N-acetyltransferase Activity Research Articles

Expression and genotype-dependent catalytic activity of N-acetyltransferase 2 (NAT2) in human peripheral blood mononuclear cells and its modulation by Sirtuin 1

N-acetyltransferase 2 (NAT2) catalyzes the biotransformation of numerous arylamine and hydrazine drugs and carcinogens. Genetic polymorphisms of NAT2 modify drug efficacy and toxicity and susceptibility to diseases such as cancer and type 2 diabetes. Expression of NAT2 has been documented in the liver and gastrointestinal tract but not in other tissues. Deacetylation of cytosolic proteins by sirtuins is a post-translational modification important in regulatory networks of diverse cellular processes. The aim of the present study was to investigate NAT2 expression in peripheral blood mononuclear cells (PBMC) and the effects of NAT2 genotype and Sirtuin 1 (SIRT1). Both NAT2 and SIRT1 proteins were expressed on PBMC. Their expression was more prevalent on CD3+ compared to CD19+ and CD56+ cell populations. N-acetylation capacity of PBMC exhibited a NAT2 gene-dose response toward the N-acetylation of isoniazid. Subjects with rapid NAT2 genotype showed an apparent Vmax of 42.1 ± 2.4; intermediate NAT2 genotypes an apparent Vmax of 22.6 ± 2.2; and slow acetylator NAT2 genotypes an apparent Vmax of 19.9 ± 1.7 nM acetyl-isoniazid/24 h/million cells. The N-acetylation capacity of NAT2 in the presence of SIRT1 enhancer was significantly decreased (p < 0.001), conversely, the transient silencing of SIRT1 resulted in an increase of N-acetylation capacity (p < 0.001). These findings are the first report of NAT2 genotype-dependent expression on PBMC and post-translational modification by SIRT1. These findings constitute a substantial advance in our understanding of human N-acetyltransferase expression and a new much less invasive method for measurement of human NAT2 expression and phenotype.

Berberine: A potential adjunct for the treatment of gastrointestinal cancers?

Gastrointestinal cancers are among the most prevalent cancers in the general population. Despite effective early diagnostics and intervention, the gastrointestinal cancer-related mortality still remains elevated. Berberine (BBR) is a benzyl tetra isoquinoline alkaloid exracted from several plants. BBR is nontoxic to human normal cells, but suppresses the growth of different tumor cells: melanoma, epidermoid carcinoma, hepatoma, oral carcinoma, glioblastoma, prostatic carcinoma, and gastric carcinoma. In particular, BBR seems to suppress the proliferation of gastrointestinal cancers in a number of preclinical models. Several mechanisms of action have been hypothesized and demonstrated: immunomodulation, inhibition of topoisomerase enzymes, suppression of the EGF receptor, Her2/neu, and the VEGF receptor, induction of p53, Cip1/p21, Kip1/p27, Rb expression, induction of apoptosis (by regulation of MMPs pathway, caspases, Bax, and Smac/DIABLO), inhibition of arylamin N-acetyltransferase activity, and regulation of microRNAs expression. The aim of this review is to summarize the pharmacological effects of BBR on animal and human gastrointestinal cancers.

Knockout of human arylamine N-acetyltransferase 1 (NAT1) in MDA-MB-231 breast cancer cells leads to increased reserve capacity, maximum mitochondrial capacity, and glycolytic reserve capacity.

Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. NAT1 can also hydrolyze acetyl-coenzyme A (acetyl-CoA) in the absence of an arylamine substrate. Expression of NAT1 varies between individuals and is elevated in several cancers including estrogen receptor positive (ER+) breast cancers. To date, however, the exact mechanism by which NAT1 expression affects mitochondrial bioenergetics in breast cancer cells has not been described. To further evaluate the role of NAT1 in energy metabolism MDA-MB-231 breast cancer cells with parental, increased, and knockout levels of NAT1 activity were compared for bioenergetics profile. Basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured followed by programmed sequential injection of Oligomycin (ATP synthase inhibitor), FCCP (ETC uncoupler), Antimycin A (Complex III inhibitor), and Rotenone (Complex I inhibitor) to evaluate mitochondrial bioenergetics. Compared to the cell lines with parental NAT1 activity, NAT1 knockout MDA-MB-231 cell lines exhibited significant differences in bioenergetics profile, while those with increased NAT1 did not. Significant increases in reserve capacity, maximum mitochondrial capacity, and glycolytic reserve capacity were observed in NAT1 knockout MDA-MB-231 cell lines compared to those with parental and increased NAT1 activity. These data indicate that NAT1 knockout in MDA-MB-231 breast cancer cells may enhance adaptation to stress by increasing plasticity in response to energy demand.

Chemical composition and biological activities of the essential oil from Cleome rutidosperma DC

Cleome rutidosperma DC, commonly known in Jamaica as ‘consumption-weed’ is a plant traditionally used in folklore for treating tuberculosis and other infectious and chronic ailments. We evaluate for the first time the chemical composition and biological activities of the essential oil components of the complete aerial parts of this plant. The essential oil obtained by steam distillation (0.02%) was analyzed by a combination of gas chromatography-flame ionization detector (GC-FID), gas chromatography-mass spectroscopy (GC–MS) and retention index (RI). The volatile oil of C. rutidosperma was dominated by oxygenated diterpenes (67.6%); with (Z)-phytol (65.1%) being the single most abundant constituent. C. rutidosperma aerial essential oil exhibited moderate inhibition against the activity of recombinant arylamine N-acetyltransferase (NAT) from Mycobacterium marinum (IC50 22.20 ± 1.80 μg/μL), while, racemic phytol had an inhibition with an IC50 of 22.33 μg/μL ± 0.50 μg/μL, thus accounting for the NAT inhibition imparted by the crude oil. Inhibition of NAT, a key enzyme in mycobacterial growth may be the pathway in which phytol, shown in this study to interact with the active site using in-silico methods, renders its previously demonstrated anti-tubercular properties. The phytol rich essential oil also demonstrated antimicrobial activity against all nine human pathogenic bacteria and the fungus strain assayed, with the most significant inhibitory activity against Bacillus cereus and justifies the need for further evaluation and development of the essential oils from this plant.

Chronic treatment with dexamethasone alters clock gene expression and melatonin synthesis in rat pineal gland at night.

BackgroundMelatonin is a neuroendocrine hormone that regulates many functions involving energy metabolism and behavior in mammals throughout the light/dark cycle. It is considered an output signal of the central circadian clock, located in the suprachiasmatic nucleus of the hypothalamus. Melatonin synthesis can be influenced by other hormones, such as insulin and glucocorticoids in pathological conditions or during stress. Furthermore, glucocorticoids appear to modulate circadian clock genes in peripheral tissues and are associated with the onset of metabolic diseases. In the pineal gland, the modulation of melatonin synthesis by clock genes has already been demonstrated. However, few studies have shown the effects of glucocorticoids on clock genes expression in the pineal gland.ResultsWe verified that rats treated with dexamethasone (2 mg/kg body weight, intraperitoneal) for 10 consecutive days, showed hyperglycemia and pronounced hyperinsulinemia during the dark phase. Insulin sensitivity, glucose tolerance, melatonin synthesis, and enzymatic activity of arylalkylamine N-acetyltransferase, the key enzyme of melatonin synthesis, were reduced. Furthermore, we observed an increase in the expression of Bmal1, Per1, Per2, Cry1, and Cry2 in pineal glands of rats treated with dexamethasone.ConclusionThese results show that chronic treatment with dexamethasone can modulate both melatonin synthesis and circadian clock expression during the dark phase.

Retrospective analysis of estrogen receptor1 and N‑acetyltransferase gene expression in normal breast tissue, primary breast tumors, and established breast cancer cell lines.

The expression levels of estrogen receptor 1 (ESR1), arylamine N-acetyltransferase 1 (NAT1), and arylamine N-acetyltransferase 2 (NAT2) are implicated in breast cancer; however, their co-expression profiles in normal breast tissue, primary breast tumors and established breast cancer cell lines are undefined. NAT1 expression is widely reported to be associated with ESR1 expression and is frequently investigated in breast cancer etiology. Furthermore, the NAT2 phenotype has been reported to modify breast cancer risk in molecular epidemiological association studies. Understanding the relationships between the expression levels of these genes is essential to understand their role in breast cancer etiology and treatment. In the present study, NAT1, NAT2 and ESR1 expression data were accessed from repositories of RNA-Seq data covering 57 breast cancer cell lines, 1,043 primary breast tumors and 99 normal breast tissues. The relationships between gene expression, and between NAT1 activity and RNA expression in breast cancer cell lines were evaluated using non-parametric statistical analyses. Differences in gene expression in each dataset, as well as gene expression differences in normal breast tissue compared to primary breast tumors, and stratification by estrogen receptor status were determined. NAT1 and NAT2 mRNA expression were detected in normal and primary breast tumor tissues; NAT1 expression was much higher than NAT2. NAT1 and ESR1 expression were strongly associated, whereas NAT2 and ESR1 expression were not. Although NAT1 and NAT2 expression were associated, the magnitude was moderate. NAT1, NAT2, and ESR1 expression were increased in primary breast tumor tissue compared with normal breast tissue; however, the magnitude and significance of the differences were lower for NAT2. Analysis of NAT1, NAT2, and ESR1 expression in normal and primary breast tissues and breast cancer cell lines suggested that NAT1 and NAT2 expression are regulated by distinctive mechanisms, whereas NAT1 and ESR1 expression may have overlapping regulation. Defining these relationships is important for future investigations into breast cancer prevention.

Hyphal development in Candida albicans from different cell states.

Candida albicans is an important opportunistic fungal pathogen of immunocompromised individuals. The ability to switch between yeast, pseudohyphal, and hyphal growth forms (polymorphism) is one of the most investigated virulence attributes of C. albicans. The usual method for inducing hypha formation in the lab is by diluting cells from a saturated culture into fresh medium at 37°C. The molecular mechanism at action under these conditions has been previously investigated. C. albicans can also form hyphae in growing cells without dilution. The ability of C. albicans to form hyphae in different cell states facilitates the fungus to adapt varied host environments during infection. A recent study by Su et al. uncovered the molecular mechanism for how C. albicans develops hyphae under the condition without inoculation. N-Acetylglucosamine (GlcNAc) stimulates filamentation in log phase cells through transcriptional down-regulation of NRG1, the major repressor of hyphal development. Instead of cAMP-PKA pathway, GlcNAc sensor Ngs1 is responsible for this process. Ngs1 binds to GlcNAc to activate its N-acetyltransferase activity, leading to the induction of BRG1 expression. The increased level of BRG1 could repress NRG1 transcripts, resulting in hyphal growth. Hyphal development in log phase cells induced by serum or neutral pH also requires activation of BRG1 to down-regulate NRG1 transcription. Therefore, hyphal induction under the condition without inoculation is trigged by Brg1-mediated removal of Nrg1 inhibition. This review describes our current understanding of the molecular mechanism underlying hyphal development, the best studied virulence factor in C. albicans. These will expand the number of potential drug targets with novel modes of action for anti-virulence therapeutics.

Hyphal induction under the condition without inoculation in Candida albicans is triggered by Brg1-mediated removal of NRG1 inhibition.

Candida albicans can switch between yeast and hyphae growth forms, which is critical for its pathogenesis. Diluting from saturated cells into fresh medium at 37°C is routinely used to induce hyphae, which depends on the cAMP-PKA pathway-activated transcriptional down-regulation of NRG1 and degradation of Nrg1 protein triggered by inoculation. It is reported that N-acetylglucosamine (GlcNAc), serum or neutral pH could stimulate filamentation in log phase cells, whereas how C. albicans develops hyphae without inoculation remains unknown. Here, we show that NRG1 down-regulation is necessary for hyphal growth under this condition. Instead of cAMP-PKA pathway, GlcNAc sensor Ngs1 is responsible for the down-regulation of NRG1 upon GlcNAc induction in log phase cells through its N-acetyltransferase activity. From a genetic screen, Brg1 is found to be essential for hyphal development without inoculation. Ngs1 binds to BRG1 promoter to induce its expression in GlcNAc. Importantly, constitutively expressed BRG1 induces NRG1 down-regulation even in the absence of GlcNAc or Ngs1. Serum or neutral pH-induced filamentation in log phase cells is also through Brg1-mediated NRG1 down-regulation. Our study provides a molecular mechanism for how C. albicans forms hyphae in different cell states. This flexibility may facilitate C. albicans to adapt varied host environment during infection.

The effect of α-solanine on the activity, gene expression, and kinetics of arylamine N-acetyltransferase in HepG2 cells.

α-solanine is one of the major components of Solanumnigrum Linn., the fruits of which are used in China for food. In the present study, α-solanine was selected to assess the inhibition of arylamine N-acetyltransferase (NAT) activity and mRNA expression of NAT and kinetics in HepG2 cells. NAT activity was examined by HPLC. The double-reciprocal plot was contrived to yield a regression equation to calculate Km and Vmax. NAT mRNA expression was determined by PCR. The results revealed that α-solanine could significantly decrease NAT activity in intact HepG2 cells or the cytoplasm. Km did not differ either for intact HepG2 cells or for the cytoplasm, however Vmax was significantly different. α-solanine could decrease the expression of NAT1 mRNA and NAT2 mRNA. In summary, α-solanine was a non-competitive inhibitor of NAT in HepG2 cells. It decreased NAT activity through non-competitive inhibition of NAT activity and by decreasing the expression of NAT1 mRNA and NAT2 mRNA.

The Apicomplexa-specific glucosamine-6-phosphate N-acetyltransferase gene family encodes a key enzyme for glycoconjugate synthesis with potential as therapeutic target

Apicomplexa form a phylum of obligate parasitic protozoa of great clinical and veterinary importance. These parasites synthesize glycoconjugates for their survival and infectivity, but the enzymatic steps required to generate the glycosylation precursors are not completely characterized. In particular, glucosamine-phosphate N-acetyltransferase (GNA1) activity, needed to produce the essential UDP-N-acetylglucosamine (UDP-GlcNAc) donor, has not been identified in any Apicomplexa. We scanned the genomes of Plasmodium falciparum and representatives from six additional main lineages of the phylum for proteins containing the Gcn5-related N-acetyltransferase (GNAT) domain. One family of GNAT-domain containing proteins, composed by a P. falciparum sequence and its six apicomplexan orthologs, rescued the growth of a yeast temperature-sensitive GNA1 mutant. Heterologous expression and in vitro assays confirmed the GNA1 enzymatic activity in all lineages. Sequence, phylogenetic and synteny analyses suggest an independent origin of the Apicomplexa-specific GNA1 family, parallel to the evolution of a different GNA1 family in other eukaryotes. The inability to disrupt an otherwise modifiable gene target suggests that the enzyme is essential for P. falciparum growth. The relevance of UDP-GlcNAc for parasite viability, together with the independent evolution and unique sequence features of Apicomplexa GNA1, highlights the potential of this enzyme as a selective therapeutic target against apicomplexans.

Genetic and small molecule inhibition of arylamine N-acetyltransferase 1 reduces anchorage-independent growth in human breast cancer cell line MDA-MB-231.

Arylamine N-acetyltransferase 1 (NAT1) expression is reported to affect proliferation, invasiveness, and growth of cancer cells. MDA-MB-231 breast cancer cells were engineered such that NAT1 expression was elevated or suppressed, or treated with a small molecule inhibitor of NAT1. The MDA-MB-231 human breast cancer cell lines were engineered with a scrambled shRNA, a NAT1 specific shRNA or a NAT1 overexpression cassette stably integrated into a single flippase recognition target (FRT) site facilitating incorporation of these different genetic elements into the same genomic location. NAT1-specific shRNA reduced NAT1 activity in vitro by 39%, increased endogenous acetyl coenzyme A levels by 35%, and reduced anchorage-independent growth (sevenfold) without significant effects on cell morphology, growth rates, anchorage-dependent colony formation, or invasiveness compared to the scrambled shRNA cell line. Despite 12-fold overexpression of NAT1 activity in the NAT1 overexpression cassette transfected MDA-MB-231 cell line, doubling time, anchorage-dependent cell growth, anchorage-independent cell growth, and relative invasiveness were not changed significantly when compared to the scrambled shRNA cell line. A small molecule (5E)-[5-(4-hydroxy-3,5-diiodobenzylidene)-2-thioxo-1,3-thiazolidin-4-one (5-HDST) was 25-fold more selective towards the inhibition of recombinant human NAT1 than N-acetyltransferase 2. Incubation of MDA-MB-231 cell line with 5-HDST resulted in 60% reduction in NAT1 activity and significant decreases in cell growth, anchorage-dependent growth, and anchorage-independent growth. In summary, inhibition of NAT1 activity by either shRNA or 5-HDST reduced anchorage-independent growth in the MDA-MB-231 human breast cancer cell line. These findings suggest that human NAT1 could serve as a target for the prevention and/or treatment of breast cancer.

Heritability of the melatonin synthesis variability in autism spectrum disorders

Autism Spectrum Disorders (ASD) are heterogeneous neurodevelopmental disorders with a complex genetic architecture. They are characterized by impaired social communication, stereotyped behaviors and restricted interests and are frequently associated with comorbidities such as intellectual disability, epilepsy and severe sleep disorders. Hyperserotonemia and low melatonin levels are among the most replicated endophenotypes reported in ASD, but their genetic causes remain largely unknown. Based on the biochemical profile of 717 individuals including 213 children with ASD, 128 unaffected siblings and 376 parents and other relatives, we estimated the heritability of whole-blood serotonin, platelet N-acetylserotonin (NAS) and plasma melatonin levels, as well as the two enzymes arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT) activities measured in platelets. Overall, heritability was higher for NAS (0.72 ± 0.091) and ASMT (0.59 ± 0.097) compared with serotonin (0.31 ± 0.078), AANAT (0.34 ± 0.077) and melatonin (0.22 ± 0.071). Bivariate analyses showed high phenotypic and genetic correlations between traits of the second step of the metabolic pathway (NAS, ASMT and melatonin) indicating the contribution of shared genetic factors. A better knowledge of the heritability of the melatonin synthesis variability constitutes an important step to identify the factors that perturb this pathway in individuals with ASD.

Role of N-acetyltransferase 2 acetylation polymorphism in 4, 4′-methylene bis (2-chloroaniline) biotransformation

Arylamine N-acetyltransferase 1 (NAT1) and 2 (NAT2) catalyze the acetylation of arylamine carcinogens. Single nucleotide polymorphisms in the NAT2 coding exon present in NAT2 haplotypes encode allozymes with reduced N-acetyltransferase activity towards the N-acetylation of arylamine carcinogens and the O-acetylation of their N-hydroxylated metabolites. NAT2 acetylator phenotype modifies urinary bladder cancer risk following exposures to arylamine carcinogens such as 4-aminobiphenyl. 4, 4′-methylene bis (2-chloroaniline) (MOCA) is a Group 1 carcinogen for which a role of the NAT2 acetylation polymorphism on cancer risk is unknown. We investigated the role of NAT2 and the genetic acetylation polymorphism on both MOCA N-acetylation and N-hydroxy-MOCA O-acetylation. MOCA N-acetylation exhibited a robust gene dose response in rabbit liver cytosol and in cryopreserved human hepatocytes derived from individuals of rapid, intermediate and slow acetylator NAT2 genotype. MOCA exhibited about 4-fold higher affinity for recombinant human NAT2 than NAT1. Recombinant human NAT2*4 (reference) and 15 variant recombinant human NAT2 allozymes catalyzed both the N-acetylation of MOCA and the O-acetylation of N-hydroxy-MOCA. Human NAT2 5, NAT2 6, NAT2 7 and NAT2 14 allozymes catalyzed MOCA N-acetylation and N-hydroxy-O-acetylation at rates much lower than the reference NAT2 4 allozyme. In conclusion, our results show that NAT2 acetylator genotype has an important role in MOCA metabolism and suggest that risk assessments related to MOCA exposures consider accounting for NAT2 acetylator phenotype in the analysis.

Global transcriptional response of Aspergillus niger in the process of glucoamylase fermentation

BackgroundIn the phase of oxygen limitation during Aspergillus niger fermentation, the cell growth decreased, while the yield of glucoamylase increased continuously and significantly. Explanation on the changes of transcriptome profile during this process may improve our understanding on mechanisms of cell adaption and enzyme production in A. niger.ResultsThe transcriptomic data from 4 time points in oxygen limitation process of a glucoamylase production A. niger strain were analyzed. Hierarchical clustering of all samples showed that the strongest transcriptional response occurred between the early and middle stage of oxygen limitation. 515 differentially expressed genes (DEGs) were identified and were clustered into 12 expression patterns. Continuously down-regulated DEGs were significantly enriched in GO terms of the ribosome, translation, and aminoacyl-tRNA biosynthesis, and continuously up-regulated DEGs were mainly involved in GO terms of fatty acid catabolism, N-acetyltransferase activity, lipase activity, and carboxylesterase activity. Pyruvate kinase and asparagine synthetase which related to the biosynthesis of the main amino acid composition of the enzyme were significantly up-regulated. Sterol-regulatory element-binding proteins (SREBP) transcription factor SrbB was one of the most up-regulated proteins, indicating its important roles in hypoxia fermentation of A. niger.ConclusionComparative transcriptome data analysis of the fermentation revealed that the overall reduced biosynthesis of translation machine and fatty acid, acceleration of fatty acid catabolism, and increased synthesis of main amino acid compositions of glucoamylase are the key transcriptional changes during the oxygen limitation fermentation process of A. niger.

N-Acetyltransferase 2 Genotype-Dependent N-Acetylation of Hydralazine in Human Hepatocytes.

Hydralazine is used in the treatment of essential hypertension and is under investigation for epigenetic therapy in the treatment of neoplastic and renal diseases. N-acetyltransferase (NAT) 2 exhibits a common genetic polymorphism in human populations. After recombinant expression in yeast, human NAT2 exhibited an apparent Lineweaver-Burk constant (K-m) value (20.1 ± 8.8 μM) for hydralazine over 20-fold lower than the apparent K-m value (456 ± 57 μM) for recombinant human NAT1 (P = 0.0016). The apparent Vmax value for recombinant human NAT1 (72.2 ± 17.9 nmol acetylated/min/mg protein) was significantly (P = 0.0245) lower than recombinant human NAT2 (153 ± 15 nmol acetylated/min/mg protein), reflecting 50-fold higher clearance for recombinant human NAT2. Hydralazine NAT activities exhibited a robust acetylator gene dose response in cryopreserved human hepatocytes both in vitro and in situ. Hydralazine NAT activities in vitro differed significantly with respect to NAT2 genotype at 1000 (P = 0.0319), 100 (P = 0.002), and 10 μM hydralazine (P = 0.0029). Hydralazine NAT activities differed significantly (P < 0.001) among slow acetylator hepatocytes, (NAT2*5B/*5B > NAT2*5B/*6A > NAT2*6A/*6A). The in situ hydralazine N-acetylation rates differed significantly with respect to NAT2 genotype after incubation with 10 (P = 0.002) or 100 µM (P = 0.0015) hydralazine and were higher after incubation with 100 μM (10-fold) than with 10 μM (4.5-fold) hydralazine. Our results clearly document NAT2 genotype-dependent N-acetylation of hydralazine in human hepatocytes, suggesting that hydralazine efficacy and safety could be improved by NAT2 genotype-dependent dosing strategies.

SPECTROPHOTOMETRIC DETERMINATION OF IZONIAZID AND ITS DERIVATIVES BY REACTION OF FORMATION THE POLYMETHINE DYE WITH 4-OXOURACIL

The process of tuberculosis treatment requires monitoring of the concentration of antituberculosis drugs in the blood, as well as their distribution and elimination from the body to prevent overdose of antituberculosis drugs. Individualizing the dose of drugs possibly including individual activity of the enzyme N-acetyltransferase, may be determined indirectly by biotransformation product of antituberculosis drugs.&#x0D; Currently existing methods for detecting the content of the test drug acetylation of isoniazid - mass spectrometry, high performance liquid chromatography have several major limitations such as the high cost, complexity of tests methods in experimentation.&#x0D; This article demonstrates the results of development of unified methods of quantitative determination of antituberculosis drug isoniazid and its conjugate - acetylizoniazid. The basis of spectrophotometric method is the cleavage reaction of isoniazid pyridine ring to form glutaconic aldehyde and its subsequent combination with the 4-oxouracil with the formation of a polymethine dye.&#x0D; The developed method allows to carry out quantitative determination of isoniazid in the pharmaceuticals forms as well as isoniazid and its derivatives in body fluids (urine), which indirectly makes it possible to evaluate the activity of N-acetyltransferase during pharmacokinetic studies.

SPECTROPHOTOMETRIC DETERMINATION OF IZONIAZID AND ITS DERIVATIVES BY REACTION OF FORMATION THE POLYMETHINE DYE WITH 4-OXOURACIL

The process of tuberculosis treatment requires monitoring of the concentration of antituberculosis drugs in the blood, as well as their distribution and elimination from the body to prevent overdose of antituberculosis drugs. Individualizing the dose of drugs possibly including individual activity of the enzyme N-acetyltransferase, may be determined indirectly by biotransformation product of antituberculosis drugs.&#x0D; Currently existing methods for detecting the content of the test drug acetylation of isoniazid - mass spectrometry, high performance liquid chromatography have several major limitations such as the high cost, complexity of tests methods in experimentation.&#x0D; This article demonstrates the results of development of unified methods of quantitative determination of antituberculosis drug isoniazid and its conjugate - acetylizoniazid. The basis of spectrophotometric method is the cleavage reaction of isoniazid pyridine ring to form glutaconic aldehyde and its subsequent combination with the 4-oxouracil with the formation of a polymethine dye.&#x0D; The developed method allows to carry out quantitative determination of isoniazid in the pharmaceuticals forms as well as isoniazid and its derivatives in body fluids (urine), which indirectly makes it possible to evaluate the activity of N-acetyltransferase during pharmacokinetic studies.

Pharmacokinetics of the antimicrobial drug Sulfanilamide is altered in a preclinical model of vascular calcification.

In vascular smooth muscle, calcium overload is linked to advancing age. The pharmacokinetics of Sulfanilamide (SA), a compound with antibacterial properties, was evaluated in a preclinical model of vascular calcification. SA was used since it is useful to study possible modifications in the renal and hepatic management of drugs. Vascular calcification was induced by administration of a single high dose of vitamin D3 to rats (treated group) 10days before the experiments. A parallel control group was processed. The decrease of renal blood flow due to calcification of the renal arteries explains, at least in part, the decrease in the renal clearance of SA observed in treated rats. The liver metabolic function increased in treated rats as demonstrated by increases in plasma appearance rate of acetylated-Sulfanilamide (ASA), hepatic ASA content and hepatic N-acetyltransferase activity. The decrease in renal excretion of SA was not completely compensated by the hepatic metabolism increase, since the elimination rate of SA from the central compartment (K1-0 ) decreased in the treated group. In summary, in this experimental model with sustained arterial calcinosis induced by a single high dose of vitamin D3 10days before the experiments, the pharmacokinetics of an aminobenzenesulfonamide is modified, at least in part, by the increase in the activity of hepatic N-acetyltransferase and the decrease in renal blood flow. This study emphasizes the importance of considering the presence of vascular calcification when a drug dose scheme is performed, in order to optimize pharmacotherapeutic results.

Structural and functional effects of nucleotide variation on the human TB drug metabolizing enzyme arylamine N-acetyltransferase 1

The human arylamine N-acetyltransferase 1 (NAT1) enzyme plays a vital role in determining the duration of action of amine-containing drugs such as para-aminobenzoic acid (PABA) by influencing the balance between detoxification and metabolic activation of these drugs. Recently, four novel single nucleotide polymorphisms (SNPs) were identified within a South African mixed ancestry population. Modeling the effects of these SNPs within the structural protein was done to assess possible structure and function changes in the enzyme. The use of molecular dynamics simulations and stability predictions indicated less thermodynamically stable protein structures containing E264K and V231G, while the N245I change showed a stabilizing effect. Coincidently the N245I change displayed a similar free energy landscape profile to the known R64W amino acid substitution (slow acetylator), while the R242M displayed a similar profile to the published variant, I263V (proposed fast acetylator), and the wild type protein structure. Similarly, principal component analysis indicated that two amino acid substitutions (E264K and V231G) occupied less conformational clusters of folded states as compared to the WT and were found to be destabilizing (may affect protein function). However, two of the four novel SNPs that result in amino acid changes: (V231G and N245I) were predicted by both SIFT and POLYPHEN-2 algorithms to affect NAT1 protein function, while two other SNPs that result in R242M and E264K substitutions showed contradictory results based on SIFT and POLYPHEN-2 analysis. In conclusion, the structural methods were able to verify that two non-synonymous substitutions (E264K and V231G) can destabilize the protein structure, and are in agreement with mCSM predictions, and should therefore be experimentally tested for NAT1 activity. These findings could inform a strategy of incorporating genotypic data (i.e., functional SNP alleles) with phenotypic information (slow or fast acetylator) to better prescribe effective treatment using drugs metabolized by NAT1.

Season-dependent effects of photoperiod and temperature on circadian rhythm of arylalkylamine N-acetyltransferase2 gene expression in pineal organ of an air-breathing catfish, Clarias gariepinus

Arylalkylamine N-acetyltransferase (AANAT) activity, aanat gene expression and melatonin production have been reported to exhibit prominent circadian rhythm in the pineal organ of most species of fish. Three types of aanat genes are expressed in fish, but the fish pineal organ predominantly expresses aanat2 gene. Increase and decrease in daylength is invariably associated with increase and decrease in temperature, respectively. But so far no attempt has been made to delineate the role of photoperiod and temperature in regulation of the circadian rhythm of aanat2 gene expression in the pineal organ of any fish with special reference to seasons. Therefore, we studied effects of various lighting regimes (12L-12D, 16L-8D, 8L-16D, LL and DD) at a constant temperature (25°C) and effects of different temperatures (15°, 25° and 35°C) under a common photoperiod 12L-12D on circadian rhythm of aanat2 gene expression in the pineal organ of Clarias gariepinus during summer and winter seasons. Aanat2 gene expression in fish pineal organ was studied by measuring aanat2 mRNA levels using Real-Time PCR. Our findings indicate that the pineal organ of C. gariepinus exhibits a prominent circadian rhythm of aanat2 gene expression irrespective of photoperiods, temperatures and seasons, and the circadian rhythm of aanat2 gene expression responds differently to different photoperiods and temperatures in a season-dependent manner. Existence of circadian rhythm of aanat2 gene expression in pineal organs maintained in vitro under 12L-12D and DD conditions as well as a free running rhythm of the gene expression in pineal organ of the fish maintained under LL and DD conditions suggest that the fish pineal organ possesses an endogenous circadian oscillator, which is entrained by light-dark cycle.