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contaminated with mycotoxins. Poor harvest practices and importance as a demulcent and febrifuge and also possess
[2]
[31]
inadequate conditions during drying, handling, packaging, anti-inflammatory, analgesic, and antimicrobial activities. [32,33]
storing and transporting can contribute to the growth of The aim of the current research was to assess possible
fungi and an increased risk of production of mycotoxins. The protective effects of C. olitorius extracts against cytotoxic
[3]
importance of co-occurrence of mycotoxins lies in the changes effects and disruption of DNA integrity induced by FB and
1
that might occur in the combined toxicity of mycotoxins. In AFB in the rat hepatoma cell line (H4IIE-luc).
[4]
1
addition, the existence of relationships in the occurrences of
mycotoxins allows predictions of the presence of individual METHODS
mycotoxins from the presence of others. [1]
Chemicals
Among these mycotoxins, aflatoxin B (AFB ) is the Aflatoxin B and FB (98% purity) were purchased from
1
1
1
1
predominant contaminant in cereals and oilseed and Sigma Chemicals (St. Louis, MO, USA). The DNA extraction
presents a significant risk, due to being hepatotoxic and kit (DNeasy Blood and Tissue Kit) was obtained from
[5]
carcinogenic to humans and animals. [4,6-8] AFB is classified Qiagen (Hilden, Germany). A DNA ladder, polymerase chain
1
by the International Agency of Research on Cancer (IARC) as reaction (PCR) master mix containing 100 base pairs and
a Group 1 carcinogen. This mycotoxin is also mutagenic, RNAse free water were obtained from Fermentas Inc.,
[9]
teratogenic, and immunosuppressive in farm, and laboratory (Glen Burnie, MD, USA). Supertherm Taq polymerase was
animals, [10-12] and primarily affects cell-mediated immunity. purchased from JMR Holdings (London, UK). Forty primers
[13]
AFB is also able to induce reactive oxygen species (ROS), [8,14-16] were obtained from Operon Technologies (Alameda, CA, USA).
1
possibly requiring activation of cytochrome P450. All solvents used were analytical grade from Burdick and
Jackson (Muskegon, MI, USA).
Fumonisins, mainly produced by Fusarium verticillioides
and F. proliferatum, are mycotoxins commonly found on corn. Plant materials
The most toxic and abundant of these is fumonisin B (FB ), Stems and leaves of C. olitorius were collected from a
1
1
which causes esophageal and hepatic cancer in humans and residential garden in the city of Potchefstroom, North West
liver and kidney cancer in rodents. [17-19] IARC evaluated FB and Province, South Africa. The plant material was freeze-dried,
1
classified it as probably carcinogenic to humans (Group 2B). pulverized, and 1 g was infused with 10 mL water for 24 h
[20]
Moreover, FB modulates immunity in animals and decreases at room temperature. After centrifugation, the supernatant
1
viability of lymphocytes in poultry. [21] was freeze-dried and stored at 4 °C until used.
Humans and animals are constantly exposed to small Cytotoxicity
concentrations of these mycotoxins, either individually Rat hepatoma cells (H4IIE-luc) were used as the mammalian
or in combination. Mycotoxicoses occur seasonally in model. This cell line had been stably transfected with a firefly
[22]
areas that have not implemented effective prophylactic luciferase reporter gene under control of the dioxin response
measures. While interactions between mycotoxins had element and the aryl hydrocarbon receptor mechanism. [34-37]
[23]
been discussed, few studies have been conducted with These cells were originally developed as a reporter gene
[24]
these combinations. assay to determine the presence of, and to semi-quantify
the concentrations of certain groups of persistent organic
[38]
Cochorus olitorius (C. olitorius, Tiliaceae family) is indigenous to pollutants including mixtures. [39]
the Middle East, including Egypt and South Africa. Young leaves
of C. olitorius are regarded to be a healthy vegetable in East H4IIE-luc cells were seeded at a density of 10,000 cells/mL
Asia and Japan, typically known as moroheiya. [25,26] Its health media (Dulbecco’s Modified Eagle’s Medium, Sigma: D2902;
benefits have been reported to include antitumor activity St. Louis, MO, USA) in the inner 60 wells of a 96-well
by inhibiting tumorigenesis, antioxidant properties, microplate. A volume of 250 μL of culture medium,
[27]
[28]
and antibacterial activity. Young leaves of C. olitorius are supplemented with 0.044 mol/L NaHCO and 10% fetal
[29]
3
rich in calcium, potassium, phosphate, iron, ascorbic acid, bovine serum (Life Technologies, Carlsbad, CA, USA), was
carotene and other nutrients, and contain a large amount added to each well. To avoid edge effects and to create a
of mucilaginous polysaccharides. [28,30] It has also been homogenous microclimate across all wells containing cells,
reported that compounds such as carotenoids, flavonoids, outer cells received 250 μL Dulbecco’s phosphate buffered
and vitamin C, isolated from leaves of C. olitorius, exhibit saline (PBS) (Tewksbury, MA, USA). Two sets of plates were
significant antioxidant characteristics. In addition, leaves incubated, at 37 °C in humidified air with 5% CO , with one
[30]
2
of C. olitorius have been reported to have ethno-medicinal set for 24 h and the other set for 48 h. After incubation,
76 Hepatoma Research | Volume 1 | Issue 2 | July 15, 2015