Elsevier

Toxicology in Vitro

Volume 22, Issue 5, August 2008, Pages 1184-1190
Toxicology in Vitro

Arsenite and cadmium, but not chromium, induce NAD(P)H:quinone oxidoreductase 1 through transcriptional mechanisms, in spite of post-transcriptional modifications

https://doi.org/10.1016/j.tiv.2008.03.010Get rights and content

Abstract

NAD(P)H:quinone oxidoreductase (Nqo1)-mediated detoxification of quinones plays a critical role in cancer prevention. Metals alter the carcinogenicity of AhR ligands, such as TCDD, by modulating the induction of Nqo1, but the mechanism(s) remain unresolved. To decipher the molecular mechanisms involved in the alteration of Nqo1, we analyzed the effect of the metals As3+ (5 μM), Cd2+ (5 μM), and Cr6+ (25 μM) on the transcriptional activation of the Nqo1 gene and post-transcriptional modifications, in Hepa 1c1c7 cells. Both As3+ and Cd2+ induced Nqo1 mRNA in a time-dependent manner and potentiated TCDD-induced Nqo1 mRNA. Cr6+ on the other hand, completely inhibited the induction of Nqo1 mRNA by TCDD. The induction of Nqo1 mRNA by the metals was completely inhibited with the DNA transcription inhibitor actinomycin-D, indicating a requirement for de novo mRNA synthesis for the induction. Furthermore, the protein synthesis inhibitor cycloheximide decreased Nqo1 mRNA induction, suggesting a role for a labile protein in the transcriptional induction of Nqo1 mRNA by metals. Surprisingly, all three metals decreased Nqo1 mRNA stability while having no effect on Nqo1 protein half-life. Meanwhile, As3+ and Cd2+ induced constitutive Nqo1 activity and potentiated the induction by increasing concentrations of TCDD. On the other hand, Cr6+ inhibited inducible Nqo1 activity. It is apparent that metals alter Nqo1 expression at the transcriptional level, through a labile protein-mediated pathway.

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.

References (39)

  • R.T. Mulcahy et al.

    Constitutive and beta-naphthoflavone-induced expression of the human gamma-glutamylcysteine synthetase heavy subunit gene is regulated by a distal antioxidant response element/TRE sequence

    The Journal of Biological Chemistry

    (1997)
  • D.W. Nebert et al.

    How knockout mouse lines will be used to study the role of drug-metabolizing enzymes and their receptors during reproduction and development, and in environmental toxicity, cancer, and oxidative stress

    Biochemical Pharmacology

    (1997)
  • P. Nioi et al.

    Contribution of NAD(P)H:quinone oxidoreductase 1 to protection against carcinogenesis, and regulation of its gene by the Nrf2 basic-region leucine zipper and the arylhydrocarbon receptor basic helix-loop-helix transcription factors

    Mutation Research

    (2004)
  • J. Pi et al.

    Transcription factor Nrf2 activation by inorganic arsenic in cultured keratinocytes: involvement of hydrogen peroxide

    Experimental Cell Research

    (2003)
  • T.H. Rushmore et al.

    Glutathione S-transferases, structure, regulation, and therapeutic implications

    The Journal of Biological Chemistry

    (1993)
  • D. Stewart et al.

    Degradation of transcription factor Nrf2 via the ubiquitin-proteasome pathway and stabilization by cadmium

    The Journal of Biological Chemistry

    (2003)
  • H.I. Swanson

    DNA binding and protein interactions of the AHR/ARNT heterodimer that facilitate gene activation

    Chemico-Biological Interactions

    (2002)
  • P. Talalay et al.

    Role of nicotinamide quinone oxidoreductase 1 (NQO1) in protection against toxicity of electrophiles and reactive oxygen intermediates

    Methods in Enzymology

    (2004)
  • Y.D. Wei et al.

    Chromium inhibits transcription from polycyclic aromatic hydrocarbon-inducible promoters by blocking the release of histone deacetylase and preventing the binding of p300 to chromatin

    The Journal of Biological Chemistry

    (2004)
  • Cited by (0)

    View full text