Aflatoxin B1-induced Cytotoxicity Research Articles

Pathological Role of Oxidative Stress in Aflatoxin-Induced Toxicity in Different Experimental Models and Protective Effect of Phytochemicals: A Review.

Aflatoxin B1 is a secondary metabolite with a potentially devastating effect in causing liver damage in broiler chickens, and this is mainly facilitated through the generation of oxidative stress and malonaldehyde build-up. In the past few years, significant progress has been made in controlling the invasion of aflatoxins. Phytochemicals are some of the commonly used molecules endowed with potential therapeutic effects to ameliorate aflatoxin, by inhibiting the production of reactive oxygen species and enhancing intracellular antioxidant enzymes. Experimental models involving cell cultures and broiler chickens exposed to aflatoxin or contaminated diet have been used to investigate the ameliorative effects of phytochemicals against aflatoxin toxicity. Electronic databases such as PubMed, Science Direct, and Google Scholar were used to identify relevant data sources. The retrieved information reported on the link between aflatoxin B1-included cytotoxicity and the ameliorative potential/role of phytochemicals in chickens. Importantly, retrieved data showed that phytochemicals may potentially protect against aflatoxin B1-induced cytotoxicity by ameliorating oxidative stress and enhancing intracellular antioxidants. Preclinical data indicate that activation of nuclear factor erythroid 2-related factor 2 (Nrf2), together with its downstream antioxidant genes, may be a potential therapeutic mechanism by which phytochemicals neutralize oxidative stress. This highlights the need for more research to determine whether phytochemicals can be considered a useful therapeutic intervention in controlling mycotoxins to improve broiler health and productivity.

Proteomics reveals the alleviation of zinc towards aflatoxin B1-induced cytotoxicity in human hepatocyes (HepG2 cells).

Aflatoxin B1 (AFB1) is a known carcinogen found in contaminated food and designated by the World Health Organization as a class I carcinogenic substance. AFB1 presents with carcinogenicity, teratogenicity, and mutagenicity, and the liver is the human organ most susceptible to AFB1. Zinc (Zn), which is one of the essential nutrient elements that could protect the cells from biological toxins, heavy metals, hydrogen peroxide, metal chelators and radiation, is assessed in this study for its potential to alleviate AFB1-induced cytotoxicity. Samples were divided into three groups, namely CK, AFB1, and AFB1+Zn. Protein expressions were analyzed by two-way electrophoresis combined with flight mass spectrometry, with 41 differentially expressed proteins identified in the results, mainly related to oxidative stress, cell apoptosis, DNA damage, and energy metabolism. Zn was found to regulate the expression of peroxidases (peroxiredoxin-1, peroxiredoxin-5, peroxiredoxin-6) to relieve AFB1-induced oxidative stress. Moreover, Zn could decrease the expression of pro-apoptotic genes (cleaved-caspase-3, caspase-9, and Bax) and increase the expression of anti-apoptotic genes (Bcl-2 and Bcl-xl) to alleviate the cell apoptosis induced by AFB1. In addition, AFB1 reduced intracellular ATP levels, whereas Zn supplementation boosted ATP levels and maintained homeostasis and a steady state of cellular energy metabolism by modulating AMPK-ACC phosphorylation levels, while many zinc finger proteins changed after AFB1 treatment. These results, therefore, indicate that Zn could alleviate AFB1-induced cytotoxicity by changing the expressions of zinc finger proteins in liver hepatocellular carcinoma (HepG2 cells).

Quercetin protects the buffalo rat liver (BRL-3A) cells from aflatoxin B1-induced cytotoxicity via activation of Nrf2-ARE pathway

Aflatoxin B1 (AFB1) is the most toxic mycotoxin widely presented in agricultural products, and the protective effect of quercetin (QUE), a natural antioxidant, against AFB1-induced cytotoxicity to the buffalo rat liver (BRL-3A) cells was investigated. With an IC50 of 23 μM, AFB1 induced a significant oxidative stress to BRL-3A cells evidenced by a dose-dependent reduction of mitochondria membrane potential (MMP), ATP content, and activities of endogenous antioxidant enzymes along with increased levels of reactive oxygen species (ROS) and lipid peroxidation biomarker of malondialdehyde (MDA). The activity of CYP1A2, the key enzyme to convert AFB1 to reactive AFB1 exo-8,9- epoxide, was also increased, which, probably in together with ROS, led to cell apoptosis with DNA fragmentation, chromatin condensation and increased lactate dehydrogenase release. After the BRL cells were pre-treated by low level QUE (2.5 and/or 5 μM) for 24 h and then exposed to AFB1, the activities of antioxidant enzymes including haeme oxygenase-1, glutathione S-transferase, superoxide dismutase, and the ratio of reduced to oxidised glutathione were significantly increased whereas the levels of intracellular ROS and MDA were reduced. The QUE pre-treatment also increased the levels of MMP, ATP and DNA integrity, and reduced the expression of apoptosis related genes ofBax andCaspase-3. The Western blotting study revealed increased content of phosphorylated Akt and nuclear NF-E2-related factor 2 (Nrf2), indicating an activation of Nrf2-ARE pathway in counteracting oxidative stress and cytotoxicity of AFB1. Thus, the QUE pre-treatment enhanced the anti-stress capacity of the cells through the activation of the Nrf2-ARE pathway, and QUE-based measures could be developed to ameliorate the toxicity caused by AFB1.

Protective Effects of Purple Waxy Corn on Aflatoxin B1-induced Oxidative Stress and Micronucleus in HepG2 Cells

The purpose of this study was to investigate the protective effects of purple waxy corn extract (Zea may L.) on aflatoxin B1-induced cytotoxicity, oxidative stress and micronuclei in HepG2 cells. Purple waxy corn extract contained phenolics (187.15-341.65 µg gallic acid/mg), flavonoids (0.55-21.35 µg quercetin/mg) and anthocyanins (43.70-220.43 mg cyanidin-3-glucoside/l). Antioxidant activities of the extracts were measured using oxygen radical absorbance capacity (1410.70-2214.49 µM trolox/mg) and ferric reducing/antioxidant power (292.38-612.38 µM trolox/mg) assays. There were significant correlations between phenolic, flavonoid and anthocyanin content and antioxidant activities (R= 0.740-0.778) of purple waxy corn extract. Studies in HepG2 cells found that 0.5 and 1 mg/ml purple waxy corn extract increased cell viability in AFB1-treated HepG2 cells. Purple waxy corn extract reduced intracellular ROS production, increased GSH content and antioxidant enzyme GR, GPx and GST activities and micronucleus in aflatoxin B1-treated HepG2 cells. Therefore, purple waxy corn extract reduced aflatoxin B1 toxicity through induction of antioxidant activities and, thus, inhibited oxidative stress-induced DNA damage caused by aflatoxin B1.

Resveratrol inhibits aflatoxin B1-induced oxidative stress and apoptosis in bovine mammary epithelial cells and is involved the Nrf2 signaling pathway

Aflatoxins are widely occurring food contaminants that are particularly harmful to dairy products and cows. The plant polyphenol resveratrol has been reported to have a good effect on increasing the resistance of cells toward toxins. Therefore, we measured the effects of aflatoxin B1 and resveratrol on the viability of the MAC-T cow mammary epithelial cell line. The appropriate treatment concentrations were assayed (12.81 μM aflatoxin B1 and 43.81 μM resveratrol) to verify the protective effect of resveratrol toward mammary epithelial cells. The results showed that resveratrol alleviates aflatoxin B1-induced cytotoxicity, including the increase in ROS and the decrease in mitochondrial membrane potential (MMP) and apoptosis in MAC-T cells. The expression of mRNA transcripts (including Nrf2, Keap1, NQO1, HO-1, SOD2 and HSP70) for components of the Nrf2 signaling pathway was evaluated by real-time fluorescent quantitative PCR, with resveratrol also exhibiting a good regulatory effect. Thus, resveratrol was shown to have an ameliorating effect on aflatoxin toxicity in MAC-T cells.

Zinc inhibits aflatoxin B1-induced cytotoxicity and genotoxicity in human hepatocytes (HepG2 cells)

Aflatoxin B1 (AFB1) has strong carcinogenicity. Consumption of AFB1-contaminated agricultural products and the occurrence of hepatocellular carcinoma have received widespread attention. The aim of this paper was to investigate whether zinc supplementation could inhibit AFB1-induced cytotoxicity and genotoxicity in HepG2 cells and the mechanism of this inhibition. Our data suggest that zinc sources can relieve a certain degree of AFB1-induced cytotoxicity and genotoxicity by protecting against apoptotic body formation and DNA strand breaks, affecting S phase cell cycle arrest, reducing 8-OHdG formation, inhibiting global DNA hypomethylation and regulating gene expression in antioxidation, zinc-association and apoptosis processes. Consequently, zinc stabilizes the integrity of DNA and improves cell survival.These data provides new insights into the protective role of zinc in alleviating AFB1-induced cytotoxicity and mediating epigenetic changes in hepatocytes, demonstrating that zinc sources have detoxification properties in mycotoxin-induced toxicity.

Cytochrome P450 2A13 mediates aflatoxin B1-induced cytotoxicity and apoptosis in human bronchial epithelial cells

Cytochrome P450 (CYP) 2A13 is mainly expressed in the respiratory system and has the ability to metabolize aflatoxin B1 (AFB1). However, the role of CYP2A13-mediated AFB1 metabolism and its consequences in human lung epithelial cell is not clear. Therefore, the objectives of this study were to investigate the significance of CYP2A13 in AFB1-induced cytotoxicity, DNA adducts, and apoptosis. To achieve these objectives, CYP2A13 was stably over-expressed in immortalized human bronchial epithelial BEAS-2B cells (B-2A13) and its significance in AFB1-induced cytotoxicity, DNA adducts, and apoptosis was compared to cells with stably expression of CYP1A2 (B-1A2), the predominant AFB1 metabolizing enzyme in liver, as well as CYP2A6 (B-2A6) as controls. AFB1 induced B-2A13 cytotoxicity and apoptosis in a dose- and time-dependent manner. The cytotoxic and apoptotic effects of AFB1 were significantly remarkable in B-2A13 cells than those of B-1A2 and B-2A6 cells. The increased expression of pro-apoptotic proteins, such as C-PARP, C-caspase-3, and Bax, and decreased expression of anti-apoptotic proteins, such as caspase-3, Bcl-2, and p-Bad further confirmed the data of AFB1-induced cytotoxicity and apoptosis. Furthermore, increased DNA adduct was observed in B-2A13 after AFB1 treatment as compared to B-1A2 cells and B-2A6 cells. Finally, treatment with nicotine, a competitor of AFB1, and 8-methoxypsoralen (8-MOP), an inhibitor of CYP enzyme, further confirm the critical role of CYP2A13 in AFB1-induced cytotoxicity and apoptosis. Collectively, these findings suggest adverse effects of AFB1 in respiratory diseases mediated by CYP2A13.

Protective Effect of Ebselen on Aflatoxin B1-Induced Cytotoxicity in Primary Rat Hepatocytes

Protective Effect of Ebselen on Aflatoxin B1-Induced Cytotoxicity in Primary Rat Hepatocytes

Cis-Terpenones as an Effective Chemopreventive Agent against Aflatoxin B1-Induced Cytotoxicity and TCDD-Induced P450 1A/B Activity in HepG2 Cells

Aflatoxin B1 (AFB1) is a potent carcinogen, which can significantly increase the risk of hepatocellular carcinoma development through food contamination. In past decades, chemopreventive agents, such as oltipraz and chlorophyllins, have demonstrated that chemo-intervention is an effective approach to reduce hepatotoxicity by AFB1. However, because of the potential adverse effects of these agents, alternative novel mechanism-based chemopreventive agents are needed. We report here that novel cis-terpenones 1-3, which were synthesized as the precursors of natural product analogues in our laboratory, showed promising protective effects against AFB1-induced cytotoxicity in HepG2 cells. Chemo-protection was observed with increasing concentrations of cis-terpenones in the co-treatment of AFB1, and no cytotoxicity was observed with cis-terpenones alone. In addition, cis-terpenones 1-3 at 10 and microM effectively inhibited induced cytochrome P450 1A/1B activity by 50% in HepG2 cells, as indicated by an EROD assay. P450 1A/B is involved in the activation of many pre-carcinogens and is highly inducible in liver cells. These results suggested that novel terpenones 1-3 are candidates for the development of novel mechanism-based chemopreventive agents against AFB1 and other carcinogenic stimuli.

Protective effect of ebselen on aflatoxin B1-induced cytotoxicity in primary rat hepatocytes.

Recent studies have shown that aflatoxin B1 enhances reactive oxygen species formation and causes oxidative damage, which may ultimately contribute to the cytotoxicity and carcinogenic effect of aflatoxin B1. Ebselen, 2-phenyl-1,2-benzoisoseleazol-3(H)-one, a synthetic seleno-organic compound has been shown to possess glutathione peroxidase-like activity and free radical scavenging ability. Thus present study was designed to investigate the protective effect of ebselen on aflatoxin B1-induced cytotoxicity in primary rat hepatocytes. Aflatoxin B1-induced cytotoxicity and lipid peroxidation were determined by lactate dehydrogenase leakage and malondialdehyde generation, respectively. Intracellular reactive oxygen species level was measured using the fluorescent probe 2',7'-dichlorofluorescin diacetate, and the intracellular reduced glutathione concentration was determined with a fluorometric method. Ebselen was found to display a dose-dependent protective effect on lactate dehydrogenase leakage and malondialdehyde generation caused by aflatoxin B1 exposure. The results also demonstrate that ebselen efficiently inhibits the intracellular reactive oxygen species formation in aflatoxin B1-treated hepatocytes in a dose and time-dependent manner. It was also noted that ebselen was able to increase the intracellular reduced glutathione concentration, both in the control and in aflatoxin B1-treated hepatocytes. The protection of ebselen against aflatoxin B1 cytotoxicity, however, was not affected by lowering the concentration of intracellular reduced glutathione. The overall data indicate that ebselen possesses a potent protective effect against aflatoxin B1-induced cytotoxicity, and the main mechanism involved in the protection may be its strong capability in inhibiting intracellular reactive oxygen species formation and preventing oxidative damage.

Differential response of primary cultures of human and rat hepatocytes to aflatoxin B1-induced cytotoxicity and protection by the hepatoprotective agent (+)-cyanidanol-3

Differential response of primary cultures of human and rat hepatocytes to aflatoxin B1-induced cytotoxicity and protection by the hepatoprotective agent (+)-cyanidanol-3

Prolonged Maintenance of Active Cytochrome P-450 in Adult Rat Hepatocytes Co-Cultured with Another Liver Cell Type

The cytochrome P-450 content promptly fell in adult rat hepatocytes cultured in conventional conditions whereas no obvious change was detected over a 10-day period when these cells were co-cultured with another rat liver epithelial cell type. A concomitant maintenance of the amino-pyrine N-demethylase activity and a high aflatoxin B1-induced cytotoxicity was observed, and addition of phenobarbital to the culture medium produced about a 2-fold increase in cytochrome P-450 level. These results indicate that when placed in co-culture, adult hepatocytes remain differentiated for several days and suggest that such a co-culture system is an appropriate model for pharmacotoxicological studies.

Effect of inhibitors of microsomal enzymes on aflatoxin B1-induced cytotoxicity and inhibition of RNA synthesis in isolated rat hepatocytes

Previous studies conducted in this laboratory demonstrated that AFB1 activation and deactivation was effectively inhibited by metyrapone and TCPO in isolated hepatocytes. The present study was undertaken to study the toxic effect of AFB1 on hepatocyte and RNA synthesis, and to assess the influence of the inhibitors on AFB1-induced cytotoxicity and AFB1-inhibited RNA synthesis. AFB1 at 50 μM was toxic and inhibited macromolecular synthesis by greater than 70% at 180 min of incubation whereas at lower concentrations of AFB1 (0.05–10 μM) dose-and time-dependent decreases in cell viability, protein and RNA synthesis were observed. Using [3H]-AFB1 (0–1.5 μM), the uptake and covalent binding of the toxin were also dose-and time-dependent. Initial rates of these processes to reach half-maximum was found to be 0.25 μM AFB1. In cells treated with AFB1 (5 μM) and metyrapone (1.0 mM) or SKF-525A (10 μM), the cell viability was similar to the control and [3H]-uridine incorporation was significantly higher than AFB1 treated cells. AFB1 and TCPO (0.5 mM) treated cells exhibited further decreases in cell viability and RNA synthesis. Results suggest that the binding of AFB1 to DNA and impairment of transcriptional activity may lead to cell death.

A comparison of aflatoxin B1-induced cytotoxicity, mutagenicity and prophage induction in Salmonella typhimurium mutagen tester strains TA1535, TA1538, TA98 and TA100

Treatment of Ames mutagen tester strains with aflatoxin B1 (AFB1) and S9 mix results not only in the production of a poten mutagen, but induces a pathway that leads to the induction of prophages present in all Ames tester strains. Characterization of the prophage induction and mutagenic response following AFB1 treatment showed that plasmid pKM101 dramatically enhances mutagenesis, but suppressed prophage induction. Spontaneous release of phage by TA98 and TA100 was also lower than in TA1535 and TA1538. In addition to mutagenesis and prophage induction, survival of all 4 tester strains was quantitated after AFB1 treatment. The data show that the frameshift tester strains (TA1538 and TA98) are more sensitive to the bactericidal action of AFB1 than the base-pair tester strains (TA1535 and TA100), survival being significantly affected above 100 ng. One of several hypotheses examined was the difference in the number and types of prophages present in base-pair tester strains that are not detectable in the frame-shift tester strains. These data suggest that prophage induction can detect DNA damage that is non-mutagenic; and that it is important to characterize the lysogenic nature of the Ames strains since it may influence the observed histidine revertant rate and the survival of the tester strain.