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194 ENTEROCOCCUS FAECALIS GENERATES CHROMOSOMAL INSTABILITY IN EUKARYOTIC CELLS: POTENTIAL ROLE FOR REDOX-ACTIVE INTESTINAL COMMENSALS IN COLORECTAL CARCINOGENESIS.
  1. X. Wang.,
  2. M. M. Huycke
  1. Department of Veterans Affairs Medical Center and University of Oklahoma Health Sciences Center, Oklahoma City, OK

Abstract

Enterococcus faecalis is a human intestinal commensal that produces extracellular superoxide and can damage colonic epithelial cell DNA. To investigate whether E. faecalis can induce chromosomal instability (CIN), the most common form of genomic instability in sporadic human colorectal cancer, we measured the mutagenic effects of E. faecalis strain OG1RF on ALN Chinese hamster ovary cells. These cells contain one copy of human chromosome 11 that encodes CD59, a cell surface marker. Following 2 hr exposure to OG1RF, ALN cells deficient in CD59 were selected by lysing CD59 expressing cells with anti-CD59 antibody and complement. OG1RF caused CIN in a dose-dependent manner (average mutant fraction per 200,000 cells [6 SD] of 35.6 6 8.6 for no bacteria, and 47 6 12.7, 66.5 6 10.3, and 84.1 6 8.4 for OG1RF at 1 3 107, 1 3 108, and 1 3 109 cfu ml21, respectively). In comparison, 2 Gray of g irradiation resulted in a mutant fraction of 89.5 6 7.5 per 200,000 cells. Loss of CD59 was verified by PCR. Large deletions in chromosome 11 (12-110 Mb) were found in 4 of 10 selected clones using SNP GeneChip mapping. The mutant fraction for ALN cells exposed to OG1RF at 1 3 109 cfu ml21 decreased 45% after addition of MnSOD (p < .0001) but not catalase (p = .875). OG1RF-induced CIN was augmented by polyunsaturated fatty acids. The mutant fraction increased 28% with 10 μM arachidonic acid (p = .009) and 50% with 10 μM docosahexaenoic acid (p = .0003). Furthermore, OG1RF-induced CIN decreased 48% with 100 μM g-carboxyethyl-hydroxychroman (g-CEHC), a metabolite of g-tocopherol, compared to no effect using 100 μM a- or g-tocopherol. g-CEHC is a potent COX-2 inhibitor. Reverse transcriptase PCR showed rapid induction of COX-2 in ALN cells following exposure to OG1RF. g-CEHC may protect against CIN through this mechanism. Finally, to assess the ability of E. faecalis to induce CIN indirectly, ALN cells were exposed to murine macrophages (RAW 264.7 cells) grown on a permeable support following infection with OG1RF (ie, no direct contact between cells). An increased mutant fraction (87.7 6 7.5 mutants per 200,000 cells) was observed. These data support the hypothesis that E. faecalis directly and indirectly induces CIN in target cells possibly through peroxidation of membrane lipids and/or induction of COX-2 to produce clastogens. These findings suggest that redox-active intestinal microbiota can contribute to the induction of CIN and thereby promote colorectal adenomas and cancer.

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