Effects of ionizing radiation on biological molecules--mechanisms of damage and emerging methods of detection

Abstract

Significance: The detrimental effects of ionizing radiation (IR) involve a highly orchestrated series of events that are amplified by endogenous signaling and culminating in oxidative damage to DNA, lipids, proteins, and many metabolites. Despite the global impact of IR, the molecular mechanisms underlying tissue damage reveal that many biomolecules are chemoselectively modified by IR.

Recent advances: The development of high-throughput "omics" technologies for mapping DNA and protein modifications have revolutionized the study of IR effects on biological systems. Studies in cells, tissues, and biological fluids are used to identify molecular features or biomarkers of IR exposure and response and the molecular mechanisms that regulate their expression or synthesis.

Critical issues: In this review, chemical mechanisms are described for IR-induced modifications of biomolecules along with methods for their detection. Included with the detection methods are crucial experimental considerations and caveats for their use. Additional factors critical to the cellular response to radiation, including alterations in protein expression, metabolomics, and epigenetic factors, are also discussed.

Future directions: Throughout the review, the synergy of combined "omics" technologies such as genomics and epigenomics, proteomics, and metabolomics is highlighted. These are anticipated to lead to new hypotheses to understand IR effects on biological systems and improve IR-based therapies.

FIG. 1.

IR generates the potent intracellular oxidants H₂O₂, O₂^•−, and ^•OH, along with reductants H^• and e_aq⁻. Endogenous ROS propagation occurs through the mitochondrial ETC and the increased expression of signaling enzymes such as NOX and iNOS. Reductive stress induced by IR leads to loss of sulfur in protein methionine and cysteine residues. ^•OH, hydroxyl radical; O₂^•−, superoxide; e_aq⁻, hydrated electron; H^•, hydrogen radical; H₂O₂, hydrogen peroxide; iNOS, inducible nitric oxide synthase; IR, ionizing radiation; NOX, NADPH oxidase; ROS, reactive oxygen species. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

Effects of IR on cellular DNA: oxidative damage to nucleobases and riboses mediated by ^•OH.

FIG. 3.

Effects of IR on cellular lipids. (A) Oxidative cis-trans isomerization of polyunsaturated fatty acids. (B) Reactive aldehydes generated by lipid peroxidation. (C) Chemical probes for the detection of intracellular ROS (DPPP) and reactive aldehydes (DNPH; CHH). CHH, 7-(diethylamino)coumarin-3-carbohydrazide; DNPH, 2,4-dinitrophenylhydrazine; DPPP, diphenyl-1-pyrenylphosphine.

FIG. 4.

Detection of reactive aldehydes and carbonylated proteins. Derivatization of protein carbonyls using DNPH and mechanistically similar Girard's P reagent and a biotinylated hydroxylamine.

FIG. 5.

Radiation-induced sulfur oxidation in proteins, affecting (A) methionine and (B) cysteine residues. Oxoforms in gray are readily reduced to thiol by DTT and other reductants in vitro.

FIG. 6.

Impact of IR-induced reductive stress on protein sulfur, including methionine, cysteine, and cystine.

FIG. 7.

Capture of cysteine sulfenic acid by 1,3-dicarbonyl compounds. (A) Dimedone (top), Alk-β-KE (bottom); click chemistry (not shown) installs a biotin affinity tag; the ketoester enables hydroxylamine-mediated ester cleavage, generating MS-friendly isoxazolone. (B) MS² spectrum for the Alk-β-KE-labeled peptide after Asp-N digestion reveals MS compatibility. (C) Accumulation of intracellular ROS in response to probes is lower for Alk-β-KE compared with dimedone (10 mM each). (D) Click chemistry attaches biotin tag; labeling by Alk-β-KE is specific to CySOH and abolished in the presence of TCEP. (E) Addition of Alk-β-KE to cells, followed by lysis, click to attach the biotin moiety and immunoblotting illustrates membrane permeability. (F) Cellular toxicity of Alk-β-KE is reduced compared with dimedone as measured by the DCF assay. Adapted from Qian et al. (272). DCF, dichlorodihydrofluorescein; MS, mass spectrometry; TCEP, tris(2-carboxyethyl)phosphine. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 8.

General workflow for in vitro labeling of CySOH using biotinylated 1,3-dicarbonyl probes. Proteins are precipitated to remove unreacted probe; then, labeled proteins are enriched using avidin-based methods. After proteolytic digestion, protein identifications are obtained by LC-MS/MS analysis of the full peptide mixture, while site mapping requires a second round of enrichment. LC, liquid chromatography. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars