Low androgen status inhibits erectile function by inducing eNOS uncoupling in rat corpus cavernosum.

BACKGROUND: The number of erectile dysfunction (ED) patients is increasing annually. How to improve the effectiveness of ED treatment is an important issue for the field of andrology. OBJECTIVES: To investigate whether low androgen status impairs the erectile function of rats by regulated endothelial nitric oxide synthase (eNOS) uncoupling. MATERIALS AND METHODS: Thirty-six 8-week-old male Sprague Dawley (SD) rats were randomly divided into six groups as follows: 4-week sham-operated group (4w-sham), 4-week castration group (4w-cast), 4-week castration + testosterone (T) group (4w-cast + T), 8-week sham-operated group (8w-sham), 8-week castration group (8w-cast), and 8-week castration + T group (8w-cast + T). Three mg/kg of T was subcutaneously injected every other day in castration + T groups. The ratio of the maximum intracavernous pressure/the mean arterial pressure (ICPmax/MAP), the level of serum T, dihydrobiopterin(BH2 ), tetrahydrobiopterin (BH4 ), nitric oxygen(NO), 3-nitrotyrosine(3NT), dihydrofolate reductase (DHFR), guanosine triphosphate cyclohydrolase 1 (GTPCH1), nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), and eNOS monomers/dimers in the corpus cavernosum were detected. RESULTS: The ratio of ICPmax/MAP and BH4 /BH2 , the level of serum T, NO, and GTPCH1 decreased significantly in castration groups compared with sham-operated groups and castration + T groups (P < .05) and decreased significantly in 8w-cast group compared with 4w-cast group (P < .05). The expression of 3NT and NOX2 and the ratio of eNOS monomers/dimers increased significantly in castration groups compared with sham-operated groups and castration + T groups (P < .01) and increased significantly in 8w-cast group compared with 4w-cast group (P < .01). The expression of DHFR in 4w-cast group was significantly higher than that in 4w-sham group and 4w-cast + T group (P < .01) and in 8w-cast group was significantly lower than that in 8w-sham group and 8w-cast + T group (P < .01). DISCUSSION AND CONCLUSION: Low androgen status induces eNOS uncoupling by reducing BH4 /BH2 and increasing 3NT. Due to the decreased NO production, the erectile function of the rats was impaired.

Plasma B-vitamins and one-carbon metabolites and the risk of breast cancer in younger women.

PURPOSE: We examined the association of plasma B-vitamins and metabolites, and related genetic variants, with risk of breast cancer among predominantly premenopausal women. METHODS: We conducted a nested case-control study within the Nurses' Health Study II. From blood samples collected in 1996-1999 and follow-up through 2007, plasma measures were available for 610 cases and 1207 controls. Unconditional multivariable logistic regression was used to estimate relative risks (RR) of breast cancer and 95% confidence intervals (CIs). We examined whether associations varied by methylenetetrahydrofolate reductase (MTHFR) and dihydrofolate reductase polymorphisms, breast cancer risk factors, or tumor characteristics. RESULTS: Plasma vitamin B12 was associated with a 64% higher risk of breast cancer comparing the highest versus lowest quintile (95% CI 1.17-2.29, p-trend = 0.02). Plasma folate (comparable RR = 1.18, 95% CI 0.84-1.66), pyridoxal 5'-phosphate (RR = 1.18, 95% CI 0.85-1.64), homocysteine (RR = 0.93, 95% CI 0.67-1.28), cysteine (RR = 1.14, 95% CI 0.81-1.62), and cysteinylglycine (RR = 0.93, 95% CI 0.66-1.31) were not associated with overall breast cancer risk. Folate was significantly positively associated with invasive and estrogen receptor-positive/progesterone receptor-positive breast cancer, and this association was suggestively stronger for bloods collected post-fortification. Several nutrient/breast cancer associations varied across subgroups defined by age, smoking, alcohol, multivitamin use, and MTHFR status (p-interaction < 0.05). CONCLUSIONS: Overall, plasma B-vitamins and metabolites were not associated with lower breast cancer risk. Plasma vitamin B-12 was positively associated with higher risk of overall breast cancer, and plasma folate was positively associated with risk of invasive breast cancer. Additionally, there may be associations in subgroups defined by related genetic variants, breast cancer risk factors, and tumor factors. Further studies in younger women and in the post-fortification era are needed to confirm these findings.

A phase 1 clinical trial of sequential pralatrexate followed by a 48-hour infusion of 5-fluorouracil given every other week in adult patients with solid tumors.

BACKGROUND: Pralatrexate (PDX) is an inhibitor of dihydrofolate reductase that was rationally designed to improve cellular uptake and retention of the drug. Preclinical data have shown synergy with the sequential administration of a dihydrofolate reductase inhibitor followed 24 hours later by 5-fluorouracil (5-FU). METHODS: Twenty-seven patients were enrolled at 1 of 5 PDX dose levels from 75 to 185 mg/m(2) on day 1 followed 24 hours later by 5-FU at a dose of 3000 mg/m(2) /48 hours every 2 weeks with folic acid and vitamin B12 supplementation. Baseline blood was collected for pharmacogenetic analysis of polymorphisms of methylenetetrahydrofolate reductase and thymidylate synthase. RESULTS: Mucositis was the most common dose-limiting toxicity. When the worst toxicities across all cycles were considered, grade 3 to 4 neutropenia, anemia, and thrombocytopenia were found to have occurred in 14.8%, 14.8%, and 0% of patients, respectively. Grade 2 to 3 toxicities included mucositis (66.6%), dehydration (33.3%), fatigue (25.9%), and diarrhea (22.2%). Version 3.0 of the National Cancer Institute Common Toxicity Criteria was used to grade toxicities The median progression-free survival (PFS) was 112 days (range, 28-588 days). Seven patients (26%) had a PFS of >180 days (5 patients with colorectal cancer, 1 patient with pancreatic cancer, and 1 patient with non-small cell lung cancer). Polymorphisms in methylenetetrahydrofolate reductase and thymidylate synthase did not correlate with toxicity. CONCLUSIONS: The recommended dose of PDX was 148 mg/m(2) . A subset of heavily pretreated patients had PFS durations of ≥6 months with this regimen.

Analyses of copy number variation reveal putative susceptibility loci in MTX-induced mouse neural tube defects.

Copy number variations (CNVs) are thought to act as an important genetic mechanism underlying phenotypic heterogeneity. Impaired folate metabolism can result in neural tube defects (NTDs). However, the precise nature of the relationship between low folate status and NTDs remains unclear. Using an array-comparative genomic hybridization (aCGH) assay, we investigated whether CNVs could be detected in the NTD embryonic neural tissues of methotrexate (MTX)-induced folate dysmetabolism pregnant C57BL/6 mice and confirmed the findings with quantitative real-time PCR (qPCR). The CNVs were then comprehensively investigated using bioinformatics methods to prioritize candidate genes. We measured dihydrofolate reductase (DHFR) activity and concentrations of folate and relevant metabolites in maternal serum using enzymologic method and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Three high confidence CNVs on XqA1.1, XqA1.1-qA2, and XqE3 were found in the NTD embryonic neural tissues. Twelve putative genes and three microRNAs were identified as potential susceptibility candidates in MTX-induced NTDs and possible roles in NTD pathogenesis. DHFR activity and 5-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), and S-adenosylmethionine (SAM) concentrations of maternal serum decreased significantly after MTX injection. These findings suggest that CNVs caused by defects in folate metabolism lead to NTD, and further support the hypothesis that folate dysmetabolism is a direct cause for CNVs in MTX-induced NTDs.

Increased frequency of expression of elevated dihydrofolate reductase in T-cell versus B-precursor acute lymphoblastic leukemia in children.

The relationships between dihydrofolate reductase (DHFR) levels or methotrexate membrane transport and acute lymphoblastic leukemia (ALL) immunophenotype were evaluated on 51 T-cell and 44 B-precursor ALL specimens from 90 pediatric ALL patients at diagnosis and relapse, using a fluorescent methotrexate analog (PT430) and flow cytometry assay (Matherly et al, Blood 85:500, 1995). For T-cell ALL, 35 of 45 (78%) of newly diagnosed patients' specimens exhibited elevated DHFR relative to DHFR levels in ALL blasts from methotrexate-responsive patients. For 30 of 45 diagnostic T-ALL specimens, DHFR expression was heterogeneous, with up to 3 separate subpopulations covering a 44-fold range; the DHFR-overproducing fractions comprised 10% to 88% of the total blasts. Elevated DHFR was less common in B-precursor ALL at diagnosis, being detected in only 17 of 36 specimens (47%); 11 of these samples exhibited DHFR heterogeneity. Median maximal DHFR levels were fourfold higher in T-cell than B-precursor ALL at diagnosis. Within a particular phenotypic group, there were no correlations between DHFR levels and patient prognostic features, including age, sex, chromosomal abnormalities, white blood cell counts (WBCs), and percentage of S-phase. However, for B-precursor ALL, there was a notably higher fraction of African-American than white patients with elevated DHFR. For patients (both phenotypes) with low WBCs (<50,000/ microL), event-free survival times were significantly shorter for those expressing DHFR above a threshold level than for patients expressing DHFR below this level (P < .016); this relationship was not seen for patients with high WBCs (>50,000/microL). Elevated DHFR was detected in 11 of 14 relapse specimens (5 of 6 T-cell and 6 of 8 B-precursor). Two of five paired relapse specimens (both T-cell) from patients treated with methotrexate exhibited increased DHFR levels over those at diagnosis (2.5- to 5-fold); the fraction of DHFR-overproducing blasts was also increased in 4 of 5 paired relapse specimens (2 B-precursor and 2 T-cell). In contrast to the variations in DHFR, highly impaired methotrexate transport was detected in only 6 of 95 ALL specimens, including both diagnosis and relapse. There was no correlation between phenotype and impaired transport. These data provide further rationale for the use of mechanistically based prognostic factors to complement known biologic or disease-based prognostic indicators in the design of ALL therapy including methotrexate, particularly with patients presenting with low WBCs. The finding of a markedly increased frequency of DHFR overexpression in T-cell over B-precursor ALL suggests that this difference is associated with the poorer prognosis of patients with T-cell ALL treated with standard-dose antimetabolite therapy and implies that higher-dose methotrexate (> or = 1 g/m2) during consolidation therapy may be useful in the treatment of this disease.

Biochemical responses of broiler chicks to folate deficiency.

The effects of folate sufficiency and deficiency in three pathways of folate metabolism were studied in 2-and 3-week-old broiler chicks. Erythrocyte phosphoribosylpyrophosphate concentrations and dihydrofolate reductase (EC 1.5.1.3) activity were significantly elevated in folate deficiency. Percentage incorporation of deoxyuridine into bone marrow DNA was reduced in folate deficiency. There was a trend towards reduced liver dihydrofolate reductase activity in deficient chicks. These studies identify further biochemical criteria that can be used to assess folate status of chicks.

The disposition and metabolism of [14C]piritrexim in dogs after intravenous and oral administration.

The disposition of [14C]piritrexim ([14C]PTX) in male dogs after iv and po doses of 1.8 mg/kg was examined. After either route of administration, greater than 90% of the dose was recovered in the exreta within 72 hr; approximately 20% was recovered in urine and 70% in feces. [14C]PTX was extensively metabolized by dogs; unchanged drug accounted for less than 15% of the dose in the excreta. The O-demethylated metabolites, 2'- and 5'-demethyl PTX, the glucuronide conjugate of 2'-demethyl PTX, and the sulfate conjugate of 5'-demethyl PTX were the major metabolites. Unchanged drug accounted for a large proportion of the drug-related radiocarbon in plasma. The average plasma half-life of PTX after iv administration was 2.6 +/- 0.3 hr, and the average total body clearance was 0.33 +/- 0.13 liter/hr/kg. After po administration, peak plasma concentrations of 0.9 +/- 0.3 micrograms/ml occurred about 1.1 hr after the dose; the absolute oral bioavailability of PTX was 0.63 +/- 0.14. Because the O-demethyl metabolites were active dihydrofolate reductase inhibitors, 2'- and 5'-demethyl PTX were synthesized, and the pharmacokinetics and bioavailability of these compounds in dogs after iv and po administration (5 mg/kg) were examined. The plasma concentration-time data for both compounds after iv doses were described by a two-compartment model, with t1/2 beta = 1.3 and 0.8 hr for the 2'- and 5'- demethyl compounds, respectively. Neither compound showed significant advantages over PTX in terms of pharmacokinetics or bioavailability.

Action of intermediate doses of methotrexate on dihydrofolate reductase in malignant diseases.

This report describes the effect of intermediate methotrexate (MTX) doses on dihydrofolate reductase (DHFR) activity in vivo in the leukocytes of 16 children with malignant diseases. The authors used a cytochemical technique, and the enzyme was studied in intact cells. The treatment protocols included MTX 500 mg-2 g/m2 weekly with leucovorin rescue. The above doses of MTX partially inhibit DHFR. The reduction of enzyme activity was observed in leukocytes within 24 h after MTX infusion, and it was more obvious in the polymorphonucleas and the monocytes. Complete inhibition of enzyme activity was not observed. These results do not agree with those of previous reports using biochemical techniques, which showed that small amounts of MTX inhibit DHFR activity. Even the large doses of MTX used in this study do not completely inhibit enzyme activity. It would be worthwhile to test the effect of even larger doses of MTX to find out if DHFR activity is inhibited.

High-performance liquid chromatographic and enzyme-inhibition assays of methotrexate concentrations in serum from patients receiving high-dose therapy: tight binding of some methotrexate to a protein that could be dihydrofolate reductase.

In the serum of patients receiving high-dose methotrexate (MTX), concentrations of the drug were monitored using both high-performance liquid chromatographic (HPLC) and enzyme-inhibition assays. In samples obtained greater than or equal to 24 hours after drug application, the HPLC assay measured higher MTX concentrations than the enzyme-inhibition assay. In 72-hour samples, the increase was usually greater than 200%. This difference was not observed in serum spiked with MTX. While the HPLC assay needs sample cleanup, ie, protein precipitation, the enzyme-inhibition assay routinely employs native serum. When MTX was measured in the supernatant with the enzyme inhibition assay, the results equaled those obtained with the HPLC procedure. In patient serum, MTX eluted from a Sephadex G-75 column in two fractions. One had the same retention volume as free MTX and could be assayed directly. The other had a retention volume like dihydrofolate reductase. In this fraction, MTX could only be determined after denaturing proteins. Only small amounts of tightly bound MTX were found in the serum of patients on intermediate-dose therapy (500-600 mg/m2). The observation that after high-dose MTX therapy part of the MTX in patient serum is strongly bound to a protein which could be DHFR raises the question as to the source and fate of this complex. Furthermore, the present finding has clinical relevance to the extent that the accepted limits for risk of MTX toxicity are based on methods using native serum. However, any procedure employing protein precipitation for sample cleanup may grossly overestimate active MTX serum concentrations, especially in the 72-hour serum samples.

Antifolate effect of triamterene on human leucocytes and on a human lymphoma cell line.

The inhibitory effect of triamterene and its metabolites on human leucocyte dihydrofolate reductase has been studied. Under test conditions with dihydrofolic acid 0.5 X 10(-5) M, triamterene 7 X 10(-5) M produced total enzyme inhibition, whereas the metabolites hydroxytriamterene and the sulphate ester of hydroxytriamterene were less effective inhibitors; at their maximum attainable concentration of 5 X 10(-5) M, dihydrofolate reductase was inhibited by 80% and 50%, respectively. Cultures of the BJAB-B95-8 human lymphoma cell line were incubated with various concentrations of triamterene. Because of their increased specific activity of dihydrofolate reductase, the cells were able to maintain normal DNA metabolism, as measured by the ratio of the incorporation rates of 3H-deoxyuridine and 3H-thymidine, as well as normal cell growth at 1 X 10(-6) M, and in some cases at 1 X 10(-5) M triamterene. At 8 X 10(-5) M triamterene, the strong inhibitory effect caused severe impairment of DNA metabolism and subsequently dissolution of the cell culture. The results are discussed in relation to the possible toxic side effects of long-term triamterene treatment in patients suffering from alcoholic cirrhosis, who may have impaired metabolism of triamterene and a concomitant severe folate deficiency.

Trimethoprim-induced elevation of dihydrofolate reductase activity in human leukocytes.

The administration of trimethoprim (TMP)--a diamino benzylpyrimidine compound which binds very tightly the bacterial dihydrofolate reductase--was accompanied by the appearance of measurable levels of dihydrofolate reductase in peripheral leukocytes from patients with nonhematological diseases. In all instances, enzyme activity rose rapidly between the fourth and eighth day after TMP. The time course of the rise and fall of dihydrofolate activity approaches cellular life span and is similar to that obtained after methotrexate or triamterene administration. Dihydrofolate reductases, partially purified from leukocytes of patients treated with TMP, bone marrow and leukemic leukocytes, had simila molecular weights, pH optima, Ki of inhibitor (methotrexate); they were stimulated to the same degree by KCl and urea. Electrophoresis of the enzyme on cellulose acetate strip resulted in the separation of two enzymatically active protein components. No differences in the electrophoretic behavior of the three blood cell enzymes were noted. The findings noted above are consistent with the suggestion that the observed rise in dihydrofolate reductase activity is a quantitative one. Moreover, the effect of TMP in vivo is discussed in comparison with the currently held hypothesis for methotrexate action (stabilization by the drug of a previously synthetized enzyme).

Tetrahydrofolate dehydrogenase in blood cells and in hematopoietic organs of different vertebrate species.

Some general aspects in cytochemical demonstration of the tetrahydrofolate dehydrogenase, concerning the final reaction product, are studied. Steady and variable factors are detected in a comparative study of Vertebrate hemopoiesis: the enzyme exhibits peculiar features in different cell types. The reactivity progressively decrease in the erythropoietic series, in concomitance with hemoglobin synthesis. Conversely an increase in the intensity of reaction is found in the granulocytopoietic series; the correspondence of positive material with the specific (eosinophil and heterophil) granulations can be discussed. The thrombocytopoietic series is also labelled by this reaction.

Cytochemical demonstration of dihydrofolate reductase in leukaemia and other haematological diseases.

The dihydrofolate reductase activity has been studied cytochemically in various haematological diseases. The variation between normal controls, Hodgkin's disease, myeloma, polycythaemia vera, chronic lymphocytic leukaemia and chronic myeloid leukamia was not significant, comparing the same type of cells. In acute myeloid leukaemia and acute lymphoblastic leukaemia the blast cells were weakly positive or negative. This finding is very interesting as the blast cells are capable of division. Probably the dihydrofolate reductase appears in the blast cells in some stage of mitosis. Lymphocytes stimulated by phytohaemagglutinin showed increased enzyme activity compared with normal non-stimulated lymphocytes. The "blast like" cells were more strongly positive than the blast cells of leukaemic patients. The patients with acute lymphoblastic leukaemia or acute myeloid leukaemia treated with methotrexate showed increased dihydrofolate reductase activity cytochemically.

Blood leucocyte enzymes. II. Activities at 8-9 a.m. in cells of normal subjects, chronic lymphatic leukaemia and chronic myeloid leukaemia patients.

The activities of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, G6PD), 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP oxidoreductase, 6PGD), hexokinase (ATP: D-hexose 6-phosphotransferase, Hx), lactate dehydrogenase (D-lactate: NAD oxidoreductase, LDH). glutamate oxaloacetate transaminase (L-aspartate: 2 oxoglutarate aminotransferase, GOT) and dihydrofolate reductase (DHFR) were measured at 8 a.m. in leucocytes of healthy individuals and patients with chronic myeloid leukaemia (CML), chronic lymphatic leukaemia (CLL), myelofibrosis with myeloid metaplasia and polycythaemia vera. In view of the heterogeneity of the leucocyte populations in these conditions, the enzyme activities were correlated to the number of immature cells in CML and to the percentage of lymphocytes in CLL. No differences in the enzyme activities were found between the white cells of healthy individuals, myelofibrosis with myeloid metaplasia and polycythaemia vera. In CML the activities of all enzymes except GOT correlated directly with the number of immature cells; an inverse correlation with the number of lymphocytes was observed in CLL. GOT was the only enzyme whose activity correlated with the number of lymphocytes in the cell suspension. Furthermore, a significantly higher activity of this enzyme was found in Ficoll-isolated CLL lymphocytes as compared to normal lymphocytes.