Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring.

Folate is an essential vitamin for vertebrate embryo development. Methotrexate (MTX) is a folate antagonist that is widely prescribed for autoimmune diseases, blood and solid organ malignancies, and dermatologic diseases. Although it is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, the effect of paternal MTX exposure on their offspring has been largely unexplored. Here, we found MTX treatment of adult medaka male fish (Oryzias latipes) causes cranial cartilage defects in their offspring. Small non-coding RNA (sncRNAs) sequencing in the sperm of MTX treated males identify differential expression of a subset of tRNAs, with higher abundance for specific 5' tRNA halves. Sperm RNA methylation analysis on MTX treated males shows that m5C is the most abundant and differential modification found in RNAs ranging in size from 50 to 90 nucleotides, predominantly tRNAs, and that it correlates with greater testicular Dnmt2 methyltransferase expression. Injection of sperm small RNA fractions from MTX-treated males into normal fertilized eggs generated cranial cartilage defects in the offspring. Overall, our data suggest that paternal MTX exposure alters sperm sncRNAs expression and modifications that may contribute to developmental defects in their offspring.

Chronobiology in nephrology: the influence of circadian rhythms on renal handling of drugs and renal disease treatment.

INTRODUCTION: Chronobiology studies the phenomenon of rhythmicity in living organisms. The circadian rhythms are genetically determined and regulated by external synchronizers (the daylight cycle). Several biological processes involved in the pharmacokinetics and pharmacodynamics of drugs are subjected to circadian variations. Chronopharmacology studies how biological rhythms influence pharmacokinetics, pharmacodynamics, and toxicity, and determines whether time-of-day administration modifies the pharmacological characteristics of the drug. Chronotherapy applies chronopharmacological studies to clinical treatments, determining the best biological time for dosing: when the beneficial effects are maximal and the incidence and/or intensity of related side effects and toxicity are minimal. Most water-soluble drugs or drug metabolites are eliminated by urine through the kidney. The rate of drug clearance in the urine depends on several intrinsic variables related to renal function including renal blood flow, glomerular filtration rate, the ability of the kidney to reabsorb or to secrete drugs, urine flow, and urine pH, which influences the degree of urine acidification. Curiously, all these variables present a circadian behavior in different mammalian models. CONCLUSION: The circadian rhythms have influence in the renal physiology, pathophysiology, and pharmacology, and these data should be taken into account in clinical nephrology practice.

G80A Single Nucleotide Polymorphism in Reduced Folate Carrier-1 Gene in a Mexican Population and its Impact on Survival in Patients with Acute Lymphoblastic Leukemia.

BACKGROUND: Hyper-CVAD is the treatment for patients with acute lymphoblastic leukemia in our institution. OBJECTIVE: To evaluate the impact of single nucleotide polymorphisms at genes associated with methotrexate metabolism on survival. METHODS: The presence of the single nucleotide polymorphisms G80A at reduced folate carrier-1 gene and C677T in the methylenetetrahydrofolate reductase gene was determined by denaturing high performance liquid chromatography and validated by sequencing. Both single nucleotide polymorphisms were evaluated in 71 healthy donors and in an exploratory pilot trial with acute lymphoblastic leukemia patients to determine the influence of these single nucleotide polymorphisms on clinical outcome. Clinical characteristics, response, and outcome were registered. A Cox regression analysis was done to evaluate factors influencing response and overall survival. RESULTS: There were no differences in the frequency of single nucleotide polymorphisms between volunteers and acute lymphoblastic leukemia patients according to the Hardy-Weinberg test. Sensitivity and specificity were 72 and 91% for the G80A, and 64 and 75% for the C677T, respectively. The multivariate analysis showed that the T-immunophenotype and the presence of single nucleotide polymorphism G80A reduced folate carrier-1 were associated with a shorter relapse-free survival and overall survival. CONCLUSIONS: The presence of G80A single nucleotide polymorphism at reduced folate carrier-1 gene in acute lymphoblastic leukemia patients was associated with a poorer prognosis.

PAMAM-grafted TiO2 nanotubes as novel versatile materials for drug delivery applications.

PAMAM-grafted TiO2 nanotubes (PAMAM-TiO2NT) have been synthesized and evaluated as new drug nanocarriers, using curcumin (CUR), methotrexate (MTX), and silibinin (SIL) as model therapeutic compounds. TiO2NT were surface-modified using a silane coupling agent and subsequently conjugated with PAMAM dendrimer of the third generation. The characterization of PAMAM-TiO2NT nanomaterials was performed by FTIR, TEM, N2 adsorption-desorption isotherms, XRD, and TGA techniques, which accounted for a 2.6wt.% of PAMAM grafting in the prepared materials. The drug loading capacity, drug release properties, and cytotoxicity of PAMAM-TiO2NT showed a significant improvement compared to pristine TiO2NT, thus revealing the promising properties of these new materials for drug delivery purposes.

Polimorfismo do gene timidilato sintase e nível plasmático total de homocisteína em um grupo de pacientes turcos com artrite reumatoide: relação com a atividade da doença e toxicidade ao metotrexato / Thymidylate synthase genetic polymorphism and plasma total homocysteine level in a group of Turkish patients with rheumatoid arthritis: relationship with disease activity and methotrexate toxicity

Resumo Introdução: Relata-se que o polimorfismo do gene timidilato sintase (TS) e a homocisteína têm relação com o metabolismo do metotrexato (MTX), com achados conflitantes. O objetivo deste estudo foi determinar os níveis de homocisteína e a frequência de polimorfismos de repetição tripla (TS3R) e dupla (TS2R) do gene TS em um grupo de pacientes turcos com AR e avaliar sua associação com a toxicidade ao MTX e a atividade da doença. Métodos: Foram incluídos no estudo 64 pacientes com AR e 31 indivíduos no grupo controle, com média de 48,7 ± 12,5 e 46,2 ± 13,4 anos. Foram obtidas as características demográficas e foi registrado o número de pacientes que relataram efeitos adversos ao MTX no grupo AR. Foram analisados os níveis de homocisteína e os polimorfismos TS2R/TS3R. Foi determinada a distribuição de genótipos de acordo com a toxicidade ao MTX e a atividade da doença. Resultados: Os dados demográficos foram semelhantes entre os pacientes e controles. Todos faziam suplementação de ácido fólico a uma dose média de 5 mg/semana. Dos 64 pacientes, 36 apresentaram efeitos adversos ao tratamento com MTX. Encontrou-se uma frequência de polimorfismos TS2R e TS3R semelhante nos grupos AR e controle. Encontrou-se que os polimorfismos TS2R e TS3R eram semelhantes em pacientes com e sem eventos adversos relacionados com o MTX. O nível médio de homocisteína também foi similar em pacientes com e sem polimorfismo do gene TS, mas era mais elevado (12,45 μmol/L vs. 10,7 μmol/L) em pacientes com do que sem efeitos adversos relacionados com o MTX. O nível médio de homocisteína se correlacionou com o VHS no grupo AR. Conclusões: Os níveis de homocisteína podem afetar a atividade da doença e a toxicidade ao MTX, mas os polimorfismos 2 R e 3 R no gene TS não se correlacionaram com a toxicidade ao MTX em pacientes com AR que recebem suplementação de ácido fólico. São necessários mais estudos para esclarecer os polimorfismos em outras enzimas que podem ser responsáveis pela toxicidade ao MTX em pacientes com AR.

Abstract Background: The polymorphism of thymidylate synthase (TS) gene and homocysteine are reported to have a relationship to methotrexate (MTX) metabolism, with conflicting results. The aim of this study was to determine homocysteine levels and the frequency of TS gene triple repeat (TS3R) and double repeat (TS2R) polymorphisms in a group of Turkish RA patients and evaluate its association with MTX toxicity and disease activity. Methods: Sixty-four patients with RA and 31 control subjects with a mean age of 48.7 ± 12.5 and 46.2 ± 13.4 years were enrolled for the study. Demographic characteristics were obtained and a number of patients with MTX-related adverse affects were recorded in the patient group. The homocysteine levels and TS2R/TS3R polymorphisms of the TS gene were analyzed and the distribution of genotypes according to MTX toxicity and disease activity was determined. Results: The demographic properties were similar between the patient and control subjects. Folic acid supplementation with a mean dose of 5 mg folic acid/week was present in all patients. Thirty-six of the 64 patients showed adverse effects to MTX treatment. The respective frequency of TS2R and TS3R polymorphisms was found to be similar in the patient and control groups. TS2R and TS3R gene polymorphisms were found to be similar in patients with and without MTX-related adverse events. The mean homocysteine level was also similar in patients with and without TS gene polymorphism, but was found to be higher (12.45 μmol/L vs 10.7 μmol/L) in patients with MTX-related side effects than in patients without side effects. The mean level of homocysteine was correlated with levels of ESR in the patient group. Conclusions: In conclusion, homocysteine levels might affect the disease activity and toxicity of MTX but 2R and 3R polymorphisms in the TS gene were not related with MTX-related toxicity in RA patients receiving folate supplementation. Further studies are needed to illuminate the polymorphisms in other enzymes that might be responsible for the MTX toxicity in patients suffering from RA.

Adenosine: an endogenous mediator in the pathogenesis of psoriasis

Abstract: It is known that inflammatory and immune responses protect us from the invasion of micro-organisms and eliminate "wastes" from the injured sites, but they may also be responsible for significant tissue damage. Adenosine, as a purine nucleoside, which is produced in inflamed or injured sites, fulfills its role in limiting tissue damage. Although, it may have a pleiotropic effect, which signals it with a proinflammatory state in certain situations, it can be considered a potent anti-inflammatory mediator. The effects of adenosine, which acts through its receptors on T cell, on mast cell and macrophages, on endothelial cells, on neutrophils and dendritic cells, as they indicate TNF-alpha and cytokines, show that this mediator has a central role in the pathogenesis of psoriasis. The way it acts in psoriasis will be reviewed in this study.

Thymidylate synthase genetic polymorphism and plasma total homocysteine level in a group of Turkish patients with rheumatoid arthritis: relationship with disease activity and methotrexate toxicity.

BACKGROUND: The polymorphism of thymidylate synthase (TS) gene and homocysteine are reported to have a relationship to methotrexate (MTX) metabolism, with conflicting results. The aim of this study was to determine homocysteine levels and the frequency of TS gene triple repeat (TS3R) and double repeat (TS2R) polymorphisms in a group of Turkish RA patients and evaluate its association with MTX toxicity and disease activity. METHODS: Sixty-four patients with RA and 31 control subjects with a mean age of 48.7 ± 12.5 and 46.2 ± 13.4 years, were enrolled to the study. Demographic characteristics were obtained and number of patients with MTX-related adverse affects, were recorded in the patient group. The homocysteine levels and TS2R/TS3R polymorphisms of the TS gene were analyzed and the distribution of genotypes according to MTX toxicity and disease activity, were determined. RESULTS: The demographic properties were similar between the patient and control subjects. Folic acid supplementation with a mean dose of 5mg folic acid/week, was present in all patients. Thirty-six of the 64 patients showed adverse effects to MTX treatment. The frequency of TS2R and TS3R polymorphisms were found to be similar in the patient and control groups. TS2R and TS3R gene polymorphisms were found to be similar in patients with and without MTX-related adverse events. The mean homocysteine level was also similar in patients with and without TS gene polymorphism, but was found to be higher (12.45µmol/L vs 10.7µmol/L) in patients with MTX-related side effects than in patients without side effects. The mean level of homocysteine was correlated with levels of ESR in the patient group. CONCLUSIONS: In conclusion, homocysteine levels might effect the disease activity and toxicity of MTX but 2R and 3R polymorphisms in the TS gene, were not related with MTX-related toxicity in RA patients receiving folate supplementation. Further studies are needed to illuminate the polymorphisms in other enzymes that might be responsible from the MTX toxicity in patients suffering from RA.

Adenosine: an endogenous mediator in the pathogenesis of psoriasis.

It is known that inflammatory and immune responses protect us from the invasion of micro-organisms and eliminate "wastes" from the injured sites, but they may also be responsible for significant tissue damage. Adenosine, as a purine nucleoside, which is produced in inflamed or injured sites, fulfills its role in limiting tissue damage. Although, it may have a pleiotropic effect, which signals it with a proinflammatory state in certain situations, it can be considered a potent anti-inflammatory mediator. The effects of adenosine, which acts through its receptors on T cell, on mast cell and macrophages, on endothelial cells, on neutrophils and dendritic cells, as they indicate TNF-alpha and cytokines, show that this mediator has a central role in the pathogenesis of psoriasis. The way it acts in psoriasis will be reviewed in this study.

Histological and histochemical studies on the effects of methotrexate on the liver of adult male albino rat / Estudios Histológico e Histoquímico del Efecto del Metotrexato en el Hígado de Rata Macho Albina Adulta

El metotrexato es ampliamente usado en la terapia de varias enfermedades malignas. El presente trabajo fue diseñado para investigar los cambios histológicos e histoquímicos del hígado de rata albina, después de administrar dicho fármaco. Se usaron 15 ratas albinas, machos, adultas, que fueron divididas en 3 grupos: El grupo I no tuvo tratamiento correspondiendo al control. A los grupos II y III se les administró, por vía intraperitoneal, una solución salina normal y metotrexato, respectivamente, con una dosificación de 0,5 mg por Kg de peso, dos veces por semana, con una duración total de 3, 6 y 9 semanas. Las ratas fueron sacrificadas y los hígados extraídos y procesados para los estudios histológico e histoquímico. El examen de los hígados del grupo III mostró infiltración celular mononuclear y un incremento en la cantidad de fibras colágenas en la vía portal. Hubo áreas focales de necrosis de células hepáticas con distorsión de la arquitectura hepática normal. Además, hubo un gradual y progresivo decrecimiento del contenido de glicógeno en los hepatocitos. La actividad de deshidrogenasa succínica y fosfatas alcalinas también disminuyó, pero sí hubo un aumento de la actividad de las fosfatasas ácidas en las áreas degeneradas y pérdida de actividades en áreas de necrosis celular masiva. En conclusión, inyecciones repetidas de metotrexato causan daño hepático de maginitud definida. Esta hepatotoxicidad progresó a medida que las dosis se fueron acumulando. El presente estudio muestra evidencias claras de la potencia citotóxica de este medicamento.

Methotrexate (MTX) is widely used in the therapy of various types of malignancy. The present work was designed to investigate the histological and histochemical changes in the liver of albino rat following methotrexate administration. Fifteen adult male albino rats were used in the present work. They were divided into three main groups: Group I was kept without treatment and served as control. Groups II and III were given intraperitoneal injections of normal saline and MTX, respectively, at a dosage of (0.5 mg/Kg) twice weekly for total durations of 3, 6 and 9 weeks. The rats were sacrificed and the livers were excised and processed for histological and histochemical study. Examination of sections of the livers of group III showed mononuclear cell infiltration and an increase in the amount of collagen fibers in the portal tracts. There were focal areas of liver cell necrosis with distortion of the normal hepatic architecture. Moreover, there was a gradual and progressive decrease of glycogen content in the hepatocytes. Furthermore, succinic dehydrogenase and alkaline phosphatase activity were also decreased. In addition there was an increase in acid phosphatase activities in the degenerated areas and loss of activities in areas of massive cellular necrosis. It was concluded that repeated injections of MTX causes hepatic damage of a definite magnitude. This hepatotoxicity progressed with increasing cumulative doses of methotrexate. The present study provided further evidence to the cytotoxic potency of this antifolate.

Methotrexate bioavailability after oral and intramuscular administration in children.

Although methotrexate is one of the most commonly used drugs for maintenance therapy in childhood acute lymphocytic leukemia (ALL), its oral absorption is highly variable and its intramuscular bioavailability at dosages used for ALL therapy has not been assessed in children. We therefore determined the absolute bioavailability of orally and intramuscularly administered methotrexate in 12 pediatric patients receiving 13 to 120 mg/m2 methotrexate every week as maintenance therapy for ALL. Mean bioavailability, as determined by comparing the area under the concentration-time curve after oral or intramuscular administration with that produced by the same dosage given intravenously, was 33% (range 13% to 76%) for oral (n = 11) and 76% (54% to 112%) for intramuscular (n = 7) administration (P less than 0.01). Median bioavailability (with orally administered dosages less than or equal to 40 mg/m2 (range 13 to 40 mg/m2) was 42% (19% to 76%); at dosages greater than 40 mg/m2 (43 to 76 mg/m2), bioavailability was significantly lower, 17.5% (12.7% to 22.3%, p less than 0.02). Conversely, there was no significant relationship between dosage and bioavailability with intramuscularly administered drug. The substantially higher bioavailability for intramuscularly injected methotrexate may warrant its consideration as an alternative to oral administration, especially for dosages greater than 40 mg/m2.