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...

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and procarcinogens that require activation by host metabolism. Metabolic activation of PAHs by AldoKeto-Reductases (AKRs) leads to formation of reactive and redox active o-quinones, which may cause oxidatively generated DNA damage. Spectrophotometric assays showed that NADPH caused PAH o-quinones to enter futile redox-cycles, which result in the depletion of excess cofactor. Copper (II) amplified NADPH-dependent redox-cycling of the o-quinones. Concurrent with NADPH oxidation, molecular oxygen was consumed, indicating the production of ROS. To determine whether PAH o-quinones can cause 8-oxo-dGuo formation in salmon testis DNA, three pre-requisite experimental conditions were satisfied. Quantitative complete enzymatic hydrolysis of DNA was achieved, adventitious oxidation of dGuo was eliminated by the use of chelex and desferal, and basal levels of less than 2.0 8-oxo-dGuo/10 5 dGuo were obtained. The HPLC-ECD analytical method was validated by spiking the DNA with standard 8-oxo-dGuo and demonstrating quantitative recovery. HPLC-ECD analysis revealed that in the presence of NADPH and Cu(II), submicromolar concentrations of PAH o-quinones generated > 60.0 8-oxo-dGuo adducts/10 5 dGuo. The rank order of 8-oxo-dGuo generated in isolated DNA was NP-1,2-dione > BA-3,4-dione > 7,12-DMBA-3,4-dione > BP-7,8-dione. The formation of 8-oxo-dGuo by PAH o-quinones was concentrationdependent. It was completely or partially inhibited when catalase, tiron or a Cu(I) specific chelator, bathocuproine were added, indicating the requirement for H 2 O 2 , O 2 − and Cu(I), respectively. Methional which is a copper-hydroperoxo complex (Cu(I)OOH) scavenger also suppressed 8-oxodGuo formation. By contrast, mannitol, sodium benzoate and sodium formate, which act as hydroxyl radical scavengers, did not block its formation. Sodium azide which can act as both a hydroxyl radical and 1 O 2 scavenger abolished the formation of 8-oxo-dGuo. These data showed that the production of 8-oxo-dGuo was dependent on Cu(II) /Cu(I) catalyzed redox cycling of PAH o-quinones to

Reactive and redox-active polycyclic aromatic hydrocarbon (PAH) o-quinones produced by Aldo-Keto Reductases (AKRs) have the potential to cause depurinating adducts leading to the formation of abasic sites and oxidative base lesions. The aldehyde reactive probe (ARP) was used to detect these lesions in calf thymus DNA treated with three PAH o-quinones (BP-7,8-dione, 7,12-DMBA-3,4-dione, and BA-3,4-dione) in the absence and presence of redox-cycling conditions. In the absence of redox-cycling, a modest amount of abasic sites were detected indicating the formation of a low level of covalent o-quinone depurinating adducts (>3.2 x 10(6) dNs). In the presence of NADPH and CuCl2, the three PAH o-quinones increased the formation of abasic sites due to ROS-derived lesions destabilizing the N-glycosidic bond. The predominant source of AP sites, however, was revealed by coupling the assay with human 8-oxoguanine glycosylase (hOGG1) treatment, showing that 8-oxo-dGuo was the major lesion caused by PAH o-quinones. The levels of 8-oxo-dGuo formation were independently validated by HPLC-ECD analysis. Apyrimidinic sites were also revealed by coupling the assay with Escherichia coli (Endo III) treatment showing that oxidized pyrimidines were formed, but to a lesser extent. Different mechanisms were responsible for the formation of the oxidative lesions depending on whether Cu(II) or Fe(III) was used in the redox-cycling conditions. In the presence of Cu(II)-mediated PAH o-quinone redox-cycling, catalase completely suppressed the formation of the lesions, but mannitol and sodium benzoate were without effect. By contrast, sodium azide, which acts as a *OH and 1O2 scavenger, inhibited the formation of all oxidative lesions, suggesting that the ROS responsible was 1O2. However, in the presence of Fe(III)-mediated PAH o-quinone redox-cycling, the *OH radical scavengers and sodium azide consistently attenuated their formation, indicating that the ROS responsible was *OH.

Polycyclic aromatic hydrocarbon (PAH)oPolycyclic aromatic hydrocarbons (PAHs) 2 are ubiquitous environmental pollutants that include over 200 compounds with two or more fused benzene rings. PAHs are formed as a result of incomplete combustion of fossil fuels (e.g. coal and oil) and are present in car and diesel exhaust and smoked or charbroiled food (1-3). They are also found in cigarette smoke condensate and tobacco products and are suspect agents in the causation of human lung cancer (4, 5). PAHs must be metabolically activated to reactive genotoxins to cause their mutagenic and carcinogenic effects.Two major metabolic activation pathways are possible starting from the proximate PAH carcinogen (Ϫ)B[a]P-7,8-transdihydrodiol (Fig. 1). The P4501A1/1B1 pathway converts (Ϫ)B[a]P-7,8-trans-dihydrodiol to yield (ϩ)-anti-7,8-dihydroxy-9␣,10␤-epoxy-7,8,9,10-tetrahydroB[a]P (6 -8). This diol epoxide forms stable N 2 -2Ј-deoxyguanosine (dGuo) adducts in vitro and in vivo (9, 10) and leads to mutation in H-ras (11) and may account for mutations in "hot spots" in p53 observed in lung cancer (12). The G to T transversions most often observed in these genes might arise because of the action of one or more trans-lesional by-pass DNA polymerases that read through stable diol-epoxide DNA adducts with low processivity and fidelity (13,14).As an alternative, human aldo-keto reductases (AKR1A1 and AKR1C1-AKR1C4) catalyze the NADP ϩ -dependent oxidation of (Ϯ)B[a]P-7,8-trans-dihydrodiol to produce the electrophilic and redox-active B[a] P-7,8-dione (15, 16). In this pathway, AKRs convert B[a]P-7,8-trans-dihydrodiol to form a ketol that rearranges to a catechol. The catechol then undergoes two subsequent one-electron oxidations to yield the fully oxidized o-quinone. Once formed, B[a]P-7,8-dione amplifies reactive oxygen species (ROS) by entering futile redox cycles that deplete cellular reducing equivalents (e.g. NADPH) (17). PAH * This work was supported, in whole or in part, by National Institutes of Health Grants RO1-CA39504 and P30-ES013508 (to T. M. P.) and RO1-CA130038 (to I. A. B.

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Since Korean mistletoe (Viscum album) has been used for alleviating metabolic diseases, it may also prevent the impairment of energy, glucose, lipid, and bone metabolisms in an estrogen-deficient animal model. We determined that long-term consumption of Korean mistletoe water extract (KME) can alleviate menopausal symptoms such as hot flush, increased abdominal fat mass, dyslipidemia, hyperglycemia, and decreased bone mineral density in ovariectomized (OVX) rats fed a high-fat diet, and explored the mechanisms of the effects. OVX rats were divided into four groups and fed high-fat diets supplemented with either 0.6% dextrin (control), 0.2% lyophilized KME + 0.4% dextrin (KME-L), or 0.6% lyophilized KME (KME-H). Sham rats were fed with the high-fat diets with 0.6% dextrin as a normal-control without estrogen deficiency. After eight weeks, OVX rats exhibited impaired energy, glucose and lipid metabolism, and decreased uterine and bone masses. KME-L did not alleviate energy dysfunction. However, KME-H lowered serum levels of total-, LDL-cholesterol, and triglycerides and elevated serum HDL-cholesterol levels in OVX rats with dyslipidemia, to similar levels as normal-control rats. Furthermore, KME-H improved HOMA-IR, an indicator of insulin resistance, in OVX rats. Surprisingly, KME-H fed rats had greater lean mass in the abdomen and leg without differences in fat mass but neither dosage of KME altered bone mineral density in the lumbar spine and femur. The increased lean mass was related to greater phosphorylation of mTOR and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) in the quadriceps muscles. Hepatic triglyceride contents were lowered with KME-H in OVX rats by increasing carnitine palmitoyltransferase-1 (CPT-1) expression and decreasing fatty acid synthase (FAS) and sterol regulatory element-binding protein-1c (SREBP-1c) expression. In conclusion, KME may be useful for preventing some menopausal symptoms such as hot flushes, dyslipidemia, hepatic steatosis, and loss of muscle mass in post-menopausal women.

PAHs (polycyclic aromatic hydrocarbons) are suspect lung cancer carcinogens that must be metabolically converted into DNA-reactive metabolites. P4501A1/P4501B1 plus epoxide hydrolase activate PAH to (+/-)- anti-benzo[ a]pyrene diol epoxide ((+/-)- anti-BPDE), which causes bulky DNA adducts. Alternatively, aldo-keto reductases (AKRs) convert intermediate PAH trans-dihydrodiols to o-quinones, which cause DNA damage by generating reactive oxygen species (ROS). In lung cancer, the types or pattern of mutations in p53 are predominantly G to T transversions. The locations of these mutations form a distinct spectrum characterized by single point mutations in a number of hotspots located in the DNA binding domain. One route to the G to T transversions is via oxidative DNA damage. An RP-HPLC-ECD assay was used to detect the formation of 8-oxo-dGuo in p53 cDNA exposed to representative quinones, BP-7,8-dione, BA-3,4-dione, and DMBA-3,4-dione under redox cycling conditions. Concurrently, a yeast reporter system was used to detect mutations in the same cDNA samples. Nanomolar concentrations of PAH o-quinones generated 8-oxo-dGuo (detected by HPLC-ECD) in a concentration dependent manner that correlated in a linear fashion with mutagenic frequency. By contrast, micromolar concentrations of (+/-)- anti-BPDE generated (+)- trans- anti-BPDE-N (2)-dGuo adducts (detected by stable-isotope dilution LC/MS methodology) in p53 cDNA that correlated in a linear fashion with mutagenic frequency, but no 8-oxo-dGuo was detected. Previous studies found that mutations observed with PAH o-quinones were predominately G to T transversions and those observed with (+/-)- anti-BPDE were predominately G to C transversions. However, mutations at guanine bases observed with either PAH-treatment occurred randomly throughout the DNA-binding domain of p53. Here, we find that when the mutants were screened for dominance, the dominant mutations clustered at or near hotspots primarily at the protein-DNA interface, whereas the recessive mutations are scattered throughout the DNA binding domain without resembling the spectra observed in cancer. These observations, if extended to mammalian cells, suggest that mutagenesis can drive the pattern of mutations but that biological selection for dominant mutations drives the spectrum of mutations observed in p53 in lung cancer.