The author tried to review and summarize low-dose effects of endocrine disrupting chemicals (EDCs) through an extensive literature survey of toxicological studies with bisphenol A (BPA), taking BPA as an example for which many studies were published. Data on low-dose effects with BPA, especially on neurobehavioral effects after fetal or early postnatal exposures, suggested that there would be new aspects to be considered. Specific mention for future tasks was made. Firstly, toxicity tests should be designed with more elaboration to ensure a sufficient number of animals with careful handling of litters to allow adequate statistical analysis and appropriate selection of dosages to obtain insight in dose-response relationship. Secondly, precise measurement of plasma levels in both humans and rodents and construction of relevant physiologically-based pharmacokinetic models would help obtain quantitative estimates of intake and target-organ exposure relationship. Thirdly, biological backgrounds, particularly differences and similarities in endocrinological, neurological and immunological aspects among species, should be revisited. Fourthly, mechanistic deliberations on the possibilities of epigenetic mechanism and examinations of putative neurobehavioral effects or a presumptive link of miscarriage with BPA exposures are requested. Finally, general public concerns must be addressed in a thoughtful way so that a simple precautionary approach is not pursued, but uncertainties of the new toxicological aspects should be carefully explained. Further researches and internationally concerted efforts on elucidating risk of low-dose effects by integrating knowledge will contribute to setting new directions in toxicology and improving chemical risk assessments.

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The aryl hydrocarbon receptor (Ahr), a ligand-activated transcriptional factor, mediates the transcriptional activation of a battery of genes encoding drug metabolism enzymes. In the present study, we investigated the hepatic mRNA expression profile in Ahr-null (Ahr KO) mice compared to wild-type mice by microarray analysis to find new Ahr target genes. Pooled total RNA samples of liver extracted from 7- and 60-week-old Ahr KO or wild-type mice were studied by DNA microarray representing 19,867 genes. It was demonstrated that 23 genes were up-regulated and 20 genes were down-regulated over 2 fold in Ahr KO mice compared with wild-type mice commonly within the different age groups. We focused on insulin-like growth factor binding protein-1 (Igfbp-1) and lipoprotein lipase (Lpl) that were up-regulated in Ahr KO mice. The higher expression in Ahr KO mice compared to wild-type mice were confirmed by real-time RT-PCR analysis. In the wild-type mice but not in the Ahr KO mice, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment increased the Igfbp-1 and Lpl mRNA levels. The expression profile of Igfbp-1 protein was consistent with that of Igfbp-1 mRNA. Since Lpl is the primary enzyme responsible for hydrolysis of lipids in lipoproteins, the serum triglyceride levels were determined. Indeed, the serum triglyceride levels in Ahr KO mice was lower than that in wild-type mice in accordance with the Lpl mRNA levels. Contrary to our expectation, TCDD treatment significantly increased the serum triglyceride levels in wild-type, but did not in Ahr KO mice. These results suggest that serum triglyceride levels are not correlated with hepatic Lpl expression levels. In the present study, we found that Ahr paradoxically regulates Igfbp-1 and Lpl expressions in the liver.

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Uncontrolled exposure of active and passive smokers to trace metals causes increase in health risks. The primary objective of this study was to determine whether local and imported cigarette brands used in Pakistan, have elevated levels of metals or not. Six metals manganese (Mn), cobalt (Co), copper (Cu), cadmium (Cd), lead (Pb) and zinc (Zn) were determined in tobacco of twenty cigarette brands (local and imported) used in Pakistan by flame atomic absorption Spectrophotometry. To overcome contamination chances and for complete digestion of analytes a microwave digester was used. The analytical results showed highest concentration of Mn (84.78 µg/g dry weight), Cd (0.525 µg/g dry weight) and Zn (14.34 µg/g dry weight) metals in imported brands in relation to counterparts from the local brands. Certain elevated levels were observed for Co (3.344 µg/g dry weight), Pb (14.16 µg/g dry weight) and Cu (7.889 µg/g dry weight) metals in local brands. The inter-metal relationships in the tobacco of local and imported cigarette brands showed some integrated variation in the selected metal levels. In view of health risk associated with the above metals, there should be a strict quality control over monitoring of heavy metals during growing, processing and smoking of tobacco. Therefore, it is prudent to minimize exposure to toxic substances whenever possible because smoking and exposure to cigarette smoke is a confounder to be taken into account when carrying out epidemiological studies on human exposure to metals.

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DNA is damaged by reactive oxygen species (ROS) and such damage is age-dependent. Blood chemical parameters also change age-dependently. Glutathione (GSH) plays an important role as an antioxidant. However, the effects of GSH on DNA damage and blood chemistry are unclear. Therefore, this study was aimed to evaluate GSH contribution to DNA damage and changes of blood chemical parameters in aged and young rats. The GSH content in the livers and kidneys of aged rats (20 months) were lower than that in young rats (9 weeks of age) with higher DNA damage detected by a comet assay. There was a negative correlation between the GSH content and the DNA damage in the liver and kidney. L-buthionine (S,R)-sulfoximine (BSO; 0, 5, 20 mM), which inhibits GSH synthesis, was administered in drinking water for 28 days to young and aged rats (8 weeks and 19 months of age at the start of the administration). The treatment significantly decreased GSH levels in the heart, liver, lung and kidney of either the young or aged rats without causing DNA damage in those organs. When compared with young rats, aged rats showed higher levels in aspartate aminotransferase, alanine aminotransferase, total bilirubin, total cholesterol, globulin, creatinine, sodium and chloride and lower levels in alkaline phosphatase, triglyceride, albumin/globulin and inorganic phosphorus. However, BSO did not change these parameters in young or aged rats. These results showed that there was a negative correlation between GSH and DNA damage during aging, but the BSO-induced GSH depletion did not affect DNA damage or blood chemistry levels in young and aged rats under these study conditions.

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Oral toxicity of 4-methylbenzoic acid in male and female Sprague-Dawley rats was profiled through a twenty-eight-day repeated dose toxicity study (the 28-day study) and a screening test for reproductive/developmental toxicities (the reproduction/developmental study) conducted under Organisation for Economic Co-operation and Development (OECD) test guidelines. Daily administration of 4-methylbenzoic acid, at a dose level of 0, 100, 300 or 1,000 mg/kg, did not show any adverse effect on reproductive organs of animals in the 28-day study. In the reproductive/developmental study, however, 1,000 mg/kg/day of the compound reduced epididymal weights and increased incidence of cauda epididymal oligo/azoospermia. While the compound did not affect estrous cycle or mating performances, 1,000 mg/kg of the compound reduced fertility. Furthermore, 300 mg/kg or more of the compound increased pre-implantation loss, which resulted in a decrease in the number of offspring, and reduced body weight gain of the dams during the latter period of gestation. From these results, the no-observed-effect-level (NOEL) for reproductive/developmental toxicities is considered to be 100 mg/kg, whereas 1,000 mg/kg did not show any effect on neonates. In the 28-day study, NOEL is considered to be 300 mg/kg for male and female rats, since 1,000 mg/kg of the compound caused, in both sexes, a few minor changes, such as temporal salivation, a slight increase in food consumption and a moderate increase in blood aspartate aminotransferase (AST) activity. Thus, 4-methylbenzoic acid has the potential for reproductive toxicity, with diverse adverse effects on the epididymis, after repeated administration, observed in the two studies.

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Expression of hepatic cytochrome P450 (CYP) isoforms was compared in Sprague-Dawley (SD) and Wistar (WI) rats, which are commonly used strains in preclinical studies. Basal CYP1A1, CYP1A2, and CYP3A2 mRNA levels were higher in WI rats than in SD rats (by 8-, 3- and 2-fold, respectively). Treatment with phenobarbital, a potent CYP inducer, increased the predominance of expression of these three mRNAs in WI rats (by 26-, 4-, and 2-fold, respectively) along with the predominance of increased microsomal total P450 contents and smooth-surface endoplasmic reticulum in the centrilobular hepatocytes. CYP1A enzymatic activity was also higher in WI rats than in SD rats. No strain differences were observed in phenobarbital induction of CYP2B1/2, CYP2C6, or CYP3A1. CYP3A2 mRNA was more strongly induced by dexamethasone, a typical inducer of CYP3A, together with CYP3A1 mRNA, in WI rats than in SD rats (by 2-fold), whereas the CYP1A1 and CYP1A2 mRNA expression induced by β-naphtoflavone, a typical inducer of CYP1A, did not differ between the two strains. Furthermore, WI rats exhibited predominantly arylhydrocarbon receptor, pregnane X receptor, and constitutive androstane receptor mRNAs, responsible for CYP1A or CYP3A induction, with phenobarbital or dexamethasone induction. In conclusion, significant, predominant expression of hepatic CYP1A and CYP3A mRNAs in WI rats was observed, possibly related to nuclear receptor-mediated induction. Considering the pharmacokinetic and toxicological importance of CYP1A and CYP3A, different outcomes might arise depending on the rat strains used in preclinical studies of drugs metabolized typically or mainly by both isoforms.

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Diabetic cardiomyopathy can progress toward overt heart failure with increased mortality. The hexosamine biosynthesis pathway has been implicated in signaling for fibrosis by the kidney. Thiamine (vitamin B₁) is an indispensable coenzyme and required at intracellular glucose metabolism. In this study, we assessed if decrease of flux through the hexosamine biosynthesis pathway induced by high-dose thiamine therapy counteracts diabetes-induced cardiac fibrosis. The diabetes model used was the streptozotocin-induced diabetic rat. Normal control and diabetic rats were studied for 2 weeks with and without thiamine, and followings were analyzed; plasma biochemicals (total cholesterol and triglycerides), morphological changes, mRNA abundance relevant to cardiac failure (brain natriuretic peptide) and fibrosis (transforming growth factor-ß1, thrombospondine, fibronectin, plasminogen activator-I and connective tissue growth factor) as well as and matrix metalloproteinase activity were investigated. Thiamine repletion prevented diabetes-induced cardiac fibrosis without changes in plasma glucose concentration. This was achieved by prevention of thiamine depletion, increased pro-fibrotic mRNA abundance and decreased metalloproteinase activity in the heart of diabetic rats. O-glycosylated protein was significantly higher in the left ventricular of diabetic rats compared to control rats, which was decreased by thiamine administration. Thiamine repletion prevented diabetes-induced cardiac fibrosis in experimental diabetes, probably by suppression of hexosamine biosynthesis pathway.

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Although thyroid hormones are crucial for cerebellar development, and several thyroid hormone-dependent genes are known to be correlated with morphological development of the cerebellum, the precise mechanisms of morphological cerebellar changes in hypothyroidism (HT) remain unknown. To investigate these mechanisms in experimental rat HT induced by the anti-thyroid drug methimazole (MMI-HT rat), we carried out gene expression analysis (sonic hedgehog (Shh), reelin, and Bax) using quantitative real-time PCR. Histological examination revealed cerebellar abnormalities, including reductions in the thickness of the molecular layer and delayed disappearance of the external granular layer (EGL), as well as excess bulges or sublobules in the internal granular layer (IGL). At Postnatal Day (P) 6, Shh expression in MMI-HT rat was comparable to that in controls, thus suggesting that Shh expression was sufficient to form the lobes in the initial phase. However, Shh expression decreased in the later phases, as compared with age-matched controls. This demonstrated that stronger and sustained signaling is necessary for partitioning of the cardinal lobes into lobes and sublobes. Although reelin expression was not clearly different from that in controls, Bax expression decreased at P 15. The attrition of Bax at P 15 as well as Shh in the later phase may be related to irregularities in the IGL and the relatively large numbers of internal granular cells. Taken together, these results suggest that Shh expression is related to the morphological cerebellar changes in experimental hypothyroidism and that sustained signaling by Shh may play a key role in normal development, particularly lobulation, in the cerebellum.

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In order to elucidate the role of metallothionein (MT) in preventing the adverse effects of X-ray irradiation, we examined the susceptibility of MT-I/II null mice to bone marrow injury caused by X-irradiation and effects of pretreatment with MT-inducing metals on X-ray injury. Eight-week-old male mice were exposed to a single bout of whole-body X-irradiation at a dose between 0.1 and 6.0 Gy. The numbers of leukocytes, reticulocytes with micronuclei (MNRET) in the blood, and polychromatic erythrocytes with micronuclei (MNPCE) in the bone marrow were determined 24 hr after X-irradiation. X-irradiation significantly decreased the total number of leukocytes in MT-I/II null mice and wild-type mice in a dose-dependent manner, but the total number of leukocytes was significantly lower in MT-I/II null mice than in wild-type mice at a low dose of irradiation, between 0.1 and 1.0 Gy. X-irradiation (0.1 and 0.5 Gy) significantly increased the appearance of MNRET and MNPCE in both strains, but the increase was greater in the MT-I/II null mice than in the wild-type mice. Additional groups of mice were pre-administered bismuth nitrate or zinc sulfate to induce MT in the bone marrow cells prior to X-irradiation; the X-ray injury was prevented by such treatments in wild-type mice only. Thus, the present results suggest that MT plays a protective role against a low dose of X-ray injury.

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An outbreak of food poisoning that affected at least ten people in various regions of Japan was traced to exposure to Chinese dumplings contaminated with the organophosphate insecticide Methamidophos. We experienced the most serious case, a five years old girl, who suffered coma. She presented with features of cholinergic overactivity and her serum cholinesterase activity was 9 U/l. We started intravenous treatment with pralidoxime iodide, atropine sulfate, and midazolam. Her symptoms improved gradually and she was discharged on day 25 without any sequelae. Though poisoning attributed to organophosphate insecticides has become less common in recent years, it is even more important to diagnose the problem rapidly based on the characteristic symptoms and to start specific treatment at the earliest possible stage after poisoning.

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Epidemiological and experimental studies have suggested that exposure to metalloid arsenic constitutes a risk factor for vascular disease associated with atherosclerosis. Since in atherosclerosis, types of proteoglycans (PGs) present change depending on the stage, we investigated the effect of 2 chemical forms of inorganic arsenic—a trivalent sodium arsenite and a pentavalent sodium arsenate—on the synthesis of PGs in cultured arterial smooth muscle cells. The results indicate that arsenite but not arsenate, at a noncytotoxic level, inhibits general PG synthesis independent of cell density. Arsenite may be one of the chemical forms of inorganic arsenic that influences the PG composition in blood vessel walls during the progression of vascular disorders such as atherosclerosis.

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CByB6F1-Tg(HRAS)2Jic mice (brand name: rasH2 mouse) are produced by two breeding facilities, CLEA Japan, Inc. (Fuji, Shizuoka, Japan) and Taconic (Germantown, NY, USA), and supplied world wide. To confirm carcinogenic conformity of both mice, a 26-week carcinogenicity test was performed on a total of 120 mice obtained from both facilities under the same protocol and same timing in our facility. All mice were divided into a vehicle (citrate buffer at pH 4.5, 10 ml/kg, single intraperitoneal injection) group and a MNU (N-methyl-N-nitrosourea, 75 mg/kg, single intraperitoneal injection) group. Fifteen mice of each sex were assigned to each group. The survival rate of the vehicle group was maintained at 100% for mice from both facilities at completion of the test. In the MNU group, MNU-induced tumor death occurred from 9 to 12 weeks after administration, and the final survival rate for both facilities was 6.7%. In the pathological examination, only benign tumors of lungs, spleen, forestomach and skin were observed in a few mice in the vehicle group of both facilities. In the MNU group, the incidence of forestomach papilloma/squamous cell carcinoma in mice from both facilities was 100%. The incidences of malignant lymphoma in CLEA Japan mice and Taconic mice were 86.7% and 93.3%, respectively, and no significant difference was observed (Fisher’s exact probability test). Although lung adenoma and skin papilloma/keratoacanthoma, which are major MNU induced tumors in this strain, were observed in several mice from both facilities, no significant differences were found. Consequently, carcinogenic conformity of rasH2 mice derived from two breeding facilities was confirmed by the present study.

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The histamine H4 receptor (H4R) is the newest receptor identified of four histamine receptors. Its expression in numerous immune and inflammatory organs has been implicated in relation to immune systems and allergic diseases. In the present study, we demonstrate the expression of H4R in human dermal fibroblasts and investigate changes in its expression level when stimulated by histamine, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), dexamethasone and indomethacin. Histamine and PMA showed no effects on H4R expression. LPS and indomethacin up-regulated H4R mRNA expression, and 20 µM dexamethasone increased H4R protein levels. These results indicate a good prospective for this new receptor in the development of effective treatments of inflammatory diseases and pruritus or for the appropriate prevention of toxicities.

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Sudden deaths of F344 rats (F344/Du Crj (Fischer)) have occurred frequently in the late stage of carcinogenicity studies using stomach tubes. To reduce the sudden deaths, the incidence of sudden deaths was compared in the control groups from 104-week carcinogenicity studies using two different stomach tubes (metal and Teflon) and feeds (pellet and powder). The results indicate that replacing metal tubes with Teflon tubes from the first administration or after week 41 of administration was not effective in reducing the sudden deaths. On the other hand, sudden deaths did not occur at all after changing the feed from pellets to powder after week 44 or 79 of administration. In addition, although decreased body weight and retention of feed in the oral, pharyngeal and laryngeal cavities were observed in the animals that died suddenly, there were no abnormalities in histopathological examination. Therefore, it is suggested that changing the feed from pellets to powder should be effective in reducing the sudden deaths of F344 rats in long-term oral gavage studies or carcinogenicity studies.

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