Commentary Benzene, NQO1, and genetic susceptibility to cancer

NAD(P)H:quinone oxidoreductase 1 (NQO1; EC 1.6.99.2), originally called DT-diaphorase (1), is an enzyme that has attracted considerable attention because of its ability to detoxify a number of natural and synthetic compounds and, conversely, to activate certain anticancer agents (2, 3). It is also a highly inducible enzyme. Synthetic antioxidants, such as butylated hydroxyanisole, and extracts of cruciferous vegetables, including broccoli, have been shown to be potent inducers of NQO1 (4, 5). This inducibility has led to the suggestion that NQO1 plays an important role in cancer chemoprevention (6). In 1980, Edwards et al. (7) reported that 4% of a British population completely lacked NQO1 activity, but the reasons for and implications of this finding were unclear at the time. In the early 1990s, as part of their studies on the bioactivation of quinone anticancer agents, Ross, Gibson, and their colleagues were characterizing the NQO1 activities of various colon and lung carcinoma cell lines (8). They noticed that two of the lines, the BE colon carcinoma line and the nonsmall cell lung cancer H596 cell line, were different in that they showed no demonstrable NQO1 activity. By using DNA sequencing analysis, they established the presence of a homozygous C to T point mutation at position 609 of the NQO1 cDNA from the BE cell line (8). This mutation conferred a proline-to-serine substitution at position 187 of the NQO1 protein, which they suggested was responsible for the lack of NQO1 activity in BE cells. Sequencing of the coding region of NQO1 from lung H596 cells subsequently showed the presence of the identical homozygous point mutation found in BE cells (9). Thus, the lack of NQO1 activity in certain cell lines and subjects in the Edwards et al. study was most likely the result of homozygous inheritance of two mutant alleles at position 609 in the NQO1 gene. Confirmation of this idea came from the development of a simple PCR-restriction fragment length polymorphismbased method for detecting the 609 C 3 T polymorphism by Sies and coworkers in Germany (10). NQO1 activity was shown to be absent in three renal carcinoma patients who were homozygous for the mutant allele (11). Recent genotype– phenotype studies in vivo have further confirmed that the homozygous C609T change results in a lack of NQO1 enzyme activity and protein (12). The development of a simple method for detecting the polymorphism meant that it could be examined in human populations. In 1992, together with investigators from the National Cancer Institute and the Chinese Academy of Preventive Medicine, we collected samples of blood from subjects in a case-control study of benzene hematotoxicity in Shanghai, China (13). Benzene is metabolized in the liver to phenol, hydroquinone, and catechol, which then travel to the bone marrow and can be activated by peroxidases to highly toxic quinones (14). NQO1 is capable of maintaining these quinones in their reduced form, thereby detoxifying them. We therefore hypothesized that NQO1 would protect against benzene toxicity and that individuals lacking NQO1 would be at higher risk of benzene poisoning. Analysis of DNA isolated from the subjects in Shanghai by the Ross laboratory (15) revealed that subjects who were homozygous for the 609 C 3 T polymorphism were significantly more likely to be poisoned by benzene (measured as decreased blood cell counts) (odds ratio 5 2.6; 95% confidence intervals, 1.1–6.6) and were at elevated risk of contracting benzene-induced leukemia. This work built on a body of evidence from studies in vitro by Smart and Zannoni (16) and in animals and cell lines by Trush, Twerdok, and coworkers (17, 18), which suggested that NQO1 protected against benzene toxicity. Our case-control study also revealed the high incidence of the mutant NQO1 allele in the Chinese population with approximately 20% of the population being homozygous mutants, a finding that has been confirmed in other Asian populations (19). The reasons for this high incidence are intriguing, as it is not known what selective pressures are responsible. A potential problem with our finding of NQO1’s protective effect against benzene toxicity in a human epidemiological study was the anomalous observation from the Ross laboratory that freshly isolated human bone marrow cells lacked expression of NQO1 (20). A protective role for NQO1 against benzene-derived quinones in the marrow was difficult to reconcile with this observation. A likely explanation of this apparent anomaly is offered in this issue of the Proceedings by Moran, Siegel, and Ross (21), who demonstrate that the benzene metabolite hydroquinone induces high levels of NQO1 activity in bone marrow cells, including CD341 progenitor cells, with the wild-type (CyC) genotype. Exposure to noncytotoxic doses of hydroquinone induced intermediate levels of NQO1 activity in heterozygous (CyT) cells, but had no effect in cells with the homozygous mutant (TyT) genotype. Thus, failure to induce functional NQO1 in cells with homozygous mutant alleles may make them susceptible to the toxic effects of benzene metabolites and thereby may explain the increased risk of benzene poisoning in individuals with the (TyT) genotype. Numerous questions remain, however, about the role NQO1 plays in protecting the body against chemical exposures, the mechanism of its induction by hydroquinone and other chemicals, and the susceptibility of individuals with mutant alleles to various cancers, including leukemia. There is also the interesting biochemical question of why homozygous mutant cells have no NQO1 activity. Ross and coworkers have shown that cells with the homozygous mutant genotype still express significant quantities of NQO1 mRNA but have little or no NQO1 protein (9). Transfection of NQO1 cDNA containing the C609T mutation into Escherichia coli and COS-1 cells resulted in expression of mutant NQO1 protein. However, recombinant mutant NQO1 purified from E. coli had only 2–4% of the activity of the wild-type enzyme. The reasons for the low activity of the mutant protein are currently under investigation and may be related to its instability. NQO1 was first called DT-diaphorase after its discovery as a cytosolic diaphorase by Ernster and colleagues in 1958 (2).