Hibiscus manihot L. flower extract induces anticancer activity through modulation of apoptosis and autophagy in A549 cells.

Lung cancer is a major public health issue and heavy burden in China and worldwide due to its high incidence and mortality without effective treatment. It's imperative to develop new treatments to overcome drug resistance. Natural products from food source, given their wide-ranging and long-term benefits, have been increasingly used in tumor prevention and treatment. This study revealed that Hibiscus manihot L. flower extract (HML) suppressed the proliferation and migration of A549 cells in a dose and time dependent manner and disrupting cell cycle progression. HML markedly enhanced the accumulation of ROS, stimulated the dissipation of mitochondrial membrane potential (MMP) and that facilitated mitophagy through the loss of mitochondrial function. In addition, HML induced apoptosis by activation of the PTEN-P53 pathway and inhibition of ATG5/7-dependent autophagy induced by PINK1-mediated mitophagy in A549 cells. Moreover, HML exert anticancer effects together with 5-FU through synergistic effect. Taken together, HML may serve as a potential tumor prevention and adjuvant treatment for its functional attributes.

NR2F2 alleviates pulmonary fibrosis by inhibition of epithelial cell senescence.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fatal, and aging-associated interstitial lung disease with a poor prognosis and limited treatment options, while the pathogenesis remains elusive. In this study, we found that the expression of nuclear receptor subfamily 2 group F member 2 (NR2F2), a member of the steroid thyroid hormone superfamily of nuclear receptors, was reduced in both IPF and bleomycin-induced fibrotic lungs, markedly in bleomycin-induced senescent epithelial cells. Inhibition of NR2F2 expression increased the expression of senescence markers such as p21 and p16 in lung epithelial cells, and activated fibroblasts through epithelial-mesenchymal crosstalk, inversely overexpression of NR2F2 alleviated bleomycin-induced epithelial cell senescence and inhibited fibroblast activation. Subsequent mechanistic studies revealed that overexpression of NR2F2 alleviated DNA damage in lung epithelial cells and inhibited cell senescence. Adenovirus-mediated Nr2f2 overexpression attenuated bleomycin-induced lung fibrosis and cell senescence in mice. In summary, these data demonstrate that NR2F2 is involved in lung epithelial cell senescence, and targeting NR2F2 may be a promising therapeutic approach against lung cell senescence and fibrosis.

ACSS3 regulates the metabolic homeostasis of epithelial cells and alleviates pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease of unknown etiology. The emerging evidence demonstrates that metabolic homeostatic imbalance caused by repetitive injuries of the alveolar epithelium is the potential pathogenesis of IPF. Proteomic analysis identified that Acetyl-CoA synthetase short chain family member 3 (ACSS3) expression was decreased in IPF patients and mice with bleomycin-induced fibrosis. ACSS3 participated in lipid and carbohydrate metabolism. Increased expression of ACSS3 downregulated carnitine palmitoyltransferase 1A (CPT-1A) and resulted in the accumulation of lipid droplets, while enhanced glycolysis which led to an increase in extracellular lactic acid levels in A549 cells. ACSS3 increases the production of succinyl-CoA through propionic acid metabolism, and decreases the generation of acetyl-CoA and ATP in alveolar epithelial cells. Overexpression of Acss3 inhibited the excessive deposition of ECM and attenuated the ground-glass opacity which determined by micro-CT in vivo. In a nutshell, our findings demonstrate that ACSS3 decreased the fatty acid oxidation through CPT1A deficiency and enhanced anaerobic glycolysis, this metabolic reprogramming deactivate the alveolar epithelial cells by lessen mitochondrial fission and fusion, increase of ROS production, suppression of mitophagy, promotion of apoptosis, suggesting that ACSS3 might be potential therapeutic target in pulmonary fibrosis.

Tuftelin1 drives experimental pulmonary fibrosis progression by facilitating stress fiber assembly.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease (ILD) with unknown etiology, characterized by sustained damage repair of epithelial cells and abnormal activation of fibroblasts, the underlying mechanism of the disease remains elusive. METHODS: To evaluate the role of Tuftelin1 (TUFT1) in IPF and elucidate its molecular mechanism. We investigated the level of TUFT1 in the IPF and bleomycin-induced mouse models and explored the influence of TUFT1 deficiency on pulmonary fibrosis. Additionally, we explored the effect of TUFT1 on the cytoskeleton and illustrated the relationship between stress fiber and pulmonary fibrosis. RESULTS: Our results demonstrated a significant upregulation of TUFT1 in IPF and the bleomycin (BLM)-induced fibrosis model. Disruption of TUFT1 exerted inhibitory effects on pulmonary fibrosis in both in vivo and in vitro. TUFT1 facilitated the assembly of microfilaments in A549 and MRC-5 cells, with a pronounced association between TUFT1 and Neuronal Wiskott-Aldrich syndrome protein (N-WASP) observed during microfilament formation. TUFT1 can promote the phosphorylation of tyrosine residue 256 (Y256) of the N-WASP (pY256N-WASP). Furthermore, TUFT1 promoted transforming growth factor-ß1 (TGF-ß1) induced fibroblast activation by increasing nuclear translocation of pY256N-WASP in fibroblasts, while wiskostatin (Wis), an N-WASP inhibitor, suppressed these processes. CONCLUSIONS: Our findings suggested that TUFT1 plays a critical role in pulmonary fibrosis via its influence on stress fiber, and blockade of TUFT1 effectively reduces pro-fibrotic phenotypes. Pharmacological targeting of the TUFT1-N-WASP axis may represent a promising therapeutic approach for pulmonary fibrosis.

ADRB2 inhibition combined with antioxidant treatment alleviates lung fibrosis by attenuating TGFß/SMAD signaling in lung fibroblasts.

Idiopathic pulmonary fibrosis is a progressive and fatal interstitial lung disease with a poor prognosis and limited therapeutic options, which is characterized by aberrant myofibroblast activation and pathological remodeling of the extracellular matrix, while the mechanism remains elusive. In the present investigation, we observed a reduction in ADRB2 expression within both IPF and bleomycin-induced fibrotic lung samples, as well as in fibroblasts treated with TGF-ß1. ADRB2 inhibition blunted bleomycin-induced lung fibrosis. Blockage of the ADRB2 suppressed proliferation, migration, and invasion and attenuated TGF-ß1-induced fibroblast activation. Conversely, the enhancement of ADRB2 expression or functionality proved capable of inducing fibroblast-to-myofibroblast differentiation. Subsequent mechanistic investigation revealed that inhibition of ADRB2 suppressed the activation of SMAD2/3 in lung fibroblasts and increased phos-SMAD2/3 proteasome degradation, and vice versa. Finally, ADRB2 inhibition combined with antioxidants showed increased efficacy in the therapy of bleomycin-induced lung fibrosis. In short, these data indicate that ADRB2 is involved in lung fibroblast differentiation, and targeting ADRB2 could emerge as a promising and innovative therapeutic approach for pulmonary fibrosis.

Cellular Senescence: A Troy Horse in Pulmonary Fibrosis.

Pulmonary fibrosis (PF) is a chronic interstitial lung disease characterized by myofibroblast abnormal activation and extracellular matrix deposition. However, the pathogenesis of PF remains unclear, and treatment options are limited. Epidemiological studies have shown that the average age of PF patients is estimated to be over 65 years, and the incidence of the disease increases with age. Therefore, PF is considered an age-related disease. A preliminary study on PF patients demonstrated that the combination therapy of the anti-senescence drugs dasatinib and quercetin improved physical functional indicators. Given the global aging population and the role of cellular senescence in tissue and organ aging, understanding the impact of cellular senescence on PF is of growing interest. This article systematically summarizes the causes and signaling pathways of cellular senescence in PF. It also objectively analyzes the impact of senescence in AECs and fibroblasts on PF development. Furthermore, potential intervention methods targeting cellular senescence in PF treatment are discussed. This review not only provides a strong theoretical foundation for understanding and manipulating cellular senescence, developing new therapies to improve age-related diseases, and extending a healthy lifespan but also offers hope for reversing the toxicity caused by the massive accumulation of senescence cells in humans.

Single-Cell RNA Sequencing Provides New Insights into Therapeutic Roles of Thyroid Hormone in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fatal interstitial lung disease without an effective cure. Herein, we explore the role of 3,5,3'-triiodothyronine (T3) administration on lung alveolar regeneration and fibrosis at the single-cell level. T3 supplementation significantly altered the gene expression in fibrotic lung tissues. Immune cells were rapidly recruited into the lung after the injury; there were much more M2 macrophages than M1 macrophages in the lungs of bleomycin-treated mice; and M1 macrophages increased slightly, whereas M2 macrophages were significantly reduced after T3 treatment. T3 enhanced the resolution of pulmonary fibrosis by promoting the differentiation of Krt8+ transitional alveolar type II epithelial cells into alveolar type I epithelial cells and inhibiting fibroblast activation and extracellular matrix production potentially by regulation of Nr2f2. In addition, T3 regulated the crosstalk of macrophages with fibroblasts, and the Pros1-Axl signaling axis significantly facilitated the attenuation of fibrosis. The findings demonstrate that administration of a thyroid hormone promotes alveolar regeneration and resolves fibrosis mainly by regulation of the cellular state and cell-cell communication of alveolar epithelial cells, macrophages, and fibroblasts in mouse lungs in comprehensive ways.

Serum Proteomics Identifies Biomarkers Associated With the Pathogenesis of Idiopathic Pulmonary Fibrosis.

The heterogeneity of idiopathic pulmonary fibrosis (IPF) limits its diagnosis and treatment. The association between the pathophysiological features and the serum protein signatures of IPF currently remains unclear. The present study analyzed the specific proteins and patterns associated with the clinical parameters of IPF based on a serum proteomic dataset by data-independent acquisition using MS. Differentiated proteins in sera distinguished patients with IPF into three subgroups in signal pathways and overall survival. Aging-associated signatures by weighted gene correlation network analysis coincidently provided clear and direct evidence that aging is a critical risk factor for IPF rather than a single biomarker. Expression of LDHA and CCT6A, which was associated with glucose metabolic reprogramming, was correlated with high serum lactic acid content in patients with IPF. Cross-model analysis and machine learning showed that a combinatorial biomarker accurately distinguished patients with IPF from healthy individuals with an area under the curve of 0.848 (95% CI = 0.684-0.941) and validated from another cohort and ELISA assay. This serum proteomic profile provides rigorous evidence that enables an understanding of the heterogeneity of IPF and protein alterations that could help in its diagnosis and treatment decisions.

Artificial neural network identified the significant genes to distinguish Idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease that causes irreversible damage to lung tissue characterized by excessive deposition of extracellular matrix (ECM) and remodeling of lung parenchyma. The current diagnosis of IPF is complex and usually completed by a multidisciplinary team including clinicians, radiologists and pathologists they work together and make decision for an effective treatment, it is imperative to introduce novel practical methods for IPF diagnosis. This study provided a new diagnostic model of idiopathic pulmonary fibrosis based on machine learning. Six genes including CDH3, DIO2, ADAMTS14, HS6ST2, IL13RA2, and IGFL2 were identified based on the differentially expressed genes in IPF patients compare to healthy subjects through a random forest classifier with the existing gene expression databases. An artificial neural network model was constructed for IPF diagnosis based these genes, and this model was validated by the distinctive public datasets with a satisfactory diagnostic accuracy. These six genes identified were significant correlated with lung function, and among them, CDH3 and DIO2 were further determined to be significantly associated with the survival. Putting together, artificial neural network model identified the significant genes to distinguish idiopathic pulmonary fibrosis from healthy people and it is potential for molecular diagnosis of IPF.

Fenbendazole Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice via Suppression of Fibroblast-to-Myofibroblast Differentiation.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal interstitial lung disease with unknown etiology. Despite substantial progress in understanding the pathogenesis of pulmonary fibrosis and drug development, there is still no cure for this devastating disease. Fenbendazole (FBZ) is a benzimidazole compound that is widely used as an anthelmintic agent and recent studies have expanded the scope of its pharmacological effects and application prospect. This study demonstrated that FBZ treatment blunted bleomycin-induced lung fibrosis in mice. In vitro studies showed that FBZ inhibited the proliferation and migration of human embryo lung fibroblasts. Further studies showed that FBZ significantly inhibited glucose consumption, moderated glycolytic metabolism in fibroblasts, thus activated adenosine monophosphate-activated protein kinase (AMPK), and reduced the activation of the mammalian target of rapamycin (mTOR) pathway, thereby inhibiting transforming growth factor-ß (TGF-ß1)-induced fibroblast-to-myofibroblast differentiation and collagen synthesis. In summary, our data suggested that FBZ has potential as a novel treatment for pulmonary fibrosis.

Inhibition of MAD2L1 Mediates Pulmonary Fibrosis through Impairment of Mitochondrial Function and Induction of Cell Senescence.

Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible, and progressive interstitial lung disease characterized by recurrent alveolar epithelial cell injury, fibroblast hyperproliferation, and cumulative deposition of extracellular matrix leading to alveolar destruction in the lungs. Mitotic arrest deficient 2 like 1 (MAD2L1) is a component of the mitotic spindle assembly checkpoint that prevents the onset of anaphase until all chromosomes are properly aligned at metaphase and is a potential therapeutic target in cancers. However, the role of MAD2L1 in pulmonary fibrosis has not been explored. We analyzed the expression of MAD2L1 in lung tissues from control subjects, IPF patients, and mice with bleomycin-induced fibrosis via IHC, qRT-PCR, and Western blot analysis. We examined the roles of MAD2L1 in ROS production, mitochondrial function, cell senescence, and the establishment of a profibrotic microenvironment. We found that MAD2L1 was highly upregulated in alveolar epithelial cells in fibrotic lung tissues from both patients with IPF and mice with bleomycin-induced fibrosis. Loss of MAD2L1 expression or activity led to decreases of cell viability and proliferation in A549 cells. Subsequent mechanistic investigation demonstrated that inhibition of MAD2L1 damaged mitochondria, which led to augmented ROS production and cellular senescence, and thus promoted the establishment of a profibrotic microenvironment. Taken together, these results reveal that alleviation of alveolar epithelial cell mitochondrial damage arising from augmentation of MAD2L1 may be a novel therapeutic strategy for mitigating pulmonary fibrosis.

Progesterone affects the transcription of genes in the circadian rhythm signaling and hypothalamic-pituitary-gonadal axes and changes the sex ratio in crucian carp (Carassius auratus).

Progesterone (P4) is an extensively applied progestin in human and veterinary medicine that has been widely detected in ambient aquatic environments, which can be detrimental to the health of aquatic organisms. Here we investigate the long-term effects of P4 on the transcription of genes related to the circadian rhythm signaling pathway and hypothalamic-pituitary-gonadal (HPG) axes in the crucian carp, which may have a potentially negative on endocrine-disrupting and sex differentiation impacts. Our results suggest that the expression of genes associated with the circadian rhythm signaling pathway are altered following exposure for 10, 20, 30, 40, 50 and 60 d, leading to disorders in the endocrine system disorders and the regulation of HPG axes-related gene expression. These maladies may affect gonadal development and the reproductive systems of crucian carp and provide a plausible mechanism for the observed change in sex ratio toward females after 180 d.

Cytotoxicity, genotoxicity, oxidative stress, and apoptosis in HepG2 cells induced by the imidazole ionic liquid 1-dodecyl-3-methylimidazolium chloride.

This study purposes to assess the cytotoxicity of 1-dodecyl-3-methylimidazolium chloride ([C12 min]Cl) in human hepatocellular carcinoma (HepG2) cells. To this end, HepG2 cells were exposed to a range concentration of [C12 min]Cl and evaluated cell viability, genotoxicity, oxidative stress, apoptosis, cell cycle, and apoptosis-related gene expression to determine cytotoxicity. The outcomes showed that [C12 min]Cl curbed HepG2 cell growth and reduced cell viability in a concentration- and time-dependent manner. Moreover, our assay results also revealed that exposure to [C12 min]Cl prompted DNA damage and apoptosis, reduced SOD and GSH content, enhanced MDA level, and changed the cell cycle of HepG2 cells. In addition, [C12 min] Cl caused alters in the expression levels of p53, Bax, and Bcl-2, indicating that p53 and Bcl-2 family may be involved in the cytotoxicity and apoptosis of HepG2 cells induced by [C12 min]C1. In summary, these results indicate that [C12 min]Cl exerts genotoxicity, physiological toxicity and prompts apoptosis in HepG2 cells, and is not an alleged green solvent.

Progesterone affects sex differentiation and alters transcriptional of genes along circadian rhythm signaling and hypothalamic-pituitary-gonadal axes in juvenile Yellow River Carp (Cyprinus carpio var.).

Progesterone (P4) is a biologically active steroid hormone that is involved in the regulation of oocyte growth and maturation, as well as development of the endometrium and implantation in the uterus of humans. It can also stimulate oocyte maturation in female fish, as well as spermatogenesis and sperm motility in male fish. Thus, P4 has been extensively used in human and animal husbandry as a typical progestin. However, P4 remaining in the water environment will pose a potential hazard to aquatic organisms. For example, it can interfere with sex differentiation and reproduction in aquatic vertebrates such as fish. Therefore, we investigated the effects of prolonged progesterone exposure on the expression of genes related to circadian rhythm signaling and the hypothalamic-pituitary-gonadal (HPG) axes in Yellow River Carp, which may have a potential impact on their sex differentiation. Our results suggested that P4 exposure altered the expression of genes related to circadian rhythm signaling, which can lead to disorders in the endocrine system and regulate the HPG axes-related activities. Furthermore, the expression of genes related to the HPG axes was also altered, which might affect gonadal development and the reproductive systems of Yellow River Carp. In addition, these changes may provide a plausible mechanism for the observed shifts in their sex ratio toward females.

Molecular cloning, characterization of dax1 gene and its response to progesterone in Misgurnus anguillicaudatus.

Progesterone (P4) are aquatic contaminants that can impair fish reproduction even in low concentrations. The aim of this study was to investigate the effects of P4 on the sex differentiation, by quantitative determination of transcriptional changes of a candidate target gene (dax1, has a function in the sex determination and gonadal differentiation of several vertebrate species) in Misgurnus anguillicaudatus. We first cloned and characterized the full-length cDNAs for the dax1 in M. anguillicaudatus (designated as Ma-dax1). Sequence analysis reveals that Ma-dax1 shares high homology with dax1 in other species. Quantitative real-time PCR (qRT-PCR) and in situ hybridization showed that Ma-dax1 gene was highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis, central nervous system development and gonad development. For the P4 administration assay, groups of mature fish were exposed for 1, 7, 14, 21 and 28 days to nominal concentrations of 10, 100, and 1000 ng/L P4 in a flow-through system. Quantification of Ma-dax1 transcripts revealed the expression of Ma-dax1 mRNA is altered after P4 treatment in mature gonads. Those showed that P4 could influence the sexual development and sex differentiation in M. anguillicaudatus by disturbing sex differentiation-associated gene expression, and dax1 can be used as a sensitive molecular biomarker for early warning to monitor the environmental progestins chemicals in fresh water environment.

Molecular cloning and mRNA expression pattern of Sox4 in Misgurnus anguillicaudatus.

Sox4, a member of the SoxC subfamily which of the Sox family, plays important roles in the development of the vertebrate gonad and nervous system. We have cloned a Sox4 homologue fromthe brain of Misgurnus anguillicaudatus using homologous cloning and rapid amplification of cDNA ends. We named the cloned gene as MaSox4. The full-length cDNA was 2122 bp, containing a 718 bp 5'-untranslated region and a 267 bp 3'-untranslated region. The open-reading frame of the cloned gene encoded 378 amino acids and contained a characteristic HMG-box DNA-binding domain with the specific motif (RPMNAFMVW). Phylogenetic analysis indicated that MaSox4 is highly homologous to Sox4 in different species. Protein sequence analysis showed that MaSox4 is a nonsecretory hydrophilic protein. Quantitative real-time reverse transcription polymerase chain reaction and in situ hybridization assay revealed that MaSox4 was ubiquitously expressed during embryogenesis and is present in various adult tissues, especially in the central nervous system. Our study suggests that MaSox4 is highly conserved among vertebrates' evolution and might be involved in developmental processes such as embryogenesis, neurogenesis and gonad development.

Cloning, characterization and function analysis of DAX1 in Chinese loach (Paramisgurnus dabryanus).

The mechanisms of sex determination and differentiation have not been elucidated in most fish species. In this study, the full-length cDNAs of DAX1 was cloned and characterized in aquaculture fish Chinese loach (Paramisgurnus dabryanus), designated as Pd-DAX1. The cDNA sequence of Pd-DAX1 was 1261 bp, including 795 bp open reading frame (ORF) encoding 264 amino acids. Pd-DAX1 shares highly identical sequence with DAX1 homologues from different species. The expression profiles of Pd-DAX1 in different developmental stages and diverse adult tissues were analyzed by quantitative real-time RT-PCR and in situ hybridization (ISH). Pd-DAX1 was continuously expressed during embryogenesis, with the extensive distribution in the development of the central nervous system. Tissue distribution analysis revealed that Pd-DAX1 expressed widely in adult tissues, with the highest expression level found in testis, moderate level in ovary, showing a sex-dimorphic expression pattern. Pd-DAX1 mainly located in spermatogonia cells, spermatocytes, primary oocytes and previtellogenic oocyte cells, implying that Pd-DAX1 may involve in gametogenesis. These preliminary findings suggest that Pd-DAX1 gene is highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis, central nervous system development and gonad development.

Toxicity of imidazoles ionic liquid [C16mim]Cl to Hela cells.

Our study aimed to evaluate the toxicity of 1-hexadecyl-3-methylimidazolium chloride ([C16min]Cl) on the human cervical carcinoma (Hela) cells. We evaluated toxicity, cell viability, genotoxicity, oxidative stress, apoptosis, and apoptosis-related gene expression in Hela cells following exposure to [C16min]Cl. The results indicated that [C16min]Cl inhibited the growth of Hela cells, decreased cell viability, induced DNA damage and apoptosis, inhibited superoxide dismutase, decreased glutathione content, as well as increased the cellular malondialdehyde level of Hela cells. Moreover, [C16min]Cl induced changes in the transcription of p53, Bax and Bcl-2, suggesting that the p53 and Bcl-2 family might have been involved in the cytotoxicity and apoptosis induced by [C16min]Cl in Hela cells. Taken together, these results revealed that [C16min]Cl imparts oxidative stress, genotoxicity, and induces apoptosis in Hela cells; hence, it is not a green solvent.

Toxic effects of cyhalofop-butyl on embryos of the Yellow River carp (Cyprinus carpio var.): alters embryos hatching, development failure, mortality of embryos, and apoptosis.

As a universal environmental contaminant, the herbicide cyhalofop-butyl is considered to have infested effects on the embryonic development of aquatic species. The present study focused on an assessment of the impacts of cyhalofop-butyl on Yellow River carp embryos. It was found that cyhalofop-butyl inhibited the hatching of the embryos, and the hatching rate decreased with higher concentrations of the herbicide. The mortality rate was increased on exposure to cyhalofop-butyl and was significantly higher in the 1.6 and 2 mg/L treatment groups over 48 h. All of the embryos of the 2 mg/L treatment group died within the 48 h post-hatching stage. And the transcription of several embryos related to apoptosis was also influenced by cyhalofop-butyl exposure. Further, cyhalofop-butyl exposure leads to a series of morphological changes (pericardial edema, tail deformation, and spine deformation) in embryos, which were consistent with significant modifications in the associated genes. These results provided a scientific basis for further studies into the effects of cyhalofop-butyl on aquatic organisms.

Toxicity of imidazoles ionic liquid [C16mim]Cl to HepG2 cells.

Ionic liquids have garnered increasing attention due to their capacity for low vapor pressure, lack of flammability, designability, good stability, and as a asubstitute for conventional organic solvents. However, their toxicity to various organisms has caused growing concern in recent years. Our study aims to evaluate the toxicity of 1-hexadecyl-3-methylimidazolium chloride ([C16min]Cl) to human hepatocellular carcinoma (HepG2) cells, including cell viability, genotoxicity, oxidative stress, apoptosis, cell cycle, and apoptosis-related gene expression. Our results with HepG2 cells suggested that [C16min]Cl inhibited cellular growth, decreased cell viability, induced DNA damage and apoptosis, inhibited superoxide dismutase, decreased glutathione content, increased cellular malondialdehyde levels as well as altering the cell cycle. Moreover, the induction of [C16min]Cl altered the transcription of p53, Bax and Bcl-2, which are critical for controlling cell cycles progression and death, which suggests its involvement with cytotoxicity and apoptosis induced by [C16min]Cl in HepG2 cells. Taken together, these results revealed that [C16min]Cl exerted genotoxicity, oxidative stress and induced apoptosis in HepG2 cells; hence, it is not a healthy solvent.