Elsevier

Aquatic Toxicology

Volume 83, Issue 4, 1 August 2007, Pages 295-305
Aquatic Toxicology

Receptor activated C kinase is down-regulated in the male gonad of the marine bivalve mollusc Mya arenaria exposed to tributyltin (TBT)

https://doi.org/10.1016/j.aquatox.2007.05.003Get rights and content

Abstract

This study was aimed at investigating the molecular mechanisms by which tributyltin (TBT) impairs the reproductive processes in the marine bivalve Mya arenaria. The suppression polymerase chain reaction subtractive hybridization (SSH) method was used to identify differentially expressed transcripts in the gonads of adult M. arenaria 72 h after a single injection of 160 ng TBT in the adductor muscle. Subtractive cDNA libraries comprising 322 clones were obtained. These clones were sequenced and corresponded to 55 female and 26 single male non-redundant cDNAs. Following similarity searches in genome databases, some of the transcripts could be assigned to cellular functions including mitochondrial respiration, structural proteins, structure of cytoskeleton, nucleic acid regulation, general metabolism and signal transduction. Among the potentially differentially regulated transcripts, Receptor for activated C kinase 1 (RACK1) represented 6% of the total down-regulated clones in males and the corresponding protein exhibited a high degree of similarity (80%) with the human polypeptide. The RACK1 cDNA from M. arenaria consists of 1085 bp, encoding a 318 deduced polypeptide which contains five internal tryptophan-aspartate (WD) repeats, six putative PKC phosphorylation sites, one tyrosine kinase site, four putative N-myristoylation sites as well as a transmembrane segment spanning amino acid 228–251. A significant down-regulation (by ∼30% (p < 0.05)) of RACK1 expression in male gonads exposed to TBT was confirmed by quantitative real-time RT-PCR. Transcript levels of RACK1 were higher in the female gonads than in the mantle, gills and male gonads. Gene expression as detected by in situ hybridization was strong in mature oocytes comparatively to primary germ cells. RACK1 may be a useful biomarker for TBT exposure in the reproductive system of bivalve molluscs.

Introduction

It is now well established that a variety of chemical compounds capable of disrupting the reproductive endocrine pathways are present in the aquatic environment. The antifouling compound tributyltin (TBT), one of the most potent endocrine disruptors contaminating the aquatic environment, provokes imposex in aquatic neogastropods (Matthiessen and Gibbs, 1998). TBT has been used in marine antifouling paints as a biocide to prevent attachment and growth of organisms on the hulls of vessels (De Mora and Pelletier, 1997). Despite legislation banning the use of TBT for ships shorter than 25 m, several studies have shown a persistent presence of TBT in the marine environment. For example, in the St. Lawrence River, organotins were detected in sediments (Regoli et al., 1999) as well as in zebra mussel (Regoli et al., 2001), sea star (Pelletier and Normandeau, 1997) and beluga whale (Saint-Louis et al., 2000) tissue samples.

In vertebrates, it has been demonstrated that TBT affects many cellular functions. It inhibits energy production (Stridh et al., 1999), disrupts calcium homeostasis (Reader et al., 1993) and induces apoptosis in rainbow trout hepatocytes (Reader et al., 1999). In the adult marine bivalve Mytilus edulis, TBT induces genotoxic damage at environmentally realistic concentrations as shown by using both the comet assay and the micronucleus test (Hagger et al., 2005). One of the mechanisms proposed for explaining the genotoxic effects of TBT is the disruption of intracellular calcium homeostasis (Orrenius et al., 1992). The increase in store-dependent calcium influx may be triggered by TBT interaction with calcium regulating proteins, such as calmodulin (Cima et al., 2002) or by inhibition of sarcoplasmic endoplasmic reticulum Ca2+ ATPase (Kass and Orrenius, 1999).

Soft-shell clams, Mya arenaria, which live buried in sediments, play a key role in the ecosystem of the intertidal zone biota of the St. Lawrence River (Desrosiers and Brêthes, 1984). In the St. Lawrence River, M. arenaria were able to concentrate TBT in their gonads up to 120 ng/g dry weight (Gagné et al., 2003). Previous studies have shown that TBT affects the reproductive physiology of M. arenaria by delaying sexual maturation (Gagné et al., 2003) and by reducing steroid hormone levels (Siah et al., 2003).

Several attempts have been made to unravel the mechanism of imposex observed in neogastropods. In Nucella lapillus females, TBT was shown to disrupt the hormone metabolism by increasing the level of free testosterone and to induce penis formation in females (Spooner et al., 1991). The inhibition of aromatase activity has been investigated and could result in the increase of free testosterone levels in two neogastropod species (Nassarius reticulates and N. lapillus) (Bettin et al., 1996). In snails, it was shown that the levels of testosterone and estradiol are regulated by esterification of these steroid hormones with fatty acids (Gooding et al., 2003, Janer et al., 2005). In a following study, Janer et al. (2006) showed that TBT interferes with the level of esterified testosterone and therefore increases the levels of free testosterone. TBT as a neurotoxic xenobiotic has also been investigated in snails and it was demonstrated that it induces imposex in Ilyanassa obsoleta by stimulating the release of the neuropeptide APGWamide (Oberdörster and McClellan-Green, 2000, Oberdörster and McClellan-Green, 2002).

To date, the molecular mechanisms by which TBT induces reproductive endocrine disruption in M. arenaria remain unknown. The aim of this study was to identify the transcripts up- or down-regulated by TBT in the gonads of M. arenaria by using suppression PCR subtractive hybridization (SSH). This constitutes a means of identification of the molecular actors of the gonadal response to TBT exposure as well as a way towards understanding the action mechanisms of TBT as an endocrine disruptor.

Section snippets

Animals and TBT treatment

In the present investigation, adult M. arenaria, 5–6 cm in length, were sampled at Métis Bay (48°40′44″; 68°02′17″) in the St. Lawrence River maritime estuary (Québec, Canada). Back in the laboratory, clams were kept in tanks (volume, 50 L) where they were buried in sediment for a 22-day acclimatizing period prior to the experiments. Sediment originated from a “clean” reference site located in Métis Bay on the banks of the St. Lawrence River. The animals were then divided into control (n = 60) and

Suppression subtractive hybridization

The suppression subtractive hybridization technique was used to select differentially expressed transcripts in the gonads of male and female M. arenaria exposed to TBT. A total of 322 clones potentially regulated by TBT were sequenced. These clones, ranging from approximately 200–500 bp in length, were analyzed for redundancy by multiple alignments. Respectively, 55 and 26 potentially differentially regulated singleton clones were identified as independent in females and males and the remaining

Conclusion

This study was conducted to determine differential gene expression induced by the exposure of M. arenaria to TBT. We have identified RACK1 transcript as down-regulated in the male gonads using SSH technique and this result was confirmed by real-time quantitative PCR. Due to its ability to interact with key signalling molecules, such as PKC isoforms, RACK1 is generally considered to play a central role in pivotal physiological processes, such as cell protection (Mourtada-Maarabouni et al., 2005

Acknowledgements

Ahmed Siah was the recipient of a postdoctoral fellowship of the Région Haute-Normandie (France) and of the Fonds Québécois de Recherche sur la Nature et les Technologies (Québec, Canada).

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