DNA damage and repair in intact animals. Single-stranded regions in rat liver DNA following administration of dimethylnitrosamine.

Rat liver DNA was radioactively labelled by administration of [3H]thymidine following partial hepatectomy. Two weeks later, the rats were treated with the carcinogen, dimethylnitrosamine. DNA was isolated and fractionated by elution from benzoylated DEAE-cellulose with NaCl and caffeine solution. The caffeine-eluted fraction was increased by administration of dimethylnitrosamine. This increase was proportional to the dose of carcinogen injected and persisted for at least 24 h after administration of the carcinogen. These data, together with the results of hydroxyapatite chromatography, suggest that the DNA contains short single-stranded sections associated with much longer regions of native DNA.     

DNA single-strand breaks and toxicity induced by 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone or N-nitrosodimethylamine in hamster and rat liver

Syrian golden hamsters and F344 rats display contrasting susceptibilitiesto hepatocarcinogenesis induced by the tobacco-specific nitrosamine4-(methylnitrosamino)-1-(3-pyri-dyl)-1-butanone (NNK) and N-nitrosodimethylamine(NDMA). In this study, the time courses of DNA single-strandbreaks (SSB) and toxicity induced by NNK and NDMA in hamsterand rat liver were compared. DNA SSB reached a maximum 12 hafter carcinogen treatment, partially correlating with previousreports on time courses of DNA methyiation. The persistenceof DNA SSB up to 2–3 weeks after NNK or NDMA treatmentreflects a deficient repair of some DNA lesions. No significantspecies differences in the kinetics of DNA SSB induction andrepair were observed following NNK or NDMA (0.39 mmol/kg) treatment.However, NNK induced slightly more DNA SSB than NDMA in bothspecies. This could reflect the formation of intermediates withmore DNA-damaging capacity or inhibiting DNA processes. A significanthepatoxic effect of NNK, evaluated by plasma markers of liverinjury, was observed in rats and hamsters 12–24 h post-treatment.In contrast, NDMA induced earlier (<12 h) enzyme elevations.Maximum hepatotoxic effects were observed 24 h (NNK-treatedhamsters and rats, NDMA-treated rats) or 2 weeks (NDMA-treatedhamsters) after carcinogen administration. Three weeks aftertreatment, hepatotoxicity of NNK and NDMA was still detectedin hamsters, but not in rats. These results suggest that thetoxic effects of NNK and NDMA initiate a regenerative processthat occurs faster in rat than in hamster liver. Since hepatocarcinogenesisoccurs in NNK- but not in NDMA-treated rats, promutagenic lesionsgenerated from NNK might be fixed preferentially during cellproliferation.     

Persistent structural change in replicating hepatic DNA isolated from diethylnitrosamine-treated rats

Structural analysis, by benzoylated O-(diethylaminoethyl) (DEAE)-cellulose chromatography, was made of DNA from the livers of rats receiving 100 mg/kg diethylnitrosamine and subsequently subjected to partial hepatectomy. Under these conditions, different DNA labelling procedures permit damage to be associated with pre-existing or newly synthesised DNA. Persistent single stranded regions could be detected in DNA isolated more than 3 days after carcinogen treatment only if the animals were subjected to hepatectomy. This damage was attributable to lesions impeding DNA replication. Induction of proliferative activity up to at least 14 days after nitrosamine treatment made manifest DNA damage, the extent of which was not decreased as the interval between carcinogen treatment and surgery was increased.     

Comparative tumor initiating activities of N-nitrosomethylbenzylamine and N-nitrosodimethylamine in rat liver

The tumor initiating activities of nitrosomethylbenzylamine(NMBzA), an esophageal carcinogen, and nitrosodimethylamine(NDMA), a hepatocarcinogen, were compared in rat liver usingmodifications of the initiation assays of Tsuda et al. (CancerRes., 40, 1157, 1980) and Pitot et al. (Nature, 271, 456, 1978).Equimolar doses of NMBzA or NDMA (33.5 µmol/kg) were injectedi.p. into male Sprague-Dawley rats 18 h after partial hepatectomy.Following selection, foci of preneoplastic hepatocytes weredetected by histochemical staining for / glutamyltranspeptidase.Nitrosomethylamylamine (NMAmA), 115 µmol/kg), also anesophageal carcinogen, was tested in the assay of Tsuda et al.,and nitrosodiethylamine (NDEA, 33.5 µmol/kg), a hepaticand esophageal carcinogen, was tested in the assay of Pitotet al. Both NDMA and NDEA induced significant increases in thenumber of preneoplastic foci above background. In contrast,neither NMBzA nor NMAmA increased the number of foci above background.Microsomes from regenerating liver had a lower capacity to metabolizeboth NDMA and NMBzA compared to microsomes from intact liver,but the decrease in activity was similar for both compounds.Neither NDMA nor NMBzA significantly inhibited the first waveof DNA synthesis in vivo in the regenerating liver. The resultsdemonstrate that in contrast to NDMA and NDEA, NMBzA and NMAmAlack tumor initiating activity in the liver.     

Formation of 5-hydroxymethyl-2'-deoxyuridine in hepatic DNA of rats treated with gamma-irradiation, diethylnitrosamine, 2-acetylaminofluorene or the peroxisome proliferator ciprofibrate

The peroxisome proliferator ciprofibrate was tested for its ability to induce DNA damage in the form of 5-hydroxymethyl-2'-deoxyuridine (HMdU), an adduct that results from the reaction of thymine in DNA with hydroxyl radicals. In order to quantify HMdU, DNA containing [3H]thymidine of high specific activity had to be obtained. Since hepatocytes normally have a very low rate of DNA synthesis, rats were subjected to partial hepatectomy to stimulate DNA synthesis and then were administered [methyl-3H]thymidine by three p.o., i.p. or i.v. injections 20, 22 and 24 h after partial hepatectomy; or by slow infusion through the portal vein, starting 20 h after partial hepatectomy for 4 h. The specific activity of DNA in rats receiving [3H]thymidine through the portal vein was considerably higher than in rats receiving p.o., i.p. or i.v. injections. Rats were then exposed to various doses of gamma-irradiation after partial hepatectomy and infusion of [6-3H]thymidine through the portal vein. DNA from the liver was extracted, enzymatically hydrolyzed and analyzed by HPLC. The percentage of HMdU in DNA increased in a dose-dependent manner. Rats were then treated with the carcinogens 2-acetylaminofluorene (AAF) or diethylnitrosamine (DEN) in conjunction with partial hepatectomy and infusion of [methyl-3H]thymidine. There was an increase in HMdU formation after a single administration of DEN or AAF. Another group of rats was fed a diet containing the peroxisome proliferator ciprofibrate for 3 weeks. After partial hepatectomy and infusion of [6-3H]thymidine, these rats were fed the same ciprofibrate-containing diet for 2-4 more weeks. HMdU was detected in DNA at 2-4 weeks after [6-3H]thymidine infusion, but the level at 4 weeks was nearly 50% less than at 2 weeks. This study shows that oxidative DNA damage in the form of HMdU is induced in the liver by gamma-irradiation, DEN, AAF and peroxisome proliferation.     

In vivo and in vitro genotoxicity of several N-nitrosamines in extrahepatic tissues of the rat.

Toxicological mechanisms involved in organotropism of tumor induction may include cell-specific metabolic activation of the carcinogen, in vivo distribution of active metabolites and persistance of induced DNA damage. In order to elucidate which factors are involved in the organotropic action of environmentally relevant N-nitrosamines, we have studied their genotoxic and cytotoxic effects within primary intact cells of lung and kidney. The end-points determined were cytotoxicity by trypan blue exclusion and DNA single-strand break (SSB) induction by alkaline filter elution. The assays were performed in vitro to determine organ-specific metabolic activation by incubating the cells with the test compounds. The results obtained with N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodiethanolamine (NDEIA), N-nitrosoethylvinylamine (NEVA), N-nitrosodibutylamine (NDBA), N-nitrosobutylbutanolamine (NBBOH), N-nitrosobutylcarboxypropylamine (BCPN), N-nitrosomethylbenzylamine (NMBzA) and N-nitrosodibenzylamine (NDBzA) indicate that several compounds may be activated to reactive metabolites by cells of kidney and lung, NDBzA revealing the highest degree of cytotoxicity. In contrast, genotoxicity in kidney cells was induced only by NBBOH and BCPN and at relative low levels. Primary lung cells could not be employed as indicators for genotoxic effects in vitro because the cell yield was not sufficient to perform the alkaline elution assay. To assess the distribution of NDMA in the whole rat and the persistence of the induced DNA damage in the two organs, further studies were carried out after oral application of 1, 2, 4, 10, 20, 32 and 40 mg/kg to the animals. Following 1 h exposure of rats to NDMA, the lowest effective genotoxic dose for lung and kidney was 2 mg NDMA/kg body wt. A plateau was achieved after a dose of 20 mg/kg in both organs. Furthermore, the persistence of DNA damage was studied in the lung. After 4 h exposure, DNA damage was still detectable at 32 mg NDMA/kg, but for the lower doses it was reduced nearly to control levels. After 16 h exposure the SSB rate in lung cells was reduced for all dose levels except for the highest dose of 40 mg/kg.     

Comparative tumorigenicity and DNA methylation in F344 rats by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosodimethylamine

The tumorigenic activities and DNA methylating abilities in F344 rats of the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the structurally related nitrosamine N-nitrosodimethylamine (NDMA) were compared. Groups of 30 male rats were given 60 s.c. injections of 0.0055 mmol/kg of either NNK or NDMA over a 20-week period (total dose, 0.33 mmol/kg). The experiment was terminated after 104 weeks. The numbers of rats with tumors were as follows for NNK and NDMA, respectively: liver, 10 and 6; lung 13 and 0; and nasal cavity, 6 and 1. NNK was significantly more tumorigenic than was NDMA toward the lung (P less than 0.01) and nasal cavity (P less than 0.05). Groups of rats were treated with a single s.c. injection of 0.39 mmol/kg or 0.055 mmol/kg of NNK or NDMA and the levels of 7-methylguanine and O6-methylguanine were measured in liver, lung, and nasal mucosa 1-48 h after treatment. In liver and lung, levels of 7-methylguanine and O6-methylguanine in DNA were 3-22 times (P less than 0.001) greater in NDMA treated rats than in NNK treated rats. Levels of methylation induced by NDMA and NNK in the nasal mucosa were similar. The results of this study demonstrate that NNK is a more potent tumorigen than NDMA in the F344 rat and suggest that DNA methylation alone does not account for its strong tumorigenicity in rat lung and nasal mucosa.     

Formation of O6-methylguanine in regenerating rat liver by N-nitrosomethylbenzylamine is not sufficient for initiation of preneoplastic foci

The DNA methylating activities of N-nitrosodimethylamine (NDMA),an initiator of hepatic -glutamyltranspeptidase-positive foci,and N-nitrosomethylbenzylamine (NMBzA), which does not initiate,were studied in regenerating rat liver. Equimolar doses of ¹⁴C-labelledNDMA and NMBzA (33.5 µmol/kg) were administered to maleSprague-Dawley rats 18 h after partial hepatectomy. NDMA andNMBzA both produced 7-methylguanine and O⁶-methylguanine. Theresults suggest that although the formation of O⁶-methylguaninemay be necessary it is not sufficient for initiation of preneoplasticfoci.     

Effect of food restriction on the metabolism of dimethylnitrosamine (NDMA) in rats

The influence of food restriction on the macromolecular interactions of the hepatocarcinogen dimethylnitrosamine (NDMA) in the livers of male Sprague Dawley rats was investigated. Two-three month old rats were food restricted (FR) (40% with respect to ad libitum fed rats) for three weeks. The liver weight, total protein, microsomal and cytoplasmic protein, cytochrome P-450 and DNA content per whole liver were all reduced significantly in food restricted rats. Five hours after a single dose of (14C) NDMA (28 mg/k.b.w., 21 microCi/rat) the levels of 7-methylguanine increased in restricted rats by 32%. Cytochrome P-450 mediated generation of HCHO from NDMA (1.8 fold) in restricted rats was greater. Binding of NDMA derived radioactivity to total hepatic proteins decreased by 46% in restricted rats. These results suggest that food restriction enhances the metabolic activation of dimethylnitrosamine in Sprague Dawley rats.     

Effect of magnesium on nickel-induced genotoxicity and cell transformation

Raising the extracellular level of magnesium ions inhibitednickel-induced DNA strand breaks, DNA-protein crosslinks, sisterchromatid exchanges, chromosomal aberrations and cell transformation.Carcinogenic nickel ions preferentially damaged centromeresand other heterochromatic regions of Chinese hamster ovary cellchromosomes. Elevation of extracellular magnesium levels preventedthe effects of nickel on heterochromatin and inhibited celltransformation, but did not substantially reduce the DNA damageinduced by nickel in euchromatic regions. This study suggeststhat heterochromatic DNA damage may be important to the nickel-inducedneoplastic transformation process.     

二甲基亚硝胺诱导大鼠DNA O^6—甲基鸟嘌呤的免疫组化研究

本文应用双PAP法对二甲基亚硝胺(N-nitrosodjmethylamine,NDMA)诱导大鼠DNAO~6—甲基鸟嘌呤(O~5—methylguanine,O~6—mG)进行了研究。分别给大鼠一次腹腔注射NDMA 1,2,3,5,10,30,50mg/kg,5小时后大鼠产生O~6—mG具有明显的剂量反应关系以及器官和细胞特异性。随着NDMA剂量的增加,产生O~6—mG的器官种类和细胞类型增加,阳性细胞形成O~6—mG的量也增加。小剂量(1—5mg/kg)NDMA仅见肝组织的细胞形成O~6—mG,NDMA剂量达50mg/kg时,肝、肾、肺、气管,食管和鼻咽组织的细胞均检出O~6—mG。     

The relationship between N-nitrosodimethylamine metabolism and DNA methylation in isolated rat hepatocytes

The metabolism of N-nitrosodimethylamine (NDMA) and its methylationof DNA were simultaneously determined in hepatocytes isolatedfrom untreated and saline- and pyrazole-treated male Sprague-Dawleyrats. Metabolism of NDMA was directly measured by monitoringits disappearance via gas chromatography coupled with a sensitiveand specific detector for N-nitrosamines. DNA methylation wasdetermined in the same cells employed in the metabolism studiesusing a monoclonal antibody-based competitive ELISA procedurespecific for O⁶-methyldeoxyguanosine (6-Me-dG). The apparentK_m and V_max, for NDMA metabolism are 61 µM and 56 pmol/min/10⁶cells respectively for hepatocytes isolated from untreated rats.It was found that the addition of pyrazole to the in vitro hepatocyteincubations caused a dose-dependent inhibition of both metabolismand DNA methylation. However, when DNA methylation is expressedas a function of NDMA metabolized, there is no significant differencebetween hepatocyte incubations without or with pyrazole, withan average value of 79 nmol 6-Me-dG/mol dG/nmol NDMA metabolized.Based on the pyrazole inhibition studies, cyto-chrome P450IIE1is responsible for at least 60% of the DNA methylation in rathepatocytes. In pyrazole-pretreated rats there was an inconsistentincrease in NDMA metabolism, but when metabolism was elevatedso was DNA methylation. In contrast, microsomes isolated frompyra zole-pretreated rats consistently showed elevated metabolismof NDMA. Based on the simultaneous determination of adduct levelsand metabolism, there is 1 6-Me-dG adduct formed/133 000 NDMAmolecules metabolized in the uninduced hepatocytes.     

The effect of N-nitrosodimethylamine and N-nitroso-N-benzylmethylamine on [3H]thymidine incorporation into the DNA of target and non-target tissues in the zinc-deficient rat

The influence of dimethylnitrosamine (NDMA), a liver carcinogen and nitrosobenzylmethylamine (NBMA) as esophageal carcinogen on [3H]thymidine incorporation into DNA was studied in the esophagus, liver, forestomach and gastric-stomach of fasted zinc-deficient and pair-fed zinc-sufficient rats, measured 1 h after the thymidine injection. In the untreated animals, dietary zinc deficiency significantly depressed [3H]thymidine incorporation (89%) into the DNA of forestomach only. NDMA, administered 4 h before death at 30 mg/kg, produced 50-55% inhibition in [3H]thymidine incorporation in the esophagus of rats of both dietary groups. This inhibition became more pronounced in the forestomach, reaching 90-94% in the zinc-deficient forestomach and 63-86% in their zinc-sufficient counterparts at NDMA levels ranging from 5 to 20 mg/kg. NBMA at 2 mg/kg produced 60% inhibition in the DNA synthesis of zinc-deficient esophagus and 40% in the corresponding zinc-sufficient ones, this difference being significant at P less than 0.01. On the other hand, [3H]thymidine incorporation in the forestomach DNA was markedly lowered in the presence of NBMA. Recovery of DNA synthesis in the 4 tissues from a single dose of NDMA or NBMA was monitored up to 12 days. Following NDMA injection, [3H]thymidine incorporation in the forestomach of both dietary groups remained inhibited (3% of untreated control) for 5 days, a significant recovery (45% of untreated control) was observed only in the zinc-sufficient animals. Following NBMA injection, [3H]thymidine incorporation was also inhibited in the zinc-deficient esophagus for a longer time than in the zinc-sufficient ones. In autoradiographic studies, the percentage of cells showing 30 or more grains/nucleus was significantly decreased (P less than 0.001) in the NBMA-treated and marginally decreased (P less than 0.05) in the NDMA- or NBMA-treated zinc-deficient and zinc-sufficient rats as compared with the saline-treated zinc-sufficient controls. These results were discussed in the light of our previous findings that NBMA enhanced esophageal tumorigenesis in the zinc-deficient rats and that NDMA, a liver carcinogen produced forestomach tumors in the zinc-deficient but not in the zinc-sufficient rats.     

The nature of microsomal N-nitrosodimethylamine demethylase and its role in carcinogen activation

The subcellular localization of N-nitrosodimethylamine (NDMA)demethylase in rat liver cells and the role of this enzyme inthe activation of NDMA were studied. The enzyme activity waspredominantly located in the microsomal fraction and the additionof cytosol to the microsomes did not produce a synergistic effect.The microsomal NDMA de-methylase activity appeared to correspondto the ability of the microsomes to convert NDMA to the alkylatingspecies. The alkylation of DNA was assayed by the measurementof O⁶-methylguanine and 7-methylguanine by fluorescence afterisolation by h.p.l.c. The induction of microsomal NDMA de-methylaseactivity by isopropanol, ethanol, acetone, and fasting was closelyrelated to the increase of DNA methylation by NDMA in an incubationsystem in vitro. Pretreatment of rats with ethanol, which inducedheaptic NDMA demethylase activity, enhanced DNA alkylation invivo with a high dose of NDMA (75 mg/kg body weight) but notwith low doses of NDMA (25 mg/kg body weight). The role of NDMAde-methylase in the activation of NDMA is discussed.     

Nafenopin-induced rat liver peroxisome proliferation reduces DNA methylation by N-nitrosodimethylamine in vivo

The hypolipidaemic drug nafenopin (NAF) has been shown to enhancethe hepatocarcinogenic effect of N-nitrosodimethylamine (NDMA)and N-nitrosodiethylamine in rats. We have investigated whetherthe NAF-induced peroxisome proliferation in hepatocytes interfereswith NDMA's metabolism and interaction with DNA. Adult maleWistar rats received a single i.p. injection of [¹⁴C]NDMA (2mg/kg) and were killed 4 h later. DNA was isolated from liverand kidney, hydrolysed in 0.1 N HCI and analysed by Sephasorbchromatography. In rats pre-treated with NAF (0.2% in the dietover a period of 3 weeks), the concentration of N7-methylguaninein hepatic DNA (µmol/mol guanine) was 46% below controlvalues. This is probably due to the greater amount of targetDNA, as NAF caused a marked hepatomegaly with a 50% increasein total liver DNA content. Concentrations of N7-methylguaninein kidney DNA were twice as high in NAF-pre-treated animalswhen compared to control rats. This is unlikely to result froma shift in the metabolism of NDMA from liver to other rat tissuessince the time course and extent of the conversion of [¹⁴C]NDMAto ¹⁴CO₂ and ¹⁴C-labelled urinary metabolites were identicalin NAF-treated and control animals. There was no indicationthat NAF inhibits the activity of the hepatic O⁶-alkylguanine-DNAalkyltransferase.     

Effect of diallyl sulfide on aristolochic acid-induced forestomach carcinogenesis in rats

In this study the development of aristolochic acid (AA) inducedtumors in rats with and without diallyl sulfide (DAS) was studied.Experiments were also conducted to establish the effects ofDAS administration on AA-derived DNA single-stranded regionsand DNA adduct formation in the forestomach of such animals.Forestomach, urinary bladder and thymus tumors were inducedin male BD-6 rats after oral treatment for 12 weeks with AA(2 x 10 mg/kg/week). Administration of 150 mg/kg DAS intragastrically4 h prior to AA treatment reduced significantly the number ofrats that developed forestomach tumors (6–9 months afterthe start of experiment). The incidence of AA-induced forestomachtumors was 10% (two out of 20 rats) after co-administrationof DAS and 60% (12 out of 20 animals) when AA was administeredalone. The high dose of DAS (2 x 150 mg/kg) markedly inhibitedthe formation of squamous cell carcinomas in the forestomach.However, the thioether did not prevent the formation of forestomachand urinary bladder papillomatosis. Additionally, DAS co-administrationdecreased the accumulation of single-stranded regions in ratforestomach DNA. Using the nuclease P1 enhancement method ofthe ³²P-postlabeling assay, a decrease in the level of AA-derivedadducts was also detected after co-administration of DAS. Weconclude that the decrease of DNA damage after DAS co-administrationis associated with the delay in conversion of papillomas tomalignant forestomach tumors.     

Nitrosodimethylamine metabolism in rat ovaries. Interactions of its metabolites with nucleic acids and proteins

N-Nitrosodimethylamine (NDMA) was metabolized by ovarian slices of noninbred Sprague-Dawley rats (200-250 g body wt, to CO2 and to reactive metabolites that bind covalently to nucleic acids. That ability was about 10 or 5 times smaller than the one observed in liver slices, respectively. Both ovarian microsomes and mitochondria were able to biotransform NDMA to formaldehyde and to reactive metabolites that bind covalently to proteins. Formaldehyde formation by microsomes was significantly higher than that by ovarian mitochondria but of the same order of magnitude. Ability to lead to covalent binding to proteins in microsomes was not significantly different from that in the respective mitochondrial fraction. DNA isolated from ovarian slices activating NDMA revealed the presence of the altered bases 7-methylguanine (7-MeGua) and O6-methylguanine (O6-MeGua) resulting from NDMA reactive metabolites' attack. Results suggest potential, mutagenic, carcinogenic and reproductive risks derived from women's exposure to NDMA present in tobacco smoke, food, beverages, workplace or other environmental sources.     

A possible mechanism for the dose-response relationship observed for renal mesenchymal tumours induced in the rat by a single dose of N-nitrosodimethylamine

The incidence of renal mesenchymal tumours induced in rats by N-nitrosodimethylamine (NDMA) is related to the dose in a sigmoidal dose-response curve. Each kidney bears only one or two tumours at 20-24 months. In contrast, one week after dosing, a large number of preneoplastic proliferative foci is present, the incidence of which is linearly related to dose and directly proportional to methylation of DNA by NDMA. It is suggested that most of these foci are removed by host defense mechanisms before they can progress to tumour, thus accounting for the sigmoid shape of the dose-response curve for the tumours.     

Estimation of the fraction of the dose of N-nitrosodimethylamine metabolized to methylamine in rats

Despite many years of research on the metabolism of N-nitrosodimethylamine (NDMA) in rats, the significance of enzymatic denitrosation as a pathway remains unclear. To assess the role of this pathway of metabolism in rats, animals were administered NDMA by intravenous infusion at two infusion rates until steady state was achieved and the concentrations of NDMA (Css,NDMA) and methylamine (MA) (Css,MA), a product of the enzymatic denitrosation pathway, were determined in plasma. The clearance of NDMA (ClNDMA) from plasma was determined by dividing the infusion rate by Css,MA. The plasma clearance of MA (ClNDMA) was determined in a separate experiment. The fraction of the dose of NDMA metabolized by enzymatic denitrosation (fm) was calculated using the equation fm = (Css,MA*ClMA)/(Css,NDMA*ClNDMA). By this method it was estimated that 29% of the dose of NDMA was metabolized via the enzymatic denitrosation pathway. Thus enzymatic denitrosation is an important pathway in the metabolism of NDMA in rats.     

Inhibition of poly(ADP-ribose) synthesis may affect DNA repair prior to ligation

The effects of modification of poly(ADP-ribosyl)atlon reac tionshave been examined In normal (F107) and ataxia telangieclasia(AT23) flbroblasts following damage by methyl methanesuiphonate(MMS) and u.v. light. The technique of benzoylated DEAE (BD)-cellulosechromatography was util ized to estimate both the extent andnature of the damage to DNA Induced by these agents and to examinethe effects of an inhibitor of poly(ADP-ribose) synthetase,3-aminobenz- amide (3AB), on these parameters. Single strandbreakage, determined by nucleoid sedimentation, and levels ofpoly ADP(ribose) synthesis were monitored. Increase in the proportion of DNA containing single-stranded regions, as m by stepwiseelution from BD-cellulose, was observed follow ing MMS damageIn both cell types. In the presence of 3AB, a further accumulationof DNA containing single-stranded regions occurred, with theeffect being more prominent in AT23 fibroblasts. U.v. lightdamage did not induce increased binding to BD-cellulose in normalcells, and the increase observed in AT23 cells was much lessthan that seen follow ing alkylatlon damage. Examination ofthe nature of single- stranded damage by caffeine gradient elutlonfrom BD-cell ulose following MMS treatment revealed discretestructural lesions, which were enhanced in the presence of 3AB.A similar effect was exerted by arabinofuranosyl cytosine. Thebehaviour of these intermediates, which could be associatedwith repair, was not in accord with the suggestion that 3ABInhibits only the ligation stage of the repair process. Ourresults suggest that specific intermediate stages in DNA repairare sensitive to 3AB, and it seems likely that these stagesoccur prior to ligation.