N-3 polyunsaturated fatty acids (PUFAs) improve endothelial function. The arachidonic acid-derived metabolites (epoxyeicosatrienoic acids (EETs)) are part of the endothelial hyperpolarization factor and are vasodilators independent of nitric oxide. However, little is known regarding the regulation of EET concentration by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in blood vessels. Sprague-Dawley rats were fed either a control or fish oil diet for 3 weeks. Compared with the control, the fish oil diet improved acetylcholine-induced vasodilation and reduced the protein expression of soluble epoxide hydrolase (sEH), a key EET metabolic enzyme, in aortic strips. Both DHA and EPA suppressed sEH protein expression in rat aorta endothelial cells (RAECs). Furthermore, the concentration of 4-hydroxy hexenal (4-HHE), a lipid peroxidation product of n-3 PUFAs, increased in n-3 PUFA-treated RAECs. In addition, 4-HHE treatment suppressed sEH expression in RAECs, suggesting that 4-HHE (derived from n-3 PUFAs) is involved in this phenomenon. The suppression of sEH was attenuated by the p38 kinase inhibitor (SB203580) and by treatment with the antioxidant N-acetyl-L-cysteine. In conclusion, sEH expression decreased after n-3 PUFAs treatment, potentially through oxidative stress and p38 kinase. Mild oxidative stress induced by n-3 PUFAs may contribute to their cardio-protective effect.
Previously, we reported that implantation of isolated single bone marrow-derived cells into radiation-injured urinary bladders could restore structure and function. However, injections of isolated single cells had some limitations. Thus, in this study, we produced bone marrow-derived cell sheets in temperature-responsive culture dishes that release the monolayer sheets intact. We then determined whether the produced cell sheets could restore function to irradiated urinary bladders. Twenty female 10-week-old Sprague-Dawley (SD) rats were irradiated with 2 gray once a week for 5 weeks. Bone marrow cells harvested from two male 17-week-old green fluorescence protein-transfected SD rats were placed in primary culture for 7 days. Bone marrow cell-derived outgrowths were harvested by enzymatic digestion and transferred into the atelocollagen-coated temperature-responsive culture dishes for 2 days. To harvest the secondarily cultured cells as monolayer sheets, a support membrane was put in each culture dish, and then the temperature was reduced to 20 degrees C. Each released cell sheet was then patched onto the irradiated anterior bladder wall (n=10). As controls, cell-free sheets were similarly patched (n=10). After 4 weeks, transplanted cells were detected on the bladder walls. The cell sheet-transplanted bladders had smooth muscle layers and acetylcholinesterase-positive nerve fibers in proportions that were significantly larger than those of the control bladders. In addition, the cell sheet-transplanted bladders had reduced prolyl 4-hydroxylase beta (P4HB)-positive regions of collagen synthesis and apoptosis within the smooth muscle layers. In cystometric investigations, threshold pressures, voiding interval, micturition volume, and bladder capacity in the cell sheet-transplantation group were significantly higher than those in the control group. Residual volume of the cell sheet-transplantation group was significantly lower compared with the control. There were 24 growth factor mRNAs in the cell sheet-transplanted urinary bladders that were expressed greater than or equal to two-fold over the controls. In conclusion, cell sheet engineering has great potential to restore damaged urinary bladders.
Polyploidy has played a most important role in speciation and evolution of plants and animals. It is thought that low frequency of polyploidy in mammals is due to a dosage imbalance that would interfere with proper development in mammalian polyploids. The first tetraploid mammal, Tympanoctomys barrerae (Octodontidae), appears to be an exception to this rule. In this study we investigated X chromosome inactivation (XCI) and genomic imprinting in T. barrerae, two epigenetic processes usually involved in dosage control in mammalian genomes. The imprinting status of the Peg1 gene was determined by Peg1 allelic expression studies. The inactive X chromosome was identified on interphase nuclei by immunofluorescence using specific antisera raised against Met3H3K27 and macroH2A1. Quantitative PCR was used to compare the Peg1/Dmd ratio in T. barrerae and in its most closely related diploid species, Octomys mimax. Our data demonstrate that parental-specific silencing of at least one gene and normal X chromosomal dosage mechanism are conserved in the tetraploid genome. We hypothesize a concerted action of genetic and epigenetic mechanisms during the process of functional diploidization of this tetraploid genome.
Title: Dynamic CpG and non-CpG methylation of the Peg1/Mest gene in the mouse oocyte and preimplantation embryo Imamura T, Kerjean A, Heams T, Kupiec JJ, Thenevin C, Paldi A Ref: Journal of Biological Chemistry, 280:20171, 2005 : PubMed
In somatic tissues, the CpG island of the imprinted Peg1/Mest gene is methylated on the maternal allele. We have examined the methylation of CpG and non-CpG sites of this differentially methylated CpG island in freshly ovulated oocytes, in vitro aged oocytes, and preimplantation embryos. The CpG methylation pattern was heterogeneous in freshly ovulated oocytes, despite the fact that they all were arrested in metaphase II. After short in vitro culture, Peg1/Mest became hypermethylated, whereas prolonged in vitro culture resulted in demethylation in a fraction of oocytes. Non-CpG methylation also occurred in a stage-specific manner. On alleles that were fully methylated at CpG sites, this modification was found, and it became reduced in two-cell stage embryos and blastocysts. These observations suggest that the process of establishment of the methylation imprint at this locus is more dynamic than previously thought.
Donepezil has been licensed for use in Japan to improve cognitive function since 1999. Among 94 patients with probable Alzheimer's disease who we treated with donepezil, seven patients developed urinary incontinence, although this event was transient in most patients.
Title: Mutagenic and alkylating activities of organophosphate impurities of commercial malathion Imamura T, Talcott RE Ref: Mutat Res, 155:1, 1985 : PubMed
The purpose of this study was to determine if 4 major organophosphate impurities of malathion were active as alkylators of nitrobenzylpyridine (NBP) or as mutagens in the Salmonella typhimurium bioassay. Malathion, isomalathion, O,O,O-trimethyl phosphorothioate, O,O,S-trimethyl phosphorothioate, and O,S,S-trimethyl phosphorodithioate produced alkylated NBP at varying rates. In order of increasing NBP reactivity, the compounds ranked: O,O,O-trimethyl phosphorothioate = O,O,S-trimethyl phosphorothioate less than O,S,S-trimethyl phosphorodithioate less than isomalathion = malathion. At 37 degrees C, the most reactive compounds produced an NBP alkylation rate equal to approximately 25% of the rate produced by methyl methanesulfonate, a potent Salmonella mutagen. However, none of the organophosphates were mutagenic in S. typhimurium TA97, TA98 and TA100 when tested by the standard plate-incorporation method or by the preincubation modification of the plate-incorporation method. The possible relationships between NBP reactivity and the biological activities of these organophosphates are discussed.
Title: Sequential and dose-dependent alterations in rat bronchiolar epithelium during O,O,S-trimethyl phosphorothioate induced delayed toxicity Gandy J, Fukuto TR, Imamura T Ref: Journal of Pathology, 143:127, 1984 : PubMed
The sequential and dose-dependent effects of O,O,S-trimethyl phosphorothioate (OOS-Me) on rat lung bronchiolar epithelium were investigated using scanning electron microscopy. At 12 h after oral treatment (20 mg/kg), there was a small increase in debris in the bronchioles and, by 24 h, there was much debris as well as phagocytic cells in the bronchioles. After 3 days, there was a decrease in the number of bronchiolar Clara cells as determined by the loss of their characteristic apical bulges. Concomitantly, there was a significant increase in lactate dehydrogenase activity in bronchopulmonary lavage fluid. By day 7, the Clara cells appeared to be reforming and, by day 14, the morphology of bronchiolar epithelium had returned to normal. Dose-dependent studies revealed a threshold dose level of OOS-Me between 10 and 20 mg/kg which produced the observed effects in Clara cells. Both sequential and dose-dependent effects of OOS-Me on Clara cells were correlated with changes in bronchopulmonary lavage lactate dehydrogenase activity.
Title: Lung injury and delayed toxicity produced by O,S,S-trimethyl phosphorodithioate, an impurity of malathion Konno N, Fukuto TR, Imamura T Ref: Toxicol Appl Pharmacol, 75:219, 1984 : PubMed
O,S,S,-Trimethyl phosphorodithioate (OSS-Me), an impurity present in organophosphorus insecticides, produced morphological alteration of rat and mouse terminal nonciliated bronchiolar epithelial (Clara) cells. The effects of OSS-Me on pulmonary and hepatic microsomal enzymes were studied following its po administration to rats. Oral 28-day LD50 of OSS-Me for rats was 67 mg/kg. The animals were treated with OSS-Me at 40, 100, and 160 mg/kg and killed 24 hr later. The relative lung weights were not affected at this time. Pulmonary microsomal benzo[a]pyrene hydroxylase decreased significantly; activities were less than 36% of control at the lowest dose. In contrast, the effect of OSS-Me treatment on hepatic monooxygenase activity was moderate. Benzo[a]pyrene hydroxylase, p-nitroanisole demethylase, or 7-ethoxycoumarin deethylase were not affected by OSS-Me treatment at any dose. Pulmonary and hepatic malathion carboxylesterase activities decreased following OSS-Me treatment. The decrease was more marked in liver. Time course effects of OSS-Me treatment on these parameters were examined by treating rats at 40 mg/kg, and the animals were killed at 6, 12, 24, and 72 hr after treatment. The lung relative wet weight was increased markedly at 72 hr. The benzo[a]pyrene hydroxylase activity of pulmonary microsomes was decreased at 24 and 72 hr after treatment, the maximum decrease being observed at 72 hr. At this time, glutathione of the lungs was depleted markedly. Significant decreases were not observed in hepatic monooxygenase activities. Liver glutathione content was not reduced at 72 hr. Pulmonary and hepatic malathion carboxylesterase activities decreased throughout the time course. OSS-Me selectively inhibited benzo[a]pyrene hydroxylase activity, depleted glutathione, and caused morphological alteration of Clara cells in lungs of rats. Although the toxic mechanism(s) produced by OSS-Me remains unknown, these results support the view that the lung is a target organ of OSS-Me-induced delayed toxicity.
Title: Phenobarbital pretreatment protects against morphologic changes in rat bronchiolar epithelium caused by an impurity of malathion Gandy J, Talbot P, Fukuto TR, Imamura T Ref: American Journal of Pathology, 111:350, 1983 : PubMed
Oral administration (20 mg/kg) of O,O,S-trimethyl phosphorothioate (OOS) causes delayed toxicity in rats; ie, death occurs as late as 28 days after treatment. OOS-treated rats show morphologic changes in the bronchiolar epithelium of the lung; nonciliated (Clara) cells are fewer but larger 3 days after treatment. We have now found that pretreatment with the P-450-dependent mixed-function oxidase inducer, phenobarbital, protects against the morphologic changes caused by OOS. These results support the view that the lung is a target organ of the delayed toxicity caused by OOS and that OOS detoxification is mediated by P-450-dependent metabolism.
Title: Selective inhibition of rat pulmonary monooxygenase by O,O,S-trimethyl phosphorothioate treatment Imamura T, Gandy J, Fukuto TR Ref: Biochemical Pharmacology, 32:3191, 1983 : PubMed
The effects of oral administration of O,O,S-trimethyl phosphorothioate (OOS), an impurity present in widely used organophosphorus insecticides, were studied using pulmonary and hepatic microsomal enzymes of rats. The animals were treated with OOS at 10,20 and 40 mg/kg, and were killed on day 3 after treatment. Their relative lung weights increased markedly at 20 and 40 mg/kg, increasing 94% at the highest dose, whereas the weight of liver decreased. At 20 mg/kg OOS, the cytochrome P-450 content of the lung and liver decreased to 83 and 80% of the control levels respectively. Pulmonary microsomal 7-ethoxycoumarin (7-Ec) O-deethylase decreased in a dose-dependent manner; activities were less than 10% of control at the 40 mg/kg dose. The activity of pulmonary coumarin hydroxylase also decreased following OOS treatment, but the decrease was not dose-dependent since no activity was detectable at doses over 10 mg/kg. In contrast, the effect of OOS treatment on hepatic monooxygenase activity was moderate. 7-Ec deethylase activity was not affected by OOS treatment at any dose level, while p-nitroanisole (p-NA) demethylase activity was decreased only at the 40 mg/kg dose of OOS. Pulmonary malathion carboxylesterase activity was not affected by OOS treatment. In contrast, a dose-dependent decrease was observed in the liver carboxylesterase. Time course effects of OOS treatment on these parameters were examined by treating rats at 20 mg/kg. The animals were killed 0.5, 1,3 and 7 days after the treatment. The 7-Ec deethylase activity of pulmonary microsomes was decreased on days 0.5, 1 and 3 after treatment, the maximum decrease being observed on day 1. Significant decreases were not observed in hepatic microsomal activities of 7-Ec deethylase or p-nitroanisole demethylase throughout the experimental period; rather, these activities were higher on day 7. Hepatic microsomal malathion carboxylesterase was lower on days 0.5, 1 and 3 after OOS treatment.
Title: An impurity of malathion alters the morphology of rat lung bronchiolar epithelium Imamura T, Gandy J, Fukuto TR, Talbot P Ref: Toxicology, 26:73, 1983 : PubMed
Oral administration of O,O,S-trimethyl phosphorothioate (OOS), an impurity in technical malathion, caused morphological changes in the bronchiolar epithelium of rat lungs. OOS-treated rat lungs had fewer but larger Clara (non-ciliated) cells than lungs from control rats given either corn oil or purified malathion. Moreover, lactate dehydrogenase (LDH) activity in bronchopulmonary lavage fluid was significantly higher in OOS than in control rats. We interpret these data to mean that OOS, and/or its metabolite(s) causes a lesion in the lung. Because of the widespread agricultural use of technical malathion, future work should address the significance of our findings and the possible toxic effect of OOS on lung tissue.
Title: Effect of drug metabolism inducer and inhibitor on O,O,S-trimethyl phosphorothioate-induced delayed toxicity in rats Imamura T, Hasegawa L, Gandy J, Fukuto TR Ref: Chemico-Biological Interactions, 45:53, 1983 : PubMed
Oral administration of O,O,S-trimethyl phosphorothioate (OOS), an impurity present in widely used organophosphorus insecticides, causes delayed toxicity in rats, i.e., death occurring as late as 28 days after the treatment. The signs of toxicity include body weight loss (maximum on day 3), red staining around the nose, mouth and eyes, and an increased level of lactate dehydrogenase (LDH) in bronchopulmonary lavage fluid accompanied by morphological alteration of non-ciliated bronchiolar epithelial Clara cells. Pretreatment with phenobarbital, piperonyl butoxide (2 h), SKF 525-A, or small multiple doses of OOS protected against the OOS-induced elevated level of bronchopulmonary lavage LDH, and the other signs of delayed toxicity including morphological alteration of Clara cells. These studies support the view that OOS-induced delayed toxicity is mediated by the cytochrome P-450 dependent metabolism of OOS, and the lung may be the major target organ of delayed toxicity produced by OOS.
Title: Malathion and phenthoate carboxylesterase activities in pulmonary alveolar macrophages as indicators of lung injury Imamura T, Schiller NL, Fukuto TR Ref: Toxicol Appl Pharmacol, 70:140, 1983 : PubMed
Malathion and phenthoate carboxylesterase activities were investigated in pulmonary alveolar macrophages (PAM) in Sprague-Dawley rats. PAM was found to be capable of hydrolyzing phenthoate at a faster rate than malathion. Oral administration to rats with O,O,S-trimethyl phosphorothioate (OOS-Me), a pneumotoxic impurity present in technical grades of malathion and phenthoate, increased the activities of these esterases in PAM without affecting an activity in lung microsomal carboxylesterase. The time course study indicated that this increase was maximal on Day 1 following treatment with OOS-Me at 20 and 40 mg/kg of doses. To assess the usefulness of measuring these esterases in PAM as an indicator of lung damage, paraquat and bromobenzene were administered to rats with treatment regimens which have been shown previously to result in histopathologically demonstrable pneumotoxicity. Malathion and phenthoate carboxylesterase activities in PAM were increased by two- to threefold following treatment with paraquat or bromobenzene. These treatments also increased lung microsomal malathion carboxylesterase activity by threefold. Furthermore, infection of rats with Pseudomonas aeruginosa by intratracheal inoculation increased malathion and phenthoate carboxylesterase activities in PAM by two- to threefold without increasing these activities in lung microsomes. These results indicate that PAM may play a significant role in detoxifying airborne malathion and phenthoate when inhaled. Furthermore, the activities of malathion and phenthoate carboxylesterases may be useful for detecting lung injury produced by pneumotoxic chemicals as well as bacterial infection.
