History of Japanese Society of Toxicology.

Founded in 1981, the Japanese Society of Toxicology (JSOT) has grown into an organization of nearly 3,000 members working together to advance the nation's scientific knowledge and understanding of toxicology through the implementation of planning that ensures a systematic and efficient expenditure of energies and resources, and is closely aligned with a strategy for accomplishing the Society's long-range plans. To promote public education in toxicology, the Society organizes public lectures during each year's annual meeting. Other activities include hosting scientific conferences, promoting continuing education, and facilitating international collaboration.

Extracellular recombinant annexin II confers UVC-radiation resistance and increases the Bcl-xL to Bax protein ratios in human UVC-radiation-sensitive cells.

In this study, we found that refractoriness to ultraviolet (UVC) light-induced cell death was increased in UVC-radiation-sensitive cells derived from Cockayne syndrome patients when the cells were precultured in medium supplemented with recombinant annexin II (rANX II). In CS3BES cells, an immortal cell line derived from Cockayne syndrome patients, the rANX II supplementation-induced UVC-radiation resistance was suppressed by treatment with an anti-annexin II antibody and EGTA. The amount of biotinylated annexin II on the cell surface increased in the rANX II-supplemented cells but did not increase in the cells that were cotreated with rANX II and EGTA.

Expression levels of drug-metabolizing enzyme, transporter, and nuclear receptor mRNAs in a novel three-dimensional culture system for human hepatocytes using micro-space plates.

We evaluated a novel three-dimensional primary culture system using micro-space plates to determine the expression levels of 61 target (drug-metabolizing enzymes, transporters, and nuclear receptors) mRNAs in human hepatocytes. We measured mRNA expression levels of many target genes in four lots of cryopreserved human hepatocyte primary cells after 120 h of culture and compared differences in mRNA expression levels between cultures using traditional plates and those using micro-space plates. In this study, we show that the mRNA levels of many experimental targets in human hepatocytes before inoculation resemble the levels inside the human liver.

Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins.

Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for human, mouse, and rat carboxylesterase genes that identifies homolog gene families and allocates a unique name for each gene.

Effects of strain differences and vehicles on results of local lymph node assays.

The Local Lymph Node Assay (LLNA) is now regarded as the worldwide standard. The analysis of accumulated LLNA data reveals that the animal strains and vehicles employed are likely to affect LLNA results. Here we show that an obvious strain difference in the local lymph node response was observed between DMSO-treated CBA/CaOlaHsd and CBA/CaHsdRcc mice.

Genotoxicity of acrylamide in vitro: Acrylamide is not metabolically activated in standard in vitro systems.

The recent finding that acrylamide (AA), a genotoxic rodent carcinogen, is formed during the frying or baking of a variety of foods raises human health concerns. AA is known to be metabolized by cytochrome P450 2E1 (CYP2E1) to glycidamide (GA), which is responsible for AA's in vivo genotoxicity and probable carcinogenicity. In in-vitro mammalian cell tests, however, AA genotoxicity is not enhanced by rat liver S9 or a human liver microsomal fraction.

Beta-glucuronidase activity is a sensitive biomarker to assess low-level organophosphorus insecticide exposure.

Acetylcholinesterase and butyrylcholinesterase (BChE) activities in blood are widely used as the biomarkers for organophosphorus insecticide (OP) exposure. In the present study, we conducted a cross-sectional study to evaluate plasma beta-glucuronidase (BG), a sensitive biomarker candidate for OP exposure, BChE activities and urinary dialkyl phosphates (DAPs), OP metabolites. We assessed the relationship between these biomarker levels in the following groups: 32 controls (control), 21 pest control operators and their co-workers who had not sprayed OPs within 3 days prior to sample collection (PCO1), and 21 pest control operators who sprayed OPs within those 3 days (PCO2).

Tissue-specific mRNA expression profiles of drug-metabolizing enzymes and transporters in the cynomolgus monkey.

Pairs of forward and reverse primers and TaqMan probes specific to each of 19 drug-metabolizing enzymes (cytochrome P450s, UDP-glucuronosyltransferases, glutathione S-transferases, and sulfotransferases) and 5 transporters (ABC and SLC transporters) in the cynomolgus monkey were prepared. The expression level of each target mRNA was analyzed in total RNA obtained from three specimens of various cynomolgus monkey tissues (adrenal gland, brain, heart, kidney, large intestine, liver, lung, pancreas, prostate, small intestine, spleen, testis, and thymus) by real-time reverse transcription PCR using an Applied Biosystems 7500 Fast Real-Time PCR System. The data obtained in the present study provide useful information on tissue-specific profiles of the expression of these target mRNAs in the cynomolgus monkey, and the results are expected to be valuable in establishing drug metabolism- and transporter-mediated screening systems using the cynomolgus monkey for the evaluation of new chemical entities in new drug development.

Tissue-specific mRNA expression profiles of human solute carrier 35 transporters.

Pairs of forward and reverse primers and TaqMan probes specific to each of 23 human solute carrier 35 (SLC35) transporters were prepared. The mRNA expression level of each target transporter was analyzed in total RNA from single and pooled specimens of adult human tissues (adipose tissue, adrenal gland, bladder, bone marrow, brain, cerebellum, colon, heart, kidney, liver, lung, mammary gland, ovary, pancreas, peripheral leukocytes, placenta, prostate, retina, salivary gland, skeletal muscle, small intestine, smooth muscle, spinal cord, spleen, stomach, testis, thymus, thyroid gland, tonsil, trachea, and uterus), from pooled specimens of fetal human tissues (brain, heart, kidney, liver, spleen, and thymus), and from three human cell lines (HeLa cell line ATCC#: CCL-2, human cell line Hep G2, and human breast carcinoma cell line MDA-435) by real-time reverse transcription PCR using an Applied Biosystems 7500 Fast Real-Time PCR System. The mRNA expression of SLC35As, SLC35Bs, SLC35Cs, SLC35D1, SLC35D2, SLC35Es, and SLC35F5 was found to be ubiquitous in both adult and fetal tissues.

Comparison of inducibility of multidrug resistance (MDR)1, multidrug resistance-associated protein (MRP)1, and MRP2 mRNAs by prototypical microsomal enzyme inducers in primary cultures of human and cynomolgus monkey hepatocytes.

This study investigated the changes in the mRNA levels of the ATP binding cassette (ABC) transporters multidrug resistance 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), and multidrug resistance-associated protein 2 (MRP2) following exposure to the prototypical microsomal enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) in primary cultures of cryopreserved human and cynomolgus monkey hepatocytes. Analysis was performed by real-time reverse transcription-polymerase chain reaction using primers and TaqMan probes. First, the time course of the mRNA expression of these transporters in primary cultures of human and cynomolgus monkey hepatocytes was examined in detail.

Comparison of inducibility of sulfotransferase and UDP-glucuronosyltransferase mRNAs by prototypical microsomal enzyme inducers in primary cultures of human and cynomolgus monkey hepatocytes.

We investigated the change of the mRNA levels of sulfotransferase and UDP-glucuronosyltransferase isoforms by the prototypical microsomal enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) in primary cultures of cryopreserved human and cynomolgus monkey hepatocytes. Real-time RT-PCR analysis was performed using primers and TaqMan probes. Rif, Dex, and Ome increased SULT2A1 mRNA level in both human and cynomolgus monkey hepatocytes in dose-dependent manner, but not SULT1A1 mRNA level.

Comparison of inducibility of CYP1A and CYP3A mRNAs by prototypical inducers in primary cultures of human, cynomolgus monkey, and rat hepatocytes.

This study was conducted to investigate the effects of treatment with the prototypical inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on the mRNA levels of drug-metabolizing enzymes in primary cultures of cryopreserved human, cynomolgus monkey, and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. Treatment with Ome substantially increased the mRNA levels of both CYP1A1 and CYP1A2 in human hepatocytes, but increased only the mRNA level of CYP1A1 in monkey hepatocytes, whereas it had no marked effect on the mRNA levels of CYP1A1 or CYP1A2 in rat hepatocytes.

Brain regional acetylcholinesterase activity and muscarinic acetylcholine receptors in rats after repeated administration of cholinesterase inhibitors and its withdrawal.

Activity of acetylcholinesterase (AChE) and specific binding of [(3)H]quinuclidinyl benzilate (QNB), [(3)H]pirenzepine (PZP) and [(3)H]AF-DX 384 to muscarinic acetylcholine receptor (mAChR) preparations in the striatum, hippocampus and cortex of rats were determined 1, 6 and 11 days after the last treatment with an organophosphate DDVP, a carbamate propoxur or a muscarinic agonist oxotremorine as a reference for 7 and 14 days. AChE activity was markedly decreased in the three regions 1 day after the treatment with DDVP for 7 and 14 days with a gradual recovery 6 to 11 days, and much less decreased 1, 6 and 11 days after the treatment with propoxur for 7 days but not for 14 days in the hippocampus and cortex. The binding of [(3)H]-QNB, PZP and AF-DX 384 in the three regions was generally decreased by the treatment with DDVP for 7 and 14 days.

Is plasma beta-glucuronidase a novel human biomarker for monitoring anticholinesterase pesticides exposure? A Malaysian experience.

A cross-sectional study was conducted to investigate the effects of acute and chronic pesticide exposure on the plasma beta-glucuronidase enzyme activity among five patients of acute pesticide poisoning in Tengku Ampuan Rahimah Hospital, Klang, 230 farmers in the MADA area, Kedah and 49 fishermen in Setiu, Terengganu. The duration of pesticide exposure among the patients was unknown, but the plasma samples from patients were collected on day one in the hospital. The duration of pesticide exposure among the farmers was between 1 and 45 years.

Regulation of mRNA expression of MDR1, MRP1, MRP2 and MRP3 by prototypical microsomal enzyme inducers in primary cultures of human and rat hepatocytes.

The mRNA induction of various transporters by rifampicin (Rif), dexamethasone (Dex) and omeprazole (Ome) was investigated in primary cultures of cryopreserved human and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. In primary cultures of human hepatocytes, mRNA levels of MDR and MRP1 were increased by about 1.

Structure, function and regulation of carboxylesterases.

This review covers current developments in molecular-based studies of the structure and function of carboxylesterases. To allay the confusion of the classic classification of carboxylesterase isozymes, we have proposed a novel nomenclature and classification of mammalian carboxylesterases on the basis of molecular properties. In addition, mechanisms of regulation of gene expression of carboxylesterases by xenobiotics and involvement of carboxylesterase in drug metabolism and enzyme induction are also described.

Effects of prototypical drug-metabolizing enzyme inducers on mRNA expression of housekeeping genes in primary cultures of human and rat hepatocytes.

Quantitative real-time RT-PCR was used to investigate the effects of prototypical drug-metabolizing enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on mRNA expression levels of the housekeeping genes beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-glucuronidase (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolylisomerase A (PPIA), TATA box binding protein (TBP), and transferrin receptor (TFRC) in primary cultures of cryopreserved human and rat hepatocytes. The mRNA levels of ACTB, GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in human hepatocytes were constant at all concentrations of inducers. However, the mRNA level of GAPDH relative to HPRT1 in rat hepatocytes was markedly increased by Rif.

Assessment of induction of cytochrome P450 by NO-1886 (ibrolipim), a lipoprotein lipase-promoting agent, in primary cultures of human hepatocytes and in female rat liver.

The mRNA levels of human cytochrome P450 (CYP)2Cs and CYP3As in primary cultures of freshly isolated human hepatocytes were assessed after exposure to NO-1886 and rifampicin, a typical inducer of CYP3As. mRNA levels were analyzed by real-time RT-PCR using an ABI PRISM 7700 Sequence Detector system. Exposure to NO-1886 for 24 hr at a concentration of less than 10 microM showed only a tendency to reduce or increase the expression levels of CYP2C8, CYP2C9, CYP2C19, CYP3A4, or CYP3A5 mRNA.

Hepatocyte nuclear factor-4alpha plays pivotal roles in the regulation of mouse carboxylesterase 2 gene transcription in mouse liver.

The mouse carboxylesterase 2 isozyme, mCES2, is thought to play important roles in lipid metabolism and is expressed in the liver, kidney, and small intestine at high levels. In this study, we examined the molecular mechanisms controlling this tissue-specific expression of mCES2, and demonstrated that hepatocyte nuclear factor-4alpha (HNF-4alpha) could enhance transcription of the mCES2 gene in vitro and in vivo. It was found that effects of HNF-4alpha on the level of mCES2 promoter activity were repressed by small heterodimer partner (SHP) and chenodeoxycholic acid (CDCA) in luciferase assays.

Extremely sensitive biomarker of acute organophosphorus insecticide exposure.

Egasyn-beta-glucuronidase complex is located at the luminal site of liver microsomal endoplasmic reticulum. When organophosphorus insecticides (OP) are incorporated into the liver microsomes, they become tightly bound to egasyn, a carboxylesterase isozyme, and subsequently, beta-glucuronidase (BG) is dissociated and released into blood. Consequently, the increase in plasma BG activity becomes a good biomarker of OP exposure.

Toxicological implications of esterases--from molecular structures to functions.

This article reports on a keynote lecture at the 10th International Congress of Toxicology sponsored by the International Union of Toxicology and held on July 2004. Current developments in molecular-based studies into the structure and function of cholinesterases, carboxylesterases, and paraoxonases are described. This article covers mechanisms of regulation of gene expression of the various esterases by developmental factors and xenobiotics, as well as the interplay between physiological and chemical regulation of the enzyme activity.

Salmonella/human S9 mutagenicity test: a collaborative study with 58 compounds.

A large and extensive body of data on the use of human liver S9 fractions in the Salmonella mutagenicity test (Ames test) is presented; the data were obtained from a collaborative study by JEMS/BMS (Bacterial Mutagenicity Test Study Group) members and the Human and Animal Bridging Research Organization (HAB). In this study, the mutagenicity of 58 chemicals, many of which were judged to be human carcinogens by the IARC, was determined by the Ames test (the pre-incubation method at 37 degrees C for 20 min) in the presence of a selected human liver S9 fraction with a high drug-metabolic activity or a pooled human liver S9 fraction with a moderate drug-metabolic activity. For reference, mutagenicity was also examined in the presence of a phenobarbital/5,6-benzoflavone-pretreated rat liver S9 fraction, which is normally used in mutagenicity testing systems.

Different effects of desipramine on bufuralol 1''-hydroxylation by rat and human CYP2D enzymes.

Inhibitory effects of desipramine (DMI) on rat and human CYP2D enzymes were studied using bufuralol (BF) 1''-hydroxylation as an index. Inhibition was examined under the following two conditions: 1) DMI was co-incubated with BF and NADPH in the reaction mixture containing rat or human liver microsomes or yeast cell microsomes expressing rat CYP2D1, CYP2D2 or human CYP2D6 (co-incubation); 2) DMI was preincubated with NADPH and the same enzyme sources prior to adding the substrate (preincubation). When either rat liver microsomes or recombinant CYP2D2 was employed, the preincubation with DMI (0.

Dexamethasone-induced methylprednisolone hemisuccinate hydrolase: its identification as a member of the rat carboxylesterase 2 family and its unique existence in plasma.

Carboxylesterases (CESs) play important roles in the metabolism of many ester-drugs. In the present study, we identified and characterized dexamethasone-induced methylprednisolone hemisuccinate (MPHS) hydrolase in rat liver microsomes. Intraperitoneal injection of dexamethasone resulted in a significant increase in the level of MPHS hydrolase activity accompanied by induction of a specific CES isozyme.

Impact of warm ischemic time on microsomal P450 isoforms in a porcine model of therapeutic liver resection.

Human microsomes and hepatocytes obtained from non-transplantable livers of brain-dead donors are very useful in predicting the in vivo metabolism of xenobiotics in humans. Fresh liver specimens obtained from therapeutic liver resection are also useful for research in cases where non-transplantable livers are not readily available. In the present study, the effect of warm ischemic duration, in the course of hepatic surgery, on the activities of liver cytochrome P450 (CYP) CYP1A, CYP2C, CYP2D, CYP2E1 and CYP3A were evaluated in a porcine model.