Glutathione S-transferase expression and isoenzyme composition during cell differentiation of Caco-2 cells.

The human colon adenocarcinoma cell line Caco-2 is frequently used to study human intestinal metabolism and transport of xenobiotica. Previous studies have shown that both Caco-2 cells and human colon cells constitutively express the multigene family of detoxifying enzymes glutathione S-transferases (GSTs), particularly GST alpha and GST pi. GSTs may play a fundamental role in the molecular interplay between phase I, II enzymes and ABC-transporters. The gut fermentation product, butyrate, can modulate the potential for detoxification. The aim of this study was to investigate the basal expression of further cytosolic GSTs in Caco-2 cells during cell differentiation. In addition, a comparison was made with expression levels in MCF-7 and HepG2, two other cell types with barrier functions. Finally, the butyrate-mediated modulation of gene and protein expression was determined by real time PCR and western blot analysis. In Caco-2, gene and protein expression levels of GST alpha increased during cell differentiation. High levels of GSTO1 and GSTP1 were constantly expressed. No expression of GSTM5 and GSTT1 was detected. HepG2 expressed GSTO1 and MCF-7 GSTZ1 most intensively. No expression of GSTA5, GSTM5, or GSTP1 was detected in either cell. Incubation of Caco-2 cells with butyrate (5 mM) significantly induced GSTA1 and GSTM2 in proliferating Caco-2 cells. In differentiated cells, butyrate tended to increase GSTO1 and GSTP1. The results of this study show that a differentiation-dependent expression of GSTs in Caco-2 cells may reflect the in vivo situation and indicate the potential of butyrate to modify intestinal metabolism. GSTA1-A4 have been identified as good markers for cell differentiation. The Caco-2 cell line is a useful model for assessing the potential of food-related substances to modulate the GST expression pattern.

Two common single nucleotide polymorphisms in the gene encoding beta-carotene 15,15'-monoxygenase alter beta-carotene metabolism in female volunteers.

The key enzyme responsible for beta-carotene conversion into retinal is beta-carotene 15,15'-monoxygenase (BCMO1). Since it has been reported that the conversion of beta-carotene into vitamin A is highly variable in up to 45% of healthy individuals, we hypothesized that genetic polymorphisms in the BCMO1 gene could contribute to the occurrence of the poor converter phenotype. Here we describe the screening of the total open reading frame of the BCMO1 coding region that led to the identification of two common nonsynonymous single nucleotide polymorphisms (R267S: rs12934922; A379V: rs7501331) with variant allele frequencies of 42 and 24%, respectively. In vitro biochemical characterization of the recombinant 267S + 379V double mutant revealed a reduced catalytic activity of BCMO1 by 57% (P<0.001). Assessment of the responsiveness to a pharmacological dose of beta-carotene in female volunteers confirmed that carriers of both the 379V and 267S + 379V variant alleles had a reduced ability to convert beta-carotene, as indicated through reduced retinyl palmitate:beta-carotene ratios in the triglyceride-rich lipoprotein fraction [-32% (P=0.005) and -69% (P=0.001), respectively] and increased fasting beta-carotene concentrations [+160% (P=0.025) and +240% (P=0.041), respectively]. Our data show that there is genetic variability in beta-carotene metabolism and may provide an explanation for the molecular basis of the poor converter phenotype within the population.

Identification and characterization of a mammalian enzyme catalyzing the asymmetric oxidative cleavage of provitamin A.

In vertebrates, symmetric versus asymmetric cleavage of beta-carotene in the biosynthesis of vitamin A and its derivatives has been controversially discussed. Recently we have been able to identify a cDNA encoding a metazoan beta,beta-carotene-15,15'-dioxygenase from the fruit fly Drosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving beta-carotene to give two molecules of retinal. Mutations in the corresponding gene are known to lead to a blind, vitamin A-deficient phenotype. Orthologs of this enzyme have very recently been found also in vertebrates and molecularly characterized. Here we report the identification of a cDNA from mouse encoding a second type of carotene dioxygenase catalyzing exclusively the asymmetric oxidative cleavage of beta-carotene at the 9',10' double bond of beta-carotene and resulting in the formation of beta-apo-10'-carotenal and beta-ionone, a substance known as a floral scent from roses, for example. Besides beta-carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the beta,beta-carotene-15,15'-dioxygenases, and the two enzyme types have several conserved motifs. To establish its occurrence in different vertebrates, we then attempted and succeeded in cloning cDNAs encoding this new type of carotene dioxygenase from human and zebrafish as well. As regards their possible role, the apocarotenals formed by this enzyme may be the precursors for the biosynthesis of retinoic acid or exert unknown physiological effects. Thus, in contrast to Drosophila, in vertebrates both symmetric and asymmetric cleavage pathways exist for carotenes, revealing a greater complexity of carotene metabolism.

[Interstitial laser-induced coagulation of the prostate for therapy of benign hyperplasia]. / Die interstitielle laserinduzierte Koagulation (ILK) der Prostata zur Therapie der benignen Hyperplasie (BPH).

Interstitial laser-induced coagulation (ILC) is a new, minimally invasive, procedure for the treatment of benign prostatic hyperplasia (BPH). It is based on the placement of special light guides in the prostatic tissue, either transurethrally or perineally. The radiation source is a Nd-YAG laser, which is active for 3 or 5 min per fiber placement, respectively, with the power reduced stepwise. The coagulation necroses caused shrink during follow-up, leading to a decrease in the obstruction. In 239 patients treated in this way the mean I-PSS-Score fell from 25.4 to 8.1 points and the quality of life index from 4.1 to 1.6, the urinary peak flow rate increased from 7.7 to 16.3 ml/s, the residual urine volume fell from 151 to 32 ml, and the mean prostate volume declined from 47.4 to 32.2 ml in the first 3 months of follow up; 9.2% of the patients required further treatment within 1 year because of persistent obstruction. The complication rate was low. Statistical analysis showed no factors allowing prediction of the outcome. ILC is an effective method with few side effects and complications, and it has a wide range of indications for all BPH patients.

[Interstitial laser coagulation of benign prostatic hyperplasia]. / Die interstitielle Laserkoagulation der benignen Prostatahyperplasie.

We report on the new method of interstitial laser coagulation for the treatment of benign prostatic hyperplasia (BPH). The procedure is based on the interstitial application of Nd:YAG laser irradiation, delivered through a new light guide system. Such light applicators coagulate constant tissue volumes in a homogeneous manner, as proven by in vitro studies in different tissues, including surgically removed prostate adenoma. The extent of the coagulation is determined by laser power and irradiation time. At 5 W, for example, and during a 10-min period, this zone reached a diameter of up to 20 mm. Temperatures generated in the process were over 100 degrees C, as measured by time/space resolution. These results were confirmed by in vivo studies in canine prostates. In the course of 7 weeks, the coagulated areas formed scars with degeneration and fibrosis, accompanied by marked shrinking. Neighbouring organs were not affected. The method was successfully transferred to clinical practice. The application of the light guides to the lateral lobes was performed percutaneously from the perineum under transrectal ultrasound guidance. The median lobe was punctured transurethrally under direct vision. Twenty-seven patients with an average age of 67.7 years were treated between July 1991 and March 1992. At the time of evaluation 15 patients had a follow-up of more than 2 months. They experienced a mean increase of peak flow rate from 6.6 to 15.2 ml/s and a mean decrease of residual volume from 206 to 38 ml. This was accompanied by a marked lessening of symptoms. The average prostate weight decreased from 63 to 44 g. Sexually active patients did not experience retrograde ejaculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Fine structure of zonal changes in experimental Nd:YAG laser-induced interstitial hyperthermia.

Interstitial thermotherapy using Nd:YAG-laser induced hyperthermia is a new stereotactic method for the treatment of brain tumors in poorly accessible regions. To provide a basis for the underlying tissue alterations, we have analyzed the spatial and temporal pattern of interstitial laser hyperthermia lesions in the normal rat brain by histological, immunohistochemical, and electron microscopical methods. The acute changes corresponded to the temperature gradient surrounding the laser probe and showed a distinct zonal architecture. Membrane destruction on a cellular and subcellular level appears to be of major significance in the pathogenesis of the laser lesion. The tissue reaction followed the course known for coagulation necrosis and resulted in a well-defined defect. These results, although limited by the choice of the experimental model, may be helpful in the interpretation of images obtained in future applications of interstitial thermotherapy.

Thermal effects and histologic changes from Nd:YAG laser irradiation on normal and diseased aortic tissue using a novel angioplasty catheter with a mobile optical fiber: an in vitro assessment.

Although various laser angioplasty devices are currently being examined, thermal damage and perforation of the vessel wall remains the major acute complication of vascular laser recanalization. Consequently, the aim of this study was to investigate the thermal effects and histologic changes from laser irradiation (Nd:YAG, 1064 nm) on normal and diseased aortic tissue using a novel angioplasty device. During laser emission the coaxially guided optical fiber tip was positioned in reference to the end hole of the metallic capped probe as follows: (1) at the end hole (metal-cap position), (2) protruding 10 mm from that end hole (bare-fiber position), (3) withdrawn 5 mm into the metal cap's lumen (hot-tip position). In total, 96 laser impacts (25 joule: 5 W, 5 s, each) were produced on normal and atherosclerotic aorta in air through a 0.2-mm-core-diameter silica quartz fiber, with direct contact on the intimal surface of the target tissue by both the fiber and the metal cap and by either the fiber or the metal cap (n = 32 each). Tissue temperature was measured by means of special sensors positioned opposite the irradiated intimal spot in direct contact with the adventitial surface. Morphohistologic evaluation of lesions was performed and injury indexes were determined.

In vitro ablation of normal and diseased vascular tissue by a fiber-transmitted holmium laser.

An in vitro study was performed to test the ablative potential of a fiber-transmitted pulsed holmium laser (2.1 microns) applied to normal vascular wall, lipomatous plaques, and calcified plaques. Different fluences per pulse of 42, 80, 205, and 315 J/cm2 were used. Ablation of normal tissue and lipomatous plaques was achieved with all power settings. Effective ablation of calcified plaques, however, occurred only after a minimum fluence of 205 J/cm2, and the effect was significantly less pronounced compared to ablation of soft lesions.