Akihiro Koide

Modification by curcumin of mutagenic activation of carcinogenic N-nitrosamines by extrahepatic cytochromes P-450 2B1 and 2E1 in rats

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)

Enhancement by Cigarette Smoke Exposure of 2-Amino-3,8-dimethylimidazo(4,5- f )quinoxaline-induced Rat Hepatocarcinogenesis in Close Association with Elevation of Hepatic CYP1A2

The modifying effects of cigarette smoke (CS) exposure on a heterocyclic amine (HCA) 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx)‐induced carcinogenesis were investigated in male F344 rats. Groups 1 and 2 were fed MeIQx at a dose of 300 ppm, and simultaneously received CS and sham smoke (SS) for 16 weeks, respectively. Groups 3–5 were given the MeIQx diet for 4 weeks, and simultaneously exposed to CS for 4 weeks (group 3), exposed to CS for 12 weeks after the MeIQx treatment (group 4) or received SS for 16 weeks (group 5). Groups 6 and 7 were fed basal diet and respectively received CS and SS for 16 weeks. In terms of the mean number or area, the development of glutathione S‐transferase placental form‐positive (GST‐P+) liver cell foci was significantly (P<0.01) greater in group 1 than in group 2. The mean number of colonic aberrant crypt foci (ACFs) per animal was increased by continuous CS exposure regardless of MeIQx feeding, the differences between groups 4 and 5 (P<0.05), and between groups 6 and 7 (P<0.05) being significant. Immunoblot analysis confirmed that the hepatic CYP1A2 level in group 6 was remarkably increased as compared to that in group 7. In addition, liver S9 from rats in group 6 consistently increased the mutagenic activities of six HCAs including MeIQx as compared to those in group 7. Thus, our results clearly indicate that CS enhances hepatocarcinogenesis when given in the initiation phase via increasing intensity of metabolic activation for MeIQx and possibly colon carcinogenesis when given in the post‐initiation phase in rats induced by MeIQx.

Effect of α-naphthyl isothiocyanate on 2-amino-3-methylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in rats

The modifying effects of α‐naphthyl isothiocyanate (ANIT) on 2‐amino‐3‐methylimidazo[4,5‐b]pyridine (PhIP)‐induced mammary carcinogenesis were investigated in female Sprague‐Dawley (SD) rats, and the hepatic activities of the phase II detoxifying enzymes glutathione S‐transferase (GST) and quinone reductase (QR) were also assayed. Ninety‐eight rats were divided into 4 groups. Starting at 6 weeks of age, rats were fed the high‐fat diet without ANIT (Groups 1 and 4) or the experimental diet (high‐fat diet mixed with 400 ppm ANIT, Groups 2 and 3). At 7 weeks of age, Groups 1 and 2 were given PhIP in corn oil (85 mg/kg body weight, 8 times for 11 days) by intragastric intubation. One week after the last PhIP injection, 5 rats in each group were sacrificed to assay GST and QR activities, and the experimental diets for Groups 2 and 3 were switched to the high‐fat diet without ANIT until termination of the experiment. Group 4 served as the vehicle control. All rats were sacrificed at 24 weeks after the start of the experiment. At termination of the experiment, mammary tumours were detected in Groups 1 (PhIP alone) and 2 (PhIP + ANIT) and were shown histologically to be adenocarcinomas; their incidences (multiplicities) were 56.3% (1.66 ± 2.31/rat) in Group 1 and 6.7% (0.07 ± 0.25/rat) in Group 2 (p < 0.001). Mean sizes of the tumours were 10.6 ± 5.3 mm in Group 1 and 6.5 mm in Group 2. No mammary tumours were observed in rats of Groups 3 and 4. In addition, ANIT treatment significantly increased the activities of GST and QR in the livers of rats in Groups 2 and 3 as compared to Groups 1 and 4. These results imply that the isothiocyanate compound ANIT shows potent inhibitory effects on mammary carcinogenesis induced by PhIP in female SD rats when administered during the initiation stage.

Differences in susceptibility to N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine‐induced urinary bladder carcinogenesis between SD/gShi rats with spontaneous hypospermatogenesis and SD/cShi rats with spontaneous hydronephrosis

Differences in susceptibility to N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN)‐induced urinary bladder carcinogenesis between two substrains of male Sprague–Dawley rats were examined. One substrain was SD/gShi, which has spontaneous hypospermatogenesis, and the other was SD/cShi, which is a sister strain of SD/gShi, and has normal testis but spontaneous hydronephrosis. SD/gShi rats had a lower incidence of urinary bladder tumors and had lower 5‐bromo‐2′‐deoxyuridine labeling indices in the urinary bladder epithelium than SD/cShi rats when BBN was given. SD/gShi rats had significantly lower urinary concentrations of N‐butyl‐N‐(3‐carboxypropyl)nitrosamine (BCPN), which is a metabolite and proximate carcinogen of BBN. In vitro analysis also showed significantly less BCPN formation, using an S9 mix derived from the liver and kidney, in SD/gShi rats than in SD/cShi rats. BCPN formation in vitro was markedly inhibited by non‐selective cytochrome P450 (CYP) inhibitors, but not alcohol dehydrogenase inhibitor. However, analysis of CYP proteins including hepatic CYP1A1/2, 2B1/2, 2E1, and 3A2 and renal CYP2E1 and 3A2 revealed no significant variation in levels in either tissue in the groups. There were also no significant intergroup differences in the mutagenicity of carcinogens, including heterocyclic amines and N‐nitrosamines, activated by CYP1A1/2 and CYP2E1 and/or CYP2B1/2, respectively. These results suggest that SD/gShi rats are less susceptible to BBN, possibly because less BCPN is produced by CYP isoforms other than those investigated. A contribution of CYP4B1 to the strain difference is also possible. (Cancer Sci 2005; 96: 637 – 644)

Effect of cigarette smoke on mutagenic activation of environmental carcinogens by cytochrome P450 2A8 and inactivation by glucuronidation in hamster liver

To elucidate the mechanism underlying suppression of N-nitrosobis(2-oxopropyl)amine (BOP)-induced hamster pancreatic carcinogenesis by cigarette smoke (CS), hepatic levels of microsomal cytochrome P450 (CYP) enzymes, mutagenic activation of environmental carcinogens and three types of uridine diphosphate-glucuronyltransferase (UDPGT) and sulphotransferase (ST) activities were assayed in male Syrian golden hamsters and F344 rats exposed to CS. Immunoblot analyses of microsomal CYP proteins revealed induction of constitutive CYP1A2 (2.6-fold increase) and 2A8 (4.0-fold increase) and induction of CYP1A1 and constitutive CYP1A2 (3.9-fold increase) in rats following exposure to CS for 4 weeks using a Hamburg type II smoking machine. CS exposure enhanced mutagenicities of four heterocyclic amines in the presence of liver S9 in both species, whereas the mutagenicities of aflatoxin B(1) (AFB(1)), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were significantly increased by CS in hamsters but not in rats. However, no CS-induced alterations in the mutagenic activities of other carcinogens, including BOP and other pancreatic carcinogens, were observed in either species. Application of several CYP inhibitors revealed that the mutagenic activities of MeAαC, AFB(1) and NNK in the hamster liver S9 were partly associated with CYP2A8, whereas those of the three pancreatic carcinogens were selectively associated with CYP2B. CS enhanced UDPGT activities towards 4-nitrophenol (4-NP) (1.9- to 2.0-fold) but did not affect those of bilirubin, testosterone UDPGTs and three STs in both species. Together with the previous findings that BOP does not induce tumourigenesis in rats and that the glucuronidation of β-oxypropylnitrosamines is higher in rats than in hamsters, suppression of BOP-induced pancreatic carcinogenesis by CS might be attributed to increased detoxification by 4-NP UDPGT and not decreased CYP2B activation. This is the first demonstration of the induction of CYP2A protein by CS; CYP2A protein polymorphisms have been associated with oral and pulmonary carcinogenesis in smokers.

The enhancing effect of ethanol on the mutagenic activation of N-nitrosomethylbenzylamine by cytochrome P450 2A in the rat oesophagus

Alcohol consumption is frequently associated with various cancers and the enhancement of the metabolic activation of carcinogens has been proposed as a mechanism underlying this relationship. The ethanol-induced enhancement of N-nitrosodiethylamine (DEN)-mediated carcinogenesis can be attributed to an increase in hepatic activity. However, the mechanism of elevation of N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis remains unclear. To elucidate the mechanism underlying the role of ethanol in the enhancement of NMBA-induced oesophageal carcinogenesis, we evaluated the hepatic and extrahepatic levels of the cytochrome P450 (CYP) and mutagenic activation of environmental carcinogens by immunoblot analyses and Ames preincubation test, respectively, in F344 rats treated with ethanol. Five weeks of treatment with 10% ethanol added to the drinking water or two intragastric treatments with 50% ethanol, both resulted in elevated levels of CYP2E1 (1.5- to 2.3-fold) and mutagenic activities of DEN, N-nitrosodimethylamine and N-nitrosopyrrolidine in the presence of rat liver S9 (1.5- to 2.4-fold). This was not the case with CYP1A1/2, CYP2A1/2, CYP2B1/2 or CYP3A2, nor with the activities of 2-amino-3-methylimidazo[4,5-f]quinoline, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, aflatoxin B(1) or other N-nitroso compounds (NOCs), including NMBA. Ethanol-induced elevations of CYP2A and CYP2E1 were observed in the oesophagus (up to 1.7- and 2.3-fold) and kidney (up to 1.5- and 1.8-fold), but not in the lung or colon. In oesophagus and kidney, the mutagenic activities of NMBA and four NOCs were markedly increased (1.3- to 2.4-fold) in treated rats. The application of several CYP inhibitors revealed that CYP2A were likely to contribute to the enhancing effect of ethanol on NMBA activation in the rat oesophagus and kidney, but that CYP2E1 failed to do so. These results showed that the enhancing effect of ethanol on NMBA-induced oesophageal carcinogenesis could be attributed to an increase in the metabolic activation of NMBA by oesophageal CYP2A during the initiation phase, and that this occurred independently of CYP2E1.