Genotoxic and oxidative stress effects of 2-amino-9H-pyrido[2,3-b]indole in human hepatoma G2 (HepG2) and human lung alveolar epithelial (A549) cells

Abstract

2-Amino-9H-pyrido[2,3-b]indole (AαC), which is present in high quantities in cigarette smoke and also in fried food, has been reported to be a probable human carcinogen. However, few studies have reported on the genotoxicity and oxidative stress induced by AαC. This study investigated the genotoxic effects of AαC in human hepatoma G2 (HepG2) and human lung alveolar epithelial (A549) cells using the comet assay. Significant increases in DNA fragment migration indicated that AαC causes serious DNA damage in HepG2 and A549 cells. The role of oxidative stress in the mechanism of AαC-induced genotoxicity was clarified by measuring the level of intracellular reactive oxygen species (ROS), the GSH/GSSG ratio and the formation of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage. The results showed that the levels of ROS and 8-OHdG increased, whereas the GSH/GSSG ratio decreased. The concentration of 8-OHdG was positively related to DNA damage. Taken together, these results indicate that AαC can induce genotoxicity and oxidative stress and that AαC likely exerts genotoxicity in HepG2 and A549 cells through ROS-induced oxidative DNA damage. This is the first report to describe AαC-induced genotoxic and oxidative stress in HepG2 and A549 cells.     

Genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells

The recent finding that acrylamide (AA), a carcinogen in animal experiments and a probable human carcinogen, is formed in foods during cooking raises human health concerns. The relevance of dietary exposure for humans is still under debate. The purpose of the study was to evaluate the possible genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells, a cell line of great relevance to detect genotoxic/antigenotoxic substances, using single cell gel electrophoresis (SCGE) assay and micronucleus test (MNT). In order to clarify the underlying mechanism(s) we evaluated the intracellular generation of reactive oxygen species (ROS) and the level of oxidative DNA damage by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG). The involvement of glutathione (GSH) in the AA-induced oxidative stress was examined through treatment with buthionine sulfoximine (BSO) to deplete GSH. The results indicate that AA caused DNA strand breaks and increase in frequency of MN in HepG2 cells in a dose-dependent manner. The possible mechanism underlies the increased levels of ROS, depletion of GSH and increase of 8-OHdG formation in HepG2 cells treated with AA. We conclude that AA exerts genotoxic effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS and depletion of GSH.     

Apoptotic effects of dichloro stearic and dichloro myristic acid in human hepatoma cells (HepG2)

Chlorinated fatty acids represent the major fraction of extractable organically bound chlorine in fish. After dietary intake such fatty acids may accumulate in adipose tissue, and even be transferred from mother to child via breast milk. We have previously reported that 9,10-dichloro stearic acid and 5,6-dichloro myristic acid inhibited cell growth. The aim of the present work was to investigate whether the growth inhibitory effect of these modified fatty acids might involve apoptosis. Human hepatoma cells (HepG2) were cultured for 4 days before addition of chlorinated fatty acids, and then cultured for another day before harvested. Morphological analysis was mainly done by light microscopy. In addition, fluorescence microscopy and electrophoretic analysis of DNA were carried out. The effect of 0.3 and 0.6 mmol/l was studied. Both chlorinated fatty acids seemed to cause a concentration-dependent increase in the relative abundance of pycnotic and broken nuclei, as well as nuclear fragments, with the strongest effect of dichloro stearic acid. Apoptosis by the chlorinated fatty acids was however less than that of docosahexaenoic acid, a known apoptosis effector. In conclusion, chlorinated fatty acids seem to possess the ability to cause apoptosis.     

DNA damage and metallothionein synthesis in human hepatoma cells (HepG2) exposed to cadmium.

Cadmium is an important heavy metal environmental toxicant, which is classified as a human carcinogen. The comet assay was used to evaluate the levels of DNA damage in a metabolically competent HepG2 cell line after treatment with low, non-cytotoxic and physiologically relevant concentrations of cadmium, alone and in combination with the dietary mutagen 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and with the environmental mutagen benzo[a]pyrene (B(a)P). After exposure of the cells to 10, 100 and 1000 nM CdCl(2), a dose- and time-dependent increase of DNA damage was detected. Maximal damage was found after 12 h of treatment, but declined with further incubation with CdCl(2). The increased synthesis of metallothioneins on exposure to CdCl(2) up to 12 h suggests that they are responsible for the adaptation of HepG2 cells to the DNA damaging effects of CdCl(2). Co-treatment of the cells with CdCl(2) (10-1000 nM) and IQ (300 microM) induced a dose-dependent increase of DNA damage compared to cells treated with IQ alone. Co-genotoxic activity was also observed by increased formation of micronuclei in cells exposed to IQ and 1000 nM CdCl(2); at this concentration, CdCl(2) alone also induced micronuclei in HepG2 cells. Our results support the hypothesis that direct and indirect mechanisms are involved in cadmium-induced DNA damage.     

Genotoxic effects induced by beta-myrcene following metabolism by liver HepG2/C3A human cells

Beta-myrcene [or myrcene (1,6-Octadiene, 7-methyl-3-methylene-)] and the essential oils containing this monoterpene have been widely used in cosmetics, detergents, and soaps, and as flavoring additives for food and beverages. Due to the potentially high level of human exposure to beta-myrcene, and absence of studies involving its genotoxicity in human cells, the aim of this study was to investigate the cytotoxic and genotoxic potential of this terpenoid in non-metabolizing cells (leukocytes) and liver metabolizing cells (HepG2/C3A cells). Prior to the genotoxic assessment by the comet and micronucleus (MN) assays, a range of beta-myrcene concentrations was tested in a preliminary MTT assay. Regarding the MTT assay, the results showed cytotoxic effects for leukocytes at 250 µg/ml and higher concentrations, while for HepG2/C3A cells, absence of cytotoxicity was noted relative to all tested concentrations (after 24 hr exposure). Thus, the concentrations of 2.5, 10, 25, 50, and 100 µg/ml for leukocytes, and 2.5, 100, and 1000 µg/ml for HepG2/C3A cells were selected for subsequent assays. Genotoxicity evaluation demonstrated significant DNA damage in the comet assay and significant chromosomal abnormalities including nucleoplasmic bridges and nuclear buds in HepG2/C3A cells at beta-myrcene concentrations of 100 and 1000 µg/ml. Under our experimental conditions, caution is recommended in the use of beta-myrcene, since this compound produced genotoxic effects especially after metabolic activation using human HepG2/C3A cells, which may be associated with carcinogenic and teratogenic effects previously reported in the literature.     

Copper toxicity affects proliferation and viability of human hepatoma cells (HepG2 line)

In Wilson's disease and Indian childhood cirrhosis (ICC) copper accumulates in the liver resulting in poor hepatocyte regeneration and fibrosis. An inhibition of hepatocyte proliferation and an increase in cell death could account for these outcomes. To establish how the toxicity of this metal ion impacts upon the proliferation and viability of the HepG2 cells they were cultured in 4-32 microM copper(II) sulphate (CuSO4)). These levels were comparable to the circulatory and tissue concentrations of copper recorded for these two diseases. Specific uptake comparable to levels of copper recorded in the livers of patients with Wilson's disease and ICC was measured in the HepG2 cells. After 48 h acid vesicle function increased from 4 to 32 microM Cu2+ but significantly declined at 64 microM compared to the controls. Lysosomal acid phosphatase showed a concentration dependent decline in activity at 72 h. Cellls exposed to 64 microM Cu2+ had a potential doubling time (Tpot) 21 h longer than the control cells due to a prolonged DNA synthesis phase. At 64 microM Cu2+, increases of necrosis up to 18% were seen whereas comparable levels of apoptotic and necrotic cells (<5%) were seen below this concentration. Chronic exposure over 8 weeks impaired colony-forming efficiency at concentrations of 16 microM Cu2+ and above. This study suggests that when liver cells sequester large amounts of copper, the toxic effects include delayed cell-cycle progression, a gradual loss of replicative capacity, and an increased incidence of cell death.     

Lipid lowering effects of Momordica charantia (Bitter Melon) in HIV-1-protease inhibitor-treated human hepatoma cells, HepG2

1. Hyperlipidemic effects of HIV-1-protease inhibitors (PI) are associated with increased hepatic production of triglyceride (TG)-rich lipoproteins, rather than lipoprotein clearance. PI are known to increase apolipoprotein B (apoB) secretion, apoC-III mRNA expression and decrease apoA-1 secretion. Nutritional therapy remains an important strategy to manage PI-associated hyperlipidemia. 2. This study investigated the in vitro efficacy of Asian vegetable, Momordica charantia or bitter melon (BM) to ameliorate PI-associated apoB and lipid abnormalities in HepG2 cells. 3. Our study demonstrates that bitter melon juice (BMJ) significantly reduced apoB secretion and apoC-III mRNA expression and normalized apoA-I expression in PI-treated HepG2 cells. BMJ also significantly reduced cellular TG and microsomal TG transfer protein, suggesting that lipid bioavailability and lipidation of apoB assembly may play a role in decreased apoB secretion. 4. Identifying molecular targets of BM may offer alternative dietary strategies to decrease PI-associated hyperlipidemia and improve quality of life among HIV-1-infected patients.     

Genotoxic effects of silver nanoparticles with/without coating in human liver HepG2 cells and in mice

With the rapid expansion of human exposure to silver nanoparticles (AgNPs), the genotoxicity screening is critical to the biosafety evaluation of nanosilver. This study assessed DNA damage and chromosomal aberration in the human hepatoma cell line (HepG2) as well as the effects on the micronucleus of bone marrow in mice induced by 20 nm polyvinylpyrrolidone‐coated nanosilver (PVP‐AgNPs) and 20 nm bare nanosilver (AgNPs). Our results showed that the two types of AgNPs, in doses of 20‐160 μg/mL, could cause genetic toxicological changes on HepG2 cells. The DNA damage degree of HepG2 cells in 20 nm AgNPs was higher than that in 20 nm PVP‐AgNPs, while the 20 nm PVP‐AgNPs caused more serious chromosomal aberration than 20 nm AgNPs. Both kinds of AgNPs caused genetic toxicity in a dose‐dependent manner in HepG2 cells. In the micronucleus test on mouse bone marrow cells, in doses of 10, 50 and 250 mg/kg body weight administered orally for 28 days once a day, the two kinds of AgNPs have no obvious inhibitory effect on the mouse bone marrow cells, and the effect of chromosome aberration could be documented at the high dose of 250 mg/kg. These results suggest that AgNPs have genotoxic effects in HepG2 cells and limited effects on bone marrow in mice; both in vitro and in vivo tests could be of great importance on the evaluation of genotoxicity of nanosilver. These findings can provide useful toxicological information that can help to assess genetic toxicity of nanosilver in vitro and in vivo.     

Genotoxic effects of the cyanobacterial hepatotoxin cylindrospermopsin in the HepG2 cell line

The cyanobacterial alkaloid cylindrospermopsin (CYN) is being increasingly identified in drinking water supplies worldwide. It is a potent protein synthesis inhibitor and causes human intoxications and animal mortality. The few genotoxicity studies available indicate that CYN is genotoxic, generally implying that it is pro-genotoxic. We evaluated CYN genotoxicity in the human hepatoma cell line, HepG2, analyzing the induction of DNA strand breaks, with the alkaline comet assay, and micronuclei (MNi), nuclear bud (NBUD), and nucleoplasmic bridge (NPB) formation, with the cytokinesis block micronucleus (CBMN) assay. In addition, changes in the expression of genes involved in the response to DNA damage (P53, CDKN1A, GADD45α, and MDM2) and genes presumably involved in CYN metabolism (genes from the Cytochrome P450 family: CYP1A1 and CYP1A2) were determined, using quantitative real-time PCR. Non-cytotoxic concentrations of CYN induced increased DNA damage after 12 and 24 h of exposure and increased the frequency of MNi, NBUDs, and NPBs after 24 h exposure. Moreover, CYN up-regulated the expression of the CYP1A1 and CYP1A2 genes. Although no changes in the expression of the P53 tumor-suppressor gene were found, CYN up-regulated the expression of the P53 downstream-regulated genes CDKN1A, GADD45α, and MDM2. Our results provide new evidence that CYN is genotoxic and strongly suggest that it needs to be considered in the human health risk assessment.     

大黄素诱导人肝癌HepG2细胞线粒体凋亡作用研究

目的 研究大黄素对人肝癌HepG2细胞线粒体凋亡的影响。方法 培养人肝癌HepG2细胞,与5、10、20、40、60、80、100 μmol/L的大黄素作用24、48 h,MTS法检测细胞增殖;40、80、160 μmol/L大黄素作用HepG2细胞24 h,AO/EB双荧光染色法观察细胞凋亡的形态学改变;Annexin V/PI染色经流式细胞仪检测细胞凋亡;分光光度法检测caspase 3活性;ATP试剂盒检测细胞ATP含量,不同荧光探针加载后流式细胞仪测定大黄素对HepG2细胞内活性氧（ROS）含量、Ca²⁺浓度、线粒体膜电位（MMP）变化的影响。结果 大黄素抑制HepG2细胞生长,且呈时间、浓度相关性,半数抑制浓度（IC₅₀）为（77.42±1.25）μmol/L;随着大黄素浓度升高,AO/EB双染观察到细胞核浓缩、碎裂、凋亡小体等凋亡形态;与对照组比较,大黄素40、80、160 μmol/L作用于HepG2细胞24 h后细胞凋亡率显著增加,caspase 3活性显著增强,ROS水平、Ca²⁺浓度明显增加（P<0.05、0.01、0.001）,80、160 μmol/L组线粒体膜电位明显降低,ATP含量显著下降（P<0.05、0.01、0.001）。结论 大黄素造成HepG2细胞内ROS堆积,ATP合成功能障碍,线粒体膜电位明显下降,进而诱导线粒体通透转运孔开放,导致钙离子和细胞色素C外流,活化caspase蛋白家族,导致细胞凋亡。     

3-Nitrobenzanthrone (3-NBA) induced micronucleus formation and DNA damage in human hepatoma (HepG2) cells

3-Nitrobenzanthrone (3-NBA), identified in diesel exhaust and in airborne particulate matter, is a potent mutagen in Salmonella, induces micronuclei formation in mice and in human cells and DNA adducts in rats. In the present study, we investigated the genotoxic potency of 3-NBA in human HepG2 cells using the micronucleus (MN) assay and the single cell gel electrophoresis (SCGE). 3-NBA caused a genotoxic effect at concentrations > or =12 nM in both assays. In the micronucleus assay, we found 98.7+/-10.3 MN/1000 BNC at a concentration of 100 nM 3-NBA in comparison to 27.3+/-0.6 MN/1000 BNC with the negative control. At the same concentration, the DNA-migration (SCGE) showed an Olive tail moment (OTM) of 2.7+/-0.45 and %DNA in the tail of 8.28+/-0.76; OTM and %DNA in the tail of cells treated with the negative control were 0.73+/-0.08 and 2.81+/-0.30, respectively. The results are discussed under consideration of former studies.     

Genotoxic and mutagenic effects of erythrosine B,a xanthene food dye,on HepG2 cells

Erythrosine (ErB) is a xanthene and an US Food and Drug Administration approved dye used in foods, drugs and cosmetics. Although its utilization is permitted, ErB is described as inhibitor of enzymes and protein–protein interactions and is toxic to pituitary and spermatogenesis processes. However, the genotoxicity and mutagenicity of ErB is inconclusive in the literature. This study aimed to analyze the genotoxicity of this dye using the alkaline comet assay and is the first investigation to evaluate ErB mutagenicity using the cytokinesis block micronucleus cytome (CBMN-Cyt) assay in HepG2 cells. These cells were chosen because they produce phase I and phase II enzymes that can mimic in vivo metabolism. The cells were treated with seven concentrations (0.1–70.0 μg mL⁻¹) of ErB, and the results showed genotoxicity at the two highest concentrations and mutagenicity at six concentrations. Furthermore, as micronuclei result from clastogenic and aneugenic processes, while comet assay is often considered more sensitive and detects DNA single strain breaks, we suggest that an aneugenic is responsible for the observed damage. Although ErB is approved for use in the food, cosmetic and pharmaceutical industries, it must be used carefully because it damages the DNA structure.     

Uptake characteristics of galactosylated emulsion by HepG2 hepatoma cells

Galactosylated (Gal) emulsions containing various molar ratios of cholesten-5-yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol) as a ligand for asialoglycoprotein receptors were prepared to study the effect of the galactose content of Gal-emulsions labeled with [3H]cholesteryl hexadecyl ether on their targeted delivery to hepatocytes. The uptake characteristics of Gal-emulsions having Gal-C4-Chol of 1, 3, 4, 6, and 9 mol% were evaluated in HepG2 cells which possess asialoglycoprotein receptors and NIH3T3 cells which are lack of asialoglycoprotein receptors. The uptake and internalization by HepG2 cells was enhanced by the addition of Gal-C4-Chol to the Gal-emulsions whereas the uptake of Gal-emulsions by NIH3T3 cells was not much and was comparable with that of bare-emulsions. In the presence of excess Gal-BSA, the uptake of Gal-emulsions having Gal-C4-Chol of 4, 6, and 9% was inhibited suggesting asialoglycoprotein receptor mediated uptake. Moreover, Gal-emulsions having Gal-C4-Chol of 4, 6, and 9% showed a slight increase in surface binding and exhibited extensive uptake and internalization into HepG2 cells. The present study strongly suggested that the Gal-emulsions are taken up by the asialoglycoprotein receptor-mediated endocytosis and galactose density of Gal-emulsions is important for effective recognition and cell internalization.     

Genotoxic effects of (-)-cubebin in somatic cells of mice

(?)‐Cubebin belongs to the dibenzylbutyrolactone lignan group, which is widely distributed in the plant kingdom. Because this compound shows interesting biological activities, it is extremely important to evaluate its possible genotoxic activity to allow its safe use in humans. Thus, the present study was performed to investigate the genotoxicity potential activity of (?)‐cubebin assessed by two assays: micronucleus in bone marrow cells and comet test in peripheral blood leukocytes of Swiss mice. In the (?)‐cubebin dose range‐finding assays, the maximum tolerated dose was greater than 2000 mg kg^?1. The compound was administered by an oral route at single doses of 250, 500 and 2000 mg kg^?1 body weight. Cytotoxicity was assessed by scoring 200 consecutive total polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). Under our experimental conditions, micronucleus and comet assays, respectively, showed that (?)‐cubebin caused dose‐related clastogenic and genotoxic effects in the somatic cells investigated. PCE/NCE ratio showed no cytotoxicity for the three doses of the compound. The data suggest caution in the ingestion of (?)‐cubebin by humans, especially at high doses. Copyright © 2010 John Wiley & Sons, Ltd.     

Genotoxic effects of vanadium(IV) in human peripheral blood cells

Vanadium has been considered an aneuploidogen; however, there is controversial information about the clastogenic effects of vanadium compounds. In this study, the genotoxicity of vanadium(IV) tetraoxide (V(2)O(4)) was evaluated in human cultured lymphocytes and leukocytes using the mitotic index (MI), the replicative index (RI), chromosome aberrations (CA), sister chromatid exchanges (SCE), satellite associations (SA) and the single cell gel electrophoresis (SCGE) assay. This chemical induced a clear dose-response in MI inhibitions and modifications in the RI. In the CA, including breaks and exchanges and in the SCE, a significant increase appeared in the treated group compared with the controls. The SA test did not reveal an important difference. For the detection of genotoxic properties of vanadium(IV) using the SCGE assay, the 2 h evaluation period was not long enough for the chemical to enter the cell. These results indicate that vanadium(IV) tetraoxide is capable of inducing cytotoxic and cytostatic effects and chromosomal damage.     

Mitochondria permeability transition-dependent tert-butyl hydroperoxide-induced apoptosis in hepatoma HepG2 cells

Tert-butyl hydroperoxide (t-BHP) has been demonstrated to induce apoptosis in hepatoma cell line HepG2, but poor data were available on the signaling pathway initiated by t-BHP. In this work, we studied in details the apoptotic pathways induced in HepG2 cells by t-BHP. DNA fragmentation, activation of caspases and cytochrome c release were demonstrated. Permeability transition pore inhibitors prevented the DNA fragmentation and caspase activation induced by t-BHP. In addition, changes in the mitochondrial membrane potential were detected: hyperpolarization preceded loss of membrane potential. It also preceded caspase activation which occurred before the induction of DNA fragmentation. Taken together, these results emphasize the central role played by mitochondria in the initiation of apoptosis in HepG2 cells exposed to oxidant agents.