Biotransformation in the zebrafish embryo -temporal gene transcription changes of cytochrome P450 enzymes and internal exposure dynamics of the AhR binding xenobiotic benz[a]anthracene.

Not much is known about the biotransformation capability of zebrafish (Danio rerio) embryos. For understanding possible toxicity differences to adult fish, it might be crucial to understand the biotransformation of chemicals in zebrafish embryos i.e. as part of toxicokinetics. The biotransformation capabilities were analysed for two different stages of zebrafish embryos in conjunction with the internal concentrations of a xenobiotic. Zebrafish embryos of the late cleavage/early blastula period (2-26 hpf) and the early pharyngula period (26-50 hpf) were exposed for 24 h to the AhR binding compound benz[a]anthracene (BaA). Time dependent changes in cyp transcription (cyp1a, cyp1b1, cyp1c1 and cyp1c2) as well as concentration & time-dependent courses of BaA in the fish embryo and the exposure medium were analysed. Additionally, the CYP mediated formation of biotransformation products was investigated. We found correlations between transcriptional responses and the internal concentration for both exposure types. These correlations were depending on the start of the exposure i.e. the age of the exposed embryo. While no significant induction of the examined gene transcripts was observed in the first 12 h of exposure beginning in the blastula period a correlation was apparent when exposure started later i.e. in the pharyngula period. A significant induction of cyp1a was detected already after 1.5 h of BaA exposure. Gene transcripts for cyp1b1, cyp1c1 and cyp1c2 showed expressions distinctly different from cyp1a and were, in general, less inducible by BaA in both exposure windows. The toxicokinetic analysis showed that the biotransformation capability was fivefold higher in the older fish embryos. Biotransformation products of phase I reactions were found between 32 hpf and 50 hpf and were tentatively identified as benz[a]anthracene-phenol and benz[a]anthracene-dihydrodiol-epoxide. In conclusion, not only duration but also onset of exposure in relation to the developmental stage of zebrafish embryos is important in the analysis and interpretation of effects due to different biotransformation capabilities.

A toxicokinetic study of specifically acting and reactive organic chemicals for the prediction of internal effect concentrations in Scenedesmus vacuolatus.

The toxic potency of chemicals is determined by using the internal effect concentration by accounting for differences in toxicokinetic processes and mechanisms of toxic action. The present study examines toxicokinetics of specifically acting and reactive chemicals in the green algae Scenedesmus vacuolatus by using an indirect method. Concentration depletion in the exposure medium was measured for chemicals of lower (log KOW < 3: isoproturon, metazachlor, paraquat) and moderate (log KOW 4-5: irgarol, triclosan, N-phenyl-2-naphthylamine) hydrophobicity at 7 to 8 time points over 240 min or 360 min. Uptake and overall elimination rates were estimated by fitting a toxicokinetic model to the observed concentration depletions. The equilibrium of exposure concentrations was reached within minutes to hours or was even not observed within the exposure time. The kinetics of bioconcentration cannot be explained by the chemical's hydrophobicity only, but influential factors such as ionization of chemicals, the ion trapping mechanism, or the potential susceptibility for biotransformation are discussed. Internal effect concentrations associated with 50% inhibition of S. vacuolatus reproduction were predicted by linking the bioconcentration kinetics to the effect concentrations and ranged from 0.0480 mmol/kg wet weight to 7.61 mmol/kg wet weight for specifically acting and reactive chemicals. Knowing the time-course of the internal effect concentration may promote an understanding of toxicity processes such as delayed toxicity, carry-over toxicity, or mixture toxicity in future studies.

The internal concentration of organic substances in fish embryos--a toxicokinetic approach.

In ecotoxicity assessment, the ambient exposure concentration is typically applied to quantify the toxic potential of xenobiotic substances. However, exposure and organism-related differences in bioconcentration often cause a considerable variability of toxicity data. This can be minimized by using the internal organism concentration, because toxicokinetic modifying factors are considered implicitly. In the present study, the relationship between ambient and internal concentration-time profiles was investigated for zebrafish (Danio rerio) embryos. The aim was to gain a better understanding and interpretation of exposure-based methods using this model organism. For this purpose, a simple and effective approach to determine the internal concentration was developed. Embryos were exposed to a series of 4 neutral organic substances (naphthalene, fluorene, fluoranthene, benz[a]anthracene) of different hydrophobicity for 72 h. The internal and ambient concentrations were measured at 8 to 9 time points. Kinetics of uptake and elimination were modeled using a first-order 1-compartment model. Biotransformation processes appeared to influence the internal concentrations of fluoranthene and benz[a]anthracene after 48 h. The bioconcentration factors (BCFs) obtained are in excellent agreement with those determined in previous studies using radiolabeled substances. The method demonstrated in the present study is a further step toward a refined ecotoxicity assessment using fish embryos, which links toxicity to the chemical concentration within the organism. This system may also be considered as an alternative to animal testing for BCF determination.