# Presentation: 049867

## Steroidogenesis:

**Detailed Review Paper**

**Jerome Goldman**
**Endocrinology Branch**
**Reproductive Toxicology Division**
**NHEERL, ORD**
**U.S. Environmental Protection Agency**

- 1

## 2

**  ****- ****Strengths  /  Limitations**

******   ****Considerations in the Selection of a Screening Approach **

******  ****Use of Tissues vs. Primary Cell Preparations**

******  ****Cell Lines**

******  ****Candidate Steroidogenesis Protocol**

******  ****Candidate Chemicals**

**  ****-**** Review of Strengths / Limitations as a Screening Approach**

**Overview**

******  ****Recommendations**

## 3

## 4

## 5

******  ****Cost (Personnel / Equipment)**

******  ****Predictiveness**

******  ****Sensitivity**

******  ****Ease of Use**

******  ****Standardization**

******  ****Animal Use (Refine / Reduce / Replace)**

******  ****Variability (Intra- & Inter-laboratory)**

******  ****Multiple Samples Evaluated (Throughput)**

******  ****Metabolic Activation**

******  ****Time Requirements (Personnel Time / Stability of Prep)**

## Exposure

**Ex vivo**

**In vitro**

- Biological
- Material

**Cells: **
**Primary culture / Established cell line**

**Tissues**

- Sampling

**Flow-through Approach**

**Single Sample**

**Multiple Samples: **
**Media Replacement**
**or Cumulative Sampling**

**Perifusion**

**Perfusion**

- Gender

**Female**

**Male**

**Incubation Vial**

**Organs**

- 6

## 7

**Strengths**

**Limitations**

**Approach**

**In Vitro**

**-****Exposure limited to tissues/ cells of interest-  specificity of response**

**-****Random assignment of tissues/   cells to treatments reduces variability**

**-****Reduction in animals use**

**In Vivo**

**-****Allows for more extended periods of exposure**

**-****Systemic exposures allow for metabolism / normal interactions among organs &/or tissues.**

**-****Standard routes of exposure**

**-****If cell cultures employed, maintenance can add an additional level of complexity**

**-****Issues of general toxicity of compound in vitro**

**-****Solubility of the compound in culture**

**-****Shorter exposure times / higher throughput**

**-****Less material needed**

**-****Lack of metabolic activation**

**-****Sophisticated equipment may be required**

**-****Increased costs / animal usage**

**-****Indirect effects on steroidogenesis: hypothalamic-pituitary effects, changes in body weight, systemic toxicity**

**-****Positive response in vitro, but failure to reach target tissue in vivo**

**-****Lower costs**

## 8

**Strengths**

**Limitations**

**Type of Exposure**

**In Vitro**
**In vitro exposure**
**In vitro sampling**

**-****Exposure limited to tissues/cells of interest**

**-****Random assignment of tissues/cells to treatment conditions reduces variability**

**-****Reduction in number of animals required / shorter exposure times**

**Ex Vivo**
**In vivo exposure**
**In vitro sampling**

**-****Allows for more extended periods of exposure**

**-****Systemic exposures allow for normal interactions among organs &/or tissues.**

**-****Standard routes of exposure**

**-****Movement of compound out from the cells/tissues in culture may alter the response characteristics present in vivo**

**-****Maintenance of cell cultures can add an additional level of complexity**

**-****Added level of concern about general toxicity of compound in vitro**

**-****Solubility of the compound in culture**

**-****More limited control of exposure levels compared to in vitro approach**

## *

**0**

**100**

**250**

**500**

**750**

**1000**

**0**

**5**

**10**

**15**

**20**

**25**

**30**

**Control**

**Pair-fed **

**Control**

**C8**

**0**

**200**

**400**

**600**

**800**

**1000**

**1200**

**- hCG**

**+ hCG**

**% Increase in hCG-stimulated**

**Testosterone Production**

**Testosterone (ng/ml)**

**Ammonium Perfluorooctanoate (C8- **

****

**M)**

- 25 mg/kg/d -

- 15days

**Ex Vivo Testosterone Production from**
**Isolated Rat Leydig cells in Response to **
**Ammonium Perfluorooctanoate**

**Effects of In Vitro Exposure to**
**Ammonium Perfluorooctanoate****1**** on**
**Testosterone Production**

- Data from Biegel et al. Toxicol. Appl. Pharmacol. 134:18-25, 1995.  Presented as Steroidogenesis DRP- Fig. 4-17.

- 1Derivative of perfluorocarboxylic acid plasticizers

- 9

## Steroidogenesis DRP: In Vitro Approaches Reviewed

******Isolated Organs   ****(Perfusion  /  Perifusion)**

******    ****Sectioned / Minced Tissue**

******    ****Primary Cell Preparations**

******    ****Cell Lines**

******  ****Testis  /  Ovary**

******  ****Testis  /  Ovary**

******   ****Leydig cells  /  Granulosa cells**

- 10

## 11

- 1979b)

- Steroid

- hormones

- (deduced)

- Steroid

- hormones (5)

- (Gurler &

- Steroid

- hormones (5)

- (Bambino & Hsueh,

- 1981)

- Steroid hormones

- (4)

- Enzyme Act.

- Histology

- (Kelce et al., 1991;

- Biegel et al., 1995;

- Klinefelter et al.,

- 1991)

- Steroid hormones

- (2)

- (Hoelscher and

- Ascoli, 1996)

**Specificity**

****

- 

- 

****

****

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****

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- 

- 

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- 

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- 

****

****

- 

- 

- (

- (

****

- )

- )

- General

- Specialized

- Specialized

- General

- General

- Specialized

- Specialized

****

****

- 

- 

****

- 

- 

****

****

- 

- 

****

- 

- 

****

****

****

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- 

****

****

- 

- 

****

- 

- 

****

****

- 

- 

****

****

****

****

****

****

****

**None**

- (deduced)

- Donatsch, 1979)

****

- 

- 

****

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- 

****

****

- 

- 

****

**Comparison Summary of In Vitro Methods – Table 4-11**

## 12

- Steroidogenesis DRP-  Figure 5-1

## 13

**Minced Tissue Steroidogenesis Assay**

**Strengths**

**Limitations**

******  ****Not Difficult to perform**

**********Tissue quickly obtained & readied for incubation.  Less personnel time involved.**

******   ****In vitro or ex vivo exposures.**

******  ****Use of non-stimulated or stimulated conditions under varying concentrations of compound.**

****** ****Use of whole minced tissue increases variability.**

****** ****For ovarian tissue, female cycling status can affect results.  Steroid release from variable numbers of preovulatory follicles, corpora lutea.**

****** ****Need to eliminate early traumatic hormonal release when obtaining baseline values (Correctable)**

**-****   Ex vivo approach allows for any metabolic activation to occur.**

****** ****Use of animals-  can vary depending on  ex vivo or in vitro exposure designs.**

## Strengths

**Strengths**

**Limitations**

**Biological Material**

**Cells**

**-****Uniform cell type can be employed that may well reduce interassay variability & increase magnitude of response **

**Tissues**

**-****Maintenance of architectural integrity / interaction among different cell types**

**-****Maintenance of cell cultures can add an additional level of complexity**

**-****Characterized cell line could reduce interlab variability**

**-****If cells isolated from toxicant-exposed animals, will increase assay time considerably**

**-****Loss of tissue structural integrity**

**-****For ex vivo exposure, less time to removal of tissue & placement in medium than for isolated cells**

**-****In vitro penetration of compound into tissue will vary, depending on nature and size of tissue**

**-****Discrepancies among cell lines in ease of maintenance & char-acteristics of steroid secretion**

**-****Compared to isolated cells or cell lines, less uniformity of test samples can add to variability**

**-****Improved penetration of compound**

- 14

## Assessment of Cell / Tissue Viability 

_**Cells**_

******  ****Dye Exclusion (trypan blue)**

******  ****Tetrazolium Dye Based Assays (e.g., MTT reduction)**

******  ****ATP Bioluminescence Assay**

_**Tissues**_

******  ****Cytokine Release**

******  ****Lactic Dehydrogenase**

******  ****ATP Bioluminescence Assay**

- 15

## 16

**Control Group Coefficients of Variation:  Testosterone Secretion**

**Sliced Testis**

**Crude Leydig Cell Prep**
**(~12-15%)**

**Purified Leydig Cell Prep**
**(80-95%)**

**9**

**Nagata et al. (1999) FEBS Lett. 444:160.**

_**Preparation**_

_**Non-stim.**_

_**LH**__**/hCG-stim.**_

**6**

**Romanelli et al. (1997) Life Sci. 61:557.**

**8**

**Klinefelter et al. (1991) TAP 107:460.**

**12**

**26**

**Kan et al. (1985) J. Steroid Biochem. 23:**
**   ****1023**

**27**

**23**

**Gray et al. (1995) TAP 130:248.**

**29**

**Powlin et al. (1998) Tox. Sci. 46:61.**

**28**

**15**

**Laskey & Phelps (1991) TAP 108:296.**

**12**

**11**

**Ronco et al. (2001) Toxicology 159:99.**

**23**

**22**

**Wilker et al. (1995) Toxicology 95:93.**

**23**

_**Reference**_

**13**

**Guillou et al. (1985) FEBS Lett. 184:6.**

**12**

**Banczerowski et al. (2001) Br.Res. 906:25.**

**50**

**40**

**Raji & Bolarinwa (1997) Life Sci. 61:1067.**

**25**

**45**

**Chambon et al. (1985) Andrologia 17:172.**

**30**

- Incubation parameters: 105-106 cells/well; 3-4h collection period; 100mIU hCG or 50 ng/ml oLH stimulation

**-**

**-**

**-**

**-**

## 17

******     ****Endpoints of Interest / Appropriateness of Cell Type**

******  ****Availability / Cost  **

******  ****Assessment of Enhanced and Diminished Secretion**

** ********   ****Non-stimulated vs. Stimulated Release**

**********Primary Culture vs. Characterized Cell Line**

******     ****Steroidogenically Active**

******  ****Ease of Maintenance **

## 18

**Examples of Cell Lines Employed in Studies of Steroidogenesis**

**MA-10****	****(mouse Leydig cell tumor line)**

**R2C****	****(rat Leydig cell tumor line)**

**H540****	****(rat Leydig cell tumor line)**

**mLTC-1****	****(mouse Leydig cell tumor line)**

**H295R****	****(human adrenocortical cell tumor line)**

**HO-23****	****(immortalized human granulosa cell line)**

**Jc-410****	****(stable porcine granulosa cell line)**

**Comments**

- Employed for pregnenolone / P4 production & StAR expression. Low basal P4; marked stimulated release. Very low T- recent report; induced by db-cAMP & hCG.

- High basal P4; limited stimulated release; high levels of P450arom & 5-reductase.

- Employed for evaluation of early steps in pathway (cholesterol **** progesterone). Can produce androgens with db-cAMP pretreatment. Loss of responsiveness to hCG/LH.

- Aromatase evaluations.  High basal 3-HSD; lower basal 17-hydroxylase. Possibly useful to study entire pathway.  Ease of maintenance?

- Primarily P4, some E2 measurements; loss of responsiveness to gonadotropins.

- P4 secretion.

- P4 & T. Loss of receptors under hCG.

**KGN****	****(human granulosa-like tumor cell line)**

- Relatively high aromatase (stimulated by db-cAMP and FSH.  P4 secretion responsive to db-cAMP stimulation.  Minimal (if any) baseline secretion of DHEA, androstenedione or estradiol (17-hydroxylation appears absent).

## 19

- Pregnenolone
**9.2** nmol/106 cells/48h

- 17OH-PREG
**52.1**

- DHEA
**5.1**

- Progesterone
**48.6**

- 17OH-PROG
**88.6**

- Androstenedione
**283**

- Testosterone
**13.4**

- 3-HSD/5-4 isomerase

- 17-hydroxylase/17,20-lyase

- 17-hydroxylase/17,20-lyase

- 17-hydroxylase/17,20-lyase

- 17-HSD

- 17-hydroxylase/17,20-lyase

**Steroid Production by H295R Cells**

**Adapted from Logi******** et al. (1999) J. Molec. Endocrinol. 23: 23-32.**

- P450scc

## 20

- 17OH-PREG
**0.34**
**0.48**

- DHEA
**<0.2**
**<0.2**

- Progesterone
**0.24**
**2.47**

- Testosterone

- 3-HSD/5-4 isomerase

- 17-hydroxylase/17,20-lyase

- 17-hydroxylase/17,20-lyase

- 17-hydroxylase/17,20-lyase

- 17-HSD

- 17-hydroxylase/17,20-lyase

**Steroid Production by KGN Cells**

**Adapted from Nishi et al. (2001)  Endocrinology 142:437-445.**

- P450scc

**Non-stim.-**
**db-cAMP-**

- Pregnenolone
**0.41** ng/ml/106 cells/24h
**5.45****             “               **

- 17OH-PROG
**<0.2**
**0.25**

- Androstenedione
**<0.2**
**<0.2**

## 21

**********Sliced Testis    (***Quartered, in vitro exposure***) -  Testosterone**

******   ****Explore feasibility of using a cell line as an alternative**

**-****H295R – possibility that entire steroidogenic pathway**
**	****(including aromatase activity) can be evaluated.**
**	****[ATCC availability]**

**  ****-****Advantages:  Ease of use,  reduced preparation / personnel **
**	****           ****time, reduced animal use, female cyclicity not **
**	****           ****an issue.**

**  ****-****Limitations:  Variability, lower sensitivity compared to **
**	****          ****purified cell preps.**
****** ****Inclusion of assessments of tissue viability.**

**DRP Recommendation**

**Alternative**

**-****MA-10 – Commonly employed for progesterone release, **
**	****so good database available.  [M. Ascoli, Univ. Iowa]**

## 22

**Ketoconazole (Mixed P450 inhibitor)**

**Cyanoketone  (3********-HSD inhibitor)**

**Dimethoate  (pesticide; Inhibits StAR expression / **
**	****	****Suppression of cholesterol side-chain cleavage)**

**Trilostane  (3********-HSD inhibitor)**

**Aminoglutethimide (Aromatase inhibitor / Cholesterol**
**  ****	****	****          ****side-chain cleavage inhibitor)**

**Prochloraz (fungicide; Aromatase inhibitor)**

## 23

**Ketoconazole (various P450  sites)**

**Cyanoketone [****CKT****]**

**Dimethoate [****DMTH****]**

**Trilostane  [****TLS****]**

**Aminoglutethimide [****AMG****]**

**Prochloraz [****PRO****]**

**CKT**
**TLS**

**CKT**
**TLS**

**CKT**
**TLS**

**DMTH**
**AMG**

**AMG**
**PRO**

**AMG**
**PRO**

## Appendices

**Selected Examples of Hormonal Actions**

- A1

## Actions of Testosterone: Selected Examples

**     ****genitalia during fetal development.  Sexual differention of CNS.**

******  ****Maturation of internal reproductive tract and external genitalia **
**     ****at puberty**

******  ****Accessory sex gland function (with conversion to dihydro-**
**     ****testosterone)**

******  ****Stimulation of spermatogenesis**

******  ****Anabolic action, growth of long bones**

******  ****Regulation of gonadotropin secretion**

**Actions of Testosterone: Selected Examples**
**(Male)**

- A2

## Actions of Progesterone: Selected Examples

****** ****Maternal ovarian maintenance of pregnancy.   Subsequent placental ****	****production. **

****** ****Together with estradiol, regulates cyclicity-  feedback effects on ****	****GnRH, LH, FSH secretion.**

** ******** ****Increases mammary gland alveolar-lobular formation**

**Actions of Progesterone: Selected Examples**
**(Female)**

- A3

## Actions of Estradiol: Selected Examples

**(Female)**

****** ****Growth / maintenance of female reproductive tract.  Pubertal ****	****development**

****** ****Increases granulosa cell proliferation.**

****** ****Increases progesterone receptors in endometrium.**

****** ****Regulation of LH surge / cyclicity.**

****** ****Increases development of secondary sex characteristics.**

****** ****Increases growth of endometrium and myometrium.**

****** ****Stimulates duct development in mammary tissue.**

****** ****Effects on behavior**

****** ****Functions as a neuroprotectant**

- A4