Co-treatment with deoxycholic acid and azoxymethane accelerates secretion of HMGB1 in IEC6 intestinal epithelial cells.

OBJECTIVES: High-mobility group box 1 (HMGB1) is a nuclear protein that acts as a ligand of the receptor for advanced glycation end products (RAGE) and its expression enhances progression of cancer. However, the mechanism underlying HMGB1 secretion is still unclear. In this study, we examined the effect of deoxycholic acid (DCA), a promoter of colon carcinogenesis, on HMGB1 secretion. MATERIALS AND METHODS: We used an in vitro transformation model comprised of IEC6 intestinal epithelial cells treated with azoxymethane (AOM) and/or DCA. HMGB1 expression and secretion were examined by Western and Northern blot analyses, and ELISA. Intracellular translocation of HMGB1 was examined by protein fractionation. RESULTS: AOM + DCA-treated IEC6 cells showed upregulation of HMGB1 mRNA expression and increased level of HMGB1 protein in culture medium, but decreased level of HMGB1 protein in the nucleus. AOM + DCA treatment increased level of histone H4 acetylation, which induced translocation of HMGB1 from the nucleus to the cytoplasm and increased HMGB1 secretion. Leptomycin B inhibited extranuclear translocation and secretion of the HMGB1 protein. CONCLUSION: These findings suggest that DCA affects intracellular localization and secretion of HMGB1.

Suppressive effects of a selective cyclooxygenase-2 inhibitor, etodolac, on 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis.

The present study was conducted to examine effects of a clinically available selective cyclooxygenase (COX)-2 inhibitor, etodolac, on the development of rat tongue squamous cell carcinomas (SCCs) induced by 4-nitroquinoline 1-oxide (4-NQO), and on the immunohistochemically demonstrable expression of COX-2. Fischer 344 rats, 6 weeks old at the commencement, were administered 4-NQO at the doses of 20-30 ppm in their drinking water for 12 weeks. Then, etodolac was supplemented into the diet at doses of 150 and 300ppm for 16 weeks. Rats were sacrificed at 28 weeks and tongue lesions were histologically examined. The incidence and the multiplicity of SCCs induced by 4-NQO were dose-dependently reduced by etodolac, with significance at the highest dose of 300 ppm. Etodolac did not significantly affect the immunohistochemical expression of COX-2 in the lesions which did develop. These results indicate that etodolac can inhibit the development of rat tongue SCCs, probably by inhibiting COX-2 activity rather than its expression. Thus, etodolac may be a promising candidate chemopreventive agent for individuals at high risk of oral cancer.

Interleukin-15 expression is associated with malignant potential in colon cancer cells.

Interleukin 15 (IL-15 mRNA expression was detected in human colorectal cancer cells (Colo320, WiDr, TCO and DLD1) by the reverse transcriptase-polymerase chain reaction (RT-PCR). Only Colo320 and WiDr cells secreted IL-15 culture medium. With IL-15 treatment, all cell lines grew at a rate of 120-180% of that of nontreated cells. A binding assay with (125)I-labeled IL-15 showed binding activity to IL-15 in Colo320 (K(d): 0.098 nM) cells. IL-15 also reversed the growth inhibition caused by serum starvation in Colo320 cells. IL-15-induced cell growth in regular and serum-free media was abrogated by anti-IL-15 antibody treatment in Colo320 cells. Moreover, IL-15 treatment reduced doxorubicin-induced cytostasis and cytolysis in Colo320 cells by 50%. The invasion capacity of IL-15-treated Colo320 cells was 5.3 times that of untreated cells. Immunoblotting showed that IL-15-treated Colo320 cells exhibited downregulation of p21Waf1 and Bax, and upregulation of Bcl-2, phospho-AKT, MMP9/MMP2, and VEGF. Finally, immunostaining of human colon cancer revealed that 33 (70%) of 47 Dukes' C cases showed IL-15 expression in cancer cells, whereas only 16% of Dukes' B cases did (p < 0.0001). IL-15 may play important roles in cell proliferation, invasion, and metastasis of human colorectal cancer.

Increased expression of cyclooxygenase-2 protein in 4-nitroquinoline-1-oxide-induced rat tongue carcinomas and chemopreventive efficacy of a specific inhibitor, nimesulide.

Expression of cyclooxygenase (COX)-2 protein in 4-nitroquinoline-1-oxide (4-NQO)-induced rat tongue lesions and the postinitiation chemopreventive potential of a selective COX-2 inhibitor, nimesulide (NIM), were examined in Fischer 344 male rats. NIM was administered in the diet at doses of 150, 300, and 600 ppm for 14 weeks after treatment with 25-35 ppm 4-NQO in the drinking water for 12 weeks. Western blot analysis revealed COX-2 protein to be barely expressed in the normal tongue epithelia, whereas it was increased approximately 6-fold in squamous cell carcinomas (SCCs). Immunohistochemically, COX-2 protein was diffusely present in SCCs and dysplasia but expressed only in basal cells in hyperplasia and papillomas. In basal cells of normal epithelia, it was also occasionally weakly stained. NIM dose-dependently decreased at doses of 150 and 300 ppm, the incidences of SCCs to 4 of 12 (33.3%) and 1 of 13 (7.7%) and their multiplicity to 0.33+/-0.49 and 0.08+/-0.28 per rat, respectively, as compared with 4-NQO alone group values of 9 of 11 (81.8%) and 1.00+/-0.77. A lesser decrease was observed with 600 ppm, the values being 5 of 12 (41.7%) and 0.50+/-0.67. NIM did not significantly affect the development of hyperplasias, dysplasias, and papillomas. These results clearly indicate chemopreventive potential of a selective COX-2 inhibitor against the postinitiation development of SCCs in rat tongue carcinogenesis.

Enhancement of hepatocarcinogenesis initiated with diethylnitrosamine or N-nitrosobis(2-hydroxypropyl)amine by a choline-deficient, L-amino acid-defined diet administered prior to the carcinogen exposure in rats.

Effects of pre-administration of a choline-deficient, L-amino acid-defined (CDAA) diet on hepatocarcinogenesis initiated with diethylnitrosamine (DEN) or N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats were investigated. A pre-administrating period was set as 1 week, because CDAA diet induces liver injuries by this time-point. In a time-course study, male Fischer 344 rats, 6 weeks old, received a 1-week pre-administration of choline-supplemented, L-amino acid-defined (CSAA) or CDAA diet, DEN at a dose of 100 mg/kg body weight by a single intraperitoneal injection, then CSAA or CDAA diet for up to 8 weeks, and were sacrificed 4, 6 and 8 weeks after DEN. CDAA diet administered only after DEN significantly increased the numbers of glutathione S-transferase placental form (GST-P)-positive lesions 4, 6 and 8 weeks after DEN and their sizes 6 and 8 weeks after DEN. CDAA diet administered both before and after DEN similarly increased the numbers and sizes of GST-P-positive lesions, but with a significantly greater degree than obtained by the diet administered only after DEN. In a dose response study, rats received vechicle or DEN, at a dose of 0.001, 0.01, 0.1, 1, 10, 20, 50, 100 or 200 mg/kg body weight, 1 week after the commencement of CSAA or CDAA diet, and sacrificed 8 weeks after vehicle or DEN. The significant increases of the numbers of GST-P-positive lesions were obtained after 50-200 mg/kg body weight of DEN under the CSAA diet administration, whereas those were detected after 10-200 mg/kg under CDAA diet administration. Sizes became significantly larger with only 200 mg/kg body weight of DEN in the CSAA case but with 50-200 mg/kg in the CDAA case. Male Wistar rats received a 1-week pre-administration of CSAA or CDAA diet, vehicle or BHP, at a dose of 600 or 1200 mg/kg body weight, by a single intraperitoneal injection, then CSAA or CDAA diet for 8 weeks, and were then sacrificed. The numbers of GST-P-positive lesions demonstrated significant increment with 1200 mg/kg body weight of BHP by CDAA diet administered only after BHP and, to a significantly greater degree, by the diet administered both before and after BHP. While CDAA diet administered only after BHP did not alter the sizes of GST-P-positive lesions, the diet administered both before and after 600 and 1200 mg/kg body weight of BHP significantly increased the sizes of the lesions. These results indicate that the pre- plus post-administration of CDAA diet enhances hepatocarcinogenesis initiated with DEN or BHP, more than the post-administration only, thus providing a sensitive model to detect weak liver carcinogenic potency of environmental chemicals.

Genetic resistance to chemical carcinogen-induced preneoplastic hepatic lesions in DRH strain rats.

DRH strain rats were established by inbreeding a closed colony of Donryu rats continuously fed the chemical hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene for over 10 years. They are highly resistant to chemical induction of liver cancer and preneoplastic lesions. We studied the genetic basis of DRH resistance to preneoplastic lesions by analyzing 108 (F344 x DRH)F2 male rats fed 3'-methyl-4-dimethylaminoazobenzene for 7 weeks. Five parameters of preneoplastic liver lesions were selected for quantitative analysis: (a) number of glutathione S-transferase placental form-positive foci per unit area of liver section; (b) percentage area occupied by the foci; (c) average size of foci; (d) glutathione S-transferase placental form mRNA level; and (e) gamma-glutamyltranspeptidase mRNA level. Furthermore, O6-methylguanine DNA methyltransferase and mannose 6-phosphatase/insulin-like growth factor 2 receptor mRNA levels were quantified. Composite interval mapping analysis showed that there were two remarkably significant clusters of quantitative trait loci affecting preneoplastic liver lesions on chromosomes 1 and 4. These clusters were designated collectively as Drh1 and Drh2, respectively. The functions of the recessive DRH allele of Drh1 and the semidominant DRH allele of Drh2 were to suppress the phenotypes of precancerous lesions. Each cluster showed two to three subpeaks in linkage likelihood plots, suggesting the presence of several closely linked quantitative trait loci affecting preneoplastic lesions. Possible candidate genes at each locus will be discussed. Expression of O6-methylguanine DNA methyltransferase and mannose 6-phosphatase/insulin-like growth factor 2 receptor did not affect DRH resistance to hepatocarcinogenesis, although they were polymorphic between DRH and F344 rats.

Increased expression of cyclooxygenase-2 protein in rat lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine.

Expression of cyclooxygenase (COX)-2 protein in preneoplastic and neoplastic lung lesions induced by the administration of 2000 ppm of N-nitrosobis(2-hydroxypropyl)amine (BHP) in the drinking water to Wistar male rats, was examined immunohistochemically. The majority of alveolar/bronchiolar adenomas (ADs) and all adenocarcinomas (ADCs) examined, stained positive or strongly positive for COX-2. In contrast, only a minority of alveolar/bronchiolar hyperplasias demonstrated immunoreactivity and half of the squamous cell carcinomas examined, were only weakly positive. Western blotting analysis also revealed expression of COX-2 protein in the resected ADs and ADCs. These results clearly indicate up-regulated expression of COX-2 in lung neoplastic lesions, particularly ADs and ADCs, induced by BHP in rats.

Increased expression of cyclooxygenase-2 protein in rat urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl) nitrosamine.

The anti-inflammatory drugs, aspirin and piroxicam, are known to possess chemopreventive potential against rat superficial urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Recently, we found similar inhibitory effects with a selective cyclooxygenase (COX)-2 inhibitor, nimesulide. In order to clarify the inhibitory mechanisms, we have further studied the expression of COX-2 protein in urinary bladder tumors induced by BBN in Fischer 344 male rats. For comparison, papillomatosis caused by uracil-induced urolithiasis, and normal epithelial cells, were also investigated. Western blot analysis revealed COX-2 protein to be barely expressed in the normal epithelial cells, whereas it was increased 13-22-fold in varying sizes of urinary bladder tumors and 7-fold in papillomatosis. Immunohistochemically, COX-2 protein was diffusely expressed in transitional cell carcinomas and nodulo-papillary hyperplasia but weakly expressed only in basal cells in simple hyperplasia and normal-looking surrounding epithelia. In papillomatosis, it was moderately expressed only in endothelial cells in stroma. These results indicate that COX-2 plays important roles in the development of preneoplastic and neoplastic lesions in the rat urinary bladder, and therefore could be a good target for chemoprevention of superficial lesions.

Genetic properties for the suppression of development of putative preneoplastic glutathione S-transferase placental form-positive foci in the liver of carcinogen-resistant DRH strain rats.

The post-initiation stage of hepatocarcinogenesis was investigated in carcinogen-resistant inbred DRH rats and the parental strain, carcinogen-sensitive Donryu rats. Male rats at 5 weeks of age from both strains were treated with N-nitrosodiethylamine (200 mg/kg i.p.) followed by feeding with a diet containing 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) from 2 weeks later and were then subjected to partial hepatectomy at 1 week later. At 8 weeks after the start of treatment, the mean area occupied by glutathione S-transferase placental form (GST-P)-positive lesions was about 30% in Donryu rats but less than 4% in DRH rats despite the presence of comparable numbers of foci in the livers of both strains. These observations suggested that clonal expansion of GST-P-positive foci in DRH rat liver was significantly suppressed under these conditions. Furthermore, this genetic property was dominantly inherited in the F1 rats by crosses of DRH and carcinogen-sensitive inbred F344 rats; that is, the induction of GST-P mRNA in the livers of F344 x DRH F1 rats was dominantly suppressed after administration of 3'-Me-DAB for 8 weeks as compared with parental F344 rats under the same conditions. We compared the intrinsic properties related to growth potential of liver cells between adult DRH and Donryu rats. DRH rat liver showed retarded and/or reduced DNA synthesis after partial hepatectomy or a single i.v. injection of lead nitrate and lower activity of telomerase induced by 3'-Me-DAB administration for 1 week, as compared with the Donryu rat liver. The intrinsic properties observed in this study may be related, at least in part, to the low incidence of liver tumors induced by hepatocarcinogens in DRH rats.

Prevention by 1'-acetoxychavicol acetate of the induction but not growth of putative preneoplastic, glutathione S-transferase placental form-positive, focal lesions in the livers of rats fed a choline-deficient, L-amino acid-defined diet.

The effects of 1'-acetoxychavicol acetate (ACA) on endogenous rat liver carcinogenesis because of chronic feeding of a choline-deficient, L-amino acid-defined (CDAA) diet were examined. Male Fischer 344 rats, 6 weeks old, received the CDAA diet containing ACA at doses of 0, 0.005, 0.010 and 0.050% for 12 weeks and were then killed. ACA decreased the numbers of putative preneoplastic, glutathione S-transferase placental form (GST-P)-positive, focal lesions developing in the livers of rats fed the CDAA diet but did not alter their sizes. At the same time, ACA reduced the levels of 8-hydroxyguanine, a parameter of oxidative DNA damage, but did not significantly affect generation of 2-thiobarbituric acid-reacting substances, indicators of oxidative extra-DNA damage, or hepatocyte proliferation. Furthermore, ACA did not exert any significant effects on the numbers or sizes of GST-P-positive lesions in the livers of rats when administered between weeks 2 and 8 after initiation with a single i.p. dose of 200 mg/kg body wt of N-nitrosodiethylamine. These results indicate that ACA prevents the CDAA diet-associated induction of putative preneoplastic lesions by reduction of oxidative DNA damage but does not affect their subsequent growth.

Inhibition by phenyl N-tert-butyl nitrone of early phase carcinogenesis in the livers of rats fed a choline-deficient, L-amino acid-defined diet.

Male Wistar rats were fed a choline-deficient, L-amino acid-defined (CDAA) diet alone or in combination with a nitrone-based free radical trapping agent, phenyl N-tert-butyl nitrone (PBN) in the drinking water at the concentrations of 0.013, 0.065, and 0.130% for 12 weeks. PBN inhibited the changes that are normally induced in the livers of rats by the CDAA diet feeding, i.e., development of putative preneoplastic lesions, proliferation of connective tissue, reduction of glutathione S-transferase activity, formation of 8-hydroxyguanine in DNA, and an increase in inducible cyclo-oxygenase (COX2) activity. PBN, however, did not prevent the increases in the COX2 mRNA or protein levels brought on by the CDAA diet These results indicate that the loss of glutathione S-transferase activity and COX2 induction may play significant roles in rat liver carcinogenesis by the CDAA diet and that PBN prevents neoplasia not only by its radical scavenging activity but also by inhibiting COX2 activity at the catalytic level.

Chemopreventive effects of nimesulide, a selective cyclooxygenase-2 inhibitor, on the development of rat urinary bladder carcinomas initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine.

The chemopreventive potential of a selective cyclooxygenase-2 inhibitor, nimesulide (NIM), against the development of rat superficial urinary bladder carcinomas after initiation with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was examined. Six-week-old Fischer 344 male rats were given 0.05% BBN in their drinking water for 8 weeks, followed by diets supplemented with 0, 100, 200, or 400 ppm NIM for 12 weeks, and they were then sacrificed. NIM decreased, in a dose-dependent manner, the incidence of transitional cell carcinoma (TCC) to 12 of 20 (60.0%), 8 of 16 (50.0%), and 5 of 19 (26.3%) and the multiplicity of TCCs to 0.75 +/- 0.79, 0.56 +/- 0.63, and 0.37 +/- 0.78 per rat at 100, 200, and 400 ppm, respectively, as compared with the BBN alone group values of 18 of 20 (90.0%) and 2.35 +/- 1.23. NIM did not significantly affect the cell differentiation or invasiveness of TCCs. These results indicate clear chemopreventive potential of a selective cyclooxygenase-2 inhibitor against postinitiation development of superficial rat urinary bladder carcinomas.

Prevention by inhibitors of arachidonic acid cascade of liver carcinogenesis, cirrhosis and oxidative DNA damage caused by a choline-deficient, L-amino acid-defined diet in rats.

Effects of inhibitors of arachidonic acid (AA) cascade on the development of fatty liver, cirrhosis, glutathione S-transferase placental form (GST-P)-positive preneoplastic nodules, neoplastic nodules and generation of 8-hydroxydeoxyguanosine (8-OHdG), caused by a choline-deficient, L-amino acid-defined (CDAA) diet, were examined in Fischer 344 male rats by feeding CDAA diet supplemented with the inhibitors for 12 and 30 weeks. None of the inhibitors affected fatty liver. Among cyclooxygenase (COX) inhibitors, an irreversibly acting acetylsalicylic acid and a long-acting piroxicam, and to a much lesser extent the short-acting ibuprofen but not indomethacin, inhibited the development of cirrhosis, GST-P-positive and neoplastic nodules and generation of 8-OHdG. A phospholipase A2 inhibitor p-bromophenacylbromide (BPB) also exerted similar but lesser extent of inhibitory effects. Lipoxygenase inhibitors quercetin and nordihydroguiaretic acid inhibited GST-P-positive nodules but not cirrhosis or 8-OHdG. Present results suggest that perturbed AA cascade, particularly augmented COX pathway, might play key roles in the causation of liver lesions in the CDAA diet model.

Inhibitory effect of tomato juice on rat urinary bladder carcinogenesis after N-butyl-N-(4-hydroxybutyl)nitrosamine initiation.

The effects of tomato juice on urinary bladder carcinogenesis were studied in male Fischer 344 rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in rats. The animals (6 weeks old) were given 0.05% BBN in their drinking water for 8 weeks, followed by diluted tomato juice for 12 weeks, and killed at 20 weeks after the beginning of the experiment. Lycopene concentrations in the livers of rats given tomato juice were elevated. Histopathological analysis of urinary bladder lesions revealed the numbers, but not incidences, of urinary bladder transitional cell carcinomas (TCCs) to be decreased in the group given tomato juice. No influence on the incidence of simple and nodullopapillary hyperplasias, invasion or differentiation of TCC was noted. These results indicate that tomato juice, presumably the contained lycopene and other anti-oxidants in combination, exerts an inhibitory effect on the development of TCCs in the rat urinary bladder.

Induction of telomerase activity during regeneration after partial hepatectomy in the rat.

The regulation of telomerase activity during regeneration induced by two-thirds partial hepatectomy (PH) was investigated in 6-week-old male F344 rats. Groups of animals were serially sacrificed 0, 6, 16, 24, 36 and 72 h after the operation and telomerase activity was determined by the telomeric repeat amplification protocol (TRAP) assay followed by densitometric quantification. DNA synthesis was immunohistochemically quantified in terms of bromodeoxyuridine (BrdU) incorporation. The expression levels of telomerase RNA were examined by Northern blot analysis. Telomerase activity was increased significantly from 6 to 36 h but had decreased to close to the normal levels after 72 h. DNA synthesis reached a maximum 24 h after PH. However, the expression levels of telomerase RNA did not change during regeneration. The results suggest that telomerase is actively regulated by unknown mechanisms throughout the cell cycle in regenerating rat hepatocytes.

Inhibition of early-phase exogenous and endogenous liver carcinogenesis in transgenic rats harboring a rat glutathione S-transferase placental form gene.

Hepatocarcinogenesis initiated with N-nitrosodiethylamine (DEN) and that initiated by feeding of a choline-deficient, L-amino acid-defined (CDAA) diet were compared in transgenic male Wistar rats harboring a rat glutathione S-transferase placental form (GST-P) gene (GST-P-Tg rats) and non-transgenic (N-Tg) rats. Eight-week-old GST-P-Tg and N-Tg rats were administered DEN intraperitoneally at 100 mg/kg body weight, subjected to a selection procedure with 2-acetylaminofluorene and CCl4, and killed at the end of weeks 5 and 12. Other groups were fed the CDAA diet for 12 weeks and killed. Five weeks after the DEN treatment, numbers and sizes of gamma-glutamyltransferase (GGT)- or GST-P-positive lesions and 8-hydroxyguanine (8-OHG) levels in the livers were significantly less in GST-P-Tg rats than in N-Tg rats. The lesion numbers were unchanged between the ends of weeks 5 and 12 in GST-P-Tg rats, but decreased in N-Tg rats. The lesion sizes were increased in GST-P-Tg rats, but unchanged in N-Tg rats. While the proliferating cell nuclear antigen labeling indices (PCNA L.I.) in and surrounding the lesions were decreased, more prominently in GST-P-Tg rats than in N-Tg rats, the 8-OHG levels were also decreased but similarly in both cases. After 12 weeks on the CDAA diet, the lesion incidences, numbers and sizes, 8-OHG levels, PCNA L.I. in and surrounding the lesions, and liver injury were significantly less in GST-P-Tg rats than in N-Tg rats. These results indicate that insertion of a rat GST-P transgene alters the early phase of exogenous and endogenous rat hepatocarcinogenesis, presumably due to enhanced detoxification by GST-P expressed both transiently during the initiation and chronically in the altered hepatocyte populations.

Inhibition by N-(4-hydroxyphenyl)retinamide and all-trans-retinoic acid of exogenous and endogenous development of putative preneoplastic, glutathione S-transferase placental form-positive lesions in the livers of rats.

The effects of N-(4-hydroxyphenyl)retinamide (4-HPR) and all-trans-retinoic acid (tRA) on the exogenous and endogenous models of rat liver carcinogenesis respectively using diethylnitrosamine (DEN) and a choline-deficient, L-amino acid-defined (CDAA) diet were studied. For the exogenous study, male Fischer 344 rats, 6 weeks old, were given a single i.p. dose of 200 mg/kg body wt of DEN, partially hepatectomized at week 3, administered 4-HPR at doses of 0, 0.04, 0.08 and 0.16% or tRA at 0, 0.004, 0.008 and 0.015% in diet from week 2 for 6 weeks, and killed at the end of week 8. For the endogenous study, rats were fed the CDAA diet containing 4-HPR or tRA for 12 weeks and killed at the end of week 12. 4-HPR decreased the numbers and sizes of the glutathione S-transferase placental form-positive foci, assayed as putative preneoplastic lesions, the levels of 8-hydroxyguanine (8-OHG), a parameter of oxidative DNA damage, and the bromodeoxyuridine labeling indices (BrdU L.I.) by all three doses in the DEN-initiated case and, more prominently, in the CDAA diet-associated case. In contrast, while tRA failed to exert inhibitory effects apparently on foci development, 8-OHG formation or BrdU labeling in the DEN-initiated case, it reduced the numbers and sizes of the foci, the 8-OHG levels and the BrdU L.I. by all three doses in the CDAA diet-associated case. Furthermore, both 4-HPR and tRA inhibited the CDAA diet-associated induction of hepatocyte necrosis and connective tissue increase but not intrahepatocellular fat accumulation. These results indicate that 4-HPR exerts chemopreventive effects against the exogenous and endogenous rat liver carcinogenesis, while tRA can inhibit only the latter.

Inhibition by piroxicam of oxidative DNA damage, liver cirrhosis and development of enzyme-altered nodules caused by a choline-deficient, L-amino acid-defined diet 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.

Chemopreventive efficacy of piroxicam administered alone or in combination with lycopene and beta-carotene on the development of rat urinary bladder carcinoma after N-butyl-N-(4-hydroxybutyl)nitrosamine treatment.

The effects of the non-steroidal anti-inflammatory drug (NSAID) piroxicam and the carotenoids lycopene and beta-carotene, alone or in combination, on the development of rat superficial urinary bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were studied. Male Fischer 344 rats, 6 weeks old, were given 0.05% BBN in the drinking water for 8 weeks followed by administration of piroxicam (0.0075% in the diet), lycopene (0.0025% in the drinking water) and/or beta-carotene (0.0025% in the drinking water) for 12 weeks, then killed for histological analysis of urinary bladder lesions. Cell proliferation potential was analyzed by immunohistochemical staining of the proliferative cell nuclear antigen (PCNA). Piroxicam alone, piroxicam+lycopene, and piroxicam +lycopene+ beta-carotene all significantly decreased the incidences and numbers of transitional cell carcinomas (TCCs), but the combination of piroxicam with carotenoids did not result in a clear improvement in the preventive potential of piroxicam. Piroxicam+ beta-carotene also caused a significant reduction and lycopene alone a slight but not significant reduction in the number of TCCs. In contrast, beta-carotene alone and lycopene+ beta-carotene were without inhibitory influence on any of the lesion categories examined, and the latter significantly increased the proportion of high-grade TCCs. Nevertheless, all of the chemopreventive agents, either alone or in combination, significantly decreased the TCC PCNA index, the effect extending to the surrounding epithelium in the piroxicam+lycopene and piroxicam+lycopene+beta-carotene groups. These results indicate that the NSAID piroxicam may be a more effective chemopreventive agent than lycopene and beta-carotene for superficial urinary bladder carcinogenesis.

Inhibition by green tea extract of diethylnitrosamine-initiated but not choline-deficient, L-amino acid-defined diet-associated development of putative preneoplastic, glutathione S-transferase placental form-positive lesions in rat liver.

The effects of green tea extract (GTE) on exogenous and endogenous models of rat liver carcinogenesis using diethylnitrosamine (DEN) and a choline-deficient, L-amino acid-defined (CDAA) diet were studied. For the exogenous carcinogenesis study, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal dose of 200 mg/kg body weight of DEN, partially hepatectomized at week 3, and administered GTE at doses of 0, 0.01 and 0.1% in the drinking water from week 2 for 10 weeks. For the endogenous carcinogenesis study, rats were fed the CDAA diet and simultaneously given GTE for 12 weeks. All rats were killed at the end of week 12. After DEN-initiation, the apparent numbers of glutathione S-transferase placental form-positive foci, assayed as putative preneoplastic lesions, were decreased by the administration of GTE, though their sizes were not altered. In contrast, GTE did not significantly reduce the numbers of the lesions induced by the CDAA diet or affect their sizes. While the levels of 8-hydroxyguanine, a parameter of oxidative DNA damage, were reduced by the GTE administration in both experimental models, GTE did not protect against the CDAA-diet-associated liver tissue damage in terms of either histology or plasma marker enzyme levels. We conclude that, while GTE may be a possible chemopreventive agent for nitrosamine-initiated hepatocarcinogenesis in the absence of chronic hepatocyte damage, it does not significantly inhibit lesion development in hepatocarcinogenesis associated with the CDAA diet, a cirrhosis-associated model.