Description of pain associated with persistent postoperative pain after total knee arthroplasty.

After total knee arthroplasty (TKA), approximately 20% of patients experience persistent postoperative pain (PPP). Although preoperative and postoperative pain intensity is a relevant factor, more detailed description of pain is needed to determine specific intervention strategies for clinical conditions. This study aimed to clarify the associations between preoperative and postoperative descriptions of pain and PPP. Fifty-two TKA patients were evaluated for pain intensity and description of pain preoperatively and 2 weeks postoperatively, and the intensities were compared. In addition, the relationship between pain intensity and PPP at 3 and 6 months after surgery was analyzed using a Bayesian approach. Descriptions of arthritis ("Throbbing" and "aching") improved from preoperative to 2 weeks postoperative. Several preoperative ("Shooting", "Aching", "Caused by touch", "Numbness") and postoperative ("Cramping pain") descriptors were associated with pain intensity at 3 months postoperatively, but only "cramping pain" at 2 weeks postoperatively was associated with the presence of PPP at 3 and 6 months postoperatively. In conclusion, it is important to carefully listen to the patient's complaints and determine the appropriate intervention strategy for the clinical condition during perioperative pain management.

Evaluation of the novel liver micronucleus assay using formalin-fixed tissues.

BACKGROUND: The repeated-dose liver micronucleus (RDLMN) assay is an effective and important in vivo test for detecting genotoxic compounds, particularly for those that require metabolic activation to show genotoxicity. In a collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS), micronucleus induction of 22 chemicals with the RDLMN assay employing the collagenase digestion method was examined and reported on. Recently, we have developed a method which enables retrospective evaluation of micronucleus induction in formalin-fixed liver tissues (the formalin-fixed method) obtained in general toxicity studies completed in the past. Using this method, we were able to easily evaluate clastogenic potential of chemicals from the formalin-fixed tissues obtained in the general toxicity studies.In this study, to evaluate the usefulness of the formalin-fixed method, we have conducted a liver micronucleus assay using the formalin-fixed liver samples obtained from the above collaborative study (18 of 22 test chemicals) and carried out a comparison with the results obtained by the collagenase digestion method. RESULTS: Comparison of the collagenase digestion and formalin-fixed methods was conducted using the results of the micronucleus assays with a total of 18 test chemicals which included 12 genotoxic hepatocarcinogens (Group A), 4 genotoxic carcinogens but not liver targeted (Group B), and 2 nongenotoxic hepatocarcinogens (Group C). The formalin-fixed method obtained the similar results as the collagenase digestion method in 10 out of the 12 chemicals of Group A, and all chemicals of Group B and Group C. Although the results were statistically contradictive due to different levels of concurrent negative control, the 2 other chemicals of Group A showed comparable responses between the two methods. CONCLUSION: The present study shows that the formalin-fixed method is capable of detecting liver carcinogens with sensitivity equal to or higher than that of the collagenase digestion method. We recommend use of the formalin-fixed method because of its capability of enabling retrospective evaluation of micronucleus induction in the formalin-fixed liver tissues obtained in general toxicity studies completed in the past.

Identification of novel plasminogen activator inhibitor-1 inhibitors with improved oral bioavailability: Structure optimization of N-acylanthranilic acid derivatives.

Novel plasminogen activator inhibitor-1 (PAI-1) inhibitors with highly improved oral bioavailability were discovered by structure-activity relationship studies on N-acyl-5-chloroanthranilic acid derivatives. Because lipophilic N-acyl groups seemed to be important for the anthranilic acid derivatives to strongly inhibit PAI-1, synthesis of compounds in which 5-chloroanthranilic acid was bound to a variety of highly lipophilic moieties with appropriate linkers was investigated. As the result it appeared that some of the derivatives possessing aryl- or heteroaryl-substituted phenyl groups in the acyl chain had potent in vitro PAI-1 inhibitory activity. Oral absorbability of typical compounds was also evaluated in rats, and compounds 40, 55, 60 and 76 which have diverse chemical structure with each other were selected for further pharmacological evaluation.

Gene expression profiles in auricle skin as a possible additional endpoint for determination of sensitizers: A multi-endpoint evaluation of the local lymph node assay.

The murine local lymph node assay (LLNA) is widely used to test chemicals to induce skin sensitization. Exposure of mouse auricle skin to a sensitizer results in proliferation of local lymph node T cells, which has been measured by in vivo incorporation of H3-methyl thymidine or 5-bromo-2'-deoxyuridine (BrdU). The stimulation index (SI), the ratio of the mean proliferation in each treated group to that in the concurrent vehicle control group, is frequently used as a regulatory-authorized endpoint for LLNA. However, some non-sensitizing irritants, such as sodium dodecyl sulfate (SDS) or methyl salicylate (MS), have been reported as false-positives by this endpoint. In search of a potential endpoint to enhance the specificity of existing endpoints, we evaluated 3 contact sensitizers; (hexyl cinnamic aldehyde [HCA], oxazolone [OXA], and 2,4-dinitrochlorobenzene [DNCB]), 1 respiratory sensitizer (toluene 2,4-diisocyanate [TDI]), and 2 non-sensitizing irritants (MS and SDS) by several endpoints in LLNA. Each test substance was applied to both ears of female CBA/Ca mice daily for 3 consecutive days. The ears and auricle lymph node cells were analyzed on day 5 for endpoints including the SI value, lymph node cell count, cytokine release from lymph node cells, and histopathological changes and gene expression profiles in auricle skin. The SI values indicated that all the test substances induced significant proliferation of lymph node cells. The lymph node cell counts showed no significant changes by the non-sensitizers assessed. The inflammatory findings of histopathology were similar among the auricle skins treated by sensitizers and irritants. Gene expression profiles of cytokines IFN-γ, IL-4, and IL-17 in auricle skin were similar to the cytokine release profiles in draining lymph node cells. In addition, the gene expression of the chemokine CXCL1 and/or CXCL2 showed that it has the potential to discriminate sensitizers and non-sensitizing irritants. Our results suggest that multi-endpoint analysis in the LLNA leads to a better determination of the sensitizing potential of test substances. We also show that the gene expression of CXCL1 and/or CXCL2, which is involved in elicitation of contact hypersensitivity (CHS), can be a possible additional endpoint for discrimination of sensitizing compounds in LLNA.

The PIGRET assay, a method for measuring Pig-a gene mutation in reticulocytes, is reliable as a short-term in vivo genotoxicity test: Summary of the MMS/JEMS-collaborative study across 16 laboratories using 24 chemicals.

The in vivo mutation assay using the X-linked phosphatidylinositol glycan class A gene (Pig-a in rodents, PIG-A in humans) is a promising tool for evaluating the mutagenicity of chemicals. Approaches for measuring Pig-a mutant cells have focused on peripheral red blood cells (RBCs) and reticulocytes (RETs) from rodents. The recently developed PIGRET assay is capable of screening >1×106 RETs for Pig-a mutants by concentrating RETs in whole blood prior to flow cytometric analysis. Additionally, due to the characteristics of erythropoiesis, the PIGRET assay can potentially detect increases in Pig-a mutant frequency (MF) sooner after exposure compared with a Pig-a assay targeting total RBCs (RBC Pig-a assay). In order to test the merits and limitations of the PIGRET assay as a short-term genotoxicity test, an interlaboratory trial involving 16 laboratories was organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagenicity Society (MMS/JEMS). First, the technical proficiency of the laboratories and transferability of the assay were confirmed by performing both the PIGRET and RBC Pig-a assays on rats treated with single doses of N-nitroso-N-ethylurea. Next, the collaborating laboratories used the PIGRET and RBC Pig-a assays to assess the mutagenicity of a total of 24 chemicals in rats, using a single treatment design and mutant analysis at 1, 2, and 4 weeks after the treatment. Thirteen chemicals produced positive responses in the PIGRET assay; three of these chemicals were not detected in the RBC Pig-a assay. Twelve chemicals induced an increase in RET Pig-a MF beginning 1 week after dosing, while only 3 chemicals positive for RBC Pig-a MF produced positive responses 1 week after dosing. Based on these results, we conclude that the PIGRET assay is useful as a short-term test for in vivo mutation using a single-dose protocol.

Measuring reproducibility of dose response data for the Pig-a assay using covariate benchmark dose analysis.

The reproducibility of the in vivo Pig-a gene mutation test system was assessed across 13 different Japanese laboratories. In each laboratory rats were exposed to the same dosing regimen of N-nitroso-N-ethylurea (ENU), and red blood cells (RBCs) and reticulocytes (RETs) were collected for mutant phenotypic analysis using flow cytometry. Mutant frequency dose response data were analysed using the PROAST benchmark dose (BMD) statistical package. Laboratory was used as a covariate during the analysis to allow all dose responses to be analysed at the same time, with conserved shape parameters. This approach has recently been shown to increase the precision of the BMD analysis, as well as providing a measure of equipotency. This measure of equipotency was used here to demonstrate a reasonable level of interlaboratory reproducibility. Increased reproducibility could have been achieved by increasing the number of cells scored, as this would reduce the number of zero values within the mutant frequency data. Overall, the interlaboratory trial was successful, and these findings support the transferability of the in vivo Pig-a gene mutation assay.

Evaluation of red blood cell Pig-a assay and PIGRET assay in rats using chlorambucil.

The Pig-a assay is a novel method to assess the in vivo mutagenicity of compounds, and it is expected to be useful for the detection of genotoxicity. In this study, to assess the performance of the Pig-a assay targeting red blood cells (RBCs; RBC Pig-a assay) and reticulocytes (RETs; PIGRET assay), chlorambucil, which is a genotoxicant, was orally administered to male rats once at 10, 20 and 40mg/kg on Day 1, and the mutant frequencies (MFs) of RBCs and RETs were examined periodically. In the RBC Pig-a assay, significant increases in MFs were observed at 40mg/kg on Day 15 and at 20mg/kg or higher on Day 29. In the PIGRET assay, MFs increased significantly at all dose levels on Day 8 and only at 20mg/kg on Day 15, but there was no increase in MFs in the treatment groups on Day 29. In conclusion, the RBC Pig-a assay and PIGRET assay in rats have sufficient sensitivity to detect the mutagenicity of chlorambucil, and the PIGRET assay could detect its mutagenicity earlier and at a lower dose than the RBC Pig-a assay.

Evaluation of the repeated-dose liver micronucleus assay using 2,4-dinitrotoluene: a report of a collaborative study by CSGMT/JEMS.MMS.

The liver micronucleus assay using young adult rats has the potential to detect liver carcinogens by repeated dosing, and could be expected to be integrated into repeated-dose toxicity studies using a hepatocyte isolation method without the traditional in situ collagenase perfusion. In this study, to assess the performance of the repeated-dose liver micronucleus assay, 2,4-dinitrotoluene (DNT), which is a rodent liver carcinogen, was administered orally to male rats at doses of 50, 100 and 200 mg/kg/day once daily for 14 or 28 consecutive days, and the frequencies of micronucleated hepatocytes (MNHEPs) and micronucleated immature erythrocytes (MNIMEs) were examined. Significant increases in the MNHEPs were observed at 50 mg/kg/day or more in the 14-day treatment, and 50 and 100 mg/kg/day in the 28-day treatment. These increases were dependent on both the dose and the number of administrations, which indicates the possibility that the MNHEPs accumulate as a result of repeated dosing. In contrast, no increase in the MNIMEs was observed. In conclusion, the repeated-dose liver micronucleus assay using young adult rats is sufficiently sensitive to detect the genotoxicity of 2,4-DNT at a low dose.

Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS).

The repeated-dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect hepatocarcinogens. We conducted a collaborative study to assess the performance of this assay and to evaluate the possibility of integrating it into general toxicological studies. Twenty-four testing laboratories belonging to the Mammalian Mutagenicity Study Group, a subgroup of the Japanese Environmental Mutagen Society, participated in this trial. Twenty-two model chemicals, including some hepatocarcinogens, were tested in 14- and/or 28-day RDLMN assays. As a result, 14 out of the 16 hepatocarcinogens were positive, including 9 genotoxic hepatocarcinogens, which were reported negative in the bone marrow/peripheral blood micronucleus (MN) assay by a single treatment. These outcomes show the high sensitivity of the RDLMN assay to hepatocarcinogens. Regarding the specificity, 4 out of the 6 non-liver targeted genotoxic carcinogens gave negative responses. This shows the high organ specificity of the RDLMN assay. In addition to the RDLMN assay, we simultaneously conducted gastrointestinal tract MN assays using 6 of the above carcinogens as an optional trial of the collaborative study. The MN assay using the glandular stomach, which is the first contact site of the test chemical when administered by oral gavage, was able to detect chromosomal aberrations with 3 test chemicals including a stomach-targeted carcinogen. The treatment regime was the 14- and/or 28-day repeated-dose, and the regime is sufficiently promising to incorporate these methods into repeated-dose toxicological studies. The outcomes of our collaborative study indicated that the new techniques to detect chromosomal aberrations in vivo in several tissues worked successfully.

Immunosuppressive potential of bardoxolone methyl using a modified murine local lymph node assay (LLNA).

2-Cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid methyl ester (CDDO-Me; bardoxolone methyl) is one of the synthetic oleanane triterpenoids (SOs). It is known that it is the strongest Nrf2/ARE signaling inducer of SOs and slightly inhibits immune response. Little was known about the immunomodulatory action of CDDO-Me in vivo. We assessed its immunosuppressive potential by using the modified mouse lymph node assay (LLNA) including immunosuppression-related gene expression analysis. In the modified LLNA, CDDO-Me showed a significant decrease in lymph node weight and changes in expressions of the immunosuppression-related genes, Zfp459 and Fmo2. It has been already reported that a decrease in lymph node weight was induced by several types of immunosuppressive chemicals such as calcineurin inhibitors, antimetabolites, steroids, and alkylators. In addition, changes in Zfp459 and Fmo2 expression was reported in response after only treatment of antimetabolites. From these results, CDDO-Me is considered to have an immunosuppressive action and similar mechanism to antimetabolites.

Discrimination of genotoxic and non-genotoxic hepatocarcinogens by statistical analysis based on gene expression profiling in the mouse liver as determined by quantitative real-time PCR.

The general aim of the present study is to discriminate between mouse genotoxic and non-genotoxic hepatocarcinogens via selected gene expression patterns in the liver as analyzed by quantitative real-time PCR (qPCR) and statistical analysis. qPCR was conducted on liver samples from groups of 5 male, 9-week-old B6C3F(1) mice, at 4 and 48h following a single intraperitoneal administration of chemicals. We quantified 35 genes selected from our previous DNA microarray studies using 12 different chemicals: 8 genotoxic hepatocarcinogens (2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane) and 4 non-genotoxic hepatocarcinogens (1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, di(2-ethylhexyl)phthalate and furan). A considerable number of genes exhibited significant changes in their gene expression ratios (experimental group/control group) analyzed statistically by the Dunnett's test and Welch's t-test. Finally, we distinguished between the genotoxic and non-genotoxic hepatocarcinogens by statistical analysis using principal component analysis (PCA) of the gene expression profiles for 7 genes (Btg2, Ccnf, Ccng1, Lpr1, Mbd1, Phlda3 and Tubb2c) at 4h and for 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb2c) at 48h. Seven major biological processes were extracted from the gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response pathway, resulting from signal transduction by a p53-class mediator leading to the induction of apoptosis. Eight genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Phlda3 and Plk2) that are directly associated with Trp53 contributed to the PCA. The current findings demonstrate a successful discrimination between genotoxic and non-genotoxic hepatocarcinogens, using qPCR and PCA, on 12 genes associated with a Trp53-mediated signaling pathway for DNA damage response at 4 and 48 h after a single administration of chemicals.

Effect of RNA interference of BID and BAX mRNAs on apoptosis in granulosa cell-derived KGN cells.

In mitochondrion-dependent type II apoptosis, BH3-interacting domain death agonist (BID) and BCL-2-associated X protein (BAX) promote death ligand and receptor-mediated cell death. In porcine ovaries, the levels of BID and BAX increase in follicular granulosa cells during atresia. In the present study, to confirm the pro-apoptotic activity of BID and BAX in granulosa cells, we examined the effect of RNA interference of BID or BAX on apoptosis using a human ovarian granulosa tumor cell line, KGN. By reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, expression of BID and BAX was detected in KGN cells. Then, we suppressed BID and BAX mRNA expression in KGN cells using small interfering RNA (siRNA). When BID or BAX was suppressed, a significant decrease in the apoptotic cell rate was noted. In granulosa-derived cells, BID and BAX showed pro-apoptotic activity. These results suggest that BID and BAX act as signal-transducing factors in mitochondrion-dependent type II apoptosis.

Novel gene markers of immunosuppressive chemicals in mouse lymph node assay.

The murine local lymph node assay (LLNA) is an immunologically based test of the sensitizing potential of immunotoxicants, but also evaluates immunosuppressive potential with good sensitivity and specificity. We conducted the LLNA with calcineurin inhibitors (tacrolimus and cyclosporin A), antimetabolites (methotrexate and azathioprine), steroids (dexamethasone and prednisolone), and an alkylator (cyclophosphamide). We performed a comprehensive analysis of results of gene expression in lymph nodes obtained in the LLNA using a highly sensitive DNA chip, 3D-Gene™, and the quantitative reverse transcription-polymerase chain reaction (qPCR). Zfp459 expression increased in all animals treated with immunosuppressive chemicals. Ltf, Cbll1 and Lias expression changed specifically in response to the calcineurin inhibitors, Fmo2 and 9630033F20Rik in response to the antimetabolites, Krt8, Gjb1, Hmha1 and Sfrs7 in response to the steroids, and Gbp1 and Mup5 in response to the alkylator. Therefore, these genes were considered novel markers for immunosuppression and their expression could be used to evaluate the mechanism of action of immunosuppressive chemicals.

Bid and Bax are involved in granulosa cell apoptosis during follicular atresia in porcine ovaries.

More than 99% of follicles undergo "atresia" during follicular development and growth. Follicular atresia is predominantly regulated by granulosa cell apoptosis. However, the intracellular signaling pathway of apoptosis in granulosa cells has not been revealed. In the present study, we examined changes in the expression of BH3-interacting domain death agonist (Bid) and Bcl-2-associated X protein (Bax), which are considered to promote the cell death ligand/receptor-mediated process in mitochondrion-dependent type II apoptosis, in porcine granulosa cells during atresia. Levels of mRNA and protein of Bid and Bax were determined by the reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting techniques, respectively. Levels of Bid and Bax mRNA and protein were markedly increased in granulosa cells of early atretic follicles compared with those of healthy follicles. In situ hybridization and immunohistochemical staining revealed that mRNA and protein of Bid and Bax were present in the granulosa cells, though only traces were found in healthy follicles; however, strong staining was noted in atretic follicles. These results indicate that Bid and Bax appear to be signal transduction factors in granulosa cells during follicular atresia and appear to play proapoptotic roles and confirm that the porcine granulosa cell is a mitochondrion-dependent type II apoptotic cell.

Toxicological effect of emodin in mouse testicular gene expression profile.

Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a herbal medicine extracted from the rhizomes of Rheum palmatum, and is known as an inhibitor of casein kinase II (CK2). The CK2α' knockout mice are known to be male-infertile; however, there have been no reports on the toxicity of emodin in male reproductive organs/tissues. To evaluate the toxicological effects of emodin on differential gene expression profiles of the testis as compared with acrylamide, mice were orally administered emodin and acrylamide for 5 days at a dose of 1000 and 50 mg kg(-1) per day, respectively, and euthanized 24 h after the final administration. Both chemicals induced hypospermatogenesis, eosinophilic change and apoptosis of germ cell. A DNA microarray analysis showed that the IGF-1 receptor signaling was most closely related to the above testicular toxicity induced by emodin, and the RhoA regulation, TGF/WNT and cytoskeletal remodeling, TNFR1 signaling and adenosine A2A receptor signaling were commonly associated with the two chemicals. We selected 36 genes associated with CK2, apoptosis and spermatogenesis and determined their expression by quantitative reverse transcription-polymerase chain reaction (qPCR). Both chemicals perturbed the expression of genes associated with CK2. Genes related to spermatogenesis were also affected, as evidenced by hypospermatogenesis, and eosinophilic change and apoptosis of germ cell. The results suggest that emodin causes testicular toxicity, including apoptosis with related the IGF-1 receptor signaling pathway, and the two chemicals commonly affect CK2, spermatogenesis and sperm motility via four pathways, such as TNFR1 signaling.

Expression and function of apoptosis initiator FOXO3 in granulosa cells during follicular atresia in pig ovaries.

In mammalian ovaries, most follicles are lost by atresia before ovulation. It has become apparent that the apoptosis of granulosa cells induces follicular atresia. Forkhead box O3 (FOXO3), also called FKHRL1 (forkhead in rhabdomyosarcoma-like 1), is a proapoptotic molecule that belongs to the FOXO subfamily of forkhead transcription factors. Foxo3-deficient female mice were reported to be infertile because of abnormal ovarian follicular development, but the precise influences of FOXO3 on follicular atresia of mature ovary have not been determined. Therefore, we examined the expression and function of FOXO3 in porcine ovarian follicles and granulosa-derived cells. FOXO3 mRNA levels in granulosa cells of porcine ovaries increased during atresia, while FOXO3 protein was abundant in granulosa cells of early atretic follicles. By immunohistochemistry, the inner surface area of the granulosa layer in early atretic follicles was strongly stained with anti-FOXO3 antibody. The granulosa cells expressing FOXO3 coincided with apoptotic cells, indicating a role of FOXO3 as a proapoptotic factor in granulosa cells of porcine ovaries. In porcine (JC-410) and human (KGN) granulosa-derived cells, cell death was induced by transfection of FOXO3 expression vectors. Expression of the proapoptotic factors Fas ligand (FASLG) and BCL2-like 11 (BCL2L11) was upregulated by FOXO3 in KGN cells. In conclusion, FOXO3 is expressed in porcine ovarian follicles and induces apoptosis in granulosa cells, suggesting that it is a candidate for the initiator of follicular atresia.

Evaluation of a liver micronucleus assay in young rats (IV): a study using a double-dosing/single-sampling method by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS).

A collaborative study was conducted to evaluate whether a liver micronucleus assay using four-week-old male F344 rats can be used to detect genotoxic rat hepatocarcinogens using double-dosing with a single-sampling 4 days after the second dose. The assay methods were thoroughly validated by the seven laboratories involved in the study. Seven chemicals, 2,4-diaminotoluene, diethyl nitrosamine, p-dimethylaminoazobenzene, 1,2-dimethylhydrazine dihydrochloride, 2,4-dinitrotolunene, 2,6-dinitrotoluene and mitomycin C, known to produce positive responses in the single-dosing/triple-sampling method were selected for use in the present study, and each chemical was examined in two laboratories with the exception of 2,4-dinitrotolunene. Although several of the compounds were examined at lower doses for reasons of toxicity than in the single-dosing/triple-sampling method, all chemicals tested in the present study induced micronuclei in liver cells indicating a positive result. These findings suggest that the liver micronucleus assay can be used in young rats to detect genotoxic rat hepatocarcinogens using a double-dosing/single-sampling procedure. Further, the number of animals used in the liver micronucleus assay can be reduced by one-third to a half by using the double-dosing/single-sampling method. This reduction in animal numbers also has significant savings in time and resource for liver perfusion and hepatocyte isolation.

Changes in expression and localization of X-linked inhibitor of apoptosis protein (XIAP) in follicular granulosa cells during atresia in porcine ovaries.

Follicular selection predominantly depends on granulosa cell apoptosis in porcine ovaries, but the molecular mechanisms regulating the induction of apoptosis in granulosa cells during follicular selection remain incompletely understood. To determine the role of X-linked inhibitor of apoptosis protein (XIAP), which suppresses caspase-3, -7 and -9 activities and acts as an endogenous inhibitor of apoptotic cell death, in the regulation of granulosa cell apoptosis during follicular atresia, we examined the changes in the expression level and localization of XIAP mRNA and protein in granulosa cells during follicular atresia using reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, Western blotting and immunohistochemistry, respectively. High levels of XIAP mRNA and protein were noted in the granulosa cells of healthy follicles, and decreased levels were noted during follicular atresia. In situ hybridization and immunohistochemistry demonstrated that XIAP mRNA and protein were strongly expressed in the granulosa cells of healthy follicles, but negative/trace stainings were noted in those of atretic follicles. The present findings strongly indicate that XIAP is a candidate molecule which acts as an anti-apoptotic/pro-survival factor by inhibiting intracellular apoptosis signaling and is involved in the regulation of apoptosis in porcine granulosa cells.

cFLIP regulates death receptor-mediated apoptosis in an ovarian granulosa cell line by inhibiting procaspase-8 cleavage.

More than 99% of follicles in mammalian ovaries undergo atresia, but the mechanisms regulating the strict selection process are still unclear. Granulosa cell apoptosis is considered the trigger of follicular atresia, which occurs in advance of the death of an oocyte. Cellular FLICE-like inhibitory protein (cFLIP), a homologue of procaspase-8 (also called FLICE), is an intracellular anti-apoptotic protein. It is expressed in granulosa cells of porcine ovaries, where its levels decreases during follicular atresia. We hypothesized that cFLIP regulates granulosa cell apoptosis by acting as a pro-survival factor. In the present study, to further reveal the function of cFLIP in granulosa cells, we examined the anti-apoptotic mechanism of cFLIP using KGN, a human granulosa tumor cell line. Fas-mediated apoptosis was induced by co-treatment with anti-Fas antibody (CH-11), which acts as an agonist of Fas-ligand, and cycloheximide (CHX). When cFLIP was stably expressed in KGN cells following transfection of an expression vector, the Fas-mediated apoptosis was inhibited. Suppression of cFLIP by small interfering RNA (siRNA) spontaneously induced cell death. Silencing of cFLIP promoted cleavage of procaspase-8, and the cell death caused by cFLIP siRNA was completely blocked by a caspase-8 inhibitor (Z-IETD-FMK), indicating that cFLIP regulates apoptosis in KGN cells by inhibiting cleavage of procaspase-8. In conclusion, cFLIP is an essential pro-survival factor for granulosa cells, and it prevents granulosa cell apoptosis by inhibiting procaspase-8 activation.

Molecular cloning of a porcine (Sus scrofa) apoptosis inhibitory ligand, netrin-1, and its receptor, p53RDL1.

The apoptosis inhibitory ligand (Netrin-1) and its receptor (p53-regulated receptor for death and life: p53RDL1) play an important role in the regulation of selective apoptosis. When Netrin-1 binds to p53RDL1, p53-dependent apoptosis is inhibited. We identified porcine (Sus scrofa) cDNAs encoding Netrin-1 [pNetrin-1; 1,803 base pairs (bp) and 600 amino acids (aa)] and p53RDL1 (pp53RDL1; 2,838 bp and 945 aa). Porcine p53RDL1 (pp53RDL1) contains a death domain (DD), a tandem specific amino acid region, in its C-terminal, suggesting that it mediates death signaling by binding with other pro-apoptotic factors via the DD. Porcine Netrin-1 (pNetrin-1), pp53RDL1 and the DD in pp53RDL1 showed high levels of identity in aa sequence with human and murine Netrin-1 (98 and 97%, respectively), p53RDL1 (94 and 91%, respectively) and the DD in p53RDL1 (96 and 95%, respectively). Reverse transcription-polymerase chain reaction (RT-PCR) revealed that the levels of pNetrin-1 and pp53RDL1 mRNAs were moderate in granulosa cells compared with their expression in other tissues and that their levels during follicular atresia were stable. The Netrin-1 and p53RDL1 system may regulate the induction of apoptosis in porcine granulosa cells.