N-acetyl transferase 2 polymorphisms associated with isoniazid pharmacodynamics: molecular features for ligand interaction.

BACKGROUND: Isoniazid is mainly metabolized by arylamine N-acetyltransferase 2 (NAT2). Rapid acetylator types have NAT2*4/*4 alleles. Intermediate acetylator types have any of the following alleles: NAT2*4/*5, *4/*6, or *4/*7. Slow acetylator types do not have the NAT2*4 allele. We examined molecular features of these NAT2 molecules. MATERIALS AND METHODS: Structures of NAT2*5, *6, and *7 were constructed based on X-ray data of human NAT2*4 using a molecular modeling technique. RESULTS: The NAT2*4 molecule mostly occupied a positive electrostatic potential field. Ile(114) and Arg(197), which are mutation sites of NAT2*4 to NAT2*5 and NAT2*6, were located in the peripheral part of the positive field. Gly(286), the mutation site from NAT2*4 to NAT2*7, was located near coenzyme A (CoA) in the boundary of the positive and negative fields. CONCLUSION: Nonbinding energies between NAT2s and isoniazid were larger than those of CoA. Molecular polymorphism appears to influence the reactivity between NAT2 and the external ligand.

Effects of N-acetyltransferase 2 (NAT2), CYP2E1 and Glutathione-S-transferase (GST) genotypes on the serum concentrations of isoniazid and metabolites in tuberculosis patients.

For the purpose of a side-effect monitoring of isoniazid (INH), we investigated the relationship between the genotypes of drug-metabolizing enzymes involved in INH metabolism and the serum concentrations of INH and its metabolites in 129 tuberculosis patients hospitalizing in the National Hospital Organization Chiba-East Hospital. Genotype distributions of N-acetyltransferase 2 (NAT2), CYP2E1*5B, CYP2E1*6, Glutathione-S-transferase (GST) M1 and GST T1 were similar to those already reported in Japanese populations. Acetylating pathway of INH to acetyl isoniazid (AcINH) tended to shift to the hydrolytic pathway generating hydrazine (Hz) with the increase of mutant alleles in NAT2 gene. Serum concentration of Hz was significantly higher in slow acetylators than in rapid acetylators of NAT2. And also, serum concentration of Hz was significantly higher in the group that showed a high concentration of rifampicin (RFP) than in which RFP was not detected. The effect of CYP2E1 gene polymorphisms on the serum concentration of Hz was rarely observed, while that of GST gene polymorphism was observed in intermediate acetylators of NAT2. Hz tended to accumulate in patients with GST M1 null genotype. Therefore, it is conceivable that the risk factors of Hz accumulation are as follows: NAT2 slow acetylator phenotype, high concentration of serum RFP, and GST M1 null genotype. In these cases, we think it's necessary to pay attention to the development of hepatic disorder caused by Hz.

Functional analysis of transcriptional activity of cytosine and adenine (CA) repeats polymorphism in the estrogen receptor beta gene.

Cytosine and adenine (CA) repeats polymorphism (D14S1026) iterating "cytosine and adenine" nucleotide motifs is one of the genomic microsatellites in intron 5 of the estrogen receptor beta (ER beta) gene (14q22-24). Relations between CA repeats polymorphism and several diseases have been shown. Although the relation between number of CA repeats and gene transcription has been actively studied using several genes, results have remained contradictory until this time. In this study, we examined the functional effects of CA repeats polymorphism on transcriptional activity based on our knowledge of the ER beta gene. After preparing four types of reporter gene constructs containing 15, 18, 24 or 27 CA repeats, luciferase reporter gene assays were performed. Relative luciferase activities of these constructs were not significantly different from that of the no inserted vector and variation of CA repeats did not affect these activities. Our results indicate that CA repeats polymorphism might not only affect transcriptional activity but also other processes of gene expressions. Further studies are needed to clarify the specific functions of CA repeats polymorphism in the ER beta gene.

Methylenetetrahydrofolate reductase and reduced folate carrier-1 genotypes and methotrexate serum concentrations in patients with rheumatoid arthritis.

We investigated the genotypes of methylenetetrahydrofolate reductase (MTHFR) and reduced folate carrier-1 (RFC-1), and the serum concentrations of methotrexate (MTX) in 100 outpatients with rheumatoid arthritis (RA). Frequencies of MTHFR C677T and A1298C were similar to those reported in Japanese RA patients, while frequencies of RFC-1 G80A genotypes differed from those reported in RA patients in the United States. No correlations were found between these genotypes and serum MTX levels.

Sex differences in PPARgamma expressions in rat adipose tissues.

Clinical studies had suggested that there were sex differences in pharmacological and side effects of pioglitazone. However, there are few studies on the sex differences of peroxisome proliferator-activated receptor (PPAR) gamma expressions in rat adipose tissues. We investigated the sex differences in peroxisome proliferator-activated receptor (PPAR) gamma expressions in rat adipose tissues. Subcutaneous abdominal adipose tissues and perigonadal adipose tissues were obtained from male and female Wistar rats (12 weeks of age). Expressions of PPARgamma protein were determined by Western blot analysis with anti-PPARgamma antibody. Vaginal smear check was performed in female rats. We obtained adipose tissues from females, according to the different phases of the estrous cycle. Both PPARgamma1 and gamma2 subtypes were expressed in subcutaneous adipose tissues. Expressions of PPARgamma2 in subcutaneous adipose tissues were significantly higher in males than in females. On the other hand, expressions of PPARgamma2 in perigonadal adipose tissues were significantly higher in females than in males. Expressions of PPARgamma2 in perigonadal adipose tissues in females significantly decreased during diestrus. It can be suggested that the sex hormones might affect the expressions of PPARgamma2 in adipose tissues.

A comparative study of histamine activities on differentiation of osteoblasts and osteoclasts.

The effects of histamine and its receptor antagonists on mouse bone marrow cells (MBMC) and MC3T3-E1 cells were studied to elucidate the precise molecular mechanisms underlying histamine activities in the respective cell types. The studied parameters were osteoclast differentiation and expressions of receptor activator of nuclear factor kappaB ligand (RANKL), histamine receptors (HR), and osteoblast differentiation markers. The osteoclastogenesis was assessed by TRAP-dye method. Expressions of RANKL, HR and the osteoblast differentiation markers were evaluated by RT-PCR analysis. In MBMC, 1 microM histamine doubled the number of osteoclast-like cells in a dose-dependent manner. Expressions of RANKL peaked at histamine concentrations of 1 microM and 0.1 microM in MBMC and MC3T3-E1, respectively. H(1)R antagonist, but not H(2)R antagonist, inhibited RANKL expressions induced by histamine in MC3T3-E1. Histamine induced expressions of cell differentiation markers in MC3T3-E1, but not in MBMC, under the conditions that RANKL expressions were induced by histamine in both types of cells. These results indicate the following: (1) Histamine induction of osteoclastogenesis is mediated by RANKL expressed via H(1)R, but not via H(2)R in mouse osteoblast-like cells; (2) and the major target of histamine action is the RANKL-RANK signaling pathway in osteocytes. This observation is consistent with the traditionally recognized histamine action of bone resorption at the osteoclast site.