Quantification of Urinary Free and Total Oxidative Stress Biomarkers and Their Differential Associations with Polycyclic Aromatic Hydrocarbon Exposure: A Bushfire Smoke Study

Abstract Urinary excretion profiles of free and metabolically conjugated oxidative stress biomarkers (OSBs) remain poorly characterized, potentially underestimating total excretion and biasing exposure–effect interpretation. In this study, the profiles of free, glucuronidated, and sulfated forms of eight urinary OSBs reflecting nucleic acid, protein, lipid, and glycoxidation damage were characterized using enzymatic hydrolysis. Free and total (sum of the three measured forms) OSBs, along with hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) as smoke exposure biomarkers, were analyzed in 47 urine samples from a bushfire smoke cohort. Sulfate conjugates were detected for both 3-chlorotyrosine (Cl-Tyr) and 3-bromotyrosine (Br-Tyr), accounting for median proportions of 37% (IQR: 28%–56%) and 89% (IQR: 71%–94%); glucuronide conjugates were detected for 8-iso-prostaglandin F2α (8-iso-PGF2α), with a median proportion of 57% (IQR: 49%–80%). Other OSBs were mostly in free forms. 2-Hydroxynaphthalene was significantly associated with increased levels of 4-hydroxynonenal mercapturic acid (β = 0.42, 95% CI: 0.087–0.75) and total Cl-Tyr (β = 0.35, 95% CI: 0.048–0.66), suggesting potential links of PAH exposure with lipid peroxidation and neutrophil-mediated protein chlorination. However, such association was not observed for free Cl-Tyr. These findings indicate that phase II metabolism contributes substantially to the urinary excretion of Cl-Tyr, Br-Tyr, and 8-iso-PGF2α. Urinary total Cl-Tyr, rather than free Cl-Tyr, may serve as a more sensitive biomarker for assessing protein damage associated with smoke-related PAH exposure.