Biomarker induction in tropical fish species on the Northwest Shelf of Australia by produced formation water.

Normal operation of oil well platforms results in the discharge of produced formation water (PFW). The expression of CYP1A, CYP2M1- and 2K1-like proteins was examined for use as possible biomarkers of PFW exposure. A pilot study on the Northwest Shelf of Australia had indicated that PFW contamination possibly contributes to induction of CYP1A-like proteins in Gold-Spotted Trevally (Carangoides fulvoguttatus). The pilot study samples were re-examined for CYP1A, and, in addition, CYP2K1/2M1-like proteins. In a subsequent caged fish study in the same location a second species, Stripey seaperch (Lutjanus carponotatus), caught at a clean site, were distributed to three caging sites in a PFW gradient from the Harriet A production platform: A (near-field), B (far-field) and C (a non-impacted reference site). Fish were sampled at time (T) T = 0, T = 3 and T = 10 days. Significant increases of CYP1A, one CYP2K1- and two CYP2M1-like proteins were noted at Site A at T = 10d. For another CYP2K1-like protein, a significant increase was observed at Site A only at T = 3d. These results support a previous study indicating that CYP1A protein is sensitive to PFW exposure. Importantly, statistically significant environmental induction of both CYP2M1- and CYP2K1-like proteins in tropical fish due to PFW exposure had not previously been described and induction of enzymes in the CYP2 family suggest new biomarkers for PFW. In addition, the novel response of one CYP2K-like protein requires further verification, but offers promise for improved monitoring of sub-lethal responses in marine organisms.

Toxicity of an engineered nanoparticle (fullerene, C60) in two aquatic species, Daphnia and fathead minnow.

Water-soluble fullerene (nC60) has been shown to induce lipid peroxidation (LPO) in brain of juvenile largemouth bass (LMB, Micropterus salmoides) [Oberdörster, E., 2004. Manufactured nanomaterials (fullerenes, c60) induce oxidative stress in brain of juvenile largemouth bass. Environ. Health Persp. 112, 1058-1062]; and upregulate genes related to the inflammatory response and metabolism, most notably CYP2K4 [. Nanotoxicology: an emerging discipline evolving from 116 studies of ultrafine particles. Environ. Health Persp. 113, 823-839]. The initial study in LMB was performed using tetrahydrofuran (THF)-solubilized nC60, although C60 can also be solubilized by stirring in water. The current study investigates differences in acute toxicity to Daphnia magna between THF-solubilized and water-stirred-nC60 as a range-find for further assays in adult male fathead minnow (FHM, Pimephales promelas). The daphnia 48-h LC50 for THF-nC60 was at least one order of magnitude less (0.8 ppm) than that for water-stirred-nC60 (> 35 ppm). FHM were dosed with either 0.5 ppm of THF- or water-stirred-nC60 for 48 h. There was 100% mortality in the THF-nC60-exposed fish between 6 and 18 h, while the water-stirred-nC60-exposed fish showed no obvious physical effects after 48 h. Water-stirred-nC60 elevated LPO in brain, significantly increased LPO in gill, and significantly increased expression of CYP2 family isozymes in liver as compared to control fish.

Environmental induction of CYP1A-, CYP2M1- and CYP2K1-like proteins in tropical fish species by produced formation water on the northwest shelf of Australia.

Normal operation of oil well platforms results in the discharge of "produced formation water" (PFW). The expression of CYP1A, CYP2M1- and 2K1-like proteins was examined for use as possible biomarkers of PFW exposure. A pilot study at the Harriet A production platform, on the Northwest Shelf of Australia, had indicated that PFW contamination possibly contributes to induction of CYP1A- and 2K1/2M1-like proteins in Gold-Spotted Trevally (Carangoides fulvoguttatus). In a subsequent caged fish study in the same location, Stripey seaperch (Lutjanus carponotatus) were caught at a clean site, then distributed to three caging sites: A (near-field), B (far-field) and C (a non-impacted reference site). Fish were sampled at time T = 0, 3 and 10 days. Significant increases of CYP1A, one CYP2K1- and two CYP2M1-like proteins were noted at Site A at T = 10. For another CYP2K1-like protein, a significant increase was observed at Site A only at T = 3. Prevailing winds changed between days 6 and 8 of sampling, moving the surface water plume directly west, possibly impacting in situ PFW exposure. The results indicate that tropical fish CYP1A protein is sensitive to PFW exposure. Importantly, statistically significant environmental induction of both CYP2M1- and CYP2K1-like proteins in tropical fish due to PFW exposure had not previously been described and CYP2 family induction may represent possible new biomarkers (other than CYP1A) of PFW exposure. In addition, the novel fraction-specific response of CYP2K-like proteins requires further verification but offers promise for improved monitoring of sub-lethal responses in marine organisms. (Supported by Australian Institute of Marine Science, Apache Energy Ltd. and the Environmental Toxicology Research Program at The University of Mississippi).

Japanese medaka (Oryzias latipes): developmental model for the study of alcohol teratology.

BACKGROUND: Animal models are necessary to investigate the mechanism of alcohol-induced birth defects. We have used Japanese medaka (Oryzias latipes) as a non-mammalian model to elucidate the molecular mechanism(s) of ethanol teratogenesis. METHODS: Medaka eggs, within 1 hr post-fertilization (hpf) were exposed to waterborne ethanol (0-1000 mM) in hatching solution for 48 hr. Embryo development was observed daily until 10 days post-fertilization (dpf). The concentration of embryonic ethanol was determined enzymatically. Cartilage and bones were stained by Alcian blue and calcein, respectively and skeletal and cardiovascular defects were assessed microscopically. Genetic gender of the embryos was determined by PCR. Levels of two isoenzymes of alcohol dehydrogenase (Adh) mRNAs were determined by semi-quantitative and real-time RT-PCR. RESULTS: The concentration of ethanol required to cause 50% mortality (LC50) in 10 dpf embryos was 568 mM, however, the embryo absorbed only 15-20% of the waterborne ethanol at all ethanol concentrations. The length of the lower jaw and calcification in tail fin cartilaginous structures were reduced by ethanol exposure. Active blood circulation was exhibited at 50+ hpf in embryos treated with 0-100 mM ethanol; active circulation was delayed and blood clots developed in embryos treated with 200-400 mM ethanol. The deleterious effects of ethanol were not gender-specific. Moreover, ethanol treatment was unable to alter the constitutive expression of either Adh5 or Adh8 mRNA in the medaka embryo. CONCLUSIONS: Preliminary results suggested that embryogenesis in medaka was significantly affected by ethanol exposure. Phenotypic features normally associated with ethanol exposure were similar to that observed in mammalian models of fetal alcohol syndrome. The results further indicated that medaka embryogenesis might be used as an alternative non-mammalian model for investigating specific alterations in gene expression as a means to understand the molecular mechanism(s) of ethanol-induced birth defects.

Summary results from a pilot study conducted around an oil production platform on the Northwest Shelf of Australia.

The Australian Institute of Marine Science (AIMS) conducted a pilot study around the Harriet A oil production platform on the Northwest Shelf of Australia. We evaluated hepatic ethoxyresorufin-O-deethylase (EROD) activity, fluorescent aromatic compounds (FACs) in bile and immunodetection of CYP1A-like proteins in two Australian tropical fish species, Gold-Spotted Trevally (Carangoides fulvoguttatus) and Bar-Cheeked Coral Trout (Plectropomus maculatus) to assess exposure to petroleum hydrocarbons associated with produced formation water (PFW). Additionally, the incidence of hydrocarbon-degrading bacteria isolated from the liver and bile of all fish captured was examined. Low EROD activity was found in both species, with EROD activity in C. fulvoguttatus showing significant site differences. FACs and CYP1A protein levels in C. fulvoguttatus showed a clear trend in hydrocarbon exposure consistent with hydrocarbon chemistry data: Harriet A>Harriet C>reference site. P. maculatus showed elevated levels of FACs at Harriet A as compared to the reference site and demonstrated detectable levels of CYP1A-like proteins at these two sites. Hydrocarbon-degrading bacteria were found in the liver and bile of both species, yet there was no correlation by sites. Our results demonstrate that C. fulvoguttatus and P. maculatus have potential as indicator species for assessing the effects from exposure to petroleum hydrocarbons. Both FACs and CYP1A are providing warning signs that there is potential for biological effects on fish populations exposed to PFW around the Harriet A production platform.

Expression of Adh8 mRNA is developmentally regulated in Japanese medaka (Oryzias latipes).

We cloned two full-length alcohol dehydrogenase (ADH) cDNAs from the liver tissue of adult Japanese medaka (Oryzias latipes). The coding regions spanned 1134 and 1137 nucleotides (nt) and the deduced amino acid sequences shared 63.6% identity between them. Phylogenetic analysis of the deduced amino acid sequence data identified the 1137nt as an orthologue of mammalian Adh5 (Class III) and the 1134 nt as an ortholog of zebrafish Adh8 genes. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis further showed that adult medaka Adh5 mRNA was expressed in all the organs tested (brain, eye, gill, GI, heart, liver, kidney, muscle, skin, spleen, testis and ovary) while Adh8 mRNA showed tissue-specific expression (eye, GI, liver, kidney, muscle and skin). Comparison of the Adh5 and Adh8 mRNA expression in eye, gill, liver, kidney and skin indicate that Adh8 mRNA copy numbers are higher in all these tissues compared to Adh5 mRNA expression. Both Adh5 and Adh8 mRNAs are expressed during embryonic development with Adh5 mRNA transcripts present with very high copy number throughout the development. However, Adh8 mRNA is expressed in very low copy numbers initially ( approximately 1 h post fertilization; hpf) but begin to increase from 48 hpf to a level of approximately 200-fold higher at hatching. Therefore, it appears that in Japanese medaka, the expression of Adh8 mRNA, not Adh5 mRNA, is developmentally regulated.

Combined effects of ethanol and cinnamaldehyde in the Japanese medaka embryo-larval assay (MELA).

The Japanese medaka (Oryzias latipes) was used in the medaka embryo-larval assay (MELA) to determine possible adverse developmental effects of ethanol and the spice component, cinnamaldehyde (CAD). Fish may be exposed to waterborne ethanol and a variety of natural products from non-point sources or leaks during ethanol use as a fuel and from point source processing plant effluents. Consumption of ethanol during human pregnancy is known to cause fetal alcohol syndrome (FAS), a collection of birth defects including craniofacial abnormalities thought to be caused by the generation of free radicals during ethanol metabolism by both alcohol and aldehyde dehydrogenase(s). Fish are also susceptible to FAS (Dasmahapatra et al., meeting abstract). The activity of aldehyde dehydrogenase is inhibited by CAD [Biochem. J. 282 (1992) 353-360; Biochem. Pharmacol. 45 (1993) 1621-1630], and CAD is known to cause developmental abnormalities in the rat [Food Chem. Toxicol. 27 (1989) 781-786]. Therefore, the combined effects of treatment with both ethanol and CAD would be expected to produce additive or greater than additive effects in the MELA assay. Medaka were exposed to ethanol at 100 mM, CAD at 10, 1.0, 0.67 or 0.50 mM, to ethanol and CAD combined, or were non-treated controls. Ethanol at 100 mM was without effect. CAD alone at 10 mM and 1.0 mM was lethal by 1 dpf. Embryos exposed to 100 mM ethanol and 0.67 mM CAD exhibited cardiovascular and pigmentation defects and delayed hatching. Embryos exposed to 0.50 mM CAD alone had less severe cardiovascular problems as compared to the combined ethanol and CAD treatment. Taken together the results indicate that the combined effects of ethanol and CAD are greater than the individual effects and indicate the need to monitor effluents in fish nursery areas to protect natural fish populations. Supported by PHS/NIH ES07929.