Inhibition of Glutathione S-Transferase by Ethacrynic Acid Augments Ischemia-Reperfusion Damage and Apoptosis and Attenuates the Positive Effect of Ischemic Postconditioning in a Bilateral Acute Hindlimb Ischemia Rat Model.

AIMS: We studied the effects of the inhibition of the endogene antioxidant glutathione-S-transferase (GST) by ethacrynic acid (EA) on ischemia-reperfusion (IR) injury and postconditioning (PC) in the lower extremities. We aimed to examine the oxidative stress parameters (OSP), inflammatory response and activation of proapoptotic signaling proteins (PSP) after revascularization surgery. METHODS: Sixty Wistar rats were divided into 6 groups: control, IR, PC, EA-control, IR and administration of EA (IR/EA) and PC and administration of EA (PC/EA). The IR, PC, IR/EA and PC/EA groups underwent 60 min of infrarenal aortic cross-clamping. After that, PC was performed in the PC and PC/EA groups. In 3 of the groups, the animals were treated with EA (EA-control, IR/EA and PC/EA groups) as well. The ischemia was followed by 120 min of reperfusion. Blood samples and biopsy specimens were collected from the quadriceps muscle. Plasma malondialdehyde, reduced glutathione, thiol/sulfhydryl group levels, TNF-α and IL-6 concentrations and superoxide-dismutase enzyme activity were measured. RESULTS: The levels of the OSP and the inflammatory proteins were higher in the EA-administered groups. The ratio of phosphorylated PSP was higher in the EA-administered groups and the protective effect of PC did not develop. CONCLUSIONS: Inhibition of GST by EA augmented the IR damage. GST inhibition was associated with a different activation of the mitogen-activated protein kinases and the PSP, regulating these pathways in the process of apoptosis and PC.

Genotoxic effect of ethacrynic acid and impact of antioxidants.

It is known that ethacrynic acid (EA) decreases the intracellular levels of glutathione. Whether the anticipated oxidative stress affects the structural integrity of DNA is unknown. Therefore, DNA damage was assessed in EA-treated HCT116 cells, and the impact of several antioxidants was also determined. EA caused both concentration-dependent and time-dependent DNA damage that eventually resulted in cell death. Unexpectedly, the DNA damage caused by EA was intensified by either ascorbic acid or trolox. In contrast, EA-induced DNA damage was reduced by N-acetylcysteine and by the iron chelator, deferoxamine. In elucidating the DNA damage, it was determined that EA increased the production of reactive oxygen species, which was inhibited by N-acetylcysteine and deferoxamine but not by ascorbic acid and trolox. Also, EA decreased glutathione levels, which were inhibited by N-acetylcysteine. But, ascorbic acid, trolox, and deferoxamine neither inhibited nor enhanced the capacity of EA to decrease glutathione. Interestingly, the glutathione synthesis inhibitor, buthionine sulfoxime, lowered glutathione to a similar degree as EA, but no noticeable DNA damage was found. Nevertheless, buthionine sulfoxime potentiated the glutathione-lowering effect of EA and intensified the DNA damage caused by EA. Additionally, in examining redox-sensitive stress gene expression, it was found that EA increased HO-1, GADD153, and p21mRNA expression, in association with increased nuclear localization of Nrf-2 and p53 proteins. In contrast to ascorbic acid, trolox, and deferoxamine, N-acetylcysteine suppressed the EA-induced upregulation of GADD153, although not of HO-1. Overall, it is concluded that EA has genotoxic properties that can be amplified by certain antioxidants.

Effect of combined treatment with diuretics and gabapentin on convulsive threshold in mice.

Research data show that diuretics can have anticonvulsant properties. This study examined effects of ethacrynic acid, a loop diuretic, and hydrochlorothiazide, a thiazide-type diuretic, on the anticonvulsant activity of gabapentin, a newer antiepileptic drug, in the maximal electroshock seizure threshold test in mice. Diuretics were administered intraperitoneally (ip.) both acutely (single dose) and chronically (once daily for seven days). Electroconvulsions were produced by an alternating current (50 Hz, 500 V, 0.2 s stimulus duration) delivered via ear-clip electrodes by a generator. Additionally, the influence of combined treatment with the diuretics and gabapentin on motor performance in the chimney test has been assessed. In the current study, ethacrynic acid at the chronic dose of 12.5 mg/kg and the single dose of 100 mg/kg did not affect the anticonvulsant activity of gabapentin. Similarly, hydrochlorothiazide (100 mg/kg), both in acute and chronic experiments, had no effect on the gabapentin action. On the other hand, in the chimney test, the combined treatment with ethacrynic acid (100 mg/kg) and gabapentin (50 mg/kg) significantly impaired motor performance in mice. Based on the current preclinical findings, it can be suggested that the diuretics should not affect the anticonvulsant action of gabapentin in epileptic patients. However, the combination of ethacrynic acid with gabapentin may cause neurotoxicity.

Topical application of the antiapoptotic TAT-FNK protein prevents aminoglycoside-induced ototoxicity.

We previously demonstrated that an artificial protein, TAT-FNK, has antiapoptotic effects against cochlear hair cell (HC) damage caused by ototoxic agents when applied systemically. To examine the feasibility of topical protein therapy for inner ear disorders, we investigated whether gelatin sponge soaked with TAT-FNK and placed on the guinea pig round window membrane (RWM) could deliver the protein to the cochlea and attenuate aminoglycoside (AG)-induced cochlear damage in vivo. First, we found that the immunoreactivity of TAT-myc-FNK was distributed throughout the cochlea. The immunoreactivity was observed from 1-24 h after application. When Tat-FNK was applied 1 h before ototoxic insult (a combination of kanamycin sulfate and ethacrynic acid), auditory brainstem response threshold shifts and the extent of HC death were significantly attenuated. When cochlear organotypic cultures prepared from P5 rats were treated with kanamycin, TAT-FNK significantly reduced the extent of caspase-9 activation and HC death. These findings indicate that TAT-FNK topically applied on the RWM can enter the cochlea by diffusion and effectively prevent AG-induced apoptosis of cochlear HCs by suppressing the mitochondrial caspase-9 pathway.

Inhibitory effects of ethacrynic acid analogues lacking the α,ß-unsaturated carbonyl unit and para-acylated phenols on human cancer cells.

A series of ethacrynic acid analogues, lacking the α,ß-unsaturated carbonyl unit, was synthesized and subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, Hs578Ts(i)8 as well as of human prostate cancer cells, C4-2B. These cell lines provide a good model system to study migration and invasion, since they represent metastatic cancer. Our studies show that ethacrynic acid analogues with methyl substituents at the aromatic ring demonstrate no inhibitory effect on the migration of both cancer cell lines, whereas a precursor in the synthesis of these ethacrynic acid analogues (II-1, a para-acylated m-cresol) is an excellent inhibitor of the migration of both cancer cell lines.

Non-sensory cells in the deafened organ of Corti: approaches for repair.

After moderate cochlear trauma, hair cells degenerate and their places are taken by phalangeal scars formed by differentiated supporting cells. A short time after trauma, these supporting cells can respond to an induced expression of genes which signal hair cell differentiation during normal development and transdifferentiate into new hair cells. However, these non-sensory cells often lose their differentiated features after severe insults or prolonged hearing loss and become a simple, flat epithelium. The flat organ of Corti can serve as a substrate for gene- and stem cell-based therapies. Despite its prevalence, the flat epithelium is not well characterized. Recent data show that cells of the flat epithelium can divide and maintain the structural confluence of the membranous labyrinth. The mitotic potential of these cells should facilitate production of cells for therapies based on recapitulation of development or insertion of stem cells.

Ethacrynic acid. Effects on the cochlear potentials in normal and high blood oxygen.

The effect of ethacrynic acid (EA) at different blood O(2) saturations on cochlear potentials of guinea pigs was investigated. All 18 young healthy guinea pigs received 50 mg/kg/h of EA intravenously and were divided into three groups: first group, normal (90.00+/-6.30-86.17+/-4.83 mm Hg); second group, lower Po(2) (78.00+/-4.74-70.00+/-4.42 mm Hg); and third group, high Po(2) (174.40+/-13.41-179.00+/-26.15 mm Hg). The partial pressure of oxygen (Po(2)), the partial pressure of carbon dioxide (Pco(2)), and the pH of the blood were measured before EA administration and at the end of the experiment (3 h later) by drawing blood samples from the contralateral carotid artery. Cochlear potentials-endocochlear potential (EP), cochlear microphonics (CM), and action potentials (AP)-were recorded by standard methods from the first turn of the cochlea. Experimental data seem to indicate that elevation of the Po(2) to 174-179 mm Hg during relatively high doses of EA treatment prevents the declines in cochlear potentials which were observed in the first and second groups (normal and lower Po(2)), and preserves active ion transport which is responsible for the generation of cochlear potentials. These data suggest a means by which to reduce the ototoxic effect of EA and possibly indicates a method of treatment for hearing loss which developed after the administration of EA.

Depletion of reduced glutathione enhances motor neuron degeneration in vitro and in vivo.

The mechanism of selective and age-dependent motor neuron degeneration in human amyotrophic lateral sclerosis (ALS) has not been defined and the role of glutathione (GSH) in association with motor neuron death remains largely unknown. A motor neuron-like cell culture system and a transgenic mouse model were used to study the effect of cellular GSH alteration on motor neuron cell death. Exposure of NSC34 motor neuron-like cells to ethacrynic acid (EA) or l-buthionine sulfoximine (BSO) dramatically reduced the cellular GSH levels, and was accompanied by increased production of reactive oxygen species (ROS) measured by the dichlorofluorescin (DCF) fluorescent oxidation assay. In addition, GSH depletion enhanced oxidative stress markers, AP-1 transcriptional activation, c-Jun, c-Fos and heme oxygenase-1 (HO-1) expression in NSC34 cells analyzed by a luciferase reporter, Western blotting and quantitative PCR assays respectively. Furthermore, depletion of GSH decreased mitochondrial function, facilitated apoptosis inducing factor (AIF) translocation, cytochrome c release, and caspase 3 activation, and consequently led to motor neuron-like cell apoptosis. In an ALS-like transgenic mouse model overexpressing mutant G93A-Cu, Zn-superoxide dismutase (SOD1) gene, we showed that the reduction of GSH in the spinal cord and motor neuron cells is correlated with AIF translocation, caspase 3 activation, and motor neuron degeneration during ALS-like disease onset and progression. Taken together, the in vitro and in vivo data presented in the current report demonstrated that decreased GSH promotes multiple apoptotic pathways contributing, at least partially, to motor neuron degeneration in ALS.

Cell death after co-administration of cisplatin and ethacrynic acid.

Ethacrynic acid (EA) significantly enhances the ototoxic effects of cisplatin. To gain insights into the mechanisms underlying Cis/EA ototoxicity, cochleas were labeled with several apoptotic markers. Cis/EA treatment caused extensive outer hair cell (OHC) and inner hair cell (IHC) damage; OHC lesions decreased from the base towards apex of the cochlea whereas the IHC lesion was relatively constant (25-60%) along the length of the cochlea. Propidium iodide labeled OHC nuclei appeared relatively normal at 6h post-treatment, were condensed and fragmented at 12h post-treatment and were frequently missing 48 h post-treatment. Initiator caspase 8, associated with membrane death receptors, and TRADD, a protein that recruits caspase 8, were present in OHC at 6h post-treatment. Caspase 8 labeling increased from 6 to 24h, but was largely absent at 48 h post-treatment. Executioner caspase 3 and caspase 6, which lie downstream of caspase 8, were expressed in OHC 12-24h post-treatment. Initiator caspase 9, associated with mitochondrial damage, was only expressed at low levels at 48 h post-treatment. These results suggest that the rapid onset of Cis/EA induced programmed cell death is initiated by membrane death receptors associated with TRADD and caspase 8.

Characterization of hepatic glutathione S-transferases in coho salmon (Oncorhynchus kisutch).

The glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes which protect against chemical injury. In contrast to mammals, GST expression in fish has not been extensively characterized, especially in the context of detoxifying waterborne pollutants. In the Northwestern United States, coho salmon (Oncorhynchus kisutch) are an important species of Pacific salmon with complex life histories that can include exposure to a variety of compounds including GST substrates. In the present study we characterized the expression of coho hepatic GST to better understand the ability of coho to detoxify chemicals of environmental relevance. Western blotting of coho hepatic GST revealed the presence of multiple GST-like proteins of approximately 24-26kDa. Reverse phase HPLC subunit analysis of GSH affinity-purified hepatic GST demonstrated six major and at least two minor potential GST isoforms which were characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI MS-MS) and Fourier transform-ion cyclotron resonance (FT-ICR) MS analyses. The major hepatic coho GST isoforms consisted of a pi and a rho-class GST, whereas GSTs representing the alpha and mu classes constituted minor isoforms. Catalytic studies demonstrated that coho cytosolic GSTs were active towards the prototypical GST substrate 1-chloro-2,4-dinitrobenzene, as well as towards ethacrynic acid and nitrobutyl chloride. However, there was no observable cytosolic GST activity towards the pesticides methyl parathion or atrazine, or products of oxidative stress, such as cumene hydroperoxide and 4-hydroxynonenal. Interestingly, coho hepatic cytosolic fractions had a limited ability to bind bilirubin, reflecting a potential role in the sequestering of metabolic by-products. In summary, coho salmon exhibit a complex hepatic GST isoform expression profile consisting of several GST classes, but may have a limited a capacity to conjugate substrates of toxicological significance such as pesticides and endogenous compounds associated with cellular oxidative stress.

Oxidative stress and Mrp2 internalization.

Oxidative stress in the liver is sometimes accompanied by cholestasis. We have described the internalization of multidrug resistance-associated protein 2/ATP-binding cassette transporter family 2 (Mrp2/Abcc2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress in rat liver. However, the signaling pathway and regulatory molecules have not been investigated. In the present study, we investigated the mechanism of EA-induced Mrp2 internalization using isolated rat hepatocyte couplets (IRCHs). The Mrp2 index, defined as the ratio of Mrp2-positive canalicular membrane staining in IRCHs per number of cell nuclei, was significantly reduced by treatment with EA. This reduction was abolished by a nonspecific protein kinase C (PKC) inhibitor Gö6850, a Ca(2+) chelator, EGTA, but not by a protein kinase A (PKA)-selective inhibitor, a Ca(2+)-dependent conventional PKC (cPKC) inhibitor Gö6976, or a protein kinase G (PKG) inhibitor (1 microM). Moreover, an increase in the intracellular Ca(2+) level and NO release into medium were observed shortly after the EA treatment. Both of these increases, as well as Mrp2 internalization, were completely blocked by EGTA. In conclusion, EA produced a reduction in GSH, Ca(2+) elevation, NO production, and nPKC activation in a sequential manner, finally leading to Mrp2 internalization.

The fate of outer hair cells after acoustic or ototoxic insults.

In epithelial sheets, clearance of dead cells may occur by one of several routes, including extrusion into the lumen, phagocytic clearance by invading lymphocytes, or phagocytosis by neighboring cells. The fate of dead cochlear outer hair cells is unclear. We investigated the fate of the "corpses" of dead outer hair cells in guinea pigs and mice following drug or noise exposure. We examined whole mounts and plastic sections of normal and lesioned organ of Corti for the presence of prestin, a protein unique to outer hair cells. Supporting cells, which are devoid of prestin in the normal ear, contained clumps of prestin in areas of hair cell loss. The data show that cochlear supporting cells surround the corpses and/or debris of degenerated outer hair cells, and suggest that outer hair cell remains are phagocytosed by supporting cells within the epithelium.

Selective toxicity of ethacrynic acid towards lymphocytes of chronic lymphocytic leukemia in vitro.

We have used the MTT colorimetric assay to determine the sensitivity to ethacrynic acid of lymphocytes from normal donors and of peripheral blood cells from leukaemia patients. Whereas normal lymphocytes and cells from acute or chronic myeloid leukaemia showed similar sensitivities (median inhibitory dose, ID50 = 20-22 microM), lymphocytes from chronic lymphocytic leukaemia patients were much more sensitive (ID50 = 6 microM). This result was found irrespective of the assay duration.

Ethacrynic-acid-induced glutathione depletion and oxidative stress in normal and Mrp2-deficient rat liver.

Oxidative stress in the liver is sometimes accompanied by cholestasis. We investigated the localization and role of multidrug-resistance-associated protein (Mrp) 2, a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress. Normal Sprague-Dawley rat (SDR) and Mrp2-deficient Eisai hyperbilirubinemic rat (EHBR) livers were perfused with 500 microM EA. The release of glutamic pyruvic transaminase (GPT) and thiobarbituric-acid-reactive substances (TBARS) from EHBR liver was markedly delayed compared with that from SDR liver. This is mainly due to the higher basal level of glutathione (GSH) in EHBR liver (59.1 +/- 0.3 nmol/mg protein) compared with SDR liver (39.7 +/- 1.5 nmol/mg protein). EA similarly induced a rapid reduction in GSH followed by mitochondrial permeability transition in the isolated mitochondria from both SDR and EHBR. Internalization of Mrp2 was detected before nonspecific disruption of the canalicular membrane and GPT release in SDR liver perfused with 100 microM EA. SDR liver preperfused with hyperosmolar buffer (405 mosmol/L) for 30 min induced internalization of Mrp2 without changing the basal GSH level, while elimination of hepatic GSH by 300 microM EA perfusion was significantly delayed thereafter. Concomitantly, hepatotoxicity assessed by the release of GPT and TBARS was also significantly attenuated under hyperosmolar conditions. In conclusion, preserved cytosolic and intramitochondrial GSH is the key factor involved in the acute hepatotoxicity induced by EA and its susceptibility could be altered by the presence of Mrp2.

Cyclic GMP and cell death in rat cerebellar slices.

Incubated slices of young rat cerebellum were used to examine the possible relationship between the neurotoxic effects of excitatory amino acids and their ability to elicit large increases in the levels of cyclic GMP in this tissue. No cell death was detectable following exposure of the slices to the guanylate cyclase activator, nitroprusside (up to 0.3 mM), the phosphodiesterase inhibitor, isobutylmethylxanthine (0.5 mM), or to cyclic GMP (10 mM) and its dibutyryl and 8-bromo derivatives (0.5 mM). However, incubation of the slices with tbe guanylate cyclase inhibitors, N-methylhydroxylamine and hydroxylamine (0.1-1 mM), methylene blue (10-100 microM), ethacrynic acid (300 microM) and retinol (1 mM) caused a progressive destruction of the differentiating cells. The damage induced by N-methylhydroxylamine and hydroxylamine was inhibited by nitroprusside, cyclic GMP and isobutylmethylxanthine. It could also be reduced by lowering the partial pressure of oxygen, by oxygen radical scavenging enzymes and by omitting Ca2+ from the medium. Oxygen radical generating enzyme systems mimicked the pattern of toxicity of the guanylate cyclase inhibitors but their effects were not reduced by nitroprusside or omission of Ca2+. The results indicate that guanylate cyclase/cyclic GMP does not mediate amino acid neurotoxicity but, instead, may be part of a protective mechanism against oxygen free radicals.

An investigation of the sex difference in the diuretic response to ethacrynic acid in mice.

1. Female mice of the Hough/Porton and Tuck/TO strains were found to be more sensitive than male mice to the diuretic effects of oral and intravenous doses of ethacrynic acid. 2. The sensitivity of Hough/Porton male mice to ethacrynic acid was increased after pretreatment with stilboestrol and the sensitivity of female Hough/Porton mice decreased after pretreatment with testosterone. 3. There were no significant sex differences in the diuretic response to frusemide, acetazolamide, aminophylline, bendrofluazide, and Su 15049A although a small, but significant, increase in the sensitivity of male Tuck/TO mice to triamterene was noted. 4. The sex difference in diuretic response to ethacrynic acid may be related to an effect of sex hormone balance on its metabolism or on the sensitivity of its renal receptor.

Inhibition of soluble glutathione S-transferase by diuretic drugs.

Glutathione transferases are believed to play an important protective role in the various tissues of animals and man by catalysing the glutathione conjugation of electrophilic drugs and electrophilic drug metabolites. Many of these compounds have the potential to react with vital cellular macromolecules in the absence of this enzyme system. We have investigated the interaction of a number of high ceiling diuretics with the glutathione transferases contained in the cytosolic fraction of the rat liver. Of bumetanide, ethacrynic acid, furosemide, indacrynic acid and tienilic acid, only ethacrynic acid was conjugated with glutathione. Further experiments revealed that ethacrynic, indacrynic and tienilic acids are all potent inhibitors of glutathione S- aryltransferase . Glutathione S- alkyltransferase and glutathione S-epoxide transferase were also inhibited by the diuretics, but to a lesser extent than glutathione S- aryltransferase . The diuretics giving the greatest inhibition of these reactions were chemically related to ethacrynic acid. The concept where inhibition of glutathione-S-transferase by a drug may enhance its own toxicity is considered. This mechanism has also the potential of enhancing the toxicity of other concurrently administered drugs which normally require glutathione S-transferase for detoxication.

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.

Glutathione depletion in the guinea pig and its effect on the acute cochlear toxicity of ethacrynic acid.

There is controversy as to whether or not the acute cochlear toxicity of ethacrynic acid (EA) is dependent upon its metabolic conversion to EA-cysteine via conjugation with glutathione. In order to investigate this we examined the acute effects of EA on cochlear potentials in guinea pigs in which glutathione levels were decreased by prior administration of (+/-)-buthionine sulphoximine (BSO), an inhibitor of glutamylcysteine synthetase. First, we determined the effects of BSO on hepatic and renal glutathione levels in the guinea pig. Guinea pigs (pigmented animals of both sexes or male albino animals) were killed at intervals up to 72 hr after i.p. administration of 1.6 g kg-1 BSO. Livers, and also kidneys in the case of pigmented guinea pigs, were removed and total glutathione (GSH + GSSG) measured. Glutathione levels reached a nadir in the liver at 24-48 hr (11% of control) and in the kidneys at 24 hr (14% of control) after administration of BSO. Hepatic but not renal levels approached control values by 72 hr. There were no sex or strain differences. Pigmented guinea pigs were anaesthetised and their endocochlear potential and a.c. cochlear potential in response to a 4 kHz tone were measured using an intracochlear microelectrode. The depression of these potentials by i.v. administration of 60 mg kg-1 EA was not affected by administration of 1.6 g kg-1 BSO 24 hr earlier, despite profound depletion of glutathione. Also prior p.o. administration of N-acetyl-L-cysteine did not affect hepatic glutathione levels nor modify the toxicity of EA. These results suggest that the acute cochlear toxicity of EA is not altered by glutathione depletion, a finding which argues against a role for the metabolic activation of EA in its ototoxicity.

Toxicity of uricosuric diuretics in rat hepatocyte culture.

Several aryloxyacetic acid diuretics have shown hepatotoxicity in humans, yet there continues to be interest in developing these compounds because of the uricosuric properties of some of them. This study was designed to test the utility of the hepatocyte monolayer culture as a model for studying these compounds. In addition, an attempt was made to define the structural components that are common to hepatotoxicity. Ticrynafen, indacrinone, ethacrynic acid and A-49816, an investigational compound, were found to be toxic in hepatocyte cultures; thus, with the exception of indacrinone, paralleling the experience in humans. The toxic compounds share a ketodichlorophenoxyacetic acid chemical structure. A-56234, an investigational uricosuric, was also found to be toxic in cultures but has not been demonstrated to be hepatotoxic in humans in limited clinical experience. It does not possess the ketodichlorophenoxyacetic acid structure proper but may be metabolized to a closely related structure. Furosemide, which does not have the ketodichlorophenoxyacetic acid structure, was not toxic in hepatocyte cultures and has not been hepatotoxic in humans. Thus, the structure common to the toxic compounds is ketodichlorophenoxyacetic acid or a closely related compound. The hepatocyte monolayer system appears to be a good model for demonstrating toxicity and, perhaps, for predicting toxicity of new compounds under development.