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Biomedical subjects

L Richert

Publications and source records attributed to L Richert.

At least 19 recordsLinked to original sources
PubMed2007

A metabolic screening study of trichostatin A (TSA) and TSA-like histone deacetylase inhibitors in rat and human primary hepatocyte cultures.

Hydroxamic acid (HA)-based histone deacetylase (HDAC) inhibitors, with trichostatin A (TSA) as the reference compound, are potential antitumoral drugs and show promise in the creation of long-term primary cell cultures. However, their metabolic properties have barely been investigated. TSA is rapidly inactivated in rodents both in vitro and in vivo. We previously found that 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxyamide or 4-Me2N-BAVAH (compound 1) is metabolically more stable upon incubation with rat hepatocyte suspensions. In this study, we show that human hepatocytes also metabolize TSA more rapidly than compound 1 and that similar pathways are involved. Furthermore, structural analogs of compound 1 (compounds 2-9) are reported to have the same favorable metabolic properties. Removal of the dimethylamino substituent of compound 1 creates a very stable but 50% less potent inhibitor. Chain lengthening (4 to 5 carbon spacer) slightly improves both potency and metabolic stability, favoring HA reduction to hydrolysis. On the other hand, Calpha-unsaturation and spacer methylation not only reduce HDAC inhibition but also increase the rate of metabolic inactivation approximately 2-fold, mainly through HA reduction. However, in rat hepatocyte monolayer cultures, compound 1 is shown to be extensively metabolized by phase II conjugation. In conclusion, this study suggests that simple structural modifications of amide-linked TSA analogs can improve their phase I metabolic stability in both rat and human hepatocyte suspensions. Phase II glucuronidation, however, can compensate for their lower phase I metabolism in rat hepatocyte monolayers and could play a yet unidentified role in the determination of their in vivo clearance.

Animals↗
PubMed2005

Comparison of clearance predictions using primary cultures and suspensions of human hepatocytes.

Various incubation conditions of human hepatocytes were compared for their accuracy in predicting the in vivo hepatic clearance (CL(H)) of model compounds. The test compounds were the highly cleared, low protein bound naloxone (in vivo CL(H) = 25 ml min(-1) kg(-1); free fraction = 0.6), the medium clearance, highly protein bound midazolam (CL(H) = 12 ml min(-1) kg(-1); free fraction = 0.04) and the low clearance, highly protein bound bosentan (CL(H) = 3.9 ml min(-1) kg(-1); free fraction = 0.02). Each compound was tested in three 'hepatocyte systems', using resections from three donors, in the presence and absence of human serum. Those hepatocyte systems were: conventional primary cultures, freshly isolated suspensions and cryopreserved suspended hepatocytes. Except for a twofold overestimated CL(H) for bosentan from conventional primary cultures, and despite variable cryopreservation recoveries, similar predictions of CL(H) were recorded with all hepatocyte systems. Moreover, the CL(H) values obtained with cryopreserved suspended hepatocytes were similar to those obtained with freshly isolated suspensions. For midazolam and bosentan, the predicted in vivo CL(H) was markedly higher in the presence of serum, whereas serum had little influence on the scaled-up CL(H) of naloxone. In vivo, CL(H) was properly approached for naloxone and bosentan (particularly from experiments in the presence of serum), but it was strongly underestimated for midazolam (particularly in the absence of serum). Additional compounds need to be investigated to confirm the above findings as well as to assess why the clearances of some highly protein-bound compounds are still considerably underestimated.

Algorithms↗
PubMed2004

[Inpatient treatment costs of skin diseases. Diagnosis-based cost calculation in a university dermatology clinic].

BACKGROUND AND OBJECTIVE: With the advent of the DRG system, German hospitals are being forced to improve cost transparency and to uncover inefficient structures. METHODS: Diagnosis-based costs were calculated in the Department of Dermatology in the University Hospital of Freiburg for ten samples, each containing 20 cases. By using in-patient files, cost and productivity information for the fiscal year 2000 was attributed to each single case in a bottom-up-approach. The resulting total costs per case were contrasted to their remuneration under the DRG system. RESULTS: Average case costs were determined between EUR 1,339.83 and EUR 5,714.73. With 19.3% to 28.4% of total costs, nursing costs were the biggest single cost factor. Prolonging the length of stay incurred average extra costs of EUR 144.26 to EUR 199.98 per case and day. The correlation of case costs and their corresponding DRG cost weights was modest with r=0,48. There was considerable cost variance within individual DRGs. CONCLUSIONS: The diagnosis-related cost calculation reveals striking cost differences relating to specific diagnoses. This method is more suitable for cost calculations in the hospital than more general approaches.

Academic Medical Centers↗
PubMed2004

How could aortic arginase activity enhancement be involved in DOCA-salt hypertension?

This study was to examine whether the increase in aortic arginase activity observed in DOCA-salt hypertensive rats is involved in the mechanism of physiological hypertension by participating to vessel hypertrophy and/or to the impairment of endothelium-dependent relaxation to acethylcholine. We measured polyamine content and relaxation-response to acethylcholine in aortic rings isolated from control and DOCA-salt treated Sprague-Dawley rats after in vitro modification of arginase activity. Polyamine content was significantly increased in aorta from DOCA-salt hypertensive rats compared with controls. In the normotensive rats, the addition of L-valine (an inhibitor of arginase) decreased the relaxation response to acethylcholine whereas the addition of arginase increased the relaxation dependent response. On the contrary, in DOCA-salt hypertensive rats, the addition of L-valine or of arginase did not change the endothelium dependent relaxation. The results obtained suggest that the increase in aortic arginase activity in DOCA-salt hypertension could contribute to vascular hypertrophy but not to the impairment of endothelium-dependent relaxation.

Acetylcholine↗
PubMed2004

Qualitative and quantitative assessment of drug-drug interaction potential in man, based on Ki, IC50 and inhibitor concentration.

Strategies used to screen new drug entities as potential inhibitors of CYP450 enzymes are now widely used to select candidates in the drug discovery process. However, the information obtained based on IC50 values are usually more of qualitative nature. The aim of this study was to find out whether a more quantitative assessment of interaction potential could be achieved on the basis of the ratio I/Ki (I corresponds to inhibitor concentration). Ki values, in vivo data, namely plasma exposures under control condition vs in presence of inhibitors, were obtained from literature for 36 compounds. For a quantitative assessment, the following inhibitor concentrations were considered: I max and I in,max (respectively, maximum I in systemic circulation and in portal vein), I max,u and I in,max,u (respectively, maximum unbound I in systemic circulation and in portal vein). The predicted interaction was calculated as AUCinhibitor/AUCcontrol = 1 + I/Ki, where AUCcontrol and AUCinhibitor represent, respectively, the area under curve of the plasma concentration vs time profile under control conditions (ie without inhibitor) and with inhibitor. The use of I/Ki allowed a more quantitative estimation of the interaction potential. In this context, protein binding appeared to be a key parameter to be considered to avoid overestimation of DDI potential. Thus, 60% successful predictions could be achieved based on the ratio I max,u/Ki. Yet, some major deviations between in vivo DDI were obtained with this approach and the observations on the relevance of the inhibitor concentrations and the impact of binding need to be interpreted very cautiously in the absence of information on additional parameters such as fm and fh for example.

Area Under Curve↗
PubMed2003

Time course of cytochromes P450 decline during rat hepatocyte isolation and culture: effect of L-NAME.

The present work describes an isozyme-related effect of collagenase perfusion on hepatocyte microsomal cytochrome (CYP)-dependent monooxygenase activities: CYP 1A1/2-, 2B1/2-, 3A1/2- and 2E1-dependent activities in microsomes from rat hepatocytes after isolation were about 60% of that of liver microsomes, and CYP 4A1-dependent activity was equivalent to liver microsomes. In contrast, the microsomal protein content of the various CYP isoforms was not affected by hepatocyte isolation. This is in accordance with the hypothesis of CYP inactivation during the process of hepatocyte isolation by collagenase digestion. L-NAME (1 mM) was found unable to protect from the decline of CYP-dependent monooxygenase activities following hepatocyte isolation. It is possible that the decrease in glutathione peroxidase activity observed in the presence of L-NAME, namely depression of defense against peroxynitrite, could counteract the beneficial effect of L-NAME on nitric oxide synthesis inhibition. The present work also shows that L-NAME could not avoid the progressive, isoform-specific, most probably turnover-related, decline of CYP proteins and related monooxygenase activities in cultured hepatocytes. Dysregulations in the mechanisms of CYP expression in rat hepatocyte cultures, presently unknown but nitric oxide independent, thus appear to occur in cultured rat hepatocytes.

Animals↗
PubMed2002

Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers.

The structure of poly(l-lysine) (PLL)/hyaluronan (HA) polyelectrolyte multilayers formed by electrostatic self-assembly is studied by using confocal laser scanning microscopy, quartz crystal microbalance, and optical waveguide lightmode spectroscopy. These films exhibit an exponential growth regime where the thickness increases exponentially with the number of deposited layers, leading to micrometer thick films. Previously such a growth regime was suggested to result from an "in" and "out" diffusion of the PLL chains through the film during buildup, but direct evidence was lacking. The use of dye-conjugated polyelectrolytes now allows a direct three-dimensional visualization of the film construction by introducing fluorescent polyelectrolytes at different steps during the film buildup. We find that, as postulated, PLL diffuses throughout the film down into the substrate after each new PLL injection and out of the film after each PLL rinsing and further after each HA injection. As PLL reaches the outer layer of the film it interacts with the incoming HA, forming the new HA/PLL layer. The thickness of this new layer is thus proportional to the amount of PLL that diffuses out of the film during the buildup step, which explains the exponential growth regime. HA layers are also visualized but no diffusion is observed, leading to a stratified film structure. We believe that such a diffusion-based buildup mechanism explains most of the exponential-like growth processes of polyelectrolyte multilayers reported in the literature.

Chemical Phenomena↗
PubMed2002

Cryopreservation of adult human hepatocytes obtained from resected liver biopsies.

Isolated human hepatocytes have been shown to represent a valuable in vitro model to investigate the metabolism and cytotoxicity of xenobiotics. In addition, human hepatocyte transplantation and artificial liver support systems using isolated human hepatocytes are currently investigated as treatment for acute and chronic hepatic failure. In this regard, human hepatocyte banking by cryopreservation would be of great interest. In the present study, freshly isolated hepatocytes from resected liver biopsies of 28 separate donors (viability: 88 +/- 2%; plating efficiency: 79 +/- 5%) were cryopreserved using two different protocols, stepwise freezing (SF) or progressive freezing (PF), in combination (PF(+), SF(+)) or not (PF(-), SF(-)) with a 30 min preincubation in culture medium at 37 degrees C. Total recovery was higher after PF (38 +/- 3%) than after SF (12 +/- 2%). Preincubation prior to SF had no effect on plating efficiency of thawed hepatocytes (SF(-): 38 +/- 6% versus SF(+): 46 +/- 7%) while preincubation prior to PF increased plating efficiency of thawed hepatocytes (PF(-): 42 +/- 6% versus PF(+): 64 +/- 4%, p < 0.05). In attached cultured human cryopreserved/thawed hepatocytes (CH) from the PF(+) group, albumin production and glutathione content were not significantly different from those of the freshly isolated hepatocyte (FIH) cultures. Cells in CH monolayers appeared smaller than cells in FIH monolayers. In addition, the pattern of cytochrome P450- and UDP-glucuronosyl transferase-dependent isoenzyme activities and GST activity were different, suggesting a variability in the resistance to cryopreservation of the various liver hepatocyte populations. Taken all together, the results of the present study suggest that recovery of human hepatocytes after isolation prior to progressive freezing should allow human hepatocyte banking for use in pharmacotoxicology and cell therapy research purposes.

Adult↗
PubMed2002

Influence of pre-, intra- and post-operative parameters of donor liver on the outcome of isolated human hepatocytes.

The aim of the present study was to analyse, retrospectively on a large panel of patients (149), the influence of the donor liver characteristics on the outcome of human hepatocyte isolation obtained from resected liver biopsies from surgical waste after hepatectomy. Among the pre-operative parameters, the type of disease, age and sex of the patient, previous chemotherapy, alcohol or tobacco consumption did not affect the yield, viability, attachment rate and function of the isolated human hepatocytes. Pre-operative biological and anatomopathological data indicated that, while mild steatosis (</=10% steatotic hepatocytes) did also not affect the outcome of hepatocyte isolation, stronger steatosis (>10% steatotic hepatocytes) tended to decrease hepatocyte yield. Cholestasis, as assessed by gamma-glutamyl transferase serum values, significantly negatively correlated with the percentage of digested liver and the yield of viable cells. Intra-operative clamping time, that is, warm ischaemia, longer than 30 min was found to decrease both the percentage of digested liver and cell yield. Among the post-operative parameters, the percentage of digested liver decreased when biopsy weights were higher than 100 g, the use of glue tended to increase both the percentage of digested tissue and the yield of viable cells.In conclusion, human diseased livers appear to be a valuable source of isolated functional human hepatocytes. We recommend, for an optimal isolation, to use liver biopsies weighing less than 100 g, to glue the section surfaces of the biopsies and to avoid the use of moderate steatotic livers (>10% steatotic hepatocytes) and cholestatic livers, as well as livers undergoing warm ischaemia or clamping during resection due to the decrease in cell yield.

Journal Article↗
PubMed2001

Strain difference (WKY, SPRD) in the hepatic antioxidant status in rat and effect of hypertension (SHR, DOCA). Ex vivo and in vitro data.

We assessed the hepatic antioxidant status of spontaneously (SHR) and desoxicorticosterone acetate (DOCA)-induced hypertensive rats and that of respective normotensive Wistar Kyoto (WKY) and Sprague-Dawley (SPRD) rats. For this we evaluated, ex vivo in liver cytosols, reduced glutathione (GSH) content, glutathione-related enzyme (peroxidase, reductase and transferase) activities as well as the rate of lipid peroxidation in 9-11 week-old rats. The antioxidant status and the cytotoxicity of acetaminophen, a radical- and hydrogen peroxide-mediated hepatotoxic compound, were also assessed in vitro in cultured hepatocytes isolated from hypertensive (SHR, DOCA) and normotensive control (WKY, SPRD) rats. Our results suggest that a difference exists in the hepatic antioxidant status between rat strains, with GSH levels being lower (-15%) and lipid peroxidation rate higher (+30%) in WKY compared to SPRD rats. In hepatocyte cultures from WKY rats, both GSH content and catalase activity were lower (-30 and -70% respectively) compared to hepatocyte cultures from SPRD rats. This was associated with a 35% higher cytotoxicity of acetaminophen in cultured hepatocytes from WKY rats compared to that in hepatocytes from SPRD rats. Hypertension in DOCA rats (mmHg: 221+/-9 vs. 138+/-5 in control SPRD rats) was associated with decreases (about 30%) in both glutathione peroxidase (GSH-Px) and catalase activities, ex vivo in livers and in vitro in hepatocyte cultures. Hypertension in SHR (mmHg: 189+/-7 vs. 130+/-5 in control WKY rats) was also associated with decreases (about 50%) in GSH-Px activity, ex vivo in livers and in vitro in hepatocyte cultures but catalase activity was not modified. The IC50 of acetaminophen was also lower in hepatocytes from hypertensive rats compared to respective controls, which could be related to the weakened antioxidant status in hepatocytes from hypertensive rats. Our data thus suggest that hepatocyte cultures are appropriated tools in which to assess hepatotoxicity and hepatoprotection in hypertension.

Acetaminophen↗
PubMed2001

A bioassay using the human hepatoblastoma cell line HepG2 for detecting phototoxicity of furocoumarins.

We successfully evaluated the human hepatoblastoma cell line HepG2 as a model to assess phototoxicity of coumarins. Five natural furocoumarins were tested and their phototoxic activities, obtained by measuring cell viability in the presence of UV using the MTT test, were as follows: xanthotoxin (8-MOP) >> heraclenol = trichoclin = imperatorin >> peucedanin, both in growing and confluent cell cultures. This easy-to-perform, miniaturised, quantitative and sensitive method could therefore be used as a primary screening test for phototoxicity of a large number of compounds and plant extracts.

Cell Line↗
PubMed2001

Comparative phenotype and immunogenicity of freshly isolated and immortalized rat hepatocytes.

Immortalized hepatocytes are an attractive cell source for hepatocyte transplantation and gene transfer. We compared the phenotype and immunogenicity of freshly isolated (FIH) and immortalized (IMH) rat hepatocytes. Effect of culture and proinflammatory cytokines (TNF-alpha, IFN-gamma) was studied on phenotype. FIH were isolated by collagenase digestion. Two SV40 immortalized hepatocyte cell lines were tested (RH1 and P9). Immunophenotyping was performed by FACS analysis using anti-rat-specific antibodies. Immunogenicity was evaluated by a mixed lymphocyte hepatocyte reaction (MLHR). FIH suspension was an almost homogeneous parenchymal cell population with few (1-2%) CD8+ cells. FIH showed a positive staining for ICAM-1 (20-35%) and for Class I (RT1A, 30-60%) but no staining for Class II (RT1B). After 48 h of culture, the already ICAM-1-positive cells were more strongly stained and additionally 3.6% of the cells (possibly endothelial cells) were Class II positive. IMH showed a consistent expression of Class I (93-97%) and ICAM-1 (95-97%) but no expression of Class II. Culture of IMH for 48 h had no effect on Class II expression but increased ICAM-1 expression. Addition of TNF-alpha at 1000 UI/ml to cultures of FIH or IMH increased Class I and ICAM-1 expression whereas IFN-gamma (50 or 1000 UI/ml) had no evident effect. Hepatocyte immunogenicity, assessed in MLHR and appreciated by the stimulation index (SI) test/SI syngeneic control, was similar for IMH (RH1: 2.68+/-0.89; P9: 2.37+/-0.78) and FIH (2.52+/-0.18). In conclusion, despite some quantitative immunophenotypic differences, FIH and IMH induced the same proliferation rate of allogeneic T lymphocytes. Thus, immortalized hepatocytes may constitute an appropriate cellular model to study the prevention of hepatocyte rejection by gene transfer.

Animals↗
PubMed2000

Effects of model inducers on thyroxine UDP-glucuronosyl-transferase activity in vitro in rat and mouse hepatocyte cultures.

Thyroxine (T(4))-UDP-glucuronosyltransferase (UGT) activity was measured directly in cultured male Sprague-Dawley rat and OF-1 mouse hepatocyte monolayers. The activity of T(4)-UGT (pmol/min/g liver) in vitro in hepatocyte cultures was, after 24 hr in culture, equivalent to that previously measured in vivo in rat and mouse liver microsomes (Viollon-Abadie et al., 1999). A progressive decline in T(4)-UGT activity occurred over time in both rat and mouse hepatocyte cultures. Treatment of cultures with various model inducers such as phenobarbital (PB), beta-naphthoflavone (NF) and clofibric acid (CLO) induced a strong increase in T(4)-UGT activity in rat hepatocyte monolayers. In addition, and as expected from available in vivo data, treatment of rat hepatocyte cultures with NF also increased p-nitrophenol (PNP)-UGT activity and treatment with PB or CLO increased bilirubin (Bili)-UGT activity. In contrast, T(4)-UGT activity in mouse hepatocyte monolayers was not affected by the treatments, neither were PNP- and Bili- UGT activities. These in vitro data confirm our previous in vivo observations that these inducers increase rat but not mouse liver T(4)-UGT activities (Viollon-Abadie et al., 1999). The present study thus demonstrates that hepatocyte monolayers are appropriated for the evaluation and inter-species comparison of the effects of xenobiotics on T(4)-UGT activities.

Animals↗
PubMed2000

Antioxidant status, lipid peroxidation, mixed function oxidase and UDP-glucuronyl transferase activities in livers from control and DOCA-salt hypertensive male Sprague Dawley rats.

The effects of DOCA-salt hypertensive treatment on hepatic glutathione-dependent defense system, antioxidant enzymes, lipid peroxidation, mixed function oxidase and UDP-glucuronyl transferase activities were investigated in male Sprague Dawley rats. Compared with controls, DOCA-salt hypertensive rats had lower body weights (linked to liver hypertrophy). Mixed function oxidase and p-nitrophenol-UGT activities were not affected by the treatment but a significant lower rate of the glucuronoconjugation rate of bilirubin (p < 0.001) was observed in DOCA-salt hypertensive rats. While cytosolic glutathione contents and glutathione reductase activity were not affected, glutathione peroxidase (p < 0.001), glutathione transferase (p < 0.001) and catalase (p < 0.01) activities were decreased and associated with higher malondialdehyde contents (p < 0.001) in treated rats. The imbalance in liver antioxidant status (increasing generation of cellular radical species), associated with increases in lipid peroxidation, suggests that oxidative stress might be directly related to arterial hypertension in DOCA-salt treated male Sprague Dawley rats.

Animals↗
PubMed2000

Clofibric acid or diethylmaleate supplemented diet decrease blood pressure in DOCA-salt treated male Sprague Dawley rats--relation with liver antioxidant status.

The effects of 8-week diethylmaleate (DEM) and clofibric acid (CFA) supplemented diet on blood pressure, body and liver weights, liver antioxidant status and nitric oxide synthase (NOS) activity were investigated in 8-week DOCA-salt treated and untreated Sprague-Dawley male rats. It appeared that DEM and particularly CFA treatments were associated with a significant decrease in blood pressure in DOCA-salt treated rats, and an accentuation of the decreases in body weights in both diet supplemented groups. This was not associated with increases in NO production in the liver. In contrast, hepatic lipid peroxidation was significantly decreased in both DOCA-salt treated and untreated groups on DEM and particularly on CFA supplemented diet. The protective effects of CFA and DEM against hepatic cellular damage could be involved in the decreases in blood pressure in DOCA-salt treated rats, where CFA was more efficient than DEM. In CFA supplemented groups, there was a strong increase in hepatic superoxide dismutase (SOD), glutathione-peroxidase (GSH-Px), and catalase (CAT) activities and in DEM supplemented groups, increases in SOD and CAT activities and in GSH levels were observed. Our data suggest that normalization of blood pressure in DOCA-salt treated rats by CFA was due to an enhancement of the half-life of NO while DEM increased its availability.

Animals↗
PubMed2000

Increased arginase activity in aorta of mineralocorticoid-salt hypertensive rats.

The present study was designed, first to investigate aortic arginase activity during the development and the establishment of mineralocorticoid-salt (DOCA-salt) hypertension, and second, to determine the relationship between arginase activity and blood pressure by giving a protein-supplemented diet (50% casein) known to increase hepatic arginase activity. Our results showed that aortic arginase activity in established hypertension of DOCA-salt rats was higher than in normotensive rats. The protein-supplemented diet (50% casein) accelerated the development of DOCA-salt hypertension. There was a positive correlation between arginase activity and the level of blood pressure in these DOCA-salt hypertensive rats fed 50% casein but not in DOCA-salt hypertensive rats on a normal (20% casein) diet. In normotensive rats, the protein-supplemented diet decreased aortic arginase activity and produced no change in systolic blood pressure. Our data suggest that aortic arginase activity is modified in established DOCA-salt hypertension and could participate in the physiopathology of arterial hypertension.

Animals↗
PubMed2000

Peroxisome proliferators induce apoptosis and decrease DNA synthesis in hepatoma cell lines.

We examined the effects of various peroxisome proliferators (PPs) such as the hypolipidaemic agents clofibric acid (CLO), bezafibrate (BEZA), ciprofibrate (CIPRO) and nafenopin (NAFE) and the plasticizer di-(2-ethylhexyl)phthalate (DEHP) on peroxisomal enzyme activities, apoptosis and DNA synthesis in rat FaO and human HepG2 hepatoma cell lines. Both growing and confluent cultures were treated with PPs (250 microM) for 48 or 72 h. In accordance with our previous observations in PP-treated primary hepatocyte cultures of rat and human origin, the various PPs increased peroxisomal enzyme activities in rat FaO cells but not in human HepG2 cells. PPs strongly induced apoptosis in FaO cells. They did not affect TGFbeta-induced apoptosis, with the exception of DEHP and NAFE, respectively blocking and increasing induced apoptosis in confluent cultures. Moreover, PPs produced a minor, but significant, decrease in DNA synthesis in FaO cells. PPs also decreased DNA synthesis in growing HepG2 cells, and CLO, CIPRO and NAFE induced apoptosis in confluent HepG2 cultures. This is in opposition with the effects of PPs on primary hepatocyte cultures, i.e. inhibition of both spontaneous and TGFbeta-induced apoptosis and increases in DNA synthesis in rat hepatocytes, and unchanged mitosis-apoptosis balance in human hepatocytes.

Acyl-CoA Oxidase↗