Assessment of membrane toxicity using [3H]arachidonic acid release in U937 cells

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Toxic. in Vitro Vol. 8, No. 4. pp. 775 777, 1994 Copyright © 1994 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0887-2333/94 $7.00 + 0.00

ASSESSMENT OF MEMBRANE TOXICITY USING [3H]ARACHIDONIC ACID RELEASE IN U937 CELLS H.-P. KLOCKING, U. SCHLEGELMILCHand R. KLOCKING* Department of Toxicology, Institute of Pharmacology and Toxicology and *Institute of Medical Microbiology, Medical School Erfurt, PO Box 595, 99012 Erfurt, Germany Abstract--The release of arachidonic acid (AA) from membrane phospholipids is part of a signal system with which cells respond to environmental changes of various kinds. Using the promonocytic cell line U937, the kinetics of AA release was studied in [3H]AA-labelled U937 cells under the influence of various chemicals. Membrane-toxic agents such as lysolecithin and sodium dodecyl sulfate (SDS) cause pronounced enhancement of [3H]AA release in U937 cells. The effect occurred immediately after exposure and proved to be dose-dependent, non-enzymatic and irreversible. With regard to mean [3H]AA-releasing rate in untreated U937 cells, the following maximum tolerable concentrations were found: lysolecithin, 0.5 # g/ml; SDS, 2 p g/ml; dimethoate, 31.3 # g/ml; chloramine, 160 # g/ml; 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg/ml; formamide, 50 mg/ml and dimethyl sulfoxide, 100 mg/ml. AA release represents a highly sensitive bioindicator for the membrane-toxic effect of a chemical substance.

INTRODUCTION As the cell membrane represents the site of primary contact of chemicals acting on cells and organisms, it may have already suffered damage that either is not detected at a sufficiently early stage or is completely omitted from the general toxicity assessment. To detect membrane lesions, therefore, a number of in vitro tests have been developed, the results of which reflect different types of damage at different times after exposure (Calvin, 1992). Arachidonic acid is a particularly suitable marker for early membrane changes because, first, it is an integral part of cell membrane phospholipids and, secondly, it is rapidly released, mostly catalysed by enzymes, thus indicating changes in the cell environment. Furthermore, arachidonic acid represents the substrate for inflammation mediators--leucotrienes and prostagland i n s - - t h a t are formed according to the metabolic route. The release of arachidonic acid in the promonocytic cell line U937 under the influence of various chemicals--including well-known membrane-toxic substances--was studied in order to find an equivalent in vitro test to assess the risk of inflammation of skin and mucous membrane provoked by cell membrane lesions.

MATERIALSANDMETHODS Chemicals. [3H]Arachidonic acid (sp. act. 23.8 GBq/mg) was obtained from Amersham Buchler Abbreviations: AA = arachidonic acid; 2,4-D = 2,4dichlorophenoxyacetic acid; DMSO = dimethyl sulfoxide; F C S = foetal calf serum; MTC=maximum tolerable concentration; SDS = sodium dodecyl sulfate.

G m b H & Co. K G (Braunschweig, Germany); chloramine was a product of Fahlberg-List Pharma G m b H (Magdeburg, Germany); 2,4-dichlorophenoxyacetic acid (2,4-D) was obtained from Aldrich Chemic (Steinheim, Germany); dimethyl sulfoxide (DMSO) was purchased from Merck-Schuchardt (Hohenbrunn, near Munich, Germany); Bi58EC (Chemic AG, Bitterfeld-Wolfen, Bitterfeld, Germany) with a content of 380 g dimethoate/litre was used in these experiments. Other chemicals were obtained from the following sources: formamide from Merck, Darmstadt, Germany, lysolecithin from CalbiochemNovabiochem G m b H , Bad Soden/Ts., Germany; sodium dodecyl sulfate (SDS) from Ferak, Berlin, Germany. Cells. The promonocytic cell line U937 was grown in R P M I 1640 medium, containing 10% foetal calf serum (FCS), for 2-3 days. For A A release studies, cell cultures were washed twice with serum-free R P M I by centrifugation (200g, 5min) and resuspending in serum-free R P M I (usually at a density of 106 cells/ml). After incubation at 37°C and 5% CO2 for 1 hr, 10% FCS and 1/tCi/ml [3H]AA were added and kept at 37°C for 24 hr. The labelling medium was then removed by washing and cells were resuspended in cold serum-free R P M I as described above. In all experiments, the 24 hr uptake of [3H]AA was over 80%. Determination o f [3H]AA release. Aliquots (ml) of [3H]AA-prelabelled U937 cells were placed in sterile 8 ml polystyrene tubes (Falcon) and test substances dissolved in cold serum-free R P M I were added at graduated concentrations. Control cells received an appropriate volume of R P M I without test substances. Cell cultures were kept for 1 hr at 4°C, after which the temperature was raised to 37°C. Depending 775

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concentration ( pg/ml ) Fig. 1. Influence of different chemicals on the release of [3H]arachidonic acid in U937 cells: --IS]-lysolecithin; --II--sodium dodecyl sulfate (SDS); - - © ~ i m e t h o a t e ; --O--chloramine; • (2,4-D); - - x --formamide; - - O ~ i m e t h y l sulfoxide.

on the type of experiment, the cells were further incubated for 1 hr (dose-response relationship) or 24 hr (kinetics). After incubation, [3H]AA was determined in the 200g supernatants of cultures by liquid scintillation counting. Three experiments were carried out in any given series. All assays were performed in triplicate. Calculation of the maximum tolerable concentration (MTC). To determine the M T C of the test substance,

a confidence interval was calculated by means of variance analysis for both the R P M I cell control and the dose-response curve up to the 10-fold [3H]AA release of the cell control. The first concentration was determined at which a significant difference was found between A A release of the test substance and that of the cell control. The next lower concentration of the test substance was taken as maximum tolerable concentration (MTC).

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Time (hr) after temperature shift from 4°C to 37°C Fig. 2. Influence of different chemicals on the kinetics of [3H]-arachidonic acid in U937 cells; (a) lysolecithin (1/~g/ml); (b) SDS (10/tg/ml); (c) dimethoate (100 pg/ml); (d) chloramine (500 #g/ml); (e) formamide (I00 mg/ml).

Membrane toxicity and arachidonic acid release RESULTS Figure 1 illustrates the [3H]AA release of U937 cells under the influence of lysolecithin, SDS, dimethoate, chloramine, 2,4-D, formamide and DMSO. The dose-response curve clearly demonstrates that the cells responded to all the substances with a concentration-dependent increase in [3H]AA release. However, the effective ranges of concentration of the test substances differed by some orders of magnitude. Correspondingly, this was also true for the MTC values. For lysolecithin and SDS, the MTC values amounted to 0.5 and 2.0#g/ml, respectively; for formamide and DMSO these values amounted to 50 and 100 mg/ml, respectively. The MTC values of the remaining substances (dimethoate, chloramine and 2,4-D: 31, 160 and 1000#g/ml) demonstrated their position between the extremely toxic and the particularly well-tolerated substances. The kinetic studies of [3H]AA release proved that U937 cells suspended in serum-free RPMI show only very slight changes within the first 2 hr of incubation. The [3H]AA release was only slightly enhanced after a temperature shift from 4 to 37°C. The membranedisintegrating effect of lysolecithin and SDS was corroborated by the markedly enhanced [3H]AA release that it caused. The effect was apparent even at 4°C, which means that it is temperature-independent and thus non-enzymatic. The 24hr value demonstrated the irreversibility of the membrane lesion, so that lysolecithin and SDS are rightly considered to be membrane-toxic substances. Dimethoate also enhanced [3H]AA release; the effect, which was apparent even at 4°C, was even greater after an increase in temperature to 37°C. Chloramine (500 #g/ml) did not influence [3H]AA release, either at 4°C or at 37°C; only after 24 hr was a significant increase in [3H]AA release found, compared with the control cells. Similar reactions were found with

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formamide, which did not provoke an increase in [3H]AA release before 2hr after the increase in temperature (Fig. 2). DISCUSSION

Stark et al. (1983) have already demonstrated, for the irritants n-butanol and isopropyl myristate, the enhanced release of radioactive material from [3H]AA-prelabelled hepatoma cells. These findings have been confirmed and extended by the studies described here on the release by other chemicals of [3H]AA from [3H]AA-prelabelled U937 cells. As shown by the results of our studies, the membrane toxicity of chemicals can be demonstrated by [3H]AA release, in a highly sensitive manner immediately after cell contact, whereas the results of the chromium-51 release test (Eichhorn et al., 1987) are usually available only after 24 hr. As inflammation represents the predominant type of damage caused by irritants, the arachidonic acid release test may be useful in prescreening of irritant effects of substances. Acknowledgement--The authors wish to thank the Fritz

Thyssen-Stiftung in Cologne, Germany, for technical support. REFERENCES

Calvin G. (1992) New approaches to the assessment of eye and skin irritation. Toxicology Letters 64]65, 157-164. Eichhorn U., K16cking R., Schweizer H. and Klfcking H.-P. (1987) Cell membrane toxicity detected with the chromium-51 release test. Archives of Toxicology Suppl. 11, 334-337. Stark D. M., Shopsis C., Borenfreund E. and Walberg I. (1983) Alternative approaches to the Draize assay: chemotaxis cytology, differentiation, and membrane transport studies. In Alternative Methods in Toxicology 1: Product Safety Evaluation. Edited by A. M. Goldberg, pp. 179-203. Mary Ann Liebert, New York.