Genes involved in cysteine metabolism of Chironomus tepperi are regulated differently by copper and by cadmium

Abstract

Freshwater invertebrates are often exposed to metal contamination, and changes in gene expression patterns can help understand mechanisms underlying toxicity and act as pollutant-specific biomarkers. In this study the expressions of genes involved in cysteine metabolism are characterized in the midge Chironomus tepperi during exposures to sublethal concentrations of cadmium and copper. These metals altered gene expression of the cysteine metabolism differently. Both metals decreased S-adenosylhomocysteine hydrolase expression and did not change the expression of S-adenosylmethionine synthetase. Cadmium exposure likely increased cystathionine production by up-regulating cystathionine-β-synthase (CβS) expression, while maintaining control level cysteine production via cystathionine-γ-lyase (CγL) expression. Conversely, copper down-regulated CβS expression and up-regulated CγL expression, which in turn could diminish cystathionine to favor cysteine production. Both metals up-regulated glutathione related expression (γ-glutamylcysteine synthase and glutathione synthetase). Only cadmium up-regulated metallothionein expression and glutathione S-transferase d1 expression was up-regulated only by copper exposure. These different transcription responses of genes involved in cysteine metabolism in C. tepperi point to metal-specific detoxification pathways and suggest that the transsulfuration pathway could provide biomarkers for identifying specific metals.

Introduction

Gene expression patterns are being increasingly used to identify exposure to stressful environmental conditions. In aquatic environments, they are used to examine how organisms respond to toxicants and are favored because they have the potential to identify sublethal stress that can lead to environmental degradation (Adams et al., 1989, Galay-Burgos et al., 2003). As a result, the US EPA now accepts gene expression data as part of a weight-of-evidence approach for environmental assessment (Van Aggelen et al., 2010). Several studies have investigated transcriptomic responses to pollution in freshwater biota (e.g. Hook et al., 2008, Marinkovic et al., 2012) and for these data to be fully interpreted the underlying mechanisms causing changes in the transcriptome of studied species need to be understood.

Cysteine metabolism plays a central role in detoxification against environmental stressors (Jones et al., 2004, Sugiura et al., 2005) (Fig. 1). Demethylation and remethylation of methionine are essential to cellular repair and function (James et al., 2002, Chen et al., 2010). Cysteine can only be produced via the transsulfuration pathway and is required for proteins, including metallothionein (MT), that protect cells directly from metal stress. Cysteine is also a precursor for glutathione (GSH) and is involved in several antioxidant responses (Hughes et al., 2009). Several genes regulate cysteine metabolism and its intermediates, the transcription of these genes can report on dynamics of the cysteine metabolic pathway, which in turn can inform how an organism responds to stressors.

The Chironomidae (Diptera), particularly species from the genus Chironomus, are commonly used in toxicological testing as several species can be cultured in the laboratory (for example: Dawson et al., 2000, Martin et al., 2008, Stevens, 1993, Watts and Pascoe, 2000). Furthermore, Chironomus larvae tend to live on or in the sediments and are exposed to hydrophobic toxicants in sediment (Burton, 1991). Chironomus riparius (Chironomidae) is a standard test organism (US-EPA, 1996, OECD, 2004) and Chironomus tentans is widely used in toxicity testing (Martinez et al., 2003, Rakotondravelo et al., 2006, Lee and Choi, 2009). The transcriptome of C. riparius was recently investigated (Marinkovic et al., 2012), although only the responses of a few genes to different pollutants have been described in these species (Lee et al., 2006, Park and Kwak, 2008, Park et al., 2009, Planello et al., 2010). Neither of these species occurs in Australia, however Chironomus tepperi (Skuse) is widespread in the Australian mainland, can be easily cultured in the laboratory, and occurs in metal-contaminated habitats. While C. tepperi is used in toxicological testing, there is little sequence information and no gene expression data yet available for this species.

In this study, we isolate eight genes involved in the cysteine metabolism of C. tepperi using sequence alignments of conserved genes from other chironomid and dipteran species. The genes investigated encode enzymes involved in the remethylation cycle (S-adenosylmethionine synthetase; SAM and S-adenosylhomocysteine hydrolase; SAH), the transsulfuration pathway (cystathionine-γ-lyase; CγL, cystathionine-β-synthase; CβS, γ-glutamylcysteine synthase; GCS, and glutathione synthetase; GS) and two representing resulting stress response proteins (glutathione S-transferase delta 1; GSTd1 and metallothionein; Mtn). The transcriptional responses of these genes were characterized after 24 h exposures to two sublethal concentrations of copper (Cu: 0.05 mg/L and 0.5 mg/L) and cadmium (Cd: 0.25 mg/L and 0.5 mg/L). We show that these metals induce different changes in gene expression regulating for cysteine metabolism intermediates and products.