Andres Caro - Academia.edu (original) (raw)
Papers by Andres Caro
Archives of Biochemistry and Biophysics, 2007
Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid... more Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (α-tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1-AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by α-tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or α-tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1expressing cells: (i) is associated with release of Ca 2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; iii) does not depend on increased influx of extracellular calcium, and iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1.
Journal of Biological Chemistry, 2001
The objective of this work was to investigate whether CYP2E1-and oxidative stress-dependent toxic... more The objective of this work was to investigate whether CYP2E1-and oxidative stress-dependent toxicity in HepG2 cells is mediated by an increase of cytosolic Ca 2؉ and activation of Ca 2؉-modulated processes. HepG2 cells expressing CYP2E1 (E47 cells) or control cells not expressing CYP2E1 (C34 cells) were preloaded with arachidonic acid (AA, up to 10 M) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 M) for variable periods (up to 12 h). Toxicity was greater in E47 cells than in C34 cells at all times and combinations of iron/AA tested. Cytosolic calcium increased with incubation time in both cell lines, but the increase was higher in E47 cells than in C34 cells. The rise in calcium was an early event and preceded the developing toxicity. Toxicity in E47 cells and the increase in Ca 2؉ were inhibited by omission of Ca 2؉ from the extracellular medium, and toxicity was restored by reincorporation of Ca 2؉. An inhibitor of Ca 2؉ release from intracellular stores did not prevent the toxicity or the increase in Ca 2؉ , reflecting a role for the influx of extracellular Ca 2؉ in the toxicity. Reactive oxygen production was similar in media with or without calcium, indicating that calcium was not modulating CYP2E1-dependent oxidative stress. Toxicity, lipid peroxidation, and the increase of Ca 2؉ in E47 cells exposed to iron-AA were inhibited by ␣-tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40fold). The toxicity in E47 cells mirrorred calpain activation and was inhibited by calpeptin, suggesting that calpain activation plays a causal role in toxicity. These results suggest that CYP2E1-dependent toxicity in this model depends on the activation of lipid peroxidation, followed by an increased influx of extracellular Ca 2؉ and activation of Ca 2؉-dependent proteases.
![Research paper thumbnail of Oxidative stress affects [alpha]-tocopherol content in soybean embryonic axes upon imbibition and following germination](https://attachments.academia-assets.com/79653246/thumbnails/1.jpg)
](Plant physiology, 1993
l h e content of a-tocopherol (a l) in isolated soybean (Glycine mar, var Hood) embryonic axes wa... more l h e content of a-tocopherol (a l) in isolated soybean (Glycine mar, var Hood) embryonic axes was measured upon germination. Dry, high-vigor axes contained 1.2 f 0.1, nmol/axis and after an increase during the initial 6 h of imbibition, there was a decline to 1.0 f 0.1 nmol/axis at 24 h of incubation. lncubation in the presence of the redox-cycling agent paraquat (4 mM) for 24 h increased the a1 content to 1.9 2 0.2 nmol/axis. When the incubation medium was supplemented with 500 f i~ Fe-EDTA over 24 h, the content of a1 increased to 1.8 f 0.1 nmol/axis. lsolated axes from soybean seeds stored for 56 months contained 6.5-C 0.3 nmol of crl/axis after 24 h of imbibition as compared to 1.0 f 0.1 nmol of al/axis in axes from soybean seeds stored for 8 months. In all of these experimental situations, oxidant production as assessed in vivo by a fluorometric assay was increased by 4 mM paraquat (8fold), 500 f i~ iron (2-fold), and 56 months of storage (4-fold) after 24 h of imbibition. l h e data presented here suggest that the cellular content of a1 is physiologically adjusted as a response to conditions of oxidative stress. The set of intracellular or extracellular conditions that leads to an increase in the steady-state concentration of reactive oxygen species such as superoxide radical (O2-), hydrogen peroxide (H202), hydroxyl radical (. OH), singlet oxygen ('O2), lipid hydroperoxides, or related species is termed oxidative stress (Chance et al., 1979). Physiologically, metabolic activity in conjunction with the structural organization of the cell is able to minimize adverse effects of oxidative stress. Susceptibility to oxidative stress is a function of the overall balance between the factors that increase oxidant generation and those substances that exhibit antioxidant capability. Tocopherols (with vitamin E activity) are critica1 antioxidants in eukaryotic organisms, ubiquitous in higher plants, and essential nutrients for animals (Tramontano et al., 1992). However, little is known about the endogenous content in plant tissues. aT (5,7,8-trimethyltocol) is the predominant tocopherol in vegetative tissue (Newton and Pennock, 1971; Hardy et al., 1991). Early studies in which two-dimensional chromatography was used showed that aT was predominant in young maize, wheat, barley, and peas and that other tocopherols appeared as the plant aged (Tramontano et al.,
Archives of Biochemistry and Biophysics, 2007
Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid... more Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (α-tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1-AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by α-tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or α-tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1expressing cells: (i) is associated with release of Ca 2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; iii) does not depend on increased influx of extracellular calcium, and iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1.
Journal of Biological Chemistry, 2001
The objective of this work was to investigate whether CYP2E1-and oxidative stress-dependent toxic... more The objective of this work was to investigate whether CYP2E1-and oxidative stress-dependent toxicity in HepG2 cells is mediated by an increase of cytosolic Ca 2؉ and activation of Ca 2؉-modulated processes. HepG2 cells expressing CYP2E1 (E47 cells) or control cells not expressing CYP2E1 (C34 cells) were preloaded with arachidonic acid (AA, up to 10 M) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 M) for variable periods (up to 12 h). Toxicity was greater in E47 cells than in C34 cells at all times and combinations of iron/AA tested. Cytosolic calcium increased with incubation time in both cell lines, but the increase was higher in E47 cells than in C34 cells. The rise in calcium was an early event and preceded the developing toxicity. Toxicity in E47 cells and the increase in Ca 2؉ were inhibited by omission of Ca 2؉ from the extracellular medium, and toxicity was restored by reincorporation of Ca 2؉. An inhibitor of Ca 2؉ release from intracellular stores did not prevent the toxicity or the increase in Ca 2؉ , reflecting a role for the influx of extracellular Ca 2؉ in the toxicity. Reactive oxygen production was similar in media with or without calcium, indicating that calcium was not modulating CYP2E1-dependent oxidative stress. Toxicity, lipid peroxidation, and the increase of Ca 2؉ in E47 cells exposed to iron-AA were inhibited by ␣-tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40fold). The toxicity in E47 cells mirrorred calpain activation and was inhibited by calpeptin, suggesting that calpain activation plays a causal role in toxicity. These results suggest that CYP2E1-dependent toxicity in this model depends on the activation of lipid peroxidation, followed by an increased influx of extracellular Ca 2؉ and activation of Ca 2؉-dependent proteases.
Plant physiology, 1993
l h e content of a-tocopherol (a l) in isolated soybean (Glycine mar, var Hood) embryonic axes wa... more l h e content of a-tocopherol (a l) in isolated soybean (Glycine mar, var Hood) embryonic axes was measured upon germination. Dry, high-vigor axes contained 1.2 f 0.1, nmol/axis and after an increase during the initial 6 h of imbibition, there was a decline to 1.0 f 0.1 nmol/axis at 24 h of incubation. lncubation in the presence of the redox-cycling agent paraquat (4 mM) for 24 h increased the a1 content to 1.9 2 0.2 nmol/axis. When the incubation medium was supplemented with 500 f i~ Fe-EDTA over 24 h, the content of a1 increased to 1.8 f 0.1 nmol/axis. lsolated axes from soybean seeds stored for 56 months contained 6.5-C 0.3 nmol of crl/axis after 24 h of imbibition as compared to 1.0 f 0.1 nmol of al/axis in axes from soybean seeds stored for 8 months. In all of these experimental situations, oxidant production as assessed in vivo by a fluorometric assay was increased by 4 mM paraquat (8fold), 500 f i~ iron (2-fold), and 56 months of storage (4-fold) after 24 h of imbibition. l h e data presented here suggest that the cellular content of a1 is physiologically adjusted as a response to conditions of oxidative stress. The set of intracellular or extracellular conditions that leads to an increase in the steady-state concentration of reactive oxygen species such as superoxide radical (O2-), hydrogen peroxide (H202), hydroxyl radical (. OH), singlet oxygen ('O2), lipid hydroperoxides, or related species is termed oxidative stress (Chance et al., 1979). Physiologically, metabolic activity in conjunction with the structural organization of the cell is able to minimize adverse effects of oxidative stress. Susceptibility to oxidative stress is a function of the overall balance between the factors that increase oxidant generation and those substances that exhibit antioxidant capability. Tocopherols (with vitamin E activity) are critica1 antioxidants in eukaryotic organisms, ubiquitous in higher plants, and essential nutrients for animals (Tramontano et al., 1992). However, little is known about the endogenous content in plant tissues. aT (5,7,8-trimethyltocol) is the predominant tocopherol in vegetative tissue (Newton and Pennock, 1971; Hardy et al., 1991). Early studies in which two-dimensional chromatography was used showed that aT was predominant in young maize, wheat, barley, and peas and that other tocopherols appeared as the plant aged (Tramontano et al.,