Comparison of the metabolism of 10 chemicals in human and pig skin explants.

Skin metabolism is important to consider when assessing local toxicity and/or penetration of chemicals and their metabolites. If human skin supply is limited, pig skin can be used as an alternative. To identify any species differences, we have investigated the metabolism of 10 chemicals in a pig and human skin explant model. Phase I metabolic pathways in skin from both species included those known to occur via cytochrome P450s, esterases, alcohol dehydrogenases and aldehyde dehydrogenases. Common Phase II pathways were glucuronidation and sulfation but other conjugation pathways were also identified. Chemicals not metabolized by pig skin (caffeine, IQ and 4-chloroaniline) were also not metabolized by human skin. Six chemicals metabolized by pig skin were metabolized to a similar extent (percentage parent remaining) by human skin. Human skin metabolites were also detected in pig skin incubations, except for one unidentified minor vanillin metabolite. Three cinnamyl alcohol metabolites were unique to pig skin but represented minor metabolites. There were notable species differences in the relative amounts of common metabolites. The difference in the abundance of the sulfate conjugates of resorcinol and 4-amino-3-nitrophenol was in accordance with the known lack of aryl sulfotransferase activity in pigs. In conclusion, while qualitative comparisons of metabolic profiles were consistent between pig and human skin, there were some quantitative differences in the percentage of metabolites formed. This preliminary assessment suggests that pig skin is metabolically competent and could be a useful tool for evaluating potential first-pass metabolism before testing in human-derived tissues.

From genomics to metabolomics, moving toward an integrated strategy for the discovery of fungal secondary metabolites.

Fungal secondary metabolites are defined by bioactive properties that ensure adaptation of the fungus to its environment. Although some of these natural products are promising sources of new lead compounds especially for the pharmaceutical industry, others pose risks to human and animal health. The identification of secondary metabolites is critical to assessing both the utility and risks of these compounds. Since fungi present biological specificities different from other microorganisms, this review covers the different strategies specifically used in fungal studies to perform this critical identification. Strategies focused on the direct detection of the secondary metabolites are firstly reported. Particularly, advances in high-throughput untargeted metabolomics have led to the generation of large datasets whose exploitation and interpretation generally require bioinformatics tools. Then, the genome-based methods used to study the entire fungal metabolic potential are reported. Transcriptomic and proteomic tools used in the discovery of fungal secondary metabolites are presented as links between genomic methods and metabolomic experiments. Finally, the influence of the culture environment on the synthesis of secondary metabolites by fungi is highlighted as a major factor to consider in research on fungal secondary metabolites. Through this review, we seek to emphasize that the discovery of natural products should integrate all of these valuable tools. Attention is also drawn to emerging technologies that will certainly revolutionize fungal research and to the use of computational tools that are necessary but whose results should be interpreted carefully.

Stable isotope ratio analysis for verifying the authenticity of balsamic and wine vinegar.

In this paper, we investigate whether the analysis of stable isotope ratios D/H and ¹³C/¹²C in ethanol and acetic acid and of ¹8O/¹6O in water can be applied to the ingredients of "aceto balsamico di Modena IGP" (ABM) to evaluate their authenticity. We found that impurities in the extraction solution do not affect the ¹³C/¹²C of acetic acid and the D/H values of acetic acid are not affected under a composite NMR experiment. The standard deviation of repeatability and standard deviation of reproducibility are comparable in wine vinegar and ABM and generally lower than those quoted in the official methods. This means that the validation parameters quoted in the official methods can also be applied to the ingredients of ABM. In addition, we found no changes in the isotopic values from wine to vinegar and to ABM, and from the original must to the ABM must, providing experimental evidence that reference data from wine databanks can also be used to evaluate the authenticity of vinegar and ABM.

Effect of skin metabolism on dermal delivery of testosterone: qualitative assessment using a new short-term skin model.

The skin is a metabolically active organ expressing biotransformation enzymes able to metabolize both endogenous molecules and xenobiotics. We investigated the impact of metabolism on the delivery of testosterone through the skin with an ex vivo pig ear skin system as an alternative model for human skin. Penetration, absorption and metabolic capabilities were investigated up to 72 h after application of [(14)C]-testosterone doses of 50-800 nmol on either fresh or frozen skin, with the latter model being metabolically inactive. Testosterone absorption and metabolite production were monitored by radio-HPLC and gas chromatography-mass spectrometry. Testosterone absorption through frozen skin was much lower, irrespective of the dose of testosterone applied, compared to fresh skin. Using fresh skin samples, >95% of the radioactivity recovered in culture media, as well as the skin itself, corresponded to metabolites. These results were compared with the metabolic data obtained from other in vitro systems (liver and skin microsomes). The present work leads to the conclusion that most of the enzymatic activities expressed in liver fractions are also expressed in pig and human skin. The metabolic activity of the skin can modulate the biological activity of pharmaceuticals (and xenobiotics). Consequently, it can also greatly affect transdermal drug delivery.

Use of the γH2AX assay for assessing the genotoxicity of polycyclic aromatic hydrocarbons in human cell lines.

The development of in vitro genotoxic assays as an alternative method to animal experimentation is of growing interest in the context of the implementation of new regulations on chemicals. However, extrapolation of toxicity data from in vitro systems to in vivo models is hampered by the fact that in vitro systems vary in their capability to metabolize chemicals, and that biotransformation can greatly influence the experimental results. Therefore, much attention has to be paid to the cellular models used and experimental conditions. Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic ubiquitous pollutants. Human exposure to PAHs is mainly from food origin. In this study, a detailed analysis of the biotransformation capabilities of three human cell lines commonly used for in vitro testing (HepG2, ACHN and Caco-2) was undertaken using 3 model PAHs (benzo(a)pyrene [B(a)P], fluoranthene [FLA] and 3-methylcholanthrene [3-MC]). Concomitantly the genotoxicity of these PAHs was investigated in different cell lines, using a new genotoxic assay (H2AX) in 96-well plates. The metabolic rates of B(a)P, FLA and 3-MC were similar in HepG2 and Caco-2 cell lines, respectively, though with the production of different metabolites. The ACHN cell line was shown to express very limited metabolic capabilities. We demonstrated that the PAHs having a high metabolic rate (B(a)P and 3-MC) were genotoxic from 10(-7) molar in both HepG2 and Caco-2 cells. The present study shows that H2AX measurement in human cell lines competent for the metabolism, is an efficient and sensitive genotoxic assay requiring less cells and time than other currently available tests.

Assessment of metastable atom bombardment (MAB) ionization mass spectrometry for the fast determination of heterocyclic aromatic amines in cooked meat.

An investigation of metastable atom bombardment (MAB) ionization mass spectrometry for the fast characterization of mutagenic/carcinogenic heterocyclic aromatic amines (HAAs) formed during heating processes of meats is presented. The aim of our study was to use the selective ionization of MAB to develop a detection method for HAAs in non-purified meat extracts, thus avoiding purification and concentration steps and reducing analysis time. Sample introduction into the MAB ion source was achieved by pyrolysis, allowing the direct and fast insertion of complex food extracts into the mass spectrometer. Analysis conditions were optimized on standard HAAs by using different ionization gases for the MAB process. Metastable nitrogen was selected as the best MAB gas for the analysis of HAAs. Ionization selectivity is shown by the detection of heterocyclic amines in non-purified chicken meat extracts spiked with HAAs. A quantitative approach is also presented by using pyrograms as chromatograms for quantification purposes. HAAs determination using Py-MAB-ToF was finally performed on cooked chicken breast extracts and compared to an LC-APCI-MS/MS method. Although Py-MAB-ToF sensitivity remains to be improved in the present state of development of our prototype device, only 2 h from the cooking were required to obtain quantitative results in good agreement with HAAs concentrations measured by LC-MS/MS in 36 h. Figure Experimental set-up for pyrolysis-MAB-ToF mass spectrometry experiments.

Chemical investigation and authenticity of Indian vanilla beans.

Representative and validated samples taken from a 500 acre vanilla (Vanilla planifolia Andrews) plantation in India have shown significant deviations in aromatic profile, especially the relative amounts of vanillin (high) and p-hydroxybenzaldehyde (low) and the deuterium isotopic (SNIF-NMR) values. However, the carbon isotopic values (carbon 13 profiles) were generally in accordance with the previous findings on vanilla from other geographic origins.

Geographic origin determination of heroin and cocaine using site-specific isotopic ratio deuterium NMR

SNIF-NMR (Site-specific natural isotopic fractionation measured by deuterium NMR) was employed on 36 heroin samples from seven different known origins, and two cocaine samples from two different known origins. Heroin has two "synthetic" deuterium labeled sites (the two acetyls from acetic anhydride, each representing three equivalent nuclei) and 15 "natural" deuterium labeled sites (originating from the morphine produced in the opium plant). The "natural" sites have the potential for determining geographic location of the original opium plant, while the "synthetic" sites could assist in giving information about the commercial source of acetic anhydride used to convert morphine to heroin. Cocaine has 15 "natural" deuterium labeled sites. This study shows that SNIF-NMR has some use in determining the geographic origin of heroin and also has good potential for determining the geographical origin of cocaine.

Specific natural isotope profile studied by isotope ratio mass spectrometry (SNIP-IRMS): (13)C/(12)C ratios of fructose, glucose, and sucrose for improved detection of sugar addition to pineapple juices and concentrates.

The delta(13)C values of fructose, glucose, and sucrose have been determined in authentic pineapple juices. The sugar fraction is separated from the organic acids by an anionic exchange process. Then the individual components (fructose, glucose, and sucrose) are isolated on a preparative HPLC device using a NH(2)-type column. It is demonstrated that no significant isotope fractionation occurs when close to 100% of material is recovered and when the hydrolysis of sucrose is avoided. The control of the recovery rates and of the sucrose hydrolysis rate after purification is recommended for a reliable interpretation of the results. Correlations between the delta(13)C values of fructose (delta(13)Cf), glucose (delta(13)Cg), and sucrose (delta(13)Csu) can be characterized by systematic differences between these values. For the set of measurements on authentic pineapple juices and concentrates, the mean and the standard deviation of the differences are delta(13)Cf - delta(13)Cg = -0.6 +/- 0.6 per thousand, delta(13)Cf - delta(13)Csu = -1.3 +/- 0. 6 per thousand, and delta(13)Cf - delta(13)Csu = -0.7 +/- 0.5 per thousand. The determinations of the (13)C content of fructose, glucose, and sucrose enable a refinement of the detection of added sugars in fruit juices, re-enforcing the SNIP-IRMS method.