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Purpose: Drug-induced liver injury is becoming an increasingly important topic in drug research and clinical practice. Due to a lack of experimental animal models, predicting drug-induced liver injury in humans is challenging. Azathioprine (AZA) is a classical immunosuppressant with hepatotoxic adverse effects. The present study aimed to address the hepatoprotective effect of carvedilol (CAR) against AZA-induced hepatocellular injury via assessing redox-sensitive signals., Method: To achieve this purpose, rats were allocated into four groups: control, CAR only, AZA only, and CAR plus AZA groups. The induction of hepatic injury was induced by a single intraperitoneal injection of AZA at a dose of 50 mg/kg on the 6th day of the experiment. Each experimental protocol was approved and supervised by the Ethics Committee for Animal Experiments., Results: The results of the present study revealed that CAR administration significantly diminished AZA-induced hepatic dysfunction, as evidenced by relief of hepatic function biomarkers and histopathological aberration induced by AZA injection. Besides, CAR restored oxidant/antioxidant balance as well as NRF2 expression. In addition, CAR suppressed inflammatory response induced by AZA challenge as evidenced by downregulation of TLR4, TNF-α, MPO, and eNOS/iNOS levels in hepatic tissue. Moreover, CAR recovered apoptotic/anti-apoptotic status by modulation of caspase-3/Bcl2 expression., Conclusion: Taken together, CAR protects against AZA-induced hepatic injury via antioxidant, anti-inflammatory, and anti-apoptotic activities. These findings revealed that CAR could be a good candidate for hepatic injury protection and can be added to AZA therapeutic regimen to reduce their adverse effect., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Thiopurines by their glucocorticoid-sparing property help in maintaining remission for patients with inflammatory bowel disease (IBD), when glucocorticoids are reduced and withdrawn. However, due to bone marrow suppression, it cannot be used in various conditions where it is indicated. A 17-year-old patient presented with pancytopenia with neutropenic sepsis and alopecia after 3 weeks of starting azathioprine for her underlying Crohn's disease. Thiopurine S-methyltransferase (TPMT;*2, *3A, *3C) analysis resulted in a wild-type genotype, whereas homozygous Nudix hydrolase 15 (NUDT 15 C415T) variant was positive. Azathioprine was stopped immediately, and she was started on broad-spectrum antibiotics that led to some clinical improvements initially, but later on, the patient developed intestinal obstruction along with postoperative complications leading to death. In this report, we highlight a case of serious hematological toxicity associated with azathioprine use in a patient with Crohn's disease with homozygous NUDT 15 variant, thus favoring the implementation of a pharmacogenomic approach before starting azathioprine, particularly in the Asian population. [ABSTRACT FROM AUTHOR]

Introduction: Crohn's disease is a chronic, relapsing and inflammatory condition. Azathioprine (AZA) is an immunosuppressive drug used for maintenance of the disease remission. However, the side effects this drug causes to patients, makes it questionable, as to its safety, for health. Purpose: To determine the type and severity of side effects caused by AZA treatment in Crohn's disease. Materials and methods: Through systematic review of literature, 85 studies were found, 10 of which were selected. The selection criteria were: a) articles, which were primary studies, reviews or meta-analyzes, b) available in full text, c) written in English and d) finally articles, referring to humans. Results: Studies show that AZA, is drug of choice, for treating Crohn's disease. However, side effects from its use are numerous and increasing in severity. According to the survey results, people who used this drug, developed at some point in their treatment, side effects such as hepatotoxicity, myelotoxicity, acute pancreatitis (p<0.001), gastrointestinal intolerance, general hardship, blood disorders, fever wave, itching and arthralgia. Conclusions: Recommendations from these studies show that side effects immerge from almost all systems in the patient's body, but it is not proven if all of them have to do exclusively with the drug or the disease's nature. As AZA holds a prominent role in disease's treatment, the use of more thorough controls is recommended for simultaneous treatment of side effects. [ABSTRACT FROM AUTHOR]

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.

Methotrexate (MTX) and azathioprine (AZA) are chemotherapeutic, antimetabolic, and immunosuppressive agents with substantial risks such as oxidative lesions to the liver. This study examined the effect of grape seed extract (GSE; gervital) in attenuating hepatotoxicity caused by MTX or AZA treatment. Rats were divided into six groups (six rats per group): Group I, normal control group; Group II, GSE (150 mg/kg/day); Group III, MTX (8 mg/kg/week); Group IV, AZA (15 mg/kg/day); Group V, GSE (150 mg/kg/day) + MTX (8 mg/kg/week); and Group VI, GSE (150 mg/kg/day) + AZA (15 mg/kg/day). After 35-day experimental period, all rats were sacrificed and blood was collected for biochemical study and hemoglobin (Hb) assessment. The liver was weighed and triaged for histological, ultrastructural, and biochemical studies. MTX and AZA treatment decreased Hb levels, increased relative liver weight, increased the activity of glutamate pyruvate transaminase (ALT) and glutamate oxaloacetate transaminase (AST) aminotransferase (ALT) and aspartate aminotransferase (AST) values, and displayed histopathological and ultrastructural alterations. These changes included the disorganization of hepatocytes, pyknosis, karyolysis of some nuclei, and mononuclear leukocytic infiltration. The liver with significant oxidative stress (OS) showed decreased reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) and increased malondialdehyde (MDA) levels. In contrast, GSE administration ameliorated ALT, AST, and all histopathological and ultrastructural changes. GSE treatment also reduced MDA levels but increased the antioxidant parameters. In conclusion, it was concluded that GSE supplementation could be considered as a promising antioxidant in reducing OS, histopathological and ultrastructural alterations induced by MTX and AZA., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Azathioprine (Aza) is a purine antimetabolite immunosuppressant that is widely employed for immunosuppressive therapy in post-transplant recipients or patients with autoimmune diseases. Chronic use of immunosuppressants might produce several side effects, including a high rate of neoplasms in these patients. Considering that genotoxic effects are associated with an increased risk of developing cancer, the aim of this study was to examine the recombinogenic, genotoxic, and cytotoxic effects of Aza using Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster , as well as comet and micronucleus assays in mouse bone marrow cells. Further, the adverse effects of Aza were determined in mouse hepatic and renal tissues using histopathological analysis. Data demonstrated that Aza induced significant increased genotoxicity in D. melanogaster and mouse bone marrow cells at all concentrations tested. Homologous recombination was the predominant genotoxic event noted for the first time to be initiated by Aza in SMART. In histopathological analysis, Aza did not show any marked toxic activity in mouse hepatic and renal tissues. Therefore, the high rate of neoplasms reported in patients with long-term use of Aza may be attributed, at least partially, to the genotoxic action of this drug.

Background: The male reproductive system is a sensitive and intricate process that can be distressed following exposure to various toxicants. Therapeutic drugs, especially chemotherapeutics, can also adversely affect male fertility by instigating hormonal changes leading to testicular cells injury. Azathioprine (AZA) is an effective anticancer drug, but some cases of testicular toxicity have been reported. The aim of this work was to investigate the protective effects of taurine chloramine (TAU-Cl), a reported antioxidant and antiinflammtory peptide, against AZA-induced testicular dysfunction in male rats and ascertain the contributing mechanisms., Methods: Forty male rats were allocated into four equal groups; (i) normal control rats, (ii) TAU-Cl group (100 mg/kg b.w/day for 10 weeks, (iii) AZA group (5 mg/day for 4 weeks); (iv) TAU-Cl/AZA group., Results: AZA caused increased DNA damage in the testes, and alterations in sex hormones and sperm quality, including sperm count, viability, and motility. Moreover, testicular tissue from the AZA-treated group had increased levels of oxidative stress indicator, MDA, and decreased activity of the antioxidant enzymes as superoxide dismutase (SOD), reduced glutathione (GSH) and catalase (CAT) levels. These deleterious events were accompanied by upregulated levels of the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), and protein expression of iNOS and NFκB-p65, interleukin-1beta (IL-1β), and proapoptotic marker; caspase-9, together with decreased Bcl-2, NrF2 and hemeoxygenase (HO-1) expression. In contrast, TAU-Cl pretreatment significantly abrogated these toxic effects which were confirmed histologically., Conclusion: Pretreatment with TAU-Cl exerts a protective effect against AZA-induced male reproductive testicular atrophy. This finding could open new avenues for the use of TAU-Cl as a complementary approach to chemotherapy supportive care.

Background and Aim: Interindividual variation seen in the thiopurine metabolism is attributed to the genetic variant in thiopurine methyltransferase (TPMT) gene leading to myelosuppression. In Asians, the thiopurine-induced toxicity is not completely explained by TPMT variants. Literature indicates that a newer genetic variant in nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) gene is associated with thiopurine intolerance. We aimed to determine the risk allele frequency of NUDT15 genetic variant and its association with thiopurine-induced toxicity in Indian patients., Methods: In this pilot study, 69 patients on thiopurine therapy were analyzed. The frequencies of thiopurine-induced leukopenia were recorded. NUDT15 (C415T) and TPMT (*2, *3A, *3B, and *3C) genotyping was performed using amplification refractory mutation system-polymerase chain reaction and restriction fragment length polymorphism technique. Results were validated by DNA sequencing., Results: The NUDT15 CC, CT, and TT genotypes were found to be 86.9%, 11.5%, and 1.5%, respectively, whereas TPMT genetic variants were absent. Of 60 patients without NUDT15 variant, none developed leukopenia, whereas of nine patients with NUDT15 variant, six developed leukopenia (P-value < 0.0001). The mean thiopurine dose of 1.01 and 0.73 mg/kg/day for patients with wild and mutant NUDT15 alleles, respectively, was statistically significant (P < 0.01). The sensitivity and specificity for NUDT15 variant were 100% and 95.2%, respectively., Conclusions: The NUDT15 risk allele frequency was 7.2%. There are 6/69 (8.7%) patients who developed leukopenia and harbored NUDT15 variant, thus showing a strong association for thiopurine-induced toxicity. Hence, NUDT15 genotyping may be considered before thiopurine therapy in Indian patients., (© 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Understanding central mechanisms underlying drug-induced toxicity plays a crucial role in drug development and drug safety. However, a translation of cellular in vitro findings to an actual in vivo context remains challenging. Here, physiologically based pharmacokinetic (PBPK) modeling was used for in vivo contextualization of in vitro toxicity data (PICD) to quantitatively predict in vivo drug response over time by integrating multiple levels of biological organization. Explicitly, in vitro toxicity data at the cellular level were integrated into whole-body PBPK models at the organism level by coupling in vitro drug exposure with in vivo drug concentration-time profiles simulated in the extracellular environment within the organ. PICD was exemplarily applied on the hepatotoxicant azathioprine to quantitatively predict in vivo drug response of perturbed biological pathways and cellular processes in rats and humans. The predictive accuracy of PICD was assessed by comparing in vivo drug response predicted for rats with observed in vivo measurements. To demonstrate clinical applicability of PICD, in vivo drug responses of a critical toxicity-related pathway were predicted for eight patients following acute azathioprine overdoses. Moreover, acute liver failure after multiple dosing of azathioprine was investigated in a patient case study by use of own clinical data. Simulated pharmacokinetic profiles were therefore related to in vivo drug response predicted for genes associated with observed clinical symptoms and to clinical biomarkers measured in vivo. PICD provides a generic platform to investigate drug-induced toxicity at a patient level and thus may facilitate individualized risk assessment during drug development., Competing Interests: L. K. is employee of Bayer Technology Services GmbH, the company developing the PBPK modeling tools PK-Sim and MoBi.

A new in vivo gene mutation assay has been developed based on the phosphatidylinositol glycan anchor biosynthesis, Class A gene (Pig-a in rodents) as an endogenous reporter. Using this Pig-a assay, the in vivo mutagenicity of a single dose of azathioprine (Aza) was investigated in red blood cells (RBC Pig-a assay) and reticulocytes (PIGRET) of rats. Eight-week old male rats were orally dosed once with Aza at 50, 100 and 200mg/kg or ethylnitrosourea (ENU) at 10 and 40mg/kg as a positive control. Because 4 out of 6 animals at 200mg/kg of Aza died 3days after the dosing, this dose group was excluded for analyses. The frequencies of Pig-a mutants in RBCs and reticulocytes (RET) were evaluated once a week for 4 weeks after the treatment. With a single exposure to ENU, the frequencies of Pig-a mutants in both RBCs and RETs increased in a time- and dose-dependent manner. In contrast, with Aza small effects that were not statistically significant were observed in rats at 21 and 14days in the RBC Pig-a and PIGRET assays respectively. Based on the present results, the mutagenic potential of Aza is negligible after single oral administration in rats., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mismatch-repair (MMR)-deficient cells show increased in vitro tolerance to thiopurines because they escape apoptosis resulting from MMR-dependent signaling of drug-induced DNA damage. Prolonged treatment with immunosuppressants including azathioprine (Aza), a thiopurine prodrug, has been suggested as a risk factor for the development of late onset leukemias/lymphomas displaying a microsatellite instability (MSI) phenotype, the hallmark of a defective MMR system. We performed a dose effect study in mice to investigate the development of MSI lymphomas associated with long term Aza treatment. Over two years, Aza was administered to mice that were wild type, null or heterozygous for the MMR gene Msh2. Ciclosporin A, an immunosuppressant with an MMR-independent signaling, was also administered to Msh2(wt) mice as controls. Survival, lymphoma incidence and MSI tumor phenotype were investigated. Msh2(+/-) mice were found more tolerant than Msh2(wt) mice to the cytotoxicity of Aza. In Msh2(+/-) mice, Aza induced a high incidence of MSI lymphomas in a dose-dependent manner. In Msh2(wt) mice, a substantial lifespan was only observed at the lowest Aza dose. It was associated with the development of lymphomas, one of which displayed the MSI phenotype, unlike the CsA-induced lymphomas. Our findings define Aza as a risk factor for an MSI-driven lymphomagenesis process.

Data in a toxicity test are evaluated generally per parameter. Information on the response per animal in addition to per parameter can improve the evaluation of the results. The results from the six studies in rats, described in the paper by Kemmerling, J., Fehlert, E., Rühl-Fehlert, C., Kuper, C.F., Stropp, G., Vogels, J., Krul, C., Vohr, H.-W., 2015. The transferability from rat subacute 4-week oral toxicity study to translational research exemplified by two pharmaceutical immunosuppressants and two environmental pollutants with immunomodulating properties (In this issue), have been subjected to principal component analysis (PCA) and principal component discriminant analysis (PC-DA). The two pharmaceuticals azathioprine (AZA) and cyclosporine A (CSA) and the two environmental pollutants hexachlorobenzene (HCB) and benzo(a)pyrene (BaP) all modulate the immune system, albeit that their mode of immunomodulation is quite diverse. PCA illustrated the similarities between the two independent studies with AZA (AZA1 and AZA2) and CSA (CSA1 and CSA2). The PC-DA on data of the AZA2 study did not increase substantially the information on dose levels. In general, the no-effect levels were lower upon single parameter analysis than indicated by the distances between the dose groups in the PCA. This was mostly due to the expert judgment in the single parameter evaluation, which took into account outstanding pathology in only one or two animals. The PCA plots did not reveal sex-related differences in sensitivity, but the key pathology for males and females differed. The observed variability in some of the control groups was largely a peripheral blood effect. Most importantly, PCA analysis identified several animals outside the 95% confidence limit indicating high-responders; also low-to-non-responders were identified. The key pathology enhanced the understanding of the response of the animals to the four model compounds., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Exposure to chemicals may have an influence on the immune system. Often, this is an unwanted effect but in some pharmaceuticals, it is the intended mechanism of action. Immune function tests and in depth histopathological investigations of immune organs were integrated in rodent toxicity studies performed according to an extended OECD test guideline 407 protocol. Exemplified by two immunosuppressive drugs, azathioprine and cyclosporine A, and two environmental chemicals, hexachlorobenzene and benzo[a]pyrene, results of subacute rat studies were compared to knowledge in other species particular in humans. Although immune function has a high concordance in mammalian species, regarding the transferability from rodents to humans various factors have to be taken into account. In rats, sensitivity seems to depend on factors such as strain, sex, stress levels as well as metabolism. The two immunosuppressive drugs showed a high similarity of effects in animals and humans as the immune system was the most sensitive target in both. Hexachlorobenzene gave an inconsistent pattern of effects when considering the immune system of different species. In some species pronounced inflammation was observed, whereas in primates liver toxicity seemed more obvious. Generally, the immune system was not the most sensitive target in hexachlorobenzene-treatment. Immune function tests in rats gave evidence of a reaction to systemic inflammation rather than a direct impact on immune cells. Data from humans are likewise equivocal. In the case of benzo[a]pyrene, the immune system was the most sensitive target in rats. In the in vitro plaque forming cell assay (Mishell-Dutton culture) a direct comparison of cells from different species including rat and human was possible and showed similar reactions. The doses in the rat study had, however, no realistic relation to human exposure, which occurs exclusively in mixtures and in a much lower range. In summary, a case by case approach is necessary when testing immunotoxicity. Improvements for the translation from animals to humans related to immune cells can be expected from in vitro tests which offer direct comparison with reactions of human immune cells. This may lead to a better understanding of results and variations seen in animal studies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Azathioprine (AZA), 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG) are antimetabolite drugs, widely used as immunosuppressants and anticancer agents. Despite their proven efficacy, a high incidence of toxic effects in patients during standard-dose therapy is recorded. The aim of this study is to explain, from a mechanistic point of view, the clinical evidence showing a significant role of glutathione-S-transferase (GST)-M1 genotype on AZA toxicity in inflammatory bowel disease patients. To this aim, the human nontumor IHH and HCEC cell lines were chosen as predictive models of the hepatic and intestinal tissues, respectively. AZA, but not 6-MP and 6-TG, induced a concentration-dependent superoxide anion production that seemed dependent on GSH depletion. N-Acetylcysteine reduced the AZA antiproliferative effect in both cell lines, and GST-M1 overexpression increased both superoxide anion production and cytotoxicity, especially in transfected HCEC cells. In this study, an in vitro model to study thiopurines' metabolism has been set up and helped us to demonstrate, for the first time, a clear role of GST-M1 in modulating AZA cytotoxicity, with a close dependency on superoxide anion production. These results provide the molecular basis to shed light on the clinical evidence suggesting a role of GST-M1 genotype in influencing the toxic effects of AZA treatment.

Aim: To investigate if azathioprine could reduce adenoma formation in Apc(Min/+) , a mouse model of sporadic intestinal tumorigenesis., Methods: Azathioprine was administered via drinking water (estimated 6-20 mg/kg body weight per day) to Apc(Min/+) and wildtype mice. Control animals received vehicle only (DMSO) dissolved in drinking water. At 15 wk of age all mice were sacrificed and intestines of Apc(Min/+) were harvested for evaluation of polyp number. Azathioprine induced toxicity was investigated by immunohistochemical analysis on spleens., Results: All azathioprine treated mice showed signs of drug-associated toxicity such as weight loss and development of splenic T-cell lymphomas. Although this suggests that the thiopurine concentration was clearly in the therapeutic range, it did not reduce tumor formation (48 ± 3.1 adenomas vs 59 ± 5.7 adenomas, P = 0.148)., Conclusion: We conclude that in the absence of inflammation, azathioprine does not affect intestinal tumorigenesis.

Azathioprine, an immunosuppressant, has gained a prominent position in the clinic for prevention of graft rejection in organ transplants, as well as dermatological autoimmune diseases. However, according to a number of research reports, hepatotoxicity, as one of the side effects, is a major obstacle in azathioprine therapy. In this study, an integrated toxicoproteomic and toxicotranscriptomic analysis was performed using rat primary hepatocytes, in order to gain insight into the in-depth pathway map related to azathioprine-induced hepatotoxicity. For proteomic and transcriptomic analysis, rat primary hepatocytes were exposed to azathioprine at IC20 concentration for 24 h. In particular, 2D LC-MS/MS and informatics-assisted label-free strategy for proteomic analysis were applied in order to increase the number of identified proteins and to improve the confidence of the quantitation results. Among 119 differentially identified protein species, 69 were upregulated and 50 were downregulated in the azathioprine-treated group. At the mRNA level, results of transcriptomic analysis showed increased transcription of 340 genes and decreased transcription of 63 genes in the azathioprine-treated group. Based on the analysis of transcriptomic and proteomic results using the DAVID program, drug metabolism/oxidative stress enzymes, xenobiotic metabolism by cytochrome P450, fatty acid metabolism, primary bile acid biosynthesis, contraction, inflammation metabolism, and mitogen-activated protein kinase (MAPK) kinase (ERK/JNK/p38 kinase) pathways were affected in azathioprine-treated hepatotoxicity. The effects on genes and proteins related to several important pathways were confirmed by real-time PCR and immunoblot analysis, respectively. This study is the first to report on relevant pathways related to azathioprine-induced hepatotoxicity through performance of integrated transcriptomic and proteomic analyses., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Drug-induced liver injury (DILI) is a growing concern in the fields of drug development and clinical drug therapy because numerous drugs have been linked to hepatotoxicity. However, it is difficult to predict DILI in humans due to the lack of experimental animal models. Although azathioprine (AZA), which is a widely used immunosuppressive drug, is generally well tolerated, a small number of patients prescribed AZA develop severe hepatitis. However, the mechanism underlying this process has not yet been elucidated. In this study, we developed a mouse model of AZA-induced liver injury and investigated the mechanisms responsible for the hepatotoxicity of AZA. Female BALB/c mice were orally administered AZA. After AZA administration, the plasma levels of alanine aminotransferase and aspartate aminotransferase were increased, and liver damage was confirmed through a histological evaluation. In addition, the hepatic glutathione levels and superoxide dismutase activity were significantly decreased. The plasma levels of reactive oxygen species were significantly increased during the early phase of AZA-induced liver injury, and the hepatic mRNA levels of immune- and inflammation-related factors were also significantly changed. In conclusion, oxidative stress and the subsequently activated immune- and inflammation-related factors are involved in AZA-induced liver injury., (Copyright © 2013. Published by Elsevier Ireland Ltd.)

In the present study, we investigated the potential protective effects of royal jelly against azathioprine-induced toxicity in rat. Intraperitoneal administration of azathioprine (50 mg/kgB.W.) induced a significant decrease in RBCs count, Hb concentration, PCV%, WBCs count, differential count and platelet count, hepatic antioxidant enzymes (reduced glutathione and glutathione s-transferase) and increase of serum transaminases (alanine aminotransferase and aspartate aminotransferase enzymes) activities, alkaline phosphatase and malondialdehyde formation. Azathioprine induced hepatotoxicity was reflected by marked pathological changes in the liver. Oral administration of royal jelly (200 mg/kgB.W.) was efficient in counteracting azathioprine toxicity whereas it altered the anemic condition, leucopenia and thrombocytopenia induced by azathioprine. Furthermore, royal jelly exerted significant protection against liver damage induced by azathioprine through reduction of the elevated activities of serum hepatic enzymes. Moreover, royal jelly blocked azathioprine-induced lipid peroxidation through decreasing the malondialdehyde formation. In conclusion, royal jelly possesses a capability to attenuate azathioprine-induced toxicity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The results of extraction of 6-mercaptopurine and azathioprine from aqueous saline solutions with organic solvents of different chemical nature and their mixtures are presented. Synergism between the distribution coefficients was demonstrated during the extraction of the above compounds with 1,4-dioxane and chloroform mixtures. The use of a chloroform--1,4-dioxane system in the molar ratio of 0.8:0.2 as a solvent resulted in the maximum values of distribution coefficients of 6-mercaptopurine and azathioprine.

Background: Infliximab-induced hepatotoxicity is reported in several case studies involving patients with inflammatory bowel disease (IBD) and a direct hepatotoxic effect has been proposed., Objective: The aim of this study was to determine the direct in vitro toxicity of infliximab. As a proof of principle the in vitro toxicity of thiopurines and methotrexate was also determined., Methods: Cell survival curves and the half maximal inhibitory concentrations (IC(50)) were obtained after 24, 48 and 72 h of incubation in HepG2 cells with the IBD drugs azathioprine, 6-mercaptopurine, 6-thioguanine, methotrexate or infliximab by using the WST-1 cytotoxicity assay., Results: No in vitro hepatotoxicity in HepG2 cells was seen with infliximab, while concentration-dependent cytotoxicity was observed when HepG2 cells were incubated with increasing concentrations of azathioprine, 6-mercaptopurine and 6-thioguanine., Conclusion: Infliximab alone or given in combination with azathioprine showed no direct hepatotoxic effect in vitro, indicating that the postulated direct hepatotoxicity of infliximab is unlikely.

Background: Oral thiopurines are effective and widely used in treatment of inflammatory bowel disease (IBD) in humans, although their use is limited due the development of adverse events. Here, we examine the efficacy and toxicity of oral treatment with 6-tioguanine (6-TG) and azathioprine (AZA) in a murine model of IBD., Methods: We induced acute or chronic colitis in BALB/c mice by one or four cycles of 3% dextran sulphate sodium (DSS), respectively. Mice were treated by daily gavages of various dosages of 6-tioguanine, azathioprine, or by phosphate buffered saline (PBS) starting the first day of DSS or after two cycles of DSS, respectively. We monitored the efficacy and toxicity by measuring the weight change and serum alanine aminotransferase (ALT) activity and by disease severity and histology, at the end of the experiment. Moreover, we measured cytokine production after colon fragment cultivation by enzyme-linked immunoabsorbent assay and numbers of apoptotic cells in the spleen by flow cytometry., Results: 6-TG is effective in the treatment of acute DSS-induced colitis in a dose-dependent manner and 40 μg of 6-TG is significantly more effective in the treatment of acute colitis than both AZA and PBS. This effect is accompanied by decrease of IL-6 and IFN-γ production in colon. We did not observe histological abnormalities in liver samples from control (PBS) or 6-TG treated mice. However, liver samples from most mice treated with AZA showed mild, yet distinct signs of hepatotoxicity. In chronic colitis, all thiopurine derivatives improved colitis, 20 μg of 6-TG per dose was superior. High doses of 6-TG led to significant weight loss at the end of the therapy, but none of the thiopurine derivatives increased levels of serum ALT. Both thiopurine derivatives reduced the proportion of apoptotic T helper cells, but a high production of both IL-6 and TGF-β was observed only in colon of AZA-treated mice., Conclusions: Use of 6-TG in the treatment of experimental colitis in mice appears superior to AZA administration and placebo. In contrast to 6-TG, the use of AZA resulted in histological liver abnormalities.

Background: The thiopurine prodrug azathioprine is used extensively in cancer therapy. Exposure to this drug results in the selection of DNA mismatch repair-deficient cell clones in vitro. It has also been suggested that thiopurine drugs might constitute a risk factor for the emergence of human neoplasms displaying microsatellite instability (MSI) because of deficient DNA mismatch repair., Methods: Azathioprine was administered via drinking water (6-20 mg/kg body weight per day) to mice that were null (Msh2⁻(/)⁻; n = 27), heterozygous (Msh2(+/)⁻; n = 22), or wild type (Msh2(WT); n = 18) for the DNA mismatch repair gene Msh2. Control mice (45 Msh2⁻(/)⁻, 38 Msh2(+/)⁻, and 12 Msh2(WT)) received drinking water lacking azathioprine. The effect of azathioprine on tumorigenesis and survival of the mice was evaluated by Kaplan-Meier curves using log-rank and Gehan-Breslow-Wilcoxon tests. Mouse tumor samples were characterized by histology and immunophenotyping, and their MSI status was determined by polymerase chain reaction analysis of three noncoding microsatellite markers and by immunohistochemistry. Msh2 status of tumor samples was assessed by loss of heterozygosity analyses and sequencing after reverse transcription-polymerase chain reaction of the entire Msh2 coding sequence. All statistical tests were two-sided., Results: Most untreated Msh2(WT) and Msh2(+/)⁻ mice remained asymptomatic and alive at 250 days of age, whereas azathioprine-treated Msh2(WT) and Msh2(+/)⁻ mice developed lymphomas and died prematurely (median survival of 71 and 165 days of age, respectively). Azathioprine-treated Msh2(+/)⁻ mice developed diffuse lymphomas lacking Msh2 expression and displaying MSI due to somatic inactivation of the functional Msh2 allele by loss of heterozygosity or mutation. By contrast, azathioprine-treated Msh2(WT) mice displayed no obvious tumor phenotype, but histological examination showed microscopic splenic foci of neoplastic lymphoid cells that retained Msh2 expression and did not display MSI. Both untreated and azathioprine-treated Msh2⁻(/)⁻ mice had a reduced lifespan compared with untreated Msh2(WT) mice (median survival of 127 and 107 days of age, respectively) and developed lymphomas with MSI., Conclusion: Azathioprine-induced carcinogenesis in mice depends on the number of functional copies of the Msh2 gene.

Azathioprine (Aza) is an important drug commonly used in the therapy of autoimmune system disorders. It induces hepatotoxicity and hazard effects that restrict its use. The effects of administration of grape seed extract and folic acid on Aza toxicity by gavage (simultaneously) daily for 4 weeks were studied by determining the changes in some hematological parameters and liver histology. The glutathione level (GSH) and lipid peroxidation content as malondialdehyde (MDA) in the liver tissue were measured. The repeated intake of Aza (25 mg/kg body weight) induced anemia characterized by decreased erythrocyte and leukocyte counts and reticulocyte and hematocrit percentages, while the prothrombin time was significantly increased. Moreover, Aza caused a significant decrease in phagocytic activity and lymphocyte percentage. Aza induced hepatic damage as indicated by pronounced changes in the histological structure, a significant increase in serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), and MDA content in the liver tissue. Meanwhile, the GSH activity was significantly decreased. Co-treatment with grape seed extract and Aza minimized the previously mentioned hazard effects of Aza and significantly protected the hepatic tissue by ameliorating the antioxidant activity. Folic acid administration, simultaneously, with Aza only improved the anemia. It may be concluded that grape seed extract is a useful herbal remedy, especially for controlling oxidative damages and is considered as a potent protective agent against Aza hepatotoxicity., (Copyright © 2010 John Wiley & Sons, Ltd.)

The prediction of the carcinogenic risk for humans is mostly based on animal experiments. For the last 20 years, however, the scientific community has paid great attention to alternative strategies in compliance with common moral and ethical values. The new European chemical regulation REACH (Reg. EC 1907/2006) requires the performance of new studies in vertebrates only as a last resort. REACH asks for the development of validated in vitro protocols that can replace, in the medium to the long term, animal bioassays. An in vitro cell transformation assay (CTA) is proposed as an alternative to in vivo carcinogenicity testing. This assay is reported in the list of accepted methods for REACH (Reg. EC 440/2008). The BALB/c 3T3 model represents one of the most well-known CTAs and is regarded as a useful tool to screen single chemicals or complex mixtures for carcinogenicity prediction. In this study we used a modified protocol to highlight the transforming potential of three single compounds, ethinylestradiol (EE), azathioprine (AZA-T), melphalan, and two polychlorinated biphenyls (PCBs) mixtures, which are known or suspected to be human carcinogens. We also evaluated the activity of the antioxidant alpha-lipoic acid (ALA), a promising tumor chemopreventive. A significant increase in transformation frequency was observed when the BALB/c 3T3 cells were exposed to EE, AZA-T or melphalan as well as after PCBs treatment. On the contrary, ALA did not induce any increase of foci occurrence. Our results confirm the suitability of the improved protocol to discriminate carcinogenic compounds and support the use of BALB/c 3T3 cell transformation assay as a possible alternative to predict carcinogenic risk to humans., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background and Objective: Individuals with intermediate or no thiopurine S-methyltransferase (TPMT) activity are at risk of hematotoxicity when treated with standard doses of thiopurines, thus, pretreatment identification of these individuals is of major importance. The purpose of this study was to determine the frequency and distribution of TPMT polymorphic variants, known to functionally impair TPMT activity, in the highly heterogeneous Israeli population., Methods: TPMT genotyping of individuals representing three major demographic groups in Israel was carried out by PCR restriction fragment length polymorphism and high-resolution melting., Results: Frequencies of TPMT risk alleles differed significantly among the screened Israeli subpopulations: Druze showed fivefold and twofold higher frequencies than Jews and Moslems, respectively. Specifically, allelic frequencies of TPMT*3A were 0.73% (95% CI 0.34-1.45%), 0.79% (95% CI 0.16-2.39%), and 3.19% (95% CI 1.78-5.58%) in Jews, Moslems, and Druze, respectively. Although not found in Jews, TPMT*3C was found at an allelic frequency of 1.05% (95% CI 0.31-2.76%) and 0.75% (95% CI 0.02-2.84%) in Moslems and Druze. TPMT*2 and TPMT*3B were not detected in any of the Israeli subpopulations studied., Conclusion: These data indicate that the Israeli population displays a distinct TPMT genetic variability that is comprised of a mix of three major genetically diverse subpopulations, each with its unique TPMT allelic frequency distribution pattern and likelihood of developing an adverse reaction to thiopurine drugs.

The survival and growth rate of the Indian freshwater mussel, Lamellidens marginalis, (Lamarck) was ascertained in cultivation by using certain drugs in CIFA, fish farm, BBSR (India) during June 1998 to February 1999. Two sets of experiments were carried out to evaluate the effects of drugs like Betamethasone, Calcium, Azathioprine, Stanazolol, and Folic acid. Chloramphenicol was added with each treatment as prophylaxis to prevent the bacterial growth. In the first set, the inactiveness and mortality of the mussels in different drugs were studied through two different dosages and in subsequent tests the fixation of dosage was employed. The study in the second set was regarding the survival, increment of shell length, its thickness, and wet weight in response to different drugs therapy. The drugs were administered parenterally in "fixed dosage" at a regular interval of 21-23 days. The survival rate was good with Betamethasone and Azathioprine that is 75%, whereas it was 16.66% in Folic acid treatment. But the mussels originating from the control site had the significant survival rate though the growth rate was average. Calcium treatment had shown a marked increment of shell thickness and luster. The culture was lasted for 160 days. The wet weight gain of mussels in all the treatments were significant, p<0.0001 whereas increment of shell thickness was significant only in treatment B (Calcium) and treatment D (Azathioprine), p<0.0001 but with regard to the increment of length of mussel, treatment E (Stanazolol) was not significant, p>0.05. The regression analysis was adopted to find out the coefficient of determination (R(2)=0.90, being the best) from the relationship between length and weight of mussels and to establish the LWR equation with condition factor k=W/L(b).

Thiopurines (azathioprine, 6-mercaptopurine and 6-thioguanine) are therapeutic compounds widely administered in the clinic for their multiple uses (autoimmune diseases, post-transplant immunosuppression and cancer). Despite these advantages, their therapeutic potential is limited by occasional adverse effects (myelotoxicity and hepatotoxicity) and by a relatively frequent lack of efficacy. Previous studies have demonstrated that azathioprine decreased the viability of rat hepatocytes. In order to investigate cytotoxic effects of thiopurines in human liver, we used primary human hepatocytes and a highly differentiated human hepatoma cell line, HepaRG, treated or not with azathioprine, 6-mercaptopurine and 6-thioguanine. In parallel, expression of the genes involved in the metabolism of thiopurines, glutathione synthesis and antioxidant defences was measured by quantitative PCR. We clearly demonstrate that human liver parenchymal cells were much less sensitive than rat hepatocytes to thiopurine treatments. The toxic effects appeared after 96 h of treatment while ATP depletion was observed after a 24 h incubation with azathioprine and 6-mercaptopurine. Toxic effects were more pronounced for azathioprine and 6-mercaptopurine, when compared to 6-thioguanine, and might explain glutathione synthesis and antioxidant enzyme induction only by these two drugs. Finally, we also demonstrate for the first time an up-regulation by azathioprine and 6-mercaptopurine of inosine monophosphate dehydrogenase which might have consequences on the de novo biosynthesis of guanine nucleotides and thiopurines metabolism.

Azathioprine (AZA) is a cytotoxic immunosuppressive drug used in the prevention of rejection in organ transplants and the treatment of auto-immune diseases. However, AZA is haemotoxic causing significant bone marrow depression. The present studies were to characterize the haemotoxicity of AZA in the female CD-1 mouse. In Experiment 1, a dose-ranging study, with AZA gavaged daily for 10 days, clinical evidence of toxicity was evident at 125 mg/kg and above. Experiment 2 was a dose-response study with AZA gavaged daily for 10 days at 40-120 mg/kg. At day 1 after the final dose, AZA induced a dose-related pancytopaenia, reduced femoral marrow cellularity, increases in serum levels of the cytokine fms-like tyrosine kinase 3 ligand, reduction in granulocyte-monocyte colony-forming units and erythroid colonies, and increased bone marrow apoptosis. Histology demonstrated hepatocyte hypertrophy, thymic atrophy, reduced splenic extramedullary haemopoiesis, and reduced cellularity of sternal bone marrow. In Experiment 3, AZA was dosed for 10 days at 100 mg/kg with autopsies at 1, 3, 9, 22, 29, 43 and 57 days postdosing. At 1, 3 and 9 days, haematological parameters reflected changes in Experiment 2. At 22/29 days, many blood parameters were returning towards normal; at 43/57 days, most parameters compared with controls. However, there was some evidence of a persistent (i.e. residual/late-stage) mild reduction in RBC and erythroid progenitor cell counts at day 43/57. We conclude that the CD-1 mouse provides an acceptable model for the haemotoxicity of AZA in man.

Objective: The critical dose of chloroquine/hydroxychloroquine leading to a maculopathy or generalised retinopathy remains undetermined. In the literature, 100 g is considered the dose at which regular vision checks should be performed. Generally, chloroquine is said to be more toxic than hydroxychloroquine. A young patient presenting with toxic maculopathy after 57 g of hydroxychloroquine and a daily dosage of 2 mg/kg body weight prompted us to retrospectively look at our patients examined in this respect over about 1 year., Methods: The data of patients who were examined because of chloroquine/hydroxychloroquine intake or a respective maculopathy/retinopathy were retrospectively analysed. The time period was January 2005 until March 2006. Retinal damage was defined by fundus changes and alteration of the multifocal electroretinogram (ERG)., Results: Twenty-one patients--18 women and three men--were examined. The mean age was 51 years (range 6-71). Five of the nine chloroquine-treated patients developed a maculopathy, and one of them developed an additional generalised retinopathy. Of the patients treated by hydroxychloroquine, three of 12 suffered from a maculopathy and one from an additional generalised retinopathy. The cumulative doses leading to retinal damage ranged from 170 g to 1650 g for chloroquine and from 57 g to 1190 g for hydroxychloroquine. The highest cumulative doses without leading to signs of retinopathy were 790 g for chloroquine and 1200 g for hydroxychloroquine., Conclusions: There is a high variability of cumulative doses of chloroquine/hydroxychloroquine that lead to a toxic retinopathy. Therefore, early and regular ophthalmologic examinations are recommended. Electrophysiological testing should be performed once a year, corresponding to about 60 g of base with one tablet a day. For electrophysiology, the multifocal ERG has turned out to be the most important test in this regard. However, visual acuity and funduscopy should be performed more frequently.

Background: The aim of the study was to evaluate the incidence of abnormality of liver tests (LTs) or hepatotoxicity in a large group of inflammatory bowel disease (IBD) patients and, specifically, to assess the incidence of azathioprine (AZA)/mercaptopurine (MP)-induced liver injury in a long-term follow-up study., Methods: All consecutive IBD patients followed for at least 5 years were included in this retrospective study. LTs including alanine transaminase, aspartate transaminase, alkaline phosphatase, gamma-glutamyl transferase, and bilirubin were periodically monitored. "Abnormality-of-LTs" was defined as LTs between N (upper limit of the normal range) and 2 N, and "liver injury/hepatotoxicity" as LTs>2 N., Results: A total of 786 patients were included, and 138 received AZA/MP; 120 patients (15%) and 39 (5%) presented abnormality of LTs or hepatotoxicity, respectively, during follow-up. The most frequent explanations were AZA/MP treatment and fatty liver disease. Among AZA/MP-treated patients (690 patient-years follow-up) the incidence of abnormal LTs and hepatotoxicity was, respectively, 7.1% and 2.6% per patient-year. Most patients spontaneously normalized LTs despite maintaining AZA/MP. These drugs were withdrawn due to hepatotoxicity (LTs>5 N and lack of decrease despite 50% dose reduction) in 3.6% of the patients and all of them normalized LTs., Conclusions: In IBD patients, AZA or MP treatment induces abnormality of LTs in a relatively high proportion of the cases, but the development of true hepatotoxicity/liver injury is exceptional. Moreover, most of the cases of thiopurine-induced hepatotoxicity in IBD patients are mild, and the abnormalities in LTs spontaneously return to normal values despite AZA/MP being maintained, therapy withdrawal being necessary in only approximately 4% of the patients.

This study aimed to evaluate the responses of human hepatocytes to azathioprine hepatotoxicity in comparison with the well-studied azathioprine hepatotoxicity in rat hepatocytes and the effects of protective agents to suppress azathioprine hepatotoxicity. Azathioprine presented its hepatotoxicity at clinically relevant concentrations (lower than 10 microm) in primary rat hepatocytes after 48 h of treatment as shown by a severe decrease in cell viability as well as intracellular GSH depletion. However, primary human hepatocytes exhibited only significant intracellular GSH depletion after treatment with azathioprine at these clinically relevant concentrations, while a reduction in cell viability by 29% was only evidenced after 48 h of treatment with azathioprine at the high concentration of 50 microm. In addition, a monolayer culture of primary rat hepatocytes was used as an in vitro model to examine the protective effects of antihepatotoxic drugs including glutathione (GSH), N-acetylcysteine (NAC, a GSH precursor), liquorice and glycyrrhizic acid (GA), a major bioactive component of liquorice, against hepatotoxicity of 1 microm azathioprine. It was found that both liquorice and GA showed substantial protection according to assays of cell viability and intracellular GSH, while neither GSH nor NAC had such a protective function. Similarly, GA protected human hepatocytes from intracellular GSH depletion on exposure to 1 microm azathioprine. These results implied that GA or liquorice could be considered as potent protection agents against azathioprine hepatotoxicity.

Background: We wished to investigate the toxicity of four immunosuppressant and antimetabolic drugs, which are known to influence postoperative wound healing, on three different human ocular cell lines., Methods: Acute toxicity to cyclosporin A, azathioprine, mitomicyn C and daunorubicin was assessed in Chang cells by monitoring their uptake of propidium iodide during a 3-h period. Chronic toxicity was assessed by monitoring the proliferation and viability of subconfluent cultures of Chang cells, human corneal endothelial cells (HCECs) and retinal pigmented epithelial (RPE) cells after continuous exposure to the drugs for 7 days., Results: Acute toxicity testing revealed no obvious effects. However, the chronic toxicity tests disclosed a narrow concentration range over which cell proliferation decreased dramatically but calcein metabolism was sustained. Although the three lines reacted similarly to each agent, HCECs were the most vulnerable to daunorubicin and mitomycin. At a daunorubicin concentration of 0.05 microg/ml, a 75% decrease in calcein metabolism (P < 0.001) and a > or = 95% cell loss (P < 0.001) were observed. At a mitomycin concentration of 0.01 mug/ml, cell density decreased by 61% (P < 0.001) without a change in calcein metabolism, but at 0.1 microg/ml, the latter parameter decreased to 12% (P = 0.00014). At this concentration the proliferation of Chang and RPE cells decreased by more than 50%, whilst calcein metabolism was largely sustained. Cyclosporin inhibited cell proliferation moderately at lower concentrations (< 5 microg/ml; P=0.05) and substantially at higher ones, with a corresponding decline in calcein metabolism. Azathioprine induced a profound decrease in both parameters at concentrations above 5 microg/ml., Conclusion: Daunorubicin, cyclosporin and azathioprine could be used to inhibit excessive intraocular scarring after glaucoma and vitreoretinal surgery without overly reducing cell viability. The attributes of immunosuppressants lie in their combined antiproliferative and immunomodulatory effects.

Many patients undergoing chronic therapy with the purine analogue Azathioprine (Aza) have highly elevated HPRT lymphocyte mutant frequencies (MFs), and it is likely that these increases are due to selection of pre-existing HPRT mutant lymphocytes. A similar selection in germ cells might result in an increased frequency of the Lesch-Nyhan syndrome. In this study, a mouse model for Aza mutant selection was developed and Aza toxicity was evaluated in the germ cells of treated mice. Groups of 20 male C57BL/6 mice were treated by gavage three times/week with 0, 5, 10, 25, 50, or 100mg/kg Aza, and three to eight mice from each group were sacrificed at various times for up to 23 weeks. Mice treated with 25-100mg/kg Aza were all dead by 14 weeks of treatment. Hprt lymphocyte MF assays indicated that the treated mice had reduced numbers of spleen lymphocytes. Most treated mice had Hprt MFs similar to those of control mice (2.1+/-1.6 x 10(-6)), however, highly elevated MFs were detected in one out of three mice given 5mg/kg for 10 weeks, one out of three mice given 10mg/kg for 10 weeks, and one out of eight mice given 10mg/kg for 23 weeks (e.g., 233 x 10(-6) after 10 weeks of 5mg/kg). Sequence analysis of Hprt cDNA indicated that all mutant clones from one of these mice had a T-->A transversion in the initiation codon. Multiplex-PCR on mutant clones from the other two mice indicated that all the clones from one had a deletion of Hprt exons 2 and 3, while most of the mutants from the other had lost all of the Hprt exons. Measurements of testicular weight, and of sperm count, viability, morphology, and motility found that Aza produced low levels of toxicity in sperm, with the most consistent effect being a reduction in the testicular weight. The data suggest that mice chronically treated with 5 and 10mg/kg Aza (doses similar to those used in humans) have elevated Hprt MFs due to clonal amplification of selected Hprt mutants. The results also suggest that mice treated with these doses of Aza retain reasonable fertility, and will be useful for breeding experiments to examine the possibility of increasing the germ-line transmission of Hprt mutations.

As an anti-metabolite, Azathioprine inhibits the de novo and salvage pathways of purine synthesis. Intraperitoneal injection of this drug results in not only lymphocyte suppression but also toxicity to bone marrow, gastrointestinal tract, and liver. This Azathioprine-induced hepatotoxicity was found to be associated with oxidative damage. Plants with antioxidative properties have been traditionally used to prevent diseases associated with free radicals. In this report, we used water extracts of three herbal plants that have been commonly used for treating many illnesses (Hibiscus sabdariffa, Rosmarinus officinalis and Salvia officinalis). Here we show their novel hepatoprotective effects against Azathioprine-induced hepatotoxicity in rats. Typically, administration of Azathioprine induces oxidative stress through depleting the activities of antioxidants and elevating the level of malonialdehyde in liver. This escalates levels of alanine aminotransferase, and aspartate aminotranferase in serum. Pretreatment with any of the three herbal plants used in this investigation proved to have a protective effect against Azathioprine-induced hepatotoxicity. Animals pretreated with water extracts from any of the three herbs under investigation not only failed to show necrosis of the liver after azathioprine administration, but also retained livers that, for the most part, were histologically normal. In addition, these herbs blocked the induced elevated levels of alanine aminotransferase and aspartate aminotranferase in serum. The Azathioprine-induced oxidative stress was relieved to varying degrees by the examined herbal extracts. This effect was evident through reducing malonialdehyde levels and releasing the inhibitory effect of Azathioprine on the activities of glutathione, catalase and superoxide dismutase. To our knowledge, this report is the first that shows hepatoprotective effects of Hibiscus, Rosmarinus and Salvia species against Azathioprine-induced acute liver damage.

Azathioprine (AZA) is an immunosuppressive prodrug that undergoes metabolism by thiopurine S-methyltransferase (TPMT). Eighty to ninety-five percent of low or deficient TPMT enzyme activity is genetically determined by the presence of three nonfunctional mutant alleles: TPMT*2, TPMT*3A and TPMT*3C. Using TPMT as a pharmacogenetic paradigm, we explored the association between these genetic mutations and development of adverse drug effects in an ethnically diverse renal transplant population receiving azathioprine. Biochemical and clinical data were retrospectively evaluated during the first four weeks after kidney transplantation. TPMT nonfunctional mutant alleles were identified by polymerase chain reaction-based methods. Of 89 patients initially consented, 36 met inclusion criteria for this retrospective study. Five patients possessing a single TPMT nonfunctional mutant allele were identified: TPMT*3A: n = 2 Caucasians; TPMT*3B: n = 1 Caucasian; TPMT*3C: n = 2 African-Americans. TPMT nonfunctional mutant alleles were associated with significant reductions in hematological indices and a significant increase in cyclosporine plasma concentrations in the first month post-transplant. TPMT genotype was an independent predictor for hemoglobin, hematocrit and red blood cell changes while mean azathioprine dose (mg/kg/day), azathioprine dose (mg/kg/day) at day 30 and cyclosporinemia at day 30 were not. Prospective application of pharmacogenetic principles may assist in optimization of immunosuppressive drug therapy and minimize drug toxicities.

The purpose of this study was to examine the predictive value of humoral and cellular immune parameters in determining the immunotoxic effects of the oral administration of azathioprine (AZA), cyclophosphamide (CY), or cyclosporin A (CsA) at doses of 25/17, 10, or 25 mg/kg per day, respectively, for 30 days in F344 female rats. The effect of these known immunosuppressive compounds on the immune response was assessed in a humoral model that consisted of the administration of nitrophenyl-chicken gamma globulin (NP-CgammaG) and keyhole limpet hemocyanin (KLH) antigens during immunosuppressive treatment and the measurement of resulting rat antigen-specific IgG and IgM, as well as total IgG, levels. Cellular assessment parameters were collected from the same groups of animals as the humoral parameters and included organ weights and cellularity, hematology, lymphocyte phenotype characteristics, spleen cell mitogen stimulation (T and B cell-dependent), splenic natural killer (NK) cell cytotoxicity, and bone marrow cellularity and lymphocyte phenotype differential. Although decreases in several of the cellular assay parameters were observed, the only functional assays to demonstrate a statistically significant immunosuppressive effect by all three immunosuppressive agents were the antigen-specific serum IgG levels. The primary (day 10; 15 days post-immunization) and secondary (day 25; 5 days post-rechallenge) nitrophenyl (NP) responses were significantly suppressed by > or =60%. The use of NP hapten provided consistent responses when analyzed with a sensitive, well developed, ELISA methodology. Absolute lymphocyte phenotyping and lymphocyte hematology were also predictive of T cell immunosuppression for all three compounds. The data presented herein suggests that these two parameters, NP-IgG humoral response and lymphocyte phenotyping, are sufficient for identifying immunosuppressive compounds.

Azathioprine (AZA) is an important drug used in the therapy of autoimmune system disorders. It induces hepatotoxicity that restricts its use. The rationale behind this study was the proven efficacy of N-acetylcysteine (NAC; a replenisher of sulfhydryls) and reports on the antioxidant potential of aminoguanidine (AG; an iNOS inhibitor), that might be useful to protect against the toxic implications of AZA. AG (100 mg/kg; i.p.) or NAC (100 mg/kg; i.p.) were administered to the Wistar male rats for 7 days and after that AZA (15 mg/kg, i.p.) was given as a single dose. This caused an increase in the activity of hepatic aminotransferases (AST and ALT) in the serum 24 h after AZA treatment. AZA (7.5 or 15 mg/kg, i.p.) also caused an increase in rat liver lipid peroxides and a lowering of reduced glutathione (GSH) contents. In the other part of experiment, protective effects of AG and NAC were observed on AZA induced hepatotoxicity. NAC significantly protected against the toxic effects produced by AZA. Pretreatment with NAC prevented any change in the activities of both the aminotransferases after AZA. This pretreatment also resulted in a significant decline in the contents of lipid peroxides and a significant elevation in GSH level was evident after AZA treatment. In the group with AG pretreatment the activities of AST and ALT did not increase significantly after AZA when compared to control. However, the lipid peroxides and GSH levels did not have any significant difference when compared to AZA group. These observations also indicate that the improvement in the GSH levels by NAC is the most significant protective mechanism rather than any other mechanistic profile. The protective effect of AG against the enzyme leakage seems to be through the liver cell membrane permeability restoration and is independent of any effects on liver GSH contents.

Comparative study of disturbances of intramural duodenal lymphoid tissue in mice, which were induced by the action of "classical" immunosuppressing drug azathioprine and by herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) showed similar immunosuppressing effect that was more potent after herbicide treatment. Significant reduction in the number of the cells of lymphoid series found in the duodenal wall was accompanied by inflammatory-destructive processes and adipose tissue outgrowth. Administration of hypolipidemic plant extract to animals immunosuppressed with either azathioprine or herbicide 2,4-D, promoted the restoration of intramural duodenal morphological structures and the restitution of lymphoid cell parameters in lamina propria practically to the level of control values. The results obtained indicate the immuno-correcting effect of the plant extract, which is determined by a rich complex of biologically active substances of its components. Active thymic fraction (AFT-2) was also found to possess an immuno-correcting effect, promoting the restoration of cellular composition of diffuse lymphoid tissue in duodenal lamina propria. Immuno-correcting effect of AFT-2 seems to result from the activity of its component thymic peptides.

Azathioprine (AZA) is used clinically sometimes at high doses for short-term therapy to treat acute rejection of kidney allograft or to desensitize hypersensitive patients to it. The delayed consequences of this approach had not been well investigated. Therefore, in this study we have investigated the delayed consequences of high-dose short-term AZA administration in rabbits. Our results showed that oral administration of AZA (10 mg/kg/day) to rabbits for two weeks induced reversible thrombocytosis and delayed fatal macrocytic anemia. Moreover, neither the hemoglobin level nor the white blood cell count was affected by AZA. The solvent of AZA had no effect on blood cell counts and hemoglobin levels. We can conclude that although high-dose AZA therapy may not induce immediate and significant changes in blood picture, delayed fatal macrocytosis may occur.

Modulatory effect of quercetin on azathioprine induced toxic changes was studied in spleen of experimental animals. Azathioprine treatment caused an increase in serum albumin/globin ratio and a decrease in total protein in spleen tissue. An increase in a membrane bound ATPases was also noted. Supplementation of quercetin with azathioprine increased the protein content and lowered the activities of membrane ATPase in spleen. There was a decrease in serum albumin globulin ratio. It was concluded that quercetin modulated the protein and membrane bound ATPase activities and protected the spleen from azathioprine induced membrane damage.

Antimetabolites such as methotrexete and 6-mercaptopurine have been shown to have circadian variations in their toxicities. However, chronopharmacological profiles of mizoribine (Miz) that is newly synthesized as an anti-metabolic agent for immunosuppression, have not been evaluated. In this study, we examined the dosing time-dependent alterations in the pharmacokinetics and pharmacodynamics of Miz. In addition, chronopharmacology of azathiopurine (Aza) was also evaluated to compare with that of Miz. Initially, Miz (10 and 20 mg/kg) or Aza (20 mg/kg) was orally administered at 8:00 hr or 20:00 hr for 3 weeks to rats. To reveal the dosing time-dependent difference of pharmacokinetics, Miz (20 mg/kg) was orally given at 8:00 hr or 20:00 hr and blood was obtained for 12 hours. Finally, Miz (20 mg/kg) or Aza (20 mg/kg) was administered at 8:00 hr or 20:00 hr to rats with heterotopic allogeneic heart grafts. The Miz group treated at 8:00 hr and Aza group treated at 20:00 hr showed severe myelosuppression compared with their each opposite dosing time. AUC of Miz in the morning trial was twice as high as that in the evening trial. The graft survival durations of the Miz- and Aza-treated groups were significantly longer than those of the respective control groups, but were not affected by dosing time of each agent. These results suggest that the toxicity, but not efficacy of Miz is varied with the dosing time. The chronotoxicological phenomenon of Miz might be, at least in part, explained by the dosing time-dependent difference in serum drug concentrations and apparent clearance.

The influence of liposomal preparation obtained from the Baikal seal fat on lymphoid structures in mouse coecum wall on the background of immunodepressive state induced by asathioprine was studied using microscopic methods and statistic analysis. It was demonstrated that asaphioprine administration caused decline in lymphoid serie cells, outgrowth of adipose tissue, causes destructive changes of lymphoid structures in the coecal wall. After the effect of liposomes on the background on the immune suppression morphological state of lymphoid structures was approximated to the normal state while the indexes of cellular composition returned to the level of control values which was conditioned by immunocorrecting properties of the drug studied.

Azathioprine (AZA) is used in the treatment of patients with refractory inflammatory bowel disease; however, its use is limited because of systemic toxicity associated with long-term use. Ileocecal delivery of AZA might be advantageous if local intestinal therapeutic effects could be provided with decreased systemic side effects. Decreased cecal systemic absorption would allow higher dosages of AZA to be administered. A two-phase study was performed to compare the systemic exposure of AZA and 6-mercaptopurine (6-MP) following administration of AZA into the stomach, jejunum, and cecum and to compare the systemic exposure to AZA and 6-MP following administration of three different dosages of AZA into the cecum. In phase I, six healthy male volunteers received three 50 mg sequential doses of AZA via an oral tube directly placed into the stomach, jejunum, and cecum, respectively. In phase II, six healthy male volunteers received three different dosages (50, 300, 600 mg of AZA) into the cecum. Plasma concentrations of AZA and 6-MP at various times were quantified and area under the plasma concentration-time curve (AUC) and mean residence time (MRT) were determined. No significant differences in the AUC of AZA were seen at the different sites. The AUC of 6-MP following administration of AZA into the jejunum (67.0 +/- 30.1 ng x hr/ml) was higher compared to the stomach (39.9 +/- 38.1 ng/hr/ml) and cecum (29.2 +/- 10.9 ng x hr/ml). Jejunal absorption was 68% higher than absorption from the stomach and 129% higher than that of the cecum. Gastric absorption was 27% higher than that of the cecum. Increased dosages given into the cecum resulted in increased AUCs of AZA and 6-MP. The AUCs of AZA following 50, 300, and 600 mg dosages were 16.9 +/- 7.4, 52.3 +/- 67.2, and 132 +/- 151 ng x hr/ml, respectively, and the AUCs of 6-MP were 22.2 +/- 14.9, 63.4 +/- 50.6, and 104 +/- 115 ng x hr/ml, respectively. Systemic exposure to 6-MP is reduced following administration of AZA into the cecum, most likely secondary to reduced absorption of 6-MP from the colon. Higher dosages of AZA presented to the cecum do result in increased systemic absorption, but may still allow more drug to be administered with less toxicity than the same dose received orally.

Azathioprine, an anti-neoplastic drug and therapeutic immunosuppressant, was administered intraperitoneally at 10.0 and 50.0 mg/kg to 3-6-month-old tilapia (Oreochromis niloticus). Consistent alterations in immune cellular parameters of the blood, pronephros (hematopoietic kidney) and spleen were observed. Peripheral blood total cellularity decreased as the azathioprine dose increased, to approximately half that of the control. Differential analysis of white blood cells indicated a decline in lymphocyte number, in particular, with increased dosage of azathioprine. Pronephric total cellularity was depressed in fish receiving the 10.0 or 50.0 mg/kg dose. In contrast, both splenic weight and splenic total cellularity increased proportionately with the increase in the drug dosage. Histopathologic examination of the spleens showed normal patterns for both control and 10.0 mg/kg dose groups. At 50.0 mg/kg, spleens were characterized by marked expansion of the white pulp, although lymphocytes were rare. Melanomacrophage centers at the higher dose were also larger and more numerous than in the control group. Evaluation of splenic and pronephric leukocytes with apoptotic markers showed an increase in apoptotic cells in the pronephros with increasing drug dose. These changes in fish are consistent with those seen in humans and laboratory rodents dosed with azathioprine, suggesting that fish may be potentially useful as preliminary models for detecting immunosuppressive compounds.