Douglas W. Hoffman

Potentiation of ototoxicity by glutathione depletion.

The combination of 10 mg/kg ethacrynic acid (ETA) and 100 mg/kg kanamycin (KA) caused neither morphologic damage to the cochlea nor change in the auditory brain stem response of the chinchilla. However, after pretreatment with a single dose of buthionine sulfoximine (BSO; 800 mg/kg intraperitoneally) to reduce intracellular glutathione (γ-glutamylcysteinylglycine; GSH) levels, the above single administration of ETA and KA resulted in complete deafness and severe morphologic damage. The kidney, which has a rapid GSH turnover and is therefore especially susceptible to GSH depletion by BSO, also demonstrated severe damage after this treatment. A similar rapid turnover of GSH and resulting limited capacity to detoxify reactive metabolites and free radicals may determine cochlear and renal vulnerability to this toxicity. These findings may explain the clinical observations of enhanced ototoxicity in patients administered aminoglycoside antibiotics concomitantly with loop diuretics.

Neurotransmitters of the cochlea and cochlear nucleus: Immunocytochemical evidence

Many neurotransmitter candidates have been identified in the cochlea and cochlear nucleus with the use of immunocytochemical techniques. Choline acetyltransferase immunoreactivity suggests acetylcholine as a transmitter of medial and lateral efferent systems in the cochlea. Immunoreactivities to enkephalins, dynorphins, calcitonin gene-related peptide, and tyrosine hydroxylase (a marker for dopamine) are also found in lateral efferents. Choline acetyltransferase, enkephalin, and dynorphin immunoreactivities are co-contained in neurons of the lateral system. In the anteroventral cochlear nucleus, the inhibitory amino acid transmitters, gamma aminobutyric acid (GABA), and glycine, as well as the presumed excitatory amino acid transmitter of the auditory nerve, have been directly or indirectly localized, immunocytochemically, to discrete populations of terminals on spherical cells with distinct morphologic characteristics.

Therapeutic drug monitoring of risperidone and 9-hydroxyrisperidone in serum with solid-phase extraction and high-performance liquid chromatography

This laboratory developed a simple and efficient solid-phase extraction method that is combined with high-performance liquid chromatography for rapid and precise therapeutic monitoring of risperidone (Risperdal) in blood concentrations. The solid-phase extraction uses a mixed bed column. Sensitivity of the chromatographic method is 0.5 ng/ml (180 pmol/ml) of drug in serum, and separations can be performed in a 15-minute chromatographic run. Advantages of this approach include enhanced speed, sensitivity, and efficiency. A high level of sensitivity may be achieved because of the absence of interference from other drugs, metabolites, or serum components.

Effects of opioid be drugs on auditory evoked potentials suggest a role of lateral olivocochlear dynorphins in auditory function

Multiple gene products of opioid peptide families (e.g., enkephalins, dynorphins) with differing opioid receptor specificities are present within olivocochlear efferent terminals. Enkephalins activate mu- and delta-opioid receptors, and are generally inhibitory in the nervous system, and dynorphins are kappa-receptor agonists, which may be excitatory to postsynaptic neurons. We have examined the effects of intravenously administered opioid agonists and antagonists on click-evoked N1 and N2 amplitudes and latencies of the compound action potential in the chinchilla recorded at the round window. Parenteral administration of the opioid receptor antagonist naloxone or the potent mu-receptor agonist fentanyl did not alter N1 and N2 amplitudes or latencies. The kappa-receptor agonist, mu-receptor antagonist pentazocine caused marked increases in N1 and N2 amplitudes over baseline values at threshold intensities. These effects were not abolished by naloxone. No effects were seen on the cochlear microphonic, supporting a site of action of these effects at the lateral olivocochlear efferent terminals on auditory nerve dendrites under inner hair cells. Similar results were obtained when far field auditory evoked responses were recorded. Results were obtained under ketamine/pentobarbital anesthesia, which provided stable recording baselines in contrast to tiletamine/zolezepam/pentobarbital, with which an upward drift in auditory potentials was observed. This stimulatory action of kappa-agonists on auditory-evoked potential amplitudes appears to represent a physiological role of the lateral olivocochlear efferent innervation. The different neurotransmitters of the olivocochlear efferents (e.g. enkephalins, dynorphins, acetylcholine) may have antagonistic actions on auditory potentials, as may the lateral and medial systems themselves.

Several distinct receptor binding enkephalins in olivocochlear fibers and terminals in the organ of corti

Biochemical studies centering on the use of reverse-phase high-performance liquid chromatography (HPLC) and radioimmunoassays (RIA) demonstrate the presence in the guinea pig organ of Corti of at least 3 enkephalin-related peptides, two of which are identified as Met- and Leu-enkephalin, respectively. Enkephalins were identified and quantitated by HPLC-RIA in the isolated second turn of the organ of Corti, but were not found in stria vascularis or auditory nerve dissected from the cochlea. Three enkephalin-immunoreactive HPLC fractions inhibited the binding of labeled naloxone to rat brain membranes. All enkephalins identified by the combined HPLC-RIA procedure had an apparent molecular weight similar to that of Met- and leu-enkephalin peptide standards. Immunocytochemistry, performed with the best-characterized Met-enkephalin antiserum used in the RIAs, localized the enkephalin-like immunoreactivity to lateral efferent fibers and terminals under inner hair cells of the organ of Corti. Other antisera raised against Met-enkephalin, not used for RIA, visualized enkephalin-like immunoreactivity in medial efferent fibers under outer hair cells as well. This enkephalin-like immunoreactivity may reflect the presence in the medial efferent system of other structurally similar peptides in addition to those detected biochemically. Efferent fiber lesion, by evulsion of the vestibular nerve close to the vestibulocochlear anastomosis in which the olivocochlear fibers run, eliminated enkephalin-like immunoreactivity and the enkephalin-related peptides identified by HPLC-RIA.

Electrochemical detection for high-performance liquid chromatography of ketoconazole in plasma and saliva

Ketoconazole, cis-1-acetyl-4-[4[[2-(2,4-dichlorophenyl)-2-(1H-imidazol- 1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine, a clinically used antifungal agent, is also an inhibitor of steroid hormone biosynthesis. A high-performance liquid chromatographic method is described which resolves ketoconazole with selectivity and high sensitivity provided by the use of electrochemical detection. Ketoconazole can be detected in high-performance liquid chromatography by electrochemical oxidation at a glassy carbon electrode at a potential of +1.0 V. Electrochemical detection offers improved sensitivity and selectivity over ultraviolet absorbance or fluorescence detection after derivatization. The method utilizes a volatile buffer system compatible with postcolumn analyses and an internal standard which is electrochemically active. This technique provides a simple method to assay ketoconazole. Ketoconazole can be detected in human plasma and saliva after a single oral therapeutic dose.

Inhibition of glutathione-related enzymes and cytotoxicity of ethacrynic acid and cyclosporine

Glutathione (GSH) is an endogenous thiol that detoxifies active oxygen and reactive species formed during intermediary metabolism and drug detoxification. Compounds with a range of potential toxicities were tested for their abilities to affect GSH reductase and GSH S-transferase activities, which are each components of the two principal detoxification pathways in which GSH participates. A high performance liquid chromatographic method for determining oxidized and reduced GSH was modified to assay GSH reductase activity. With this method it was possible to demonstrate that ethacrynic acid, which inhibits GSH S-transferase, also inhibits the activity of GSH reductase. Inhibition of GSH reductase by ethacrynic acid was similar to that seen with carmustine (BCNU). GSH reductase activity was not affected by cis- or transplatin, buthionine sulfoximine, other loop diuretics, cyclosporine A or aminoglycosides. Cyclosporine inhibited GSH S-transferase at 50 microM and higher concentrations. These results support a role for GSH-mediated detoxification mechanisms in ethacrynic acid- and cyclosporine-associated cytotoxicity, which may mediate their toxicities and their potential as adjunctive agents in antineoplastic therapy. A better understanding of the mechanism of their toxicity can greatly extend the clinical usefulness of these agents, as this toxicity is the basis of both their therapeutic and antitherapeutic actions.

Comparison of cyclosporine and FK506 effects on glutathione levels in rat cochlea, brain, liver and kidney

Cyclosporine treatment (50 mg/kg/day, p.o.) caused increases in rat renal reduced glutathione (GSH) levels of 205 and 673%, respectively, after 5 and 10 days. No changes were seen in liver GSH with either dose of cyclosporine. FK506 (2.5 mg/kg/day, p.o., for 7 days) caused an approximately 200% increase in kidney GSH, and an approximately 250% increase in hepatic GSH levels. Oxidized glutathione (GSSG) was never more than 1-2% of the level of the reduced form in any tissue from control animals. Small increases in the ratios of oxidized to reduced glutathione were seen in livers and kidneys from both cyclosporine- and FK506-treated animals. No changes in GSH or GSSG levels were seen in brains or cochleas from any animal.

Age dependent changes in choline uptake of the chick iris

The kinetics of the uptake of choline, the rate-limiting substrate in the in vivo synthesis of acetylcholine, were studied during the period 1--7 years in the iris of the chick. These changes were correlated to the endogenous levels of acetylcholine and choline in the same organ. Vmax values per iris decrease significantly at 5 years and continue to decrease at 7 years, to 64% and 37% of the one year value, respectively. If the variation in Vmax is calculaed per protein the decline is 34% between 1 and 5 years. Km does not change significantly during the period 1--7 years. Total acetylcholine and choline levels follow a similar trend, decreasing progressively from 1 to 7 years. Sensitivity to hemicholinium (5.5 X 10(-5) M) decreases significantly between 5 days of incubation (d.i.) and 5 years. Inhibition by ouabain (10(-4) M) shows an opposite trend, increasing significantly from 20% at 5 d.i. to 52% at 3 months. At 5 years sensitivity to ouabain is the same as at 3 months. Nardependence decreases significantly between 5 d.i. and 5 years but no significant changes are seen between 5 d.i. and 3 months. Uptake at 27 degrees C decreases from 59% of control at 5 d.i. to 45% at 3 months, and is not changed at 5 years. Our observations suggest that the effect of aging on peripheral cholinergic neurons is not generalized, but is specifically directed toward the neuronal periphery (terminals) as opposed to cell bodies.

Localization of preproenkephalin mRNA-expressing cells in rat auditory brainstem with in situ hybridization

Hair cells and auditory nerve dendrites in the inner ear are innervated by pontine neurons that have been demonstrated by immunochemical techniques to contain several neurotransmitters, including acetylcholine and the opioid peptide enkephalins and dynorphins. The functions of these nerve fibers are not known, but may involve modifying auditory sensitivity to low intensity stimuli. In the guinea pig the opioid pathways originate in the lateral superior olivary region. A recent study in the gerbil has reported cells expressing preproenkephalin mRNA present only in the ventral nucleus of the trapezoid body, and not in the superior olivary region. In the present study, a non-radioisotopically labeled in situ hybridization method was used to identify cells expressing mRNA coding for preproenkephalin in rat pontine neurons, specifically in the ventral nucleus of the trapezoid body. These cells may represent an enkephalin-containing medial olivocochlear system in the rat, the origin of the lateral system in the rat that differs markedly from the better-studied guinea pig and cat, or a non-olivocochlear enkephalin-containing system.