Isovaleric acid accumulation in odontocete melon during development.

Biosonar in odontocetes is a highly complex process for gathering information about the surrounding environment. The forehead melon lipid and mandibular lipid tissues, which comprise the region known as the acoustical window for cetacean sound production and reception, have a unique biochemical composition that is made up of unusual fatty deposits rich in isovaleric acid. Although the structure of these acoustical lipids was elucidated three decades ago, little work has been done to determine their origin during cetacean development. The objective of this research was to examine development of the acoustical region by characterizing the accumulation of isovaleroyl lipids throughout cetacean early life stages. Biochemical analyses of melon tissue of Phocoena phocoena and Tursiops truncatus of different sizes (as an indicator of age) demonstrated that the proportion of isovalerate increased significantly with length. These results indicate that the acoustic system is not fully developed at birth and that its biochemical structure changes throughout development.

Organochlorines in stranded pilot whales (Globicephala melaena) from the coast of Massachusetts.

Pilot whales strand periodically along the U.S. coast, and these strandings offer an opportunity for the collection of tissues for biomonitoring of contaminant exposure in cetaceans, as well as for specimen archiving. Concentrations of organochlorine (OC) contaminants (e.g., PCB congeners, pesticides, DDTs) were measured in tissue samples from pilot whales that stranded in 1986 and 1990 along the Massachusetts coast. Adult and fetal samples of blubber, liver, brain, and kidney were collected, as well as ovaries from mature female whales. Many of the OCs found in maternal tissues were detected in corresponding fetal tissues indicating maternal transfer of OCs to the fetus. The concentrations of individual OCs in tissues varied considerably among the animals. Statistically significant differences were found between females and males for the concentrations of certain analytes (e.g., SigmaPCBs, p,p'-DDE) and these differences may be partially due to contaminants being transferred by the female whales during gestation and lactation. The concentrations of OCs in different tissues were similar when based on total lipid weight, except for the brain, which contained the lowest lipid-normalized OC concentrations. The low concentrations in brain may be related to the disparate lipid compositions in this tissue as well as the presence of the blood-brain barrier. The availability of data on these archived and biomonitoring samples provides a baseline for future retrospective studies.

Environmental contaminants and the prevalence of hemic neoplasia (leukemia) in the common mussel (Mytilus edulis complex) from Puget Sound, Washington, U.S.A.

The relationship between hemic neoplasia, a blood cell disorder in bivalve molluscs, and chemical contaminants was evaluated in the common mussel (Mytilus edulis complex). Hemic neoplasia (HN) is endemic to mussel populations in Puget Sound. The prevalence of hemic neoplasia ranged from 0 to 30% in mussels from nine sites in Puget Sound, Washington. Organic chemical contamination in sediment from these sites range from 0.1 to 64.0 ppm of polycyclic aromatic hydrocarbons (PAHs) and 0.07 to 0.50 ppm chlorinated hydrocarbons. No relationship between the body burden of environmental contaminants and the prevalence of HN in mussels was identified. To evaluate the short-term ability of chemical contaminants to induce HN in mussels, mussels, from a site where mussels were previously determined to be HN free, were fed microencapsulated PAHs (composed of a mixture of phenanthrene, flouranthene, and benzo[a]pyrene) or PCBs (Aroclor 1254) and the prevalence of HN was assessed after 30 days of exposure. Although an apparent increase in HN prevalence (20 to 30%) was observed in all treatments groups except the untreated controls, no significant difference in the prevalence of HN was observed between the control group of mussels fed corn oil (vehicle) and mussels fed either PAHs or PCBs in corn oil. A long-term (180-day) exposure study was conducted to evaluate the influence of PAHs or PCBs in modulating the prevalence of HN in a mussel population already exhibiting a moderate HN prevalence. Mussels, from a site where mussels were previously determined to exhibit a background prevalence of HN, fed microencapsulated PAHs, PCBs, and corn oil (vehicle) over a long time period (180 days), revealed an apparent increased prevalence of HN (30 to 40%) above the low levels (20%) initially present. However, no significant difference in the prevalence of HN was observed between the control group of mussels fed corn oil (vehicle) and mussels fed either PAHs or PCBs in corn oil. Although chemical contaminants have been proposed as a modulating factor in the development and promotion of HN in bivalve molluscs from environmentally stressed and degraded habitats, we find no evidence that chemical contaminants induce or promote the development of HN in the mussel M. edulis complex.

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.

The Cyc8 (Ssn6)-Tup1 corepressor complex is composed of one Cyc8 and four Tup1 subunits.

The Cyc8 (Ssn6)-Tup1 corepressor complex is required for repression in several important regulatory systems in yeast cells, including glucose repression and mating type. Cyc8-Tup1 is recruited to target genes by interaction with diverse repressor proteins that bind directly to DNA. Since the complex has a large apparent molecular mass of 1,200 kDa on nondenaturing gels (F. E. Williams, U. Varanasi, and R. J. Trumbly, Mol. Cell. Biol. 11:3307-3316, 1991), we used a variety of approaches to determine its actual subunit composition. Immunoprecipitation of epitope-tagged complex and reconstitution of the complex from in vitro-translated proteins demonstrated that only the Cyc8 and Tup1 proteins were present in the complex. Hydrodynamic properties showed that these proteins have unusually large Stokes radii, low sedimentation coefficients, and high frictional ratios, all characteristic of asymmetry which partly accounts for the apparent high molecular weight. Calculation of native molecular weights from these properties indicated that the Cyc8-Tup1 complex is composed of one Cyc8 subunit and four Tup1 subunits. This composition was confirmed by reconstitution of the complex from Cyc8 and Tup1 expressed in vitro and analysis by one- and two-dimensional gel electrophoresis.

Identification of a peroxisome proliferator-responsive element upstream of the human peroxisomal fatty acyl coenzyme A oxidase gene.

Peroxisome proliferators cause a rapid and coordinated transcriptional activation of genes encoding the enzymes of the peroxisomal beta-oxidation pathway in rats and mice. Cis-acting peroxisome proliferator responsive elements (PPREs) have been identified in the 5'-flanking region of H202-producing rat acyl-CoA oxidase (ACOX) gene and in other genes inducible by peroxisome proliferators. To gain more insight into the purported nonresponsiveness of human liver cells to peroxisome volume density and in the activity of the beta-oxidation enzyme system, we have previously cloned the human ACOX gene, the first and rate-limiting enzyme of the peroxisomal beta-oxidation system. We now present information on a regulatory element for the peroxidase proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers. The PPRE, consists of AGGTCA C TGGTCA, which is a direct repeat of hexamer half-sites interspaced by a single nucleotide (DR1 motif). It is located at -1918 to -1906 base pairs upstream of the transcription initiation site of this human ACOX gene. This PPRE specifically binds to baculovirus-expressed recombinant rat PPAR alpha/RXR alpha heterodimers. In transient transfection experiments, the maximum induction of luciferase expression by ciprofibrate and/or 9-cis-retinoic acid is dependent upon cotransfection of expression plasmids for PPAR alpha and RXR alpha. The functionally of this human ACOX promoter was further demonstrated by linking it to a beta-galactosidase reporter gene or to a rat urate oxidase cDNA and establishing stably transfected African green monkey kidney (CV1) cell lines expressing reporter protein. The human ACOX promoter has been found to be responsive to peroxisome proliferators in CV1 cells stably expressing PPAR alpha, whereas only a basal level of promoter activity is detected in stably transfected cells lacking PPAR alpha. The presence of a PPRE in the promoter of this human peroxisomal ACOX gene and its responsiveness to peroxisome proliferators suggests that factors other than the PPRE in the 5'-flanking sequence of the human ACOX gene may account for differences, if any, in the pleiotropic responses of humans to peroxisome proliferators.

Butyltin contamination of sediments and benthic fish from the East, Gulf and Pacific coasts of the United States.

Butyltin concentrations were determined in sediments, tissues and stomach contents of fish collected in 41 embayments on the East, Gulf and Pacific coasts of the U.S.A. between 1986 and 1991 as part of the National Oceanic and Atmospheric Administration's (NOAA) National Benthic Surveillance Project (NBSP). A total of 99 sediments, 108 fish liver samples from 11 fish species, and 10 composites of fish stomach contents were analyzed for tetrabutylin, tributylin, dibutylin and monobutylin. Tributyltin (TBT) was detected (i.e. > 10 ng/g) in 38 of the sediments samples analyzed and was generally the predominant bulytin present; concentrations of total butyltins ranged from 15 to 1600 ng/g wet weight. The highest concentrations were found in sediments from urban sites, especially sites on the West coast. Many of the fish liver and stomach contents samples also contained butyltins. Tributyltin represented 83 (7.1)% [mean (SEM); n=15], 64 (6.6)% (n=12) and 36 (7.8)% (n=12) of the total butyltins in livers from white croaker, winter flounder and Atlantic croaker, respectively, suggesting possible species differences in biotransformation of TBT. The concentrations of butyltins in stomach contents indicated that diet can be a significant route of exposure of fish to butyltins. Between 1986 and 1991 butyltin concentrations in sediments and fish generally appeared to be declining; however, no statistically significant temporal trends were observed at individual sites or for the sites overall.

Overexpression and characterization of the human peroxisomal acyl-CoA oxidase in insect cells.

Human liver peroxisomes contain two acyl-CoA oxidases, namely, palmitoyl-CoA oxidase and a branched chain acyl-CoA oxidase. The palmitoyl-CoA oxidase (ACOX) oxidizes the CoA esters of straight chain fatty acids and prostaglandins and donates electrons directly to molecular oxygen, thereby producing H2O2. The inducibility of this H2O2-generating ACOX in rat and mouse liver by peroxisome proliferators and the postulated role of the resulting oxidative stress in hepatocarcinogenesis generated interest in characterizing the structure and function of human ACOX. We have constructed a full-length cDNA encoding a 660-amino acid residue human ACOX and produced a catalytically active human ACOX protein at high levels in Spodoptera frugiperda (Sf9) insect cells using the baculovirus vector. Immunoblot analysis demonstrated that the full-length 72-kDa polypeptide (component A) was partially processed into its constituent 51-kDa (component B) and 21-kDa (component C) products, respectively. Recombinant protein (approximately 20 mg/l x 10(9) cells) was purified to homogeneity by a single-step procedure on a nickel-nitrilo-triacetic acid affinity column. Using the purified enzyme, Km and Vmax values for palmitoyl-CoA were found to be 10 microM and 1.4 units/mg of protein, respectively. The maximal activities for saturated fatty acids were observed with C12-18 substrates. The overexpressed human ACOX protein was identified in the cytoplasm of the insect cells by immunocytochemical staining. Individual expression of either the truncated ACOX 51-kDa (component B) or the 21-kDa (component C) revealed lack of enzyme activity, but co-infection of the insect cells with recombinant viruses expressing components B and C resulted in the formation of an enzymatically active heterodimeric B+C complex which could subsequently be inactivated by dissociating with detergent.

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)

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.

Screening for planar chlorobiphenyl congeners in tissues of marine biota by high-performance liquid chromatography with photodiode array detection.

A rapid method has been developed to screen for planar chlorobiphenyl (CB) congeners, as well as certain other CBs and DDTs, in tissue samples from marine biota. The analytes were extracted from tissue matrices with 1:1 hexane/pentane (v/v) and interfering compounds were separated from the CBs on a gravity-flow column packed with acidic, basic and neutral silica gel eluted with 1:1 hexane/methylene chloride (v/v). Subsequently, the planar CB congeners were resolved from the DDTs and other CBs by HPLC on Cosmosil PYE analytical columns cooled to 9 degrees C and were measured by an ultraviolet (UV) photodiode array (PDA) detector. Two important advantages of PDA over conventional UV detection were the ability to identify individual analytes by comparing their UV spectra to those of reference standards and the ability to establish the spectral homogeneity (purity) of the analytes by comparing spectra within a peak to the apex spectrum. The HPLC/PDA method was tested with tissue samples from fish, shellfish and marine mammals; concentrations of certain CBs and DDTs in samples determined by screening compared favorably with those in the same samples analyzed by a comprehensive method (e.g., gas chromatography/high resolution mass spectrometry). However, the HPLC/PDA method was about an order of magnitude less sensitive than determinations by comprehensive methods.

Chemical contaminants in gray whales (Eschrichtius robustus) stranded along the west coast of North America.

The concentrations of selected chlorinated hydrocarbons (e.g. PCBs, DDTs, DDEs, chlordanes) and essential (e.g. zinc, selenium, copper) and toxic (e.g. mercury, lead, arsenic) elements were measured in tissues and stomach contents from 22 gray whales (Eschrichtius robustus) stranded between 1988 and 1991 at sites from the relatively pristine areas of Kodiak Island, AK, to more urbanized areas in Puget Sound, WA, and San Francisco Bay, CA. The majority of animals were stranded at sites on the Washington outer coast and in Puget Sound. The gray whale has the unique feeding strategy among Mysticeti of filtering sediments to feed on benthic (bottom dwelling) invertebrates. Thus, the wide geographical distribution of the stranded whales allowed (1) an initial assessment of whether concentrations of chemical contaminants in these whales exhibited region specific differences and (2) whether toxic chemicals that accumulate in sediments may have contributed to the mortality and stranding of gray whales near the more polluted urban areas. Analyses for chlorinated hydrocarbons in blubber from 22 animals showed no apparent significant differences among stranding sites. The concentrations of sigma PCBs and sigma DDEs in blubber, for example, ranged from 120 to 10,000 and 9 to 2100 p.p.b. (ng/g) wet weight, respectively. Additionally, analyses of chlorinated hydrocarbons and selected elements in liver (n = 10) also showed no apparent significant differences between whales stranded in Puget Sound and whales stranded at more pristine sites (Alaska, Washington outer coast and Strait of Juan de Fuca and Strait of Georgia). For example, the concentrations of sigma PCBs and sigma DDEs in liver ranged from 79 to 1600 and 7 to 280 p.p.b., respectively, and the concentrations of the toxic elements, mercury and lead ranged from 9 to 120 and 20 to 270 p.p.b., respectively. Analyses of stomach contents revealed low concentrations of chlorinated hydrocarbons, but high concentrations (wet weight) of aluminum (1,700,000 +/- 450,000 p.p.b.), iron (320,000 +/- 250,000 p.p.b.), manganese (23,000 +/- 15,000 p.p.b.), and chromium (3400 +/- 1300 p.p.b.), but no significant differences were observed between whales stranded in Puget Sound compared to whales stranded at the more pristine sites. The relative proportions of these elements in stomach contents of stranded whales were similar to the relative proportions in sediments, which is consistent with a geological source of these elements from the ingestion of sediment during feeding. Thus, overall, the concentrations of anthropogenic chemicals in stranded gray whales showed little relation to the levels of chemical contaminants at the stranding sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation of the human peroxisomal acyl-CoA oxidase gene: organization, promoter analysis, and chromosomal localization.

Peroxisomal acyl-CoA oxidase (ACOX; EC 1.3.3.6) is the first enzyme of the fatty acid beta-oxidation pathway, which catalyzes the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs, and it donates electrons directly to molecular oxygen, thereby producing H2O2. The discovery of carcinogenic peroxisome proliferators, which markedly increase the levels of this H2O2-producing ACOX in rat and mouse liver, generated interest in peroxisomal beta-oxidation system genes. The present study deals with the structural organization of human ACOX gene. This gene spans approximately 33 kb and consists of 14 exons and 13 introns. Primer-extension analysis revealed three principal cap sites, which were mapped at 50, 52, and 53 nt upstream of the initiator methionine codon. The 5' flanking region of the ACOX gene was sequenced up to 500 bp upstream of the cap sites. This promoter region is G + C-rich and contains three copies of the "GC box" hexanucleotides. Multiple GC boxes are a characteristic feature of the rat ACOX and bifunctional protein genes of the beta-oxidation system. A + T-rich TATA-boxlike sequences, TTTATTT and TTATT, have also been identified in this human ACOX gene, but typical CCAAT motifs are absent. This ACOX gene has been mapped to chromosome 17q25 by in situ hybridization, using a biotinlabeled probe.

Relationships between toxicopathic hepatic lesions and exposure to chemical contaminants in English sole (Pleuronectes vetulus), starry flounder (Platichthys stellatus), and white croaker (Genyonemus lineatus) from selected marine sites on the Pacific Coast, USA.

Relationships between hepatic lesions and chemical contaminant concentrations in sediments, stomach contents, liver tissue, and bile were statistically evaluated in three species of bottomfish, English sole (Pleuronectes vetulus), starry flounder (Platichthys stellatus), and white croaker (Genyonemus lineatus), captured from 27 urban and nonurban sites on the Pacific Coast from Alaska to southern California. Lesions detected were neoplasms, preneoplastic foci of cellular alteration, nonneoplastic proliferative lesions, unique or specific degenerative/necrotic lesions, nonspecific degenerative/necrotic lesions, and hydropic vacuolation of biliary epithelial cells and hepatocytes. In general, lesion prevalences were significantly higher in all three species captured at chemically contaminated urban sites, and certain lesions had significantly higher relative risks of occurrence at urban sites in Puget Sound, San Francisco Bay, the vicinity of Los Angeles, and San Diego Bay. Concentrations of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, DDT and its derivatives, and chlordanes and dieldrin in sediment, stomach contents, liver, and fluorescent aromatic compounds in bile were significant risk factors for the occurrence of neoplastic, preneoplastic, nonneoplastic proliferative, and specific degenerative/necrotic lesions, as well as hydropic vacuolation. Fish age also had a significant influence on occurrence of several hepatic lesions, but gender was rarely a significant risk factor. These relationships provide strong evidence for the involvement of environmental contaminants in the etiology of hepatic lesions in several marine bottomfish species and clearly indicate the utility of these lesions as biomarkers of contaminant-induced effects in wild fish.

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.

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.

The metabolism of benzo(a)pyrene by English sole (Parophrys vetulus): comparison between isolated hepatocytes in vitro and liver in vivo.

1. Metabolites and DNA adducts of 3H-benzo(a)pyrene (BaP) formed by isolated hepatocytes from English sole (Parophrys vetulus) in vitro were compared to those in bile and liver of sole exposed i.m. to 3H-BaP. 2. English sole liver was perfused with a collagenase solution and hepatocytes were isolated with greater than 95% viability. Determination of kinetic parameters for metabolism of 3H-BaP showed a Km of 29 +/- 10 microM and an apparent Vmax of 1300 pmol BaP metabolized/10(6) cells per h. 3. Analysis of medium from hepatocyte cultures and bile by ion-pair h.p.l.c. showed significant amounts of radioactivity in regions where glucuronide and glutathione conjugates of BaP metabolites elute. No sulphate conjugates of BaP metabolites were detected. The major unconjugated metabolite formed by hepatocytes was the BaP-9,10-dihydrodiol. 4. Hydrolysis of glucuronide conjugates by beta-glucuronidase and reversed-phase h.p.l.c. analysis of chloroform-soluble metabolites showed the presence of BaP-7,8-dihydrodiol, 1-hydroxyBaP and 3-hydroxyBaP. The identities of these metabolites were confirmed by comparing their fluorescence spectra with those of standard BaP metabolites. 5. Analysis by 32P-postlabelling of the BaP-DNA adducts formed in isolated hepatocytes and liver revealed that major adducts detected are derived from the anti-7,8-dihydrodiol-9,10-epoxideBaP (anti-BaPDE) and syn-BaPDE. 6. Results show that the types of conjugated metabolites and BaP-DNA adducts formed in primary hepatocyte culture were similar to those in bile and liver of English sole exposed to BaP. Thus, isolated hepatocytes from English sole afford a reliable alternative to live fish for studies of the mechanisms of hepatic xenobiotic metabolism and DNA adduct formation in a species shown to be susceptible to induction of hepatocarcinogenesis by PAHs.

Hepatic xenobiotic metabolizing enzymes in two species of benthic fish showing different prevalences of contaminant-associated liver neoplasms.

English sole (Parophrys vetulus) and starry flounder (Platichthys stellatus) are closely related benthic fish which show substantial differences in prevalences of contaminant-associated hepatic neoplasms and putatively preneoplastic foci of cellular alteration when captured from estuaries containing a variety of organic chemical contaminants, including polycyclic aromatic hydrocarbons (PAH) and chlorinated hydrocarbons. Because PAH are strongly implicated as causative agents in the etiology of these lesions, several of the hepatic enzymes involved in activation and detoxication of PAH were studied in these two species. Hepatic aryl hydrocarbon hydroxylase (AHH), epoxide hydrolase (EH), and glutathione S-transferase (GST) activities were measured in animals sampled from both contaminated and reference areas. English sole, the species showing higher prevalences of contaminant-associated hepatic lesions, had higher (1- to 2-fold) hepatic activities of AHH and lower activities of EH (0.8-fold) and GST (1.8-fold) than those of starry flounder, regardless of site of capture. These results are largely consistent with increased activation and decreased detoxication of PAH by English sole in comparison to starry flounder. Both laboratory and field data suggested that the observed species differences in enzyme activities were constitutive and not related to differential exposure to contaminants. There were also substantial differences between these species with respect to expression of GST isoenzymes, in that starry flounder expressed two highly anionic GST isoenzymes which did not correspond to any GST isoenzymes expressed in English sole liver; a previous study in an elasmobranch fish showed that an anionic GST was most active toward PAH oxides. These differences in enzyme activities and isoenzyme profiles suggest a toxicological basis which may help to explain, at least in part, the differences in prevalences of contaminant-associated liver neoplasms between these two species.

The CYC8 and TUP1 proteins involved in glucose repression in Saccharomyces cerevisiae are associated in a protein complex.

Mutations of yeast CYC8 or TUP1 genes greatly reduce the degree of glucose repression of many genes and affect other regulatory pathways, including mating type. The predicted CYC8 protein contains 10 copies of the 34-amino-acid tetratricopeptide repeat unit, and the predicted TUP1 protein has six repeated regions found in the beta subunit of heterotrimeric G proteins. The absence of DNA-binding motifs and the presence of these repeated domains suggest that the CYC8 and TUP1 proteins function via protein-protein interaction with transcriptional regulatory proteins. We raised polyclonal antibodies against TrpE-CYC8 and TrpE-TUP1 fusion proteins expressed in Escherichia coli. The CYC8 and TUP1 proteins from yeast cells were detected as closely spaced doublets on Western immunoblots of sodium dodecyl sulfate-polyacrylamide gels. Western blots of nondenaturing gels revealed that both proteins are associated in a high-molecular-weight complex with an apparent size of 1,200 kDa. In extracts from delta cyc8 strains, the size of the complex is reduced to 830 kDa. The CYC8 and TUP1 proteins were coprecipitated by either antiserum, further supporting the conclusion that they are associated with each other. The complex could be reconstituted in vitro by mixing extracts from strains with complementary mutations in the CYC8 and TUP1 genes.

Hepatic activities of xenobiotic metabolizing enzymes and biliary levels of xenobiotics in English sole (Parophrys vetulus) exposed to environmental contaminants.

English sole (Parophrys vetulus) are susceptible to the development of hepatic disease, including neoplasia, as a result of environmental exposure to polycyclic aromatic hydrocarbons (PAHs). The metabolism of PAHs, believed to be an essential factor in the development of neoplasia, has received considerable study in English sole, except that xenobiotic metabolizing enzymes (XMEs) have not been well-studied in this species. In the present work, the activities of hepatic aryl hydrocarbon hydroxylase (AHH), glutathione-S-transferase (GST), and epoxide hydrolase (EH) were measured in English sole exposed to several organic xenobiotics. These studies included an examination of the effects of captivity, the short-term responses of hepatic XME activities to several xenobiotic compounds, and detailed studies of the time- and dose-responses of hepatic XME activities to both a representative carcinogenic PAH (benzo[a]pyrene) and to a complex mixture of contaminants extracted from a sediment collected from a polluted area of Puget Sound, WA. Additionally, during the captivity and time- and dose-response studies, the levels of fluorescent aromatic compounds (FACs) were measured in the bile of the fish, both to provide an estimation of contaminant exposure and to evaluate the time- and dose-responses of this measure. The results of the captivity studies showed that the levels of FACs in bile were most affected by captivity, primarily as a result of changes in feeding status. The results of the exposure studies showed that xenobiotic metabolism, as reflected in hepatic activities of XMEs and levels of FACs in the bile, is altered by exposure to environmental contaminants. Whereas hepatic AHH activity could be rapidly and substantially increased by such exposure, activities of GST and EH were not affected, even up to 42 days after exposure. Moreover, because fish were exposed to a wide range of doses of chemicals or mixtures of chemicals which are known to be present in contaminated estuaries, and the responses of the hepatic AHH system and the levels of FACs in bile were measured at several time periods after exposure, the results provided substantial validation for the use of these two measures as bioindicators of exposure to environmental contamination in benthic fish.