The Formation of g=2.49-Species of Cytochrome P450 in the Rat Liver by PCB126 Oral Administration: Identification of Heme Axial Ligands by EPR Spectroscopy

Rat livers and microsomes were subjected to electron paramagnetic resonance (EPR) measurements at 77 K. The EPR spectra of the livers from the control group, carbon tetrachloride-, 3-methylcholanthrene-, and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126)-treated rats exhibited an EPR spectrum at g=2.40, 2.24, and 1.93, which is characteristic of P450 in a resting state. The liver of the PCB126-treated rats showed an additional distinct EPR spectrum at g=2.49, 2.26, and 1.87 (g=2.49-species). The heme environmental structure of g=2.49-species was identified by crystal field analysis using three EPR g-values of the microsome treated with various chemicals. These results indicated that g=2.49-species is a hemeprotein with cysteine thiolate at the 5th coordination site, and a nitrogenous ligand at the 6th site.     

Metabolic disturbances in fish exposed to sodium pentachlorophenate (NaPCP) and 3,3',4,4',5-pentachlorobiphenyl (PCB126), individually or combined

The Swan River Estuary is the recipient of multiple urban and agricultural contaminants which have the potential to induce liver detoxication enzymes as well as altering the metabolism of aquatic organisms. To test if altered liver metabolism would influence liver detoxication capacities, pink snapper (Pagrus auratus) were i.p. injected with peanut oil (controls), or pentachlorobiphenyl #126 (PCB126), with sodium pentachlorophenate (NaPCP), or PCB126+NaPCP. Relative to controls, ethoxyresorufin-O-deethylase (EROD) activity was induced in the PCB126 and PCB126+NaPCP fish, but not in the NaPCP group. In the liver, cytochrome c oxidase (CCO) activity was enhanced by the treatments while citrate synthase (CS) activity remained unchanged and lactate dehydrogenase (LDH) activity was increased in the NaPCP treatment only. The results suggest that liver CCO activity may be a suitable biomarker of effect following exposure to PCBs or phenolic compounds. In the white muscle, only the PCB126+NaPCP treatment enhanced CCO activity, with all other enzymatic activities remaining unchanged. It appears that the resilience to metabolic perturbations is greater for white muscle than for liver. Low serum sorbitol dehydrogenase (sSDH) activity and histopathology of the liver indicated no significant alteration of cellular structure, albeit the lipid droplet size was increased in the PCB126 and in the PCB126+NaPCP treatments. It is concluded that the hepatic metabolic changes correspond to histopathological observations, but an altered metabolic capacity do not influence the metabolism of xenobiotics by liver enzymes, as measured by EROD activity.     

Oral exposure to Microcystis increases activity-augmented antioxidant enzymes in the liver of loach (Misgurnus mizolepis) and has no effect on lipid peroxidation

Recently, eutrophication has induced severe cyanobacterial blooms in the Naktong River, the second largest river of Korea. In the present study, lipid peroxidation and the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, were evaluated in the liver of loach (Misgurnus mizolepis) that were orally exposed to a low dose of Microcystis through dietary supplementation with bloom scum. Loach received 75 mg of dry cells/kg body weight mass (equal to 10 microg microcystin-RR/kg body mass), for 28 days under controlled conditions. Antioxidant enzymatic activity and lipid peroxidation were measured after termination of exposure. The activities of antioxidant enzyme were significantly increased in the livers of toxin-exposed loach after 28 days of exposure, as compared to control fish. However, lipid peroxidation remained stable in both groups. These results suggest that antioxidant enzymes were able to eliminate oxidative stress induced by low concentrations of microcystins and to prevent increased lipid peroxidation in the liver of loach.     

Antioxidant responses and lipid peroxidation following intranasal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in rats: increased susceptibility of olfactory bulb

We evaluated an alternative method to investigate a possible involvement of environmental toxins in the pathology of Parkinson's disease (PD). There is considerable evidence supporting the role of oxidative stress in the toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin largely used to modeling PD in primates and rodents. We have recently demonstrated that rats treated with intranasal (i.n.) infusion of MPTP suffer from progressive signs of PD that are correlated with time-dependent degeneration in dopaminergic neurons. In the present study, we investigated the time-dependent (2 h to 7 days) effect of a single i.n. administration of MPTP (0.1 mg/nostril) on the glutathione-related antioxidant status and lipid peroxidation (TBARS) in the adult Wistar rat brain. The effects were more pronounced in the olfactory bulb at 6 h after i.n. MPTP administration, as indicated by an increase in TBARS and total glutathione (GSH-t) levels, and also in the gamma-glutamyl transpeptidase (GGT) activity. Increased levels of TBARS, GSH-t and GGT activity were also observed at 6 h post-MPTP infusion in some structures (e.g. striatum, hippocampus and prefrontal cortex). No difference regarding glutathione reductase activity was observed in any of the brain structures analyzed, while a marked decrease in glutathione peroxidase activity was specifically observed in the substantia nigra 7 days after MPTP treatment. These results demonstrate that a single i.n. infusion of MPTP in rats induces significant alterations in the brain antioxidant status and lipid peroxidation, reinforcing the notion that the olfactory system represents a particularly sensitive route for the transport of neurotoxins into the central nervous system that may be related to the etiology of PD.     

Age-related changes in antioxidant enzyme activities, fatty acid composition and lipid peroxidation in whole body Gammarus locusta (Crustacea: Amphipoda)

The main aim of this work was to provide baseline data on aspects of pro-oxidant and antioxidant processes in different life-stages of the marine amphipod Gammarus locusta. The activities of antioxidant enzymes and levels of lipid peroxidation were determined in whole body juveniles, subadults, and male and female adults of a laboratory population of G. locusta. Fatty acid composition of individuals at these different stages of development was also characterised in order to examine the contribution that polyunsaturated fatty acids (PUFA) might make to the peroxidation status of animals. The antioxidant enzymes, measured in whole body 100,000 supernatants, comprised catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD; EC 1.15.1.1) and glutathione peroxidase (GPX; EC 1.11.1.9). Fatty acids were analysed as fatty acid methyl esters (FAME). Lipid peroxidation was examined in terms of the levels of lipid peroxides determined as thiobarbituric acid-reactive malondialdehyde equivalents. Age-related changes were seen in antioxidant enzyme status: levels of SOD (p<0.01) and GPX (p<0.001) activities decreased progressively during development from juveniles to adults. Sex-related changes in GPX activity were also seen, the levels being higher in adult males than females (p<0.001). The amount of FAME present in whole body amphipod also changed over the life span. Among PUFA, the eicosapentaenoic (C20:5n-3), arachidonic (C20:4n-6) and docosahexaenoic (C22:6n-3) were the most abundant acids in this species, and both their individual concentrations and total PUFA increased progressively with age (up to 3.3-fold; p<0.001). The latter changes may contribute to the explanation of the observed differences in peroxidation status of the animals with age; thus, levels of lipid peroxides increased up to 40% in adult males compared to other age-classes (p<0.01). Overall, the decline in antioxidant enzyme activities, coupled with increased levels of PUFA, as the individual grows older, may render the older animals more susceptible to lipid peroxidation and oxidative stress.     

Oxidative stress induced by beta-amyloid peptide(1-42) is involved in the altered composition of cellular membrane lipids and the decreased expression of nicotinic receptors in human SH-SY5Y neuroblastoma cells

The neurotoxic effects and influence of beta-amyloid peptide (Aβ)_1–42 on membrane lipids and nicotinic acetylcholine receptors (nAChRs) in human SH-SY5Y neuroblastoma cells were investigated in parallel. Exposure of the cultured cells to varying concentrations of Aβ_1–42 evoked a significantly decrease in cellular reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide), together with enhanced lipid peroxidation and protein oxidation. Significant reductions in the total contents of phospholipid and unbiquinone-10, as well as in the levels of the 3 and 7 subunit proteins of nAChRs were detected in cells exposed to Aβ_1–42. In contrast, such treatment had no effect on the total cellular content of cholesterol. Among these alterations, increased lipid peroxidation and decreased levels of cellular phospholipids were most sensitive to Aβ_1–42, occurring at lower concentrations. In addition, when SH-SY5Y cells were pretreated with the antioxidant Vitamin E, prior to the addition of Aβ_1–42, these alterations in neurotoxicity, oxidative stress, composition of membrane lipids and expression of nAChRs were partially prevented. These findings suggest that stimulation of lipid peroxidation by Aβ may be involved in eliciting the alterations in membrane lipid composition and the reduced expression of nAChRs associated with the pathogenesis of AD.     

Protective effects of lazaroid U74389F (16-desmethyl tirilazad) on endrin-induced lipid peroxidation and DNA damage in brain and liver and regional distribution of catalase activity in rat brain

Endrin, a poly-halogenated cyclic hydrocarbon, induces hepatic lipid peroxidation, modulates calcium homeostasis, decreases membrane fluidity, and increases nuclear DNA damage. Little information is available on the neurotoxicity of endrin. The effects of endrin on lipid peroxidation, DNA damage, and regional distribution of catalase activity were assessed in rat brain and liver 24 h following an acute oral dose of 4.5 mg endrin/kg. Lipid peroxidation associated with whole brain mitochondria increased 2.4-fold, whereas microsomal lipid peroxidation increased 2.8-fold following endrin administration. Lipid peroxidation also increased 2.0-fold both in hepatic mitochondria and microsomes. Catalase activity decreased 24% in the hypothalamus, 23% in the cortex, 38% in the cerebellum, and 11% in the brain stem in response to endrin. A 4.3-fold increase in brain nuclear DNA-single strand breaks (SSB) was observed in endrin-treated rats. Pretreatment of rats intraperitoneally with the lazaroid U74389F (16-desmethyl tirilazad) (10 mg/kg in two doses) attenuated the biochemical consequences of endrin-induced oxidative stress. The administration of U74389F in citrate buffer (pH 3.8) provided better protection than administering the lazaroid in corn oil, decreasing endrin-induced lipid peroxidation by 50–80% and DNA-SSB by approximately 72% in liver and 85% in brain, while ameliorating the suppressed catalase activity. The data suggest an involvement of an oxidative stress in the neurotoxicity and hepatotoxicity induced by endrin, which can be attenuated by the lazaroid U74389F.     

Relationship between glutathione S-transferase, catalase, oxygen consumption, lipid peroxidation and oxidative stress in eggs and larvae of Boophilus microplus (Acarina: Ixodidae)

Glutathione S-transferases (GSTs) are enzymes that act in excretion of physiologic and xenobiotic substances, protecting cells against chemical toxicity and stress. In this work, we characterized the enzymatic activity of GST in eggs and larvae of cattle tick Boophilus microplus, on different days after oviposition and eclosion. The results showed that the GST activity varied depending on the time elapsed after oviposition and eclosion. Molecules involved in mechanism of protection from oxidative stress are correlated with the increase in GST activity. The oxygen consumption kinetics showed a positive correlation with the increase in GST activity during embryogenesis. A high content of thiobarbituric acid reactive substances were observed in egg and larva extracts, indicating that ticks face high oxidative stress during embryogenesis and aging. In eggs and larvae, GST activity can be correlated to kinetic parameters of oxidative stress such as catalase and glutathione. In addition, GST activity showed strong positive correlation with lipid peroxidation, an indication that it plays a role in oxidant defences in eggs.