Application of confocal laser scanning microscopy to detect oxidative stress in human colon cells

Introduction Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modula te gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells a s a tool to study such associations in nutritional toxicology Methods We have measured H2O2-induced oxidative stress in different colon c ell lines, in freshly isolated human colon crypts, and, for comparative pur poses, in NIH3T3 mouse embryo fibroblasts. Detection was performed by load ing the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2',7'-d ichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H2O2 and fluorescence detection with confocal laser s canning microscopy (CLSM). Using the microgel electrophoresis ("Comet") Ass ay, we also examined HT29 stem and clone 19A cells and freshly isolated pri mary colon cells for their relative sensitivity toward H2O2-induced DNA dam age and for steady-state levels of endogenous oxidative DNA damage. Results A dose-response relationship was found for the H2O2-induced dye dec omposition in NIH3T3 cells (7.8-125 mu M H2O2) whereas no effect occurred i n the human colon tumour cell lines HT29 stem and HT29 clone 19A (62-1000 m u M H2O2) Fluorescence was significantly increased at 62 mu M H2O2 in the h uman colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive toward s H2O2 than the more differentiated upper crypt cells. In contrast to the C LSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A , followed by the primary colon cells and HT29 stem cells. Conclusions Oxidative stress in colon cells leads to damage of macromolecul es which is sensitively detected in the Comet Assay The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM met hod is a sensitive technique to detect very low concentrations of H2O2-indu ced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it pr ovides the unique possibility of assessing cell specific levels of oxidativ e stress in explanted human tissues. Our results demonstrate that the actua l target cells of colon cancer induction are indeed susceptible to the oxid ative activity of H2O2.