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Neuropathic pain is one of the foremost adverse effects that worsens quality of life for patients undergoing an antiretroviral treatment. Currently, there are no effective analgesics for relieving it; thus, there is an urgent need to develop novel treatments for neuropathic pain. Previously, we described and validated F11 cells as a model of DRG (dorsal root ganglia) neurons. In the current work, we employed F11 cells to identify regulators of antiretroviral-induced neuropathic pain combining functional and transcriptomic analysis. The antiretroviral zalcitabine (ddC) increased the excitability of differentiated F11 cells associated with calcium signaling without morphological changes in the neuronal phenotype, mimicking the observed increase of painful signaling in patients suffering from antiretroviral-induced neuropathic pain. Employing RNA sequencing, we observed that zalcitabine treatment upregulated genes related with oxidative stress and calcium homeostasis. The functional impact of the transcriptomic changes was explored, finding that the exposure to zalcitabine significantly increased intracellular oxidative stress and reduced store-operated calcium entry (SOCE). Because the functional and transcriptomic evidence points toward fundamental changes in calcium signaling and oxidative stress upon zalcitabine exposure, we identified that NAD(P)H quinone dehydrogenase and the sarcoplasmic/endoplasmic reticulum calcium ATPase 3 were involved in zalcitabine-induced hyperexcitability of F11 cells. Overexpression of those genes increases the calcium-elicited hyperexcitability response and reduces SOCE, as well as increases intracellular ROS levels. These data do not only mimic the effects of zalcitabine but also highlight the relevance of oxidative stress and of calcium-mediated signaling in antiretroviral-induced hyperexcitability of sensory neurons, shedding light on new therapeutic targets for antiviral-induced neuropathic pain.

Highly active antiretroviral therapy (HAART) works effectively in inhibiting HIV replication in patients. However, the use of nucleoside reverse transcriptase inhibitors (NRTIs) often causes side effects of neuropathic pain, and its mechanism remains to be elucidated. Therefore, we aim to explore the mechanism of NRTIs-induced neuropathic pain at the transcriptome level. C57BL/6 J mice were given intraperitoneal injection of zalcitabine (ddC) or saline (control) for 2 weeks, during which the mechanical pain threshold of the mice was detected by von Frey test. Then the L3~L5 spinal segments of the mice were isolated and subsequently used for RNA sequencing (RNA-seq) on the last day of treatment. The mechanical pain threshold of mice given ddC decreased significantly. Compared with the control group, ddC caused significant changes in the expression of 135 genes, of which 66 upregulated and 69 downregulated. Enrichment analysis showed that the functions of these genes are mainly enriched in regulation of transcription, multicellular organism development, and cell differentiation, and the pathway is mainly enriched in the cGMP-PKG signaling pathway and AMPK signaling pathway. Furthermore, key genes such as Gabrd, Kcnd3, Npcd, Insr, Lypd6, Scd2, and Mef2d were also identified. These may serve as drug targets for the prevention or treatment of NRTI-induced neuropathic pain.

Nucleoside reverse transcriptase inhibitors (NRTIs) were the first drugs used to treat human immunodeficiency virus infection, and their use can cause mitochondrial toxicity, including mitochondrial DNA (mtDNA) depletion in several cases. The first-generation NRTIs, including 2',3'-dideoxycytidine (ddC), were originally and are still pursued as anticancer agents. NRTI-sensitive DNA polymerases localizing to mitochondria allow for the opportunity to poison proliferating cancer cell mtDNA replication as certain cancers rely heavily on mitochondrial functions. However, mtDNA replication is independent of the cell cycle creating a significant concern that toxicants such as ddC impair mtDNA maintenance in both proliferating and nonproliferating cells. To examine this possibility, we tested the utility of the HepaRG cell line to study ddC-induced toxicity in isogenic proliferating (undifferentiated) and nonproliferating (differentiated) cells. Following ddC exposures, we measured cell viability, mtDNA copy number, and mitochondrial bioenergetics utilizing trypan blue, Southern blotting, and extracellular flux analysis, respectively. After 13 days of 1 μM ddC exposure, proliferating and differentiated HepaRG harbored mtDNA levels of 0.9% and 17.9% compared with control cells, respectively. Cells exposed to 12 μM ddC contained even less mtDNA. By day 13, differentiated cell viability was maintained but declined for proliferating cells. Proliferating HepaRG bioenergetic parameters were severely impaired by day 8, with 1 and 12 μM ddC, whereas differentiated cells displayed defects of spare and maximal respiratory capacities (day 8) and proton-leak linked respiration (day 14) with 12 μM ddC. These results indicate HepaRG is a useful model to study proliferating and differentiated cell mitochondrial toxicant exposures., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The direct neurotoxicity of HIV and neurotoxicity of combination antiretroviral therapy medications both contribute to the development of neuropathic pain. Activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) plays a crucial role in mechanical and thermal hyperalgesia. The P2Y 12 receptor expressed in SGCs of the DRG is involved in pain transmission. In this study, we explored the role of the P2Y 12 receptor in neuropathic pain induced by HIV envelope glycoprotein 120 (gp120) combined with ddC (2',3'-dideoxycytidine). A rat model of gp120+ddC-induced neuropathic pain was used. Peripheral nerve exposure to HIV-gp120+ddC increased mechanical and thermal hyperalgesia in gp120+ddC-treated model rats. The gp120+ddC treatment increased expression of P2Y 12 receptor mRNA and protein in DRG SGCs. In primary cultured DRG SGCs treated with gp120+ddC, the levels of [Ca 2+ ] i activated by the P2Y 12 receptor agonist 2-(Methylthio) adenosine 5'-diphosphate trisodium salt (2-MeSADP) were significantly increased. P2Y 12 receptor shRNA treatment inhibited 2-MeSADP-induced [Ca 2+ ] i in primary cultured DRG SGCs treated with gp120+ddC. Intrathecal treatment with a shRNA against P2Y 12 receptor in DRG SGCs reduced the release of pro-inflammatory cytokines, decreased phosphorylation of p38 MAPK in the DRG of gp120+ddC-treated rats. Thus, downregulating the P2Y 12 receptor relieved mechanical and thermal hyperalgesia in gp120+ddC-treated rats.

Human immunodeficiency virus (HIV) patients treated with nucleoside reverse transcriptase inhibitors (NRTIs), have been known to develop neuropathic pain. While there has been a major shift away from some neurotoxic NRTIs in current antiretroviral therapy, a large number of HIV patients alive today have previously received them, and many have developed painful peripheral neuropathy. The exact mechanisms by which HIV with NRTIs contribute to the development of neuropathic pain are not known. Previous studies suggest that cytoplasmic polyadenylation element-binding protein (CPEB), reactive oxygen species (ROS), and cAMP-response element-binding protein (CREB)-binding protein (CBP), are involved in the neuroimmunological diseases including inflammatory/neuropathic pain. In this study, we investigated the role of CPEB, mitochondrial ROS (mtROS), or CBP in neuropathic pain induced by HIV envelope protein gp120 combined with antiretroviral drug. The application of recombinant gp120 into the sciatic nerve plus systemic ddC (one of NRTIs) induced mechanical allodynia. Knockdown of CPEB or CBP using intrathecal antisense oligodeoxynucleotide (AS-ODN) reduced mechanical allodynia. Intrathecal mitochondrial superoxide scavenger mito-tempol (Mito-T) increased mechanical withdrawal threshold. Knockdown of CPEB using intrathecal AS-ODN, reduced the up-regulated mitochondrial superoxide in the spinal dorsal horn in rats with gp120 combined with ddC. Intrathecal Mito-T lowered the increased expression of CBP in the spinal dorsal horn. Immunostaining studies showed that neuronal CPEB positive cells were co-localized with MitoSox positive profiles, and that MitoSox positive profiles were co-localized with neuronal CBP. Our studies suggest that neuronal CPEB-mtROS-CBP pathway in the spinal dorsal horn, plays an important role in the gp120/ddC-induced neuropathic pain in rats., (Copyright © 2016. Published by Elsevier Inc.)

Painful peripheral neuropathy due to the antiretroviral therapy used to treat HIV is one of the most prevalent side effects occurring in at least 30% of patients living with this infection. We have evaluated the electrophysiological and behavioral effects of d4T and ddC on peripheral large and small nerve fibers in male rats treated with d4T (Sprague-Dawley, 50 mg/kg, twice within 1 week), ddC (Wistar, 50 mg/kg, 3 times per week for 3 weeks), or vehicle. The effect of the interventions was assessed using behavioral measures of mechanical sensitivity, conventional nerve conduction studies, and microneurographic single nerve C-fiber recordings. To mimic as much as possible the human clinical condition, all treated animals were included in the study. No statistically significant differences were observed in behavioral parameters of mechanical sensitivity. Nerve conduction studies did not reveal any significant change in the ddC-treated group. In contrast, we observed electrophysiological evidence of significant demyelinating neuropathy 1 week after the start of d4T treatment. Additionally, spontaneous activity in mechanoinsensitive C-nociceptors was observed in both drug-treated groups. No relationship could be established between measures of spontaneous activity in C-nociceptors and the results of the behavioral tests. Our results show that both models of antiretroviral-induced neuropathy differ in their effects on peripheral nerves. However, both groups present abnormal spontaneous activity in mechanoinsensitive C-nociceptors that can be used as a model for pharmacological intervention.

The human DNA polymerase gamma (Pol γ) is responsible for DNA replication in mitochondria. Pol γ is particularly susceptible to inhibition by dideoxynucleoside-based inhibitors designed to fight viral infection. Here, we report crystal structures of the replicating Pol γ-DNA complex bound to either substrate or zalcitabine, an inhibitor used for HIV reverse transcriptase. The structures reveal that zalcitabine binds to the Pol γ active site almost identically to the substrate dCTP, providing a structural basis for Pol γ-mediated drug toxicity. When compared to the apo form, Pol γ undergoes intra- and inter-subunit conformational changes upon formation of the ternary complex with primer/template DNA and substrate. We also find that the accessory subunit Pol γB, which lacks intrinsic enzymatic activity and does not contact the primer/template DNA directly, serves as an allosteric regulator of holoenzyme activities. The structures presented here suggest a mechanism for processivity of the holoenzyme and provide a model for understanding the deleterious effects of Pol γ mutations in human disease. Crystal structures of the mitochondrial DNA polymerase, Pol γ, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol γ-mediated drug toxicity., (© 2015 The Authors.)

Today HIV-1 infection is recognized as a chronic disease with obligatory lifelong treatment to keep viral titers below detectable levels. The continuous intake of antiretroviral drugs however, leads to severe and even life-threatening side effects, supposedly by the deleterious impact of nucleoside-analogue type compounds on the functioning of the mitochondrial DNA polymerase. For detailed investigation of the yet partially understood underlying mechanisms, the availability of a versatile model system is crucial. We therefore set out to develop the use of Caenorhabditis elegans to study drug induced mitochondrial toxicity. Using a combination of molecular-biological and functional assays, combined with a quantitative analysis of mitochondrial network morphology, we conclude that anti-retroviral drugs with similar working mechanisms can be classified into distinct groups based on their effects on mitochondrial morphology and biochemistry. Additionally we show that mitochondrial toxicity of antiretroviral drugs cannot be exclusively attributed to interference with the mitochondrial DNA polymerase.

The antiretroviral toxic neuropathy, a distal sensory polyneuropathy associated with antiretroviral treatment, is a frequently occurring neurological complication during treatment of patients with AIDS and often leads to discontinuation of antiretroviral therapy. The mechanisms by which antiretroviral drugs contribute to the development of neuropathic pain are not known. Using drugs that reduce intracellular calcium ions (Ca(2+)), we investigated the hypothesis that altered cytosolic Ca(2+) concentration contributes to the 2',3'-dideoxycytidine (ddC)-evoked painful neuropathy. Administration of ddC induced mechanical and cold allodynia, which were abolished by intrathecal administration of TMB-8, a blocker of Ca(2+) release from intracellular stores, and by ryanodine, a RyR antagonist. Treatment with the IP3R antagonist heparin prevented mechanical allodynia with no effect on thermal response. To further clarify the pathway involved, we investigated the role of HuD, a RNA binding protein involved in neuronal function. HuD silencing reverted both mechanical and cold allodynia inducing, a phenotype comparable to that of ryanodine-exposed mice. HuD binding to the RyR2 mRNA, the most abundant RyR isoform in the spinal cord, was demonstrated and RyR2 silencing prevented the ddC-induced neuropathic pain. A positive regulation of gene expression on CaMKIIα by HuD was also observed, but sequestration of CaMKIIα had no effect on ddC-induced allodynia. The present findings identify a spinal RyR2 pathway activated in response to ddC administration, involving the binding activity on RyR2 mRNA by HuD. We propose the modulation of the RyR2 pathway as a therapeutic perspective in the management of antiretroviral painful neuropathy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Nucleoside reverse transcriptase inhibitors (NRTIs) are known to produce painful neuropathies and to enhance states of pain hypersensitivity produced by HIV-1 infection in patients with AIDS leading to discontinuation of antiretroviral therapy, thus limiting viral suppression strategies. The mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In the current study, we tested the hypothesis that HuD, an RNA binding protein known to be an essential promoter of neuronal differentiation and survival, might be involved in the response to NRTI-induced neuropathy. Antiretroviral neuropathy was induced by a single intraperitoneal administration of 2',3'-dideoxycytidine (ddC) in mice. HuD was physiologically expressed in the cytoplasm of the soma and in axons of neurons within DRG and spinal cord and was considerably overexpressed following ddC treatment. ddC up-regulated spinal GAP43 protein, a marker of neuroregeneration, and this increase was counteracted by HuD silencing. GAP43 and HuD colocalize in DRG and spinal dorsal horn (SDH) axons and administration of an anti-GAP43 antibody aggravated the ddC-induced axonal damage. The administration of a protein kinase C (PKC) inhibitor or the PKCγ silencing prevented both HuD and GAP43 increased expression. Conversely, treatment with the PKC activator PDBu potentiated HuD and GAP43 overexpression, demonstrating the presence of a spinal PKC-dependent HuD-GAP43 pathway activated by ddC. These results indicated that HuD recruitment and GAP43 protein increase are mechanistically linked events involved in the response to antiretroviral-induced neurodegenerative processes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: HIV-associated sensory neuropathy affects over 50% of HIV patients and is a common peripheral nerve complication of HIV infection and highly active antiretroviral therapy (HAART). Evidence shows that painful HIV sensory neuropathy is influenced by neuroinflammatory events that include the proinflammatory molecules, MAP Kinase, tumor necrosis factor-α (TNFα), stromal cell-derived factor 1-α (SDF1α), and C-X-C chemokine receptor type 4 (CXCR4). However, the exact mechanisms of painful HIV sensory neuropathy are not known, which hinders our ability to develop effective treatments. In this study, we investigated whether inhibition of proinflammatory factors reduces the HIV-associated neuropathic pain state., Results: Neuropathic pain was induced by peripheral HIV coat protein gp120 combined with 2',3'-dideoxycytidine (ddC, one of the nucleoside reverse transcriptase inhibitors (NRTIs)). Mechanical threshold was tested using von Frey filament fibers. Non-replicating herpes simplex virus (HSV) vectors expressing interleukin 10 (IL10) were inoculated into the hindpaws of rats. The expression of TNFα, SDF1α, and CXCR4 in the lumbar spinal cord and L4/5 dorsal root ganglia (DRG) was examined using western blots. IL-10 expression mediated by the HSV vectors resulted in a significant elevation of mechanical threshold. The anti-allodynic effect of IL-10 expression mediated by the HSV vectors lasted more than 3 weeks. The area under the effect-time curves (AUC) in mechanical threshold in rats inoculated with the HSV vectors expressing IL-10, was increased compared with the control vectors, indicating antinociceptive effect of the IL-10 vectors. The HSV vectors expressing IL-10 also concomitantly reversed the upregulation of p-p38, TNFα, SDF1α, and CXCR4 induced by gp120 in the lumbar spinal dorsal horn and/or the DRG at 2 and/or 4 weeks., Conclusion: The blocking of the signaling of these proinflammatory molecules is able to reduce HIV-related neuropathic pain, which provide a novel mechanism-based approach to treating HIV-associated neuropathic pain using gene therapy.

Individuals infected with the HIV and taking certain antiretroviral drugs to suppress viral replication have a high prevalence of neuropathic pain that is not alleviated by analgesic/adjuvant drugs that are often efficacious for the relief of other types of neuropathic pain. There is therefore a great need for new analgesics to alleviate the pain of antiretroviral toxic neuropathy (ATN). Small-molecule angiotensin II type 2 receptor (AT2R) antagonists, with ≥1000-fold selectivity over the angiotensin II type 1 receptor, produced analgesia in the chronic constriction injury of the sciatic nerve rat model of peripheral nerve trauma. Hence, the present study was designed to assess their analgesic efficacy in a rat model of ATN. The analgesic efficacy of small-molecule AT2R antagonists (EMA200 and EMA300) was assessed in a rat model of dideoxycytidine (ddC)-induced ATN. Single intraperitoneal bolus doses of EMA200 (0.3-10 mg/kg) induced dose-dependent analgesia in ddC-rats; the mean ED50 was 3.2 mg/kg. Twice-daily intraperitoneal administration of EMA300 at 30 mg/kg to ddC-rats for 3 days produced significant analgesia on days 2 and 3 of the treatment period. Therefore, small-molecule AT2R antagonists should be investigated further as novel analgesics for the relief of ATN.

Dideoxycytidine (zalcitabine, ddC) produces neurotoxic effects. It is particularly important to understand the toxic effects of ddC on different subpopulations of dorsal root ganglion (DRG) neurons which express distinct tyrosine kinase receptor (Trk) and to find therapeutic factors for prevention and therapy for ddC-induced peripheral sensory neuropathy. Insulin-like growth factor-1 (IGF-1) has been shown to have neurotrophic effects on DRG sensory neurons. However, little is known about the effects of ddC on distinct Trk (TrkA, TrkB, and TrkC) expression in DRG neurons and the neuroprotective effects of IGF-1 on ddC-induced neurotoxicity. Here, we have tested the extent to which the expression of TrkA, TrkB, and TrkC receptors in primary cultured DRG neurons is affected by ddC in the presence or absence of IGF-1. In this experiment, we found that exposure of 5, 25, and 50 μmol/L ddC caused a dose-dependent decrease of the mRNA, protein, and the proportion of TrkA-, TrkB-, and TrkC-expressing neurons. IGF-1 (20 nmol/L) could partially reverse the decrease of TrkA and TrkB, but not TrkC, expression with ddC exposure. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (10 μmol/L) blocked the effects of IGF-1. These results suggested that the subpopulations of DRG neurons which express distinct TrkA, TrkB, and TrkC receptors were affected by ddC exposure. IGF-1 might relieve the ddC-induced toxicity of TrkA- and TrkB-, but not TrkC-expressing DRG neurons. These data offer new clues for a better understanding of the association of ddC with distinct Trk receptor expression and provide new evidence of the potential therapeutic role of IGF-1 on ddC-induced neurotoxicity.

Background: In the human immunodeficiency virus (HIV)-associated sensory neuropathy, neuropathic pain associated with the use of nucleoside reverse transcriptase inhibitors (NRTIs) in patients with HIV/acquired immunodeficiency syndrome is clinically common. While evidence demonstrates that neuropathic pain is influenced by neuroinflammatory events that include the proinflammatory molecules, tumor necrosis factor-α (TNF-α), stromal cell-derived factor 1-α (SDF1-α), and C-X-C chemokine receptor type 4 (CXCR4), the detailed mechanisms by which NRTIs contribute to the development of neuropathic pain are not known. In this study, we investigated the role of these proinflammatory molecules in the dorsal root ganglion (DRG) and the spinal dorsal horn in NRTIs-mediated neuropathic pain state., Methods: Neuropathic pain was induced by intraperitoneal administration of 2',3'-dideoxycytidine (ddC, one of the NRTIs). Mechanical threshold was tested using von Frey filament fibers. Nonreplicating herpes simplex virus (HSV) vectors expressing p55 TNF soluble receptor (p55TNFSR) were inoculated into hindpaw of rats. The expression of TNF-α, SDF1-α, and CXCR4 in both the lumbar spinal cord and the L4/5 DRG was examined using Western blots. Intrathecal CXCR4 antagonist was administered., Results: The present study demonstrated that (1) systemic ddC induced upregulation of TNF-α, SDF1-α, and CXCR4 in both the lumbar spinal cord and the L4/5 DRG; (2) p55TNFSR mediated by a nonreplicating HSV vector reversed mechanical allodynia induced by systemic ddC; (3) intrathecal administration of the CXCR4 antagonist AMD3100 increased mechanical threshold; and (4) HSV vector expressing p55TNFSR reversed upregulation of TNF-α, SDF1-α, and CXCR4 induced by ddC in the lumbar spinal dorsal horn and the DRG., Conclusions: Our studies demonstrate that TNF-α through the SDF1/CXCR4 system is involved in the NRTIs-related neuropathic pain state and that blocking the signaling of these proinflammatory molecules is able to reduce NRTIs-related neuropathic pain. These results provide a novel mechanism-based approach (gene therapy) to treating HIV-associated neuropathic pain.

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl-DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs-acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)-we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3'-ends. We also show that Tdp1-/- cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1-/- cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs.

The roles and actions of the tumor suppressor protein p53 have been extensively studied with regard to nuclear events, including transcription and DNA damage repair. However, the direct roles of p53 in mitochondrial DNA (mtDNA) replication and function are less well understood. Studies herein used a mitochondrial-targeted p53 (MTS-p53) to determine its effects on both mtDNA abundance and mitochondrial function. MTS-p53 decreased cellular proliferation and mtDNA abundance in HepG2 cells transfected with wild-type (WT) human p53. When MTS-p53 cells were treated with the nucleoside reverse transcriptase inhibitor (NRTI), 2',3'-dideoxycytidine or 2',3'-dideoxyinosine, mtDNA depletion that resembled untransfected controls was observed in both instances. p53-Overexpressing cells showed reduced mitochondrial function by oximetry, including a reduction in maximal respiratory capacity and reserve capacity. A truncated p53 (MTS-p53-290) was generated for localization exclusively to the mitochondria. MTS-p53-290 cells proliferated at control levels but displayed decreased mtDNA abundance and mitochondrial function with NRTI treatment. The MTS-p53-290 cells demonstrated that only the nuclear fraction of p53 controlled cellular proliferation, which was supported by the MTS-p53 results. Data herein indicate that overexpression of p53 in the mitochondria reduces mtDNA abundance and increases the sensitivity of mammalian cells to NRTI exposure by reducing mitochondrial function., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

While oxidative stress has been implicated in small-fiber painful peripheral neuropathies, antioxidants are only partially effective to treat patients. We have tested the hypothesis that Drp1 (dynamin-related protein 1), a GTPase that catalyzes the process of mitochondrial fission, which is a mechanism central for the effect and production of reactive oxygen species (ROS), plays a central role in these neuropathic pain syndromes. Intrathecal administration of oligodeoxynucleotide antisense against Drp1 produced a decrease in its expression in peripheral nerve and markedly attenuated neuropathic mechanical hyperalgesia caused by HIV/AIDS antiretroviral [ddC (2',3'-dideoxycytidine)] and anticancer (oxaliplatin) chemotherapy in male Sprague Dawley rats. To confirm the role of Drp1 in these models of neuropathic pain, as well as to demonstrate its contribution at the site of sensory transduction, we injected a highly selective Drp1 inhibitor, mdivi-1, at the site of nociceptive testing on the dorsum of the rat's hindpaw. mdivi-1 attenuated both forms of neuropathic pain. To evaluate the role of Drp1 in hyperalgesia induced by ROS, we demonstrated that intradermal hydrogen peroxide produced dose-dependent hyperalgesia that was inhibited by mdivi-1. Finally, mechanical hyperalgesia induced by diverse pronociceptive mediators involved in inflammatory and neuropathic pain-tumor necrosis factor α, glial-derived neurotrophic factor, and nitric oxide-was also inhibited by mdivi-1. These studies provide support for a substantial role of mitochondrial fission in preclinical models of inflammatory and neuropathic pain.

Objective: Long-term antiretroviral treatment with nucleoside analogue reverse transcriptase inhibitors (NRTI) may result in a cardiomyopathy due to mitochondrial DNA (mtDNA) depletion. An intact mitochondrial function is required for the synthesis of intramyocardial pyrimidine nucleotides, which in turn are building blocks of mtDNA. We investigated if NRTI-related cardiomyopathy can be prevented with pyrimidine precursors., Methods: Mice were fed with zidovudine or zalcitabine with or without simultaneous Mitocnol, a dietary supplement with high uridine bioavailability. Myocardia were examined after 9 weeks., Results: Both NRTI induced a cardiomyopathy with mitochondrial enlargement, a disrupted cristal architecture on electron microscopy and diminished myocardial mtDNA copy numbers. The myocardial mtDNA-encoded cytochrome c-oxidase I subunit was impaired more profoundly than the nucleus-encoded cytochrome c-oxidase IV subunit. The myocardial formation of reactive oxygen species and mtDNA mutations was enhanced in zidovudine and zalcitabine treated animals. Mitocnol attenuated or normalized all myocardial pathology when given with both NRTI, but by itself had no intrinsic effects and no apparent adverse effects., Conclusions: Zidovudine and zalcitabine induce a mitochondrial cardiomyopathy, which is antagonized with uridine supplementation, implicating pyrimidine pool depletion in its pathogenesis. Pyrimidine pool replenishment may be exploited clinically because uridine is well tolerated.

Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes phosphorylation of thymidine monophosphate to thymidine diphosphate. TMPK also mediates phosphorylation of monophosphates of thymidine nucleoside analog (NA) prodrugs on the pathway to their active triphosphate antiviral or antitumor moieties. Novel transgenic mice (TG) expressing human (h) TMPK were genetically engineered using the alpha-myosin heavy chain promoter to drive its cardiac-targeted overexpression. In '2 by 2' protocols, TMPK TGs and wild-type (WT) littermates were treated with the NA zidovudine (a deoxythymidine analog, 3'-azido-3'deoxythymidine (AZT)) or vehicle for 35 days. Alternatively, TGs and WTs were treated with a deoxycytidine NA (racivir, RCV) or vehicle. Changes in mitochondrial DNA (mtDNA) abundance and mitochondrial ultrastructure were defined quantitatively by real-time PCR and transmission electron microscopy, respectively. Cardiac performance was determined echocardiographically. Results showed TMPK TGs treated with either AZT or RCV exhibited decreased cardiac mtDNA abundance. Cardiac ultrastructural changes were seen only with AZT. AZT-treated TGs exhibited increased left ventricle (LV) mass. In contrast, LV mass in RCV-treated TGs and WTs remained unchanged. In all cohorts, LV end-diastolic dimension remained unchanged. This novel cardiac-targeted overexpression of hTMPK helps define the role of TMPK in mitochondrial toxicity of antiretrovirals.

Background: Real-time PCR has quantified decreased mitochondrial DNA levels in association with nucleoside reverse transcriptase inhibitor (NRTI) therapy of HIV-infected populations. However, real-time PCR is best suited to distinguish log differences in an analyte. In an effort to monitor individuals in more detail, we developed a flow cytometric assay to gauge mitochondrial function., Methods: Flow cytometric quantification of a mitochondrial DNA-encoded mitochondrial protein (cytochrome c oxidase subunit I (COX-I)) and a nuclear DNA-encoded mitochondrial protein [ATP synthase subunit D (Sub-D)] was optimized and validated., Results: Intra-assay and interassay variability was low using peripheral blood mononuclear cells (PBMCs) (CV of 6.15% for COX-I and 7.11% Sub-D, and 9.38% and 9.83% for COX-I and Sub-D, respectively). Mitochondrial protein depletion was evident with in vitro treatment of cells with ethidium bromide (EtBr) and zalcitabine (ddC). Mitochondrial protein expression in 40 healthy adults clustered tightly. Depletion of mitochondrial protein, however, was neither detected in cryopreserved PBMC from NRTI-treated children (n = 9) nor in adults with a history of symptoms consistent with mitochondrial toxicity or ongoing treatment with didanosine (ddI) or stavudine (d4T) (n = 51)., Conclusions: A validated flow cytometric assay allows simultaneous detection of mitochondrial DNA and nuclear DNA encoded proteins at the single cell level, offering a method to monitor for mitochondrial function. Prospective studies are required to evaluate whether mitochondrial protein loss is observed in at-risk patients prior to the onset of symptoms from mitochondrial dysfunction., ((c) 2008 Clinical Cytometry Society.)

Several lines of evidence suggest that descending serotoninergic facilitatory pathways are involved in neuropathic pain. These pathways may involve 5-HT2A receptors known to play a role in spinal and peripheral sensitization. The implication of this receptor in neuropathy was investigated in a model of peripheral neuropathy induced by 2',3'-dideoxycytidine, a nucleoside analogue with reverse transcriptase inhibitory properties used in HIV/AIDS therapy. Four days after a single 100mg/kg i.v. administration in the tail vein, mitochondrial alterations in nociceptive and non-nociceptive dorsal root ganglion cells were observed at the lumbar level. These alterations were not associated with TUNEL labelling or with modification of the total number of dorsal root ganglion cells. At the same time point, 5-HT2A receptor immunolabelling was increased throughout the dorsal horn (by 49.5% in layer II and 57.8% in layer III). The number of 5-HT2A receptor immunoreactive neurons in the dorsal root ganglion was also increased by 30.7%. Four days after 2',3'-dideoxycytidine administration, rats had developed thermal allodynia as well as mechanical hyperalgesia and allodynia, which dose-dependently decreased after epidural injection of MDL 11,939, a 5-HT2A receptor antagonist. Moreover, 5-HT2A receptor knock-out mice did not develop 2',3'-dideoxycytidine-induced neuropathy whereas their control littermates displayed a neuropathy comparable to that observed in rats. Our data show that 2',3'-dideoxycytidine-induced neuropathy is associated with alterations of nociceptive and non-nociceptive peripheral cells and that the 5-HT2A receptor is involved in the peripheral sensitization of nociceptors as well as in a wide central sensitization of dorsal horn neurons.

The nucleoside analog 2',3'-dideoxycytidine (ddC) has been used for treatment of human immunodeficiency virus (HIV) infections. ddC causes delayed toxicity when cells are exposed to the drug at low concentration for prolonged periods of time. The delayed toxicity is due to inhibition of mitochondrial DNA (mtDNA) replication, which results in mtDNA depletion and mitochondrial dysfunction. In the present study we have cultured CEM T-lymphoblast cells in the presence of low concentrations of ddC to generate two cell lines resistant to the delayed toxicity of the drug. Both cell lines were resistant to mtDNA depletion by ddC. The mechanism of ddC resistance was investigated and we showed that the resistant cells had decreased mRNA expression of the nucleoside kinases deoxycytidine kinase and thymidine kinase 2. We also studied the mitochondrial DNA in the cells and showed that the ddC-resistant cells had structurally intact mtDNA but 1.5-2-fold increased mtDNA copy-number as well as increased levels of the mitochondrial transcription factor A (Tfam). Our study suggests that cells may increase their level of mtDNA to counteract mtDNA depletion induced by ddC, while keeping pronounced antiviral activity of the drug.

Nucleoside analog reverse transcriptase inhibitors (NRTIs) are known to directly inhibit mitochondrial complex I activity as well as various mitochondrial kinases. Recent observations that complex I activity and superoxide production are modulated through cAMP-dependent phosphorylation suggests a mechanism through which NRTIs may affect mitochondrial respiration via kinase-dependent protein phosphorylation. In the current study, we examine the potential for NRTIs to inhibit the cAMP-dependent phosphorylation of complex I and the associated NADH:CoQ oxidoreductase activities and rates of superoxide production using HepG2 cells. Phosphoprotein staining of immunocaptured complex I revealed that 3'-azido-3'-deoxythymidine (AZT; 10 and 50 microM), AZT monophosphate (150 microM), and 2',3'-dideoxycytidine (ddC; 1 microM) prevented the phosphorylation of the NDUFB11 subunit of complex I. This was associated with a decrease in complex I activity with AZT and AZT monophosphate only. In the presence of succinate, superoxide production was increased with 2',3'-dideoxyinosine (ddI; 10 microM) and ddC (1 microM). In the presence of succinate+cAMP, AZT showed an inverse dose-dependent effect on superoxide production. None of the NRTIs examined inhibit PKA activity suggesting that the observed effects are due to a direct interaction with complex I. These data demonstrate a direct effect of NRTIs on cAMP-dependent regulation of mitochondrial bioenergetics independent of DNA polymerase-gamma activity; in the case of AZT, these observations may provide a mechanism for the observed long-term toxicity with this drug.

Objective: Zidovudine is an antiretroviral nucleoside analog reverse transcriptase inhibitor that induces mitochondrial myopathy by interfering with the replication of mitochondrial DNA (mtDNA). Because zidovudine inhibits thymidine kinases, the mechanism of mtDNA depletion may be related to an impairment of the de novo synthesis of pyrimidine nucleotides, which are required building blocks of mtDNA. This study was undertaken to determine whether mitochondrial myopathy is a class effect of antiretroviral nucleoside analogs, and whether the muscle disease can be prevented by treatment with uridine as a pyrimidine nucleotide precursor., Methods: BALB/c mice were treated with zidovudine or zalcitabine. Some of the mice were cotreated with mitocnol, a dietary supplement with high uridine bioavailability. Mice hind limb muscles were examined after 10 weeks., Results: Zidovudine induced muscle fiber thinning, myocellular fat deposition, and abnormalities of mitochondrial ultrastructure. In mice treated with zidovudine, organelles contained low mtDNA copy numbers and reduced cytochrome c oxidase activity. The expression of the mtDNA-encoded cytochrome c oxidase I subunit, but not of nucleus-encoded mitochondrial proteins, was impaired. Zidovudine also increased the levels of myocellular reactive oxygen species and blood lactate. Uridine supplementation attenuated or normalized all pathologic abnormalities and had no intrinsic effects. Zalcitabine did not elicit muscle toxicity., Conclusion: Our findings indicate that zidovudine, but not zalcitabine, induces mitochondrial myopathy, which is substantially antagonized by uridine supplementation. These results provide proof of the importance of pyrimidine pools in the pathogenesis of zidovudine myopathy. Since uridine supplementation is tolerated well by humans, this treatment strategy should be investigated in clinical trials.

COX (cytochrome c oxidase) deficiency is one of the main causes of genetic mitochondrial disease and presents with multiple phenotypes, depending on whether the causative mutation exists in a mitochondrial or nuclear gene and on whether it involves an altered catalytic or structural component or an assembly factor for this membrane-embedded 13-subunit enzyme complex. COX deficiency is routinely observed in AD (Alzheimer's disease), although there is continuing debate about whether this is a causative or a secondary consequence of the condition. Altered levels of COX and reduced oxidative phosphorylation capacity have been reported in other common diseases, including cancer, and are seen as unwanted side effects in a number of drug treatments, particularly with antiretroviral and antibiotic treatments. Here, we introduce a simple, rapid, high-throughput 96-well plate protocol that uses a multiplex approach to determine the amount and activity of COX, which should find widespread use in evaluating the above diseases and in drug safety studies. Importantly, the method uses very small amounts of cell material or tissue and does not require the isolation of mitochondria. We show the utility of this approach by example of the analysis of fibroblasts from patients with COX activity deficiency and the effect of the antiretroviral drug ddC (2',3'-dideoxycytidine) on the biogenesis of the enzyme.

A distal symmetrical sensory peripheral neuropathy is frequently observed in people living with Human Immunodeficiency Virus Type 1 (HIV-1). This neuropathy can be associated with viral infection alone, probably involving a role for the envelope glycoprotein gp120; or a drug-induced toxic neuropathy associated with the use of nucleoside analogue reverse transcriptase inhibitors as a component of highly active anti-retroviral therapy. In order to elucidate the mechanisms underlying drug-induced neuropathy in the context of HIV infection, we have characterized pathological events in the peripheral and central nervous system following systemic treatment with the anti-retroviral agent, ddC (Zalcitabine) with or without the concomitant delivery of HIV-gp120 to the rat sciatic nerve (gp120+ddC). Systemic ddC treatment alone is associated with a persistent mechanical hypersensitivity (33% decrease in limb withdrawal threshold) that when combined with perineural HIV-gp120 is exacerbated (48% decrease in threshold) and both treatments result in thigmotactic (anxiety-like) behaviour. Immunohistochemical studies revealed little ddC-associated alteration in DRG phenotype, as compared with known changes following perineural HIV-gp120. However, the chemokine CCL2 is significantly expressed in the DRG of rats treated with perineural HIV-gp120 and/or ddC and there is a reduction in intraepidermal nerve fibre density, comparable to that seen in herpes zoster infection. Moreover, a spinal gliosis is apparent at times of peak behavioural sensitivity that is exacerbated in gp120+ddC as compared to either treatment alone. Treatment with the microglial inhibitor, minocycline, is associated with delayed onset of hypersensitivity to mechanical stimuli in the gp120+ddC model and reversal of some measures of thigmotaxis. Finally, the hypersensitivity to mechanical stimuli was sensitive to systemic treatment with gabapentin, morphine and the cannabinoid WIN 55,212-2, but not with amitriptyline. These data suggests that both neuropathic pain models display many features of HIV- and anti-retroviral-related peripheral neuropathy. They therefore merit further investigation for the elucidation of underlying mechanisms and may prove useful for preclinical assessment of drugs for the treatment of HIV-related peripheral neuropathic pain.

Background and Purpose: Cannabinoids are associated with analgesia in acute and chronic pain states. A spectrum of central cannabinoid (CB(1)) receptor-mediated motor and psychotropic side effects limit their therapeutic potential. Here, we investigate the analgesic effect of the palmitoylethanolamide (PEA) analogue, palmitoylallylamide (L-29), which via inhibition of fatty acid amide hydrolase (FAAH) may potentiate endocannabinoids thereby avoiding psychotropic side effects., Experimental Approach: The in vivo analysis of the effect of L-29 on measures of pain behaviour in three rat models of neuropathic pain., Key Results: Systemically administered L-29 (10 mg kg(-1)) reduced hypersensitivity to mechanical and thermal stimuli in the partial sciatic nerve injury (PSNI) model of neuropathic pain; and mechanical hypersensitivity in a model of antiretroviral (ddC)-associated hypersensitivity and a model of varicella zoster virus (VZV)-associated hypersensitivity. The effects of L-29 were comparable to those of gabapentin (50 mg kg(-1)). The CB(1) receptor antagonist SR141716a (1 mg kg(-1)) and the CB(2) receptor antagonist SR144528 (1 mg kg(-1)) reduced the effect of L-29 on hypersensitivity in the PSNI and ddC models, but not in the VZV model. The peroxisome proliferator-activated receptor-alpha antagonist, MK-886 (1 mg kg(-1)), partially attenuated the effect of L-29 on hypersensitivity in the PSNI model. L-29 (10 mg kg(-1)) significantly attenuated thigmotactic behaviour in the open field arena without effect on locomotor activity., Conclusions and Implications: L-29 produces analgesia in a range of neuropathic pain models. This presents L-29 as a novel analgesic compound that may target the endogenous cannabinoid system while avoiding undesirable side effects associated with direct cannabinoid receptor activation.

Nucleoside analogs are associated with various mitochondrial toxicities, and it is becoming increasingly difficult to accommodate these differences solely in the context of DNA polymerase gamma inhibition. Therefore, we examined the toxicities of zidovudine (AZT) (10 and 50 microM; 2.7 and 13.4 microg/ml), didanosine (ddI) (10 and 50 microM; 2.4 and 11.8 microg/ml), and zalcitabine (ddC) (1 and 5 microM; 0.21 and 1.1 microg/ml) in HepG2 and H9c2 cells without the presumption of mitochondrial DNA (mtDNA) depletion. Ethidium bromide (EtBr) (0.5 microg/ml; 1.3 microM) was used as a positive control. AZT treatment resulted in metabolic disruption (increased lactate and superoxide) and increased cell mortality with decreased proliferation, while mtDNA remained unchanged or increased (HepG2 cells; 50 microM AZT). ddC caused pronounced mtDNA depletion in HepG2 cells but not in H9c2 cells and increased mortality in HepG2 cells, but no significant metabolic disruption in either cell type. ddI caused a moderate depletion of mtDNA in both cell types but showed no other effects. EtBr exposure resulted in metabolic disruption, increased cell mortality with decreased cell proliferation, and mtDNA depletion in both cell types. We conclude that nucleoside analogs display unique toxicities within and between culture models, and therefore, care should be taken when generalizing about the mechanisms of nucleoside reverse transcriptase inhibitor toxicity. Additionally, mtDNA abundance does not necessarily correlate with metabolic disruption, especially in cell culture; careful discernment is recommended in this regard.

2 ',3 '-dideoxycytidine (ddC) is a nucleoside analog that has been shown to produce a delayed toxicity which may be due to the depletion of mitochondrial DNA (mtDNA). In order to gain further understanding of the events involved in mitochondrial toxicity, two different CEM cell lines were selected for resistance to the delayed ddC toxicity.

Objective: To evaluate if nucleoside analogue reverse transcriptase inhibitors (NRTIs) and polymerase-gamma inhibitors deplete mitochondrial DNA (mtDNA) in cultured primary lymphocytes and if such depletion might be associated with functional defects., Methods: Primary peripheral blood CD4 and CD8 lymphocytes were purified from six healthy humans (three male and three female), stimulated mitotically (CD3/CD28) and cultured for 10 days in the presence or absence of NRTIs. Lymphocyte proliferation, mtDNA content, the expression of mtDNA-encoded cytochrome c-oxidase II (COXII) and lactate production were assessed., Results: In CD4 lymphocytes, 10-day exposure to zalcitabine (1.77 microM), didanosine (118 microM) and stavudine (361 microM) induced a time-dependent decline of mtDNA. Compared with controls, residual mtDNA levels were 25%, 21% and 40%, respectively. COXII was reduced to 55%, 35% and 70% of control values. Lactic acid production was increased (by 214%, 294% and 175%, respectively). At day 10, lymphocyte counts were reduced (to 60%, 51%, and 41%, respectively). Zidovudine (71 microM) also reduced lymphocyte counts to 34% and increased lactic acid production by 170%, but did not induce mtDNA and COXII depletion. All these changes were highly significant. Lower NRTI concentrations (0.177 microM of zalcitabine, 11.8 microM of didanosine, 3.6 microM of stavudine and 7.1 microM of zidovudine) had effects at the border of significance. Similar observations were made in CD8 lymphocytes., Conclusions: In human lymphocytes, zalcitabine, didanosine and stavudine induce dose- and time-dependent mtDNA depletion, which is associated with decreased cell proliferation and increased lactate production. Zidovudine impairs lymphocyte division without inducing mtDNA depletion.

Large scale screening for neuroprotective drugs for peripheral neuropathies requires development of a high throughput system that is reliable and reproducible. Currently most accurate outcome measures of axonal degeneration are based on time-consuming, laborious measurement of morphological changes in neurites. In order to improve on the scalability of the screening procedure we developed a real-time RT-PCR based method of gene expression that correlates very well with morphological measures of neuritic degeneration. We examined the changes in GAP-43 expression in primary dorsal root ganglion (DRG) neurons in vitro with exposure to a zalcitabine (ddC), an antiretroviral drug that causes neuropathy in human immunodeficiency virus (HIV)-infected individuals, with and without FK506, an immunophilin ligand with neuroprotective and neuroregenerative properties. Similar to morphological measures of neuritic degeneration, in ddC-treated cultures there was a reduction in the expression of GAP-43 mRNA. This was prevented, in a dose-dependent manner, by co-administration of FK506. This assay, performed in a 96-well format, can easily be scaled for high throughput screening (HTS) using robotic systems., (Copyright 2004 Elsevier B.V.)

Enantiomers of beta-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D/L-D4FC) are nucleoside analog reverse transcriptase inhibitors (NRTIs) currently under investigation as antiviral agents. One of the major problems of NRTIs is toxicity to mitochondria. It has been shown that mitochondrial toxicity of NRTIs can correlate with incorporation and removal of these compounds by mitochondrial DNA polymerase (Pol gamma). Mechanistic studies have shown that, if activated, NRTIs are incorporated more efficiently by HIV-1 reverse transcriptase (RT) and less efficiently by Pol gamma, the corresponding nucleosides are considered to be more selective. In the present study, in order to predict potential DNA Pol gamma-related mitochondrial toxicity of D- and L-D4FC, the incorporation and removal of the monophosphate form of these compounds by Pol gamma were studied using transient kinetic methods. Our cell-free results showed that Pol gamma incorporated the natural D-isomer significantly more efficiently than the unnatural L-isomer. However, the removal rates of these enantiomers from the chain-terminated primers were almost identical. While these results suggest that D-D4FC may present more mitochondrial toxicity than L-D4FC in cell-free assays, we have previously shown that HIV-1 RT prefers D-D4FC-TP as a substrate over the L-isomer, particularly in the case of mutant forms of RT associated with nucleoside drug resistance such as M184V. Since the effectiveness of NRTIs is a balance between efficiency of incorporation by wild-type and drug-resistant forms of HIV-1 RT and mitochondrial toxicity, our kinetic results suggest that both enantiomers may show promise as potential therapeutics.

Objective: To assess in vitro if uridine may be suitable to prevent or treat mitochondrial toxicity related to nucleoside analogue reverse transcriptase inhibitors (NRTIs)., Methods: Human HepG2-hepatocytes were exposed to NRTIs with or without uridine for 25 days. Cell growth, lactate production, intracellular lipids, mitochondrial DNA (mtDNA) and the ratio between the respiratory chain components COX II (mtDNA-encoded) and COX IV (nuclear-encoded) were measured., Results: HepG2 cells exposed to zalcitabine (177 nM) without uridine developed a severe depletion of mtDNA (to 8% of wild-type mtDNA levels), resulting in a decline of cell proliferation and COX II levels, with increased lactate and lipid accumulation. Uridine fully abrogated the adverse effects of zalcitabine on hepatocyte proliferation and normalized lactate synthesis, intracellular lipids and COX II levels by adjusting mtDNA levels to about 65% of NRTI-unexposed control cells. This effect was dose-dependent, with a maximum at 200 microM of uridine. Uridine also rapidly and fully restored cell function when added to cells with established mitochondrial dysfunction (zalcitabine for 15 days) despite continued zalcitabine exposure. Uridine also normalized cell proliferation in HepG2 cells exposed to 36 microM of stavudine and protected HepG2-cells exposed to 7 microM of zidovudine + 8 microM of lamivudine (pyrimidine analogues), but failed to improve cell function or mtDNA in cells exposed to 11.8 or 118 microM of didanosine (a purine analogue)., Conclusions: The pyrimidine precursor uridine may attenuate the mitochondrial toxicity of antiretroviral pyrimidine NRTIs in vitro, and its supplementation may represent a promising strategy in the prevention or treatment of mitochondrial toxicities in HIV-infected patients.

Although antiviral nucleoside analog therapy successfully delays progression of HIV infection to AIDS, these drugs cause unwelcome side-effects by inducing mitochondrial toxicity. We and others have demonstrated that the mitochondrial polymerase, DNA polymerase gamma (pol gamma), participates in mitochondrial toxicity by incorporating these chain-terminating antiviral nucleotide analogs into DNA. Here, we explore the role of three highly conserved amino acid residues in the active site of human pol gamma that modulate selection of nucleotide analogs as substrates for incorporation. Sequence alignments, crystal structures and mutagenesis studies of family A DNA polymerases led us to change Tyr951 and Tyr955 in polymerase motif B to Phe and Ala, and Glu895 in polymerase motif A was changed to Ala. The mutant polymerases were tested for their ability to incorporate natural nucleotides and the five antiviral nucleoside analogs currently approved for antiviral therapy: AZT, ddC, D4T, 3TC and carbovir. Steady-state kinetic analysis of the pol gamma derivatives with the normal and antiviral nucleotides demonstrated that Tyr951 is largely responsible for the ability of pol gamma to incorporate dideoxynucleotides and D4T-MP. Mutation of Tyr951 to Phe renders the enzyme resistant to dideoxynucleotides and D4T-TP without compromising the activity of the polymerase. Alteration of Glu895 and Tyr955 to Ala had the largest effect on overall polymerase activity with normal nucleotides, producing dramatic increases in K(m(dNTP)) and large decreases in k(cat). Mutation of Tyr955 in pol gamma causes the degenerative disease progressive external ophthalmoplegia in humans, and we show that this residue partially accounts for the ability of pol gamma to incorporate D4T-MP and carbovir. Alteration of Glu895 to Ala slightly increased discrimination against dideoxynucleotides and D4T-TP. The mechanisms by which pol gamma selects certain nucleotide analogs are discussed.

Clinical studies with tenofovir disoproxil fumarate, an oral prodrug of the nucleotide analog tenofovir, recently approved for the treatment of HIV, have demonstrated antiviral activity and good tolerability in HIV-infected patients. In order to better understand the cytotoxicity profile of tenofovir relative to the other nucleoside reverse transcriptase inhibitors (NRTIs), the in vitro effects of these agents were evaluated in various human cell types. Tenofovir inhibited the proliferation of liver-derived HepG2 cells and normal skeletal muscle cells with CC(50) values of 398 and 870 microM, respectively. In comparison, ZDV, ddC, ddI, d4T, and abacavir all showed lower CC(50) values in these two cell types. Evaluation of hematopoietic toxicity revealed that tenofovir was less cytotoxic towards erythroid progenitor cells (CC(50)>200 microM) than ZDV, d4T, and ddC (CC(50)=0.06-5 microM). Despite some degree of donor-to-donor variability, the inhibitory activity of the tested NRTIs against myeloid cell lineage, in the order of decreasing severity, was consistently ddC>ZDV>d4T>tenofovir>3TC. Finally, tenofovir showed substantially weaker effects on proliferation and viability of renal proximal tubule epithelial cells than cidofovir, a related nucleotide analog with the potential to induce renal tubular dysfunction. In conclusion, tenofovir exhibited weak cytotoxic effects in all cell types tested with less in vitro cytotoxicity than the majority of NRTIs currently used for the treatment of HIV disease.

Retroviruses may cause diseases in their vertebrate hosts. They are distinguished by their common means of replication involving reverse transcription, a process inhibited by nucleoside reverse transcriptase inhibitors (NRTIs) and other compounds used in antiretroviral chemotherapy. Previous work on NRTIs has been limited to their effect on human immunodeficiency virus (HIV) (for review see Ho & Hitchcock, 1989; Weller, 1999) and little information exists regarding the efficacy and therapeutic potential of these drugs against other retroviruses. We have tested all six NRTIs licensed for HIV treatment [didanosine (ddI), zalcitabine (ddC), lamivudine (3TC), stavudine (d4T), zidovudine (AZT) and abacavir (ABC)] against seven retroviruses representative of the traditional subfamilies: Spumavirinae, Lentivirinae and the Oncovirinae. As expected, each drug showed a range of activities against the panel of retroviruses, some drugs inhibiting other viruses at concentrations well below those required for HIV. Overall, AZT was the most active inhibitor (IC50 range, 0.032-1.0 microM), being most active against the Spuma (foamy) viruses. Abacavir was inhibitory for HIV-1, MN strain (HIV-1 MN), amphotrophic murine leukemia virus (MLV-A) and simian foamy virus type 6 (SFV-6). The least effective inhibitor, 3TC (IC50 range, 0.32->100 microM), was most potent against simian retrovirus types 1 and 2 (SRV-1, SRV-2) and HIV-1, but did not inhibit foamy viruses and MLV-A. Additionally, there were differences in the concentration of drug required to inhibit closely related viruses. Taken together, these data suggest that NRTIs have a wide spectrum of antiretroviral activity and the activity of compounds, even against closely related retroviruses, cannot be predicted.

Background: Previous guidelines for HIV-infected pregnant women have recommended zidovudine (ZDV) monotherapy during the second and third trimesters of pregnancy to prevent fetal HIV infection. New guidelines suggest that women should continue or be offered combination antiretroviral therapy (including protease inhibitors) during pregnancy. Nevertheless, little animal or human toxicity data underlie these recommendations., Methods: We used an in vitro rat whole embryo culture system to assess the embryo toxicity of various nucleoside analogues, namely, ZDV, dideoxyinosine (ddI), and 2', 3'-dideoxycytidine (ddC), and the HIV-1 protease inhibitor, indinavir, both alone and in combination., Results: Although human fetal concentrations of these compounds are unknown, no gross abnormalities were detected after incubation with these agents, either alone or in combination at concentrations that would be expected to be achievable in human maternal serum (1-50 microM). ZDV in combination with ddC at >100 microM, resulted in severe growth retardation and morphologic abnormalities not seen with either agent singly., Conclusions: We conclude that the combination of ZDV/ddC results in severe concentration-dependent embryo toxicity. No growth retardation or gross morphologic abnormalities were found for any of the agents, either singly or in combination, at clinically relevant concentrations., (Copyright 2000 Wiley-Liss, Inc.)

We have previously identified activation of ras proto-oncogenes and inactivation of tumor suppressor genes including p53, p16(INK4a) and p15(INK4b) in 2',3'-dideoxycytidine (ddC)- and/or 1,3-butadiene (BD)-induced lymphomas derived from B6C3F1 (C57BL/6xC3H/He) mice, indicating that alterations of ras signaling pathway, p53 and pRb growth control pathways are important in the development of these chemically induced lymphomas. However, there is still a subset of tumors that displayed no changes in these genes. Thus, we investigated whether the Raf1, Mdm2, c-Myc, Cdc25a and Cdc25b proto-oncogenes, which are implicated in the ras or p53 or pRb pathways, are alternative oncogenic target genes. Analyses of gross genomic alterations by Southern blots failed to reveal rearrangement or amplification in any of the tumors examined. Frequent point mutations on the substrate binding domain of the Raf1 gene has been reported in 1-ethyl-1-nitrosourea (ENU)-induced murine lymphomas and lung tumors, along with a conspicuous lack of ras mutations [U. Naumann, I. Eisenmann-Tappe, U.R. Rapp, The role of raf kinases in development and growth of tumors, Recent Results Cancer Res., 143 (1997) 237-244]. To investigate whether Raf1 mutation is involved in our set of tumor especially those without ras mutations, the PCR-based single-strand conformation analyses (SSCA) and direct DNA sequencing were employed. No mutations but four genetic polymorphisms between C57BL/6 and C3H/He were found, with two of them reported as point mutations previously (op. cit.). The polymorphisms were utilized for allelic loss study of Raf1 locus. Losses of heterozygosity were found in six of 31 BD-induced lymphomas. These results indicate that genetic alterations of c-Myc, Cdc25, Raf1 and Mdm2 proto-oncogenes may not be involved in the development of ddC- and BD-induced lymphomas and the inactivation of tumor suppressor gene(s) located close to Raf1 gene might be important in the development of a subset of BD-induced lymphomas.

The short-term cardiac side effects of 2',3'-dideoxycytidine (ddC, zalcitabine) were studied in rats in order to understand the biochemical events contributing to the development of ddC-induced cardiomyopathy. In developing animals, ddC treatment provoked a surprisingly rapid appearance of cardiac malfunctions characterized by prolonged RR, PR, and QT intervals and J point depression. The energy metabolism in the heart was compromised, characterized by a decreased creatine phosphate/creatine ratio (from 2.05 normal value to 0.75) and a decreased free ATP/ADP ratio (from 332 normal value to 121). The activity of respiratory complexes (NADH: cytochrome c oxidoreductase and cytochrome oxidase) also decreased significantly. Southern blot and polymerase chain reaction analysis did not show deletions or a decrease in the quantity of mitochondrial DNA (mtDNA) deriving from ddC-treated rat hearts, indicating that under our experimental conditions, ddC-induced heart abnormalities were not the direct consequence of mtDNA-related damage. The ddC treatment of rats significantly increased the formation of reactive oxygen species (ROS) in heart and skeletal muscle as determined by the oxidation of non-fluorescent dihydrorhodamine123 to fluorescent rhodamine123 and the oxidation of cellular proteins determined from protein carbonyl content. An activation of the nuclear poly-(ADP-ribose) polymerase (EC 2.4.2.30) and an increase in the mono-ADP-ribosylation of glucose-regulated protein and desmin were observed in the cardiac tissue from ddC-treated animals. A decrease in the quantity of heat shock protein (HSP)70s was also detected, while the level of HSP25 and HSP60 remained unchanged. Surprisingly, ddC treatment induced a skeletal muscle-specific decrease in the quantity of three proteins, one of which was identified by N-terminal sequencing as myoglobin, and another by tandem mass spectrometer sequencing as triosephosphate isomerase (EC 5.3.1.1). These data show that the short term cardiotoxicity of ddC is partially based on ROS-mediated signalling through poly- and mono-ADP-ribosylation reactions and depression of HSP70 levels, whose processes represent a new mtDNA independent mechanism for ddC-induced cell damage.

Exposure of a human lymphocytic cell line, H9 cells, to 0.5 microM and 5.0 microM dideoxycytidine (ddC) resulted in isolation of ddC-resistant H9-ddC0.5w and H9-ddC5.0w cell lines. In addition, these cell lines were also resistant to azidothymidine and had reduced deoxycytidine kinase and thymidine kinase activities. We now show that these cell lines are 4-fold and 2000-fold collaterally resistant to 5-fluoro-2'-deoxyuridine (FdUR), respectively, but not to 5-fluorouracil (FU). Biochemical evaluations show that, compared to the parental cells, the FdUR phosphorylation was reduced to 36.3% and 9.2% and the FdUMP levels were decreased to 48.1% and 1.2% in these cell lines. Taken together, the data suggest that ddC, an antiviral agent, is capable of inducing resistance to FdUR-a drug that is not its analog and which has a different metabolism, target site, and mechanism of action., (Copyright 1999 Academic Press.)

The effect of 2',3'-dideoxycytidine (ddC) treatment on two human glioblastoma cell lines was characterized. ddC treatment (10 nM-100 microM) of glioblastoma cells was associated with enlargement and shortening of processes within 2-3 days. Assessment of mitochondrial membrane potential showed an early decrease in the number of polarized mitochondria in the glioma cells, at between 16 to 84% of the untreated control. With chronic exposure to varying doses of ddC, the tumor cells underwent apoptosis within 5-32 days. This apoptosis was dramatic, with a complete loss of cell viability in < 6 hours after cells were noted to begin detaching from the tissue culture flask. Supplementation with uridine, pyruvate and glucose could delay cellular death at the lower doses of ddC (10 nM to 15 microM) but not at the higher doses. However, regardless of supplementation, all cells eventually underwent apoptosis. ddC is a potent inducer of apoptosis in human glioblastoma cells. Assessments of efficacy in in vivo models and clinical trials should be performed to determine the value of ddC in the treatment of malignant brain tumors.

Human macrophages when cultured for several weeks in the presence of therapeutically relevant 2',3'-dideoxycytidine (ddC) concentrations show a time-dependent decay in mitochondrial DNA content. This decay is associated with a reduction of Rhodamine 123 fluorescence, a marker for mitochondrial membrane potential suggesting that impairment of mitochondrial functions occurs. Mitochondrial metabolic impairment was confirmed by direct evaluation of lactate production, which is markedly increased in cells treated with ddC. The activity of protein kinase C and intracellular free Ca2+ upon addition of phorbol 12-myristate 13-acetate (PMA) were lower in the drug-treated cells compared to controls. A 50% reduction in O2-release was also found upon PMA stimulation. Fluorescent latex beads, yeast and bacteria phagocytosis were normal, but intracellular bacteria killing was markedly impaired in ddC-exposed macrophages. Thus, ddC exerts a delayed mitochondrial toxicity also on differentiated macrophages with impairment of several metabolic properties and O2 production causing a reduced ability of these phagocytic cells to kill phagocytosed bacteria.

Zidovudine (AZT), didanosine (ddI) and zalcitabine (ddC) are the reference antiretroviral therapy in patients with AIDS. A toxic mitochondrial myopathy can be observed in patients treated with AZT, but not with ddI and ddC. All 3 compounds can inhibit mitochondrial (mt)DNA polymerase and cause termination of synthesis of growing mtDNA strands and mtDNA depletion. The propensity to injure particular target tissues is unexplained. In our work, cultured muscle cells prepared from human muscle biopsies, were exposed to various concentrations of AZT (4-5000 micromol/l), ddI (5-1000 micromol/l) and ddC (1-1000 micromol/l) for 10 days. We evaluated cell proliferation and differentiation and measured lipid droplet accumulation, lactate production and respiratory chain enzyme activities. All 3 compounds induced a dose-related decrease of cell proliferation and differentiation. AZT seemed to be the most potent inhibitor of cell proliferation. AZT, ddI and ddC induced cytoplasmic lipid droplet accumulations, increased lactate production and decreased activities of COX (complex IV) and SDH (part of complex II). NADHR (complex I) and citrate sinthase activities were unchanged. Zalcitabine (ddC) and, to a lesser extent, ddI, were the most potent inhibitors of mitochondrial function. In conclusion, AZT, ddI and ddC all exert cytotoxic effects on human muscle cells and induce functional alterations of mitochondria possibly due to mechanisms other than the sole mtDNA depletion. Our results provide only a partial explanation of the fact that AZT, but not ddI and ddC, can induce a myopathy in HIV-infected patients. AZT myopathy might not simply result from a direct mitochondrial toxic effect of crude AZT.

Carbovir (CBV) is a guanine nucleoside analog with potent in vitro anti-HIV activity. A prodrug of CBV is currently being evaluated in clinical trials as a potential agent for the treatment of AIDS. The ability of CBV to inhibit mitochondrial DNA synthesis in intact CEM cells was evaluated in the present study, because most of the currently available anti-HIV nucleoside analogs have significant toxicities that result from their inhibition of mitochondrial DNA synthesis. No delayed cytotoxicity was observed in CEM cells treated with 50 microM CBV for 4 weeks. In addition, CBV at concentrations as high as 1 mM did not cause a decline in mitochondrial DNA levels and only minimally increased the concentration of lactic acid in the medium. In contrast to these results with CBV, treatment of CEM cells with 0.5 microM 2',3'-dideoxycytidine resulted in delayed cytotoxicity, a decrease in mitochondrial DNA content and increases in lactic acid levels in the medium. These results indicated that treatment of CEM cells with CBV did not result in the inhibition of mitochondrial DNA synthesis and suggested that treatment of AIDS patients with CBV, or a prodrug of CBV, would not result in some of the toxicities seen with the other anti-HIV nucleoside analogs.

2',3'-dideoxycytidine (ddC) is a synthetic pyrimidine nucleoside analogue approved for treatment of HIV-positive patients. Previous studies indicated that ddC has the potential to cause thymic lymphoma in C57BL/6 x C3H F1 (hereafter called B6C3F1) mice. In this study, we evaluated the carcinogenic potential of ddC in two different mouse models. B6C3F1 hybrid mice carry ecotropic endogenous proviral sequences that may be activated to cause lymphoma, whereas NIH Swiss mice lack proviral sequences that can be expressed. The mice were treated with ddC by gavage at 500 and 1000 mg/kg/day for up to 6 months (human dose, 2.25 mg/day) and evaluated for toxicity, plasma levels of ddC, and pathological changes. Lymphocyte cell markers from the thymic lymphomas were assessed by immunophenotyping. Expression of p53 protein was evaluated using immunohistochemical staining. Treatment-related thymic lymphomas were present in both mouse models with a higher incidence in NIH Swiss than in B6C3F1 mice. The lymphomas were more prevalent in females than in males of both mouse models. Most mice with thymic lymphoma died during the course of the study. In addition to the thymus, lymphoma was often present in lymph nodes, spleen, and other organs. Lymphomas arose more frequently in mice that lack endogenous ecotropic retroviral sequences and thus were not due to activation of endogenous provirus. During the third month of the study, a few NIH Swiss mice that died had granulosa cell tumors of the ovary. Treatment-related but reversible thymic atrophy was observed in both mouse models. There was a very high correlation between the internal dose of ddC and the incidence of thymic lymphoma in both mouse models. Most of the lymphocytes from control thymuses and ddC-induced lymphomas were positive for Thy-1.2 (pan-T), heat stable antigen, and CD4 and CD8 markers, with no marked differences in the lymphocyte markers of the tumors between sexes or dose groups. p53 protein was detected in only 20% (23/115) of the ddC-induced lymphomas with mostly minimal expression in scattered cells. Because ddC induced lymphomas in two different mouse models, the potential carcinogenic risk should be considered in long-term treatment of HIV-positive patients, especially children and adolescent patients treated with ddC.

The combinations of foscarnet plus 2',3'-dideoxyinosine and foscarnet plus 2',3'-dideoxycytidine synergistically inhibit the replication of human immunodeficiency virus type 1 isolates, including two 3'-azido-3'-deoxythymidine-resistant isolates. The combination of 2',3'-dideoxyinosine plus 2',3'-dideoxycytidine showed additive inhibition of the majority of the human immunodeficiency virus isolates tested. All three combinations showed pronounced antagonistic cytotoxicity and thus were less toxic to the growth of peripheral blood mononuclear cells than the separate drugs.

Inhibition of in vitro colony formation of human hematopoietic progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid) by the antiviral nucleoside drugs alovudine, zalcitabine, zidovudine, ganciclovir, stavudine, didanosine, lamivudine, and acyclovir was measured. Significant correlations between in vitro 50% inhibitory concentrations and the daily human exposures (area under the concentration-time curve from 0 to 24 h; in micromolar.hour) of these chronically administered drugs in human immunodeficiency virus-positive patients that induced neutropenia or anemia were demonstrated by both linear regression and Spearman rank-order analyses. These quantitative correlations allow estimation of the exposure at which bone marrow toxicity may occur with candidate compounds.

Groups of 10 male and 20 female B6C3F1 mice received 0, 500, or 1000 mg/kg/day 2'3'-dideoxycytidine (ddC) by gavage for 13 weeks. At the end of the 13-week exposure period all males and 10 females per group were necropsied while the remaining females were held for 1 month without further treatment. Thymic atrophy was present at the 13-week necropsy in male and female mice administered 1000 mg/kg/day and in females administered 500 mg/kg/day, but was not present in females following 1 month of recovery. Thymic lymphoma was present in 1 female that received 500 mg/kg/day and 1 female that received 1000 mg/kg/day. In a follow-up study groups of 70 female mice received 0, 500, or 1000 mg/kg/day for 13 weeks. At the end of the 13-week exposure period 20 mice per group were necropsied and the remaining animals held for 3 months without further treatment. Thymic atrophy was observed in ddC-exposed groups at the 13-week necropsy but not in mice allowed to recover for 13 weeks. Thymic lymphoma occurred in 3/50 mice that received 500 mg/kg/day and in 17/50 mice that received 1000 mg/kg/day but did not occur in mice from the vehicle control group.