Search

Heterocyclic amines (HCAs) are formed when cooking meats at high temperatures or for long periods of time, and their major metabolic pathway includes hepatic N‑hydroxylation by CYP1A2 followed by O-acetylation by N-acetyltransferase 2 (NAT2). NAT2 is a highly polymorphic gene involved in metabolism of drugs and xenobiotics, and its single nucleotide polymorphisms (SNPs) produce phenotypes characterized as slow, intermediate, or rapid acetylator. Recent studies have documented associations between HCAs and insulin resistance and between certain NAT2 phenotypes and insulin resistance. In this study, we demonstrated that NAT2 activity and exposure to certain HCAs may alter cellular glucose and lipid homeostasis. Additionally, these results demonstrate changes in sensitivity to insulin following exposure to some HCAs. These findings are significant because they indicate a genetic component (NAT2 polymorphism) and an environmental component (dietary HCA exposure via consumption of cooked meat) in the pathogenesis of insulin resistance in hepatocytes of human origin.

Background: Tuberculosis (TB) is one of the top ten causes of death by a single infectious agent. Extensively Drug-Resistant TB (XDR-TB) is a type of MDR TB, defined as TB resistant to at least isoniazid and rifampicin, plus any fluoroquinolone and at least one of three injectable second-line drugs. XDR-TB is considered a significant public health threat in Thailand because of the rising of reported XDR-TB cases, the more financial burden for both patient and provider sites, and more adverse events in XDR-TB treatment. This dissertation examines the biopsychosocial factors, such as a) whether non-adherence to the MDR-TB treatment regimen is a leading cause of development to XDR-TB, b) which NAT2 diplotype or HLA class 2 alleles associated with XDR-TB cases, c) the pattern of initial culture conversion in XDR-TB group while on MDR-TB treatment compare to MDR-TB group.Methods: We conducted a matched case-control study and recruited all secondary XDR-TB cases that were reported to the Bureau of Tuberculosis from October 2014 to June 2019. A total of 45 secondary XDR-TB cases were enrolled at 32 original hospital sites, across 29 provinces in Thailand. MDR-TB controls were randomly selected from the same hospital, where XDR-TB cases were treated. The proportion of cases to controls was one XDR-TB to two MDR-TB matched by geographical region. In the first two studies (Chapter 2-3), nine of 45 secondary XDR-TB cases died before we started. Then, only 36 XDR-TB cases and 75 MDR-TB controls were available for interviews and genetic tests. The conditional logistic regression was used to assess the magnitude of the non-adherence effect. Fisher exact test and conditional logistic regression were also used to explore the association between NAT2 diplotype, HLA class 2 alleles, and XDR-TB cases. In the third study (Chapter 4), we could not find the record of sputum culture in seven of 45 secondary XDR-TB cases. We used Cox proportional hazard regression for the difference in time to initial culture conversion in 38 XDR-TB cases and 76 MDR-TB controls. We also used the sensitivity and specificity of initial culture conversion at the beginning of MDR-TB treatment to 15-month in the prediction of the XDR-TB susceptibility and MDR-TB treatment outcome.Results: We found that patients with non-adherence while on MDR-TB treatment were ten times more likely to develop XDR-TB (AOR 10.08, 95%CL 1.36, 74.87) while adjusted for type of Directly Observed Treatment, age, gender, socioeconomic status, and harmful alcohol use which was less than the magnitude from improper regimen (AOR = 26.89, 95%CL 7.39, 97.81) while adjusted for culture sending and region. For the genetic study, NAT2*4/5B (OR 3.9, 95%CI 1.2 13.0) and the heterozygosity of HLA-DRB1 16:02 were about four times associated with XDR-TB cases (OR 3.7, 95%CI 1.2, 11.9). None of the associations was found in HLA DQB1. For the third study, the improper regimen for MDR-TB treatment was found to significantly reduce the chance of initial sputum culture conversion (aHR 0.24, 95%CL 0.15, 0.38) adjusted for wrong drug administration and dosage, and follow-up sputum culture. Furthermore, positive culture at the end of four-month of treatment was a good predictor for failure outcome (sensitivity 67.7%, specificity 93.8%) and the development of XDR-TB (sensitivity 63.2%, specificity 94.7%). Conclusion: The main factor that causes XDR-TB was not non-adherence; the more significant factor was improper regimens for MDR-TB treatment, which mainly came from the provider site. From the aspect of host genetic, the result of this study was found significant in NAT2 *4/5B and heterozygosity of HLA-DRB1 16:02. There was no clinically significant for NAT2. However, HLA-DRB1 16:02 is associated with acquired immunodeficiency that can make the host more likely to develop XDR-TB. For the follow-up of sputum culture conversion, the improper MDR-TB treatment regimen also played a significant role in reducing the chance of sputum culture conversion. Also, the lack of sputum culture conversion at month four would give us the best prediction for the development of XDR-TB. Overall, the finding from these three studies suggested the probable risk and protective factors of XDR-TB. From a policy perspective, further work is needed to examine the impact of the new DR-TB guidelines on the actual MDR-TB treatment regimen and assess the effectiveness of the treatment audit system when it starts.

Cigarette smoking in women has been associated with adverse reproductive outcomes such as reduced ovarian reserve, poorer in vitro fertilization (IVF) outcomes and increased adverse pregnancy outcomes. This study examined the association of smoking with ovarian reserve in a cross-sectional study of women seeking fertility treatment, and potential effect modification by race and NAT2 acetylator phenotype. Data from 265 women from the Louisville Tobacco Smoke, Genetic Susceptibility, and Infertility (LOUSSI) Study were analyzed. A total of 265 women were recruited through a single infertility clinic between September 2016 and June 2018. Information on current smoking status was assessed using a structured questionnaire and confirmed by cotinine assay. Single nucleotide polymorphisms in NAT2 were genotyped to determine acetylator status and serum anti-Müllerian hormone (AMH) level was used to assess ovarian reserve. The association of smoking with ovarian reserve was assessed using linear and logistic regression models with adjustment for potential confounders. Effect modification by race and NAT2 phenotype were assessed by including interaction terms in the regression models. Overall, smoking was not significantly associated with ovarian reserve. Results suggest that heavy smoking and higher pack-years of exposure may decrease ovarian reserve. Although most associations were not statistically significant, the effect of smoking on ovarian reserve was more pronounced among non-Hispanic Black women and slow NAT2 acetylators. These results are based on a small clinical population and require replication in a larger and more representative study population.

The present work explores the androgen regulation of N-acetyltransferase 2 (Nat2*) gene expression in CD1 mouse kidney. COS-1 expression of mouse Nat1*, Nat2*8, and Nat3* revealed (a) that p-aminobenzoic acid (PABA), p-aminobenzoylglutamate acid, 5-aminosalicylic acid (5-ASA), and 2-aminofluorene are Nat2-selective substrates, (b) that isoniazid and sulfamethazine are Nat1-selective substrates, and (c) that mouse Nat3 only exhibits N-acetylating activity towards 5-ASA. To explore tissue- and gender-specific expression of Nat2 in CD1 mouse strain, the developmental pattern of kidney and liver acetylating activity was studied from birth to 80 days of age. In the liver, PABA-Nat2 activity showed developmental but no gender-specific differences. The kidney Nat2 activity showed a 2.5- and 5.8-fold increase in female and male mice, respectively, at 80 days. Immunoblot analysis revealed an increase in kidney Nat protein (2-fold) only in males at 80 days of age. Although, liver Nat protein levels showed a modest increase during development, we found no differences between males and females. The Nat2 mRNA levels determined by ribonuclease protection assay showed that the Nat2 mRNA levels in mouse kidney of males increased significantly during development while the Nat2 transcript levels remained unchanged in females. There were no gender-associated differences of Nat2 mRNA levels in mouse liver. These observations suggest that the androgenic modulation of kidney Nat2 is accomplished by an increase in transcript levels which is translated into an increase in Nat2 enzymatic protein. In addition, the $5\sp\prime$-untranslated region of mouse Nat2* was sequenced and characterized. Primer extension analysis and RNase protection assays exposed multiple transcription initiation sites ($-$112 to $-$151nt). Computer sequence analysis revealed a promoter-like region consisting of TATA boxes, a CAAT box, Sp1 binding site, a palindromic hormone response element (HRE), and an AP-1 binding site. Transient expression of CAT reporter constructs of the mouse Nat2*-HRE demonstrated positive regulation of the HSV-tk1 promoter which is dependent on the presence of hormone and steroid receptor. Thus, these results suggest that the HRE found in the promoter region of Nat2* is a potential candidate for the mediation of androgenic regulation of Nat2* in mouse kidney.

N-Acetyltransferase (NAT) N-acetylates various arylamine drugs (e.g., p-aminosalicylate, p-aminobenzoate, and isoniazid), and carcinogens (e.g., $\beta$-naphthylamine, 2-aminofluorene, and benzidine). It is well established that a polymorphism is associated with the human NAT2* locus. Phenotypic dissimilarities in acetylator status between individuals may have undesirable clinical consequences, such as impaired therapeutic efficacy in rapid acetylators and increased drug toxicity in slow acetylators. The aim of this thesis is twofold: (1) to provide molecular genetic and biochemical evidence for the genotypic and acetylator phenotypic identity of congenic mice with the parental strains of origin, and to further characterize the mouse NAT2*9 polymorphism; and (2) to rigorously investigate the biochemical events associated with structurally variant N-acetyltransferase alleles at the human NAT1* locus. Congenic mouse strains were developed in order to eliminate differences due to other polymorphic background genes, and these strains may give us an indication of the influence of the genetic background on the expression of the individual NAT* genes. The data demonstrate that congenic acetylator mouse strains were actually constructed, since the congenic strains were genotypically and phenotypically identical to the corresponding parental strains of origin. A novel mechanism of slow acetylation was disclosed, namely, that the Asp99Ile change in NAT2*9 from slow acetylator mice does not hinder the synthesis of hepatic NAT2 9 protein, but, rather, leads to the production of a conformationally modified NAT2 molecule that resists degradation by tissue proteases but is labile and catalytically impaired. Four human NAT1* variants have been identified in our laboratory, namely, NAT1*4, *10, *11, and *16. NAT1*4, *10, and *16 have identical coding regions, whereas NAT1*11 contains two amino acid substitutions; each NAT1* variant is associated with a unique 3$\sp\prime$ untranslated region. The coding region and 3$\sp\prime$ untranslated region events were studied in a eukaryotic expression system, whereas the coding region events were studied in a prokaryotic expression system. The results indicate that the 3$\sp\prime$ untranslated region may determine the NAT1 acetylator status of an individual by producing less NAT1 protein, this represents a novel mechanism of slow acetylation.