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.

Study of NAT2 genetic polymorphism in West African subjects: example of an healthy non-smoker Senegalese population.

The NAT2 genetic polymorphism determines the individual acetylator status and, consequently, the capacity to metabolize, or not, drugs and xenobiotics which are substrates of NAT2. As the nature and frequency of the NAT2 polymorphisms vary remarkably between populations of different ethnic origins, genotyping strategies used to predict the acetylation phenotype need to be adapted for each particular population regarding their genetic backgrounds at this locus. As few data on the genetic polymorphism of NAT2 are available in the Senegalese population, we performed an extensive identification of NAT2 variants in 105 healthy non-smoker Senegalese subjects by direct PCR sequencing of the coding region. Eleven previously described SNPs were identified in this Senegalese population. Upon allele analysis, the four most frequent alleles were of the NAT2*5- (35.7 %), NAT2*6- (21.0 %), NAT2*12- (16.7 %) and NAT2*14- (10.0 %) type, the remaining alleles, including the wild-type NAT2*4, having each a frequency lower than 10 %. According to the observed genotypes, 51 and 50 subjects were predicted to be of the rapid (48.6 %) and slow (47.6 %) acetylator phenotype, respectively, while four individuals (3.8 %) were considered of unknown phenotype as they carry at least one allele with a yet unknown functional effect. These baseline data would be of particular interest to set up an efficient genotyping strategy to predict the acetylation status of Senegalese patients with tuberculosis and, thus, to optimize their isoniazid treatment.

Determination of a quantitative relationship between hepatic CYP3A5*1/*3 and CYP3A4 expression for use in the prediction of metabolic clearance in virtual populations.

The creation of virtual populations allows the estimation of pharmacokinetic parameters, such as metabolic clearance in extreme individuals rather than the 'average human'. Prediction of variability in metabolic clearance within genetically diverse populations relies on understanding the covariation in the expression of enzymes. A number of statistically significant positive correlations have been observed in the hepatic expression of cytochrome P450 drug metabolising enzymes. However, these rarely provided a quantitative description of the relationships which is required in creating virtual populations. Collation of data from 40 human liver microsomal samples in the current study indicated a significant positive relationship between hepatic microsomal CYP3A5*1/*3 and CYP3A4 content. Having developed a model describing the relationship between hepatic CYP3A4 and CYP3A5*1/*3, the Simcyp Population-based Simulator(®) was used to investigate the consequences of either incorporating or ignoring the relationship between the two enzymes on estimates of drug clearance. Simulations indicated that for a compound with greater metabolism by CYP3A5 than CYP3A4, such as tacrolimus, incorporation of the correlation between CYP3A4 and CYP3A5 does have an impact on the prediction of oral clearance. Failure to consider the relationship between CYP3A4 and CYP3A5 when creating the virtual population led to a 32% lower estimate of oral clearance in individuals possessing both the CYP3A5*1/*3 genotype and high basal concentrations of CYP3A4. Potential clinical implications may include an inadequate dose estimation during clinical study design, the consequences of which may include organ rejection in transplant recipients using immunosuppressants such as tacrolimus or toxicity due to elevated concentrations of circulating metabolites.

Individual exposure level following indoor and outdoor air pollution exposure in Dakar (Senegal).

The consequences of indoor and outdoor air pollution on human health are of great concern nowadays. In this study, we firstly evaluated indoor and outdoor air pollution levels (CO, CO2, NO, NO2, PM10) at an urban site in Dakar city center and at a rural site. Then, the individual exposure levels to selected pollutants and the variations in the levels of biomarkers of exposure were investigated in different groups of persons (bus drivers, traders working along the main roads and housemaids). Benzene exposure levels were higher for housemaids than for bus drivers and traders. High indoor exposure to benzene is probably due to cooking habits (cooking with charcoal), local practices (burning of incense), the use of cleaning products or solvent products which are important emitters of this compound. These results are confirmed by the values of S-PMA, which were higher in housemaids group compared to the others. Urinary 1-HOP levels were significantly higher for urban site housemaids compared to semirural district ones. Moreover, urinary levels of DNA oxidative stress damage (8-OHdG) and inflammatory (interleukin-6 and -8) biomarkers were higher in urban subjects in comparison to rural ones. The air quality measurement campaign showed that the bus interior was more polluted with PM10, CO, CO2 and NO than the market and urban or rural households. However, the interior of households showed higher concentration of VOCs than outdoor sites confirming previous observations of higher indoor individual exposure level to specific classes of pollutants.

Prevalence of new psychoactive substances and prescription drugs in the Belgian driving under the influence of drugs population.

Driving under the influence of drugs (DUID) is a worldwide problem. Several countries have adopted DUID legislations which prove their deterrent effect and impact on road safety. However, the use of new psychoactive substances (NPS) and prescription drugs is not known, as the applied roadside screening tests have not yet been adapted for these compounds. In this study, 558 blood samples obtained during roadside controls in Belgium (January to August 2015) after a positive Drugwipe 5S® test and 199 oral fluid (OF) samples obtained from negatively screened test pads were analyzed. The NPS positivity rate was 7% in blood, while it reached 11% in OF. NPS detected were: diphenidine, ketamine, 4-fluoroamphetamine, 2-amino-indane, methoxetamine, α-PVP, methiopropamine, a mix of 5-MAPB/5-EAPB, TH-PVP, mephedrone, methedrone, 4-methylethylcathinone, 5-MeO-DALT, 4-Acetoxy-DiPT, AB Fubinaca, FUB-JWH018, JWH020, trifluoromethylphenylpiperazine, and ethylphenidate. Moreover, 17% of blood samples (and 5% of OF) contained an analgesic drug, 10% (0.5%) a benzodiazepine/hypnotic, 5% (2%) an antidepressant, 2% (3%) an antipsychotic, 2% an antiepileptic drug, and 1% methylphenidate. The presence of NPS in the young (and predominately male) DUID population is proven. Furthermore, a high level of poly-drug use including combinations of NPS, licit, and drugs of abuse was observed. Further research concerning the development of on-site NPS detection techniques should be established. Meanwhile, the effects of combined drug use on driving ability and the physical/psychological signs after NPS use should be performed to improve the on-site DUID detection of NPS by police officers, so they can engage in blood sampling for a general unknown screening.

[Molecular pharmacogenetics in hospital laboratories in France: current data and future prospects]. / La pharmacogénétique moléculaire hospitalière en France: données actuelles et perspectives.

Molecular pharmacogenetic units have recently been established in several hospital laboratories in France. The clinical impact of these units is still limited and numerous problems of organizational, ethical, legal, technical, social and economical nature remain to be resolved. However, an increasing number of these units, a rise in their activities and an enlargement of their scope of application are foreseeable in the future. Ultimately, these units would significantly contribute to limit the public health problem caused by interindividual variabilities in drug effects. In view of these prospects, it seems essential that such hospital activity should be quickly recognised by the authorities and the various health sectors in France. It is also essential that the problems that arise from such pharmacogenetic activities should be considered by the authorities and would profit from the organization of a national network and from financial guarantees.

Ameliorative effect of vitamin C against hepatotoxicity induced by emamectin benzoate in rats.

In the present study, we aimed to assess the potential protective effect of ascorbic acid (AA) against emamectin benzoate (EMB)-induced hepatotoxicity. For this purpose, biochemical, histopathological and analytical investigations were performed. Male Wistar rats were distributed into three groups, that is, a control group, an EMB group given 10 mg EMB/kg body weight (BW) by gavage and an EMB + AA group given 10 mg EMB/kg BW and vitamin C intraperitoneally (200 mg/kg). The duration of the treatment was 28 days and the duration of the study was 42 days. There was a statistically significant increase of all hepatic biomarkers, that is, aspartate aminotransferase, alanine aminotransferase and gamma-glutamyltransferase activities, and glycemia, in EMB-treated group when compared with the control group. Light microscopic observations revealed variable signs of hepatotoxicity in the EMB group, which were represented by alteration of normal hepatic architecture, inflammatory cell infiltration, hepatocellular steatosis and foci of necrosis at 28 and 42 days post-treatment. However, co-treatment with vitamin C reduced EMB-related liver toxicity and diminished the abnormal biochemical and architectural damage. Emamectin B1a and B1b residues were detectable in all plasma samples of treated rats at 14, 21 and 28 days of treatment. The drug liver tissue concentration was significantly lower in EMB + AA group compared with EMB group at 28 and 42 days. In conclusion, the findings of the present study clearly indicate a significant protective action of vitamin C against EMB hepatotoxicity.

[Therapeutic failure: importance of genes?]. / Echec thérapeutique: peut-être une affaire de gènes?

Drug management can be a difficult task in certain situations because of the variable response observed from one patient to another. Genetic factors affecting the pharmacokinetics and pharmacodynamics of drug reactions could explain the interindividual variability in drug response. Pharmacogenetic analysis provides insight into the molecular mechanisms involved in drug response, with the ultimate goal of achieving optimal drug efficacy and safety. Numerous polymorphisms have been described in genes encoding drug-metabolising enzymes, transporters, and receptors. For some drugs, the impact on drug bioavailability and effect has been elucidated. We review here the molecular basis of interindividual variation in drug response and the methods used to identify individual risk of drug failure or toxicity. Clinical applications, concerning enzymes metabolising drugs (cytochrome P4502D6, thiopurine S-methyltransferase and N-acetyltransferase) provide an illustrative demonstration of the usefulness of pharmacogenetic tests in improving patient management. Clinical validation of these tests and new technologies (real-time PCR, DNA chips) should, in the future promote pharmacogenetics in clinical practice and may be lead to more individualized drug therapy.

Prevention of isoniazid toxicity by NAT2 genotyping in Senegalese tuberculosis patients.

Isoniazid (INH), recommended by WHO (World Health Organization) in the treatment of tuberculosis (TB), is metabolized primarily by the genetically polymorphic N-acetyltransferase 2 (NAT2) enzyme. The human population is divided into three different phenotypic groups according to acetylation rate: slow, intermediate, and fast acetylators. The objective of this study was to explore the relationship between NAT2 genotypes and the serum concentrations of INH. Blood samples from 96 patients with TB were taken for the analysis. NAT2 polymorphisms on coding region were examined by polymerase chain reaction (PCR) direct sequencing; the acetylation status was obtained by measuring isoniazid (INH) and its metabolite, acetylisoniazid (AcINH) in plasma was obtained by using the liquid chromatography coupled to mass spectrometry. TB patients were distributed into two groups of fast and slow acetylators according to the acetylation index calculated based on the plasma concentration of INH in the 3rd hour (T3) after an oral dose. Our PCR analysis identified several alleles, where NAT2*4, NAT2*5A, NAT2*6A, and NAT2*13A were the most important. The concentrations of INH varied between 1.10 mg/L and 13.10 mg/L at the 3rd hour and between 0.1 and 9.5 mg/L at the 6th hour. The use of the acetylating index I3 allowed the classification of tested patients into two phenotypic groups: slow acetylators (44.3% of TB patients), and rapid acetylators (55.7%). Patient's acetylation profile provides valuable information on their therapeutic, pharmacological, and toxicological responses.

Acute Methiopropamine Intoxication After "Synthacaine" Consumption.

Use of methiopropamine (MPA), a synthetic metamfetamine analog, has been detected since 2011 in Europe, but there is limited information on its acute toxicity. A 30-year-old man was admitted to the emergency department in a confused state, with paranoid delusion, auditory and visual hallucinatory experiences, and incoherent speech following the use of "synthacaine" (a slang term derived from "synthetic" and "cocaine"). Toxicological screening for pharmaceuticals and drugs of abuse by liquid chromatography-diode-array detector, gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS-MS) detected MPA, which was subsequently quantified by a specific LC-MS-MS method. Of note, 13 h after presentation to the emergency department, the plasma concentration of MPA was 14 ng/mL. This case report confirms the toxicity of MPA and the need for toxicological analysis to confirm the substance actually ingested by users of new psychoactive substances.

Characterization of the in vivo immune network of IDO, tryptophan metabolism, PD-L1, and CTLA-4 in circulating immune cells in melanoma.

In melanoma, both the induction of immunosuppression by tumor cells and the inflammatory antitumor response can induce an upregulation of counter-regulatory mechanisms such as indoleamine 2,3-dioxygenase (IDO), programmed death-ligand 1 (PD-L1) and CTLA-4+ regulatory T-cells (Tregs) in the tumor microenvironment. Even though these immunosuppressive mediators are targets for immunotherapy, research investigating their expression in the peripheral blood is lacking. We therefore, performed flow cytometry on PBMCs of stage I-IV melanoma patients. IDO expression was detected in plasmacytoid dendritic cells (pDC) and monocytic myeloid-derived suppressor cells (mMDSC), and increased in advanced disease stage (p = 0.027). Tryptophan breakdown confirmed the functional activity of IDO and was linked with increased PD-L1+ cytotoxic T-cells (p = 0.009), relative lymphopenia (p = 0.036), and a higher mDC/pDC ratio (p = 0.002). High levels of circulating PD-L1+ cytotoxic T-cells were associated with increased CTLA-4 expression by Tregs (p = 0.005) and MDSC levels (p = 0.033). This illustrates that counter-regulatory immune mechanisms in melanoma should be considered as one interrelated signaling network. Moreover, both increased PD-L1+ T-cells and CTLA-4 expression in Tregs conferred a negative prognosis, indicating their in vivo relevance. Remarkably, circulating CTLA-4, IDO, and pDC levels were altered according to prior invasion of the sentinel lymph node and IDO expression in the sentinel was associated with more IDO+ PBMCs. We conclude that the expression of IDO, PD-L1, and CTLA-4 in the peripheral blood of melanoma patients is strongly interconnected, associated with advanced disease and negative outcome, independent of disease stage. Combination treatments targeting several of these markers are therefore likely to exert a synergistic response.

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.

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.

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.

[Pharmacogenetics or the promise of a personalized medicine: variability in drug metabolism and transport]. / La pharmacogénétique ou la promesse d'une médecine personnalisée : variations du métabolisme et du transport des médicaments.

There is much variability in the manner individuals respond to drugs, such that the management of some drugs is problematic. In France, the incidence of hospital admissions related to adverse drug reactions is estimated to be 3.2 %, at an annual cost of over 300 millions euros. Genetic factors affecting the pharmacokinetics and pharmacodynamics of drugs partly explain interindividual variability in drug response. Pharmacogenetic focuses on the molecular mechanisms involved in drug response, and its ultimate goal is the optimisation of drug treatments, both in terms of efficacy and safety. Numerous polymorphisms in genes encoding drug-metabolising enzymes, transporters and receptors have been described and their consequences on disposition and effect of a substantial number of medications have been elucidated. This review focuses on variability of drug metabolism and transport to define the objectives of pharmacogenetics, the molecular bases of interindividual variation in drug response and the methods used for the evaluation of the individual risk of drug failure or toxicity. Some clinical applications of pharmacogenetics have already been developed in routine medicine resulting in significant improvement in patient treatment. The clinical validation of an increasing number of pharmacogenetic tests, as well as the development of new highly efficient technologies for genotyping (real-time PCR, DNA chips) should further promote pharmacogenetics in clinical practice and lead to the development of a patient-tailored drug therapy.