Biomedical subjects

M Ishidate

Publications and source records attributed to M Ishidate.

At least 55 records · Page 3Linked to original sources

PubMed1990

A comparison of chromosome aberration induction by 25 compounds tested by two Chinese hamster cell (CHL and CHO) systems in culture.

Twenty-five chemicals were tested for the induction of chromosomal aberrations in 2 cultured mammalian cell systems, Chinese hamster lung cells (CHL) and Chinese hamster ovary cells (CHO). This study was carried out to provide a data set that would permit an assessment of the extent to which the 2 systems agree in the results produced. Results presented for the 2 systems in this paper are not based on the same criteria but rather on the criteria standardly used in each of the systems. In tests conducted in the absence of S9 mix, 7 chemicals gave positive results in both systems and 12 were negative in both. In tests with S9 mix, 5 were positive in both systems and 9 were negative in both. When the overall results including tests both with and without S9 mix were considered, the 2 systems agreed on 15 results, 11 positives and 4 negatives. A review of the test conditions and data suggests that disagreements in test results were more often due to differences in the protocols used in these 2 systems than to a difference in the sensitivities of the 2 cell lines.

Animals↗

PubMed1990

Sensitivity of Salmonella typhimurium YG1024 to urine mutagenicity caused by cigarette smoking.

Salmonella typhimurium YG1024, an O-acetyltransferase-overproducing derivative of TA98, was found to be more sensitive in detecting mutagenicity in human urine caused by cigarette smoking, than the conventional strain TA98, in Ames test in the presence of S9 mix. YG1021, another derivative of TA98 with elevated nitroreductase levels, did not show increased sensitivity. These results suggest that the mutagenic compounds concentrated by Blue Rayon from smokers' urine are aromatic amino compounds.

Animals↗

PubMed1990

The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides.

Ultra-vital staining with acridine orange (AO) is introduced into the micronucleus assay with mouse peripheral blood cells. Peripheral blood was stained vitally by dropping whole blood on an AO-coated slide and covering the sample with a coverslip. With this method, reticulocytes are identified easily by their red fluorescing reticulum structure. The distinction between young and mature erythrocytes was clearer and less subjective than the distinction between polychromatic and normochromatic erythrocytes by Giemsa staining or by conventional AO fluorescent staining. Although the induction of micronucleated peripheral reticulocytes (MNRETs) was delayed by about 12 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optimal sampling time. It is concluded that bone marrow cells can be replaced by peripheral blood as material for the micronucleus assay.

Acridine Orange↗

PubMed1990

Synthesis, chemical properties and mutagenicity of 1,6- and 3,6-dinitrobenzo[a]pyrenes.

Nitration of benzo[a]pyrene (BaP) with HNO3 (d = 1.38) produced a mixture of dinitroBaPs (1,6- and 3,6-isomers) and mononitroBaPs (1-, 3- and 6-isomers). Pure 1,6-dinitroBaP and 3,6-dinitroBaP were obtained by the reduction of the dinitroBaPs mixture with NaSH to yield the separable products 1-amino-6-nitroBaP and 3-amino-6-nitroBaP, followed by conversion to dinitroBaPs via the the diazonium salts. The half-wave potentials (E1/2) corresponding to the one-electron reduction of dinitroBaPs were measured and the relationship of these values to the mutagenicity is discussed.

Benzopyrenes↗

PubMed1989

A procedure for data analysis of the rodent micronucleus test involving a historical control.

No standard procedure of data analysis for rodent micronucleus tests involving historical controls has been established. In the present paper, under the presumption that the distribution of the historical control is stable and reliable, a procedure with three statistical steps is proposed to analyze the frequency of micronucleated polychromatic erythrocytes (MNPCEs). In the first step, the frequencies of MNPCEs in negative and positive control groups of a current experiment of the micronucleus test are compared with the distribution of historical negative and positive controls to examine the technical validity of the current experiment. In the second step, the frequency of MNPCEs in each treatment group is compared with the distribution of the historical negative control. In the third step, the dose-response relation is tested with the Cochran-Armitage trend test. A Monte Carlo stimulation study shows that the power of this procedure is acceptable and also this procedure is robust. An application of this procedure on real data reveals that it is effective in detecting clastogenic chemicals when the probability of a type I error is nearly .01.

Animals↗

PubMed1989

Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes. II. Chromosomal aberration tests with cultured mammalian cells.

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on ptaquiloside and its related compounds, hypoloside B, hypoloside C, illudin M and illudin S. Ptaquiloside induced chromosomal aberrations at doses as low as 4.5 micrograms/ml (0.0113 mM). The clastogenic effect was pH-dependent. The same activity was observed at a 90-fold higher dose at pH 5.3 in the culture medium compared with the activity at pH 7.4 or pH 8.0. Both hypoloside B and hypoloside C were also clastogenic at almost the same dose level as that of ptaquiloside. Illudin M and illudin S were also potent clastogens and induced aberrations at much lower doses than ptaquiloside. These results suggest that the clastogenic effect is involved in the mechanism of carcinogenic potency of ptaquiloside in animals.

Animals↗

PubMed1989

A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella typhimurium strains TA98 and TA100.

'Classical nitroreductase' is an enzyme involved in the intracellular metabolic activation of mutagenic nitroarenes. The nitroreductase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1021 and YG1026) had more than 50 times higher nitrofurazone-reductase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic action of 2-nitrofluorene, 1-nitropyrene and 2-nitronaphthalene. These results indicate that the new strains permit the efficient detection of mutagenic nitroarenes.

Cloning, Molecular↗

PubMed1989

Micronucleus test with 1-beta-D-arabinofuranosylcytosine administered by intraperitoneal injection and oral gavage.

The effect of route of administration on the outcome of the micronucleus test was evaluated in 2 laboratories by administering the model chemical, 1-beta-D-arabinofuranosylcytosine (Ara-C), by intraperitoneal injection (i.p.) and oral gavage (p.o.) to 2 mouse strains, MS/Ae and CD-1. On the basis of a small-scale acute toxicity study and a pilot experiment for the micronucleus test, a full-scale test was performed with a 24-h sampling time at doses of 12.5, 25, 50, and 100 mg/kg i.p. and 25, 50, 100, and 200 mg/kg p.o. In both strains, MNPCEs were induced at lower dose levels by the i.p. treatment, as determined not only on the basis of mg/kg but also as a ratio of the LD50. When compared with other chemicals tested in this collaborative study, the effective dose levels of this chemical based on the LD50s were exceptionally low by both routes and in both strains, e.g., less than 0.3% of the LD50 by the i.p. treatment. The maximum frequencies of MNPCEs induced were, however, identical (MS/Ae) or even higher (CD-1) by the p.o. treatment.

Administration, Oral↗

PubMed1989

Negative micronucleus tests on caprolactam and benzoin in ICR/JCL male mice.

Male ICR/JCL mice were given a single intraperitoneal injection of 125, 250, and 500 mg/kg of caprolactam (CAP), or 250, 500, and 1000 mg/kg of benzoin (ZOIN). Bone marrow preparations were made 24, 30, and 48 h after treatment with the maximum dose, and 30 h after treatment with the other doses. The slides were coded before microscopic examination. No significant increase was found in the incidence of micronucleated polychromatic erythrocytes after treatment with either CAP or ZOIN.

Animals↗

PubMed1989

Characterization of three forms of cytochrome P-450 inducible by 3-methylcholanthrene in Golden hamster livers with special reference to aflatoxin B1 activation.

Three forms of cytochrome P-450 of liver microsomes of 3-methylcholanthrene-treated Golden hamsters were purified and characterized as regards their catalytic activity toward aflatoxin B1-related hepatocarcinogenic mycotoxins. These include two major forms, designated as cytochrome P-450-AFB (P-450-I) and P-450-II, and one minor form, P-450-III. Cytochromes P-450-AFB, P-450-II, and P-450-III have their absorption maximum in the carbon monoxide-complex of the reduced form at 448.5, 447.0, and 448.0 nm, have apparent molecular weights of 56,000, 58,000, and 59,500, and are in the low spin, high spin, and low spin state, respectively. Of these, cytochrome P-450-AFB was shown to be highly active in the mutagenic activation of aflatoxin B1-related hepatocarcinogens such as sterigmatocystin and O-methylsterigmatocystin. Activation of aflatoxin B1 by hepatic microsomes of 3-methylcholanthrene-treated hamsters was inhibited almost completely by the antibody against P-450-AFB but not by the antibody against P-450-II, indicating that P-450-AFB is the major component responsible for the activation of aflatoxin B1 by hamster liver. Western blot analysis demonstrated that no protein cross-reacted with the antibody to P-450-AFB in the liver microsomes from guinea pig, rat, mouse, and house musk shrew (Suncus murinus) treated with 3-methylcholanthrene, while one or two proteins cross-reacted with the antibody to P-450-II in the liver microsomes of these animals.

Aflatoxin B1↗

PubMed1989

[Mutagenicity of steviol: an analytical approach using the Southern blotting system].

Steviol is the aglycone of stevioside, which is a non-caloric sugar substitute commonly used in Japan. Our previous studies and Pezzuto et al. have demonstrated that steviol is mutagenic after metabolic activation in the forward mutation assay using Salmonella typhimurium TM677 (TM677), whereas it is non-mutagenic in the reverse mutation assay (Ames test) using S. typhimurium TA 100, TA98, TA102 and TA97. There is the possibility, therefore, that activated steviol selectively induces a deletion or insertion of more than one base pair which cannot be detected by strains commonly used in the Ames test. In this study, we confirmed first that the 8-azaguanine (8-AG) resistance of the TM677 mutants appears to reside in the chromosomal gpt gene, since it can be complemented by the gpt gene of E. coli (Ecogpt). The chromosomal DNA of TM677 and TM677 mutants were digested by several restriction enzymes: BamHI, Sau3AI, AluI, TaqI, HaeIII, HpaII and RsaI, and analyzed by the Southern blot hybridization technique with a probe to the gpt gene DNA of E. coli. No significant differences in DNA fragment length, however, were formed between the wild type and spontaneous or steviol-induced mutants.

Azaguanine↗

PubMed1988

Mutagenicity to bacteria, cultured cells, and rodents of the human carcinogen chlornaphazine.

The human carcinogen and nitrogen mustard chlornaphazine (CN) has been confirmed to be mutagenic to Salmonella and, unexpectedly, the more so when evaluated in the presence of liver S9 mix. It also has been established as clastogenic to Chinese hamster lung cells exposed in vitro to dose levels greater than 2.5 micrograms/ml. Chlornaphazine subdued mice at doses of 5 g/kg, but only the occasional death occurred during the 4 days following oral administration of this dose in corn oil. Consequently, a median lethal dose level was not established. Nonetheless, dose levels of 500 mg/kg or greater gave a clear positive response in both the mouse and the rat bone marrow micronucleus assay. Although depression of erythropoeisis was observed in mice, a clastogenic response still was observed in the bone marrow 24 hr after dosing. The positive response in the rat was greater than that observed in the mouse. The present data provide a further instance of an established human carcinogen being readily detected by standard in vitro and in vivo mutagenicity assays.

Animals↗

PubMed1988

Human embryonic cell growth assay for teratogens with or without metabolic activation system using microplate.

In vitro microassay for the screening of teratogens was investigated on cancer chemotherapeutic agents sterigmatocystins and benzimidazoles using human embryonic palatal mesenchymal (HEPM) cells. Five thousand cells were inoculated into each well of 96-well microtiter plates, and cultivated for 24 hr, after which the media were changed with new ones that contained various amounts of chemicals; after cultivation for an additional 72 hr, the media were discarded, and cells attached to the tissue plate were fixed and stained with Giemsa's solution; the cell number then was counted by colony counter with three readings for each well. For the metabolic activation, the liver S9 obtained from rats pretreated with phenobarbital and 5,6-benzoflavone and cofactors (S9 mix) were added directly to the HEPM cell cultures along with chemicals. After 6 hr, the cultures were exchanged with a fresh medium and incubated for a further 72 hr. The final IC50 (the concentration that inhibits growth 50%) concentration-finding run had 7 to 11 concentration points (mean, three to four wells). Concentrations of the cancer chemotherapeutic agents that inhibited growth by 50% ranged from 0.001 to 10 micrograms/ml. Sterigmatocystins indicated strong inhibition; among three derivatives, O-acetyl sterigmatocystin was the most potent inhibitor. Benzimidazoles also exhibited an inhibitory action on HEPM cell growth; nitro and chloro groups at the 5 position in 2-(2-pyridyl)benzimidazole were found to be potent substituents. As for the activation of cyclophosphamide in the HEPM cell culture, IC50 was decreased to 1.0 ug/ml by the incubation with S9 mix for 6 hr under our experimental conditions, and sterigmatocystin was found to be activated by S9 mix.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzimidazoles↗

PubMed1988

Induction of chromosomal aberrations in active oxygen-generating systems. I. Effects of paraquat in Chinese hamster cells in culture.

A possible role for the superoxide anion radical (O2-) in the clastogenicity of paraquat (PQ) was investigated in cultured Chinese hamster cells. When cells were treated with 0.8 mg/ml of PQ for 3 h followed by 21 h of recovery time, structural chromosome aberrations were induced in about 50% of the metaphases examined. Almost all aberrations were of the chromatid-type and involved exclusively gaps and breaks. The induction of chromosomal aberrations by PQ was enhanced by a 1-h pretreatment with diethyldithiocarbamate, an inhibitor of superoxide dismutase. Diethyl maleate, a glutathione scavenger, also enhanced the induction of chromosomal aberrations, but 3-aminotriazole, an inhibitor of catalase, showed no such effects. Enhanced induction of chromosomal aberrations was also observed when PQ-treated cells were cultured at a high oxygen concentration (80%). The present results suggest that the production of chromosomal aberrations by PQ may be directly or indirectly related to the generation of O2-, but not to the formation of hydrogen peroxide by the dismutation reaction of O2- or of other active oxygen species including the hydroxyl radical and singlet oxygen.

Amitrole↗

PubMed1988

Induction of chromosomal aberrations in active oxygen-generating systems. II. A study with hydrogen peroxide-resistant cells in culture.

Cells hyper-resistant to hydrogen peroxide (H2O2) were obtained from a Chinese hamster cell line (CHL) by repeated treatments with H2O2 at stepwise increasing concentrations. A clonal line (R-8) was approximately 10 times more resistant to H2O2 than the parental cells, and retained its resistance for about 2 months in normal medium. However, with further passages after the completion of the present study, the elevated resistance gradually decreased. Although the concentration of H2O2 required to induce chromosomal aberrations in 50% of treated cells was about 10 times higher in R-8 than in the parental cells, there were no distinct differences between the cells in the induction of chromosomal aberrations by 3 alkylating agents (N-methyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea and mitomycin C). The catalase activity of R-8 was 10-fold in comparison with the parental cells, but no obvious differences were seen in the activities of superoxide dismutase (SOD), glutathione peroxidase and glutathione reductase. Therefore, the elevated H2O2-resistance seemed to be associated with the enhanced catalase activity. The induction of chromosomal aberrations in two O2- generating systems--xanthine oxidase plus hypoxanthine (XO + HX), and paraquat--was compared between R-8 cells and the ordinary CHL cells. XO + HX produced chromosomal aberrations in the parental cells but not in the R-8, while paraquat induced almost the same level of aberrations in both cell lines. This finding suggests that different active oxygens are responsible for the induction of aberrations in these two O2- generating systems, i.e., H2O2 in XO + HX and O2- in paraquat.

Animals↗

PubMed1988

A comparative analysis of data on the clastogenicity of 951 chemical substances tested in mammalian cell cultures.

A literature review was conducted using original papers published during 1964-1985 on the in vitro clastogenicity of chemical substances. Results of tests on 951 chemical substances were abstracted from over 240 reports to form the database. The evaluation of these data relied on each author's original conclusion on a positive or negative outcome. Of these 951 substances, 447 (47%) were consistently positive either with or without activation; 417 (44%) were negative in the direct test but not tested with metabolic activation systems; 4 were negative but tested only with activation; and 30 (3%) were clearly negative both with and without activation. The remaining 53 substances gave variable results when tested under different experimental protocols or in different cell types, but were positive in at least one test. Although discrepant results were found associated with some cell types, the addition of metabolic activation systems tended to eliminate such variability. No one cell appeared to be superior in response to all clastogens. For screening purposes, the choice of cell may thus depend more on the general usefulness and reliability of a cell type than on a strong response to a particular chemical. However, the use of a suitable metabolic activation system does appear to be of critical importance. The concentration at which clastogenic effects were detected varied extensively for different test substances, ranging from a minimum of 4.3 X 10(-8) to 6.9 X 10(2) mM. Possible mechanisms of action for substances active at only high levels are discussed, but no satisfactory explanation is available at this time. The relevance of tests conducted at concentrations high enough to alter significantly the osmolarity and other culture conditions is considered, and caution urged in the interpretation of test results obtained under physiologically stressful conditions. The clastogenic potential was compared quantitatively using an index of effective concentration (D20) and one which estimates the number of cells with exchange aberrations expected per mg/ml (TR) for data obtained by using a uniform protocol and cultures of Chinese hamster lung (CHL) cells. Both values were distributed over a wide range, demonstrating the variety of genotoxic potential in chemicals. In general, a substance which was active at only high concentrations produced fewer exchange-type aberrations. In vivo activity, as measured by tumourigenic effect and formation of micronuclei in bone marrow, tended to be greater for substances with a D20 below 10(-2) mg/ml and a TR value over 10(3).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Micronucleus tests in mice on 39 food additives and eight miscellaneous chemicals.

A total of 47 chemical substances including 32 synthetic food additives, seven additives from natural sources, three trihalogenated methanes, two fluoro-compounds for dental use, one insecticide, and two other compounds were subjected to the micronucleus test in mice. Five compounds, i.e. chlorine dioxide, maltol, potassium bromate, sodium chlorite and sodium dehydroacetate, were found to induce micronuclei after a single ip injection. Potassium bromate, sodium chlorite and sodium dehydroacetate were tested further by oral administration, and potassium bromate showed a clearly positive result. Almost all the compounds which were negative with the single treatment were additionally tested by four or five multiple treatments, but none showed any indication of micronucleus induction.

Animals↗

PubMed1988

Isolation and characterization of ras-transfected BALB/3T3 clone showing morphological transformation by 12-O-tetradecanoyl-phorbol-13-acetate.

The transformation frequency of mouse BALB/3T3 cells was significantly enhanced after transfection with an activated ras oncogene (v-Ha-ras) followed by treatment with a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), suggesting that the ras oncogene acted as an initiator in two-stage carcinogenesis. A cell clone (Bhas42) containing the ras oncogene was isolated from the ras-transfected BALB/3T3 cells. Bhas42 cells were flat and showed contact inhibition, but the addition of TPA to quiescent Bhas42 cultures resulted in a dramatic change of cell morphology to spindle shape, doubling of the cell population, and increased DNA synthesis.

Animals↗