The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinest... more The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinesterase action. Recently, we have shown that, at noncholinergic doses (1 to 10 nM), POX (the bioactive metabolite of parathion) causes apoptotic cell death in murine EL4 T-lymphocytic leukemia cell line through activation of caspase-3. In this study, by employing caspase-specific inhibitors, we extend our observations to elucidate the sequence of events involved in POX-stimulated apoptosis. Pretreatment of EL4 cells with the caspase-9-specific inhibitor zLEHD-fmk attenuated POX-induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor zIETD-fmk had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to POX treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in POX-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, POX triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that POX disrupted mitochondrial transmembrane potential. Neither this effect nor cytchrome c release was dependent on caspase activation, since the general inhibitor of the caspase family zVAD-fmk did not influence both processes. Finally, POX treatment also resulted in a time-dependent up-regulation and translocation of the proapoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that POX induces apoptosis in EL4 cells through a direct effect on mitochondria by disrupting its transmembrane potential, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide a useful strategy to minimize organophosphate-induced poisoning.
Introduction Methotrexate (MTX) is an antineoplastic agent widely used in low dose to treat patie... more Introduction Methotrexate (MTX) is an antineoplastic agent widely used in low dose to treat patients with rheumatoid arthritis (RA). Its side effects can partly be explained by folate antagonism. Folinic acid (Leucovorin) is generally administered with MTX to decrease MTX-induced toxicity. However information regarding the inhibitory effect of folinic acid against cytogenetic damage caused by MTX is limited. The aim of this study was to assess the protective effect of folinic acid against MTX-genotoxicity. Methods This study was done on Wistar albino rats and in patients with RA. Forty rats of both sexes were randomized into four equal groups and dosed in the following way: Group-I, distilled water vehicle; Group-II, 0.5mg/kg folinic acid; Group-III, 0.5 mg/kg MTX; Group-IV, 0.5mg/kg folinic acid plus 0.5mg/kg MTX. Doses were given i.p., once daily for 8 consecutive days. A bone marrow chromosomal study and a micronucleus test were performed for each rat. Twenty patients with RA (5males and 15 females) on a 10mg weekly dose of MTX, i.m., for one month, were administered the same dose of MTX in addition to 10mg of folinic acid as a single dose 4 hours after MTX administration, i.m., every week for another 4 weeks. Chromosomal studies as well as a micronucleus test were evaluated for each patient. Results MTX produced a significant genetic injury as proved by the increased incidence of chromosomal aberration and micronuclei formation in Group-III animals. Inversely, folinic acid (group IV) produced a significant protection against genetic damages induced by MTX. In RA patients, folinic acid provides satisfactory improvement of MTX-induced genetic damage. Conclusion Folinic acid has a protective affect against MTX genotoxicity in human as well as in animal models.
The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinest... more The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinesterase action. Recently, we have shown that, at noncholinergic doses (1 to 10 nM), POX (the bioactive metabolite of parathion) causes apoptotic cell death in murine EL4 T-lymphocytic leukemia cell line through activation of caspase-3. In this study, by employing caspase-specific inhibitors, we extend our observations to elucidate the sequence of events involved in POX-stimulated apoptosis. Pretreatment of EL4 cells with the caspase-9-specific inhibitor zLEHD-fmk attenuated POX-induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor zIETD-fmk had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to POX treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in POX-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, POX triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that POX disrupted mitochondrial transmembrane potential. Neither this effect nor cytchrome c release was dependent on caspase activation, since the general inhibitor of the caspase family zVAD-fmk did not influence both processes. Finally, POX treatment also resulted in a time-dependent up-regulation and translocation of the proapoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that POX induces apoptosis in EL4 cells through a direct effect on mitochondria by disrupting its transmembrane potential, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide a useful strategy to minimize organophosphate-induced poisoning.
Introduction Methotrexate (MTX) is an antineoplastic agent widely used in low dose to treat patie... more Introduction Methotrexate (MTX) is an antineoplastic agent widely used in low dose to treat patients with rheumatoid arthritis (RA). Its side effects can partly be explained by folate antagonism. Folinic acid (Leucovorin) is generally administered with MTX to decrease MTX-induced toxicity. However information regarding the inhibitory effect of folinic acid against cytogenetic damage caused by MTX is limited. The aim of this study was to assess the protective effect of folinic acid against MTX-genotoxicity. Methods This study was done on Wistar albino rats and in patients with RA. Forty rats of both sexes were randomized into four equal groups and dosed in the following way: Group-I, distilled water vehicle; Group-II, 0.5mg/kg folinic acid; Group-III, 0.5 mg/kg MTX; Group-IV, 0.5mg/kg folinic acid plus 0.5mg/kg MTX. Doses were given i.p., once daily for 8 consecutive days. A bone marrow chromosomal study and a micronucleus test were performed for each rat. Twenty patients with RA (5males and 15 females) on a 10mg weekly dose of MTX, i.m., for one month, were administered the same dose of MTX in addition to 10mg of folinic acid as a single dose 4 hours after MTX administration, i.m., every week for another 4 weeks. Chromosomal studies as well as a micronucleus test were evaluated for each patient. Results MTX produced a significant genetic injury as proved by the increased incidence of chromosomal aberration and micronuclei formation in Group-III animals. Inversely, folinic acid (group IV) produced a significant protection against genetic damages induced by MTX. In RA patients, folinic acid provides satisfactory improvement of MTX-induced genetic damage. Conclusion Folinic acid has a protective affect against MTX genotoxicity in human as well as in animal models.
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