Abstract 3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) is a porphyrinogenic compound that induces hepatic formation of N-methyl protoporphyrin IX (NmePPIX), a potent endogenous inhibitor of ferrochelatase (FECH). Inhibition of FECH by NmePPIX blocks the conversion of protoporphyrin IX (PPIX) into heme, resulting in PPIX accumulation and PPIX-mediated cholestatic liver injury. Although cytochrome P450 (CYP)-mediated transfer of a methyl group from DDC has long been proposed as the mechanism underlying DDC-induced NmePPIX formation, the specific CYP enzymes responsible for DDC metabolism and NmePPIX formation remain poorly characterized. Here, a metabolomics-based approach identified six CYP-dependent DDC metabolites (M1–M6) in the incubation with mouse liver S9 fractions. Notably, no demethylated DDC metabolite was detected. We further demonstrated that CYP3A contributes to four metabolic pathways of DDC, including a previously unrecognized pathway involving deamination and intramolecular cyclization. Given the important role of CYP3A in DDC metabolism and its high hepatic expression, we investigated whether CYP3A contributes to NmePPIX formation. Our in vivo study found that Cyp3a deficiency had no effect on DDC-induced NmePPIX formation, PPIX accumulation, or PPIX-mediated liver injury. Collectively, these findings provide the first comprehensive characterization of CYP-dependent DDC metabolism and demonstrate that CYP3A is not essential for DDC-induced NmePPIX formation, indicating that the major CYP-dependent metabolic pathways of DDC are mechanistically distinct from the pathway leading to NmePPIX formation.
Abstract The use of gadolinium-based compounds as contrast agents in magnetic resonance imaging scans has been popularized owing to the superior anatomical images they produce and tracking of a broad spectrum of clinical conditions. While these compounds are considered safe when administered at appropriate dose levels, toxicity has been reported for patients with impaired renal functions. Toxicogenomic and intracellular accumulation studies have also implicated genes relevant to lipid synthesis, disruption of cytoplasmic membrane integrity, and accumulation in the neuronal interstitium, kidney, liver, and skin. These findings have prompted widespread studies into the mechanism of intracellular accumulation and cytotoxicity of gadolinium-based contrast agents (GBCAs). In the present work, neuroblastoma SH-SY5Y cells were treated with macrocyclic Gd chelate gadoterate meglumine, linear Gd chelate gadopentetic acid, and GdCl3 salt to decipher their ability to activate the release of danger-associated molecular pattern signals (DAMPs), key steps in the induction of immunologic cell death (ICD). Furthermore, intracellular accumulation and localization of the GBCAs in SH-SY5Y cells were studied using inductively coupled plasma optical emission spectroscopy (ICP-OES) and transmission electron microscopy (TEM). Our results showed no significant cytotoxicity to SH-SY5Y cells until 5 mM for all compounds tested except gadopentetic acid. Cells undergoing early apoptosis were observed with both gadoterate meglumine and gadopentetic acid at 1 and 10 mM compared to nontreated control samples. Evidence for mild induction of immunogenic cell death was observed for both linear and macrocyclic GBCAs but not in GdCl3-treated SH-SY5Y cells, with a concentration-dependent increase in extracellular adenosine triphosphate (ATP) level, high mobility group box 1 (HMGB1) protein, and relocation of calreticulin. Furthermore, uptake inhibition studies suggest clathrin-dependent endocytosis by blocking GTPase activity of dynamin proteins in linear gadopentetic acid and accumulation along the cell membrane as seen in TEM images.
Abstract Epidemiological studies suggest a strong association between phthalate exposure and proliferative diseases of the prostate. However, experimental evidence for phthalate-induced malignant transformation in normal prostate epithelial cells is lacking. Therefore, the objective of this study was to evaluate the effects of both the parent di-2-ethylhexyl phthalate (DEHP) and its primary metabolite mono-2-ethylhexyl phthalate (MEHP) on the proliferation and malignant transformation of RWPE-1 normal prostate epithelial cells. Cells were exposed to DEHP and MEHP separately at concentrations of 1 μM and 100 μM for 72 h to evaluate acute effects and for a long term (8 weeks) at 1 μM to evaluate carcinogenic potential. Cell viability and oxidative stress were determined by MTT and DCFH assays, respectively. Cell cycle analysis was performed using flow cytometry, whereas gene expression changes were analyzed at the transcript level by qRT-PCR and at the protein level by Western blot. Phthalate-induced malignant transformation of cells was evaluated by soft agar assay. Results revealed that both DEHP and MEHP caused a decrease in cell viability and increased the intracellular levels of reactive oxygen species (ROS) at 72 h. However, long-term exposure to DEHP and MEHP resulted in increased proliferation and malignant transformation of RWPE-1 cells. Significant changes in the expression of selected genes not only confirmed increased proliferation and oxidative stress but also revealed the activation of a genotoxic estrogen metabolic pathway as an underlying mechanism for phthalate-induced malignant transformation. Decreased expression of the tumor suppressor PTEN and the increased expression of oncogenic c-Myc as well as its upstream regulator β-Catenin further suggest that phthalates cause malignant transformation through the activation of the c-Myc-β-Catenin pathway. This study reveals for the first time that long-term exposure to both DEHP and its metabolite can induce malignant transformation in prostate epithelial cells through activation of the genotoxic estrogen metabolic pathway and oncogenic c-Myc-β-Catenin signaling.
Abstract Perfluorooctanoic acid (PFOA), a representative persistent organic pollutant (POP), has been linked to numerous adverse health effects. Ulcerative colitis (UC), characterized by recurrent colon inflammation, is known to have environmental exposure as a primary etiological factor; however, the toxicological mechanisms underlying PFOA-induced UC remain unclear. This study therefore aims to comprehensively investigate the association between PFOA exposure and UC. A meta-analysis was conducted to evaluate the association between PFOA exposure and UC onset, and network toxicology, cross-species transcriptomics, protein–protein interaction networks, and weighted gene coexpression network analysis were employed to identify potential targets and mechanisms. Single-cell RNA sequencing, molecular docking, and Mendelian randomization were used to pinpoint and validate hub genes, while in vivo DSS-induced colitis and in vitro inflammation models were applied to assess the functional impact of PFOA. The meta-analysis of four studies confirmed a significant positive association between PFOA exposure and UC risk (Fisher z = 0.20, 95% CI: 0.13–0.27). We identified 170 overlapping targets linking PFOA to UC, predominantly enriched in immune-inflammatory activation and metabolic disruption. Seven hub genes (IRF1, CXCL1, CXCL9, CXCL10, CXCL11, CASP1, and IL1B) constituted a UC-associated coexpression module, with monocytes/macrophages as primary sources by single-cell profiling. Molecular docking confirmed PFOA binding to these proteins, and Mendelian randomization established IRF1 as a causal risk factor. In vivo, PFOA exacerbated DSS-induced colitis, impairing barrier function, promoting apoptosis and mitochondrial dysfunction. In conclusion, PFOA exposure is associated with increased UC risk and may promote colonic inflammation by affecting the chemokine–inflammasome network and epithelial function. IRF1 may be a key hub in this process, but its upstream role needs validation. These findings highlight the potential importance of reducing environmental pollutant exposure in UC prevention.
Palladium nanoparticles (Pd NPs), extensively used in automobile catalytic converters, are increasingly released into the environment and represent an emerging nanopollution concern for aquatic ecosystems. This study examined the chronic effects of environmentally relevant Pd NP exposure on the freshwater vertebrate model , integrating bioaccumulation analysis, oxidative stress profiling, histopathology, and bulk RNA-seq transcriptomics with computational cell-type inference analyses. Adult zebrafish were exposed for 42 days to low (0.4 ng/L) and high (22 ng/L) Pd NP concentrations. Inductively coupled plasma-mass spectrometry confirmed dose-dependent Pd bioaccumulation in whole-body tissues. Biochemical analyses indicated a disruption of gonadal redox homeostasis, characterized by altered activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase, and lipid peroxidation, indicating sustained oxidative stress. Histological examination of ovaries and testes demonstrated progressive structural damage, including follicular atresia, delayed oocyte maturation, and impaired spermatogenesis, highlighting reproductive vulnerability. Transcriptomic profiling showed concentration-dependent transcriptional changes under Pd NP exposure, including reduced expression of mitochondrial energy metabolism genes and increased expression of DNA repair, cell cycle regulation, steroid biosynthesis, and stress-response pathways. High-dose Pd exposure strongly increased the expression of cell cycle and stress-response genes, including ccnb1 (41 to 8296 TPM), cdc25b (41 to 1937 TPM), and tp53 (110 to 604 TPM), while mitochondrial energy metabolism genes were consistently suppressed. Notably, PI3K-AKT-mTOR, p53, and cell cycle signaling axes exhibited biphasic regulation, reflecting compensatory and maladaptive stress responses. This study identifies potential ecological and human health risks associated with palladium nanoparticle dispersal and emphasizes the need for safer catalyst design and stricter environmental management of platinum group nanoparticles.
