Time and dose-related ultraviolet B damage in viable pig skin explants held in a newly developed organ culture system

Photochemistry and Photobiology, May 2001 by Rijnkels, Jolanda M, Whiteley, Larry O, van Henegouwen, Gerard M J Beijersbergen

Time and Dose-related Ultraviolet B Damage in Viable Pig Skin Explants Held in a Newly Developed Organ Culture System(para)

ABSTRACT

For facilitating photochemical and toxicological studies an ex vivo skin model was developed in our laboratory using skin from domestic pigs. The model comprised the use of a complete skin piece, including the dermis and stratum corneum, of bigger areas to make future topical applications easier. Fully differentiated skin explants (5 X 50 mm, thickness 5 mm) were irradiated with ultraviolet B (UVB; 1-10 kj/m^sup 2^; 6 W/m^sup 2^). Directly thereafter they were brought in culture (Dulbeccos modified Eagles medium containing hydrocortisone; air/liquid interface) for a maximum of 144 h. In nonirradiated skin explants, signs of tissue degeneration were observed after 48 h in culture (hematoxylin and eosin, light microscope). However, keratinocytes, isolated enzymatically (thermolysin and trypsin) at different time intervals in culture from nonirradiated skin explants showed negligible loss in viability (trypan blue exclusion) and increased apoptosis (terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphatase nick end labeling assay) for up to 72 h. Explants irradiated with a single dose of UVB showed a clear and reproducible dose- and time-dependent tissue degeneration, loss in keratinocyte viability and increase in apoptosis compared with nonirradiated explants at the same time interval. In conclusion, the presently designed ex vivo pig skin model can be a useful and cheap tool for future investigations of short-term UV-induced effects in combination with phototoxic and photoprotective compounds.

?Abbreviations: DMEM, Dulbeccos modified Eagles medium; DNase, deoxyribonuclease; EDTA, ethylenediamine-tetraacetic acid; Hepes, N-2-hydroxythylpiperazine-N'-2-ethane-sulfonic acid; MED, minimal erythema dose; PBS, phosphate buffered saline; PI, propidium iodide; RNase, ribonuclease; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphatase nick end labeling; UVB, ultraviolet B.

INTRODUCTION

Prolonged sun exposure is strongly related to phototoxic effects in the skin, such as irritation or sunburn (1), photoallergy (2), immune suppression (3,4), photoaging (5,6) and skin cancer (7,8). In the last few decades much attention has been given to skin cancer because of its high incidence in the Caucasian population. The deleterious effects of sunlight are mainly ascribed to ultraviolet B (UVB^; 280-320 nm) radiation (9), of which the majority is absorbed within the epidermis (10). It is clear that UVB effects may be enhanced or prevented by photosensitive (e.g. certain antibiotics) or photoprotective (e.g. antioxidants) compounds, respectively (11-13).

In understanding phototoxic skin effects and the interference of photosensitive or photoprotective compounds, various clinical and in vivo/vitro laboratory skin models are used. Interesting is the increasing use of domestic pigs. Over the years numerous studies have revealed a stronger physiological and morphological resemblance of pig skin with human skin than skins of other origin, such as rodents (14-- 16). A few research groups used in vivo domestic pigs for studying short-term cutaneous phototoxic UVB effects (17-- 20). Ethical, practical and financial considerations, however, urge the need of using ex vivo/in vitro models. Skin organ culture models are interesting because the epidermis is fully differentiated at the time of biopsy. Furthermore, uniform pig skin biopsies are easily and cheaply obtained from an abattoir, because pig skin is considered to be a waste product. Several ex vivo organ culture systems, sometimes preceded by in vivo oral or topical applications, with skin biopsies originating from humans (21-23), guinea pigs (24), mice (25) and even domestic pigs (26-29) have been developed over the years.

Notwithstanding these developments, most of these ex vivo skin culture systems are used for more clinical purposes, such as human tissue expansion, skin biology and skin diseases, and for pharmacological or toxicological purposes, such as percutaneous absorption studies and skin irritation tests. Because of the many benefits such ex vivo pig skin models may have for phototoxicology, we have implemented and adjusted an ex vivo pig skin organ culture model for short-term photochemical and toxicological studies. The adjustments were focused toward the use of the complete skin, consisting of the complete dermis, epidermis and stratum corneum, and of relatively big skin areas (1.5-2 cm^sup 2^ per explant). With these adjustments eventual dermal UV effects on epidermis are not ruled out and topical application of photosensitive or photoprotective compounds are made easier. For comparison, in most skin organ culture systems, most of the dermis is removed and areas used mostly range between 0.04 and 0.25 cm^sup 2^ per explant. To assess the quality and structural integrity of the skin explants during culture and after irradiation with UVB, the tissue morphology and possible appearance of epithelial necrotic cell death and apoptosis were monitored. With the help of these biological parameters an UVB dose-effect relationship was established.