Antioxidants and total oxyradical scavenging capacity during grass shrimp, Palaemonetes pugio, embryogenesis.

During embryogenesis in grass shrimp the capacity to scavenge oxyradicals increased as measured by the Total Oxyradical Scavenging Capacity (TOSC) assay. The increase in TOSC during embryogenesis was associated with increasing concentrations of a number of antioxidants, including coenzyme Q (ubiquinone), alpha-tocopherol and reduced glutathione. Glutathione concentrations ranged from 0.004 to 0.005 nmol/embryo in early embryo stages and reached concentrations between 0.16 to 0.23 nmol/embryo in late embryo stages. Ascorbate remained essentially constant (0.16-0.20 nmol/embryo) throughout embryogenesis and may provide the preponderance of TOSC during early embryo development. Carotenoids were associated with yolk lipovitellin and these antioxidants decreased as yolk was absorbed during embryogenesis. Astaxanthin and beta-carotene were identified in embryos with astaxanthin always the principal carotenoid. In early embryo stages there are maternally derived antioxidants but as embryogenesis proceeds there is an assembly of a complex antioxidant system by newly formed cells and tissues.

Antioxidant defenses in killifish (Fundulus heteroclitus) exposed to contaminated sediments and model prooxidants: short-term and heritable responses.

A population of killifish (Fundulus heteroclitus) inhabiting a Superfund site on the Elizabeth River (VA, USA) is tolerant of the acute toxicity of the sediments from the site; previous work suggests that this tolerance is based both on genetic adaptation and physiological acclimation. In this study, larval first- and second-generation (F1 and F2) offspring of Elizabeth River killifish were more resistant to the toxicity of t-butyl hydroperoxide (a model prooxidant) than were King's Creek (reference site) offspring, indicating a heritable tolerance of exposure to oxidative stress. In laboratory experiments designed to elucidate the mechanistic basis for this increased tolerance, we exposed laboratory-raised F1 and F2 offspring from Elizabeth River and King's Creek killifish to Elizabeth River sediments, menadione, or t-butyl hydroperoxide, and measured the following antioxidant parameters: total oxyradical scavenging capacity (TOSC); glutathione content (total and disulfide); activities of glutathione reductase (GR); glutathione peroxidase (GPx); and glutamate cysteine ligase (GCL) activities and protein levels of copper-zinc superoxide dismutase (CuZnSOD); and protein levels of manganese superoxide dismutase (MnSOD). Exposure to Elizabeth River sediments lead to consistent increases in total glutathione concentrations, GR activities, and MnSOD protein levels, and in some cases increased GPx and GCL activities, in both populations. In addition, Elizabeth River offspring (larvae) showed higher basal TOSC values, glutathione concentrations, and MnSOD protein levels. These data suggest that upregulated antioxidant defenses play a role in both short-term (physiological) and heritable (multigenerational/evolutionary) tolerance of the toxicity of these Superfund sediments. The responses of specific antioxidant parameters, including sex-specific responses in the cases of glutathione concentrations and GR activity, are also discussed.

Integrating enzymatic responses to organic chemical exposure with total oxyradical absorbing capacity and DNA damage in the European eel Anguilla anguilla.

In this work, susceptibility to oxidative stress was analyzed under laboratory conditions in the European eel Anguilla anguilla. Eels were treated with increasing concentrations of benchmark environmental pollutants, namely, benzo[a]pyrene ([BaP], at 0, 0.1, 1, 10, and 50 mg/kg), beta-naphthoflavone ([BNF], at 0, 0.1, 1, 10, and 50 mg/kg), Arochlor 1254 (at 0, 0.1, 1, 10, and 50 mg/kg), and 2,3,7,8-tetrachlorodibenzo p-dioxin ([TCDD], at 0, 0.01, 0.1, 1, and 2 microg/kg). The integral relationships were analyzed between induction of ethoxyresorufin O-deethylase (EROD) activity, its involvement in perturbing oxyradical metabolism, and the role of cytochrome P450 and/or oxidative stress in mediating genotoxic effects. To reveal whether the oxidative status in exposed organisms was altered as a result of chemical exposure, measurements of the main endogenous antioxidant defenses were integrated with the measurement of total oxyradical scavenging capacity (TOSC) toward peroxyl radicals and hydroxyl radicals (*OH). This approach permits discriminating the resistance of a tissue toward different forms of oxyradicals, thereby indicating a differential role for specific reactive oxygen species (ROS) in perturbing the balance between prooxidant and antioxidant mechanisms. All the analyzed chemicals promoted EROD induction (reflective of CYP1A) and altered either the levels or the activities of the antioxidants studied, which might be anticipated to exert alterations in oxyradical metabolism. Analysis of TOSC suggested the prevalence of metabolic oxidative pathways leading to the more reactive *OH on exposure to the chemicals studied. Of these chemicals, enhanced EROD activity correlated with genotoxic damage only in the cases of the nonhalogenated hydrocarbons BaP and BNF. The highest degree of genotoxic damage was consistently observed in organisms in which the capacity to absorb or scavenge OH was lowest. These data suggest a general relationship between oxidative stress and loss of DNA integrity in juvenile eels exposed to the chemicals studied herein.

Application of biomarkers for assessing the biological impact of dredged materials in the Mediterranean: the relationship between antioxidant responses and susceptibility to oxidative stress in the red mullet (Mullus barbatus).

In the period 1997-2000, approximately 1,800,000 m3 of material dredged from the Port of Leghorn was discharged into a sea dumping site located 14 miles from the coast. The red mullet (Mullus barbatus) was used as a bioindicator species for monitoring the biological impact of these discharges on a geographical and temporal scale. Organisms were sampled over three years (1998-2000) at different stations and several biomarkers, both of exposure and effect, were analyzed. Bioavailability of specific classes of pollutants was evaluated by analyzing levels of metallothioneins, the activity of cytochrome P450 1A (CYPIA) and of glutathione S-transferases. Among biomarkers of effect, special attention was paid to the balance between prooxidant challenge and antioxidant defenses, and to the appearance of damage caused by oxidative stress. The analyses of the main components of the antioxidant system included superoxide dismutase, catalase, glutathione peroxidases, glutathione reductase, glyoxalase I and II, and total glutathione. These data were integrated with the measurement of total oxyradical scavenging capacity (TOSC) as an indication of the overall biological resistance to toxicity of different forms of oxyradicals (peroxyl radicals, hydroxyl radicals and peroxynitrite). Results indicated a biological impact in organisms sampled near the disposal site; the impact was particularly evident during 1999 and mainly related to organic chemicals such as PAH. Exposure to these pollutants also caused variations in the levels and activity of several antioxidants. The analysis of TOSC, however, revealed that the overall capacity of specific tissues of organisms to absorb various oxidants was not seriously compromised when challenged with increased prooxidant pressures. Variations of single antioxidants were useful in revealing early warning "biological responses", while integration with TOSC analyses indicated if such changes also reflect a more integrated and functional "biological effect" with possible consequences at the organisms level. The red mullet appears to be a useful sentinel species for a biomarker approach to monitoring impact caused by dredged materials.

Bioorganic mechanisms of the formation of free radicals catalyzed by glucose oxidase.

In this communication, we have described the activation of several xenobiotics by glucose oxidase from Aspergillus niger. The following compounds are readily reduced by d-glucose, in the presence of glucose oxidase: p-nitroso-N,N-dimethylaniline, methyl-1,4-benzoquinone, and 7,7,8,8-tetracyano-quinodimethane. In each case, the products of enzymatic reduction undergo a dismutation reaction with the parent compound and thus afford the formation of free radicals. In some cases, and at an appropriate pH value, the transformation of a parent compound into free radicals is almost quantitative. Under optimal conditions, free radicals are stable for several minutes in aqueous solutions under physiological conditions.