Simultaneous measurement of nucleated cell counts and cellular differentials in rat bone marrow examination using flow cytometer.

The purpose of this study was to establish the simultaneous measurement of nucleated cell counts and cellular differentials in rat bone marrow examination. The bone marrow cells were stained with an anthraquinone fluorescent DNA stain (DRAQ5) and fluorescence-labeled antibodies, and were analyzed quantitatively using a flow cytometer in the presence of internal standard beads. DRAQ5 distinguished populations of nucleated cells. The absolute counts of nucleated cells were determined using an internal standard, and were equivalent to that measured by the electrical resistance method. The population of nucleated cells was classified into myeloids and erythroids by labeling with CD11b/c and CD71 antibodies, respectively. In a separate examination, T- and B-lymphocytes were also classified by labeling with CD3 and CD45RA antibodies, respectively. The classification of each cell lineage was identical with that of the alternative flow-cytometric method in which cells were differentiated according to cellular size and the fluorescence of a peroxidase indicator, 2',7'-dichlorofluorescin. The ratios of cell lineage, together with myeloid/erythroid ratio (ME), were the same as those obtained by a manual microscopic method. The present flow cytometric method enables the simultaneous measurement of the total nucleated cell counts and cellular differentials of rat bone marrow cells, allowing for rapid and highly quantitative bone marrow examination in rats.

Correlation among clinicopathological parameters of myocardial damage in rats treated with isoproterenol.

The correlation among clinicopathological parameters of myocardial damage was investigated in rats administered a single subcutaneous dose of isoproterenol at 0 (saline), 0.04, 0.4 and 4 mg/kg. Total lactate dehydrogenase (LDH), creatine kinase (CK), and their isoenzymes (LDH-1, LDH-2 and CK-MB), as well as troponin I and tropinin T, were measured 4 h after the administration of the drug. Troponin I was determined by a chemiluminescence method using Bayer Centaur and DPC Immulyze, as well as by ELISA. Troponin T was assayed semi-quantitatively using Trop T Sensitive. A high correlation was found among LDH isoenzymes, troponin I (Centaur) and troponin T. The present result provides a baseline for interpreting changes in the different parameters of myocardial damage assayed by different methods in toxicity studies.

Battery of tests for profiling abnormalities of vitamin K-dependent coagulation factors in drug-toxicity studies in rats.

A battery of simple tests for profiling abnormalities of vitamin K-dependent coagulation factors encountered in drug-toxicity studies was verified in rats treated with warfarin (3 and 10 mg/kg, p.o). The thrombotest, or hepaplastin-test, is useful as a follow-up test after routine screening tests for coagulation abnormalities based on PT and APTT, to rule out other coagulation-factor abnormalities. Measurement of coagulation factor activities (factors II, VII, IX and X) using factor-deficient human plasmas provides direct evidence of decreased activities of vitamin K-dependent factors. Furthermore, Echis carinatus venom coagulation time, together with factor II activity, allows us to confirm the generation of PIVKA-II.

Lack of elevated liver carcinogenicity of aminophenylnorharman in p53-deficient mice.

The hepatocarcinogenic potential of 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH) was investigated using male and female p53 deficient mice. Incidence of oval cell hyperplasia was 2/14 (14.3%), 14/23 (60.9%), and 2/10 (20%) in p53 nullizygous (-/-), heterozygous (+/-), and wild type (+/+) mice, respectively, exposed to 30 ppm APNH for 15 weeks, while hepatocellular anisonucleosis was observed only in APNH-treated p53 (-/-) mice. At 40 weeks, hepatocellular carcinomas had developed in 16/46 (34.8%) and 10/27 (37.0%) of female p53 (+/-) and (+/+) mice in contrast to only 1/45 (2.2%) and 2/12 (16.7%) in their male counterparts, respectively, without any detectable p53 gene mutations. Dose-dependent APNH-DNA adduct formation and transcriptional induction of CYP 1A1, but not CYP 1A2, was revealed with 7-day APNH treatment using female C57BL/6J mice. These results suggested hepatocarcinogenicity of APNH in mice could be linked to the liver microenvironment including hormonal milieu but independent of p53 expression and p53 gene mutations.

High susceptibility of nullizygous p53 knockout mice to colorectal tumor induction by 1,2-dimethylhydrazine.

PURPOSE: The susceptibility of male p53 nullizygote (-/-), heterozygote (+/-), and wild-type (+/+) mice to 1,2-dimethylhydrazine (DMH) induction of colon carcinogenesis was investigated. METHODS: In a preliminary short-term experiment, male mice of three genotypes were given s.c. of 20 mg/kg DMH once weekly for 5 weeks. In a medium-term experiment, mice were given weekly s.c. of DMH for 15 weeks. In a long-term experiment, male p53 (+/-) and (+/+) mice were given weekly injections of DMH for 15 weeks, and killed at week 30. RESULTS: In the medium-term experiment, carcinomas were observed in 70% of p53 (-/-) mice, although there were no carcinomas in p53 (+/+) and (+/-) mice. In the long-term experiment, there was no significant difference in incidences of adenomas and carcinomas between p53 (+/+) and (+/-) mice. PCR-single strand conformation polymorphism analysis of exons 5-8 of p53 gene revealed four mutations in one focal atypia, one adenoma, and two carcinomas, out of 56 colonic proliferative lesions in the medium- and long-term experiments. CONCLUSIONS: These results suggest that p53 might not be a direct target of DMH but complete loss of p53 might elevate susceptibility to DMH-induced colorectal carcinogenesis.

Elevated susceptibility of the p53 knockout mouse esophagus to methyl-N-amylnitrosamine carcinogenesis.

Mutations of the p53 tumor suppressor gene constitute one of the most frequent molecular changes in a wide variety of human cancers, including those in the esophagus. Mice deficient in p53 have recently attracted attention for their potential to identify chemical genotoxins. In this study we investigated the susceptibility of p53 nullizygous (-/-), heterozygous (+/-) and wild-type (+/+) mice to methyl-N-amylnitrosamine (MNAN), which specifically induces esophageal tumors in mice and rats. The p53 (+/-) and (+/+) mice were treated with 5 or 15 p.p.m. MNAN in their drinking water for 8 weeks then maintained without further treatment for an additional 7 or 17 weeks, being killed at experimental weeks 15 or 25. An additional group of p53 (-/-) mice were given 5 p.p.m. MNAN for 8 weeks and killed at week 15. At 15 weeks in the 5 p.p.m. groups, squamous cell carcinomas (SCCs) were observed in 10/12 (83.3%) p53 (-/-) and 1/15 (6.7%) p53 (+/-) mice, but in none of the p53 (+/+) mice. In the animals receiving 15 p.p.m., 2/14 (14.3%) p53 (+/-) and 1/11 (9.1%) p53 (+/+) mice developed SCCs. At 25 weeks, the incidence of SCCs was 7/16 (43.8%) and 8/14 (57.1%) in p53 (+/-) mice and 1/13 (7.7%) and 2/10 (20.0%) in p53 (+/+) mice at 5 and 15 p.p.m., respectively. Of the SCCs examined by PCR-single strand conformation polymorphism analysis, 61% (14/23) from p53 (+/-) and 50% (6/12) from p53 (+/+) mice demonstrated mutations in the p53 gene (exons 5-8). These results indicate the order of susceptibility to MNAN-induced esophageal tumorigenesis to be as follows: nullizygotes (-/-) > heterozygotes (+/-) > wild-type (+/+), and provide strong evidence of involvement of p53 mutations in the development of esophageal SCCs.

Summation of initiation activities in the liver after partial hepatectomy.

Summation of initiation activities of different carcinogens in the liver after partial hepatectomy (PH) was investigated with reference to induction of glutathione S-transferase placental form (GST-P) positive foci. Firstly, effects of repeated administration of 1,2-dimethylhydradine (DMH) were compared with the results of a single administration of the same total dose (Expt. I). Subsequently, we studied summation of initiation potential with serial administration of DMH with diethylnitrosamine (DEN) or N-bis (2-hydroxpropyl)-nitrosamine (DHPN). In Expt. I, induction of GST-P-positive foci by multiple low-dose administration was equal to that with the single large-dose treatment. In order to avoid toxicity in hepatectomized rats, the low repeated-dose approach appeared superior. In Expt. II, the numbers of GST-P-positive foci in the groups treated with DMH plus DHPN or DMH plus DEN were significantly higher than those in the groups receiving the carcinogens singly. It is concluded that there is summation of initiation potential with doses of a single or multiple carcinogens. These results suggest that the present initiation assay model is useful to investigate summation of initiation activities of various environmental chemicals.