Ataru Okumura

Troglitazone, a Ligand for Peroxisome Proliferator-activated Receptor γ Inhibits Chemically-induced Aberrant Crypt Foci in Rats

The biological roles of peroxisome proliferator‐activated receptors (PPARs) in various diseases, including inflammation and cancer, have been highlighted recently. Although PPARγ ligand is suspected to play an important role in carcinogenesis, its effects on colon tumorigenesis remain undetermined. The present tune‐course study was conducted to investigate possible modifying effects of a PPARγ ligand, troglitazone, on the development and growth of aberrant crypt foci (ACF), putative precursor lesions for colon carcinoma, induced by azoxymethane (AOM) or dextran sodium sulfate (DSS) in male F344 rats. Oral troglitazone (10 or 30 mg/kg body weight (b.w.)) significantly reduced AOM (two weekly subcutaneous injections, 20 mg/kg b.w.)‐induced ACF. Treatment with troglitazone increased apoptosis and decreased polyamine content and ornithine decarboxylase (ODC) activity in the colonic mucosa of rats treated with AOM. Gastric gavage of troglitazone also inhibited colitis and ACF induced by DSS (1% in drinking water), in conjunction with increased apoptosis and reduced colonic mucosal polyamine level and ODC activity. Our results suggest that troglitazone, a synthetic PPARγ ligand, can inhibit the early stage of colon tumorigenesis with or without colitis.

Inhibitory effects of benzyl isothiocyanate and benzyl thiocyanate on diethylnitrosamine-induced hepatocarcinogenesis in rats.

The effects of two aromatic thiocyanates, benzyl isothiocyanate (BITC) and benzyl thiocyanate (BTC), on diethylnitrosamine (DEN)‐induced hepatocarcinogenesis were examined in rats. A total of 108 male ACI/N rats, 5 weeks old, were divided into 6 groups (18 rats in each). Group 1 was given a single i.p, injection of DEN (200 mg/kg body weight) one week after the start of the experiment and then kept on the basal diet until the end of the experiment (1 year). Groups 2 and 3 were treated with DEN and received dietary BITC (100 ppm) or BTC (100 ppm), respectively, throughout the experimental duration. Groups 4 and 5 were not given the carcinogen and were fed the diet containing BITC or BTC, respectively. Group 6 was kept on the basal diet alone and served as a control. Liver neoplasms were seen in Groups 1, 2 and 3. Incidence and average number of liver neoplasms in Group 2 were significantly smaller than in Group 1 (P<0.0005 and P<0.001, respectively). The incidence of liver neoplasms in Group 3 was slightly lower than in Group 1, although the difference was not statistically significant. The numbers of glutathione S‐transferase placental form (GST‐P)‐positive foci in Group 2 and γ‐glutarnyltranspepridase (GGT)‐positive foci in Groups 2 and 3 were significantly smaller than those in Group 1 (P<0.001). The average and unit areas of GST‐P‐ or GGT‐positive foci in Group 2 or 3 were also significantly smaller than those in Group 1 (P<0.05). These results suggest that BITC and BTC are chemopreventive agents for DEN‐induced liver tumorigenesis.

Inhibitory effect of the non-steroidal anti-inflammatory drugs, indomethacin and piroxicam on 2-acetylaminofluorene-induced hepatocarcinogenesis in male ACIN rats

The modifying effects of the non-steroidal anti-inflammatory drugs, indomethacin (IMC) and piroxicam (PC) on hepatocarcinogenesis induced by 2-acetylaminofluorene (AAF) were investigated in male ACI/N rats. Rats were divided into 6 groups: group 1 was fed a diet containing 200 ppm AAF for 16 weeks, starting at 6 weeks of age; group 2 was fed an AAF together with 130 ppm PC-containing diet; group 3 received an AAF diet and IMC (10 ppm) in their drinking water; group 4 was fed a PC diet alone; group 5 was given IMC alone; and group 6 served as controls. The PC diet, or the drinking water containing IMC, was given to the rats starting at 5 weeks of age until 1 week after the carcinogen exposure. At termination of the experiment (week 36), the incidences of iron-excluding altered liver cell foci (24.2 +/- 5.2/cm2) and liver cell tumors (1/10, 10%), and the tumor multiplicity (0.10/rat) in rats of group 2 were significantly smaller than those of group 1 (foci incidence, 42.6 +/- 6.7/cm2; tumor incidence, 10/10, 100%; and multiplicity, 4.00/rat) (P < 0.05). Similarly, the incidence of iron-excluding hepatocellular foci (27.4 < 1.2/cm2) and liver cell tumors (1/10, 10%) and the tumor multiplicity (0.10/rat) in rats of group 3 were significantly lower than those of group 1 (P < 0.05). There were no liver cell lesions (foci and neoplasms) in rats of groups 4, 5 and 6. Thus, PC and IMC inhibited the hepatocarcinogenesis induced by AAF when administered concurrently with the carcinogen and the results may indicate possible involvement of altered arachidonic metabolism in the initiation phase of AAF-induced liver carcinogenesis.

Acute and Subacute Toxicity Tests of Madder Root, Natural Colorant Extracted From Madder (Rubia Tinctorium), in (C57Bij6 x c3H)F1 Mice

As part of the safety assessment of madder root (MR), a food colorant extracted from madder (Rubia tinctorium), toxicity tests were undertaken using (C57BL/6 x C3H)F1 mice of both sexes. An acute toxicity test was performed by 14-day administration of MR dissolved in distilled water by gavage at doses of 0, 500, 2000, 3500, and 5000 mg/kg body weight to groups of each sex. One male mouse dosed at 5000 mg/kg body weight was dead before the end of the study, indicating that the maximum tolerated dose of MR was between 3500 and 5000 mg/kg body weight. A subacute toxicity test of MR was performed using 62 mice of each sex, mixing their diets with MR at concentrations of 0, 0.3, 0.6, 1.25, 2.5, and 5% for 90 days. All mice tolerated these doses of MR well. The body weight gains of either sex were not affected by the treatment. None of the mice treated with MR showed clinical signs of toxicity. Histopathological examinations showed retention cysts of the kidneys and epidermal vaginal cysts in a few of the treated or control mice. No hyperplastic, preneoplastic, and neoplastic lesions and no pathological findings of toxicity were found. These results suggest that dietary exposure of MR at these doses has no acute or subacute toxic effects on mice.

Carcinogenicity study of cochineal in B6C3F1 mice

The carcinogenicity of cochineal, a red colouring used in food and other products, was studied in a 2-yr bioassay in B6C3F1 mice. Groups of 50-55 mice of each sex were given 0, 3 or 6% cochineal in the diet for 2 yr. Mice of all groups developed tumours including hepatocellular adenomas or carcinomas, pulmonary adenomas or adenocarcinomas and lymphomas or lymphatic leukaemias, and the incidences of these tumours were not significantly different in treated and control groups. The results indicate that cochineal lacks carcinogenicity in mice and are consistent with those of in vitro short-term assays of cochineal and of carminic acid, an active principle of cochineal.

Juxtaglomerular cell tumor cell line producing active renin and its precursors

SummaryWe have established tumor cell lines from a rare juxtaglomerular cell tumor (JGCT) of the kidney. The original tumor contained both neoplastic cells and a tubular component. The tumor cell line (JX-G) maintained a capacity for renin production after the 20th passage. Cells at the fourth and 16th passage strongly reacted immunocytochemically with an antihuman renin antibody. Western blot analysis at the 9th passage revealed a positive reaction for renin at Mr 45000 and Mr 62000 which corresponded to renin precursors. However, the total amount of active renin secreted into the culture medium was high in the primary culture, but undetectable after the 4th passage. These findings indicate that the JGCT cell lines produced both active and inactive renin and the primary cultured JGCT cells secreted active renin, but that the secretion of renin had diminished after several passages. Additional stimuli thus appear necessary for maturation of renin and its subsequent secretion. These findings and the presence of a tubular component in the original tumor suggest that some signal transduction system is maintained in JGCTs.The physiological relationship of JGCT to the juxtaglomerular apparatus (JGA) and possible applications of the new cell lines are also discussed.

Simultaneous Measurement of Unscheduled and Replicating DNA Synthesis by Means of a New Cell Culture Insert DNA Retention Method: Rapid Induction of Replicating DNA Synthesis in Response to Genotoxic Carcinogens

In order to measure simultaneously replicating DNA synthesis (RDS) and unscheduled DNA synthesis (UDS) in rat hepatocytes responding to exposure to carcinogens, a new method, namely the “cell culture insert DNA retention (CDR)” method, was developed. All CDR procedures for cell culture, digestion of cytoplasm and retention of DNA were performed on membranes attached to cell culture containers. Four subgroups of primary cultures of hepatocytes prepared from rats were exposed to a genotoxic or non‐genotoxic carcinogen with or without 10 mM hydroxyurea and incubated for 4 h with 10 μCi/ml [3H]thymidine. The membranes were then processed for both liquid scintillation and autoradiography. Among seven tested chemicals, three genotoxic agents, 3,2′‐dimethyl‐4‐aminobiphenyl, 2‐acetylaminofluorene and diethylnitrosamine, and two non‐genotoxic carcinogens, nafenopin and phenobarbital, induced RDS within 4 h after the exposure, indicating that these carcinogenic agents induce cell proliferation in non‐proliferating rat hepatocytes prior to the emergence of genotoxic changes. Several indices were devised to characterize the genotoxicity of the tested chemicals. The induction patterns obtained showed a wide variation in the individual characteristics of carcinogen‐induced genotoxicity and mitogenicity in the early phase of initiation. This is the first report of simultaneous measurement, by using a combination of autoradiography and liquid scintillation, of UDS and RDS induced in rat hepatocytes. The described CDR approach will be useful for risk assessment and characterization of carcinogenic and tumor‐promoting agents.

Genotoxicity of pyrene oxide and 1-nitropyrene oxides in hepatocyte primary culture/DNA repair test

The genotoxicity of a pyrene oxide, 1-nitropyrene (NP) oxides and other related compounds was examined in the hepatocyte primary culture (HPC)/DNA repair test. Pyrene 4,5-oxide and both 1-NP-4,5-oxide and 1-NP-9,10-oxide elicited clearly positive responses of DNA repair. In this assay, 1-NP itself was weakly positive. However, other related chemicals such as pyrene, 1-nitro-3-hydroxypyrene, 1-nitro-6-hydroxypyrene, and 1-nitro-8-hydroxypyrene did not generate positive responses.