Genotoxic potential of Microcystin-LR and nodularin in vitro in primary cultured rat hepatocytes and in vivo in rat liver

Microcystin-LR (MCYST-LR) and nodularin (NOD) are known as tumor promoters in experimental animals and so present potential health threats for humans. Although their hepatotoxic mechanisms have been very well documented, many other effects of these toxins are relatively undescribed, indeed controversial, notably those related to their genotoxicity. In the present investigation, we examined how these toxins could induce DNA damage using a combination of in vitro and in vivo approaches. We first used the (32)P-postlabeling assay to test hydrophobic adduct formation on DNA from primary cultured rat hepatocytes treated with noncytotoxic concentrations of MCYST-LR and NOD (2 and 10 ng/mL). Analysis of the autoradiograms of DNA digests isolated from the hepatocytes did not show any hydrophobic DNA adduct formation. However, these toxins significantly decreased the amount of hydrophobic endogenous adducts, termed I compounds. We next investigated oxidative DNA damage by using the (32)P-postlabeling assay to analyze 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) content as a biomarker of possible DNA lesions. Both MCYST-LR and NOD significantly enhanced 8-oxo-dG in time- and dose-dependent manner in vitro in primary cultured hepatocytes and in vivo in rat liver cells. Thus, it appears that the depletion of endogenous DNA adducts (I compounds) and/or the increase of 8-oxo-dG levels by MCYST-LR and NOD could be involved in the formation of hepatic tumors during long-term exposure to these cyanobacterial hepatotoxins.     

Critical role of reactive oxygen species formation in microcystin-induced cytoskeleton disruption in primary cultured hepatocytes.

Cyanobacteria (blue-green algae)-contaminated water is a worldwide public health problem. Microcystins are a group of liver-specific toxins generated by cyanobacteria. It is generally believed that the protein phosphorylation that leads to the disruption of intermediate filaments plays an important role in microcystin-induced hepatotoxicity. However, the mechanisms that contribute to the microcystin-induced alterations of microtubules and microfilaments are not fully understood. In the present study, the effects of microcystin-fR (M-LR), the most common microcystin, were examined on the organization of cellular microtubules and microfilaments in primary cultured rat hepatocytes. Our results indicate that M-LR initiated reactive oxygen species (ROS) formation followed by altering the cytoskeleton structures, which eventually led to significant LDH leakage. These effects were completely prevented by TEMPOL, a superoxide dismutase mimic, and also partially prevented by desferoxamine. These findings provide further evidence that ROS formation, especially superoxide radical, plays a crucial role in M-LR-induced disruption of cytoskeleton organization and consequent hepatotoxicity.     

Photoirradiation of dehydropyrrolizidine alkaloids--formation of reactive oxygen species and induction of lipid peroxidation

Pyrrolizidine alkaloid (PA)-containing plants are widespread in the world and are probably the most common poisonous plants affecting livestock, wildlife, and human. PAs require metabolic activation to generate pyrrolic metabolites (dehydro-PAs) that bind cellular protein and DNA, leading to hepatotoxicity and genotoxicity, including tumorigenicity. In this study we report that UVA photoirradiation of a series of dehydro-PAs, e.g., dehydromonocrotaline, dehydroriddelliine, dehydroretrorsine, dehydrosenecionine, dehydroseneciphylline, dehydrolasiocarpine, dehydroheliotrine, and dehydroretronecine (DHR) at 0-70 J/cm² in the presence of a lipid, methyl linoleate, resulted in lipid peroxidation in a light dose-responsive manner. When irradiated in the presence of sodium azide, the level of lipid peroxidation decreased; lipid peroxidation was enhanced when methanol was replaced by deuterated methanol. These results suggest that singlet oxygen is a photo-induced product. When irradiated in the presence of superoxide dismutase, the level of lipid peroxidation decreased, indicating that lipid peroxidation is also mediated by superoxide. Electron spin resonance (ESR) spin trapping studies confirmed that both singlet oxygen and superoxide anion radical were formed during photoirradiation. These results indicate that UVA photoirradiation of dehydro-PAs generates reactive oxygen species (ROS) that mediated the initiation of lipid peroxidation. UVA irradiation of the parent PAs and other PA metabolites, including PA N-oxides, under similar experimental conditions did not produce lipid peroxidation. It is known that PAs induce skin cancer and are secondary (hepatogenous) photosensitization agents. Our results suggest that dehydro-PAs are the active metabolites responsible for skin cancer formation and PA-induced secondary photosensitization.     

Roles of reactive oxygen species and MAP kinases in the primary rat hepatocytes death induced by toosendanin

Toosendanin (Tsn), a triterpenoid extracted from Melia toosendan Sieb et Zucc, possesses different pharmacological effects in human and important values in agriculture. However, liver injury has been reported when toosendanin or Melia-family plants, which contain toosendanin are applied. The mechanism by which toosendanin induces liver injury remains largely unknown. Here we reported that toosendanin induced primary rat hepatocytes death by mitochondrial dysfunction and caspase activation. Toosendanin led to decrease of mitochondrial membrane potential, fall in intracellular ATP level, release of cytochrome c to cytoplasm, activation of caspase-8, 9, and 3 and ultimately cell death. Level of reactive oxygen species (ROS) was also increased in hepatocytes after incubation with toosendanin. Catalase, the H2O2-decomposing enzyme, can prevent the reduction in ATP level and protect hepatocytes from toosendanin-induced death. The ERK1/2 (p44/42 MAP kinases) and JNK (c-Jun N-terminal kinase) were activated, but p38 MAPK was not activated by toosendanin. Inhibition of ERK1/2 activation sensitized hepatocytes to death and increased activity of caspase-9 and 3 in response to toosendanin. Inhibition of JNK attenuated toosendanin-induced cell death. These results suggested that toosendanin causes death of primary rat hepatocytes by mitochondrial dysfunction and caspase activation. Generation of ROS and MAP kinases activation might be involved in this process.     

Relationships between intracellular vitamin E, lipid peroxidation, and chemical toxicity in hepatocytes

The cellular content of vitamin E was measured in isolated rat hepatocytes exposed to various types of chemical injury. Vitamin E was determined as alpha-tocopherol by HPLC with in-line uv and electrochemical detection. The cytotoxicity of diquat, a redox cycling compound, was accompanied by a decrease in cellular alpha-tocopherol and a stimulation of lipid peroxidation. Both the loss of alpha-tocopherol and the accumulation of lipid peroxidation products could be prevented by addition of either the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) or the reducing agent dithiothreitol (DTT). DTT also prevented the oxidation of soluble and protein thiols and completely protected against cytotoxicity, while DPPD addition only delayed the onset of hepatocyte death. Cytotoxic doses of the naphthoquinone, menadione, and the pyridine compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-pyridinium ion did not deplete alpha-tocopherol levels, nor did they result in significant lipid peroxidation. On the other hand, a peroxidizing, but noncytotoxic dose of ADP-Fe3+ rapidly decreased cellular alpha-tocopherol levels. These data demonstrate that cellular alpha-tocopherol loss is neither a prerequisite for, nor a necessary consequence of toxicity. Moreover, a substantial depletion (ca. 50%) of alpha-tocopherol does not necessarily result in cell death. Although alpha-tocopherol protects against the oxidation of cellular lipids, the maintenance of hepatocyte alpha-tocopherol content does not prevent the oxidation of soluble and protein thiols. These other targets of oxidative damage seem to play a more critical role in hepatocyte toxicity.     

Roles of reactive oxygen species,NF-kappaB,and peroxiredoxins in glycochenodeoxycholic acid-induced rat hepatocytes death

The aim of this study was to determine the roles of reactive oxygen species (ROS), NF-kappaB and antioxidants in glycochenodeoxycholic acid (GCDC, 0-400 micromol/l, 0.5- 3 h)-induced hepatocytes death. The differential uptake of ethidium bromide and acridine orange revealed that apoptotic death occurred dose-dependently in GCDC-treated hepatocytes whereas necrotic death was prominent especially at higher GCDC concentrations (> or =200 micromol/l). ROS generation measured fluorometrically either by a confocal laser microscope or by a microplate fluorescence reader was increased dose-dependently. The dose-dependent NF-kappaB activation with the significant IkappaB-alpha decrease preceded both hepatocyte cell death and the alteration of antioxidant enzymes. The Cu/Zn-SOD level among several antioxidants, we checked, remained unchanged. In contrast, the catalase level and its enzymatic activity were markedly decreased only at 400 micromol/l. The Prx I and Prx II, newly defined antioxidant enzymes reducing H(2)O(2) levels were decreased at the 200 and 400 micromol/l. These observations point to ROS generation in the GCDC-treated hepatocyte as the proximate event that triggers NF-kappaB activation, IkappaB-alpha proteolysis, Prx depletion, and finally cell death. And oxidative stress may be more related to necrotic cell death in GCDC-treated hepatocytes.     

Cadmium toxicity and lipid peroxidation in isolated rat hepatocytes

The toxicity of cadmium may be due to alteration in membrane structure which may be caused by peroxidation of the composite lipids. Isolated hepatocytes provide a suitable system to examine the role of lipid peroxidation in a toxic response at the cellular level. Therefore, isolated rat hepatocytes were incubated with varying cadmium concentrations (50–400 μm) for up to 75 min. An increase in lipid peroxidation due to cadmium was observed. The integrity of the cell membrane, as measured by loss of intracellular potassium ion and leakage of aspartate aminotransferase, was adversely affected in the presence of cadmium. The lactate to pyruvate ratio of hepatocyte suspensions was increased upon incubation with cadmium. Several chelating compounds were found to reduce intracellular accumulation of cadmium, cellular toxicity and lipid peroxidation. However, amelioration of toxicity was not consistently associated with inhibition of the lipid peroxidation response. The antioxidant compounds, sodium diethyldithiocarbamate and N,N′-diphenyl-p-phenylene-diamine were found to inhibit the lipid peroxidation attributable to cadmium, but did not have any consistent protective effect against loss of intracellular potassium ion. The results of this study show that the toxicity induced by cadmium in isolated rat hepatocytes can be dissociated from the concurrently observed lipid peroxidation, which indicates that the toxic response is not caused by the lipid peroxidation.     

The effect of lipid peroxidation products on reactive oxygen species formation and nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophages

Lipid peroxidation induced by oxidants leads to the formation of highly reactive metabolites. These can affect various immune functions, including reactive oxygen species (ROS) and nitric oxide (NO) production.The aim of the present study was to investigate the effects of lipid peroxidation products (LPPs) - acrolein, 4-hydroxynonenal, and malondialdehyde - on ROS and NO production in RAW 264.7 macrophages and to compare these effects with the cytotoxic properties of LPPs. Macrophages were stimulated with lipopolysaccharide (0.1 μg/ml) and treated with selected LPPs (concentration range: 0.1-100 μM). ATP test, luminol-enhanced chemiluminescence, Griess reaction, Western blotting analysis, amperometric and total peroxyl radical-trapping antioxidant parameter assay were used for determining the LPPs cytotoxicity, ROS and NO production, inducible nitric oxide synthase expression, NO scavenging, and antioxidant properties of LPPs, respectively.Our study shows that the cytotoxic action of acrolein and 4-hydroxynonenal works in a dose- and time-dependent manner. Further, our results imply that acrolein, 4-hydroxynonenal, and malondialdehyde can inhibit, to a different degree, ROS and NO production in stimulated macrophages, partially independently of their toxic effect. Also, changes in enzymatic pathways (especially NADPH-oxidase and nitric oxide synthase inhibition) and NO scavenging properties are included in the downregulation of reactive species formation.     

Cytotoxic and cytoprotective activities of curcumin. Effects on paracetamol-induced cytotoxicity, lipid peroxidation and glutathione depletion in rat hepatocytes

The cytoprotective effect of curcumin, a natural constituent of Curcuma longa, on the cytotoxicity of paracetamol in rat hepatocytes was studied. Paracetamol was selected as a model-toxin, since it is known to be bioactivated by 3-methylcholanthrene inducible cytochromes P450 presumably to N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite which upon overdosage causes protein- and non-protein thiol-depletion, lipid peroxidation and cytotoxicity measured as LDH-leakage. At low concentrations curcumin was found to protect significantly against paracetamol-induced lipid peroxidation, without protection against paracetamol-induced LDH-leakage and without protection against paracetamol-induced GSH-depletion. At a 100 times higher concentration of curcumin the observed protective effect on lipid peroxidation was accompanied with a tendency to increase cellular GSH-depletion and LDH-leakage. No time-dependency was found as to the curcumin-induced effects: treatment of the hepatocytes 1 hr before, concomitantly or 1 hr after the addition of paracetamol to the cells had similar effects. In contrast to what was expected on the basis of previous in vivo experiments, at higher concentrations curcumin itself was found to be slightly cytotoxic. Curcumin-induced LDH-leakage was accompanied by a significant depletion of GSH. It has been concluded that the observed cytoprotective and cytotoxic activities of curcumin may be explained by a strong anti-oxidant capacity of curcumin and the capability of curcumin to conjugate with GSH. Furthermore, it has been concluded that lipid peroxidation is not playing a causal role in cell-death induced by paracetamol or by curcumin.     

Age-related changes in reactive oxygen species production in rat brain homogenates

The generation of reactive oxygen species (ROS) and resultant oxidative stress have been implicated in the mechanism of brain dysfunction due to age-related neurodegenerative diseases or exposure to environmental chemicals. We have investigated intrinsic age-related differences in the ability of the various brain regions to generate ROS in the absence and presence of Fe(2)+. ROS production in crude brain homogenates from adult rats was linear with respect to time and tissue concentration, and was stimulated to a greater extent by Fe(2)+ than was TBARS production. ROS production was then determined in homogenates from cerebral cortex, striatum, hippocampus, and cerebellum of 7-day-old, 14-day-old, 21-day-old, adult (3-6-month old), and aged (24-month-old) rats using the fluorescent probe 2',7'-dichlorodihydrofluorescin (DCFH). Basal levels of ROS production were similar in 7-, 14-, and 21-day olds, increased in adults, and highest in aged rats, and did not differ between brain regions. ROS production was stimulated by Fe(2)+ (0. 3-30 microM) in a concentration-dependent manner in all brain regions. However, the stimulation of ROS production by Fe(2)+ varied with age. ROS production was greater in 14- and 21-day-old rats compared with adult and aged animals. ROS production in 7-day-old rats was decreased at low Fe(2)+ concentrations and increased at high Fe(2)+ concentrations compared to adult and aged rats. These data show that brain homogenates from neonatal rats respond differently to Fe(2)+, and suggest that developing animals may be more sensitive to oxidative stress in the brain after exposure to toxicants. Published by Elsevier Science Inc.     

CCl4-induced lipid peroxidation in isolated rat hepatocytes with different oxygen concentrations

It has been difficult to demonstrate convincingly the occurrence of lipid peroxidation by increases in the concentration of thiobarbituric acid (TBA) reactants in response to carbon tetrachloride (CCl₄) in hepatocytes isolated from fed, nontreated rats. This study was undertaken to determine whether or not the incubation atmosphere and the content of the incubation media could be contributing factors for the inhibition of CCl₄-induced lipid peroxidation. Isolated hepatocytes incubated in either Eagle's minimum essential medium (EMEM) or Tris buffer were incubated with and without CCl₄ (1 or 5 μl/4 ml hepatocyte suspension) under an atmosphere of either air or carbogen (95% O₂:5% CO₂). Samples were taken at 30, 60, or 120 min for determination of ethane (in the gas phase), TBA reactants concentration, and leakage of lactate dehydrogenase (LDH). Under air, CCl₄ stimulated ethane formation and increased the concentration of TBA reactants in hepatocytes isolated from fed, nontreated rats. This stimulatory effect was found irrespective of the incubation medium, although absolute values were generally lower in Tris buffer than in EMEM. Losses of LDH were also observed with the higher concentration of CCl₄. When carbogen was the atmosphere for incubation, no significant increases in the lipid peroxidation parameters could be found in response to CCl₄. Again, this response was irrespective of the incubation medium. Under carbogen, CCl₄-induced LDH leakage was no longer observed. Thus, these data provide a possible explanation as to why it has been difficult to observe lipid peroxidation, as evidenced by elevated TBA reactants concentration in hepatocytes isolated from fed, nontreated rats.     

Perfluoroalkylated compounds induce cell death and formation of reactive oxygen species in cultured cerebellar granule cells

The present communication investigates the effects of different perfluoroalkylated compounds (PFCs) on formation of reactive oxygen species (ROS) and cell death in cultured cerebellar granule cells. This allows direct comparison with similar effects found for other environmental contaminants like polychlorinated biphenyls and brominated flame-retardants. The increase in ROS formation and cell death was assayed using the fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA) and the trypan blue exclusion assay. The effects of the PFCs were structure dependent. Cell death was induced at relatively low concentrations by perfluorooctyl sulfonate (PFOS), perfluorooctane sulfonylamide (PFOSA) and the fluorotelomer alcohol 1H, 1H, 2H, 2H-perfluorodecanol (FTOH 8:2) with EC₅₀-values of 62 ± 7.6, 13 ± 1.8 and 15 ± 4.2 μM (mean ± SD) respectively. PFOS, perfluorooctanoic acid (PFOA) and PFOSA induced a concentration dependent increase in ROS formation with EC₅₀-values of 27 ± 9.0, 25 ± 11 and 57 ± 19 μM respectively. Reduced cell viability and ROS formation were observed at concentration level close to what is found in serum of occupationally exposed workers. The effect of PFCs on ROS formation and cell viability was compared with other halogenated compounds and future investigations should emphasize effects of mixtures and how physical chemical properties of the compounds influence their toxicity.     

6-Hydroxydopamine-induced glutathione alteration occurs via glutathione enzyme system in primary cultured astrocytes

Aim: To define the role of enzymes involved in glutathione metabolism in 6-hydroxydopamine (6-OHDA)-induced glutathione alteration in primary cultured astrocytes. Methods: Total glutathione (GSx) levels were determined using the modified enzymatic microtiter plate assay. The mRNA levels of T-glutamylcyste inesynthetase (γGCS), T-glutamyltransferase (γGT), glutathione peroxidase (GPx), GR (glutathione reductase), and glutathione transferases (GST) were determined using RT-PCR. γGT activity was determined using γGT assay kits. Results: In primary cultured astrocytes, 6-OHDA induced a significant elevation of cellular GSx levels after treatment for 24 h. However, the GSx levels decreased after 24 h and the values were even lower than the value in the control group without 6-OHDA at 48 h. RT-PCR data showed that the mRNA levels of γGCS, the ratelimiting enzyme of γ-L-glutamyl-L-cysteinylglycine (GSH) synthesis, were increased by 6-OHDA after treatment for 24 h and 48 h; the mRNA levels of GPx, GR, and GST did not alter in 6-OHDA-treated astrocytes after treatment for 24 h and 48 h; and 6-OHDA increased the mRNA levels and the activity of γGT after treatment for 48 h, which induced a decrease in GSx levels, despite the up-regulation of γGCS aftere xposure to 6-OHDA for 48 h. Conclusion: The change in γGCS correlated with the increase in GSH levels induced by 6-OHDA after treatment for 24 h. GSx levels decreased because of increased γGT mRNA levels and γGT activity induced by 6-OHDA after treatment for 48 h.     

Ethanol-induced changes in lipid peroxidation and glutathione content in rat brain

The effect of acute and chronic ethanol administration on brain lipid peroxide and glutathione levels was investigated in rats. Acute ethanol administration (5 g/kg, i.p.) led to an increase in lipid peroxide levels and a decrease in glutathione levels in whole brain homogenates without cerebellum. However, there was no change in brain lipid peroxide and glutathione levels of rats chronically treated with ethanol.     

Has reactive oxygen a role in methylglyoxal toxicity? A study on cultured rat hepatocytes

The toxicity of methylglyoxal and its ability to generate reactive oxygen species were investigated in cultured rat hepatocytes. Under aerobic and anaerobic conditions methylglyoxal increased lactate dehydrogenase (LDH) release and trypan blue uptake in a concentration dependent manner. Those concentrations of methylglyoxal causing cell injury (1 mM<) also caused the release of reactive oxygen species as indicated by peroxidase-catalyzed luminol chemiluminescence. Release of reactive oxygen was detectable only under aerobic conditions, and only became significant when a large portion of the cells had already lost their viability. It is concluded that methylglyoxal injures cultured rat hepatocytes and induces the generation of reactive oxygen species. The reactive oxygen species, however, are essentially not involved in methylglyoxal hepatotoxicity but are released by already severely injured cells.     

Arsenic induces mitochondria-dependent apoptosis by reactive oxygen species generation rather than glutathione depletion in Chang human hepatocytes

This study was conducted to evaluate the possible involvement of mitochondrial pathway in NaAsO₂-induced apoptosis and the role of reactive oxygen species (ROS) and reduced glutathione (GSH) in the apoptotic effect in Chang human hepatocytes. The MTT assay demonstrated that sodium arsenite (NaAsO₂) treatment for 24 h caused a dose-dependent decrease of cell viability. NaAsO₂ treatment (0–30 μM) was also found to induce phosphatidylserine externalization, a hallmark of apoptosis; to disrupt the mitochondrial membrane potential (Δψ _m ); to cause the release of cytochrome c into the cytosol, and to trigger cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner. All these changes were accompanied with the enhanced generation of intracellular ROS and malondialdehyde (MDA). Increase of intracellular GSH also coincided unexpectedly. Moreover, the extracellular addition of N-acetyl-l-cysteine (NAC, 5 mM) effectively reduced the generation of ROS and MDA, and rescued the cells from NaAsO₂ induced apoptosis and related alteration of mitochondria. These data suggest that the arsenic-induced cell apoptosis occurs though the mitochondrial pathway, and is mostly dependent on generation of ROS rather than GSH depletion in Chang human hepatocytes.     

Hydroxylated polychlorinated biphenyls increase reactive oxygen species formation and induce cell death in cultured cerebellar granule cells

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that bioaccumulate in the body, however, they can be metabolized to more water-soluble products. Although they are more readily excreted than the parent compounds, some of the metabolites are still hydrophobic and may be more available to target tissues, such as the brain. They can also cross the placenta and reach a developing foetus. Much less is known about the toxicity of PCB metabolites than about the parent compounds. In the present study, we have investigated the effects of eight hydroxylated (OH) PCB congeners (2′-OH PCB 3, 4-OH PCB 14, 4-OH PCB 34, 4′-OH PCB 35, 4-OH PCB 36, 4′-OH PCB 36, 4-OH PCB 39, and 4′-OH PCB 68) on reactive oxygen species (ROS) formation and cell viability in rat cerebellar granule cells. We found that, similar to their parent compounds, OH-PCBs are potent ROS inducers with potency 4-OH PCB 14 < 4-OH PCB 36 < 4-OH PCB 34 < 4′-OH PCB 36 < 4′-OH PCB 68 < 4-OH PCB 39 < 4′-OH PCB 35. 4-OH PCB 36 was the most potent cell death inducer, and caused apoptotic or necrotic morphology depending on concentration. Inhibition of ERK1/2 kinase with U0126 reduced both cell death and ROS formation, suggesting that ERK1/2 activation is involved in OH-PCB toxicity. The results indicate that the hydroxylation of PCBs may not constitute a detoxification reaction. Since OH-PCBs like their parent compounds are retained in the body and may be more widely distributed to sensitive tissues, it is important that not only the levels of the parent compounds but also the levels of their metabolites are taken into account during risk assessment of PCBs and related compounds.     

Methyl-thiophanate increases reactive oxygen species production and induces genotoxicity in rat peripheral blood

Methylthiophanate is one of the widely used fungicides to control important fungal diseases of crops. The aim of this study was to elucidate the short-term hematoxicity and genotoxicity effects of methylthiophanate administered by intraperitoneal way at three doses (300, 500 and 700?mg/kg of body weight) after 24, 48 and 72?h. Our results showed, 24?h after methylthiophanate injection, a hematological perturbation such as red blood cells (p?<?0.05, p?<?0.05 and p?<?0.01) and hemoglobin content (p?<?0.05), respectively, and a noticeable genotoxic effect in WBC evidenced by a significant increase in the frequency of the micronuclei and a decrease in cell viability. An increase in erythrocyte osmotic fragility was also noted after 24 and 48?h of methylthiophanate treatment at graded doses. A significant increase in hydrogen peroxide, advanced oxidation of protein products and malondialdehyde levels, in erythrocytes of methylthiophanate-treated rats with 300, 500 and 700?mg/kg of body weight, was also observed after 24?h of treatment (p?<?0.05, p?<?0.01 and p?<?0.001, respectively), suggesting the implication of oxidative stress in its toxicity. Antioxidants activities of superoxide dismutase and glutathione peroxidase in erythrocytes significantly increased (p?<?0.001) 24?h after the highest dose injected. While all these parameters were improved after 72?h of methylthiophanate injection (300, 500 and 700?mg/kg body weight). In conclusion, these data showed that the exposure of adult rats to methylthiophanate resulted in oxidative stress leading to hematotoxicity and the impairment of defence system, confirming the pro-oxidant and genotoxic effects of this fungicide.     

Formation of active oxygen species by dithranol, III. Dithranol, active oxygen species and lipid peroxidation in vivo

At pH 7.8 the dithranol anion 1a is oxidized in the dark to yield the bianthrone 3 besides dithranol brown, whilst O₂ is reduced to O₂^·? and H₂O₂. O₂^·? was detected by the reduction of nitroblue-tetrazolium (NBT, 4 ). The formation of the formazan 5 was inhibited by superoxide dismutase (SOD). Exclusion of O₂ and addition of SOD reveal a direct electron transfer from 1a to 4 and to cytochrome c which was not affected in the presence of SOD. — In vivo experiments indicate a low rate of lipid peroxidation which was determined on the basis of ethane exhalation.