Mitochondrial DNA methylation profiling of the human prefrontal cortex and nucleus accumbens: correlations with aging and drug use.

BACKGROUND: Despite the brain's high demand for energy, research on its epigenetics focuses on nuclear methylation, and much of the mitochondrial DNA methylation remains seldom investigated. With a focus on the nucleus accumbens (NAcc) and the prefrontal cortex (PFC), we aimed to identify the mitochondrial methylation signatures for (1) distinguishing the two brain areas, (2) correlating with aging, and (3) reflecting the influence of illicit drugs on the brain. RESULT: We collected the brain tissue in the NAcc and the PFC from the deceased individuals without (n = 39) and with (n = 14) drug use and used whole-genome bisulfite sequencing to cover cytosine sites in the mitochondrial genome. We first detected differential methylations between the NAcc and the PFC in the nonusers group (P = 3.89 × 10-9). These function-related methylation differences diminished in the drug use group due to the selective alteration in the NAcc. Then, we found the correlation between the methylation levels and the chronological ages in the nonusers group (R2 = 0.34 in the NAcc and 0.37 in the PFC). The epigenetic clocks in illicit drug users, especially in the ketamine users, were accelerated in both brain regions by comparison with the nonusers. Finally, we summarized the effect of the illicit drugs on the methylation, which could significantly differentiate the drug users from the nonusers (AUC = 0.88 in the NAcc, AUC = 0.94 in the PFC). CONCLUSION: The mitochondrial methylations were different between different brain areas, generally accumulated with aging, and sensitive to the effects of illicit drugs. We believed this is the first report to elucidate comprehensively the importance of mitochondrial DNA methylation in human brain.

Simultaneous Determination and Quantitation of Paraquat, Diquat, Glufosinate and Glyphosate in Postmortem Blood and Urine by LC-MS-MS.

A simple method, incorporating protein-precipitation/organic backwashing and liquid chromatography-tandem mass spectrometry (LC-MS-MS), has been successfully developed for the simultaneous analysis of four highly water-soluble and less volatile herbicides (paraquat, diquat, glufosinate and glyphosate) in ante- and postmortem blood, urine and gastric content samples. Respective isotopically labeled analogs of these analytes were adopted as internal standards. Acetonitrile and dichloromethane were used for protein precipitation and organic solvent backwashing, respectively, followed by injecting the upper aqueous phase into the LC-MS-MS system. Chromatographic separation was achieved using an Agilent Zorbax SB-Aq analytical column, with gradient elution of 15 mM heptafluorobutyric acid and acetonitrile. Mass spectrometric analysis was performed under electrospray ionization in positive-ion multiple reaction monitoring mode. The precursor ions and the two transition ions (m/z) adopted for each of these four analytes were paraquat (185; 169 and 115), diquat (183; 157 and 78), glufosinate (182; 136 and 119) and glyphosate (170; 88 and 60), respectively. Analyte-free blood and urine samples, fortified with the analytes of interest, were used for method development/validation and yielded acceptable recoveries of the analytes; interday and intraday precision and accuracy data; calibration linearity and limits of detection and quantitation. This method was successfully incorporated into an overall analytical scheme, designed for the analysis of a broad range of compounds present in postmortem samples, helpful to medical examiners' efforts to determine victims' causes of death.