Toxicity, toxicology and pharmacology study of butenolide and isocyanide as antifouling compounds

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2011

In the oceans, all submerged surfaces are colonized by microbes and large fouling organisms. Biofouling on shiphulls and underwater pipelines can lead to huge maintenance cost. To control biofouling, antifouling compounds are used in paints to protect the painted area. However, the rising concentration of several antifouling compounds in the sea caused serious environmental problems, which led to the restriction or complete ban on their usage. Alternative antifouling compounds are being developed. Detailed characterizations of their toxicity, toxicology and pharmacology are important for their risk assessment.Butenolide [5-octylfuran-2(5H)-one] is a promising antifouling compounds that have been developed in recent years. In this study, the acute toxicity of butenolide was assessed in several non-target organisms, including micro algae, crustaceans, and fish. Results were compared with previously reported results on the effective concentrations used on fouling (target) organisms. According to OECD’s guideline, the predicted no effect concentration (PNEC) was 0.168 μg l-1, which was among one of the highest in representative new biocides. Mechanistically, the phenotype of butenolide-treated Danio rerio (zebrafish) embryos was similar to the phenotype of the pro-caspase-3 over-expression mutant with pericardial edema, small eyes, small brains, and increased numbers of apoptotic cells in the bodies of zebrafish embryos. Butenolide also induced apoptosis in HeLa cells, with the activation of c-Jun N-terminal kinases (JNK), Bcl-2 family proteins, and caspases and proteasomes/lysosomes involved in this process.The effects of butenolide on larval behavior and histology were compared in two major fouling organisms: the barnacle Balanus amphitrite cyprid larvae and the bryozoan Bugula neritina swimming larvae. Butenolide diminished the positive phototactic behavior of B. amphitrite (EC50 = 0.82 μg ml-1) and B. neritina (EC50 = 3 μg ml-1). Its effect on B. amphitrite attachment was influenced by temperature, and butenolide increased attachment of B. neritina larvae on the bottom of the experimental wells. At concentrations of 4 μg ml-1 and 10 μg ml-1, butenolide decreased attachment of B. amphitrite and B. neritina, respectively, but the effects were reversible within a certain treatment time. Morphologically, butenolide inhibited the swelling of B. amphitrite cyprid secretory granules, and altered rough endoplasmic reticulum (RER) in the cement gland. In B. neritina swimming larvae, butenolide reduced the number of secretory granules in the pyriform-glandular complex.Several butenolide-binding proteins were found in three different target organisms: 1) the B. amphitrite ACAT1 (also known as T2 thiolase), which is a mitochondrial tetrameric enzyme involved in ketone body synthesis and degradation. Acetyl-CoA and acetoacetyl- CoA, which are substrate and product of ACAT1, increased the settlement rate of B. amphitrite under butenolide treatment, suggesting the involvement of ACAT1 in the butenolide’s antifouling effect. The rescuing effect could only be observed after 4 d of treatment, by when a lot of butenolide would have been degraded already (unpublished result), suggesting that these metabolites were helping more larvae to recover after the butenolide concentration dropped. 2) The B. neritina ACADVL (very long chain acyl-CoA dehydrogenase), the first enzyme of the very long chain fatty acid beta-oxidation pathway. Alternative energy sources acetoacetate and pyruvate increased the settlement rate of B. neritina under butenolide treatment, suggesting the decreased settlement rate induced by butenolide was because of the energy shortage, which would result from the inhibition of ACADVL. Staining of lipid with Sudan Black B showed that the butenolide treated B. neritina larvae catalyzed their lipid at a rate similar to their control counterparts, suggesting that when the function of ACADVL was insufficient as a result of butenolide treatment, ACOX1 (acyl-CoA oxidase) catalyzed the dehydrogenation of very long chain fatty acyl-CoA without generating energy. 3) The B. neritina actin. The involvement of B. neritina actin in butenolide-induced change is suggested by the failure of larvae to successfully metamorphose after they attach under butenolide/acetoacetate or butenolide/pyruvate treatment. 4) two B. neritina GSTs (glutathione S-transferase), which belong to a group of enzymes involved in phase II detoxification or the isolation, transport and synthesis of endogenous hydrophobic compounds; and 5) marine bacterium Vibrio sp. 010 succinyl-CoA synthetase subunit beta (SCSβ), an enzyme in the citric acid cycle. The growth of marine bacterium Vibrio sp. 010 was inhibited by butenolide, which could be the result of the inhibition of succinyl-CoA synthetase. The ACAT1 and ACADVL are acyl-CoA binding enzymes involved in fatty acid metabolism; the actin and SCSβ are NTP-binding proteins; the two GSTs are glutathione binding proteins. No known proteins involved in cell cycles, cell proliferations, cell differentiations, cell death and neuronal transmissions were found to bind butenolide in these species.Isocyanide (2,2-dimethyldodec-11-enenitrile) is another promising antifouling compound. Its mode of action was investigated with the settlement-competent larvae (which are ready to settle) of two major marine fouling species, B. neritina and B. amphitrite, and with the embryo of an important non-target organism zebrafish D. rerio. In B. neritina swimming larvae, the isocyanide did not affect the total attachment rate (≤ 50 μg ml-1), but made more to attach at the bottom of the container. The isocyanide binding proteins in B. neritina include two proteins bands. The 30 KD protein band contains two proteins similar to voltage dependent anion channels (VDAC), which control the direct coupling of the mitochondrial matrix to the energy maintenance of the cytosol and the release of apoptogenic factors from mitochondria of mammalian cells. The 39 KD protein could not be identified in the database by LC-MS/MS. In B. amphitrite cyprid, the isocyanide binding protein was similar to NADH-ubiquinone oxidoreductase, which is the 'entry enzyme' of oxidative phosphorylation in the mitochondria (complex I). In Danio rerio embryos, the isocyanide caused “wavy” notochord, hydrocephalus, pericardial edema, bad blood circulation, and defect in pigmentation and hematopoiesis. The isocyanide treated zebrafish embryos phenocopied copper deficiency, suggesting that isocyanide induced a similar pathophysiological process. This is the first report on the isocyanide binding proteins in fouling organisms, and the first toxicology study of this compound on zebrafish.