Organ-specific, carcinogen-induced increases in cell proliferation in p53-deficient mice.

Transgenic mice with germ-line p53 alleles disrupted by gene targeting are sensitive to the development of some spontaneous tumors and have provided researchers with much information with respect to cancer. In the present study, to cast light on the organ specificity of chemically induced carcinogenesis, we evaluated carcinogen-induced cell proliferation in target organs in heterozygote p53 knockout mice (p53-deficient mice). Groups of 9- or 10-week-old wild-type(+/+) and p53-deficient mice were respectively treated with one of the following carcinogens for 4 weeks: N-butyl-N-(4-hydroxybutyl)nitrosamine (0.0075% in drinking water); dimethylnitrosamine (0.001% in drinking water); dihydroxy-di-N-propylnitrosamine (0.1% in drinking water); 1,2-dimethylhydrazine (10 mg/kg body weight s.c. injection once a week); 4-nitroquinoline 1-oxide (4-NQO, 10 mg/kg b.w. s.c. injection once a week); or 7,12-dimethylbenz(a)anthracene (25 microg/kg body weight dermal application once a week). Cell proliferation was evaluated by measuring the 5-bromo-2'-deoxyuridine labeling indices in each target organ. The p53 and p21 statuses were evaluated by comparing the expressions of p53 protein, p21waf1/cip1 mRNA, and p21waf1/cip1 protein between the mice. 5-Bromo-2'-deoxyuridine labeling indices of the urinary bladder and the skin were significantly increased in p53-deficient mice as compared with the wild-type(+/+) mice. In the all organs examined, carcinogen-induced p21waf1/cip1 mRNA overexpression was detected with levels obviously lower in the p53-deficient animals. These data suggest that p53-deficient mice have an organ-specific increased sensitivity to the induction of cell proliferation in the urinary bladder and the skin. These are the same organs for which sensitivity to carcinogenesis has been reported. Because a decrease of p21waf1/cip1 protein overexpression was also observed in the organs in which cell proliferation did not appreciably differ from the level in wild-type(+/+) mice, this decrease might have no effect on sensitivity to cell proliferation and carcinogenesis. Alternatively, it might play an important role in the cell cycle regulation of only the sensitive organs.

Cyclin D1 overexpression in rat two-stage bladder carcinogenesis and its relationship with oncogenes, tumor suppressor genes, and cell proliferation.

Overexpression of cyclin D1 has been implicated in the malignant transformation of a variety of human cancers, including urinary bladder carcinomas. However, few reports have addressed the significance of cyclin D1 overexpression in chemical carcinogenesis in rodents. In the present study, we evaluated the oncogenic potential of cyclin D1 in experimental rat urinary bladder carcinogenesis and its relationships to the oncogenes cyclin E, K-ras, and H-ras as well as tumor suppressor genes p53 and p21WAF1/Cip1. In addition, proliferation status of preneoplastic lesions and tumors was assessed by proliferating cell nuclear antigen immunohistochemistry. Fisher 344 rats were initiated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in the drinking water for 4 weeks and then administered 5% sodium L-ascorbate in diet. Animals were sacrificed at weeks 4, 8, 12, 18, and 24. Preneoplastic lesions such as papillary or nodular hyperplasia and neoplastic lesions of the urinary bladder were observed during carcinogenesis. By immunohistochemical examination, overexpression of cyclin D1 protein was observed in 17% of papillary or nodular hyperplasias, 66% of papillomas, and 69% of transitional cell carcinomas, whereas nuclear accumulation of p53 was observed in none of the preneoplastic lesions and in fewer than 2% of transitional cell carcinomas. Overexpression of cyclin D1 in preneoplastic lesions and tumors was not dependent on the size of the tumors or their proliferation status. Quantitation of mRNA in tumors by multiplex reverse transcription-PCR showed that average mRNA expression of cyclin D1 and cyclin E was increased, whereas average p21WAF1/Cip1 mRNA expression was decreased. More than 2-fold overexpression of cyclin D1 mRNA was observed in 50 and 60% of tumors at weeks 18 and 24, respectively. Localization of cyclin D1 mRNA expression was demonstrated by in situ hybridization, and the results were comparable to immunohistochemistry findings. None of the 25 tumors we examined by PCR-single-strand conformational polymorphism analysis harbored p53 mutations, H-ras mutations, or K-ras mutations. Thus, during the promotion phase of two-stage bladder carcinogenesis, overexpression of cyclin D1 in tumor cells may provide yet another mechanism by which tumors can gain a growth advantage. In contrast, tumors with mutated p53 may not have a growth advantage. Our results suggest that overexpression of cyclin D1 plays a critical role during urinary bladder carcinogenesis.

High susceptibility of p53(+/-) knockout mice in N-butyl-N-(4-hydroxybutyl)nitrosamine urinary bladder carcinogenesis and lack of frequent mutation in residual allele.

The loss of p53 functions is considered to compromise the growth-suppression machinery of the cell and facilitate neoplastic change. In humans, genetic alteration in the p53 gene is one of the most frequently observed molecular changes in tumors, including urinary bladder carcinomas. We have investigated the susceptibility of heterozygote p53 knockout mice to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in terms of urinary bladder tumor induction. Both p53(+/-) knockout mice and C57BL/6 original parent strain were administered 0, 0.002, 0.004, 0.0075 and 0.025% BBN in the drinking water for 20 weeks. As compared with the C57BL/6 strain, greater lesion yields were observed in knockout mice after 20 weeks of treatment. Transitional cell carcinomas were found in 9 (75%) and 12 (100%) of each 12 mice of the 0.0075 and 0.025% BBN treatment groups, respectively, whereas only 1 (11%) and 6 (67%) of each 9 of the C57BL/6 mice demonstrated tumors. Preneoplastic lesions (dysplasia) were also observed more frequently in the lower dose groups in the knockout mice than C57BL/6 mice. PCR single-strand conformation polymorphism analysis followed by DNA direct sequencing of the p53 gene (exons 5-8) extracted from bladder tumors demonstrated mutations in 3 of 11 (27.3%; exon 7) and 8 of 29 (27.6%; exons 5-8) tumors in C57BL/6 and knockout mice, respectively. There was no significant difference in the mutation rates at the residual p53 gene between the two cases. All mutations observed in knockout mice were restricted to the normal allele, and none were present in the gene-targeted null allele. In a separate experiment, 5-bromo-2'-deoxyuridine labeling indices after treatment with BBN for 2 or 4 weeks were significantly higher in knockout mice than wild-type mice. Measurement of the urinary concentration of N-butyl-N-(3-carboxypropyl)nitrosamine, a proximate carcinogenic metabolite, revealed no significant differences between knockout and original parent strain after administration of 0.0075% BBN in the drinking water for 4 weeks. In conclusion, knockout mice are distinctly more sensitive to urinary bladder carcinogenesis induced by BBN than their original parent strain, as evidenced by elevated DNA synthesis during carcinogen administration and an increased tumor yield. The high susceptibility of p53 knockout mice appeared to be related to the high level of cell proliferation rather than that of N-butyl-N-(3-carboxypropyl)nitrosamine in the urine or that of mutations at the p53 gene.

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.

Is the Watanabe heritable hyperlipidemic rabbit a suitable experimental model for percutaneous transluminal coronary angioplasty in humans? A light microscopic, immunohistochemical and ultrastructural study.

OBJECTIVES: This study was designed to assess an experimental model for the study of mechanisms that underlie restenosis after percutaneous transluminal coronary angioplasty. BACKGROUND: The Watanabe heritable hyperlipidemic (WHHL) rabbit lacks the receptor for low density lipoproteins, produces atherosclerotic lesions very similar to those in humans and, therefore, could serve as a suitable model. METHODS: Percutaneous transluminal angioplasty was performed on the left subclavian artery of 10 homozygous rabbits. The animals were killed at a few hours or 3, 7, 14 or 28 days after the procedure. The artery was fixed by perfusion, and the site of angioplasty was examined by both light and electron microscopy with the use of conventional and immunohistochemical staining techniques. RESULTS: Angioplasty had caused a flap-like or dissecting tear into the media. At day 3, cells within the preexisting media adjacent to the injury had the ultrastructural characteristics of synthetic smooth muscle cells. At day 7, spindle cells at the site of injury stained either negative or very weakly positive with a marker for actin; ultrastructurally, these cells had the synthetic phenotype. At day 14, the spindle cells showed a mix of contractile and synthetic phenotypes. The surface was partially covered by endothelial cells. At day 28, the dominant cell type was the contractile smooth muscle cell and the surface was completely covered by endothelial cells. CONCLUSIONS: Both the injury and the response to injury after percutaneous transluminal angioplasty were almost identical to that seen in humans after coronary angioplasty. Thus, the WHHL rabbit appears to be an appropriate experimental model for use in further studies.

Angiotensin blockade improves cardiac and renal complications of type II diabetic rats.

Using Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a new model of human non-insulin-dependent diabetes mellitus (NIDDM), we examined the role of local angiotensin II in cardiovascular and renal complications of NIDDM. OLETF rats were orally given cilazapril (an angiotensin-converting enzyme inhibitor, 1 or 10 mg/kg), E4177 (an angiotensin AT1 receptor antagonist, 10 mg/kg), or vehicle for 26 or 40 weeks (from the age of 20 to 46 or 60 weeks). Cardiac mRNAs were measured by Northern blot analysis, and the thickening of the coronary arterial wall and the degree of perivascular fibrosis were determined by an image analyzer. Cilazapril or E4177 did not significantly affect body weight or plasma glucose and insulin levels of OLETF rats, indicating the minor effects on diabetes itself. However, both drugs significantly and similarly prevented coronary microvascular remodeling (the increase in wall thickening and perivascular fibrosis in coronary arterioles and small coronary arteries) in OLETF rats, and they were associated with the suppression of cardiac transforming growth factor-beta1 expression. Both drugs suppressed not only the increase in left ventricular weight but also the downregulation of cardiac alpha-myosin heavy chain expression in OLETF rats. Glomerulosclerosis and glomerular hypertrophy in OLETF rats were improved by cilazapril and E4177 to a comparable extent. These results, taken together with the fact that OLETF rats show normal plasma renin levels, support that the AT1 receptor is involved in the pathogenesis of cardiac and renal complications in NIDDM.

Characteristics of diabetes, blood pressure, and cardiac and renal complications in Otsuka Long-Evans Tokushima Fatty rats.

To characterize the molecular mechanism of cardiac and renal complications in non-insulin-dependent diabetes mellitus (NIDDM), we examined the gene expression of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a new animal model for human NIDDM, at the ages of 14 weeks (prediabetic stage), 30 weeks (NIDDM stage), and 54 weeks (IDDM stage). Tissue mRNA levels were measured by Northern blot analysis. In 14-week-old OLETF rats, cardiac mRNAs for transforming growth factor-beta1 (TGF-beta1) and extracellular matrix, including collagen types I, III, and IV and laminin, were significantly increased compared with control rats (Long-Evans Tokushima Otsuka rats). Cardiac beta-myosin heavy chain (MHC) mRNA of OLETF was increased at 30 and 54 weeks of age, whereas alpha-MHC mRNA of OLETF was inversely decreased at 54 weeks. Marked perivascular fibrosis was seen in the hearts of OLETF rats from 30 weeks of age. In the kidney of OLETF rats, glomerular TGF-beta1 expression was temporally increased at 30 weeks of age, followed by glomerulosclerosis characterized by mesangial proliferation, thickening of the basement membrane, and nodular lesions. Blood pressure of OLETF rats remained higher than that of control rats from the prediabetic stage to the IDDM stage. Thus, in OLETF rats, cardiac fibrosis-related gene expressions were already enhanced at the prediabetic stage, which supports the involvement of these gene expressions in cardiac perivascular fibrosis. The antithetical change in beta- and alpha-MHC expressions seems to participate in the decreased cardiac contractility seen in diabetes. Furthermore, TGF-beta1 may also contribute to glomerulosclerosis of OLETF rats. OLETF rats seem to be a useful model to study the mechanism of hypertension and cardiac and renal complications in NIDDM.

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.

Dose-dependent inhibition of glutathione S-transferase placental form-positive hepatocellular foci induction in the rat by methyl propyl disulfide and propylene sulfide from garlic and onions.

Two organosulfur compounds, methyl propyl disulfide (MPD) and propylene sulfide (PS) from garlic and onions, were studied for their modifying effects on hepatocarcinogenesis in the F344 rats. Modifying potential was scored by comparing the number and area per cm2 of induced glutathione S-transferase placental form (GST-P)-positive foci in the liver. MPD and PS significantly reduced both these parameters of GST-P-positive foci in a dose-dependent manner. To investigate possible mechanisms of inhibition, ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SAT) activities were measured. In MPD and PS-high dose-treated liver tissue there was a tendency for their decrease, albeit non-significant, which suggested that the inhibitory effect might have been caused by decreased cell proliferation associated with decreased polyamine biosynthesis. In evaluating relationships between diet and cancer, it is thus necessary to consider various effects in assessing possible protective roles of garlic and onions.

Promotion of NCI-Black-Reiter male rat bladder carcinogenesis by dimethylarsinic acid an organic arsenic compound.

Dimethylarsinic acid (DMAA) is a major metabolite of inorganic arsenicals in mammals. In the present study, we investigated its promoting effects on urinary bladder carcinogenesis in NCI-Black-Reiter (NBR) rats, which lack alpha2u-globulin synthesizing ability. Male 9-14-week-old NBR rats were treated sequentially with 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) for 4 weeks and then given 100 ppm DMAA in their drinking water (group 1) for 32 weeks. Induction of preneoplastic lesions (papillary or nodular hyperplasia) in this DMAA-treated group was significantly increased as compared to the carcinogen alone control group (P < 0.01). The development of carcinomas was also enhanced and a significant increase in the 5-bromo-2'-deoxyuridine (BrdU) labeling index of the urinary bladder epithelial cells was observed for the DMAA treatment group. These results indicate that DMAA has promoting effects on urinary bladder carcinogenesis even in NBR rats, so its effects are not dependent on the presence of alpha2u-globulin.

Enhancement of urinary bladder carcinogenesis in nullizygous p53-deficient mice by N-butyl-N-(4-hydroxybutyl)nitrosamine.

We recently reported p53 mutations to be frequent in mouse invasive urinary bladder carcinomas, with and without metastasis. However, the role of p53 dysfunctions during carcinogenesis remains unclear. In the present study, heterozygous and nullizygous p53-deficient mice and their littermates were treated with the urinary bladder carcinogen, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), at a concentration of 0.01% in the drinking water throughout the experiment. This markedly accelerated urinary bladder carcinogenesis but not development of other tumors in the nullizygous p53-deficient mice. Thus the appearance of neoplastic urothelial lesions in nullizygotes (at day 60 of the experiment) was earlier than in wild-type mice and heterozygotes (at day 125). Moreover, malignant vascular tumors (hemangiosarcomas (HS)) were found in all four nullizygotes killed later than day 108. Mutational inactivation of the wild-type allele was not apparent in either the single transitional cell carcinoma observed in a wild-type mouse and a hemangiosarcoma in a heterozygote. Overall, it can be concluded that the number of normal p53 alleles is a significant determining factor in the susceptibility of urothelial cells to carcinogens. The role of the p53 defect in mouse urinary bladder carcinogenesis may thus be to diminish the threshold for occurrence of additional genetic alterations.

Epithelial cell proliferation in the digestive tract induced by space restriction and water-immersion stress.

The effects of space restriction and water-immersion stress on epithelial cell proliferation in the digestive tract, with special attention to the esophagus, stomach and duodenum, in 8-week-old SD male rats were examined. Histological assessment revealed spotted hemorrhagic lesions in the fundus of the glandular stomach, accompanied by statistically increased 5-bromo-2'-deoxyuridine (BrdU) labeling index in the fundic and pyloric regions. Furthermore, biochemical analysis demonstrated an increased activity of ornithine decarboxylase (ODC) and spermidine/spermine N1-acetyltransferase (SAT), known as key late-limiting enzymes of the polyamine pathway, in the gastric fundus. The stress may induce a remarkable increase in expression of c-fos, c-jun and c-myc mRNAs in both fundic and pyloric regions of the glandular stomach. There were no remarkable changes in the esophagus. These results indicate that space restriction and water-immersion stress induced cell proliferation in the glandular stomach through overexpression of proto-oncogenes and increased ODC and SAT activities that might be related to the promotion of gastric carcinogenesis.

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.

Dimethylarsinic acid induces 8-hydroxy-2'-deoxyguanosine formation in the kidney of NCI-Black-Reiter rats.

Dirnethylarsenic peroxyl radical [(CH(3))(2)AsOO] has been postulated to be responsible for DNA damage induced by dimethylarsinic acid (DMA). In an effort to elucidate the possible mechanism of tumor-inducing potential of DMA, an experiment was designed to investigate the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a specific marker of oxidative base damage in the kidney tissues of NCI-Black Reiter (NBR) rats. Animals were divided into four groups and administered the vehicle - saline, 5, 10 and 20 mg/kg body weight respectively of DMA by gavage, once a day, 5 days a week, for a period of 4 weeks. DMA induced increase of 8-OHdG levels in the kidney of the rats treated, with the highest level at the dose of 10 mg/kg body weight. Analysis of the kidney for cell proliferation employing PCNA-positive index showed greater proliferation in the tissues of treated rats. However, DMA did not have any influence on apoptosis in this regimen. Histopathological examination of the kidney selections revealed the presence of vacuolated degeneration and dilation of the proximal tubule cells in two groups (10 and 20 mg/kg body weight). This study provides evidence to substantiate the role of DMA in inducing oxidative DNA damage in the kidney.

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.

Inhibition of liver glutathione S-transferase placental form-positive foci development in the rat hepatocarcinogenesis by Porphyra tenera (Asakusa-nori).

The effects of Asakusa-nori, Porphyra tenera (PT), a popular edible seaweed in Japan, on the development of putative preneoplastic lesions, glutathione S-transferase placental form (GST-P)-positive foci, in the male F344 rat liver were examined using a medium-term bioassay system. PT significantly decreased both the number and area of GST-P-positive foci in rat livers initiated with diethylnitrosamine (DEN). To investigate possible mechanisms of inhibition, effects of PT on 5-bromo-2'-deoxyuridine (BrdU) labeling in GST-P-positive foci and the surrounding area of hepatocytes were studied. The ratio of the GST-P-positive foci to surrounding tissue labeling indices was decreased in the PT-treated group as compared with the DEN alone group. Ornithine decarboxylase activity in the liver was slightly increased and spermidine/spermine N'-acetyltransferase activity was slightly decreased in the PT-treated animals. These results suggest that PT possesses chemopreventive effects against DEN-induced hepatocarcinogenesis.

Infrequent involvement of microsatellite instability in urinary bladder carcinomas of the NON/Shi mouse treated with N-butyl-N-(4-hydroxybutyl)nitrosamine.

Variation in the frequency of microsatellite instability (MSI) has been reported in different kinds of human malignant tumors, with less than one-third of invasive urinary bladder carcinoma cases estimated to be affected. Here we investigated the MSI for 27 microsatellite sequences in invasive urinary bladder carcinomas of the NON/Shi mouse induced by N-butyl-N-(4-hydroxybutyl)nitrosamine. A total of 28 urinary bladder carcinomas of both transitional cell and squamous cell types were studied. All were invasive (greater than pT3) and high-grade and 10 of them had metastasis. Only two (11%) of 18 primary bladder carcinomas without metastasis foci showed alterations in one or two loci. None of 10 pairs of urinary bladder carcinomas and metastasis foci demonstrated any alterations. In conclusion, MSI which represents a defect in the DNA mismatch repair system is infrequent and therefore unlikely to be a critical step in genesis of invasive mouse urinary bladder carcinomas.

Presence of a no-observed effect level for enhancing effects of development of the alpha-isomer of benzene hexachloride (alpha-BHC) on diethylnitrosamine-initiated hepatic foci in rats.

The dose dependence of the promoting effects of the alpha-isomer of benzene hexachloride (alpha-BHC) on hepatocarcinogenesis was investigated in a medium-term rat liver bioassay (Ito test). A total of 195 F344 male rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at the start of the experiment and subjected to two-thirds partial hepatectomy at week 3. Two weeks after the administration of DEN, alpha-BHC were fed to rats at doses of 0, 0.01, 0.1, 0.5, 1, 2, 4, 7.5, 15, 30, 60, 125 and 500 ppm in diet for 6 weeks. All surviving animals were killed at week 8, and their livers were examined immunohistochemically for detection of glutathione S-transferase placental form (GST-P)-positive foci, surrogate preneoplastic lesions. Quantitative values for numbers and areas were dose-dependently increased in rats given alpha-BHC at 0.5-500 ppm. However, those for groups treated with 0.01 and 0.1 ppm were decreased, albeit not significantly in comparison to the controls. Cytochrome P450 3A2 (CYP3A2) protein levels and activities showed a good correlation to the number and area of GST-P-positive foci. These results support evidence of hormesis and indicate a no-observed effect level for alpha-BHC promoting potentials may exist regarding rat liver carcinogenesis, which correlates with expression of CYP3A2 in the liver.

The response to percutaneous transluminal coronary angioplasty: An ultrastructural study of smooth muscle cells and endothelial cells.

We investigated the ultrastructure of the repair tissue formed after percutaneous transluminal coronary angioplasty (PTCA). Four hearts (6 coronary arteries) were investigated; 5 arteries after single PTCA and 1 after repeated PTCA. In the earliest lesion (5 days after PTCA), all smooth muscle cells were of a synthetic phenotype, with abundant rough endoplasmic reticulum and few myofilaments, whereas the oldest lesion (259 days after PTCA) was composed of contractile smooth muscle cells, characterized by abundant myofilaments and small amounts of rough endoplasmic reticulum. In lesions taken at intermediate times (74 and 77 days), the smooth muscle cells were of an intermediate phenotype, with rough endoplasmic reticulum and myofilaments present in approximately equal portions. Regenerating endothelial cells observed at 74 days after PTCA were characterized by a rounded shape and a paucity of intercellular connections in the form of simple junctions, which suggested an immature state. At 259 days after PTCA, the endothelial cells were more mature, as indicated by a flat shape and the presence of many tight junctions and a complete basement membrane. The findings suggest a relation between the stage of endothelial cell regeneration and the phenotype of the smooth muscle cells.

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.