Modification of transplacental tumorigenesis by 3-methylcholanthrene in mice by genotype at the Ah locus and pretreatment with beta-naphthoflavone

Transplacental lung and liver tumorigenesis in the mouse by 3-methylcholanthrene (MC) was assessed as a function of inducibility of MC metabolism in fetus and in mother, and of pretreatment of the mothers with a noncarcinogenic inducer, beta-naphthoflavone (beta NF). Pregnant (C57BL/6 X DBA/2)F1 females (genotype Ahb Ahd, inducer responsive) mated to DBA/2 males received 45 or 100 mg/kg MC on gestation day 17, and DBA/2 females (genotype Ahd Ahd, nonresponsive) mated to F1 males were given 5 or 30 mg MC/kg. These crosses generated both responsive and nonresponsive offspring. Phenotype and tumor incidences were determined at 13 months of age. The transplacental action of MC was dose dependent and resulted in more lung and liver tumors in induction-responsive offspring than in nonresponsive littermates in most comparisons. beta NF alone did not result in increased numbers of tumors. Significant, complex effects were seen when the mothers were pretreated with beta NF (150 mg/kg) on gestation day 15, before MC on day 17. The beta NF pretreatment protected the fetuses of the F1 mothers: there was a significant overall 30 to 50% reduction in numbers of lung and liver tumors. The greatest effect was seen in the induction-responsive males, who experienced a 50% reduction in both incidence and multiplicity of lung tumors after 100 mg MC/kg, compared with males exposed to MC only. By contrast, beta NF pretreatment of DBA mothers had no general effect but rather potentiated the action of the 5 mg MC/kg dose on multiplicity of lung tumors in inducible males, causing a significant 4-fold increase. It also caused a 60% increase in inducible male liver tumor multiplicity when given before the 30 mg MC/kg dose. Thus, beta NF pretreatment was protective when the mother was inducible, especially in the inducible fetuses of such a mother, but when the mother was noninducible the beta NF pretreatment had no effect in some situations and potentiated the action of the carcinogen in others, mainly in inducible fetuses. These results underscore the fact that induced maternal and fetal metabolism contribute to risk of transplacental tumorigenesis by MC in qualitatively opposite ways.     

Alkoxyresorufin O-dealkylases: association with the murine Ah locus

The hepatic microsomal dealkylation of a series of alkoxyresorufins and the oxidation of phenoxazone to resorufin were investigated in C57BL/6 and DBA/2 mice of both sexes. In both strains of mice and in both sexes the dealkylation rate decreased with increasing length of the alkyl chain. With all alkoxyresorufins the dealkylation rates were higher in the C57BL mice than the DBA mice, whereas the rate of phenoxazone hydroxylation was higher in the latter. In the C57BL mice, and to a lesser extent in the DBA mice, females were more efficient in dealkylating the resorufin ethers. Treatment with 3-methylcholanthrene (3MC) enhanced the rates of dealkylation of all alkoxyresorufins in the C57BL mice but not in the DBA mice, the extent of stimulation being highest for the propoxy- and butoxyresorufins and least for pentoxy-, heptoxy- and benzyloxyresorufins. The same treatment had no effect on the oxidation of phenoxazone in either strain of mice. It is concluded that the dealkylation of alkoxyresorufins, not the oxidation of phenoxazone, is associated with the murine Ah locus.     

Immunohistochemical determination of inducibility phenotype with a monoclonal antibody to a methylcholanthrene-inducible isozyme of cytochrome P-450

A monoclonal antibody (MAb) to a methylcholanthrene (MC)-induced cytochrome P-450, designated MAb 1-7-1, was used for immunohistochemical staining of formalin-fixed tissues from oil- and MC-treated C57BL/6, DBA/2, and [(C57BL/6 X DBA/2) F1 X DBA/2] F2 mice. An avidin-biotin-peroxidase complex immunohistochemical technique was used. For controls, the tissues were also exposed to MAbs 1-48-5 and HyHel-9 (to egg white lysozyme). In liver, MAb 1-7-1 specifically stained the cytoplasm of centrilobular hepatocytes of C57BL/6 mice treated with MC (80 mg/kg) 48 h before kill; staining was not observed with vehicle-treated C57BL/6 mice, with oil- or MC-treated DBA/2 mice, or with comparable antibody concentrations of control MAbs 1-48-5 or HyHel-9. In the F2 mice, about 50% were expected to be MC inducible (AhbAhd). Inducibility phenotype was determined by measuring the conversion of [14C]MC to oxidized and conjugated products by liver homogenates. In freshly fixed material from MC-treated mice, those livers shown by the determination of phenotype to be inducible also stained with MAb 1-7-1, whereas those not induced were immunohistochemically negative. Furthermore, there was a significant positive correlation between degree of staining and the level of MC-metabolizing activity measured biochemically. The immunohistochemical procedure was also accurate in determination of inducibility phenotype of livers that had been in paraffin blocks for up to 2 yr if more concentrated antibody was used. In lung, MAb 1-7-1 stained specifically the alveolar walls and endothelium of blood vessels in MC-induced C57BL/6 mice only; the control MAbs and other mice gave negative results. Similarly, in kidney MAb 1-7-1 stained only glomeruli and interstitial tissue of MC-induced C57BL/6 mice and only endothelium of blood vessels in the colons of these mice. These observations are consistent with induction of the cytochrome P-450 recognized by MAb 1-7-1 in the endothelial cells of extrahepatic tissue. Immunohistochemical staining with MAb thus shows great promise for highly specific localization of particular species of cytochromes P-450 in tissues, for in situ quantification of these enzymes, and for determination of inducibility phenotype with fixed material.     

Persistence of benzo(a)pyrene metabolite:DNA adducts in lung and liver of mice

The persistence of benzo(a)pyrene (BP) metabolite:DNA adducts has been studied in lung and liver of A/HeJ and C57BL/6J mice after a dose of BP (6 mg/mouse) which induces pulmonary adenomas in A/HeJ mice but not in C57BL/6J mice. BP is not a hepatic carcinogen in either strain. Following p.o. administration of [3H]BP, animals were killed at times ranging from 10 hr to 28 days, and BP metabolite:DNA adducts were analyzed by high-pressure liquid chromatography. The major adduct identified in each tissue was the (+)-7 beta-8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct. A 7 beta, 8 alpha-dihydroxy-9 beta,10 beta,epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, a (-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct, and an unidentified adduct were also observed. The disappearance of (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP adduct in A/HeJ mice followed first-order kinetics over the time period examined, with a half-life of 18 and 9 days in lung and liver, respectively. The decay of this adduct in C57BL/6J mice was biphasic in both tissues. Our data on cell turnover suggest that there is active removal of adducts in liver, but that normal DNA turnover can account for the partial or possibly total observed disappearance of adducts in lung. These results suggest that the tissue specificity for BP-induced neoplasia in A/HeJ mice may be related to the relative persistence of adducts and high cell turnover rates in lung. In contrast, the results on formation and persistence of adducts and cell turnover do not provide an explanation for the strain difference in susceptibility to BP-induced pulmonary adenomas. It was also shown that the rates of removal of BP metabolite:DNA adducts in A/HeJ mice are not significantly different at a 500-fold lower BP dose.     

2-aminofluorene-hepatic DNA adducts in congenic mouse lines differing in Ah responsiveness

The influence of beta-naphthoflavone (BNF) pretreatment on 2-aminofluorene (2-AF)-hepatic DNA adduct formation was evaluated in Ah-responsive and non-responsive congenic mouse lines through use of HPLC analysis of 32P-postlabeled nucleotides. C57BL/6J (B6) mice were used as an example of BNF-responsive mice while B6.D-Ahd, a line congenic with B6, was used as the non-responsive line. Induction at the Ah locus with BNF increased adduct levels in hepatic DNA in B6 mice but not in B6.D mice 3 h after a 60 mg/kg i.p. dose of 2-AF. The slow acetylator counterparts of B6 and B6.D, namely B6.A and B6.A.D-NatsAhd (a new congenic line produced from B6.A and B6.D), had lower adduct levels than the rapid acetylators before induction. Although adduct levels in B6.A and B6.A.D were increased following BNF induction, the level of adducts remained below those of induced B6 mice. In the three lines that responded to BNF induction, male mice had a greater relative increase in hepatic DNA adduct levels than females. For all four lines, with or without BNF pretreatment, greater adduct levels were found in the females. These results imply that responsiveness to aromatic hydrocarbon induction, as well as rapid acetylation, may be risk factors in hepatic DNA damage following arylamine exposure. Female mice appear to be more susceptible to such damage than males.     

Strain-specific tumorigenesis in mouse skin induced by the carcinogen, 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one, and its relation to DNA adduct formation and persistence

The incidence of skin tumors has been studied in three strains of mice, namely, TO, C57BL, and DBA/2, after treatment with the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one. After either a single dose followed by croton oil promotion or a continual dose of the carcinogen, tumors were observed in the TO and C57BL strains, with the TO mice having the shorter mean latent period. The DBA/2 mice, however, appeared to be resistant to tumor formation by either treatment. To understand the mechanism of resistance, several criteria have been investigated. Metabolism of the carcinogen was assessed in terms of the total DNA adduct formation and the pattern of individual adducts after separation by high-pressure liquid chromatography, and no major differences between the three strains was found. Similarly, the rates of disappearance of the individual adducts when measured over 14 days posttreatment were not strain specific. Persistent binding of the carcinogen after 2 months was found in all three strains and could be reduced markedly if croton oil was administered throughout this period. The ability of the phorbol esters to cause biochemical changes in both sensitive and resistant strains was indicated by the induction of ornithine decarboxylase in each of the three strains after treatment with either croton oil or its active component, 12-O-tetradecanoylphorbol-13-acetate.     

Investigation of the formation and accumulation of liver DNA adducts in mice chronically exposed to tamoxifen

Tamoxifen was administered to three strains of female mice (B6C3F1, C57BL/6 and DBA/2) in short- and long-term studies to determine their ability to activate tamoxifen and cause hepatic DNA damage. 32P- Postlabelling of liver DNA from mice treated for 4 days showed a group of major adducts that increased in a dose-dependent manner and co- chromatographed with the major adducts detected in rat liver. On cessation of dosing, the majority of adducts were cleared within 3 days. Binding of [14C]tamoxifen to DNA nucleotides was demonstrated by the use of accelerator mass spectrometry. In long-term studies of 12 months to 2 years duration, dependent on strain, tamoxifen was administered continuously in the diet to give a daily dose of approximately 40 mg/kg. DNA adducts were detected after 3 months, although the number of adducts decreased with time and by 2 years were not detectable in the tamoxifen treated mice. None of the treated groups showed a significantly increased incidence of liver tumours, with or without phenobarbital promotion and there was no sustained liver cell proliferation. Tamoxifen was detected in the mouse livers, but at levels 50 times lower than those reported in a comparable rat study. These results suggest that, in contrast to the rat, tamoxifen is non-carcinogenic in mice because it does not cause sufficient cumulative DNA damage, or act as a promoter by causing cell proliferation.      

Effect of polycyclic aromatic compounds and phorbol esters on ornithine decarboxylase and aryl hydrocarbon hydroxylase activities in mouse liver

Single i.p. injections of 3-methylcholanthrene (MC; 50 mg/kg) administered to inbred C57BL/6 mice or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 micrograms/kg) to DBA/2 mice gave an increase in the hepatic activities of ornithine decarboxylase (ODC) and aryl hydrocarbon hydroxylase (AHH) with peaks occurring by 12 and 48 hr, respectively. A single i.p. dose of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 micrograms/kg) enhanced the activity of ODC about 70-fold within 12 hr in C57BL/6 mice and 18-fold within 24 hr in DBA/2 mice without affecting AHH activity markedly. 4-O-Methyl-12-O-tetradecanoylphorbol-13-acetate (100 micrograms/kg) raised ODC activity to about 25% of the TPA-treated value in C57BL/6 mice; in DBA/2 mice, TPA and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate induced ODC activity to roughly the same level. Benzo(e)pyrene (50 mg/kg) failed to affect ODC and AHH activities significantly in either strain. The inducing effect of TPA on ODC activity was potentiated by a simultaneous administration of MC to C57BL/6 mice; combined TPA and TCDD to DBA/2 mice exerted an additive effect on hepatic ODC activity. Difluoromethylornithine administered i.p. effectively inhibited the induction of ODC activity elicited by TPA, MC, or TCDD either alone or in various combinations but did not interfere with AHH induction. These data indicate that different regulatory factors are involved in the ODC induction process elicited by TPA and polycyclic aromatic compounds and that MC and TCDD may induce ODC activity by different mechanisms. The results also confirm our earlier findings in rat skin and cells in culture which suggest that the ODC and AHH induction processes can occur independently of each other. Additionally, there is a strain-related difference in sensitivity with regard to ODC-inducing activity of TPA in the livers of C57BL/6 and DBA/2 mice.     

2-Aminofluorene metabolism and DNA adduct formation by mononuclear leukocytes from rapid and slow acetylator mouse strains

Following exposure of mice to the arylamine carcinogen 2-aminofluorene,DNA-carcinogen adducts can be found in the target tissues liverand bladder, and also in circulating leukocytes. Evidence ispresented here that mouse mononuclear leukocytes (MNL) are capableof metabolizing 2-aminofluorene to DNA-binding metabolites whichgive rise to the adducts found in the MNL. Both lymphocytesand monocytes were able to acetylate arylamines during 18 hof culture. The degree of acetytation was determined by theN-acetyltransferase genotype of the mice as shown through useof acetylator congenic strains which differ only in the Nat-2gene. Cultured MNL from rapid acetylator mice (C57BL/6J andA.B6-Nat¹) produced about twice as much N-acetylaminofluorenefrom 2-aminofluorene and 6- to 8-fold as much N-acetyl-p-amino-benzoicacid from p-aminobenzoic acid as cells from slow acetylatormice (B6.A-Nat⁵ and A/J). Other differences in arylamine metabolismby MNL in culture were observed and shown to be due to geneticfactors, currently unidentified, other than N-acetyltransferase.DNA adduct formation following incubation of MNL with the arylaminecarcinogen 2-aminofluorene was related to both acetylation capacityand to other genetic metabolic factors in the mouse genome.MNL from rapid acetylator mice with the C57BL/6J background(B6) had 3-fold the DNA adduct levels of cells from the correspondingslow acetylator congenic (B6.A-Nat^$). Similarly, MNL from rapidacetylator mice with the A/J background (A.B6-Nat^r) had twicethe DNA adduct levels of those from their corresponding slowcongenic (A). Adduct levels in MNL from C57BL/6J were nearlythe same as those of MNL from A/J, again indicating the involvementof loci other than acetylation in DNA adduct formation. Thefinding of genetically dependent arylamine carcinogen metabolismand DNA adduct formation in cultured MNL suggests the possibilityof using cultured MNL for assessing individual susceptibilityto arylamine-induced DNA damage.     

Initiation of hepatocarcinogenesis in infant male B6C3F1 mice by N-hydroxy-2-aminofluorene or N-hydroxy-2-acetylaminofluorene depends primarily on metabolism to N-sulfooxy-2-aminofluorene and formation of DNA-(deoxyguanosin-8-yl)-2-aminofluorene adducts

Previous work from this laboratory provided strong evidencethat N-sulfooxy-2-aminofluorene is the major ultimate electro-philicand carcinogenic metabolite of N-hydroxy-2-acetyl-aminofluorene(N-hydroxy-AAF) in the livers of infant male B6C3F₁ (C57BL/6Jx C3H/HeJ F₁ mice. Over 90% of the hepatic DNA adducts in thesemice consisted of N-(deoxyguan-osin-8-yl)-2-aminofluorene [N-(dGuo-8-yl)]and<10% were deoxyguanosinyl adducts containing 2-acetylaminofluor-ene(AAF) residues. In the present study hepatic DNA adduct formationand tumor initiation by N-hydroxy-2-aminofluor-ene (N-hydroxy-AF)were examined in these mice. N-(dGuo-8-yl)-AF was the only adductdetected in the hepatic DNA; the level at 9 h after a singlei.p. dose of 0.04 or 0.06 µmol/g body wt of [³H]N-hydroxy-AFwas 1.0 or 1.7 pmol/mg DNA. Pre-treatment with a single i.p.dose (0.04 µmol/g body wt) of the sulfotransferase inhibitorpentachlorophenol (PCP) decreased the DNA adduct level by >80%.Similar levels of this adduct were found by ³²P-postlabelinganalysis of DNA from mice treated with unlabeled N-hydroxy-AF.The liver DNA of in-fant male brachyinorphic B6C3F₂ mice [deficientin 3'-phos-phoadenosine-5'-phosphosulfate (PAPS)] containedonly 0.3 pmol/mg DNA of N-(dGuo-8-yl)-AF after an i.p. doseof 0.06 µmol of N-hydroxy-AF/g body wt, while their phenotypi-callynormal (PAPS-sufficient) male littermates had 1.9 pmol/mg DNA.A single i.p. dose of 0, 0.015, 0.03, 0.06 or 0.12 µmol/body wt of N-hydroxy-AF in infant male B6C3F mice induced by10 months an average of 0.2, 2.5, 7, 11 or 14 hepatomas/mouse.Pretreatment with PCP reduced the liver tumor multiplicity ateach dose level by >80%. Essen-tially the same average tumormultiplicities and inhibitions of tumor formation by PCP pretreatmentwere obtained following injections of N-hydroxy-AF or N-hydroxy-AAFat the three lower dose levels. Collectively these data stronglyindicated that N-sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxyAF in the livers of infant male B6C3F₁ mice. Furthermore, sinceonly N-(dGuo-8-yl)-AF adducts were found in the he atic DNAthese lesions appear to be critical in the initiation of hepatocarcinogenesisin these mice by N-hydroxy-AF.     

The covalent binding of polycyclic hydrocarbons to DNA in the skin of mice of different strains.

The binding of three tritium-labelled carcinogenic polycyclic hydrocarbons, 7,12-dimethylbenz (a) anthracene (DMBA), benzo (a) pyrene (BP) and 3-methylcholanthrene (MCA) to DNA in mouse skin has been studied in C57BL, DBA/2 and Swiss mice following topical application of the hydrocarbons. DNA isolated from the treated areas was hydrolysed to deoxyribonucleosides and chromatographed on Sephadex LH20 columns. The levels of binding of hydrocarbon to DNA were determined from the amount of radioactivity eluted from Sephadex LH20 columns in those fractions containing hydrocarbon-DNA adducts and the radioactivity shown, by rechromatography on AG5OWX4 columns, to be due to tritium incorporation into normal deoxyribonucleosides was not included. C57BL mice were treated with doses of DMBA ranging from 0.025 μmol to 1 μmol/mouse and the levels of hydrocarbon bound to DNA 19 h after treatment were determined; there was no evidence for a threshold dose below which no binding to DNA occurs, and the same hydrocarbon-DNA product peaks were obtained at all doses. The levels of binding of DMBA (1 μmol/mouse) to DNA in skin were compared in C57BL, DBA/2 and Swiss mice at times varying from 6 h to 8 days after treatment. DMBA became bound to similar extents in Swiss and C57BL mice and to a slightly greater extent in DBA/2 mice; the rate of disappearance of bound DMBA from DNA was similar in all three strains. DMBA (0.1 μmol/mouse) was bound to DNA in C57BL and DBA/2 mice to similar extents 19 h after treatment and to a slightly lesser extent in Swiss mice. The ratios of the sizes of the hydrocarbon-DNA product peaks varied with the time after treatment, but were similar at any given time for the three strains. Both BP (1 μmol/mouse) and MCA (1 μmol/mouse) were bound to DNA to similar extents 19 h and 48 h after treatment in all three strains. BP (0.1 μmol/mouse) was bound to DNA in the order DBA/2>C57BL> Swiss 19 h after treatment. The levels of binding for all three hydrocarbons in the different strains do not show a correlation with the reported susceptibilities of the three strains to polycylic hydrocarbon carcinogenesis.     

Formation and persistence of benzo[a]pyrene--DNA adducts in different tissues of C57BL/10 and DBA/2 mice

Synchronous fluorescence spectrophotometry (SFS) developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)--DNA adducts was used to measure the formation and disappearance of DNA adducts in the lung, liver, kidney, spleen and small intestine of genetically responsive C57BL/10 (B10) and nonresponsive DBA/2 (D2) mice. After single stomach intubation of 100 mg/kg of benzo[a]pyrene (B[a]P) in both strains, binding of BPDE to DNA reached a peak 48 h after treatment. However, the levels of binding in the lung, liver, kidney and spleen were higher in D2 than in B10 mice. In contrast to this, in the small intestine the higher level of BPDE binding was found in B10 mice and reached its maximum 24 h earlier. Thereafter a very rapid drop in the level of BPDE--DNA adducts to a value of approximately 50% after 48 h was observed in this tissue. In the other tissues of the B10 mice the rate of adducts removal was slower, but by 14 days after treatment 90-100% of adducts were removed. In the D2 mice up to the 4th day after treatment the rates of removal of the BPDE--DNA adducts were similar to that of the B10 mice. Thereafter the level of bound hydrocarbon decreased at a slower rate. During the whole period after B[a]P treatment distinct differences between organs in the amount of BPDE--DNA adducts were observed. In D2 mice the highest level of binding was found in the spleen followed by the lung, kidney, liver and small intestine. In B10 mice the highest level of binding was observed in the DNA of small intestine. The data suggest that the decreased rate of B[a]P metabolism in D2 mice may be at least in some tissues the reason of higher binding of BPDE--DNA adducts in comparison with B10 mice.     

The effect of pentachlorophenol on DNA adduct formation in p53 wild-type and knockout mice exposed to benzo[a]pyrene

Previous studies have shown that pentachlorophenol (PCP) has both potentiative and antagonistic effects on the genotoxicity of benzo[a]pyrene (B[a]P). It has been suggested that these effects are due to inhibition and/or induction of enzymes involved in the biotransformation of B[a]P [Carcinogenesis 16 (1995) 2643]. However, B[a]P [J. Biol. Chem. 274 (1999) 35240] and a metabolite of PCP, tetrachlorohydroquinone (TCHQ) [Chem. Biol. Interact. 105 (1997) 1], induce p53 protein synthesis in vitro. To investigate this effect further, C57BL/6Tac trp53+/+ (wild-type, WT) and C57BL/6Tac trp53-/- (knockout, KO) mice were exposed to 55 microg B[a]P/g BW alone or in combination with 25 microg/g PCP. Hepatic and lung DNA were analyzed for the major B[a]P DNA adduct, 7R,8S,9S-trihydroxy-10R-(N2-2'-deoxyguanosyl)-7,8,9,10-tetrahydro-B[a]P (BPDE-N2G) and other minor adducts using the 32P-postlabeling assay. BPDE-N2G adducts were detected in all animals exposed to B[a]P. Similar adduct levels were observed in WT mice exposed to 55 microg/g B[a]P compared with KO mice exposed to B[a]P alone or in combination with PCP. Interestingly, hepatic and lung BPDE-N2G adducts were decreased in WT mice exposed to B[a]P with PCP (P<0.05). Total DNA adducts in the liver (P<0.05) were also decreased in WT mice exposed to B[a]P and PCP. Total DNA adducts in either hepatic or lung DNA isolated from KO mice were not different in mice treated with PCP and B[a]P. These results suggest that the decrease in BPDE-N2G adducts observed in WT mice may be a result of p53 accumulation or induction of repair pathways in response to damage induced by PCP.     

Levels and membrane localization of the c-K-ras p21 protein in lungs of mice of different genetic strains and effects of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor 1254

Mutational activation of the K-ras oncogene often occurs in human and mouse lung adenocarcinomas. Since K-ras p21 functions in trans-membrane signaling, we have investigated whether the amount of this protein in lung cell membranes is a variable that could influence lung tumorigenesis, either due to genetic differences or in response to tumor promoters. The six mouse strains assessed showed little difference in the total lung K-ras p21 after immunoprecipitation and immunoblotting. However, amount of ras p21 in the membrane fraction showed significant differences, with C57BL/6 and BALB/c having 3-5-fold more than NIH Swiss, AKR and DBA mice. Interestingly, a congenic AKR strain having the Ahr(b-1) Ah receptor allele from C57BL/6 mice (designated AKR.B6Ah) had high lung membrane K-ras p21 similar to that of C57BL/6. To test for possible changes related to lung tumor promotion, mice were treated with a promotional dose of TCDD (5 nmol/kg). After 48 h C57BL/6 lungs showed an increase in p21 in both total and membrane fractions. BALB/c, DBA and Swiss mice showed an increase only in membranes. There was no change in the AKR and AKR.B6Ah. Aroclor 1254 (250 mg/kg) caused an increase in membrane/cytosol ratio in Swiss mice. Thus the membrane:cytosol K-ras p21 ratio may be influenced by the Ahr phenotype, and TCDD and PCBs can induce p21 or increase its membrane level in certain strains, but these properties are not fully dependent on Ahr receptor type. In confirmation of the relevance of these findings for the tumor target cell type, the immortalized alveolar type 2 E10 cell line presented K- ras p21 in membrane, and this was increased 4-fold by treatment with 10 nM TCDD.      

DNA adduct levels in congenic rapid and slow acetylator mouse strains following chronic administration of 4-aminobiphenyl.

4-Aminobiphenyl (4-ABP) is a human and mouse bladder carcinogen. Epidemiological studies have shown that individuals with a slow acetylator phenotype, especially those exposed to high levels of carcinogenic aromatic amines, show an increased susceptibility to bladder cancer. In order to determine if a slow acetylator phenotype results in increased DNA damage, congenic mouse strains C57BL/6J and B6.A-Nat(s), which differ genetically at the acetyltransferase (EC 2.3.1.5) locus as homozygous rapid (Natr/Natr) and homozygous slow (Nat(s)/Nat(s)) acetylators respectively, were continuously administered 4-ABP.HCl (55-300 p.p.m.) in their drinking water for 28 days. The levels of covalently bound N-(deoxyguanosin-8-yl)-4-ABP-DNA adducts, which are believed to be critical for the initiation of tumors, were quantitated in the liver and bladder by 32P-postlabeling analysis. The levels of the hepatic DNA adduct increased with dose in both sexes, but were independent of the mouse acetylator genotype. At comparable doses, however, the levels of DNA adducts were 2-fold higher in the liver of the female as compared to the male animals. The DNA adducts also increased with dose in bladder of the male mice, but in contrast to the liver, the adduct levels were approximately 2-fold lower in the bladder DNA of the female mice. Also in contrast to the liver, the levels of bladder DNA adducts were significantly higher (P < or = 0.03) in the phenotypic rapid acetylator females compared to the slow acetylators at both 75 and 150 p.p.m. doses; the median levels of adducts were 10-20% higher in the phenotypic slow acetylator male bladders compared to their rapid acetylator counterparts. The results of these studies are consistent with the increased carcinogenicity of 4-ABP to the liver of female mice and the bladder of male mice. They further suggest that factors other than acetylator phenotype limit the extent of DNA adduct formation from 4-ABP in these mice.     

Antitumor activity of lentinan in murine syngeneic and autochthonous hosts and its suppressive effect on 3-methylcholanthrene-induced carcinogenesis

The antitumor effect of lentinan in syngeneic and autochthonous tumor-host systems and its suppressive effect on 3-methylcholanthrene (MC)-induced carcinogenesis were confirmed using DBA/2 and SWM/Ms hosts. The regressive activity of lentinan against the solid form of Sarcoma 180 was the most effective in DBA/2, SWM/Ms, or A/J mice and less effective in C3H/He or C57BL/6 mice. The growth of a syngeneic MC-induced DBA/2.MC.CS-1 fibrosarcoma (native and trypsinized) was markedly inhibited, and the regression of tumors was detected by the i.p. injection of minute amounts of lentinan into DBA/2 mice, which were the most suitable host in lentinan treatment. When DBA/2 mice were used, lentinan was also effective for even autochthonous primary tumors induced within 15 weeks after MC inoculation, but less effective for tumors induced during the 16 to 36 weeks after MC treatment. Lentinan showed a prominent suppressive effect in MC-induced carcinogenesis using DBA/2 and SWM/Ms mice but not effect when BALB/c, C57BL/6, or C3H/He mice were used. The timing of lentinan administration in the latter result was examined using SWM/Ms mice, and lentinan, when it was given daily for 10 days after the third week of MC inoculation, was strikingly effective (33%), but not so effective (63%) when lentinan was given after the sixth week of MC treatment, compared with tumor-occurrence rate in the control group (88%). The reason why DBA/2, SWM/Ms, or A/J mice were suitable hosts for lentinan treatment is not clear, but the natural killer capability or phagocytic macrophage function in these strains seems to have no relation to lentinan action, because A/J mice are deficient in natural killer function, and in these strains of mice the phagocytic function of macrophages is weak. It may be quite possible that these strains of mice are most sensitive to delayed-type hypersensitivity and/or cytotoxic T-cell response in which T-cells and lentinan play important roles. The tumor-host systems presented here provide a good model in which lentinan retains an inhibitory capacity in syngeneic and autochthonous hosts, and such a model offers the possibility for further study of the host defense mechanism against cancer.     

Comparison of epidermal protein kinase C activity, ornithine decarboxylase induction and DNA synthesis stimulated by TPA or dioctanoylglycerol in mouse strains with differing susceptibility to TPA-induced tumor promotion

The purpose of this study was to examine the activity and associated kinetic parameters of epidermal protein kinase C (PKC) following stimulation by sn-1,2-dioctanoylglycerol (DIC8) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and to examine the relationship between levels of epidermal PKC activity and the induction of ornithine decarboxylase by these agents, utilizing various stocks and strains of mice. Importantly, the mouse strains and stock used in this study have known differing susceptibilities to undergo TPA-induced tumor promotion: the CD-1 stock and the DBA/2 strain (both sensitive to TPA-induced tumor promotion) and the C57BL/6 strain (resistant to TPA-induced tumor promotion). TPA-stimulated protein kinase C activity was measured in the 10(5)g supernatant fraction of epidermal homogenates using lysine-rich histone as a phosphate acceptor substrate. The maximal velocities for TPA-stimulated epidermal PKC activity in CD-1, DBA/2 and C57BL/6 were 0.28, 0.29 and 0.27 nmol PO4-histone/mg 10(5)g protein/min, respectively. TPA-stimulated epidermal PKC from CD-1, DBA/2 and C57BL/6 had similar theoretical Vmax values and the apparent concentrations of TPA yielding half-maximal stimulation of PKC were also similar. DiC8-stimulated PKC activity to a greater Vmax; however, the concentration required to yield half-maximal stimulation of PKC was one thousand times greater than that of TPA. There were no strain differences in these parameters when the enzyme was stimulated with DiC8. Thus, the levels of epidermal PKC activity in CD-1, DBA/2 and C57BL/6 mice exhibit no strain differences when stimulated by TPA or DiC8 using lysine-rich histone as a phosphate acceptor substrate. Since sn-1,2-diacylglycerols are known effective inducers of epidermal ornithine decarboxylase (ODC) activity, the induction of epidermal ODC was examined in each mouse strain 5 h after topical application of 2 nmol TPA, 5 nmol TPA or 2.5 mumol DiC8. After topical treatment with TPA, C57BL/6 demonstrated an unexpected 2- and 4-fold increase in ODC activity over CD-1 and DBA/2 mice. After treatment with DiC8, C57BL/6 demonstrated a 6- and 10-fold increase in ODC activity over CD-1 and DBA/2, respectively. Thus, the resistant strain (C57BL/6) demonstrated a 'hyperinducibility' of epidermal ODC activity by TPA or DiC8. The time course for the induction of epidermal ODC was examined in each strain, and at every time point measured (3-15 h), the C57BL/6 strain exhibited this 'hyperinducibility' of ODC relative to the other strains. Epidermal DNA synthesis was stimulated to a similar extent in C57BL/6 and CD-1 mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

Correlation of DNA adduct formation and riddelliine-induced liver tumorigenesis in F344 rats and B6C3F(1) mice

Riddelliine is a naturally occurring pyrrolizidine alkaloid that induces liver hemangiosarcomas in male and female F344 rats and male B6C3F(1) 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 B6C3F(1) 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 (32)Ppostlabeling/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.     

Comparison of the histological changes in the skin of DBA/2 and C57BL/6 mice following exposure to various promoting agents

The effects of multiple applications of 12-O-tetradecanoyl-phorbol-13-acetate(TPA, 6.8 nmol), teleocidin (6.8 nmol), 1,8-dihydroxy-3-methyl-9-anthrone(chrysarobin, 220 nmol), mezerein (6.8 nmol), 4-O-Methyl-TPA(4-O-Me-TPA, 150µg) and benzoyl peroxide (BzP, 20 mg)on the skin of DBA/2 and C57BL/6 mice were studied histologically.After four applications of TPA given over a 2-week period, theepidermis of DBA/2 mice showed a marked epidermal hyperplasiaand the presence of a much greater number of dark basal kerntinocytes(DCs) 48 h after the last treatment compared with C57BL/6 micetreated with a similar dose and protocol. A marked dermal infiltrationof polymorphonuclear leukocytes (PMNs) was observed in DBA/2mice 48 h after the last application of TPA, whereas littlePMN infiltration was observed in skin of C57BL/6 mice. At 96h after the last application of TPA, DBA/2 mice still showeda much greater degree of epidermal hyperplasia than C57BL/6mice. PMNs were virtually absent in the dermis of both DBA/2and C57BL/6 mice by 96 h after the last TPA treatment. Interestingly,treatment of both strains of mice with multiple applicationsof teleocidin induced a marked epidermal hyperplasia, a highpercentage of DCs and a high labeling index (LI), similar tothat observed in DBA/2 mice 48 h after the last treatment. Chrysarobin(given once-weekly for 4 weeks) induced a moderate sustainedhyperplasia and DC response 48 h after the last treatment inboth DBA/2 and C57BL/6 mice; however, C57BL/6 mice showed agreater epidermal hyperplasia than DBA/2 mice. Chrysarobin induceda significant infiltration of PMNs into the dermis of DBA/2mice whereas in C57BL/6 mice there only a slight dermal infiltrationof PMNs. Mezerein (given twice-weekly for 2 weeks) induced amoderate epidermal hyperplasia, DC response and LI of similarmagnitude in both DBA/2 and C57BL/6 mice, but did not inducePMN infiltration in either strain. BzP and 4-O-Me-TPA (giventwiceweekly for 2 weeks) induced only a weak sustained epidermalhyperplasia, DC response and LI of similar magnitude in bothstrains of mice, and there was little, if any, dermal infiltrationof PMNs either 48 or 96 h after the last treatment. Examinationof the relationship between the extent of induced hyperplasiaand the DC response showed an excellent linear correlation whereasthe extent of PMN infiltration into the dermis was not wellcorrelated with either parameter. The results suggest that theinduction of both sustained hyperplasia and DCs correlate wellwith the skin papillomapromoting ability of the various promotingagents examined, and that C57BL/6 mice are somewhat peculiarin their resistance to the induction of these parameters byphorbol esters.     

Polycyclic aromatic hydrocarbon-inducible DNA adducts: evidence by 32P-postlabeling and use of knockout mice for Ah receptor-independent mechanisms of metabolic activation in vivo

There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are potent carcinogens in laboratory animals and are suspected human carcinogens. The PAHs are bioactivated by cytochrome P450 (CYP)1A1/1B1 enzymes to reactive intermediates that bind to DNA, a critical step in the initiation of carcinogenesis. The Ah receptor (AHR) plays a critical role in the induction of CYP1 enzymes (i.e., CYP1A1, 1A2 and 1B1) by PAHs such as benzo[a]pyrene (BP) and 3-methylcholanthrene (MC). In our investigation, we tested the hypothesis that AHR-null animals are less susceptible to PAH-induced DNA adduct formation than wild-type animals. Wild-type [AHR (+/+)] mice or mice lacking the gene for the AHR were treated with a single dose (100 micromol/kg) of BP or MC, and hepatic DNA adducts were analyzed by (32)P-postlabeling. BP induced multiple hepatic DNA adducts in wild-type as well as AHR-null animals, suggesting the existence of AHR-independent mechanisms for BP metabolic activation. On the other hand, DNA adduct formation was markedly suppressed in AHR-null animals exposed to MC, although the major MC-DNA adduct was produced in these animals. Hepatic activities and apoprotein contents of 7-ethoxyresorufin O-deethylase (EROD) (CYP1A1) and 7-methoxyresorufin O-demethylase (MROD) (CYP1A2) activities were markedly induced by BP and MC in the wild-type, but not, in AHR-null animals. CYP1B1 expression was also induced, albeit to a lesser extent by the PAH MC, but not BP, in the wild-type animals. In conclusion, these results demonstrate the existence of AHR- and CYP1A1-independent mechanisms of PAH metabolic activation in mouse liver, a phenomenon that may have important implications for PAH-mediated carcinogenesis.