Immediate and Early Events Associated with Photobiomodulation in RPE Cells

Abstract

Purpose : Several marketed FDA-approved devices claim beneficial effects due to the photobiomodulation (PBM) effect. Most researchers in the field believe these effects are mediated by light absorption in mitochondria but no cellular mechanisms have been definitively resolved. We performed systematic studies using ultrafast and steady-state spectroscopic methods, and fluorescence microscopy, to determine which cellular components absorb light to generate PBM effects.

Methods : We used cultured retinal pigment epithelial (RPE) cells (hTERT-RPE1), and the mitochondria isolated from them (Qiagen kit) to investigate the metabolic effects and the primary chromophores for low irradiance light at various wavelengths. Photo-oxidation occurred at wavelengths expected to generate reactive oxygen species (ROS) and some wavelengths where it was unexpected. Reduction/Oxidation (ReDox) states of active mitochondria was measured with resonance Raman (RR) spectroscopy of reduced cytochrome c. We followed oxidative phosphorylation in mitochondria using both respirometry (O₂ consumption) and FT-IR absorption of steady-state CO₂ production. Femtosecond transient absorption of purified electron transport chain (ETC) enzyme complexes was used to identify low level light-induced effects on heme Soret band electronic transitions.

Results : We show that ROS, and/or reactive nitrogen species (RNS) were generated from blue and green wavelength exposures to RPE cells, but not from the red or IR wavelengths associated with PBM effects. The RR experiments with cytochrome c supported unexpected results from green exposure to isolated mitochondria, and along with the measurement of CO₂ production was effective at measuring substrate-dependent ETC and tricarboxylic acid cycle (TCA) activities, respectively. We found that prior low level red and blue light exposures to mitochondria altered their rate of ETC substrate utilization relative to unexposed mitochondria. Ultrafast transient absorption could distinguish between oxidized and reduced forms of cytochrome c, and detected long-lived transients in Complex III of the ETC.

Conclusions : Low level green light exposures of cells unexpectedly generated ROS/RNS and altered mitochondrial activity. Low level red light efficiently increased the rate of ETC and TCA cycle, confirming that mitochondria play a key role in the PBM response.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.