Benzalkonium chloride (BAC) is a widely used disinfectant/preservative, and respiratory exposure to this compound has been reported to be highly toxic. Spray‐form household products have been known to contain BAC together with triethylene glycol (TEG) in their solutions. The purpose of this study was to estimate the toxicity of BAC and TEG mixtures to pulmonary organs using in vitro and in vivo experiments. Human alveolar epithelial (A549) cells incubated with BAC (1‐10 μg/mL) for 24 hours showed significant cytotoxicity, while TEG (up to 1000 μg/mL) did not affect cell viability. However, TEG in combination with BAC aggravated cell damage and inhibited colony formation as compared to BAC alone. TEG also exacerbated BAC‐promoted production of reactive oxygen species (ROS) and reduction of glutathione (GSH) level in A549 cells. However, pretreatment of the cells with N‐acetylcysteine (NAC) alleviated the cytotoxicity, indicating oxidative stress could be a mechanism of the toxicity. Quantification of intracellular BAC by LC/MS/MS showed that cellular distribution/absorption of BAC was enhanced in A549 cells when it was exposed together with TEG. Intratracheal instillation of BAC (400 μg/kg) in rats was toxic to the pulmonary tissues while that of TEG (up to 1000 μg/kg) did not show any harmful effect. A combination of nontoxic doses of BAC (200 μg/kg) and TEG (1000 μg/kg) promoted significant lung injury in rats, as shown by increased protein content and lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluids (BALF). Moreover, BAC/TEG mixture recruited inflammatory cells, polymorphonuclear leukocytes (PMNs), in terminal bronchioles and elevated cytokine levels, tumor necrosis factor α (TNF‐α), and interleukin 6 (IL‐6) in BALF. These results suggest that TEG can potentiate BAC‐induced pulmonary toxicity and inflammation, and thus respiratory exposure to the air mist from spray‐form products containing this chemical combination is potentially harmful to humans.

Bone differentiation is crucial for skeletal development and maintenance. Its dysfunction can cause various pathological conditions such as rickets, osteoporosis, osteogenesis imperfecta, or Paget’s disease. Although traditional two-dimensional cell culture systems have contributed significantly to our understanding of bone biology, they fail to replicate the intricate biotic environment of bone tissue. Three-dimensional (3D) spheroid cell cultures have gained widespread popularity for addressing bone defects. This review highlights the advantages of employing 3D culture systems to investigate bone differentiation. It highlights their capacity to mimic the complex in vivo environment and crucial cellular interactions pivotal to bone homeostasis. The exploration of 3D culture models in bone research offers enhanced physiological relevance, improved predictive capabilities, and reduced reliance on animal models, which have contributed to the advancement of safer and more effective strategies for drug development. Studies have highlighted the transformative potential of 3D culture systems for expanding our understanding of bone biology and developing targeted therapeutic interventions for bone-related disorders. This review explores how 3D culture systems have demonstrated promise in unraveling the intricate mechanisms governing bone homeostasis and responses to pharmacological agents.

Benzalkonium chloride (BAC), an antimicrobial agent in inhalable medications and household sprays, has been reported to be toxic to pulmonary organs. Although cell membrane damage has been considered as the main cytotoxic mechanism of BAC, its concentration- and time-dependent cellular effects on lung epithelium have not been fully understood. In the present study, human lung epithelial (H358) cells were exposed to 0.2–40 μg/mL of BAC for 30 min or 21 days. Cell membranes were rapidly disrupted by 30 min exposure, but 24 h incubation of BAC (4–40 μg/mL) predominantly caused apoptosis rather than necrosis. BAC (2–4 μg/mL) induced mitochondrial depolarization, which may be associated with increased expression of pro-apoptotic proteins (caspase-3, PARP, Bax, p53, and p21), and decreased levels of the anti-apoptotic protein Bcl-2. The protein expression levels of IRE1α, BiP, CHOP, and p-JNK were also elevated by BAC (2–4 μg/mL) suggesting the possible involvement of endoplasmic reticulum stress in inducing apoptosis. Long-term (7–21 days) incubation with BAC (0.2–0.6 μg/mL) did not affect cell viability but led to epithelial-mesenchymal transition (EMT) as shown by the decrease of E-cadherin and the increase of N-cadherin, fibronectin, and vimentin, caused by the upregulation of EMT transcription factors, such as Snail, Slug, Twist1, Zeb1, and Zeb2. Therefore, we conclude that apoptosis could be an important mechanism of acute BAC cytotoxicity in lung epithelial cells, and chronic exposure to BAC even at sub-lethal doses can promote pulmonary EMT.

Overdose of acetaminophen (APAP) can cause severe liver injury. Although alcohol is considered a risk factor for APAP toxicity, the mechanism underlying the interaction between alcohol and APAP remains unclear. Binge alcohol (5 g/kg every 12 h, 3 doses) reduced the concentration of cysteine and glutathione (GSH) and decreased expression of cystathionine β-synthase (CβS), cystathionine γ-lyase (CγL), and glutamate cysteine ligase catalytic subunit (GCLC) in the livers of male C57BL/6 mice. Furthermore, the levels of GSH S-transferase (GST) and GSH peroxidase (GPx) were decreased. To evaluate the effect of binge drinking on APAP-induced liver injury, 300 mg APAP was administered following alcohol binges. APAP in the binge group significantly amplified the serum ALT more than two fold and enhanced the pro-apoptotic proteins with a severe centrilobular necrosis compared to APAP alone. APAP treatment after alcohol binges caused lower levels of hepatic cysteine and GSH than APAP alone over 24 h, indicating that alcohol binges reduced GSH regenerating potential. Exposure to APAP after binge treatment significantly increased oxidative stress (lipid peroxidation) and endoplasmic reticulum (ER) stress (Grp78 and ATF6) markers at 6 h after treatment. Notably, the IRE1α/ASK1/MKK4/JNK pathway was activated, whereas CHOP expression was reduced by APAP administration in mice with pre-exposed alcohol binges compared with APAP alone. Thus, pretreatment with binge alcohol decreases GSH-mediated antioxidant capacity and contributes to augmentation of liver injury caused by subsequent APAP administration through differential ER stress signaling pathway.

Polyhexamethyleneguanidine phosphate (PHMG-P) is a widely used polymeric antimicrobial agent known to induce significant pulmonary toxicity. Several studies have reported that the liver also can be a target organ of polyhexamethyleneguanidine (PHMG) toxicity, but the exact effect of this compound on liver cells is not well understood. To identify the mechanism of PHMG hepatotoxicity, HepG2 cells were exposed to PHMG-P for 72 h. The cell viability was significantly decreased by PHMG-P in a time- and concentration-dependent manner. The mitochondrial membrane potential was markedly reduced by PHMG-P and the apoptotic signaling cascade was activated. The increases observed in C/EBP homologous protein (CHOP), p-IRE, and p-JNK levels in PHMG-P-treated cells indicated the induction of endoplasmic reticulum stress. To verify the role of ER stress in PHMG-P-induced cytotoxicity, HepG2 cells were pretreated with the chemical chaperone, tauroursodeoxycholic acid (TUDCA) and then co-treated with TUDCA and PHMG-P for 24 h. Interestingly, TUDCA inhibited PHMG-P-induced ER stress and cytotoxicity in a dose-dependent manner. The apoptotic cell death and mitochondrial depolarization were also prevented by TUDCA. The proteins involved in the apoptotic pathway were all normalized to their control levels in TUDCA-treated cells. In conclusion, the results suggest that PHMG-P induced significant cytotoxicity in liver cells and ER stress-mediated apoptosis, which may be an important mechanism mediating this hepatotoxicity.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver dysfunction characterized by excess lipid accumulation; non-alcoholic steatohepatitis can transform into more severe conditions, such as cirrhosis and hepatocellular carcinoma. Although several pharmacologic approaches have been evaluated in clinical trials, there are no approved therapies for NAFLD. Previous studies have suggested that taurine supplementation alleviates fatty liver; however, the underlying mechanism remains obscure. In this study, we investigated the beneficial effects of taurine on fatty liver injury in vivo induced by tunicamycin, a chemical endoplasmic reticulum (ER) stressor. The mice were administered 2% taurine for 2 weeks prior to intraperitoneal tunicamycin injection; after 72 h of treatment, the mice were euthanized. Tunicamycin treatment significantly increased the levels of serum ALT and AST and hepatic triglycerides. Notably, these changes were alleviated by taurine supplementation. Taurine normalized the protein and/or mRNA levels involved in ER stress signaling (IRE1a, p-IRE1a, ATF6, XBP1, BiP, and CHOP) and lipid metabolism (CD36, MTTP, and ApoB), which were dysregulated by tunicamycin treatment. The stimulation of hepatic lipid export by taurine was evidenced by the recovery of blood VLDL levels. Furthermore, taurine supplementation prevented tunicamycin-induced lipid peroxidation and decreased glutathione (GSH) levels by correcting abnormal cysteine catabolism involved in the production of both taurine and GSH. Therefore, taurine supplementation can prevent tunicamycin-induced liver injury by counteracting oxidative and ER stress.