Correlation between incidence of 1,2-dimethylhydrazine-induced colon carcinomas and DNA damage in six genetically different mouse strains

Mice from strains with different susceptibility to the colon-specific carcinogen 1,2-dimethylhydrazine (DMH) were tested for DNA damage in liver, kidney and colon after administration of the compound at a dosage that has been reported to induce a high incidence of adenocarcinoma in the colon of rodents. DNA breaks were evaluated from their elution rate constant according to the alkaline elution technique. We found that 4 h after administration of the carcinogen there was a substantial and comparable DNA damage in liver and kidney of all strains examined. Conversely, colon DNA damage was hardly above control levels in the carcinogen-resistant strains. The highest DNA damage was detected in the most susceptible strain and was slightly lower in the two other susceptible strains. We propose that the extent of DNA breakage in a target organ could be one of the factors determining organ-specific and strain-specific susceptibility to DMH.     

Azoxymethane-induced colon tumors and aberrant crypt foci in mice of different genetic susceptibility

Azoxymethane (AOM) is an organotropic colon carcinogen that is commonly used to induce colon tumors in rodents. Unlike its parent compound, 1,2-dimethylhydrazine (DMH), a tumor susceptibility phenotype in inbred mice with respect to AOM has not been established. Thus, this study was undertaken to determine whether genetic susceptibility extends to this carcinogen. SWR/J, A/J (both susceptible to DMH carcinogenesis) and AKR/J (resistant) mice were treated with 10 mg/kg AOM i.p. once a week for 8 weeks. Twenty-five weeks after the initial injection, tumor yield was determined. With a single exception, only SWR/J and A/J mice developed tumors, with a distribution that was limited to the distal colon (16.3±1.1 and 36.4±2.4, respectively). The formation of aberrant crypt foci (ACF), putative preneoplastic lesions, was also assessed in whole-mount colons using Methylene Blue staining. Consistent with tumor multiplicity, the total number of ACF was highest in A/J mice, followed by SWR/J mice. In addition, A/J mice had a significantly greater number of large ACF (five or more crypts per foci) than the other strains. Despite the absence of colon tumors, however, AKR/J mice did develop a significant number of ACF. This finding suggests that ACF in resistant mice are persistent but do not progress to tumors.     

DNA damage in liver,colon, stomach,lung and kidney of BALB/c mice treated with 1,2-dimethylhydrazine

DNA single-strand breaks induced in various organs of BALB/c mice by treatment with a single dose of 1,2-dimethylhydrazine (DMH) were studied by means of the alkaline elution method modified in order to allow the evaluation of DNA damage in vivo with no need of radioactive prelabelling. DNA damage was detected in liver, lung, kidney, stomach and colon mucosa, with the liver showing the greatest amount of damage. Its degree was dependent on the dose and route of administration. A differential effect was evident in colon mucosa from Swiss and C57BL/6 mice which are respectively susceptible and resistant to the induction of bowel tumors by DMH. The higher degree of DNA damage found in liver in comparison with colon mucosa is consistent with the previously reported higher degree of DNA methylation, but does not correlate with the specificity of this carcinogen in inducing tumors of the large intestine in mice given repeated subcutaneous injections.     

Sequential and morphological analyses of aberrant crypt foci formation in mice of differing susceptibility to azoxymethane-induced colon carcinogenesis

Aberrant crypt foci (ACF), putative preneoplastic lesions, are early morphological changes induced by the colon carcinogen azoxymethane (AOM). Although inbred mice differ markedly in their susceptibility to AOM carcinogenesis, we have previously shown that ACF develop in both resistant and sensitive mouse strains after AOM treatment. The purpose of this study was to examine the sequential development and identify the morphological characteristics of ACF induced by AOM in the distal colon of sensitive and resistant mice. A/J (highly susceptible), SWR/J (relatively susceptible) and AKR/J (resistant) mice were treated with 10 mg/kg AOM or saline i.p. once a week for 6 weeks and were killed at 1, 2, 4, 6, 9 and 24 weeks after the last injection. The distal colons were stained with methylene blue and the numbers of ACF and tumors determined. Tumors were present as early as 4 weeks after AOM exposure in SWR/J and A/J mice and increased in frequency throughout the study in both strains. No tumors developed in the AKR/J mice. ACF, however, formed in all strains of mice. The greatest difference between susceptible and resistant strains was in the number of large ACF that developed at later time points. Furthermore, morphometric analysis revealed that A/J mice had the highest percentage of dysplastic ACF, followed by SWR/J mice. These data indicate that the difference in cancer risk from AOM may be due to the lack of progression of smaller ACF in the resistant mice and to the development of dysplasia in a higher percentage of ACF from susceptible strains.     

Susceptibility to 1,2-dimethylhydrazine-induced colonic tumors and epithelial cell proliferation characteristics of F1, F2, and reciprocal backcrosses derived from SWR/J and AKR/J parental mouse strains

Hybrid crosses were performed between SWR/J, a strain highly sensitive to 1,2-dimethylhydrazine (DMH), and AKR/J, a strain highly resistant to the carcinogen. F1 and F2 and reciprocal backcrosses were tested to determine if proliferative characteristics such as high activity, wide compartment (PC), and a large S-phase population in the middle third of crypts were linked to susceptibility and inherited as a dominant autosomal trait as was reported for DMH tumor response. A blend of resistant and sensitive tumor and proliferative characteristics was observed in the F1 and F2 crosses. A tumor incidence of 43.7% in the F1 and 52% in the F2 was obtained rather than the respective 100% and 75% expected frequencies. One week after the sixth injection of DMH, the incidence of focal areas of atypism (FAA) in the backcross to resistance (BCR) and the backcross to sensitivity (BCS) was the same (4.1 per FAA-bearing animal). This suggested that the response to the carcinogen was similar in both groups up to this point. Yet 20 weeks later, the BCR had a 7.3% tumor incidence, far lower than the 50% incidence expected. The BCS had an incidence of 98.6%, not unlike SWR frequencies and close to the expected 100% tumor incidence. Proliferative characteristics in the backcrosses, however, did not revert to parental levels. Instead, the labeling index (LI) or percentage of S-phase cells to total cells scored was significantly higher in the BCR than in the BCS (10.2% +/- 3.2% versus 8.1% +/- 2.2%, P less than 0.02). This study has shown that in crosses between these two strains (SWR/J and AKR/J), susceptibility to DMH-induced tumor is not inherited as a dominant trait. Neither are the proliferative characteristics of the colonic mucosa inherited in a simple Mendelian manner nor are the kinetic properties of the epithelial cells linked to DMH tumor susceptibility. It is suggested that the parental AKR/J strain may contribute a protective or resistant factor, that is, a repressor gene, which impedes the progression of carcinogen-induced foci of dysplasia to colonic neoplasia.     

The relationship between 1,2-dimethylhydrazine dose and the induction of colon tumours: tumour development in female SWR mice does not require a K-ras mutational event

In this study we have investigated the relationship between the dose of 1,2-dimethylhydrazine (DMH) and the yield (and location) of tumours in a mouse strain susceptible to colon tumour induction. Female SWR mice were injected with 6.8 mg/kg DMH i.p. once a week for 1, 5, 10 and 20 weeks and the animals were followed for almost 2 years. Administration of increasing doses of DMH resulted in a dose-dependent decrease in survival time. Colon tumours developed in 26, 76 and 87% of mice given a total dose of 34, 68 and 136 mg/kg DMH, respectively: no tumours were detected in animals treated with a total dose of 6.8 mg/kg. Most colon tumours (79%) were located in the distal colon with the remainder being found in the mid colon and none were detected in either the proximal colon or small intestine. As mutations in the K-ras gene are thought to be key events in the pathogenesis of human and rodent colon tumours, we determined the frequency of codon 12 and 13 K-ras mutations in these tumours by restriction site mutation analysis and/or DNA sequencing. A total of 50 colon tumour samples were analysed for codon 12 mutations and of these 29 were also screened for codon 13 mutations. No mutations were detected in either of these codons. The mutational activation of the K-ras gene is not an essential step in the development of DMH-induced colon tumours in female SWR mice and if similar considerations apply to humans, then the aetiological role of alkylating agents may be underestimated from the prevalence of K-ras GC-->AT transitions in human tumours.     

1,2-Dimethylhydrazine-induced nuclear aberrations in A/J and C57BL/6J mouse colonic crypts

A single exposure to 1,2-dimethylhydrazine [(DMH) CAS: 540-73-8] produces several forms of aberrant nuclei in the crypts of the murine colon. The frequency of nuclear aberrations (NAs) was examined in the distal colonic crypts in DMH-sensitive A/J mice and relatively DMH-resistant C57BL/6J mice before and after a single exposure to DMH. NAs, mitotic figures, and crypt column heights were scored for all animals as a function of time following administration of DMH. In both strains there was a significant increase in the absolute and relative frequency of NAs by 12 hours, with a corresponding drop and subsequent overshoot in the mitotic index by 48 hours after DMH. The temporal changes in crypt column height correlate closely with the temporal changes in frequency of NAs in both strains. The results showed that both inbred strains respond to acute DMH exposure in a similar and parallel fashion over time. It was concluded that the NA index assay is a sensitive method for detecting early DMH exposure. However, this assay does not relate to ultimate outcome after chronic DMH exposure and should not be used as a predictor of eventual neoplastic transformation of colonic mucosa with this carcinogen.     

Expression analysis of the group IIA secretory phospholipase A(2) in mice with differential susceptibility to azoxymethane-induced colon tumorigenesis

The murine non-pancreatic secretory phospholipase A(2) (sPLA(2)) has been proposed as a tumor modifier of multiple intestinal neoplasia (Min). A genetic polymorphism in the mouse gene that causes a disruption in exon 3 results in loss of functional protein. Mouse strains with a disrupted sPLA(2) gene are susceptible to the Min phenotype and develop numerous intestinal polyps, whereas mice with normal sPLA(2) develop only a limited number of polyps. The following study was undertaken to test the hypothesis that sPLA(2) plays an equivalent role in murine susceptibility to the colon carcinogen azoxymethane (AOM). sPLA(2) status was confirmed by sequencing in mice that are highly susceptible (A/J), susceptible (SWR/J) and resistant (AKR/J) to AOM-induced tumorigenesis. Constitutive expression of sPLA(2) mRNA was compared in small intestine and colon of untreated mice using semi-quantitative RT-PCR. Whereas mRNA expression was nearly absent in A/J mice, AKR/J mice exhibited extensive expression throughout the intestine. Despite the wild-type sPLA(2) gene, colonic mRNA expression in SWR/J mice was significantly lower relative to AKR/J. Immunohistochemical analysis of sPLA(2) protein confirmed the mRNA data. The effect of AOM on colonic sPLA(2) expression was also examined. Twenty-four weeks after the last of six weekly injections of AOM (10 mg/kg i.p.), RT-PCR analysis of distal colons revealed a significant increase in mRNA in normal-appearing epithelium and tumor tissue from AOM-treated mice relative to controls. However, there was no corresponding increase in protein expression in A/J mice. The absence of sPLA(2) expression within control colons of tumor-susceptible A/J mice together with low expression in SWR/J colons is consistent with its potential role as an intestinal tumor modifier, but the carcinogen-induced increase in expression raises doubts as to the significance of sPLA(2) in inhibiting carcinogenesis.     

Effect of retinoids on the induction of colon cancer in F344 rats by N-methyl-N-nitrosourea or by 1,2-dimethyihydrazine

The possible modifying effect of synthetic and natural retinoidson the incidence of colon cancer in rats induced by 2 intrarectaldoses of 2.5 mg of N-methyl-N-nitrosourea (MNU) given once aweek for 2 successive weeks or a single 150 mg/kg body weightdose of 1,2-dime-thylhydrazine (DMH), s.c. was investigated.Emphasis was on the effect of the development of early tumorsas visualized by endoscopy. With the retinoids N-ethyl-retinamide,N-2-hydroxyethylretinamide, N-(4-hydro- xyphenyl)-all-trans-retinamide(RAHA), and retinyl acetate (RA) administered orally after thecarcinogens, significant differences in early developing tumorswere not found. At histopathological examination of the tumorsthe RAHA + DMH group had significantly fewer adenomas per animal.The percentage of adenoma bearing rats was significantly lowerin groups receiving RAHA + DMH or RA + DMH. However, food consumptionwas lower in rats consuming either RAHA or RA. Retinyl palmitate(RP) and RAHA was administered intrarectally to MNU-inducedrats either before or after the carcinogen. When administeredbefore MNU, RP caused a significant increase in the percentageof tumor bearing animals and the average number of tumors peranimal as visualized endos copically. At histopathological examination,all retinoid groups except RAHA given after the carcinogen,produced significantly more adenomas per animal and a significantlygreater adenoma incidence than did the control groups. Thus,in two systems, the oral administration of retinoids did notclearly inhibit the early or later stages of colon tumor development.Inirarectal infusion of two retinoids had no effect on colonicmor phology but at histopathological examination of later stagetumors there was an enhanced adenoma response.     

Supplemental dietary calcium fails to alter the acute effects of 1,2-dimethylhydrazine on O6-methylguanine, O6-alkylguanine-DNA alkyltransferase and cellular proliferation in the rat colon

Prior studies from our laboratory have demonstrated that K-rasG to A mutations were detectable in a high percentage of carcinomaswhich developed in the colons of animals treated with the knowncolonic procarcinogen, 1,2-dimethyl-hydrazine (DMH). Moreover,in this model, the incidence of these mutations was decreasedby a supplemental dietary calcium regimen which concomitantlydecreased the frequency of rats with multiple tumors as wellas tumor size. In an attempt to clarify the possible mechanism(s)involved in this antimutagenic effect of supplemental calcium,two groups of Sprague—Dawley rats were fed semisyntheticdiets containing either 0.87 or 1.80% cakium by weight for 3weeks, s.c. injected with 100 mg/kg of DMH and killed priorto and at various time periods (16-144 h) after injection. Thecolons of animals were analyzed and compared with respect toO⁶-methylguanine content in DNA, O⁶-alkylguanine-DNA alkyltransferaselevels as well as cellular proliferation, as assessed by immunohistochemicalstaining of colonic crypts by bromodeoxyundine. In certain experiments,these parameters were also analyzed in the proximal and distalcolon before and at various times after administration of DMH.The results of these experiments demonstrated that supplementaldietary calcium was not found to influence significantly O⁶-methylguaninelevels, alkyltransferase levels or cellular proliferation inthe entire colon or in either colonic segment before or afterthe acute administration of DMH. DMH did, however, differentiallyalter all three of these biochemical parameters in the colonicsegments (distal > proximal), possibly due to a greater degreeof metabolic activation in the distal colon.     

Cyclophosphamide induced DNA strand breaks in mouse embryo cephalic tissue in vivo

Pregnant C3H mice were exposed to teratogenic doses of cyclophosphamide(CPA), on the 11th day after copulation. The effects of thisagent on embryonal cephalic DNA strand breaks were assessedbetween 3 and 40 h after drug administration. Administrationof 15, 30 and 60 mg CPA/kg body weight resulted in conversionof 23,30 and 44% of the DNA to the single-stranded form, respectively.No detectable DNA damage was evident 3 h after drug administration,but after 6 h significant DNA damage had occurred, reachinga maximum after 9 h. However, no evidence of DNA strand breakswas present at 22, 30 and 40 h after CPA treatment, suggestingthat these lesions had been repaired. These findings demonstratethat cephalic DNA damage Induced by CPA in the developing mouseembryo occurs in a time and concentration dependent manner,and provide some insight into the kinetics of formation andremoval of DNA strand breaks caused by CPA in vivo     

Dietary n-3 PUFA increases the apoptotic response to 1,2-dimethylhydrazine, reduces mitosis and suppresses the induction of carcinogenesis in the rat colon

The effect of dietary fish oil on colonic crypt cell apoptosis and proliferation was examined in male Wistar rats, 24 and 48 h after administration of 1,2-dimethylhydrazine (DMH), and its influence on the induction of aberrant crypt foci (ACF) in the distal colon was assessed. Rats (125-150 g) fed a high-fat semi-synthetic diet containing corn oil (CO) were given DMH (30 mg/kg body wt) or a sham injection of EDTA/NaCl. Animals were then fed either the CO diet or a diet in which fish oil (EPA 18.7%; DHA 8%) was substituted for corn oil. Subgroups of rats (n = 5) were killed after 24 and 48 h, and crypt cell apoptosis and proliferation were quantified by morphological criteria in isolated intact crypts from the mid and distal colon. Consumption of the fish oil diet (FO) was associated with increased apoptotic cell death (P < 0.001) and suppression of proliferation (P < 0.05) in colonic crypts both 24 and 48 h after DMH. In a second experiment, animals were given three injections of DMH or sham injections of carrier at weekly intervals. For 48 h after each injection animals were fed either the CO or FO diet, but otherwise maintained on the CO throughout. The number and crypt multiplicity of ACF in the distal colon were determined after 18 weeks, and animals given the FO diet for the 48 h period following carcinogen administration were found to have significantly fewer ACF than rats fed the CO diet (P < 0.05). The data demonstrate that the fatty acid composition of the diet is an important determinant in the induction of carcinogenesis by DMH. The proliferative and apoptotic response of the colonic crypt to carcinogen and fish oil, coupled with the reduced incidence of ACF, suggest n-3 PUFA can protect against the carcinogenic effects of DMH by mediating changes in the balance proliferation and cell death.     

Proliferative characteristics of chronic crypt cells in C57BL/6J and A/J mice as predictors of subsequent tumor formation

It has been proposed that the number and extent of tumors formed after chronic exposure to dimethylhydrazine (DMH) can be predicted by the indigenous number and distribution of DNA-synthesizing cells in the murine colonic mucosa, and that this sensitivity to DMH is genetically determined. In order to test this hypothesis we studied two genetically distinct inbred strains of mice; the DMH-sensitive A/J (A) mouse, and the relatively DMH-resistant C57BL/6J (B) mouse before and after a single exposure to DMH. The untreated A strain had the longer crypt column [33.2 +/- 0.8 (SD) cells versus 28.8 +/- 0.9 cells], a higher absolute number of labeled cells per crypt column (4.4 +/- 0.6 versus 2.6 +/- 0.9), a greater labeling index (13.4 +/- 1.6% versus 9.1 +/- 2.9%), a wider proliferative compartment, and a greater number and percentage of labeled cells in the middle and upper thirds of the crypt than the untreated B strain. After acute exposure to DMH the A strain lost 14 +/- 3% of their total body weight, while the B strain lost 0.5 +/- 2% total body weight 48 h post-DMH. There was an initial loss of cryptal cells, a drop in the labeling index, and a subsequent increase and overshoot in the number of labeled cells and the labeling index. This pattern of cell loss and recovery over time was parallel in both strains, and thus cannot explain the differences in ultimate tumor formation after chronic exposure to the carcinogen. The data are consistent with the theory that the susceptibility to DMH carcinogenesis can be predicted by the indigenous proliferative characteristics of the murine colonic mucosa. The acute proliferative response to DMH in these strains is similar and parallel; thus ultimate tumor load may depend on long term effects such as the establishment of stable transmissible mutations.     

Methylene chloride-induced DNA damage: an interspecies comparison

DNA single-strand (ss) breaks were detected in the livers ofB6C3F1 mice immediately following exposure to 4000–8000p.p.m. methylene chloride (MC) for 6 h. This damage was undetectable2 h after exposure, suggesting an active DNA repair process.Similarly, DNA ss breaks were detected in whole lung homogenatestaken from mice exposed to 2000–6000 p.p.m. MC. The DNAof mouse Clara cells incubated in vitro with MC was also damagedat concentrations of 5 mM MC and above. Pre-treatment of micewith the glutathione depletor buthionine sulphoximine (BSO)caused a decrease in the amount of DNA damage detected, suggestinga GST-mediated mechanism. DNA damage was also reduced in Claracells when incubated in vitro with MC in the presence of BSO.In CHO cells induction of DNA damage was dependent upon exogenousMC metabolism by mouse liver S100 fraction (but not microsomes)in the presence of GSH. DNA ss breaks were not induced by MCin hamster hepatocytes in vitro at concentrations from 5 to90 mM MC, nor in eight individual samples of normal human hepatocytesexposed to MC at similar concentrations. The ability of MC toinduce DNA ss breaks in the four species studied is entirelycompatible with the known carcinogenicity of this chemical inanimals and offers experimental evidence to suggest that humanswould not be susceptible to MC-induced liver cancer. The DNAss breaks correlate with the metabolism of MC by the GST pathwayand provide an explanation for the lack of sensitivity of hamstersand rats to MC-induced liver cancer.     

Role of cytochrome P450 in DNA damage produced by treatment of colon cells with 1,2-dimethylhydrazine

Human colon cells (LS174T) were treated with the model colon carcinogen 1,2-dimethylhydrazine (DMH) to determine the production of O(6)-methylguanine DNA adducts. Three known P450 inducers (benzanthracene, pyrazole and phenobarbital) were used to produce different P450 environments in each group of cells prior to treatment with DMH. An increased level of DNA damage of different degrees above uninduced levels was observed in all treated groups. Inhibition of the natural protection systems (glutathione and O(6)-methyltransferase) were also included in the study. Glutathione apparently is not of significant protection against DMH damage in colon cells challenged with DMH. In contrast methyltransferase does exert a protective role in this type of cells by reducing the extent of DNA O(6)-methylguanine adduct formation in colon cells following induction of different panels of cytochrome P450 isoforms.     

Effect of acetylator genotype on 3, 2'-dimethyl-4-aminobiphenyl induced aberrant crypt foci in the colon of hamsters

Aberrant crypt foci (ACF) are assumed to be preneoplastic lesionsin both rodent and human carcinogenesis. The colon carcinogen3,2'-dimethyl-4-aminobiphenyl (DMAB), like other arylamines,undergoes N-acetylation and O-acetylation by polymorphic acetyltransferase(NAT2). In the present study we characterized ACF in hamstercolon for the first time and compared the ability of DMAB toinduce ACF in homozygous rapid and slow acetylator congenicSyrian hamsters (Bio 1.5/H-NAT2^r and Bio 1.5/H-NAT2^s, respectively),differing only at the NAT2 gene locus and other closely linkedloci. The animals received DMAB (75 mg/kg body weight s.c.)or vehicle (PBS/DMSO 1:1) as a control, twice weekly for 2 weeks,then once a week for 4 weeks. Ten weeks after the first injectionACF were observed in the DMAB treated hamsters, but not in thecontrols. However, the number of ACF was three times higher(P = 0.016) in the colons of the NAT2^r hamsters compared withthe colons of the NAT2^shamsters. In the two congenic hamsterlines we also studied the induction of ACF with 1, 2-dimethythydrazine(DMH) treatment, a colon carcinogen not metabolized by NAT2.Hamsters given DMH (25 mg/kg body weight s.c.), once a weekfor 3 weeks, showed ACF induction in the colon after 10 weeks,but there was no difference between the NAT2^r and NAT2^s hamsters.Further scanning electron microscopic and histological examinationof ACF observed with the light microscope, revealed the samegross morphology and therefore confirmed the basis for the scoringof ACF. The ACF in hamster colons were in principle similarto the lesions observed in other species.     

Dibenz[a,h]anthracene-induced subcutaneous tumors in mice. Strain sensitivity and the role of carcinogen metabolism

Four inbred strains of mice (C3H/HeJ, C57BL/6J, AKR/J, and DBA/2J)were injected s.c. with 150 µg of dibenz[a,h]-anthraceneand followed for 9 months. Strains C3H/HeJ and C57BL/6J weremost susceptible to dibenz[a,h]anthracene-induced carcinogenesiswith 80% and 53% incidence of tumors, respectively. StrainsAKR/J and DBA/2J were much less sensitive with only one tumorobserved out of a total of 60 treated mice. Dibenz[a,h]anthracenewas shown to induce hepatic aryl hydrocarbon hydroxylase activityin the two sensitive strains, C3H/HeJ and C57BL/6J, but notin the two resistant strains, AKR/J and DBA/2J. When 3-methyl-cholanthrene-treatedliver microsomes from the four strains were studied for dibenz[a,h]anthracenemetabolism in vitro, the two sensitive strains not only demonstrateda 3- to 4-fold greater overall rate of metabolism than the tworesistant strains, but also showed a quantitative shift witha greater percentage of the total metabolites being the 3,4-diolof dibenz[a,h]anthracene. This diol is the presumed precursorto the apparent ultimate carcinogen, dibenz[a,h]anthracene 3,4-diol-1,2-epoxide.     

Tumor formation is correlated with expression of beta-catenin-accumulated crypts in azoxymethane-induced colon carcinogenesis in mice

We have reported that β-catenin-accumulated crypts (BCAC) are independent of aberrant crypt foci (ACF) in the colonic mucosa of rats exposed to colorectal carcinogens, and we suggested that they may be premalignant lesions. In the present study, we performed a comparative study on the formation of the two types of early-appearing lesions (BCAC and ACF), and tumors of the colon in two mouse strains with different susceptibility to azoxymethane (AOM). SWR/J mice are known to be relatively susceptible to AOM, whereas AKR/J mice are reported to be virtually resistant. Both AKR/J and SWR/J mice, 6 weeks old, received subcutaneous injections of AOM (15 mg/kg body weight) once a week for 3 weeks, and were sacrificed at 16 and 41 weeks of age. Colons of the animals sacrificed at 16 and 41 weeks of age were processed to examine expression of the early-appearing lesions and neoplasms. Although AKR/J mice had a lower incidence of colonic tumors than SWR/J mice did, AKR/J mice showed a similar frequency of ACF to that in SWR/J mice. In both strains, ACF were detected at high frequency in the proximal colon, whereas tumors developed mainly in the distal colon. Importantly, the incidence of BCAC in SWR/J mice was significantly higher than that in AKR/J mice, and the highest frequency was observed in the distal segments of the colon. These results support the idea that BCAC are a reliable surrogate endpoint for colon carcinogenesis in 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.