Radical Mechanisms in Nitrosamine- and Nitrosamide-Induced Whole-Genome Gene Expression Modulations in Caco-2 Cells

Abstract

N-nitroso compounds (NOCs) may be implicated in human colon carcinogenesis, but the toxicological mechanisms involved have not been elucidated. Because it was previously demonstrated that nitrosamines and nitrosamides, representing two classes of NOC, induce distinct gene expression effects in colon cells that are particularly related to oxidative stress, we hypothesized that different radical mechanisms are involved. Using electron spin resonance spectroscopy, we investigated the radical-generating properties of genotoxic NOC concentrations in human colon adenocarcinoma cells (Caco-2). Cells were exposed to nitrosamides (N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea) or nitrosamines (N-nitrosodiethylamine, N-nitrosodimethylamine, N-nitrosopiperidine, and N-nitrosopyrrolidine). Nitrosamines caused formation of reactive oxygen species (ROS) and carbon-centered radicals, which was further stimulated in the presence of Caco-2 cells. N-methyl-N-nitrosourea exposure resulted in a small ROS signal, and formation of nitrogen-centered radicals (NCRs), also stimulated by Caco-2 cells. N-methyl-N'-nitro-N-nitrosoguanidine did not cause radical formation at genotoxic concentrations, but at increased exposure levels, both ROS and NCR formation was observed. By associating gene expression patterns with ROS formation, several cellular processes responding to nitrosamine exposure were identified, including apoptosis, cell cycle blockage, DNA repair, and oxidative stress. These findings suggest that following NOC exposure in Caco-2 cells, ROS formation plays an important role in deregulation of gene expression patterns that may be relevant for the process of chemical carcinogenesis in the human colon, in addition to the role of DNA alkylation.