Comparisons of the effects of chemical carcinogens in mixed cultures of rat hepatocytes and human fibroblasts

Unscheduled DNA synthesis (UDS) was measured simultaneouslyin rat hepatocytes and human fibroblasts when combined culturesof the 2 cell types were exposed to procarcinogens. Human fibroblastswere preincubated with 5-bromo-2'-deoxyuridine (BRdU) to substitutefor thymidine in the DNA. Hepatocyte DNA was separated fromthe heavier BRdU-substituted fibroblast DNA by isopycnic centrifugationsin neutral cesium chloride and the specific activities of theDNA's were determined. In the presence of hepatocytes, benzo[a]pyrene(BP) induced more UDS in the fibroblasts than in the hepatocytes.BP induced no UDS in the fibroblasts in the absence of hepatocytes.Diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) stimulateda significant amount of UDS only in the hepatocytes. Thus, theco-cultures of hepatocytes and fibroblasts responded with UDSto these chemical carcinogens in a manner that parallels thetissue specificity of the carcinogenicity of these chemicalsin vivo. That is, the known hepatocarcinogens DEN and AAF, onlystimulated significant UDS in the hepatocytes, whereas the non-hepatocarcinogen,BP, though activated in these cultures mainly by the hepatocytes,stimulated more UDS in the fibroblasts than in the hepatocytes.The amount of [³H]BP bound to DNA was investigated in the co-culturesand in cultures of fibroblasts alone. When co-cultures wereexposed to [³H]BP the fibroblast DNA had approximately 3 timesmore BP bound to it (per µg DNA) than did the hepatocyteDNA. The amount of [³H]BP bound to the DNA of cultures of fibroblastsalone was 29% of the amount bound to the DNA of the fibroblastsfrom the co-cultures. Thus, although the hepatocytes were mainlyresponsible for the activation of BP, more [³H]BP was boundto the fibroblast DNA. It is suggested that the intercellulardistribution of carcinogenic metabolites may be a significantdeterminant of the carcinogenic effect.     

SHORT COMMUNICATION: Time-Related introduction of DNA repair synthesis in rat hepatocytes following in vivo treatment with cyproterone acetate

Cyproterone acetate (CPA), a synthetic steroid hormone widelyused as a human pharmaceutical, has recently been shown to induceunscheduled DNA synthesis (UDS) in vitro in primary culturesof rat and human hepatocytes. In the present study CPA was evaluatedfor its ability to initiate UDS in the liver of female ratsin vivo by means of the in vivo/in vitro hepatocyte DNA repairtest. Using the standard sampling time of 16 h after singleoral dosing, a dose-related UDS responce at >50 mg CPA/kgbody wt was observed. In order to examine the time course ofCPA induced UDS, different sampling times (4, 16, 48, 72, 96,and 144 h) after a single oral administration of 100 mg CPA/kgbody wt were used. Whereas no UDS was induced in liver cellsisolated 4 h after treatment, continuous DNA repair activitywas observed after 16 h, with a maximum effect of     

Failure of the peroxisome proliferator WY-14,643 to induce unscheduled DNA synthesis in rat hepatocytes following in vivo treatment

Peroxisome proliferator hepatocarcinogens lack genotoxic activityin numerous in vitro assays using non-target cells which donot respond with peroxisome proliferation. Therefore, the effectof in vivo treatment with WY-14,643 on DNA repair was quantitatedin rat hepatocytes, the target cell for carcinogenesis. PalmitoylCoA oxidase and carnitine acetyltransferase activities in isolatedhepatocytes were elevated by WY-14,643 (50 mg/kg/day by gavagefor up to 5 consecutive days) and by WY-14,643 (0.1%) or di(2-ethylhexyl)phthalate (DEHP) (1.2%) feeding (for up to 28 days), indicatingperoxisome proliferation had occurred. DNA repair as unscheduledDNA synthesis (UDS) was measured autoradiographically as netnuclear grains following thymidine incorporation in primaryhepatocyte cultures. Treatment of rats with WY-14,643 (gavageor feeding) or DEHP (feeding) did not induce UDS. Addition of2-acetylaminofluorene to replicate cultures demonstrated thatWY-14,643 or DEHP treatment did not prevent repair response.Additional cultures were treated with H₂O₂ (0.8 mM H₂O₂ 3 xat 1-h intervals) to evaluate the ability of UDS to detect anyrepair which may be induced by peroxisomal metabolism. H₂O₂did not induce UDS at this concentration, nor did it prevent2-acetylaminofluorene- induced repair. UDS was, however, observedin a separate experiment using a higher concentration of H₂O₂.In summary, a highly carcinogenic peroxisome proliferator didnot induce UDS in the target cell for carcinogenesis in spiteof peroxisome proliferation following in vivo treatment.     

Induction of DNA repair in rat spermatocytes and hepatocytes by 1,2-dibromoethane: the role of glutathione conjugation

1,2-Dibromoethane (EDB) is a widely used industrial chemical,and a well-known mutagen and carcinogen. EDB is biotransformedeither by cytochrome P450-dependent oxidation, leading to theformation of bromoacetaldehyde, or by enzyme-catalyzed conjugationwith glutathione, giving rise to reactive half-sulfur mustardcompounds and their derivatives. In vitro mutagenicity and DNAbinding studies suggest that the latter pathway is the primarysource of genotoxic metabolites from EDB. In this study we haveexamined EDB -induced unscheduled DNA synthesis (UDS) in F-344rat pachytene spermatocytes and hepatocytes. EDB (10–100µM) induced UDS in both hepatocytes and spermatocytesin vitro. In contrast, only hepatocytes exhibited UDS when isolatedfrom rats given EDB (100 mg/kg) 2 h earlier, and only then ifthe compound was given i.p. rather than orally. Preincubationof hepatocytes or spermatocytes with inhibitors of cytochromeP450-mediated oxidation had no effect on EDB induction of UDSin vitro. In contrast, depletion of cellular glutathione stronglyinhibited EDB-induced UDS in both cell types in vitro. Treatmentof rats with 175 mg metyrapone/kg (an inhibitor of hepatic mixed-functionoxidases) 1 h prior to administration of EDB in vivo had noeffect on EDB-induced UDS in hepatocytes, but led to a positiveUDS response to EDB in spermatocytes in vivo. This suggeststhat the mixed-function oxidase pathway of metabolism is theprimary route of clearance of EDB and that inhibition of cytochromeP450-mediated oxidation led to a more extensive tissue distributionof the parent compound. These data also suggest that the pathwaywhich produces genotoxic metabolites from EDB in hepatocytesand spermatocytes, in vitro and in vivo, involves the conjugationof EDB to glutathione and its subsequent metabolism.     

Cell growth state determines susceptibility of repair DNA synthesis to inhibition by hydroxyurea and 1-beta-D-arabinofuranosylcytosine

The effects of inhibitors of replicative DNA synthesis on repairDNA synthesis have been examined by autoradiography in severaldifferent cell types and in cells in different growth states.Hydroxyurea (HU) and 1-ß-D-arabino-furanosylcytosine(ara C), administered together, influence unscheduled DNA synthesis(UDS) in a manner which is independent of the status of thecell culture (normal or transformed) and of the species, butwhich is strongly affected by whether the cells are proliferatingor quiescent. In proliferating human, Chinese hamster and Microtuscell cultures, UDS is not inhibited by HU and ara C, and mayeven appear to be stimulated. In quiescent cultures of thesecells UDS is reduced by HU and ara C. In cells reseeded froma confluent culture and followed during proliferation and backto quiescence the effect of inhibitors parallels the growthpattern. The results are interpreted in terms of changes inthe sizes of endogenous DNA precursor pools; they underlinethe potential problems associated with quantitating UDS in thepresence of inhibitors.     

肝癌高发区饮用塘水浓缩物诱发人肝细胞非程序DNA合成的研究

利用人原代肝细胞非程序ＤＮＡ合成试验（ＵＤＳ）对广西某肝癌高发区的居民饮用塘水浓缩物进行检测，结果发现：塘水浓缩物的浓度在０．１０ｍｇ／ｍｌ－０．５０ｍｇ／ｍｌ时，可诱导人肝细胞ＵＤＳ反应，对ＤＮＡ具有明显的损伤作用，且显示出一定的剂量一效应关系，表明塘水中存在有基因毒性物质，提示该地区居民的肝癌高发与长期饮用糖水有关。     

Effect of glutathione depletion and inhibition of glucuronidation and sulfation on 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism, PhIP-DNA adduct formation and unscheduled DNA synthesis in primary rat hepatocytes

The potent rat colon carcinogen 2-amino-l-methyl-6-phenylimidazo[4,5-d]pyridine (PhIP), unlike other food-borne heterocyclicamines, does not induce tumors in rat liver. This correlateswith an extremely low level of PhIP-DNA adducts formed in thistissue, and together these observations suggest that PhIP isefficiently detoxified in the liver. In order to identify possibledetoxification mechanisms, we assessed the effect of inhibitionof glucuronidation, glutathione (GSH) conjugation and sulfationon PhIP metabolism and PhlP-induced DNA damage in rat hepatocytes.Hepatocytes isolated from rats pretreated with Aroclor 1254metabolized PhIP to the same products found in vivo. N-Hydroxy-PhIPN3-glucuronide and N-hydroxy-PhIP N²-glucuronide were majorand minor metabolites respectively. ³²P-Postlabeling analysisof DNA from the PhIP-treated hepatocytes indicated the presenceof two major adducts, one of which was identified as N-(deoxyguanosin-8-yl)-PhIP,and one minor adduct. There was no unscheduled DNA synthesis(UDS) in these cells. However, pretreatment of the hepatocyteswith 1-bromoheptane and buthionine sulfoximine, which depletesGSH and prevents its resynthesis, resulted in a 15-fold increasein the formation of PhIP-DNA adducts, as well as in a high levelof UDS. GSH depletion had no effect on the formation of detectablePhIP metabolites. Hepatocyte pretreatment with D-galactosamine,which inhibits glucuronidation, increased the formation of DNAadducts two-fold and UDS was increased similarly. D-Galactosaminedecreased the formation of the two N-glucuronides of N-hydroxy-PhIPby 50–60%, but had no effect on other metabolites. Pentachlorophenol,which strongly inhibits sulfotransferases, decreased adductformation slightly, but had essentially no effect on UDS oron the formation of PhIP metabolites. These results indicatethat metabolic conjugation pathways involvingGSH and glucuronidationmay play an important role in protecting rat liver against PhIPcarcinogenesis.     

Examination of genotoxicity, toxicity and morphologic alterations in hepatocytes following in vivo or in vitro exposure to methapyrilene

The antihistamine methapyrilene (MP) was widely used as a componentof cold, allergy and sleep-aid medications in the 1970s untilit was identified as a potent rat liver carcinogen. MP doesnot induce positive responses in most short-term genotoxicityassays, which suggests that it is carcinogenic by a non-genotoxicmechanism. We have evaluated the potential of MP to induce unscheduledDNA synthesis (UDS), a genetic end point and S-phase synthesis(SPS), an indicator of cell proliferation, in Fischer-344 (F344)rat and B6C3F1 mouse liver. We also examined the response ofMP in hepatocytes from two species treated in vitro. MP failedto induce UDS in rat or mouse liver following in vivo treatment,or in hepatocytes from rat and adult human treated in vitro.Control rats and mice yielded <0.3% of cells in S-phase (%S).In contrast, MP induced significant elevations in SPS both inmale F344 rat (6.3%S) and female B6C3F1 mice (1.4%S). In themale rat, sorbitol dehydrogenase (SDH), bilirubin, serum glutamicoxaloacetic transaminase (SGOT) and serum glutamic pyruvatetransaminase (SGPT) showed elevations of 9-, 10-. 17- and 28-foldover controls respectively, indicating that significant hepatotoxicitywas induced by MP. This was confirmed by histopathologic examination,which revealed significant periportal and focal necrosis followedby an increased presence of mitotic figures. These results indicatethat MP is not genotoxic in rat liver, but is a potent inducerof hepatic cell proliferation by inducing toxicity and subsequentregeneration, which may be an important mechanism of hepatocarcinogenesis.     

Sequential analysis of DNA damage and repair during the development of carcinogen-induced rat liver hyperplastic lesions

The level of DNA fragmentation, as evaluated by alkaline elution, and of unscheduled DNA synthesis (UDS), as measured by autoradiography, was determined in the parenchymal cells from the entire liver during the development of hyperplastic lesions induced in the rat by the following treatment: diethylnitrosamine (DEN) (200 mg/kg i.p.) on Day 0; CCl4 (2 ml/kg intragastrically) on Day 21; dietary administration of 0.02% 2-acetylaminofluorene during the third and the fourth wk; and of 0.05% phenobarbital from the sixth wk. Both DNA fragmentation and UDS were constantly detected, concomitantly with the presence of gamma-glutamyltransferase (gamma-GT)-positive hepatocytes, in the primary cultures derived from the liver of rats of this experimental group sacrificed at 4, 5, 6, and 7 wk after DEN injection, their amount being approximately the same at the fourth and at seventh wk. Moreover, evidence of DNA alterations was still present, albeit diminished, 22 wk after the beginning of treatment. In contrast, DNA fragmentation and UDS did not persist past the fifth wk, and gamma-GT-positive hepatocytes were very few or totally absent in hepatocyte primary cultures from control rats treated with DEN alone, 2-acetylaminofluorene alone, or 2-acetylaminofluorene:CCl4. CCl4 alone, and phenobarbital alone caused only a modest, albeit statistically significant, increase in DNA elution rate and UDS, respectively. In a comparison performed on hepatocyte primary cultures obtained from rats of the experimental group sacrificed at the fifth wk after the injection of DEN, the level of UDS was higher in gamma-GT-positive than in gamma-GT-negative hepatocytes. These results indicate that the regimen used to induce the selective proliferation of initiated hepatocytes actually produces extensive DNA lesions which can give rise to additional carcinogenic initiations.     

Carcinogen-induced unscheduled DNA synthesis in xenotransplanted human tracheobronchial epithelium: comparison with rat tracheal epithelium

Extrapolation from rodent genotoxicity data to humans is complicatedby variables such as interspecies differences in carcinogenmetabolism and DNA repair. A xenograft system containing humanbronchial epithelial cells was used to assess the inductionof unscheduled DNA synthesis (UDS) by carcinogens and to comparethe response with that of rat tracheal epithelium. Cells fromhuman bronchus were grown in explant culture, inoculated intode-epithelialized rat tracheas and implanted subcutaneouslyinto nude mice. Within six weeks, a differentiated mucociliaryepithelium lined the xenografted tracheas. Fresh rat tracheasand human xenografts were cut into rings and incubated in mediacontaining [³H]thymidine and either the direct-acting car cinogen,N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 3–1000 µM),or a carcinogen requiring metabolic activation, 4-nitroquinoline-1-oxide(4-NQO, 3–100 pM). Tissues were then fixed, sectioned,processed for autoradiography and the number of nuclear grains(NG) determined for 100 epithelial cells lining the tracheain each section. A time- and concentration-dependent increasein NG was observed in both human xenografts and rat tracheasafter treatment with MNNG or 4-NQO, indicating induction ofUDS by these agents. The UDS response to MNNG in the human xenograftswas similar to that observed in the rat tracheas, whereas theresponse to 4-NQO was greater in rat tracheas. These studiesindicate that the human xenograft system should have applicationsfor the study of carcinogen-induced damage in normal human bronchialepithelial cells.     

Formation and repair of benzo[a]pyrene--DNA adducts in cultured hamster tracheal epithelium determined by 32P-postlabeling analysis and unscheduled DNA synthesis

Hamster tracheal organ cultures were used to investigate therelationship between DNA adduct formation measured directlyby the ³²P-postlabeling assay, and the DNA damage measured indirectlyby the unscheduled DNA synthesis (UDS) assay. Hamster tracheaswere treated with three concentrations of benzo[a]pyrene (B[a]P)for 2 days. Postlabeling and UDS assays were also carried outa few days after removal of the B[a]P. Furthermore, the typesof B[a]P—DNA adducts formed in the in vitro organ culturewere qualitatively compared with those formed in vivo afterintratracheal intubation of B[a]P attached to Fe₂O₃ particles.In vivo only one adduct was detected by ³²P-postlabeling. Thisadduct co-chromatographed with the trans-addition produce ofdG and (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE). In vitro, a clear B[a]P-DNA adduct pattern was alsofound with the ³²P-postlabeling assay. Four different adductswere found. The main adduct spot migrated to the same positionon the thin-layer chromatogram as the in vivo adduct. B[a]P-DNAadduct formation was both time-and dose-dependent. During thefirst day after removal of B[a]P the adduct levels still increased,thereafter they decreased at all B[a]P concentrations. A time-and dose-dependent increase in UDS was observed in the trachealepithelial cells treated with B[a]P in vitro. After removalof the B[a]P, UDS decreased immediately, in contrast to theformation of DNA adducts. The results of the present study showthat B[a]P induces time- and dose-dependently both DNA adductsand UDS in hamster tracheal organ culture. Moreover, the mainDNA adduct formed in vitro, dG-(+)-anti-BPDE, was the same asthat found in vivo.     

Lack of genotoxic activity of di(2-ethylhexyl)phthalate (DEHP) in rat and human hepatocytes

Di(2-ethylhexyl)phthalate (DEHP) is a widely used plasticizerwhich has been reported to induce a statistically significantincrease in the incidence of hepatocellular carcinomas in femaleFischer-344 rats (8/50) when administered in the diet at 12000 p.p.m. for two years. Numerous studies with cells in culturehave failed to show any genotoxic activity associated with DEHP.Because DEHP induces multiple changes in the liver, such asperoxisomal proliferation, it was possible that these alterationscould result in genotoxic effects in the treated whole animalthat would not be seen in cells in culture. Accordingly, theability of DEHP to induce DNA damage or repair was examinedin rat hepatocytes in vivo and in vitro and in human hepatocytesin vitro. Unscheduled DNA synthesis was measured by incorporationof [³H]thy-midine into primary hepatocyte cultures immediatelyisolated from treated animals or hepatocyte cultures incubateddirectly with DEHP. DNA damage was measured by alkaline elutionof cellular DNA from the same cultures. In vivo-in vitro treatmentregimens were: (i) female rats, 12 000 p.p.m. DEHP in the dietfor 30 days; (ii) female rats, 12 000 p.p.m. in the diet for30 days, followed by 500 mg/kg DEHP by gavage 2 h before sacrifice;(iii) male rats, 500 mg/kg DEHP by gavage 2, 12, 24, or 48 hbefore sacrifice; and (iv) male rats, 150 mg/kg/day by gavagefor 14 days. In vitro conditions were 0.1, 1.0 and 10.0 mM DEHPin the cultures for 18 h. Primary cultures of human hepatocyteswere prepared from freshly discarded surgical material and exposedto the same concentration of DEHP. Concentrations up to 0.5mM mono(2-ethylhexyl)phthalate, a principal metabolite of DEHP,were also examined in the human hepatocyte assay. No chemicallyinduced DNA damage or repair was observed in vivo or in vitroin rat or human hepatocytes under any of the conditions employed.However, an increase in the percentage of cells in S-phase inthe animals given DEHP was observed. These data indicate thatDEHP does not exhibit direct genotoxic activity in the animalseven with a treatment regimen which eventually produced tumorsin a long term bioassay, and that both rat and human hepatocytesare similar in their lack of a genotoxic response to DEHP exposurein culture.     

Role of cytochrome P450 in DNA damage induced by N-nitrosodialkylamines in cultured rat hepatocytes

The involvement of cytochrome P450 in the cytotoxicity and DNAdamage-repair induced by N-nitrosodipropylamine (NDPA), N-nitroso-n-butyl-n-propylamine(NBPA) and N-nitrosodibutylamine (NDBA) was investigated incultured hepatocytes isolated from untreated, phenobarbital(PB)-and pyridine (PYR)-pretreated rats. Pretreatment of ratswith PB caused a 10-fold increase in the sensitivity of hepatocytesto the cytotoxic actions of NDPA, NBPA and NDBA as measuredby trypan blue exclusion, whereas PYR pretreatment increasedthe sensitivity of hepatocytes to NDPA and NBPA, but not toNDBA. This elevated sensitivity correlated well with increased7-pentoxyresorufin depentylase activity catalyzed by P450 2B1in cultures from PB-pretreated rats and enhanced p-nitrophenolhydroxylase activity of P450 2E1 in cultures of hepatocytesfrom PYR-pretreated rats. Unscheduled DNA synthesis showed thatDNA damage-repair was significantly increased in freshly isolatedhepatocytes from PB- and PYR-pretreated rats. With increasingtime in culture, however, there was a marked reduction in theDNA damage repair response, concomitant with a decrease in thecytotoxicity of NDPA, NBPA and NDBA in primary cultures of hepatocytes.Coincident with this, a rapid loss in the specific activitiesof P450 2B1 and 2E1 was detected during the first 48 h in allprimary cultures. Although N-nitrosodimethylamine (NDMA), usedas a positive control, produced high nuclear grain counts incultures from PYR-pretreated rats, the toxic effect of NDMAin rat hepatocytes was much weaker than that observed with NDPA,NBPA and NDBA. This result suggests that the type of DNA damageor repair efficiency induced by NDPA, NBPA or NDBA might differfrom that due to NDMA.     

Occupational and in vitro exposure to styrene assessed by unscheduled DNA synthesis in resting human lymphocytes

Lymphocytes from 38 individuals occupationally exposed to styreneconcentrations in workroom air of 1 p.p.m. to 40 p.p.m. wereexamined for any genotoxic effects using unscheduled DNA synthesis(UDS) as the indicator of DNA damage. The mean level of N-acetoxy-2-acetylaminofluorene(NA-AAF) induced UDS was significantly increased (p < 0.001)for the styrene exposed group when compared to the mean levelfor the unexposed controls. There was no significant effecton u.v.-induced UDS from the in vivo styrene exposure. Lymphocytecultures exposed in in vitro to styrene concentrations up to100 µM have confirmed the UDS data collected on individualsoecupationally exposed to styrene. In addition, the in vitrostudy has also shown that the increased NA-AAF induced UDS resultingfrom styrene exposure was paralleled by a similar increase inNA-AAF binding to DNA. Taken together these results indicatethat styrene exposure does not inhibit DNA repair synthesis,but rather it predisposes lymphocytes to an increased risk forDNA damage induction from subsequent genotoxic exposures.     

Genotoxic activity of 1-chloromethylpyrene in stomach epithelium in vivo: insensitivity of the stomach scintillation UDS assay

An acknowledged weakness of current testing programmes for genotoxichazard has been the potential insensitivity of the establishedmouse bone marrow micronucleus test and rat liver unscheduledDNA synthesis (UDS) assays to direct-acting or short-lived mutagens,which may be consumed at the site of initial contact. In suchcases, in vivo test systems sampling tissues such as the skinor the stomach would provide valuable data. To test these principlesa stomach UDS assay was evaluated using the potent locally activemutagen 1-chloromethylpyrene (1-CMP). Contrary to expectations,no UDS response was observed 16 h following 1-CMP dosage byoral gavage. To confirm the integrity of the 1-CMP used forthe stomach UDS assay, a sample of the stored chemical was re-evaluatedin vitro and shown to be still strongly positive in the Amesassay and to have alkylating activity at least 15 min afterincubation at stomach acid pH. No UDS response was observedwhen test dose levels were reduced or when earlier samplingtimes were used. Other genotoxic endpoints were examined instomach. ³²P-Postlabelling analysis revealed high levels ofadduct formation in gastric DNA. An assay utilizing electrophoresisof DNA (the comet assay) showed the occurrence of DNA damagefollowing dosing with 1-CMP in vivo. These positive resultsconfirmed that 1-CMP should be regarded as a potential in vivogenotoxin. The failure to detect a UDS response to 1-CMP instomach was investigated; a strong UDS response was observedin an in vitro hepatocyte UDS assay of 1-CMP indicating thatthe rat was capable of repairing 1-CMP-derived DNA adducts.Pretreatment of rats with hydroxyurea depressed the level ofincorporation of thymidine into DNA both in negative and positive[methyl-N-nitrosoguanidine (MNNG)] controls. The results ofthese studies indicated that the protease digestion method employeddid not selectively or efficiently sample those cells with anyUDS response to 1-CMP or MNNG, and the activity seen for thelatter was most likely due to the presence of S phase cellswithin the digests. As a result of the finding that UDS responseswere not demonstrated for the potent direct-acting mutagens1-CMP and MNNG, the protease digestion/scintillation methodfor stomach UDS does not appear to have general value in a screeningprogramme for locally active genotoxic agents.     

Correlation between induction of unscheduled DNA synthesis in the liver and excretion of mutagenic metabolites in the urine of rats exposed to the carcinogenic air pollutant 2-nitrofluorene

The genotoxic effects of 2-nitrofluorene (NF) have been studiedin vivo by measuring the induction of DNA repair, i.e. unscheduledDNA synthesis (UDS), in hepatocytes from male rats pretreatedby oral gavage with NF. During the NF exposure, Urine was collectedfor 24 h, and its mutagenicity was investigated in the plateincorporation assay, using Salmonella TA98 as tester strain.The urine samples were also used for the identification of excretedmetabolites of NF. Rats treated with 2-acetylaminofluorene (AAF)were studied simultaneously. A positive UDS response was observed12 and 24 h following a single gavage exposure to 12.5, 25 and50 mg/kg NF, with the response returning to near control levelsby 36 h. The positive control AAF induced approximately twicethe response observed with NF, and both compounds gave a UDSresponse that was 2–3 times higher in Wistar rats relativeto Sprague-Dawley rats. A potent directacting mutagenic effectwas observed in urine samples after NF treatment, while AAFexposure only gave rise to a weak mutagenic effect, the NF/AAFratio being 10/1. The stronger urinary mutagenicity after NFtreatment relative to AAF treatment was associated with thepresence of hydroxylated NFs. The genotoxic effect observedin the liver after NF treatement is, on the other hand, morelikely due to the same AAF metabolites that are also formedafterin vivo treatment with AAF.     

Induction of CYP1A and glutathione S-transferase activities by 2,3,7,8-tetrachlorodibenzo-p-dioxin in human hepatocyte cultures

Induction of CYP1A and glutathione S-transferase activitieswith 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studiedin human hepatocytes in primary culture to investigate the variabilityof inducibility and the potency of TCDD. Determining inductionof 7-ethoxyresorufin O-deethylase activity, preferentially catalyzedby CYP1A isozymes, we obtained concentration—responsediagrams in TCDD-treated hepatocyte cultures from transplantdonors and patients undergoing hepatic surgery. At a concentrationof 10^–10 M TCDD approximately half-maximal induction ofCYP1A was observed. Northern analysis of CYP1A gene expressionshowed a similar concentration — response relationship.In comparison with rat hepatocytes, human hepatocytes were about10-fold less sensitive towards the CYP1A-inducing effect ofTCDD. No pronounced interindividual differences in the inducingpotency of TCDD (concentration which leads to half-maximal induction)were obvious in the six human individuals studied, whereas theefficacy of CYP1A induction was highly variable. In addition,inducibility of glutathione S-transferase (GST) activity alsorevealed a considerable degree of interindividual variation,i.e. a complete lack of induction in three out of six hepatocytepreparations and a highly variable efficacy of GST inductionamong responders which was not related to CYP1A inducibility.     

SHORT COMMUNICATION: In vitro colonization ability appears soon after initiation of hepatocarcinogenesis in the rat

Hepatocytes were isolated from rat livers at various intervalsafter initiating hepatocarcinogenesis by a single administrationof methyl(acetoxymethyl)nitrosamine after a partial hepatectomy.Proliferative hepatocyte colonies were regularly observed inprimary cultures derived from initiated livers when these cultureswere incubated in medium containing the liver tumor promoter,phenobarbital. Fewer colonies generally were observed in initiatedcell cultures that were incubated without phenobarbital andin phenobarbital-induced cultures derived from non-initiatedcontrol livers. Because in vitro proliferative activity is acharacteristic of malignant hepatocytes these results suggestthat the feature of in vitro colonization may characterize apopulation of carcinogen-altered hepatocytes.     

Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with dinitrotoluene

The purpose of this study was to examine the induction of unscheduledDNA synthesis (UDS) by the potent hepatocarcinogen technicalgrade dinitrotoluene (tgDNT; 76% 2, 4-DNT, 19% 2, 6-DNT) usingthe in vivo-in vitro hepatocyte DNA repair assay. Male Fischer-344rats were treated by gavage and hepatocytes were isolated byliver perfusion and cultured with [³H]thymidine. UDS was measuredby quantitative autoradiography as net grains/nucleus (NG);5 NG was considered positive. Controls consistently had –3 to – 6 NG. A dose-related increase in UDS was observed12 h after treatment, with 200 mg/kg tgDNT producing 26 NG.A 50-fold increase in the number of cells in S-phase was observedat 48 h after treatment. This increase in S-phase cells couldbe suppressed in the presence of 10–20 mM hydroxyurea(HU), while the same levels of HU did not affect the level ofUDS at 12 h after treatment. 2, 4-DNT produced only a weak response,in contrast to 2, 6-DNT which was a potent inducer of UDS. Treatmentof female rats with tgDNT yielded only modest increases in UDSand DNA replication relative to males. These results are consistentwith the carcinogenicity studies and indicate that tgDNT isa potent genotoxic agent, with 2, 6-DNT contributing the majorportion of the effect.