RESUMEN
Some colonic luminal molecules resulting from bacterial metabolism of alimentary or endogenous compounds are believed to exert various effects on the colonic epithelial cell physiology. We isolated surface epithelial cells and intact colonic crypts in order to test bacterial metabolites in the pig model, which is often considered relevant for extrapolation to the physiopathology of the human gastrointestinal tract. Using colonocytes isolated with EDTA, we found that the initial cell viability, estimated by the membrane integrity and oxidative capacity measurement, fell rapidly despite several experimental attempts to preserve it such as the use of a medium designed to increase the adherence of epithelial cells and of a coated extracellular matrix, the presence in the culture medium of the oxidative substrate butyrate, and the use of an inhibitor of the caspases involved in cell apoptosis. In contrast, using dispase and collagenase as proteolytic agents, we were able to obtain pig colonic crypts that maintain an excellent membrane integrity after 4 h. Using this preparation, we were able to test the presumably cytotoxic luminal compounds hydrogen sulfide, ammonia, and deoxycholic acid on colonic crypt viability. Of these, only deoxycholic acid was found to significantly alter the cellular membrane integrity. It is concluded that pig colonic crypts can be useful for the in vitro appraisal of the cytotoxic properties of luminal compounds.
Asunto(s)
Amoníaco/toxicidad , Membrana Celular/efectos de los fármacos , Colon/citología , Ácido Desoxicólico/toxicidad , Sulfuro de Hidrógeno/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colon/efectos de los fármacos , Colon/ultraestructura , Células Epiteliales/ultraestructura , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestructura , PorcinosRESUMEN
Butyrate is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibers in the colon lumen which severely affects the proliferation of colon cancer cells in in vitro experiments. Although butyrate is able to interfere with numerous cellular targets including cell cycle regulator expression, little is known about butyrate metabolism and its possible involvement in its effect upon colon carcinoma cell growth. In this study, we found that HT-29 Glc-/+ cells strongly accumulated and oxidized sodium butyrate without producing ketone bodies, nor modifying oxygen consumption nor mitochondrial ATP synthesis. HT-29 cells accumulated and oxidized sodium acetate at a higher level than butyrate. However, sodium butyrate, but not sodium acetate, reduced cell growth and increased the expression of the cell cycle effector cyclin D3 and the inhibitor of the G1/S cdk-cyclin complexes p21/WAF1/Cip1, demonstrating that butyrate metabolism downstream of acetyl-CoA synthesis is not required for the growth-restraining effect of this SCFA. Furthermore, HT-29 cells modestly incorporated the 14C-labelled carbon from sodium butyrate into cellular triacylglycerols and phospholipids. This incorporation was greatly increased when D-glucose was present in the incubation medium, corresponding to the capacity of hexose to circulate in the pentose phosphate pathway allowing NADPH synthesis required for lipogenesis. Interestingly, when HT-29 cells were cultured in the presence of sodium butyrate, their capacity to incorporate 14C-labelled sodium butyrate into triacylglycerols and phospholipids was increased more than twofold. In such experimental conditions, HT-29 cells when observed under an electronic microscope, were found to be characterized by an accumulation of lipid droplets in the cytosol. Our data strongly suggest that butyrate acts upon colon carcinoma cells upstream of acetyl-CoA synthesis. In contrast, the metabolism downstream of acetyl-CoA [i.e. oxidation in the tricarboxylic acid (TCA) cycle and lipid synthesis] likely acts as a regulator of butyrate intracellular concentration.
Asunto(s)
Acetilcoenzima A/biosíntesis , Butiratos/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Western Blotting , Butiratos/farmacología , División Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Respiración de la Célula/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Neoplasias del Colon/enzimología , Ciclina D3 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Cuerpos Cetónicos/metabolismo , Ornitina Descarboxilasa/metabolismo , Fosfolípidos/metabolismo , Acetato de Sodio/metabolismo , Acetato de Sodio/farmacología , Triglicéridos/metabolismo , Células Tumorales CultivadasRESUMEN
The genes encoding (2Fe-2S) plant-like ferredoxins were studied in the widely used cyanobacterium Synechocystis PCC6803. The fedl gene (ssI0020) coding for the most abundant ferredoxin product was found to be expressed strongly as a light-induced monocistronic transcript, whereas the other fed genes appeared to be silent (sIr1828) or moderately expressed as polycistronic transcripts regulated by either light fluence (sIr0150, negative control) or glucose availability (sII1382). fedl was found to be critical to Synechocystis PCC6803 viability in spite of sIr0150, sII1382 or flavodoxin induction, even after the addition of glucose that compensates for the loss of photosynthesis. Nevertheless, fedl could be deleted from all chromosome copies in cells propagating a fedl gene (even of heterologous origin) on a replicating plasmid. This strain was used as the host for the subsequent introduction of fedl mutant alleles propagated on a second vector. Analysis of the fedl mutant strains generated after plasmid exchange showed that the C18-C85 disulphide bridge is not central either to the tight compaction of ferredoxin I or to its reduction by photosystem I and demonstrated that the length of the Fedl carboxy terminus is important for effective PSI/FedI interactions. The plasmid-shuffling strategy presently described has general applicability for mutational analysis of essential genes in many organisms, as it is based on promiscuous plasmids.