AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function.

UNLABELLED: The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4-phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell-surface-resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of α- and µ2-adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin-dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 3×FLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of α-adaptin inhibits BSEP internalization from the plasma membrane and increases its cell-surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S-transferase pulldown assay, and time-lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes. CONCLUSION: AP2 mediates the internalization and subsequent degradation of CM-resident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis.

Microphysiological systems in early stage drug development: Perspectives on current applications and future impact.

Microphysiological systems (MPS) are making advances to provide more standardized and predictive physiologically relevant responses to test articles in living tissues and organ systems. The excitement surrounding the potential of MPS to better predict human responses to medicines and improving clinical translation is overshadowed by their relatively slow adoption by the pharmaceutical industry and regulators. Collaboration between multiorganizational consortia and regulators is necessary to build an understanding of the strengths and limitations of MPS models and closing the current gaps. Here, we review some of the advances in MPS research, focusing on liver, intestine, vascular system, kidney and lung and present examples highlighting the context of use for these systems. For MPS to gain a foothold in drug development, they must have added value over existing approaches. Ideally, the application of MPS will augment in vivo studies and reduce the use of animals via tiered screening with less reliance on exploratory toxicology studies to screen compounds. Because MPS support multiple cell types (e.g. primary or stem-cell derived cells) and organ systems, identifying when MPS are more appropriate than simple 2D in vitro models for understanding physiological responses to test articles is necessary. Once identified, MPS models require qualification for that specific context of use and must be reproducible to allow future validation. Ultimately, the challenges of balancing complexity with reproducibility will inform the promise of advancing the MPS field and are critical for realization of the goal to reduce, refine and replace (3Rs) the use of animals in nonclinical research.

Current status and future perspective of computational toxicology in drug safety assessment under ontological intellection.

For the safety assessment of pharmaceuticals, initial data management requires accurate toxicological data acquisition, which is based on regulatory safety studies according to guidelines, and computational systems have been developed under the application of Good Laboratory Practice (GLP). In addition to these regulatory toxicology studies, investigative toxicological study data for the selection of lead compound and candidate compound for clinical trials are directed to estimation by computational systems such as Quantitative Structure-Activity Relationship (QSAR) and related expert systems. Furthermore, in the "Go" or "No-Go" decision of drug development, supportive utilization of a scientifically interpretable computational toxicology system is required for human safety evaluation. A pharmaceutical safety evaluator as a related toxicologist who is facing practical decision needs not only a data-driven Artificial Intelligence (AI) system that calls for the final consequence but also an explainable AI that can provide comprehensive information necessary for evaluation and can help with decision making. Through the explication and suggestion of information on the mechanism of toxic effects to safety assessment scientists, a subsidiary partnership system for risk assessment is ultimately to be a powerful tool that can indicate project-vector with data weight for the corresponding counterparts. To bridge the gaps between big data and knowledge, multi-dimensional thinking based on philosophical ontology theory is necessary for handling heterogeneous data for integration. In this review, we will explain the current state and future perspective of computational toxicology related to drug safety assessment from the viewpoint of ontology thinking.

Human hepatocytes can repopulate mouse liver: histopathology of the liver in human hepatocyte-transplanted chimeric mice and toxicologic responses to acetaminophen.

A human hepatocyte-transplanted chimeric mouse has been established by transplantation of human hepatocytes to urokinase-type plasminogen activator transgenic/severe combined immunodeficiency (uPA(+/+)/SCID) mice. These chimeric mice have various amounts of human hepatocytes that proliferate extensively and progressively replace mouse hepatocytes. In the chimeric liver, hepatic cords and sinusoid-like structures were observed. The human hepatocytes expressed human albumin, human cytochrome P450 enzymes, and human transporter proteins. Furthermore, electron microscopic analysis demonstrated bile canaliculi associated with human hepatocytes in the chimeric mouse livers. These results indicate that the chimeric mouse livers contain functionally intact and differentiated human hepatocytes. Additionally, the toxicologic response of hepatocytes to acetaminophen (APAP) administration was compared in normal and chimeric mouse livers. Following 1,400 mg/kg APAP, mild hepatocellular degeneration was observed in the human hepatocyte areas in the chimeric mice, compared with severe centrilobular hepatocellular necrosis in the ICR mouse livers. In conclusion, these chimeric livers contain functionally differentiated human hepatocytes, and are less susceptible to APAP toxicity, compared to ICR mice.

Methodological validation of an existing telemetry system for QT evaluation in conscious guinea pigs.

INTRODUCTION: Guinea pigs are suitable for in vivo QT assessment of newly discovered drugs at the pre-clinical stage because of the ease with which these animals can be handled, the lower amount of compound required for testing, and the similarity of the ion channels between the guinea pig heart and the human. Our purpose was to provide detailed methodological information on an existing telemetry recording system for use in evaluating QT interval prolongation in guinea pigs. METHODS: Hartley guinea pigs weighing 400-700 g were used to investigate the appropriate configuration of electrodes to record defined T-waves and the influence of the surgical implantation of a transmitter on the QT interval, as well as to determine the appropriate formula for QT correction. In addition, the validity of using telemetry-monitored guinea pigs was tested by using compounds with (positive references) or without (vehicles) a QT-prolonging effect. RESULTS: A lead with the negative pole placed between the scapulas and the positive pole positioned close to the sternum was found to be the most appropriate to obtain well-defined T-waves. The period for recovery from transmitter implantation was estimated to be at least 1 week. The best-fit formula for our telemetry guinea pig model was a modified Bazett's formula. QTc was prolonged significantly in guinea pigs given positive references, and the QTc was unaffected when the animals were given vehicles. DISCUSSION: We believe that the information provided herein will be a quite helpful guide for researchers to evaluate the QT interval reliably and reproducibly in this telemetry guinea pig model.

Early changes in sperm motility, acrosome reaction, and gene expression of reproductive organs in rats treated with sulfasalazine.

Previously, we reported that decreased epididymal expression of CD59 and decay accelerating factor (DAF) genes may affect sperm motility and the acrosome reaction in rats treated long-term (28 days) with sulfasalazine. To investigate the early effects of sulfasalazine on the male reproductive tract, we presently examined sperm motility, the acrosome reaction, and gene expression in the testes and epididymides of rats treated with sulfasalazine for 1, 7 or 14 days. Reduced sperm motility and acrosome reactions were noted on day 7, however, there were no remarkable changes in testicular gene expression. On the other hand, attenuated epididymal gene expression of CD59 and DAF was observed as early as day 1. As CD59 and DAF are secreted from the epididymis and play a role in sperm maturation, we hypothesize that sulfasalazine affects sperm maturation as an early effect and that CD59 and DAF genes are related to the negative effect.

Integrated NMR-based metabonomic investigation of early metabolic effects of ethylene glycol monomethyl ether (EGME) on male reproductive organs in rats.

High-resolution Magic Angle Spinning (Hr-MAS) (1)H-NMR spectroscopy was used to analyze intact testicular tissues ex vivo and to investigate the toxicological effects of ethylene glycol monomethyl ether (EGME), a well-known spermatocytes toxicant, on male reproductive organs by NMR-based metabonomic analysis. Especially, we reported the first Hr-MAS (1)H-NMR spectra of epididymis. Sexually matured male rats were treated with 50 and 2,000 mg/kg EGME, and body weight, reproductive organs weight, histopathology and plasma biochemistry were examined at 6 and 24 hr after administration. Two multivariate statistical methods, namely, unsupervised PCA and supervised PLS-DA, indicated that the balance of endogenous metabolites was perturbed in both reproductive organs and biofluids. In the testes, lactate, creatine and glutathione were mainly affected by EGME treatment. In urine and plasma, altered excretions of the TCA cycle intermediates (2-oxoglutarate, citrate and succinate) and the ketone-bodies (acetoacetate and beta-hydroxybutyrate) were also observed. The finding in current integrated metabonomic analysis of both intact tissues and biofluids suggested that EGME-induced testicular toxicity was attributed to perturbation of the energy supply processes, suppression of the TCA cycle, or oxidative stress. Furthermore, Hr-MAS (1)H-NMR proved useful to investigate the molecular snapshot of biological tissues and the mechanism of toxicity.

Simultaneous measurement of nucleated cell counts and cellular differentials in rat bone marrow examination using flow cytometer.

The purpose of this study was to establish the simultaneous measurement of nucleated cell counts and cellular differentials in rat bone marrow examination. The bone marrow cells were stained with an anthraquinone fluorescent DNA stain (DRAQ5) and fluorescence-labeled antibodies, and were analyzed quantitatively using a flow cytometer in the presence of internal standard beads. DRAQ5 distinguished populations of nucleated cells. The absolute counts of nucleated cells were determined using an internal standard, and were equivalent to that measured by the electrical resistance method. The population of nucleated cells was classified into myeloids and erythroids by labeling with CD11b/c and CD71 antibodies, respectively. In a separate examination, T- and B-lymphocytes were also classified by labeling with CD3 and CD45RA antibodies, respectively. The classification of each cell lineage was identical with that of the alternative flow-cytometric method in which cells were differentiated according to cellular size and the fluorescence of a peroxidase indicator, 2',7'-dichlorofluorescin. The ratios of cell lineage, together with myeloid/erythroid ratio (ME), were the same as those obtained by a manual microscopic method. The present flow cytometric method enables the simultaneous measurement of the total nucleated cell counts and cellular differentials of rat bone marrow cells, allowing for rapid and highly quantitative bone marrow examination in rats.

Changes of micro-RNA expression in rat liver treated by acetaminophen or carbon tetrachloride--regulating role of micro-RNA for RNA expression.

Recently, microRNAs, involved in RNA interference, were discovered as a new gene regulation, with little is known in the filed of toxicology. In this study, a toxic dose of acetaminophen or carbon tetrachloride was administered singly to male rats, and microarry analysis using mirVana miRNA bioarray was performed. Partial least squares-discriminant analysis of the microarray data revealed that microRNAs expression was specifically changed by treatments at 6 hr after dosing. Furthermore, we focused on miR298 and miR370 among the microRNAs commonly affected by hepatotoxicants, because they were speculated to regulate an oxidative stress-related gene. From real-time RT-PCR analysis, microRNAs expression was suppressed by hepatotoxicants at 6 and 24 hr. Regarding acetaminophen, the decreases were found even though there were no morphological changes in the liver at 6 hr. To investigate these 2 microRNAs in more detail, we measured their expression, WST-1 for mitochondrial function and LDH release for cell collapse in primary cultured hepatocytes exposed to several concentrations of acetaminophen for 3 hr. At more than 5 mM, the microRNA expression and WST-1 decreased, whereas LDH was unchanged. Therefore, the change in microRNA expression occurred at the time when mitochondrial function was damaged prior to cell collapse. From all the above findings, we conclude that microRNAs were affected by hepatotoxicants and that the changes were found in the early phase of toxicity. Thus, our data suggest microRNAs have an important role for toxicological mechanism and we proposed that the changes in microRNA expression might be key molecules for toxicity expression.

Evaluation of human hepatocyte chimeric mice as a model for toxicological investigation using panomic approaches--effect of acetaminophen on the expression profiles of proteins and endogenous metabolites in liver, plasma and urine.

Toxicological responses to acetaminophen (APAP) overdose were evaluated in human hepatocytes transplanted chimeric mice using 2-dimensional gel electrophoresis (2DE)-based proteomics and (1)H-nuclear magnetic resonance (NMR)-based metabonomics. Huge variations, which were supported by histopathological findings, were observed in proteins expression in chimeric mice liver. The proteomic analysis of the livers showed that the proteins involved in the pathways of lipid/fatty acid metabolism, glycolysis and energy metabolism/production were affected. In addition, oxidative stress-related proteins showed altered expression. The metabonomic analysis of urine and plasma revealed alterations of endogenous metabolites, which were the intermediates involved in the tricarboxylic acid (TCA) cycle. Those findings were already confirmed in normal mice. We hypothesized that the mechanism of APAP-induced effects on chimeric mice liver was in accordance with the mechanism observed in normal mice. Therefore, these toxicopanomic approaches successfully revealed that the mechanisms in humans were identical with "known" APAP-induced hepatotoxicity detected in chimeric mice. Further investigations are needed to detect idiosyncratic hepatotoxicity in humans using chimeric mice.

Effect of dose regimen on the toxicity of 2'-deoxy-2'-methylidenecytidine (DMDC) in monkeys.

2'-deoxy-2'-methylidenecytidine (DMDC) is a potential anticancer deoxycytidine analog of cytosine arabinoside. Using monkeys, we conducted a 4-week toxicity study with toxicokinetics of DMDC at 1, 3, and 10 mg/kg/day and a dose-regimen study of three different schedules of once-daily administration (5 mg/kg/day) for 1 week every 2 weeks, 2 weeks every 4 weeks, and 3 weeks every 4 weeks. Deaths, myelosuppression, intestinal toxicity, and swelling of palm and sole skin were observed by oral DMDC treatment at 10 mg/kg/day in 4-week repeated toxicity study; however, no skin disorders have been reported in humans. No notable changes were observed at 1 and 3 mg/kg/day. The curves of dose vs. AUC and the AUC at MTD in monkey are similar to those in humans. In the dose-regimen study, all the toxicities were reversible but more severe toxicity was observed with the longer administration periods. One-week interruption showed sufficient recovery of decreased WBC in dosing regimens of 1-week-on/1-week-off and 2-weeks-on/2-weeks-off. A 2-week recovery period was almost sufficient for the recovery of decreased RBC, HCT, and skin disorders in the 2-weeks-on/2-weeks-off regimen. Therefore, once-daily for 2 weeks every 4 weeks was concluded to be the optimal dose regimen. In summary, myelosuppression, intestinal toxicity, and skin disorders were observed in DMDC treatment in monkeys, the relationship between AUC and toxicity in monkeys was close to that in humans, and in preclinical studies, it is advantageous to investigate optimal dose regimens using the appropriate species.

Physicochemical and cell-based approach for early screening of phospholipidosis-inducing potential.

Some of the principal requisites of toxicity screening methods in drug discovery are their ease to perform and high throughput, as well as the possibility to predict the occurrence of clinical events. Phospholipidosis is one of the toxicities often induced by potential drugs. Several physicochemical methods for the prediction of phospholipidosis have been reported. The purpose of the present study was to examine the predictability of methods based on lipophilicity and charge parameters. We employed a test set of 33 compounds including 11 in-house compounds. The phospholipidosis-inducing potential (PLIP) of the test set compounds was determined by the fluorescence-labeled lipid accumulation assay using isolated rat hepatocytes. This assay was verified by transmission electron microscopy (EM). The usefulness of the ClogP - most basic pK(a) (pK(a) -MB) plot to the PLIP of compounds was examined. This plot was unable to predict the PLIP of zwitterions. In order to improve its predictability, the net charge of a given molecule (NC) was introduced instead of pK(a) - MB, since the NC corresponds directly to the ionization state of compounds in the organelles. Compounds with high ClogP (> 1) and high NC (1< or =NC< or =2) tended to be positive. This finding was also confirmed using 30 additional validation set compounds obtained from the literature. The ClogP - NC plot differentiated positive and negative compounds with more than 98% accuracy (62/63), indicating its usefulness in drug discovery.

Genomic cluster and network analysis for predictive screening for hepatotoxicity.

The present study was undertaken to estimate the usefulness of genomic approaches to predict hepatotoxicity. Male rats were treated with acetaminophen (APAP), carbon tetrachloride (CCL), amiodarone (AD) or tetracycline (TC) at toxic doses. Their livers were extracted 6 or 24 hr after the dosings and were used for subsequent examinations. At 6 hr there were no histological changes noted in any of the groups except for the CCL group, but at 24 hr, such changes were noted in all but the AD group. Regarding genomic analysis, we performed hierarchical cluster analysis using S-plus software. The individual microarray data were clearly classified into 5 treatment-related clusters at 24 hr as well as at 6 hr, even though no morphological changes were noted at 6 hr. In the gene expression analysis using GeneSpring, transcription factor and oxidative stress- and lipid metabolism-related genes were markedly affected in all treatment groups at both time points when compared with the corresponding control values. Finally, we investigated gene networks in the above-affected genes by using Ingenuity Pathway Analysis software. Down-regulation of lipid metabolism-related genes regulated by SREBP1 was observed in all treatment groups at both time points, and up-regulation of oxidative stress-related genes regulated by Nrf2 was observed in the APAP and CCL treatment groups. From the above findings, for the application of genomic approaches to predict hepatotoxicity, we considered that cluster analysis for classification and early prediction of hepatotoxicity and network analysis for investigation of toxicological biomarkers would be useful.

Investigation of proteomic biomarkers in in vivo hepatotoxicity study of rat liver: toxicity differentiation in hepatotoxicants.

We investigated the overall protein expression profiles in the in vivo hepatotoxicity of rats induced by four well-recognized hepatotoxicants. Acetaminophen (APAP), amiodarone (AMD), tetracycline (TC) and carbon tetrachloride (CTC) were administered to male rats by gavages and the liver at 24 hr post-dosing was applied to the proteomic experiment. Blood biochemistry and histopathology were examined to identify specific changes related to the compounds given. Protein expression in the liver was investigated by 2-dimensional gel electrophoresis (2DE), and spots showing a significantly different expression in treated versus control group were excised from gels and identified by Q-Tof mass spectrometer. They were well characterized based on their functions related to the mechanisms of toxicity of the compounds. Among them, we focused on the 8 proteins that were affected by all 4 compounds examined. Proteins related to oxidative stress response such as carbonic anhydrase III (CA3) and 60kDa heat shock protein (HSP60), and energy metabolism such as adenylate kinase 4 (AK4) were found. Moreover, hierarchical clustering analysis using 2D-gel spots information revealed the possibility to differentiate the groups based on their toxicity levels such as severity of liver damage. These results suggested that assessing the effects of hepatotoxicants on protein expression is worth trying to screen candidate compounds at the developmental stage of drugs.

Relationship between AUC of 5'-DFUR and toxicity of capecitabine, fluoropyrimidine carbamate analogs, and 5'-DFUR in monkeys, mice, and rats.

Capecitabine is an oral fluoropyrimidine carbamate which is converted to 5-fluorouracil (5-FU) via 3 enzymatic step to 5'-deoxy-5-fluorocytidine (5'-DFCR), 5'-deoxy-5-fluorouridine (5'-DFUR), and finally 5-FU. We performed 4-week toxicity studies of capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorouridine), galocitabine (trimethoxybenzyl-5'-deoxy-5-fluorocytidine), 4 different fluoropyrimidine carbamate analogs (R=butyl, isopentyl, propyl, or phenethyl), and 5'-DFUR in cynomolgus monkeys with toxicokinetic measurements of intact molecules, 5'-DFCR, and 5'-DFUR. Four-week toxicity data for capecitabine in rats and mice were also obtained for comparison. Capecitabine, galocitabine, butyl, and isopentyl analogs showed similar toxicities in hematopoietic and intestinal organs at 1.0 mmol/kg and the AUCs of 5'-DFUR were approximately 40 to 60 microg*hr/ml. These compounds showed slight toxicity at 0.5 mmol/kg and no toxicity at 0.1 mmol/kg, and AUCs of 5'-DFUR were approximately 30 and 5 microg*hr/ml, respectively. Propyl and phenethyl analogs showed slight toxicity at 1.0 mmol/kg and no toxicity at 0.5 mmol/kg, and AUCs of 5'-DFUR were approximately 30 and 10 microg*hr/ml, respectively. On the other hand, severe and slight-to-moderate toxicity was observed at 0.5 and 0.25 mmol/kg in 5'-DFUR-treated monkeys and AUCs of 5'DFUR were 35.6 and 5.2 microg*hr/ml, respectively. In mice and rats, the toxicity of capecitabine was less than in monkeys relative to dose, but 5'-DFUR AUCs were almost the same. In conclusion, 5'-DFUR AUC correlated with toxicity following oral administration of capecitabine and its analogs in monkeys, mice, and rats, although this relationship is not seen in humans. Capecitabine was less toxic in monkeys than oral 5'-DFUR according to dose (mmol/kg) and 5'-DFUR AUC.

In vivo hepatotoxicity study of rats in comparison with in vitro hepatotoxicity screening system.

For the establishment of a high throughput screening system using primary cell cultures, investigation of elucidated toxicities to assess the correlation between in vitro and in vivo hepatotoxicity is necessary in the safety evaluation of the compound. In the previous study, we reported the usability of rat primary cultured hepatocytes for establishment of high throughput screening system. To confirm the reliability of rat primary hepatocytes culture screening system, we conducted a single-dose in vivo study with relatively high dose of hepatotoxicant in rats using 4 reference compounds (acetaminophen, amiodarone, tetracycline, carbon tetrachloride), and investigated histopathological changes and expression of oxidative stress-related proteins by immunohistochemistry. We also carried out a proteomics analysis for estimating the reliable and sensitive biomarkers. Histopathologically, compound-specific hepatotoxicity was detected at 24 hr after administration in all compounds except amiodarone, which is known to induce phospholipidosis. Immunohistochemically, oxidative stress-related proteins were increased within 6 hr after administration in all treated groups. Proteomics analysis revealed several protein biomarkers related to oxidative stress and mitochondrial metabolism-regulation, which had been previously detected by proteomics analysis in in vitro screening system. Oxidative stress-related proteins were considered as useful biomarkers of hepatotoxicity; since they were detected by immunohistochemistry and proteomics analysis prior to appearance of compound-specific histopathological changes detected by light microscopy. Considering the relevance of in vitro system to in vivo system from the aspect of new biomarkers related to the toxicogenomics/toxicoproteomics, in vitro primary cell culture system would be sufficient to detect hepatotoxicity in the early stage of drug discovery.

The principle of safety evaluation in medicinal drug - how can toxicology contribute to drug discovery and development as a multidisciplinary science?

Pharmaceutical (drug) safety assessment covers a diverse science-field in the drug discovery and development including the post-approval and post-marketing phases in order to evaluate safety and risk management. The principle in toxicological science is to be placed on both of pure and applied sciences that are derived from past/present scientific knowledge and coming new science and technology. In general, adverse drug reactions are presented as "biological responses to foreign substances." This is the basic concept of thinking about the manifestation of adverse drug reactions. Whether or not toxic expressions are extensions of the pharmacological effect, adverse drug reactions as seen from molecular targets are captured in the category of "on-target" or "off-target", and are normally expressed as a biological defense reaction. Accordingly, reactions induced by pharmaceuticals can be broadly said to be defensive reactions. Recent molecular biological conception is in line with the new, remarkable scientific and technological developments in the medical and pharmaceutical areas, and the viewpoints in the field of toxicology have shown that they are approaching toward the same direction as well. This paper refers to the basic concept of pharmaceutical toxicology, the differences for safety assessment in each stage of drug discovery and development, regulatory submission, and the concept of scientific considerations for risk assessment and management from the viewpoint of "how can multidisciplinary toxicology contribute to innovative drug discovery and development?" And also realistic translational research from preclinical to clinical application is required to have a significant risk management in post market by utilizing whole scientific data derived from basic and applied scientific research works. In addition, the significance for employing the systems toxicology based on AOP (Adverse Outcome Pathway) analysis is introduced, and coming challenges on precision medicine are to be addressed for the new aspect of efficacy and safety evaluation.

Effects of sulfasalazine on sperm acrosome reaction and gene expression in the male reproductive organs of rats.

Sulfasalazine (SASP) has been reported to depress the fertility in men and experimental male animals, but the fundamental mechanisms of infertility caused by SASP are still unknown. This study was designed to investigate the mechanisms of infertility in rats treated with SASP at a dose of 600 mg/kg for 28 days, including monitoring of sperm motility using computer associated sperm analysis system and acrosome reaction by FITC-concanavalin A lectin staining. The sperm motility and acrosome reaction, which are important for fertilization, were significantly reduced by SASP. Furthermore, to investigate the molecular mechanisms of infertility induced by SASP, mRNA expression analysis in the testes was performed using cDNA microarray as a first screening. It was revealed that CD59, which is located on the acrosomal membrane and is known to be important for the reproductive function of sperm, was affected in the testes; this was also confirmed by real-time PCR analysis, but the spermatogenesis-related genes examined in this study were not affected. Therefore, we focused on CD59 and two other acrosome membrane related-genes: MCP and DAF. CD59, MCP, and DAF in the epididymides of SASP-treated rats were significantly decreased as assessed by real-time RT-PCR analysis and additionally, the expression of CD59 protein was found to be decreased by Western blotting. These results allowed us to hypothesize that the suppression of epididymal acrosomal membrane proteins synthesis with their consequent reduced incorporation to the sperm membrane leads to a depressed sperm motility and acrosome reaction, and thereby leads to infertility in SASP treated male rats.

Identification of oxidative stress-related proteins for predictive screening of hepatotoxicity using a proteomic approach.

We investigated the effects of three hepatotoxicants, acetaminophen (APAP), amiodarone (AD) and tetracycline (TC), on protein expression in primary cultured rat hepatocytes with toxicoproteomic approach, which is two-dimensional gel electrophoresis (2DE) and mass spectrometry. The objectives of this study were to search for alternative toxicity biomarkers which could be detected with high sensitivity prior to the appearance of morphological changes or alterations of analytical conventional biomarkers. The related proteins in the process of cell degeneration/necrosis such as cell death, lipid metabolism and lipid/carbohydrate metabolism were mainly affected under exposure to APAP, AD and TC, respectively. Among the differentially expressed proteins, several oxidative stress-related proteins were clearly identified after 24-hr exposure, even though they were not affected for 6-hr exposure. They were glutathione peroxidase (GPX) as a down-regulated protein as well as peroxiredoxin 1 (PRX1) and peroxiredoxin 2 (PRX2) as up-regulated proteins, which are known to serve as antioxidative enzymes in cells. These findings suggested that the focused proteins, GPX and PRXs, could be utilized as biomarkers of hepatotoxicity, and they were useful for setting high throughput screening methods to assess hepatotoxicity in the early stage of drug discovery.

Protein expression analysis of rat testes induced testicular toxicity with several reproductive toxicants.

The utilization of safety biomarkers to predict the possibility of compound-related toxicity provides several advantages for drug discovery and development, especially at an early stage. The objectives of this study were to investigate the effects of male reproductive toxicants on protein expression profiles in the rat testes and to identify potential biomarker candidates. Four well-known reproductive toxicants, ethylene glycol monomethyl ether (EGME), cyclophosphamide (CP), sulfasalazine (SASP) and 2,5-hexanedione (2,5-HD), were administered to male rats in a single dose, and protein expression profiles were investigated after 24 hr by two-dimensional gel electrophoresis (2DE). Histopathological examination of the testes and serum concentration analysis were also performed. From the results of the comparison of 2D-gels among different doses of a compound and among compounds, 52, 20, 24 and 111 spots were nominated as differentially expressed spots with EGME, CP, SASP and 2,5-HD treatments, respectively. Several spermatogenesis-involved proteins were identified, including glutathione S-transferase (GST), testis-specific heat shock protein 70-2 (HSP70-2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphatidylethanolamine-binding protein (PEBP). Some of them were altered by more than one compound. In summary, remarkable histopathological findings were observed only in the EGME high-dose group, and most of the protein changes were detected before histopathological changes occurred. Therefore, the proteins identified in this study could potentially serve as biomarkers to evaluate male reproductive toxicity at an early stage of drug discovery and development.