Formation of the adduct 6-(deoxyguanosin-N2-yl)-3-amino-benzo[a]pyrene from the mutagenic environmental contaminant 3-nitrobenzo[a]pyrene

3-Nitrobenzo[a]pyrene (3-nitro-B[a]P) is a potent bacterialmutagen as a result of nitroreduction. Reaction of N-hydroxy-3-amino-B[a]P,prepared in situ from reduction of 3-nitro-B[a]P with calf thymusDNA, was studied. After enzymatic digestion of the DNA, theresulting modified nucleosides were analyzed by thermosprayHPLC-MS and high-resolution proton NMR spectroscopy. The majoradduct was identified as 6-(deoxyguanosin-N²-yl)-3-amino-B[a]P.The same adduct was obtained from incubation of DNA with 3-nitro-B[a]Pin the presence of the mammalian nitroreductase xanthine oxidase,and hypoxanthine. These data indicate that a mammalian nitroreductasecan metabolize 3-nitro-B[a]P to an activated derivative thatreacts with DNA to give a novel adduct distant from the siteof N-hydroxylation.     

Differential effects of phorbol esters on c-fos and c-myc and ornithine decarboxylase gene expression in mouse skin in vivo

Hyperplasiogenic and tumor-promoting phorbol esters such as12-O-tetradecanoylphorbol-13-acetate or l2-O-retinoylphorbol-13-acetateinduce the sequential transient expression of the proto-oncogenesc-fos and c-myc and the ornithine decarboxylase gene in mouseskin in vivo. This sequence of biochemical events probably dependson an activation of protein kinase C by these agents. The non-irritantskin mitogens 4-O-methyl-12-O-tetradecanoylphorbol-13-acetateand ethyl phenyl propiolate do not increase the expression ofthese genes to a comparable extent. Thus, 12-O-tetradecanoylphorbol-13-acetateand 12-O-retinoylphorbol-13-acetate induce epidermal hyperproliferationby different biochemical mechanisms as do 4-O-methyl-12-O-tetradecanoylphorbol-13-acetateand ethyiphenyipropiolate.     

The biological activity and activation of 15,l6-dihydro-1,11-methanocyclopenta [a] penanthren-17-one, a carcinogen with an obstructed bay region

The proposal that an unobstructed bay region is a prerequisitefor tumorigenic activity in cydopenta[a]phenanthrene-17-onesis not supported by the observation of the tumorigenicity of15,16-dihydro-1,ll-methanocydopenta[a]- phenanthrene-17-onetowards the skin of T.O. mice. The title compound is oxidisedin vitro by a mixed function oxidase to produce, inter alia,a trans-3,4-dihydrodiol, postulated as the proximate tumorigen.Unequivocal identification of a second metabolite as a trans-1,2-dihydrodiolderivative demonstrates the potential for enzymatic oxidationwithin the obstructed bay region and supports the proposal thatthe ultimate tumorigen is a trans-3,4dihydrodiol-anti-l,2-oxide.This is further substantiated by the chromatographic behaviourof the major hydrocarbon-nucleoside adduct derived from mouseskin treated with the parent compound in vivo. The structuresof certain others of the metabolite produced in vitro are alsoconsidered.     

N-Acetylbenzidine-N'-glucuronidation by human, dog and rat liver

While N-glucuronidation is an important pathway for metabolismof aromatic amines, it has not been demonstrated for N-acetylbenzidine.A glucuronide of N-acetylbenzidine was synthesized and identifiedby mass spectrometry as N-acetylbenzidine-N'-glucuronide. ThisN'-glucuronide is acid labile with a t     

Increased urinary nitrosamine excretion in patients with urinary diversions

Tumor development at the site of ureterointestinal anasto mosisis a recognized complication in patients with continent urinarydiversions. Aerobic cultures of rectal urine samples from 30patients with urinary diversions (26 uretero sigmoidostomies,two colon conduits, one ileal conduit and a Gersuny bladder)showed a complex bacterial flora containing nitrate-reducingorganisms (Escherichia coli, Proteus and Kiebsiella spp.). Incomparison to normal bladder urine samples from control volunteers(n = 20), rectal urine samples from ureterosiginoidostomy patients(n = 26) showed a significant decrease (P < 0.0001) in urinarynitrate (0.93 ? 0.39 versus 0.27 ? 0.23 mmol/l), a significantincrease (P < 0.0001) in urinary nitrite (not detected versus29.24 ? 39.93 µmol/l) as well as a significant increase(P = 0.013) in urinary N-nitroso compound excretion (57.33 ?33.87 versus 93.96 ? 65.76 mnol/l). Significant increases werealso found for the urinary excretion of individual volatileand non-volatile N-nitroso compounds, clearly demonstratinga bacterially mediated in vivo formation of N-nitroso compoundsin the ‘colon’ bladders of patients with urointestinaldiversions that may be an important etiological risk factorfor colon carcinogenesis in this patient group.     

Real-time PCR analysis of the N-acetyltransferase NAT1 allele *3, *4, *10, *11, *14 and *17 polymorphism in squamous cell cancer of head and neck

Although tobacco smoke has been established as a main risk factorin the development of head and neck squamous cell cancer (HNSCC),genetic polymorphisms of xenobiotic metabolizing enzymes aresupposed to modulate an individual's susceptibility to smoking-relatedHNSCC. N-acetyltransferase (NAT) 1 gene is known to be polymorphicand its protein product is implicated in the activation anddetoxification of carcinogens, such as aromatic amines, presentin tobacco smoke. We developed a rapid and reproducible LightCycler^TM-assistedreal-time polymerase chain reaction (PCR) for NAT1 genotyping,which allowed the parallel differentiation of NAT1*3, *4, *10and *11 alleles and separately of NAT1*14 and *17 alleles within60 min without the need for further post-PCR processing. Inorder to investigate the role of the NAT1 gene polymorphismas a risk-modifying factor in HNSCC, we tested for the presenceof NAT1*3, *4, *10, *11, *14 and *17 alleles in a case-controlstudy of 291 HNSCC patients and 300 healthy controls of Caucasianorigin. Our findings suggest that in Caucasians, the risk ofHNSCC is not associated with NAT1 polymorphism. The overalldistribution of NAT1 allele frequencies was not significantlydifferent among cases and controls. The presence of the fastacetylator NAT1*10 and NAT1*11 alleles did not significantlyincrease the risk of HNSCC and no modifying effect of NAT1*10was observed among smokers. This new approach in NAT1 genotypingsubstantially increases throughput of sample analysis and, therefore,enhances opportunities to study NAT1 as a risk factor in differentcancers in large-scale studies.     

Tumor-initiating activity of the bay-region dihydrodiols and diolepoxides of dibenz[a,j]anthracene and cholanthrene on mouse skin

The tumor-initiating activity of dibenz[a,j]anthracene, (±)dibenz[a,j]anthracene-trans-3,4-dioland (±)dibenz[a,j]anthracene-anti-3,4-diol-1,2-epoxidein mouse skin was examined and compared to that of cholanthrene,(±)cholanthrene-trans-9, 10-diol and (± )cholanthrene-anti-9,10-diol-7,8-epoxide.The tumor-initiating activity of these compounds dissolved inacetone or in tetrahydrofuran (THF) was also compared. In acetone,dibenz[a,j]anthracene was a weak tumor initiator with maximaltumor yields of 1.27 and 3.00 per mouse at 400 and 800 nmoldoses, respectively. At the 400 nmol dose, the diol of thiscompound was slightly more active than the parent compound whilethe tumorigenic activity of the diol-epoxide was significantlyhigher. The diol-epoxide was almost three times more activethan the parent compound as a tumor-initiator. Cholanthrenewas a moderate tumor-initiator with maximal tumor yields of6.90 and 8.86 tumors per mouse at 200 and 600 nmol doses, respectively,after 20 weeks of promotion. At comparable doses, (±)cholanthrene-trans-9,10-diolwas 50% as potent as cholanthrene as a tumor initiator whereasthe diol-epoxide was only minimally active. Replacing the acetonewith THF as solvent vehicle increased the tumor-initiating activityof cholanthrene-diol-epoxide; however, the parent compound stillretained higher tumor-initiating activity than its bay-regiondiol-epoxide. The low tumorigenic activity of cholanthrene-diol-epoxideis thought to reflect the high chemical reactivity and low stabilityof this derivative, which may prevent it from penetrating toepidermal targets. In contrast, the bay-region diol-epoxidederivative of dibenz-[a,j]anthracene appears to be stable enoughto exert greater biologic activity when applied to mouse skin.     

Mutagenic potential of DNA adducts formed by diol-epoxides, triol-epoxides and the K-region epoxide of chrysene in mammalian cells

The syn- and anti-tsomers of chrysene-l,2-diol-3,4oxide (syn-diol-epoxideand anti-diol-epoxide) and of 9-hydroxychry-sene-l, 2-diol-3,4-oxide (syn-triol-epoxide and anti-triol-epoxide) and chrysene-5,6-oxide,the K-region epoxide, were tested for their ability to induce6-thioguanine-resistant mutants in V79 Chinese hamster cells.The levels of DNA adducts formed by each compound in the V79cells were determined by ³²P-post-labelling analysis. The mostpotent mutagen, in terms of the mutation frequency/nmol compoundadministered, was the anti-triol-epoxide, which was 1.7 timesas active as the anti-triol-epoxide. The anti-diol-epoxide was10 times more active than both the syn-triol-epoxide and thesyn-diol-epoxide, which in turn were several times more activethan the K-region epoxide. However, when the results were expressedas mutations/pmol total adducts formed, the anti-triol-epoxideand anti-diol-epoxide were shown to be of similar potency andapproximately twice as active as the other three compounds.Thus differences in the conformation of adducts formed withDNA by syn- and anti-isomers may be responsible for their differentmutagenic potentials; the presence of a phenolic OH-group atthe 9-position of a chrysene-l,2-diol-3,4-oxide appears to increaseits chemical reactivity.     

Investigations on the carcinogenicity of fusarin C -- a mutagenic metabolite of Fusarium moniliforme

The cancer initiating potential of fusarin C, a mutagen producedby Fusarium moniliforme strain MRC 826 was investigated on mouseskin using 12-O-tetradecanoyl-phorbol-13-acetate as promoterand in rat liver using pheno-barbital as promoter. In neitherof these models did fusarin C act as a cancer initiator. Culturematerial of strain MRC 826, which previously was found to behepatocarcinogenic in rats, exhibited cancer promoting activityin rat liver using diethylnitrosamine (DEN) as initiator andthe induction of gamma glutamyltranspeptidase (GGT)-positivefoci as end-point. The culture material of this fungus couldalso induce the formation of GGT positive foci without DEN initiation.These results seem to indicate that fusarin C is not involvedin the hepatocarcinogenic activity of F. moniliforme strainMRC 826.     

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.     

Bacterial and mammalian cell mutagenicity of four optically active bay-region 10,11-diol-8,9-epoxides of the nitrogen heterocycle dibenz (a,h) acridine

The mutagenk activities of the enantiomers of the diastereo-mericpair of bay-region 10, 11-diol-8, 9-epoxides of dibenz-[a,h]acridine(DB[a,h]ACR) were evaluated in histidine-dependent strains ofSalmonella typhimurium and in cultured Chinese hamster V79 cells.In strains TA98 and TA100 of S.typhimurium, the (–)-[8S,9R,10R,11S]diol-epoxide was the most mutagenic compound, inducing 1200and 6900 His⁺ revertants/nmol respectively. The mutagenic activityof each of the remaining three isomers was essentially independentof the bacterial strain used and had 14–72% of the activityof the [S,R,R,S] isomer. However, in Chinese hamster V79 cells,the (+)-[8R,9S,10S,11R] diol-epoxide was the most mutageniccompound (68 8-azaguanine resistant variants/ nmol/10⁵ cells),inducing from 2 to 11 times as many mutations as the other threeisomers. These results are analogous to previous studies withthe bay-region diol-epoxides of other polycyclic hydrocarbonsin that the isomer with [R,S,S,R] absolute configuration hashad variable activity in the bacterial assays, but has generallybeen the most active in the mammalian cells. Furthermore, thisisomer has almost always been highly tumorigenic in the mouse.     

Urinary excretion of nitrate, nitrite and N-nitroso compounds in Schistosomiasis and bilharzia bladder cancer patients

Saliva and 24-h urine samples were collected from male Schistosomiasis(bilharzia) patients with S. haematobium infection and possibleconcurrent S.mansoni infection without diagnosed bladder cancer(n = 27), bilharzia patients with diagnosed bladder cancer (n= 23) as well as a comparative control group (n = 27) of healthyEgyptian volunteers with no current bilharzia infection and/orbacterial urinary tract infections from the Nile Delta areaof Egypt. Saliva samples were analysed for the presence of nitrateand nitrite; urine samples were analysed for the presence ofnitrate, nitrite, volatile and non-volatile N-nitroso compounds.Bilharzia patients prior to, and after, diagnosed bladder cancerregularly excreted free nitrite as well as volatile nitrosamines(N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitroso-piperidineand N-nitrosopyrrolidine) in addition to which elevated concentrationsof non-volatile N-nitrosamino acids (N-nitrosoproline, N-nitrososarcosine,N-nitrosothiazolidine-4-carboxylic acid and its 2-methyl derivative)were also present. Total urinary excretion of volatile N-nitrosocompounds (0.32 ± 0.64 µg/day; mean ± SD)and nonvolatile N-nitroso compounds (31.20 ± 22.07 µg/day)was observed in the Egyptian control group. Significantly higherconcentrations were found in bilharzia patients: 3.47 ±6.42 (P < 0.05) and 62.91 ± 21.96 (P < 0.05); aswell as in bilharzia patients with diagnosed bladder cancer:1.71 ± 1.96 (P < 0.02) and 44.94 ± 7.31 respectively.Free nitrite was found in the urine of two volunteers in theEgyptian control group (1.7 and 3.0 µg/day), urinary nitritewas significantly increased in bilharzia patients (5.18 ±9.11 µg/day, P < 0.02) and in bladder cancer patients(1.75 ± 2.81 µg/day, P < 0.05). Nitrate concentrationswere elevated from 139.3 ± 82.2 in the control groupto 143.6 ± 136.3 and 175 ± 190 in the bilharziaand bladder cancer groups respectively. These results indicatethat significant in vivo formation of nitrite and volatile N-nitrosocompounds occurs in the urinary bladder of bilharzia patientsand this may be an oetiological factor in the induction of bilharzialbladder cancer associated with S.haematobium infection.     

Bacteremia Complicating Acute Leukemia with Special Reference to Its Incidence and Changing Etiological Patterns

Over the 15-yr period, 1972–1986, 194 episodes of bacteremiaoccurred in 132 patients wtih acute leukemia at the Third Departmentof Medicine, Kanazawa University Hospital, giving an incidenceof 478 episodes per 1,000 hospital admissions. This incidencewas at least twice as high as that in patients with chronicleukemia, malignant lymphoma, multiple myeloma or aplastic anemia,and about 40-fold higher than that in patients with all otherinternal diseases. The rate of occurrence of bacteremia, whetherunimicrobial or polymicrobial, remained almost unchanged throughoutthe study period. The frequency of gram-negative bacilli decreasedsignificantly, however, from 81% of the total isolates for thefirst 10-yr period to 50% for the second 5-yr period. Escherichiacoli and Klebsiella pneumoniae were isolated in markedly decreasingfrequency, but Pseudomonas aeruginosa and Enterobacter cloacaein relatively constant frequency. The majority of P. aeruginosaisolatesbelonged to a limited number of O-antigen groups, suggestingthe possibility of nosocomial infection. On the other hand,the frequency of gram-positive cocci increased from 9 to 36%.Staphylococcus epidermidis,Enterococcus species, and Staphylococcusaureus emerged as important pathogens. Such a change in thespectrum of organisms was considered to coincide with the commonuse of the so-called second- and third-generation cephalosporinsand central venous catheters. It is thus suggested that vancomycinbe added to empiric antibiotic therapy, especially when gram-positiveinfections are clinically or microbiologically suspected, andthat reducing the acquisition of P. aeruginosa from the hospitalenvironment remains a priority in infection prevention.     

Formation and removal of dibenzo[a,e]fluoranthene-DNA adducts in mouse embryo fibroblasts

In vivo binding of dibenzo[a, e]fluoranthene (DBF) to mouseembryo fibroblast DNA was compared with that observed previouslyin vitro on calf thymus DNA incubated with mouse liver microsomes.The h.p.l.c. elution patterns of the adducts formed by DBF metaboliteswith DNA and obtained in vivo at the optimal exposure time of42–48 h were qualitatively very similar to the patternsobtained in vitro, but their amplitude was quantitatively reduced.There are two striking differences between the in vivo and invitro results. Firstly, the most polar peak A, very abundantin vitro, was absent in vivo. Secondly, the reactivity of thetwo major proximate metabolites of DBF, the bay and pseudo-bayregion dihydrodiols, was very different in intact cells comparedwith the results in vitro. When incubated in vitro, pseudo-bayregion dihydrodiol DBF was twice as reactive as bay region dihydrodiolDBF. The opposite reactivities were observed in vivo. The majorDBF-DNA adducts formed in vivo were collected in the peaks E,B and C. The predominant peak E contained DNA adducts of bothbay and pseudo-bay region dihydrodiolepoxides which are themajor ultimate metabolites of DBF in vivo and in vitro. Theother two prominent peaks B and C contained DNA adducts of 3-hydroxyDBF pseudo-bay region dihydrodiolepoxide and the 7-hydroxy DBFbay region dihydrodiolepoxide, respectively. After adduct formation,post incubation of fibroblasts for a further 48 h, in the absenceof DBF, eliminated half the amount of adducts present. PeakB adducts were repaired more efficiently than those of peaksE, C D and F. The carcinogenic initiating activity of DBF appearsto be a complex process in which several DNA adducts play arole.     

Ascorbic acid may protect against human gastric cancer by scavenging mucosal oxygen radicals

High dietary ascorbic acid intake appears to protect againstgastric cancer. This may be due to its action as a scavengerof reactive radical species formed in the gastric mucosa, resultingin a reduced level of radical-mediated DNA damage. We have studied82 patients, of whom 37 had Helicobacter pylori-associated gastritis,a condition which predisposes to gastric cancer. Using electronparamagnetic resonance (EPR) spectroscopy we have demonstrated,for the first time, that ascorbyl radicals are generated inhuman gastric mucosa, presumably as a result of scavenging offree radicals by ascorbic acid. Quantification of ascorbyl radicalsdemonstrates that there is a higher concentration in those patientswith H.pylori gastritis compared with subjects with normal histology(P < 0.01). We also found gastric mucosal luminol-enhancedchemiluminescence and malondialdehyde concentrations (whichare believed to be markers of radical generation and tissuedamage) to be higher in patients with H.pylori gastritis comparedwith those with normal histology (P < 0.001 and P < 0.01respectively). The observed concentrations of the ascorbyl radicalcorrelate with the level of luminol-enhanced chemiluminescence(r = 0.41, P < 0.001), but not with malondialdehyde concentrations(r = 0.08, P = 0.47). Mucosal ascorbic acid and total vitaminC concentrations did not vary between histological groups, nordid they correlate with mucosal levels of the ascorbyl radical,chemiluminescence or malondialdehyde. These data suggest thatascorbic acid is acting as a scavenger of free radicals generatedin human gastric mucosa. The experiments therefore provide directsupportive evidence for the hypothesis that ascorbic acid protectsagainst gastric cancer by scavenging reactive radical specieswhich would otherwise react with DNA, with resultant geneticdamage.     

Enhanced repair of O6-methylguanine in liver DNA of rats pretreated with phenobarbital, 2,3,7,8-tetrachlorodibenzo-p-dioxin, ethionine, or N-alkyl-N-nitrosoureas

Rats were pretreated for a number of weeks with the liver tumourpromoters phenobarbital and 2, 3, 7, 8-tetrachlorodi-benzo-p-dioxin,the direct alkylating agents N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea,and the hepatocardnogens ethionine and diethylnitrosamine. Asubsequent challenge with a single, low dose of radioactivelylabelled dimethyl-nitrosamine was given to assay the capacityof the liver for O⁶-methylguanine repair. Pretreatment with0.05% phenobarbital in the diet for 8 weeks (a promoting regimen)resulted in significantly enhanced O⁶-methylguanine repair;shorter pretreatment periods (3 days or 2 weeks) had no significanteffect. Repeated injection of another liver tumour promoter,2,3, 7, 8-tetrachlorodibenzo-/>-dioxin, also resulted inenhancement of O⁶methylguanine repair. Pretreatment for 2 weekswith N-ethyl-N‘-nitrosourea resulted in strongly enhancedO6-methylguanine repair, as did a similar pretreatment withdiethylnitrosamine, which was included as a positive control.The same pretreatment scheme which was highly effective in thecase of N’-ethyl-N-nitrosoiirea, was found to be totallyin effective in the case of N-methyl-N-nitrosourea. When N methyl-N-nitrosoureawas administered for 8 weeks instead of 2, a small but statisticallysignificant increase in O⁶-methyl-guanine repair was observed.It is concluded that two factors are responsible for the loweffectivity of N-methyl-N-nitroso-urea. The first is the relativelylow extent of liver DNA methylation by this compound when comparedwith dimethyl-nitrosamine. The second is the low efficiencyof methylating agents (expressed per extent of DNA alkylation)to induce O⁶-methylguanine repair in rat liver when comparedwith ethylating agents. Pretreatment for 2 weeks with a dietcontaining DL-ethionine also resulted in a substantially increasedO⁶-methylguanine repair capacity. Neither this enhancement,nor that induced by a pretreatment with diethylnitrosamine,could be inhibited by simultaneous feeding of a methionine-enricheddiet. Our results indicate that neither increased hepatocellularproliferation nor direct interaction with DNA are necessaryfor the induction of O⁶-methylguanine repair enhancement. Itis concluded that the capacity of an agent to enhance O⁶-methylguaninerepair in rat liver reflects the hepato(co)carcinogenic capacityof that agent.     

Cytotoxicity, DNA adduct formation and DNA repair induced by 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline and 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in cultured human mammary epithelial cells

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP) are heterocyclic amines (HAs) found in cooked meats.Both compounds are mammary gland carcinogens in rats. The initiationof carcinogenesis by the HAs is believed to be associated withtheir DNA adduct formation that occurs after metabolic activationof the parent amines via cytochrome P-450-mediated N-hydroxylationand esterification. To assess the capacity of the human mammaryepithelium to metabolically activate the HAs, we used the ³²P-postlabelingmethod to measure the levels of DNA adducts in a culture ofhuman mammary epithelial cells exposed to IQ, PhIP or theirN-hydroxylamine metabolites. Whereas 50 µM parent aminesdid not form detectable levels of DNA adducts in cultured humanmammary epithelial cells after 24 h incubations, concentrationsas low as 1 µM N-hydroxylamines produced detectable levelsof adducts after 2 h incubations. N-Hydroxy-PhIP formed higheradduct levels than N-hydroxy-IQ at all concentrations tested.For example, following a 2 h incubation at 50 µM, adductlevels (per 10⁷ nucleotides) were 674 and 16 for N-hydroxy-PhIPand N-hydroxy-IQ, respectively. At similar initial adduct levels(10–11/10⁷ nucleotides), 60–80% of IQ- and PhIP—DNAadducts were removed after 24 h, indicating that the mammaryepithelial cell culture showed efficient repair of HA adducts.Whereas neither IQ nor PhIP was cytotoxic, both N-hydroxy-IQand N-hydroxy-PhIP were cytotoxic as assessed by a dose-dependentinhibition of colony formation. After exposure to 0.1, 1, 10or 50 µM N-hydroxy-PhIP (or N-hydroxy-IQ), colony formationwas 103 (94), 84 (74), 37 (29) and 3 (2)% of the control values,respectively. Only N-hydroxy-PhIP (at 10 and 50 µM), however,was cytotoxic as assessed by the MTT cell survival assay (whichmeasures the capacity of mitochondria to metabolize a tetrazoliumsalt). The ability of the N-hydroxylamines to form DNA adductsand to be cytotoxic in a culture of human mammary epithelialcells may implicate these metabolites as proximate carcinogenicforms of IQ and PhIP in the human mammary gland. However, whetherthere are inter-individual differences in N-hydroxylamine metabolism,adduct formation and repair in human mammary epithelial cellsrequires further study. The results from this study support the usefulness of culturedhuman mammary epithelial cells for studies on the genotoxicityand metabolism of the N-hydroxylamines of HA food mutagens.     

Re: Yang, J. and Duerksen-Hughes, P. (1998) A new approach to identifying genotoxic carcinogens: p53 induction as an indicator of genotoxic damage. Carcinogenesis, 19, 11171125.

Dear Sir, In a recent issue of the Journal, Yang and Duerksen-Hughes presenta new in vitro approach for identifying genotoxic carcinogens.In this study they used p53 induction in vitro as an indicatorof genotoxic damage. The authors suggest that this endpointcould be used as an effective tool for identifying environmentalcarcinogens. In our opinion, p53 induction has clear weaknesses as an endpointfor in vitro genotoxicity testing. It is induced by many types     

Glucuronidation of N-hydroxy heterocyclic amines by human and rat liver microsomes

The food-borne carcinogenic and mutagenic heterocyclic aromaticamines undergo bioactivation to the corresponding N-hydroxy(OH)-arylamines and the subsequent N-glucuronidation of thesemetabolites is regarded as an important detoxification reaction.In this study, the rates of glucuronidation for the N-OH derivativesof 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ), 2-amino-l-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), 2-amino-6-methyi-dipyrido[l,2-a:3',2'-d]imidazole(Glu-P-1) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MelQx) by liver microsomal glucuronosyltransferase were comparedto that of the proximate human urinary bladder carcinogen, N-OH-aminobiphenyl(N-OH-ABP) and the proximate rat colon carcinogen N-OH-3,2'-dimethyl-4-aminobiphenyl(N-OH-DMABP). Human liver microsomes catalyzed the uridine 5'-diphosphoglucuronicacid (UDPGA)-dependent glucuronidation of N-OH-IQ, N-OH-PhIP,N-OH-Glu-P-1 and N-OH-MeIQx at rates of 59%, 42%, 35% and 27%,respectively, of that measured for N-OH-ABP (11.5 nmol/min/mg).Rat liver microsomes also catalyzed the UDPGA-dependent glucuronidationof N-OH-PhIP, N-OH-Glu-P-1 and N-OH-IQ at rates of 30%, 20%and 10%, respectively of that measured for N-OH-DMABP (11.2nmol/min/mg); activity towards N-OH-MelQx was not detected.Two glucuronide(s) of N-OH-PhIP, designated I and II, were separatedby HPLC. Conjugate II was found to be chromatographically andspectrally identical with a previously reported major biliarymetabolite of PhlP in the rat, while conjugate I was identicalwith a major urinary metabolite of PhIP in the dog. Hepaticmicrosomes from rat, dog and human were found to catalyze theformation of both conjugates. The rat preferentially formedconjugate II (I to II ratio of 1:15), while the dog and humanformed higher relative amounts of conjugate I (I to II ratioof 2.5:1.0 and 1.3:1.0 respectively). Fast atom bombardmentmass spectrometry of conjugates I and II gave the correspondingmolecular ions and showed nearly identical primary spectra.However, collision-induced spectra were distinct and were consistentwith the identity of conjugates I and II as structural isomers.Moreover, the UV spectrum of conjugate I exhibited a