Toxicity of iron nanoparticles towards primary cultures of human bronchial epithelial cells.

Air pollution is a public health issue and the toxicity of ambient particulate matter (PM) is well-recognized. Although it does not mostly contribute to the total mass of PM, increasing evidence indicates that the ultrafine fraction has generally a greater toxicity than the others do. A better knowledge of the underlying mechanisms involved in the pathological disorders related to nanoparticles (NPs) remains essential. Hence, the goal of this study was to determine better whether the exposure to a relatively low dose of well-characterized iron-rich NPs (Fe-NPs) might alter some critical toxicological endpoints in a relevant primary culture model of human bronchial epithelial cells (HBECs). We sought to use Fe-NPs representative of those frequently found in the industrial smokes of metallurgical industries. After having noticed the effective internalization of Fe-NPs, oxidative, inflammatory, DNA repair, and apoptotic endpoints were investigated within HBECs, mainly through transcriptional screening. Taken together, these results revealed that, despite it only produced relatively low levels of reactive oxygen species without any significant oxidative damage, low-dose Fe-NPs quickly significantly deregulated the transcription of some target genes closely involved in the proinflammatory response. Although this inflammatory process seemed to stay under control over time in case of this acute scenario of exposure, the future study of its evolution after a scenario of repeated exposure could be very interesting to evaluate the toxicity of Fe-NPs better.

Cadmium-Induced Renal Cell Toxicity Is Associated With MicroRNA Deregulation.

Cadmium is an environmental pollutant well known for its nephrotoxic effects. Nevertheless, mechanisms underlying nephrotoxicity continue to be elucidated. MicroRNAs (miRNAs) have emerged in recent years as modulators of xenobiotic-induced toxicity. In this context, our study aimed at elucidating whether miRNAs are involved in renal proximal tubular toxicity induced by cadmium exposure. We showed that cadmium exposure, in 2 distinct renal proximal tubular cell models (renal proximal tubular epithelial cell [RPTEC]/human telomerase reverse transcriptase [hTERT] and human kidney-2), resulted in cytotoxicity associated with morphological changes, overexpression of renal injury markers, and induction of apoptosis and inflammation processes. Cadmium exposure also resulted in miRNA modulation, including the significant upregulation of 38 miRNAs in RPTEC/hTERT cells. Most of these miRNAs are known to target genes whose coding proteins are involved in oxidative stress, inflammation, and apoptosis, leading to tissue remodeling. In conclusion, this study provides a list of dysregulated miRNAs which may play a role in the pathophysiology of cadmium-induced kidney damages and highlights promising cadmium molecular biomarkers that warrants to be further evaluated.

Toxicological effects of ambient fine (PM2.5-0.18) and ultrafine (PM0.18) particles in healthy and diseased 3D organo-typic mucocilary-phenotype models.

The knowledge of the underlying mechanisms by which particulate matter (PM) exerts its health effects is still incomplete since it may trigger various symptoms as some persons may be more susceptible than others. Detailed studies realized in more relevant in vitro models are highly needed. Healthy normal human bronchial epithelial (NHBE), asthma-diseased human bronchial epithelial (DHBE), and COPD-DHBE cells, differentiated at the air-liquid interface, were acutely or repeatedly exposed to fine (i.e., PM2.5-0.18, also called FP) and quasi-ultrafine (i.e., PM0.18, also called UFP) particles. Immunofluorescence labelling of pan-cytokeratin, MUC5AC, and ZO-1 confirmed their specific cell-types. Baselines of the inflammatory mediators secreted by all the cells were quite similar. Slight changes of TNFα, IL-1ß, IL-6, IL-8, GM-CSF, MCP-1, and/or TGFα, and of H3K9 histone acetylation supported a higher inflammatory response of asthma- and especially COPD-DHBE cells, after exposure to FP and especially UFP. At baseline, 35 differentially expressed genes (DEG) in asthma-DHBE, and 23 DEG in COPD-DHBE, compared to NHBE cells, were reported. They were involved in biological processes implicated in the development of asthma and COPD diseases, such as cellular process (e.g., PLA2G4C, NLRP1, S100A5, MUC1), biological regulation (e.g., CCNE1), developmental process (e.g., WNT10B), and cell component organization and synthesis (e.g., KRT34, COL6A1, COL6A2). In all the FP or UFP-exposed cell models, DEG were also functionally annotated to the chemical metabolic process (e.g., CYP1A1, CYP1B1, CYP1A2) and inflammatory response (e.g., EREG). Another DEG, FGF-1, was only down-regulated in asthma and specially COPD-DHBE cells repeatedly exposed. While RAB37 could help to counteract the down-regulation of FGF-1 in asthma-DHBE cells, the deregulation of FGR, WNT7B, VIPR1, and PPARGC1A could dramatically contribute to make it worse in COPD-DHBE cells. Taken together, these data contributed to support the highest effects of UFP versus FP and highest sensitivity of asthma- and notably COPD-DHBE versus NHBE cells.

Trace elements in e-liquids - Development and validation of an ICP-MS method for the analysis of electronic cigarette refills.

Electronic cigarette use has rapidly increased in recent years. In assessing their safety, and in view of coming regulations, trace elements (TE) are among the potentially toxic compounds required to be evaluated in electronic cigarette refill fluids ("e-liquids"). An analytical method using inductively coupled plasma with mass spectrometric detection (ICP-MS) was developed and rigorously validated in order to determine concentrations of 15 TE in 54 e-liquids from a French brand. Despite a significant matrix effect from the main e-liquid constituents, and difficulties related to the current lack of reference materials, our method demonstrated satisfactory linearity, precision and robustness, and permitted the quantification of low concentrations of these 15 elements: lower limits of quantification (LLQ) obtained were ≤4 ppb for all elements except for Ni, Cu and Zn (16 ppb, 20 ppb and 200 ppb, respectively). All TE concentrations in all tested samples were <510 ppb, mostly near or below the LLQs. This method is transposable and is timely for laboratories seeking to meet a prospective demand in light of current or future regulations.

Metal enriched quasi-ultrafine particles from stainless steel gas metal arc welding induced genetic and epigenetic alterations in BEAS-2B cells.

Recent evidence has supported welding fume (WF)-derived ultrafine particles (UFP) could be the driving force of their adverse health effects. However, UFP have not yet been extensively studied and are currently not included in present air quality standards/guidelines. Here, attention was focused on the underlying genetic and epigenetic mechanisms by which the quasi-UFP (Q-UFP, i.e., ≤ 0.25 µm) of the WF emitted by gas metal arc welding-stainless steel (GMAW-SS) exert their toxicity in human bronchial epithelial BEAS-2B cells. The Q-UFP under study showed a monomodal size distribution in number centered on 104.4 ± 52.3 nm and a zeta potential of -13.8 ± 0.3 mV. They were enriched in Fe > Cr > Mn > Si, and displayed a relatively high intrinsic oxidative potential. Dose-dependent activation of nuclear factor erythroid 2-related factor 2 and nuclear factor-kappa B signaling pathway, glutathione alteration, and DNA, protein and lipid oxidative damage were reported in BEAS-2B cells acutely (1.5 and 9 µg/cm2, 24 h) or repeatedly (0.25 and 1.5 µg/cm2, 3 × 24 h) exposed to Q-UFP (p < 0.05). Alterations of the Histone H3 acetylation were reported for any exposure (p < 0.05). Differentially regulated miRNA and mRNA indicated the activation of some critical cell signaling pathways related to oxidative stress, inflammation, and cell cycle deregulation towards apoptosis. Taken together, these results highlighted the urgent need to better evaluate the respective toxicity of the different metals and to include the Q-UFP fraction of WF in current air quality standards/guidelines relevant to the occupational settings.

Mitochondrial alterations triggered by repeated exposure to fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) fractions of ambient particulate matter.

Nowadays ambient particulate matter (PM) levels still regularly exceed the guideline values established by World Health Organization in most urban areas. Numerous experimental studies have already demonstrated the airway toxicity of the fine fraction of PM (FP), mainly triggered by oxidative stress-induced airway inflammation. However, only few studies have actually paid close attention to the ultrafine fraction of PM (UFP), which is likely to be more easily internalized in cells and more biologically reactive. Mitochondria are major endogenous sources of reactive oxygen species (ROS) through oxidative metabolism, and coordinate many critical cellular signaling processes. Mitochondria have been often studied in the context of PM toxicity and generally associated with apoptosis activation. However, little is known about the underlying adaptation mechanisms that could occur following exposure at sub-apoptotic doses of ambient PM. Here, normal human bronchial epithelial BEAS-2B cells were acutely or repeatedly exposed to relatively low doses (5 µg.cm-2) of FP (PM2.5-0.18) or quasi-UFP (Q-UFP; PM0.18) to better access the critical changes in mitochondrial morphology, functions, and dynamics. No significant cytotoxicity nor increase of apoptotic events were reported for any exposure. Mitochondrial membrane potential (ΔΨm) and intracellular ATP content were also not significantly impaired. After cell exposure to sub-apoptotic doses of FP and notably Q-UFP, oxidative phosphorylation was increased as well as mitochondrial mass, resulting in increased production of mitochondrial superoxide anion. Given this oxidative boost, the NRF2-ARE signaling pathway was significantly activated. However, mitochondrial dynamic alterations in favor of accentuated fission process were observed, in particular after Q-UFP vs FP, and repeated vs acute exposure. Taken together, these results supported mitochondrial quality control and metabolism dysfunction as an early lung underlying mechanism of toxicity, thereby leading to accumulation of defective mitochondria and enhanced endogenous ROS generation. Therefore, these features might play a key role in maintaining PM-induced oxidative stress and inflammation within lung cells, which could dramatically contribute to the exacerbation of inflammatory chronic lung diseases. The prospective findings of this work could also offer new insights into the physiopathology of lung toxicity, arguably initiate and/or exacerbate by acutely and rather repeated exposure to ambient FP and mostly Q-UFP.

Study of in vitro and in vivo genotoxic effects of air pollution fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) particles on lung models.

Air pollution and particulate matter (PM) are classified as carcinogenic to humans. Pollutants evidence for public health concern include coarse (PM10) and fine (PM2.5) particles. However, ultrafine particles (PM0.1) are assumed to be more toxic than larger particles, but data are still needed to better understand their mechanism of action. In this context, the aim of our work was to investigate the in vitro and in vivo genotoxic potential of fine (PM2.5-018) and quasi ultra-fine (PM0.18) particles from an urban-industrial area (Dunkirk, France) by using comet, micronucleus and/or gene mutation assays. In vitro assessment was performed with 2 lung immortalized cell lines (BEAS-2B and NCI-H292) and primary normal human bronchial epithelial cells (NHBE) grown at the air-liquid interface or in submerged conditions (5 µg PM/cm2). For in vivo assessment, tests were performed after acute (24 h, 100 µg PM/animal), subacute (1 month, 10 µg PM/animal) and subchronic (3 months, 10 µg PM/animal) intranasal exposure of BALB/c mice. In vitro, our results show that PM2.5-018 and PM0.18 induced primary DNA damage but no chromosomal aberrations in immortalized cells. Negative results were noted in primary cells for both endpoints. In vivo assays revealed that PM2.5-018 and PM0.18 induced no significant increases in DNA primary damage, chromosomal aberrations or gene mutations, whatever the duration of exposure. This investigation provides initial answers regarding the in vitro and in vivo genotoxic mode of action of PM2.5-018 and PM0.18 at moderate doses and highlights the need to develop standardized specific methodologies for assessing the genotoxicity of PM. Moreover, other mechanisms possibly implicated in pulmonary carcinogenesis, e.g. epigenetics, should be investigated.

Air pollution-derived PM2.5 impairs mitochondrial function in healthy and chronic obstructive pulmonary diseased human bronchial epithelial cells.

In order to clarify whether the mitochondrial dysfunction is closely related to the cell homeostasis maintenance after particulate matter (PM2.5) exposure, oxidative, inflammatory, apoptotic and mitochondrial endpoints were carefully studied in human bronchial epithelial BEAS-2B, normal human bronchial epithelial (NHBE) and chronic obstructive pulmonary disease (COPD)-diseased human bronchial epithelial (DHBE) cells acutely or repeatedly exposed to air pollution-derived PM2.5. Some modifications of the mitochondrial morphology were observed within all these cell models repeatedly exposed to the highest dose of PM2.5. Dose- and exposure-dependent oxidative damages were reported in BEAS-2B, NHBE and particularly COPD-DHBE cells acutely or repeatedly exposed to PM2.5. Nuclear factor erythroid 2-p45 related factor 2 (NRF2) gene expression and binding activity, together with the mRNA levels of some NRF2 target genes, were directly related to the number of exposures for the lowest PM2.5 dose (i.e., 2 µg/cm2), but, surprisingly, inversely related to the number of exposures for the highest dose (i.e., 10 µg/cm2). There were dose- and exposure-dependent increases of both nuclear factor kappa-B (NF-κB) binding activity and NF-κB target cytokine secretion in BEAS-2B, NHBE and particularly COPD-DHBE cells exposed to PM2.5. Mitochondrial ROS production, membrane potential depolarization, oxidative phosphorylation, and ATP production were significantly altered in all the cell models repeatedly exposed to the highest dose of PM2.5. Collectively, our results indicate a cytosolic ROS overproduction, inducing oxidative damage and activating oxygen sensitive NRF2 and NF-kB signaling pathways for all the cell models acutely or repeatedly exposed to PM2.5. However, one of the important highlight of our findings is that the prolonged and repeated exposure in BEAS-2B, NHBE and in particular sensible COPD-DHBE cells further caused an oxidative boost able to partially inactivate the NRF2 signaling pathway and to critically impair mitochondrial redox homeostasis, thereby producing a persistent mitochondrial dysfunction and a lowering cell energy supply.

Genetic and epigenetic alterations in normal and sensitive COPD-diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM2.5.

Even though clinical, epidemiological and toxicological studies have progressively provided a better knowledge of the underlying mechanisms by which air pollution-derived particulate matter (PM) exerts its harmful health effects, further in vitro studies on relevant cell systems are still needed. Hence, aiming of getting closer to the human in vivo conditions, primary human bronchial epithelial cells derived from normal subjects (NHBE) or sensitive chronic obstructive pulmonary disease (COPD)-diseased patients (DHBE) were differentiated at the air-liquid interface. Thereafter, they were repeatedly exposed to air pollution-derived PM2.5 to study the occurrence of some relevant genetic and/or epigenetic endpoints. Concentration-, exposure- and season-dependent increases of OH-B[a]P metabolites in NHBE, and to a lesser extent, COPD-DHBE cells were reported; however, there were more tetra-OH-B[a]P and 8-OHdG DNA adducts in COPD-DHBE cells. No increase in primary DNA strand break nor chromosomal aberration was observed in repeatedly exposed cells. Telomere length and telomerase activity were modified in a concentration- and exposure-dependent manner in NHBE and particularly COPD-DHBE cells. There were a global DNA hypomethylation, a P16 gene promoter hypermethylation, and a decreasing DNA methyltransferase activity in NHBE and notably COPD-DHBE cells repeatedly exposed. Changes in site-specific methylation, acetylation, and phosphorylation of histone H3 (i.e., H3K4me3, H3K9ac, H3K27ac, and H3S10ph) and related enzyme activities occurred in a concentration- and exposure-dependent manner in all the repeatedly exposed cells. Collectively, these results highlighted the key role played by genetic and even epigenetic events in NHBE and particularly sensitive COPD-DHBE cells repeatedly exposed to air pollution-derived PM2.5 and their different responsiveness. While these specific epigenetic changes have been already described in COPD and even lung cancer phenotypes, our findings supported that, together with genetic events, these epigenetic events could dramatically contribute to the shift from healthy to diseased phenotypes following repeated exposure to relatively low doses of air pollution-derived PM2.5.

Differential responses of healthy and chronic obstructive pulmonary diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM4.

While the knowledge of the underlying mechanisms by which air pollution-derived particulate matter (PM) exerts its harmful health effects is still incomplete, detailed in vitro studies are highly needed. With the aim of getting closer to the human in vivo conditions and better integrating a number of factors related to pre-existing chronic pulmonary inflammatory, we sought to develop primary cultures of normal human bronchial epithelial (NHBE) cells and chronic obstructive pulmonary disease (COPD)-diseased human bronchial epithelial (DHBE) cells, grown at the air-liquid interface. Pan-cytokeratin and MUC5AC immunostaining confirmed the specific cell-types of both these healthy and diseased cell models and showed they are closed to human bronchial epithelia. Thereafter, healthy and diseased cells were repeatedly exposed to air pollution-derived PM4 at the non-cytotoxic concentration of 5 µg/cm2. The differences between the oxidative and inflammatory states in non-exposed NHBE and COPD-DHBE cells indicated that diseased cells conserved their specific physiopathological characteristics. Increases in both oxidative damage and cytokine secretion were reported in repeatedly exposed NHBE cells and particularly in COPD-DHBE cells. Diseased cells repeatedly exposed had lower capacities to metabolize the organic chemicals-coated onto the air-pollution-derived PM4, such as benzo[a]pyrene (B[a]P), but showed higher sensibility to the formation of OH-B[a]P DNA adducts, because their diseased state possibly affected their defenses. Differential profiles of epigenetic hallmarks (i.e., global DNA hypomethylation, P16 promoter hypermethylation, telomere length shortening, telomerase activation, and histone H3 modifications) occurred in repeatedly exposed NHBE and particularly in COPD-DHBE cells. Taken together, these results closely supported the highest responsiveness of COPD-DHBE cells to a repeated exposure to air pollution-derived PM4. The use of these innovative in vitro exposure systems such as NHBE and COPD-DHBE cells could therefore be consider as a very useful and powerful promising tool in the field of the respiratory toxicology, taking into account sensitive individuals.

NAT2 genotyping and efficacy of sulfasalazine in patients with chronic discoid lupus erythematosus.

Sulfasalazine is an effective agent for chronic discoid lupus erythematosus (CDLE) but the response to treatment is considerably variable between patients and is also unpredictable. The reason for this might relate to differences in metabolism of the drug which is extensively acetylated by the polymorphic enzyme N-acetyltransferase 2 (NAT2). To test this possibility, the N-acetylation phenotype of eleven patients with CDLE and treated by standard doses of sulfasalazine was retrospectively determined by genotyping. A clear-cut difference in the outcome of treatment was observed according to whether the patients were slow acetylators (SA) or rapid acetylators (RA). Eight out of 11 patients responded to treatment with a complete or marked remission of the disease. Seven of them were RA. The three other patients who did not respond at all to the drug were SA. In addition, SA seem to be more prone to toxic events. These findings strongly suggest that the genetic polymorphism of NAT2 is responsible for differences in the response to sulfasalazine in patients with CDLE. Therefore, candidates for sulfasalazine therapy should be genotyped to identify those patients who might benefit from the drug.

A novel CYP2D6 allele with an abolished splice recognition site associated with the poor metabolizer phenotype.

A novel loss-of function allele of the CYP2D6 gene was characterized in a PM individual using exon-by-exon PCR-SSCP analysis. This allele, we termed CYP2D6(F), harbours four mutations including a new mutation (D6-F) which abolishes the splice acceptor site of the 1st intron and results in a premature stop codon. DNA samples from a large population of healthy unrelated volunteers were tested for D6-F using a PCR-assay we developed for the specific identification of the mutation in genomic DNA. The prevalence of D6-F was very low. However, its identification combined with that of the previously reported gene inactivating mutations would further increase the phenotype prediction rate by genotyping.

Genetic analysis of the cytochrome P450 CYP2D6 polymorphism in patients with systemic lupus erythematosus.

Previous reports of an association between the polymorphic cytochrome P450 CYP2D6 and systemic lupus erythematosus are conflicting. Following the elucidation of the molecular basis of the CYP2D6 genetic polymorphism, we re-examined the hypothesis of an association of this gene with a susceptibility to system lupus erythematosus by analysing the complete CYP2D6 coding sequence. For this purpose, we studies the occurrence of 16 mutations in genomic DNA from 69 systemic lupus erythematosus patients and a large control group using a previously described polymerase chain reaction-single strand confirmation polymorphism analysis. In addition, we studied the occurrence of 11 alleles and 21 genotypes in the same individuals by the combined use of restriction fragment length polymorphism and allele-specific polymerase chain reaction followed by polymerase chain reaction-single strand confirmation polymorphism analysis. No significant differences in the distribution of overall genotypes and predicted phenotypes were observed between system lupus erythematosus patients and controls. The only new finding of our study is the higher frequency of one non functional allele, namely the CYP2D6*4A, in systemic lupus erythematosus versus control individuals (P = 0.007). This increased frequency was not statistically significant in multiple comparison analysis and was not related to any specific clinical features of systemic lupus erythematosus. These results suggest that CYP2D6 genotype as well as CYP2D6 phenotype are not determinant of susceptibility to systemic lupus erythematosus but the presence of the inactive CYP2D6*4A allele may be a contributory factor.

Polymorphism of the cytochrome P450 CYP2D6 gene in a European population: characterization of 48 mutations and 53 alleles, their frequencies and evolution.

The polymorphic cytochrome P450 CYP2D6 is involved in the metabolism of various drugs of wide therapeutic use and is a presumed susceptibility factor for certain environmentally-induced diseases. Our aim was to define the mutations and alleles of the CYP2D6 gene and to evaluate their frequencies in the European population. Using polymerase chain reaction-single strand conformation polymorphism analysis, 672 unrelated subjects were screened for mutations in the 9 exons of the gene and their exon-intron boundaries. A total of 48 point mutations were identified, of which 29 were novel. Mutations 1749 G-->C, 2938 C-->T and 4268 G-->C represented 52.6%, 34.3% and 52.9% of the mutations in the total population, respectively. Of the eight detrimental mutations detected, the 1934 G-->A, the 1795 Tdel and the 2637 Adel accounted for 65.8%, 6.2% and 4.8% respectively, within the poor metabolizer subgroup. Fifty-three different alleles were characterized from the mutation pattern and by allele-specific sequencing. They are derived from three major alleles, namely the wild-type CYP2D6*1A, the functional CYP2D6*2 and the null CYP2D6*4A. Five allelic variants (CYP2D6*1A, *2, *2B, *4A and *5) account for about 87% of all alleles, while the remaining alleles occur with a frequency of 0.1%-2.7%. These data provide a solid basis for future epidemiological, clinical as well as interethnic studies of the CYP2D6 polymorphism and highlight that the described single strand conformation polymorphism method can be successfully used in designing such studies.

Molecular analysis of the N-acetyltransferase 1 gene (NAT1*) using polymerase chain reaction-restriction fragment-single strand conformation polymorphism assay.

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. Considering the increasing number of mutations in the NAT1 gene that are detected, NAT1* genotyping using conventional polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) or allele-specific amplification assays has become complicated. We developed a rapid and powerful strategy allowing the full characterization of NAT1* alleles. This method, based on single-strand conformation polymorphism analysis of a unique PCR product encompassing the entire intronless NAT1*-coding region along with additional flanking segments in the 5' and 3' untranslated regions, was then applied to DNA samples from 270 individuals. Nine NAT1* allelic variants, including two novel (NAT1*28 and NAT1*29), and 15 different genotypes were identified. This approach could be advantageously used in epidemiological studies to provide more definite data on suspected associations between NAT1* genotype and certain pathological processes.

An efficient strategy for detection of known and new mutations of the CYP2D6 gene using single strand conformation polymorphism analysis.

To detect mutations in the cytochrome P450 CYP2D6 gene (CYP2D6), we developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The efficiency of the method was evaluated by analysing DNA samples from extensive metabolizers (EM) and poor metabolizers (PM) of debrisoquine. Haplotypes, alleles and mutations of CYP2D6 had previously been characterized in each individual using PCR assays, Xba I restriction fragment length polymorphism (RFLP) and sequencing. PCR-SSCP results were in complete agreement with those obtained using established methods. All previously characterized mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments allowing their identification. We further tested the efficiency of PCR-SSCP for detecting new CYP2D6 mutations. DNA from a PM subject presumed to carry an unknown non-functional mutant allele of CYP2D6 was amplified and bands with aberrant migration patterns were observed on SSCP gels. Sequence analysis of the corresponding DNA fragments revealed the causative mutations. In this way, a novel non-functional allele of the gene, carrying three previously reported mutations and a new mutation in the third exon which results in a premature termination codon, was characterized. Finally, CYP2D6 SSCP analysis was performed on DNA amplified with fluorescent primers and an automated DNA sequencer was used for SSCP analysis of products. We conclude that the PCR-SSCP approach is a powerful method of identifying simultaneously known and new mutations of the CYP2D6 gene.

In-vitro analysis of the contribution of CYP2D6.35 to ultra-rapid metabolism.

From 10 to 30% of CYP2D6 ultra-rapid metabolizers of Caucasian origin harbor alleles with duplicated or amplified functional CYP2D6 genes. Recently, the CYP2D6*35 allele has been reported to be more frequent in ultra-rapid metabolizing subjects than in extensive metabolizers, suggesting a possible role of this variant in CYP2D6 duplication-negative ultra-rapid metabolizing subjects. In this study, we examined the functional consequences of the Val11Met, Arg296Cys and Ser486Thr amino acid substitutions associated with the CYP2D6*35 on the expression and catalytic activity of the variant enzyme, heterologously expressed in yeast. Our results indicate that the functional activity and level of expression of recombinant CYP2D6.35 are comparable with those of the wild-type enzyme, thus precluding the hypothesis that the high level of enzyme activity in CYP2D6 duplication-negative ultra-rapid metabolizing subjects is a consequence of the expression of a more catalytically effective CYP2D6.35 enzyme.

Characterization of a variable number tandem repeat region in the thiopurine S-methyltransferase gene promoter.

Characterization of the genetic polymorphism of thiopurine S-methyltransferase enzyme (TPMT; EC 2.1.1.67) is required because of its clinical importance for patients exposed to thiopurine drugs. A number of point mutations have already been characterized in exons and introns of the TPMT gene. Here we report the identification of a polymorphic locus within the promoter region of the gene. This polymorphism was detected by polymerase chain reaction - single strand conformation polymorphism analysis of DNA samples from 54 unrelated European individuals. A total of five alleles with length variations were distinguished through the 5'-flanking region involved in the TPMT gene expression. Sequence analysis revealed that these variations were due to a variable number of tandem repeats (VNTR), ranging from four to eight repeats. Each repeat consists of 17 or 18 bp units and contains putative binding sites for transcription factors. The most frequent alleles harbour four or five tandem repeats, a heterozygosity rate of 0.44 was calculated, and a stable Mendelian inheritance of alleles was demonstrated. Analysis of the effect of each VNTR allele on promoter activity of a reporter gene was further performed in various cell lines by transient transfection assay. A modulatory effect of VNTR alleles was observed in vitro, but the repeat polymorphism did not display a significative role in TPMT gene regulation in vivo. Further studies need to be carried out to support the hypothesis that VNTR may contribute to the large interindividual variations of TPMT activity.

Genotypic and phenotypic analysis of the polymorphic thiopurine S-methyltransferase gene (TPMT) in a European population.

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.

Identification of genetic variants in the human thromboxane synthase gene (CYP5A1).

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.