Cooling of Cells and Organs Confers Extensive DNA Strand Breaks Through Oxidative Stress and ATP Depletion

Cooling at 4 degrees C is routinely used to lower metabolism and preserve cell and tissue integrity in laboratory and clinical settings, including organ transplantation. However, cooling and rewarming produce cell damage, attributed primarily to a burst of reactive oxygen species (ROS) upon rewarming. While DNA represents a highly vulnerable target of ROS, it is unknown whether cooling and/or rewarming produces DNA damage. Here, we show that cooling alone suffices to produce extensive DNA damage in cultured primary cells and cell lines, including double-strand breaks (DSBs), as shown by comet assay and pulsed-field gel electrophoresis. Cooling-induced DSB formation is time- and temperature-dependent and coincides with an excess production of ROS, rather than a decrease in ATP levels. Immunohistochemistry confirmed that DNA damage activates the DNA damage response marked by the formation of nuclear foci of proteins involved in DSB repair, gamma-H2Ax, and 53BP1. Subsequent rewarming for 24 h fails to recover ATP levels and only marginally lowers DSB amounts and nuclear foci. Precluding ROS formation by dopamine and the hydroxychromanol, Sul-121, dose-dependently reduces DSBs. Finally, a standard clinical kidney transplant procedure, using cold static storage in UW preservation solution up to 24 h in porcine kidney, lowered ATP, increased ROS, and produced increasing amounts of DSBs with recruitment of 53BP1. Given that DNA repair is erroneous by nature, cooling-inflicted DNA damage may affect cell survival, proliferation, and genomic stability, significantly impacting cellular and organ function, with relevance in stem cell and transplantation procedures

Temperature effects on DNA damage during hibernation

During multiday torpor, deep-hibernating mammals maintain a hypometabolic state where heart rate and ventilation are reduced to 2%–4% of euthermic rates. It is hypothesized that this ischemia-like condition may cause DNA damage through reactive oxygen species production. The reason for intermittent rewarming (arousal) during hibernation might be to repair the accumulated DNA dam-age. Because increasing ambient temperatures (Ta’s) shortens torpor bout duration, we hypothesize that hibernating at higher Ta’swill result in a faster accumulation of genomic DNA damage. To test this, we kept 39 male and female garden dormice at a Ta of either 57C or 107C and obtained tissue at 1, 4, and 8 d in torpor to assess DNA damage and recruitment of DNA repair markers in splenocytes. DNA damage in splenocytes measured by comet assay was significantly higher in almost all torpor groups than in sum-mer euthermic groups. Damage accumulates in the first days of torpor at Ta = 57C (between days 1 and 4) but not at Ta = 107C. At the higher Ta, DNA damage is high at 24 h in torpor, indicating either a faster buildup of DNA damage at higher Ta’soranin-complete repair during arousals in dormice. At 57C, recruitment of the DNA repair protein 53BP1 paralleled the increase in DNA damage over time during torpor. In contrast, after 1 d in torpor at 107C, DNA damage levels were high, but 53BP1 was not re-cruited to the nuclear DNA yet. The data suggest a potential mis-match in the DNA damage/repair dynamics during torpor at higher Ta’s.</p

Efficacy and Safety of Undenatured Type II Collagen in The Treatment of Osteoarthritis of The Knee: A Randomized, Double-blind, Placebo-controlled Trial

BACKGROUND: Available medication for pain and joint stiffness release in osteoarthritis (OA) often gives considerable side effects. Undenatured type II collagen (UC-II) has been considered as a treatment for OA for its ability to prevent the progress of articular cartilage damage. Hence, this study aimed to evaluate the efficacy and safety of UC-II in modulating knee joint function. METHODS: This was a randomized, double-blind, placebo-controlled study involving 102 OA subjects. Subjects were randomized into two groups: receiving an oral daily dose of 40 mg/day UC-II or placebo containing microcrystalline cellulose for 90 days. Efficacy was evaluated by using the Western Ontario McMaster Osteoarthritis Index (WOMAC), Lequesne’s Functional Index (LFI), and Visual Analogue Scale (VAS) score on day-1, -7, -30, -60, and -90. Safety was evaluated by assessing the adverse events (AEs) and abnormal laboratory findings. RESULTS: The WOMAC total score showed a significant difference between the UC-II group vs. the placebo group from day-7 (p<0.05) to day-90 (p<0.01). UC-II was more effective in reducing the WOMAC total scores by 81.6% compared to 19.2% in the placebo group after 90 days. The total LFI and VAS score was significantly reduced in subjects supplemented with UC-II compared to the placebo group (75.8% vs. 7.8%; 67.9% vs. 12.2%, respectively). No significant changes were observed in vital signs and clinical laboratory tests compared to the placebo. The UC-II had a good safety profile with no serious adverse events among participants. CONCLUSION: UC-II significantly improved the knee pain, stiffness, and functional mobility of OA patients and was well-tolerated. KEYWORDS: osteoarthritis, undenatured type II collagen, WOMAC, VAS, LF