Characterization of the liver cytochrome P450 in the marine fish Mugil cephalus and effects of some hydrocarbons on the mixed-function oxidase components

The marine fish Mugil cephalus was caught in the Bay of Marseilles (France) in 1985 and the liver microsomal cytochrome P₄₅₀ was identified at a concentration of about 0.230 nmol mg^-1 protein. Optimal assay conditions were established to measure two mixed-function cytochrome P₄₅₀-dependent oxidases: aniline hydroxylase and p-nitroanisole O-demethylase. Induction of cytochrome P₄₅₀ occurred after exposure of mullet to an aromatic hydrocarbon and a water-soluble petroleum fraction. The liver cytochrome P₄₅₀ was solubilized and purified. The monomer molecular weight of partially purified cytochrome P₄₅₀, determined by sodium dodecyl-sulfate-gel electrophoresis, was 51 000.     

Studies on cytochrome P450 in Mytilus galloprovincialis: induction by Na-phenobarbital and ability to biotransform xenobiotics

Mytilus galloprovincialis has a hepatopancreatic monooxygenase activity cytochrome P₄₅₀-dependent. The present paper studies the induction of this activity in the hepatopancreatic microsomes after treatment of M. galloprovincialis with Na-phenobarbital. Mussels were collected from the Gulf of La Spezia in 1985. We measured the increase in levels of cytochrome P₄₅₀, b₅, and P₄₂₀ and the 7-ethoxycoumarin deethylase activity. Furthermore, the fraction of mouse liver was replaced by the correspondent mussels' hepatopancreatic fraction in the yeast genotoxicity test to determine the ability of mussels to biotransform cyclophosphamide and styrene oxide. The results suggest that edible mussels are capable of detoxicating styrene oxide but not of bioactivating cyclophosphamide.     

Preliminary data on PAH metabolism in the marine mussel Mytilus galloprovincialis from Arcachon Bay,France

Oxidation of polycyclic aromatic hydrocarbons (PAH) and epoxide-conjugation capacities were examined from mussels, Mytilus galloprovincialis, collected from a clean site at the entrance to Arcachon Bay (south-west France) in 1984, using subcellular preparations from whole bodies and digestive glands. The existence of low-cytochrome P-450-dependent oxygenase activities [cytochrome P-450 content, NADPH cytochrome c reductase and benzopyrene monooxygenase (BaPMO) activities] was confirmed, as well as the presence of a comparatively important capacity for epoxide conjugation [epoxide hydrolase (EH), glutathione S epoxide-transferase (GST) activities and total cytosolic-glutathione content]. The optimal incubation temperatures for the individual in vitro enzyme activities in the mussel (25°C for BaPMO, 31°C for EH and GST) were lower than that required for optimal mammalian enzyme activities. Except for cytochrome P-450 content, there was no significant difference in enzyme activities recorded in whole-body and digestive gland fractions.     

Chronic inhalation effects of trichloroethylene on hepatic microsomal mixed function oxidase system in rats

Inhalation effects of trichloroethylene, at levels of 0, 200 and 600 ppm for a period of 4 and 12 weeks, on the contents of hepatic microsomal protein, cytochrome P₄₅₀, cytochrome b₅, and on the activities of NADPH cytochrome C reductase, aminopyrine N‐demethylase and aniline hydroxylase were investigated. Body weight remained unaltered at 200 ppm level, whereas at 600 ppm level a significant decrease in body weight was observed. Liver weight showed increases at an exposure level of 200 ppm by the end of 4 and 12 weeks. Relative liver weight (% body weight) showed an increase at 200 and 600 ppm level by the end of week 4, whereas, after 12 weeks increase was observed only in animals exposed to the 600 ppm level. Hepatic microsomal protein was decreased by the end of 4 weeks at both the exposure levels. Cytochrome P₄₅₀ and cytochrome b, contents were increased at the 200ppm level after 4 and 12 weeks. NADPH cytochrome C reductase activity showed a marked increase at both the 200 and 600 ppm levels after 4 and 12 weeks. Aminopyrine N‐demethylase activity was increased at the 200 ppm level and decreased at the 600 ppm level by the end of 4 weeks, whereas, at the end of 12 weeks the aminopyrine N‐demethylase activity was near control values. Aniline hydroxylase activity was increased at the 200 ppm level after 4 and 12 weeks. Similarly, at the 600 ppm level aniline hydroxylase activity was increased after 12 weeks.     

Mutagenicity of polyaromatic hydrocarbons by chemical models for cytochrome P450 in Ames assay

DNA damage is an important step in carcinogenesis. The Ames assay is a short-term screening of carcinogens that induce DNA damage. Most carcinogens require enzymatic activation through oxidation by cytochrome P450 (CYP450) in the presence of S9 mix. A combination of iron (Fe)(III) porphyrin and an oxidant is also able to oxidize compounds as an alternative metabolic pathway to CYP450. Previously it was reported that a chemical model containing a water-soluble 5,10,15,20-tetrakis(1-methylpyridinium4-yl)porphyrinatoiron(III) chloride (4-MPy) and tert-butyl hydroperoxide (t-BuOOH) activated aromatic amines and amides. In this study, a chemical model composed of an Fe porphyrin, water-insoluble 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride (F₅P) or water-soluble 4-MPy was optimized with an oxidant – t-BuOOH, magnesium monoperoxyphthalate (MPPT), or iodosylbenzene (PhIO). Subsequently the mutagenicity of benzo[a]pyrene (B[a]P) and chrysene in Salmonella typhimurium TA strains was compared. B[a]P was activated by a combination of F₅P or 4-MPy plus MPPT or PhIO in S. typhimurium TA1538. The B[a]P-induced mutagenicity with F₅P plus oxidant was higher than 4-MPy plus oxidant. Mutagenicity of chrysene, a tetracyclic aromatic hydrocarbon, was not detected in the presence of F₅P/PhIO in S. typhimurium TA98, but was activated in the presence of F₅P/MPPT. The F₅P/MPPT activated other polyaromatic hydrocarbons (PAH) in the S. typhimurium TA98 assay including dibenz[a,c]anthracene, dibenz[a,h]anthracene, 3-methylcholanthrene, and benzo[a]anthracene. The results indicated that the F₅P/MPPT was the most efficient model for detecting PAH-induced mutagenicity in the Ames assay.     

Cytochrome P-450 monooxygenase system and benzo(a)pyrene metabolism in echinoderms

A comparative study on mixed-function oxygenase (MFO) systems was carried out on four echinoderm species: the asteroidsAsterias rubens andMarthasterias glacialis, a holothurianHolothuria forskali and an echinoidEchinus esculentus. Cytochromes P-450 andb ₅ and the MFO system-associated NADH-ferricyanide reductases NADH-cytochromec reductase, NADPH-cytochromec reductase and benzo(a)pyrene (BP) hydroxylase activities were present in microsomal fractions of pyloric caeca ofA rubens andM. glacialis and in the haemal plexus ofH. forskali. In contrast, cytochrome P-450 and BP hydroxylase activity were not detectable in the gonads ofE. esculentus. The tissue and subcellular distribution of the MFO system was studied inA. rubens. MFO system components were found in the stomachs and gonads, although detection of cytochrome P-450 in the latter tissue was difficult. Sex-related differences were not significant. The contents of the MFO system components and BP hydroxylase activities were highest in the microsomal (100 000 ×g) fractions, but MFO system components were also found in the mitochondrial (12 000 ×g) and cytosolic fractions. The BP hydroxylase activity in pyloric caeca microsomes ofA. rubens was NADPH-dependent and was inhibited by several agents known to be inhibitors of vertebrate cytochromes P-450. In the former respect, the characteristics of the MFO system were more like those of vertebrates and crustaceans than that of molluscs.     

Saturated and unsaturated hydrocarbons in marine benthic algae

Saturated and olefinic hydrocarbons were determined in 24 species of green, brown and red benthic marine algae from the Cape Cod area (Massachusetts, USA). Among the saturated hydrocarbons, n-pentadecane predominates in the brown and n-heptadecane in the red algae. A C₁₇ alkyleyclopropane has been identified tentatively in Ulvalactuca and Enteromorpha compressa, two species of green algae. Mono-and diolefinic C₁₅ and C₁₇ hydrocarbons are common. The structures of several new C₁₇, C₁₉ and C₂₁ mono-to hexaolefins have been elucidated by gas chromatography, mass spectrometry and ozonolysis. In fruiting Ascophyllum nodosum, the polyunsaturated hydrocarbons carbons occur exclusively in the reproductive structures. The rest of the plant contains n-alkanes from C₁₅ to C₂₁. A link between the reproductive chemistry of benthic and planktonic algae and their olefin content is suggested. An intriguing speculation is based on Paffenhöfer's (1970) observation that the sex ratio of laboratory reared Calanus helgolandicus depends upon the species of algae fed to the nauplii. The percentage of males produced correlates with our analyses of heneicosahexaene in the algal food. Our analyses of the hydrocarbons in benthic marine algae from coastal environments should aid studies of the coastal food web and should enable us to distinguish between hydrocarbon pollutants and the natural hydrocarbon background in inshore waters.Contribution No. 2582 of the Woods Hole Oceanographic Institution; and Contribution No. 227, Systematics-Ecology Program.     

Hydrocarbons in benthic algae from the eastern Gulf of Mexico

Thirty-six samples of benthic algae were collected from the continental shelf along the eastern Gulf of Mexico. The algae contain 82.8±143 ppm aliphatic hydrocarbons by dry weight and 11.8±22.7 ppm aromatic and polyolefinic hydrocarbons. The aliphatic constituents of red algae are composed almost entirely of n-C₁₇ (70 to 95% of aliphatic weight); green algae have varying amounts of n-alkanes in the range of n-C₁₅ to n-C₁₉, with homologous series of odd carbon number n-alkenes. Phytadienes occur as high as 740 ppm dry weight in the green algae and do not appear to be related to time or place of collection or to presence of any petroleum pollutants. The single brown algal species contains predominantly n-C₁₅. About 30% of the samples have hydrocarbons resembling degraded petroleum residues, but no petroleum pollution of recent origin was detected in any specimen.     

Benzo[a]pyrene oxidation and microsomal enzyme activity in the mussel (Mytilus edulis) and other bivalve mollusc species from the Western North Atlantic

Analysis of subcellular fractions revealed a complement of microsomal electron transport components including reductases and heme proteins in several organs of the three bivalve species Mytilus edulis, Macrocallista maculata and Area zebra. Dithionite difference spectroscopy of CO-treated microsomes yielded spectra typical of cytochrome P-450 in digestive gland and gill, with absorption maxima at 450 nm. A time-dependent reduction of cytochrome P-450 was also observed. The levels of these components and rates of microsomal benzo[a]pyrene (BP) metabolism were highest in the digestive gland, and were very similar between species. In M. edulis there was a suggested seasonal variation in BP metabolism but no population differences in this activity or in levels of other components. Digestive gland microsomal metabolites of BP identified by HPLC retention and UV spectroscopy included BP-1.6-quinone, BP-3,6-quinone and BP-6,12-quinone, which comprised 65% of the total metabolites, and dihydrodiols and phenols, the latter products consistent with cytochrome P-450 monooxygenation and expoxide hydrolase function. However, the inconsistent dependence of BP metabolism on NADPH, and inconsistent inhibition by CO suggest that catalyst(s) additional to cytochrome P-450 may be acting in BP metabolism. Based on these results and the prominent quinone formation, we speculate that peroxidative mechanism(s) may be involved. The role of peroxidative as well as well as monooxygenase reactions in the in-vivo disposition and effects of foreign chemicals in bivalves, and also the major function of cytochrome P-450 in these bivalves, remain to be established.Some of these results have appeared in preliminary form; J. J. Stegeman, Sea Grant Annual Report, Words Hole Oceanographic Institution, p 15, 1981     

(<Emphasis Type="Italic">Z</Emphasis>)-9-Pentacosene − contact sex pheromone of the locust borer, <Emphasis Type="Italic">Megacyllene robiniae</Emphasis>

Summary. Male locust borers, Megacyllene robiniae (Förster), responded to females only after contacting them with their antennae, indicating that mate recognition was mediated by a contact sex pheromone. GC-MS analyses of whole-body extracts of males and females determined that the profiles of compounds in the extracts were qualitatively similar, but differed considerably in the ratios of compounds between sexes. Biological activities of reconstructed blends of the most abundant straight-chain (nC₂₃, nC₂₄, nC₂₅, nC₂₆), methyl-branched (3me-C₂₃, 3me-C₂₅), and unsaturated (Z9:C₂₃, Z9:C₂₅, Z9:C₂₇ compounds in extracts from females were assessed in arena bioassays, assessing four distinct steps in the mating behavior sequence of males (orientation, arrestment, body alignment, mounting and attempting to couple the genitalia). Males were unresponsive to freeze-killed, solventwashed females treated with blends of straight-chain and methyl-branched alkanes, but responded strongly to females treated with the blend of alkenes. Further trials determined that the complete sequence of mating behaviors, up to and including coupling the genitalia, was elicited by Z9:C₂₅ alone. Z9:C₂₅ comprised 16.4 ± 1.3% of the total hydrocarbons in whole-body hexane extracts of females and was co-dominant with two other hydrocarbons that were not active. In contrast, in solid phase microextraction (SPME) wipe samples from several areas of the cuticle, Z9:C₂₅ appeared as the single dominant peak, comprising 34.6 – 37.8% of the sampled hydrocarbons. Our data indicate that Z9:C₂₅ is a contact sex pheromone of M. robiniae, being the most abundant hydrocarbon on the surface of the cuticular wax layer of females where it is readily accessible to the antennae of males.     

Effect of nutrient enrichment on growth of the eelgrass Zostera marina in the Chesapeake Bay,Virginia, USA

The addition of two commerical fertilizers, one 5% NH₄NO₃, 10% P₂O₅, 10% K₂O, and the other 10% NH₄NO₃, 10% P₂O₅, 10% K₂O, ahd a dramatic effect on the growth of Zostera marina in the Chesapeake Bay. There was a significant increase in the length, biomass and total number of turions of fertilized plots compared with controls during a 2 to 3 month period. Data from this short-term field experiment suggest that Z. marina beds in the Chesapeake Bay are nutrient-limited, that the grwoth form of Z. marina may be related to the sediment nutrient supply, and that Z. marina may competitively exclude Ruppia maritima by light-shading.     

Evidence for the stratification of hydrocarbons in the epicuticular wax layer of female Megacyllene robiniae (Coleoptera: Cerambycidae)

Contact pheromones mediate mate recognition and play important roles in mating systems of longhorned beetles (Coleoptera: Cerambycidae). One common bioassay of contact chemoreception in cerambycids involves presenting a freeze-killed female to a male in a Petri dish arena. If the male attempts to mate with the female carcass, it confirms that mate recognition signals are present and intact and behavior is not involved. Cuticular hydrocarbons are then stripped from the female with successive solvent washes, rendering her unattractive to males and also resulting in a crude extract containing the cuticular hydrocarbons. To test the bioactivity of the crude extract, the same female is then treated with the extract and presented again to the male. Males of some species, including Megacyllene robiniae (F?rster), respond less readily to reconstituted females than to those same beetles before they were solvent-extracted. In the present study, we test the hypothesis that the contact pheromone of M. robiniae, Z9:C₂₅, exists as a layer on the surface of the epicuticle. We used solid phase microextraction (SPME) to sample cuticular hydrocarbons of female beetles after they were freeze-killed, solvent washed, and treated with crude cuticular extracts. We found that extracting cuticular hydrocarbons from females and applying the resulting crude extract back onto the solvent-washed cadaver scrambles the wax layer and decreases the abundance of the contact pheromone presented on the surface of the insect.     

Endemism and dispersal: comparative phylogeography of three surgeonfishes across the Hawaiian Archipelago

To evaluate the hypothesis that a general correlation exists between species range size and dispersal ability, we surveyed mitochondrial cytochrome b sequence variation in three surgeonfish species with vastly different ranges: Ctenochaetus strigosus, Hawaiian endemic, N = 531; Zebrasoma flavescens, North Pacific, N = 560; Acanthurus nigrofuscus, Indo-Pacific, N = 305. Collections were made throughout the 2,500 km expanse of the Hawaiian Archipelago and adjacent Johnston Atoll. Analyses of molecular variance demonstrate that all three species are capable of maintaining population connectivity on a scale of thousands of km (all species global Φ_ST = NS). However, rank order comparison of pairwise Φ_ST results and Exact test P-values revealed modest but significantly different patterns of gene flow among the three species surveyed, with the degree of genetic structure increasing as range size decreases (P = 0.001). These results are consistent with mtDNA surveys of four additional Hawaiian reef fauna in which a wide-spread Indo-Pacific species exhibited genetic homogeneity across the archipelago, while three endemics had significant population subdivision over the same range. Taken together, these seven cases invoke the hypothesis that Hawaii’s endemic reef fishes evolved from species with reduced dispersal ability that, after initial colonization, could not maintain contact with parent populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hydrocarbons in the antennal gland secretion of female European beewolves, Philanthus triangulum (Hymenoptera, Crabronidae)

The antennae of female European beewolves (Philanthus triangulum, Hymenoptera: Crabronidae) contain unique gland reservoirs that are inhabited by the symbiotic bacterium ‘Candidatus Streptomyces philanthi’. The reservoir content is secreted into the subterranean brood cells where it serves a dual function: (1) It provides the larvae with spatial information about the orientation of cocoon spinning, and (2) it supplies the larva with the symbiotic bacteria that are needed for the protection of the cocoon against pathogenic microorganisms. We analyzed the hydrocarbon composition of the antennal gland secretion (AGS) by solid phase micro-extraction–gas chromatography–mass spectrometry. We detected 28 substances, the majority of which were saturated and unsaturated hydrocarbons, with minor amounts of some branched alkanes, two ketones, and three unknown substances. There was a strong dimorphism in the chemical composition of the AGS among beewolf females. The two morphs had either (Z)-9-pentacosene (C₂₅-type) or (Z)-9-heptacosene (C₂₇-type) as the main component, and they differed significantly in the relative amounts of most of the other AGS substances. The hydrocarbon composition of the AGS is very similar to that of the postpharyngeal gland of female beewolves, suggesting that the hydrocarbons are universally used for different purposes and transported into the gland from the hemolymph. The putative function of the hydrocarbons in the AGS is discussed.     

Hydrocarbons characterization in coastal sediments of the Argentine Patagonia using the nuclear magnetic resonance (NMR) spectroscopy

Hydrocarbons in Patagonian sediments were analyzed to evaluate their biogenic or petrogenic origins. Structural determination by ¹H-NMR spectrometry and principal component analysis (PCA) show that the first two principal components accounted for more than 75.7% of the variance. The results indicate that biogenic hydrocarbons have mainly linear hydrocarbon chains and polar groups (high H₂, H₄, H₅, and H_COOH), while petrogenic hydrocarbons show more branched chains and mainly mono-aromatic components, e.g., crude oil (high H₁, H_1A, and H₃) or mainly poly-aromatic components (high H₄, and H_2A) in weathering oil.     

Low levels of global genetic differentiation and population expansion in the deep-sea teleost <Emphasis Type="Italic">Hoplostethus atlanticus</Emphasis> revealed by mitochondrial DNA sequences

The orange roughy Hoplostethus atlanticus is a well-known commercial species with a global distribution. There is no consensus about levels of connectivity among populations despite a range of techniques having been applied. We used cytochrome c oxidase subunit I (COI) and cytochrome b sequences to study genetic connectivity at a global scale. Pairwise Φ_ST analyses revealed a lack of significant differentiation among samples from New Zealand, Australia, Namibia, and Chile. However, low but significant differentiation (Φ_ST = 0.02–0.13, P < 0.05) was found between two Northeast Atlantic sites and all the other sites with COI. AMOVA and the haplotype genealogy confirmed these results. The prevalent lack of genetic differentiation is probably due to active adult dispersal under the stepping-stone model. Demographic analyses suggested the occurrence of two expansion events during the Pleistocene period.     

Respiration and nitrogen excretion by the squid Loligo forbesi

The amphipod Corophium volutator (Pallas) of the Bay of Fundy is a major food resource for migrating shorebirds. A relatively low total lipid content (ca 1.7%) was found in summer and fall samples of 1986. Analysis of triacylglycerides which would normally reflect an animal diet through fatty acid input, revealed high proportions of fatty acids characteristic of algae. C. volutator contains hydrocarbons (0.2% of total lipids) with a unimodal distribution in the range C₁₈ to C₃₅ with maximum at C₂₅, and a weak odd-carbon preference (carbon preference index, C.P.I. =1.22). This hydrocarbon pattern shown by C. volutator seems to be biogenic, but due to both a weak odd-carbon preference and a significant content of the isoprenoid phytane, the presence of petroleum hydrocarbons cannot be ruled out. Comparison of the hydrocarbon composition be ruled out. Comparison of the hydrocarbon composition found in C. volutator with hydrocarbons described in the literature for its most likely sources of food suggests that highly degraded material and aged detritus are components of the diet of this deposit feeder, and as important as the local diatom population. No organohalogen pesticides (DDT, dichlorodiphenyl-trichloroethane, or derivatives) or polychlorinated biphenyls (PCBs) were detected in the lipid extracts of C. volutator. Absence of these materials and low hydrocarbon levels explain the continued successful exploitation of this food resource by migrating shorebirds.     

Pollutant effects on the mixed function oxygenase (MFO) and reproductive systems of the marine polychaeteNereis virens

The cytochrome P-450 dependent mixed-function oxygenase (MFO) system ofNereis virens, found in the endoplasmic reticulum of the intestinal epithelium, has similar components and cofactor requirements when compared to other invertebrates. Worms exposed to food containing benzo(a)pyrene or a mixture of polychlorinated biphenyls (Aroclor 1254) had significantly higher MFO activity and cytochrome P-450 content than unexposed ones.N. virens from a site in Porland, Maine, USA, which was heavily contaminated with oil, and nearby clean reference sites were studied. The weight, MFO activity and presence or absence of gametes were determined. The MFO activity in worms from the oil-contaminated site was approximately six times that of worms from the reference sites; worms from the reference areas were six times larger than oil-contaminated worms. Worms from the oil-contaminated area lacked or had undeveloped gametes in spring, summer and fall. In contrast, many of the worms collected from the reference sites at the same times had fully developed gametes.     

Hydrocarbons contamination and microbial degradation in mangrove sediments of the Niger Delta region (Nigeria)

The distribution of aliphatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs), and their degradability by bacteria in epipelic and benthic sediments from Qua Iboe Estuary mangrove ecosystem and associated creeks were investigated. The research findings revealed that total aliphatic hydrocarbons in sediments ranged from 16.82 mg·kg ^?1 to 210 mg kg ^?1, wheras total PAHs ranged from 6.30 to 35.55 mg·kg ^?1 dry weight of sediment. Low molecular mass (i.e. the 2–3-ring) PAHs were predominant in almost all the sampling points, whereas the higher molecular masses (4-, 5- and 6-ring PAHs) had the lowest concentrations. In general, the sediment samples ES ₂ (39.7%), ES ₃ (24.8%), BS ₁ (46.7%), BS ₂ (49.9%) and BS ₃ (44.2%) showed<50% contributions of Σ combustion–derived PAH (COMP-PAH) concentration to the Σ PAH concentrations, whereas ES ₁ (57%) contained>50% of COMP-PAHs. Our results have also shown that many mangrove bacteria have strong capacity to utilise Qua Iboe Light (QL) crude oil as the sole source of carbon and energy, while lower number of bacterial species including Bacillus sp., Micrococcus sp., Pseudomonas aeruginosa, Alcaligenes sp. and Flavobacterium sp. exhibited detectable PAHs degradability; and as such may serve as efficient degraders of QL crude oil contamination of mangrove ecosystem.     

An assessment of the air pollution data from two monitoring stations in Kuwait

This article assesses the air pollution data from two monitoring stations in Kuwait. The measurements cover major pollutants, i.e., CO, CO₂, methanated and non-methanated hydrocarbons, NO _x , SO₂, O₃, and particulate matter (PM10). The data also includes meteorological parameters, i.e., solar intensity, temperature, wind speed, and wind direction, and has been collected over a period 4 years, from 2001 to 2004. Data analysis includes the assessment of annual hourly averages and 1-h maxima. Typical pollutant concentration trends, similar to those previously reported for Kuwait and for other locations around the world, are observed except for particulate matter measurements, which have higher values because of proximity to the desert. Emissions of nitrogen oxides show a consistent increase over the years. This is caused by the increase in the number of motor vehicles and the expansion in power generation and industrial activities. The data collected is a subset of the air quality criteria, as defined by the US EPA (United States Environmental Protection Agency).