Carcinogen-DNA adducts in cultures of rat and human hepatocytes.

Exposure to chemical carcinogens can often be identified by detection of DNA adduct lesions. Primary cultures of isolated rat and human hepatocytes were exposed to 2-acetyl-aminofluorene (AAF), 4-aminobiphenyl (ABP), or benzo[a]pyrene (BP). The isolated DNA from the exposed cells was analyzed using the 32P-post-labeling assay. A greater total of carcinogen-DNA adducts, 2-12-fold, were observed in human hepatocytes than rat hepatocytes at the same concentrations. The predominant DNA adducts for each carcinogen were the same between rat and human cells. The N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the major AAF-DNA adduct. The N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) was the major ABP-DNA adduct. In the rat N2-[10 beta-(7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl] deoxyguanosine (dG-N2-BP) and two unidentified adducts were nearly equivalent in amount, while the major BP-DNA adducts in the humans was the dG-N2-BP. The rat hepatocyte in vitro results are comparable to the predominant adducts found with rats exposed in vivo. The two different cultures of human hepatocytes demonstrated qualitative and quantitative differences in specific DNA adducts from rat hepatocytes. This study and others using human hepatocyte cultures demonstrate that this in vitro system can provide useful information for assessing human carcinogenic hazards.     

Diaziquone-induced cytotoxicity in isolated rat hepatocytes

2,5-Diaziridinyl-3,6-bis(carboethoxyamine)-1,4-benzoquinone (AZQ) is a lipid-soluble antitumor agent. The following evidence using isolated rat hepatocytes as a model for cytotoxicity studies suggests that, under aerobic conditions, AZQ participates in futile oxidation-reduction cycling and oxygen activation. The H2O2 formed can mediate oxidative stress cytotoxicity in compromised cells. (a) Addition of AZQ to hepatocytes causes the stoichiometric oxidation of glutathione (GSH) to oxidized glutathione. No subsequent reduction back to GSH occurred. This was found to be the result of reversible inactivation of glutathione reductase by AZQ. The extent of GSH oxidation increased with AZQ concentration. (b) Cytotoxicity occurred when AZQ concentrations were sufficient to completely deplete GSH. (c) Addition of AZQ to hepatocytes enhanced cyanide-resistant respiration. (d) If the hepatocytes were compromised with azide or cyanamide to inhibit catalase, cytotoxicity was increased 10-fold or 100-fold if ascorbate was also present. (e) AZQ readily induced Ca2+ release by energized mitochondria. Ascorbate markedly enhanced the effectiveness of AZQ, and catalase delayed Ca2+ release. H2O2 formed by aerobic oxidation-reduction cycling of AZQ may therefore cause mitochondrial Ca2+ release.     

Metabolism of acrylonitrile by isolated rat hepatocytes

Several of the pathways of metabolism of the suspected carcinogen acrylonitrile (AN) were identified previously in this laboratory with the use of subcellular fractions and purified enzymes (Guengerich, F.P., Geiger, L.E., Hogy, L.L., and Wright, P.L., Cancer Res., 41: 4925-4933, 1981). In order to establish the relative contributions of the various pathways leading to activated and detoxicated products, we examined AN metabolism in isolated Fischer 344 rat hepatocytes as a model. Reduced glutathione (GSH) was depleted, and cell viability was lost in an AN concentration-dependent manner. The major GSH adduct formed at all AN concentrations was identified as S-(2-cyanoethyl)GSH using thin-layer and high-performance liquid chromatography. Acid hydrolysis and amino acid analysis of labeled hepatocellular, protein revealed S-(2-carboxyethyl)-cysteine as the major adduct formed, indicating direct alkylation of cysteinyl residues by AN. 2-Cyanoethylene oxide accumulated in the hepatocyte incubations but did not appear to contribute extensively to alkylation of GSH or protein. Cyanide, resulting from hydrolysis of 2-cyanoethylene oxide, appeared to be completely converted to thiocyanate, which was identified by gel exclusion chromatography and mass spectrometry of the methyl derivative. The concentration of thiocyanate formed was directly proportional to the concentration of AN used. Cyanide does not appear to play a role in AN-mediated cell death. Alkylation of hepatocellular DNA and RNA and extracellular DNA was not observed to an extent greater than one base in 3.5 X 10(5). The relative rates of the various pathways were compared, and more than 97% of the metabolites can be accounted for by the described reactions. These results are of use in evaluating the contribution of the various pathways and modes of binding of AN to toxicity and carcinogenicity in liver and extrahepatic target tissues.     

Colloidal gold ultraimmunocytochemical localization of DNA and RNA adducts in rat hepatocytes.

The localization of DNA and RNA adducts was studied at the ultrastructural level using antibodies directed against O6-methylguanine (O6-metG) and the protein A-gold technique. Primary rat hepatocyte cultures were exposed for 2 h to 5 mM N-nitrosodimethylamine (NDMA). In NDMA-treated cells, the O6-metG-induced immunoreactive sites do not appear at random but seem to be concentrated in the nucleus, and in the cytoplasm, in areas rich in rough endoplasmic reticulum (RER) elements. Mitochondria were not significantly labelled. Untreated control preparations showed no specific immunogold labelling. After RNase digestion of ultrathin sections obtained from cells exposed to NDMA and subsequent immunogold labelling, most of the immunolabelling in the cytoplasm had disappeared, and that over the nucleus had only been slightly reduced, as compared to undigested specimens from NDMA-treated cultures. After similar digestion with DNase, a strong reduction of the labelling of the nucleus was observed, but labelling of the cytoplasm was practically unaffected by this enzymatic treatment, as compared to what was observed in undigested preparations of NDMA-treated hepatocytes. The results provide evidence of preferential formation of O6-metG at the DNA and RNA levels, in the nucleus and cytoplasm RER, respectively. Furthermore, this study demonstrates the applicability of the high-resolution protein A-gold technique for ultrastructural detection of nucleic acid adducts in NDMA-treated hepatocytes using affinity-purified anti-O6-metG polyclonal antibodies.     

Sulfation-dependent formation of N-acetylated and deacetylated DNA adducts of N-hydroxy-4-acetylaminobiphenyl in male rat liver in vivo and in isolated hepatocytes.

Administration of 3H-labeled N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) to male Wistar rats with or without prior partial hepatectomy (PH) resulted in covalent binding of 3H activity to liver macromolecules. Pretreatment with the sulfotransferase inhibitor pentachlorophenol (PCP) 45 min before administration of the arylhydroxamic acid strongly decreased the covalent binding. Analysis of aminobiphenyl adducts after TFA hydrolysis of DNA and RNA showed that PCP decreased the formation of both the N-acetylated adduct N-[deoxy)guanosin-8-yl)-4-acetylaminobiphenyl [(d)G-C8-AABP] and the deacetylated adduct N-[deoxy)-guanosin-8-yl)-4-aminobiphenyl [(d)G-C8-ABP] by 60-80%. In incubations with hepatocytes from male Wistar or Sprague-Dawley rats, omission of inorganic sulfate also strongly decreased the covalent binding of 3H-labeled N-OH-AABP to RNA and protein. Analysis of RNA adducts showed a 70-80% decrease in the formation of G-C8-ABP in the absence of sulfate. Another, as yet unidentified, adduct was only slightly decreased. Similar results were obtained with the structurally related carcinogen N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP). Pretreatment with PCP decreased the incidence of gamma-glutamyltranspeptidase-positive foci in the liver of male rats when analyzed 30 days after a single injection of N-OH-AABP or N-OH-FAABP by 60 and 80% respectively. Thus, both N-acetylated and deacetylated RNA and DNA adducts of N-OH-AABP in rat liver are formed by sulfation and this metabolic activation pathway is responsible for the formation of genotoxic metabolites involved in the generation of preneoplastic cells.     

Metabolism of N-nitrosodi-n-propylamine and N-nitrosodiallylamine by isolated rat hepatocytes

Isolated rat hepatocytes were incubated with approximately equimolar amounts of N-nitrosodi-n-propylamine (NDPA) and N-nitrosodiallylamine (NDAA) in order to compare their metabolism. The principal metabolite of NDPA was N-nitroso-(2-hydroxypropyl)propylamine, which was present as a glucuronide. N-Nitroso-(3-hydroxypropyl)propylamine and N-nitrosopropyl-(carboxyethyl)amine were minor metabolites; no N-nitrosomethylpropyl-amine (NMPA) was detected. A single N-nitroso metabolite of NDAA was found and identified as N-nitroso-(2,3-dihydroxypropyl)allylamine. These data indicate that the allyl group of N-nitrosodiallylamine is readily oxidized by hepatocytes in vitro. It appears unlikely that N-nitrosomethylpropylamine is an intermediate in N-nitrosodi-n-propylamine metabolism.     

Metabolism and activation of 2-acetylaminofluorene in isolated rat hepatocytes.

The metabolism of 2-acetylaminofluorene (AAF) as well as the activation of AAF to covalently bound and mutagenic intermediates were studied in isolated rat hepatocytes. The cell system readily formed oxidized, deacetylated, and conjugated AAF metabolites. Pretreatments of animals with the inducer beta-naphthoflavone led to increases in phenolic and conjugated as well as covalently protein-bound products. Addition of 4-nitrophenol, a substrate for conjugation, increased the levels of free phenols and inhibited the formation of water-soluble metabolites. At the same time, the rates of covalent protein binding were decreased. Formation of 9-hydroxy-2-acetylaminofluorene could also be demonstrated. The pathway leading to this alicyclic hydroxylated AAF metabolite was not induced by prior beta-naphthoflavone treatment, nor was it inhibited by 4-nitrophenol addition. The cells converted AAF as well as aminofluorene and 2,4-diaminoanisole to mutagenic intermediates which were released into the incubation medium. 2-Aminofluorene was considerably more mutagenic than was AAF in this system. Addition of microsomes increased the mutagenicity of AAF, but not that of 2-aminofluorene or 2,4-diaminoanisole, presumably by deacetylation of N-hydroxy-2-acetylaminofluorene to N-hydroxy-2-aminofluorene.     

Sphingolipids suppress preneoplastic rat hepatocytes in vitro and in vivo

Sphingolipids can modulate cell growth, differentiation and apoptosis. In the present investigation, selective death of hepatocytes localized in enzyme-altered foci (EAF hepatocytes) was shown to be induced by sphingolipids. Sphingosine (20 micro M) caused rapid cell death predominantly of EAF hepatocytes in vitro. During 4 h of such exposure, cytochrome c was released from the mitochondria into the cytoplasm and the number of cells demonstrating cleaved caspase-9 activity increased. The selective sensitivity of EAF cells to sphingolipid-induced death was attenuated by tumor necrosis factor-alpha. In previous studies we have demonstrated that EAF hepatocytes are resistant to Fas-mediated apoptosis, a resistance shown here to be reversed by low concentrations of sphingosine. Immunohistological staining revealed higher levels of glucosylated ceramide in EAF than in the surrounding tissue. Furthermore, an inhibitor of glucosylation enhanced the toxicity of ceramide towards EAF cells. TLC analysis suggested low levels of sphingosine in preneoplastic lesions. In in vivo experiments EAF-bearing rats were fed a diet supplemented with 0.1% sphingomyelin for 2 weeks. Sphingolipid feeding reduced the number of EAF and EAF area in the liver by 40-50% as compared with rats fed a control diet. These studies indicate that the turnover of sphingolipids in preneoplastic EAF hepatocytes is altered. This alteration may explain not only the increased sensitivity of EAF cells towards sphingolipid-induced cell death, but also the resistance of these hepatocytes to cell death involving sphingolipids as second messengers. Furthermore, sphingomyelin in the diet may prevent EAF development. It is suggested that the altered turnover of sphingolipids might be a target for chemoprevention of hepatocellular carcinoma.     

Metabolism of 1- and 2-naphthylamine in isolated rat hepatocytes.

The liver probably plays a major role in the metabolic activation of the bladder carcinogen 2-naphthylamine (2-NA) and in the inactivation of the non-carcinogenic isomer 1-naphthylamine (1-NA). However, metabolic profiles of these compounds (including primary metabolites and directly determined conjugates) in hepatocytes are not available. Therefore metabolism of 1- and 2-NA was compared in freshly isolated hepatocytes from 3-methylcholanthrene (MC)-treated and untreated rats. At 10 microM, 2-NA was found to be mainly N-acetylated (66% of total metabolites after 1 h incubation) and N-glucuronidated (19%). Minor pathways led to C-oxidation (7%) and N-oxidation (3%; 2% present as the N-glucuronide). In hepatocytes from MC-treated rats total metabolism was slightly affected (1.5-fold increase). However, C- and N-oxidation were markedly increased (63 and 18% respectively), while N-acetylation and N-glucuronidation were diminished (5 and 2% respectively). Similar experiments were carried out with 1-NA. Its N-glucuronide was the predominant metabolite (68%) followed by the N-acetylated compound (15%) while C-oxidation was low and N-oxidized metabolites could not be detected, even after induction. The results demonstrate that MC treatment markedly shifted 2-NA metabolism from N-acetylation and N-glucuronidation to N- and C-oxidation. In the case of 1-NA metabolism extensive N-glucuronidation together with the lack of N-oxidation may prevent carcinogenesis.     

Interactions of vinblastine and vincristine with methotrexate transport in isolated rat hepatocytes

The accumulation of methotrexate (MTX) in the presence of vinblastine (VBL) and vincristine (VCR) was studied in isolated rat hepatocytes. In accordance with our recent study on vindesine (VDS), we found VBL and VCR to reduce net MTX accumulation significantly at 15 min after MTX addition. Drug concentrations of 100 M VBL and 500 M VCR led to 67% and 82% reductions in intracellular MTX, respectively. Since there was only a slight inhibition of MTX efflux by 100 M VBL, the accumulation data demonstrate that the major effect of VBL is on MTX influx. Dixon-plot analyses are suggestive of competitive inhibition of the MTX influx, yielding inhibition constants (K _i values) of 55 M for VBL and 110 M for VCR. Since theK _i values correspond grossly to plasma levels obtained in humans shortly after the infusion of therapeutic doses of the vinca alkaloids studied herein, the interaction with MTX uptake could serve to diminish the toxicity of MTX to nonmalignant cells.This study was supported financially by the Norwegian Cancer Society     

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.     

Measurement of in vitro cellular pharmacokinetics of 5-fluorouracil in human and rat cancer cell lines and rat hepatocytes using a flow-through system

Summary A flow-throught system was used to study the cellular pharmacokinetics of 5-fluorouracil (5-FU) in four human cell lines (squamous-cell carcinoma HEp-2, colon carcinoma WiDr, hepatoma Hep G2, and breast carcinoma MCF-7) as well as in the rat hepatoma H35 cell line and in freshly isolated rat hepatocytes. The system made it possible to restrict the decrease in the concentration of 5-FU in the medium, to keep the volume in which the metabolites accumulated relatively small, and to study the dynamics of a response during and after a change in the composition of the eluent. Clearance of 5-FU from the eluent was achieved predominantly (>95%) by its catabolism to dihydrofluorouracil in the tumor cell lines and to 2-fluoro--alanine in the hepatocytes. Not only rat hepatocytes but also HEp-2 cells showed relatively high clearance values. A concentration-dependent 5-FU elimination was observed, indicating saturation of 5-FU elimination according to Michaelis-Menten kinetics (K_m 14–22 M). The maximal velocity (V_max) values ranged from 0.025 to 0.13 nmol 5-FU/10⁶ cells per minute. For HEp-2 cells, high-concentration pulse injections of 5-FU, thymine, uridine, or uracil immediately led to a reduction in 5-FU conversion, followed by recovery within 5 min. The flow-through system proved to be adequate for the study of the non-linear pharmacokinetics of 5-FU in different intact cells and for the comparison of various manipulations of these pharmacokinetics.Abbreviations 5-FU 5-fluorouracil - FUR 5-fluorouridine - F-DHU dihydrofluorouracil - F--ala 2-fluoro--alanine - F-UPA -fluoroureidopropionic acid - HEPES 4-(2-hydroethyl)-1-piperazine-ethane sulfonic acid - MEM modified minimal essential medium - HBSS Hanks' balanced salt solution - HPLC high-performance liquid chromatography Supported by grant IKA 87-16 from the Netherlands Cancer Foundation. One author (G. J. P.) is the recipient of a senior research fellowship from the Royal Netherlands Academy of Arts and Sciences (KNAW)     

Metabolic studies of 15N-labeled N-nitrosoproline in isolated rat hepatocytes and subcellular fractions

The metabolism of the non-carcinogenic N-nitrosoproline (NPRO) was investigated in vitro using both S9 preparations and isolated hepatocytes from F344 rats. The studies were performed using 15N-labeled nitrosamine and the reaction mixtures were examined mass spectrometrically for the presence of 15N2 or other 15N-labeled gaseous products. In addition, the metabolism of NPRO was monitored by capillary gas chromatography. The results indicated no 15N2 production from either the hepatocyte or S9 preparations, as well as no detectable loss of substrate from the reaction mixtures. Mass spectrometric analysis failed to reveal any metabolites of NPRO. The results suggest that NPRO may be refractory to the normal nitrosamine activating enzymes, confirming its suitability for use in human epidemiological studies of endogenous nitrosation.     

Quantitation of aflatoxin B1-DNA adducts in woodchuck hepatocytes and rat liver tissue by indirect immunofluorescence analysis

A quantitative indirect immunofluorescence technique was developed utilizing a monoclonal antibody (6A10) recognizing the imidazole ring-opened form of the major N-7 guanine adduct of aflatoxin B1 (AFB1). This method was used to investigate adduct formation in woodchuck hepatocytes treated in culture and in liver tissue of rats treated i.p. with AFB1. Fluorescein isothiocyanate-labeled secondary antiserum was used for adduct localization in conjunction with 4',6-diamidino-2-phenylindole dihydrochloride staining to localize nuclei. Quantitation of AFB1-DNA adducts was carried out by densitometric analysis of photographic slides. Specific nuclear staining was observed in both woodchuck hepatocytes and rat liver tissue. There was a dose-response relationship between fluorescence intensity and AFB1 dose in treated animals. Turnover of adducts could also be followed in animals over 48 h with this method. DNA was isolated from liver tissue of treated animals and adduct levels were quantitated by competitive enzyme-linked immunosorbent assay with antibody 6A10 and by fluorescence spectroscopy. There was a significant correlation of the quantitative immunofluorescence intensity with levels of AFB1 adducts detected by enzyme-linked immunosorbent assay (r = 0.61, P less than 0.05) and spectrofluorescence (r = 0.78, P less than 0.01). This immunohistochemical method should be applicable to the detection of adducts in liver tissues of humans exposed to high levels of dietary AFB1.     

Metabolic activation of 6-nitrochrysene in explants of human bronchus and in isolated rat hepatocytes

It has previously been shown that 6-nitrochrysene can be activated to electrophilic species capable of reacting with DNA through metabolic pathways that form N-hydroxy-6-aminochrysene or trans-1,2-dihydroxy-1,2-dihydro-6-aminochrysene as critical intermediates. Since the lung is a known target tissue for the carcinogenic action of polycyclic nitroaromatic hydrocarbons, we investigated the metabolism and DNA binding of [3H]6-nitrochrysene in 11 specimens of human bronchus. Analysis of medium from [3H]6-nitrochrysene-treated explants indicated the presence of trans-9,10-dihydroxy-9,10-dihydro-6-nitrochrysene (0.04-330 pmol/mg epithelial DNA), trans-1,2-dihydroxy-1,2-dihydro-6-nitrochrysene (12-1700 pmol/mg epithelial DNA), 6-aminochrysene (1.6-2200 pmol/mg epithelial DNA), and trans-1,2-dihydroxy-1,2-dihydro-6-aminochyrsene (3.6-610 pmol/mg epithelial DNA). Both the levels and the relative proportions of these metabolites varied widely in explants from different individuals. The amount of DNA recovered and the level of DNA modification were sufficient for adduct analysis in eight of the 11 cases for which metabolite data were obtained. Five additional bronchial specimens for which metabolite data were not obtained were also analyzed for carcinogen-DNA adducts. The levels of binding varied from 0.06 to 30.5 pmol [3H]6-nitrochrysene bound/mg DNA (two adducts per 10(8) nucleotides-10 adducts per 10(6) nucleotides). HPLC analyses of enzymatic hydrolysates of the explant DNA indicated that 11 of 13 cases contained adducts with retention times identical to those of adducts derived from trans-1,2-dihydroxy-1,2-dihydro-6-aminochrysene or N-hydroxy-6-aminochrysene. The adduct derived from trans-1,2-dihydroxy-1,2-dihydro-6-aminochrysene was the major adduct detected in eight of 13 cases. The reasons for the variation in metabolism and adduct formation observed in [3H]6-nitrochrysene-treated explants of bronchus from different donors are not known but may reflect differences in the activities of enzymes responsible for the metabolism of this compound. The influence of induction of drug metabolizing enzymes on the activation pathway of 6-nitrochrysene in an intact cell system was tested using rat hepatocytes. 6-Nitrochrysene was incubated with freshly isolated hepatocytes from rats that were either untreated or pretreated with phenobarbital, 3-methylcholanthrene or Aroclor 1254. Although the levels of adducts were similar in all cases, the pattern of DNA adducts formed in these hepatocytes was dependent on the nature of the pretreatment of the rats. As previously reported, hepatocytes from untreated rats contained adducts derived from N-hydroxy-6-aminochrysene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Consequences of 3-methylcholanthrene-type induction for the metabolism of 4-aminobiphenyl in isolated rat hepatocytes

Carcinogenic aromatic amines such as 4-aminobiphenyl (4-ABP)are extensively metabolized by both oxidative and conjugationreactions. Thus the burden of genotoxic metabolites of 4-ABPin a target organ is probably influenced by the balance of N-hydroxylationand alternative metabolic pathways in the hepatocyte. In freshlyisolated rat hepatocytes, 4-ABP (at a substrate concentrationof 10 µM) was mainly N-acetylated (54% of total metabolites),while 2% Ar-hydroxy-4-ABP-N-glucuronide and 21% of unconjugatedN-hydroxylated metabolites were detectable. Ring-hydroxylatedmetabolites and the primary N-glucuronide of 4-ABP accountedfor 8% and 4%, respectively. Pretreatment of rats with 3-methylcholanthrene(MC), a dioxin-type inducer of CYP1A isozymes and phenol UDP-glucuronosyltransferase(UGT1A1), led to a dramatic decrease of W-acetylated (2% oftotal metabolites) and an increase of N-hydroxylated (54% asfree and glucuronidated compound) and ring-hydroxylated (35%)metabolites. Essentially similar effects were seen at a substrateconcentration of 50 µM. Consistently, MC-type inductionwith ß-naphthoflavone resulted in a significant increasein the formation of DNA adducts of 4-ABP, detected by ³²P-postlabellingof hepatocellular DNA. The results suggest that, similar toa previous study with 2-naphthylamine (2-NA), MC treatment leadsto a marked shift from conjugation to N-oxidation. However,N-hydroxy-4-ABP (in contrast to N-hydroxy-2-NA) is mostly releasedfrom hepatocytes in the unconjugated form.     

In vitro mutagenicity studies on cyproterone acetate using female rat hepatocytes for metabolic activation and as indicator cells

The synthetic sex steroid cyproterone acetate (CPA) was shownto induce DNA repair in primary hepatocytes from female rats,confirming previous reports about a sex- specific genotoxicpotential of CPA in female rat liver. CPA did not induce genemutations or chromosomal aberrations in V79 Chinese hamstercells co-cultivated with hepatocytes from female rats as themetabolizing system. Hepatocytes from the same rats under identicalexposure conditions as in the positive unscheduled DNA synthesis(UDS) test were used In these experiments. The maximum concentrationstested in the mutagenicity assays were 30–100 times highercompared with the lowest observed effect concentration of 3x10^–6mol/l in the DNA repair test. The requirement for cell-celltransfer of the putative genotoxic metabolites in the co-cultureexperiments may be the reason for the discrepancy between positiveUDS effects and negative mutagenicity and clastogenicity results.To further investigate if CPA can induce chromosomal mutationswithin the same cells that provide metabolic activation an invitro micronucleus assay with proliferating female rat hepatocytes was performed. The results gave some indications of amicronucleus-inducing potential of CPA. However, under the conditionsof the micronucleus assay CPA was also shown to increase theproliferative activity of hepatocytes. Since micronucleus formationis also dependent on mitotic activity, it cannot be determinedwhether the increase in micronucleus formation after CPA exposureindicates a clastogenic potential or whether it is just a consequenceof the mitogemc potential of CPA. Although CPA or its metabolitesobviously have a DNA damaging potential and stimulate considerableDNA excision repair, our findings do not establish a clear mutagenicpotential.     

2-Nitropropane induces DNA repair synthesis in rat hepatocytes in vitro and in vivo

The genotoxicity of 2-nitropropane (2-NP) and 1-nitropropane(1-NP) was investigated by measuring the induction of DNA repairsynthesis in rat liver cells in vitro and in vivo. 2-NP stronglyinduced DNA repair synthesis in both cases. When applied invivo, 2-NP was considerably more effective in hepatocytes frommales than in those from females. 1-NP was not active in vitroor in vivo. 2-NP and 1-NP did not induce repair in cell linesof extrahepatic origin derived from rat, mouse, hamster andman. The results are consistent with the reported carcinogenicityof 2-NP in rat liver and suggest that the formation of hepatocarcinomasby 2-NP is due to the generation of a genotoxic metabolite from2-NP by liver-specific metabolism.     

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.     

Excretion kinetics of the DNA adducts of cis-diamminedichloroplatinum(II) formed in vitro in rat urine

The main adduct of cis-diamminedichloroplatinum(ll) (cis-Pt)with DNA, cis-[Pt(NH₃)₂(dGpdG)], was administered i.p. to rats.Urine was collected daily for 4 days. The adduct was purifiedby a weak cation exchanger and quantitated by HPLC with UV detection.The recovery of the adduct was the chemical instability of cis-[Pt(NH₃₂(dGpdG)]in urine as shown in an in vitro incubation. Adjusted for thisinstability the recovery in urine was > 70% of the dose.When cis-Pt-DNA (the molar ratio of cis-Pt to nucleotide = 1:50) was administered i.p. to rats only 1.25 ± 0.23% ofplatinum was excreted in urine in the form of cis-[Pt(NH₃)₂(dGpdG)]and cis-[Pt(NH₃)₂(dApdG)] during the first 4 days. If the removalof the cis-Pt-DNA adducts from human tissues is to be followed,their possible slow excretion and chemical instability in urineneeds to be considered.