Concentration and age-dependent elimination kinetics of polychlorinated dibenzofurans in Yucheng and Yusho patients.

Half-life estimates of three polychlorodibenzofurans (PCDFs) were calculated using serial blood samples collected over a 15 to 19-year period. Blood fat PCDFs were modeled in eight individuals who were exposed to contaminated rice oil in Japan (Yusho, n = 5) and in Taiwan (Yucheng, n = 3). The elimination kinetics of PCDFs were concentration-dependent, with faster rates observed at higher concentrations and the apparent transition to slower rates occurring at about 1-3 ppb. Average half-lives of 1.1, 2.3, and 1.5 years above the transition concentration and 7.2, 5.7, and 3.5 years below it were estimated for 2,3,4,7,8-pentaCDF, 1,2,3,4,7,8-hexaCDF, and 1,2,3,4,6,7,8-heptaCDF, respectively. A positive linear correlation of half-life with age was observed for the combined group, with a rate of increase of 0.19, 0.12, and 0.05-year half-life per year of increase in age for penta-, hexa-, and hepta-CDF, respectively. The distinctly younger Yucheng patients exhibited far lower variability in half-lives and age-related trends that were quite consistent with the corresponding data on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for younger persons exposed in the Seveso incident. These age- and concentration-dependent half-lives for PCDFs may have important risk assessment implications for estimating body burdens. The current study provides limited additional evidence that PCDFs, like TCDD, are more rapidly eliminated in younger individuals.

A glycoside of Nicotina tabacum affects mouse dopaminergic behavior.

Climbing in the forced swimming test is considered a dopaminergic-specific behavior. A substance of Nicotina tabacum affecting dopamine neuronal activity was investigated using the mouse behavioral system. The substance was found to be a glycoside with the peripheral sugar chain structures Fuc alpha 1-2Gal, Gal beta 1-4GlcNAc and GalNAc alpha 1-3GalNAc and with basic polymannoses. The glycoside dose-dependently increased behavior via D2 neuronal activity, but not D1 activity. This suggests that smoking can affect human brain function not only via the nicotinic cholinergic neuron, but also via the D2 neuron.

Behavioral effect of herbal glycoside in the forced swimming test.

We investigated the behavioral effects of Chinese herbal medicines in the forced swimming test. One of these medicines, Kami-shoyo-san, induced an antidepressive climbing behavior in mice. An effective substance detected to be an O-linked glycoside with the sugar chain structure GalNAc alpha 1-3GalNAc was separated. The behavioral effect was dose-dependently decreased by the dopamine 2 antagonist sulpiride, but not by the dopamine 1 antagonist SCH-233960. Investigated Chinese herbal medicines, including Kami-shoyo-san, have been used for human depression. These facts suggest that glycoside is one of the antidepressant-like substances of Chinese herbal medicines.

[The changes of PCBs and PCDFs as well as symptoms in Yusho patients for 30 years].

Two mass-food poisonings called Yusho and Yucheng occurred in Western Japan and Taiwan in 1968 and 1979, respectively. Blood samples from 5 Yusho patients and 3 Yucheng patients were collected several times from 1982 to 1998 and from 1980 to 1995, respectively, and analyzed for PCB and PCDF congeners. Fat base concentrations of 2,3,4,7,8-penta-CDF in the Yusho patients, which were responsible for about 70% of the dioxin toxicity, are estimated to be decreased from 60 ppb in 1969 to 0.8 ppb in 1997 with the median half-life of 2.9 years in the first 15 years after the onset and 7.7 years in the next stage of 15 years. Fat base concentrations of total PCBs in the blood of Yusho patients are estimated to be decreased from 75 ppm just after the onset to 2.3 ppm after passage of 30 years. Typical Yusho symptoms of acneiform eruption, dermal pigmentation and increased eye discharge were very gradually recovered with lapse of ten years. However, enzyme and/or hormone mediated sign of high serum triglyceride, high serum thyroxin, immunoglobulin disorder and others are persistently maintained for 30 years.

[Prostatic cancer developing after transurethral resection of the prostate for benign prostatic hyperplasia].

From January 1993 to June 1998, 319 cases were histopathologically diagnosed as prostatic cancer. In 7 of the 319 cases (2.2%) transurethral resection of the prostate (TUR-P) had been performed and a diagnosis of benign prostatic hyperplasia had been made with the resected specimens. The interval between TUR-P and the diagnosis of prostatic cancer ranged from 22 months to 15 years. All the cases showed an elevation of the prostate specific antigen (PSA) value (6.4-399 ng/ml, Tandem-R: RIA) at the time of cancer diagnosis. In 2 cases, PSA was measured in cancer screening. The clinical stage was stage B1 in 2 cases, stage B2 in 2 and D2 in 3. Only one case had been regularly followed-up after TUR-P, in which cancer was diagnosed by needle biopsy 22 months after TUR-P, because of the sustained high PSA values. Since most of such patients have an advanced stage of prostate cancer, it is of importance to have periodical follow-up examinations after TUR-P. The measurement of PSA appears the most reliable means in this way.

Cardiac dysfunction because of secondary hemochromatosis caused by congenital non-spherocytic hemolytic anemia.

Most patients diagnosed with secondary hemochromatosis have had repeated blood transfusions. Cardiac failure accounts for approximately one-third of the deaths associated with hemochromatosis. Liver dysfunction or hormonal disorders such as diabetes generally precede cardiac failure. A 23-year-old woman with hemochromatosis had, despite significant left ventricular dysfunction, liver function within the normal range on biochemical evaluation. She was treated for congestive heart failure and given desferoxamine intravenously. She did not have primary hemochromatosis, and had not received multiple blood transfusions or iron supplement. As a child the patient had been diagnosed with congenital non-spherocytic hemolytic anemia not requiring transfusion; thus, this is a unique case of secondary hemochromatosis.

Murine and human SDF2L1 is an endoplasmic reticulum stress-inducible gene and encodes a new member of the Pmt/rt protein family.

We isolated murine and human cDNAs for SDF2L1 (stromal cell-derived factor 2-like1) and characterized the genomic structures. Northern blot analysis of the gene expression in various tissues revealed that both murine Sdf2l1 and human SDF2L1 genes are expressed ubiquitously, with particularly high expression in the testis. The SDF2L1 protein has an endoplasmic reticulum (ER)-retention-like motif, HDEL, at the carboxy (C)-terminus. Interestingly, SDF2L1 protein also shows significant similarity to the central hydrophilic part of protein O-mannosyltransferase (Pmt) proteins of Saccharomyces cerevisiae, the human homologues of Pmt (POMT1 and POMT2) and Drosophila melanogaster rotated abdomen (rt) protein. In a murine hepatocellular carcinoma cell line, Sdf2l1 was strongly induced by tunicamycin and a calcium ionophore, A23187, and weakly induced by heat stress but was not induced by cycloheximide. In conclusion, SDF2L1 protein is a new member of Pmt/rt protein family and Sdf2l1 is a new ER stress-inducible gene.

Ibotenic acid-induced lesions of the medial septum increase hippocampal membrane associated protein kinase c activity and reduce acetylcholine synthesis: prevention by a phosphatidylcholine/vitamin B12 diet.

Ibotenic acid infusion into the medial septum (MS) results in biochemical alterations in the hippocampus. The biochemical events involved in this neuronal lesion are poorly understood. We investigated the effect of a purified diet supplemented with egg phosphatidylcholine (PC) and vitamin B(12) on ibotenic acid-medicated biochemical changes in the rat hippocampus and crude synaptosomal membranes. Male Wistar rats with this MS lesion were fed a purified diet (control diet) or a purified diet supplemented with 5.7 g PC and 125 microg vitamin B(12) per 100 g (experimental diet) for 18 days. Sham-operated rats were fed the control diet. Compared with the sham-operated rats, MS-lesioned rats fed the control diet showed increased activity of membrane-bound protein kinase C (PKC), decreased activity of choline acetyltransferase, and decreased concentrations of acetylcholine in the hippocampus. The ratio of cholesterol to phospholipid in the crude synaptic membrane was lower in the lesioned rats than in the sham-operated rats, but this was not accompanied by any alteration in membrane lipid fluidity. MS-lesioned rats fed the experimental diet showed lowered PKC activity and elevated acetylcholine concentrations than did rats fed the control diet, but there were no significant effects on choline acetyltransferase activity and the lipid ratio. The ibotenic acid-mediated elevation of PKC activity was observed as early as 2 days postinjury in the control diet-fed rats but not in the experimental diet-fed rats. We propose that ibotenic acid mediates pathophysiologic actions through the activation of PKC and that PC combined with vitamin B(12) ameliorates the second messenger-mediated injury.

[Analysis of 209 PCB congeners by high separation gas chromatography/low resolution mass spectrometer].

All PCB congeners were analyzed by high separation gas chromatography/low resolution mass spectrometer, using 209 PCB congeners as standards. They were separated into 169 peaks, including 28 peaks containing 2 PCBs and 8 peaks containing 3 PCBs. The rice oil samples of Fukuoka and Taiwan Poisonings were analyzed for PCB congeners. They showed 115 separated peaks of PCB congeners. Total PCB concentrations were 879 and 769 ppm in 2 samples of Fukuoka rice oil and 57 and 83 ppm in 2 samples of Taiwan rice oil.

[Analysis of all PCB congeners in breast milk and blood of Yusho patients].

Breast milk and blood of Yusho patients were analyzed for polychlorinated biphenyl (PCB) congeners by High separation gas chromatography/Low resolution mass spectrometer. Seventy-one and forty-nine PCB congeners were identified and quantified in the breast milk and blood, respectively. Total PCB concentrations (Whole base) in breast milk of 2 Yusho patients were 69.9 and 15.1 ppb, respectively, being 11.6 and 2.5 times higher than those of 4 control breast milk. Average total PCB concentration of 5.0 ppb in whole blood of 13 Yusho patients was 3.6 times higher than those of control persons. Fourteen PCB congeners, such as 99, 117, 130, 137, 138, 156, 157, 164, 170, 171, 172, 189, 191 and 195, were particularly higher concentration in breast milk and blood of Yusho patients than in those of control persons. Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and coplanar PCBs were also analyzed in the breast milk of Yusho patients and control persons. Large portions, 83 and 74%, of Dioxin toxic equivalency (TEQ) in the 2 Yusho breast milk were consisted of TEQ of 2,3,4,7,8-pentaCDF only, while total TEQ in the breast milk of control persons was consisted of PCDFs 48%. PCDDs 29% and coplanar PCBs 23%.

Induction of apoptosis by bufalin in human tumor cells is associated with a change of intracellular concentration of Na+ ions.

In an attempt to characterize the mechanisms that are operative at the early stages of the induction of apoptosis by bufalin, a component of the traditional Chinese medicine chan'su, we examined the effects of bufalin on plasma membrane potential, as determined by monitoring the uptake by cells of rhodamine 123. Bufalin induced apoptosis in human monocytic leukemia THP-1 cells, in human lymphoblastic leukemia MOLT-3 cells, and in human colon adenocarcinoma COLO320DM cells but not in normal human leukocytes, for example, polymorphonuclear cells and lymphocytes, and not in murine leukemia P388D1 and M1 cells. Treatment for 3 h with bufalin at 10(-6) M caused a decrease in the plasma membrane potential in several lines of human tumor cells but not in murine leukemia cells. No changes in mitochondrial membrane potential, as monitored with the fluorescent dye JC-1, and no release of cytochrome c were observed within at least 6 h after the start of treatment with bufalin. Moreover, overexpression of bcl-2 in human leukemia HL60 cells that had been transfected with cDNA for bcl-2 prevented bufalin-induced apoptosis but had no significant effect on the change in plasma membrane potential induced by bufalin. Since bufalin specifically inhibits the Na+,K(+)-ATPase of human but not murine tumor cells, and since this inhibition leads to a change in intracellular concentration of Na+ ions, our findings suggest that bufalin induces apoptosis in human tumor cells selectively via inhibition of the Na+,K(+)-ATPase, which acts upstream of the bcl-2 protein.

Induction of food intake by a noradrenergic system using clonidine and fusaric acid in the neonatal chick.

To clarify noradrenergic systems on food intake of the neonatal chicks, we examined the effects of i.c.v injection of clonidine (CLON), an alpha2-receptor agonist, and fusaric (5-butylpicolinic) acid (FA), a dopamine (DA)-beta-hydroxylase (DBH) inhibitor. Although a high dose (250 ng) of CLON induced a narcoleptic response and reduced food intake, food intake at 30 min post-injection was enhanced by lower doses (25 and 50 ng) of CLON. Central administration of FA (25, 50 and 100 microg) increased food intake in a dose-dependent fashion. It is suggested that feeding behavior is stimulated by low levels of CLON and decreased by further production of norepinephrine (NE), and FA may play the disturbance of sleeping and then enhance food intake.

Effects of repeated selective serotonin reuptake inhibitor paroxetine treatments on mouse forced swimming.

Studies were performed in the mouse forced swimming model, a well known experimental depression model, in order to detect the mechanism of the antidepressive effects induced by repeated serotonin reuptake inhibitor (SSRI) dosing. Five-day repeat dosing of a typical SSRI, paroxetine, increased climbing, a distinctive antidepressive behavior, 1 h after but not 1 h before treatment. The coinjection of paroxetine and serum in mice treated with four repeated doses of paroxetine distinctively increased the behavior, but the coinjection of paroxetine and serum in mice without paroxetine did not. These results indicate that repeated dosing of paroxetine produces a serum substance related to the antidepressive effects induced by serotonin neuron activities. Furthermore, the behavior induced by 5-day repeated dosing of paroxetine was decreased by 100 and 10 micrograms/kg of ketanserin (5-HT2 antagonist) and 100 micrograms/kg of LY-278584 (5-HT3 antagonist). The present findings strongly suggest that repeated dosing of paroxetine produces a serum substance stimulating the antidepressive neuronal pathway sensitively mediated by 5-HT2 and 5-HT3 receptor activity.

Concentrations of PCBs, PCDFs and PCDDs in the blood of Yusho patients and their toxic equivalent contribution.

Yusho blood and control serum were collected in around 1991 and analyzed for congeners of PCDDs, PCDFs and PCBs. In Yusho blood, TEQ was mainly contributed by 2,3,4,7,8-penta-CDF (77-248 ppt), 1,2,3,4,7,8-hexa-CDF (15-37 ppt) and 2,3,3',4,4',5-hexa-CB (16-77 ppt). Total TEQ in Yusho blood (185-441 ppt) was only 3-14 times higher than that of control serum (31-61 ppt). It is notable that the concentrations of 3,3',4,4',5-, 2,3,3',4,4'- and 2,3',4,4',5-penta-CBs in Yusho blood were comparable to or lower up to 3-5 times than those in control blood.

Effects of single and repeated administrations of Toki-shakuyaku-san on the concentrations of brain neurotransmitters in mice.

The amounts of monoamine-related substances (NE, MHPG, DA, DOPAC, HVA, 5-HT, and 5-HIAA and acetylcholine (ACh) in the cerebral cortex, corpus striatum and hippocampus of mice treated orally with a powdered extract of Toki-shakuyaku-san (TSS) were measured using the HPLC-ECD method. Single administration of TSS at 50 mg/kg decreased the content of NE in the hippocampus, but increased the contents of DA, DOPAC and HVA. Single administration of TSS at 50 mg/kg increased the contents of DA and HVA in the cerebral cortex and those of DA, DOPAC and HVA in the hippocampus, but decreased the content of NE. Repeated administration (twice a day, for 14 days) of TSS at 50 mg/kg increased the contents of DA, DOPAC and 5-HIAA in the cerebral cortex, while 500 mg/kg increased the contents of NE, MHPG, DOPAC, 5-HT and 5-HIAA in the cerebral cortex, and the NE and DA in the corpus striatum. With regard to ACh content, single and repeated administrations of TSS at 50 and 500 mg/kg had no influence in the three regions of mouse brain. These results suggest that single administration of TSS stimulates the function of the dopaminergic nervous system in the hippocampus in mice and inhabits the function of the adrenergic nervous system, and that repeated administration of TSS stimulates the function of the adrenergic, dopaminergic and serotonergic nervous systems in the cerebral cortex. TSS, however, did not show any influence on the brain ACh content in mice.

EGG phosphatidylcholine combined with vitamin B12 improved memory impairment following lesioning of nucleus basalis in rats.

We investigated the effects of egg phosphatidylcholine (PC) combined with vitamin B12 on memory in the Morris water maze task, and on choline and acetylcholine (ACh) concentrations in the brain of rats. Animals with nucleus basalis Magnocellularis (NBM) lesion received intragastric administration of egg PC or vitamin B12, or both for 18 days. Memory acquisition and retention were remarkably impaired in NBM lesioned rats compared with in sham-operated control. NBM lesioned group had lower choline and ACh concentrations than control group in the frontal cortex. High dose of egg PC alone significantly increased choline concentration, but did not change ACh concentration in the frontal cortex. High dose of vitamin B12 alone did not change choline and ACh concentrations in the brain. Either egg PC or vitamin B12 did not improve memory acquisition and retention. However, low dose of egg PC combined with vitamin B12 significantly increased ACh concentration and improved memory acquisition and retention in the NBM lesioned rats. We concluded that egg PC combined with vitamin B12 improved the memory impairment of NBM lesioned rats through the action on the cholinergic neurons.

[The condition of PCBs and PCDFs in the blood of Yusho patients 20 years after the onset].

Blood samples of Yusho and control persons were analyzed for individual congeners of PCDDs, PCDFs, and PCBs by high resolution GC/MS. Concentrations of 2,3,4,7,8-penta-CDF, 1,2,3,4,7,8-hexa-CDF and 2,3,3',4,4',5-hexa-CB in Yusho blood were up to 56 times higher than the corresponding concentrations in the control blood. These high concentrations have persisted for 23 years after the incident. Concentrations of 3,3',4,4',5-penta-CB and 2,3',4,4',5-penta-CB in some Yusho blood were lower than the control blood. In Yusho blood, 2,3,4,7,8-penta-CDF contributed the highest toxicity (TEQ 77-248 ppt in lipid) among the congeners determined and toxic contribution of PCDFs was very large (41-77%) in the chlorinated pollutants. Thirty PCB congeners were identified in the blood of Yusho patients in 1996 by GC/MS. The average total PCB concentration in Yusho blood were 4.9 times higher than that of the controls. Characteristic PCB congeners in Yusho patients were 2,2',3,4,4',5-hexa-CB, 2,3,3',4,4',5-hexa-CB and 2,3,3',4,4',5'-hexa-CB and their concentration ratios to the controls were 8-19.

[Tissue distribution of methylsulfonyl metabolites derived from Kanechlor 400 in mice].

Kanechlor 400, which caused the "Yusho disease", was i.p. administered to mice and methylsulfonyl (MeSO2) metabolites were investigated with respect to the concentration in liver and lung during 28 days after the administration. Major components were 3- and 4-MeSO2 derivatives from seven PCBs (IUPAC no. #31, #49, #64, #70, #101, #110, #132). In the liver, similar concentration ratio of 3- and 4-MeSO2 derivatives was observed, whereas seven 4-MeSO2 derivatives were selectively retained in the lung. Methylsulfone metabolites of triCB (#31) were rapidly formed and eliminated. The highest concentration of the metabolites in the lung was 4-MeSO2-2, 2', 4', 5-tetraCB. Concentration ratio of MeSO2-CBs to residual PCBs was 1:2.1 in the liver whereas 4.6:1 in the lung 28 days after the administration.

Normal human fibroblasts immortalized by introduction of human papillomavirus type 16 (HPV-16) E6-E7 genes.

This report demonstrates that normal human fibroblasts can be immortalized by the introduction of HPV-16 E6-E7 genes. We designed zinc-inducible expression plasmids with HPV-16 E6, E7 or both. Each plasmid was introduced into normal human fibroblasts (TIG-3 cells) using lipofection methods. Only transfectants with the HPV-16 E6-E7 zinc-inducible expression plasmid, which were cultured in medium supplemented with 100 microM ZnSO4, overcame crisis and could be cultured over 200 population doubling levels (PDLs). These cell lines showed the reactivation of telomerase after crisis, and morphological alterations were also observed.

Suppression of gravitropic response of primary roots by submergence.

Primary roots of six plant species were placed horizontally either in humid air or under water, and their growth and gravitropic responses were examined. In air, all the roots showed a normal gravitropic curvature. Under water without aeration, roots of rice (Oryza sativa L.), oat (Avena sativa L.), azuki bean (Vigna angularis Ohwi et Ohashi), and cress (Lepidium sativum L.) curved downward at almost same rate as in air, whereas the curvature of roots of maize (Zea mays L.) and pea (Pisum sativum L.) was strongly suppressed. Submergence did not cause a decrease in growth rate of these roots. When roots of maize and pea were placed horizontally under water without aeration and then rotated in three dimensions on a clinostat in air, they showed a significant curvature, suggesting that the step suppressed by submergence is not graviperception but the subsequent signal transmission or differential growth process. Constant bubbling of air through the water partly restored the gravitropic curvature of maize roots and completely restored that of pea roots. The curvature of pea roots was also partly restored by the addition of an inhibitor of ethylene biosynthesis, aminooxyacetic acid. In air, ethylene suppressed the gravitropic curvature of roots of maize and pea. Furthermore, the level of ethylene in the intercellular space of the roots was increased by submergence. These results suggest that the accumulation of ethylene in the tissue is at least partly involved in suppression of transmission of the gravity signal or of differential growth in maize and pea roots under conditions of submergence.