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Dibutyl phthalate (DBP) contamination has raised global concern for decades, while its health risk with toxic mechanisms requires further elaboration. This study used zebrafish ZF4 cells to investigate the toxicity of ferroptosis with underlying mechanisms in response to DBP exposure. Results showed that DBP induced ferroptosis, characterized by accumulation of ferrous iron, lipid peroxidation, and decrease of glutathione peroxidase 4 levels in a time-dependent manner, subsequently reduced cell viability. Transcriptome analysis revealed that voltage-dependent anion-selective channel (VDAC) in mitochondrial outer membrane was upregulated in ferroptosis signaling pathways. Protecting mitochondria with a VDAC2 inhibitor or siRNAs attenuated the accumulation of mitochondrial superoxide and lipid peroxides, the opening of mitochondrial permeability transition pore (mPTP), and the overload of iron levels, suggesting VDAC2 oligomerization mediated the influx of iron into mitochondria that is predominant and responsible for mitochondria-dependent ferroptosis under DBP exposure. Furthermore, the pivotal role of activating transcription factor 4 (ATF4) was identified in the transcriptional regulation of vdac2 by ChIP assay. And the intervention of atf4b inhibited DBP-induced VDAC2 upregulation and oligomerization. Taken together, this study reveals that ATF4-VDAC2 signaling pathway is involved in the DBP-induced ferroptosis in zebrafish ZF4 cells, contributing to the in-depth understanding of biotoxicity and the ecological risk assessment of phthalates. [Display omitted] • Dibutyl phthalate (DBP) exposure induces ferroptosis in zebrafish cell. • Voltage-dependent anion-selective channel 2 (VDAC2) is responsible for ferroptosis. • Activating transcription factor 4 (ATF4) regulates vdac2 expression. • ATF4-VDAC2 signaling pathway participates in the DBP-induced ferroptosis. [ABSTRACT FROM AUTHOR]

Dibutyl phthalate (DBP) is commonly applied plasticizer in plastic products such as face masks, easily leaches or migrates into environment and its widespread contamination posed profound health risks. Further concerns rise regarding to the toxicity of DBP at subcellular level, while little is known about the ranging effects on mitochondrial susceptibility. Present study investigated the mitochondrial impairments with implicated cell death upon DBP exposure on zebrafish cells. Elevated mitochondrial oxidative stress reduced its membrane potential and count, enhanced fragmentation, and impaired ultrastructure that showed smaller size and cristae rupture. Afterwards, the critical function of ATP synthesis was damaged and the stabilized binding capacity between DBP with mitochondrial respiratory complexes was simulated by the molecular docking. And the top pathways enrichment of mitochondrion and metabolism by transcriptome analyses verified the mitochondrial dysfunction that indicated the human diseases risks. The mitochondrial DNA (mtDNA) replication and transcription with DNA methylation modifications were also disrupted, reflecting the genotoxicity on mtDNA. Moreover, the activated autophagy and apoptosis underlying mitochondrial susceptibility integrated into cellular homeostasis changes. These findings provide the first systemic evidence broadening and illustrating the mitochondrial toxicity of DBP exposure on zebrafish model that raise concern on phthalates contamination and ecotoxicological evaluation. [Display omitted] • DBP exposure elevated oxidative stress, impaired mitochondrial function and ultrastructure. • DBP showed stabilized binding capacity with the mitochondrial respiratory complexes. • DBP induced mtDNA instability including hypermethylation, depressed replication and transcription. • Mitochondrial susceptibility indicated the risks of neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

• The ox-mtDNA levels were elevated in maternal fish and offspring under DBP exposure. • The mtDNA in the high energy-consuming tissues are more susceptible. • The ox-mtDNA of D-loop region in blood exhibit a prompt response. • DBP exposure impaired the offspring fitness associated with maternal ox-mtDNA damage. Dibutyl phthalate (DBP) is a widely-used plasticizer that is dispersed in various environments, causing significant pollution and health risks. The toxic mechanism of DBP has been discussed in recent years, while the susceptibility of mitochondrial DNA (mtDNA) to DBP exposure and the resulting damage remain unclear. In this study, maternal zebrafish were exposed to environmentally relevant concentration of DBP for 0, 2, 4, and 6 weeks. Results showed that DBP exposure impaired health status, leading to the reduced body length and weight, condition factor, hepatosomatic index, and gonadosomatic index. Furthermore, DBP exposure induced oxidative stress and ATP deficiency in the gill and liver in a time-dependent manner. The oxidized mtDNA (ox-mtDNA) levels in the D-loop and ND1 regions were assessed in different tissues, showing distinct response patterns. The high energy-consuming tissues such as heart, brain, gill, and liver exhibited elevated susceptibility to mitochondrial damage, with a rapid increase in ox-mtDNA levels in the short term. Conversely, in muscle, ovary, eggs, and offspring, ox-mtDNA gradually accumulated over the exposure period. Notably, the ox-mtDNA levels in the D-loop region of blood showed a prompt response to DBP exposure, making it convenient for evaluation. Additionally, decreased hatching rates, increased mortality, lipoperoxidation, and depressed swimming performance were observed in offspring following maternal DBP exposure, suggesting the inherited impairments of maternal mtDNA. These findings highlight the potential for ox-mtDNA to serve as a convenient biomarker for environmental contamination, aiding in ecological risk assessment and forewarning systems in aquatic environment. [ABSTRACT FROM AUTHOR]