Rats exposed to liquid mosquito repellent (LMR) containing allethrin (3.6% w/w) 8-hrs/ day for a period of ninety days did not produce any signs of toxicity or death. Significant increases in relative weight of liver and adrenal in males, brain and thyroid in females were observed. No significant changes were noticed in clinical enzyme profile, gonadal enzymes and histopathology of vital organs except mild changes in the activities of liver and serum alkaline phosphates (ALP), testicular glucose-6-phosphate dehydrogenase (G-6PDH) and epididymal sorbitol dehydrogenase (SDH). Rats exposed to LMR for one generation had not produced changes in their reproductive indices such as fertility index, gestation index, live pups/ dam and sex ratio. There was no change in the preweanling evaluation of pups such as survival and growth index on post-natal days (PND) 0, 4, 7,14 and 21. No significant pathomorphological changes were observed in liver, brain, kidney and gonads of PND 21 pups. Absences of any major adverse effects in the adult as well as weanling rats suggest the safe use of allethrin-based LMR.
Para- and ortho-chloronitrobenzene (p- and o-CNB) were compared for subchronic toxicity by feeding F344 rats and BDF1 mice of both sexes p-CNB-or o-CNB-containing diets at 5 different concentrations for 13 weeks. The two isomers induced hematotoxicity and hepatotoxicity of different toxic potencies. p-CNB produced an anemic sign of external appearance in rats and mice, while o-CNB did not. Significant increases in the incidences of increased erythropoiesis in the bone marrow and increased extramedullary hematopoiesis in the spleen and liver, and in serum total bilirubin in rats and mice appeared at lower dose levels of p-CNB than o-CNB. A significant increase in serum ALT activity appeared at lower dose levels of o-CNB than p-CNB, together with appearance of both necrosis and hydropic degeneration of hepatocytes only in the o-CNB-fed rats and nuclear enlargement with atypia of hepatocytes only in the o-CNB-fed mice. BMDL10s of p- and o-CNB for the hematotoxic endpoint, substitutes for NOAELs, were 0.177 mg/kg/day and 1.03 mg/kg/day for the rats, respectively. For the mice, the NOAELs of p-and o-CNB for the hematotoxic endpoint were 10.5 mg/kg/day and 10.4 mg/kg/day, respectively. A NOEL of o-CNB for the hepatotoxic endpoint resulted in 13.8 mg/kg/day for the rats and 12.2 mg/kg/day for the mice. These results suggest that p-CNB is a more potent hematotoxicant than o-CNB, whereas o-CNB is a more potent hepatotoxicant than p-CNB, and that the rat hematopoietic system is more susceptible to p-CNB than the mouse hematopoietic system.
For the establishment of a high throughput screening system using primary cell cultures, investigation of elucidated toxicities to assess the correlation between in vitro and in vivo hepatotoxicity is necessary in the safety evaluation of the compound. In the previous study, we reported the usability of rat primary cultured hepatocytes for establishment of high throughput screening system. To confirm the reliability of rat primary hepatocytes culture screening system, we conducted a single-dose in vivo study with relatively high dose of hepatotoxicant in rats using 4 reference compounds (acetaminophen, amiodarone, tetracycline, carbon tetrachloride), and investigated histopathological changes and expression of oxidative stress-related proteins by immunohistochemistry. We also carried out a proteomics analysis for estimating the reliable and sensitive biomarkers. Histopathologically, compound-specific hepatotoxicity was detected at 24 hr after administration in all compounds except amiodarone, which is known to induce phospholipidosis. Immunohistochemically, oxidative stress-related proteins were increased within 6 hr after administration in all treated groups. Proteomics analysis revealed several protein biomarkers related to oxidative stress and mitochondrial metabolism-regulation, which had been previously detected by proteomics analysis in in vitro screening system. Oxidative stress-related proteins were considered as useful biomarkers of hepatotoxicity; since they were detected by immunohistochemistry and proteomics analysis prior to appearance of compound-specific histopathological changes detected by light microscopy. Considering the relevance of in vitro system to in vivo system from the aspect of new biomarkers related to the toxicogenomics/toxicoproteomics, in vitro primary cell culture system would be sufficient to detect hepatotoxicity in the early stage of drug discovery.
We purified male rat urinary α2u-globulin, prepared the antibody in rabbits, and improved an immunohistochemical detection method using this antibody for male rat-specific α2u-globulin accumulation appearing as hyaline droplets in the kidneys. Our prepared antibody reacted specifically with α2u-globulin in both immunohistochemical and Western blotting analyses, furthermore, and the graded immuno-reactivities on the slide were well associated with computational image analyzing results. Using this method, we retrospectively analyzed the renal sections from the toxicity studies of 12 nephrotoxic chemicals, which had already been conducted under the Japanese Existing Chemicals Survey Program. We demonstrated that the hyaline droplets induced by treatment with 10 chemicals (1,4-dibromobenzene, dicyclopentadiene, 3,4-dimethylaniline, 1,4-dicyanobenzene, tetrahydrothiophene-1,1-dioxide, 1,3-dicyanobenzene, acenaphthene, 3,4-dichloro-1-butene, 3a,4,7,7a-tetrahydro-1H-indene and 3,5,5-trimethylhexan-1-ol) were directly associated with α2u-globulin accumulation. This immunohistochemical method is convenient for applying, even retrospectively, paraffin sections from general toxicity studies and could be useful for qualifying male rat-specific hyaline droplets consisting of α2u-globulin and renal risk in humans.
We investigated the overall protein expression profiles in the in vivo hepatotoxicity of rats induced by four well-recognized hepatotoxicants. Acetaminophen (APAP), amiodarone (AMD), tetracycline (TC) and carbon tetrachloride (CTC) were administered to male rats by gavages and the liver at 24 hr post-dosing was applied to the proteomic experiment. Blood biochemistry and histopathology were examined to identify specific changes related to the compounds given. Protein expression in the liver was investigated by 2-dimensional gel electrophoresis (2DE), and spots showing a significantly different expression in treated versus control group were excised from gels and identified by Q-Tof mass spectrometer. They were well characterized based on their functions related to the mechanisms of toxicity of the compounds. Among them, we focused on the 8 proteins that were affected by all 4 compounds examined. Proteins related to oxidative stress response such as carbonic anhydrase III (CA3) and 60kDa heat shock protein (HSP60), and energy metabolism such as adenylate kinase 4 (AK4) were found. Moreover, hierarchical clustering analysis using 2D-gel spots information revealed the possibility to differentiate the groups based on their toxicity levels such as severity of liver damage. These results suggested that assessing the effects of hepatotoxicants on protein expression is worth trying to screen candidate compounds at the developmental stage of drugs.
Gentamicin (GM) has been widely used as an antibiotic and its nephrotoxicity has been recognized. However, the alternation of heat shock protein (HSP) 72 as an inductive protein in proximal tubular cells treated with GM is still unclear. In this study, GM cytotoxicity and its effect on the expression of HSP72 in human kidney proximal tubular (HK-2) cells were measured. HK-2 cells were incubated for 24 hr, 48 hr, 72 hr, and 96 hr with GM only and GM plus MnCl2, respectively. Cytotoxicity was determined by the release of lactate dehydrogenase (LDH). Activity of N-acetyl-β-D-glucosaminidase (NAG) and effects of GM on oxidation in HK-2 cells were investigated by measurements of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, and the ability of viable cells to reduce a tetrazolium-based compound (MTT). The expression of HSP72 was measured by immunocytochemistry, Western blotting and RT-PCR. Cells were exposed to GM at a concentration of 100 μg/ml. After 24 hr MTT uptake decreased significantly and then gradually until 96 hr. LDH release increased time-dependently from 24 hr to 72 hr, but decreased at 96 hr compared with the data at 72 hr when cells were treated with GM only. Both results of NAG and SOD activities and results of MDA content were similar to that of the LDH release. The amount of HSP72 positive cells increased at 24 hr after exposure to GM up to 72 hr. HSP72 expression increased significantly from 24 hr, and reached its peak at 72 hr when cells were treated with GM only. Furthermore, the change of the HSP72 gene transcription was similar to the expression of HSP72. These results demonstrated that GM treatment could induce damage to HK-2 cells and that the expression of HSP72 increased when cells were injured by GM.
The immunotoxicity of anticancer drugs has been measured with whole blood TNF-α production induced by LPS-stimulation. Cis-platinum (CDDP) or 5-fluorouracil (5-FU) was given intravenously to mice once a day for three days. The amount of produced-TNF-α lowered significantly in CDDP 2 mg/kg and 5 mg/kg, while the number of leukocytes scarcely changed even when a significant decrease in body weight was observed at the dose of 5 mg/kg. On the other hand, 5-FU 20 mg/kg also caused a significant decline in the amount of produced TNF-α but it showed no apparent effects on the number of leukocytes and the body weight. These results indicate that LPS-induced TNF-α production is a more sensitive measure than leukocyte count used conventionally to detect the immunotoxicity of two anticancer drugs, CDDP and 5-FU, and suggest that it might be a more practical index for hazard identification in clinical treatment with anticancer drugs.
Semen samples were obtained from 30 volunteers who had never consumed betel quid. Swim-up spermatozoa from the 30 seminal samples of non-betel quid chewers and also non-smokers, usually not exposed to passive smoking, were treated in vitro with arecoline at different concentrations to evaluate the action of these drugs on sperm motility. Highly motile sperms were collected and divided into 5 equal fractions. Four fractions were supplemented with various concentrations of arecoline and one as control. The study was carried out at time 0 and +1, +2, +3 and +4 hr of incubation. Sperm cells were also extracted and blotted with COX-2 antibody after arecoline treatment after 4 hr incubation. The sperm motility parameters, i.e., motility, average path velocity, curvilinear velocity, straight-line velocity and linearity, were significantly decreased after arecoline treatment. In vitro, arecoline induces the COX-2 expression of sperm cells in a dose-dependent manner. This is the first report to demonstrate that arecoline may mediate COX-2 expression in human sperms, resulting in inflammation response. This situation may act on the structure responsible for the flagellar motion and cause the reduction of sperm motility.
The present study was undertaken to elucidate restraint effects of protecting jackets that are used to protect against biting the sites of percutaneous application in pregnant rabbits. Animals wore protecting jackets from gestational Day (GD) 0 to 19 (GD 0 group) or from GD 6 to 19 (GD 6 group). Major restraint effects were decreases in body weight gain and food consumption, which were marked during the first 2 to 13 days in jackets, and diminished thereafter. Therefore, the animals in the GD 0 group had almost recovered from the effects on GD 16, but those in the GD 6 group were still under the effects of restraint on GD 19. The above findings indicate that early start of habituation to protection jackets will provide better results in practical reproduction studies using percutaneous application in rabbits.