Regular Usage of Acetaminophen Resulted in Significant Effects on Signaling Pathways in Female Mouse Hearts

Acetaminophen (APAP) is a common medication used worldwide to treat pain and fever. When taken as directed, it is regarded as safe and effective. Yet, current research is showing that consistent APAP use can cause cardiovascular issues and increase blood pressure. Studies on the effect of non-steroidal anti-inflammatory drugs (NSAIDs) found that regular usage caused an increase in reactive oxygen species (ROS), leading to cardiac dysfunction. Since APAP has a mechanism of action similar to NSAIDs, we investigated what effects it had on mouse hearts. We hypothesize that regular acetaminophen usage increases ROS production in cardiomyocytes, causing dysfunction in the heart. 6-month-old female mice were treated with either water or acetaminophen dissolved in water daily for seven days. The concentration of APAP dissolved was 103 mg/kg/day, equivalent to a 500 mg caplet for humans. The hearts were collected and pulverized, then homogenized in urea lysis buffer. Afterward, the samples were centrifuged to separate the supernatant from the pellet, which was discarded. The remaining supernatant was treated with acetone overnight to allow proteins to precipitate. After precipitation, the pellet was kept, and the supernatant was discarded. The pellet was resuspended, treated with Tandem Mass Tag (TMT) label reagents, and quenched. The samples were then combined into one sample and analyzed with liquid chromatography (LC) mass spectroscopy (MS) to determine protein changes. The LC/MS/MS data was analyzed using power analysis and Sequest-HT to compare the sample proteins to all mouse sequences. Then, Target Decoy PSM was used to exclude decoy sequences from the samples, and Scaffold Q+ was used to quantify the sample peptides and proteins identified from the MS and LC analysis. Analysis of the various affected signaling pathways showed significant protein expression changes in multiple pathways, including increased and decreased regulation of enzymes in the purine de novo synthesis pathway and a decrease in the purine degradation pathways in the hearts of the acetaminophen-treated mice compared to the hearts of vehicle-treated mice. Acetaminophen also caused a decrease in glutathione oxidation and reduction in the antioxidant pathway. Overall, our results display significant effects on signaling pathways in the hearts of female mice regularly treated with APAP. NIEHS/Superfund Research Program (P42 ES004699) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.