Dihydrofolic Acid Reductase Research Articles

An electrochemical biosensor for the determination of folic acid in pregnant women based on DHFR/c-MWCNTs/TiO2NPs modified gold electrode

The present research article describes a newly designed sensing method for determining folic acid levels. Folic acid is a B vitamin that helps the body makes healthy new cells. It is also known as vitamin B9 or folic acid. A new folic acid biosensor was constructed based on dihydrofolic acid reductase (DHFR) immobilized on c-MWCNT/TiO2NPs modified gold electrode. Prepared titanium dioxide nanoparticles have been characterized by Transmission electron microscope (TEM), X-Ray Diffractometer (XRD), and Fourier Transform Infrared spectroscopy (FTIR). Fabricated working gold electrode (i.e. DHFR/c-MWCNTs/TiO2NPs/AuE) was characterized at various stages of its construction by Scanning electron microscope (SEM), electrochemical impedance spectroscopy (EIS), FTIR, and cyclic voltammetry to confirm its fabrication. The biosensor showed an optimum response at pH 7.5 and 35 ​°C temperature. The maximum current of the constructed folic acid biosensor was due to electrons generated at 0.125 ​V against Ag/AgCl electrode. The proposed biosensor showed a low detection limit (11.48 ​nM), a wide linear range (5 ​nM–50 ​nM), a sensitivity of 0.42 ​μA/nM/cm2, and good storage stability. Folic acid biosensor was evaluated and utilized for folic acid level quantification in serum samples of pregnant women.

Single Low-Dose Methotrexate-Induced Fatal Pancytopenia: Case Report and Review of the Literature

Methotrexate (MTX) is a disease-modifying anti-rheumatic and anti-folate drug which inhibits dihydrofolic acid reductase.

Development and Validation of Rapid and Sensitive HPLC Method for the Determination of Methotrexate in Human Serum

Methotrexate competitively inhibits dihydrofolic acid reductase and thereby inhibits DNA synthesis and cellular replication.This study describes a simple and fast high-performance liquid chromatography method for the determination of methotrexate [MTX] in serum.samples were collected from adult cancer patients receiving high dose MTX at Mahathma Gandhi Memorial hospital (Warangal,AP.India) at various time intervals after the end of each infusion. Serum was deproteinized with trichloroacetic acid and the supernatant was injected into a 250×4.6 mm octadecylsilane column. Mobile phase was made of TRIS-phosphate buffer (pH 5.7): methanol: acetonitrile (70:20:10) with a flow rate of 1ml/min. Ultraviolet detection was done at 313 nm and at ambient temperature. Para aminoacetophenone was used as internal standard. Methotrexate and internal standard retention times were 4.6 and 9.5 minutes, respectively. Results showed that reproducibility (precision) of method within a day was 2.6 to 6 percent and between days was 5.5 to 9.5 percent. The recovery of the method was between 61.5 and 72.7 percent. The quantitation limit of the method for methotrexate was 0.1μM. This method is suitable for quantitation of methotrexate after infusion of high doses of this drug and has good accuracy, precision and quantitation limit. Key Words: Methotrexate; HPLC; Serum Concentration. DOI: 10.3329/sjps.v2i1.1693Stamford Journal of Pharmaceutical Sciences Vol.2(1) 2009: 8-13

Over-expression, Purification, and Characterization of Recombinant Human Arylamine N-Acetyltransferase 1

Human arylamine N-acetyltransferase 1 (NAT1) has been overexpressed in E. coli as a mutant dihydrofolic acid reductase (DHFR) fusion protein with a thrombin sensitive linker. An initial DEAE anion-exchange chromatography resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin, and human rNAT1 was purified with a second DEAE column. A total of 8 mg of human rNAT1 from 2 1 of cell culture was purified to homogeneity with this methodology. Arylamine substrate specificities were determined for human rNATI and hamster rNAT2. With both NATs, the second order rate constants (k(cat)/ Kmb) for p-aminobenzoic acid (PABA) and 2-aminofluorene (2-AF) were several thousand-fold higher than those for procainamide (PA), consistent with the expected substrate specificities of the enzymes. However, p-aminosalicylic acid (PAS), previously reported to be a human NAT1 and hamster NAT2 selective substrate, exhibits 20-fold higher specificity for hamster rNAT2 (k(cat)/Kmb 3410 microM(-1) s(-1)) than for human rNAT1 (k(cat)/Kmb 169.4 microM(-1) s(-1)). p-aminobenzoyl-glutamic acid (pABglu) was acetylated 10-fold more efficiently by human rNAT1 than by hamster rNAT2. Inhibition studies of human rNAT1 and hamster rNAT2 revealed that folic acid and methotrexate (MTX) are competitive inhibitors of both the unacetylated and acetylated forms of the enzymes, with K(I) values in 50 - 300 micro range. Dihydrofolic acid (DHF) was a much poorer inhibitor of human rNAT1 than of hamster rNAT2. The combined results demonstrate that human rNAT1 and hamster rNAT2 have similar but distinct kinetic properties with certain substrates, and suggest that folic acid, at least in the non-polyglutamate form, may not have an effect on human NAT1 activity in vivo.

Clinical Studies of Atovaquone, Alone or in Combination with other Antimalarial Drugs, for Treatment of Acute Uncomplicated Malaria in Thailand

The therapy of Plasmodium falciparum malaria continues to be a problem in many parts of Southeast Asia because of multidrug resistance to nearly all existing antimalarial drugs. Atovaquone is a novel hydroxynaphthoquinone with broad spectrum anti-protozoal activity. We recently evaluated the antimalarial activity of atovaquone in a series of dose-ranging studies in 317 patients with malaria at the Bangkok Hospital for Tropical Diseases. Originally, the drug was administered alone. Using atovaquone alone resulted in satisfactory, initial clinical responses in all patients; the mean parasite and fever clearance times were 62 and 53 hr, respectively. However, irrespective of the duration of therapy, overall cure rates were approximately 67%. In vitro sensitivity studies on parasites taken from patients prior to treatment and at the time of recrudescence showed a marked decrease in susceptibility to atovaquone in the recrudescent parasites. To improve cure rates, atovaquone was administered in combination with other drugs with antimalarial activity. Proguanil and tetracycline were chosen due to laboratory evidence of potentiation; doxycycline was selected because it has a longer half-life than tetracycline. Although pyrimethamine did not show laboratory evidence of potentiation with atovaquone, it was chosen as an alternative inhibitor of dihydrofolic acid reductase with a longer half-life than proguanil. The clinical studies with these drug combinations confirmed the laboratory results with marked improvement in cure rates for proguanil, tetracycline, and doxycycline; pyrimethamine showed only minimal improvement. Proguanil was subsequently selected as the preferred drug partner because of its long record of safety and the ability to use the drug in pregnant women and children. Of the 104 patients with falciparum malaria treated with atovaquone plus proguanil for 3-7 days, 101 were cured and had virtually no adverse side effects. The combination of atovaquone and proguanil also was effective in eliminating erythrocytic forms of P. vivax, but parasitemia recurred in most patients.

Densely Methylated DNA Islands in Mammalian Chromosomal Replication Origins

Densely methylated DNA sequence islands, designated DMIs, have been observed in two Chinese hamster cell chromosomal replication origins by using a PCR-based chemical method of detection. One of the origins, oriS14, is located within or adjacent to the coding sequence for ribosomal protein S14 on chromosome 2q, and the other, ori-beta, is approximately 17 kbp downstream of the dhfr (dihydrofolic acid reductase) locus on chromosome 2p. The DMI in oriS14 is 127 bp long, and the DMI in ori-beta is 516 bp long. Both DMIs are bilaterally methylated (i.e., all dCs are modified to 5-methyl dC) only in cells that are replicating their DNA. When cell growth and DNA replication are arrested, methylation of CpA, CpT, and CpC dinucleotides is lost and the sequence islands display only a subset of their originally methylated CpG dinucleotides. Several possible roles for DMI-mediated regulation of mammalian chromosomal origins are considered.

Densely methylated DNA islands in mammalian chromosomal replication origins.

Densely methylated DNA sequence islands, designated DMIs, have been observed in two Chinese hamster cell chromosomal replication origins by using a PCR-based chemical method of detection. One of the origins, oriS14, is located within or adjacent to the coding sequence for ribosomal protein S14 on chromosome 2q, and the other, ori-beta, is approximately 17 kbp downstream of the dhfr (dihydrofolic acid reductase) locus on chromosome 2p. The DMI in oriS14 is 127 bp long, and the DMI in ori-beta is 516 bp long. Both DMIs are bilaterally methylated (i.e., all dCs are modified to 5-methyl dC) only in cells that are replicating their DNA. When cell growth and DNA replication are arrested, methylation of CpA, CpT, and CpC dinucleotides is lost and the sequence islands display only a subset of their originally methylated CpG dinucleotides. Several possible roles for DMI-mediated regulation of mammalian chromosomal origins are considered.

Phenotypic resistance to methotrexate and N-phosphonacetyl L-aspartate is induced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA).

Three different 3T6 mouse fibroblast cell clones with intrinsically different sensitivities to methotrexate (MTX) have been isolated from an originally heterogeneous population. When these 3 different clones were exposed to MTX in the presence of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) the degree of enhancement of recovery of methotrexate resistant (MTXR) colonies was greatest in the most sensitive clone. MTXR colonies isolated and cultured in the presence and absence of MTX and TPA were analysed for dihydrofolic acid reductase (dhfr) levels by flow cytometry after binding of fluorescent methotrexate. None of the 58 clones showed major changes in dhfr levels. Dot-blot analysis of 12 clones indicated no increases in dhfr gene copy number or mRNA levels consistent with gene amplification. Southern analysis of 6 further clones indicated that only 1 clone isolated by multi-step selection had amplified dhfr sequences. TPA-enhanced mouse 3T6 N-phosphonacetyl-L-aspartate (PALA)-resistant colony recovery and mouse 3T3 MTXR colony recovery were also shown, by dot-blot analysis, not to be due to gene amplification. The data indicate that TPA can have a profound effect on drug-resistant colony recovery by mechanisms other than induction of gene amplification.

7-Hydroxy-methotrexate and clinical toxicity following high-dose methotrexate therapy.

7-Hydroxy-MTX production after consecutive high-dose MTX therapy (12 g/m2) was measured in 7 patients with osteosarcoma by HPLC. 7-Hydroxy-MTX serum values in the last cycle were found to be significantly lower compared with the first high-dose MTX treatment of the adjuvant chemotherapy protocol (COSS 80). Moreover, in another patient highly reduced 7-hydroxy-MTX production was correlated with severe clinical toxicity. As 7-hydroxy-MTX is a 200 fold less potent dihydrofolic acid reductase inhibitor compared with MTX decreased production of the metabolite may lead to enhanced clinical toxicity which may not be predictable monitoring MTX serum levels alone.

Trimethoprim, failure to penetrate intoPseudomonas aeruginosacells

Permeability mutants of Escherichia coli and Pseudomonas aeruginosa demonstrated a significantly higher susceptibility for trimethoprim (TMP). Although the target enzyme of this drug, dihydrofolic acid reductase, expressed about the same activity, it was comparably inhibited by TMP in permeability mutants and in wild-type strains. The weak activity in wild-type strains depends on the decreased uptake.

A mechanism of resistance to methotrexate NADPH but not NADH stimulation of methotrexate binding to dihydrofolate reductase

Characteristics of methotrexate (MTX) inhibition of dihydrofolic acid reductase (DHFR) enzyme activity and the effects of NADPH and NADH on enzyme-drug interaction were studied. Two highly sensitive assay procedures were used. The first utilized tritium-labeled MTX to measure direct binding properties of the enzyme and the second utilized tritium-labeled dihydrofolate (H 2PteGlu) and folate (PteGlu) to analyze kinetics of reduction of these substrates. NADPH was found to enhance DHFR binding of MTX ( K d = 2.6 × 1 O −11 M), whereas NADH was found to have no effect ( K d = 3.7 × 10 −9 M). However, NADH proved to be a good substrate for folate reduction compared to NADPH, especially in low salt buffer. The observation that NADH supports the reduction of folate and dihydrofolate but not MTX binding suggests that natural resistance to MTX could exist if NADH replaces NADPH as the main cofactor for DHFR.

The nucleotide sequence of the cDNA coding for the human dihydrofolic acid reductase

The nucleotide sequence of the human dihydrofolic acid reductase (DHFR) reading frame has been derived from the analysis of human DHFR cDNA. This sequence and the corresponding amino acid sequence have been compared with those available for the enzyme and its coding segment from other organisms. There is an 89% nucleotide sequence homology between the human DHFR reading frame and the mouse coding sequence. Furthermore, amino acid-sequence homologies of 74%, 81% and 89% has been found between human DHFR and chicken, bovine and mouse DHFR, respectively.

Variable content of double minute chromosomes is not correlated with degree of phenotype instability in methotrexate-resistant human cell lines.

Several variants resistant to 1.8 x 10(-4) M DL-methotrexate (MTX) have been isolated from the human cell lines HeLa BU25 and VA2-B by exposing them to progressively increasing concentrations of the drug. A striking variability of phenotype and chromosome constitution was observed among the different variants. All resistant cell lines exhibited a greatly increased dihydrofolic acid reductase (DHFR) activity and DHFR content; however, the DHFR activity levels varied considerably among the variants, ranging between about 35 and 275 times the parental level. In the absence of selective pressure, the increased DHFR activity was unstable, and in all cell lines but one was completely lost over a period ranging in different variants between 25 and 200 days. The MTX-resistant cells lines showed anomalies in their chromosome constitution, which involved the occurrence of a duplicated set of chromosomes in most cells of some of the variants and the presence of double minute chromosomes in all cell lines. An analysis of the correlation of loss of double minute chromosomes and loss of DHFR activity in the absence of MTX has given results consistent with the idea that the double-minute chromosomes contain amplified DHFR genes. However, the most significant finding is that, in contrast to what has been reported in the mouse system, the recognizable double-minute chromosomes varied greatly in number in different variants without any relationship to either the level of DHFR activity or the degree of instability of MTX resistance in the absence of selective pressure. These and other observations point to the occurrence in the human MTX-resistant variants of another set of DHFR genes, representing a varied proportion of the total, which is associated with the regular chromosomes, and which may be unstable in the absence of selective pressure.

Variable content of double minute chromosomes is not correlated with degree of phenotype instability in methotrexate-resistant human cell lines.

Several variants resistant to 1.8 x 10(-4) M DL-methotrexate (MTX) have been isolated from the human cell lines HeLa BU25 and VA2-B by exposing them to progressively increasing concentrations of the drug. A striking variability of phenotype and chromosome constitution was observed among the different variants. All resistant cell lines exhibited a greatly increased dihydrofolic acid reductase (DHFR) activity and DHFR content; however, the DHFR activity levels varied considerably among the variants, ranging between about 35 and 275 times the parental level. In the absence of selective pressure, the increased DHFR activity was unstable, and in all cell lines but one was completely lost over a period ranging in different variants between 25 and 200 days. The MTX-resistant cells lines showed anomalies in their chromosome constitution, which involved the occurrence of a duplicated set of chromosomes in most cells of some of the variants and the presence of double minute chromosomes in all cell lines. An analysis of the correlation of loss of double minute chromosomes and loss of DHFR activity in the absence of MTX has given results consistent with the idea that the double-minute chromosomes contain amplified DHFR genes. However, the most significant finding is that, in contrast to what has been reported in the mouse system, the recognizable double-minute chromosomes varied greatly in number in different variants without any relationship to either the level of DHFR activity or the degree of instability of MTX resistance in the absence of selective pressure. These and other observations point to the occurrence in the human MTX-resistant variants of another set of DHFR genes, representing a varied proportion of the total, which is associated with the regular chromosomes, and which may be unstable in the absence of selective pressure.

Multiple forms of human dihydrofolate reductase messenger RNA: Cloning and Expression in Escherichia coli of their DNA coding sequence

The programming capacity for the synthesis of human dihydrofolic acid reductase in a rabbit reticulocyte lysate has been found to be greatly enhanced in the polysomal poly(A)-containing RNA from a methotrexate-resistant human cell variant (6A3), as compared to the RNA from its parental line (VA 2-B). A major fraction of this promoting activity is associated with a 3.8 × 10 3 base RNA species detectable as a band in the ethidium bromide-stained electrophoretic pattern of the RNA from 6A3 cells, but not in the RNA from VA 2-B cells. Furthermore, sucrose gradient fractionation experiments have indicated that another substantial portion of the messenger activity is associated with RNA components around 10 3 bases in size. Double-stranded complementary DNA synthesized from total poly(A)-containing RNA of 6A3 cells has been size fractionated, and both large (1400 to 3800 base-pairs) and small size complementary DNA (600 to 1400 base-pairs) species have been used separately to transform Escherichia coli χ2282 with pBR322 as a vector. Of 76 transformants obtained with the large size complementary DNA, identified by a differential colony hybridization assay, none has expressed the dihydrofolic acid reductase coding sequence in E. coli, as judged by resistance to trimethoprim. By contrast, eight trimethoprim-resistant transformants have been obtained using the small size complementary DNA, and their plasmids have been shown to contain the dihydrofolic acid reductase coding sequence by restriction mapping and DNA sequencing; moreover, immunoautoradiographic experiments have revealed the presence in the extracts of two of these transformants of a protein with the electrophoretic mobility and immunoreactivity of human dihydrofolic acid reductase. Restriction mapping and DNA transfer hybridization experiments have further indicated that the inserts of the chimaeric plasmids conferring trimethoprim resistance upon the host and of those lacking this capacity cover together a complementary DNA region of about 3.35 × 10 3 base-pairs, in which the 564 base-pair dihydrofolic acid reductase coding stretch is located near the 5′ end of the sense strand. RNA transfer hybridization experiments using different cloned complementary DNA fragments as probes have shown the presence of three species of dihydrofolic acid reductase-specific messenger RNAs, with sizes of 3.8 × 10 3, 1.0 × 10 3 and 0.8 × 10 3 bases, differing in the length of the 3′ untranslated region, in the poly(A)-containing RNA from two methotrexate-resistant variants, 6A3 and 10B3, and, in greatly reduced amounts, in the RNA from their respective parents, VA 2B and HeLa BU25.

Isolation and characterization of dihydrofolic acid reductase from methotrexate-sensitive and -resistant human cell lines.

Dihydrofolic acid reductase has been purified by affinity chromatography to apparent homogeneity from the human HeLa BU-25 cell line and from two methotrexate-resistant variants, one deriving from HeLa BU-25 and the other from the human VA2-B cell line. The purified enzymes from the three sources have been characterized in their physical and enzymatic properties. They were not found to differ significantly as concerns their electrophoretic mobility in polyacrylamide gels under a variety of conditions, their specific dihydrofolic acid reductase and folic acid reductase activities, their Km values for folic acid and TPNH, their sensitivity to methotrexate, and the pH dependence of their folic acid reductase activity. The human dihydrofolic acid reductase has an apparent molecular weight of 21,000 to 22,000, a Km for folic acid of 6.1 to 7.6 X 10(-6) M and a Km for TPNH of 1.6 to 1.7 X 10(-4) M, turnover numbers of about 500 and 65 mol/min/mol of enzyme for the dihydrofolic acid reductase and the folic acid reductase activity, respectively. The values of the above mentioned physical and kinetic parameters are comparable to those reported for the dihydrofolic acid reductase from other animal cell systems. The dihydrofolic acid reductase content of the two-resistant cell lines is at least 200-fold higher than that of the methotrexate-sensitive HeLa BU-25 cell line. The available evidence indicates that this increased dihydrofolic acid reductase content results from a hyperproduction of an enzyme identical or similar to that of the sensitive cells, presumably due to a selective dihydrofolic acid reductase gene amplification, as previously reported for other cell lines of rodent origin.

Effects of trimethoprim on leukaemic cells in vitro.

Trimethoprim-Sulfamethoxazole (TMP-SMZ) is a fixed combination of antibiotics which is widely used for prophylaxis and treatment of infections in patients undergoing cancer chemotherapy. TMP has been reported to inhibit growth of haemopoietic stem cells in vitro. If TMP-SMZ inhibits leukaemic cell growth, it could interfere with antileukaemic treatment, especially with S phase specific agents. TMP-SMZ at a concentration of 10 micrograms/ml (TMP) produced 50% inhibition of incorporation of 3H-deoxyuridine in DNA of L1210 and human lymphoblastic leukaemia cell. TMP-SMZ (1 g/ml TMP) produced 30% prolongation of doubling time of L1210 in vitro. Pure TMP (10 micrograms/ml) but not SMZ (50 micrograms/ml) produced the same effect as TMP-SMZ. Cell inhibitory effects could be completely reversed by folinic acid. These findings suggest that TMP produces some degree of inhibition of dihydrofolic acid reductase in mammalian cells and can potentially influence the effects of chemotherapy on tumour and/or host cells.

Principal component regression analysis applied in structure‐activity relationships. 1. selective dihydrofolic acid reductase inhibitors

AbstractThe principal component regression analysis is used when the predicting variables correlate significantly against the regressor variables and, when a collinearity and multicollinearity exist among regressor variables. The example adapted from enzymology shows that no loss of information occurs with respect to the chemical constants in comparison with the reduced model of multivariate regression analysis.

A Rapid Enzymatic Assay for Methotrexate in Serum

A kinetic enzymological assay for methotrexate in serum is described; the results are available after 1.5 hours. The lower limit of sensivity is 50 nmol/1; the day-to-day variation coefficients compare favourably with other procedures (about 7% in the 200-600 nmol/1 range). The method uses purified dihydrofolic acid reductase from bovine liver. The problem of its instability in the assay is circumvented by several measures, e.g. the use of a reaction rate analyser.

Thymidylate synthetase and dihydrofolic acid reductase in the stimulated human lymphocyte.

The phytohemagglutinin (PHA)-stimulated human lymphocytes demonstrated trace or no activity (dihydro) folate acid reductase using three methods including a radioassay, but demonstrated ample activity of thymidylate synthetase. This was true regardless of the day of harvest, (first through seventh) of the stimulated lymphocyte. The lymphocyte extracts revealed no inhibitor to the reductase enzyme when these extracts were added before the liver extracts to the assay system. When methotrexate (MTX) was added to the culture media of the lymphocytes, there was, as expected, an increase in the synthetase activity, but the expected rise in the reductase activity did not occur, it remained nil. On the other hand, MTX did influence the incorporation of nucleosides by the stimulated lymphocytes in a fashion similar to its action on the incorporation of the same nucleosides by other cells.