Optimizing the Use of Zebrafish Feeding Trials for the Safety Evaluation of Genetically Modified Crops.

In Europe, the toxicological safety of genetically modified (GM) crops is routinely evaluated using rodent feeding trials, originally designed for testing oral toxicity of chemical compounds. We aimed to develop and optimize methods for advancing the use of zebrafish feeding trials for the safety evaluation of GM crops, using maize as a case study. In a first step, we evaluated the effect of different maize substitution levels. Our results demonstrate the need for preliminary testing to assess potential feed component-related effects on the overall nutritional balance. Next, since a potential effect of a GM crop should ideally be interpreted relative to the natural response variation (i.e., the range of biological values that is considered normal for a particular endpoint) in order to assess the toxicological relevance, we established natural response variation datasets for various zebrafish endpoints. We applied equivalence testing to calculate threshold equivalence limits (ELs) based on the natural response variation as a method for quantifying the range within which a GM crop and its control are considered equivalent. Finally, our results illustrate that the use of commercial control diets (CCDs) and null segregant (NS) controls (helpful for assessing potential effects of the transformation process) would be valuable additions to GM safety assessment strategies.

From mRNA Expression of Drug Disposition Genes to In Vivo Assessment of CYP-Mediated Biotransformation during Zebrafish Embryonic and Larval Development.

The zebrafish (Danio rerio) embryo is currently explored as an alternative for developmental toxicity testing. As maternal metabolism is lacking in this model, knowledge of the disposition of xenobiotics during zebrafish organogenesis is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this study was to assess cytochrome P450 (CYP) activity in zebrafish embryos and larvae until 14 d post-fertilization (dpf) by using a non-specific CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR) and a CYP1-specific substrate, i.e., 7-ethoxyresorufin (ER). Moreover, the constitutive mRNA expression of CYP1A, CYP1B1, CYP1C1, CYP1C2, CYP2K6, CYP3A65, CYP3C1, phase II enzymes uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) and sulfotransferase 1st1 (SULT1ST1), and an ATP-binding cassette (ABC) drug transporter, i.e., abcb4, was assessed during zebrafish development until 32 dpf by means of quantitative PCR (qPCR). The present study showed that trancripts and/or the activity of these proteins involved in disposition of xenobiotics are generally low to undetectable before 72 h post-fertilization (hpf), which has to be taken into account in teratogenicity testing. Full capacity appears to be reached by the end of organogenesis (i.e., 120 hpf), although CYP1-except CYP1A-and SULT1ST1 were shown to be already mature in early embryonic development.

Gene transcription ontogeny of hypothalamic-pituitary-thyroid axis development in early-life stage fathead minnow and zebrafish.

The hypothalamic-pituitary-thyroid (HPT) axis is known to play a crucial role in the development of teleost fish. However, knowledge of endogenous transcription profiles of thyroid-related genes in developing teleosts remains fragmented. We selected two model teleost species, the fathead minnow (Pimephales promelas) and the zebrafish (Danio rerio), to compare the gene transcription ontogeny of the HPT axis. Control organisms were sampled at several time points during embryonic and larval development until 33 days post-fertilization. Total RNA was extracted from pooled, whole fish, and thyroid-related mRNA expression was evaluated using quantitative polymerase chain reaction. Gene transcripts examined included: thyrotropin-releasing hormone receptor (trhr), thyroid-stimulating hormone receptor (tshr), sodium-iodide symporter (nis), thyroid peroxidase (tpo), thyroglobulin (tg), transthyretin (ttr), deiodinases 1, 2, 3a, and 3b (dio1, dio2, dio3a and 3b), and thyroid hormone receptors alpha and beta (thrα and ß). A loess regression method was successful in identifying maxima and minima of transcriptional expression during early development of both species. Overall, we observed great similarities between the species, including maternal transfer, at least to some extent, of almost all transcripts (confirmed in unfertilized eggs), increasing expression of most transcripts during hatching and embryo-larval transition, and indications of a fully functional HPT axis in larvae. These data will aid in the development of hypotheses on the role of certain genes and pathways during development. Furthermore, this provides a background reference dataset for designing and interpreting targeted transcriptional expression studies both for fundamental research and for applications such as toxicology.

Permanent Deiodinase Type 2 Deficiency Strongly Perturbs Zebrafish Development, Growth, and Fertility.

Iodothyronine deiodinases are selenocysteine-containing enzymes that activate or inactivate thyroid hormones (THs). Deiodinase type 2 (Dio2) catalyzes the conversion of the prohormone T4 into the transcriptionally active T3 and is the predominant activating deiodinase in zebrafish. Using zinc finger nucleases, we generated two different dio2(-/-) mutant zebrafish lines to investigate the physiological function of this TH activator. The first line contains a deletion of 9 bp, resulting in an in-frame elimination of three conserved amino acids. The other line is characterized by an insertion of 4 bp, leading to the introduction of a premature stop-codon. Both lines completely lack Dio2 activity, resulting in a strong reduction of T3 abundancy in all tissues tested. Early development is clearly perturbed in these animals, as shown by a diverse set of morphometric parameters, defects in swim bladder inflation, and disturbed locomotor activity tested between 1 and 7 days after fertilization. Permanent Dio2 deficiency also provokes long-term effects because growth and especially fertility are severely hampered. Possible compensatory mechanisms were investigated in adult dio2(-/-) mutants, revealing a down-regulation of the inactivating deiodinase Dio3 and TH receptor transcript levels. As the first nonmammalian model with permanent Dio2 deficiency, these mutant zebrafish lines provide evidence that Dio2 is essential to assure normal development and to obtain a normal adult phenotype.