Human keratinocyte cultures as models of cutaneous esterase activity

Pergamon

Toxicology in Vitro 11 (1997) 637-640

Human

Keratinocyte Cultures as Models Cutaneous Esterase Activity C. L. BARKER

of

and R. H. CLOTHIER

FRAME Alternatives Laboratory, Department of Human Queens Medical Centre, University of Nottingham,

Morphology, Nottingham

The Medical School, NC7 2UH, UK

Abstract-A reproducible, quantifiable assay has been developed for the measurement of esterase activity in human keratinocyte cultures, using the model substrate 4-methyl umbelliferyl heptanoate (MUH) which is hydrolysed to the fluorescent metabolite 4-methyl umbelliferone (MU). Activity was assessed in two human keratinocyte cell lines, NCTC 2544 and SVK-14, and in freshly isolated human activity in breast keratinocytes from primary culture to passage 3. V,., values for MUH hydrolysing the two cell lines showed that the less differentiated cell line NCTC 2544 (V,,, = 23.00 + 2.84) expresses a much higher activity than SVK-14s (V,,, = 13.28 + 1.42) which are more differentiated and able to form a cornified envelope. Activity in the freshly isolated human breast keratinocytes decreased with time in culture in all three donors tested, which is also likely to relate to the extent of cell differentiation. In human skin, xenobiotic esters penetrating the stratum corneum may be exposed to changing levels of hydrolysing esterases as they are absorbed across the epidermal cell layers. The assay for MUH hydrolysis will be a useful tool for the study of esterase activity in populations of human keratinocytes in vitro. ~0 1997 Published by Elsevier Science Ltd Abbreviations: HBSS = Hanks’ umbelliferyl heptanoate. Keywords: keratinocyte;

balanced

esterase;

salt solution;

umbelliferone;

MUH

= 4-methyl

metabolism.

INTRODUCTION

skin is increasingly being recognized as a major route of entry of chemicals into the systemic circulation. Moreover, it is no longer considered a largely inert barrier but an actively metabolizing organ (Hotchkiss, 1995). Frequent exposure to a wide range of xenobiotics including pharmaceutical products/pro-drugs, cosmetic and sunscreen ingredients, environmental pollutants and pesticides make the skin a major target for chemical toxicity. For a compound to be exposed to significant quantities of cutaneous metabolizing enzymes, it must first be absorbed across the lipophilic stratum corneum into the viable ceil layers. Metabolism by epidermal keratinocytes may then further influence the permeability kinetics of the compound, since parent chemical and metabolites may be absorbed at different rates depending on their physicochemical properties. Esterases are known to be present in skin at significant levels such that hydrolysis of xenobiotic esters may occur following topical exposure (McCracken et al., 1993). Hydrolysis during percutaneous absorption may influence not only the permeability kinetics of the compound, but also the The

0887-2333/97/$17.00 + 0.00 0 SSDI SO887-2333(97)00037-4

MU = 4-methyl

1997 Published

therapeutic or toxic actions of the parent molecule and its metabolites. Hydrolysis in skin is therefore an important aspect of hazard identification after topical exposure to esterified compounds. Difficulties in measuring metabolism in the skin in vivo have led in recent years to a search for appropriate in vitro alternatives (for review see 1996). Cultured keratinocytes with Bronaugh, esterase activity reflecting in vivo levels have a number of potential uses. They would allow a more informed prediction of the disposition of a given chemical within the skin to be made, including the influence of hydrolysis on the therapeutic and toxicological profile, and possible induction or inhibition of cutaneous esterases. MUH is an ideal model substrate for the study of esterase activity as it is cleaved by intracellular esterases to a fluorescent metabolite, MU, which is easily detected in situ (Stadler et ul., 1989). In this study, MUH hydrolysis was measured in two human keratinocyte cell lines, NCTC 2544 (with limited differentiating capacity) and SVK-14 (able to differentiate and form a cornified envelope). Activity was compared with that of freshly isolated human breast keratinocytes in primary culture and at passage numbers one, two and three.

by Elsevier Science

Ltd. All rights

reserved.

Printed

in Great

Britain

C.

638 IMATERIALS

L.

Barker

and R. H. Clothier

AND METHODS

Cell culture media and Hanks‘ balanced salt solution (HBSS) were obtained from Gibco,‘Life Technologies; Collagen I solution was obtained from ICN Biomedicals: all other reagents were purchased from Sigma Chemical Company. Krratirwq~tc~

isolutiotl

Skin was collected during breast reduction surgery and stored in keratinocyte serum free media at 4 C for approximately 24 hr before use. In a sterile environment, the skin pieces were placed into rinse solution A (20 pgjml gentamicin: IO klg.‘ml Fungizone in phosphate buffered saline) for 2 3min. The skin was then transferred to rinse solution B (2 ilg:rnl gentamicin; I fig/ml Fungizone in HBSS) until ready for use.

The skin was removed from rinse solution B using sterile forceps and stretched over a curved surface. Split-thickness sections were taken using a sterile skin graft knife. Stretching the skin over a curved surface resembles more closely its orientation irl r?ro. and allows for more satisfactory split-thickness sections to be taken without the formation of wrinkles. The sections of skin were then incubated at 4 C in protease (Dispase. 25 Uml) for IX 20 hr. The skin sections were removed to a petri dish containing a small amount of rinse solution B. The epidermis was peeled away from the dermis and placed into trypsin/EDTA at 37 C. After approximately I5 min. the cells which were in suspension were removed using a Pasteur pipette and centrifuged in keratinocyte media containing 10% foetal calf serum for

5 min at 1000 rpm. The cell pellet was resuspended in keratinocyte serum free media and seeded into culture flasks at a density of 4 x IO4 per cm’. The culture flasks had been precoated with 5.3 pg/cm’ Type I collagen to aid cell attachment. Cells were subcultured when approaching confluency to provide cells of increasing passage number for measurement of esterase activity without cryopreservation between experiments.

On day I. cells were seeded onto 24-well culture plates at a density of I x lO’/well (cell lines) or 3 x IO’:well (freshly isolated keratinocytes). The isolated cells were cultured on plates which had been precoated with 5.3 pg/cm’ type 1 collagen to aid cell attachment. The plates were maintained in a cell culture incubator at 37°C with 5% carbon dioxide. On day 3, cells were re-fed with fresh media. On day 4. media was aspirated and cells washed with pre-warmed HBSS. Test solutions of MUH were applied to triplicate wells at a range of concentrations (from 20 to 700 PM). Stock MUH was prepared in dimethyl sulfoxide then diluted I: 100 in a sodium bicarbonate buffer (pH 7.4) made up in HBSS and supplemented with 20 tnM HEPES. Investigations showed this buffering system to be necessary to resist pH fluctuations and therefore to maintain a stable fluorescence of MU. Control wells: MUH only (no cells): cells only (no MUH). The plates were returned to the cell culture incubator. Formation of the fluorescent hydrolysis product MU was measured using the CytofluorTM plate

600 [MUHI

= 350 FM

A

0

Time (min) Fig. I. MUH

hydrolysis

by human

keratinocyte

cell lines.

NCTC 2544

639

Esterase activity in keratinocytes Table I, Hydrolysis of MUH by human keratinwyte cell lines: comparison of V,, values

NCTC 2544

Cell line

V.,,,,(nmol

MlJ/mg protein/mitt) 23.00 + 2.84 (n = 5)

SVK-I4 13.28 + 1.42 (n = 3)

(360 nm,,/460 nm,,) at the following time points (min): 2, 5, 10, 15, 20, 30, 45, and 60. Substrate solutions were aspirated from the plate and all wells washed with HBSS. Sodium hydroxide (1 ml, 0.5 M) was added to each well and the plates were kept frozen until protein determination was carried out by the method of Lowry (Lowry et al., 1951). The amount of MU produced was calculated against a standard curve (at 37°C pH 7.4, in bicarbonate buffer). Esterase activity was expressed as nmol MU formed per mg protein per min. reader

RESULTS

In all cells tested (NCTC 2544, SVK-14 and freshly isolated human breast keratinocytes), an initial rapid rate of MUH hydrolysis, which was dependent on substrate concentration, was followed by a slower secondary rate which became evident after 20-30 min. Controls confirmed that MU formation in the absence of cells was negligible. A substrate concentration of 350 PM was found to saturate the enzyme, since increasing the concentration beyond

this level did not lead to a further increase in the initial rate of hydrolysis (data not shown). Therefore the initial rate of reaction at 350 PM MUH was used to calculate V,,, values (expressed as nmol MU produced per mg protein per min). Expression of results per mg protein allowed the amount of MUH hydrolysis to be standardized between experiments. A separate experiment with isolated keratinocytes (data not shown) had demonstrated that the total protein content per cell did not vary significantly between the differentiated and non-differentiated cells used in these studies. A comparison of V,,, values gave an indication of differing levels of the esterases responsible for MUH hydrolysis between the different cell types. The concentration-time profiles for MU production shown in Fig. 1 demonstrate a marked difference in activity between the two cell lines NCTC 2544 and SVK-14. The mean V,,, values shown in Table 1 confirm a much higher level of MUH hydrolysing activity in NCTC 2544 than in SVK-14 cells. Figure 2 demonstrates that the freshly isolated human breast keratinocytes displayed a fall in MUH hydrolysing activity with passage number in all three donors tested. The most noticeable drop occurred between primary culture and the passage 1 cells, where activity appeared to fall by over 50%. V,,, values were in a similar range to those of the keratinocyte cell lines but were variable between donors.

35

25

Donor 1

Donor 2

Fig. 2. Hydrolysis of MUH by human breast keratinocytes: V,,!, values.

Donor 3

C. L. Barker and R. H. Clothier

640 DISCUSSION

Mean values for V,,,,,, calculated for the two human keratinocyte cell lines, NCTC 2544 and SVK-14. showed low variability between experiments, indicating that the described assay for the hydrolysis of MUH is a reproducible measure of intracellular esterase activity. A clear advantage of this assay over previously published methods for the measurement of MUH hydrolysis (e.g. see Stadler rt al.. 1989). is the employment of a buffer system that maintains pH at 7.4 throughout the experiment-a crucial factor since the fluorescence of MU fluctuates considerably with relatively small changes in pH (unpublished results). Additionally, quantitation of MUH hydrolysis as nM MU formed per mg protein relates the measured changes to cell number, rather than quoting the reaction in fluorescence units only. The data presented may currently be interpreted in either of two ways. The clear difference in esterase activity between the two cell lines suggests that in human epidermis, the basal keratinocytes may express a higher level of esterase activity than cells in the more differentiated layers. The less differentiated cell line NCTC 2544 displayed a much higher rate of MUH conversion than SVK-14---a cell line which is thought to be more differentiated and can be stimulated to form a cornified envelope. In freshly isolated human breast keratinocytes. the decrease in esterase activity with passage number may also support this hypothesis. since all ceils in the population became visibly more differentiated with each passage. However. current knowledge of skin physiology strongly suggests that a different conclusion is more plausible. Keratinocytes in the granular layer are known to undergo a burst of hydrolytic activity just prior to and during their transition into a corneocyte (Holbrook, 1994). Hence it would be expected that esterase activity is higher in more differentiated keratinocytes. The data do not necessarily contradict this theory, since it is probable that a primary culture of human epidermal keratinocytes is comprised of a population of cells derived from all epidermal layers, that is, at various stages of differentiation.

However, the subculture procedure favours retention of keratinocytes derived from the basal layer, as a result of their mitotic capability. Hence, a selective retention of basal cells may explain the sudden drop in overall esterase activity observed between the primary culture and that of the passage I cells. The degree of heterogeneity in freshly isolated human keratinocytes in primary culture through to passage .7 is~ currently being investigated using immunohistochemical techniques. If the latter of the two theories described above is correct. the suitability of the two human keratinocyte cell lines, NCTC 2544 and SVK-14, for studies of epidermal esterase activity must be questioned until a more detailed assessment of their morphological characteristics and differentiation status has been carried out. The measurement of MUH hydrolysis by the method described here appears to be a useful tool for the study of overall esterase activity in human keratinocytes in vitro. Atknoll,ledgemen/~~The sponsors of FRAME

financial support of the industrial is gratefully acknowledged.

REFERENCES

Bronaugh R. L. (1996) Methods for in cirro skin metabolism studies. In Dermaroro.uicolog,. 5th Ed. Edited by F.N. Marzulli and H.I. Maibach. DD. 383-388. Tavlor & Francis. Washington. DC. Holbrook K. A. (1994) Ultrastructure of the epidermis. In T/IO Keratinoq~tc Handbook. Edited by 1. M. Leigh, E. Birgitte Lane and F. M. Watt. pp. 43-51. Cambridge University Press. Cambridge. Hotchkiss S. A. M. (1995) Cutaneous toxicity. Kinetic and metabolic determinants. To\-icolo~,~ and Ecoto.~icologj ,Yem 2, I&18. Lowry 0. H.. Rosebrough N. J.. Farr A. L. and Randall R. J. (195 I) Protein measurement with the Folin phenol rcagen t Jour-nal of’ Biologicnl Clwmistr~ 193, 265-275. McCracken N. W., Blain P. G. and Williams F. M. (1993) Nature and role of xenobiotic metabolizing esterases in rat liver. lung. skin and blood. Bioclwmical Pharmacolog? 45, 31 36. Stadler R.. Detmar M., Stephanek K., Bangemann C. and Orfanos C. (1989) A rapid fluorimetric assay for the determination of keratinocyte proliferation in oitro. Jrumal

of Inwstigatiw

Dermatology

93, 532-534.