Claudine Deloménie

Genotyping of the polymorphic N-acetyltransferase (NAT2*) gene locus in two native African populations.

The hepatic N-acetyltransferase enzyme encoded by the NAT2* gene locus is responsible for the human polymorphic acetylation of numerous arylamine or hydrazine-containing drugs and xenobiotics including AIDS-related therapeutic agents such as isoniazid and sulphonamides. The genetic basis underlying the human acetylation polymorphism has been extensively studied in several populations but native African populations were poorly documented. In the present study, 117 unrelated black Africans, namely Dogons from Mali and Gabonese, were investigated for NAT2* allelic variability and genotype distribution. Thirteen NAT2* alleles were unambiguously identified by combined use of allele-specific reamplifications and restriction endonuclease digestions. Our results confirm the African origin of G191->A substitution in the NAT2* coding region which was previously associated with slow acetylation in African-Americans. The finding of high allelic diversity in the studied populations is consistent with the hypothesis of a single African origin for NAT2*-associated polymorphism. Finally, no excess of the slow acetylator phenotype is predicted in these populations, implying no need for fitting NAT2* polymorphism-sensitive therapies to black Africans, compared to Caucasians.

Identification and Functional Characterization of Arylamine N-Acetyltransferases in Eubacteria: Evidence for Highly Selective Acetylation of 5-Aminosalicylic Acid

Arylamine N-acetyltransferase activity has been described in various bacterial species. Bacterial N-acetyltransferases, including those from bacteria of the gut flora, may be involved in the metabolism of xenobiotics, thereby exerting physiopathological effects. We characterized these enzymes further by steady-state kinetics, time-dependent inhibition, and DNA hybridization in 40 species, mostly from the human intestinal microflora. We report for the first time N-acetyltransferase activity in 11 species of Proteobacteriaceae from seven genera: Citrobacter amalonaticus, Citrobacter farmeri, Citrobacter freundii, Klebsiella ozaenae, Klebsiella oxytoca, Klebsiella rhinoscleromatis, Morganella morganii, Serratia marcescens, Shigella flexneri, Plesiomonas shigelloides, and Vibrio cholerae. We estimated apparent kinetic parameters and found that 5-aminosalicylic acid, a compound efficient in the treatment of inflammatory bowel diseases, was acetylated with a catalytic efficiency 27 to 645 times higher than that for its isomer, 4-aminosalicylic acid. In contrast, para-aminobenzoic acid, a folate precursor in bacteria, was poorly acetylated. Of the wild-type strains studied, Pseudomonas aeruginosa was the best acetylator in terms of both substrate spectrum and catalytic efficiency. DNA hybridization with a Salmonella enterica serovar Typhimurium-derived probe suggested the presence of this enzyme in eight proteobacterial and four gram-positive species. Molecular aspects together with the kinetic data suggest distinct functional features for this class of microbial enzymes.

Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop.

Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560-564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29-131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122-131 in NAT1 model and residues 122-133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.

Association of GSTT1 non-null and NAT1 slow/rapid genotypes with von Hippel-Lindau tumour suppressor gene transversions in sporadic renal cell carcinoma.

The von Hippel-Lindau (VHL) tumour suppressor gene is commonly mutated in renal cell carcinoma of clear cell type (CCRCC). We investigated the possible relationship between VHL mutations in sporadic CCRCC and polymorphism of genes encoding enzymes involved in carcinogen metabolism: two cytochrome P450 monooxygenases (CYP1A1 and CYP2D6), one NAD[P]H:quinone oxidoreductase (NQO1), three glutathione S-transferases (GSTM1, GSTT1 and GSTP1) and two arylamine N-acetyltransferases (NAT1 and NAT2). We analysed DNA from tumour and nontumoural kidney tissue from 195 CCRCC patients. Single VHL mutations were identified in 88 patients and double mutations were present in two patients. Nine of 18 transversions were GC to TA, four were AT to TA, four were GC to CG and one was AT to CG. Ten of 19 transitions were GC to AT and nine were AT to GC. We also identified 53 frameshifts and two GC to AT at CpG. An excess of transversions was observed in a subset of patients with active GSTT1 [GSTT1 (+) genotype] and probably defective NAT1 (NAT1 S/R variant genotype). All 18 transversions were in GSTT1 (+) patients, whereas only 76% of transitions (P = 0.05) and 81% of the other mutations (P = 0.06) occurred in this genotype. We found that 28% of the transversions were in the NAT1 S/R genotype versus 12% of the transitions (P = 0.40) and 4% of the other mutations (P = 0.01). This suggests that pharmacogenetic polymorphisms may be associated with the type of acquired VHL mutation, which may modulate CCRCC development.

Hyaluronic acid-conjugated lipoplexes for targeted delivery of siRNA in a murine metastatic lung cancer model.

We have investigated the impact of hyaluronic acid (HA)-coating on the targeting capacity of siRNA lipoplexes to CD44-overexpressing tumor cells. Cellular uptake and localization of HA-lipoplexes were evaluated by flow cytometry and fluorescence microscopy and both methods showed that these lipoplexes were rapidly internalized and localized primarily within the cytoplasm. Inhibition of luciferase expression on the A549-luciferase lung cancer cell line was achieved in vitro using an anti-Luc siRNA. 81% of luciferase gene expression inhibition was obtained in vitro with HA-lipoplexes at +/- ratio 2. In vivo, in a murine A549 metastatic lung cancer model, the treatment with HA-lipoplexes carrying anti-luciferase siRNA led to a statistically significant decrease of luciferase expression as opposed to progressive increase with non-modified lipoplexes or NaCl 0.9%. The reduction of the expression of luciferase mRNA tumor of mice treated with HA-lipoplexes supported the inhibition effect due to siRNA. These results highlight the potential of HA-lipoplexes in CD44-targeting siRNA delivery.