This study investigated whether a polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene (C677T) modifies responses to methotrexate (MTX) in patients undergoing bone marrow transplantation. About 10% to 12% of the population carry the MTHFR TT genotype (enzyme activity, 30% of wild type [CC]). Patients (n ‫؍‬ 220) with chronic myelogenous leukemia underwent marrow allografts and were given a short course of MTX. MTX toxicity measures included the oral mucositis index (OMI), speed of engraftment (platelet and granulocyte counts), and bilirubin. Patients with lower MTHFR activity (TT genotype) had 36% higher mean OMI during days 1 to 18 (؉5.7, P ‫؍‬ .046) and 20% higher OMI between days 6 and 12 (؉3.8, P ‫؍‬ .27). Platelet counts recovered more slowly among patients with the TT genotype compared to wild type (24% slower recovery to 10 000 platelets/L, P ‫؍‬ .23; 34% slower to 20 000/ L, P ‫؍‬ .08 Folate is essential for nucleotide synthesis. The effectiveness of MTX is largely attributable to its role as an inhibitor of dihydrofolate reductase. Its metabolites also inhibit other folate enzymes, including 5,10-methylenetetrahydrofolate reductase (MTHFR), 1,3,4 which converts 5,10-methylenetetrahydrofolate to 5,10-methyltetrahydrofolate.A common MTHFR polymorphism (C677T) results in reduced activity. 5 The variant TT genotype, associated with about 30% of wild-type (CC) activity, is present in about 10% to 12% of white and Asian populations. Heterozygotes (CT) (about 60% activity) constitute approximately 40% of the population. Variations are seen in risk of acute lymphocytic leukemia, 6 colorectal neoplasia, 7-10 neural tube defects, 11,12 and possibly cardiovascular disease, 13 largely in the presence of low folate levels. Because MTX induces a low folate state, we hypothesized that toxicity would be aggravated among patients with the TT and, possibly, the CT genotype. Study design Study design and patient populationWe undertook a study of patients undergoing marrow transplantation at the Fred Hutchinson Cancer Research Center (FHCRC) from 1992 to 1999.The following criteria applied: (1) first chronic phase of chronic myelogenous leukemia 14 ; (2) first transplantation; (3) conditioning regimen: either busulfan/cyclophosphamide 15 or cyclophosphamide/total body irradiation, as described 15,16 ; (4) age at transplantation, at least 18 years; and (5) available DNA. All patients received 4 doses of MTX (intravenously, day 1, at 15 mg/m, 2 and days 3, 6, and 11, at 10 mg/m 2 ) and cyclosporine (as previously described 15 ) for prevention of GVHD. 2 All patients gave informed consent. The study was approved by the FHCRC Institutional Review Board. Data collectionData were abstracted by a single abstractor (blinded to genotypes) on previous interferon treatment, smoking, MTX administration, and, if applicable, MTX serum levels and leucovorin administration.Data from the patient database included: (1) conditioning regimen; (2) donor/matching status; (3) demographics; (4) weight, height, and calculated ...

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Acetaminophen overdose is a leading cause of drug-related acute liver failure in the United States. Glutathione, a tripeptide antioxidant protects cells against oxidative damage from reactive oxygen species and plays a crucial role in the detoxification of xenobiotics, including acetaminophen. Glutathione is synthesized in a two-step enzymatic reaction. Glutamate-cysteine ligase carries out the rate-limiting and first step in glutathione synthesis. We have generated C57Bl/6 mice that conditionally overexpress glutamate-cysteine ligase, and report here their resistance to acetaminophen-induced liver injury. Indices of liver injury included histopathology and serum alanine aminotransferase activity. Male transgenic mice induced to overexpress glutamate-cysteine ligase exhibited resistance to acetaminophen-induced liver injury when compared with acetaminophen-treated male mice carrying, but not expressing glutamate-cysteine ligase transgenes, or to female glutamatecysteine ligase transgenic mice. We conclude that glutamatecysteine ligase activity is an important factor in determining acetaminophen-induced liver injury in C57Bl/6 male mice. Because people are known to vary in their glutamate-cysteine ligase activity, this enzyme may also be an important determinant of sensitivity to acetaminophen-induced liver injury in humans.The tripeptide antioxidant glutathione (GSH; ␥-glutamylcysteinylglycine) is one of the most abundant cellular thiols. It protects cells against oxidative damage from reactive oxygen species, maintains cellular redox status, promotes cell growth, and plays a crucial role in the detoxification of xenobiotics. GSH can directly scavenge free radicals, act as an antioxidant in GSH-mediated reduction of peroxides and act as a co-substrate for glutathione S-transferase-mediated detoxification of reactive intermediates formed during phase I metabolism (1).GSH plays a major role in detoxifying many hepatotoxicants including acetaminophen (APAP), 3 an over-the-counter analgesic and antipyretic (2-5). APAP overdose is responsible for nearly 50% of the acute liver failure cases in the United States (6) and is thus of high public health concern. APAP metabolism has been well defined, making it a good model for drug-induced liver toxicity. APAP is primarily metabolized through sulfation and glucuronidation pathways (7-9). However, a fraction of APAP is bioactivated by cytochrome P-450s to n-acetyl-p-benzoquinoneimine (NAPQI), which can bind to cellular proteins (3, 7, 10 -12). NAPQI also covalently binds to GSH and is either converted back to APAP, or forms the non-toxic APAP-GSH conjugate. APAP overdose results in depletion of hepatic GSH (by as much as 90%) (2). As GSH stores are depleted, increased levels of NAPQI-protein adducts form, and such adducts are thought to be an important contributor to APAP hepatotoxicity (3,7,8,10,11).Pretreatment with N-acetylcysteine (7, 9, 10), a source of cysteine for GSH biosynthesis, attenuates APAP-induced hepatotoxicity. N-Acetylcysteine given soon after APAP (within...

Background: Chronic progressive lung disease is the most serious complication of cystic fibrosis (CF). Glutathione plays an important role in the protection of the CF lung against oxidant-induced lung injury. Objectives: We hypothesized that a polymorphism in a novel candidate gene that regulates glutathione synthesis might influence CF lung disease. Methods: In a cross-sectional study, subjects were recruited from CF clinics in Seattle and multiple centers in Canada. We tested for an association between CF lung disease and a functional polymorphism in the glutamate-cysteine ligase catalytic subunit (GCLC) gene. Multiple linear regression was used to test for association between polymorphisms of GCLC and severity of CF lung disease while adjusting for age, Pseudomonas aeruginosa infection, and cystic fibrosis transmembrane conductance regulator (CFTR) genotype. Analysis was repeated for patients with CF stratified by CFTR genotype. Measurements and Main Results