Glutathione protects heavy metal-induced inhibition of hepatic microsomal ethoxyresorufin O-deethylase activity in Dicentrarchus labrax L.

doi:10.1016/j.ecoenv.2004.03.003

Ecotoxicology and Environmental Safety

Volume 58, Issue 3, July 2004, Pages 379-385

https://doi.org/10.1016/j.ecoenv.2004.03.003 Get rights and content

Abstract

The in vitro effects of chromium (Cr(VI)), copper (Cu²⁺), iron (Fe²⁺), mercury (Hg²⁺), and zinc (Zn²⁺) were assessed on liver microsomal ethoxyresorufin O-deethylase (EROD) activity from a sea bass (Dicentrarchus labrax L.) preexposed under laboratory conditions to 2.7 μM β-naphthoflavone. The reduced glutathione (GSH) protection potential against heavy metal effects was also studied. The heavy metal concentration ranges used for this study were as follows: 10 pM–5 mM Cr(VI), 10 pM–100 μM Cu²⁺, 10 pM–1 mM Fe²⁺, 10 pM–10 μM Hg²⁺, and 10 pM–100 μM Zn²⁺. Liver microsomal EROD activity was significantly inhibited after in vitro exposure to Cr(VI) (500 μM), Cu²⁺ (1 μM), Fe²⁺ (100 μM), Hg²⁺ (100 pM), and Zn²⁺ (10 μM). Heavy metals inhibitory effect on liver EROD activity was ordered as follows: Hg²⁺>Cu²⁺>Zn²⁺>Fe²⁺>Cr(VI). Protective effects against Hg²⁺ (1 and 10 μM), Cu²⁺ (1, 10, and 100 μM), and Zn²⁺ (10, 50, and 100 μM) were observed in the presence of 0.5 mM GSH by a decrease in liver microsomal EROD activity inhibition. However, 0.5 mM GSH did not protect liver microsomal EROD activity from Cr(VI), and Fe²⁺-induced inhibition. The effect of metal mixtures (Cu²⁺+Zn²⁺, Zn²⁺+Fe²⁺, Zn²⁺+Cr(VI), and Cr(VI)+Fe²⁺) (100 μM) on liver microsomal EROD activity was also assessed, revealing a synergistic interaction.

Introduction

The presence of industrial, agricultural, and other chemicals in the aquatic environment has been recognized as a potential threat to the aquatic environment. Among water contaminants, heavy metals reach the highest concentrations in sediments and in aquatic organisms (Gumgum et al., 1994). Mercury (Hg), lead (Pb), and cadmium (Cd) are considered the most dangerous metals in the aquatic environment (Goyer et al., 1995). Essential metals, such as chromium (Cr), copper (Cu), zinc (Zn), and iron (Fe) have also shown their toxic effects when their intake is excessively high (Pipe et al., 1999; Suresh et al., 1992). Being indestructible in biological tissues, heavy metals when incorporated into an organism have their toxicological effects regulated by two general mechanisms, namely the specific binding to ligands and excretion.

Ethoxyresorufin O-deethylase (EROD) measurement in fish is used as an in vivo biomarker of exposure to polycyclic aromatic hydrocarbons and structurally related compounds (Stegeman and Hahn, 1994; Buchelli and Fent, 1995). Fish liver microsomal EROD activity may be inhibited by heavy metals (Viarengo et al., 1997). Our previous studies have clearly shown a significant liver microsomal EROD activity inhibition in eel's liver organ culture exposed to Cr(VI), after in vivo β-naphthoflavone (BNF) exposure (Oliveira et al., 2003).

Glutathione (GSH) is a very important modulator of cellular homeostasis, including detoxification of metals (transport, storage, and metabolism), such as inorganic mercury, methylmercury, and oxyradicals (Ballatori (1991), Ballatori (1994); Costa and Klein, 1999; Mates and Sanchez-Jimenez, 2000). This compound is the most abundant nonprotein thiol in almost all the aerobic species (Wang and Ballatori, 1998). The effects of GSH on metal-mediated DNA oxidation (Yuann et al., 1999) and the inhibition of free radical formation by Cu ions (Pandey et al., 2001) have been examined. Moreover, the protective effect of GSH has also been attributed to its ability to stabilize metal in its neutralized state, preventing redox cycling and free radicals generation (Wang and Ballatori, 1998). A previous study concerning the in vitro effects of GSH physiological concentrations (0.5–10 mM) on Dicentrarchus labrax liver microsomal EROD activity showed that 0.5 mM GSH had no effect on EROD activity (Oliveira and Santos, 2003). However, 1 mM GSH significantly increased EROD activity, whereas concentrations higher than 2 mM significantly inhibited it.

Therefore, this study was designed to study the in vitro effects of heavy metals (Cr(VI), Cu²⁺, Fe²⁺, Hg²⁺, and Zn²⁺), individually or combined, on D. labrax liver microsomal EROD activity previously induced by BNF. Moreover, the in vitro protective potential of glutathione on sea bass liver microsomal EROD activity was also assessed.

Section snippets

Chemicals

β-naphthoflavone, dimethyl sulfoxide (DMSO), reduced glutathione, and resorufin were purchased from Sigma Chemical Co.; 7-ethoxyresorufin and NADPH were from Roche; Hg²⁺ (HgCl₂) was obtained from M & BM (England); Cr(VI) (K₂Cr₂O₇), Cu²⁺ (CuCl₂), Fe²⁺ (FeSO₄.7H₂O), and Zn²⁺ (ZnSO₄.7H₂O) were from E. Merck-Darmstadt (Germany). The salt mixture to prepare artificial seawater was obtained from Sera Meersaltz (Heinsberg, Germany).

Animals

The experiment was carried out using one juvenile D. labrax L.

Results

The liver microsomal in vitro exposure to Hg²⁺ caused significant EROD activity inhibition at 100 pM (16%) and 1 nM (16%) (P<0.05), at 10 nM (34%) and 100 nM (34%) (P<0.01), and at 1 μM (89%) and 10 μM (100%) (P<0.001) (Fig. 1A). However, liver microsomal EROD activity inhibition by Hg²⁺ at 1 and 10 μM significantly decreased (P<0.01, P<0.001) in the presence of 0.5 mM GSH (Fig. 1B).

A significant liver microsomal EROD activity inhibition (P<0.001) was observed in the presence of 1 μM Cu²⁺ corresponding

Discussion

Previous in vivo studies on fish demonstrated that PAH treatments combined with simultaneous or delayed administration of metals resulted in significantly reduced EROD and benzo(a)pyrene hydroxylase activity (George and Young, 1986; George, 1989; Viarengo and co-workers, 1997). According to Eggens et al. (1992), heavy metal contamination is a possible explanation for the discrepancies between the organic xenobiotic levels sometimes observed in fish liver tissue and its EROD activity. This

Conclusion

D. labrax liver microsomal EROD activity is inhibited in vitro by heavy metals. Sea bass liver microsome sensitivity may be ordered as follows: Hg²⁺>Cu²⁺>Zn²⁺>Fe²⁺>Cr(VI). Heavy metal mixtures showed a synergistic liver microsomal EROD inhibition. GSH has a very important protective role concerning Hg²⁺, Cu²⁺, and Zn²⁺ in sea bass liver microsomal EROD activity inhibition.

Acknowledgments

The authors express appreciation to Aveiro University Research Institute (CESAM).

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Glutathione protects heavy metal-induced inhibition of hepatic microsomal ethoxyresorufin O-deethylase activity in Dicentrarchus labrax L.

Abstract

Introduction

Section snippets

Chemicals

Animals

Results

Discussion

Conclusion

Acknowledgments

Adv. Pharmacol.

Aquat. Toxicol.

Comp. Biochem. Physiol.

J. Biol. Chem.

Chemosphere

Toxicology

Trends Biochem. Sci.

Int. J. Biochem. Cell Biol.

Water Res.

Ecotoxicol. Environ. Saf.

Aquat. Toxicol.

Free Radicals Biol. Med.

Mar. Pollut. Bull.

Comp. Biochem. Physiol.

Mar. Environ. Res.

Comp. Biochem. Physiol.

Comp. Biochem. Physiol.

Comp. Biochem. Physiol.

Statistical Methods in Biology

Mechanisms of metal transport across liver cell plasma membranes

Drug Metabol. Rev.

Induction of cytochrome P450 as a biomarker for environmental contamination in aquatic ecosystems

Crit. Rev. Environ. Sci. Technol.

Ethoxyresorufindirect fluorimetric assay of a microsomal O-deethylation which is preferentially inducible by 3-methylcholantrene

Drugs Metab. Dispos.