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Androgens
- Role in males similar to the of estrogens in females
- development of male sexual characteristics
- stimulating protein synthesis, growth of bones
- cell differenciation, spermatogenesis
- male type of behaviour
Androgens
- Endogenous ligands ­ androgen hormones
- testosterone
- dihydrotestosterone (DHT)
- androstanediol
- dehydroepiandrosterone
- androstenedione
OH
O
Testosterone
Hypothalamo-pituitary axis
- Regulation of testosterone
synthesis
- Hypothalamus ­
Gonadotropin releasing
hormone
- Pituitary ­ folicle
stimulating and
luteineising hormone
Hypothalamo-pituitary axis
- Folicle stimulating hormone
- Stimulates synthesis of
androgen binding proteins
and spermatogenesis in
Sertoli cells (testis)
- Luteineizing hormone
- Stimulates testosterone
production in Leydig cells
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Testosterone
- synthetized in testis (Leydig
cells)
- in lesser extent in adrenals
Dihydrotestosterone
- The most important derivative of testosterone
- Formed extratesticulary from testosterone
- 5-reductase
Dihydrotestosterone
- In several tissues (seminal vesicles, prostate, skin)
higher affinity to androgen receptor than
testosterone
- Daily production 5-10% of testosterone OH
O
Dihydrotestosterone
Mechanism of action
Androgens
AR
Androgen-responsive
elements
Gene
expression
AR
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Mechanisms of
androgen signalling disruption
- Illegitimate activation of AR
- Binding to AR without activation
- Decrease of AR cellular levels
- FSH/LH signalling disruption
- Changes in androgen metabolism
Mechanisms of androgen
signalling disruption
Binding to AR
- Mostly competitive inhibition ­ xenobiotics do
mostly NOT activate AR-dependent transcription
- Few compounds are able to activate AR in absence
of androgen hormones x in presence of T/DHT
antiandrogenic (metabolites of funghicide
vinclozoline, some PAHs)
Mechanisms of androgen
signalling disruption
Decrease of AR levels
- Under normal circumstances, DHT treatment leads
to increase of AR level
BUT no effect observed during co-treatment with
Cyproterone acetate or hydroxyflutamide
(drugs/pesticides)
Mechanisms of androgen
signalling disruption
FSH/LH (gonadotropins) signalling disruption
- FSH/LH expression - regulation via negative
feedback by testosterone
- Suppressing leads to alterastions of spermatogenesis
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Mechanisms of androgen
signalling disruption
Alterations of testosterone synthesis
- Inhibition of P450scc needed for side chain
cleavage of cholesterol (fungicide ketoconazol)
- Inhibition of 17- -hydroxylase and other CYPs - ­
enzymes needed for testosterone synthesis
(ketoconazol)
Mechanisms of androgen
signalling disruption
Testosterone metabolic clearance
- Induction of UDP-glucuronosyltransferase or
monooxygenases CYP1A, 1B involved in androgen
catabolism
- Pesticides Endosulfan, Mirex, o-p´-DDT
Effects of male exposure to
antiandrogens
Exposure during prenatal development:
-malformations of the reproductive tract
- reduced anogenital distance
- hypospadias (abnormal position of the urethral
opening on the penis)
- vagina development
- undescendent ectopic testes
- atrophy of seminal vesicles and prostate gland
Effects of male exposure to
antiandrogens
Exposure in prepubertal age:
- delayed puberty
- reduced seminal vesicles
- reduced prostate
Exposure in adult age:
- oligospermia
- azoospermia
- libido diminution
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AR-binding - potencies
(Ref: DHT EC50 ~ 0.1 uM)
Compound IC50 (M)
Benz[a]anthracene 3,2
Benzo[a]pyrene 3,9
Dimethylbenz[a]anthracene 10,4
Chrysene 10,3
Dibenzo[a,h]anthracene activation in range 0,1-10M
Bisphenol A 5
vinclozolin metabolites 9,7
hydroxyflutamide 5
Aroclor typical values 0,25-1,11
Individual PCBs typical values 64 - 87
tris-(4-chlorophenyl)-methanol 0,2
Antiandrogenic compounds
tris-(4-chlorophenyl)-methanol
- Ubiquitous contaminant of uncertain origin
- Probable metabolite of DDT-mixture contaminant
- Levels in human blood serum cca. 50nM
- EC50 ­ cca. 200nM
In vivo antiandrogenicity assessment
Hershberger assay
- castrated rats treated with substance examined
- Endpoint ­ after 4-7 days ­ seminal vesicles and
ventral prostate weight
Measurement of testosterone concentration in serum
In vitro
antiandrogenicity assessment
Most often employed ­ prostatic cell lines
Cell proliferation assays ­ cell lines with androgen-
dependent growth;
- Treatment with tested chemical only (androgenicity)
or cotreatment with DHT (antiandrogenicity)
- mammary carcinoma cell lines
- prostatic carcinoma cell lines
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In vitro antiandrogenicity assessment
Receptor-reporter assays
- Gene for luciferase or GFP synthesis under
transcriptional control of AR
- Luciferase:
- AR-Calux (human breast carcinoma T47D)
- PALM (human prostatic carcinoma PC-3)
- CHO515 (Chinese hamster ovary CHO)
AR-binding - potencies
(Ref: DHT EC50 ~ 0.1 uM = 100nM)
In vitro antiandrogenicity assessment
GFP
- Possibility of nondestructive measurement
(fluorescence of intact cells)
X
Less sensitive ­ lack of enzymatic amplification
- Human prostatic cell lines
In vitro antiandrogenicity assessment
Yeast assays
- Mostly -galactosidase as reporter enzyme
- Easy cultivation and experimental design
X
- Cell wall may obstruct transport of chemical into cell=>
=> false negatives