Oxidative stress in ecotoxicology: from the analysis of individual antioxidants to a more integrated approach.

An integrate approach will be discussed for investigations on oxidative stress in xenobiotic toxicity. While the analysis of individual antioxidants is useful for their sensitivity and to understand the mode of action of a stressor, the integration with the analysis of the total antioxidant capacity provides a more holistic assessment of the overall biological significance of such variations. TOSC has a greater predictive value on the health condition of the organisms and allows to discriminate the different role of specific ROS in oxidative stress syndrome.

Seasonal variations of susceptibility to oxidative stress in Adamussium colbecki, a key bioindicator species for the Antarctic marine environment.

The area of free radical biology is of increasing interest for marine organisms since the enhanced formation of reactive oxygen species (ROS) is a common pathway of toxicity induced by stressful environmental conditions. In polar environments responses of the antioxidant system could be useful as an early detection biomarkers of unforeseen effects of human activities which are progressively increasing in these remote areas. However, the characterization of antioxidant defences in appropriate sentinel species is of particular value also in terms of a possible adaptation to this extreme environment. The scallop, Adamussium colbecki, is a key species for monitoring the Antarctic environment and, besides single antioxidants, the total oxyradical scavenging capacity (TOSC) assay has been recently used for quantifying the overall ability of this organism to neutralize peroxyl radicals (ROO*), hydroxyl radicals (*OH) and peroxynitrite (HOONO). The aim of this work was to obtain a better characterization of these biological responses which can indicate the occurrence of biological disturbance; in this study the total oxyradical scavenging capacity was further analyzed to assess the presence of seasonal fluctuations in the susceptibility to oxidative stress in this species. The capability to neutralize peroxyl radicals and hydroxyl radicals increased at the end of December, while resistance towards peroxynitrite did not show any significant variations during the Antarctic summer. These results suggest the occurrence of metabolic changes which mainly influence intracellular formation of ROO* and *OH, with more limited effects on HOONO. Despite the limited time window analyzed, as a typical constraint in Antarctic research at Terra Nova Bay, an increased resistance to these specific oxyradicals might be related to the period of highest feeding activity, or to other intrinsic factors in the animals' physiology such as the phase of reproductive cycle.

Sex-related responses to oxidative stress in primary cultured hepatocytes of European flounder (Platichthys flesus L.).

Effects of oxidative stress induced by xenobiotic compounds were studied in primary cultures of isolated hepatocytes of immature European flounder (Platichthys flesus L.) of both sexes caught in a relatively unpolluted area of the German Bight (North Sea). Cells were exposed to oxidative stressors such as 100 microM hydrogen peroxide (H2O2), 100 microM benzo[a]pyrene (B[a]p) and 50 microM nitrofurantoin (N-(5-nitro-2-furfurylidene)-1-aminohydantoin; NF) for 2 and 24 h. Cell mortality was determined with the use of the fluorescent ethidium homodimer-1 and calcein. Oxidative stress response was assessed by quantitative analysis of (1) intracellular reactive oxygen species (ROS) formation with dihydrorhodamine 123, (2) lipid peroxidation on the basis of concentrations of lipid hydroperoxides and the lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) and (3) cellular total oxidant-scavenging capacity (TOSC) using the TOSC assay (Winston et al., 1998). An increase in ROS formation was detected as early as 2 h after exposure to H2O2, B[a]p and NF. After 24 h, stress responses were lower, except following exposure to NF. The pattern of responses differed with the different oxidative stressors. Lipid peroxidation and the capacity to scavenge ROS were increased significantly in both sexes only after exposure to H2O2, whereas B[a]p and NF provoked sex-dependent responses. B[a]p-induced lipid peroxidation and increase in scavenging capacity were observed only in hepatocytes of females, whereas NF initiated these responses only in cells of males. Sex differences in oxidative stress response only after exposure to pro-oxidants that require enzymatic activation infer the importance of biotransformation pathways in stress responses. Because of their sensitivity to oxidative stress, flounder hepatocytes provide a useful model for early risk assessment of xenobiotics.

Oxidation of thiodiglycol (2,2'-thiobis-ethanol) by alcohol dehydrogenase: comparison of human isoenzymes.

Sulfur mustard is a chemical warfare agent that causes blistering of the skin and damages the eyes and airway after environmental exposure. We have previously reported that thiodiglycol (TDG, 2,2'-bis-thiodiethanol), the hydrolysis product of sulfur mustard, is oxidized by alcohol dehydrogenase (ADH) purified from horse liver or present in mouse liver and human skin cytosol. Humans express four functional classes of ADH composed of several different isozymes, which vary in their tissue distribution, some occurring in skin. To help us evaluate the potential contribution of the various human isozymes toward toxicity in skin and in other tissues, we have compared the catalytic activity of purified human class I alphaalpha-, beta1beta1-, beta2beta2-, and gamma1gamma1-ADH, class II pi-ADH, class III chi-ADH, and class IV sigma-ADH with respect to TDG oxidation and their relative sensitivities to inhibition by pyrazole. Specific activities toward TDG were 123, 79, 347, 647, and 12 nmol/min/mg for the class I alphaalpha-, beta1,beta1-, beta2beta2-, and gamma1gamma1-ADH and class II pi-ADH, respectively. TDG was not a substrate for class III chi-ADH. The specific activity of class IV sigma-ADH was estimated at about 1630 nmol/min/mg. 1 mM pyrazole, a potent inhibitor of class I ADH, inhibited the class I alphaalpha, beta1beta1, beta2beta2, and gamma1gamma1 ADH and class IV sigma-ADH by 83, 100, 56, 90, and 73%, respectively. The class I alphaalpha- and beta1beta1-ADH oxidized TDG with kcat/Km value of 7-8 mM(-1) min(-1), beta2beta2-ADH with a value 19 mM(-1) min(-1) and class I gamma1gamma1-ADH with a value of 176 mM(-1) min(-1). The kcat/Km value for class IV sigma-ADH was estimated at 4 mM(-1) min(-1). The activities of class IV sigma-ADH and class I gamma1gamma1-ADH are of significant interest because of their prevalence in eyes, lungs, stomach, and skin, all target organs of sulfur mustard toxicity.

Interaction of alcohol dehydrogenase with tert-butylhydroperoxide: stimulation of the horse liver and inhibition of the yeast enzymes.

Preincubation of horse liver alcohol dehydrogenase (HLADH) with the oxidative agent, tert-butyl hydroperoxide (tBOOH) results in a twofold stimulation of the ethanol dehydrogenase activity of this enzyme. This stimulation was dependent on tBOOH concentration up to 100 mM; above this concentration tBOOH did not further stimulate ethanol oxidation by HLADH. Active-site-directed reagents and classical ADH binary complexes were used to probe the possible mechanism of this activating effect. The rate and extent of stimulation by tBOOH is strongly reduced by binary complexes with NAD(+) or NADH, whose pyrophosphate groups bind to Arg-47 and Arg-369. In contrast stimulation by tBOOH was not prevented by AMP or the sulfhydryl reagents dithiothreitol and glutathione, suggesting, respectively, a lack of role for Lys-228 and sulfhydryl group oxidation in the stimulation by tBOOH. In contrast to the liver enzyme, treatment of yeast ADH (YADH) with tBOOH irreversibly inhibited its ethanol dehydrogenase activity. Inhibition of YADH by tBOOH approximated first-order rate kinetics with respect to enzyme at fixed concentrations of tBOOH between 0.5 to 300 mM. Four -SH groups per molecule of YADH were modified by tBOOH, whereas only two -SH groups were modified in HLADH. The stimulation of HLADH by tBOOH is suggested to be due to destabilization of the catalytic Zn-coordination sphere and amino acids associated with coenzyme binding in the active site, while inactivation of YADH appears to be associated with -SH group oxidation by the peroxide.

Evaluation of the total peroxyl radical-scavenging capacity of flavonoids: structure-activity relationships.

The antioxidant activity of a series of flavonoids against peroxyl radicals generated from thermal homolysis of 2, 2'-azobis-amidinopropane was determined by the Total Oxyradical Scavenging Capacity (TOSC) assay. Seven flavonoids with hydroxy and/or methoxy substitution were analyzed and compared to the water-soluble vitamin E analogue Trolox. The most active compound was the flavonol quercetin, followed by its 3-glycoside derivative rutin; these were 7 and 5 times, respectively, better scavengers of peroxyl radical than Trolox. Among the flavones with both hydroxy and methoxy substitution, the most active against peroxyl radicals was the 5,6,4'-trihydroxy-7,8,3'-trimethoxyflavone (thymonin), with a TOSC value 1.5 times greater than that of Trolox. The activity of the remaining flavones was in the following relative order: 5, 4'-dihydroxy-6,7,8,3'-tetramethoxyflavone > 5-hydroxy-3,6,7,3', 4'-pentamethoxyflavone (artemetin) > 5,4'-dihydroxy-3,6, 7-trimethoxyflavone > 5,6,7,8,2',3',4',5'-octamethoxyflavone (agehoustin A). The data suggest a potential role for dietary intake of flavonoid-containing foods in lowering the risk of certain pathophysiologies that have been associated with free-radical-mediated events.

Distribution of microsomal CO-binding chromophores and EROD activity in sea anemone tissues.

Our previous studies indicated that sea anemone microsomes contain cytochrome P450 (CYP) and have ethoxyresorufin O-dealkylation (EROD) activity. Other marine invertebrates have discrete organs which concentrate cytochromes P450, whereas cnidarians have evolved only to the tissue level of development. To examine the distribution of CYP in sea anemones, microsomes were prepared from the following tissue regions of two sea anemones, Anthopleura xanthogrammica: outer (heavy muscular wall), inner (imperfect and perfect mesentery, and retractor muscle), soft (digestive sac, gonads, and mesentery filaments), and tentacular (including algal/diatom symbiont). The cytochrome P450 content was distributed relatively evenly among the tissue regions. In contrast, the 418-nm CO-binding chromophore was approximately 10 times greater in the outer region than in any other region. Further, the 490-nm peak (which interferes with quantification of CYP in sea anemones) was greater in the outer region. In general, the EROD activity was comparable in the inner and soft regions and highest in the tentacles. However, the EROD results may have been complicated by the presence of the algal/diatom symbiont.

Microsomal deacetylation of ring-hydroxylated 2-(acetylamino)fluorene isomers: effect of ring position and molecular mechanics considerations.

Metabolism of arylamides such as 2-(acetylamino)fluorene to mutagenic products is catalyzed by various liver microsomal and cytosolic enzymes. Deacylation is believed to be a deactivating pathway, and the activity of the microsomal deacetylase toward N-hydroxy-2-AAF is exceedingly greater than toward the parent 2-AAF. Another deactivating pathway is cytochrome P450-catalyzed ring hydroxylation. We have studied the effect of ring hydroxyl substitution on the activity of the liver microsomal deacetylase from Aroclor 1254-treated rats in vitro. The deacetylase activity was generally decreased toward ring-hydroxylated derivatives in the order of 2AAF approximately 1-OH-AAF > 3-OH-AAF > 7-OH-AAF > 5-OH-AAF approximately 9-OH-AAF. The difference in activity between 2-AAF and 5-OH- and 9-OH-AAF was about eightfold. Molecular mechanics calculations reveal that structural and geometrical parameters are more important than the energies associated with the different isomers. We show that the greater the distance of the hydroxyl group on the fluorenyl ring structure from the acetylamino group, the slower the rate of deacetylation. The difference in reactivity between the 1-hydroxy-2-AAF and the other hydroxy-2-AAF isomers is due to the lack of planarity of the 1-hydroxy isomer as compared to the essentially planar configuration of the other isomers. The relative contribution of microsomal ring hydroxylation and deacetylation to detoxification of arylamides remains to be established.

Immunochemical analysis of liver microsomal cytochromes P450 of the American alligator, Alligator mississippiensis.

Ten antibodies raised against various mammalian and fish cytochromes P450 (CYP) enzymes were used to probe the effects of xenobiotic pretreatment on liver microsomes of the American alligator, Alligator mississippiensis. Pretreatment with phenobarbital (PB), 3-methylcholanthrene (3MC), and PB plus 3MC elicited significant induction of multiple CYP enzymes in alligator, as detected by antibodies to CYP1A, CYP2B, CYP2C, CYP2E, CYP2K, and CYP3A. In contrast to the rat, 3MC treatment induced alligator liver microsomes that were immunoreactive with antibodies to CYP2 family enzymes. Induction of CYP enzymes was not as apparent with the Aroclor 1254 (ARO), and 2,2',4,4' tetrachlorobiphenyl (TCB) pretreatment used; fewer CYP enzymes primarily detected with antibodies against CYP2C or CYP2E were observed. Clofibrate (CLO; 80 mg/kg Days 1-4), markedly induced CYP4A in rat but this induction was not apparent in alligator. A purified PB-induced alligator liver microsomal CYP enzyme cross-reacted with several antibodies raised against CYP2 family enzymes but did not cross-react with antibodies raised against other CYP families. This indicates the PB-inducible CYP in alligator shares some epitope homology with several CYP2-family enzymes from other animals. These experiments demonstrate the usefulness and limitations of using antibodies across phylogenetic classes. While indicating the presence of CYP enzymes that have epitope homology with CYP1A, CYP2, CYP3 and CYP4 enzymes in alligator, it remains to be established whether these CYP forms are alligator orthologues of mammalian enzymes. In all cases, the relative abundance of alligator liver microsomal CYP as determined by immunoblot analysis appeared lower than found in rat. The presence and induction of CYP indicated by immunochemical analysis, corroborated previously reported enzymatic studies of the same microsomal preparations (Ertl et al., 1998a). Thus, increases in CYP protein by the various inducers employed were paralled by the increases in CYP enzyme-specific or selective activities, e.g., induction of CYP1A protein corresponded with induction of EROD.

Enzymatic and nonenzymatic production of free radicals from the carcinogens 4-nitroquinoline N-oxide and 4-hydroxylaminoquinoline N-oxide.

The anion radicals of 4-nitroquinoline N-oxide (4-NQO) and 4-nitrosoquinoline N-oxide (4-NOQO) carcinogens were detected and characterized by electron spin resonance (ESR) spectroscopy. The structures of the radical intermediates were examined by density functional theory (DFT) at the level of hybrid unrestricted uBecke3LYP. The formation of superoxide anion radical catalyzed by flavin-containing enzymes such as cytochrome P450 reductase or xanthine oxidase in the presence of 4-NQO or 4-nitroquinoline N-oxide was studied by spin-trapping experiments. In this case, the ESR signal of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-superoxide radical adduct was observed, and its formation was inhibited by superoxide dismutase (SOD). No ESR signal was detected when the two-electron-transferring flavoenzyme DT-diaphorase (NADPH-quinone oxidoreductase) was used. The above is consistent with a one-electron reduction in the metabolism of these nitro compounds to anion free radicals by various flavoenzyme reductases.

Quantification of total oxidant scavenging capacity of antioxidants for peroxynitrite, peroxyl radicals, and hydroxyl radicals.

We have extended the application of our previously reported total oxidant scavenging capacity (TOSC) assay (Winston et al., Free Radical Biol. Med. 24, 480-493, 1998) to permit facile quantification of the absorbance capacity of antioxidants toward three potent oxidants, i.e., hydroxyl radicals, peroxyl radicals, and peroxynitrite. Respectively, these oxidants were generated by the iron plus ascorbate-driven Fenton reaction, thermal homolysis of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (ABAP), and 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). Each of these oxidants reacts with alpha-keto-gamma-methiolbutyric acid (KMBA), which is oxidized and yields ethylene. The antioxidant capacity of the compounds tested is quantified from their ability to inhibit ethylene formation relative to a control reaction. Assay conditions were established in which control reactions give comparable yields of ethylene with each of the oxidants studied. Thus, the relative efficiency of various antioxidants could be compared under conditions of quantitatively similar KMBA oxidizing capability by the three oxidants. Reduced glutathione was an efficient scavenger of peroxyl radicals, but scavenged peroxynitrite and hydroxyl radicals relatively poorly. Uric acid, Trolox, and ascorbic acid were comparable scavengers of peroxynitrite and peroxyl radicals. Uric acid and Trolox were approximately an order of magnitude less efficient as scavengers of hydroxyl radicals. The classical hydroxyl radical scavenging agents mannitol, dimethyl sulfoxide, and benzoic acid had much higher TOSC values with hydroxyl than with peroxyl radicals or peroxynitrite. The very different chemical reactivity toward KMBA by the SIN-1- and iron-ascorbate-generated oxidants indicates that hydroxyl radical is not a major oxidant produced by the SIN-1 system. The data show that the TOSC assay is useful and robust in distinguishing the reactivity of various oxidants and the relative capacities of antioxidants to scavenge these oxidants.

Nitroarene reduction and generation of free radicals by cell-free extracts of wild-type, and nitroreductase-deficient and -enriched Salmonella typhimurium strains used in the umu gene induction assay.

Studies of the enzymatic properties of cell-free extracts prepared from overnight cultures of the normal, and nitroreductase-deficient and -enriched strains of Salmonella typhimurium, designed for use in the umu gene induction assay of Oda et al. (1992), were undertaken in an effort to clarify the nature of nitroreductase deficiency in relation to mutagenicity. The ability of these strains to promote oxygen consumption and free radical intermediates of representative nitroarene substrates was measured, respectively, by oxygen polarography and electron spin resonance (ESR) spectroscopy. The substrates 4-nitropyridine N-oxide (4NPO) and 4-nitroquinoline N-oxide (4NQO) stimulated the rate and extent of NADH-dependent oxygen consumption catalyzed by cell-free extracts prepared from wild-type, and nitroreductase-deficient and -enriched strains. The extent of oxygen consumption was greater than stoichiometric with respect to the amount of nitroaromatic substrate, which implied one-electron reduction of 4NQO by these bacterial extracts and subsequent redox cycling with oxygen. ESR spectroscopy confirmed the production of free radical metabolites of the nitroarene substrates, which were inferred by the oxygen consumption studies. At equal protein concentrations the cell-free extracts of each strain catalyzed univalent reduction of 4NPO yielding the 59 line signal characteristic of the 4NPO nitro anion radical. This ESR signal was potently inhibited by the flavoprotein inhibitors CuSO4 and PCMB, albeit a twofold or higher concentration of both inhibitors was required to inhibit the signal produced by extract from the nitroreductase-deficient strain than that produced by the other strains. The results indicate that the nitroreductase-deficient strain of Salmonella typhimurium developed for use in the umu gene induction assay is not deficient in either one-electron nitro group or quinone reductase activity.

Liver microsomal cytochromes P450-dependent alkoxyphenoxazone O-dealkylation in vitro by alligator and rat: activities, inhibition, substrate preference, and discrimination factors.

Six substituted alkoxyphenoxazones (resorufins) and four inhibitors of P450-dependent mixed-function oxygenases (MFO) were used to probe the breadth and extent of P450 metabolism induced by pretreatment with five xenobiotic chemicals in liver microsomes of the American alligator, Alligator mississippiensis. Phenobarbital (PB), 3-methylcholanthrene (3MC), and PB-3MC co-pretreatment elicited major induction of alligator MFO activity measured by alkoxyresorufin O-dealkylation (AROD). The induced levels of activities observed with appropriate substrate, 7-ethoxy, 7-methoxy, 2-phenylbenzyloxy, 7-pentoxy, or 7-benzyloxyresorufin (EROD, MROD, PBROD, PROD and BROD, respectively), were 10 to 100 times lower in alligator as compared to rat. The exception was a higher level of isopropoxyresorufin O-dealkylation (IPROD) in alligator. The induction regimes used in alligator and rat revealed marked differences in substrate preference, discrimination factors (DF) for various inducible P450 isoforms. EROD, a classic indicator of CYP1A activity in rat, had a low DF in alligator. MROD was the best discriminator in alligator of CYP1A-type induction. In contrast to rats, pretreatment of alligators with Aroclor 1254, 2,2',4,4' tetrachlorobiphenyl, and clofibrate caused minor alterations in AROD relative to untreated controls. The inhibitors, alpha-napthaflavone, 1-ethynylpyrene, SKF 525A, and 9-ethynylphenanthrene, inhibited AROD activity of the expected P450 isoform. For example, 10 microM alpha-napthaflavone inhibited liver microsomal EROD catalyzed by 3MC-inducible isoforms from alligator by 90% and from rat by 97%. Similarly, 10 microM SKF 525A inhibited PROD catalyzed by PB-inducible isoforms by 63% and 79% in alligator and rat liver microsomes, respectively. To the best of our knowledge, the present studies are the first to show PB induction of P450 activities typical of the mammalian CYP2 family and their inhibition with classical inhibitors in alligator liver. While our data indicate metabolism of P450 substrates with preferences to certain isoforms, it remains to be established which isoforms exert catalytic function in alligator and whether these are homologues or orthologues of mammalian isoforms.

Beta carotene and its oxidation products have different effects on microsome mediated binding of benzo[a]pyrene to DNA.

The effects of beta-carotene (betaC) and its oxidation products on the binding of benzo[a]pyrene (BaP) metabolites to calf thymus DNA was investigated in the presence of rat liver microsomes. Mixtures of betaC oxidation products (betaCOP) as well as separated, individual betaC oxidation products were studied. One set of experiments, for example, involved the use of the mixture of betaCOP obtained after a 2-h radical-initiated oxidation. For this data set, the incorporation of unoxidized betaC into microsomal membranes caused the level of binding of BaP metabolites to DNA to decrease by 29% over that observed in the absence of betaC; however, the incorporation of the mixture of betaCOP caused the binding of BaP metabolites to DNA to increase 1.7-fold relative to controls without betaC. Two variations of this experiment were studied: (1) When no NADPH was added, betaC decreased the binding of BaP metabolites to DNA by 19%, but the mixture of betaCOP increased binding by 3.3-fold relative to that observed in the absence of betaC. (2) When NADPH was added under near-anaerobic conditions, betaC caused an almost total (94%) decrease in binding whereas betaCOP had no effect on the amount of binding relative to that observed in the absence of betaC. Both betaCOP and cumene hydroperoxide caused BaP metabolites to bind to DNA even when NADPH was omitted from the incubation mixture. Separation of the mixture of betaC oxidation products into fractions by HPLC allowed preliminary testing of individual betaC oxidation products separately; of the various fractions tested, the products tentatively identified as 11,15'-cyclo-12,15-epoxy-11,12,15,15'-tetrahydro-beta-carotene and beta-carotene-5,6-epoxide appeared to cause the largest increase in BaP-DNA binding. Microsomes from rats induced with 3-methylcholanthrene (3MC) or Aroclor 1254 produced different levels of binding in some experimental conditions. We hypothesize that, under some conditions, the incorporation of betaC into microsomal membranes can be protective against P450-catalyzed BaP binding to DNA; however, the incorporation of betaCOP facilitates the formation of BaP metabolites that bind DNA, although only certain P450 isoforms catalyze the binding process.

Antibiotic effects on cytochromes P450 content and mixed-function oxygenase (MFO) activities in the American alligator, Alligator mississippiensis.

There are no Food and Drug Administration (FDA)-approved antimicrobial agents for use in cultured American alligators (Alligator mississippiensis) destined for human consumption yet some producers administer antibiotics for prophylaxis. The cytochromes P450-dependent mixed-function oxygenases (MFO) catalyze the oxidation of xenobiotic compounds such as drugs, pesticides and polycyclic aromatic hydrocarbons. Herein, we describe the effects of oxytetracycline, ceftazidime and enrofloxacin on the MFO system of the American alligator, Alligator mississippiensis. Juvenile alligators (4 animals/treatment) were administered these antibiotics intraperitoneally in an effort to induce hepatic microsomal cytochromes P450. Alligators treated with enrofloxacin exhibited emesis and convulsive spasms within 5 min of the initial injection. Total hepatic cytochromes P450 contents were significantly decreased in oxytetracycline-and enrofloxacin-pretreated alligators. In vitro hepatic microsomal benzyloxyresorufin O-dealkylase (BROD) activity was significantly decreased by enrofloxacin pretreatment. Western blots of proteins from antibiotic-pretreated alligator hepatic microsomes incubated with several mammalian and fish cytochromes P450 (CYP) antibodies exhibited little or no induction of CYP1A1, 2B, 2C and 2E1. In vitro incubation with enrofloxacin and oxytetracycline caused a concentration-dependent decrease in alkyl-substituted phenoxazone dealkylase activities catalyzed by phenobarbital- and 3-methylcholanthrene-induced alligator hepatic microsomes.

Single electron reduction of xenobiotic compounds by glucose oxidase from Aspergillus niger.

Various species of fungi express glucose oxidase that catalyzes formation of gluconolactone from glucose with concomitant, direct divalent reduction of molecular oxygen to hydrogen peroxide. A physiological function ascribed to this extracellular enzyme is production of hydrogen peroxide for use in lignin degradation catalyzed by lignin peroxidases. Herein, we show that glucose oxidase can catalyze one-electron reduction of several different classes of xenobiotic compounds resulting in generation of free radical products. Electron spin resonance (ESR) spectroscopy was used to visualize the one-electron reduction products of 4-nitropyridine-N-oxide (4NPO), 1,4-naphthoquinone (1,4NQ), and dichlorophenolindolphenol (DCPIP). Hyperfine splitting constants were used to generate computer simulations of the spectra confirming the presence of free radical products.

A rapid gas chromatographic assay for determining oxyradical scavenging capacity of antioxidants and biological fluids.

Herein, we report a new, rapid,and reliable method for measuring the protective antioxidant potential of pure antioxidant solutions or biological tissues. Peroxyl radicals generated by thermal homolysis of 2,2'-azobis-amidinopropane (ABAP) cause the oxidation of alpha-keto-gamma-methiolbutyric acid (KMBA) to ethylene; ethylene formation is monitored by gas chromatographic analysis of head space from the reaction vessel. The partial inhibition of ethylene formation in the presence of antioxidants that compete with KMBA for oxyradicals is the basis of the Total Oxyradical Scavenging Capacity Assay (TOSCA). The assay is shown to be reliable for quantifying ROS scavenging potential. The quantifiable parameters are consistent with the relative order of those predicted by the fluorescence- and oxygen electrode-based assays reported in the literature. Antioxidants competing for peroxyl radicals influenced the rate of KMBA oxidation in different ways, but the calculation of TOSC was not affected by such variations. Responses were linear over a wide range of sample concentrations and the TOSC values of classical soluble antioxidants showed the following relative order: Trolox > uric acid > ascorbic acid > GSH. The KMBA method was reliable for biological tissues; the TOSC for 1 microg rat liver cytosolic protein was 0.40 +/- 0.02 and for the microsomal membrane, 0.15 +/- 0.03. Soluble antioxidants accounted for 77% of the protective antioxidant potential in rat liver cytosol. When incorporated into the microsomal membrane, alpha-tocopherol markedly enhances antioxidant protection against peroxyl radical; thus, the assay is suitable for the assessment of fat-soluble antioxidants.

Induction time course of cytochromes P450 by phenobarbital and 3-methylcholanthrene pretreatment in liver microsomes of Alligator mississippiensis.

Alligator mississippiensis has at least two classes of inducible hepatic microsomal cytochromes P450 (CYP): (1) those induced by 3-methylcholanthrene (3MC), and (2) those induced by phenobarbital (PB). The rates of induction by these xenobiotic compounds are significantly slower than those reported for mammals. Carbon monoxide binding, western blots, and enzymatic activity measurements indicated that at least 48-72 hr are required to reach full induction. A methoxy-, ethoxy-, pentoxy, and benzyloxyphenoxazone (resorufin) O-dealkylation (MROD, EROD, PROD, and BROD) profile was indicative of substrate selectivity typical of 3MC- and PB-induced P450s. MROD and BROD showed the greatest ability to discriminate between alligator hepatic microsomes induced by 3MC and PB, respectively. This is in contrast to mammals, in which EROD is a biomarker of polycyclic aromatic hydrocarbon exposure because of its ability to discriminate the induction of CYP 1A. In a similar manner, PROD is a highly preferred activity of CYP 2B in mammals; thus, it is used to indicate CYP 2B induction. The induction of P450 by PB is a general phenomenon in mammals and birds. To the best of our knowledge, this is the first report demonstrating PB induction of P450 activities typical of the mammalian CYP 2 family isoforms in alligator or any reptilian liver. The importance of this finding to the evolution of CYP 2 family regulation by PB is heightened by the fact that induction by this xenobiotic is not common to fish and other lower vertebrates (Ertl RP and Winston GW, Comp Biochem Physiol, in press). Although indicating the presence of CYP 1A- and CYP 2B-like isoforms in alligator, it remains to be established how closely related these alligator P450s are to mammalian isoforms.

The microsomal mixed function oxidase system of amphibians and reptiles: components, activities and induction.

This article reviews current research in amphibian and reptilian cytochromes P450, important to the overall understanding of xenobiotic metabolism in the ecosystem and the evolution of P450s. Amphibians and reptilians contain the normal mixed function oxidase system (MFO). In general the MFO content and activities are less than those found in mammals, but only a few of the known activities have been examined in these vertebrate classes. Research to date has focused on two families of cytochromes P450, CYP1 and 2. The isoforms examined catalyze the classic activities but there have been notable absences. The total number of isoforms present and the breadth of substrates metabolized are yet unknown. Induction by foreign compounds (xenobiotics) is lengthier and yields lower levels of induced activity than is typically found in mammals. When these animals are pretreated with 3-methylcholanthrene (3MC) and beta-naphthaflavone (BNF), which are known to induce the same isoform in mammals, multiple isoforms are induced with different activities. Phenobarbital-pretreatment in turtles and alligators induces cytochromes P450 and suggestive data indicates induction in the lizard Agama lizard and the newt Pleurodeles waltl. In amphibians and reptiles a CYP2B protein does appear to be present along with constitutive activities associated with the 2 family of cytochromes P450. The markedly different response to classic inducers combined with lower or absent activities alters the view of how amphibians and reptilians respond to xenobiotic challenges.

Spectral analysis and catalytic activities of the microsomal mixed-function oxidase system of the sea anemone (phylum: Cnidaria).

Cnidarians are primarily marine organisms with a wide and diverse habitat worldwide. Previous studies from our laboratory demonstrated the presence of proteins in the molecular mass range of 50-60 KDa that were recognized by several antibodies raised against fish cytochromes P450 of the CYP 1, 2, and 3 families in microsomes prepared from the columnar regions of 5 species of sea anemones (Heffernan et al. Mar Environ Res 1996;42:353-357). Pursuant to those studies we report herein results of spectral analyses, NAD(P)H-oxidoreductase and ethoxyresorufin O-dealkylase (EROD) of sea anemone microsomal fractions. The predominant feature in the difference spectrum of dithionite (DTN)-reduced, CO-liganded anemone microsomes was a peak with lambda max of approximately 418 nm, which slowly increased for about 20 min and decreased after about 40 min. A relatively lower amplitude 450 nm peak was attained within 5 min of CO addition and was stable for up to 90 min. The 450 nm peak did not increase concomitant to the decrease in the 418 nm peak suggesting that the latter is not denatured P450. A significantly larger 450 nm peak was attained in CO-difference spectra when DTN was added prior to CO. NADPH-dependent cytochrome c (P450) reductase of the sea anemones was 1.8-3.9 nmol/min/mg protein, which is at the lower end of the range observed in invertebrates. NADH-cytochrome c reductase was 9-25 nmol/min/mg protein, while the NADH-ferricyanide (b5) reductase ranged from 73-232 nmol/min/mg protein. When microsomal EROD activity was measured under conditions in which the reaction was linear with respect to protein concentration, a decrease in fluorescence was typically observed for the initial 15 min of the time course and then increased linearly for up to 90 min; initial velocities were determined from the increasing linear region. NADPH was the preferred cofactor for EROD activity and the NAD(P)H-EROD activity was higher in Anthopleura elegantissima than Anthopleura xanthogrammica. The Bunodosoma cavernata NADPH-EROD activity was barely noted at the detection limit of the assay and NADH-EROD activity was not detected. These results are consistent with a functional P450-dependent MFO system in cnidarians, with characteristics both similar to and unique from other marine invertebrates.