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Objectives Chronic stress and burnout are key health issues for office workers that may contribute to a myriad of poor health outcomes. The presence of natural elements may improve psychological well-being in workers but the number of existing studies is relatively low, and more longitudinal research is specifically needed to assess how characteristics of workers’ day-to-day environments may impact mental health outcomes like affect, depression and stress. This report outlines a multi-study investigation of workers at Amazon, a multinational e-commerce company based in Seattle, Washington, USA, and the mental health benefits associated with exposure to nature. Methods In Study 1, participants (n = 153) responded to a cross-sectional survey that assessed the association of self-reported visitation to an indoor company greenspace with psychological well-being including symptoms of depression, anxiety, positive and negative affect, and stress. In Study 2, a subset of participants from Study 1 (n = 33) completed multiple surveys in a 2-week period that assessed the association of the naturalness of their current environments with their state levels of psychological well-being. Results We found contact with more natural outdoor environments was significantly associated with reduced state anxiety, after adjusting for activity type, location, and participants’ trait levels of nature relatedness. Conclusions Findings demonstrate that nature contact in everyday life is significantly associated with decreased levels of state anxiety. More research is needed to investigate the role of nature contact as a potential intervention in the workplace for improved mental health.

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 shortterm 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. r 1999 Academic Press

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.

Liver neoplasms are rarely detected in young wild fish. Therefore, other lesions occurring early in the histogenesis of hepatic neoplasia need to be considered as biomarkers of chemical contaminant exposure effects in monitoring studies, especially where adult fish are not available. Moreover, exposure effects may be more reliably assessed in younger fish that have not yet migrated extensively. Accordingly, livers of subadult English sole were histologically examined from nine sites in Puget Sound, WA and the same fish were assessed for contaminant exposure by measurement of fluorescent aromatic compounds (FACs) in bile, hepatic levels of polychlorinated biphenyls (PCBs) and cytochrome P4501A (CYP1A) as catalytic activity of aryl hydrocarbon hydroxylase (AHH), and Hydrophobic DNA adducts in liver by 32P-postlabelling. Although neoplasms were rare, higher prevalences of preneoplastic, regenerative, and unique degenerative/ necrotic lesions were detected in sole from contaminated sites. Prevalences of these early histopathologic biomarkers were significantly higher at the more contaminated sites, and concentration of mean biliary FACs at each capture site was a significant risk factor for most lesions, as determined by stepwise logistic regression. By this statistical method, we also demonstrated that several measures of bioaccumulation or biochemical response to contaminants were significant and near-significant risk factors for prevalences of most hepatic lesion categories. For example, mean hepatic AHH activity was a significant risk factor for prevalence of all lesion types, except neoplasms; hepatic PCB and xenobiotic-DNA adduct concentrations were significant risk factors for the most frequently detected lesion category, hepatocellular nuclear pleomorphism/megalocytic hepatosis, and the inclusive category ‘any early toxicopathic lesion’. These findings further support the utility of certain non-neoplastic liver lesions as early indicators of biological damage in subadult as well as adult fish exposed to xenobiotics in the marine environment.

Cytochemical responses in the digestive tissue of Mytilus edulis complex fed a microencapsulated mixture of PAHs (composed of phenanthrene, fluoranthene and benzo[a]pyrene) or PCBs (Aroclor 1254) were evaluated. Lysosomal membrane labilization period and lipofuscin content of digestive tissue were significantly decreased and increased, respectively, after 30 days of exposure to PAHs but not PCBs. NADPH-ferrihemoprotein reductase activity in the digestive tissue was significantly increased after 30 days exposure to both PAHs and PCBs, whereas catalase activity was significantly increased only after exposure to PCBs. Neutral lipid content and gamma-glutamyl transpeptidase activity of digestive tissue of mussels were significantly increased and decreased, respectively, after 30 days exposure to both PAHs and PCBs when compared to untreated mussels, but were not significantly different when compared to mussels treated with com oil representing the vehicle control. Exposure to PAHs induced peroxisome proliferation in the digestive tissue of M. edulis complex. The observed cytochemical responses in the digestive tissue of M. edulis complex exposed to PAHs and PCBs in the laboratory are comparable to the cytochemical responses observed in the digestive tissue of mussels collected in the field from sites contaminated with predominantly PAHs and PCBs.

Experiments with three species of infaunal invertebrates (a polychaete, Armandia brevis and amphipods, Rhepoxynius abronius and Eohaustorius washingtonianus) with different modes of feeding gave a wide range in toxic response to sediment associated tributyltin (TBT), while exhibiting consistent lethal tissue residues. These studies showed that bioaccumulation and toxicity of sediment-associated TBT were strongly controlled by the organic carbon content of the sediment, which we concluded was primarily due to its influence on interstitial water (IW) concentrations of TBT. Major differences in the response to sediment-associated TBT were found between species, which was attributed to differences in their rates of uptake and elimination of this compound. Predictions for bioaccumulation and toxicity for each species based on these toxicokinetic rates were matched closely by observed values. Based on comparisons of water-only and IW exposures (when water and sediment concentrations of TBT were in equilibrium) and predictions made with toxicokinetic rates, the major route of uptake for each of the species tested appeared to be from dissolved TBT. We determined the mean (sd) organic-carbon normalized sediment-water partition coefficient (Koc) to be approximately 25 100 (5500) for TOC values ranging from 0.3 to 1.0%, which was five times higher than the reported Kow. Additionally, we determined the dissolved organic carbon—water partition coefficient (Kdoc) to be 1652, which was three fold lower than the Kow. The results also showed that the Koc could be influenced by infaunal organisms, presumably by reducing IW concentrations below predicted values, which raises questions about the environmental relevance of sediment bioassays using these organisms and the expected IW concentrations present in field sediments.

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.

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.

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 ΣPCBs and ΣDDEs in blubber, for example, ranged from 120 to 10000 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 ΣPCBs and ΣDDEs 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 (1700 000 ± 450 000 p.p.b.), iron (320000 ± 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. Further, the results showed that the concentrations of potentially toxic chemicals in tissues were relatively low when compared with the concentrations in tissues of marine mammals feeding on higher trophic level species, such as fish. The lack of data from apparently healthy gray whales, however, limits the assessment of whether the levels of anthropogenic contaminants found in tissues may have deleterious effects on the health of gray whales.

The relationship between exposure to xenobiotic compounds and ovarian development was evaluated in prespawning female winter flounder (Pleuronectes americanus) sampled from eleven sites on the northeast coast of the United States during the 1988 and 1989 spawning seasons. Three sites were located in Boston Harbor, MA, four sites were in Raritan Bay, NJ and four sites were in nearby embayments. Sediments from these sites exhibited a wide range in concentrations of xenobiotic compounds (e.g. aromatic hydrocarbon (PAH) concentrations ranged from 20 to 50 000 ng/g dry wt. and polychlorinated biphenyl (PCB) concentrations ranged from 2 to 1400 ng/g dry wt.), with the sites in Boston Harbor and Raritan Bay the most heavily contaminated. The following parameters associated with ovarian development were measured: ovarian developmental stage, ovarian atresia, gonadosomatic index, plasma estradiol, fecundity and egg weight. Contaminant exposure was assessed by measuring concentrations of fluorescent aromatic compounds (FACs) in the bile, hepatic aryl hydrocarbon hydroxylase (AHH) activity, concentrations of PCB in liver, ovary and brain and concentrations of xenobiotic-DNA adducts in liver tissue. Additionally, liver tissue was examined histologically for the presence of suspected toxicopathic lesions. In general, indicators of contaminant exposure were elevated and prevalences of suspected toxicopathic lesions were highest in fish from sites within Boston Harbor and Raritan Bay. Hepatic AHH activity, however, was heavily influenced by the reproductive cycle and showed little correlation with other indicators of contaminant exposure. Evidence of decreased egg weight and increased atresia in fish with high tissue concentrations of PCB or high levels of FACs in bile was observed. However, contaminant exposure had no clear negative impact on gonadal recrudescence, gonadosomatic index, plasma estradiol concentrations, or fecundity in female winter flounder. These results are in contrast to results with another Pleuronectid species, English sole (Pleuronectes vetulus), which shows inhibited gonadal development and lower plasma estradiol concentrations at contaminated sites in Puget Sound, WA. The apparent difference between English sole and winter flounder in susceptibility to contaminant-induced reproductive dysfunction could be related to a number of factors, including the differences in the migratory behavior of the two species during the reproductive season. English sole reside in contaminated estuaries throughout vitellogenesis and move offshore to spawn, while winter flounder often remain offshore for extended periods during early vitellogenesis and move into contaminated estuaries prior to spawning. Because of these contrasting migration patterns, both the duration and timing of exposure to contaminants during gonadal recrudescence may differ substantially in these two species and may contribute, in part, to the observed differences in the prevalence of reproductive dysfunction in fish from contaminated estuaries.

In order to monitor the quality of coastal waters that provide habitats for living marine resources, samples of sediment and biota must be analyzed to assess the degree and distribution of anthropogenic contamination. Analytical time and costs can be greatly reduced by first employing methods that screen for contaminants before selecting samples for rigorous analyses. In this paper, we review the applications of rapid high-performance liquid chromatographic (HPLC) methods to screen for aromatic compounds in sediment, bile and tissue samples. These methods have been used to assess damage to natural resources after the Exxon Valdez oil spill. In addition, the bile screening method has also been used to evaluate contaminants in fish sampled for a national monitoring program. The rapid screening of sediment or bile provides an estimate of contaminant concentrations that can then be confirmed in selected samples by more complicated and expensive analyses by gas chromatography/3-mass spectrometry (GC-MS). Furthermore, HPLC and GC-MS chromatographic patterns from sediment and bile can provide information about the source of contamination, e.g., crude oil, diesel fuel or pyrogenic contaminants. We also discuss the important role screening methods will play in the future in assessing the quality of aquatic habitats, the safety of seafood, and other important issues related to anthropogenic contamination.

A suite of chemical and biochemical parameters was measured in three species of benthic flatfish (English sole, Parophrys vetulus; rock sole, Lepidopsetta bilineata; and starry flounder, Platichthys stellatus) sampled from up to five sites in Puget Sound, WA, USA, to assess the sensitivity of the parameters to differences in levels of contaminant exposure and the relative merit of the use of a suite of indices for assessing exposure and sublethal effects. The indices examined were hepatic levels of polychlorinated biphenyls (PCBs) and biliary fluorescent aromatic-compound concentrations, hepatic activities of aryl hydrocarbon hydroxylase and ethoxyresorufin-O-deethylase, and levels of total hepatic GSH and hydrophobic DNA-xenobiotic adducts; the last-mentioned were determined by using the 32P-postlabeling assay. The results showed that the indices examined could discriminate among sites exhibiting different degrees of chemical contamination; however, species differences in the range of response of some indices were observed. Additionally, the use of multiple indices appeared to enhance the assessment of contaminant exposure and sublethal effects.

Our studies have shown that early stages of the reproductive process in female English sole (Parophrys vetulus) are disrupted by contaminant exposure. To determine the potential for contaminants to affect later stages of the reproductive cycle, we examined the effects of maternal contaminant exposure on egg and larval viability in this species, in a field study conducted on running ripe fish collected for two consecutive years from spawning grounds. Maternal contaminant exposure was assessed by several methods. Stepwise multiple regression of the data showed that contaminant exposure was only a minor factor in determining egg and larval viability. However, because the range of contaminant exposure in the spawning fish did not encompass the high levels seen in non-spawning fish captured from contaminated areas, it can be hypothesized that female English sole exposed to substantial levels of contaminants might be at least partially excluded from the spawning population.

Field studies on ovarian development in English sole from urban and nonurban sites in Puget Sound, Washington, USA, demonstrated that animals with elevated levels of fluorescent aromatic compounds (FACs) in bile were less likely to enter vitellogenesis and had lower plasma concentrations of estradiol than female sole with low levels of contaminant exposure. Biliary FACs were positively correlated with hepatic P450 activity, which was also elevated in sole showing inhibited ovarian development. These findings suggest that contaminant exposure may disrupt vitellogenesis in female fish. Results of the field study were supported by laboratory experiments showing that pretreatment of gravid female English sole with extracts of contaminated sediment decreased levels of endogenous estradiol. A similar response was observed in rock sole and flathead sole treated with Prudhoe Bay crude oil. However, exposure of English sole to sediment extracts had little effect on the activity of hepatic-steroid-metabolizing enzymes, indicating that some mechanism other than enhanced steroid metabolism may be responsible for reductions in endogenous circulating-steroid levels. Our recent studies suggest that reductions in endogenous estradiol levels may result from depressed ovarian steroidogenesis, as contaminant-associated reductions in in-vitro ovarian estradiol production were observed in English sole, rock sole, and flathead sole. Preliminary studies on winter flounder and white croaker indicate that they may also experience reproductive impairment as a result of exposure to contaminants, but the phase of the reproductive cycle that is most severely impacted varies from species to species.

Approximately 1 year after Kuwaiti crude oil was spilled and burned during the Gulf War, scientists on board the NOAA R/V Mt Mitchell sampled sediment and biota in the Gulf to look for evidence of petroleum contamination. Subtidal sediments and fish bile were analysed for petroleum-related aromatic compounds (ACs) on board the Mt Mitchell using cost-effective and rapid high-performance liquid chromatographic (HPLC) screening methods. Parent ACs and their alkyl homologs were measured in sediments. However, because fish extensively metabolize ACs in their livers to form polar metabolites that are concentrated in bile for excretion, metabolites of ACs were measured in bile. HPLC screening analyses found ACs in many of the sediment and fish bile samples collected. The results of HPLC screening were confirmed in selected samples by gas chromatography/mass spectrometry (GC/MS) analyses that identified petroleum-related ACs (e.g., alkyl phenanthrenes or dibenzothiophenes) in sediments and their metabolites in bile.

Our studies have shown that early stages of the reproductive process in female English sole (Parophrys vetulus) are disrupted by contaminant exposure. To determine the potential for contaminants to affect later stages of the reproductive cycle, we examined the effects of maternal contaminant exposure on egg and larval viability in this species, in a field study conducted on running ripe fish collected for two consecutive years from spawning grounds. Maternal contaminant exposure was assessed by measuring levels of polychlorinated biphenyls (PCBs) in ovarian and hepatic tissue, biliary levels of fluorescent aromatic compounds (FACs), and hepatic aryl hydrocarbon hydroxylase (AHH) activities. Additionally, the presence of significant liver disease or ovarian atresia was documented, and levels of plasma estradiol were measured. Stepwise multiple regression of the data showed that contaminant exposure was only a minor factor in determining both egg and larval viability. Hepatic AHH activity showed a slight negative correlation with fertilization success, explaining only 4% of the variability. Increased levels of PCBs in ovaries of spawning female English sole were not associated with reduced reproductive success. However, because the range of contaminant exposure in the spawning fish did not encompass the high levels seen in non-spawning fish captured from contaminated areas, it can be hypothesized that female English sole exposed to substantial levels of contaminants might be at least partially excluded from the spawning population.

Aromatic hydrocarbons (AHs) are extensively metabolized into oxidized products by fish and mammals. Some metabolites are more toxic than the parent AHs; however, currently there are no methods to screen tissues of fish or other marine species for the presence of AH metabolites. In the present study, a relatively rapid, semiquantitative method that combines enzymatic hydrolysis, methylene chloride extraction and hexane/potassium hydroxide partitioning with HPLC/fluorescence analysis, was developed for the estimation of the concentrations of polar fluorescent aromatic compounds (polar FACs; e.g., AH metabolites) in fish tissues. Liver and muscle from rock sole (Lepidopsetta bilineata) exposed to Prudhoe Bay crude oil in the laboratory were analyzed. Tissues from English sole (Parophrys vetulus) sampled at two sites (one nonurban reference and one AH-contaminated) in Puget Sound, WA were also analyzed. The concentrations of polar FACs in liver and muscle of PBCO-exposed rock sole were linearly proportional to dose. Also, in both species exposed to contaminants, the concentrations of polar FACs in liver were ∼15 times as great as those in muscle. Polar FACs, however, were not detected in muscle of English sole collected at either the contaminated or reference site, suggesting that polar FACs in naturally exposed animals may be present only at low concentrations unless exposed to high levels of contaminants such as AHs. Thus, the present method can detect, estimate and rank the relative concentrations of polar FACs (e.g., AH metabolites) in tissues, such as liver and muscle of fish. The method augments our ability to assess environmental exposure to potentially toxic contaminants such as AHs.

We are currently analyzing hepatic cytochrome P4501A and associated monooxygenase activities in fish sampled in several regional and national monitoring programs, including the National Benthic Surveillance Project of NOAA's Status and Trends Program, damage assessment studies of the Exxon Valdez oil spill, and intensive surveys of specific embayments, such as Puget Sound, Washington. Thus far, apparent contaminant-related increases in the activities of cytochrome P4501A-dependent monooxygenases have been readily measured in most test species. The results presented in this paper show that, for II species of fish, there is excellent concordance between hepatic activities of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (EROD); moreover, levels of cytochrome P4501A determined by an enzyme-linked immunosorbent assay (ELISA) are also generally concordant with results from catalytic assays. The use of both a catalytic assay and immunoquantitation is recommended, because of the additional quality assurance provided by concurrent use of an immunoquantitation technique, which is desirable in large monitoring programs.

Liver neoplasms are rare in young wild fish; therefore, it is essential to consider toxicopathic liver lesions that occur earlier than neoplasms as biomarkers of contaminant exposure in studies where targeted or available fish are not adults. Also some exposure effects may be more reliably assessed in younger fish that have not yet migrated extensively. We addressed these issues by histologically examining subadult English sole, rock sole, and starry flounder captured from eight sites in Puget Sound, and by measuring fluorescent aromatic compounds (FACs) in bile and polychlorinated hiphenyls (PCBs) in liver. Neoplasms were detected rarely; higher prevalences of preneoplastic, regenerative, and degenerative lesions were detected in most species, primarily from contaminated sites, including hydropic vacuolation of hepatocytes and biliary cells in rock sole and starry flounder. These nomeoplastic lesion types are inducible in fish by exposure to various toxicants in the laboratory, and/or (in the case of hydropic vacuolation) are associated with contaminant exposure and liver neoplasia in adult wild fish. Prevalences of these ‘early’ lesions were significantly higher at the more contaminated sites, as determined by several statistical approaches, including logistic regression. Moreover, prevalences of most lesions in all three species correlated with mean bile FAC levels at the sites, and prevalences of some lesions in English sole correlated with liver PCB levels. The present findings further support the utility of certain liver lesions other than neoplasms as early indicators of biological damage in subadult as well as adult fish exposed to xenobiotics in the marine environment.

In this present study, juvenile chinook salmon were collected from a contaminated urban estuary, the Duwamish Waterway, as well as from a non-urban estuary, the Nisqually River estuary, to determine if exposure to toxic chemicals affects immunocompetency. Juvenile chinook salmon were also sampled from the two hatcheries that release salmon into these estuaries. The ability of anterior kidney (AK) and splenic (SP) leucocytes from primed and unprimed juvenile salmon from the Duwamish Waterway, Nisqually River estuary, and their respective hatcheries to produce a primary and secondary in vitro plaque-forming cell (PFC) response to the hapten, trinitrophenyl (TNP) was examined. Trinitrophenyl-keyhole limpet haemocyanin (TNP-KLH) was added in vitro to AK leucocytes or as TNP-lipopolysaccharide (TNP-LPS) to both AK and SP leucocytes. The primary AK and SP plaque-forming cell response to TNP in salmon from the estuaries or hatcheries was not significantly different. Primed AK leucocytes from salmon collected from the hatcheries and the non-urban estuary were able to produce a heightened secondary response to TNP-KLH; in contrast, primed AK leucocytes from salmon collected from the urban estuary were unable to produce a secondary PFC response to TNP-KLH. Anterior kidney leucocytes from salmon collected from all four areas were able to produce a heightened secondary PFC response to TNP-LPS. However, the AKs plaque-forming cell response generated in primed chinook salmon collected from the urban estuary to TNP-LPS was significantly lower than that produced in salmon from the hatchery. This suppressed PFC response in primed AK cells to TNP-LPS was not observed in primed AK leucocytes from salmon collected from the non-urban estuary and its hatchery. These results suggest that the cells involved in generating immunological memory to TNP-KLH and TNP-LPS were affected in salmon exposed to contaminants in the urban estuary, with cells which generate memory to TNP-KLH being more affected. The PFC responses of primed SP leucocytes from salmon collected from the Duwamish Waterway and Nisqually River estuaries, stimulated in vitro with TNP-LPS were not significantly different. The consequence of suppressed immunological memory in disease resistance of juvenile salmon is currently unknown.

1. Effects of a contaminated sediment extract on the disposition and metabolism of 3H-17β-estradiol in juvenile English sole and on plasma levels of endogenous estradiol in gravid females were examined. 2. Plasma estradiol levels were reduced in both extract-treated juvenile and gravid sole compared to controls, and in juvenile sole this decrease preceeded an increase in bile levels of 3H-17β-estradiol. 3. Extract treatment had no significant effect on types or proportions of 17β-estradiol metabolites or levels of related hepatic enzymes. 4. Thus, lower plasma estradiol levels in extract-treated sole were apparently due to alterations in physiological factors (e.g. increased bile flow) and not to enhanced metabolism of 17β-estradiol.

Vitellogenic female English sole were sampled from four areas in Puget Sound that varied in the nature and degree of chemical contamination. The fish were then injected with an analogue of Luteinizing Hormone Releasing Hormone (LHRH) in the laboratory to induce spawning. Ability to spawn, time to spawn, larval viability, and initial concentrations of plasma estradiol and vitellogenin [measured as alkaline-labile protein associated phosphate (ALP)] were assessed. In general, low initial plasma estradiol and ALP concentrations and subsequent reproductive impairment were most common in English sole from sites where levels of sediment contaminants [e.g. polycyclic aromatic hydrocarbons and polychlorinated biphenyls (PCB)] and measures of contaminant exposure in fish (hepatic PCB concentrations and aryl hydrocarbon hydroxylase activity) were highest, and where pollution-associated liver lesions in fish (i.e. neoplasms, foci of cellular alterations, specific degeneration/necrosis, and storage disorders) were most prevalent. Additionally, the time to spawn was found to be inversely correlated with initial plasma estradiol concentrations. Spawning success was found to be positively correlated with initial plasma estradiol and ALP concentrations as determined by logistic regression analysis; nearly 62% of the variability in the spawning response could be accounted for by these factors. Fertilization success was also found to be positively correlated with initial ALP concentrations, whereas fish captured from contaminated sites produced lower proportions of normal larvae. Overall, these findings suggest that contaminant exposure may result in poor reproductive success of female English sole. This may be related to a hormone imbalance or to slower ovarian development (non-synchronous timing) of female English sole from contaminated sites.

Peroxisome proliferators cause a rapid and coordinated transcriptional activation of genes encoding the enzymes of the peroxisomal β-oxidation pathway in rats and mice. Cis-acting peroxisome proliferator responsive elements (PPREs) have been identified in the 5′-flanking region of H2O2-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 proliferator-induced increases in peroxisome volume density and in the activity of the β-oxidation enzyme system, we have previously cloned the human ACOX gene, the first and rate-limiting enzyme of the peroxisomal β-oxidation system. We now present information on a regulatory element for the peroxisome 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 PPARα/RXRα 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 PPARα and RXRα. The functionality of this human ACOX promoter was further demonstrated by linking it to a β-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 PPARα, whereas only a basal level of promoter activity is detected in stably transfected cells lacking PPARα. 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.

A method for the determination of tetra-, tri-, di- and monobutyltin in marine sediments and in tissues from English sole was developed. The method utilized dichloromethane/tropolone extraction of the butyltins, derivatization with hexylmagnesium bromide, and a silica/alumina column cleanup prior to gas chromatographic/mass spectrometric (GC/MS) analysis. A number of quality assurance measures were incorporated in the method. Mean recoveries of the surrogate spike tripentyltin from sediment and tissue reference materials ranged from 96% to 110%. The method was applied to sediments and livers of English sole collected in Puget Sound, Washington state. Total butyltin concentrations in sediments ranged from 1000 ng/g were found in some sediments. Butyltins were also determined in livers from English sole captured at sites where sediments were contaminated with butyltins. Dibutyltin (at concentrations up to 870 ng/g dry weight as Sn) was the predominant butyltin species found in the livers.

Application of a new microtechnique is described for the derivation of acoustic properties (ultrasonic velocity, adiabatic compressibility and acoustic impedance) of lipids. The technique utilizes very small volumes (4 × 10−3 −4 × 10−6 ml) of lipids and thus overcomes the requirement for large volumes inherent in the use of conventional velocimeters. The use of the microtechnique allows acoustic properties to be included among the variety of other physical properties routinely measured in the characterization of lipids, including those isolated in small amounts from complex biological mixtures.

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.

Tritiated benzo[a]pyrene (BaP) and (+/-)-7,8-dihydroBaP (BaP 7,8-dihydrodiol) were incubated with English sole liver microsomes in the presence of salmon testes DNA. The modified deoxynucleosides were isolated by Sephadex LH-20 column chromatography and analyzed by reverse-phase high-pressure liquid chromatography (HPLC). A single, major adduct (60-68% of the total modified deoxynucleosides) was formed when either BaP or BaP 7,8-dihydrodiol was incubated with sole liver microsomes and DNA. Although other minor BaP-DNA adducts were formed, none represented greater than 3% of the total adducts. The major adduct had a retention time on HPLC identical to that of the N2-[10 beta (7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]-deoxy-guanosine (7R-anti-BPDE/trans-dG) adduct formed when anti-BPDE, the ultimate carcinogen of BaP in mammals, was incubated with DNA. Analysis of the Bay region tetrols showed that only the 7 alpha, 8 beta, 9 beta, 10 alpha-tetrahydroxy-7,8,9,10-tetrahydroBaP, a hydrolysis product of the anti-BPDE, was formed when BaP was incubated with sole liver microsomes. When BaP 7,8-dihydrodiol was used as the substrate, the 7 alpha, 8 beta, 9 beta, 10 alpha-, 7 alpha, 8 beta, 9 alpha, 10 beta-, and 7 alpha, 8 beta, 9 alpha, 10 alpha-tetrahydroxy-7,8,9,10-tetrahydroBaP's were formed, indicating the formation of both anti- and syn-BPDE. The ratio of tetrols of anti-BPDE/syn-BPDE was 2; however, the ratio of adducts of anti-BPDE/syn-BPDE was 20. Thus, the findings show that hepatic microsomes of English sole, a fish species having a high incidence of liver neoplasia in chemically contaminated estuaries, metabolized BaP and BaP 7,8-dihydrodiol stereoselectively to form predominantly the 7R-anti-BPDE/trans-dG adduct.

English sole (Parophrys vetulus) were exposed to [3H]benzo[a]pyrene (B[a]P) and [14C]naphthalene (NPH) in sediment containing 1% Prudhoe Bay crude oil (PBCO). Bioeoncentration values (pmoles of hydrocarbon equivalents in g of dry tissue/pmoles of hydrocarbon equivalents in g of sedimentassociated water (SAW)) for NPH were greater than corresponding values for B[a]P in tissues of fish exposed for 24 h. However, from 24 to 168 h of the exposure, a substamial decline (P < 0.05) in NPH-derived radioactivity and a significant (P < 0.05) increase in B[a]P-derived radioactivity occurred in most of the tissues examined. When fish were transferred for 24 h to sediment free of radioactivity and PBCO, the retention of B[a]P-derived radioactivity in the tissues of fish was considerably greater than that for NPH. Metabolites of B[a]P and NPH in sediment, SAW, liver and bile of fish were characterized by thinlayer chromatography (TLC). For liver, a two-dimensional TLC was devised to separate B[a]P and its metabolites from liver lipids. An important finding was that liver of English sole metabolized B[a]P to a far greater extent than NPH; at 24 h after the exposure, the ratio of concentrations of B[a]P to its metabolites was 1 to 49 whereas that for NPH was 6 to 1. Larger proportions of glucuronide conjugates than sulfate conjugates of both NPH and B[a]P were present in bile of English sole. Naphthalene was largely converted into a glucuronide conjugate of l,2-dihydro-l,2-dihydroxynaphthalene. A number of metabolites of B[a]P known to be toxic to mammals were detected in both liver and bile including 7,8-dihydro-7,8-dihydroxy B[a]P and its conjugates. These findings of extensive metabolism of B[a]P by fish liver very probably explain why B[a]P is usually not detected in liver of fish even when considerable concentrations of B[a]P are detected in the environment of the fish.

Metabolic activation and covalent binding of benzo[ a ]pyrene (BP) to deproteinized salmon sperm DNA by supernatant fractions (10,000 g ) of liver homogenates isolated from untreated, 3-methylcholanthrene (3-MC) or BP-treated starry flounder ( Platichthys stellatus ) and coho salmon ( Oncorhynchus kisutch ) were investigated. The influence of temperature, pH, time and concentrations of protein, BP, NADPH and DNA on covalent binding was investigated to obtain optimum conditions for in vitro binding (pmoles of BP equivalents bound/mg DNA/mg protein) of [ 3 H]BP to DNA for each of the two fish species. When the supernatant fractions from untreated starry flounder were used, the covalent binding of BP to DNA was 7.5 and 2.5 times greater than the values obtained with the supernatant fractions from untreated coho salmon or rat respectively. Treatment of both fish species with 3-MC or BP resulted in a marked (10- to 53-fold) increase in the binding. Ethyl acetate-extractable metabolites formed by fish liver supernatant fractions consisted of BP dihydrodiols (4,5-, 7,8-, and 9,10-dihydrodiols), phenols (3-OH, 9-OH, and 7-OH), quinones (3,6-, 1,6- and 6,12-Q) and BP 4,5-oxide. For both fish species, BP 9,10-dihydrodiol and BP 7,8-dihydrodiol were the major metabolites comprising as much as 48–72 per cent of the total ethyl acetate-extractable metabolites; 3-hydroxy BP was also present in significant amounts. The ratio of the non-K region dihydrodiols to phenols was significantly greater for both fish species compared to rat.

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

Controle des teneurs en tetrabutyl-, tributyl-, dibutyl et butyl stannanes dans les sediments de l'estuaire Puget sound

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.

Phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine from the melon of the porpoise Tursiops gilli were shown to contain 7.9,7.2, and 0.8 moles % of long-chain iso acids (C14-C16), respectively. The occurrence of the long-chain iso acids is an anomalous finding in the phospholipids of higher animals. Isovaleric acid was not detected in the melon phospholipids, despite the presence of large amounts of neutral lipids rich in this short-chain, branched acid. Both isovaleric and the longchain iso acids were virtually absent from the phospholipids of the post-cranial blubber. The positional distribution of the acids in phosphatidylcholine indicated that the iso acids show a strong preference for position 2. This finding is consistent with the general rule that structural modifications in the acyl chains of phospholipids are usually accommodated at position 2, rather than at position 1. The long-chain triacylglycerols (C10-C18 acyl chains) contained 31.6 moles % of iso acids which were confined to chain lengths of C12-C16. These iso acids may replace C16-C20 monoenoic acids commonly present in the lipids of other mammalian systems. Positional analyses of these glycerides revealed that, with the exception of isopentadecanoic acid, no distinct tendency existed to deposit the iso acids at position 2 in preference to positions 1 and 3. Moreover, no preference was shown for the deposition of the polyenoic acids at position 2. Such a lack of positional specificity is shown by the polyenoic acids in triacylglycerols of some marine mammals

The toxicity of a reference sediment supplemented with mixtures of seven aromatic hydrocarbons (fluoranthene, phenanthrene, benz[a]anthracene, benzo[a]pyrene (BaP), 2,6-dimethylnaphthalene (DMN), 1-methylnaphthalene, and 2-methylnaphthalene, or four chlorinated hydrocarbons (polychlorinated biphenyls (PCBs, Aroclor 1254), hexachlorobutadiene, 2,2-bis( p -chlorophenyl)-1,1,1-trichloroethane and hexachlorobenzene) was evaluated in amphipod ( Rhepoxynius abronius ) lethality bioassays. Significant mortality occurred among amphipods exposed for 10 days to a reference sediment (46% fines, 0·9% total organic carbon) supplemented with a mixture of chlorinated hydrocarbons at concentrations (0·05–1·0 μg g −1 dry wt) approximating those measured in contaminated sediments of Puget Sound, WA, as well as at concentrations five times as great (5 ×). In contrast, sediments supplemented with the aromatic hydrocarbon mixture were toxic only at the higher (5 ×) concentrations (0·5–15·0 μg g −1 dry wt). Sediment concentrations of the mixture components and their bioavailability to amphipods were assessed using radiolabeled PCBs, DMN and BaP. Measurement of radioactivity incorporated by R. abronius after the 10-day exposure period indicated a dose-related uptake of xenobiotic compounds from both the chlorinated and aromatic hydrocarbon-supplemented sediments. Dimethylnaphthalene and the metabolically refractive PCBs were bioaccumulated to a greater extent than BaP, which is known to be metabolized extensively by R. abronius .

English sole ( Parophrys vetulus ) were exposed to environmentally realistic levels of sediment-associated 3 H-benzo[a]pyrene (3 μg BaP/g sediment, dry weight) and 14 C-Aroclor 1254 (1 μg PCBs/g), separately and together, for up to 10 days. BaP and its metabolites in tissues reached steady-state concentrations by the first day of the exposure, whereas PCBs did not approach steady-state concentrations until the tenth day of exposure. Simultaneous exposure of sole to BaP and PCBs, relative to separate exposure to the xenobiotics, significantly increased the concentrations of BaP-derived radioactivity and decreased the concentrations of PCB-derived radioactivity in some tissues and bile. Accumulation of PCB-derived radioactivity, estimated as the burden in tissues, was significantly greater (4- to 13-fold) than that of BaP-derived radioactivity throughout the experiments and regardless of the type of exposure. The rank of the concentrations of PCB-derived radioactivity in tissues and bile was: bile ∼ liver > brain > kidney ∼ gill > skin ∼ blood ∼ muscle and for BaP-derived radioactivity the order was: bile > liver > gill > kidney > skin ∼ blood > muscle > brain. BaP-derived radioactivity in liver and bile was present primary (85–99%) as metabolites, whereas PCB-derived radioactivity was present equally as parent compounds and metabolites in bile and primarily (98 %) as parent compounds in liver. Hydrolysis of bile from PCB/BaP-exposed sole with β-glucuronidase/arylsulfatase released 35 % of the BaP-derived radioactivity and 32 % of the PCB-derived radioactivity as primary metabolites. A much higher proportion of the BaP-derived radioactivity (64 %) than the PCB-derived radioactivity (13 %) in bile was unaffected by the enzyme treatment, indicating differences in the conjugation of the primary metabolites of PCBs and BaP. The results suggest that in contaminated environments the tissue to sediment concentration ratios for PCBs would exceed that for BaP and its metabolites; however, BaP would be continually absorbed and metabolized by sole to potentially carcinogenic and mutagenic compounds. In addition, the tissue levels of these toxic compounds may be increased by simultaneous exposure to PCBs.

Physical properties (e.g. specific gravity, adiabatic compressibility and sound velocity) of lipids isolated from tissues from contiguous areas of the fatty melon of an echo-locating porpoise (Delphinus delphis) were determined to elucidate relations between lipid composition and structure, and sound transmission in the head. Lipid content varied greatly within the melon (13.6-77.6% of the tissue weight) and triacylglycerols (80-100%) were the major lipid components. This lipid class was composed of diisovaleroylglycerides (triacylglycerols containing two isovaleroyl moieties and a long-chain acyl moiety), monoisovaleroyldiacylglycerols and triacylglycerols consisting of long-chain acids. The lipid-rich (greater than 45%) areas in the melon contained a high proportion (greater than 45% of total triacylglycerols) of diisovaleroylglycerides. There were gradations of sound velocities within the melon; the lowest sound velocities were associated with high concentrations of diisovaleroylglycerides (less than 1400 m/s) and the highest with high concentrations of long-chain triacylglycerols. Assuming an average sound frequency of 75 kHz, and considering dimensions of melon (path length and width of 12-14 cm and 5 cm, respectively), a forward radiating lobe of 15-25 degrees is produced. Thus, the deposition of lipids of different acoustic properties in a three-dimensional matrix within the porpoise melon results in a lens for the projection of sound into the marine environment.

The whole brain of a porpoise (Delphinus delphis) comprised 23.1 wt% of phospholipids on a dry weight basis. Ethanolamine phosphoglycerides (36.6 wt%), choline phosphoglycerides (27.3 wt%), and serine phosphoglycerides (16.9 wt%) were the major components of the phospholipids. A unique feature of the data was the occurrence of large amounts of isovaleric acid in choline phosphoglucerides (28.1 mol%) and ethanolamine phosphoglycerides (6.4 mol%), together with 11.6 and 15.2 mol% of long-chain (C11--C16) iso-acids, respectively. Interestingly, serine phosphoglycerides did not contain detectable amounts of isovaleric acid although trace amounts of long-chain iso-acids were present. No previous evidence exists to show that appreciable amounts of a short-chain acid can be accommodated in animal phospholipids. The occurrence of isovaleric acid in the principal phosphoglycerides of the porpoise brain elicits an interest in how such an anomalous structure is accommodated in the lipid bilayers of the neural membranes.

Benzo[a]pyrene became bound to the hepatic DNA in juvenile English sole ( Parophrys vetulus ) force fed tritiated benzo[a]pyrene. No statistically significant change was observed in the level of the binding from 16 h to 2 wk after the single exposure. Specific activities of binding were similar for both DNA and protein. Moreover, a binding index was calculated to represent the number of benzo[a]pyrene molecules bound per 106 nucleotides after administration of a theoretical dose of 1 mmole of hydrocarbon per kg body weight. The value for English sole liver DNA was of the same order of magnitude as the values reported for mouse skin and mammary gland in which benzo[a]pyrene is carcinogenic.

Rainbow trout (Salmo gairdneri) held at 7 degrees and 16 degrees were given Aroclor 1254 (PCB) (10 mg/kg body wt) via intraperitoneal injections. The binding of [3H]benzo[a]pyrene (BaP) to deproteinized salmon sperm DNA was assayed (pmoles BaP equivalents per mg DNA per mg protein) using the post-mitochondrial supernatant (S 10) fractions from livers of fish at 24-168 hr after the PCB exposure. Liver enzymes from the untreated fish acclimated at 7 degrees yielded an average binding value (0.37 +/- 0.17) which was significantly greater (P less than 0.05) than the value (0.07 +/- 0.03) for untreated fish at 16 degrees. Liver supernatant fractions from PCB-induced fish acclimated at 16 degrees and sampled at 24-120 hr showed a substantial increase (P less than 0.05) in the binding (average value 2.4 +/- 1.8) compared to the value obtained with untreated fish at 16 degrees. At 24, 48 and 120 hr after the PCB treatment of fish held at 7 degrees, there was no significant increase in the binding value or extent of metabolism of BaP compared to that obtained with the untreated fish at 7 degrees. However, at 168 hr, three of four fish at 7 degrees responded to the PCB treatment with significantly (P less than 0.05) increased binding values (3.3 +/- 1.6). Chromatographic analyses of the ethyl acetate-soluble metabolites revealed that 3-hydroxy BaP and 7,8- and 9,10-dihydrodiols were the major metabolites; K-region metabolites were formed in trace amounts in untreated and PCB-treated fish at both temperatures. No marked qualitative differences were observed in metabolite profiles after the PCB treatment; however, overall metabolism of BaP and production of reactive metabolites by liver enzymes were considerably (P less than 0.05) enhanced in the PCB-induced fish at both 7 degrees and 16 degrees.

1. 1. Diisovaleroylglycerides were found to be preferentially deposited in a discrete region (acoustic window) of the external mandibular tissue of the porpoise (Tursiops tmncatus), through which sound waves arc directed to the interior mandibular waveguide. The diisovaleroylglycerides may play a significant role in modifying the properties of sound received from the aquatic environment. 2. 2. Mass spectral analyses revealed that the diisovaleroylglycerides from the acoustic tissues arc almost exclusively the 1,3-isomers. 3. 3. The previously reported active mctabolismof the isovaleroyl chains in the diisovaleroyl glycerides of acoustic tissues is discussed in relation to the occurrence of essentially a single isomeric form.

1. 1. Acyl moieties of triacyl glycerols and wax esters from acoustic (mandibular canal and melon) and blubber tissues of the porpoise ( Tursiops gilli ) were compared to investigate the possible role of lipid structures in bio-acoustics. Similar comparisons were made of alcohol moieties derived from wax esters. 2. 2. Acyl moieties of triacyl glycerols from the mandibular canal, melon and blubber contained 43.0 mole %, 50.5 mole % and 8.7 mole % of isovaleric acid, and 20.4 mole %, 21.5 mole % and 3.2 mole % saturated long-chain iso acids, respectively. 3. 3. Acyl moieties of wax esters from the mandibular canal, melon and blubber contained 43.6 mole %, 58.4 mole % and 18.9 mole % isovaleric acid, and 17.5 mole %, 21.0 mole % and 10.5 mole % saturated long-chain iso acids. The alcohols of wax esters from the mandibular canal, melon and blubber comprised 47.5 mole %, 61.4 mole % and 18.8 mole % saturated iso alcohols, respectively. Wax esters from acoustic tissues contained 2–3 mole % isopentanol. This alcohol was not detected in wax esters from blubber. 4. 4. Triacyl glycerols and wax esters from the blubber contained relatively high percentages of straight-chain unsaturated acids and alcohols. 5. 5. The data reveal that acoustic tissues deposit saturated branched acids and alcohols at the expense of straight-chain unsaturated acids and alcohols common to most terrestrial and aquatic species.