Xing-yu Liu

Crotonaldehyde induces oxidative stress and caspase-dependent apoptosis in human bronchial epithelial cells

Crotonaldehyde is a widespread environmental pollutant and lipid peroxidation product. Crotonaldehyde is a risk factor for many diseases (e.g., chronic pulmonary inflammation). However, its toxicity and its mechanism of action have not been thoroughly investigated. The purpose of this study is to investigate crotonaldehyde-induced oxidative stress and mechanism of cell death in BEAS-2B cells. Crotonaldehyde caused decreases of intracellular reduced glutathione levels and increases of reactive oxygen species in a dose-dependent manner. Crotonaldehyde induced cell death by apoptosis, and gradually transitioned to necrosis at high dose of crotonaldehyde, as demonstrated by Annexin V-FITC/PI staining and cell morphology analysis. Crotonaldehyde-induced ATP decline observed in the study might partially account for the switch from apoptosis to necrosis. Mitochondria membrane potential, cytochrome c release, caspase-9, and caspase-3/7 activity were investigated, and the results suggest that crotonaldehyde-induced apoptosis was activated in a caspase-dependent way. Collectively, these results demonstrate crotonaldehyde induces cell oxidative stress and caspase-dependent apoptosis.

Mutagenicity of acrolein and acrolein-induced DNA adducts.

Acrolein mutagenicity relies on DNA adduct formation. Reaction of acrolein with deoxyguanosine generates α-hydroxy-1, N2-propano-2′-deoxyguanosine (α-HOPdG) and γ-hydroxy-1, N2-propano-2′-deoxyguanosine (γ-HOPdG) adducts. These two DNA adducts behave differently in mutagenicity. γ-HOPdG is the major DNA adduct and it can lead to interstrand DNA-DNA and DNA-peptide/protein cross-links, which may induce strong mutagenicity; however, γ-HOPdG can be repaired by some DNA polymerases complex and lessen its mutagenic effects. α-HOPdG is formed much less than γ-HOPdG, but difficult to be repaired, which contributes to accumulation in vivo. Results of acrolein mutagenicity studies haven’t been confirmed, which is mainly due to the conflicting mutagenicity data of the major acrolein adduct (γ-HOPdG). The minor α-HOPdG is mutagenic in both in vitro and in vivo test systems. The role of α-HOPdG in acrolein mutagenicity needs further investigation. The inconsistent result of acrolein mutagenicity can be attributed, at least partially, to a variety of acrolein-DNA adducts formation and their repair in diverse detection systems. Recent results of detection of acrolein-DNA adduct in human lung tissues and analysis of P53 mutation spectra in acrolein-treated cells may shed some light on mechanisms of acrolein mutagenicity. These aspects are covered in this mini review.

Crotonaldehyde-exposed macrophages induce IL-8 release from airway epithelial cells through NF-κB and AP-1 pathways

Crotonaldehyde, a highly toxic α, β-unsaturated aldehyde, is a major component of cigarette smoke and a ubiquitous environmental pollutant. Crotonaldehyde exposure is known to have adverse effects on respiratory health, but the underlying mechanisms remain obscure. To examine the interaction between macrophages and airway epithelial cells after exposure to crotonaldehyde, BEAS-2B and A549 cells were treated with conditioned media from a human monocytic leukemia cell line (THP-1) cells stimulated with crotonaldehyde. We demonstrate that conditioned media from THP-1 cells stimulated with crotonaldehyde increased interleukin (IL)-8 production, enhanced nuclear factor (NF)-κB and AP-1 DNA-binding activity in BEAS-2B and A549 cells. Analysis of these conditioned media revealed marked increases in tumor necrosis factor (TNF)-α, IL-1β and IL-8 levels. Preincubation of conditioned media with either TNF-α- or IL-1β-neutralizing antibodies reduced IL-8 production. Furthermore, BEAS-2B and A549 cells directly treated with crotonaldehyde induced increase in IL-8 production. These data suggest that crotonaldehyde is capable of directly stimulating the production of IL-8 in both macrophages and airway epithelial cells. Crotonaldehyde-stimulated macrophages also amplify the inflammatory response by enhancing IL-8 release from airway epithelial cells mediated by NF-κB and AP-1 pathways through a mechanism involving TNF-α and IL-1β. These findings indicate that crotonaldehyde can cause lung inflammatory response via multiple mechanisms, and may contribute to chronic airway inflammation in smokers.

Gene expression profile and cytotoxicity of human bronchial epithelial cells exposed to crotonaldehyde

Crotonaldehyde is an environment pollutant and lipid peroxidation product. Crotonaldehyde produces adverse effects to humans and serves as a risk factor for human pulmonary diseases. Like acrolein and 4-hydroxynonenal, crotonaldehyde seems likely to alter many cell signaling cascades, including inflammatory responses. The purpose of this study was to investigate the genome-wide transcriptional responses of normal human bronchial epithelial cells exposed to crotonaldehyde. Using microarrays technology, the global changes in transcriptional level were analyzed. Prior to RNA extraction, cells were exposed to crotonaldehyde at 40 or 80 microM for 3 or 6h. Real-time quantitative polymerase chain reaction (qPCR) was performed to validate microarray data and cell cycle arrest was determined. The commonly differentially regulated genes in many biological processes were dysregulated including inflammatory responses, exogenous metabolism, cell cycle, heat shock responses, and antioxidant responses. Results in the present study screen out the important roles of HMOX1 in regulating other signaling cascades and ALDH1A3 in detoxifying exogenous toxicants. Collectively, our study demonstrated that crotonaldehyde altered gene expression profile in the genome-wide transcriptional level in normal human bronchial epithelial cells. And many of them represented potential mechanisms of crotonaldehyde causing cytotoxicity and tissue injury in the human lung.

Crotonaldehyde induces apoptosis and immunosuppression in alveolar macrophages.

Crotonaldehyde, a highly toxic α, β-unsaturated aldehyde, is a major component of cigarette smoke (CS) and a ubiquitous environmental pollutant. Exposure to crotonaldehyde-rich pollutants such as CS is associated with suppression of respiratory host defense against infections. The aim of this study was to evaluate the apoptotic and immunological effects of crotonaldehyde exposure in a rat alveolar macrophage (AM) cell line, NR8383. Our studies showed that crotonaldehyde induced AM cell death mainly via the apoptotic process. Crotonaldehyde also decreased the phagocytic activity of AMs. Crotonaldehyde caused inhibition of NO, TNF-α, IL-1β and IL-12 production in AMs treated with lipopolysaccharide (LPS), which is probably related to inhibition of NF-κB activation. These results indicate that crotonaldehyde can cause adverse effects in AMs via multiple mechanisms, and may contribute to compromised lung immunological response in smokers.

Crotonaldehyde induces autophagy-mediated cytotoxicity in human bronchial epithelial cells via PI3K, AMPK and MAPK pathways ☆

Crotonaldehyde is an ubiquitous hazardous pollutant in the environment which can be produced naturally, artificially and endogenously. Acute exposure of crotonaldehyde was reported to induce severe lung injury in humans and experimental animals. However, the exact toxicity mechanisms of crotonaldehyde in organisms have not been fully explored. In the present study, we explored the role autophagy played in the cytotoxicity induced by crotonaldehyde in human bronchial epithelial cells (BEAS-2B), and the pathways that mediated autophagy, including the phosphatidylinositol 3-kinase (PI3K) pathway, the AMP-activated protein kinase (AMPK) pathway and the mitogen-activated protein kinase (MAPK) pathways, were examined and validated. We found that crotonaldehyde induced cytotoxicity and autophagy simultaneously in BEAS-2B cells, and blockage of autophagic flux significantly elevated the viability of BEAS-2B exposed to high concentrations of crotonaldehyde. Crotonaldehyde down-regulated the activity of PI3K pathway, and elevated the activities of AMPK and MAPK pathways. Pretreatment of specific agonist or antagonist of these pathways could inhibit autophagy and partly improve the viability. These results suggested that acute exposure of crotonaldehyde induced cell death mediated by autophagy, which might be helpful to elucidate the toxicity mechanisms of crotonaldehyde and contribute to environmental and human health risk assessment.

Acute exposure to crotonaldehyde induces dysfunction of immune system in male Wistar rats

Crotonaldehyde is a ubiquitous air pollutant in the environment. It is reported to be harmful to the biosystems in vivo and in vitro. The exposure to crotonaldehyde irritates the mucous membranes and induces edema, hyperemia, cell necrosis, inflammation, and acute respiratory distress syndrome in the lungs. However, the effects of crotonaldehyde on the immune system have not been reported. In the present study, 6-8 weeks old male Wistar rats were exposed to crotonaldehyde by intratracheal instillation at doses of 4, 8, and 16 μL/kg body weight (b.w.). The general damage in the animals was investigated; the cell counting and the biochemical analysis in the peripheral blood were tested. Furthermore, we investigated the functions of alveolar macrophages (AMs), the alterations of the T-lymphocyte subsets, and the cell composition in the bronchoalveolar lavage fluid (BALF). We found that the activities of the animals were changed after exposure to crotonaldehyde, the cellular ratios and the biochemical components in the peripheral blood were altered, the ratio of mononuclear phagocytes decreased, and the ratios of lymphocytes and granulocytes elevated significantly in BALF. Meanwhile, crotonaldehyde altered the ratio of the T-lymphocyte subsets, and the phagocytic rates and indices of AMs increased obviously. In conclusion, crotonaldehyde induces dysfunction of immune system in male Wistar rats.