Cytotoxic effect of aminoglycoside antibiotics on the mammalian cell lines.

Aminoglycoside antibiotics have been used for treating serious but also routine infections in veterinary and human medicine for many years. The basic aim of this work is to evaluate the cytotoxicity of dihydrostreptomycin and neomycin in vitro on three cell cultures - BHK-21 (Syrian golden hamster kidney fibroblast), VERO (African green monkey kidney fibroblast) and FEA (feline embryonic fibroblast) cells. The morphological changes were examined by Giemsa staining. Cells were dried and visualized under fluorescence microscope. After the exposure to different experimental doses of dihydrostreptomycin (812.5-20000 µg/mL) and neomycin (1000-20000 µg/mL) during 24 h, the viability of BHK-21, FEA and VERO cell lines were evaluated by MTT assay. Viability of BHK-21 cells significantly (P < 0.001) decreased after treatment with 3500; 5500 and 7500 µg/mL of dihydrostreptomycin and 9000; 10000 and 20000 µg/mL of neomycin. The FEA cell viability decreased significantly (P < 0.001; P < 0.01) at 2500 and 3000 µg/mL dihydrostreptomycin and at 3000 µg/mL of neomycin treatment. Only the highest concentration of dihydrostreptomycin (20000 µg/mL) reduced VERO cell viability significantly (P < 0.01). Based on or results we can assume the effect of different antibiotics in different concentrations on cell lines is various. Detection of antibiotic toxicity to animal cells is very important because of the increasing resistance of bacteria. One of the solutions is drug dose increasing, but only to a certain concentration, since the toxic effect over the therapeutic one will prevail, which we have also shown in this work.

The binding site on cochlear stereocilia for antisera raised against renal Na+ channels is blocked by amiloride and dihydrostreptomycin.

The mechanoelectrical transduction channels on hair cells have been suggested to be operated by tip links that are stretched when the hair bundle is deflected in the direction of the tallest row of stereocilia. Localising these channels is therefore an important test of this hypothesis. The transduction channels are known to be amiloride-sensitive and immunogold labelling with antibodies raised against the amiloride-sensitive epithelial Na+ channel from kidney (alpha NaCh), has suggested that sites with similar characteristics are located in the region where the tips of the shorter stereocilia appear to come into contact with the sides of the adjacent taller stereocilia rather than being associated directly with the tip links. Now, further immunocytochemical experiments have been performed to determine if amiloride and dihydrostreptomycin, both of which can block transduction, can affect this labelling. Immunofluorescent labelling of the stereocilia is obtained when surface preparations of the organ of Corti are fixed and incubated with alpha NaCh followed by an appropriate secondary antibody. This labelling is abolished by trypsinization prior to fixation but retained if the tissue is pretreated with amiloride and then trypsinized in its presence. Because amiloride is known to protect amiloride-binding sites from degradation by trypsin, these results suggest that alpha NaCh is revealing amiloride-binding sites on the stereocilia. Similarly, immunofluorescent labelling of the stereocilia is abolished if cochlear tissue is pretreated with dihydrostreptomycin (DHS) and fixed in its presence prior to incubation with alpha NaCh. Quantitative analysis of colloidal gold labelling using transmission electron microscopy shows that DHS treatment produces a significant reduction in the number of gold particles on stereocilia, especially in the region of contact between them. These results suggest that anti-Na+ recognises a site with characteristics similar to the mechanoelectrical transduction channels.

[The cochlea in short-term culture].

In order to establish organotypic culture of the cochlea, the dissection of the early postnatal mouse cochlea and its morphology in short-term culture were investigated. The viability of hair cells in the culture was assessed using differential interference contrast (DIC) microscopy and double-staining with fluorescein diacetate (FDA) and propidium iodide (PI). The hair cells remained viable and did not show any signs of damage for up to 8-18 hours. When incubated in Hank's solution containing 1 mmol/L dihydrostreptomycin, the hair cells initially deteriorated in 0.5-4.5 hours and progressed to entire degeneration after 10-20 hours. In addition to its utilization in organotypic culture, the cochlea in short-term culture can be used as a valuable model for studying cellular and molecular neurobiology of the inner ears.

Ototoxic antibiotics: a review.

Fifty years of experience with aminoglycoside antibiotics has confirmed their usefulness in many infections with Gram-negative bacteria. The ototoxic action has been investigated clinically in patients and in numerous animal studies. The increased knowledge about molecular structure, pharmacology and pharmacokinetics as well as information of the site of action in the labyrinth has resulted in reduced risks for severe toxic damage in kidneys and in the ear. Nevertheless, a number of cases of severe hearing loss and a few cases of deafness still occur every year. Further studies on modified molecular structure as well as site of action at the molecular level and ways of avoiding toxicity will make this group of antibiotics still more useful.

Ototoxicity in vitro: effects of neomycin, gentamicin, dihydrostreptomycin, amikacin, spectinomycin, neamine, spermine and poly-L-lysine.

The effects that the aminoglycoside-aminocyclitol antibiotics amikacin, dihydrostreptomycin, gentamicin, neomycin, and spectinomycin, the neomycin fragment neamine, and the polybasic compounds spermine and poly-L-lysine, have on outer hair cells in cochlear cultures prepared from the early post-natal mouse have been assessed using both scanning and transmission electron microscopy (SEM and TEM). The antibiotics were used at concentrations ranging from 0.25-1.0 mM, spermine from 10 microM to 3.0 mM, and poly-L-lysine from 0.05-2 microM. Qualitative assessment of apical surface damage allows the antibiotics to be ranked in the following order: neomycin > gentamicin > dihydrostreptomycin > amikacin > neamine > spectinomycin. At a concentration of 1 mM spectinomycin is essentially non-toxic and the effects of neamine are marginal. Poly-L-lysine and spermine also cause surface damage, with poly-L-lysine being substantially more toxic than any of the antibiotics, and spermine ranking, on the basis of SEM observations, between dihydrostreptomycin and amikacin. TEM indicates that although all toxic compounds cause damage to the apical surface of the hair cell, only neomycin, poly-L-lysine and spermine induce the formation of whorls of tightly packed membrane resembling myelin within the apical surface lesions to any great extent. Apical-surface changes induced by dihydrostreptomycin and amikacin are simply large distensions of the cell filled with cytoplasmic organelles of normal appearance. Although the effects of the aminoglycoside antibiotics are largely limited to the apical surface of the cell, poly-L-lysine induces complete necrosis of the cell, and spermine causes a dramatic increase in cytoplasmic electron density and condensation of the nuclear chromatin.

[Mechanical stimulation of isolated outer hair cells as a test system. Inhibition of transduction by streptomycin treatment]. / Mechanische Reizung isolierter äusserer Haarzellen als Testsystem. Hemmung der Transduktion durch Streptomycinbehandlung.

Deflection of the hair bundle of isolated outer hair cells from the guinea pig cochlea can induce a receptor potential. Outer hair cells from the third and fourth turns of the guinea pig cochlea were isolated according to the method described by Zenner et al. Cells were maintained in Hank's balanced salt solution at room temperature. The whole cell potential was measured by the patch-clamp technique with soda glass capillaries (resistance 3-5 M omega) filled with KCl-Ringer solution. After compensation for the junction potential the stable resting potential of 46 cells was -63 mV +/- 5 mV. The hair bundle was deflected sinusoidally, with amplitudes ranging from 6 degrees to a maximum of 31 degrees in the positive direction (i.e., in the direction of the longest stereocilium). The stimulus was a piezo-driven glass capillary with an opening diameter of 5 microns. This mechanical stimulation induced in 33% of all stimulated cells (n = 46) a receptor potential response of 2.1 +/- 1.4 mV (maximally 5.5 mV). Deflection of the hair bundle in the opposite direction led to no change in the membrane potential, i.e. the cells were not hyperpolarized (minimal resolution 0.5 mV). Since the resting potential of the cells was more positive than the potassium equilibrium potential under our experimental conditions, the receptor current was most likely mediated by an influx of Na+ ions into the cell. The receptor potential response could be completely and reversibly blocked by the addition of dihydro-streptomycin.(ABSTRACT TRUNCATED AT 250 WORDS)

Does streptomycin cause an error catastrophe?

We have examined the interpretation that streptomycin kills a bacterial culture by initiating the so-called error catastrophe. In particular, we asked whether the increased translational error rate induced by the antibiotic gives rise to an autocatalytic loss of functional fidelity of the devices responsible for gene expression, which ultimately causes the death of the culture. We have analyzed the performance characteristics of one of these devices, namely the ribosome in streptomycin-treated bacteria. We find that, although the treated ribosomes are constructed from error-containing proteins, they are not significantly different in elongation rate and fidelity from those ribosomes taken from untreated bacteria. We conclude that the bacteriocidal effect of streptomycin is not due to the initiation of an error catastrophe.

The species-specific nature of the ototoxicity of dihydrostreptomycin in the patas monkey.

The remarkable susceptibility of the inner ear of the patas monkey (Erythrocebus patas) to the ototoxic action of dihydrostreptomycin (DHSM) (and streptomycin (SM)) is well established in this paper and affords a rare example of a species-specific reaction to a restricted class of compounds within the aminoglycoside group of antibiotics. In a series of experiments, behavioral and morphological observations together provided the following profile of DHSM ototoxicity in the patas monkey: Sudden onset of hearing loss beginning after 7-9 weeks of treatment; Substantial, though often partial, hearing impairment beginning at the high frequencies and progressing with or without continued treatment to the low frequencies; In the inner ear, a corresponding and selective loss of nerve fibers and of outer hair cells, relative to inner hair cells, beginning in the base of the cochlea and proceeding toward the apex; Continued and progressive loss of hearing for several months after cessation of drug treatment; and Non-auditory effects in some animals on the kidney and vestibular system. Results from control experiments confirmed this special relationship between the patas monkey and DHSM: Other nonhuman primates (macaques and vervet monkeys) were essentially unaffected by DHSM; The patas showed no equivalent sensitivity to other aminoglycosides such as kanamycin or to other forms of ototraumatic insult such as intense noise.

Dihydrostreptomycin toxicity in the Mongolian gerbil, Meriones unguiculatus.

A fatal syndrome of acute toxicity was produced in Mongolian gerbils following the injection of a penicillin-dihydrostreptomycin-procaine combination. The toxicity was determined to be due to the dihydrostreptomycin component. Fifty milligrams of dihydrostreptomycin produced 80-100% mortality in 55-65 g gerbils.

Behavioral ototoxicology.

Methods for the evaluation in experimental animals of toxic substances that produce hearing impairment are described. In the experiments reported here, animals were trained by positive reinforcement operant conditioning procedures so that their hearing could be examined by behavioral means. When normal hearing was established, aminoglycosidic antibiotics (kanamycin and dihydrostreptomycin) were given daily and hearing tests administered in order that the course of hearing loss could be closely followed. Initial loss of sensitivity to the high frequencies always progressed in time to impairment at the low frequencies, and these changes in hearing were correlated with a loss of receptor cells in the inner ear which started in the basal region of the cochlea and advanced toward the apex. Although such behavioral procedures are moderately expensive to instrument and relatively time-consuming to apply, they are shown to yield valid quantitative measures of hearing. Further, they provide for reliable early detection of the toxic process and a measure of behavioral impairment that can be precisely related to the histopathological changes that occur simultaneously in the inner ear and auditory nerve.

The patas monkey as a model for dihydrostreptomycin ototoxicity.

Although the cochlear toxicity of dihydrostreptomycin (DHSM) is well-recognized in man, it has always proved difficult to demonstrate in animals. Hearing thresholds in M. nemestrina monkeys remained essentially unchanged after DHSM 100 mg/kg im daily for 8 months, but E. patas monkeys were severely deafened by DHSM 20 mg/kg for 90 days, a regimen formerly used in treating human tuberculosis. The patas monkey may prove to be the animal model of choice for evaluating aminoglycoside ototoxicity.