High content analysis: A sensitive tool to detect and quantify the cytotoxic, synergistic and antagonistic effects of chemical contaminants in foods

Aflatoxin B1 (AFB1), ochratoxin A (OTA) and fumonisin B1 (FB1) are important mycotoxins in terms of<br/>human exposure via food, their toxicity and regulatory limits that exist worldwide. Mixtures of toxins can frequently be present in foods, however due to the complications of determining their combined toxicity,<br/>legal limits of exposure are determined for single compounds, based on long standing toxicological<br/>techniques. High content analysis (HCA) may be a useful tool to determine total toxicity of complex<br/>mixtures of mycotoxins. Endpoints including cell number (CN), nuclear intensity (NI), nuclear area (NA),<br/>plasma membrane permeability (PMP), mitochondrial membrane potential (MMP) and mitochondrial<br/>mass (MM) were compared to the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium<br/>bromide (MTT) and neutral red (NR) endpoints in MDBK cells. Individual concentrations of each<br/>mycotoxin (OTA 3mg/ml, FB1 8mg/ml and AFB11.28mg/ml) revealed no cytotoxicity with MTTor NR but<br/>HCA showed significant cytotoxic effects up to 41.6% (p0.001) and 10.1% (p0.05) for OTA and AFB1,<br/>respectively. The tertiary mixture (OTA 3mg/ml, FB1 8mg/ml and AFB1 1.28mg/ml) detected up to 37.3%<br/>and 49.8% more cytotoxicity using HCA over MTT and NR, respectively. Whilst binary combinations of<br/>OTA (3mg/ml) and FB1 (8mg/ml) revealed synergistic interactions using HCA (MMP, MM, NI endpoints)<br/>not detected using MTT or NR. HCA is a highly novel and sensitive tool that could substantially help<br/>determine future regulatory limits, for single and combined toxins present in food, ensuring legislation is based on true risks to human health exposure.