phenanthrenes and Fish-Diseases

The simultaneous exposure of organisms to toxicants and disease causing agents poses a serious risk to important stocks. Worldwide, aquatic animal disease outbreaks have been increasing in both frequency and severity, and many have been associated with anthropogenic environmental change. Little is known about the complex interactions of the immune system and biotransformational pathways of vertebrates; however, urbanization and coastal development create a scenario in which a wide range of species are exposed to chemical pollutants in conjunction with a wide spectrum of ubiquitous, opportunistic pathogens. These interactions can severely compromise organismal health. Potential effects include decreased fitness, increased predation, decreased fecundity, reduced metabolic activity, suppressed immune function and mortality. Recent attention has been paid to immunomodulation in toxicant exposed fishes. In our current study we investigated the effects of the common polycyclic aromatic hydrocarbon phenanthrene in conjunction with Mycobacterium marinum infection in the zebrafish, Danio rerio. The goal of our study was to elucidate the interactions between stressors in the host organism. Fish were exposed to either a high or low dose of phenanthrene, infected with M. marinum or received a combination exposure of toxicant and bacteria. Results of our study were evaluated using survivorship analysis, toxicant body burden, and histology. Our data show an interaction between M. marinum infection and exposure to a high dose of phenanthrene in the zebrafish. Survivorship was significantly reduced for animals only exposed to the high dose of phenanthrene as compared to all other experimental groups. The increased survivorship for fish exposed to both Mycobacterium and a high dose of phenanthrene suggests an antagonistic interaction between stressors. Body burden data, which show significant differences in the ratio of phenanthrene:metabolites between experimental groups, suggests a disruption of the biotransformational pathway. We postulate that the inflammatory response, initiated by bacterial infection, is impeding the ability of the zebrafish to completely metabolize phenanthrene. In addition, the correlation between reduced metabolite production and increased survival indicates that phenanthrene metabolites are more toxic than the parent compound. Our study underscores the importance of investigating multiple stressor interactions as a way to better understand compl

Topics: Animals; Dose-Response Relationship, Drug; Fish Diseases; Mycobacterium Infections, Nontuberculous; Mycobacterium marinum; Phenanthrenes; Stress, Physiological; Water Pollutants, Chemical; Zebrafish

Alkylated polycyclic aromatic hydrocarbons, such as retene (7-isopropyl-1-methylphenanthrene), induce cytochrome P450 1A (CYP1A) enzymes and produce dioxin-like toxicity in the embryo-larval stages of fish characterized by the signs of blue sac disease (BSD). The signs of toxicity are well characterized; however, the mechanism is not well understood. To elucidate the role of CYP1A in retene toxicity, larval rainbow trout (Oncorhynchus mykiss) were co-treated with a range of concentrations of alpha-naphthoflavone (ANF), a known CYP1A inhibitor. The co-treatment produced synergistic toxicity at 3.2-100 microg/L ANF, after which toxicity at 180 microg/L ANF dropped to levels typical of retene-only. At 320 microg/L ANF, toxicity increased with or without retene, indicating that ANF alone was capable of inducing BSD. In addition, the additive toxicity of retene-only and 320 microg/L ANF-only approximately equalled that of the co-exposed larvae (100 microg/L retene+320 microg/L ANF), indicating response addition. Thus, two mechanisms of action occurred in co-exposed larvae at different concentrations of ANF. In trout larvae, there was a correlation between toxicity and CYP1A protein concentrations, and in juvenile trout, ANF produced a concentration-dependent inhibition of ethoxyresorufin-O-deethylase (EROD) activity without a measurable drop in CYP1A protein. Taken together, the mechanism underlying the synergistic toxicity is EROD-independent and may be AhR-dependent. This study demonstrated that multiple, exposure-dependent mechanisms can occur in mixture toxicity, suggesting that current risk assessment models may drastically underestimate toxicity, particularly of mixtures containing both CYP1A inducers and inhibitors.

Topics: Animals; Benzoflavones; Blotting, Western; Cytochrome P-450 CYP1A1; Fish Diseases; Liver; Oncorhynchus mykiss; Phenanthrenes; Receptors, Aryl Hydrocarbon; Statistics, Nonparametric; Survival Analysis; Toxicity Tests

Retene (7-isopropyl-1-methyl phenanthrene) is a polycyclic aromatic hydrocarbon (PAH), that causes dioxin-like toxicity to early life stages of fish, including increased rates of mortality, developmental defects characterized as blue sac disease (BSD), and induction of CYP1A enzymes. This study determined whether toxicity is associated with retene, or with its metabolism by CYP1A enzymes to hydroxylated derivatives. Larval rainbow trout (Oncorhynchus mykiss) were co-exposed to four concentrations of waterborne retene and four concentrations of waterborne alpha-naphthoflavone (ANF), a compound that antagonizes CYP1A induction and inhibits oxygenation reactions. The prevalence of mortality and BSD increased in an exposure-dependent manner for larvae exposed to retene alone. Tissue concentrations of CYP1A protein and retene metabolites also increased, but no un-metabolized retene (i.e., the parent compound) was measurable. At low concentrations of ANF, toxicity increased dramatically, while tissue concentrations of polar hydroxylated metabolites of retene decreased, and concentrations of less polar metabolites, and of parent retene, increased. At the highest concentration of ANF, retene toxicity was eliminated, and parent retene was the predominant form in tissue; no concentration of ANF was toxic by itself. The inhibition of retene hydroxylation and toxicity by ANF suggests that toxicity was caused by specific retene metabolites, and not by parent retene. The potentiation of retene toxicity at low concentrations of ANF, and the antagonism at high concentrations is a unique, non-linear interaction based on modulating CYP1A enzyme activity and retene metabolism. It demonstrates that effects on fish of different complex mixtures of hydrocarbons may not be easily predicted.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Benzoflavones; Blotting, Western; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP1A1; Drug Interactions; Enzyme Induction; Enzyme Inhibitors; Fish Diseases; Larva; Methanol; Oncorhynchus mykiss; Phenanthrenes; Water Pollutants, Chemical

Retene (7-isopropyl-1-methylphenanthrene) causes blue sac disease (BSD) in early life stages of fish, an effect similar to that of 2,3,7,8-tetrachlorodibenzo(p)dioxin. The signs of BSD include cytochrome P450 (CYP1A) induction, edema, hemorrhaging, and craniofacial deformities, indicating membrane damage, circulatory failure, and impaired development. To test if the underlying cause was oxidative stress, rainbow trout (Oncorhynchus mykiss) larvae were exposed to waterborne retene or to known prooxidants (paraquat, t-butyl hydroperoxide, and carbon tetrachloride) in the presence or absence of vitamin E, an antioxidant. Fish exposed to retene showed an increased prevalence of BSD, reduced tissue concentrations of vitamin E and total glutathione, and a lower percentage of glutathione in a reduced form. Coexposure to vitamin E reduced the prevalence of BSD and restored tissue concentrations of vitamin E, but it did not affect retene uptake or tissue concentrations of glutathione. These responses are consistent with oxidative stress as a mode of action of retene. However, retene did not affect whole-body lipid peroxide concentrations, and prooxidants did not affect the prevalence of BSD and had only minimal effects on tissue glutathione and vitamin E. Possible explanations for these conflicting results include prooxidant exposures were insufficient to generate oxidative stress; lipid peroxidation may not be measurable in whole-body homogenates of retene-exposed fish if effects are localized to endothelial cells, where CYP1A enzymes are most induced; or retene may have an alternate mode of action (e.g., adduction of retene metabolites to lipids, protein, or DNA).

Topics: Animals; Cytochrome P-450 CYP1A1; Enzyme Induction; Fish Diseases; Glutathione; Larva; Lipid Peroxides; Oncorhynchus mykiss; Oxidative Stress; Phenanthrenes; Vitamin E; Water Pollutants, Chemical

Early life stages of rainbow trout were exposed to different regimes of water-borne retene (7-isopropyl-1-methylphenanthrene) to determine if there is an ontogenic stage particularly sensitive to retene toxicity, and if cytochrome P-4501A (CYP1A) induction is a forerunner to blue sac disease (BSD), the syndrome of toxicity. CYP1A protein concentrations, measured by immunohistochemistry, were first detected during organogenesis, when organ and enzyme systems are first being developed, and steadily increased until swim-up. The prevalence of signs of BSD rose 1 wk following a marked increase in CYP1A activity after hatch, suggesting that CYP1A induction is related to BSD. The larval stage was the most sensitive to retene toxicity, based on CYP1A induction and a high prevalence of BSD. The most common signs of BSD were hemorrhaging, yolk-sac edema, and mortality, but hemorrhaging was the first and most frequently observed response. Tissue concentrations of retene were elevated just after fertilization, but decreased steadily as fish developed to the swim-up stage, most likely due to the establishment of more efficient metabolic and excretory systems in later stages of development.

Topics: Animals; Biomarkers; Chorion; Cytochrome P-450 CYP1A1; Disease Models, Animal; Environmental Exposure; Environmental Monitoring; Fetal Diseases; Fish Diseases; Fishes; Geologic Sediments; Immunohistochemistry; Larva; Neurotoxicity Syndromes; Oncorhynchus mykiss; Organogenesis; Phenanthrenes; Prevalence; Tissue Distribution; Water Pollutants, Chemical

An h.p.l.c.-fluorescence technique was used to estimate relative concentrations of metabolites of xenobiotics in bile of 103 English sole (Parophrys vetulus) from both polluted and minimally polluted (reference) sites in Puget Sound, WA. Fish from polluted sites had concentrations of xenobiotics in bile with naphthalene-, phenanthrene- and benzo[a]pyrene-like fluorescence that averaged 9, 14 and 19 times, respectively, those of fish from reference sites. Within a polluted site, fish with liver lesions had significantly higher bile concentrations of xenobiotics with benzo[a]pyrene-like fluorescence than did fish without liver lesions. Individual metabolites of fluorene, phenanthrene, anthracene, biphenyl and dimethylnaphthalene were determined by g.l.c.-mass spectrometry in extracts of hydrolysed bile of three English sole from polluted waterways; concentrations ranged from 90 to 19000 ng/g, wet wt. Other xenobiotics were tentatively identified, but not quantified.

Topics: Animals; Benzo(a)pyrene; Bile; Chemical and Drug Induced Liver Injury; Chromatography, High Pressure Liquid; Fish Diseases; Fishes; Hydrocarbons; Liver Diseases; Naphthalenes; Phenanthrenes; Spectrometry, Fluorescence; Water Pollutants; Water Pollutants, Chemical