William L. Reichert

Exposure of two species of deposit-feeding amphipods to sediment-associated [3H]benzo[a]pyrene: Uptake, metabolism and covalent binding to tissue macromolecules

Abstract Two species of deposit-feeding marine gammaridian amphipods, Rhepoxynius abronius and Eohaustorius washingtonianus, were exposed to sediment-associated [3H]benzo[a]pyrene (BaP) at 12±1°C. Concentrations of BaP-derived radioactivity increased with time in both E. washingtonianus and R. abronius, and the levels of radioactivity were similar in both species after 7 days of exposure. A significantly (P

Uptake and metabolism of lead and cadmium in Coho salmon (oncorhynchus kisutch)

Abstract 1. Coho salmon exposed to water-borne lead and cadmium accumulated relatively high concentrations of each metal in gills, liver and kidney (primarily the posterior segment). 2. Cadmium concentrations in kidney increased for at least a month after termination of exposure; however, concentrations of lead remained about the same. 3. Highest concentrations of cadmium in kidney and liver were in cytosolic fractions, where substantial proportions were bound to proteins of 8900 molecular weight. Gills sequestered cadmium in the form of this readily-inducible metal-binding protein. 4. No specific lead-binding protein was identified in cytosolic fractions. This metal was primarily associated with proteins exceeding 55000 mol.wt.

Effect of environmental temperature on naphthalene metabolism by juvenile starry flounder (Platichthys stellatus).

Juvenile starry flounder (Platichthys stellatus) maintained at 4° or 12°C were forced-fed3H-1-naphthalene. At 24 hr, after the initiation of exposure, significantly (p < 0.05) higher concentrations (2 to 15 times) of naphthalene were present in tissues of starry flounder at 4°C than those present in fish held at 12°C. The influence of lowering of water temperature on naphthalene retention was even more marked after one week. At this time, muscle and liver of fish at 4°C contained 26 and 34 times, respectively, more naphthalene than did muscle and liver of fish at 12°C. Concentrations of total metabolites, in most tissues were not substantially higher at the lower temperature either 24 or 168 hr after the naphthalene-exposure.Thin-layer Chromatographic separation of the metabolites revealed that at 24 hr, 1,2-dihydro-1,2-dihydroxynaphthalene (dihydrodiol) was the major component in liver (40 to 50% of extracted metabolites) and muscle (∼-80% of extracted metabolites) regardless of the temperature. Bile contained, primarily, conjugates (e.g., glucuronides), which yielded the dihydrodiol as the principal metabolite on enzymatic hydrolysis. From 24 to 168 hr, the concentrations of each metabolite class did not vary directly with the concentrations of total metabolites. Accordingly, at 168 hr, the ratio of total metabolite concentrations in liver of fish at 4°C compared to 12°C was 1.6, whereas the ratios for the dihydrodiol, sulfate/glucoside conjugates and glucuronide conjugates were 4.5, 0.6 and 3.8 respectively.Generally, lowered water temperature increased tissue concentrations of the parent hydrocarbon and its metabolites. However, the magnitude of the increase was dependent upon the compound, the tissue, and the time after the initiation of the exposure. The results emphasize the importance of determining concentrations of individual metabolites together with parent hydrocarbons in tissues of fish when assessing effects of environmental parameters on xenobiotic toxicity.

Improved flatfish health following remediation of a PAH-contaminated site in Eagle Harbor, Washington

Eagle Harbor in Puget Sound, WA became a Superfund site in 1987 due to polycyclic aromatic hydrocarbons (PAHs) released chronically from a nearby creosoting facility. Early studies here (1983-1986) demonstrated up to an approximately 80% prevalence of toxicopathic liver lesions, including neoplasms, in resident English sole (Parophrys vetulus). These lesions in English sole are consistently associated with PAH exposure in multiple field studies, and one laboratory study. Later studies (1986-1988) incorporated biomarkers of PAH exposure and effect, including hepatic CYP1A expression and xenobiotic-DNA adducts, and biliary fluorescent aromatic compounds (FACs). Before site remediation, lesion prevalences and other biomarker values in this species from Eagle Harbor were among the highest compared to other sites in Puget Sound and the US Pacific Coast. To sequester PAH-contaminated sediments, in 1993-1994, a primary cap of clean sediment was placed over the most-contaminated 54acres, with a 15-acre secondary cap added from 2000-2002. Lesion prevalences and biomarker values before primary capping were reduced compared to 1983-1986, consistent with facility closure in 1988 and shore-based source controls begun in 1990. Liver lesion risk, hepatic CYP1A activities, and levels of biliary FACs from fish collected immediately after and at regular intervals up to 2 years after primary capping were variable relative to pre-capping. Over the entire monitoring period since primary capping (128 months), but particularly after 3 years, there was a significantly decreasing trend in biliary FACs, hepatic DNA adducts and lesion risk in English sole. In particular, lesion risk has been consistently low (<0.20) compared to primary cap initiation (set at 1.0), from approximately 4 years after primary capping through April 2004. These results show that the sediment capping process has been effective in reducing PAH exposure and associated deleterious biological effects in a resident flatfish, and that longer term monitoring of pollutant responses in biological resources, such as resident fish, is needed in order to demonstrate the efficacy of this type of remediation.

DNA adducts in hematopoietic tissues and blood of the mummichog (Fundulus heteroclitus) from a creosote-contaminated site in the Elizabeth River, Virginia.

Hydrophobic DNA adducts were examined in liver, anterior kidney, spleen, and blood of tumor-prone mummichog (Fundulus heterclitus) from the creosote-contaminated Atlantic Wood (AW) site (Elizabeth River, Virginia). DNA adducts eluted in a diagonal radioactive zone, characteristic of polycyclic aromatic hydrocarbon exposure, in all examined tissues of AW fish. Mummichog demonstrated significantly higher levels of DNA adducts in spleen (394 +/- 109 nmol adducts/mol nucleotides) than in liver (201 +/- 77 nmol adducts/mol nucleotides) or anterior kidney (211 +/- 68 nmol adducts/mol nucleotides; P = 0.036). The levels of DNA adducts in the pooled blood (pool of four) were 142 nmol adducts/mol nucleotides. DNA adducts were not detected in the liver, anterior kidney, spleen and blood of fish collected from the reference site (< 2 nmol adducts/mol nucleotides). The high levels of DNA adducts detected in tissues of AW mummichog may be linked to the increased cancer incidence and immunosuppression in this population.

Persistence of benzo(a)pyrene -DNA adducts in hematopoietic tissues and blood of the mummichog, Fundulus heteroclitus

The formation and persistence of benzo[a]pyrene (B[a]P)-DNA adducts were investigated in blood, liver and two hematopoietic tissues (anterior kidney and spleen) of the mummichog (Fundulus heteroclitus). Fish were injected with a single, sublethal dose of B[a]P (12 mg/kg body weight) and sampled from 8 to 96 days post-injection. 32P-Postlabeling analysis and storage phosphor imaging were used to resolve and quantify hydrophobic DNA adducts. One major DNA adduct was present in each of the examined tissues at all sampling times. This adduct had similar chromatographic characteristics to those of the adduct standard, 7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl-3-phosphate)-7,8,9,10-tetrahydro-benzo[a]pyrene (B[a]PDE-dG). Minor DNA adduct spots, representing less than 2% of the total DNA adducts, were observed in some liver, anterior kidney and spleen samples for up to 32 days post-injection. The B[a]P-DNA adducts reached maximal levels at 32 days post-injection and persisted for at least 96 days in all examined tissues. B[a]P-DNA adduct levels were significantly higher in the liver and anterior kidney than in the spleen from 16 to 96 days (P<0.001), although liver and anterior kidney DNA adduct levels were not significantly different at any time. This is the first controlled study to demonstrate the formation and persistence of B[a]P-DNA adducts in hematopoietic tissues and blood of fishes exposed to the prototypical polycyclic aromatic hydrocarbon, B[a]P. Although persistent DNA adducts are generally recognized as potential initiators of carcinogenic processes, adducts in these vital tissues may also lead to disruption of physiological functions such defense mechanisms and hematopoiesis.

32p-Postlabeling Of DNA: A Sensitive Method For Assessing Environmentally Induced Genotoxicity

In contaminated environments organisms are exposed to complex mixtures of genotoxic compounds, many of which have not yet been identified. Moreover, for certain genotoxic compounds metabolism to a reactive metabolite(s) is necessary before binding to the genetic material, DNA, can occur. Thus, nonspecific procedures are needed that can detect damage to DNA by a variety of different compounds or their metabolites in animals exposed to these compounds in contaminated environments. The level of covalent binding of genotoxic compounds to DNA in organisms from highly contaminated areas, however, occurs at extremely low levels; thus, central issues for the use of any technique for measuring DNA adducts are the sensitivity of the assay and its applicability to animals exposed to complex mixtures. Presently, the 32P-postlabeling assay (1,2) shows particular promise, because it has a very low limit of detection (1 adduct in 109-1010 nucleotides) and does not require characterization of individual adducts before they are measured, which is an important feature for monitoring studies of marine organisms exposed to complex mixtures of contaminants. There are several versions of the 32P-postlabeling assay reported in the literature (I), and we have used the version that separates xenobiotic-modified nucleotides from normal nucleotides by n-butanol extraction (3 ) ; this increases the sensitivity of the assay by allowing analysis of larger quantities of DNA, and the recovery of adducts with this version of the assay is less dependent on the chemical nature of the adducts than in other versions (4).

Metabolism of orally administered naphthalene in spawning English sole (Parophrys vetulus)

Abstract English sole (gravid females and ripe males) were force-fed [3H]naphthalene (NPH) in salmon oil and tissues were examined at 24, 48, and 168 hr. NPH was detected in all tissues and fluids examined (e.g., liver, blood, muscle, bile, testes, and ovaries) at 24 hr. There were no statistically significant differences in NPH concentrations, based on dry weight, between comparable tissues and fluids of the sexes, except for the gonads. Ovarian NPH concentrations were approximately three times higher than those in testes. The highest percentage (5%) of the total dose at 24 hr was present in the ovaries; liver and muscle contained about 1–2% of the dose. Concentrations of NPH in all tissues declined markedly from 24 to 168 hr. At 24 hr, male English sole had significantly higher hepatic NPH hydroxylase activity than did female sole. However, no significant sex differences were evident in tissue concentrations of total metabolites of NPH. In liver, blood, and testes, the metabolites at 24 hr were composed of approximately equal proportions of conjugates and nonconjugates. Nonconjugates were the predominant metabolites in the ovaries (>88% of the total). Thin-layer chromatography (TLC) of metabolites from liver, blood, ovaries, and testes revealed that the major nonconjugate metabolite was 1,2-dihydro-1,2-dihydroxynaphthalene (diol) and glucuronides were the major conjugate class present. Both TLC and high-performance liquid chromatography indicated that the glucuronide conjugate in the liver was derived from the diol. The naphthol conjugates were not detected. The ability of fish to rapidly accumulate orally administered NPH and its metabolites in the gonads may have significant implications with respect to fertilization and subsequent development of the embryo.

Exposure of Marine Mammals to Genotoxic Environmental Contaminants: Application of the 32P-Postlabeling Assay for Measuring DNA-Xenobiotic Adducts♯

There is little information on exposure of marine mammals to genotoxic environmental contaminants. The 32P-postlabeling assay has been successfully used to assess exposure to genotoxic polycyclic aromatic compounds in fish and humans. In the present study, a preliminary investigation showed that polycyclic aromatic compound-like DNA adducts were present in hepatic tissues of harbor seals (Phoca vitulina richardsi) exposed to petroleum following the Exxon Valdez oil spill. However, for marine mammals, effects from changes in tissue condition on DNA recovery and quality is of concern, because tissue samples are often collected from animals that have been dead for unknown periods of time. To assess the effects of postmortem thermal history on DNA recovery from tissue and on DNA adduct quantitation, samples of harbor porpoise (Phocoena phocoena) hepatic tissue were incubated for up to 10 d at 4 and 30 °C. Only traces (<4 μg) of hepatic DNA were recovered from 200 mg of tissue after incubation at 30 °C for 36 h. At 4 °C, DNA (50–130 μg) was recovered from tissue incubated for up to 6 d; whereas DNA recovery at 10 d was minimal. Chromatograms of 32P-labeled DNA digests of liver tissue held at 4 and 30 °C and salmon sperm DNA held at 30 °C for 2 d had comparable profiles, suggesting that alteration of DNA bases had occurred during incubation of porpoise liver tissue. Moreover, the chromatograms of DNA extracted from liver tissues of harbor porpoises caught incidentally in a northwest Atlantic fishery, packed in ice and sampled several days later also exhibited similar altered DNA structures. Although, altered DNA structures that can interfere with the DNA adduct quantitation were present in autolyzed tissue, changes in the 32P-postlabeling chromatography conditions can decrease the interference. Moreover, in a study with tissues taken from California sea lions (Zalophus californianus) immediately postmortem and stored at –80 °C until processing, DNA structures associated with tissue breakdown were not observed. The DNA from sea lions, however, had putative age-dependent hepatic DNA modifications, which have a distinctive profile, and must be considered when evaluating exposure of marine mammals to polycyclic aromatic compounds. Overall, the findings showed that with attention to the postmortem thermal history of the tissue samples hepatic DNA adducts, as measured by 32P-postlabeling, have the potential to serve as a biological indicator of exposure of marine mammals to environmental genotoxic compounds.

Biomarker and histopathologic responses in flatfish following site remediation in Eagle Harbor, WA

Abstract Eagle Harbor is a designated Superfund site due to high sediment concentrations of creosote-derived polycyclic aromatic hydrocarbons (PAHs) released chronically from a nearby creosoting facility. Studies with English sole Pleuronectes vetulus from this site (1984–1986) demonstrated high prevalences of toxicopathic liver lesions including neoplasms in resident sole. Inducibility of neoplasia-related lesions by injections of a PAH-rich fraction extracted from Eagle Harbor sediment has also been shown. Further studies (1986–1988) also sampled starry flounder Platichthys stellatus and rock sole Lepidopsetta bilineata, and incorporated biomarkers of PAH exposure and effect, including hepatic CYP1A expression, biliary fluorescent aromatic compounds (FACs), and hydrophobic DNA adducts in liver. Hepatic lesion prevalences and biomarker values in these species from Eagle Harbor were among the highest found at Puget Sound sites. A cap of uncontaminated sediment was placed (September 1993–March 1994) over the most contaminated portions of Eagle Harbor in an attempt to sequester PAH-contaminated sediments. Lesion prevalences and biomarker values just before capping began were generally reduced compared to historical data, consistent with creosoting facility closure and site-based source controls. Similar data from fish collected immediately after capping, and, at 3, 6, 12, 16, 19, 21, 31, 44, and 49 months after cap completion, are presented to determine the efficacy of the capping in ameliorating PAH exposure and associated effects in resident flatfish species.