Elsevier

Aquatic Toxicology

Volume 185, April 2017, Pages 121-128
Aquatic Toxicology

Disrupting effects of azocyclotin to the hypothalamo-pituitary-gonadal axis and reproduction of Xenopus laevis

https://doi.org/10.1016/j.aquatox.2017.02.010Get rights and content

Highlights

  • Azocyclotin delayed the metamorphosis of Xenopus laevis.

  • Azocyclotin disrupted the gonadal differentiation of Xenopus laevis.

  • Azocyclotin altered the expression of genes involved in the HPG axis of X. laevis.

  • Steroidogenesis involved in the HPG axis was interfered after azocyclotin exposure.

Abstract

Over the past few decades, the hazards associated with the extensive use of organictin compounds have become an issue of extreme concern, while at present the effects of these substances on amphibians remain poorly understood. In the present study, we chose azocyclotin, one of common use acaricides in China. We focused on sexual development and steroidogenesis disrupting effects of azocyclotin in the Xenopus laevis. Tadpoles were exposed to azocyclotin (0.05 and 0.5 μg/L) for long-term (4 months) study. Results showed that exposure to azocyclotin caused developmental toxicity, including decreased survival, body weight, body length, gonadosomatic index, hepatosomatic index and female phenotype. At the same time, statistical increase in mean age at completion of metamorphosis was observed in azocyclotin treatments in comparison with control group. Furthermore, hormone concentrations, and steroidogenesis genes expression of adult frog were further evaluated in 28 days exposure. Results demonstrated that the key regulating hormones, e.g. testosterone and pregnenolone, were significantly upregulated. The expression levels of selected steroidogenic genes were also significantly altered. Our study demonstrated that azocyclotin could delay the metamorphosis and disrupt the gonadal differentiation of X. laevis. Steroidogenesis and the expression of genes involved in the hypothalamus-pituitary-gonadal-liver axis in frogs were disrupted after azocyclotin exposure. Azocyclotin showed both androgenic and antiestrogenic activity for X. laevis. Those findings emphasized the influence of azocyclotin on non-target species in the context of ecotoxicological risk assessment.

Introduction

Organictin compounds (OTCs) have been introduced into the environment mainly or exclusively through anthropogenic sources in the past decades (Cao et al., 2009). The environmental concerns on OTCs increased due to the wide application in agriculture and industry, such as polyvinyl chloride stabilizers, antifouling paints, timber preservatives, fungicides, molluscicides, nematocides, ovicides, rodent repellants and miticides, primarily containing tributyl- (e.g. tributyltin) as toxic additives (Cao et al., 2009, Hoch, 2001). Consequently, considerable amount of these compounds have dispersed into environmental media and biota via multiple transport processes, which increases risks for aquatic organisms (Kördel and Stein, 1997). Environmental monitoring has frequently detected OTCs in seawater, surface water, sediments and biota (Barbara and Andrzej, 2013, Furdek et al., 2012, Gui et al., 2016, Kördel and Stein, 1997). In seawater, for example, OTCs concentrations are generally at ng Sn L−1 levels (Furdek et al., 2012). Particularly high levels of OTCs were found in the ports of China, with concentrations reach up to 977 ng Sn L−1 (Gao et al., 2006, Jiang et al., 2001, Hung et al., 2001). In biota, up to 1675 ng Sn g−1 of OTCs were found in mussels, despite the fact that TBT (tributyltin) – containing antifouling paints have been banned since 2008 (Furdek et al., 2012). Besides detected in commercial marine products, OTCs have also been found in several other types of food in China (Liu and Jiang, 2002, Ma et al., 2015, Zhou et al., 2004). It is thus suggested that there is still severe organotin contamination and effective actions should be taken as soon as possible to prevent future human health risks.

It is known that tributyltin shows high acute toxicity at environmentally relevant concentrations and could act as endocrine disruptors to non-target organisms (Guo et al., 2010, Wu et al., 2014). Abundant data linked tributyltin with the sexual abnormality of gastropods (Kolpin et al., 2002). In addition, previous studies demonstrated that exposure to tributyltin caused adverse growth and development effects in wood frog larvae (Higley et al., 2013). Tributyltin could significantly delay the metamorphosis and disrupt the gonadal differentiation of Xenpous laevis at environmentally relevant concentrations (Shi et al., 2014). Although these studies have shown metamorphic and gonadal development disrupting effects of OTCs in amphibian, the impacts of OTCs on X. laevis sexual development still poorly understood, particularly with regard to the relation with steroidogenesis. Some OTCs were act as enzymes inhibitor in several species (Iguchi et al., 2008, Shimasaki et al., 2003). Enzymes of the cytochrome P450 monooxygenase super family (CYPs) are found in all prokaryotes and eukaryotes (Rendic and Di, 1997) and are active in the steroid metabolism pathways. OTCs have the potential to interact with these steroidogenic CYPs and signals, and consequently cause endocrine disruption.

In the present study, we focused on the organotin acaricide, azocyclotin [tri(cyclohexyl)-1H-1,2,4-triazol-1-yltin], which is widely used in agriculture, specifically in the production of fruits and vegetables (Ma et al., 2015, Niu et al., 2011). Additionally, azocyclotin is quickly hydrolyzed to cyhexatin after application (Kördel and Stein, 1997). The metabolite cyhexatin has been banned in 1987 in China, which is also a non-systemic acaricide and it has been suggested more toxic than the parent compound (Sinclair and Boxall, 2003). Previous studies reported that azocyclotin applied as soil slurry at concentrations of 135 μg/L had severe effects on the biocenoses in aquatic microcosms (Kördel and Stein, 1997). Exposure to azocyclotin during metamorphosis resulted in thyroid endocrine disruption in X. laevis (Li et al., 2016a). However, there is still limited information available about the toxic effects of azocyclotin currently. Previous studies in vivo have shown that X. laevis is sensitive to endocrine disruptors (Hayes et al., 2010a, Hayes et al., 2010b, Qin et al., 2007), therefore, we chose X. laevis as the model organism to study the potential endocrine disruption of azocyclotin. The hypothalamus-pituitary-gonad axis is a system highly conserved across vertebrates (Perkins et al., 2013), which is responsible for maintaining homeostasis of steroid hormones by regulating their synthesis, secretion, transport and metabolism (Payne and Hales, 2004). Hence, in the present study, we investigated the endocrine disrupting effects of the azocyclotin on the steroid and transcriptional profiles of the key pathway within the X. laevis gonad. The effects of azocyclotin on the gonadal development were further examined. Our results showed that exposure to azocyclotin can affect balance of hormone levels in both female and male frog, as well as gonadal development, thereby may cause adversely impact on reproduction.

Section snippets

Chemicals, reagents and materials

Azocyclotin standard was obtained from Zhejiang Heben Pesticide & Chemicals Co., Ltd (CAS: 41083-11-8; purity = 95%). Azocyclotin was dissolved in dimethyl sulfoxide (DMSO) and stored in −20 °C. All other chemicals used in this study were of analytical grade. 3-aminobenzoic acid ethyl ester, methanesulfonate salt (MS-222) was purchased from Sigma (St. Louis, MO, USA). TRIzol, RNase-free water, PrimeScript RT reagent Kit and SYBR Premix Ex TapTM II were purchased from Takara (Dalian, China). A

Concentrations of azocyclotin in water and in animal tissue

The concentrations of azocyclotin in water were monitored. No traces of azocyclotin were found in the control groups. Exposure solutions measurements showed that azocyclotin levels after renewal were slightly changed compared to the nominal concentrations (Table S2). Alterations of azocyclotin concentrations at 0 h were observed compared with those at 24 h, the degradation rates of azocyclotin ranged from 14.2% to 18.5% during a refreshing interval (Table S2), which suggested the fluctuations of

Discussion

Great concerns have been raised in recent years over the potential adverse effects of OTCs on reproductive endocrine system (Cao et al., 2009, Furdek et al., 2012, Kolpin et al., 2002, Shimasaki et al., 2003). In the present study, we focused on investigating the sexual development and steroidogenesis disrupting effects of azocyclotin in the X. laevis. Our study demonstrated that azocyclotin could delay the metamorphosis, disrupt the gonadal differentiation of X. laevis, and alter

Conflict of interest

The authors have declared no conflict of interest.

Acknowledgement

This work was financial supported the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20120101110074).

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