Received for publication April 29, 2005.
Revised October 18, 2005.
Accepted for publication October 19, 2005.

Transcriptional Regulation of The NAD(P)H:Quinone Oxidoreductase and Glutathione S-Transferase Ya Genes by Mercury, Lead, and Copper

Abstract

Recently, we demonstrated the ability of heavy metals, particularly, Hg²⁺, Pb²⁺, and Cu²⁺ to differentially modulate in Hepa 1c1c7 cells the expression of the phase II xenobiotic metabolizing enzymes, Nqo1 and Gst ya genes, yet the mechanisms involved remain unknown. To investigate the molecular mechanisms involved in the regulation of Nqo1 and Gst ya genes by heavy metals, Hepa 1c1c7 cells were treated with Hg²⁺, Pb²⁺, or Cu²⁺ in the presence and absence of TCDD, a potent inducer of Nqo1, Gst ya, and Cyp1a1 genes. Analysis of the time-dependent effect of heavy metals revealed that Hg²⁺ and Pb²⁺ increased, while Cu²⁺ inhibited the constitutive and inducible expression of Nqo1 and Gst ya mRNAs in a time-dependent manner. The RNA synthesis inhibitor, actinomycin D, significantly inhibited the Nqo1 and Gst ya mRNAs induction in response to metals, indicating a requirement of de novo RNA synthesis. The protein synthesis inhibitor, cycloheximide, significantly inhibited metal-mediated induction of Nqo1 and Gst ya mRNAs, which coincided with a decrease in the Nrf2 protein expression, implying the requirement of Nrf2 protein synthesis for the induction of these genes. Furthermore, inhibition of Nrf2 protein degradation by MG-132, a 26S proteasome inhibitor, significantly reversed the cycloheximide-mediated inhibition of Nqo1 and Gst ya mRNAs, which coincided with an increase in the expression of Nrf2, confirming that a transcriptional mechanism is involved. Nqo1 and Gst ya mRNA and protein decay experiments revealed lack of posttranscriptional and posttranslational mechanisms. This is the first demonstration that heavy metals regulate the expression of Nqo1 and Gst ya genes through a transcriptional mechanism.

Key words: Ah receptor, antioxidants, carcinogen metabolism, glutathione transferases, heavy metals, NAD(P)H-quinone oxidoreductase, phase II drug metabolism, toxicology

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