RESEARCH ANALYSIS

and

UTILIZATION SYSTEM

Marijuana Effects on the

Endocrine and

Reproductive Systems

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service • Alcohol, Drug Abuse, and Mental Health Administration
Marijuana Effects on

the Endocrine and

Reproductive Systems

Editors:

Monique C. Braude, Ph.D.

Jacqueline P. Ludford, M.S.

National Institute on Drug Abuse

NIDA Research Monograph 44

A RAUS Review Report

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Public Health Service

Alcohol, Drug Abuse, and Mental Health Administration

National Institute on Drug Abuse

5600 Fishers Lane

Rockville, Maryland 20857
NIDA Research Monographs are prepared by the research divisions of the

National Institute on Drug Abuse and published by its Office of Science. The

primary objective of the scenes is to provide critical reviews of research

problem areas and techniques. the content of state of-the-art conferences, Integrative research reviews and signifigant original research Its

dual publication emphasis IS rapid and targeted dissemination to the

scientific and professional community

Editorial Advisory Board

Avram Goldstein, M.D.

Addiction Research Foundation

Palo Alto. Colifornia

Jerome Jaffe, M.D.

University of Connecticut School of Medicine

Formington, Connecticut

Reese T. Jones, M.D.

Langley Porter Neuropsychiatric lnstitute

University of California

San Francisco, California

Jack Mendelson, M.D.

Alcohol once Drug Abuse Research Center

Harvard Medical School

McLeon Hospital

Belmont, Massachusetts

Helen Nowlis, Ph.D.

Rochester. New York

Lee Robins, Ph.D.

Washington University School of Medicine

St Louis, Missouri

NIDA Research Monograph Series

William Pollin, M.D.

DIRECTOR, NIDA

Jack Durell, M.D.

ASSOCIATE DIRECTOR, OFFICE OF SCIENCE, NIDA

EDITOR-IN-CHIEF

Eleanor W. Waldrop

MANAGING EDITOR

Parklawn Building, 5600 Fishers Lane Rockville Maryland 20857
Marijuana Effects on

the Endocrine and

Reproductive Systems
ACKNOWLEDGMENT

This monograph is based upon papers and discussion from the RAUS

Review Conference on the Endocrine and Reproductive Effects of

Marijuana, held March 1 and 2, 1983, in Rockville, Maryland. The

conference was sponsored by the Office of Science and the Division

of Preclinical Research, National Institute on Drug Abuse.

COPYRIGHT STATUS

The National Institute on Drug Abuse has obtained permission from

the copyright holders to reproduce certain previously published

material as noted in the text. Further reproduction of this

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holders. All other material in this volume except quoted passages

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Citation of the source is appreciated.

Opinions expressed in this volume are those of the authors and do

not necessarily reflect the opinions or official policy of the

National Institute on Drug Abuse or any other part of the U.S.

Department of Health and Human Services.

The U.S. Government does not endorse or favor any specific

commercial product or commodity. Trade or proprietary names

appearing in this publication are used only because they are

considered essential in the context of the studies reported herein.

Library of Congress catalog card number 83-,600600

DHHS publication number (HDM)84-1278

Printed 1984

NIDA Research Monographs are indexed in the Index Medicus. They

are selectively included in the coverage of American Statistics

Index, Biosciences Information Service, Chemical Abstracts, Current

Contents, Psychological Abstracts, and Psychopharmacology Abstracts.

iv
Preface

The Research Analysis and Utilization System (RAUS) is designed to

serve four functions:

Collect and systematically classify the findings of all

intramural and extramural research supported by the

hational institute on Drug Abuse (NIDA);

Evaluate the findings in selected areas of particular

interest and formulate a state-of-the-art review by a

panel of scientific peers;

Disseminate findings to researchers in the field and to

administrators, planners, instructors, and other

interested persons;

Provide a feedback mechanism to NIDA staff and planners so

that the administration and monitoring of the NIDA

research program reflect the very latest knowledge gleaned

from research in the field.

Since there is a limit to the number of reseach findings that can

be intensively reviewed annually, four subject areas are chosen

each year to undergo a thorough examination. Distinguished

scientists in the selected field are provided with copies of

reports from NIDA-funded research and invited to add any

information derived from the literature and from their own research

in order to formulate a comprehensive vick of the field. Each

reviewer is charged with writing a state-of-the-art paper in his or

her particular subject area. These papers, together with a summary

of the discussions and recommendations which take place at the

review meeting, make up a RAUS Review Report in the NIDA Research

Monograph series.

v
The subject of the effects of marijuana on the endocrine and reproductive systems was chosen for a RAUS review in Fiscal Year 1983

because marijuana use is so widespread among American youth and,

therefore, is of great programmatic importance to NIDA. Increased

prevalence of marijuana use over the past decade has been accompanied

by decreasing age of first use, and there is grave public health

concern about its effects on youth who are undergoing maturation of

their reproductive systems at about the same time as they are likely

to begin using marijuana. Since there is a growing body of research

on the subject, it became incumbent upon NIDA to gather the knowledge

that was available, evaluate it, and disseminate it. The results of

the RAUS review are presented in this monograph.

Dr. Monique C. Braude served as the scientific chairperson for the

meeting . Jacqueline P. Ludford is the RAUS coordinator.

vi
Contents

Preface v

Executive Summary

Jacqueline P. Ludford and Monique C. Braude 1

Effects of Cannabinoids on Gene Expression

Gary S. Stein and Janet L. Stein 5

Effects of Cannabis and Natural Cannabinoids on

Chromosomes and Ova

Akira Morishima 25

Endocrine Effects of Marijuana in the Male: Preclinical Studies

Jack Harclerode 46

Endocrine Aspects of Cannabinoid Action in Female Subprimates:

Search for Sites of Action

LeeTyrey 65

Acute, Short-Term, and Chronic Effects of Marijuana

on the Female Primate Reproductive Function

Carol Grace Smith and Ricardo H. Asch 82

Effects of Marijuana on Neuroendocrine Hormones

in Human Males and Females

Jack H. Mendelson and Nancy K. Mello 97

Effect of Marijuana on Pregnancy and Fetal Development

in the Human

Katherine Tennes 115

Discussion and Recommendations

Moniaue C. Braude 124

List of NIDA Research Monographs 130

vii

Executive Summary

Jacqueline P. Ludford, M.S., and Monique C. Braude, Ph.D.

Isolated reports of impaired sexual behavior, lowered hormone

levels, and abnormal offspring in animals after administration of

marijuana or its active principles, such as delta-9-tetrahydrocannabinol (THC), prompted a review of current findings

relevant to the effects of marijuana on genetics and reproduction.

A RAUS review meeting was held on March l-2, 1983, and reviewers

were charged with evaluating the state of the art in the following

areas:

Effects of Cannabinoids

on Gene Expression

Effects of Cannabis and

Natural Cannabinoids on

Chromosomes and Ova

Effects of Marijuana in the

Male: Preclinical Studies

Endocrine Aspects of Cannabinoid

Action in Female Subprimates:

Search for Site of Action

Acute, Short Term, and Chronic

Effects on the Female

Primate Reproductive Function

Effects of Marijuana on Dr. Jack Mendelson

Neuroendocrine Function McLean Hospital/

in Human Males and Females Harvard University

Marijuana: Prenatal

Exposure in the Human

Dr. Gary Stein

University of

Florida

Dr. Akira Morishima

Columbia University

Dr. Jack Harclerode

Bucknell University

Dr. Lee Tyrey

Duke University

Dr. Carol Smith

Uniformed Services

University of the

Health Sciences

Dr. Katherine Tennes

University of

Colorado

1
Dr. Stein first discussed the importance of the preferential

expression of specific genes which can be associated with

modifications in the organization and/or representation of genetic

sequences. To address regulation of eukaryotic genetic sequences,

one must consider control at several cellular levels in the nucleus

and cytoplasm. Gene expression encompasses an extensive range of

cellular structures and biochemical processes starting in the

nucleus with DNA and terminating with the RNA molecule. Dr. Stein

then reviewed his studies on the effect of cannabinoids on the

genome, and on gene expression. To assess more definitively the

influences of cannabinoids on gene expression, Stein's group

examined the effect of Delta-9-THC on the representation of RNA

transcripts from two defined genetic sequences, histone genes and

ribosomal genes, in several human cell lines. They found that THC

causes a dose-dependent reduction in the cellular representation of

histone mRNA sequences at the higher concentrations used in their

assay. The extent to which cannabinoids affect the expression of

specific genetic sequences other than histone sequences is still an

open-ended question. Understanding the manner in which druginduced alterations in gene expression are brought about should,

Stein believes, provide insight into the molecular basis of

cannabinoid-related modifications in cellular function.

Dr. Morishima reviewed the various reports on studies of the

effects of marijuana and natural cannabinoids on chromosomes. The

evidence from the available cytogenetic studies suggests that

cannabis and cannabinoids are extremely weak clastogens, i.e.,

produce little chromosome breakage and that their clastogenic

effects become apparent only in appropriately sensitive test

systems such as primary spermatocytes and bone marrow cells,

whereas the human lymphocyte system is relatively insensitive to

their clastogenic effects. He then reported the results of his

recent studies on the effects of THC on mice ova, showing that

chronic administration of THC to sexually developing mice produced

an increase in abnormal ova, although the percentage of increase

was small. It appears that this increase in degenerated ova was

caused by their inability to successfully undergo first cleavage

division, probably affecting the process of meiosis. Following his

review of the studies which reported errors of chromosome

separation (ECS) and of this recent data, Morishima now proposes a

new concept that cannabis and cannabinoids in vitro and in vivo act

by disrupting the meiotic as well as the mitotic processes.

Dr. Harclerode focused on the effect of chronic exposure of

laboratory animals to cannabinoids, with emphasis on the male

reproductive system. The reports in the literature of reduction in

reproductive organ weights are accompanied by reports that show

that the auality and ouantity of sperm produced by the testis are

affected by cannabinoids. Treatment of mice with THC for as little

as 5 days resulted in reduction of sperm production and appearance

of abnormal sperm. This was often accompanied by a decrease in

testicle and seminal vesicle weights. Two gonadotropins, LH and

FSH, secreted by the pituitary gland are of major importance to

reproduction in the male. A single hypothalamic factor, the

2
gonadotropin-releasing hormone (GnRH) is believed to be responsible

for the release of LH and FSH. THC induces a block of GnRH release

which results in lowered LH and FSH, thus reducing testosterone

production by the Leydig cells of the testis. Other hormones that

might have a synergistic or antagonistic effect upon reproduction

in the male are the adrenal cortical hormones, thyroid hormones,

growth hormones, and prolactin. THC appears to depress prolactin,

thyroid gland function, and growth hormone while elevating adrenal

cortical steroids.

Dr. Tyrey reviewed the effects of cannabinoids, primarily THC, on

the female reproductive function in subprimates and discussed

cannabinoid action on the target organs (uterus and ovary) as well

as on the CNS and the hypothalamic-pituitary axis. He concluded

that the search for a site of cannabinoid action in subprimates has

raised the possibility of cannabinoid effects at each level of the

female reproductive system. He feels that the early suggestion

that THC may have a direct "estrogen-like action on the uterus" has

not been substantiated by later studies which failed to show that

THC interacts with the estrogen cytoplasmic receptor. However,

there is now evidence that THC alters the secretion of reproductive

pituitary hormones (LH, prolactin) and of ACTH through effects in

the brain.

Dr. Smith reviewed the acute and chronic effects of THC on the

reproductive function of the female primate. She pointed out that

studies in these species show that cannabinoids inhibit secretion

of LH and FSH as well as prolactin. These changes in pituitary

hormones produce decreases in sex steroid hormones and cause

changes in ovulation. Dr. Smith also emphasized that the principal

site of action of cannabinoids is the hypothalamus. Her recent

findings show, however, that these cannabinoid effects are

reversible in sexually mature animals when drug treatment is

terminated and that there is development of tolerance to the

effects of THC after chronic administration. This may explain why

evidence of disrupting effects on female reproductive function has

been scarce. She observed that, in humans, it is not yet known how

much disruption of reproductive hormone levels is necessary for

changes in human fertility and sexual function to become apparent,

and she emphasized the need for clinical studies in female

marijuana smokers.

There are technical problems in obtaining data on the effect of

marijuana on human reproductive systems, but Dr. Mendelson reported

on the available data and on his own considerable work in this

area. In his study of the effects of marijuana on pituitarygonadal hormones in human males, he found that the use of marijuana

alone did not suppress testosterone levels. Similar carefully

controlled studies of human female hormone levels are scarce,

although reports from animals suggest, as mentioned above, that THC

may produce a significant decrease in prolactin and LH. There have

been reports that marijuana users had shorter menstrual cycles and

lower prolactin levels than age-matched nonusers. A residential

ward study in human females is now underway, and some preliminary

observations were reported at the meeting.

3
Dr. Tennes reviewed the current knowledge about the effect of

marijuana on human pregnancy and fetal development. Although 10%

to 37% of pregnant women report use of marijuana, evidence

regarding its effect is confounded by the use of other substances,

nutrition, truthfulness of the woman's recall and report of the

amount of use, changes in use during pregnancy and the trimester

when these changes occurred, and a host of other technical

problems. There is suggestive evidence that marijuana may alter

the delivery process, reduce intrauterine weight gain by the fetus,

or affect visual and neurological excitatory responses. All of

these findings need to be confirmed in their relationship to

marijuana use, especially since marijuana is freauently used in

conjunction with tobacco and alcohol, which have their own

deleterious effects on the fetus.

These data will be further discussed in the Discussion and

Recommendations Section.

AUTHORS

Jacqueline P. Ludford, M.S.

Research Analysis Branch

Office of Science

National Institute on Drug Abuse

Rockville, Maryland 20857

Monique C. Braude, Ph.D.

Biomedical Branch

Division of Preclinical Research

National Institute on Drug Abuse

Rockville, Maryland 20857

4
Effects of Cannabinoids on Gene

Expression

Gary S. Stein, Ph.D., and Janet L. Stein, Ph.D.

INTRODUCTION

In this article we will consider approaches that have been taken

and can be taken to assess the influence of cannabinoids and other

abused substances on the genome and on gene expression. This is a

problem central to understanding drug-induced effects on a broad

spectrum of biological processes since numerous modifications in

cell structure and function, which have been reported to be

associated with abused substances, either a) affect expression of

genetic sequence or b) are a reflection of modifications in gene

expression. Within this context we should emphasize that

drug-induced perturbations in gene expression can result from

alterations in the genome itself or from modifications in the

transcription, processing, or translation of genetic information.

This article will be divided into three parts. First, by way of

introduction, we will summarize the experimental basis for our

current concepts of the eukaryotic genome and eukaryotic gene

control. Second, we will review approaches that nave been taken

to address the influence of cannabinoids on gene expression. We

will then consider approaches, which can be taken and should be

pursued, to further define in molecular terms cannabinoid-induced

effects on the structure, organization, and regulation of specific

genes.

It is our strong conviction that there are many long-standing and

to date unresolved questions related to cannabinoid-induced

effects on genes and gene control. Answers to these questions are

essential to understand the influence of abused substances from

the standpoints of immediate health hazards and, perhaps even more

important, of hereditary effects. It is encouraging tnat during

the past several years our understanding of genes and gene

regulation in cells has evolved dramatically, largely through a

number of highly innovative cellular and molecular approaches that

have been taken to address the organization and regulation of

eukaryotic genes. We are therefore now in a position,

conceptually and technologically, to apply these approaches to

assessing the effects of abused substances on the genome and on

gene expression--particularly in human cells.

5
I. Genes and Gene Regulation

Several of the experimental observations which historically have

served as the basis for our current concept of gene expression are

summarized in table 1. While in general terms, these classical

observations have a direct bearing on the manner in which

eukaryotic genes are controlled, a number of subtle qualifications

based on recent results provide explanations for long-standing

inconsistencies in our understanding of eukaryotic gene regulation.

TABLE 1

Gene Expression in Eukaryotic Cells

1 . All diploid cells in an organism contain the same amount of

DNA.

2. All diploid cells contain identical genetic information.

3. Limited expression of genes in all cells.

4. Differences and similarities in expression of specific genes

in differentiated cells.

5. Ability to modify expression of specific genes.

The initial experimental Observations which led to models for

eukaryotic gene control were that all diploid cells of an organism

contain the same amount of DNA and that the DNA sequences present

in all diploid cells are identical. Equally important were the

observations that all cells express only a limited number of

genetic sequences and that those genes expressed reflect general

metabolic requirements shared by all living cells as well as

specialized requirements of differentiated cells. For example,

almost all cells express genes encoding enzymes involved in

intermediary metabolism while expression of globin genes is

restricted to erythropoietic cells. Superimposed upon this

preferential expression of specific genes, which permits cells to

execute their specialized biological/biochemical functions, is the

flexibility to permit variation in those genes expressed in

response to modifications of cellular activities or cellular

requirements. It was these observations that led to experimental

pursuit of the mechanisms by which defined genetic sequences are

selectively expressed while others are held in a nontranscribed

structure, conformation, and transcriptional state. What we must

now additionally take into consideration is that expression of

genes can be associated with modifications in the organization

and/or the representation of genetic sequences.

6
Our views of eukaryotic genes and eukaryotic gene regulation are

constantly evolving. Structural and functional properties of

genes are largely inseparable, as reflected by a functional

relationship between the organization and expression of genetic

sequences. The eukaryotic genome is a protein-DNA complex, both

chromosomal proteins and DNA being essential for genome structure,

and alterations in the interactions of chromosomal proteins with

DNA in turn affect transcription or the transcriptional potential

of specific genes. It is becoming increasingly apparent that the

eukaryotic genome is not a static macromolecular complex, but

rather is subject to modifications in organization, structure, and

conformation which influence expression. There are different

types of genes, those which encode proteins and those for which

the products are ribosomal or transfer RNAs. Moreover, there are

substantial differences in the organization of various genetic

sequences, ranging in complexity from genes whose encoded proteins

are represented by contiguous nucleotide sequences to genes from

which the transcripts must undergo numerous splicing steps to

generate functional messenger RNAs. It has been well documented

that different genes are under different types of regulation.

Likewise, there may be some differences in the structure and

regulation of the same genes in different biological situations.

It therefore follows that to address regulation of eukaryotic

genetic sequences it is necessary to consider control at several

levels, which have been delineated in table 2. By definition gene

expression encompasses an extensive range of cellular structures

TABLE 2

Regulation of Gene Expression

TRANSCRIPTION

.Deletion-Addition

NUCLEUS DNA .Rearrangement

.Amplification

.Methylation

TRANSCRIPT PROCESSING

.Splicing

Nucleoplasm .5' Capping

.3" Polyadenylation

.Methylation

.RNA-Protein complexes

TRANSPORT TO CYTOPLASM

CYTOPLASM TRANSLATION

POST-TRANSLATIONAL MODIFICATIONS

7
and biochemical processes, beginning in the nucleus at the DNA

double helix and terminating with a completely processed and

functional protein or RNA molecule. This presents a problem of an

extremely complex nature, and cannabinoid-induced lesions may

reside at any one or a combination of cellular levels.

Within the nucleus key steps in control of gene readout reside at

the level of the genome and in the nucleoplasm. Cannabinoids may

influence the structure and/or function of DNA nucleotide

sequences which constitute structural genes or their components,

in which case regions of the genome coding for defined proteins

would not be transcribed or the transcripts would not be appropriately processed and translated into functional proteins. In

addition, cannabinoid-induced alterations in genetic sequences

coding for the synthesis of ribosomal RNAs, tRNAs, or purported

"regulatory RNAs" must be considered. Cannabinoio-induced

alterations may also become apparent in the nucleotides contained

within regulatory sequences or within those sequences involved in

punctuating the genetic code. In an overall evaluation of tne

mechanisms by which cannabinoids may modify genes, one must bear

in mind that there are four general categories of changes in the

nucleotide bases which are prevalent--base substitutions,

modifications of preexisting bases, base additions, and base

deletions. Recent evidence for additions, deletions, and

amplification of nucleotide sequences, as well as rearrangements

of genetic sequences in conjunction with expression, necessitates

serious consideration of quantitative and qualitative modifications in DNA as potential regulatory events, and hence targets for

drug-induced perturbations in gene expression. Within this

context drug-mediated effects on DNA methylation, which has been

implicated in structural/transcriptional properties of genetic

sequences, should not be overlooked.

In evaluating the implications of cannabinoid-associated DNA

sequence modifications, one must critically determine the

influence of these drugs on the capability of the cell to repair

its DNA correctly. The repair process may itself introduce or

amplify errors.

Cannabinoid-induced modifications in gene expression may also

result from changes in macromolecules, principally chromosomal

proteins, which interact with DNA and are intimately involved with

the structural and transcriptional properties of the genome.

Variations of these proteins and their mode of association with

other genome components may be attributable to alterations in

amino acid sequences as well as to post-translational modifications such as acetylation, methylation, phosphorylation, and

ADP-ribosylation. It should be kept in mind that cannabinoidinduced changes in the metabolism of acetate, methyl, phosphate,

and ADP-ribose groups may be caused by variations in genetically

coded enzymes which are responsible for the addition and removal

of these moieties from genome-associated proteins. In addition,

some of these post-translational modifications of chromosomal

proteins may occur, at least in part, by nonenzymatic mechanisms.

8
Another class of macromolecules which possess the ability to

influence readout as as function of cannabinoid treatment are the

RNA polymerases. Here, cannabinoid-induced changes may reside in

any one or several of the polymerases, in any one or several of

the subunits of the given polymerase, or in "factors" which

influence the specificity or efficiency of the enzyme.

A complex system which contains numerous focal points for

cannabinoid-induced lesions in the expression of genetic

information is that which is utilized in the processing of RNA

molecules. This is a multicomponent system consisting of:

a) endo- and exonucleases which cleave and degrade ribonucleotide

sequences during RNA precursor processing; b) enzymes modifying

ribonucleotide bases; c) nucleotidyl exotransferases which utilize

the 3' and 5' ends of RNA molecules as primers for addition of

nontemplated ribonucleotides; and a) proteins which complex with

RNAs or precursors thereof and are involved with enzymatic

modifications of transcripts, export of transcripts from the

nucleus to the cytoplasm, or assembly of functional translational

complexes. Such processing occurs in the three principal classes

of RNA molecules--ribosomal RNAs, messenger RNAs, and transfer

RNAs. While these reactions generally occur in the nucleoplasm,

they have also been reported to take place, to some extent, in the

cytoplasm.

Cannabinoid-induced aberrations in gene expression may also result

from perturbations in the equally complex cellular protein synthesizing and processing machinery which resides primarily in the

cytoplasm. This may involve lesions in ribosomal and transfer

RNAs, in ribosomal proteins, in the extensive range of "translational factors," and in enzymes involved in the assembly and/or

activation of proteins. Enzymes involved with protein turnover

constitute targets often overlooked when considering potentially

important sites for cannabinoid-induced lesions in gene expression.

From the preceding discussion it should be apparent that

cannabinoid-induced modifications in gene expression may result

from perturbations in a broad spectrum of macromolecular,

biosynthetic processes in the nucleus as well as in the

cytoplasm. Any step in the elaboration and processing of genetic

information is a potential target for a drug-induced lesion. Do

cannabinoids modify the structure or composition of the genome?

Do cannabinoids modify which genes are transcribed and which

remain silent? Do cannabinoids affect the efficiency or fidelity

of transcription? Are RNA processing steps modified by

cannabinoids? Do these drugs act at the translational level? The

key to addressing these questions is availability of high

resolution procedures for detecting cannabinoid-induced changes in

gene expression at various levels, and equally important, for

determining if drug-induced perturbations in gene expression are

functional or nonfunctional.

9

446-384 O - 84 - 2
II. Effect of Cannaoinoids on the Genome

A. Composition of the Genome

The eukaryotic genome exists in the form of a protein-DNA complex

(Stein et al. 1974, 1975); hence, an assessment of cannabinoidinduced effects on the composition of the genome requires

evaluating the influence of cannabinoids on both DNA and

chromosomal proteins. It is also necessary to consider the

influence of cannabinoids on both chromatin and on chromosomes

since these represent interchangeable modes of genome packaging.

Several laboratories have investigated the effects of cannabinoids

on chromosome morphology and on the cellular representation of

specific chromosomes. Yet, to date this remains an area where

considerable controversy exists. The critical issues are whether

cannabinoids exhibit clastogenic activity, that is, induce

chromosome breaks, and/or whether cannabinoids act as mitotic

poisons. The latter effect would imply drug-induced action,

direct or indirect, on the mitotic apparatus or on the region of

the chromosome where attachment of spindle fibers occurs--

centromeric DNA or centromere-associated chromosomal proteins.

The mutagenic nature of cannabinoid-induced chromosomal lesions

also remains to be resolved. An indepth review of these

chromosome-related effects of cannabinoids is covered in the

chapter by Morishima in this volume.

An examination of the influence of cannabinoids on chromosomal

proteins indicates that the relative composition of both histones

and nonhistone chromosomal proteins is not significantly altered.

However, psychoactive and nonpsychoactive cannabinoids appear to

bring about a dose-dependent decrease in the synthesis of some

chromosomal polypeptides (Mon et al. 1981a,b). These results tend

to suggest that while cannabinoids do not affect the relative

cellular levels of specific histones, which are the molecules

primarily responsible for DNA packaging, these drugs may affect

the ability of cells to express genes which code for histone

proteins and/or affect histone protein turnover. Nonhistone

chromosomal proteins, which are involved in structural, enzymatic,

and regulatory action at the level of the genome, may be similarly

affected following cannabinoid treatment. Variations observed in

the extent to which chromosomal proteins are acetylated following

cannabinoid treatment can be related to changes in the nature of

chromosomal protein-DNA interaction, which may in turn reflect

drug-induced modification in chromatin structure and/or in

transcriptional properties of the genome. The studies carried out

to date do indeed suggest possible drug-induced changes in genome

composition, structure, and function but the data are of a

correlative nature. Although as discussed above, cannabinoidinduced alterations in gene organization are not an unrealistic

expectation, experimental data to substantiate or eliminate such a

possibility are lacking.

10
B. Gene Expression

Two approaches have been undertaken in several laboratories,

including ours, to study cannabinoid-induced effects on gene

expression (Blevins and Regan 1976; Carchman et al. 1976a,b;

Desoize et al. 1979; End et al. 1977; Green et al. 1983; Nahas et

al. 1974a,b, 1977; Lemberger 1973; McClean and Zimmerman 1976; Mon

et al. 1978, 1981a,b; Nahas and Desoize 1974; Nahas and Paton

1979; White et al. 1976; Zimmerman and McClean 1973; Zimerman and

Zimmerman 1976; Zimmerman et al. 1979). Early in vivo studies

suggested that cannaoinoid treatment brings about dose-dependent

inhibition of 3H-thymidine incorporation into DNA, 3H-uridine

incorporation into RNA, and 3H-leucine incorporation into

protein. However, these results, particularly the 3H-uridine

and 3H-leucine results, are complicated by the influence of

cannabinoids on the ribonucleotide and amino acid precursor pools--

perhaps in part a reflection of cannabinoid-induced effects on

cellular membranes. In vitro transcription stuaies carried out

using isolated nuclei, DNA, or chromatin suggest that such

preparations from untreated control and cannabinoid-treated cells

do not differ significantly with respect to their ability to

synthesize RNA. Interpretation of the latter studies is not

complicated by drug-related effects on precursor pools; however,

from these in vitro experiments it is possible to conclude only

that the overall transcriptional capacity of the genome is

refractory to cannabinoid treatment, and no indication of possible

cannabinoid-induced effects on the qualitative nature of gene

transcription can be gleaned. Furthermore, caution should be

exercised in interpreting results from in vitro studies because

the fidelity of the transcription process and the transcripts by

necessity must be carefully evaluated.

Recently, to assess more definitively the influence of

cannabinoids on gene expression, we examined the effect of

THC on the representation of RNA transcripts from two defined

genetic sequences, histone genes and ribosomal genes, in several

human cell lines. Levels of cellular histone mRNAs and ribosomal

RNAs were assayed by hybridization with cloned genomic human

histone and ribosomal genes under conditions where quantitation

was not influenced by nucleotide precursor pools. Our results

suggest that -THC causes a dose-dependent reduction in the

cellular representation of histone mRNA sequences. This

drug-induced reduction is at least to some extent selective

because cellular levels of ribosomal RNAs are not affected. We

have also observed that the cannabinoid-induced effect on histone

gene expression is less pronounced in human cells with active

drug-metabolizing systems.

Human histone and ribosomal genes represent two distinct types of

genetic sequences which differ with respect to their organization,

regulation, and functions. Human nistone genes are a family of

moderately reiterated genetic sequences--approximately 40 copies

per haploid genome. Each histone mRNA is transcribed from a set

of contiguous nucleotide sequences (unspliced), and histone gene

11
expression is related to cell proliferation. The gene products,

the histone proteins, are required for packaging several yaras of

DNA into "nucleosomes" where they are contained in a nucleus only

several microns in diameter. These histone proteins are necessary

for genome replication (to package newly replicated DNA) and

additionally play a role in the control of gene expression. The

human ribosomal genes are represented as a reiterated set of

sequences and the final gene products are the major structural RNA

species associated with large and small ribosomal subunits. In

contrast to the histone genes, where the primary transcripts

undergo a minimal amount of processing, the 5.8S, 18S, and 28S

ribosomal RNAs are derived from a 45S precursor via a series of

post-transcriptional cleavages.

Initially, the steady state levels of histone mRNAs were

determined in exponentially growing human cervical carcinoma

cells, HeLa S3 cells, following treatment with increasing

concentrations of -THC. Total cellular RNAs were fractionated

electrophoretically in 1.5% agarose gels (Rave et al. 1979),

transferred to nitrocellulose (Southern 1975) and hybridized with

32p-labeled [nick-translated (Maniatis et al. 1975)] cloned

genomic human histone sequences (Sierra et al. 1982). The levels

of histone mRNAs were then assayed autoradiographically.

Isolation of total celluar RNA permits greater than 90% recovery,

circumventing loss of RNA through nuclease activity and physical

manipulations which generally occur during subcellular fractionation. Because the hybridization probe is radiolabeled

in vitro rather than the cellular RNAs in vivo, quantitation of

RNAs is not complicated by the intracellular ribonucleotide

precursor pools. RNA samples are quantitated spectrophotometrically prior to electrophoretic fractionation and the extent

of transfer to nitrocellulose is monitored by ethidium bromide

staining and/or ultraviolet shadowing prior to and following

diffusion transfer. The efficiency of transfer to nitrocellulose

by the procedure used in these experiments has been monitored by

transfer of 32p-labeled DNA and shown to be greater than 95%.

The data in figures 1 and 2 clearly indicate that -THC brings

about a dose-dependent decrease in the representation of mRNAs for

the four core histone proteins, H2A, H2B, H3, and H4. Shown in

figure 1A is a hybridization signal obtained when 50 μg of

nitrocellulose-immobilized, total cellular HeLa cell RNAs from

control, and -THC treated, cells are hybridized with a cloned

human DNA sequence (pFF435) encoding H2A, H2B, and H3 histone

mRNAs. While the levels of H2A, H2B, an H3 histone mRNAs

isolated from cells treated with 10 μM -THC are not below

those from nondrug-treated or vehicle-treated controls, a marked

inhibition (greater than 80%--see table 3) is observed in cells

treated with 30 μM and 40 μM drug concentrations. Verification

that equivalent amounts of all' RNA samples were fractionated can

be gleaned from figure 1B which shows similar levels of ethidium

bromide staining of all RNAs and from figure 1C which shows

similar levels of all RNAs by ultraviolet shadowing. It should be

12
TABLE 3

Effect of 9-THC on Cellular Levels

of Human (HeLa) Histone mRNAs

Treatment Drug Conc. % Inhibition

-THC 10 μM 0.0

-THC 30 μM 78.1

-THC 40 μM 81.0

Vehicle Control 0 0.0

Control 0 0.0

FIGURE 1A

A) Effects of varying concentrations (10 μM, 30 μM, 40 μM, VCvehicle treated control and C-control) of -THC on the representation of mRNAs for three of the four core histone proteins, H2A, H2B,

and H3. The signals shown were obtained when 50 μg of electrophoretically fractionated nitrocellulose-imobilized total cellular

HeLa cell RNAs were hybridized to a cloned human DNA sequence

(FF435) encoding H2A, H2B, and H3 histone mRNAs.

13
B.) Ethidium bromide stain of 1.5% (w/v) agarose gel with 6% (w/v)

formaldehyde, containing 10 μg of each of the -THC treated and

control samples of total cellular RNAs from HeLa cells. The gel was

stained for one hour in 0.1 M anmonium acetate containing 0.1 μg/ml

ethidium bromide and destained overnight in water. The gel was

placed on a shortwave ultraviolet transilluminator and photographed

with Polaroid type 57 film using an orange filter.

C) Ultraviolet shadowing of 1.% (w/v) agarose gel with 6% (w/v)

formaldehyde, containing 50 μg of each of the -THC-treated and

control samples of total cellular RNAs from HeLa cells. The gel

was placed on a cellulose-fluorescent thin layer chromatography

plate and illuminated from above by shortwave ultmviolet ligkt.

The gel was photographed with polaroid type 57 film using an orange

filter.

D) Densitometric scan of autoradiographic hybridization signals

obtained when 50 μg of electrophoretically fractionated nitrocellulose immobilized total cellular RNAs from HeLa cells treated with

varying concentrations of -THC were hybridized to a cloned human

DNA sequence (pFF435) encoding H2A, H2B, and H3 histones. The top

portion of the scan meusures the absorbance of the signal which is

determined electronically within the densitometer based on the measured optical density. The lower portion is the Zig-Zag time base

integrator and is used to quantitate the area under the curve and

thus, the concentration of the sample.

14
FIGURE 2A FIGURE 2B

Effects of varying concentrations (10 μM, 30 μM, 40 μM, VC-vehicle

treated control and C-control) of -THC on the representation of

mRNAs for histones H3 and H4. The signals shown were obtained when

50 μg of electrophoretically fractionated, nitrocellulose-immobilized

total cellular HeLa cell RNAs were hybridized to cloned hwnan DNA

sequences encoding: A) H3 histone (pF0422) and B) H4 histone

(pF0108A).

noted that because equivalent amounts of RNA from control and

drug-treated cells were analyzed, the data in figure 1A reflect a

dose-dependent, -THC-mediated inhibition in the relative

representation of three core histone mRNA species. A

dose-dependent inhibition of the absolute amounts of H2A, H2B, and

H3 histone mRNA/cell, with pronounced inhibition evident at 30 and

40 uM drug concentrations, was also observed when equivalent

aliquots (by volume) of RNA extracts from equivalent numbers of

control and -THC-treated cells were similarly analyzed (see

figure 1D). The data in figure 2 are results from experiments in

which total cellular RNAs from control and -THC-treated

exponentially growing HeLa S3 cells were analyzed by hybriaization

with cloned genomic H3 (pF0 422) (figure 2A) or H4 (pF0 108A)

(figure 2B) histone sequences. Consistent with the results shown

in figure 1, a greater than 80% inhibition in the representation

of H3 and H4 histone mRNAs was observed following treatment with

30 and 40 μM drug concentrations.

15
The influence of -THC on the levels of histone mRNAs was then

studied in normal human diploid cells (WI38 human diploid fibroblasts) and in SV40-transformed WI38 cells. A dose-dependent,

drug-induced decrease in the levels of all four core nistone mRNAs

was observed in both normal human diploid fibroblasts and in

SV40-transformed human diploid fibroblasts--a cannabinoid-induced

inhibition similar to that seen in HeLa S3 cells. As shown in

figures 3A and B, when total cellular RNAs from control and

Ag-THC-treated WI38 cells are hybriaized with 32p-labeled

pFF435, a plasmid containing cloned human genomic H2A, H2B, and H3

histone coding sequences, decreased levels of histone mRNAs are

observed in both normal WI38 and in SV40-transformed WI38 cells

treated with 30 and 40 μM drug concentrations. Confirmation of

the -THC-mediated inhibition of core histone mRNA levels in

normal and SV40-transformed WI38 human diploid fibroblasts can be

seen in figures 3C and 3D as well as in figures 3E and 3F where

similar drug-induced inhibitions in the representation of H3 and

H4 mRNAs, respectively, were observed.

The levels of H2A, H2B, H3, and H4 histone mRNAs were similarly

assayed in A549 human lung carcinoma cells after treatment with

A

g

-THC. These cells have been reported to have active drug

metabolizing systems and to efficiently metabolize polycyclic

hydrocarbon-containing carcinogens. A pronounced decrease in the

inhibitory effect of 9-THC on the representation of core

histone mRNAs was observed in A549 cells compared With HeLa S3

cells and WI38 cells (normal and SV40-transformed). It is

unlikely that the reduced sensitivity of A549 cells to cannabinoid

treatment is attributable to changes in drug uptake. Tne

intracellular levels of -THC in SV40-transformed WI38 cells

and in A549 cells, when monitored by intracellular incorporation

of 3H THC (table 4) do not reflect the differences seen in

histone mRNA levels (figures 3 and 4).

TABLE 4

Cellular Uptake and Subcellular

Distribution of 3H- -THC

cell type cpm/l07

 cells % nucleus % cytoplasm

SV-40-WI-38 1.2 x 105

32.6% 67.4%

A549 1.3 x 105

37.9% 62.1%

16
FIGURE 3E FIGURE 3F

Effects of varying concentrations (10 μM, 30 μM, 40 μM, VC-vehicle

treated control and C-control) of -THC on the representation of

mRNAs for the four core histones. The Signals shown were obtained

when 50 μg of electrophoretically fractionated nitrocelluloseimmobilized total cellular RNAs were hybridized to cloned human DNA

sequences. A) WI38 and B) SV40-WI38 total cellular RNA hybridized

to a DNA probe (pFF435) encoding H2A, H2B and H3 histones; C) WI38

and D) SV40-WI38 total cellular RNA hybridized to a DNA probe

(pFO422) encoding H3 histone; E) WI38 and F) SV40-WI38 total cellular RNA hybridized to a DNA probe (pF0108A) encoding H4 histone.

17
FIGURE 4B FIGURE 4C

Effects of varying concentrations (10 μM, 30 μM, 40 μM, VC-vehicle

treated control and C-control) of -THC on the representation of

mRNAs for the four core histones. The signals shown were obtained

when 50 μg of electrophoretically fractionated, nitrocelluloseimmobilized total cellular RNAs from A549 cells were hybridized to

cloned human DNA sequences coding for: A) H2A, H2B, and H3 histones

(pFF435); B) H3 histone (pFF422); C) H4 histone (pF0108A).

Several lines of experimental evidence suggest that the -THCinduced reductions in histone mRNA levels we have observed in

normal and transformed human cells are not merely a reflection of

a general, nonspecific cannabinoid-induced inhibition in RNA

synthesis. As reported previously, the cannabinoid-induced

inhibition of 3H-uridine incorporation into total cellular RNAs

largely reflects a drug-induced influence on the intracellular

nucleotide precursor pool rather than an effect on cellular RNA

metabolism (Mon et al. 1981a,b). The absence of a significant

quantitative effect of psychoactive and nonpsychoactive

cannabinoids on levels of nuclear (Mon et al. 1981a,b) or

chromatin (Mon et al. 1981a,b) transcription in vitro further

suggests that these drugs do not interfere with the general levels

or rates of cellular RNA synthesis.

18
The inability of -THC, at concentrations between 10 and 40 μM,

to modify the levels of ribosomal RNAs provides more direct

evidence for some extent of specificity to the cannabinoidmediated decrease in histone mRNA levels. In all experiments

reported in this paper, the representation of 18S and 28S

ribosomal RNAs was monitored in control, and in -THC-treated,

cells by staining gels with ethidium bromide and by ultraviolet

shadowing. A typical example of a gel showing the levels of the

major ribosomal RNAs in control and in drug-treated cells is shown

in figures 1B and 1C. Additionally, when electrophoretically

fractionated cellular RNAs from control and -THC-treated cells

were hybridized with 32P-labeled cloned human 18S (LS-2) and 28s

(LS-6) ribosomal RNA coding sequences, a dose-dependent decrease

in the representation of these RNAs was not observed. Figures 5A

and 5B show no change in the levels of 28S ribosomal RNAs from

-THC-treated HeLa and SV40-transformed WI38 cells in the same

RNA samples where greater than 80% reduction was observed for the

representation of core histone mRNAs in treated cells. Figure 5C

shows unchanged levels of 18S ribosomal RNA in these same cells

following hybridization with 32P-labeled human 18s ribosomal DNA.

A long-standing question has been whether cannabinoids influence

the expression of specific genetic sequences. While cannabinoidinduced effects on cell structure and function, coupled with

cannabinoid-mediated modification in macromolecular biosynthesis,

are consistent with such a contention, direct experimental

evidence for an effect of cannabinoids on expression of specific

genes has to date not been reported. In this paper we present

data which indicate that treatment of exponentially growing normal

diploid and transformed human cells with Ag-THC results in a

dose-dependent decrease in the representation of histone mRNAs,

with a decreased sensitivity of cells with highly developed drug

metabolizing systems. This cannabinoid-mediated reduction of

cellular histone mRNA levels does not simply reflect a general

decrease in cellular mRNA levels or in cellular RNA metabolism.

We also present data indicating that the levels of ribosomal RNAs

are not altered by the concentrations of -THC used in our

studies, and we have reported previously that general levels of in

vitro and in vivo RNA synthesis are not quantitatively affected by

either psychoactive or nonpsychoactive cannabinoids.

While our results clearly indicate that -THC preferentially

inhibits expression of histone genes, the levels at which

regulation is perturbed and the biological implications of this

cannabinoid-mediated effect remain to be resolved. The reduction

in cellular levels of histone mRNAs after cannabinoid treatment

may be attributable to alterations in mRNA stability, transcription, or processing of histone transcripts. Additionally,

drug-induced structural modifications in the histone genes and in

their flanking regulatory sequences should also be considered

within this context. By analogy with other moderately reiterated

eukaryotic sequences which have been shown to unaergo structural

modifications in conjunction with phenotypic changes, cannabinoidinduced effects on the structural features of human histone genes

19
Effects of varying concentrations (10 μM, 30 μM, 40 μM, VC-vehicle

treated control and C-control) of -THC on the representation of

28S and 18S ribosomal RNAs. The signals shown were obtained when

10 μg of electrophoretically fractionated, nitrocellulose-immobilized

total cellular RNAs were hybridized to cloned human DNA sequences.

A) HeLa and B) SV40-WI38 total cellular RNA hybridized to a DNA probe

(LS-6) encoding 28S RNA; C) HeLa total cellular RNA hybridized to a

DNA probe (LS-2) encoding 18S RNA.

could be a possibility. The extent to which the expression of

specific genetic sequences other than histone sequences is

affected by cannabinoids is also an open-ended question--one which

is particularly important because the organization and regulation

of the moderately reiterated human histone genes differ considerably from those of the more complex spliced single copy genes.

From a biological standpoint the selective effect of -THC on

expression of histone genes may be understandable. Expression of

histone genes has been shown to be temporally and functionally

coupled with DNA replication (Stein et al. 1979; Stein and Borun

1972; Wu and Bonner 1981), and cannabinoids have been shown to

bring about a dose-dependent inhibition in cell proliferation

(Blevins and Regan 1976; Carchman et al. 1976a,b; Desoize et al.

1979; End et al. 1977; Green et al. 1983; Nahas et al. 1974a,b,

1977; Lemberger 1973; McClean and Zimmerman 1976; Mon et al. 1978,

1981a,b; Nahas and Desoize 1974; Nahas and Paton 1979; White et

20
al. 1976; Zimmerman and McClean 1973; Zimmerman and Zimmerman

1976; Zimmerman et al. 1979). In fact, in the normal and

transformed cell lines we have examined, the extent to which

histone mRNA levels are affected by -THC is paralleled by the

extent to which proliferative activity is affected by

cannabinoids. It remains to be determined whether expression of

other genetic sequences, whose expression is prerequisite for DNA

replication or mitotic division, are preferentially inhibited by

cannabinoids. Equally important is whether the cannabinoidmediated modifications in cellular histone mRNA levels are

attributable to a direct effect on the histone genes or the

transcripts, or alternatively, whether the effects of cannabinoids

on histone gene expression are indirect, e.g., acting initially on

other genetic sequences or cellular macromolecules.

III. Approaches to Defining Effects of Cannabinoids on Specific

Genes

Cannabinoid-induced modifications in cell structure and function

have been well-documented as have a series of physiological

effects resulting from such drug-induced cellular changes. Two

pivotal biological processes which have been shown to be

dramatically influenced by cannabinoids are endocrine function and

cell proliferation--both of which have been reviewed in this

monograph. Moreover, these are not unrelated processes since in

many cases proliferation is responsive to hormonal control.

Understanding the manner in which drug-induced alterations in gene

expression are brought about should provide insight into the

molecular basis of cannabinoid-related modifications in cellular

function.

Since cannabinoid-induced alterations in gene expression can

result from changes in the organization of genetic sequences

and/or in the manner in which genetic information is transcribed

and processed, a critical and systematic evaluation of the

influence of cannabinoids on the structure and expression of

specific genetic sequences, particularly in human cells, should be

a high priority. Understanding cannabinoid-induced effects on

human gene organization and expression is prerequisite to

evaluating possible short-term, long-term, and hereditable

disorders that may arise from the use of these drugs either

therapeutically or as abused substances. Of equal importance,

despite the history of fragmentary and often controversial reports

of cannabinoid-induced modification in genome-related phenomena

(e.g., chromosomal changes, alterations in RNA synthesis, etc.),

we are now in a position to address these issues directly and

definitively. Availability of a series of cloned human genes

permits evaluation of drug-related effects on specific genes, on

defined regions of genes, and on transcription and processing of

genetic information. Examples of ways in which cloned genetic

sequences can be utilized as high resolution probes for the

identification and quantitation of several specific human gene

transcripts were presented in the previous section of this chapter.

21
It will be instructive to focus efforts where possible on human

studies; for example, drug-induced effects on the organization of

specific genetic sequences or regions thereof can be performed

using DNA from only 20 ml of blood. Thereby the opportunity is

available to determine the effects of cannabinoias on the genomes

of subjects participating in endocrine function and behavior

studies. A number of normal and tumor-derived human cell lines

are available and should be utilized to complement such an

approach. By combining both intact organism and cell culture

approaches, it is possible to draw on the physiological reality of

the organism and the biochemical simplicity of isolated cells. It

would also be appropriate to concentrate efforts on evaluating

drug-induced effects on a limited series of genetic sequences,

those related to proliferation and endocrine function, where

phenotypic effects are well understood and the information

obtained can be integrated with other ongoing investigations.

In summary, we have attempted to discuss a series of high

resolution approaches and procedures which can provide important

information regarding the influence of cannabinoids on genome

structure and function. Examples of the applications of several

of these approaches and procedures have been presented in an

attempt to document the feasibility of their implementation. We

are highly optimistic that in the next few years our understanding

of the genetic effects of cannabinoids at the cellular and

molecular level will be significantly enhanced. These same

approaches can be implemented for assaying the influence of

unfractionated marijuana extracts, psychoactive and

nonpsychoactive components of marijuana, natural and synthetic

cannabinoids, and cannabinoid metabolites as well as other abused

substances, individually or in conjunction with cannabinoids.

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of macromolecular synthesis in human and other mammalian cells.

In: Nahas, G.G., ed. Marihuana: Chemistry, Biochemistry and

Cellular Effects. New York: Springer, 1976 p. 213.

Carchman, R.A.; Harris, L.S.; and Munson, A.E. The inhibition of

DNA synthesis by cannabinoids. Cancer Research 36:95, 1976a.

Carchman, R.A.; Warner, W.; White, A.C.; and Harris, L.S.

Cannabinoids and neoplastic growth. In: Nahas, G.G., ea.

Marihuana: Chemistry, Biochemistry and Cellular Effects.

New York: Springer, 1976b, p. 329.

Desoize, B.; Leger, C.; and Nahas, G. Plasma membrane inhioition

of macromolecular precursor transport by THC. Biochem Pharmac

28:1113, 1979.

End, D.W.; Thoursen, K.; Dewey, W.L.; and Carchman, R.A. A

comparative study of the disposition of (-)- -tetrahydrocannabinol in neuroblastoma and glioma cells in tissue culture:

Relation to cellular impairment. Molec Pharmacol 13:864, 1977.

22
Green, L.; Marashi, F.; Stein, J.L.; and Stein, G.S. A decreased

influence of cannabinoids on macromolecular biosynthesis and

cell proliferation in human cells which metabolize polycyclic

hydrocarbon carcinogens. Anticancer Research, in press, 1983.

Lemberger, L. Tetrahydrocannabinol metabolism in man. Drug

Metabolism and Disposition 1:461, 1973.

Maniatis, T.; Jeffrey, A.; and Kleid, D.G. Nucleotide sequence of

the rightward operator of phage. Proc Natl Acad Sci USA

72:1184-1188, 1975.

McClean, D.K., and Zimmerman, A.M. Action of -tetrahydrocannabinol on cell division and macromolecular synthesis in

division-synchronized protozoa. Pharmacology 14:307-321, 1976.

Mon, M.J.; Haas, A.E.; Stein, J.L.; and Stein, G.S. Influence of

psychoactive and nonpsychoactive cannabinoids on cell

proliferation and macromolecular biosynthesis in human cells.

Biochem Pharmacol 30:31, 1981a.

Mon, M.J.; Haas, A.E.; Stein, J.L.; and Stein, G.S. Influence of

psychoactive and nonpsychoactive-cannabinoids on cnromatin

structure and function in human cells. Biochem Pharmacol 30-45

1981b.

Mon, M.J.; Jansing, R.L.; Doggett, S.; Stein, J.L.; and Stein, G.S.

Influence of -tetrahydrocannabinol on cell proliferation and

macromolecular biosynthesis in human cells. Biochem Pharmacol

27:1759-1765, 1978.

Nahas, G.G., and Desoize, B. Effect inhibiteur du 5 n-amylresorcinol sur la transformation lymphoblastique. C R Acad Sci

Paris Series D 279:1607-1608, 1974.

Nahas, G.C., and Paton, W.D.M. Marihuana: Biological Effects.

Oxford: Pergamon Press, 1979.

Nahas, G.G.; Armand, J.P.; and Hsu, J. Inhibition in vitro de la

blastogenese des lymphocytes T par le -tetrahydrocannabinol.

C R Acad Sci Paris Series D 278:679, 1974a.

Nahas, G.G.; Desoize, B.; Armand, J.P.; Hsu. J.; and Morishima, A.

Inhibition in vitro de-la transformation iymphocytaire par

divers cannaibinoids naturels. C R Acad Sci Paris Series D

279:785-787, 1974b.

Nahas, G.G.; Morishima, A.; and Desoize, B. Effects of cannabinoids of macromolecular synthesis and replication of cultured

lymphocytes. Fed Proc 36:1748-1752, 1977.

Rave, N.; Crkvenjakov, R.; and Boedtker, H. Identification of

procollagen mRNAs transferred to diazobenzyloxymethyl paper from

formaldehyde agarose gels. Nucleic Acids Res 6:3559-3567, 1979.

Sierra, F.; Lichtler, A.; Marashi, F.; Rickles, R.; Van Dyke, T.;

Clark, S.; Wells, J.; Stein, G.; and Stein, J. Organization of

human histone genes. Proc Natl Acad Sci USA 79:1795, 1982.

Southern, E.M. Detection of specific sequences among DNA

fragments separated by gel electrophoresis. Jour Mol Biol

98:503, 1975.

Stein, G.S., and Borun, T.W. The synthesis of acidic chromosomal

proteins during the cell cycle of HeLa S3 cells. I. The

accelerated accumulation of acidic residual nuclear protein

before the initiation of DNA replication. Jour Cell Biol

52:292, 1972.

23
Stein, G.S.; Hochhauser, S.; and Stein, J.L. Histone genes: Their

structure and control. In: Busch, H.; ed. The Cell Nucleus.

Chromatin, Part D. New York:

Stein, G.S.; Spelsberg, T.C.;

Academic Press 1979, p. 259

and Kleinsmith, L.J. Nonhistone

chromosomal proteins and gene regulation. Science 183:817, 1974.

Stein, G.S.; Stein, J.L.; and Kleinsmith, L.J. Chromosomal

proteins and gene regulation. Scientific Amer 232.46, 1575.

White, A.C.; Munson, J.A.; Munson, A.E.; and Carchman, R.A.

Effects of -tetrahydrocannabinol in lewis lung adenocarcinoma

cells in tissue culture. Jour Natl Cancer Inst 56:6556, 1976.

Wu, R.S., and Bonner, W.M. Separation of salistone syntnesis

from S-phase histone synthesis in dividing cells. Cell

27:321-331, 1981.

Zimmerman, A.M., and McClean, D.K. Action of narcotic and

hallucinogenic agents on the cell cycle. In: Zimmerman. A.M.:

Padilla, G.M.; and Cameron, I.L., eds. Drugs and the Cell Cycle

New York: Academic press, 1973, pp. 67-94.

Zimmerman, A.M., and Zimmerman, S.B. The influence of marihuana on

eukaryote cell growth and development. In: Nahas, G.G.. ed.

Marihuana: Chemistry, Biochemistry and Cellular Effects.

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ACKNOWLEDGMENTS

These studies were supported by research grants DA-01188 and

DA-02033 from the National Institute on Drug Abuse. We are

indebted to Drs. Roy Schmickel and Golder Wilson for providing us

with cloned human ribosomal genes.

AUTHORS

Gary S. Stein, Ph.D.

Department of Biochemistry and Molecular Biology

University of Florida College of Medicine

Gainesville, Florida 32610

Janet L. Stein, Ph.D.

Department of Immunology and Medical Microbiology

University of Florida College of Medicine

Gainesville, Florida 32610

24
Effects of Cannabis and Natural

Cannabinoids on Chromosomes and

Ova

Akira Morishima, M.D., Ph.D.

EFFECTS OF CANNABIS AND NATURAL CANNABINOIDS ON CHROMOSOMES

Introduction

During the past dozen years, a number of cytogenetic investigations

on the effects of cannabis and natural cannabinoids have been reported in the literature. These studies have yielded contradictory

results, leading to a controversy as to whether or not these agents

are capable of inducing chromosonal abnormalities and, therefore,

whether marijuana abuse carries any risk of producing chromosome

damage. As recently as 1980, Zimmerman and Yesoda Raj (1980, p.

258) stated, in discussing their cytogenetic observations, "Although

the current investigation further supports the concept of a detrimental action of cannabinoids on somatic cells, there remains some

doubt concerning a direct action of THC on chromosomes." The purpose of this paper is to review these published studies in an attempt

to understand better the basis from which this controversy arose.

An environmental agent, including chemical substances, capable of

modifying the structure of chromosomes or the numbers of chromosomes

of a normal nuclear complement falls into the broad definition of a

chromosomal mutagen. However, the majority of investigations on

chromosomal mutagens during the past decade, have focused on induction of alterations in the chromsomal structure, mainly chromosome

breaks. This was largely due to the initial finding on lysergic

acid diethylamide (LSD-25) which was reported to induce breaks in

chromosomes of human lymphocytes (Cohen et al. 1967). A number of

chemical agents which can induce chromosome breakages have been

identified. These clastogens, e.g., mitomycin C (Cohen and Shaw

1964, Adler 1976), generally induce a marked increase in the incidence of chromatic and isochromatid breaks.

The standard technique for examining chromosome breaks utilizes a

rapidly proliferating tissue, such as culture of peripheral lymphocytes or fibroblasts, or bone marrow aspirates. The specimens are

usually, but not always, exposed to a mitotic arrestant to increase

the number of observable metaphases. The cells are then exposed to

25

446-384 O - 84 - 3
a hypotonic solution in order to disperse the chromosomes so that

the overlaps are minimizd when chromosomes are examined microscopically. Although these preparations yield suitable specimens for

morphological observation of individual chromosanes, the use of both

the mitotic arrestant and hypotonic solution disrupts the natural

spacial relationship of chromosomes. Thus, these preparations

cannot be used to examine the segregational process of chromosomes.

Further, because the technique tends to induce metaphase plates

from which some chromosomes are lost due to an excessive dispersion,

metaphases are usually scanned under a low-power mangnification in

order to choose only those plates which appear to contain the full

complement of well spread chromosomes before they are subjected to

a detailed analysis under a high-power microscopic observation. A

trained observer, using the standard procedure, is usually capable

of discriminating those metaphase plates containing several chromosomes less than the modal number, under a low-power magnification.

Thus, the usual method for observation of chromosome breakage precludes detection of hypoploid metaphases with a greatly reduced

number of chromosomes.

Other methods used to examine clastcgenic substances include the

dominant lethal method (Rohrborn 1970), examination of sister

chromatid exchanges (Kato 1974) and of unscheduled DNA repair synthesis (Stich et al. 1971). Although these tests may detect events

related to chromosome breakage, there is no proof that the results

can be correlated directly with the clastogenic effect of an agent.

Further, few studies on marijuana have utilized these methods of

investigation (Legator et al. 1976, Zimmerman et al. 1978).

Clastogens may affect chranosanes at various stages of a cell cycle

(Röhrborn 1970). However, the vast majority of induced chromosome

breaks should be repaired at the original breaking point leaving no

detectable abnormality at metaphase of the succeeding cell cycle.

Chromatid and isochromatid breaks probably represent breakage which

occurred during the cell cycle in which the metaphase is observed.

Only occasional breaks may result in non-repair or simultaneous

cooperative repair between two or more breaking points, involving

one or more chromosomes, resulting in structural rearrangements

(Evans et al. 1967). These events would be detectable at a subsequent metaphase, provided that the cell bearing such an abnormality

is capable of surviving and undergoing the mitotic process. Thus,

detection of even a relatively low incidence of chromosomal rearrangements would imply a much greater clastogenic effect of an

agent. Morphologically observable structural rearrangements of

chromosomes include fragments, chromatid exchanges between two

chromosomes, chain and ring formations, translocations, deletions,

and inversions.

Some chromosomes mutagens are known to have little or no clastogenic effect, but principally act by disrupting the normal mitotic

process. Thus, there are at least two classes of chemical mutagens.

When HeLa cells were exposed to nitrous oxide, formation of microtubules became anomalous and the mitoses were arrested at metaphase

(Brinkley and Rao 1973). Olivetol, 5-n-amylresorcinol, which has

a molecular structure of the C-ring common to all cannabinoids, was

26
found to induce marked hypoploidy and abnormal chromosomal segregation in human lymphocytes when added to the culture medium

(Morishirra et al. 1976a, 1976b). Halogenated inhalation anesthetics (Sturrock and Nunn 1976, Kusyk and Hsu 1976) and other

volatile liquids such as benzene, toluene and chloroform (Liang

and Hsu 1983) have been found to act in a similar fashion. These

mitotic disrupters seem to have little effect on interphase chromo

somes, but affect the mitotic apparatus through their effect on

spindle and microtubular formation during a cell division

(Morishima et al. 1976b, Grant et al. 1977). Their mutagenic

effects are not likely to be detected by use of prokaryotes which

are commonly employed in screening tests for environmental mutagens, since these organisms do not utilize a mitotic apparatus

for cell division (Kusyk and Hsu 1976).

The effects of a mitotic disrupter mainfest themselves as abnormal

movements of chromosomes errors of chromosome segregation (FCS),

during mitosis, and may give rise to aneuploidy or gross imbalance

of genetic material of the cell (Kusyk and Hsu 1976, Henrich et al.

1980). Rapidly proliferating tissue specimens can be fixed for

microscopic examination without the use of a mitotic arrestant or

a hypotonic solution. In these preparations, spindles are not

artificially disrupted and chromosomes are not dispersed. Therefore, these specimens are suitable for observation of metaphase,

anaphase, and telophase chromosomes in more or less natural

spacial relationships, and are referred to as anaphase preparations. Although the morphology of individual chromosomes is not

readily discernible due to overlaps, ECS can be examined directly

in these preparations (Fig. 1). These include bridge formation,

anaphase lag, misalignment of chromosomes (chromosomes out of

phase with others), unequal segregation in bipolar division and

multipolar division (Henrich et al. 1980). ECS commonly leads to

formation of aneuploid cells.

The effects of a mitotic disrupter may also be examined indirectly

by observation of unselected metaphase plates prepared in the usual

fashion with the use of a mitotic arrestant and hypotonic solution

(Morishima et al. 1976b). This method allows detection of hypoploidy, including those cells which are missing a large number of

chromosomes from the complement. However, because of the artefactual loss of chromosomes attendant to the hypotonic treatment,

an extremely large number of metaphases must be examined in both

the control and the experimental groups of cells before a valid

conclusion can be drawn (Morishima et al. 1976a).

Induction of aneuploidy by a mitotic disrupter also may be detected

by measuring the DNA content of cells using microspectrography

(Leuchtenberger et al. 1973a). An euploid metaphase should contain

4DNA (tetraploid amounts of DNA), and an euploid telophase cell

should contain 2DNA (diploid amounts of DNA). A normal cell in

S-phase (synthetic period of DNA in the interphase) would contain

DNA in amounts between 2DNA and 4DNA.

An acentric chromosome fragment, produced by a break in the chromosome or as a sequela of anaphase bridge formation, will fail to

27
Figure 1. Errors of chromosome segregation (ECS). From Henrich et al. (1980). (A) Anaphase bridge.

(B) Severed bridge. (C) Anaphase Zag. (D) Micronucleus (arrow) and multiple bridges. (E) Chromosome out of phase with others in metaphase. (F) Unequal segregation in bipolar diversion.

(G) Multipolar division. (H) Multipolar division and bridges.
move toward a pole during the anaphase movement. The resultant

anaphase lag leads to the exclusion of the fragment from either

daughter nucleus, although it is retained within the cytoplasm.

This cytoplasmic chromosomal material is referred to as a micronucleus. Since polychromatic erythrocytes of bone marrow lack the

nucleus, presence of a micronucleus can be detected readily in

these cells. The micronucleus test in polychromatic erythrocytes

has been developed as a useful in vivo test system for detecting

chromosomal fragments induced by chemical mutagens (Schmid 1975).

That a positive result may arise from either chromosomal breakage

or from ECS should be kept in mind.

The disparate results of cytogenetic studies on the effects of

cannabis and cannabinoids are reviewed, below, by considering

their ability to act as clastogens separately from their effects

as mitotic disrupters. Some published data were retabulated in

order to make them compatible with the format of presentation and

for easier comparison with the data of others. In a number of

publications, different types of observations were made, or multiple types of studies were performed. These data were considered

separately.

Cannabis and Cannabinoids as Clastogens

Chromosome breakage has been examined extensively in human lymphocytes because of their ready accessibility. For some in vivo

studies, lymphocytes of marijuana abusers were cultured, and the

incidence of chromosome breaks in metaphase preparations was compared with that found in lymphocytes of non-marijuana users who

served as the controls (Dorrance et al. 1970, Gilmour et al. 1971),

Herha and Obe 1974, Martin et al. 1974, Stenchever et al. 1974).

In other studies, marijuana users were asked to abstain from its

use for a period of time, and then were given cannabis or delta-9-

tetrahydrocannabinol (THC), the principal psychoactive component

of cannabis, by mouth, or were given marijuana cigarettes to smoke

under a controlled environment (Nichols et al. 1974, Matsuyama et

al. 1976, 1977). In these experiments, the users served as their

own control during the period of abstinence, and the incidence of

chromosome breakage during the exposure to cannabis or THC was

compared with that observed during the control period. The results

of these studies are summarized in table 1. Of the 9 studies, 7

yielded no significant increase in the incidence of chomosome

breaks. When the results were positive (Gilmour et al. 1971,

Stenchever et al. 1974), the increased incidence was a few percent

over the controls.

For in vitro studies, human lymphocytes from non-marijuana smokers

were cultured in the presence of THC, cannabis resin or marijuana

extracts (Table 1). These studies uniformly yielded negative results (Neu et al. 1970, Stenchever and Allen 1972, Martin et al.

1974, Stenchever et al. 1976).

From these studies, it may be concluded that the human lymphocyte

system is relatively insensitive to the clastogenic effects of

29
TABLE 1. The effects of cannabis and natural cannabinoids as

clastogens* in human lymphocytes

Experimental system and design

In vivo studies

Marijuana smokers

Marijuana smokers, light

Marijuana smokers, heavy + LSD

Cannabis users + LSD

Marijuana smokers

Cannabis or THC given by mouth

Marijuana smokers

Marijuana smokers; controlled

smoking

Marijuana smokers; controlled

smoking

In vitro studies

THC added to cultures

THC added to cultures

Cannabis resin added to cultures

Marijuana extract or THC added

Neg

Neg

Neg

Neg

Neg

to cultures

Results

Magnitude of effect

(%)

Control/Experiment

Neg

Neg

0.52/2.18

Neg

Neg

Neg

1.2/3.4

Neg

Reference

Dorrance et al. 1970

Gilmour et al. 1971

Gilmour et al. 1971

Herha and Obe 1974

Martin et al. 1974

Nichols et al. 1974

Stenchever et al. 1974

Matsuyama et al. 1976

Matsuyama et al. 1977

Neu et al. 1970

Stenchever and Allen 1972

Martin et al. 1974

Stenchever et al. 1976

* Includes observations on single chromatid and isochromal breaks, fragments,

chromatid exchanges, chain and ring formations, translocations, deletions, and

inversions, but excludes chromosome gaps.

± LSD--With or without history of abuse of Lysergic acid diethylamide.

30
cannabis and cannabinoids, or that these substances have little or

no clastogenic effects.

Studies on induction of chromosome breakage by cannabis or cannabinoids in tissues other than human lymphocytes are summarized in

table 2. When rats were exposed to THC, and the lymphocytes of

exposed mothers and their offspring were examined, no increase in

chromosome breaks was observed (Pace et al. 1971). From the

cultured fibroblasts of embryos whose mothers were injected with

cannabis resin, a similar result was obtained (Martin et al. 1974).

However, when mice were exposed to THC, cannabinol (CBN), cannabidol (CBD), or marijuana extracts, and their primary spermatocytes

were examined, a significant increase in the chromosomal rearrangements was observed (Zimmerman et al. 1979, Dalterio et al. 1982).

A similar increase in structural rearrangements of chromosomes was

found in bone marrow cells of the treated mice (Zimmerman et al.

1980). The magnitude of the clastogenic effect ranged from several

percent to 15% over the controls. The effects of THC, CBN, CBD,

and marijuana extracts were essentially the same. A study on dominant lethal test, using THC-treated male mice mated with untreated

females, yielded negative results (Legator et al. 1976).

When various types of somatic cells in tissue culture were exposed

to marijuana smoke (Leuchtenberger et al. 1973a), cannabis resin

(Martin et al. 1974), or THC (Zimnerman et al. 1978), disparate

results were obtained (Table 2). In one study, chromosome breaks

in metaphase preparations, the sister chromatid exchanges, and the

unscheduled DNA repair synthesis were examined without detecting

mutagenic effect of THC (Zimmerman et al. 1978). Thus, the in

vitro studies on the clastogenic effects were mostly negativeReviewing all of the above studies as a group, it appears that

cannabis and cannabinoids are extremly weak clastogens, and that

their clastogenic effects become apparent only in appropriately

sensitive test systems such as primary sperm-atocytes and bone

marrow cells, whereas the human lymphocyte system is relatively

insensitive to their clastogenic effects (Table 1 and 2). Varying

sensitivity of different tissues to a single chemical mutagen has

been noted previously (Röborn 1970).

Cannabis and cannabinoids as mitotic disrupters

In vivo studies on induction of ECS by cannabis and cannabinoids

are summarized in table 3. The parameters studied included observations on hypoploidy in metaphase preparations (Morishima et al.

1976a, 1979), ECS in anaphase preparations (Morishima et al. 1979,

Zimmerman and Yesoda Raj 1980), aneuploidy and univalent sex chromosomes in primary spermatocytes (Zimmerman et al. 1979, Dalterio

et al. 1982) and micronuclei in the polychromatic erythrocytes

(Legator et al. 1976, Zimmerman and Yesoda Raj 1980). The systems

utilized in these studies varied from human lymphocytes to bone

marrow cells and primary spermatocytes of the mouse. Nevertheless,

all but 2 out of 9 studies yielded statistically significant increases in the ECS. The effect was apparent for marijuana smoking,

31
TABLE 2. The effects of cannabis and natural cannabinoids as

clastogens* in other than human lymphocyte system

Experimental system and design

In vivo studies

THC injections into pregnant

rats; dams and offspring

examined

Neg

Cannabis resin injections into Neg

pregnant rats; embryos

examined

Zimmerman and Yesoda Raj

1980

Dalterio et al. 1982

Leuchtenberger et al.

1973a 

Cannabinoids injected into mice;

primary spermatocytes examined

Cannabinoids injected into mice;

bone marrow cells examined

Marijuana extract or cannabinoids

fed to mice; primary spermatocytes examined

In vitro studies

Cultured human lung cells

exposed to marijuana smoke

Cannabis resin added to rat embryonic fibroblast cultures

THC added to fibroblast and XP

cell cultures; metaphase and

sister chromatid exchange

examined

Results

Magnitude of effect

(%)

Control/Experiment

6.30/11.45-THC

6.30/10.72-CBN

6.30/11.72-RD

2.0/12.3-THC

2.0/17.O-CBN

2.0/14.3-CBD

0.81/5.74-Extract

0.81/4.54-THC

0.81/6.49-CBN

Pos

(Quantitative data

not available)

Neg

Reference

Pace et al. 1971

Martin et al. 1974

Zimmerman et al. 1979

Martin et al. 1974

Neg Zimmerman et al. 1978

* Includes observations on single chromatid and isochromatid breaks, fragments,

chromatid exchanges, chair and ring formations, translations, deletions,

inversions, and sister chromatid exchanges, but excludes chromasome gaps.

32
TABLE 3. The effects of cannabis and natural cannabinoids as

mitotic disrupters*: in vivo studies

Experimental system and design

THC fed to mice; micronuclei in

bone marrow cells examined

Neg

Hypoploid metaphases in lymphocytes of marijuana smokers,

heavy

Hypoploid metaphases in lymphocytes of marijuana smokers,

light

Hypoploid metaphases in lympho

cytes of marijuana smokers,

controlled smoking

ECS in lymphocytes of marijuana

smokers; controlled smoking

Cannabinoids injected into mice;

aneuploidy in primary sperma

tocytes examined

Cannabinoids injected into mice;

micronuclei in bone marrow

cells examined

Cannabinoids injected into mice;

ECS in bone marrow cells

examined

Cannabinoids given by mouth to

mice; aneuploidy and univalent

sex chromosomes in primary

spermatocytes examined

Results

Magnitude of effect

(%)

Control/Experiment

15.52/36.23

31.19/37.09

12.83/35.66

1.20/2.01

Neg

0.9/3.7-THC

0.9/5.3-CBN

0.9/5.0-CBD

8.0/17.3-THC

8.0/12.0-CBN

8.0/12.0-CBD

8.22/13.87-Extract

8.22/34.38-THC

8.22/17.32-CBN

Reference

Legator et al. 1976

Morishima et al. 1976a

Morishima et al. 1976a

Morishima et al. 1979

Morishima et al. 1979

Zimmerman et al. 1979

Zimmerman and Yesoda Raj

1980

Zimmerman and Yesoda Raj

1980

Delterio et al. 1982

* Includes observations on hypoploid cells, aneuploidy, polyploidy, micronuclei

in polychromatic erythrocytes of the bone marrow, univalent chromosomeS during

meiosis and errors of chromosome segregation (ECS) as seen in metaphase or

anaphase preparations.

33
THC, CBN, CBD, and marijuana extract. The magnitude of effects

ranged from a few percent to as high as 26% over the controls.

In vitro studies on induction of ECS are summarized in table 4.

Again, methods used to examine the property of mitotic disrup

tion varied from one study to another. These included observations

on abnormal DNA contents of somatic and germ cells (Ieuchtenberger

and Leuchtenberger 1971, Ieuchtenberger et al. 1973a, 1976), ECS

in anaphase preparations (Ieuchentenberger and Ieuchentenberger

1971, Ieuchtenberger et al. 1973a, 1979, Henrich et al. 1980), and

aneuploid and hypoploid metaphases (Ieuchtenberger et al. 1973b,

Morishim et al. 1976a, Stenchever et al. 1976). The systems

utilized for the experiments were also variable, including lung

cells of man and mouse in culture, explants of testis of the mouse,

and human lymphocytes or human breast cancer cells in tissue culture. Nevertheless, all 8 studies resulted in statistically significant increases of abnormal cells. Marijuana smoke, THC, and

marijuana extracts were all effective in includiug the ECS. The

magnitude of the effects ranged from a few percent to about 30%

over the controls.

Conclusions

The contrast between the two groups of studies, one in which mainly

the clastogenic effects were examined (Table 1 and 2) and another

in which principally the mitotic disruptive effects were observed

(Table 3 and 4), is striking. Taking all the studies together in

which mitotic or meiotic movements of chromosomes were examined, it

is apparent that cannabis and cannabinoids are mitotic disrupters

capable of preventing the normal segregation of chromosomes (Table

3 and 4). The apparent contradictory results on the effects of

cannabis and cannabinoids on induction of chromosomal abnormalities

were explained previously as possibly being due to variations in

the criteria for microscopic observations from one laboratory to

another, effects of drugs other than marijuana being used by

marijuana abusers, or variations in amounts of cannabinoids used.

However, by separating those studies which examined mainly the

clastogenic effects from those which examined the property of

mitotic disruption, the controversy can be reconciled. It may be

concluded that cannabis and cannabinoids possess the property of

a mitotic disrupter, and that they may also have extremely weak

clastogenic activity.

Whether or not cannabis and cannabinoids can produce stable chromosome alterations which can be transmitted through many cell generations is a major concern to all cytogenetic investigators. Although

in all probability the majority of cells bearing observed chromosome

abnormalities are incapable of surving more than a few cell cycles,

it seems reasonable and prudent to assume that occasional cells

affected by these chromosomal mutagens will bear stable chromosomal

aberrations which will not be incompatible with cellular proliferation. Induction of such cells could lay a foundation for oncogenesis, teratogenesis, and mutagenesis. The observation of induced

chromosomal abnormalities in primary spermatocytes (Zimmerman et al.

34
TABLE 4. The effects of cannabis and natural cannabinoids as

mitotic disrupters*: in vitro studies

Experimental system and design

Cultured lung cells of mice

exposed to marijuana and

cigarette smoke; DNA contents and ECS examined

Cultured human lung cells exposed to marijuana smoke;

DNA content and ECS examined

Cultured human lung cells

exposed to marijuana smoke;

aneuploidy examined

Explants of testes of mice

exposed to marijuana smoke;

DNA contents in spematids

examined

THC added to human lymphocyte

cultures; hypoploidy

examined

Marijuana extract added to

human lymphocyte culture;

aneuploidy examined

Cultured human breast cancer

cells exposed to marijuana

make; abnormal mitosis

examined

THC added to human lymphocyte

cultures; ECS examined

Results

Magnitude of effect

(%)

control/Experiment

5 fold increase in

tetraploid DNA

contents.

Increased ECS

5-7 fold increase

in aneuploid

amounts of DNA

or ECS

44/69

20/50

22.56/33.13

2.0-4.0/9.0-12.0

8.0/21.5

1.11/3.54

Reference

Leuchtenberger and

Leuchtenberger 1971

Leuchtenterger et al.

1973a

Leuchtenterger et al.

1973b

Leuchtenberger et al. 1976

Morishima et al. 1976

Stenchever et al. 1976

Leuchtenberger et al. 1979

Henrich et al. 1980

* Includes observations on aneuploid DNA contents per cell, aneuploidy, abnormal

mitoses, and errors of chromosome segregation (FCS) as see in metaphase or

anaphase preparations.

35
1979, Dalterio et al. 1982) suggests that induction of chromosome

abnormalities transmissible to offspring is still a possibility.

EFFECTS OF THC ON OVA

Although there is a growing body of evidence indicating that cannabis and cannabinoids can adversely affect the germ cells of males,

little information is available on their effects upon female germ

cells. Chronic administration of cannabis and cannabinoids has

resulted in decreased sperm counts and motility (Dixit at al. 1974,

Hembree et al. 1976, 1979, Huang et al. 1979, Dalterio et al. 1982).

Although these findings may be mediated through the suppressive

effects of cannabis and cannabinoids on the secretion of gonadotropins (Kolodny et al. 1974, Cohen 1976, Smith et al. 1976, 1980,

Halclerode et al. 1979), there is evidence to indicate that these

substances can directly affect germ cells. Chromosomal abnormalities have been induced in primary spermatocytes in in vivo experiments (Zimmerman et al. 1979, Dalterio et al. 1982), as well as in

an in vitro study (Leuchtengerger et al. 1976).

Studies on female gem cells are, as yet, meager. In vitro

steroidogenesis by isolated follicles was inhibited by THC, and

resumption of meiosis occurred without the normal accompaniment of

detachment of cumulus-granulosa cells from the ova, as assessed by

the germinal vesicle breakdown (Reich et al. 1982).

The Effect of THC on Early Embryogenesis

In our recent studies the effects of chronic administration of THC

on early embryogenesis were investigated in mice treated with THC

prior to sexual maturation (Nogawa et al. 1983, Shinohara et al.

1983). The study was designed to imitate prolonged abuse of marijuana by sexually immature and adolescent girls. Female mice were

injected, intraperitoneally, with THC 5 m/kg/d. or with the vehicle

for 21 consecutive days, starting on the 30th day of life. Vaginal

opening generally occurred in these mice on the 35th day of life.

The dose of THC was approximately equivalent to that absorbed by man

smoking 1 to 2 marijuana cigarettes per day (Rosenkrantz 1976). The

mice were injected intraperitoneally with 5 I.U. of pregnant mare

serum gonadotropin on the last day of THC injection, and 48 hours

later with 5 I.U. of human chorionic gonadotropin (hCG) in order

to induce superovulation. The time of hCG injection was taken as

time zero. Females were placed with untreated young adult males

for 16 hours immediately after hCG injection and allowed to mate.

One group of females was sacrificed 22 to 26 hours after hCG injection. Another group of female mice was sacrificed at 49 to 52

hours. The ova were recovered from the oviducts and immediately

examined microscopically. After the morphological examination

either metaphase or anaphase preparations of these ova were made.

The rate of mating, ovulation, and fertilization did not differ

between the control and the experimental group.

36
From 85 control and 95 THC-treated mice sacrificed between 22 and

26 hours, 2,495 and 3,238 ova were recovered, respectively. Only

1.0% and 1.4% of these from the control and the THC-treated mice,

respectively, were at the 2-cell or later stages of development and

therefore had completed the first cleavage division. Thus, these

samples represented ova just prior to the first cleavage division.

The incidence of morphologically abnormal ova, 12.3% for the control and 11.2% for the THC-treated animals, did not differ between

the two groups.

From 261 control and 201 THC-treated mice sacrificed between 49 and

52 hours, 8,001 and 6,345 ova were obtained, respectively. The

majority of ova were in the 2-cell stage, but a significant proportion of the ova were in the process of or had completed the second

cleavage division (Table 5). Thus, these samples represented ova

at the time of the second cleavage division. The incidence of

morphologically abnormal ova (Fig. 2) was 13.6% in the control and

19.1% in the THC-treated group (Table 5). This difference was

significant at p<0.00l. Therefore, it was concluded that the incidence of abnormal ova increased from the period just before the

first cleavage division to the time of the second cleavage division in the THC-treated animals. It appeared that morphological

degeneration of ova occurs progressively between these two periods,

resulting in an accumulation of abnormal ova at the time of the

second cleavage division. The incidence of ova that had successfully completed the first cleavage division, those in the 2-cell

or later stages of development, was 77.2% for the control and 71.7%

for the THC-treated group (Table 5). This difference was significant at p<0.005. Therefore, it was concluded that THC induced an

increased rate of failure in completing the first cleavage division. In order to assess the success rate of the second cleavage

division, the ova in the 1-cell stage and the morphologically

abnormal ova were eliminated from tabulation (Table 6). There were

a total of 6,183 ova in the control and 4,552 in the THC-treated

group, representing apparently healthy ova that had successfully

completed the first cleavage division. Of these ova, 15.4% in

the control and 14.3% in the THC-treated group, were at either the

3-cell or 4-cell stage of developmnt, thus having successfully

undergone the process of the second cleavage division. The difference between the two groups was not significant. Therefore,

THC treatment did not appear to interfere with the second cleavage

division for those ova that had completed the first cleavage

division.

Among 2,215 ova from the control and 2,078 from the THC-treated

group examined in the anaphase preparations, 365 and 301, respectively, were at metaphase, anaphase, or telophase of the second

cleavage division. The only type of ECS found in these cells was

anaphase bridge formation found in 5 cells from the controls and

in 2 cells from the THC-treated group. Thus, THC did not induce

ECS at the time of the second cleavage division.

When 2,426 ova obtained from the control animals and 1,018 ova

from the THC-treated group were examined in the metaphase preparations, the mitotic index was 6.7% for the control and 7.5% for

37
TABLE 5. The incidence of morphologically normal and abnormal ova

of the muse, 49 to 52 hours after the hCG injection

(at the time of the second cleavage division)

Normal (%) Abnormal (%) Total No.

Cell Stage of ova

1 2 3 4

Control 9.1 65.3 4.2 7.7 13.6 8,001

THC-treated 9.2 61.4 3.2 7.1 19.1 6,345

TABLE 6. The success rate of the second cleavage division in

apparently health ova that had completed the first

cleavage division, 49 to 52 hours after the hCG injection

Stages of development

of ova (%)

3- and 4-cell 2-cell

Total No.

of ova

Control 15.4 84.6 6,183

THC-treated 14.3 85.7 4,552

38
Figure 2. Normal and Abnormal Ova. From Nogawa et al. (1983). (A), (B), (C) Normal 1-cell,

2-cell, and 4-cell stage ova. (D) Liquefaction degeneration of the left ovum, and a normal ovum

on the right. (E) Fragmentation of ooplasm in 2 ova. (F) Unequal cleavage. (G) Premature loss

of zona pellucida in a 1-cell and 2-cell stage ova. (H) Zona only. (I) Premature hatching.
the THC-treated group. No obvious aneuploidy was observed in 114

metaphases from the controls or in 69 metaphases from the THC

treated animals, although dispersion of chromosomes a number

of metaphases was less than ideal.

Discussion

Chronic administration of THC, at a modest dose, to presumably

sexually developing mice and continuing through their early adulthood induced a significant increase in morphologically abnormal

ova. Vaginal opening, which takes place under the influence of

estrogen, occurred at about the 35th day of life in the mice used

in this study. Therefore, it is reasonable to assume that the

protocol for THC administration covered the prepubertal stages of

sexual maturation.

Although the absolute percentage of increase in abnormal ova was

relatively small at 5.5%, the order of magnitude was similar to

the increased cytogenetic abnormalities induced by THC in the

primry spermatocytes of the mouse (Zimmerman et al. 1979), and

to the 2.4% increase in ECS induced in human lymphocytes by THC

in vitro (Henrich et al. 1980).

Although there is no clear evidence of increased fertility or fetal

wastage among chronic marijuana abusers, the effect of the drug may

be difficult to detect by epidemiological studies, even if the

observed effect of THC in the mouse were to occur in man. Natural

loss of concepti in humans has been estimated to be as high as 78%

(Roberts and Lowe 1975), but recognizable spontaneous abortion

accounts for only 11.5% of known pregnancies (Shapiro et al. 1962).

Therefore, if marijuana abuse increases the wastage of early concepti by the same order of magnitude as that observed in the mouse,

the extremely high incidence of natural loss would readily mask

the increase and prevent clinical detection.

Despite the relatively small percentage of increase in degenerating

ova induced by THC, the biological significance of this observation

may differ greatly from the adverse effect of THC on other tissues.

In contrast to most somatic tissues and sperm, de novo formation of

oocytes does not occur after birth (Zuckerman 1956, Tsafriri 1978).

Thus, any damage inflicted upon oocytes is likely to be irreversible

and permanent.

The observed increase in degenerating ova was caused by their inability to undergo successfully the first cleavage division. The

pathogenesis, therefore, is likely to lie in the process of meiosis,

which takes place during the time-frame just prior to ovulation and

shortly after fertilization. It is reasonable to assume that some

24 to 48 hours would elapse before morphologic changes of degeneration can be detected in an ovum that undergoes aberrant meiosis.

Fragmentation of ooplasm, for example, occurs 24 hours after an

oocyte reaches full maturity (Zenzes and Engel 1976), when not

fertilized. That the THC prevented the ova from completing the

first cleavage division was further supported by our failure to

detect an increase in ECS or aneuploidy during the second cleavage

40
division. Presumably only those ova that were not significantly

affected during meiosis would have completed the first cleavage

division successfully and would have survived to undergo the second

cleavage division.

The pathogenic process leading to the observed degeneration of ova

is likely to lie in meiosis. The evidence implicating THC as a

mitotic disrupter, which interfers with the normal segregation of

chromosomes, has been reviewed above. Currently, studies are in

progress to examine directly the effects of THC on the meiotic

process in ova of the mouse.

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ACKNOWLEDGEMENT

This work was supported by a grant from NIDA, lR0 DA02833.

AUTHOR

Akira Morishima, M.D., Ph.D.

Department of Pediatrics

College of Physicians & Surgeons

Columbia University

630 West 168th Street, New York, NY 10032

45
Endocrine Effects of Marijuana in

the Male: Preclinical Studies

Jack Harclerode, Ph.D.

INTRODUCTION

The research efforts of many investigators in the recent past have

made it abundantly clear that exposure to marijuana has significant

effects upon the reproductive system and the effects of cannabinoid

treatment are equally significant on both male and female reproductive systems. Among the effects of cannabinoid treatment on the

male reproductive system that have been reported arc altered

testicular function, in the form of depressed male hormone secretion, and changes in both the quantity and quality of the sperm

produced by the seminiferous tubules. There have been changes

reported in the weight and in certain of the enzymes associated

with the reproductive organs. Much research effort focused on the

ability of THC to depress the secretion of the gonadotropins from

the pituitary that are responsible for stimulating testosterone

production by the Leydig cells of the testis and the action on the

hypothalamus to depress gonadotropic releasing hormone (GnRH).

Maintenance and regulation of normal reproductive capacity in the

male is a complex and highly integrated phenomenon. It requires

proper nutritional and hormonal support, not only by the hormones

directly involved in reproduction, hut also by the synergistic

action of hormones produced by other endocrine organs throughout

the body. The regulation of many endocrine organs in the body is

though the hypothalamus, an area which is sensitive to chemical,

hormonal, and sensory input from all parts of the body. Thus, it

is easy to understand why psychoactive drugs which alter neural

function in various parts of the brain have so much of an effect

on the output of trophic hormones from the hypothalamus. This

review will attempt to summarize the effect that marijuana and its

various constituents have on the reproductive system of laborator

animals. Although this review emphasizes effects on the male

reproductive system, some female data are included for comparative

purposes.

THE EFFECTS OF THC ON GONADOTROPINS

In 1973 Marks reported that luteinizing hormone levels in ovariectomized rats were reduced sharply by tetrahydrocannibinol (THC)

46
(1, 3, and 10 mg/kg I.V.). Marks concluded that the action of THC

was not related to activation of muscarinic receptors but that it

might be due to the observed ability to increase the uptake and

retention of catecholamines by brain tissue, which would in effect

produce an inhibition of GnRH release from the hypothalamus.

Symons et al. (1976) showed that both acute and chronic treatment

of rats with THC (5 mg/kg 2 times a week for 6 weeks i.m.) produced

a decrease in both plasma luteinizing hormone (LH) and testosterone

levels. They also observed that the effect of an acute dose was

more dramatic than the chronic treatment and that THC somehow

decreased the pituitary's response to GnRH.

Collu et al. (1975) reported that injection of prepubertal male

rats with 10 mg/kg THC three times a week for 30 days resulted in

reduced levels of LH in the plasma while pituitary levels of

luteinizing hormone were unmodified. They similarly reported

that plasma levels of follicle stimulating hormone (FSH) were not

affected by the drug treatment. Dalterio et al. (1978) showed

that oral administration of THC (50 mg and 100 mg/kg) to male mice

resulted in a reduction of plasma testosterone, LH, and FSH levels,

but that single doses of cannabinol (CBN) had no effect on any of

these three hormones. They concluded that the reduction in plasma

hormone levels was due to inhibition of pituitary LH release, as

well as to a direct effect on the testis to alter the responsiveness to LH stimulation.

Smith et al. (1976) injected male Rhesus monkeys with THC (5 mg/kg

I.M. acute) and produced a 65% reduction in serum testosterone

levels that returned to normal over the next 3-day period. They

found a depression in LH levels that was comparable in magnitude

and duration to the depression in testosterone levels. Fran these

data they postulated that the THC inhibition of male sex hormones

was due to its interaction with the central nervous system. Thus

it appears that THC affects the hypothalamus and pituitary of males

in a similar manner to the way it affects females.

In female ovariectomized monkeys, Snith et al. (1979a) compared the

effects of various doses of THC on the levels of LH and on the

levels of follicle stimulating hormone. Five mg/kg produced a 68%

reduction in LH levels and a 56% reduction in FSH levels. The

maximum decrease occurred at the same time for both gonadotropins.

Smith et al. (1979a) have also reported that the decrease in both

LH and FSH were equivalent in response to equal doses of THC whether

the THC was administered as a pure substance or as the same percent

in crude marijuana extract (CME). These data suggest that the

inhibitory action of marijuana on gonadotropin levels is produced

by THC and that the other cannabis derivatives contained in marijuana do not contribute to the effect, since neither cannabidol (CBD)

nor CBN had any significant effect on gonadotropin levels, even up

to dosages of 10 mg/kg. Thus they have concluded that the inhibitory effect of cannabis derivatives on gonadotropins is related to

their psychoactivity. It should be pointed out, however, that

certain non-psychoactive cannabinoids which may not be involved in

altering hormonal changes in the hypothalamus, the pituitary, or

the testis, may contribute to a direct effect on other reproductive

47
functions such as sperm production by the seminiferous tubules,

augmentation of hormonal effects on growth and secretory activity

of accessory sex organs (such as prostate and seminal vesicles),

interaction with hormones at the target tissue level, or altering

receptors for hormones on target tissues.

THE EFFECT OF MARIJUANA ON GONADOTROPIN RELEASING HORMONE

The hypothalamus contains neurosecretory neurons which are responsible for the synthesis and secretion of factors that regulate the

release of the hormones elaborated by the anterior lobe of the

pituitary gland. A single hypothalamic factor seems to be responsible for the release of both LH and FSH and this factor is

gonadotropin releasing hormone. The secretion of GnRH is affected

by a variety of factors including neural, chemical, sensory, hormonal, and various drug treatments of the animal. Normally,

release of GnRH is regulated by the neural transmitters of the

hypothalamus. Thus, factors which alter dopamine and norepinephrine concentrations also alter GnRH release. As a general rule,

things which enhance the release of adrenergic or dopaminergic substances in the hypothalamus should stimulate the release of GnRH,

and those substances, such as drugs, which antagonize adrenergic

and dopaminergic release should inhibit the release of GnRH.

Several studies have indicated that the pharmacological effect of

THC on gonadotropin levels involves the alteration of gonadotropin

releasing hormone. Smith et al. (1979a) using the overiectomized

monkey, showed that administration of GnRH to monkeys that had received THC 6 hours before (2.5 mg/kg I.M.), was able to reverse

THC-induced reduction in LH and FSH within 30 minutes after GnRH

administration. Tyrey (1978) similarly showed that administration

of GnRH to ovariectomized rats previously treated with THC reversed

the effects of THC induced depression of LH and FSH. Nir et al.

(1973) had shown earlier that GnRH might be involved, in a study

that examined the effect; of THC-induced suppression of ovulation in

rats by the administration of GnRH; and Asch et al. (1979a) also

reported that GnRH could reverse the THC-induced suppression of

ovulation in rabbits.

Thus it appears that THC blocks GnRH release by the hypothalamus.

Since the pituitary remains capable of responding to exogenous GnRH

in the presence of THC, it seems likely that the observed THCinduced suppression of gonadotropin output arises indirectly from

an action of THC upon the hypothalamus and not through a direct

effect upon the pituitary.

In contrast to the acute studies reported above, Rosenkranz and

Esber (1980) reported that prolonged oral administration of THC to

young rats (2, 10, or 50 mg/kg for 14 to 180 days) tended to increase gonadotropins, indicating that tolerance to the effects of

THC may develop in males. They also reported that cannabidiol

treated male monkeys had increased follicle stimulating hormones

(CBD, 20, 100, and 300 mg/kg oral), but that the steroid hormones

were essentially unchanged.

48
It is not surprising that the psychoactive ingredient in marijuana,

THC, has as a major effect the depressing of gonadal hormone levels

by acting through the hypothalamus to suppress GnRH release. In

view of the psychoactive nature of THC, it is possible that it

alters neural transmitter substances throughout the brain, but

especially in that region of the central nervous system that is

important in regulating GnRH release from the hypothalmus. Very

few studies have examined whether hypothalamic neural transmitters

are in fact affected by marijuana smoke or THC treatment. There

are, however, some studies which have shown that THC affects neural

transmitter substances in other areas of the brain. Kramer and

Ben-David (1974) have shown that both serotonin and dopamine are

probably responsible for the inhibitory effect of THC on prolactin,

and Marks (1973) indicated that hypothalamic cholinergic mechanisms

are probably not involved in the THC depression of LH release.

Several early studies (Truitt and Anderson 1972; Fuxe and Janssen

1972; and Welch et al. 1971), reported that biogenic amines in the

CNS are altered by THC treatment. Others (Howes and Osgood 1974;

and Hershkowitz et al. 1977) showed that THC prevents reuptake of

dopamine, norepinephrine, and serotonin into the respective nerve

endings throughout the brain. Other authors (Revuelta et al. 1979)

have shown that THC depressed cholinergic activity in the rat

hippocampus.

These reports indicate that serotonin levels in certain areas of

the brain may increase with concomittant decrease in catecholamines

in other areas of the brain. Thus it appears that future studies,

which address themselves to clarifying the effect of marijuana or

THC on neurotransmitters in the hypothalamic area and their sub

sequent effect on GnRH and other factors involved in regulating

pituitary function, would be quite important.

There are other possible mechanisms to partially explain the observed effects of THC on pituitary horn-one output other than

through altering hypothalamic regulation of GnRH. Jakubovic and

McGeer (1972) showed that THC (but not nonpsychoactive cannabinoids) decreased the synthesis of protein and nucleic acid in the

infant rat brain. Cannabinoids also may affect the cell membranes

of nerves, for Greenberg et al. (1978) have shown an inhibition

of LPC acyltransferase, a plasma membrane bound enzyme, which may

be involved in neurotransmitter uptake mechanisms in the synpatosomes of mouse brain. Also, marijuana may produce morphological

changes, at the ultrastructural level in the neural tissue, such

as synaptic cleft widening and clumping of synaptic vesicles in

the synaptic cleft (Harper et al. 1977).

EFFECT OF MARIJUANA ON THE TESTIS AND ACCESSORY REPRODUCTIVE ORGANS

Roth crude marijuana extract (CME) and THC produced a decrease in

the weight of the reproductive organs of rats when given over a

prolonged period of time and over several dosage ranges. The

greatest effects were reported for high dosages (15 and 75 mg/kg

orally daily for 77 days). Fujimoto et al. (1978) showed significant reduction in ventral prostate, seminal vesicles, and

epididymal weight, which was correlated with a decrease in plasma

49
testosterone levels and was accompanied by a reduced number of sperm

in the fluids of the epididymus. Treatment of the rats for only 5

days produced none of these changes. The effects of the cannabinoids appeared to be reversible, because there was a return to

control levels of organ weights 30 days after cessation of drug

treatment.

Similarly, Dixit and Lohiya (1975) report that cannabis extract

produced a marked reduction in the weights of seminal vesicles,

ventral prostate, epididymus, and preputial glands of castrated

adult male mice. Further, if cannabis extract was administered in

combination with testosterone propionate, the growth stimulation

produced by testosterone propionate alone was inhibited, indicating

that cannabis extract may be antiandrogenic. However, there was

no indication as to which compounds in the extract might produce

the antiandrogenic effect. Vyas and Singh (1976) report that

cannabis administered to pigeons for 30, 60, or 90 days produced a

significant decrease in the gross weight of the testis, decrease

in the diameter of seminiferous tubules, degenerative changes in

the seminiferous tubules, and a complete cessation of sperm production. Dixit et al. (1977) also showed that CME (10 mg/kg I.P.

for 10 days) produced degenerative changes in the testis of a toad

including decreased Leydig cell nuclei, a reduction of RNA and

protein contents in the testis, and an indication of reduced androgen production.

Purohit et al. (1979) report that THC administered to hypophysectomized rats was able to block the stimulatory effect of human chorionic gonadotropin on ventral prostate and seminal vesicle weights

but that serum testosterone and dihydroxytestosterone levels were

unaffected. They concluded that THC must have both a direct tissue

effect and an indirect hypothalamic-hypophyseal effect to reproduce

the changes observed in the testis. Ghosh et al. (1981) report

that THC (10 mg/kg for 7 days) prevented the testosterone induced

changes of DNA, RNA and protein content, acid phosphatase, and an

isoenzyme variant of acid phosphatase in the ventral prostate tissues of adult castrated rats. They theorized that THC acted antiandrogenically directly at the level of the male accessory sex

organs.

The reports in the literature of reduction in reproductive organ

weights are accompanied by reports that show that the quality and

quantity of sperm produced by the testis is affected by cannabinoids. Fosenkrantz and Hayden (1979) report that rats exposed to

single daily doses of Turkish marijuana smoke, containing .25% THC,

exhibited seminiferous tubule degeneration with an interference

with sperm maturation. Zimmerman et al. (1979a, 1979b) have shown

that treatment of mice for as few as 5 days with THC (5 mg/kg I.P.)

or cannabidiol (CBD) (10 and 25 mg/kg I.P.) produced an increase

in the number of abnormal sperm, including sperm which had an increased number of ring and chain translocations when the cells were

evaluated 16 days after the last dose of cannabinoids. When calls

were evaluated 35 days after the last of 5 consecutive days of cannabinoid treatment, among the abnormal sperm reported where those

which had heads without hooks, banana shaped heads, amorphous

50
heads, and folded heads. Huang et al. (1979) reported that rats

receiving .4 or 3 mg THC/kg as marijuana smoke for 75 days exhibited a decreased number of epididymal sperm, and an increased

number of sperm with head to tail dissociations. These sperm

changes were accanpanied by a decrease in testicle weight, a decrease in the weight of the seminal vesicles, and an increase in

adrenal gland weight.

Dalterio et al. (1982) treated male mice with THC, CBD, or CME

(25 mg/kg) daily for 5 days. The mice were killed 50 to 60 days

after cessation of drug treatment and meiotic chromosomes were prepared for microscopic examination from the testes. They reported

that THC treated animals had a higher frequency of unpaired sex

chromosomes at metaphase than did controls. Other chromosomal

aberrations reported for cannabinoids include: ring and chain

translocations, an increased incidence of aneupolidy, and a higher

frequency of polyploidy. They also report that the cannabinoid

treated males impregnated significantly fewer females, that the

females successfully impregnated had a higher pre- and post-natal

death rate and an increased percentage of pregnancies with fetal

loss. The Fl male offspring, of these cannabinoid exposed males,

who successfully impregnated females produced litters that had a

higher incidence of developmental anomalies or that exhibited

chromsomal rearrangements.

Several studies have been reported that may help to explain the

reduced sperm production as well as the abnormalities that are seen

in the semen after treatment of animals with various cannabinoids.

Jakubovic and McGeer (1977) studied the effects of THC and various

water insoluble cannabinoids on metabolism in testicular slices and

testicular cell suspensions. Cannabinoids (.1 mM) caused significant diminution in the amount of radioactivity that was incorporated into nucleic acids, lipids, and proteins in the testis slices.

The cannabinoids inhibited the synthesis of labeled nucleotides and

the incorporation of radioactive amino acids into proteins. They

concluded that cannabinoid treatment produces a direct inhibitory

effect of the cannabinoids on the kinases and/or polymerases involved in the RNA or DNA synthesis and that the inhibition of protein synthesis may be, in turn, a result of decreased chromosomal

DNA, which could ultimately lead to alteration in spermatogenesis.

Jakubovic et al. (1979) also examined the effects of cannabinoids

on testosterone and protein synthesis in rat testis Leydig cells in

vitro. Various cannabinoids were added to the Leydig cell preparation at .15 mM concentration. It was found that HOG stimulated

preparations incurred a 50% reduction in testosterone synthesis.

However, the nonstimulated cultures had no effect on basal steroidogenesis even at cannabinoid concentrations of 15 mM. They concluded that cannabinoids at this high concentration may interfere

directly with the Leydig cells to inhibit both protein synthesis

and testosterone synthesis, and that the consequence of this inhibition might be affected spermatogenesis.

Several investigators have examined the effect that cannabinoids

have on the enzymes of the testis in both in vivo and in vitro

51
systems. There is evidence that cannabinoids affect the enzymes

found in Sertoli cells in the seminiferous tubules which are important in sperm maturation. Schwartz et al. (1977) have shown that

treatment of rats with THC (2 mg/kg I.P. for 9 days) or with cannabidiol (2 mg/kg I.P. for 9 days), produced a decrease in both

microsomal cytochrome P-450 Leydig cells of the testis (an enzyme

which is involved in the biosynthesis of testosterone) and gammaglutanlyl transpeptidase (a marker protein for Sertoli cells in the

seminiferous tubules of the testis). Normal levels of cytochrome

P-450 and gamma-glutamyl transpeptidase were restored when exogenous LH and FSH were supplied to the THC treated animals. These

results indicate that some enzymatic defects caused by cannabinoid

exposure may be prevented if normal levels of the gonadotropins

are maintained and that cannabinoid effects on the testis might be

due to defects of pituitary hormones rather than a direct action

of the drugs on the testis. Goldstein et al. (1977) reported that

CBD (2 mg/kg/day, I.F.) and THC (2 mg/kg/day I.P.) depressed an

esterase isozyrne of the rat Ieydig cells after 10 days of treatment with the cannabinoids. Also, Hubbard et al. (1979) reported

a reduced cholesterol esterase activity in cultured rat Leydig

cells at a concentration of 16 mM. This enzyme hydrolyzes the

cholesteryl esters of oleic, arachadonic, and palmitic acids.

A group of investigators (Burstein et al. 1978a, 1978b, 1979 and

Shoupe et al. 1980) has examined the effect of various cannabinoids

on the enzymes, involved in the synthesis of testosterone, that

are located in the Leydig cells. An early study (Burstein 1978a)

indicated that neither THC nor CBN (1 and 5 micrograms/ml) altered

the transformation of either pregnenalone or progesterone to testosterone, a dose of cannabinoid that caused a 60% decrease in

overall testosterone synthesis in vitro. They postulated that

the cannabinoids either blocked the conversion of cholesterol to

pregnenalone or somehow affected the availability of precursorcholesterol. Burstein et al. (1979) used isolated mouse Leydig

cells as a model to examine the stepwise steroidogenic pathway for

the site of action of THC and concluded that THC inhibition was

effected at the release of precursor cholesterol from its ester

storage. Shoupe et al. (1980) showed that THC inhibited crude

Leydig cell cholesterol esterase preparations with a mixed type of

inhibition.

Cannabinoids are relatively insoluble in the aqueous medium used

to culture testicular cells which raises the question of what the

concentration of cannabinoids is in the culture media and how cannabinoids may affect other constituents of the culture media. A

further complication is the fact that the actual tissue concentration in vivo may never approach the levels that were added to

cultures to cause the results reported in vitro. At least one

study (Nahas et al. 1981) indicated that there was no selective

concentration of radioactive -THC in the cells of the testis of

mice administered the compound. Thus, some of the observations

reported for alteration in protein synthesis and testosterone synthesis may be an artiface of an in vitro system and may not be

representative of what was actually occurring in vivo.

52
THE EFFECTS OF THC ON PROLACTIN SECRETION

The reports of research which examined the effect of THC on prolactin secretion in the male are somewhat controversial and contain conflicting reports. Some of the apparent conflict can be

attributed to the method of administration of the THC, and perhaps

the dosage of THC as well. Collu (1976) administered THC (20μg)

intraventricularly for a week to prepubertal and to adult rats.

He reported that pituitary levels of prolactin were increased in

both prepubertal and adult animals, and noted that there were no

changes in brain levels of noradrenaline, dopamine, or serotonin

following drug treatment. Kramer and Ben-David (1974) had reported

a suppression of prolactin release by acute THC treatment. The

administration of the serotonin antagonist, cyprohepatidine, or

the dopamine antagonists, perphenazine and chemozine, was able to

abolish the THC induced suppression of prolactin. They also questioned whether the ability of THC to decrease TRH, a hormone known

to cause prolactin release, might be responsible for the THCinduced suppression of prolactin release.

Bromley et al. (1977) administered a rather high dose of THC (30

mg/kg I.P.) to male rats and showed a marked suppression of prolactin as quickly as 15 minutes after THC injection. They reported

that THC blocked the release of prolactin in ether-stressed rats,

and also found that THC impaired, but did not prevent, a significant pituitary response to perphenazine.

Dalterio et al. (1981) reported a reduction of plasma prolactin

levels in stressed and non-stressed male mice after a single dose

of THC (50 mg/kg P.O.). However, chronic treatment had no effect

on prolactin under these conditions (3 times/week/3 weeks).

Chronic exposure to CBN resulted in reduced plasm prolactin levels

in stresssed mice, indicating that perhaps non-psychoactive ingredients in marijuana, at least in the dosages used in this study, can

exert effects on endocrine function. Daley et al. (1974) reported

that male rats injected with THC (4 and 16 mg/kg I.P.) had heavier

pituitary gland weights than controls at both dosage levels and

that, although serum prolactin was significantly increased, there

was no increase in the total piuitary concentration of prolactin.

Smith et al. (1979b) and Asch et al. (1979b) have shown that administration of THC (2.5 or 2 mg/kg I.M.) produced a short-lived

inhibition of prolactin levels in the serum of male Rhesus monkeys.

Administration of thyrotropin releasing hormone (TRH) was able to

increase prolactin levels in THC treated monkeys, indicating that

the hypothalamus was the site of action in the THC-induced depression of prolactin release fran the pituitary.

Although there are conflicting reports on the ability of THC to

either increase or decrease prolactin levels in the pituitary and

in the serum, it appears that the effect of THC and perhaps other

non-psychoactive ingredients in marijuana is to produce a prompt

depression of prolactin secretion from the pituitary gland in the

male. The exact mechanism of this inhibition is not known at this

53
time; however, marijuana probably has some action on the hypothalamus to alter secretion of the several neural transmitters which are

probably involved in eliciting prolactin release fran the pituitary

gland. The ability of THC to inhibit the release of prolactin,

LH, and FSH fran the pituitary would appear to be unique, and in

apparent contrast to many other psychoactive drugs which stimlate

prolactin release while inhibiting the secretion of both LH and

FSH. It is also apparent that the male pattern of response to THC

with repressed prolactin levels is the same as has been shown to

occur in female animals and it is probable that the reduction is

caused by the same mechanism that cause the reduction in females.

EFFECT OF MARIJUANA THE ADRENAL GLAND

Cortical Hormones

Exposure to stressful situations elicits a prompt secretion of

adrenocortical steriods which help the organisms to counteract the

stress. The adrenal cortex responds to acute cannabinoid treatment with a prompt rise in corticosterone levels in the plasma.

Exposure to a wide range of dosages of THC ranging fran 2 to 50 mg/

kg body weight produced increased corticosterone levels in the

plasm of both the rat and the mouse. Dewey et al. (1970) showed

that ascorbic acid, which is inversely correlated to adrenal cortical hormne secretion, was depleted fran the adrenal cortex of

laboratory rats. Maier and Maitre (1975) demonstrated that. the

increased corticosterone in plasma of rats pretreated with THC was

accompanied by a decrease in adrenal cortical cholesterol, a precursor to adrenal cortical hormones, and an increase in unesterified fatty acids; however, the rabbit did not respond to THC with

a similar increase in wrtisol.

Birmingham and Bartova (1976) showed that the response of elevated

plasma corticosterone to THC disappeared after 8 days of treatment

with a dose of 3 mg/kg body weight. Pertwee (1974) also showed

that tolerance developed to the effect of THC on corticosterone

levels in muse plasm and did so without impairing the effect of

stress in mobilizing wrticosterone release. Mitra et al. (1977)

found no evidence of tolerance after 21 days of THC (10 mg/kg/day)

administration.

Bromley and Zimmerman (1976) examined the possibility that THC

acts as a general systemic stressor. Generally, stress causes an

increase in both prolactin and corticusterone levels in male rats.

However, their results indicated that, unlike most stressful stimuli, acute administration of THC caused a depression in prolactin

while stimulating corticosterone release.

Several studies have indicated that the wrticosterone response to

THC was mediated through the pituitary by the action of THC on

central nervous structures to modify the secretion or release of

ACTH from the hypothalamus. Barry et al. (1973) ruled out the

possibility that THC acted directly on the adrenal cortex to elicit corticosterone release. Their study employed hypophysectomized

rats which had been administered THC at 2 mg/kg body weight, a

54
dosage which significantly increases plasma corticosterone levels.

After hypophysectcmy, THC was unable to increase plasma corticosterone levels. Puder et al. (1982) further showed that adult male

rats, bearing complete hypothalamic deafferentations, who were

injected with THC (5 mg/kg) had no significantly altered serum

concentration of either ACTH or corticosteroids. These results

demonstrate that extrahypothalamic sites and/or neural pathways

mediate the effect of THC to decrease corticosterone secretion.

Further evidence for a central action of THC comes fran the study

of Malor et al. (1978). Mice treated with THC had a dosedependent

rise in plasma nonesterified fatty acids (NEFA) due to THC's action

in elevating corticosterone. The THC induced rise in plasma NEFA

was blocked by prior administration of the dopamine receptor antagonists, perphenazine or pimozide. Thus the elevation of plasma

NEFA produced in mice, by THC, is probably centrally mediated and

requires the presence of functional dopaminergic receptors which

presumably cause ACTH to be released to effect the corticosterone

induced rise in plasma NEFA. Moreover, pentobarbital (75 mg/kg)

(Mitra et al. 1977) and dexamethasone (Kokka and Garcia 1974), two

agents which block ACTH secretion, also prevent the adrenocortical

response to THC (10 mg/kg).

Several investigators have examined the accmulation of radioactive

THC by various brain structures. Erdmann et al. (1976) showed that

the amount of radioactive THC taken up by the preoptic areas, hypothalamus and pituitary, structures which are important for the

regulation and release of hormones, did not concentrate more radioactive compound than other brain structures. Also there was a

reduction in the amount of radioactive wrticosterone that was

accumulated by the hypothalamus and thalamus if rats were treated

with THC at high doses (9 mg/kg I.P.). Interestingly, and perhaps

in apparent contrast, smaller doses (3 mg/kg I.P.) actually increased corticosterone uptake (Drew and Slagle 1973). However,

Johnson et al. (1978) showed that high doses of cannabinoids (30

and 100 mg/kg) increased uptake of radioactive wrticosterone by

the whole brain. The cannabinoids examined were 11-hydroxy-delta9-THC, THC, and cannabinol (CBN). Pretreatment with THC at 3, 10,

or 100 mg/kg SC increased the affinity of the hippocampus for tritiated corticosterone, but its concentration was decreased in the

hypothalamus, mid-brain, pons, and medulla.

Collu (1976) injected THC (20 μg) directly into the ventricles of

the rat brain and produced an increase in the general activity of

cells in the adrenal and pituitary glands which was correlated with

an increased corticoceterone production. Chronic treatment of rats

and mice with THC produced an increased adrenal weight, which returned to control levels upon cessation of drug treatment (Dixit

et al. 1974). Pats pretreated with THC (5 mg/kg I.P.) were still

able to release corticosterone in response to stress (Jacobs et al.

1979).

Apparently not all of the effects of cannabinoids on adrenocortical

function are due to the effect that they have to stimlate ACTH and

corticosterone production by a direct endocrine route. Several

55
investigators have examined the effect of THC, CNB, and CBD on muse

and rat adrenal cortical cells grown in tissue culture. Addition of

10-6 to 10-4M cannabinoid to the incubation medium in which the cultured adrenal cortical cells were being grown prevented the cells

from responding to added ACTH (Carchman et al. 1976, Warner et al.

1977). The cannabinoids appear to depress the synthesis of adrenal

cortical steroids at a site between synthesis of cAMP and of pregnenalone (Warner et al. 1977). Delta-9-tetrahydrocannibinol (3.2

and 16 μM) added to an incubation medium that contained homogenized

rat adrenal cells produced an inhibition of cholesterol esterase

activity similar to that found in Leydig cells (Burstein et al.

1978a).

Adrenal Medulla

Several studies have examined the effect of THC on other cannabinoids on catecholamines and 5-hydroxytryptamine turnover in brain

and adrenals. Johnson et al. (1981) showed that 30 mg/kg of THC

increased the ammount of tritiated tryptophane found in the brains

of mice and increased the amount of tritiated 5-hydroxytryptamine

that was synthesized during the lo-minute period before decapitation. Previous studies have shown that this effect of THC was

mediated by increased levels of plasma corticosterone in the mice

produced by treatment with THC, since adrenalectomy inhibited it.

Thus, there is a possibility that wrticosterone may mediate the

effect of THC on tryptophane disposition and metabolism.

Mazurkiewics-Kwilecki and Filczewski (1973) showed that administration of THC (2 mg/kg daily) for 1 week caused an increase in the

synthesis of tritiated catecholamines in the brain and adrenals.

However, the endogenous norepinephrine and dopamine concentration

in the brain, and epinephrine and norepinephrine levels in the

adrenals, were apparently unaltered. The ability of THC to increase serotonin in the brain of mice has been confirmed by Welch

et al. (197l), who showed that intraperitoneal injection of 10 mg/

kg elevated serotonin concentration in the telencephalon by 30%

within 10 minutes after injection but had no effect on the endogenous levels of brain norepinephrine or dopmine. Delta-9-tetrahydrocannabinol, on the other hand, depleted adrenal epinephrine by

25% within 10 minutes. Mitra et al. (1976) showed that administration of a single dose of THC (10 or 50 mg/kg) produced a dosedependent increase in the content of norepinephrine and epinephrine

in the rat adrenal gland and that THC given acutely both at low

and high doses decreased the rate of synthesis and increased the

rate of depletion of dopamine, decreased the rate of depletion of

norepinephrine, and increase the rate of depletion of epinephrine.

Biswas et al. (1975) also showed that THC affected the rat adrenal

medulla; acute treatment (10 mg/kg) caused a decrease in catecho

lamines, and chronic treatment (10 mg/kg for 30 days) produced a

similar decrease in total catecholamine content of the adrenal

medulla, as well as an hypertrophy of chromaffin substance.

56
THE EFFECT OF MARIJUANA ON THYROID GLAND

The function of thyrotropin releasing hormone (TRH) is, to elicit

thyrotropin (TSH) secretion from the pituitary which in turn stimulates the thyroid gland to concentrate iodide from the blood, and

to synthesize and secrete the thyroid hormones. TRH is secreted

from the hypothalamus in response to dopamine and norepinephrine

stimulation. Grimm and Reichlin (1973) showed that incubation of

the hypothalamic tissue from mouse brain with either neurotransmitter, dopamine, or norepinephrine, was effective in causing a

release of TRH. However, agents which blocked conversion of

dopamine to norepinephrine were unable to elicit this response.

They also showed that whereas acetylcholine had no effect on TRH

release, serotonin tended to inhibit TRH release

The first indication that thyroid gland function might be affected

by marijuana was reported in 1965 by Miras, who showed that cannabis resin administered to rats produced a depression of radioactive

iodine accumulation in the thyroid gland. He was, however, unable

to show whether the cannabinoids in the extract affected thyroid

gland function directly at the site of the thyroid gland or whether

the action of the cannabinoids was by alteration of hypothalamic

output of TSH. Lomax (1970) injected marijuana distillate extract

into rats and found a decreased release of radioactive iodine from

the thyroid glands of rats. He showed that the hypothalamus was

probably the site of action by injecting TSH during the period when

marijuana was maximally depressing the radioactive iodine release

and reversed the inhibition of marijuana on radioactive iodine

release.

Nazar et al. (1977) reported that acute administration of THC at

2.5 mg/kg, and chronic treatment for 3 days, depressed serum thyroxine concentration in the rat. Single injections depressed

thyroxine in the serum for 6 hours following drug treatment.

Administration of TSH elevated serum thyroxine, indicating that

THC probably had its action on thyroid function at the level of the

hypothalamus. Under chronic THC treatment, however, twice daily

for 14 days, an apparent tolerance to the thyroid depressant action

of THC appeared to develop. They further showed that the presence

of the adrenal hormone was not required for the depressant action

of THC on thyroid gland function. Esber et al. (1976) administered

2 and 4 mg of THC per day by inhalation to rats and produced a

significant lowering of triicdothyronine (T3) in the serum; serum

thyroxine was not affected. Oral administration of THC to rats

(10 mg/kg/day for 14 days) had a more dramatic effect on serum

hormonal levels than administration by inhalation of marijuana

smoke. Oral administration significantly depressed both triiodothyronine and thyroxine.

Administering THC or smoking marijuana produced a decrease in body

temperature, with a peak response observed between 1 and 2 hours

after drug administration, which lasted from 5 to 6 hours. Bhargava

(1980) was able to antagonize the hypothermic response to THC in

mice by either intracerebral or intraperitoneal administration of

TRH prior to the THC injection. Similar effects were produced by

57

446-384 O - 84 - 5
histidyl proline diketopiperazine (HPD), postulated as a metabolite

of TRH, if given intracerebrally; however, it was completely ineffective if given intraperitoneally. Thus, the antagonism of THCinduced hypothermia by TRH may be mediated by its conversion to HPD

in the central nervous sytem.

EFFECT OF MARIJUANA ON GROWTH HORMONE

There is a body of evidence to indicate that the exposure of animals to marijuana, a marijuana extract, cannabinoids, or the

psychoactive ingredient, THC, produced depressed body weights and

altered organ sizes. There are a number of reasons to believe

that marijuana affects the normal growth processes. Growth of the

organism and its organ systems is a complex and highly integrated

system affected by many external and internal factors such as

nutrition, heredity, and hormonal regulation. Although many hormones interact to affect growth processes, one of the more important hormones is growth hormone; for this reason several research

workers have examined the effect that THC has on growth hormone.

Collu et al. (1975) injected prepubertal male rats intraperitoneally three times a week for a month with either 1 or 10 mg/kg

of THC. Treatment with 10 mg/kg produced rats with smaller tails

and prostates than non-treated animals and, in addition, plasma

levels of growth hormone and LH were decreased at all drug dosages.

While pituitary quantities of GH and LH remain unchanged, they

reported no effect on FSH and brain levels of norepinephrine,

epinephrine, serotonin, and 5-hydroxyindole acetic acid which were

comparable to those of the control group.

Collu (1976) administered THC (20 μg) each day for a week to prepubertal and adult rats intraventricularly. Prepubertal rats had

lower prostate weight; however, they had increased plasma and pituitary levels of growth hormone. Kokka and Garcia (1974) showed

that administration of 5 to 20 μm/kg of THC to adult rats caused

an inhibition of growth hormone secretion and a stimulation of

ACTH secretion. Dexamethasone, a potent supressor of ACTH secretion, blocked the stimulatory affect of an acute injection of THC

on ACTH secretion but did not affect its inhibitory action of GH

secretion. Treatment of rats with pentobarbital usually induces

a rise of plasma growth hormone; however, THC was able to suppress

the pentobarbital induced rise of plasma GH. Dalterio et al. (1981)

report that growth hormone levels in the plasma of male mice that

received a single dose of THC were reduced only in non-stressed

animals, whereas chronic treatment with THC did not affect prolactin or growth hormone levels under either stressed or non-stressed

conditions. In apparent contrast to the effects of THC, acute

administration of CBD, a non-psychoactive ingredient in marijuana,

produced increased plasma GH levels in non-stressed mice, while

repeated CBN treatments reduced GH levels in stressed animals.

SUMMARY

Marijuana affects a variety of hormones that are regulated by hypothalamic function and it appears that the psychoactive ingredient,

THC, is the major compound responsible for this action. It is

58
probable that THC affects these hormones through its ability to

alter various neural transmitters in the hypothalamus or neural

transmitters in the CNS which impinge on the hypothalarms. The

dopaminergic and serotonergic fibers seem to be particularly important. The two gonadotropins, LH and FSH, secreted by the pituitary

gland are of major importance to reproduction in the male. Both

gonadotropins appear to respond to a single releasing factor fran

the hypothalmus, GnRH, which is sensitive to catecholamine neurotransmitters. The THC-induced block of GnRH release results in

lowered LH and FSH which is responsible for reduced testosterone

production by the Leydig cells of the testis. Other hormones that

might have a synergistic or antagonistic effect upon reproduction

in the male are the adrenal cortical hormones, prolactin, thyroid

hormones, and growth hormones. THC appears to depress prolactin,

thyroid gland function, and growth hormone while elevating adrenal

cortical steriods.

Chronic exposure of laboratory animals, such as rats, mice, and

monkeys to marijuana and to the various cannabinoids in marijuana

has altered the function of several of the accessory reproductive

organs. Reports of reduced prostate and seminal vesicle weights,

as well as altered testicular function, have been partially explained by the effect of marijuana in lowering serum testosterone

needed for proper function and support. Although some of the

change in organ weight may be due to lowered testosterone production by the Leydig cells of the testis, sane of the weight changes

may be due to a direct action of THC, and perhaps some of the

other nonpsychoactive cannabinoids in marijuana, on the tissue

themselves. Also, of concern are the reports that acute cannabinoid treatments affects the quality and quantity of spermatozoa

produced by the testis. The question is still unanswered as to

whether or not the effects observed on spermatozoa are due to a

direct action of the cannabinoids on spermatogenesis, or whether

some of the observed effects may be due to altered hormone levels

which are necessary for the support of spermatogenesis. Reduced

testosterone and FSH may be important in producing the observed

changes in sperm production by the seminiferous tubules.

Many of the effects on the endocrine system caused by chronic

treatment of animals with THC are completely reversible with time

and there is reason to believe that tolerance develops to these

effects with acute exposure to THC. Still, many unanswered questions remain regarding the long-term consequence of marijuana use

on such important functions of male reproduction as sperm formation and maturation, and the long-term effects on the function

of the sexual organs. Until the time that answers to these and

other questions are forthcoming, it is questionable whether marijuana should be consumed by adolescent males or those males with

marginal fertility problems.

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AUTHOR

Jack Harclerode, Ph.D.

Department of Biology

Bucknell University

Lewisburg, Pennsylvania 17837

64
Endocrine Aspects of Cannabinoid

Action in Female Subprimates:

Search for Sites of Action

Lee Tyrey, Ph.D.

INTRODUCTION

There is no longer any doubt that marijuana and the cannabinoids

which it contains have pronounced effects on endocrine function

in humans and a variety of experimental animals. The increased

availability of delta-9-tetrahydrocannabinol (THC), the primary

psychoactive constituent of marijuana (Mechoulam 1970), and other

cannabinoids has greatly stimulated research into the physiological

effects of these unique agents. The results of this research

clearly indicate that THC administration alters the plasma levels

of several pituitary hormones, presumably by altering their

secretion. Among these hormones are the gonadotropins and

prolactin, the pituitary hormones that regulate reproductive

function. The effects of THC on these reproductive hormones have

been most pronounced in the female at least in part because the

rather wide fluctuations in their secretion and the consequent

pronounced cyclic changes in target tissues make almost any perturbation of this system easily discernible. At the same time, the

fact that changes in a target tissue such as the uterus depend upon

ovarian hormones, which in turn depend upon appropriate stimulation

from the pituitary whose activity is regulated by the brain makes

this system particularly complex. The functional dependencies

make it difficult to determine dhere cannabinoids may exert their

influence in the chain of interrelated events from the brain to the

target tissue. It is not surprising, therefore, that questions

remain regarding the extent of cannabinoid action on the uterus,

ovary, pituitary, and brain. What follows is an attempt to review

the evidence for the impact of cannabinoids, primarily THC, on these

potential sites of action.

THE UTERUS

The possibility that THC may have a direct “estrogenic” effect on

the uterus was raised by Solomon et al. (1976) who treated young

adult ovariectomized rats with daily intraperitoneal injections of

THC in dosages of 1, 2.5, or 10 mg/kg body weight (BW). Inject ions

started on the day of ovariectomy and continued for 14 days. The

reproductive tracts were removed for examination. All three doses

65
of THC had significant uterotropic effects as indicated by the

occurrence of uterine weights greater than those in vehicletreated ovariectomized rats. Nonetheless, the uterine stimulation

was substantially below that produced by 2 ug/kg doses of estradiol

benzoate. Of the three THC doses, the 2.5 mg/kg dose appeared to

have a somewhat greater stimulatory effect than the lower or higher

doses. Subsequently, Solomon et al. (1977) reported that cytologic

examination of the reproductive tracts removed from the THC-treated

ovariectomized animals indicated hyperplasia and hypertrophy of the

uterus which was most pronounced in those animals treated with the

intermediate 2.5 mg/kg dose. The uteri were reported to show

growth of the surface epithelium, endometrial stroma, and

myometrium, as welI as proliferation of the endometrial glands. The

vaginal epithelium showed increased stratification and cornification, frequently with a superficial layer of mucoid cells. These

findings contrasted to those with control animals in which the uteri

were small and poorly vascularized as typical for the ovariectomized

rat. While these interesting findings suggested that THC may have

direct estrogenic activity, a possibility subsequently supported by

a report that THC competitively inhibited the binding of 3H-estradiol to estrogen receptors (Rawitch et al. 1977), the bulk of the

evidence detracts from that hypothesis.

Several studies in which intact or ovariectomized rats and mice were

treated chronically with cannabis extract or THC (Dixit et al. 1975;

Rosenkrantz et al. 1975; Okey and Truant 1975; Virgo 1979) indicated

that uterine weights were either reduced or unaltered, even when

treatment was continued as long as 180 days (Rosenkrantz et al.

1975). Okey and Truant (1975) found no evidence of uterine

stimulation in immature or ovariectomized female rats after treatment with cannabis resin administered either in the diet or by

intraperitoneal injection. Moreover, cannabis resin added to rat

uterine cytosol preparations failed to compete with the binding of

3H-estradiol to estrogen receptors. In later studies, Okey and

Bondy (1977; 1978) conducted a detailed assessment of the potential

3H-THC binding to rodent mammary and uterine cytosol receptors, as

well as of the ability of unlabeled cannabinoids to compete with

estradiol for binding to receptor sites. They found that THC added

to cytosol preparations even at the limit of its solubility did not

compete with 3H-estradiol for receptor binding. Similar results

were obtained with the THC metabolite, 11-0H-THC. Studies

utilizing sucrose density gradients indicated that while the 3Hestradiol binding peak was typically in the 8s region, 3H-THC binding was restricted to the 4-5s region and was not inhibited by

excess amounts of either unlabeled THC or estradiol. These data

provide compelling evidence that THC does not exert a direct

estrogen effect on target tissues, since binding to the specific

high-affinity estradiol receptor is a necessary precondition for

such action.

66
Because THC does not interact with the uterine cytoplasmic estrogen

receptor, the earlier findings of Solomon et al. (1976; 1977) await

satisfactory explanation. Since THC activates the pituitary-adrenal

axis and increases plasma corticosterone concentration (Kubena et

al. 1971; Jacobs et al. 1979), Solomon et al. (1977) suggested that

a presumptive increase in the secretion of adrenal steroids induced

by THC treatment in their study may have altered the response of

the castrate’s reproductive tract to THC. It seems possible,

however, that insofar as the adrenal cortex is capable of producing

sex steroids which could act on the uterus, including considerable

amounts of progesterone (Holzbauer et al. 1969), the adrenal

activation by THC might in fact explain its “estrogenic” activity.

Perhaps an examination of the uterine effects of THC in adrenalectomized-ovariectomized rats would settle the issue

THE OVARY

The possibility that cannabinoids exert direct effects on the ovary

was raised by the observation that THC or its metabolites

concentrate in the corpora lutea of mice (Freudenthal et al. 1972).

Although this accumulation could be related merely to the

lipophilic character of cannabinoids, the possibility of direct

action was given physiological support by the observation of Nir

et al. (1973). These investigators found that exogenous LH

administration in a dose which was adequate to cause full ovulation

in rats whose normal ovulation had been prevented by pentobarbital

treatment was able to induce ovulation in only 40% of the animals

in which ovulation was blocked by THC. Subsequently, the same

group (Ayalon et al. 1977) found that two- to four-fold greater

doses of ovine LH were required to restore ovulation in THC-treated

animals as compared with those blocked with pentobarbital. Since

the ovulatory surge of gonadotropins in the rat is followed by

prostaglandin accumulation in the ovarian follicles (Bauminger et

al. 1975), and since prostaglandins have been implicated in the

mechanism of follicle rupture at ovulation (Tsafrifi et al. 1972),

Ayalon and his colleagues (1977) measured the accumulation of

ovarian prostaglandins following the injection of LH into

proestrous rats whose spontaneous LH surges were blocked with either

pentobarbital or THC. Consistent with the reduced ovulatory

response to LH, the ovaries of the THC-treated animals had

substantially reduced levels of prostaglandin E in comparison with

the ovaries of pentobarbital-treated rats. The fact that higher

doses of LH brought about a graded increase in prostaglandin

accumulation and ovulation suggested that THC or its metabolites

may interfere with the ovulatory process through an inhibition of

ovarian prostaglandin synthesis.

Ovarian steroid synthesis and secretion also seem to be subject to

direct inhibition by cannabinoids. Consistent with the inhibitory

effects of cannabinoids on steroidogenesis in testicular Leydig

cells (Burstein et al. 1978b) and adrenal tumor cells (Warner et

al. 1977), hCG-stimulated progesterone secretion from dispersed

rat luteal cells was reported to be inhibited by the addition of

67
THC or cannabinol to the incubation medium in micromolar

concentrations (Burstein et al. 1979). Later work extended the

observation of these THC inhibitory effects to isolated granulosa

cells (Moon et al. 1982). FSH-stimulated progesterone secretion

from incubated porcine granulosa cells was inhibited in a doserelated fashion by THC, 11-0H-THC, and SP-111A, a water soluble

derivative of THC. The latter also was shown to inhibit basal as

well as FSH-stimulated progesterone secretion from incubated rat

granulosa celIs. When entire rat follicles, which include the

thecal compartment in addition to the granulosa cells, were

cultured (Reich et al. 1982). THC and cannabidiol inhibited the

release not only of progesterone but also of testosterone and

estradiol. The inhibition was observed for both basal steroid

secretion and that stimulated by the addition of LH to the explant

incubation medium. It is interesting to note that the nonpsychoactive cannabinoids are somewhat more potent than THC in inhibiting

steroidogenesis in ovarian cells (Burstein et al. 1979; Reich et

al. 1982).

A unifying hypothesis to explain the similar actions of cannabinoids

on steroidogenesis in the testis, ovary and adrenal was suggested

by Burstein et al. (1978a) based on their observation that THC

inhibits cholesterol esterase activity over the same range of

concentrations effective in inhibiting steroid secretion. Since

steroidogenesis is to a major extent dependent upon the

availability of precursor free cholesterol, the inhibition of

esterase activity can be viewed as the likely explanation of reduced

steroid secretion from cells and tissues incubated with

cannabinoids. Consistent with this hypothesis are observations

that cannabinoids do not reduce LH-stimulated cyclic AMP production

in rat follicle explants and that the addition of cholesterol to

explant culture medium at least partially restores progesterone

production in the presence of cannabinoids (Reich et al. 1982).

Thus there is clear evidence that both psychoactive and nonpsychoactive cannabinoids exert a direct inhibitory action on

steroidogenesis. Whether the cellular site of action is cholesterol

esterase or is ultimately shown to be elsewhere, it seems that the

anti-steroidogenic activity of cannabinoids is unrelated to the

unique psychoactive properties possessed by THC. The physiological

consequences of these direct ovarian effects of cannabinoids, of

course, remain to be demonstrated. Attempts to do this in estrous

rabbits whose ovarian steroid secretion was stimulated by hCG

injection failed to find any effect of THC on the secretory patterns

of progesterone, testosterone, or dihydrotestosterone (Asch et al.

1979b). Of course, it is possible that the large dose of hCG (100

IU) overcame direct steroidogenic inhibitory effects of THC. In

any case, one may speculate that the direct ovarian effects of THC

identified in vitro are unlikely to be of major consequence in the

functional sense view of the profound effects of THC on the

ovarian regulatory hormones emanating from the pituitary.

68
THE HYPOTHALAMIC-PITUITARY AXIS

In the intact animal, the effects of cannabinoid treatment on

ovarian function, as well as those on the adrenal (Kubena et al.

1971; Jacobs et al. 1979) and thyroid (Nazar et al. 1977), can be

accounted for by the action of THC at the level of the brain or

pituitary. The suppression of plasma thyroxine subsequent to THC

treatment is readily overcome by the administration of thyroid

stimulating hormone (Nazar et al. 1977), and the corticosterone

elevation is accompanied by a marked elevation in ACTH levels

(Puder et al. 1982). In the case of those pituitary hormones which

act on the ovary, especially LH and prolactin, there is now abundant

evidence that THC exposure can alter their secretion to an

extraordinarv extent.

Luteinizing Hormone

The acute intravenous administration of THC to ovariectomized rats

in doses of 1 to 10 mg/kg BW was found to significantly reduce the

serum concentration of LH in samples taken 1 hr after treatment

(Marks 1973). Since the secretion of LH from the pituitary occurs

in an episodic fashion, consisting of semi-regular periodic

pulsatile releases, a lowering of serum LH concentrations by THC

could result either from an attenuation or reduced frequency of the

secretory pulses or from a reduction in the basal secretion upon

which such pulses occur. Subsequent work has shown that the decline

in serum LH following THC treatment results not merely from an

attenuation, but from a complete suppression of the episodic LH

secretory pulses (Tyrey 1978). Serial blood sampling of individual

animals after intravenous injections of THC in doses of 0.5 to 8

mg/kg BW revealed a complete, but transient, inhibition of LH

pulses. The consequent rapid decline in serum LH occurred at a

rate consistent with estimates for the serum half-life of endogenous

LH in the ovariectomized rat (Weick 1977). It was thus not possible

to discriminate between serum LH levels following THC treatment and

those observed 20 and 45 min after hypophysectomy (Tyrey 1978) an

observation implying that for a time following THC treatment little

new LH was secreted into the circulation.

In later experiments with ovariectomized rats, the

secretion of LH was inhibited by acute intravenous

with doses as low as 62.5 ug/kg BW (Tyrey 1980).

the earlier experiments with higher THC dosages no

apparent between the THC dosage and the degree of

episodic

THC treatment

In both these and

relationship was

LH inhibition

produced; however, the duration of the inhibitory effect clearly

depended upon the amount of THC administered. Although THC doses

of 62.5 ug/kg suppressed LH secretion for less than 30 min, the

duration of suppression increased with larger doses in a doserelated manner, continuing for 1 to 2 hrs when the dose reached

4 mg/kg BW (Tyrey 1978; 1980). The finding that the duration, but

not the degree, of THC-induced LH suppression in the ovariectomized

rat is dose-dependent is consistent with observations made in the

ovariectomized monkey (Besch et al. 1977; Smith et al. 1979). The

69
rat and monkey data differ, however., in that THC administration to

monkeys by intramuscular injection in doses equivalent to those

utilized in the rat studies produced a considerably longer period of

LH suppression, continuing for as long as 24 hrs in some cases.

Whether this difference results solely from the different routes

of drug administration used in these studies remains to be

determined.

While the elevated gonadotropin secretion in the ovariectomized

animal makes this model system particularly useful for

investigating the inhibitory actions of THC, serum LH in intact

cycling female rats also is suppressed following acute (Chakravarty

et al. 1975) or chronic (Chakravarty et al. 1979) THC treatment.

Moreover, experiments with intact cyclic female rats have shown

that, in addition to suppressing circulating LH levels, THC also

inhibits the spontaneous increase in LH secretion which brings about

ovulation and conversion of the ruptured follicle to a corpus

luteum. Nir et al. (1973) found that intraperitoneal administration

of THC in doses which approximated 10 mg/kg BW would prevent the

proestrous surge of LH and thereby block ovulation. Similar effects

have been noted for the rabbit, in which the ovulatory surge of

gonadotropins is not released spontaneously, but rather is induced

by mating. In this species, the acute intramuscular injection of

THC in doses of 2.5 or 5 mg/kg BW at 2 hrs before mating completely

blocked the reflex LH surge and ovulation (Asch et al. 1979a).

In spontaneous ovulation, the ovulatory surge of gonadotropin is

believed to be initiated by the positive feedback action of ovarian

estrogen on brain centers regulating LH secretion. It is possible

to demonstrate this positive feedback action of estrogen in

ovariectomized rats which have been estrogen primed. In this model,

a second estrogen injection 3 days after the priming injection

induces a surge of LH not unlike that occurring on the afternoon of

proestrus. Thus it is noteworthy that the estrogen-induced LH surge

in the estrogen-primed ovariectomized rat is also blocked by THC

administration (Steger et al. 1980). Treatment with THC therefore

appears to prevent expression of the cyclic gonadotropin release

mechanism responsive to estrogen or, in the case of reflex ovulators

such as the rabbit, neural activation. Whether cannabinoid

exposure actually prevents activation of this cyclic release

mechanism or, alternatively, simply prevents its expression by

inhibiting LH secretion through an action which may be equivalent to

that operating to suppress the tonically elevated LH secretion in

the ovariectomized animal remains a matter for speculation. Of

course, in either case, the actual mechanism of THC action is

unknown.

Although cannabinoids may have direct effects on steroid secretion

from the ovary or other steroid secreting glands, it is not possible

to explain the inhibition of LH secretion on the basis of a direct

effect on the pituitary. Several studies have established that the

administration of exogenous GnRH readily overcomes the suppression

of LH secretion induced by THC exposure (Ayalon et al. 1977; Tyrey

1978; Smith et al. 1979; Asch et al. 1979a). Moreover, the

70
magnitude of the LH response to an injection of exogenous GnRH in

ovariectomized rats during THC-induced LH suppression was equivalent

to that induced by GnRH in ovariectomized rats in which LH secretion

was suppressed by estrogen and progesterone pretreatment rather than

by THC (Tyrey 1978). In contrast to these studies, there is a

single report of reduced LH responsiveness to exogenous GnRH in

male rats after acute or chronic THC treatment (Symons et al. 1976).

Follicle Stimulating Hormone

The effect of cannabinoid treatment on FSH secretion is neither as

clear nor as extensively studied as that on LH. While serum FSH

levels in ovariectomized monkeys were suppressed in a fashion

parallel to that of LH following THC treatment (Smith et al. 1979),

THC treatment of ovariectomized rats in doses that suppressed LH

concentrations had no effect on serum FSH levels (Steger et al.

1981). However, the proestrous surge of FSH in the cyclic rat, like

that of LH, can be prevented by appropriately timed THC injections

(Ayalon et al. 1977). Additional studies are required to clarify

this apparent difference in the FSH responses to THC treatment in

rats and monkeys.

Prolactin

Prolactin, which has lactogenic and, in the rat, luteotropic

actions, also is suppressed by cannabinoid treatment. While one

group (Daley et al. 1974) has reported a modest increase in serum

prolactin in male rats at 24 hrs after the last of 4 daily THC

injections, most reports indicate reduced serum prolactin levels

after THC treatment. Studies with intact male (Kramer and BenDavid 1974; 1978) and female (Chakravarty et al. 1975) rats,

ovariectomized rats (Hughes et al. 1981), and male and

ovariectomized female monkeys (Asch et al. 1979) all revealed a

clear suppression of basal prolactin secretion by THC. This is of

particular interest since, unlike LH whose release requires active

stimulation from the hypothalamus, prolactin secretion is under

tonic inhibitory control. Dopamine plays a central role in this

inhibitory control by acting directly on the prolactin secreting

cells of the pituitary as a prolactin inhibiting factor (PIF), and,

perhaps, by acting in the hypothalamus to bring about the release

of some other PIF. Thus basal serum prolactin concentrations,

normally maintained at relatively low levels as a result of tonic

inhibition, can be further decreased by THC treatment. This raises

the question as to whether active increases in prolactin secretion,

which would require withdrawal of the tonic inhibition or

stimulation by a releasing factor, perhaps TRH, also might be

suppressed by THC. In the rodent there are three recognized

physiological stimuli for prolactin secretion: rising estrogen

titers, stimulation of the uterine cervix, and suckling of the

lactating female. As indicated below, the secretion of prolactin

induced by any of these stimuli can be blocked by THC treatment.

The estrogen provoked rise in serum prolactin occurring coincident

with the proestrous gonadotropin surge in the rat is blocked, as is

71
the gonadotropin surge itself, by THC administration (Ayalon et al.

1977). Thus THC inhibits the surge secretion of LH, FSH and

prolactin during proestrus.

The secretion of prolactin induced by cervical stimulation occurs in

a pattern quite distinct from that of proestrus and without the

coincident surges in LH and FSH. Stimulation of the uterine cervix,

whether resulting from mating or some artificial means, initiates a

series of twice daily prolactin surges, one nocturnal and one

diurnal, which continue for several days. The effect of THC treatment on these surges was studied in rats in which pseudopregnancies

were induced by electrical stimulation of the cervix (Hughes and

Tyrey 1982). When such animals were given a single intravenous

injection of THC in a dose of 4 mg/kg BW at a time just

before the beginning of a nocturnal surge was expected, the

initiation of that surge was delayed for approximately 1 hr; but not

otherwise affected. On the other hand, when the THC was

administered in hourly doses of 1 mg/kg BW throughout the interval

during which the prolactin rise normally occurs, the surge was

prevented altogether. It should be noted that even though the

hourly THC injections continued into the next day, there was no

apparent inhibition of the presumably basal prolactin secretion

occurring at that time. The reason for the failure of these

continued injections to suppress prolactin below control levels is

not clear, but conceivably could be related to the repetitive

treatment at rather close intervals leading to the development of

tolerance to continued drug action. Additional investigation is

required to clarify this issue.

An early indication of the potential inhibitory influence of THC on

the increase in prolactin secretion induced by the suckling stimulus

was provided by the report (Borgen et al. 1971) of marked postnatal

mortality among rat pups born to mothers chronically treated with

THC during pregnancy. These investigators suggested that this

effect resulted from inadequate maternal lactation, rather than from

some defect in the newborn pups. Cross-fostering of the offspring

of THC-treated mothers to control mothers and of the control

offspring to THC-treated mothers allowed the drug-exposed offspring

to survive, while the cross-fostered control pups did poorly.

Starvation was apparently the cause of the increased pup mortality,

for milk was not always visible in the stomachs of pups suckling

treated mothers. Similar effects associated with perinatal THC

treatment have since been reported for mice (Szepsenwol et al. 1979;

Hatoum et al. 1981).

The reports of lactational problems in THC-treated rodents suggested

that THC treatment may interfere with the increased prolactin

secretion required for normal lactation. Indeed a delay in the

post-partum prolactin rise in mice chronically treated with THC

during pregnancy and the early post-partum period (Raine et al.

1978) indicated that prolactin secretion is altered in treated

mothers. Of particular importance was the question of whether the

prolactin surge induced by each suckling episode, necessary for the

continuation of adequate lactation, might be sensitive to the

72
inhibitory actions of JHC. An early attempt to answer this question

(Bromley et al. 1978) was compromised by the immediate disruption

of maternal behavior resulting from THC administration.

Consequently it was necessary to remove pups from the mother at the

time of THC administration and to judge the effects of THC treatment on suckling-induced prolactin secretion by comparisons of the

relative rates of decline in serum prolactin in the THC-treated and

control mothers. A somewhat more rapid decline of prolactin

concentrations in THC-treated animals was interpreted as indicating

an inhibition of suckling-induced prolactin secretion, since it was

assumed that prolactin secretion in the controls would continue for

a time even after pup removal. Later studies utilized urethane

anesthesia to circumvent the disruption of nursing behavior by THC

administration and, since this procedure allows the neuroendocrine

reflex response to suckling to remain intact, a clear demonstration

of the inhibitory effect of THC on suckling-induced prolactin

secretion was possible (Tyrey and Hughes 1983). The intravenous

injection of THC into lactating rats just prior to suckling

prevented the reflex prolactin surge in most animals. When THC

administration was delayed until after the surge was under way,

prolactin secretion was nonetheless curtailed and the serum

prolactin concentration declined even though suckling continued.

As in the case of LH, there is no evidence that cannabinoids inhibit

prolactin secretion through direct action on the pituitary. THC

added to hemi-pituitary incubations in concentrations of 10-6 or

10-4 M had no detectable effect on the release of prolactin into

the medium (Hughes et al. 1981). That these incubated hemipituitaries could respond to agents which have a direct suppressive

action was demonstrated by the clear inhibition of prolactin

secretion following the addition of the dopamine agonist

bromocryptine (CB-154) to the incubation medium. Since the in vitro

studies did not exclude the possibility that prolactin suppression in

vivo resulted from the direct pituitary action of a THC metaboliterather than from THC itself, Hughes et al. (1981) also investigated

the potential effect of THC on prolactin secretion from ectopic

pituitary autografts. The elevated prolactin secret ion from

autografts placed beneath the kidney capsule in hypophysectomized

rats was unaffected by the intravenous injection of THC in a dose

of 1 mg/kg BW, even though this dose was maximally effective in

suppressing serum prolactin from the pituitary remaining under CNS

control. The failure of THC to inhibit prolactin secretion from

these pituitary autografts or from pituitary tissue incubated in

vitro provides strong evidence against the possibility that THC

inhibits prolactin secretion through action at the pituitary level.

A similar conclusion has been drawn from the observation in the

primate that TRH administration stimulates prolactin release during

THC suppression (Asch et al. 1979c). Thus, the effect of THC on

both gonadotropin and prolactin secretion appears to involve some

alteration of the central neuroendocrine control of the pituitary.

73

446-384 O - 84 - 6
CENTRAL NERVOUS SYSTEM

If THC does not act directly on the pituitary to alter hormone

secret ion, the next logical potential site of action would be the

medial basal hypothalamus (MBH). This region of the hypothalamus

contains the greatest abundance of nerve endings containing the

hypothalamic releasing and inhibiting factors which form the

functional link between the brain and anterior pituitary. While it

is reasonable to suggest that THC may disrupt anterior pituitary

function through a direct effect on these neurosecretory elements,

the most recent evidence indicates that this is probably not the

case.

Hughes et al. (1983) recently utilized microsurgical procedures in

female rats to produce complete deafferentation of the MBH and then

tested the ability of THC to inhibit prolactin secretion in these

animals. The deafferentation procedure disconnects the MBH from

other parts of the brain, but leaves it anatomically and

functionally connected to the pituitary. Low serum prolactin

concentrations are maintained in these animals as a result of

continued tonic inhibitory control by the remaining hypothalamic

“island.” When such animals were treated with intravenous THC in

a dose which caused profound inhibition of prolactin secretion in

the intact animal, there was no detectable decrease in serum

prolactin concentrations. The hypothalamic region included within

the surviving “islands”, determined histologically, included, in

addition to the median eminence, most of the arcuate nucleus and

the ventral portions of the ventromedial nuclei. Since this region

of the hypothalamus is generally acknowledged to be directly

involved in the regulation of prolactin secretion, the lack of THC

effectiveness in suppressing serum prolactin levels in these rats

suggests that THC does not act at a site within the MBH, and thus

presumably not on those elements (most directly involved in

prolactin regulation.

Since isolation of the MBH from the remainder of the brain

prevented expression of the THC inhibitory effect on prolactin,

other animals were subjected to partial deafferentation of the MBH

by the placement of cuts in the frontal plane at the level of the

suprachiasmatic nuclei or just rostral to these nuclei. These cuts

were intended to interrupt rostral afferent pathways into the MBH

believed to be important in the regulation of prolactin secretion.

Both the frontal cuts at the suprachiasmatic level and those placed

slightly more rostrally prevented the suppression of prolactin

secretion by THC. Moreover, in some animals with the more rostral

presuprachiasmatic cuts, a brief rise in serum prolactin was noted,

the significance of which remains to be explained. Nonetheless,

the failure of THC to bring about the expected suppression of

prolactin secretion in animals with these frontal cuts implies that

the inhibitory action of THC on prolactin secretion depends either

upon the anterior hypothalamic region destroyed by these cuts, or

upon the rostral fiber pathways which pass through this region to

reach the MBH.

74
Additional experiments utilizing more selective lesioning techniques

were conducted in an attempt to identify an afferent system which

might be involved in conveying the THC inhibitory effect. Septal

lesions interrupting the lateral corticohypothalamic tract which

includes a suspected prolactin regulatory pathway from the orbital

frontal neocortex to the preoptic area (Tindal and Knaggs 1977)

failed to prevent suppression of serum prolactin after THC

administration. Similarly, although lesions of the amygdala

influence prolactin secretion in the rat (Peters and Gala 1980),

destruction of amygdalar projections into the preoptic and

hypothalamic areas by way of the diagonal band of Broca and the

stria terminalis failed to prevent expression of the THC inhibitory

effect on prolactin secretion. Finally, destruction of the

periventricular region at the rostral midbrain level, intended to

interrupt a presumptive ascending prolactin regulatory pathway

(Tindal and Knaggs 1977), also failed to alter the suppressive

effect of THC.

The deafferentation studies thus provide evidence that the THCinduced suppression of prolactin secretion in the female rat is

not attributable to site of action within the MBH. Furthermore,

prevention of the THC suppressive effect by frontal cuts at the

suprachiasmatic level suggests that rostral pathways into the MBH

convey the THC inhibitory effect. If this is the case, those

pathways carrying hypothalamic projections from the orbitofrontal

neocortex, amygdalar, and midbrain periventricular areas thought to

be involved in prolactin regulation do not appear critical.

In addition to the evidence that THC acts at a site outside of the

MBH in inhibiting prolactin secretion, other evidence indicates

that this is also the case for THC stimulation of ACTH secretion

(Puder et al. 1982). The ACTH surge normally induced in the rat

by THC administration does not occur in rats previously subjected

to complete hypothalamic deafferentation. Thus extra-hypothalamic

sites appear to be involved in the mediation of the stimulatory

effects of THC on ACTH secretion as well as its inhibitory effects

on prolactin.

SUMMARY

The search for a site of cannabinoid action in non-primate

experimental animals has raised the possibility of drug action at

each level of the female reproductive system. The early suggestion

that THC may have a direct “estrogen-like” action on the uterus

has not been substantiated by subsequent investigations indicating

that THC does not interact with the estrogen cytoplasmic receptor.

Since receptor recognition is a fundamental requirement for

hormone action, it is unlikely that THC acts as an estrogen.

The experiments suggesting such action should be repeated under

conditions where the potential confounding effects of steroids

secreted from non-gonadal sources are controlled,

Direct ovarian effects of THC on the ovulatory process in the

rodent as well as on steroid secretion from the cells of both the

75
corpus luteum and the preovulatory follicle have been demonstrated.

Whether these effects have significance with respect to

physiological function remains a question, however, in view of the

rather pronounced effects of cannabinoids on the secretion of

those pituitary hormones regulating these ovarian events. Only

additional investigation in vivo of the more subtle gonadal effects

of THC treatment can clarify this issue.

With respect to the pituitary hormones, there is clear evidence for

the profound effects of cannabinoid exposure, of which the most

pronounced may be those on the secretion of LH and prolactin.

Effects on these reproductive hormones carry the threat of

potential disturbance of the reproductive process, especially in

the female where there is great dependence upon the appropriate

cyclic changes in hormone levels. The full biologic impact of the

pituitary effects of cannabinoids requires careful and thorough

assessment.

It can be concluded with reasonable confidence that THC alters the

secretion of the pituitary reproductive hormones, and that of ACTH

as well, through actions in the brain. While it would be

reasonable to suggest that this site of action may reside in the

MBH, the region of the hypothalamus most intimately associated with

pituitary function, that does not seem to be the case for effects

on prolactin and ACTH. Prevention of the expression of THC effects

on these hormones by MBH deafferentation points to a more distant

site for THC action. Some of the more likely possible sites for

THC inhibition of prolactin secretion have been investigated, but

direct evidence for their involvement was not forthcoming.

Similarly, the pineal apparently is not involved in the

suppression of LH secretion by THC (Johnson et al. 1980). Thus the

search for a site of THC action which had narrowed to a focus upon

the hypothalamic-pituitary axis must again be expanded into a

probing of higher brain regions. This means, of course, that the

search for a site of drug action, and ultimately a mechanism, has

been enormously complicated. Nevertheless, the need for a more

complete understanding of cannabinoid actions on central neuroendocrine regulation clearly justifies the effort.

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ACKNOWLEDGMENT

The preparation of this review was supported by grant DA 02006

from the National Institute on Drug Abuse.

AUTHOR

Lee Tyrey, Ph.D.

Department of Obstetrics & Gynecology

Duke University Medical Center

Durham, North Carolina 27710

87
Acute, Short-Term, and Chronic

Effects of Marijuana on the Female

Primate Reproductive Function

Carol Grace Smith, Ph.D., and Ricardo H. Asch, M.D.

INTRODUCTION

The reproductive effects of marijuana and delta-9-tetrahydrocannabinol (THC) have received much attention fran the scientific

community for the past several years. Early clinical reports

indicated that chronic marijuana use might be associated with

decreased hormone levels and infertility. later studies failed to

confirm these findings. Studies in laboratory animals clearly

demonstrate that THC has pronounced effects on reproductive

hormones and on ovulation and spermatogenesis, and have provided

much informatiom on how these effects are produced. These studies

have not, however, provided much insight into the apparent discrepancy in the pronounced drug effects reported in laboratory animal

studies and the less impressive effects or absence of disruptive

effects reported in clinical studies. Since much of our knowledge

of human reproductive physiology has been obtained from studies in

laboratory animals, it is difficult to ascribe these differences

to species variations. It is more likely that the discrepancies

in laboratory animal studies and the clinical studies are based on

differences in experimental designs and failure to consider the

role of the development of drug tolerance in the conclusions of

these studies.

The sexually mature rhesus monkey is one of the best experimental

animal models for studying the human reproductive system. The

female commonly has a 28-day menstrual cycle that is controlled by

negative and positive feedback mechanisms between gonadal steroids

and pituitary gonadotropins identical to those found in the human

menstrual cycle. Specific ammounts of marijuana derivatives can be

administered to these animals and reproductive paramters can be

examined directly. The purpose of this review is to sumarize the

effects of acute, short-term (less than 30 days), and chronic

treatment with THC on the primate menstrual cycle.

82
ACUTE EFFECTS OF MARIJUANA (THC) OF FEMALE REPRODUCTIVE HORMONES

Acute Effects of THC on Pituitary Hormones

There is evidence that hypothalamic nervous pathways that control

the secretion of ganadotropins are inhibited by such CNS drugs as

marijuana, narcotics, barbiturates and tranquilizers. The neuroendocrine effects of marijuana and its constituent compounds are

exceptimal in several ways and have provided interesting insights

into the study of drug effects on hypothalamic-pituitary function.

Since the initial report by Marks (1973) of the inhibitory effect

of THC on luteinizing hormone levels in ovariectanized rats, a

ber of studies have appeared which confirm this observation and

attempt to describe the mechanism. Studies in the ovariectanized

rhesus monkey have defined the dose-response relationship of the

effect (Smith et al. 1979a). In these studies, THC (0.6-5.0

mg/kg) was given to ovaraiectomized monkeys by an intramscular

injection. The result was a prompt and significant&crease in LH

levels (average 50-80 percent decrease) that lasted for 12-24

hours depending upon the dose level of THC. These results are in

agreement with studies in ovariectomized rats (Tyrey 1978) which

showed a suppression in episodic LH secretion following THC

administration. In both the monkey and rat studies, the magnitude

of the decreases appeared not to be directly related to the dose

of THC; but the duration of the responses was shown to be related

to the dose, with larger doses of THC producing longer lasting

depression in gonadotropin levels. The suppression of LH secretion appeared to be complete, but the effect was completely

reversible.

A comparision of the effects of the various doses of THC on the

levels of LH and the levels of FSH in ovariectanized monkeys

showed great difference between the two gonadotropins (Smith

et al. 1979b). For example, the average maximum decreases in LH

and FSH following the 5.0 mg/kg dose of THC were 68 percent and 56

percent respectively. Comparison of the time course of the

effects on gonadotropin levels in individual monkeys showed that

the maximum decrease in hormone levels occurred at generally the

same times for LH and FSH.

It is important to note, at this point, that these studies have

examined the effect of delta-9-tetrahydrocannabinol on pituitary

hormone levels. One study has compared the effects of THC and

other marijuana derivatives, including the crude alcohol extract

of marijuana (CME) containing 25 to 30 percent THC (Smith et al.

1980). THC and CME were administered to ovariectomized monkeys at

equivalent dose levels (based on total ammount of THC). The

decreases in both LH and FSH levels were equivalent in response to

equal doses of THC and CME. That is, the 4.16 mg/kg dose of CME

produced an average 40.8 percent inhibition of LH levels that

lasted for 6 hours. The equivalent dose of THC (1.25 mg/kg)

produced an average 35.9 percent decrease in LH that also lasted

for 6 hours. Other cannabis derivatives that were examined for

83
effects on pituitary hormones included cannabidiol (CBD) and

cannabinol (CBN). Neither CBD nor CSN had any statistically

significant effect on gonadotropin levels at dose levels up to 10

mg/kl.

The comparison of the inhibition of gonadotropins produced by THC,

marijuana extract, and the other cannabis derivatives indicates

that the inhibitory action of marijuana on gonadotropin levels is

producedby THC, andthatthe other cannabis derivatives contained

in marijuana do not contribute to the effect (Smith et al. 1980).

This is particularly evident because the relative doses of CBN and

CBD used in this study were much larger than would be contained in

the doses of marijuana extract. In addition, these results

suggest, but do not prove, that the inhibitory effect of cannabis

derivatives on gonadotropins is related to their psychoactivity.

Further studies need to be done with other psychoactive cannabinoids and with cannabis derivatives devoid of psychoactive properties. It should also be noted that while certain cannabis derivatives may not contribute significantly to the endocrine changes

caused by marijuana, they may have other direct effects on spermatogenesis, ovulation, and other reproductive functions.

The pharmacological site of action of a single dose of THC on

gonadotropin lwels was also investigated in ovariectomized

monkeys (Smith et al. 1979b). Synthetic gonadotropin releasing

hormones (GnRH)was administered to the monkeys 6 hours after the

administration of 2.5 mg/kg of THC. This dose of THC produces a

statistically significant depression in gonadotropins that lasts

for at least 12 hours. GnRH administration resulted in a stimulation of the blood levels of both LH and FSH. The stimulation of

LH and FSH levels measured after GnRH administration could be

considered as a reversal of the effect of THC since all of the

stimulated gonadotropin levels were within the 95 percent confidence interval established for each monkey's control values. The

response to releasing hormone in ovariectomized rats was the same

as the response in monkeys (Tyrey 1978). Further, the suppression

of ovulation in rats (Nir et al 1973) and in rabbits (Asch 1979a)

caused by THC can be reversed by the administration of GnRH.

These results show that the pituitary gland can respond to hypothalamic releasing hormones by releasing gonadotropins in the presence

of THC. This indicates a hypothalamic site of action for THC, and

while the exact mechanism of the inhibitory effect of THC on

gonadotropin secretion remains unknown, the involvement of hypothalamic neuroendocrine processes seems very likely.

While there are a number of drugs, including narcotics, phenothiazine tranquilizers, and sex steroids, that will inhibit LH and FSH

levels, these drugs have a stimlatory effect on prolactin levels.

Studies in male and female rhesus monkeys have shown that the

acute effect of THC on prolactin levels is a significant, but

short-lived inhibition (Asch 1979b). THC or vehicle was administered to the monkeys, and blood was drawn at 30, 60, 90, 120 and

180 minutes after injection. While vehicle administration produced

84
no cmsistent change in prolactin levels, THC produced a prompt

and sigficant decrease in prolactin level. The decrease in

prolactin levels was significant for both male and female monkeys

(average 84 percent decrease) at all of the time intervals from 30

to 180 minutes. The site of action of the inhibitory effect of

THC on prolactin was determined by using thyrotropin releasing

hormone (TRH). TRH administration stimulated the release of

prolactin fran the pituitary by a direct actim on the pituitary

gland. The results indicated that the effect of THC on prolactin

levels is mediated by a hypothalamic site of actim, since the

administration of TRH at 30 minutes after THC injectim reverses

the inhibitory effect on prolactin levels. This inhibitory actim

of THC on prolactin secretim has also been shown in the rodent

(Daley et al. 1974 and Raine et al. 1978). Further, this respmse

can be abolished by cyproheptadine, a serotonin antagonist or

perphenazine and pimozine, dopamine antagonists (Kramer and

Ben-David 1974). While the exact mechanism remains unclear, the

evidence now shows that the acute effect of THC on prolactin is

inhibitim.

It is frequently difficult to distinguish between direct drug

actims on the hypothalamic-pituitary axis and subsequent effects

on ggonadal hormones and sex accessory gland function. The distinction is an important one,however. Most neuroactive drugs produce

only transient effects on the central nervous pathways necessary

for normal gonadotropin secretion. The disruptive effects of

these drugs are likely to be transient and canpletely reversible.

Under these circumstances, normal adults may experience only

subtle changes in sexual function. However individuals with

compromised reproductive function may exhibit major problems. It

is also likely that adolescents may risk reproductive damage,

since the endocrine events associated with puberty are dependent

upon the developing hypothalamic-pituitary axis.

Acute Effects of THC on Neuroendocrine Function

It has been known for several decades that the secretions of the

anterior pituitary gland are regulated by substances generated by

the hypothalamus. The best evidence for this came from the

demonstration of the existence of a factor in the hypothalamus

that causes the release of the gonadotropins from the anterior

pituitary gland. This releasing factor actually brings about the

release of both the gonadotropins LH and FSH, so it is also

referred to as gmadotropin releasing hormone (GnRH). It is now

recognized that all of the effects of the hypothalamus on the

release of both FSH and LH can be explained by variations in dose,

time course, and steroid hormone interactims at the pituitary

level with a single releasing factor, CnRH. The mechanisms behind

basal or tonic levels of secretion of gonadotropins, the generation of gonadotropin surges, or even the timing of the onset of

puberty are not well understood.

There is considerable evidence in experimental animals that the

85
hypothalamic neurons that release GnRH are regulated by biogenic

amines. For example, dopamine andnorepinephrine cmtent in the

hypothalamus change with different stages of the estrous cycle in

the rat, and dopamine can stimulate GnRH release when incubated

with hypothalamic fragments in vitro (Schneider and McCann 1976).

Little is know, however, about the role of biogenic amines in LH

or FSH cmtrol in primates. Primates and rodents differ in the

development of the cyclic LH release mechanism, in that androgen

levels early in life suppress that resonse in the rodentbutnot

in the primate. In the rhesus monkey, both thetmic and episodic

secretary mechanisms appeartobelocalizedwithinthemedial

basal hypothakus (Knobil 1974), while in rodents, neural pathwaysthatariseelsewhere in the brain inpinge upon the GnRH

system. These differences may help explain the differing susceptibility of gonadotropins in primates and rodents to pharmacological

manipulation. In general, it can be said that the neuropticological studies in primates indicate that norepinephrine, and

perhaps dopanine, may be involved in GnFN release. Adrenergic and

dopaminergic agonists probably enhance the release of GnRH, and

adrenergic and dopaninergic antagonists probably inhibit the

release of GnRH. More studies need to be dme in primates and

humans before the exact role of these transmitters can be defined.

Of the hypothalamic mechanism for the control of pituitary

hormones, the mechanisms for the control of prolactin have been

most extensively studied. Prolactin secretion is probably controlled by two systems: a prolactin inhibitory factor (PIF) and

one or more prolactin releasing factors. Current evidence indicates that dopamine is the inhibitory substance of hypothalamic

origin that cmtrols prolactin secretion. The existence of a

prolactin releasing factor or factors is less well established.

Serotonin (5-HT), in cmtrast to dopamine, has been shown to

produce an increase in prolactin release (Meites et al. 1972).

Current evidence gives no clear picture of the effects of THC on

hypothalanic neurotransmitters and GnRH concentrations. Studies

by Doms et al. (1981) show that acute admistration of THC causes

an accumulation of GnRH containing granules in the medial basal

hypothalamus in rats. In recently completed studies, Steger et

al. (personal communication) examined levels of the hypothalamic

transmitters dopamine, norepinephrine, and serotonin, and of GnRH,

in ovariectomized rhesus mmkeys that were sacrificed 12 hours

after a single dose of THC (2.5mg/kg) or vehicle. The brains were

removed and frozen within 5 to 7 minutes. The median eminence,

medial basal hypothalamus and preoptic area and a portion of the

frontal cortex were dissected and prepared for neurotransmitter

analysis. The results showed no effect of THC on norepinephrine

or dopanine levels inanyofthebrainareas studied. GnRH levels

were significantly elevated in the median eminence and the medial

basal hypothalamus in the THC treated monkeys. There were no

significant changes in serotonin levels in any of the brainareas

studied but 5-hydroxyindoleacetic acid (5-HIAA) levels were

significantly decreased in the preoptic area in the THC treated

86
monkeys. These results indicate that acute THC treatment muses

an accumulation of GnRH in the hypothalamus when gonadotropin

levels are markedly depressed. If the decline in 5-HIAA levels

indicates decreased serotmergic activity, this may explain the

decrease in prolactin caused by acute THC treatment. Additional

experiments are needed before the mechanism of the effects of THC

on hypothalamic function can be described.

Acute Effects of THC on Gonadal Hormones

The acute effects of THC on menstrual cycle hormones have been

Studied in the rhesus monkey using both in vivo and in vitro

techniques (Almirez et al. 1983). In the in vivo studies, THC or

vehicle was administered by an intramuscular injection to rhesus

monkeys on day 20, 21 or 22 of the menstrual cycle. Progesterone

levels were measured at 6 hour intervals for the first 24 hours

after treatment. THC caused a significant decrease in

progesterone levels during this 24 hour period. This decrease was

reversed by the administration of human chorionic gonadotropin

(HCG) at 6 hours after THC administration. This stimulatory

effect of HCG could be observed as early as 30 minutes after

injection, and normal progesterone levels were observed from 180

minutes to 48 hours after the HCG injection. These results

indicate that the acute effect of THC on progesterone levels is

mediated by an indirect effect on pituitary gonadotropins rather

than direct effects on the ovarian synthesis and secretion of

progesterone. This cmclusimis supportedby in vitro studies of

basal progesterme production by dispersed luteal cells from the

rhesusmmkey. The corporaluteawere surgically removed from the

ovaries six to eight days after ovulation. The dispersed cells

will continue to produce progesterone for several hours without

adding gonadotropin to the medium. THC or marijuana extract was

added to the cell suspension to a final cmcentratim of up to 50

micromolar. Neither THC nor marijuana extract had any effect on

progesterone production at any cmcentratim up to 50 micromolar

(limit of drug solubility).

Other in vitro studies with cannabinoids have shown that these

drugs disrupt gonadal steroidogenesis, protein and nucleic acid

synthesis, glucose utilization and prostaglandin synthesis, and

reduce cyclic AMP concentrations in various species including

mice, rats and pigs and in various tissues including Leydig cells,

granulosa cells and luteal cells (Ayalm et al. 1977; Burstein et

al. 1979; Dalterio et al. 1976; Jakubovic and McGeer 1976; and

Reich et al. 1982). Because of the cmflict of the in vivo

studies that show no direct effect on gonadal steroid production

with these in vitro results, special attentim to the details of

the in vitro methods warranted. All of the cannabinoids

studies in vitro systems are poorly water soluble. If

the solubility of the compound is exceeded, the actual tissue

level may be greater than the cmcentratim added to the medium.

Unlike the studies with dispersed luteal cells from rhesus

monkeys, most of the other in vitro systems use tissues that

87
require the addition of a gonadotropin to the incubation medium to

stimulate steroid production. The integrity of the in vitro

environment and the function of the gonadotropins (polypeptide

hormones)added to the incubations may be compromised in the

presence of these drugs. Until adequate informatim is available

on the actual levels of these drugs in the media and in the

tissues and on the chemical identity of the drugs throughout the

incubatim period, the cmclusims drawn fran all in vitro studies

will remain tentative.

SHORT-TERM EFFECTS OF THC ON FEMALE REPRODUCTIVE HORMONES

The effects of short-term administration of THC on the menstrual

cycle in the rhesus monkey has been studied in our laboratories.

One such study examined the effect of THC administration

follicular development and ovulation (Asch el al. 1981). The

rhesus monkeys used in these studies had nonral menstrual cycles.

The first day of menses was designated as day 1 of the cycle, and

ovulation normally occurs about days 15. In order to determine

whether THC administration would affect ovulatim in these monkeys,

THC or vehicle was injected daily from day 1 to day 18 of the

cycle. Blood was drawn daily from day 8 to day 18 of the cycle

and then every other day until the occurrence of menses. The

hormones that were measured included total estrogens, LH, and

progesterme. Serial lapsroscopies were dme twice weekly to

observe follicular maturation, ovulation, and corpus luteun

formation. None of the five THC-treated monkeys exhibited estrogen

rises or LH surges, and nme of the THC-treated monkeys ovulated

before the next menses. The period of disruptim that followed

the short-term THC treatment ranged from 55 to 145 days. After

this period, normal hormone levels and ovulation were observed in

all monkeys. This study shows that the inhibitory effect of THC

on gonadotropin secretion is sufficient to disrupt ovulatim and

that the subsequent disruptim of the menstrual cycle may last as

long as several months. When exogenous gonadotropins were administered to these monkeys treated with THC during the first 18 days

of the cycle, ovulatim was restored and normal luteal function

followed. The successful induction of ovulation and normal luteal

function using exogenous gonadotropins in the presence of antiovulatory doses of THC clearly supports the hypothesis of a central

action of the drug rather than an action directly at the gonad.

These results are consistent with studies from other laboratories

that show that THC treatment causes an inhibition of ovulation in

rats (Nir- et al. 1973) and rabbits (Asch et al. 1979a) by a

reversible inhibition of gonadotropin secretim.

Similar results were obtainedin studies in which THC was administered to rhesus monkeys during the luteal phase of normal ovulatory

cycles (Asch et al. 1979c). The daily administration of THC (2.5

mg/kg) had no effect on serum progesterone levels or on the length

of the luteal phase. Again, the post-treatment period was marked

by an absence of normal levels of estrogens, gonadotropins, and

progesterone. The prolactin levels recorded during the post-treat88
ment period were 4 to 5 times greater than prolactin levels in

normal ovulatory cycles. An additional study was done in which

increasing doses of HCG were administered from day 6 to day 10

after ovulation. This treatment with HCG during the luteal phase

in cmtrol animals resulted in augmentation of the progesterone

levels to 4 to 5 times greater than those of control cycles. The

daily injections of THC (2.5 mg/kg) had no effect on the

HCG-induced progesterone rise when compared to either vehicle

treatment or cmtrol responses. These results show that THC has

no direct effect on luteal functim during normal cycles.

Further, THC has no direct effect on corpus luteum function

stimulated by HCG administration. The lack of an effect of THC on

HCG-stimulated corpus luteum function is particularly important,

since this experimental condition mimics progesterone secretion in

early pregnancy.

CHRONIC EFFECTS OF THC ON THE FEMALE REPRODUCTIVE HORMONES

Drug tolerance can be defined as a decrease in pharmacologic

respmse that results from prior exposure to the drug. Tolerance

to the behavioral and cardiac effects of THC administration has

been reported in man and laboratory animals. Tolerance can

develop by several mechanisms including metabolic tolerance or an

increased metabolic clearance of the drug from the body. Cellular

or adaptive tolerance occurs when the organ system through

homeostatic mechanisms loses its sensitivity to the drug's

actims. Either of these mechanisms could be involved in the

development of tolerance to the reproductive effects of THC.

The chronic studies described here were designed to study the

effects of chronic THC on the primate menstrual cycle and to

examine the mechanisms involved in the development of tolerance

(Smith et al. 1983). The study was designed to continue drug

treatment for at least one year or until tolerance developed and

normal cycles were restored. Five female rhesus monkeys with

normal menstrual cycles were used in this study. Ovulation was

detected by monitoring plasma estrogen, LH and progesterme levels

and by laparoscopic examination. Daily vaginal swabbings were

utilized to detect the onset of vaginal bleeding and duration of

menses. Each monkey was followed for one control cycle and me

vehicle treatment cycle before the THC treatment began. On day l

of the third cycle the monkeys began to receive thrice weekly

injections of 2.5 mg/kg THC or 1.25 mg/kg THC. The injections

were given on a M-W-F schedule (at noon), and blood was sampled on

each treatmnt day immediately before injectim. The drug or

vehicle was administered by an intrarmuscular injectim. The blood

level of THC obtained with the 2.5 mg/kg dose of THC 3 times per

week in monkeys is equivalent to moderate to heavy use of

marijuana (5 to 6 joints per day; 3 times per week). Blood levels

of THC were measured by RIA (Cook et al. 1982) where adequate

session was available after hormone measurements. The maximum blood

level of THC was an average of 300 ng/ml at 60 minutes after

injection. The blood level had decreased to an average of 20

89

446-384 0 - 84 - 7
ng/ml by 12 hours, and this through level was maintained until the

next dose at 48 hours. These parameters did not change

significantly throughout the studies. The average minimum THC

blood level during the anovulatory cycles was 409 ng/ml (range

288-542 ng/ml) and 378 ng/ml (range 250-640 ng/ml) after cycles

were restored. The average trough level of THC at 48 hours after

drug injection was 15.3 ng/ml (range 12.1-16.5 ng/ml) during the

anovulatory cycles and 18.8 ng/ml (range 13.0-24.0 ng/ml) after

menstrual cycles were restored.

All monkeys exhibited normal hormone levels and ovulation during

the control and vehicle cycles. Cycle lengths were within normal

limits for the colony. After the drug injections began, none of

the monkeys ovulated or shoved normal hormone levels. Results of

this study are shown for five monkeys in Figure 1. The durations

of the drug effects (days until next normal menstruation) were

135, 110 and 103 days for the monkeys treated with the 2.5 mg/kg

dose and 120 and 70 days for monkeys treated with the 1.25 mg/kg

dose of THC. After the tolerance to the drug effects developed,

normal cycles were re-established. Ovulation was again detected

by laparoscopy and normal hormone levels were observed. Comparisons

were made between pre-treatment prolactin values from control and

vehicle cycles, prolactin values during the period of disruption,

and after menstrual cycles were restored. There appeared to be

sane decrease in the average prolactin levels during the period of

disruption produced by the chronic drug treatment for several of

the monkeys. When the data for the 5 monkeys were combined, there

was no significant decrease in prolactin levels. It was clear,

however, that the previously observed elevations in prolactin

levels following discontinuatian of short-term drug treatment are

not observed with chronic drug treatment.

This chronic study demonstrates the disruptive effects of chronic

THC administration on the primate menstrual cycle. Since both

gonadotropins and sex steroids are at basal levels during the

period of disruption, it is likely that there is a direct suppression of hypothalamic-pituitary activity. This study also demonstrates that with chronic drug treatment, tolerance develops to

the inhibitory effect of THC and normal cycles are re-established.

The mechanism for the tolerance to the effect of THC is not known.

Tolerance develops to other pharmacologic effects of THC including

euphoria andtachycardia. Behavioral tolerance has been reported

in rhesus monkeys and was observed in this chronic study as well.

Preliminary data from this study and complete pharmacokinetic

studies in man and laboratory animals indicate that increased drug

metabolism or clearance is not a major factor in the development

of tolerance (Lemberger and Rubin 1978). It is likely that the

tolerance that develops to the reproductive effects of THC is due

to adaptation of neural mechanisms in the hypothalamus rather than

to increased metabolism of the drug.

The results of this chronic study are consistent withone clinical

90
FIGURE 1. Tolerance develops to the disruptive effects of delta-9

tetrahydrocannabinol on the primate menstrual cycle.

91
study of young women who regularly used marijuana (Bauman 1980).

These women experienced changes in menstrual cycles associated

with decreased prolactin levels. However, the developnent of

tolerance and return to apparently normal menstrual cycles may

mean that normally fertile young women who use marijuana regularly

may not notice much change in their menstrual cycles. Drug

effects may be obvious during adolescence in young women who have

some other menstrual irregularities, or if pregnancy occurs. This

study also demonstrates that the development of tolerance must be

considered available in reproductive studies in young men and

women who use marijuana and may help to explain sane of the

conflicting data in human and laboratory animal studies.

One proposed mechanism for the disruption of the reproductive

system produced by marijuana suggests a direct action of THC at

the cellular level of the reproductive target organs. Studies by

Shoemaker and Harmon (1977) indicate that THC may compete with sex

steroids for their receptor proteins in the target organ cells.

The binding of THC to these receptors would either antagonize the

trophic effects of steroids in these tissues or produce the same

trophic effect as the sex steroid. Solomon and coworkers (1977)

have shown a positive trophic effect of THC on reproductive

tissues in ovariectomized rats when THC was administered by

intraperitoneal injection. Their work, however, was criticized by

Okey and Bondy (1978) who claim that experiments in which THC is

administered by intraperitoneal injection give erratic and unreliable results, caused by inflammation of abdominal organs.

Several of the cannabis derivatives including THC have been

examined in the rhesus monkey for estrogenic activity. The

compounds do not interact with estrogen receptors in reproductive

tissues (Smith, R.G. et al. 1979) nor do they have any inherent

estrogenic activity in vivo (Smith et al. 1979a). THC interferes

with the growth and development of the sex accessory organs in

both male and female animals but not by a direct action on the

tissues (Husain and Lame 1982). The postulated direct effects of

these and other drugs by a tissue receptor mechanism are not as

unlikely as they may appear. A number of plant sterols and

synthetic compounds have inherent estrogenic activity. One such

synthetic compound, diethylstilbestrol (DES), has estrogenic

activity that exceeds the most potent endogenous estrogen in many

assays. Certainly, there are other chemicals in marijuana that

may have hormonal activity, but none has yet been described. It

is important to point out that the steroid receptor mechanism is

highly stereospecific. Therefore, it is not likely that many

drugs will interfere at this level of cellular activity.

SUMMARY

Studies with laboratory animals clearly show that the crude drug

marijuana and delta-9-THC, the principal psychoactive ingredient,

inhibit secretion of the pituitary hormones LH and FSH as well as

prolactin. These changes in pituitary hormone levels produce

decreases in sex steroid hormones and cause disruption of ovulation

92
and spermatogenesis. With chronic drug use, disruption of sex

accessory organs has also been observed. A principal site of THC

action is the hypothalamus, because THC effects on pituitary

hormones productim can be reversed with hypothalamic releasing

factors. It is now known that drug effects in sexually mature

animals are reversible when drug treatment stops. In adults,

tolerance develops to hormone changes brought on by the use of

marijuana. Clinical studies on human subjects generally agree

with the animal findings, although conflicting results have been

reported as well. It is likely that the differences in results

obtained in experiments with laboratory animals and with humans

are caused, at least in part, by differences in experimental

design. Further, it is not known how much disruption of

reproductive hormone levels is necessary for changes in human

fertility and sexual function to occur.

The use of marijuana by pregnant women or by women are

attempting to became pregnant is cause for special concern.

Studies with laboratory animals and retrospective studies on women

who have used marijuana during pregnancy show that the risks of

pregnancy loss and other adverse effects on the fetus are increased

by marijuana use. THC crosses the placental barrier and while the

potent teratogenic and mutagenic effects suggested for marijuana

some years ago have not been confirmed, significant changes

consistent with retardation of fetal growth and development have

been observed. Effects of THC on the proper functioning of the

placenta may be responsible for these effects on pregnancy.

Pregnancy that occurs after the development of tolerance with

chronic marijuana use may involve an ovum that has been damged by

exposure to the drug during critical developmental stages. More

studies need to be done before the mechanisms of toxic effects on

pregnancy and fetal development can be described.

While there have been no clinical studies relating marijuana use

to adolescent development, studies in laboratory animals show that

the developing reproductive system during adolescence is

particularly vulnerable. Single casereports in the clinical

literature strongly suggest that the effects observed in animals

may also occur in young people. Marijuana inhibits events during

adolescence that are crucial for normal reproductive function, so

that a delay in normal sexual development may result and the

possibility of permanent infertility also exists.

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Reich, R., Laufer, N., Lewysohn, O., Cordva,.T., Ayalon. D., and

Tsafriri, A. In vitro effects of cannabinoids on follicular

function in the rat. Biol Reprod 27:223-231, 1982.

Raine, J.M., Wing, D.R., and Paton, W.D. The effects of

delta-1-tetrahydrocannabinol on mammary gland growth, enzyme

activity and plasma prolactin levels in the mouse. Eur J

Pharmacol 51:11-17, 1978.

Schneider, H.P.G. and McCam, S.M. Possible role of dopamine as

transmitter to promote discharge of LH-releasing factor.

Endocrinology 85:121-132, 176.

Shoemaker, R.H. and Harmon, J.W. Suggested Mechanism for the

demasculinizing effect of marihuana. Fed Proc 36:345, 1977.

Smith, C.G., Besch, N.F., and Asch, R.H. Effects of marihuana on

the reproductive system. In: Thomas, J.A. and Singhal, R.

(Eds.) Advances in Sex Hormone Research Vol. 4, Baltimore:

Urban and Schwartzenberg, 1980, pp.373-294.

Smith, C.G., Smith, M.T., Besch, N.F., Smith, R.G., and Asch,

R.H. Effect of delta-9-tetrahydrocannabinol (THC) on female

reproductive function. In: Nahas, G.C. and Patton, W.D.M.

(Eds.) Marihuana: Biolical Effects. Advances in the

Biosciences, Vol. 22-23, New York: Pergamon Press, 1979a,

pp. 449-461

Smith, C.G., Besch N.F. Smith, R.G., and Besch, P.K. Effect of

tetrahydrocannibinol the hypothalmic-pituitary axis in the

ovariectomized rhesus monkey. Fertil Steril 31:335-339, 1979b.

Smith, C.G., Almirez, R.G., Berenberg Asch, R.H.

Tolerance develops to the disruptive effects of

delta-9-tetrahydrocannabinol on the primate menstrual cycle.

Science 219:1453-1455, 1983.

Smith., R.G., Besch, N.F., Besch, P.K., and Smith, C.G.

Inhibition of gonadotropin by delta-9-tetrahydrocannabinol:

mediation by steroid receptors. Science 204:325-327, 1979.

Solomon, J., Cocchia, M.A., and DiMartino, R. Effect of

delta-9-tetrahydrocannabinol in uterine and vaginal cytology of

ovariectomized rats. Science 195:875-877, 1977.

Tyrey, L. Delta-9-tetrahydrocannibinol suppression of episodic

luteinizing hormone secretion in ovariectanized rat.

Endocrinology 102:1808-1814, 1978.

95
AUTHORS

Carol Grace Smith, Ph.D.

Deparmtment of Pharmacology

Uniformed Services University School of Medicine

4301 Jones Bridge Road

Bethesda, Maryland 20814

Ricardo H. Asch, M.D.

Department of Obstetrics and Gynecology

University of Texas Health Science Center

7703 Floyd Curl Drive

San Antonio, Texas 78284

96
Effects of Marijuana on

Neuroendocrine Hormones in

Human Males and Females

Jack H. Mendelson, M.D., and Nancy K. Mello, Ph.D.

I. MARIJUANA EFFECTS ON PITUITARY-GONADAL HORMONES IN MALES

In 1971, our laboratory began clinical research on marijuana effects in humans following a request by the National Commission on

Marijuana for acute and chronic studies of marijuana smoking in

human males (Mendelson et al. 1972; Mendelson et al. 1974a). During the past 12 years we have examined the biological and behavioral consequences of marijuana use in over 200 human male volunteer subjects. Many of these studies have been multi-disciplinary and have involved investigators from internal medicine, psychiatry, psychology, biochemistry, physiology, sociology, neuropsychology and neuroradiology (Mendelson et al. 1974a and b;

1976a and b; Rossi et al. 1977, 1978; Kuehnle et al. 1977).

Numerous experimental animal studies, primarily conducted with

rodents, have shown that cannabis compounds suppress plasma testosterone levels (Collu et al. 1975; Mascarinec et al. 1978;

Symons et al. 1976; Thompson et al. 1974, 1973). Varying effects

of cannabis have been reported in vitro studies of leydig cell

testosterone synthesis from tissues obtained from rat and mice

testes (Burstein et al. 1978, 1979; Dalterio et al, 1977;

Jakubovic et al. 1979). The manner in which cannabis preparations affect leydig cell function in the intact animal remains

unclear since leydig cell regression has been found in mice

(Dixit et al. 1974) but no leydig cell changes were observed in

pigeons (Vyas and Singh 1976). There are also conflicting data

concerning the effects of cannabis compounds on HCG stimulation

of plasma testosterone, with both inhibitory and lack of inhibitory effects reported (Jakubovic et al. 1979).

Luteinizing hormone levels have been found to be suppressed in

male rodents following injection of cannabis compounds (Collu et

al. 1975; Marks 1973; Symons et al. 1976), but it is not clear

if luteinizing hormone levels were depressed antecedent to changes

in plasma testosterone levels. It is unfortunate that parametric

data are not available for cannabis effects on sequential determination of luteini zing hormone and testosterone levels in plasma

97
samples obtained from experimental animals. If cannabis compounds suppress testosterone levels via a central mechanism it

would be anticipated that decrements in luteinizing hormone

levels would occur prior to changes in plasma testosterone

levels. On the other hand, if decrements in plasma testosterone

levels occurred initially without an antecedent fall in LH levels

the most parsimonious explanation for this phenomena is that cannabis acts directly to inhibit testosterone production, increase

testosterone clearance, or both. But if cannabis compounds directly affect production or clearance rate of gonadal steroids,

negative feedback stimulation should result in an increment

rather than a decrement in LH values. An unfortunate lack of

attention to well known feedback control mechanisms for regulation of hypothalamic-pituitary-gonadal function in cannabis related studies with experimental animals has been critically commented upon by Abel in his recent review on marijuana and sexual

function (Abel 1981). An excellent comprehensive review of the

effects of marijuana and cannabis on reproduction and endocrine

function has been prepared by Dr. Eric Bloch (1983).

Studies of marijuana effects on pituitary gonadal hormones in

human males were stimulated by Harmon and Aliapoulios’ (1972)

clinical observations of gynecomastia in 3 men who reported smoking large quantities of marijuana over a long period of time.

Subsequently, Kolodny and his associates (1974) reported that

plasma testosterone levels in 20 male marijuana smokers (aged

18-28) were significantly lower than testosterone levels in males

who reported no past history of marijuana use. In 1974, we reported minimal effects of chronic marijuana smoking on testosterone levels in men studied under controlled conditions, on a

clinical research ward (Mendelson et al. 1974b). Since antecedent use of other drugs (alcohol, opiates) has been shown to depress male plasma testosterone levels (cf. Mendelson et al. 1978a)

we felt it was essential to evaluate the direct effects of marijuana in an otherwise drug-free environment. Twenty-seven men

(aged 21-26) were designated as heavy or casual marijuana users

on the basis of their past history of marijuana use. Twelve subjects reported smoking marijuana for an average of 5.3 years and

smoking an average of 11.5 times per month during the year before

the study. These subjects were designated as casual users. Fifteen subjects reported smoking marijuana for 5.6-years and smoked

a mean of 42 marijuana cigarettes per month during the year before the study. These subjects were designated as heavy users.

The study consisted of 3 phases: a drug-free baseline period of

5 days; a 21-day period during which subjects could acquire and

smoke marijuana cigarettes; and a postmarijuana smoking period of

5 days duration. During the 21-day marijuana smoking phase, subjects had an opportunity to work at a simple operant task to earn

points that were exchangeable for either money or marijuana cigarettes. Blood samples were obtained daily between 8:30 and 9:30

a.m. throughout the 31-day study for analysis of plasma testosterone levels.

98
The effects of marijuana on plasma testosterone levels in casual

and heavy marijuana smokers are shown in figures 1 and 2. Casual

users smoked an average of 54.3 marijuana cigarettes during the

21 day period. Casual users smoked slightly more marijuana during successive 7 day periods of the study (figure 1). However,

plasma testosterone levels were not significantly different from

baseline, and no statistically significant differences in testosterone levels were found between any phases of the study (fig. 1).

The heavy users smoked a mean of 119.5 marijuana cigarettes during the 21 day smoking period (figure 2). The heavy users

smoked more marijuana cigarettes each day than the casual users

and the heavy users also smoked progressively more marijuana over

successive phases of the study (figure 2). Despite heavy marijuana smoking, plasma testosterone levels did not change appreciably from baseline. No statistically significant differences

in plasma testosterone lwels of heavy smokers were found during

any phases of the study. Moreover, there were no statistically

significant differences in plasma testosterone levels between

heavy and casual users during any phase of the study.

In 1976, Kolodny and his associates reported findings obtained in

studies of acute changes of plasma testosterone levels in normal

males after marijuana smoking. Plasma testosterone levels were

measured at 15, 30, 60, 120 and 180 minutes after marijuana smoking as compared with values ohtained for the same individuals during a control period when no marijuana smoking took place. Kolodny

and his colleagues (1976) also reported that statistically significant lower plasma luteinizing hormone values were found 180

minutes after marijuana smoking in the same group of men.

We subsequently undertook a more detailed investigation of the

interrelationships between marijuana use, luteinizing hormone and

testosterone levels in healthy adult males, studied under controlled research ward conditions (Mendelson et al. 1978b). A

major technological advance allowed us to collect continuous

plasma samples over 24 hours from a chronic intravenous catheter

attached to a battery-operated portable non-thrombogenic pump.

This technique permitted determination of integrated plasma

values for testosterone and luteinizing hormone before, during,

and after a period of chronic marijuana use. Again, no systematic relationships were found between marijuana smoking and increases or decreases in plasma testosterone levels and no correlation was obtained between changes in luteinizing hormone levels

with marijuana use. Figure 3 (middle) shows the relationship between marijuana cigarette smoking and plasma testosterone and

IH values in a representative subject. Comparison of these data

with plasma testosterone and LH values prior to and following marijuana use (rows 1 and 3, figure 3) shows that there were episodic

fluctuations in testosterone levels and smaller fluctuations in

plasma LH levels on all study days. But these episodic variations could not be correlated with antecedent or concurrent marijuana use (Mendelson et al. 1978b). The episodic secretory patterns of these pituitary-gonadal hormones attest to the importance

99
Figure 1. Plasma Testosterone Values for 12 “Casual Users before, during and after a 21 -Day Marihuana-Smoking Period

From: Mendelson, J.H., Kuehnle, J., Ellingboe, J., and Babor, T.F. Plasma

testosterone levels before, during and after chronic marihuana smoking.

N. Eng. J. Med., 291:1051-1055, 1974b. Copyright, The Massachusetts

Medical Society. Reprinted by permission.
Figure 2. Plasma Testosterone Levels for 15 “Heavy Users” before, during and after a 21-Day Marihuana-Smoking Period

From: Mendeison, J.H., Kuehnle, J., Ellingboe, J., and Babor, T.F. Plasma

testosterone levels before, during and after chronic marihuana smoking.

N. Engl. J. Med., 291:1051-1055, 1974b. Copyright, The Massachusetts

Medical Society. Reprinted by permission.
Fig. 3. Plasma lestosterone and luteinizing hormone values for a 26-year-old healthy adult male before during

and after marihuana smoking Days 5. 26 and 29 refer to experimental day (day 26 was the 21st consecutive

day of marihuana smoking) The 11:00 AM sample for testosterone and LH analysis on day 26 was lost and

therefore not reported

Mendelson, J.H.; Ellingboe, J.; Kuehnle, J.C.; and Mello, N.K. Effects of chronic marihuana use on integrated plasma testosterone and luteinizing hormone levels. J Pharmacol

Exp Ther, 207:611-617, 1978. © 1978, American Society for Pharmacology andrxperimental

Theraputics. Reprinted by permission.

102
of using an integrated plasma sampling procedure which yields a

true mean value for each collection interval.

II. MARIJUANA EFFECTS ON PITUITARY GONADAL HORMONES IN FEMALES

Despite the considerable clinical evidence that certain drugs of

abuse (alcohol, heroin, methadone) suppress reproductive hormones

and compromise sexual function in males (Mendelson et al. 1978a;

Gordon et al. 1976; Elendelson et al. 1977) and disrupt menstrual

cycles in females (Wallach et al. 1969; Mello 1980) there have

been relatively few systematic studies of drug effects on pituitary gonadal hormones essential for reproductive function in

women. Since disruption of normal hormonal function in women

could adversely affect fetal growth and development, it is of particular importance to acquire objective information about the

interaction between drug use and abuse and reproductive hormone

function in human females.

Although precise estimates of incidence and prevalence of marijuana smoking among young men and women are not available, the

presumed frequency of marijuana use among young women argues

strongly for an empirical analysis of the direct effects of marijuana on reproductive hormones. Although studies of marijuana

effects on male reproductive hormones have yielded conflicting

data, there is accumulating evidence that THC, the psychoactive

component of marijuana, adversely affects female reproductive

function.

THC Effects on LH Secretion and Ovulation

The administration of THC ( tetrahydrocannabinol) to female

rodents has been consistently shown to suppress luteinizing hormone (LH) secretion (Marks 1973; Nir et al. 1973; Tyrey 1978,

1980). A dose dependent suppression of LH in ovariectomized rats

occurred rapidly, within one hour after THC administration

(Marks 1973). THC suppression of LH could be blocked by relatively small doses of estradiol which suggests that THC suppressed

LH secretory activity at suprapituitary, and probably hypothalamic

sites.

THC was found to suppress ovulation as well as cyclic luteinizing

hormone secretion in the rat (Nir et al. 1973). However, normal

patterns of spontaneous ovulation could be restored in THC treated

rats by gonadotropin releasing hormone administration. This provides further evidence that the suppressant effects of THC on LH

occur in the hypothalamus (Nir et al. 1973).

More recently Tyrey (1978, 1980) has reported a dose dependent

suppression of LH secretion in ovariectomized rats at relatively

high doses (0.5 to 8 mg/kg/bwt)(Tyrey 1978) and at doses more

comparable to those attained by human smokers (62.5 to 500 mcg/

kg/bwt) (Tyrey 1980). Administration of THC in doses of 62.5 mcg/

kg/bwt or above rapidly suppressed episodic LH release and

reduced serum LH concentration by 42 to 68 % (Tyrey 1980). The

103
disruption of LH secretion persisted longer at higher doses of

THC, but the degree of LH suppression was comparable across the

dose range studied (Tyrey 1980).

The administration of THC to ovariectomized rhesus monkeys has

also been shown to significantly suppress LH secretion over 12-

24 hours (Besch et al. 1977). These observations have been confirmed and extended in a series of well controlled studies in

ovariectomized monkeys (Smith et al. 1979a). In contrast to findings in rats (Tyrey 1980), the degree of THC induced suppression

of LH was not correlated with the THC dose. However, the dura

tion of LH suppression was dose dependent in monkeys and III was

reversed within 48 hours after THC administration.

In both rhesus monkey (Smith et al. 1979a) and rat (Tyrey 1978)

administration of LH/RH reversed the inhibitory effects of THC on

LH secretion. The investigators interpret these results to indicate that THC suppression of LH secretion occurred at hypothalamic sites.

Although chronic inject ion of THC suppressed ovulation and

decreased gonadotropin, gonadal steroids, and prolactin levels

in mature rhesus monkey for a period of several months, this

suppression did not persist (Smith et al. 1983). Continued expo--

sure of the female rhesus monkey to THC (beyond 2-3 months)

was associated with a remission of menstrual cycle and pituitary

gonadal hormonal abnormalities and thus female monkeys develop

tolerance to disruptive effects of marijuana on reproductive

function.

The rabbit model has been especially useful in studies of reproductive function since ovulation can be induced. Asch and coworkers (1979) attempted to determine if THC induced suppression

of ovulation in the rabbit was due to hypothalamic, pituitary or

gonadal mechanisms. It was found that THC administration did not

directly affect ovarian function and did not directly suppress

pituitary release of luteinizing hormone (Asch et al. 1979).

Rather, gonadotropin secretory activity from the pituitary appeared to be inhibited by THC effects which were mediated at the

hypothalamic level.

In summary studies of THC effects on LH episodic secretory pattern

in several animal models agree that THC inhibits or suppresses

the release of gonadotropin releasing hormone at the hypothalamus.

THC does not appear to directly affect either the production or

release of ovarian steroids (Asch et al. 1979; Smith et al. 1979b).

The extent to which the duration and degree of LH suppression by

THC is dose dependent has been inconsistent across species (e.g.

Tyrey 1980; Smith et al. 1979b). Finally, tolerance to THC effects upon reproductive hormones occurs in monkey (Smith et al.

1983).

THC Effects on Prolactin

Studies of the effects of THC on prolactin have yielded incon104
sistent results. THC has been reported to increase serum prolactin levels (Daley et al. 1974) and to suppress prolactin secretion (Kramer and Ben-David 1974) in male rats. Subsequent studies in humans and rat have yielded comparable conflicting results

(Lemberger et al. 1975; Raine et al. 1978).

Recent carefully controlled studies in monkey have shown that an

acute dose of THC produces a significant decrease in prolactin

levels in both male and female rhesus monkeys (Smith et al. 1979b).

However, the THC induced decrease in prolactin is of relatively

short duration. Further studies by Smith and associates indicate

that THC induced suppression of prolactin levels is probably

mediated at a suprapituitary site. The finding that THC produces

a suppression of prolactin levels in male and female rhesus monkeys and also suppress LH levels in female monkey is of considerable interest since psychoactive drugs which have potent effects

in suppressing LH levels, such as opiate agonists, (Tolis et al.

1975) stimulate rather than inhibit prolactin secretion.

Implications

It is clear from the foregoing that THC consistently produces significant changes in pituitary gonadal hormones which are essential

for normal reproductive function in experimental animal models.

The major unanswered question is what are the relevance of these

data for human females? There are often marked species differences even within animal models and the degree to which THC induced disruption of pituitary gonadal hormones in animals can be

extrapolated to humans is an empirical question. Despite the

predictive values (and relative economy) of studying drug effects

in animals, the ultimate significance of these findings can only

be determined in human studies. Tyrey (1980) has argued for the

importance of human studies in view of his observations in rats.

Tyrey concludes that studies of LH patterns in human females

“may reveal a more striking deviation from normal than thus far

has been apparent. This possibility together with a broader implication of possible associated disturbances in reproductive

function is worthy of further evaluation” (Tyrey 1980, p. 308).

In recent years animal models have appeared to gain some ascendancy over human studies and economic considerations have lent

special weight to the familiar arguments often advanced in favor

of rodent studies: i.e. activity, nutritional, genetic control;

feasibility of surgical intervention (e.g. ovariectomy, castration); toxicological analysis of post mortem tissue, and relative

expendability. Since both animal and clinical studies are essential in many spheres of biology and medicine, it seems appropriate

to comment on some seldom acknowledged limitations of animal

studies, which specifically apply to animal studies of marihuana.

It is well known that differences in drug metabolism and disposition as well as species variations in hormonal mechanisms may limit

the validity of ad hoc comparisons between animal and human pharmacology. The significance of experimental animal studies for

human pharmacology may be limited by factors such as non105

446-384 0 - 84 - 8
physiological levels of drug dosage, differences in route of

administration, and species differences in time and duration of

peak drug dose effects. For example, Smith and co-workers (1979a)

reported that parenteral administration of THC in single doses

of 2.5 to 5.0 mg/kg/day significantly suppressed LH in female

rhesus monkeys. However, a single parenteral dose of 2.5 mg/kg

of THC is equivalent to an inhalation dose of 150 mg of THC for

a 60 kilogram female. High quality marijuana contains approximately 2% THC and a 1 gram marijuana cigarette contains about

20 mg of THC. Therefore, a 60 kilogram female would have to

smoke at least 7 to 8 one gram marijuana cigarettes containing

2% THC over a time course equivalent to a bolus injection of THC

in order to receive a peak dose effect comparable to the acute

administration of 2.5 mg/kg in monkey over a comparable time

interval.

Investigators who are conducting studies to determine the effects

of cannabis on pituitary gonadal hormones should be very careful

to specify the rationale, for extrapolating the relevance of

doses of THC which they have used with animals to humans. Obviously, they should be extremely careful in justifying doses employed in experimental animal studies to human pharmacology.

Unfortunately, problems appear to consistently arise in this

area. For example, in a recent study (Dalterio et al. 1983) the

dose of THC administered to mice was 50 mg/kg of body weight.

The authors rationalized selection of this dose as follows: “This

dose would correspond to about 4 mg/kg orally in the human user

or 3 marijuana cigarettes of 1% THC based on a body surface conversion of 12 for mouse.” The selection of a body surface conversion factor of 12 for mouse in comparison to human may be considered appropriate although it should be emphasized that different conversion factors may be appropriate for different drugs

based upon the pharmacokinetics, metabolism, and excretion of

specific compounds. Nevertheless, if we accept the conversion

factor of 12 we concur that 50 mg/kg for the mouse would be equivalent to 4 mg/kg for the human. But is 4 mg/kg the dose of marijuana which is contained in 3 marijuana cigarettes with 1% THC as

the authors state? If a 70 kg human ingested 4 mg of THC/kg of

body weight, the total amount ingested would be 280 mg. A 1 gram

marijuana cigarette containing 1% THC contains 10 mg of THC. A

simple calculation reveals that 3 marijuana cigarettes contain

30 mg of THC. However, 280 mg of THC would actually be present

in 28 1 gram marijuana cigarettes which contain 1% THC. Surely

there is a significant difference between human consumption of

3 vs. 28 marijuana cigarettes. Although the error in calculation

of dosage was undoubtedly a computation problem, pharmacologists

should exercise special care in computing dose equivalents for

animal and human studies.

Given the problems associated with experimental animal studies

it is important to determine if the acute and chronic use of marijuana by human females produces any alterations or abnormalities

in those pituitary gonadal hormones which are essential for reproductive function. Moreover, we believe it is essential to study

106
marijuana self-administration by smoking and inhalation since

this route of administration most closely approximates marijuana

use in real life circumstances. It is also important to examine

the effects of marijuana on female reproductive hormones under

conditions where other psychoactive drugs which are known to significantly alter pituitary gonadal hormones in males (e.g. alcohol and opiates) do not confound data on marijuana effects on hormonal functions. It is likely that variations in the degree of

other drug use may account, in part, for some of the discrepant

findings concerning marijuana effects on male pituitary gonadal

hormones.

Only one study has been published on the effects of marijuana on

reproductive hormones in human females. Women were studied on an

outpatient basis without control of other drug use, the results

obtained were congruent with animal studies of the effects of THC.

Bauman and co-workers (1979) compared 26 women who reported using

marijuana at least 4 times a week with 16 age-matched control

women who reported never using marijuana. A blood sample was

collected on days 1, 5, 10, 12-18, 20, 25 and 30 of the menstrual

cycle in each group. Thirty-seven of the 42 women were studied

over 2 complete menstrual cycles. Samples were analyzed for

estrone, estradiol, luteinizing hormone, follicle stimulating

hormone, prolactin and testosterone. It was found that marijuana

users had significantly shorter menstrual cycles and a significantly shorter luteal phase than non-marijuana smokers. Prolactin levels were consistently and significantly lower in marijuana

users and testosterone levels were consistently and significantly

higher. However, there were no statistically significant differences in other hormones measured. The two groups of women had an

equivalent number of anovulatory cycles (Bauman 1979). These

data further attest to the importance of determining the direct

effects of marijuana use on pituitary gonadal hormones essential

for reproductive function in healthy, young marijuana users,

under controlled conditions.

At the time of preparation of this review article our laboratory

is at approximately the midpoint of a series of studies designed

to assess acute and chronic effects of marijuana on reproductive

hormones in women. These studies involve comprehensive assessment of menstrua1 cycle function and pituitary gonadal hormone

status in women who use marijuana daily, weekly, or less frequently. A novel portion of these studies is a 35-day residential research ward program where detailed evaluations can be carried out

on marijuana use and covariance of menstrual cycle function. Hormonal data are currently being analyzed and results should be

available for statistical analysis utilizing appropriate computer

programs in the near future. Several behavioral findings which

appear to be quite robust may be described at this time.

1) First, approximately 20% of all women who responded to advertisements of solicitation for participation in the study were

found to have galactorrhea during initial medical screening examinations. The exact incidence of galactorrhea is unknown in

107
the general population. Whether or not galactorrhea present

in 20% of a volunteer population represents a pathological phenomena attributable to regular marijuana use or is unrelated to

marijuana or other drug use remains to be determined. The majority of the women who had galactorrhea did not have elevated or

abnormal serum prolactin levels. However, hyperprolactinemia is

not necessarily a concomitant or a unique causal factor in galactorrhea. Serial determinations of prolactin levels which were

obtained during the course of chronic marijuana smoking in our

studies should provide better information about possible concordance between marijuana use and galactorrhea.

2) Marijuana use by females as reported by past history, ongoing

use in naturalistic settings assessed by daily diary reports and

actual observed use on the research ward is less (both in terms

of dose and frequency) than that found in males of similar age

and socioeconomic status. This finding is not particularly surprising since drug use (including alcohol) by females is usually

not as great as observed for males. However, it should be noted

that important exceptions may exist. For example, the heaviest

marijuana user observed in any of our studies conducted on our

research ward with over 150 individuals was a female. Moreover,

this subject developed unequivocal signs and symptoms of cannabis

withdrawal following cessation of marijuana smoking. Thus,

trends toward light or heavy marijuana use as a function of gender may misleadingly suggest that very heavy or exceptionally

heavy use of marijuana is not likely to occur in female users.

Given the very large number of women who smoke marijuana in contemporary American society it may be anticipated that very heavy

or extreme use may occur in a small proportion of total female

users but nevertheless may involve a surprisingly large number

of individuals. For example, if our current findings that 10%

of female marijuana users smoke 4-8 marijuana cigarettes per day

are applied to the general population, there are perhaps 250,000

to 300,000 females who are engaging in this heavy degree of marijuana use.

3) We have completed preliminary analysis of acute effects of

marijuana smoking on pituitary gonadal hormones during the periovulatory period of the menstrual cycle. The major purpose of

discussing these data is to highlight some of the problems associated with interpretation of studies of acute marijuana effects

on hormone function in women.

Table 1 presents LH levels prior to and following (at 0 time)

smokin a 1 gram marijuana cigarette containing approximately

1.8% THC (standardized marijuana cigarettes were obtained

from the National Institute on Drug Abuse). The women who participated in the study were carefully monitored over three consecutive menstrual cycle phases to precisely establish the timecourse

of the periovulatory period. A criterion of 200 ng/ml commonly

accepted in endocrinological practice was employed to define the

lower limit of luteinizing hormone levels associated with the

midcycle, periovulatory, LH surge. These studies were carried

108
TABLE I

TIME(minutes) LH (ng/ml) X ± S.D.

-120 380 ± 128

-90 351 ± 132

-60 345 ± 134

-30 335 ± 137

0 293 ± 110

1.5 322 ± 126

20 361 ± 135

25 343 ± 114

30 339 ± 113

45 298 ± 113

60 289 ± 125

90 281 ± 147

120 240 ± 118

150 223 ± 127 *

180 216 ± 83 *

*lower than 0 time value (p < .05)

109
out under research ward conditions to ensure adequate safety and

comfort for research subjects and also to assure that no other

substance use confounded interpretation of the III data.

Plasma specimens were collected from an indwelling intravenous

catheter from 120 minutes prior to and 180 minutes following

marijuana smoking. Subjects smoked an entire 1 gram marijuana

cigarette containing 1.8% THC over a 15 minute period by deeply

inhaling the pyrolized material at a frequency of once per 30

seconds.

Statistical analysis of LH levels shown in Table 1, revealed that

statistically significant decrements in LH levels (when compared

with 0 time values) occurred at 150 and 180 minutes (p < .05).

Curiously these statistically significant findings of decrements

in LH values are similar to data reported by Kolodny and his

associates for marijuana induced decrements in LH levels in males

(Kolodny et al. 1976). Do the findings we have obtained with

females have any biological significance?

If we examine data for all LH values shown in Table 1, it is

apparent that there is a steady decrement in LH levels from -120

minutes to 0 time prior to marihuana smoking (2 way ANOVA

p = 0.026). Since the periovulatory period is relatively short

(24 to 36 hours), it may be possible that we are simply observing a normal decrease in LH levels through time. We have attempted

to carry out assessments of LH levels during the periovulatory

period following placebo marijuana cigarette smoking (placebo

marijuana cigarettes were provided by the National Institute on

Drug Abuse). However, because of the short duration of the periovulatory LH surge, we have, as of this time, been only able to

successfully study one female who was administered a marijuana

placebo cigarette during the periovulatory LH surge. Findings

obtained with this subject were almost identical to those observed

for the 5 subjects who received the active marijuana cigarettes.

We therefore believe that great caution must be exercised in interpreting changes in H levels measured at ovulation after concomitant use of marijuana.

Hopefully, discrepancies between putative cannabis effects on

neuroendocrine function in humans as contrasted with data obtained

in experimental animal studies will be resolved as a consequence

of improved experimental design and methods. However, very recent

studies suggest that the complexities in interpreting data obtained from basic studies may be increasing rather than diminishing . Sauer and his associates (1983) have studied the interaction

of cannabis compounds with the estrogen receptor. They found

that both crude marijuana extract and condensed marijuana smoke

competed with estradiol for binding to the estrogen receptor of

rat uterus cytosol. However, pure THC and ten THC metabolites did not compete with estradiol for receptor binding. Moreover in in vivo studies cannabis extract in large doses had

neither estrogenic nor anti-estrogenic effects. Sauer and his

110
colleagues (1983) concluded, “thus, although estrogen receptor

binding activity was observed in crude marijuana extract, marijuana smoke condensate and several known components of cannabis,

direct estrogenic activity of cannabis extract could not be demonstrated in vivo. ” It therefore appears that lack of concordance

of findings in experimental animal and human studies, (with respect to marijuana effects on hormonal function) is paralleled by

equally discordant data obtained from in vitro basic receptor

binding studies compared to effects observed the intact animal.

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ACKNOWLEDGEMENT

Supported by NIDA Research Grant No. 5 R01 DA 02905, "Marijuana

Effects on Reproductive Hormones in Women," and ADAMHA Research

Scientist Award No. 5 K05 DA 00064, "Biobehavioral Studies of

Narcotics Abuse in Humans."

AUTHORS

Jack H. Mendelson, M.D.

Nancy K. Mello, Ph.D.

Alcohol and Drug Abuse Research Center

McLean Hospital/Harvard Medical School

Belmont, Massachusetts 02178

114
Effects of Marijuana on Pregnancy

and Fetal Development in the

Human

Katherine Tennes, M.A.

The first systematic studies of the effects of marijuana on

pregnancy and on fetal growth were published in the past two

years. Results of the investigations are suggestive of an

effect, but none of the investigators thus far has reported

consistently significant effects upon pregnancy, nor have they

found signs or symptoms which could be considered a syndrome

characteristic of neonates exposed to marijuana. Major difficulties

are encountered in conducting clinical studies of this nature

which may account, in part, for the tentativeness of the results.

This paper will review the evidence linking maternal and fetal

risk factors with marijuana use during pregnancy and consider

the methodological problems inherent in the research designs.

PREGNANCY, LABOR, AND DELIVERY

One group of investigators conducted two studies to test the

hypothesis that women who smoke marijuana during pregnancy will

experience greater difficulty at delivery than non-users (Greenland

et al. 1982a, 1982b, 1983 in press). The hypothesis was based

upon evidence from animal studies that cannabinoids (or their

metabolites) affect uterine contractility.

In the first study, conducted at three prenatal care centers in

the Los Angeles area, Greenland et al. (1982a, 1982b) compared 35

women who used marijuana more than once a month during pregnancy

with 36 non-users. A significantly higher proportion of users

was found to have precipitate labor (37% users versus 3% nonusers, p < .01) and meconium passage (57% users versus 25% nonusers, p < .05). They also examined differences between marijuana

users who tested positive and those who tested negative for THC

at delivery. Precipitate labor was observed in half of the 16

women who had positive urine or serum tests for THC and none of

the women who had negative results. This finding suggested that

the effect upon delivery was related to recency of use before

delivery.

Other apparent differences between users and non-users, not

reaching statistical significance, were: more prolonged or

arrested labor (31% users, 19% non-users), operative deliveries

(31% users, 14% non-users), abnormal fetal tests (11% users, 0%

non-users), and manual removal of the placenta (17% users, 8% nonusers).

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In an attempt to verify these findings, 313 women enrolled in a

low-risk home-delivery program, were studied (Greenland et al. 1983

in press). In this group, precipitate labor was again more common

among the 41 marijuana users (13% users, 8% non-users) as was

dysfunctional active labor (43% users, 35% non-users), meconium passage

(17% users, 13% non-users), and manual removal of the placenta (10%

users, 3% non-users). However, the magnitude of the differences was

much smaller than in the first study and statistical significance was

reached only in the case of manual removal of the placenta (p < .05).

The investigators considered the results inconclusive due to the

small sample size and the possibility that chance alone could account

for finding a few significant relationships when several hundred

variables were examined. Nevertheless, they pointed out that there

appeared to be a consistent pattern indicating that marijuana users

generally experienced more difficult labor and their infants passed

more meconium.

The predominant effect of marijuana on uterine contractility is a

clinically relevant but currently unresolved issue. In vitro,

studies have shown both an inhibitory and a stimulatory effect on

prostaglandin synthesis (Burstein and Raz 1972; Burstein et al. 1973;

Burstein and Hunter, 1981). Since prostaglandins have been implicated in the onset of labor (Quilligan et al. 1981) and are used

clinically as uterine stimulants, further research is warranted to

clarify the overall effect of marijuana on labor.

FETAL GROWTH AND DEVELOPMENT

Two studies designed to investigate the effect of maternal alcohol

consumption upon fetal growth and development considered marijuana

as one of the variables in the data base.

From 1977 to 1979, a sample of 1,690 women and infants were surveyed

at Boston City Hospital (Alpert et al. 1981; Hingson et al. 1982).

The primary focus of the infant examinations was upon growth

retardation and physical anomalies associated with the Fetal Alcohol

Syndrome. Examination of birthweight by multivariate analysis

indicated that 43% of the variance was accounted for by gestational

age, mother’s age, mother’s weight change during pregnancy, weight

prior to pregnancy, race, cigarettes per day, infant sex, and

marijuana. The variables entered the equation in the order given

and all were significant at p < .05. It was of interest that

marijuana but not alcohol made a significant contribution to the

variance in birthweight. If the difference in birthweight was

calculated by comparing 181 marijuana users with non-users, without

accounting for confounding factors, the infants were found to

weigh less by 300 grams (p < .001). When the effect of marijuana

upon infant weight was calculated from the regression equation, the

infants exposed to marijuana were lighter by 105 grams (p < .01).

Thus, the investigators pointed out that multivariate analyses are

essential for estimating the effects of a single variable upon

outcome measures in order to avoid attributing to marijuana an effect

partially accounted for by confounding factors.

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A second investigation of the effects of prenatal exposure to

alcohol was conducted at about the same time in 1978-1979 in Denver

on a smaller population of 278 women (Tennes and Blackard 1980).

The variance in birthweight was analyzed by stepwise multiple

regression. The variables entered in the regression were:

gestational age, mother’s pre-pregnancy weight, number of minor

physical anomalies, nicotine, parity, marijuana, caffeine, mother’s

age, socio-economic status, and alcohol. Only the first five

variables were significant at p < .05. Thus, marijuana was not

regarded as making a significant contribution to the variance

in birthweight although it was the sixth variable accepted by the

regression. Nevertheless, since the Boston City Hospital study

indicated that marijuana may contribute to a reduction in fetal

weight gain, the Denver study was considered to be suggestive of

a similar trend but limited by an inadequate sample size.

TERATOGENIC EFFECTS

A second effect of marijuana upon fetal development suggested by

the Boston City Hospital study was an increase in dysmorphology.

Of 1,341 infants, 31 were found to have features compatible with

the Fetal Alcohol Syndrome (Hanson et al. 1978). Women who smoked

marijuana were found to be five times more likely than non-users

to deliver a child with these features (p < .001). Low maternal

weight gain during pregnancy and exposure to roentgenograms also

contributed to the risk but at a lower rate than marijuana.

Recently, Qazi et al. (1982 and personal communication), during

clinical examinations of infants for congenital anomalies, found

six infants with fetal-alcohol-syndrome-like features. The infants’

mothers denied using alcohol but smoked tobacco moderately and

used marijuana regularly, from two to 14 times daily. The infants

also were of low birthweight for gestational age, These retrospect -

ive observations corroborate the findings in the Boston City Hospital

prospective study. The finding that criteria developed to judge

an alcohol effect was associated with marijuana exposure suggests

a lack of specificity for either alcohol or marijuana. Furthermore,

the presumptive evidence implicating marijuana does not rule out

the possibility that the causative factor may be an unidentified

variable common to some heavy alcohol and some heavy marijuana users.

Retrospective observations by clinicians have been an important meam

of detecting the teratogenic potential of a drug. The likelihood

of noticing a cause-and-effect relationship between a specific

malformation and a drug is increased if the abnormality is rare,

if the drug usage is widespread, or if there is a sudden incremental

change in the incidence of drug use. Despite the reported increased

use of marijuana by young women in the past decade, teratogenic

effects have rarely been reported by clinicians. Three cases of

infants with malformations described in the literature (Carakushansky

et al. 1969; Bogdanoff et al. 1972; Hecht et al. 1968) were born to

polydrug users and the contribution of marijuana to the defects could

not be isolated.

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NEUROBEHAVIORAL EFFECTS

The influence of prenatal exposure to a drug upon neurological and

behavioral functioning is difficult to measure in the neonatal

period because of instability due to the birth process, to

extrauterine adjustments and to rapid neonatal growth (Lancioni

et al. 1980). Nevertheless, examinations of infants born soon

after birth are useful in isolating acute intrauterine effects,

for monitoring withdrawal symptoms and for establishing a baseline

for later developments. The seriousness of intrauterine drug

exposure upon central nervous system functioning can be determined

only by assessments of the children at older ages.

Before neurobehavioral effects can be evaluated, gestational age and

birthweight need to be taken into account. In an ongoing prospective

study in Ottawa (Fried et al. 1980a, 1980b, 1981), 420 women were

interviewed and only those who said they used no illicit drugs other

than marijuana were accepted into the study. Of 89 infants who were

examined, nine were born to women who were heavy users of marijuana

throughout pregnancy. Fried compared the nine heavy marijuana users

with controls matched for nicotine and alcohol. He found that women

who used marijuana gained significantly less weight during pregnancy

than non-users (p < .02) and a significantly higher proportion

delivered before 40 weeks’ gestation (p < .05). No effect of

marijuana exposure upon infants’ birthweight was detected when

gestational age was taken into account. Given the number of

variables that influence birthweight and the relatively small impact

expected from marijuana, an effect of marijuana on birthweight would

be difficult to detect in a sample this small.

In a preliminary report of neurobehavioral examinations of the

infants based upon 27 items in the Brazelton Neonatal Assessment

Scale (Brazelton 1973), Fried found three items that distinguished

infants of 11 heavy and moderate marijuana users from an equal

number of controls matched for nicotine and alcohol. First, there

was an alteration in response to a visual stimulus, second, an

increase in tremulousness, and third, a peculiar high-pitched cry.

When a light is directed at the eyes of the sleeping infant, the

expected response is a large body movement or marked blinking,

without awakening, followed by diminishing responses or "habituation"

as the light is presented repetitively. Five of the infants born

to the moderate or heavy users, as compared with two of the controls,

failed to give the motoric response to the light; and two of the

users but none of the controls failed to habituate.

Fried speculated that the infants’ altered visual responses might be

a manifestation of effects similar to those reported in rabbits

and primates immediately after injection of THC. In rabbits, the

absolute visual thresholds were found to be elevated (Rose et al.

1979). The primate study suggested that THC inhibits light sensitive

neurons in the lateral geniculate nucleus of the thalamus (McIsaac

et al. 1971). Though the association to animal studies is intriguing,

the finding of both greater sensitivity and greater insensitivity to

a light in the infants with heavy marijuana exposure is puzzling.

118
By contrast with the measurement of sensitivity, no difference was

found between the infants of users and non-users in visual

orientation, focusing or following during awake, alert states.

Heightened tremulousness was observed in infants of users during

crying, wakefulness, or sleep. Based on the entire sample of 89

infants, a significant dose response relationship between tremors

and marijuana use was found (r = .25, p < .002) and the relationship

did not change when nicotine and alcohol were partialed out. The

third difference, a high-pitched cry similar to a “cri de chat,”

was noted among one-third of the infants of regular marijuana

users and never among the controls. Tremors and a peculiar cry

are characteristics of infants undergoing narcotic withdrawal

(Lipsitz 1975). However, infants undergoing narcotic withdrawal

have an adversive reaction to light, rather than insensitivity,

and they are extremely irritable and hypertonic, characteristics

not found in infants of marijuana users.

Repeated examinations of the infants at later ages in Fried’s study

suggested that diminished responsiveness to visual stimuli and

heightened tremulousness persisted in half the infants of heavy

users at nine days of age but were no longer present at 30 days.

No difference between infants of seven users and seven matched

controls was found in scores on an infant development test at

one year of age.

Because of the large number of variables being considered and the

small sample size, these results may not be regarded as conclusive.

Elaboration of the findings is expected to result from the

investigation still in progress in Ottawa and from a similar study

being conducted in Denver. In addition to assessments of growth,

neurological status and behaviors, infants in both studies are being

evaluated for the presence of major and minor physical anomalies.

METHODOLOGICAL PROBLEMS

Naturalistic clinical studies are subject to a number of

methodological problems that need to be considered in designing

research and interpreting results. This paper will discuss problems

with data collection and verification, and treatment of confounding

variables with reference to the studies reviewed above.

Self-Report of Drug Use

A major concern of clinical studies dependent upon self-reported

data of illegal drug use is that underreporting will obscure real

effects. Greenland et al. (1982a) made an important contribution

toward establishing the veracity of self-report as a method of

data collection by radioimmunoassay of blood and urine specimens

for cannabinoids. He found no positive tests among those denying

usage.

Both Greenland et al, (1982a) and Fried et al. (1980a) prescreened

subjects for use of illegal drugs other than marijuana during pregnancy and confirmed the self-reports by chemical determinations for

119
amphetamines, opiates, etc. No contradiction between self-reports

and bioassays was found.

The validation of self-report data may be applicable only to subjects

enrolled in research studies. In the majority of studies, the women

choose whether or not to participate. Refusal rate in public prenatal clinics in Boston and Denver was approximately 15%. Women choosing

not to participate may be different from participants in their

willingness to reveal actual drug use.

Not only is the data base dependent upon the woman’s report, but in

naturalistic studies, the woman is in control of administration of

the drug. A proportion of women who use marijuana prior to pregnancy

and in the first trimester decrease use in the course of the

pregnancy. In the Denver studies we found that about one-third of

the heavy users of marijuana sharply decreased their use early in

pregnancy when they learned they were pregnant. A second abrupt drop

in use occurred in some women after they were interviewed for the

study although no advice was given regarding its use. Since the

effects during embryogenesis may be different from later effects on

fetal growth and brain development, separate assessments need to be

made of early and chronic exposure.

An additional problem in data gathered by self-report is in

estimating actual amounts of fetal exposure to cannabis. Assay

of the infants’ cord bloods at time of birth for THC is of very

limited value since blood levels do not reflect tissue levels in

the infant and assays are limited to -9-THC and its metabolites

which may not be the only effective components of marijuana.

Confounding Factors

Perhaps the most difficult methodological problem confronting

investigation of marijuana use during pregnancy is the need to

account for potentially confounding effects.

Variables known to co-vary with marijuana use include age, cigarette

smoking, alcohol, and socio-economic status. Greenland et al.

(1982a) addressed the issue when selecting subjects by matching a

group of marijuana users with a group of non-users for age, number

of children, and the medical center providing care. To examine the

effect of other covariants of marijuana use, the investigators

calculated the proportion of difference in each outcome variable

that could be attributed to the other risk factors. Confidence in

the results of the study was heightened by the finding that in no

instance was the association between marijuana and the outcome

variable altered by the adjustment.

In the Boston City Hospital study, identification of marijuana as

a factor contributing to the variance in birthweight was a result

of analysis by multivariate techniques. The usefulness of this

excellent method, used to account for confounding variables, is

limited by the need for a relatively large sample size, in order to

fulfill the assumptions underlying the statistic.

120
A different set of confounding variables may be appropriate when

examining specific effects of marijuana use on delivery or on

neonatal characteristics. Correlates of these outcome measures

need to be considered in addition to correlates of maternal

marijuana use. Meconium-stained fluid during delivery, for example,

is known to be associated with small and with large infants and

to be influenced by postmaturity. To gain clinical credibility,

the association between marijuana use and meconium passage require

systematic examination of all known correlates of meconium staining.

Likewise, other factors co-vary with tremulousness in the neonate.

One of the conditions associated with tremulousness is infection

which may vary as a function of life-style rather than directly

with marijuana use.

One benefit of the pioneering studies of marijuana use during

pregnancy is that they suggest specific effects which can then be

subjected to systematic indepth scrutiny. As was pointed out by

Hingson et al. (1982), studies that attempt to relate a specific

drug such as marijuana to abnormal fetal development without

examining a host of confounding variables “may best be regarded as

identifying factors that are symptomatic of a higher risk pregnancy,

rather than a definite cause of pregnancy risk.”

CONCLUSION

The rate of marijuana use by pregnant women is sufficient to

represent a health hazard if marijuana is found to have a deleterious

effect upon maternal well-being or fetal development. The reported

use among pregnant women, varying in the localities sampled from

10% to 37%, is comparable to an estimated 26% of women between ages

18 and 25 surveyed in the general population (Fishbume et al. 1980).

Inconclusive evidence from the first exploratory studies in the

human suggest that marijuana use may alter the delivery process,

reduce intrauterine weight gain by the fetus or affect visual and

neurological excitatory responses. Confirmation of these findings

among the studies is lacking as is evidence for the unconfounded

or direct action of marijuana. Nevertheless, the studies serve an

important function in providing directions for new and ongoing

investigations.

Finally, three issues might be considered of importance for future

investigations of the effects of marijuana as used by pregnant

women. First, the degree of placental transfer of -9-THC or its

metabolites, including the possibility that rates are variable over

the course of pregnancy, has not been determined in the human

(Abel 1980). Second, the synergistic or interactive effects of

illicit drugs or medications with marijuana as taken by pregnant

women has been given little attention. Third, the immediate clinical

relevance of findings as well as the long-term effects need to be

assessed in evaluating the impact of the drug.

121
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1973, 66 pp.

Burstein, S. and Raz, A. Inhibition of prostaglandin E 2

biosynthesis by delta-1-tetrahydrocannabinol. Prostaglandins,

2(5):369-374. 1972.

Burstein S., Levin, E., and Varanell, C. Prostaglandins and

cannabis - II. Inhibition of biosynthesis by the naturally

occurring cannabinoids. Biochem Pharmacol, 22(4):2905-2910,

1973.

Burstein, S. and Huntcr, S. Prostaglandins and cannabis - VIII.

Elevation of phospholipase A2 activity by cannabinoids in whole

cells and subcellular preparations. J Clin Pharmacol 21:240S248S, 1981.

Carakushansky, G., New, R., and Gardner, L,. Lysergide and cannabis

as possible teratogens in man. Lancet l, 586:150-151, 1969.

Fishbume, P., Abelson, H., and Cisin,. National Survey on

Drug Abuse: Main Findings 1979. National Institute on Drug

Abuse. DHHS Pub. No. (ADM) 80-976. Rockville, Maryland:

Office of Medical and Professional Affairs.

Supt. of Documents, U.S. Government Printing Office, 1980.

250 pp.

Fried, P. Marijuana use by pregnant women: neurohehavioral

effects in neonates. Drug Alcohol Depend, 6:415-424, 1980(a).

Fried, P., Watkinson, B., Grant, A., abd Knights, R. Changing

patterns of soft drug use prior to and during pregnancy: a

prospective study. Drug Alcohol Depend, 6:323-343, 1980(b).

Fried, P. Marihuana use by pregnant women and effects on offspring:

an update. Neurobehav Toxicol Teratol, 3:195-201, 1981.

Greenland, S., Staisch, K., Brown, N., and Gross, S. The effects

of marijuana use during pregnancy - I. A preliminary epidemiologic

study. Am J Obstet Gynecol, 143:408-413

Greenland, S., Staisch, K., Brown, N., and Gross, S. Effects of

marijuana on human pregnancy, labor: and delivery. Neurobehav

Toxicol Teratol, 4:447-450, 1982(b).

Greenland, S., Richwald, G., and Honda, G. The effects of

marijuana use during pregnancy - II. A study in a low-risk

home-delivery population, Drug Alcohol Depend, 1983 (in press)

Hanson, J., Streissguth, A., and Smith, D. The effects of moderate

alcohol consumption during pregnancy on fetal growth and

morphogenesis. J Pediatr, 92:457-460, 1978.

Hecht, F., Beals, R., M., Jolly, H., and Roberts, P.

Lysergic-acid-diethylamide and cannabis as possible teratogens

in man. Lancet 2, 577:1037, 1968.

122
Hingson, R., Alpert, J., Day, N., Pooling, E., Kayne, H., Morelock,

S., Oppenheimer, E., and Zuckerman, B. Effects of maternal

drinking and marijuana use on fetal growth and development.

Pediatrics, 70:539-546, 1982.

Lancioni, G., Horowitz, F., and Sullivan, J. The NBAS-K: I. A

study of its stability and structure over the first month of life.

Infant Behav Dev, 3:341-359, 1980.

Lipsitz, P. A proposed narcotic withdrawal score for use with

newborn infants. Clin Ped, 14:92-594, 1975.

McIsaac, W., Fritchie, G., Idänpään-Heikkilä, J., Ho, B., Englert, L.

Distribution of marihuana in monkey brain and concomitant behavioral effects. Nature, 230:593-594, 1971.

Qazi, Q., Marlane, Beller, E., Milman, D., and Crumbleholme, W.

Is marijuana smoking fetotoxic? Pediatr Res, 16:272A, 1982.

Quilligan, E., Little, A., Oh., W., Catz, C., Alexander, D., and

Cornblath, M. Pregnancy, birth and the infant. Child Health

and Human Development: An Evaluation and Assessment of the

State of the Science. NIH Pub.

Rose, S. Dwyer, W., and Yehle, A. Delta 9-tetrahydro-cannabinol:

elevation of absolute visual thresholds of rabbits. Pharmacol

Biochem Behav, 10:851-853, 1979.

Tennes, K. and Blackard, C. Maternal alcohol consumption, birthweight, and minor physical anomalies. Am J Obstet Gynecol, 138:

774-780, 1980.

AUTHOR

Katherine Tennes, M.A.

Department of Psychiatry

University of Colorado Health Sciences Center

4200 East Ninth Avenue

Box C-264

Denver, Colorado 80262

123
Discussion and Recommendations

Monique C. Braude, Ph.D.

Historically, the National Institute on Drug Abuse (NIDA) and its

predecessor, the National Institute of Mental Health (NIMH) have

been committed to research on the biomedical effects of marijuana

and its major constituents, the cannabinoids. This commitment has

extended through the last 15 years to become an essential part of

the preclinical and clinical program of marijuana research.

A significant part of this marijuana program has been devoted to

studies of the effects of marijuana on the endocrine and reproductive systems in view of its great potential impact for the

health of future generations. Early preclinical as well as

clinical studies, reviewed in the previous chapters, pointed out

significant effects of marijuana or its constituents on the

hormonal and reproductive systems usually after acute or shortterm administration. After long-term administration, the hormonal

changes seen after acute administration often disappear, probably

due to the development of tolerance.

A great deal of discussion during the meeting centered on the doses

or concentrations which should be given to experimental animals

versus those used by man. One should not forget that animals are

usually less susceptible to drugs than humans as shown in

behavioral and pharmacological testing. Furthermore, early studies

could not correlate plasma levels of cannabinoids with biological

effects as the analytical methodology to assay cannabinoids in

biological fluids was not yet sufficiently developed. Today, the

availability of rapid and economical assays, such as the EMIT assay

and the RIAs allow aualitative and quantitative assessment of

marijuana constituents in biological fluids. It should therefore

be easier to correlate plasma levels across species to endocrine

effects in future studies.

Much of the important early research on the effects of marijuana on

the reproductive system was done on the male in part due to the

relative simplicity of the male system compared to the more complex

hormonal interactions that occur in the female. The early work

which focused attention on the fact that marijuana depresses

reproductive hormones, such as LH and testosterone, has led to

124
important observations of changes in the sexual organs such as

decreases in testis, seminal vesicles and prostate weights. The

reports of reduction in reproductive organ weights are often

accompanied by reports of oligospermia and sperm abnormalities, in

rodents as well as in primates. Chronic use of marijuana can

produce decrements in the function of many endocrine organs

including the pituitary, testis, thyroid, and adrenal cortex. The

initial fears of prolonged depression in LH and testosterone have

been tempered with time, as it now appears that some degree of

tolerance to the effect of marijuana on the endocrine system in the

adult animal may occur.

Studies of the effects of marijuana on the endocrine system in vivo

have been often narrowed to studies of its major psychoactive

ingredient, delta-9-THC (THC). In vitro, it appears that many

organ systems respond not only to the direct addition of THC to the

incubation medium but also to the addition of various other

cannabinoids as well. Whether such actions of the cannabinoids

occur in vivo yet remain to be proven.

The elucidation of the mechanisms involved in the effects of

cannabinoids on the target organs (ovary, testis) have led to

numerous discussions, some believing that these effects are

produced by a direct action on the target organs, others believing

that the cannabinoids exert an indirect action via the hypothalamic

pituitary gonadal (HPG) axis. There is some evidence to indicate

that there might be a direct action of the cannabinoids on the

reproductive tissues themselves in addition to the reproductive

decrements caused by the altered hormonal environment, but the

debate is still open.

The search for the site of cannabinoid action on the reproductive

system has led to a number of studies of the effects of

cannabinoids on various parts of the HPG axis and the brain. It is

now believed that THC does not act directly on the pituitary to

alter hormone secretion of LH and prolactin, for instance. A major

site of action is the hypothalamus, as it has been shown that THC

effects on pituitary hormone production can be reversed by

hypothalamic releasing factors. It now also appears that higher

neural centers may be affected, which could alter the neuronal

input to the hypothalamus.

RECOMMENDATIONS

The members of the review panel were asked to present their ideas

for future research which would build upon our current knowledge

base. Most of the question and answer sessions following the oral

presentation of each paper, as well as most of the discussion

session at the end of the meeting, centered on this issue.

The following is a synthesis of the highlights of some of their

recommendations, which is not intended as, and should not be

considered to be, an exhaustive list.

125
1. Basic research on the effects of cannabinoids on the genome and

on gene expression

Cannabinoid-induced modifications in cell structure and function

have been well documented as have a series of physiological effects

resulting from such drug-induced cellular changes. Two pivotal

processes which have been shown to be significantly influenced by

cannabinoids are endocrine function and cell proliferation.

Moreover, these are not unrelated processes since in many cases

proliferation is responsive to hormone control. In view of recent

advances in molecular biology, an understanding of the manner in

which drug-induced alterations in gene expression are brought about

is now possible and should provide insight into the molecular basis

of cannabinoid-related modifications in cellular functions.

Cannabinoid-related alterations in gene expression can result from

changes in the organization of genetic sequences and/or in the

manner in which genetic information is transcribed and processed.

Therefore, a critical and systematic evaluation of the influence of

cannabinoids on the structure and expression of specific genetic

sequences, particularly in human cells, should be a high priority.

Understanding cannabinoid effects on human gene organization and

expression at doses/concentrations approximating usual human use is

prerequisite to evaluating possible short-term or long-term

inheritable disorders that may arise from the use of these drugs

either therapeutically or as abused substances. Of particular

importance, despite the history of fragmentary and often

contradictory reports of cannabinoid-induced modifications in

genome-related phenomena (e.g. chromosomal changes, alterations in

RNA metabolism, etc.), we are now in a position to address these

issues directly and definitively. The availability of a series of

cloned human genes permits evaluation of drug-induced effects on

specific genes and defined regions of genes as well as on the

transcription and processing of genetic information. Cloned

genetic sequences can be used as high resolution probes for

identification and quantitation of specific human gene transcripts

in several human cell types following cannabinoid administration.

It would be instructive to focus where possible on human studies

since, for example, drug-induced effects on the organization of

specific genetic sequences or regions thereof can be performed

utilizing DNA from white blood cells isolated from only a 20 ul

blood sample. Thereby, the opportunity is available to determine

the effects of cannabinoids on the genome of subjects participating

in endocrine function and/or behavioral studies. A number of

normal and tumor-derived human cell lines are available and should

be utilized to complement such an approach. By combining both

intact organism and cell culture approaches it is possible to draw

on the physiological reality of the organism and the biochemical

simplicity of isolated cells. It would also be appropriate to

concentrate efforts on evaluating drug-induced effects on a limited

series of genetic sequences, those related to proliferation and

endocrine function, where definitive effects are well understood

and the information obtained from such studies can be integrated

with ongoing investigations. This approach can be implemented for

assaying unfractionated marijuana extracts, psychoactive and

126
nonpsychoactive components of marijuana, natural and synthetic

cannabinoids, and cannabinoid metabolites, as well as other abused

substances individually or in conjunction with cannabinoids.

2. Effects in the female

Up to 1978, studies of cannabinoids with women who were or might

become pregnant were not allowed by the Food and Drug Administration in view of the early preclinical reports of malformations

in offspring of cannabis treated animals. However, as reviewed in

this volume and also in the proceedings of an ARF/WHO scientific

meeting published in 1983 on adverse consequences of cannabis use,

further studies indicate that cannabinoids are, at most, weakly

teratogenic in rodents and rabbits and there are so far no reports

of gross malformations in children of marijuana-using parents. As

studies in women are now allowed, the following recommendations

were made:

a. Surveys of pregnant women have shown that the incidence of

marijuana use among pregnant women is approximately the same as

in young adult women in the general population. Because of the

high incidence of marijuana use by pregnant women, it is now

important to investigate the effects directly upon pregnancy

and upon the development and functioning of their offspring's

reproductive function.

b. Dosage and time of marijuana use during gestation are

difficult to control in clinical studies of pregnant women,

but correlation of marijuana with the cannabinoid content of

biological fluids should be made periodically. Research using

the primate as a model appears to be the most direct approach

to investigating physiologic effects upon maintenance of

pregnancy, initiation of delivery, and neonatal risk factors.

c. Naturalistic studies of pregnant women's use of marijuana

are needed to establish patterns of use and to identify

confounding variables. Since a proportion of women who use

marijuana regularly prior to pregnancy reduce their use after

they become aware of being pregnant, research should be

directed toward determining effects upon development of the

embryo early in pregnancy as well as the effects upon fetal

development. Since a proportion of pregnant marijuana users

are polydrug users, it is important to investigate the

incidence and patterns of specific illegal drugs of choice used

in conjunction with marijuana by pregnant women.

d. Because of the difficulties involved in identifying a

cohort of women who are chronically heavy users of marijuana

throughout pregnancy, it might be appropriate to organize a

consortium of researchers who have such a cohort identified in

order to pool information from medical records of the women and

neonates born to them.

e. Knowledge is scarce regarding the effects of marijuana upon

the mother's postnatal care of the infant in women who are

chronic users after pregnancy. Both the exposure of the

127
infants through breastfeeding and the effect upon women's

motivation to give maternal care need to be examined. Physiologic

effects may be investigated through animal models but only research

involving mother-infant dyads will be able to account for effects

upon the child's behavior and subsequent development.

3. Effects of THC in the male

Although studies in the last 10 years have concentrated on male

subjects, there are still a number of questions to be resolved

about the effects of marijuana and its constituents on the male

endocrine and reproductive systems. For instance:

a. Are there any specific direct effects of THC on the testis

and other accessory reproductive organs? There are many

reports in the preclinical literature of reduced weight of

seminal vesicles, prostate, and epididymis of animals which

have been treated chronically with THC. There is also some

evidence to indicate that even after cessation of treatment and

a return to normal hormonal levels, the reproductive organs

never make up the weight deficit that is incurred during the

period of drug treatment. Is this due to hormonal involvement

or are nutritional factors involved?

b. How does THC and/or marijuana affect sperm production and

auality? Many possibilities exist here. Do cannabinoids have

a direct effect on the germinal epithelium of the seminiferous

tubules or are the effects indirect, i.e., produced by altered

hormonal and nutritional environment induced by THC within the

seminiferous tubules themselves?

c. What is the effect of THC on FSH output by the pituitary

and what effect does this have on spermatogenesis in the male?

In essence, changes in FSH have been poorly documented. More

information is needed about the effect of THC on FSH production

by the pituitary.

d. What are the specific sites of action of cannabinoids

within the central nervous system which could alter the

secretion of the hypothalamic releasing hormones that regulate

pituitary hormone production? Recent evidence, as reviewed in

this volume by Tyrey, has indicated that THC is probably acting

through higher CNS sites to alter the neural transmitters in

the hypothalamus that are responsible for secretion of

releasing hormones.

e. What are the long-term effects of exposure of juvenile

males to marijuana or THC? How does exposure affect them

during maturation? Are there changes incurred in juveniles

exposed to marijuana which alter reproductive function as

adults? What changes occur through perinatal exposure to

marijuana? This search must be at all levels including

neuroendocrine, change in function of reproductive structures,

and perhaps alterations in sexual behavior.

128
In conclusion, the acute and short-term effects of THC and

marijuana on the neuroendocrine and reproductive systems have been

fairly well elucidated, as reviewed in this monograph. However,

the effects of long-term marijuana smoking and of marijuana

components other than THC need to be further investigated and

should be pursued with vigor.

AUTHOR

Monique C. Eraude, Ph.D.

Biomedical Branch

Division of Preclinical Research

National Institute on Drug Abuse

Rockville, Maryland 20857

129
While limited supplies last, single copies of the monographs may

be obtained free of charge from the National Clearinghouse for

Drug Abuse Information (NCDAI). Please contact NCDAI also for

information about availability of coming issues and other

publications of the National Institute on Drug Abuse relevant to

drug abuse research.

Additional copies may be purchased from the U.S. Government

Printing Office (GPO) and/or the National Technical Information

Service (NTIS) as indicated. NTIS prices are for paper copy.

Microfiche copies, at $4.50, are also available from NTIS.

Prices from either source are subject to change.

Addresses are:

NCDAI

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GPO

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1 FINDINGS OF DRUG ABUSE RESEARCH. Not available from NCDAI.

Vol. 1: GPO out of stock NTIS PB #272 867/AS $32.50

Vol. 2: GPO out of stock NTIS PB #272 868/AS $29.50

2 OPERATIONAL DEFINITIONS IN SOCIO-BEHAVIORAL DRUG USE RESEARCH

1975. Jack Elinson, Ph.D., and David Nurco, Ph.D., eds. Not

available from NCDAI.

GPO out of stock NTIS PB #246 338/AS $16

3 AMINERGIC HYPOTHESES OF BEHAVIOR: REALITY OR CLICHE? Bruce J.

Bernard, Ph.D., ed. Not available from NCDAI.

GPO Stock #017-024-00486-3 $6.50 NTIS PB #246 687/AS $16

130
4 NARCOTIC ANTAGONISTS: THE SEARCH FOR LONG-ACTING PREPARATIONS.

Robert Willette, Ph.D., ed.

GPO out of stock NTIS PB #247 096/AS $8.50

5 YOUNG MEN AND DRUGS: A NATIONWIDE SURVEY. John A. O'Donnell,

Ph.D., et al. Not available from NCDAI.

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6 EFFECTS OF LABELING THE "DRUG ABUSER": AN INQUIRY. Jay R.

Williams, Ph.D. Not available from NCDAI.

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7 CANNABINOID ASSAYS IN HUMANS. Robert Willette, Ph.D., ed.

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8 Rx: 3x/WEEK LAAM - ALTERNATIVE TO METHADONE. Jack Blaine, M.D.,

and Pierre Renault, M.D., eds.

Not available from GPO NTIS PB #253 763/AS $14.50

9 NARCOTIC ANTAGONISTS: NALTREXONE PROGRESS REPORT. Demetrios

Julius, M.D., and Pierre Renault, M.D., eds. Not available from

NCDAI.

GPO Stock #017-024-00521-5 $7.00 NTIS PB #255 833/AS $17.50

10 EPIDEMIOLOGY OF DRUG ABUSE: CURRENT ISSUES. Louise G. Richards,

Ph.d., and Louise B. Blevens eds. Not available from NCDAI.

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11 DRUGS AND DRIVING. Robert Willette, Ph.D., ea. Not available

from NCDAI.

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12 PSYCHODYNAMICS OF DRUG DEPENDENCE. Jack D. Blaine, M.D., and

Demetrios A. Julius, M.D., eds. Not available from NCDAI.

GPO Stock #017-024-00642-4 $5.50 NTIS PB #276 084/AS $17.50

13 COCAINE: 1977. Robert C. Petersen, Ph.D., and Richard C.

Stillman, M.D., eds. Reports the extent and limits of current

knowledge about cocaine, its use and misuse.

GPO Stock #017-024-00592-4 $6.00 NTIS PB #269 175/AS $19

14 MARIHUANA RESEARCH FINDINGS: 1976. Robert C. Petersen, Ph.D.,

ed. Technical papers on which the 6th Marihuana and Health report

to Congress was based.

GPO out of stock NTIS PB #271 279/AS $22

15 REVIEW OF INHALANTS: EUPHORIA TO DYSFUNCTION. Charles Wm.

Sharp, Ph.D., and Mary Lee Brehm, Ph.D., eds. Review of inhalant

abuse, including an extensive bibliography.

GPU Stock #017-024-00650-5 $7.50 NTIS PB #275 798/AS $28

16 THE EPIDEMIOLOGY OF HEROIN AND OTHER NARCOTICS. Joan Dunne

Rittenhouse, Ph.D., ed. Not available from NCDAI.

GPO Stock #017-024-00690-4 $6.50 NTIS PB #276 357/AS $20.50

131
17 RESEARCH ON SMOKING BEHAVIOR. Murray E. Jarvik, M.D., Ph.D.,

et al., eds. Includes epidemiology, etiology, consequences of

use, and approaches to behavioral change. From a NIDA-supported

UCLA conference.

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18 BEHAVIORAL TOLERANCE: RESEARCH AND TREATMENT IMPLICATIONS.

Norman A. Krasnegor, Ph.D., ed. Theoretical and empirical studies

of nonpharmacologic factors in development of drug tolerance.

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19 THE INTERNATIONAL CHALLENGE OF DRUG ABUSE. Robert C. Petersen,

Ph.D., ed. Papers from the VI World Congress of Psychiatry .

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20 SELF-ADMINISTRATION OF ABUSED SUBSTANCES: METHODS FOR STUDY.

Norman A. Krasnegor, Ph.D., ed. Techniques used to study basic

processes underlying abuse of drugs, ethanol, food, and tobacco.

GPO Stock #017-024-00794-3 $6.50 NTIS PB #288 471/AS $22

21 PHENCYCLIDINE (PCP) ABUSE: AN APPRAISAL. Robert C. Petersen,

Ph.D., and Richard C. Stillman, M.D., eds. For clinicians and

researchers, assessing the problem of PCP abuse.

GPO Stock #017-024-00785-4 $7.00 NTIS PB #288 472/AS $25

22 QUASAR: QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIPS OF

ANALGESICS, NARCOTIC ANTAGONISTS, AND HALLUCINOGENS. Gene

Barnett, Ph.D.; Milan Trsic, Ph.D.; and Robert Willette, Ph.D.;

eds. Not available from NCDAI.

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23 CIGARETTE SMOKING AS A DEPENDENCE PROCESS. Norman A.

Krasnegor, Ph.D., ed. Discusses factors involved in the onset,

maintenance, and cessation of the cigarette smoking habit.

Includes an agenda for future research.

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24 SYNTHETIC ESTIMATES FOR SMALL AREAS: STATISTICAL WORKSHOP

PAPERS AND DISCUSSION. Jos. Steinberg, ed. Papers from a workshop on statistical approaches that yield needed estimates of

data for States and local areas. Not available from NCDAI.

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25 BEHAVIORAL ANALYSIS AND TREATMENT OF SUBSTANCE ABUSE. Norman

A. Krasnegor, Ph.D., ed. Papers on commonalities and

implications for treatment of dependency on drugs, ethanol, food,

and tobacco.

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26 THE BEHAVIORAL ASPECTS OF SMOKING. Norman A. Krasnegor, Ph.D.,

ed. Reprint of the behavioral section of the 1979 Report of the

Surgeon General on Smoking and Health; introduction by editor.

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27 PROBLEMS OF DRUG DEPENDENCE, 1979: PROCEEDINGS OF THE 41ST

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG DEPENDENCE, INC. L.S. Harris, Ph.D., ed. Not available from NCDAI.

GPO Stock #017-024-00981-4 $9.00 NTIS PB #80-175482 $37

28 NARCOTIC ANTAGONISTS: NALTREXONE PHARMACOCHEMISTRY AND

SUSTAINED-RELEASE PREPARATIONS. Robert Willette, Ph.D., and

Gene Barnett, Ph.D., eds. Papers report research on sustainedrelease and long-acting devices for use with the narcotic antagonist naltrexone.

GPO Stock #017-024-01081-2 $7.00 NTIS PB #81-238875 $23.50

29 DRUG ABUSE DEATHS IN NINE CITIES: A SURVEY REPORT. Louis A.

Gottschalk, M.D., et al. Not available from NCDAI.

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30 THEORIES ON DRUG ABUSE: SELECTED CONTEMPORARY PERSPECTIVES.

Dan J. Lettieri, Ph.D.; Mollie Sayers; and Helen Wallenstein

Pearson, eds. Volume presents summaries of major contemporary

tneories of drug abuse by each of 43 leading theorists.

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31 MARIJUANA RESEARCH FINDINGS: 1980. Robert C. Petersen, Ph.D.,

ed. The text of the 8th Marijuana and Health report to Congress

and the background scientific papers on which it was based.

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32 GC/MS ASSAYS FOR ABUSED DRUGS IN BODY FLUIDS. Rodger L. Foltz,

Ph.D.; Allison F. Fentiman, Jr., Ph.D.; and Ruth B. Foltz. A

collection of methoas for auantitative analysis of several

important drugs of abuse by gas chromatography- mass spectrometry.

GPO Stock #017-024-01015-4 $6.00 NTIS PB #81-133746 $19

33 BENZODIAZEPINES: A REVIEW OF RESEARCH RESULTS, 1980. Stephen

I. Szara, M.D., D.Sc., and Jacqueline P. Ludford, M.S., eds.

A RAUS (Research Analysis and Utilization System) Review Report

on the aouse liability of the Denzodiazepine “tranquilizers.”

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34 PROBLEMS OF DRUG DEPENDENCE, 1980: PROCEEDINGS OF THE 42ND

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. Not available from

NCOAI.

GPO Stock #017-024-01061-8 $8.00 NTIS PB #81-194847 $34

35 DEMOGRAPHIC TRENDS AND DRUG ABUSE, 1980-1995. Louise G.

Richards, Ph.D., ed. Estimates of probable extent and nature of

nonmeoical drug use, 1980-1995, based on age structure and other

characteristics of U.S. population.

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36 NEW APPROACHES TO TREATMENT OF CHRONIC PAIN: A REVIEW OF MULTIDISCIPLINARY PAIN CLINICS AND PAIN CENTERS. Lorenz K.Y. Ng, M.D.,

ed. Discussions by active practitioners in the treatment of pain.

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37 BEHAVIORAL PHARMACOLOGY OF HUMAN DRUG DEPENDENCE. Travis

Thompson, Ph.D., and Chris E. Johanson, Ph.D., eds. Presents a

growing body of data, systematically derived, on the behavioral

mechanisms involved in use and abuse of drugs.

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38 DRUG ABUSE AND THE AMERICAN ADOLESCENT. Dan J. Lettieri,

Ph.D., and Jacqueline P. Ludford, M.S., eds. A RAUS Review

Report, emphasizing use of marijuana: epidemiology, sociodemographic and personality factors, family and peer influence,

delinouency, and biomedical consequerces.

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39 YOUNG MEN AND DRUGS IN MANHATTAN: A CAUSAL ANALYSIS.

Richard R. Clayton, Ph.D., and Harwin L. Voss, Ph.D. Examines

tne etiology and natural history of drug use, with special focus

on heroin. Includes a Lifetime Drug Use Index.

GPO Stock #017-024-01097-9 $5.50 NTIS PB #82-147372 $19

40 ADOLESCENT MARIJUANA ABUSERS AND THEIR FAMILIES. Herbert

Hendin, M.D., Ann Pollinger, Ph.D., Richard Ulman, Ph.D., and

Arthur Carr, Ph.D. A psychodynamic study of adolescents

involved in neavy marijuana use, to determine what interaction between family and adolescent gives rise to drug abuse.

GPO Stock #017-024-01098-7 $4.50 NTIS PB #82-133117 $13

41 PROBLEMS OF DRUG DEPENDENCE, 1981: PROCEEDINGS OF THE 43RD

ANNUAL SCIENTIFIC MEETING, THE COMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. Not available from

NCDAI.

Not available from GPO NTIS PB #82-190760 $41.50

42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL FLUIDS. Richard L.

Hawks, Ph.D., ed. Not available from NCDAI.

GPO Stock #017-024-01151-7 $5 NTIS PB #83-136044 $1643

43 PROBLEMS OF DRUG DEPENDENCE, 1982: PROCEEDINGS OF THE 44TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. Not available from

NCDAI.

GPO Stock #017-024-01162-2 $8.50 NTIS PB #83-252-692 $40

44 MARIJUANA EFFECTS ON THE ENDOCRINE AND REPRODUCTIVE SYSTEMS.

Monique C. Braude, Ph.D., and Jacqueline P. Ludford, M.S., eds.

A RAUS Review Report of animal studies and preclinical and

clinical studies of effects of cannabinoids on human endocrine

and reproductive functions.

45 CONTEMPORARY RESEARCH IN PAIN AND ANALGESIA, 1983. Roger M.

Brown, Ph.D.; Theodore M. Pinkert, M.D., J.D.; and Jacqueline P.

Ludford, M.S., eds. A RAUS Review Report on the anatomy,

physiology, and neurochemistry of pain and its management.

GPO Stock #017-024-01191-6 $2.75 NTIS PB #84-184670 $11.50

134
46 BEHAVIORAL INTERVENTION TECHNIQUES IN DRUG ABUSE TREATMENT.

John Grabowski, Ph.D.; Maxine L. Stitzer, Ph.D., and Jack E.

Henningfield, Ph.D., eds. Reports on a variety of behavioral

contingency management procedures used in research/treatment

environments.

GPO Stock #017-024-01192-4 $4.25 NTIS PB #84 184688 $16

47 PREVENTING ADOLESCENT DRUG ABUSE: INTERVENTION STRATEGIES.

Thomas J. Glynn, Ph.D.; Carl G. Leukefeld, D.S.W.; and

Jacqueline P. Ludford, M.S., eds. A RAUS Review Report on a

variety of approaches to prevention of adolescent drug abuse, how

they can be applied, their chances for success, and needed future

research.

Not available from GPO NTIS PB to be assigned

48 MEASUREMENT IN THE ANALYSIS AND TREATMENT OF SMOKING

BEHAVIOR. John Grabowski, Ph.D., and Catherine S. Bell, M.S.,

eds. Based upon a meeting cosponsored by NIDA and the National

Cancer Institute to delineate necessary and sufficient measures

for analysis of smoking behavior in research and treatment

settings.

GPO Stock #017-024-01181-9 $4.50 NTIS PB 84-145-184 $14.50

IN PRESS

49 PROBLEMS OF DRUG DEPENDENCE, 1983: PROCEEDINGS OF THE 44TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. A collection of

papers which together record a year's advances in drug abuse

research; also includes reports on tests of new compounds for

efficacy and dependence liability.

51 DRUG ABUSE TREATMENT EVALUATION: STRATEGIES, PROGRESS, AND

PROSPECTS. Frank M. Tims, Ph.D., ed. A state-of-the-art review

of drug abuse treatment evaluation, identifying research needs,

promising approaches, and emerging issues.

52 TESTING DRUGS FOR DEPENDENCE POTENTIAL AND ABUSE LIABILITY.

Joseph V. Braay, Ph.D., and Scott E. Lukas, Ph.D., eds.

Describes animal and human test procedures for assessing

dependence potential and abuse liability of opioids, stimulants,

depressants, hallucinogens, cannabinoids, and dissociative

anesthetics.

IN PREPARATION

50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF ABUSE. John

Grabowski, Ph.D., ed. Content ranges from an introductory

overview through neuropharmacology, pharmacology, animal and

human behavioral pharmacology, patterns of use in the natural

environment of cocaine users, treatment, through commentary on

societal perceptions of use.

135

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