Stress responses and biological residues of sulfanilamide antibiotics in Arabidopsis thaliana.

Sulfonamides (SAs) are antibiotics widely used in clinical practice, livestock and poultry production, and the aquaculture industry. The compounds enter the soil environment largely through livestock and poultry manure application to farmland. SAs not only affect plant growth, but also pose a potential threat to human health through SA residues in plant tissues. In particular, sulfamethoxazole (SMZ) has been classified as a Category 3 carcinogen by the World Health Organization, and thus its soil ecological toxicity and possible health risks are of concern. Using A. thaliana as a model plant, stress responses and biological residues of sulfadiazine (SD), sulfametoxydiazine (SMD), and SMZ were investigated in the present study. Root length and aboveground plant biomass were significantly inhibited by the three types of SA, whereas lateral roots exposed to SMD grew vigorously. The contents of chlorophyll a and chlorophyll b and photosystem II maximum photochemical quantum yield declined with increase in drug concentration, which indicated that exposure to SAs affected photosynthesis and inhibited chlorophyll synthesis in A. thaliana. With increase in drug concentration, reactive oxygen species (ROS) accumulation in the leaves increased significantly. Activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were activated at low SA concentrations, but increased lipid peroxidation occurred with increase in SA concentration. Of the three compounds, SMZ was the most toxic to A. thaliana, followed by SD, and SMD was the least toxic. The results indicated that the risk of SMD entering an organism through the food chain is greater than that for SMZ and SD.

Direct photo transformation of tetracycline and sulfanomide group antibiotics in surface water: Kinetics, toxicity and site modeling.

The direct photo-transformation of widely used antibiotics, including Tetracycline (TTC), chlortetracycline (CTC), sulfamethoxazole (SMX) and sulfamethazine (SMZ) were quantified for surface water by using artificial UV irradiation. The photolysis rate is directly proportional to the overlap between the absorption spectrum of the solution and the spectrum of the terrestrial sunlight. Increasing overlap fraction of Tetracycline (TC) group than Sulfanomide (Sulfa) group, the transformation of TC group is certified much faster than the sulfa group. The speciation of TC and Sulfa group antibiotics are pH-dependent and consequently influence its light adsorption spectrum. And the toxicity of the four target antibiotics along the photo-transformation was assessed. In field aquatic environment, a temporal- and spatial half-life model described the behavior of the antibiotics in water column of victoria harbour could be validated by using experimentally obtained quantum yield with the target field meteorological data. The modeling results indicated the photolysis rate of different kind of antibiotics varied differently along season, daily time and water depth. Summer, midday and surface layer of water body would be the time- and space-highlight spot in which the phototransformation are the dominant process for antibiotics concentration depletion. Seasonal variety would be enhanced for sulfa-group kind antibiotics, which having only tail overlapped with irradiation spectrum. Daily averaged half-lives of TC group were relatively stable, while the sulfa group antibiotics were found to vary from about 300 to 750 h, dependent on the seasonal change.

Design and Comparative Evaluation of the Anticonvulsant Profile, Carbonic-Anhydrate Inhibition and Teratogenicity of Novel Carbamate Derivatives of Branched Aliphatic Carboxylic Acids with 4-Aminobenzensulfonamide.

Epilepsy is one of the most common neurological diseases, with between 34 and 76 per 100,000 people developing epilepsy annually. Epilepsy therapy for the past 100+ years is based on the use of antiepileptic drugs (AEDs). Despite the availability of more than twenty old and new AEDs, approximately 30% of patients with epilepsy are not seizure-free with the existing medications. In addition, the clinical use of the existing AEDs is restricted by their side-effects, including the teratogenicity associated with valproic acid that restricts its use in women of child-bearing age. Thus, there is an unmet clinical need to develop new, effective AEDs. In the present study, a novel class of carbamates incorporating phenethyl or branched aliphatic chains with 6-9 carbons in their side-chain, and 4-benzenesulfonamide-carbamate moieties were synthesized and evaluated for their anticonvulsant activity, teratogenicity and carbonic anhydrase (CA) inhibition. Three of the ten newly synthesized carbamates showed anticonvulsant activity in the maximal-electroshock (MES) and 6 Hz tests in rodents. In mice, 3-methyl-2-propylpentyl(4-sulfamoylphenyl)carbamate(1), 3-methyl-pentan-2-yl-(4-sulfamoylphenyl)carbamate (9) and 3-methylpentyl, (4-sulfamoylphenyl)carbamate (10) had ED50 values of 136, 31 and 14 mg/kg (MES) and 74, 53, and 80 mg/kg (6 Hz), respectively. Compound (10) had rat-MES-ED50 = 13 mg/kg and ED50 of 59 mg/kg at the mouse-corneal-kindling test. These potent carbamates (1,9,10) induced neural tube defects only at doses markedly exceeding their anticonvuslnat-ED50 values. None of these compounds were potent inhibitors of CA IV, but inhibited CA isoforms I, II and VII. The anticonvulsant properties of these compounds and particularly compound 10 make them potential candidates for further evaluation and development as new AEDs.

Teratology study of amide derivatives of branched aliphatic carboxylic acids with 4-aminobenzensulfonamide in NMRI mice.

BACKGROUND: Valproic acid (VPA), widely used to treat epilepsy, bipolar disorders, and migraine prophylaxis, is known to cause neural tube and skeletal defects in humans and animals. Aminobenzensulfonamide derivatives of VPA with branched aliphatic carboxylic acids, namely 2-methyl-N-(4-sulfamoyl-phenyl)-pentanamide (MSP), 2-ethyl-N-(4-sulfamoyl-phenyl)-butyramide (ESB), 2-ethyl-4-methyl-N-(4-sulfamoyl-phenyl)-pentanamide (EMSP), and 2-ethyl-N-(4-sulfamoyl-benzyl)-butyramide (ESBB), have shown more potent anticonvulsant activity than VPA in preclinical testing. Here, we investigated the teratogenic effects of these analogous compounds of VPA in NMRI mice. METHODS: Pregnant NMRI mice were given a single subcutaneous injection of either VPA at 1.8 or 3.6 mmol/kg, or MSP, ESB, EMSP, or ESBB at 1.8, 3.6, or 4.8 mmol/kg on gestation day (GD) 8. Cesarean section was performed on GD 18, and the live fetuses were examined for external and skeletal malformations. RESULTS: Compared with VPA, which induced neural tube defects (NTDs) in fetuses at 1.8 and 3.6 mmol/kg, the analog derivatives induced no NTDs at dose levels up to 4.8 mmol/kg (except for a single case of exencephaly at 4.8 mmol/kg MSP). Skeletal examination showed several abnormalities mainly at the axial skeletal level with VPA at 1.8 mmol/kg. Fused vertebrae and/or fused ribs were also observed with MSP, ESB, EMSP, and ESBB, they were less severe and seen at a lower incidence that those induced by VPA at the same dose level. CONCLUSIONS: In addition to exerting more potent preclinical antiepileptic activity, teratology comparison indicates that aminobenzensulfonamide analogs are generally more weakly teratogenic than VPA.

An evaluation of the chick cardiomyocyte micromass system for identification of teratogens in a blind trial.

The chick micromass culture system has advantages over the validated rat system - ready availability and non-culling of the donor parent - but needs to give comparable results. This study confirmed comparability and the ability to extend the system to cover cardiac effects. It was also compared with the validated embryonic stem cell cardiomyocyte model. A teratogen and paired non-teratogen with known in vivo effects were used. Differential effects were measured via changes in cell protein content, cell viability (resazurin reduction and neutral red uptake), and cell contractility. Results showed that teratogens [L-ethionine, 5-fluorouracil and sulphisoxazole] could be distinguished from non-teratogens [DL-methionine, 6-methyluracil and sulphanilamide respectively]. Dichloroacetone and dichloropropanol affected embryonic stem cells but not the micromass; dichloropropanol had a greater effect than dichloroacetone. This approach revealed differential effects on contractility independent of effects on activity/viability, whilst the total cell protein remained unchanged. We suggest that pre-validation of this system should be examined.

Oryzalin bodies: in addition to its anti-microtubule properties, the dinitroaniline herbicide oryzalin causes nodulation of the endoplasmic reticulum.

Oryzalin is a much-used pre-emergence herbicide which causes microtubules (Mt) to depolymerize. Here, we document that this dinitroaniline herbicide also leads to characteristic changes in the morphology of the endoplasmic reticulum (ER) and Golgi apparatus. These effects, which are reversible upon washing out the herbicide, are already elicited at low concentrations (2 microM) and become most pronounced at 20 microM. For our studies, we have employed roots of Arabidopsis thaliana, tobacco leaf epidermal cells, and BY-2 suspension cultures, all expressing the luminal ER marker GFP::HDEL. In all cell types, the typical cortical network of the ER assumed a pronounced nodulated morphology with increasing oryzalin concentrations. This effect was enhanced through subsequent application of brefeldin A (BFA). Thin sections of Arabidopsis roots observed in the electron microscope revealed the nodules to consist of a mass of anastomosing ER tubules. Oryzalin also caused the cisternae in Golgi stacks to increase in number but reduced their diameter. Oryzalin retarded ER mobility but did not prevent latrunculin B-induced clustering of Golgi stacks on islands of cisternal ER. While the mechanism underlying these changes in endomembranes remains unknown, it is specific for oryzalin since these effects were not elicited with other Mt-depolymerizing herbicides, e.g., trifluralin, amiprophosmethyl, or colchicine.

Postanoxic damage of microglial cells is mediated by xanthine oxidase and cyclooxygenase.

Brain ischemia and the following reperfusion are important causes for brain damage and leading causes of brain morbidity and human mortality. Numerous observations exist describing the neuronal damage during ischemia/reperfusion, but the outcome of such conditions towards glial cells still remains to be elucidated. Microglia are resident macrophages in the brain. In this study, we investigated the anoxia/reoxygenation caused damage to a microglial cell line via determination of energy metabolism, free radical production by dichlorofluorescein fluorescence and nitric oxide production by Griess reagent. Consequences of oxidant production were determined by measurements of protein oxidation and lipid peroxidation, as well. By using site-specific antioxidants and inhibitors of various oxidant-producing pathways, we identified major sources of free radical production in the postanoxic microglial cells. The protective influences of these compounds were tested by measurements of cell viability and apoptosis. Although, numerous free radical generating systems may contribute to the postanoxic microglial cell damage, the xanthine oxidase- and the cyclooxygenase-mediated oxidant production seems to be of major importance.

Surflan and oryzalin impair reproduction in the teleost medaka (Oryzias latipes).

We conducted studies to determine if the xenoestrogens Surflan and its active ingredient oryzalin, affect indices of reproductive fitness in medaka (Oryzias latipes). Oryzalin (0.5, 0.25mg/l) or Surflan (2.0 microl/l) and oryzalin (0.5mg/l) significantly increased the mean number of non-fertilized eggs produced by treated females paired with untreated males, or by untreated females paired with treated males. Oryzalin (1.0, 0.5, 0.25mg/l) and Surflan (3.8, 2.0, 1.0 microl/l) significantly affected the time to hatch of eggs from treated females paired with untreated males, and from untreated females paired with treated males. Surflan (3.8, 2.0, 1.0 microl/l) induced intersex lesions in 80-100% of males. Oryzalin-exposed males exhibited a significant increase in the incidence of necrotic spermatids and necrotic spermatogonia, while oryzalin-exposed females had significantly fewer immature oocytes and an increase in the occurrence of hyperplastic ovaries. Our results indicate that Surflan and oryzalin affect both reproduction and gonadal histology in male and female medaka.

Toxicity of herbicides in highway runoff.

Previous field monitoring at two highway sites found highway-applied herbicides in storm water runoff at maximum concentrations ranging from 10 microg/L for glyphosate and diuron to as high as 200 microg/L for oryzalin. To determine whether these herbicides at these concentrations can cause any toxicity to aquatic organisms, a standard toxicity study was conducted. Storm water was collected along Highway 37, Sonoma County, California, USA, and the herbicides isoxaben, oryzalin, diuron, clopyralid, and glyphosate were spiked into the storm water at the highest concentrations observed during the five previous field-monitoring campaigns. Three different toxicity studies were conducted and the results showed the following: No significant reduction in reproduction or increase in mortality relative to the control for an 8-d Ceriodaphnia (water flea) toxicity test; no significant increase in mortality or decrease in biomass compared to the control during a 7-d Pimephales (fish) toxicity test; and, in a 96-h Selenastrum (algae) toxicity test, both the 10-microg/L diuron treatment and the combined 50-microg/L isoxaben plus 200-microg/L oryzalin treatment produced significant (p < 0.05) reductions in algal growth compared to the controls, although the 30-microg/L clopyralid or 10-microg/L glyphosate treatments did not exhibit any toxic effects.

Identification of the herbicide Surflan and its active ingredient oryzalin, a dinitrosulfonamide, as xenoestrogens.

Numerous environmental contaminants have been identified as endocrine disruptors (EDs)--substances that alter endocrine homeostasis by interfering with the biological action, production, or pharmacokinetics of endogenous hormones. Xenoestrogens are those EDs whose biological activity is similar to endogenous estrogen. This report presents data that identified Surflan, a proprietary herbicide emulsion, and its active ingredient oryzalin as xenoestrogens. In vitro, Surflan and oryzalin activated an estrogen-inducible reporter gene, and oryzalin competitively displaced 17beta-estradiol from the estrogen receptor. In vivo, Surflan and oryzalin induced expression of estrogen-regulated high-molecular-weight choriogenin genes in medaka (Oryzias latipes). These results are consistent with the characteristics of previously identified xenoestrogens and indicate that Surflan and oryzalin have the potential to adversely affect numerous estrogen-regulated biological processes.

Evaluation of antimicrobial agents for veterinary use in the ecotoxicity test using microalgae.

The influence of antimicrobial agents approved as veterinary drugs in Japan on the growth of green algae, Selenastrum capricornutum and Chlorella vulgaris, was studied in accordance with the OECD guidelines for testing chemicals. Among the agents tested, growth inhibitory activity was very varied, i.e. erythromycin showed the strongest activity (EC50, 50% effective concentration, = 0.037 mg/l), sulfa drugs had activity to some extent (EC50s of sulfamethoxazole, sulfadiazine, and sulfadimethoxine were 1.5, 2.2, and 2.3 mg/l, respectively), but ampicillin and cefazolin did not inhibit growth (EC50s>1000 mg/l). We also investigated synergistic effect of combining sulfa drugs with trimethoprim or pyrimethamine, which are commonly used as a combined drug. By adding trimethoprim, the growth inhibitory activity of sulfamethoxazole and sulfadiazine was significantly enhanced. Growth inhibition by sulfa drugs was reduced by the addition of folic acid, indicating that they inhibit folate synthesis in green algae.

Role of endotoxin in 6-sulfanilamidoindazole(6SAI)-induced arthritis in rats.

6-Sulfanilamidoindazole (6SAI) induces selflimiting arthritis in rats. Since close relationships exist between arthritis and endotoxin, four experiments were conducted to clarify the relationship between endotoxin and 6SAI-induced arthritis. Endotoxin levels in the plasma from the abdominal aorta and portal vein from rats that had 6SAI (500 mg/kg) administered orally for up to 7 days remained within the control values at day 1 and day 3, and were significantly elevated at day 7. Endotoxin levels in the synovial fluid from the same rats showed no significant change. Ankle swelling and redness in rats treated 11 consecutive days with 6SAI did not ameliorate when coadministered with an anti-endotoxin agent, polymyxin B sulfate. Histopathological examination on the ankles of rats treated orally with non-arthiritogenic sulfonamides including sulfonamide, sulfamethoxazole and sulfadimethoxin (250 and 500 mg/kg/day, each compound) for 2 weeks demonstrated no inflammatory changes, while hyperplasia/hypertrophy of thyroid epithelial cells were frequently observed. When histopathological changes in the ankles from rats coadministered with 6SAI and lipopolysaccharide (LPS, Escherihia coli O55:B5, 50 microg/kg, i.v.) were compared with those in rats treated with 6SAI or LPS alone, the ankles from the 6SAI+LPS treated animals had marked edematous inflammation in the synovium and surrounding connective tissues, whereas the LPS-group had only mild focal infiltration of polymorphonuclear leukocytes in the synovium and the 6SAI-group showed no apparent changes. These results suggest that endotoxin is not a direct cause but a possible acceralating factor of 6SAI-induced arthritis, and that the effects of 6SAI on gut bacteria is not related with the pathogenesis of this model.

Systemic histopathology of rats treated with 6-sulfanilamidoindazole, a novel arthritogenic sulfonamide.

6-Sulfanilamidoindazole (6SAI) is a sulfonamide that induces acute, self-limiting arthritis in rats, and 6SAI-induced arthritis is thought to be a model for testing anti-inflammatory agents. In this study, in order to clarify the location of arthritis and relationships between arthritis and other changes in this model, we have investigated the detailed pathologic changes in rats administered orally with 6SAI (125, 250, 500 mg/kg) daily for 4 wk in a time-course experiment. Moderate to severe arthritis was observed in rats of middle- and high-dose groups. Histologically, in the affected ankle, exudative synovitis and periarthritis were observed at 1 wk, granulation tissue formation with angiogenesis and periosteal new bone formation at 2 wk, and marked fibrosis of affected area at 4 wk, respectively. In addition to these changes, in periarticular and periosteal tissues of affected ankles, subendothelial insudation of small-sized arteries and medial fibrinoid degeneration of medium-sized arteries were observed at 1 and 2 wk and intimal thickening and medial hypertrophy at 4 wk, respectively. No arterial changes were observed in the unaffected ankles. Similar arterial changes were often observed in the liver, thyroid glands, and lungs and rarely in various organs and tissues. Acute inflammation of serous tissues such as mesentery, mediastinum, and capsule of spleen or thymus were also present in 6SAI-treated groups, and it was sometimes accompanied by arteritis. In addition, in 6SAI-treated rats, follicular hyperplasia of thyroid glands and pituitary changes, which are thought to be related to depression of thyroid hormone production by 6SAI, were observed. These results show that 6SAI induces not only arthritis but also arteritis, serositis, and thyroid change, and it is necessary to take the interaction between these changes into consideration when anti-inflammatory agents are tested in this model.

p-Aminobenzene sulphonyl morpholine, compound 82/208 a new anticonvulsant agent.

p-Aminobenzene sulphonyl morpholine, compound 82/208, was evaluated for acute toxicity and anticonvulsant action in mice against tonic seizures induced by supramaximal electroshock and pentylene tetrazole and strychnine induced seizures and for its effect on blood pressure and respiration in cat. Diphenyl hydantoin (DPH) was used as reference standard. Compound 82/208 exhibited anticonvulsant activity against electroshock induced seizures and PTZ induced tonic seizures in mice. The compound had several distinct advantages over DPH in experimental evaluation in mice.

Mutagenic activity of newly synthesized sulfa drugs to Salmonella typhimurium.

The mutagenic activity of seven newly synthesized sulfa drugs was studied in Salmonella typhimurium, using forward mutation to 8-azaguanine (8-AG) resistance and reversion mutation assays (Ames test) both in the absence and presence of Aroclor induced rat liver S9. In forward mutation assays, N1-methylsulfanilamide, N4-acetyl-N1-methylsulfanilamide and N4-acetyl-N1-diethylsulfanilamide were mutagenic to S. typhimurium TM677 both in the presence and absence of metabolic activation while N4-acetylsulfanilamide, N1-diethylsulfanilamide and 4-nitro-N-2-pyridinylbenzenesulfonamide [2-(p-nitrobenzenesulfonamido)pyridine] were mutagenic only in the presence of metabolic activation. But 2-(N4-acetylsulfanilamido)pyridine was mutagenic in neither the presence nor the absence of metabolic activation. However, none of the seven compounds had any mutagenic effect on S. typhimurium TA98 or TA100 in the absence or presence of metabolic activation, by the Ames test preincubation method. The relationship between the structure of the compounds and their mutagenic activity is also discussed.

N4-(arylmethylene)-N1-[1-(1,2,5,6-tetrahydropyridyl)]-sulphanilamide derivatives as novel possible hypoglycemic agents.

Two series of sulphanilamide derivatives (Va-i and Vla-i), comprising both azomethine and arylsulphonamidotetrahydropyridine moieties, were especially synthesized to investigate the additive effect of such moieties on the pharmacological activity of the produced sulphonamides as possible therapeutic agents for producing hypoglycemia. Most of the hitherto prepared sulphanilamide derivatives, exhibited marked hypoglycemic activity. Structure-activity relationships were investigated and discussed, not only on the basis of lipophilic (pi) and/or electronic (sigma) characters of the aromatic substituent (R), but also in light of a steric effect in these molecules. The structure of the newly synthesized compounds was inferred from elemental and spectral analysis.

Are newer scientific concepts in regulatory toxicology used timely and appropriately?

Laws regulating toxicology (e.g. toxic thresholds allowed, poison classes or definition of necessary preclinical testing) might improve health and save lives. Scientific facts will always serve as a mandatory base for political decision-making, but there will also be additional influences (perception and acceptance of risks, possible benefits, economic considerations etc.). These latter factors may vary considerably from one society to another. The Delaney clause prohibited the marketing of any product which was found to be carcinogenic in animals. Due to their benefits, exceptions were made for drugs. In other countries, too, other chemicals could be an exception due to a different perception of the risk or different scientific evaluation. Clear cases of major events always trigger changes in legislation. When in 1937 a newly-marketed sulfanilamide elixir led to severe kidney damage and 70 deaths, the FDA quickly endorsed the propositions of the investigation team set up by the American Medical Association: animal testing in two species with histopathologic examination before a marketing authorization could be granted became mandatory. A similarly rapid reaction followed in Europe when it was detected that Thalidomide was responsible for malformations in the offspring of mothers who had taken the drug in early pregnancy. When the effects are more difficult to link to a chemical, there may be time delays in regulatory actions. However, a sophisticated evaluation system was introduced for better monitoring of drug and chemical hazards. Some examples will be given in order to discuss the difficulties of timely and appropriate use of scientific findings.

Induction of reversible urothelial cell hyperplasia in rats by clorsulon, a flukicide with weak carbonic anhydrase inhibitory activity.

Clorsulon, a flukicide registered for use in treating Fasciola hepatica infections in cattle, has induced urinary bladder urothelial cell hyperplasia in rats at oral doses of 30 mg/kg/day or more. Despite previous testing at doses above this threshold, this lesion had not been found in subchronic or chronic toxicity studies in rats. After ruling out the presence of a contaminant as the causative factor in producing this lesion, a study was conducted in which clorsulon increased the pH and altered the electrolyte composition of urine, consistent with its weak carbonic anhydrase inhibitory activity. The acid/base balance of the diet markedly affected the threshold for induction of the urothelial cell hyperplasia: acidification by addition of 5% ammonium chloride to the diet reduced the incidence and severity. In additional studies it was found that the urothelial cell hyperplasia was most pronounced after 1 week of treatment compared with daily exposure for either 5 or 15 wk. The reversibility of the hyperplasia despite continued treatment confirms that the hyperplasia is not a preneoplastic lesion, a conclusion supported by negative studies of carcinogenicity in rodent bioassays of clorsulon and other drugs with carbonic anhydrase inhibitory activity.

[Evaluation of the immunotoxicity of sulfanilamide, anti-inflammatory and antihistaminic preparations]. / K otsenke immunotoksichnosti nekotorykh sul'fanilamidnykh, protivovospalitel'nykh i protivogistaminnykh preparatov.

The effects of 15 widely used drugs on the production of IgE-antibodies and the development of anaphylactic sensitization of rat skin were studied. Some drugs were shown to be able to increasing antibody titres in the blood and prolonging skin sensitization.

Quantitative evaluation of ototoxic side effects of furosemide, piretanide, bumetanide, azosemide and ozolinone in the cat--a new approach to the problem of ototoxicity.

A new method for the quantitative assessment of acute ototoxic side effects of drugs is described. It is suitable for screening purposes. The method is based on the determination of the toxic dose (TD50) which causes a defined hearing loss in 50% of the animals tested. The hearing loss is defined as a complete suppression of the compound action potential (CAP) of the auditory nerve, elicited by clicks 30 dB above threshold. This is approximately equivalent to a clinical hearing loss of 30 dB. The TD50 is used to estimate the therapeutic range. With this approach ototoxic side effects of furosemide, piretanide and bumetanide were compared quantitatively in cats. The TD50 values for CAP suppression were 18.37 mg/kg for furosemide; 4.29 mg/kg for piretanide and 2.21 mg/kg for bumetanide. As equipotent diuretic doses are 2.61 mg/kg for furosemide, 0.26 mg/kg for piretanide and 1.16 mg/kg for bumetanide, it appears that the relative ototoxicity is least for piretanide and highest for bumetanide. Plasma concentrations, determined initially and when recovery of CAP to 50% of control had occurred, indicate that bumetanide may be more slowly eliminated from the cochlear spaces than furosemide and piretanide. In addition azosemide and ozolinone were tested. The TD50 for azosemide was less than 10 mg/kg. With ozolinone where there are two isomers, only the diuretic (-)ozolinone was ototoxic; the TD50 was less than 100 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)