Catalysis of nitrosation in vitro and in vivo in rats by catechin and resorcinol and inhibition by chlorogenic acid

Measurements were made of the effects of phenolic compounds,some of which are present in the human diet, on the nitrosationof proline by nitrite to give N-nitrosoproline (NPRO). In vitro,resorcinol, catechin, p-nitrosophenol and phenol were catalystsand chlorogenic acid an inhibitor; guaiacol showed a marginalcatalytic effect. Both the catalytic and the inhibiting effectswere dependent on pH and on the concentration of phenolic compounds;catalysis by resorcinol and catechin was increased at optimalratios of [nitrite]: [phenolic compound]. Endogenous nitrosationwas examined in vivo by co-administration of nitrite, prolineand a phenolic compound to rats and by monitoring the amountof NPRO excreted in the urine. Under similar experimental conditions,the catalytic effects observed in vivo decreased in the sameorder as those observed in vitro: resorcinol > p-nitrosophenol> catechin > phenol guaiacol; chlorogenic acid acted asan inhibitor. Catalysis and inhibition of N-nitrosation in ratsin vivo appears to occur via mechanisms similar to those invitro, although the effects in vivo were smaller. The implicationsof our findings for the endogenous formation of N-nitroso compoundsand for variations in exposure due to different dietary constituentsin humans are discussed.     

Enzymatic acetylation and sulfation of N-hydroxyarylamines in bacteria and rat livers

In mammalian hepatic cytosol both acetyltransferase and sulfotransferaseare involved in the activation of N-hydroxy derivatives of arylaminesand arylamides. The role of acetyltransferase is also shownin Salmonella, whereas no rigid evidence- is provided on therole of sulfotransferase in Salmonella. In Ames mutagenesistest without S9-mix, the number of revertants of Salmonellatyphimurium TA98 induced was 10-fold higher with 2-hydroxyamino-3-methyl-imidazo[4,5-f]quinoune(N-hydroxy-IQ) than with 2-hydroxy-amino-6-niethvldipyrido[l,2-a:3',2'-d]imidazole(N-hydroxy-Glu-P-1). The extents of the binding to calf thymusDNA of N-hydroxy-Glu-P-1 were, however, 3.9 to 8.6-fold higherthan that of N-hydroxy-IQ in both acetyl CoA- and PAPS-fortifiedrat hepatic cytosol systems. To understand the mechanism causingthe apparent discrepancy between the results of the mutationand DNA binding, the activating capacities of cytosols of S.typhimuriumTA98 and TA98/1,8-DNP₆ strains on the binding of N-hydroxy-Ghu-P-1and N-hydroxy-IQ have been examined in comparison with thoseof rat livers. Although both N-hydroxyarylamines were activatedby hepatic cytosols in the presence of PAPS, no significantDNA binding of these N-hydroxyarylamines was detected in thepresence of PAPS and either one of the two strains of bacterialcytosols. In addition, both cytosols of TA98 and TA98/1,8-DNP₆strains showed no measurable activity on the sulfation of p-nitrophenol,suggesting no capacity for sulfotransferase-mediated activationof N-hydroxyarylamines in Salmonella. On the contrary, the extentsof the acetyl CoA-dependent binding of N-hydroxy-IQ in cytosolsof TA98, but not of TA98/1,8-DNP₆, were respectively 6- andWold higher than those in hepatic cytosols of male and femalerats, although the extents of the binding of N-hydroxy-Glu-P-1were rather higher in hepatic than in bacterial cytosols. Inaddition, the covalent binding of N-hydroxy-2-acetylaminofhioreneto DNA was detected in hepatic, but not in bacterial cytosob,although the binding of N-hydroxy-2-aminofluorene was detectablein both hepatic and bacterial cytosols in the presence of acetylCoA. These results indicate that the metabolic activating capacitiesof Salmonella and rat liver cytosols differ qualitatively, andthe difference in the substrate specificity of acetyltransferasebetween Salmonella and rat livers may be involved, in part,in the difference of then- DNA damage in bacteria and mammals.     

32P-Postlabeling analysis of IQ, MelQx and PhIP adducts formed in vitro in DNA and polynucleotides and found in vivo in hepatic DNA from IQ-, MelQx- and PhIP-treated monkeys

The ³²P-postlabeling method was used to examine the adductsin DNA, polynucleotides, and mononucleotides reacted in vitrowith the N-hydroxy and N-acetoxy derivatives of 2-amino-3-methylimidazo[4,5-f]quinoline(IQ), 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MelQx)or 2-amino-1-methy1-6-phenylimidazo[4,5-b]pyridine (PhIP). Adductprofiles were compared to those found in vivo in liver of cynomolgusmonkeys fed IQ, MeIQx or PhIP. The N-acetoxy derivatives ofIQ, MelQx and PhIP (generated in situ from the correspondingN-hydroxylamine in the presence of acetic anhydride) each formedthree principal adducts in DNA. Adduct 1 of IQ, MeIQx and PhlPwas chromatographically identical to the ³²P-labeled bis(phosphate)derivative of N-(deoxyguanosin-8-yI)-IQ, N-(deoxyguanosin-8-yI)-MeIQx,and N-(deoxyguanosin-8-yl)-PhIP respectively, and this adductcomprised     

Binding of deferoxamine to asbestos fibers in vitro and in vivo

We studied the binding of tritium-labeled deferoxamine, a strongiron chelator, to crocidolite asbestos fibers in vitro and invivo. In aqueous suspension of asbestos, deferoxamine bindingwas rapid and strong, suggesting specific binding to iron. Forthe in vivo experiments, diffusion chambers containing nativeasbestos fibers or deferoxamine-washed asbestos were implantedin the peritoneal cavities of mice. Five days after parenteralinjection of tritiated deferoxamine chambers were removed andthe asbestos counted. More than twice as much label(2206 ±348 c.p.m./100 mg asbestos) was bound to the native asbestosas compared to the deferoxamine-washed asbestos (1080 ±201 c.p.m./100 mg asbestos), suggesting specific binding invivo. Since deferoxamine can inhibit asbestos toxicity in vitro,these experiments suggest the feasibility of testing whetherdeferoxamine can prevent asbestos-related disease in vivo     

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.     

N-Acetylation and N-formylation of carcinogenic arylamines and related compounds in dogs

When sulfanilamide, p-amlnobenzoic acid, 4-amino- biphenyl,2-aminofluorene or 1-aminopyrene was given orally to dogs, thecorresponding N-acetyl and N-formyl derivatives were isolatedfrom urine or feces. These metabolites were identified unequivocallyby comparison with an authentic sample by UV and mass spectrometryand their behavior in TLC and HPLC. Dog intestinal flora andseveral bacterial strains exhibited both N-acetylatlng and N-formylatingactivities, in varying degrees, toward all of the arylaminestested. The metabolites formed by the intestinal bacteria werealso isolated and identified unequivocally. The results suggestthat the intestinal microflora plays an important role in theformation of N-acyl derivatives from arylamines in dogs.     

32P-Postlabelling analysis of the covalent binding of benzo[ghi]perylene to DNA in vivo and in vitro

Benzo[ghi]perylene (B[ghi]P) is a polycyclic aromatic hydrocarbon(PAH), present in complex combustion products, and evidencefor its carcinogenic activity in experimental animals is equivocal,and yet it has demonstrable mutagenic activity in vitro. Inorder to investigate the possible DNA binding properties ofB[ghi]P, groups of male Parkes mice were treated topically with1.0 µmol of B[ghi]P. Mice were killed up to 3 months aftertreatment, DNA was isolated from the treated areas of skin andanalysed for adducts by ³²P-postlabelling. Maximum levels ofbinding (0.57 fmol/µg DNA) were detected 2 days aftertreatment and adducts were found to persist for at least 12weeks after treatment, a property of many PAHs whh known tumor-initiatingactivity. B[ghi]P also became bound to DNA in vitro in the presenceof 3-methylcholanthrene-induced rat liver microsomal preparations.When chromatographed on PEI–cellulose, the major adductsformed by B[ghi]P in vivo and in vitro appeared to be identical.However, they were found to be different when compared by reversed-phaseHPLC. These differences might explain, in part, the differencesin the biological activity of B[ghi]P in vivo and in vitro,.The behaviour of B[ghi]P when present in a mixture was alsoexamined. B[ghi]P was applied topically to mouse skin with sixother PAHs at a dose level of 0.25 µmol/PAH/mouse. DNAisolated 24 h after treatment was analysed for adducts by ³²P-postlabelllng.Whilst the total level of binding was 30% lower than expectedfrom the sum of the binding levels that resulted when the hydrocarbonswere applied singly, the formation of B[ghi]P–DNA adductsdid not appear to be inhibited. The results have demonstratedthat B[ghi]P has significant DNA binding ability in vivo andin vitro and on the basis of its DNA binding ability in mouseskin it would be predicted to be at least a weak tumour initiator.The formation of DNA adducts by B[ghi]P when present in an artificialmixture of PAHs suggests that B[ghi]P may contribute to theDNA binding activity of more complex carcinogenic mixtures.     

The neu-oncogene product in serum and tissue of patients with breast carcinoma

BACKGROUND:: A soluble 105 kD neu-related protein is detectable in conditionedmedium from breast cancer cells expressing the neu-oncogeneproduct and in serum of nude mice bearing tumors that overexpressneu-oncogene PATIENTS AND METHODS:: In 100 patients with primary (n - 33) relapse-free (n - 6) andmetastatic (n - 61) breast carcinoma the serum levels of thesoluble new-related protein were investigated by ELISA techniques.Median age was 57 years, range 26–89 years. RESULTS:: The neu-protein serum levels were below 40 HNU/ml (human neu-antigenunit) in 72 patients and 40 or more HNU/ml in 28 patients. In30 patients with primary breast carcinoma, tested before mastectomy,all serum- neu-protein samples were negative. However, 26 of61 metastazised patients (43%) were serum-neu-protein-positive.In disseminated disease (n – 61), serum-neu-protein-positivitywas more likely to be seen in patients with visceral metastases(18/33 – 54%), than in patients with nonvisceral metastases(8/28 – 28%). Furthermore, monitoring of the serum-neu-proteinlevels reflected clinical course. For 53 patients original paraffin-embeddedtumor material was available for studying immunohistochemicalneu-protein expression. In 39/53 (73%) patients immunohistochemicaland ELISA data showed corresponding results. In 27/30 (90%)patients, from whom sera and tissue could be obtained at thesame time at primary mastectomy, results of immunohistochemistryin primary tumor and serum ELISA were negative and mutuallyconfirmatory. However, the other three patients were positivefor immunohistochemical neu-protein expression in primary tumorbut negative for serum-neu-protein expression. CONCLUSIONS:: Our results suggest that patients with advanced breast cancerand an elevated serum-neu-protein level may have a poor clinicaloutcome. This test might be a useful tool for monitoring patientswith advanced breast carcinoma, but not those with early disease.Further prospective studies are warranted to elucidate the questionof whether this test can contribute to determining prognosisand treatment strategies. breast carcinoma, c-erb-B2, HER-2, neu, oncogene, pl85     

Formation and persistence of O6-ethylguanine in genomic and transgene DNA in liver and brain of {lambda}lacZ transgenic mice treated with N-ethyl-N-nitrosourea

LacZ transgenic mice are suitable for short-term muta-genicitystudies in vivo. Mutagenicity in these mice is determined inthe lacZ transgene. Since the lacZ gene is of bacterial originthe question has been raised whether DNA-adduct formation andrepair in the transgene are comparable to those in total genomicDNA. Mice were treated with N-ethyl-N-nitrosourea (ENU) andkilled at several time points following treatment. Some micewere pretreated with O⁶-benzylguanine to inactivate the repairprotein O⁶-alkylguanine-DNA alkyltransferase (AGT). O⁶-ethylguanine(O⁶-EtG) was determined in lacZ in liver and brain by meansof a monoclonal antibody-based immuno-affinity assay. In addition,O⁶-EtG and N7-ethylguanine (N7-EtG) were assayed in total genomicDNA of liver and brain with an immunoslotblot procedure. Inliver, the initial O⁶-EtG level in total genomic DNA was 1.6times that in lacZ. The extent of repair of O⁶-EtG during thefirst 1.5 h after treatment was 2.1 times that in lacZ. At latertime points, O⁶-EtG repair was the same. N7-EtG repair in genomicDNA was evident. In contrast to the liver, little repair ofO⁶-EtG in total genomic and lacZ DNA occurred in the brain whileN7-EtG was repaired. No initial difference in O⁶-EtG levelswere found in lacZ and genomic brain DNA. These findings indicatethat in the liver, total genomic DNA is more accessible thanlacZ to ENU and/or the AGT protein, during the first 1.5 h followingtreatment. Because the difference in O⁶-EtG levels in the transgeneand genomic DNA in the liver is restricted to the first 1.5h after treatment, while the fixation of mutations occurs atlater time points, O⁶-EtG-induced mutagenesis most likely isalso very similar in both types of DNA.     

32P-Postlabeling and HPLC separation of DNA adducts formed by diesel exhaust extracts in vitro and in mouse skin and lung after topical treatment

Diesel exhaust extracts contain many carcinogenic compoundswhich have been shown to form polycyclic aromatic hydrocarbon(PAH)— and nitrated PAH—DNA adducts in rodent skinand lung. The aim of this study was to characterize by ³²P-postlabeling,TLC and HPLC the primary postlabeled PAH-DNA adduct(s) formedin vitro and in vivo by diesel extracts. The diesel particleextracts had known concentrations of benzo[a]pyrene, benzo[b,j,k]-fluoranthenes(B[b,j,k]F) and chrysene. DNA adducts were analyzed in calfthymus DNA incubated in vitro with PAHs activated by S9 mixand in skin and lung DNA from topically treated mice. The maindiesel-derived DNA adduct formed in vitro and in vivo did notco-migrate on HPLC and large TLC plates with ()-r-7, t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE)—,B[b]F—, B[j]F—, B[k]F— or chrysene—DNAadduct standards. By co-chromatography DNA adducts formed bychrysene from both in vitro and in vivo samples were identified.Nissan diesel extract containing higher PAH concentrations thanVolkswagen automobile extract formed skin DNA adducts that co-migratedwith chrysene— and anti BPDE—DNA-derived adducts.We conclude that the use of a highly sensitive ³²P-postlabelingmethod combined with HPLC improves the identification of PAHadducts formed by complex mixtures such as diesel exhaust extracts.     

Nitrate, nitrite and volatile N-nitroso compounds in the urine of Schistosoma haematobium and Schistosoma mansoni infected patients

The present study presents, for the first time, the amountsof nitrate, nitrite and volatile N-nitroso compounds in salivaand urine samples of Schistosoma haematobium and Schistosomamansoni infected patients. Mid-morning saliva and 24 h urinesamples were collected from male patients infected with S.haematobium(n = 129 saliva and 79 urine samples) and S.mansoni (n = 64saliva and 65 urine samples) and in a comparative control groupof healthy individuals (n = 27) from the Nile Delta region ofEgypt. Saliva samples were analyzed for the presence of nitrateand nitrite; while urine samples were analyzed for the presenceof nitrate, nitrite and volatile N-nitroso compounds. In thecontrol group, N-nitroso-dimethylamine (NDMA) was detected atconcentrations (mean ± SD) of 0.27 ± 0.47 µg/day.N-Nitrosopyrrolidine (NPIP; 0.6 µg/day) and N-nitrosopyrrolidine(NPYR; 0.4 µg/day) were also present in one sample. S.mansoniinfected subjects showed significantly (P < 0.001) higherlevels of 2.9 ± 2.9 µg/day NDMA and a higher frequencyof NPIP (in 40/65 samples; 0.4 ± 0.3 µg/day) andNPYR occurrence (in 59/65 samples; 0.9 ± 0.9 µg/day).Significant further increases in the excretion of volatile N-nitrosocompounds were found in S.haematobium-infected patients withmean daily excretion of 19.2 ± 21 µg/day NDMA (inall samples; P < 0.001), 1.6 ± 2.3 µg/day NPIP(in 56/79 samples; P < 0.001) and 1.3 ± 1.9 µg/dayNPYR (in 58/79 samples; P < 0.1). The differences eitherin salivary nitrite/nitrate or in urinary nitrite between thethree distinct groups were not significant. However, the urinaryexcretion of nitrate was elevated from 139 ± 82mg/dayin the control group to 249 ± 126 mg/day in S.mansoniinfected patients (P < 0.001) and to 174 ± 176 mg/dayin S.haematobium infected subjects (P < 0.005 in comparisonto S.mansoni infected group). These results suggest a possiblerole of N-nitroso compounds in the etiology of schistosome-associatedbladder cancer and imply a partial participation of S.mansoniin the multistage process of urinary schistosomiasis-associatedbladder carcinogenesis.     

High Rates of Ki-ras Point Mutation in Both Intra- and Extra-hepatic Cholangiocarcinomas

The presence of the Ki-ras gene mutations in 14 cases of intrahepaticcholangiocarcinoma (IHC) and nine cases of extrahepatic cholangiocarcinoma(EHC) were investigated by polymerase chain reaction-singlestrand conformation polymorphism analysis. To obtain enrichedtumor cell DNA, the microdissection method was used on formalin-fixedparaffin-embedded tissue sections. Point mutations at codon12 of the Ki-ras gene were detected in seven (50%) of the 14cases of IHC and six (67%) of the nine EHC cases. In all butone of the ras gene mutation cases, G to A transitions in thesecond position of codon 12 were detected, the exception beinga G to T transition in the same position in one IHC. No pointmutations were detected at codon 13 or 61 in either IHC or EHC.Furthermore, there was no demonstrable correlation between Ki-rasmutation and patient age, tumor Stage, histological findingsor prognosis. The present results demonstrated a higherparticipationof Ki-ras gene mutations in EHC than found in previous studies,and provided a confirmation and extension of the results earlierreported by Tada et al, and Tsuda et al, for IHC.     

Genotoxic effects of cyclopenta-fused polycyclic aromatic hydrocarbons in isolated rat hepatocytes and rabbit lung cells

Benz[j]aceanthrylene (B[j]A) and benz[l)aceanthrylene (B[l]A),two isomeric cyclopenta polycyclic aromatic hydrocarbons (CP-PAH)structurally related to 3-methylcholanthrene, were studied withrespect to their genotoxic effects in isolated liver and lungcells. Both compounds were found to cause DNA adducts measuredby the ³²P-postlabelling technique. The level of DNA-adductsin rat hepatocytes exposed to 30 µg/ml B[l]A and B[j]Afor 4 h were 46.5 ± 22.0 and 8.3 ± 5.1 fmol/µgDNA respectively. Using butanol extractions, the major and oneof the minor B[l] A adducts co-chromatographed with B[j]A-l,2-oxide adducts of 2'-deoxyadenosine and 2'-deoxyguanosine.Thus, oxidation at the cyclopenta-ring of B[j]A appears to bean important activation pathway. In hepatocytes, 3-30 µg/mlof B[j]A and B[j]A induced DNA damage and repair measured bothas increased alkaline elution of DNA and as increased incorporationof [³H]TdR in the DNA. B[l]A was somewhat more potent than B[j]Ain inducing DNA repair. Reactive CP-PAH intermediates formedin the hepatocytes caused mutations in Salmonella typhimuriumTA98 upon co-incubation. DNA adducts were also observed in isolatedrabbit lung cells exposed to 30 µg/ml B[l]A or B[j]A for2 h. A total of 14.5 ± 6.9, 2.9 ± 2.1 and 0.2± 0.6 fmol B[l] A adducts/µg DNA were observedin Clara cells, type II pneumocytes and alveolar macrophagesrespectively. The main B[l]A adduct observed in the liver cellswas not found in the lung cells. On the other hand, the levelsof B[j]A adducts in the lung cells were in the range 4-14% ofthat found in liver cells, and no major differences betweenthe various lung cells were observed. Neither B[l]A nor B[j]Ainduced DNA damage measured by alkaline elution in the lungcells, indicating that these adducts are not alkali labile.     

c-H-ras and c-K-ras gene hypomethylation in the livers and hepatomas of rats fed methyl-deficient, amino acid-defined diets

The extent of methylation of the c-H-ras and c-K-ras oncogeneswas compared in neoplastic and preneoplastic livers of ratsfed one of several methyl-deficient, amino acid-defined dietsfor 18 months, with or without a preceding initiating dose ofdiethylnitrosamine (DEN). The restriction endonucleases MspI,HpaII, HhaI, PaeR71 and XhoI were used for studying the extentand pattern of DNA methylation. The results indicated that bothc-H-ras and c-K-ras oncogenes were hypomethylated in all DNAsamples derived from both neoplastic and preneoplastic liversof rats fed any of the methyl-deficient diets used, regardlessof whether or not the rats had received an initiating dose ofDEN. It thus appears that dietary methyl deficiency does indeedlead to hypomethylation of ras genes in the DNAs of the resultingtumors. However, the significance of this hypomethylation inthe tumorigenic process is not clearly understood.     

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.     

Repair of O6-propylguanine and O6-butylguanine in DNA by O6-alkylguanine-DNA alkyltransferases from rat liver and E. coli

DNA substrates containing O⁶-n-butylguanine, O⁶-iso-butylguanine,O⁶-n-propylguanine and O⁶-iso-propylguanine were prepared byreaction of calf thymus DNA with the appropriate N-alkyl-N-nitrosourea.These substrates were used to test the ability of O⁶-alkylguanine-DNAalkyltransferases from Escherichia coli and rat liver to removesuch alkyl groups from the O⁶-position of guanine. It was foundthat all of these adducts were removed by the alkyltransferases,but the branched alkyl chain iso-butyl- and iso-propyl adductswere removed very slowly. Also, when tested with a DNA substratecontaining both O⁶-n-propylguanine and O⁶-iso-propylguanine,the alkyltransferases removed almost all of the n-propyl-adductbefore the iso-propyl-adduct was attacked. Both alkyltransferasesshowed a decreasing rate of reaction as the size of the alkylgroup increased, but there was a significant difference betweenthe rat liver and E. coli alkyltransferase in the relative rates.The rat liver alkyltransferase repaired O⁶-methylguanine moreslowly than the E. coli protein, but was considerably more rapidthan the bacterial equivalent when acting on n-propyl- and n-butyl-adducts.The relative rates of repair were methyl > ethyl > n-propyl> n-butyl >iso-propyl, iso-butyl for the E. coli alkyltransferaseand methyl > ethyl, n-propyl > n-butyl > iso-propyl,iso-butyl > 2-hydroxyethyl for the rat liver protein. Theseresults indicate that differential rates of repair may contributeto the relative risks of carcinogenesis and mutagenesis by exposureto alkylating agents of different size and that rates of repairmay be species specific and must be determined from specificmeasurements rather than extrapolated from data on other organisms.     

Metabolism of aromatic amines: relationships of N-acetylation, O-acetylation, N,O-acetyltransfer and deacetylation in human liver and urinary bladder

N-Acetoxyarylamines are reactive metabolites that are implicatedin the initiation of the carcinogenic process by some N-substitutedaryl compounds. The objective of this study was to explore therelationship between the production of these reactive speciesand N-acetylation (NAT), a reaction previously demonstratedto be polymorphic in the human. Human liver and urinary bladdermucosa samples were frozen within 4–8 h post mortem. Thesetissues were assayed for the (i) O-acetylation (OAT) of N-hydroxy-3,2'-di-methyl-4-aminobiphenyl (N-OH-DMABP) by acetyl CoA, (ii)intramolecular N,O-acetyltransfer (AHAT) of N-hydroxy-2-acetylaminofluorene(N-OH-AAF), (iii) NAT of 2-aminofluorene (2-AF) and p-aminobenzoicacid (PABA) by acetyl CoA and (iv) deacetylation of N-OH-AAF.Cytosolic AHAT and OAT showed partial inhibition by paraoxon.The ratio of paraoxon insensitive AHAT to OAT to NAT of PABAto NAT of 2-AF appears to be 1:2:11:22 using freshly made cytosolsfrom frozen livers. Freezing of the cytosol resulted in extensiveloss of activities. All four of these cytosolic enzyme activitiesexhibited a similar polymorphic response. Microsomal deacetylationshowed a monomorphic response. Similar to the liver, urinarybladder epithelial cells also catalyzed the same reactions.However, the OAT and AHAT activities were detected mainly inmicrosomes. These data suggest that phenotypically rapid acetylatorshave a greater biochemical potential for the metabolic activationof aromatic amines by pathways that involve O-acetylation.