Di-n-butyl Phthalate-Induced Cardiovascular Effects Following Long-Term In Vitro Exposure

Introduction:
Plasticizers used in polymers are closely linked to cellular toxicity and, more recently, to cardiovascular health risks. Among these, di-n-butyl phthalate (DBP)—a low-molecular-weight phthalic acid ester used in adhesives—poses a significant concern. While DBP toxicity is documented, most in vitro studies focus on short-term effects, leaving the consequences of chronic, prolonged exposure poorly understood. This study aimed to optimize a long-term in vitro methodology to evaluate DBP’s impact on the cardiovascular system by simulating exposure periods in vascular smooth muscle cells (A7r5) ranging from 1 to 10 days.

Methods:
Cells were cultured in 96-well microplates and exposed to ten DBP concentrations (0.001 to 1000 µM). The experimental design included two control groups (culture medium and an ethanol vehicle) to ensure baseline accuracy. Cell viability was assessed using MTT assays, followed by ANOVA statistical analysis.

Results:
Results revealed a significant toxicity peak 96 hours post-exposure. This effect was most prominent at the highest dosages (500–1000 µM) and, notably, at the lowest concentration (0.001 µM)—a value 10⁵ times lower than the maximum—suggesting a complex concentration-response relationship. Between 96 and 240 hours, cellular recovery was limited, indicating persistent cytotoxic effects throughout the observation period. Furthermore, physical changes were observed in both the culture appearance and the plastic surface of the plates.

Discussion/Conclusions:
These structural impacts will be further investigated using scanning electron microscopy (SEM). In conclusion, DBP exposure induces time and dose-dependent responses that may contribute to vascular dysfunction following prolonged exposure. These findings underscore the necessity of long-term models to accurately assess the persistent risks posed by phthalate exposure.