Medico-legal assessment of methamphetamine and amphetamine serum concentrations-what can we learn from survived intoxications?

Medico-legal experts are increasingly enlisted to assess the methamphetamine and amphetamine serum concentrations after a criminal offense. However, since criminal users rarely provide useful information to medico-legal experts regarding the substances abused, when the substance(s) was/were used, dose of ingestion tools are needed to interpret the analytical data, which can be used as objective evidence in such cases. A comparative series of methamphetamine and amphetamine serum concentrations were used to analyze the frequency of concentrations, to determine methamphetamine/amphetamine concentration ratios, and prove them as a tool to distinguish pure methamphetamine from mixed amphetamine/methamphetamine ingestion. Additionally, two cases of survived accidental methamphetamine intoxication, resulting from ingestion smuggling which was longitudinally monitored, and pharmacokinetic parameters were assessed. In a series of 628 samples where the most frequent concentration of methamphetamine exceeded the therapeutic level, there was a strong correlation suggesting pure methamphetamine consumption, when the ratios of methamphetamine/amphetamine concentrations were within the range between 3 and 10. In the two cases of methamphetamine bodypacking, the relevant serum concentrations of methamphetamine and amphetamine, which could be measured up to 9 days after ingestion, indicated a decrease of the methamphetamine/amphetamine ratios in an exponential manner. However, the ratios were not always within the range between 3 and 10. Lastly, the course of the serum concentrations suggested an increase of the apparent elimination half-life of methamphetamine. In terms of the objective evidence required in criminal law, calculating methamphetamine/amphetamine concentration ratio is not a suitable to means to distinguish pure methamphetamine intake and that of mixed amphetamine/methamphetamine abuse in an individual case. Instead, methamphetamine high serum concentrations and the possible increase in apparent elimination half-life suggest that an extended detection period may be used to distinguish between "illicit use" as compared to "therapeutic use" of methamphetamine.

A quantitative HPLC-MS/MS method for studying internal concentrations and toxicokinetics of 34 polar analytes in zebrafish (Danio rerio) embryos.

An analytical method using high-performance liquid chromatography-tandem mass spectrometry was developed to determine internal concentrations of 34 test compounds such as pharmaceuticals and pesticides in zebrafish embryos (ZFE), among them, cimetidine, 2,4-dichlorophenoxyacetic acid, metoprolol, atropine and phenytoin. For qualification and quantification, multiple reaction monitoring mode was used. The linear range extends from 0.075 ng/mL for thiacloprid and metazachlor and 7.5 ng/mL for coniine and clofibrate to 250 ng/mL for many of the test compounds. Matrix effects were strongest for nicotine, but never exceeded ±20 % for any of the developmental stages of the ZFE. Method recoveries ranged from 90 to 110 % from an analysis of nine pooled ZFE. These findings together with the simple sample preparation mean this approach is suitable for the determination of internal concentrations from only nine individual ZFE in all life stages up to 96 h post-fertilization. Exemplarily, the time course of the internal concentrations of clofibric acid, metribuzin and benzocaine in ZFE was studied over 96 h, and three different patterns were distinguished, on the basis of the speed and extent of uptake and whether or not a steady state was reached. Decreasing internal concentrations may be due to metabolism in the ZFE.

Distinct metabolic differences between various human cancer and primary cells.

Recent years have seen resurging interest in cancer cell metabolism and the role of secreted cancer metabolites in modulating the tumor stroma. Using a combination of nontargeted and targeted LC and GC-MS methods, the exometabolomes of three leukemia, two melanoma, three renal cell carcinoma, two colorectal adenocarcinoma, four hepatocellular carcinoma, three breast cancer, two bladder carcinoma, and one glioblastoma cell line, as well as five primary cultures of human melanocytes, hepatocytes, monocytes, CD4 and CD8 lymphocytes, that had been all cultivated under identical conditions, were investigated. Unsupervised affinity propagation clustering of the metabolic footprints yielded five distinct clusters that grouped the investigated cell cultures mainly according to the tissue of origin. A common expected feature of all neoplastic cells was high lactate production. Extracellular arginine and nicotinamide were major discriminants between normal and neoplastic hepatocytes. Further, significant differences in the assimilation of di- and tripeptides were observed. This finding appears to underscore the importance of peptides for meeting the increased bioenergetic and biosynthetic demands of many cancers.

Suppressive effects of tumor cell-derived 5'-deoxy-5'-methylthioadenosine on human T cells.

The immunosuppressive tumor microenvironment represents one of the main obstacles for immunotherapy of cancer. The tumor milieu is among others shaped by tumor metabolites such as 5'-deoxy-5'-methylthioadenosine (MTA). Increased intratumoral MTA levels result from a lack of the MTA-catabolizing enzyme methylthioadenosine phosphorylase (MTAP) in tumor cells and are found in various tumor entities. Here, we demonstrate that MTA suppresses proliferation, activation, differentiation, and effector function of antigen-specific T cells without eliciting cell death. Conversely, if MTA is added to highly activated T cells, MTA exerts cytotoxic effects on T cells. We identified the Akt pathway, a critical signal pathway for T cell activation, as a target of MTA, while, for example, p38 remained unaffected. Next, we provide evidence that MTA exerts its immunosuppressive effects by interfering with protein methylation in T cells. To confirm the relevance of the suppressive effects of exogenously added MTA on human T cells, we used an MTAP-deficient tumor cell-line that was stably transfected with the MTAP-coding sequence. We observed that T cells stimulated with MTAP-transfected tumor cells revealed a higher proliferative capacity compared to T cells stimulated with Mock-transfected cells. In conclusion, our findings reveal a novel immune evasion strategy of human tumor cells that could be of interest for therapeutic targeting.

Influence of the perivitelline space on the quantification of internal concentrations of chemicals in eggs of zebrafish embryos (Danio rerio).

The chorion and the perivitelline space which surround unhatched zebrafish embryos (ZFE, Danio rerio) may affect the determination of internal concentrations of study compounds taken up in early life-stages of ZFE. Internal concentration-time profiles were gathered for benzocaine, caffeine, clofibric acid, metribuzin and phenacetin as study compounds over 96 h of exposure starting with ZFE at 4h post-fertilization. Liquid chromatography coupled to tandem-mass spectrometry (LC-MS/MS) was used to determine the concentration of the study compounds from intact (i.e. unhatched), dechorionated and from hatched ZFE. The mass of the study compounds per ZFE was 5-30 ng higher for intact ZFE compared to dechorionated ones. Thus, internal concentrations were overestimated if only intact ZFE were analyzed. Dechorionation of unhatched ZFE after their exposure is proposed to determine the true internal concentration in the embryo. For the compounds studied here the mass of the study compounds determined in unhatched ZFE after a short term (5 min) exposure provided a reasonable estimate of the mass taken up by the chorion and the PVS. This mass can be subtracted from the total mass found in unhatched ZFE to calculate the true internal mass. Estimating the mass in the chorion and the PVS from the concentration of the study compound in the external exposure medium and the volume of the PVS provided no reasonable results.

Transient overexpression of adh8a increases allyl alcohol toxicity in zebrafish embryos.

Fish embryos are widely used as an alternative model to study toxicity in vertebrates. Due to their complexity, embryos are believed to more resemble an adult organism than in vitro cellular models. However, concerns have been raised with respect to the embryo's metabolic capacity. We recently identified allyl alcohol, an industrial chemical, to be several orders of magnitude less toxic to zebrafish embryo than to adult zebrafish (embryo LC50 = 478 mg/L vs. fish LC50 = 0.28 mg/L). Reports on mammals have indicated that allyl alcohol requires activation by alcohol dehydrogenases (Adh) to form the highly reactive and toxic metabolite acrolein, which shows similar toxicity in zebrafish embryos and adults. To identify if a limited metabolic capacity of embryos indeed can explain the low allyl alcohol sensitivity of zebrafish embryos, we compared the mRNA expression levels of Adh isoenzymes (adh5, adh8a, adh8b and adhfe1) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for adh8a and adh8b expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of adh8a, but not adh8b mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC50 = 0.42 mg/L in adh8a mRNA-injected embryos). Furthermore, GC/MS analysis of adh8a-injected embryos indicated a significant decline of internal allyl alcohol concentrations from 0.23-58 ng/embryo to levels below the limit of detection (< 4.6 µg/L). Injection of neither adh8b nor gfp mRNA had an impact on internal allyl alcohol levels supporting that the increased allyl alcohol toxicity was mediated by an increase in its metabolization. These results underline the necessity to critically consider metabolic activation in the zebrafish embryo. As demonstrated here, mRNA injection is one useful approach to study the role of candidate enzymes involved in metabolization.