Elsevier

Aquatic Toxicology

Volume 147, February 2014, Pages 140-150
Aquatic Toxicology

Modulation of aromatase activity as a mode of action for endocrine disrupting chemicals in a marine fish

https://doi.org/10.1016/j.aquatox.2013.12.023Get rights and content

Highlights

  • Effects of four EDCs on reproduction and aromatase in a marine fish were studied.

  • Aromatase activity in brain and ovary were affected differently by the four EDCs.

  • Changes in male brain aromatase activity were associated with decreased reproduction.

  • GSI, but not plasma vitellogenin, mirrored changes in male brain aromatase activity.

Abstract

The steroidogenic enzyme aromatase catalyzes the conversion of androgens to estrogens and therefore plays a central role in reproduction. In contrast to most vertebrates, teleost fish have two distinct forms of aromatase. Because brain aromatase activity in fish is up to 1000 times that in mammals, fish may be especially susceptible to negative effects from environmental endocrine-disrupting chemicals (EDCs) that impact aromatase activity. In this study, the effects of estradiol (E2), ethynylestradiol (EE2), octylphenol (OP), and androstatrienedione (ATD) on reproduction and aromatase activity in brains and gonads from the marine fish cunner (Tautogolabrus adspersus) was investigated. The purpose of the study was to explore the relationship between changes in aromatase activity and reproductive output in a marine fish, as well as compare aromatase activity to two commonly used indicators of EDC exposure, plasma vitellogenin (VTG) and gonadosomatic index (GSI). Results with E2, EE2, and ATD indicate that aromatase activity in cunner brain and ovary are affected differently by exposure to these EDCs. In the case of E2 and EE2, male brain aromatase activity was signficantly increased by these treatments, female brain aromatase activity was unaffected, and ovarian aromatase activity was significantly decreased. Treatment with the aromatase inhibitor ATD resulted in significantly decreased aromatase activity in male and female brain, but had no significant impact on ovarian aromatase activity. Regardless of test chemical, a decrease or an increase in male brain aromatase activity relative to controls was associated with decreased egg production in cunner and was also correlated with significant changes in GSI in both sexes. E2 and EE2 significantly elevated plasma VTG in males and females, while ATD had no significant effect. Treatment of cunner with OP had no significant effect on any measured endpoint. Overall, results with these exposures indicate EDCs that impact aromatase activity also affect reproductive output in spawning cunner.

Introduction

Endocrine disruption occurs when an exogenous chemical interferes with the normal functioning of the endocrine signaling pathways in an organism. Endocrine-disrupting chemicals (EDCs) have the potential to affect normal reproduction and development because these processes are controlled by an array of hormone signals. Over the past decade, EDCs have been found in freshwater, estuarine, and marine environments worldwide (Campbell et al., 2006, Liu et al., 2009, Kumar and Xagoraraki, 2010). Concern over the effects that EDCs may have on natural fish populations has inspired numerous studies to determine whether EDC exposure impacts fish reproduction (see Mills and Chichester, 2005, for review). The majority of this research has focused on EDCs that interact with steroid hormone receptors, such as estrogens, alkylphenols, bisphenol A, DDT and metabolites, polynuclear aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs). However, some EDCs are known to act through mechanisms other than interaction with steroid hormone receptors. One such mechanism is interference with normal hormone biosynthesis. An example of this type of EDC is tributyltin (TBT), a compound used in anti-fouling paints applied to ships and marine structures. TBT gained notoriety as the chemical that induced masculinization of female gastropod molluscs (‘imposex’). Rather than interacting with steroid hormone receptors, TBT appears to interfere with steroid hormone biosynthesis (see Matthiessen and Gibbs, 1998, for review) and specifically has been shown to modulate activity of the steroidogenic enzyme aromatase in fish (Lyssimachou et al., 2006, McAllister and Kime, 2003). There is some evidence that another widespread EDC, the herbicide atrazine, also alters the activity of aromatase (Crain et al., 1997, Keller and McClellan-Green, 2004, Sanderson et al., 2000).

Aromatase, a steroidogenic enzyme encoded by the CYP19 gene, is a member of the cytochrome P450 family. Aromatase is a rate-limiting enzyme complex that catalyzes the conversion of androgens (androstenedione and testosterone) to estrogens (estrone and estradiol) during steroidogenesis. In fish, aromatase activity occurs in a number of tissues, but invariably is highest in brains and ovaries (Callard and Tchoudakova, 1997, Choi et al., 2005, González and Piferrer, 2003, Sawyer et al., 2006). In contrast with most vertebrates, two distinct isozymes of aromatase have been characterized in teleost fish (Choi et al., 2005, Greytak et al., 2005, Kishida and Callard, 2001, Tchoudakova and Callard, 1998). One form, P450aromA, predominates in fish ovaries, while the other form, P450aromB, prevails in fish brains. For example, P450aromA accounts for greater than 98% of the total aromatase mRNA transcripts in the ovaries of fathead minnows (Pimephales promelas), while P450aromB accounts for 99.8% of the transcripts in the brain (Villeneuve et al., 2006). In adult European sea bass (Dicentrarchus labrax), the ratio of P450aromA:P450aromB transcripts was 100:1 in ovaries, while in brain the ratio was 1:1000 (Blázquez et al., 2008). The two isozymes derive from separate gene loci, CYP19A and CYP19B, and are controlled by different regulatory mechanisms (Callard and Tchoudakova, 1997, Tchoudakova and Callard, 1998). Within a single species, the CYP19A and CYP19B genes have only about a 60% sequence homology, while the homology for the CYP19A gene between different fish species is about 85% (Blázquez and Piferrer, 2004, Guiguen et al., 2010).

Studies on the function of aromatase in fish gonads indicate that ovarian aromatase controls circulating levels of estrogens and is critical to female differentiation and development (Chang et al., 2005, Guiguen et al., 2010, Kitano et al., 1999, Kumar et al., 2000, Patil and Gunasekera, 2008, Piferrer et al., 2005). In the fish brain, aromatase is localized in the forebrain and pituitary, which are both associated with reproduction (Piferrer and Blázquez, 2005). Gelinas et al. (1998) documented that seasonal changes in brain aromatase mRNA expression in goldfish (Carassius auratus) were associated with seasonal changes in reproductive status, supporting that brain aromatase is important in fish reproduction. Aromatization of testosterone into estrogen locally in the brain has been shown to activate male sexual behaviors in a variety of vertebrate species (reviewed by Balthazart et al., 2006). Others have determined that a decrease in brain aromatase activity appears to be necessary to shift steroidogenesis in gonads to the maturation inducing hormone (MIH) that is critical for final maturation of sperm and eggs (Afonso et al., 2000, Patino et al., 2001). Interestingly, brain aromatase activity in fish is one hundred to one thousand times that in mammals (Callard et al., 1981, Pasmanik and Callard, 1985).

While we were unable to locate any studies in the current literature that explored the relationship between reproductive success and aromatase activity, there are several reports of alterations in aromatase activity in fish collected from EDC-contaminated environments. Orlando et al. (2002) report that aromatase activity was significantly elevated in both brains and ovaries of female Eastern mosquitofish (Gambusia holbrooki) from the Fenholloway River in Florida, which receives a large volume of paper mill effluent. Reduced ovarian aromatase activity was found in carp (Cyprinus carpio) collected downstream from both a sewage treatment plant and an agricultural area in the Ebro River, Spain (Lavado et al., 2004). Significantly decreased brain aromatase activity was observed in bream (Abramis brama) from stretches of the Elbe River in Germany that receive organic and metal contamination (Hecker et al., 2007). In Sweden, female perch (Perca fluviatilis) from a lake contaminated with dump leachate showed significantly lower brain aromatase activity than perch from a control lake (Noaksson et al., 2003). Laboratory studies have also indicated that a number of environmental EDCs are capable of modulating aromatase activity in fish. For example, Kuhl and Brouwer (2006) found that TBT exposure dramatically inhibits P450aromB activity in juvenile medaka (Oryzias latipes), while exposure to o,p′-dichlorodiphenyltrichloroethane (o,p′-DDT) increases P450aromB activity. Patel et al. (2006) report that benzo(a)pyrene decreases ovarian aromatase activity but increases female brain aromatase activity in adult killifish (Fundulus heteroclitus) exposed for 15 days. Other researchers have investigated how various EDCs (e.g., diethylstilbestrol (DES), bisphenol A, nonylphenol, and benzo(a)pyrene) affect the expression of aromatase by measuring changes in P450arom mRNA in zebrafish (Danio rerio) embryos and juveniles (Kazeto et al., 2004, Kishida et al., 2001). However, there is not a consistent correlation between P450arom mRNA and aromatase activity (Gelinas et al., 1998, Gen et al., 2001, Kortner et al., 2009, Kuhl and Brouwer, 2006, Montserrat et al., 2004, Park et al., 2006, Villeneuve et al., 2006). Because the activity of aromatase can be regulated post-transcriptionally (Balthazart et al., 2001), changes in P450arom mRNA do not necessarily predict changes in aromatase catalytic activity. Such mRNA changes have even been suggested to reflect an opposite compensatory effect to offset alterations in actual aromatase enzyme activity (Sanderson, 2006).

In this study, we investigated the effects of four EDCs (e.g., E2, EE2, OP, and ATD) on reproduction and aromatase activity in brains and gonads from the marine fish cunner (Tautogolabrus adspersus) treated in vivo in the laboratory. Cunner, a northern member of the Labridae (wrasse) family, is a temperate reef fish with a narrow home range and commonly inhabits shallow estuarine and marine areas among sheltered rock substrates, pilings, piers, wrecks, and reefs (Green, 1975). Because of their habitat preference, cunner are likely to be found in nearshore waters that receive sewage treatment effluent or agricultural runoff, which are both sources of environmental EDCs. Cunner exhibit a well-characterized courtship behavior that culminates in a sudden rush to the surface of the water with simultaneous release of gametes by male and female fish (Martel and Green, 1987, Pottle and Green, 1979, Pottle et al., 1981). Cunner are a good marine model species for reproductive studies in the laboratory because these fish are easily collected, are amenable to laboratory holding, and spawn daily during their reproductive season (Gutjahr-Gobell et al., 2002).

The steroidal estrogens E2 and EE2 enter water bodies in effluent from sewage treatment works and in runoff from concentrated animal feeding operations (CAFOs). They are found in sewage effluent discharges in low ng/L concentrations (Baronti et al., 2000, Desbrow et al., 1998, Snyder et al., 1999). E2 and EE2 bind with an organism's estrogen receptors with an affinity similar to the endogenous hormone 17β-estradiol and elicit the same responses, which in fish includes production of the egg yolk protein precursor VTG (Sumpter and Jobling, 1995, Christiansen et al., 1998). OP, a biodegradation product of alkyl polyethoxylate detergents, is present in sewage effluent at low μg/L concentrations (Lye et al., 1999, Snyder et al., 1999, Rudel et al., 1998) and is considered a weak estrogen receptor ligand (Laws et al., 2000). OP has been shown to bind in vitro with estrogen receptors in trout liver cytosol (White et al., 1994) and primary cultures of trout hepatocytes (Jobling and Sumpter, 1993), as well as to induce vitellogenesis in a number of fish species at μg/L concentrations (Andreassen et al., 2005, Gronen et al., 1999, Jobling et al., 1996, Routledge et al., 1998). The fourth chemical tested, ATD, does not have environmental relevance, but is a specific aromatase inhibitor thought to block the synthesis of aromatase at the transcriptional level (Balthazart, 1997). ATD was tested to determine if modulation of aromatase activity alone is sufficient to alter reproduction in cunner. The focus of this study was to investigate the relationship between aromatase activity and reproductive output in a marine fish, as well as examine any correlation between aromatase activity and two established indicators of endocrine disruption, GSI and VTG.

Section snippets

Chemicals

17β-Estradiol (1,3,5(10)-estratrien-3,17β-diol; CAS # 50-28-2) was obtained from Steraloids, Inc., Wilton, NH. 17α-Ethynylestradiol (17α-ethynyl-1,3,5(10)-estratrien-3,17β-diol; CAS # 57-63-6) and 1,4,7-androstatriene-3,17-dione (CAS # 633-35-2) were purchased from Sigma-Aldrich Co., St. Louis, MO. Octylphenol (4-tert-Octylphenol; CAS # 140-66-9; 95% purity) was obtained from Aldrich Chemical Company, Milwaukee, WI. For preparation of slow-release implants, methylene chloride (CAS # 75-09-2)

Results

Table 1 shows the mean numerical values (±SD) for all reproductive endpoints, brain and ovarian aromatase activity, GSI and VTG. For GSI, liver weight was subtracted from the wet weight of each fish to eliminate any temporary effects that accumulation of VTG in liver tissue might have on body weight, since our goal was to discern changes in gonad size that may occur due to a test chemical treatment. If liver weight was not subtracted, an increase in liver weight due to VTG accumulation would

Discussion

Changes in aromatase activity have prevously been suggested to be a possible indicator of endocrine disruption or reproductive dysfunction in fish (Hallgren et al., 2006, Hecker et al., 2007, Hinfray et al., 2006, Hinfray et al., 2010, Lyssimachou et al., 2006, Sanderson, 2006). Hecker et al. (2007) found a strong linear relationship between decreased brain aromatase activity and suppression of reproductive maturity in wild bream collected from the Elbe River in Germany. Hinfray et al. (2010)

Conclusions

Modulation of normal male brain aromatase activity in fish shows promise not only as an indicator of endocrine disruption, but also as an indicator of reproductive dysfunction. Additonal studies looking at both aromatase activity and reproductive endpoints need to be conducted with a wider range of EDCs to see if the relationship noted here holds. In addition, results obtained in cunner treated with E2, EE2, or the aromatase inhibitor ATD provide some insight into the respective roles that

Acknowledgments

The authors thank Mr. Walker Modic, Ms. Janet Ferrell, and Ms. Alycia Collins for their technical assistance. We thank Ms. Martha Simoneau for her assistance in data analysis and her technical edit of the manuscript. We also are grateful to Dr. James Heltshe, Dr. Suzanne Ayvazian, Dr. Monique Perron, and Dr. Brenda Rashleigh, as well as anonymous reviewers, who provided valuable comments to an earlier version of this manuscript. Statistical advice was provided by Dr. James Heltshe. Plasma VTG

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