Toxicokinetics of a developmental toxicity test in zebrafish embryos and larvae: Relationship with drug exposure in humans and other mammals.

To study the effects of drugs on embryo/fetal development (EFD), developmental and reproductive toxicity studies in zebrafish (Danio rerio) embryos is expected to be an accepted alternative method to animal studies using mammals. However, there is a lack of clarity in the relationship between the concentration of developmental toxicity agents in whole embryos or larvae (Ce) and that in aqueous solution (Cw), and also between the amount of drug exposure required to cause developmental toxicity in zebrafish embryos or larvae and that required in mammals. Here, we measured Ce for developmental toxicity agents every 24 h starting at 24 h post fertilization (hpf). We found a high correlation (R 2: 0.87-0.96) between log [Ce/Cw] and the n-octanol-water distribution coefficient at pH 7 (logD) of each drug at all time points up to 120 hpf. We used this relationship to estimate the Ce values of the 21 positive-control reference drugs listed in ICH guidelines on reproductive and developmental toxicity studies (ICH S5). We then calculated the area under the Ce-time curve in zebrafish (zAUC) for each drug from the regression equation between log [Ce/Cw] and logD and compared it with the AUC at the no-observed-adverse-effect level in rats and rabbits and at the effective dose in humans described in ICH S5. The log of the calculated zAUC for the 14 drugs identified as positive in the zebrafish developmental toxicity test was relatively highly positively correlated with the log [AUC] for rats, rabbits, and humans. These findings provide important and positive information on the applicability of the zebrafish embryo developmental toxicity test as an alternative method of EFD testing. (267 words).

Augmentation of Pectoral Fin Teratogenicity by Thalidomide in Human Cytochrome P450 3A-Expressing Zebrafish.

The pharmacological and toxicological effects of active metabolites of enzymes including cytochrome P450 (CYP) are important. While it has been believed for a long time that thalidomide causes characteristic limb malformation only in rabbits and primates including humans, the involvement of their CYP3A subtypes (CYP3As) has been suggested. Recently, however, it was reported that zebrafish were sensitive to thalidomide, showing defects of pectoral fins, homologous organs of forelimbs in mammals, as well as other deformities. In this study, we prepared human CYP3A7 (hCYP3A7)-expressing zebrafish (F0) using a transposon system. Thalidomide caused pectoral fin defects and other malformations including pericardial edema in hCYP3A7-expressing embryos/larvae but not in wild-type and hCYP1A1-expressing embryos/larvae. Thalidomide also reduced the expression of fibroblast growth factor 8 in pectoral fin buds in only hCYP3A7-expressing embryos/larvae. The results suggest the involvement of human-type CYP3A in thalidomide teratogenicity.

Transgenic Zebrafish Expressing Rat Cytochrome P450 2E1 (CYP2E1): Augmentation of Acetaminophen-Induced Toxicity in the Liver and Retina.

Metabolic activation is the primary cause of chemical toxicity including hepatotoxicity. Cytochrome P450 2E (CYP2E) is involved in this process for many hepatotoxicants, including acetaminophen (APAP), one of the most common analgesics and antipyretics. Although the zebrafish is now used as a model for toxicology and toxicity tests, the CYP2E homologue in zebrafish has not been identified yet. In this study, we prepared transgenic zebrafish embryos/larvae expressing rat CYP2E1 and enhanced green fluorescent protein (EGFP) using a ß-actin promoter. Rat CYP2E1 activity was confirmed by the fluorescence of 7-hydroxycoumarin (7-HC), a metabolite of 7-methoxycoumarin that was specific for CYP2 in transgenic larvae with EGFP fluorescence (EGFP [+]) but not in transgenic larvae without EGFP fluorescence (EGFP [-]). APAP (2.5 mM) caused reduction in the size of the retina in EGFP [+] larvae but not in EGFP [-] larvae, while APAP similarly reduced pigmentation in both larvae. APAP at even 1 mM reduced the liver size in EGFP [+] larvae but not in EGFP [-] larvae. APAP-induced reduction of liver size was inhibited by N-acetylcysteine. These results suggest that rat CYP2E1 is involved in some APAP-induced toxicological endpoints in the retina and liver but not in melanogenesis of the developing zebrafish.

Cytochrome P450 Expression and Chemical Metabolic Activity before Full Liver Development in Zebrafish.

Zebrafish are used widely in biomedical, toxicological, and developmental research, but information on their xenobiotic metabolism is limited. Here, we characterized the expression of 14 xenobiotic cytochrome P450 (CYP) subtypes in whole embryos and larvae of zebrafish (4 to 144 h post-fertilization (hpf)) and the metabolic activities of several representative human CYP substrates. The 14 CYPs showed various changes in expression patterns during development. Many CYP transcripts abruptly increased at about 96 hpf, when the hepatic outgrowth progresses; however, the expression of some cyp1s (1b1, 1c1, 1c2, 1d1) and cyp2r1 peaked at 48 or 72 hpf, before full liver development. Whole-mount in situ hybridization revealed cyp2y3, 2r1, and 3a65 transcripts in larvae at 55 hpf after exposure to rifampicin, phenobarbital, or 2,3,7,8-tetrachlorodibenzo-p-dioxin from 30 hpf onward. Marked conversions of diclofenac to 4'-hydroxydiclofenac and 5-hydroxydiclofenac, and of caffeine to 1,7-dimethylxanthine, were detected as early as 24 or 50 hpf. The rate of metabolism to 4'-hydroxydiclofenac was more marked at 48 and 72 hpf than at 120 hpf, after the liver had become almost fully developed. These findings reveal the expression of various CYPs involved in chemical metabolism in developing zebrafish, even before full liver development.

Receptor-binding affinities of bisphenol A and its next-generation analogs for human nuclear receptors.

An endocrine-disrupting chemical Bisphenol A (BPA) binds specifically to a nuclear receptor (NR) named ERRγ. Although the importance of receptor-binding evaluation for human NRs is often stressed, the binding characteristics of so-called next-generation (NextGen) bisphenol compounds are still poorly understood. The ultimate objective of this investigation was to evaluate BPA and its NextGen analogs for their abilities to bind to 21 human NRs, the greatest members of NRs for which tritium-labeled specific ligands were available. After establishing the detailed assay conditions for each NR, the receptor binding affinities of total 11 bisphenols were evaluated in competitive binding assays. The results clearly revealed that BPA and the NextGen bisphenols of BPAF, BPAP, BPB, BPC, BPE, and BPZ were highly potent against one or more of NRs such as CAR, ERα, ERß, ERRγ, and GR, with IC50 values of 3.3-73 nM. These bisphenols were suggested strongly to be disruptive to these NRs. BPM and BPP also appeared to be disruptive, but less potently. BPF exhibited only weak effects and only against estrogen-related NRs. Surprisingly, most doubtful bisphenol BPS was supposed not to be disruptive. The NRs to which BPA and NextGen bisphenols did not bind were RARα, RARß, RARγ, and VDR. PPARγ, RORα, RORß, RORγ, RXRα, RXRß, and RXRγ, exhibited very weak interaction with these bisphenols. The ten remaining NRs, namely, ERRγ, ERß, ERα, CAR, GR, PXR, PR, AR, LXRß, and LXRα, showed distinctly strong binding to some bisphenols in this order, being likely to have consequential endocrine-disruption effects.

Comparing time-series of chemical concentrations in zebrafish (Danio rerio) embryos/larvae exposed to teratogens with different hydrophobicity; caffeine, sodium valproate, and diethylstilbestrol.

Developmental toxicity is an adverse developmental outcome, i.e., death, malformation, growth retardation, or functional deficiency. Recently, alternative methods of assessing developmental toxicity using zebrafish (Danio rerio) as a preliminary screening have attracted attention because of their low cost and high throughput. However, most toxicity evaluations have been based on a chemical concentration in an aqueous solution, and the chemical concentrations in embryos/larvae and their temporal behavior have in most cases been unclear, regardless of differences of chemical hydrophobicity. In the present study, we selected three teratogens with different hydrophobicities (caffeine, CA, log Kow -0.07; sodium valproate, VA, log Kow 0.26 (pH 7.4); and diethylstilbestrol, DES, log Kow 5.07), and we measured their concentrations in embryos/larvae exposed to these chemicals every 24 hr post-fertilization (hpf) until 144 hpf. Kinetic analysis based on a one-compartment fish model that yields first order kinetics for CA and VA revealed that concentrations of both CA and VA in embryos/larvae increased gradually and became saturated by around 100 hpf. In contrast, DES concentrations in embryos/larvae reached a maximum at 48 or 72 hpf and then decreased gradually. The present study suggests that the temporal pattern of chemical concentrations is a function of the hydrophobicity of the chemicals.