Blue light-triggered photochemistry and cytotoxicity of retinal

Highlights

• Blue light-excited retinal generates ROS, induces oxidation of unsaturated lipids.

• Photoexcited-retinal disrupts subcellular localization of lipidated signaling molecules.

• Similar to blue light, retinal also triggers cellular damage with sunlight too.

• Retinal accumulation in cell membranes supports its photosensitization.

• Retinal photoexcitation induces DNA and mitochondrial damage, and cell death.

Abstract

The chemical- and photo- toxicity of chromophore retinal on cells have long been debated. Although we recently showed that retinal and blue light exposure interrupt cellular signaling, a comprehensive study examining molecular underpinnings of this perturbation and its consequences to cellular fate is lacking. Here, we report molecular evidence for blue light excited-retinal induced oxidative damage of polyunsaturated lipid anchors in membrane-interacting signaling molecules and DNA damage in cells using live-cell imaging and in vitro experimentation. The incurred molecular damage irreversibly disrupted subcellular localization of these molecules, a crucial criterion for their signaling. We further show retinal accumulation in lipid-bilayers of cell membranes could enhance the lifetime of retinal in cells. Comparative response-signatures suggest that retinal triggers reactions upon photoexcitation similar to photodynamic therapy agents and generate reactive oxygen species in cells. Additionally, data also shows that exposing retinal-containing cells to sunlight induces substantial cytotoxicity. Collectively, our results explain a likely in vivo mechanism and reaction conditions under which bio-available retinal in physiological light conditions damages cells.

Introduction

Toxicity of all-trans-retinal (ATR or retinal) has been implicated in vision disorders [[1], [2], [3]]. Though ATR and its condensation products (lipofuscins) are considered as factors for light-induced oxidative stress [[4], [5], [6]] and subsequent ocular photodamage [[7], [8], [9], [10]], their molecular underpinnings require investigation. ATR mediated NADPH oxidase activation, and the resultant reactive oxygen species (ROS) generation are attributed to oxidative mitochondrial damage in retinal pigment epithelial cells [1] while antioxidants have been shown to reduce the phototoxicity [5,6]. Additionally, ligand-binding G-protein coupled receptor (GPCR) activation by ATR, through an unknown mechanism, was proposed for oxidative DNA and mitochondrial damage [1,11]. Recently, we showed evidence for Blue-Light-Excited-retinal (BLE-retinal) induced distortion of phosphatidylinositol 4,5 bisphosphate (PIP2) on the plasma membrane (PM), and subsequent cellular signaling perturbation [12].

The possibility of ATR accumulation in lipid bilayers of photoreceptor disk-membranes, despite efficient retinal transport mechanisms, is likely to allow continuous exposure of cells to photoexcited retinal [13]. Additionally, defects in the retinal clearance mechanism may also allow ATR accumulation in RPE [14]. Non-visual cells of the body such as skin-cells are also exposed to retinal that enters circulation, although at much lower concentrations [15,16]. Retinal is also synthesized in situ in some cells via enzymatic conversion of retinol (vitamin A), which is abundant in the blood through dietary intake [17]. Therefore, cells in peripheral tissues can also be influenced by photoexcited retinal.

Ultraviolet (UV) light has sufficient energy to excite and damage a minor fraction of DNA in exposed cells, triggering sunburn and causing conditions including melanoma [[18], [19], [20], [21]]. Blue light, with its relatively lower energy than UV, however, has been shown to induce cellular, physiological and behavioral complications in animals, including macular degeneration, skin aging, and sleep-deprivation [[22], [23], [24]]. In addition, indoor-tanning associated extensive UV and blue light exposure are directly linked to non-melanoma and melanoma skin cancers [25]. Though blue light is extensively used in dentistry, polymer composites in implant medicine and in curing adhesives, potential health implications of such light exposures are yet to be investigated [26]. The upsurge of smartphones and digital screen usage is a major public concern since these devices emit blue light to generate colour images, and causes conditions including insomnia and Transient Smartphone Blindness (TSB) [27]. However, whether the prolonged-usage of these devices induces permanent vision damage is unclear.

Free retinal absorbs light spanning across far-UV to blue light with an absorption maximum of 381 nm [12]. Therefore, the blue region of visible light and the absorption spectrum of retinal significantly overlap. Unlike UV, neither cornea nor lens of the eye block this blue spectral window, and thus likely facilitates photoexcitation of bioavailable retinal. Therefore, molecular underpinnings of BLE-retinal in living cells are essential to comprehend the long-debated cytotoxic effects of retinal as well as blue light.

Here, we report mechanisms and reaction conditions for retinal photoexcitation upon blue light illumination in the cellular environment. Our results indicate that, upon blue-light-exposure, retinal perturbs subcellular localization of selected lipidated G-proteins including farnesylated Gγ and Ras G-proteins that may contribute to cellular photodamage. We also show retinal and blue light-induced ROS generation in lipid-bilayers of cell membranes induces peroxidation of polyunsaturated lipid anchors of signaling proteins. Additionally, we demonstrate extensive phototoxicity in cells exposed to sunlight, a natural source of blue light, only in the presence of retinal. Overall, the data presented here elaborate molecular underpinnings of cytotoxicity associated with blue light excitation of retinal in living cells and its broader physiological consequences.