P450 1A Research Articles

Modulation of carcinogen metabolizing enzymes by chromanone A; a new chromone derivative from algicolous marine fungus Penicillium sp.

A marine fungal isolate, Penicillium sp. fungus isolated from seaweed, Ulva sp., led to the isolation of a new chromone derivatives, 2-(hydroxymethyl)-8-methoxy-3-methyl-4H-chromen-4-one (chromanone A). The structure was determined by interpretation of their spectroscopic data (1D and 2D NMR, MS, UV and IR). At the nitiation stage of carcinogenesis, carcinogens is activated by cytochrome P-450 1A (CYP1A) and detoxified by glutathione S-transferases (GST), quinine reductase (QR), and epoxide hydrolase (mEH). We tested the modulatory effect of chromanone A on these carcinogen metabolizing enzymes. The results indicated that chromanone A (4 μg/ml) is a promising inhibitor of CYP1A activity up to 60% of the stimulated-CYP1A in murine hepatoma cells (Hepa1c1c7), and it significantly induced GST but not total thiols at low concentrations. Chromanone A had no influence on QR activity, while it resulted in a significant dose-dependant enhancement mEH activity in Hepa1c1c7 cells ( P < 0.05–0.01). Additionally, chromanone A possessed a potent specific radical scavenging activity against hydroxyl radicals more than peroxyl radicals that may be responsible for the inhibitory effect of chromanone A on the induced-DNA damage in cells. In conclusion, this study proved that chromanone A may act as an active tumor anti-initiating via modulation of carcinogen metabolizing enzymes and protection from DNA damage.

Hepatic P450 Enzyme Activity, Tissue Morphology and Histology of Mink (Mustela vison) Exposed to Polychlorinated Dibenzofurans

Dose- and time-dependent effects of environmentally relevant concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQ) of 2,3,7,8-tetrachlorodibenzofuran (TCDF), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a mixture of these two congeners on hepatic P450 enzyme activity and tissue morphology, including jaw histology, of adult ranch mink were determined under controlled conditions. Adult female ranch mink were fed either TCDF (0.98, 3.8, or 20 ng TEQTCDF/kg bw/day) or PeCDF (0.62, 2.2, or 9.5 ng TEQPeCDF/kg bw/day), or a mixture of TCDF and PeCDF (4.1 ng TEQTCDF/kg bw/day and 2.8 ng TEQPeCDF/kg bw/day, respectively) for 180 days. Doses used in this study were approximately eight times greater than those reported in a parallel field study. Activities of the cytochrome P450 1A enzymes, ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) were significantly greater in livers of mink exposed to TCDF, PeCDF, and a mixture of the two congeners; however, there were no significant histological or morphological effects observed. It was determined that EROD and MROD activity can be used as sensitive biomarkers of exposure to PeCDF and TCDF in adult female mink; however, under the conditions of this study, the response of EROD/MROD induction occurred at doses that were less than those required to cause histological or morphological changes.

Biochemical changes in rockfish, Sebastes schlegeli, exposed to dispersed crude oil

This paper describes the response of the ovoviviparous rockfish, Sebastes schlegeli, to hydrocarbons in the water-accommodated fraction (WAF) of crude oil, in the presence or absence of oil dispersants. Concentrations of cytochrome P-450 1A (CYP1A) and levels of its catalytic activity ethoxyresorufin O-de-ethylase (EROD) in rockfish exposed to WAF at concentrations of 0.1% and 1% were significantly increased by the addition of a dispersant, Corexit 9500 after 48 h exposure. After 72 h exposure, the levels of CYP1A and EROD activity were significantly increased in 0.1% and 0.01% chemically enhanced WAF (CEWAF) (Corexit 9500 and Hiclean II dispersant). Bile samples from fish exposed to WAF alone had low concentrations of hydrocarbon metabolites, exemplified by 1-hydroxypyrene. After 72 h exposure, hydrocarbon metabolites in bile from fish exposed to WAF in the presence of either Corexit 9500 or Hiclean II were significantly higher compared with fish exposed to WAF alone or control fish. These experiments confirm that the use of oil dispersants will increase the exposure of ovoviviparous fish to hydrocarbons in oil.

PCB126 enhanced the genotoxicity of BaP in HepG2 cells by modulating metabolic enzyme and DNA repair activities

Both of benzo(a)pyrene (BaP) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) are ubiquitous and persistent environmental pollutants. These two chemicals coexist in various environmental media and human samples and thus may have combined effects on human health. However, the toxic effects and related mechanism of co-exposure to BaP and PCB126 remain unknown. In a series of experiments using the HepG2 cells exposed to BaP (50microM) or/and PCB126 (0.01, 0.1, 1 and 10nM), we measured the rate of micronucleus (MN) formation, CYP1A1 activity and expression of nucleotide excision repair (NER) proteins (XPA and XPC). We found that the exposure to BaP or PCB126 alone could effectively increase the CYP1A1 activity and the XPA expression. BaP alone had a profound enhancement of MN formation. Compared with BaP alone, co-exposure to both BaP and PCB126 significantly enhanced the CYP1A1 activity and the formation of MN but reduced the expression of both XPA and XPC. The synergistic effect of PCB126 on BaP-induced MN formation was inhibited by alpha-naphthoflavone (ANF), an inhibitor of CYP1A1. Our findings suggest that PCB126 may enhance BaP-induced DNA damage and genotoxicity by increasing cytochrome P450 1A activity and decreasing the NER capacity.

EROD and MROD as Markers of Cytochrome P450 1A Activities in Hepatic Microsomes from Entire and Castrated Male Pigs

In the present study, we characterized the kinetic parameters of 7-ethoxy-resorufin O-deethylation (EROD) and 7-methoxyresorufin O-demethylation (MROD) in hepatic microsomes from entire and castrated male pigs. Validation parameters of an HPLC-based method to analyse EROD and MROD activities are also described. Eadie-Hofstee plot analysis demonstrated a biphasic kinetic of EROD, indicating that at least two forms of cytochrome P450 are involved in this reaction. MROD followed monophasic kinetic, suggesting that a single enzyme, or enzymes with similar affinities, is responsible for the reaction. Inhibitory effects of α-naphthoflavone (ANF), ellipticine and furafylline were studied using microsomes from entire and castrated male pigs. ANF is a known inhibitor of both cytochrome P450 1A1 and 1A2 (CYP1A1 and CYP1A2); the presence of ANF in the incubations resulted in the inhibition of both EROD and MROD activities in porcine liver microsomes. EROD activities in porcine liver microsomes were also inhibited by selective CYP1A1 inhibitor ellipticine, but not by CYP1A2 inhibitor furafylline. MROD activities were strongly inhibited by ellipticine and to a much lesser extent by furafylline. Further studies are needed to evaluate substrate specificities of porcine CYP1A1 and CYP1A2.

In vitro cancer chemopreventive properties of polysaccharide extract from the brown alga, Sargassum latifolium

Polysaccharides of edible algae attracted extensive interest due to their numerous biological activities. Sargassum latifolium (Turner) C. Agardh, belongs to Sargassaceae, is a brown algae in red sea shores in Egypt. This work is a novel attempt to explore the cancer chemopreventive activity of different fractions of water-soluble polysaccharide extract derived from S. latifolium. Estimation of cancer chemopreventive activity, specifically anti-initiation, including the modulation of carcinogen metabolism and the antioxidant capacity, revealed that E1 and E4 were potent anti-initiators, where they lead not only to an inhibition in the carcinogen activator cytochrome P450 1A (IC50 2.54 and 10.30μg/ml, respectively), but also to an induction in the carcinogen detoxification enzymes glutathione-S-transferases (144% and 225% of the control, respectively). E1 and E4 inhibited 59% and 63% of the induced-DNA damage, as measured by comet assay. Similarly both E1 and E4 possessed potential anti-promoting properties as indicated by their anti-inflammatory activity. E1 and E4 enhanced the macrophage proliferation; however they dramatically inhibited the stimulated NO (30.7% and 59.3%), TNF-α (38.2% and 54.9) and COX-2 (20% and 18%), respectively. E3 showed a selective cytotoxicity against lymphoblastic leukemia (1301 cells), while other fraction extracts had no cytotoxic effect against all tested cell lines. E3 led to a major disturbance in cell cycle including arrest in both S-phases in 1301 cells. This disturbance was associated with an induced-cell death due to apoptosis, but not necrosis. In conclusion, E1 and E4 are promising cancer chemopreventive fractions, since they had tumor anti- initiating activity via their protective modulation of carcinogen metabolism, and tumor anti-promoting activity via their anti-inflammatory activity, while E3 can be considered as a promising anti-cancer agent against leukemia.

Are Atlantic Cod in Store Lungegårdsvann, a Seawater Recipient in Bergen, Affected by Environmental Contaminants? A qRT-PCR Survey

The aim of this study was to examine the transcriptional levels of selected genes in liver and head kidney of Atlantic cod Gadus morhua sampled in Store Lungegårdsvann, a seawater recipient situated in the middle of the city of Bergen, Norway, for effects of contaminants released from municipal sewage effluents and former dump sites. Five males and six females were caught with fish traps in Store Lungegårdsvann in 2006. Cod from a location near Jondal in the Hardanger Fjord were used as controls (five males and four females). The following 12 genes were picked as potential markers of contaminant exposure: cytochrome P-450 1A (CYP1A), cytochrome P-450 2C33-like (CYP2C33-like), cytochrome P-450 3C (CYP3C), glutathione S-transcriptase π (GST) (detoxification and biotransformation), Mn superoxide dismutase (Mn SOD), glutathione reductase (GR), heat-shock protein 70 (HSP70) (oxidative stress), vitellogenin A (VtgA), vitellogenin B (VtgB), zona pellucida 2 (ZP2) (effects of estrogen disruptors), B-cell lymphoma 2 (Bcl-2), and cyclin-dependent kinase inhibitor 1A (CDKN1A) (radiation). The results showed that two males caught in Store Lungegårdsvann possessed high transcriptional levels of VtgA, VtgB, and ZP2 mRNA in the liver. In addition, CYP1A was 4.9-fold higher expressed in males from Store Lungegårdsvann compared to males from the reference population. CYP2C33-like mRNA expression was significantly higher (1.8-fold) in females from Store Lungegårdsvann than in females from the reference population. CYP1A was significantly lower (4.7-fold) expressed in head kidney of females from Store Lungegårdsvann than in females from Hardanger Fjord. In a follow-up examination with sexually mature cod sampled in Store Lungegårdsvann in 2007, the livers were shown to contain high levels of polychlorinated biphenyls (PCB) and dioxin-like PCB. In conclusion, fish inhabiting Store Lungegårdsvann are exposed not only to endocrine disruptors but also to other contaminants that affect the transcription of phase I biotransformation genes.

Crude cacao theobroma cacao extract reduces mutagenicity induced by benzo[a]pyrene through inhibition of CYP1A activity in vitro

Polyphenols have been shown to have potent antioxidant activity, and therefore, food containing polyphenols is expected to contribute to the prevention of cancer. However, food contains not only polyphenols but also various other constituents. We used the Ames test to investigate the effects of crude extracts of whole cacao products, which are known to be rich in polyphenols, on the mutagenicity of benzo[a]pyrene (B[a]P) in Salmonella typhimurium strain TA 98 and tert-butyl hydroperoxide (t-BuOOH) in S. typhimurium strain TA 102. B[a]P induces mutagenicity by metabolic activation and t-BuOOH induces it by generation of free radicals. While white chocolate did not modulate the numbers of revertant colonies produced by B[a]P treatment, milk chocolate and cacao powder extracts did. On the other hand, surprisingly, none of the cacao products tested affected the number of revertant colonies when t-BuOOH was used as the mutagen. At maximum concentration (13.25 mg cacao powder/ml), the crude cacao powder extract reduced ethoxyresorufin O-deethylase activity to 17.4% of the control, suggesting that whole cacao products inhibit cytochrome P450 (CYP) 1A activity. In conclusion, inhibition of CYP1A activity by cacao products may prevent DNA damage by reducing metabolic activation of carcinogens.

Assessment of metabolic capabilities of PLHC-1 and RTL-W1 fish liver cell lines

Metabolic capabilities of PLHC-1 and RTL-W1 cell lines were investigated since to date, cytochrome P450 (CYP) 1A and glutathione-S-transferase have been almost the unique biotransformation enzymes reported in these cells. Functionality of CYP3A-, CYP2M- and CYP2K-like enzymes was assessed by studying the hydroxylation of testosterone (T) and lauric acid (LA), and glucuronidation and sulfation capacity was assessed by looking at 1-naphthol (1-N) and T conjugation. Only PLHC-1 cells showed the ability to hydroxylate T at 6beta-position (a CYP3A-like catalysed pathway) and LA at (omega-1)-position (a CYP2K-like catalysed pathway). Hydroxysteroid dehydrogenase and steroid reductase enzymes showed comparatively higher activities than CYPs: 5alpha-dihydrotestosterone, androstenedione and 3beta-androstanediol were the major metabolites of T detected in both cell lines. Regarding phase II activities, both cell lines metabolised 1-N to glucuronide and sulfate conjugates. In contrast, when using T as substrate, RTL-W1 formed the glucuronide, whilst PLHC-1 formed the corresponding sulfate. Overall, the observed enzymatic activities are much lower (up to 17.5 x 10(3) times) than those reported in primary cultures of fish hepatocytes. The present study highlights the need of developing new fish cell lines that could be used as alternative in vitro tools for studying xenobiotic metabolism and toxicity in fish.

Cytochrome P450-depedent Drug Oxidation Activity of Liver Microsomes from Microminipigs, A Possible New Animal Model for Humans in Non-clinical Studies

Small minipigs (Bland name, Micromini Pig; registered as a novel variety of pig in the Japanese Ministry of Agriculture, Forestry and Fisheries) were developed with the aim of non-clinical pharmacological/toxicological use. They were principally mated with<10 kg body weight at 7 months-old resulting in good handling. Cytochrome P450 (P450)-and flavin-containing monooxygenases (FMO)-dependent drug oxidation activity of liver microsomes prepared from male Microminipigs (8 months-old) was compared with that for pooled dogs, monkeys, and humans. High P450 2D-dependent bufuralol 1'-hydroxylation and FMO-dependent benzydamine N-oxygenation activity was observed in liver microsomes from Microminipigs. Typical P450 1A, 2B, 2C, 2E, and 3A-dependent drug oxidation activity was also seen in Microminipigs. However, occasional differences might give undetected low P450 2A-dependent coumarin 7-hydroxylation in Microminipigs at 8-months-old, in contrast to liver microsomes from one 10-days-old Microminipis and commercially available pooled minipigs which had low but detectable coumarin 7-hydroxylation activity. The present results suggest that there is some overlap in Microminipig and human P450 substrate specificity. These findings should provide important information for greater understanding of drug metabolism in Microminipigs, as an experimental animal model for non-clinical use.

Molecular Cloning of Novel Cytochrome P450 1A Genes from Nine Japanese Amphibian Species

Novel cytochrome P450 1A (CYP1A) cDNA fragments were isolated from the liver of nine Japanese amphibian species using reverse transcription polymerase chain reaction (RT-PCR). Degenerate PCR primers were used to amplify 122-bp fragments of CYP1A cDNAs. Construction of a phylogenetic tree revealed that urodele and anuran amphibians formed two branches. Within the anuran species, three branches were formed: 1) Ranidae and Rhacophoridae, 2) Bufo japonicus formosus and Hyla japonica, and 3) Xenopus laevis. The cDNA nucleotide sequence of these CYP1A fragments showed identities ranging 72-98% (all), 72-78% (Anura vs. Urodela), 75 to 98% (Anura), 81% (Urodela), 74-80% (Xenopus laevis vs. nine Japanese amphibians).

Assessing and monitoring local and long-range-transported hydrocarbons as potential stressors to fish stocks

The Arctic is poorly described and understood compared to the North Sea and other boreal areas. We need to learn how knowledge obtained at boreal conditions may be applied at Arctic conditions. Increasing activity of the hydrocarbon industry may cause exposure stress from discharges. Within a joint industry research programme (Biosea) we studied how responses in fish from oil exposure may differ at North Sea boreal and Barents Sea Arctic conditions. Hydrocarbon uptake, metabolites, and enzymatic and genotoxic biomarker type of effect responses were measured in cod (Gadus morhua L.). Hydrocarbon metabolites remain longer in fish bile than original hydrocarbons, which are eliminated fast from tissues. The metabolites may be measured to background concentrations. They describe exposure and they constitute a link to other effects. Body burden, biliary polyaromatic hydrocarbon (PAH) metabolite concentration, and quantities of Cytochrome P-450 1A (CYP1A) and DNA adducts increased with oil in water concentration. The extent of biomarker expression was lower for some biomarkers and elimination was slower at the lowest temperature. The results show that several factors have to be accounted for if warm-water biomarker data are to be applied in cold water.

Potential effects of perfluorinated compounds in common cormorants from Lake Biwa, Japan: An implication from the hepatic gene expression profiles by microarray

Contamination levels of perfluorinated compounds (PFCs), including perfluorooctanesulfonate (PFOS), perfluorononanoic acid (PFNA), perfluorooctane sulfonamide (PFOSA), perfluorohexanesulfonate (PFHS), and perfluorooctanoic acid (PFOA), were determined in the livers of wild common cormorants (Phalacrocorax carbo) from Lake Biwa, Japan. Potential effects of PFCs alone and combined effects by complex mixture of PFCs and dioxins and related compounds (DRCs) were also assessed by gene expression profiling using a cormorant oligo array. Perfluorooctanesulfonate, PFNA, and PFOSA were detected in almost all liver samples analyzed, while concentrations of PFHS and PFOA were below the limit of quantification. The microarray data analyses revealed that hepatic PFC levels were correlated with the expression of 74 genes. Real-time reverse-transcript polymerase chain reaction data demonstrated that PFOS concentration was positively correlated with mRNA levels of glutathione peroxidase 1 and glutathione S-transferase alpha 3 and negatively correlated with levels of heat shock 70-kDa protein 8 and tumor rejection antigen 1 mRNAs. These results suggest the induction of antioxidant enzymes in response to oxidative stress caused by PFCs and the suppression of molecular chaperones, leading to reduction in protein stability. Moreover, multiple regression analyses identified seven significant models in which certain genes showed expression levels altered by accumulation of PFCs and DRCs. The regression models explained associations with cytochrome P450 1A mRNA and protein expression levels, and its catalytic activity, ethoxyresorufin-O-deethylase of both PFNA and the 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalent levels. Thus, the regression models suggested the potential of PFCs to enhance toxicities of DRCs. Since mixture toxicity is an urgent issue, further study is required to understand the effects of mixtures of PFCs and DRCs in wild cormorants.

Protective effects of vitamin E against 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) induced toxicity in zebrafish embryos

3,3′,4,4′,5-Pentachlorinated biphenyls 126 (PCB126) is a global environmental contaminant that can induce cellular oxidative stress. We investigated whether vitamin E can protect against toxicity from PCB126 during zebrafish ( Danio rerio) development. Zebrafish embryos were exposed to 100 nM PCB126 and compared with a second group that was co-exposed with 100 μM vitamin E until 5 days post fertilization. PCB126 induced pericardial sac edema, yolk sac edema, and growth retardation in zebrafish embyos. In contrast, vitamin E co-exposure group did not show any gross changes. Real-time PCR results showed that vitamin E co-exposure group were restored to control group for the expression levels of heat shock protein 70 Cognate, aryl hydrocarbon receptor type-2, cytochrome P450 1A, and superoxide dismutase-1. These data give insights into the use of vitamin E to reduce PCB126-mediated toxicity and into the use of zebrafish embryos for exploring mechanisms underlying the oxidative potential of AHR agonists.

Monodictyochromes A and B, Dimeric Xanthone Derivatives from the Marine Algicolous Fungus Monodictys putredinis

Investigations of the marine-derived fungus Monodictys putredinis led to the isolation of two novel dimeric chromanones (1, 2) that consist of two uniquely modified xanthone-derived units. The structures were elucidated by extensive spectroscopic measurements including NOE experiments and CD analysis to deduce the configuration. The compounds (1, 2) were examined for their cancer chemopreventive potential and shown to inhibit cytochrome P450 1A activity with IC(50) values of 5.3 and 7.5 μM, respectively. In addition, both compounds displayed moderate activity as inducers of NAD(P)H:quinone reductase (QR) in cultured mouse Hepa 1c1c7 cells, with CD values (concentration required to double the specific activity of QR) of 22.1 and 24.8 μM, respectively. Compound 1 was slightly less potent than compound 2 in inhibiting aromatase activity, with IC(50) values of 24.4 and 16.5 μM.

Oxidative Metabolism of the Bioactive and Naturally Occurring β-Carboline Alkaloids, Norharman and Harman, by Human Cytochrome P450 Enzymes

Norharman and harman are naturally occurring beta-carboline alkaloids exhibiting a wide range of biological, psychopharmacological, and toxicological actions. They occur in foods and tobacco smoke and also appear endogenously in humans. In this research, metabolic and kinetic studies with cytochrome P450 enzymes and human liver microsomes showed that beta-carbolines were efficiently oxidized to several ring-hydroxylated and N-oxidation products that were subsequently identified and quantified. 6-Hydroxy- beta-carboline (6-hydroxynorharman and 6-hydroxyharman) was a major metabolite efficiently produced (high kcat and low Km) by P450 1A2 and 1A1 and to a minor extent by P450 2D6, 2C19 and 2E1. 3-Hydroxy-beta-carboline (3-hydroxynorharman and 3-hydroxyharman), another major metabolite, was specifically produced by P450 1A2 and 1A1, whereas beta-carboline-N(2)-oxide (harman-2-oxide and norharman-2-oxide) was produced by P450 2E1. The same pattern of metabolism was confirmed for human liver microsomes. Oxidative metabolism for harman was slightly higher than norharman, but norharman showed lower Km values. The oxidation of beta-carbolines is a detoxication route performed mainly by P450 1A2 and 1A1, with the participation of P450 2D6, 2C19, and 2E1, as additional contributors. Then, individual variations in the levels and activity of these P450s may influence biotransformation of beta-carboline alkaloids and their ultimate biological effects. beta-Carbolines were previously reported as comutagens and/or inhibitors of mutagens activated by P450 1A enzymes such as heterocyclic amines and polycyclic hydrocarbons. Results in this work show that beta-carbolines are good ligands and substrates for P450 1A2/1A1, contributing to the explanation of some of their toxicological effects.

Biological effect monitoring in dab (Limanda limanda) using gene transcript of CYP1A1 or EROD—a comparison

Gene expression analyses with real-time (RT)-polymerase chain reaction (PCR) gains importance in marine monitoring. This new technique has to be compared to the classical approaches like the well known biomarker ethoxyresorufin-O-deethylase (EROD) to test their suitability for monitoring programmes. The goal of the present study is to compare EROD activity and CYP1A1 mRNA expression in the important monitoring fish species dab (Limanda limanda) and to answer the question of whether these parameters reflect the polycyclic aromatic hydrocarbon (PAH) contamination of the fish. Further on, glyceraldehyd-3-phosphate dehydrogenase (GAPDH) was investigated as a potential housekeeping gene. Female dab were caught in the summer of 2004 in the North Sea and in the Baltic. EROD activity was determined in liver samples by a kinetic fluorimetric assay according to a standard protocol. The gene expression of CYP1A (cytochrome P450 1A) and GAPDH were determined by means of RT-PCR. Results were compared to gonado somatic index and to the concentration of PAH metabolite 1OHPyr (1-hydroxypyrene) analysed in the bile fluids of the fish, respectively. Dab from all stations showed a considerable individual variation in the levels of both CYP1A mRNA and EROD. Highest mean values for CYP1A mRNA and EROD were detected in the northern part of the sampling area. In contrast, the PAH metabolite 1OHPyr was found at the highest concentration in fish caught near the German coast. CYP1A mRNA and EROD showed only a minor but significant correlation (r = 0.32, p < 0.05, n = 123). 1OHPyr in bile correlated significantly (p < 0.05) with the amount of GAPDH mRNA content in the liver. The significant but low correlation of CYP1A mRNA and EROD activity on an individual basis illustrates that these two parameters are apparently not closely linked. However, maximum EROD values correspond with maximum CYP1A mRNA concentrations when station means are regarded. Because EROD and CYP1A mRNA in dab follow different physiological principles, their application will lead to related but not identical monitoring results. This should be taken into account when future marine monitoring programmes are designed. The results also indicate that PAH are not the crucial factor for CYP1A and EROD levels in dab from the off-shore areas in the North Sea. This is remarkable because the PAH metabolism is known to be CYP1A-dependent and the widely used biomarker EROD has been recommended for monitoring PAH-related effects in fish from the North Sea. Due to a correlation between GAPDH and 1OHPyr, GAPDH was not suitable as housekeeping gene for dab. Neither the results from EROD nor from CYP1A1 mRNA measurements in dab reflected their exposure to PAH as measured by the PAH metabolite 1OHPyr. Thus, the question arises of whether EROD or CYP1A mRNA is a suitable biomarker at all to indicate PAH exposure in dab from the open North Sea. For future biological effect monitoring, it is advisable to measure more and predominately independent parameters by RT-PCR and to incorporate more components of the detoxification system.

Effects of atrazine on hepatic metabolism and endocrine homeostasis in rainbow trout ( Oncorhynchus mykiss)

The herbicide atrazine (ATZ) is one of the most widely used pesticides in the world and is now under scrutiny for its alleged capacity to disrupt the endocrine system. Exhibiting negligible interaction with the estrogen receptor (ER), ATZ's mode of action remains to be elucidated. ATZ may act as an inducer of the enzyme aromatase, which converts androgens to estrogens, although other mechanisms should also be taken into consideration such as impairment of hepatic metabolism. Therefore we administered juvenile rainbow trout ( Oncorhynchus mykiss) a dose of either 2 or 200 μg ATZ/kg, or of carrier control phosphate buffered saline (PBS) and we measured plasma concentrations of testosterone (T), 17beta-estradiol (E2) and vitellogenin (Vtg) 6 days after exposure. Simultaneously we analyzed hepatic gene expression of cytochrome P450 ( CYP) 1A and pi-class glutathione S-transferase ( GST-P), and catalase (CAT) activity. Although sex steroid levels showed no significant alterations, we found a dose-dependent increase in Vtg and a concomitant decrease in CYP1A. There was no effect of ATZ on GST-P mRNA levels but GST-P was positively correlated with CYP1A. Also, CYP1A was negatively correlated with liver CAT and E2, and varied with T concentrations in a hormetic manner. The results showed that ATZ can alter hepatic metabolism, induce estrogenic effects and oxidative stress in vivo, and that these effects are linked.

High performance liquid chromatography determination of cytochrome P450 1A and 2C activities in bovine liver microsomes

This study reports fluorescence high performance liquid chromatography (HPLC) and UV-Vis HPLC methods for the determination of 7-ethoxyresorufin O-deethylase (EROD) and tolbutamide methylhydroxylase (TMH) activities, respectively, using bovine liver microsomes. The detection limits were 0.022 and 5.5 pmol on the column, respectively; intra-day and inter-day precisions (expressed as relative standard deviation) were <10%. Both methods showed enough sensitivity to allow for an accurate determination of enzyme kinetic parameters according to Michaelis-Menten plots and the results were: K(m)=0.23+/-0.051 microM, V(max)=0.488+/-0.035 nmol/min/mg protein for EROD activity, and K(m)=1010+/-155.7 microM, V(max)=0.089+/-0.006 nmol/min/mg protein for TMH activity. An Eadie-Hofstee plot analysis showed that in bovine liver microsomes, EROD and TMH activities followed a monophasic kinetic pattern. alpha-Naphthoflavone, a cytochrome P450 1A1/2 (CYP1A1/2) inhibitor, and sulfaphenazole, a cytochrome P450 2C9 (CYP2C9) inhibitor, decreased EROD and TMH activities, respectively. The sensitivity of the methods allowed the use of microsomes with low enzyme activity, such as those from veal calf liver. Thus, EROD and TMH activities may be adopted as markers for the evaluation of CYP1A and CYP2C9-like activities in liver microsomes from veal and beef cattle.

Differential gene expression and biomarkers in zebrafish ( Danio rerio) following exposure to produced water components

The main effluent from oil and gas production is produced water (PW), a waste that contains low to moderate concentrations of oil-derived substances such as polycyclic aromatic hydrocarbons (PAHs) and alkylphenols (APs). PW components may be present in seawater at low concentrations over large areas in the vicinity of oil and gas production facilities. In this study, zebrafish ( Danio rerio) were exposed to control and three treatments (high-, pulsed-, low-dose) of a synthetic PW mixture for 1, 7 and 13 weeks. The aim was to investigate the development of transcriptome and biomarker responses as well as relationships between early responses and population-relevant effects. The synthetic PW contained a mixture of low-molecular-weight PAHs (<5 ring) and short-chain APs (C1–C4). The water-borne exposure levels (sum PAH) ranged from 0.54 ppb (low dose) to 5.4 ppb (high dose). Bile pyrene metabolites ranged from 17–133 ng g −1 bile in the control group to 23–1081 ng g −1 bile in the high exposure group. Similar levels have been observed in wild fish, confirming an environmentally relevant exposure. The expression of mRNAs of hepatic genes was investigated in the high exposure group using the Zebrafish OligoLibrary™ from Compugen. Functional clustering analysis revealed effects in the reproductive system, the nervous system, the respiratory system, the immune system, lipid metabolism, connective tissue and in a range of functional categories related to cell cycle and cancer. The majority of differentially expressed mRNAs of genes were down-regulated, suggesting reduction in gene transcription to be as relevant as up-regulation or induction when assessing biological responses to PW exposure. Biomarkers for effects of PAHs (cytochrome P450 1A) and environmental estrogens (vitellogenin) did not appear to be affected by the chronic exposure to low concentration of PW components. Effects at the population level included a reduction in condition factor in male fish from all exposed groups and spinal column deformations in the F1 generation of exposed groups. The different exposure regimes did not produce any significant differences in reproduction or recruitment. The results from this study demonstrate that environmentally relevant concentrations of PW affect gene expression and population-relevant endpoints in zebrafish, although links between the two were not obvious.