No abstract

In order to elucidate the mechanism underlying enhancement by ethanol of N-nitrosodiethylamine (DEN)- and N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in rats, hepatic levels of cytochrome P-450 (CYP) enzymes, mutagenic activation of several N-nitrosamines and three kinds of UDP-glucuronyltransferase (UDPGT) activities were assayed in F344 rats. Immunoblot analyses of microsomal CYP proteins revealed induction of CYP2E1 (approximately 2-fold), but not CYP2B1/2, 1A1/2 or 3A2, by treatment with 10% ethanol in the drinking water for 2 weeks. In contrast, s.c. treatment with 0.5 mg/kg NMBA three times per week for 2 weeks produced no significant alterations in the levels of these CYP species. Ethanol treatment also elevated the mutagenic activities of N-nitrosodimethylamine (DMN), DEN and N-nitrosopyrrolidine (NPYR) in strain TA100 up to 2.1-, 1.6- and 2.3-fold above each control, respectively. However, this was not the cases for four N-nitrosamines, including NMBA, in strain TA100 and two heterocyclic amines and aflatoxin B(1) in strain TA98. In addition, ethanol did not affect UDPGT activities towards 4-nitrophenol, bilirubin and testosterone. Hepatic CYP species responsible for mutagenic activation of selected N-nitrosodialkylamines were confirmed by use of specific CYP inducers and inhibitors with the liver from F344 and Wistar rats, indicating that DMN, DEN and NMBA are selectively activated by CYP2E1, predominantly by CYP2E1 with a slight contribution by CYP2B2 and selectively by CYP2B1/2, respectively. These results demonstrate that ethanol exerts an enhancing effect on mutagenic activation by CYP2E1 of DMN, DEN and NPYR, but does not affect that of NMBA and the other carcinogens by CYP2B1/2, 1A1/2 and 3A2 and UDPGT1A1, 1A6 and 2B1 activities. Consequently, this suggests that enhancement by ethanol of DEN-induced esophageal carcinogenesis in F344 rats can be attributed to an increase in hepatic activation during the initiation phase, but that of NMBA-induced tumorigenesis is not attributable to metabolic activation and inactivation via glucuronidation in liver.

To elucidate the mechanism underlying suppression by curcumin of esophageal carcinogenesis induced by NMBA, we evaluated the CYP level and mutagenic activation of environmental carcinogens, by immunoblot analyses and Ames preincubation test, respectively, and bilirubin, 4-nitrophenol and testosterone UDPGT activities in F344 rats treated with curcumin and/or NMBA. No significant alterations in the hepatic levels of constitutive CYP proteins, mutagenic activation by liver S9 or hepatic UDPGT activities were produced by subcutaneous treatment with 0.5 mg/kg NMBA for 5 weeks and/or feeding of 0.05% and 0.2% curcumin for 6 weeks. In contrast, gavage of 0.2% curcumin decreased esophageal CYP2B1 and 2E1 by up to 60%, compared with vehicle control. Similarly, intragastric treatment with 270 mg/kg curcumin decreased esophageal and gastric CYP2B1 and CYP2E1, but not in lung, kidney or intestine. Conversely, large intestinal CYP2B1 was 2.8-fold higher in the treated rats than in control rats. Mutagenic activities of NOC, including NMBA, in the presence of esophagus and stomach S9 were markedly decreased in the treated rats, whereas those in the presence of large intestine S9 were 2.2-3.0-fold above control. These results show that modifying effects of curcumin on esophageal carcinogenesis can be attributed to a decrease in metabolic activation of NMBA by esophageal CYP2B1 during the initiation phase, without the contribution of metabolic activation and inactivation by liver. Further, the present findings suggest the potential of curcumin for modification of gastric and intestinal carcinogenesis initiated with NOC. (Cancer Sci 2006; 97: 896-904) H uman esophageal cancer has been closely associated with exposure to nitrate and nitrite, which are precursors of NOC, and exposure to NMBA is also associated with an increased risk of human cancer in the esophagus in China. (1)(2)(3)(4) In fact, NMBA is known to be the most potent carcinogen for rat esophagus, irrespective of its mode of administration. (5,6 ) It has been shown that esophageal mucosa microsomes from male Sprague-Dawley rats can form benzaldehyde and formaldehyde from NMBA at rates 1/5 and 1/60 of hepatic levels of both metabolites, respectively, and the metabolisms are inhibited more than 95% by CO and 70% by SKF525A, typical CYP inhibitors.(7) O 6 -methylated guanine level in rats given NMBA is six times higher in esophagus DNA than in liver DNA, (8) and isothiocyanates markedly decrease the incidence and multiplicity of NMBA-induced esophageal tumors, with inhibition of esophageal DNA methylation in F344 rats.(9) Further, it has been shown that ethanol has an enhancing effect on DEN-induced esophageal tumorigenesis in F344 rats, (10) with enhancement of metabolic activation of DEN by hepatic CYP2E1.(11) These findings indicate the importance of metabolic activation of NMBA by the target organ and/or liver during the initiation phase, and some NOC are known to be metabolized in the esophagus at a relatively high rate, often leading to high levels of esophageal D...

We have previously shown that p53(+/-) knockout mice are highly sensitive to urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in spite of a lack of effects of p53 heterozygosity on N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN) excretion in urine. To determine the influence of p53 deficiency on in vitro formation of BCPN, mutagenicity of BBN and BCPN and levels of several cytochrome P450 (CYP) isozymes, groups of five p53(+/-) knockout and wild-type mice (littermates), as well as animals of the C57BL/6 parental strain, were administered 0.025% BBN in their drinking water for 4 weeks. The livers and kidneys were then used for analyses of BBN metabolism, western immunoblotting and Ames liquid incubation. BBN treatment caused a slight decrease in BCPN formation in the livers of C57BL/6 mice, but there was no significant difference between p53 knockout, wild-type and C57BL/6 mice. In kidney BCPN formation in p53 knockout mice was 33-46% less than that in their wild-type counterparts. Using anti-rat CYP antibodies, CYP1A2, 2B9/10, 2E1 and 3A11/13 were constitutively detected in liver microsomes and CYP2E1 and 3A11/13 in the kidney. Densitometric determination of these CYP proteins revealed no significant variation in levels detected in both tissues among the four groups of mice. BBN and BCPN were not mutagenic for Salmonella typhimurium TA100 in either the absence or presence of liver S9 from untreated mice and rats and from p53 knockout mice treated with BBN. In conclusion, p53 deficiency and BBN had no enhancing effects on metabolism of BBN to BCPN and expression of the CYP isozymes typically responsible for activation of environmental carcinogens, including both of the N-nitrosamines tested, and their mutagenicity, indicating that the high susceptibility of p53(+/-) knockout mice is not attributable to metabolic activation in liver and kidney by CYP isozymes or urinary excretion of BCPN.

In order to elucidate the mechanism underlying enhancement by cigarette smoke (CS) of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced rat hepatocarcinogenesis, hepatic levels of cytochrome P-450 (CYP) enzymes, mutagenic activation of various carcinogens and UDP-glucuronyltransferase (UDPGT) activities were assayed in male F344 rats. Immunoblot analyses for microsomal CYP proteins revealed induction of CYP1A1 and constitutive CYP1A2 (2.3- to 2.7-fold), but not CYP2B1/2, 2E1 or 3A2, by CS exposure for 1, 12 or 16 weeks using a Hamburg type II smoking machine; the enhancement of CYP1A2 was 4.7-5.7 times that of CYP1A1. CS exposure also elevated the mutagenic activities of MeIQx and five other heterocyclic amines (HCAs) 1.4- to 3.7-fold, but not those of benzo[a]pyrene (BP) and aflatoxin B(1) in strain TA98 and N-nitrosodimethylamine, N-nitrosopyrrolidine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in strain TA100. In contrast, feeding 300 p.p.m. MeIQx in the diet for 1 or 16 weeks produced no significant alterations in the levels of these CYP species and mutagenic activities. However, i.g. administration of 50 or 100 mg/kg MeIQx in a single dose selectively increased CYP1A1 and 1A2 (2.6-fold) levels and mutagenic activities of five HCAs (1.7- to 3.3-fold), but not BP. On the other hand, feeding of MeIQx for 16 weeks enhanced UDPGT activities towards 4-nitrophenol and testosterone (2.9- and 1.5-fold, respectively), but not bilirubin, while CS exposure induced that towards 4-nitrophenol (1.6-fold); combined treatment with CS and MeIQx showed a summation effect on induction of UDPGT1A6 activity (3.5-fold). Consequently, these results demonstrate that CS and MeIQx have a bifunctional action, with similar induction patterns of specific CYP proteins, mutagenic activity and UDPGT activity. In conjunction with the finding of N-hydroxy-MeIQx being a poor substrate for rat liver UDPGT, our results clearly indicate that enhancement by CS of MeIQx-induced hepatocarcinogenesis in F344 rats can be attributed to an increase in metabolic activation of MeIQx by hepatic CYP1A2 during the initiation phase.