PMC:3824683 / 18622-28125 JSONTXT

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abuse such as MA is thought to be an additional risk factor in worsening the HIV-associated neurodegeneration.9 Viral proteins, such as gp120, have been shown to interplay cooperatively with MA and damage BBB integrity in mouse brain.17 In addition, MA alone is also known to induce behavioral impairment in gp120 transgenic mice.40 However, there is no direct evidence that elucidates the mechanism responsible for the combined effect of MA- and gp120-induced toxicities in the brain. We have recently shown that MA and gp120 can induce proinflammatory cytokine IL-6 via common signaling mechanism involving PI3K/Akt and NF-κB pathways.6, 41 On the other hand, an increasing body of literatures clearly suggests oxidative stress as one of the mediators of neurotoxicity induced by HIV-1 gp120 as well as MA independently. However, no attempt has been made to determine the fate of oxidative stress in a possible situation where HIV-infected individuals were MA user as well. This study was undertaken with the idea of determining whether gp120 and MA-mediated oxidative stress would cause increased apoptosis in astrocytes and whether CYP450 has any role in oxidative stress.\nOxidative stress is associated with cell toxicity in various neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease.42 In addition, several reports have highlighted the role of oxidative stress in the pathology of HIV-associated neurocognitive disorders (HAND).43 This was further confirmed by our studies with morphine, wherein we showed morphine-mediated increased virus replication,44 accelerated disease progression,45 and increased oxidative stress46, 47 in macaque model of HIV/AIDS. The oxidative stress produced by either MA or gp120 has also been shown to have neurotoxic potential via various intermediates. MA, in particular, is found to increase oxidative stress predominantly via dopaminergic and glutamatergic mechanisms.10 The dopaminergic toxicity observed with MA is attributed to its structural similarity with dopamine. Furthermore, gp120 is shown to increase oxidative stress via glutathione and lipid peroxidation.8, 48 Concurrent with the existing literatures, we also observed increased oxidative stress in astrocytes treated with gp120 and MA individually, in time- and concentration-dependent manners.\nRecently, dopamine has been shown to regulate the expression of various CYP isozymes in the liver.49 Furthermore, Bromek et al.50 has shown that CYP2D increases dopamine formation in the brain. In addition to the metabolism of xenobiotics including substances of abuse, the role of CYPs in oxidative stress is unequivocally accepted in various tissues/organs including the brain.51 Specifically, the roles of CYP2E1 for alcohol metabolism-mediated toxicity and CYP2A622 for tobacco/nicotine metabolism-mediated toxicity52 have been implicated through the oxidative stress pathway in many tissues/organs. Our group along with others has previously reported that astrocytes also express many CYPs at variable levels, and they have demonstrated the role of CYPs in alcohol- and nicotine-mediated oxidative stress and toxicities.52, 53 Similarly, MA has been shown to induce mRNA expression of CYP2E1 and activity of CYP2C6 in rats.54, 55 Our observations with regard to increased expressions of CYP2A6, 2B6, and 2D6 by MA and CYP2E1, 2B6, and 2D6 by gp120 are important to elucidate the roles of these CYPs in oxidative stress, metabolism of certain xenobiotics, and homeostasis of other resident molecules such as dopamine in the brain. More importantly, the additive increase in the expressions of CYP2D6 and CYP2E1 by gp120+MA is intriguing, which suggests a possibility that CYPs might have been involved in oxidative stress. This is the first evidence that shows an additive effect between MA or gp120 and CYP expression in the CNS.\nAmphetamine analogs including MA are known to interact with CYP2D6 and alters its activity,56 which is further supported by the finding that selective inhibitors of CYP2D6 reduces the metabolism of ecstacy (MA analog) in vitro.57 However, the role of CYP2E1 in the metabolism of MA is largely unknown, except a few recent reports that have shown an involvement of CYP2E1 and CYP2B in the metabolism of either MA or pyrolysis product of MA.58, 59 CYP2E1 is mainly known for the metabolic activity in the presence of ethanol; however, its involvement in MA-mediated production of ROS leading to increased cell death is not known. We are able to confirm involvement of CYP2E1 in oxidative stress because DAS, a selective inhibitor for CYP2E1, significantly abrogated not only gp120/MA-mediated production of ROS but also cell death caused by these two stimulants individually or in combination. Unlike CYP2E1 inhibitor, the CYP2D6 and CYP2B6 inhibitors increased MA/gp120-induced ROS, suggesting lack of its role in MA/gp120-mediated oxidative stress. The increased oxidative stress by CYP2D6 inhibitors has been observed before and has been attributed to the direct effect of these inhibitors.60\nThe interaction of various CYPs with NADPH system is tightly regulated by NOX enzymes.61 CYP2E1 is specifically involved in the metabolism of R-OH compounds into aldehydes, which is coupled with the conversion of NADPH to NADP+ and the released electron is consumed in the process. This NADP+ is converted via non-CYP mechanism into NADPH, which is recycled back to NADPH by NOX family of enzymes (Figure 8). The NOX family of enzymes, particularly NOX2 and NOX4, is potentially known to increase ROS in astrocytes independently as well as in association with FWH chemistry.31, 36 Furthermore, the superoxides and peroxides generated via NOX enzymes can result in increased expressions of ferritin and protein carbonylation38 as shown in this study as well. The role of NOX in substance abuse such as alcohol62 and MA18 is known in neuronal toxicity. Furthermore, the NOX enzymes have been implicated in therapeutic intervention of CNS disorders such as strokes and Alzheimer's disease.63, 64 Our observations with NOX2 and NOX4 knockdown to rescue cells from oxidative damage support the possibility of using NOX as a target for developing potential therapeutics for the treatment of neuroAIDS, especially among MA users.\nVarious free radicals, generated during oxidative stress, may lead to multifactorial physiological effects in the cells via DNA damage, lipid peroxidation, or activation of apoptotic mechanisms. The ROS-mediated BBB damage has been shown to cause lipid peroxidation and loss of tight junction protein.13, 17 MA and gp120 in combination are specifically shown to alter the levels of tight junction proteins to create a ‘leaky' BBB, which may enhance the invasion of infected monocytes into the brain.17 Similarly, cocaine in association with gp120 has also been shown to increase ROS, which involves caspase-3 and NF-κB activation to induce apoptosis in astrocytes.11 In our prior studies, we have shown an overlapping mechanism between MA and gp120 involving NF-κB activation.41 We have further shown the induction of proinflammatory cytokines as a result of NF-κB activation. In the present study, we observed an increase in the expression of cleaved caspase-3 protein in MA and/or gp120, which were further confirmed with the increase in the caspase-3 cleavage activity. Our results are consistent with the recent finding that apoptosis is involved in gp120-mediated toxicity.23, 65 Furthermore, caspase-3 cleavage activity was inhibited by selective inhibitors for CYP2E1, NOX, and FWH reaction. Taken together, these findings suggest that the increase in caspase-3 expression and activity via ROS and cytokines via NF-κB activation may lead to toxicity in astrocytes. Thus, there is a possibility of a cross-talk between these two mechanisms and therefore further studies are needed to investigate the effect of CYP-mediated ROS on cytokine production or vice versa. Our TUNEL staining was also consistent with this notion as both MA and gp120 independently as well as in combination led to increased DNA damage. Our observations with MTT assay further confirmed the toxic consequences of ROS production as a result of MA/gp120 interaction. In addition, inhibitors for CYP2E1, NOX, and FWH reaction abolished caspase-3 activity and apoptotic cell death, which clearly suggested that ROS, induced by gp120/MA interaction, was responsible for the apoptotic cell death.\nIn summary, results from our present study indicate that in the astrocytes, MA potentiates the gp120-mediated oxidative stress that may worsen the neurodegenerative complications in HIV-infected MA users. The involvement of CYP2E1 in interaction between gp120 and MA presents a novel opportunity to explore their further roles in neurotoxicity. Furthermore, it provides an opportunity to use this pathway as a target to find potentially novel pharmaceuticals. Specific inhibitors such as DAS, which is also a food additive,22 may potentially be used as food supplements/drugs in HIV-infected MA users to reduce neurotoxicity. The oxidative stress-mediated increased toxicity by MA/gp120 in astrocytic cells also provides an opportunity to potentially use antioxidants in reducing MA-mediated HIV-1 pathogenesis in the patients who abuse MA. Finally, as peptide inhibitors against NOX have been shown to be protective in animal models, these inhibitors may potentially be used to reduce MA-induced HIV-1 pathogenesis in the humans."}

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We have recently shown that MA and gp120 can induce proinflammatory cytokine IL-6 via common signaling mechanism involving PI3K/Akt and NF-κB pathways.6, 41 On the other hand, an increasing body of literatures clearly suggests oxidative stress as one of the mediators of neurotoxicity induced by HIV-1 gp120 as well as MA independently. However, no attempt has been made to determine the fate of oxidative stress in a possible situation where HIV-infected individuals were MA user as well. This study was undertaken with the idea of determining whether gp120 and MA-mediated oxidative stress would cause increased apoptosis in astrocytes and whether CYP450 has any role in oxidative stress.\nOxidative stress is associated with cell toxicity in various neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease.42 In addition, several reports have highlighted the role of oxidative stress in the pathology of HIV-associated neurocognitive disorders (HAND).43 This was further confirmed by our studies with morphine, wherein we showed morphine-mediated increased virus replication,44 accelerated disease progression,45 and increased oxidative stress46, 47 in macaque model of HIV/AIDS. The oxidative stress produced by either MA or gp120 has also been shown to have neurotoxic potential via various intermediates. MA, in particular, is found to increase oxidative stress predominantly via dopaminergic and glutamatergic mechanisms.10 The dopaminergic toxicity observed with MA is attributed to its structural similarity with dopamine. Furthermore, gp120 is shown to increase oxidative stress via glutathione and lipid peroxidation.8, 48 Concurrent with the existing literatures, we also observed increased oxidative stress in astrocytes treated with gp120 and MA individually, in time- and concentration-dependent manners.\nRecently, dopamine has been shown to regulate the expression of various CYP isozymes in the liver.49 Furthermore, Bromek et al.50 has shown that CYP2D increases dopamine formation in the brain. In addition to the metabolism of xenobiotics including substances of abuse, the role of CYPs in oxidative stress is unequivocally accepted in various tissues/organs including the brain.51 Specifically, the roles of CYP2E1 for alcohol metabolism-mediated toxicity and CYP2A622 for tobacco/nicotine metabolism-mediated toxicity52 have been implicated through the oxidative stress pathway in many tissues/organs. Our group along with others has previously reported that astrocytes also express many CYPs at variable levels, and they have demonstrated the role of CYPs in alcohol- and nicotine-mediated oxidative stress and toxicities.52, 53 Similarly, MA has been shown to induce mRNA expression of CYP2E1 and activity of CYP2C6 in rats.54, 55 Our observations with regard to increased expressions of CYP2A6, 2B6, and 2D6 by MA and CYP2E1, 2B6, and 2D6 by gp120 are important to elucidate the roles of these CYPs in oxidative stress, metabolism of certain xenobiotics, and homeostasis of other resident molecules such as dopamine in the brain. More importantly, the additive increase in the expressions of CYP2D6 and CYP2E1 by gp120+MA is intriguing, which suggests a possibility that CYPs might have been involved in oxidative stress. This is the first evidence that shows an additive effect between MA or gp120 and CYP expression in the CNS.\nAmphetamine analogs including MA are known to interact with CYP2D6 and alters its activity,56 which is further supported by the finding that selective inhibitors of CYP2D6 reduces the metabolism of ecstacy (MA analog) in vitro.57 However, the role of CYP2E1 in the metabolism of MA is largely unknown, except a few recent reports that have shown an involvement of CYP2E1 and CYP2B in the metabolism of either MA or pyrolysis product of MA.58, 59 CYP2E1 is mainly known for the metabolic activity in the presence of ethanol; however, its involvement in MA-mediated production of ROS leading to increased cell death is not known. We are able to confirm involvement of CYP2E1 in oxidative stress because DAS, a selective inhibitor for CYP2E1, significantly abrogated not only gp120/MA-mediated production of ROS but also cell death caused by these two stimulants individually or in combination. Unlike CYP2E1 inhibitor, the CYP2D6 and CYP2B6 inhibitors increased MA/gp120-induced ROS, suggesting lack of its role in MA/gp120-mediated oxidative stress. The increased oxidative stress by CYP2D6 inhibitors has been observed before and has been attributed to the direct effect of these inhibitors.60\nThe interaction of various CYPs with NADPH system is tightly regulated by NOX enzymes.61 CYP2E1 is specifically involved in the metabolism of R-OH compounds into aldehydes, which is coupled with the conversion of NADPH to NADP+ and the released electron is consumed in the process. This NADP+ is converted via non-CYP mechanism into NADPH, which is recycled back to NADPH by NOX family of enzymes (Figure 8). The NOX family of enzymes, particularly NOX2 and NOX4, is potentially known to increase ROS in astrocytes independently as well as in association with FWH chemistry.31, 36 Furthermore, the superoxides and peroxides generated via NOX enzymes can result in increased expressions of ferritin and protein carbonylation38 as shown in this study as well. The role of NOX in substance abuse such as alcohol62 and MA18 is known in neuronal toxicity. Furthermore, the NOX enzymes have been implicated in therapeutic intervention of CNS disorders such as strokes and Alzheimer's disease.63, 64 Our observations with NOX2 and NOX4 knockdown to rescue cells from oxidative damage support the possibility of using NOX as a target for developing potential therapeutics for the treatment of neuroAIDS, especially among MA users.\nVarious free radicals, generated during oxidative stress, may lead to multifactorial physiological effects in the cells via DNA damage, lipid peroxidation, or activation of apoptotic mechanisms. The ROS-mediated BBB damage has been shown to cause lipid peroxidation and loss of tight junction protein.13, 17 MA and gp120 in combination are specifically shown to alter the levels of tight junction proteins to create a ‘leaky' BBB, which may enhance the invasion of infected monocytes into the brain.17 Similarly, cocaine in association with gp120 has also been shown to increase ROS, which involves caspase-3 and NF-κB activation to induce apoptosis in astrocytes.11 In our prior studies, we have shown an overlapping mechanism between MA and gp120 involving NF-κB activation.41 We have further shown the induction of proinflammatory cytokines as a result of NF-κB activation. In the present study, we observed an increase in the expression of cleaved caspase-3 protein in MA and/or gp120, which were further confirmed with the increase in the caspase-3 cleavage activity. Our results are consistent with the recent finding that apoptosis is involved in gp120-mediated toxicity.23, 65 Furthermore, caspase-3 cleavage activity was inhibited by selective inhibitors for CYP2E1, NOX, and FWH reaction. Taken together, these findings suggest that the increase in caspase-3 expression and activity via ROS and cytokines via NF-κB activation may lead to toxicity in astrocytes. Thus, there is a possibility of a cross-talk between these two mechanisms and therefore further studies are needed to investigate the effect of CYP-mediated ROS on cytokine production or vice versa. Our TUNEL staining was also consistent with this notion as both MA and gp120 independently as well as in combination led to increased DNA damage. Our observations with MTT assay further confirmed the toxic consequences of ROS production as a result of MA/gp120 interaction. In addition, inhibitors for CYP2E1, NOX, and FWH reaction abolished caspase-3 activity and apoptotic cell death, which clearly suggested that ROS, induced by gp120/MA interaction, was responsible for the apoptotic cell death.\nIn summary, results from our present study indicate that in the astrocytes, MA potentiates the gp120-mediated oxidative stress that may worsen the neurodegenerative complications in HIV-infected MA users. The involvement of CYP2E1 in interaction between gp120 and MA presents a novel opportunity to explore their further roles in neurotoxicity. Furthermore, it provides an opportunity to use this pathway as a target to find potentially novel pharmaceuticals. Specific inhibitors such as DAS, which is also a food additive,22 may potentially be used as food supplements/drugs in HIV-infected MA users to reduce neurotoxicity. The oxidative stress-mediated increased toxicity by MA/gp120 in astrocytic cells also provides an opportunity to potentially use antioxidants in reducing MA-mediated HIV-1 pathogenesis in the patients who abuse MA. Finally, as peptide inhibitors against NOX have been shown to be protective in animal models, these inhibitors may potentially be used to reduce MA-induced HIV-1 pathogenesis in the humans."}