The potential toxicological insights about the anti-HIV drug azidothymidine-derived monoselenides in human leukocytes: Toxicological insights of new selenium-azidothymidine analogs.

Acquired immunodeficiency syndrome (AIDS) is a worldwide disease characterized by impairments of immune function. AIDS can be associated with oxidative stress (OS) that can be linked to selenium (Se) deficiency. Se is fundamental for the synthesis of selenoproteins, such as glutathione peroxidase and thioredoxin reductase. These enzymes catalyze the decomposition of reactive oxygen species and contribute to maintain equilibrium in cell redox status. Literature data indicate that organoselenium compounds, such as ebselen and diphenyl diselenide, have antioxidant properties in vitro and in vivo models associated with OS. Nevertheless, selenocompounds can also react and oxidize thiols groups, inducing toxicity in mammals. Here, we tested the potential cytotoxic and genotoxic properties of six analogs of the prototypal anti-HIV drug azidothymidine (AZT) containing Se (5'-Se-(phenyl)zidovudine; 5'-Se-(1,3,5-trimethylphenyl)zidovudine; 5'-Se-(1-naphtyl)zidovudine; 5'-Se-(4-chlorophenyl)zidovudine) (C4); 5'-Se-(4-methylphenyl)zidovudine (C5); and 5'-(4-methylbenzoselenoate)zidovudine). C5 increased the rate of dithiothreitol oxidation (thiol oxidase activity) and C2-C4 and C6 (at 100 µM) increased DNA damage index (DI) in human leukocytes. Moreover, C5 (200 µM) decreased human leukocyte viability to about 50%. Taken together, these results indicated the low in vitro toxicity in human leukocytes of some Se-containing analogs of AZT.

Is the lobster cockroach Nauphoeta cinerea a valuable model for evaluating mercury induced oxidative stress?

Organic and inorganic forms of mercury are highly neurotoxic environmental contaminants. The exact mechanisms involved in mercury neurotoxicity are still unclear. Oxidative stress appears to play central role in this process. In this study, we aimed to validate an insect-based model for the investigation of oxidative stress during mercury poisoning of lobster cockroach Nauphoeta cinerea. The advantages of using insects in basic toxicological studies include the easier handling, rapid proliferation/growing and absence of ethical issues, comparing to rodent-based models. Insects received solutions of HgCl2 (10, 20 and 40mgL(-1) in drinking water) for 7d. 24h after mercury exposure, animals were euthanized and head tissue samples were prepared for oxidative stress related biochemical determinations. Mercury exposure caused a concentration dependent decrease in survival rate. Cholinesterase activity was unchanged. Catalase activity was substantially impaired after mercury treatment 40mgL(-1). Likewise, GST had a significant decrease, comparing to control. Peroxidase and thioredoxin reductase activity was inhibited at concentrations of 20mgL(-1) and 40mgL(-1) comparing to control. These results were accompanied by decreased GSH levels and increased hydroperoxide and TBARS formation. In conclusion, our results show that mercuric compounds are able to induce oxidative stress signs in insect by modulating survival rate as well as inducing impairments on important antioxidant systems. In addition, our data demonstrates for the first time that Nauphoeta cinerea represents an interesting animal model to investigate mercury toxicity and indicates that the GSH and thioredoxin antioxidant systems plays central role in Hg induced toxicity in insects.

Evidences for a role of glutathione peroxidase 4 (GPx4) in methylmercury induced neurotoxicity in vivo.

We evaluated the activity and expression of antioxidant enzymes in the cerebellum and cortex of Swiss adult male mice exposed to methylmercury (MeHg) in drinking water (40mg/L) during 21 days. The activity of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD) and thioredoxin reductase (TrxR) were determined spectrophotometrically. The expression (protein levels) of GPx1 and GPx4 isoforms, TrxR1 as well as heat shock protein 70 (HSP70) were evaluated using specific antibodies and normalized by actin levels. The exposure of mice to MeHg caused a significant impairment in locomotors performance in the open field test (crossings and rearing). This result was followed by a significant reduction of GPx and TrxR activities in the cerebellum and cortex when compared to untreated animals. We also observed a substantial decrease in GPx1, GPx4 and TrxR1 protein levels in the cerebellum, while in the cerebral cortex, only GPx4 and TrxR1 were decreased after MeHg treatment. The activities of the antioxidant enzymes GR, GST, CAT and SOD were increased in the cerebellum after MeHg administration to mice. In contrast, only CAT was increased in the cerebral cortex of MeHg-treated animals. The expression of HSP70 was up-regulated only in the cerebellum where MeHg-exposed mice showed a significant increase in the immunocontent of HSP70 when compared to controls. This is the first report showing a role for GPx4 in the neurotoxicity induced by MeHg in vivo. In addition, our data indicates that the selenoproteins GPx and TrxR as main targets during MeHg exposure, which may be considered in biomarker studies.

Protective effects of organoselenium compounds against methylmercury-induced oxidative stress in mouse brain mitochondrial-enriched fractions

We evaluated the potential neuroprotective effect of 1-100 µM of four organoselenium compounds: diphenyl diselenide, 3’3-ditri-fluoromethyldiphenyl diselenide, p-methoxy-diphenyl diselenide, and p-chloro-diphenyl diselenide, against methylmercury-induced mitochondrial dysfunction and oxidative stress in mitochondrial-enriched fractions from adult Swiss mouse brain. Methylmercury (10-100 µM) significantly decreased mitochondrial activity, assessed by MTT reduction assay, in a dose-dependent manner, which occurred in parallel with increased glutathione oxidation, hydroperoxide formation (xylenol orange assay) and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS). The co-incubation with diphenyl diselenide (100 µM) completely prevented the disruption of mitochondrial activity as well as the increase in TBARS levels caused by methylmercury. The compound 3’3-ditrifluoromethyldiphenyl diselenide provided a partial but significant protection against methylmercury-induced mitochondrial dysfunction (45.4 ± 5.8 percent inhibition of the methylmercury effect). Diphenyl diselenide showed a higher thiol peroxidase activity compared to the other three compounds. Catalase blocked methylmercury-induced TBARS, pointing to hydrogen peroxide as a vector during methylmercury toxicity in this model. This result also suggests that thiol peroxidase activity of organoselenium compounds accounts for their protective actions against methylmercury-induced oxidative stress. Our results show that diphenyl diselenide and potentially other organoselenium compounds may represent important molecules in the search for an improved therapy against the deleterious effects of methylmercury as well as other mercury compounds.

Protective effects of organoselenium compounds against methylmercury-induced oxidative stress in mouse brain mitochondrial-enriched fractions.

We evaluated the potential neuroprotective effect of 1-100 µM of four organoselenium compounds: diphenyl diselenide, 3'3-ditri-fluoromethyldiphenyl diselenide, p-methoxy-diphenyl diselenide, and p-chloro-diphenyl diselenide, against methylmercury-induced mitochondrial dysfunction and oxidative stress in mitochondrial-enriched fractions from adult Swiss mouse brain. Methylmercury (10-100 µM) significantly decreased mitochondrial activity, assessed by MTT reduction assay, in a dose-dependent manner, which occurred in parallel with increased glutathione oxidation, hydroperoxide formation (xylenol orange assay) and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS). The co-incubation with diphenyl diselenide (100 µM) completely prevented the disruption of mitochondrial activity as well as the increase in TBARS levels caused by methylmercury. The compound 3'3-ditrifluoromethyldiphenyl diselenide provided a partial but significant protection against methylmercury-induced mitochondrial dysfunction (45.4 ± 5.8% inhibition of the methylmercury effect). Diphenyl diselenide showed a higher thiol peroxidase activity compared to the other three compounds. Catalase blocked methylmercury-induced TBARS, pointing to hydrogen peroxide as a vector during methylmercury toxicity in this model. This result also suggests that thiol peroxidase activity of organoselenium compounds accounts for their protective actions against methylmercury-induced oxidative stress. Our results show that diphenyl diselenide and potentially other organoselenium compounds may represent important molecules in the search for an improved therapy against the deleterious effects of methylmercury as well as other mercury compounds.

Genotoxicity of organoselenium compounds in human leukocytes in vitro.

DNA damage and cell viability of human leukocytes cells were examined as simple tests for screening the potential toxicity of organoselenium compounds. Leukocytes were incubated with different organoselenium compounds at 4, 10, 40 and 100 microM or vehicle (DMSO) for 3h at 37 degrees C before of in vitro assays. Cell viability was determined by Trypan blue exclusion. DNA damage was assessed using the alkaline comet assay with silver staining. The exposure of leukocytes to (S)-tert-butyl 1-diselenide-3-methylbutan-2-ylcarbamate, (S)-tert-butyl 1-diselenide-3-phenylpropan-2-ylcarbamate, (S)-2-amino-1-diselenide-3-methylpropanyl, (S)-2-amino-1-diselenide-3-phenylpropanyl, 3',3-ditrifluoromethyl diphenyl diselenide, 4',4-dimethoxy diphenyl diselenide, 4',4-dichloro diphenyl diselenide and 2',2,4',4,6',6-hexamethyl diphenyl diselenide, in the range of 10-100muM, induced a significant increase in Damage Index (DI). The genotoxic effect of all compounds was associated with high frequencies of cells with damage level 4 and all compounds caused a decrease in cell viability. Our results suggest that the selenium compounds tested were genotoxic and cytotoxic to human leukocytes cells in vitro and that the organoselenium amino acid derivatives ((S)-tert-butyl 1-diselenide-3-methylbutan-2-ylcarbamate, (S)-tert-butyl 1-diselenide-3-phenylpropan-2-ylcarbamate, (S)-2-amino-1-diselenide-3-methylpropanyl and (S)-2-amino-1-diselenide-3-phenylpropanyl) were more genotoxic than aromatic derivatives (3',3-ditrifluoromethyl diphenyl diselenide, 4',4-dimethoxy diphenyl diselenide, 4',4-dichloro diphenyl diselenide and 2',2,4',4,6',6-hexamethyl diphenyl diselenide). These effects may be linked to the pro-oxidant activity exhibited by selenium compounds when used in relatively high concentrations.

Hemolytic and genotoxic evaluation of organochalcogens in human blood cells in vitro.

This study investigated the hemolytic and genotoxic effect of different organoselenium and organotellurium compounds in human blood cells, as simple tests for screening the toxicity of organochalcogenides. For osmotic fragility (OF) test, samples of total blood were incubated with the organochalcogens at 4, 8, 50, 75 and 100 microM or vehicle (DMSO) for 90 min at 37 degrees C. The EC(50) values for hemolysis were significantly increased in erythrocytes exposed to diphenyl selenide (II), diphenyl diselenide (III), diphenyl telluride (IV), diphenyl ditelluride (V), (S)-2-amino-1-diselenide-3-methylpropanyl (IX), butyl(styryl)telluride (XIII) and 2-(butyltellurium)furan (XIV) at higher concentrations tested. The exposure of erythrocytes to organochalcogens diphenyl diselenide (II) and butyl(styryl)telluride (XIII), which had greater hemolytic effect, did not modify catalase activity, reactive oxygen species (ROS) production and -SH content. On the other hand, Na(+)/K(+) ATPase activity of erythrocyte ghosts was significantly inhibited by the compounds diphenyl diselenide (II) and butyl(styryl)telluride (XIII) (P<0.05) in a concentration-dependent manner. The inhibition of Na(+)/K(+) ATPase activity was completely reversed by dithiothreitol (DTT); indicating reaction of these organochalcogens with thiol groups of the enzyme. The thiol oxidase activity of the compounds II and XIII was supported by the fact that the rate of DTT oxidation was increased significantly by both chalcogens. In the higher concentrations, the compounds (II) and (XIII) were strongly genotoxic and cytotoxic to human leukocytes cells, as verified by the DNA damage and cell viability evaluation. Our results suggest that at relatively high concentration organochalcogenides exhibit hemolytic and genotoxic action in human blood cells, which are probably linked to their thiol oxidase activity and preferential interaction with sulfhydryl groups critical to enzymes.