Careers in toxicology in Europe--options and requirements. Report of a workshop organized on behalf of the Individual Members of EUROTOX during the EUROTOX Congress 2000 in London (September 17-20, 2000).

In view of the lack of information regarding careers for toxicologists in Europe, the Individual Members of EUROTOX organised a workshop on careers in toxicology during the EUROTOX Congress 2000 in London. Toxicologists are mainly employed in academia, regulatory agencies, contract research organisations (CROs) and the chemical and pharmaceutical industries. There are also a few governmental institutes involved with toxicological work other than teaching or regulation. Toxicologists can also work as independent consultants, especially for commercial organisations. The requirements for starting a career in any of the above organisations, the need and the advantages and disadvantages of specialisation, and further career prospects are summarised and briefly discussed. The organisations, and also working as an independent toxicology consultant, offer interesting professional work of relevance to modern-day society. There is currently a shortage of toxicologists not only in the traditional field of risk assessment but also especially in new areas, e.g. toxicogenomics. This shortage may be at least in part due to insufficient training opportunities. Further consideration of career opportunities is planned and will be published in due course.

Localization of the nephron site of gentamicin-induced hypercalciuria in the rat: a micropuncture study.

In vivo renal micropuncture techniques were used to locate the nephron site of hypercalciuria induced by acute gentamicin infusion in anaesthetized Sprague Dawley rats. Three series of experiments were conducted. The effect of gentamicin on calcium reabsorption in the proximal tubule (Series I) and loop of Henle (Series II) was investigated using in vivo microperfusion whereas the effect on distal calcium handling (Series III) was studied using in vivo microinfusion. In all three experimental series, acute systemic gentamicin infusion at 0.28 mg kg(-1) min(-1) caused significant hypercalciuria within 30 min of commencing drug infusion. Gentamicin had no effect on the rates of urine flow or sodium excretion. Acute gentamicin infusion had no effect on unidirectional calcium reabsorption in the proximal tubule or loop of Henle despite a simultaneous and highly significant hypercalciuria at the whole kidney level. Net fluid reabsorption was also unaffected by the drug in these nephron segments. Acute gentamicin infusion significantly increased the urinary recovery of calcium following microinfusion into early distal tubules, whereas urinary calcium recovery was decreased after microinfusion into late distal tubules. We conclude that acute gentamicin-induced hypercalciuria is mediated by a decrease in calcium reabsorption in the early distal tubule. Thus, the acute hypercalciuric effect of gentamicin occurs at a different nephron site to the nephrotoxic effects associated with longer-term administration of the drug. It is, therefore, unlikely that gentamicin-induced hypercalciuria is involved in the pathogenesis of subsequent proximal tubular cell injury.

Gadolinium chloride toxicity in the mouse.

1. Groups of five male and five female CD-1 mice received a single intravenous injection of gadolinium chloride at dosages of 0 (saline control), 0.05, 0.1 and 0.2 mmol/ kg. All mice were necropsied 48 h post dose. 2. Plasma analysis showed increases in concentrations of lactate dehydrogenase (both sexes), aspartate aminotransferase and alanine aminotransferase (females only) in the 0.2 mmol/kg group. Cholesterol was elevated at all dosages in both sexes whilst globulin was raised in both sexes at 0.1 and 0.2 mmol/kg. 3. Histological lesions were present at all dosages and increased in severity in a dose-related fashion. The most common lesions were: mineral emboli in capillaries, accumulation of mineral in the mononuclear phagocytic system, hepatocellular necrosis, and lymphoid depletion, necrosis and mineralisation in the spleen. 4. Such observations are similar to those in rats given gadolinium chloride and should be assessed when evaluating the toxicological profile of gadolinium containing compounds being developed for nuclear magnetic resonance imaging.

Neurotoxic potential of gadodiamide after injection into the lateral cerebral ventricle of rats.

PURPOSE: Results of a previous report showed that, if administered by intraventricular injection to access tissue normally protected by the blood-brain barrier, gadopentetate dimeglumine produced acute excitation, persistent ataxia, and widespread brain lesions in rats at 5-micromol/g brain but not at 3.8-micromol/g brain. The present study using gadodiamide was undertaken to see whether the effects were agent-specific. METHODS: Rats, surgically prepared with a lateral ventricular cannula, were administered a slow injection at 2 microL/min of gadodiamide into the lateral ventricle, and behavioral and neuropathologic changes were noted. RESULTS: Both gadodiamide and gadopentetate dimeglumine produced focal and generalized myoclonus over several hours. Gadodiamide did not produce the medium-term tremor or persistent ataxia seen after treatment with gadopentetate dimeglumine. Neuropathologic changes developed over 1 to 3 days and took three distinct forms: vacuolated thalamic lesions closely resembling those produced by gadopentetate dimeglumine; small but similar vacuolated symmetrical caudate lesions not produced by gadopentetate dimeglumine; and severe swelling and astrocytic hypertrophy and hyperplasia in the cerebellar vermis, again not produced by gadopentetate dimeglumine. Unlike gadopentetate dimeglumine, gadodiamide produced no spinal cord lesions. The cerebellar changes were seen at 1.25-micromol/g brain and above, behavioral changes at 2.5-micromol/g brain and above, and thalamic and caudate lesions at 10-micromol/g brain, the maximal dose used. Markedly reducing the rate of injecting the same volume over 28 hours prevented the acute excitation but did not reduce the severity of the morphologic effects. CONCLUSION: The acute excitatory effects of high intraventricular doses of gadopentetate dimeglumine and gadodiamide are similar and appear to be attributable to local action at the infusion site, but differences exist between the two agents in the character and topography of the distant morphologic changes. The cerebellum was the brain area most sensitive to gadodiamide in this experimental model. It is unlikely that gadodiamide would gain access to the brain at these tissue doses when used intravenously for conventional clinical imaging, but our experimental model suggested that it had some unexpectedly specific neuropathologic potential.

The effect of gentamicin and furosemide given in combination on cochlear potentials in the guinea pig.

Single doses of gentamicin and furosemide given in combination result in a rapid and profound loss of cochlear function. In this study, measurement of three gross cochlear potentials (cochlear microphonics, compound action potentials and the endocochlear potential) were carried out in order to determine the ototoxic sites of action of the drugs given in combination. The rapidity and severity of the cochlear deficit is dose dependent and with the doses employed in this study (80 mg/kg gentamicin i.v. 80 mg/kg furosemide i.v.), complete loss of cochlear function is seen after about 72 hours. Twenty-four hours after i.v. administration of the drugs, significant increases in compound action potential thresholds between 6 and 32 kHz were seen. In contrast, over this frequency range the generation of cochlear microphonics in response to stimulus levels of 70 dB SPL appeared to be unaffected. The endocochlear potential remained unaffected at 24 and 72 hours after administration. These findings are taken as evidence that the primary site of ototoxic action of the two drugs in combination may be at the level of the inner hair cells and/or the afferent synapse.

Acute gentamicin-induced hypercalciuria and hypermagnesiuria in the rat: dose-response relationship and role of renal tubular injury.

1. Standard renal clearance techniques were used to assess the dose-response relationship between acute gentamicin infusion and the magnitude of hypercalciuria and hypermagnesiuria in the anaesthetized Sprague-Dawley rat. Also investigated were whether these effects occurred independently of renal tubular cell injury. 2. Acute gentamicin infusion was associated with a significant hypercalciuria and hypermagnesiuria evident within 30 min of drug infusion. The magnitude of these responses was related to the dose of drug infused (0.14-1.12 mg kg(-1) min[-1]). Increased urinary electrolyte losses resulted from a decreased tubular reabsorption of calcium and magnesium. 3. A rapid dose-related increase in urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion was also observed in response to gentamicin infusion. However, there was no evidence of renal tubular cell injury and no myeloid bodies were observed within the lysosomes of the proximal tubular cells. Gentamicin may thus interfere with the mechanisms for cellular uptake and intracellular processing of NAG causing increased NAG release into the tubular lumen. 4. The absence of changes in renal cellular morphology indicates that the excessive renal losses of calcium and magnesium were an effect of gentamicin per se and not the result of underlying renal tubular injury. The renal effects described in this paper were apparent after administration of relatively low total drug doses, and with plasma concentrations calculated to be within the clinical range. These findings suggest that disturbances of plasma electrolyte homeostasis could occur in the absence of overt renal injury in patients receiving aminoglycoside antibiotics.

Gadolinium chloride toxicity in the rat.

Groups of 10 male and 10 female Sprague-Dawley rats were given a single intravenous injection of gadolinium chloride solution at dosages of 0 (saline vehicle), 0.07, 0.14, and 0.35 mmol/kg. Apart from 1 top-dose female, which died during dosing, 5 rats/sex/ group were necropsied 48 hr postdose, and the remaining 5 rats/sex/group were necropsied 14 days postdose. Macroscopic, hematological, and clinical chemistry analyses were undertaken on all animals that were necropsied. Histopathological examination was undertaken on all organs from high-dose and control animals necropsied 48 hr postdose and on tissues that showed treatment-related changes from all other rats necropsied either 48 hr or 14 days postdose. Major lesions related to gadolinium chloride administration consisted of mineral deposition in capillary beds (particularly lung and kidney), phagocytosis of mineral by the mononuclear phagocytic system, hepatocellular and splenic necrosis followed by dystrophic mineralization, mineralization of the fundic glandular mucosa in the absence of necrosis followed by mucous cell hyperplasia, decreased platelet numbers and increased prothrombin time, and activated partial thromboplastin time. Electron microscopy and x-ray microanalysis of the spleen and liver revealed electron-dense deposits in splenic macrophages, Kupffer cells, and hepatocytes composed of gadolinium, calcium, and phosphate.

Time course of stomach mineralization, plasma, and urinary changes after a single intravenous administration of gadolinium(III) chloride in the male rat.

In a previous experiment it was reported that the intravenous administration of gadolinium chloride (GdCl3) to rats results in a discrete band of interstitial mineralization in the fundic glandular mucosa of the stomach. To investigate the time course for the development of this lesion and its relationship to plasma calcium and phosphate concentrations, 2 experiments were carried out in male Sprague-Dawley rats given a single intravenous dose of 0.07 mmol/kg GdCl3. Plasma calcium and phosphate concentrations approximately doubled between 30 min and 12 hr postdose but had regressed back to near normal values by 24 hr. However, there were no observable clinical signs in treated animals. Histologically, there was progressive mineralization of the lamina propria of the neck region of the fundic glands from 6 hr postdose, forming a distinctive mineral band by 12 hr postdose. At 7 and 14 days postdose the mineral deposits were accompanied by mucous cell hyperplasia, interstitial fibrosis, and a very sparse infiltration of inflammatory cells. By 56 days postdose only occasional mineral deposits remained. Transmission electron microscopy showed mineral first nucleated on collagen in the interstitium, but there was no evidence of cell necrosis. X-ray microanalysis showed that the interstitial mineral was composed of calcium and phosphate in the form of hydroxyapatite; gadolinium (Gd) was only very rarely identified. These findings are consistent with metastatic mineralization. The source, cause, and the exact nature of the excess plasma calcium and phosphate are unknown, and the possible significance of this effect for clinical use of Gd-containing chelates in nuclear magnetic resonance imaging requires further investigation.

A study of the nephrotoxicity of three cephalosporins in rabbits using 1H NMR spectroscopy.

Male New Zealand White rabbits received a single intravenous injection of 125 mg/kg cephaloridine, 500 mg/kg cefoperazone or 1000 mg/kg cephalothin. Histological examination of kidneys at 48 h post-dose confirmed the presence of bilateral necrosis of the proximal convoluted tubules in the cephaloridine-treated animals. 1H-NMR urinalysis of cephaloridine-treated rabbits detected drug-related resonances, decreased hippurate and increased glucose at 0-24 h post-dose accompanied by elevated levels of lactate, glycine, citrate, glutamine/glutamate and alanine at 24-48 h post-dose. No histopathological changes were observed following administration of cefoperazone or cephalothin. 1H-NMR spectra of urine collected from these animals showed drug-related resonances and decreased hippurate levels at 0-24 h post-dose, and increased glucose levels at 24-48 h post-dose. Analysis of urine by conventional clinical-chemistry failed to reveal any statistically significant differences between the treatment groups. Under the conditions of this study, the nephrotoxic effects of cephaloridine and the minimal effects of cefoperazone and cephalothin could be clearly distinguished by 1H-NMR urinalysis but not by conventional urinalysis.

The relative hypolipidaemic activity and hepatic effects of ciprofibrate enantiomers in the rat.

The aim of this study was to establish whether the individual enantiomers of racemic ciprofibrate, a potent hypolipidaemic agent and peroxisome proliferator, differ significantly in either pharmacological potency or toxic potential. After a single oral dose to male Fischer F344 rats at dosages below 10 mg/kg, S(-) ciprofibrate produced slightly, but statistically significantly, greater reductions in plasma concentrations of cholesterol than R(+) ciprofibrate. Similarly, at low concentrations in F344 rat hepatocyte cultures, S(-) ciprofibrate produced slightly, but statistically significantly, greater inductions of peroxisomal beta-oxidation activity than R(+) ciprofibrate. However, after seven daily doses, the differences in pharmacological effects of the two enantiomers were no longer apparent. Furthermore, in contrast to its effects in vitro, R(+) ciprofibrate produced slightly, but statistically significantly, greater inductions of peroxisomal beta-oxidation activity in vivo than S(-) ciprofibrate. These observations may be possibly explained on the basis that following multiple dosing, plasma concentrations of R(+) ciprofibrate 24 hr post-dose were greater than those of its optical antipode. Thus the slightly greater potency of the S(-) enantiomer after a single dose may have been overcome by the greater plasma concentrations of the less potent enantiomer. Both enantiomers produced similar reductions in plasma concentrations of thyroxine. The data indicate that at low dosages S(-) ciprofibrate is a slightly more potent hypolipidaemic agent after a single dose in rats and a slightly more potent peroxisome proliferator at low concentrations in vitro. However, following multiple dosing, both enantiomers produced changes in plasma concentrations of lipids, hepatic enzyme activities and plasma concentrations of thyroxine which were of comparable magnitude to those produced by the racemate. Since these early changes have been linked mechanistically to the chronic toxicity of the racemate in the rat, it could be predicted that the individual enantiomers of ciprofibrate under conditions employed in chronic safety studies, would produce the same spectrum of rodent toxicity as the racemate.

A critical review of the optimum duration of chronic rodent testing for the determination of non-tumourigenic toxic potential: a report by the BTS Working Party on Duration of Toxicity Testing.

This review indicates that for the detection of non-neoplastic toxic effects: 1. Four decades of accumulated literature provide no lead as to the optimum duration of repeat dose toxicity testing required for all classes of chemicals, although 6 months repeated administration appears adequate for pharmaceuticals. 2. Three month studies predicted the 2 year outcome for 70% of the compounds evaluated in this pilot study using data published by the US National Toxicology Program. 3. In spite of the limitations of this pilot study, this finding is considered encouraging as it is close to that generated previously on more detailed confidential pharmaceutical data. This suggests that the exercise should now be expanded using confidential surveys of industrial data to determine the concordance resulting from the evaluation of a larger group of chemicals.

Effects of gentamicin, neomycin and tobramycin on renal calcium and magnesium handling in two rat strains.

1. Standard renal clearance techniques were used to compare the acute effects of gentamicin, neomycin and tobramycin on renal calcium and magnesium handling in Sprague-Dawley and Fischer 344 rats. 2. Significant hypercalciuric and hypermagnesiuric responses to all three drugs (P < 0.01) were apparent within 30 min of the onset of drug infusion. 3. The magnitude of the acute hypercalciuric and hypermagnesiuric response to the three aminoglycosides was comparable. This contrasts with their nephrotoxic action where neomycin >> gentamicin > tobramycin. The magnitude of the acute physiological responses to these drugs do not therefore reflect their nephrotoxic potential. 4. Sprague-Dawley rats were at least as responsive as Fischer rats in their acute renal responses to gentamicin. If Fischer rats are more sensitive to aminoglycoside nephrotoxicity than Sprague-Dawley rats, this is not reflected in their acute responses to gentamicin.

The role of investigative toxicology in pharmaceutical industry.

In summary, the benefits of a mechanistic toxicology approach include increased understanding of the processes underlying primary pathology, the development of predictive capability, enhanced decision making and improved responsiveness to regulatory agencies. The overall objective of toxicology in the pharmaceutical industry is risk benefit assessment in order to support product registration. This requires both that studies be conducted in accordance with regulatory guidelines and that the biological processes underlying pathology be understood. These should be regarded as inseparable components of the risk assessment process.

In vitro toxicity of cephalosporin and aminoglycoside antibiotics to LLC-PK(1) cells assessed by changes in glucose uptake.

Nephrotoxin-induced damage to cultured kidney cells (LLC-PK(1)) was investigated using assays of general cytotoxicity (neutral red uptake) and of functional impairment (glucose accumulation). The latter correctly ranked both the cephalosporin and aminoglycoside antibiotics (acute and chronic proximal tubule toxicants respectively) in terms of their relative in vivo toxicities. Functional impairment after low-dose long-term exposure to the aminoglycosides occurred at sublethal drug concentrations. Assessment of general cytotoxicity correctly ranked only the aminoglycoside antibiotics.

Preclinical safety assessment and pharmacokinetics of gadodiamide injection, a new magnetic resonance imaging contrast agent.

In a wide range of preclinical studies of gadodiamide injection (Omniscan, Sanofi Winthrop, New York, NY, and Nycomed AS, Oslo, Norway), the pharmacokinetics of the compound have been delineated and its safety demonstrated. The pharmacokinetic behavior of gadodiamide was consistent with its extracellular distribution. Its half-life in rats, rabbits, and monkeys was short, 18, 38, and 75 minutes, respectively. Gadodiamide was shown to be excreted rapidly, primarily through the kidneys. In rats, 94% of the administered dose was excreted in the urine within the first 24 hours after administration. Approximately 1% to 4% appeared in the feces during the same period. Gadodiamide injection has been shown to have a remarkably low acute lethal toxicity, superior to that of gadopentetate dimeglumine injection (Magnevist, Berlex Laboratories, Wayne, NJ, and Schering AG, Berlin, Germany) or gadoterate meglumine (Dotarem, Laboratoire Guerbet, Aulnay-Sous-Bois, France). In comparison with gadopentetate dimeglumine injection, gadodiamide injection had fewer effects on cardiovascular and hemodynamic function after rapid intravenous injection in anesthetized dogs and, in vitro at high concentrations, on erythrocyte fragility and arterial wall tension. The lesser effects might be attributable, at least in part, to the lower osmolality of gadodiamide injection, although it remains to be seen whether this will translate into any advantage for gadodiamide injection at the lower doses used for imaging procedures in patients. Similar to all known intravenously administered diagnostic imaging agents, gadodiamide injection produces vacuolization of the proximal tubular cells in the kidney, without any change in renal function. However, the single-dose threshold for this effect is greater than 0.5 mmol/kg in the rat; even after a dose of 10 mmol/kg, the vacuolization was only "moderate" in degree and was shown to have regressed partially during the 7 days after administration. In monkeys, administration of 0.25 mmol/kg daily for 28 days had no effect on the kidney, thus providing reassurance of the wide margin of safety for any effect of this compound on the kidney. Although intended for single administration in patients, gadodiamide injection has been studied extensively in a range of subchronic studies in rats and monkeys. The compound was well tolerated in monkeys even when administered at doses up to 1.25 mmol/kg daily for 28 consecutive days. In rats, significant toxicity occurred only at high doses, particularly in male animals, and the pattern of toxicity (involving the stomach, testes, and skin) suggested a disturbance of zinc metabolism. Gadodiamide injection produced no significant irritation when administered by a variety of intravascular and extravascular routes.

Toxicology of sensory systems: a perspective.

This brief review is the result of a recent meeting of the British Toxicology Society (Toxicology of Sensory Systems, University of York, April 2-3, 1992). The meeting provided the opportunity to discuss the anatomy, physiology and function of the eye, ear, nasal epithelium and peripheral sensation and the methods that are available to detect injury or dysfunction both in the preclinical and clinical situation. In addition, the mechanism whereby certain chemicals can perturb some of these organs was discussed. The aim of this short article is to highlight some of the recent advances in understanding in these areas with regard to their relevance or impact on toxicology. For convenience the areas will be discussed under separate headings.

Gentamicin-induced hypercalciuria in the rat: assessment of nephron site involved.

Two independent techniques were used in anesthetized rats in an attempt to locate the nephron site of the reduced tubular calcium reabsorption accompanying acute gentamicin infusion. The first technique was that of lithium clearance used to assess proximal sodium (and secondarily calcium) handling. Observations that lithium clearance was comparable in control and gentamicin-treated animals (1.83 +/- 0.39 vs. 1.46 +/- 0.14 ml.min-1 for first experimental period) suggests a lack of proximal effect of the drug. The second technique was that of tracer microinjection whereby superficial nephrons were injected with 45Ca and tubule calcium transport was assessed from the recovery of radioactivity in the final urine. 45Ca recovery values from distal microinjections were comparable in control and gentamicin-treated groups (81.1 +/- 2.0 vs. 77.7 +/- 4.6%). However, 45Ca recovery values from proximal microinjections were significantly higher in the gentamicin group (9.4 +/- 1.0 vs. 3.5 +/- 0.8%; P < .001). These data suggest that the effects of gentamicin on renal calcium handling are mediated at a nephron site proximal to the distal tubule (i.e., loop of Henle or proximal tubule itself). Closer examination of individual proximal micropuncture data may point to an effect occurring predominantly in the pars recta of the proximal tubule or loop of Henle. Taken together, the results of both parts of the present study suggest that the early physiological effects of gentamicin on the kidney occur in a different nephron segment from any subsequent nephrotoxicity.

An investigation of the acute effect of gentamicin on the renal handling of electrolytes in the rat.

Standard renal clearance techniques were used to investigate the acute effects of gentamicin on renal electrolyte handling in anesthetized rats. Data were obtained before substantial changes in tubular integrity would be expected to occur, thus permitting a distinction between the actions of the drug per se and renal changes resulting from underlying tubular damage. Infusion of gentamicin over a 3-hr period resulted in an immediate and sustained calciuresis and magnesiuresis, with no significant change in the renal clearance of sodium or potassium. Within 60 min of the onset of drug infusion at 0.28 mg/kg/min, renal calcium clearance (ml/min) increased from 0.095 +/- 0.013 to 0.210 +/- 0.016 (P less than .001) and renal magnesium clearance (ml/min) increased from 0.538 +/- 0.059 to 0.662 +/- 0.053 (P less than .01). Because it was subsequently shown that infusion of gentamicin did not alter glomerular filtration rate, it is clear that both responses resulted from a reduced tubular reabsorption of the respective ions; they were rapidly reversible when gentamicin infusion ceased. A higher dose of gentamicin (0.56 mg/kg/min for 3 hr) also significantly decreased plasma magnesium concentrations (0.40 +/- 0.01 vs. 0.49 +/- 0.02 mmol/l, treated vs. control; P less than .01). Gentamicin-induced changes in the renal handling of calcium and magnesium, therefore, occur independently of and before the development of nephrotoxicity. They may be at least partially responsible for the alteration in electrolyte homeostasis seen in humans during aminoglycoside treatment (e.g., hypomagnesemia). It is interesting to speculate on the possibility that they may also in some way contribute to the subsequent cellular injury that is known to occur with prolonged use of gentamicin.

Comparison of LDH, (51)Cr, and BCECF efflux as indices of non-steroidal anti-inflammatory drug-induced toxicity in human gastro-intestinal (HGT-1, HCT-8 and T84) cell lines: BCECF efflux is not an index of plasma membrane integrity.

Toxicity induced by non-steroidal anti-inflammatory drugs (NSAIDs) was assessed in vitro in three human gastro-intestinal epithelial cell lines (gastric HGT-1, ileo-caecal HCT-8 and colonic T84) using three proposed indices of cell plasma membrane integrity. Lactate dehydrogenase (LDH) and (51)CR efflux from cells over 5 hr under control conditions was small (about 3 and 9%, respectively). In contrast, the efflux of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was much greater, ranging from 45% in HGT-1 cells to 76% in HCT-8 and T84 cells over 5 hr. Indomethacin increased LDH and (51)Cr efflux at concentrations >/= 5 mm. The rank order for the toxicity of four NSAIDs, assessed by (51)Cr efflux in HCT-8 cells, was indomethacin > sulindac > ketoprofen = aspirin. In contrast, the effects of indomethacin on BCECF efflux were bimodal. At concentrations ranging from 10 mum to 5 mm, depending on the cell line, indomethacin reduced BCECF efflux to a minimum of approximately 20% of the control efflux rate. With higher concentrations of indomethacin, BCECF efflux increased to levels similar to, but not exceeding, control rates. Sulindac (0.01-10 mm) and ketoprofen (1-10 mm), but not aspirin (0.01-10 mm) also reduced BCECF efflux. The reduction in BCECF efflux observed with low concentrations of NSAIDs, and the very high rate of BCECF efflux under control conditions, negates the use of this tracer as an index of plasma membrane integrity. However, this inhibition could itself be a sensitive index of toxicity. The mechanism of inhibition of BCECF efflux is suggested to be due to inhibition of transport-mediated flux, and this is a possible site for NSAID action.