Transcriptional regulation of glutathione biosynthesis genes, γ-glutamyl-cysteine ligase and glutathione synthetase in response to cadmium and nonylphenol in Chironomus riparius.

We characterized Chironomus riparius glutathione (GSH) biosynthesis genes, γ-glutamyl-cysteine ligase catalytic subunit (cr-gcl) and glutathione synthetase (cr-gs) and studied their expression after cadmium (Cd) and nonylphenol (NP) exposure. The full length cDNA of the Cr-GCL catalytic subunit was 2185 base pair (bp) in length containing an open reading frame of 1905bp, a 13bp 5' and 267bp 3' untranslated regions. The theoretical molecular mass of the deduced amino acid sequence (633) was 72.65kDa with an estimated pI of 5.42. The partial cDNA of Cr-GS was 739bp in length consisting 221 amino acids. The deduced amino acid sequence of Cr-GCL and Cr-GS cDNAs showed high conservation with homologs from other species. In phylogenetic analysis Cr-GCL and Cr-GS were grouped with equivalent genes from insects belonging to the dipteran order. The expression of cr-gcl and cr-gs was measured using quantitative real-time PCR after exposure to sub lethal concentrations of Cd (2, 10 and 20mg/L) and NP (10, 50 and 100μg/L) for 12, 24, 48 and 72h using real-time PCR methods. The mRNA expression of Cr-GCL and Cr-GS was significantly modulated after exposure to different concentrations of Cd and NP for different time periods. Total GSH levels showed a non-significant decrease after exposure to Cd for 24h. However, no change in GSH levels was observed after exposure to NP for 24h. These results suggest that Cr-GS and Cr-GCL expression is modulated by Cd and NP stress and may play an important role in detoxification of xenobiotics and antioxidant defense. We conclude that Cr-GS and Cr-GCL could be used as biomarkers of Cd and NP stress in aquatic environment for the studied species.