Differential effects of 808-nm light on electron transport chain enzymes in isolated mitochondria: Implications for photobiomodulation initiation.

Photobiomodulation is a term for using low-power red to near-infrared light to stimulate a variety of positive biological effects. Though the scientific and clinical acceptance of PBM as a therapeutic intervention has increased dramatically in recent years, the molecular underpinnings of the effect remain poorly understood. The putative chromophore for PBM effects is cytochrome c oxidase. It is postulated that light absorption at cytochrome c oxidase initiates a signaling cascade involving ATP and generation of reactive oxygen species (ROS), which subsequently results in improved cellular robustness. However, this hypothesis is largely based on inference and indirect evidence, and the precise molecular mechanisms that govern how photon absorption leads to these downstream effects remain poorly understood. We conducted low-power PBM-type light exposures of isolated mitochondria to 808 nm NIR light, at a number of irradiances. NIR exposure was found to enhance the activity of complex IV, depress the activity of complex III, and had no effect on the activity of complex II. Further, examining the dose-response of complex IV we found NIR enhancement did not exhibit irradiance reciprocity, indicating the effect on complex IV may not have direct photochemical basis. In summary, this research presents a novel method to interrogate the earliest stages of PBM in the mitochondria, and a unique window into the corresponding molecular mechanism(s) of induction.

Wavelength- and irradiance-dependent changes in intracellular nitric oxide level.

SIGNIFICANCE: Photobiomodulation (PBM) refers to the beneficial effects of low-energy light absorption. Although there is a large body of literature describing downstream physiological benefits of PBM, there is a limited understanding of the molecular mechanisms underlying these effects. At present, the most popular hypothesis is that light absorption induces release of nitric oxide (NO) from the active site of cytochrome c oxidase (COX), allowing it to bind O2 instead. This is believed to increase mitochondrial respiration, and result in greater overall health of the cell due to increased adenosine triphosphate production. AIM: Although NO itself is a powerful signaling molecule involved in a host of biological responses, less attention has been devoted to NO mechanisms in the context of PBM. The purpose of our work is to investigate wavelength-specific effects on intracellular NO release in living cells. APPROACH: We have conducted in-depth dosimetry analyses of NO production and function in an in vitro retinal model in response to low-energy exposure to one or more wavelengths of laser light. RESULTS: We found statistically significant wavelength-dependent elevations (10% to 30%) in intracellular NO levels following laser exposures at 447, 532, 635, or 808 nm. Sequential or simultaneous exposures to light at two different wavelengths enhanced the NO modulation up to 50% of unexposed controls. Additionally, the immediate increases in cellular NO levels were independent of the function of NO synthase, depended greatly on the substrate source of electrons entering the electron transport chain, and did not result in increased levels of cyclic guanosine monophosphate. CONCLUSIONS: Our study concludes the simple model of light-mediated release of NO from COX is unlikely to explain the wide variety of PBM effects reported in the literature. Our multiwavelength method provides a novel tool for studying immediate and early mechanisms of PBM as well as exploring intracellular NO signaling networks.

Hyperthermia sensitizes pigmented cells to laser damage without changing threshold damage temperature.

We studied the efficacy of mild hyperthermia as a protective measure against subsequent laser-induced thermal damage. Using a well established in vitro retinal model for laser bioeffects, consisting of an artificially pigmented human retinal pigment epithelial (RPE) cell culture (hTERT-RPE1), we found both protection and sensitization to laser damage that depended upon the location of pigment granules during the hyperthermia preconditioning (PC). Photothermal challenge of cell monolayers consisted of 16 independent replicate exposures of 65 W/cm2 at 514 nm and post laser damage was assessed using fluorescence indicator dyes. Untreated cells had 44% damage, but when melanosome particles (MPs) were intracellular or extracellular during the hyperthermia treatment, laser-induced cell damage occurred 94% or 25% of the time, respectively. Using a recently published method called microthermography, we found that the hyperthermia pretreatment did not alter the threshold temperature for cell death, indicating an alteration in absorption or localization of heat as the mechanism for sensitization and protection. Raman microspectroscopy revealed significant chemical changes in MPs when they were preconditioned within the cytoplasm of cells. Our results suggest intracellular pigment granules undergo chemical modifications during mild hyperthermia that can profoundly affect absorption or heat dissipation.