Arsenite and cadmium, but not chromium, induce NAD(P)H:quinone oxidoreductase 1 through transcriptional mechanisms, in spite of post-transcriptional modifications
Introduction
Aromatic compounds with oxygen containing substituents can be converted enzymatically and non-enzymatically to cytotoxic, quinone-containing byproducts. Quinones exert their toxic effects by multiple mechanisms, including the generation of reactive oxygen species (ROS) and their conversion to DNA-binding semiquinone free radicals. Arylating quinones also react with cellular thiols, forming quinone–thiol adducts (Monks and Jones, 2002). Polycyclic aromatic hydrocarbons (PAHs) represent a class of compounds whose toxicity may be attributed to the formation of quinone- and epoxide-containing metabolites. PAHs are ubiquitous and persistent environmental contaminants, often released into the air, soil, and water from natural and anthropogenic sources. Forest fires and volcano emissions are examples of natural sources of airborne PAHs (Baek et al., 1991). On the other hand, the production of coal tar (Baek et al., 1991), industrial power generation (Dor et al., 1999), wood treatment plants, and automotive exhaust (Baek et al., 1991) serve as major anthropogenic sources.
Interestingly, PAHs induce a host of biological responses, including the induction of enzymes responsible for their metabolism, through binding to and activating the aryl hydrocarbon receptor (AhR). The AhR is a cytoplasmic ligand-activated transcription factor that translocates into the nucleus upon binding its cognate ligands. In the nucleus, the AhR dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) and the complex then binds to the xenobiotic responsive element (XRE), resulting in subsequent transcriptional events and the up-regulation of a host of responsive genes (Swanson, 2002). Among these genes are those encoding a number of drug metabolizing enzymes, including four phase I enzymes: cytochrome P4501a1 (Cyp1a1), Cyp1a2, Cyp1b1, and Cyp2s1; and four phase II enzymes: NAD(P)H: quinone oxidoreductase (Nqo1), glutathione S-transferase alpha, cytosolic aldehyde dehydrogenase-3 and UDP-glucuronosyltransferase 1a6 (Nebert and Duffy, 1997, Rivera et al., 2002).
Metabolic activation of PAHs into their quinone-containing metabolites is mediated primarily by members of the phase I drug metabolizing enzymes, specifically Cyp1a1 (Gelboin, 1980). The induction of the phase II enzymes, such as Nqo1, serves as an adaptive mechanism to decrease the deleterious effect of these mutagenic metabolites.
Nqo1 is a homodimeric flavoprotein that catalyses the detoxication of quinones through a single step, two-electron reduction process (Nioi and Hayes, 2004, Talalay and Dinkova-Kostova, 2004). In addition to detoxifying quinones, Nqo1 also helps to maintain endogenous antioxidants, specifically ubiquinone and α-tocopherol-quinone, in their reduced, and hence, active forms (Landi et al., 1997, Siegel et al., 1997). Thus, the oxidoreductase plays an important cytoprotective role.
Consequently, chemicals capable of inducing Nqo1 and other detoxifying enzymes, or inhibiting Cyp1a1, may modulate the mutagenicity and carcinogenicity of these environmental pollutants (Zhang et al., 1992, Zhang et al., 1994). Interestingly, AhR ligands co-exist in the environment with metal contaminants, typified by arsenite (As3+, a metalloid commonly referred to as a metal), cadmium (Cd2+), and chromium (Cr6+). In our previous studies, we demonstrated the ability of these metals to modulate the induction of Nqo1 by various AhR ligands through the induction of oxidative stress. However, the molecular mechanisms mediating the interaction between AhR ligands and metals remain to be discovered.
The role of oxidative stress in the transcriptional regulation of Nqo1 is well established. Widespread studies have unveiled the presence of not only the XRE, but another regulatory element in the 5′ flanking region of the Nqo1 gene: the antioxidant response element (ARE). The ARE mediates the basal expression as well as induction of Nqo1 in response to ROS, antioxidants, and tumor promoters (Jaiswal, 1994, Li and Jaiswal, 1994). In fact, regulation of Nqo1 through the ARE is part of a cellular defense mechanism responsible for the induction of a host of enzyme proteins in response to oxidative stress (Jaiswal, 1994, Rushmore and Pickett, 1993). These enzymes include UDP-glucuronosyl transferases (UDP-GT) and those involved in glutathione homeostasis, such as glutathione S-transferases (GSTs) and γ-glutamylcysteine synthetase (γ-GCS) (Mulcahy et al., 1997).
Although the transcriptional regulation of Nqo1 is well documented, very little information about possible posttranscriptional regulation of the Nqo1 gene products, and the susceptibility of Nqo1 to modifiers of its activity, is available. In an attempt to identify the molecular targets and pathways susceptible to modification by the metals, we examined the effect of As3+, Cd2+, and Cr6+ on Nqo1 expression at the various pathways required for full expression of Nqo1 protein activity. Hepa 1c1c7 cells were used as they are a highly responsive cell line to AhR ligands and have been extensively used to elucidate pathways and mechanisms involving the AhR. Northern blot analysis was used to construct the time course of Nqo1 mRNA. To assess the effect of the metals on transcriptional regulation, mRNA levels were assessed in the presence of actinomycin D (Act-D) or cycloheximide (CHX). Studies with Act-D and CHX were also carried out to determine the effect of the metals on Nqo1 mRNA and protein stability, respectively. Here, we report for the first time transcriptional and posttranscriptional modifications of Nqo1 by metals.
Section snippets
Materials
Sodium arsenite, cadmium chloride, chromium trioxide, cycloheximide, anti-rabbit IgG peroxidase secondary antibody, and protease inhibitor cocktail were purchased from Sigma Chemical Co. (St. Louis, MO). 2,3,7,8-Tetrachlorodibenzo-p-dioxin, >99% pure, was purchased from Cambridge Isotope Laboratories (Woburn, MA). TRIzol reagent, and the random primers DNA labeling system were purchased from Invitrogen Co. (Grand Island, NY). Actinomycin D was purchased from Calbiochem (San Diego, CA).
Effect of metals on the time-dependent induction of Nqo1 mRNA
We analyzed the time course of Nqo1 gene expression at various time points up to 24 h after treatment, at first, with the metals alone. All three metals caused a time-dependent induction of Nqo1 mRNA, albeit in a dissimilar manner (Fig. 1A). An increase in Nqo1 mRNA was apparent as early as 1 h after treatment of Hepa 1c1c7 cells with each of the metals, and the degree of induction was similar with the three metals. The statistically significant, but rather small induction of Nqo1 mRNA by Cr6+
Discussion
Nqo1 is a detoxifying enzyme that catalyzes the detoxification of quinones through a single step, two-electron reduction, mechanism (Joseph et al., 1994). Nqo1 is also an important regulator of intracellular redox status, by virtue of its ability to maintain antioxidants in their active forms. Hence, Nqo1 protects against cellular oxidative stress and PAH-induced carcinogenicity (Long et al., 2000) and tight regulation of this enzyme is critical for cellular protection against oxidative stress,
Acknowledgments
This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Grant RGPIN 250139 to A.O.S. R.H.E. is the recipient of a Canada Graduate Scholarship (CGS Doctoral) and a Izaak Walton Killam Memorial Scholarship.
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