M40403, a superoxide dismutase mimetic,protects cochlear hair cells from gentamicin,but not cisplatin toxicity

Gentamicin, an aminoglycoside antibiotic, and cisplatin, a platinum-based anticancer drug, are two commonly used clinical drugs with ototoxic side effects. The ototoxicity of gentamicin and cisplatin has been linked to the production of reactive oxygen species (ROS), although the specific ROS pathways have not been identified. One ROS that might play a role in ototoxicity is the superoxide radical, which is enzymatically dismutated to molecular oxygen and hydrogen peroxide by endogenous superoxide dismutase (SOD) enzymes. M40403, a manganese-based nonpeptidyl molecule that mimics the activity of SOD, was tested for its ability to protect against gentamicin and cisplatin toxicity in cochlear organotypic cultures from neonatal C57BL/10J mice. Cultures were treated with gentamicin or cisplatin alone or in combination with M40403. M40403 alone had no effect on outer hair cell (OHC) or inner hair cell (IHC) survival at doses of 1, 5, and 10 microM, but a high dose of 30 microM reduced hair cell numbers by approximately 30%. Gentamicin alone and cisplatin alone killed OHCs and IHCs in a dose-dependent manner. The addition of M40403 to gentamicin-treated cultures significantly increased OHC and IHC survival in a dose-dependent manner, whereas M40403 failed to protect hair cells in cisplatin-treated cultures at any dose. The results suggest that the toxicity of gentamicin and cisplatin to cochlear hair cells are mediated by different pathways. Clinically, increased levels of SOD or SOD mimetics might provide significant protection against aminoglycoside ototoxicity.     

Assessing cisplatin-induced ototoxicity and otoprotection in whole organ culture of the mouse inner ear in simulated microgravity

Cisplatin is a widely used anti-cancer drug. Ototoxicity is a major dose-limiting side-effect. A reproducible mammalian in-vitro model of cisplatin ototoxicity is required to screen and validate otoprotective drug candidates. We utilized a whole organ culture system of the postnatal mouse inner ear in a rotating wall vessel bioreactor under “simulated microgravity” culture conditions. As previously described this system allows whole organ culture of the inner ear and quantitative assessment of ototoxic effects of aminoglycoside induced hair cell loss. Here we demonstrate that this model is also applicable to the assessment of cisplatin induced ototoxicity. In this model cisplatin induced hair cell loss was dose and time dependent. Increasing exposure time of cisplatin led to decreasing EC₅₀ concentrations. Outer hair cells were more susceptible than inner hair cells, and hair cells in the cochlear base were more susceptible than hair cells in the cochlear apex. Initial cisplatin dose determined the final extent of hair cell loss irrespective if the drug was withdrawn or continued. Dose dependant otoprotection was demonstrated by co-administration of the antioxidant agent N-acetyl l-cysteine. The results support the use of this inner ear organ culture system as an in vitro assay and validation platform for inner ear toxicology and the search for otoprotective compounds.     

Cellular pool of transient ferric iron, chelatable by deferoxamine and distinct from ferritin, that is involved in oxidative cell injury.

A cellular pool of transient ferric iron that is chelatable by deferoxamine, distinct from ferritin, and required for oxidative cell injury has been identified in cultured rat hepatocytes labeled with 59FeCl3. Pretreatment of hepatocytes with deferoxamine depleted the cellular pool of chelatable iron and protected the cells from an oxidative injury. Incubation of deferoxamine-pretreated hepatocytes in serum-free medium restored both the chelatable iron pool and the susceptibility to oxidative injury. Furthermore, inhibition of protein degradation with chymostatin prevented the restoration of both the chelatable pool and susceptibility to oxidative injury. The deferoxamine-chelatable iron pool was distinguished kinetically and immunochemically from the larger cellular pool of ferritin iron. The labeled iron in the deferoxamine-chelatable pool was transient, unlike either the total cellular uptake of 59Fe or its incorporation into ferritin, both of which increased with time of labeling. With pulse-chase labeling, the percentage of the total uptake of 59Fe that was represented by the deferoxamine-chelatable pool decreased. At the same time, the percentage represented by radioactivity immunoprecipitable as ferritin increased. Furthermore, immunoprecipitation of ferritin from the labeled lysates enriched the resulting immunosupernatants in deferoxamine-chelatable iron. The degree of enrichment for chelatable iron correlated with the percentage of the cellular label that was immunoprecipitable as ferritin. The deferoxamine-chelatable iron appears to represent a metabolically common pool of iron that is rapidly in transit through the cell. Extracellular iron entering the pool can be utilized for heme synthesis or stored in ferritin, whereas protein degradation releases storage iron into this pool.     

Ototoxicity: mechanisms of cochlear impairment and its prevention

Aminoglycosides, cisplatin, and non-steroidal anti-inflammatory drugs (NSAIDs) are widely used pharmacological agents. There is a possibility, however, that the use of these agents may induce transient or permanent hearing loss and tinnitus as side effects. Recent animal studies have clarified mechanisms leading to the ototoxicity induced by these agents, at least in part. The permanent hearing loss caused by aminoglycosides and cisplatin is suggested to be predominantly associated with the apoptotic death of outer hair cells. Both drugs generate reactive oxygen species (ROS) in the inner ear. ROS can activate cell-death pathways such as the c-Jun Nterminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways, which in turn, induce hair cell apoptosis. On the other hand, the abuse of NSAIDs may transiently cause tinnitus and mild to moderate hearing loss. NSAIDs impair the active process of the outer hair cells and affect peripheral and central auditory neurons. Conversely, recent reports clarified that NSAIDs are potential therapeutic agents against cochlear injuries. In this review, recent findings from animal studies regarding the ototoxicity induced by aminoglycosides, cisplatin, and NSAIDs are summarized. Their ototoxic mechanisms are focused on.     

Ototoxicity of cisplatin administered to guinea pigs via the round window membrane

Animal models of ototoxicity represent an elementary tool in otolaryngologic research. Such models are usually created via the consecutive injection of ototoxic drugs or the co-administration of ethacrynic acid and low-dose ototoxic drugs. Injection via the round window membrane (RWM) is one approach that allows for local drug delivery into the inner ear. In this study, 47 guinea pigs received an injection of varying doses of cisplatin via the RWM, and data concerning the animals' auditory brainstem responses, hair cells, and spiral ganglion neurons were analyzed. Our results indicate the high efficiency and generally small reaction of the subjects, suggesting that the application of cisplatin via the RWM is an effective animal model for ototoxicity research.     

Potential role for oxidative stress in 2,2'-dichlorobiphenyl-induced inhibition of uterine contractions but not myometrial gap junctions.

Previously, we reported that 2,2'-dichlorobiphenyl (2,2'-DCB)-induced decreases of amplitude and synchronization of uterine contractions are dependent on MAPK-induced phosphorylation of Connexin43 (Cx43) and inhibition of myometrial gap junctions. Recent studies show that oxidative stress inhibits uterine contractions and myometrial gap junctions also. The present study examines the hypothesis that 2,2'-DCB-induced modification of uterine contraction is dependent on oxidative stress-mediated inhibition of myometrial gap junctions via activation of mitogen-activated protein kinase (MAPK) and phosphorylation of Cx43. In uterine strips treated with alpha-tocopherol (100 microM), deferoxamine mesylate (Def, 50 microM), or superoxide dismutase (SOD, 1000 U) after a 1-h exposure to 100 microM 2,2'-DCB, modification of uterine contractions reversed 1 h after initiating antioxidant treatment. Treatment of uterine strips with 100 microM 2,2'-DCB for 1 h lowered total SOD activity and also induced a surge of superoxide generation after 5 min of exposure. However, myometrial cells exposed in culture to 100 microM 2,2'-DCB did not produce reactive oxygen species as determined by the lack of superoxide anion generation measured by the cytochrome c reduction assay, reactive species by the formazan assay, hydrogen peroxide by the 2',7'-dichlorofluorescein assay, and lipid peroxidation by the thiobarbituric acid-reactive substance assay. Furthermore, cotreatment with SOD or Def was unable to prevent 2,2'-DCB-induced phosphorylation of Cx43, activation of MAPK, and inhibition of myometrial gap junctions. Although antioxidants reversed 2,2'-DCB-induced inhibition of uterine contraction force and synchronization, the myometrial cell culture experiments failed to support oxidative stress as a mechanistic link between 2,2'-DCB-induced inhibition of myometrial gap junctions and modification of uterine contraction.     

In Vitro Ototoxicity of aminoglycosides and platin derivatives. A semi-automatic assay for sensory hair cell damage in explanted rat organ of corti

The ototoxic damage that drugs such as neomycin, kanamycin, colistin, cisplatin, transplatin and carboplatin cause on outer and inner hair cells in postnatal day 3 rat cochlear explants was investigated. Phalloidin–fluorescein conjugate-stained stereocilia bundles of sensory hair cells were quantified by video image analysis as a measurement of ototoxic effect. The video image quantification system established dose–response curves for ototoxic drugs (e.g. calculation of an IC₅₀) and allowed comparisons between several ototoxins from the same family. This methodology provided the means to assess the efficacy of otoprotectant agents in preventing ototoxicity. Poly-l-aspartate (10⁻⁵ ) and poly-l-glutamate (10⁻⁵ ) protected auditory hair cells from neomycin (10⁻³ ) toxicity while reduced glutathione (10⁻³ ) provided protection against cisplatin (10⁻⁴ )-induced hair cell damage.     

Alterations in cellular Ca(2+) and free iron pool by sulfur amino acid deprivation: the role of ferritin light chain down-regulation in prooxidant production

Deficiency of sulfur amino acids occurs in certain pathophysiological states such as protein-calorie malnutrition. Sulfur amino acid deprivation (SAAD) increases oxidative stress through a decrease in GSH. Ferritin expression is induced by oxidative stress, which confers resistance to oxidative insults. The effects of SAAD on the changes in cellular Ca(2+) and free iron pool, prooxidant production and the ferritin light chain (FLC) expression were comparatively evaluated in Hepa1c1c7 and Raw264.7 cells. [Ca(2+)](i) was rapidly increased by SAAD. Sulfhydryl-containing compounds prevented the increase in [Ca(2+)](i) in cells under SAAD, supporting the role of redox-state in the regulation of [Ca(2+)](i). Thapsigargin or Ca(2+)-free medium inhibited the increase in [Ca(2+)](i), showing that Ca(2+) originated from endoplasmic reticulum as well as from extracellular source. Inhibition of Ca(2+) mobilization decreased the fluorescence of Phen Green SK inside cells, representing the inhibition of free iron release. Both inhibition of Ca(2+) mobilization and iron chelation decreased dichlorofluorescein oxidation, indicating the possibility that the increase in [Ca(2+)](i) affected that in cellular free iron and prooxidant production. FLC protein level was immunochemically detectable in Raw264.7 cells, but not in Hepa1c1c7 cells. SAAD alone (or in combination with FeSO(4)) down-regulated FLC protein expression, while SAAD increased the FLC mRNA level in both Hepa1c1c7 and Raw264.7 cells. Calcium or iron chelators prevented increases in the FLC mRNA. These results provided evidence that changes in cellular Ca(2+) and iron pool by SAAD increased cellular oxidative stress and that the down-regulation of FLC protein by SAAD would further enhance prooxidant production in spite of the increase in FLC mRNA.     

Protective effects of apocynin on cisplatin‐induced ototoxicity in an auditory cell line and in zebrafish

Cisplatin is a very effective anticancer drug and generates reactive oxygen species (ROS) such as superoxide anions that can deplete antioxidant protective molecules in the cochlea. These processes result in the death of cochlear hair cells by induction of apoptosis. Apocynin, which is used as a specific nicotinamide adenine dinucleotide phosphate oxidase inhibitor, has a preventive effect for intracellular ROS generation. In this study, the effect of apocynin was investigated in a cochlear organ of Corti‐derived cell line, HEI‐OC1 cells, and in transgenic zebrafish (Brn3C: EGFP). To investigate the protective effects of apocynin, HEI‐OC1 cells were treated with various concentrations of apocynin and a 20 µm concentration of cisplatin, simultaneously. An in vivo study of transgenic zebrafish (Brn3C: EGFP) was used to investigate the protective effects of apocynin on cisplatin‐induced hair cell death. In an in vitro study, apocynin appeared to protect against cisplatin‐induced apoptotic features on Hoechst 33258 staining in the HEI‐OC1 cells. Treatment of the HEI‐OC1 cells with 100 µm of apocynin, significantly decreased caspase‐3 activity. Treatment of the cells with a 100 µm concentration of apocynin and a 20 µm concentration of cisplatin significantly decreased the intracellular ROS production. In the in vivo study, apocynin significantly decreased the TUNEL reaction and prevented cisplatin‐induced hair cell loss of the neuromasts in the transgenic zebrafish at low concentrations (125 and 250 µm ). These findings suggest that apocynin has antioxidative effects and prevents cisplatin‐induced apoptotic cell death in HEI‐OC1 cells as well as in zebrafish. Copyright © 2011 John Wiley & Sons, Ltd.     

Advances in iron overload therapies. prospects for effective use of deferiprone (L1), deferoxamine, the new experimental chelators ICL670, GT56-252, L1NA11 and their combinations

Effective new therapies and mechanisms have been developed for the targeting and prevention of iron overload and toxicity in thalassaemia and idiopathic haemochromatosis patients. A new era in the development of chelating drugs began with the introduction of deferiprone or L1, which as a monotherapy or in combination with deferoxamine can be used universally for effective chelation treatments, rapid iron removal, maintenance of low iron stores and prevention of heart and other organ damage caused by iron overload. Several experimental iron chelators such as deferasirox (4-[3,5-bis (2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid) or ICL670, deferitrin (4,5-dihydro-2- (2,4-dihydroxyphenyl)-4-methylthiazole-4 (S)-carboxylic acid) or GT56-252, 1-allyl-2-methyl-3-hydroxypyrid-4-one or L1NAll and starch deferoxamine polymers have reached different stages of clinical development. The lipophilic ICL670, which can only be administered once daily is generally ineffective in causing negative iron balance but is effective in reducing liver iron. It is suspected that it may increase iron absorption and the redistribution of iron from the liver to the heart and other organs. The experimental iron chelators do not appear to have significant advantages in efficacy and toxicity by comparison to deferiprone, deferoxamine or their combination. However, the prospect of combination therapies using deferiprone, deferoxamine and new chelators will provide new mechanisms of chelator interactions, which may lead to higher efficacy and lower toxicity by comparison to monotherapies. A major disadvantage of the experimental chelators is that even if they are approved for clinical use, they are unlikely to be as inexpensive as deferiprone and become available to the vast majority of thalassaemia patients, who live in developing countries.     

Deferasirox (ICL670A) effectively inhibits oesophageal cancer growth in vitro and in vivo

Background and Purpose

Growing evidence implicates iron in the aetiology of gastrointestinal cancer. Furthermore, studies demonstrate that iron chelators possess potent anti-tumour activity, although whether iron chelators show activity against oesophageal cancer is not known.

Experimental Approach

The effect of the iron chelators, deferoxamine (DFO) and deferasirox, on cellular iron metabolism, viability and proliferation was assessed in two oesophageal adenocarcinoma cell lines, OE33 and OE19, and the squamous oesophageal cell line, OE21. A murine xenograft model was employed to assess the effect of deferasirox on oesophageal tumour burden. The ability of chelators to overcome chemoresistance and to enhance the efficacy of standard chemotherapeutic agents (cisplatin, fluorouracil and epirubicin) was also assessed.

Key Results

Deferasirox and DFO effectively inhibited cellular iron acquisition and promoted intracellular iron mobilization. The resulting reduction in cellular iron levels was reflected by increased transferrin receptor 1 expression and reduced cellular viability and proliferation. Treating oesophageal tumour cell lines with an iron chelator in addition to a standard chemotherapeutic agent resulted in a reduction in cellular viability and proliferation compared with the chemotherapeutic agent alone. Both DFO and deferasirox were able to overcome cisplatin resistance. Furthermore, in human xenograft models, deferasirox was able to significantly suppress tumour growth, which was associated with decreased tumour iron levels.

Conclusions and Implications

The clinically established iron chelators, DFO and deferasirox, effectively deplete iron from oesophageal tumour cells, resulting in growth suppression. These data provide a platform for assessing the utility of these chelators in the treatment of oesophageal cancer patients.

Linked Article

This article is commented on by Keeler and Brookes, pp. 1313–1315 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12093     

Toxicity of dipyridyl compounds and related compounds

Five dipyridyl isomers, 2,2'-, 2,3'-, 2,4'-, 3,3'-, and 4,4'-dipyridyl, are products resulting from the pyrolytic degradation of tobacco products and degradation of the herbicide paraquat, and therefore may be present in the environment. In this article, the toxicological properties of these dipyridyl isomers in humans and animals are reviewed. Epidemiological studies suggest that cancerous skin lesions in workers involved in the manufacturing of paraquat may be associated with exposure to dipyridyl compounds. Experimental animal studies suggest that dipyridyl isomers may have several toxicological effects. Three of the dipyridyl isomers (the 2,2', 2,4', and 4,4' isomers) appear to be inducers of some metabolic enzymes. The 2,2'-dipyridyl isomer, an iron chelator, appears to influence vasospasm in primate models of stroke. The cytotoxic effects of 2,2'-dipyridyl on several leukemia cell lines have been reported, and a potent teratogenic effect of 2,2'-dipyridyl has been observed in rats. Based on the results of paraquat studies in experimental animal models, it has been proposed that paraquat may have deleterious effects on dopaminergic neurons. These findings support the epidemiological evidence that paraquat exposure may be associated with the development of Parkinson's disease. Studies designed to determine an association between paraquat exposure and Parkinson's disease are complicated by the possibility that metabolic changes may influence the neurotoxicity of paraquat and/or its metabolites. Preliminary unpublished data in mice show that 300-mg/kg doses of 2,2'-dipyridyl are neurotoxic, and 300-mg/kg doses of 2,4'- and 4,4'-dipyridyls are lethal. These results are consistent with earlier studies in Sherman rats using high 2,2'- and 4,4'-dipyridyl doses. New studies are needed to further explore the toxicological properties of dipyridyls and their potential public health impact.     

Novel synthetic protective compound,KR‐22335, against cisplatin‐induced auditory cell death

Cisplatin [cis‐diammine‐dichloroplatinum (II)] is a widely used chemotherapeutic agent, and one of its most severe side effects is ototoxicity. In the course of developing a new protective agent against cisplatin‐induced ototoxicity, we have been interested in a novel synthetic compound, 3‐Amino‐3‐(4‐fluoro‐phenyl)‐1H‐quinoline‐2,4‐dione (KR‐22335). We evaluated the effectiveness of KR‐22335 as an otoprotective agent against cisplatin‐induced toxicity. The otoprotective effect of KR‐22335 against cisplatin was tested in vitro in cochlear organs of Corti‐derived cell lines, HEI‐OC1, and in vivo in a zebrafish (Danio rerio) model. Cisplatin‐induced apoptosis, cell cycle arrest and an increase in intracellular reactive oxygen species (ROS) generation were demonstrated in HEI‐OC1 cells. KR‐22335 inhibited cisplatin‐induced apoptosis and mitochondrial injury in HEI‐OC1 cells. KR‐22335 inhibited cisplatin‐induced activation of JNK, p‐38, caspase‐3 and PARP in HEI‐OC1 cells. Scanning and transmission electron micrographs showed that KR‐22335 prevented cisplatin‐induced destruction of kinocilium and stereocilia in zebrafish neuromasts. Tissue TUNEL of neuromasts in zebrafish demonstrated that KR‐22335 blocked cisplatin‐induced TUNEL positive hair cells in neuromasts. The results of this study suggest that KR‐22335 may prevent ototoxicity caused by the administration of cisplatin through the inhibition of mitochondrial dysfunction and suppression of ROS generation. KR‐22335 may be considered as a potential candidate for protective agents against cisplatin‐induced ototoxicity. Copyright © 2013 John Wiley & Sons, Ltd.     

Enhancement of radiation-induced apoptosis by Podophyllum hexandrum

The aqueous extract of Podophyllum hexandrum (RP-1), which has been recently reported to manifest radioprotective and anti-tumour properties, has been investigated for its mode of action. RP-1, under in-vitro conditions dose-dependently chelated metal ions, inhibited radiation or metal ion-induced hydroxyl radicals and lipid peroxidation and scavenged superoxide anions. Intraperitoneal administration of RP-1 to mice pre-irradiation (10 Gy) induced more DNA fragmentation and lipid peroxidation in thymocytes maximally at 4 and 8 h, respectively, in comparison with RP-1 treatment or irradiation. Flow-cytometric quantification of sub-diploid peak, oligonucleosomal cleavage assay (ladder) and depletion of total thiols also corroborated the ability of RP-1 to enhance radiation-induced apoptosis. RP-1 in presence of 100 microM CuSO(4) induced strand breaks in plasmid DNA and addition of metal chelators (EDTA and deferoxamine) inhibited the strand scission. Treatment with a major constituent of RP-1, podophyllin, did not cause strand breaks, but isolated constituents of RP-1, quercetin or podophyllotoxin, induced strand breaks. Depending on its concentration in the milieu, RP-1 acted as a pro- or antioxidant modifying the radiation-induced apoptosis and therefore could be exploited for cancer management.     

Expression of myeloperoxidase in the inner ear of cisplatin-treated guinea pigs

Cisplatin is known to cause inner ear damage (ototoxicity). The role of myeloperoxidase (MPO) in the inner ear of the guinea pigs after injections of cisplatin i.p. was examined immunohistochemically. Three days after the injection, the animals were sacrificed with intracardiac perfusion of fixative, and temporal bones were removed and processed for immunohistochemistry with the anti-MPO antibody. MPO could be detected after 3 days in the lateral wall, the organ of Corti, supporting cells of the sensory epithelium and dark cells. These results suggest that MPO and reactive oxygen species are involved in the inner ear dysfunction after the application of cisplatin.     

New chelation therapies and emerging chelating drugs for the treatment of iron overload

Iron chelation therapy using deferoxamine or deferiprone (L1) is effective for the treatment of most transfused iron-loaded patients. The combination administration of deferiprone in the daytime and deferoxamine in the night appears to be universally effective in rapidly achieving negative iron balance. The cardiac iron removal effect of deferiprone increases the prospects of longer survival in beta-thalassaemia patients. New chelators have reached the stage of clinical development such as deferitrin, 1-allyl-2-methyl-3-hydroxypyrid-4-one (L1NAll) and the starch deferoxamine polymers. Deferasirox has received a conditional approval in the US under the FDA-accelerated approval regulations, but needs further verification of its efficacy and safety. Future iron chelation therapies are likely to be based on combinations of chelating drugs.     

Apotransferrin protects cortical neurons from hemoglobin toxicity

The protective effect of iron chelators in experimental models of intracerebral hemorrhage suggests that nonheme iron may contribute to injury to perihematomal cells. Therapy with high affinity iron chelators is limited by their toxicity, which may be due in part to sequestration of metals in an inaccessible complex. Transferrin is unique in chelating iron with very high affinity while delivering it to cells as needed via receptor-mediated endocytosis. However, its efficacy against iron-mediated neuronal injury has never been described, and was therefore evaluated in this study using an established cell culture model of hemoglobin neurotoxicity. At concentrations similar to that of CSF transferrin (50-100 micrograms/ml), both iron-saturated holotransferrin and apotransferrin were nontoxic per se. Overnight exposure to 3 μM purified human hemoglobin in serum-free culture medium resulted in death, as measured by lactate dehydrogenase release assay, of about three-quarters of neurons. Significant increases in culture iron, malondialdehyde, protein carbonyls, ferritin and heme oxygenase-1 were also observed. Holotransferrin had no effect on these parameters, but all were attenuated by 50-100 micrograms/ml apotransferrin. The effect of apotransferrin was very similar to that of deferoxamine at a concentration that provided equivalent iron binding capacity, and was not antagonized by concomitant treatment with holotransferrin. Transferrin receptor-1 expression was localized to neurons and was not altered by hemoglobin or transferrin treatment. These results suggest that apotransferrin may mitigate the neurotoxicity of hemoglobin after intracerebral hemorrhage. Increasing its concentration in perihematomal tissue may be beneficial.     

Protection against cisplatin ototoxicity by adenosine agonists

Cisplatin is a commonly used antineoplastic agent that causes ototoxicity through the formation of reactive oxygen species (ROS). Previous studies have shown that cisplatin causes an upregulation of A(1) adenosine receptor (A(1)AR) in the cochlea, and that application of the adenosine agonist, R-phenylisopropyladenosine (R-PIA), to the round window (RW) results in significant increases in cochlear glutathione peroxidase and superoxide dismutase. These data suggest that adenosine receptors (ARs) are an important part of the cytoprotective system of the cochlea in response to oxidative stress. The purpose of the current study was to investigate the effect of various adenosine agonists on cisplatin ototoxicity using RW application. Auditory brainstem response (ABR) thresholds were recorded in anesthetized chinchillas at 1, 2, 4, 8 and 16kHz. The auditory bullae were surgically opened, and 1mM R-PIA, 10microM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)/R-PIA (1mM) cocktail, 100microM 2-chloro-N-cyclopentyladenosine (CCPA), 2-[4-(2-p-carboxy-ethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS) or vehicle were applied to the RW. After 90min, the remaining solution was removed and cisplatin was applied to the RW. The bullae were closed and the animals recovered for 72h, after which, follow-up ABRs were performed. Cochleae were harvested for scanning electron microscopy (SEM) and for lipid peroxides. Pre-administration of the A(1)AR agonists R-PIA or CCPA significantly reduced cisplatin-induced threshold changes at all but the highest test frequency. In addition, A(1)AR agonists protected against cisplatin-induced hair cell damage and significantly reduced cisplatin-induced lipid peroxidation. Co-administration of the A(1)AR antagonist, DPCPX, completely reversed the protective effects of R-PIA. In contrast, pretreatment with CGS-21680, an A(2A) adenosine receptor (A(2A)AR) agonist, significantly increased cisplatin-induced threshold changes. Our findings are consistent with the notion that the A(1)AR contributes significantly to cytoprotection in the cochlea, and thereby protects against hearing loss.     

Nitric oxide synthase inhibitor suppresses the ototoxic side effect of cisplatin in guinea pigs

Cisplatin is known to cause inner ear damage (ototoxicity). The role of inducible nitric oxide synthase (iNOS) in the cochlea of guinea pigs after injections of cisplatin or a combination of cisplatin and NOS inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) i.p. was examined electro-and immunohistochemically. The auditory brain stem responses (ABR) were measured prior to injection and 3 days after the injection. Three days after injection, the cochleas were examined immunohistochemically for iNOS. We found that iNOS was expressed in the cisplatin- and L-NAME/ cisplatin-treated cochlea. The threshold shift of ABR was significant in the cisplatin group, whereas it was decreased in the L-NAME/cisplatin group. iNOS catalyzed high NO levels lead to inner ear dysfunction. Our results indicate that iNOS mediates the ototoxicity of cisplatin.     

Mobilization of iron from cells by hydroxyquinoline-based chelators

With the aim of identifying an iron (Fe) chelator which is effective at mobilizing intracellular Fe, two novel ligands were synthesized and tested. Hydroxyquinoline is known to possess a high affinity for Fe and was thus chosen as the Fe binding motif for the hexadentate chelators, C1 (2,2'-[ethane-1,2-diylbis(iminomethylene)]diquinolin-8-ol) and C2 (2,2'-[cyclohexane-1,2-diylbis(iminomethylene)]diquinolin-8-ol). Both chelators are lipophilic, with Fe3+ complexes slightly more hydrophilic than the free ligands. C1 and C2 were equally toxic to K562 cells, and partial protection was afforded by supplementing the culture medium with human holotransferrin, suggesting that some of the toxicity of the ligands is due to cellular Fe depletion. Micromolar concentrations of both ligands effectively mobilized 59Fe from reticulocytes and K562 cells. In reticulocytes, 50 microM C1 caused the release of 60% of the cells' initial 59Fe uptake after a 4h incubation. Under the same conditions, C2 revealed a release of 50% of the 59Fe. Overall, both ligands merit in vivo study for oral activity. Their effectiveness at low concentrations makes them candidates for therapeutic use.