Investigations of the causes and effects of increased arachidonic 12-lipoxygenase metabolite levels in an intestinal epithelial cell culture model
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Background. Arachidonate 12-lipoxygenase (ALOX15) introduces a molecular oxygen at carbon 12 of arachidonic acid to generate a 12-hydroperoxy eicosatetraeneoic acid (12HpETE) and related derivatives which have significant biological activity. Although the structures and enzymatic properties of ALOX15 have been elucidated, the physiological roles of ALOX15 are not yet fully understood. There is evidence that iron deficiency induces ALOX15 expression in rat gastrointestinal mucosa. Gene expression profiles using microarray analysis indicate that ALOX15 mRNA is upregulated in both dietary and genetic iron-deficiency rat models and increased ALOX15 protein and metabolite levels have been demonstrated to coincide with morphological changes in the GI epithelium. The signal(s) which induces these events are unclear and may be a direct result of iron deficiency or may be a secondary mechanism such as hypoxia induced by iron deficiency anemia. Rat GI mucosa is a complex tissue and detailed examination of possible mechanisms in the rat model is difficult. The focus of this thesis is to employ an intestinal epithelial cell (IEC-6) culture model to explore potential signals leading to the induction of ALOX15: iron deficiency and hypoxia; and to investigate the effects of the major ALOX15 metabolite, 12 hydroxy-octadecadieneoic acid (12-HETE), on IEC-6 cell proliferation and differentiation. Methods. IEC-6 cells were made iron deficient with 0.25-1.0 mmol/L deferoxamine (DFO); an iron chelator) or hypoxic in a 1% O 2 environment. ALOX15 induction was screened by analysis of cell lysate 12-HETE levels by HPLC. Cell counts and MTT assay were used to assess the effects of 12-HETE on pre-confluent IEC-6 cell proliferation. Microscopic western blue dye deposition and cellular alkaline phosphatase enzyme kinetic assay were used as markers to assess the effects of 12–HETE on post-confluent IEC-6 cell differentiation. Results. Cellular total iron assay indicated a 50% reduction in cellular iron levels after DFO treatment without a significant difference in cell growth. HPLC analysis of cell lysate and conditioned media 12-HETE levels showed no increases in levels in DFO or hypoxia treated cells. Treatment of IEC-6 cells with 0.31 nm 12-HETE resulted in significantly increased proliferation rate and differentiation as measured by alkaline phosphatase activity markers. Conclusion. Neither iron chelation nor hypoxia appear to induce production of ALOX15 metabolites in IEC-6 cells however treatment of IEC-6 cells with 12-HETE induces both increased cell proliferation and differentiation.