Pyrene Monooxygenase Activity Research Articles

Biomarkers employment in planktonic copepods for early management and conservation of aquatic ecosystems: The case of the ‘Capo Peloro’ lakes (southern Italy)

Transitional water ecosystems can be exposed to high levels of anthropic pressure. The main aim of this study was to present the ‘Capo Peloro’ lakes (southern Italy) case study, which highlights the advantageous use of ecotoxicological biomarkers in key copepod species, to reveal potential contamination in transitional water ecosystems. The focus was on changes that occur after the summer season, as during the summer, the surrounding human population increases significantly, with the consequent enhancement of anthropic pressures. Biomarkers were defined to monitor the zooplankton copepods Paracartia latisetosa, Acartia tonsa and Oithona brevicornis. Acetylcholinesterase, lipid peroxidation, and benzo(a)pyrene-monooxygenase activities were determined. P. latisetosa, A. tonsa and O. brevicornis show linear increases in their acetylcholinesterase activities with increasing concentrations in sample homogenates, which defines their high metabolic rates, and thus the suitability of these species as bioindicators. Biochemical alterations in these species induce changes in their biological attributes that can also lead to their disappearance, with a cascade of consequences for the higher trophic levels in the food chain. These data indicate that for the two lakes of Capo Peloro there is contamination by pesticides, carbamates and polycyclic and polyhalogenated aromatic hydrocarbons. This occurs mainly at the end of the summer season, and only affects the species of the low trophic levels. This case study shows the benefits provided by this biomarker approach as applied to zooplankton species, to monitor transitional water ecosystems and to estimate their toxicological risk, and thus to provide an early warning signal for loss of ecosystem integrity.

Potential use of ecotoxicological biomarkers in Serratella ignita (Ephemeroptera) larvae for Alcantara river (Sicily, Italy) water quality assessment

Benthic macroinvertebrates are important components of aquatic river ecosystems. These organisms are often used for biological monitoring since they are good indicators of the aquatic freshwater environment health status which can be negatively affected by human, agricultural and industrial activities. Many studies focused on the use of observed changes in macroinvertebrate communities or populations, but studies using biochemical biomarkers in these species are almost absent. The aim of this paper was to test the employment of ecotoxicological biomarkers in Ephemeroptera larvae to assess the water quality of the Alcantara river (Sicily) from its headwater to its mouth. This river represents the main source of potable water for all the counties situated in the Alcantara valley and for the city of Messina, and is the primary irrigation source for the large surrounding agricultural lands. Acetylcholinesterase activity (AChE) and Benzo(a)pyrene monooxygenase (BPMO) activity were evaluated in Serratella ignita (Ephemerellidae) larvae from different sampling sites along the river to show the potential presence of xenobiotic substances. Environmental parameters, such as temperature, dissolved oxygen, pH, conductivity and flow velocity, were also measured at different sampling sites. The biomarker approach in invertebrates represents an early warning signal of ecotoxicological alterations, providing information on which contaminants exert toxic effects on different biota. The results showed that the highest inhibition of AChE activity in S. ignita larvae was in Randazzo and Castiglione di Sicilia villages (i.e. high and medium level of the river). Here, agricultural activities are intense, with high production of fruit, olives, citrus and the heaviest usage of fertilizers, pesticides, and neurotoxic substances like organophosphate insecticides and carbammates in all the Alcantara valley. Regarding BPMO activity, the highest value was recovered in S. ignita larvae from the river mouth, which was characterised by the highest entry of water run off of streets and loaded with xenobiotic lipophilic compounds. Both AChE and BPMO were not linked to water temperature values. The data reported in the present work represent the first attempt to monitor levels of two widely recognised enzymatic biomarkers in benthic macroinvertebrates. Results indicate the possible use of this approach in macroinvertebrate larvae to study the health status of Alcantara river. The river appears to be impacted by contamination mainly from waste discharges and, upstream, from large pasture areas. Considering the world-wide distribution of S. ignita, the biomarker approach used to assess water quality may be applicable not only to Alcantara river, but also to other lotic ecosystems.

Screening of ecotoxicological, qualitative and reproductive variables in male European sea bass Dicentrarchus labrax (L.) reared in three different fish farms: Facility location and typology

The aim of this study was to investigate the influence of both facility location and typology of fish farm on some ecotoxicological, qualitative and reproductive variables in European sea bass Dicentrarchus labrax L. Several variables were investigated: gonado-somatic index (GSI), liver-somatic index (LSI); 7-ethoxyresorufin-O-deethylase (EROD), benzo(a)pyrene monooxygenase and acetylcholinesterase activities; glutathione (GSH), testosterone, 17β-estradiol, total lipid, phospholipid (PL) and triglyceride contents. In addition, the histological sections of gonads were examined. Results suggest that LSI, EROD activity, GSI, GSH, PL, hormone levels and gonad morphology were influenced by different facility locations and typologies of fish farm.

Detection of mutagens and BaPMO inducers in river water using the Blue Cotton adsorption technique

Induction of Mixed Function Oxidase (MFO) activity and bioactivation potential were measured in experimental carp and in native fish species from two rivers with different pollution level (Sava and Mrežnica). The experimental carp were intraperitoneally exposed to various water volume equivalents of Blue Cotton extracts from both rivers. The induction of MFO measured as a Benzo(a)pyrene monooxygenase (BaPMO) activity was increased up to 9.3-fold in experimental carp and up to 11.3-fold in native fish from Sava River, whereas the values from Mrežnica River showed only a slight increase when compared with the control (highest increase of 1.8-fold in nose carp). Accordingly, bioactivation potential using modified Ames test was higher in both experiments with Sava River. Both measured parameters in experimental carp increased in a dose-dependent manner in accordance to river volume equivalents. Different induction potential of native fish species comparable between two rivers confirmed the known possibility of their usage in biomonitoring studies. These results give qualitatively a new support to the idea of using Blue Cotton extraction technique combined with induction of MFO activity and Ames test in fish as a good biomarker for assessing risk of exposure to mutagens/carcinogens such as Polycyclic Aromatic Hydrocarbons (PAHs), especially due to the simplicity and cost-effectiveness of these methods.

DNA adducts, benzo( a)pyrene monooxygenase activity, and lysosomal membrane stability in Mytilus galloprovincialis from different areas in Taranto coastal waters (Italy)

The aim of this study was to investigate the impact of environmental pollution at different stations along the Taranto coastline (Ionian Sea, Puglia, Italy) using several biomarkers of exposure and the effect on mussels, Mytilus galloprovincialis, collected in October 2001 and October 2002. Five sampling sites were compared with a “cleaner” reference site in the Aeronautics Area. In this study we also investigated the differences between adduct levels in gills and digestive gland. This Taranto area is the most significant industrial settlement on the Ionian Sea known to be contaminated by polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc. Exposure to PAHs was evaluated by measuring DNA adduct levels and benzo( a)pyrene monooxygenase activity (B( a)PMO); DNA adducts were analyzed by 32P-postlabeling with nuclease P1 enhancement in both gills and digestive glands to evaluate differences between DNA adduct levels in the two tissues. B( a)PMO was assayed in the microsomal fraction of the digestive glands as a result of the high expression of P450-metabolizing enzymes in this tissue. Lysosomal membrane stability, a potential biomarker of anthropogenic stress, was also evaluated in the digestive glands of mussels, by measuring the latent activity of β- N-acetylhexosaminidase. Induction of DNA adducts was evident in both tissues, although the results revealed large tissue differences in DNA adduct formation. In fact, gills showed higher DNA adduct levels than did digestive gland. No significant differences were found in DNA adduct levels over time, with both tissues providing similar results in both years. DNA adduct levels were correlated with B( a)PMO activity in digestive gland in both years ( r=0.60 in 2001; r=0.73 in 2002). Increases were observed in B( a)PMO activity and DNA adduct levels at different stations; no statistical difference was observed in B( a)PMO activity over the two monitoring campaigns. The membrane labilization period in mussels from some stations was decreased in both years. No statistical differences were established in the membrane labilization times from 2001 to 2002. Our results suggest the existence of different sources and amounts of environmental contaminants at the stations investigated. The formation of DNA adducts confirms the existence of activation pathways in mussels and shows the importance of DNA adduct analysis in the gill tissue in addition to the more commonly used digestive gland; these results confirm the utility of lysosomal membrane stability as a biomarker of general stress. Overall, the integrated use of biomarkers of exposure and the effects of environmental contaminants on living marine organisms may help to better interpret the impact of pollutants in a marine coastal environment.

The use of a non-lethal tool for evaluating toxicological hazard of organochlorine contaminants in Mediterranean cetaceans: new data 10 years after the first paper published in MPB

In the Mediterranean Sea, top predators, and particularly cetacean odontocetes, accumulate high concentrations of organochlorine contaminants and toxic metals, incurring high toxicological risk. In this paper we investigate the use of the skin biopsies as a non-lethal tool for evaluating toxicological hazard of organochlorines in Mediterranean cetaceans, presenting new data 10 years after the paper published by Fossi and co-workers [Mar. Poll. Bull. 24 (9) (1992) 459] in which this new methodology was first presented. Some organochlorine compounds, now with worldwide distribution, are known as endocrine disrupting chemicals (EDCs). Here the unexplored hypothesis that Mediterranean cetaceans are potentially at risk due to organochlorines with endocrine disrupting capacity is investigated. High concentrations of DDT metabolites and PCB congeners (known as EDCs) were found in the different Mediterranean species ( Stenella coeruleoalba, Delphinus delphis, Tursiops truncatus and Balaenoptera physalus). In this paper we also propose benzo(a)pyrene monooxygenase (BPMO) activity in marine mammal skin biopsies (non-lethal biomarker) as a potential indicator of exposure to organochlorines, with special reference to the compounds with endocrine disrupting capacity. A statistically significant correlation was found between BPMO activity and organochlorine levels (DDTs, pp ′DDT, op ′DDT, PCBs and PCB99) in skin biopsies of males of B. physalus. Moreover a statistical correlation was also found between BPMO activity and DDT levels in skin biopsies of the endangered Mediterranean population of D. delphis. These results suggest that BPMO induction may be an early sign of exposure to organochlorine EDCs and can be used for periodic monitoring of Mediterranean marine mammal toxicological status.

Cytochrome P450, acetylcholinesterase and gonadal histology for evaluating contaminant exposure levels in fishes from a highly eutrophic brackish ecosystem: the Orbetello Lagoon, Italy

Biochemical markers and ovarian histology were investigated in prespawning females of grass goby ( Zosterisessor ophiocephalus) and grey mullet ( Mugil cephalus) collected, respectively, in late spring and summer 2000 in four sites of a highly eutrophic brackish ecosystem of central Italy, the Orbetello Lagoon. Exposure to chlorinated and aromatic hydrocarbons was evaluated in fish livers by the somatic liver index (SLI) and by measuring 7-ethoxyresorufin- O-deethylase (EROD) and benzo(a)pyrene monooxygenase (BaPMO) activities. Acetylcholinesterase (AChE) activity was measured in brain and gills to evaluate exposure to organophosphates (OPs) and carbamates (CBs). The gonad somatic index (GSI) was used to confirm ovarian maturation and ovarian histology was investigated as a potential biomarker for environmental effects. Samples from the Western Basin, near a sewage treatment plant (STP) off the town of Orbetello, showed higher SLI values and higher EROD and BaPMO activities than those collected from the Ansedonia Canal (AC) in the Eastern Basin ( p<0.05) and respect to those from reference sites: the Albegna River (AR) Delta for grass goby and the Nassa Canal (NC), connected with the sea, for grey mullet both located in the Western Basin as well. Low brain AChE activity was observed in both species from the reference sites (AR and NC) in association with the presence of anomalies in developing oocytes: unexpectedly small in grass goby and irregular disintegrated cytoplasm in grey mullet. The results indicate that the Western Basin is more polluted than the Eastern Basin particularly in the Orbetello where the sewage treatment plant may be a source of aromatic and chlorinated compounds while the Albegna River and the Nassa Canal may be sources of OPs and CBs.

Bioavailability of PAHs in the Biobio river (Chile): MFO activity and biliary fluorescence in juvenile Oncorhynchus mykiss

Cytochrome P450-dependent monooxygenase (MFO) activity and levels of bile PAH metabolites were measured in fish ( Oncorhynchus mykiss) exposed to Biobio river (Chile) water. Experiments were performed simultaneously in both the field and laboratory to investigate whether the river water contained notable bioavailable PAH fractions. The field experiment was conducted using fish caged for 41 days at La Mochita (Biobio river mouth) whereas the laboratory experiment involved a 21-day exposure treatment with water collected in the same area. Induction of hepatic MFO, assayed by benzo(a)pyrene monooxygenase activity (BaPMO), and levels of PAH metabolites in the bile were measured in groups of eight (field experiment) and four specimens (laboratory experiment) sampled after 5, 10, 21 and 41 days of exposure. BaPMO was induced by a factor of 23 and 25 in fish caged for 21 and 41 days, respectively. Likewise, a significant BaPMO induction was found in the fish group experimentally exposed to river water for 21 days. Biliary fluorescence measurements, recorded by fixed wavelength fluorescence (FF) and synchronous fluorescence spectroscopy (SFS), revealed significantly high levels of PAH metabolites in bile samples of fish caged for 21 and 41 days. A positive relationship was found between BaPMO activity and biliary PAH metabolites. This study provides evidences that (i) Biobio river water contains a significant bioavailable PAH fraction in terms of marked effects on fish and (ii) the biological indicators MFO activity and bile PAH metabolites represent an excellent screening methodology to assess PAH exposure.

Modification of Benzo(a) pyrene Monooxygenase Activity by Phenolic Antioxidants BHA and BHT and by their UV-Irradiation Products in 3T3 Fibroblasts

Abstract The phenolic antioxidants BHA and BHT, widely used in foods, drugs and cosmetics, are able to modulate initiation and promotion of chemical carcinogenesis by decreasing the binding to DNA of active metabolites. Since these compounds can be substrates for activating (MFO) as well as for detoxifying (Trans-ferases) enzymes it was of interest to study the effect of BHA and BHT in intact cells. Microspectrofluorometric studies of kinetics of B(a)P metabolism in single living fibroblasts 3T3 were then performed to study the effect of BHA and BHT on MFO activity in single living cells. It appeared that in intact cellular systems BHA and BHT were unable to inhibit directly MFO activity and that the inhibition, observed after 18 hours cell treatment, should be due to the production of oxidized derivatives by cellular metabolism of BHA or BHT. Furthermore photochemical (UV) oxidation of BHA and BHT also led to derivatives able to inhibit MFO activity. The mechanism of action and the nature of these active products remain to be elucidated.

Effect of Benzo(a)pyrene on Hepatic Biotransformation Activities: Time Course of Induction in Aquaculture European Sea Bass (Dicentrarchus labrax)

Abstract The responses of phase I and phase II enzymes to benzo(a)pyrene (BaP) exposure were characterized in liver of fish farm European sea bass (Dicentrarchus labrax). Treatment of sea bass with BaP by intraperitoneal injection produced a rapid putative MFO induction. Highest ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin-O-deethylase (ECOD) and benzo(a)pyrene monooxygenase (BaPMO) activities were observed in microsomes 14–24 h after BaP injection. EROD was induced 14.5 fold, ECOD 3.9 and BaPMO 13 fold. Spectrophotometrically measured microsomal cytochrome P-450 and NADPH-cytochrome c reductase activity were not induced by BaP injection, whereas a low increase in cytochrome b5 level was observed (induction factor 1.6 fold). Induction of phase II enzyme activities was lower than that of phase I enzymes: microsomal uridine diphospho-glucuronyl transferase (UDPGT) and cytosolic glutathion-S-transferase (GST) were induced 3.9 and 1.5 fold, respectively. The results indicate that sea bass is a sensitive species with respect to exposure to polycyclic aromatic hydrocarbons, exhibiting early marked responses of phase I and phase II biotransformation enzymes. The species offers potential as a tool for both laboratory studies and pollution monitoring. In the latter context it could be employed in the fish farms that occur alongside coastal regions.

The potential of carp to bioactivate benzo[ a]pyrene to metabolites that bind to DNA

We have investigated the formation of DNA adducts in starved, fed and 5,6-benzoflavone-pretreated carp following i.p. administration of benzo(a)pyrene. 32P-postlabeling analysis of the liver DNAs showed the presence of one predominant (>92%) adduct in all three groups. Cochromatography experiments revealed that the main adduct was identical to authentic BPDEI-dG (10β-(deoxyguanosin-N 2-yl)-7β,8α,9α-trihydroxy-7,8,9,10 -tetrahydrobenzo[a]pyrene). The formation of the adduct was evident as early as 1.5 h post-treatment and the levels increased steadily up to 7 days, reaching about 125, 110 and 102 attomole/μg DNA in starved, fed and benzoflavone-pretreated carp, respectively. During this period, the benzo[a]pyrene-induced benzo[a]pyrene monooxygenase activity increased from the uninduced, natural level of about 3 pmol/mg per min to levels of 35, 62 and 79 pmol/mg per min in starving, fed and 5,6-benzoflavone pretreated fish, respectively. A slow but steady formation of the diolepoxide-dG adduct was also observed in the liver DNA of carp following p.o. treatment. These results indicate that carp can biotransform polycyclic aromatic hydrocarbons such as benzo[a]pyrene to ‘reactive’ metabolites that bind to DNA.

Dose-related induction of rat hepatic drugmetabolizing enzymes by diuron and chlorotoluron, two substituted phenylurea herbicides

Dose-related induction of various hepatic drug-metabolizing enzymes has been investigated after shortterm treatment of rats by diuron and chlorotoluron, a dichlorinated and a monochlorinated phenylurea herbicide, respectively. Results suggest that ‘saturation’ of the induction system of benzo( a)pyrene monooxygenase, 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase activities may occur in the same range of the molar doses of both compounds, and with the dichlorinated herbicide at much higher activities. Induction of epoxide hydrolase, UDP-glucuronyltransferase and glutathione S-transferases also shows saturation curves in the function of molar doses. However, the structural difference is not reflected in the enhancement of enzyme activities.

The effects of prochloraz on some microsomal parameters in rat liver.

The antifungal agent prochloraz [l-n-propyl-N(2,4,6-trichlorophenoxy ethylcarbamoyl)-imidazole] induces cytochrome P-450 in the liver of vertebrates and interacts with lipid metabolism (Riviere et al. 1984; Laignelet et al. 1986). A study was undertaken to detect changes in lipid composition and fluidity of microsomal membranes. Sprague-Dawley rats (180-190g) were injected i.p. with either 600 Ixmol/kg prochloraz (95% pure; Schering Co.) in corn oil or with corn oil only, 3 days before sacrifice. In the liver homogenates vitamin A was measured (Olson 1979). The following assays were carried out with the microsomal suspensions: benzo(a)pyrene monooxygenase activity (BaPMO) (Depierre et al. 1975), epoxide hydrolase activity (EH) (Oesch et al. 1971), microsomal protein content (Lowry et al. 1951) and cytochrome P-450 content (Omura and Sato 1964). Microsomal lipids were extracted (Bligh and Dyer 1959) and quantitatively determined (Canal et al. 1973); cholesterol content was also measured (Boehringer Mannheim standard kit). Individual phospholipids were separated by two-dimensional TLC (Wolff et al. 1984) and total lipid phosphorus as well as individual phospholipids were thus determined (Ames 1966). Fatty acids were derivatized to their methyl esters (Morrison and Smith 1964) and analysed by GLC. Microsomal fluidity was studied at 37~ by fluorescence depolarization measurement of diphenyl hexatriene (DPH) according to Schinitzki and Barenholdz (1978). The results are summarized in Table 1 and all the differences indicated below are significant (at p < 0.01 level; t test calculation). Prochloraz significantly increased liver weight (+ 27%) and decreased the vitamin A liver burden (-17%). In treated animals the microsomal protein content of whole liver was significantly increased (nearly 40%). In microsomes of treated animals cytochrome P-450 content and BaPMO activity were significantly increased (+21% and + 121%, respectively), although EH activity was not altered. Prochloraz did not change the total lipid and phospholipid contents in microsomal membranes. However, the cholesterol level in these membranes and thus the phospholipid/cholesterol molar ratio were decreased ( -22% and -20%, respectively). Consequently, the microviscosity, evaluated in terms of fluorescence with the probe DPH, was significantly decreased ( 12%). It was reported that prochloraz inhibited lanosterol 14-demethylation (a cytochrome P-450 dependent reaction) in fungi (Schwinn 1983) and increased lanosterol liver content in Japanese quails (Riviere et al. 1984). More-

Benzo(a)pyrene mono-oxygenase activity in the shore CRAB, Carcinus maenas, in response to salinity stress

1. The cytochrome P-450 mono-oxygenase system is involved in the detoxification of xenobiotic compounds and, as such, has been suggested as a possible indicator of certain hydrocarbon pollutants. Microsomal benzo(a)pyrene mono-oxygenase (BPM) activity was assayed in the antennary gland, eyestalk, testes and heart of Carcinus maenas exposed to constant and “cycling” salinity regimes over a range of 5–36% 0. 2. In all tissues examined, an inverse relationship between BPM activities and hypo-salinity stress was noted, with lower BPM levels monitored at salinities between 25 and 36% 0, approximately 75–110% seawater. The enzyme activity-salinity pattern was not considered quantifiable, however, as there was significant variation in assays of BPM activity. 3. Of the tissues examined, BPM activities were highest in the antennary gland and the BPM differential between salinity extremes was also greatest in this tissue. 4. A seasonal pattern, associated with ambient temperature, was superimposed at all salinities, with higher BPM activities recorded during the winter. 5. In view of the significant and inconsistant variations in BPM activity during exposure to salinity levels normally associated with the habitat of Carcinus, the usefulness of BPM as an indicator of organic pollution is questionable.

3-Methylcholanthrene does induce mixed function oxidase activity in hepatopancreas of spiny crab Maja crispata

1. 1. Type II inducers (7,8-benzoflavone, benzo(a)-pyrene and 3-methylcholanthrene) as well as Aroclor 1254, significantly increase benzo(a)pyrene monooxygenase activity in crab hepatopancreas while type I inducer (phenobarbital) does not enhance benzo(a)pyrene monooxygenase activity. 2. 2. 3-methylcholanthrene and benzo(a)pyrene treatment of crabs significantly increase cytochrome P-450 content. 3. 3. Benzo(a)pyrene monooxygenase induction in hepatopancreas of 3-methylcholanthrene treated crabs was inhibited by simultaneous treatment with cycloheximide but not by actinomycin D. 4. 4. Actinomycin D insensitivity can be explained involving a regulatory pattern of induction on the posttranscriptional and/or translational, rather than transcriptional level.

Metabolism of some carcinogenic aromatic amines in four species of marine sponges

1. Postmitochondrial fractions from marine sponges Geodia cydonium, Tethya aurantium, Verongia aerophoba and Pellina semitubulosa activate precarcinogenie aromatic amine 2-aminoanthracene, but not precarcinogenie polycyclic aromatic hydrocarbon benzo(a)pyrene, to Salmonella typhimurium TA 98 mutagens. 2. All four sponge species lack a benzo(a)pyrene monooxygenase activity, but possesses the enzyme activity whose characteristics (selective activation of aromatic amines, NADPH-dependency, pH optimum at 8.4) are similar to FAD-containing monooxygenase. 3. Tethya postmitochondrial fraction possesses an UDP-glucuronyl transferase activity which catalyzes the conjugation of a considerable part of metabolized 2-acetylamino [9- 14C]fluorene to water soluble glucuronides. 4. The possible ecological significance of exuded aromatic amine metabolites as well as the significance of the presence of the selective potential for the activation of aromatic amines to mutagens among sponges for our understanding of the fate and effects of carcinogens in the marine environment are discussed.

Effects of pretreatment with 2,3,7,8-tetrachlorodibenzofuran on microsomal monooxygenase activity in Drosophila melanogaster

The effects of tetrachlorodibenzofuran pretreatment was studied in microsomes from adult Drosophila of the Karsnäs 60 w strain. Pretreatment for 18 h with 0.2 or 1 mg TCDBF did not increase benzo[ a]pyrene monooxygenase activity, 7-ethyoxyresorufin deethylase activity or the formation of 2-, 3- or 4-hydroxylated metabolites from biphenyl, in accordance with previous results with α-naphthoflavone pretreatment. The results suggest that adult Drosophila is non-responsive to induction by polycyclic aromatic hydrocarbons. The role of the TCDD receptor, which has been reported to be present in Drosophila, is obscure against this background. The implications for mutagenicity testing in Drosophila is discussed.

Purification and characterization of a single form of cytochrome P-450 from the spiny crab Maja crispata

1. 1. Microsomes from Maja crispata hepatopancreas contain all the components of the functional mixed function oxidase system: cytochrome P-450 (0.47 nmol/mg), the activity of NADPH cytochrome c reductase (12.25 nmol/mg/min) and benzo[a]pyrene monooxygenase activity (6.58 pmol/mg/min). 2. 2. Solubilization of hepatopancreas microsomes with sodium cholate, and affinity chromatography on omega-amino- n-octyl Sepharose 4B, gave a single cytochrome P-450 peak eluting with 0.2% Emulgen 913. 3. 3. DEAE cellulose chromatography of this cytochrome peak gave rise to a single haemoprotein peak, with apparent monomer M τ = 53,500, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis.

DNA damage benzo[α]pyrene in the liver of mosquito fish Gambusia affinis

Exposure of Gambusia affinis to water containing different concentrations of benzo[α]pyrene (bαP) causes an increase in benzo[α]pyrene monooxygenase (BPMO) activity which reaches a maximum on the second day. Concomitantly, the DNA is altered in such a way that nuclease S 1-sensitive sites (SSS) become measurable. The size distribution of liver DNA treated with nuclease S 1 in control fish shows two populations of DNA by length, with means of 30 × 10 6 and 60 × 10 6 Daltons, respectively. In fish treated with 100 ppb BαP, the population with longer molecules of DNA disappears and shorter molecules increase in number. This may be explained in terms of the introduction of an additional 0.31–0.46 DNA nicks per control DNA molecule caused by metabolically activated BαP derivatives.

Tissue distribution, seasonal variation and induction of benzo[ a] pyrene monooxygenase activity in the crab Maja crispata

The mixed function oxygenase activities (benzo[ a]pyrene monooxygenase; E.C. 1.14.14.1; aryl hydrocarbon hydroxylase, AHH) from six organs of the crab Maja crispata have been characterized as stemming from one single form of enzyme. Its activity optimum was found at 27°C, pH 7.4, with NADPH, oxygen and magnesium present, and is similar to the one previously described for insects and vertebrates. The enzyme activity levels are: pyloric stomach > gills > heart > reproductive organs > hepatopancreas > muscles. There are no significant differences between male and female crabs. Recovery of enzyme activities from the hepatopancreas could be increased by a factor of 2.5 with proteinase inhibitor, and by 14 with a phospholipase inhibitor present during the preparation. During summer months the specific mixed function oxygenase (MFO) activities were generally high, but dropped to low values during the winter and correlated with ambient temperature and not with the reproductive cycle. In hepatopancreas, oral intubation with benzo[ a]pyrene produced a ten-fold increase of the enzyme activity, while external exposure to Diesel-2 oil, or feeding benzo[ a]pyrene-contaminated prey, increased the enzyme activity in the pyloric stomach. Stomach enzyme activity in crabs from three different locations differed significantly, possibly due to different histories of pollution.