Chemoprevention by Trans-Resveratrol used for the reduction of in vitro toxicity of environmental chemicals

A Tompa MD PhD DSc, Z Kocsis MS, Z Marcsek PhD, M Jakab MS, X Szende MD PhD DSc

NCPH - National Institute of Chemical Safety, Budapest, Hungary

Aim: There is a growing concern in the primary cancer prevention to use natural compounds in food, which are able to reduce the potency of environmental carcinogens exposed through occupation or life style. In our present work we studied the possible chemopreventive effect of trans-resveratrol (a naturally occurring unsaturated polyphenol antioxidant from plants with phytoestrogen character, 3,4',5-trihydroxy-trans-stilbene, RES) on two widely used chloracetamide herbicides (alachlor and acetochlor) on the cytotoxicity in estrogen receptor positive (ER+) and negative (ER-) human cell lines. In parallel, apoptosis induction and the inhibition of the cell cycle in S-phase caused by RES treatment was determined in the cell lines by morphological and flow cytometric analysis. Methods: Cell lines: MCF 7 (ER+) human mammary adenocarcinoma, HepG2 (ER+) human hepatocellular carcinoma and Vero (ER-) monkey kidney fibroblast origin. RES treatments were in the range of 0,4-200 M, alachlor and acetochlor in the range of 1 500g/ml. Cytotoxicity was measured by MTT assay, the percentage of mitoses and apoptosis was determined with hematoxilin/eosin histological staining and by tunnel reaction. Flow cytometry was performed after bromo-deoxy-uridine (BrdU) labeling in the last hour of cell cultivation. Results: Cytotoxicity of RES was dependent on dose and time course in all cell lines examined in our studies. At 48 hrs the cytotoxic dose of RES on ER+ cell lines increased to 100 M while on the ER- Vero to 50 M and the cytotoxicity increase continued at 72 hrs. Interestingly, the low doses of RES (0,4-10 M) exhibited a slight stimulatory effect on both ER+ cell lines. The cytotoxicity of alachlor and acetochlor was significantly decreased by the addition of low dose (10M) RES on Vero cells. Morphological analysis showed that low doses (<M) of RES treatment in ER+ MCF-7 cells increased the percentage of mitotic cells. At RES concentrations of 50M and above, the percentage of mitotic cells were significantly reduced and the percentage of apoptotic cells was increased. Flow cytometric analysis showed, that RES treatment decreased the ratio of S-phase only above 100 mM in both ER+ and ER- cell lines. Apoptosis and cell death were increased in ER+ cells at 50 mM after 48h of RES treatment, while this increase in ER – cells occurred earlier, already at the 24th hour of treatment. Conclusions: RES exhibits phytoestrogen activity as it increases cell proliferation in lower doses (0,4-10 M) on ER+ cells, while RES had no such effect on ER- cells. In higher doses (>50 M) RES decreases S-phase and mitotic index, and induces apoptosis and cell death, respectively.Our results suggest that the toxic effect of low dose nutritional contaminants (alachlor, acetochlor) may be reduced by the intake of low dose RES in food.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 997 (Chemoprevention).