Studies in gastric carcinogenesis. II. Absence of elevated concentrations of N-nitroso compounds in the gastric juice of Greek hypochlorhydric individuals

The concentrations of nitrate, nitrite, N-nitrate compoundsand bacteria were measured in 96 samples of fasting gastricjuice, pH 0.90–8.50, obtained from 56 individuals justbefore or at various times (8 days –1 year) after gastricoperation. The mean pH of the post-operative samples [4.66 ±0.39 (SEM)] was significantly higher than that of the pre-operativeones [3.29 ± 0.33 (SEM]. A positive correlation withpH was observed for the concentrations of total and nitrate-reducingbacteria (median values 5.0 x 10⁵ organisms/ml and 9.2 x 10⁴organisms/ml, respectively, for samples with pH6.5, and 1.2x 10³ organisms/ml and 0 organisms/ml, respectively, for sampleswith pH 2.5) and nitrite [mean values 22.5 ± 3.1 (SEM)µM and 3.20 ± 0.5 (SEM) uM for samples with pH6.5 and pH 2.5, respectively]. No correlation with pH was seenfor the concentrations of nitrate [mean value 0.48 ±0.06 (SEM) mM] or N-nitroso compounds [mean value 0.30 ±0.06 (SEM) µM]. The concentrations of bacteria and nitrite,although increased in hypochlorhydric individuals, were lowerthan those reported for corresponding individuals in other,primarily British, studies. It is suggested that the relativelylow concentrations of nitrite observed in our hypochlorohydricpopulation may account for the absence of elevated concentrationsof N-nitroso compounds and that the latter phenomenon may berelated to the relatively low frequency of gastric cancer inGreece.     

Coexposure to ethanol with N-nitrosodimethylamine or 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone during lactation of rats: marked increase in O(6)-methylguanine-DNA adducts in maternal mammary gland and in suckling lung and kidney

Use of alcoholic beverages increases risk of cancer at several target sites, including the breast. Of several possible mechanisms for this effect, competitive inhibition by ethanol of hepatic clearance of nitrosamines, resulting in increased dose delivery to posthepatic tissues, gives the quantitatively most pronounced enhancement. We investigated whether this effect would pertain to the mammary gland, and to ethanol and nitrosamines delivered translactationally to sucklings. Ethanol (1.6 g/kg) was administered by gavage to nursing Sprague-Dawley rats 10 min before 5 mg/kg N-nitrosodimethylamine (NDMA) or 50 mg/kg 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK); treatment was on postnatal days 1, 7, or 14. Tissues taken 4 h later for analysis of O(6)-methylguanine in DNA were liver, blood, and mammary glands from the mothers, and liver, lung, kidney, and blood from the sucklings. Ethanol cotreatment resulted in a marked, 10-fold increase in O(6)-methylguanine adducts from NDMA in mammary gland, as well as smaller but significant increases in this tissue from NNK and in maternal blood cells from both chemicals; adducts in maternal liver decreased slightly. In the sucklings, ethanol cotreatment also lowered adducts in liver after NDMA or NNK treatment. After NDMA, adducts were also detected in suckling lung and kidney and were increased five- to 10-fold after ethanol coexposure. Adducts from either chemical, with or without ethanol, decreased markedly in all suckling tissues with development from postnatal day 1 to day 14. Thus ethanol coexposure with nitrosamines increases O(6)-methylguanine DNA adducts in mammary gland and strongly influences adduct formation in suckling tissues after translactational delivery.     

Investigations of the DNA-damaging activity of human gastric juice

Human gastric juice previously treated with nitrite was examined for its ability to cause O6-alkylguanine-type modifications to 2'-deoxyguanosine or DNA in vitro. Analysis by radioimmunoassay indicated that, in five out of ten cases, incubation with 5 mM 2'-deoxyguanosine resulted in the formation of 375-1350 fmol/ml O6-ethyl-2'-deoxyguanosine (O6-etdGuo) or, in one case, 110 pmol/ml O6-methyl-2'-deoxyguanosine O6-medGuo). When gastric juice-treated calf-thymus DNA was examined for its ability to consume (through suicide repair of O6-alkylguanine-type damage) O6-alkylguanine-DNA alkyltransferase (AAT) from rat liver, eight out of eight samples could not. However, in four out of eight cases, a reduction in the rate of removal of O6-[3H]methylguanine from a 3H-methylated DNA substrate was observed. This finding is compatible with the presence, in gastric juice-treated DNA, of damage capable of binding to, but not undergoing repair by, the AAT.     

Studies in gastric carcinogenesis. IV. O6-Methylguanine and its repair in normal and atrophic biopsy specimens of human gastric mucosa. Correlation of O6-alkylguanine-DNA alkyltransferase activities in gastric mucosa and circulating lymphocytes

DNA extracted from biopsies of normal or atrophic gastric mucosaobtained from 20 individuals was analysed for the presence ofthe precarcinogenic alkylation lesion O⁶-methylguanine by arecently developed, highly sensitive assay based on repair bythe Escherichia coli O⁶-alkylguanine-DNA alkyltransferase (AGT)enzyme in competition with a radiolabelled oligonucleotide containingO⁶-methylguanine (O⁶-meG). With a limit of detection of 0.5fmol O⁶-meG in 10 µg DNA, only one DNA sample (derivedfrom a region of the stomach with advanced chronic atrophicgastritis) was found marginally positive, containing 0.52 fmol/10µg DNA (8.3 x 10^-8 mol O⁶-meG/mol guanine). Measurementsof AGT in 49 biopsies of normal, atrophic, hyperplastic or dysplasticmucosa obtained from the gastric antrum or corpus of 18 individualsdid not reveal any significant effects of mucosal Histologyon AGT. The average AGT value found was 6.9 ± 3.5 (SD)fmol/µg DNA, which is lower than the values reported fora number of other human tissues (liver, small intestine andlung). Measurement of AGT levels in gastric mucosa and circulatinglymphocytes of the same individuals revealed a psitive correlation(P < 0.005), suggesting that lymphocytes may serve as a usefulsurrogate marker for AGT activity in gastric mucosa in studiesof the epidemiology of this important repair enzyme.     

Detection and quantitation of benzo[a]pyrene-derived DNA adducts in mouse liver by liquid chromatography-tandem mass spectrometry: comparison with 32P-postlabeling

The polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P) is a proven animal carcinogen that is potentially carcinogenic to humans. B[a]P is an ubiquitous environmental pollutant and is also present in tobacco smoke, coal tar, automobile exhaust emissions, and charred food. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using electrospray ionization and selected reaction monitoring (SRM) has been developed for the detection of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE-N(2)dG) adducts formed in DNA following the metabolic activation of B[a]P to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE). The method involves enzymatic digestion of the DNA sample to 2'-deoxynucleosides following the addition of a stable isotope internal standard, [(15)N(5)]B[a]PDE-N(2)dG, and then solid phase extraction to remove unmodified 2'-deoxynucleosides prior to analysis by LC-MS/MS SRM. The limit of detection of the method was 10 fmol (approximately 3 B[a]PDE-N(2)dG adducts per 10(8) 2'-deoxynucleosides) using 100 microg of calf thymus DNA as the matrix. Calf thymus DNA reacted with B[a]PDE in vitro and mouse liver DNA samples at different time points following dosing intraperitoneally with 50, 100, and 200 mg/kg B[a]P was analyzed. Three stereoisomers of the B[a]PDE-N(2)dG adduct were detected following the reaction of calf thymus DNA with B[a]PDE in vitro. The levels of B[a]PDE-N(2)dG DNA adducts in the mice livers were found to increase in a dose-dependent manner with adducts reaching maximal levels at 1-3 days and then gradually decreasing over time but still detectable after 28 days. A very good correlation (r = 0.962, p < 0.001) was observed between the results obtained for the mouse liver DNA samples using LC-MS/MS SRM as compared to those obtained using a (32)P-postlabeling method. However, the levels of adducts observed following (32)P-postlabeling using butanol enrichment were approximately 3.7-fold lower. The LC-MS/MS method allowed the more precise quantitation of DNA adduct levels that were structurally characterized, in addition to a reduction in the time taken to perform the analysis when compared with the (32)P-postlabeling method.     

Polar, functionalized guanine-O6 derivatives resistant to repair by O6-alkylguanine-DNA alkyltransferase: implications for the design of DNA-modifying drugs

The protein O6-alkylguanine-DNA alkyltransferase (Atase) is responsible for the repair of DNA lesions generated by several clinically important anti-cancer drugs; this is manifest as active resistance in those cancer cell lines proficient in Atase expression. Novel O6-substituted guanine analogues have been synthesized, bearing acidic, basic and hydrogen bonding functional groups. In contrast to existing O6-modified purine analogues, such as methyl or benzyl, the new compounds were found to resist repair by Atase even when tested at concentrations much higher than O6-benzylguanine, a well-established Atase substrate active both in vitro and in vivo. The inactivity of the new purines as covalent substrates for Atase indicates that agents to deliver these groups to DNA would represent a new class of DNA-modifying drug that circumvents Atase-mediated resistance.     

Interactions between CYP1A1 polymorphisms and exposure to environmental tobacco smoke in the modulation of lymphocyte bulky DNA adducts and chromosomal aberrations

CYP1A1 plays an important role in the metabolic activation of polycyclic aromatic hydrocarbons (PAH), carcinogenic components of air pollution. The influence of CYP1A1 genotype (*2A, *2B and *4) on the levels of lymphocyte bulky DNA adducts and the frequency of cells with aberrant chromosomes was assessed in 194 non-smoking subjects in whom recent exposure to environmental tobacco smoke (ETS) and airborne particulate-associated PAH were measured during two consecutive seasons (winter and summer). While CYP1A1*4 had no consistent effect on either biomarker of genetic damage, the levels of both biomarkers responded in a parallel fashion to changes in exposure/CYP1A1*2A genotype combinations during both seasons. Specifically, the levels of both biomarkers were increased in carriers of at least one CYP1A1*2A allele, as compared with CYP1A1*1 homozygotes, in subjects with ETS exposures >0.8 h/day during the previous 4 days and mean personal exposure to benzo[a]pyrene <0.9 ng/m3 during the previous 24 h (all P < 0.05). Outside these exposure limits the differential effect in CYP1A1*2A variants was lost. Although the numbers of subjects with the CYP1A1*2B polymorphism was small, the same trend appeared to be followed in this case. These effects are interpreted as resulting from differential induction of CYP1A1 expression in CYP1A1*2A and CYP1A1*2A/*2B carriers by components of ETS-polluted air at levels of exposure readily suffered by large segments of the general population and suggest that subjects with these genotypes may have increased susceptibility to the genotoxic effects of ETS.