Fd Chem. Toxic. Vol. 24, No. 6/7, pp. 465--466, 1986 0278-6915/86 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1986 Pergamon Journals Ltd

ADENOSINE TRIPHOSPHATE AS AN INDICATOR OF CELLULAR TOXICITY IN VITRO

R. B. KEMP, D. M. CROSS and R. W. J. MEREDITH Zoology Department, University College of Wales, Penglais, Aberystwyth, Dyfed, SY23 3DA, Wales

Abstract--Using a mouse fibroblast line (L929-derived LS) in suspension culture and adenosine triphosphate (ATP) levels as the endpoint, a highly predictive screen for toxicity has been developed and subjected to validation. Cells were exposed to chemicals of known toxicity for 4 hr and levels of ATP were determined using the luciferin/luciferase bioluminescent system, emitted light being measured in an automated Packard luminometer. Concentrations producing a 50% reduction in cellular ATP (ATPs0) were compared with those causing 50% cell death (CDs0) as established with the fluorescent dyes, fluorescein diacetate and ethidium bromide. Ranking of the chemicals according to the ATPs0 was generally similar to that based on the CDs0 determinations and demonstrated a range of toxicity from very mild to severe.

Introduction

It has been advocated that cell cultures in vitro provide a plausible screen, and even replacement, for toxicity studies in animals. Probably the two most controversial uses of animals in this respect are the Draize rabbit eye irritation test and LDs0 determinations.

Kemp, Meredith, Gamble & Frost (1983) showed that cell death (CDs0) in LS cells derived from L929 mouse fibroblasts was predictive of ocular irritancy (as demonstra ted by the Draize eye test) for an " u n k n o w n " series of 11 shampoos, which varied in effect from mildly to severely damaging to the eye. The cells in suspension were exposed to the toxicant for 4 hr and their viability was then assessed using a combinat ion of two fluorescent dyes, fluorescein

diacetate and ethidium bromide, to demonstra te the status of the permeability barrier.

While this method was easy to use, endpoint determinat ions were t ime-consuming and, to a certain extent, subjective. Biochemical assays, on the other hand, are quantitative and readily automated in terms of machinery. Adenosine t r iphosphate (ATP) is an excellent indicator o f cellular 'heal th ' because it is the prime energy donor. Moreover, with the purification of the firefly luciferin/luciferase system (Giick, 1977), spectroscopic techniques permit a rapid and highly sensitive (104 cells) detection of ATP. Kemp, Meredith & Gamble (1985), with financial support from Packard Instrument Co. (Downers Grove, IL, USA), proved the value of this technique using a series of pure chemicals and com- mercial formulations, some of which were insoluble or immiscible in the culture medium.

Table I. Toxicity of 20 test compounds to LS cells as determined from intracellular adenosine triphosphate (ATP) concentrations and cell viability after 4-hr exposures

ATP method Viability assay

ATPso* CDsot Test chemi :al Use (,u g/ml) r (#g/ml) •

Diethylstilboestrol Synthetic oestrogen 16.6 0.88 16.3 0.84 p-Aminophenol Photography/dyes 30.5 0.89 39.6 0.76 Sodium dodecyl sulphate Detergent 51.4 0.85 51.2 0.95 p-Chloromercuribenzoic a c i d Analytical chemical 72.6 0.76 73.1 0.78 Butylated hydroxyanisole Food antioxidant 76.6 0.91 79.1 0.72 6-Methylcoumarin Flavouring 170.9 0.91 241.1 0.89 Bendiocarb Insecticide 225.8 0.73 345.9 0.99 Methylparaben Food preservative 341.1 0.95 635.8 0.98 Propanolol Cardiac depressant 378.6 0.89 438.9 0.89 2,4-Dinitrophenol Metabolic uncoupler 619.3 0.88 743.6 0.66 Phenytoin Anticonvulsant 638.6 0.73 1320.0 0.82 2,4-Dichlorophenoxyacetic ac id Herbicide 741.3 0.73 1000.0 0.80 Phenobarbital Sedative 1620.0 0.91 1590.0 0.91 L-Ascorbic acid Food industry 2490.0 0.81 2510.0 0.70 Chloroquine sulphate Antimalarial 4050.0 0.93 3650.0 0.85 Paracetamol Analgesic 5210.0 0.83 8030.0 0.89 6-Mercaptopurine Immunosuppresant 5390.0 0.93 6050.0 0.79 Sodium chloride - - 7040.0 0.88 7130.0 0.70 o-Glucose Metabolite 70 × 10 - - 70 x 10 - - n-Fructose Metabolite 70 x 10 -- 70 x 10 - -

r ffi Coefficient of variation *Concentration of test substance that reduces cellular ATP by 50%. tConcentration of test substance producing 50% cell death.

465

466 R.B. KEMP et al.

Farther validation study

A further series of 20 'blind' chemicals, supplied as part of the FRAME Alternative Test Validation Scheme, has been tested in the last 6 months and the data are presented in Table 1. Cells were exposed to the chemicals for 4 hr at 37°C and levels of ATP were determined using the luciferin/luciferase bio- luminescent system, emitted light being measured in an automated Packard luminometer (model 6500). Using changes in cellular ATP levels as the index, these substances, which have a variety of uses, were found to range in toxicity to LS cells from very mild to extremely harmful. It will not be possible fully to assess these results until animal lethality data become available, but ranking for the chemicals in terms of ATPs0 values (the concentrations of test substances reducing cellular ATP by 50%) was similar to that for the CDso (concentrations causing 50% cell death) determinations. Some of the substances had to be dissolved as stock solutions in organic solvents (dimethylsulphoxide, methanol or ethanol), but none of these solvents was at all toxic at the concentration

used in the cell suspensions and all gave CDs0 and ATPs0 values above 50 mg/ml. Nevertheless, there is an increasing awareness of the 'knock-on' effects of solvents.

Further analysis has already revealed that ATP measurements gave indications of harmful effects at lower concentrations of toxicant than did fluorescence changes. Studies of the effects of possible activation of these compounds by incubation with a liver S-9 preparation are now required.

REFERENCES

Glick D. (1977). Microchemical analytical techniques of potential clinical interest. III. Single cell analysis. Clin. Chem. 23, 1465.

Kemp R. B., Meredith R. W. J. & Gamble S. H. (1985). Toxicity of commercial products on cells in suspension culture: a possible screen for the Draize eye irritation test. Fd Chem. Toxic. 23, 267.

Kemp R. B., Meredith R. W. J., Gamble S. & Frost M. (1983). A rapid cell culture technique for assessing the toxicity of detergent-based products in vitro as a possible screen for eye irritancy in vivo. Cytobios 36, 153.