In vivo effects of zearalenone on the expression of proteins involved in the detoxification of rat xenobiotics

Zearalenone (ZEN) is a lactone derivative of the resorcylic acid produced by various Fusarium species that are widely found in foods and animal feeds. ZEN exerts species‐specific estrogenic effects, possibly because of the metabolism differences arising from reduction, hydroxylation, or glucuro‐conjugation. The main objective of this study was to determine the levels of expression of rat proteins that are involved in the ZEN detoxification pathway upon acute ZEN treatment. This was achieved by monitoring the mRNA associated with 25 genes using RT‐PCR upon ZEN uptake. These genes code for a variety of proteins that are involved in cellular detoxifying pathways, transporters, cytochromes P450 (CYPs), hydroxysteroid dehydrogenases, and transferases, and receptors that are involved in CYP expression or steroid metabolism. Liver samples from rats treated with ZEN were compared to untreated rats or animals treated with classical CYP inducers (phenobarbital, dexamethasone, β‐naphtoflavone, and clofibrate). Significant changes of mRNA expression were observed for the efflux transporter, P‐glycoprotein, monooxygenases (CYP2C7, CYP2E1, CYP3A1, CYP3A2, and aromatase), steroid dehydrogenases, and Uridine diphospho–glucuronyl transferases (UGTs). Following a single ZEN treatment, the initial modifications in mRNA levels indicate a close association with microsomal enzyme activity of the CYP2B, CYP2C, and CYP3A protein families. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Cytotoxicity of Fusarium mycotoxins to mammalian cell cultures as determined by the MTT bioassay.

Fusarium mycotoxins occur worldwide in cereal grains and animal feeds and cause outbreaks of Fusarium mycotoxicoses in humans and animals. In this study mammalian cell cultures were used to screen the cytotoxicity of the most common Fusarium mycotoxins; deoxynivalenol (DON), zearalenone (ZEN), fumonisin B(1) (FB(1)) and moniliformin (MON). The most sensitive cell line for each Fusarium mycotoxin was determined for further toxicological investigations as an alternative to whole animal testing. Chinese hamster ovary cells (CHO-K1) were found to be the most sensitive for DON and FB(1) with IC(50) values of 0.27 and 85.5 microg/ml, respectively, after 48-h exposure. The hepatocellular carcinoma cells (HepG2) showed the highest sensitivity to MON with IC(50) values of 39.5 for 48 h and 26.8 microg/ml for 72-h exposure. Balb/c mice keratinocyte cell line (C5-O) was found to be the most sensitive to ZEN with IC(50) of 24.1 microg/ml after 72-h exposure. DON was found the most cytotoxic to the cell cultures of all the mycotoxins tested, followed by MON, ZEN, and FB(1). The results indicated that CHO-K1, C5-O, and HepG2 cells were found to be the sensitive cell lines for preliminary screening of DON, ZEN and MON contaminated feed and food extracts, respectively.     

Comparative mechanisms of zearalenone and ochratoxin A toxicities on cultured HepG2 cells: Is oxidative stress a common process?

Zearalenone (ZEN) and Ochratoxin A (OTA) are structurally diverse fungal metabolites that can contaminate feed and foodstuff and can cause serious health problems for animals as well as for humans. In this study, we get further insight of the molecular aspects of ZEN and OTA toxicities in cultured human HepG2 hepatocytes. In this context, we have monitored the effects of ZEN and OTA on (i) cell viability, (ii) heat shock protein (Hsp) 70 and Hsp 27 gene expressions as a parameter of protective and adaptive response, (iii) oxidative damage, and (iv) cell death pathways. Our results clearly showed that both ZEN and OTA inhibit cell proliferation. For ZEN, a significant induction of Hsp 70 and Hsp 27 was observed. In the same conditions, ZEN generated an important amount of reactive oxygen species (ROS). Antioxidant supplements restored the major part of cell mortality induced by ZEN. However, OTA treatment downregulated Hsp 70 and Hsp 27 protein and mRNA levels and did not induce ROS generation. Antioxidant supplements did not have a significant effect on OTA‐induced cell mortality. Using another cell system (Vero monkey kidney cells), we demonstrated that OTA downregulates three members of HSP 70 family: Hsp 70, Hsp 75, and Hsp 78. Our findings showed that oxidative damage seemed to be the predominant toxic effect for ZEN, while OTA toxicity seemed to be rather because of the absence of Hsps protective response. Furthermore, the two mycotoxins induced an apoptotic cell death. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009.     

Zearalenone exposure affects mouse oocyte meiotic maturation and granulosa cell proliferation

Zearalenone (ZEN) is a metabolite of Fusarium and is a common contaminant of grains and foodstuffs. ZEN acts as a xenoestrogen and is considered to be cytotoxic, tissue toxic, and genotoxic, which causes abortions and stillbirths in humans and animals. Since estrogens affect oocyte maturation during meiosis, in this study we investigated the effects of ZEN on mouse oocyte meiotic maturation and granulosa cell proliferation. Our results showed that ZEN‐treated oocyte maturation rates were decreased, which might be due to the disrupted cytoskeletons: (1) ZEN treatment resulted in significantly more oocytes with abnormal spindle morphologies; (2) actin filament expression and distribution were also disrupted after ZEN treatment, which was confirmed by the aberrant distribution of actin regulatory proteins. In addition, cortical granule‐free domains (CGFDs) were disrupted after ZEN treatment, which indicated that ZEN may affect mouse oocyte fertilization capability. ZEN reduced mouse granulosa cell proliferation in a dose‐dependent manner as determined by MTT assay and TUNEL apoptosis analysis, which may be another cause for the decreased oocyte maturation. Thus, our results demonstrated that exposure to zearalenone affected oocyte meiotic maturation and granulosa cell proliferation in mouse. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1226–1233, 2015.     

NO-1886 ameliorates glycogen metabolism in insulin-resistant HepG2 cells by GSK-3β signalling

Objectives The aim of the study was to elucidate the possible role and mechanism of NO‐1886 (ibrolipim, a lipoprotein lipase activator) in ameliorating insulin resistance induced by high palmitate. Methods HepG2 cells were cultured in RPMI 1640 medium and were treated with palmitate to induce insulin resistance. Free fatty acids (FFAs), glucose, glycogen, cell viability and mRNA and protein levels were analysed separately. Key findings We found that HepG2 cells treated with 0.5 mm palmitate for 48 h led to a significant decrease of insulin‐induced glucose consumption (from 2.89 ± 0.85 mm in the control to 0.57 ± 0.44 mm in palmitate). Insulin resistance (IR) of HepG2 cells was induced by 0.5 mm palmitate for 48 h. NO‐1886 stimulated glucose consumption, glycogen synthesis and FFA absorption in insulin‐resistant HepG2 cells. Maximum stimulation effects were observed with 10 µm NO‐1886 for 24 h. Compared with the dimethyl sulfoxide‐treated group, 2.5 µm NO‐1886 or higher could induce the mRNA expression of lipoprotein lipase. Meanwhile, NO‐1886 increased the protein content of P‐GSK‐3βser⁹ and decreased the protein level of GSK‐3β in insulin‐resistant HepG2 cells, but NO‐1886 didn't change the protein levels of PI3‐Kp85 and Akt2. Conclusion Lipoprotein lipase activator NO‐1886 could increase glycogen synthesis in HepG2 cells and could ameliorate the insulin resistance, which was associated with GSK‐3 signalling.     

Effects of water soluble PM2.5 extracts exposure on human lung epithelial cells (A549): A proteomic study

Exposure to airborne particulate matter (PM)2.5, a PM with aerodynamic diameter of less than 2.5 µm, is known to be associated with a variety of adverse health effects. However, the molecular mechanisms involved in fine PM toxicity are still not well characterized. The present study aims to provide new insights into the cytotoxicity of PM2.5 on human lung epithelial cells (A549) at the proteomic level. Two‐dimensional difference gel electrophoresis revealed a total of 27 protein spots, whose abundance were significantly altered in A549 cells exposed to water‐soluble PM2.5 extracts (WSPE). Among these, 12 spots were upregulated while 15 were downregulated. Twenty‐two proteins were further identified by matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass/mass spectrometry and database search. The results revealed that oxidative stress, metabolic disturbance, dysregulation of signal transduction, aberrant protein synthesis and degradation, as well as cytoskeleton disorganization are major factors contributing to WSPE‐mediated toxicity in human lung cells. It is further proposed that induction of apoptosis through p53, c‐Myc and p21 pathways may be one of the key toxicological events occurred in A549 cells under WSPE stress. The data obtained here will aid our understanding of the toxic mechanisms related to PM2.5, and develop useful biomarkers indicative of inhalable PM2.5 exposure. Copyright © 2013 John Wiley & Sons, Ltd.     

Proteomic analysis of liver cancer cells treated with 5‐Aza‐2′‐deoxycytidine (AZA)

5‐Aza‐2′‐deoxycytidine (AZA) is a potent inhibitor of DNA methylation that exhibits anti‐tumor activity in a variety of tumor cells via reactivation of tumor suppressor genes. However, few studies have been done on the biological and clinical significance of AZA in human hepatocellular carcinoma. To identify potential genes that may be aberrantly methylated and confer growth advantage to neoplastic cells and to better understand the molecular mechanism(s) underlying AZA anti‐tumor activity, a proteomics approach was used to annotate global gene expression changes of HepG2 cell line pre‐ and post‐treatment with AZA. A total of 56 differentially expressed proteins were identified by 2D gel analysis, 48 of which were up‐regulated while the remaining 8 were down regulated. Among the identified proteins, eight of these showed marked changed proteins, including seven up‐regulated proteins: glutathione S‐transferase P, protein DJ‐1, peroxiredoxin‐2, UMP‐CMP kinase, cytochrome c‐type heme lyase, enhancer of rudimentary homolog, profilin‐1, and one down‐regulated protein, heat‐shock protein β?1. The possible implication of these proteins in hepatocarcinogenesis is discussed. We tested two up‐regulated proteins, glutathione S‐transferase P and peroxiredoxin‐2, using RT‐PCR and their expression was consistent with the results obtained in the protein level. Both of these genes were methylated when methylation‐specific PCR was used against their promoter regions. Following treatment with AZA, the gene promoter regions were found to be unmethylated, concomitant with overexpression of the proteins compared to HepG2 cells without treatment. These data provide useful information in evaluating the therapeutic potential of AZA for the treatment of HCC. Drug Dev Res 69, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of CYP3A4 by 6′,7′‐dihydroxybergamottin in human CYP3A4 over‐expressed hepG2 cells

Objectives We previously established HepG2‐GS‐3A4, a cell line from hepatoblastoma with overexpression of human CYP3A4 and glutamine synthetase (GS). We further reported that these cells can be applied for screening inhibitors of CYP3A4 in vitro. The purpose of this study was to determine whether our CYP3A4‐overexpresed cell could be applied to evaluate mechanisms of CYP3A4 inhibition by 6′,7′‐dihydroxybergamottin (DHB), which is one of the major furanocoumarins in grapefruit juice, by using these cells. Methods Nifedipine oxidation, activity and protein expression of NADPH‐cytochrome reductase (POR) of HepG2‐GS‐3A4 cell were measured. CO‐binding spectrumassay in microsomal fraction of the cells was also evaluated. Key findings DHB and ketoconazole, a well‐known inhibitor of CYP3A4, inhibited nifedipine oxidation in a concentration‐dependent manner. DHB at a concentration of 3.0 µm , sufficient to inhibit the nifedipine oxidation, decreased POR activity; however, ketoconazole at a concentration of 0.9 µm , sufficient to inhibit the oxidation, did not affect the activity. The expression of POR protein in HepG2‐GS‐3A4 cells was not changed by either DHB or ketoconazole. The expression of CYP3A4 mRNA and protein was not changed by the addition of DHB or ketoconazole. DHB also reduced the absorption rate at 450 nm in a CO‐binding spectrum assay without alteration of the wavelength of maximum absorption. The mean absorption value at 450 nm slightly decreased with ketoconazole; however, the difference was not significant. Conclusions We concluded that inhibition of CYP3A4 activity by DHB includes the inhibition of POR activity. HepG2‐GS‐3A4 might be a good tool to evaluate the mechanisms.     

Reactive oxygen species‐dependent JNK downregulated olaquindox‐induced autophagy in HepG2 cells

Autophagy plays an important role in response to intracellular and extracellular stress to sustain cell survival. However, dysregulated or excessive autophagy may lead to cell death, known as “type II programmed cell death,” and it is closely associated with apoptosis. In our previous study, we proposed that olaquindox induced apoptosis of HepG2 cells through a caspase‐9 dependent mitochondrial pathway. In this study, we investigated autophagy induced by olaquindox and explored the crosstalk between apoptosis and autophagy in olaquindox‐treated HepG2 cells. Olaquindox‐induced autophagy was demonstrated by the accumulation of monodansylcadervarine, as well as elevated expression of autophagy‐related MAP‐LC3 and Beclin 1 proteins. The autophagy inhibitor 3‐methyladenine significantly increased the apoptotic rate induced by olaquindox, which was correlated with increased ratio of Bax/Bcl‐2. The further studies showed that olaquindox increased the levels of reactive oxygen species (ROS), and antioxidant N‐acetyl‐L ‐cysteine (NAC) effectively blocked the accumulation of ROS but failed to block autophagy. Moreover, olaquindox induced the activation of c‐Jun N‐terminal protein kinase (JNK), and JNK inhibitor SP600125 failed to block autophagy. Instead, olaquindox‐induced autophagy was enhanced by NAC or SP600125. Meanwhile, JNK activation was remarkably blocked by NAC, indicating that ROS may be the upstream signaling molecules of JNK activation and involved in the negative regulation of olaquindox‐induced autophagy. These results suggest that olaquindox induces autophagy in HepG2 cells and that olaquindox‐induced apoptosis can be enhanced by 3‐methyladenine. Olaquindox‐induced autophagy in HepG2 cells is upregulated by Beclin 1 but downregulated by ROS‐dependent JNK. Copyright © 2014 John Wiley & Sons, Ltd.     

Metaproteomics Reveals Alteration of the Gut Microbiome in Weaned Piglets Due to the Ingestion of the Mycotoxins Deoxynivalenol and Zearalenone

The ingestion of mycotoxins can cause adverse health effects and represents a severe health risk to humans and livestock. Even though several acute and chronic effects have been described, the effect on the gut metaproteome is scarcely known. For that reason, we used metaproteomics to evaluate the effect of the mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) on the gut microbiome of 15 weaned piglets. Animals were fed for 28 days with feed contaminated with different concentrations of DON (DONlow: 870 μg DON/kg feed, DONhigh: 2493 μg DON/kg feed) or ZEN (ZENlow: 679 μg ZEN/kg feed, ZENhigh: 1623 μg ZEN/kg feed). Animals in the control group received uncontaminated feed. The gut metaproteome composition in the high toxin groups shifted compared to the control and low mycotoxin groups, and it was also more similar among high toxin groups. These changes were accompanied by the increase in peptides belonging to Actinobacteria and a decrease in peptides belonging to Firmicutes. Additionally, DONhigh and ZENhigh increased the abundance of proteins associated with the ribosomes and pentose-phosphate pathways, while decreasing glycolysis and other carbohydrate metabolism pathways. Moreover, DONhigh and ZENhigh increased the abundance of the antioxidant enzyme thioredoxin-dependent peroxiredoxin. In summary, the ingestion of DON and ZEN altered the abundance of different proteins associated with microbial metabolism, genetic processing, and oxidative stress response, triggering a disruption in the gut microbiome structure.     

The role of oxidative stress in citreoviridin‐induced DNA damage in human liver‐derived HepG2 cells

We hypothesize that citreoviridin (CIT) induces DNA damage in human liver‐derived HepG2 cells through an oxidative stress mechanism and that N‐acetyl‐l ‐cysteine (NAC) protects against CIT‐induced DNA damage in HepG2 cells. CIT‐induced DNA damage in HepG2 cells was evaluated by alkaline single‐cell gel electrophoresis assay. To elucidate the genotoxicity mechanisms, the level of oxidative DNA damage was tested by immunoperoxidase staining for 8‐hydroxydeoxyguanosine (8‐OHdG); the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH) were examined; mitochondrial membrane potential and lysosomal membranes' permeability were detected; furthermore, protective effects of NAC on CIT‐induced ROS formation and CIT‐induced DNA damage were evaluated in HepG2 cells. A significant dose‐dependent increment in DNA migration was observed at tested concentrations (2.50–10.00 µM) of CIT. The levels of ROS, 8‐OHdG formation were increased by CIT, and significant depletion of GSH in HepG2 cells was induced by CIT. Destabilization of lysosome and mitochondria was also observed in cells treated with CIT. In addition, NAC significantly decreased CIT‐induced ROS formation and CIT‐induced DNA damage in HepG2 cells. The data indicate that CIT induces DNA damage in HepG2 cells, most likely through oxidative stress mechanisms; that NAC protects against DNA damage induced by CIT in HepG2 cells; and that depolarization of mitochondria and lysosomal protease leakage may play a role in CIT‐induced DNA damage in HepG2 cells. © 2014 The Authors. Published by Wiley Periodicals Inc. Environ Toxicol 30: 530–537, 2015.     

Gene and protein responses of human lung tissue explants exposed to ambient particulate matter of different sizes

Context: Exposure to ambient particulate air pollution is associated with increased cardiovascular and respiratory morbidity and mortality. It is necessary to understand causal pathways driving the observed health effects, particularly if they are differentially associated with particle size.

Objectives: To investigate the effect of different size ranges of ambient particulate matter (PM) on gene and protein expression in an in vitro model.

Materials and methods: Normal human tracheobronchial epithelium (NHTBE) three-dimensional cell constructs were exposed for 24?h to washed ambient PM of different sizes (size 1: 7–615?nm; size 2: 616 nm–2.39 µm; size 3: 2.4–10 µm) collected from a residential street. A human stress and toxicity PCR array was used to investigate gene expression and iTRAQ was used to perform quantitative proteomics.

Results: Eighteen different genes of the 84 on the PCR array were significantly dysregulated. Treatment with size 2 PM resulted in the greatest number of genes with altered expression, followed by size 1 and lastly size 3. ITRAQ identified 317 proteins, revealing 20 that were differentially expressed. Enrichment for gene ontology classification revealed potential changes to various pathways.

Discussion and conclusions: Different size fractions of ambient PM are associated with dysregulatory effects on the cellular proteome and on stress and toxicity genes of NHTBE cells. This approach not only provides an investigative tool to identify possible causal pathways but also permits the relationship between particle size and responses to be explored.     

Flavonoids in Scutellaria immaculata and S. ramosissima (Lamiaceae) and their biological activity

Objectives The aim of this study was to investigate the flavonoid composition of Scutellaria immaculata and S. ramosissima (Lamiaceae) and the in‐vitro biological activity of their extracts and flavonoids. Methods The flavonoid composition of S. immaculata (Si) and S. ramosissima (Sr) were analysed using LC‐MS. Antimicrobial activity was studied in vitro against a range of bacteria and fungi using diffusion and microdilution methods. Anti‐trypanosomal and cell proliferation inhibitory activity of the extracts and flavonoids was assessed using MTT. The antioxidant activity of the flavonoids and extracts were evaluated using DPPH* test. Key findings LC‐MS investigation of Si and Sr plants allowed the identification, for the first time, of an additional 9 and 16 flavonoids, respectively. The methanol, chloroform and water extracts from these plants and six flavonoids (scutellarin, chrysin, apigenin, apigenin‐7‐O‐glucoside, cynaroside and pinocembrine) exhibited significant inhibition of cell growth against HeLa, HepG‐2 and MCF‐7 cells. The chloroform extract of Sr showed potent cytotoxic effects with IC50 (drug concentration which resulted in a 50% reduction in cell viability) values of 9.25 ± 1.07 µg/ml, 12.83 ± 1.49 µg/ml and 17.29 ± 1.27 µg/ml, respectively. The highest anti‐trypanosomal effect against T. b. brucei was shown by the chloroform extract of Sr with an IC50 (drug concentration which resulted in a 50% inhibition of the biological activity) of 61 µg/ml. The pure flavonoids showed an IC50 range between 3 and 29 µm , with cynaroside as the most active compound with an IC50 value of 3.961 ± 0.133 µm . The chloroform extract of Sr has potent antimicrobial activity against Streptococcus pyogenes (minimum inhibitory concentration, MIC = 0.03 mg/ml). Pinocembrine exhibited a strong activity against the all bacteria except Escherichia coli and yeasts. Water extracts of Sr and Si exhibited potent antioxidant activity with IC50 values of 5.62 ± 0.51 µg/ml and 3.48 ± 0.02 µg/ml, respectively. Scutellarin exerted stronger antioxidant activity than other flavonoids. Conclusions This is the first study reporting an in‐vitro biological investigation for Si and Sr. Especially the chloroform extract of Sr showed potent anticancer and antimicrobial activity. Cynaroside had a highly selective and strong cytotoxicity against T. b. brucei while showing only mild effects against cancer cells.     

Study on the uptake and deglycosylation of the masked forms of zearalenone in human intestinal Caco-2 cells

This study investigated for the first time the intestinal transfer of two modified forms of zearalenone (ZEN), zearalenone-14-glucoside (ZEN14Glc) and zearalenone-16-glucoside (ZEN16Glc), using polarized monolayers of Caco-2 cells. The cells were apically exposed to 40 μM of ZEN14Glc, ZEN16Glc and ZEN, separately. Results showed that, after apical administration, ZEN14Glc and ZEN16Glc can be detected in cellular extracts indicating uptake by intestinal cells. Moreover, the glucosylated forms were cleaved to release ZEN, with a different cleavage ability for the two conjugated isomers. In particular, ZEN16Glc seems to be less prone to deglycosylation compared to ZEN14Glc, probably on account of an increased steric hindrance. We could show that human cytosolic β-glucosidase is able to cleave ZEN14Glc liberating ZEN, but is unable to cleave ZEN16Glc.All metabolites could cross the cell membrane and be detected in the apical compartment, while ZEN was also found in the basolateral compartment together with the modified mycotoxin form.     

Downregulation of human paraoxonase 1 (PON1) by organophosphate pesticides in HepG2 cells

Paraoxonase 1 (PON1) is a calcium‐dependent esterase synthesized primarily in the liver and secreted into the plasma where it is associated with high‐density lipoproteins (HDL). PON1 hydrolyzes and detoxifies some toxic metabolites of organophosphorus compounds (OPs) such as methyl parathion and chlorpyrifos. Thus, PON1 activity and expression levels are important for determining susceptibility against OPs poisoning. Some studies have demonstrated that OPs can modulate gene expression through interactions with nuclear receptors. In this study, we evaluated the effects of methyl parathion and chlorpyrifos on the modulation of PON1 in Human Hepatocellular Carcinoma (HepG2) cells by real‐time PCR, PON1 activity assay, and western blot. The results showed that the treatments with methyl parathion and chlorpyrifos decreased PON1 mRNA and immunoreactive protein and increased inflammatory cytokines in HepG2 cells. The effects of methyl parathion and chlorpyrifos on the downregulation of PON1 gene expression in HepG2 cells may provide evidence of OPs cytotoxicity related to oxidative stress and an inflammatory response. A decrease in the expression of the PON1 gene may increase the susceptibility to OPs intoxication and the risk of diseases related to inflammation and oxidative stress. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 490–500, 2017.     

The human fatty acid synthase: a new therapeutic target for coxsackievirus B3-induced diseases?

Coxsackievirus is linked to a large variety of severe human and animal diseases such as myocarditis. The interplay between host factors and virus components is crucial for the fate of the infected cells. However, host proteins which may play a role in coxsackievirus-induced diseases are ill-defined. Two-dimensional gel electrophoresis of protein extracts obtained from coxsackievirus B3 (CVB3)-infected and uninfected HeLa or HepG2 cells combined with spot analysis revealed several proteins which are exclusively up-regulated in infected cells. One of these proteins was identified as the fatty acid synthase (FAS). By using cerulenin and C75, two known inhibitors of FAS we were able to significantly block CVB3 replication. FAS appears to be directly involved in CVB3-caused pathology and is therefore suitable as a therapeutic target in CVB3-induced diseases.     

Cytotoxicity of various chemicals and mycotoxins in fresh primary duck embryonic fibroblasts: a comparison to HepG2 cells

To screen cost‐effectively the overall toxicity of a sample, particularly in the case of food and feed ingredient quality control, a sensitive bioassay is necessary. With the wide variety of cytotoxicity assays, performance comparison between assays using different cells has become of interest. Fresh primary duck embryonic fibroblasts (DEF) were hypothesized to be a sensitive tool for in vitro cytotoxicity screening; cell viability of DEF in response to various cytotoxins was determined and compared with response of HepG2 cells. The IC₅₀ values by the alamar blue assay in the DEF cells had a high correlation (R² = 0.96) with those obtained in HepG2 cells. Within the same toxin, primary DEF yielded significantly lower IC₅₀ values than that obtained from HepG2 cells using the MTT and alamar blue assay. Additionally, primary DEF responded to all mycotoxins tested using the alamar blue assay, while HepG2 was less sensitive, particularly at short exposure times. The estimated IC₅₀ for aflatoxin B₁, fumonisins B₁ and deoxynivalenol in DEF after 72 h incubation were 3.69, 4.19 and 1.26 μg ml^–1, respectively. Results from the current study suggest that primary DEF are more sensitive to cytotoxins and mycotoxins compared to HepG2, and thus may have great potential as an effective tool for cytotoxicity assessment. The question remains whether in vitro IC₅₀ values can accurately predict in vivo toxicity; however, the current study accentuates the need for further attention to identify sensitive cell models for in vitro cytotoxicity screening and subsequent exploration of species‐specific prediction models for in vivo toxicity. Copyright © 2016 John Wiley & Sons, Ltd.     

Antiapoptotic effects of dietary antioxidants towards N‐nitrosopiperidine and N‐nitrosodibutylamine‐induced apoptosis in HL‐60 and HepG2 cells

The aim of this work was to determine the effect of vitamin C, diallyl disulfide (DADS) and dipropyl disulfide (DPDS) towards N‐nitrosopiperidine (NPIP) and N‐nitrosodibutylamine (NDBA)‐induced apoptosis in human leukemia (HL‐60) and hepatoma (HepG2) cell lines using the terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling assay. None of the vitamin C (5–50 µm ), DADS and DPDS (1–5 µm ) concentrations selected induced a significant percentage of apoptosis. In simultaneous treatments, vitamin C, DADS and DPDS reduced the apoptosis induced by NPIP and NDBA in HL‐60 and HepG2 cells (around 70% of reduction). We also investigated its scavenging activities towards reactive oxygen species (ROS) produced by NPIP and NDBA using 2',7'‐dichlorodihydrofluorescein diacetate in both cell lines. ROS production induced by both N‐nitrosamine was reduced to control levels by vitamin C (5–50 µm ) in a dose‐dependent manner. However, DADS (5 µm ) increased ROS levels induced by NPIP and NDBA in HL‐60 (40 and 20% increase, respectively) and HepG2 cells (18% increase), whereas DPDS was more efficient scavenger of ROS at the lowest concentration (1 µm ) in both HL‐60 (52 and 25% reduction, respectively) and HepG2 cells (24% reduction). The data demonstrated that the scavenging ability of vitamin C and DPDS could contribute to inhibition of the NPIP‐ and NDBA‐induced apoptosis. However, more than one mechanism, such as inhibition of phase I and/or induction of phase II enzymes, could be implicated in the protective effect of dietary antioxidants towards NPIP‐ and NDBA‐induced apoptosis in HL‐60 and HepG2 cells. Copyright © 2009 John Wiley & Sons, Ltd.     

An ecofriendly synthesized gold nanoparticles induces cytotoxicity via apoptosis in HepG2 cells

Microbes have long been used for the synthesis of a variety of nanoparticles. Hepatocellular carcinoma (HCC) is the primary liver cancer and it is the second leading cause of cancer‐related mortality worldwide. In this study, we have synthesized Enterococcus mediated gold nanoparticles (AuNPs) and investigated their cytotoxic potential against human hepatocellular cancer cell line (HepG2). AuNPs were synthesized using Enterococcus sp. RMAA. HepG2 cells were treated with different concentrations of AuNPs for 24 hours and cytotoxicity was analyzed by MTT ((4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay. AuNPs induced reactive oxygen species expression was analyzed by 2′,7′‐dichlorodihydrofluorescein diacetate staining. Morphological changes related to apoptosis was analyzed by annexin V/propidium iodide staining. Protein expression of proliferating cell nuclear antigen (PCNA) was done by western blotting analysis. Bacterial‐mediated AuNPs caused significant cytotoxicity in HepG2 cells. AuNPs treatment also caused the significant expression of ROS and morphological damage related to apoptosis. AuNPs treatments were responsible for the dislocation of cytochrome c from mitochondria to cytosol. The protein expression of PCNA was significantly decreased upon AuNPs treatment. These findings suggest that Enterococcus‐mediated AuNPs can inhibit the proliferation of HepG2 cells via intracellular ROS mediated apoptosis, decreased PCNA expressions, and it may have the potential to treat HCC.