Tissue expression of aquaporin-5 and cytochrome c as potential forensic biomarkers of drowning: Evidence from an experimental rat model.

Determining whether a person died from drowning remains a major forensic challenge, particularly in cases involving advanced decomposition where only skeletal remains are available. Traditional methods, such as diatom analysis and macroscopic autopsy findings, often lack sufficient specificity and reliability. Although several molecular biomarkers have been investigated, none has yet demonstrated sufficient accuracy for routine forensic application. This prospective experimental study evaluated a novel dual-biomarker approach using Aquaporin 5 (AQP5), a water channel protein, and Cytochrome c (CYCS), an apoptosis-related protein, as potential indicators of drowning. Thirty-eight adult Wistar rats were randomly assigned to experimental drowning groups or non-drowning control groups (exposed to freshwater from Boračko and Jablaničko lakes. Lung, femoral bone, and dental tissues were collected postmortem, and AQP5 and CYCS concentrations were quantified using commercially available ELISA kits. Statistical analyses included the Shapiro-Wilk test and the Mann-Whitney U test, with statistical significance set at p < 0.05. AQP5 expression in dental tissue differed significantly between drowning and control groups in both freshwater environments (p = 0.0184 and p = 0.0235), whereas no significant differences were observed in lung or bone tissues. CYCS expression did not differ significantly between drowning and control groups in any tissue examined. These findings suggest that AQP5 expression in dental tissue may represent a promising supplementary biomarker for the forensic diagnosis of drowning, particularly in cases where only skeletal remains are available. Further validation in human tissues and larger experimental cohorts is warranted.