Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that can be activated by xenobiotics and natural fatty acids. To assess the potential physiological activity of PPAR ligands on cardiac muscular cells, the effects of PPARα agonist, WY-14,643, on both rat hearts and a rat cardiomyocyte cell line (H9c2 cells) were investigated. Male F344 rats were fed a diet containing WY-14,643 at a concentration of 100 ppm for 26 weeks. Cardiac muscular hypertrophy was revealed by morphometric analysis in which the diameter of the muscular fibers in WY-14,643-treated rats was larger than those of control rats. Using H9c2 cells in vitro, the protein content per cell was increased in a dose-dependent manner with the treatment of WY-14,643. The transcription of myosin light chain-2(MLC-2), a parameter of myocardial hypertrophy, was increased in H9c2 cells transfected with the rat MLC-2/luciferase fusion gene by WY-14,643 as well as other peroxisome proliferators, clofibrate and di(2-ethylhexyl) phthalate. In addition, accumulation of myosin light chain protein was confirmed in H9c2 cells treated with WY-14,643 at 10 μg/ml for 7 days or more by immunohistochemistry. These results suggest that PPARα ligands have a potential to regulate MLC-2, which is a contractile protein in cardiomyocytes and may play a part of role in the pathogenesis of cardiac hypertrophy.
This collaborative study was conducted to determine the utility and sensitivity of nine sperm motion parameters generated by a Hamilton-Thorne Sperm Analyzer (HTM-IVOS) for detecting adverse effects of chemicals on sperm motion in rats. The efficacy of sperm motion parameters was investigated using nine reproductive toxicants: adriamycin, α-chlorohydrin (3 different studies were carried out), dinoseb, ethylene glycol monoethyl ether, 2,5-hexanedione, sulfasalazine, trimethyl phosphate, and ornidazole. The percentage of motile sperm (% motile sperm), the only parameter expressing the status of semen containing non-motile sperm, detected adverse effects on sperm motion in 9 out of 10 studies. However, weak effects on sperm motion were not detected by this parameter in 4 out of 7 studies in which sperm motion disorders were noted at medium or low dosages. The percentage of progressively motile sperm (% progressive sperm) and the sperm velocity parameters (average path velocity, straight line velocity, and curvilinear velocity) detected adverse effects on sperm motion in all studies. In 7 studies which noted sperm motion disorders at medium or low dosages, weak effects on sperm motion were detected by the % progressive sperm in 5 studies and by the sperm velocity parameters in 6 studies. In 10 studies, amplitude of lateral head displacement (ALH) did not detect adverse effects on sperm motion in 4 studies, and beat cross frequency (BCF) failed to detect adverse effects on sperm motion in 3 studies. Because ALH and BCF show the swimming pattern of spermatozoa as head movement, the characteristics of these parameters are different from the % progressive sperm and the sperm velocity parameters. Straightness (STR) and linearity (LIN), which are secondary parameters calculated from sperm velocity parameters, could not detect adverse effects on sperm motion when the sperm velocity parameters did not detect adverse effects. On the basis of these results, we concluded that the % progressive sperm and sperm velocity parameters are useful and sensitive indicators for detecting adverse effects on sperm motion. However, in the % progressive sperm, setting up a suitable threshold of VAP and/or STR is important to gain further sensitivity for detecting adverse effects on sperm motion. The % motile sperm is useful for assessment of sperm motion disorder, and ALH and BCF are useful for evaluating the swimming pattern of sperm. STR and LIN are not very useful for detecting adverse effects on sperm motion.
The toxicities of 4-nitrophenol and 2,4-dinitrophenol in newborn and young rats was examined and the susceptibility of newborn rats was analyzed in terms of presumed unequivocally toxic and no observed adverse effect levels (NOAELs). In the 18-day repeated dose newborn rat study, 4-nitrophenol was orally given from Day 4 to Day 21 after birth but did not induce any toxicity up to 160 mg/kg in the main study, although it induced death in one of six males at 160 mg/kg, and three of six males and one of six females at 230 mg/kg in a prior dose-finding study. In the 28-day repeated dose oral toxicity study starting at 6 weeks of age, 4-nitrophenol caused the death of most males and females at 1,000 mg/kg but was not toxic at 400 mg/kg except for male rat-specific renal toxicity. As unequivocally toxic levels were considered to be 230 mg/kg/day in newborn rats and 600 to 800 mg/kg/day in young rats, and NOAELs were 110 mg/kg/day in newborn rats and 400 mg/kg/day in young rats, the susceptibility of the newborn to 4-nitrophenol appears to be 2.5 to 4 times higher than that of young animals. In the newborn rat study of 2,4-dinitrophenol, animals died at 30 mg/kg in the dose-finding study and significant lowering of body and organ weights was observed at 20 mg/kg in the main study. In the 28-day young rat study, clear toxic signs followed by death occurred at 80 mg/kg but there was no definitive toxicity at 20 mg/kg. As unequivocally toxic levels and NOAELs were considered to be 30 and 10 mg/kg/day in newborn rats and 80 and 20 mg/kg/day in young rats, respectively, the toxicity of 2,4-dinitrophenol in newborns again seems to be 2 to 3 times stronger than in young rats. Abnormalities of external development and reflex ontogeny in the newborn were not observed with either chemical. Based on these results, it can be concluded that the toxic response in newborn rats is at most 4 times higher than that in young rats, at least in the cases of 4-nitrophenol and 2,4-dinitrophenol.
The usefulness of sperm analyses in dogs, including sperm motion analysis, was investigated. Sperm motion analysis was performed with the CellSoft-4000 computer-assisted sperm analysis (CASA) system. First, we examined the conditions for preservation of optimal semen quality. We found that sperm retained more of their motility at 4°C than at 37°C. Secondly, we observed sperm motion, concentration and morphology in dog semen continuously for 11 weeks. We collected semen samples during the test period, and the samples retained sperm motion, concentration and morphology. Finally, we administrated α-chlorohydrin, which decreases rodent sperm motion, at a single oral dose of 100 mg/kg or 150 mg/kg to dogs. Sperm motion was inhibited immediately after α-chlorohydrin treatment, and recovered after 2 weeks. None of the experimental animals were sacrificed in the above-mentioned examinations. We thus confirmed that sperm analyses including motion analysis in dogs are useful in male fertility studies.
We previously reported that both hyperthermia and hypothermia induced micronuclei in mouse bone marrow cells (Asanami and Shimono, 1997a, 1997b, 1999). To investigate the effects of temperature on chromosome aberration in vitro, we conducted chromosome aberration and micronucleus tests under hyper- and hypothermic conditions using Chinese hamster lung (CHL) cells. In the chromosome aberration test, we observed positive responses at 40°C and 41°C for 24 hr, and at 42°C for 6 hr and over. In the micronucleus test, we observed positive responses at 31°C, 33°C, and 40°C for 24 hr, and at 42°C for 2 hr. The results suggest that in CHL cells, hypothermic conditions can induce micronuclei while hyperthermic conditions can induce both chromosome aberrations and micronuclei.
The levels of 20 dioxin congeners, 7 polychlorinated dibenzo-p-dioxins (PCDDs), 10 polychlorinated dibenzofurans (PCDFs) and three coplanar polychlorinated biphenyls (Co-PCBs), in bile were examined in comparison with those in the blood and liver, in 27 autopsy cases. Total-TEQ values were the same in the bile (43.2±30.9 pg TEQ/g lipid) and blood (43.1±24.2 pg TEQ/g lipid), and three times higher in the liver (127.8±57.4 pg TEQ/g lipid). Highly chlorinated PCDDs and PCDFs have a tendency to accumulate in the liver, and their levels in bile and blood were relatively low compared with those in the liver, with 1,2,3,4,6,7,8-heptachlorodibenzofuran having the highest tendency among the 20 congeners. Daily excretion in bile was calculated to be 54 pg TEQ at age 65, by assuming that daily bile secretion is 750 ml and is concentrated 7.5-fold in the gallbladder. The correlation between bile and blood total-TEQ was high, with a correlation coefficient of 0.89 among the 27 autopsy cases. Thus, the regression equation of y=1.14x−6.02 will provide us levels in bile (total TEQ per g lipid), knowing the blood total-TEQ level, where x is total-TEQ per g lipid of the blood. Furthermore, accumulation of dioxins was estimated to be 0.99, 0.70 and 1.91 pg TEQ/g lipid/year in bile, blood and liver, respectively.
To clarify toxic effects of long-term oral administration of low dose cadmium (Cd) on the liver and kidney, six groups of female Sprague-Dawley rats were fed a diet containing Cd-polluted rice or CdCl2 at concentrations up to 40 ppm, and killed after 12,18, and 22 months. With toxicological parameters, including histopathology, there was no evidence of Cd-related hepato-renal toxicity, despite a slight decrease of mean corpuscular volume and mean corpuscular hemoglobin of red blood cells with 40 ppm CdCl2. Dose-dependent accumulation of Cd was observed in the liver and kidneys with peak levels of 130±42 μg/g and 120±20 μg/g, respectively, at 18 months in animals treated with 40 ppm CdCl2. A dose-dependent increase in urinary Cd levels became evident with time. Induction of metallothionein (MT) was also observed in the liver and kidney with a high correlation to the corresponding Cd levels. In the proximal renal tubular epithelia of 40 ppm CdCl2-treated rats at 22 months, prominent accumulation of Cd was observed in secondary lysosomes associated with MT deposits in their exocytotic residual bodies. The results demonstrated that, in contrast to the case with high-dose Cd-administration, renal toxicity is not induced by long-term oral administration of low amounts of Cd, although tissue accumulation does occur. Possible protective mechanisms may be operating.