Seiko Tamano

A medium‐term rat liver bioassay for rapid in vivo detection of carcinogenic potential of chemicals

A reliable medium‐term bioassay system for rapid detection of carcinogenic potential of chemicals in the human environment has been developed. The 8‐week‐protocol consists of 2 stages; male F344 rats are given a single intraperitoneal injection of diethylnitrosamine (200 mg/kg) for initiation of liver carcinogenesis, followed by a 6‐week test chemical treatment starting 2 weeks thereafter. Test chemicals are usually given in the diet or the drinking water and in the 2nd week of test chemical treatment, all rats are subjected to two‐thirds partial hepatectomy in order to induce regenerative cell replication. The end‐point marker is the glutathione S‐transferase placental form (GST‐P)‐positive hepatic focus, the numbers and sizes of which are analyzed using an image‐analyzer and expressed as values per unit liver section (1 cm2). When the yield of GST‐P‐positive foci is significantly enhanced (P<0.05) over the control value, a chemical is judged to possess carcinogenic or promotion potential for the liver. Among 313 chemicals already tested in this system in our laboratory, 30/31 (97%) mutagenic hepatocarcinogens and 29/33 (88%) non‐mutagenic hepatocarcinogens gave positive results. Ten out of 43 (23%) agents known to be carcinogenic in organs other than the liver were also positive. It is particularly important that only one of 48 non‐carcinogens gave a very weak positive result, so that the system has a very low false‐positivity rate. It is now well documented that the assay system is highly effective for detecting hepatocarcinogens, bridging the gap between traditional long‐term carcinogenicity tests and short‐term screening assays. At the Fourth International Conference on Harmonization, our medium‐term liver bioassay based on an initiation and promotion protocol was recommended in the guidelines as an acceptable alternative to the long‐term rodent carcinogenicity test. (Cancer Sci 2003; 94: 3–8)

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.

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.

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.

Lack of Carcinogenicity of Quercetin in F344/DuCrj Rats

Quercetin was administered at dietary levels of 0(control), 1.25 and 5.0% to groups of 50 male and 50 female rats for 104 weeks, and then all animals were maintained without quercetin supplement for a further 8 weeks. At 5.0% quercetin, both sexes showed growth retardation throughout the study. There were no treatment‐ascribed effects regarding clinical signs, mortality, urinalyses or hematology. Although serum glucose in 5.0% quercetin‐treated males was significantly decreased and some relative organ weights in 5.0% groups showed statistically significant increases, these latter changes seemed to be related to the growth retardation. An increased incidence of non‐neoplastic hyperplastic polyps in the cecum was noted in the 5.0% males. The incidences of cystic changes and fibroadenomas of the mammary gland, and foci (areas) of hepatocellular alteration in the 5.0% females, and liver bile duct proliferations in the 5.0% males were significantly decreased. No proliferative lesions of the urinary bladder related to treatment with quercetin were found in any rats. The incidences of several other nonneoplastic and neoplastic lesions which demonstrated statistically significant changes appeared to be related to the growth retardation or to be within the normal range, and therefore none was considered to be significant biologically. Thus, the investigation did not demonstrate any clear carcinogenic effect of quercetin on F344 rats at dietary levels of up to 5.0%.

Spontaneous Tumors in Aging (C57BL/6N X C3H/HeN)F1 (B6C3F1) Mice

Spontaneous tumors in untreated (C57BL/6N x C3H/HeN)F1 (B6C3F1) mice used as controls in carcinogenicity tests were recorded. In both sexes, the development of spontaneous tumors was age-related. In 244 male mice, the most common tumors were hyperplastic nodules of the liver, hepatocellular carcinomas, malignant lymphomas/leukemias, lung adenomas, and adenocarcinomas. In 246 female mice, the most common tumors were malignant lymphomas/leukemias, pituitary adenomas, neoplastic nodules of the liver, hepatocellular carcinomas, and lung adenomas. Hepatocellular carcinomas metastasized in 20.3% of the animals with these tumors. Few other tumors except malignant lymphomas and leukemias metastasized. Various tumors of other organs and/or tissues were found at low incidences.

Modification of N-methyl-N-nitrosourea initiated carcinogenesis in the rat by subsequent treatment with antioxidants, phenobarbital and ethinyl estradiol

The modifying effects of butylated hydroxyanisole (BHA), sodium L-ascorbate (SA), phenobarbital (PB) and ethinyl estradiol (EE) were studied by their administration to F344 rats subsequent to initiation with N-methyl-N-nitrosourea (MNU), a wide-spectrum carcinogen inducing tumors in many organs. Rats were initially given 4 doses of MNU (50 mg/kg) intraperitoneally within a 2-week period and then placed on a diet containing BHA (1%), SA (5%), PB (0.05%) or EE (0.001%) for 23 weeks prior to killing. Since the experiment was based on a whole body concept of carcinogenesis, all major organs were examined histologically and histochemically for any preneoplastic lesions. BHA enhanced forestomach and urinary bladder carcinogenesis as did SA also for the urinary bladder, whereas PB enhanced the induction of gamma-glutamyl transpeptidase positive (gamma-GT+) foci in the liver and also the incidence of thyroid carcinoma and forestomach carcinoma. In contrast, EE inhibited the induction of thyroid tumors, malignant lymphoma or leukemia. Thus these compounds, when given after initiation of many organs by a single carcinogen, exert an influence on the site of tumor development by, as yet unknown, organotropic modifying effects.

Lack of Adverse Effects of Whole-Body Exposure to a Mobile Telecommunication Electromagnetic Field on the Rat Fetus

Abstract The recent steep increase in the number of users of cellular phones is resulting in marked increase of exposure of humans to radiofrequency electromagnetic fields (EMFs). Children are of particular concern. Our goal was to evaluate potential adverse effects of long-term whole-body exposure to EMFs simulating those from base stations for cellular phone communication. Pregnant rats were given low, high or no exposure. At the high level, the average specific absorption rate (SAR)for the dams was 0.066–0.093 W/kg. The SAR for the fetuses and the F1 progeny was 0.068–0.146 W/kg. At the low level, the SARs were about 43% of these. The 2.14 GHz signals were applied for 20 h per day during the gestation and lactation periods. No abnormal findings were observed in either the dams or the F1 generation exposed to the EMF or in the F2 offspring. Parameters evaluated included growth, gestational condition and organ weights for dams and survival rates, development, growth, physical and functional development, hormonal status, memory function and reproductive ability of the F1 offspring (at 10 weeks of age) along with embryotoxicity and teratogenicity in the F2 rats. Thus, under our experimental conditions, whole-body exposure to 2.14 GHz for 20 h per day during gestation and lactation did not cause any adverse effects on pregnancy or the development of rats.

Participation of urinary Na+, K+, pH, and L-ascorbic acid in the proliferative response of the bladder epithelium after the oral administration of various salts and/or ascorbic acid to rats

Changes in urinary parameters (particularly electrolyte levels and pH), and DNA synthesis and the morphology of the bladder epithelium were investigated in rats that were fed for 4 or 8 wk on diets containing various Na, K, Mg or Ca carbonate salts, with or without L-ascorbic acid (AsA). [The carbonate salts were fed at a level of 3% in the diet, and AsA or AsA-Na was administered at 5% in the diet. NH4Cl was at 1% in the diet.] The effects of treatment with NH4Cl (used as a urine acidifier), and of combined treatment with sodium ascorbate (AsA-Na) and NH4Cl were also investigated. Urinary pH was significantly elevated in groups given NaHCO3, K2CO3, AsA + NaHCO3, AsA + K2CO3 and AsA-Na, whereas treatment with AsA or NH4Cl alone caused a significant drop in urinary pH. An increase in urinary electrolytes or ascorbic acid was associated with the corresponding dosing regimen. DNA synthesis in the bladder epithelium was increased in groups given NaHCO3, K2CO3, AsA + NaHCO3, AsA + K2CO3 or AsA-Na. Furthermore, all treatments that induced an elevation of DNA synthesis also induced some morphological alterations in the bladder epithelium. The administration of AsA in conjunction with NaHCO3 or K2CO3 induced levels of change greater than those with either salt alone. In contrast, the degree of response in the bladder epithelium of rats given AsA-Na was reduced by the simultaneous administration of NH4Cl. These results suggest that the degree of DNA synthesis and/or morphological alteration in the rat-bladder epithelium after treatment with various bases may depend on changes in urinary concentrations of Na+ or K+ ions and/or pH, and the presence of ascorbic acid in the urine. The results are discussed in relation to the possible promotion by various treatment regimens (salts +/- AsA) of urinary bladder carcinogenesis.

Involvement of macrophage inflammatory protein 1α (MIP1α) in promotion of rat lung and mammary carcinogenic activity of nanoscale titanium dioxide particles administered by intra-pulmonary spraying

Titanium dioxide (TiO(2)) is evaluated by World Health Organization/International Agency for Research on Cancer as a Group 2B carcinogen. The present study was conducted to detect carcinogenic activity of nanoscale TiO(2) administered by a novel intrapulmonary spraying (IPS)-initiation-promotion protocol in the rat lung. Female human c-Ha-ras proto-oncogene transgenic rat (Hras128) transgenic rats were treated first with N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water and then with TiO(2) (rutile type, mean diameter 20 nm, without coating) by IPS. TiO(2) treatment significantly increased the multiplicity of DHPN-induced alveolar cell hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas, confirming the effectiveness of the IPS-initiation-promotion protocol. TiO(2) aggregates were localized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm. To investigate the underlying mechanism of its carcinogenic effects, TiO(2) was administered to wild-type rats by IPS five times over 9 days. TiO(2) treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide dismutase activity and macrophage inflammatory protein 1alpha (MIP1alpha) expression in the lung. MIP1alpha, detected in the cytoplasm of TiO(2)-laden alveolar macrophages in vivo and in the media of rat primary alveolar macrophages treated with TiO(2) in vitro, enhanced proliferation of human lung cancer cells. Furthermore, MIP1alpha, also detected in the sera and mammary adenocarcinomas of TiO(2)-treated Hras128 rats, enhanced proliferation of rat mammary carcinoma cells. These data indicate that secreted MIP1alpha from TiO(2)-laden alveolar macrophages can cause cell proliferation in the alveoli and mammary gland and suggest that TiO(2) tumor promotion is mediated by MIP1alpha acting locally in the alveoli and distantly in the mammary gland after transport via the circulation.