REPORT

NAD(P)H : quinone Oxidoreductase 1 Deficiency and Increased Susceptibility to 7,12-Dimethylbenz[a]-anthracene-Induced Carcinogenesis in Mouse Skin

Delwin J. Long, II, Rebekah L. Waikel, Xiao-Jing Wang, Dennis R. Roop, Anil K. Jaiswal

Affiliations of authors: D. J. Long II, A. K. Jaiswal (Department of Pharmacology), R. L. Waikel, D. R. Roop (Department of Molecular and Cellular Biology), X.-J. Wang (Department of Dermatology), Baylor College of Medicine, Houston, TX.

Correspondence to: Anil K. Jaiswal, Ph.D., Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 (e-mail: ajaiswal{at}bcm.tmc.edu).


    ABSTRACT
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The phase II enzyme NAD(P)H : quinone oxidoreductase 1 (NQO1) catalyzes quinone detoxification, protecting cells from redox cycling, oxidative stress, mutagenicity, and cytotoxicity induced by quinones and its precursors. We have used NQO1-/- C57BL/6 mice to show that NQO1 protects them from skin cancer induced by the polycyclic aromatic hydrocarbon benzo[a]pyrene. Herein, we used NQO1-/- mice to investigate whether NQO1 also protects them against 7,12-dimethylbenz[a]anthracene (DMBA), where methyl substituents diminish primary quinone formation. Methods: Dorsal skin of NQO1-/- or wild-type C57BL/6 mice was shaved. When tested as a complete carcinogen, DMBA (500 or 750 µg in 100 µL of acetone) alone was applied to the shaved area. When tested as a tumor initiator, DMBA (200 or 400 nmol in 100 µL of acetone) was applied to the shaved area; 1 week later, twice-weekly applications of phorbol 12-myristate 13-acetate (PMA)—10 µg dissolved in 200 µL of acetone—to the same area began and were continued for 20 weeks. Tumor development was monitored in all mice (12–15 per group). All statistical tests were two-sided. Results: When DMBA (750 µg) was tested as a complete carcinogen, about 50% of the DMBA-treated NQO1-/- mice but no DMBA-treated wild-type mouse developed skin tumors. When DMBA (both concentrations) was used as a tumor initiator, NQO1-/- mice developed larger tumors at a greater frequency than their wild-type littermates. Twenty-three weeks after the first PMA treatment in the tumor initiator test, all 30 NQO1-/- mice given 400 nmol of DMBA had developed skin tumors, compared with 33% (10 of 30) of treated wild-type mice (P<.001). Conclusions: NQO1-/- mice are more susceptible to DMBA-induced skin cancer than are their wild-type littermates, suggesting that NQO1 may protect cells from DMBA carcinogenesis.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons (PAHs), such as 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene, are abundant environmental contaminants that are formed during the incomplete combustion of organic materials, such as wood, coal, and mineral oil and products derived from them (1,2). PAHs are also found in motor vehicle exhaust and tobacco smoke and can be produced by pyrolysis of amino acids, fatty acids, and carbohydrates during cooking (1,2). PAHs can be transformed into carcinogenic compounds by the following enzyme pathway: Cytochrome P450-dependent monooxygenases form epoxides in the bay region of PAHs (14). Epoxide hydrolase then transforms these epoxides into less reactive diols. Finally, another cytochrome P450 activates these diols by epoxidation, converting them to the carcinogenic diol epoxides (5,6). However, in another pathway containing protective (phase II) enzymes, such as glutathione transferases, sulfotransferases, and uridine diphosphate (UDP)-glucuronosyltransferases (14), diols can be conjugated and thus removed.

NAD(P)H : quinone oxidoreductase 1 (NQO1) is a phase II enzyme that catalyzes the two-electron reduction and detoxification of quinones and their precursors (79). By catalyzing two-electron reductions, NQO1 converts quinones directly into hydroquinones and bypasses the one-electron-reducing pathways, catalyzed by enzymes such as cytochrome P450 reductase, which generate semiquinone intermediates that lead to reactive oxygen species. These reactive oxygen species can contribute to the bioactivation, cytotoxicity, and mutagenicity of PAHs (1013). Consequently, NQO1 activity reduces quinone-induced DNA adduct formation and DNA mutagenicity (1216), including that induced by benzo[a]pyrene quinone. Many compounds that block the toxic, mutagenic, and/or neoplastic effects of carcinogens also elevate the levels of phase II enzymes, including NQO1 (79,1719).

Two percent to 4% of humans lack a functional NQO1 gene and thus have little or no NQO1 activity relative to that of the general population (20,21). As a model system for NQO1 deficiency and PAH carcinogenesis in humans, we generated NQO1-/- mice, which do not express NQO1 (22). NQO1-/- mice are more susceptible to menadione-induced toxicity and PAH (benzo[a]pyrene)-induced skin cancer than are their wild-type (NQO1+/+) littermates (22,23). DMBA is a potent PAH carcinogen (24) in which methyl substituents block primary quinone formation (25). The metabolically activated bay-region epoxides of DMBA can react with purines in DNA and may induce tumorigenesis and carcinogenesis (26). A study (27) has shown that sulforaphane, a compound isolated from 3-day-old broccoli (Brassica oleracea italica) sprouts, can protect Sprague-Dawley rats from DMBA-induced mammary tumors. Sulforaphane stimulates the expression of phase II enzyme genes, including NQO1, without substantially changing the synthesis of cytochrome P450 (28), but it was not clear whether this effect was mediated by NQO1 or by other detoxifying enzymes induced by sulforaphane.

In this report, we investigate whether NQO1 protects against DMBA by determining whether NQO1-/- mice are more susceptible to DMBA-induced skin carcinogenesis than are wild-type mice.


    MATERIALS AND METHODS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemicals

DMBA and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemical Company (St. Louis, MO). Acetone and 10% neutral buffered formalin were purchased from Fisher Scientific (Houston, TX).

C57BL/6 NQO1–/– and Wild-Type Mice

NQO1-/- C57BL/6 mice were generated in our laboratory by homologous recombination and replacement of NQO1 exon 6 with a neomycin-resistance gene (22). For this study, mice were housed in polycarbonate shoebox cages, with a 12-hour light/12-hour dark cycle, a temperature of 24 °C ± 2 °C, a relative humidity of 55% ± 10%, and a negative atmospheric pressure. The mice received standard rodent chow and acidified tap water ad libitum. Seven- to 9-week-old mice were used because their cells were actively growing and experiments might run for more than 50 weeks. Animals received humane care throughout the experiment according to the guidelines of the National Institute of Environmental Health Sciences for animal welfare.

Chemical Carcinogenesis Protocol

The back of each 7- to 9-week-old NQO1-/- or wild-type C57BL/6 mouse was shaved with hair clippers, and DMBA dissolved in acetone was applied topically at various doses to one place on the shaved area (29). Each group consisted of 12–15 mice. When DMBA was tested as a complete carcinogen, 750 µg (2925 nmol) or 500 µg (1950 nmol) in 100 µL of acetone was applied once to the shaved area. When DMBA was tested as a tumor initiator, 200 nmol (51 µg) or 400 nmol (102 µg) in 100 µL of acetone was applied once to the shaved area; 1 week later, 10 µg of PMA dissolved in 200 µL of acetone was applied to the same place twice a week for 20 weeks. Control mice for both experiments received acetone alone.

Histologic Examination and Immunochemical Analysis

Hematoxylin–eosin (H&E) analysis. Skin tumors were fixed in neutral buffered formalin, embedded in paraffin, sectioned (5 µm), and stained with H&E or immunostained with keratin 1 antibody (Vector Laboratories Inc., Burlingame, CA).

Bromodeoxyuridine (BrdUrd) analysis. To determine the proportion of proliferating cells in tumors, we gave tumor-bearing mice intraperitoneal injections of BrdUrd (125 mg/kg) in phosphate-buffered saline. One hour later, tumors (papillomas) of the same size were excised, fixed, and processed as described previously (30). The sections were incubated with fluorescein isothiocyanate-conjugated anti-BrdUrd monoclonal antibodies (Becton-Dickinson, Franklin Lakes, NJ) and guinea pig anti-mouse keratin-14 antiserum, which reacts with the epithelial component of papillomas. Keratin-14 was visualized with biotinylated anti-guinea pig immunoglobulin G and streptavidin–Texas Red (Vector Laboratories Inc.).

Statistical Analysis

Statistical significance was determined for the rate of tumor formation in the wild-type and NQO1-/- mice with a two-sided Fisher's exact test.


    RESULTS
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
DMBA as a complete carcinogen.

DMBA was tested as a complete carcinogen by the application of 750 or 500 µg in 100 µL of acetone to a shaved area on the back of a mouse. NQO1-/- mice began to develop skin tumors approximately 9 weeks after DMBA treatment, but no wild-type mouse had developed a tumor by 30 weeks after DMBA treatment. By 12 weeks after treatment, 58% of the female and 50% of the male NQO1-/- mice given 750 µg of DMBA had developed skin tumors (Table 1Go; for females, P = .005; for males, P = .014), and 25% of the NQO1-/- mice given 500 µg of DMBA had developed skin tumors (P = .22). The frequency of tumors was one tumor per mouse. More or less similar results were obtained for male and female mice. All of the mice were followed for 30 weeks after DMBA treatment. All of the tumors in NQO1-/- mice were relatively small, well-differentiated papillomas that were identified by H&E staining and keratin 1 immunostaining (data not shown).


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Table 1. Mouse skin tumors that developed after DMBA treatment*
 
DMBA as a tumor initiator.

To test DMBA as a tumor initiator, we applied 200 or 400 nmol in 100 µL of acetone to a shaved area on the back of a mouse and then, 1 week later, applied 10 µg of PMA dissolved in 200 µL of acetone to the same area twice a week for 20 weeks. Approximately 17 weeks after the DMBA treatment, NQO1-/- and wild-type mice began to develop skin tumors. The tumors in NQO1-/- mice were larger (8 mm versus 2 mm) and more numerous (two to four tumors versus one tumor) than in the wild-type littermates (Fig. 1Go). By 19 weeks after the DMBA treatment, 67% of the NQO1-/- mice given 400 nmol of DMBA had developed skin tumors (Table 2Go; P = .14) compared with 33% of the wild-type mice treated similarly. Twenty-three weeks after the initial PMA treatment, all of the NQO1-/- mice given 400 nmol of DMBA had developed skin tumors (P<.001) compared with 33% of the wild-type mice treated similarly. By BrdUrd incorporation, there were 40% more proliferating cells in NQO1-/- tumors than in wild-type tumors (P = .05; Fig. 2Go, A). Thirty-six percent (20 of 55) of the NQO1-/- papillomas converted to carcinomas, as demonstrated by H&E staining (Fig. 2Go, B), but only one wild-type tumor of 18 converted to a carcinoma.



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Fig. 1. Gross appearance of skin tumors from NQO1+/+ (upper panel) and NQO1-/- (lower panel) mice. The skin tumors developed 17 weeks after a single application of 400 nmol of 7,12-dimethylbenz[a]anthracene followed by application of phorbol 12-myristate 13-acetate twice a week for 20 weeks. NQO1 = NAD(P)H : quinone oxidoreductase 1.

 

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Table 2. Incidence of mouse skin tumors after a single topical application of DMBA*
 


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Fig. 2. A) Bromode-oxyuridine (BrdUrd) analysis of 7,12-dimethylbenz[a]anthracene (DMBA)/phorbol 12-myristate 13-acetate (PMA)-induced papillomas from wild-type (left) and NQO1-/- (right) mice. Papillomas from NQO1-/- mice exhibited a 40% increase in the number of BrdUrd-labeled nuclei compared with papillomas from wild-type mice. Counterstain is keratin 14, which highlights the epithelial component of the papillomas. B) Histologic type of a typical NQO1-/- tumor induced by DMBA/PMA and stained with hematoxylin–eosin. Histologic analysis of this tumor reveals a moderately to poorly differentiated squamous cell carcinoma. Keratin pearls (KP) are present throughout the carcinoma. NQO1 = NAD(P)H : quinone oxidoreductase 1. Bar = 100 µm.

 

    DISCUSSION
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The phase II enzyme NQO1 protects against quinone-induced redox cycling, oxidative stress, cytotoxicity, and mutagenicity (79) and also protects skin against benzo[a]pyrene-induced carcinogenesis (23). In this report, we provide evidence that NQO1 also protects skin against DMBA-induced carcinogenesis. When DMBA was tested as a complete carcinogen, NQO1-/- mice developed skin tumors, but wild-type mice expressing NQO1 did not. When DMBA was tested as a tumor initiator, NQO1-/- mice developed tumors that were larger and more numerous and that incorporated more BrdUrd than those of wild-type mice. Thus, this study and previous studies with benzo[a]pyrene (23) clearly demonstrate that NQO1 protects against PAH-induced carcinogenesis, even when primary quinones are not generated.

Three possible mechanisms by which NQO1 could protect against PAH are as follows:

First, NQO1 could detoxify PAH quinone metabolites. This mechanism, however, can probably be ruled out because benzo[a]pyrene-3,6-quinone, which is detoxified by NQO1, produced no skin tumors in NQO1-/- mice (31).

Second, NQO1 could act as an antioxidant to reduce the oxidative stress (79) that is increased by metabolic products of both DMBA and benzo[a]pyrene (2,32). This mechanism is supported by the observations that NQO1 can reduce cellular coenzyme Q and {alpha}-tocopherolquinone to generate strong antioxidants, reduced coenzyme Q and {alpha}-tocopherolhydroquinone (33,34); that carcinogenesis induced by DMBA and benzo[a]pyrene is inhibited by several free-radical scavengers and antioxidants (27,32); and finally that mice deficient in metallothionein, a cysteine-rich metal-binding free-radical scavenger, developed skin tumors after treatment with DMBA (29). Thus, a mechanism of action for NQO1 involving the reduction of oxidative stress has some merit.

Third, a more likely mechanism of action for NQO1, however, may involve stabilization of the tumor suppressor p53. Inhibition of NQO1 with dicoumarol leads to degradation of p53 by proteosomes (35). Thus, an animal with decreased NQO1 activity might also have decreased levels of p53, which could then increase its susceptibility to PAH-induced carcinogenesis.

It is not known whether NQO1-/- mice are more sensitive in the initiation or the promotion phase of carcinogenesis. Because NQO1-/- mice were more sensitive to DMBA as a complete carcinogen (this report) and developed more DNA adducts when treated with menadione (31), these mice are likely to be more sensitive in the initiation phase.

NQO1 activity is present in all tissues. However, large variations in NQO1 activity have been observed (79) in different individuals, different tissue types of the same individual, and normal and tumor tissues. NQO1 gene expression is induced in response to xenobiotics, antioxidants, oxidants, heavy metals, and UV and ionizing radiation (79,36,37). The NQO1 gene is one of more than two dozen phase II cellular defense genes induced by electrophilic and/or oxidative stress (36, 37). These genes include glutathione S-transferases, which conjugate hydrophobic electrophiles and reactive oxygen species with glutathione; UDP-glucuronosyltransferases, which catalyze the conjugation of glucuronic acid with xenobiotics and drugs for their excretion; epoxide hydrolase, which inactivates epoxides; and {gamma}-glutamylcysteine synthetase, which plays a key role in the regulation of glutathione metabolism (79). Therefore, the coordinated induction of these genes, including NQO1, presumably provides the necessary protection for cells against free-radical damage, oxidative stress, and neoplasia. Sulforaphane is a chemical that induces detoxifying enzymes (including NQO1) and protects against mammary tumor development in rats fed DMBA (27). Our results suggest that sulforaphane protects against DMBA-induced cancer, at least in part, by its ability to induce NQO1.

In conclusion, we demonstrated that NQO1-/- mice are more susceptible than their wild-type littermates to DMBA-induced skin cancer and, to our knowledge, we demonstrate for the first time that NQO1 protects against PAH-induced cancer, whether or not quinones are generated. We also show that NQO1 acts as an endogenous factor that protects against DMBA-induced cancer. We believe that these results are particularly important because 2%–4% of the human population does not express NQO1. Our results with DMBA and previous results with benzo[a]pyrene (23) suggest that NQO1-deficient humans may be more sensitive to PAH-induced cancer.


    NOTES
 
Supported by Public Health Service grant R01ES07943 from the National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services.

We thank Harris Busch, Janet L. Stringer, and Ramachandra Reddy at Baylor College of Medicine, Houston, TX, for helpful discussions and suggestions. We also thank Bharat Aggarwal at The University of Texas M. D. Anderson Cancer Center, Houston, for critical evaluation.


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Manuscript received January 31, 2001; revised May 23, 2001; accepted June 7, 2001.


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