Complementary Analysis of Colorectal Cancer-Associated DNA Using Square Wave Voltammetry and LC-MS/MS in a Murine Model

Abstract

Mitochondrial dysfunction has emerged as a critical factor in colorectal cancer development, contributing to altered cellular metabolism, increased oxidative stress, and genomic instability that can drive carcinogenic processes. To study mitochondrial dysfunction's role in carcinogenesis, colorectal cancer model NDUFS4 knockout (KO) mice were analyzed alongside wild-type (WT) controls. Electrochemical methods coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied to investigate DNA modifications in colorectal cancer under conditions of mitochondrial dysfunction. Square wave voltammetry (SWV) at layer-by-layer modified electrodes with immobilized DNA allowed detection of guanine oxidation changes catalyzed by the electrochemical mediator, Ru(byp)32+. Peak current differences of tumor samples compared to normal samples indicated DNA structural alterations, which were validated through comprehensive LC-MS/MS base compositional analysis of acid-hydrolyzed DNA samples. Applying the electrochemical approach, DNA from WT tumor samples displayed statistically significant (p < 0.05) decreased oxidative currents versus normal tissue, a response consistent with guanine depletion. In contrast, KO sample sets showed heterogeneous responses, with some exhibiting increased currents suggesting guanine preservation, while others showed decreased currents similar to WT trends. Subsequent LC-MS/MS composition analysis validated these findings, allowing quantification of all four DNA bases (guanine, adenine, cytosine, thymine) and detection of oxidatively damaged bases (8-oxoguanine) and epigenetic modifications (5-methylcytosine). The electrochemical-LC-MS/MS analysis approach revealed three potential carcinogenic pathways: a classical oxidative damage pathway in WT tumors characterized by G:C to A:T transitions and global hypomethylation; a protective adaptation pathway in the KO Set 1 tumor sample showing enhanced G:C content preservation and stable methylation; and a compromised repair pathway in KO Set 2 tumor samples exhibiting 8-oxoguanine accumulation with nucleotide depletion. The results suggest a hypothesis that mitochondrial complex I deficiency fundamentally alters the mechanisms of DNA modification in colorectal cancer. This work provides insight into the complex relationship between mitochondrial dysfunction and genomic instability, opening new avenues for understanding and treating metabolically-driven cancers. Strong correlations between guanine content changes and electrochemical signals validated the reliability of the electrochemical method for rapid DNA modification screening. Combining electrochemical and mass spectrometry techniques provided validation of DNA modifications observed in cancer progression while offering a versatile, cost-effective approach to investigate metabolic influences on genomic stability.