Expression profiles of seven glutathione S-transferase (GST) genes from Venerupis philippinarum exposed to heavy metals and benzo[a]pyrene

Abstract

Glutathione S-transferases (GSTs) are phase II enzymes that facilitate the detoxification of xenobiotics, and also play important roles in antioxidant defense. In this study, we reported the cloning and molecular characteristics of seven genes of the GST family (VpGSTS1, VpGSTS2, VpGSTS3, VpGSTO, VpGSTMi, VpGSTM and VpGSTR) from Venerupis philippinarum together with mRNA tissue distribution patterns and temporal expression profiles in response to cadmium, copper and benzo[a]pyrene (B[a]P) exposures. The deduced amino acid sequences of VpGSTs showed high similarities to counterparts of other species that clustered into the same clades in the phylogenetic analysis. At basal levels of tissue expression, most VpGSTs were highly expressed in hepatopancreas compared with other tissues. All VpGSTs showed differential response profiles depending on the concentrations of various toxicants and exposure times. More notably, the expressions of VpGSTS2 and VpGSTS3 transcripts were significantly up-regulated in hepatopancreas from Cu and B[a]P-exposed animals, indicating that these two sigma VpGSTs were highly sensitive to Cu and B[a]P exposure. However, the expressions of VpGSTM and VpGSTR were significantly induced by Cu or B[a]P exposure, respectively. These findings suggested the role of VpGSTS2, VpGSTS3, VpGSTM and VpGSTR in defense against oxidative stress and highlighted their potential as biomarkers to Cu or B[a]P exposure.

Introduction

Aquatic organisms are frequently exposed to numerous environmental pollutants including polycyclic aromatic hydrocarbons (PAHs) and heavy metals (Rainbow, 1995, Xue and Warshawsky, 2005, Wan et al., 2009). Heavy metals such as copper (Cu) and cadmium (Cd) can induce excessive production of reactive oxygen species (ROS) in cells, which cause oxidative modification of the major cellular macromolecules (Cecconi et al., 2002). Benzo[a]pyrene (B[a]P) is an extensively studied prototype of PAH and has been diffused into marine environment as an persistent organic pollutant, inducing cytotoxic, mutagenic and carcinogenic effects in various animal species (Gonzalez and Gelboin, 1994). In recent years, research on the gene expression of some detoxification and antioxidant enzymes has been recognized as a powerful tool to assess the ecotoxicological effects of certain chemicals (Woo et al., 2009, Won et al., 2011).

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a superfamily of multifunctional phase II enzymes primarily involved in detoxification of both endogenous and exogenous electrophiles. Generally, the detoxification mechanisms of GSTs lie in the conjugation of reduced glutathione with toxic compounds or their metabolites to increase the hydrophilicity and facilitate the excretion of toxicants (Ketterer et al., 1983). Moreover, GSTs have been found to play a critical role in mitigating oxidative stress in all life forms (Lee et al., 2008), and GST activities have been widely used as potential biomarkers for the monitoring of environmental pollution as well (Hoarau et al., 2002, Shailaja and D'Silva, 2003, Cunha et al., 2007).

GSTs have been found in all the prokaryotic and eukaryotic organisms investigated so far, in which they have three intra-cellular locations: in the cytosol, in mitochondria, and in microsomes (Hayes et al., 2005). Currently, at least 15 different classes of GSTs (alpha, beta, delta, epsilon, kappa, lambda, mu, omega, phi, pi, sigma, tau, theta, zeta, and rho) have been identified from numerous phylogenetically diverse organisms based on the differences in structural, catalytic and immunological characters (Hayes et al., 2005). In the previous studies, the mRNA expression of multiple specific GSTs had been investigated in several aquatic organisms after exposure to environmental pollutants (Boutet et al., 2004, Lee et al., 2008, Wan et al., 2008a, Li et al., 2009, Kim et al., 2010, Nail and Choi, 2011, Won et al., 2011). These studies provided the possibility to explore the correlation of specific GST responses with the detoxification of various toxicants, which were perhaps useful to evaluate the potential of different GST isoforms as molecular biomarkers to monitor marine environmental pollution. However, phylogenetically diverse organisms do not always respond similarly with regard to the transcriptional response of GSTs, therefore it is necessary to analyze the expression profiles of these genes across species.

Manila clam, Venerupis philippinarum is widely distributed and occurs at high densities around the heavily polluted Bohai Sea (Liang et al., 2004). It has been listed as a sentinel organism in “Mussel Watch Programs” launched in China. Our previous studies had demonstrated that this species exhibited distinct biochemical and genetic responses when exposed to heavy metals and B[a]P (Liu et al., 2011, Wang et al., 2011, Zhang et al., 2011a, Zhang et al., 2011b), possessing the requisite features of a promising indicator species. Because GST isoenzymes were considered useful biomarkers in mollusk species (Boutet et al., 2004, Hoarau et al., 2006, Wan et al., 2008a, Wan et al., 2008b, Wan et al., 2009), investigation on the expression profiles of VpGSTs isoforms to specific environmental pollutants would be helpful to identify potential biomarkers for future environmental biomonitoring. To our knowledge, only one GST-pi gene was cloned and characterized from V. philippinarum (Xu et al., 2010). However, information about different classes of VpGSTs and their expression profiles to various toxicants exposure is currently unavailable. In this study, we reported the identification, characterization and distribution of seven VpGSTs from our ESTs database. To further identify promising VpGST isoforms as biomarkers, the expression patterns of seven VpGSTs were also investigated after exposure to three typical environmental contaminants in the Bohai Sea.