Safety assessment of a new bioactive compound, extracted from Lupinus albus seeds, through the analysis of its cytotoxic and genotoxic properties

Bischoff, Nicolaj; Schwerdtle, Tanja; Silva, Maria João

http://hdl.handle.net/10400.18/6854

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Authors

Bischoff, Nicolaj

Schwerdtle, Tanja

Silva, Maria João

Abstract(s)

Colorectal cancer (CRC) is the third most common type of cancer worldwide, being highly metastatic and mostly resistant to anticancer treatment. Especially due to its high incidence in young people and a lack of adequate treatment CRC is imposing an increasing health risk for future generations. Patients who develop CRC often suffer from Inflammatory Bowel Diseases (IBD), such as Chron’s Disease (CD) or Ulcerative Colitis (UC), before the on-set of tumorgenesis. One of the most feared complications in CRC is the cancer metastasis from the colon into secondary tissues, which is primarily responsible for the high lethality of CRC. The induction of inflammation, as well as the metastasis formation, are strongly associated with an over-expression of a subgroup of matrixmetalloproteinases (MMP), the so-called gelatinases (MMP-2/MMP-9). Deflamin is a novel matrixmetallopoteinase-inhibitor (MMPI), extracted from the seeds of Lupinus albus (tremoço), which has shown anti-inflammatory properties in the gastrointestinal tract, and has been pointed as a promising antiinflammatory and cancer preventive agent. The mechanism of action from this plantbased protein shows a high inhibitory activity against MMP-9 and/or MMP-2. This makes deflamin a great candidate to become a valuable anti-inflammatory nutraceutical agent, as well as a powerful asset for the treatment and prevention of IBD and CRC. However, potential secondary adverse effects must be avoided and an early stage safety evaluation of its potential toxic effects on human colon cancer cells is needed. This work is aimed at contributing to the safety assessment of deflamin through the analysis of cytotoxic and genotoxic properties of the purified deflamin and a Lupinus extract in Caco-2 colon cancer cells. Furthermore, its bioavailability and transport via a polarized and differentiated cell monolayer was assessed, to get further information of possible uptake mechanisms. The cytotoxic effects were analyzed by the MTT assay, Neutral Red Uptake (NRU) assay, and monolayer integrity (TEER) following an incubation time of 24, 48 and 72 hours at a concentration range from 10-640 μg/ml. The cytotoxic assessment showed a dose- and time-dependent decrease of cell viability in Caco-2 cells that did not reach statistical significance. Genotoxicity was assessed by using the alkaline comet assay in combination with the cytokinesis-block micronucleus (CBMN) assay at the three non-cytotoxic concentrations of 20, 80 and 320 μg/ml. The evaluation of DNA damage with the alkaline comet assay did not show a dose- or time-dependent increase in the level of DNA damage. The exposure of Caco-2 cells to extract did not increase the frequency of micronuclei after 24 hours exposure to the extract. Preliminary data obtained from the CBMN assay, for the purified deflamin did show a dose-dependent increase in the number of micronuclei but further investigation to confirm these results is require.