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