Cytochrome P450 Catalytic Activities Research Articles

Decoding the Cytochrome P450 Catalytic Activity in Divergence of Benzophenone and Xanthone Biosynthetic Pathways

Plant benzophenones and xanthones have an impressive spectrum of biological activities. Here, we characterized three cytochrome P450 (CYP) enzymes from the Garcinia xanthochymus tree that convey 2,4,6-trihydroxybenzophenone (2,4,6-triHB) toward the diversification between the biosynthesis of benzophenones and xanthones. The CYP3 catalyzes the transformation of 2,4,6-triHB to 2,3′,4,4′,6-pentahydroxybenzophenone (2,3′,4,4′,6-pentaHB) through two consecutive hydroxylations, while the paralog CYP1 catalyzes the synthesis of 1,3,7-trihydroxyxanthone (1,3,7-triHX). To understand the product diversity, we performed homology modeling and active site-directed reciprocal mutations on CYP3. The mutant enzyme assays revealed the key role of the V375 residue. Although the V375A mutation favors the 1,3,7-triHX formation, the V375L enhances further the yield of 2,3′,4,4′,6-pentaHB. The engineered triple mutant CYP3-V375L/S479A/K480H demonstrated an improved product specificity and catalytic transformation toward 2,3′,4,4′,6-pentaHB. Our results represent an advance in the metabolism of benzophenones and xanthones, laying the foundation for discovery of downstream genes and even the production of new derivatives. The engineering of CYP enzymes that stand in divergence of biosynthetic pathways for benzophenones and xanthones provides the basis for designing new drugs and facilitates future synthetic biology applications.

APPROACHES TO INCREASING THE EFFICIENCY OF THE ELECTROCATALYSIS OF CYTOCHROME P450 3A4

An electrochemical system based on cytochrome P450 and riboflavin has been developed, which makes it possible to increase the yield of the product in a model system using cytochrome P450 substrate - erythromycin. Achieved an increase in the catalytic activity of cytochrome P450 by 28-36%.

Nanostructures of diamond, graphene oxide and graphite inhibit CYP1A2, CYP2D6 and CYP3A4 enzymes and downregulate their genes in liver cells.

Introduction and objectiveCurrently, carbon nanostructures are vastly explored materials with potential for future employment in biomedicine. The possibility of employment of diamond nanoparticles (DN), graphene oxide (GO) or graphite nanoparticles (GN) for in vivo applications raises a question of their safety. Even though they do not induce a direct toxic effect, due to their unique properties, they can still interact with molecular pathways. The objective of this study was to assess if DN, GO and GN affect three isoforms of cytochrome P450 (CYP) enzymes, namely, CYP1A2, CYP2D6 and CYP3A4, expressed in the liver.MethodsDose-dependent effect of the DN, GO and GN nanostructures on the catalytic activity of CYPs was examined using microsome-based model. Cytotoxicity of DN, GO and GN, as well as the influence of the nanostructures on mRNA expression of CYP genes and CYP-associated receptor genes were studied in vitro using HepG2 and HepaRG cell lines.ResultsAll three nanostructures interacted with the CYP enzymes and inhibited their catalytic activity in microsomal-based models. CYP gene expression at the mRNA level was also downregulated in HepG2 and HepaRG cell lines. Among the three nanostructures, GO showed the most significant influence on the enzymes, while DN was the most inert.ConclusionOur findings revealed that DN, GO and GN might interfere with xenobiotic and drug metabolism in the liver by interactions with CYP isoenzymes responsible for the process. Such results should be considered if DN, GO and GN are used in medical applications.

ANTICANCEROGENIC ACTIVITY OF BRASSINOSTEROIDS IN LIVER TUMOR CELLS

In this study, we first characterized the effect of natural brassinosteroids, 24-epibrassinolide (EBl) and 28-homocastasterone (HCS), and synthetic analogs, (22S,23S)-24-epibrassinolide and (22S,23S)-28-homocastastone, on the growth of the cancer cell line Hep G2 (hepatocellular carcinoma), as well as on the catalytic activity of cytochrome P450, which participates in the metabolism of most procarcinogens. All four compounds at high concentrations suppressed the proliferation of the test cell line. It is also interesting that at low concentrations, 24-EBl, (22S,23S)-24-EBl and (22S,23S)28-HCS activated significantly the Hep G2 cell growth. All studied brassinosteroids, except for 28-HCS, inhibited the activity of CYP1A1 and CYP1B1. The effect depended on the structure of the side chain and was more pronounced in the case of the SS orientation of the hydroxyl groups at the positions C22 and C23 ((22S,23S)-28-homocastasterone). The results of this work suggest that the studied brassinosteroids (especially (22S,23S)-28-homocastasterone) can be used to create effective drugs for tumor prevention and treatment.

Hard coral (Porites lobata) extracts and homarine on cytochrome P450 expression in Hawaiian butterflyfishes with different feeding strategies

Dietary specialists tend to be less susceptible to the effects of chemical defenses produced by their prey compared to generalist predators that feed upon a broader range of prey species. While many researchers have investigated the ability of insects to detoxify dietary allelochemicals, little research has been conducted in marine ecosystems. We investigated metabolic detoxification pathways in three species of butterflyfishes: the hard coral specialist feeder, Chaetodon multicinctus, and two generalist feeders, Chaetodon auriga and Chaetodon kleinii. Each species was fed tissue homogenate of the hard coral Porites lobata or the feeding deterrent compound homarine (found in the coral extract), and the expression and catalytic activity of cytochrome P450 (CYP) 3A-like and CYP2-like enzymes were examined after one-week of treatment. The P. lobata homogenate significantly induced content and catalytic activity of CYP2-like and CYP3A-like forms, by 2–3 fold and by 3–9 fold, respectively, in C. multicinctus. Homarine caused a significant decrease of CYP2-like and CYP3A-like proteins at the high dose in C. kleinii and 60–80% mortality in that species. Homarine also induced CYP3A-like content by 3-fold and catalytic activity by 2-fold in C. auriga, while causing non-monotonic increases in CYP2-like and CYP3A-like catalytic activity in C. multicinctus. Our results indicate that dietary exposure to coral homogenates and the feeding deterrent constituent within these homogenates caused species-specific modulation of detoxification enzymes consistent with the prey selection strategies of generalist and specialist butterflyfishes.

Electrochemical methods in biomedical studies

Own experimental studies on the development of highly sensitive methods of electrochemical analysis applicable for biochemical research in the postgenomic era as well as electrochemical sensor systems for analysis of various biological objects have been summarized. Electroanalysis of catalytic activity of cytochrome P450 resulted in the development of a system for screening potential substrates and/or inhibitors of this class hemoproteins, as well as biologically active compounds modulating the catalytic function of this protein. The study of kinetics of bioaffinity troponin I/anti-troponin I (antibody to TnI) interactions in human plasma resulted in the development of a highly sensitive piezoelectric immunosensor, performing direct registration of biochemical interactions based on the difference of the kinetic parameters of specific and nonspecific bioaffinity interactions without additional administration of labels and without chemical modifications. The developed methods of direct registration of the electrochemical activity of bacterial cells Escherichia coli JM109 are applicable for real time evaluation of antibacterial activity of drug substances; this requires minimal volumes of cells (106 CFU/electrode). Special attention is paid to experimental data on preparation of polymers with molecular imprints (molecularly imprinted polymers, MIP) as analogues of antibodies and biorecognizing elements, carrying selective complementary analytes binding based on the “lock and key” principle.

Significance of CYP2C9 genetic polymorphism in inhibitory effect of Δ9-tetrahydrocannabinol on CYP2C9 activity

We recently reported that Δ9-tetrahydrocannabinol (Δ9-THC), the primary psychoactive constituent in marijuana, potently inhibits the catalytic activity of cytochrome P450 (CYP) 2C9.1, a wild-type variant. However, effects of CYP2C9 genetic polymorphisms on the inhibitory potency of Δ9-THC have not been investigated. In this study, in vitro inhibitory effects of Δ9-THC on catalytic activities of two major allelic variants, CYP2C9.2 and CYP2C9.3, were examined with the recombinant enzymes. Δ9-THC inhibited S-warfarin 7-hydroxylation, diclofenac 4′-hydroxylation, and tolbutamide 4-hydroxylation by CYP2C9.2 and CYP2C9.3 in a concentration-dependent manner. The IC50 values for the catalytic activities toward S-warfarin, diclofenac, and tolbutamide were 2.39, 1.04, and 1.12 μM, respectively, for CYP2C9.2, and 2.91, 1.38, and 1.09 μM, respectively, for CYP2C9.3. The inhibitory potencies against these mutant variants were similar to those against the wild-type variant. In contrast, Δ9-THC inhibited 7-methoxy-4-(trifluoromethyl)coumarin O-demethylase activity of CYP2C9.1, CYP2C9.2, and CYP2C9.3 to various extents, showing the IC50 values of 1.02, 4.20 and >10 μM, respectively. Our recent study showed that a preincubation of Δ9-THC in the presence of NADPH decreased the inhibitory effect on CYP2C9.1 activity. Then the effect of preincubation on Δ9-THC-mediated inhibition of S-warfarin 7-hydroxylation was investigated with CYP2C9.2 and CYP2C9.3. The preincubation of Δ9-THC with CYP2C9.2 attenuated the inhibitory effect of the phytocannabinoid, whereas preincubation with CYP2C9.3 did not. These results indicate that the inhibitory effects of Δ9-THC on CYP2C9 variants depend on the substrates used and the preincubation time.

Abstract A136: Nonclinical disposition, metabolism, and in vitro drug-drug interaction assessment of DM1, a component of trastuzumab emtansine (T-DM1).

Abstract Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate in clinical development for the treatment of human epidermal growth factor receptor 2 (HER2)-positive cancers. T-DM1 is composed of trastuzumab, a stable linker, and the microtubule polymerization inhibitor DM1, a derivative of maytansine. After binding to HER2 on tumor cells, T-DM1 undergoes receptor-mediated internalization and is expected to go through proteolytic degradation and catabolism, resulting in the directed delivery of DM1 to the tumor cells overexpressing HER2. Although catabolism and elimination of T-DM1 have been reported previously, little is known about the metabolism and elimination of the DM1 component. This abstract summarizes aspects of the metabolism and distribution of DM1 to further the understanding of its disposition and potential for T-DM1 drug-drug interactions. In vivo disposition and metabolism in rats: [3H]-DM1 at 91 μCi/kg (200 μg/kg) was administered to rats via a bolus intravenous injection. Blood, various tissues, urine, bile and feces were collected over the following 3 to 5 days and analyzed for radioactivity. DM1 distributed rapidly and extensively to the lungs, liver, kidneys, spleen, heart, gastrointestinal tract, and adrenal glands. The major route of excretion of DM1 was through the bile/feces, with a minimal amount excreted in the urine. A good mass balance (∼100% recovery of the injected dose) was achieved over 5 days. DM1 was extensively metabolized to multiple metabolites through oxidation, hydrolysis, methylation, and glutathione conjugations. In vitro metabolism and potential for drug-drug interactions: To identify the major human cytochrome P450 (CYP) isoforms responsible for the metabolism of DM1 and determine the metabolites formed, DM1 was incubated in human liver microsomes (HLMs) in the presence of selective chemical inhibitors and with human recombinant CYP. The results of these studies demonstrated that DM1 was metabolized mainly by CYP3A4 and, to a lesser extent, by CYP3A5. The oxidative and hydrolysis metabolites of DM1 were detected in the in vitro incubation. The induction and inhibitory properties of DM1 on CYP were evaluated using cryopreserved human hepatocytes and HLMs, respectively. DM1 was found to be neither an inducer nor an inhibitor of major CYP isoforms up to the highest concentrations tested at 1 μM (738 ng/mL) and 0.7 μM (500 ng/mL), respectively. In addition, a monolayer assay using MDCKII-MDR1 cells assessed the potential of DM1 as a substrate and inhibitor of the P-glycoprotein (P-gp) transporter. DM1 was found to be a substrate, but not an inhibitor, of P-gp when tested at 0.5 M (369 ng/mL). Conclusion: Collectively, the in vitro data suggest that the DM1 component of T-DM1 has little potential for drug-drug interactions as a perpetrator, especially considering the low systemic exposure of DM1 (averaging 5 ng/mL or 0.007 μM) achieved in clinical studies and its lack of inhibition of CYP catalytic activities and P-gp activity at clinically relevant concentrations. This is consistent with the lack of evidence of drug-drug interaction observed in phase Ib/II clinical studies of T-DM1 combined with pertuzumab and taxanes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A136.

Astaxanthin can alter CYP1A-dependent activities via two different mechanisms: Induction of protein expression and inhibition of NADPH P450 reductase dependent electron transfer

Astaxanthin (Ax), a xanthophyll carotenoid, is reported to induce cytochrome P450 (CYP) 1A-dependent activity. CYP1A is one of the most important enzymes participating in phase I metabolism for chemicals, and it can activate various mutagens. To investigate the effect of Ax on the metabolic activation of a typical promutagen, benzo[a]pyrene by CYP1A, we orally administrated Ax-containing oil (100mg Ax/kg body weight/day for 3days) to male Wistar rats. In the treated rat liver, expression of CYP1A1 mRNA, protein, and its activity were significantly increased (5.5-, 8.5-, and 2.5-fold, respectively). In contrast, the activities of phase II enzymes (glutathione S-transferase and glucuronosyl-transferase) were not modulated by Ax-containing oil. As a consequence, the mutagenicity of benzo[a]pyrene was more enhanced in Ax-treated rats, compared with controls in the Ames assay. On the other hand, NADPH P450 reductase activity was decreased in liver microsomes from the treated group. This result suggests the possibility that Ax inhibits the electron supply necessary for CYP catalytic activities and decreases CYP1A activity indirectly. In conclusion, Ax-containing oil intake can alter CYP1A-dependent activities through two different mechanisms: (1) induction of CYP1A1 mRNA, protein expression, and activity; and (2) inhibition of the electron supply for the enzyme.

In Vitro inhibitory potential of decursin and decursinol angelate on the catalytic activity of cytochrome P-450 1A1/2, 2D15, and 3A12 isoforms in canine hepatic microsomes

Danggui is one of the most popular herbal medicines consumed by patients in different clinical settings in Asian countries. In this study, the two major pyranocoumarin compounds extracted from the Korean Angelica gigas root decursin (DC) and decursinol angelate (DA) were examined in vitro with regard to their abilities to inhibit hepatic CYP1A1/2, CYP2D15, and CYP3A12 catalytic activities in canine liver microsomes. The two components were capable of inhibiting CYP1A1/2, CYP2D15, and CYP3A12 catalytic activities, but the potencies varied. DC and DA selectively and noncompetitively inhibited CYP1A1/2 activity, with K ( i ) values of 90.176 and 67.560 microM, respectively. On the other hand, they exhibited slight inhibitory effects on CYP2D15 and CYP3A12 with K ( i ) values of 666.180 and 872.502 microM, 990.500 and 909.120 microM (1'hydroxymidazolam, MDZ1'H), and 802.800 and 853.920 microM (4-hydroxymidazolam, MDZ4H), respectively. Additionally, they showed increased inhibition after preincubation, which suggests the involvement of a mechanism-based inhibition. In sum, this in vitro data should be heeded as a signal of possible in vivo interactions. The use of human liver preparations would considerably strengthen the practical impact of the data generated from this study.

MG-132 inhibits the TCDD-mediated induction of Cyp1a1 at the catalytic activity but not the mRNA or protein levels in Hepa 1c1c7 cells

Previous studies have shown that 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced degradation of aryl hydrocarbon receptor (AhR) is inhibited by MG-132, a potent inhibitor of the 26S proteasome. Therefore, the current study aims to address the effect of MG-132 on the AhR-regulated gene, cytochrome P450 1a1 ( Cyp1a1), using murine hepatoma Hepa 1c1c7 cells. Our results showed that MG-132 at the highest concentration tested, 10 μM significantly increased the Cyp1a1 at mRNA, protein and catalytic activity levels through a transcriptional mechanism. On the other hand, MG-132 further potentiated the TCDD-mediated induction of Cyp1a1 at mRNA but not at protein level. In contrast, MG-132 significantly inhibited the TCDD-mediated induction of Cyp1a1 catalytic activity. In addition, we showed that the decrease in Cyp1a1 catalytic activity is not Cyp specific, as MG-132 significantly inhibited Cyp2b1 and total cytochrome P450 catalytic activities. These results prompted us to examine the effect of MG-132 on total cellular heme content and heme oxygenase-1 (HO-1) mRNA, a rate limiting enzyme of heme degradation. Our results showed that MG-132 significantly induced HO-1 mRNA in a concentration-dependent manner. Furthermore, MG-132 potentiated the induction of HO-1 mRNA by TCDD in a concentration-dependent manner. The induction of HO-1 mRNA level coincided with a decrease in total cellular heme content. In conclusion, the present study demonstrates for the first time that MG-132, despite of increasing Cyp1a1 mRNA expression, it decreases its activity probably through decreasing its heme content.

Catalytic Activity of Cytochrome P-450 using NADP+ Reduced by an Anionic Hydride Organosiloxane

Abstract Cytochrome P-450 (P450) catalyzes a wide variety of chemical reactions; however, its use for in vitro assays has several limitations, the most striking one is the use of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) coenzyme. In this work, the P450 activity using NADP+ reduced by an anionic organosiloxane, commonly named silica hydride, was evaluated. The results showed that the reduction of NADP+with silica hydride was concentration- and time-dependent. P-450 activity was maintained when NADP+ and silica hydride were added during the reaction; however, it was lower than when commercial NADPH was employed. This is due to the ability of silica hydride to reduce P450 iron atom as corroborated by the electronic paramagnetic resonance (EPR). Furthermore, this compound possibly chelates FeII because, in its presence, the P450 affinity for aniline diminishes. However, the P450 activity was the best when NADP+ was reduced by silica hydride before the former was added to the reaction. Therefore, this system could be apt for studying biotransformation reactions.

Cytochrome P450 3A, NADPH cytochrome P450 reductase and cytochrome b5 in the upper airways in horse

Gene and protein expression as well as catalytic activity of cytochrome P450 (CYP) 3A were studied in the nasal olfactory and respiratory mucosa and the tracheal mucosa of the horse. We also examined the activity of NADPH cytochrome P450 reductase (NADPH P450 reductase), the amount of cytochrome b 5 and the total CYP content in these tissues. Comparative values for the above were obtained using liver as a control. The CYP3A related catalytic activity in the tissues of the upper airways was considerably higher than in the liver. The CYP3A gene and protein expression, on the other hand, was higher in the liver than in the upper airway tissues. Thus, the pattern of CYP3A metabolic activity does not correlate with the CYP3A gene and protein expression. Our results showed that the activity of NADPH P450 reductase and the level of cytochrome b 5 in the relation to the gene and protein expression of CYP3A were higher in the tissues of the upper airways than in the liver. It is concluded that CYP3A related metabolism in horse is not solely dependent on the expression of the enzyme but also on adequate levels of NADPH P450 reductase and cytochrome b 5.

Effect of silymarin on pyrogallol- and rifampicin-induced hepatotoxicity in mouse

Rifampicin and pyrogallol, besides beneficial effects, elicit hepatotoxicity in experimental animals and humans. The present investigation was undertaken to elucidate the role of drug/toxicant-metabolizing enzymes in rifampicin- and pyrogallol-induced hepatotoxicity and the effect of silymarin, a herbal antioxidant, on rifampicin- and pyrogallol-induced alterations in mouse liver. Male Swiss albino mice were treated intraperitoneally with and without rifampicin (20 mg/kg) and/or pyrogallol (40 mg/kg) for 1, 2, 3 and 4 weeks. In some experiments, animals were treated with silymarin (40 mg/kg), 2 h prior to rifampicin and/or pyrogallol. The differential expression and catalytic activity of cytochrome P-450 ( CYP) 1A1, CYP1A2 and CYP2E1, the activity of glutathione- S-transferase, glutathione peroxidase and glutathione reductase, and lipid peroxidation were measured in the liver of control and treated groups. CYP1A1 expression and catalytic activity were not altered following individual or combinational treatment. A significant augmentation in the expression and activity of CYP1A2 and CYP2E1 was observed following pyrogallol and rifampicin + pyrogallol treatment; however, rifampicin exhibited a significant induction of CYP2E1 only. Attenuation of glutathione- S-transferase, glutathione reductase and glutathione peroxidase activities and augmentation of lipid peroxidation were observed following rifampicin and/or pyrogallol treatment and a cumulative effect was seen when the two drugs were administered in combination. Silymarin restored the rifampicin- and/or pyrogallol-induced alterations in the expression and activity of CYP1A2 and CYP2E1, the activity of glutathione- S-transferase, glutathione reductase, and glutathione peroxidase, and lipid peroxidation. The results demonstrate the role of CYP1A2, CYP2E1, glutathione- S-transferase, glutathione reductase and glutathione peroxidase in rifampicin- and pyrogallol-induced hepatotoxicity and provide evidence for the involvement of silymarin in attenuation of drug-induced hepatotoxicity.

The Role of Cytochrome P450 1B1 in Dibenzo[ a,l ]pyrene-induced Carcinogenesis

In human cells, the most carcinogenic polycyclic aromatic hydrocarbon dibenzo[ a,l ]pyrene (DB[ a,l ]P) forms high levels of DNA adducts through formation of the ( m )- anti -(11 R ,12 S )-diol (13 S ,14 R )-epoxide (DB[ a,l ]PDE) and its metabolic precursor, the ( m )-(11 R ,12 R )-diol. Generation of these adducts results from the catalytic activity of cytochrome P450 (P450) 1A1 and 1B1. Additional adducts such as (+)- syn -DB[ a,l ]PDE-DNA or more polar DNA adducts were detected only after increasing exposure doses of the parent compound or in cells that express P450 1A1. At low concentrations (·;100 nM) exclusively ( m )- anti -DB[ a,l ]PDE-DNA adducts were formed by P450 1B1, which is constitutively expressed in many mammalian tissues. Measurement of DNA binding and mutagenicity of DB[ a,l ]P in V79 cells expressing human P450 enzymes revealed a higher activity of P450 1B1 compared to 1A1 at low concentrations. Treatment of P450 1B1 knockout mice and DNA binding studies with fibroblasts isolated from these animals provided further evidence for the central role of P450 1B1-catalyzed formation of ( m )- anti -DB[ a,l ]PDE-DNA adducts in DB[ a,l ]P-induced carcinogenesis.

The Role of Cytochrome P450 1B1 in Dibenzo[ a,l ]pyrene-induced Carcinogenesis

In human cells, the most carcinogenic polycyclic aromatic hydrocarbon dibenzo[ a,l ]pyrene (DB[ a,l ]P) forms high levels of DNA adducts through formation of the ( m )- anti -(11 R ,12 S )-diol (13 S ,14 R )-epoxide (DB[ a,l ]PDE) and its metabolic precursor, the ( m )-(11 R ,12 R )-diol. Generation of these adducts results from the catalytic activity of cytochrome P450 (P450) 1A1 and 1B1. Additional adducts such as (+)- syn -DB[ a,l ]PDE-DNA or more polar DNA adducts were detected only after increasing exposure doses of the parent compound or in cells that express P450 1A1. At low concentrations (·;100 nM) exclusively ( m )- anti -DB[ a,l ]PDE-DNA adducts were formed by P450 1B1, which is constitutively expressed in many mammalian tissues. Measurement of DNA binding and mutagenicity of DB[ a,l ]P in V79 cells expressing human P450 enzymes revealed a higher activity of P450 1B1 compared to 1A1 at low concentrations. Treatment of P450 1B1 knockout mice and DNA binding studies with fibroblasts isolated from these animals provided further evidence for the central role of P450 1B1-catalyzed formation of ( m )- anti -DB[ a,l ]PDE-DNA adducts in DB[ a,l ]P-induced carcinogenesis.

Macrolide – induced clinically relevant drug interactions with cytochrome P‐450A (CYP) 3A4: an update focused on clarithromycin, azithromycin and dirithromycin

Macrolide – induced clinically relevant drug interactions with cytochrome P‐450A (CYP) 3A4: an update focused on clarithromycin, azithromycin and dirithromycin

Glutathione-dependent defence system and monooxygenase enzyme activities in Arctic charr Salvelinus alpinus (L.) exposed to ozone

One of the major obstacles of the increasing usage of ozone in aquaculture is the lack of relevant risk assessment in culture conditions. Before the apparent advantages of ozonation can be utilised efficiently, the safety margins and biological basis of ozone toxicity should be assessed. In this research, 1-year-old Arctic charr (Salvelinus alpinus (L.)) were exposed to an ozone concentration high enough to inactivate Aeromonas sp. bacteria in freshwater, but too low to be directly lethal to the fish themselves. The effects of ozone exposure on the activity of glutathione-dependent antioxidant enzymes and monooxygenase reactions were studied in blood and in liver. The fish were acclimated for 2 months at 10.3°C and 14.1°C followed by an ozone exposure for 15 and 30 min. The fish were exposed to ozone at both temperatures giving a calculated O3 dose of 0.34 and 0.69 mg/l min during the above sampling times. Oxidised glutathione (GSSG) and oxidative stress index ([2×GSSG/total glutathione]×100) were significantly elevated in whole blood by ozone exposure at both temperatures indicative of oxidative stress. At the same time, no signs of oxidative stress were detected in liver. However, total glutathione (tGSH) concentrations in liver were elevated at 14°C indicating increased ability of liver tissue to defend against reactive oxygen species (ROS). Glutathione S-transferase (GST), GSSG and cytochrome P-450 catalytic activity (ethoxyresorufin-O-deethylase, EROD) in liver were not influenced by ozone exposure. Initially, both hepatic glutathione reductase (GR) and GSH were elevated at 10°C, possibly indicating ability to compensate for temperature. This study demonstrates that even the lowest dose (0.34 mg/l min) of ozone was high enough to cause oxidative stress in the blood of Arctic charr and the signs of oxidative stress occur throughout the exposure. Furthermore, a blood sample is a reliable source of information when assessing the status of the glutathione-dependent antioxidant system after a short-term ozone exposure.

Study of abzymes with cytochrome P-450 catalytic activity.

Study of abzymes with cytochrome P-450 catalytic activity.

Characterization of the critical amino acids of an Aspergillus parasiticus cytochrome P-450 monooxygenase encoded by ordA that is involved in the biosynthesis of aflatoxins B1, G1, B2, and G2.

The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B1, G1, B2, and G2 requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B1. Complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restored the ability to produce aflatoxins B1, G1, B2, and G2. The ordA gene placed under the control of the GAL1 promoter converted exogenously supplied OMST to aflatoxin B1 in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the ordA1 gene had three point mutations which resulted in three amino acid changes (His-400-->Leu-400, Ala-143-->Ser-143, and Ile-528-->Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the ordA gene in the synthesis of aflatoxins G1, and G2 in A. parasiticus suggests that enzymes required for the formation of aflatoxins G1 and G2 are not present in A. flavus. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. The ordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee.