Oxidative Stress leads to Increase, DNA strand breaks

Modulating Factors 

MF Details 

Effects on the KER 

References 

Age 

Reduced antioxidant capacities have been linked to aged lenses (in humans >30 years old). The development of a chemical barrier between the cortex and the nucleus is partially responsible, as it prevents GSH from protecting aged lens cells from ROS. 

Prevention of RONS-mediated damage is primarily achieved by antioxidants, so a lowered capacity would likely lead to reduced damage mitigation abilities. 78% of lens over 30 had a low level of GSH in the center compared to 14% of lens under 30. Lens epithelial cells have an associated 3-fold increase in γ-H2AX (marker of DNA damage) when GSH-PX decreases by 2-fold. 

Taylor & Davies, 1987; Cabrera & Chihuailaf, 2011; Quinlan & Hogg, 2018; Sweeney & Truscott, 1998; Meng & Fang, 2021 

Free radical scavengers 

ROS-scavengers are essential components of the body’s natural defense against oxidative damage. Increased ROS production leads to increased incidence of electron donation by scavengers, thus reducing the overall level of free radical scavengers available to deal with ROS. 

Isothiocyanates, such as sulforaphane (SFN), activate the release of more enzymatic scavengers. When SFN was added to in vitro LECs, LDH decreased to near unexposed cell levels and was associated with 3.3x less DNA strand breaks compared to the non-SFN cells following stressor exposure. Epigallocatechin-3-gallate (EGCG) also has antioxidant properties and was shown to alleviate radiation-induced increases in oxidative stress and DNA strand breaks within rat hippocampi. 

Taylor et al., 1987; Cabrera et al., 2011; Liu et al., 2013; El-Missiry et al., 2018 

Media 

Mesenchymal stem cell-conditioned medium (MSC-CM), which has self-renewal, differential and proliferation capacities. 

MSC-CM treatment has also been shown to improve ROS levels and decrease radiation-induced DNA strand breaks within mouse hippocampal neuronal cells. 

Huang et al., 2021 

Reference  

Experiment Description 

Result 

Cencer et al., 2018 

In vitro, human LECs exposed to UVB and tested for 120 min post exposure with fluorescent probes to detect ROS production and mitochondrial superoxide, and tetramethylrhodamine-dUTP (TMR) red assay to detect strand breaks. 

Both ROS and DNA strand breaks were increased by both 0.014 J/cm² and 0.14 J/cm² UVB radiation. At 0.014 J/cm², cellular ROS increased a maximum of 15 fluorescence units above the control at 5 minutes post-UVB, while DNA strand breaks increased about 115 fluorescence units above the control at this time. At 0.14 J/cm², cellular ROS increased a maximum of about 35 fluorescence units above the control at 90 minutes post-UVB, while mitochondrial superoxide increased about 30 fluorescence units above the control and DNA strand breaks increased about 125 fluorescence units above the control at this time. 

Ahmadi et al., 2021  

In vitro, human LECs exposed to 0.065-0.3 Gy/min gamma radiation, with dihydroethdium (DHE) fluorescent probes to measure ROS levels and comet assay to measure strand breaks. 

Human LECs exposed in vitro to 0.1 - 0.5 Gy gamma rays showed a gradual increase in ROS levels and a corresponding gradual increase in DNA in the tail from the comet assay (indicative of increased DNA strand breaks) with the maximum dose displaying a 10% increase in ROS levels and a 17% increase in DNA strand damage. 

Li et al., 1998  

In vitro, bovine LECs were exposed to 40 and 400 µM H₂O₂ with an alkaline unwinding assay to determine strand break levels. 

Immediately after LECs were exposed to 40 µM and 400 µM H₂O₂, there were ~145% and ~150% DNA strand breaks compared to the unexposed control level, respectively. The amounts of DNA strand breaks in cells exposed to both concentrations were reduced to ~105% of the unexposed control level after 30 mins. After 400 µM H₂O₂, oxidative stress as measured by LDH was 1200% of control in neuroblastoma cells. 

Spector et al., 1997  

In vitro, rat LECs exposed to 100 and 125 µM H₂O₂ with alkaline elution assay to determine single strand break level. 

Exposure to 125 µM of H₂O₂ to lens epithelial cells resulted in reduction of intact DNA to near 1% by 9 hr post-exposure. Exposure to 100 µM H₂O₂ reduced SOD and GSH levels by 2-fold. 

El-Missiry et al., 2018 

In vivo, albino Wistar rats were exposed to 4 Gy of γ radiation (¹³⁷Cs source) at 0.695 rad/s. Kits were used to measure 4-HNE (secondary product of lipid peroxidation) and protein 

carbonyl group levels as markers of oxidative stress. Antioxidants including GSH, GPx 

and GR were also assessed. The comet assay was used to analyze DNA strand breaks by visualizing DNA tail %, tail length and tail moment. 

4-HNE and protein carbonyl levels increased by approximately 2- and 3-fold after radiation exposure. GSH and GPx levels decreased by approximately 3-fold each, whereas GR levels decreased by approximately 5-fold. Tail DNA %, tail length and tail moment increased by approximately 2-, 3- and 6-fold after exposure to 4 Gy. 

Ungvari et al., 2013 

In vitro. CMVECs and rat hippocampal neurons were irradiated with 2-8 Gy ¹³⁷Cs gamma rays. 5(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) staining, and flow cytometry were used to measure ROS production. DNA damage was quantified by measuring the tail DNA content (as a percentage of total DNA) using the Comet Assay-IV software. 

Day 1 post-irradiation showed increased cellular peroxide production and increased mitochondrial oxidative stress in CMVECs in a dose-dependent manner, increasing a maximum of ~3-fold at 8 Gy. Tail DNA content also increased in a dose-dependent manner with an approximate increase from 0 to 45% at 8 Gy. 

Huang et al., 2021 

In vitro, HT22 cells (mouse hippocampal neuronal cell line) were exposed to 10 Gy of X-irradiation at 6 Gy/min. ROS levels were measured using H2-DCFDA staining and fluorescence microscope analysis, whereas western blotting was used to detect γ-H2AX. 

At 10 Gy, intracellular ROS generation increased by 5-fold and γ-H2AX increased by 3-fold. 

Zhang et al., 2017 

In vitro. HT22 cells were exposed to 8 and 12 Gy X-rays. Relative intracellular ROS levels were determined by DCFDA. p-ATM, γ-H2AX were measured with Western blot. 

Following 8 Gy irradiation, intracellular ROS levels increased ~1.8-fold. Phosphorylation of ATM and γ-H2AX were increased 4.4-fold and 3.2-fold, respectively, 30 minutes after 12 Gy. 

Cervelli et al., 2014 

In vitro. HUVECs were irradiated with single doses (0.125, 0.25, 0.5 Gy), or fractionated doses (2 × 0.125 Gy, 2 × 0.250 Gy) of X-rays. Intracellular ROS generation was measured with a fluorescent dye, C-DCFDA, using a spectrofluorometer. Immunofluorescence microscopy was used to measure γ-H2AX foci. 

Intracellular ROS production was significantly increased in a dose-dependent manner (1.6-, 2- and 2.8-fold at 0.125, 0.25, 0.5 Gy, respectively). When HUVECs were exposed to fractionated doses, no increase in ROS generation was observed, compared with respective single doses. 24h post-irradiation the percentage of foci-positive cells exposed to 0.125 Gy, 2 × 0.125 Gy, 0.250 Gy, 2 × 0.250 Gy and 0.5 Gy, was 1.68, 1.48, 3.53, 2.59, 8.74-fold over the control, respectively. 

Sakai et al., 2017 

In vitro. HAECs were exposed to 100uM H₂O₂. Intracellular ROS was measured by CM-H2DCFDA. DNA DSBs were detected by immunofluorescent analysis with γ-H2AX as a marker. 

Intracellular ROS increased by ~3.7-fold 

p-ATM increased by ~4.7-fold. γ-H2AX increased by ~3.4-fold. 

Reference  

Experiment Description 

Result 

Meng et al., 2021 

In vitro, human LECs exposed to 50 µM H₂O₂ with DCFH-DA fluorescent probe to detect ROS levels and immunofluorescence and western blot assay to detect γ-H2AX. 

50 µM H₂O₂ exposure to lens epithelial cells increased oxidative stress, with ROS measured by LDH, by 4-fold and decreased the level of antioxidants by 2-fold as measured by SOD and GSH-PX. This resulted in 3-fold increase in γ-H2AX. 

Smith et al., 2015 

In vitro, human LECs exposed to 30 µM H₂O₂ with alkaline comet assay to determine amount of strand breaks. 

Treatment of lens epithelial cells to 30 µM H₂O₂ induced DNA strand breaks by 55% at 0.5 hr after exposure and increased the level of LDH by ~1.4 fold at 24 hr post-exposure. 

Liu et al. 2013 

In vitro, human LECs exposed to 30 µM H₂O₂ with alkaline comet assay determination of strand breaks. 

LDH increased by ~1.4 fold at 24 hr post-exposure, with a 5x increase from control levels in DNA strand breaks. 

Reference  

Experiment Description 

Result 

Yang et al., 1998  

In vitro, rabbit LECs exposed to H₂O₂ with TCA addition and thiol assay to determine non-protein thiol (NP-SH) level and alkaline elusion assay to determine strand breaks. 

 In rabbit LECs exposed in vitro to 125 µM H₂O₂, non-protein thiol levels decreased to <5% control (indicates oxidative stress) 30 min post-irradiation, and % DNA retained using alkaline elution decreased by 1.6 log (indicates increased DNA fragmentation) within the next 8.5 h.