Brian Pittman

Tea polyphenols as inhibitors of mutagenicity of major classes of carcinogens

Previous research suggested that the mutagenicity of some genotoxic carcinogens, mainly heterocyclic amines, was decreased by green or black tea extracts, or tea polyphenol fractions. Thus, it seemed important to test a variety of genotoxic carcinogens with distinct chemical structures and means of biochemical activation as regards modification of mutagenicity in appropriate strains of Salmonella typhimurium by 3 concentrations of polyphenols 60, 100, or B, standard commercial polyphenol preparations from green or black tea. Polyphenols sharply decreased the mutagenicity of a number of aryl- and heterocyclic amines, of aflatoxin B1, benzo[a]pyrene, 1,2-dibromoethane, and more selectively, of 2-nitropropane, all involving an induced rat liver S9 fraction. Good inhibition was found with 2 nitrosamines that required a hamster S9 fraction for biochemical activation. No effect was found with 1-nitropyrene, and with the direct-acting (no S9) 2-chloro-4-methyl-thiobutanoic acid. Thus, with some exceptions, polyphenols considerably decreased the mutagenicity of diverse types of carcinogens.

Tea polyphenols inhibit the formation of mutagens during the cooking of meat

Powerful mutagens are formed during the broiling or frying of meat. These mutagens cause specific cancers in animal models, and epidemiological studies suggest that they increase the risk of breast and colon cancer. It is important, therefore, to inhibit the formation of these mutagens. Application of tea polyphenols, polyphenon 60 from green tea, and polyphenon B from black tea, to both surfaces of ground beef before cooking inhibits the formation of the mutagens in a dose-related fashion. This procedure is simple and effective, and utilizes inexpensive tea, a product that deserves consideration for practical use.

Effect of Tea Extracts, Polyphenols, and Epigallocatechin Gallate on Azoxymethane-induced Colon Cancer

Abstract Studies were conducted to determine the chemopreventive efficacy of several types of tea extracts on azoxymethane-induced colon cancer in male F344 rats. After determining the maximally tolerated dosage of the tea products, their effect in a colon cancer model was investigated. Groups of 36 male F344 rats Received 2 subcutaneous doses of 15 mg/kg azoxymethane (AOM) at Weeks 6 and 7. Experimental groups also Received as drinking fluids 3600 ppm of black or green tea extracts, 1800 ppm of EGCG, or 1800 ppm of black or green tea polyphenols beginning at 5 weeks of age. Additional groups drank a lower dose of 360 ppm of the five tea products. The experiments were terminated 43 weeks after the first tea exposure. No evidence of toxicity was observed since the body weight gain of all groups was similar. The rats given AOM had carcinoma of the small intestine and of the colon, classified histologically as in situ carcinoma, exophytic, invasive, and Peyers patch carcinoma. In the small intestine, most of the neoplasms were classified as invasive, but in the colon, most were exophytic. The various tea products failed to produce a significant difference in the incidence of the several types of colon and small intestine carcinoma. The multiplicity of colon cancers ranged from 1.2-2.8 in all groups. The group on 3600 ppm of green tea had a significantly higher tumor multiplicity than the control group on AOM and water. Also, the group on 3600 ppm of green tea had a significantly higher tumor multiplicity than the group on 360 ppm. The tea products did not affect the development aspects of the tumors in most groups. The mechanisms underlying these findings rest on the fact that azoxymethane is metabolized mainly by cytochrome P450 2E1, and this enzyme system is not affected by tea.

Diethylnitrosamine exposure-responses for DNA ethylation, hepatocellular proliferation, and initiation of carcinogenesis in rat liver display non-linearities and thresholds

Abstract In previous exposure-response studies, we have documented non-linearities for some of the early effects in rat liver of diethylnitrosamine (DEN) and a near no-effect levels for initiation of promotable liver neoplasms at the lowest cumulative exposure of 0.5 mmol/kg body weight; this in spite of formation of DNA adducts and induction of hepatocellular altered foci (HAF). To extend these investigations, in an initiation segment, young male F344 rats were administered four exposures of DEN ranging from a cumulative total of 0.25 mmol, which is half of the previously used low exposure, up to 2 mmol per kg body weight, an effective initiating exposure. These exposures were achieved by once weekly intragastric instillations of one-tenth the total exposures for up to 10 weeks. The initiation segment was followed by a 4 week recovery segment, to allow for remission of acute and subchronic effects of DEN, after which the groups were maintained on 0.06% phenobarbital in the diet for 24 weeks to promote liver tumor development in order to assess initiation. During and after initiation and at the end of recovery, selected groups were studied for several crucial effects involved in hepatocarcinogenicity. The low exposure produced a low-level of DNA ethylation at both 5 and 10 weeks of exposure, measured as O4-ethylthymidine, the most persistent promutagenic ethylation product. At the 5 week interval, the adduct values of the higher exposures were less than proportional to the increment of exposure, suggestive of nonlinearity. Assessment of cellular proliferation by staining for proliferating cell nuclear antigen revealed that the lowest exposure did not increase the replicating fraction of hepatocytes during the initiation (10 weeks) or recovery (4 weeks) segments, whereas in the three higher exposure groups, proliferation was increased in relation to dose and time. Preneoplastic HAF expressing glutathione S-transferase-placental-type were present at low multiplicity in control livers and their multiplicity was increased in all exposure groups by the end of exposure, at which time the increase in the high exposure group was disproportionately greater than the increment of exposure. After phenobarbital administration in the promotion segment, all exposure groups exhibited further HAF increases at 39 weeks. At the end of the promotion segment, no hepatocellular neoplasm was found in 80 controls or in 40 rats in the low exposure group. In the mid-low exposure group, which was the previously studied low exposure, only one adenoma was found, yielding a 3% incidence, while in the two higher exposure groups, 32 and 80% of rats exhibited liver neoplasms, which were increased disproportionately greater than the increments of exposure. Thus, the findings document non-linearities of early DEN effects and at the lowest cumulative dose, a no-effect level (NEL) or threshold for initiation of promotable liver neoplasms. These findings provide a conceptual basis for understanding why low-level exposures to DNA-reactive carcinogens may convey no cancer risk.

On the occurrence of Leydig cell tumors in the F344 rat.

Dunning began inbreeding, what is now the Fischer F344 rat, in 1931. Because of her publications showing a low incidence of spontaneous tumors to 35 months of age, we selected the F344 rat for most of the studies in the National Cancer Institute (NCI) Bioassay Program, beginning in 1964. We were surprised by the finding that untreated male F344 rats displayed a high incidence of Leydig cell tumors of the testes beginning at about 17 months of age. The key difference between the results of Dunning and the NCI Bioassay Program was that her animals were retired breeders, whereas the NCI studies utilized virgin rats. The question of breeding was, therefore, examined as a possible protective factor. Groups of male F344 CDF/Crl rats were kept as virgin animals, or permitted access to bilaterally tube ligated female F344/Crl rats that were replaced twice over a 52-week period. At that time, all males from both groups were housed three per cage to 85 weeks when they were killed in a CO(2) atmosphere and necropsied. Sections were prepared from the fixed tissues, stained and studied by histopathology. The results were evaluated by appropriate statistical methods. Virgin and sexually active F344 rats displayed monolateral or bilateral Leydig cell tumors. There was no statistical difference between the two groups. Despite the early difference between breeding and virgin F344 rats, a control experiment failed to disclose an effect of sexual activity on the occurrence of Leydig cell cancers. This disease displays some difference in incidence in various parts of the world, with the higher socioeconomic groups having a greater incidence. Etiological factors on the occurrence of this disease in animals and in humans remain to be discovered.

Oral enzyme therapy and experimental rat mammary tumor metastasis

Although there has been much interest over the years in the medical use of orally administered proteolytic enzymes, there is considerable controversy about their efficacy against advanced stages of cancer. In light of this, the goal of the present study was to assess the inhibitory effects of different doses of an orally administered porcine pancreas preparation on the growth and metastasis of the R13762 transplantable rat mammary tumor. Five groups of 12 F-344 female retired breeders were inoculated orthotopically with a 2mm3 tumor implant and placed on the following diets: (1) AIN-76A diet + 20% porcine pancreas preparation (PPP); (2) AIN-76A + 20% PPP + 10 mg Mg citrate/rat/day; (3) AIN-76A + 2% PPP; (4) AIN-76A + 2% PPP + 10 mg Mg citrate and (5) AIN-76A only (control). Primary tumor development was monitored for 40 days and following sacrifice, lungs were excised, stained and metastatic foci quantitated. Metastatic foci were sorted into 3 groups based on their radii: small (<1mm), medium (1-3mm) and large (>3mm), and volumes calculated. The oral enzyme preparation had no effect on primary tumor growth or on body weight change over the duration of the study. The percent (incidence) of rats with pulmonary metastases among the five groups were not significantly different. However, among the three size categories of pulmonary foci, decreased incidence was found only in the large (>3mm) volume subset of the 2% PPP group supplemented with Mg++. When assessed in terms of mean number of pulmonary foci/rat, the 20% PPP group exhibited the highest and controls the lowest frequency with the important exception of the 2% PPP + Mg++ group (large volume) which exhibited the lowest frequency of all treatment groups. In general, the presence of Mg++ resulted in marked decreases in mean number of pulmonary foci/rat compared to groups fed PPP without the Mg++ supplement. Similar results were obtained when foci were quantitated in terms of metastatic volume rather than frequency. The results of this laboratory animal study suggest that to show effective inhibition of metastatic dissemination of the R13762 tumor by PPP, lower doses of PPP and larger numbers of animals, to account for the high variability in the model, will be required.