Allyl alcohol toxicity in different life stages of zebrafish.

<p>Zebrafish embryos were exposed for the indicated periods (hpf  =  hours post fertilization). For the toxicity studies we used 1 embryo per well, 10 wells per exposure concentration. Exposure from 24–72 hpf was performed with and without chorion. Error bars indicate the asymmetric 95% confidence intervals of the concentration-response curves (n = 3; for acrolein no replicates have been performed and therefore no confidence intervals have been calculated). In comparison to a previously published LC₅₀ for zebrafish embryos obtained from Lammer <i>et al.</i> 2009 (open circle), acrolein toxicity based on an exposure from 2–50 hpf (n = 1) is depicted in the grey shaded area (closed symbol). Molecular weights of allyl alcohol and acrolein: 58.08 and 56.06 g/mol, respectively.</p

Transient Overexpression of <i>adh8a</i> Increases Allyl Alcohol Toxicity in Zebrafish Embryos

<div><p>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 <i>in vitro</i> 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 (<i>adh5</i>, <i>adh8a</i>, <i>adh8b</i> and <i>adhfe1</i>) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for <i>adh8a</i> and <i>adh8b</i> expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of <i>adh8a</i>, but not <i>adh8b</i> mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC₅₀ = 0.42 mg/L in <i>adh8a</i> mRNA-injected embryos). Furthermore, GC/MS analysis of <i>adh8a</i>-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 <i>adh8b</i> nor <i>gfp</i> 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.</p></div

Analysis of internal allyl alcohol concentration in zebrafish embryos by GC-MS.

<p>For each replicate 50 embryos were used. AAL, allyl alcohol. n.d., not detectable.</p

Overexpression of <i>adh8a</i> did not alter embryonic development.

<p>Lateral views of embryo at 72–2 hpf with <i>gfp</i>, <i>adh8a</i>, or <i>adh8b</i> mRNAs.</p

Effect of the injection of <i>adh8a</i> and <i>adh8b</i> mRNA on the LC₅₀ of allyl alcohol.

<p>Error bars represent 95% asymmetric confidence intervals of LC₅₀ data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090619#pone.0090619.s005" target="_blank">Table S1</a> for dose-response modeling). <i>gfp</i> mRNA was injected as an unspecific control.</p

Compound Specific and Enantioselective Stable Isotope Analysis as Tools To Monitor Transformation of Hexachlorocyclohexane (HCH) in a Complex Aquifer System

Technical hexachlorocyclohexane (HCH) mixtures and Lindane (γ-HCH) have been produced in Bitterfeld-Wolfen, Germany, for about 30 years until 1982. In the vicinity of the former dump sites and production facilities, large plumes of HCHs persist within two aquifer systems. We studied the natural attenuation of HCH in these groundwater systems through a combination of enantiomeric and carbon isotope fractionation to characterize the degradation of α-HCH in the areas downstream of a former disposal and production site in Bitterfeld-Wolfen. The concentration and isotope composition of α-HCH from the Quaternary and Tertiary aquifers were analyzed. The carbon isotope compositions were compared to the source signal of waste deposits for the dumpsite and highly contaminated areas. The average value of δ¹³C at dumpsite was −29.7 ± 0.3 ‰ and −29.0 ± 0.1 ‰ for (−) and (+)­α-HCH, respectively, while those for the β-, γ-, δ-HCH isomers were −29.0 ± 0.3 ‰, −29.5 ± 0.4 ‰, and −28.2 ± 0.2 ‰, respectively. In the plume, the enantiomer fraction shifted up to 0.35, from 0.50 at source area to 0.15 (well T1), and was found accompanied by a carbon isotope enrichment of 5 ‰ and 2.9 ‰ for (−) and (+)­α-HCH, respectively. The established model for interpreting isotope and enantiomer fractionation patterns showed potential for analyzing the degradation process at a field site with a complex history with respect to contamination and fluctuating geochemical conditions