Detection of radiation-induced senescence by the Debacq-Chainiaux protocol: Improvements and upgrade in the detection of positive events.

Senescent cells are blocked in the cell cycle but remain metabolically active. These cells, once engaged in the senescence process, fail to initiate DNA replication. Due to the shortening of telomeres, replicative senescence can be triggered by a DNA damage response. Moreover, cells can also be induced to senesce by DNA damage in response to elevated reactive oxygen species (ROS), activation of oncogenes, cell-cell fusion or after ionizing radiation. There are multiple experimental ways to detect senescent cells directly or indirectly. Senescence-associated cellular traits (SA ß-Gal activity, increase in cell volume and lysosome content, appearance of γ-H2AX foci, increase of ROS and oxidative damage adducts, etc.) can be identified by numerous methods of detection (flow cytometry, confocal imaging, in situ staining, etc.). Here, we improved an existing flow cytometry protocol and further developed a new one specifically tailored to ionizing radiation-induced endothelial senescence. Thus, we have upgraded the Debacq-Chainiaux protocol and added improvements in this protocol (i) to better detect positive events (ii) to offer a compatibility to simultaneously analyze various intracellular molecules including phosphorylated signaling proteins and cytokines, whether related or not to senescence processes.

RadioTransNet, Radiotherapy Translational and Preclinical Research Network: Results from the dedicated French cancer institute (INCa) call for projects.

PURPOSE: The RadioTransNet project is a French initiative structuring preclinical and translational research in radiation therapy for cancer at national level. The network's activities are organized around four chosen priorities, which are: target definition, normal tissue, combined treatments and dose modelling. The subtargets linked to these four major priorities are unlimited. They include all aspects associated with fundamental radiobiology, preclinical studies, imaging, medical physics research and transversal components clearly related to these scientific areas, such as medical oncology, radio-diagnostics, nuclear medicine and cost-effectiveness considerations. METHOD: During its first phase of activity, four workshops following the consensus conference model and based on scientific and medical state of the art in radiotherapy and radiobiology were organized on the four above-mentioned objectives to identify key points. Then a road map has been defined and served as the basis for the opening in 2022 of a dedicated call, SEQ-RTH22, proposed by the French cancer national institute (INCa). RESULTS: Four research projects submitted by RadioTransNet partners have been selected to be supported by INCa: the first by Professor Anne Laprie from Oncopole Claudius-Regaud and Inserm ToNic in Toulouse on neurocognition and health after pediatric irradiation, the second submitted by Fabien Milliat from IRSN aims to study decryption and targeting of endothelial cell-immune cells interactions to limit radiation-induced intestinal toxicity, the third project, submitted by Yolanda Prezado from institut Curie-CNRS on proton minibeam radiotherapy as a new approach to reduce toxicity, and the latest project proposed by R. de Crevoisier from centre Eugène-Marquis in Rennes on predictive multiscale models of head and neck radiotoxicity induced for optimized personalized radiation therapy. Topics of each of these projects are presented here. CONCLUSION: RadioTransNet project has been launched in 2018, supported by INCa, in order to structure and promote preclinical research in oncology radiotherapy and to favor collaboration between the actors of this research. INCa relied on RadioTransNet initiatives and activities, resulting in the opening of dedicated call for projects. Beyond its first main goals, RadioTransNet network is able to help to fund the human and technical resources necessary to conduct optimal translational and preclinical research in radiation oncology.

[Interactions between vascular endothelium and immune cells: A key control point of radiation-induced digestive lesions]. / Interactions endothélium vasculaire ­ cellules immunitaires : un point de contrôle clef des lésions digestives radio-induites.

Radiation-induced toxicity of the digestive tract is a major clinical concern as many cancer survivors have received radiotherapy for tumours of the abdominopelvic area. The coordination and orchestration of a tissue's response to stress depend not only on the phenotype of the cells that make up the tissue but also on cell-cell interactions. The digestive system, i.e., the intestine/colon/rectum, is made up of a range of different cell populations: epithelial cells, stromal cells, i.e. endothelial cells and mesenchymal lineages, immune cells and nerve cells. Moreover, each of these populations is heterogeneous and presents very significant plasticity and differentiation states. The pathogenesis of radiation-induced digestive lesions is an integrated process that involves multiple cellular compartments interacting in a complex sequence of events. Understanding all the cellular events and communication networks that contribute to the tissue's response to stress is therefore a major conceptual and methodological scientific challenge. The study of heterogeneous populations of cells in a tissue is now possible thanks to "single cell' RNA sequencing and spatial transcriptomics techniques, which enable a comprehensive study of the transcriptomic profiles of individual cells in an integrated system. In addition, the mathematical and bioinformatics tools that are now available for the large-scale analysis of data allow the inference of cell-cell communication networks. Such approaches have become possible through advances in bioinformatics algorithms for the analysis and deciphering of interaction networks. Interactions influence the tissue regeneration process through expression of various molecules, including metabolites, integrins, junction proteins, ligands, receptors and proteins secreted into the extracellular space. The vascular network is viewed as a key player in the progression of digestive lesions, which are characterised by infiltration of a range of immune cells. A better characterisation of endothelium/immune cell interactions in suitable preclinical models, as well as in humans, may help to identify some promising therapeutic targets for the prediction, prevention or treatment of digestive toxicity after radiotherapy.

Variation of 4 MV X-ray dose rate in fractionated irradiation strongly impacts biological endothelial cell response in vitro.

PURPOSE: Even though X-ray beams are widely used in medical diagnosis or radiotherapy, the comparisons of their dose rates are scarce. We have recently demonstrated in vitro (clonogenic assay, cell viability, cell cycle, senescence) and in vivo (weight follow-up of animals and bordering epithelium staining of lesion), that for a single dose of irradiation, the relative biological effectiveness (RBE) deviates from 1 (up to twofold greater severe damage at the highest dose rate depending on the assay) when increasing the dose rate of high energy X-ray beams. MATERIAL AND METHODS: To further investigate the impact of the dose rate on RBE, in this study, we performed in vitro fractionated irradiations by using the same two dose rates (0.63 and 2.5 Gy.min-1) of high-energy X-rays (both at 4 MV) on normal endothelial cells (HUVECs). We investigated the viability/mortality, characterized radiation-induced senescence by using flow cytometry and measured gene analysis deregulations on custom arrays. RESULTS: The overall results enlighten that, in fractionated irradiations when varying the dose rate of high-energy X-rays, the RBE of photons deviates from 1 (up to 2.86 for viability/mortality experiments performed 21 days postirradiation). CONCLUSION: These results strengthen the interest of multiparametric analysis approaches in providing an accurate evaluation of the outcomes of irradiated cells in support of clonogenic assays, especially when such assays are not feasible.

Tumour and normal tissue radiosensitivity.

The place of personalized treatments is highly increasing in medical and radiation oncology. During the last decades, a huge number of assays have been developed to predict responses of normal tissues and tumours. These tests have not yet been included into daily clinical practice but the recent developments of radiation oncology are paving the way of personalized strategies including the risk of tumour recurrence and normal tissue reactions. Concerning tumor radiosensitivity prediction, no test are currently used, even if the radiosensitivity index and the genome-based model for adjusting radiotherapy dose assays seem the most promising with level II of evidence. Commercial developments are under progress. Concerning normal tissue radiosensitivity prediction, single nucleotide polymorphims of prostate cancer patients and radiation-induced CD8 T-lymphocyte apoptosis breast and prostate assays are of level I of evidence. They can be proposed before the beginning of radiotherapy in order to propose personalized treatments according to both risks of tumour and normal tissue radiosensitivity. Commercial developments are also under way.

Variation of 4 MV X-ray dose rate strongly impacts biological response both in vitro and in vivo.

Whereas an RBE > 1 is described for very low-energy X-ray beams (in the range of 25-50 kV), there is a consensus that the RBE of X-rays (from 0.1 to 3 MeV) is equal to 1, whatever the energy or dose rate of the beam. Comparisons of X-ray beam dose rates are scarce even though these beams are widely used in medical diagnosis or radiotherapy. By using two dose rates (0.63 and 2.5 Gy.min-1) of high-energy X-rays on normal endothelial cells (HUVECs), we have studied the clonogenic assay, but also viability/mortality, cell cycle analysis and measured cellular senescence by flow cytometry, and have performed gene analysis on custom arrays. In order to consolidate these data, we performed localized irradiation of exteriorized small intestine at 0.63 and 2.5 Gy.min-1. Interestingly, in vivo validation has shown a significantly higher loss of weight at the higher dose when irradiating to 19 Gy a small fragment of exteriorized small intestine of C57Bl6J mice. Nevertheless, no significant differences were observed in lesioned scores between the two dose rates, while bordering epithelium staining indicated twofold greater severe damage at 2.5 Gy.min-1 compared to 0.63 Gy.min-1 at one week post-irradiation. Taken together, these experiments systematically show that the relative biological effectiveness of photons is different from 1 when varying the dose rate of high-energy X-rays. Moreover, these results strongly suggest that, in support of clonogenic assay, multiparametric analysis should be considered to provide an accurate evaluation of the outcome of irradiated cells.

JC human polyomavirus is associated to chromosomal instability in peripheral blood lymphocytes of Hodgkin's lymphoma patients and poor clinical outcome.

BACKGROUND: B cells are potential sites for latency and reactivation of the human neurotropic JC polyomavirus (JCV). We investigated JCV and Epstein-Barr virus (EBV) status in peripheral blood lymphocytes (PBL) from 74 Hodgkin's lymphoma (HL) and 91 B-cell non-Hodgkin's lymphoma (B-NHL) patients. PATIENTS AND METHODS: JCV and EBV DNA were assessed by PCR, and FISH technique was used to localize viral infection and to estimate chromosomal instability (rogue cells, 'chromosomal aberrations') throughout evolution. The influence of viral infection and chromosomal instability on freedom from progression (FFP) was investigated in HL patients. RESULTS: PCR product sequencing of PBL identified JCV in 42 (57%) circulating lymphocytes of HL patients. FISH analysis revealed that the presence of cells with a high JCV genome copy number--associated to the presence of rogue cells and 'higher frequency of chromosomal aberrations'--increased from 15% before treatment to 52% (P < 10(-5)) after. The co-activation of JCV and EBV was independent of known prognostic parameters and associated with a shorter FFP (JCV and EBV co-activation P < 0.001, rogue cells P < 0.002). CONCLUSION: In HL, JCV activation and chromosomal instability have been identified in PBL and associated with a poorer prognosis, especially in EBV+.

[Low doses of ionizing radiation and risk of cardiovascular disease: A review of epidemiological studies]. / Irradiations à faibles doses et risque de pathologie cardiovasculaire : revue des études épidémiologiques.

BACKGROUND: While cardiovascular risks associated with high level of ionizing radiation are well-established, long-term effects of low and medium levels of exposure, between 0 and 5 gray (Gy), on the cardiovascular system are debated. METHODS: Available literature was reviewed considering various populations, such as survivors of atomic bombs, nuclear workers, Chernobyl liquidators, radiologists and radiological technologists and patients exposed for medical reasons. RESULTS: A significant increased risk of cardiovascular diseases associated with low doses of ionizing radiation was observed in 13 studies among the 27 analyzed. The ischemic heart diseases risk was detailed in 16 studies and seven of them showed a significant increase. The cerebrovascular risk was significantly increased in five studies among the 12 considered. CONCLUSION: Some epidemiological and experimental data are clearly in favour of an increased cardiovascular risk associated with exposure to low doses. However, given the multifactorial origin of cardiovascular diseases and the lack of a clear pathophysiologic mechanism, epidemiological results have to be carefully interpreted. Further research should be conducted in this area.

Cooperative effect of roscovitine and irradiation targets angiogenesis and induces vascular destabilization in human breast carcinoma.

Angiogenesis is considered as an essential process for tumour development and invasion. Previously, we demonstrated that cyclin-dependent kinase inhibition by roscovitine induces a radiosensitization and a synergistic antitumoral effect in human carcinoma but its effect on the microenvironment and tumour angiogenesis remains unknown. Here, we investigated the effect of the combination roscovitine and ionizing radiation (IR) on normal cells in vitro and on tumour angiogenesis in MDA-MB 231 tumour xenografts. We observed that the combination roscovitine and IR induced a marked reduction of angiogenic hot spot and microvascular density in comparison with IR or roscovitine treatments alone. The Ang-2/Tie-2 ratio was increased in presence of reduced vascular endothelial growth factor level suggesting vessel destabilization. In vitro, no radiosensitization effect of roscovitine was found in endothelial, fibroblast, and keratinocyte cells. IR potentiated the antiproliferative effect of roscovitine without inducing apoptosis in endothelial cells. Roscovitine decreased IR-stimulated vascular endothelial growth factor secretion of MDA-MB 231 and endothelial cells. A reduction in the endothelial cells invasion and the capillary-like tube formation in Matrigel were observed following the combination roscovitine and IR. This combined treatment targets angiogenesis resulting in microvessel destabilization without inducing normal cell toxicity.

[Role of endothelium in radiation-induced normal tissue damages]. / Rôle de l'endothélium dans les dommages radio-induits aux tissus sains.

More than half of cancers are treated with radiation therapy alone or in combination with surgery and/or chemotherapy. The goal of radiation therapy is to deliver enough ionising radiation to destroy cancer cells, without exceeding the level that the surrounding healthy cells can tolerate. Unfortunately, radiation-induced normal tissue injury is still a dose limiting factor in the treatment of cancer with radiotherapy. Early and late side-effects not only limit radiation dose escalation, but might also affect the patient's quality of life. Vascular injury is one of the most common effects of radiotherapy on normal tissues. Radiation-induced fibrogenesis is characterized by an orchestrated pathological wound-healing response in which the radiation-induced endothelium dysfunction plays a critical role. Irradiated endothelial cells acquire a proinflammatory, procoagulant and prothrombotic phenotype. The knowledge of molecular mechanisms involved in endothelium dysfunction following radiation is needed to identify therapeutic targets and develop strategies to prevent and /or reduce side-effects of radiation therapy.

Inhibition of Rho kinase modulates radiation induced fibrogenic phenotype in intestinal smooth muscle cells through alteration of the cytoskeleton and connective tissue growth factor expression.

BACKGROUND: Late radiation enteritis in humans is associated with accumulation of extracellular matrix and increased connective tissue growth factor (CTGF) expression that may involve intestinal muscular layers. AIMS: We investigated the molecular pathways involved in maintenance of radiation induced fibrosis by gene profiling and postulated that alteration of the Rho pathway could be associated with radiation induced fibrogenic signals and CTGF sustained expression. PATIENTS AND METHODS: Ileal biopsies from individuals with and without radiation enteritis were analysed by cDNA array, and primary cultures of intestinal smooth muscle cells were established. Then, the effect of pharmacological inhibition of p160 Rho kinase, using Y-27632, was studied by real time reverse transcription-polymerase chain reaction, western blot, and electrophoretic mobility shift assay. RESULTS: Molecular profile analysis of late radiation enteritis showed alterations in expression of genes coding for the Rho proteins. To investigate further the involvement of the Rho pathway in intestinal radiation induced fibrosis, primary intestinal smooth muscle cells were isolated from radiation enteritis. They retained their fibrogenic differentiation in vitro, exhibited a typical cytoskeletal network, a high constitutive CTGF level, increased collagen secretory capacity, and altered expression of genes coding for the Rho family. Rho kinase blockade induced a simultaneous decrease in the number of actin stress fibres, alpha smooth muscle actin, and heat shock protein 27 levels. It also decreased CTGF levels, probably through nuclear factor kappaB inhibition, and caused decreased expression of the type I collagen gene. CONCLUSION: This study is the first showing involvement of the Rho/Rho kinase pathway in radiation fibrosis and intestinal smooth muscle cell fibrogenic differentiation. It suggests that specific inhibition of Rho kinase may be a promising approach for the development of antifibrotic therapies.

Plasma lipids, lipoprotein composition and profile during induction and treatment of hepatic lipidosis in cats and the metabolic effect of one daily meal in healthy cats.

Anorexia in obese cats may result in feline hepatic lipidosis (FHL). This study was designed to determine plasma lipids and lipoprotein profiles in queens at different stages during experimental induction of FHL (lean, obese, FHL), and after 10 weeks of treatment. Results were compared with those obtained from lean queens of same age fed the same diet but at a maintenance level, once a day. Hepatic lipidosis led to an increase in plasma triacylglycerol (TG), very low density lipoprotein (VLDL) and low density lipoprotein (LDL), and an enrichment of LDL with TG and of high density lipoprotein (HDL) with cholesterol, suggesting that VLDL secretion is enhanced, VLDL and LDL catabolism is lowered, and lipoprotein exchanges are impaired in FHL. This study also showed that cholesterolaemia is increased in cats fed at a dietary rhythm of one meal per day compared to ad libitum feeding.

'In-field' and 'out-of-field' functional impairment during subacute and chronic phases of experimental radiation enteropathy in the rat.

PURPOSE: To investigate subacute and chronic functional consequences of localized irradiation of rat small intestine on exposed and shielded segments (proximal and distal). MATERIALS AND METHODS: The surgical model of a scrotal hernia was used. The ileal loop was exposed to single doses of 18, 21 or 29.6 Gy X-irradiation. Epithelial structure and transport capacity were followed 2 and 26 weeks post-exposure. RESULTS: Irradiated segments showed mucosal ulceration followed by transmural fibrosis. Transport capacity was impaired from 2 to 26 weeks. Subacute functional impairment was noticed in the proximal segment, without either morphological alteration or neutrophil influx. At 26 weeks, both proximal and distal segments showed impaired epithelial transport capacity, with neutrophil influx in the submucosa in cases of 21-Gy exposure and in the submucosa and muscularis propria after 29.6 Gy. CONCLUSIONS: Radiation enteritis was characterized by functional impairment, within as well as outside, the irradiation field. During the subacute phase, the irradiated segment may be a source of mediators which might influence intestinal function outside the site of injury via the blood stream and/or enteric nervous system. The development of an intestinal occlusion syndrome during the chronic phase might be responsible for intestinal dysfunction but it does not rule out a possible inflammatory process developing in the shielded parts of the small intestine.

Intralipid 10%: physicochemical characterization.

OBJECTIVES: Parenteral fat emulsions contain two populations of particles: artificial chylomicrons rich in triacylglycerols (TAG), and liposomes (bilayer of phospholipids [PL] enveloping an aqueous phase). Centrifugation permits isolating the liposomes in the infranatant called mesophase. The aim of the present work was to better characterize this mesophase chemically and to view the particles it contains by electron microscopy. METHODS: Electron microscopy (Philips 410) was performed after cryofracture on native 10% Intralipid, mesophase (centrifugation for 1 h at 27 000 g), and a liposome-enriched fraction (ring of density 1.010-1.030 g/l obtained after centrifuging mesophase in a KBr density gradient at 100 000 g for 24 h). The TAG and protein content of the mesophase was analyzed and the proteins partially characterized by immunodetection (Western-blot). RESULTS: This electron microscope study of 10% Intralipid gives evidence for the coexistence of artificial chylomicrons (mean diameter, 260 nm) and liposomes (43 nm), the latter being smaller than expected and containing 8% w/w TAG after purification. The solubilization of TAG in PL bilayers (reported to be < or = 3.1% w/w) might have been increased in parenteral emulsions by the manufacturing process or/and the high TAG/PL ratio. Minute amounts of proteins have also been detected and partially characterized using a specific antibody raised against the human 7 kDa Anionic Polypeptide Factor (APF), known to strongly interact with PL in bile. CONCLUSIONS: This work has shown that the size (mean diameter, 43 nm) of the liposomes present in 10% Intralipid is smaller than that usually assumed. Traces of hydrophobic proteins in the emulsion may account for certain allergic reactions sometimes observed in infused patients.

Plasma lipoprotein distribution in the turkey (Meleagris gallopavo).

The plasma lipoprotein profile has been determined in fasted 7-week-old male turkeys. Lipoprotein classes were subfractionated by density gradient ultracentrifugation. According to phospholipid concentration over the density gradient, an initial peak was visible in the usual LDL density range, whereas two peaks were detected in that of HDL. As density increased, the lipid composition of particles showed an increase in cholesteryl esters and decrease in triglycerides. VLDL were recovered in the first fraction (d<1.013) on the top of the gradient and IDL in fractions 2-5 (d=1.013-1.028 g/ml). The LDL and HDL populations in the density range 1.028-1.090 (fractions 6-12) differ from that found in the other bird species analyzed under the same experimental conditions. LDL predominated in fractions 6-8 with mostly beta-motility and apoB100 as the major protein component. HDL predominated in fractions 10-12 (d=1.055-1.090 g/ml) and corresponded to the first HDL peak (HDL-(A)), with mostly alpha-mobility and apoA-I as the major protein component. Both LDL- and HDL-like particle populations were present in fractions 6-12, making the separation between the two classes of lipoproteins difficult. The second peak in the HDL density range (HDL-(B), d=1.076-1.146 g/ml) contained only HDL-type particles above d=1.090 g/ml. This points out the specificity of the lipoprotein distribution in the turkey that is unique among animals. The density limit at d=1.048 g/ml is a good compromise for the separation of LDL from HDL; however, the presence of HDL-like particles in the LDL density range, and the existence of two, and even three HDL subclasses should be taken into account in the design of further metabolic studies.

Hamsters predisposed to sucrose-induced cholesterol gallstones (LPN strain) are more resistant to excess dietary cholesterol than hamsters that are not sensitive to cholelithiasis induction.

We compared the effects of cholesterol feeding in male hamsters from two strains with different propensities to sucrose-induced cholelithiasis; Laboratoire de Physiologie de la Nutrition (LPN) hamsters are predisposed to developing biliary cholesterol gallstones, whereas Janvier (JAN) hamsters are not. When fed a basal control diet, LPN hamsters had a lower cholesterolemia (-21%, P = 0.01) than JAN hamsters, and a higher activity of 3-hydroxy-3-methyl glutaryl coenzyme A reductase in liver (+148%, P = 0.018) and intestine (+281%, P < 0.0001). After feeding the same diet enriched with 0.3% cholesterol for 5 wk, cholesterolemia increased more dramatically in JAN hamsters (+235%, P < 0.001) than in LPN hamsters (+108%, P < 0.001), as did the liver concentration of cholesterol, which reached 152.30 +/- 13.00 and 44.41 +/- 9.06 micromol/g, respectively. Only JAN hamsters displayed hepatomegaly, with an increased cholesterol saturation index of the gallbladder bile (+100%, P < 0.01), due to the cholesterol challenge. In liver, cholesterol feeding reduced cholesterol 7alpha-hydroxylase activity and mRNA level, and stimulated sterol 27-hydroxylase and oxysterol 7alpha-hydroxylase activities. Hepatic levels of LDL receptor decreased by approximately 60% in both strains, whereas HDL receptor scavenger class B type 1 (SR-BI) levels were unaffected by dietary cholesterol. The greater resistance of LPN hamsters to the hypercholesterolemic diet can be explained by a lower capacity to store cholesterol in the liver and greater efficiency in reducing the activity of 3-hydroxy-3-methyl glutaryl coenzyme A reductase in response to cholesterol feeding [from 11263 to 261 pmol/(min x organ) in LPN hamsters and from 4530 to 694 pmol/(min x organ) in JAN hamsters]. These results highlight the usefulness of this two-strain model, which offers some analogy with the inverse association between the predisposition to cholelithiasis and the risk of atherosclerosis in humans.

Overexpression of SR-BI in hamsters treated with a novel ACAT inhibitor (F12511).

The effect of a novel acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor on cholesterol metabolism was studied in hamsters. Oral administration of F12511 (10 mg/kg/d) for 4 weeks produced a decrease in dietary cholesterol absorption (-18%) and in the liver concentration of esterified cholesterol (-75%), as compared with control values in untreated hamsters. While the hepatic expression of LDLr was unchanged by the treatment, that of SR-BI was increased (+142%), which suggests that the hepatic expression of SR-BI could be upregulated by a depletion of the cholesterol stores, due to ACAT inhibition. This SR-BI overexpression, however, did not induce a fall in plasma HDL-cholesterol concentration, in contrast with previous reports in transgenic mice overexpressing SR-BI at a higher extent.

Cholesterol, bile acid, and lipoprotein metabolism in two strains of hamster, one resistant, the other sensitive (LPN) to sucrose-induced cholelithiasis.

A comprehensive study of cholesterol, bile acid, and lipoprotein metabolism was undertaken in two strains of hamster that differed markedly in their response to a sucrose-rich/low fat diet. Under basal conditions, hamsters from the LPN strain differed from Janvier hamsters by a lower cholesterolemia, a higher postprandial insulinemia, a more active cholesterogenesis in both liver [3- to 4-fold higher 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMG-CoAR) activity and mRNA] and small intestine, and a lower hepatic acyl-coenzyme A:cholesterol acyltransferase activity. Cholesterol saturation indices in the gallbladder bile were similar for both strains, but the lipid concentration was 2-fold higher in LPN than in Janvier hamsters. LPN hamsters had a lower capacity to transform cholesterol into bile acids, shown by the smaller fraction of endogenous cholesterol converted into bile acids prior to fecal excretion (0.34 vs. 0.77). In LPN hamsters, the activities of cholesterol 7alpha-hydroxylase (C7OHase) and sterol 27-hydroxylase (S27OHase), the two rate-limiting enzymes of bile acid synthesis, were disproportionably lower (by 2-fold) to that of HMG-CoAR. When fed a sucrose-rich diet, plasma lipids increased, dietary cholesterol absorption improved, hepatic activities of HMG-CoA reductase, C7Ohase, and S27OHase were reduced, and intestinal S27OHase was inhibited in both strains. Despite a similar increase in the biliary hydrophobicity index due to the bile acid enrichment in chenodeoxycholic acid and derivatives, only LPN hamsters had an increased lithogenic index and developed cholesterol gallstones (75% incidence), whereas Janvier hamsters formed pigment gallstones (79% incidence). These studies indicate that LPN hamsters have a genetic predisposition to sucrose-induced cholesterol gallstone formation related to differences in cholesterol and bile acid metabolism.

Short and long-term effects of streptozotocin on dietary cholesterol absorption, plasma lipoproteins and liver lipoprotein receptors in RICO rats.

Adult male genetically hypercholesterolemic RICO rats were studied 6 and 28 days after streptozotocin (STZ) administration together with untreated RICO controls. The absorption coefficient of dietary cholesterol was determined using dual-isotope blood ratio method. Plasma lipoproteins as well as fecal neutral sterols and bile acids were analysed at both experimental times. Liver lipid parameters were measured and lipoprotein receptors (LDLr, SR-BI and HB2) were assayed by immunodetection. Six days after STZ administration, dietary cholesterol absorption was more efficient (+49%) in treated rats than in controls, and stayed higher (+68%) in the diabetic rats sacrificed at day 28. Fecal neutral sterol elimination decreased soon after STZ administration (by 35% at day 6), due to a higher cholesterol absorption coefficient, then increased to control level at day 28, due to installed diabetes-induced hyperphagia. Comparison of the lipoprotein profiles indicated that the concentration of HDL1. which is typically high in control Rico rats, fell significantly in diabetic rats at both experimental times, whereas that of HDL2 increased only at day 28. In diabetic rats, an early and strong enhancement of the hepatic expression of SR-BI appeared at day 6 (+415%) and persisted at day 28, but at a lesser extent (+85%). The expression of LDLr and HB2 was unchanged at day 6, but was significantly modified at day 28 (+140% for LDLr and -50% for HB2). These data show that streptozotocin-induced diabetes in Rico rats results in modifications of the expression of liver lipoprotein receptors which can contribute to alterations of the lipoprotein profile.

Antilithiasic effect of beta-cyclodextrin in LPN hamster: comparison with cholestyramine.

Beta-Cyclodextrin (BCD), a cyclic oligosaccharide that binds cholesterol and bile acids in vitro, has been previously shown to be an effective plasma cholesterol lowering agent in hamsters and domestic pigs. This study examined the effects of BCD as compared with cholestyramine on cholesterol and bile acid metabolism in the LPN hamster model model for cholesterol gallstones. The incidence of cholesterol gallstones was 65% in LPN hamsters fed the lithogenic diet, but decreased linearly with increasing amounts of BCD in the diet to be nil at a dose of 10% BCD. In gallbladder bile, cholesterol, phospholipid and chenodeoxycholate concentrations, hydrophobic and lithogenic indices were all significantly decreased by 10% BCD. Increases in bile acid synthesis (+110%), sterol 27-hydroxylase activity (+106%), and biliary cholate secretion (+140%) were also observed, whereas the biliary secretion of chenodeoxycholate decreased (-43%). The fecal output of chenodeoxycholate and cholate (plus derivatives) was increased by +147 and +64%, respectively, suggesting that BCD reduced the chenodeoxycholate intestinal absorption preferentially. Dietary cholestyramine decreased biliary bile acid concentration and secretion, but dramatically increased the fecal excretion of chenodeoxycholate and cholate plus their derivatives (+328 and +1940%, respectively). In contrast to BCD, the resin increased the lithogenic index in bile, induced black gallstones in 34% of hamsters, and stimulated markedly the activities of HMG-CoA reductase (+670%), sterol 27-hydroxylase (+310%), and cholesterol 7alpha-hydroxylase (+390%). Thus, beta-cyclodextrin (BCD) prevented cholesterol gallstone formation by decreasing specifically the reabsorption of chenodeoxycholate, stimulating its biosynthesis and favoring its fecal elimination. BCD had a milder effect on lipid metabolism than cholestyramine and does not predispose animals to black gallstones as cholestyramine does in this animal model.