Sex-dependent promoting effect of polychlorinated biphenyls on enzyme-altered islands induced by diethylnitrosamine in rat liver

The promoting effect of Clophen A 50, a commercial mixture ofpolychlorinated biphenyis (PCBs) on preneoplastic islands, initiatedby diethylnitrosamine (DEN), was studied in male and femaleSprague-Dawley rats. The islands were identified histochemicallyby loss of adenosine-5'-triphosphatase (ATPase) and/or emergenceof gamma-glutamyltranspeptidase (GGTase). Treatment with 12x 8 mg DEN/kg body wt./day initiated a similar number and totalarea of islands in males and females. Additional weekly applicationof Clophen A 50 (50 or 100 mg/kg body week, for 7 weeks) enhancedthe number of ATPase-deficient islands 3-fold in males and 9-foldin females. The total area was increased 4-fold in males and15-fold in females. Number and area of GGTasepositive islandswere similarly enhanced. The emergence of a small number ofislands after application of Clophen A 50 alone may indicatea weak carcinogenic potency. PCB treatment caused an increasein liver weight, which amounted to 55% in males and 20% in femalescompared to controls. This increase is partly due to cell hypertrophy,as indicated by determination of cell size. The mitogenic activityof Clophen A 50 was evaluated by measurement of the mitoticindex of unaltered hepatocytes at 24, 48 h, and 7 days afterapplication of a single dose (100/mg/kg body wt.) of ClophenA 50. The mitotic index in control animals of both sexes was0.3%, and was enhanced 8-fold in males, 24 h after PCB treatment.In females only a slight, non-significant increase was observed.The results indicate that the sex-dependent promoting effectof Clophen A 50 is independent from its mitogenic action.     

Correlation of O4-ethyldeoxythymidine accumulation, hepatic initiation and hepatocellular carcinoma induction in rats continuously administered diethylnitrosamine

Recent experiments have demonstrated that O⁶-ethyldeoxy-guanosine(O⁶-EtdG) is efficiently repaired while O⁴-ethyl-deoxythymidine(O⁴-EtdT) accumulates in hepatocyte DNA of 8-week-old F-344rats during continuous diethylnitrosamine (DEN) administration.To determine if O⁴-EtdT accumulation correlates with hepaticinitiation, we have quantitated O⁴-EtdT concentrations, andthe incidence of-glutamyl transferase positive (GGT+) foci andhepatocellular carcinoma induced by increasing duration of exposureto DEN in the drinking water (40 p.p.m.). In 8-week-old F-344rats the number of GGT + foci increased non-linearly with durationof exposure and reached a maximum of 500 foci/cm³ after 10 weeks.Administration of DEN to 8-week-old F-344 rats for 6 weeks followedby a 15-week administration of 0.05% phenobarbital (PB) in thediet did not result in the induction of hepatocellular carcinoma.Exposure of 4-week-old F-344 rats to DEN for up to 10 weeksproduced an O₄-EtdT steady-state concentration (7–10 x10^–6 mol O⁴-EtdT/mol dT) similar to that previously observedafter administration of DEN to 8-week-old F-344 rats. However,the maximal concentration of O⁴-EtdT was detectable after ashorter period of DEN administration in the younger rats. Theincidence of GGT+ foci also increased more rapidly in 4-week-oldrats, but again plateaued at 500 foci/cm³ after 4, 6 or 8 weeksof DEN administration. A 100% incidence of hepatocellular carcinomaoccurred in 4-week-old rats administered DEN for 6, 8 or 10weeks, followed by promotion with 0.05% PB in the diet untilweek 22 of the study. Lower incidences of bepato-cellular carcinoma(89 and 6%) were observed following PB-promotion of rats administeredDEN for 4 and 2 weeks, respectively. The influence of age onDEN-induced hepatic initiation was examined further by quantitatingGGT + foci induced by 4 weeks of DEN administration in groupsof rats which were 4–14 weeks old at the start of thecarcinogen exposure. The results demonstrated that the youngerrats were 15-fold more susceptible than the older rats to theinitiating effects of DEN. This growth-dependent effect on hepaticinitiation in the presence of nearly equivalent amounts of pro-mutagenicDNA damage further implicates the necessity of replication forhepatic initiation.     

Persistent effect of a low dose of preadministered diethylnitrosamine on the induction of enzyme-altered foci in rat liver

The effect of a low dose of preadministered diethylnitrosamine(DEN) on the induction of enzyme-altered foci in the liversof male full-grown Fischer 344 rats was studied. As a pretreatment,DEN at a dose of 10 mg/kg body wt was injected i.p. At varioustimes after DEN pretreatment a complete initiation, consistingof administration of the same dose of DEN by the same routein rats subjected to partial hepatectomy (PH), was performed,followed by application of selection pressure. Enzyme-alteredfoci stained with -glutamyltrans-peptidase (-GTP) and glutathioneS-transferase placental form (GST-P) were then assayed. Decreasesin the numbers and areas of foci in the rats which receivedsaline + PH 14 or 28 days after DEN pretreatment were observedin comparison with rats which received saline + PH immediatelyafter DEN. On the other hand, the numbers and areas of fociwere not decreased in rats which received the complete initiation,consisting of DEN + PH, at various times after DEN pretreatmentwhen compared with rats which received these at the same timeas the DEN pretreatment. This persistent effect of DEN pretreatmenton the complete initiation lasted up to 182 days after the timeof DEN pretreatment. In this experiment, GST-P was found tobe a more sensitive marker for the detection of putative preneoplasticliver-cell foci than -GTP.     

Pancreatic acinar cell tumors in rats induced by 3, 2'-dimethyl-4-aminobiphenyl

Pancreatic acinar cell lesions, including foci, nodules, adenomasand in situ carcinomas, were found in male F344 rats given s.c.injections of 3, 2'-dimethyl-4-aminobiphenyl (DMAB) at dosesranging from 50 to 167 mg/kg body weight in two 60-week experiments.The pancreatic lesions induced by DMAB were essentially thesame as those reported in rats treated with 4-hydroxyaminoquinoline-1-oxideand azaserine. DMAB at high doses induced pancreas acinar cellas well as fat necrosis, and tumorigenicity for the pancreaswas most effective at high necrogenic levels of carcinogen exposure.Mean labeling indices in basophilic foci, acidophilic foci,nodules and adenomas assessed by bromodeoxyuridine incorporationwere elevated to0.69, 2.17, 1.43 and 0.96% respectively, incontrast to the value of 0.39% observed for the surroundingnormal acinar cells. The increase was significant in the acidophilicfoci and nodules and the fact that these lesions had very highlabeling indices suggests that they may have potential for progressionto tumors.     

1,N6-etheno-2' -deoxyadenosine and 3,N4-etheno-2'-deoxycytidine detected by monoclonal antibodies in lung and liver DNA of rats exposed to vinyl chloride

1,N⁶-Etheno-2'-deoxyadenosine (dAdo) and 3,N⁴-Etheno-2'-deoxycytidine(dCyd) are formed in vitro by reaction of DNA with the electrophilicmetabolites of vinyl chloride (VC), chloroethylene oxide andchloroacetaldehyde. To detect and quantitate these DNA adductsin vivo, we have raised a series of specific monoclonal antibodies(Mab). Among those, Mab EM-A-1 and Mab EM-C-1, respectively,were used for detection of dAdo and dCyd by competitive radioimmunoassay(RIA), following pre-separation of the etheno adducts from DNAhydrolysates by high perfonnance liquid chromatography. At 50%inhibition of tracer-antibody binding, both Mab had a detectionlimit of 187 fmol and antibody affinity constants (K) of 2 x10⁹ l/mol. The levels of dAdo and dyd were quantitated in theDNA of lung and liver tissue of young Sprague-Dawley rats exposedto 2000 p.p.m. of VC for 10 days. The dAdo/2'-deoxyadenosineand dCyd/2'-deoxycytidine molar ratios were 1.3 x 10^–7and 3.3 x 10^–7 respectively, in lung DNA, and 5.0 x 10^–8and 1.6 x 10^–7 in liver DNA. When hydrolysates of 3 mgof DNA were analyzed by RIA at 25% inhibition of tracer-antibodybinding, dAdo and dCyd were not detected in liver DNA from untreatedrats above the limiting dAdo/2'-deoxyadenosine and dCyd/2'-deoxycytidinemolar ratios of 2.2 x 10^–8 and 3.1 x 10^–8, respectively.     

Repair of premutagenic DNA lesions in human fetal tissues: evidence for low levels of O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase activity in some tissues

The activities of the DNA repair enzymes O⁶-methylguanine-DNAmethyltransferase and uracil-DNA glycosylase, and the replicativeenzyme DNA polymerase , were measured in extracts of human fetaltissues at 18–20 weeks of gestation. In general, O⁶-methylguanine-DNAmethyltransferase activities in fetal tissues were in the samerange as in the corresponding adult tissues, except for fetalliver which had 5-fold lower activity. Uracil-DNA glycosylasewas, surprisingly, 4-fold lower in fetal tissues compared withadult tissues. Since a critical factor in carcinogenesis maybe the rate of repair relative to DNA replication, the activitiesof O⁶-methyl-guanine-DNA methyltransferase and uracil-DNA glycosylasewere compared with the DNA polymerase activity in the sameextract. When expressed in this way, O⁶-methylguanine-DNA methyltransferaseactivity was lowest in liver and brain and 2- to 14-fold higherin kidney, lung, colon, stomach, small intestine and pancreas.The ratio of uracil-DNA glycosylase to DNA polymerase variedless between different organs. These findings indicate thatseveral fetal organs may be more sensitive than adult organsto some alkylating agents that are known to occur in the environment.Furthermore, the lower capacity of DNA repair is not restrictedto repair of alkylation damage, since the activity of uracil-DNAglycosylase is also lower than in adult tissues.     

Promotion of preneoplastic lesions and induction of CYP2B by unleaded gasoline vapor in female B6C3F1 mouse liver

An initiation-promotion protocol was used to test the hypothesisthat unleaded gasoline (UG) vapor acts as a liver tumor promoterin female mice under exposure conditions in which UG was hepatocarcinogenicin a cancer bioassay. Twelve day old female B6C3F1 mice wereinjected with N-nitrosodiethylamine (DEN, 5 mg/kg, i.p.) orvehicle. Starting at 5–7 weeks of age, mice were exposedby inhalation 6 h/day, 5 days/week for 13 weeks to 0 or 2039p.p.m. of PS-6 blend UG, the same gasoline blend used in thecancer bioassay. Putative preneoplastic lesions in liver, characterizedmainly as basophilic foci in H&E-stained liver sections,were found exclusively in mice treated with DEN. While similarnumbers of altered hepatic foci were found in DEN-initiatedmice treated with 0 or 2039 p.p.m. UG, UG treatment significantlyincreased both the mean volume (3.2-fold) and the volume fraction(3.6-fold) of the foci. To determine if UG induced CYP2B, asubfamily of cytochrome P450 commonly induced by liver tumorpromoters in rodents, pentoxyresorufin-O-dealkylase (PROD) activitywas assayed in hepatic microsomes derived from the above livers.UG vapor increased hepatic PROD activity 8-fold, while increasingcytochrome P450 content only 30%. To ascertain if a more recentblend of UG, API 91-1, would have similar biological effectsas PS-6, female B6C3F1 mice were gavaged for 3 days with cornoil or 1800 mg/kg/day PS-6 or API 91–1 blend UG. PS-6and API 91-1 blend UG induced similar increases in relativeliver weight (25%), PROD activity (9-fold) and hepatocyte labelingindex (8-fold) relative to controls. These data demonstratethat PS-6 blend UG vapor promotes preneoplastic lesions andinduces CYP2B in female mouse liver under exposure conditionsin which it causes liver tumors, and suggest that a more recentblend of UG may have similar effects.     

Inhibition of carcinoma formation and of vascular invasion in grafts of radiation-initiated thyroid clonogens by unirradiated thyroid cells

Quantitative transplantation techniques have been employed tostudy radiogenic cancer initiation frequency and cell interactionsduring promotion/progression in grafted clonogenic rat thyroidepithelial cells. The graft recipients were surgically thyroidectomizedand maintained on a diet containing <50 ng iodine per g.The results confirm that radiogenic initiation is a common cellularevent; one of 32 surviving 5-Gy-irradiated thyroid clonogensgave rise to cancer in grafts initially containing 11 clonogensper transplantation site. The data demonstrate that the efficiencyof promotion/progression is inversely related to grafted irradiatedcell number. As the number of transplanted surviving irradiatedclonogens was increased progressively from 11 to 720 clonogensper graft site, the carcinoma frequency per grafted clonogenprogressively decreased to one per920. Addition of un-irradiatedthyroid cells to the transplant inocula further suppressed promotion/progressionof radiation-initiated thyroid clonogens. Furthermore, the probabilityof vascular invasion, a reflection of metastatic potential incarcinomas which arose from irradiated grafted thyroid clonogens,was reduced by addition of unirradiated thyroid cells to thetransplant inocula. Assays of thyroid stimulating hormone (TSH)titers in the sera of thyroidectomized rats 44 weeks after transplantationof clonogenic thyroid cells indicate that the suppression ofneoplastic promotion/progression observed with increased numbersof cells per graft site is due at least in part to feed-backinhibition of TSH production by thyroid hormone of graft origin.Whether local cellular interactions are also involved in thisinhibitory process is currently under investigation.     

Specific binding of the tumor promoter TPA in various mouse organs as measured by a 'cold acetone-filter assay'

Rapid, specific, saturable and partially reversible bindingof the tumor promoter 12-O-tetradecanoylphorbol-13-acetate ([³H]TPA)to a particulate fraction of mouse brain can be demonstratedby means of a ‘cold acetone-filter assay’; by washingwith cold acetone at -20°C, nonspecific binding of the highlylipophilk [³H]TPA to membranes can be reduced to 10% of thetotal binding. A comparative study of homogenates of severalorgans with this test revealed specific [³H]TPA binding/ µgDNA in the order brain > lung spleen Over kidney thymus ovaries. Specific binding sites were also detected hi 600 xg supernatant fractions of homogenates from epidermis, forestomach,glandular stomach and small intestine. Competition experimentsshowed displacement of [³H]TPA by TPA > phorbol-12,13-didecanoate> 12-deoxyphorbol-13-tetradecano-ate > phorbol-12,13-dibutyrate(PDBu) > phorbol-12,13-diacetate > 4-O-methyl-TPA >4-phorbol-12,13-didecanoate in the brain particulate fraction.Specific [³H]TPA binding is sensitive to heat, periodate anddithiothreitol, but is relatively insensitive to urea or to1–5% solutions of several common detergents. It is estimatedthat the present binding test is 500 times more sensitive thanthe widely-accepted [³H]PDBu assay; perhaps more important,the present method employs TPA, which is extremely effectiveboth as a tumor promoter in vivo and as a pleiotropic effectorof cells in vivo and in vitro.     

Differential regulation of two microsomal epoxide hydrolases in hyperplastic nodules from rat liver

Two microsomal epoxide hydrolases of the rat liver were foundto be differentially regulated in hyperplastic nodules. Whilstthe activity for substrates of the well-known microsomal epoxidehydrolase with a broad substrate specificity (EHb), benzo[a]pyrene4,5-oxide and androstene oxide (16,17-epoxyandrosten-3-one),was greatly (5-fold) increased in the nodule microsomes andmoderately (2-fold) increased in the surrounding tissue, thatfor the substrate of the novel microsomal epoxide hydrolase,cholesterol 5, 6-oxide (EH_ch) remained unchanged. Since bothenzymes convert endogenous steroid epoxides but with distinctstructural features, this differential regulation may indicatea role of endogenous steroid epoxide(s) of a defined structureduring hepatocarcinogenesis. Alternatively, this differentialregulation may serve as a marker during hepatocarcinogenesis.     

Sensitivity of rat and mouse peripheral blood lymphocytes to BaP adduction and SCE formation

Both mice and rats were injected i.p. with doses of benzo[a]pyrene(BaP) ranging from 10 to 100 mg/kg to and compare species sensitivityto and the relationship between sister chromatid exchange (SCE)induction and DNA adduct formation. Twenty-four hours afterinjection, blood was removed by cnrdiac puncture and the peripheralblood lymphocytes (PBLs) were analyzed for both DNA adduct formationby ³²P-postlabeling and SCE induction following lymphocyte culture.BaP induced similar, but not identical, SCE dose-response curvesfor each species. After BaP administration, the major DNA adduct,N²-[10ß-(7ß, 8, 9-trihydroxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene)y1]deoxy-guanosine(BPDEI-dGuo), was 10-fold more prevalent in the PBLs of themouse than those of the rat. Thus, for equivalent amounts ofBPDEI-dGuo, a greater number of SCEs are induced in the ratthan the mouse.     

Inhibitory effect of {alpha}-tocopherol on the formation of nitrosomorpholine in mice treated with morpholine and exposed to nitrogen dioxide

The possibility that -tocopherol (vitamin E) inhibits the formationof nitrosomorpholine (NMOR) in vivo was investigated in miceorally pretreated with -tocopherol (2.5–100 mg/kg bodywt) once daily for 6 days. Twenty-four hours later, the animalswere injected i.p. with 2 mg of morpholine (MOR) per animalfollowed by exposure txo 47 p.p.m. of NO₂ for 2 h. Under theseconditions, low oral doses of -tocopherol (2.5–5 mg/kgbody wt) significantly decreased NMOR formation in vivo. Astotal body -tocopherol levels increased, in vivo NMOR formationdecreased, and a maximal 50–70% inhibition of NMOR formationoccurred at -tocopherol levels of 5 µg/g body wt. Additionalresults showed that NMOR was rapidly eliminated in mice, sothat studies which measure the levels of NMOR found in animalstreated with MOR and then exposed to NO₂ may underestimate theamount of NMOR that is actually formed. Finally, oral pretreatmentof up to 100 mg of -tocopherol/kg body wt had no effect on NMORelimination.     

Immunohistochemical localization of O6-ethyldeoxyguanosine and deoxyguanosin-8-yl-(acetyl)aminofluorene in liver sections of rats treated with diethylnitrosamine, ethylnitrosourea or N-acetylaminofluorene

Antibodies raised in rabbits against the bovine serum albuminconjugates of O⁶-ethylguanosine (O⁶-EtGuo) and N-(guanosin-8-yl)-N-acetyl-2-aminofluorene(Guo-8-AAF) have been used in a double peroxidase-antiperoxidasestaining assay to visualize the localization of DNA-O⁶-ethyldeoxyguanosine(O⁶-EtdGuo) and some of the interaction products of N-acetyl-2-aminofluorene(AAF) with DNA guanine in liver sections of rats treated withdiethylnitrosamine (DEN), ethylnitrosourea (ENU), or AAF respectively.O⁶-EtdGuo could be detected in nuclei of parenchymal cells afterinjection of DEN (12 – 50 mg/kg) or ENU (140 mg/kg). Cleartime and dose dependencies were observed. The lowest dose ofDEN which, at 5 h after injection, resulted in immunohistochemicallydetectable levels of O⁶-EtdGuo, was 12 mg/kg; 5 h after 6 mg/kgno consistent difference between treated and untreated ratscould be observed. A striking heterogeneity in staining patternwas observed after DEN: centrilobular regions were stained muchmore than peripheral zones. At 7 days after a single DEN injectionof 50 mg/kg small rims of significantly stained hepatocytescould still be observed around the central veins. No heterogeneityof staining pattern was observed 2 h after ENU. ENU, in contrastwith DEN, also resulted in a significant staining of nonparenchymalcells. At 24 h after ENU no significant staining of hepatocytescould be detected, but positive staining was still present inbile duct cells, vascular endothelial cells and sinusoidal cells.The results indicate a detection level of 5 µmol O⁶-EtdGuo/molDNA-P, i.e., 5 x 10⁴ O⁶-EtdGuo residues per diploid genome.Using the anti-Guo-8-AAF antiserum, positive results were obtained6 days after a single AAF dose of 0.5 – 10 mg/kg, correspondingto a detection limit of 0.4 µmol dGuo-8-(A)AF/mol DNA-P.Staining was rather homogenously distributed over the liverlobules. Persistency of the AAF-DNA interaction products wasinvestigated both after 10 and 2 mg/kg AAF. Increased nuclearstaining could be observed up to 8 weeks after 10 mg/kg and4 weeks after 2 mg/kg.     

A combination of palytoxin with 1-oleoyl-2-acetyl-glycerol (OAG) or insulin or interleukin-1 synergistically stimulates arachidonic acid metabolism, but combinations of 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-type tumor promoters with OAG do not

The combination of palytoxin and 1-oleoyl-2-acetyl-glycerol(OAG) synergistically stimulates production of 6-keto-PGF₁ andPGF₂ by rat liver cells (the C-9 cell line). In contrast, thecombination of 12-O-tetradecanoylphorbol-13-acetate (TPA)-typetumor promoters (TPA, dihydroteleocidin B, aplysiatoxin, phorbol-12,13-didecanoate)and OAG does not. Production of 6-keto-PGF₁ by palytoxin addedwith recombinant murine interleukin-1 (IL-1) or with insulinis also greater than the sum of the two effects taken independently.Palytoxin and OAG individually stimulate the release of radio-labeledcompounds from the rat liver cells pre-labeled with [³H]arachidonicacid and also act synergistically to release labeled metabolites.After separation by h.p.l.c., these materials co-chromatographwith authentic 6-keto-PGF₁ and arachidonic acid. The synergisticstimulation by palytoxin and OAG is biphasic; a rapid synergisticproduction of 6-keto-PGF₁ or release of radiolabel from [³H]arachidonicacid prelabeled cells is followed, after 2 –4 h, by aprolonged synergistic response.     

Glutathione conjugation and DNA-binding of ({+/-})-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and ({+/-})-7{beta},8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in isolated rat hepatocytes

Isolated rat liver hepatocytes, previously depleted of glutathione(GSH) by treatment with diethylmaleate, were allowed to incorporate[³H]glycine into their GSH. Incubation of ³H-labelled cellswith ¹⁴C-labelled (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene((±)-BP-7,8-dihydrodiol) or (±)7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene(()-BPDE) revealed the formation of double labelled products.This together with evidence from amino acid analysis indicatesformation of GSH-conjugates of the highly carcinogenic BP-derivatives.Incubation of hepatocytes isolated from 3-methylcholanthrene(MC) treated rats with ³H-labelled (±)-BP-7,8-dihydrodiolor (±)-BPDE resulted in binding of radioactivity to DNA.Reduction of the intracellular level of GSH to 40% of the normallevel resulted in an approximate 2-fold increase in the DNA-bindingof either substrate. In addition there was a concurrent decreasein the amount of GSH-conjugates formed. These data clearly demonstratethat GSH participates in conjugation reactions with carcinogenic(±)-BP-7,8-dihydrodiol and (±)-BPDE and that theintracelluilar level of GSH is important in preventing reactiveintermediates from reacting with the DNA in intact cells.     

Enhancement of DEN initiation of liver carcinogenesis by inhibitors of NAD+ ADP ribosyl transferase in rats

The effect of inhibitors of NAD⁺ ADP ribosyl transferase (ADPRT)on the early stage of liver carcinogenesis of diethylnitrosamine(DEN) was studied by estimating the number and size of -glutamyltranspeptidase(-GTP) positive foci assayed as markers of cell populationsinitiated by DEN in the rat liver. ADPRT inhibitors investigatedwere 3-amino-benzamide (ABA), 5-methylnicotinamide (MNAM), andthymidine. A single i.p. injection of ABA at > 150 mg/kgbody weight (B.W.) enhanced dose-dependently the induction of-GTP positive foci in rat liver initiated by 20 mg/kg B.W. ofDEN. The magnitude of the effect was similar to that observedwhen partial hepatectomy (PH) was performed instead of ABA administration.Single i.p. injections of MNAM or thymidine at a dose of 600mg/kg B.W. also enhanced the induction of foci in rat liverinitiated by the 20 mg/kg dose of DEN. Based on the above results,ABA was used as a representative ADPRT inhibitor for clarifyingthe mechanisms underlying the effects. Administration of ABAat a dose of 600 mg/kg B.W. was effective in enhancing the inductionof foci if given 1 day before DEN, simultaneously to DEN, and1 day after DEN initiation but it was ineffective if it wasgiven 3 days after DEN or thereafter. Liver cell necrosis wasnot detectable either by analysis of serum enzymes or histologically1, 3, 5, 7 and 14 days after 20 mg/kg B.W. of DEN with or withoutadministration of 600 mg/kg B.W. of ABA. No initiating activitywas observed for ABA administered at doses of 600 and 1200 mg/kgB.W. as assayed by development of -GTP positive foci. Long termABA administration in the diet at concentrations of 0.05, 0.1and 0.2% did not show any promoting activity for liver carcinogenesisinitiated by DEN. Furthermore, chronic administration of 0.2%ABA in the diet did not result in detectable toxicity and/orcarcinogenic effects. These results suggest that ADPRT and associatedDNA repair plays an important role in the early initiating stageof liver carcinogenesis and provide the basis for a new experimentalapproach to the analysis of the mechanisms of chemical carcinogenesisand in establishing a more sensitive assay system for livercarcinogenesis in rats.     

Utilization of 1, N6-Etheno-2'-deoxyadenosine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli

To test whether vinyl chloride-induced mutagenesis might involveambiguous base pairing of 1, N⁶-etheno-adenine (A) during DNAsynthesis, we examined the base pairing potential of dATP duringDNA synthesis catalyzed by Escherichia coli DNA polymerase I(Klenow fragment). An electrophoretic assay of chain elongationwas used to assess the degree to which dATP could substitutefor each of the normal dNTPs during elongation of a primer annealedto a bacteriophage template. Despite the fact that the ethenobridge completely blocks normal Watson-Crick pairing of A withT, we observed that dATP could substitute for dATP during primerelongation (although inefficiently). In addition, detectablesubstitution of dATP for dGTP and dCTP occurred, indicatingthat A exhibits ambiguous base pairing properties. The relativeease of dAMP incorporation (opposite template T, C and G) appearedto vary considerably at different positions along the template.The major, form of eA incorporation (replacement of A) was confirmedby measurements of dATP-dAMP turnover (a commonly used methodfor detecting misincorporation), and also by the demonstrationthat A was present in enzymatic hydrolysates prepared from DNAthat was synthesized with dATP replacing dATP. A model for ambiguousbase pairing of dATP is proposed, in which incorporation occursvia the protonated, syn form of dATP.     

Development of monoclonal antibodies specific for 1,N2-ethenodeoxyguanosine and N2,3-ethenodeoxyguanosine and their use for quantitation of adducts in G12 cells exposed to chloroacetaldehyde

Monoclonal antibodies specific for N²3-ethenodeoxyguanosine(N²,3-dGuo) and 1,N²-ethenodeoxyguanosine (1,N²-dGuo) were developed.In a competitive ELISA, 50% inhibition of binding of the N²,3-dGuospecific antibody (ETH1) was achieved with 18 fmol of N²,3-dGuo.Fifty per cent inhibition of the 1,N²-dGuo-specific antibody(ETH2) required 11 pmol 1,N²-dGuo. Immunoassays for N²,3-dGuoand 1,N²-dGuo in single-stranded DNA were developed using theseantibodies. The immunoassays could detect as little as 48 fmolof N²,3-dGuo or 340 fmol 1,N²-dGuo in 25 µg of singlestranded DNA. These assays and previously developed immunoassaysfor 1,N⁶-ethenodeoxyadenosine (1,N⁶-dAdo) and 3,N⁴-ethenodeoxycytidine(3,N⁴-dCyd) were used to measure etheno adduct levels in DNAof cells exposed to chloroacetaldehyde. The cells used wereV79 cells with an inactivated hprt gene and a single copy ofthe bacterial gpt gene (G12 cells). The most abundant ethenoadduct was 1,N⁶-dAdo, followed by 3,N⁴-dCyd and N²,3-dGuo. 1,N²-dGuowas not detected in chloroacetaldehyde-treated G12 cells. Chloroacetaldehydewas also shown to be mutagenic in these same cells.     

Viscometric analysis of DNA damage in kidney and lung following exposure of rats to small doses of chemical carcinogens

A new viscometric technique has been used lo detect DNA damagein kidney and lung of rats treated with six chemical carcinogens.In alkaline conditions (pH 12.5) the reduced viscosity (_red)of kidney and lung DNA from control rats increased slowly withtime reaching a maximum, (_red)^max, after 9–12 h. Carcinogens,by inducing DNA strand breaks either chemically or indirectlyby excision repair or during incubation in alkali, cause a reductionof DNA supercoiling which can be sensitively measured by monitoringchanges in viscosity. Computerized analysis of time-viscositycurves showed that a statistically significant reduction ofthe time required for (_red) to reach 95% of its maximum value(t-95) was induced by the following single i.p. doses: N-nitrosodimethylamine(DMN), kidney 0.07 mg/kg, lung 0.28 mg/kg; N-nitrosodiethylamine(DEN), kidney 3.2 mg/kg, lung 12.8 mg/kg; N-nitroso-N-methylurea,kidney and lung 0.5 mg/kg; 1,2-dimethyIhydrazine (DMH), kidney1 mg/kg, lung 16 mg/kg; 4-nitroquinoline-l-oxide (NQO), kidney2.5 mg/kg, lung 0.63 mg/kg; 2-acetylamino-fluorene, kidney andlung 12.5 mg/kg. The decrease of t-95 was constantly dose-related.The comparison with data previously obtained from liver demonstratesthat DMN, DEN, DMH and NQO caused the greatest amount of DNAdamage in the organ most susceptible to tumor induction. Viscositychanges elicited by DMN, DEN and DMH are quantitatively wdlcorrelated with the extent of DNA alkyla-tion.