Safrole-2',3'-oxide induces cytotoxic and genotoxic effects in HepG2 cells and in mice.

Safrole-2',3'-oxide (SAFO) is a reactive electrophilic metabolite of the hepatocarcinogen safrole, the main component of sassafras oil. Safrole occurs naturally in a variety of spices and herbs, including the commonly used Chinese medicine Xi xin (Asari Radix et Rhizoma) and Dong quai (Angelica sinensis). SAFO is the most mutagenic metabolite of safrole tested in the Ames test. However, little or no data are available on the genotoxicity of SAFO in mammalian systems. In this study, we investigated the cytotoxicity and genotoxicity of SAFO in human HepG2 cells and male FVB mice. Using MTT assay, SAFO exhibited a dose- and time-dependent cytotoxic effect in HepG2 cells with TC(50) values of 361.9µM and 193.2µM after 24 and 48h exposure, respectively. In addition, treatment with SAFO at doses of 125µM and higher for 24h in HepG2 cells resulted in a 5.1-79.6-fold increase in mean Comet tail moment by the alkaline Comet assay and a 2.6-7.8-fold increase in the frequency of micronucleated binucleated cells by the cytokinesis-block micronucleus assay. Furthermore, repeated intraperitoneal administration of SAFO (15, 30, 45, and 60mg/kg) to mice every other day for a total of twelve doses caused a significant dose-dependent increase in mean Comet tail moment in peripheral blood leukocytes (13.3-43.4-fold) and in the frequency of micronucleated reticulocytes (1.5-5.8-fold). Repeated administration of SAFO (60mg/kg) to mice caused liver lesions manifested as a rim of ballooning degeneration of hepatocytes immediately surrounding the central vein. Our data clearly demonstrate that SAFO significantly induced cytotoxicity, DNA strand breaks, micronuclei formation both in human cells in vitro and in mice. More studies are needed to explore the role SAFO plays in safrole-induced genotoxicity.

Aristolochic acid suppresses DNA repair and triggers oxidative DNA damage in human kidney proximal tubular cells.

Aristolochic acid (AA), derived from plants of the Aristolochia genus, has been proven to be associated with aristolochic acid nephropathy (AAN) and urothelial cancer in AAN patients. In this study, we used toxicogenomic analysis to clarify the molecular mechanism of AA-induced cytotoxicity in normal human kidney proximal tubular (HK-2) cells, the target cells of AA. AA induced cytotoxic effects in a dose-dependent (10, 30, 90 microM for 24 h) and time-dependent manner (30 microM for 1, 3, 6, 12 and 24 h). The cells from those experiments were then used for microarray experiments in triplicate. Among the differentially expressed genes analyzed by Limma and Ingenuity Pathway Analysis software, we found that genes in DNA repair processes were the most significantly regulated by all AA treatments. Furthermore, response to DNA damage stimulus, apoptosis, and regulation of cell cycle, were also significantly regulated by AA treatment. Among the differentially expressed genes found in the dose-response and time-course studies that were involved in these biological processes, two up-regulated (GADD45B, NAIP), and six down-regulated genes (TP53, PARP1, OGG1, ERCC1, ERCC2, and MGMT) were con-firmed by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). AA exposure also caused a down-regulation of the gene expression of anti-oxidant enzymes, such as superoxide dismutase, glutathione reductase, and glutathione peroxidase. Moreover, AA treatment led to increased frequency of DNA strand breaks, 8-hydroxydeoxyguanosine-positive nuclei, and micronuclei in a dose-dependent manner in HK-2 cells, possibly as a result of the inhibition of DNA repair. These data suggest that oxidative stress plays a role in the cytotoxicity of AA. In addition, our results provide insight into the involvement of down-regulation of DNA repair gene expression as a possible mechanism for AA-induced genotoxicity.

Microarray analysis reveals the inhibition of nuclear factor-kappa B signaling by aristolochic acid in normal human kidney (HK-2) cells.

AIM: To study the molecular mechanism underlying the effect of aristolochic acid (AA), a major active component of plants from the Aristolochiaceae family using microarray analysis. METHODS: Human kidney (HK-2) cells were treated with AA (0, 10, 30, and 90 micromol/L) for 24 h, and the cell viability was measured by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Complementary DNA microarrays were used to investigate the gene expression pattern of HK-2 cells exposed to AA in triplicate. A quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay was used to verify the microarray data for selected nuclear factor kappa B (NF-kappaB)-regulated genes. Furthermore, the subcellular localization of NF-kappaB p65 was visualized by immunofluorescence confocal microscopy in HK-2 cells. The NF-kappaB activity was examined by a luciferase reporter assay in HK-2/NF-kappaB transgenic cells. RESULTS: AA exhibited a dose-dependent cytotoxic effect in HK-2 cells and induced alterations in the gene expression profiles related to the DNA damage response, DNA repair, macromolecule metabolic process, carbohydrate metabolic process, DNA metabolic process, apoptosis, cell cycle, and transcription. In addition, 9 biological pathways associated with immunomodulatory functions were down-regulated in AA-treated HK-2 cells. A network analysis revealed that NF-kappaB played a central role in the network topology. Among NF-kappaB-regulated genes, 8 differentially expressed genes were verified by qRT-PCR. The inhibition of NF-kappaB activity by AA was further confirmed by immunofluorescence confocal microscopy and by NF-kappaB luciferase reporter assay. CONCLUSION: Our data revealed that AA could suppress NF-kappaB activity in normal human cells, perhaps partially accounting for the reported anti-inflammatory effects of some plants from the genus Aristolochia.

Relationship Between San-Huang-Xie-Xin-Tang and its herbal components on the gene expression profiles in HepG2 cells.

Traditional Chinese medicine (TCM) has been used for thousands of years. Most Chinese herbal formulae consist of several herbal components and have been used to treat various diseases. However, the mechanisms of most formulae and the relationship between formulae and their components remain to be elucidated. Here we analyzed the putative mechanism of San-Huang-Xie-Xin-Tang (SHXXT) and defined the relationship between SHXXT and its herbal components by microarray technique. HepG2 cells were treated with SHXXT or its components and the gene expression profiles were analyzed by DNA microarray. Gene set enrichment analysis indicated that SHXXT and its components displayed a unique anti-proliferation pattern via p53 signaling, p53 activated, and DNA damage signaling pathways in HepG2 cells. Network analysis showed that most genes were regulated by one molecule, p53. In addition, hierarchical clustering analysis showed that Rhizoma Coptis shared a similar gene expression profile with SHXXT. These findings may explain why Rhizoma Coptis is the principle herb that exerts the major effect in the herbal formula, SHXXT. Moreover, this is the first report to reveal the relationship between formulae and their herbal components in TCM by microarray and bioinformatics tools.

Berberine suppresses inflammatory agents-induced interleukin-1beta and tumor necrosis factor-alpha productions via the inhibition of IkappaB degradation in human lung cells.

Pulmonary inflammation is a characteristic of many lung diseases. Increased levels of pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), have been correlated with lung inflammation. In this study, we demonstrated that various inflammatory agents, including lipopolysaccharide, 12-o-tetradecanoylphorbol-13-acetate, hydrogen peroxide, okadaic acid and ceramide, were able to induce IL-1beta and TNF-alpha productions in human lung epithelial cells (A-549), fibroblasts (HFL1), and lymphoma cells (U-937). Berberine, the protoberberine alkaloid widely distributed in the plant kingdom, was capable of suppressing inflammatory agents-induced cytokine production in lung cells. Inhibition of cytokine production by berberine was dose-dependent and cell type-independent. Moreover, the suppression of berberine on the cytokine production resulted from the inhibition of inhibitory kappaB-alpha phosphorylation and degradation. In conclusion, our findings suggested the potential role of berberine in the treatment of pulmonary inflammation.

Acetaldehyde induces matrix metalloproteinase-9 gene expression via nuclear factor-kappaB and activator protein 1 signaling pathways in human hepatocellular carcinoma cells: Association with the invasive potential.

Alcohol consumption is a significant risk factor for hepatocellular carcinoma (HCC). Alcohol also increases the prevalence of invasion in HCC patients. However, the molecular mechanism on the metastatic effect of alcohol is unclear so far. Herein we demonstrated that acetaldehyde, the primary metabolite of ethanol, increased matrix metalloproteinase-9 (MMP-9) gelatinolytic activity and promoted cell invasion through the up-regulation of MMP-9 gene transcription in HepG2 cells. The transcription of MMP-9 gene was regulated by 10 microM acetaldehyde via inductions of nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1) activities. Acetaldehyde stimulated the translocation of NF-kappaB into nucleus through inhibitory kappaB-alpha (IkappaB-alpha) and c-Jun N-terminal kinase (JNK)/beta-transducin repeat-containing protein (beta-TrCP) signaling pathways. Acetaldehyde also induced AP-1 activity via the phosphorylation of p38 kinase. In conclusion, our findings demonstrated for the first time that acetaldehyde activated NF-kappaB and AP-1 activities via IkappaB, JNK/beta-TrCP, and p38 signaling pathways, resulting in MMP-9 gene expression and hepatocarcinoma cells invasion. These results suggested that acetaldehyde might be a potential factor involved in the invasiveness of HCC in alcoholic patients.