Morphology of the male rat gonad after in Utero exposure to immunosuppressants.

The protocol for immunosuppression of pregnant women is based on immunosuppressant panels. The aim of the study was to determine the influence of commonly applied combinations of immunosuppressants to pregnant rats on the morphology of the offspring' testes. Pregnant rats were treated with cyclosporin A (CsA), mycophenolate mofetil (MMF) and prednisone (Pred) (CMG); tacrolimus (Tc), MMF and Pred (TMG); CsA, everolimus (Ev) and Pred (CEG). Testes of mature offspring underwent morphological analysis. Mainly in the testes of CMG and TMG rats the morphological and functional changes were observed: immature germ cells (GCs) in the seminiferous tubule (ST) lumen, invaginations of the basement membrane, infolding to the seminiferous epithelium (SE), the ST wall thickening, increased acidophilia of Sertoli cells' (SCs) cytoplasm, large residual bodies near the lumen, dystrophic ST and tubules look like the Sertoli cell-only syndrome, Leydig cells with abnormal cell nucleus, hypertrophy of the interstitium, blurring of the boundary between ST wall and interstitium, a reduced number of GCs in the SE, vacuolation of the SE. In the CEG there were only a reduced number of GCs in some tubules and vacuolization of SCs. The safest combination of drugs was CEG, while the TMG and CMG were gonadotoxic.

Schisandrin A alleviates mycophenolic acid-induced intestinal toxicity by regulating cell apoptosis and oxidative damage.

The gastrointestinal side effects of mycophenolic acid affect its efficacy in kidney transplant patients, which may be due to its toxicity to the intestinal epithelial mechanical barrier, including intestinal epithelial cell apoptosis and destruction of tight junctions. The toxicity mechanism of mycophenolic acid is related to oxidative stress-mediated, the activation of mitogen-activated protein kinases (MAPK). Schisandrin A (Sch A), one of the main active components of the Schisandra chinensis, can protect intestinal epithelial cells from deoxynivalenol-induced cytotoxicity and oxidative damage by antioxidant effects. The aim of this study was to investigate the protective effect and potential mechanism of Sch A on mycophenolic acid-induced damage in intestinal epithelial cell. The results showed that Sch A significantly reversed the mycophenolic acid-induced cell viability reduction, restored the expression of tight junction protein ZO-1, occludin, and reduced cell apoptosis. In addition, Sch A inhibited mycophenolic acid-mediated MAPK activation and reactive oxygen species (ROS) increase. Collectively, our study showed that Sch A protected intestinal epithelial cells from mycophenolic acid intestinal toxicity, at least in part, by reducing oxidative stress and inhibiting MAPK signaling pathway.

Study on alleviate effect of Wuzhi capsule (Schisandra sphenanthera Rehder & E.H. Wilson extract) against mycophenolate mofetil-induced intestinal injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Schisandra sphenanthera Rehder & E.H. Wilson (S. sphenanthera) is a botanical medicine included in the 2020 edition of the ChP that has a variety of medicinal activities, including hepatoprotective, anticancer, antioxidant and anti-inflammatory properties. Wuzhi capsule (WZ) is a proprietary Chinese medicine made from an ethanolic extract of S. sphenanthera that is commonly used to treat drug-induced liver injury. However, there are no research reports exploring the effects of WZ on the prevention of mycophenolate mofetil (MMF)-induced intestinal injury and its underlying mechanisms. AIM OF THE STUDY: This experiment aimed to evaluate the ameliorative effect of WZ on MMF-induced intestinal injury in mice and its underlying mechanisms. MATERIALS AND METHODS: A mouse model of MMF-induced intestinal injury was established and treated with WZ during the 21-day experimental period. The pathological characteristics of the mouse ileum were observed. Tight junction (TJ) protein changes were observed after immunofluorescence staining and transmission electron microscopy, and ROS levels were measured by using DHE fluorescent dye and the TUNEL assay for apoptosis. The expression of p65, p-p65, IκBα, p-IκBα, the TJ proteins occludin and ZO-1 and the apoptosis-related proteins Bax, Bcl-2, cleaved caspase-3 and caspase-3 were analysed by Western blot. Levels of DAO, ET, TNF-α, IL-1ß, IL-6, IFN-γ, MDA and SOD were analysed by using kits. RESULTS: MMF activated the NF-κB signaling pathway to cause intestinal inflammation, increased intestinal permeability, changed the expression of TJ protein in the intestinal epithelium, and increased oxidative stress and apoptosis levels. WZ significantly downregulated the expression of p-p65 and p-IκBα to relieve the inflammatory response, reduced intestinal permeability, maintained intestinal TJ protein expression, and reduced intestinal oxidative stress and apoptosis. CONCLUSION: Our research suggested that MMF can cause intestinal injury; by contrast, WZ may exert anti-inflammatory, antioxidant and apoptosis-reducing effects to alleviate MMF-induced intestinal injury.

In vitro study of the toxic and teratogenic effects of prednisolone, azathioprine and mycophenolate mofetile on embryological development of rats.

This study aimed to evaluate the effects of the glucocorticoid prednisolone, the mycophenolic acid prodrug, azathioprine, and the fungi fermentation end product, mycophenolate mofetile on the embryological development of rats. Nine day-old rat embryos were cultured in rat serum containing prednisolone at varying concentrations (5-30 µg/ml) for 48 h. The test groups were cultured separately in rat serum containing 0.3-10 µg/ml azathioprine and 1-10 µg/ml mycophenolate mofetile. Embryonic development parameter effects of both drugs in combination with prednisolone (20 µg/ml) were studied using morphological methods, with special attention given to the incidence of malformations. The genotoxic effects of agents evaluated with the TUNEL test revealed that prednisolone is not a cause of developmental toxicity. The maximum safe dose of prednisolone that could be used in combination with other immunosuppressive agents was determined to be 20 µg/ml. Azathioprine was found to be toxic and teratogenic for the rat embryos beginning at a dose of 1 µg/ml. Dose-dependent toxic and teratogenic effects of mycophenolate mofetile were detected at doses lower than normal clinical ones.

Mycophenolate Mofetil Hepatotoxicity Associated With Mitochondrial Abnormality in Liver Transplant Recipients and Mice.

OBJECTIVES: Mycophenolate mofetil (MMF) is a widely used immunosuppressive agent. MMF hepatotoxicity has been reported in non-transplant and renal transplant patients with minimal histologic description. This is the first study describing detailed histology and ultrastructure of MMF hepatotoxicity. METHODS: Four liver-transplant recipients (Cases 1-4) were suspected to have MMF hepatotoxicity. Cases 1-3 (two females and one male; 4-17 years) had multiple biopsies for liver function test (LFT) abnormalities. Case 4 (female; 16 years) had a surveillance biopsy. Electron-microscopic examination (EM) was requested on Cases 1-3 for unexplained, persistent LFT elevation and histologic abnormalities despite therapy and Case 4 for unexplained histologic abnormalities despite a stable clinical course. To confirm the pathologic changes in the human allografts, livers from MMF-treated and untreated mice were also reviewed. RESULTS: While the allograft biopsies showed nonspecific histologic changes, EM revealed unequivocal mitochondrial abnormalities similar to those seen in primary and secondary mitochondrial disorders. In Cases 1 and 2, LFTs improved after stopping and reducing MMF, respectively. In Case 3, pre- and post-MMF treatment biopsies were performed and only the post-MMF biopsy demonstrated mitochondrial abnormalities. Mitochondrial abnormality in Case 4 was subclinical. The mouse study confirmed that MMF caused various stress changes in the mitochondria; number of mitochondria/cell (mean ± standard deviation; untreated group: 58.25 ±â€Š8.426; MMF-treated group: 76.37 ±â€Š18.66), number of lipid droplets/cell (untreated: 0.9691 ±â€Š1.150; MMF-treated: 3.649 ±â€Š4.143) and sizes of mitochondria (µm, untreated: 0.8550 ±â€Š0.3409; MMF-treated: 0.9598 ±â€Š0.5312) were significantly increased in hepatocytes in the MMF-treated mice compared with the untreated mice (P < 0.0001). CONCLUSIONS: Although MMF is safe for the majority of patients, MMF can cause mitochondrial stress, which may trigger more severe mitochondrial abnormalities in a small subset. MMF hepatotoxicity should be considered for MMF-treated patients with unexplained, persistent LFT abnormalities and nonspecific histologic findings. EM should be requested for these cases.

A systematic overview of the spermatotoxic and genotoxic effects of methotrexate, ganciclovir and mycophenolate mofetil.

INTRODUCTION: Immunosuppressant drugs are increasingly being used in the reproductive years. Theoretically, such medications could affect fetal health either through changes in the sperm DNA or through fetal exposure caused by a presence in the seminal fluid. This systematic overview summarizes existing literature on the spermatotoxic and genotoxic potentials of methotrexate (MTX), a drug widely used to treat rheumatic and dermatologic diseases, and mycophenolate mofetil (MMF), which alone or supplemented with ganciclovir (GCV) may be crucial for the survival of organ transplants. MATERIAL AND METHODS: The systematic overview was performed in accordance with the PRISMA guidelines: A systematic literature search of the MEDLINE and Embase databases was done using a combination of relevant terms to search for studies on spermatotoxic or genotoxic changes related to treatment with MTX, GCV or MMF. The search was restricted to English language literature, and to in vivo animal studies (mammalian species) and clinical human studies. RESULTS: A total of 102 studies were identified, hereof 25 human and 77 animal studies. For MTX, human studies of immunosuppressive dosages show transient effect on sperm quality parameters, which return to reference values within 3 months. No human studies have investigated the sperm DNA damaging effect of MTX, but in other organs the genotoxic effects of immunosuppressive doses of MTX are fluctuating. In animals, immunosuppressive and cytotoxic doses of MTX adversely affect sperm quality parameters and show widespread genotoxic damages in various organs. Cytotoxic doses transiently change the DNA material in all cell stages of spermatogenesis in rodents. For GCV and MMF, data are limited and the results are indeterminate, for which reason spermatotoxic and genotoxic potentials cannot be excluded. CONCLUSIONS: Data from human and animal studies indicate transient spermatotoxic and genotoxic potentials of immunosuppressive and cytotoxic doses of MTX. There are a limited number of studies investigating GCV and MMF.

Keratinocyte growth factor ameliorates mycophenolate mofetil-induced intestinal barrier disruption in mice.

OBJECTIVES: Although mycophenolate mofetil-induced (MMF) effectively improves long-term graft survival, the gastrointestinal (GI) side effects due to MMF-induced GI barrier damage limit its use in clinic. Keratinocyte growth factor (KGF) plays a crucial role in the intestinal protection and repair process. This study is designed to investigate the protective effect of KGF on MMF-induced intestinal mucosal barrier disruption and the potential mechanism. METHODS: Thirty adult male C57BL/6 mice were assigned to one of the following groups: the MMF group, the MMF + KGF group, and the control group (n = 10 in each group). Animals in the MMF group received MMF (500 mg/kg) by gavage once daily for 15 consecutive days; animals in the MMF + KGF group received MMF (500 mg/kg) by gavage and KGF (5 mg/kg) by intraperitoneal injection once daily for 15 consecutive days; and control mice were given an equal volume of vehicle during the 15-day experimental period. In each group, intestinal paracellular permeability, histopathological changes and shifts in tight junction (TJ) protein were evaluated; further, proliferation and apoptosis of intestinal epithelial cells (IECs) were assessed, and intraepithelial lymphocytes (IELs) were isolated and analyzed by flow cytometry. RESULTS: MMF caused intestinal mucosal injury, increased intestinal mucosal permeability, and altered expression of TJ protein. Moreover, MMF treatment inhibited IEC proliferation and increased apoptosis. MMF treatment resulted in a lower proportion of γδ+ T cells in IELs (γδ+ IELs). Conversely, concurrent administration of KGF with MMF effectively alleviated MMF-induced intestinal mucosal disruption, inhibited the increase in intestinal permeability, and maintained TJ protein expression. KGF also reversed the MMF-mediated inhibition of proliferation and promotion of apoptosis in IECs. In addition, KGF significantly enhanced the proportion of γδ+ IELs. CONCLUSION: Our findings suggest that MMF induces intestinal epithelial barrier disruption in mice. KGF may play a protective role to ameliorate the disruption and provide a therapeutic intervention for gastrointestinal disorders induced by MMF.

Drug screening of food and drug administration-approved compounds against Babesia bovis in vitro.

Babesia (B.) bovis is one of the main etiological agents of bovine babesiosis, causes serious economic losses to the cattle industry. Control of bovine babesiosis has been hindered by the limited treatment selection for B. bovis, thus, new options are urgently needed. We explored the drug library and unbiasedly screened 640 food and drug administration (FDA) approved drug compounds for their inhibitory activities against B. bovis in vitro. The initial screening identified 13 potentially effective compounds. Four potent compounds, namely mycophenolic acid (MPA), pentamidine (PTD), doxorubicin hydrochloride (DBH) and vorinostat (SAHA) exhibited the lowest IC50 and then selected for further evaluation of their in vitro efficacies using viability, combination inhibitory and cytotoxicity assays. The half-maximal inhibitory concentration (IC50) values of MPA, PTD, DBH, SAHA were 11.38 ± 1.66, 13.12 ± 4.29, 1.79 ± 0.15 and 45.18 ± 7.37 µM, respectively. Of note, DBH exhibited IC50 lower than that calculated for the commonly used antibabesial drug, diminazene aceturate (DA). The viability result revealed the ability of MPA, PTD, DBH, SAHA to prevent the regrowth of treated parasite at 4 × and 2 × of IC50. Antagonistic interactions against B. bovis were observed after treatment with either MPA, PTD, DBH or SAHA in combination with DA. Our findings indicate the richness of FDA approved compounds by novel potent antibabesial candidates and the identified potent compounds especially DBH might be used for the treatment of animal babesiosis caused by B. bovis.

Vancomycin relieves mycophenolate mofetil-induced gastrointestinal toxicity by eliminating gut bacterial ß-glucuronidase activity.

Mycophenolate mofetil (MMF) is commonly prescribed and has proven advantages over other immunosuppressive drugs. However, frequent gastrointestinal side effects through an unknown mechanism limit its use. We have found that consumption of MMF alters the composition of the gut microbiota, selecting for bacteria expressing the enzyme ß-glucuronidase (GUS) and leading to an up-regulation of GUS activity in the gut of mice and symptomatic humans. In the mouse, vancomycin eliminated GUS-expressing bacteria and prevented MMF-induced weight loss and colonic inflammation. Our work provides a mechanism for the toxicity associated with MMF and a future direction for the development of therapeutics.

Accuracy, discriminative properties and reliability of a human ESC-based in vitro toxicity assay to distinguish teratogens responsible for neural tube defects.

The poor correlation of developmental toxicity studies in animals with human outcome data has emphasized the need for complementary assays based on human cells and tissues. As neural tube defects represent an important proportion of congenital malformations, we evaluated here the accuracy of a human embryonic stem cell (hESC)-based assay to predict chemically induced disruption of neural tube formation. As teratogenic compounds, we used cyclopamine (CPA), valproic acid (VPA), ochratoxin A (OTA) and mycophenolic acid (MMF), all suspected or known inducers of human neural tube defects, as well as theophylline and saccharin as negative control compounds. We analyzed their effects on the ability of hES cells to give rise to neural precursors (expressing specific marker Nestin), to form neural tube-like structures (rosettes), and to express specific markers (Sox1, Otx2, Lix1, EvI1, Rspo3) during rosette formation. The results showed that various effects of the selected compounds on early neural development could be specifically revealed in vitro through related alterations of neurogenic differentiation of hESC. Furthermore, it was possible to discriminate toxicants acting at different time points during embryonic development and, therefore, responsible for distinct adverse effects on neural tube formation. By comparing four different hESC lines, we observed a significant (up to fivefold) variability of the line-dependent response to toxicants. We highlight at least two sources of variability: one related to the heterogeneity of hESC lines in culture (stemness/commitment profiles); the second to possible genetically determined differences in individual sensitivity to teratogens.

An intact microbiota is required for the gastrointestinal toxicity of the immunosuppressant mycophenolate mofetil.

BACKGROUND: Mycophenolate mofetil (MMF) is commonly prescribed after transplantation and has major advantages over other immunosuppressive drugs, but frequent gastrointestinal (GI) side-effects limit its use. The mechanism(s) underlying MMF-related GI toxicity have yet to be elucidated. METHODS: To investigate MMF-related GI toxicity, experimental mice were fed chow containing MMF (0.563%) and multiple indices of toxicity, including weight loss and colonic inflammation, were measured. Changes in intestinal microbial composition were detected using 16S rRNA Illumina sequencing, and downstream PICRUSt analysis was used to predict metagenomic pathways involved. Germ-free (GF) mice and mice treated with orally administered broad-spectrum antibiotics (ABX) were utilized to interrogate the importance of the microbiota in MMF-induced GI toxicity. RESULTS: Mice treated with MMF exhibited significant weight loss, related to loss of body fat and muscle, and marked colonic inflammation. MMF exposure was associated with changes in gut microbial composition, as demonstrated by a loss of overall diversity, expansion of Proteobacteria (specifically Escherichia/Shigella), and enrichment of genes involved in lipopolysaccharide (LPS) biosynthesis, which paralleled increased levels of LPS in the feces and serum. MMF-related GI toxicity was dependent on the intestinal microbiota, as MMF did not induce weight loss or colonic inflammation in GF mice. Furthermore, ABX prevented and reversed MMF-induced weight loss and colonic inflammation. CONCLUSIONS: An intact intestinal microbiota is required to initiate and sustain the GI toxicity of MMF. MMF treatment causes dynamic changes in the composition of the intestinal microbiota that may be a targetable driver of the GI side-effects of MMF.

Effect of Penicillium roqueforti mycotoxins on Caco-2 cells: Acute and chronic exposure.

Penicillium roqueforti is a common food and feed contaminant. However, it is also worldwide renowned for its use as a technological culture responsible for the typicity of blue-veined cheese. Members of the P. roqueforti species are also known to be able to produce secondary metabolites including mycophenolic acid (MPA) and roquefortine C (ROQ C) mycotoxins. In order to more closely simulate the reality of mycotoxin exposure through contaminated food consumption, this work investigated the toxicological effects of MPA and ROQ C not only in acute but also in chronic (i.e. 21-days continuous exposure) conditions on Caco-2 cells. Acute exposure to high MPA or ROQ C concentrations induced an increase of IL-8 secretion. Effects of 21-days continuous exposure on barrier integrity, based on concentrations found in blue-veined cheese and mean of blue cheese intake by French consumers, were monitored. Concerning exposure to ROQ C, no alteration of the intestinal barrier was observed. In contrast, the highest tested MPA concentration (780 µM) induced a decrease in the barrier function of Caco-2 cell monolayers, but no paracellular passage of bacteria was observed. This study highlighted that exposure to MPA and ROQ C average concentrations found in blue-veined cheese does not seem to induce significant toxicological effects in the tested conditions.

Validation of the cell line LS180 as a model for study of the gastrointestinal toxicity of mycophenolic acid.

1. Gastrointestinal (GI) intolerability is a concern for drugs such as mycophenolic acid (MPA) and drug metabolism may play a role. Few in vitro models exist that allow for the preclinical evaluation of a potential role of drug metabolism in intestinal drug toxicity. Thus, we sought to develop an in vitro model based on the human colon adenocarcinoma cell line LS180 to investigate MPA's negative effects on intestinal cells. 2. Stability of expression of key enzymes of MPA metabolism (UGT1A7, UGT1A9, UGT1A10, UGT2B7, CYP3A4 and CYP3A5), transporters (OATP1B1, OATP1B3, OATP2B1, MRP1, MRP2 and MDR1) and the nuclear receptor PXR over 12 passages in combination with guanosine supplementation to counter MPA's antiproliferative effects (determined by western blot analysis and proliferation assays, respectively) was established. 3. Expression of LS180 key enzymes remained stable over passages 47-59 and MPA-induced growth inhibition was circumvented by exogenous guanosine over a period of three days. MPA was not cytotoxic at concentrations up to 250 µM, a concentration that intestinal cells adjacent to the dissolving capsule or tablet are exposed to. 4. We concluded that LS180 cells are suitable to study the potential association between MPA metabolism and its negative effects on intestinal cells.

Synthesis and Immunosuppressive Activity of New Mycophenolic Acid Derivatives.

BACKGROUND: Immunosuppressive drugs are widely used to prevent and treat allograft rejection and autoimmune diseases. Mycophenolic acid (MPA) and its derivatives are currently one of the most prescribed immunosuppressive drugs; however, metabolic drawbacks and variable interand intrapatient responses limit their use. OBJECTIVE: In order to find out new safe and effective immunosuppressive compounds, we report here the synthesis and pharmacological evaluation of hybrid MPA derivatives containing the thalidomide/ phthalimide subunits. RESULTS: All compounds 3a-d exhibited an enhanced ability to reduce the levels of pro-inflammatory cytokines compared to the parental drugs MPA and thalidomide. The mixed lymphocyte reaction assay has demonstrated that compound 3d - (E)-(3-(1,3-dioxoisoindolin-2-yl)-2,6-dioxopiperidin-1- yl)methyl-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4- enoate - has superior activity compared to that of MPA. In addition, compound 3d was less cytotoxic against Jurkat cells than MPA and did not demonstrate in vivo genotoxic effect. CONCLUSION: All these data have shown that compound 3d is a promising lead compound useful in the immunosuppressive therapy.

Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli.

BACKGROUND: Transplant recipients are treated with immunosuppressive (IS) therapies, which impact host-microbial interactions. We examined the impact of IS drugs on gut microbiota and on the expression of ileal antimicrobial peptides. METHODS: Mice were treated for 14 days with prednisolone, mycophenolate mofetil, tacrolimus, a combination of these 3 drugs, everolimus, or water. Feces were collected before and after treatment initiation. Ileal samples were collected after sacrifice. Fecal and ileal microbiota were analyzed by pyrosequencing of 16S rRNA genes and enumeration of selected bacteria by culture, and C-type lectins were assessed in ileal tissues by reverse transcriptase-quantitative polymerase chain reaction. RESULTS: Prednisolone disrupted fecal microbiota community structure, decreased Bacteroidetes, and increased Firmicutes in the feces. Prednisolone, tacrolimus, and mycophenolate mofetil modified fecal microbiota at the family level in each experimental replicate, but changes were not consistent between the replicates. In ileal samples, the genus Clostridium sensu stricto was dramatically reduced in the prednisolone and combined IS drug groups. These modifications corresponded to an altered ileal expression of C-type lectins Reg3γ and Reg3ß, and of interleukin 22. Interestingly, the combined IS treatment enabled a commensal Escherichia coli to flourish, and dramatically increased colonization by uropathogenic E. coli strain 536. CONCLUSIONS: IS treatment alters innate antimicrobial defenses and disrupts the gut microbiota, which leads to overgrowth of indigenous E. coli and facilitates colonization by opportunistic pathogens.

Birth defects in juvenile Wistar rats after exposure to immunosuppressive drugs during pregnancy.

INTRODUCTION: Immunosuppressive drugs and their active metabolites can cross the placental barrier and enter fetal circulation. The adverse effects on the fetus include chromosomal aberrations, structural malformations, organ-specific toxicity and intrauterine growth retardation. The aim of our study was to investigate the impact of "safe" and "contraindicated" immunosuppressive drugs on birth defects in juvenile Wistar rats after exposure of pregnant female rats to these drugs. MATERIAL AND METHODS: The study was conducted on 32 female Wistar rats, subjected to immunosuppressive regimens most commonly used in therapy of human kidney transplant recipients. The animals received drugs by oral gavage 2 weeks before pregnancy and during 3 weeks of pregnancy. RESULTS: Treatment with mycophenolate mofetil and everolimus turned out to be toxic. We have noticed a significantly reduced number of live births in all pregnant rats exposed to these drugs in combination with calcineurin inhibitors and prednisone. Malformations and histological changes of fetal organs were confirmed after mycophenolate mofetil exposure during pregnancy. CONCLUSIONS: Mycophenolate mofetil turned out to be more toxic when used with tacrolimus than with cyclosporin (delivery of live offspring was possible only in the latter group). Everolimus in combination with cyclosporin effectively suppressed the fetal maturation in utero, but did not contribute to the development of malformations.

Mycophenolic Acid-Induced Developmental Defects in Zebrafish Embryos.

With the increasing use of mycophenolic acid (MPA) in solid organ transplantation, some clinical studies indicate that it is also a human teratogen. However, it is unknown by which mechanism MPA acts as a teratogen. Mycophenolic acid was a selective blocker of de novo purine synthesis, and its immunosuppressive effect is mediated by the inhibition of inosine monophosphate dehydrogenase, which could be a target for MPA-induced toxicity as well. The aim of our study was to examine the direct influence of MPA exposure on zebrafish (Danio rerio) embryos. Morphological defects including tail curvature and severe pericardial edema in zebrafish embryos caused by MPA (3.7-11.1 µmol/L) were found in a dose-dependent manner. The teratogenic index (25% lethal concentration value (LC25)/no observed adverse effect level ratio) was 16, which indicated MPA as a teratogen. Quantitative polymerase chain reaction analysis revealed that the expression level of impdh1b and impdh2 was significantly reduced by MPA treatment at 8 µmol/L (equals to LC25 level). All the toxic effects could be partially reversed by the addition of 33.3 µmol/L guanosine. Our results indicated that MPA impairs the development of zebrafish embryos via inhibition of impdh activity, which subsequently caused a guanosine nucleotide depletion in vivo.

Circadian variation of cytotoxicity and genotoxicity induced by an immunosuppressive agent "Mycophenolate Mofetil" in rats.

Immunosuppressive drugs such as Mycophenolate Mofetil (MMF) are used to suppress the immune system activity in transplant patients and reduce the risk of organ rejection. The present study investigates whether the potential cytotoxicity and genotoxicity varied according to MMF dosing-time in Wistar Rat. A potentially toxic MMF dose (300 mg/kg) was acutely administered by the i.p. route in rats at four different circadian stages (1, 7, 13 and 19 hours after light onset, HALO). Rats were sacrificed 3 days following injection, blood and bone marrow were removed for determination of cytotoxicity and genotoxicity analysis. The genotoxic effect of this pro-drug was investigated using the comet assay and the micronucleus test. Hematological changes were also evaluated according to circadian dosing time. MMF treatment induced a significant decrease at 7 HALO in red blood cells, in the hemoglobin rate and in white blood cells. These parameters followed a circadian rhythm in controls or in treated rats with an acrophase located at the end of the light-rest phase. A significant, thrombocytopenia was observed according to MMF circadian dosing time. Furthermore, abnormally shaped red cells, sometimes containing micronuclei, poikilocytotic in red cells and hypersegmented neutrophil nuclei were observed with MMF treatment. The micronucleus test revealed damage to chromosomes in rat bone marrow; the comet assay showed significant DNA damage. This damage varied according to circadian MMF dosing time. The injection of MMF in the middle of the dark-activity phase produced a very mild hematological toxicity and low genotoxicity. Conversely, it induced maximum hematological toxicity and genotoxicity when the administration occurred in the middle of the light-rest phase, which is physiologically analogous to the end of the activity of the diurnal phase in human patients.

Mycophenolic acid, an immunomodulator, has potent and broad-spectrum in vitro antiviral activity against pandemic, seasonal and avian influenza viruses affecting humans.

Immunomodulators have been shown to improve the outcome of severe pneumonia. We have previously shown that mycophenolic acid (MPA), an immunomodulator, has antiviral activity against influenza A/WSN/1933(H1N1) using a high-throughput chemical screening assay. This study further investigated the antiviral activity and mechanism of action of MPA against contemporary clinical isolates of influenza A and B viruses. The 50 % cellular cytotoxicity (CC50) of MPA in Madin Darby canine kidney cell line was over 50 µM. MPA prevented influenza virus-induced cell death in the cell-protection assay, with significantly lower IC50 for influenza B virus B/411 than that of influenza A(H1N1)pdm09 virus H1/415 (0.208 vs 1.510 µM, P=0.0001). For H1/415, MPA interfered with the early stage of viral replication before protein synthesis. For B/411, MPA may also act at a later stage since MPA was active against B/411 even when added 12 h post-infection. Virus-yield reduction assay showed that the replication of B/411 was completely inhibited by MPA at concentrations ≥0.78 µM, while there was a dose-dependent reduction of viral titer for H1/415. The antiviral effect of MPA was completely reverted by guanosine supplementation. Plaque reduction assay showed that MPA had antiviral activity against eight different clinical isolates of A(H1N1), A(H3N2), A(H7N9) and influenza B viruses (IC50 <1 µM). In summary, MPA has broad-spectrum antiviral activity against human and avian-origin influenza viruses, in addition to its immunomodulatory activity. Together with a high chemotherapeutic index, the use of MPA as an antiviral agent should be further investigated in vivo.

PRMT3: new binding molecule to RhoGDI-α during mycophenolic acid-induced ß-cell death.

Mycophenolic acid (MPA)-induced beta cell toxicity limits islet graft survival. However, the signal transduction mechanisms underlying MPA-induced ß-cell toxicity have not been fully elucidated. Previously, we showed that MPA-induced pancreatic ß-cell apoptosis proceeds via RhoGDI-α down-regulation linked to Rac1 activation. In the present study, we investigated factors affecting RhoGDI-α during MPA-induced ß-cell apoptosis. The presence of RhoGDI-α-related protein was determined with the use of yeast 2-hybrid (Y2H) analysis. Y2H screening of RhoGDI-α was performed in yeast PBN204 strain containing 3 reporters (URA3, lacZ, and ADE2) under the control of different GAL promoters. INS-1E cells (an insulin-secreting pancreatic ß-cell line) were treated with MPA for 12, 24, and 36 hours. Eighty-three real positives were obtained by Y2H analysis, and of these, arginine N-methyltransferase 3 (PRMT3) protein interacted with RhoGDI-α in INS-1E cells. PRMT3 gene expressions and its protein levels were significantly decreased during MPA-induced apoptosis. In summary, PRMT3 and RhoGDI-α were found to interact in INS-1E cells. Furthermore, MPA was found to regulate this interaction in INS-1E cells by down-regulating the gene expression of PRMT3. These findings suggest that control of the interaction between PRMT3 and RhoGDI-α could be used to prevent MPA-induced ß-cell death.