Effects of Exercise Intervention on Habitual Physical Activity Above Lactate Threshold Under Free-living Conditions: A Randomized Controlled Trial.

The purpose of the present investigation was to examine the effects of an exercise-based intervention on the daily physical activity (PA) above the lactate threshold under free-living conditions. A total of 81 middle-aged to older males (51±7 years) were randomly assigned into one of 2 groups. The subjects' body weight, visceral fat area, lactate threshold (LT), and PA levels were measured before and after the 8-week lifestyle intervention. The PA levels were assessed using a pedometer with a uni-axial accelerometer (Lifecorder, Kenz, Nagoya). During the intervention, 300 min/week of exercise at LT was targeted in the exercise group. The LT was determined by the log-log transformation method. The subjects in the control group were instructed to maintain their usual lifestyles. In the exercise group, the body weight, visceral fat area and LT significantly improved after the intervention. In contrast, the duration of PA at an intensity of LT, >3 METs and >6 METs did not differ significantly. The present investigation did not find significant changes in the PA at LT under free-living conditions as a result of an 8-week exercise-based intervention, although the participants successfully improved their aerobic capacity and body composition.

Accumulation of short bouts of non-exercise daily physical activity is associated with lower visceral fat in Japanese female adults.

The purpose of the present investigation was to assess the relationship between bouts of very short daily physical activity (PA) lasting <10 min with obesity and abnormal fat distributions. A total of 42 females (age 50±6 years, height 156±5 cm, body weight 54±8 kg, body mass index 22±3 kg/m2) participated in the present investigation. Computed tomography was used to evaluate the area of visceral adipose tissue and subcutaneous adipose tissue (VAT and SAT). All participants wore a pedometer with a one-axial accelerometer (Lifecorder, Kenz, Japan) in order to determine their frequency (bouts/day) of PA and moderate to vigorous intensity PA (MVPA). The total frequency of PA and MVPA, including all bout durations, was not significantly associated with the body fat distribution. The frequency of PA lasting longer than 3 min and 5 min, and MVPA lasting longer than 1 min and 3 min were significantly associated with the area of the VAT (p<0.05). A smaller area of VAT was associated with a higher frequency of PA and MVPA lasting 1-5 min. The present investigation did not find that very short bouts of PA lasting<1 min played a significant role in controlling abdominal fat distribution.

Peroxisome proliferator activated receptor gamma in colonic epithelial cells protects against experimental inflammatory bowel disease.

INTRODUCTION: Peroxisome proliferator activated receptor gamma (PPARgamma) is expressed in epithelial cells, macrophage, and T and B lymphocytes. Ligand induced activation of PPARgamma was reported to attenuate colitis activity but it is not clear whether this protection is mediated by epithelial or leucocyte PPARgamma. METHODS: Mice with targeted disruption of the PPARgamma gene in intestinal epithelial cells, generated using a villin-Cre transgene and floxed PPARgamma allele and designated PPARgamma(DeltaIEpC), were compared with littermate mice having only the PPARgamma floxed allele with no Cre transgene that expressed PPARgamma in the gut, designated PPARgamma(F/F). Colitis was induced by administering dextran sodium sulphate (DSS) and the two mouse lines compared for typical symptoms of disease and expression of inflammatory cytokines. RESULTS: PPARgamma(DeltaIEpC) mice displayed reduced expression of the PPARgamma target genes ADRP and FABP in the gut but were otherwise normal. Increased susceptibility to DSS induced colitis, as defined by body weight loss, colon length, diarrhoea, bleeding score, and altered histology, was found in PPARgamma(DeltaIEpC) mice in comparison with PPARgamma(F/F) mice. Interleukin (IL)-6, IL-1beta, and tumour necrosis factor alpha mRNA levels in colons of PPARgamma(DeltaIEpC) mice treated with DSS were higher than in similarly treated PPARgamma(F/F) mice. The PPARgamma ligand rosiglitazone decreased the severity of DSS induced colitis and suppressed cytokine production in both PPARgamma(F/F) and PPARgamma(DeltaIEpC) mice. CONCLUSIONS: These studies reveal that PPARgamma expressed in the colonic epithelium has an endogenous role in protection against DSS induced colitis and that rosiglitazone may act through a PPARgamma independent pathway to suppress inflammation.

Sparse and wavy hair: a new model for hypoplasia of hair follicle and mammary glands on rat chromosome 17.

Mutant animals in the skin and hair have been used to identify important genes in biomedical research. We describe a new mutant rat, sparse and wavy hair (swh), that spontaneously arose in a colony of inbred WTC rats. The mutant phenotype was characterized by sparse and wavy hair, which was most prominent at age 3-4 weeks, and was inherited in an autosomal recessive manner. The swh/swh rats showed impaired gain of body weight, and their hair follicles were reduced both in number and size, associated with hypoplasia of the sebaceous glands and the subcutaneous fat tissue. Female swh/swh rats were unable to suckle their offspring. Their mammary glands were hypoplastic, and differentiation of mammary epithelial and myoepithelial cells was impaired. Linkage analysis of 579 backcross rats localized the swh locus to a .35-cM region between D17Rat131 and D17Rat50 in the distal end of rat Chr 17. The swh locus spanned the 3.7-Mb genomic region where 24 genes have been mapped and corresponded to the centromere region of the mouse Chr 2 or the region of the human Chr 10p11.1-p14. None of the genes or loci described in mouse or human hair and skin diseases mapped to these regions. These findings suggest that the rat swh is a novel mutation associated with impaired development of the skin appendages, such as hair follicles, sebaceous glands, and mammary glands, and will provide an experimental model to clarify a gene and mechanisms for development of skin appendages.

Existence of a threshold for induction of aberrant crypt foci in the rat colon with low doses of 2-amino-1-methyl-6-phenolimidazo[4,5-b]pyridine.

Until recently it has been generally considered that genotoxic carcinogens have no threshold in exerting their potential for cancer induction. However, the nonthreshold theory can be challenged with regard to assessment of cancer risk to humans. In the present study we show that a food derived, genotoxic hepatocarcinogen, 2-amino-1-methyl-6-phenolimidazo[4,5-b]pyridine (PhIP), does not induce aberrant crypt foci (ACF) as preneoplastic lesions at low dose (below 50 ppm) or 8-hydroxy-2'-deoxyguanosine (below 400 ppm) in the rat colon. Moreover PhIP-DNA adducts were not formed at the lowest dose (below 0.01 ppm). Thus, the dose required to initiate ACF is approximately 5000 times higher than that needed for adduct formation. The results imply a no-observed effect level (existence of a threshold) for colon carcinogenesis by a genotoxic carcinogen.

p53 and H-ras mutations and microsatellite instability in renal pelvic carcinomas of NON / Shi mice treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine: different genetic alteration from urinary bladder carcinoma.

We previously reported p53 mutations to be frequent (greater than 70%), whereas both H-ras mutations and microsatellite instability (MSI) were infrequent (about 10%), in urinary bladder carcinomas (UBCs) and their metastatic foci in the N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced mouse urothelial carcinogenesis model. In the present study, an analysis of p53 and H-ras mutations as well as MSI was performed on 12 renal pelvic carcinomas (RPCs) and 8 metastatic or invading foci produced by the same experimental procedure. Histologically, 10 of the RPCs were transitional cell carcinomas and the remaining 2 were squamous cell carcinomas. p53 mutations were infrequent and only found in one primary RPC (8%), its metastatic foci and an invading lesion in another animal (in a total 2 of 12; 17%). H-ras mutations were slightly more frequent (found in 3 of 12 animals; 25%), 4 of 5 involving codon 44, GTG to GCG, not a hot-spot reported for human cancers. In two cases, H-ras mutations were confined to lung metastasis and not detectable in their primary RPCs. MSI analysis was available for 6 pairs of primary RPCs and their metastatic foci, and 4 animals (67%) had MSI at one or more microsatellite loci. Overall, the distribution of genetic alterations differed from that in UBCs produced by the same experimental protocol. The results thus suggest that different genetic pathways may participate in carcinogenesis of the upper and lower urinary tract due to BBN.

Promotion of chemically induced rat esophageal tumorigenesis with post-initiation ethanol modification.

Post-initiation ethanol modification on N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal carcinogenesis model was investigated in male, 6-week-old, F344 rats that received s.c. injections, 3 times per week, of 0.5 mg/kg NMBA for the first 5 weeks and then were treated with 0% (Group 1), 3.3% (Group 2), and 10% (Group 3) ethanol in the drinking water for up to 20 weeks. Group 4 received 10% ethanol without NMBA administration and Group 5 was maintained without any chemical treatment. There were no statistical differences in the incidence and multiplicity of esophageal tumors among Groups 1 to 3. However, the multiplicity of hyperplasias was statistically greater in Group 3 than in Groups 1 or 2. Esophageal epithelia of all rats in Groups 4 and 5 demonstrated a normal histology. BrdU labelling indices of tumors and hyperplasias in NMBA-treated groups were essentially similar, although cycline D1 was overexpressed to a greater extent in tumors and also hyperplasias of Group 3 than in Groups 1 or 2. The results indicated ethanol to exert weak promotion effects through cycline D1 overexpression on rat esophageal tumorigenesis initiated with NMBA.

Detection of p53 gene mutations in human esophageal squamous cell carcinomas using a p53 yeast functional assay: possible difference in esophageal carcinogenesis between the young and the elderly group.

A p53 yeast functional assay, which cannot only detect p53 gene mutations but also can assess p53 gene function, was used to screen for p53 gene dysfunction in human esophageal squamous cell carcinomas. Surgically resected frozen tissues of esophageal squamous cell carcinomas from 57 patients were examined for p53 gene mutation. Because the mean age of the patients diagnosed with esophageal squamous cell carcinoma was 64 years, we classified those who were <65 years of age as the Young Group and classified the others as the Elderly Group. The incidence of p53 gene mutations was 43 of 57 (75%). The incidence of p53 gene mutations observed in the Young Group was significantly higher than in the Elderly Group (P = 0.0007). Alcohol and smoking status did not relate to p53 gene mutation expression. Survival rate after surgery was not significantly associated with the presence of p53 gene mutation. However, in the Young Group with p53 gene mutation, those who had null mutations had a significantly shorter survival than those without null mutations (P = 0.0455). No other clinicopathological factors were associated with p53 gene mutations. Possibly, there may be a difference in esophageal carcinogenesis between the Young and the Elderly groups, because the incidence of p53 gene mutations is different between the two groups. In the Young Group, p53 gene mutation may cause esophageal carcinogenesis, and null mutation for p53 gene is a significant prognostic factor.

Possible prevention by abieslactone of development of diethylnitrosamine-initiated GST-P positive foci in the rat liver.

Triterpenoid compounds, isolated from plants of Abies genus (Pinceae), are known to exert anti-tumor promotion activities in mouse skin carcinogenesis. In the present study, we investigated whether AVB-1 and acid and acid methyl ester derivatives have inhibitory effects on rat hepatocarcinogenesis by using a liver medium-term bioassay for carcinogens (Ito's test), immunohistochemically assessing the numbers and areas per cm(2) of preneoplastic lesions, glutathione S-transferase placental form (GST-P)-positive foci. In experiment 1, 6-week-old male Fisher 344 rats were given a single intraperitoneal injection of diethylnitrosamine (200 mg/kg b.w.) and subjected to two-thirds partial hepatectomy at week 3. From weeks 2 to 8, the compounds were given three times a week at a dose of 1 mg/kg b.w. by i.g. gavage and found to significantly decrease the number of GST-P-positive foci in the liver. In experiment 2, AVB-1 was given three times a week at doses of 3, 1, or 0.3 mg/kg b.w. by i.g. gavage from weeks 2 to 8. All doses of AVB-1 significantly decreased the numbers of GST-P-positive foci. Thus, our results suggest that AVB-1 is a chemopreventive agent for rat hepatocarcinogenesis.

Chemopreventive effects of S-methylcysteine on rat hepatocarcinogenesis induced by concurrent administration of sodium nitrite and morpholine.

The aim of the present study was to examine the chemopreventive efficacy of S-methylcysteine (SMC) on rat hepatocarcinogenesis induced by concurrent administration of sodium nitrite (NaNO(2)) and morpholine (Mor) using a medium-term rat liver carcinogenesis bioassay (Ito test). Administration of SMC caused significant reduction in the areas of glutathione S-transferase placental form positive foci along with a significant decrease of hepatocyte 5-bromo-2'-deoxyuridine (BrdU) labeling indices. These results demonstrated potent chemopreventive effects of SMC against hepatocarcinogenesis due to concurrent administration of Mor and NaNO(2). SMC could thus be an effective chemopreventive agent for decreasing the risk of carcinogenicity from environmental precursors of N-nitroso compounds.

Increased oxidative stress with gene alteration in urinary bladder urothelium after the Chernobyl accident.

We have previously shown that bladder urothelium of people living in the cesium-137 ((137)Cs)-contaminated areas of Ukraine demonstrates accumulation of stable p53 and p53 mutational inactivation, preferentially through G:C to A:T transition mutations at CpG dinucleotides, with a codon 245 hot spot. In the present study, we analyzed immuno-histochemically the relationship between oxidative stress markers and over-expression of p53 and H-ras in urinary bladder urothelium from 42 men with benign prostatic hyperplasia. Bladder mapping biopsies were obtained from 15 patients from a highly radiocontaminated area (group I), 14 patients from the less contaminated city of Kiev (group II) and 13 patients as a control group from "clean" (without radiocontamination) areas of Ukraine (group III). Irradiation cystitis with multiple foci of severe dysplasia and carcinoma in situ were observed in 15 of 15 (100%, group I) and 9 of 14 (64%, group II) cases, with 4 small transitional-cell carcinomas incidentally detected in groups I and II. Markedly elevated levels of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2) and 8-hydroxy-2;-deoxyguanosine (8-OHdG) were noted in these bladder urothelial lesions from groups I and II, accompanied by strong over-expression of p53 and less H-ras expression. These findings support the hypothesis that iNOS, COX-2 and 8-OHdG in bladder urothelium are induced by long-term exposure to low-dose radiation with a close relationship to p53 over-expression that could predispose to bladder carcinogenesis.

Promotion of skin carcinogenesis by dimethylarsinic acid in keratin (K6)/ODC transgenic mice.

Dimethylarsinic acid (DMA) is a major metabolite of inorganic arsenicals in mammals, and arsenic exposure is associated with tumor development in a wide variety of human tissues, particularly the skin. Transgenic mice with ornithine decarboxylase (ODC) targeted to hair follicle keratinocytes are much more sensitive than littermate controls to carcinogens. In this study we investigated the promoting effect of DMA on skin carcinogenesis in such K6 / ODC transgenic mice. The back skin of female C57BL / 6J K6 / ODC transgenic mice, 10 to 14 weeks old, was initiated with topical application of 7, 12-dimethylbenz[alpha]anthracene (DMBA) at a dose of 50 microg or acetone alone on day 1 of the experiment, followed by treatment with 3.6 mg of DMA, 5 microg of 12-O-tetradecanoylphorbol-13-acetate (TPA) or neutral vehicle (control) twice a week for 18 weeks. Mice were killed 1 week after the end of the treatment. Induction of skin tumors was significantly accelerated in the DMA-treated group, as well as in the TPA-treated group, indicating that DMA has a promoting effect on skin tumorigenesis in K6 / ODC transgenic mice.

Inhibitory effects of 1,3-diaminopropane, an ornithine decarboxylase inhibitor, on rat two-stage urinary bladder carcinogenesis initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine.

Overexpression of ornithine decarboxylase (ODC) has been shown to be characteristic of tumor development and progression in humans and experimental animals. Therefore, we have examined the effects of 1, 3-diaminopropane dihydrochloride (DAP), a potent inhibitor of ODC, on rat two-stage urinary bladder carcinogenesis initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). In experiment 1 (36 weeks), 6-week-old F344 male rats were administered 0.05% BBN in drinking water for 4 weeks and then divided into four groups. Animals of groups 1 and 2 received basal diet and drinking water supplemented with or without DAP (2 g/l). Groups 3 and 4 were given diet containing 5% sodium L-ascorbate (NaAsA), a typical urinary bladder tumor promoter, and drinking water with or without DAP. Administration of DAP to group 1 significantly reduced tumor size, multiplicity and incidence, particularly of papillomas, when compared with group 2 values. DAP together with NaAsA (group 3) also decreased tumor size relative to the group 4 case. To determine the effects of DAP on the early stages of bladder carcinogenesis and its mechanisms, a similar protocol was conducted (experiment 2) with death after 20 weeks. DAP treatment caused complete inhibition (0% incidence) of papillary and/or nodular hyperplasia in group 1 but was without influence in group 3, as compared with the respective controls. Moreover, the ODC activity, bromodeoxyuridine labeling indices and mRNA expression levels of cyclin D1 in the urinary bladder mucosa, determined by northern blotting, were markedly lower in group 1 than in group 2, but values were comparable for both groups administered NaAsA. Assessment of mRNA expression levels of the angiogenic vascular endothelial growth factor suggested no involvement in the inhibitory effects of DAP on urinary bladder carcinogenesis. The results indicate that inhibition of ODC could reduce urinary bladder carcinogenesis in rats, particularly in the early stages, through antiproliferative mechanisms.

Low-dose-dependent carcinogenic potential of 2-amino-3-methylimidazo[4,5-f]quinoline in the immunodeficient (SCID) mouse colon.

The carcinogenic potential of 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ), one of the most potent mutagenic heterocyclic aromatic amines in food, for the colon was assessed in mice with severe combined immunodeficiency (SCID). In Experiment I, 60 male animals, 7-8 weeks old, were administered 300, 100, or 0 ppm IQ in the diet for 20 weeks. The incidence of aberrant crypt foci (ACF), preneoplastic lesions, was 100% in the two IQ-administered groups, whereas no ACF were found in the controls. Larger lesions, at least four aberrant crypts per focus, were noted in the colons of both treated groups. Most ACF were located in the proximal colon, and the bromodeoxyuridine-labeling indexes were elevated in a dose-dependent manner, especially in this region. In Experiment II, IQ was administered in the diet at 50, 10, 2, or 0 ppm to 60 female and male 7- to 8-week-old SCID mice for 30 and 23 weeks, respectively. The incidence of ACF was dose dependent in both sexes: 100%, 100%, and 63% in the females administered 50, 10, and 2 ppm, respectively, and 100%, 83%, and 38%, respectively, in the males. Lesions of at least four aberrant crypts per focus were again evident with the 50-ppm dose. The long-term or higher dose administration of IQ in the diet might thus be applied to elucidate colon carcinogenesis in the SCID mouse.

Specific p53 gene mutations in urinary bladder epithelium after the Chernobyl accident.

After the Chernobyl accident, the incidence of urinary bladder cancers in the Ukraine population increased gradually from 26.2 to 36.1 per 100,000 between 1986 and 1996. Urinary bladder epithelium biopsied from 45 male patients with benign prostatic hyperplasia living in radiocontaminated areas of Ukraine demonstrated frequent severe urothelial dysplasia, carcinoma in situ, and a single invasive transitional cell carcinoma, combined with irradiation cystitis in 42 cases (93%). No neoplastic changes (carcinoma in situ or transitional cell carcinoma) were found in 10 patients from clean areas (areas without radiocontamination). DNA was extracted from the altered urothelium of selected paraffin-embedded specimens that showed obviously abnormal histology (3 cases) or intense p53 immunoreactivity (15 cases), and mutational analysis of exons 5-8 of the p53 gene was performed by PCR-single-strand conformational polymorphism analysis followed by DNA sequencing. Nine of 17 patients (53%) had one or more mutations in the altered urothelium. Urine sediment samples were also collected from the patients at 4-27 months after biopsy and analyzed by PCR-single-strand conformational polymorphism analysis or yeast functional assay, and identical or additional p53 mutations were found in four of five cases. Interestingly, a relative hot spot at codon 245 was found in five of nine (56%) cases with mutations, and 11 of the 13 mutations determined (73%) were G:C to A:T transitions at CpG dinucleotides, reported to be relatively infrequent (approximately 18%) in human urinary bladder cancers. Therefore, the frequent and specific p53 mutations found in these male patients may alert us to a future elevated occurrence of urinary bladder cancers in the radiocontaminated areas.

Dose-dependent induction of aberrant crypt foci in the colons but no neoplastic lesions in the livers of heterozygous p53-deficient mice treated with low dose 2-amino-3-methylimidazo [4,5-f]quinoline.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a food derived heterocyclic amine which induces aberrant crypt foci (ACF) and tumors in the livers in mice. However, most previous studies of carcinogenicity were carried out with high dose treatments, so the practical risk associated with the low dose exposure is unclear. We, therefore, assessed whether low dose IQ causes ACF formation in the colons of mice constitutively hemizygous for functional p53. Simultaneously, we screened for development of preneoplastic foci in the liver. A total of 60 heterozygous p53-deficient mice as well as 60 wild-type mice were divided into five groups and administered IQ in the diet at concentrations of 50, 10, 2, 0.4 and 0 ppm until the end of the experiment. ACF were detected in the 50, 10 and 2 ppm-treated groups and the numbers of those comprising one aberrant crypt (AC) in p53-deficient mice treated with 10 or 2 ppm were significantly increased, compared to counterpart wild-type values. A dose-dependent increase of ACF was also observed in transgenic mice groups but no large ACF developed. In spite of extensive examination, no preneoplastic foci could be detected in either transgenic or wild-type mice. The results suggested that germline p53 deficiency may slightly enhance the development of ACF in colons but not in the liver. The fact that no ACF were detected in the lowest, 0.4 ppm, treated groups may imply a practical non-effective level of IQ for tumor induction.

LOH and mutational analysis of p53 alleles in mouse urinary bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl) nitrosamine.

In human urinary bladder carcinogenesis, alterations in the p53 tumor suppressor gene are common events. We have previously reported that they are also frequent in invasive urinary bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in NON/Shi mice. To further investigate the significance of the p53 gene status for mouse urinary bladder carcinogenesis, we examined both allele loss and mutational alterations in urinary bladder cancers of (NON/Shi x C3H/He/Shi) F1 hybrid mice exposed to the carcinogen for 12 weeks and then maintained for a further 9 weeks without treatment. An intragenic silent polymorphism within exon 7 of the p53 gene between NON/Shi and C3H/He/Shi mice allows assessment of allele loss of the p53 gene and determination of the parental origin of mutated and/or lost alleles. A tissue microdissection method was employed to obtain carcinoma samples without excessive contamination with normal tissue. Allele losses were detected in one of 14 tumors (7.1%) and nine mutations in eight of 14 (57%) tumors were found in exons 5-8 by polymerase chain reaction-single strand conformation polymorphism followed by DNA direct sequencing analysis. All mutations involved one base substitution with an amino acid change, although the types of base substitution were random. In conclusion, the high incidence of p53 alterations suggests a significant role in the genesis of invasive urinary bladder tumors in BBN-treated mice.

Concentration dependent promoting effects of sodium L-ascorbate with the same total dose in a rat two-stage urinary bladder carcinogenesis.

Sodium L-ascorbate (Na-AsA) has been demonstrated to be a strong promoter of rat urinary bladder tumor development initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). In the present study, we investigated variation in its promoting activity when the same total dose was given with different concentrations and exposure times. After 4 weeks administration of 0.05% BBN, group 1 served as a control without any post-initiation treatment. The rats in groups 2-4 received 1.25% Na-AsA diet for 36 weeks, 2.5% Na-AsA for 18 weeks and 5% Na-AsA for 8 weeks, respectively. Tumor number (papillomas and carcinomas) was greatest in group 3, and area in group 4 (P < 0.05). However, no enhancement was noted in group 2, although preneoplastic lesions were significantly increased. These results suggest that with the same total administration dose, high concentration of Na-AsA has the strongest promoting effects on tumor development in urinary bladder carcinogenesis.

Genetic instability and p53 mutations in metastatic foci of mouse urinary bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl)nitrosamine.

In a variety of human malignancies, alteration of the p53 tumour suppressor gene is known as a significant indicator of late progression events including invasion and metastasis, with a possible close relationship to genetic instability. Mutational analysis of the p53 and H-ras genes was performed for 10 pairs of N-butyl-N-(4-hydroxybutyl)nitrosamine-induced invasive mouse urinary bladder carcinomas and metastatic foci. p53 Mutations were found in nine of 10 (90%) primary carcinomas and seven of 10 (70%) metastatic foci. A total of eight p53 mutations in primary carcinomas were common in metastatic foci in six pairs. Additional p53 or H-ras mutations which were not identified in the primary carcinomas were found in three metastatic foci. Evaluation of the allelic distribution of the p53 mutations using RT-PCR, PCR and subcloning, further indicated possible intra-tumour genomic heterogeneity or excess copy numbers of the p53 gene due to genetic instability. Overall, p53 alterations were frequent in mouse urinary bladder carcinomas demonstrating progression. The results suggest that genetic instability might underlie generation of additional genetic alterations in this animal model.