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Title: Steroidogenesis:


1
Steroidogenesis Detailed Review Paper
Jerome Goldman Endocrinology Branch Reproductive
Toxicology Division NHEERL, ORD U.S.
Environmental Protection Agency
1
2
Overview
l Considerations in the Selection of a
Screening Approach
l In Vitro Approaches to Evaluating
Steroidogenesis
- Strengths / Limitations
l Use of Tissues vs. Primary Cell Preparations
l Candidate Steroidogenesis Protocol
- Review of Strengths / Limitations as a
Screening Approach
l Cell Lines
l Recommendations
l Candidate Chemicals
2
3
3
4
4
5
Selection of an Screening Approach to Evaluate a
Toxicant Effect on Steroidogenesis
Considerations
l Predictiveness
l Sensitivity
l Variability (Intra- Inter-laboratory)
l Animal Use (Refine / Reduce / Replace)
l Ease of Use
l Standardization
l Cost (Personnel / Equipment)
l Time Requirements (Personnel Time / Stability
of Prep)
l Multiple Samples Evaluated (Throughput)
l Metabolic Activation
5
6
Steroid Evaluation Using In Vitro Approaches
Gender
Female
Male
Exposure
Ex vivo
In vitro
Biological Material
Cells Primary culture / Established cell line
Organs
Tissues
Flow-through Approach
Incubation Vial
Sampling
Single Sample
Multiple Samples Media Replacement or
Cumulative Sampling
Perifusion
Perfusion
6
7
Strengths
Approach
Limitations
In Vitro
-Exposure limited to tissues/ cells of interest-
specificity of response
-Lack of metabolic activation
-Issues of general toxicity of compound in vitro
-Random assignment of tissues/ cells to
treatments reduces variability
-Solubility of the compound in culture
-If cell cultures employed, maintenance can add
an additional level of complexity
-Reduction in animals use
-Shorter exposure times / higher throughput
-Sophisticated equipment may be required
-Less material needed
-Positive response in vitro, but failure to reach
target tissue in vivo
-Lower costs
In Vivo
-Standard routes of exposure
-Increased costs / animal usage
-Systemic exposures allow for metabolism / normal
interactions among organs /or tissues.
-Indirect effects on steroidogenesis
hypothalamic-pituitary effects, changes in body
weight, systemic toxicity
-Allows for more extended periods of exposure
7
8
Strengths
Limitations
Type of Exposure
-Maintenance of cell cultures can add an
additional level of complexity
-Exposure limited to tissues/cells of interest
In Vitro In vitro exposure In vitro sampling
-Random assignment of tissues/cells to treatment
conditions reduces variability
-Added level of concern about general toxicity of
compound in vitro
-Reduction in number of animals required /
shorter exposure times
-Solubility of the compound in culture
-More limited control of exposure levels compared
to in vitro approach
-Allows for more extended periods of exposure
Ex Vivo In vivo exposure In vitro sampling
-Systemic exposures allow for normal interactions
among organs /or tissues.
-Movement of compound out from the cells/tissues
in culture may alter the response characteristics
present in vivo
-Standard routes of exposure
8
9
Ex Vivo Testosterone Production from Isolated Rat
Leydig cells in Response to Ammonium
Perfluorooctanoate
Effects of In Vitro Exposure to Ammonium
Perfluorooctanoate1 on Testosterone Production
1200
30

1000
25
- hCG
20
hCG
800
15
Testosterone (ng/ml)
600
Increase in hCG-stimulated
Testosterone Production
10
400
5
0
200
0
100
0
250
500
750
Control
Pair-fed
C8
1000
Ammonium Perfluorooctanoate (C8-
Control
25 mg/kg/d -
m
M)
15days
1Derivative of perfluorocarboxylic acid
plasticizers
9
Data from Biegel et al. Toxicol. Appl. Pharmacol.
13418-25, 1995. Presented as Steroidogenesis
DRP- Fig. 4-17.
10
Steroidogenesis DRP In Vitro Approaches Reviewed
l Isolated Organs (Perfusion / Perifusion)
l Testis / Ovary
l Sectioned / Minced Tissue
l Testis / Ovary
l Primary Cell Preparations
l Leydig cells / Granulosa cells
l Cell Lines
10
11
Comparison Summary of In Vitro Methods Table
4-11
General
Specialized
Specialized
General
General
Specialized
Specialized































None






Steroid
Steroid
Steroid
Steroid hormones
Steroid hormones
hormones
hormones (5)
hormones (5)
(4)
(2)
1979b)
(Gurler
(Bambino Hsueh,
(Hoelscher and
(deduced)
Enzyme Act.
(deduced)
1981)
Ascoli, 1996)
Donatsch, 1979)
11
Histology
(Kelce et al., 1991
Biegel et al., 1995
Klinefelter et al.,
1991)
























Specificity




























(
(
)
)
12
12
Steroidogenesis DRP- Figure 5-1
13
Minced Tissue Steroidogenesis Assay
Strengths
Limitations
l Use of whole minced tissue increases
variability.
l Not Difficult to perform
l Tissue quickly obtained readied for
incubation. Less personnel time involved.
l For ovarian tissue, female cycling status can
affect results. Steroid release from variable
numbers of preovulatory follicles, corpora lutea.
l In vitro or ex vivo exposures.
l Need to eliminate early traumatic hormonal
release when obtaining baseline values
(Correctable)
- Ex vivo approach allows for any metabolic
activation to occur.
l Use of non-stimulated or stimulated conditions
under varying concentrations of compound.
l Use of animals- can vary depending on ex vivo
or in vitro exposure designs.
13
14
Biological Material
Strengths
Limitations
-Uniform cell type can be employed that may well
reduce interassay variability increase
magnitude of response
-Maintenance of cell cultures can add an
additional level of complexity
Cells
-Discrepancies among cell lines in ease of
maintenance char-acteristics of steroid
secretion
-Characterized cell line could reduce interlab
variability
-Improved penetration of compound
-If cells isolated from toxicant-exposed animals,
will increase assay time considerably
-Loss of tissue structural integrity
Tissues
-In vitro penetration of compound into tissue
will vary, depending on nature and size of tissue
-Maintenance of architectural integrity /
interaction among different cell types
-For ex vivo exposure, less time to removal of
tissue placement in medium than for isolated
cells
-Compared to isolated cells or cell lines, less
uniformity of test samples can add to variability
14
15
Assessment of Cell / Tissue Viability
Cells
l Dye Exclusion (trypan blue)
l Tetrazolium Dye Based Assays (e.g., MTT
reduction)
l ATP Bioluminescence Assay
Tissues
l Lactic Dehydrogenase
l ATP Bioluminescence Assay
l Cytokine Release
15
16
Control Group Coefficients of Variation
Testosterone Secretion
Non-stim.
LH/hCG-stim.
Reference
Preparation
-
23
Gray et al. (1995) TAP 130248.
Sliced Testis
29
Powlin et al. (1998) Tox. Sci. 4661.
28
22
Wilker et al. (1995) Toxicology 9593.
23
Banczerowski et al. (2001) Br.Res. 90625.
-
50
45
Chambon et al. (1985) Andrologia 17172.
30
Crude Leydig Cell Prep (12-15)
26
Kan et al. (1985) J. Steroid Biochem. 23 1023
27
40
Raji Bolarinwa (1997) Life Sci. 611067.
25
15
Laskey Phelps (1991) TAP 108296.
12
11
Ronco et al. (2001) Toxicology 15999.
23
Purified Leydig Cell Prep (80-95)
9
Nagata et al. (1999) FEBS Lett. 444160.
-
6
Romanelli et al. (1997) Life Sci. 61557.
8
Klinefelter et al. (1991) TAP 107460.
12
-
13
Guillou et al. (1985) FEBS Lett. 1846.
12
16
Incubation parameters 105-106 cells/well 3-4h
collection period 100mIU hCG or 50 ng/ml oLH
stimulation
17
Isolated Cells Considerations in Selection
l Steroidogenically Active
l Endpoints of Interest / Appropriateness of
Cell Type
l Primary Culture vs. Characterized Cell Line
l Assessment of Enhanced and Diminished
Secretion
l Non-stimulated vs. Stimulated Release
l Availability / Cost
l Ease of Maintenance
17
18
Examples of Cell Lines Employed in Studies of
Steroidogenesis
Comments
MA-10 (mouse Leydig cell tumor line)
Employed for pregnenolone / P4 production StAR
expression. Low basal P4 marked stimulated
release. Very low T- recent report induced by
db-cAMP hCG.
R2C (rat Leydig cell tumor line)
High basal P4 limited stimulated release high
levels of P450arom 5a-reductase.
H540 (rat Leydig cell tumor line)
Employed for evaluation of early steps in pathway
(cholesterol a progesterone). Can produce
androgens with db-cAMP pretreatment. Loss of
responsiveness to hCG/LH.
mLTC-1 (mouse Leydig cell tumor line)
P4 T. Loss of receptors under hCG.
H295R (human adrenocortical cell tumor line)
Aromatase evaluations. High basal 3b-HSD lower
basal 17a-hydroxylase. Possibly useful to study
entire pathway. Ease of maintenance?
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
(17a-hydroxylation appears absent).
HO-23 (immortalized human granulosa cell line)
P4 secretion.
Jc-410 (stable porcine granulosa cell line)
Primarily P4, some E2 measurements loss of
responsiveness to gonadotropins.
18
19
Steroid Production by H295R Cells
Adapted from LogiJ et al. (1999) J. Molec.
Endocrinol. 23 23-32.
Cholesterol
P450scc
17a-hydroxylase/17,20-lyase
17a-hydroxylase/17,20-lyase
Pregnenolone 9.2 nmol/106 cells/48h
17OH-PREG 52.1
DHEA 5.1
3b-HSD/D5-4 isomerase
Progesterone 48.6
17OH-PROG 88.6
17a-hydroxylase/17,20-lyase
17a-hydroxylase/17,20-lyase
Androstenedione 283
17b-HSD
Testosterone 13.4
19
20
Steroid Production by KGN Cells
Adapted from Nishi et al. (2001) Endocrinology
142437-445.
Cholesterol
P450scc
17a-hydroxylase/17,20-lyase
17a-hydroxylase/17,20-lyase
17OH-PREG 0.34 0.48
DHEA lt0.2 lt0.2
Pregnenolone 0.41 ng/ml/106 cells/24h 5.45

Non-stim.- db-cAMP-
3b-HSD/D5-4 isomerase
Progesterone 0.24 2.47
17OH-PROG lt0.2 0.25
17a-hydroxylase/17,20-lyase
17a-hydroxylase/17,20-lyase
Androstenedione lt0.2 lt0.2
17b-HSD
20
Testosterone
21
Recommendations
DRP Recommendation
l Sliced Testis (Quartered, in vitro exposure)
- Testosterone
-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. l
Inclusion of assessments of tissue viability.
Alternative
l Explore feasibility of using a cell line as
an alternative
-H295R possibility that entire steroidogenic
pathway (including aromatase activity) can be
evaluated. ATCC availability
-MA-10 Commonly employed for progesterone
release, so good database available. M.
Ascoli, Univ. Iowa
21
22
Candidate Chemicals for Prevalidation
Ketoconazole (Mixed P450 inhibitor)
Cyanoketone (3b-HSD inhibitor)
Trilostane (3b-HSD inhibitor)
Dimethoate (pesticide Inhibits StAR expression
/ Suppression of cholesterol side-chain
cleavage)
Aminoglutethimide (Aromatase inhibitor /
Cholesterol side-chain cleavage
inhibitor)
Prochloraz (fungicide Aromatase inhibitor)
22
23
DMTH AMG
CKT TLS
CKT TLS
CKT TLS
AMG PRO
Ketoconazole (various P450 sites)
Cyanoketone CKT
Trilostane TLS
AMG PRO
Dimethoate DMTH
Aminoglutethimide AMG
Prochloraz PRO
23
24
Appendices
Selected Examples of Hormonal Actions
A1
25
Actions of Testosterone Selected Examples (Male)
l Differentiation of internal reproductive tract
and external male genitalia during fetal
development. Sexual differention of CNS.
l Maturation of internal reproductive tract and
external genitalia at puberty
l Accessory sex gland function (with conversion
to dihydro- testosterone)
l Stimulation of spermatogenesis
l Anabolic action, growth of long bones
l Regulation of gonadotropin secretion
A2
26
Actions of Progesterone Selected
Examples (Female)
l Together with estradiol, regulates cyclicity-
feedback effects on GnRH, LH, FSH secretion.
l Maternal ovarian maintenance of pregnancy.
Subsequent placental production.
l Secretion by corpus luteum

-preparation of uterine endometrium for
possible pregnancy -inhibits new follicular
development and uterine contractions
during pregnancy

l Increases mammary gland alveolar-lobular
formation
A3
27
Actions of Estradiol Selected Examples (Female)
l Growth / maintenance of female reproductive
tract. Pubertal development
l Increases granulosa cell proliferation.
l Increases growth of endometrium and myometrium.
l Increases progesterone receptors in endometrium.
l Regulation of LH surge / cyclicity.
l Increases development of secondary sex
characteristics.
l Stimulates duct development in mammary tissue.
l Effects on behavior
l Functions as a neuroprotectant
A4
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