Red meat intake-induced increases in fecal water genotoxicity correlate with pro-carcinogenic gene expression changes in the human colon

Red meat consumption is associated with an increased colorectal cancer (CRC) risk, which may be due to an increased endogenous formation of genotoxic N-nitroso compounds (NOCs). To assess the impact of red meat consumption on potential risk factors of CRC, we investigated the effect of a 7-day dietary red meat intervention in human subjects on endogenous NOC formation and fecal water genotoxicity in relation to genome-wide transcriptomic changes induced in colonic tissue. The intervention showed no effect on fecal NOC excretion but fecal water genotoxicity significantly increased in response to red meat intake. Colonic inflammation caused by inflammatory bowel disease, which has been suggested to stimulate endogenous nitrosation, did not influence fecal NOC excretion or fecal water genotoxicity. Transcriptomic analyses revealed that genes significantly correlating with the increase in fecal water genotoxicity were involved in biological pathways indicative of genotoxic effects, including modifications in DNA damage repair, cell cycle, and apoptosis pathways. Moreover, WNT signaling and nucleosome remodeling pathways were modulated which are implicated in human CRC development. We conclude that the gene expression changes identified in this study corroborate the genotoxic potential of diets high in red meat and point towards a potentially increased CRC risk in humans.

Colorectal cancer and the relationship between genes and the environment

Colorectal cancer (CRC) is a significant cause of morbidity and mortality in developed countries, with both genetic and environmental factors contributing to the etiology and progression of the disease. Several risk factors have been identified, including positive family history, red meat intake, smoking, and alcohol intake. Protective factors include vegetables, calcium, hormone replacement therapy, folate, nonsteroidal anti-inflammatory drugs, and physical activity. The interaction between these environmental factors, in particular diet and genes, is an area of growing interest. Currently, oncogenes, tumor suppressor genes, and mismatch repair genes are believed to play an essential role in colorectal carcinogenesis. When considering the genetics of CRC, only 10% of cases are inherited and only 2-6% can be ascribed to the highly penetrant genes, such as APC, hMLH and hMSH2. Lower penetrance genes combined with a Western-style diet contribute to the majority of sporadic CRCs. The purpose of this article is to give a brief overview of the epidemiologic studies that have been conducted and present the major findings. Here, we examine the molecular events in CRC, with particular focus on the interaction between genes and environment, and review the most current research in this area.

Exchangeability of mammalian DNA ligases between base excision repair pathways

In mammalian cells, DNA ligase IIIalpha and DNA ligase I participate in the short- and long-patch base excision repair pathways, respectively. Using an in vitro repair assay employing DNA ligase-depleted cell extracts and DNA substrates containing a single lesion repaired either through short-patch (regular abasic site) or long-patch (reduced abasic site) base excision repair pathways, we addressed the question whether DNA ligases are specific to each pathway or if they are exchangeable. We find that immunodepletion of DNA ligase I did not affect the short-patch repair pathway but blocked long-patch repair, suggesting that DNA ligase IIIa is not able to substitute DNA ligase I during long-patch repair. In contrast, immunodepletion of DNA ligase IIIa did not significantly affect either pathway. Moreover, repair of normal abasic sites in wild-type and X-ray cross-complementing gene 1 (XRCC1)-DNA ligase IIIalpha-immunodepleted cell extracts involved similar proportions of short- and long-patch repair events. This suggests that DNA ligase I was able to efficiently substitute the XRCC1-DNA ligase IIIa complex during short-patch repair.

Repair of abasic sites in DNA

Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase beta adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase delta/epsilon and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase delta/epsilon is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions. (C) 2003 Elsevier B.V. All rights reserved.

Mechanism of the Escherichia coli DNA T:G-mismatch endonuclease (Vsr protein) probed with thiophosphate-containing oligodeoxynucleotides

The mechanism of the Escherichia coli DNA T:G mismatch endonuclease (Vsr) has been investigated using oligodeoxynucleotides substituted, at the scissile phosphate, with isomeric phosphorothioates and a 3'-phosphorothiolate. Binding and kinetic data with the phosphorothioates/phosphorothiolate indicate that the two magnesium ions, which constitute essential co-factors, are required to stabilise the extra negative charge developed on the phosphate as the transition state is formed. Additionally one of the magnesium ions serves to activate the leaving group (the non-bridging 3'-oxygen atom of the scissile phosphate) during the hydrolysis reaction. Stereochemical analysis, using the R-p phosphorothioate isomer, indicates that Vsr carries out a hydrolytic reaction with inversion of stereochemistry at phosphorus, compatible with an in-line attack of water and a pentacovalent transition state with trigonal bipyramidal geometry. In conjunction with structures of Vsr bound to its products, these data allow the reconstruction of the enzyme-substrate complex and a comprehensive description of the hydrolysis mechanism. (c) 2005 Elsevier Ltd. All rights reserved.

Effects of aluminium chloride and aluminium chlorohydrate on DNA repair in MCF10A immortalised non-transformed human breast epithelial cells

Use of underarm aluminium (Al)-based antiperspirant salts may be a contributory factor in breast cancer development. At the 10th Keele meeting, Al was reported to cause anchorage-independent growth and double strand DNA breaks in MCF10A immortalised non-transformed human breast epithelial cells. We now report that exposure of MCF10A cells to Al chloride or Al chlorohydrate also compromised DNA repair systems. Longterm (19–21 weeks) exposure to Al chloride or Al chlorohydrate at a 10−4 M concentration resulted in reduced levels of BRCA1 mRNA as determined by real-time RT-PCR and BRCA1 protein as determined by Western immunoblotting. Reduced levels of mRNA for other DNA repair genes (BRCA2, CHK1, CHK2, Rad51, ATR) were also observed using real-time RT-PCR. Loss of BRCA1 or BRCA2 gene function has long been associated with inherited susceptibility to breast cancer but these results suggest that exposure to aluminium-based antiperspirant salts may also reduce levels of these key components of DNA repair in breast epithelial cells. If Al can not only damage DNA but also compromise DNA repair systems, then there is the potential for Al to impact on breast carcinogenesis.

Structural characterization of a new crystal form of the four-way Holliday junction formed by the DNA sequence d(CCGGTACCGG)2: sequence versus lattice?

DNA-strand exchange is a vital step in the recombination process, of which a key intermediate is the four-way DNA Holliday junction formed transiently in most living organisms. Here, the single-crystal structure at a resolution of 2.35 Å of such a DNA junction formed by d(CCGGTACCGG)2, which has crystallized in a more highly symmetrical packing mode to that previously observed for the same sequence, is presented. In this case, the structure is isomorphous to the mismatch sequence d(CCGGGACCGG)2, which reveals the roles of both lattice and DNA sequence in determining the junction geometry. The helices cross at the larger angle of 43.0° (the previously observed angle for this sequence was 41.4°) as a right-handed X. No metal cations were observed; the crystals were grown in the presence of only group I counter-cations.