Use of Early Juvenile Zebrafish Danio Rerio for In-Vivo Assessment of Endocrine Modulation by Xenoestrogens

Reliable and cost-effective early-life stage (ELS) bioassays incorporating practical experimentation without compromising scientific relevance are crucial in chemical risk assessment. This study investigated the use of 20 days- post-fertilization life stage ( 20dpfZF ) of zebrafish Danio rerio to screen environmental chemicals known to be estrogenic in adult fish. Firstly, studies with key genes in steroidogenesis were conducted; the brain isoform of aromatase gene ( cyp19a1b ) being the most prominently expressed biomarker. Regulation of mRNA levels of molecular biomarkers, vitellogenin 1 gene ( vtg1) and cyp19a1b were selected to assess the endocrine modulation by xenoestrogens, 17α-ethinylestradiol (EE2), 4- n -nonylphenol (NP), 4- t -octylphenol (OP) and bisphenol A (BPA). Groups of 20dpfZF (n=15) as three replicates were exposed to chemicals over a five-day period in aerated static setups. Exposure of 20dpfZF to sediment spiked with EE2 (nominal 3μg g -1 dw) was also conducted to assess the sensitivity of this life-stage to sediment with estrogenic potency. Whole body homogenates of exposed juveniles showed the estrogenic potential of chemicals in the order: EE2 > OP > BPA > NP. Higher relative expression of cyp19a1b was noticed at lower ambient concentrations of EE2, although vtg1 showed more pronounced expression to it. The 20dpfZF responded in a dose-related way to sediment spiked with EE2, expanding its use as a general aquatic animal model. The suitability of 20dpfZF as an in vivo model, along with stable expression of reference genes was established. In addition to consistent expression pattern of key target genes on xenoestrogenicity, it serves as a practical screening model for the risk assessment of environmental chemicals and samples with estrogenic potential.peerReviewe

Responses to ultraviolet radiation in larval pike, Esox lucius, of two origins and ages

Ultraviolet radiation (UVR)-induced mortality and behavioural disorder were studied in larval northern pike of two origins and ages. Newly hatched larvae of two differently coloured populations and six-day-old larvae of one population were exposed to four fluence rates of UVR, resulting in total doses from 11.5 to 63 kJ m–2, and monitored for mortality and behaviour. The rate of mortality and the severity of behavioural disorder differed by origin and age of the animals, but the effect was fluence-rate dependent. Total melanin concentration of newly hatched larvae was measured to assess if sensitivity to UVR correlated with pigmentation, but no differences in melanin concentration between larvae from different origins were found.peerReviewe

UV-B Exposure Causes DNA Damage and Changes in Protein Expression in Northern Pike (Esox lucius) Posthatched Embryos

The ongoing anthropogenically caused ozone depletion and climate change has increased the amount of biologically harmful UV-B radiation, which is detrimental to fish in embryonal stages. The effects of UV-B radiation on the levels and locations of DNA damage manifested as cyclobutane pyrimidine dimers (CPDs), heat shock protein 70 (HSP70) and p53 protein in newly hatched embryos of pike were examined. Pike larvae were exposed in the laboratory to current and enhanced doses of UV-B radiation. UV-B exposure caused the formation of CPDs in a fluence rate-dependent manner, and the CPDs were found deeper in the tissues with increasing fluence rates. UV-B radiation induced HSP70 in epidermis, and caused plausible p53 activation in the brain and epidermis of some individuals. Also at a fluence rate occurring in nature, the DNA damage in the brain and eyes of pike and changes in protein expression were followed by severe behavioral disorders, suggesting that neural molecular changes were associated with functional consequences.peerReviewe

Photoinduced lethal and sublethal toxicity of retene, a polycyclic aromatic hydrocarbon derived from resin acid, to coregonid larvae

A comparative investigation on the acute phototoxicity of retene to vendace (Coregonus albula) and whitefish (C. lavaretus), both having pelagial larvae in spring, was conducted. To test the concept of early warning of sublethal biomarkers in relation to lethality to posthatch stages, we examined the effects of ultraviolet-B (UV-B) and retene on the levels of cytochrome P4501A (CYP1A) and heat shock protein 70 (HSP70) by exposing the animals to elevated levels of these factors for 48 and 72 h, respectively. Whereas UV-B and retene on their own were not lethal, simultaneous retene and UV-B exposure caused very high mortality to both species. The median lethal concentration (LC50; i.e., the concentration at which half of the larvae died) of retene as a precursor was 41 μg/L for vendace and 15 to 16 μg/L, depending on the UV-B dose, for whitefish. Retene evoked substantial induction of CYP1A in larvae of both species, and UV-B induced CYP1A in whitefish. In vendace, no effect on HSP70 levels by any factor was observed. In whitefish, however, UV-B radiation and water retene alone upregulated HSP70, but no additive response was detected. The CYP1A is a biomarker of exposure to retene in both species. The HSP70 is an early warning signal of UV-B exposure in whitefish. As a species, vendace appears to be more resistant than whitefish to the phototoxicity of retene, as indicated by the higher tolerance.peerReviewe

Fluence rate or cumulative dose? : Vulnerability of larval northern pike (Esox lucius) to ultraviolet radiation

Newly hatched larvae of northern pike were exposed in the laboratory to four fluence rates of ultraviolet radiation (UVR; 290–400 nm) over three different time periods, resulting in total doses ranging from 3.0 ± 0.2 to 63.0 ± 4.4 kJ·m−2. Mortality and behavior of the larvae were followed for 8–12 days, and growth measured at the end of the experiment. Also, the principle of reciprocity—that the UVR-induced mortality depends on the cumulative dose, independent of fluence rate—was tested. Fluence rates higher than 1480 ± 150 mW·m−2 caused mortality and growth retardation. The highest fluence rate (3040 ± 210 mW·m−2) caused 100% mortality in 5 days. All fluence rates caused behavioral disorders, which led to death at fluence rates higher than 1480 mW·m−2. Reciprocity failure occurred with the lowest and highest dose (550 ± 45 and 3040 ± 210 mW·m−2, respectively). The results show that fluence rate is of primary importance when assessing the UVR-related risk.peerReviewe