Margaret M. Krahn

Associations between metabolites of aromatic compounds in bile and the occurrence of hepatic lesions in English sole (Parophrys vetulus) from Puget Sound, Washington.

Statistical comparisons were made between the relative mean concentrations of metabolites of aromatic compounds in bile (as estimated by a high-performance liquid chromatography/fluorescence detection method) and idiopathic liver lesions in English sole (Parophrys vetulus) from eleven Puget Sound sites. A significant positive correlation was found between prevalences of neoplasms, foci of cellular alteration, megalocytic hepatosis and total hepatic lesions and the concentrations of metabolites of aromatic compounds. These findings provide further evidence of the putative relationship between aromatic compounds and serious idiopathic liver diseases in bottom-dwelling fish.

Relationships between hepatic neoplasms and related lesions and exposure to toxic chemicals in marine fish from the U.S. West Coast.

English sole (Parophrys vetulus) inhabiting polluted waterways and embayments of Puget Sound, Washington, are affected with a variety of multiple, co-occurring idiopathic hepatic lesions, including unique degenerative conditions, putatively preneoplastic foci of cellular alteration, and neoplasms. Results of a statistical analysis of the patterns of co-occurrence of these lesions in wild English sole are consistent with the concept that these lesions represent morphologically identifiable steps forming a sequence of progression ultimately leading to the development of hepatic neoplasms. This progressive sequence parallels the pattern identified in experimental models of chemically induced hepatocarcinogenesis in rodents. The rationale for the hypothesis that these lesions in wild English sole can be caused by exposure to certain hepatoxic and hepatocarcinogenic xenobiotic compounds in the marine environment is based on the demonstration of significant and consistent statistical associations between levels of aromatic hydrocarbons (AHs) in sediment and prevalences of these idiopathic liver lesions; a significant contribution by sediment AHs to the variability in hepatic neoplasm prevalence in a logistic regression model; significantly increased probabilities for several idiopathic lesions in sole from chemically contaminated sites in Puget Sound; significant correlations between uptake of polycyclic aromatic hydrocarbons, as measured by levels of fluorescent metabolites of aromatic compounds in bile of sole, and prevalences of several hepatic lesion types; and experimental induction of unique degenerative, proliferative, and putatively preneoplastic focal lesions in English sole injected with either benzo(a)pyrene or a polycyclic aromatic hydrocarbons (PAH) enriched fraction of an extract from a contaminated urban sediment from Puget Sound.(ABSTRACT TRUNCATED AT 250 WORDS)

Analytical methods for determining metabolites of polycyclic aromatic hydrocarbon (PAH) pollutants in fish bile. A Review.

The determination of polycyclic aromatic hydrocarbon (PAH) metabolites in bile can serve as a tool for assessing environmental PAH exposure in fish. Biliary PAH metabolite levels can be measured using several analytical methods, including simple fluorescence assays (fixed fluorescence detection or synchronous fluorescence spectrometry); high-performance liquid chromatography with fluorescence detection (HPLC-F); gas chromatography-mass spectrometry (GC-MS) after deconjugation, extraction and derivatization of the bile sample, and finally by advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) methods. The method alternatives are highly different both with regard to their analytical performance towards different PAH metabolite structures as well as in general technical demands and their suitability for different monitoring strategies. In the present review, the state-of-the-art for these different analytical methods is presented and the advantages and limitations of each approach as well as aspects related to analytical quality control and inter-laboratory comparability of data and availability of certified reference materials are discussed.

Effects of age, sex and reproductive status on persistent organic pollutant concentrations in “Southern Resident” killer whales

Southern Resident killer whales (Orcinus orca) that comprise three fish-eating pods (J, K and L) were listed as endangered in the US and Canada following a 20% population decline between 1996 and 2001. Blubber biopsy samples from Southern Resident juveniles had statistically higher concentrations of certain persistent organic pollutants than were found for adults. Most Southern Resident killer whales, including the four juveniles, exceeded the health-effects threshold for total PCBs in marine mammal blubber. Maternal transfer of contaminants to the juveniles during rapid development of their biological systems may put these young whales at greater risk than adults for adverse health effects (e.g., immune and endocrine system dysfunction). Pollutant ratios and field observations established that two of the pods (K- and L-pod) travel to California to forage. Nitrogen stable isotope values, supported by field observations, indicated possible changes in the diet of L-pod over the last decade.

Evidence for exposure of fish to oil spilled into the Columbia river

Abstract On March 19, 1984, more than 170000 gallons of oil were spilled into the Columbia River. We had recently developed analytical methods for estimating the exposure of fish to aromatic compounds by measuring the concentrations of metabolites of these contaminants in fish bile. The oil spill provided an opportunity to field test our methods in assessing the exposure of fish to petroleum aromatic compounds from the spilled oil. Our findings indicated that, within 5 days after the spill, mean concentrations of metabolites of aromatic compounds in the bile of white sturgeon (Acipenser transmontanus) captured 57 miles downstream from the spill were significantly higher than those of sturgeon caught upriver.

Toxic chemicals, including aromatic and chlorinated hydrocarbons and their derivatives, and liver lesions in white croaker (Genyonemus lineatus) from the vicinity of Los Angeles

High concentrations of toxic chemicals in sediment and white croaker (Genyonemus lineatus), as well as liver diseases (e.g., carcinomas) in this species, were found in the Los Angeles area. The highest concentrations of aromatic hydrocarbons (AHs) in the sediment were in San Pedro Bay, and the highest concentrations of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) derivatives were in sediment from near the White Point sewer outfall. Concentrations of AHs, polychlorobiphenyls (PCBs), and DDT derivatives were generally higher in food organisms from the croakers stomach than in sediment. Moreover, croaker from San Pedro Bay and White Point were substantially contaminated with DDT derivatives and metabolites of aromatic compounds, compared to croaker from the Hyperion outfall and Dana Point. The evidence suggest that the observed pathological conditions of the liver were associated with exposure of the croaker to toxic chemicals, which occurred, at least in part, through the ingestion of contaminated food organisms.

Aromatic hydrocarbon metabolites in fish: authomated extraction and high-performance liquid chromatographic separation into conjugate and non-conjugate fractions

An automated extractor-concentrator was used to extract metabolites of naphthalene, 2,6-dimethylnaphthalene, and benzo[a]pyrene from serum, bile and liver homogenate of rainbow trout (Salmo gairdneri). The extracts were analyzed by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection. Recoveries of naphthalene and 2,6-dimethylnaphthalene metabolites from all matrices were generally greater than 90%; however, the recoveries of benzo[a]pyrene metabolites from serum ranged from 37-99%. In addition, conjugated metabolites of polycyclic aromatic hydrocarbons (PAHs) were separated from non-conjugated metabolites and parent PAHs by using two diol columns with normal-phase HPLC. The extraction and separation techniques were also applied to isolate metabolites in samples from fish fed 2,6-dimethylnaphthalene.

Sediment-associated contaminants and liver diseases in bottom-dwelling fish

High concentrations of chemicals have been found in sediments from urban areas of Puget Sound. Hundreds of organic chemicals (including certain aromatic hydrocarbons [AHs] and various chlorinated compounds) were identified. Statistical methods were used to evaluate possible relationships between the chemistry data and fish diseases. Positive correlations were found between the frequencies of liver neoplasms (e. g., hepatocellular carcinoma) and other liver lesions in English sole (Parophrys vetulus) and concentrations of AHs in sediment; such correlations were not found with chlorinated hydrocarbons. Strong evidence was also obtained to show that many organic chemicals in sediment are bioavailable to bottom-dwelling fish. Stomach contents (consisting mainly of benthic invertebrates) from English sole had concentrations of a number of AHs similar to those in the sediment from which the fish were taken. In these same fish, metabolites of many aromatic compounds were found in bile using a procedure combining HPLC with fluorescence detection. Further, the concentrations of certain xenobiotic metabolites in bile were correlated positively with the occurrence of liver neoplasms in English sole.

Determination of mixtures of benzo(a)pyrene, 2,6-dimethylnaphthalene and their metabolites by high-performance liquid chromatography with fluorescence detection

Abstract Nonradiometric techniques were used to identify and quantitate two aromatic hydrocarbons and several of their metabolites in biological samples. High-performance liquid chromatographic separation combined with ultraviolet fluorescence detection provided a sensitive and selective method of trace analysis. Metabolites of benzo[ a ]pyrene and 2,6-dimethylnaphthalene were prepared by in vitro incubation of these substrates, singly and together, with liver microsomes of coho salmon ( Oncorhynchus kisutch ). Individual metabolites were separated by high-performance liquid chromatography and quantitated at fluorescence excitation and emission wavelengths characteristic of the corresponding parent hydrocarbon. Future refinement of these techniques may allow the determination of more complex mixtures of xenobiotics and their metabolites in marine organisms.

Rapid analysis of naphthalene and its metabolites in biological systems: Determination by high-performance liquid chromatography/fluorescence detection and by plasma desorption/chemical ionizations mass spectometry

A rapid procedure for the determination of naphthalene and its metabolites in bile of rainbow trout and mice is described. The integrated analytical techniques combine high-performance liquid chromatography/ultraviolet fluorescence detection and plasma desorption/chemical ionization mass spectrometry for identification and quantitation. After separation by reverse-phase liquid chromatography, naphthalene and its metabolites are detected and quantitated by ultraviolet fluorescence spectrometry. Identification of two metabolites is confirmed by mass spectrometry. A direct insertion probe tip for a conventional chemical ionization mass spectrometer was modified to obtain spectra of thermally labile compounds. A spectrum of less than 100 ng of naphthyl glucuronide, a labile glucuronic acid conjugate of 1-naphthol, was obtained with this system.