Reactive oxygen species (ROS) are mutagenic and may thereby promote cancer1. Normally, ROS levels are tightly controlled by an inducible antioxidant program that responds to cellular stressors and is predominantly regulated by the transcription factor Nrf2 and its repressor protein Keap12-5. In contrast to the acute physiological regulation of Nrf2, in neoplasia there is evidence for increased basal activation of Nrf2. Indeed, somatic mutations that disrupt the Nrf2-Keap1 interaction to stabilize Nrf2 and increase the constitutive transcription of Nrf2 target genes were recently identified, suggesting that enhanced ROS detoxification and additional Nrf2 functions may in fact be pro-tumorigenic6. Here, we investigated ROS metabolism in primary murine cells following the expression of endogenous oncogenic alleles of K-Ras, B-Raf and Myc, and find that ROS are actively suppressed by these oncogenes. K-RasG12D, B-RafV619E and MycERT2 each increased the transcription of Nrf2 to stably elevate the basal Nrf2 antioxidant program and thereby lower intracellular ROS and confer a more reduced intracellular environment. Oncogene-directed increased expression of Nrf2 is a novel mechanism for the activation of the Nrf2 antioxidant program, and is evident in primary cells and tissues of mice expressing K-RasG12D and B-RafV619E, and in human pancreatic cancer. Furthermore, genetic targeting of the Nrf2 pathway impairs K-RasG12D-induced proliferation and tumorigenesis in vivo. Thus, the Nrf2 antioxidant and cellular detoxification program represents a previously unappreciated mediator of oncogenesis.

Analysis of cellular 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dGuo) as a biomarker of oxidative DNA damage has been fraught with numerous methodological problems. This is primarily due to artifactual oxidation of dGuo that occurs during DNA isolation and hydrolysis. Therefore, it has become necessary to rely on using the comet assay, which is not necessarily specific for 8-oxo-dGuo. A highly specific and sensitive method based on immunoaffinity purification and stable isotope dilution liquid chromatography (LC)-multiple reaction monitoring (MRM)/mass spectrometry (MS) that avoids artifact formation has now been developed. Cellular DNA was isolated using cold DNAzol (a proprietary product that contains guanidine thiocyanate) instead of chaotropic- or phenol-based methodology. Chelex-treated buffers were used to prevent Fenton chemistry-mediated generation of reactive oxygen species (ROS) and artifactual oxidation of DNA bases. Deferoxamine was also added to all buffers in order to complex any residual transition metal ions remaining after Chelex treatment. The LC-MRM/MS method was used to determine that the basal 8-oxo-dGuo level in DNA from human bronchoalveolar H358 cells was 2.2 ± 0.4 8-oxo-dGuo/107 dGuo (mean ± standard deviation) or 5.5 ± 1.0 8-oxo-dGuo/108 nucleotides. Similar levels were observed in human lung adenocarcinoma A549 cells, mouse hepatoma Hepa-1c1c7 cells, and human HeLa cervical epithelial adenocarcinoma cells. These values are an order of magnitude lower than is typically reported for basal 8-oxo-dGuo levels in DNA as determined by other MS- or chromatography-based assays. H358 cells were treated with increasing concentrations of potassium bromate (KBrO3) as a positive control or with the methylating agent methyl methanesulfonate (MMS) as a negative control. A linear dose−response for 8-oxo-dGuo formation (r2 = 0.962) was obtained with increasing concentrations of KBrO3 in the range of 0.05 mM to 2.50 mM. In contrast, no 8-oxo-dGuo was observed in H358 cell DNA after treatment with MMS. At low levels of oxidative DNA damage, there was an excellent correlation between a comet assay that measured DNA single strand breaks (SSBs) after treatment with human 8-oxo-guanine glycosylase-1 (hOGG1) when compared with 8-oxo-dGuo in the DNA as measured by the stable isotope dilution LC-MRM/MS method. Availability of the new LC-MRM/MS assay made it possible to show that the benzo[a]pyrene (B[a]P)-derived quinone, B[a]P-7,8-dione, could induce 8-oxo-dGuo formation in H358 cells. This most likely occurred through redox cycling between B[a]P-7,8-dione and B[a]P-7,8-catechol with concomitant generation of DNA damaging ROS. In keeping with this concept, inhibition of catechol-O-methyl transferase (COMT)-mediated detoxification of B[a]P-7,8-catechol with Ro 410961 caused increased 8-oxo-dGuo formation in the H358 cell DNA.

8-oxo-dGuo ͉ DNA strand breaks ͉ tobacco carcinogens ͉ reactive oxygen species P olycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, which are produced as a result of fossil-fuel combustion and are found in car exhaust and charbroiled and smoked foods (1, 2). They are also present as mixtures in tobacco smoke and are implicated in the causation of human lung cancer (3). To exert their carcinogenic effects, PAHs must be metabolically activated to DNA-damaging agents that will result in the signature mutations in lung cancer. These mutations are G-to-T transversions that either activate the K-ras protooncogene at the 12th and 61st codon (4) or inactivate the p53 tumor suppressor gene at hot spots in its DNA binding domain (5).Using benzo[a]pyrene (B[a]P) as a representative PAH, three pathways of activation have been proposed that lead to these mutations. The first pathway involves the formation of (ϩ)-anti-7␣,8␤-dihydroxy-9␣,10␤-epoxy-7,8,9,10-tetrahydroB[a]P {(Ϯ)- anti-B[a]PDE}.In this pathway there is sequential monoxygenation catalyzed by cytochrome P450 (P450) 1A1/1B1 and hydration to form 7␣,8␤-dihydroxy-7,8-dihydroxy-B[a]P, which undergoes a secondary monoxygenation to form (ϩ)-anti-B[a]PDE (6). This diol-epoxide forms stable (ϩ)-anti-trans-B[a]PDE-N 2 -2Ј-deoxyguanosine (dGuo) adducts, which via trans-lesional bypass DNA polymerases, yield G-to-T transversions (7).The second pathway involves metabolic activation by P450 peroxidases to yield radical cations (8), which can form depurinating adducts that lead to abasic sites. Apurinic/apyrimdinic (AP) sites, if not repaired, can give rise to G-to-T transversions (9). However, it is unlikely that radical cations are sufficiently long-lived to damage DNA in intact cells.The third pathway of PAH activation is the NAD(P ϩ )-dependent oxidation of PAH-trans-dihydrodiols to PAH oquinones catalyzed by dihydrodiol dehydrogenase members of the aldo-keto reductase (AKR) superfamily (10). AKRs divert PAH trans-dihydrodiols to form ketols that spontaneously rearrange to catechols (Scheme 1). The catechols undergo two one-electron oxidation events to produce the corresponding redox-active and electrophilic o-quinones. PAH o-quinones can form stable and depurinating DNA adducts in vitro (11,12), and these adducts may provide a route to G-to-T transversion mutations.In the presence of NAD(P)H, PAH o-quinones also undergo nonenzymatic reduction back to catechols. This event establishes futile redox cycles, which amplify the generation of reactive oxygen species (ROS) at the expense of NADPH and may lead to a prooxidant cellular state. Because a prooxidant state has been associated with tumor initiation and promotion (13), the AKR pathway of PAH activation is attractive in that it could explain how PAHs act as complete carcinogens. In addition, ROS may cause oxidative DNA damage such as 7,8-dihydro-8-oxo-2Ј-deoxyguanosine (8-oxo-dGuo) lesions, which can lead to G-to-T transversions (14). Amplification of ROS by catechol-oquinone interconversion has...

Benzo [a]pyrene (B[a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically activated by three enzymatic pathways; by peroxidases (e.g. cytochrome P450-peroxidase) to yield radical cations; by P4501A1/1B1 monoxygenation plus epoxide hydrolase to yield diol-epoxides; and by P4501A1/1B1 monoxygenation, epoxide hydrolase plus aldo-keto reductases (AKRs) to yield o-quinones. In humans, a major exposure site for environmental and tobacco smoke PAH is the lung, however, the profile of B[a]P metabolites formed at this site has not been well characterized. In this study, human bronchoalveolar H358 cells were exposed to B[a]P, and metabolites generated by peroxidase (B[a]P-1,6-and B[a]P-3,6-diones), from cytochrome P4501A1/1B1 monooxygenation (3-hydroxyl-B[a]P, B[a]P-7,8-and 9,10-trans-dihydrodiols, and B[a]P -r-7,t-8,t-9,c-10-tetrahydrotetrol (B[a]P -tetrol-1)), and from AKRs (B[a]P-7,8-dione) were detected and quantified by RP-HPLC-with in line photo-diode array and radiometric detection, and identified by LC-MS. Progress curves showed a lag-phase in the formation of 3-hydroxy-B[a]P, B[a]P-7,8-transdihydrodiol, B[a]P-tetraol-1 and B[a]P-7,8-dione over 24 h. Northern blot analysis showed that B [a]P induced P4501B1 and AKR1C isoforms in H358 cells in a time-dependent manner providing an explanation for the lag-phase. Pretreatment of H358 cells with 10 nM 2,3,7,8-tetrachlorodibenzop-dioxin, (TCDD) eliminated this lag-phase, but did not alter the levels of the individual metabolites observed, suggesting that both B[a]P and TCDD induction ultimately yield the same B[a]P-metabolic profile. The one exception was B[a]P-3,6-dione which was formed without a lag-phase in the absence and presence of TCDD, suggesting that the peroxidase responsible for its formation was neither P4501A1 nor 1B1. Candidate peroxidases that remain include PGH synthases and uninduced P450 isoforms. This study shows that the P4501A1/1B1 and AKR pathways are inducible in human lung cells and that the peroxidase pathway was not. It also provides evidence that each of the pathways of PAH-activation yield their distinctive metabolites in H358 human lung cells and that each pathway may contribute to the carcinogenic process.