Cisplatin-induced Neurotoxicity Research Articles

APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226+177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons.

Attenuation of Cisplatin-Induced Neurotoxicity by Cyanidin, a Natural Inhibitor of ROS-Mediated Apoptosis in PC12 Cells.

Cisplatin-based chemotherapy in clinic is severely limited by its adverse effect, including neurotoxicity. Oxidative damage contributes to cisplatin-induced neurotoxicity, but the mechanism remains unclearly. Cyanidin, a natural flavonoid compound, exhibits powerful antioxidant activity. Hence, we investigated the protective effects of cyanidin on PC12 cells against cisplatin-induced neurotoxicity and explored the underlying mechanisms. The results showed that cisplatin-induced cytotoxicity was completely reversed by cyanidin through inhibition of PC12 cell apoptosis, as proved by the attenuation of Sub-G1 peak, PARP cleavage, and caspases-3 activation. Mechanistically, cyanidin significantly inhibited reactive oxygen species (ROS)-induced DNA damage in cisplatin-treated PC12 cells. Our findings revealed that cyanidin as an apoptotic inhibitor effectively blocked cisplatin-induced neurotoxicity through inhibition of ROS-mediated DNA damage and apoptosis, predicating its therapeutic potential in prevention of chemotherapy-induced neurotoxicity. Cisplatin caused DNA damage, activated p53, and subsequently induced PC12 cells apoptosis by triggering ROS overproduction. However, cyanidin administration effectively inhibited DNA damage, attenuated p53 phosphorylation, and eventually reversed cisplatin-induced PC12 cell apoptosis through inhibition ROS accumulation.

The preventive effect of oxytocin to Cisplatin-induced neurotoxicity: an experimental rat model.

Peripheral neurotoxicity is a frequent dose-limiting side effect of the chemotherapeutic agent cisplatin. This study was conducted to investigate the preventive effect of oxytocin (OT) on cisplatin-induced neurotoxicity in rats. Forty-four adult female rats were included in the study. Thirty-six rats were administered intraperitoneally (i.p.) single dose cisplatin 10 mg/kg and divided in to 3 groups. The first group (n = 12) received saline i.p., whereas the second group (n = 12) and the third group (n = 12) were injected with 80 µg/kg and 160 µg/kg OT, respectively, for 10 days. The remaining 8 rats served as the control group. Electromyography (EMG) studies were recorded and blood samples were collected for the measurement of plasma lipid peroxidation (malondialdehyde; MDA), tumor necrosis factor (TNF)-α, and glutathione (GSH) levels. EMG findings revealed that compound muscle action potential amplitude was significantly decreased and distal latency was prolonged in the nontreated cisplatin-injected rats compared with the control group (P < 0.005). Also, nontreated cisplatin-injected rats showed significantly higher TNF-α and MDA levels and lower GSH level than control group. The administration of OT significantly ameliorated the EMG alterations, suppressed oxidative stress and inflammatory parameters, and increased antioxidative capacity. We suggest that oxytocin may have beneficial effects against cisplatin-induced neurotoxicity.

Riboflavin Arrests Cisplatin-Induced Neurotoxicity by Ameliorating Cellular Damage in Dorsal Root Ganglion Cells.

Cis-Diamminedichloroplatinum II- (CP-) induced neurotoxicity is one of the least explored aspects of this drug. Dorsal root ganglia (DRG) cells are considered as the primary target, and their damage plays a vital role in pathogenesis and etiology of CP-induced neurotoxicity. The present study is aimed at confirming if riboflavin (RF) has any protective role in shielding the DRG from CP-induced toxicity. After conducting the established treatment strategy on mice under photoillumination, it was observed that, despite the fact that RF alone is partially toxic, its combination with CP significantly ameliorated the drug-induced damage in DRG cells as evidenced by histological analysis. In addition, it was interesting to observe that the combination group (RF + CP) was able to induce apoptosis in the target cells up to a significant extent which is considered as the most preferred way of countering cancer cells. Therefore, RF can act as an effective adjuvant compound in CP-based chemoradiotherapy to improve clinical outcomes in the contemporary anticancer treatment regimes.

Electron Microscopy in Rat Brain Slices Reveals Rapid Accumulation of Cisplatin on Ribosomes and Other Cellular Components Only in Glia

Cisplatin is a widely used, effective anticancer drug. Its use, however, is associated with several side effects including nephrotoxicity and neurotoxicity. It is known that cisplatin is accumulated in cells by the organic cation transport system and reacts with nucleotides, damaging them, but the precise target of cisplatin-induced neurotoxicity remains obscure. Here we report direct visualization of cisplatin inside brain cells using in vivo “cisplatin staining,” a technique that takes advantage of the high electron density of cisplatin, which contains platinum (atomic mass = 195). After applying 0.1% cisplatin to living brain slices for 30 min, we fixed the tissue and observed the accumulated cisplatin using electron microscopy. We found that cisplatin was localized mainly to ribosomes associated with endoplasmic reticulum (EPR) in glial cells and to the myelin sheath formed by oligodendrocytes around neuronal axons. Staining of nuclear DNA was moderate. Our in vivo “cisplatin staining” method validated that the main target of cisplatin is a direct attack on myelin and the RNA contained in ribosomes.

Selenium partially prevents cisplatin-induced neurotoxicity: A preliminary study

Cisplatin is an anticancer drug and it has neurotoxic effects. On the other hand, the neuroprotective effect of selenium was observed in previous studies. However, the effect of selenium on cisplatin-induced neurotoxicity has not been studied yet. Therefore, we aimed to investigate whether selenium prevent cisplatin-induced neurotoxicity. Twenty-one male Wistar albino rats were divided into three groups: control (C), cisplatin (CS), cisplatin and selenium (CSE, n=7 in each group). Cisplatin (12mg/kg/day, i.p.) was administered for 3 days to CS and CSE groups. Also, CSE group received via oral gavage 3mg/kg/day (twice-a-day as 1.5mg/kg) selenium 5 days before of cisplatin injection and continued for 11 consecutive days. The same volumes of saline were intraperitoneally and orally administered to C group at same time. At the end of experimental protocol, electrophysiological and histopathological examinations were performed. The nerve conduction velocity, amplitude of compound action potential and number of axon of CS group were significantly lower than the C group. However, the same parameters of CSE group were significantly higher than the CS group. Although, cisplatin has a peripheral neurotoxic effect in rats, this effect was partially prevented by selenium treatment. Thus, it appears that co-administration of selenium and cisplatin may be a useful approach to decrease severity of peripheral neurotoxicity.

Azadirachta indica attenuates cisplatin-induced neurotoxicity in rats.

Objective:The objective of this study is to investigate the neuroprotective effects of Azadirachta indica leaves against cisplatin (CP)-induced neurotoxicity.Materials and Methods:Female Wistar rats were treated with vehicle (control); a single intraperitoneal 5 mg/kg CP (CP group); neem leaves (orally 500 mg/kg) for 5 and 10 days, N5 and N10 groups, respectively; neem leaves (500 mg/kg) for 5 days after CP injection, collagenous protein nitrogen (CPN) group; neem leaves (500 mg/kg) for 5 days before CP injection, noncollagenous protein group and neem leaves in a dose of 500 mg/kg for 5 days before and after CP injection, noncollagenous protein nitrogen group. Rats were sacrificed 5 days after CP injection to determine neural lipid peroxidation (LPO), nitric oxide (NO), and glutathione (GSH) levels. The neuronal antioxidant enzymes were evaluated in brain homogenates.Results:CP injection increased LPO, NO levels and decreased GSH level, whereas neem reversed these effects. Morphological brain damage and apoptosis induction were apparent in the CP group. In the CPN group, the histological damage and apoptosis induction caused by CP was improved, whereas morphological findings of neem before and after CP injection implied a well preserved brain tissue. No changes, in biochemical parameters were observed with neem treated groups.Conclusion:This study suggests that methanolic extract of neem leaves may be of therapeutic benefit when used with CP.

Fish oil protects the peripheral and central nervous systems against cisplatin-induced neurotoxicity

ObjectiveThe protective effects of fish oil (FO) on cisplatin (CP)-induced central and peripheral neurotoxicity were investigated in rats.MethodsRats (n = 28) were divided equally into four groups, the first group was kept as a control. In the second and third groups, CP and FO were given at doses of 7 mg/kg and 1 softgel/rat/day, respectively. In the fourth group, CP and FO were given together at the same doses.ResultsAlthough CP caused significant oxidative damage, via induction of lipid peroxidation and reduction in the antioxidant defense system potency, FO treatment largely reversed these effects. CP also resulted in histopathological damage, such as apoptosis, and electromyographical changes in the sciatic nerve. FO treatment partially prevented the histopathological and electromyographical effects of CP.DiscussionCP has severe central and peripheral neurotoxic effects in rats and these effects were largely prevented by FO treatment. Thus, it appears that co-administration of FO with CP may be a useful approach to attenuate the negative effects of CP on the nervous system.

Flavaglines as Potent Anticancer and Cytoprotective Agents

Flavaglines represent a family of plant natural products that display potent anticancer, cardioprotective, and neuroprotective activities. Novel flavagline derivatives were synthesized and examined for their cytotoxicity on a panel of human cancer cell lines, their cardioprotection against doxorubicin-induced apoptosis in cardiomyocytes, and their neuroprotection in culture models of Parkinson's disease and cisplatin-induced neurotoxicity. The structural requirements of flavaglines for cardio- and neuroprotection were for the first time unraveled and appeared to be slightly different from those for cytotoxicity on cancer cells. We provide also the first evidence that flavaglines may alleviate cisplatin-induced neurotoxicity, suggesting a prophylactic potential of these compounds to prevent this frequently encountered adverse effect of cancer chemotherapies.

Curcumin reduces cisplatin-induced neurotoxicity in NGF-differentiated PC12 cells

The potential neuroprotective benefits of curcumin against cisplatin neurotoxicity were investigated. Curcumin is a polyphenol derived from the rhizome of Curcuma longa whose pharmacological effects include antioxidant, anti-inflammatory and anti-cancer properties. Cisplatin is a potent chemotherapeutic drug with activity against a wide variety of tumors, although it has notorious side effects. Cisplatin neurotoxicity is clinically evident in patients that have undergone a full course of chemotherapy and develop a peripheral neuropathy that may affect the treatment regimen and the patient's qualify of life. In this study, we examined whether curcumin can protect against cisplatin neurite outgrowth inhibition in PC12 cells, which is an indicator of the protective potential against neuropathy. We also investigated whether curcumin affects cisplatin effectiveness by analyzing the modulation of p53 gene expression and its effect on cisplatin cytotoxicity in HepG2 tumor cells. Non-cytotoxic concentrations of curcumin reduced in vitro neurotoxicity of cisplatin in PC12 cells. The treatment of PC12 cells with cisplatin (10μg/mL) significantly reduced neurite outgrowth. The tested concentration of curcumin (1.0 and 10μg/mL) did not result in neurite toxicity but nevertheless diminished cisplatin-induced inhibition of neurite outgrowth by up to 50% (p<0.05). Our results indicate that curcumin does not compromise cisplatin's anticancer activity. Curcumin neither suppressed p53 mRNA transcription nor protected tumor cells against cisplatin cytotoxicity. These results indicate that curcumin may reduce cisplatin-induced neurotoxicity, and clinical studies should potentially be considered.

Walnut consumption protects rats against cisplatin-induced neurotoxicity

Walnut is extensively used in traditional medicine for treatment of various ailments. It is described as an anticancer, anti-inflammatory, blood purifier and antioxidant agent. In this study, we investigated whether or not Walnut could protect neurons against cisplatin-induced neurotoxicity in rats. Dietary walnut (6%) was assessed for its neuroprotective effects through the alteration in performance of hippocampus- and cerebellum-related behaviors following chronic cisplatin treatment (5mg/kg/week for 5 consecutive weeks) in male rats. We also evaluated the effect of cisplatin and walnut administration on nociception. We showed that exposure of adolescent rats to cisplatin resulted in significant decrease in explorative behaviors and memory retention. Walnut consumption improved memory and motor abilities in cisplatin treated rats, while walnut alone did not show any significant changes in these abilities compared to saline. Cisplatin increased latency of response to nociception, and walnut reversed this effect of cisplatin. We conclude that walnuts in the diet following anticancer drugs such as cisplatin might have a protective effect against cisplatin-induced disruptions in motor and cognitive function. However, further studies are needed to elucidate the exact mechanisms of this protective effect of walnut and to explore underlying mechanisms.

D-Methionine protects against cisplatin-induced neurotoxicity in cortical networks

Cisplatin is a platinum-based chemotherapeutic agent widely used for the treatment of various types of cancer. Patients undergoing cisplatin treatment often suffer from a condition known as “chemobrain”, ototoxicity, peripheral neuropathy, weight loss, nausea, vomiting, nephrotoxicity, seizures, hearing loss and tinnitus. d-Methionine (d-Met), a sulfur-containing nucleophilic antioxidant, has been shown to prevent cisplatin-induced side effects in animals without antitumor interference. In this study, we have used an in vitro model of cortical networks (CNs), enriched in auditory cortex cells; to quantify cisplatin neurotoxicity and the protective effects of d-Met. Dissociated neurons from auditory cortices of mouse embryos were grown on microelectrode arrays with 64 transparent indium–tin oxide electrodes, which enabled continuous optical and electrophysiological monitoring of network neurons. Cisplatin at 0.10–0.25mM induced up to a 200% increase in spontaneous spiking activity, while concentrations at or above 0.5mM caused irreversible loss of neuronal activity, accompanied by cell death. Pretreatment with d-Met, at a concentration of 1.0mM, prevented the cisplatin-induced excitation at 0.10–0.25mM, caused sustained excitation without occurrence of cell death at 0.5mM, and delayed cell death at 0.75mM cisplatin. l-Methionine, the optical isomer, showed lower potency and less efficacy than d-Met, was less protective against 0.1mM cisplatin, and proved ineffective at a concentration of 0.5mM cisplatin. Pre-exposure time of d-Met was associated with the protective effects at 0.1 and 0.5mM cisplatin, with longer pre-exposure times exhibiting better protection. This study quantifies as a function of concentration and time that d-Met protects central nervous system tissue from acute cisplatin toxicity.

Regulation of renal organic anion and cation transporters by thymoquinone in cisplatin induced kidney injury

In previous studies, we have demonstrated the biological activity of thymoquinone (TQ), an active compound extracted from the Nigella sativa plant, against cisplatin-induced neurotoxicity. Recenty, it was observed that there is an inherent lack in regulation of renal organic anion and cation transporters in cisplatin-induced nephrotoxicity. Here, we report, for the first time, the effect of TQ on alterations in the renal expression of organic anion transporters (OATs) and organic cation transporters (OCTs), as well as multidrug resistance-associated proteins (MRPs) in rats treated with cisplatin. Twenty-eight 8-week-old male Wistar rats were divided into four groups of control, TQ treated (10mg/kg b.w. in drinking water for 5days), cisplatin (7mg/kg b.w., i.p.) and TQ and cisplatin combination treatment. Cisplatin-induced malondialdehyde (MDA) and 8-isoprostane increase was found to be markedly reduced in rats treated with TQ. In cisplatin only treated rats, the induced renal injury increased protein levels of the efflux transporters MRP2 and MRP4 while expression of OAT1, OAT3, OCT1 and OCT2 was reduced. In combination TQ- and cisplatin-treated rats, expression of MRP2 and MRP4 proteins was decreased in the kidneys. Conversely, TQ treatment increased levels of OCT1, OCT2, OAT1 and OAT3 and decreased levels of 8-isoprostane and MDA levels in cisplatin-treated rats. In conclusion, the present study shows that the TQ synergizes with its nephroprotective effect against cisplatin-induced acute kidney injury in rats.

Interactions of neurotransmitter systems during postnatal development of the rat hippocampal formation: Effects of cisplatin

The distribution of neuroimmunohistochemical markers for the serotoninergic, noradrenergic, glutamatergic and GABAergic systems (respectively, 5HT2AR, β1AR, GluR 2/3 and GAD65/67) was determined in the hippocampal formation at stages PD11, PD17 and PD30 of postnatal development of untreated rats and cisplatin-treated rats after a single injection of the drug at 10days of life. In the different time points the neurons of the dentate gyrus and Cornu Ammonis progressively acquire mature morphological characteristics, and cell genesis, migration of interneurons and differentiation of mossy cells occur.Cisplatin induced decrease in immunoreactivity for most of the selected neurotransmitter markers, thereby altering the postnatal development of circuits in the hippocampal formation. Cisplatin also brought out clear evidence for an interaction between excitatory and inhibitory neurotransmitter markers during the postnatal maturation of cells and fiber projections containing GluR2/3 and GAD65, despite the fact that glutamatergic neurons and GABAergic interneurons are divergent in their source of genesis and in their mode of migration. In fact, GluR2/3 immunofluorescence was increased in the principal cells early, at PD11, possibly to reduce the calcium influx into the cell. Moreover, cisplatin might cause a loss of GABAergic interneurons early and reduction of fiber projections to hippocampal layers due to altered cell migration or to cell injury; late changes, particularly in GAD67 cell number in the dentate gyrus did not result in redistribution or recovery in treated rats. With the use of cisplatin it has been demonstrated here for the first time that the critical differentiation of dentate gyrus hilar β1AR and GluR2/3 mossy cells takes place between PD11 and PD17. Changes in neurotransmitter marker immunopositivity occurred subsequently to cytoarchitectural changes in the dentate gyrus and Cornu Ammonis which were already evident one day after cisplatin injection, suggesting that degeneration and cell loss may have occurred.Cisplatin was found to be a useful tool for following CNS development and for understanding how hippocampal neuronal networks react to injury. Furthermore, cisplatin-induced neurotoxicity may be used to reveal useful information on the genesis, migration and distribution, and differentiation of distinct types of hippocampal neurons.

P-774 - Protective effects of mirtazapine on cisplatin-induced oxidative stress in rat brains: a biochemical and genetic evaluation

Cisplatin treatment is associated with neurotoxicity and it is a limitation to effective anti-cancer treatment as it may lead to dose-reduction. Therefore, effective strategies to reduce the severity of neurotoxicity are intensively being searched for. Oxidative stress might play an important role here. Moreover, several antioxidants have been used successfully for prevention. Interestingly, mirtazapine may be one of promising agents for the avoidance of cisplatin-induced neurotoxicity via its less-known antioxidant properties. We aimed to examine a possible protective effect of mirtazapine against the cisplatin-induced neurotoxicity in rat brains. Eighteen rats were divided into 3 groups equally: a control group (CG), an untreated group (UG), and a treated group (TG). The TG was first given 30 mg/kg mirtazapine by gastric gavage while the others received solely distilled water. And then, 10 mg/kg cisplatin was injected intraperitoneally every day for 14 days to the animals in the UG and the TG while the rest of them were given only isotonic saline solution. While tGSH and NO mean scores were found to be statistically higher in the TG when compared with the UG, MDA and MPO mean scores were statistically lower in the TG when compared with the UG. Similarly, there was a statistical difference both between the UG and the TG for 8-OH GUA. These findings suggest that mirtazapine might be a cytoprotective agent and this might justify investigating whether cisplatin at the desired doses can be administered concurrently with mirtazapine in the absence of other considerations.

Impact of Long-Term Serum Platinum Concentrations on Neuro- and Ototoxicity in Cisplatin-Treated Survivors of Testicular Cancer

Cisplatin-induced neurotoxicity and ototoxicity (NTX) are important adverse effects after chemotherapy for testicular cancer (TC). Although serum platinum is measurable years after therapy, its impact on NTX has not been evaluated. In all, 169 cisplatin-treated survivors of TC provided blood samples at Survey I and reported NTX during Survey I (1998-2002) and Survey II (2007-2008). Serum platinum was quantified by inductively coupled plasma mass spectrometry. Patient-reported outcomes were evaluated with the Scale for Chemotherapy-Induced Neurotoxicity (SCIN), regarding the extent of symptom bother as 0, "not at all"; 1, "a little"; 2, "quite a bit"; or 3, "very much." Summing the six symptom scores yielded a total SCIN score of 0 to 18. Categorizing total SCIN scores into quartiles yielded similar-sized groups with increasing symptoms. Multivariate ordinal logistic regression analyses evaluated associations between NTX and long-term serum platinum levels, adjusting for cisplatin dose, dosing schedule, and age. At Survey I, a significant four- to five-fold association with total SCIN score emerged for the highest serum platinum quartile (odds ratio [OR], 4.69; 95% CI, 1.82 to 12.08). Paresthesias and Raynaud's syndrome (hands and feet) showed significant two- to four-fold increased risks with the highest platinum quartile. At Survey II, total SCIN score remained significantly associated with the highest platinum quartile (OR, 4.28; 95% CI, 1.36 to 13.48). Paresthesias (hands and feet) and tinnitus showed significant three- to four-fold increased risks for the highest platinum quartile. Cumulative cisplatin dose was not associated with total SCIN score or individual SCIN symptoms in multivariate analyses. Here we document a significant relationship between increasing levels of residual serum platinum and NTX severity after adjusting for initial cisplatin dose.

Drosophila melanogaster: A new model to study cisplatin-induced neurotoxicity

Platinum-based compounds are widely used and effective chemotherapeutic agents; however, sensory peripheral neuropathy is a dose-limiting and long term side effect for 20–30% of patients. A critical question is whether the mechanisms of cell death underlying clinical efficacy can be separated from the effects on neurons in order to develop strategies that prevent platinum-induced neuropathy. In rodent dorsal root ganglion neurons (DRG), cisplatin has been shown to bind and damage neuronal DNA, inducing apoptosis; however genetic manipulation in order to study mechanisms of this phenomenon in the rodent model system is costly and time-consuming. Drosophila melanogaster are commonly used to study neurological disorders, have DNA damage-apoptosis mechanisms homologous to mammalian systems, and have readily-available, inexpensive tools for rapid genetic manipulation. We therefore sought to develop adult Drosophila as a new model to study cisplatin-induced neurotoxicity. Adult Drosophila were exposed to 10, 25, 50, 100, 200 and 400 μg/ml cisplatin for 3 days and observed for fly survival and geotactic climbing behavior, cisplatin-DNA binding and cellular apoptosis. On day 3, 50 μg/ml cisplatin reduced the number of flies able to climb above 2 cm to 43% while fly survival was maintained at 92%. 100% lethality was observed at 400 μg/ml cisplatin. Whole fly platinum-genomic DNA adducts were measured and found to be comparable to adduct levels previously measured in rat DRG neurons. Brain, ovaries, kidney and heart harvested from cisplatin treated flies were stained for active caspase 3. Apoptosis was found in ovaries and brain but not in heart and kidney. Brain apoptosis was confirmed by transmission electron microscopy. Expression of the anti-apoptotic baculoviral protein, p35, in neurons using the GAL4-UAS system prevented cisplatin-induced apoptosis in the brain and restored climbing behavior. In conclusion, cisplatin-induced behavioral and apoptotic changes in Drosophila resemble those seen in mammals. Furthermore, the use of lethality and climbing assays combined with powerful gene manipulation, make Drosophila a suitable model to study mechanisms of cisplatin neurotoxicity.

Cisplatin induced Mitochondrial DNA damage in dorsal root ganglion neurons

Cisplatin is a platinum-based chemotherapeutic agent that induces peripheral neuropathy in 30% of patients. Peripheral neuropathy is the dose limiting side effect, which has no preventative therapy. We have previously shown that cisplatin induces apoptosis in dorsal root ganglion (DRG) sensory neurons by covalently binding to nuclear DNA (nDNA), resulting in DNA damage, subsequent p53 activation and Bax-mediated apoptosis via the mitochondria. We now demonstrate that cisplatin also directly binds to mitochondrial DNA (mtDNA) with the same binding affinity as nDNA. Cisplatin binds 1 platinum molecule per 2166 mtDNA base pairs and 1 platinum molecule per 3800 nDNA base pairs. Furthermore, cisplatin treatment inhibits mtDNA replication as detected by 5-bromo-2′-deoxy-uridine (BrdU) incorporation and inhibits transcription of mitochondrial genes. The relative reduction in mtDNA transcription is directly related to the distance the gene is located from the transcription initiation point, which implies that randomly formed platinum adducts block transcription. Cisplatin treated DRG neurons exhibit mitochondrial vacuolization and degradation in vitro and in vivo. Taken together, this data suggests that direct mtDNA damage may provide a novel, distinct mechanism for cisplatin-induced neurotoxicity separate from the established nDNA damage pathway.

Comparative Effects of Alpha Lipoic Acid and Melatonin on Cisplatin-Induced Neurotoxicity

ABSTRACTCisplatin is a carcinogenic agent having important cytotoxic effects. Cisplatin treatment increases the levels of free oxygen radicals in neurologic tissues. We investigated the effects of alpha lipoic acid (ALA) and melatonin (MEL) on the electrophysiological parameters and on activities of nerve fibers having different conduction properties on cisplatin neurotoxicity. Neurotoxicity was induced by a single injection of 10 mg/kg intraperitoneal (ip) cisplatin. Supplementation was started 1 day before cisplatin injection with either 100 mg/kg/day ip ALA or 4 mg/kg/day ip MEL for 7 days. Compound action potentials were recorded from isolated sciatic nerves in vitro, and numerical analyses were conducted. Cisplatin-induced neurotoxicity resulted in a significant decrease (p <.05) in maximum depolarization (mV), areas (mV·ms), and maximum and minimum upstroke velocity values (mV/ms). Although these decrements were restored by ALA and MEL, ALA was found to be more effective. Conventional conduction velocity measurements and conduction velocity distribution histograms have shown that ALA supplementation can recover the effects of cisplatin while MEL cannot. The conduction velocity distribution histograms have shown that antioxidant supplementation results in a restoration on contribution of fast-conducting fibers (51.8–77.7 m/s), which is deteriorated by cisplatin. Consequently, ALA has more potential to make up for the deleterious effects of cisplatin-induced neurotoxicity.

Implications of Apurinic/Apyrimidinic Endonuclease in Reactive Oxygen Signaling Response after Cisplatin Treatment of Dorsal Root Ganglion Neurons

Peripheral neuropathy is one of the major side effects of the anticancer drug cisplatin. Although previous work suggests that this neuropathy correlates with formation of DNA adducts in sensory neurons, growing evidence suggests that cisplatin also increases the generation of reactive oxygen species (ROS), which could cause DNA damage. Apurinic/apyrimidinic endonuclease/redox factor-1 (Ape1/Ref-1) is a multifunctional protein involved in DNA base excision repair of oxidative DNA damage and in redox regulation of a number of transcription factors. Therefore, we asked whether altering Ape1 functions would influence cisplatin-induced neurotoxicity. Sensory neurons in culture were exposed to cisplatin for 24 hours and several end points of toxicity were measured, including production of ROS, cell death, apoptosis, and release of the immunoreactive calcitonin gene-related peptide (iCGRP). Reducing expression of Ape1 in neuronal cultures using small interfering RNA (siRNA) enhances cisplatin-induced cell killing, apoptosis, ROS generation, and cisplatin-induced reduction in iCGRP release. Overexpressing wild-type Ape1 attenuates all the toxic effects of cisplatin in cells containing normal endogenous levels of Ape1 and in cells with reduced Ape1 levels after Ape1siRNA treatment. Overexpressing the redox deficient/repair competent C65-Ape1 provides partial rescue, whereas the repair-deficient Ape1 (N226A + R177A) does not protect neurons from cisplatin toxicity. We also observe an increase in phosphorylation of p53 after a decrease in Ape1 levels in sensory neuronal cultures. These results strongly support the notion that Ape1 is a potential translational target such that protecting Ape1 levels and particularly its DNA repair function could reduce peripheral neuropathy in patients undergoing cisplatin treatment.