Phototoxicity

Phototoxicity is a phenomenon known in live-cell, where illuminating a fluorescent molecule (the fluorescently active site is called a fluorophore) causes the selective death of the cells expressing it.

In fluorescence microscopy

While not completely understood, it seems to be clear that the main cause for phototoxicity is the formation of oxygen radicals due to non-radiative energy transfer.

Typically in fluorescence, photons of a certain wavelength excite electrons of the illuminated fluorophore to higher energy states. When these excited electrons return to a lower energy state, they emit a photon with a lower energy level thus causing the emission of light of a longer wavelength. This principle of fluorescence is also known as Stokes shift.

Unfortunately for microscopists, in many cases some of the energy is not used for this radiative energy transfer but is transferred to oxygen causing the formation of oxygen radicals. These radicals are highly toxic to living cells, sometimes killing cells in seconds.

Phototoxicity in live cells depends strongly on the kind of fluorescent molecule used. The isolation and characterization of fluorescent proteins such as green fluorescent protein (GFP) has provided biologists with fluorochromes which show a much weaker phototoxic effect compared to most smaller chemically synthesized fluorescent molecules such as FITC or rhodamine. Still, the energy level of excitation light as well as the duration of illumination must be minimized to ensure long-term survival of living cells during fluorescent imaging.

In humans

Phototoxic substances

A phototoxic substance is a chemical compound which becomes toxic only when exposed to light.

• Some medicines: Tetracycline antibiotics, Methyl aminolevulinate
• Some cold pressed essential oils: bergamot oil
• Some plant juices: from parsley, Hogweed

Toxicology Testing

3T3 Neutral Red Phototoxicity Test - An in vitro toxicological assessment test used to determine the cytotoxic and photo(cyto)toxicity effect of a test article to murine fibroblasts in the presence or absence of UVA light.

"The 3T3 Neutral Red Uptake Phototoxicity Assay (3T3 NRU PT) can be utilized to identify the phototoxic effect of a test substance induced by the combination of test substance and light and is based on the comparison of the cytotoxic effect of a test substance when tested after the exposure and in the absence of exposure to a non-cytotoxic dose of UVA/vis light. Cytotoxicity is expressed as a concentration-dependent reduction of the uptake of the vital dye - Neutral Red.

Substances that are phototoxic in vivo after systemic application and distribution to the skin, as well as compounds that could act as phototoxicants after topical application to the skin can be identified by the test. The reliability and relevance of the 3T3 NRU PT have been evaluated and has been shown to be predictive when compared with acute phototoxicity effects in vivo in animals and humans." Taken with permission from [1]

This is a relatively new assay that was recently adopted by regulatory agencies such as OECD and FDA as an accepted method for the assessment of phototoxic potential of test substances.

Other testing methods for the assessment of phototoxic potentional are available, such as in vitro toxiciology models are currently being developed and explored that use reconstituted tissues and new photo-sensitization testing models also exist that are used to determine how a compound may react after being applied to the skin and irradiated with solar simulated light.

Related External Links:

In Vitro Phototoxicity Test

ICCVAM 3T3 Neutral Red Phototoxicity Testing Page

3T3 NRU Phototoxicity Test