Immunodetection of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human tissues1

https://doi.org/10.1016/S0891-5849(00)00310-5Get rights and content

Abstract

Despite the extensive interest in NADPH:quinone oxidoreductase (NQO1, DT-diaphorase), there is little immunohistochemical information regarding its distribution in either normal human tissues or in human tumors. Using immunohistochemistry (IHC), we have examined cell-specific expression of NQO1 in many normal tissues and tumors as a step toward defining the distribution of NQO1 in humans. NQO1 was detected by IHC in respiratory, breast duct, thyroid follicle, and colonic epithelium, as well as in the corneal and lens epithelium of the eye. NQO1 was also detected by IHC in vascular endothelium in all tissues examined. NQO1 could also readily be detected in the endothelial lining of the aorta but was not detected using immunoblot analysis in the myocardium. Adipocytes stained positive for NQO1, and the enzyme was also detected by both IHC and immunoblot analysis in parasympathetic ganglia in the small intestine and in the optic nerve and nerve fibers. NQO1 was not highly expressed in five different human liver samples using immunoblot analysis, whereas studies using IHC demonstrated only trace NQO1 staining in isolated bile duct epithelium. NQO1 expresion was also examined by IHC in a variety of solid tumors. Marked NQO1 staining was detected in solid tumors from thyroid, adrenal, breast, ovarian, colon, and cornea and in non–small cell lung cancers. The NQO1 content of many solid tumors supports the use of NQO1-directed anticancer agents for therapeutic purposes, but the distribution of NQO1 in normal tissues suggests that potential adverse effects of such agents need to be carefully monitored in preclinical studies.

Introduction

DT-diaphorase (NADPH:quinone oxidoreductase, NQO1) was discovered in the late Professor Ernster’s laboratory, and the first reports of this discovery were published in 1958 [1], [2]. The enzyme was detected accidentally during studies of NAD and NADP dehydrogenases, and the historical aspects of the discovery have been described elsewhere [3]. NQO1 has attracted interest over the years as an enzyme involved in the detoxification of xenobiotics such as quinones and quinone-imines [4], [5], [6] and an enzyme associated with protection against mutagenesis and carcinogenesis [7], [8], [9], [10]. Recently, NQO1 has been characterized as being capable of generating antioxidant forms of ubiquinone and vitamin E after free radical attack [11], [12], providing conclusive evidence that this enzyme forms part of the body’s antioxidant defense system.

The role of NQO1 in chemoprotection has also been supported by the increased incidence of disease and xenobiotic-induced toxicity in individuals carrying a polymorphism in NQO1. The polymorphism is a homozygous C to T change at position 609 of the cDNA, which codes for a proline to serine change in the amino acid structure of the enzyme. The homozygous C609T change results in either nondetectable or, at best, trace amounts of mutant NQO1 protein and a lack of NQO1 activity [13], [14]. Because the homozygous change at position 609 results in an essentially null phenotype, it provides a convenient molecular tool with which to assess the role of NQO1 in vivo. The NQO1 609 polymorphism has been associated with an increased risk of urothelial tumors [15], therapy-related acute myeloid leukemia [16], urolithiasis [17], cutaneous basal cell carcinomas [18], and pediatric leukemias [19], as well as with increased benzene-induced hematopoietic toxicity [20]. Recent work suggests that a lack of NQO1 due to this polymorphism is not a risk factor for prostate cancer [21]. The role of NQO1 in susceptibility to many other forms of cancer is currently under investigation, and our own work suggests it is a risk factor for pharyngeal cancer in a Japanese population (M. Yano, D. Siegel, and D. Ross, unpublished results).

Many enzymes which have been generalized as detoxification systems may also bioactivate certain substrates, and NQO1 also falls into this category. NQO1 has been demonstrated to bioactivate nitropyrenes contained in tobacco smoke [22], and this has been suggested as a possible explanation of an association of the wild-type NQO1 allele with an increased incidence of lung cancer in certain ethnic groups [23], [24]. NQO1 has been found to be expressed at high levels in many human tumors [25], [26], [27], [28], [29], which has led to the possibility of developing compounds bioactivated by NQO1 as chemotherapeutic agents [30], [31], [32], [33]. Chemotherapeutic agents that are bioactivated by NQO1 include mitomycin C, streptonigrin [33], [34], and newly developed agents that are highly efficient substrates for NQO1, such as the aziridinylbenzoquinone RH1 [35]. RH1 is currently under consideration for clinical trial, and efforts are underway to correlate clinical response of established agents, such as mitomycin C, to patient NQO1 status.

Despite the extensive interest in NQO1, there is little information regarding its distribution in either normal human tissues or in human tumors. We have recently localized NQO1 in human lung tumors and uninvolved tissues by using immunohistochemistry [36]. In this manuscript, we have extended these data to many other tissues and tumors as a step toward defining the distribution of NQO1 in humans.

Section snippets

Human tissues

Archival samples of formalin-fixed, paraffin-embedded tissues (thyroid, adrenal, breast, ovary, lung) were supplied by the University of Colorado Cancer Center. Archived paraffin blocks of normal eye and corneal tumors were supplied by Dr. Matthew W. Wilson, Departments of Ophthalmology and Pathology, University of Colorado Health Sciences Center. Fresh frozen aorta and myocardial tissues were supplied by Dr. Kathleen Stringer, Department of Pharmacy Practice, School of Pharmacy, University of

Results

Using IHC, we examined cell-specific expression of NQO1 in normal human tissues. In previous work [36], we have reported a high level of NQO1 expression in respiratory epithelial cells (Fig. 1A). In this study, NQO1 was also detected by IHC in the epithelium of other tissues. Positive staining for NQO1 was observed in breast duct epithelium (Fig. 1B); thyroid follicle epithelium (Fig.1C); epithelial lining of the colon (Fig. 1D); and in the eye in corneal and lens epithelia (Figs. 1E and 1F).

Discussion

We have previously demonstrated the presence of NQO1 in human respiratory epithelium and endothelium [36], and our present data extend these observations to a number of other human tissues. NQO1 was detected in the epithelium of thyroid, breast, colon, and eye tissues, and immunostaining in these tissues was consistent with a high level of NQO1 expression. We have also observed that NQO1 is expressed at high levels in vascular endothelial cells [36], which is consistent with our findings in

Acknowledgements

The work described was supported by NIH grants RO1 CA 51210 and RO1 ES 09554.

References (51)

  • T. Cresteil et al.

    High levels of expression of the NAD(P)H:quinone oxidoreductase (NQO1) gene in tumor cells compared to normal cells of the same origin

    Biochem. Pharmacol.

    (1991)
  • L. Ernster et al.

    Soluble diaphorase in animal tissues

    Acta Chem. Scand.

    (1958)
  • L. Ernster

    Diaphorase activities in liver cytoplasmic fractions

    Fed. Proc.

    (1958)
  • L. Ernster

    DT-diaphorasea historical review

    Chemica Scripta

    (1987)
  • P.L. Chesis et al.

    Mutagenicity of quinonespathways of metabolic activation and detoxification

    Proc. Natl. Acad. Sci. USA

    (1984)
  • C. Huggins et al.

    Induced protection of adrenal cortex against 7,12- dimethylbenz(a)anthracene. Influence of ethionine. Induction of menadione reductase. Incorporation of thymidine-H3

    J. Exp. Med.

    (1964)
  • A.M. Benson et al.

    Increase of NAD(P)H:quinone reductase by dietary antioxidantspossible role in protection against carcinogenesis and toxicity

    Proc. Natl. Acad. Sci. USA

    (1980)
  • P. Talalay et al.

    Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis

    Proc. Natl. Acad. Sci. USA

    (1988)
  • M.A. Trush et al.

    Analysis of target cell susceptibility as a basis for the development of a chemoprotective strategy against benzene-induced hematotoxicities

    Environ. Health Perspect.

    (1996)
  • R.E. Beyer et al.

    The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems

    Proc. Natl. Acad. Sci. USA

    (1996)
  • D. Siegel et al.

    The reduction of alpha-tocopherolquinone by human NAD(P)H:quinone oxidoreductasethe role of alpha-tocopherol hydroquinone as a cellular antioxidant

    Mol. Pharmacol.

    (1997)
  • R.D. Traver et al.

    Characterization of a polymorphism in NAD(P)H:quinone oxidoreductase (DT-diaphorase)

    Br. J. Cancer

    (1997)
  • D. Siegel et al.

    Genotype-phenotype relationships in studies of a polymorphism in NAD(P)H:quinone oxidoreductase 1

    Pharmacogenetics

    (1999)
  • W.A. Schulz et al.

    Increased frequency of a null allele for NAD(P)H:quinone oxidoreductase in patients with urological malignancies

    Pharmacogenetics

    (1997)
  • W.A. Schulz et al.

    Predisposition toward urolithiasis associated with the NQO1 null-allele

    Pharmacogenetics

    (1998)
  • Cited by (263)

    • Hypoxia

      2022, Comprehensive Pharmacology
    • Fate of drug-metabolizing enzymes in cardiovascular diseases: Concepts and challenges

      2022, Biochemistry of Drug Metabolizing Enzymes: Trends and Challenges
    View all citing articles on Scopus
    1

    This work is dedicated to the memory of Professor Lars Ernster, who provided us with enthusiastic support, scientific insight, and constant encouragement in our many interactions.

    View full text