Cytotoxic Effects Of Main Mycotoxins In Milk And Feed On Caco-2 Cells

This study was carried out to investigate the cytotoxic, oxidative damage and DNA damage effect on Caco-2 cell lines of some main common mycotoxins in food or feed and be divided into two experiment.ExperimentⅠ: Aflatoxin M1(AFM1), a classed 2B human carcinogen, mainly exists in milk and is harmful to human health. The studies on the toxicity of AFM1 mixed with other mycotoxins, however, are extremely few compared to the many study of its matrix, aflatoxin B1(AFB1). This paper for the first time investigated the cytotoxicity of AFM1 combined with ochratoxin A(OTA), zearalenone(ZEA) and α-zearalenol(α-ZOL) against human colon adenocarcinoma cell lines(Caco-2). The cell viability was suppressed by the all individual mycotoxins, in a dose- and time-dependent manner. The results from tetrazolium salt(MTT) assay demonstrated that AFM1 had the similar cytotoxicity as OTA(IC50= 4.10 μM) upon exposure for 72 h, which was much higher than that of ZEA and α-ZOL. The cytotoxic effect of all the mycotoxins combinations increased in the following order: OTA+α-ZOL < ZEA+α-ZOL < OTA+ZEA < AFM1+ZEA < AFM1+α-ZOL < AFM1+OTA < OTA+ZEA+α-ZOL < AFM1+ZEA+OTA < AFM1+ZEA+α-ZOL< AFM1+OTA+α-ZOL< AFM1+ZEA+OTA+α-ZOL. Isobologram analysis was further applied, which indicated that the existence of AFM1 brought additive and synergistic effect,except for AFM1+α-ZOL for antagonism effect. The synergistic effect is the highest(CI=0.17±0.05 at IC25) in the quaternary combination of all mytotoxin-combination investigated.Experiment Ⅱ :Aflatoxin B1(AFB1) and aflatoxin M1(AFM1) are natural mycotoxins that frequently present in food and feed and pose serious risks to human health. There are few data in the literature regarding the impairment of AFB1 and AFM1 on intestine. Therefore, the present study was undertaken to investigate their cytotoxic effect on Caco-2 cells, especially the differentiated ones that resemble mature small intestinal enterocytes. Both undifferentiated(UC) and differentiated(DC) cell were treated with AFB1 and AFM1 at various concentrations for up to 72 h. Cell viability, lactate dehydrogenase(LDH) release, reactive oxygen species(ROS) production and DNA damage were determined. Data showed that AFB1 and AFM1 significantly inhibited UC and DC cell growth, increased LDH release and caused genetic damage in a time- and dose-dependent manner(p<0.05). In comparison, AFB1 was found more toxic over AFM1 on both UC and DC. All these cytotoxic outcome might associate with the intracellular ROS generation, leading to cell membrane damage and DNA strand break. Additionally, DC was found more sensitive to aflatoxins, which might due to the alteration of enzymes during cell differentiation. The present study provided the first in vitro evidence of DNA damage of DC induced by AFB1 and AFM1.