Complementary Square-Wave Voltammetry and LC-MS/MS Analysis to Elucidate Induced Damaged and Mutated Mitochondrial and Nuclear DNA from in Vivo Knockdown of the BRCA1 Gene in the Mouse Skeletal Muscle

Abstract

Breast cancer 1 gene (BRCA1) DNA mutations impact skeletal muscle functions. Inducible skeletal muscle specific Brca1 homozygote knockout (Brca1KOsmi, KO) mice accumulate mitochondrial DNA (mtDNA) mutations resulting in loss of muscle quality.1 Complementary electrochemical andmass spectrometry analyses were utilized to rapidly assess mtDNA or nuclear DNA (nDNA) extracted directly from mouse skeletal muscles. Oxidative peak currents (I p) from DNA immobilized layer by layer (LbL) were monitored using square-wave voltammetry (SWV) via Ru­(bpy)3 2+ electrocatalysis. I p significantly decreased (p < 0.05) for KO mtDNA compared to heterozygous KO (Het) or wild type (WT), indicative of decreases in the guanine content. nDNA I p significantly increased in KO compared to WT (p < 0.05), suggesting an accumulation of damaged nDNA. Guanine or oxidatively damaged guanine content was monitored via appropriate m/z mass transitions using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Guanine in both KO mtDNA and nDNA was significantly lower, while oxidatively damaged guanine in KO nDNA was significantly elevated versus WT. These data demonstrate a loss of guanine content consistent with mtDNA mutation accumulation. Oxidative damage in KO nDNA suggests that repair processes associated with Brca1 are impacted. Overall, electrochemical and LC-MS/MS analysis can provide chemical-level answers to biological model phenotypic responses as a rapid and cost-effective analysis alternative to established assays.

Skeletal Muscle Function Is Dependent Upon BRCA1 to Maintain Genomic Stability

“…To date, our data suggest that induced deletion of BRCA1 in the skeletal muscle leads to visible heterogeneity of mitochondrial size and reduced respiratory kinetics, both of which are associated with an accumulation of mtDNA mutations. It should be noted that we have also identified the accumulation of damaged DNA, specifically the increased presence of oxidatively damaged guanine, in the nucleus of skeletal muscle from BRCAKO1 ism compared with the WT groups (35). Thus, the data collectively indicate a strong association between the accumulation of DNA damage and mutations both in the mitochondria and nucleus, which seem to contribute to altered mitochondrial respiration and reduced muscle quality.…”

“…To test this hypothesis, we used a random mutation capture assay where we identified an accumulation of mutations in the mitochondrial DNA isolated from the BRCAKO1 smi compared with the WT groups (29). Using two additional analytical approaches to assess DNA structural changes to seek chemically based answers, we have since confirmed that mitochondrial DNA from BRCAKO1 smi accumulates mutations compared with mitochondrial DNA from the WT groups (35). Our data specifically demonstrate that BRCAKO1 smi mtDNA exhibits losses in guanine content, consistent with mutations stemming from initial damage.…”

Non-Targeted Metabolomics Combined with Chemometrics by UHPLC–Orbitrap–HRMS and Antioxidant Activity of Atractylodes chinensis (DC.) Koidez. from Eight Origins

“…MS2 fragmentation resulted in main fragments at m / z 135.03018, 110.03527, and 109.05110, originating from the neutral loss of NH 3 , CH 2 N 2 , and CHON, respectively. According to its mass spectrum information and the information from the previous literature [ 30 ], it was identified as Guanine.…”