Cutaneous toxicity testing in organ culture: Neutral red uptake and reduction of tetrazolium salt (MTT)

Toxic. in Vitro Vol. 7, No. 1, pp. 81-86, 1993 Printed in Great Britain.All fights reserved

0887-2333/93$6.00+ 0.00 Copyright © 1993PergamonPress Ltd

CUTANEOUS TOXICITY TESTING IN ORGAN CULTURE: NEUTRAL RED UPTAKE AND REDUCTION OF TETRAZOLIUM SALT (MTT) J. J. M. VANDE SANDT,A. A. J. J. L. RUTTEN and H. B. W. M. KO'~'ER* TNO Toxicology and Nutrition Institute, Department of Biological Toxicology, PO Box 360, 3700 AJ Zeist, The Netherlands (Received 27 May 1992; revisions received 3 August 1992)

Abstract--Rabbit full thickness skin can be maintained viable for 7 days using a two-compartment organ culture model. We report that this model is useful for assessingcutaneous toxicity. 11 chemicalswere each applied topically and then cytotoxicity was determined using two different assays. Mitochondrial activity was assessed by measuring reduction of the tetrazolium salt MTT, while uptake of the vital dye neutral red was used as a parameter of membrane damage. Conversion of MTI" was inhibited in a dose-dependent way by all of the chemicals tested except dimethyl sulphoxide. Furthermore, when the skin explants were kept in culture after exposure to the test agents, both repair of initial damage and delayed toxicity could be observed to some extent. Uptake of neutral red was affected dose-dependently by six out of the 11 chemicals tested. The data indicate that a strong correlation exists between the cytotoxicity of chemicals and their effect on the conversion of MTT in skin organ culture. The MTT assay reported here offers the possibility of studying both acute and delayed cytotoxicity, and to some extent, recovery from initial damage.

INTRODUCTION

cellular enzymes into culture medium correlated well with histological changes of the skin after topical application of chemicals (Bartnik et al., 1990; Helman et al., 1986; Kao et al., 1983). Dermal toxicity was also associated with the inhibition of [14C]leucine incorporation into proteins of skin explants (Kao et al., 1983; Moore et al., 1986). Oliver and Pemberton (1985) developed a method to identify corrosive compounds by measuring changes in electrical resistance of epidermal slices. Uptake of neutral red is a well known parameter of cytotoxicity. Neutral red (NR) is a vital dye which accumulates in the lysosomes of viable cells. Treatments causing membrane damage inhibit the accumulation of this dye. NR uptake is used in cell cultures to assess chemosensitivity (Borenfreund and Puerner, 1985; Hockley and Baxter, 1986), viral cytopathogenicity (Finter, 1969) and immunotoxicity (Miillbacher et al., 1984). Another assay that is widely used to assess cytotoxicity in monolayer cultures is the MTT assay (Mossman, 1983). The tetrazolium salt MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is reduced in viable cells to a purple coloured MTT-formazan precipitate by mitochondrial dehydrogenase enzymes. Recently, we reported a two-compartment organ culture, in which rabbit skin discs remain viable for 7 days (Rutten et al., 1990). Dermal and epidermal histomorphology, as well as on terminal differentiation of the keratinocytes, were observed in a degree similar to the situation in vivo. Using this model, several parameters of toxicity were examined,

Skin toxicity testing of chemicals for registration purposes and labelling is currently being carried out in vivo according to national and international guidelines based on the method described by Draize et al. (1944). In order to reduce animal discomfort, there is a need for reliable, preferably simple and expensive, in vitro alternatives to animal testing. A variety of in vitro systems has been developed to study the effects of chemicals on the skin. These range from keratinocyte cultures (Babich and Borenfreund, 1990; Cohen et al., 1991) or keratinocytes seeded on a substrate containing fibroblasts (Triglia e t al., 1991) to organ cultures (Bartnik et al., 1990). However, conventional cell cultures are limited to testing compounds in an aqueous environment. Organ cultures and reconstructed epidermis have the advantages of the presence of differentiating keratinocytes and the possibility of topical application of test compounds. Furthermore, an intact stratum corneum provides a physiological barrier between chemicals and living cells. Different parameters for cutaneous toxicity have been described using organ cultures. Leakage of *Present address: OECD, Environment Directorate, 2 rue Andrr-Paseal, 75775 Paris Cedex 16, France. Abbreviations: CDNB = 1-chloro-2-4-dinitrobenzene; DMSO = dimethyl sulphoxide; FCS = foetal calf serum; MTT-70 =concentration of test substance that decreased MTT conversion by 70%; NR = neutral red; NR-70 = concentration of test substance that decreased NR uptake by 70%; PBS = phosphate buffered saline; SDS = sodium dodecyl sulphate. 81

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including leakage of lactate dehydrogenase and transdermal penetration of fluorescein (van de Sandt et al., 1991). The objective of the present study was to evaluate further the usefulness of organ culture for assessing cutaneous toxicity. Both the N R and the MTT assay were modified for the organ culture model. Dose- and time-response relationships were investigated after the topical application of test agents from a variety of different classes. MATERIALS AND METHODS

Chemicals. The following chemicals were selected for testing: ethanol (Nedalco, Bergen op Zoom, The Netherlands), hexachlorophene (Aldrich Chemic, Brussels, Belgium), and sodium hydroxide (NaOH), hydrochloric acid (HC1), citric acid, dimethyl sulphoxide (DMSO), formaldehyde, glutaraldehyde, sodium dodecyl sulphate (SDS), l-chloro-2,4-dinitrobenzene (CDNB) and Tween-20 (all purchased from Merck-Schuchardt, Amsterdam, The Netherlands). All chemicals were dissolved in demineralized water, except for CDNB and hexachlorophene which were dissolved in propylene glycol (Boom b.v., Meppel, The Netherlands). Skin organ culture. Organ culture of rabbit skin was carried out according to the method described by Rutten et al. (1990). In short, the dorsal skin of 2.5-3.0 kg male rabbits (New Zealand White, E N K I Rabbit Farm, Someren, The Netherlands) was clipped free of hair 24 hr before the animal was killed by an iv overdose of Nembutal (Ceva, Paris, France). After dissecting the skin, subcutaneous fat was carefully removed. Circular discs (2 cm 2) were punched out and were washed three times in RPMI-1640 medium (Flow Laboratories, Rickmansworth, Herts., UK) supplemented with 2.5 mg Fungizone/ litre, 50 mg gentamicin/litre and 2 mM-L-glutamine (all from Flow). Skin explants were cultured on Millicell-HA inserts (Millipore Corp., Bedford, MA, USA) placed in six-well plates in a humidified incubator gassed with 5% Co2, 40% 02 and 55% N 2. The six-well plates were rocked approximately nine times per minute. Only the dermal side of the skin was in contact with the culture medium (RPMI-1640 supplemented with 10% foetal calf serum (FCS; Flow), 2 mM-L-glutamine and 50 mg gentamicin/litre). The culture medium was renewed every 24 hr. Optimizing o f the M T T and NR assays. Before testing different chemical compounds for their cutaneous toxicity, the N R and MTT assays were adapted for organ culture. The incubation period and the concentration of the dyes were optimized. During the incubation of the skin explants with MTT or NR, culture medium without FCS, but with L-glutamine and gentamicin, was used. After 1 day of culture, non-exposed skin explants were incubated in 3 ml culture medium, containing either MTT or NR. MTT (Sigma, St Louis, MO, USA) was tested at final concentrations of 0.5, 1.0,

2.0 or 4.0 mg/ml. N R (Sigma) was tested at three concentrations. Culture medium containing 0.02% (w/v) N R was incubated overnight at 37°C, after which NR crystals were removed by centrifugation (10 minutes, 400g). The saturated supernatant was diluted 0, 2.5 or 5 times with culture medium. After 2, 4, 8 or 24 hr of incubation, the skin discs were washed twice in 0.1 u-phosphate buffered saline (pH 7.4; PBS). The MTT-formazan precipitate was extracted from the tissue in a polycarbonate/ polypropylene tube (Nunc Products, Roskilde, Denmark) containing 5ml acidified isopropanol (0.04 N-HC1). After an extraction period of at least 48 hr, the optical density at 560 nm (OD560) was measured on an SP8-400 UV/VIS spectrophotometer (Pye Unicam Ltd). N R was extracted from the tissue in the same way as MTT, using an acidified aqueous solution with 50% ethanol (0.02 N-acetic acid); optical density was measured at 540 nm (OD540). Application of the test substances. Test substances were applied topically 1 day after isolation of the skin. The epidermal side of the skin discs was carefully dried using prefilters (Millipore, filter type AP10). Subsequently, fresh prefilters were put on the epidermal side together with 160 pl of the test solution. This volume is equivalent to that used in the in vivo test (0.48 ml/cm2). After 4 hr, the filters were removed. Each skin disc was washed three times with 0.1 M-PBS while remaining in the insert units. When skin explants were cultured for a prolonged time period after exposure, fresh culture medium was added to the wells. The MTT assay was carried out 0, 24 and 48 hr after removal of the test compound. The skin discs were incubated with 2.0 mg MTT/ml culture medium for 4 hr. The NR assay was performed directly after removal of the test substance. A concentration of 0.02% (w/v) N R was used; the incubation period was 4 hr. In all experiments, three skin pieces were used to determine the value at each experimental point. The mean OD560 or OD~o of the control (vehicle) was set to represent 100% viability. Results were plotted as percentage of untreated control versus the concentration of the test agent on a log scale, and MTT-70 and NR-70 were determined directly from the graphs. The mean coefficient of variation for the MTT assay was 13.2+9.6 ( n = 4 3 ) ; for the NR assay it was 8.4 + 4.3 (n = 48). RESULTS

The MTT and N R assays were optimized for organ culture conditions. MTT conversion clearly increased with time and concentration (Fig. I). When combinations of long incubation times and high MTT concentrations were used, the formazan product was not restricted to the skin tissue, but was also released into the culture medium. Therefore, medium containing 2.0 mg MTT/ml and an incubation period of 4 hr

In vitro cutaneous toxicity testing 3.5"

83

Table 1. Concentrations of selected chemical compounds, extrapolated from dose-response relationships, giving 70% of MTT conversion (MTT-70) or 70% reduction in neutral red uptake (NR-70) in comparison with the controls (measurements were performed directly after removal of the test compound) Dose range MTT-70 NR-70 (%, w/v) (%) (%)

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Severe Severe Moderate Mild Mild Moderate Severe Moderate Severe Mild Mild

DMSO = dimethyl sulphoxide NC = not calculated SDS = sodium dodecyl sulphate CDNB = 1-chloro-2,4-dinitrobenzene *Data from Registry of Toxic Effects of Chemical Substances (on-line database, 1992). #MTT-70 or NR-70 could not be calculated.

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Fig. I. Effect of the incubation time on MTT-formazan production. Skin discs, unexposed to test chemicals, were incubated in medium containing MTT at 0.5 (O), 1.0 (O), 2.0 ( V ) or 4.0 (F'l) mg/ml. Results are expresseeJ as the mean of three measurements and range bars indicate the SEM. w a s selected, providing a combination of high sensi-

tivity of the assay and feasibility. Uptake of N R was also dependent on the concentration in the culture medium (Fig. 2). However, consistently less N R was

extracted after 24 hr of incubation than after 8 hr. In further studies, 0.02% N R was used, with an incubation period of 4 hr. Directly after exposure to the test chemicals, cytotoxicity was measured and expressed as the concentration of these test compounds that resulted in a 70% reduction in MTT conversion (MTT-70) or NR uptake (NR-70) in comparison with control values (Table 1). Dose-response relationships for several test compounds are given in Fig. 3 (MTT assay) and Fig. 4 (NR assay). MTT-formazan production was 110100-

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Fig. 2. Effect of the incubation time on neutral red absorption. Skin discs, unexposed to test chemicals, were incubated in medium containing neutral red (NR) at 0.02 (O), 0.008 ( 0 ) or 0.004 (~7) %, w/v. Results are expressed as the mean of three measurements and range bars indicate the SEM.

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Fig. 3. Effect of various chemicals on the reduction of MTT. Increasing concentrations (%, w/v) of test compounds were applied topically to skin explants ( n = 3 ) for 4hr. MTT-formazan production was determined immediately after the removal of the test compound ( l l ethanol, A formaldehyde, O glutaraldehyde, • SDS, • CDNB, + Tween-20, + hexachlorophene).

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Fig. 4. Effect of various chemicals on the absorption of neutral red. Increasing concentrations (%, w/v) o f test c o m p o u n d s were applied topically to skin explants (n = 3) for 4 hr. Neutral red uptake was determined immediately after the removal of the test c o m p o u n d ( I ethanol, A formaldehyde, O glutaraldehyde, • SDS, • C D N B , + Tween-20).

i n h i b i t e d in a d o s e - d e p e n d e n t w a y b y all o f t h e chemicals tested except DMSO. Hexachlorophene e x p o s u r e i n d u c e d o n l y a slight d e c r e a s e o f M T T formazan formation; application of a 10% solution c a u s e d a 1 3 % r e d u c t i o n o f t h e c o n t r o l v a l u e (Fig. 3). W h e n t h e test c o m p o u n d s were a s s e s s e d in t h e N R a s s a y , five o u t o f 11 c h e m i c a l s d i d n o t r e d u c e t h e r e s p o n s e in a d o s e - d e p e n d e n t w a y . T h e s e c o m p o u n d s were h y d r o c h l o r i c acid, citric acid, D M S O , C D N B and hexachlorophene.

Table 2. Concentrations of selected chemical compounds, extrapolated from dose-response relationships, giving 70% of the MTT conversion (MTT-70), in comparison with the vehicle control (the test was carried out at 0, 24 and 48 hr after removal of the test compound)

Fig. 5. Effect of citric acid on M T T reduction at several timepoints after removal of the chemical from the skin cultures. Citric acid was applied topically to skin explants for 4 h r at 0.01 (O), 0.I (O), 1.0 ( V ) or 10.0 (lq) % (w/v). Results are expressed as the mean of three measurements and range bars indicate the SEM. I n a d d i t i o n to m e a s u r e m e n t o f toxicity directly a f t e r e x p o s u r e , t h e M T T a s s a y w a s c a r r i e d o u t at 24 a n d 48 h r a f t e r r e m o v a l o f t h e test c o m p o u n d s . T h e 110 100 o

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DMSO = dimethyl sulphoxide NC = not calculated SDS = sodium dodecyl sulphate CDNB ~ l-chloro-2,4-dinitrobenzene ND = not determined *MTT-70 could not be calculated.

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Fig. 6. Effect of SDS on M T T reduction at several time points after removal o f the chemical from the skin cultures. SDS was applied topically to skin explants at 0. l (O), 1.0 ( 0 ) or l0 ( ~ ) % (w/v). Results are expressed as the mean o f three measurements, and range bars indicate the SEM.

In vitro cutaneous toxicity testing

results showed that, depending on the time period between exposure and testing, MTT-70 values could vary considerably (Table 2). Repair of inflicted mitochondrial damage was observed after exposure to 1% citric acid: the initial 55% reduction of MTT conversion was restored to control values within 48 hr (Fig. 5). In contrast, with SDS (Fig. 6) or hexachlorophene (Table 2), cytotoxicity increased with time after exposure. DISCUSSION MTT-formazan production by the skin explants increased with increasing incubation time and increasing concentration of MTT in the culture medium. Extraction of NR from the skin tissue also increased with increasing concentration, but was consistently lower after 24 hr of incubation than after 8 hr of incubation. This may be due to lysosomal degradation of the dye. These results indicate that incubation time must be exactly defined in order to make comparison between tests possible. After topical exposure to test compounds, MTT conversion was inhibited in a dose-dependent way by all of the chemicals tested except DMSO. This solvent is known to have a low irritation capacity in vivo (Smyth et al., 1962). In the NR assay, five of the selected test compounds did not show a cytotoxic response: not only DMSO, but also CDNB, hexachlorohene, HCl and citric acid did not reduce N R uptake in a dose dependent manner. The reason for this difference in outcome between the two assays is uncertain, but may be the result of specific mitochondrial damage inflicted by these four chemicals. Another possible explanation is that macroscopicai swelling of the skin tissue, occurring after exposure to these chemicals, may enhance the extracellular retention of neutral red by the skin explants. For the test chemicals that were found to be clearly cytotoxic in both assays, MTT-70 and NR-70 values were of the same order of magnitude. This observation is in agreement with results of others. Borenfreund et al. (1988) found a good correlation between the two parameters for most of their test agents, using 3T3 fibroblasts. Triglia et al. (1991) tested several chemicals in a three-dimensional dermal model, containing human keratinocytes and fibroblasts. After application of the chemicals in the medium, no differences were found between NR-50 and MTT-50 values in this system. In both systems, however, cytotoxicity was induced at 30-270% lower concentrations than in our model, using SDS, Tween-20 and ethanol as test compounds. This difference may be due to the fact that when the test compound is applied topically in organ culture, the stratum corneum forms a barrier between the test compound and the living cells. Moreover, we applied the test chemicals for 4 hr, whereas both Triglia et al. (1991) and Babich and Borenfreund (1990) performed a f

85

much more extended exposure time (12 and 24hr, respectively). The MTT test was carried out at several time intervals after removal of test compounds. MTT-70 values decreased significantly with time for hexachlorophene and SDS (Table 2). Directly after exposure to hexachlorophene, a maximum of 13% inhibition of MTT conversion was observed at the concentration range between 0 and 10% (Fig. 3). After an additional culture period of 24 hr, however, MTT-70 had decreased to 0.1% (Table 2). This delayed toxic effect is also described in vivo for hexachlorophene, where irritation increased with time and reached a peak 96-120 hr after application (Morikawa et al., 1974). Subsequent to SDS exposure in our model, an increase in effect was also observed when the skin was cultured for an additional time of 48 hr (Fig. 6). Small amounts of the applied chemicals, left in the skin tissue after washing, may cause cell death at a later time. In contrast to this, repair of the initial damage was observed after application of citric acid (Table 2); 1% of this compound caused a decrease of 55% in MTT conversion, but after 48 hr, mitochondrial activity was restored to control values (Fig. 5). These results imply that it may not be sufficient to assess an in vitro response to a toxicant at only one time point. The assessment of dermal toxicity of chemicals may be improved by including more time points, similar to those used in the in vivo Draize test. Conclusions

The two-compartment skin organ culture model is applicable for assessing dermal toxicity. MTT conversion was found to be a simple and reliable parameter for dermal toxicity after topical application of several selected test chemicals. Both repair of initial damage and delayed toxicity could be detected to some extent when skin explants were cultured for a prolonged time after exposure. NR uptake was affected by only six of the 11 compounds tested, and may therefore not be the most suitable parameter for assessing toxicity in skin organ culture. authors wish to thank B. G. A. G. G. Be~luet-Passelecq, J. M. Westenend, and A. M. Tieleman for their excellent technical assistance, and Professor Dr V. J. Feron and Dr A. H. Penninks for helpful discussions. This study was supported by the Dutch Interdepartmental Platform to Alternatives to Animal Studies. Acknowledgements--The

REFERENCES

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