Cloning of glycerol-3-phosphate dehydrogenase genes (ZrGPD1 and ZrGPD2) and glycerol dehydrogenase genes (ZrGCY1 and ZrGCY2) from the salt-tolerant yeast Zygosaccharomyces rouxii.

The ZrGPD1 and ZrGPD2 genes encoding putative glycerol-3-phosphate dehydrogenases were isolated from the salt-tolerant yeast, Zygosaccharomyces rouxii. Both genes are homologous to GPD1 of Saccharomyces cerevisiae and are constitutively expressed in Z. rouxii cells. Putative glycerol dehydrogenase genes, ZrGCY1 and ZrGCY2, which are highly homologous to GCY1 of S. cerevisiae, were also isolated. Since the level of transcripts of ZrGCY1 and ZrGCY2 increased in Z. rouxii cells subjected to salt stress, it is suggested that the pathway of the signal transduction of salt stress controls the expression of these genes. The Accession Nos of these sequences in GenBank are as follows: ZrGPD1, AB047394; ZrGPD2, AB047395; ZrGCY1, AB047396; ZrGCY2, AB047397.

Spontaneous passage of a colon cast in the absence of abdominal aneurysm.

The spontaneous passage of colon cast from a 76-year-old Japanese female patient is reported. Macroscopically, the colon case was shaped like the airbladder of a fish. Histopathologically, the cast consisted of degenerated colonal mucosa, including glands. No inflammatory reaction was apparent. The patient lacked any evidence of abdominal aneurysm. Since there have been only five reported cases of colon cast in the literature, and since in all of those association with abdominal aneurysms was always described, the present study represents the first report demonstrating the formation of a colon cast in the absence of associated abdominal aneurysm. However, the patient was found to exhibit several risk factors for ischemic colitis, such as arteriosclerosis on the wall of the abdominal aorta, chronic constipation, and colonic stenosis. Her colonal mucosal surface, indeed, suggested ischemic colitis. This case report, therefore, indicates that ischemic colitis, due to various causes, may be responsible for the formation of colon casts, and that the presence of an abdominal aneurysm is not necessarily a prerequisite for colon cast formation. This report may contribute to a better understanding of the pathogenesis of colon casts.

Chronic toxicity carcinogenicity studies of triethanolamine in B6C3F1 mice.

The chronic toxicity and carcinogenic potential of triethanolamine was examined in B6C3F1 mice. Triethanolamine, dissolved in distilled water at levels of 0 (control), 1, and 2%, was given to groups of 50 males and 50 females ad libitum in drinking water for 82 weeks. Neoplasms developed in all groups, including the control group, but no dose-related increase of the incidence of any tumor was observed in treated groups of both sexes. There were no adverse effects as regards survival of the mice, organ weights, and specific incidence of neoplasms in the treated, compared to the control group. This chronic toxicity test provides no evidence of carcinogenic potential of triethanolamine in B6C3F1 mice.

Carcinogenic potency of N-nitrosomethyl(2-hydroxypropyl)amine and other metabolic relatives of N-nitrosobis(2-hydroxypropyl)amine by single intraperitoneal injection on the lung of rats.

The carcinogenic effects of a single intraperitoneal injection of N-nitrosobis(2-hydroxypropyl)amine (BHP) or its metabolic relatives, N-nitrosomethyl(2-hydroxypropyl)amine (MHP), N-nitrosobis(2-oxopropyl)amine (BOP), N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) and N-nitroso-2,6-dimethylmorpholine (NDMM), were studied in male Wistar rats. The main target organ of these nitrosamines proved to be the lung, followed by the thyroid. Lung lesions were induced in a dose-dependent manner with total lung tumor incidences reaching 55% to 100%. BHP, MHP, HPOP and NDMM all caused lung carcinomas to develop (22% to 44% incidence), whereas BOP was only associated with adenomas. On the basis of dose administered and incidence of carcinomas, MHP appeared to be the most potent lung carcinogen of the five nitrosamines investigated. Smaller numbers of neoplasms were also induced in the kidney, urinary bladder, esophagus and intestine at differing rates by these nitrosamines.

Comparative histochemical investigation of the glutathione S-transferase placental form and gamma-glutamyltranspeptidase during N-nitrosobis(2-hydroxypropyl)amine-induced lung carcinogenesis in rats.

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.

Effects of 3-aminobenzamide on the induction of gamma-glutamyl-transpeptidase-positive foci by various chemicals in rat liver.

The effects of 3-aminobenzamide (ABA), a representative inhibitor of poly(ADP-ribose)polymerase, on the induction of gamma-glutamyl-transpeptidase (GGT)-positive foci, an early lesion occurring during hepatocarcinogenesis, in rat liver by various chemicals were studied in Fischer 344 and Wistar rats. ABA exhibited an enhancing effect on the induction of GGT-positive foci by benzo[a]pyrene, but no effects on induction by a methylating agent, N-methyl-N-nitrosourea, in both rat strains. In contrast, the induction of GGT-positive foci by another methylating agent, 1,2-dimethylhydrazine, was enhanced by ABA in Wistar rats, but not affected in Fischer rats. There were no effects of ABA on the induction of GGT-positive foci by N-bis(2-hydroxypropyl) nitrosamine in Wistar rats. These results indicate a differential effect of ABA on the induction of GGT-positive foci by different chemicals in different rat strains.

Studies of carcinogenicity of sodium chlorite in B6C3F1 mice.

The carcinogenic activities of sodium chlorite in B6C3F1 mice were examined. Sodium chlorite was given at concentrations of 0 (control), 0.025% (low dose), or 0.05% (high dose) in the drinking water of 150 female and 150 male mice for 80 weeks, after which time the animals were returned to distilled water without sodium chlorite. All mice were sacrificed 85 weeks from the beginning of the experiment. The incidence of tumor-bearing animals was 32% (control), 34% (low dose), and 26% (high dose) in female mice, and 46% (control), 57% (low dose), and 53% (high dose) in male mice. The types and incidence of neoplasms that occurred frequently in each group of both sexes were similar to those observed spontaneously in B6C3F1 mice. The incidence of lymphomas/leukemias in the high dose group of females (2%), however, was lower than that in the control group (15%). Furthermore, the incidence of pulmonary adenomas in the high dose group of males (12%) was higher than that in the control group (0%), but neither dose-related increases in the adenoma incidences nor increased incidences of the adenocarcinomas were observed. These results indicated no clear evidence of a carcinogenic potential of sodium chlorite in B6C3F1 mice.

Effect of vitamin E on the induction and evolution of enzyme-altered foci in the liver of rats treated with diethylnitrosamine.

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.

Induction of gamma-glutamyltranspeptidase-positive foci in rat liver by deoxycholic acid.

Initiating activities of the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA), were examined in terms of the induction of gamma-glutamyltranspeptidase (GGT)-positive foci in rat liver. A rapid induction model of enzyme-altered foci was utilized. GGT-positive foci were clearly induced by DCA ranging from 0.05% to 0.5% in the diet. In contrast, the induction of GGT-positive foci by LCA was less pronounced and was not dose-dependent. These results suggest that DCA possesses initiating activity.

Lung carcinogenesis by N-nitrosobis(2-hydroxypropyl)amine-related compounds and their formation in rats.

Lung carcinogenesis by a single intraperitoneal injection of N-nitrosobis(2-hydroxypropyl)amine (NDHPA) and related compounds was studied in male Wistar rats. NDHPA, N-nitrosomethyl(2-hydroxypropyl)amine (NMHPA), N-nitrosobis(2-oxopropyl)amine (NDOPA), N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (NHPOPA) and N-nitroso-2,6-dimethylmorpholine (NDMMOR) induced high incidences of lung neoplasms in rats. The formation of NDHPA, NDMMOR and NMHPA in the stomach of rats treated with precursor amines and sodium nitrite was detected by high-performance liquid chromatography (HPLC).

Histochemical studies on gamma-glutamyltranspeptidase activity of pancreatic acinar cell lesions induced by 4-hydroxyaminoquinoline 1-oxide and/or azaserine in rats.

The utility of gamma-glutamyltranspeptidase (gamma-GTP) as an enzyme marker during pancreatic acinar cell carcinogenesis in rats was assessed by measuring its enzyme-histochemical performance in pancreatic acinar cell lesions induced by 4-hydroxyaminoquinoline 1-oxide (4-HAQO) and/or azaserine in partially-pancreatectomized Fischer 344 and Wistar rats. Rats were given a single intravenous injection of 4-HAQO (10 or 7 mg/kg body weight) 3 days after partial pancreatectomy followed by intraperitoneal injections of azaserine (30 mg/kg) once a week for 10 weeks, or the same treatment without azaserine. The animals were sacrificed at 3, 6, 10, 12 and 18 months. 4-HAQO predominantly induced basophilic foci in Fischer rats, while in Wistar rats acidophilic foci and acidophilic hyperplastic nodules were predominant. A preferential enhancement of the induction of acidophilic foci and hyperplastic nodules was exhibited in Fischer rats following co-administration with azaserine. Normal acinar cells were positive for gamma-GTP. 90 to 100% of basophilic foci were either negative or slightly positive for gamma-GTP, whilst 68 to 98% of acidophilic foci were positive. The gamma-GTP activities of acidophilic hyperplastic nodules were more variable between nodules than within nodules, and either co-administration of azaserine or extension of experimental duration time appeared to increase the gamma-GTP positive nodules. Between the gamma-GTP positive and decreased nodules, no histological but some morphometrical differences were observed. As far as the nodules induced by 4-HAQO in Fischer rats were concerned, all of the gamma-GTP decreased nodules had thin fibrous capsules and exhibited ultrastructurally more atypia than the positive ones. Present study thus revealed that gamma-GTP is neither a useful nor invariable enzyme marker during pancreatic acinar cell carcinogenesis in rats.

Ileal atresia and absence of appendix.

A case of ileal atresia with absence of vermiform appendix in a female newborn is reported. At the region about 105 cm from the Treitz's arch, intestinal atresia was observed with an associated V-shaped deformity of the mesentery. Polyp-like protuberance was observed in the area a few centimeters from the blind end of the distal intestine. Histologically, keratinizing squamous cells, bile pigments, lanugo hair, and alcian-blue stained materials and cells were observed in the serosal layer of the tip of the proximal blind segment and mesenterium connecting to this portion. Thrombi were not observed in the mesenterium connecting to the proximal blind segment, gap segment, and distal blind segment. Squamous cells and lanugo hair were found in the green meconium taken from the large intestine. Polyp-like protuberance was surfaced by normal intestinal mucosa, and in its inside there were two layers consisting of folded muscular tissue. In this report, we describe the detailed pathology of ileal atresia and discuss its possible cause.

Comparative histochemical investigation of the glutathione S-transferase placental form and gamma-glutamyltranspeptidase during N-nitrosobis(2-hydroxypropyl)amine-induced pancreatic carcinogenesis in hamsters.

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 putative preneoplastic lesions and adenocarcinomas in the pancreas of Syrian golden hamsters treated with N-nitrosobis(2-hydroxypropyl)amine (BHP). Areas with ductular proliferation, ductal hyperplasia, and intraductal carcinoma were strongly positive for GST-P binding and negative for gamma-GT. Cystic adenoma, microcarcinoma, and carcinomas were constantly positively stained by GST-P and partially positive for gamma-GT. GST-P appears to be useful as a positive marker for putative preneoplastic lesions in pancreatic carcinogenesis. Since normal acinar cells are strongly positive for gamma-GT, the findings might suggest that acinar cells contribute to the development of cystic adenoma, microcarcinoma, and carcinomas.

4-Hydroxyaminoquinoline 1-oxide metabolism and DNA adducts in the early stage of tumorigenesis in rats: comparison of target organ pancreas with non-target organ liver.

In order to probe key early molecular events which might be responsible for the initiation of rat pancreatic tumorigenesis by 4-hydroxyaminoquinoline 1-oxide (4-HAQO), the uptake and metabolism of carcinogen and the formation and subsequent repair of DNA adducts were monitored under conditions of high and low tumorigenicity, respectively in partially pancreatectomized and non-operated animals, and in the liver, a non-target organ for this carcinogen. Although uptake of radioactively labelled 4-HAQO was higher in the liver than in the pancreas, generation of DNA adducts was 20 times greater in the latter organ. This discrepancy was probably due to a difference in the metabolic profile of 4-HAQO. The spectrum of the adducts was qualitatively similar in both organs. No qualitative or quantitative differences could be established under the high and low tumorigenicity conditions with regard to DNA adduct formation or persistence. The major difference was the presence of a relatively large extent of pancreatic DNA replication under the high tumorigenic condition. The results indicated that metabolic profile of 4-HAQO, quantity of DNA adducts and levels of DNA replication are key factors involved in initiation of tumorigenesis.

Mutagenic activation of carcinogenic N-nitrosopropylamines by rat liver: evidence for a cytochrome P-450 dependent reaction.

Mutagenic potential of carcinogenic N-nitrosopropylamines was examined by the Ames's liquid incubation assay, using rat liver 9000 g supernatant (S9) fraction for metabolic activation. N-Nitrosobis(2-hydroxypropyl)amine, N-nitroso(2-hydroxypropyl)-(2-oxopropyl)amine (HPOP), N-nitrosobis(2-oxopropyl)amine (BOP), N-nitrosobis(2-acetoxypropyl)amine, N-nitroso-2,6-dimethylmorpholine, N-nitrosomethyl-(2-hydroxypropyl)amine and N-nitrosomethyl(2-oxopropyl)amine all showed positive mutagenicity in strain TA100 in the presence of liver S9 while being negative in strain TA98. With the exception of HPOP and BOP, which were also mutagenic in TA100 without S9 metabolic activation, these N-nitrosopropylamines required the presence of microsomes as a source of enzymes as well as NADP+ as a cofactor for mutagenic activation. Treatment of rats with polychlorinated biphenyls or phenobarbital (PB) resulted in a marked increase in the ability of S9 to activate the seven N-nitrosamines tested whereas 3-methylcholanthrene (3-MC) induction was not effective. All the mutagenic activities were considerably decreased by preincubation in an atmosphere of either carbon monoxide or nitrogen gas or by adding cytochrome c to the S9 mixture. Metyrapone, a specific inhibitor of PB-inducible major cytochrome P-450, considerably inhibited mutagenicity, whereas 7,8-benzoflavone, a specific inhibitor of 3-MC-inducible major cytochrome P-448, was totally lacking this effect. These results demonstrate a correlation between rat liver S9 dependent mutagenicity of six N-nitrosopropylamines and their known carcinogenicity in rat in vivo experiments, and that the PB-inducible major cytochrome P-450 is involved in the mutagenic activation. BOP was also shown to be activated by extrahepatic (lung, kidney, pancreas) tissue S9, blood S9 and bovine serum albumin (BSA) to the extent of 50% of that activity obtained with liver S9. A possible mechanism of BSA-mediated activation of BOP is discussed.

Distribution, metabolism and excretion of N-nitrosobis(2-hydroxypropyl)amine in Wistar rats.

The metabolic fate of the carcinogen N-nitrosobis(2-hydroxypropyl)amine (BHP) in male Wistar rats was studied. The blood level of [1-14C]BHP after a single intraperitoneal injection, administered at a carcinogenic dose of 3 g/kg body weight, reached a maximum within 1 h. Whereas a relatively high concentration of 14C was found in the blood and target organs, such as the lung, liver, thyroid gland and kidney 1 h after the treatment, most of the radioactive labelling had disappeared from the tissues by 24 h after injection. Most of the administered 14C was eliminated via the urine; 90.8% was excreted in the urine within the 24 h period, 5.5% in the feces and 3.2% by way of expired air. Studies in rats with exteriorized bile flow demonstrated that about 11% of the intraperitoneally administered 14C was excreted via the bile in 24 h. Analysis by h.p.l.c. detected BHP (78.1% of the dose), HPOP (1.5%), glucuronides of BHP (4.3%) and HPOP (0.16%), MHP (0.03%) and unknown metabolites (6.0%) in the urine 24 h after the treatment. Besides these metabolites, BOP and two unidentified metabolites were also detected in the blood, lung, liver or kidney of rats 3 h after the treatment. These results suggest the involvement of BHP metabolites, HPOP, MHP and BOP, in carcinogenesis and in particular lung carcinogenesis induced by BHP in rats.

Enhancement of DEN initiation of liver carcinogenesis by inhibitors of NAD+ ADP ribosyl transferase in rats.

The effect of inhibitors of NAD+ ADP ribosyl transferase (ADPRT) on the early stage of liver carcinogenesis of diethylnitrosamine (DEN) was studied by estimating the number and size of gamma-glutamyltranspeptidase (gamma-GTP) positive foci assayed as markers of cell populations initiated by DEN in the rat liver. ADPRT inhibitors investigated were 3-aminobenzamide (ABA), 5-methylnicotinamide (MNAM), and thymidine. A single i.p. injection of ABA at greater than 150 mg/kg body weight (B.W.) enhanced dose-dependently the induction of gamma-GTP positive foci in rat liver initiated by 20 mg/kg B.W. of DEN. The magnitude of the effect was similar to that observed when partial hepatectomy (PH) was performed instead of ABA administration. Single i.p. injections of MNAM or thymidine at a dose of 600 mg/kg B.W. also enhanced the induction of foci in rat liver initiated by the 20 mg/kg dose of DEN. Based on the above results, ABA was used as a representative ADPRT inhibitor for clarifying the mechanisms underlying the effects. Administration of ABA at a dose of 600 mg/kg B.W. was effective in enhancing the induction of foci if given 1 day before DEN, simultaneously to DEN, and 1 day after DEN initiation but it was ineffective if it was given 3 days after DEN or thereafter. Liver cell necrosis was not detectable either by analysis of serum enzymes or histologically 1, 3, 5, 7 and 14 days after 20 mg/kg B.W. of DEN with or without administration of 600 mg/kg B.W. of ABA. No initiating activity was observed for ABA administered at doses of 600 and 1200 mg/kg B.W. as assayed by development of gamma-GTP positive foci. Long term ABA administration in the diet at concentrations of 0.05, 0.1 and 0.2% did not show any promoting activity for liver carcinogenesis initiated by DEN. Furthermore, chronic administration of 0.2% ABA in the diet did not result in detectable toxicity and/or carcinogenic effects. These results suggest that ADPRT and associated DNA repair plays an important role in the early initiating stage of liver carcinogenesis and provide the basis for a new experimental approach to the analysis of the mechanisms of chemical carcinogenesis and in establishing a more sensitive assay system for liver carcinogenesis in rats.

Enzyme histochemical studies on transplantable pancreatic adenocarcinomas in Syrian golden hamsters.

Transplantable pancreatic adenocarcinomas were established in subcutaneous tissue of Syrian golden hamsters and examined by enzyme histochemistry. The original tumors were found at week 40 after subcutaneous injection of N-nitrosobis(2-hydroxypropyl)amine (DHPN) at a dose of 250 mg/kg body weight once a week for the first 20 weeks of the experiment. They were well differentiated adenocarcinomas and their histology was not changed by serial transfer for 18 generations. Their transplantability was 67 to 100% and their average doubling time was 4.1 +/- 0.82 d. The enzymes gamma-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), alkaline phosphatase (ALPase), and acid phosphatase (ACPase) were examined in tumor cells at the 14th and 15th generations and in ductal cells and acinar cells of normal pancreas of hamsters. Increased GGT activity was found in the tumor cells, and a strongly positive reaction was observed in acinar cells but not ductal cells of normal pancreas. LDH and SDH were found in the tumor cells, acinar cells and ductal cells. No ALPase was detected in the tumor cells, acinar cells, or ductal cells. ACPase was found in acinar cells and ductal cells, but not in the tumor cells.

Effects of chrysotile asbestos on lung and pleural carcinogenesis of N-bis(2-hydroxypropyl)nitrosamine in rats.

The effects of chrysotile asbestos on lung and pleural carcinogenesis by N-bis(2-hydroxypropyl)nitrosamine (DHPN) in male Wistar rats were studied. Chrysotile, 30 mg per rat, was injected into the left pleural cavity and 3 g/kg body wt. DHPN was injected once into the abdominal cavity. Lung tumors (adenoma, adenocarcinoma, squamous cell carcinoma, and combined carcinoma) occurred at the highest incidence (100%). Adenocarcinoma was seen in 4 of 11 (36%) rats killed at 35 weeks and in 6 of 12 (50%) rats killed at 52 weeks, squamous cell carcinoma occurred in 1 of 11 (9%) rats killed at 35 weeks and 3 of 12 (25%) rats killed at 52 weeks, and mixed carcinoma was seen in 1 of 12 (8%) rats killed at 52 weeks, which received chrysotile and DHPN. Adenocarcinoma was seen in 9 of 11 (82%) rats which received DHPN only and killed at 52 weeks. Mesotheliomas were seen in 2 of 11 (18%) rats, killed at 35 weeks, and 3 of 12 (25%) rats, killed at 52 weeks, which received chrysotile and DHPN. Hyaline thickening of the pleura was seen in 100% of rats receiving chrysotile. Mesothelial cell hyperplasia and adenomatous and/or fibromatous growth of the mesothelium were seen in the pleura on both sides, ranging from 36% to 50% and 31% to 64% in rats receiving chrysotile and DHPN, respectively. Asbestos bodies were seen in the pleura on both sides and in the lung.

Effects of caffeine on pancreatic tumorigenesis by 4-hydroxyaminoquinoline 1-oxide in partially pancreatectomized rats.

The effects of caffeine on pancreatic tumorigenesis by 4-hydroxyaminoquinoline 1-oxide (4-HAQO) and on pancreatic DNA synthesis were studied in partially pancreatectomized male Wistar rats. 4-HAQO was injected i.v. as a single dose of 7 mg/kg body weight 3 days after partial pancreatectomy. Caffeine was injected s.c. every 12 h at the maximum tolerated dose (m.t.d.) of 120 mg/kg body weight, half the m.t.d., and one quarter the m.t.d. from 12 to 72 h before and 0 to 72, 72 to 132, and 0 to 132 h after 4-HAQO treatment. Post-treatment with caffeine from 0 to 132 h had a dose-dependent biphasic effect on pancreatic tumorigenesis: post-treatment with the m.t.d. of caffeine decreased the total number of nodules, whereas treatment with one quarter the m.t.d. of caffeine increased their number. Decrease in the number of nodules was also observed on post-treatment with the m.t.d. of caffeine from 0 to 72 or from 72 to 132 h. Pretreatment with the m.t.d. of caffeine had no significant effect on the number of nodules. Recovery of pancreatic DNA synthesis was slower after simultaneous treatment with the m.t.d. of caffeine and 4-HAQO than after treatment with 4-HAQO alone. The possible mechanism of the effect of caffeine on pancreatic tumorigenesis induced by 4-HAQO in rats is discussed.