Antioxidants attenuate gentamicin-induced free radical formation in vitro and ototoxicity in vivo: D-methionine is a potential protectant

We have recently suggested antioxidant therapy against aminoglycoside-induced hearing loss based on the hypothesis of a redox-active aminoglycoside-iron complex causing ototoxicity. The present study compares seven antioxidants and iron chelators for their ability to attenuate gentamicin-induced free radical generation in vitro and ototoxicity in guinea pig in vivo.Free radical formation by gentamicin was measured by chemiluminescence detection both in a non-enzymatic system in vitro and in cell culture. Deferoxamine, 2,3-dihydroxybenzoate, or salicylic acid suppressed gentamicin-induced luminescence in both tests. This indicated the usefulness of the assay as a screen for potential protectants since these agents had previously been shown to attenuate gentamicin-induced ototoxicity in vivo. Histidine and D-methionine, amino acids with chelating and antioxidant properties, also suppressed gentamicin-mediated luminosity both in vitro and in cell culture. In contrast, the metal chelators succimer (2, 3-dimercaptosuccinic acid (DMSA)) and trientine (N, N'-bis[2-aminoethyl]-1,2 ethanediamine) promoted free radical formation and were excluded from further studies. Histidine and D-methionine were then administered to guinea pigs receiving concurrent treatment with gentamicin (120 mg/kgx19 days). Threshold shifts induced by gentamicin were significantly attenuated by twice-daily injections of D-methionine. Once-daily injections of histidine or D-methionine were less effective, pointing to the importance of pharmacokinetics in antioxidant protection in vivo.The study presents a simple screening system for agents with the potential to attenuate gentamicin-induced hearing loss. It also supports the hypothesis of free radical formation as an underlying cause of gentamicin ototoxicity.     

Glutathione limits noise-induced hearing loss

The generation of reactive oxygen species (ROS) is thought to be part of the mechanism underlying noise-induced hearing loss (NIHL). Glutathione (GSH) is an important cellular antioxidant that limits cell damage by ROS. In this study, we investigated the effectiveness of a GSH supplement to protect GSH-deficient animals from NIHL. Pigmented guinea pigs were exposed to a 4 kHz octave band noise, 115 dB SPL, for 5 h. Group 1 had a normal diet, while groups 2, 3 and 4 were fed a 7% low protein diet (leading to lowered tissue levels of GSH) for 10 days prior to noise exposure. One hour before, immediately after and 5 h after noise exposure, subjects received either an intraperitoneal injection of 5 ml/kg body weight of 0.9% NaCl (groups 1 and 2), 0.4 M glutathione monoethyl ester (GSHE; group 3) or 0.8 M GSHE (group 4). Auditory thresholds were measured by evoked brain stem response at 2, 4, 8, 12, 16 and 20 kHz before and after noise exposure. Ten days post exposure, group 1 showed noise-induced threshold shifts of approximately 20 dB at 2, 16 and 20 kHz and 35 to 40 dB at other frequencies. Threshold shifts in group 2 were significantly greater than baseline at 2, 4, 16 and 20 kHz. GSHE supplementation in a dose-dependent fashion attenuated the threshold shifts in the low protein diet animals. Hair cell loss, as evaluated with cytocochleograms, was consistent with the auditory-evoked brainstem response results. Group 2 exhibited significantly more hair cell loss than any of the other groups; hair cell loss in group 3 was similar to that seen in group 1; group 4 showed less loss than group 1. These results indicate that GSH is a significant factor in limiting noise-induced cochlear damage. This is compatible with the notion that ROS generation plays a role in NIHL and that antioxidant treatment may be an effective prophylactic intervention.     

Aminoglycoside antibiotics

In the 50 years since their discovery, the aminoglycoside antibiotics have seen unprecedented use. Discovered in the 1940s, they were the long-sought remedy for tuberculosis and other serious bacterial infections. The side effects of renal and auditory toxicity, however, led to a decline of their use in most countries in the 1970s and 1980s. Nevertheless, today the aminoglycosides are still the most commonly used antibiotics worldwide thanks to the combination of their high efficacy with low cost. This review first summarizes the history, chemistry, antibacterial actions and acute side effects of the drugs. It then details the pathophysiology of aminoglycoside ototoxicity including experimental and clinical observations, risk factors and incidence. Pharmacokinetics, cellular actions and our current understanding of the underlying molecular mechanisms of ototoxicity are discussed at length. The review concludes with recent advances towards therapeutic intervention to prevent aminoglycoside ototoxicity.     

阿司匹林预防庆大霉素耳毒性的随机对照试验

目的观察阿司匹林对庆大霉素耳毒性的预防作用。方法设计实施多中心的随机双盲对照试验，预防组在应用庆大霉素的同时口服阿斯匹林2周，对照组口服安慰剂。用药前后检测纯音测听3次，对比听力损伤发生率。结果两组的血清庆大霉素水平相当，预防组的听力损伤发生率为3％（3／89），明显低于对照组的13％（14／106）（P＝0．013）。试验中阿司匹林没有影响到庆大霉素的血清药物水平和时程。结论同时伍用阿司匹林可以有效减轻庆大霉素的耳毒性损伤，氨基甙类抗生素的耳毒性防护研究已从动物实验走向临床。     

The effect of noise on deoxyglucose uptake into inner ear tissues of the mouse

Summary The uptake of deoxyglucose into inner ear tissues was studied in the mouse. Sixty minutes after a single i.v. injection of 5 mCi 2-deoxy-D-[³H]glucose/kg body weight, stria vascularis/spiral ligament and organ of Corti as well as other body tissues were dissected and analyzed for radioactivity. Uptake into inner ear tissues was three to five times lower than into brain or heart. The ratio of deoxyglucose-6-phosphate to deoxyglucose was 6040 and the compounds were eliminated from the inner ear with a half life of approximately 60 min. Exposure to 100 dB of white noise during the radioactive pulse decreased uptake of deoxyglucose into both stria vascularis/spiral ligament and organ of Corti by 50%.This work was supported by grant NS 12881 from the National Institutes of Health and Program Project grant NS 05785 and training grant NS 07106     

Characteristics and drug responses of cochlear and vestibular adenylate cyclase

Summary Adenylate cyclase activity of dissected cochlear and vestibular structures was assayed with the ATP-analog adenylyl imidodiphosphate as substrate. High activities (per mg protein) were found in stria vascularis and in vestibular preparations, lower activities in spiral ligament, VIIIth nerve, and organ of Corti. The enzyme from all structures was stimulated by fluoride, guanylyl imidodiphosphate or manganese(II) ions, and strongly inhibited by ethacrynate and lead ion. The anti-cancer drug cis-diammine-dichloro platinum significantly inhibited adenylate cyclase from stria vascularis.Presented at the 16th Workshop of Inner Ear Biology in Bern 1979     

Cardiac biopsy in childhood

Endomyocardial biopsy was attempted in 18 children aged 5 months to 15 years with 82% success. Biopsies obtained from 15 children were examined by light and electron microscope making positive morphological diagnoses in 3 cases. The biopsy findings were actively helpful in 7 other cases, which contrasts with experience in adult biopsy series. This is a low risk procedure which does not add to the hazards of cardiac catheterization in children.     

Treatment of menorrhagia with a novel 'frameless' intrauterine levonorgestrel-releasing drug delivery system: a pilot study.

OBJECTIVE: To evaluate the effect on menstrual blood loss of a novel 'frameless' intrauterine drug delivery system, the FibroPlant levonorgestrel intrauterine system (IUS), releasing 14 microg of levonorgestrel/day. An ancillary objective was to evaluate the contraceptive performance. STUDY DESIGN: This was an open-label, non-comparative ongoing pilot study. Thirty-two insertions were performed in fertile women aged between 31 and 51 years for the treatment of menorrhagia, as well as for contraceptive purposes. Fifteen women who developed excessive bleeding were fitted with the FibroPlant levonorgestrel IUS immediately following the removal of a copper-bearing intrauterine device (IUD), the GyneFix IUD. To discriminate between menorrhagia and normal menstrual blood loss, women were evaluated using a simple visual assessment technique. The trial covered a period from a minimum of 1 month up to 23 months. RESULTS: At the time of study analysis, the total number of woman-months was 361. Fourteen of the women had had the FibroPlant levonorgestrel IUS in place for more than 1 year, and 29 women for 6 months or more. All women reported greatly reduced bleeding. However, no cases of amenorrhea resulting from endometrial suppression were encountered. The reduction of bleeding was substantial after 1 month of treatment and decreased further over the next months to remain stable thereafter. The mean bleeding score before treatment was 338 (range 185-740) in the group who had had no prior use of an IUD and 368 (range 185-890) in the group with prior IUD use. The mean bleeding score dropped to a mean score of 70 (range 5-210) in the 'no prior IUD use' group and to a mean score of 52 (range 3-150) in the 'prior IUD use' group, after 1-23 months of follow-up. This result is highly statistically significant (p < 0.001). There were no statistical differences in bleeding scores before and during treatment between the two groups of women, with or without prior use of the copper IUD. Significant spotting was rare after the first 3 months following insertion. No complications (e.g. infection, expulsion or perforation) or pregnancies occurred. The FibroPlant levonorgestrel IUS was well tolerated by all women involved in the study and no systemic hormonal side-effects were reported.CONCLUSION: The FibroPlant levonorgestrel IUS is effective in significantly reducing the amount of menstrual blood loss in women with menorrhagia. Strong endometrial suppression is the principal mechanism explaining both the effect on menstrual blood loss and the contraceptive performance of the IUS. There were no differences in bleeding scores before and during treatment between the two groups of women with or without prior use of the copper IUD, suggesting that the development of heavy bleeding was not related to the use ofthe IUD. The therapeutic effect of this contraceptive method is highly desirable, particularly in women with heavy bleeding or anemia in developing countries, as other treatment modalities are less effective, more costly, more invasive or inaccessible. The low daily release rate of levonorgestrel from the FibroPlant levonorgestrel IUS results in a low incidence of hormonal side-effects and reduces the likelihood of amenorrhea. The simple design characteristics and revolutionary anchoring system minimize the occurrence of complaints of pain and the incidence of expulsion.     

Chronic electrical stimulation reverses deafness-related depression of electrically evoked 2-deoxyglucose activity in the guinea pig inferior colliculus

The [¹⁴C]-2-deoxyglucose (2-DG) autoradiographic technique was used to study how auditory-related metabolic activity changes with deafness, and how chronic electrical stimulation of the deafened system may modify these changes. Guinea pigs were deafened by administration of kanamycin and ethacrynic acid. After nine weeks of deafness, the basal unstimulated uptake of 2-DG in the inferior colliculus (IC) was lower than in normal hearing control animals. 100 μA of acute cochlear electrical stimulation significantly increased 2-DG uptake in normal hearing animals but did not evoke a significant increase in four or nine week deafened animals. Electrically elicited 2-DG uptake in the IC is therefore depressed by prolonged deafness. In a second series of experiments, after four weeks of deafness, animals were chronically electrically stimulated via a cochlear implant 2.5-3.5 h a day, five days a week for five weeks at 100 μ,A. Acute cochlear electrical stimulation following this chronic stimulation significantly increased 2-DG uptake in the contralateral IC over unstimulated levels. This suggests that some depressive effects of profound deafness on the auditory brain stem may be reduced or reversed with chronic electrical stimulation by a cochlear implant.     

Inhibition of inner ear ornithine decarboxylase by neomycin in-vitro

We quantitated the activity of ornithine decarboxylase (ODC) in homogenates and subcellular fractions of inner ear tissues from the rat and guinea pig and demonstrate inhibition of cochlear ODC by the aminoglycoside neomycin. Subcellular fractionation showed the enzyme associated with the post-mitochondrial supernatant fraction in each of the tissues: Specific activities of ODC, defined as alpha-difluoromethylornithine (DFMO)-sensitive decarboxylation of ornithine, in the supernatant fractions of combined inner ear tissues were: guinea pig = 44 +/- 4 pmoles CO2 produced/hour/mg protein, and rat = 133 +/- 30. In the guinea pig, supernatant fractions of the lateral wall tissues (stria vascularis and spiral ligament) had specific activities of 62 +/- 25, those of the organ of Corti (plus VIIIth nerve) 64 +/- 41. The ototoxic aminoglycoside neomycin produced a dose-dependent inhibition of ODC with half-maximal inhibition observed at 50 microM drug and almost complete inhibition at 100 microM. This is the first report of the presence of ODC in the inner ear and its inhibition by neomycin. Since both the ODC-inhibitors, DFMO and neomycin, can cause hearing loss in patients and experimental animals it is suggested that inhibition of ODC may be an important factor in the ototoxicity of these drugs.