Alice Baynes, Gemma Montagut Pino, Anne E Lockyer, Hanna Belschner, Edwin Routledge, Susan Jobling. Biology at Brunel 2 nd May 2018

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1 Unexpected Endpoints: Pharmaceutical 5αreductase inhibitors, designed to treat prostate cancer in men, disrupt gastropod morphogenesis during embryo development. Alice Baynes, Gemma Montagut Pino, Anne E Lockyer, Hanna Belschner, Edwin Routledge, Susan Jobling. Biology at Brunel 2 nd May 2018

2 In mammals and other vertebrates, 5α-reductase (5αR) converts the steroid androgen, testosterone (T) to the more potent dihydrotestosterone (DHT) DHT plays a vital role in vertebrate sexual development DHT it is also involved in many androgen dependent disorders Two main pharmaceuticals are used to inhibit 5αR and reduce DHT >Finasteride (FIN) and Dutasteride (DUT) 5αR

3 5αR inhibition in vertebrate test species Finasteride (FIN) utero exposure - male rat Decreases in AGD Increased nipple retention, hypospadias, ectopic testes, and prostate malformations Finasteride (FIN) treated Silurana (Xenopus) tropicalis early larval development Sex ratios/ phenotypes skewed + intersex Dutasteride (DUT) treated adult fathead minnow (pair breeding) Reduce reproductive output, disrupted male and female gametes Bowman et al Duart-Guterman et al Langlois et al Margiotta-Casaluci et al

4 So why molluscs? Gastropod molluscs have recently been introduced into OECD chemical testing guidelines for reproductive toxicity screening 3Rs initiatives support the use of invertebrates in screening /mechanistic research We have significant knowledge-gaps in fundamental developmental and reproductive mechanisms in these animals en;jsessionid=1pu1rbkqsptc3.x-oecd-live-03 en;jsessionid=1pu1rbkqsptc3.x-oecd-live-03

5 So why molluscs? 5α-reductase homologs identified in mollusc genomes (e.g. Biomphalaria glabrata, Aplysia californica, Mizuhopecten yessoensis) Gastropods and bivalves can convert exogenous testosterone (T) to 5αdihydrotestosterone (DHT) Janner et al Schwarz et al

6 However, B. glabrata does not use vertebrate steroid androgens in their reproductive development or have a nuclear androgen receptor (AR) in its genome.

7 So what, if anything, does 5αR do in gastropods?

8 Experimental design Early development window often sensitive to disruption Exposure from blastula 20hrs after being laid Water exposure: Control, Solvent control and100 µg/l DUT Six replicate egg masses per treatment (1 per well, embryos/mass) Snails normally hatch ~5-7 days after being laid 27 C)

9 CONTROL SOLVENT CONTROL 100 µg/l DUT did not hatch Elongated banana or polo (donut) phenotype Inhibition of 5αR by dutasteride causes developmental abnormalities 9

10 Really? Is it Real? Repeatable? Dose response? Is it just DUT or can FIN cause same response? Can other enzyme inhibitors cause the same response? Is it just toxicity?

11 % phenotype /response Dutasteride dose-response 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% DEATH OTHER MALFORMATIONS 'BANANA' SHAPE HATCHED/OK 0% Control Solvent DUT5 DUT10 DUT20 DUT40 DUT80 DUT160 Treatment µg/l Percentage of different phenotypes/responses. Day 6 of exposure. Average of four independent experiments, each with six replicate egg masses per treatment. (DUT 160µg/L = 304 nm) 11

12 % Phenotype/ response Finasteride dose-response 100% 90% 80% DEATH 70% 60% 50% OTHER MALFORMATIONS 'BANANA' SHAPE 40% 30% HATCHED/OK 20% 10% 0% Control Solvent DUT100 FIN300 FIN450 FIN675 FIN1013 FIN1520 Treatment µg/l Percentage of different phenotypes/responses. Day 6 of exposure. Average of four independent experiments, each with six replicate egg masses per treatment. DUT 100 µg/l positive control. (FIN 1520µg/L = 4µM) 12

13 Pharmaceutical steroidogenesis inhibitors Steroidogenesis in Humans Häggström M, Richfield D (2014). "Diagram of the pathways of human steroidogenesis". WikiJournal of Medicine 1 (1). DOI: /wjm/ ISSN , CC BY-SA 3.0,

14 % Phenotype / response % Phenotype/ response Enzyme inhibitors 100% 90% DEATH Galeterone, inhibitor of CYP17A1, 17,20- lyase, AR antagonist Trilostane, inhibitor of 3 β-hydroxysteroid dehydrogenase Atorvastatin, inhibitor of HMG- CoA reductase Chenodeoxycholic acid, 5β-reductase inhibitor 80% 70% 60% 50% 40% 30% 20% 10% 0% 100% 90% 80% 70% 60% 50% 40% 30% Control Solvent DUT100 GAL 1500 CHEN 3000 Treatment µg/l OTHER MALFORMAT IONS 'BANANA' SHAPE HATCHED/ OK DEATH OTHER MALFORMAT IONS 'BANANA' SHAPE 20% 10% HATCHED/ OK 0% CONTROL SOLVENT DUT100 ATOR 3000 TRIL 3000 Treatment µg/l 14

15 Initial conclusions 5αR enzymes may have a physiological role in gastropods Don t (yet) understand the molecules (substrate) and pathways involved Endpoints in B. glabrata different to fish/frogs, BUT sensitivity to 5αR disruption at similar concentrations The differences in potency between DUT and FIN are also reflected in other species e.g. 0.5mg/day DUT vs 5mg/day FIN (human therapeutic dose)

16 Conclusions continued 5αR genes are conserved between molluscs and vertebrates (and plants) but substrates and impact of disruption can be very different More research is required to better understand fundamental biology of molluscs

17 What next...? Short-term: See if I can grow banana snails beyond embryo/larvae stage Monitor for other possible abnormalities Western blots (5αR1/5αR2) of control and treated embryos Publish initial findings! 17

18 What next...? Long-term: New collaboration Dr Carmel McDougall, Griffith University, Australian Rivers Institute Comparative and functional genomics Mollusc biomineralisation Sustainable aquaculture Possible role of 5alpha reductase in molluscan shell growth/mantle patterning? Pearl industry Aquaculture Materials science 18

19 Unexpected Endpoints: Pharmaceutical 5αreductase inhibitors, designed to treat prostate cancer in men, disrupt gastropod morphogenesis during embryo development Thank you. Questions..? Alice Baynes, Gemma Montagut Pino, Anne E Lockyer, Hanna Belschner, Edwin Routledge, Susan Jobling. Alice.baynes@brunel.ac.uk 19

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22 Bg Bg Dr Bg Bg Dr β-tubulin 5αR-1 22

23 Other Malformations Normal Banana 23

24 % Phenotype/ response Other suspected 5αR inhibitors? Benzo[f]quinoline, (Experimental compound - not pharmaceutical) possible 5αR1 inhibitor γ-linolenic acid (Polyunsaturated fatty acid) possible 5αR inhibitor 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Control Solvent DUT100 BENZO 1500 Treatment µg/l BENZO 3000 LINO 3000 DEATH OTHER MALFORMATI ONS BANANA SHAPE HATCHED/OK 24

25 % Phenotype / response % Phenotype / response Other straight snail compounds Dorsomorphin (bone morphogen protein inhibitor) SB (inhibitor of transforming growth factor-β type I activin receptor-like kinase) Both previously reported to induce straight (non-coiling) phenotypes in gastropods, but not in our test system. SB431542: low solubility - increased solvent to 1% v/v compared to 0.01% (in all treatments), this level was toxic 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Control Solvent DUT100 DORS200 DORS300 DORS400 Treatment µg/l Control Solvent DUT100 SB 1900 SB 3800 Treatment µg/l DEATH OTHER MALFORMA TIONS 'BANANA' SHAPE HATCHED/ OK DEATH OTHER MALFORMA TIONS 'BANANA' SHAPE HATCHED/ OK Grande C, Patel NH. Nodal signalling is involved in left-right asymmetry in snails. Nature Shimizu K, et al. Possible functions of Dpp in gastropod shell formation and shell coiling. Dev Genes 25 Evol. 2011