Transient Overexpression of adh8a Increases Allyl Alcohol Toxicity in Zebrafish Embryos

Abstract

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.

Is fish embryo test (FET) according to OECD 236 sensible enough for delivering quality data for effluent risk assessment?

“…In addition to the processes described, embryos may have the protective effect of the chrorion against chemicals, which has been the topic of exhaustive research (Ozoh ; Braunbeck et al ; Henn and Braunbeck, ; Klüver et al ). An attempt to overcome this protective effect observed on FET was extension of exposure time from 48 to 96 h, allowing the posthatch larvae to enter into contact with the test solution (Braunbeck et al ).…”

Applicability of the fish embryo acute toxicity (FET) test (OECD 236) in the regulatory context of Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH)

“…However, the validation studies as well as some other published studies reported substances with weaker toxicity to fish embryos compared with adult/juvenile fish (Knöbel et al ; Busquet et al ; Klüver et al , ). Recently, Glaberman et al () analyzed 161 pesticides and also found an underestimation of acute toxicity for chemicals with specific mode of actions, particularly neurotoxicants.…”

“…For one substance (allyl alcohol), experimental evidence indicates that a weak or lack of biotransformation by alcohol dehydrogenase resulted in the observed 323‐fold lower toxicity in fish embryos (Klüver et al ). The biotransformation product of allyl alcohol—acrolein—exhibited a 500‐fold higher toxicity than its parent substance (with respect to data available for the 48‐h exposure of acrolein).…”

AEO-7 Surfactant is “super Toxic” and Induces Severe Cardiac, Liver and Locomotion Damage in Zebrafish Embryos

“…Zebra sh have been shown to be a good model for predicting hepatotoxicity [68][69][70], probably because the enzymes and pathways involved in xenobiotic metabolism (e.g., the aryl hydrocarbon receptor and isozymes of cytochrome P-450 and aldehyde dehydrogenase) are all evolutionarily conserved and all functional from the early stages of the development of zebra sh embryos, including our experimental window [63,71,72].…”