Katsumi Imaida

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.

RAPID BIOASSAY METHODS FOR CARCINOGENS AND MODIFIERS OF HEPATOCARCINOGENESIS

It is very important to detect environmental carcinogens in a short period. For this purpose, a rapid bioassay system based on two-step hepatocarcinogenesis has been developed in our laboratory. Rats were initially given a single dose (200 mg/kg) of diethylnitrosamine (DEN) i.p. and, starting 2 weeks later, were treated with test compounds for 6 weeks and then sacrificed, all rats being subjected to a two thirds partial hepatectomy at week 3. Carcinogenic potential was scored by comparing the glutathione S-transferase placental form-positive foci in the liver with those of the corresponding control. More than 90% of hepatocarcinogens showed positivity, and none of the compounds reported as noncarcinogenic demonstrated positivity. Furthermore, this system also detected inhibitory effects. In order to detect nonhepatocarcinogens, other appropriate systems also have been developed, for example, using methylnitrosourea or other multispectrum carcinogens. These rapid bioassay systems are particularly useful for the screening of environmental carcinogens.

Medium-term liver and multi-organ carcinogenesis bioassays for carcinogens and chemopreventive agents

To bridge the gap between long-term carcinogenicity tests and short-term screening assays such as the Ames test, several types of medium-term bioassay for rapid detection of carcinogenic agents have been developed using male F344 rats. The liver model, in which diethylnitrosamine initiation and acceleration of carcinogenesis by partial hepatectomy are essential components, requires only 8 weeks of animal experimentation and a few weeks for quantitative analysis of hepatic preneoplastic lesions. Using the model, a total of 250 chemicals have been analyzed and the efficacy of the system for hapatocarcinogens has thereby been well established. Other models are so-called multi-organ bioassays for detection of carcinogenic agents in multiple organs within relatively short periods. Among these, the DMBDD bioassay with 5 known carcinogens as initiators has been found to be most applicable and has now been introduced for practical use. Data from these bioassays and several single organ carcinogenesis systems have demonstrated that carcinogenic and modifying effects of individual exogenous agents may markedly differ from organ to organ. Therefore, research into chemoprevention should be based on a whole body level analysis. The present medium-term systems are very useful for this purpose.

Carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the rat.

A total of 10 highly-mutagenic heterocyclic amines have been identified to be carcinogenic in rodents. Among these, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), generally the most abundant with normal cooking procedures, induces mammary and colon carcinomas in rats in a clear dose-dependent manner. In a two-generation exposure (transplacental and trans-breast milk) experiment using Sprague-Dawley rats, an increased risk of mammary adenocarcinoma development was found in the second generation. Excretion of PhIP into the milk and transfer of PhIP to fetuses and neonates with resultant hepatic PhIP-DNA adduct formation were also confirmed. On the other hand, PhIP mammary carcinogenesis was significantly inhibited by coadministration of chlorophyllin or a synthetic antioxidant, 1-O-hexyl-2,3,5-trimethylhydroquinone, in long-term experiments using female F344 rats. The available findings strongly suggest that this food-derived carcinogen might be of importance as an environmental factor in the production of human cancers and that its carcinogenicity could be largely avoided by reducing intake of such compounds or by adoption of appropriate chemopreventive measures.

Diet and prostate cancer

The importance of dietary factors for prostate carcinogenesis has been proven by epidemiological studies of immigrants from Asia into the USA. Intake of foodstuffs rich in fat, including meat, is suggested to be a risk factor. Experimentally, while some studies demonstrated high fat intake to promote rat prostate carcinogenesis, others did not. Charcoal-cooked red meat and fish have been demonstrated to contain heterocyclic amines that are carcinogenic in rodents and non-primates. Among them, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been shown to induce cancers in the mammary glands, colon and prostate of rats. Although there are epidemiological data showing that PhIP could contribute to the development of breast cancer, equivalent evidence for prostate cancer is lacking. However, as protective dietary factors, micronutrients such as selenium, zinc, isoflavones, carotenoids and lycopenes and vitamins E and D have been listed. Animal experimentation on prostate cancer has consistently supported preventive potential for carotenoids and isoflavones, in contrast to the inconsistent results with high fat diets. Although the diet has apparently an important influence on prostate carcinogenesis in man, further research is necessary for clarification of specific factors in man.

Experimental prostate carcinogenesis - rodent models.

A number of rodent models of prostate carcinoma development have been established to study mechanisms and modifying potential. All except for transgenic mouse models need long experimental periods for generation of a high yield of cancers. Spontaneous prostate tumor models, while not practical in terms of time and tumor incidences, allow the natural course of multistep neoplasia to be followed without a need for chemical exposure. Carcinogens, especially in combination with testosterone, can induce prostate carcinomas in rats, but none are prostate-specific, so that tumor development in other organs is a complicating factor. Induction of invasive prostate carcinomas in the rat frequently requires long-term administration of a pharmacological dose of testosterone with or without application of a chemical carcinogen. While there are several transgenic mouse models, each also has strong and weak points, and it is therefore necessary to select the best model for the purpose of any experimental study.

Chronic toxicity of butylated hydroxytoluene in Wistar rats

Abstract Groups of 57 Wistar rats of each sex were maintained on diet containing 0.25 or 1% butylated hydroxytoluene (BHT) for 104 wk; control groups comprised 36 rats of each sex. Treated rats of both sexes showed reduced body-weight gain, relative spleen weight and white-blood-cell count while in the males there was also a reduction in serum triglyceride. BHT-treated animals of both sexes showed increased relative liver weight and total blood cholesterol but increases in red-blood-cell count could be seen only in females and only the males showed increased γ-glutamyltransferase. No significant histological changes were observed in the liver or haematopoietic system to explain these haematological and biochemical changes. Tumours were found in the liver, pancreas, mammary glands, uterus, pituitary gland, adrenal glands and in some other organs of some of the treated rats, but their incidence was not significantly different from that in controls. This experiment showed no carcinogenic effect of BHT on rats.

Lack of a Dose-response Relationship for Carcinogenicity in the Rat Liver with Low Doses of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline or N-Nitrosodiethylamine

For a long period, it has been generally considered that carcinogens, particularly genotoxic ones, have no threshold in exerting their potential for cancer induction. However, the non‐threshold theory can be challenged with regard to assessment of cancer risk to humans. Here we show that a food‐derived, genotoxic hepatocarcinogen, 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline, forms DNA adducts at low doses, but does not induce glutathione S‐transferase placental form (GST‐P)‐positive foci (considered to be preneoplastic lesions) or 8‐hydroxy‐2′‐deoxyguanosine in rat liver. Moreover a N‐nitroso compound, N‐nitrosodiethylamine, at low doses was also found not to induce GST‐P‐positive foci in rat liver. These results imply that there is a no‐observed effect level for hepatocarcinogenesis by these genotoxic carcinogens.

Early detection of carcinogenic substances and modifiers in rats

Over the past 20 years, we have been developing in vivo medium-term bioassay systems in rats for detecting carcinogenic and modifying effects of test compounds. The systems are based on the two-step hypothesis of carcinogenesis. In a liver model, male F344 rats are initially given a single dose of diethylnitrosamine (DEN, 200 mg/kg, i.p.) and starting 2 weeks later are treated with test compounds for 6 weeks and then killed, all rats being subjected to two-thirds partial hepatectomy at week 3. Carcinogenic potential is scored by comparing the numbers and areas per cm(2) of induced glutathione S-transferase placental form (GST-P) positive foci in the livers of groups of about 15 rats with those of corresponding control groups given DEN alone. A positive response is defined as a significant increase in the quantitative values of GST-P-positive foci, such a negative response as no change or a decrease. The results obtained have been compared with reported Salmonella/microsome and long-term carcinogenicity test findings for the same compounds. Of the liver carcinogens, 30 out of 31 (97%) mutagenic and 29 out of 33 (88%) non-mutagenic compounds gave positive results. Carcinogens other than hepatocarcinogens gave a lower proportion of positive results (9 out of 42, 21%). This bioassay also provides information concerning inhibitory potential. The practical utility and benefits of a multi-organ medium-term experimental protocol for early detection of carcinogenic agents and modifiers acting at sites other than the liver are also discussed.

The 1.5 GHz electromagnetic near-field used for cellular phones does not promote rat liver carcinogenesis in a medium-term liver bioassay.

We have recently established that local exposure to a 929.2 MHz electromagnetic near‐field, used for cellular phones, does not promote rat liver carcinogenesis in a medium‐term bioassay system. In the present study, a 1.439 GHz electromagnetic near‐field (EMF), another microwave band employed for cellular phones in Japan, was similarly investigated. Time division multiple access (TDMA) signals for the Personal Digital Cellular (PDC) Japanese cellular telephone standard system were directed to rats through a quarter‐wavelength monopole antenna. Numerical dosimetry showed that the peak SARs within the liver were 1.91–0.937 W/kg, while the whole‐body average specific absorption rates (SARs) were 0.680–0.453 W/kg, when the time‐averaged antenna radiation power was 0.33 W. Exposure was for 90 min a day, 5 days a week, over 6 weeks, to male F344 rats given a single dose of diethylnitrosamine (200 mg/kg, i.p.) 2 weeks previously. At week 3, all rats were subjected to a two‐thirds partial hepatectomy. At week 8, the experiment was terminated and the animals were killed. Carcinogenic potential was scored by comparing the numbers and areas of the induced glutathione S‐transferase placental form (GST‐P)‐positive foci in the livers of exposed (48) and sham‐exposed rats (48). Despite increased serum levels of corticosterone, adrenocorticotropic hormone (ACTH) and melatonin, the numbers and the areas of GST‐P‐positive foci were not significantly altered by the exposure. These findings clearly indicated that local body exposure to a 1.439 GHz EMF, as in the case of a 929.2 MHz field, has no promoting effect on rat liver carcinogenesis in the present model.