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 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.
Footnotes
-
This work was supported by the Natural Sciences and Engineering Council of Canada (NSERC) Grant RGPIN 250139 to A.O.S.E. H.M.K. is the recipient of the Canada Institute of Health Research (CIHR)/Rx&D Health Research Foundation Graduate Scholarship.
-
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
-
doi:10.1124/dmd.105.005397.
-
ABBREVIATIONS: XME, xenobiotic metabolizing enzyme; Nqo1, NAD(P)H:quinone oxidoreductase 1; Gst ya, glutathione S-transferase subunit Ya; XRE, xenobiotic responsive element; ARE, antioxidant responsive element; AhR, aryl hydrocarbon receptor; Nrf2, nuclear factor erythroid 2-related factor 2; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TEMED, N,N,N′,N′-tetramethylethylenediamine; Act-D, actinomycin D; CHX, cycloheximide; MG-132, carbobenzoxy-l-leucyl-l-leucyl-leucinal; PBS, phosphate-buffered saline; DCPIP, 2,6-dichlorophenolindophenol; CHP, cumene hydroperoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PAGE, polyacrylamide gel electrophoresis.
- Received April 28, 2005.
- Accepted October 19, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|