Akio Ogata

Effects of Maternal Exposure to Imazalil on Behavioral Development in F1‐Generation Mice

Female mice were exposed maternally to imazalil through diet to provide levels of 0 (control), 0.0006, 0.0018, and 0.0054% during gestation and lactation periods, and selected reproductive and neurobehavioral parameters were measured in F₁ generation. There was no adverse effect of imazalil on litter size, litter weight, or sex ratio at birth. With respect to behavioral developmental parameters, surface righting on postnatal day 4 of male offspring was delayed significantly in a dose-related manner (p < 0.05). Regarding exploratory behavior in the F₁ generation, movement time was significantly long (p = 0.0206) in the low-dose group of males at 8 weeks of age. Spontaneous behavior examination in males indicated that movement time increased but in females decreased in the low-dose groups in the F₁ generation. The dose levels of imazalil in the present study produced some adverse effects in neurobehavioral parameters in mice.

Inhibition of aromatase activity by green tea extract catechins and their endocrinological effects of oral administration in rats.

We orally administered polyphenone-60 (P-60), green tea extract catechins, in the diet (0, 1.25 and 5%) to male rats for 2, 4 and 8 weeks initiated at 5 weeks old. It was found that a 5% dose to male rats for 2-8 weeks induced goiters and decreased weights of the body, testis and prostate gland. Endocrinologically, elevating plasma thyroid stimulating hormone (TSH), luteinizing hormone (LH) and testosterone levels and decreasing tri-iodothyronine (T(3)) and thyroxine (T(4)) levels were induced by this treatment. We also found that P-60 as a whole and some of its constituents exhibited inhibitory effects on human placental aromatase activity by in vitro assay. The concentration of P-60 that required producing 50% inhibition of the aromatase activity (IC(50) value) was 28 microg/ml. The IC(50) values of (-)-catechin gallate (Cg), (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCg) and (-)-gallocatechin gallate (GCg) were 5.5 x 10(-6), 1.0 x 10(-4), 6.0 x 10(-5) and 1.5 x 10(-5) M, respectively. (-)- Epicatechin gallate (ECg) at 1.0 x 10(-4) M produced 20% inhibition. (-)-Epicatechin (EC) and (+)-catechin (CT) exhibited no effects on aromatase activity. The endocrinological changes observed in vivo were in conformity with antithyroid effects and aromatase inhibition effects of P-60 and its constituents.

Multi-walled carbon nanotubes translocate into the pleural cavity and induce visceral mesothelial proliferation in rats

Multi‐walled carbon nanotubes have a fibrous structure similar to asbestos and induce mesothelioma when injected into the peritoneal cavity. In the present study, we investigated whether carbon nanotubes administered into the lung through the trachea induce mesothelial lesions. Male F344 rats were treated with 0.5 mL of 500 μg/mL suspensions of multi‐walled carbon nanotubes or crocidolite five times over a 9‐day period by intrapulmonary spraying. Pleural cavity lavage fluid, lung and chest wall were then collected. Multi‐walled carbon nanotubes and crocidolite were found mainly in alveolar macrophages and mediastinal lymph nodes. Importantly, the fibers were also found in the cell pellets of the pleural cavity lavage, mostly in macrophages. Both multi‐walled carbon nanotube and crocidolite treatment induced hyperplastic proliferative lesions of the visceral mesothelium, with their proliferating cell nuclear antigen indices approximately 10‐fold that of the vehicle control. The hyperplastic lesions were associated with inflammatory cell infiltration and inflammation‐induced fibrotic lesions of the pleural tissues. The fibers were not found in the mesothelial proliferative lesions themselves. In the pleural cavity, abundant inflammatory cell infiltration, mainly composed of macrophages, was observed. Conditioned cell culture media of macrophages treated with multi‐walled carbon nanotubes and crocidolite and the supernatants of pleural cavity lavage fluid from the dosed rats increased mesothelial cell proliferation in vitro, suggesting that mesothelial proliferative lesions were induced by inflammatory events in the lung and pleural cavity and likely mediated by macrophages. In conclusion, intrapulmonary administration of multi‐walled carbon nanotubes, like asbestos, induced mesothelial proliferation potentially associated with mesothelioma development.

Teratogenicity of thiabendazole in ICR mice.

Thiabendazole (TBZ) was tested for teratogenicity using Jcl:ICR mice. TBZ suspended in olive oil was given orally to pregnant mice at different stages of organogenesis. All foetuses were removed from the uterus on day 18 of gestation, and were examined for external and skeletal anomalies. In mice given 700, 1300 or 2400 mg TBZ/kg body weight/day on days 7-15 of gestation, dose-dependent external and skeletal anomalies, especially cleft palate and fusion of vertebrae, were observed. In mice given a single dose of TBZ (2400 mg/kg) on any one of days 6-13, an increased number of malformations was observed. Various types of malformation occurred, especially in the mice treated on day 9. Reduction deformity of limbs was found in mice given TBZ on days 9-12, a change that has not previously been observed to occur spontaneously in normal ICR mice in our laboratory. In order to determine a dose-response relationship, groups of mice were given one of 17 doses of TBZ (30-2400 mg/kg) on day 9 of gestation. The number of litters having foetuses with reduction deformity of limbs and of those having foetuses with skeletal fusion increased in proportion to the dose of TBZ. The regression lines of Y (probit response) on X (log dose) for reduction deformity of limbs and for skeletal fusion were Y = 2.47X - 3.65 and Y = 1.54X + 0.48, respectively. The effective doses (ED1) for the two malformations were 362.0 and 26.4 mg/kg, respectively.

Effects of tartrazine on exploratory behavior in a three-generation toxicity study in mice

Tartrazine was given to mice in the diet at levels of 0 (control), 0.05%, 0.15%, and 0.45% from 5 weeks of age of the F(0) generation to 9 weeks of age of the F(2) generation, and selected reproductive and neurobehavioral parameters were measured. In the F(1) generation, the development of swimming direction at postnatal day (PND) 7 was accelerated significantly in male offspring in a dose-related manner. Surface righting at PND 7 was affected significantly in female offspring in dose-related manner. Several variables in exploratory behavior showed significant tendencies to be affected in the treatment groups in male offspring at 3 weeks of age. In the F(2) generation, the development of swimming direction at PND 7 was accelerated significantly in the high-dosed group in male offspring. Time taken of olfactory orientation at PND 14 was accelerated significantly in male offspring in a dose-related manner. Several variables in exploratory behavior showed significant tendencies to be affected in the treatment groups in male offspring at 3 weeks of age, and in males at 8 weeks of age. The dose levels of tartrazine in the present study produced a few adverse effects on neurobehavioral parameters throughout generations in mice.

Sub-acute toxicity of piperonyl butoxide in F344 rats

Piperonyl butoxide, alpha-[2-(2-Butoxyethoxy)ethoxy]-4,5-methylenedioxy- 2-propyltoluene, is a pesticide synergist. F344 rats of both sex were maintained on diets containing 0, 0.6, 1.2 or 2.4% of piperonyl butoxide for 13 weeks. At the end of experimental period, they were necropsied. Selected organs were weighted and serum was analyzed by clinical chemistry. In male and female rats of the 2.4%-group, body weight gains were depressed, macroscopically, hepatomegaly was marked and liver weights were significantly higher than those of the control group. In male and female rats of all treated groups, relative kidney weights were significantly increased in a dose-dependent manner. Rats of the 2.4%-group had increased levels of albumin, cholesterol, urea nitrogen and gamma-glutamyl transpeptidase. Examination of livers of the male 2.4%-group by light microscopy showed enlarged hepatocytes with glassy cytoplasm and fatty deposition. On occasion, there was coagulative necrosis of a few hepatocytes in the periportal area and oval cell proliferation. The kidney of treated rats showed atrophy of epithelium in the proximal convoluted tubules. These results indicated that toxicity of piperonyl butoxide in rats was directed primarily to the liver and kidney.

Effects of prenatal exposure to styrene trimers on genital organs and hormones in male rats.

Styrene trimers migrate from polystyrene food container into foods. We evaluated the estrogenic activity of styrene trimers such as 2,4,6-triphenyl-1-hexene (ST-1), 1a-phenyl-4a-(1′-phenylethyl)tetralin (ST-2), 1a-phenyl-4e-(1′-phenylethyl)tetralin(ST-3), 1e-phenyl-4a-(1′-phenylethyl)tetralin (ST-4), and 1e-phenyl-4e-(1′-phenylethyl)tetralin (ST-5) using the reporter-gene assay with MVLN cells stably expressing the estrogen-stimulated reporter gene, and it was confirmed that ST-1, ST-3, and ST-4 had estrogen-like activity. On the other hand, ST-2 and ST-5 had anti-estrogen-like activity. We examined the estrogenic activity in vivo of ST-1, ST-3, and ST-4. The styrene trimers were administered to pregnant rats, and the effects on the offspring were examined. ST-1, ST-3, or ST-4 (0, 10, 100, 1000 μg/kg body wt/day) were subcutaneously injected into pregnant rats from gestational Day 11 through 17, and the male offspring were sacrificed on postnatal days (PND) 101–103. In the ST-4 treatment groups, the relative anogenital distance on PND 3 was significantly shortened. The relative testis weight was remarkably decreased in all styrene trimer treatment groups. Relative weights of the prostate and epididymides significantly decreased in the ST-4 treatment groups. The relative brain weight was markedly reduced in the ST-3 and ST-4 treatment groups. A significant decrease of the Sertoli cell count was observed in the ST-1 and ST-4 treatment groups. The serum follicle stimulating hormone level was remarkably reduced in all styrene trimer treatment groups. The luteinizing hormone level was significantly decreased and the testosterone level increased in the ST-1 and ST-4 groups. These results suggest that prenatal exposure to estrogenic styrene trimers at low levels obstructed genital organ development, and disrupted the endocrine systems of male rat offspring.

Flame retardant tetrabromobisphenol A induced hepatic changes in ICR male mice

Tetrabromobisphenol A (TBBPA) is widely used throughout the world as flame retardant for electronic equipment or building materials, and is detected in air at the dismantling plant, sewage sludge, sediment or human serum samples. In the present study, we examined the effects of TBBPA on the liver when administered to mice for 14 consecutive days. Groups of 7 (control group) or 8 (treated group) Crlj:CD1 (ICR) male mice were given 0 (control), 350, 700 or 1400mg/kg body weight/day TBBPA (99.1% pure) in olive oil for 14 days. The serum concentration of total-cholesterol in high-dose (1400mg/kg BW) group was higher than those of the control group. Absolute and relative liver weights were dose-dependently increased, and were significantly increased in high-dose (1400mg/kg BW) group. The histological findings showed that the slight enlargement of the hepatocytes, inflammatory cell infiltrations and focal necrosis of hepatocytes were more marked in liver of treated groups (from 350mg/kg BW) than in control group. The present data suggest the possibility of inducing hepatic lesion by TBBPA dosing.

The maternal-foetal distribution of thiabendazole administered in two different vehicles to pregnant mice

We have compared the effects of two vehicles on the maternal-foetal distribution of 14C-thiabendazole ([14C]TBZ) given orally to Jcl: ICR mice on day 9 or 16 of gestation. TBZ, either suspended in olive oil (TBZ-O) or in a 0.5% aqueous solution of gum arabic (TBZ-G), was given orally on day 9 of gestation at a dose of 1 g/kg body weight (1 microCi/mouse). The mice were killed 0.5, 1, 3, 6, 12, 24 or 72 hr later. In mice treated with TBZ-O maximum levels of radioactivity in plasma and conceptuses were observed at 0.5 hr, whereas in those treated with TBZ-G maximum 14C levels were observed at 6 hr. The uptake of radioactivity from TBZ-O into the plasma and conceptuses was significantly higher than that from TBZ-G. Only trace levels of radioactivity were detected at 72 hr in both treatment groups. The placental transfer of [14C]TBZ was examined in mice treated with doses of 1 g/kg body weight (1 microCi/mouse) given as TBZ-O or TBZ-G on day 16 of gestation. The radioactivity in foetuses, placentas and maternal plasma was higher in mice treated with TBZ-O than those given TBZ-G. The placental transfer of [14C]TBZ was also examined by whole-body autoradiography in mice treated on day 16 of gestation.

Teratogenicity of thujaplicin in ICR mice

Beta-thujaplicin (TP) was studied by in vitro and in vivo tests for teratogenicity using ICR mice. In the in vitro study, TP (0, 3.125, 6.25, 12.5 microg/ml medium) dissolved in dimethyl sulfoxide (DMSO) was administered to cultured embryos on 9 day of gestation. After 24 hr of exposure to TP, the embryos were examined for developmental parameters and external anomalies. Growth retardation and embryos with facial dysplasia or hydrocyst of the tail tip were observed among the embryos given 12.5 microg/ml. In the in vivo study, TP (0, 420, 560, 750 or 1000 mg/kg) dissolved in olive oil was administered orally to pregnant mice on day 9 of gestation. All foetuses were removed from the uterus on day 18 of gestation, and were examined for external and skeletal anomalies. Various types of malformations were observed in the mice given 560 mg/kg or more. The number of litters having foetuses with external or skeletal anomalies increased in proportion to the dose of TP. The regression lines of Y (probit response) on X (log dose) for external anomalies was Y = 4.87X-8.43 . The 1% effective dose (ED1) for the malformation was 190 mg/kg. The present study shows that TP has teratogenic effects on mice.