Aqueous extract of Corchorus olitorius decreases cytotoxicity of aflatoxin B1 and fumonisin B1 in H4IIE-luc cells

 Introduction

Co-occurrence of various mycotoxins in foodstuffs and animal feed is common because each toxigenic fungus can produce more than one mycotoxin and foodstuff can be colonized by several fungi either while growing in the field or during storage or transport. Processed products are often composed of various raw materials which might be contaminated with mycotoxins. Poor harvest practices and inadequate conditions during drying, handling, packaging, storing and transporting can contribute to the growth of fungi and an increased risk of production of mycotoxins. The importance of co-occurrence of mycotoxins lies in the changes that might occur in the combined toxicity of mycotoxins. In addition, the existence of relationships in the occurrences of mycotoxins allows predictions of the presence of individual mycotoxins from the presence of others.

Among these mycotoxins, aflatoxin B 1 (AFB 1 ) is the predominant contaminant in cereals and oilseed and presents a significant risk, due to being hepatotoxic and carcinogenic to humans and animals. AFB 1 is classified by the International Agency of Research on Cancer (IARC) as a Group 1 carcinogen. This mycotoxin is also mutagenic, teratogenic, and immunosuppressive in farm, and laboratory animals, [ and primarily affects cell-mediated immunity. AFB 1 is also able to induce reactive oxygen species (ROS), possibly requiring activation of cytochrome P450.

Fumonisins, mainly produced by Fusarium verticillioides and F. proliferatum, are mycotoxins commonly found on corn. The most toxic and abundant of these is fumonisin B 1 (FB 1 ), which causes esophageal and hepatic cancer in humans and liver and kidney cancer in rodents. IARC evaluated FB 1 and classified it as probably carcinogenic to humans (Group 2B). Moreover, FB 1 modulates immunity in animals and decreases viability of lymphocytes in poultry.

Humans and animals are constantly exposed to small concentrations of these mycotoxins, either individually or in combination. Mycotoxicoses occur seasonally in areas that have not implemented effective prophylactic measures. While interactions between mycotoxins had been discussed, few studies have been conducted with these combinations.

Cochorus olitorius (C. olitorius, Tiliaceae family) is indigenous to the Middle East, including Egypt and South Africa. Young leaves of C. olitorius are regarded to be a healthy vegetable in East Asia and Japan, typically known as moroheiya. Its health benefits have been reported to include antitumor activity by inhibiting tumorigenesis, antioxidant properties, and antibacterial activity. Young leaves of C. olitorius are rich in calcium, potassium, phosphate, iron, ascorbic acid, carotene and other nutrients, and contain a large amount of mucilaginous polysaccharides. It has also been reported that compounds such as carotenoids, flavonoids, and vitamin C, isolated from leaves of C. olitorius, exhibit significant antioxidant characteristics. In addition, leaves of C. olitorius have been reported to have ethno-medicinal importance as a demulcent and febrifuge and also possess anti-inflammatory, analgesic, and antimicrobial activities. The aim of the current research was to assess possible protective effects of C. olitorius extracts against cytotoxic effects and disruption of DNA integrity induced by FB 1 and AFB 1 in the rat hepatoma cell line (H4IIE-luc).

  Methods

Chemicals Aflatoxin B 1 and FB 1 (98% purity) were purchased from Sigma Chemicals (St. Louis, MO, USA). The DNA extraction kit (DNeasy Blood and Tissue Kit) was obtained from Qiagen (Hilden, Germany). A DNA ladder, polymerase chain reaction (PCR) master mix containing 100 base pairs and RNAse free water were obtained from Fermentas Inc., (Glen Burnie, MD, USA). Supertherm Taq polymerase was purchased from JMR Holdings (London, UK). Forty primers were obtained from Operon Technologies (Alameda, CA, USA). All solvents used were analytical grade from Burdick and Jackson (Muskegon, MI, USA).Plant materials Stems and leaves of C. olitorius were collected from a residential garden in the city of Potchefstroom, North West Province, South Africa. The plant material was freeze-dried, pulverized, and 1 g was infused with 10 mL water for 24 h at room temperature. After centrifugation, the supernatant was freeze-dried and stored at 4 °C until used.Cytotoxicity Rat hepatoma cells (H4IIE-luc) were used as the mammalian model. This cell line had been stably transfected with a firefly luciferase reporter gene under control of the dioxin response element and the aryl hydrocarbon receptor mechanism. [34],[35],[36],[37] These cells were originally developed as a reporter gene assay to determine the presence of, and to semi-quantify the concentrations of certain groups of persistent organic pollutants [38] including mixtures. [39]H4IIE-luc cells were seeded at a density of 10,000 cells/mL media (Dulbecco's Modified Eagle's Medium, Sigma: D2902; St. Louis, MO, USA) in the inner 60 wells of a 96-well microplate. A volume of 250 μL of culture medium, supplemented with 0.044 mol/L NaHCO 3 and 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA), was added to each well. To avoid edge effects and to create a homogenous microclimate across all wells containing cells, outer cells received 250 μL Dulbecco's phosphate buffered saline (PBS) (Tewksbury, MA, USA). Two sets of plates were incubated, at 37 °C in humidified air with 5% CO 2 , with one set for 24 h and the other set for 48 h. After incubation, the medium was removed and replaced with medium containing C. olitorius extract at either of two concentrations (20 or 40 μg/mL) and incubated for another 24 h. The medium was replaced with medium containing varying concentrations of AFB 1 (50, 25, 2.5, 0.25, 0.025 μmol/L) dissolved in methanol, or of FB 1 (200, 100, 10, 1, 0.1 μmol/L) dissolved in methanol. A combination of the already mentioned concentrations of AFB 1 and FB 1 were also tested: 50 μmol/L AFB 1 + 200 μmol/L FB 1 ; 25 μmol/L AFB 1 + 100 μmol/L FB 1 , and so on. Exposure to mycotoxins was carried out in triplicates. Cells in six wells in each plate were exposed only to the aqueous extract of C. olitorius and cells in 11 wells were not exposed to anything except the growth medium.To determine the viability, based on metabolic activity of cells, a colorimetric assay was performed using the yellow dye 3-(4,5-dimethyltiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT, Montigny-le-Bretonneux, France). In this assay, MTT is converted to formazan (blue) by mitochondrial reductase enzymes in living cells. [40] A final concentration of 500 μg/mL MTT was added to each well and incubated for 30 min. Blue formazan crystals that were formed by reduced MTT were dissolved with dimethylsulfoxide and absorbance by the formazan was measured spectrophotometrically at 560 nm. The amount of blue formazan produced is proportional to the amount of viable cells, and the percentage of viable to dead cells was calculated by comparison with a control (untreated and solvent control). Viability among various C. olitorius treatments described above were compared to the viability of cells treated only with mycotoxins by applying the same protocol described before, but omitting aqueous extract of C. olitorius.Extraction of DNA Harvested cells were washed with PBS to remove the nonadherent dead cells. The adherent cells were removed by trypsinizing (0.25% trypsin, 0.1% versene EDTA; purchased from Thermo Scientific, Rockford, IL, USA) and activity was stopped by addition of media. The cell suspension was centrifuged at 3,000 g for 5 min at room temperature. Genomic DNA was extracted from cells according to the Qiagen instruction manual and concentrations determined spectrophotometrically by use of the NanoDrop ND-1,000 Spectrophotometer. Purity of DNA was assessed by examining the 260/280 nm ratio. [41]Random amplification of polymorphic DNA-polymerase chain reaction analysis Amplification of DNA fragments was carried out using an ICycler (Bio-Rad, Herts, UK) thermal cycler using 20 primers from the Operon Biotechnologies (BioCampus Colonge Nattermannalle, Germany). PCR amplification was conducted in 25 mL reaction volumes containing 10 ng genomic DNA, 12.5 pmol/L master mix (×2) (Thermo Fisher Scientific, Carlsbad, CA, USA), 1.0 units of Supertherm Taq polymerase and 50 pmol/L primer. The PCR reactions were carried out in a thermocycler (Bio-Rad C1000, Bio-Rad, Hercules, CA, USA), programed with a first denaturation for 5 min at 95 °C, followed by 40 cycles for 30 s denaturation at 95 °C, 30 s annealing at 37 °C and 1 min extension at 72 °C. Final extension at 72 °C for 5 min was allowed before holding at 4 °C for 5 min. Reaction products were stored at -80 °C prior to electrophoresis.Gel electrophoresis Amplified products together with marker (100 bp DNA) were resolved by gel electrophoresis (60 V/cm for 135 min) on 2% agarose gel in tris-acetate-EDTA buffer containing 0.001 mg/mL ethidium bromide purchased from Sigma Chemical Co. (St. Louis, MO, USA). Gels were photographed by Gel Documentation System (Gensnap) software (Synegen, UK).Band analysis The gels for control and exposed DNA were run for each of the 20 primers [Table 1]. A DNA ladder of 100 bp was also run in each gel. The bands for PCR products were analyzed by TotalLab Quant (V11.5: TL100-LX59-7YF4-EX). The fluorimetric profiles of each amplification reaction were studied both qualitatively and quantitatively by comparing profiles from control and DNA exposed to the extracts. Each change observed in random amplification of polymorphic DNA (RAPD) profiles of treated groups (disappearances and appearance of bands in comparison to the control RAPD profiles) was given the arbitrary score of +1. The mean was then calculated for each experimental group exposed to the mycotoxins for varying time periods. Template genomic stability (%) was calculated as "100 - (100a/n)" where "a" is the average number of changes in DNA profiles and "n" is the number of bands selected in control DNA profiles. [42]Statistical analysis All data were statistically analyzed with the Graphpad Prism 4.02 Inc. (La Jolla, CA, USA). The significance of the differences among treatment groups was determined with two-way analysis of variance. The assumptions of parametric statistics were confirmed. Normality was confirmed by the Kolmogorov-Smirnov test and homogeneity of variance was confirmed by use of Levine's test. All statements of significance were based on a probability of P≤ 0.05.

  Results

The results of cell viability assay revealed that H4IIE-luc cells that were treated with both concentrations (20 and 40 μg/mL) of C. olitorius extract were statistically significantly more viable after 24 h exposure than the ones that were not treated with the plant extract. However, there was no significant protection by the plant extract against FB 1 after 48 h of exposure. Furthermore, C. olitorius extract could not protect the cells from the AFB 1 concentration series or the combination exposure (AFB 1 + FB 1 ) irrespective of the exposure period (24 or 48 h) . A dose-dependent decrease of cell viability after exposure to increasing amounts of AFB 1 was observed only after 48 h a. After 24 h exposure, the response was not linear (hormetic effect). Except for the lowest concentration, cytotoxicity was more pronounced after 48 h. However, protective effects of the C. olitorius extract were observed after both 24 h and 48 h of exposure to AFB 1 . After 48 h, viability, expressed as a percentage of H4IIE-luc cells affected by FB 1 , was approximately 40% less than that of cells exposed to FB 1 alone. After 48 h, there was also no dose-dependence, but cytotoxicity was less pronounced. Protective effects of 20 or 40 μg/mL C. olitorius extract were observed. After both 24 and 48 h of exposure, production of MTT formazan was greater in the presence of both concentrations of C. olitorius extract at all tested doses of FB 1 compared to those in the absence of C. olitorius extract b. No significant differences were found between 20 and 40 μg/mL of C. olitorius extract after 24 h of exposure.

Incubation of H4IIE-luc cells with AFB 1 + FB 1 for 24 h resulted in greater cytotoxicity to cells as measured by the MTT assay, with significant toxicity at the sum of the two mycotoxin concentrations 12.5 and 125 μmol/Lc. The cells were least viable when they were exposed to the mixture of 250 μmol/L mycotoxin. Addition of C. olitorius extract to cells resulted in slightly greater viability. At lesser concentrations of AFB 1 (1.25 μmol/L) + FB 1 (12.5 μmol/L), protective effects of aqueous extracts of C. olitorius on viability of cells was greater relative to the cells that did not receive plant extract

In cases where bands were gained after exposure to C. olitorius extract at the shorter duration of exposure, 21 new bands out of 75 (28%) were amplified. A similar trend was observed during the longer exposure, where 25 of 75 bands (33.3%) appeared [Figure 3]c. Protective effects of C. olitorius extract were observed as new bands appeared during the 24 h, since 32 of 75 bands (42.7%) appeared; during the longer exposure, 23 of 75 bands (30.7%) disappeared [Figure 3]d.When OPD 9 primer was used, a maximum of 10 RAPD-PCR disappeared when cells were exposed to the mixture of FB 1 and AFB 1 + aqueous extract of C. olitorius for 48 h [Table 4]. However, when with OPD 15 was used as the primer, the maximum appearance of new bands showed the same number of bands lost (10) that was observed in cells exposed to AFB 1 + aqueous extract of C. olitorius after 24 h.There was a significant difference in stability of the DNA template between control and each of the treated groups [Figure 4]. However, no significant difference was observed in stability of the DNA template between control and cells exposed to the aqueous extract of C. olitorius alone. The protective effect of the aqueous extract of C. olitorius on DNA was observed in the cells exposed to FB 1 and AFB 1 . 

Discussion

Aflatoxin B 1 and FB 1 are the most frequently observed mycotoxins in food and animal feed. In African and European countries, both mycotoxins are found in maize. [43] Toxicity and carcinogenicity of AFB 1 , which has been classified as Group 1 carcinogen are thought to be directly linked to its bioactivation, resulting in a reactive form of AFB 1 , the 8, 9-epoxide. Bioactivation of AFB 1 occurs primarily by a microsomal cytochrome P450-dependent epoxidation of the terminal furan ring of AFB 1 , which is responsible for binding to cellular macromolecules such as DNA, RNA and other protein constituents. [44],[45],[46],[47] The MTT assay is more sensitive and reproducible than testing intact animals and is valuable in determining the modes of action of toxins. In the current study, H4IIE-luc cells responded to FB 1 and AFB 1 as well as a mixture of the two mycotoxins. Cytotoxic effects of FB 1 have been previously observed for murine microglial cells and primary astrocytes, [48] rat glioblastoma cells, [49],[50] human keratinocytes and esophageal epithelial cells, [51] primary rat hepatocytes and rat liver in vivo.[52] In human and rat glioblastoma cells and mouse hypothalamic cells, production of ROS was increased after exposure to 10-100 μmol/L of FB 1 for 48-144 h. [53] Exposure to 10-100 μmol/L of FB 1 for 72 h had no effect on production of ROS in human fibroblasts, or in primary cultures of rat astrocytes exposed to the same concentrations of FB 1 for as long as 6 days. [54],[55] Exposure to concentrations as high as 20 μmol/L of FB 1 did not significantly reduce the viability of IPEC-J2 cells. [56] In the current study, EC 50 could not be calculated for FB 1 because viability of cells exposed to 200 μmol/L was reduced only 41.6%, which is consistent with previously published results. [44] In yet another study, FB 1 was only weakly cytotoxicity. [57] The EC 50 for AFB 1 was 1.87 μmol/L, which is similar to that observed previously by others, [58],[59],[60] who reported EC 50 values ranging from 0.065 μmol/L for B-CMV1A2 cells to 14 μmol/L in BE12-6 cells. Exposure of H4IIE-luc cells to greater concentrations of AFB 1 and FB 1 resulted in lethality that was a concentration- and time-dependent. This effect was greater in cells treated with AFB 1 or AFB 1 + FB 1 . The interaction of FB 1 and AFB 1 in the induction of DNA damage and its correlation with biomarkers of cellular oxidative status has previously been reported to occur in vivo.[4],[8],[22],[61] These reports suggested that genotoxicity and carcinogenicity of AFB 1 were enhanced by exposure to FB 1 . [8] The in vivo results indicated that these effects were due to the production of ROS, which resulted in lipid peroxidation. [4],[61] AFB 1 is a well-known genotoxicant. When the mechanism by which the aqueous extract of C. olitorius protected H4IIE-luc rat hepatoma cells against genetic damage caused by AFB 1 and/or FB 1 was investigated by use of RAPD analysis, there were statistically significant differences in the profiles of expression of the investigated genes, between the control and the treated cell lines at all concentrations tested. [33] Differences in the profile between the control and the treated samples were due to point mutations and/or base modifications of the genome caused by AFB 1 and/or FB 1 . [62] Changes were observed for all genes for which primers were used. In our study, both qualitative and quantitative analyses showed that both mycotoxins increased instability of DNA templates of cells, in time- and concentration-dependent manners. This result supports the conclusion that both mycotoxins are direct-acting, genotoxicants that have the potential to attack hotspots present in DNA. The number of stable bands increased as a function of time and dose. Inconsistency in profiles of bands in RAPD analyses might have been observed because the two mycotoxins are acting directly as genotoxicants. However, they might act as genotoxicants through generation of free radicals during metabolism of the toxins through reactions of either electrophiles or nucleophiles with DNA. This interaction creates changes in their sequences that ultimately results in the formation of new priming sites and/or disappearances of existing priming sites for the RAPD primers. Thus, it gives different RAPD profiles for cells exposed to toxins. [63] Random amplification of polymorphic DNA-PCR suffers from inherent limitations such as a lack of reproducibility and occurrence of pseudo-bands which prevent its routine application. [64] However, if conditions of the assays are properly optimized, these limitations can be resolved. [65],[66] By optimizing conditions of the analysis, cloning the PCR products and further sequencing the products, RAPD can be useful in analyzing the nature and mode of action of the genotoxicants. [65],[66] While in the present study RAPD could detect toxin-induced DNA damage, further studies would be needed before it could be used regularly as a tool in the detection of alterations in DNA sequence due to the genotoxicants.Previous studies have demonstrated that certain compounds in the diet can offer protection against toxicity of mycotoxins. [67] Natural vitamins, carotenoids, polyphenol and trace elements are potentially beneficial in protection against mycotoxicosis. [68] Green leafy vegetables are known to be dietary sources of minerals, trace elements and phytochemicals that contribute to health. [69] Molecular evidence has suggested that trace elements and antioxidant molecules in green, leafy vegetables lessen risks of cancer and cardiovascular diseases through mechanisms that modulate free radical attack on nucleic acids, proteins, and polyunsaturated fatty acids. [70] C. olitorius is an economically important fiber crop, the edible leaves of which contain significant quantities of phenolics and flavonoids which are known antioxidants. [33],[71],[72],[73],[74] Although in the current study the active compound(s) in the aqueous extract of C. olitorius were not isolated or identified, flavonoids are possible candidates among the active compound(s) in C. olitorius. C. olitorius contains abundant amounts of a number of flavonoids that could act as antioxidants, including: 5-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, quercetin 3-galactoside, quercetin 3-glucoside, quercetin 3-(6-malonylglucoside), quercetin 3-(malonylgalactoside), ascorbic acid, a-tocopherol, and chlorophyll. [29] Furthermore, C. olitorius contains relatively high levels of quercetin glycosides. Several novel flavonol glycosides named corchorusides A and B, in addition to a major component, capsugenin-25, 30-O-β-diglucopyranoside have been isolated from C. olitorius. [26] Recently, several flavonoids, such as rutin, and quercetin and phenolic compounds, including gallic acid, chlorogenic acid, p-cumaric acid, ferulic acid, and ellagic acid have been isolated from extracts of C. olitorius. [75] Consequently, protective effects of the aqueous extract of C. olitorius against cytotoxicities of AFB 1 and FB 1 in H4IIE-luc might be due to the antioxidant capacity and the abundant occurrence of the flavonoid compounds. Another candidate for the active compound(s) is chlorophyll. Numerous in vitro studies have indicated that derivatives of chlorophyll, including chlorophyllide A and B and pheophorbide A and B can attenuate chemical genotoxicity by forming a molecular complex with pro-mutagens, [74],[75],[76] which might involve strong chlorophyll-AFB 1 and/or FB 1 interaction via their planar unsaturated cyclic rings. [75] Derivatives of pheophorbide A and B provided additional protection not only by direct trapping, but also by increasing glutathione S-transferase activity against hepatic AFB 1 metabolites. [76] In conclusion, both AFB 1 and FB 1 induced oxidative stress, which resulted in cytotoxicity and fragmentation of DNA of H4IIE-luc rat hepatoma cells after various durations of exposure to these toxins singly or in combination. Exposure to these mycotoxins resulted in appearance of new bands in the RAPD analysis, in addition to DNA damage. Treatment with an aqueous extract of C. olitorius resulted in a significant improvement in viability of cells and reduced damage to DNA in H4IIE-luc cells exposed to mycotoxins. Due to these effects, C. olitorius is suggested to be a traditional edible plant containing potential chemo-preventive agents for human cancers. However, additional studies on the uptake, metabolism, and disposition of the active ingredients in C. olitorius need to be further studied. Currently, the active ingredient (s) are unknown, and it is also not known whether these constituents that are effective in vitro can have similar effects in vivo.Acknowledgments

The Morogo Research Program gratefully acknowledges the National Research Foundation of South Africa (Focus Area Grant FA2004050600064) and National Research Center, Cairo, Egypt Project #10070112 for financial support of this study. Prof. Giesy was supported by the Canada Research Chair program, a Visiting Distinguished Professorship in the Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, the 2012 "High Level Foreign Experts" (#GDT20143200016) program, funded by the State Administration of Foreign Experts Affairs, the P.R. China to Nanjing University and the Einstein Professor Program of the Chinese Academy of Sciences.  References

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