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Protocol no. 44
CELL CULTURE PHOTOTOXICITY TEST

Human A431 cells and mouse 3T3 cells are exposed in culture to UV light both in the presence and absence of test compound. Phototoxicity is expressed as a decrease in cell viability as determined by the MTT assay.

CONTACT

Dr. P.A. Duffy
I.C.I. Pharmaceuticals Safety of Medicines Department Mereside Alderley Park Cheshire, SK10 4TG UK Tel: England - 625 512758

RATIONALE

Phototoxicity can be classified as an abnormal cutaneous response which occurs as a result of exposure to ultraviolet (UV) light or visible radiation and is associated with the presence of xenobiotic (which may be present locally or systemically in the body). This test is designed to mimic such a response by subjecting cell cultures to increasing periods of exposure to UV illumination in the absence and presence of test compounds (at concentrations previously demonstrated to have no cytotoxic effect). Subsequent, post-exposure, cell growth is determined using the MTT assay (the basis for which is outlined below). Increased exposure to UV light leads to an inhibition of cell growth and a decrease in cell viability. If this detrimental effect is significantly potentiated in the presence of a test chemical it may be considered a likely phototoxic agent. Use of the MTT assay The tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), is taken up into cells and reduced by a mitochondrial dehydrogenase enzyme to yield a purple formazan product which is largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. Solubilisation of the cells results in the liberation of the product which can readily be detected using a simple colorimetric assay. The intracellular reduction of MTT is thus indicative of the normal functional biochemistry of energy-requiring mitochondrial enzyme reactions and, more broadly, provides a means to estimate the number of viable cells.

BASIC PROCEDURE

Cytotoxicity Assay Monolayers of human A431 epidermal cells or mouse 3T3 cells are exposed to increasing concentrations of test chemical for 24 hours, after which time the cell number/viability is determined by the colorimetric MTT assay. The highest concentration of each chemical at which no reduction in cell viability is observed (compared to the control situation) is recorded as being the "no effect" concentration. Phototoxicity Assay Monolayers are incubated for 4 hours then exposed to UVA or a combination of UVA and UVB light for increasing periods of time, both in the absence and presence (at the "no effect" concentration) of test chemical. The cultures are incubated for a further 20 hours, after which viability is determined by the MTT assay. The formation of formazan product is plotted against the time of exposure to UV and the ID50 value, i.e. that dose of UV that reduces cell viability to 50% of control levels, calculated from the curves. The ID50 values in the absence and presence of test compounds are then compared. A compound is considered of phototoxic potential when the ID50 value in the presence of test compound is significantly less than that which occurs in its absence.

CRITICAL ASSESSMENT

This method provides a rapid, relatively inexpensive, simple to perform method for screening many compounds for potential phototoxicity. The test conditions can be strictly controlled with regard to time period of exposure and wavelength of light. UV light is used because it contains the wavelengths most often associated with phototoxic reactions. There is, however, a very small number of chemicals activated by light from within the visible part of the spectrum. The test avoids a number of problems associated with human testing such as variability due to skin thickness, pigmentation and pharmacokinetic and metabolic handling of the compound. These factors must, however, be considered when attempting to extrapolate and interpret results in relation to likely in vivo phototoxicity. The main difference to the in vivo situation is that the test is based on the exposure of test chemical in contact with only a monolayer of one type of cell. The cell lines used reflect the types of cell present at the site of activity, i.e. the skin. While the phototoxic insult is a biophysical one, and might therefore be expected to produce the same reaction in most types of cell, significant differences have nevertheless been observed in the sensitivities of A431 epidermal cells and 3T3 fibroblasts. This could be due to cell lineage specific biochemical characteristics. The use of two cell types reduces the chances of missing a specific phototoxin. A further consideration is the lack of a metabolic activation component. However, it appears from the literature that hepatic metabolism is not greatly involved in the production of molecules that are subsequently activated by UV light to phototoxic products. The porphyria type of photoinduced dermal reactions are one exception. If, however, hepatic metabolism was considered to be of potential significance in relation to a given test compound, the inclusion of an hepatic S9 microsomal metabolic activation system would only require slight modification of the protocol. The MTT assay as an end-point Formation of the formazan product has been found to correlate well with cell viability in terms of normal functioning of mitochondrial energy-requiring biochemical reactions. The assay compares favourably with several other methods used in the determination of cell number/viability e.g. dye exclusion. It is a rapid, sensitive, relatively simple to perform and lends itself to semi-automation. It should be noted, however, that the MTT assay is not readily adaptable for use with static cell populations or those of low mitochondrial activity. Certain compounds may selectively affect the mitochondria of the cells resulting in a greatly overestimated level of toxicity. A recent paper (Plumb et al., 1989) draws attention to the possible influence of pH on the MTT reaction. However, this effect should not be of any significance in the assay as used in the Phototoxicity Test.

TEST STATUS

Undergoing in-house development

CHEMICALS TESTED

p-Aminobenzoic acid (PABA)
Amiodarone
Aspirin
Benoxaprofen
Benzalkonium chloride
Benzoic acid
Chlorpromazine
Demeclocycline
Diazepam
Dinitrochlorobenzene (DNCB)
Diphenhydramine
Dithranol
Doxycycline
Ethynyloestradiol
Griseofulvin
Hexachlorophene
Hydrochlorothiazide
Hydrocortisone
Imipramine
7-Methoxycoumarin
5-Methoxypsoralen
8-Methoxypsoralen
Methyl-DOPA
Nalidixic acid
Piroxicam
Promethazine
Protryptyline
Tetracycline
Vinblastine
Vitamin A

REFERENCES

  1. Barnes, D. (1987) In vitro growth of A431 human epidermoid carcinoma. Methods in Enzymology, 146, 88-92.
  2. Duffy, P.A., Bennett, A., Roberts, M. & Flint, O.P. (1987) Prediction of phototoxic potential using human A431 cells and mouse 3T3 cells. Molecular Toxicology, 1, 579-587.
  3. Duffy, P.A., Bennett, A., Roberts, M. & Flint, O.P. (1989) The prediction of phototoxic potential using human A431 cells and mouse 3T3 cells. In: In Vitro Toxicology : New Directions (Alternative Methods in Toxicology, Vol. 7) (ed. Goldberg, A.M.) Mary Ann Liebert Inc., New York. pp. 327-335.
  4. Giard, D.J., Aranson, S.A. & Todaro, G.J. (1973) In vitro cultivation of human tumours: Established cell lines derived from a series of solid tumours. J. Natl. Cancer Inst., 1, 1417-1427.
  5. Hawk, J.L.M. (1984) Photosensitizing agents used in the United Kingdom. Clin. & Exp. Dermatol., 9, 300-302.
  6. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55-63.
  7. Plumb, J.A., Milroy, R & Kaye, S.B. (1989) Effects of the pH dependence of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide - Formazan absorption on chemosensitivity. Cancer Research, 49, 4435-4440
  8. Todaro, G.J. & Green, H. (1963) Qualitative studies of the growth of mouse embryo cells in culture and their development into established cell lines. J. Cell Biol., 17, 299-313.

IP-44 © June 199