Reproductive Toxicity of Crude oil-Dispersant Mixture in Caenorhabditis elegans

As crude oil remains a vital natural resource for the energy need of the world, environmental crude oil spills continue to be a health risk to human beings and ecological systems. During clean-up efforts, surfactant-like dispersants are used to break down big oil slicks into small droplets. Therefore it is necessary to investigate the health impacts of dispersed oil as a mixture entity rather than based on the toxicological profile of individual chemicals. Since reproductive stages of organisms are generally being more sensitive to the effects of toxicants than other stages, investigation of crude oil/dispersant exposure effects on reproduction is critically important. However, studies on the reproductive effects of crude oil-dispersant mixture exposure and its mechanism remain insufficient. The nematode Caenorhabditis elegans (C. elegans) has been a useful tool for environmental toxicity studies, and it is a well-known animal model to study the reproduction system. Therefore in this study, we employed the nematode C. elegans to test impacts of crude oil/dispersant exposure on basic biological processes growth, reproduction, microRNAs and protein-coding gene expression and its underlying mechanisms. In Chapter 1, we employed parallel experiments to test the effects of crude oil from the DWH oil well, chemical dispersant Corexit 9500A, and dispersant-oil mixture on growth and reproduction in the model organism Caenorhabditis elegans. Both the crude oil and the dispersant significantly inhibited the reproduction of C. elegans. Dose-dependent inhibition of hatched larvae production was observed in worms exposed to both crude oil and dispersant. Importantly, the chemical dispersant Corexit 9500A potentiated crude oil effects; the dispersant-oil mixture induced more significant effects than oil or dispersant-alone exposures. While oil-alone exposure and dispersant-alone exposure have none to moderate inhibitory effects on hatched larvae production, respectively, the mixture of dispersant and oil induced much more significant inhibition of offspring production. The production of hatched larvae was almost completely inhibited by several high concentrations of the dispersant-oil mixture. We also investigated the effects of crude oil/dispersant exposure at the molecular level by measuring the expressions of 31 functional genes. Results showed that the dispersant and the dispersant-oil mixture induced aberrant expressions of 12 protein-coding genes. These 12 genes are associated with a variety of biological processes, including egg-laying, oxidative stress, muscle contraction, and neurological functions. In Chapter 2, we showed that crude oil-dispersant mixture affected reproduction by inducing abnormal sperm during the process of spermatogenesis. Results showed that the abnormal immature sperm were significantly increased in the gonad arms of Dis-Oil mixture treated animals compared to controls (K-medium). We further explored the oil-dispersant mixture toxicity effects on spermatogenesis by using a male C. elegans strain. After 48h exposure to Dis-Oil mixture, spermatids appeared abnormal morphology including irregular shape of the spermatid membrane and unexpected tails induced by dispersed oil. Moreover we utilized puf-8; lip-1 tumor sensitive strain to test the cell fate of immature sperm induced by Dis-Oil mixture treatment. We found increased tumor occurrence in dispersed oil treatments compared to control. Results also suggest that the immature sperm may undergo dedifferentiation and become tumor-like cells in puf-8; lip-1 mutants through the MAPK-independent pathway. Based on the genome-wide investigation of microRNA profile, in Chapter 3, we found that the aberrant expression of miRNAs was induced. The KEGG pathway enrichment analyses indicated that those significantly changed miRNAs regulate many biological processes in C. elegans. Many affected pathways are related to environmental information processing, such as ABC transporters, MAPK signaling pathway, Erbb signaling pathway, JAK-STAT signaling pathway, MTOR signaling pathway and calcium-signaling pathway. Some pathways are related to oil uptake and metabolism, such as endocytosis, fatty acid biosynthesis and phosphatidylinositol signaling system. In summary, based on our studies, both crude oil and dispersant can induce the reproductive toxicity, and the dispersant enhanced the crude oil toxicity. Since the currently identified proteins and microRNAs in C.elegans show remarkable conservation with mammals including humans, the oil/dispersant may also induce similar change at physiological and molecular levels and affect many biological processes in mammals