The roles of reactive oxygen species and antioxidants in cryopreservation

Abstract

Cryopreservation has facilitated advancement of biological research by allowing the storage of cells over prolonged periods of time. While cryopreservation at extremely low temperatures would render cells metabolically inactive, cells suffer insults during the freezing and thawing process. Among such insults, the generation of supra-physiological levels of reactive oxygen species (ROS) could impair cellular functions and survival. Antioxidants are potential additives that were reported to partially or completely reverse freeze-thaw stress-associated impairments. This review aims to discuss the potential sources of cryopreservation-induced ROS and the effectiveness of antioxidant administration when used individually or in combination.

Keywords: Cryopreservation; antioxidant; oxidative stress; reactive oxygen species.

Figure 1. Metabolism and sources of ROS

(A) Detoxification and metabolism of reactive oxygen/nitrogen species. (B) Sources of ROS, and localization of enzymes that counteracts ROS in the mitochondria, endoplasmic reticulum (ER), peroxisome, cytosol and the extracellular space. SOD1 is localized in both the mitochondria intermembrane space and cytosol, SOD3 is located extracellularly and SOD2 is found exclusively mostly in the mitochondria matrix. Catalase that reduces hydrogen peroxide (H₂O₂) into H₂O is mostly located in the peroxisomes. Glutathione peroxidase (GPx) is found in the mitochondria and cytosol. Peroxiredoxins (Prx) and thioredoxins (Trx) which constitute the Peroxiredoxin–Thioredoxin (Prx/Trx) system can be found in the nucleus, mitochondria, ER, peroxisome and the extracellular environment. Electron transport chain (ETC), Cytochrome P450 family of enzymes (Cyps), xanthene oxidase (XO) and NADPH oxidases (NOX) are potential sources of O₂^•−, while ERO1 and acetyl CoA oxidases (AcoX) produce H₂O₂. Nitric oxide synthase (NOS) is a potential source of NO^•. Aquaporins (Aqp) facilitate the movement of H₂O₂ across membranes. Single snowflake indicates ROS detected while two snowflakes indicate an implication with cryopreservation. Cu²⁺/Fe³⁺ (); Cu¹⁺/Fe²⁺ (); Source of ROS (); Enzyme (); O₂^•− (); O₂ (); H₂O (); H₂O₂ (); •OH (); ONOO⁻ (); NO^• (); NO₂⁻ (); H⁺ (); Detected during cryopreservation (); Implicated during cryopreservation ().

Figure 2. Effects of different levels of reactive oxygen/nitrogen species on cellular biomolecules

Protein can react with ONOO⁻, H₂O₂, NO^•, ^•OH and aldehydes such as 4-Hydroxynonenal (4-HNE) can react with protein side chains (e.g. amino acids such as lysine). The formation of oxo-histidine and disulfide bonds are mostly reversible and mediate redox signaling under mild oxidative stress and may not be deleterious. High level of ROS lead to protein aggregation, denaturation and fragmentation. Mitochondrial/nuclear DNA can react with O₂^•−, ONOO⁻ and ^•OH. Mutations and double/single-strand breaks mediated by ROS are minimized by the DNA-Damage Response (DDR). Proteins such as p53, RAD51 and yH2AX are DDR constituents involved in cryopreservation. Severe oxidative stress can overwhelm the DDR, resulting in mutations and double/single strand breaks. Lipids can react with ONOO⁻ and ^•OH to cause lipid peroxidation and form lipid peroxides (LPOs). LPOs can decompose into aldehydes (Ald) such as 4-HNE and malondialdehyde (MDA). At low levels of ROS, cells are quiescent. Moderate levels of ROS facilitates beneficial redox signaling to modulate cellular survival, growth and division. Overwhelming levels of ROS can initiate cell death.