Analysis of DNA Adducts and Mutagenic Potency and Specificity in Rats Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in multiple organs/tissues of rats by unresolved mechanisms. For this report, evidence for ACN-induced direct/indirect DNA damage and mutagenesis was investigated by assessing the ability of ACN, or its reactive metabolite, 2-cyanoethylene oxide (CEO), to bind to DNA in vitro, to form select DNA adducts [N7-(2'-oxoethyl)guanine, N2,3-ethenoguanine, 1,N6-ethenodeoxyadenosine, and 3,N4-ethenodeoxycytidine] in vitro and/or in vivo, and to perturb the frequency and spectra of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene in rats exposed to ACN in drinking water. Adducts and frequencies and spectra of Hprt mutations were analyzed using published methods. Treatment of DNA from human TK6 lymphoblastoid cells with [2,3-14C]-CEO produced dose-dependent binding of 14C-CEO equivalents, and treatment of DNA from control rat brain/liver with CEO induced dose-related formation of N7-(2'-oxoethyl)guanine. No etheno-DNA adducts were detected in target tissues (brain and forestomach) or nontarget tissues (liver and spleen) in rats exposed to 0, 3, 10, 33, 100, or 300 ppm ACN for up to 105 days or to 0 or 500 ppm ACN for ∼15 months; whereas N7-(2'-oxoethyl)guanine was consistently measured at nonsignificant concentrations near the assay detection limit only in liver of animals exposed to 300 or 500 ppm ACN for ≥2 weeks. Significant dose-related increases in Hprt mutant frequencies occurred in T-lymphocytes from spleens of rats exposed to 33-500 ppm ACN for 4 weeks. Comparisons of "mutagenic potency estimates" for control rats versus rats exposed to 500 ppm ACN for 4 weeks to analogous data from rats/mice treated at a similar age with N-ethyl-N-nitrosourea or 1,3-butadiene suggest that ACN has relatively limited mutagenic effects in rats. Considerable overlap between the sites and types of mutations in ACN-exposed rats and butadiene-exposed rats/mice, but not controls, provides evidence that the carcinogenicity of these epoxide-forming chemicals involves corresponding mutagenic mechanisms.

Interindividual Differences in DNA Adduct Formation and Detoxification of 1,3-Butadiene-Derived Epoxide in Human HapMap Cell Lines.

Smoking-induced lung cancer is a major cause of cancer mortality in the US and worldwide. While 11-24% of smokers will develop lung cancer, risk varies among individuals and ethnic/racial groups. Specifically, African American and Native Hawaiian cigarette smokers are more likely to get lung cancer as compared to Caucasians, Japanese Americans, and Latinos. It is important to identify smokers who are at the greatest risk of developing lung cancer as they should be candidates for smoking cessation and chemopreventive intervention programs. Among 60+ tobacco smoke carcinogens, 1,3-butadiene (BD) is one of the most potent and abundant (20-75 µg per cigarette in mainstream smoke and 205-361 µg per cigarette in side stream smoke). BD is metabolically activated to 3,4-epoxy-1-butene (EB), which can be detoxified by glutathione S-transferase theta 1 (GSTT1)-mediated conjugation with glutathione, or can react with DNA to form 7-(1-hydroxy-3-buten-2-yl)guanine (EB-GII) adducts. In the present study, we employed EBV-transformed human lymphoblastoid cell lines (HapMap cells) with known GSTT1 genotypes to examine the influence of the GSTT1 gene on interindividual variability in butadiene metabolism, DNA adduct formation/repair, and biological outcomes (apoptosis). We found that GSTT1- HapMap cells treated with EB in culture produced lower levels of glutathione conjugates and were more susceptible to apoptosis but had similar numbers of EB-GII adducts as GSTT1+ cells. Our results suggest that GSTT1 can influence an individual's susceptibility to butadiene-derived epoxides.

DNA Adduct Formation in the Lungs and Brain of Rats Exposed to Low Concentrations of [13C2]-Acetaldehyde.

Air pollution is a major environmental risk for human health. Acetaldehyde is present in tobacco smoke and vehicle exhaust. In this study, we show that [13C2]-acetaldehyde induces DNA modification with the formation of isotopically labeled 1, N2-propano-2'-deoxyguanosine adducts in the brain and lungs of rats exposed to concentrations of acetaldehyde found in the atmosphere of megacities. The adduct, with the addition of two molecules of isotopically labeled acetaldehyde [13C4]-1, N2-propano-dGuo, was detected in the lung and brain tissues of exposed rats by micro-HPLC/MS/MS. Structural confirmation of the products was unequivocally performed by nano-LC/ESI+-HRMS3 analyses. DNA modifications induced by acetaldehyde have been regarded as a key factor in the mechanism of mutagenesis and may be involved in the cancer risks associated with air pollution.

Effects of Black Raspberry Extract and Berry Compounds on Repair of DNA Damage and Mutagenesis Induced by Chemical and Physical Agents in Human Oral Leukoplakia and Rat Oral Fibroblasts.

Black raspberries (BRB) have been shown to inhibit carcinogenesis in a number of systems, with most studies focusing on progression. Previously we reported that an anthocyanin-enriched black raspberry extract (BE) enhanced repair of dibenzo-[a,l]-pyrene dihydrodiol (DBP-diol)-induced DNA adducts and inhibited DBP-diol and DBP-diolepoxide (DBPDE)-induced mutagenesis in a lacI rat oral fibroblast cell line, suggesting a role for BRB in the inhibition of initiation of carcinogenesis. Here we extend this work to protection by BE against DNA adduct formation induced by dibenzo-[a,l]-pyrene (DBP) in a human oral leukoplakia cell line (MSK) and to a second carcinogen, UV light. Treatment of MSK cells with DBP and DBPDE led to a dose-dependent increase in DBP-DNA adducts. Treatment of MSK cells with BE after addition of DBP reduced levels of adducts relative to cells treated with DBP alone, and treatment of rat oral fibroblasts with BE after addition of DBPDE inhibited mutagenesis. These observations showed that BE affected repair of DNA adducts and not metabolism of DBP. As a proof of principle we also tested aglycones of two anthocyanins commonly found in berries, delphinidin chloride and pelargonidin chloride. Delphinidin chloride reduced DBP-DNA adduct levels in MSK cells, while PGA did not. These results suggested that certain anthocyanins can enhance repair of bulky DNA adducts. As DBP and its metabolites induced formation of bulky DNA adducts, we investigated the effects of BE on genotoxic effects of a second carcinogen that induces bulky DNA damage, UV light. UV irradiation produced a dose-dependent increase in cyclobutanepyrimidine dimer levels in MSK cells, and post-UV treatment with BE resulted in lower cyclobutanepyrimidine dimer levels. Post-UV treatment of the rat lacI cells with BE reduced UV-induced mutagenesis. Taken together, the results demonstrate that BE extract reduces bulky DNA damage and mutagenesis and support a role for BRB in the inhibition of initiation of carcinogenesis.

Tribbles 2 mediates cisplatin sensitivity and DNA damage response in epithelial ovarian cancer.

Aim was to identify methylated genes with functional involvement in cisplatin-resistance development of epithelial ovarian cancer (EOC). Genome-wide analyses of hypermethylated CpG-islands in resistant cell lines in combination with qRT-PCR analyses were used to identify epigenetically silenced genes. EOC-Type-II tumors were analyzed for gene methylation and expression and TCGA data were interrogated in-silico. Experiments revealed 37 commonly hypermethylated genes in resistant cells of which Tribbles 2 (TRIB2) showed the most pronounced downregulation on mRNA level and was characterized further. TRIB2 showed a reactivation after 5'-Aza-Cytidine treatment in resistant cells but a cisplatin-dependent, prominent upregulation on mRNA level in sensitive cells, only. Re-expression in resistant A2780 cells increased the sensitivity to cisplatin and other DNA-damaging agents, but not taxanes. Contrary, knockdown of TRIB2 increased resistance to cisplatin in sensitive cells. TRIB2 was involved in the induction of a cisplatin-dependent cell cycle arrest and apoptosis by influencing p21 and survivin expression. An increased Pt-DNA-adduct formation in TRIB2 re-expressing cells did not translate in higher levels of dsDNA damage (yH2AX-foci). Thus, TRIB2 is potentially involved in the signal transduction from nucleotide excision repair of intrastrand cross links. Importantly, patient stratification of two homogenous cohorts of EOC-Type-II patients from Jena (n = 38) and the TCGA (n = 149) by TRIB2 mRNA expression consistently revealed a significantly decreased PFS for patients with low TRIB2 levels (log-rank p < 0.05). Tumors from resistant patients expressed the lowest levels of TRIB2. Downregulation of TRIB2 contributes to platin-resistance and TRIB2 expression should be validated as prognostic and predictive marker for EOC.

AFB1 hepatocarcinogenesis is via lipid peroxidation that inhibits DNA repair, sensitizes mutation susceptibility and induces aldehyde-DNA adducts at p53 mutational hotspot codon 249.

Aflatoxin B1 (AFB1) contamination in the food chain is a major cause of hepatocellular carcinoma (HCC). More than 60% of AFB1 related HCC carry p53 codon 249 mutations but the causal mechanism remains unclear. We found that 1) AFB1 induces two types of DNA adducts in human hepatocytes, AFB1-8,9-epoxide-deoxyguanosine (AFB1-E-dG) induced by AFB1-E and cyclic α-methyl-γ-hydroxy-1,N2-propano-dG (meth-OH-PdG) induced by lipid peroxidation generated acetaldehyde (Acet) and crotonaldehyde (Cro); 2) the level of meth-OH-PdG is >30 fold higher than the level of AFB1-E-dG; 3) AFB1, Acet, and Cro, but not AFB1-E, preferentially induce DNA damage at codon 249; 4) methylation at -CpG- sites enhances meth-OH-PdG formation at codon 249; and 5) repair of meth-OH-PdG at codon 249 is poor. AFB1, Acet, and Cro can also inhibit DNA repair and enhance hepatocyte mutational sensitivity. We propose that AFB1-induced lipid peroxidation generated aldehydes contribute greatly to hepatocarcinogenesis and that sequence specificity of meth-OH-PdG formation and repair shape the codon 249 mutational hotspot.

4-Hydroxynonenal metabolites and adducts in pre-carcinogenic conditions and cancer.

4-hydroxy-2-nonenal (HNE) is an amazing reactive compound, originating from lipid peroxidation within cells but also in food and considered as a "second messenger" of oxidative stress. Due to its chemical features, HNE is able to make covalent links with DNA, proteins and lipids. The aim of this review is to give a comprehensive summary of the chemical properties of HNE and of the consequences of its reactivity in relation to cancer development. The formation of exocyclic etheno-and propano-adducts and genotoxic effects are addressed. The adduction to cellular proteins and the repercussions on the regulation of cell signaling pathways involved in cancer development are reviewed, notably on the Nrf2/Keap1/ARE pathway. The metabolic pathways leading to the inactivation/elimination or, on the contrary, to the bioactivation of HNE are considered. A special focus is given on the link between HNE and colorectal cancer development, due to its occurrence in foodstuffs and in the digestive lumen, during digestion.

Glycoconjugated Site-Selective DNA-Methylating Agent Targeting Glucose Transporters on Glioma Cells.

DNA-alkylating drugs continue to remain an important weapon in the arsenal against cancers. However, they typically suffer from several shortcomings because of the indiscriminate DNA damage that they cause and their inability to specifically target cancer cells. We have developed a strategy for overcoming the deficiencies in current DNA-alkylating chemotherapy drugs by designing a site-specific DNA-methylating agent that can target cancer cells because of its selective uptake via glucose transporters, which are overexpressed in most cancers. The design features of the molecule, its synthesis, its reactivity with DNA, and its toxicity in human glioblastoma cells are reported here. In this molecule, a glucosamine unit, which can facilitate uptake via glucose transporters, is conjugated to one end of a bispyrrole triamide unit, which is known to bind to the minor groove of DNA at A/T-rich regions. A methyl sulfonate moiety is tethered to the other end of the bispyrrole unit to serve as a DNA-methylating agent. This molecule produces exclusively N3-methyladenine adducts upon reaction with DNA and is an order of magnitude more toxic to treatment resistant human glioblastoma cells than streptozotocin is, a Food and Drug Administration-approved, glycoconjugated DNA-methylating drug. Cellular uptake studies using a fluorescent analogue of our molecule provide evidence of uptake via glucose transporters and localization within the nucleus of cells. These results demonstrate the feasibility of our strategy for developing more potent anticancer chemotherapeutics, while minimizing common side effects resulting from off-target damage.

Pre-steady-state kinetic investigation of bypass of a bulky guanine lesion by human Y-family DNA polymerases.

3-Nitrobenzanthrone (3-NBA), a byproduct of diesel exhaust, is highly present in the environment and poses a significant health risk. Exposure to 3-NBA results in formation of N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA), a bulky DNA lesion that is of particular importance due to its mutagenic and carcinogenic potential. If not repaired or bypassed during genomic replication, dGC8-N-ABA can stall replication forks, leading to senescence and cell death. Here we used pre-steady-state kinetic methods to determine which of the four human Y-family DNA polymerases (hPolη, hPolκ, hPolι, or hRev1) are able to catalyze translesion synthesis of dGC8-N-ABAin vitro. Our studies demonstrated that hPolη and hPolκ most efficiently bypassed a site-specifically placed dGC8-N-ABA lesion, making them good candidates for catalyzing translesion synthesis (TLS) of this bulky lesion in vivo. Consistently, our publication (Biochemistry 53, 5323-31) in 2014 has shown that small interfering RNA-mediated knockdown of hPolη and hPolκ in HEK293T cells significantly reduces the efficiency of TLS of dGC8-N-ABA. In contrast, hPolι and hRev1 were severely stalled by dGC8-N-ABA and their potential role in vivo was discussed. Subsequently, we determined the kinetic parameters for correct and incorrect nucleotide incorporation catalyzed by hPolη at various positions upstream, opposite, and downstream from dGC8-N-ABA. Notably, nucleotide incorporation efficiency and fidelity both decreased significantly during dGC8-N-ABA bypass and the subsequent extension step, leading to polymerase pausing and error-prone DNA synthesis by hPolη. Furthermore, hPolη displayed nucleotide concentration-dependent biphasic kinetics at the two polymerase pause sites, suggesting that multiple enzyme•DNA complexes likely exist during nucleotide incorporation.

The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells.

Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD>KO>WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N'-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were more sensitive to the cytotoxicity of 4-nitroquinoline N-oxide, styrene oxide, cisplatin, methyl methanesulfonate, and ethyl methanesulfonate than WT cells. However, the KO cells displayed sensitivity camptothecin, etoposide, bleomycin, hydroxyurea, crotonealdehyde, and methylglyoxal in a manner similar to the WT cells. Our results suggest that Pol ζ plays an important role in the protection of human cells by carrying out TLS across bulky DNA adducts and cross-links, but has no or limited role in the protection against strand-breaks in DNA.

Y-family DNA polymerase-independent gap-filling translesion synthesis across aristolochic acid-derived adenine adducts in mouse cells.

Translesion DNA synthesis (TLS) operates when replicative polymerases are blocked by DNA lesions. To investigate the mechanism of mammalian TLS, we employed a plasmid bearing a single 7-(deoxyadenosine-N6-yl)-aristolactam I (dA-AL-I) adduct, which is generated by the human carcinogen, aristolochic acid I, and genetically engineered mouse embryonic fibroblasts. This lesion induces A to T transversions at a high frequency. The simultaneous knockouts of the Polh, Poli and Polk genes did not influence the TLS efficiency or the coding property of dA-AL-I, indicating that an unknown DNA polymerase(s) can efficiently catalyze the insertion of a nucleotide opposite the adduct and subsequent extension. Similarly, knockout of the Rev1 gene did not significantly affect TLS. However, knockout of the Rev3l gene, coding for the catalytic subunit of polζ, drastically suppressed TLS and abolished dA-AL-I to T transversions. The results support the idea that Rev1 is not essential for the cellular TLS functions of polζ in mammalian cells. Furthermore, the frequency of dA-AL-I to T transversion was affected by a sequence context, suggesting that TLS, at least in part, contributes to the formation of mutational hot and cold spots observed in aristolochic acid-induced cancers.

Translesion Synthesis of the N(2)-2'-Deoxyguanosine Adduct of the Dietary Mutagen IQ in Human Cells: Error-Free Replication by DNA Polymerase κ and Mutagenic Bypass by DNA Polymerases η, ζ, and Rev1.

Translesion synthesis (TLS) of the N(2)-2'-deoxyguanosine (dG-N(2)-IQ) adduct of the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated in human embryonic kidney 293T cells by replicating plasmid constructs in which the adduct was individually placed at each guanine (G1, G2, or G3) of the NarI sequence (5'-CG1G2CG3CC-3'). TLS efficiency was 38%, 29%, and 25% for the dG-N(2)-IQ located at G1, G2, and G3, respectively, which suggests that dG-N(2)-IQ is bypassed more efficiently by one or more DNA polymerases at G1 than at either G2 or G3. TLS efficiency was decreased 8-35% in cells with knockdown of pol η, pol κ, pol ι, pol ζ, or Rev1. Up to 75% reduction in TLS occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N(2)-IQ bypass. Mutation frequencies (MFs) of dG-N(2)-IQ at G1, G2, and G3 were 23%, 17%, and 11%, respectively, exhibiting a completely reverse trend of the previously reported MF of the C8-dG adduct of IQ (dG-C8-IQ), which is most mutagenic at G3 ( ( 2015 ) Nucleic Acids Res. 43 , 8340 - 8351 ). The major type of mutation induced by dG-N(2)-IQ was targeted G → T, as was reported for dG-C8-IQ. In each site, knockdown of pol κ resulted in an increase in MF, whereas MF was reduced when pol η, pol ι, pol ζ, or Rev1 was knocked down. The reduction in MF was most pronounced when pol η, pol ζ, and Rev1 were simultaneously knocked down and especially when the adduct was located at G3, where MF was reduced by 90%. We conclude that pol κ predominantly performs error-free TLS of the dG-N(2)-IQ adduct, whereas pols η, pol ζ, and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ζ and κ showed that the former was inefficient in full-length primer extension on dG-N(2)-IQ templates, whereas the latter was efficient in both error-free and error-prone extensions. We believe that the observed differences between the in vitro experiments using purified DNA polymerases, and the cellular results may arise from several factors including the crucial roles played by the accessory proteins in TLS.

Mutational signature of aristolochic acid: Clue to the recognition of a global disease.

Mutational signatures associated with specific forms of DNA damage have been identified in several forms of human cancer. Such signatures provide information regarding mechanisms of tumor induction which, in turn, can reduce exposure to carcinogens by shaping public health policy. Using a molecular epidemiologic approach that takes advantage of recent advances in genome sequencing while applying sensitive and specific analytical methods to characterize DNA damage, it has become increasingly possible to establish causative linkages between certain environmental mutagens and disease risk. In this perspective, we use aristolochic acid, a human carcinogen and nephrotoxin found in Aristolochia herbs, to illustrate the power and effectiveness of this multidisciplinary approach. The genome-wide mutational signature for this toxin, detected initially in cancers of the upper urinary tract, has subsequently been associated with cancers of the liver and kidney. These findings have significant implications for global public health, especially in China, where millions of individuals have used Aristolochia herbal remedies as part of traditional Chinese medicine and, thus, are at risk of developing aristolochic acid nephropathy and/or upper urinary tract carcinomas. The studies reported here set the stage for research into prevention and early detection, both of which will be required to manage a potentially devastating global disease.

Induction of cytochromes P450 1A1 and 1A2 suppresses formation of DNA adducts by carcinogenic aristolochic acid I in rats in vivo.

Aristolochic acid I (AAI) is a natural plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. One of the most efficient enzymes reductively activating AAI to species forming AAI-DNA adducts is cytosolic NAD(P)H: quinone oxidoreductase 1. AAI is also either reductively activated or oxidatively detoxified to 8-hydroxyaristolochic acid (AAIa) by microsomal cytochrome P450 (CYP) 1A1 and 1A2. Here, we investigated which of these two opposing CYP1A1/2-catalyzed reactions prevails in AAI metabolism in vivo. The formation of AAI-DNA adducts was analyzed in liver, kidney and lung of rats treated with AAI, Sudan I, a potent inducer of CYP1A1/2, or AAI after pretreatment with Sudan I. Compared to rats treated with AAI alone, levels of AAI-DNA adducts determined by the (32)P-postlabeling method were lower in liver, kidney and lung of rats treated with AAI after Sudan I. The induction of CYP1A1/2 by Sudan I increased AAI detoxification to its O-demethylated metabolite AAIa, thereby reducing the actual amount of AAI available for reductive activation. This subsequently resulted in lower AAI-DNA adduct levels in the rat in vivo. Our results demonstrate that CYP1A1/2-mediated oxidative detoxification of AAI is the predominant role of these enzymes in rats in vivo, thereby suppressing levels of AAI-DNA adducts.

DNA adducts induced by in vitro activation of extracts of diesel and biodiesel exhaust particles.

CONTEXT: Biodiesel and biodiesel-blend fuels offer a renewable alternative to petroleum diesel, but few data are available concerning the carcinogenic potential of biodiesel exhausts. OBJECTIVES: We compared the formation of covalent DNA adducts by the in vitro metabolic activation of organic extracts of diesel-exhaust particles (DEP) from petroleum diesel and soy biodiesel and correlated DNA adduct levels and mutagenicity in Salmonella TA100. METHODS: We examined two different DEP from petroleum diesel (C-DEP and B0), one from soy bean oil biodiesel (B100) and one from combustion of a blend of 20% B100 and 80% B0 (B20) for in vitro DNA adduct-forming potential under oxidative or nitroreductive conditions in the presence of calf thymus DNA as well as in vivo in Salmonella TA100. The modified DNA was hydrolyzed and analyzed by (32)P-postlabeling using either butanol extraction or nuclease P1 pre-enrichment. RESULTS: Multiple DNA adducts were produced with chromatographic mobilities consistent with PAH and nitro-PAH adducts. The types and quantities of DNA adducts produced by the two independent petroleum diesel DEP were similar, with both polycyclic aromatic hydrocarbon (PAH)- and nitro-PAH-derived adducts formed. Relative potencies for S9-mediated DNA adduct formation, either per mass of particulate or per MJ(th) energy consumed were B100 > B0 > B20. CONCLUSIONS: Soy biodiesel emissions induced DNA damage in the form of presumptive PAH and nitro-PAH DNA adducts that correlated with mutagenicity in Salmonella. B20 is the soy biodiesel used most commonly in the US, and it produced the lowest DNA adduct-emission factor, ∼50% that of petroleum diesel.

Nucleotides with altered hydrogen bonding capacities impede human DNA polymerase η by reducing synthesis in the presence of the major cisplatin DNA adduct.

Human DNA polymerase η (hPol η) contributes to anticancer drug resistance by catalyzing the replicative bypass of DNA adducts formed by the widely used chemotherapeutic agent cis-diamminedichloroplatinum (cisplatin). A chemical basis for overcoming bypass-associated resistance requires greater knowledge of how small molecules influence the hPol η-catalyzed bypass of DNA adducts. In this study, we demonstrated how synthetic nucleoside triphosphates act as hPol η substrates and characterized their influence on hPol η-mediated DNA synthesis over unmodified and platinated DNA. The single nucleotide incorporation efficiency of the altered nucleotides varied by more than 10-fold and the higher incorporation rates appeared to be attributable to the presence of an additional hydrogen bond between incoming dNTP and templating base. Finally, full-length DNA synthesis in the presence of increasing concentrations of synthetic nucleotides reduced the amount of DNA product independent of the template, representing the first example of hPol η inhibition in the presence of a platinated DNA template.

Metformin inhibits 7,12-dimethylbenz[a]anthracene-induced breast carcinogenesis and adduct formation in human breast cells by inhibiting the cytochrome P4501A1/aryl hydrocarbon receptor signaling pathway.

Recent studies have established that metformin (MET), an oral anti-diabetic drug, possesses antioxidant activity and is effective against different types of cancer in several carcinogen-induced animal models and cell lines. However, whether MET can protect against breast cancer has not been reported before. Therefore, the overall objectives of the present study are to elucidate the potential chemopreventive effect of MET in non-cancerous human breast MCF10A cells and explore the underlying mechanism involved, specifically the role of cytochrome P4501A1 (CYP1A1)/aryl hydrocarbon receptor (AhR) pathway. Transformation of the MCF10A cells into initiated breast cancer cells with DNA adduct formation was conducted using 7,12-dimethylbenz[a]anthracene (DMBA), an AhR ligand. The chemopreventive effect of MET against DMBA-induced breast carcinogenesis was evidenced by the capability of MET to restore the induction of the mRNA levels of basic excision repair genes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1), and the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). Interestingly, the inhibition of DMBA-induced DNA adduct formation was associated with proportional decrease in CYP1A1 and in NAD(P)H: quinone oxidoreductase 1 (NQO1) gene expression. Mechanistically, the involvements of AhR and nuclear factor erythroid 2-related factor-2 (Nrf2) in the MET-mediated inhibition of DMBA-induced CYP1A1 and NQO1 gene expression were evidenced by the ability of MET to inhibit DMBA-induced xenobiotic responsive element and antioxidant responsive element luciferase reporter gene expression which suggests an AhR- and Nrf2-dependent transcriptional control. However, the inability of MET to bind to AhR suggests that MET is not an AhR ligand. In conclusion, the present work shows a strong evidence that MET inhibits the DMBA-mediated carcinogenicity and adduct formation by inhibiting the expression of CYP1A1 through an AhR ligand-independent mechanism.

The shifting perception on antioxidants: the case of vitamin E and ß-carotene.

Antioxidants are vital for aerobic life, and for decades the expectations of antioxidants as health promoting agents were very high. However, relatively recent meta-analyses of clinical studies show that supplementation of antioxidants does not result in the presumed health benefit, but is associated with increased mortality. The dilemma that still needs to be solved is: what are antioxidants in the end, healthy or toxic? We have evaluated this dilemma by examining the presumed health effects of two individual antioxidants with opposite images i.e. the "poisonous" ß-carotene and the "wholesome" vitamin E and focused on one aspect, namely their role in inducing BPDE-DNA adducts. It appears that both antioxidants promote DNA adduct formation indirectly by inhibition of the protective enzyme glutathione-S-transferase π (GST π). Despite their opposite image, both antioxidants display a similar type of toxicity. It is concluded that, in the appreciation of antioxidants, first their benefits should be identified and substantiated by elucidating their molecular mechanism. Subsequently, the risks should be identified including the molecular mechanism. The optimal benefit-risk ratio has to be determined for each antioxidant and each individual separately, also considering the dose.

Prevalence of visual impairment in El Salvador: inequalities in educational level and occupational status / Prevalencia de deficiencia visual en El Salvador: desigualdades según el nivel educativo y la situación laboral

OBJECTIVE: To examine the prevalence of blindness, visual impairment, and related eye diseases and conditions among adults in El Salvador, and to explore socioeconomic inequalities in their prevalence by education level and occupational status, stratified by sex. METHODS: Based upon the Rapid Assessment of Avoidable Blindness (RAAB) methodology, this nationwide sample comprised 3 800 participants (3 399 examined) ≥ 50 years old from 76 randomly selected clusters of 50 persons each. The prevalence of blindness, visual impairment and related eye diseases and conditions, including uncorrected refractive error (URE), was calculated for categories of education level and occupational status. Multiple logistic regression models were fitted to calculate odds ratios (ORs) and 95% confidence intervals (CIs) and stratified by sex. RESULTS: Age-adjusted prevalence was 2.4% (95% CI: 2.2-2.6) for blindness (men: 2.8% (95% CI: 2.5-3.1); women: 2.2% (95% CI: 1.9-2.5)) and 11.8% (95% CI: 11.6-12.0) for moderate visual impairment (men: 10.8% (95% CI: 10.5-11.1); women: 12.6% (95% CI: 12.4-12.8)). The proportion of visual impairment due to cataract was 43.8% in men and 33.5% in women. Inverse gradients of socioeconomic inequalities were observed in the prevalence of visual impairment. For example, the age-adjusted OR (AOR) was 3.4 (95% CI: 2.0-6.4) for visual impairment and 4.3 (95% CI: 2.1-10.4) for related URE in illiterate women compared to those with secondary education, and 1.9 (95% CI: 1.1-3.1) in cataract in unemployed men. CONCLUSIONS: Blindness and visual impairment prevalence is high in the El Salvador adult population. The main associated conditions are cataract and URE, two treatable conditions. As socioeconomic and gender inequalities in ocular health may herald discrimination and important barriers to accessing affordable, good-quality, and timely health care services, prioritization of public eye health care and disability policies should be put in place, particularly among women, the unemployed, and uneducated people.

OBJETIVO: Analizar la prevalencia de la ceguera, la deficiencia visual, y las enfermedades y afecciones oculares relacionadas en adultos de El Salvador, y explorar las desigualdades socioeconómicas en cuanto a su prevalencia según el nivel educativo y la situación laboral, estratificados por sexos. MÉTODOS: Se adoptó el método de Evaluación Rápida de la Ceguera Evitable, y se escogió una muestra a escala nacional de 3 800 participantes (de ellos se examinaron 3 399) de 50 años de edad o mayores, pertenecientes a 76 agrupamientos seleccionados aleatoriamente y constituidos por 50 personas cada uno. Se calculó la prevalencia de la ceguera, la deficiencia visual y las enfermedades y afecciones oculares relacionadas, incluido el error de refracción no corregido, según las diferentes categorías de nivel educativo y situación laboral. Se emplearon modelos de regresión logística múltiple para calcular las razones de posibilidades (OR) y los intervalos de confianza (IC) de 95%, y se estratificaron por sexos. RESULTADOS: La prevalencia ajustada por edad fue de 2,4% (IC de 95%: 2,2-2,6) para la ceguera (hombres: 2,8% [IC de 95%: 2,5-3,1]; mujeres: 2,2% [IC de 95%: 1,9-2,5]) y de 11,8% (IC de 95%: 11,6-12,0) para la deficiencia visual moderada (hombres: 10,8% [IC de 95%: 10,5-11,1]; mujeres: 12,6% [IC de 95%: 12,4-12,8]). La proporción de deficiencias visuales debidas a catarata fue de 43,8% en los hombres y de 33,5% en las mujeres. En la prevalencia de la deficiencia visual se observaron gradientes inversos de desigualdades socioeconómicas. Por ejemplo, la OR ajustada por edad fue de 3,4 (IC de 95%: 2,0-6,4) para la deficiencia visual y de 4,3 (IC de 95%: 2,1-10,4) para el error de refracción no corregido relacionado en las mujeres analfabetas, en comparación con las que tenían un nivel de educación secundaria, y fue de 1,9 (IC de 95%: 1,1-3,1) para la catarata en los hombres desempleados. CONCLUSIONES: La prevalencia de ceguera y deficiencia visual es alta en la población adulta de El Salvador. Las principales afecciones asociadas son la catarata y el error de refracción no corregido, ambas tratables. Puesto que las desigualdades socioeconómicas y de género en materia de salud ocular pueden ser indicativas de discriminación y de la existencia de barreras importantes para obtener acceso a servicios de atención de salud asequibles, de buena calidad y oportunos, es preciso dar prioridad a la atención oftalmológica pública y a las políticas dirigidas a corregir la discapacidad, en particular en las mujeres y en las personas desempleadas y sin formación.

In vivo detection of a novel endogenous etheno-DNA adduct derived from arachidonic acid and the effects of antioxidants on its formation.

Previous studies showed that 7-(1',2'-dihydroxyheptyl)-substituted etheno DNA adducts are products of reactions with the epoxide of (E)-4-hydroxy-2-nonenal, an oxidation product of ω-6 polyunsaturated fatty acids (PUFAs). In this work, we report the detection of 7-(1',2'-dihydroxyheptyl)-1,N(6)-ethenodeoxyadenosine (DHHedA) in rodent and human tissues by two independent methods: a (32)P-postlabeling/HPLC method and an isotope dilution liquid chromatography-electrospray ionization-tandem mass spectrometry method, demonstrating for the first time that DHHedA is a background DNA lesion in vivo. We showed that DHHedA can be formed upon incubation of arachidonic acid with deoxyadenosine, supporting the notion that ω-6 PUFAs are the endogenous source of DHHedA formation. Because cyclic adducts are derived from the oxidation of PUFAs, we subsequently examined the effects of antioxidants, α-lipoic acid, Polyphenon E, and vitamin E, on the formation of DHHedA and γ-hydroxy-1,N(2)-propanodeoxyguanosine (γ-OHPdG), a widely studied acrolein-derived adduct arising from oxidized PUFAs, in the livers of Long Evans Cinnamon (LEC) rats. LEC rats are afflicted with elevated lipid peroxidation and prone to the development of hepatocellular carcinomas. The results showed that although the survival of LEC rats was increased significantly by α-lipoic acid, none of the antioxidants inhibited the formation of DHHedA, and only Polyphenon E decreased the formation of γ-OHPdG. In contrast, vitamin E caused a significant increase in the formation of both γ-OHPdG and DHHedA in the livers of LEC rats.