Point mutations of the Ki-ras and p53 genes in rat lung lesions induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) were investigated by polymerase chain reaction-single strand conformation polymorphism analysis followed by direct sequencing using paraffin-embedded tissues. Male Wistar rats 6 wk old were given 2000 ppm BHP in drinking water for 15 wk. Another group was given drinking water without BHP. The rats were killed 20-27 wk after the beginning of the experiment. Lung adenomatous and squamous lesions, including carcinomas, were induced. The frequencies of Ki-ras mutations were 40% (six of 15) in alveolar hyperplasias, 36% (five of 14) in adenomas, 72% (18 of 25) in adenocarcinomas, 20% (three of 15) in squamous metaplasias, 50% (three of six) in squamous cell carcinomas, and 50% (five of 10) in adenosquamous carcinomas. The mutations were all G-->A transitions at the second position of codon 12; no other mutations were detected. However, Ha-ras mutations in exons 1 and 2 and p53 mutations in exons 5, 6, and 7 were not detected in adenocarcinomas and squamous cell carcinomas. These results indicate that Ki-ras mutation is an early genetic event in some adenomatous and squamous lung carcinogeneses and that Ki-ras mutations can cause benign lesions to convert to malignant lesions. The results also show that Ha-ras and p53 mutations are not involved in rat lung carcinogenesis induced by BHP.

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The effects of methionine on hepatocarcinogenesis induced by Coadministration of a choline‐deflcient L‐amino acid‐defined (CDAA) diet and ethionine were examined. F344 male rats were divided into 4 experimental groups. Groups 1 and 2 received the CDAA diet and a choline‐supplemented L‐amino acid‐defined (CSAA) diet, respectively. Group 3 received the CDAA diet containing 0.05% ethionine, and group 4 the CDAA diet containing 0.05% ethionine and 0.47% methionine. Animals were killed after 12 weeks of treatment. Histologically, the CDAA diet induced intracellular fat accumulation and foci. In contrast, ethionine caused not only foci, but also hyperplastic nodules, cholangiofibrosis and the proliferation of oval cells without such fat accumulation. Methionine abolished the development of all of the liver lesions induced by Coadministration of the CDAA diet and ethionine. To investigate the effects of methionine on induction of c‐myc and c‐Ha‐ras expression, as well as generation of 8‐hydroxyguanine (8‐OHGua) and 2‐thiobarbituric acid‐reacting substances (TBARS), by Coadministration of the CDAA diet and ethionine, subgroups of 3 to 5 animals were killed at 2, 4, 8 or 11 days after the beginning of the experiment. Coadministration of the CDAA diet and ethionine markedly enhanced the level of expression of c‐myc and c‐Ha‐ras, 8‐OHGua formation and TBARS generation as compared with the CDAA or CSAA diet within 11 days, and methionine blocked these actions. These results indicate that addition of methionine prevents the induction of c‐myc and c‐Ha‐ras expression, 8‐OHGua formation and TBARS generation, as well as hepatocellular lesions, by Coadministration of the CDAA diet and ethionine in rats, and suggest a possible involvement of oxidative stress and gene expression in hepatocarcinogenesis by these agents.

The effect of cell cycle disturbance due to colchicine on the induction of enzyme‐altered foci during liver regeneration in rats was studied. For initiation, diethylnitrosamine (DEN) at a dose of 10 mg/ kg was injected intraperitoneally and partial hepatectomy (PH) was performed 4 h thereafter. Colchicine at doses of 0, 0.1, 0.25 and 0.5 mg/kg was injected intraperitoneally 1 and 3 days after the initiation, followed by application of selection pressure consisting of 2‐acetylaminofluorene (AAF) and carbon tetrachloride (CCl4) administration. As end point lesions, γ–glutamyltransferase (GGT)‐positive enzyme‐altered foci were assayed at week 5. There was no significant effect of colchicine on numbers of foci. However, a significant, dose‐dependent increase in the area of GGT‐positive lesions in the groups treated with colchicine was observed. Bromodeoxyuridine labeling indices were higher in foci induced in colchicine‐treated rats than in the untreated rats. In a separate experiment, serum glutamic pyruvic transaminase was not increased significantly after DEN and colchicine treatment, and the mitotic index at 6 days after PH was increased in the liver of colchicine‐treated rats. These results suggest that the cell cycle disturbance induced by colchicine causes more pronounced selective growth of cells initiated by DEN and colchicine, and this experimental model may be useful for analyzing the mechanisms underlying that growth advantage and the effects of cell cycle abnormalities in liver 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.