Polymorphisms of XRCC1 and XRCC2 DNA Repair Genes and Interaction with Environmental Factors Influence the Risk of Nasopharyngeal Carcinoma in Northeast India

Multiple genetic and environmental factors have been reported to play key role in the development of nasopharyngeal carcinoma (NPC). Here, we investigated interactions of XRCC1 Arg399Gln and XRCC2 Arg188His polymorphisms and environmental factors in modulating susceptibility to NPC in Northeast India. One-hundred NPC patients, 90 first-degree relatives of patients and 120 controls were enrolled in the study. XRCC1 Arg399Gln and XRCC2 Arg188His polymorphisms were determined using PCR-RFLP, and the results were confirmed by DNA sequencing. Logistic regression (LR) and multifactor dimensionality reduction (MDR) approaches were applied for statistical analysis. The XRCC1 Gln/Gln genotype showed increased risk (OR=2.76; P<0.024) of NPC. However, individuals with both XRCC1 and XRCC2 polymorphic variants had 3.2 fold elevated risk (P<0.041). An enhanced risk of NPC was also observed in smoked meat (OR=4.07; P=0.004) and fermented fish consumers (OR=4.34, P=0.001), and tobacco-betel quid chewers (OR=7.00; P=0.0001) carrying XRCC1 polymorphic variants. However, smokers carrying defective XRCC1 gene showed the highest risk (OR = 7.47; P<0.0001). On MDR analysis, the best model for NPC risk was the five-factor model combination of XRCC1 variant genotype, fermented fish, smoked meat, smoking and chewing (CVC=10/10; TBA=0.636; P<0.0001); whereas in interaction entropy graphs, smoked meat and tobacco chewing showed synergistic interactions with XRCC1. These findings suggest that interaction of genetic and environmental factors might increase susceptibility to NPC in Northeast Indian populations.     

Effect of herbicide 2,4,5-trichlorophenoxyethanol (TCPE) containing dioxin on mutation and induction of sister chromatid exchanges

Previous studies have indicated that both herbicide 2,4,5-trichlorophenoxyethanol(TCPE) and its contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) enhance liver tumor incidence in male Swiss mice in adosedependent manner. In this report the mutagenicity of TCPE(containing 0.1 p.p.m. TCDD) in the Salmonella/microsome assaywas studied, and the effect of this herbicide on the colony-formingability and on the frequency of sister chromatid exchange (SCE)in Chinese hamster cells were determined. TCPE (1–500µg/plate) appeared non-mutagenic in strains TA 1535, TA1537, TA 1538, TA 98 and TA 100 either in the absence or presenceof S-9 mix from male Wistar rat, male Swiss mouse and male hamsterliver pre-treated by Aroclor 1254, using the standard plateincorporation and the pre-incubation assay. Male mouse urine,after a single administration of TCPE to the animal, also provedto be non-mutagenic in strains TA 98 and TA 100. In mammaliancell systems, however, TCPE directly induced a dose relatedincrease in SCE frequency. The dose required to double the controlSCE frequency was in the slightly toxic range of the survivalcurve for TCPE. The cytotoxicity was diminished both in bacterialand mammalian systems in the presence of S-9 mix from male Swissmouse liver. This S-9 mix strongly reduced SCE frequency inducedby TCPE. The induction of SCE observed cannot be attributedto the contaminant TCDD alone, because the SCE production wasnot influenced by the appropriate low concentration of pureTCDD.     

Evaluation of Risk Factors for Nasopharyngeal Carcinoma in a High-risk Area of India,the Northeastern Region

Northeastern India is a major nasopharyngeal carcinoma (NPC) high risk-area although the rest of thecountry has very low incidence. A case-control study of 105 NPC cases and 115 controls was conducted toidentify the potential risk factors for NPC development in this region. Information was collected by interviewerabout socio-demographic characteristics, cigarette smoking, alcohol consumption, dietary history, occupationalhistory, and a family history of cancer. Epstein-Barr viral load was assayed from the blood DNA by real timePCR. Associations between GSTs genotypes, cytochrome P450 family including CYP1A1, CYP2E1 and CYP2A6polymorphisms and susceptibility to relationship between the diseases were studied using PCR-RFLP assay.Results indicate that Epstein-Barr virus load was significantly higher in patients compared to controls (p<0.0001).Furthermore, concentration of blood EBV-DNA was significantly higher in advanced stage disease (Stage III andIV) than in early stage disease (Stage I and II) (p<0.05). Presence of CYP2A6 variants that reduced the enzymeactivity was significantly less frequent in cases than controls. Smoked meat consumption, exposure to smoke,living in poorly ventilated house and alcohol consumption were associated with NPC development among thepopulation of Northeastern India. Thus, overall our study revealed that EBV viral load and genetic polymorphismof CYP2A6 along with living practices which include smoked meat consumption, exposure to smoke, living inpoorly ventilated houses and alcohol consumption are the potential risk factors of NPC in north eastern region ofIndia. Understanding of the risk factors and their role in the etiology of NPC are helpful forpreventive measuresand screening.     

Mutagenicity of smoke condensate of bidi--an indigenous cigarette of India

The possible mutagenicity of bidi smoke condensate (BSC), anindigenous form of an Indian cigarette has been studied usingthree short-term test systems. It was seen that BSC caused frame-shiftmutations in Salmonella typhimurium strains TA 98 and TA 1538and required metabolic activation. In the mammalian test systems,BSC induced 8 azaguanine resistant mutations in V79 Chinesehamster cells in the presence of S9 mixture and induced elevatedfrequencies of micronucleated erythrocytes in the bone marrowof Swiss mice.     

Major routes of metabolism of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the rat

The metabolic fate of 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline(MeIQx), a carcinogen formed in cooked meat and fish, has beeninvestigated in male Sprague-Dawley rats. Five metabolites wererecovered from bile of animals given an intragastric dose of{2-¹⁴C]MeIQx. These accounted for nearly all of the radioactivityin bile. The chemical structures of these metabolites were elucidatedby proton NMR, UV and mass spectroscopy. Three structures maybe assigned unambiguously: two sulfamates, N-(3,8.dimethylimidazo[4,5f]quinoxalin-2-yl)sulfamic acid and N-(8-hydroxymethyl-3-methylimidazo[4,5f]quinoxalin-2-yl)sulfamic acid, and N-(8-one glucuronide, N²(ß-1-glucosiduronyl)-2-amino-3,8-dimelhyliinidazo[4,5f]quinoxaline In addition, an acetyl and a glucosiduronylconjugate of 5-hydroxy-MeIQx were observed. The spectral evidencedid not allow an unambiguous assignment of the site of conjugation.The two glucuronides were excreted in urine and the sulfamateof MeIQx was found in feces as well as urine. All five metaboliteswere found to be non-mutagenic to Salmonella typhimurium TA98with or without metabolic activation. The glucuronide conjugateswere found also to be non-mutagenic when ß- glucuronidasewas incorporated with S-9 mixture in the mutation assay, andthus all appear to be detoxification products. The previouslyreported metabolite, 2-amino-8-hydroxymethyl-3-methylimidazo[4,5f]quinoxalinewhich is mutagenic to Salmonella typhimurium TA98 with metabolicactivation, was identified as a minor component in both urineand feces.     

Formaldehyde as a possible mutagenic metabolite of N-nitrodimethylamine and of other agents which are suggested to yield non-alkylating species in vitro

N-Nitramines are biologically active compounds of environmentalsignificance. In this study the suggested bioactivation of N-nitrodimethylaminevia oxidation at the methyl-group was confirmed, as was indicatedby formaldehyde liberation. N-Nitrodimethylamine and formaldehydeas well as the suggested metabolites, N-nitrohydroxymethylethylamineand N-nitromethylamine were tested for mutagenicity in histidineauxotrophic Salmonella typhimurium strains in a variety of conditions.N-Nitrodimethylamine was mutagenic only in S. typhimurium TA100 after pre-incubation with bacteria and a complete metabolizingmixture containing 9000 g liver supernatant and NADPH-regeneratingcofactors. N-Nitrohydroxymethylmethylamine and formaldehydewere approximately equally mutagenic without the metabolizingmixture in TA 100 and TA 98, but not in TA 1535. The additionof the 9000 g supernatant of homogenized liver increased theyield of his⁺ revertants induced by the two compounds. N-Nitromethylaminewas not mutagenic with or without the metabolic activation system.The results suggest that formaldehyde Is possibly the mutagenicallyactive intermediate formed during in vitro metabolism of N-nitrodimethylamine.Furthermore the participation of formaldehyde as the mutagenicintermediate of other non-alkylating N-nitro and N-nitroso compoundsis demonstrated.     

Urinary excretion of mutagens in coke oven workers

The influence of occupational exposure to polycyclic aromatichydrocarbons (PAHs) on urinary mutagenic activity was assessedin 75 coke oven workers, using a highly sensitive bacterialmutagen technique (extraction with C18 resin and liquid micro-preincubationtest on strain TA98 of Salmonella typhimurium in the presenceof metabolizing and deconjugating enzymes). Exposure to PAHswas assessed according to the urinary excretion of 1-pyrenol;the main confounding factors were checked by the number of cigarettessmoked per day and the levels of nicotine and its metabolitesin urine, or by ascertaining whether recommended dietary restrictionshad been followed. Of the 20 urine samples which turned outto be positive (producing at least double the number of spontaneousrevertants), 19 (95%) belonged to smokers. Only one non-smokerhad obvious urinary mutagenic activity, and was highly exposedoccupationally to PAHs (urinary 1-pyrenol of 3.930 µmol/molof creatinine). Of the five urine samples from subjects whohad not followed the recommended diet, two (40%) were clearlymutagenic. Multiple regression analysis (n = 67) showed thatthe presence of samples positive for urinary mutagenic activitydepended only on smoking habits, if this confounding factorwas assessed according to the number of cigarettes smoked perday, while the significant influence of exposure to PAH couldbe shown when the confounding factor was objectively estimatedaccording to the urinary levels of nicotine and its metabolites.Assessment of the mutagenic potency of urinary extracts (netrevertants/mmol creatinine) confirmed the strong influence ofsmoking habits on urinary mutagenic activity (all smokers 2156± 2691 versus non-smokers 939 ± 947 net revertants/mmolcreatinine; Mann-Whitney test: P < 0.01). In smokers highlyexposed to PAHs, greater excretion of mutagens with respectto low-exposure smokers was revealed (3548 ± 4009 versus1552 ± 1227 net revertants/mmol creatinine; Mann-Whitneytest: P < 0.01). Multiple regression analysis showed thatthe mutagenic potency of urinary extracts of coke oven workersdepended on exposure to PAHs, tobacco smoking habits, and consumptionof fried, grilled or barbecued meat. Increased urinary mutagenicactivity strengthens epidemiological evidence of the increasedrisk of renal and urinary tract tumours in these workers. Thepresence of mutagenic metabolites in urine as a result of occupationalexposure to PAH may be demonstrated only by using highly sensitivetechniques for assessing urinary mutagenic activity in studieswhich include careful checking of the main confounding factors.     

The synthesis and mutagenicity of the N-formyl analog of N-hydroxyphenacetin

The synthesis and purification of N-hydroxy-N-formyl-p-phenetidine(N-OH-FP) is described. This new compound was subjected to mutagenicitytesting using Salmonella typhimurium strains TA98, TA100, TA1535,TA1537 and TA1538 both in the presence and absence of the post-mitochondrialfraction of rat liver homogenate. Simultaneous mutagenicitytesting of the known phenacetin metabolite, N-hydroxy-phenacetin(N-OH-AP), was conducted with the same tester strains. The N-formylderived hydroxamic acid (N-OH-FP) was found to be a much strongermutagen than N-hydroxy-phenacetin (N-OH-AP). Furthermore, N-OH-FPalso behaved as a direct-acting mutagen unlike N-OH-AP. Thechemical stabilities of N-OH-AP and N-OH-FP were studied inphosphate buffer in the pH range of 3–8; and both thehydroxamic acids were found to be stable to the conditions employed.The results of this study support the hypothesis that enzymaticdeacylation is an activation process for the expression of mutagenicityby hydroxamic acids.     

Mutagenicity of N-hydroxy-2-acetylaminofluorene and N-hydroxy-phenacetin and their respective deacetylated metabolites in nitroreductase deficient Salmonella TA98FR and TA100FR

The mutagenicity of N-hydroxy-2-acetylaminofluorene and N-hydroxyphenacetinand their respective deacetylated metabolites, N-hydroxy-2-aminofluoreneand 2-nitrosofluorene, and N-hydroxyphenetidine and p-nitrosophenetolewas determined in nitroreductase deficient Salmonella testerstrains TA 98FR and TA100FR. The mutagenicity of N-hydroxy-2-acetylaminofluorenemediated by either rat liver microsomes or rat liver 105 000g supernatant fractions was no different in either TA98 (nitroreductaseproficient) or TA98FR (nitroreductase deficient). Similarlythe mutagenicity of N-hydroxyphenacetin mediated by hamsterliver microsomes was not affected by either the presence orabsence of nitroreductase activity in TA100. N-Hydroxy-2-aminofluoreneand 2-nitrosofluorene were equipotent direct acting mutagensin both TA98 and TA98FR, as were both N-hydroxyphenetidine andp-nitrosophenetole in TA100 and TA 100FR. Ascorbate (5 mM) andNADPH (1 mM) had no significant effect on the mutagenidty ofeither N-hydroxy-2-acetylaminofluorene, N-hydroxy-2-aminofluorene,or 2-nitrosofluorene in TA98 or TA98FR whereas ascorbate andNADPH markedly inhibited the mutagenicity of both N-hydroxyphenetidineand p-nitrosophenetole in both TA100 and TA100FR. Ascorbateappears to be inhibiting the mutagenicity of N-hydroxyphenetidineand p-nitrosophenetole as a result of the nonenzymatic chemicalreduction of these compounds to non-mutagenic derivatives.     

Levels of mutagens in the urine of smokers of black and blond tobacco correlate with their risk of bladder cancer

Levels of urinary mutagens, thioethers, N-nitrosamino acids,nitrate, nicotine, cotinine and creatinine were measured in21 non-smokers, 26 smokers of blond tobacco, 9 smokers of blacktobacco and 5 smokers of both types of tobacco, all eating asimilar diet. Results were expressed either per 24 h urine orper mmol creatinine. The sum of urinary nicotine and cotininelevels (N+C) was used as a measure of exposure to the numberof cigarettes smoked. Statistically significant positive dose-effectrelationships were obtained between the urinary N+C levels and(i) the number of revertants (Salmonella typhimurium TA98, witha metabolic activation system); (ii) the concentration of thioethers;(iii) the levels of N-nitrosoproline or the sum of all nitrosaminoacids excreted and (iv) the amount of urinary nitrate. No suchcorrelation was found between N+C levels and induction potencyin the SOS chromotest. A linear dose-effect relationship betweenurinary mutagenicity (i.e. log revertants of S.typhimurium TA98)and N+C levels or number of cigarettes per day was establishedfor smokers of blond tobacco. After adjustment for N+C levels,the urine of smokers of black tobacco contained twice as muchmutagenic material as did the urine of blond tobacco smokers(P = 0.02). For other exposure markers, no statistically significantdifference was found between the two types of smokers. Epidemiologicalstudies have shown that the risk of urinary bladder cancer is2.5 times higher in smokers of black tobacco than in blond tobacco.Therefore, our findings on urinary mutagenicity provide experimentalevidence that the type of tobacco is the factor responsiblefor the observed difference in risk and that smoking of blackas compared to blond tobacco results in a higher exposure ofthe urinary bladder to genotoxic hence potentially carcinogenicsubstances.     

DNA binding by [2, 5-14C]N7-nitrosopyrrolidine in excision-repair proficient and deficient strains of Salmonella. Evidence for a major premutagenic adduct

Little is known about the nature and possible genotoxic effectsof the DNA adducts formed by N-nitrosopyrrolidine (NPYR) inwhole animals. DNA binding in DNA isolated from [2, 5-¹⁴C]NPYR-treatedSalmonella was studied and attempts were made to monitor DNAadducts and correlated DNA binding with mutagenesis. NPYR wasmetabolized by hamster liver S-9 fraction in the presence ofS.typhimurium TA1535 (uvrB^–) or TA1975(uvrB⁺ DNA isolatedfrom TA1535 contained about three times as much radioactivityas that isolated from TA1975, and NPYR-induced mutagenesis wasseveral-fold higher in TA1535. The fraction of radioactivityincorporated into TA1535 was {small tilde}10^-5. Thermal hydrolysisof the ¹⁴C-containing DNA at neutral pH, followed by precipitation,released {small tilde}2/3 of the radioactivity into the supernatant.HPLC analysis of the supernatant revealed one major peak. Thispeak was absent in DNA from TA1975. Acid hydrolysis of the DNAprecipitate after neutral hydrolysis released most of the residualradioactivity. Several small peaks were observed after HPLCanalysis of the TA1535 acid hydrolysate or the TA1975 acid hydrolysate.These results demonstrate that NPYR is capable of binding toSalmonella DNA yielding one major product after hydrolysis andthis DNA binding product appears to be repaired by the excisionrepair system. The fact that the major peak of radioactivityreleased from Salmonella is only found in the strain which isefficiently reverted by NPYR suggests that mutagenesis is dependenton the DNA modification leading to this peak.     

Metabolic activation to a mutagen of 3-hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, a secondary metabolite of benzo[a]pyrene

3-Hydroxy-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (3-OH-BP-7,8-diol)wag isolated from arylsulfatase/ß-glucuronidase-treatedbile of rats to which 3-hydroxybenzo[a]pyrene (3-OH-BP) hasbeen administered. This triol was investigated for mutagenicityin Salmonella typhimurium (reversion to histidine prototrophyof strains TA 97, TA 98, TA 100 and TA 1537) and in V79 Chinesehamster cells (acquisition of resistance to 6-thioguanine).When no exogenous metabolizing system was added the triol wasinactive, while 3-OH-BP showed weak mutagenic effects with allfour bacterial strains. In the presence of NADPH-fortified postmitochondrialsupernatant fraction (S9 mix) of liver homogenate from Aroclor1254-treated rats, the mutagenicity of 3-OH-BP was potentiated,and the triol was activated to a mutagen(s). In the presenceof S9 mix, the triol was 5—18 times more mutagenic than3-OH-BP in strains TA 97, TA 100 and TA 1537, but both compoundsshowed similar mutagenic potencies with strain TA 98. Thesestrain differences strongly suggest that the mutagenicity of3-OH-BP in the S9 mix-mediated test was not exclusively dueto metabolites of 3-OH-BP-7, 8-diol. Trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-7,8-diol), like the triol, showed mutagenic effects onlyin the presence of S9 mix. Strain TA 1537 was reverted by thetriol but not by the diol. In the other bacterial strains thediol was more mutagenic than the triol, the difference in potencybeing largest in strain TA 100 (2.5-to 10-fold, depending onthe experimental conditions). In V79 cells, the diol was a potentmutagen, while the triol showed only very weak mutagenic effects.However the triol was more cytotoxic than the diol. High cytotoxicityof the triol was observed even in the absence of S9 mix. Theresults of the present study demonstrate that metabolites of3-OH-BP-7, 8-diol) are biologically-active derivatives of benzo[a]pyrene.Comparison of the mutagenic effectiveness in different bacterialstrains also reveals that metabolites of 3-OH-BP-7, 8-diol andof BP-7, 8-diol substantially differ in the kind of geneticalterations they evoke.     

The differential mutagenicity of isoniazid in fluctuation assays and Salmonella plate tests

The anti-tuberculostatic drug, isoniazid (INH) was evaluatedfor its mutagenic potential using Salmonella plate tests andfluctuation assays with various strains of bacteria, and differentmetabolic activation systems. In the Salmonella plate test INHproved to be a weak directly-acting base-substitution mutagenwhich was detoxified by S9-mix. S. typhimurium TA 1530 and TA1535 were the sensitive strains, and this result confirmed someof the published data. In the present studies mutagenic activitywas further diminished in the presence of larger concentrationsof rat liver S9-mix. Furthermore, the reduction in mutagenicactivity was observed with S9-mix derived from untreated, Aroclor1254-treated or phenobarbitone/ß-naphthoflavone treatedrats. In direct contrast, using the microtitre fluctuation assay,the mutagenic activity of INH was elevated in the presence ofrat liver S9-mix, and continued to increase with increasingS9-concentration. This result was obtained irrespective of theS9-source. S. typhimurium strains TA 1530, TA 1535 and his G46,and E. coli strains TA 85, TA 86 and WP2 uvrA were all sensitiveto the mutagenicity of INH after metabolic activation. The primarystep in the metabolic activation of INH in the fluctuation testwas mediated by a cytosolic enzyme, and the activity of dapsoneas a competitive substrate implicated the involvement of anN-acetyl transferase. The rapid diffusion of the cytosolic enzymeinto the basal agar layer, or the nonspecific binding of theenzyme (or the active mutagenic INH metabolite) to componentsof the agar, may explain the contradictory data obtained inthe Salmonella plate test. The modifying effects of agar onthe distribution of drug metabolising enzymes within liver S9fractions should be carefully considered when evaluating datafrom Salmonella plate tests.     

Dysfunction of mitochondria due to environmental carcinogens in nasopharyngeal carcinoma in the ethnic group of Northeast Indian population

Nasopharyngeal carcinoma (NPC) is a rare cancer worldwide, but in India, NPC is uncommon in its subcontinent except in the north-eastern part of the country. NPC is thought to be caused by the combined effects of environmental carcinogens, genetic susceptibility and Epstein-Barr virus (EBV). This is the first study that aimed to examine the selected risk factors, mostly dietary, viral environmental, metabolic gene polymorphisms, mitochondrial DNA (mtDNA) copy number variation and their risk, in subjects who are highly prone to NPC in the ethnic groups of Northeast India, which has included cases, first-degree relatives and controls. The cases and controls were selected from three ethnic groups (Manipuri, Naga and Mizo) of Northeast India with high prevalence of NPC. This case–control family study includes 64 NPC patients, 88 first-degree relatives and 100 controls having no history of cancer. PCR-based detection was done for EBV–latent membrane protein 1 (LMP1) gene and glutathione S-transferase Mu 1 (GSTM1)–glutathione S-transferase theta 1 (GSTT1) polymorphism. A comparative ΔCt method was used for the determination of mtDNA content. An increased risk of 2.00–6.06-folds to NPC was observed with those who intake smoked meat and fish, salted fish and fermented fish; betel nut chewers; tobacco smokers; alcohol drinkers; and those who have kitchen inside the living room, glutathione S-transferase null genotype and EBV infection. The risk of NPC increased in cases with decreased mtDNA copy number (P _trend?=?0.007). A significant difference between GST null genotypes and EBV infection with mtDNA content was found in the cases (P?<?0.0001). The understandings of environment–genetic risk factors and their role in the etiology of NPC are helpful as preventive measures and screening.     

Heterocyclic aromatic amine-DNA-adducts in bacteria and mammalian cells detected by 32P-postlabeling analysis

The formation of DNA adducts by the fried meat mutagen and carcinogen2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was studied bymeans of ³²P-postlabeling of DNA digests and four-directionalt.l.c. Three major and five minor adducts were detected in assaysof DNA digests obtained from Salmonella typhimurium TA98 cellsafter treatment with IQ and rat liver postmitochondrial supernatant(S9). A qualitatively identical adduct pattern was obtainedwith nitro-IQ (3-methyl-2-nitrounidazo[4,5-f]quinoline), a newanalogue of IQ with a nitro instead of the amino group. Thesetwo compounds, therefore, form the same ultimate metabolite.The same adduct pattern was also found after TA98/1,8-DNP⁶ (acetyltransferase-deficient)cells were treated with nitro-IQ; this is probably due to aresidual acetyltransferase activity in this strain. Upon treatmentof TA98 cells with 1 mM IQ for 3 h one adduct was detected in4.7 x 10⁵ total bases; a considerably higher adduct frequency,one in 4.2 x 10³, was induced by nitro-IQ (70 µM, 30 min).The IQ isomer 2-amino-1-methyliniidazo[4,5-f]quinoline (isoIQ)and its nitro-analogue nitro-isoIQ (1-methyl-2-nitroimidazo[4,5-f]-quinoline)also produced identical adducts. Their common adduct patternwas very similar to the IQ adduct pattern but was located ina position different from that of the IQ adduct pattern. DNAfrom Syrian hamster embryo (SHE) cells treated with IQ and S9exhibited adducts apparently identical with those of SalmonellaDNA.     

CARCINOGENSIS: Formation of DNA adducts by the co-mutagen norharman with aromatic amines

Norharman, widely distributed in our environment, is alone notmutagenic to Salmonella typhimurium TA98 and TA100 either withor without S9 mix, but becomes mutagenic to S. Typhimurium TA98with S9 mix when non-mutagenic aromatic amines like anilineor o- or m-toluidine are added. Thus norharman has been calleda ‘co-mutagen’. In the present study we examinedwhether or not DNA adducts are formed in DNA of S.typhimuriumTA98 by treatment with norharman and aromatic amines using ³²P-post-labelinganalysis under modified adduct intensification conditions. Whena sample of norharman (8 mg) and aniline (4 mg) was incubatedwith 4 ml of overnight culture of S.typhimurium TA98 in thepresence of 20 ml S9 mix for 6 h at 37°C, three adduct spotswere detected at a total relative adduct labeling (RAL) of 10.8± 2.27/10⁸ nucleotides. Under the same conditions, amixture of norharman (8 mg) and o-toluidine (4 mg) yielded threeadduct spots at a RAL of 3.74 ± 1.71/10⁸ nucleotides.With a combination of norharman and m-toluidine, a single adductspot was seen at a RAL of 0.04 ± 0.01/10⁸ nucleotides.In contrast, norharman with p-toluidine did not produce adductspots. Furthermore, neither norharman nor the aromatic aminesthemselves gave any evidence of adducts. Thus DNA adduct formationby norharman with aromatic amines correlates with the co-mutagenicaction of norharman in S.typhimurium TA98.     

Microsomal metabolism of 1-nitrobenzo[e] to a highly mutagenic K-region dihydrodiol

Aerobic metabolism of 1-nitrobenzo [e](1-nitro-BeP) by rat livermicrosomes produced 1-nitro-BeP trans-4,5-dihydrodiol, 6-hydroxy-1-nitro-BeP,and 8-hydroxy-1-nitro-BeP. When 3,3,3-trichioropropylene 1,2-oxidewas incorporated into the metabolism, 1-nitro-BeP 4,5-oxidewas the predominant metabolite, and 1-nitro-BeP trans-4,5-dlhydrodiolwas not detected. All of the metabolites were purified by bothreversed- and normal-phase HPLC and characterized by analysisof their mass and 500 MHz proton NMR spectral data. 1-Nitro-BePwas not metabolized under hypoxic conditions. 1-Nitro-BeP andits four metabolites were assayed in Salmonella typhimuriumtester strains TA98, TA98NR and TA98/1,8-DNP₆, both in the presenceand absence of S9 activation. As predicted, 1-nitro-BeP wasa weak mutagen without S9 (2 revertants/µg in TA98); theaddition of S9 resulted in {small tilde}18, 17 and 4 revertants/µin TA98, TA98NR and TA98/1,8-DNP₆ respectively. The two phenolicmetabolites were mutagenic both in the presence and absenceof S9, producing moderate responses (19–84 revertants/µg)In addition, while the 1-nitro-BeP 4,5-oxide was only weaklymutagenic in TA98 (6–14 revertants/µg), 1-nitro-BePtrans 4,5-dihydrodiol was unexpectedly potent ({small tilde}300revertants/µg both with and without S9). These resultsindicate that microsomal epoxidation of 1-nitro-BeP followedby epoxide hydrolase-catalyzed hydrolysis of the resulting epoxideto the 1-nitro-BeP trans-4,5-dihydrodiol results in the mostpotent mutagenic derivatives. The weak mutagenicity of 1-nitro-BeP4,5-oxide demonstrates that not all epoxides of nitrated polycyclic aromatic hydrocarbons (PAHs) are more mutagenic thanthe corresponding parent nitro-PAHs. Also, the lower S9-mediatedmutagenicity of 1-nitro-BeP in TA98/1,8-DNP₆ compared with TA98indicates that the mutagenicity of 1-nitro-BeP is dependentupon nitroreduction and transesterification. Finally, we previouslyhypothesized that nitrated PAHs with their nitro substituentsperpendicular or nearly perpendicular to the aromatic ringsare very weak or non-direct-acting mutagens in Salmonella typhimuriumtester strains. The results reported in this communication demonstratethat ring-oxidized derivatives of nitro-PAHs do not always followthis structure— mutagenicity correlation.     

Mutagenicity of dibromodulcitol (DBD), an alkylating anticancer drug) and its mono- and bifunctional conversion products studied by the Salmonella/microsome assay

Dibromodulcitol (DBD) and one of its most important bifunctionaltransformation products, dianhydrogalactitol (DAG) with similarcytostatic effect, were tested by the Salmonella/microsome assayon strains TA 1535, TA 1538, TA 98 and TA 100 using the plateincorporation technique. Both drugs were direct mutagens instrains TA 1535 and TA 100 and non-mutagenic in other strains.Their mutagenic effect was not influenced by S-9 mix from ratliver. Mutagenicity of DAG was very limited because of its markedtoxicity. The other monofunctional alkylating derivatives, i.e.,1-bromo-3, 6-anhydrodulcitol and 1,2-epoxy-3,6-anhydrodulcitolwere highly mutagenic in strains TA 1535 and TA 100 with andwithout S-9 mix despite having no anticancer effect. Anticanceractivity was exerted only by the bifunctional alkylating hexitols(DBD, DAG) which showed moderate mutagenic activity comparedto the monofunctional derivatives. No correlation could be establishedbetween the mutagenic and anticancer effect of the four structurallyrelated hexitols. Mutagenicity of urine and bile from rats aftera single administration of the maximum tolerated (450 mg/kg)dose of DBD was also examined, and the hexitol components ofthe same urine sample were identified by t.l.c. DBD and itsmutagenic transformation products were excreted in urine andnot through the bile. The mutagenic effect of DBD observed cannotbe attributed exclusively to DBD itself, because the parentmolecule, like other alkylating agents, easily undergoes spontaneousdecomposition under in vitro and in vivo conditions to releaseboth bi- and monofunctional alkylating solvolysis products andthese highly reactive derivatives may play a role in this effect.No significant difference in the relative mutagenicity was detectedbetween DBD and cyclophosphamide, used as a reference substance.     

Risk factors for nasopharyngeal carcinoma

In an effort to assess the relative importance of various risk factors of nasopharyngeal carcinoma (NPC), which includes antibodies to Epstein-Barr virus capsid antigen (anti-VCA) and early antigen (anti-EA) as well as other environmental factors, a multivariate logistic regression method was applied to analyze previously collected data from an epidemiologic study on 343 cases with NPC and 1017 neighborhood controls. Anti-VCA and anti-EA titers were found significantly associated with NPC. The relative risk increased with the increase of antibody titers. Individuals who smoked 30 or more cigarettes per day had more than 3.4 times higher risk than those who never smoked, while no increase in the risk was observed for those smoking less than 20 cigarettes per day and ex-smokers. Use of herb drugs, working under poor ventilation and nativity were also found to increase the NPC risk. In cases other than smoking 20 or more cigarettes per day and the frequent use of herb drugs, the synergistic interaction was not observed. In addition, male NPC individuals and Mainland Chinese were found to have relatively lower antibody titers as compared with female individuals or native Taiwanese.     

Effects of cytosol on mutagenesis induced by N-nitrosodimethylamine, N-nitrosomethylurea and {alpha}-acetoxy-N-nitrosodimethylamine in different strains of Salmonella: evidence for different ultimate mutagens from N-nitrosodimethylmine

N-Nitrosodimethylamine (NDMA), but not N-nitroso-N-methylurea(MNU) was more mutagenic in the Salmonella hisG428 strain, TA104,than in the hisG46 strain, TA100 in the presence of rat or hamsterliver S-9 mix. As both NDMA and MNU can give rise to methyldiazoniumion (MDI) it appears that NDMA can be metabolized to an additionalmutagen with a higher activity in TA104. The effects of UV anderror-prone repair on NDMA and MNU-induced mutagenesis in TA104were also different. -Acetoxy-NDMA, which gives rise to theNDMA metabolite, -hydroxy-NDMA, was more mutagenic in TA104than TA100, under certain conditions. Several metabolites ofNDMA (formaldehyde, 1,1-dimethylhydrazine and nitrite) werenot significantly mutagenic at the concentrations that couldhave been generated from NDMA. It was previously reported thatthe microsomal-mediated mutagenesis induced by NDMA is greatlyincreased by cytosol in TA104, but not in TA100. The currentstudy found that when cytosol was separated into a high anda low mol. wt fraction, neither greatly enhanced microsomal-mediatedmutagenesis by NDMA in TA104. Addition of NAD to the high, butnot the low mol. wt fraction resulted in greatly enhanced activationof NDMA to a mutagen in TA104. The enhancement by cytosol ofNDMA-induced mutagenesis in hisG428 was only observed when bothmicrosomes and cytosol were simultaneously present. These observationsindicate that (i) the precursor to the ultimate mutagen is relativelyshort-lived; and (ii) the metabolism of -hydroxy-NDMA to a secondarymutagenic metabolite, possibly N-nitroso-N-methylformamide,by alcohol dehydrogenase may be responsible for the ultimatemutagen with relatively high activity in TA104.