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Aop: 289

AOP Title

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Inhibition of 5α-reductase leading to impaired fertility in female fish

Short name:

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5α-reductase,female fish

Graphical Representation

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Click to download graphical representation template

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Authors

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Chang Seon Ryu, KIST Europe, Germany

Baeckkyoung Sung, KIST Europe, Germany

Seungyun Baik, KIST Europe, Germany 

Young Jun Kim, KIST Europe. Germany

Yongoh Lee, KIST Europe.Germany

Point of Contact

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Young Jun Kim   (email point of contact)

Contributors

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  • Young Jun Kim

Status

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Author status OECD status OECD project SAAOP status
Open for citation & comment Under Development 1.81 Included in OECD Work Plan


This AOP was last modified on March 28, 2020 06:39

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Revision dates for related pages

Page Revision Date/Time
5α-reductase, inhibition April 18, 2019 19:48
Decrease, dihydrotestosterone (DHT) level April 10, 2019 05:22
Reduction, Plasma 17beta-estradiol concentrations September 26, 2017 11:30
Reduction, Plasma vitellogenin concentrations September 16, 2017 10:14
Reduction, Cumulative fecundity and spawning March 20, 2017 17:52
5α-reductase, inhibition leads to Decrease, DHT level April 18, 2019 19:54
Decrease, DHT level leads to Reduction, Plasma 17beta-estradiol concentrations April 18, 2019 19:55
Reduction, Plasma 17beta-estradiol concentrations leads to Reduction, Plasma vitellogenin concentrations October 18, 2018 11:02
Reduction, Plasma vitellogenin concentrations leads to Reduction, Cumulative fecundity and spawning September 18, 2018 20:55
finasteride, dutasteride, epristeride April 18, 2019 19:56

Abstract

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This AOP is designed to detect changes in cumulative fecundity and spawning resulted from the inhibition of 5α-reductases by 5α-reductase inhibitors. 5α-reductase catalyzes a 3-oxo-5α-steroid to a 3-oxo-Δ4-steroid.  Major reaction is the conversion of testosterone to 5α-dihydrotestosterone (DHT) which is a strong endogenous androgen receptor agonist.  Inhibition of 5α-reductase can be caused by chemical inhibitors such as finasteride, dutasteride, epristeride, and etc. 5α-reductase inhibition (KE 790), the MIE for this AOP, results in decreasing levels of DHT and possibly 3β-androstanediol (3β-diol, agonist of estrogen receptor β), metabolite of DHT, followed by increasing of the level of testosterone in female fish (L.Mariotta-Calsaluci et al., 2013 Aquatic Toxicol). Whereas inhibition of 5α-reductase leads to decrease in the level of 17β-estradiol (E2) (KE 219) in a female by the unknown mechanism, which corresponds to decreased egg production and spawning.

 


Background (optional)

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This AOP describes an adverse outcome that results from the inhibition of 5α-reductase (3-Oxo-5α-steroid 4-dehydrogenase) in female fish. 5α-reductase catalyzes a 3-oxo-5α-steroid to a 3-oxo-Δ4-steroid.  Major reaction is the conversion of testosterone to 5α-dihydrotestosterone (DHT) which is a strong endogenous androgen receptor agonist.  Inhibition of 5α-reductase can be caused by chemical inhibitors such as finasteride, dutasteride, epristeride, and etc. 5α-reductase inhibition (KE 790), the MIE for this AOP, results in decreasing levels of DHT and possibly 3β-androstanediol (3β-diol, agonist of estrogen receptor β), metabolite of DHT, followed by increasing of the level of testosterone in female fish (L.Mariotta-Calsaluci et al., 2013 Aquatic Toxicol). Whereas inhibition of 5α-reductase leads to decrease in the level of 17β-estradiol (E2) (KE 219) in a female by the unknown mechanism, which corresponds to decreased egg production and spawning. There have been a few studies on the evaluation of the inhibition of 5α-reductase in fish (L.Mariotta-Calsaluci et al., 2013 Aquatic Toxicol.;García-García et al. 2017  J Steroid Biochem Mol Biol) and these studies did not clarify the mechanism of the inhibition of 5α-reductase to decrease 17β-estradiol (E2) in female fish. Ornostay et al. (2016) reported DHT increased the level of E2 and steroidogenesis gene expression in fathead minnow ovary. The level of E2 is highly correlated with the synthesis of vitellogenin (VTG), having significant roles in reproduction. Reduced VTG (KE 221) in fish has been used as an endpoint for adverse effects on fertility and reproduction (Toxicol Sci, 2013. 132(2):284-297; Environ Toxicol Chem, 2016. 35(8): 2117-2224; Environ Toxicol, 2017. 32(7):1869-1877; Aquat Toxicol, 2018. 200:206-216). Additionally, possible KE is the inhibition of 5α-reductase affects the level of the other endogenous substrate steroids such as androstenedione, progesterone, cortisol, and aldosterone. The physiological responses of the reduction of these steroids and the inhibition of 5α-reductase are not fully understood (Azzouni et al. 2012). Furthermore, key event relationship (KER) to the levels of reduced aromatase (expression/activity) or reduced VTG by the inhibition of 5α-reductase was not well defined. Cumulative fertility is the major endpoint for the evaluation of reproductive toxicity caused by endocrine disruption with the exposure to endocrine disrupting chemicals (Ecotoxicol Environ Saf, 2018. 162:438-445; Environ Pollut, 2018. 240:403-411; J Appl Toxicol, 2018.38(4):544-551).


Summary of the AOP

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Events: Molecular Initiating Events (MIE)

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Key Events (KE)

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Adverse Outcomes (AO)

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Sequence Type Event ID Title Short name
1 MIE 1617 5α-reductase, inhibition 5α-reductase, inhibition
2 KE 1613 Decrease, dihydrotestosterone (DHT) level Decrease, DHT level
3 KE 219 Reduction, Plasma 17beta-estradiol concentrations Reduction, Plasma 17beta-estradiol concentrations
4 KE 221 Reduction, Plasma vitellogenin concentrations Reduction, Plasma vitellogenin concentrations
5 AO 78 Reduction, Cumulative fecundity and spawning Reduction, Cumulative fecundity and spawning

Relationships Between Two Key Events
(Including MIEs and AOs)

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Title Adjacency Evidence Quantitative Understanding
5α-reductase, inhibition leads to Decrease, DHT level adjacent High High
Decrease, DHT level leads to Reduction, Plasma 17beta-estradiol concentrations adjacent Low Low
Reduction, Plasma 17beta-estradiol concentrations leads to Reduction, Plasma vitellogenin concentrations adjacent High High
Reduction, Plasma vitellogenin concentrations leads to Reduction, Cumulative fecundity and spawning adjacent High High

Network View

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Stressors

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Name Evidence Term
finasteride, dutasteride, epristeride High

Life Stage Applicability

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Life stage Evidence
3 to < 6 months Moderate

Taxonomic Applicability

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Term Scientific Term Evidence Link
fish fish Moderate NCBI

Sex Applicability

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Sex Evidence
Female Moderate

Overall Assessment of the AOP

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Attached file: Overall assessment

WP 1: Development of metabolomics platform for evaluation of endocrine metabolism and disruption

Molecular initiating events (MIEs) and key events (KEs) will be identified for depicted Dutasteride and finesteride on steroidogenesis. This will allow us to build up an AOP for these substances. After exposure of organisms to EDCs in non-lethal concentrations, a metabolomics study using high resolution mass spectrometry will help to identification MIEs and the KEs on mitochondrial or endoplasmic reticulum CYP450, hydroxysteroid dehydrogenase, short-chain dehydrogenase/reductase. Protein and mRNA expression level will be measure for verification and identification of pathway.

  • Development of analytic methods using mass spectrometry for cholesterol metabolism including steroids under endocrine disruption chemicals treatment. Selection of target enzymes in steroidogenesis and prediction of biological effects on knock-out and knock-in cell models.

*Potential target enzymes: Steroid 5α-reductase, CYP11A1, CYP17, CYP19, CYP21, CYP11B1, CYP11B2, 3β-Hydroxysteroid dehydrogenase, and 17β-Hydroxysteroid dehydrogenases, aldoketo reductase, membrane-associated progesterone receptors

 

WP 2: Quantitative evaluation of inhibition assay in the developed platform

The effects of depicted quantitative metabolomics will be tested in certain in vitro cell models experiments using knockout/knock-in system. These experiments will be clarified possible effects on the toxicity pathway.

  • Evaluation of inhibition effects using chemical inhibition on in vitro cell models
  • Development of stable 5α reductase knockout/knock-in system using CRISPR/Cas9 system (dCas9/CRISPR-SAM)
  • Identification of pathway using knockout/knock-in models

 

WP 3: Development of the quantitative adverse outcome pathway

Dose-response based qAOPs can be developed by systems toxicology approach

  • Quantification of endocrine disruption effects using in vivo models which are invertebrates (water flea of brine shrimp) and vertebrates. To integrate the quantitative relationships generated by CYP450 expression or in vivo testing, quantitative AOPs (qAOPs) providing dose-time responses are more critical than AOPs. We will compare previous approaches to qAOP building
  • Identification of interspecies comparison of new target toxicity pathway in humans, rodents, fish cell line and vertebrates model.
  • Quantitative comparison of key event relationship

Domain of Applicability

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Chemical: This AOP applies to anti-androgenic effects. Compounds which can inhibit the 5α reductase in vitro can be used for this AOP such as Flutamide, Dutasteride, and Finasteride

Sex: The AOP applies to female fish only.

Life stages: The relevant life stages for this AOP are reproductively mature adults. 

Taxonomic: teleost fish species.


Essentiality of the Key Events

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Although a few results for female fish have presented the proposed key event relationship, a low level of DHT resulted in low level of E2 in fish. Essentiality of proposed the metabolites from DHT such as 3β diol, an estrogenic compound can be supported for the reduction of Vtg expression. Lower E2 level by reduced DHT can be supported by negative feedback as anti-androgenic activities. Dutasteride in repeated dose caused a concentration-dependent decrease in E2, Vtg level by non-aromatizable estrogenic hormone level.

Concordance of dose-response relationships

Chemicals

Species

Exposure period

Range of dose

Effects

Reference

Flutamide

FHMs

14 and 21 days

100, 320, 1000 µg/L

Plasma T level (↑) in female

(Panter, Hutchinson et al. 2004)

Flutamide

Juvenile asian catfish

50 days

33 µg/L

Plasma T level (↓)

Plasma 11KT level (↓)

(Rajakumar, Singh et al. 2012)

Flutamide

FHMs

21 days

50, 500 µg/L

Plasma Vtg level (↑) in male

Plasma Vtg level (↑) in female

(Jensen, Kahl et al. 2004)

Tamoxifen and flutamide

Eel hepatocyte

6 days

1 µM, 10 µM

Vtg level in medium (↓)

(Kwon, Choi et al. 2005)

Finasteride

Gilthead seabream

7, 15, 21 days

1 µg/g-body mass

Activity of 5α-reductase after 7 days (↓)

DHT serum level (↓), Plasma E2 (↓), spermatogenesis (↓)

(García-García, Sánchez-Hernández et al. 2017)

Finasteride

Medaka

12, 21 days

50, 500, 5000 µg/L

Gonad weight (↑) in male (F1 generation)

(Lee, Loux-Turner et al. 2015)

Dutasteride

Fish; fathead minnow (Pimephales promelas)

21 days

10, 32, and 100 µg/L

Treatment of dutasteride → decreaded egg production, spawning, expression of secondary sexual characteristics

In male: E2 (little bit decreased), T (↓), KT (↓)

In female: E2 (↓), T (↑), KT (no effect)

Vitellogenin (↓) in female

Hatching rate (↓)

(Margiotta-Casaluci, Hannah et al. 2013)

Linuron

FHMs

21 days

1, 10, 100 µg/L

Plasma Vtg (↓) in female

(Marlatt, Lo et al. 2013)

Fadrozole

FHMs

21 days

2-50 µg/L

Plasma E2 (↓) and Plasma Vtg (↓) in female; Plasma T (↑) and plasma 11KT (↑) in male

(Ankley, Kahl et al. 2002)

Fadrozole

Fathead minnows

8 days

3, 30 µg/L

Plasma E2 level (↓) after 1 days but, it recovered  after 8 days at 3 µg/L

Plasma Vtg level (↓)

(Villeneuve, Mueller et al. 2009)

DHT

Mature female tilapia (Oreochromis mossambicu) hepatocytes

48 h

0.1-10 µM

Vtg level in medium (↑) in female

Vtg level in medium (↑) in male

(Kim, Takemura et al. 2003)

Androstenedione

Juvenile rainbow trout

14 days

0.05-50 mg/kg per day

Plasma E2 (↑)

Plasma Vtg (↑)

(Bowman, Kroll et al. 2000)

Testosterone

Fish; Zebrafish (Danio rerio)

60 days

100, 320, 1000 ng/L

In male: Vtg (↓), Maturity (↑)

In female: Vtg (↓), Maturity (↓)

Increased ratio of males

Decreased ratio of females

(Baumann, Holbech et al. 2013)

 


Evidence Assessment

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The steroidogenesis pathways for androgenic and estrogenic activity are well known. However, the indirect linkage between anti-androgenic and estrogenic effects is not a feasible concept. It is not clear whether 5α reductase inhibition limits the Vtg uptake and oocyte maturation. At present, the links between DHT and E2 or Vtg can be supported for this AOP. Furthermore, the negative feedback loop and plasma level of DHT metabolite 3β diol can be potentially covered as an estrogenic activity. So far, this mechanism can support the uncertainties regarding the plausible concepts through the function of 5α reductase including three metabolites via reduced NADPH in steroidogenesis (Dihydrotestosterone, Androstanedione, Pregnandione).


Quantitative Understanding

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The quantitative understanding of the AOP is insufficient to directly link a measure of chemical potency as a 5α reductase inhibition to a predicted effect concentration at the level of cumulative fecundity. However, knock out gene techniques can be analyzed quantitatively.


Considerations for Potential Applications of the AOP (optional)

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This AOP is designed to detect changes in cumulative fecundity and spawning resulted from the inhibition of 5α-reductases by 5α-reductase inhibitors. Alteration of fecundity and spawning in fish is the critical endpoint for reproductive toxicity caused by endocrine disruption. This endpoint is essential and useful for screening of the potential endocrine disrupting chemicals and/or risk assessment for the possible contaminated sites by these chemicals. Therefore, this AOP can be applied to the prediction of reproductive toxicity caused by the inhibition of 5α-reductase .


References

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Ankley, G. T., M. D. Kahl, K. M. Jensen, M. W. Hornung, J. J. Korte, E. A. Makynen and R. L. Leino (2002). "Evaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas)." Toxicological sciences : an official journal of the Society of Toxicology 67(1): 121-130.

Baumann, L., H. Holbech, S. Keiter, K. L. Kinnberg, S. Knörr, T. Nagel and T. Braunbeck (2013). "The maturity index as a tool to facilitate the interpretation of changes in vitellogenin production and sex ratio in the Fish Sexual Development Test." Aquatic toxicology (Amsterdam, Netherlands) 128-129: 34-42.

Bowman, C. J., K. J. Kroll, M. J. Hemmer, L. C. Folmar and N. D. Denslow (2000). "Estrogen-induced vitellogenin mRNA and protein in sheepshead minnow (Cyprinodon variegatus)." General and comparative endocrinology 120(3): 300-313.

Diotel, N., J.-L. Do Rego, I. Anglade, C. Vaillant, E. Pellegrini, H. Vaudry and O. Kah (2011). "The brain of teleost fish, a source, and a target of sexual steroids." Frontiers in neuroscience 5: 137-137.

García-García, M., M. Sánchez-Hernández, M. P. García-Hernández, A. García-Ayala and E. Chaves-Pozo (2017). "Role of 5α-dihydrotestosterone in testicular development of gilthead seabream following finasteride administration." The Journal of steroid biochemistry and molecular biology 174: 48-55.

Jensen, K. M., M. D. Kahl, E. A. Makynen, J. J. Korte, R. L. Leino, B. C. Butterworth and G. T. Ankley (2004). "Characterization of responses to the antiandrogen flutamide in a short-term reproduction assay with the fathead minnow." Aquatic toxicology (Amsterdam, Netherlands) 70(2): 99-110.

Kim, B.-H., A. Takemura, S. J. Kim and Y.-D. Lee (2003). "Vitellogenin synthesis via androgens in primary cultures of tilapia hepatocytes." General and comparative endocrinology 132(2): 248-255.

Kwon, H. C., S. H. Choi, Y. U. Kim, S. O. Son and J. Y. Kwon (2005). "Androgen action on hepatic vitellogenin synthesis in the eel, Anguilla japonica is suppressed by an androgen receptor antagonist." The Journal of steroid biochemistry and molecular biology 96(2): 175-178.

Lee, M. R., J. R. Loux-Turner and K. Oliveira (2015). "Evaluation of the 5α-reductase inhibitor finasteride on reproduction and gonadal development in medaka, Oryzias latipes." General and comparative endocrinology 216: 64-76.

Margiotta-Casaluci, L., R. E. Hannah and J. P. Sumpter (2013). "Mode of action of human pharmaceuticals in fish: the effects of the 5-alpha-reductase inhibitor, dutasteride, on reproduction as a case study." Aquatic toxicology (Amsterdam, Netherlands) 128-129: 113-123.

Marlatt, V. L., B. P. Lo, A. Ornostay, N. S. Hogan, C. J. Kennedy, J. R. Elphick and C. J. Martyniuk (2013). "The effects of the urea-based herbicide linuron on reproductive endpoints in the fathead minnow (Pimephales promelas)." Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 157(1): 24-32.

Martyniuk, C. J., S. Bissegger and V. S. Langlois (2013). "Current perspectives on the androgen 5 alpha-dihydrotestosterone (DHT) and 5 alpha-reductases in teleost fishes and amphibians." General and comparative endocrinology 194: 264-274.

Panter, G. H., T. H. Hutchinson, K. S. Hurd, A. Sherren, R. D. Stanley and C. R. Tyler (2004). "Successful detection of (anti-)androgenic and aromatase inhibitors in pre-spawning adult fathead minnows (Pimephales promelas) using easily measured endpoints of sexual development." Aquatic toxicology (Amsterdam, Netherlands) 70(1): 11-21.

Rajakumar, A., R. Singh, S. Chakrabarty, R. Murugananthkumar, C. Laldinsangi, Y. Prathibha, C. C. Sudhakumari, A. Dutta-Gupta and B. Senthilkumaran (2012). "Endosulfan and flutamide impair testicular development in the juvenile Asian catfish, Clarias batrachus." Aquatic toxicology (Amsterdam, Netherlands) 110-111: 123-132.

Sullivan, C. V. and O. Yilmaz (2018). Vitellogenesis and Yolk Proteins, Fish. Encyclopedia of Reproduction (Second Edition). M. K. Skinner. Oxford, Academic Press: 266-277.

Villeneuve, D. L., N. D. Mueller, D. Martinović, E. A. Makynen, M. D. Kahl, K. M. Jensen, E. J. Durhan, J. E. Cavallin, D. Bencic and G. T. Ankley (2009). "Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor." Environmental health perspectives 117(4): 624-631.