Mee Song

Inflammation in methotrexate-induced pulmonary toxicity occurs via the p38 MAPK pathway.

Methotrexate (MTX) has been widely used for the treatment of inflammatory diseases and rheumatoid arthritis (RA), as well as a variety of tumors. However, MTX-induced toxicity is a serious and unpredictable side effect of this therapy and an important clinical problem. We used microarray analysis to examine MTX-induced gene expression in a human lung epithelial cell line (BEAS-2B) and identified 10 differentially expressed genes related to the p38 mitogen-activated protein kinase (MAPK) pathway, including IL-1beta, MKK6, and MAPKAPK2. Differential gene expression was confirmed via real-time RT-PCR. To determine the functional significance of MTX-induced p38 MAPK activation, we used a p38 MAPK inhibitor (SB203580) to block the p38 MAPK cascade. We also used protein array technology to investigate the modulated expression of pro- and anti-inflammatory cytokines in BEAS-2B cells. MTX activated IL-1beta expression and induced the phosphorylation of various proteins in the p38 MAPK cascade, including TAK1, MKK3/MKK6, p38 MAPK, MAPKAPK2, and HSP27. Finally, HSP27 activation may increase IL-8 secretion, resulting in a pulmonary inflammatory response such as pneumonitis. Although IL-1beta and IL-8 expression increased, the expression of IL-4, IL-6, IL-12, TNF-alpha, MIP-1alpha, and MIP-1beta decreased in a dose-dependent manner. These results suggest that the modulation of cytokine expression may play an important role in MTX-induced pulmonary toxicity.

Polycyclic aromatic hydrocarbons induce migration in human hepatocellular carcinoma cells (HepG2) through reactive oxygen species‐mediated p38 MAPK signal transduction

Although polycyclic aromatic hydrocarbons (PAHs) are carcinogenic and have been extensively studied with regard to tumor formation, few studies have investigated the involvement of these environmental chemicals in tumor migration and invasion. Polycyclic aromatic hydrocarbons induce reactive oxygen species (ROS) and activate MAPK signal transduction. The p38 signaling transduction pathway, one of the most typical MAPK pathways, plays an essential role in regulating cell migration. Therefore, we investigated whether three PAHs, benzo[a]anthracene (B[a]A), benzo[k]fluoranthene (B[k]F), and indeno[1,2,3‐c,d]pyrene (IND), induce migration in human hepatocellular carcinoma cell line HepG2 through ROS‐mediated p38 MAPK signal transduction. Reactive oxygen species generation and p38 MAPK activity both increased in a dose‐dependent manner and were prevented by SB203580, an inhibitor of p38 MAPK, and N‐acetylcysteine (NAC), a ROS scavenger. Expression of migration‐related genes was also increased by B[a]A, B[k]F, and IND in a dose‐dependent manner and was inhibited by SB203580 and NAC. The migration of HepG2 cells, observed using the Transwell migration assay, also increased in a dose‐dependent manner and was prevented by SB203580 and NAC. Our results indicate that the ROS‐mediated p38 MAPK signaling pathway plays an essential role in the PAH‐induced migration of HepG2 cells. (Cancer Sci 2011; 102: 1636–1644)

Identification of molecular signatures predicting the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs).

Assessing the potential carcinogenicity of human toxins represents an ongoing challenge. Chronic rodent bioassays predict human cancer risk with limited reliability, and are expensive and time-consuming. To identify alternative prediction methods, we evaluated a transcriptomics-based human in vitro model to classify carcinogens by their modes of action. The aim of this study was to determine the transcriptomic response and identify specific molecular signatures of polycyclic aromatic hydrocarbons (PAHs), which can be used as predictors of carcinogenicity of environmental toxins in human in vitro systems. We found that characteristic molecular signatures facilitate identification and prediction of carcinogens. To evaluate the change in gene expression levels, human hepatocellular carcinoma (HepG2) cells were exposed to nine different PAHs (benzo[a]pyrene, dibenzo[a,h]anthracene, 3-methylcholanthrene, naphthalene, chrysene, phenanthrene, benzo[a]anthracene, benzo[k]fluoranthene, and indeno[1,2,3-c,d]pyrene) for 48 h. Unsupervised gene expression analysis resulted in a characteristic molecular signature for each toxin, and a supervised analysis identified 31 outlier genes as distinct molecular signatures distinguishing carcinogens from noncarcinogens. Further analysis and multi-classification revealed 430 genes as surrogate markers for predicting carcinogenic potencies of each PAH with 100% accuracy. Our results suggest that these expression signatures can be used as predictable and discernible surrogate markers for detecting and predicting PAH exposure, and their carcinogenic potential. Furthermore, the use of these markers can be more widely applied in combination with traditional techniques for assessing and predicting toxic exposure to PAHs.

Mechanisms underlying methotrexate-induced pulmonary toxicity

Background: Methotrexate (MTX) has been widely used for the treatment of inflammatory diseases and rheumatoid arthritis, as well as a variety of tumors. However, MTX-induced pulmonary toxicity is a serious and unpredictable side effect of the therapy, which includes allergic, cytotoxic or immunologic reactions, and is a major clinical problem. Objective: To summarize the mechanisms of action involved in MTX-induced pulmonary toxicity. Methods: We reviewed the literature describing MTX-induced adverse pulmonary effects and the mechanisms of action underlying MTX-induced pulmonary toxicity. Conclusion: The mechanisms underlying MTX toxicity are complex. The clinical effects may be attributable to both the anti-inflammatory and immunosuppressive effects of MTX. The mechanisms causing the side effects of MTX include mutation of the genotype, inhibition of transport, MTX-polyglutamates and P-glycoprotein binding with MTX. The p38 MAPK-signaling pathway is especially associated with a pulmonary inflammatory response. These mechanisms can be applied to optimize drug treatment.

Genotoxicity of nano-silica in mammalian cell lines

Nanomaterials are defined by the U.S. National Nanotechnology Initiative as materials that have at least one dimension in the 1- to 100-nm range. Due to their unique physical and chemical characteristics, nanotechnology has become one of the leading technologies over the past 10 years. This study represents data on genotoxic effects of nanoparticles and their application for assessing human health risks. Silica (SiO2) is a multi-functional ceramic material that is being used in various industries to improve surfaces and mechanical properties of diverse materials, such as paints and coatings, plastics, synthetic rubber, adhesives, sealants, or insulation materials. However, recent studies have shown that nano-sized silica (nano-silica) (10 nm in diameter) can generate adverse effects, like liver injury and inflammation. The cytotoxicity and genotoxicity of nano-silica were investigated using the dye exclusion assay, comet assay, and mouse lymphoma thymidine kinase (tk+/−) mouse lymphoma assay (MLA). IC20 of nano-silica in L5178Y cells was determined to be of 2,441.41 μg/mL and 2,363.28 μg/mL without and with S-9, respectively. Also IC20 of nano-silica in BEAS-2B cells was determined to be of 2,324.23 μg/mL and 537.11 μg/mL without and with S-9, respectively. In the comet assay, treating L5178Y cells and BEAS-2B cells with nanosilica treatment induced approximately 2-fold increases in tail moment (P<0.05) without and with S-9. Also, the mutant frequencies in the nano-silica treated L5178Y cells were not significantly increased compared to the solvent controls. The results of this study indicate that nano-silica can cause primary DNA damage and cytotoxicity but not mutagenicity in cultured mammalian cells.

Induction of apoptosis in human leukemia cells through the production of reactive oxygen species and activation of HMOX1 and Noxa by benzene, toluene, and o-xylene.

Whereas benzene (BZ) is a well-known human carcinogen, toluene (TOL) and o-xylene (o-XY) are not; however, all three compounds are important environmental pollutants. Although BZ, TOL, and o-XY have been shown to induce apoptosis in vitro, their mechanism of toxicity remains unclear. In this study, we sought to identify the apoptotic pathway(s) activated by BZ, TOL, and o-XY in human HL-60 promyelocytic leukemia cells. Cell cycle analysis by propidium iodide (PI) staining and flow cytometric analyses of Annexin V/PI double-stained cells revealed similar patterns of apoptosis following BZ, TOL, and o-XY exposure. Though reactive oxygen species (ROS) production contributes significantly to BZ metabolite-induced apoptotic cell death, we hypothesized that BZ, TOL, and o-XY can themselves trigger ROS production, leading to the activation of apoptotic signaling. Dose-dependent increases in ROS production and significant tail moments were observed in HL-60 cells exposed to all three compounds. Real-time RT-PCR revealed increased HMOX1 and Noxa expression in BZ-, TOL-, and o-XY-treated HL-60 cells, confirming the results of previous microarray analyses. Similar expression profiles were found in human K562 erythromyeloblastoid leukemia cells and human U937 leukemic monocyte lymphoma cells. Pretreatment with the ROS scavenger N-acetyl cysteine decreased the effects of exposure to BZ, TOL, and o-XY. In summary, this study provides useful insights into the mechanism of BZ-, TOL-, and o-XY-induced apoptosis in leukemia cells.

Gene Expression Analysis Identifies DNA Damage-related Markers of Benzo(a)pyrene Exposure in HepG2 Human Hepatocytes

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) that is carcinogenic to humans. Although the environmental distribution and metabolism of BaP have been reported and many researchers are performing risk-assessment and toxicological studies of BaP by means of physical and chemical measurements, only a few studies have examined the expression of mRNAs and their functions in BaP-induced toxicity. Toxicogenomic technology, a new paradigm in toxicity screening, is a useful approach for evaluating the toxic properties of environmental pollutants. We analyzed gene expression profiles using human oligonucleotide chips and identified genes in human hepatocellular carcinoma (HepG2) cells whose expression changed > 1.5-fold after exposure to BaP. The expression of 4,048 and 3,926 genes was up-and down-regulated > 1.5-fold (P < 0.01), respectively, after exposure. Gene ontology (GO) analysis of these genes revealed significant enrichment in several key biological processes related to DNA damage, including DNA repair, cell cycle arrest, and apoptosis. We also performed a contrastive study of cellular effects in HepG2 cells exposed to BaP, and identified increased expression of related genes, cell cycle arrest, and apoptotic cells. These results suggest that genetic markers of BaP-induced toxicity may be molecular blueprints that can be more widely implemented in combination with more traditional techniques for assessment and prediction.

Gene expression analysis identifies potential biomarkers of phenanthrene in human hepatocytes (HepG2)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquious in the environment both as natural products and as environmental contaminants. Among PAHs, phenanthrene (PH) that is ubiquitously distributed throughout the environment was subjected in this study. Although environmental distribution and metabolism of PH have been well reported, there are only a few studies examined the expression of mRNA and their functions on PH-induced toxicity. A new paradigm in toxicity screening, toxicogenomic technology represents a useful approach for evaluating the toxic properties of environmental pollutants. In this respect, we elicited the genes which were changed more than 2-fold by analysis of gene expression profiles in human hepatocellular carcinoma (HepG2) cells, exposed to PH by using human oligonucleotide chip. 913 up- and 814 down-regulated genes changed their expression by more than 2-fold and p-values 0.05 through PH exposure. Gene Ontology (GO) analysis on these genes revealed significant enrichments in the several key biological processes related to the hepatotoxicity such as cell migration, wound healing, cytoskeleton organization, microtubule-based process, apoptosis, and cell cycle checkpoint. In conclusion, the present study suggests that PH exerts its toxicity by modulating the mRNA expression in HepG2 cells. we suggest that genes expressed by PH as a molecular signature which can be used more widely implemented in combination with more traditional techniques for assessment and prediction of toxicity by exposure to PH.

Phospholipidosis Induced by PPARγ Signaling in Human Bronchial Epithelial (BEAS-2B) Cells Exposed to Amiodarone

Phospholipidosis (PL), a disorder characterized by an accumulation of phospholipids in lysosome-derived multilamellar vesicles owing to abnormal lipid metabolism. Amiodarone (AM), an antiarrhythmic drug, can induce pulmonary PL. First, to evaluate potential mechanisms of phospholipidosis, we found lipid metabolism--related genes by microarray. PPARG, FADS2, and SCD out of these genes were key genes in lipid metabolism and PPAR signaling by AM. The messenger RNA (mRNA) levels of PPARG, FADS2, and SCD were upregulated by AM. The PPARγ antagonist GW9662 was used to investigate the possible involvement of PPARγ as a mediator of AM-induced PL, and FADS2 and SCD small interfering RNAs (siRNAs) were used to examine the involvement of FADS2 and SCD in AM-induced PL. The inhibition of PPARγ by GW9662 significantly attenuated the AM-induced upregulation of SCD and slightly decreased the AM-induced upregulation of FADS2. And the pretreatment of GW9662 significantly decreased the AM-induced uptake of the fluorescent phospholipid analog NBD-PC. The siRNA-mediated gene silencing of FADS2 and SCD also decreased the AM-induced NBD-PC uptake. These results suggest that the activation of the PPARγ signaling pathway, including FADS2 and SCD, may play an important role in AM-induced PL. PPARG, FADS2, and SCD are AM-induced PL-related genes and may serve as potential biomarkers for PL caused by pulmonary toxicity. We also provide evidence for a possible mechanism of PL, the accumulation of phospholipid in the induction of FADS2 and SCD by PPARγ, in AM-induced pulmonary toxicity.

Formation of a 3,4-diol-1,2-epoxide metabolite of benz[a]anthracene with cytotoxicity and genotoxicity in a human in vitro hepatocyte culture system

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that require metabolic activation to exert their carcinogenic effects. This study investigated the 3,4-diol-1,2-epoxide formation of benz[a]anthracene (BA) and its toxic effects in a human in vitro hepatocyte culture system. Both mRNA and protein expression of metabolic enzymes which can activate PAHs to carcinogenic forms increased after BA exposure in HepG2 cells and our quantitative analysis showed that the formation of BA-3,4-diol-1,2-epoxide in medium extracts increased in a time-dependent manner. We also performed several comparative studies which show that much lower concentrations of BA-3,4-diol-1,2-epoxide had stronger cytotoxicity and genotoxicity than higher doses of BA. These results suggest that BA is activated as the major carcinogenic metabolite 3,4-diol-1,2-epoxide, in human in vitro culture systems by metabolic enzymes and that this metabolite has stronger cytotoxic and genotoxic effects than its parent compound.