Adverse outcome pathway on photochemical toxicity initiated by light exposure

Satomi Onoue, Yoshiki Seto

Laboratory of Biopharmacy, School of Pharmaceutical Sciences, University of Shizuoka

Corresponding Author: Satomi Onoue

Phototoxicity is an adverse reaction in the light-exposed tissues triggered by normally harmless doses of sunlight (Moore, 1998; Moore, 2002; Roberts, 2001).  Recently, high-intensity UV rays from the sun have reached the Earth’s surface with the destruction of the ozone layer, and interest in phototoxic events has increased enormously.  Notably, phototoxic reactions against exogenous agents are caused by the combined effects of environmental light and external agents, including drugs, cosmetics, and foods (Epstein, 1983; Stein and Scheinfeld, 2007).

In this AOP, the primary trigger for a compound to be considered with respect to potential to create photochemical and photobiological reactions is the absorption of photon energy from light ranging from 290 to 700 nm.  The extent of absorption depends on the wavelength of light and the type of absorbing chromophores in the light-exposed tissues.  A molecule is excited by absorption of photon energy, and the photoactivated molecule induces photochemical reactions via energy transfer (type I photochemical reaction) and free radical generation (type II photochemical reaction).  These photochemical reactions result in generation of radicals and reactive oxygen species, and the reactive species react with biomolecules.  Generated radicals of a target chemical bind to DNA and proteins, resulting in formation of these photo-adducts, and reactive oxygen species (ROS), including singlet oxygen and superoxide, induce oxidation of biomolecules.  These key events bring inflammatory events in the light-exposed tissues (Brendler-Schwaab et al. , 2004; Epstein and Wintroub, 1985; Quintero and Miranda, 2000).

This AOP describes the pathway of photochemical toxicity between attack of ROS generated from photoactivated chemicals to membranes and inflammatory events in light-exposed tissues.

The primary event in any photosensitization process can be the absorption of photons of the appropriate wavelength, which allows chromophore to reach an excited state.  The excitation energy is often transferred to oxygen molecules, followed by generation of ROS.  These appear to be the principal intermediate species in the phototoxic response.  In cells, this cascade gives rise to local oxidative stress and damage to genomic DNA, proteins, and lipids within cell membranes.  From the standpoint of risk assessment, ROS generation from photoirradiated chemicals has been considered to be one of key determinants in recognizing their phototoxic potential.  The ROS assay has been designed to assess photoreactivity of pharmaceuticals, of which the principle is to monitor types I and II photochemical reactions of the test chemicals when exposed to simulated sunlight.  This simple analytical test could be used to screen potential chemical scaffolds, leads, and candidate drugs to identify and/or select away from those having phototoxic potential.  The validation study for the ROS assay has been carried out by the Japan Pharmaceutical Manufacturers Association (JPMA), supervised by the Japanese Center for the Validation of Alternative Methods (JaCVAM).  The validation study indicates satisfactory outcomes in terms of transferability, intra- and inter-laboratory variability, and predictive capacity.  Thus, a negative result in this ROS assay would indicate a very low probability of phototoxicity, whereas a positive result would be a flag for follow-up assessment.  ROS assay was successfully adopted as ICH S10 guideline (2014) and OECD test guideline 495 (2019).

The focus of this AOP is on photochemical toxicity, especially photoactivation of target chemicals followed by generation of ROS.  ROS generated from photoirradiated chemicals can react with molecules on the membranes, including lipids and proteins, and the reactions may lead to inflammatory events in the UV-exposed tissues. 

Phototoxicity is an adverse reaction triggered by normally harmless doses of sunlight.  There are two types of photosensitive disorders, endogenous and exogenous phototoxicity, and the observable changes to the sunlight-exposed tissues are essentially detrimental, and include the following appearance; (i) immediate faint erythema during exposure, (ii) delayed erythemal responses, (iii) abnormal keratinisation and vacuolated cells, (iv) formation of desquamating layer, and (v) desquamation (peeling) (Moore, 1998; Moore, 2002; Roberts, 2001).  Recently, high-intensity UV rays from the sun have reached the Earth’s surface with the destruction of the ozone layer, and interest in phototoxic events has increased enormously.  Notably, phototoxic reactions against exogenous agents are caused by the combined effects of UV irradiation and external agents, including drugs, cosmetics and foods (Stein and Scheinfeld, 2007).  Phototoxic skin responses after administration of photosensitive drugs, so-called drug-induced phototoxicity, have been recognized as undesirable side effects, and several classes of drugs, even when not toxic by themselves, may become reactive under exposure to environmental light, inducing undesired phototoxic responses (Epstein, 1983).

The primary trigger for a compound to be considered with respect to potential to create photochemical and photobiological reaction is the absorption of UV and visible light ranging from 290 to 700 nm.  The extent of absorption depends on the wavelength of light and the type of absorbing chromophores in the UV-exposed tissues.  UV radiation is usually divided into several ranges based on its physiologic effects: (1) UVA (near UV): 320–400 nm (UVA I: 340–400 nm and UVA II: 320–340 nm), (2) UVB (middle UV): 290–320 nm, and (3) UVC (far UV): 180–290 nm (Svensson et al., 2001; Vassileva et al., 1998).  The sun emits ultraviolet radiation in the UVA, UVB, and UVC bands, but because of absorption by the atmosphere's ozone layer, the main ultraviolet radiation that reaches the Earth's surface is UVA (Dubakiene and Kupriene, 2006).  Absorption of light through the skin and eyes, primarily in the 290–700 nm range, varies with wavelength, such that light in the red region of the spectrum reaches well into the subcutis layer; whereas at 300 nm or shorter wavelength, only an estimated 10% passes through the epidermis (Epstein, 1989).  Thus, penetration and absorption of light in the UV-exposed tissues is important factor in drug-induced phototoxicity as Grotthus-Draper law of photobiology states; only light that is absorbed can be active in photochemical and photobiological processes.

When a drug molecule absorbs a photon energy, electrons can be prompted from occupied orbitals (the ground state) to an unoccupied orbital (S1, S2) depending upon bond type and associated energy level.  Furthermore, unpaired singlet state electrons (opposite spin) may be converted to triplet state (parallel spin) by inversion of the spin via intersystem crossing of the absorbed energy.  To return to the ground state from S1, S2/T1, T2, energy must be dissipated by internal conversion, fluorescence (from singlet state), phosphorescence (from triplet state) or via chemical reaction, giving rise to photoproducts and/or potential external reactions with biomolecules.

In addition, molecular oxygen, a triplet radical in its ground state, appears to be the predominant acceptor of excitation energy as its lowest excited level (singlet state) has a comparatively low value.  An energy transfer from excited triplet photosensitizer to the oxygen (type II photochemical reaction) could produce excited singlet oxygen which might, in turn, participate in a lipid- and protein-membrane oxidation or induce DNA damage.  An electron or hydrogen transfer could lead to the formation of free radical species (type I photochemical reaction), producing a direct attack on the biomolecules or in the presence of oxygen, to evolve towards secondary free radicals such as peroxyl radicals or the very reactive hydroxyl radical, a known intermediate in the oxidative damage of biomolecules.  This toxic pathway corresponds to successive reactions which involve the appearance of superoxide anion radical, its dismutation to from hydrogen peroxide followed with the hydrogen peroxide reduction to form hydroxyl radical.  Herein, excitation of the drug by light may give rise to ROS such as singlet oxygen and superoxide, which may be one of causative molecules for the drug-induced phototoxicity (Brendler-Schwaab et al., 2004; Epstein and Wintroub, 1985).

The MIE and KEs in this AOP could contribute to assays development for photosafety evaluation and an AOP-based IATA construction.  AOP-based IATA can be applied for various aims including screening of chemicals, prioritization of chemicals for further testing, and risk assessment. 

The regulatory applicability of the AOP would be to use experimental results from assays based on MIE and KEs as indictors for the risk of phototoxic reactions. 

Combined use of photobiochemical properties and tissue exposure data would be of help for photosafety evaluation of chemicals.  Risk assessment would be possible when exposure data in light-exposed tissues combined with assay data based on AOP.

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