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.

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.

Microarray analysis of 6-mercaptopurine-induced-toxicity-related genes and microRNAs in the rat placenta.

MicroRNAs (miRNAs) are small single-stranded RNAs of 19-25 nucleotides and are important in posttranscriptional regulation of genes. Recently, the role of miRNAs in toxicity incidence is reported to be a regulator of key-stopper of gene expression, however the detailed mechanism of miRNAs is not well known yet. 6-Mercaptopurine (6-MP), the anti-leukemic and immunosuppressive drug, produced teratogenicity and pregnancy loss. We focused on the placenta to evaluate toxicity in embryo/fetal development produced by 6-MP treatment. MiRNA expression in the placenta was analyzed by miRNA microarray. Fifteen miRNAs were upregulated on GD13 and 5 miRNAs were downregulated on GD15 in 6-MP treatment rat placentas. Some miRNAs may have functions in apoptosis (miR-195, miR-21, miR-29c and miR-34a), inflammation (miR-146b), and ischemia (miR-144 and miR-451). In the maternal plasma, expression of miR-144 was significantly reduced by 6-MP treatment when examined by real-time RT-PCR. We determined toxicity-related gene expression in the rat placenta. Gene expression analysis was carried out by DNA oligo microarray using rat placenta total RNAs. Compared between predicted targets of miRNAs and microarray data in 6-MP-treated rat placenta, expressions of hormone receptor genes (estrogen receptor 1; Esr1, progesterone receptor; Pgr, and prolactin receptor; Prlr), xanthine oxidase (Xdh), Slc38a5 and Phlda2 genes were changed. The histopathologically found increase in trophoblastic giant cells and reduced placental growth by 6-MP treatment were well correlated to these gene expressions. These data suggest that some miRNAs may link to toxicological reactions in 6-MP-induced placental toxicity.

6-Mercaptopurine-induced histopathological changes and xanthine oxidase expression in rat placenta.

The placenta secures the embryo and fetus to the endometrium and releases a variety of steroid and peptide hormones that convert the physiology of a female to that of a pregnant female. Chemical-induced alteration or deviation of placental function in the maternal and extraembryonic tissue can ultimately lead to pregnancy loss, congenital malformation and fetal death. The 6-mercaptopurine (6-MP), an anti-leukemic drug, is known to produce undesired effects on some organs, then the placenta/embryo toxicity of 6-MP was investigated in pregnant rats given 60 mg/kg with two intraperitoneal injections on gestation days (GD) 11 and 12. The rats were sacrificed and their placentas were collected on GD13 or 15. On GD15 small and limb-defected embryos were found in the 6-MP-treated rats. Placental weights were significantly reduced on GD15, as well as a reduced number of cells was detected in the labyrinth zone with both the labyrinth and basal zones having thinned. Cleaved caspase-3-positive cells increased in number in the labyrinth zone, while in the basal zone, glycogen cells reduced with cytolysis. The number of spongiotrophoblasts and trophoblastic giant cells also increased by 6-MP treatment. The 6-MP-treatment resulted in the increased xanthine oxidase (Xdh) expression in the placenta, which gene is related to the ischemic condition of tissues. These data suggest that apoptosis of the labyrinth zone cells may lead to decreased materno-fetal exchange. Moreover, subsequent ischemia in the placental tissue may occur and induce Xdh expression.

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.

MicroRNAs expression in the ethylene glycol monomethyl ether-induced testicular lesion.

Ethylene glycol monomethyl ether (EGME) induces testicular lesion in rats and human. To investigate miRNAs expression in EGME testicular lesion, miRNA array assay and real-time RT-PCR analysis were conducted by using testis in rats treated with 50 and 2,000 mg/kg EGME for 6 and 24 hr. The expression of corresponding target gene for miRNAs was also examined. At 50 mg/kg, there were no changes in the gene expression and histopathological examination. At 2,000 mg/kg, slight decrease of phacytene spermatocytes with cell shrinkage and nucleus pyknosis at 6 hr and remarkable decrease (or cell death) of phacytene spermatocytes with Sertoli cell vacuolation at 24 hr were observed. After 24 hr, miR-449a and miR-92a decreased obviously and, miR-320, miR-134 and miR-188 increased, while only miR-760-5p increased after 6 hr. Above these miRNAs are reported to have an important role for spermatogenesis. The gene expression of Bcl-2, target for miR-449a, increased and therefore it is considered anti-apoptotic reaction has started in this stage. The expression of high mobility group AT-hook 2 (target of miR-92a) which regulates histone structure, was increased. Furthermore, histone deacethylase 4, targets for miR-320, was also affected. Above prohibiting apoptosis or activating epigenetic genes might be protective reaction to spermatocytes death under the miRNAs regulation in EGME testicular lesion.

Toxic effect onset and evaluations of medicinal drugs--horizon for Darwinian toxicological thought.

The theory of Darwinian Medicine linked to an extension of Darwin's evolutionary theory is based on the approach from the aspect of "why we become ill?".This theory enables us to understand the relationship between humans and diseases by thinking from evolutional perspective, shows an important help for preventive medicine, and is meaningful to consider the future human healthcare. Toxicology has been defined as a research of adverse effect of xenobiotic substances backed up by diverse-sciences. Toxic effects are basically responses to xenobiotic substances, and expressed as triggering or additional accelerating adverse effects toward abnormal condition. Toxic effects, biological adverse responses, are interpreted as protective responses of living body, and the adverse effects caused by drugs are also considered to be protective responses. This logic can be translated as "Darwinian Toxicology" corresponding to "Darwinian Medicine", replying to "why we get into toxic condition by xenobiotics exposure". This paper refers to the meaning of toxic effects based on mechanisms underlying and comprehensive drug safety evaluation from Darwinian Medicine perspectives.

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.

In vitro gene expression analysis of hepatotoxic drugs in rat primary hepatocytes.

The study examined the feasibility of screening for hepatotoxicity by an in vitro gene expression analysis using rat primary hepatocytes and Affymetrix Rat Toxicology U34 arrays. Hepatocytes were exposed for 6 or 24 h to eight drugs, with different mechanisms of hepatotoxicity, at one third of the cytotoxic concentration TC50, i.e. acetaminophen, cyclophosphamide, clofibrate, chlorpromazine, lithocholic acid, cisplatin, diclofenac and disulfiram. The types of transcriptional changes observed in this study were generally consistent with previously reported in vivo data, although there were some differences. In hierarchical cluster analysis, drugs formed clusters depending on their mode of toxicity against cells. The number of transcripts affected by the cholestatic hepatotoxicants (lithocholic acid and chlorpromazine) or the drugs that rarely cause of hepatotoxicity (cisplatin, diclofenac and disulfiram) were limited compared with the other drugs (acetaminophen, clobifibrate and cyclophosphamide), where they did not induce transcriptional changes apparently related to toxicity. It is concluded that in vitro gene expression analysis of hepatocytes using microarray is a useful tool for evaluating the toxicological profile of drugs and in screening for the direct toxicity of drugs against hepatocytes.

In vitro gene expression analysis of nephrotoxic drugs in rat primary renal cortical tubular cells.

Rat primary renal cortical tubular cells were exposed to seven test substances, some with, and some without, known direct renal tubular cell toxicity. Cells were exposed to the substances at either one-third or one-tenth of the TC50 for cytotoxicity for 6 h or 24 h, so as not to induce cytotoxicity but to cause some transcriptional changes. Transcriptional profiles were investigated by using the Affymetrix Rat Toxicology U34 arrays, containing probes for more than 850 genes and ESTs. Four direct toxicants, cisplatin (CDDP), its less nephrotoxic analogue carboplatin (CBDCA), cephaloridine and gentamicin, were grouped together in a hierarchical clustering. In addition, the four direct toxicants affected more than 32 transcripts at their subcytotoxic concentrations at either 6 h or 24 h exposure. On the other hand, diclofenac, cyclosporine A and zinc, which are not considered to be directly toxic to tubules, affected less than 12 transcripts. Decreased Map3k12 and increased Hmox1 were commonly observed among the four direct toxicants, which appeared to be responses to cellular damage. Two platinum complexes, CDDP and CBDCA, induced similar changes, regardless of exposure duration or concentration. The types of transcriptional changes observed in this study were consistent with previously reported in vivo data, although there were some differences. These observations suggest that an in vitro gene expression analysis approach using GeneChip is feasible for screening for direct tubular toxicity of drugs and may help to clarify the underlying mechanisms of tubular toxicity.

Aging-related changes of QT and RR intervals in conscious guinea pigs.

INTRODUCTION: Although guinea pigs are suitable for in vivo QT assessment of newly discovered drugs at the pre-clinical stage because of the similarity of the ion channels between the guinea pig heart and the human, there is limited data available regarding the characteristics of QT interval in conscious guinea pigs. Aging is one of several factors which have been shown to affect the QT interval in humans and animals. In the present study, we examined the influence of age on QT and RR intervals in conscious guinea pigs. METHODS: Electrocardiograms were recorded from female Hartley guinea pigs at the age of 6 weeks (young; n=6) and 23 months (old; n=4) via a telemetry system. The QT and RR intervals were measured during daytime and nighttime, and following intravenous bolus injection of E-4031 (0.1 mg/kg) or terfenadine (4 mg/kg). Comparisons were made to determine group differences in: (1) the normal values of the QT and RR intervals, (2) the best-fit QT-correction formula, (3) the circadian rhythm of QT and RR intervals, and (4) drug effects on repolarization. RESULTS: The normal values of QT and RR intervals in the old group were significantly longer than those in the young group. The best-fit formula for correcting QT interval was a modified Bazett's formula for both young and old groups. The old group did not show the nocturnal variation of either QT or RR interval. Terfenadine caused significantly greater QTc prolongation in the old group compared to the young. DISCUSSION: Aging affects resting QT and RR intervals in conscious female guinea pigs, a factor which should be considered when examining the effects of compounds on cardiac repolarization. Also, the present study suggests a possibility that age can affect QTc prolongation induced by some IKr blockers.

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.

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.

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.

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.