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Le site de Sanofi situé à Mourenx, dans le bassin industriel de Lacq (64), produit un médicament antiépileptique, la Dépakine®, dont le principe actif est le valproate de sodium (VPS). Ce site est une installation classée pour la protection de l’environnement (ICPE) soumise à autorisation au titre de l’article L.511-1 et suivants du Code de l’environnement. Les rejets dans l’environnement d’une telle installation sont encadrés par des limites établies sur la base d’une évaluation quantitative des risques sanitaires (EQRS). En 2017, Sanofi Chimie a réalisé une évaluation de l’impact sur la santé et l’environnement des rejets atmosphériques de valproate, en utilisant des valeurs toxicologiques de référence (VTR) à seuil par voie orale, cutanée et respiratoire proposées par le bureau d’étude CEHTRA (Consultancy for Environmental & Human Toxicology and Risk Assessment). Parmi les différentes VTR proposées par CEHTRA, Sanofi a utilisé les VTR fondées sur des effets tératogènes. L’EQRS conduite par la société AECOM pour le compte de Sanofi, en vue d’estimer les risques pour les riverains et les professionnels travaillant à proximité du site, a conclu que « les risques sanitaires liés aux rejets actuels et passés de valproate [étaient] inférieurs aux valeurs de référence » proposées par CEHTRA. L’Anses a été saisie le 29 juin 2018 par la DGS et la DGPR afin de mener, en urgence, une analyse critique des VTR du valproate élaborées pour le compte de Sanofi et utilisées dans le cadre de cette EQRS. Dans son avis du 12 juillet 2018, l’Anses n’a pas remis en cause le choix de construire une VTR à seuil mais n’a pas retenu les VTR élaborées par les deux bureaux d’études, EQUITOX (2015) et CEHTRA (2017), à partir des données animales et/ou humaines car de nombreuses données chez l’Homme disponibles dans la littérature n’avaient pas été prises en compte (Anses, 2018). Dans ce contexte, et au titre de la sécurité des professionnels exposés au valproate, une campagne de mesures de la concentration d’acide valproïque (VPA) dans le sang des employés de l’usine a été organisée par le service de santé au travail de l’entreprise du 27 novembre à la mi-décembre 2018. Le groupe d'alerte en santé travail (GAST) de Nouvelle-Aquitaine a été associé au suivi de cette campagne. Pour l’analyse de cette campagne de mesures, l’entreprise Sanofi s’est référée à une valeur biologique repère dans le sang de 5 mg.L-1. Ainsi, la DGT, la DGS et la DGPR ont saisi l’Anses, le 5 avril 2019, pour mener, en urgence, une analyse critique de la valeur biologique utilisée comme repère par Sanofi pour évaluer l’imprégnation de ses travailleurs. Au vu des données disponibles, le choix du dosage du valproate dans le sang n’a pas été remis en cause. Néanmoins, au regard de la cinétique d’élimination plasmatique de cette substance et de l’existence de métabolites urinaires, il n’a pas été exclu qu’un autre biomarqueur puisse être un meilleur témoin de l’exposition agrégée sur une période plus longue. Concernant le calcul de la valeur limite biologique (VLB), l’approche basée sur la dose thérapeutique est à privilégier mais la dose thérapeutique de 1200 mg.j-1 retenue pour le calcul de la VLB est critiquable compte tenu des données actuellement disponibles. En effet, des effets indésirables, notamment reprotoxiques, pourraient être observés à des doses inférieures à 1200 mg.j-1. Par ailleurs, plusieurs incohérences ou un manque de justification de certains choix ont été relevés lors du calcul de VLB, en particulier au niveau de l’application des facteurs d’incertitude/de protection. Par conséquent, la VLB de 5 mg.L-1 utilisée par Sanofi n’a pas été retenue. Enfin, ne disposant pas de suffisamment d’informations, il n’a pas été possible d’apporter un regard critique et de se prononcer sur la méthode d’analyse et les modalités de prélèvement. L’Anses a, par conséquent, recommandé : ‐ de réaliser une revue approfondie de la littérature prenant en compte les données les plus récentes, en particulier chez l’Homme ; ‐ d’évaluer de manière approfondie la possibilité de recommander des valeurs de référence pouvant être utilisées pour la surveillance biologique des expositions professionnelles au valproate (Anses, 2019). Au regard des différents éléments, l’Anses a été saisie le 28 septembre 2018 par la DGS et la DGPR pour élaborer une VTR chronique par inhalation pour le valproate de sodium, puis en avril 2019 par la DGT, la DGS et la DGPR pour définir des valeurs de référence pouvant être utilisées pour la surveillance des expositions professionnelles au valproate.

We showed previously that tungsten carbide–cobalt (WC–Co) nanoparticles (NP) can be used as a nanoparticulate positive control in some in vitro mammalian genotoxicity assays. Here, we investigate the mechanisms of action involved in WC–Co NP genotoxicity in L5178Y mouse lymphoma cells and primary human lymphocytes, in vitro. Data from the micronucleus assay coupled with centromere staining and from the chromosome-aberration assay show the involvement of both clastogenic and aneugenic events. Experiments with the formamidopyrimidine DNA glycosylase (FPG)-modified comet assay showed a slight (non-significant) increase in FPG-sensitive sites in the L5178Y mouse lymphoma cells but not in the human lymphocytes. Electron paramagnetic resonance spin-trapping results showed the presence of hydroxyl radicals (radical dotOH) in WC–Co NP suspensions, with or without cells, but with time-dependent production in the presence of cells. However, a significant difference in radical dotOH production was observed between human lymphocytes from two different donors. Using H2O2, we showed that WC–Co NP can participate in Fenton-like reactions. Thus, radical dotOH might be produced either via intrinsic generation by WC–Co NP or through a Fenton-like reaction in the presence of cells.

The CYP4A subfamily is known to ω-hydroxylate the endogenous arachidonic acid into 20-hydroxyeicosatetranoic acid, which has renovascular and tubular functions. The aim of this work was to report a comprehensive investigation of the CYP4A11 and CYP4A22 genetic polymorphisms in a French population. Using PCR-SSCP and sequencing strategies, a total of 26 sequence variations were identified comprising 3 missense mutations for CYP4A11 (Ser404Phe, Phe434Ser and Arg505His) and 7 missense mutations for CYP4A22 (Arg126Trp, Gly130Ser, Asn152Tyr, Val185Phe, Cys231Arg, Leu428Pro and Leu509Phe). In comparison with SNPs reported in the database (dbSNP) of the National Center for Biotechnology information (NCBI), 6 and 3 novel polymorphisms were identified in CYP4A11 and CYP4A22, respectively. The potential impact of the amino acid substitutions on the structure and/or catalytic activity of the enzymes has been estimated by the construction and validation of the CYP4A 3D models. These results could be helpful for further investigations of the potential role of CYP4A variants in the genetic susceptibility to cardiovascular diseases in humans such as arterial hypertension.

The genetic polymorphism affecting the CYP3A5 enzyme is responsible for interindividual and interethnic variability in the metabolism of CYP3A5 substrates. The full extent of the CYP3A5 genetic polymorphism was analysed in French Caucasian, Gabonese and Tunisian populations using a polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) strategy. In the three populations, eight, 17 and ten single nucleotide polymorphisms (SNPs), respectively, were identified, among which nine correspond to rare new mutations. Also identified were 16 alleles including eight new allelic variants. Significant differences were observed in the distribution of these alleles. Particularly, the frequency of the CYP3A5*3C null allele in French Caucasians (81.3%) and in Tunisians (80.0%) is higher than in the Gabonese population (12.5%) (p < 0.001). Considering the CYP3A5 genotypes of the tested individuals, only 10.4% of French Caucasians and 30.0% of Tunisians were identified as CYP3A5 expressors. In contrast, 90.0% of Gabonese subjects appear to express the CYP3A5 protein.

The human cytochrome CYP4F12 has been shown to be active toward inflammatory mediators and exogenous compounds such as antihistaminic drugs. In the present study, we report the first investigation of polymorphisms in the human CYP4F12 gene. A screening for sequence variations in the 5'-flanking region was performed by a Polymerase Chain Reaction-Single Strand Conformational Polymorphism (PCR-SSCP) strategy, using DNA samples from 53 unrelated French individuals of Caucasian origin. Several polymorphisms were identified, comprising a large deletion located in intron 1 (CYP4F12*v1), two isolated substitutions -402G>A (CYP4F12*v3) and -188 T>C (CYP4F12*v4) and nine combined mutations, -474T>C, -279A>C, -224A>G, -173G>A, -145C>G, -140T>C, -126T>C, -56T>C, and -21T>G (CYP4F12*v2). Considering the nature and location of the polymorphisms characterizing the CYP4F12*v1 and *v2, the functional relevance of those two allelic variants was further examined by transfecting different cell lines with constructs of the related region of the CYP4F12/luciferase reporter gene. Both alleles lead to a significant decrease of CYP4F12 gene expression in HepG2 cell line and, therefore, are likely to determine interindividual differences in CYP4F12 gene expression.

Le but de cette etude etait de mettre au point une methode rapide, sensible et specifique de quantification du thromboxane B2 (TXB2) par chromatographic liquide haute performance couplee a une interface d'ionisation electrospray et a une detection par spectrometrie de masse (CLHP-ESI-SM), a partir de microsomes de levure exprimant la thromboxane synthase (CYP5A1) humaine. Ce metabolite est le produit d'hydratation stable du thromboxane A2 (TXA2) produit par le CYP5A1 a partir de son substrat, la prostaglandine H2 (PGH2). La quantification du TXB2 a ete realisee a l'aide d'un standard interne deutere, le 6-ceto-PGF1 α-d4, ajoute avant une extraction par le diethylether. La separation chromatographique s'effectue en phase inverse sur colonne XTerra MS C18. L'elution est realisee en mode isocratique (60 % acetonitrile contenant 0,05 % d'acide formique - 40 % tampon formiate d'ammonium 5 mM, pH 3,0). Le mode d'ionisation electrospray negatif a ete retenu pour une meilleure detection du TXB2 et du 6-ceto-PGF1 α-d4. Cette technique appliquee avec succes aux microsomes exprimant le CYP5A1 sauvage montre une production croissante et lineaire de TXB2 pour la gamme de concentrations de PGH2 utilisee (0-120 μM). Cette methode pourra etre appliquee a l'analyse de l'activite enzymatique de nouveaux variants de la thromboxane synthase, recemment mis en evidence au laboratoire, et exprimes dans des microsomes de levure.

Human thiopurine S-methyltransferase (TPMT) is an enzyme responsible for the detoxification of widely used thiopurine drugs such as azathioprine (Aza). Its activity is inversely related to the risk of developing severe hematopoietic toxicity in certain patients treated with standard doses of thiopurines. DNA samples from four leucopenic patients treated with Aza were screened by PCR-SSCP analysis for mutations in the 10 exons of the TPMT gene. Four missense mutations comprising two novel mutations, A83T (TPMT*13, Glu(28)Val) and C374T (TPMT*12, Ser(125)Leu), and two previously described mutations, G430C (TPMT*10, Gly(144)Arg) and T681G (TPMT*7, His(227)Gln) were identified. Using a recombinant yeast expression system, kinetic parameters (K(m) and V(max)) of 6-thioguanine S-methylation of the four TPMT variants were determined and compared to those obtained with wild-type TPMT. This functional analysis suggests that these rare allelic variants are defective TPMT alleles. The His(227)Gln variant retained only 10% of the intrinsic clearance value (V(max)/K(m) ratio) of the wild-type enzyme. The Ser(125)Leu and Gly(144)Arg variants were associated with a significant decrease in intrinsic clearance values, retaining about 30% of the wild-type enzyme, whereas the Glu(28)Val variant produced a more modest decrease (57% of the wild-type enzyme). The data suggest that the sporadic contribution of the rare Glu(28)Val, Ser(125)Leu, Gly(144)Arg, and His(227)Gln variants may account for the occurrence of altered metabolism of TPMT substrates. These findings improve our knowledge of the genetic basis of interindividual variability in TPMT activity and would enhance the efficiency of genotyping methods to predict patients at risk of inadequate responses to thiopurine therapy.

Aims To identify the molecular basis for a low CYP1A2 metabolic status, as determined by a caffeine phenotyping test, in a 71-year-old, nonsmoking, Caucasian woman who presented with very high clozapine concentrations despite being administered a standard dose of the drug. Methods The nucleotide sequence of the 7 exons, exon-intron boundaries and 5′-flanking region of the CYP1A2 gene was analysed by direct sequencing. Results Only one heterozygous point mutation was identified in the donor splice site of intron 6 (3534G > A) of CYP1A2. This mutation could cause abnormal RNA splicing and therefore lead to a truncated nonfunctional enzyme. No other carrier of this mutation was identified in a population of 100 unrelated healthy Caucasians. Conclusions This is the first report of a splice-site mutation affecting the CYP1A2 gene. This polymorphism is a likely explanation for the low CYP1A2 activity associated with high clozapine concentrations in this patient.

In the present study, we report the first systematic investigation of polymorphism in the human CYP4B1 gene. Using a strategy based on single-strand conformation polymorphism analysis of PCR products (PCR-SSCP), we analyzed the twelve exons of the gene, as well as their 5'- and 3'- proximal flanking sequences, in DNA samples from 190 French Caucasians. In addition to the wild-type CYP4B1* allele (CYP4B1*1), four variants, namely CYP4B1*2, *3, *4 and *5, were characterized. The CYP4B1*3, *4 and *5 alleles encode missense mutations Arg173Trp, Ser322Gly and Met331Ile, respectively. The fourth variant, CYP4B1*2, harbors three missense mutations (Met331Ile, Arg340Cys and Arg375Cys) and a double nucleotide deletion (AT881-882del) that causes a frameshift and premature stop codon in the second third of the coding sequence of the gene. This latter mutation can be assumed to lead to the synthesis of a severely truncated protein and, therefore, probably contributes to interindividual variability of CYP4B1 expression and enzymatic activity. In order to investigate the extent of the CYP4B1*2 allele in a large population, a rapid genotyping test, based on restriction analysis of PCR products, was developed and applied to 2082 French Caucasians. Forty-two subjects were found homozygous for the AT881-882 deletion, which suggests that about 2% of individuals should be unable to develop metabolic reactions mediated by CYP4B1. Given the relatively high frequency and the functional consequences of the CYP4B1*2 allele, associations between CYP4B1 polymorphism and certain pathological processes should be considered.

Inheritable interindividual differences in prostacyclin production may be implicated in the pathogenesis of several human vascular diseases. Using a polymerase chain reaction/single-strand conformation polymorphism strategy, we screened for mutations in the gene encoding cytochrome P450 prostacyclin synthase (CYP8A1). DNA samples from healthy French volunteers (n = 130) of Caucasian origin were examined. Five mutations, comprising two previously reported silent mutations and three novel rare missense mutations (P38L, S118R, and R379S), were identified in the coding sequence of the gene. In the 5'-proximal region, we also found a variable number of tandem repeats (VNTR) polymorphism that consisted of four different alleles with 4-6 tandem repeats of a 9-bp unit containing a putative Spl transcriptional factor binding site. One of these (R6), a frequent allele (23.6% of alleles tested) harboring six repeats, is novel, whereas the other three are known. In vitro analysis of the effect of each VNTR allele on promoter activity of a reporter gene was performed by a transient transfection assay. Data confirmed the modulator effect of the VNTR polymorphism on reporter gene transcription. Furthermore, the data demonstrate that allele R6 has the most potent inducing effects in the A549 cell line and, after IL-6 stimulation, in human pulmonary artery endothelial cells. Overall, the data demonstrate that CYP8A1 is polymorphic in Caucasians, and that a polymorphism affecting the 5'-proximal region may result in interindividual differences in CYP8A1 transcriptional regulation in vivo. Additional factors, such as the presence of inflammatory mediators, may be required to modulate transcription of the CYP8A1 gene.

Thromboxane synthase (CYP5A1) catalyzes the conversion of prostaglandin H2 to thromboxane A2, a potent mediator of platelet aggregation, vasoconstriction and bronchoconstriction. It has been implicated in the patho-physiological process of a variety of diseases, such as atherosclerosis, myocardial infarction, stroke and asthma. On the basis of the hypothesis that variations of the CYP5A1 gene may play an important role in human diseases, we performed a screening for variations in the human CYP5A1 gene sequence. We examined genomic DNA from 200 individuals, for mutations in the promoter region, the protein encoding sequences and the 3'-untranslated region of the CYP5A1. Eleven polymorphisms have been identified in the CYP5A1 gene including eight missense mutations R61H, D161E, N246S, L357V, Q417E, E450K, T451N and R466Q. This is the first report of genetic variants in the human CYP5A1 altering the protein sequence. The effect of these variants on the metabolic activity of CYP5A1 remains to be further evaluated.

One major interest to analyse the extent of N-acetyltransferase 1 (NAT1*) allelic variation in the human population stems to a great extent from the possible association of interindividual differences in the metabolism of aromatic amines with certain chemically induced diseases, including cancer. Co

1. Characterization of allelic variants of the TPMT gene (TPMT) responsible for changes in TPMT activity, and elucidation of the mechanism by which these alleles act, are required because of the clinical importance of this polymorphism for patients receiving thiopurine drugs. 2. We defined the mutational and allelic spectrum of TPMT in a group of 191 Europeans. Using PCR-SSCP, we screened for mutation the entire coding sequence, the exon-intron boundaries, the promoter region and the 3'-flanking region of the gene. Six mutations were detected throughout the ten exons and seven TPMT alleles were characterized. Four of them, TPMT*2, *3A, *3C and *7, harbouring the known mutations, G238C, G460A, A719G or T681G, were nonfunctional and accounted for 0.5, 5.7, 0.8 and 0.3% of the allele totality, respectively. 3. Within the promoter region, six alleles corresponding to a variable number of tandem repeats (VNTR), were identified. VNTR*V4 and *V5a which harbour four or five repeats of a 17-18 bp unit, were the most frequent (55% and 34%, respectively). The other VNTR alleles, having from five to eight repeats, were rarer. 4. The TPMT phenotype was correctly predicted by genotyping for 87% of individuals. A clear negative correlation between the total number of repeats from both alleles and the TPMT activity level was observed, indicating that VNTRs contribute to interindividual variations of TPMT activity. Therefore, additional analysis of the promoter region of TPMT can improve the phenotype prediction rate by genotyping.

To detect mutations in the thiopurine S-methyltransferase gene (TPMT), we have developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The sensitivity of the method was first evaluated by analyzing DNA samples from five individuals, including two high methylators (HMs), two intermediate methylators (IMs), and one deficient methylator (DM). TPMT alleles and mutations in each of these individuals had previously been characterized by conventional PCR-based assays and direct sequencing analysis. All mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments, allowing their identification. We further tested the efficiency of the strategy to detect new TPMT mutations. For this purpose, additional DNAs from 15 IMs and 15 HMs were submitted to PCR-SSCP analysis. A total of 7 alleles were characterized, including two new alleles. The first one, termed TPMT*1A, harbors a single mutation C-->T at nucleotide -178 in exon 1 and was detected in a HM subject. The second one, termed TPMT*7, was characterized by a T-->G transversion at nucleotide 681 in exon 10. This allele should be a nonfunctional allele of the TPMT gene since it was observed in combination with a wild-type allele in an intermediate methylator. We conclude that the PCR-SSCP strategy we developed could be advantageously used to fully characterize the extent of allelic variation at the TPMT gene locus in populations and thus to improve our understanding of the genetic polymorphism of TPMT activity, which has considerable consequences for the toxicity and efficacy of therapeutically important and widely used drugs.

With the increasing human exposure to nanoparticles (NP), the evaluation of their genotoxic potential is of significant importance. However, relevance for NP of the routinely used in vitro genotoxicity assays is often questioned, and a nanoparticulate reference positive control would therefore constitute an important step to a better testing of NP, ensuring that test systems are really appropriate. In this study, we investigated the possibility of using tungsten carbide-cobalt (WC-Co) NP as reference positive control in in vitro genotoxicity assays, including 2 regulatory assays, the mouse lymphoma assay and the micronucleus assay, and in the Comet assay, recommended for the toxicological evaluation of nanomedicines by the French Agency of Human Health Products (Afssaps). Through these assays, we were able to study different genetic endpoints in 2 cell types commonly used in regulatory genotoxicity assays: the L5178Y mouse lymphoma cell line and primary cultures of human lymphocytes. Our results showed that the use of WC-Co NP as positive control in in vitro genotoxicity assays was conceivable, but that different parameters have to be considered, such as cell type and treatment schedule. L5178Y mouse lymphoma cells did not provide satisfactory results in the 3 performed tests. However, human lymphocytes were more sensitive to genotoxic effects induced by WC-Co NP, particularly after a 24-h treatment in the in vitro micronucleus assay and after a 4-h treatment in the in vitro Comet assay. Under such conditions, WC-Co could be used as a nanoparticulate reference positive control in these assays.

The human Cytochrome P450 4A11 (CYP4A11) is a major ω-hydroxylase involved in the regulation of blood pressure in the kidney through the conversion of arachidonic acid into 20-hydroxyeicosatetraenoic acid (20-HETE). Previous studies have reported a significant association between the 8590T>C genetic variant of CYP4A11 and hypertension. Interestingly, several population-based studies have reported ethnic differences in the prevalence of hypertension, with the highest prevalence in African populations. The aim of this work was to determine the frequency and inter-ethnic comparison of the CYP4A11 (8590T>C) functional polymorphism, in five new ethnic groups: European (99 French Caucasians), African (36 Gabonese and 50 Senegalese), South American (60 Peruvians) and North African (53 Tunisians) populations, using polymerase chain reaction-single strand conformational polymorphism and sequencing strategies. We confirmed that the CYP4A11 (8590T>C) functional polymorphism exhibits inter-ethnic frequency differences. Noteworthy, the highest 8590C allele frequency was observed in the Tunisian (30.2%), followed by Senegalese (20%) populations. In addition, the CC genotype was only found in the Gabonese and Tunisian populations (5.6% and 8.4%, respectively). These populations may be of major interest to help to clarify the linkage between hypertension and CYP4A11 (8590T>C) genotype in African populations. These findings provide data for further studies that investigate the potential association of CYP4A11 (8590T>C) variant with an incidence of hypertension genesis in respect of ethnicity.

In humans, the glycine N-acyltransferase enzyme (GLYAT) is thought to be important in the detoxification of endogenous and xenobiotic compounds which contain a carboxylic acid group, such as benzoic, isovaleric, or acetylsalicylic acids. The aim of this work was to report a comprehensive investigation of GLYAT genetic polymorphisms in DNA samples from 55 subjects of French Caucasian origin, using polymerase chain reaction-single-strand conformation polymorphism and sequencing strategies. Seven different polymorphisms of the GLYAT gene were identified, including two polymorphisms in the 5' flanking region of the gene (g.-8457C>T and g.-8010A>G), two polymorphisms in intron 5 (g.13931A>G and g.13944C>T) and three missense mutations in exon 2 (g.49T>A; p.Ser17Thr), exon 5 (g.13886A>G; p.Asn156Ser) and exon 6 (g.14435C>T; p.Arg199Cys). In addition to the wild-type allele GLYAT*1 (2.7%), four novel alleles were identified: GLYAT*2A (75.5%), *2B (4.5%), *3 (16.4%) and *4 (0.9%), and five different genotypes. Localisation of the p.Ser17Thr and p.Arg199Cys missense mutations in predicted secondary structures suggest that these variants might have a potential role on the GLYAT protein activity. These results could be helpful in investigating the potential association of GLYAT variants with an incidence of reduced efficiency in xenobiotic carboxylic acids detoxification in humans.

The human cytochrome P450 2U1 (CYP2U1) has been described as a novel extrahepatic P450. CYP2U1 is a highly conserved gene mainly expressed in brain and thymus, but also at lower levels in kidney, lung or heart. This selective tissue distribution suggests important endogenous functions, in particular in the conversion of arachidonic acid into two bioactive compounds, the 19- and 20-HETE. To investigate the extent of CYP2U1 genetic polymorphism in 70 French individuals, a screening for sequence variations in the 5′-flanking and protein encoding regions was performed using PCR-SSCP and sequencing strategies. Four polymorphisms were identified and correspond to −204C>A and –241T>C in the 5′-flanking region, −37G>A in the 5′-untranslated region, and IVS2-17T>C in the intron 2. The most frequent mutations, –241T>C (59.7%) and IVS2-17T>C (66.0%), did not seem to alter CYP2U1 lung expression. These results suggest that CYP2U1 exhibits few genetic variations and support a probable role in endogenous processes.

Since human cytochrome P450 2F1 (CYP2F1) is predominantly expressed in lung tissue and is involved in the metabolism of various pneumotoxicants with potential carcinogenic effects, variations in the nucleotidic sequence of its gene may contribute to interindividual and interethnic differences in the susceptibility to lung tumorigenesis. The aim of the current study was to compare the frequency of a previously reported frameshift mutation, namely c.14_15insC, responsible for the synthesis of a severely truncated protein, between several populations of different ethnic origins. The frequencies of this polymorphism were 26.1, 51.6, 42.7 and 22.9% in French, Gabonese, Senegalese, and Tunisian population samples, respectively, thereby representing a substantial inter ethnic variation in the CYP2F1 gene. These findings provide data for further studies that investigate the potential association of CYP2F1 haplotypes with an incidence of lung cancer genesis in respect of ethnicity.

The CYP2F1 is a human cytochrome P450 that is selectively expressed in lung tissue and involved in the metabolism of various pneumotoxicants with potential carcinogenic effects. In the present study, we report the first systematic investigation of the genetic polymorphism of this enzyme. We analyzed the nucleotidic sequence of the CYP2F1 gene in DNA samples from 90 French Caucasians consisting in 44 patients with lung cancer and 46 control individuals, using single-strand conformation polymorphism analysis of PCR products (PCR-SSCP). We identified 24 novel mutations distributed in the promoter region of the gene, as well as in the coding regions and their flanking intronic sequences. In addition to the wild-type CYP2F1*1 allele, seven allelic variant, CYP2F1*2A, *2B, *3, *4, *5A, *5B and *6, were characterized. The most frequent allelic variant, CYP2F1*2A (25.6%), harbors a combination of 9 mutations, including 2 missense mutations (Asp218Asn and Gln266His) and a 1-bp insertion (c.14_15insC) that creates a premature stop codon in exon 2, probably leading to the synthesis of a severely truncated protein with no catalytic activity. The identification of around 7% of homozygotes for the frameshift mutation in our Caucasian population suggests the existence of an interindividual variation of the CYP2F1 activity and, consequently, the possibility of interindividual differences in the toxic response to some pneumotoxicants and in the susceptibility to certain chemically induced diseases. However, our preliminary results did not show any evidence that the CYP2F1 genetic polymorphism has implications in the pathogenesis of lung cancer.

Rhodanese or thiosulfate sulfurtransferase (TST) is a mitochondrial matrix enzyme that plays roles in cyanide detoxification, the formation of iron-sulfur proteins and the modification of sulfur-containing enzymes. Transsulfuration reaction catalyzed by TST is also involved in H(2)S detoxification. To date, no polymorphism of the human TST gene had been reported. We developed a screening strategy based on a PCR-SSCP method to search for mutations in the 3 exons of TST and their proximal flanking regions. This strategy has been applied to DNA samples from 50 unrelated French individuals of Caucasian origin. Eleven polymorphisms consisting in seven nucleotide substitutions in non-coding regions, two silent mutations and two missense mutations were characterized. The functional consequences of the identified mutations were assessed in vivo by measurement of erythrocyte TST activity and/or in vitro using heterologous expression in Saccharomyces cerevisiae or transient transfection assay in HT29 and Caco-2 cell lines. The P(285)A variant appears to encode a protein with a 50% decrease of in vitro intrinsic clearance compared to the wild-type enzyme. Additionally, the six polymorphisms located upstream the ATG initiation codon are responsible for a significant decrease (ranging from 40% to 73%) in promoter activity of a reporter gene compared to the corresponding wild-type sequence. This work constitutes the first report of the existence of a functional genetic polymorphism affecting TST activity and should be of great help to investigate certain disorders for which impairment of CN(-) or H(2)S detoxification have been suggested to be involved.

Mercaptopyruvate sulfurtransferase (MPST) plays a central role in both cysteine degradation and cyanide detoxification. Moreover, deficiency in MPST activity has been suggested to be responsible for a rare inheritable disorder known as mercaptolactate-cysteine disulfiduria (MCDU). To date, no mutation of the human MPST gene has been reported. We developed a screening strategy to search for mutations in the MPST gene of 50 unrelated French individuals. Two intronic polymorphisms (IVS1 − 110C > G and IVS2 + 39C > T) and a nonsense mutation (Tyr85Stop) were identified and their functional consequences were assessed in vivo by measurement of erythrocyte MPST activity and/or in vitro using heterologous expression or transient transfection assay. The nonsense mutation likely leads to the synthesis of a severely truncated protein without enzymatic activity, as supported by our in vitro data. This work constitutes the first report of the existence of a functional genetic polymorphism affecting MPST and should be of great help to investigate certain disorders such as MCDU.

Since human CYP2A13 is expressed in the respiratory tract and is involved in the activation of tobacco-specific nitrosamines, some of the previously reported sequence variations may contribute to inter-individual and inter-ethnic differences in the susceptibility of tobacco-related tumorigenesis. The aim was to compare the frequencies of the 578C > T (Arg101Stop), 3375C > T (Arg257Cys) and 7520C > G (3′-untranslated region) mutations in several populations. The frequencies of the 578C > T polymorphism were 3.8, 0 and 1.0% in French Caucasians, Gabonese and Tunisians, respectively. In the same populations, the frequencies of the 3375C > T mutation were 0, 15.3 and 4.2%, respectively, whereas the frequencies of the 7520C > G mutation were 1.0, 20.8 and 7.3%, respectively. Marked inter-ethnic variations in CYP2A13 were identified and confirmed. These findings provide data for further studies that associate CYP2A13 haplotypes with an incidence of smoking-related tumours in respect of ethnicity.

The cytochrome P450 3A5 (CYP3A5) has been shown to be highly involved in the metabolism of many therapeutic agents. To date, several polymorphisms affecting the CYP3A5 gene have been identified but few studies have shown a complete description of the variability of the CYP3A5 in the French population. Therefore, the extent of CYP3A5 genetic polymorphism was investigated in a French population of 114 patients. The screening of the coding region with their intron–exon boundaries and the proximal flanking regions was performed using a PCR-SSCP strategy. Eighteen polymorphisms were identified, including four new mutations. They correspond to −19T > C upstream of the exon 1, 7360T > C in intron 4, 12991T > C in intron 5 and 29788delG in exon 12. We also identified 13 alleles including six new alleles. As expected, the most frequent allelic variant is CYP3A5*3 , with a frequency of 87% of all alleles. These data confirmed that CYP3A5 gene is highly polymorphic. Furthermore, it will be now interesting to evaluate the impact of this polymorphism on the pharmacokinetic parameters of different drugs.

Cytochrome P450 2D6 (CYP2D6) is an important human drug-metabolizing enzyme that exhibits a marked genetic polymorphism. Numerous CYP2D6 alleles have been characterized at a functional level, although the consequences for expression and/or catalytic activity of a substantial number of rare variants remain to be investigated. One such allele, CYP2D6*31, is characterized by mutations encoding three amino acid substitutions: Arg296Cys, Arg440His and Ser486Thr. The identification of this allele in an individual with an apparent in vivo poor metabolizer phenotype prompted us to analyze the functional consequence of these substitutions on enzyme activity using yeast as a heterologous expression system. We demonstrated that the Arg440His substitution, alone or in combination with Arg296Cys and/or Ser486Thr, altered the respective kinetic parameters [Km (microM) and kcat (min(-1))] of debrisoquine 4-hydroxylation (wild-type, 25; 0.92; variants, 43-68; 0.05-0.11) and dextromethorphan O-demethylation (wild-type, 1; 4.72; variants, 12-23; 0.64-1.43), such that their specificity constants (kcat/Km) were decreased by more than 95% compared to those observed with the wild-type enzyme. The rates of oxidation of rac-metoprolol at single substrate concentrations of 40 and 400 microM were also markedly decreased by approximately 90% with each CYP2D6 variant containing the Arg440His substitution. These in vitro data confirm that the CYP2D6*31 allele encodes an enzyme with a severely impaired but residual catalytic activity and, furthermore, that the Arg440His exchange alone is the inactivating mutation. A homology model of CYP2D6 based on the crystal structure of rabbit CYP2C5 locates Arg440 on the proximal surface of the protein. Docking the structure of the FMN domain of human cytochrome P450 reductase to the CYP2D6 model suggests that Arg440 is a key member of a cluster of basic amino acid residues important for reductase binding.

The human cytochrome CYP4F12 has been shown to be metabolically active toward inflammatory mediators and exogenous compounds such as antihistaminic drugs. We recently identified a genetic polymorphism within the promoter region, associated with a decreased level of enzyme expression. In the present study, we report the further identification of single nucleotide polymorphisms in the coding sequence of the CYP4F12 gene. A polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of DNA samples from 53 unrelated French Caucasians, allowed the identification of ten mutations, comprising seven missense mutations, 31C>T (Leu11Phe), 38C>T (Pro13Leu), 47C>T (Met16Thr), 4759G>A (Asp76Asn), 4801G>A (Val90Leu), 8896C>T (Arg188Cys) and 23545G>A (Gly522Ser). Their functional impact toward ebastine hydroxylation was evaluated using heterologous expression in Saccharomyces cerevisiae cells of site-directed mutated cDNA variants. Five out seven variants did not exhibit any significant difference in CYP4F12 catalytic activity, whereas two variants, Val90Ile and Arg188Cys, displayed significant changes in their Michaelis-Menten (Km, Vm) parameters. These data on CYP4F12 genetic polymorphism provide tools for further studies of association with pathological processes involving an inflammatory component and with variations in anti-histaminic drug response.

The human cytochrome CYP2A13, which is mainly expressed in the respiratory tract, has been shown to be highly efficient in vitro in the metabolism of tobacco-smoke carcinogens and procarcinogens such as 4-methylnitroso-1-(3-pyridyl)-1-butanone (NNK). In order to investigate the extent of CYP2A13 genetic polymorphism in a French Caucasian population of 102 individuals, a screening for sequence variations in the 5'-untranslated and protein encoding regions of its gene was performed using a polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) strategy. Six polymorphisms in the coding region were identified, including two rare missense mutations (C474G or Asp158Glu, G967T or Val323Leu) and one nonsense mutation (Arg101Stop). This deleterious mutation, the most frequent (5%) in our population, presumably encodes a severely truncated protein. The influence of the nonsense mutation in lung cancer susceptibility was examined by PCR-SSCP using peripheral blood DNA from 204 cases of lung cancer and 201 controls. The CYP2A13*7 allele, which harbours the C301T mutation, was present in 2.0% of controls and 3.4% of cases. However, multivariate analysis showed an elevated risk for small cell lung cancer in subjects heterozygous for the null allele (odds ratio OR=9.9; 95% confidence interval CI=1.9-52.2). This increased risk was not linked to other histological types of lung cancer.

In the present study, we report the first investigation of polymorphisms in the human CYP3A43 gene. A screening for sequence variations in the 5′-flanking and protein coding regions of the CYP3A43 gene was performed by a Polymerase Chain Reaction – Single Strand Conformational Polymorphism (PCR-SSCP) strategy, using DNA samples from 48 unrelated French individuals. Three polymorphisms in the coding region were identified, comprising two nucleotide substitutions, one silent (c.1047C>T) and one missense mutation (c.1018C>G/P340A), and a frame shift mutation (c.74delA), leading to a premature stop codon and, presumably, to a severely truncated protein. In order to evaluate the extent of the frame shift mutation in a larger population, 352 individuals were further genotyped. Thirty-four samples (4.83%) were found to be heterozygous and one homozygous (0.14%) for the nucleotide deletion, which suggests that, although the potential significance of this polymorphism remains to be further evaluated, some individuals are deficient for CYP3A43 activity. © 2003 Wiley-Liss, Inc.

The prostacyclin synthase enzyme (CYP8A1, EC 5.3.99.4) is the unique member of family 8 in the cytochrome P450 superfamily. Inheritable interindividual differences in prostacyclin production may be implicated in the pathogenesis of human vascular diseases. Recently, we functionally characterized a variable number of tandem repeat (VNTR) polymorphism in the 5′-proximal regulatory region of CYP8A1. In this study, we extended the CYP8A1 VNTR polymorphism analysis using a panel of DNA samples from distinct ethnic populations: Tunisians, Gaboneses and French Caucasians. A total of nine VNTR were detected, three of which represent new variants in the CYP8A1 promoter region. Differences among the three ethnic panels in the frequency of the VNTR variants were observed. This study represents the first multi-population-based analysis of the frequency and distribution of VNTR polymorphism affecting the CYP8A1 promoter.

Le site de Sanofi situé à Mourenx, dans le bassin industriel de Lacq (64), produit un médicament antiépileptique, la Dépakine®, dont le principe actif est le valproate de sodium (VPS). Ce site est une installation classée pour la protection de l’environnement (ICPE) soumise à autorisation au titre de l’article L.511-1 et suivants du Code de l’environnement. Les rejets dans l’environnement d’une telle installation sont encadrés par des limites établies sur la base d’une évaluation quantitative des risques sanitaires (EQRS). En 2017, Sanofi Chimie a réalisé une évaluation de l’impact sur la santé et l’environnement des rejets atmosphériques de valproate, en utilisant des valeurs toxicologiques de référence (VTR) à seuil par voie orale, cutanée et respiratoire proposées par le bureau d’étude CEHTRA (Consultancy for Environmental & Human Toxicology and Risk Assessment). Parmi les différentes VTR proposées par CEHTRA, Sanofi a utilisé les VTR fondées sur des effets tératogènes. L’EQRS conduite par la société AECOM pour le compte de Sanofi, en vue d’estimer les risques pour les riverains et les professionnels travaillant à proximité du site, a conclu que « les risques sanitaires liés aux rejets actuels et passés de valproate [étaient] inférieurs aux valeurs de référence » proposées par CEHTRA. L’Anses a été saisie le 29 juin 2018 par la DGS et la DGPR afin de mener, en urgence, une analyse critique des VTR du valproate élaborées pour le compte de Sanofi et utilisées dans le cadre de cette EQRS. Dans son avis du 12 juillet 2018, l’Anses n’a pas remis en cause le choix de construire une VTR à seuil mais n’a pas retenu les VTR élaborées par les deux bureaux d’études, EQUITOX (2015) et CEHTRA (2017), à partir des données animales et/ou humaines car de nombreuses données chez l’Homme disponibles dans la littérature n’avaient pas été prises en compte (Anses, 2018). Dans ce contexte, et au titre de la sécurité des professionnels exposés au valproate, une campagne de mesures de la concentration d’acide valproïque (VPA) dans le sang des employés de l’usine a été organisée par le service de santé au travail de l’entreprise du 27 novembre à la mi-décembre 2018. Le groupe d'alerte en santé travail (GAST) de Nouvelle-Aquitaine a été associé au suivi de cette campagne. Pour l’analyse de cette campagne de mesures, l’entreprise Sanofi s’est référée à une valeur biologique repère dans le sang de 5 mg.L-1. Ainsi, la DGT, la DGS et la DGPR ont saisi l’Anses, le 5 avril 2019, pour mener, en urgence, une analyse critique de la valeur biologique utilisée comme repère par Sanofi pour évaluer l’imprégnation de ses travailleurs. Au vu des données disponibles, le choix du dosage du valproate dans le sang n’a pas été remis en cause. Néanmoins, au regard de la cinétique d’élimination plasmatique de cette substance et de l’existence de métabolites urinaires, il n’a pas été exclu qu’un autre biomarqueur puisse être un meilleur témoin de l’exposition agrégée sur une période plus longue. Concernant le calcul de la valeur limite biologique (VLB), l’approche basée sur la dose thérapeutique est à privilégier mais la dose thérapeutique de 1200 mg.j-1 retenue pour le calcul de la VLB est critiquable compte tenu des données actuellement disponibles. En effet, des effets indésirables, notamment reprotoxiques, pourraient être observés à des doses inférieures à 1200 mg.j-1. Par ailleurs, plusieurs incohérences ou un manque de justification de certains choix ont été relevés lors du calcul de VLB, en particulier au niveau de l’application des facteurs d’incertitude/de protection. Par conséquent, la VLB de 5 mg.L-1 utilisée par Sanofi n’a pas été retenue. Enfin, ne disposant pas de suffisamment d’informations, il n’a pas été possible d’apporter un regard critique et de se prononcer sur la méthode d’analyse et les modalités de prélèvement. L’Anses a, par conséquent, recommandé : ‐ de réaliser une revue approfondie de la littérature prenant en compte les données les plus récentes, en particulier chez l’Homme ; ‐ d’évaluer de manière approfondie la possibilité de recommander des valeurs de référence pouvant être utilisées pour la surveillance biologique des expositions professionnelles au valproate (Anses, 2019). Au regard des différents éléments, l’Anses a été saisie le 28 septembre 2018 par la DGS et la DGPR pour élaborer une VTR chronique par inhalation pour le valproate de sodium, puis en avril 2019 par la DGT, la DGS et la DGPR pour définir des valeurs de référence pouvant être utilisées pour la surveillance des expositions professionnelles au valproate.

Nanomaterials are used in many industrial sectors, and many nanomaterial-containing consumer products are already available. In this context of increasing human exposure to nanomaterials, the evaluation of their genotoxicity is of significant importance. However, the relevance of routinely used genotoxicity assays, developed for non-nanoparticular products, is often questioned for the evaluation of nanomaterials. A nanoparticulate reference positive control would therefore constitute an important step to a better testing of nanomaterials genotoxicity, ensuring that test systems are actually appropriate and/or allowing the validation of new ones.Firstly, we studied the possibility of using commercially-available tungsten carbide-cobalt (WC-Co) nanoparticles, previously characterized for physicochemical properties (size distribution and charge in used media), as positive control in three in vitro genotoxicity assays. The mouse lymphoma thymidine kinase gene mutation assay, the micronucleus assay studying chromosomal aberrations and the comet assay detecting primary DNA damage were performed. The last two assays were realized in two cell types, the mouse lymphoma cell line L5178Y and primary cultures of human lymphocytes. Our results show that WC-Co nanoparticles could be used as positive control in these in vitro genotoxicity assays, according to cell type and treatment schedule.Secondly, we investigated the mechanisms of action involved in WC-Co nanoparticles genotoxicity. Detection of centromeres in micronuclei using fluorescence in situ hybridization (FISH) show the involvement of both clastogenic and aneugenic activities. This was correlated with the results of a chromosome aberration assay on human lymphocytes blocked in metaphase, showing chromatid breaks and polyploid cells. Moreover, as oxidative mechanisms are the most described for nanomaterials, we studied oxidative DNA damage using the modified in vitro comet assay with the DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG). We also detected a production of hydroxyl radicals using electron paramagnetic resonance in suspensions of WC-Co nanoparticles with and without cells. While performing high-throughput assays on WC-Co nanoparticles in three human cell lines corresponding to the main target organs for nanomaterials (A549 lung cell line, Hep3B liver cell line and Caki-1 kidney cell line) it was confirmed that oxidative stress play a significant role in the toxicity of WC-Co nanoparticles. Indeed, the production of reactive oxygen species in cells exposed to WC-Co nanoparticles was correlated to the observation of cytotoxicity and genotoxicity, studied using the detection of γH2AX foci.Finally, we carried out the most relevant genotoxicity assays to study nanodiamonds and lipid nanocapsules, which constitute promising nanovectors for drug delivery. The in vitro comet and micronucleus assays were performed on other cell types mimicking target organs: the T84 intestinal epithelial cell line and the 16-HBE bronchial epithelial cell line exposed to nanodiamonds of three different sizes, and human lymphocytes exposed to lipid nanocapsules of three different sizes and three different charges., Les nanomatériaux sont utilisés dans de nombreux secteurs industriels, et plusieurs produits de consommation contenant des nanomatériaux sont d’ores et déjà commercialisés. Dans ce contexte d’exposition humaine croissante aux nanomatériaux, l’évaluation de leur potentiel génotoxique est d’une importance significative. Cependant, la pertinence des tests classiques de génotoxicité, développés pour des produits non nanoparticulaires, est fréquemment remise en question pour l’évaluation des nanomatériaux. Un témoin positif de référence sous forme nanoparticulaire pourrait donc constituer une avancée importante pour l’évaluation de la génotoxicité des nanomatériaux, permettant de s’assurer que les systèmes d’essais sont appropriés et/ou d’en valider de nouveaux.Dans un premier temps, nous avons étudié la possibilité d’utiliser des nanoparticules de carbure de tungstène – cobalt (WC-Co) commerciales, préalablement caractérisées sur le plan physico-chimique (distribution de taille et charge dans les milieux utilisés), comme témoin positif dans trois essais de génotoxicité in vitro. Le test de mutations géniques au locus thymidine kinase sur cellules de lymphome de souris, le test des micronoyaux étudiant les dommages chromosomiques et le test des comètes détectant les dommages primaires à l’ADN ont ainsi été réalisés, les deux derniers essais dans deux types cellulaires, la lignée de lymphome de souris L5178Y et des cultures primaires de lymphocytes humains. Nos résultats montrent que les nanoparticules de WC-Co pourraient être utilisées comme témoin positif dans ces essais de génotoxicité in vitro, selon le type cellulaire et le schéma de traitement.Nous avons ensuite étudié les mécanismes d’action impliqués dans la génotoxicité des nanoparticules de WC-Co. Le marquage des centromères dans les micronoyaux grâce à la technique d’hybridation in situ en fluorescence (FISH) montre l’implication d’évènements clastogènes et aneugènes. Ces résultats ont été confirmés par un essai d’aberrations chromosomiques sur lymphocytes humains bloqués en métaphase, avec l’observation de cassures de chromatides et de cellules polyploïdes. Par ailleurs, les mécanismes oxydants étant les plus décrits pour les nanomatériaux, nous avons étudié les lésions oxydatives à l’ADN en utilisant le test des comètes in vitro modifié avec l’enzyme de réparation de l’ADN formamidopyrimidine DNA glycosylase (FPG). Nous avons également détecté par résonance paramagnétique électronique une production de radicaux hydroxyles après mise en suspension des nanoparticules de WC-Co en présence et en absence de cellules. Dans le cadre d’études haut-débit des nanoparticules de WC-Co réalisées dans trois lignées cellulaires humaines correspondant aux principaux organes cibles pour les nanomatériaux (la lignée pulmonaire A549, la lignée hépatique Hep3B et la lignée rénale Caki-1), il a été confirmé que le stress oxydant joue un rôle important dans la toxicité des nanoparticules de WC-Co. En effet, la production d’espèces réactives de l’oxygène dans les cellules traitées avec les nanoparticules de WC-Co était corrélée avec l’observation d’une cytotoxicité et de génotoxicité, étudiée à l’aide du test de détection des foyers γH2AX.Finalement, nous avons appliqué les tests de génotoxicité les plus pertinents à l’étude de nanodiamants et de nanocapsules lipidiques, qui constituent des candidats prometteurs pour la vectorisation de principes actifs. Les tests des comètes et des micronoyaux in vitro ont ainsi été réalisés sur d’autres types cellulaires mimant des organes cibles : la lignée intestinale T84 et la lignée bronchique 16-HBE exposées à des nanodiamants de trois tailles différentes et des lymphocytes humains exposés à des nanocapsules lipidiques de 3 tailles et 3 charges différentes.

Through evolution, in order to adapt to its chemical environment, the human organism has developed enzymatic systems that can transform exogenous molecules or xenobiotic (drugs, toxins, carcinogens…), generally of hydrophobic nature, in metabolites more easily excretable via urinary or biliary tract. Some of these enzymes are also involved in catabolic processes or in the biosynthesis of endogenous compounds (fatty acids, retinoids, steroids, prostaglandins…). These enzymes thus play a major role in the protective response of the body toward chemicals and in essential physiological processes. The existence of anomalies in the sequence or structure of the genes encoding these enzymes can expose carriers of these anomalies to particular susceptibility toward xenobiotics or to impairment of essential biological reactions. In a first step, we investigated the nature and extent of the sequence variability of three genes coding for the enzymes CYP4A11, CYP4A22 and Glycine N-acyltransferase (GLYAT). In a second step, functional analyses of sequence variations were carried out, by in silico and in vitro experiments. The CYP4A11 and CYP4A22 genes are the only members of the human CYP4A subfamily. The activity of the recently identified CYP4A22 isoform is still unknown, but the CYP4A11 isoform is know as a ω-hydroxylase of the arachidonic acid, which converted into 20-hydroxyeicosatetraenoic acid (20-HETE). Several studies have shown that genetic anomalies of CYP4A are likely to contribute for susceptibility to hypertension in humans. We analyzed the sequence variations of the CYP4A11 and CYP4A22 genes in genomic DNA samples of healthy volunteers. A total of 26 polymorphisms were identified and 5 novel CYP4A* alleles were characterized for each CYP4A gene. The CYP4A 3D models were built and validated to analyse the potential impact of sequence variations identified. This work represents the first description and characterisation of genetic polymorphism of the human CYP4A genes in a French population. The glycine N-acyltranferase or GLYAT plays an important role in the detoxification of xenobiotics containing a carboxylic group via conjugation with a glycine residue. Seven sequence variations of the GLYAT gene were identified and four novel GLYAT* alleles were characterized. Localisation of missense mutations in predicted secondary structures suggest that these variants might have a potential role on the GLYAT protein activity. These results could be helpful in investigating the potential association of GLYAT variants with an incidence of reduced efficiency in xenobiotic carboxylic acids detoxification in humans, such as acetylsalicylic acid, pesticides, and solvents (Toluene)., Afin de s'adapter à son environnement chimique, l'organisme a développé au cours de l'évolution des systèmes enzymatiques capables de transformer de nombreuses molécules étrangères ou xénobiotiques (médicaments, composés toxiques, carcinogènes...), le plus souvent de nature hydrophobe, en métabolites suffisamment hydrophiles pour être plus facilement excrétés par voie urinaire et/ou biliaire. Certaines de ces enzymes sont également impliquées dans des processus cataboliques ou de biosynthèses de composés endogènes (acides gras, rétinoïdes, stéroïdes, prostaglandines…). Ces enzymes jouent ainsi un rôle fondamental à la fois dans la défense de l'organisme face à son environnement chimique et dans des processus physiologiques essentiels. On comprend dès lors que s'il existe, chez certains individus, des anomalies de séquence ou de structure des gènes codant pour ces enzymes, une partie de la population présentera une susceptibilité particulière à certaines molécules de l'environnement, voire des dysfonctionnements de certaines réactions biologiques indispensables. Les travaux de cette thèse s'inscrivent dans cette démarche. Dans un premier temps, ils ont consisté à évaluer la nature et l'étendue de la variabilité de la séquence nucléotidique de trois gènes codants pour les enzymes CYP4A11, CYP4A22 et la Glycine N-acyltransférase (GLYAT). Dans un deuxième temps, les analyses fonctionnelles des variations de séquence identifiées ont été abordées par des approches in silico et in vitro. Les cytochromes P450 CYP4A11 et CYP4A22, participent à la biotransformation de composés endogènes et sont impliqués plus particulièrement dans la voie d’activation de l’acide arachidonique. Des travaux récents suggèrent que des anomalies génétiques de ces enzymes constituent des facteurs de susceptibilité à l’hypertension artérielle chez l’homme. Nous avons ainsi analysé les variations de séquence du gène CYP4A11 et CYP4A22 dans des échantillons d'ADN provenant de volontaires sains. Au total, 26 polymorphismes ont été identifiés et 5 nouveaux CYP4A* allèles ont été caractérisés pour chaque isoforme CYP4A. Les structures 3D des protéines CYP4A ont été construites et validées pour l’analyse de l’impact des mutations identifiées. Bien que des travaux supplémentaires soient nécessaires pour confirmer le lien entre le polymorphisme génétique du CYP4A11 et du CYP4A22 et l’hypertension artérielle, ce travail représente la première description et caractérisation du polymorphisme génétique des isoformes CYP4A dans une population Française. De plus, nous avons mise en évidence une variabilité interethnique de ce polymorphisme génétique dans différentes populations testées. La glycine N-acyltransférase ou GLYAT est une enzyme impliquée dans la détoxication de xénobiotiques contenant un groupement carboxylique par conjugaison d’un résidu de glycine. Sept variations de séquence de la GLYAT ont été identifiées et quatre nouveaux GLYAT* allèles ont été caractérisés. La localisation des certaines mutations dans des structures secondaires très conservées de la protéine suggère un impact sur l’activité catalytique de cette enzyme. Bien que les conséquences cliniques potentielles de ces variations restent encore à étudier, ces résultats seront utiles pour de futures études d’association de ce polymorphisme génétique de la GLYAT avec les altérations de détoxications de xénobiotiques contenant un groupement carboxylique comme l’aspirine, certains pesticides ou le toluène.