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This thesis presents the development of three new bioassays for the detection of compounds disrupting the early development of echinoid larvae from hatching to metamorphosis, and the interference with cellular efflux pumps. These assays come in addition to the already existing sea urchin fertilization assay and the short term ELS assay (48 or 96 hours. This chapter summarizes the contents of the thesis.
In Chapter 1, background information and objectives of the thesis are presented. Firstly the risks that POPs pose to the marine environment are introduced, as well as the need to develop tools to assess toxic effects of exposure during the most sensitive period of the life cycle of organisms, their early life development. Secondly, echinoids are presented as an invertebrate marine animal model to develop early life development bioassays. Lastly, the aim of the thesis and the experimental approach chosen is outlined.
Chapter 2 describes the development ofa new 16-day echinoid prolonged early life stage (p-ELS) bioassay that includes prolonged observation for the detection of possible delayed adverse effects during embryogenesis and larval development of the sea urchin Psammechinus miliaris. Subsequently, the newly developed bioassay was applied to study the effects of key marine POPs. Mortality, morphological abnormalities and larval development stages were quantified at specific time points during the 16-day experimental period. In contrast to amphibians and fish, P. miliaris early life development was not sensitive to dioxin-like toxicity in the p-ELS test. Triclosan (TCS) levels higher than 500 nM were acutely toxic during embryo development. Morphological abnormalities were induced at concentrations higher than 50 nM hexabromocyclododecane (HBCD) and 1000 nM tetrabromobisphenol A (TBBPA). Larval development was delayed above 25 nM HBCD and 500 nM TBBPA. Heptadecafluorooctane sulfonic acid (PFOS) exposure slightly accelerated larval development at 9 days post fertilization (dpf). However, the accelerated development was no longer observed at the end of the test period (16 dpf). The newly developed 16-day echinoid p-ELS bioassay proved to be sensitive to toxic effects of POPs and effects can be monitored for individual echinoid larvae. The most sensitive and dose related endpoint was the number of developmental penalty points. By manipulation of the housing conditions, the reproductive season could be extended from 3 to 9 months per year and the p-ELS experiments could be performed in artificial sea water as well.
In Chapter 3 a metamorphosis assaywas developed, using the sea urchin P. miliaris, to detect and quantify the potency of persistent organic pollutants (POPs) to disrupt thyroid hormone (TH) induced metamorphosis. Similar to vertebrates, echinoids have a TH induced metamorphosis, making them a potential model organisms to study TH disruption. Larvae were exposed to test compounds from the 8-armed pluteus stage until metamorphosis completion. Thyroxine (T4) accelerated metamorphosis (EC50 0.12 and 0.09 nM experiment A and B, respectively), whereas the TH synthesis inhibitor thiourea (TU) (IC50 0.1 and 0.04 mM experiment A and B, respectively) or the iodine uptake inhibitor potassium thiocyanate (KSCN) delayed metamorphosis (IC50 <0.1 mM). Polybrominated diphenyl ethers (PBDEs) strongly accelerated metamorphosis (EC50 219 nM), while TBBPA and TCS delayed it (IC50 97 and 418 nM, respectively). It was concluded that echinoids are promising marine model organisms for ecotoxicological studies and further insight into TH function may contribute to reduce the use of vertebrates to study TH disruption.
Chapter 4 focusses onthe interaction of POPs with the Multi Xenobiotic Resistance (MXR) mechanism, an important first line of defense against contaminants by pumping contaminants out of the cells. If compounds would impair the MXR mechanism, this could result in increased intracellular levels of other compounds, thereby potentiating their toxicity. A calcein-AM based larval cellular efflux pump inhibition assay (CEPIA) was developed for echinoid (P. miliaris) larvae and the effects of several contaminants in this assay were quantified. The MXR mechanism in P. miliaris may be mediated by the action of e.g. P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) which are also present in vertebrates. The larval echinoid CEPIA revealed that TCS and the nanoparticles P-85 (P-85) were 124 and 155 times more potent inhibitors (IC50 0.5 ± 0.05 and 0.4 ± 0.1 µM, respectively) of efflux pumps than the model inhibitor Verapamil (VER). PFOS (heptadecafluorooctane sulfonic acid) and pentachlorophenol also were more potent than VER, 24 and 5 times, respectively. Bisphenol A (BPA) and o,p’-dichlorodiphenyltrichloroethane (o,p’-DDT) inhibited efflux pumps with a potency 3 times greater than VER. In a 48 h early life stage bioassay with P. miliaris, exposure to a non-lethal concentration of the inhibitors TCS, VER, the model MRP inhibitor MK-571, the nanoparticles P-85 and the model P-gp inhibitor PSC-833, increased the toxicity of the toxic model substrate for efflux pumps vinblastine by a factor of 2, 4, 4, 8 and 16, respectively. The findings reveal that several contaminants accumulating in the marine environment can inhibit cellular efflux pumps, which may potentiate toxic effects of efflux pumps substrates.
The newly develop P. miliaris p-ELS and metamorphosis bioassays were applied in Chapter 5to investigate the effects of a field-relevant mixture of POPs. This is particularly relevant since these contaminants occur in the marine environment as mixtures of compounds that could influence each others effects. In these studies two field-based mixtures (FM) were tested. FM1 was composed of the following seven compounds: BDE-47 (2,2’,4,4’-tetrabromodiphenyl ether); PFOS (heptadecafluorooctane sulfonic acid); PCB-153 (2,2',4,4',5,5'-hexachlorobiphenyl); PCB-126 (3,3’,4,4’,5-pentachlorobiphenyl); HBCD (hexabromocyclododecane); DBT (dibutyltin); TPT (triphenyltin). FM2 was the same mixture without TPT and DBT. In addition TPT and DBT also were tested alone. Effects observed in the p-ELS bioassay show a significant increase in larval morphological abnormalities and delayed development at concentrations ≥FM1/81 (corresponding to a 81 times dilution of the highest test concentration). FM2 (without TPT and DBT) only induced morphological abnormalities at the highest concentration. TPT and, to a lesser extent, DBT alone also induced a statistically significant increase in morphological abnormalities at concentrations ≥0.2 and ≥32 µg/l, respectively. This corresponds to a TPT concentration approximately comparable to a 50 times diluted FM1 (FM1/50), while this DBT concentration is 10 times higher than that in FM1 (3 µg/l). Therefore, the addition of TPT to the FM2 would add more to the total toxicity than the addition of DBT. In the metamorphosis assay, FM1 induced a statistically significant metamorphosis acceleration and morphological abnormalities in juveniles at concentrations ≥FM1/27 and ≥FM1/9, respectively, while FM2 did not affect metamorphosis at all. TPT and DBT alone significantly accelerated metamorphosis at ≥1.7 and ≥4 µg/l, respectively, and caused an increase in juvenile morphological abnormalities at ≥0.1 and ≥32 µg/l, respectively. TPT can account for approximately 100% of the metamorphosis acceleration observed in larvae exposed to FM1. TPT is an strong inducer of the RXR (retinoic X receptor) which is known to synergize TH and retinoic acid dependent mediated mechanisms, both known to be crucial for early development and metamorphosis. As RXR genes are expressed in echinoids it is speculated that the strong enhancement of the toxicity of FM2 by TPT, and possibly DBT, is mediated via the RXR. Given the high environmental levels of TPT it is important to further elucidate the mechanism behind this mixture effect.
Chapter 6discusses the relevance of developing and applying bioassays for the evaluation of toxic effects of POPs during the most sensitive developmental periods of echinoids. Acute and sub-chronic effects, disruption of TH induced metamorphosis and inhibition of the MXR defense system were detected and quantified. Compounds revealed to be most toxic for the early life development of P. miliaris were: TPT, a biocide used in agriculture as fungicide, molluscicide as well as rodent and insect repellant; TCS, widely use in household hygiene products, such as toothpastes, soaps, detergents, and disinfectants; HBCD, an additive brominated flame retardant (BFRs) applied in high impact polystyrene foams, in upholstery textiles and to a less extent in electrical equipment housings; and PBDEs, which also are additive BFRs used in plastics such as high impact polystyrene, in electrical and electronic equipment and textile back-coating in furniture. Furthermore, its discussed the importance to assess (eco)toxicological effects resulting from exposure to mixtures of POPs, for example with exposure to inhibitors of the MXR defense system and alkyltin compounds. These outcomes, together with the molecular and physiological commonalities between echinoids and vertebrates, open possibilities for echinoid bioassays when aiming at the reduction of vertebrate species used in toxicological studies.
|Qualification||Doctor of Philosophy|
|Award date||18 Dec 2012|
|Place of Publication||S.l.|
|Publication status||Published - 2012|
- persistent organic pollutants
- neonatal development