TRANSCRIPTIONAL REGULATION OF THE NAD(P)H:QUINONE OXIDOREDUCTASE 1 AND GLUTATHIO

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TRANSCRIPTIONAL REGULATION OF THE NAD(P)H:QUINONE OXIDOREDUCTASE 1

AND GLUTATHIONE S-TRANSFERASE YA GENES BY MERCURY, LEAD, AND COPPER

Hesham M. Korashy, and Ayman O. S. El-Kadi

Faculty of Pharmacy and Pharmaceutical Sciences, University of

Alberta, Edmonton, Alberta, Canada

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 NAD(P)

H:quinone oxidoreductase 1 (Nqo1) and glutathione S-transferase

subunit Ya (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

2,3,7,8-tetrachlorodibenzo-p-dioxin, 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 whereas 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 mRNA 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 nuclear factor

erythroid 2-related factor 2 (Nrf2) protein expression, implying the

requirement of Nrf2 protein synthesis for the induction of these

genes. Furthermore, inhibition of Nrf2 protein degradation by

carbobenzoxy-L-leucyl-L-leucyl-leucinal (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 post-transcriptional and post-

translational mechanisms. This is the first demonstration that heavy

metals regulate the expression of Nqo1 and Gst ya genes through a

transcriptional mechanism.