Effects of weathered oil and dispersants on hatchling morphology, apical and molecular endpoints of Menidia beryllina embryo

Transcription

1 Effects of weathered oil and dispersants on hatchling morphology, apical and molecular endpoints of Menidia beryllina embryo Adeyemo, OK, Kroll, KJ, Garcia-Reyero, N, Denslow, ND

2 OUTLINE Introduction Experimental set-up Results-Apical endpoint Results-Molecular endpoint Key Findings Further work

3 Introduction Oil spill episodes necessitate dispersion. Corexits are chemical dispersants, initially 9527 and then 9500 The toxicity of corexits to aquatic organisms still being debated in literature (George-Ares and Clark (1999), Berninger et al, 2011, Khan and Payne, 2005) With lots of submissions on impact of oil/dispersants combination..are they more toxic? In this study, we looked into this using Menidia, a sensitive EPA-approved estuarine species

4 Hypothesis Exposure of Menidia embryos to WAF of oil and dispersants, singly, and as oil/dispersant combination will: 1. Disrupt tissue differentiation and hence hatching 2. Cause abnormal development of hatched embryos 3. Disrupt the transcripts of genes associated with sexual differentiation, growth and stress response

5 EXPERIMENTAL SET-UP Oil (1000µl/L) and 100µl/L dispersants (Corexit 9500 and 9527) were weathered singly and in oil/dispersant (10:1) combination for 7 days in glass bottles using stir plates and bars Weathering (for 7 days) The WAF was extracted postweathering WAF Diluted 1:5 for the exposures

6 EXPERIMENTAL SET-UP embryos at 30-48hrs post-fertilization were exposed for 72 hours as follows in quadruplicate: Control: 1 liter saltwater (25ppt) Oil- 1:5 (WAF: saltwater) C9500-1:5 (WAF: saltwater) C9527-1:5 (WAF: saltwater) Oil:9500-1:5 (WAF: saltwater) Oil:9527-1:5 (WAF: saltwater) Daily, embryos were observed for hatching and mortality. Heart rate was taken at the end.

7 Method-Molecular endpoint RNA was extracted and qpcr assay carried out for target genes: sexual differentiation and sex determination (CYP19b, Vitellogenin, Choriogenin L, StAR, AMH and DMRT1) growth regulation (GHR and IGF) stress response (CYP1A and HSP90) GAPDH served as the housekeeping gene Relative quantification log2 (-ΔΔCt) method differences in gene expression by ANOVA

8 Method-Apical endpoint A modified graduated severity index (Hose et al, 1996) score was used to determine the normalcy of embryos. Similarly, the severity of differentiation abnormality was also scored as follows Statistics was with ANOVA and post hoc analyses (p<0.05)

9 Results-Heartbeat Average temperature (± SD) was 21 ± 1.5 C Mean heartbeat was significantly lowest in Corexit (67.5 beats/min) and (67.1beats/min) exposed embryos compared with controls (82.7 beats/min)

10 Contr ol 9500 Oil/950 0 Oil 9527 Oil/952 7 Morphological abnormalities Contr ol Oil Oil/950 0 Oil/952 7 Deterioration of unhatched Skeletal abnormalities in embryos hatchlings

11 Other abnormalities were head malformation, pericardial and abdominal edema

12 Results-Embryo Normalcy Significantly more (P<0.008) abnormal embryos were observed than the control except for oil and oil/9527 combination Significantly (p=0.007) more embryos exposed to oil and dispersants were in a state of arrested differentiation, oil group was not statistically significant

13 Results-Molecular Endpoint AMH, VTG, CHG, CYP19B, StAR, DMRT were differentially expressed

14 Results-Molecular Endpoint IGF and GHR associated with growth regulation displayed non-significant differential expression

15 Results-Molecular Endpoint Significant up regulation was observed for CYP1A Down regulations was observed for HSP90 in all with 9527, Oil/9500 and Oil/9527 being significant

16 Key Findings Heart beat was significantly lowered, more embryos deteriorated and failed to hatch or had abnormalities in dispersants and oil/dispersant exposed embryos: PAHs directly disrupt fish s cardiac function (Carls et al. 1999, Incardona et al, 2004, 2005, 2011) Oil/dispersants has been reported to induce abnormal tissue differentiation and developmental abnormalities (Fucik et al, 1995)

17 Key findings The significantly impaired down regulation of StAR has potential consequences for the vitellogenic process and fecundity (Vang et al, 2007) Modulation of this process will eventually affect testicular, ovarian and adrenocortical functions Respectively, up and down regulations of CYP1A and HSP90 was induced by the combinations of oil and dispersants; CYP1A has been reported to cause developmental abnormalities when inappropriately activated in fish embryos (Antkiewicz et al, 2006, Clark et al, 2010) While the disruption of Hsp90 s function leads to lethal damage and modulates phenotype in vertebrates (Yeyati et al, 2007, Vos et al, 2000)

18 Further work Further work will look at transcription factors associated with cardiac development and function (Nkx2-5, Mef2a, GATA4, Srf, and Tbx5,)

19 ACKNOWLEDGEMENT Dr. Nancy Denslow Mr. Kevin Kroll Graduate students and Post Docs in Denslow s Lab

20 References Carls, M.G., Rice, S.D., Hose, J.E Sensitivity of fish embryos to weathered crude oil: Part 1. Low-level exposure during incubation causes malformations, genetic damage, and mortality in larval Pacific herring (Clupea pallasi). Environmental Toxicology and Chemistry 18, Fucik, K.W., Carr, K.A., Balcom, B.J Toxicity of oil and dispersed oil to the eggs and larvae of seven marine fish and invertebrates from the Gulf of Mexico. Pages in Lane P, ed. The Use of Chemicals in Oil Spill Response. American Society for Testing and Materials. ASTM Report no. STP Incardona JP, Collier TK, Scholz NL (2004) Defects in cardiac function precede morphologica abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons. Toxicol Appl Pharmacol 196(2): Incardona, J.P., Collier, T.K., Scholz, N.L. (2011). Oil spills and fish health: Exposing the heart of the matter. J Expo Sci Environ Epidemiol., 21(1), 3 4. Incardona JP, Carls MG, Teraoka H, Sloan CA, Collier TK, et al. (2005) Aryl hydrocarbon receptor-independent toxicity of weathered crude oil during fish development. Environmental Health Perspectives 113: Major, D., Zhang, Q., Wang, G., Wang, H., Oil-dispersant mixtures: understanding chemical composition and its relation to human toxicity. Toxicol. Environ. Chem. 94, Place, B., Anderson, B., Mekebri, A., Furlong, E.T., Gray, J.L., Tjeerdema, R., Field, J., A role for analytical chemistry in advancing our understanding of the occurrence, fate, and effects of corexit oil dispersants. Environ. Sci. Technol. 44(16), Yeyati PL, Bancewicz RM, Maule J, van Heyningen V (2007) Hsp90 selectively modulates phenotype in vertebrate development. PLoS Genet 3: e43. Vang, S.H., Kortner, T.M., Arukwe, A Steroidogenic acute regulatory (StAR) protein and cholesterol side-chain cleavage (P450scc) as molecular and cellular targets for 17alpha-ethynylestradiol in salmon previtellogenic oocytes. Chem Res Toxicol. 20(12), Voss AK, Thomas T, Gruss P (2000) Mice lacking HSP90beta fail to develop a placental labyrinth. Development 127: 1 11.