Aminoglycoside ototoxicity in adult CBA, C57BL and BALB mice and the Sprague-Dawley rat.

The availability of genetic information, transgenic and knock-out animals make the mouse a primary model in biomedical research. Aminoglycoside ototoxicity, however, has rarely been studied in mature mice because they are considered highly resistant to the drugs. This study presents models for kanamycin ototoxicity in adult CBA/J, C57BL/6 and BALB/c mouse strains and a comparison to Sprague-Dawley rats. Five-week-old mice were injected subcutaneously twice daily with 400-900 mg kanamycin base/kg body weight for 15 days. Kanamycin induced dose-dependent auditory threshold shifts of up to 70 dB at 24 kHz as measured by auditory brain stem-evoked responses. Vestibular function was also affected in all strains. The functional deficits were accompanied by hair cell loss in both cochlear and vestibular neurosensory epithelia. Concomitant administration of the antioxidant 2,3-dihydroxybenzoate significantly attenuated the kanamycin-induced threshold shifts. In adult male Sprague-Dawley rats, doses of 1 x 500 mg or 2 x 300 mg kanamycin base/kg body weight/day x 14 days induced threshold shifts of approximately 50 dB at 20 kHz. These were accompanied by loss of outer hair cells. The order of susceptibility, BALB>CBA>C57, was not due to differences in the pharmacokinetics of kanamycin. It also did not correlate with the presence of Ahl/Ahl2 genes which predispose C57 and BALB strains, respectively, to accelerated age-related hearing loss. Pigmentation, however, paralleled this rank order suggesting an influence of melanin on cochlear antioxidant status.

Investigation of two mercury vapor collection techniques.

OBJECTIVES: The purpose of this investigation was to develop and test two in vitro mercury vapor collection techniques: a closed bottle technique (CB) and an intraoral flow (IOF) technique. METHODS: Amalgam samples were prepared in acrylic first molars (#30) with standardized Class I preparations. In the CB technique, samples were placed in either a 25, 100 or 500 ml bottle (n = 5). Vapor was analyzed with the Jerome M-411 using a syringe method over a 7 day period. In the IOF technique an impression of the lower right quadrant of a Typodont was taken with PVS impression material leaving a 5 mm space over #30. Samples were analyzed with the Jerome M-411 connected to the impression tray via tygon tubing at the buccal surface. Average mercury vapor release rates and standard deviations were calculated for each method. Data were analyzed by two-way ANOVA followed by Tukey HSD pairwise analysis for significant findings (alpha = 0.05). RESULTS: Both techniques indicated mercury vapor release was dependent on volume. The largest bottle, 500 ml, yielded a significantly greater (p < or = 0.00) amount of mercury vapor within the CB systems. In the IOF technique, the addition of air flow over the restoration demonstrated a significant increase (p < or = 0.05) in mercury vapor released compared to the sealed IOF technique. SIGNIFICANCE: A method for mercury vapor analysis was developed for possible intraoral application. The IOF method with direct air flow removes possible saturation effects found in a CB system, while limiting external variables, which may contribute to errors associated with in vivo measurements.

In vivo release of neuroactive amino acids from the inferior colliculus of the guinea pig using brain microdialysis.

Microdialysis techniques were used to measure in vivo release of neuroactive amino acids from the central nucleus of the inferior colliculus (ICC) in anesthetized guinea pigs. Concentric dialysis probes were implanted in the ICC and perfused with Ringer solution of various compositions at a flow rate of 2.0 microliters/min. Consecutive 10-min fractions of the dialysate were collected for up to 3 h under different experimental conditions, frozen and assayed for amino acid content by high performance liquid chromatography (HPLC). There was an initial high outflow of amino acids which declined to stable baseline levels after 2 h. Following this stabilization period, perfusion with a medium containing 100 mM KCl produced an increase in the extracellular levels of aspartate (Asp), glutamate (Glu), gamma-aminobutyric acid (GABA) and glycine (Gly). Only the increases in GABA and Gly were statistically significant. None of the increases occurred in the presence of 2.0 mM cobalt suggesting the release of amino acids is calcium dependent. Histological examination revealed that tissue damage was minimal and largely confined to the immediate vicinity of the probes. We were also able to show that the blood brain barrier (BBB) appeared to heal 2 h after probe implantation. Thus, following intravenous injection of [3H]alpha-aminoisobutyric acid (AIB), which does not cross the intact BBB, no isotope was recovered in the dialysate. These results demonstrate that microdialysis is a unique and suitable method to monitor changes in the extracellular levels of amino acid neurotransmitters in a central auditory structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of a toxic metabolite from gentamicin by a hepatic cytosolic fraction.

We have demonstrated recently that incubation of the aminoglycoside gentamicin with an hepatic post-mitochondrial fraction produces a compound toxic to sensory cells from the inner ear in short-term culture; in contrast, the parent aminoglycoside was non-toxic in vitro (Huang MY and Schacht J, Biochem Pharmacol 40: R11-R14, 1990). In the present study, we investigated the subcellular distribution of the enzymatic activity and the nature of the metabolite. Isolated outer hair cells from the guinea pig cochlea were used to assay for cytotoxicity. The enzyme(s) responsible for this novel reaction of aminoglycosides was exclusively localized to the cytosolic fraction of guinea pig liver. No activity was detected in nuclear, lysosomal/mitochondrial or microsomal preparations. Furthermore, the toxin-forming enzymatic activity was associated with the high molecular weight fraction of the cytosol and did not require low molecular weight components. Filtration of the toxin through molecular weight cut-off membranes showed a molecular size of approximately 500. This evidence is consistent with the toxin being a gentamicin derivative.

Potassium-induced release of endogenous glutamate and two as yet unidentified substances from the lateral line of Xenopus laevis.

The release of endogenous glutamate and other primary amines from the lateral-line of Xenopus laevis was studied using an in vitro superfusion technique and high performance liquid chromatography. Potassium stimulation (50 mM KCl) applied to 60 or 120 lateral-line organs dissected from the skin and pooled in a perfusion chamber induced the release of glutamate and aspartate. The release of aspartate was smaller than that of glutamate and more variable. A variable release of two, as yet, unidentified substances was also detected. In low calcium (0.1 mM CaCl2), high magnesium (10 mM MgCl2) solution, 50 mM potassium failed to induce an increase in glutamate, aspartate and the two unknowns, suggesting they are released in a transmitter-like manner. The technique presents a new and simple method for studying transmitters in hair-cell systems. Although other interpretations are possible, the results are consistent with the hypothesis that glutamate is a hair-cell transmitter and suggest a potential role for other substances in the transduction process, perhaps as neuromodulators.

Pleiotropic mutants of Chinese hamster cells with altered cytidine 5'-triphosphate synthetase.

Following chemical mutagenesis and multiple-step indirect selection, four clones of Chinese hamster V79 cells were isolated which exhibited auxotrophy for thymidine, deoxycytidine, or deoxyuridine but not for cytidine or uridine. All were resistant to uridine, 3-deazauridine, 5-fluorouridine, thymidine, and cytosine arabinoside at concentrations that were toxic to wild-type V79 cells. The cytidine 5'-triphosphate (CTP) and deoxycytidine 5'-triphosphate (dCTP) pools in the mutants were expanded, but the uridine 5'-triphosphate (UTP) pool either decreased or remained unchanged relative to the wild-type level. Furthermore, since the parental cells appear to be deficient in dCMP deaminase activity and CTP (or one of its metabolites) has been shown to inhibit uridine 5'-diphosphate (UDP) reduction, an elevated CTP level should lead to the observed thymidine auxotrophy. It also explains the joint resistance of mutant clones to thymidine and cytosine arabinoside. The change in the ratio of intracellular dCTP to thymidine 5'-triphosphate (dTTP) may be responsible for the elevation in the rates of spontaneous mutations in these mutants.

The kinetics and feedback inhibition of cytidine 5'-triphosphate synthetase in wild-type and mutant Chinese hamster cells.

The kinetics and cytidine 5'-triphosphate (CTP) feedback inhibition of CTP synthetase in wild-type and four mutants of Chinese hamster V79 cells have been studied. The enzymes of the wild type and three of the four mutants exhibited positive cooperativity with the substrate uridine 5'-triphosphate (UTP). Three of the mutants had Kmapp and S50 values distinctly greater than those of the wild type, while the fourth mutant had values similar to those of the wild type. All four mutants exhibited resistance to CTP feedback inhibition, while the wild type was sensitive to such inhibition. It is postulated that a single mutational event in each mutant had caused a concomitant change of the enzyme in its binding both to the substrate UTP and to the end-product CTP.

Apparent synergism between amino donors for CTP synthesis in Chinese hamster fibroblasts.

The synergistic effects of potential amino donors were studied in the assay of CTP synthetase in extracts of Chinese hamster fibroblasts. We found that L-glutamine was not effective as the sole amino donor, but combinations of L-glutamine with NH4HCO3, L-arginine or potassium phosphate did result in the conversion of UTP to CTP. L-arginine or potassium phosphate were also not effective when used alone, and NH4HCO3 was only slightly effective. Our studies demonstrate that the individual synergistic combinations were not additive; multiple combinations of components decreased rather than increased the formation of CTP. The synergistic combinations of L-glutamine with either NH4HCO3 or L-arginine had an absolute requirement for ATP; when ATP and PEP were absent no conversion of UTP to CTP occurred. The presence of GTP in a reaction mixture slightly increased the formation of CTP when L-glutamine and NH4HCO3 were used and substantially increased CTP formation when L-glutamine and L-arginine were used. De novo CTP synthesis was greatly reduced when nonradioactive CTP was added to an assay mixture, suggesting feedback inhibition. A TLC procedure has been developed that allows for the direct separation of UTP and CTP without requiring prior conversion to the mononucleotide or nucleoside level.

Functional and metabolic studies of platelets from patients with Lesch-Nyhan syndrome.

Platelet function was investigated in three patients with the Lesch-Nyhan syndrome. Platelet count, morphology and size distribution was normal in all patients. Platelet turnover was normal. Electron microscopy did not reveal any ultrastructural abnormality. Template bleeding times were normal and prolonged after aspirin ingestion in two out of the three patients: the patient that failed to respond to the aspirin challenge also had decreased retention of platelets on a glass bead column. Biochemical studies revealed that total platelet ATP was reduced by 34% in the presence of a normal level of ADP in the storage pool. These platelets failed to incorporate radioactive hypoxanthine but did incorporate radioactive adenine to produce adenine nucleotides and a trace amount of guanine nucleotides. The results indicate that normal platelets have a functionally intact pathway for utilizing hypoxanthine as a source of preformed purine, and that the failure to salvage this purine, as in the Lesch-Nyhan syndrome, results in a decreased level of total platelet ATP. These findings suggest that platelets can function normally despite a one third reduction in total ATP content.

Incorporation of hypoxanthine into adenine and guanine nucleotides by human platelets.

1. Incubation (1-4 h) of normal human washed platelets (5-11-10-8 per ml) with [8-14C] hypoxanthine at a concentration of 10-5 M resulted in a linear incorporation of radioactivity into adenine and guanine nucleotides. 2. Washed platelets from patients with Lesch-Nyhan syndrome, deficient in hypoxanthine: guanine phosphoribosyltransferase, failed to demonstrate any significant incorporation of [8-14C] hypoxanthine but did incorporate [8-14C] adenine like normal platelets under the same incubation condition. 3. These findings are taken to indicate that normal platelets have the enzymes necessary for salvage of hypoxanthine and that hypoxanthine: guanine phosphoribosyltransferase is the obligatory first step in this pathway.