Genetic and

Biological Markers

in Drug Abuse

and Alcoholism

U. S. DEPARTMENT OF HEALTH AND HUMAN SERVICES • Public Health Service • Alcohol, Drug Abuse, and Mental Health Administration
Genetic and Biological

Markers in Drug Abuse

and Alcoholism

Editors:

Monique C. Braude, Ph.D.

Division of Preclinical Research

National Institute on Drug Abuse

Helen M. Chao, Ph.D.

Division of Extramural Research

National Institute on Alcohol Abuse and Alcoholism

NIDA Research Monograph 66

1986

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 series is to provide critical reviews of research problem

areas and techniques, the content of state-of-the-art conferences, and integrative research reviews. Its dual publication emphasis is rapid and targeted dissemination to the scientific and professional community.

Editorial Advisors

Martin W. Adler, Ph.D.

Temple University School of Medicine

Philadelphia, Pennsylvania

Sydney Archer, Ph.D.

Rensselaer Polytechnic Institute

Troy. New York

Richard E. Belleville, Ph.D.

NB Associates, Health Sciences

Rockville. Maryland

Karst J. Besteman

Alcohol and Drug Problems Association

of North America

Washington, DC.

Gilbert J. Botvin, Ph.D

Cornell University Medical College

New York, New York

Joseph V. Brady, Ph.D

The Johns Hopkins Unlversity School of

Medicine

Baltimore. Maryland

Theodore J. Cicero, Ph.D.

Washington University School of

Medicine

St Louis, Missouri

Sidney Cohen, M.D.

Los Angeles, California

Mary L. Jacobson

National Federation of Parents for

Drug-Free Youth

Omaha, Nebraska

Reese T. Jones, M.D.

Langley Porter Neuropsychiatric Institute

San Francisco, California

Denise Kandel, Ph.D.

College of Physicians and Surgeons of

Columbia University

New York, New York

Herbert Kleber, M.D.

Vale University School of Medicine

New Haven, Connecticut

Richard Russo

New Jersey State Department of Health

Trenton, New Jersey

NIDA Research Monograph Series

Charles R. Schuster, Ph.D.

DIRECTOR, NIDA

Jack Durell, M.D.

ASSOCIATE DIRECTOR FOR SCIENCE. NIDA

EDITOR-IN-CHIEF

For sale by the Superintendent of Documents

U.S. Government Printing Office

Washington, D.C. 20402

Parklawn Building, 5600 Fishers Lane. Rockville, Maryland 20857
Genetic and Biological Markers

in Drug Abuse and Alcoholism
ACKNOWLEDGMENT

This monograph is based upon papers and discussion from the

technical review on genetic and biological markers for drug abuse

and alcoholism which was held on November 28 and 29, 1984, at

Rockville, Maryland. The review was jointly sponsored by the

Division of Preclinical Research, National Institute on Drug Abuse,

and the Division of Extramural Research, National Institute on

Alcohol Abuse and Alcoholism.

COPYRIGHT STATUS

The National Institute on Drug Abuse has obtained permission from

Elsevier Biomedical Press B.V. to reproduce the figures on pages 15

and 16 and from the American Medical Association to reproduce the

figures on pages 100, 101, 102, and 104. Further reproduction of

these figures without specific permission of the copyright holders

is prohibited. All other material in this volume except quoted

passages from copyrighted sources is in the public domain and may be

used or reproduced without permission from the Institute or the

authors. Citation of the source is appreciated.

DHHS publication number (ADM)86-1444

Printed 1986

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

Alcohol and drug abuse have become major health and social problems

in the United States. Ten million American adults and 3.3 million

youths in the 14 to 17 age range may be problem drinkers.(1) In a

recent survey of high school seniors, nearly two-thirds of a

seniors report illicit drug use at some time in their lives.(2)

A wealth of literature documents that genetic factors play a

significant role in the transmission of at least some forms of

alcohol abuse and alcoholism. Studies in humans and animals further

suggest a genetic influence in individual variations of behavioral

and biological responses, as well as differential organ

sensitivities to the damaging effects of alcohol. The genetic

influence in drug abuse is, however, less well documented. There

are reports suggesting that genetics play a role in the user's

performance after opiate use. Several studies suggest that

preference for morphine is genetically related and that animal

responses to cocaine and morphine also appear to vary with genetic

background.

It is generally agreed that the genetic component in alcohol and

drug abuse is multifactorial and there may be identifiable traits or

markers that can single out individuals at risk. To foster research

in this important area, the National Institute on Drug Abuse (NIDA)

and the National Institute on Alcohol Abuse and Alcoholism (NIAAA)

cosponsored a technical review entitled: "Genetic and Biological

Markers in Drug Abuse and Alcoholism."

This meeting brought together researchers from the alcohol and drug

abuse research communities to discuss the state of knowledge

regarding genetic and biological markers, to identify research gaps,

and to recommend future directions. Topics discussed represented a

multidisciplinary approach ranging from family studies to animal

models and from behavioral traits to gene mapping.

This monograph includes the 10 papers presented at this workshop and

a summary of the presentations, prepared by Dr. Warren Nichols.

Areas in need of additional efforts or further development and

future directions for research recommended by workshop participants

v
are listed in a concluding chapter by the editors. The publication

represents the beginning of a coordinated effort between NIDA and

NIAAA to advance the field of genetic research in substance abuse.

We hope that it will stimulate collaboration among scientists in

various disciplines from both the alcoholism and the drug abuse

research communities.

Monique C. Braude, Ph.D.

National Institute on Drug Abuse

Helen M. Chao, Ph.D.

National Institute on Alcohol

Abuse and Alcoholism

(1) Noble, E.P., ed.: "Third Special Report to the U.S. Congress on

Alcohol and Health." June, 1978. National Institute on Alcohol

Abuse and Alcoholism. DHHS Publication No. (ADM) 79-832.

Rockville, MD: NIAAA, 1979.

(2) Johnston, L.D.; O'Malley, P.M.; and Bachman, J.G.: "Drugs and

American High School Students 1975-1983." National Institute on

Drug Abuse. DHHS Publication No. (ADM) 85-1374. Rockville, MD:

NIDA, 1984.

vi
Contents

Preface

Pharmacogenetic Approaches to the Prediction of Drug Response

Individual Differences in Opiate-Induced Alterations at the

Cytogenetic, DNA Repair, and Immunologic Levels:

Opportunity for Genetic Assessment

Arthur Falek; John J. Madden; David A. Shafer; and

Robert M. Donahoe. . . . . . . . . . . . . . . . 11

Elliot S. Vesell . . . . . . . . . . . . . . . . 25

Genetics as a Tool for Identifying Biological Markers

of Drug Abuse

Allan C. Collins . . . . . . . . . . . . . . . . 57

Studies on an Animal Model of Alcoholism

Ting-Kai Li; Lawrence Lumeng; William J. McBride;

Marshall B. Waller; and James M. Murphy . . . . . . . . 41

Development of DNA Probes to Investigate Genetic Variation

of Alcohol Metabolizing Enzymes

Moyra Smith; Gregg Duester; and G. Wesley Hatfield. . . . . 50

Robert S. Sparkes. . . . . . . . . . . . . . . . 5

Warren W. Nichols. . . . . . . . . . . . . . . . 1

Genetic and Biological Markers in Drug Abuse and Alcoholism:

A Summary

Monique C. Braude and Helen M. Chao. . . . . . . . . . v

Polymorphic Gene Marker Studies

Genetic Markers of Drug Abuse in Mouse Models

Louis Shuster . . . . . . . . . . . . . . . . . 71

Inheritance of Risk to Develop Alcoholism

C. Robert Cloninger; Soren Sigvardsson; Theodore Reich; and

Michael Bohman. . . . . . . . . . . . . . . . . 86

Recommendations for Future Research on Genetic and Biological

Markers in Drug Abuse and Alcoholism

Monique C. Braude and Helen M. Chao. . . . . . . . . .109

List of NIDA Research Monographs . . . . . . . . . . .112

Genetic and Biological Markers in Alcoholism and Drug Abuse

Marc A. Schuckit . . . . . . . . . . . . . . . . 97

vii
Genetic and Biological Markers in

Drug Abuse and Alcoholism:

A Summary

Warren W. Nichols, M.D., Ph.D.

The workshop on "Genetic and Biological Markers in Drug Abuse and

Alcoholism" reported in this monograph was jointly sponsored by

the National Institute on Drug Abuse (NIDA) and the National

Institute on Alcohol Abuse and Alcoholism (NIAAA). It brought

together individuals from both abuse areas with expertise in

clinical research, animal models, and molecular systems. The

aims for examining genetic and biologic markers of substance

abuse were twofold. The first aim was to develop methods for the

use of markers that could predict individuals with a hereditary

predisposition or susceptibility to alcoholism or drug addiction,

as well as predict hereditary alterations that could enhance the

toxic effects of these substances. The second aim was to examine

the use of biologic and genetic parameters to detect damage or

alterations produced by alcohol and drug abuse.

The difficulties encountered in these areas because of imprecision

and variability of diagnostic criteria and classification were

pointed out and discussed several times throughout the meeting.

It was clear that bringing together interdisciplinary groups,

such as those attending this workshop, was one good method of

reducing this variability, and it was also clear that a series

of separate meetings designed to unify diagnostic criteria and

classification would benefit the entire area of drug and alcohol

research. Further problems encountered are produced by the

almost certain heterogeneity and multifactional nature of

individuals abusing drugs and alcohol.

The markers or phenomena that may be of use in recognizing or

detecting alcohol and drug abuse that were discussed at the

meeting included: phenotypic and genotypic areas from behavioral

observations and clinical genetic studies; polymorphisms in

gene products, such as pharmacogenetic differences in metabolizing enzymes, isoenzyme variations, antigenic markers as blood

groups, and the HLA systems; and DNA polymorphisms detected by

restriction enzyme fragment length differences.

1
Strong evidence for a genetic factor or factors in alcoholism

was presented in two clinical research papers by Drs. C. Robert

Cloninger and Marc Schuckit. Both studies reported a marked

increase in the prevalence of alcoholism in a population which

had a first-degree relative that was alcoholic versus the

general population. Various demographic means, including

adoption studies, demonstrated that this was related to a

genetic trait rather than to environmental exposure. However, both

studies agreed that there were multiple genetic and environmental

influences in this complex phenotype. Dr. Cloninger also

reported a large excess of male over female alcoholics in both

the general population and the population with first-degree

alcoholic relatives. These differences appeared to have a

genetic basis and could not be explained by socioenvironmental

factors alone. Similar studies on opiates and other drugs of

abuse are not currently available, but it is believed that

from the alcohol experience similar studies could be

established to determine the role of genetic factors in drug

abuse.

Dr. Arthur Falek presented clinical research studies designed to

examine opiate-induced alterations in chromosomes, DNA repair,

and immunologic functions. Studies of peripheral lymphocytes of

addicts demonstrated a significant increase in the levels of

chromosome aberrations and sister chromatid exchange (SCE)

events. There was also an increased response in these parameters

after treatment with ultraviolet light. Formation of T lymphocytes E-rosettes was also reduced in these addicts. Some

recovery of all parameters was observed in long-term methadone

patients and marked individual variation occurred. The large

number of variables in street drug users and the importance of

separating effects of abuse substances from susceptibility to

abuse substances was emphasized by Dr. Falek. He pointed out

that there is at present no study that indicates a genetic

potential to altered response to opiates, and that twins may be

a suitable way to explore this hypothesis if a sufficiently

large addict population is available for study.

Animal models were discussed in separate presentations by Drs.

T.K. Li, Allan Collins, and Louis Shuster. Dr. Li described

his studies on alcoholism using a rat model. By selecting and

breeding rats that had a preference for alcohol over water, Dr.

Li has developed animals that self-administer alcohol by mouth

and achieve pharmacologically significant blood levels for the

pharmacologic effect rather than for the caloric value, taste,

or smell. The ethanol is positively reinforcing and leads to

tolerance and physical dependence. Animals bred for these

characteristics exhibit lower levels of serotonin and 5-hydroxyindolacetic acid in various areas of the brain. Other biochemical

and physiologic parameters are being sought with the long-term

goal of eventual human application.

2
Drs. Collins and Shuster discussed the use of various strains of

mice in studies of alcohol and drug abuse. They pointed out

that some markers of abuse substances that are of interest may

require the introduction of the substance to initiate the

marker. Also, important markers may be found in tissues not

readily accessible in humans. In both situations, animal models

may overcome these difficulties. The wealth of inbred strains.

recombinant inbred strains, congenic lines, and selectively bred

lines offer excellent starting material for analysis of genetic

predispositions. Marked differences in responses to opiates,

ethanol, and nicotine between C57B1/6 and DBA/2 mice are being

utilized to study differences in receptors, neurotransmitters,

and endogenous opiates. Evidence was cited that high-affinity

receptors are present for opiates but have not been found for

ethanol. Evidence that animals can become addicted to endogenous

opiates based on withdrawal symptoms after naloxone treatment

was also described. Publication of a compendium of quantitative

differences in drug responses of the many inbred lines and mutants

as they are determined would be very helpful to investigators in

this field.

Dr. Vesell's discussion of pharmacogenetic approaches to the

prediction of drug response served as a bridge between the

clinical studies, the animal models, and the molecular studies.

He described genetic factors subject to environmental modifications that are capable of producing variations up to fortyfold

in drug clearance, plasma drug half-life, and steady-state blood

drug concentrations. Several hereditary conditions with unusual

drug reactions based on pharmacogenetic differences were described,

as were associations with phenotypic markers such as the HLA

type. Dr. Vesell demonstrated mistakes in evaluating genetic

metabolic polymorphisms in family studies if only the parent

compound is evaluated in the absence of metabolic products.

Dr. Robert S. Sparkes listed the variety of polymorphic gene

markers that are available for studying disease association and

genetic linkage. Association refers to a close relationship

between a trait or a disease and a specific allelic variant

revealed by the study of unrelated individuals, and it is probably

important in the pathogenesis of the disease or trait. Genetic

linkage refers to the physical location of genes close to each

other on the same chromosome and requires family studies for

elucidation. Linkage studies can be used to determine which

members of an at-risk family possess a gene which has been

proven to be linked to increased susceptibility to a disease

state, while association studies may be important in determining

whether a specific genetic variant is associated with a reaction

to alcohol or another drug. Of the various polymorphic gene

markers available, Dr. Sparkes pointed out the several advantages

of DNA restriction fragment length polymorphisms (RFLP). These

include the fact that the gene or genes in the polymorphic DNA

fragments need not be expressed or even identified, and that all

3
regions of the genome can be covered rather than the approximately

20% that occurs with other phenotypic markers. This means that

any gene under question should be linkable.

Dr. Moyra Smith-Wright described the preparation of a cDNA clone

for the alcohol dehydrogenase 1 gene (ADH1) from messenger RNA

for the polypeptide gene product. This probe was used to study

the closely homologous three class I ADH genes for polymorphism

at the DNA level. The results suggest that polymorphisms occur

in at least two of the ADH genes and that the polymorphisms are

heritable. By the use of somatic cell hybrids, it has also been

possible to map these genes to the long arm of chromosome 4.

There are indications that the fetal alcohol syndrome has genetic

factors that may be approachable through these polymorphisms in

genes coding for metabolic enzymes and that this may be a good

starting point for work with these probes.

All in all, this workshop was very productive with excellent

interaction between the various interdisciplinary groups. It is

certainly time, and this is an excellent method to select wellthought-out studies in new areas that may contribute to both

prevention and treatment of these large problems.

AUTHOR

Warren W. Nichols, M.D., Ph.D.

Merck Sharp & Dohme

Research Laboratory

West Point, Pennsylvania 19486

4
Polymorphic Gene Marker Studies

Robert S. Sparkes, M.D.

INTRODUCTION

Common genetic variations are called polymorphisms and refer to

genetic traits for which at least two allelic variants occur with

a frequency which cannot be attributed to recurrent mutation alone;

therefore, each variant usually occurs with a frequency greater

than 2%. Man, being a diploid organism, has only two alleles at

each locus, but in a population of individuals there may be many

alleles at a locus. The biological significance of many of these

polymorphisms remains to be determined.

Currently available polymorphic genetic markers can be grouped

into phenotypic and DNA markers. The phenotypic markers include

red blood cell antigens, red blood cell isoenzymes, serum proteins,

and HLA antigens. The polymorphic variants at the DNA level are

called restriction fragment length polymorphisms (RFLPs).

APPLICATIONS OF POLYMORPHIC GENE MARKERS

Polymorphic genetic variation has applications in a number of

areas, but basically they afford means to identify or distinguish

the presence or absence of specific genetic alleles. This variation can be used for individual identification, such as in forensic

studies where blood stains are compared with fresh blood from a

known person. The oldest application of genetic polymorphic studies

in the laboratory is with blood transfusions in which there is

matching between donor and recipient for polymorphic red blood cell

antigens, especially in the ABO and the Rh systems. Similar application is made in tissue transplantation using the HLA polymorphic

variation. In bone marrow transplantation, the other polymorphic

markers, such as isoenzymes, can be used to identify the presence

of donor cells in a recipient following a transplant; this is based

upon differences in any of the polymorphic markers between donor

and recipient. These polymorphisms are also used for paternity

evaluation in which these markers are compared between the child,

its mother, and the putative father, searching for evidence that

there is a genetic trait that the child could not have received from

the man in question. Such information is also needed in genetic

5
linkage studies (see below). Isoensyme polymorphisms have been

used to demonstrate cross-contamination of tissue culture cell lines.

It is not uncommon to find a mixup in cultures and that the investigator is not working with the expected culture: isoenzyme studies

have demonstrated that upwards of 30% of such cultures are of an unexpected type. Major applications have also been made to the study

of human gene mapping using polymorphic traits in linkage analysis.

Finally, studies have been made to evaluate possible associations of

diseases or other traits with specific allelic types, particularly

with the ABO blood group and the HLA white blood cell system.

GENETIC LINKAGE

Human gene mapping has made major advances in recent years. This

has largely been achieved through the use of interspecific somatic

cell hybridization. This approach is based upon the nonrandom loss

of human chromosomes from the somatic cell hybrids formed between

established rodent cell lines and human cells. A correlation is

made between which human genes and which human chromosomes are present in a panel of different hybrid clones. Although each clone

usually has more than one human chromosome, the different clones are

chosen so that the total panel will permit assignment of a specific

gent to a specific chromosome. This technique can also be used to

regionally map genes on a chromosome by starting with human cells

which contain a chromosome rearrangement, usually a translocation.

The correlation is then made between the gene in question and that

part of a chromosome which remains in the somatic cell hybrid. One

of the advantages of this technique is that it does not depend upon

polymorphism for the gene which is being mapped. Much of the past

work with somatic cell hybrids has utilized species differences in

enzymes which can be seen on electrophoresis or isoelectric focusing.

In contrast to the somatic cell hybridization approach, human gent

mapping through linkage analysis does depend on genetic polymorphism.

With genetic linkage, one measures gene map distance between two genes

as reflected in the number of cross-overs which occur in meiosis

between the two genes (Conneally and Rivas 1980). Linkage is the

tendency of genes on the same chromosomes to segregate together, i.e.,

linked gents are transmitted to the same gamete more than 50% of the

time. The closer genes art to each other, the more frequently they

will be transmitted together. On the other hand, genes that are far

apart on same of the larger chromosomes may segregate randomly in

relation to each other, even though they are on the same chromosome.

All genes that are located on the same chromosome art said to be

syntenic. While all genes that are linked are syntenic, the converse

may not be true. Linkage analysis requires the study of families.

Generally, families in which a genetic trait of interest occurs are

examined, Until recently, most linkage studies have used the phenotypic polymorphic traits of the red blood cell antigens, red blood

cell isoenzymes, serum proteins, and HLA variants. Through the use

of statistical analysis, lod scores can be obtained and these reflect

the likelihood that two genes are linked to each other and how close

they may be. Because of the relative limited number of polymorphic

phenotypic markers available and because these cover only a fraction

6
of the total human genome (about 20%), genetic linkage analysis

has not been highly successful. However, with the introduction of

the DNA RFLPs, a new era has arrived in human gene mapping and, in

theory, it is now possible to map any human gene through this

approach.

All genetic variation reflects differences at the DNA level. This

is true for the phenotypic markers which reflect genetic changes

based upon differences between alleles that can be determined by

electrophoretic techniques, immunological techniques, or other

biochemical methods. With the RFLPs of DNA, one detects these

differences directly in the DNA. Study of these polymorphisms is

based upon the following. Restriction endonucleases split DNA at

specific base sequences. These base sequences at the same site in

the DNA may vary between persons. With the use of different

restriction endonucleases, potentially a large number of different

base sequences may be detected in a population of individuals. By

isolating DNA from cells of individuals and treating the DNA with

restriction endonucleases, one can separate the DNA fragments by

molecular size using agarose gel electrophoresis. One then hybridizes radioactively labeled DNA probes of specific function or DNA

probes of nonspecific or unidentified functions (anonymous probes)

to the DNA which has been treated with the restriction endonucleases

and separated by the electrophoresis. Differences in the size of

the DNA segments can be detected with the probes. These differences

can then be treated by the standard linkage approach described above.

The DNA RFLPs have a number of advantages (table 1). With the

availability of a large number of restriction endonucleases, it is

very likely that each and every gene will be found to demonstrate

polymorphisms following treatment with one or more of these restriction endonucleases. It has been estimated that perhaps only 165 DNA

probes could span or map the human genome (Botstein et al. 1980).

This would be true if the probes were mapped at random. However,

it has been estimated that many more markers may have to be studied

and evaluated in order to find the minimum number which would be

randomly distributed over the genome (Lange and Boehnke 1982).

Nevertheless, it appears at this time that with sufficient effort

the appropriate probes can be isolated and used to eventually map

the whole human genome. It should be noted that these DNA

probes can also be used in conjunction with the somatic cell hybridization approach referred to above. In addition, they can be used

in a new approach called in situ hybridization in which the radioactive probe is hybridized directly to chromosomes on a microscope

slide.

Many human genes are being rapidly mapped through the use of the DNA

probes using these three methods: linkage, somatic cell hybridization, and in situ hybridization. For example, one prominent example

using this recombinant DNA approach has been the demonstration of

linkage of the gene for Huntington's disease to an anonymous gene

identified by the DNA probe, G8 (Gusella et al. 1983). Although

still not applicable at the clinical level, these developments offer

the prospect in the near future of being able to enhance genetic

counseling for families in which Huntington's disease occurs. Poten7
tially, such an approach could be used to make prenatal diagnosis to

identify persons at risk not only before the symptoms start, but

even before the individual is born.

1.

2.

3.

Gene or DNA sequence need not be expressed;

RFLPs can be found in all regions of the genome;

Probably all genes or DNA sequences can be "made"

polymorphic with use of different restriction

endonucleases;

4. Same technique is used for all markers;

5. May lead to isolation of a gene of interest; and

6. Any gene should be "linkable."

TABLE 1

Advantages of DNA RFLPs

ASSOCIATION

Genetic linkage and association are terms that are often used interchangeably, but they describe different phenomena with different

implications (Vogel and Motulsky 1979; Hedge and Spence 1981). However, within a single family it may not be possible to distinguish

linkage from association. Evaluation of each requires the use of

genetic polymorphisms, which may in part contribute to the confusion.

Linkage relates to genetic loci, and the alleles at these loci are

useful only as markers for the loci. Evaluation of linkage requires

the study of families, especially large families. Association refers

to a concurrence greater than predicted by chance between a specific

allele at a locus and another, trait which may or may not have an

obvious genetic basis. Evaluation of association requires the study

of unrelated individuals. Association may give an insight into susceptibility and pathogenesis of a trait or disease. Thus, association

studies may be used to identify a genetic factor in a disease. With

the exception of linkage disequilibrium, association is not due to

linkage. Association may result from: pleiotrophic effects of a

single gene; epistatic interaction; selection in relation to environmental factors (for example, sickle cell gene and malarial resistance);

and population stratification.

The early examples of association are related to the ABO blood groups,

because this was the earliest polymorphic trait available for study.

Examples of significant associations between the ABO blood groups and

diseases include a higher frequency of blood type A in persons with

neoplasia of the intestinal tract, cervix, and breast, while there

is increased frequency of blood type O in persons with duodenal and

gastric ulcers. However, these associations are not sufficiently

8
strong to have clinical importance. When the highly polymorphic HLA

system became available, it was applied to a large number of diseases

in a search for associations. Some of the significant associations

between HLA and diseases include type B27 with ankylosing spondylitis and Reiter's disease, and type DR3/4 with insulin-dependent

diabetes mellitus.

In addition to the association of the sickle cell gene and the

resistance to malaria noted above, a similar resistance has been

noted for G6PD deficiency and malaria. Another association that

has been described in relation to malaria is the Duffy blood group

system in which individuals of the Fy(a-b-) phenotype demonstrate

resistance to malaria. Evidence has been presented that the Duffy

a and b antigens may act as red cell receptors for malaria parasites.

As expected, the genomic types for the resistance to malaria have

been found to occur with a high frequency in malarial areas. These

are also nice examples of how environmental factors may contribute

to specific allelic frequencies.

Demonstration of associations may help to: identify a genetic component to a disease; clarify pathogenesis of a disease; facilitate

preclinical testing to identify persons at risk so that appropriate

counsel may be given; and recognize disease heterogeneity.

Thus, associations may be important in determining whether a specific

genetic variant is associated with reaction to a drug or alcoholism.

On the other hand, genetic linkage may be useful to identify a gene

or genes which may be important in determining reactions to a drug or

alcohol, but whose function is not known. From linkage to a testable

polymorphic gene marker, one may be able to identify who in a family

may be at risk to have a gene for drug abuse or alcoholism.

REFERENCES

Botstein, D.; White, R.L.; Skolnick, M.; and Davis, R.W. Construction of a genetic linkage map in man using restriction fragment

length polymorphisms. Am J Hum Genet 21:214-331, 1980.

Conneally, P.M., and Rivas, M.L. Linkage analysis in man. In:

Harris, H., and Hirschhorm, K., eds. Advances in Human Genetics.

vol. 10. New York: Plenum Press, 1980. pp 209-266.

Gusella, J.F.; Wexler, N.S.; Conneally, P.M.; Naylor, S.L., Anderson,

M.A.; Tanzi, R.E.; Watkins, P.C.; Ottina, K.; Wallace, M.R.;

Sakaguchi, A.Y.; Young, A.B.; Shoulson, I.; Bonilla, E.; and Martin,

J.B. A polymorphic DNA marker genetically linked to Huntington's

disease. Nature 306:234-238, 1983.

Hodge, S.E., and Spence, M.A. Some epistatic two locus models of

disease. II. The confounding of linkage and association. Am J

Hum Genet 33:396-406, 1981.

Lange, K., and Boehnke, M. How many polymorphic genes will it take

to span the human genome? Am J Hum Genet 34:842-845, 1982.

Vogel, F., and Motulsky, A.G. Human Genetics. New York: SpringerVerlog, 1979. 700 pp.

9
AUTHOR

Robert S. Sparkes, MD

Division of Medical Genetics

Department of Medicine

UCLA Medical School and UCLA Center for the Health Sciences

LOS Angeles, California 90024

10
Individual Differences in Opiate-Induced

Alterations at the Cytogenetic, DNA Repair,

and Immunologic Levels: Opportunity for

Genetic Assessment

Arthur Falek, Ph.D.; John J. Madden, Ph.D.; David A. Shafer,

Ph.D.; and Robert M. Donahoe, Ph.D.

In humans there are several strategies to identify the significance of hereditary factors underlying the occurrence of a

behavioral and/or physical trait, including: a) family studies,

b) twin studies, c) linkage studies to a genetic marker, and d)

adoption studies. In particular, where expression of a trait

may be influenced by sociocultural factors, consistent findings

are required from several strategies to support the importance

of genetic factors. For example, family, twin, genetic marker

and adoption studies (Goodwin 1981) have provided consistent

support for the importance of a genetic component for

alcoholism.

Linkage studies of specific traits with genetic markers have

recently been expanded from linkage by blood groups, HLA

haplotypes, and other such chromosomally located protein markers

to those linked by specific DNA probes such as the recently

announced linkage between a polymorphic DNA probe on the short

arm of chromosome 4 and Huntington’s disease (Gusella et al.

1983; Vesell 1984). This new approach is limited at present to

the study of clearly identified single gene inherited disorders

but may in the future be appropriate for studies of apparent

polygenetic traits with strong environmental components such as

alcoholism.

While genetic studies employing different strategies have

demonstrated the importance of the genetic component in

alcoholism. unfortunately no such self-consistent data have been

obtained for opiate abuse. As reported by Liston and associates

(Liston et al. 1981) and reaffirmed most recently in our search

of the drug abuse literature, there is a paucity of information

on the contribution made by genetic factors to individual

differences in response to opiates among humans. On the other

hand, as noted by Liston and associates, there have been studies

of genetic influences on performance after opiate use as well as

on preference in animals for morphine. These studies demonstrated that there were significant differences in locomotor

11
activity among different strains of rats following morphine

administration (Eidelberg et al. 1975). They also found that

rats could be bred with a preference for morphine (Nichols and

Hsiao 1967) and that mice inbred for alcohol use also preferred

morphine (Eriksson and Kiianmaa 1971). These animal studies

demonstrating genetic mechanisms underlying variability in

response to morphine are reviewed by Dr. Shuster (this volume).

In humans, the physiological (cold pressor test) and

psychological parameters of responses to pain and to morphine in

10 sets of normal monozygotic male twins were measured (Liston

et al. 1981). This study (from the VA Hospital in Los Angeles)

provided no evidence for a genetic component in response to pain

(either pain threshold or pain tolerance) before or after

administration of morphine. Although these investigators did

find significant twin pair effects on two measures of anxiety,

they indicated that the number of twin sets in their study was

too small to produce significant differences between pairs as

compared with differences between the twin partners. The only

other report in the literature on genetic aspects of opiates in

humans is on one pair of monozygotic twins discordant for heroin

abuse (Grumet 1983).

Our own study of cellular genetic aspects of opiate abuse was

initiated to determine whether there was an impact on the genome

among humans using opiates. Initial studies were conducted to

examine levels of chromosome damage (CD) among opiate addicts as

compared with controls, since an increased level of such

chromosome alterations implies DNA damage and often may be

correlated with an increase in mutational events. For the first

study, we compared leukocyte chromosomes of 16 addicts with 16

controls (Falek et al. 1972). Our findings revealed 32 random

chromosome aberrations among the 16 drug patients for a rate of

2.7% (calculated in the first study as the number of aberrations

found divided by the number of metaphase chromosome complements

scanned) as compared with 4 chromosome breaks in the leukocytes

of the 16 control subjects for a rate of 0.4%. We consistently

found a frequency of 0.4% chromosome damage among normal

populations during the time we were conducting these studies.

There were some opiate addicts who showed no evidence of

chromosome damage and obviously there were others who displayed

more than 2 damaged chromosomes to provide the 32 damaged

chromosomes among the almost 1,200 metaphase complements

analyzed. There was no correlation of the chromosome damage

level in the addicts with the length of time of drug use, the

type of street opiate, or the abuse of other drugs. Further

studies in which addicts were followed longitudinally for a

period of more than 1 year through methadone treatment or

nonchemical detoxification provided evidence that the increased

number of damaged chromosomes at time of admission also

reflected a significantly increased frequency of cells with at

least one damaged chromosome (Falek and McFadden 1973; Falek and

Hollingsworth 1980). Inter- and intra-individual variability

was clearly evident from one time point to the next (table 1).

12
After a year the CD levels were reduced to about 0.6% for those

remaining in the study, which was not significantly different

from the 0.4% found for normal controls. Evidence that the

increase in CD was opiate induced rather than a result of other

environmental factors was provided in cytogenetic studies in

which street opiates or morphine were given to rhesus monkeys

(Fischman et al. 1983). As with the human addicts, there was a

significant increase in CD in the opiate treated monkeys as

compared with controls, proving that the CD increase was not

merely ascribable to the human addict milieu.

TABLE 1

INDIVIDUAL VARIABILITY IN DRUG ADDICTS FOLLOWED FOR

8 WEEKS IN METHADONE TREATMENT

Cells with Chromosome Breakage

Cases

1

PreMethadone

1

24 hours 8 weeks

Post- Methadone

Methadone Maintenance

1 5

2

3

4

5

6

7

8

9

10

4 1

1 0

1 1

1 6

5 0

0 1

0 6

2 6

3 6

1

1

1

2

0

3

2

5

2

A second method of examining the question of chromosome damage

is by the study of sister chromatid exchange frequencies (SCE).

SCE and CD are known to be the products of independent

mechanisms that result from different types or conditions of DNA

damage (Galloway and Wolff 1979; Lin and Wertelecki 1982). SCE

occurs with a much higher frequency than CD, and such analysis

has become established as a rapid, sensitive, and quantitative

assay to determine genetic damage (Shafer 1982). It is evident

from the literature that: 1) there are individual differences in

baseline frequency among people (Crossen 1982); 2) there is a

hereditary component in SCE formation indicated by significant

sib-sib and parent-child, but not parent-parent, correlations in

SCE base levels (Cohen et al. 1982); 3) there are increased SCE

base levels or specific SCE sensitivities in such genetic

diseases as Bloom's syndrome, xeroderma pigmentosum, or

Fanconi's anemia (Ray and German 1982); 4) there are

significantly elevated rates of SCE after in Vivo exposure to

13
known mutagens (Hansteen 1982); and 5) increased SCE induction

in vivo is significantly correlated with later carcinogenesis or

transformation (Popescu and Dipaolo 1982; Raposa 1984). As to

when such effects may occur in the cell cycle, SCE can only be

induced by treatments prior to completion of S phase

replication, while CD can also be induced by G2 treatments

with mutagens such as X-ray and radiomimetic agents.

Figure 1 is a histogram of SCE base levels for 47 heroin addicts

and 39 controls (Shafer et al. 1983). There were differences in

base level counts among individuals scored as SCEs/cell in the

chromosomes of about 15 metaphase cells per individual. In the

opiate addict population, the mean ranged from approximately 8.5

to 20.5 with an overall mean of 13.82 ± 4.76 SCE/cell. For

controls, individual mean SCE scores ranged from 5.5 to 15.2

with an overall mean score for controls of 11.16 ± 3.97.

Analysis of variance indicates a highly significant difference

between the opiate and control groups (F = 22.00, p 0.001).

Individual differences were observed in the frequency of SCE not

only when opiate addicts were compared with controls at base

levels, but also when the lymphocytes from both populations were

exposed to far ultraviolet light (far UV) at G1 and S in the

cell cycle. Figure 2 shows a histogram of SCE counts at base

level as well as after exposure to far UV (14J/m2 with

Morowitz correction) at G1 (18 hours before harvest) and at S

(12 hours before harvest). When base level and UV induced SCE

frequencies are compared for individual subjects, the data

demonstrate that interindividual variability plays a significant

role in SCE responses to far UV stress. Analysis of variance

with repeated measures (to simultaneously and separately

compared SCE means at base level and at the two treatment points

for each individual) as well as use of the nonparametric

Mann-Whitney U test confirmed that the SCE frequencies among

heroin addicts were significantly higher than controls and that

the increase at base level was paralleled by increases of

comparable magnitude with UV treatments at these time points.

Our studies on DNA repair synthesis (DRS) induced by far UV

likewise demonstrate individual response differences (Madden

et al. 1979). DES is defined as a mutagen induced increase in

the incorporation. of thymidine into all precipitable cellular

DNA beyond that incorporated by semiconservative replication.

DRS is presumptively an indicator of DNA excision repair and as

such has been used to identify such genetic defects as xeroderma

pigmentosum (Cleaver and Bootsma 1975). We, therefore,

investigated repair of far UV damage to determine whether there

was etiologic evidence to explain the observed increase in

chromosome damage among opiate addicts and the decrease in such

damage in many long-term methadone patients.

DNA repair synthesis fluence curves for different subjects

generally show a linear increase in thymidine incorporation with

increases in far UV doses up to 15 to 20 J/m2, but a few

14
FIGURE 1

Distribution histogram of SCE base levels in heroin and

control subjects based on all subjects analyzed. The

difference between the means was highly significant and the

variances within each group were statistically similar.

15
FIGURE 2

Distribution histogram of SCE levels for those heroin and

control subjects with data for all three treatment points:

base level, and UV at 12 and 18 hr. While significant

differences were most pronounced at the base level and at the

Gl UV treatment, even the slight difference in the means of

the early 5 UV treatment is supported by the data

distribution.

16
individuals reach maximal incorporation at <5 J/m2 (poor

repairers), while others are saturated only at doses of >50

J/m2 (super repairers). The repair capacity is defined as the

plateau level of the curve (i.e., the maximum measurable

thymidine incorporated). The control subjects demonstrated a

marked individual response to far UV, with a mean value of

14,700 dpm/A260 and a standard deviation of ±9,700 dpm/Al60.

This result supported the hypothesis of significant variability

in the capacity of leukocytes of individuals in the general

population to handle fluences of far UV. Variables such as age,

sex, or alcohol use did not significantly contribute to the

variance, but tobacco use significantly lowered the mean DRS

score to 10,300 dpm/A260, primarily as a result of the loss of

high repairers from the group. In contrast, heroin addiction

produced an even greater decrease in the mean of far UV induced

repair in comparison to controls as evidenced by a shift in the

maximal DRS response to largely the poor and low median portion

of the curve (X = 4,300 dpm/A260 ± 4,200). After long-term

methadone treatment (X = 4.8 years), there was no significant

difference between patients in treatment and controls

(X = 13,200 dpm/A260 ± 9,300). These data imply a correlation

between DNA repair and chromosome damage levels. When DNA

repair level is depressed, there is an increased frequency in

cytogenetic effects, while the cytogenetic damage is depressed

when DNA repair returns to more normal levels.

Based on the cytogenetic and DNA repair findings, we conducted

immunologic explorations to more specifically define the

leukocyte cell type or types involved in these repair and

cytogenetic changes (McDonough et al. 1980, 1981; Donahoe et al.

1985a). While there were no significant changes observed in B

lymphocytes or total white blood cell count, the T cell values

decreased significantly from approximately 69% found in the

control population to about 33% in the addict population (figure

3). The addicts in the study were on street heroin almost

exclusively for approximately 10 years. For the long-term

methadone patients, the E rosette frequency was increased to

56.7%. It is clear in both the addict and methadone populations

that there is a great diversity in individual response. When

viewed over time, as seen in figure 4, the data show that on the

average the E rosette depression becomes more severe the longer

the addict uses street opiates, but, as seen in the outliers,

this does not totally account for the variability in rosetting.

Similarly, individual differences are displayed in figure 5

regarding the effect of methadone therapy on the percent of

circulating E-rosette forming lymphocytes. This somewhat

unpredictable response to opiates and therapy was also reported

by Brown and colleagues (1974) in their study of mitogen induced

growth of lymphocytes from patients followed longitudinally

during methadone therapy.

Kinetic assay studies to assess opiate effects on rates of T

cell E-rosetting indicate that a major biological effect of the

opiates relates to their ability to modulate expression of

17
FIGURE 3

Histogram of the percent sheep erythrocyte (E) rosettes

formed by lymphocytes from control subjects, heroin addicts,

and methadone clients.

18
FIGURE 4

The effect of years of street heroin use on the percent

E rosettes formed by lymphocytes from addicts. The line shown

represents the best regression line which can be fitted to

the data.

19
FIGURE 5

The effect of years of methadone use on percent E rosettes

formed by lymphocytes from methodone clients. The great

variability seen can arise both from variability in the

previous street drug history of the client (type of opiate,

average daily dose, length of time addicted, individual

response to street heroin) and from the individual response of

the client to the methadone. While there is a general return

to control percents of E rosettes, a few subjects show

pronounced rosette depressions even after years in therapy.

Because of the rigorous screening of these clients for illicit

drug use during this period, these outliers cannot be ascribed

to illicit drug use.

20
regulatory proteins on the T cell membrane (Madden et al.

1983). We also found that opiate addiction associates with

altered expression of T cell antigenic markers which may occur

through opiate modulated enhancement of suppressor T cells,

resulting in a depression of Th/Ts ratio in long-term addicts

(Donahoe et al. 1985b). The observations that opiates have the

ability to modulate surface proteins/receptors on T cells

support the possibility that T cell constituencies among heroin

addicts are one of the bases for individual variation in the

expression of cytogenetic and DNA damage seen in our addict

populations. With the ability to secure relatively pure T cell

populations, it is now possible to investigate the genetic

aspects of such potential differences.

Our cytogenetic, biochemical, and immunologio assays provide

evidence of significant alterations in users of street opiates

as compared to controls, as well as individual differences in

response to street opiates and individual levels of amelioration

of these alterations over time with methadone. As indicated in

the introduction to this chapter, one method of genetic analysis

in humans, particularly with assays that apparently are not

directly under sociocultural influences, is based on concordance

rates in monozygotic (MZ) and dizygotic (DZ) twin partners.

Monozygotic twins are genetically alike while dizygotic twins

are no more similar than pairs of siblings in a family. In his

presidential address delivered before the Society for

Psychophysiological Research, David Lykken (1982) related that

human geneticists have concentrated on two basic mechanisms of

genetic transmission. He noted that mendelian mechanisms depend

on the presence or absence of a particular allele at a specific

gene locus, while the galtonian mechanisms are concerned with

polygenic transmission in which the value of a metrical trait

such as height is apparently determined by the additive action

of a number of genes at different loci. Dominance (between

alleles at one locus), simple epistasis (a type of dominance

between two genes at different loci), and polygene epistasis (a

nonadditive configuration resulting from interactions among more

than two independent or partly independent genes) may modify

polygenic transmission. In particular, the latter, while

genetic, may not run in a simple manner in families. To

determine whether or not such complications occur in responses

to and predilection for opiate use in humans, comparative twin

differences with regard to protein modulation or receptor number

on the T cell membrane between concordant and discordant MZ and

DZ twins may be a most fruitful approach to an examination of

the genetic aspects of this disorder.

21
REFERENCES

Brown, S.M.; Stimmel, B.; Taub, R.N.; Kochwa. S.; and Rosenfield, R.E.

Immunological dysfunction in heroin addicts. Arch Intern Med 134:1001-

1004, 1974.

Cleaver, J.E., and Bootsma, D. Xeroderma pigmentosum: Biochemical and

genetic characteristics. Annu Rev Genet 9:19-38, 1975.

Cohen, M.M.; Martin, A.O.; Ober, C; and Simpson, S.J. A family study of

spontaneous SCE frequency. Am J Hum Genet 34:294-300, 1982.

Crossen, P.E. Variation in the sensitivity of human lymphocytes to DNA

damaging agents measured by SCE frequency. Hum Genet 60:19-23, 1982.

Donahoe, R.M.; Madden, J.J.; Hollingsworth, F.; Shafer, D.A.; and Falek, A.

Morphine depression of T cell E-rosetting: Definition of the process. Fed

Proc 44:87-91, 1985a.

Donahoe, R.M.; Nicholson, J.K.A.; Madden, J.J.; Hollingsworth, F.; Falek, A.;

Shafer, D.A.; Gordon, D.; and Bokos, P. Drug abuse, leukocyte antigenic

markers and AIDS. In preparation, 1985b.

Eidelberg, E.; Ersparnir, R.; Kreinick, C.J.; and Harris, J. Differences in

locomotor activity, tolerance and dependence in response to morphine among

four inbred strains of mice. Eur J Pharmacol 32:329-336, 1975.

Eriksson, K. and Kiianmaa, K. Genetic analysis of susceptibility to morphine

addiction in inbred mice. Ann Med Exp Biol Fenn 49:73-78, 1971.

Falek, A., and Hollingsworth, F. Heroin and chromosome damage. Arch Gen

Psychiatry 37:227-228, 1980.

Falek, A., and McFadden, I.J. Cytogenetic follow-up of patients in a methadone

program: A pilot study. In: DuPont, R.I., and Freeman, R.S., eds. Fifth

National Conference on Methadone Treatment. Vol. 2. New York: National

Association for Prevention of Addiction to Narcotics, 1973. pp. 695-703.

Falek, A.; Jordan, R.B.; King, B.J.; Arnold, P.J.; and Skelton, W.D. Human

chromosomes and opiates. Arch Gen Psychiatry 27:511-515, 1972.

Fischman, H.K.; Roizan, L.; Moralishvili, E.; Albu, P.; Ross, D.; and Rainer, J.D.

Clastogenic effects of pregnant monkeys and their offspring. Mutat Res

118:77-89, 1983.

Galloway, S.M., and Wolff, S. The relation between chemically induced SCEs

and chromatid breakage. Mutat Res 6:297-307, 1979.

Goodwin, D.W. Genetic component of alcoholism. Annu Rev Med 32:93-99,

1981.

Grumet, G.W. Identical twins discordant for heroin abuse: Case report. J Clin

Psychiatry 44:457-459, 1983.

Gusella, J.F.; Wexler, N.S.; Conneally, P.M.; Naylor, S.L.; Anderson, M.A.; Tanzi,

R.E.; Watkins, P.C.; Ortina, K.; Wallace, R.M.; Sakaguchi, A.Y.; Young, A.B.;

Shoulson, I.; Bonilla, E.; and Martin, J.B. A polymorphic DNA marker

genetically linked to Huntington’s disease. Nature 306:234-238, 1983.

Hansteen, I.L. SCE as a monitor of industrial and environmental toxins. In:

Sandberg, A.A., ed. Sister Chromatid Exchange. New York: A.R. Liss, 1982.

pp. 675-698.

Lin, M.S.. and Wertelecki, W. Evidence that sister chromatid exchanges and

chromatid breaks are two independent events. Chromosome 85:413-419, 1982.

Liston, E.H.; Simpson, J.H.; Jarvik, L.F.; and Guthrie, D. Morphine and

experimental pain in identical twins. In: Gedda, L.; Parisi, P.; and Nance,

W.E., eds. Twin Research: Epidemiological and Clinical Studies. Vol. 3.

New York: A.R. Liss, 1981. pp. 105-116.

Lykken, D.T. Research with twins: The concept of emergenesis.

Psychophysiology 19:361-373, 1982.

22
Madden, J.J.; Falek, A.; Shafer, D.A.; and Glick, J. Effects of opiates and

demographic factors on DNA repair synthesis in human leukocytes. Proc Nat1

Acad Sci USA 76:5769-5773, 1979.

Madden, J.J.; Donahoe, R.M.; Smith, I.E.; Eltzroth, D.C.; Hollingsworth, F.;

Falek, A.; Bokos, P.J.; and Shafer, D.A. Kinetics of erythrocyte rosette

formation with T lymphocytes from drug addicted subjects. In: Harris, L.S.,

ed. Problems of Drug Dependence, 1982. National Institute on Drug Abuse

Monograph 43. DHHS Pub. No. (ADM) 83-1264. Washington, D.C.: Supt. of

Docs., U.S. Govt. Print. Off., 1983. pp. 375-379.

McDonough, R.J.; Madden, J.J.; Falek, A.; Shafer, D.A.; Pline, M.; Gordon, D.;

Bokos, P.; Kuehnle, J.C.; and Mendelson, J.H. Alteration of T and null

lymphocyte frequencies in the peripheral blood of human opiate addicts: In

vivo evidence of opiate receptor sites on T lymphocytes. J Immunol

125:2539-2543, 1980.

McDonough, R.J.; Madden, J.J.; Rosman, H.S.; Falek, A.; Wenger, N.K.; Shafer,

D.A.; Bokos, P.J.; Kuehnle, J.C.; and Mendelson, J.H. Opiate inhibition of

sheep erythocytes binding to T lymphocytes: Reversal by naloxone and cyclic

nucleotides. In: Harris, L.S., ed. Problems of Drug Dependence, 1980

National Institute on Drug Abuse Monograph 34. DHHS Pub. No. (ADM)

1058. Washington, D.C.: Supt. of Docs., U.S. Govt. Print. Off., 1981. pp.

159-165.

Nichols, J.R., and Hsiao, S. Addiction liability of albino rats: Breeding for

quantitative differences in morphine drinking. Science 157:561-563, 1967.

Popescu, N.C., and Dipaolo, J.A. The relevance of sister chromatid exchange to

the induction of neoplastic cell transformation. In: Sandberg, A.A., ed.

Sister Chromatid Exchange. New York: A.R. Liss, 1982. pp. 425-460.

Raposa, T. SCE induction by cytostatics and its relation to iatrogenic

leukemogenesis. In: Hollaender, A., and Tice, R., eds. Sister Chromatid

Exchanges: 25 Years of Experimental Research. New York: Plenum, 1984.

pp. 859-884.

Ray, J.H., and German, J. Sister chromatid exchange in the chromosome

breakage syndromes. In: Sandberg, A.A., ed. Sister Chromatid Exchange.

New York: A.R. Liss, 1982. pp. 353-377.

Shafer, D.A. Alternate replication bypass mechanisms for sister chromatid

exchange formation. In: Sandberg, A.A., ed. Sister Chromatid Exchange.

New York: A.R. Liss, 1982. pp. 67-98.

Shafer, D.A.; Falek, A.; Madden, J.J.; Tadayon, F.; Pline, M.; Bokos, P.J.;

Kuehnle, J.C.; and Mendelson, J. Parallel increases in SCE’s at base level and

with UV treatment in human opiate addicts. Mutat Res 109:73-82, 1983.

Vesell, E.S. Pharmacogenetic perspectives: Genes, drugs and disease.

Hepatology 4:959-965, 1984.

ACKNOWLEDGMENT

This research is supported by the National Institute on Drug Abuse grant DA01451.

23
AUTHORS

Arthur Falek, Ph.D.

John J. Madden, Ph.D.

David A. Shafer, Ph.D.

Robert M. Donahoe, Ph.D.

Department of Psychiatry and Biochemistry

Emory University School of Medicine

Box AF

Atlanta, Georgia 30322

and

Human and Behavior Genetics Research Laboratory

Georgia Mental Health Institute

1256 Briarcliff Road

Atlanta, Georgia 30306

24
Pharmacogenetic Approaches to

the Prediction of Drug Response

Elliot S. Vesell, M.D.

As the chapters in this monograph illustrate, numerous genetic and

environmental factors can influence the metabolism and effects of

ethanol. Despite this recent progress in identifying these factors,

the underlying cause or pathogenesis of alcoholism and of abuse of

other substances remains illusive and ill defined.

It follows that the primary products of putative "causative" genes

are unknown. The genetic and environmental factors thus far

Investigated appear to be peripheral rather than primary with

respect to causation of the underlying behavioral aberration

associated with drug abuse. So imprecise are our present concepts of

specific genetic causes of drug abuse that recommendations for

research in this area are hazardous and difficult to justify if

strict criteria are applied.

PHARMACOGENETICS

Pharmacogenetics is defined as the study of specific genetic factors

that cause large interindividual variations in response to some

drugs. Pharmacogenetic investigations would not be likely to reveal

the pathogenesis of substance abuse, but rather might explain why

certain subjects who abuse alcohol and other drugs may be

particularly susceptible to injury and toxicity, whereas others who

take larger doses resist damage and do not develop the disease (Kalow

1962; Motulsky 1964; LaDu 1972; Vesell 1973; Kalow 1982; Propping

1978). For example, racial differences in an isozyme of aldehyde

hydrogenase explain the greater susceptibility of Oriental subjects

to such effects of ethanol as facial flushing; this example

illustrates the capability of pharmacogenetics to elucidate the

basis of interindividual variations in susceptibility to drug

toxicity and was discovered by a distinguished investigator in

pharmacogenetics, H. Werner Goedde (Goedde et al. 1979, 1984; Harada

et al. 1980; Agarwal et al. 1981). Another more controversial

example of pharmacogenetic progress in this area is the possible

association of the immunogenetic markers HLA-B40 and DRW9 with

alcoholic cirrhosis of the liver in Japanese subjects (Miyamoto et

al. 1983). Although certain HLA haplotypes are highly associated

with several disorders of a fundamentally immunologic nature, such

25
as ankylosing spondylitis, lupus erythematosus, and rheumatoid

arthritis, several previous studies failed to observe such

associations of HLA haplotypes with alcoholic cirrhosis, a disease

not generally considered to be due to an immunologic abnormality.

However) recently, several immunologic abnormalities have been

described in patients with alcoholic cirrhosis (Sorrel1 and Leevy

1972; Tannenbaum et al. 1969; Tsuchimoto 1982).

In the area of abuse of drugs other than alcohol, no specific pharmacogenetic entities have thus far been identified. However, several

observations suggest that pharmacogenetic mechanisms might be

operative and might contribute to large lnterindividual variations

already documented to occur in the metabolism of methadone (Kreek

1973) and possibly in the metabolism of cocaine and heroin, two other

commonly abused drugs whose elimination from the body also occurs

mainly through biotransformation (Goodman and Gilman 1980).

In human subjects, pharmacokinetic values are more conveniently and

reliably measured than pharmacodynamic ones. Pharmacodynamic

variations undoubtedly also exist, and some of these may be

maintained by genetic mechanisms. However, it is not yet feasible to

obtain precise pharmacodynamic measurements noninvasively in human

subjects. Much technical progress is necessary before pharmacodynamic measurements can be gathered in humans reliably, safely, and

noninvasively.

The following review of pharmacogenetic progress and methodology is

offered to stimulate and suggest analogous studies on drugs of

abuse. It is readily acknowledged that formidible methodological

problems are posed by adapting to drugs of abuse these

pharmacogenetic approaches based on the administration of single

safe doses of various prescription drugs to normal subjects under

carefully controlled environmental conditions. Results of similarly

designed studies on drugs of abuse in addicts might be

uninterpretable because of confounding by numerous environmental

perturbations, including the smoking of cigarettes and/or marijuana,

nutritional variations, and intake of other drugs such as ethanol.

Ethical considerations render objectionable the administration to

unaddicted subjects of drugs at dosage levels usually ingested by

drug abusers, Other approaches would have to be taken in such normal

subjects. Possibilities include administration of tracer doses of

14C- or 13C-labeled drugs or growth of normal cells in culture to

investigate their pharmacokinetic and/or pharmacodynamic responses

to various drugs of abuse.

DYNAMIC INTERACTIONS AMONG THE GENETIC AND ENVIRONMENTAL FACTORS

THAT MODIFY DRUG RESPONSE

Unlike the numerous problems that beset studies to identify factors

that cause pharmacokinetic variations among subjects after

administration of drugs of abuse, much progress has been achieved in

investigating such factors that influence the disposition of

prescribed drugs eliminated primarily through hepatic metabolism.

This progress has occurred largely through availability of normal

26
human subjects who can safely ingest single doses of various model

drugs, such as antipyrine, aminopyrine, amobarbital, debrisoquine,

sparteine, mephenytoin, acetaminophen, theophylline, etc.

Antipyrine has proven particularly fruitful in disclosing numerous

environmental factors that can interact with each other as well as

with genetic factors to determine antipyrine elimination rates and

plasma antipyrine concentrations. The design of figure 1 suggests

dynamic interaction of such factors. In contrast to addicts, the

normal subjects used to identify the factors shown in figure 1 were

all carefully defined and uniform with respect to almost every

factor in the outer circle. This standardization of prevailing

environmental conditions among the subjects permitted each variable

to be investigated independently of all others. Dose-response

curves could be generated, relating increments of a specific factor

to subsequent changes in antipyrine disposition. This model drug

approach is exceedingly sensitive in detecting factors that can

affect drug disposition, mainly because each subject serves as his

or her own control (Vesell 1979). Use of subjects as their own

control eliminates extraneous contributions from many genetic and

environmental perturbations that occur in the alternative experimental design where the control and experimental subjects differ,

forming two separate groups that are compared. Nevertheless, like

all laboratory tests, the model drug approach has limitations.

Occasionally, it has disappointed some investigators whose

expectations from it were unrealistic or unwarranted (Vesell 1979).

The main limitation as well as advantage of the model drug approach

depends directly on the pharmacologic characteristics of the drug

selected for administration. With respect to antipyrine, the

principal advantage is that antipyrine decay in saliva and plasma

reflects directly antipyrine metabolism in the liver by three

separate cytochrome P-450 mediated monooxygenases. Since antipyrine

is absorbed completely from the gastrointestinal tract, distributes

in “total body water,” is not bound significantly to tissue or plasma

proteins, and is not eliminated to an appreciable extent by renal

mechanisms, antipyrine decay in plasma and in saliva sensitively

reflects its rate of hepatic biotransformation. Furthermore, since

antipyrine is a drug with low hepatic extraction, its elimination is

independent of hepatic blood flow. No other model drug yet proposed

enjoys so many advantages as an indicator of hepatic drugmetabolizing capacity. Nevertheless, antipyrine elimination can

only reflect the activities of a few of the many hepatic drugmetabolizing enzymes. Other such enzymes not directly involved in

antipyrlne biotransformation may be influenced to a greater or

lesser extent than the antipyrine-metabolizing enzymes by a

particular genetic or environmental factor. Hence, these effects

may not be accurately disclosed with antipyrine. For example, the

metabolism of debrisoqulne and sparteine occurs through a different

cytochrome P-450 mediated monooxygenase. For this reason, if many

hepatic drug-metabolizing enzymes are to be investigated

simultaneously, several model drugs in addition to antipyrine need

to be used.

27
FIGURE 1

This circular design suggests the multiplicity of either wellestablished or suspected host factors that may influence drug

response in man. A line joins all such factors in the outer circle

to indicate their close interrelationship. Arrows from each factor

in the outer circle are wavy to indicate that effects of each host

factor on drug response may occur at multiple sites and through

different processes that include drug absorption, distribution,

metabolism, excretion, receptor action, and combinations thereof.

28
PHARMACOGENETICS: GENE-DRUG INTERACTIONS THAT PRODUCE DISEASE

Pharmacogenetics, the study of genetically determined variations in

drug response, focuses on interindividual variations because they

are frequently encountered and often present clinical problems. For

example, the extent of these intersubject variations in response can

range from threefold to fortyfold, depending on the particular drug

and population. For drugs with low therapeutic indices, each

patient’s dosage regimen needs to be carefully adjusted to avoid

drug toxicity or undertreatment.

Since many drugs are plant alkaloids, it is not surprising that

genetic variations also exist in response to some dietary

constituents and that some inborn errors of metabolism are worsened

by dietary constituents. For example, galactosemia, phenylketonuria, Wilson’s disease, celiac disease, and lactose intolerance

due to lactase deficiency are exacerbated by foods containing

galactose, phenylalanine, copper, gluten, and milk, respectively.

Also, in several other inborn errors of intermediary metabolism,

crises can be precipitated by certain drugs. For example, in

diabetes mellitus, gout, and several forms of porphyria,

administration of adrenal glucocorticolds, thiazide diuretics, and

barbiturates, respectively, should be avoided.

Another group of inborn errors of metabolism exists in which the

aberrant (mutant) protein participates directly, rather than

indirectly, in drug disposition or action. These entitles, the main

focus in the past of the field of pharmacogenetics, include several

rare monogenically transmitted conditions (acatalasla, atypical

plasma cholinesterase, phenytoin sensitivity, and warfarin

resistance) as well as some commonly occurring monogenically

transmitted conditions (slow acetylation of isoniazid and other

drugs and glucose-6-phosphate dehydrogenase deficiency). Abundantly

reviewed in the past (Kalow 1962; Motulsky 1964; Vesell 1973; Vasell

and Penno 1983; LaDu 1972; Weinshilboum and Vesell 1984), these

pharmacogenetic conditions will not be described in detail here.

Each exemplifies how genetic constitution can determine drug

toxicity. In general, toxicity arises in a genetically susceptible

individual from accumulation of parent drug due to a block in its

biotransformation to inactive metabolites. Transmitted in classical

mendelian fashion by alleles at a single but different locus, each of

these metabolic blocks is described as monogenic. In most cases, a

double dose of a mutant gene is required to produce the defect

(recessive phenotype), but rarely, as in phenytoin toxicity, does a

single gene seem to suffice (dominant phenotype).

In 1968, the influence of genetic factors on drug response and

toxicity was extended well beyond the aforementioned monogenic

conditions to other entities encompassing different drugs (Penno et

al. 1981; Vesell and Page 1968a, 1968b, 1968c). These new

pharmacogenetic conditions involved drugs characterized by: (1)

extensive hepatic metabolism through cytochrome-P-450-dependent

monooxygenases and (ii) large interindividual variations in rates of

elimination. Almost a dozen different drugs were given to normal

29
uninduced twins who were nonsmokers, otherwise unmedicated, and not

chronic consumers of ethanol. These drugs included antipyrine

(Penno et al. 1981; Vesell and Page 1968a), phenylbutazone (Vesell

and Page 1968b), bishydroxycoumarin (Vesell and Page 1968c),

ethanol (Vasell et al. 1971). halothane (Cascorbi et al. 1971).

nortriptyline (Alexanderson et. al. 1969), amobarbital (Endrenyi et

al. 1976), phenytoin (Andreasen et al. 1973), salicylate (Furst et

al. 1977), and tolbutamide (Scott and Poffenbarger 1979).

Genetic control over extensive interindividual variations in rates

of elimination of these drugs was indicated by larger

pharmacokinetic differences within dizygotic than within monozygotic

twins (Penno et al. 1981; Vesell and Page 1968a, 1968b, 1968c; Vesell

et al. 1971; Cascorbi et al. 1971; Alexanderson et al. 1969; Endrenyi

et al. 1976; Andreasen et al. 1973; Furst et al. 1977. Scott and

Poffenbarger 1979). Intraindividual variations were small compared

to interindividual variations in these carefully selected, uninduced

twins living under uniform environmental conditions. Although such

observations supported a genetic basis for the large interindividual

variations in the disposition of these drugs, the precise mendelian

mode of transmission of these factors could not be elucidated just

from the twin studies on parent drugs. Thus, when pedigree analysis

was attempted solely on the basis of kinetic studies performed on the

parent drug, rather than on principal metabolites, the mode of

transmission of interindividual variations for bishydroxycoumarin

(Motulsky 1964), phenylbutazone (Whittaker and Price Evans 1970),

and nortriptyline (Asberg et al. 1971) was either unclear or else

more compatible with a polygenlc than a monogenic mechanism. In

light of the current information discussed below, a polygenic

attribution is misleading because more recent examinations of

individual metabolic pathways have disclosed monogenic transmission.

Since some twins participated in several of these studies, it was

possible to determine whether their rates of biotransformation of

different drugs were closely correlated. Although some correlations

occurred, rates of clearance of most drugs investigated were

independent of one another (Vesell and Page 1968c). This result was

notable for several reasons. It suggested that the twin studies had

disclosed several discrete genetic entities, rather than one or two

that determined large variations in the metabolism of numerous

drugs. Furthermore, the existence of separate genetic entities,

each involving a different molecular form (isozyme) of hepatic

cytochrome P-450, agreed with simultaneously emerging evidence for

extensive heterogeneity of this hepatic hemoprotein (Alvares et al.

1969; Lu and Levin 1974; Nebert et al. 1982; Ryan et al. 1975; Wang

et al. 1980).

THE DEBRISOQUINE POLYMORPHISM

In 1977, a landmark report disclosed that approximately 7% to 9% of

the population in the United Kingdom were genetically slow

metabolizers of the antihypertensive drug debrisoquine (Mahgoub et

al. 1977). After a 10-mg oral dose of debrisoquine, the phenotypes

of extensive metabolizers (EMS) and poor metabolizers (PMs) were

30
distinguished in an 8-hour urine specimen by the ratio between the

percent of the dose eliminated as the parent drug and the percent

removed as its principal metabolite, 4-hydroxydebrisoquine. A ratio

of 10 or more characterized deficient (poor) metabolizers of

debrisoquine. This phenotype was conferred by a double dose of an

autosomal gene. In patients treated with debrisoquine, toxicity

occurred predominantly among deficient metabolizers. Thus, two

alleles at a single genetic locus largely determine susceptibility

to adverse effects from this drug.

Using sparteine rather than debrisoquine, Eichelbaum’s group in Bonn

reported a monogenically controlled variation which now appears to

be identical in many respects to the debrisoquine polymorphism

(Eichelbaum et al. 1979). In German subjects, the frequency of this

inability to metabolize sparteine approximated that for debrisoquine

in the United Kingdom. This meant that in the United Kingdom and

Germany, for every 100,000 patients receiving usual doses of

debrisoquine or sparteine, approximately 7,000 to 9,000 would be at

risk of toxicity due to drug accumulation.

The group from the United Kingdom extended the soope of their

discovery by suggesting a panel drug approach (Idle et al. 1979):

slow metabolizers of debrisoquine were shown to be incapable of

blotransforming several other drugs as well. Thus, deficient (poor)

metabolism of debrisoquine was demonstrated to be associated with

poor metabolism of nortriptyline (Bertilsson et al. 1980; Mellstrom

et al. 1981); phenacetin (Sloan et al. 1978); phenformin (Shah et al.

1980); certain ß-adrenoceptor blocking drugs such as metoprolol,

alprenolol, bufuralol, timolol (Alvan et al. 1982), and propranolol

(Shah et al. 1982a); guanoxan (Sloan et al. 1978); perhexiline (Shah

et al. 1982b); encainide (Wang et al. 1984); and possibly Dpenicillamine (Panayi et al. 1983). The genetic locus controlling

debrisoquine metabolism could be designated "a gene of multiple

effect." Since genetically deficient metabolizers of debrisoquine

are also at increased risk of toxicity from decreased

biotransformation of approximately 21 drugs, a clinically useful

predictive teat based on drug-oxidizing capacity seems to be

indicated. In such genetically predisposed subjects and certain of

their sibs, doses of these drugs could then be appropriately reduced

or alternative drugs selected that are metabolized by other pathways

of hepatic drug oxidation, thereby avoiding the dose-dependent

adverse reactions that occur moat frequently in the deficient

phenotype. The metabolic conversion and pharmacological actions of

numerous drugs are affected to varying extents and in differing ways

by the debrisoquine polymorphism. Thus, the precise clinical

consequences of the PM phenotype relate directly to the special

properties of the particular drug administered (Idle et al. 1983):

(i) For drugs like the ß-adrenergic blockers whose hepatic extraction is high and whose elimination is therefore bloodflow dependent, oral bioavailability would be enhanced

perhaps fourfold or fivefold in PMS compared to EMs. The

clinical consequences in the PM phenotype consist of

exaggerated pharmacological responses to such drugs.

31
(ii) For drugs whose elimination requires metabolic oxidation,

drug accumulation in PM phenotypes could produce toxicity,

as in the neuropathy and liver damage after perhexilene

that occur primarily in the PM phenotype (Shah et al.

1982b).

(iii) For the few drugs that require conversion to a metabolite

before becoming pharmacologically active, such as encainide

Wang et al. 1984), where oxidative O-demethylatlon to

desmethyl-encainide is necessary for antiarrhythmic

effects, the PM phenotype will be resistant to therapeutic

effects and, hence, deprived of benefit from the drug.

(iv) For drugs such as phenacetin, the PM phenotype may, due to

blockage in one oxidative pathway, develop toxicity due to

enhanced production of a toxic metabolite via a minor

pathway. The PM phenotype is much more liable to toxicity

then the EM phenotype because in the PM phenotype, when Odeethylation of phenacetin to acetaminophen is blocked,

much more phenacetin than normal is subject to the aromatic

oxidation and deacetylation to form hepatotoxic end

hemolytic p-phenetidins (Sloan et al. 1978).

This list of consequences could be extended, but these examples

suffice to illustrate the wide variety of clinical outcomes to be

anticipated from polymorphic oxidation and how these clinical

sequelae closely reflect the special metabolic profile and

pharmacologic characteristics of the particular drug administered.

Due to the considerable clinical consequences of polymorphic

oxidation of several drugs, Idle et al. (1983) suggested that when

administration of some of these commonly used drugs with low therapeutic indices is contemplated, patients should first be phenotyped

to permit efficient and safe tailoring of dose to their individual

metabolic requirements. To perform this service, regional centers

were advocated (Idle et al. 1983). Furthermore, in new drug development where preclinical studies revealed metabolic oxidation as a

significant part of the total elimination process, this fact could

contraindicate further development of the drug or at least its use in

PM phenotypes (Idle et al. 1983). While this suggestion may seem

stringent, it could represent a theoretical possibility for a new

drug that produces very high toxicity or lethality in the PM

phenotype. Perhaps most such potentially dangerous new drugs never

reach clinical trial, obviating the need for phenotyping. Jack et

al. (1983) term "unwise" the suggestion that polymorphic or other

types of variable hepatic metabolism of a new drug contraindicate

its use or further development. They cite, to defend their position,

wide benefit from propranolol, isoniazid, phenytoin, and many

tricyclic antidepressants, none of which, they state, would have

been marketed under such restrictive policy. Finally, they consider it

unnecessary to establish regional centers to phenotype patients

before they receive polymorphically oxidized drugs. They believe

that most physicians can safely individualize dosage of such drugs

without prior phenotyping.

32
In addition to its numerous clinical applications, the debrisoquine

polymorphism underscores several fundamental principles of pharmacogenetic investigation. For example, the debrisoquine polymorphism

could only have been discovered after development of sensitive highperformance liquid chromatography techniques to detect and quantify

the principal metabolite, 4-hydroxydebrisoquine. In pharmacogenetic

studies, rates of formation of the main metabolites should be

measured, not just elimination rates of parent drugs, particularly

for drugs biotransformed by multiple pathways, because

identification of principal metabolites permits phenotyping of

subjects closer to their primary gene products (Kalow et al. 1977).

Another technical aspect of the debrisoquine story is facilitation

of genetic analysis by availability of a convenient, noninvasive

method, the metabolic ratio, to phenotype subjects. Use of the

metabolic ratio permitted rapid comparison of gene frequencies in

different populations.

These population data, interesting from several points of view,

demonstrate qualitative as well as quantitative variations in the

gene in different groups and countries (Kalow 1984). Not only do

allele frequencies change, but also genetic heterogeneity occurs;

that is, presence of additional alleles at this locus in subjects

from Ghana (Woolhouse et al. 1979, 1983). Comparisons of

debrisoquine phenotypes in five different populations (Canadian

Caucasian, Canadian Oriental, Saudi Arabian, Ghanian, and Nigerian)

suggested greater complexity than initially thought: all five

populations differed from one another in certain respects (see table

1 in Kalow 1984).

Genetic heterogeneity traditionally emerges after meticulous

investigation, in different countries or regions, of a locus that

appears initially, on the basis of limited population sampling, to

possess only two alleles. From the point of view of

pharmacogenetics, greater complexity of the debrisoquine

polymorphism than originally anticipated has several implications.

First, the nature of the alleles that occur commonly for this polymorphism should be characterized in a population and country before

new drugs affected by this polymorphism are widely given, at least on

only a single dosage schedule. Second, even though a screening

procedure to define phenotypes may be satisfactory in a particular

population, it may be unsatisfactory in another population where

different alleles predominate. Specifically, Kalow considers the

approach of phenotyping subjects according to their metabolic ratio

of debrisoquine to 4-hydroxydebrisoquine in a single 8-hour

collection of urine satisfactory in Caucasians but unreliable in

native Africans (Kalow 1984; Inaba et al. 1981). Careful kinetic

studies on the disposition of debrisoquine and its principal

metabolite have been performed in Caucasian PMs and EMs to teat the

validity of the metabolic ratio as a method to phenotype subjects

(Sloan et al. 1983).

Another contribution of the debrisoquine polymorphism has been

stimulation of the search for other genetically controlled

variations of drug oxidation. Extensive heterogeneity of the cyto33
chrome P-450 system also favored the premise that additional polymorphisms of drug oxidation exist. The search has been rewarding.

New polymorphisms, distinct from debrisoquine, have been described

for mephenytoin (Kupfer et al. 1981) and antipyrine (Penno et al.

1981; Penno and Vesell 1983). The antipyrine polymorphism was

uncovered by application of a two-stage method that should enable

still other polymorphisms to be identified.

POLYMORPHIC METABOLISM OF ANTIPYRINE

Even before the discovery in 1983 of a polymorphism of antipyrine

oxidation independent of that for debrisoquine (Penno and Vesell

1983), its existence was suggested by circumstantial evidence. In

the first place, twin studies in 1968 and 1981 revealed genetic

control over large variations not only of antipyrine elimination

(Vesell and Page 1968a), but also of rates of formation "of the three

main antipyrine metabolites (Penno et al. 1981). Second, PM

phenotypes for debrisoquine and sparteine biotransformed antipyrine

normally, not at reduced rates (Bertilsson et al. 1980; Danhof et al.

1981). Third, studies on induction of antipyrine metabolism in rata

suggested that each of its three principal metabolites arose from a

different hepatic cytochrome P-450 isozyme (Danhof et al. 1979;

Inaba et al. 1980).

Analysis of 13 families provided evidence for a commonly occurring

polymorphism of antipyrine metabolism maintained by two codominant

alleles at one or more loci with frequencies of 0.8 to 0.9 and 0.2 to

0.1 (Penno and Vasell 1983). A common genetic mechanism regulating

all cytochrome P-450 isozymes responsible for the three main

antipyrine metabolites was suggested not only by the similar

frequencies for the alleles that control variations in rates of

formation of each antipyrlne metabolite, but also by the significant

correlation among these different rate constants within each subject

(Penno and Vesell 1983). These observations are also oonsistent

with a more complex interpretation that genetic control is

maintained by some regulatory factors that are identical for all

three loci and other such factors that differ at these loci.

Since the method developed to identify the antipyrine polymorphisms

(Penno and Vesell 1983) permits searches for other polymorphisms and

has just been used in our laboratory to define a new polymorphism of

theophylline metabolism, It merits some discussion. The first step

is to measure rate constants of formation of principal metabolites

of a drug or of their partial clearances in a sufficient number of

unrelated subjects (approximately 100) to generate distribution

curves. Without this essential first step, pedigree analysis

becomes uncertain due to the absence of an alternative way that is

both quantitative and objective to determine the phenotype of each

family member. Selection of 100 subjects follows from principles

governing population genetics. Enough unrelated subjects need to be

included in the sample so that a mode represents the rarer phenotype,

either in homozygous or heterozygous form, i.e., the phenotype

controlled by an allele q with a gene frequency of approximately 0.1

(p = 0.9; q = 0.21). If q were much less than 0.1, phenotypes

34
corresponding to q (homozygotes) and 2 pq (heterozygotes) would be

undetectable since this method is not feasible for screening the

many hundreds of subjects that would be required. For example, if q

were 0.01, only 1 in 1,000 subjects would be homozygous at this locus

and only 2 in 100 would be heterozygotes. Some samples of 100

subjects might not contain even one heterozygote. Approximately 1

subject in 100 would be homozygous for an allele q if q = 0.1 (0.12:

but even in the absence of that single subject from several samples

of 100 subjects, the polymorphism would still be readily detected

because 18 of the 100 subjects investigated (2 pq or 2 x 0.9 x 0.1)

would theoretically be heterozygotes and phenotypically distinguishable on distribution curves and probits from the 81 subjects

homozygous (0.92) for the more frequently occurring allele p.

Once constructed on the basis of results in 100 unrelated subjects,

these distribution curves and probits serve to phenotype members of

different families, possibly drawn from index cases at the far ends

of the curve. Transmission patterns of these phenotypes which can be

objectively assigned to each family member are then traced to determine whether they conform to a mendelian mode of inheritance. If so,

this constitutes evidence for, but by no means unequivocally

establishes, a genetic polymorphism of drug oxidation. If patterns

of transmission in families are incompatible with mendelian laws, a

monogenic mechanism is rejected. However, before a monogenic

hypothesis is completely rejected, the family should be rechecked to

assure that the drug-metabolizing capacity of each member was truly

uninduced or uninhibited. For example, in the course of studies on

the antipyrine polymorphism, we encountered only one family that

contradicted a monogenic hypothesis (see figure 2 in Vesell 1984).

On questioning, the mother admitted taking cimetidine; on restudy,

after 3 weeks off cimetidine, she changed her values considerably,

thereby making the transmission pattern in the family consistent

with mendelian expectations.

When assessing the potential clinical significance of a genetic

polymorphism controlling cytochrome P-450 isozymes subject to extensive environmental perturbations, one should recall several points.

First, virtually all cytochrome P-450 isozymes are subject to

environmental perturbation. Differences are mainly in the extent of

induction. Furthermore, polymorphisms that may at first appear

resistant may show, on further study, sensitivity to initially

untested drugs, chemicals, or conditions. Second, additional

alleles not encountered at a genetic locus on early study may appear

later when different populations are tested. These new alleles

might regulate cytochrome P-450 isozymes that are much different in

inductive responsiveness. For these reasons, certain genetic polymorphisms of drug metabolism may possess more clinical significance

and complexity than initially appeared, and the search for more such

polymorphisms should be encouraged.

THERAPEUTIC AND TOXIC DRUG RESPONSES CONTROLLED BY GENES AT HLA LOCI

For reasons as yet unclear, some uncommon alleles at the HLA loci may

be much more strongly associated with certain drug responses than

35
alternative alleles. For example, in schizophrenic patients, HLA-A1

has been reported to be highly associated with a favorable response

to chlorpromazine (Smeraldi et al. 1976; Smeraldi and ScorzaSmeraldi 1976). By contrast, HLA-A2 appeared to be associated with

an unfavorable response. In patients with affective disorders

treated with lithium, HLA-A3 was associated with a high rate of

relapse (Perris et al. 1979).

With respect to adverse reactions to drugs, in patients with

rheumatoid arthritis treated with sodium aurothiomalate, adverse

reactions (particularly proteinuria) occurred 32 times more

frequently in the HLA-DR3 haplotype (Wooley et al. 1980). Also, in

patients with rheumatoid arthritis, agranulocytosis after levamisole

was encountered much more frequently in subjects with the rare

haplotype HLA-B27 (Schmidt and Mueller-Eckhardt 1977). Hydralazineinduced systemic lupus erythematosis occurred two to three times

more frequently in hypertensive patients with HLA-DR4 haplotype who

received the drug (Batchelor et al. 1980). Another genetically

determined phenotype, slow acetylation of hydralazine, also is

associated with increased susceptibility to lupus (Perry et al.

1970). Although the acetylation polymorphism itself does not appear

to be associated with any HLA haplotype (Batchelor et al. 1980),

potential associations between other HLA haplotypes and different

pharmacogenetic polymorphisms remain to be established end merit

study.

OTHER PHARMACOGENETIC AREAS OF INVESTIGATION

Intriguing pharmacogenetic observations that require further study

include increased sensitivity of certain ethnic groups to ethanol

(Wolff 1972), mydriatic agents (Chen and Poth 1929), and

diphenhydramine (Spector et al. 1980). In cultured cells, amplification of specific DNA sequences correlates highly with resistance

to certain cytotoxic drugs, such as adriamycin (Roninson et al.

1984); elaboration of the mechanisms for this gene-environment

interaction requires elucidation of the nature of the proteins

encoded by the amplified DNA. These and other examples indicate the

wide variety of ways in which genetic constitution can influence

drug response. The pharmacogenetic approaches described above could

be applied with the modifications described at the outset of the

chapter to elucidate large interindividual variations that occur in

response to certain drugs of abuse. Probably similar principles

pertain and results of such studies could be informative and

therapeutically useful.

REFERENCES

Agarwal, D.P.; Harada, S.; and Goedde, H.W. Racial differences in

biological sensitivity to ethanol: The role of alcohol

dehydrogenase and aldehyde dehydrogenase isozymes. Alcohol Clin

Exp Res 5:1, 1981.

Alexanderson, B.; Price Evans, D.A.; and Sjoqvist, F. Steady-state

plasma levels of nortriptyline in twins. Influence of genetic

factors and drug therapy. Br Med J 4:764-768, 1969.

Alvan, G.; Von Bahr, C.; Seideman, P.; and Sjoqvist, F. High plasma

36
concentrations of ß-receptor blocking drugs and deficient

debrisoquine hydroxylation. Lancet I:333, 1982.

Alvares, A.P.; Schilling, G.; Levin, W.; Kuntzman, R.; Brand, L.;

and Mark, L.C. Cytochromes P-450 and b5 in human liver

microsomes. Clin Pharmacol Ther 10:655-659, 1969.

Andreasen, P.B.; Froland, A.; Skovsted, L.; Andersen, S.A.; and

Hauge, M. Diphenylhydantoin half-life in man and its inhibition

by phenylbutazone: The role of genetic factors. Acta Med Scand

193:561-564, 1973.

Asberg, M.; Price Evans, D.A.; and Sjoqvist, F. Genetic control of

nortriptyline kinetics in man: A study of relative of propositus

with high plasma concentrations. J Med Genet 8:129-135, 1971.

Batchelor, J.R.; Welsh, K.I.; Mansilla Tinoco, R.; Dollery, C.T.;

Hughes, G.R.V.; Bernstein, R.; Ryan, P.; Naish, P.F.; Aber, G.M.;

Bing, R.F.; and Russell, G.I. Hydralazine-induced systemic lupus

erythematosus: Influence of HLA-DR and sex on susceptibility.

Lancet 1:1107-1109, 1980.

Bertilsson, L.; Eichelbaum, M.; Hellstrom, B.; Save, J.; Schulz, H.-

V.; and Sjoqvist, F. Nortriptyline and antipyrlne clearance in

relation to debrisoquine hydroxylation in man. Life Sci 27:1673-

1677, 1980.

Cascorbi, H.F.; Vesell, E.S.; Blake, D.A.; and Helrich, M. Genetic

and environmental influence on halothane metabolism in twins.

Clin Pharmacol Ther 12:50-55, 1971.

Chen, K.K., and Poth, E.J. Racial differences as illustrated by the

mydriatic action of cocaine, euphthalmine, and ephidrine. J

Pharmacol Exp Ther 36:429, 1929.

Danhof, M.; Krom, D.P.; and Breimer, D.D. Studies on the different

metabolic pathways of antipyrine in rata: Influence of

phenobarbital and 3-methylcholanthrene treatment. Xenobiotica

9:695-702, 1979.

Danhof, M.; Idle, J.R.; Tennissen, M.W.C.; Sloan, T.P.; Breimer,

D.D.; and Smith, R.L. Influence of the genetically controlled

deficiency in debrisoquine hydroxylation on antipyrlne metabolite

formation.

Eichelbaum, M. Spannbrucker, N.; Steinecke, B.; and Dengler, H. J.

Defective N-oxidation of sparteine in man: A new pharmacogenetic

defect. Eur J Clin Pharmacol 18:183-187, 1979.

Endrenyi, L.; Inaba, T.; and Kalow, W. Genetic study of amobarbital

elimination based on its kinetics in twins. Clin Pharmacol Ther

20:701-714, 1976.

Furst, D.E.; Gupta, N.; and Paulus, H.E. Salicylate metabolism in

twins: Evidence suggesting a genetic influence and induction of

salicylurate formation. J Clin Invest 60:32-42, 1977.

Goedde, H.W.; Harada, S.; and Agarwal, D.P. Racial differences in

alcohol sensitivity: A new hypothesis. Hum Genet 51:331-334,

1979.

Goedde, H.W.; Benkmann, H.G.; Kriese, L.; Bogdanski, P.; Agarwal,

D.P.; Du R.F.; Liangzhong, C.; Malying, C.; Yida, Y.; Jiujin, X.;

Shizhe, L.; and Yongfa, W. Aldehyde dehydrogenase isozyme

deficiency and alcohol sensitivity in four different Chinese

populations. Hum Hered 34:183-186, 1984.

Goodman, L.S., and Oilman, A. The Pharmaoological Basis of

Therapeutics. New York: MacMillan Publishing Co., Inc., 1980.

1843 pp.

37
Harada, S.; Misawa, S.; Agarwal, D.P.; and Goedde, H.W. Liver

alcohol dehydrogenase and aldehyde dehydrogenase in Japanese:

Isozyme variation and its possible role in alcohol intoxication.

Am J Hum Genet 32:8-15, 1980.

Idle J.R.; Sloan, T.P.; Smith, R.L.; and Wakile, L.A. Application

of the phenotyped panel approach to the detection of polymorphism

of drug oxidation in man. Br J Pharmacol 66:430-431P, 1979.

Idle, J.R.; Oates, N.S.; Shah, R.R:; and Smith, R.L. Protecting poor

metabolisers, a group at high risk of adverse drug reactions.

Lancet I:1388, 1983.

Inaba, T.; Lucassen, M.; and Kalow, W. Antipyrine metabolism in the

rat by three hepatic monooxygenases. Life Sci 26:1977-1983,

1980.

Inaba, T.; Otton, S.V.; and Kalow, W. Debrisoquine hydroxylation

capacity: Problems of assessment in two populations. Clin

Pharmacol Ther 29:218-223, 1981.

Jack, D.B.; Kendall, M.J.; and Wilkins, M.R. Drug reactions and the

poor metaboliser . Lancet II:110, 1983.

Kalow, W. Pharmacogenetics:

Philadelphia:

Heredity and the Response to Drugs.

Saunders, 1962.

Kalow, W. Ethnic difference in drug metabolism, Clin Pharmacokinet

7:373-400, 1982.

Kalow, W. Pharmacoanthropology: Drug metabolism. Fed Proc 43:2326-

2331, 1984.

Kalow, W.; Kadar, D.; Inaba, T.; and Tang, B.K. A case of deficiency

of N-hydroxylatlon of amobarbital. Clin Pharmacol Ther 21:530-

535, 1977.

Kreek. M.J. Plasma and urine levels of methadone. NY State J Med

73:2773-2777, 1973.

Kupfer, A.; Dick, D.; and Prelsig, R. Polymorphic mephenytoin

hydroxylation in man: A new phenotype in the genetic control of

hepatic drug metabolism. (Abstract). Hepatology 1:524, 1981.

LaDu. B.N. Pharmacogenetics: Defective enzymes in relation to

reactions to drugs: Annu Rev Med 23:453-468, 1972.

Lu, A.Y.H.. and Levin. W. The resolution and reconstitution of the

liver microsomal hydroxylation system. Biochim Biophys Acta

344:205-240, 1974.

Mahgoub, A.; Idle, J.R.; Dring, L.D.; Lancaster, R.; and Smith, R.L.

The polymorphic hydroxylation of debrisoquine in man. Lancet

II:584-586, 1977.

Nellstrom, B.; Bertilsson, L.; Sawe, J.; Schulz, H.-V.; and

Sjoqvist, F. E- and Z-10-hydroxylation of nortriptyline:

Relationship to polymorphic debrisoquine hydroxylation. Clin

Pharmacol Ther 30:189-193, 1981.

Miyamoto, K.; Ishii, H.; Takata, H.; Takagi, S.; Shigeta, Y.;

Sekiguchl, S.; Suyama, K.; Kohno, H.; and Tsuchiya M.

Association of HLA-B40 and DRW9 with Japanese alcoholic liver

cirrhosis. Pharmacol Biochem Behav 18(supp. 1):467-471, 1983.

Motulsky, A. Pharmacogenetics. Prog Med Genet 3:49-74, 1964.

Nebert, D.W.; Negishi, M.; Lang, M.A.; Hjejmeland, L.M.; and Eisen,

H.J. The Ah locus, a multigene family necessary for survival in a

chemically adverse environment: Comparison with the immune

system. Adv Genet 21:1-52, 1982.

Panayi, G.S. Huston, G.; Shah, R.R.; Mitchell, S.C.; Idle, J.R.;

38
Smith, R.L.; and Waring, R.H. Deficient sulphoxidation status

and D-penlcillamine toxicity. Lancet 1:414, 1983.

Penno, M.B., and Vesell, E.S. Monogenic control of variations in

antipyrine metabolite formation: New polymorphism of hepatic

drug oxidation. J Clin Invest 71:1698-1709, 1983.

Penno, M.B.; Dvorchik, B.H.; and Vesell, E.S. Genetic variation in

rates of antipyrine metabolite formation: A study in uninduced

twins. Proc Natl Acad Sci USA 78:5193-5196, 1981.

Perris, C.; Strandman, E.; and Wahlby, L. HLA antigens and the

response to prophylactic lithium. Neuropsychobiology 5:114-118,

1979.

Perry, H.M.; Tan, E.M.; Carmody, S.; and Sakamoto, A. Relationship

of acetyl transferase activity to antinuclear antibodies and

toxic symptoms in hypertensive patients treated with hydralazine.

J Lab Clin Med 76:114-125, 1970.

Propping, P. Pharmacogenetics.

83:123-173, 1978.

Rev Physiol Biochem Pharmacol

Roninson, I.B.; Abelson, H.T.; Housman, D.E.; Howell, N.; and

Varshavsky, A. Amplification of specific DNA sequences

correlates with multi-drug resistance in Chinese hamster cells.

Nature 809:626-628, 1984.

Ryan, D.; Lu, A.Y.H.; Kawalek, J.; West, S.B.; and Levin, W. Highly

purified cytochrome P-448 and P-450 from rat liver microsomes.

Biochem Biophys Res Commnun 64:1134-1141, 1975.

Schmidt, K.L., and Mueller-Eckhardt. C. Agranulocytosis,

levamisole, and HLA-B27. Lancet II:85, 1977.

Scott, J., and Poffenbarger, P.L. Pharmacogenetics of tolbutamide

metabolism in humans. Diabetes 28:41-51, 1979.

Shah, R.R.; Oates, N.S.; Idle, J.R.; and Smith, R.L. Genetic impairment of phenformin metabolism. Lancet II:1147, 1980.

Shah, R.R.; Gates, N.S.; Idle, J.R.; and Smith, B.L. Beta-blockers

and drug oxidation status. Lancet I:508-509, 1982a.

Shah, R.R.; Oates, N.S.; Idle, J.R.; Smith, R.L.; and Lockhart, J.D.

Impaired oxidation of debrisoquine in patients with perhexiline

neuropathy. Clin Res 284:295-299, 1982b.

Sloan, T.P.; Mahgoub, A.; Lancaster, R.; Idle, J.R.; and Smith, R.L.

Polymorphism of carbon oxidation of drugs and clinical

implications. Br Med J 2:655-657, 1978.

Sloan, T.P.; Lancaster, R.; Shah, R.R.; Idle, J.R.; and Smith, R.L.

Genetically determined oxidation capacity and the disposition of

debrisoquine. Br J Clin Pharmacol 15:443-450, 1983.

Smeraldi, E, and Scorza-Smeraldi, R. Interference between anti-HLA

antibodies and chlorpromazine. Nature 260:532-533, 1976.

Smeraldi, E.; Bellodi, L.; Saccheti, E.; and Cazzullo, C.L. The HLA

system and the clinical response to treatment with

chlorpromazine. Br J Psychiatry 129:486-489, 1976

Sorrell, M.F., and Leevy, C.M. Lymphocyte transformation and

alcoholic liver injury. Gastroenterology 63:1020-1025, 1972.

Spector, R.; Choudhury, A.K.; Chiang, C.K.; Goldberg, M.J.; and

Ghoneim, M.M. Diphenhydramine in Orientals and Caucasians. Clin

Pharmacol Ther 28:229-234, 1980.

Tannenbaum, J.P. Ruppert, R.D.; St. Pierre, R.L.; and Greenbarger,

N.J. The effect of chronic alcohol administration on the immune

responsiveness of the rat. J Allergy 44:272-281, 1969.

39
Tsuchimoto, K. Detection of antibody and antibody dependent cellmediated cytotoxicity (ADCC) against Chang liver cell in

alcoholic liver disease. Jpn J Gastroenterol 79:1588, 1982.

Vesell, E.S. Advances in pharmacogenetics. Prog Med Genet 9:291-

367, 1973.

Vesell, E.S. The antipyrine test in clinical pharmacology:

Concept ions and misconceptions. Clin Pharmacol Ther 26:275-286,

1979.

Vesell, E.S. Selection of subjects for investigation of host

factors affecting drug response: A method to identify new

pharmacogenetic conditions. Clin Pharmacol Ther 35:1-11, 1984.

Vesell, E.S., and Page, J.G. Genetic control of drug levels in man:

Antipyrine. Science 161:72-73, 1968a.

Vesell, E.S., and Page, J.G. Genetic control of drug levels in man:

Phenylbutazone. Science 159:1479-1480, 1968b.

Vesell, E.S., and Page, J.G. Genetic control of dicoumarol levels in

man. J Clin Invest 47:2657-2663, 1968c.

Vesell, E.S., and Penno. M.B. Assessment of methods to identify

sources of interindividual pharmacokinetic variations. Clin

Pharmacokinet 8:378-409, 1983.

Vesell, E.S.; Page, J.G.; and Passananti, G.T. Genetic and

environmental factors affecting ethanol metabolism in man. Clin

Pharmacol Ther 12:192-201, 1971.

Wang, P.; Mason, P.S.; and Guengerich, F.P. Purification of human

liver cytochrome P-450 and comparison to the enzyme isolated from

rat liver. Arch Biochem Biophys 199:206-219, 1980.

Wang, T.; Roden, Wolfenden, H.T.; Woosley, R.L.; Wood, A.J.J.;

and Wilkinsor, G.R. Influence of genetic polymorphism on the

metabolism and disposition of encainide in man. J Pharmacol Exp

Ther 228:605-611, 1984.

Weinshilboum, R.M., and Vesell, E.S. Human pharmacogenetics and new

directions in pharmacogenetics. Fed Proc Symposiums 43:2295-2342,

1984.

Whittaker, J.A., and Price Evans, D.A. Genetic control of

phenylbutazone metabolism in man. Br Med J 4:323-328, 1970.

Wolff, P.H. Ethnic differences in alcohol sensitivity. Science

175:449-450, 1972.

Wooley, P.H.; Griffin, J.; Panayi, G.S.; Batchelor, J.R.; Welsh,

K.I.; and Gibson, T.J. HLA-DR antigens and toxic reaction to

sodium aurothiomalate and D-penicillamine in patients with

rheumatoid arthritis. N Engl J Med 303:300-302, 1980.

Woolhouse, N.M.; Andoh, B.; Mahgoub, A.; Sloan, T.P.; Idle, J.R.;

and Smith, R.L. Debrisoquin hydroxylation polymorphism among

Ghanaians and Caucasians. Clin Pharmacol Ther 26:584-591, 1979.

Woolhouse, N.M.; Eichelbaum, Oates, N.S.; Idle, J.R.; and Smith,

R.L. Dissociation of control of debrisoquine and sparteine

oxidation in Ghanaians. 2nd World Conference on Clinical

Pharmacology and Therapeutics, Washington, Abstract No. 229,

1983.

AUTHOR

Elliot S. Vesell, M.D.

Department of Pharmacology

The Pennsylvania State University

College of Medicine

P.O. Box 850

Hershey, Pennsylvania 17033

40
Studies on an Animal Model of

Alcoholism

Ting-Kai Li, M.D.; Lawrence Lumeng, M.D.; William J.

McBride, Ph.D.; Marshall B. Waller, Ph.D., and James M.

Murphy, Ph.D.

INTRODUCTION

Alcoholism is a disorder characterized by a persistent and progressive pattern of aberrant alcohol-seeking behavior which,

over time, results in the loss of control over drinking and the

development of tolerance and physical dependence. As discussed

by Dr. Cloninger in this monograph, there is noW strong evidence

that genetic predisposition plays an important part in the

development of alcoholism in many people. Accordingly, the

identification of biological risk factors and their underlying

mechanisms of influence has become a high priority area of

alcohol-related biomedical research for the present and the

future.

It is noteworthy that there already exists a considerable body

of evidence both in humans and in experimental animals that a

variety of behavioral, physiological, and biochemical responses

to ethanol have genetic determinants. Alcohol drinking behavior

or preference is one such response (McClearn and Rodgers 1959;

Eriksson 1968; Li et al. 1979). Others include central nervous

system (CNS) (McClearn and Kakihana 1973; Riley et al. 1977) and

systemic sensitivity (Mizoi et al. 1979) to ethanol, alcohol

metabolizing capacity (Kopun and Propping 1977; Thurman 1980),

the acquisition of tolerance to ethanol (Grieve et al. 1979),

and the development of physical dependence on ethanol (Goldstein

1973). One experimental approach to gaining an understanding of

biological predisposition would be to study how these other responses to ethanol relate genetically and mechanistically to

normal and abnormal drinking behaviors. The validity of this

approach is supported by recent reports that individuals who

have the alcohol-flush reaction, caused by an abnormal elevation

of circulating acetaldehyde, are less prone to becoming alcoholic than those who do not (Harada et al. 1983; Yoshihara et

al. 1983). Activity of the low Km aldehyde dehydrogenase

isozyme is absent in tissues of individuals with this inherited

trait, rendering then intolerant to the consumption of even

small to moderate amounts of ethanol.

41
Investigators are searching for biological markers and potential

mechanisms of genetic influence in children of alcoholics, who

are a high-risk population. Dr. Schuckit reports his research

on phenotypic markers in sons of alcoholics elsewhere in this

monograph. The study of the relationship of alcohol drinking

behavior to other heritable responses to ethanol in experimental

animals requires the ready availability of animals willing-to

self-administer large amounts of ethanol. This feature is considered by most investigators to be a key element of any animal

model for alcoholism. It is well known that most experimental

animals avoid drinking ethanol solutions that are 10% (v/v) or

higher in concentration when food and water are concurrently

available. However, some strains of outbred laboratory rats do

show a wide range of individual variation in alcohol preference

and lines of rats exhibiting high and low alcohol preference

have been raised through bidirectional selection (Eriksson 1968;

Li et al. 1979). These successful breeding efforts have served

to substantiate the existence of genetic influence on alcoholseeking behavior, as have also the finding of differences in

alcohol preference among inbred mouse strains (McClearn and

Rodgers 1959). One line of alcohol-preferring rats, the P line,

has now been systematically characterized with respect to its

drinking behavior. The data indicate that it satisfies virtually all the preceived requirements for an animal model of alcoholism. Comparison of the effects of ethanol on P rats and on

NP rats, a line simultaneously selected for alcohol nonpreference (aversion), has revealed differences that should provide

insights into the biological basis of aberrant alcohol-seeking

behavior. This chapter reviews the work performed on these

selectively bred lines.

DEVELOPMENT OF AND STUDIES ON THE P AND NP LINES OF RATS

The suggested essential requirements of an animal model of alcoholism are (Lester and Freed 1973):

1.

2.

3.

4.

5.

Ethanol should be self-administered orally, in preference to water or other solutions having the same

caloric: value and palatibility.

The amount consumed should produce measurable blood

alcohol. concentrations (BACs).

Ethanol should be positively reinforcing, i.e., the

animals should be willing to overcome obstacles or work

to obtain the alcohol.

The positive reinforcing effect of ethanol should be

accountable in large part, if not entirely, by the CNS

pharmacological actions of ethanol or its metabolic

products and not because of its caloric value, taste,

or smell.

Consumption should lead ultimately to

a) repeated episodes of intoxication,

42
b) tolerance, and

c) physical dependence.

The P and NP lines were developed by selective breeding for high

and low alcohol preference from a foundation stock of Wistar

rats (Li et al. 1981). Testing was performed with an unflavored

10% (v/v) solution of ethanol made continuously available in a

Richter tube to individually housed animals. Water in an identical Richter tube as an alternate source of fluid and solid

food were provided ad libitum. The amounts of 10% ethanol,

water, and food consumed daily were measured for 3 weeks and

those animals exhibiting the highest and lowest consumption

scores (g ethanol/kg body weight/d), respectively, were mated to

start the next generation of P and NP lines. After 20 generations, the consumption scores (g/kg/d; mean ± SD) were: P

males, 5.5 ± 1.2; P females, 7.3 ± 1.7; NP males, 1.1 ± 0.6; NP

females 1.0 ± 0.9. The P and NP animals in the current S26

generation display similar characteristics. The P rats on test

consume between 20% and 30% of the total calories as ethanol. They

substitute the ethanol calories for a part of the food calories

and gain weight at the same rate as control animals not given

the ethanol solution as a fluid choice.

The P rats drink about 70% of the ethanol in the dark, when they

also eat most of the food. Drinking occurs in bursts at irregularly spaced intervals (Waller et al. 1982a). When blood is

sampled during the dark cycle, e.g., at the 3rd and 11th hours,

BACs ranging from 14 to 120 mg% have been obtained. Mean values

are about 60 mg%. When BACs are measured 30 minutes after the

completion of drinking episodes, as determined by a drinkometer,

values of 52 to 124 mg% have been obtained. The mean is 86

mg%. Clearly, these animals are attaining systemic alcohol concentrations that are pharmacologically active, at least for

humans. Studies have also been performed to determine what BACs

coincide with cessation of drinking and what blood levels produced by intravenous infusion would lead to curtailment of oral

intake (Waller et al. 1982b). It was found that 50 to 70 mg%

did both. It appears, therefore, that the higher BACs attained

with oral intake represent overshoot, caused by delayed gastrointestinal absorption.

The P rats will work in order to obtain ethanol through operant

responding (Penn et al. 1978). Free-feeding male animals were

trained to bar-press for sweetened milk in a dipper as reward

and the milk was then replaced with 10% ethanol. With food and

water constantly available, they bar-pressed for the ethanol

both on a continuous reinforcement schedule and when the barpress to reinforcement ratio was increased. In fact, the response to reinforcement ratio could be raised to 6 or 7 before

water intake increased, yielding a response rate of over 1,000

bar-presses/24 hours in each of the animals tested. Since food

and water were freely available to the animals, it appears unlikely that the P animals found the ethanol solutions rewarding

because of caloric needs or thirst.

43
The above studies, however, do not distinguish whether the reinforcing properties of ethanol are its pharmacological effects or

its taste and smell. To dissociate the post-ingestive effects

of ethanol from its orosensory cues, intragastric self-administration experiments were performed, using an experimental design

similar to that reported by Deutsch and coworkers (Deutsch and

Hardy 1976; Deutsch and Walton 1977). Male P and NP animals

were surgically implanted with transesophageal catheters for the

intragastric delivery of fluids. Following recovery, the

animals were trained to associate: a) the intragastric delivery

of ethanol with the drinking of a neutral-flavored (almond or

banana) water solution contained in a U-shaped drinking tube;

and b) the intragastric delivery of water with the drinking of

the other neutral-flavored solution in another U-tube. The

drinking of the fluids from the U-tubes activated a pump to

deliver an equal volume of either the water or the ethanol solution intragastrically. After training, the animals were presented with both U-tubes and allowed free-choice drinking of the

two flavored solutions on a 24-hour availability schedule. Food

was freely available throughout the experiment. It was found

that the P rats consistently self-infused greater volumes of the

ethanol solution and lesser volumes of the water than did the NP

rats (Waller et al. 1984a). ThiS difference was observed regardless of whether the concentration of infused ethanol was 10%,

20%, 30%, or 40%. The amount of ethanol infused by the NP rats

was always less then 1 g/kg/d at all concentrations of ethanol

tested. By contrast, the amount of ethanol infused by the P

rats increased from 3.0 ± 0.3 g/kg/d with 10% ethanol to 9.4 ±

1.7 g/kg/d with 40% ethanol. The BACs of animals measured 30 to

40 minutes after observed episodes of self-infusion of 20%

ethanol were 116 to 303 mg% (mean of 199 mg%) and, with the

self-administration of 40% ethanol, BACs were 92 to 415 mg%

(mean of 231 mg%). All animals repeatedly showed signs of intoxication, such as ataxia and somolence, after intragastric

ethanol self-administration. These blood ethanol levels

attained with intragastric self-administration were considerably

higher than those observed in the P rats with free-choice drinking. Interestingly, although the total amount of 40% ethanol

self-infused per day was significantly higher than the pretested

voluntary oral consumption scores of the P rats (6.5 g/kg/d), the

amount of 20% and 30% self-infused (5.8 and 7.3 g/kg/d, respectively) was not.

The above results are consistent with the notion that the postingestive, pharmacological actions of ethanol are rewarding to

the P rats, but are aversive to the NP rats. However, the

difference in BACs attained by the P rats with free-choice oral

consumption and with intragastric self-administration suggests

that orosensory cues also may be an important modulator of the

amount of ethanol self-administered per drinking episode. It is

important to note that Deutsch and coworkers (Deutsch and Eisner

1977; Deutsch and Walton 1977) were able to demonstrate intragastric self-administration of ethanol in large amounts in rats

unselected for ethanol preference only if they were first made

physically dependent by the prior forcible administration

44
of ethanol. Ethanol self-administration behavior

was quickly extinguished in maelected animals not made ethanoldependent, as was observed also in our study with the NP rats.

In contrast, the P animals in this study were not made dependent

on ethanol and, in fact, were ethanol-free for at least a month

before these experiments. Clearly, the innate ethanol preference of experimental animals is an important if not crucial

variable in studies of ethanol self-administration.

With chronic free-choice drinking of 10% ethanol, the P rats

develop metabolic tolerance. After 6 weeks, the ethanol elimination rate of ethanol-consuming animals was 15% higher than

that of control animals. Weight gain and total caloric intake

of the animals in the two groups were identical. Ethanol

represented 22% (week 1) to 30% (week 6) of the total calories

consumed daily by the experimental group. The degree of metabolic tolerance developed over this period with free-choice

drinking was the same as that developed by P rats forcibly given

a total liquid diet in which 35% of the total caloric content

was derived from ethanol (AIN liquid diet containing 5%

ethanol). Studies are in progress to determine the time course

and extent of neuronal tolerance development with free-choice

drinking by the P rats.

We have also examined whether chronic free-choice drinking by

the P rats produces physical dependence (Waller et al. 1982a).

Experimental animals were given constant access to 10% ethanol

and water for 20 weeks, while control animals received only

water. Food was available ad libitum. After 20 weeks, the

ethanol solution was taken away from experimental animals

and both groups were scored for signs of withdrawal by a blinded

observer. In the first 24 hours following removal of ethanol,

18 of the 19 ethanol-exposed animals exhibited signs of withdrawal. These included Straub tail, broad based gait, tremulousness, hyperactivity, wet dog shakes, teeth chattering, induced running, and bizarre behavior. These manifestations

abated after 72 hours. As expected, none of the control animals

showed withdrawal signs. We concluded, therefore, that chronic

free-choice drinking of 10% ethanol can produce physical dependence in the P rats.

The studies summarized above indicate that the P line of selectively bred rats should be a useful animal model for exploring

the biology of alcohol-seeking behavior and conditions that

promote and lessen this kind of behavior. lb this end, we have

performed a number of studies comparing the P and the NP lines.

One way in which the lines differ is the degree of motor impairment produced by moderate to high doses of ethanol. In these

studies, the animals were trained to jump onto a descending

platform in order to avoid foot shock. The height to which the

animals can jump was used as the measure of their motor performance. It was found that the NP rats took much longer to recover

from the sedative-hypnotic effects of ethanol than did the P

rats (Lumeng et al. 1982). This difference could not be

45
attributed to a difference in alcohol elimination rate between

the lines nor to a difference in CNS sensitivity to ethanol.

Rather, it was because the NP rats took longer to develop tolerance than did the P rats. This could be assessed by use of the

jump test after the administration of a second dose of ethanol

and the comparison of BACs at the time of recovery to criterion

performance after the first and the second dose. It was found

that, while both the P and NP rats developed acute tolerance,

the P rats did so more quickly or to a greater degree than did

the NP rats (Waller et al. 1983).

Although rapid tolerance development might be a mechanism that

promotes and sustains high levels of consumption, it is unlikely

to be a primary determinant of alcohol preference, since the

BACs attained during free-choice drinking, usually less than 150

mg%, are substantially lower than those produced in the studies

of tolerance development described above. Accordingly, a basis

for the difference in drinking behavior between the P and NP

rats was sought at lower doses of ethanol. It was found that

the P rats exhibit increased spontaneous motor activity (SMA)

following the intraperitoneal injection of ethanol, 0.065 to 0.5

g/kg. By contrast, the NP rats do not show stimulation, and

decreased SMA began to occur with doses of 0.5 to 1.5 g/kg. The

increase in SMA in the P rats is as much as 50% with 0.25 g/kg

doses of ethanol. (Waller et al. 1984b).

The combination of low-dose stimulation by ethanol and acute

tolerance development to the high-dose effects offers an attractive hypothesis of alcohol abuse. This hypothesis assumes that

the low-dose stimulation observed in the P rats is a reflection

of the positively reinforcing or rewarding features of ethanol

consumption, whereas the high-dose depressant effect is aversive

and inhibits drinking. As tolerance to the high-dose effects of

ethanol develops, the rewarding actions of ethanol become

progressively extended into the higher dosage range, leading to

increased consumption. We are currently testing this hypothesis

in the P line of rats.

Studies have also been performed to discern whether there are

neurochemical differences between the P and the NP rats. A

major discovered difference is in the regional brain content of

serotonin. Ethanol-naive P rats consistently have lower levels

of serotonin in the cerebral cortex, corpus striatum, thalamus,

hypothalamus, and hippocampus, and lower levels of 5-hydroxyindole acetic acid in the cerebral cortex and hippocampus, than

do NP rats (Murphy et al. 1982). Although the significance of

these neurochemical differences with respect to ethanol drinking

preference is yet unclear, it is intriguing that serotonin and

norepinephrine reuptake inhibitors are effective in reducing

voluntary ethanol consumption in rats (Amit et al. 1984). These

drugs display a similar action in the P rats, both on the

24-hour free-choice oral consumption schedule (Murphy et al.

1984) and with intragastric ethanol self-administration.

Clearly, we are interested in the specificity of this kind of

46
response in relationship to the postulated roles of serotonin

and/or norepinephrine in the reinforcing actions of ethanol.

SUMMARY

Past and ongoing studies indicate that the selectively bred P

line of rats satisfies virtually all the suggested criteria for

an animal model of alcoholism. They attain pharmacologically

active levels of BAC and develop tolerance and physical dependence with voluntary oral ethanol ingestion, while in the freefeeding state. Ethanol is positively reinforcing to the P rats

and consumption appears to be directed by the post-ingestive,

pharmacological effects of ethanol, as revealed by the intragastric self-administration studies.

Sane interesting differences between the P and the NP lines have

been uncovered. They differ in the content of serotonin in

several brain regions and they respond differently to ethanol.

The P rats develop acute tolerance to sedative-hypnotic doses of

ethanol more rapidly than do the NP rats, and they exhibit stimulation with low doses of ethanol. These differences suggest

hypotheses on mechanisms underlying alcohol-seeking behavior

which can now be tested experimentally.

It should be emphasized, however, that the described findings

are the product of but a single genetic experiment. Clearly,

replication is needed, and we are currently doing this, using a

better defined, heterogeneous stack of rats. This one experiment, however, has demonstrated the feasibility of developing

animal models of alcoholism and offers hope that the genetic

and biological basis of alcohol-seeking behavior can be explored

in the laboratory. The screening and testing of pharmacological

agents able to deter alcohol-seeking behavior is an obvious

practical application of this model.

REFERENCES

Amit, S.; Sutherland E.A.; Gill, K.; and Ogren, S.O.

Zimelidine: A review of its effects on ethanol consumption.

Neurosci Biobehav Rev 8:35-54, 1984.

Deutsch, J.A. and Eisner, A. Ethanol self-administration in

the rat induced by forced drinking of ethanol. Behav Biol

20:81-90. 1977.

Deutsch, J.A., and Hardy, W.T. Ethanol tolerance in the rat

measured by the untasted intake of alcohol. Behav Biol

17:379-389, 1976.

Deutsch. J.A. . and Walton, N.Y. A rat alcoholism model in a

free-choice situation. Behav Biol 19:349-360, 1977.

Eriksson. K. Genetic selection for voluntary alcohol consumption in the albino rat. Science 159:739-74l, 1968.

Goldstein, D.B. Inherited differences in intensity of alcohol

withdrawal reactions in mice. Nature 245:154-156, 1973.

47
Grieve, S.J.; Griffiths, P.J.; and Littleton, J.M. Genetic

influences on the rate of development of ethanol tolerance and

the ethanol dependence withdrawal syndrome in mice. Drug

Alcohol Depend 4:77-86, 1979.

Harada, S.; Agarwal, D.P.; Goedde, H.W.; and Ishikawa, B.

Aldehyde dehydrogenase isozyme variation and alcoholism in

Japan. Pharmacol Biochem Behav 18(Supp. 1):151-153, 1983.

Kopun, M., and Propping, P. The kinetics of ethanol absorption

and elimination in twins and supplementary repetitive experiments in singleton subjects. Eur J Clin Pharmacol 11:337-344,

1977.

Lester, D., and Reed, E.X. Criteria for an animal model of

alcoholism. Pharmacol Biochem Behav 1:103-107, 1973.

Li, T.-K.; Lumeng, L.; McBride, W.J.; and Waller, M.B. Progress

toward a voluntary oral consumption model of alcoholism. Drug

Alcohol Depend 4:45-60, 1979.

Li, T.-K.; Luneng, L.; McBride, W.J.; and Waller, M.B. Indiana

selection studies on alcohol-related behaviors. In: McClearn

G.E.; Deitrich, R.A.; and Erwin, V.G., eds. Development of

Animal Models as Pharmacogenetic Tools. National Institute on

Alcohol Abuse and Alcoholism Research Monograph 6. DHEW Pub.

No. (ACM) 79-847. Washington, D.C.: Supt. of Docs., U.S.

Govt. Print. off., 1981. pp. 171-191.

Lumeng, L.; Wallet, M.B.; McBride, W.J.; and Li, T.-K.

Different sensitivities to ethanol in alcohol-preferring and

nonpreferring rats. Pharmacol Biochem Behav 16:501-507, 1982.

McClearn, G.E., and Kakihana, R. Selective breeding for ethanol

sensitivity in mice. Behav Genet 3:409-410, 1973.

McClearn, G.E., and Rodgers, D.A. Differences in alcohol

preference among inbred strains of mice. J Stud Alcohol

20:691-695, 1959.

Mizoi, Y.; Ijiri, J.; Tatsumo, Y.; Kijima, T.; Fujiwara, S.; and

Adachi, J. Relationship between facial flushing and blood

acetaldehyde levels after ethanol intake. Pharmacol Biochem

Behav 10:303-311, 1979.

Murphy, J.M.; McBride, W.J.; Lumeng, L.; and Li, T.-K. Regional

brain levels of monoamines in alcohol-preferring and -nonpreferring lines of rats. Pharmacol Biochem Behav 16:145-149,

1982.

Murphy, J.M.; Waller, M.B.; Gatto, G.J.; McBride, W.J.; Lumeng,

L.; and Li, T.-K. wine uptake inhibitors attenuate

ethanol intake in alcohol-preferring (P) rats. Submitted to

Alcohol, 1984.

Penn, P.E.; McBride, W.J.; Lumeng, L.; Gaff, T.M.; and Li,

T.-K. Neurochemical and operant behavioral studies of a

strain of alcohol-preferring rats. Pharmacol Biochem Behav

8:475-481, 1978.

Riley, E.P.; Worsham, E.D.; Lester, D.; and Freed, E.X.

Selective breeding of rats for differences in reactivity to

alcohol: An approach

Stud Alcohol 38:1705-1717, 1977.

to an animal model of alcoholism. J

Thurman, R.G. Ethanol elimination rate is inherited in the

rat. Adv Exp Med Biol 132:655-662, 1980.

48
Waller, M.B.; McBride, W.J.; Lumeng, L.; and Li, T.-K. Induction of dependence on ethanol by free-choice drinking in

alcohol-preferring rats. Pharmacol Biochem Behav 16:501-507,

1982a.

Waller, M.B.; McBride, W.J.; Lumeng, L.; and Li, T.-K. Effects

of intravenous ethanol and of 4-methylpyrazole on alcohol

drinking of alcohol-preferring rats. Pharmacol Biochem Behav

17:763-768, 1982b.

Waller, M.B.; McBride, W.J.; Lumeng, L.; and Li, T.-K. Initial

sensitivity and acute tolerance to ethanol in the P and NP

lines of rats. Pharmacol Biochem Behav 19:683-686, 1983.

Waller, M.B.; McBride, W.J.; Gatto, G.J.; Lumeng, L.; and Li,

T.-K. Intragastric ethanol self-administration by ethanolpreferring and -nonpreferring lines of rats. Science

225:78-80, 1984a.

Waller, M.B.; Murphy, J.M.; McBride, W.J.; Lumeng, L.; and Li,

T.-K. Effect of low dose ethanol on spontaneous motor activity in the alcohol-preferring (P) and -nonpreferring (NP)

lines of rats. Submitted to Pharmacol Biochem Behav, 1984b.

Yoshihara, H.; Sato, N.; Kamada, T.; and Abe, H. Low Km ALDH

isozyme and alcoholic liver injury. Pharmacol Biochem Behav

18(Supp. 1):425-428, 1983.

ACKNOWLEDGMENT

Supported in part by PHS AA-03243 from the National Institute of Alcohol

Abuse and Alcoholism.

AUTHORS

Ting-Kai Li, M.D.

Lawrence Lumeng M.D.

Departments of Medicine and Biochemistry

The Regenstrief Institute

Indiana University School of Medicine

and

The Veterans Administration Medical Center

Indianapolis, Indiana 46223

William J. McBride, Ph.D.

Departments of Psychiatry and Biochemistry

The Institute of Psychiatric Research

Indiana University School of Medicine

Indianapolis, Indiana 46223

Marshall B. Waller, Ph.D.

James M. Murphy, Ph.D.

Departments of Medicine and Psychiatry

The Institute of Psychiatric Research

Indiana University School of Medicine

Indianapolis, Indiana 46223

49
Development of DNA Probes to

Investigate Genetic Variation of

Alcohol Metabolizing Enzymes

Moyra Smith, M.D., Ph.D.; Gregg Duester, Ph.D.; and G.

Wesley Hatfield, Ph.D.

INTRODUCTION

Alcohol dehydrogenase (ADH) is responsible for the oxidation of a

wide variety of primary, secondary, and aromatic alcohols, but is

most noted as being the enzyme primarily responsible for oxidizing

ethanol to acetaldehyde. Human ADH exists as a set of at least 15

different isoenzymes that differ in electrophoretic mobility, substrate affinities, and inhibition characteristics (Smith et al.

1973; Li 1977; Pares and Vallee 1981). All isoenzymes are similar

in that they are dimeric, each monomer having a molecular weight of

approximately 40,000 daltons. The various isoenzymes are categorized

into three classes. Class I contains a large group of isoenzymes

possessing various combinations of -ADH subunits coded by

three gene loci: ADH1, ADH2, and ADH3, respectively. Class II contains the ADH isoenzyme (Bosron et al. 1979; Li and Magnes 1975).

Class III contains the x-ADH isoenzyme, which oxidizes high molecular weight alcohols, but not ethanol (Pares and Vallee 1981).

Class I and II are primarily liver-specific enzymes, although small

amounts of activity of these isoenzymes occur in intestine, kidney,

and lung (Smith et al. 1971). Class III ADH is a constitutive

enzyme and is found in all cell types. Studies on the gene products

of human class I ADH genes have revealed genetic polymorphisms.

50
Common genetic variation occurs at the ADH2 and the ADH3 gene

loci. One of the variant forms of ADH2 is known to give rise

to an altered ADH which differs in its kinetic properties and

is implicated in differences in alcohol tolerance. The exact

role of genetic variation in relation to propensity to develop

alcoholism have been difficult to assess since this enzyme is

primarily a liver-specific enzyme and is not readily analyzable

in accessible tissues such as blood cells or serum.

We have initiated a research study aimed at isolating ADH and

aldehyde dehydrogenase (ALDH) genes so that molecular probes

may be derived to analyze genetic variation in the ADH and

ALDH genes and their flanking regions. The results presented

here relate to the derivation of DNA probes for the class I

ADH genes.

Availability of partial amino acid sequence data for the ß-ADH

isozyme, coded by the ADH2 gene, enabled us to select a sequence

of five amino acids with relatively low code degeneracy, near

the carboxy terminal region of the ADH polypeptide. An oligonucleotide probe corresponding to this sequence was commercially

obtained. The probe consisted of a mixture of 16 different

oligomers, one of which is perfectly complementary to ADH mRNA

(figure 1). The radiolabeled oligonucleotide probe was used to

screen colonies derived from the adult human liver cDNA library

of Orkin (Woods et al. 1982).

FIGURE 1

SEQUENCE OF OLIGONUCLEOTIDE PROBE (14 mer) FOR HUMAN ADH

One clone isolated from the cDNA library and designated pADH12

was found to contain an insert of 1100 base pairs. A restriction map of this clone was derived by further digesting the isolated cDNA insert by various enzymes. Restriction fragments

were then cloned into the M13 bacteriophage and sequenced

(Duester et al. 1984). Analysis of the DNA sequence of the insert in the pADH12 clone revealed that it contained a 593 base

pair 3' untranslated region in addition to a 273 base pair

translated region coding for 91 amino acids. The DNA sequence

in this region predicted exactly the amino acid sequence of the

terminal 91 amino acids in ß-ADH as derived by Bühler and

coworkers (1984).

51
This pADH12 cDNA clone has been used to derive genomic ADH clones.

The Maniatis library of human DNA in lambda phage was screened

with pADH12. Information from peptide mapping studies (Strydom

and Vallee 1982) and partial sequence information on the ADH3

polypeptide (Bühler et al. 1984) indicate that the coding

regions of the three class I ADH genes are closely homologous.

Using radiolabeled pADH12 as a probe under conditions of low

stringency, it should be possible to isolate from libraries of

human genomic or cDNA, clones corresponding to all three class I

ADH genes. One of the genomic clones, which was isolated from

the Maniatis library using pADH12, has been found to contain a

terminal exon, which is similar but not identical to that in pADH12 in

addition to an adjacent intron and 3' untranslated region

(Duester 1984). The base modifications present in the exon of

the ADH53 clone correspond to those which one would expect to

find in the ADH1 gene, based on the amino acid differences

between ß-and ADH polypeptide as determined by Jörnvall

(personal communication, 1984)

INVESTIGATION OF ADH GENETIC VARIATION USING THE pADH12 AND

THE ADH53 PROBES

Prior to using the ADH53 clone in studies of human DNA polymorphism, we ascertained that this clone did not contain any

repetitive DNA sequences. Human DNA was isolated from peripheral blood leukocytes or from cultured hymphoblastoid cells.

Human DNA was digested with a number of different restriction

endonucleases, and individual DNA digests were subjected to

agarose gel electrophoresis and Southern transfer to nitrocellulose filters (Southern 1972). Filters were hybridized

with radiolabeled ADH gene probes and then washed at different

stringencies prior to autoradiography. The restriction endonuclease MSP 1 has thus far proved most useful for detecting

DNA polymorphism in the ADH genes (figure 2).

FIGURE 2

MSPI FRAGMENTS OF HUMAN GENOMIC DNA REVEALED BY HYBRIDIZATION

TO ADH53 PROBE

52
Using the ADH53 probe and a low-stringency wash of the filters

(1 x SSC, at 55 C for 40 minutes), four or five MSP 1

fragments are seen. These fragments are 12.5, 10.0. 8.6, 6.8,

and 3.8 Kb in size (see figure 2 and figure 3). Occasional

individuals have also been found to have a 4.2 Kb fragment.

Following a high-stringency wash (0.1 x SSC/65 C/30

minutes) only the 10.0, the 8.6, and the 3.8 Kb fragments

remain, suggesting that these fragments are most analogous to

the ADH53 probe, which probably represents the ADH1 gene.

Using the pADH12 probe, 4 fragments may be seen. These are

the 12.5, 10.0, 8.6, and 6.8 Kb fragments. However, the 6.8 Kb

fragment is frequently very weak with pADH12. Following a highstringency wash, the 8.6 Kb fragment is the only fragment still

visualized, suggesting that this fragment is most analogous to

the ADH2 gene (see figure 3).

FIGURE 3

CHANGES IN PATTERNS OF DNA FRAGMENTS WHICH HYBRIDIZE TO ADH53 AND

ADH12 UNDER CONDITIONS OF LOW AND HIGH STRINGENCY

In occasional individuals, an 11 Kb band may be visualized. This

fragment is apparently more analogous to ADH2, based on the

observation that it is usually more intense with pADH12 than with

ADH53. The 12.5 Kb MSP 1 fragment hybridizes equally well with

pADH12 and ADH53 at low stringency. At high stringency, it is not

present with either probe. This finding suggests that the 12.5 Kb

fragment may be derived from a third class I ADH gene, possibly

the ADH3 gene which codes for the polypeptide. Individual variation has been observed with respect to the number of MSP 1 fragments. In some individuals, the 12.5 Kb fragment is absent, while

in other individuals the 10.0 Kb fragment is absent (see figure 2).

Individual variation has also been observed with respect to the

relative intensity of the different fragments (see figure 4).

53
FIGURE 4

PERSON TO PERSON DIFFERENCES IN THE RELATIVE INTENSITIES OF THE

DNA FRAGMENTS WHICH HYBRIDIZE TO THE ADH53 PROBE

Based on differences in the relative intensity of the MSP 1

fragments and on the fragment size differences in different

individuals, we have drawn up the following hypothesis. One

of the class I ADH genes (probably ADH3) gives rise to an

MSP 1 fragment which hybridizes to ADH53 and pADH12 and is

12.5 Kb. A polymorphic site exists so that in some individuals

a 10.0 Kb fragment results. However, the additional 2.5 Kb

fragment which arises as a result of this polymorphism may not

be visualized with ADH53 or pADH12. A second ADH gene (probably

ADH1) gives rise to a 6.8 Kb fragment. In certain individuals

an MSP 1 site is absent so that a 10.0 Kb fragment results.

Individuals who are doubly heterozygous at the two loci described

above will therefore exhibit a very intense 10.0 Kb fragment.

Examination of MSP 1 digestion patterns in a number of nuclear

families has provided evidence of heritability of certain fragments. In figure 5a, the mother is homozygous for the 10.0 Kb

fragment, while the father is homozygous for the 12.5 Kb fragment. Their son is an obligate heterozygote, having inherited

the 12.5 Kb fragment from his father and the 10.0 Kb fragment

from his mother.

54
FIGURE 5

ADH53 HYBRIDIZING MSP 1 FRAGMENTS PRESENT IN DNA FROM INDIVIDUALS

IN 3 NUCLEAR FAMILIES

In figure 5c the father and the mother were both homozygous for

the 10.0 Kb fragment. Their two sons were homozygous since each

inherited the 12.5 Kb fragment from their mother and father. Note

that in figures 5a and 5b there is also evidence of polymorphism

with respect to the 3.8 and 4.2 Kb fragments. The exact relationship of this variation and the 12.5 to 10.0 Kb fragment variation

is not clear at present. We have thus far examined DNA from 101

individuals, including 80 Caucasoids, 10 Orientals, and 11 Negroids. Polymorphism has been demonstrated in each population group.

In the analytical system described above, the presence of an MSP 1

fragment 10.0 Kb in size could result from polymorphism of either

the ADH1 or the ADH3 gene. In an attempt to further resolve this

polymorphism, we are currently carrying out double digestions of

human DNA with EcoRI and MSP 1. In addition, studies are being

carried out to derive gene-specific probes and/or gene-specific

hybridization conditions. Further studies are also required to

define the origin and significance of the 11 Kb band which occurs

in occasional individuals and which is apparently more intense

with the pADH12 (i.e., the ADH2) gene probe.

55
REFERENCES

Bosron, W.F.; Li, T.K.; Dafeldecker. W.P.; and Vallee, B.L. Human

liver II-alcohol dehydrogenase; kinetic and molecular properties.

Biochemistry 18:1101-1105, 1979.

Bühler, R.; Hempel, J.; Kaiser, R.; von Warburg, J.P.; and Vallee. B.L.

Human alcohol dehydrogenase-structural differences between the

ß and sub-units suggest parallel duplications in isoenzyme

evolution and predominate expression of separate gene descendants

in livers of different mammals. Proc Nat Acad Sci USA 81:6320-6324,

Duester, G.; Hatfield, G.W.; Büh1er, R.; E Hempel, J.; Jörnvall, H.;

and Smith. M, Molecular cloning and characterization of a cDNA for

the Beta sub-unit of human alcohol dehydrogenase. Proc Natl Acad

Sci USA 81:4055-4059, 1984.

Li, T.K., and Magnes, L.J. Identification of a distinctive molecular

form of alcohol dehydrogenase in human liver with high activity.

Biochem Biophys Res Commun 63:202-208. 1975.

Li, T.K. Enzymology of human alcohol metabolism.

427-484, 1977.

Adv Enzymol 45:

Pares, X., and Vallee, B.L. New human liver alcohol dehydrogenase

forms with unique kinetic characteristics. Biochem Biophys Res

Commun 98:122-130, 1981.

Southern, E.M. Deletion of specific sequence among DNA fragments

separated by gel electrophoresis. J Mol Biol 98:503-510, 1975.

Smith, M.; Hopkinson, D.A.; and Harris, H. Developmental changes

and polymorphism in human alcohol dehydrogenase. Ann Hum Genet

34:251-271, 1971.

Smith, M.; Hopkinson, D.A.; and Harris, H. Studies on the properties

of the human alcohol dehydrogenase isozymes determined by the

different loci ADH1, ADH2 and ADH3. Ann Hum Genet 37:49-67, 1973.

Strydom, D.J., and Vallee, B.L. Characterization of human alcohol

dehydrogenase isoenzymes by high performance liquid chromatography

peptide mapping. Anal Biochem 123:422-429, 1982.

Woods, D.E.; Markham, A.F.; Ricker, A.T.; Goldberger, G.; and

Colten. H. Isolation of cDNA clones for human phosphoglycerate

kinase. Proc Nat Acad Sci USA 79:5661-5665, 1982.

AUTHORS

Moyra Smith, M.D., Ph.D.

Department of Pediatrics

University of California, Irvine

Irvine, California 92717

Gregg Duester, Ph.D.

Department of Microbiology

University of California, Irvine

Irvine, California 92717

G. Wesley Hatfield, Ph.D.

Department of Microbiology

University of California, Irvine

Irvine, California 92717

56
Genetics as a Tool for Identifying

Biological Markers of Drug Abuse

Allan C. Collins, Ph.D.

A search for biological markers of drug abuse or

alcoholism presupposes that some link can be made

between the effects of behaviorally active drugs and

some biochemical factor that is found in readily accessible tissues. Presumably, the identification of

biological markers would be of value in that especially susceptible individuals could be diagnosed and

warned of potential consequences of drug use.

The identification of a biological marker involves a

search for some biological substance(s) which may be

correlated with the biological substrates for drug

dependence. Such a search requires, as a precondition,

that drug dependence is due, at least in part, to

biological factors. Since biological factors (enzymes, proteins, metabolic intermediates, anatomy) are

regulated by genes, the search for a biological marker

for drug abuse or alcoholism is essentially a search

for gene products that are directly linked to or

regulated by those genes that affect drug abuse related behavior. The search for such markers will be

successful only if variation exists within the population with regard to drug abuse related behaviors.

Presumably, variation will also exist within the population for some substance that is related to “drug

abuse genes.” Only if variation in susceptibility to

alcoholism or drug abuse exists does it make sense to

attempt to identify potential markers for these conditions.

The biological marker need not be influenced directly

by those genes that influence drug abuse. An adequate

marker could be the product of a gene that is closely

linked to the drug abuse genes. Alternatively, pleiotropic effects of drug abuse genes may prove to be

valuable markers. Pleiotropy is seen when a single

gene influences more than one phenotype or trait.

57
Thus, the search for a biological marker may be a

search for some gene product(s) that is directly related to drug abuse, a search for gene products that

are regulated by pleiotropic effects of drug abuse

genes, or a search for the products of genes that are

closely linked to the drug abuse genes.

The evidence that supports the notion that genetic

factors contribute to the development of alcoholism in

humans is substantial and is reviewed in other chapters in this monograph. On the other hand, the evidence that indicates other forms of substance abuse

are influenced by genetic factors is lacking--not

because studies have been carried out that indicate

that drug abuse is not influenced by hereditary

factors but, rather, because in depth studies have not

been carried out to assess this question. This suggests that a search for biological markers for alcoholism might be successful if initiated in the near

future because appropriate populations may be more

readily identified. On the other hand, a search for

biological markers of drug abuse may be premature

since the identification of adequate human populations

for drug abuse may not be possible.

ANIMAL MODELS

A potential solution to the problem of lack of availability of suitable human populations for study is to

develop animal models. This approach has been quite

successful in alcohol research, and a number of genetically defined animal models related to alcoholism are

now available. The progress related to the development of genetically defined animal models of drug

abuse is considerably less, although enough data are

currently available to indicate that successful models

could be obtained. This chapter describes some of the

progress that has been made in identifying potential

models and also makes recommendations as to how animal

models can be used to aid in the identification of

biological markers of drug abuse.

The advantages of animal models in the search for

biological markers of drug abuse and alcoholism are

numerous. Perhaps the most important of these advantages relates to the potential need for invasive

testing. Drug abuse and alcoholism are, in all likelihood, influenced to a major degree by central

nervous system factors. Therefore, some factor(s) in

brain are likely to be of primary importance in determining susceptibility to these afflictions. For

example, it may be that the amount of drug receptor in

specific brain regions is critical in determining

whether an individual will be more likely to abuse the

58
drug which affects this receptor. Obviously, the

availability of human brain tissue is not sufficient

to test easily any hypothesis which requires measuring

the brain content of some biological factor. It might

take years, for example, to test the hypothesis that

greater or lesser numbers of brain opiate receptors

promote the abuse of opiates. Research involving

humans would be totally impossible if the factor which

regulates "addictability" is the degree of change in

some neurochemical parameter, such as receptor number,

during chronic drug use. Animal models can be of

value in testing such hypotheses as well as in an

efficient systematic search for gene products which are

related to those genes that regulate changes in

receptor number during chronic drug treatment.

If an animal model of drug abuse is to be developed,

consideration must be given to what the model should

represent. Should the model encompass every aspect of

drug-related behavior or should it attempt to emulate

only specific aspects? A complete animal model of

human drug abuse might not be attainable. However, it

should be possible to develop models of specific aspects of the total human condition. For example, it

may be possible to identify or develop genetic stocks

of animals that differ in avidity for specific drugs,

initial sensitivity, tolerance development, and severity of a withdrawal syndrome. Once these models

have been developed, they can be used to test hypotheses relevant to the mechanisms underlying the

differences in avidity, sensitivity, etc., as well as

being of value in the search for biological markers.

The prerequisite for identifying a biological marker

for drug abuse is that some aspect of drug dependence

is influenced by genetic factors. Therefore, it seems

reasonable to suggest that the first step that should

be taken is to ascertain whether genetically influenced variation exists in appropriate animal populations. In this regard, perhaps the easiest approach,

because of the relative availability, is to screen

inbred strains for differences in the phenotype\

(trait) of interest.

Inbred Strains

Inbreeding is achieved by mating closely related individuals. In commonly available rodent stocks, this

has generally been achieved by brother-sister matings.

Inbreeding leads to an increase in genetic uniformity

within a strain, and after approximately 20 generations

of inbreeding a strain can be considered genetically

uniform; homozygosity will have been attained at all

loci with the possible exception of those alleles

which are carried on the sex chromosomes. The process

59
of inbreeding is nondirectional. Alleles are fixed in

a particular configuration by chance with the exception of those traits that are related to reproductive

fitness. Any differences found between inbred strains

are fortuitous. In addition, no assurance can be made

that a given inbred strain will have in its genome all

of the genes that influence a drug-related behavior.

Indeed, in view of the fact that most inbred rat and

mouse strains can be traced to a smell number of

animals, in some cases two, it is highly probable that

any strain will have only a sampling of all of the

possible alleles that will affect a trait of interest.

A highly recommended approach regarding the use of

inbred strains is to screen a number of strains. A

relationship between two traits in a given strain may

be unique. However, if this relationship is seen in a

number of strains, it is more likely that these two

traits are associated genetically. A major advantage

of inbred strains is their constancy over time and in

different laboratories. Such constancy increases

experimental reliability and may allow collaborative

studies that are carried out at different locations,

thereby facilitating the screening of a large number

of strains.

A classic example of consistent findings within inbred

strains is the alcohol preference of various mouse

strains; a number of investigators have noted that

C57BL mice uniformly prefer ethanol-containing solutions, whereas DBA mice avoid ethanol-containing

drinking fluids (McClearn and Rodgers 1959; Russell

and Stern 1973; Randall and Lester 1975; Pickett and

Collins 1975). Strain differences with respect to

other alcohol-related behaviors have also been well

documented. Included among these are strain differences in acute sensitivity to the effects of ethanol

on locomotor activity (Randall et al. 1975; Oliverio

and Eleftheriou 1976), passive avoidance conditioning

(MacInnes and Uphouse 1973), body temperature (Moore

and Kakihana 1978; Crabbe 1983), and duration of

ethanol-induced loss of the righting response (sleep

time) (Kakihana et al. 1966; Damjanovich and MacInnes

1973; Crabbe 1983). Strain differences in the development of tolerance to ethanol have also been described (Moore and Kakihana 1978). as have strain differences in the severity of an ethanol withdrawal

syndrome (Kakihana 1979; Crabbe et al. 1983). The

vast majority of the research relating to strain

analyses has been descriptive in nature. Very few of

these studies have attempted to determine such fundamental parameters as dominance relationships or an

estimate of the number of genes involved in regulating

the trait being measured; nor have the majority of

60
these studies attempted to provide biological explanations for these strain differences in ethanol-related

behaviors. Clearly, very few of the studies concerning strain differences in alcohol- related behaviors

have attempted to identify a marker that could be of

value in predicting differential sensitivity to the

trait being studied.

Strain differences in response to drugs of abuse have

also been reported. For example, strain differences

in response to amphetamines (Anisman 1975; Anisman and

Kokkinidis 1975; Bovet and Oliverio 1967), nicotine

(Morrison and Lee 1968; Hatchell and Collins 1977;

Marks et al. 1983; Miner et al. 1984), ha1lucinogens

(Lush 1975; Tilson et al. 1975), benzodiazepines

(Rambert et al. 1976), barbiturates (Randall and Lester 1974; Belknap et al. 1973), and opiates (Castellano and Oliverio 1975; Horowitz 1976; Collins et al.

1977) have been reported. In general, these studies

have involved examinations of different mouse or rat

strains and have investigated strain differences in

acute responses to these drugs. However, some of

these studies have assessed strain differences in drug

self administration (Horowitz 1976) or severity of a

withdrawal syndrome (Belknap et al. 1973). As is

the case with the research done in the alcohol field,

few of these investigations have attempted to estimate

gene number or dominance relationships and absolutely

no concerted attempt has been made to identify potential biological markers. Nonetheless, the fact that

strain differences have been identified argues that

genetic factors are important in regulating response

to drugs of abuse in animals. This finding supports

the assumption that biological markers might be found.

Genetic Crosses

Once inbred strain differences in a drug-related response have been identified, genetic crosses can be

developed. Such crosses can be invaluable in testing

hypotheses of interest. For example, it seems highly

likely that inbred strains which differ in some trait

of interest could be identified. These strains will

likely differ in a number of traits. Segregation

analysis can be used to test the association between

traits. Inbred strains could be crossed to obtain the

first filial (F1) generation. If a number of strains

are intermated in all possible ways, a diallel

analysis can be made. Such an analysis allows an

estimate of dominance relationships. Once the F1

generations have been obtained, these animals can be

intermated to obtain the F2 generation. The F2 generation is invaluable for assessing linkage in that

genes which are not closely linked will segregate in a

quasi-independent fashion into the F2 generation.

61
Pickett and Collins (1975) used such an analysis to

investigate the relationship between ethanol preference and brain serotonin content. Examples of other

investigations that have used F1 analyses to investigate genetic regulation include studies of the effects

of morphine on activity (Shuster et al. 1973; Castellano and Oliverio 1975) and the effects of amphetamine

on activity (Bovet and Oliverio 1973). None of these

studies have attempted to identify biological markers

for the drug-induced behaviors being studied. However, the potential for such studies seems highly

promising.

Recombinant Inbred Strains

Perhaps the moat powerful tool currently available to

test the relationship between two or more traits is

recombinant inbred strains. Recombinant inbreds are

constructed by first developing an F2 or preferably

the F3 or F4 generations derived from crossing two

inbred strains. Brother-sister mating is initiated in

these foundation stocks. As was the case with inbred

strains, genetic uniformity is achieved as inbreeding

proceeds. The F3 or F4 generations are recommended as

the foundation stock rather than the F2 because a

maximum reshuffling of genes is obtained. Linkage is

broken down and only genes that are very closely

linked wi11 be found in association with one another.

This, of course, would be a very desirable trait for a

biological marker. In theory, an F3 or F4 generation

could serve as an adequate test group for a correlational analysis. However, since each animal is genetically unique, any variance involved in measuring

the traits of interest will affect the correlation

detected. For many traits, it may be desirable to

have a test population where replicate samples can be

obtained. Recombinant inbreds are ideal for such a

situation because replicate samples can be obtained

from each recombinant inbred stock. Any variance seen

within each recombinant inbred provides an estimate of

environmental influence on the traits of interest.

In view of the fact that recombinant inbreds are

selected from F3 or F4 stocks derived from two inbred

strains, these animals should be invaluable in testing

linkage relationships. If two traits of interest are

found in association in the parental inbreds and none

of the recombinant inbreds demonstrate this association, it can be assumed that the traits were not

closely linked in the originating inbred strains. If

some, but not all, of the inbreds exhibit the traits

of interest, it can be assumed that the genes which

regulate these traits are closely linked. If 100% of

the recombinant inbreds demonstrate both traits, identity of genetic control, pleiotropy, or extremely

62
close linkage probably explain the genetic relationship. Because knowledge of the precise genetic relationship between traits of interest would be invaluable when attempting to generalize any findings made

in animals to the human condition, the use of recombinant inbreds in the identification of biological

markers may be the most powerful tool currently

available.

This reviewer could find no examples in the literature

where recombinant inbreds have been used to study the

actions of drugs of abuse. This is likely due to the

fact that such animals are not readily available.

However, Dr. Ben Taylor at Jackson Laboratories has

developed a number of recombinant inbred mouse

strains. The value of these animals in drug abuse

related research could prove to be considerable, not

just for the search for biological markers, but also

in any experiment that attempts to assess the relationship between two or more behavioral, biochemical,

or physiological traits.

Selectively Bred Lines

Selective breeding involves a systematic attempt to

develop an animal with a specific trait. Unlike inbreeding, selective breeding is directional. Thus,

the probability that two traits will be found in

association with one another by chance in selectively

bred stocks is diminished. Selective breeding will be

successful only if the foundation stock displays

genetically determined variation in the trait of interest. If this is the case, mating of extremes in

the population (i.e., mating the highest with the

highest and the lowest with the 1owest) will result in a

change in the mean value within the selected populations. If selection is accompanied by outbreeding,

the problems associated with inbreeding are minimized;

i.e., the high line(s) and the low line(s) will become

similar genetically only at those genetic loci that

affect the trait of interest. After approximately 20

generations of selective breeding, the high line will

be homozygous at all of the genetic loci that contribute to a high value while the low line will be homozygous at all the loci that contribute to a low value

of the trait of interest. Assuming that outbreeding

has been maintained, the lines will exhibit the same

diversity at all other genetic loci as existed in the

founding population.

The founding population used to develop selected lines

is extremely critical. The best population to use

would be one that has maximum genetic diversity. Such

a stock, usually referred to as a heterogeneous stock,

can be developed by systematically breeding together

63
inbred strains. The best examples of such a stock are

the HS mouse stock that is available at the University

of Colorado Institute for Behavioral Genetics

(McClearn 1972) and the National Institutes of Health

heterogeneous stock rat strain (Hansen and Spuhler

1984). These stocks were derived by systematically

breeding together eight inbred mouse or rat strains.

Therefore, these stocks have considerable genetic

diversity and the likelihood of identifying important

genes or gene products is maximized. If a heterogeneous stock is not available, an F3 generation

derived from two inbreds that differ maximally in the

trait of interest may be suitable.

Selective breeding has been used successfully in a

number of areas related to alcoholism. For example, a

number of stocks have been developed that differ in

alcohol preference (Hardones et al. 1950; Eriksson

1968; Li et al., this monograph). Most of these

stocks were obtained by selectively breeding two lines

that differ in free-choice ethanol consumption.

Selectively bred lines of animals have also been obtained that differ in acute sensitivity to ethanol

(McClearn and Kakihana 1981; Riley et al. 1977). One

of these selections, that of McClearn and Kakihana

(1981). involved the development of two lines of mice

--Long-Sleep (LS) and Short-Sleep (SS)—that differ

in duration of ethanol-induced anesthesia (sleep

time). Riley et al. (1977) successfully selected two

rat lines, Most Affected and Least Affected, that

differ in the effect of ethanol on motor activity.

Others (Goldstein and Kakihana 1975; McClearn et al.

1982) have developed mouse lines that differ in duration or intensity of a withdrawal syndrome following

chronic ethanol treatment. Al though these various

lines have served as valuable tools in testing hypotheses that may explain the differing phenotypes,

these animals have not been used in a systematic

search for biological markers of alcoholism.

Unfortunately, very few investigators have used the

selective breeding approach to study drugs of abuse.

The only selection that this reviewer is aware of

involving a drug of abuse involves morphine. Nichols

and Hsiao (1967) selectively bred for high and low

consumption of, or preference for, morphine after the

rats had been chronically treated with morphine and

withdrawn. This selection was quits successful in

that a fourfold difference in consumption of morphine

was detected as early as the third selected generation. Judson and Goldstein (1978) carried out an

abbreviated selection study in Swiss Webster mice

where lines that differed in levorphanol-induced

alterations in motor activity were sought. After

64
three generations, a 3.5-fold difference between the

high (runner) and low (nonrunner) lines was evident.

More recently, Belknap et al. (1983) have reported

their success in developing two lines of mice that

differ in morphine-induced analgesia as measured with

the hot-plate test. This selection is still underray. As of the fourth selected generation, complete

divergence of the high and low analgesia lines has not

been attained. Nonetheless, the high and low lines

differ to a greater degree than do any of the inbred

mouse strains that have been tested. Therefore, it

seems reasonable to suggest that such animals would be

invaluable in the identification of reasons for differences in sensitivity to morphine as well as in the

identification of potential biological markers.

Selection processes also may be invaluable in identifying pleiotropic genes. An example that validates

this assertion comes from the work of DeFries et al.

(1978) who selectively bred lines of mice that differ

in open field activity. These lnvestigators selected

duplicate high, low, and control activity mouse lines,

i.e., six separate mouse lines. The foundation stock

was an F3 generation derived from crossing BALB/cJ

mice with C57BL/6J mice. Activity in an open field

arena was measured. Two high lines, two low lines,

and two control (randomly bred) lines were selected

with outbreeding (avoiding common grandparents) being

maintained. The replicate lines avoid or minimize

potential problems associated with breeding of a

finite number of animals. A steady divergence in

activity has been obtained. Interestingly, within 10

to 15 generations, both of the low lines were nearly

100% albino. Apparently, the same gene(s) that influences coat color (albinism) also regulates locomotor activity in an open field arena. Thus, it could

be argued that albinism in the mouse is a biological

marker for a behavioral trait, i.e., low open field

activity. DeFries has argued that low open field

activity may well be an indication of high anxiety.

If so, these mouse stocks could be valuable research

tools for studying anxiety. Even if this is not the

case, the DeFries activity mice provide considerable

support for the notion that biological markers of drug

abuse may be found if appropriate selectively bred

lines are developed.

DISADVANTAGES OF ANIMAL MODELS

While animal models of drug abuse afford a number of

advantages for the identification of biological

markers, a number of potential disadvantages exist.

Perhaps the most important of these is that commonly

used laboratory animal stocks may not have the appropriate genome to develop a complete model that mimics

65
in all rays the human condition. It may not be accidental that alcoholism and drug abuse appear to be

uniquely human conditions. Species other than man may

not possess all of the genes required to elicit a fullblown dependence that resembles the human condition,

Even where more limited aspects of drug dependence are

being studied with animal models, caution must be

taken when extending any potential findings in animals

to humans. It is possible that animals differ in

avidity for alcohol or drugs for reasons that are

inoperative in humans.

Another potential disadvantage of developing animal

models relates to the expense. If recombinant inbreds

or selectively bred lines are to be developed, it will

likely be several years before valuable animals will

be generated. If selected lines are to be used,

special consideration must be given to the selection

phenotype. Unexpected results can sometimes be obtained. For example, we have recently determined that

a very important factor which influences the difference between the LS and SS mice to an acute dose of

ethanol relates to the fact that these animals were

selected for differences in sleep time following the

intraperitoneal injection of a 30% weight/volume

ethanol solution (Gilliam and Collins 1983); i.e.,

some of the difference in response to ethanol between

the LS and SS lines relates to the fact that the

ethanol was administered via a route that is unimportant in man. Thus, before selection is even initiated, the response to be measured, the route of

administration of the drug, and numerous other factors

should be considered carefully. This is especially

the case if the intent of the selection process is to

emulate the human condition.

A number of additional cautions must be considered

when a search for biological markers is undertaken.

The fact that a given gene product seems to be linked

to those genes involved in regulating drug abuse related behavior in mice or rats is no assurance that

linkage will also be seen in man. If the gene product

of drug abuse genes can be studied in both animals and

man, the chances of correctly identifying a marker

seem greater. The advantage of using animal models

will be greatest if the direct gene products of drug

abuse genes or pleiotropically regulated gene products

can be identified.

CONCLUSIONS

The search for biological markers of drug abuse can be

facilitated by the development of appropriate animal

66
models. These models offer the potential for invasive testing as well as the ability to more carefully control genotype than is the case in humans.

However, disadvantages exist. These include expense

and the possibility that no animal model will completely satisfy the requirements needed to mimic the human

condition. Nonetheless, the advantages far outweigh

the disadvantages, particularly when the potential

utility of animal models in answering important mechanistic questions regarding drug abuse is considered.

REFERENCES

Anisman, H. Differential effects of scopolamine and

d-amphetamine on avoidance: Strain interactions.

Pharmacol Biochem Behav 3:809-817, 1975.

Anisman. H., and Kokkinidis. L. Effects of scopolamine, d-amphetamine and other drugs affecting catecholamines on spontaneous alternation and locomotor

activity in mice. Psychopharmacologia 45:55-63,

1975.

Belknap, J.K.; Ondrusek, G.; and Waddingham, S. Barbiturate dependence in mice induced by a single

short-term oral procedure. Physiol Psychol 1:394-

396, 1973.

Belknap, J.K.; Halti, R.R.; Goebel, D.H.; and

Lame’, M. Selective breeding for high and low

levels of opiate-induced analgesia in mice. Behav

Genet 13:383-396, 1983.

Bovet, D., and Oliverio, A. Decrement of avoidance

conditioning performance in inbred mice subjected

to prolonged sessions: Performance recovery after

rest and amphetamine. J Psychol 65:45-55, 1967.

Bovet, D., and Oliverio, A. Pharmacogenetic aspects

of learning and memory. In: Bloom, F.E., and Acheson, G.H., eds. Brain Nerves and Synapses.

Vol. 4. Basel: Karger, 1973. pp. 18-28.

Castellano, C., and Oliverio, A. A genetic analysis

of morphine-induced running and analgesia in the

mouse. Psychopharmacologia 41:197-200, 1975.

Collins, R.L.; Horowitz, G.P.; and Passe, D.H. Genotype and test experience as determinants of sensitivity and tolerance to morphine. Behav Genet

7:50, 1977.

Crabbe, J.C. Sensitivity to ethanol in inbred mice:

Genotypic correlations among several behavioral responses. Behav Neurosci 97:280-289, 1983.

Crabbe, J.C.; Young, E.R.; and Kosobud, A. Genetic

correlations With ethanol withdrawal severity.

Pharmacol Biochem Behav 18(Supp.I):541-547, 1983.

Damjanovich. R.P.. and MacInnes, J.V. Factors involved in ethanol narcosis: Analysis in mice of

three inbred strains. Life Sci 13:55-65, 1973.

67
DeFries, J.C.; Gervais, M.C.; and Thomas, E.A. Response to 30 generations of selection for open field

activity in laboratory mice. Behav Genet 8:3-13,

1978.

Eriksson, K. Genetic selection for voluntary alcohol

consumption in the albino rat. Science 159:739-741,

1968.

Gilliam, D.M., and Collins, A.C. Concentration-dependent effects of ethanol in long-sleep and shortsleep mice. Alcohol Clin Exp Res 7:337-342, 1983.

Goldstein, D.B., and Kakihana, R. Alcohol withdrawal

convulsions in genetically different populations of

mice. Adv Exp Med Biol 59:343-352, 1975.

Hansen, C., and Spuhler, K. Development of the National Institutes of Health genetically heterogeneous rat stock. Alcohol Clin Exp Res 8:477-479,

1984.

Hatchell, P.C., and Collins, A.C. Influence of genotype and sex on behavioral tolerance to nicotine in

mice. Pharmacol Biochem Behav 6:25-30, 1977.

Horowitz, G.P. Morphine self administration by inbred

mice: A preliminary report. Behav Genet 6:109-110,

1976.

Judson, B.A., and Goldstein, A. Genetic control of

opiate-induced locomotor activity in mice. J

Pharmacol Exp Ther 206:56-60, 1978.

Kakihana, R. Alcohol intoxication and withdrawal in

inbred strains of mice: Behavioral and endocrine

studies. Behav Neur Biol 26:97-105. 1979.

Kakihana, R.; Brown, D.R.; McClearn, G.E.; and Tabershaw, I.R. Brain sensitivity to alcohol in inbred

mouse strains. Science 154:1574-1575, 1966.

Lush, I.E. A comparison of the effect of mescaline on

activity and emotional defaecation in seven strains

of mice. Br J Pharmacol 55:133-139, 1975.

MacInnes, J.W.., and Uphouse, L. L. Effects of alcohol

on acquisition and retention of passive-avoidance

conditioning in different mouse strains. J Comp

Physiol Psych 84:398-402, 1973.

Mardones, R.J.; Segovia, W.N.; and Hederra, D.A. The

inheritance of alcoholism in rats: I. Behavior of

the first generation of addicted rats reared on a

diet lacking in factor N. Bol Soc Biol Santiago 7:

61-62, 1950,.

Marks, H.J.; Burch, J.B.; and Collins, A.C. Genetics

of nicotine response in four inbred strains of mice.

J Pharmacol Exp Ther 226:291-301, 1983.

McClearn. G.E. Genetics as a tool in alcohol research. Ann N Y Acad Sci 197:26-31, 1972.

68
McClearn, G.E., and Kakihana, R. Selective breeding

for ethanol sensitivity: Short-sleep and long-sleep

mice. In: McClearn. G.E.: Deitrich. R.A.: and

Erwin, V.G., eds. Development of Animal Models as

Pharmacogenetic Tools. DHHS Pub. No. (ADM)81-1133

Washington, D.C.: Supt. of DOCS., U.S. Govt. Print.

Off., 1981. pp.147-160.

McClearn, G.E., and Rodgers, D.A. Differences in

alcohol preference among inbred strains of mice. Q

J Stud Alcohol 20:691-695, 1959.

McClearn, G.E.; Wilson, J.R.; Petersen, D.R.; and

Allen, D.L. Selective breeding in mice for severity

of the ethanol withdrawal syndrome. Subst Alcohol

Actions Misuse 3:135-143, 1982.

Miner, L.L.; Marks, E.J.; and Collins, A.C. Classical

genetic analysis of nicotine-induced seizures and

nicotinic receptors. J Pharmacol Exp Ther 231:545-

554. 1984.

Moore, J.A., and Kakihana, R. Ethanol-induced hypothermia in mice: Influence of genotype on development of tolerance. Life Sci 23:2331-2338, 1978.

Morrison. C.F.. end Lee, P.N. A comparison of the

effects of nicotine add physostigmine on a measure

of activity in the rat. Psychopharmacologia l3:210-

221, 1968.

Nichols, J.R., and Hsiao, S. Addiction liability of

albino rats: Breeding for quantitative differences

in morphine drinking. Science 157:561-563, 1967.

Oliverio, A., and Eleftheriou, B.E. Motor activity

and alcohol: Genetic analysis in the mouse. Physiol

Behav 16:577-581, 1976.

Pickett, R.A., and Collins, A.C. Use of genetic analysis to test the potential role of serotonin in

alcohol preference. Life Sci 17:1291-1296, 1975.

Rambert, F.A.; Amaluit, S.; and Dutiel, J. Problemes

pose par le choix d’ une souche de souris pur l’

etudes des anxiolytiques. J Pharmacol 7:517-530,

1976.

Randall, C.L., and Lester, D. Differential effects of

ethanol and pentobarbital on sleep times in C57BL

and BALB mice. J Pharmacol Exp Ther 188:27-33,

1974.

Randall, C.L., and Lester, D. Social modification of

alcohol consumption in inbred mice. Science 189:149-

151, 1975.

Randall, C.L.; Carpenter, J.A.; Lester, D.; and Friedman. B.J. Ethanol-induced mouse strain differences

in locomotor activity. Pharmacol Biochem Behav

3:533-535, 1975.

Riley, E.P.; Worsham, E.D.; Lester, D.; and Freed, E.

X. Selective breeding of rats for differences in

reactivity to alcohol. J Stud Alcohol 38:1705-1717,

1977.

69
Russell, K.E., and Stern, M.H. Sex and strain factors

in voluntary alcohol intake. Physiol Behav 10:641-

642, 1973.

Shuster, L.; Webster, G.W.; Yu, G.; and Eleftheriou,

B.E. A genetic analysis of the response to morphine

in mice: Analgesia and running. Psychopharmacologia

42:249-254, 1975.

Tilson, H.A.; Haisel, A.S.; Jourdan, M.G.; and Rech,

R.H. Comparison of the effects of d-amphetamine and

lysergic acid diethylamide in two strains of rats

having different behavioral baselines. Behav Biol

17:463-471, 1975.

ACKNOWLEDGMENTS

Work from the author’s laboratory was supported by

grants from the Department of Health and Human

Services (DA03194 and AA03527).

AUTHOR

Allan C. Collins, Ph.D.

School of Pharmacy and

Institute for Behavioral Genetics

University of Colorado

Boulder, Colorado 80309

70
Genetic Markers of Drug Abuse in

Mouse Models

Louis Shuster, Ph.D.

The brain is a very complex organ. This complexity presents

certain difficulties in the analysis of genetic determinants of

responses to drugs of abuse. One gram of cortical grey matter

contains about 200 million neurons. Each of these makes connections with several thousand other neurons. A single neuron can

have receptors for several neurotransmitters. Neurons also exhibit plasticity--that is, the nature of their neurotransmitters and

receptors can change with time. About 30,000 genes are expressed

specifically by the brain (Sutcliffe and Milner 1984).

These characteristics permit several generalizations about the

genetics of responses to neurotropic drugs. A drug response involves many separate components, such as drug metabolism; neurotransmitter turnover; peripheral as well as central responses; and

interactions between several neurotransmitters, brain regions, and

receptors. It is therefore reasonable to assume that a particular drug response, such as CNS stimulation, can be affected by

many different genes. A corollary assumption is that most of the

mutations that affect such a response will be pleiotropic. For

example, a change in catecholamine metabolism that affects responsiveness to amphetamine could also alter spontaneous behavior,

cardiovascular responses, etc. Furthermore, the same phenotypic

response, such as increased locomotor activity after a stimulant

drug, could result from mutations at several different loci.

In spite of these complexities, it is nevertheless possible to

distinguish specific mutations that alter various responses to

drugs of abuse. Work with mouse models provides some hope that we

will eventually be able to identify and isolate genetic determinants of drug abuse and link them to known genetic markers.

In the following discussion, it is taken for granted that proof of

a genetic component in drug responses is no longer necessary

(Nebert and Felton 1976). The suitability of the various mouse

models that are available will depend on the aims of any given

research project. These aims can be classified as follows:

71
TO PROVIDE A REPRODUCIBLE MODEL OF GENETIC DIFFERENCES

Many inbred lines of mice are readily available from commnercial

suppliers. They were produced by over 20 generations of full-sib

matings and are at least 99.9% homozygous. The simplest way to

look for a strain difference in responses to morphine, cocaine, or

ethanol is to consult Taylor's 1972 genetic similarity matrix.

There is a wide divergence between C57B1/6 and DBA/2, for example.

Several differences have been documented in literature for the

responses of these two strains to narcotics and to ethanol (table

1). Additional differences are described by Oliverio et al.

(1983).

For example, in C57B1/6 but not DBA/2 mice, morphine increases

motor activity, the release of striatal dopamine, and the level of

cyclic AMP in the striatum. DBA/2 mice, on the other hand, show

more analgesia after morphine, sleep three times longer after

ethanol than C57B1/6 mice, and are susceptible to audiogenic

seizures. Exposure to tobacco smoke or the injection of muscimol

increases the motor activity of DBA/2 mice, but decreases the

activity of C57B1/6 mice. There are also striking differences in

voluntary consumption. C57B1/6 mice prefer dilute solutions of

ethanol or morphine to water, but DBA/2 mice do not. Crabbe et

al. (1981) reported that the pituitaries of C57B1/6 mice contain

twice as much beta endorphin as those of DBA/2 mice. Sprott

(1975) has summarized behavioral differences.

Candidate strains can also be selected on the basis of differences

in neurochemical parameters, such as dopamine turnover or norepinephrine content. These may be found in the compilation of Ingram

and Corfman (1980). Mendelian analysis of differences in narcotic

responses can be carried out by making appropriate crosses and

backcrosses. Such crosses have demonstrated, for instance, that

the stimulation of motor activity by morphine behaves as an autosomal dominant trait (Castellano and Oliverio 1975). Comparisons

of C57B1/6 and DBA/2 mice have also permitted some correlations

between morphine responses and striatal dopamine activity (Racagni

et al. 1979). In general, inbred lines provide readily available

and consistently reproducible models of high responders and low

responders. However, these lines differ in thousands of genetic

loci and cannot easily be used to establish linkage or to isolate

the determinants which control drug responses.

TO ESTABLISH LINKAGE

Bailey (1971) developed homozygous inbred lines from the F2

generation of crosses between two inbred strains. These recombinant-inbred (RI) lines provide a replicable sampling of progenitor genes in fixed combinations. Linkage markers are provided by

histocompatibility testing of the RI lines and accessory congenic

lines. The advantage of using RI lines is that extensive breeding

is not required to establish linkage. However, they are most useful when the trait in question is under the control of a single

72
TABLE 1

Some Neuropharmacological Differences

Between C57BL/6 AND DBA/2 Mice

73
TABLE 1 (continued)

74
gene (Taylor 1978). Table 2 lists some RI analyses of responses

to drugs of abuse. In most cases, the establishment of linkage

has not been possible because multiple genes are involved.

One interesting benefit of testing RI lines has been the discovery

of one line, CXBK, that is characterized by a low analgesic response to morphine and a decreased number of narcotic receptors in

the brain (Baran et al. 1975). These mice also have a low analgesic response to acupuncture (Pomeranz 1978). to the injection of

D-amino acids (Cheng and Pomeranz 1979). and to stress (Miczek et

al. 1982).

Congenic lines can also be used to establish linkage. These are

developed by repeated backcrossing to one progenitor strain from

the F1 generation until homozygosity is achieved (Bailey 1975).

The resulting lines have a very small portion of parental genome

A. defined by histocompatibility testing, associated with the

other parental genome B. If a congenic line which is over 99.9%

congenic with parent B responds to a drug like parent A, then one

can establish the linkage of that drug response to a defined portion of a particular A chromosome. Successful application of this

approach to drugs of abuse has not yet been reported.

TO ISOLATE GENETIC DETERMINANTS

Selective Breeding

For careful genetic analysis of mechanisms of drug action, one

would like to have two populations of animals which differ only in

a single genetic determinant that controls a particular drug response. Selective breeding should, in theory, isolate those genes

which control the selected drug response. The foundation stock

should be genetically diverse. If necessary, such diversity can

be achieved by interbreeding up to eight inbred lines, as was done

in the selection of Long-Sleep (LS) and Short-Sleep (SS) mice for

differential responses to ethanol (McClearn and Anderson 1979).

The importance of carrying replicate lines and keeping stocks

large enough to prevent inbreeding has been emphasized by Crabbe

et al. (1983). They are developing lines that differ in the

intensity of withdrawal after ethanol. Interesting correlations

have been drawn between genetic susceptibility to the depressant

effects of ethanol and the ability of ethanol to fluidize neuronal

membranes (Goldstein et al. 1982). However, selective breeding is

not without problems. Achieving homozygosity is slow and tedious.

Until homozygosity is reached, it is almost impossible to replicate experiments, because the genotypes are continually changing.

The drug tests upon which selection is based are likely to pick up

pleiotropic genes which can affect the responses to a variety of

CNS-active agents. For instance, the LS and SS lines also differ

in their responses to barbiturates, adenosine derivatives, and

other CNS depressants (Dudek et al. 1984;

Proctor and Dunwiddie 1984).

Howerton et al. 1983;

Selective breeding for sensitivity

to anesthesia by nitrous oxide has also revealed a lack of

specificity (Koblin et al. 1984).

75
TABLE 2

The Use of Recombinant-Inbred Strains in

Genetic Analysis of Some Drug Responses

Drug Response Conclusion Reference

Scopolamine Exploratory behavior

al. 1974

Oliverio et

al. 1973

One locus on chromosome

4 controlling

exploratory behavior.

plus one on chromosome

17 determining response

to scopolamine

Chlorpromazine Exploratory behavior Two controlling genes, Castellano et

one on chromosome 9

Ethanol Motor activity

Morphine

analgesia

Morphine

hot plate

Motor activity

Morphine Tall-flick analgesia

Morphine Motor activity

Morphine

Naloxone

Sensitization of

motor response

Binding to narcotic

receptors

Opioids

Withdrawal jumping

Binding to brain

membranes

Oxotremorine Hypothermia

gene

Cocaine Liver damage

d-Amphetamine In vivo metabolism

Recessive allele

Eam on chromosome 7

determines marked

decline in activity

More than 2 loci

Bore than 2 loci

2 or more loci

no linkage

2 or more loci

no linkage

no linkage

Two or more genes

controlling

number of receptors

Multiple determinants

Multiple genes

Single controlling

Multiple genes

Multiple genes

Oliverio and

Eleftheriou

1976

Oliverio et

al. 1975

Oliverio et

al. 1975

Shuster et al.

1975

Shuster et al.

1975

Shuster 1975

Baran et al.

1975

Shuster 1984

Reith et al.

1981; Jacob et

al. 1983

Lush and

Andrews 1978

Shuster 1984

Shuster 1984

76
Defined Mutants

Hundreds of mutants of inbred mouse lines such as C57B1/6 are

available commercially and, in most cases, the locus Involved has

been established (Green 1981). Screening of these strains for

their responses to drugs of abuse would be a fairly easy way to

isolate genetic determinants. Of course, these genes would have

to be pleiotropic because most of the mutants were first identified by a change in coat color or some other descriptive phenotype l Preliminary work by Katz and Doyle (1980) and by our

laboratory (Shuster 1984) has turned up four mutants of C57B1/6

that differ in one or more responses to morphine (table 3). It is

intriguing that each of these mutations is found on a different

chromosome.

There are other mutants, such as Jimpy, that involve defective

development of the CNS (Law et al. 1978). These can differ in

their responses to morphine, but the defects in the CNS are so

widespread that these mice are not likely to be useful for

studying mechanisms of action.

Sublines

Sublines of several inbred lines have developed as a result of

genetic drift between isolated stocks. The number of gene

differences can be calculated from the number of generations of

inbreeding that took place before and after the stocks were

separated (Bailey 1978). For example, the difference between the

Jackson (J) and Bailey (By) sublines of C57B1/6 is no more than 50

gene pairs. It is extremely Improbable that more than one of

these pairs affects the response to morphine. We have found that

the By subline displays more analgesia after morphine than the J

subline. Crosses and backcrosses have shown that this trait behaves as an autosanal dominant gene (Shuster 1984). Assay of narcotic receptors in the brain has revealed a greater number of mu

receptors in the By mice, but no difference in delta or kappa receptors (Cremins and Shuster 1982). There are no obvious differences in the appearance of the two sublines. However, given the

nature of genes controlling drug responses, It is not surprising

that pleiotropic characteristics have been distinguished; e.g., By

runs more than J after morphine (Shuster 1984). Also, there are

differences in the climbing responseto apomorphine and the running

after amphetamine (table 4). Other sublines that have not been

studied extensively should also provide useful material for

investigating genetic determinants of drugs of abuse. For

instance, the Jackson subline of BALB/c differs from the NIH,

Bailey, and Texas sublines in that unrelated males of the J

subline will fight viciously when housed together. There Is a

marked difference in the enzymes of catecholamine metabolism

(Ciaranello et al. 1974) and in the sensitivity to d-amphetamine

and ethanol (Moisset 1977, 1978). Here is another Instance of a

single pleiotropic gene affecting several drug responses.

77
TABLE 3

Responses of C57Bl/6J and Some Defined Mutants to Morphine

TABLE 4

Some Differences in Drug and Stress Responses Between

the Bailey and Jackson Sublines of C57B1/6 Mice

78
TO DERIVE NEW GENETIC MODELS

New methodologies in genetic engineering hold promise for more

rapid development of new mouse strains that could be very useful

for the study of drugs of abuse. For example, once parthenogenesis has been achieved as a reproducible technique in mice,

both selective breeding and the development of new RI lines could

be carried out much more rapidly because homozygosity could be

achieved in only three or four generations (Csanyi 1984). Gynogenesis--that is, the production of diploid offspring from the

fertilization of eggs with inactivated sperm cells followed by

inhibition of the first mitotic division--has been carried out in

fish, frogs, and newts (Csanyi 1984). Hoppe and Illmensee (1977)

claimed to have produced gynogenic mice by cellular surgery, but

their findings have apparently not been replicated in other laboratories (Anonymous 1984).

The pioneering work of Mintz (1978) provides an interesting new

method for producing defined mouse mutants. Teratoma cells, cultured in vivo or in vitro, are integrated with embryonic cells of

a different strain in order to produce a chimera. If the chimera's germ cells are derived from the teratoma, the chimera can

give birth to mice bearing the teratoma genes, These mice do not

develop tumors. Cultured teratoma cells can be exposed to mutagens and the mutants can then be selected. Mintz has used this

technique to make a mouse model of hypoxanthine-guanine phosphorihosyltransferase deficiency (1978). Application of this

technique to drugs of abuse raises the problem of which responses

can be used in screening. Any tolerance or physical dependence

produced in vitro is unlikely to be a function of genes that are

specifically expressed in the CNS. However, it is conceivable

that mutations affecting the structure and function of cellular

membranes in general would be characterized by altered responses

to ethanol and, perhaps, other drugs as well.

TO STUDY THE ROLE OF ENDOGENOUS OPIOIDS

There is considerable indirect evidence that the release of endogenous opioids plays an important role in many forms of stressinduced analgesia (SIA). We have found important strain and subline-differences in defeat-induced analgesia (Shuster 1984). Repeated defeat produces tolerance and cross-tolerance to morphine

analgesia. Furthermore, tolerant animals exhibit withdrawal jumping when injected with naloxone, i.e., they are physically dependent (Shuster et al. 1983). A similar phenomenon has been reported (Christie and Chesher 1982) when mice are stressed by being

forced to swim.

These observations suggest that suitable mouse models could be

identified to test the hypothesis that repeated stress may produce

a craving for drugs of abuse. They also indicate the potential

value of testing drugs of abuse in strains of mice with different

genetically determined patterns of behavior.

79
RECOMMENDATIONS FOR FUTURE WORK

It is clear from the foregoing survey that mouse models can provide very useful genetic tools for studying the actions of drugs

of abuse. However, only a small fraction of the potentially useful material has even been surveyed for drug responses. It would

be most helpful if NIDA could support the following initiatives:

1.

2.

3.

4.

5.

6.

Publication of a compilation of the known responses of inbred and recombinant-inbred strains, sublines, and mutants

to drugs of abuse, including ethanol. This information is

scattered through the literature. A compilation would make

it much easier for researchers to choose appropriate models

for testing hypotheses about drug abuse.

A systematic survey of drug responses in currently available

strains and mutants which have not yet been tested. Perhaps

such a survey could be carried out on a contract basis. Of

the 500 or so known single gene mouse mutants, only about 15

have been examined even superficially.

Selective breeding for such parameters as voluntary consumption of ethanol and other drugs of abuse and ethanol. A

good example is the selective breeding of strains of rats

showing high and low preference for ethanol solutions (Li,

this volume). A genetic model for self-administration of

such stimulants as cocaine and amphetamine would be very

helpful.

The development of new recombinant inbred lines to be used

for genetic analysis. This development, like selective

breeding, is a slow and tedious process that is not easy to

support from short-term funding. If it could be carried out

at NIDA, important new resources could be made available to

the research community.

Increased emphasis on genetic correlations between behavioral parameters that can involve endogenous opioids and responses to drugs of abuse. These could include stressinduced analgesia, aggression, diurnal changes in activity

and pain responses, etc. Also, the effect of stress and

other pretreatments on voluntary consumption should be

examined.

The selection of teratocarcinoma-derived mutants that differ

in their susceptibility to drugs of abuse and/or neurochemical parameters, such as narcotic receptors, opioid peptides, and neurohormone turnover.

REFERENCES

Anonymous. Illmensee inquiry. Nature 308:394, 1984.

Baer, D.S.; McClearn, G.E.; and Wilson, J.R. Effects of chronic

administration of tobacco smoke to mice: Behavioral and metabolic measures. Psychopharmacology 67:131-137, 1980.

80
Bailey, D.W. Recombinant inbred strains. Transplantation

11:325-327, 1971.

Bailey, D.W. Genetics of histocompatibility in mice. I. New loci

and congenic lines. Immunogenetics 2:249-256, 1975. 

Bailey, D.W. Sources of subline divergence and their relative

importance for sublines of six major inbred strains of mice.

In: Morse, H.C., III, ed. Origins of Inbred Mice. New York:

Academic Press, 1978.

Baran, A.; Shuster, L.; Eleftherfou, B.E.; and Bailey, D.W. Opiate receptors in mice. Genetic differences. Life Sci 17:633-

640, 1975.

Brase, D.; Loh, H.H.; and Way, E.L. Comparison of the effects of

morphine on locomotor activity, analgesia and primary and protracted physical dependence in six mouse strains. J Pharmacol

Exp Ther 201:368-374, 1977.

Castellano, C. Strain-dependent effects of naloxone on discrimination learning in mice. Psychopharmacoloqy 73:152-156, 1981a.

Castellano, C. Strain-dependent effects of the enkephalin analogue FK33-824 on locomotor activity in mice. Pharmacol

Biochem Behav 15:729-734, 1981b.

Castellano, C., and Oliverio, A. A genetic analysis of morphineinduced running and analgesia in the mouse. Psychopharmacology

41:197-200, 1975.

Castellano, C., and Puglisi-Allegra, S. Effects of naloxone and

naltrexone on locomotor activity in C57B1/6 and DBA/2 mice.

Pharmacol Biochem Behav 16:561-563, 1982.

Castellano, C.; Eleftheriou, B.E.; Bailey, D.W.; and Oliverio, A.

Chlorpromazine and avoidance behavior: A genetic analysis.

Psychopharmacologia 34:309-316, 1974.

Cheng, R.S.S., and Pomeranz, B. Correlation of genetic differences in endorphin systems with analgesic effects of D-amino

acids in mice. Brain Res 177:583-587, 1979.

Christie, M.J., and Chesher, G.B. Physical dependence on physiologically released endogenous opiates. Life Sci 30:1173-1177,

1982.

Ciaranello, R.D.; Hoffman, H.J.; Shire, J.G.M.; and Axelrod, J.

Genetic regulation of catecholamine biosynthetic enzymes. II.

Inheritance of tyrosine hydroxylase, dopamine hydroxylase and

phenylethanolamine methyl transferase. J Biol Chem 249:4520-

4536, 1974.

Crabbe, J.C., Jr.; Allen, R.G.; Gaudette, N.D.; Young, E.;

Kosobud, A.; and Stack, J. Strain differences in pituitaryendorphin and ACTH content in inbred mice. Brain Res 219:219-

223, 1981.

Crabbe, J.C.; Kosobud, A.; and Young, E.R. Genetic selection for

ethanol withdrawal severity: Differences in replicate mouse

lines. Life Sci 33:955-962, 1983.

Cremins, J., and Shuster, L. A genetically controlled difference

in morphine analgesia and narcotic receptors in mice. Fed Proc

41:1314. 1982.

Csanyi, V. Neurochemical genetics. Int Rev Neurobiol 25:361-389,

1984.

Dudek, B.C.; Abbott, M.E.; and Phillips, T.J. Stimulant and depressant properties of sedative-hypnotics in mice selectively

81
bred for differential sensitivity to ethanol. Psychopharmacology 82.46-51 1984.

Ebel, A.; Durkin, T.; Ayad, G.; Mack, G.; and Handel P. Genetically determined cholinergic involvement in morphine-induced

behavioral responses in mice.

1980.

J Neuropharmacol 19:423-427,

Filibeck, U.; Castellano, C.; and Oliverio, A. Differential

effects of opiate agonists-antagonists on morphine-induced

hyperexcitability and analgesia in mice.

73:134-136, 1981.

Psychopharmacology

Goldstein. D.B.; Chin, J.H.; and Lyon, R.C. Ethanol disordering

of spin-labeled mouse brain membranes: Correlation with

genetically determined ethanol sensitivity of mice. Proc Nat

Acad Sci USA 79:4231-4233, 1982.

Green, M.C. Genetic Variants and Strains of the Laboratory Mouse.

Stuttgart: Gustav Fischer Verlag, 1981.

Gwynn, G.J., and Domino, E.F. Genotype-dependent behavioral

sensitivity to mu vs. kappa opiate agonists. II. Antinociceptive tolerance and physical dependence. J Pharmacol Exp Ther

231:312-316, 1984.

Hill, R.N.; Clemens, T.L.; Liu, D.K.; Vessel, E.S.; and Johnson,

W.D. Genetic control of chloroform toxicity in mice. Science

190:159-161, 1975.

Hoppe, P.C., and Illmensee, K. Microsurgically produced homozygous-diploid uniparental mice. Proc Nat Acad Sci USA

74:5657-5661, 1977.

Horowitz, G.P.; Whitney, G.; Smith, J.C.; and Stephan, F.K.

Morphine ingestion: Genetic control in mice. Psychopharmacol

52:119-122, 1977.

Howerton, T.C.; O'Connor, M.F.; and Collins, A.C. Lipid solubility is correlated with hypnotic and hypothermic responses of

Long-Sleep (LS) and Short-Sleep (SS) mice to various depressant

drugs. J Pharmacol Exp Ther 227:389-393, 1983.

Ingram, D.K., and Corfman, T.P. An overview of neurobiological

comparisons in mouse strains. Neurosci Biobehav Rev 4:421-435,

1980.

Jacob, J.; Michaud, G.; Nicola, W.A.; and Prudhomme, N. Genetic

differences in opioid binding sites and in antinociceptive

activities of morphine and ethylketocyclatocine. Life Sci

33(Supp. 1):645-648, 1983.

Katz, R.J. The albino locus produces abnormal responses to

opiates in the mouse. Eur J Pharmacol 68:229-232. 1980.

Katz, R.J., and Doyle, Enhanced responses to opiates produced by a single gene substitution in the mouse. Eur J

Pharmacol 67:301-303, 1980.

Kiianmaa, K., and Tabakoff, B. Neurochemical correlates of tolerance and strain differences in the neurochemical effects of

ethanol. Pharmacol Biochem Behav 18(Supp. 1):383-388, 1983.

Koblin, D.D.; Lurz, F.W.; O'Connor, B.; Nelson, N.T.; Eger, E.I.,

II; and Bainton, C.R. Potencies of barbiturates in mice

selectively bred for resistance or susceptibility to nitrous

oxide anesthesia. Anesth Anal 63:35-39, 1984.

Law, P.Y.; Harris, R.A.; Loh, H.H.; and Way, E.L. Evidence for

the involvement of cerebroside sulfate in opiate receptor bind82
ing: Studies with Azure A

Exp Ther 207:458-468, 1978.

Lush, I.E., and Andrews, K.M. Genetical differences in sensitiviand Jimpy mutant mice. J Pharmacol

ty to tremorine and oxotremorine in mice. Eur J Pharmacol

49:95-103, 1978.

Marks, M.J.; Burch, J.B.; and Collins, A.C. Genetics of nicotine

response in four inbred strains of mice. J Pharmacol Exp Ther

226:291-301. 1983.

McClearn, G.E.; and Anderson, S.M. Genetics and ethanol tolerance. Drug Alcohol Depend 4:61-76, 1979.

Miczek, K.A.; Thompson, M.L.; and Shuster, L. Opioid-like

analgesia in defeated mice.

Mintz, B. Gene expression in neoplasia and differentiation.

Harvey Society Lectures, Series 71. New York: Academic Press,

1978. pp 193-245.

Moisset, B. Genetic analysis of the behavioral response to d-amphetamine in mice. Psychopharmacology 53:263-267, 1977.

Moisset, B. Sub1ine differences in behavioral responses to

pharmacological agents. In: Horse, H.C., III, ed. Origins of

Inbred Mice. New York: Academic Press, 1978. pp. 483-484.

Muraki, T.; Uzumaki, H.; and Kato, R. Strain difference in

morphine-induced increase in plasma cyclic AMP and cyclic GMP

levels in relation to locomotor activity in male mice. Psychopharmacology 76:316-319, 1982.

Nabeshima, T., and Ho, I.K. Pharmacological responses to pentobarbital in different strains of mice. J Pharmacol EXp Ther

216:198-204. 1980.

Nebert, D.W., and Felton. J.S. Importance of genetic factors influencing the metabolism of foreign compounds. Fed Proc

35:1133-1141. 1976.

Oliverio, A., and Castellano, C. Genotype-dependent sensitivity

and tolerance to morphine and heroin: Dissociation between

opiate-induced running and analgesia in the mouse. Psychopharmacology 39:13-22, 1974.

0liverio, A., and Eleftheriou, B.E. Motor activity and alcohol:

Genetic analysis in the mouse:

1976.

Physiol Behav. 16:577-581,

Oliverio. A.; Eleftheriou, B.E.; and Bailey, D.W. Exploratory

activity: Genetic analysis of its modifications by scopolamine

and amphetamine. Physiol Behav. lO:893-899, 1973.

Oliverio, A.; Castellano, C.; and Eleftheriou, B.E. Morphine sensitivity and tolerance. A genetic investigation in the mouse.

Psychopharmacologia 42:219-224, 1975.

Oliverio, A.; Castellano, C.; and Puglisi-Allegra, S. Psychopharmaco-genetics of opioids. Trends in Pharmacol Sci 4:350-

352, 1983.

Pomeranz, B. Do endorphins mediate acupuncture analgesia? Adv

Biochem Psychopharm l8:351-360 1978.

Proctor, W.R., and Dunwiddie, T.W. Behavioral sensitivity to

purinergic drugs parallels ethanol sensitivity in selectively

bred mice.

Racagni, G.;

Science 224:519-521, 1984.

Bruno, F.; Juliano, E.; and Paoletti, R. Differential sensitivity to morphine-induced analgesia and motor activity in two inbred strains of mice: Behavioral and biochemical

83
correlations, J Pharmacol Exp Ther 209:111-116, 1979.

Randt, C.T.; Barnett, B.M. McEwen, B.S.; and Quatermain, D.

Amnesic effects of cycloheximide on two strains of mice with

different memory characteristics. Exp Neurol 30:467-474, 1971.

Reith, M.E.A.; Sershen, H.; Vadasz, C.; and Lajtha, A. Strain

differences in opiate receptors in mouse brain. Eur J Pharmaco1 74:377-380, 1981.

Rodgers, D.A. Factors underlying differences in alcohol preference among inbred strains of mice. Psychosom Med 28:498-513,

1966.

Shuster, L. Genetic determinants of responses to drugs of abuse:

An evaluation of research strategies. In: Sharp, C.W., ed.

Mechanisms of Tolerance and Dependence. National Institute on

Drug Abuse Research Monograph 54. DHHS Pub. No. (ADM) 84-1330.

Washington, D.C.: Supt. of Docs., U.S. Govt. Print. Off.,

1984. pp. 50-69.

Shuster, L.; Webster, G.W.; Yu, G.; and Eleftheriou, B.E. A

genetic analysis of the response to morphine in mice:

B.E., ed.. Psychopharmacogenetics. New

1975. pp. 73-97.

York: Plenum Press,

to the brain. In:

Amsterdam: Elsevier

Shuster, L. A pharmacogenetic approach

Leiblich, I., ed. Genetics of the Brain.

Biomedical-Press, l982. pp. 157-176.

Shuster, L. Genetic analysis of morphine effects: Activity,

analgesia, tolerance and sensitization. In: Eleftheriou,

Schlesinger, K., and Sharpless, S.K. Audiogenic seizures and

acoustic priming. In: Eleftheriou, B.E. ed. Psychopharmacogenetics. New York: Plenum Press, 1975. pp. 383-434.

Analgesia and running. Psychopharmacology 42:249-254, 1975.

Shuster, L.; Thompson, H.L.; Miczek K.A.; and Winslow, J.T.

Tolerance to and withdrawal from the effects of chronic defeat.

Neurosci Abstr 9:135, 1983.

Sorenssen, S.; Palmer. M.; Dunwiddie. T.: and Hoffer, B. Electrophysiological correlates of ethanol-induced sedation in differentially sensitive lines of mice. Science 210:1143-1145, 1980.

Sprott, R.L. Behavioral characteristics C57Bl/6J, DBA/2J and

B6D2Fl mice which are potentially useful for gerontological

research.

Spuhler, K.; Hoffer, B.; Weiner, N.; and Palmer, M. Evidence for

genetic correlation of hypnotic effects and cerebellar Purkinje

neuron depression in response to ethanol in mice. Pharmacol

Biochem Behavior 17:569-578, 1982.

Sutcliffe. J.G., and Milner. R.J. Brain specific gene expression.

TIBS 5:95-99, 1984.

Taylor B.A. Genetic relationships between inbred strains of

mice. J Heredity 63:83-86, 1972.

Taylor, B.A. Recombinant-inbred strains: Use in gene mapping.

In: Morse, H.C., III, ed. Origins of Inbred Mice. New York:

Academic Press, 1978.

Van Abeelen, J.H.F., and Boersma, H.J.L.M. A genetically controlled hippocampal transmitter system regulating exploratory

behavior in mice. J Neurogenetics 1:153-158. 1984.

84
ACKNOWLEDGMENTS

The research described here was supported by grants DA-01626 and

DA-01885 from the National Institute on Drug Abuse.

AUTHOR

Louis Shuster, Ph.D.

Department of Biochemistry and Pharmacology

Tufts University School of Medicine

Boston, MA 02111

85
Inheritance of Risk To Develop

Alcoholism

C. Robert Cloninger, M.D.; Soren Sigvardsson, Ph.D.;

Theodore Reich, M.D.; and Michael Bohman, M.D.

When alcoholism is assumed to be a discrete unitary trait, its

inheritance is compatible with multifactorial inheritance

(Cloninger et al. 1978). However, recent findings on ongoing

studies in St. Louis and in Sweden of the inheritance of alcoholism

indicate that alcoholism cannot be considered a homogeneous,

discrete clinical disorder. Our studies indicate that the risk of

alcoholism in the general population and in the relatives of

alcoholics changes between generations, coincident with changes in

social patterns of alcohol consumption. As patterns of exposure

to alcohol change, the expression of underlying risk factors

changes and the individuals at risk change. Our findings with

adoptees have demonstrated the interaction of genetic and

environmental factors in the development of alcoholism and have

also distinguished multiple subtypes of alcoholism that have

distinct genetic and environmental determinants.

Recognition of such gene-environment interaction and etiological

heterogeneity will facilitate the identification of genetic and

biological markers for alcoholism. Especially when social

patterns of alcohol and drug intake change between generations, it

becomes difficult to predict who is at risk and, more

specifically, what is the risk of particular individuals. The

implication is that our studies of biological and genetic risk

factors in alcoholism and, more generally substance abuse, must

take into account changing patterns of consumption and consider

the interaction of biological and social risk factors in the

development of clinical disorder. Thus, we must consider both

familial and nonfamilial influences on biosocial risk factors.

SECULAR TRENDS IN ALCOHOL USE AND ABUSE

There are large group differences in the prevalence of alcoholism

due to sociocultural influences . Both consumption and

complications have varied widely from one historical era to

another and currently vary from country to country, between social

classes, between persons of different occupations, and between men

and women (Cahalan 1970; Goodwin 1976). The significance of this

variation is often ignored in biological and genetic research

which presumes that alcoholism is a homogeneous disease entity

86
that is stably inherited from one generation to the next. Often

it is asserted that about 25% of the first-degree relatives of

alcoholics themselves will become alcoholic, with the implicit

assumption or suggestion that this is not much influenced by

secular or local differences in alcohol consumption.

Alcohol consumption per capita in the United States has increased

nearly 50% in the past generation (25 years). Half of the

alcohol drunk by Americans annually is consumed by about 10% of

the population, and the risk of social and medical complications

has increased along with overall consumption. This increased

consumption and complication rate is reflected in findings about

the risk of alcoholism in the general population and in the

relatives of alcoholics. Recent results from our ongoing family

study of alcoholics in St. Louis indicate that the risk of

alcoholism in the general population and in the relatives of

alcoholics is about twice the risk estimated 15 years earlier in

St. Louis (table 1).

TABLE 1

Alcoholism in First-Degree Relatives of White

Alcoholic Probands in St. Louis in 1968 and in 1983

Probands

Sex/Year

Prevalence,

% in General Prevalence of Alcoholic Relatives

Population % of men % of women

Men, 1968 11.4 36.0 6.7

(N=751) (N=111) (N=156)

Men, 1983 28.9 62.7 20.6

(N=1202) (N=319) (N=393)

Women, 1968 2.9

(N=608)

31.6

(N=38)

6.7

(N=60)

Women, 1983 4.3

(N=1802)

61.2 20.9

(N=67) (N=91)

The estimates of the lifetime risk of alcoholism in 1968 were

based on the survey of problem drinking described by Cahalan

(1970). The estimates of the lifetime risk of alcoholism in 1983

is based on the recent report of the Epidemiological Catchment

Area project concerning the St. Louis site by Robins et al.

(1984). Alcoholism was diagnosed in relatives of alcoholic

probands using criteria of Feighner et al. (1972) in a comparable

fashion for both time periods. Both definite and probable

alcoholics, using Feighner et al. criteria, are included so that

87
individuals with multiple social and medical problems are

considered affected in table 1. It is difficult to prefer any

particular diagnostic criteria over others because there is no

work that demonstrates a discrete point of rarity or qualitative

difference between alcoholic and nonalcoholic drinkers, such as

that recently demonstrated for schizophrenia (Cloninger et al.

1985a) or somatoform disorders (Cloninger et al. 1984). In any

case, the same diagnostic criteria were applied in comparable

fashion in the two family studies (Winokur et al. 1970; Cloninger

et al. 1978; Reich et al. 1980). The current pattern of sex

differences is similar to that of 15 years ago: alcoholism is

more common in men than in women, but the risk to relatives is the

same regardless of the sex of the alcoholic proband. However, now

the overall risk of alcoholism is about twice that observed less

than a generation ago.

This difference between generations can also be demonstrated

within our recent family study. This study includes personal

interviews with the family members of 184 hospitalized alcoholics,

129 convicted felons with a history of alcohol abuse, and 47 nonalcoholic controls. This yielded 1,588 personally interviewed

relatives. The risk of alcoholism in first-degree relatives of

alcoholics, regardless of ascertainment source, decreases from the

sibling generation (61% of brothers) to the parental generation

(47%) even without correction for age (Reich et al. 1980).

GENETIC HETEROGENEITY AND GENE-ENVIRONMENT INTERACTION

Rather than assuming that alcoholism is a homogeneous discrete

clinical disorder and asking whether or not it is heritable, we

have sought to clarify the role of etiological heterogeneity,

variable clinical expression, and the interaction of genetic and

environmental risk factors in the Stockholm Adoption Study

(Cloninger et al. 1981, 1984; Bohman et al. 1981, Bohman et al.

1984; Sigvardsson et al. 1984). The subjects included all 862 men

and 913 women of known paternity who were born out of wedlock in

Stockholm from 1930 to 1949 and adopted by nonrelatives at an

early age. Host of the subjects were separated from their

biological relatives in the first few months of life (average: 4

months), and all had their final placement in the adoptive home

prior to age 3 years (average: 8 months). By Swedish practice at

the time, neither the adoptee nor the adoptive parents were

informed about the identity or behavior of the biological parents.

Extensive data about alcohol abuse, criminality, occupational

status, and medical and social history were obtained for all

adoptees and their biological and adoptive parents from several

registers and medical sources. This includes the records of

Temperance Boards and the National Health Insurance Board system,

which includes local insurance agencies, hospitals, and clinics.

The Temperance Board in each community evaluates reports of

intemperance and then registers and imposes fines when

intemperance is confirmed. It also supervises the treatment of

alcoholics and may order involuntary hospitalization if needed.

Other cases of alcoholism in adoptees were identified by records

8 8
of outpatient and inpatient care. The adoptees ranged in age from

23 to 43 years at the time of last information. The data were

available for the lifetime of all subjects (adoptees and both sets

of parents).

The risk of alcohol abuse in adopted men who had biological

parents registered for alcohol abuse was compared to that in other

adoptees (table 2). Alcohol abuse was significantly higher in the

268 adopted-out sons of biological fathers registered for any

alcohol abuse than in the 571 sons of parents with no registered 

alcohol abuse (22.8% versus 14.7%. X

2

 = 8.27, P < .01). In

addition, of the 32 adopted-out sons of alcoholic biological

mothers, the percentage of alcohol abusers was twice as high as

for the 571 sons of nonalcoholic parents (28.1% versus 14.7%, x2 =

4.18, P < .05).

The effect of parental alcoholism on adopted women was more

complex (Bohman et al. 1981). The frequency of alcohol abuse was

more than 3 times greater in the 51 adopted-away daughters of

TABLE 2

Inheritance of Susceptibility to Alcohol

Abuse in the Stockholm Adoption Study

Alcohol Abuse in Alcohol Abuse in Adoptees

Biological Parents Sons Daughters

Father Mother N % N %

No No 571 14.7 577 2.8

Yes No 259 22.4 285 3.5

No Yes 23 26.0 29 10.3

Yes Yes 9 33.3 22 9.1

alcoholic mothers than in the 577 daughters of nonalcoholic

parents (9.8% versus 2.8%, x2 = 7.17, P < .01). In contrast, the

excess of alcohol abusers among the 307 daughters of all alcoholic

fathers was not significant (3.5% versus 2.8%). However, further

analysis revealed that there was a twofold excess of alcoholic

women among the 120 adopted-out daughters whose congenital

background was characterized largely by biological fathers who had

single registrations with the Temperance Board and no associated

criminality (6.7% versus 3.0%, x2 = 4.51, P < .05) (Bohman et al.

1981). Fathers with recurrent alcohol abuse or criminality had no

excess of alcoholic daughters.

Social factors influenced the expression of risk for alcohol abuse

and, in particular, the frequency of registrations with the

89
Temperance Board. Biological fathers with single registrations

for alcohol abuse did not increase the risk of their sons for

either single or recurrent registrations (table 3). However,

fathers with recurrent registrations had an excess of sons with

either single registrations (11.6% versus 6.6%) or recurrent

registrations (15.7% versus 8.6%). Thus, single registrations for

abuse appear to be an expression of the heritable risk for alcohol

abuse in the sons, but not in the fathers. This inconsistency is

largely explained by differences in the social circumstances of

the biological fathers and their adopted-away sons.

TABLE 3

Registrations for Alcohol Abuse in Biological

Fathers and Their Adopted-Away Sons

Father’s No.

Registrations

Number Registrations in Adopted-Away Sons

Sons % only one % recurrent

none 594 6.6 8.6

only one 96 5.2 9.4

recurrent 172 11.6 15.7

The occupational status of the biological fathers tended to be

lover than that of the adoptive fathers, and the occupational

status of the adoptive father influenced the expression of risk

for developing alcoholism. Table 4 shows the frequency of alcohol

abuse registrations in the sons in relation to the number of

registrations of the biological father and the occupational status

of the adoptive father. Adoptive fathers with unskilled

occupations had more sons with alcohol abuse than other adoptive

fathers. If the adoptive father had a skilled occupation, there

was an increased risk of alcohol abuse (single or recurrent) in

sons of biological fathers with recurrent, but not single,

registrations. In contrast, if the adoptive father had an

unskilled occupation, there was an excess of recurrent alcohol

abuse in sons of biological fathers with either single

registrations (17.2% versus 7.6%, x2 = 3.29, P < .06) or recurrent

registrations (19.0% versus 7.6%, x2 = 8.52, P < .01). Thus,

severity of alcohol abuse is a complex product of biological and

social determinants.

The only significant risk factor about the adoptive parents

themselves was low occupational status of the adoptive father.

Alcohol abuse in the adoptive parents was not associated with a

greater risk of alcohol abuse in the adoptees. Only 13% of the 31

90
TABLE 4

Occupational Status of the Adoptive Father and Frequency

of Registrations for Alcohol Abuse in Biological Fathers and

Their Adopted-Away Sons

Adoptive

Father’s

Job

skilled

skilled

skilled

unskilled

unskilled

unskilled

Biological

Father’s

Registered

none

once

recurrent

none

once

recurrent

Number of

Sons

406

67

109

188

29

63

Registration of Sons

% once % recurrent

6.2 7.6

4.5 6.0

12.8** 13.8**

7.4 10.6

6.9 17.2*

9.5 19.0***

* P<.10 compared to 406 low-risk sons

** P<.05 compared to 406 low-risk sons

*** P<.01 compared to 406 low-risk sons

men reared by an alcoholic adoptive parent abused alcohol

themselves compared to 18% of the other 831 sons. only 1 (3.7%)

of the 27 daughters reared by an alcoholic adoptive parent became

an alcohol abuser compared to 3.4% of the other 886 daughters.

Thus, imitation of parental alcohol abuse does not appear to be an

important cause of the familial aggregation of alcoholics.

THREE DISTINCT GENETIC TYPES OF ALCOHOL ABUSERS

By a series of independent tests, we have been able to distinguish

three types of alcohol abusers with distinct clinical features and

different genetic and environmental backgrounds (Bohman et al.

1984). These types have been called milieu-limited alcoholism,

male-limited alcoholism, and antisocial behavior disorder with

alcohol abuse. The clinical picture of men and women in each of

the three types of families is summarized in table 5.

The validation of this typology has proceeded by three steps. The

distinction between milieu-limited and male-limited alcoholism was

first identified by subdividing the male adoptees according to

severity, in accord with earlier clinical work by Kaij (1960).

Among the biological mothers and fathers of mild abusers, there

was an excess of mild abuse, but no criminality. Moderate abusers

had an excess of recurrent alcohol abuse complicated by criminal

behavior in their biological fathers, but no excess of alcohol

91
TABLE 5

Description of Three Family Types of Alcohol Abusers

Family Type Men Women

Milieu-Limited

Male-Limited

Alcoholism

usually mild

adult-onset

no criminality

Alcoholism

recurrent

teenage-onset

alcohol treatment

crime due to abuse

Alcoholism

usually mild

adult-onset

no criminality

Somatization

low frequency

diversiform

Antisocial Criminality Somatization

repeated violence high frequency

untreated abuse hypochondriacal

abuse or crime in their mothers. Severe abusers were heterogeneous: most had biological parent backgrounds indistinguishable

from mild abusers, but had more deprived adoptive parent

backgrounds than the mild abusers; other severe abusers had

biological parents with prominent criminal backgrounds.

Environmental variables did not influence the adoptees’ risk of

moderate abuse, which was highly heritable from father to son.

This male-limited group of alcoholic families was distinguished

from others in which both fathers and mothers were at increased

risk for alcoholism and the alcohol abuse was less severe and

unassociated with criminality. The latter group was called

milieu-limited because the sons were not at increased risk for

alcoholism unless they had an adoptive father with low social

status in addition to a biological parent with mild abuse.

The proposed distinction between these two types was first tested

by predictions about the biological parents of alcoholic women.

The biological. parents of the 913 adopted women were classified

according to the typology derived using discriminant analysis of

the men (Cloninger et al. 1981; Bohman et al. 1981). We predicted

that daughters in families of milieu-limited, but not malelimited, alcoholic parents would be at increased risk for

alcoholism. This prediction was confirmed. This confirmation of

different inheritance patterns in our single large population may

reconcile the apparent discrepancies between earlier adoption

studies which were too small to take such heterogeneity into

account (Cloninger et al. 1985b).

92
The women in the families of male-limited alcoholics had no excess

of alcohol abuse, so we evaluated their lifetime medical histories

and records of sick leave disability which are maintained for

nearly all Swedish citizens through their National Health

Insurance Board. We found that 10% of Swedish women account for

half of all sick leave disability, and that these "somatizers" are

characterized by recurrent complaints of headache, backache, and

vague abdominal symptoms from an early age (Sigvardsson et al.

1984). Further work revealed that these somatizers included women

with two distinct clinical disorders that were each highly

heritable but independent of one another: “high frequency”

somatizers who have frequent disability from abdominal, back, and

psychiatric complaints; and "diversiform" somatizers who have leas

frequent, but more diversely distributed, physical complaints and

rare psychiatric complaints (Cloninger et al. 1984). Using this

independently derived classification, we found an excess of women

with diversiform somatization in families of male-limited, but not

milieu-limited, alcoholics (Bohman et al. 1984). This strongly

supported the distinction between the two types of alcoholics

derived from the male sample, and indicated that the same genetic

predisposition had different expressions depending on the sex of

the individual.

High frequency somatization was not associated with either malelimited or milieu-limited alcoholism. However, the male relatives

of these women were characterized by prominent violent criminal

behavior; they often had multiple registrations for alcohol abuse,

but this seldom required medical or psychiatric treatment (Bohman

et al. 1984). These families appear similar in clinical features

and mode of inheritance to families in which antisocial

personality in men aggregates with Briquet’s syndrome in women,

using diagnostic criteria and subjects from the united States

(Cloninger et al. 1975).

IMPLICATIONS FOR FUTURE RESEARCH

Alcoholism is a complex phenotype that appears to be influenced by

multiple genetic and environmental determinants. In order to

characterize its inheritance, we must take into account changing

social patterns of consumption. Also, distinct familial subtypes

with different patterns of gene-environment interaction need to be

distinguished in order to obtain consistent replicable results.

Our demonstration of genetic heterogeneity in the Stockholm

Adoption Study makes it possible to reconcile the discrepant prior

reports of Roe et al, (1945) and Goodwin et al. (1973). Roe’s

findings are consistent with those observed in our milieu-limited

alcoholics, whereas Goodwin’s findings are similar to our malelimited alcoholics.

Likewise, prior difficulty in obtaining consistently replicable

results with biological and genetic markers in alcoholics may be

attributed to the etiological heterogeneity we have observed.

Individuals in families with alcoholic women more often have

resting electroencephalograms (EEGs) with little alpha activity

and poorly synchronized wave patterns than do individuals in

93
families in which only the men abuse alcohol (Propping et al.

1981). This pattern, called the borderline alpha EEC, is

heritable and shows a dramatic increase in alpha activity in

response to alcohol that is coincident with a pleasant feeling of

calm alertness and tension relief, thereby reinforcing the

tendency to drink alcohol in affected individuals (Propping et al.

1980). These findings are suggestive of an association with

milieu-limited alcoholism, but not male-limited alcoholism. In

contrast, preliminary work in Sweden has demonstrated an

association of low platelet monoamine oxidase activity in malelimited alcoholism, but not milieu-limited alcoholism

(von Knorring et al., in press). Inconsistent prior findings may

be due to neglect of such clinical and etiological heterogeneity.

Further work is needed to specify and refine explicit diagnostic

criteria for clinical subtypes of alcoholism and to identify the

biological and genetic markers which differentiate these

disorders. Recent advances in recombinant DNA technology

(Cloninger et al. 1983) are leading to rapid increases in our

knowledge of the human chromosome map. Quantitative methods for

pedigree analysis (Reich et al. 1980), improved specification of

clinical subtypes, and improved power to detect linkage when it is

present should allow more rapid and consistent progress than in

the past. High priority should be given to studies of families in

which detailed clinical data can be related to putative biological

and genetic markers.

REFERENCES

Bohman, M.; Sigvardsson, S.; and Cloninger, C.R. Maternal

inheritance of alcohol abuse: Cross-fostering analysis of

adopted women. Arch Gen Psychiatry 38:965-969, 1981.

Bohman, M.; Cloninger, C.R.; von Knorring, A.-L.; and Sigvardsson,

S. An adoption study of somatoform disorders. III.

Crossfostering analysis and genetic relationship to alcoholism

and criminality. Arch Gen Psychiatry, 41:872-878, 1984.

Cahalan, D. Problem Drinkers: A National Survey. San Francisco:

Jossey-Bass, 1970.

Cloninger, C.R.; Reich, T.; and Guze, S.B. The multifactorial

model of disease transmission: III. Familial relationship

between sociopathy and hysteria (Briquet’s syndrome). Br J

Psychiatry 127:23-32, 1975.

Cloninger, C.R.: Christiansen, K.O.; Reich, T.; and Gottesman,

I.I. Implications of sex differences in the prevalence of

antisocial personality, alcoholism, and criminality for models

of familial transmission. Arch Gen Psychiatry 35:941-951,

1978.

Cloninger, C.R.; Bohman, M.; and Sigvardsson, S. Inheritance of

alcohol abuse: Cross-fostering analysis of adopted men. Arch

Gen Psychiatry 38:861-868, 1981.

Cloninger, C.R.; Reich, T.; and Yokoyama, S. Genetic diversity,

genome organization,

psychiatric diseases.

and investigation of the etiology of

Psychiatr Dev 3:225-246, 1983.

94
Cloninger, C.R.; Sigvardsson, S.; von Knorring A-L.; and Bohman,

M. An adoption study of somatoform disorders: II.

Identification of two discrete somatoform disorders. Arch Gen

Psychiatry 41:863-871, 1984.

Cloninger, C.R.; Martin, R.L.; Guze, S.B.; and Clayton, P.J.

Diagnosis and prognosis in schizophrenia. Arch Gen Psychiatry

40:15-25, 1985a.

Cloninger, C.R.; Bohman, M.; Sigvardsson, S.; and von Knorring, AL. Psychopathology in adopted-out children of alcoholics: The

Stockholm adoption study. In: Galanter, M., ed. Recent

Developments in Alcoholism. Vol. III.

1985b. pp. 37-51.

New York: Plenum Press,

Feighner, J.; Robins, E.; Guze, S.; Woodruf, R.; Winokur, G.; and

Munoz, R. Diagnostic criteria for use in psychiatric

research. Arch Gen Psychiatry 26:57-63, 1972.

Goodwin, D. Is Alcoholism Hereditary? New York: Oxford

University Press, 1976.

Goodwin, D.W.; Schulsinger, F.; Hermansen, L.; et al. Alcohol

problems in adoptees raised apart from alcoholic biological

parents. Arch Gen Psychiatry, 28:238-243, 1973.

Kaij, L. Alcoholism in Twins: Studies of the Etiology and Sequels

of Abuse of Alcohol. Stockholm: Almquiat and Wiksell, 1960.

Propping, P.; Kruger, J.; and Janah, A. Effect of alcohol on

genetically determined variants of the normal EEG. Psychiatry

Research 2:85-90, 1980.

Propping, P.; Kruger, J.; and Mark, N. Genetic predisposition to

alcoholism. An EEG study in alcoholics and relatives. Hum

Genet 59:51-59, 1981.

Reich, T.; Rice, J.; Cloninger, C.R.; and Lewis, C. The

contribution of affected parents to the pool of affected

individuals: Path analysis of the segregation distribution for

alcoholism. In: Robins, L.N.; Clayton, P.; and Wing, J., eds.

The Social Consequences of Psychiatric Illness. New York: Brunner/Mazel, 1980. pp 91-113.

Robins, L.N.; Helzer, J.E.; Weissman, M.M., Orvaschel, H.;

Gruenberg, E.; Burke, J.D. Jr.; Regier, D.A. Prevalence of

specific psychiatric disorders in three sites. Arch Gen

Psychiatry 41:949-958, 1984.

Roe, A. and Burks, B. Adult adjustment of foster children of

alcoholic and psychotic parentage and the influence of the

foster home. No. 3 Memoirs of Section on Alcohol Studies. New

Haven, Connecticut, Yale University, 1945.

Sigvardsson, S.; von Knorring, A.-L.; Bohman, M.; and Cloninger,

C.R. An adoption study of somatoform disorders: I. The

relationship of somatization to psychiatric disability. Arch

Gen Psychiatry 41:853-862, 1984.

von Knorring, A.-L.; Bohman, M.; von Knorring, L.; and Oreland,

L. Platelet MAO activity as a biological marker in subgroups of

alcoholism. Acta Psych Scand, in press.

Winokur, G.; Reich, T.; Rimmer, J.; and Pitts, F.N. Diagnosis and

familial psychiatric illness in 259 alcoholic probands. Arch

Gen Psychiatry 23:104-111, 1970.

95
ACKNOWLEDGMENTS

This work was supported in part by United States Public Health

Service grants M-03539 from the National Institute of Alcoholism

and Alcohol Abuse, Research Scientist Development Award MH-00048

from the National Institute of Mental Health, and Swedish Medical

Research Council grant B82-21X-03789-11.

AUTHORS

C. Robert Cloninger, M.D.

Theodore Reich, M.D.

Washington University School of Medicine

Department of Psychiatry

4940 Audubon

St. Louis, MO 63110

Michael Bohman, M.D.

Soren Sigvardsson, Ph.D.

University of Umeå

Department of Child & Youth Psychiatry

901 85 Umeå, Sweden

96
Genetic and Biological Markers in

Alcoholism and Drug Abuse

Marc A. Schuckit, M.D.

This chapter reviews a research approach that can be used in

the search for genetic trait markers of a vulnerability toward

substance abuse. Reflecting the pattern of studies in the literature, emphasis is placed on alcoholism, but the possible application of findings to other substances is discussed.

Study of a predisposition toward substance abuse is a complex

undertaking. The human body has 70 trillion potential genotypes, only 5% to 20% of which are active at any one time, and

there is a long lag between the potential onset of gene expression and the development of the final clinical oicture which

might be labeled substance misuse (Schuckit 1985a).

The comments offered here begin with discussions of data supporting the importance of genetic factors in alcoholism and other

drugs of abuse. I then describe a specific model used in our

laboratory for searching for phenotypic markers associated with

a risk for alcoholism. Finally, I discuss the potential research and clinical implications of these findings and suggest

some future directions for research on vulnerabilities toward

misuse of substances other than alcohol.

GENETIC FACTORS IN ALCOHOLISM

Data generated in the last decade from family, twin, and adoption studies point to the importance of genetic factors in alcoholism (Schuckit 1984a). Virtually all family studies have

shown the familial nature of alcoholism, while studies of identical twins have documented a higher concordance rate for alcoholism than in fraternal twins. Other twin studies point to

genetic control of drinking behavior (Partanen 1966): alcohol

metabolism (Vesell et al. 1971); and some alcohol consequences,

such as cirrhosis and psychoses (Hrubec and Omenn 1981). Halfsibling and adoption studies document a fourfold increased risk

for alcoholism in the adopted-out sons and daughters of alcoholics separated from their biological parents in infancy and

raised without knowledge of their parents' drinking problem

(Schuckit et al. 1972; Goodwin et al. 1974; Bohman et al. 1981;

97
Cloninger et al. 1981). However, being raised by an alcoholic

parent figure, but with no alcoholic biological parent, does not

seem to increase the risk for alcoholism.

Finally, animal studies have demonstrated a genetic influence on

alcohol drinking behavior, central nervous system (CNS) sensitivity to alcohol, and the rate of development of tolerance

(Meisch 1982). Dr. T.K. Li (this volume) has discussed these studies and the development of alcohol-preferring rats, a potentially

powerful tool for alcohol research.

GENETIC STUDIES IN DRUG ABUSE

Unfortunately, information supporting the genetic influence in

abuse of substances other than alcohol is scarce and available

results are inconsistent. Several scientists have bred rat and

mouse strains for morphine-seeking behavior (Nichols and Hsiao

1967; Ericksson and Kiianma 1971). and there is some evidence

that there may be an overlap in an animal's preference for ethanol and morphine or phenobarbital (Sinclair et al. 1973). In

animals, at least, one might therefore be dealing with a preference for substances that may cross over from ethanol to barbiturates or opiates. This hypothesized interaction is consistent

with clinical observations that potentially lethal overdoses of

ethanol might be reversed by naloxone (Dole et al. 1982).

Human genetic studies in drug abuse are also relatively rare.

Family studies have revealed a trend for preference of similar

types of drugs within families, and monozygotic twins show greater similarity for drug preference or response than dizygotic

pairs, although not all researchers agree (Gershon 1980; Tennant

1976; Pederson 1981; Liston et al. 1981).

Although there have not been enough studies of the importance of

genetic factors in substance abuse other than alcohol to draw

even tentative conclusions, there are a number of tools that

might be considered for application in the substance abuse area

at the appropriate time. The next section addresses one model.

A MODEL FOR SEARCHING FOR PHENOTYPIC MARKERS IN ALCOHOLISM

There is now enough data supporting genetic influences in alcoholism to justify a search for the mechanism by which the increased vulnerability is expressed. One approach uses populations at elevated risk for the future development of alcoholism,

concentrating on individuals before the actual abuse develops.

These populations have been chosen because of demographic characteristics or because of the presence of close family members

who abused substances (Schuckit 1985a).

Our laboratory is currently using a model for searching for

phenotypic markers that might be associated with the vulnerability toward alcoholism. Our approach is based on the assumption

98
that alcoholism is probably a polygenic and multifactorial disorder. It is likely that a number of different types of genetic

vulnerabilities interact with cultural and social factors, such as

availability of alcohol (including the price of alcohol and distributing laws) and attitudes toward drinking and drunkenness.

Once alcoholism develops, there may be a different set of factors

that perpetuate ft. As an example, I will discuss the results

from our evaluation of whether the level of acute reaction to alcohol might contribute to the predisposition toward alcoholism.

Since sons of alcoholics have a significantly higher risk for

this disorder than sons of nonalcoholics, these men are the focus

of our work. Our sample was drawn from over 1,000 male university students and nonacademic staff, aged 21 to 25, who responded

to a yearly mailed survey (Schuckit 1984b; 1985b).

Individuals who were already abusing drugs or alcohol

or who had psychiatric or medical problems were excluded from

study. Of the remainder, sons of alcoholics (family history

positive or FHP) who were themselves drinkers, but without substance-related major life problems, were identified. For every

FHP man, a family history negative (FHN) control matched on age,

race, religion, sex, educational level, drinking pattern, and

drug-taking history was selected.

In Phase 2 of our study, FHP/FHN matched pairs were individually

invited to the laboratory on three occasions where they drank a

placebo, or 0.75 ml/kg of ethanol, or 1.1 ml/kg of ethanol (the

equivalent of about three and six drinks, respectively) under

standard conditions. Before testing, the drinking history was

reevaluated by interview and through analysis of blood alcohol

concentration (BAC) and a series of blood tests likely to change

during heavy drinking (e.g., gamma glutamyl transferase, mean

corpuscular volume, triglycerides, and uric acid) (Schuckit

1984c). In addition, the subjects' expectations of feelings

after three drinks were measured (Schuckit 1984b). Figure 1

shows BAC over time for 34 FHP and 34 FHN men. The time to peak

BAC and the rate of disappearance of ethanol in the two groups

were virtually identical after both doses. FHPs and FHNs also

indicated virtually identical expectations of how alcohol would

make them feel (Schuckit 1984b).

The actual levels of drug effect and feeling of intoxication experienced after consuming the beverage during the test sessions

was measured on a 36-point analog scale with 0 indicating no

high feeling at all and 36 indicating the highest possible. As

illustrated in figure 2, subjects receiving a placebo reported a

small increase in perception of an ethanol effect at 60 minutes,

while subjects receiving active ethanol doses reported marked

changes in feelings (Schuckit 1984b). The FHN men, however, reported significantly more intense subjective ethanol effects

than the FHP men after the low dose (0.75 ml/kg). Following the

higher ethanol dose (1.1 ml/kg) (figure 3), the group differences, while present, were not as marked. Therefore, despite

99
FIGURE 1

Mean blood alcohol concentrations (BACs) for 34 matched airs

with positive (close circles) and negative (open circles) family

histories after drinking 0.75 ml/kg of ethanol and 1.1 ml/kg of

ethanol. Bars indicate SEM, and B indicates baseline. From

Schuckit 1984b, Archives of General Psychiatry. Copyright 1984,

American Medical Association.

100
FIGURE 2

Mean self-ratings on a 0 to 36 scale for drug effect after placebo and after 0.75 ml/kg of ethanol for 23 matched pairs with

positive (close circles) and negative (open circles) family histories. Bars indicate SEM and B Indicates baseline. From

Schuckit 1984b. Archives of General Psychiatry. Copyright 1984,

American Medical Association.

101
FIGURE 3

Mean self-ratings on a 0 to 36 scale for drug effect after placebo and after 1.1 ml/kg of ethanol for 23 matched pairs with positive (close circles) and negative (open circles) family histories. Bars indicate SEM and B indicates baseline. From Schuckit

1984b, Archives of General Psychiatry. Copyright 1984, American

Medical Association.

102
identical group BACs, similar expectations of the effects of

ethanol, and similar drinking histories, FHNs expressed more

feelings of drug effects, especially after the lower dose challenge.

We have also evaluated whether the family groups differed on more

objective measures of the effects of alcohol. For example, each

subject's level of standing steadiness or body sway was evaluated

by asking them to stand with their feet together, eyes open; and

hands at their side. Each person was studied at baseline during

each of the three sessions, and then repeatedly every 30 minutes

or so after drinking the beverage. While there were no group

differences at baseline or following placebo, figure 4 demonstrates the percent of increase in body sway for the two groups

after the low ethanol dose. The FHNs increased their level of

body sway significantly more than the FHPs, a finding consistent

with the subjective response (Schuckit 1985b). Similar but less

intense group differences were again noted after the 1.1 ml/kg

of ethanol.

Finally, the levels of two hormones known to change after drinking were observed as indirect indicators of the body’s response

to alcohol. Serum levels of prolactin (PRL) increased after

ethanol administration for both family groups, but returned toward and below baseline more rapidly for the FHPs (Schuckit et

al. 1983). Similarly, following the 0.75 ml/kg challenge, plasma

levels of cortisol increased for the FHNs but not for the FHPs:

and overall, levels of this hormone were lower for the FHPs during most of the experiment (Schuckit 1984d). While hormone

levels were only measured after a single ethanol dose and definite conclusions could not be made in the absence of data from

a placebo session, the FHP/FHN differences were significant and

results are consistent with a less intensive reaction to ethanol

for FHPs.

In summary, our laboratory is searching for phenotypic markers

of differences between men at higher and lower risk for alcoholism. Once such markers are identified, the next step will be to

determine their level of genetic control, the actual mechanisms

involved in the phenotypic behaviors, and the level of correlation between the markers and the future development of alcoholism. While the genetic work is in its early stages, one consistent lead involves a decreased reaction to modest doses of ethanol in young men at elevated future risk for this disorder. It

is possible that one mechanism of increasing the vulnerability

toward alcoholism might occur through a decreased ability to

feel the effects of low ethanol doses, perhaps making it more

difficult to tell when it is time to stop drinking. The differences in subjective response between FHPs and FHNs have been replicated in two other laboratories (Mednick 1983; O'Malley and

Maisto, in press).

These comments were offered as a model of a research strategy

that can be used in attempting to identify mediators of a genetic,

103
FIGURE 4

Percent increase in body sway or standing steadiness following

0.75 ml/kg of ethanol for 23 matched pairs with positive (close

circles) and negative (open circles) family histories. Bars indicate SEM and B indicates baseline. From Schuckit 1985b,

Archives of General Psychiatry. Copyright 1984, American Medical Association.

104
influence in substance abuse. Perhaps similar approaches can be

adapted to the study of genetics of a vulnerability toward misuse of other substances. However, before that can be done, much

work needs to be carried out to establish the importance of genetic factors in the predisposition toward abuse of substances

other than alcohol.

FUTURE RECOMMENDATIONS

The information presented here can be used to help develop guidelines for future research in genetics of substance abuse. While

this list is not exhaustive, I have attempted to outline factors

that are worth consideration.

1.

2.

3.

4.

Much work needs to be done to establish whether various

forms of substance abuse are genetically influenced. Only

after the importance of these genetic factors has been established should studies of populations at high risk for

substance problems be carried out.

For meaningful research in genetics of substance abuse, it

is important that the definition of abuse of substances be

careful1y considered.

tween (a) use of drugs, (b) temporary and relatively mild

Investigators must distinguish beproblems associated with occasional drug use, and (c) pervasive and persistent problems that might be best termed

substance abuse (Schuckit 1984c). Different genetic factors

may contribute at differing levels of importance to each.

For instance, genetic factors may have nothing to do with

why someone decides to try a substance that is readily available (such as alcohol or marijuana), genetics might contribute slightly to the risk for occasional problems, and an

entirely different set of genetic factors could have a major

impact on the vulnerability toward more pervasive and persistent problems.

It is possible that different genetic influences might contribute to a vulnerability toward the abuse of alcohol than

those which impact on the abuse of heroin, and these might

be separate from factors involved in a predisposition toward polysubstance abuse.

It is important to distinguish between primary substance

abuse and problems related to substance abuse that occur

only in the midst of other major psychiatric disorders (secondary substance abuse) (Schuckit 1983, 1984c). For example, it is probable that important genetic factors influence the development of the antisocial personality disorder,

which in turn often is associated with abuse of multiple

substances (Vaillant 1982). Thus, the set of genetic factors associated with substance abuse in patients with a

105
primary antisocial personality disorder could be different

from those associated with substance abuse in primary drug

abusers.

REFERENCES

Bohman, M.; Sigvardsson, S.; and Cloninger, C.R. Maternal inheritance of alcohol abuse: Cross-fostering analysis of

adopted women. Arch Gen Psychiatry 38:965-969, 1981.

Cloninger, C.R.; Bohman, M.; and Sigvardsson, S. Inheritance

of alcohol abuse: Cross-fostering analysis of adopted men.

Arch Gen Psychiatry 38:861-868, 1981.

Dole, V.P; Fishman, J.; Goldfrank, L.; Jatinder. K.; and

McGivern, R.F. Arousal of ethanol intoxicated comatose

patients with naloxone. Alc: Clin Exp Res 6:275-279,

1982.

Eriksson, K.; and Kiianma, K. Genetic analysis of susceptibility to morphine addiction in inbred mice.

Fenn 45:389-392, 1971.

Ann Med Exp Biol

Gershon, E.S. "Behavioral and biological oharmacogenetics of Damphetamine." Paper presented at the Third International

Society for Twin Studies Meeting, Jerusalem, Israel, June 20,

1978), 1980. p. 2.

Goodwin; D.W.; Schulsinger, F.; Moller, N.; Hermansen, L.;

Winokur. G.; and Guze. S.B. Drinking problems in adopted

and nonadopted sons of alcoholics. Arch Gen Psychiatry 31:

164-169, 1974.

Hrubec, Z., and Omenn, G.S. Evidence of genetic predisposition

to alcoholic cirrhosis and psychosis: Twin concordances for

alcoholism and its biological end points by zygosity among

male veterans. Alcoholism: Clin Exp Res 5:207-213, 1981.

Li, T. Genetics of alcohol dehydrogenase. Paper presented

at the Dean's Interdepartmental Conference, University of

California, Davis and Martinez VA Medical Center, Martinez,

California, April 25, 1984.

Liston, E.H.; Simpson, J.; Jarvik, L.; and Guthrie, D. Morphine

and experimental pain in identical twins. In: Gedda L.;

Parisis, P.; and Nance, W.E., eds. Twin Research: Epidemiological and Clinical Studies Vol. New York: Alan R. Liss,

1981, pp. 105-116.

Mednick, S.A. Subjects at risk for alcoholism: Recent reports.

Paper presented at the 14th Annual Medical Scientific Conference of the National Alcoholism Forum (Research Society

on Alcoholism) Houston, Texas, April 1983.

Meisch, R. Animal studies of alcohol intake. Br J Psychiatry

141:113-120, 1982.

Nichols, J.R., and Hsiao, S. Addiction liability of albino rats:

Breeding for quantitative differences in morphine drinking.

Science 157:561-563, 1967.

O'Malley, S.S., and Maisto, S.A. The effects of family drinking

history on responses to alcohol: Expectancies and reactions

to intoxication. J Stud Alcohol in press.

106
Partanen, J.; Bruun, K.; and Markkanen. T. Inheritance of

Drinking Behavior: A Study of Intelligence Personality,

 Helsinki: Finnish Foundation

and

Use of Alcohol of Adult Twins.

for Alcohol Studies., 1966.

Pederson, N. Twin similarity for usage of common drugs. In:

Gedda, L.; Parisi, P.; and Nance, W., eds. Twin Research 3:

Epidemiological and Clinical Studies.

Liss, Inc., 1981. pp. 53-60

New York: Alan R.

Schuckit: M.A. Alcoholism and other psychiatric disorders. Hosp

Communit Psychiatr 34:1022-1027, 1983.

Schuckit, M.A. Genetic and biochemical factors in the etiology

of alcoholism. In: Grinspoon, L., ed. Psychiatry Update

Vol. III. Washington, D.C.: American Psychiatric Press, Inc.

1984a. pp.320-327.

Schuckit, M.A. Subjective responses to alcohol in sons of alcoholics and controls. Arch Gen Psychiatry 41:879-884,

1984b.

Schuckit, M.A. Drug and Alcohol Abuse: A Clinical Guide to

Diagnosis and Treatment, 2d ed. New York: Plenum Publishing

Corp., 1984c.

Schuckit, M.A. Differences in plasma cortisol after ethanol in

relatives of alcoholics and controls: Preliminary results.

J Clin Psychiatr 45:374-379, 1984d.

Schuckit, Y.A. Studies of populations at high risk for alcoholism. Psychiatric Dev 3:31-63, 1985a.

Schuckit, M. . Ethanol-induced changes in body sway in men at

high alcoholism risk. Arch Gen Psychiatry 42:375-379, 1985b.

Schuckit, M.A.; Goodwin, D.A.; and-Winokur, G. A study of alcoholism in half-siblings. Am J Psychiatry 128:1132-1136,

1972.

Schuckit, M.A.; Parker,D.C.; and Rossman, L.R. Prolactin responses to ethanol in men at elevated risk for alcoholism

and controls. Biol Psychiatr 18:1153-1159, 1983.

Sinclair, J.D.; Adkins, J.; and Walker, S. Morphine-induced

suppression of voluntary alcohol drinking in rats. Nature

246:425-427, 1973.

Tennant, F.S. Dependency traits among parents of drug abusers.

J Drug Educ 6:83-88, 1976.

Vaillant, G. Natural history of male alcoholism: Is alcoholism

the cart to sociopathy? Paper presented at the American

Psychiatric Association Annual Meeting, Toronto, Ontario,

May 5, 1982.

Vesell, E.S.; Page, J.F.; and Passananti, G.T. Genetic and environmental factors affecting ethanol metabolism in man.

Clin Pharmacol Ther 12:192, 1971.

ACKNOWLEDGMENT

This work was supported in part by the National Institute of

Alcohol Abuse and Alcoholism grant PHSAA05526-03.

107
AUTHOR

Marc A. Schuckit, M.D.

Professor of Psychiatry

School of Medicine

University of California, San Diego

and

Director, Alcohol Research Center

San Diego Veterans Administration Medical Center

3350 La Jolla Village Drive

San Diego, California 92161

108
Recommendations for Future

Research on Genetic and

Biological Markers in Drug Abuse

and Alcoholism

Monique C. Braude, Ph.D., and Helen M. Chao, Ph.D.

It is now generally agreed that genetic predisposition plays an

important role in the development of alcoholism in some

individuals. The participants at the joint NIDA/NIAAA Technical

Review on Genetic and Biological Markers in Drug Abuse and

Alcoholism reported that there is so far little evidence linking

drug abuse to genetic markers. However, the consensus of opinion

was that substance abuse could have a genetic component and, like

alcohol abuse, more than a single gene could be affected, i.e., it

would be polygenic in nature.

The workshop participants were asked to present their ideas for

future research which would build upon the 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.

It was recommended that a number of steps be taken by both NIDA and

NIAAA to share knowledge and to plan future collaborations. It was

felt that NIDA should increase its research effort at the

preclinical and clinical levels to remedy the paucity of information

in this field and to determine whether or not genetic predisposition

is a factor in substance abuse.

The following is a synthesis of the highlights of some of their

recommendations; it is not intended as, and should not be considered

to be, an exhaustive list.

1. Development of genetically defined animal models of alcohol and

drug abuse

Much of the empirical base of genetic information related to alcohol

and drug abuse has come from animal studies. Experimental animals

selected by genetic breeding for various responses to alcohol or

drugs are powerful tools for studying biological predisposition.

Several lines of rodents have been successfully bred for alcoholdrinking behavior and CNS sensitivity to alcohol. Research along

109
this avenue should be continued in the alcohol field. Selective

breeding approaches should also be used to develop models to study

drugs of abuse, to obtain genetic stocks of animals that differ in

avidity, in initial sensitivity, tolerance development, and severity

of withdrawal for specific drugs of abuse. The development of

strains of preferring or nonpreferring animals is usually done in

mice or rats (faster and cheaper), but care has to be exercised that

the results in rodents can be extrapolated to higher species. This

is a long-term approach; it requires considerable amounts of money

and expertise, as well as time, but has been very fruitful in the

alcohol field.

2. Studies of the genetic makeup of primates resistant to substance

abuse

A search could be initiated in the various primate studies supported

by NIDA to look for animals which are not vulnerable to substance

abuse and to determine their genetic makeup, i.e., to determine

which animals are protected and what are the limits of that

protection.

3. Screening of inbred strain for differences in the trait of

interest

which may not be directly relevant to the addiction process. NIDA

is already supportin

should add very little cost to the program.

work in this area, and this reorientation

Mice or rat strain differences in response to drugs of abuse have

been reported for a variety of drugs of abuse: alcohol,

amphetamines, barbiturates, hallucinogens, opiates, and others.

Most of these studies have investigated acute responses to drugs,

and a few have looked at drug self-administration or severity of a

withdrawal syndrome. The fact that strain differences have been

identified makes one assume that genetic factors are important in

regulating drug abuse responses in animals and that genetic markers

might be found. The various preclinical studies looking at

different genetic strains should be reoriented towards increased

emphasis to the problems of addiction rather than using as

biological markers other effects such as motor activity or analgesia

4. Adoption and twin studies

Adoption and twin studies have provided strong evidence for genetic

involvement in the development of alcoholism. Well-designed

prospective and longitudinal studies on children of alcoholics

should be continued to study inheritance patterns and predictors of

familial or clinical subtypes of alcoholism. Studies of families in

which detailed clinical data can be correlated to biological and

genetic markers need to be conducted. Attention must be given to

refinement of subtype classification as well as to the contribution

of environmental factors.

110
In regard to human studies, it is evident that NIDA's program has

not yet been fully developed. For instance, there is so far only

one small twin study published on the effects of opiates and only

one study supported by NIDA on the genetic correlates of marijuana

use. Population studies such as those conducted by the alcohol

researchers need to be pursued to establish the role of genetics in

addiction behavior.

An increased collaboration between NIDA and NIAAA was recommended,

as some of the human studies could share protocols and populations.

The alcohol researchers attending the meeting mentioned that they

would gladly collaborate with NIDA and could insert in their

questionnaires items relevant to drug abuse, thus making the human

studies less expensive than if initiated by either Institute

separately.

5. Genetic markers directly linked to alcoholism and drug abuse

Such markers need to be studied in order to understand the

relationship between the specific gene and the addiction process.

Studies should be conducted to test major locus effects on

susceptibility to alcohol and drug abuse and to assess the

contribution of identified major gene loci to such risks.

6. Increased clinical-preclinical communication

As in many other areas, the participants strongly recommended that

increased communication be developed between the clinical and

preclinical researchers through joint meetings such as workshops or

seminars, as it is clear that there is a definite lack of active

communication between the clinical and preclinical scientists.

AUTHORS

Monique C. Braude, Ph.D.

Biomedical Research Branch

Division of Preclinical Research

National Institute on Drug Abuse

Rockville, Maryland 20857

Helen M. Chao, Ph.D.

Biomedical Research Branch

Division of Extramural Research

National Institute on Alcohol Abuse and Alcoholism

Rockville, Maryland 20857

111
monograph series

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 $5.95, are also available from NTIS. Prices from either

source are subject to change.

Addresses are:

NCDAI

National Clearinghouse for Drug Abuse Information

Room lOA-43

5600 Fishers Lane

Rockville, Maryland 20857

GPO

Superintendent of Documents

U.S. Government Printing Office

Washington, D.C. 20402

NTIS

National Technical Information

Service

U.S. Department of Commerce

Springfield, Virginia 22161

1 FINDINGS OF DRUG ABUSE RESEARCH. NCDAI out of stock

Vol. 1: GPO out of stock NTIS PB #272 867/AS $34.95

Vol. 2: GPO out of stock NTIS PB #272 868/AS $28.95

2 OPERATIONAL DEFINITIONS IN SOCIO-BEHAVIORAL DRUG USE RESEARCH

1975. Jack Elinson, Ph.D., and David Nurco, Ph.D., eds.

NCDAI out of stock

GPO out of stock NTIS PB #246 338/AS $16.95

3 AMINERGIC HYPOTHESES OF BEHAVIOR: REALITY OR CLICHE? Bruce J.

Bernard, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #246 687/AS $16.95

112
4 NARCOTIC ANTAGONISTS: THE SEARCH FOR LONG-ACTING PREPARATIONS.

Robert Willette, Ph.D., ed.

GPO out of stock NTIS PB #247 096/AS $9.95

5 YOUNG MEN AND DRUGS: A NATIONWIDE SURVEY. John A. O'Donnell,

Ph.D., et al. NCDAI out of stock

GPO out of stock NTIS PB #247 446/AS $16.96

6 EFFECTS OF LABELING THE "DRUG ABUSER": AN INQUIRY. Jay R.

Williams, Ph.D. NCDAI out of stock

GPO out of stock NTIS PB #249 092/AS $9.95

7 CANNABINOID ASSAYS IN HUMANS. Robert Willette, Ph.D., ed.

GPO out of stock NCDAI out of stock

NTIS PB #251 905/AS $16.95

8 Rx: 3x/WEEK LAAM - ALTERNATIVE TO METHADONE. Jack Blaine, M.D.,

and Pierre Renault, M.D., eds. NCDAI out of stock

Not available from GPO NTIS PB #253 763/AS $16.95

9 NARCOTIC ANTAGONISTS: NALTREXONE PROGRESS REPORT. Demetrios

Julius, M.D., and Pierre Renault, M.D., eds.

GPO out of stock NCDAI out of stock

NTIS PB #255 833/AS $16.95

10 EPIDEMIOLOGY OF DRUG ABUSE: CURRENT ISSUES. Louise G. Richards,

Ph.D., and Louise B. Blevens, eds. NCDAI out of stock

GPO out of stock NTIS PB #266 691/AS $22.95

11 DRUGS AND DRIVING. Robert Willette, Ph.D., ed.

NCDAI out of stock

GPO Stock #017-024-00576-2 $5.50 NTIS PB #269 602/AS $16.95

12 PSYCHODYNAMICS OF DRUG DEPENDENCE. Jack D. Blaine, M.D., and

Demetrios A. Julius, M.D., eds. NCDAI out of stock

GPO out of stock NTIS PB #276 084/AS $16.95

13 COCAINE: 1977. Robert C. Petersen, Ph.D., and Richard C.

Stillman, M.D., eds. NCDAI out of stock

GPO Stock #017-024-00592-4 $6 NTIS PB #269 175/AS $22.95

14 MARIHUANA RESEARCH FINDINGS: 1976. Robert C. Petersen, Ph.D.,

ed. NCDAI out of stock

GPO out of stock NTIS PB #271 279/AS $22.95

15 REVIEW OF INHALANTS: EUPHORIA TO DYSFUNCTION. Charles Wm. Sharp,

Ph.D., and Mary Lee Brehm, Ph.D., eds.

GPO out of stock NTIS PB #275 798/AS $28.95

16 THE EPIDEMIOLOGY OF HEROIN AND OTHER NARCOTICS. Joan Dunne

Rittenhouse, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #276 357/AS $22.95

113
17 RESEARCH ON SMOKING BEHAVIOR. Murray E. Jarvik, M.D., Ph.D., et

al., eds. NCDAI out of stock

GPO out of stock NTIS PB #276 353/AS $28.95

18 BEHAVIORAL TOLERANCE: RESEARCH AND TREATMENT IMPLICATIONS.

Norman A. Krasnegor, Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #276 337/AS $16

19 THE INTERNATIONAL CHALLENGE OF DRUG ABUSE. Robert C. Petersen,

Ph.D., ed. NCDAI out of stock

GPO out of stock NTIS PB #293 807/AS $28.95

20 SELF-ADMINISTRATION OF ABUSED SUBSTANCES: METHODS FOR STUDY.

Norman A. Krasnegor, Ph.D., ed.

GPO out of stock NTIS PB #288 471/AS $22.95

21 PHENCYCLIDINE (PCP) ABUSE: AN APPRAISAL. Robert C. Petersen,

Ph.D., and Richard C. Stillman, M.D., eds.

GPO Stock #017-024-00785-4 $7 NTIS PB #288 472/AS $28.95

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.

GPO out of stock NCDAI out of stock

NTIS PB #292 265/AS $35.50

23 CIGARETTE SMOKING AS A DEPENDENCE PROCESS. Norman A. Krasnegor,

Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-00895-8 $6 NTIS PB #297 721/AS $22.95

24 SYNTHETIC ESTIMATES FOR SMALL AREAS: STATISTICAL WORKSHOP PAPERS

AND DISCUSSIDN. Jos. Steinberg, ed. NCDAI out of stock

GPO out of stock NTIS PB #299 OO9/AS $22.95

25 BEHAVIORAL ANALYSIS AND TREATMENT OF SUBSTANCE ABUSE. Norman A.

Krasnegor, Ph.D., ed.

GPO out of stock NTIS PB #80-112428 $22.95

26 THE BEHAVIORAL ASPECTS OF SMOKING. Norman A. Krasnegor, Ph.D.,

ed. (Reprint from 1979 Surgeon General's Report on Smoking and

Health.)

GPO out of stock NTIS PB #80-118755 $16.95

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. NCDAI out of stock

GPO Stock #017-024-00981-4 $9 NTIS PB #80-175482 $40.95

28 NARCOTIC ANTAGONISTS: NALTREXONE PHARMACOCHEMISTRY AND

SUSTAINED-RELEASE PREPARATIONS. Robert Willette, Ph.D., and

Gene Barnett, Ph.D., eds. NCDAI out of stock

GPO out of stock NTIS PB #81-238875 $22.95

29 DRUG ABUSE DEATHS IN NINE CITIES: A SURVEY REPORT. Louis A.

Gottschalk, M.D., et al. NCDAI out of stock

GPO out of stock NTIS PB #80-178882 $16.95

114
30 THEORIES ON DRUG ABUSE: SELECTED CONTEMPORARY PERSPECTIVES. Dan

J. Lettieri, Ph.D.; Mollie Sayers; and Helen W. Pearson, eds.

NCDAI out of stock

GPO Stock #017-024-00997-1 $10 Not available from NTIS

31 MARIJUANA RESEARCH FINDINGS: 1980. Robert C. Petersen, Ph.D.,

ed.

GPO out of stock NTIS PB #80-215171 $22.95

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.

GPO out of stock NTIS PB #81-133746 $22.95

33 BENZODIAZEPINES: A REVIEW OF RESEARCH RESULTS, 1980. Stephen I.

Szara. M.D., D.Sc., and Jacqueline P. Ludford, M.S., eds.

GPO Stock #017-024-01108-8 $5 NTIS PB #82-139106 $16.95

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. NCDAI out of stock

GPO Stock #017-024-01061-8 $8 NTIS PB #81-194847 $34.95

35 DEMOGRAPHIC TRENDS AND DRUG ABUSE, 1980-1995. Louise G.

Richards, Ph.D., ed.

GPO out of stock NTIS PB #82-103417 $16.95

36 NEW APPROACHES TO TREATMENT OF CHRONIC PAIN: A REVIEW OF

MULTIDISCIPLINARY PAIN CLINICS AND PAIN CENTERS. Lorenz K.Y.

Ng, M.D., ed.

GPO out of stock NTIS PB #81-240913 $22.95

37 BEHAVIORAL PHARMACOLOGY OF HUMAN DRUG DEPENDENCE. Travis

Thompson, Ph.D., and Chris E. Johanson, Ph.D., eds.

NCDAI out of stock

GPO Stock #017-024-01109-6 $7 NTIS PB #82-136961 $28.95

38 DRUG ABUSE AND THE AMERICAN ADOLESCENT. Dan J. Lettieri, Ph.D.,-

and Jacqueline P. Ludford, M.S., eds. A RAUS Review Report.

GPO Stock #017-024-01107-O $4.50 NTIS PB #82-148198 $16.95

39 YOUNG MN AND DRUGS IN MANHATTAN: A CAUSAL ANALYSIS.

Richard R. Clayton, Ph.D., and Harwin L. Voss, Ph.D.

GPO Stock #017-024-01097-9 $5.50 NTIS PB #82-147372 $22.95

40 ADOLESCENT MARIJUANA ABUSERS AND THEIR FAMILIES. Herbert

Hendin, M.D., Ann Pollinger, Ph.D., Richard Ulman, Ph.D., and

Arthur Carr, Ph.D. NCDAI out of stock

GPO out of stock NTIS PB #82-133117 $16.95

41 PROBLEMS OF DRUG DEPENDENCE, 1981: PROCEEDINGS OF THE 43RD

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

Not available from GPO NTIS PB #82-190760 $40.95

115
42 THE ANALYSIS OF CANNABINOIDS IN BIOLOGICAL FLUIDS. Richard L.

Hawks, Ph.D., ed.

GPO Stock #017-024-01151-7 $5 NTIS PB #83-136044 $16.95

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. NCDAI out of stock

GPO out of stock NTIS PB #83-252-692/AS $40.95

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.

GPO Stock #017-024-01202-5 $4 NTIS PB #85-150563/AS $16.95

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.

GPO Stock #017-024-01191-6 $2.75 NTIS PB #84-184670/AS $11.95

46 BEHAVIORAL INTERVENTION TECHNIQUES IN DRUG ABUSE TREATMENT. John

Grabowski. Ph.D.; Maxine L. Stitzer. Ph.D., and Jack E.

Henningfield, Ph.D., eds.

GPO Stock #017-024-01192-4 $4.25 NTIS PB #84-184688/AS $16.95

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.

GPO Stock #017-024-01180-1 $5.50 NTIS PB #85-159663/AS $22.95

48 MEASUREMENT IN THE ANALYSIS AND TREATMENT OF SMOKING BEHAVIOR.

John Grabowski, Ph.D., and Catherine S. Bell, M.S., eds.

GPO Stock #017-024-01181-9 $4.50 NTIS PB 84-145-184 $16.95

49 PROBLEMS OF DRUG DEPENDENCE, 1983: PROCEEDINGS OF THE 45TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-01198-3 $12 NTIS PB 85-151553/AS $34.95

50 COCAINE: PHARMACOLOGY, EFFECTS, AND TREATMENT OF ABUSE. John

Grabowski, Ph.D., ed.

GPO Stock #017-020-01214-9 $4.50 NTIS PB 85-150381/AS $16.95

51 DRUG ABUSE TREATMET EVALUATION: STRATEGIES, PROGRESS, AND

PROSPECTS. Frank M. Tims, Ph.D., ed.

GPO Stock #017-020-01218-1 $4.50 NTIS PB 85-150365/AS $16.95

52 TESTING DRUGS FOR PHYSICAL DEPENDENCE POTENTIAL AND ABUSE

LIABILITY. Joseph V. Brady, Ph.D., and Scott E. Lukas, Ph.D., eds.

GPO Stock #017-O24-0204-1 $4.25 NTIS PB 85-150373/AS $16.95

53 PHARMACOLOGICAL ADJUNCTS IN SMOKING CESSATION. John Grabowski,

Ph.D., and Sharon M. Hall, Ph.D., eds.

GPO Stock #017-024-01266-1 $3.50

116
54 MECHANISMS OF TOLERANCE AND DEPENDENCE. Charles Wm. Sharp,

Ph.D., ed. NCDAI out of stock

GPO Stock #017-024-01213-1 $8.50

55 PROBLEMS OF DRUG DEPENDENCE, 1984. PROCEEDINGS OF THE 46TH

ANNUAL SCIENTIFIC MEETING, THE COMMITTEE ON PROBLEMS OF DRUG

DEPENDENCE, INC. Louis S. Harris, Ph.D., ed.

GPO Stock #017-024-01242-4 $9.50

56 ETIOLOGY OF DRUG ABUSE: IMPLICATIONS FUR PREVENTION. Coryl LaRue

Jones, Ph.D., and Robert J. Battjes, D.S.W., eds.

GPO Stock #017-024-01250-5 $6.50

57 SELF-REPORT METHODS OF ESTIMATING DRUG USE: MEETING CURRENT

CHALLENGES TO VALIDITY. Beatrice A. Rouse, Ph.D., Nicholas J.

Kozel, M.S., and Louise G. Richards, Ph.D., eds.

GPO Stock #017-024-01246-7 $4.25

58 PROGRESS IN THE DEVELOPMENT OF COST-EFFECTIVE TREATMENT FOR DRUG

ABUSERS. Rebecca S. Ashery, D.S.W., ed.

GPO Stock #017-024-01247-5 $4.25

59 CURRENT RESEARCH ON THE CONSEQUENCES OF MATERNAL DRUG ABUSE.

Theodore M. Pinkert, M.D., J.D., ed.

GPO Stock #017-024-01249-1 $2.50

60 PRENATAL DRUG EXPOSURE: KINETICS AND DYNAMICS. C. Nora Chiang,

Ph.D., and Charles C. Lee, Ph.D., eds.

GPO Stock #017-024-01257-2 $3.50

61 COCAINE USE IN AMERICA: EPIDEMIOLOGIC AND CLINICAL PERSPECTIVES.

Nicholas J. Kozel, M.S., and Edgar H. Adams, M.S., eds.

GPO Stock #017-024-01258-1 $5

62 NEUROSCIENCE METHODS IN DRUG ABUSE RESEARCH. Roger M. Brown,

Ph.D., and David P. Friedman, Ph.D., eds.

GPO Stock #017-024-01260-2 $3.50

63 PREVENTION RESEARCH: DETERRING DRUG ABUSE AMONG CHILDREN AND

ADOLESCENTS. Catherine S. Bell, M.S., and Robert Battjes,

D.S.W., eds.

GPO Stock #017-024-01263-7 $5.50

64 PHENCYCLIDINE: AN UPDATE. Doris H. Clouet, Ph.D., ed.

GPO Stock #017-024-01281-5 $6.50

IN PRESS

65 WOMEN AND DRUGS: A NEW ERA FOR RESEARCH. Barbara A. Ray, Ph.D.,

and Monique C. Braude, Ph.D., eds.

117
DHHS Publication No. (ADM)86-1444

Printed 1986