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The constitutional t(11;22) is the most frequent recurrent non-Robertsonian translocation in humans, the breakpoints of which are located within palindromic AT-rich repeats on 11q23 and 22q11 (PATRR11 and PATRR22). Genetic variation of the PATRR11 was found to affect de novo t(11;22) translocation frequency in sperm derived from normal healthy males, suggesting the hypothesis that polymorphisms of the PATRR22 might also influence the translocation frequency. Although the complicated structure of the PATRR22 locus prevented determining the genotype of the PATRR22 in each individual, genotyping of flanking markers as well as identification of rare variants allowed us to demonstrate an association between the PATRR22 allele type and the translocation frequency. We found that size and symmetry of the PATRR22 affect the de novo translocation frequency, which is lower for the shorter or more asymmetric versions. These data lend support to our hypothesis that the PATRRs form secondary structures in the nucleus that induce genomic instability leading to the recurrent translocation.

Palindromic regions are unstable and susceptible to deletion in prokaryotes and eukaryotes possibly due to stalled or slow replication. In the human genome, they also appear to become partially or completely deleted, while two palindromic AT-rich repeats (PATRR) contribute to known recurrent constitutional translocations. To explore the mechanism that causes the development of palindrome instabilities in humans, we compared the incidence of de novo translocations and deletions at PATRRs in human cells. Using a highly sensitive PCR assay that can detect single molecules, de novo deletions were detected neither in human somatic cells nor in sperm. However, deletions were detected at low frequency in cultured cell lines. Inhibition of DNA replication by administration of siRNA against the DNA polymerase alpha 1 (POLA1) gene or introduction of POLA inhibitors increased the frequency. This is in contrast to PATRR-mediated translocations that were never detected in similar conditions but were observed frequently in human sperm samples. Further deletions were found to take place during both leading- and lagging-strand synthesis. Our data suggest that stalled or slow replication induces deletions within PATRRs, but that other mechanisms might contribute to PATRR-mediated recurrent translocations in humans.

The p16INK4A/CDKN2A (p16) tumor suppressor gene is known to be inactivated in up to 98% of human pancreatic cancer specimens. Chemically induced pancreatic tumors in Syrian golden hamsters have been demonstrated to share many morphological and biological similarities with human pancreatic tumors and represent a potentially suitable model for the evaluation of therapies targeting p16. The purpose of this study was to evaluate primary hamster pancreatic tumor specimens for potentially inactivating p16 alterations. Tumors were induced with N-nitroso-bis-(2-oxopropyl) amine, followed by two cycles of augmentation pressure, and were harvested on day 100. Foci of tumor cells were identified by light microscopy after staining with hematoxylin and eosin, and corresponding tumor tissues were excised for DNA extraction. The techniques of multiplex real-time PCR, direct sequencing and methylation-specific PCR were used to evaluate 30 tumor specimens for homozygous deletions, mutations and aberrant methylation of 5' CpG islands, respectively. Homozygous deletions were identified in 11 of 30 (36.7%) specimens, mutations were identified in four of 30 (13.3%) specimens, and aberrant methylation of 5' CpG islands was found in 14 of 30 (46.7%) specimens. The overall frequency of p16 alterations was 93.3% (28 of 30 specimens) and the majority of changes (83.3%) were noted to be secondary to methylation or homozygous deletion. The four mutations significantly impaired cyclin-dependent kinase 4 inhibitory activity, and two resulted in perturbation of the global structure of P16 protein. These findings indicate that p16 inactivation is a common event in chemically induced hamster tumors, and that this animal model is appropriate for comparative studies evaluating pancreatic cancer therapeutic strategies targeting p16.

Expression of cyclooxygenase (COX)-2 protein during rat hepatocarcinogenesis associated with fatty change, fibrosis, cirrhosis and oxidative DNA damage, caused by a choline-deficient, L-amino acid-defined (CDAA) diet were investigated in F344 male rats, along with the chemopreventive efficacy of the specific COX-2 inhibitor, nimesulide (NIM). Nimesulide, which was administered in the diet at concentrations of 200, 400, 600 and 800 p.p.m. for 12 weeks, decreased the number and size of preneoplastic enzyme-altered liver foci, levels of oxidative DNA damage, and the grade and incidence of fibrosis in a dose-dependent manner. A preliminary long-term study of 65 weeks also revealed that 800 p.p.m. NIM decreased the multiplicity of neoplastic nodules and hepatocellular carcinomas and prevented the development of cirrhosis. Western blot analysis revealed that COX-2 protein was barely expressed in control livers and increased approximately 2.9-fold in the livers of rats fed on a CDAA diet for 12 weeks and approximately 4.5-5.4-fold in tumors, with a diameter larger than 5 mm, at 80 weeks. Immunohistochemically, COX-2 protein was positive in sinusoidal and stromal cells in fibrotic septa, which were identified by immunoelectron microscopy as Kupffer cells, macrophages, either activated Ito cells or fibroblasts, after exposure to the CDAA diet for 12 weeks, whereas it was only occasionally weakly positive in sinusoidal, probably Kupffer, cells in control livers. In neoplastic nodules in rats fed on a CDAA diet for 30 and 80 weeks, sinusoidal cells and cells with relatively large round nuclei and scanty cytoplasm were strongly positive for COX-2 protein, with the neoplastic hepatocytes in the minority of the nodules, but not the cancer cells, being moderately positive. These results clearly indicate that rat hepatocarcinogenesis, along with fatty change, fibrosis and cirrhosis, is associated with increased expression of COX-2 protein, and point to the chemopreventive efficacy of a selective COX-2 inhibitor against, at least, the early stages of hepatocarcinogenesis.

Alteration of the FHIT gene was investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl) amine (BHP) in male Wistar rats. Animals at 6 weeks of age were given 2000 p.p.m. of BHP in drinking water for 12 weeks, then maintained without further treatment until killed at the end of week 25. A total of 25 lung adenocarcinomas were obtained and total RNAs were extracted from each for assessment of aberrant transcription of the FHIT gene by reverse transcription (RT)-polymerase chain reaction (PCR) analysis. Aberrant transcripts were detected in 15 adenocarcinomas (60%) as absence in the regions of nucleotides (nt) -9 to 279, -98 to 279, -98 to 348 or -98 to 447. Genomic DNAs were also extracted from all 25 adenocarcinomas and exons 5-9 were examined for mutations, using PCR-single strand conformation polymorphism (SSCP) analysis and sequencing. A mutation was detected in only one adenocarcinoma (4%), an ACC to ATC (Thr to IIe) transition at codon 76. Southern blot analysis of eight tumors did not show any evidence of gross rearrangement or deletion of the FHIT gene. Western blot analysis revealed reduced expression of Fhit protein in six out of 10 adenocarcinomas (60%). These results suggest that alteration of the FHIT gene may be involved in the development of lung adenocarcinomas induced by BHP in rats.

The 5' region for the endothelin receptor B (EDNRB) gene is a complex CpG island giving rise to four individual transcripts initiating within the island. Here, for the first time, we analyze the relationship between methylation and gene expression in a CpG island located in the 5' region of a gene with multiple transcription start sites. The CpG island was unmethylated in normal prostate and bladder tissue, whereas it became methylated in apparently normal colonic epithelium. Tumors derived from these tissues were frequently hypermethylated relative to the respective normal tissues. Analysis of 11 individual CpG sites located throughout the CpG island showed that specific sites with high methylation levels in several tumors were also methylated in normal tissues, suggesting that they might serve as foci for further de novo methylation. This region also had high levels of methylation in several cancer cell lines, and we found that a low methylation level in a small region within the 5' region correlated with expression of the 5'-most transcript. Interestingly, almost complete methylation 200-1000 bp downstream of the transcriptional start site did not block expression of this transcript. Finally, we show that treatment with 5-aza-2'-deoxycytidine can induce transcriptional activation of the four EDNRB transcripts. Our results show the existence of differential, tissue-dependent methylation at the EDNRB 5' region, suggest the existence of a spreading mechanism for de novo methylation, starting from methylation hotspots, and show that hypermethylation immediately 3' to a transcriptional start site does not prevent initiation.

The involvement of poly(ADP-ribose) polymerase-1 (Parp-1), one of the poly(ADP-ribose) polymerase family proteins, in genomic stability, DNA repair and cell death triggered by DNA damage has been well documented. However, the potential role of Parp-1 in carcinogenesis has not been well evaluated. In this study the carcinogenic activity of N:-nitrosobis(2-hydroxypropyl)amine (BHP) was studied in Parp-1(-/-) mice, generated by disrupting P:arp-1 gene exon 1. Parp-1(-/-) and Parp-1(+/+) male mice received 0, 250 and 500 p.p.m. BHP in their drinking water for 20 weeks and were then killed. The percentage of animals bearing hemangiomas and hemangiosarcomas in the liver and numbers of tumors per mouse were markedly higher in the Parp-1(-/-) groups given 250 or 500 p.p.m. BHP than in their Parp-1(+/+) counterparts. Hemangiosarcomas developed only in Parp-1(-/-) mice. In the lung the numbers of adenomas per mouse were increased in Parp-1(-/-) mice given BHP at 250 and 500 p.p.m. (P < 0.01) compared with the Parp-1(+/+) case. The results show that susceptibility to BHP is significantly elevated in Parp-1(-/-) mice, thus providing direct evidence that Parp-1 is relevant to carcinogenesis.

The effects of antibiotics and anti-inflammatory drugs on the promotion stage of lung carcinogenesis initiated with N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats were investigated in two experiments with a similar protocol. In experiment 1, rats received tap water containing 2000 p.p.m. BHP for 12 weeks followed by basal diet or basal diet containing 0.02% erythromycin (EM), 0. 04% ampicillin (ABPC), 1.5% sho-saiko-to, 0.02% EM plus 1.5% sho-saiko-to or 0.04% ABPC plus 1.5% sho-saiko-to for 8 weeks after BHP administration. The development of adenocarcinomas (AC), squamous cell carcinomas (SqC) and adenosquamous carcinomas (ASqC) was completely inhibited in rats given ABPC plus sho-saiko-to and the numbers of lung lesions including alveolar hyperplasias, adenomas and carcinomas were decreased in rats given EM plus sho-saiko-to or ABPC plus sho-saiko-to. Neutrophil and macrophage infiltration into alveolar spaces of the lung were also markedly suppressed. In experiment 2, rats received BHP in the same manner as in experiment 1 and basal diet or basal diet containing 0.04% ABPC, 0.006% piroxicam, 0.04% ABPC plus 0.006% piroxicam and 0.04% ABPC plus 0.75% ougon for 8 weeks. The incidence and number of carcinomas, including ACs, SqCs and ASqCs were decreased in rats given ABPC plus piroxicam or ABPC plus ougon. Bacteria, mainly Escherichia coli, were detected in broncho-alveolar lavage of rats receiving BHP. The results suggest that chronic inflammation might be involved in the progression of lung carcinogenesis by BHP in rats and its suppression may therefore be useful as a chemopreventive strategy in lung cancer clinics.

In order to assess the significance of changes in metalloproteinase activity in pancreatic carcinogenesis, the expression of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9, respectively), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2, and membrane-type 1 MMP (MT1-MMP) and MT2-MMP in ductal lesions in a rapid-production model for pancreatic duct carcinomas (PCs) in hamsters initiated with N-nitrosobis(2-oxopropyl)amine (BOP) and in subcutaneous transplantable tumors of hamster pancreatic duct carcinoma (HPDs) was investigated. Northern analysis revealed MMP-2, MMP-9, TIMP-2 and MT1-MMP mRNAs to be overexpressed in PCs. Immunohistochemically, elevated levels of MMP-2 were apparent in early duct epithelial hyperplasias and staining increased from atypical hyperplasias to carcinomas. Gelatin zymography demonstrated clear activation of proMMP-2 but not proMMP-9 in both of primary and HPD tumors, the MT1-MMP mRNA level and proMMP-2 activation being significantly correlated (r = 0.893, P < 0.001). In our rapid production model, 0.1 and 0.2% OPB-3206, an inhibitor of MMPs, given in the diet after two cycles of augmentation pressures for 48 days decreased the incidence and number of carcinomas. Gelatin zymography demonstrated that OPB-3206 inhibited activation of proMMP-2 in pancreatic cancer tissues. These results indicate that overexpression of MMP-2, TIMP-2 and MT1-MMP, and cell surface activation of proMMP-2 by MT1-MMP, are involved in the development of PCs, and that MMP-2 expression at the protein level appears in the early phase of pancreatic duct carcinogenesis. OPB-3206 may be a candidate chemopreventive agent for pancreatic ductal adenocarcinomas.

The expression of midkine (MK) in lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats was examined. The animals were administered 2000 p.p.m. of BHP in their drinking water for 12 weeks, then maintained without further treatment until being killed 20-28 weeks after the beginning of the experiment. MK mRNA expression of adenocarcinomas and squamous cell carcinomas assessed by means of the reverse transcriptase-polymerase chain reaction and northern blot analysis was significantly higher than in rat embryonic tissues (positive controls) and contrasted strongly with the lack in normal lungs. MK protein was detected immunohistochemically in 58.3% of alveolar hyperplasias, 92.3% of adenomas and 100% of adenocarcinomas and squamous cell carcinomas. The extent of staining significantly increased along with malignant progression in adenomatous (pre-)neoplastic lesions and tended to become more pronounced with malignant progression in squamous lesions. The results suggest that MK may play some essential roles in the development and progression of lung tumors induced by BHP in rats.

Previously, we have reported that aspirin, a cyclooxygenase (COX) inhibitor, can prevent the fibrosis, cirrhosis and generation of oxidative DNA damage, and the associated development of glutathione-S-transferase placental form (GST-P)-positive preneoplastic liver nodules, caused by a choline-deficient, L-amino acid-defined (CDAA) diet in rats. In the present study, in order to elucidate the role of COX pathway in liver lesion-induction by a CDAA diet, the modulatory effects of other distinct chemical classes of COX inhibitors were examined. A long-acting example, piroxicam (PIRO) (at doses of 0.01, 0.02, 0.04 and 0.06%) and the short-acting ibuprofen (IBU) (at doses of 0.02, 0.04 and 0.06%) and indomethacin (IND) (at doses of 0.005 and 0.008%) were administered in the CDAA diet to male F344 rats, and animals were killed after 12 and 30 weeks. In another experiment, IND was given in drinking water at doses of 0.001, 0.002 and 0.004%. None of the inhibitors affected the development of fatty liver caused by a CDAA diet, but PIRO at doses higher than 0.04%, strongly inhibited the development of GST-P-positive and neoplastic nodules as well as fibrosis, cirrhosis and formation of 8-hydroxydeoxyguanosine (8-OHdG) adducts. IBU at the highest dose also exhibited similar but much less pronounced inhibitory effects. With IND, there was only a tendency for inhibition with no clear dose-dependence. The results together with our previous findings, indicate that relatively strong COX inhibitors, acting irreversibly like aspirin or for extended periods like PIRO, can prevent the endogenous hepatocarcinogenesis associated with a CDAA diet, although not the development of a fatty liver, suggesting that an augmented COX pathway might play key roles in the causation of liver lesions in this model.

Epidemiological studies and animal experiments show an association of dietary intake of fish oils and low incidence of several types of cancers. The active ingredients of fish oils appear to be polyunsaturated fatty acids of omega-3 type such as eicosapentaenoic acid and docosahexaenoic acid (DHA). We have investigated chemopreventive effects of DHA on mouse intestinal polyposis using adenomatous polyposis coli (Apc) gene knockout mice. Damage to the human APC gene is responsible for not only familial adenomatous polyposis but also many sporadic cancers of the entire digestive tract. Using homologous recombination in embryonic stem cells, we recently constructed gene knockout mice containing a truncation mutation in the Apc gene at codon 716 (Apc delta 716). The heterozygous mice developed numerous intestinal polyps, and all microadenomas dissected from the earliest polyps had already lost the wild-type allele, indicating the loss of heterozygosity [Oshima et al. (1995), Proc. Natl Acad. Sci. USA, 92, 4482-4486]. We fed Apc delta 716 heterozygotes with AIN-76A purified diet containing 3% DHA for 7 weeks, and scored the number and size of intestinal polyps. Average DHA intakes per day were 4.1 and 4.3 g/kg body wt for males and females, respectively. DHA-fed females had only 31% of polyps compared with the control females that developed about 220 polyps, whereas DHA-fed females showed no significant decrease in the polyp number. As for the polyp size, the proportion of larger polyps decreased more significantly in females than in males. This is the first demonstration that DHA inhibits intestinal polyposis induced by an Apc mutation at both its formation and growth.

Initiation activities of endogenously formed N-nitrosobis(2-hydroxypropyl)amine (NBHPA), N-nitrosodiethanolamine (NDELA) and N-nitroso-2,6-dimethylmorpholine (NDMM) were investigated in a modified short-term assay for rat hepatocarcinogenesis. Male Wistar rats were fed 1% bis(2-hydroxypropyl)amine, 0.5% diethanolamine or 0.25% 2,6-dimethylmorpholine in the diet plus 0.3% sodium nitrite in the drinking water. Two weeks after starting the experimental regimen they underwent 2/3 partial hepatectomy and were then maintained on the respective diets for a further week. Following a 2 week recovery period on basal diet rats were subjected to a resistant hepatocyte regimen consisting of 0.02% 2-acetylaminofluorene in the diet for 2 weeks and 1 mg carbon tetrachloride/kg body wt by gavage at the midpoint. Initiation activity was assayed by measuring hepatic foci positive for gamma-glutamyltranspeptidase. Numbers of such foci per cm2 were significantly increased in the groups given the secondary amines together with nitrite compared with values for groups given each precursor or nitrite alone. Further, the numbers of lesions were essentially similar to those found in rats given carcinogenic doses of NBHPA, NDELA and NDMM. The results clearly of demonstrate hepatocyte initiation activities of endogenously formed carcinogens, presumably NBHPA, NDELA and NDMM.

The effects of dehydroepiandrosterone sulfate (DHAS), a typical hydroxysteroid sulfotransferase (HSTase) inhibitor, and of 3'-phosphoadenosine 5'-phosphate (PAP), a nonspecific sulfation inhibitor on N-nitrosobis(2-oxopropyl)-amine (BOP)-induced initiation were examined in a rapid production model for pancreatic carcinomas in hamsters in order to elucidate the involvement of sulfotransferase in the metabolic activation of beta-oxypropylnitrosamines. While neither low nor high doses of DHAS and PAP exerted any significant influence on the incidence of ductal lesions including carcinomas, the high dose of DHAS (350 mg/kg body wt) and a both low (90 mg/kg) and high (180 mg/kg) doses of PAP reduced the mean numbers of pancreatic ductal adenocarcinomas. The high dose of PAP also reduced the number of all ductal lesions combined. The results thus suggest that metabolic activation with STase is involved in BOP-induced pancreatic ductal carcinogenesis in hamsters, and support the hypothesis that BOP is metabolized to beta-hydroxyalkylnitrosamines followed by activation to proximate sulfuric acid esters by HSTase.

The effects of dietary administration of catechol (CC), paramethylcatechol (PMC) and di(2-ethylhexyl)phthalate (DEHP) were compared with that of butylated hydroxyanisole (BHA) in Syrian hamsters initiated with N-nitrosobis(2-hydroxypropyl)amine (BHP). Development of pancreatic atypical hyperplasias and adenocarcinomas in terms of combined multiplicity was significantly reduced by CC and DEHP. A similar slight but non-significant tendency was observed for BHA, while PMC was without effect. No statistically significant reduction of liver or gall bladder lesions was observed. The results thus suggest that both antioxidant and peroxisome proliferator categories of agents can inhibit pancreatic carcinogenesis in hamsters.

Effects of oxidative stress induced by redox-enzyme modulation on the progression stage of hepatocarcinogenesis were examined by monitoring both hepatocyte injury and hepatocellular carcinoma development in F344 rats bearing preneoplastic liver nodules induced by the Cayama-Farber procedure. Redox-enzyme modulation, which included increased cytochrome P450 reductase activity induced by phenobarbital-Na (100 mg/kg, i.p. for 3 days), inhibition of DT-diaphorase by dicumarol (25 mg/kg, i.p.), depletion of glutathione by phorone (200 mg/kg, i.p.), supplementation with the Fe(III) sodium salt of EDTA (50 mg/kg, i.p.) and redox-cycling activation by menadione (50 mg/kg, i.g.), exerted no prominent hepatocyte injury within nodules but did cause slight injury in the surrounding hepatocytes in nodule-bearing rats. The same treatments induced severe hepatocyte injury in non-treated normal rats. Redox-enzyme modulation performed every other week for 33 weeks significantly reduced the number of hepatocellular carcinomas developing in nodule-bearing rats. These results indicate that preneoplastic nodules are resistant to the oxidative stress induction caused by redox-enzyme modulation treatment and that, despite toxic effects in surrounding hepatocytes, no progression pressure is exerted. Indeed, the treatment rather demonstrates an inhibitory effect of the evolution of the nodules into hepatocellular carcinomas.

The modifying effects of an immunosuppressive agent, 6-mercaptopurine (6-MP), on development of focal lesions in liver cirrhosis models induced by carbon tetrachloride (CCl4) or furfural were studied in male F344 rats. Feeding of 6-MP at 50 p.p.m. for 20 weeks to animals with pre-existing liver cirrhosis caused immunosuppression, and significantly enhanced the induction of gamma-glutamyltranspeptidase (GGT)-positive foci and nodules in the CCl4 but not furfural case. Glutathione S-transferase P (GST-P)-positive preneoplastic lesions were not affected. Moreover, phenobarbital (PB) also enhanced the induction of GGT-positive hepatocellular lesions only in the CCl4-induced liver cirrhosis model, no promotion influence being exerted after treatment with the non-carcinogenic furfural. This study, therefore, suggests that 6-MP can enhance the induction of one type of preneoplastic foci and nodules and that essential differences exist between focal lesions arising in cirrhotic livers caused by CCl4 as opposed to furfural.

The modifying effects of the phenolic antioxidant catechol (CC) and its analogs hydroquinone (HQ) and resorcinol (RN) on pancreatic carcinogenesis were evaluated in 146 female Syrian golden hamsters. Groups of animals received either saline or 70 mg/kg body wt N-nitrosobis(2-oxopropyl)amine (BOP) s.c. injections, twice with a 2 week interval, followed by basal diet or diet containing 1.5% of CC, HQ or RN, and 0.75% CC from week 4. All hamsters were killed at week 20 and histopathologically examined for development of pancreatic, liver and gall bladder lesions. The total numbers of pancreatic lesions comprising carcinomas, atypical ductal hyperplasias and ductal hyperplasias per hamster were significantly decreased in animals receiving BOP followed by CC, HQ and RN when compared to those in hamsters given BOP followed by basal diet. Incidence values for atypical ductal hyperplasias were also significantly decreased by the RN or 0.75% CC treatments. The results thus suggest that pancreatic carcinogenesis initiated by BOP in Syrian hamsters can be inhibited by treatments with phenolic antioxidants such as CC, HQ and RN for a relatively short experimental period.

Inducibility of oxidative stress in rat liver in vivo by menadione-associated redox cycling activation under redox enzyme modulating conditions was examined by monitoring hepatocyte injury and 8-hydroxydeoxyguanosine (8-OHdG) levels of liver DNA. In addition, the treatment-associated liver tumor initiating activity was assessed in terms of development of gamma-glutamyl-transpeptidase (GGT)- and glutathione S-transferase placental form (GST-P)-positive foci and hyperplastic nodules. With or without following menadione treatment (50 mg/kg, i.g.), redox enzyme modulations of increased cytochrome P450 reductase activity induced by phenobarbital (PB)-Na (100 mg/kg, i.p. for 5 days), inhibition of DT-diaphorase by dicumarol (25 mg/kg, i.p.) and depletion of glutathione by phorone (200 mg/kg, i.p.), with or without further supplement of iron EDTA-Na-Fe(III) (70 mg/kg, i.p.), caused both substantial hepatocyte necrosis and 8-OHdG production in Fischer 344 male rats. Subsequent feeding with a 0.05% PB diet for 64 weeks resulted in slightly increased development of GGT-positive foci but not GST-P positive lesions or hyperplastic nodules, suggesting a lack of tumor-initiating activity of the oxidative DNA damage associated with redox enzyme modulations with or without menadione.

The effects of inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (ABA), luminol and 3-methoxybenzamide (MBA) on the rat liver tumor promotion activity of phenobarbital (PB) were assessed. Fischer 344 male rats were initiated with N-nitrosodiethylamine (200 mg/kg) and placed on either basal diet, diet containing 0.05% PB, diet containing various doses of the inhibitors alone or diet containing 0.05% PB plus various doses of inhibitors for 10 weeks, and then killed. Quantitation of the development of glutathione S-transferase placental form-positive foci revealed that ABA at doses of 2 and 1.5, but not 1%, significantly inhibited the PB promotion activity. Luminol dose-dependently reduced PB promotion at doses of 3 and 6% but exerted no effects at the 1 and 2% levels. MBA also demonstrated a dose-dependent inhibitory influence at doses of 1 and 2%. The results are thus strongly suggestive of an involvement of poly ADP-ribosylation in the mechanisms underlying liver tumor promotion by PB.

The initiating potential of the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA), for rat hepatocarcinogenesis was investigated using the development of hyperplastic nodules and/or glutathione S-transferase placental form (GST-P)-positive liver foci as the end point. Five week old male F344 rats were given either basal diet, or diets containing 0.5% DCA or 0.5% LCA for 3 weeks in conjunction with partial hepatectomy performed midway, followed by a selection regimen consisting of 2 weeks feeding of 0.02% 2-acetylaminofluorene diet and a single gastric intubation of carbon tetrachloride. The animals were then placed on either basal diet or a diet containing 0.05% phenobarbital (PB) for 52 weeks. Significantly higher numbers of hyperplastic liver nodules developed in the DCA-treated rats irrespective of PB promotion as compared with the respective control groups. No such increase was evident in the LCA-treated rats. In contrast, both DCA and LCA treatments enhanced the development of GST-P-positive liver foci with or without subsequent PB promotion. Only one hepatocellular carcinoma was diagnosed in a control group animal. The present data indicate that a short period of feeding of DCA and LCA in the initiation stage in conjunction with partial hepatectomy results in enhanced development of preneoplastic liver lesions under selection pressure conditions with or without subsequent PB promotion. They thus confirm and extend our previous finding of enhanced gamma-glutamyltranspeptidase-positive liver foci development in a short-term assay of DCA and LCA, and suggest that these secondary bile acids either possess possible initiating activity or some other priming effect for rat hepatocarcinogenesis.

The potential carcinogenic activity of acetaminophen (paracetamol, APAP) was studied in male F344 rats with pre-existing liver damage induced by a choline-devoid (CD) diet. In a short-term experiment, APAP was administered by intragastric intubation as single doses of 0.5-1.5 g/kg body wt after 4 weeks feeding of CD diet had produced fatty livers in rats. Two-thirds partial hepatectomy was performed 4 h subsequent to the initiating treatment step. After a 2 week recovery period, all rats were subjected to the selection procedure of Cayama et al. and killed at week 9 of the experiment. Quantitative analysis of placental form glutathione S-transferase (GST-P)-positive liver lesion development did not reveal any enhancement by APAP, whereas administration of a non-necrogenic dose of diethylnitrosamine (20 mg/kg body wt) in the same protocol demonstrated significant promotion, confirming the utility of the model for detection of weak carcinogenicity of chemicals. In the second long-term experiment, APAP was fed at doses of 0.45 and 0.9% for 25 weeks following 27 weeks administration of CD diet which produced liver cirrhosis in the rats. Despite a slight enhancement of focal liver lesions positive for gamma-glutamyltranspeptidase (GGT), no significant promotion of GST-P-positive altered foci or nodules was observed. In contrast, continuous feeding of CD diet or 0.5% phenobarbital treatment after generation of cirrhosis with CD diet clearly enhanced the induction of both GST-P and GGT-positive liver lesions. Thus, these results indicate that APAP does not possess significant carcinogenic activity in damaged rat liver.

The modifying potential of butylated hydroxyanisole (BHA) administration on pancreatic carcinogenesis was evaluated in 70 female Syrian golden hamsters. Groups of animals received saline, 70 mg/kg body weight of N-nitrosobis(2-oxopropyl)amine (BOP) or 70 mg/kg plus 20 mg/kg body weight of BOP followed by basal diet or diet containing 2% BHA from week 3. Although the body weights of hamsters receiving the 2% BHA supplement decreased, caloric restriction was not observed. All hamsters were killed at week 18 and histopathologically examined for lesion development. The incidences of pancreatic carcinomas in hamsters receiving 70 mg/kg plus 20 mg/kg body weight of carcinogen followed by 2% BHA was 7.1%, significantly lower than the 64.3% evident in hamsters given the same doses of BOP followed by basal diet. The total numbers of pancreatic lesions including carcinomas, atypical ductal hyperplasias and ductal hyperplasias and ductular proliferations in the liver were also significantly decreased in animals receiving BOP followed by 2% BHA. The results thus indicate that both pancreatic and cholangiocellular carcinogenesis initiated by BOP in Syrian hamsters can be inhibited by 2% BHA treatment for a relatively short experimental period.

The effects of dietary vitamin E (VE) on the steps of hepatocarcinogenesis, the induction and growth of gamma-glutamyltranspeptidase (GGT)-positive foci and their evolution into persistent nodules, were analyzed in the liver of rats treated with diethylnitrosamine (DEN). The induction of GGT-positive foci was inhibited by a diet containing 0.36-1.5% VE given after initiation with 200 mg/kg body weight (b.w.) DEN for 6 weeks with partial hepatectomy (PH) on week 3. The numbers and areas of GGT-positive foci were enhanced by diets containing 0.36 and 0.72% VE, given for 1 week after initiation with 10 mg/kg b.w. DEN and PH, followed by selection by 0.02% 2-acetylaminofluorene (AAF) and carbon tetrachloride (CCl4), but these were not enhanced by a diet containing 1.5% VE. Remodeling of hyperplastic nodules was not affected by the diet containing 0.72% VE given after initiation with DEN and selection for 12 weeks. The staining characteristics of GGT were different between remodeling and persistent nodules, except for those of the glutathione-S-transferase placental form (GST-P). The results obtained suggest that VE could prevent the very early events during hepatocarcinogenesis, the induction of phenotypically altered foci, but could no longer affect the later stages, the evolution of foci into persistent nodules.

The effect of a low dose of preadministered diethylnitrosamine (DEN) on the induction of enzyme-altered foci in the livers of male full-grown Fischer 344 rats was studied. As a pretreatment, DEN at a dose of 10 mg/kg body wt was injected i.p. At various times after DEN pretreatment a complete initiation, consisting of administration of the same dose of DEN by the same route in rats subjected to partial hepatectomy (PH), was performed, followed by application of selection pressure. Enzyme-altered foci stained with gamma-glutamyltranspeptidase (gamma-GTP) and glutathione S-transferase placental form (GST-P) were then assayed. Decreases in the numbers and areas of foci in the rats which received saline + PH 14 or 28 days after DEN pretreatment were observed in comparison with rats which received saline + PH immediately after DEN. On the other hand, the numbers and areas of foci were not decreased in rats which received the complete initiation, consisting of DEN + PH, at various times after DEN pretreatment when compared with rats which received these at the same time as the DEN pretreatment. This persistent effect of DEN pretreatment on the complete initiation lasted up to 182 days after the time of DEN pretreatment. In this experiment, GST-P was found to be a more sensitive marker for the detection of putative preneoplastic liver-cell foci than gamma-GTP.

The carcinogenic activity of endogenously synthesized N-nitrosobis(2-hydroxypropyl)amine (BHP) was investigated in male Wistar rats administered bis(2-hydroxypropyl)amine (BHPA) mixed in powder diet at a concentration of 1%, and sodium nitrite (SN) dissolved in distilled water at concentrations of 0.15 and 0.3%, for 94 weeks. Urinary excretion of BHP was detected in rats given 1% BHPA and 0.3% SN but not in the groups receiving either of these precursors alone. Nasal cavity, lung, esophagus, liver and urinary bladder tumors were found in animals treated with combinations of 1% BHPA and 0.15 or 0.3% SN, suggesting that the target organs of the endogenously synthesized BHP are similar to those affected when the carcinogen is administered exogenously. The incidences of nasal cavity and lung tumors reached 74 and 58% in rats given 1% BHPA and 0.3% SN, respectively. Tumors at sites other than target organs were only found at levels similar to those previously reported for spontaneous tumors in male Wistars. The present results clearly indicated the tumor inducibility of a nitrosatable amine, BHA, through an endogenous nitrosation by feeding to rats in conjunction with nitrite, and provide further suggestive evidence that endogenous nitrosations of environmental nitrosatable amines can be a potential risk factor in human cancer development.

The effects of clofibrate treatment on N-nitrosobis(2-hydroxypropyl)amine (BHP) induced liver, gall bladder, pancreas, lung and kidney carcinogenesis in hamsters were studied. Animals were given BHP as an initiator at a dose of 500 mg/kg body weight subcutaneously once a week for 5 weeks followed by diet containing 0.25 or 0.5% clofibrate for 30 weeks. Both doses of clofibrate promoted hepatocarcinogenesis as judged from the associated multiplicity of liver lesions including hyperplastic nodules and hepatocellular carcinomas. gamma-Glutamyltranspeptidase (gamma-GTP) activity was not expressed in those lesions in the liver of hamsters given BHP followed by a basal diet or diets containing clofibrate. Clofibrate at a dose of 0.5% in the diet, in contrast, inhibited the development of pancreatic adenocarcinomas and lung neoplasms, including adenomas and adenocarcinomas, without affecting carcinogenesis in the gall bladder and kidney. These results clearly indicate differential modification potential of clofibrate for BHP-induced liver, pancreas and lung carcinogenesis in Syrian hamsters.

Expression of glutathione S-transferase placental form (GST-pi) in human lung carcinoma tissue taken at autopsy or biopsy was investigated immunohistochemically. All of 34 cases of squamous cell carcinomas, including poorly, moderately and well-differentiated examples were shown to stain positively for GST-pi. Poorly differentiated adenocarcinomas were, however, negatively stained (0/5 cases), while moderately and well differentiated adenocarcinomas were found to stain with GST-pi at rates of 69% (9/13 cases) and 71% (5/7 cases), respectively. Six cases of small cell carcinomas examined were all negative. The results indicate that GST-pi may be a useful marker for non-small cell type lung cancer, especially squamous cell carcinoma which is in agreement with findings for rat lung neoplastic lesions reported previously.

The effects of inhibitors of arachidonic acid metabolism and antioxidants on the rat liver tumor promotion activity of phenobarbital (PB) were assessed using the enzyme-altered focus as the end-point lesion. Fischer 344 male rats were initiated with N-nitrosodiethylamine (200 mg/kg) and then divided into five groups placed on basal diet, diet containing 0.05% PB, diet containing 0.05% PB plus 0.75%, 1% or 1.5% levels of various inhibitors of arachidonic acid metabolism or antioxidants, or diet containing 1% or 1.5% inhibitors or antioxidants alone for 10 weeks, and then killed. p-Bromophenacyl bromide, an inhibitor of phospholipase A2, significantly inhibited the promotion activity of PB at dose levels of 0.75% and 1.5%, reaching plateau at 0.75%. Both quercetin, an inhibitor of lipoxygenase, and morin, a dual inhibitor of lipoxygenase-cyclooxygenase, significantly reduced the promotion activity of PB at the 1.5% but not 0.75% dose levels. Moreover, acetylsalicylic acid, an inhibitor of cyclooxygenase dose-dependently inhibited the promotion activity of PB. Among the antioxidants investigated, vitamin E did not affect, but n-propyl gallate and ethoxyquin exerted a dose-dependent inhibition of PB promotion. These results are strongly suggestive of an involvement of phospholipase A2, lipoxygenase and cyclooxygenase arachidonic acid metabolic pathways in the mechanisms underlying PB enhancement of hepatocarcinogenesis.

We previously reported a rapid production model for pancreatic carcinoma development in Syrian hamsters incorporating the principle of selection by resistance to cytotoxicity. In the present experiment, the efficacy of repeated augmentation pressure with regard to generation of pancreatic lesions in hamsters initiated with N-nitrosobis(2-oxopropyl)amine (BOP) was investigated. Forty-eight female Syrian golden hamsters were divided into four groups according to the frequency of augmentation pressure. Group 1 received 70 mg/kg body weight of BOP and three injections of 20 mg/kg BOP. Groups 2-4 received 70 mg/kg BOP followed by one, two or three cycles of augmentation pressure consisting of dl-ethionine on sugar and salt diet, l-methionine and 20 mg/kg BOP. Hamsters were killed 10 weeks after the beginning of the experiment and the resultant incidences of pancreatic carcinomas from groups 1-4 were 0, 30, 50 and 46.2% respectively, the numbers of pancreatic carcinomas increasing with the frequency of augmentation pressure. A 46.2% yield of cholangiocarcinomas was also observed in group 4. The model should be useful for investigation of potential modulating factors since large numbers of lesions can be induced within a total experimental period of only 10 weeks.

Immunohistochemical staining using anti-rat glutathione S-transferase placental form (GST-P) rabbit antibody and enzyme histochemical staining for gamma-glutamyltranspeptidase (gamma-GT) were investigated in lesions appearing during lung carcinogenesis induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Rats were given BHP at a concentration of 2000 p.p.m. in drinking water, and were killed after 12 weeks of BHP intake, after 12 weeks of BHP intake followed by 12 weeks of tap water intake or after 20 weeks of continuous BHP intake. It was found that bronchiolo-alveolar hyperplasias, adenomas, adenocarcinomas, squamous metaplasias and squamous cell carcinomas had been induced by BHP. All of the squamous metaplasias and squamous cell carcinomas were shown to stain with GST-P but not with gamma-GT. On the other hand, the hyperplasias, adenomas and adeno-carcinomas stained with gamma-GT to various degrees and in different areas, but did not stain with GST-P. The incidence of gamma-GT phenotype and the average percentage of gamma-GT-positive areas in hyperplasias and adenomas suggested that adenocarcinomas might develop from hyperplasias and adenomas. These results suggest that GST-P is a marker for squamous lesions while gamma-GT is a marker for adenomatous lesions in rat lung carcinogenesis. Furthermore, squamous metaplasias appear to be preneoplastic lesions of squamous cell carcinomas while gamma-GT-positive hyperplasias or adenomas are preneoplastic lesions of peripheral adenocarcinomas.