Equivocal colonic carcinogenicity of Aloe arborescens Miller var. natalensis berger at high-dose level in a Wistar Hannover rat 2-y study.

A 2-y carcinogenicity study of Aloe, Aloe arborescens Miller var. natalensis Berger, a food additive, was conducted for assessment of toxicity and carcinogenic potential in the diet at doses of 4% or 0.8% in groups of male and female Wistar Hannover rats. Both sexes receiving 4% showed diarrhea, with loss of body weight gain. The survival rate in the 4% female group was significantly increased compared with control females after 2 y. Hematological and biochemical examination showed increase of RBC, Hb, and Alb in the 4% males. The cause of these increases could conceivably have been dehydration through diarrhea. AST and Na were significantly decreased in the males receiving 4%, and Cl was significantly decreased in both 4% and 0.8% males. A/G was significantly increased in the 4% females, and Cl was significantly decreased (0.8%) in the female group. Histopathologically, both sexes receiving 4% showed severe sinus dilatation of ileocecal lymph nodes, and yellowish pigmentation of ileocecal lymph nodes and renal tubules. Adenomas or adenocarcinomas in the cecum, colon, and rectum were observed in 4% males but not in the 0.8% and control male groups. Similarly, in females, adenomas in the colon were also observed in the 4% but not 0.8% and control groups. In conclusion, Aloe, used as a food additive, exerted equivocal carcinogenic potential at 4% high-dose level on colon in the 2-y carcinogenicity study in rats. Aloe is not carcinogenic at nontoxic-dose levels and that carcinogenic potential in at 4% high-dose level on colon is probably due to irritation of the intestinal tract by diarrhea.

Antioxidant effects of flavonoids used as food additives (purple corn color, enzymatically modified isoquercitrin, and isoquercitrin) on liver carcinogenesis in a rat medium-term bioassay.

To clarify the effects of purple corn color, enzymatically modified isoquercitrin (EMIQ), and isoquercitrin (IQ), registered as natural food additives in Japan, on liver carcinogenesis in vivo, a medium-term bioassay was employed. A total of 100 male F344 rats were divided into 5 groups; groups 1 to 4 were given a single intraperitoneal injection of diethylnitrosamine (200 mg/kg b.w.) on day 1. From weeks 2 to 8, they were administered basal diet purple corn color, EMIQ, or IQ as containing test chemicals at doses of 1.0% (groups 1 and 5), 0.1% (group 2), 0.01% (group 3), or 0% (group 4) (experiments 1, 4, and 5). All rats were subjected to two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Purple corn color exerted no significant modifying effects on GST-P positive foci, preneoplastic foci, development in the liver. However, serum of rats treated with purple corn color provided evidence of antioxidant power significantly by potential antioxidant (PAO) test in vivo (experiment 2). And microarray analyses showed purple corn color to induce RNA expression such as P450 (cytochrome) oxidoreductase, phosphatidylinositol 3-kinase, and phospholipase A2 (experiment 3). Higher doses of EMIQ or IQ with strong antioxidant power in vivo by PAO test treated groups were correlated with smaller numbers of GST-P positive foci, with Spearman's rank correlation coefficients of P= 0.002 and P= 0.049, respectively (experiments 4 and 5). Therefore, the tested food additives may be effective as antioxidants in vivo and have chemopreventive potential against liver preneoplastic lesion development.

Promotion potential of madder color in a medium-term multi-organ carcinogenesis bioassay model in F344 rats.

A medium-term multi-organ carcinogenesis bioassay in rats was conducted to assess any possible tumor promoting effects of madder color extracted from the root of madder. Male F344 rats were divided into 5 groups of 20 each. All rats of groups 1 to 4 were given DMD treatment, consisted of multicarcinogens, N-nitrosodiethylamine (DEN), N-methyl-N-nitrosourea (MNU), and N-bis (2-hydroxypropyl) nitrosamine (DHPN), for 4 wk, while group 5 served as untreated control without carcinogens. The animals were then administered a basal diet containing madder color at doses of 5.0% (group 1), 2.5% (group 2 with 0.75% additional dextrin), or 0 (groups 3 with 1.5% additional dextrin, 4 without dextrin and 5) for the following 28 wk (total 32 wk). The total amount of dextrin in groups 1 to 3 diets was adjusted to 1.5% by extra dextrin because madder color powder contained dextrin. Key organs were observed histopathologically and glutathione S-transferase placental form (GST-P) positive foci of the liver were quantified. In the liver, 5.0% and 2.5% treated groups showed statistically significant dose-related increases in both number and area of GST-P positive foci, number: 2.81 +/- 0.90 and 1.96 +/- 0.93 (groups 1 and 2), area: 0.99 +/- 2.49 and 0.37 +/- 0.77, as compared with control, number: 0.87 +/- 0.72, area: 0.06 +/- 0.06 (group 3). In the kidneys, incidences (and numbers) of adenoma treated with 5.0% and 2.5%, 47.4% (0.20 +/- 0.24), and 47.4% (0.13 +/- 0.15) (groups 1 and 2) were significantly increased compared to control, 0% (0) (group 3). In conclusion, madder color demonstrated significant tumor promoting effects in the liver and kidneys in the DMD model.

Thirteen-week feeding study of thaumatin (a natural proteinaceous sweetener), sterilized by electron beam irradiation, in Sprague-Dawley rats.

This study was designed to evaluate and characterize any subchronic toxicity of thaumatin sterilized by electron beam irradiation (5.0 kGy) when administered at dietary levels of 0% (control), 0.3%, 1.0% and 3.0% to groups of 10 male and 10 female Crj: CD (SD) IGS rats for 13 weeks. Separate groups of both sexes received 3.0% non-irradiated thaumatin. There were no treatment-related clinical signs or adverse effects on the survival rate, body weight, food consumption, water consumption and urinalysis, ophthalmology, haematology, or blood biochemistry data. No treatment-related alterations in gross pathology or organ weights were found in any group. On histopathological examination, sporadic spontaneous lesions known to occur in this strain of rats were the only findings, with no specific relation to the test substance. Thus, the no-observed-adverse-effect-level (NOAEL) was judged to be a dietary level of at least 3.0% (2502 mg/kg body weight/day for males, 2889 mg/kg body weight/day for females) for electron beam irradiated thaumatin under the present experimental conditions. It was concluded that electron beam-irradiation of thaumatin does not cause changes of any toxicological significance.

Existence of a threshold for induction of aberrant crypt foci in the rat colon with low doses of 2-amino-1-methyl-6-phenolimidazo[4,5-b]pyridine.

Until recently it has been generally considered that genotoxic carcinogens have no threshold in exerting their potential for cancer induction. However, the nonthreshold theory can be challenged with regard to assessment of cancer risk to humans. In the present study we show that a food derived, genotoxic hepatocarcinogen, 2-amino-1-methyl-6-phenolimidazo[4,5-b]pyridine (PhIP), does not induce aberrant crypt foci (ACF) as preneoplastic lesions at low dose (below 50 ppm) or 8-hydroxy-2'-deoxyguanosine (below 400 ppm) in the rat colon. Moreover PhIP-DNA adducts were not formed at the lowest dose (below 0.01 ppm). Thus, the dose required to initiate ACF is approximately 5000 times higher than that needed for adduct formation. The results imply a no-observed effect level (existence of a threshold) for colon carcinogenesis by a genotoxic carcinogen.

p107 Expression in colorectal tumours rises during carcinogenesis and falls during invasion.

p107 Links to cyclin A/CDK2 (cyclin-dependent kinase 2) and cyclin E/CDK2 that are important cell cycle regulators. However, p107 expression remains unclear in almost all kinds of human solid tumours. To clarify the expression of p107 in colorectal tumours, 22 normal mucosae, 9 hyperplastic polyps, 60 adenomas, 198 primary carcinomas, 21 lymph-nodal metastases, and 10 hepatic metastases were immunohistochemically stained for p107, cyclin A, cyclin E, CDK2 and Ki67. Results were measured using labelling indices (LIs). p107 LIs surpassed the highest value in normal tissues in six of nine hyperplastic polyps, 54 of 60 adenomas, 144 of 198 primary cancers, 13 of 21 nodal foci and three of 10 hepatic foci. p107 LIs also apparently rose from normal through hyperplasia and adenoma to early carcinoma. However, they declined in liver-metastatic foci, and in primary cancers showing large size, mucinous type, venous invasion, lymphatic invasion, poorly differentiated type, deep invasion, lymph-nodal metastasis, hepatic metastasis or advanced stage. Low p107 LIs were also linked to a poor survival, particularly in stage-III patients. As the p107 LI gradually rose, the CDK2 (in primary cancers only), cyclin A, cyclin E and Ki67 LIs were elevated concurrently-in both adenomas and primary cancers. Thus, in colorectal tumours, p107 expression rises abnormally and gradually during carcinogenesis and then falls during invasion, and thereby probably perturbs the cell-cycle control and promotes carcinogenesis and invasion. Clinically, reduced p107 may indicate a poorer prognosis.

Organ differences in the enhancing potential of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine on carcinogenicity in the prostate, colon and pancreas.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant carcinogenic heterocyclic amine in cooked meat and fish, is speculated to be associated with human carcinogenesis. It has been shown to induce DNA adducts in a variety of organs in rodents and thus clarification of any enhancement of neoplasia is a very important subject for assessing human risk. In order to evaluate modifying effects of PhIP on carcinogenesis, several in vivo experiments in rats were performed. These featured dietary administration of PhIP at different dose levels and for different durations, and included intragastric dosing for a short period, or continuous dietary administration after initiation with other carcinogen, namely 3,2'-dimethyl-4-aminobiphenyl (DMAB) or 1,2-dimethylhydrazine (DMH). The data indicate that a short administration of PhIP is sufficient to induce prostate tumors but long-term treatment is required for effects in the colon. They also suggest tumor enhancing potential dependent on the organ, i.e. evident in the colon but not the prostate. Furthermore, promotion of colon neoplasia may depend on the initiator employed. Thus these findings suggest that the carcinogenic mechanisms of PhIP may vary in its different target organs.

Dose- and time-response studies of sodium o-phenylphenate urinary bladder carcinogenicity in rats.

Dose- and time-response studies of urinary bladder carcinogenesis due to orally administered sodium o-phenylphenate (OPP-Na) were performed using 5-week-old male Fischer 344 rats given diets containing 0 (control), 2500, 5000, 10,000, 15,000 or 20,000 ppm OPP-Na for 104 weeks and fed basal diets until 112 weeks (experiment 1). In addition, rats received diets containing 20,000 ppm OPP-Na for 0 (control), 12, 24, 52 or 104 weeks and were killed at week 112 (experiment 2). In experiment 1, the transitional cell carcinoma (TCC) was the major tumor type in the urinary bladder, and the dose-response curve was steep with many tumors occurring at the high doses of 15,000 and 20,000 ppm. The virtually safe dose at a risk level of 10(-6) for TCCs and papillomas was estimated to be 144 ppm by the Weibull model, a high value similar to that for sodium saccharin. In experiment 2, a few TCCs developed after 24 weeks of treatment, but the time-response curve was also steep with the majority of lesions occurring after longer exposure periods. Based on the observed steepness in dose- and time-responses, any implied cancer risk of OPP-Na at the low doses of interest to man must be considered to be very small.

Lack of promotion of 7,12-dimethylbenz[a]anthracene-initiated mouse skin carcinogenesis by 1.5 GHz electromagnetic near fields.

The effects of 1.5 GHz electromagnetic near fields of time division multiple access (TDMA) signal for the Personal Digital Cellular, Japanese cellular telephone standard (PDC) used for cellular phones, on mouse skin carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene (DMBA) were examined. Ten-week-old ICR female mice were treated with a single application of DMBA on shaved dorsal skin by painting at a concentration of 100 microg/100 microl acetone per mouse. One week later, mice were divided into four groups, receiving electromagnetic near fields exposure (DMBA-EMF), sham-exposure (DMBA-Sham), 12-O-tetradecanoylphorbol-13-acetate (TPA, 4 microg /200 microl acetone/mouse), as a positive control (DMBA-TPA), and no-treatment (DMBA-Control). EMF near fields exposure conditions were as follows: skin local peak specific absorption rate (SAR) 2.0 W/kg, whole body average SAR 0.084 W/kg (ratio of peak to average SAR is 24), 90 min a day, 5 days a week, for 19 weeks. At week 20, animals were killed and skin tumors were analyzed histopathologically. The incidences of skin tumors in DMBA-EMF, DMBA-Sham, DMBA-TPA and DMBA-Control groups were 0/48 (0%), 0/48 (0%), 29/30 (96.6%) and 1/30 (3.3%), respectively. Histopathologically, papilloma and squamous cell carcinoma (SCC) were observed in the DMBA-TPA group and only papilloma observed in the DMBA-Control group. The incidences of squamous cell papillomas and squamous cell carcinomas in DMBA-TPA and DMBA-Control groups were 29/30 (96.6%) and 1/30 (3.3%), respectively, numbers of tumors per mouse (tumor multiplicity) being 18.8 +/- 13.4 and 0.1 +/- 0.5. These data clearly demonstrated that near fields exposure to 1.5 GHz EMF, used for cellular phones, does not exert any enhancing effect on skin tumorigenesis initiated by DMBA.

Beef tallow, but not perilla or corn oil, promotion of rat prostate and intestinal carcinogenesis by 3,2'-dimethyl-4-aminobiphenyl.

The modifying effects of three kinds of fat (corn oil, beef tallow or perilla oil, each at 20% in the diet) on F344 rat prostate carcinogenesis induced by 3,2'-dimethyl-4-aminobiphenyl (DMAB) were investigated. Non-invasive carcinomas of the ventral prostate were induced by DMAB alone and invasive carcinomas of the other prostate lobes and seminal vesicles by DMAB and testosterone propionate (TP). Eight groups of F344 rats were initiated with 50 mg / kg body weight of DMAB at 2-week intervals for the first 20 weeks, four also receiving TP, extended until week 60. The animals received basal chow powder diet or one of three high fat diets throughout the experiment (60 weeks). One further group served as a non-carcinogen-treated control maintained on basal chow powder diet. Beef tallow significantly increased the development of ventral prostate carcinomas with DMAB alone (from 15 to 45%, P < 0.05), while perilla oil reduced the incidence of prostatic intraepithelial neoplasia (PIN) in the ventral lobe of rats given DMA + TP (from 70 to 10%, P < 0.01), but not in those given DMAB alone. No other effects of high fats were observed regarding PIN or invasive cancers of the dorsolateral and anterior prostate or seminal vesicles. A satellite experiment demonstrated that all high fat diets for 4 weeks increased the 5-bromo-2-deoxyuridine (BrdU) labeling index of prostate epithelial cells, suggesting that a high fat intake, irrespective of the fatty acid composition, may accelerate cell kinetics in the prostate. Of the three high fat diets, beef tallow was also found to increase intestinal carcinogenesis. Thus, the present data revealed carcinogenesis in the prostate and intestine to be promoted by beef tallow.

Pronounced inhibition by a natural anthocyanin, purple corn color, of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-associated colorectal carcinogenesis in male F344 rats pretreated with 1,2-dimethylhydrazine.

The potential of purple corn color (PCC), a natural anthocyanin, to modify colorectal carcinogenesis was investigated in male F344/DuCrj rats, initially treated with 1,2-dimethylhydrazine (DMH), receiving 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the diet. After DMH initiation, PCC was given at a dietary level of 5.0% in combination with 0.02% PhIP until week 36. No PCC-treatment-related changes in clinical signs, body weight and food consumption were found. Incidences and multiplicities of colorectal adenomas and carcinomas in rats initiated with DMH were clearly increased by PhIP. In contrast, lesion development was suppressed by PCC administration. Furthermore, in the non-DMH initiation groups, induction of aberrant crypt foci by PhIP tended to be decreased by the PCC supplementation. The results thus demonstrate that while PhIP clearly exerts promoting effects on DMH-induced colorectal carcinogenesis, these can be reduced by 5.0% PCC in the diet, under the present experimental conditions.

Organ dependent enhancement of rat 3,2'-dimethyl-4-aminobiphenyl (DMAB) carcinogenesis by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): positive effects on the intestine but not the prostate.

In order to evaluate tumor enhancing effects of the heterocyclic carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), doses of 100 and 300 p.p.m. PhIP were given for 40 weeks to male F344 rats, which initially received 3,2'-dimethyl-4-aminobiphenyl (DMAB). DMAB shows a similar carcinogenic organ spectrum to that of PhIP, including the prostate and colon. PhIP alone at a dose of 300 p.p.m. resulted in the development of prostate and intestine cancers. Furthermore, among the DMAB-treated group, enhancement of intestinal carcinogenesis by 300 p.p.m. PhIP was observed. However, no prostate enhancement was demonstrated in the DMAB + PhIP group. Since PhIP-DNA adduct formation in the prostate epithelial cells in a satellite experiment was not affected by pre-treatment with DMAB, it is speculated that the contradictory findings between the intestine and prostate may be due to the specific biological effects of PhIP. Taking into account previous data, that PhIP clearly enhanced rat 1,2-dimethylhydrazine-initiated colon tumorigenesis, the potential of PhIP to enhance colon carcinogenesis may be initiator dependent.

Prostate carcinomas developing in transgenic rats with SV40 T antigen expression under probasin promoter control are strictly androgen dependent.

We have generated a transgenic rat with the SV40 T antigen under probasin promoter control, allowing prostate-specific gene expression. Males demonstrate atypical epithelial cell proliferation in the prostate from 4 weeks of age and develop prostate carcinomas at 100% incidence before they are 15 weeks old. Castration at 5 weeks of age was found to inhibit the prostate tumor formation completely, whereas testosterone propionate administration induced marked cell proliferation as well as microinvasion in prostate carcinomas. Castration at 20 weeks of age, after tumor development, even with testosterone propionate treatment, induced complete tumor involution within 5 weeks. To investigate the underling processes, sequential histological changes were monitored 1, 2, 3, 7, 14, and 21 days after castration. At days 1-3, many apoptotic bodies and inflammatory cells, including foam cells, were observed, and clear glandular structures were no longer evident in the tumors. Seven days after castration, most glands were involved, and nuclei of the cells did not show atypia. After 14 and 21 days, only atrophic glands were observed. During this process, expression of caspase 3, caspase 6, BAX, bcl-x, TRPM-2, and MMP7 genes was apparently increased. Comparison of the gene expression profile between a prostate carcinoma in a transgenic animal and a normal prostate of a wild-type rat by a cDNA array technique was also conducted. The results suggested that our model is suitable to investigate mechanisms of carcinogenesis, including androgen dependence, involution, and apoptosis.

Establishment of rat hepatocellular carcinoma cell lines with differing metastatic potential in nude mice.

For better understanding of cancer metastasis, we have established an in vivo model for induction of highly metastatic hepatocellular carcinomas (HCC) in male F344 rats. From 1 tumor, 4 cell lines with differing metastatic potential (C1, C2, C6, C5F) were established by subcloning using the limited-dilution cloning technique. Two other lines, N1 and L2, arose from another primary HCC and a lung metastatic lesion, respectively. Although cell adhesion of each cell line in culture medium was different, tumors developing in the subcutis of nude mice after transplantation were all moderately differentiated HCC with a trabecular pattern. On subcutaneous injection into nude mice, all 6 cell lines proved to be tumorigenic in the injection site and C5F was highly metastatic to the lung. With injection into the tail vein, N1 and L2 formed frequent metastases in the lung as well as in lymph nodes. Using intraperitoneal injection, C1, C6, N1 and L2 showed marked disseminated growth in the abdominal cavity with bloody ascitis. Northern blot analysis revealed expression of known metastasis-related genes, KAI1 and heparanase, to be decreased in C5F, but no differences in expression of nm23-H1 were evident. A point mutation in the GSK-3beta phosphorylation site of the beta-catenin gene was found in L2. These transplantable HCC cell lines that have different metastatic ability should be useful for elucidation of mechanisms of metastasis.

Lack of chemopreventive effects of lycopene and curcumin on experimental rat prostate carcinogenesis.

The chemopreventive efficacy of lycopene and curcumin with regard to prostate carcinogenesis was investigated using 3,2'-dimethyl-4-aminobiphenol (DMAB)- and 2-amino-1-methylimidazo[4,5-b]pyridine (PhIP)-induced rat ventral prostate cancer models. Three 60 week experiments with male F344 rats were carried out. In the first DMAB was given for the first 20 weeks and lycopene or curcumin were administered concomitantly or subsequently at dietary doses of 15 and 500 p.p.m., respectively. In the second experiment lycopene and curcumin were given to rats pretreated with DMAB at doses of 5, 15 or 45 p.p.m. or 100 or 500 p.p.m. In the third PhIP was selected as an initiator for prostate carcinogenesis and administered for 20 weeks. Rats were then fed a diet containing lycopene at a dose of 45 p.p.m. or curcumin at a dose of 500 p.p.m. or both together. Chemopreventive effects of lycopene and curcumin on development of DMAB-induced ventral prostate carcinomas were observed only in the first experiment and no confirmation of inhibition potential was obtained in the following studies. Neither summational nor synergistic chemoprevention was evident. It is concluded from the present data that, overall, neither lycopene nor curcumin can consistently prevent rat prostate carcinogenesis.

Frequent c-Ha-ras gene mutations in rat mammary carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine in cooked meat and fish, is carcinogenic to the mammary glands of rats. Mutations in the H-ras gene were here examined in PhIP-induced mammary tumors of female F344 rats by the polymerase chain reaction followed by single strand conformation polymorphism analysis (PCR-SSCP) and restriction enzyme length polymorphism analysis (RFLP). Mutations in codon 12 of the H-ras gene were detected in four out of 13 tumors by PCR-SSCP. Three of them were GGA to GAA, and one was GGA to GTA. However, by RFLP analysis, four additional mutations in codon 13 were also detected in the same samples. Two had a GGC to CGC mutation, and the other shifts were GGC to GAC and GGC to GTC. Therefore, overall eight out of 13 cases had H-ras gene mutations. These results indicate that changes in H-ras function may play important roles in PhIP-induced mammary carcinogenesis.

Dose-dependent promotion by phenylethyl isothiocyanate, a known chemopreventer, of two-stage rat urinary bladder and liver carcinogenesis.

The effects of phenylethyl isothiocyanate (PEITC) on urinary bladder and liver carcinogenesis were analyzed in a rat model. Diets containing 0.1%, 0.05%, or 0.01% PEITC were administered for 32 wk to male Fischer 344 rats with and without pretreatment with an injection of diethylnitrosamine (200 mg/kg body wt i.p.) and 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in the drinking water for 4 wk for initiation. In the initiated groups, PEITC administration significantly increased the incidences of papillary or nodular hyperplasia, dysplasia, and transitional cell carcinomas at higher doses of 0.01%, 0.01%, and 0.05%, respectively, compared with the control group, given initiation alone, in a dose-dependent manner. Without initiation, administration of 0.1% and 0.05% PEITC induced simple and papillary or nodular hyperplasia and dysplasia in the urinary bladder. In the liver, induction of glutathione S-transferase placental form-positive foci was dose dependently enhanced by PEITC administration, but the incidences of liver tumors were not different among the groups. From the present experiment, we can conclude that > 0.01% PEITC enhances rat urinary bladder carcinogenesis, while weakly promoting hepatocarcinogenesis. In addition, it is suggested that > 0.05% PEITC has tumorigenic potential.

Enhancing effects of 2-amino-4,5-diphenylthiazole-induced polycystic kidneys on renal carcinogenesis in rats treated with dimethylnitrosamine.

The effects of the polycystic kidney environment on dimethylnitrosamine (DMN)-induced renal carcinogenesis were investigated in rats. In Experiment 1, male Wistar rats were given 25 or 10 ppm DMN in their drinking water and simultaneously administered 1% 2-amino-4,5-diphenylthiazole (DPT) in the diet for 30 weeks. DPT-induced polycystic kidney was associated with a significant increase in the number of renal cell tumors and incidence of mesenchymal tumors in the 25 ppm DMN + DPT group and the incidence of atypical tubules in the 10 ppm DMN + DPT group. PCNA labeling indices of cystic renal tubules in DPT-treated rats were significantly higher than for corresponding noncystic tubules. In Experiment 2, PCNA indices of renal tubules in 10 ppm + DPT rats and immunohistochemically CYP2E1-positive renal tubules in DPT-treated rats were demonstrated to be significantly increased on day 14. CYP2E1 mRNA expression in the kidneys of DPT-treated rats showed a fivefold increase over constitutive levels. The results thus indicate that DPT induction of polycystic kidneys enhances DMN-induced renal carcinogenesis in rats, with DPT-induced elevated cell proliferation and CYP2E1 expression in renal tubules as possible underlying mechanisms.