Cytotoxic responses to 405 nm light exposure in mammalian and bacterial cells: Involvement of reactive oxygen species

Praveen Ramakrishnan,Michelle Maclean,Scott J Macgregor,John G Anderson,M Helen Grant

doi:10.1016/j.tiv.2016.02.011

Praveen Ramakrishnan, Michelle Maclean + Show 3 more

Open Access

https://doi.org/10.1016/j.tiv.2016.02.011

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Abstract

Light at wavelength 405nm is an effective bactericide. Previous studies showed that exposing mammalian cells to 405nm light at 36J/cm2 (a bactericidal dose) had no significant effect on normal cell function, although at higher doses (54J/cm2), mammalian cell death became evident. This research demonstrates that mammalian and bacterial cell toxicity induced by 405nm light exposure is accompanied by reactive oxygen species production, as detected by generation of fluorescence from 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate. As indicators of the resulting oxidative stress in mammalian cells, a decrease in intracellular reduced glutathione content and a corresponding increase in the efflux of oxidised glutathione were observed from 405nm light treated cells. The mammalian cells were significantly protected from dying at 54J/cm2 in the presence of catalase, which detoxifies H2O2. Bacterial cells were significantly protected by sodium pyruvate (H2O2 scavenger) and by a combination of free radical scavengers (sodium pyruvate, dimethyl thiourea (OH scavenger) and catalase) at 162 and 324J/cm2. Results therefore suggested that the cytotoxic mechanism of 405nm light in mammalian cells and bacteria could be oxidative stress involving predominantly H2O2 generation, with other ROS contributing to the damage.

Highlights

Post-incubation, the broth was centrifuged at 3939 × g for 10 min, and the pellet re-suspended in 100 ml PBS, giving a population density of approximately 109 colony forming units (CFU)/ml. 2 ml suspension was transferred into the middle 2 wells of a 12-well plate and exposed, at room temperature, to increasing doses of 405 nm light (Table 2)
Unexposed controls were held under the same conditions but without 405 nm light exposure
While investigating the effects of 405 nm light on normal function, proliferation rate and viability of rat osteoblasts, it was found that an exposure period of more than 2 h at an irradiance level of 5 mW/cm2 (N36 J/cm2) was toxic to cells leading to an impairment in cell function and cell death (Ramakrishnan et al, 2014)

Summary

Introduction

Violet-blue light in the region of 405 nm has antimicrobial activity against a variety of medically-relevant bacterial species (Bache et al, 2012; Maclean et al, 2009, 2013, 2015; McKenzie et al, 2014; Ramakrishnan et al, 2014) and studies have demonstrated increased susceptibility of bacterial cells compared to their mammalian counterparts — potentially providing the ability to preferentially inactivate microbial contamination in wound and tissue environments (Dai et al, 2013; Zhang et al, 2014).The mechanism of the bactericidal action, and the occurrence of mammalian cell toxicity beyond a threshold exposure level (Ramakrishnan et al, 2014), has not been fully elucidated, but it is thought to involve the photo-excitation of endogenous porphyrin molecules, a process which generates reactive oxygen species (ROS). Both pathways culminate in significant oxidative damage to exposed cells

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