Aviso de demanda

The overall aim of this thesis was to assess the effects of endocrine disrupting chemicals with different modes of action on the reproductive health and general toxicity of the Murray River rainbowfish (M. fluviatilis).

M. fluviatilis was exposed to compounds resulting from agricultural and farming operations including atrazine, pyrimethanil and the androgen 17α trenbolone as well as personal care product propylparaben and mixtures of compounds that mimicked real wastewater effluents in rivers. The effects of perfluorooctanoic acid (PFOA), an emerging chemical of concern in Australia and worldwide, were also evaluated on this species.

The androgenic feedlot contaminant 17α trenbolone caused deleterious effects on female gonads of M. fluviatilis an increased number of atretic follicles and reduction in the circulatory levels of vitellogenin were observed with resulting effects reflected by a reduction in fecundity. Despite an apparent dose response in terms of inhibition of egg production and histopathological changes, the vitellogenin levels varied with time and concentration, similar to responses in aromatase mRNA expression. 17α trenbolone was seen to have effects beyond its androgenic action on M. fluviatilis acting as a genotoxic compound at concentrations commonly found in effluents from feedlots. Diverse egg and larvae deformations were observed, which could result in a diminished capacity in the early larvae of this species to compete for food in nature when exposed to trenbolone.

The unusual U-shaped dose-response curve of trenbolone observed in Chapter 3 suggested that this chemical might elicit a different behavior at low and high exposure concentrations.

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Thus, fish were exposed to low and high concentrations of 17α trenbolone mixed with the estrogen 17β estradiol, and by evaluating estrogen and androgenic receptor expression, it was demonstrated that 17α trenbolone alone is unable to activate the estrogen receptors. When circulatory levels of vitellogenin were investigated, it was clear that 500 ng/L of 17α trenbolone was partially able to counteract the effects of 50 ng/L of the estrogen 17β estradiol at measured protein levels although no interaction was observed at the molecular level (Chapter 4).

When exposed to atrazine, the gonadal development and maturation were disrupted since these effects are also under hormonal control proving that pathways other than the ‘classical’ estrogen receptor binding are also susceptible to disruption. This was observed at low atrazine exposure with effects on gonadal maturation in females, with a significant increase of spermatogonia in the gonads of fish exposed to 13 and 130 ppb of atrazine for 14 days whereas no effects were observed in the vitellogenin levels (Chapter 5).

Similarly, when fish were exposed to mixtures of chemicals, and in long-term exposures, a multiparameter approach was proven to be essential to understand the effects of chemical mixtures on the fish, with alterations in gonads and liver being observed when vitellogenin levels remained unaltered. Only estrone and EE2 induced increased levels of vitellogenin in the plasma of M. fluviatilis while deleterious effects on the gonads were evident from tissue histopathology in the exposures to pyrimethanil and atrazine. This is conclusive evidence that these chemicals have the ability to disrupt other pathways not simply the classically “expected” binding to an estrogen receptor (Chapter 6).

Finally, further evidence that some chemicals are able to disrupt multiple pathways was also demonstrated after exposure of M. fluviatilis to perfluorooctanoic acid (PFOA). Although PFOA was slightly estrogenic leading to the induction of the circulatory levels of vitellogenin in male fish after 14 days of exposure, this chemical was also able to interfere with the thyroid pathway and lead to changes in antioxidant processes (Chapter 7).

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8.1.1. Biomarkers of endocrine disruption

Over the past decades, endocrine disruption has been considered widespread phenomenon and many endpoints have been used in assessing the risks of EDCs to individuals and populations (Allner et al., 2016).

Amongst these, the induction of the egg yolk precursor protein vitellogenin (Vtg) in juvenile and male fish has long been established as a reliable biomarker for monitoring the effects of environmental estrogens. From our results, reduction in vitellogenin was a reliable biomarker for exposure to 17α trenbolone; since a significant reduction of Vtg was associated with exposure of M. fluviatilis to this chemical. Similarly, vitellogenin induction was correlated with the exposure of this species to 17α ethinylestradiol and estrone and PFOA. However, this biomarker was unchanged when fish were exposed to mixtures of compounds which contained estrone at 85 ng/L. This result raises concerns as to whether vitellogenin induction as a biomarker in M. fluviatilis would be a sensitive enough to detect the effects of estrogens in field situations where mixtures of chemicals at low levels are most likely to occur.

Moreover, despite the value of vitellogenin levels as a biomarker in the situations described, vitellogenin levels were unchanged when M. fluviatilis was exposed to atrazine and pyrimethanil; even though alteration in the gonads were observed, despite the fact that it is well known that gonad development and maturation are under hormonal control (Leatherland et al., 1982; Nishimura and Tanaka, 2014). Thus, the use of vitellogenin as the only endpoint in biomonitoring will likely underestimate endocrine disruptive effects as observed if exposure chemicals do not directly interfere with the nuclear receptors, leading to changes in the vitellogenin levels.

8.1.2. Gene expression and time of exposure

Gene expression is under dynamic control of transcript levels and many of compensatory mechanisms take place (Ekman et al., 2011). Hence, as observed in the exposures to trenbolone where the expression of brain and ovary aromatase and vitellogenin after 7 days of exposure and of trenbolone + 17β estradiol after 7 days, the observed gene expression responses might

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likely be reflecting compensatory mechanisms. As gene expression responses are often immediate, typically following a characteristic ‘impulse’-like pattern and within the first 96 h of exposure, in longer exposures most of the gene expression level responses have already taken place.

These impulse patterns are prevalent in responses to environmental changes in all organisms, from bacteria to mammals (Erez and Naama, 2004; López-Maury et al., 2008). Transcript levels spike up or down abruptly following the environmental cue, sustain a new level for a certain period of time and then transition to a new steady state, often similar to the original levels (Yosef and Regev, 2011).

In field exposures where organisms have been exposed for prolonged periods of time, gene expression per se as a tool might not be adequate to assess effects on organisms since after longer times gene expression patterns are likely to be more reflective of compensatory responses.

More holistic approaches using whole transcriptome assessments to better characterize and identify the overall responses of an organism not captured by this study targeted approach. That may allow instantaneous and even simultaneous genome-wide detection of the expression of genes helping to reveal mechanisms of action as well as ‘signatures’ of toxicity that could be essential in identifying and developing new biomarkers (Snape et al., 2004). However, the expression of the genes are subject to temporal dynamics and the ‘timing’ of sampling might affect the expression levels as observed previously (Shanthanagouda et al., 2013). This observation was endorsed in the current study where in M. fluviatilis the vitellogenin expression and the protein levels of vitellogenin did not align. It was expected the vitellogenin expression, were reduced at high concentrations of trenbolone considering its androgenic effects, however, it observed at molecular level despite at protein level the proportion of Vtg in plasma were reduced by the co-exposure of 17β estradiol and 500 ng/L of trenbolone.

Additionally, the general question remains as to whether changes in gene expression necessarily translate into changes in enzyme activity or protein expression (Baumann et al., 2014; Glanemann et al., 2003; Laier et al., 2006). The results in Chapter 4 of the current study endorses this question and demonstrates the need for proteomic assays and not simply measurement of gene expression.

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8.1.3. The endocrine system as a whole

In the future EDC research needs to include a broader range of endpoints beyond the common reproductive endocrine assessments, the endocrine system in fish is a complex system that consists of various glands located throughout the body which synthesize and secrete hormones to regulate an array of biological processes (Pait and Nelson, 2002). The results of this thesis have shown that chemicals in the environment are able to interfere with multiple systems and consequently single biomarker assessments are likely to underestimate endocrine disruptive effects.

Moreover, reproductive development is only one of the many effects that are controlled by the endocrine system in animals. As an example, PFOA exposures were shown to lead to the disruption of the normal thyroid homeostasis in M. fluviatilis. The thyroid gland secretes the hormones thyroxine (T4) and triiodothyronine (T3), which are believed to aid fish in adapting to changes in temperature and to osmotic stress. Thus, exposure to chemicals like PFOA could affect the ability of fish to adapt to climate change.