Dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is an environmental contaminant that produces potent toxic effects in humans and animals. TCDD is carcinogenic at multiorgan sites and induces disorders of reproduction, development, and immunity. It has been considered that the effects of TCDD partly involve disruption of the endocrine system, since TCDD causes progressive endometriosis in the rhesus monkey, suppresses development of the male reproductive system and sexual dimorphism of the brain, and damages the ovaries. These effects suggest alteration of sex steroidogenesis in the target organs. To clarify the endocrine disruptive action of TCDD, many studies of its effects on estrogen-responsive cell lines have been conducted. This mini-review describes recent experiments conducted in the authors' laboratory and discusses our understanding of the molecular mechanisms of interaction between dioxin signaling and sex-steroid hormones, focusing on three issues: 1) influence of estrogen on TCDD-induced cytochrome P4501A1 (CYP1A1) protein in vivo; 2) influence of estrogen on TCDD-induced xenobiotic response element (XRE) transactivation in cultured cell systems; and 3) effect of hormone-receptor status on TCDD-induced XRE transactivation and its relation to the cell cycle.

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An evaluation of the effects of rinbacin on the liver and blood of albino rats was carried out. Low (26.25 g/l) and high (52.50 g/l) dose levels of rinbacin were administered in the drinking water of albino rats for 13 weeks. Food and fluid intake were measured daily, and animal body weight taken weekly. Biochemical analysis of the liver function was carried out as well as some haematological parameters and histology of the liver. Results showed a significant (p ≤ 0.05) increase in all the liver function parameters tested at both dose levels. There was also an increase in packed cell volume (PCV) in the high dose group. Histological examination indicates that rinbacin at both dose sizes induced severe pathologic changes in the forms of degeneration of hepatocytes, necrosis, oedema, cellular infiltration, nuclear fragmentation and chromatinolysis. Administration of rinbacin, though could raise the PCV, may lead to hepatic damage, which might result in increased bilirubin and liver enzymes in rats. Rinbacin is toxic to the rat liver.

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We have reported that strenuous physical exercise causes a decrease in protein-bound sulfhydryl groups (p-SHs), such as albumin cysteine residues, in human plasma (Inayama et al., 1996, Life Sci., 59, 573-578). We further investigated the fate of plasma protein thiols after moderate exercise. Six untrained healthy female volunteers ran for 30-min at the individual ventilatory threshold. We observed an increase in protein cysteine mixed disulfides (p-S-Cys) after running, as evidenced by reducing plasma proteins with dithiothreitol to detect the increase of cysteine, along with the concomitant decrease in p-SHs in plasma. However, plasma protein-bound glutathione (GSH) and S-nitroso-protein were undetectable before and after exercise. Test tube experiments suggest that p-S-Cys are probably formed by the hydrolysis of protein GSH mixed disulfides by γ-glutamyltranspeptidase and peptidase, and/or by the oxidative addition of p-SHs to cystine.

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This study describes a long-term toxicity test within a period of 2 months using zebrafish (Brachydanio rerio) as the test species and concentrations of 0.05, 0.5, 5 and 50 μg/l DDT as a model substance. By collecting and counting the number of sperm released during separate mating events we observed that gametes are released asynchronously. Sperm is released in the form of sperm trails laid on the nest surface; subsequently active spermatozoa leave the trails and move in the water for several minutes. Sperm trails consists of bands of viscous material in which sperm is embedded. The water samples for the estimation of sperm presence were collected gradually within 180 min after 24 hr, 2 weeks, 1 month and 2 months of exposure. It was established that the reduction in count, activity of sperm and the average life span of sperm trails were significant (p < 0.05) at the tested concentrations 5 μg/l and 50 μg/l DDT after 1 month of exposure. In conclusion, this study demonstrates that long exposure time and higher DDT tested levels accelerate the occurrence of negative effect on the number and activity of sperm released as well as the life span of their trails.

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Herein reported are the detection methods of Aristolochic acid I (AA-I) and aristolochic acid II (AA-II), nephrotoxic components of the Aristolochia plants in Aristolochiaceae, in the Kampo medicine Toki-shigyaku-ka-goshuyu-shokyo-to. The confirmation of AA-I and AA-II was possible by the detection methods using a thin layer chromatography and a high performance liquid chromatography equipped with a Photodiode array detector (LC-PDA) and an electric ionization mass spectrometry detector. With the LC-PDA, it was possible to quickly detect AA-I and AA-II in the ten kinds of Kampo medicines in which the Aristolochia fangchi root or Aristolochia mandshuriensis stem may be misused. By using AA-I and AA-II purified from a commercial mixture by a preparative HPLC as reference standards, quantitative analyses of AA-I and AA-II were successfully carried out by use of the LC-PDA.

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Five fatty acid methyl esters in hexane were irradiated in order to obtain basic data regarding the detection of irradiation of fatty foods. Fifteen hydrocarbons which were formed by radiolysis of fatty acid methyl esters were detected using capillary gas chromatography accompanied by mass spectrometry. Dose response was observed at the range of 0.74 kGy to 10 kGy. The yields of the hydrocarbons increased as the dose increased. Clear dose rate effects were not observed at the range of 10 kGy/hr to 500 Gy/hr. Temperature effects on the formation of the hydrocarbons were observed at the range of -40 to 20 degrees Celsius. Their yields were increased as the temperature rose. The effects of oxygen level in the container were examined. The yields in the containers that contained oxygen absorbers were 5-41% of those under normal pressure. Eminent reduction of hydrocarbon yields were observed in the containers that contained oxygen absorbers. Remarkable solvent effects on the formation of the hydrocarbons were observed. The yields of hydrocarbons in benzene solution were reduced to 60-95% of those in hexane. Thus, radiolytic degradation of fatty acid methyl ethers were affected mainly by absorbed dose, irradiation temperature, oxygen pressure, and fatty acid components.

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In this study, we analyzed contents of phytoestrogens (genistein, daidzein, equol, and coumestrol) in two commercial fish diets [a diet for trout (TD) and a diet for ornamental carp (CD)] using Liquid Chromatography-Mass Spectroscopy/Mass Spectroscopy (LC-MS/MS), and these contents were compared with that of a casein-based formulated fish diet (FD) which does not contain soya bean or fish meal. We also analyzed phytoestrogen contents in commercial infant casein- and soya bean-based diets. The contents of phytoestorogens were generally high in CD, TD, and soya milk, and low or non-detectable in FD and casein-based milks. Among these samples, CD showed the highest phytoestrogen contents: genistein, 390800 ng/g; daidzein, 416800 ng/g; coumestrol, 1325 ng/g; equol, 6.4 ng/g. We also determined the estrogenic activity of the fish diets using male goldfish by measuring plasma vitellogenin (VTG) levels as a biomarker of estrogen exposure. When male goldfish were fed one of these diets for 31 days, plasma VTG was detected in CD-fed fish (78.01 ± 48.18 μg/ml) and TD-fed fish (3.51 ± 3.83 μg/ml), whereas plasma VTG was not detected in FD-fed fish (less than 0.040 μg/ml). These results indicate that the commercial fish diets examined contain a large amount of phytoestrogens and showed estrogenic activity that were strong enough to induce VTG production in male goldfish. It is necessary to eliminate estrogenic substances other than test chemicals in the screening test system for estrogenic endocrine-disrupting chemicals (EDCs). Since the formulated diet developed in the present study contain less phytoestrogens than the commercial fish diets and has low estrogenic activity, it is suggested that VTG production using male goldfish in combination with the low estrogen fish diet is a good in vivo system for evaluation of estrogenic effects of EDCs.

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Adult patients admitted with chronic diarrhea to the various wards of the University of Nigeria Teaching Hospital, Enugu, from August 1996 to October 1997 were evaluated further by detailed history-taking, thorough physical examination, serological testing for HIV infection, and stool microscopy. Of 189 patients with chronic diarrhea seen during the study period, 161 had human immunodeficiency virus (HIV) infection (85.2%) whereas 28 were HIV negative (14.9%). Other clinical findings such as weight loss, persistent cough, skin rash prolonged fever, genital ulcers, sore throat, chronic ear discharge, chronic vaginal discharge, oral thrush, herpes zoster, and sinusitis were significantly more frequent in the HIV-infected group. Diarrhea was equally more severe in this group than in the HIV-negative controls. Only Entamoeba histolytica (E. histolytica) was significantly more frequently identified in the HIV- infected patients compared with the HIV-negative group. However, since the E. histolytica was present mainly in its cyst form, it cannot be said to have any pathogenic role in the diarrhea. No acid-fast organism was identified. The microbial causes of chronic diarrhea in HIV-infected patients in Enugu may be significantly different from those reported in other parts of Africa and the developed world.

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Gene expression levels of choriogenin, vitellogenin, and estrogen receptor were determined using a reverse transcription-polymerase chain reaction (RT-PCR) technique after exposure to estrogenic chemicals to compare the sensitivities of the biomarkers of endocrine disruption in medaka, Oryzias latipes. Mature male medaka were treated with a single dose of 100 μ/l of 17α-ethinylestradiol, nonylphenol, and bisphenol A for 6 days, then RNA was extracted from the livers of treated fish for RT-PCR. Primers of RT-PCR for choriogenin H and L, and estrogen receptor were synthesized based on previously known cDNA sequences, and primers for vitellogenin I and II were synthesized based on the partial cDNA which was sequenced in this study. When the five biomarker genes were amplified by RT-PCR under the same condition, the mRNA induction level of each gene was elevated with different sensitivities. Conclusively, choriogenin L, which is a precursor of zona radiata protein (ZI-3) with molecular weight of 49 kD, showed the most sensitive gene expression in all the treated groups.

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We investigated the metabolism of glutathione (GSH) and sulfur amino acids in various tissues of C57BL male mice fed on a 24.8% protein diet (normal protein diet, NPD), a 7.5% protein diet (low protein diet, LPD), and an amino acid supplemented diet (ASD), which contained 7.5% protein and the same levels of sulfur amino acids as NPD. Although the hepatic total GSH (TGSH) concentration was lower in LPD-fed mice than in NPD-fed mice, that in ASD-fed mice recovered to the level in NPD-fed mice. TGSH concentrations in the brain and plasma were highest in ASD-fed mice, whereas those in the kidney and blood were similar in the three dietary groups. Concentrations of L-methionine and L-cystathionine, an intermediate in the metabolism from L-methionine to L-cysteine, in the brain, kidney and plasma were highest in ASD-fed mice, whereas hepatic concentrations were similar. Brain uptake of ¹⁴C-L-methionine for 60 min was higher in LPD-fed mice than in NPD-fed mice, and was further enhanced in ASD-fed mice. Accordingly, this would account for the highest brain concentrations of sulfur amino acids in ASD-fed mice. Thus, the concentrations of TGSH and sulfur amino acids were markedly higher in various tissues in ASD-fed mice than in not only LPD- but also NPD-fed mice. These results suggest that the metabolism of GSH and sulfur amino acids would drastically change by a supplement of sulfur amino acids with a lowered protein diet, at least partly because of their excessive levels compared with the other amino acids.

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It has been unknown whether or not endocrine disruptors exert their effects on neuronal functions, particularly leading to behavioral alterations. To address this hypothesis, we applied the Supermex system that detects the radiated body heat of an animal, representing the spontaneous motor activity of it. Intracisteral administration of tributyltin, an endocrine disruptor, into 5-day-old Wistar rats caused hyperactivity at 4-5 weeks of age. It was about 1.4 fold more hyperactive than vehicle-treated rats in the dark phase, but not in the light phase. Thus, it might be possible to apply the Supermex system to screen the behavioral deficits produced by other endocrine disruptors.

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The effect of estrogenic compounds (bisphenol A, genistein, and 17β-estradiol) on the ability of macrophages to produce superoxide and nitric oxide in response to stimulants was investigated. The assays were performed using 96-well microplates to measure multiple samples. Superoxide and nitric oxide were measured by chemiluminescence and fluorescence methods, respectively. Thioglycollate-induced mouse peritoneal macrophages incubated for 24 hr with the estrogenic compounds were stimulated with phorbol 12-myristate 13-acetate and lipopolysaccharide (LPS). The ability of the macrophages to produce superoxide was increased when treated with bisphenol A or genistein, but 17β-estradiol had little effect. Conversely, the ability of the macrophages to produce nitric oxide in response to LPS was strongly suppressed by high concentrations of bispenol A and genistein. 17β-estradiol was ineffective. These results suggest that bisphenol A and genistein similarly affect superoxide and nitric oxide production by a mechanism other than that involving estrogenic activity.

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The purpose of the present study is to address the question of whether accumulation of intracellular calcium modulates the regulation of proteoglycan synthesis in vascular endothelial cells. Bovine aortic endothelial cells were treated with calcium ionophore A23187 in the presence of [³⁵S]sulfate, and the labeled proteoglycans were analyzed by the cetylpyridinium chloride precipitation method and DEAE-Sephacel ion-exchange chromatography. The results showed that A23187 significantly decreases the accumulation of proteoglycans, especially heparan sulfate proteoglycans, in the cell layer and the conditioned medium of vascular endothelial cells. On the other hand, it was shown that lead, a heavy metal that inhibits endothelial heparan sulfate proteoglycan synthesis, significantly increased the intracellular calcium accumulation when the accumulation was assessed by calcium-45. The present data suggest that accumulation of intracellular calcium results in a suppression of heparan sulfate proteoglycan synthesis in vascular endothelial cells; the suppression may be one of the intracellular mechanisms for lead inhibition of endothelial heparan sulfate proteoglycan synthesis.

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Hydroxysteroid sulfotransferase (SULT2) is thought to be a key enzyme in the synthesis of neurosteroid sulfates, which are known to act as potent regulators of neuronal activity within the brain. We detected two SULT2 mRNAs (SULT2A1 and SULT2B1b) in the human brain. We isolated their cDNAs, and determined the location of their expression in the brain by Northern blot hybridization. Exclusive SULT2A1 expression was detected in the thalamus and hypothalamus, whereas SULT2B1b expression was detected throughout the brain in varying amounts, reaching levels two to three times greater in some regions than in others. These findings further our understanding of the physiological role of SULT2 enzymes within the human brain, particularly how they affect neurosteroid metabolism, and thus, modulate neuronal activity within the brain.

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