Effects of exposures to the plasticiser, di-n-butyl phthalate and the pharmaceutical, flutamide on the biomarkers of reproduction in Australian freshwater fish species, Murray rainbowfish (Melanotaenia fluviatilis).

Abstract

With the detection of anti-androgenicity in the effluents from the wastewater treatment plants (WWTPs), there is speculation that sexual disruption in fish is a multi-causal condition involving anti-androgens. Much of the research has focussed on deciphering the modes-of-action (MoAs) of (anti)estrogens and androgens. However, effects of androgen receptor (AR) antagonists have not been fully characterised and remain elusive in fish. The present study aimed to investigate the effects of the classic mammalian anti-androgen, flutamide and the emerging industrial pollutant, di-n-butyl phthalate (DnBP) on the biomarkers of reproduction in adult (male and female) and juvenile Murray rainbowfish (Melanotaenia fluviatilis). Flutamide is the “pure” anti-androgen designed to treat prostate cancer in men and polycystic ovarian syndrome in women. It has also been extensively used in toxicity testing in mammals. The in vitro anti-androgenic activity in the aquatic environment worldwide is measured in flutamide equivalents. Phthalates are a class of synthetic industrial chemicals commonly found in the aquatic environment worldwide. They have been recognised as anti-androgens in male mammals but little is known about their endocrine-disrupting effects in the native Australian fish species. Due to its detection in freshwater both in Australia and worldwide and considering its higher solubility in water (11 mg/L), it is important to investigate effects of DnBP on the reproductive fitness of native Australian fish species. Flutamide is not an environmental contaminant and has not been detected in freshwater. However, it is used as the reference chemical to quantify antiandrogenic activity in aquatic environment using in vitro assays. In addition, flutamide is also used as the model anti-androgen to investigate anti-androgenic effects in mammals. Adult female and male Murray rainbowfish were exposed to biologically active concentrations (nominal 125 – 1000 μg/L) of flutamide for 7 days. In females, histological investigation revealed marked atresia and absence of mature oocytes in the flutamide-treated fish at all concentrations investigated. Reduction in the sizes of the vitellogenic oocytes was found after treatment with 500 and 1000 μg/L flutamide. The plasma VTG and the activity of brain aromatase were reduced in fish treated with 500 and 1000 μg/L flutamide. Treatment with 500 and 1000 μ/L flutamide reduced the concentrations of 11-keto testosterone (11-KT) and 17β-estradiol (E2) in plasma. In males, qualitative assessment of the testes of the fish exposed to 125 – 1000 μg/L flutamide exhibited inhibition in transformation of spermatogonia to spermatozoa and increased testicular anomalies like multinucleated and pyknotic cells and interstitial fibrosis. VTG was induced in plasma after an exposure to 1000 μg/L of flutamide. The activity of brain aromatase declined after exposure to flutamide at all concentrations. Males exposed to 1000 μg/L of flutamide showed a down-regulation of the hepatic genes encoding androgen receptors α (ARα) and ARβ. The expression levels of the genes for the estrogen receptor α (ERα) were up-regulated and those of VTG were downregulated after treatment with 250 – 1000 μg/L of flutamide. Juvenile rainbowfish were exposed to the nominal concentrations 25 ng/L E2, 25 μg/L flutamide, 250 μg/L flutamide, 25 ng/L E2 + 25 μg/L flutamide and 25 ng/L E2 + 250 μg/L flutamide. Co-treatment with Flu high and E2 resulted in significant reductions in weights and lengths in males and condition factor in females. Inter-sex was noted in Flu high and E2+Flu high treated fish. The development of spermatocytes in the testes was inhibited by E2 and this effect was accentuated after co-treatment with flutamide. Exposures to E2 resulted in precocious oocyte development in the ovaries which was further up-regulated when fish were co-exposed to E2 and flutamide. The E2 levels decreased significantly in the head of both males and females after co-exposures to flutamide and E2. Flutamide and E2 alone increased the 11-KT levels in both sexes. However, E2+Flu low decreased 11-KT levels in males and increased them in females. Flutamide (low and high) induced VTG protein in the tails of both sexes. In males, VTG was induced in the tail tissue after exposure to flutamide but not E2. No significant increase of flutamide on E2-induced VTG concentration was noted. We concluded that anti-androgens do not add to the effects of estrogens due to different modes of action. However, they induce similar effects which can cause additive inhibition/stimulation of the gonad development. Sexually mature female and male Murray rainbowfish were exposed to sub-acute concentrations of 125 – 1000 μg/L DnBP for 7 days. The testes in 125 – 1000 μg/L DnBPexposed fish were in the early spermatogenic stage with a higher proportion of spermatogonia. The sizes of spermatogonia, Type A and B spermatocytes and spermatids were significantly smaller relative to the controls after treatment with 125 – 1000 μg/L of DnBP. The sizes of the previtellogenic oocytes in the 250 – 1000 μg/L treated fish were higher than those in the corresponding control fish. The early vitellogenic oocytes in the 1000 μg/L treated fish were smaller relative to those in the unexposed fish. Histological changes like chorion folding, shrunken ooplasm, impaired yolk production, granulomatous inflammation and interstitial fibrosis were observed in the ovaries of the fish treated with DnBP at all concentrations. The plasma VTG was significantly lower in the female and higher in males exposed to 500 – 1000 μg/L DnBP. An induction in the expression levels of the genes encoding for ERα and β and choriogenin L, coupled with an amplified activity of aromatase in the brain for the 1000 μg/L of DnBP treatment suggested an estrogenic MoA of DnBP in male fish. Juvenile fish were exposed to environmentally relevant concentrations (5, 15 and 50 μg/L) of DnBP for 90 days. The lowest observed effective concentration to significantly affect the condition factor after 90 days was 5 μg/L. Histological investigation revealed complete feminisation of the gonad in fish exposed to 5 μg/L for 90 days and to 15 and 50 μg/L of DnBP at all sampling times. In addition, incidences of inter-sex gonads were noted in the 15 and 50 μg/L of DnBP treatments at the end of the exposure period. After 90 days of exposure to DnBP, the ovaries were regressed and immature. Testes, present only in fish exposed to 5 μg/L of DnBP for 30 or 60 days, were vacuolated and immature. There was a significant induction in E2 concentration in fish exposed to 5 μg/L of DnBP for 90 days and in 15 and 50 μg/L DnBP treatments at all sampling times. Long-term exposure to low concentration (5 μg/L for 90 days) had similar hormonal effects as short-term exposure to higher concentration of DnBP (50 μg/L for 30 days) in causing in vivo estrogenicity. Longterm continuous exposures to 5 μg/L of DnBP for up to 30 days did not have profound effects on body growth and gonadal differentiation of fish. However, 30 days of continuous exposures to 15 μg/L could interfere with the gonad development and to 50 μg/L could compromise the hormonal profile of juvenile fish. The study, for the first time, reported the differential effects of two anti-androgens in male, female and juvenile Australian fish species. Using an integrated approach of histological, biochemical and molecular tools, the hypothetical models of effects and potential MoAs of flutamide and DnBP have been proposed. The data from the present study suggest that continuous exposures to biologically active concentrations of flutamide for 7 days can cause anti-androgenicity in male and defeminisation in female adult Murray rainbowfish. In addition, 35 day exposures to the anti-androgen, flutamide can induce feminisation in juvenile Murray rainbowfish and the effects of flutamide are cumulative in combination with E2. It was also concluded that continuous exposures to sub-acute concentrations of DnBP for 7 days can cause estrogenicity in male and anti-estrogenic effects in female adult Murray rainbowfish. Treatments with environmentally relevant concentrations of DnBP for 90 days during sensitive phases of development in juvenile Murray rainbowfish adversely affect the fish growth and gonad development. It is proposed that short-term exposures to high concentrations and long-term exposures to low concentrations of DnBP have similar reproductive endocrine effects. Australian water quality guidelines recommend DnBP concentrations should be < 9.9 μg/L for freshwater ecosystems (http://www.environment.gov.au/resource/australian-and-newzealand- guidelines-fresh-and-marine-water-quality-volume-1-guidelines). Levels of DnBP found in freshwaters in Australia (47 ng/L) do not pose a threat to the reproductive fitness of Murray rainbowfish. However, there is a strong need to revise the water quality guidelines for DnBP in freshwater in Australia for future reference. It is also recommended to detect, identify and quantify individual anti-androgens in freshwater in Australia and worldwide. In addition, identification of the genes and testing molecular tools regulating gonadal differentiation in Murray rainbowfish are needed to assess the reversibility of the effects caused by environmental chemicals. It is important to investigate the endocrine disruption, if any, in the fish thriving in this aquatic environment in Australia using field caging studies. Similar studies have been reported in Europe and the US.