Leveraging the Value of CDISC SEND Data Sets for Cross-Study Analysis: Incidence of Microscopic Findings in Control Animals.

Implementation of the Clinical Data Interchange Standards Consortium (CDISC)'s Standard for Exchange of Nonclinical Data (SEND) by the United States Food and Drug Administration Center for Drug Evaluation and Research (US FDA CDER) has created large quantities of SEND data sets and a tremendous opportunity to apply large-scale data analytic approaches. To fully realize this opportunity, differences in SEND implementation that impair the ability to conduct cross-study analysis must be addressed. In this manuscript, a prototypical question regarding historical control data (see Table of Contents graphic) was used to identify areas for SEND harmonization and to develop algorithmic strategies for nonclinical cross-study analysis within a variety of databases. FDA CDER's repository of >1800 sponsor-submitted studies in SEND format was queried using the statistical programming language R to gain insight into how the CDISC SEND Implementation Guides are being applied across the industry. For each component needed to answer the question (defined as "query block"), the frequency of data population was determined and ranged from 6 to 99%. For fields populated <90% and/or that did not have Controlled Terminology, data extraction methods such as data transformation and script development were evaluated. Data extraction was successful for fields such as phase of study, negative controls, and histopathology using scripts. Calculations to assess accuracy of data extraction indicated a high confidence in most query block searches. Some fields such as vehicle name, animal supplier name, and test facility name are not amenable to accurate data extraction through script development alone and require additional harmonization to confidently extract data. Harmonization proposals are discussed in this manuscript. Implementation of these proposals will allow stakeholders to capitalize on the opportunity presented by SEND data sets to increase the efficiency and productivity of nonclinical drug development, allowing the most promising drug candidates to proceed through development.

Suspected spontaneous hyperadrenocorticism in a young experimental beagle dog.

A 6-month-old female beagle dog, assigned to the low-dose group in a toxicity study, was evaluated for compound toxicity, and spontaneous hyperadrenocorticism was suspected. The animal had an externally apparent distended abdomen on clinical examination upon arrival. Pre-dose clinical pathology showed slightly higher erythroid parameters and stress leukogram on hematology; plasma biochemistry showed higher total protein, gamma-glutamyl transferase, total cholesterol, and triglyceride levels than the reference data. On necropsy, a prominent increase in adipose tissues of the subcutis and abdomen and increased weight of the adrenal gland and liver were observed. Histopathology revealed diffuse hyperplasia of adrenocortical cells in the zona fasciculata and reticularis, cortical atrophy of the thymus, and abundant glycogen accumulation in the hepatocytes. These findings were incidental and not test-substance-related. Electron microscopy of the adrenocortical cells in the zona fasciculata revealed decreased typical translucent lipid droplets, increased electron-dense lipid droplets, and abundant smooth endoplasmic reticulum and lysosomes. Additionally, increased numbers of various sizes and forms of mitochondria with tubular, vesicular, or lamellar cristae compared to that of normal animals were observed. These ultrastructural characteristics of the adrenocortical cells suggested hyperfunction. The pre-dose plasma cortisol levels were slightly higher than those of other females assigned to the toxicity study, while plasma adrenocorticotropic hormone levels were within the normal range. These findings indicate that hyperadrenocorticism is a possible cause of the systemic changes in this case.

SEND harmonization & cross-study analysis: A proposal to better harvest the value from SEND data.

The Standard for Exchange of Nonclinical Data (SEND) identifies an approach for representing nonclinical data in a structured format which has been widely adopted by the pharmaceutical industry as it is required for data submission to the United States Food & Drug Administration (US FDA). The SEND Implementation Guide (SENDIG) allows for considerable flexibility in how data is represented; interpretation of these guidelines has led to significant variability in the approach to SEND dataset creation. The purposes of this manuscript are to identify common variability in certain SEND domains and to describe how variability can be managed to enable valuable cross-study analysis use cases. The example of extracting a commonly used data point, animal age, is used to illustrate the complexity and variability of SEND datasets. Developing a solution framework to the variability problem that includes all stakeholders involved in the creation and use of SEND datasets may enable future, routine analysis of warehoused SEND data. Harmonizing the implementation and use of SEND is expected to benefit all involved stakeholders and to ultimately contribute to the goal of increased productivity in nonclinical research.

Cross study analyses of SEND data: toxicity profile classification.

A SEND toxicology data transformation, harmonization, and analysis platform were created to improve the identification of unique findings related to the intended target, species, and duration of dosing using data from multiple studies. The lack of a standardized digital format for data analysis had impeded large-scale analysis of in vivo toxicology studies. The CDISC SEND standard enables the analysis of data from multiple studies performed by different laboratories. This work describes methods to analyze data and automate cross-study analysis of toxicology studies. Cross-study analysis can be used to understand a single compound's toxicity profile across all studies performed and/or to evaluate on-target versus off-target toxicity for multiple compounds intended for the same pharmacological target. This work involved development of data harmonization/transformation strategies to enable cross-study analysis of both numerical and categorical SEND data. Four de-identified SEND datasets from the BioCelerate database were used for the analyses. Toxicity profiles for key organ systems were developed for liver, kidney, male reproductive tract, endocrine system, and hematopoietic system using SEND domains. A cross-study analysis dashboard with a built-in user-defined scoring system was created for custom analyses, including visualizations to evaluate data at the organ system level and drill down into individual animal data. This data analysis provides the tools for scientists to compare toxicity profiles across multiple studies using SEND. A cross-study analysis of 2 different compounds intended for the same pharmacological target is described and the analyses indicate potential on-target effects to liver, kidney, and hematopoietic systems.

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.

Adult Zebrafish Model for Screening Drug-Induced Kidney Injury.

Drug-induced kidney injury is a serious safety issue in drug development. In this study, we evaluated the usefulness of adult zebrafish as a small in vivo system for detecting drug-induced kidney injury. We first investigated the effects of typical nephrotoxicants, gentamicin and doxorubicin, on adult zebrafish. We found that gentamicin induced renal tubular necrosis with increased lysosome and myeloid bodies, and doxorubicin caused foot process fusion of glomerular podocytes. These findings were similar to those seen in mammals, suggesting a common pathogenesis. Second, to further evaluate the performance of the model in detecting drug-induced kidney injury, adult zebrafish were treated with 28 nephrotoxicants or 14 nonnephrotoxicants for up to 4 days, euthanized 24 h after the final treatment, and examined histopathologically. Sixteen of the 28 nephrotoxicants and none of the 14 nonnephrotoxicants caused drug-induced kidney injury in zebrafish (sensitivity, 57%; specificity, 100%; positive predictive value, 100%; negative predictive value, 54%). Finally, we explored genomic biomarker candidates using kidneys isolated from gentamicin- and cisplatin-treated zebrafish using microarray analysis and identified 3 candidate genes, egr1, atf3, and fos based on increased expression levels and biological implications. The expression of these genes was upregulated dose dependently in cisplatin-treated groups and was > 25-fold higher in gentamicin-treated than in the control group. In conclusion, these results suggest that the adult zebrafish has (1) similar nephrotoxic response to those of mammals, (2) considerable feasibility as an experimental model for toxicity studies, and (3) applicability to pathological examination and genomic biomarker evaluation in drug-induced kidney injury.

Trifluoromethyl Dihydrothiazine-Based ß-Secretase (BACE1) Inhibitors with Robust Central ß-Amyloid Reduction and Minimal Covalent Binding Burden.

The ß-site amyloid precursor protein cleaving enzyme 1 (BACE1, also known as ß-secretase) is a promising target for the treatment of Alzheimer's disease. A pKa lowering approach over the initial leads was adopted to mitigate hERG inhibition and P-gp efflux, leading to the design of 6-CF3 dihydrothiazine 8 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-cyanopicolinamide). Optimization of 8 led to the discovery of 15 (N-(3-((4S,6S)-2-amino-4-methyl-6-(trifluoromethyl)-5,6-dihydro-4H-1,3-thiazin-4-yl)-4-fluorophenyl)-5-(fluoromethoxy)pyrazine-2-carboxamide) with an excellent balance of potency, hERG inhibition, P-gp efflux, and metabolic stability. Oral administration of 8 elicited robust Aß reduction in dog even at 0.16 mg/kg. Reflecting the reduced hERG inhibitory activity, no QTc prolongation was observed at high doses. The potential for reactive metabolite formation of 15 was realized in a nucleophile trapping assay using [14 C]-KCN in human liver microsomes. Utilizing covalent binding (CVB) in human hepatocytes and the maximum projected human dosage, the daily CVB burden of 15 was calculated to be at an acceptable value of below 1 mg/day. However, hepatotoxicity was observed when 15 was subjected to a two-week tolerance study in dog, which prevented further evaluation of this compound.

Discovery of an Extremely Potent Thiazine-Based ß-Secretase Inhibitor with Reduced Cardiovascular and Liver Toxicity at a Low Projected Human Dose.

Genetic evidence points to deposition of amyloid-ß (Aß) as a causal factor for Alzheimer's disease. Aß generation is initiated when ß-secretase (BACE1) cleaves the amyloid precursor protein. Starting with an oxazine lead 1, we describe the discovery of a thiazine-based BACE1 inhibitor 5 with robust Aß reduction in vivo at low concentrations, leading to a low projected human dose of 14 mg/day where 5 achieved sustained Aß reduction of 80% at trough level.

Rational Design of Novel 1,3-Oxazine Based ß-Secretase (BACE1) Inhibitors: Incorporation of a Double Bond To Reduce P-gp Efflux Leading to Robust Aß Reduction in the Brain.

Accumulation of Aß peptides is a hallmark of Alzheimer's disease (AD) and is considered a causal factor in the pathogenesis of AD. ß-Secretase (BACE1) is a key enzyme responsible for producing Aß peptides, and thus agents that inhibit BACE1 should be beneficial for disease-modifying treatment of AD. Here we describe the discovery and optimization of novel oxazine-based BACE1 inhibitors by lowering amidine basicity with the incorporation of a double bond to improve brain penetration. Starting from a 1,3-dihydrooxazine lead 6 identified by a hit-to-lead SAR following HTS, we adopted a p Ka lowering strategy to reduce the P-gp efflux and the high hERG potential leading to the discovery of 15 that produced significant Aß reduction with long duration in pharmacodynamic models and exhibited wide safety margins in cardiovascular safety models. This compound improved the brain-to-plasma ratio relative to 6 by reducing P-gp recognition, which was demonstrated by a P-gp knockout mouse model.

Discovery of Potent and Centrally Active 6-Substituted 5-Fluoro-1,3-dihydro-oxazine ß-Secretase (BACE1) Inhibitors via Active Conformation Stabilization.

ß-Secretase (BACE1) has an essential role in the production of amyloid ß peptides that accumulate in patients with Alzheimer's disease (AD). Thus, inhibition of BACE1 is considered to be a disease-modifying approach for the treatment of AD. Our hit-to-lead efforts led to a cellular potent 1,3-dihydro-oxazine 6, which however inhibited hERG and showed high P-gp efflux. The close analogue of 5-fluoro-oxazine 8 reduced P-gp efflux; further introduction of electron withdrawing groups at the 6-position improved potency and also mitigated P-gp efflux and hERG inhibition. Changing to a pyrazine followed by optimization of substituents on both the oxazine and the pyrazine culminated in 24 with robust Aß reduction in vivo at low doses as well as reduced CYP2D6 inhibition. On the basis of the X-ray analysis and the QM calculation of given dihydro-oxazines, we reasoned that the substituents at the 6-position as well as the 5-fluorine on the oxazine would stabilize a bioactive conformation to increase potency.

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.

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.

Correlation among clinicopathological parameters of myocardial damage in rats treated with isoproterenol.

The correlation among clinicopathological parameters of myocardial damage was investigated in rats administered a single subcutaneous dose of isoproterenol at 0 (saline), 0.04, 0.4 and 4 mg/kg. Total lactate dehydrogenase (LDH), creatine kinase (CK), and their isoenzymes (LDH-1, LDH-2 and CK-MB), as well as troponin I and tropinin T, were measured 4 h after the administration of the drug. Troponin I was determined by a chemiluminescence method using Bayer Centaur and DPC Immulyze, as well as by ELISA. Troponin T was assayed semi-quantitatively using Trop T Sensitive. A high correlation was found among LDH isoenzymes, troponin I (Centaur) and troponin T. The present result provides a baseline for interpreting changes in the different parameters of myocardial damage assayed by different methods in toxicity studies.

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.

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.

Investigation of a hepatotoxicity screening system in primary cell cultures --"what biomarkers would need to be addressed to estimate toxicity in conventional and new approaches?".

High throughput toxicological estimation is required for safety evaluation in the early stage of drug discovery. In this context, establishment of an in vitro screening system reflecting in vivo toxicity is demanded for earlier safety assessment. We investigated LDH release and mitochondrial respiration (WST-1 reduction assay; WST-1) to detect cytotoxicity, morphological evaluation, and proteomics for estimating the reliable and sensitive biomarkers by using rat primary hepatocytes exposed to the compounds (acetaminophen, amiodarone, tetracycline and carbon tetrachloride) that are known to induce hepatotoxicity. In LDH release, no significant difference was detected between the control and compound exposed cells after exposure for 3 or 6 hr, but a dose-dependent increase was observed after exposure for 24 hr. Regarding the WST-1 assay, a dose-dependent reduction was detected after exposure for 6 and 24 hr to all of the compounds evaluated. In the proteomics analysis, 31 candidate proteins were identified from among the 103 demonstrating altered expression spots after exposure to acetaminophen. It was concluded that the cytotoxicity was detected earlier by measuring WST-1 than by measuring LDH release because the reduction of mitochondrial respiration is an expressions of earlier toxicity for cellular function, while the measured increase in the LDH release occurs after the failure of the cell membrane. Mitochondrial respiration ability was a useful parameter for cytotoxicity in in vitro hepato-toxicity screening, as cytotoxicity can be detected during the early stage of exposure. In addition to the conventional biomarkers, several protein biomarkers which relate to oxidative stress and metabolism-regulation were detected. Further comprehensive analysis of defined proteins would be necessary to estimate the more sensitive toxicology biomarker.

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.

Effects of male reproductive toxicants on gene expression in rat testes.

Predictive biomarkers of testicular toxicity are needed for an efficient development of drugs. The purpose of the present study was to obtain further insight into the toxicity mechanisms of various male reproductive toxicants and to detect genomic biomarkers for rapid screening of testicular toxicity. Four reproductive toxicants, 2,5-hexanedione (Sertoli cells toxicant), ethylene glycol monomethyl ether (EGME; spermatocytes toxicant), cyclophosphamide (spermatogonia toxicant) and sulfasalazine, were orally administered to male rats once. Six hours after the single dosing, gene expression in the testes was monitored by cDNA microarray and real-time RT-PCR and the testes were histopathologically examined. No histopathological abnormality was detected except for slight degeneration of spermatocytes in the EGME-treated testes. cDNA microarray analysis revealed differential gene expression profiles, and it was possible based on the profiles to characterize the action of the compounds in the testes. Interestingly, 3 spermatogenesis-related genes -- heat shock protein 70-2, insulin growth factor binding protein 3 and glutathione S transferase pi -- were affected by all the compounds. The above changes of gene expression were detectable within a short period after the dosing prior to the appearance of obvious pathological changes. These data suggest that cDNA microarray is a useful technique for evaluation of primary testicular toxicity. Furthermore, we propose the above 3 spermatogenesis-related genes as potential biomarkers of testicular toxicity.

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.