Dhrubajyoti Chakravarti

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene.

Treatment of SENCAR mouse skin with dibenzo[a,l]pyrene results in abundant formation of abasic sites that undergo error-prone excision repair, forming oncogenic H-ras mutations in the early preneoplastic period. To examine whether the abundance of abasic sites causes repair infidelity, we treated SENCAR mouse skin with estradiol-3,4-quinone (E2-3,4-Q) and determined adduct levels 1 h after treatment, as well as mutation spectra in the H-ras gene between 6 h and 3 days after treatment. E2-3,4-Q formed predominantly (⩾99%) the rapidly-depurinating 4-hydroxy estradiol (4-OHE2)-1-N3Ade adduct and the slower-depurinating 4-OHE2-1-N7Gua adduct. Between 6 h and 3 days, E2-3,4-Q induced abundant A to G mutations in H-ras DNA, frequently in the context of a 3′-G residue. Using a T.G-DNA glycosylase (TDG)-PCR assay, we determined that the early A to G mutations (6 and 12 h) were in the form of G.T heteroduplexes, suggesting misrepair at A-specific depurination sites. Since G-specific mutations were infrequent in the spectra, it appears that the slow rate of depurination of the N7Gua adducts during active repair may not generate a threshold level of G-specific abasic sites to affect repair fidelity. These results also suggest that E2-3,4-Q, a suspected endogenous carcinogen, is a genotoxic compound and could cause mutations.

The role of endogenous catechol quinones in the initiation of cancer and neurodegenerative diseases.

Publisher Summary This chapter explores the role of endogenous catechol quinones in the initiation of cancer and neurodegenerative diseases. Several lines of evidence point to the major role of endogenous catechol quinones in the etiology of cancer and neurodegenerative diseases. The role of catechol estrogen-3,4-quinones in the initiation of breast cancer is very clear. The rapidly-depurinating N3Ade adducts result in a burst of apurinic sites that overwhelm the repair machinery of the cell and generate tumorigenic mutations by error-prone repair. In contrast, repair of the apurinic sites that are formed by the slow depurination of N7Gua adducts can proceed correctly and leads to very few mutations. Mutations at Ade bases accumulate in breast cancer cells, presumably through this mechanism. This pathway of activation for endogenous estrogens is mirrored by the activation of the synthetic estrogens hexestrol and DES. Evolution of fundamental concepts and principles of chemical carcinogenesis are elaborated in this chapter. The chapter explains the mechanism of tumor initiation by PAHs. Formation of estrogen metabolites, conjugates and DNA adducts is described in the chapter. Imbalance in estrogen homeostasis and unifying mechanism of initiation of cancer by endogenous and synthetic estrogens are elaborated as well.

Detection of dibenzo[a,l]pyrene-induced H-ras codon 61 mutant genes in preneoplastic SENCAR mouse skin using a new PCR-RFLP method.

One of the key events in tumor initiation in mouse skin is mutational activation of the H-ras gene. Papillomas induced by the most carcinogenic environmental polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DB[a,l]P), in SENCAR mouse skin contain a specific H-ras codon 61 (CAA→CTA) mutation. We describe here detection of these mutations in preneoplastic skin by measuring the frequency of an induced XbaI RFLP, created by the mutation. Development of the PCR-XbaI RFLP method, sensitive enough to detect 1 codon 61 mutant allele among 10 000 wild-type genes, is described. The results indicate that codon 61 mutations are induced 1  day (0.1%) after DB[a,l]P treatment on mouse skin, reach a high value (5%) by day 3, rapidly decline between days 7–9 and increase again during the clonal expansion of pre-papillomas into tumors. The detection of codon 61 mutations 1 day after DB[a,l]P exposure suggests that mutations occurred by pre-replication misrepair.

Depurinating naphthalene-DNA adducts in mouse skin related to cancer initiation.

Naphthalene has been shown to be a weak carcinogen in rats. To investigate its mechanism of metabolic activation and cancer initiation, mice were topically treated with naphthalene or one of its metabolites, 1-naphthol, 1,2-dihydrodiolnaphthalene (1,2-DDN), 1,2-dihydroxynaphthalene (1,2-DHN), and 1,2-naphthoquinone (1,2-NQ). After 4 h, the mice were sacrificed, the treated skin was excised, and the depurinating and stable DNA adducts were analyzed. The depurinating adducts were identified and quantified by ultraperformance liquid chromatography/tandem mass spectrometry, whereas the stable adducts were quantified by (32)P-postlabeling. For comparison, the stable adducts formed when a mixture of the four deoxyribonucleoside monophosphates was treated with 1,2-NQ or enzyme-activated naphthalene were also analyzed. The depurinating adducts 1,2-DHN-1-N3Ade and 1,2-DHN-1-N7Gua arise from reaction of 1,2-NQ with DNA. Similarly, the major stable adducts appear to derive from the 1,2-NQ. The depurinating DNA adducts are, in general, the most abundant. Therefore, naphthalene undergoes metabolic activation to the electrophilic ortho-quinone, 1,2-NQ, which reacts with DNA to form depurinating adducts. This is the same mechanism as other weak carcinogens, such as the natural and synthetic estrogens, and benzene.

Possible genotoxic modes of action for naphthalene

This report provides a summary of deliberations conducted under the charge for members of Module D participating in the Naphthalene State-of-the-Science Symposium (NS(3)), Monterey, CA, October 9-12, 2006. The charge directed the panel to ascertain to the best of its ability a consensus judgment of the state-of-the-science concerning the potential for a genotoxic mode of action for naphthalene and its metabolites, with implications for low-dose extrapolations of cancer risk estimates for exposed populations. Where scientific uncertainties remained, the panel was asked to identify which scientific uncertainties (if any) could be resolved through targeted, timely, cost-effective research. The report provides a brief summary of naphthalene genotoxicity; identifies those areas where there is a general scientific consensus regarding the effects of naphthalene; identifies areas of uncertainty regarding the effects of naphthalene; and key questions that currently limit our ability to assess the genotoxic risks of naphthalene. The report also outlines a set of six studies that could resolve some of these key uncertainties.

NAD(P)H:quinone oxidoreductase 1 Arg139Trp and Pro187Ser polymorphisms imbalance estrogen metabolism towards DNA adduct formation in human mammary epithelial cells.

Estrogens (estrone, E(1); estradiol, E(2)) are oxidized in the breast first to catechols and then to form two ortho-quinones (E(1/2)-3,4-Q) that react with DNA to form depurinating adducts, which lead to mutations associated with breast cancer. NAD(P)H:quinone oxidoreductase 1 (NQO1) reduces these quinones back to catechols, and thus may protect against this mechanism. We examined whether the inheritance of two polymorphic variants of NQO1 (Pro187Ser or Arg139Trp) would result in poor reduction of E(1/2)-3,4-Q in normal human mammary epithelial cells (MCF-10F) and increased depurinating adduct formation. An isogenic set of stably transfected normal human breast epithelial cells (MCF-10F) that express a truncated (135Stop), the wild-type, the 139Trp variant or the 187Ser variant of human NQO1 cDNA was constructed. MCF-10F cells showed a low endogenous NQO1 activity. NQO1 expression was examined by RT-PCR and Western blotting, and catalytic activity of reducing E(2)-3,4-Q to 4-hydroxyE(1/2) and associated changes in the levels of quinone conjugates (4-methoxyE(1/2), 4-OHE(1/2)-2-glutathione, 4-OHE(1/2)-2-Cys and 4-OHE(1/2)-2-N-acetylcysteine) and depurinating DNA adducts (4-OHE(1/2)-1-N3Ade and 4-OHE(1/2)-1-N7Gua) were examined by HPLC with electrochemical detection, as well as by ultra-performance liquid chromatography with tandem mass spectrometry. The polymorphic variants transcribed comparably to the wild-type NQO1, but produced approximately 2-fold lower levels of the protein, suggesting that the variant proteins may become degraded. E(1/2)-3,4-Q toxicity to MCF-10F cells (IC50=24.74 microM) was increased (IC50=3.7 microM) by Ro41-0960 (3 microM), a catechol-O-methyltransferase inhibitor. Cells expressing polymorphic NQO1 treated with E(2)-3,4-Q with or without added Ro41-0960, showed lower ability to reduce the quinone ( approximately 50% lower levels of the free catechols and approximately 3-fold lower levels of methylated catechols) compared to the wild-type enzyme. The increased availability of the quinones in these cells did not result in greater glutathione conjugation. Instead, there was increased (2.5-fold) formation of the depurinating DNA adducts. Addition of Ro41-0960 increased the amounts of free catechols, quinone conjugates and depurinating DNA adducts. NQO1 polymorphic variants (Arg139Trp and Pro187Ser) were poor reducers of estrogen-3,4-quinones, which caused increased formation of estrogen-DNA adduct formation in MCF-10F cells. Therefore, the inheritance of these NQO1 polymorphisms may favor the estrogen genotoxic mechanism of breast cancer.

Ortho-quinones of benzene and estrogens induce hyperproliferation of human peripheral blood mononuclear cells

Benzene is a known leukemogen. It has been hypothesized that benzene and natural estrogens initiate cancer by forming ortho-quinones (catechol quinones) that react with DNA in cells. These quinones form depurinating DNA adducts that generate the mutations leading to cancer. This study examined whether the treatment of normal human peripheral blood mononuclear cells with the ortho-quinones of benzene or estradiol would form DNA adducts and elicit an alteration in the proliferation of these cells. Both estradiol-3,4-quinone and benzene ortho-quinone formed depurinating DNA adducts and significantly increased the mitogen-induced proliferation of normal blood mononuclear cells. Immunophenotyping of the estradiol-3,4-quinone-treated blood cells indicated that monocyte/macrophage, natural killer and T-cells were particularly prone to hyperproliferation. Thus, DNA damage induced by the ortho-quinones of benzene and estradiol may promote the growth of human blood mononuclear cells, including those that appear in large numbers in leukemia and lymphoma.