32P-postlabeling analysis of DNA adducts in liver of wild English sole (Parophrys vetulus) and winter flounder (Pseudopleuronectes americanus).

The 1-butanol adduct enhancement version of the 32P-postlabeling assay was used to measure the levels of hepatic DNA adducts in the marine flatfish, English sole (Parophrys vetulus), sampled from the Duwamish Waterway and Eagle Harbor, Puget Sound, WA, where they are exposed to high concentrations of sediment-associated chemical contaminants and exhibit an elevated prevalence of hepatic neoplasms. Hepatic DNA was also analyzed from English sole from a reference area (Useless Bay, WA) and from reference English sole treated with organic-solvent extracts of sediments from the two contaminated sites. Autoradiograms of thin-layer chromatograms of 32P-labeled hepatic DNA digests from English sole from the contaminated sites exhibited up to three diagonal radioactive zones, which were not present in autoradiograms of thin-layer chromatogram maps of 32P-labeled DNA digests from English sole from the reference site. These diagonal radioactive zones contained several distinct spots as well as what appeared to be multiple overlapping adduct spots. The levels (nmol of adducts/mol of nucleotides) of total DNA adducts for English sole from Duwamish Waterway and Eagle Harbor were 26 +/- 28 (DS) and 17 +/- 9.6, respectively. All autoradiograms of DNA from fish from the contaminated sites exhibited a diagonal radioactive zone where DNA adducts of chrysene, benzo(a)pyrene, and dibenz(a,h)anthracene, formed in vitro using English sole hepatic microsomes, were shown to chromatograph. English sole treated with extracts of the contaminated sediments had adduct profiles generally similar to those for English sole from the respective contaminated sites. The chromatographic characteristics of the adducts and the similarities in adduct profiles between field-sampled English sole and those treated with contaminated sediment extracts suggested that hydrophobic aromatic compounds of anthropogenic origin were adducted to hepatic DNA of sole from contaminated sites, but not in sole from the reference site. Moreover, an initial study with winter flounder (Pseudopleuronectes americanus) from Boston Harbor, MA, an area where high concentrations of sediment-associated chemicals are present, revealed a pattern of hepatic DNA-adducts (9.0 +/- 7.8 nmol of adducts/mol of nucleotides) similar to that observed for English sole from Eagle Harbor.

Comparative metabolism of benzo(a)pyrene and covalent binding to hepatic DNA in English sole, starry flounder, and rat.

Metabolism of benzo(a)pyrene (BaP) in vivo and in vitro was studied using two benthic fish species, English sole (Parophrys vetulus) and starry flounder (Platichthys stellatus), and Sprague-Dawley rats. At 24 h after administration of BaP (7.9 mumol/kg of body weight) to fish either p.o. (Experiment 1) or i.p. (Experiment 2), the specific activity of binding of BaP metabolites to hepatic DNA (pmol of BaP equivalent per mg of DNA) was higher in sole [2.1 in Experiment 1; 28 +/- 5 (SE) in Experiment 2] than in flounder (0.5 in Experiment 1; 14 +/- 4 in Experiment 2). Treatment of bile with beta-glucuronidase and arylsulfatase released a significantly higher proportion of 7,8-dihydroxy-7,8-dihydro-BaP (BaP 7,8-diol) from sole bile than from flounder bile in both experiments. However, the rate of BaP metabolism and rate of formation of BaP 7,8-diol by hepatic microsomes were comparable for both fish species. Thus, the differences in both the level of DNA binding and the concentration of BaP 7,8-diol in bile of BaP-exposed sole and flounder were apparently due to differences in detoxication, rather than formation, of BaP 7,8-oxide and BaP 7,8-diol-9,10-epoxide. The rate of formation of BaP 7,8-diol by rat liver microsomes (28 +/- 1 pmol of BaP 7,8-diol formed per min per mg of protein) was comparable to that by hepatic microsomes from both fish species (50 +/- 10 for sole and 33 +/- 6 for flounder), although the rate of BaP metabolism (600 +/- 200) was approximately 3 times greater than that by the fish species (190 +/- 60 for sole and 180 +/- 40 for flounder). Thus, greater proportion of BaP was converted to BaP 7,8-diol by liver microsomes of fish species than rat. These differences in BaP metabolism in vitro help explain, in part, the substantially lower binding (0.3 +/- 0.1; Experiment 2) for hepatic DNA in BaP-exposed rat than that in either sole or flounder.

Molecular epizootiology: assessment of exposure to genotoxic compounds in teleosts.

The recent development of techniques to measure levels of carcinogens covalently bound to DNA provides the opportunity to use DNA adducts as molecular dosimeters of exposure to environmental carcinogens and mutagens. This is especially important because epizootiologic studies have shown a positive association between environmental carcinogens, such as polycyclic aromatic hydrocarbons, and increased prevalence of neoplasms and related lesions, primarily in liver, of benthic fish species from a wide range of urban and industrialized areas. In studies with wild fish and mammalian species the 32P-postlabeling assay, as developed for aromatic compounds, has been used most extensively because of its high sensitivity and ability to detect structurally uncharacterized adducts. The results to date of field and laboratory studies show that hepatic DNA adducts detected in fish are associated with increased exposure to environmental polycyclic aromatic compounds in the preponderance of species examined, whereas in the limited studies with wild mammals, such a relationship is equivocal at present. The findings with fish suggest that DNA adducts, as measured by 32P-postlabeling, have the potential to be effective molecular dosimeters of exposure to environmental carcinogenic aromatic compounds and thereby may lead to an improved understanding of the etiology of neoplasia in wild teleosts.

A biomarker approach to assessing xenobiotic exposure in Atlantic tomcod from the North American Atlantic coast.

We determined levels of hepatic cytochrome P4501A (CYP1A) mRNA, hepatic DNA adducts, and fluorescent aromatic compounds (FACs) in bile, a measure of exposure to polyaromatic hydrocarbons, in Atlantic tomcod from six river systems ranging from highly polluted to relatively pristine on the northeast North American coast (the Hudson River, New York; the St. Lawrence River, Quebec; the Miramichi River, New Brunswick; the Saco and Royal rivers, Maine; and the Margaree River, Nova Scotia). Hudson River tomcod showed the greatest response for all parameters, and tomcod from the Margaree River exhibited the least response. Tomcod from the Miramichi River exhibited marked induction of CYP1A mRNA but low levels of hepatic DNA adducts and biliary FACs, whereas fish from the St. Lawrence River showed no induction of CYP1A mRNA and moderately elevated levels of DNA adducts and biliary FACs. In tomcod from the Hudson and Miramichi rivers, the levels of CYP1A mRNA were 28 times and 14 times, respectively, as great as the levels in fish from the St. Lawrence, Saco/Royal, and Margaree rivers. Mean levels of DNA adducts varied from 120 nmol adducts/mol bases in Hudson River tomcod to < 3 nmol adducts/mol bases in fish from the Miramichi and Margaree rivers. Concentrations of FACs in the bile of tomcod from the Hudson and St. Lawrence rivers were 8 and 1.8 times, respectively, as great as the concentrations in tomcod from the Miramichi River and Margaree River. In tomcod from the Hudson River, all three biomarkers were markedly elevated; in the St. Lawrence River two biomarkers were elevated, in the Miramichi River one was elevated, but no biomarker was substantially elevated in fish from the Saco/Royal and Margaree rivers. Elevated levels of hepatic DNA adducts and biliary FACs in tomcod from the Hudson River suggest increased exposure to PAHs, consistent with previous studies.

Chemical carcinogenesis in feral fish: uptake, activation, and detoxication of organic xenobiotics.

The high prevalence of liver neoplasms in English sole (Parophrys vetulus) and substantially lower prevalence of neoplasms in a closely related species, starry flounder (Platichthys stellatus) captured from industrialized waterways, provide a unique opportunity to compare biochemical processes involved in chemical carcinogenesis in feral fish species. Because levels of aromatic hydrocarbons (AHs) in urban sediments are correlated with prevalences of liver neoplasms in English sole, we have initiated detailed studies to evaluate the effects of endogenous and exogenous factors on uptake, activation and detoxication of carcinogenic AHs, such as benzo[a]pyrene (BaP), using spectroscopic, chromatographic, and radiometric techniques. The results obtained thus far show that sole readily takes up AHs associated with sediment from urban areas and that the presence of other xenobiotics, such as PCBs, in sediment increases tissue concentrations of BaP metabolites. Extensive metabolism of BaP occurred whether sole was exposed to this AH via sediment, per os, or intraperitoneally. Substantial modification of hepatic DNA occurred and persisted for a period of 2-4 weeks after a single exposure to BaP. The level of covalent binding of BaP intermediates to hepatic DNA was 10-fold higher in juvenile than adult sole and 90-fold higher in juvenile sole than in Sprague-Dawley rat, a species which is resistant to BaP-induced hepatocarcinogenesis. The level of chemical modification of hepatic DNA in juvenile flounder was 2-4 fold lower than that for juvenile sole and concentration of BaP 7,8-diol glucuronide in bile of sole was significantly higher than that in flounder bile, although the rate of formation of BaP 7,8-diol by hepatic microsomes was comparable for both species. Moreover, liver microsomes from both species, in the presence of exogenous DNA, metabolized BaP into essentially a single adduct, identified as (+)anti-7,8-diol-9,10-epoxy-7,8,9,10-tetrahydroBaP-dG. These results, along with our findings that hepatic GST activity in flounder was two times higher than in sole, demonstrate that microsomal metabolism of BaP does not accurately reflect the differences in the ability of these fish to form BaP-DNA adducts in vivo and also suggest that detoxication of reactive intermediates is an important factor in determining the levels of DNA modification by AHs and resulting toxic effects in feral fish.

Bioaccumulation of polycyclic aromatic hydrocarbons by marine organisms.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the marine environment, occurring at their highest environmental concentrations around urban centers. While they can occur naturally, the highest concentrations are mainly from human activities, and the primary sources are combustion products and petroleum. Two factors, lipid and organic carbon, control to a large extent the partitioning behavior of PAHs in sediment, water, and tissue; the more hydrophobic a compound, the greater the partitioning to these phases. These two factors, along with the octanol-water partition coefficient, are the best predictors of this partitioning and can be used to determine PAH behavior and its bioavailability in the environment. It is well known that the lipid of organisms contains the highest levels of hydrophobic compounds such as PAHs, and that organic carbon associated with sediment or dissolved in water can have the greatest influence on PAH bioavailability. Partitioning of combustion-derived PAHs between water and sediment may be much less than predicted, possibly because associations with particles are much stronger than expected. This reduced partitioning may produce erroneous results in predicting bioaccumulation where uptake from water is important. Accumulation of PAHs occurs in all marine organisms; however, there is a wide range in tissue concentrations from variable environmental concentrations, level and time of exposure, and species ability to metabolize these compounds. PAHs generally partition into lipid-rich tissues, and their metabolites can be found in most tissues. In fish, liver and bile accumulate the highest levels of parent PAH and metabolites; hence, these are the best tissues to analyze when determining PAH exposure. In invertebrates, the highest concentrations can be found in the internal organs, such as the hepatopancreas, and tissue concentrations appear to follow seasonal cycles, which may be related to variations in lipid content or spawning cycles. The major route of uptake for PAHs has been debated for years. For the more water-soluble PAHs, it is believed that the main route of uptake is through ventilated water and that the more hydrophobic compounds are taken in mainly through ingestion of food or sediment. There are many variables, such as chemical hydrophobicity, uptake efficiency, feeding rate, and ventilatory volume, which may affect the outcome. The route of uptake may be an important issue for short-term events; however, under long-term exposure and equilibrium conditions between water, prey, and sediment, the route of uptake may be immaterial because the same tissue burdens will be achieved regardless of uptake routes.(ABSTRACT TRUNCATED AT 400 WORDS)

32P-postlabeling analysis of DNA adduct formation and persistence in English sole (Pleuronectes vetulus) exposed to benzo[a]pyrene and 7H-dibenzo[c,g]carbazole.

The formation and persistence of benzo[a]pyrene (BaP)- and 7H-dibenzo[c,g]-carbazole (DBC)-DNA adducts in liver of English sole (Pleuronectes vetulus) were investigated. BaP is a putative hepatocarcinogen in English sole based on its ability to induce formation of preneoplastic foci, while DBC is a hepatocarcinogen in mammals but whose carcinogenicity in fish is not known. English sole liver was sampled from 2 h through 84 days after a single intermuscular injection of a BaP and DBC mixture (100 mumol of each/kg body wt.), and DNA adduct levels were measured by the nuclease P1 version of the 32P-postlabeling assay. The major BaP adducts detected were from binding of BaP-7,8-diol-9,10-epoxide to DNA, whereas multiple uncharacterized DBC-DNA adducts were detected. Total adduct levels for both BaP and DBC reached a maximum at 2 days post exposure. The levels of DBC-DNA adducts were greater than the levels of BaP adducts at all time points and increased more rapidly than did the levels of BaP-DNA adducts. The DBC to BaP adduct ratio was 33 +/- 8.8 at 2 h and declined to 4.2 +/- 0.48 by 12 h post exposure. From 2 to 28 days, the levels of both BaP and DBC adducts declined with apparent half-lives of 11 and 13 days, respectively. There was no apparent decline from 28 to 84 days in the levels of the remaining BaP or DBC adducts; these persistent adducts represented 32 and 36% of maximum levels, respectively. These results provide the first data on the kinetics of adduct formation and removal of a carcinogenic nitrogen-containing polycyclic aromatic compound in fish. The results showing greater binding and similar persistence of DBC-DNA adducts compared to BaP-DNA adducts suggest that DBC may be hepatotoxic and potentially carcinogenic in English sole. In a separate experiment, the effect of multiple doses of BaP (30 mumol/kg body wt.) on the levels of hepatic BaP-DNA adducts showed that adduct levels increased linearly (r = 0.815, P = 0.0007) with 5 successive doses administered at 2 day-intervals and sampled 2 days after the last dose. The persistence of both BaP-DNA and DBC-DNA adducts in liver, together with the increase in BaP-DNA adducts in English sole exposed to successive doses of BaP, suggest that hepatic xenobiotic-DNA adducts in English sole are molecular dosimeters of relatively longterm environmental exposure to genotoxic polycyclic aromatic compounds.

Formation and persistence of benzo[a]pyrene-diolepoxide-DNA adducts in liver of English sole (Parophrys vetulus).

The formation of DNA adducts from the carcinogenic environmental pollutant benzo[a]pyrene (BaP) was investigated in liver of English sole (Parophrys vetulus), a fish species that exhibits a high prevalence of liver neoplasms in several polycyclic aromatic hydrocarbon (PAH)-contaminated areas of Puget Sound, WA. Analysis by the 32P-postlabeling assay of hepatic DNA digests from English sole exposed parenterally to BaP showed the presence of BaP-diol epoxide (BaPDE)-DNA adducts. When English sole were injected with 2-15 mg BaP/kg body wt., one major adduct was detected and was identified as the anti-BaPDE-DNA adduct. Moreover, in English sole sampled at 1, 28 and 60 days post-exposure to 15 mg BaP/kg body wt., there was no significant change in the level of the anti-BaPDE-DNA adduct. The autoradiographs of 32P-labeled hepatic DNA digests from fish exposed to 100 mg BaP/kg body wt. showed an elongated spot suggesting the presence of more than one adduct. Chromatography on large polyethyleneimine sheets (20 x 20 cm) showed 2 spots with the same chromatographic characteristics as those of syn- and anti-BaPDE-deoxyguanosine adduct standards. Mild acid hydrolysis of hepatic DNA of English sole, exposed to 100 mg BaP/kg body wt., also revealed the presence of tetrols derived from both anti- and syn-BaPDE, thus confirming the presence of syn- and anti-BaPDE. In fish exposed to 2-100 mg BaP/kg body wt., a linear (0.996) dose response for anti-BaPDE-DNA adduct formation was observed. The results from this study offer the first direct evidence for the formation of the suspected ultimate carcinogen, BaPDE, in liver of English sole exposed to BaP in vivo and thus further support the hypothesis that exposure to PAHs is an important factor in the etiology of hepatic neoplasms in English sole from contaminated sites.

Molecular epizootiology of genotoxic events in marine fish: linking contaminant exposure, DNA damage, and tissue-level alterations.

Molecular epizootiological studies are increasingly being used to investigate environmental effects of genotoxic contaminants. The assessment of damage to DNA and linking the damage to subsequent molecular, cellular, or tissue-level alterations is a central component of such studies. Our research has focused on the refinement of the 32P-postlabeling assay for measuring covalent DNA-xenobiotic adducts arising from exposure to polycyclic aromatic compounds, using DNA adducts as molecular dosimeters of genotoxic contaminant exposure in biomonitoring studies, and investigating the relationship of DNA adduct formation to toxicopathic liver disease, including neoplastic lesions. A combination of field and laboratory studies using the 32P-postlabeling assay has shown that DNA adducts in marine fish are effective molecular dosimeters of genotoxic contaminant exposure. Investigations of the relationship of DNA adduct formation to neoplastic liver disease have shown that elevated levels of DNA adducts in certain fish species from contaminated coastal sites are associated with increased prevalences of toxicopathic hepatic lesions, including neoplasms, and that the ability to assess DNA damage has helped to explain, in part, species differences in lesion prevalence. Moreover, in a study of a site in Puget Sound contaminated with polycyclic aromatic compounds, we have shown, for the first time, that elevated levels of hepatic DNA adducts are a significant risk factor for certain degenerative and preneoplastic lesions occurring early in the histogenesis of hepatic neoplasms in feral English sole (Pleuronectes vetulus). These latter findings coupled with our current studies of mutational events in the K-ras proto-oncogene should provide further mechanistic substantiation that mutagenic events resulting from exposure to complex mixtures of genotoxic polycyclic aromatic compounds are involved in the etiology of hepatic neoplasia in English sole.

Electron spin resonance study of serum lipoproteins of salmon (Oncorhynchus kisutch): Structural alterations produced in high density lipoproteins by mercury and cadmium.

High denisty lipoproteins from the serum of salmon (Oncorhynchus kisutch), containing a maleimide spin label (4-male-imido-2,2,6,6-tetramethylpiperidinooxyl), were exposed to HgCl2, CdCl2, and CH3HgCl under physiological conditions. Electron spin resonance spectrometry revealed that HgCl2 and CdCl2 produce significant alterations at sites representing weakly and strongly immobilized label. The observed changes appear to be taking place primarily in the surface architecture. No alterations were found in the normal spectra of high density lipoproteins exposed to CH3HgCl under similar conditions. Each metal species binds strongly with the apo-high density lipoproteins; however, marked differences exist in the effects produced upon high density lipoprotein structure by the inorganic ions and CH3HgCl.

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.

Overview of studies on liver carcinogenesis in English sole from Puget Sound; evidence for a xenobiotic chemical etiology. II: Biochemical studies.

The levels of aromatic hydrocarbons in sediments of Puget Sound, Washington, are positively correlated with the prevalence of hepatic neoplasms and related lesions in English sole (Parophrys vetulus). To investigate the biochemical processes involved in chemical carcinogenesis in fish from Puget Sound, we have studied the uptake, activation, and detoxication of polycyclic aromatic hydrocarbons (PAHs) in English sole, and have compared these data to PAH metabolism in a related species, starry flounder (Platichthys stellatus), which shows a lower prevalence of hepatic neoplasms than sole. The results of both laboratory and field studies show that sediment-associated PAHs are biologically available to both flatfish species, and that both species accumulate similar levels of PAHs. Analyses of hepatic DNA from sole using the 32P-postlabeling technique indicate that xenobiotic chemicals were adducted to hepatic DNA of fish from the contaminated sites but not to the DNA of fish from reference sites. Studies of the ability of English sole and starry flounder to metabolize benzo(a)pyrene (BaP) and bind reactive BaP intermediates to hepatic DNA indicate that biochemical differences in the metabolism of carcinogenic PAHs may explain, at least in part, the apparent lower susceptibility of starry flounder than English sole to chemically induced hepatocarcinogenesis.

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.

Evidence of uptake, biotransformation and DNA binding of polyaromatic hydrocarbons in Atlantic cod and corkwing wrasse caught in the vicinity of an aluminium works.

Feral Atlantic cod (Gadus morhua) and corkwing wrasse (Symphodus melops) were investigated for polyaromatic hydrocarbon (PAH) contamination in the Karmsund strait, western Norway. This strait is highly contaminated with PAHs, and a main source is the chronic release of gas-scrubbing effluents from a local aluminium works. In both species, the level of biliary PAH metabolites and hepatic DNA adducts were higher in fish collected near the aluminium works. Interestingly, a significantly higher level of both biliary PAH metabolites and hepatic DNA adducts was found in corkwing wrasse as compared to cod, indicating a higher potential for genotoxic effects in this species. Hepatic cytochrome P4501A (CYP1A) in cod estimated by ethoxyresorufin-O-deethylase and an immunoassay technique (ELISA), seemed to be weakly induced at the contaminated sites. At the most contaminated site, skin ulcers and fin erosion were detected in about 70 and 45% of the cods, respectively. The data demonstrated that both cod and corkwing wrasse may be suitable target species for PAH pollution monitoring.

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

Juvenile starry flounder (Platichthys stellatus) maintained at 4 degrees or 12 degrees C were forced-fed 3H-1-naphthalene. At 24 hr, after the initiation of exposure, significantly (p less than 0.05) higher concentrations (2 to 15 times) of naphthalene were present in tissues of starry flounder at 4 degrees C than those present in fish held at 12 degrees 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 degrees C contained 26 and 34 times, respectively, more naphthalene than did muscle and liver of fish at 12 degrees 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 (approximately 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 degrees C compared to 12 degrees 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.

Storage phosphor imaging technique for detection and quantitation of DNA adducts measured by the 32P-postlabeling assay.

The 32P-postlabeling method has found wide application as a sensitive technique for detecting the presence of a broad range of bulky aromatic compounds covalently bound to DNA. In this method, the modified DNA is enzymatically degraded to 3'-mononucleotides and labeled with [32P]-phosphate at the 5'-position using [gamma-32P]ATP and T4 polynucleotide kinase. The 32P-labeled DNA digest is then chromatographed in two dimensions on polyethyleneimine - cellulose thin-layer plates. Screen-enhanced autoradiography is used to locate the presence of the radiolabeled adducts on the chromatogram, and the radioactive areas are generally excised and quantitated by liquid scintillation spectrometry. However, on a chromatogram with multiple adducts, it can be difficult to quantitative partially resolved adducts and evaluated background radioactivity levels. We have evaluated the use of storage phosphor imaging techniques to quantitate and map the radioactivity on chromatograms generated by the 32P-postlabeling method. The results showed that storage phosphor imaging was approximately 10 times more sensitive than screen-enhanced autoradiography at -80 degrees C for the detection of 32P, exhibits a greater linear range of response, has a resolution that compares favorably to film and has a lower background than does liquid scintillation spectrometry. Further, the generation of a digitized record of the distribution and intensity of radioactivity allows for computer-assisted assessment of adduct profiles and can facilitate quantitation of individual adducts and radioactive zones comprised of multiple overlapping adducts in complex chromatograms. Additionally, the permanent record created by the imaging technology permits facile retrospective analysis of samples, whereas with autoradiography and liquid scintillation spectrometry reanalysis of a replicate sample is required.

Metabolism and subsequent covalent binding of benzo[a]pyrene to macromolecules in gonads and liver of ripe english sole (Parophrys vetulus).

1. Ripe English sole (Parophrys vetulus) force-fed [3H]benzo[a]pyrene, contained 1% of the dose in liver, 0.2% in ovary and 0.1% in testis, after 24 h. No significant change occurred in levels of radioactivity from 24 to 168 h. 2. Gonads and blood contained substantially larger proportions of unchanged benzo[a]pyrene (15-37% of tissue radioactivity) and organic solvent-soluble metabolites (6-35%) than did liver and bile. 3. T.l.c. revealed the presence of phenols, quinones, 7,8-dihydro-7, 8-dihydroxy- and 9,10-dihydro-9,10-dihydroxy-benzo[a]pyrene in liver and gonads. 4. A small proportion (less than 10%) of the radioactivity in liver and gonads was present as glucuronides and sulphates; bile contained a higher proportion (ca. 20%) of total radioactivity as glucuronides and sulphates. 5. Benzo[a]pyrene intermediates were covalently bound to liver proteins and DNA, and to a lesser extent to gonadal proteins (male and female fish) and gonadal DNA (confirmed for testis only).