Fd Chem. Toxic. Vol. 24, No. 6/7, pp. 519 520, 1986 0278-6915/86 $3.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1986 Pergamon Journals Ltd

CULTURED HUMAN EPIDERMAL CELLS AS A TOOL IN SKIN TOXICOLOGY

M. A. E. MOL, J. VAN GENDEREN and O. L. WOLTHUIS Medical Biological Laboratory TNO, P.O. Box 45, 2280 AA Rijswijk, The Netherlands

Abstract--Cultured human epidermal cells have been used to study the effects of toxic compounds on cell growth and on the integrity of the cellular membrane. To determine the concentration causing a 50% inhibition of growth rate, keratinocyte cultures were exposed to the test compound in concentrations of 10-9-10-6M for 48 hr during the exponential growth phase and growth was calculated from the DNA content. To measure cell damage, fully grown cultures were exposed to a 10-4M concentration and the activities of two intracellular enzymes and the K ÷ concentration of the cells were determined at intervals over 4-24 hr. Data obtained so far indicate that this technique may be useful in testing for dermatotoxicity and in studying mechanisms of toxic action.

Introduction

A number of investigators of cutaneous toxicity use animal or human fibroblasts or different types of animal epithelial cells (Hopley, Benford & Bridges, 1985; Reinhardt, Pelli & Zbinden, 1985). It is sur- prising that human epidermal cells are rarely used for dermatotoxicity testing. As target cells of the irritant dermatitis reaction in vivo, it was thought that cul- tures of epidermal cells might be particularly helpful in the understanding of processes and mechanisms of cutaneous toxicity as well as of the metabolism of compounds in human skin. Hence, such cultures were used in the present experiments on epidermal cell damage and growth inhibition.

Experimental

Basal epidermal ceils obtained from human skin were seeded onto a feeder layer of 3T3 mouse fibroblasts in amounts of 104 cells/cm 2. The medium consisted of Dulbecco's M E M and Ham's F12 (3:1), 5% foetal calf serum, 0 .4pg hydrocortisone/ml, 10- l0 M-choleratoxin and 40 p g gentamycin/ml. After 3 days, epidermal growth factor was added at a level of l0 ng/mt. The cultures were gassed with 10% CO2 in air and incubated at 37°C on a rocking platform.

To measure cell damage, multilayered cultures of keratinocytes were exposed to a 10 -4 M concentration of a test compound in Ham's F l2 . The activity of lactate dehydrogenase (LDH) and N-acetyl-fl- glucosaminidase (f l -NAG), as well as the K + concen- tration in the cells at different time intervals, were expressed as a percentage of the total amount present in control cultures. For that reason the cultures were solubilized after incubation with a 0.7% Triton X- 100 solution in Tris-saline buffer.

For the growth inhibition assay, the cultures were exposed to a test chemical in complete medium for 48 hr during the exponential growth phase. The growth rate of the cultures was calculated from the D N A content (Labarca & Paigen, 1980) and the concentrat ion of chemical that reduced the growth rate to 50% of that of control cultures was estimated.

Results and Discussion

The severity of membrane lesions is estimated by the determination of leakage of markers with a small (K +) or a large (LDH, f l -NAG) molecular size. After exposure for 4-24 hr a considerable loss of enzyme activity can only be seen with chemicals that act directly on the membranes, such as tributyltin and sodium dodecyl sulphate. The high sensitivity of lysosomal membranes to sodium dodecyl sulphate (Imokawa & Mishima, 1979) is clearly reflected in a large decrease in intracellular f l - N A G activity. The intracellular level of K ÷ has been found to be a more sensitive indicator for cytotoxicity. Even when, after exposure for 0.25-2 hr, little enzyme leakage can be detected, considerable changes may occur in the K+-content of the cultures. This is in agreement with the results of Malik, Schwarz & Wiebel (1983), who used Chinese hamster V79 cells.

The concentrations that cause a 50% inhibition of growth rate in exponentially growing keratinocytes are 3 x 10 -6 M, 8 × 10 -8 M, 1 X 10 -s M and 7 x 10 -9 M for cycloheximide, tributyltin, diacetoxyscirpenol and T-2 fusarium toxin, respectively. Inhibition of cell growth may provide additional information about cytotoxic potency. With cycloheximide, for example, no decrease in K ÷ was found, but on the other hand a clear-cut effect on cellular growth was detected.

Conclusion

The data obtained so far with the human epidermal cell culture show that a further use of this in vitro technique with respect to mechanisms of action and metabolism of dermatotoxic agents may lead to useful results.

REFERENCES

Hopley J. P., Benford D. J. & Bridges J. W. (1985). Toxicity of a polychlorinated biphenyl in a differentiating skin cell-culture system (Abstract). Fd Chem. Toxic. 23, 325.

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Labarca C. & Paigen K. (1980). A simple, rapid and sensitive DNA assay procedure. Analyt. Biochem. 102, 344.

Malik J. K., Schwarz L. R. & Wiebel F. J. (1983). Assess- ment of membrane damage in continuous cultures of mammalian cells. Chemico-Biol. Interactions 45, 29.

Reinhardt Ch. A., Pelli D. A. & Zbinden G. 0985). Interpretation of cell toxicity data for the estimation of potential irritation. Fd Chem. Toxic. 23, 247.