Three groups of ICR male and female mice were exposed to 50-Hz, sinusoidal, alternating, horizontal magnetic fields of 0.0 mT (sham), 0.5 mT and 5.0 mT (rms) for 9 and 2 weeks prior to mating for males and females, respectively, through fertilization and until cesarean sectioning. Fetuses were collected by cesarean section on the 18th day of gestation. Approximately half were randomly selected for skeletal examination and the remainder used for visceral examination. No significant differences were found between the field- and the sham-exposed groups in pre-, post- and total implantation losses; number of live fetuses; sex ratio; live fetal weight; number of externally abnormal fetuses; and numbers of fetuses with skeletal and visceral anomalies. These results suggest that exposure to power-frequency magnetic fields has no major effects on reproduction and development in mice, and do not support the association of EMF exposure with adverse reproductive effects suggested by epidemiology.

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Effect of Tinospora cordifolia extract on modulation of hepatoprotective and immunostimulatory functions in carbon tetrachloride (CCl₄) intoxicated mature rats is reported here. Administration of CCl₄ (0.7 ml/kg body weight for 7 days) produces damage in the liver as evident by estimation of enzymes such as serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transminase (SGPT) and alkaline phosphatase (ALP) as well as serum bilirubin level. CCl₄ administration also causes immunosuppressive effects as indicated by phagocytic capacity, chemotactic migration and cell adhesiveness of rat peritoneal macrophages. However, treatment with T. cordifolia extract (100 mg/kg body weight for 15 days) in CCl₄ intoxicated rats was found to protect the liver, as indicated by enzyme level in serum. A significant reduction in serum levels of SGOT, SGPT, ALP, bilirubin were observed following T. cordifolia treatment during CCl₄ intoxication. Treatment with T. cordifolia extract also deleted the immunosuppressive effect of CCl₄, since a significant increment in the functional capacities of rat peritoneal macrophages (PMφ) was observed following T. cordifolia treatment. The results of our experiment suggest that treatment by T. cordifolia extract may be the critical remedy for the adverse effect of CCl₄ in liver function as well as immune functions.

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As a part of the ILSI-HESI Alternative to Carcinogenicity Testing (ACT) program, we performed a 26-week carcinogenetic study of nonmutagenic drug, ampicillin (ABPC) in Tg-rasH2 mice. ABPC was given to Tg-rasH2 mice (0, 350, 1000, 3000 mg/kg, p.o.) and Non-Tg mice (0, 3000 mg/kg, p.o.) daily for 26 weeks. As a positive control, a single dose of MNU was administered once to Tg-rasH2 mice (75 mg/kg, i.p.). In this study, Tg-rasH2 mice did not demonstrate any increases in tumor development in response to ABPC. Thus, ABPC had no carcinogenicity in the 26-week carcinogenesis study in Tg-rasH2 mice or in a 2-year carcinogenesis study in B6C3F1 mice.

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To establish a method for processing lymphoid organs suited to morphological, immunohistochemical and enzyme histochemical analyses for assessment of immunotoxicity, we examined a combination of fixation with periodate-lysine-paraformaldehyde (PLP) fixative and embedding in paraffin by the AMeX method (PLP-AMeX method). Spleen and thymus removed from monkeys and rats were fixed in PLP fixative for 6 hours at 4°C. After fixation, specimens were processed and embedded in paraffin by the AMeX method. In hematoxylin and eosin-stained sections, tissue architecture was well preserved. In immunohistochemical staining, markers of T lymphocytes (CD3, CD4, CD8), B lymphocytes (monkey: CD20cy, rat: CD45RA) and macrophage (monkey; CD68, rat: ED-1) were well identified according to their specificities, although the staining intensity of CD8 in the monkey and CD4 in the rat were somewhat weaker in PLP-AMeX-prepared sections than in those frozen. In enzyme histochemical staining, alkaline phosphatase activity was well preserved in neutrophils. In toluidine blue- and Giemsa-stained sections, eosinophil granules and the metachromasia of granules in basophil/mast cells were clearly detectable. These findings suggest that the PLP-AMeX method is a powerful tool for assessment of immunotoxicity.

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This study was performed to compare the effects of naloxone (NLX) and post-tetanic stimulation on isolated guinea-pig ileum followed by prolonged exposure to morphine and bestatin. Morphine or bestatin alone did not induce any responses. In the presence of 1 μM morphine, the challenge with 1 μM naloxone caused quick contraction and post-tetanic contraction. A longer duration of these NLX-induced contraction and post-tetanic contractions was observed at the 6th stimulation compared to those after the 2nd stimulation. By contrast, the addition of bestatin, an aminopeptidase inhibitor, did not induce any NLX-induced contraction, although the same results for post-tetanic contraction as those of morphine were observed. These different effects of morphine and bestatin on NLX-induced contraction and post-tetanic contraction in the ileum may be due to different mechanisms of action in the opioid system. That in turn may suggest the possibility that bestatin has a physical dependence liability.

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Mutagenicity of soils sampled at median strips, roadsides and a park neighboring arterial roads in Kurume City was determined by Ames test. Organic extracts of soils were mutagenic in strains TA98, TA100, YG1041 and YG1042 with and without S9mix. No sample showed mutagenic responses in strains YG3003 or YG7108. Extracts from soils of median strips and beside intersections showed higher mutagenicity and concentrations of PAHs and heavy metals than others, and mutagenic activity of soils correlated significantly with concentrations of PAHs and heavy metals. However, PAHs accounted for less than 12% of total mutagenicity in strains TA98 and TA100 of soil extracts. These extracts showed much higher mutagenicity in YG strains than in TA strains. The results indicate that these soils may be polluted with nitroarenes and aromatic amines.

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In vitro cytotoxicity assay to screen compounds for apoptosis-inducing potential on lymphocytes and neutrophils was investigated. Mouse, rat, dog, and human whole blood were incubated for 4 and 6 hr with actinomycin D, camptothecin, cortisone acetate, cycloheximide, doxorubicin, etoposide, 5-FU, mitomycin C and puromycin. Apoptotic lymphocytes and neutrophils were counted. All test compounds induced in vitro apoptosis of lymphocytes and/or neutrophils, but there were different potencies among the test compounds and there were also species differences in susceptibility. To investigate the in vivo effects of etoposide and cycloheximide which induced apoptosis of rat lymphocytes and that of rat lymphocytes and neutrophils, respectively, in in vitro assay, rats were intravenously administered either etoposide at 12.5, 25 or 50 mg/kg or cycloheximide at 1.25, 2.5 or 5 mg/kg. Etoposide caused decreases of circulating lymphocytes at 3 hr after administration in a dose-dependent manner, -16, -25 and -51%. Although cycloheximide caused neither decreased lymphocyte nor neutrophil counts, apoptosis in 30% of neutrophils was observed in rats receiving 5 mg/kg at 3 hr after administration. Etoposide at 50 mg/kg and cycloheximide at 5 mg/kg caused lymphocyte apoptosis in the spleen, thymus, mesenteric lymph nodes, bone marrow, and Peyer's patch from 1 to 6 hr after administration, with the maximum changes at 3 hr. In addition to apoptosis of these organs, cycloheximide at 5 mg/kg caused apoptosis of polymorphonuclear cells in the lamina propria of the small intestine. Therefore, it was found that the changes seen in the in vivo experiments considerably reflected the changes seen in the in vitro experiments. From these results, apoptosis is probably one of the major mechanisms for leukocyte toxicity induced by cytotoxic compounds, and the in vitro assay to screen compounds for acute apoptosis-inducing potential on lymphocytes and neutrophils would be useful as a primary screening method for animal toxicity studies. It may also be useful for risk assessments in advance of clinical trials.

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The local lymph node assay has recently been accepted by regulatory agencies as a stand-alone alternate method for predicting allergic contact dermatitis. To compare the sensitivity of non-radioisotope methods with that of the standard assay, we determined if these modified methods would affect evaluation of sensitization potency. For this reason, we used 2,4-dinitrochlorobenzene (DNCB) and benzocaine for different sensitizing criteria. Female CBA mice were treated for 3 days with a test compound or vehicle applied to each side of both ears. Bilateral auricular lymph node proliferative activity was assessed by the following endpoints with incorporation of ³H-methyl thymidine (3H-TdR), bromodeoxyuridine (BrdU) in vivo, and BrdU ex vivo, IL-2 production, and proliferating cell nuclear antigen (PCNA) expression. Ear thickness was also tested. The strong sensitizer DNCB was detectable by any of the non-radioisotope endpoints as well as by radioisotope-dependent standard assay. On the other hand, when evaluating the weak sensitizer benzocaine, significant changes were evident in BrdU incorporation ex vivo and in vivo, and IL-2 production. We believe that these non-radioisotope methods can assess allergic contact dermatitis caused by chemicals even in the laboratory, where it can be difficult to handle radioisotopes.

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The articular toxicity of garenoxacin (formerly T-3811 or BMS-284756) was experimentally examined utilizing juvenile beagle dogs. Garenoxacin and two other reference quinolones were administered at intravenous dosages of 30 and 60 mg/kg. Each group consisted of 3 male dogs (Experiment I). Oral dosages of 50 mg/kg of 3 compounds were also given daily to male only and female only groups (Experiment II) over a period of 7 days. We evaluated the articular toxicity of garenoxacin compared to ciprofloxacin and norfloxacin. In Experiment I, no articular toxicity was detected in the 30 mg/kg garenoxacin group. One animal from the 60 mg/kg garenoxacin group developed detectable histopathological lesions in the articular cartilages of the shoulder, elbow and knee joints. In the 30 mg/kg ciprofloxacin group and the 30 and 60 mg/kg norfloxacin groups, histopathological articular cartilage lesions of the shoulder, elbow, carpus, hip, knee and tarsus joints were observed in all of the dogs. The area under the plasma concentration-time curve (AUC_0→∞) values, after the first dose was administered, for the 30 mg/kg groups given garenoxacin, ciprofloxacin and norfloxacin were 164, 68.1 and 65.7 μg·hr/mL, respectively. In Experiment II, the degree of histopathological change was most significant in the ciprofloxacin group, followed by the norfloxacin group, and with comparatively the least changes in the garenoxacin group. The AUC_0→∞ values, obtained after the 6th day of antimicrobial administration, were 202 and 173 μg·hr/mL for male and female dogs, respectively, from the 50 mg/kg garenoxacin group. The AUC_0→∞ values for the garenoxacin group after the 6th daily administration were 7.8 to 17.0 times greater for male dogs and 3.8 to 13.2 times greater for female dogs than those obtained from the ciprofloxacin and norfloxacin groups. The concentrations of garenoxacin in the synovia, articular cartilage and the synovialis 4 hr following the last garenoxacin administration were 2.0 to 6.5 times higher for male dogs and 1.5 to 3.3 times higher for female dogs than the antimicrobial levels measured in the ciprofloxacin and norfloxacin groups. As discussed above, although the garenoxacin concentrations in plasma and joint tissue were higher than those for ciprofloxacin and norfloxacin, however, the articular toxicity of garenoxacin was much less than that of the other two antimicrobials.

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We investigated and compared the cytotoxicity of 16 reference compounds in four in vitro systems: primary cultured rat hepatocytes, hepatoma HepG2 cell line, non-hepatic HeLa and Balb/c 3T3 cell lines. After 24 hr of exposure to the test compounds, the water-soluble tetrazolium salts WST-1 assay was used as an endpoint to evaluate cytotoxicity. Acetaminophen, diclofenac sodium cyclophosphamide and disulfiram displayed from 2 to more than10 times higher IC₅₀ values in three cell lines than in rat primary cultured hepatocytes. The cytotoxic effects of aspirin, amiodarone, clorfibiric acid, chlorpromazine, erythomycin, lithocholic acid, cisplatin and quinidine in rat hepatocytes were similar or 2 times stronger than those observed in cell lines. Ketoconazole resulted in the lowest IC₅₀ value in the HeLa cell line. The data suggested that the compounds which are known to be metabolism-mediated liver toxicants have a differential hepatotoxicity in vitro and that primary cultured rat hepatocytes could represent a valuable tool for both screening and study of the effects of bio-transformation on the cytotoxicity of new chemical entities and xenobiotics in vitro.

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