Loss of peroxisomes causes oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase activity to detect cancer cells

Oxygen insensitivity of carcinoma cells and oxygen sensitivity of non-cancer cells in the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) enables detection of carcinoma cells in unfixed cell smears or cryostat sections of biopsies. The metabolic background of oxygen insensitivity is still not understood completely. In the present study, rat hepatocytes, rat hepatoma cells (FTO-2B), and human colon carcinoma cells (HT29) were used to elucidate these backgrounds. The residual activity in oxygen was 0%, 55%, and 80% in hepatocytes, hepatoma cells, and colon carcinoma cells, respectively. N-ethylmaleimide (NEM), a blocker of SH-groups, did not affect G6PD activity in both carcinoma cell types but reduced G6PD activity in hepatocytes by 40%. Ultrastructural localization of G6PD activity was exclusively in the cytoplasm of carcinoma cells, but in hepatocytes both in cytoplasm and peroxisomes. NEM abolished peroxisomal G6PD activity only. Histochemical assay of catalase activity demonstrated absence of peroxisomes in both carcinoma cell lines. it is concluded that absence of SH-sensitive G6PD activity in peroxisomes in cancer cells is responsible for the oxygen-insensitivity phenomeno

In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells

Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processe

Visualization of early events in tumor formation of eGFP-transfected rat colon cancer cells in liver

Colon cancer preferentially metastasizes to the liver. To determine cellular backgrounds of this preference, we generated an enhanced green fluorescent protein (eGFP)-expressing rat adenocarcinoma cell line (CC531s) that forms metastases in rat liver after administration to the portal vein. Intravital videomicroscopy (IVVM) was used to visualize early events in the development of tumors in livers of live animals from the time of injection of the cancer cells up to 4 days afterward. Based on information obtained with IVVM, tissue areas were selected for further analysis using confocal laser scanning microscopy (CLSM), electron microscopy (EM), and electron tomography. It was shown that initial arrest of colon cancer cells in sinusoids of the liver was due to size restriction. Adhesion of cancer cells to endothelial cells was never found. Instead, endothelial cells retracted rapidly and interactions were observed only between cancer cells and hepatocytes. Tumors developed exclusively intravascularly during the first 4 days. In conclusion, initial steps in the classic metastatic cascade such as adhesion to endothelium and extravasation are not essential for colon cancer metastasis in live

NADPH production by the pentose phosphate pathway in the zona fasciculata of rat adrenal gland

Biosynthesis of steroid hormones in the cortex of the adrenal gland takes place in smooth endoplasmic reticulum and mitochondria and requires NADPH. Four enzymes produce NADPH: glucose-6-phosphate dehydrogenase (G6PD), the key regulatory enzyme of the pentose phosphate pathway, phosphogluconate dehydrogenase (PGD), the third enzyme of that pathway, malate dehydrogenase (MDH), and isocitrate dehydrogenase (ICDH). However, the contribution of each enzyme to NADPH production in the cortex of adrenal gland has not been established. Therefore, activity of G6PD, PGD, MDH, and ICDH was localized and quantified in rat adrenocortical tissue using metabolic mapping, image analysis, and electron microscopy. The four enzymes have similar localization patterns in adrenal gland with highest activities in the zona fasciculata of the cortex. G6PD activity was strongest, PGD, MDH, and ICDH activity was similar to 60%, 15%, and 7% of G6PD activity, respectively. The K-m value of G6PD for glucose-6-phosphate was two times higher than the K-m value of PGD for phosphogluconate. As a consequence, virtual flux rates through G6PD and PGD are largely similar. It is concluded that G6PD and PGD provide the major part of NADPH in adrenocortical cells. Their activity is localized in the cytoplasm associated with free ribosomes and membranes of the smooth endoplasmic reticulum, indicating that NADPH-demanding processes related to biosynthesis of steroid hormones take place at these sites. Complete inhibition of G6PD by androsterones suggests that there is feedback regulation of steroid hormone biosynthesis via G6P

Preservation of steatotic livers: a comparison between cold storage and machine perfusion preservation

Liver grafts are frequently discarded due to steatosis. Steatotic livers can be classified as suboptimal and deteriorate rapidly during hypothermic static preservation, often resulting in graft nonfunction. Hypothermic machine perfusion (MP) has been introduced for preservation of donor livers instead of cold storage (CS), resulting in superior preservation outcomes. The aim of this study was to compare CS and MP for preservation of the steatotic donor rat liver. Liver steatosis was induced in male Wistar rats by a choline-methionine-deficient diet. After 24 hours hypothermic CS using the University of Wisconsin solution (UW) or MP using UW-Gluconate (UW-G), liver damage (liver enzymes, perfusate flow, and hyaluronic acid clearance) and liver function (bile production, ammonia clearance, urea production, oxygen consumption, adenosine triphosphate [ATP] levels) were assessed in an isolated perfused rat liver model. Furthermore, liver biopsies were visualized by hematoxylin and eosin staining. Animals developed 30 to 60% steatosis. Livers preserved by CS sustained significantly more damage as compared to MP. Bile production, ammonia clearance, urea production, oxygen consumption, and ATP levels were significantly higher after MP as compared to CS. These results were confirmed by histology. In conclusion, MP improves preservation results of the steatotic rat liver, as compared to C

Kupffer cells and not liver sinusoidal endothelial cells prevent lentiviral transduction of hepatocytes

Lentiviral vectors can stably transduce dividing and nondividing cells in vivo and are best suited to long-term correction of inherited liver diseases. Intraportal administration of lentiviral vectors expressing green fluorescent protein (Lenti-GFP) in mice resulted in a higher transduction of nonparenchymal cells than hepatocytes (7.32 +/- 3.66% vs 0.22 +/- 0.08%, respectively). Therefore, various treatments were explored to increase lentiviral transduction of hepatocytes. Lenti-GFP was injected into the common bile duct, which led to transduction of biliary epithelium and hepatocytes at low efficiency. Transient removal of the sinusoidal endothelial cell layer by cyclophosphamide to increase accessibility to hepatocytes did not improve hepatocyte transduction (0.42 +/- 0.36%). Inhibition of Kupffer cell function by gadolinium chloride led to a significant decrease in GFP-positive nonparenchymal cells (2.15 +/- 3.14%) and a sevenfold increase in GFP-positive hepatocytes compared to nonpretreated mice (1.48 +/- 2.01%). These findings suggest that sinusoidal endothelial cells do not significantly limit lentiviral transduction of hepatocytes, while Kupffer cells sequester lentiviral particles thereby preventing hepatocyte transduction. Therefore, the use of agents that inhibit Kupffer cell function may be important for lentiviral vector treatment of liver diseas

Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport

Progressive familial intrahepatic cholestasis type 1 (PFIC1, Byler disease, OMIM 211600) is a severe inherited liver disease caused by mutations in ATP8B1. ATP8B1 is a member of the type 4 subfamily of P-type ATPases, which are phospholipid flippases. PFIC1 patients generally develop end-stage liver disease before the second decade of life. The disease is characterized by impaired biliary bile salt excretion, but the mechanism whereby impaired ATP8B1 function results in cholestasis is unclear. In a mouse model for PFIC1, we observed decreased resistance of the hepatocanalicular membrane to hydrophobic bile salts as evidenced by enhanced biliary recovery of phosphatidylserine, cholesterol, and ectoenzymes. In liver specimens from PFIC1 patients, but not in those from control subjects, ectoenzyme expression at the canalicular membrane was markedly deficient. In isolated mouse livers Atp8b1 deficiency impaired the transport of hydrophobic bile salts into bile. In conclusion, our study shows that Atp8b1 deficiency causes loss of canalicular phospholipid membrane asymmetry that in turn renders the canalicular membrane less resistant toward hydrophobic bile salts. The loss of phospholipid asymmetry may subsequently impair bile salt transport and cause cholestasi