The essential role of microsomal deacetylase activity in the metabolic activation, DNA-(deoxyguanosin-8-yl)-2-aminofluorene adduct formation and initiation of liver tumors by N-hydroxy-2-acetylaminofluorene in the livers of infant male B6C3F1 mice

Deacetylation of N-hydroxy-2-acetylaminofluorene. (N-hydroxy-AAF) to N-hydroxy-2-aminofluorene (N-hydroxy-AF) has been proposedas one of the critical metabolic steps in the formation of hepaticDNA adducts and the initiation of liver tumors in 12-day-oldmale B6C3F₁ mice. In this study, the importance of the microsomaldeacetylase activity for N-hydroxy-AAF in the initiation ofhepatocarcinogenesis in these mice was demonstrated by usinga carboxylesterase and amidase inhibitor, bis(p-nitrophenyl)phosphate(BNPP), that is much less toxic in vivo than is paraoxon. Pre-incubationof liver microsomes from 12-day-old male B6C3F₁ mice with 10^–3M BNPP reduced the deacetylase activity by 80% while paraoxoninhibited the deacetylase activity completely at a concentrationof 10_–4 M. Pretreatment of 12-day-old male B6C3F₁ micewith 4 x 75 µg doses of BNPP/g body weight before theadministration of n-hydroxy-AAF reduced the hepatic N-(dGuo-8-yl)-AFadduct levels to 1.09 and 0.68 pmol/mg DNA compared with 2.87and 1.64 pmol/mg DNA for mice treated once with 0.06 or 0.03µmol of N-hydroxy-AAF/g body weight respectively. However,BNPP pretreatments did not affect the levels of the acetylatedDNA adducts, N-(dGuo-8-yl)-AAF and 3-(dGuo-N²-yl)-AAF, formedby these doses of N-hydroxy-AAF. The initiation of liver tumorsby n-hydroxy-AAF was also inhibited by BNPP pretreatment. Thus,for mice that received single doses of 0.12, 0.06 and 0.03 µmolof N-hydroxy-AAF/g body weight, the multiplicities of livertumors at 10 months were reduced by BNPP pretreatments to 5.6,1.0 and 0.3 compared with multiplicities of 11.8, 4.8 and 1.7without pretreatment respectively. On the other hand, BNPP pretreatmentshad no significant inhibitory effects on the levels of the hepaticDNA-N-dGuo-8-yl)-AF adduct or on the liver tumor multiplicitiesinduced by comparable doses of N-hydroxy-AF. It is concludedthat deacetylation of n-hydroxy-AAF to N-hydroxy-AF is essentialfor the metabolic activation, DNA-N-(dGuo-8-yl)-AF adduct formationand liver tumor initiation in infant male B6C3F₁ mice by N-hydroxy-AAF.     

N-Sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxy-2-acetylaminofluorene in the livers of infant male C57BL/6J x C3H/HeJ F1 (B6C3F1) mice

The hepatic DNA of 12-day-old male B6C3F₁ (C57BL/6J x C3H/HeJ)mice given an i.p. dose of 0.06 or 0.11 µmol/g body weightof N-hydroxy-[³H]-2-acetylaminofluorene (N-hydroxy-AAF) containedat 9 h 3 or 6 pmol of N-(deoxyguanosin-8-yl)-2-aminofluoreneadducts per mg. Together the level of the two acetylated adductsN-(deoxyguanosin-8-yl)-2-acetyl-aminofluorene and 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorenewas 10% of this amount. The same doses of unlabeled carcinogeninduced by 10 months a 100% incidence of hepatomas with averagesof 10 and 15 hepatomas per mouse, respectively. Injection of0.04 µmol/g body weight of pentachlorophenol (PCP) 45min before the dose of N-hydroxy-AAF decreased the number ofadducts in the DNA by 90% and the average number of hepatomasper liver by 80–90%. As compared to their normal malelittermates, male brachymorphic B6C3F₂ mice, which are deficientin hepatic 3'-phosphoadenosine-5'-phosphosulfate (PAPS), treatedwith N-hydroxy-AAF formed only 25% as many hepatic DNA adductsand developed only 10% as many hepatomas. Hepatic cytosols from12-day-old B6C3F₁ mice contained PAPS-dependent sulfotransferaseactivity for N-hydroxy-2-aminofluorene (N-hydroxy-AF), a previouslyunrecognized activity, as well as sulfotransferase activityfor N-hydroxy-AAF; both activities were inhibited 60% by 1 µMand 80% by 10 µM PCP. Cytosolic acetyl coenzyme A-dependentacetyltransferase activity for N-hydroxy-AF, cytosolic N,O-acyltransferaseactivity for N-hydroxy-AAF, and microsomal deacetylase for N-hydroxy-AAFwere not significantly inhibited by PCP under these conditions.The above data strongly indicate that N-sulfoöxy-2-aminofluoreneis the major ultimate electrophilic and carcinogenic metaboliteof N-hydroxy-AAF in the livers of infant male B6C3F₁ mice.     

The role of hyperplastic nodules in dichloroacetic acid-induced hepatocarcinogenesis in B6C3F1 male mice

Dichloroacetic acid (DCA) has recently been shown to increase significantly the incidence of hepatic adenomas (HAs) and hepatocarcinomas (HCs) in male B6C3F1 mice. Although little is known about the mechanism of DCA carcinogenesis, chronic ingestion of the compound in drinking water induces primarily hyperplastic nodules (HNs) prior to the appearance of HAs and HCs. Given the putative preneoplastic potential of the HNs, we undertook this study to determine the role of the HNs in the progression of DCA-induced hepatocarcinogenesis. This role was assessed by detecting the expression of five different tumor markers: p21 ras, p39 c-jun, phosphotyrosine, tumor-associated aldehyde dehydrogenase and alpha-fetoprotein, all known from previous studies to be expressed more often in neoplastic liver lesions than in normal liver. Tumor marker expression was detected by immunohistochemical methods using formalin-fixed, paraffin-embedded sections of normal B6C3F1 mouse liver, and DCA-induced HNs, HAs and HCs. The results demonstrated that, except for the c-jun marker, HNs expressed the markers significantly less often than either HAs or HCs. Equal expression of c-jun occurred in any of the three lesion types. Although these results could be used to argue that no relationship existed between HNs and later-appearing HAs and HCs, those HNs that were marker positive contained small nests of marker-positive hepatocytes among a field of normally appearing unstained hepatocytes. No similar nests of marker-positive cells were detected in any area of normal liver outside the HNs. Also very few altered hepatic foci (AF) were detected with these markers or with hematoxylin and eosin, or with histochemical stains for ATPase or glucose-6-phosphatase deficiencies. These results suggested that these nests within some HNs were areas of transformed, or neoplastic hepatocytes. Phenotypic heterogeneity analysis, in which the number of tumor markers co-expressed by any given lesion was examined, confirmed a significantly greater percentage of HAs and HCs expressing multiple markers than HNs. Those HNs that expressed multiple markers, however, expressed at the same frequency as HAs and HCs and the expression was confined to the same nests of cells. Taken together, these data suggest that these nests of marker-positive cells within the HNs were neoplastic and could develop into later-appearing HAs and/or HCs. The absence of marker expression in normal liver and limited expression in the few AF indicates that the HNs may be the only significant preneoplastic lesion in DCA-induced hepatocarcinogenesis.     

Ras oncogene activation during hepatocarcinogenesis in B6C3F1 male mice by dichloroacetic and trichloroacetic acids

Dichloroacetic (DCA) and trichloroacetic (TCA) acids, two majorby-products formed during chlorine disinfection of drinkingwater, increase the incidence of tumors in B6C3F1 mice by 6-and 3-fold respectively. In order to understand better the mechanismby which these two compounds induce liver tumors, the incidenceand spectrum of mutations in the K- and H-ras proto-oncogenesin these tumors were analyzed. DNA from spontaneous, DCA- andTCA-induced liver tumors from B6C3F1 male mice was evaluatedfor point mutations in exons 1, 2 and 3 of the two genes bysingle-stranded conformation polymorphism. Results demonstrateda similar incidence of mutations for exon 2 of H-ras in spontaneouscarcinomas (58%), and in carcinomas induced by DCA 3.5 g/l (50%),1.0 g/1(48%) and TCA 4.5 g/l (45%). Only four samples showedmutations in the other exons of H-ras or in K-ras. Sequenceanalysis of spontaneous tumor samples with second exon H-rasmutations revealed a change in codon 61 from CAA to AAA in 80%and CAA to CGA in 20% of tumors. In contrast, tumors with H-rasmutations from DCA-treated mice revealed a H-61 change fromCAA to AAA in 21 % at 3.5 g/l and 16% at 1.0 g/l. CAA to CGAwas observed in 50% of tumors from mice given DCA 3.5 or 1.0g/l, and CAA to CTA was present in 29% and 34% of the two dosagegroups respectively. Interestingly, TCA showed the same mutationalspectrum as the spontaneous liver tumors. The data indicatesthat induction of liver carcinoma by DCA and TCA involves activationof the H-ras proto-oncogene at a frequency similar to that observedin spontaneous tumors. However, the mechanism(s) for inducinghepatocellular carcinoma does not appear to be identical forDCA and TCA.     

Altered gene expression in spontaneous hepatocellular carcinomas from male B6C3F1 mice

In this study, we analyzed spontaneous hepatocellular carcinomas (HCCs) from male B6C3F1 mice for alterations in the expression of the genes for c-myc, insulin-like growth factor II (IGF-II), cyclin D1, transforming growth factor-α (TGF-α), and the epidermal growth factor receptor (EGFR). These genes are all important in growth control in the rodent liver, and therefore, alterations in these genes or their products may result in unregulated growth. Northern blot analysis demonstrated an increase in expression of c-myc mRNA in five of 21 (24%) spontaneous HCCs compared with nontumor tissue. Tumors that had an increase in c-myc mRNA did not have an amplified c-myc gene. Of the HCCs analyzed, 18 of 29 (62%) showed reexpression of IGF-II RNA when compared with controls. Cyclin D1 mRNA was overexpressed in seven of 27 (26%) of the tumors analyzed relative to controls. Tumors with an increase in cyclin D1 mRNA also overexpressed the cyclin D1 protein. RNA encoding for the EGFR was decreased in 21 of 23 (91%) HCCs when compared with controls. None of the 29 liver tumors analyzed for alterations in expression of TGF-α mRNA differed from controls. Also, each individual tumor had a unique set of molecular alterations even when different tumors from the same animal were analyzed. These novel findings suggest that IGF-II, cyclin D1, c-myc, and EGFR are important mediators of carcinogenesis in spontaneous mouse liver tumor formation. Mol. Carcinog. 19:31–38, 1997. © 1997 Wiley-Liss, Inc.     

Hepatocarcinogenicity of chlordane in B6C3F1 and B6D2F1 male mice: evidence for regression in B6C3F1 mice and carcinogenesis independent of ras proto-oncogene activation

Logistic regression analysis of age-specific prevalences forneoplastic and non-neoplastic liver lesions was used to examinetreatment responses for B6C3F1 and B6D2F1 male mice continuouslyexposed to chlordane (55 p.p.m.) and to determine whether neoplasmswere dependent on continuous exposure in the B6C3F1 mice. Inorder to determine if ras oncogene activation plays a role inthe carcinogenicity of chlordane and whether the activationis dependent on genetic background, liver tumors from chlordane-treatedB6C3F1 and B6D2F1 mice were analyzed for the presence of activatingmutations in the ras oncogene. The overall liver tumor prevalenceat terminal killing was nearly 100% for both strains; however,the age-specific prevalence increased more rapidly in B6C3F1mice than in B6D2F1 mice. Tumor-bearing B6C3F1 mice had an averageof two more tumors per liver than B6D2F1 mice at their respectiveterminal killings (5.4 versus 3.3). When chlordane exposurewas discontinued for a group of B6C3F1 mice (‘stop’group) at 491 days of age, overall tumor multiplicity significantlydecreased by 30% from an average of 4.4 per tumor-bearing-animalat 525 days to 3.1 at terminal killing (568 days). Over thesame time period the prevalence of hepatocellular carcinomassignificantly decreased from 80 to 54% and adenomas from 100to 93% by terminal killing in B6C3F1 ‘stop-group’mice. Chlordane induced diffuse hepatocellular centrilobularhypertrophy, frequent multinucleate hepatocytes, toxic changeand hepatoproliferative lesions composed predominantly of acidophilichepatocytes in nearly 100% of both the B6C3F1 and B6D2F1 mice.The development of histological evidence of toxicity closelyparalleled the temporal development of hepatocellular neoplasiaand decreased in severity when the tumor burden was maximal.No H- or K-ras mutations were detected in the chlordane-inducedhepatocellular tumors in B6C3F1 mice (15 adenomas and 15 carcinomas)or B6D2F1 mice (10 adenomas and 10 carcinomas). In conclusion,chlordane induced liver tumors in both B6C3F1 and B6D2F1 malemice by mechanisms independent of ras oncogene activation and30% of both benign and malignant liver tumors in the B6C3F1mice regressed after exposure was discontinued.     

Cancer prevention by adult‐onset calorie restriction after infant exposure to ionizing radiation in B6C3F1 male mice

Children are especially sensitive to ionizing radiation and chemical carcinogens, and limiting their cancer risk is of great public concern. Calorie restriction (CR) is a potent intervention for suppressing cancer. However, CR is generally not appropriate for children. This study, therefore, examined to see if adult‐onset CR influences the lifetime cancer risk in mice after early‐life exposure to ionizing radiation. Infant male mice (1‐week‐old) were exposed to 3.8 Gy X‐rays, fed a control 95 kcal/week or CR 65 kcal/week diet from 7 weeks of age (adult stage), and their lifespan and tumor development were assessed. Irrespective of CR, X‐rays shortened lifespan by 38%, and irrespective of irradiation CR extended lifespan by 20%. Thymic lymphoma (TL) and early‐occurring non‐TL were induced by radiation. The liver and Harderian gland were more susceptible to radiation‐induced tumors than the lungs and non‐thymic lymphoid tissues (late occurring). CR reduced the risk of hepatocellular carcinoma, late‐occurring non‐TL, lung tumor, Harderian tumor, and hemangioma but had less impact on TL and early‐occurring non‐TL. Most notably, the effects of X‐rays on induction of lung tumors, late‐occurring non‐TL and hemangioma were essentially canceled by CR. The ability of CR to prevent late‐occurring tumors was the same for non‐irradiated and irradiated mice, indicating that the mechanism by which CR influences cancer is independent of irradiation. Our results indicate that adult‐onset CR significantly inhibits late‐occurring tumors in a tissue‐dependent manner regardless of infant radiation exposure.     

DNA adduct formation in the bone marrow of B6C3F1 mice treated with benzene

We used P₁-enhanced ³²P-postlabeling to investigate DNA adductformation in the bone marrow of B6C3F1 mice treated intraperitoneallywith benzene (BZ). No adducts were detected in the bone marrowof controls or mice treated with various doses of BZ once aday. After twice-daily treatment with BZ, 440 mg/kg, for 1 to7 days, one major and two minor DNA adducts were detected. Therelative adduct levels ranged from 0.06–1.46x10^–7.In vitro treatment of bone marrow from B6C3F1 mice with variousdoses of hydroquinone (HQ) for 24 h also produced three DNAadducts. These adducts were the same as those formed after invivo treatment of bone marrow with BZ. Co-chromatography experimentsindicated that the principal DNA adduct detected in the bonemarrow of B6C3F1 mice was the same as that detected in HL-60cells treated with HQ. This finding suggests that HQ may bethe principal metabolite of BZ leading to DNA adduct formationin vivo. DNA adduct 2 corresponds to the DNA adduct formed inHL-60 cells treated with 1,2,4-benzenetriol. DNA adduct 3 remainsunidentified. After a 7-day treatment with BZ, 440 mg/kg twicea day, the number of cells per femur decreased from 1.6x10⁷to 0.85xlO⁷, indicating myelotoxicity. In contrast, administrationof BZ once a day produced only a small decrease in bone marrowcellularity. These studies demonstrate that metabolic activationof BZ leads to the formation of DNA adducts in the bone marrow.Further investigation is required to determine the role of DNAadducts and other forms of DNA damage in the myelotoxic effectsof exposure to BZ.     

Sulfuric acid esters as major ultimate electrophilic and hepatocarcinogenic metabolites of 4-aminoazobenzene and its N-methyl derivatives in infant male C57BL/6J x C3H/HeJ F1 (B6C3F1) mice

Liver cytosols from 12-day-old male C57BL/6 X C3H/HeJ F1 (B6C3F1) mice contain 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferase activity for N-hydroxy-4-aminoazobenzene and N-hydroxy-N-methyl-4-aminoazobenzene. No acetyl co-enzyme A-dependent transacetylase activity for these hydroxylamines was detected in the cytosols. Pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol were only moderately active inhibitors of the sulfotransferase activity; at a 100-microM concentration each compound inhibited the activity by only 50-80%. A single dose of 0.04 mumol/g body weight of PCP administered to 12-day-old male B6C3F1 mice 45 min prior to a single dose of 0.1 mumol/g body weight of [3H]4-aminoazobenzene ([3H]AB) or [3H]N,N-dimethyl-4-aminoazobenzene ([3H]DAB) inhibited DNA adduct formation by approximately 50%. Under identical conditions, PCP also reduced the average number of hepatomas induced per mouse at 9 months by AB and N-methyl-4-aminoazobenzene (MAB) by 52 and 36%, respectively. PCP strongly inhibited the hepatocarcinogenicity of DAB or AB when this agent was administered in the diet with either dye to female CD-1 mice over a 10- month period. Single doses of 0.15 mumol/g body weight of [3H]AB and [3H]DAB bound to hepatic DNA of 12-day-old brachymorphic B6C3F2 mice, which are deficient in the synthesis of PAPS, at levels 15 and 20%, respectively, of those found in their phenotypically normal litter mates. Under identical conditions, the incidence of hepatomas in brachymorphic mice at 9 months were 11 and 29%, with averages of 0.2 and 0.8 hepatomas/mouse for AB and MAB, respectively. Incidences of 77 and 86%, with averages of 6.6 and 5.4 hepatomas/mouse, respectively, were found in their phenotypically normal litter mates. These data strongly indicate that N-sulfo?xy-AB is a major ultimate electrophilic and hepatocarcinogenic metabolite of AB in mice. Similarly, this ester and N-sulfo?xy-N-methyl-4-aminoazobenzene appear to be critical metabolites for these activities of DAB and MAB.     

Activation of H-ras oncogenes in male B6C3F1 mouse liver tumors induced by vinthionine or 2-chloroethyl methyl sulfide

Vinthionine (S-vinyl-DL-homocysteine) is hepatocarcino-genicin rats and mice. [Vinyl-¹⁴C]vinthionine binds cova-ently torat liver DNA, RNA and protein in vivo, but not in vitro. Thisamino acid is directly mutagenic in Salmonella typhimurium TA100and TA1535; the mechanism of its metabolic activation in vivoin bacteria and liver is under study. In the present study livertumors were induced in 12-day-old male B6C3F1 mice by singlei.p. injections of vinthionine or the alkylating agent 2-chloroethylmethylsulfide (CEMS).At 10 months the gross tumors were examinedfor the presence of activated H-ras oncogenes. DNA was isolatedfrom single tumors per mouse from 37mice treated with vinthionineand from 31 mice treated with CEMS. These DNAs were screenedfor codon 61 mutations by restriction fragment length polymorphismof PCR-amplified H-ras gene fragments. Thirty seven of 37 vinthionine-inducedhepatomas had H-ras mutations in this codon, which consistedof seven C     

Pentachlorophenol (PCP) produces liver oxidative stress and promotes but does not initiate hepatocarcinogenesis in B6C3F1 mice.

To elucidate the mechanism of hepatocarcinogenesis of pentachlorophenol (PCP) in mice, critical effects related to carcinogenicity were studied in the livers of B6C3F1 male mice administered PCP at concentrations of 600 and 1200 p.p.m. in the diet for 8 weeks. Oxidative stress was assessed by measurements of 8-oxodeoxyguanosine (8-oxodG) in the liver nuclear DNA and hepatocyte cell proliferation was quantified by bromodeoxyuridine incorporation. Also, initiation and promotion were assessed in a two-stage hepatocarcinogenesis model in which one group of mice was given PCP at concentrations of 600 and 1200 p.p.m. as initiator for the first 13 weeks with subsequent administration of phenobarbital (PB) as promoter at a concentration of 500 p.p.m. in the drinking water for 29 weeks. A second group was initiated with diethylnitrosamine (DEN) at 20 p.p.m. in the drinking water for the first 13 weeks followed after a 4 week recovery interval by PCP at concentrations of 300 and 600 p.p.m. in the diet for 25 weeks. Significant elevations in 8-oxodG levels and cell proliferation were observed in a dose-dependent manner. Incidences and multiplicities of hepatocellular tumors in mice treated with PCP after DEN initiation were increased compared with those in mice given initiation only. In contrast, in mice given PCP as initiator followed by PB no enhancement of neoplastic lesions occurred. These findings are interpreted to demonstrate that PCP exerts a promoting action, but not an initiating effect on liver carcinogenesis and that the promoting action is related to oxidative stress and compensatory hepatocellular proliferation.     

SHORT COMMUNICATION: Detection of DNA adducts in the white blood cells of B6C3F1 mice treated with benzene

We have employed the P₁-enhanced ³²P-postlabeling procedureto detect the formation DNA of adducts in the white blood cells(WBC) of B6C3F1 mice treated by i.p. injection with benzene.Treatment twice a day with 440 mg/kg benzene for 1–7 daysresulted in the formation of one major (adduct 1) and one minor(adduct 2) DNA adduct in the WBCs of mice. The same DNA adductpattern was also found in the bone marrow (BM) of benzene treatedmice. The relative adduct levels were dependent upon both benzenedose from 100–440 mg/kg and treatment time from 1 to 7days. The relative adduct levels ranged between 0.11 and 1.33adducts in 10⁷ nucleotides for WBCs and 0.16–1.21 adductsin 107 nucleotides for BM. Following treatment with benzene,the levels of DNA adducts formed in WBCs were significantlycorrelated with the levels of DNA adducts formed in BM (r² =0.97, P <0.001). Our results suggest that measurement ofDNA adducts in WBCs may be an indicator of DNA adduct formationin BM following BZ exposure.     

DNA adduct formation and induction of micronuclei and mutations in B6C3F1/Tk mice treated neonatally with acrylamide or glycidamide

Acrylamide, a food contaminant, is carcinogenic in experimental animals, with both genotoxic and nongenotoxic pathways being proposed. To obtain information regarding mechanisms of acrylamide tumorigenesis, we compared the extent of DNA adduct formation and induction of micronuclei and mutations in mice treated neonatally with acrylamide and its electrophilic metabolite glycidamide. Male and female B6C3F₁/Tk mice were treated intraperitoneally on postnatal days (PNDs) 1, 8 and 15 or PNDs 1–8 with 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day. One day after the final dose, B6C3F₁/Tk^+/+ mice were killed to measure DNA adduct levels and peripheral blood micronuclei. Three weeks after the last treatment, B6C3F₁/Tk^+/? mice were killed to assess the Hprt and Tk mutant frequencies in spleen lymphocytes. The levels of N7‐(2‐carbamoyl‐2‐hydroxyethyl)guanine, the major glycidamide‐DNA adduct, decreased in the order 0.70 mmol glycidamide > 0.70 mmol acrylamide > 0.14 mmol glycidamide ～ 0.14 mmol acrylamide. Only glycidamide increased the frequency of micronucleated reticulocytes and normochromatic erythrocytes. In mice treated on PNDs 1, 8 and 15, the Hprt mutant frequency was increased by 0.70 mmol glycidamide. In mice dosed on PNDs 1–8, 0.70 mmol glycidamide caused extensive mortality; each of the other treatments increased the Tk mutant frequency, whereas acrylamide increased the Hprt mutant frequency. These data suggest that the mutagenic response in neonatal mice treated on PNDs 1, 8 and 15 is due to glycidamide, whereas mutations resulting from dosing on PNDs 1–8 are due to another mechanism. © 2008 Wiley‐Liss, Inc.     

Enzyme and immunohistochemical phenotyping of diethylnitrosamine-induced liver lesions of male C3H/He, B6C3F1 and C57BL/6J mice.

Male C3H/He, B6C3F1 and C57BL/6J mice were given a single injection of diethylnitrosamine (20 micrograms/g body wt) on day 15 after birth and animals were killed 17-29 (C3H/He and B6C3F1) and 29-46 weeks (C57BL/6J) after treatment. Carcinogen-induced liver lesions were identified by a deficiency in the marker enzyme glucose-6-phosphatase and the enzymatic phenotypes of these lesions were studied by enzyme and immunohistochemical methods using serial liver sections stained for seven additional histochemical markers. In all three mouse strains, liver lesions were characterized by an increased basophilia and a decreased expression of UDP-glucuronosyl-transferase, microsomal epoxide hydrolase and NADPH-cytochrome P450 reductase, while the cytochrome P450 isoenzymes 1A2, 2C6 and 2E1 were virtually unexpressed. Quantitative analyses revealed that throughout all periods of investigation, on average greater than 70% of the glucose-6-phosphatase-deficient lesions occupying up to 99% of the total volumetric fraction expressed concomitant alterations in at least one of these additional marker stainings. Upon determination of the phenotypic complexity levels, between 70 and 90% of lesions were found to contain alterations in at least six of the markers analysed, while lesions with alterations in less than three markers were comparatively infrequent. In the light of previous observations in the rat liver system, the relative homogeneity of enzyme phenotypes and the apparent lack of time-dependent changes in enzyme expression suggest that the majority of lesions of all three mouse strains possess an increased neoplastic character already from the very early beginning of the carcinogenic process in liver.     

Identification of C8-modified deoxyinosine and N2-and C8-modified deoxyguanosine as major products of the in vitro reaction of N-hydroxy-6-aminochrysene with DNA and the formation of these adducts in isolated rat hepatocytes treated with 6-nitrochrysene and 6-aminochrysene

Since 6-nitrochrysene and 6-aminochrysene have shown activityin carcinogenicity bioassays, we have begun an investigationof their metabolic activation pathways and the nature of thecarcinogen—DNA adducts that may be formed. N-Hydroxy-6-aminochrysene(N-hydroxy-AC), a candidate proximate or ultimate carcinogenand the highest polycyclic N-hydroxy arylamine homolog studiedthus far, was prepared by direct chemical synthesis and characterizedby ¹H-n.m.r. spectroscopy. Its rate and extent of reaction withDNA in vitro was 20–30 nmol bound/mg DNA/30 min, whichis 2–10 times greater than has been reported for severalother carcinogenic N-hydroxy arylamines. Three major amino-chrysene-nucleosideadducts were detected in enzymatic hydrolysates of this N-hydroxy-AC-modifiedDNA, and these were isolated and identified by mass and ¹H-n.m.r.spectroscopy as N-(deoxyinosin-8-yl)-6-aminochrysene, 5-(deoxy-guanosin-N²-yl)-6-aminochrysene,and N-(deoxyguanosin-8-yl-6-aminochrysene. These adducts accountedfor 32%, 28%, and 22% respectively, of the total DNA adductsformed. We hypothesize that the deoxyinosine adduct is derivedfrom spontaneous oxidation of the corresponding deoxyadenosineadduct prior to or during DNA isolation and adduct preparation.DNA isolated from Sprague-Dawley rat hepatocytes which had beentreated with [³H]6-aminochrysene or [³H]6-nitrochrysene containedup to 12 pmol adducts/mg DNA (4 adducts per 10⁶ nucleotides).High performance liquid chromatography (h.p.l.c.) analyses ofenzymatic hydrolysates of this DNA indicated that the majorproducts formed co-chromatographed with the C8-deoxyinosineand C8-deoxy-guanosine adducts. N-(Deoxyinosin-8-yl)-6-aminochryseneand N-(deoxyinosin-8-yl)-6-aminochrysene accounted for 45% and30% respectively, of the total DNA adducts formed in these cells.The preferential modification of deoxyadenosine by N-hydroxy-6-aminochryseneand the apparent facile oxidation of this adduct to a deoxyinosinederivative is thus far unique among the reactions of N-hydroxyarylamineswith DNA and would not be predicted on the basis of reactivityalone.     

Identification and quantitation of hepatic DNA adducts formed in B6C3F1 mice from 1'-hydroxy-2', 3'-dehydroestragole: comparison of the adducts detected with the l'-3H-labelled carcinogen and by 32P-postlabelling

The identities and levels of DNA adducts formed in mouse liverafter administration of the hepatocarcinogen [l'-³H]1'-hydroxy-2',3'-dehydroestragole obtained by analysis of the ³H-containingadducts were compared with those found by ³²P-postlabellinganalysis. As previously observed the two dia-stereomers of N²-(dehydroestragol-l'-yl)-deoxyguanosinewere the only adducts detected by use of the tritiated carcinogen.Similarly, the unresolved diastereomers of N²-(de-hydroestragol-l'-yl)-deoxyguanosine-3,5-diphosphate were the only adducts detected by the postlabellingprocedure. Analysis by ³²P-postlabelling of defined mixturesof the normal deoxynucleoside-3'-phosphates and synthetic N²-(dehydro-estragol'-l-yl)-deoxyguanosine-3'-phosphateshowed that recovery of the labelled adduct was about 60% ofthat of the normal nucleotides. Likewise, the levels of theadduct in the hepatic DNA from mice treated with l'-hydroxydehydroestra-gole,as determined by ³²P-postlabelling, were generally 60 –80% of those obtained by analysis for the tritiated adducts.Since l'-oxodehydroestragole-deoxyadenosine adducts, the majorproducts obtained on reaction of l'-oxodehydro-estragole withDNA in vitro, were not detected by ³²P-post-labelling in thehepatic DNA from mice treated with 1'-hydroxydehydroestragole,these data provide further evidence that the covalent bindingof l'-hydroxydehydroestragole to liver DNA in vivo does notinvolve thel'-oxo derivative.     

In vitro steady-state levels of hydrogen peroxide after exposure of male F344 rats and female B6C3F1 mice to hepatic peroxisome proliferators

The hypothesis that hepatocarcinogenesis resulting from treatmentof rats and mice with peroxisome proliferators is linked toincreased cellular levels of hydrogen peroxide from peroxisomalß-oxidation was investigated. Male F344 rats and femaleB6C3F2₁ mice were treated for 14 days with di(2-ethylhexyl)phthalate(DEHP) or di(2-ethylhexyl)adipate (DEHA), industrial plasticizers,or nafenopin, a hypolipi-demic drug. Activities of enzymes responsiblefor the production [peroxisomal palmitoyl CoA oxidase (PCO)]and degradation [catalase (Cat) and glutathione peroxidase (GSHPx)]of H₂O₂ were assayed in liver homogenates prepared from treatedanimals. The activities of the peroxisomal enzymes PCO and Catwere enhanced 5- to 25-fold and 1.5-to 3-fold respectively bytreatment with the peroxisome proliferators. The activity ofGSHP_x, a cytoplasmk enzyme, was decreased 40–60% in liverhomogenates prepared from treated pnimalx compared to controlanimals. A kinetic treatment of the rates of formation of hydrogenperoxide by PCO, and of degradation of hydrogen peroxide bycatalase was used to estimate steady-state hydrogen peroxideconcentrations ([H₂O₂]) during peroxisomal oxidation of palmitoylCoA. Increases in peroxisomal steady-state [H₂O₂] for the F344rat liver homogenates correlated well with the carcinogenicpotential of these chemicals, determined in previous carcinogenicitystudies. Increases in the steady-state [H₂O2] were also calculatedfor liver homogenates prepared from mice treated with thesecompounds. Decreases in liver lipid peroxidation were observedafter treatment with each chemical in both species. The resultsof these studies are consistent with an involvement of increasedperoxisomal hydrogen peroxide in the hepatocarcinogenesis ofthese compounds.     

Correlation of DNA adduct formation and riddelliine-induced liver tumorigenesis in F344 rats and B6C3F1 mice [Cancer Lett. 193 (2003) 119-125

Riddelliine is a naturally occurring pyrrolizidine alkaloid that induces liver hemangiosarcomas in male and female F344 rats and male B6C3F1 mice. We previously reported that eight dehydroretronecine (DHR)-derived DNA adducts were formed in liver DNA of rats treated with riddelliine. In order to examine the relationship between DNA adduct levels and the incidence of hemangiosarcomas, we have measured DHR-derived DNA adduct levels in purified rat and mouse liver endothelial cells, the cells of origin for the hemangiosarcomas. F344 rats and B6C3F1 mice were treated by gavage 5 days per week for 2 weeks with riddelliine at 1.0 mg/kg for rats and 3.0 mg/kg for mice. One, 3, 7, and 28 days after the last dose, liver parenchymal and endothelial cell fractions were isolated, and the quantities of DHR-derived DNA adducts were determined by 32P-postlabeling/HPLC. The DHR-derived DNA adduct levels in the endothelial cells were significantly greater than in the parenchymal cells. The DNA adduct levels in rat endothelial cells were greater than in the mouse endothelial cells. These results indicate that the levels of riddelliine-induced DNA adducts in specific populations of liver cells correlate with the preferential induction of liver hemangiosarcomas by riddelliine.     

DNA adduct formation in B6C3F1 mice and Fischer-344 rats exposed to 1, 2, 3-trichloropropane

1, 2, 3-Trichloropropane (TCP) is a multispecies, multisitecarcinogen which has been found to be an environmental contaminantIn this study, we have characterized and measured DNA adductsformed in vivo following exposure to TCP. [¹⁴C]TCP was administeredto male B6C3F1 mice and Fischer-344 rats by gavage at dosesused in the NTP carcinogenesis bioassay. Both target and nontargetorgans were examined for the formation of DNA adducts. Adductswere hydrolyzed from DNA by neutral thermal or mild acid hydrolysis,isolated by HPLC, and detected and quanti-tated by measurementof radioactivity. The HPLC elution profile of radioactivitysuggested that one major DNA adduct was formed. To characterizethis adduct, larger yields were induced in rats by intraperitonealadministration of TCP (300 mg/kg). The DNA adduct was isolatedby HPLC based on coelution with the radiolabeled adduct, andcompared to previously identified adducts. The isolated adductcoeluted with S-[1-(hydroxymethyl)-2-(N⁷-guanyl)-ethyljglutathione,an adduct derived from the structurally related carcinogen 1,2-dibromo-3-chloropropane (DBCP). Analysis by electrospray massspectrometry suggested that the TCP-induced adduct and the DBCP-derivedadduct were identical. The ¹⁴C-labeled DNA adduct was distributedwidely among the organs examined. Adduct levels varied dependingon species, organ, and dose. In rat organs, adduct concentrationsfor the low dose ranged from 0.8 to 6.6 µmol per mol guanineand from 7.1 to 47.6 µmol per mol guanine for the highdose. In the mouse, adduct yields ranged from 0.32 to 28.1 µmolper mol guanine for the low dose and from 12.2 to 208.1 µmolper mol guanine for the high dose. The relationship betweenDNA adduct formation and organ-specific tumorigenesis was unclear.Although relatively high concentrations of DNA adducts weredetected in target organs, several nontarget sites also containedhigh adduct levels. Our data suggest that factors in additionto adduct formation may be important in TCP-induced carcinogenesis.