Time-series analysis of gene expression profiles induced by nitrosamides and nitrosamines elucidates modes of action underlying their genotoxicity in human colon cells

Abstract

N-nitroso compounds (NOCs) may represent a carcinogenic risk to humans following endogenous colonic nitrosation processes. We used the colon adenocarcinoma cell line Caco-2 to investigate transcriptomic changes at three time points (1, 6, 24 h) following exposure to genotoxic concentrations of six different NOCs (two nitrosamides, four nitrosamines) with the purpose of identifying biological processes that may play a part in the carcinogenicity of these compounds. This is especially important for nitrosamide exposure where, in light of their high reactivity, important gene expression modifications may take place early in the exposure. We also analyzed NOC-induced O(6)-methylguanine adducts in relation to transcriptomics since these adducts may influence the expression of genes pivotal in NOC-associated carcinogenicity. Many modified pathways appeared related to DNA damage, cell cycle, apoptosis, growth factor signaling and differentiation, which are linked with carcinogenicity. Nitrosamides showed the strongest response at 1h of exposure, while nitrosamines had the strongest effect at 6 and 24 h. Additionally, methylation was strongly associated with processes that may contribute to the carcinogenic risk. In summary, we have found that NOC-induced gene expression changes vary over time and that many of the modified pathways and processes indicate a carcinogenic risk associated with NOC exposure.

Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells

“…For example, compounds identified as growth suppressive for the primary cells, eg, many of the fatty acids, nitrates, terpenoids, and curcuminoids, are all known to either promote differentiation or exhibit cell toxicity. 38 , 39 Glucoraphanin (#70), responsible for the bitterness of cabbage and Brussels sprouts, reduced epithelial cell numbers to near 0, consistent with its reported toxicity when consumed in large amounts. Enterolactone (#75, a microbial metabolite of flaxseed) increased cell numbers relative to that of the control, consistent with its failure to prevent intestinal adenoma formation and pose an increased cancer risk when at very high levels.…”

Novel insights into the molecular mechanisms underlying risk of colorectal cancer from smoking and red/processed meat carcinogens by modeling exposure in normal colon organoids

“…To our knowledge this is the first to study the impact of carcinogens from smoking and red/processed meats on normal epithelial cells of the colon crypt stem cell niche, the expected target cell population for the origin of neoplastic changes [ 11 ]. Previous studies aimed to determine the transcriptomic response of smoking/red meat in the colon using patients already presenting with CRC [ 9 , 10 ], while studies of the individual chemical constituents of our carcinogen cocktail have been primarily performed in CRC cell lines [ 5 – 7 , 36 , 37 ]. These are unlikely to reflect the normal response of colon crypt epithelial cells to environmental factors.…”

“…In this way, our study does not model the infrequent dosing of carcinogens likely observed through smoking or dietary intake. The selection of dose for each compound was similar to doses chosen across numerous previous studies in different cell lines [ 5 , 7 , 36 , 37 , 54 – 57 ]. However, we note that the carcinogens doses chosen for this study are likely orders of magnitude greater than would be expected to be found in the colon from tobacco smoke inhalation and/or daily consumption of red/processed meats.…”

Nitrate and nitrite in the diet: How to assess their benefit and risk for human health

“…More recent evidence reported a significant association between urinary NOC excretion and micronuclei frequency in human lymphocytes as well as gene expression changes associated with malignant cell transformation in NOC‐exposed Caco‐2 cells . Whole blood transcriptomes of 30 individuals were screened for transcription patterns correlating with NOC exposure markers and micronuclei frequency in lymphocytes .…”