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Objective: Purposes of this work were i) to develop an in silico model of tumor response to radiotherapy, ii) to perform an exhaustive sensitivity analysis in order to iii) propose a simplified version and iv) to predict biochemical recurrence with both the comprehensive and the reduced model. Methods: A multiscale computational model of tumor response to radiotherapy was developed. It integrated the following radiobiological mechanisms: oxygenation, including hypoxic death; division of tumor cells; VEGF diffusion driving angiogenesis; division of healthy cells and oxygen-dependent response to irradiation, considering, cycle arrest and mitotic catastrophe. A thorough sensitivity analysis using the Morris screening method was performed on 21 prostate computational tissues. Tumor control probability (TCP) curves of the comprehensive model and 15 reduced versions were compared. Logistic regression was performed to predict biochemical recurrence after radiotherapy on 76 localized prostate cancer patients using an output of the comprehensive and the reduced models. Results: No significant difference was found between the TCP curves of the comprehensive and a simplified version which only considered oxygenation, division of tumor cells and their response to irradiation. Biochemical recurrence predictions using the comprehensive and the reduced models improved those made from pre-treatment imaging parameters (AUC = 0.81 $\pm$ 0.02 and 0.82 $\pm$ 0.02 vs. 0.75 $\pm$ 0.03, respectively). Conclusion: A reduced model of tumor response to radiotherapy able to predict biochemical recurrence in prostate cancer was obtained. Significance: This reduced model may be used in the future to optimize personalized fractionation schedules.

International audience; Raman spectroscopy is an imaging technique that has been applied to assess molecular compositions of living cells to characterize cell types and states. However, owing to the diverse molecular species in cells and challenges of assigning peaks to specific molecules, it has not been clear how to interpret cellular Raman spectra. Here, we provide firm evidence that cellular Raman spectra (RS) and transcriptomic profiles of glioblastoma can be computationally connected and thus interpreted. We find that the dimensions of high-dimensional RS and transcriptomes can be reduced and connected linearly through a shared low-dimensional subspace. Accordingly, we were able to predict global gene expression profiles by applying the calculated transformation matrix to Raman spectra and vice versa. From these analyses, we extract a minimal gene expression signature associated with specific RS profiles and predictive of disease outcome.

International audience; Background - In head and neck squamous cell carcinoma (HNSCC) 30% of cN0 patients have occult metastasis. LN invasion is a major prognostic factor. Sentinel lymph node (SLN) is an option for cN0 neck management. One-step nucleic acid amplification (OSNA) used to analyze SLN in breast cancer is also a candidate to get more reliable intraoperative HNSCC lymph node (LN) staging. Objective - To compare OSNA analysis to pathological analysis in cN0 HNSCC. Materials and methods - 157 LN from 26 cN0 HNSCC patients were prospectively analyzed (6.3LN/patient). Exclusion criteria were previous surgery or radiotherapy. Each node was cut into 4 equal pieces alternatively sent to pathological analysis and OSNA technique. IHC CK19 was performed on the primary tumor biopsy and RT-qPCR of CK19, PVA and EPCAM on the LN lysate of discordant cases. Results - OSNA was able to provide intraoperative result in all patients. OSNA detected 21 metastases. There were 139 concordant LN (88.5%). There were 18 initial discordant LN (11.5%), 13 (8.3%) were OSNA positive/pathological analysis negative, 5 (3.2%) were OSNA negative/pathological analysis positive. After elimination of allocation bias, false negative rate was 1.3%, sensitivity and specificity were 90% and 95.6%, PPV and NPV were 75% and 98.5%. Conclusion - Our results suggest that OSNA should be considered to improve SNB analysis both for increasing micro metastasis diagnosis and offer extemporaneous results. Study registered under clinicaltrials.gov database number NCT02852343.

Chronic obstructive pulmonary disease (COPD) is an incurable and progressive disease. Emphysema is the principal manifestation of COPD, and the main cause of this condition is cigarette smoke (CS). Natural products have shown antioxidant and anti-inflammatory properties that can prevent acute lung inflammation and emphysema, but there are few reports in the literature regarding therapeutic approaches to emphysema. We hypothesized that supplementation with natural extracts would repair lung damage in emphysema caused by CS exposure. Mice were exposed to 60 days of CS and then treated or not with three different natural extracts (mate tea, grape and propolis) orally for additional 60 days. Histological analysis revealed significant improvements in lung histoarchitecture, with recovery of alveolar spaces in all groups treated with natural extracts. Propolis was also able to recovery alveolar septa and elastic fibers. Propolis also increased MMP-2 and decreased MMP-12 expression, favoring the process of tissue repair. Additionally, propolis recruited leukocytes, including macrophages, without ROS release. These findings led us to investigate the profile of these macrophages, and we showed that propolis could promote macrophage alternative activation, thus increasing the number of arginase-positive cells and IL-10 levels and favoring an anti-inflammatory microenvironment. We further investigated the participation of Nrf2 in lung repair, but no Nrf2 translocation to the nucleus was observed in lung cells. Proteins and enzymes related to Nrf2 were not altered, other than NQO1, which seemed to be activated by propolis in a Nrf2-independent manner. Finally, propolis downregulated IGF1 expression. In conclusion, propolis promoted lung repair in a mouse emphysema model via macrophage polarization from M1 to M2 in parallel to the downregulation of IGF1 expression in a Nrf2-independent manner. (C) 2017 Elsevier Ltd. All rights reserved.

Spermiogenesis, the ultimate stage of spermatogenesis, is a process involving autophagy. At this stage, the acrosome is generated by vesicular fusion and most of the cytoplasm disappears. Autophagy, literally "eating oneself", allowing the elimination and replacement of proteins and nonfunctional organelles, ensures the recycling of cellular constituents and is a highly conserved cellular mechanism within eukaryotic cells. The machinery of autophagy is present in the spermatozoon, regulating the vitality and mobility of the cells. The environmental and behavioral impact on autophagy and the consequences on spermatogenesis are beginning to be studied. The purpose of this review is to synthesize current knowledge about autophagy in the mature male gamete.Autophagie et spermatozoïde.La spermiogenèse, étape ultime de la spermatogenèse, est un processus qui fait intervenir des acteurs qui participe à l’autophagie. C’est en effet lors de cette étape que se forme l’acrosome par fusion vésiculaire et que disparaît la majeure partie du cytoplasme du spermatozoïde. L’autophagie (littéralement « se manger soi-même »), en permettant l’élimination et le remplacement continuel des protéines et des organites non fonctionnels, assure le recyclage des constituants de la cellule. C’est un mécanisme cellulaire très conservé au sein des cellules eucaryotes. La machinerie de l’autophagie est également présente dans les spermatozoïdes. Elle régule la vitalité de ces cellules et leur mobilité. Les conséquences environnementales et comportementales sur l’autophagie et sur la spermatogenèse commencent à être étudiées. Le but de cette revue est de synthétiser les connaissances actuelles concernant les processus d’autophagie dans le gamète mâle mature.

Knowledge of the anatomy of the male pelvic floor is important to avoid damaging the pelvic floor muscles during surgery. We set out to explore the structure and innervation of the smooth muscle (SM) of the whole pelvic floor using male fetuses. We removed en-bloc the entire pelvis of three male fetuses. The specimens were serially sectioned before being stained with Masson's trichrome and hematoxylin and eosin, and immunostained for SMs, and somatic, adrenergic, sensory and nitrergic nerve fibers. Slides were digitized for three-dimensional reconstruction. We individualized a middle compartment that contains SM cells. This compartment is in close relation with the levator ani muscle (LAM), rectum, and urethra. We describe a posterior part of the middle compartment posterior to the rectal wall and an anterior part anterior to the rectal wall. The anterior part is split into (1) a centro-levator area of SM cells localized between the right and left LAM, (2) an endo-levator area that upholsters the internal aspect of the LAM, and (3) an infra-levator area below the LAM. All these areas are innervated by autonomic nerves coming from the inferior hypogastric plexus. The core and the infra-levator area receive the cavernous nerve and nerves supplying the urethra. We thus demonstrate that these muscular structures are smooth and under autonomic influence. These findings are relevant for the pelvic surgeon, and especially the urologist, during radical prostatectomy, abdominoperineal resection and intersphincteric resection. Clin. Anat., 2019. © 2019 Wiley Periodicals, Inc.

International audience; Intrahepatic cholangiocarcinoma (ICC) is the second most common type of primary cancer in the liver. ICC is an aggressive cancer with poor prognosis and limited therapeutic strategies. The identification of new drug targets and prognostic biomarkers is an important clinical challenge for ICC. The presence of an abundant stroma is a histological hallmark of ICC. Given the well-established role of the stromal compartment in the progression of cancer diseases, we hypothesized that relevant biomarkers could be identified by analyzing the stroma of ICC. By combining laser capture microdissection and gene expression profiling, we demonstrate that ICC stromal cells exhibit dramatic genomic changes. We identified a signature of 1,073 nonredundant genes that significantly discriminate the tumor stroma from nontumor fibrous tissue. Functional analysis of differentially expressed genes demonstrated that up-regulated genes in the stroma of ICC were related to cell cycle, extracellular matrix, and transforming growth factor beta (TGFβ) pathways. Tissue microarray analysis using an independent cohort of 40 ICC patients validated at a protein level the increased expression of collagen 4A1/COL4A1, laminin gamma 2/LAMC2, osteopontin/SPP1, KIAA0101, and TGFβ2 genes in the stroma of ICC. Statistical analysis of clinical and pathological features demonstrated that the expression of osteopontin, TGFβ2, and laminin in the stroma of ICC was significantly correlated with overall patient survival. More important, multivariate analysis demonstrated that the stromal expression of osteopontin was an independent prognostic marker for overall and disease-free survival. CONCLUSION: The study identifies clinically relevant genomic alterations in the stroma of ICC, including candidate biomarkers for prognosis, supporting the idea that tumor stroma is an important factor for ICC onset and progression.

International audience; Registration of histopathology volumes to Magnetic Resonance Images(MRI) is a crucial step for finding correlations in Prostate Cancer (PCa) and assessing tumor agressivity. This paper proposes a two-stage framework aimed at registering both modalities. Firstly, Speeded-Up Robust Features (SURF) algorithm and a context-based search is used to automatically determine slice correspondences between MRI and histology volumes. This step initializes a multimodal nonrigid registration strategy, which allows to propagate histology slices to MRI. Evaluation was performed on 5 prospective studies using a slice index score and landmark distances. With respect to a manual ground truth, the first stage of the framework exhibited an average error of 1,54 slice index and 3,51 mm in the prostate specimen. The reconstruction of a three-dimensional Whole-Mount Histology (WMH) shows promising results aimed to perform later PCa pattern detection and staging. © 2016 IEEE.

Clinical studies suggest that moderate alcohol consumption can have beneficial effects, in particular regarding cardiovascular events, insulin resistance, and type 2 diabetes. In this study, lean and obese diabetic ob/ob mice were submitted or not to chronic ethanol intake via the drinking water for 6 months, which was associated with moderate levels of plasma ethanol. Plasma levels of alanine aminotransferase and aspartate aminotransferase were not increased by alcohol intake. Ethanol consumption progressively reduced the gain of body weight in ob/ob mice, but not in lean mice, and this was observed despite higher calorie intake. Increased plasma free fatty acids and glycerol in ethanol-treated ob/ob mice suggested peripheral lipolysis. Glycemia and insulinemia were significantly reduced, whereas adiponectinemia was increased in ethanol-treated ob/ob mice. Liver weight and triglycerides were significantly decreased in ethanol-treated ob/ob mice, and this was associated with less microvesicular steatosis. Hepatic levels of AMP-activated protein kinase and the phosphorylated form of acetyl-CoA carboxylase were higher in ethanol-treated ob/ob mice, suggesting better fatty acid oxidation. However, hepatic mRNA expression of several lipogenic genes was not reduced by ethanol consumption. Finally, mild oxidative stress was noticed in the liver of ethanol-treated mice, regardless of their genotype. Hence, our data are in keeping with clinical studies suggesting that moderate ethanol intake can have beneficial effects on type 2 diabetes and insulin sensitivity, at least in part through increased levels of plasma adiponectin. However, further studies are needed to determine whether long-term drinking of light-to-moderate amounts of ethanol is safe for the liver.

International audience; Multi-parametric magnetic resonance imaging (mMRI) is the standard exam for prostate cancer diagnosis, staging and risk assessment in current clinical routine. Correlating mMRI in-vivo observations with biological findings from radical prostatectomy specimen would improve the optimal therapy selection. Thus, we proposed a method for reconstructing and registering the prostate whole-mount histology (WMH) to the MRI, considering a thin slicing of the prostatectomy specimen. The method was evaluated on 3 patients, included in a prospective study, for which hematein-eosin-safran and immunohistochemistry stainings were performed. The registration error was assessed by measuring the Euclidean distance between landmarks, previously identified by an expert on both mMRI and histological slices. The mean error was 4.90 +/- 1.34 mm. Our method demonstrated promising results for registering prostate WMH to in-vivo mMRI, thus allowing for spatial accurate correlation between radiologic observations and biological information

International audience; Non-alcoholic fatty liver disease is associated with obesity, diabetes, and metabolic syndrome. The detection of systemic metabolic changes associated with alterations in the liver status during non-alcoholic fatty liver disease could improve patient follow-up. The aim of the present study was to evaluate the potential of mid-infrared fibre evanescent wave spectroscopy as a minimum-invasive method for evaluating the liver status during non-alcoholic fatty liver disease. Seventy-five mice were subjected to a control, high-fat or high-fat–high carbohydrate diets. We analysed the serum biochemical parameters and mRNA levels of hepatic genes by quantitative RT-PCR. Steatosis was quantified by image analysis. The mid-infrared spectra were acquired from serum, and then analysed to develop a predictive model of the steatosis level. Animals subjected to enriched diets were obese. Hepatic steatosis was found in all animals. The relationship between the spectroscopy-predicted and observed levels of steatosis, expressed as percentages of the liver biopsy area, was not linear. A transition around 10% steatosis was observed, leading us to consider two distinct predictive models (10%) based on two different sets of discriminative spectral variables. The model performance was evaluated using random cross-validation (10%). The hypothesis that additional metabolic changes occur beyond this transition was supported by the fact that it was associated with increased serum ALT levels, and Col1α1 chain mRNA levels. Our data suggest that mid-infrared spectroscopy combined with statistical analysis allows identifying serum mid-infrared signatures that reflect the liver status during non-alcoholic fatty liver disease.

International audience

The aryl hydrocarbon receptor (AhR) is involved in various processes such as cytochrome P450 (P450) 1A induction after xenobiotic exposure. It is also considered to play a major role in cell proliferation and differentiation. Recent evidences have suggested a cross-talk between AhR functions and the mitogen-activated protein kinase (MAPK) cascade. We now report that 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a specific inhibitor of MAPK kinase (MEK) MEK1/2, elicits a marked increase in CYP1A1 expression at both mRNA and protein levels associated with a significant increase of enzyme activity in primary rat hepatocytes and a human hepatoma cell line. This induction occurred independently of MEK/extracellular signal-regulated kinase (ERK) activation and in the absence of ERK1 and ERK2 expression. The effect of U0126 was mediated by its ability to transactivate xenobiotic responsive element (XRE)-driven genes, as demonstrated by transfection assays with an XRE-driven luciferase construct in the human B16A2 hepatoma cell line. CYP1A1 modulation was abolished by a cotreatment with resveratrol, an established AhR antagonist, arguing for AhR activation by U0126. Such an effect was demonstrated by direct in vitro ligand binding competition assays using rabbit liver cytosol, showing that this compound binds AhR with an EC(50) = 25 x 10(-6) M. Moreover, we demonstrated that U0126 is a substrate for several P450s including human CYP1A2, -1A1, and -1B1. We conclude that the widely used specific inhibitor of MEK/ERK, U0126, also acts as a potent AhR activator and an inducer of related genes. Such effects on the AhR may have an impact on biological functions attributed previously to MAPK inhibition.

Recent studies support the view that in addition to its effect on both phase I and phase II xenobiotic metabolizing enzymes, the synthetic chemopreventive agent oltipraz also increases the nucleotide excision repair (NER) which represents the major pathway of elimination of chemical carcinogen DNA adducts. Since most carcinogens are activated in the liver, we investigated the influence of oltipraz on NER activity of this target tissue by using two different approaches. First, we employed an assay based on the measurement of DNA repair in cisplatin-damaged plasmid DNA incubated in the presence of cell-free extracts prepared from either rat liver or human hepatoma HepG2 cells treated by oltipraz. Secondly, we analyzed the removal of aflatoxin B 1 -derived DNA adducts formed in primary human hepatocytes exposed to oltipraz after treatment with this mycotoxin. Whatever the strategy used, NER activity was not altered in liver cells. These data demonstrated that liver cells actively repair bulky DNA adducts by NER and that oltipraz does not influence their NER activity neither in vivo nor in vitro , consequently strongly suggesting that the chemopreventive agent oltipraz is acting before the initiation step of cancer development.

Background Mutations of BRAF, NRAS and c-KIT oncogenes are preferentially described in certain histological subtypes of melanoma and linked to specific histopathological features. BRAF-, MEK- and KIT-inhibitors led to improvement in overall survival of patients harbouring mutated metastatic melanoma. Objectives To assess the prevalence and types of BRAF, NRAS, c-KIT and MITF mutations in cutaneous and mucous melanoma and to correlate mutation status with clinicopathological features and outcome. Methods Clinicopathological features and mutation status of 108 samples and of 98 consecutive patients were, respectively, assessed in one retrospective and one prospective study. Clinicopathological features were correlated with mutation status and the predictive value of these mutations was studied. Results This work identified significant correlations between BRAF mutations and melanoma occurring on non-chronic sun-damaged skin and superficial spreading melanoma (P < 0.05) on one hand, and between NRAS mutations and nodular melanoma (P < 0.05) on the other hand. Younger age (P < 0.05), microscopic (P < 0.05) and macroscopic (P < 0.05) lymphatic involvement at diagnosis of primary melanoma were significantly linked to BRAF mutations. A mutated status was a positive predictive factor of a response to BRAF inhibitors (OR = 3.44). Mutated melanoma showed a significantly (P = 0.038) higher objective response rate to cytotoxic chemotherapy (26.3%) than wild-type tumours (6.7%). Conclusion Clinical and pathological characteristics of the primary melanoma differed between wild-type and BRAFor NRAS-mutated tumours. Patients with BRAF-mutated tumours were younger at diagnosis of primary melanoma. Patients carrying mutations showed better responses better to specific kinase inhibitors and interestingly also to systemic cytotoxic chemotherapy.

Rat liver epithelial cells (RLECs) isolated by trypsinization of the livers of normal 10 day old rats are largely used in co-culture with primary hepatocytes. The aim of the present study was to investigate the expression of biotransformation enzyme-encoding genes in three preparations of RLEC lines. Although no expression of cytochrome P450 1A1/2 (CYP1A1/2), CYP2B1/2, CYP2C6 or CYP3A mRNAs could be detected, we found that all of the different preparations of RLECs expressed a high level of CYP2E1 mRNA. We demonstrated the presence of the CYP2E1 apoprotein in microsomes of RLECs by immunoblot analyses, together with chlorzoxazone 6-hydroxylation, an activity known to be mainly catalyzed by CYP2E1. In addition, acetone treatment of these cells resulted in an increase in both CYP2E1 apoprotein and chlorzoxazone 6-hydroxylation activity levels. Finally, we showed the susceptibility of RLECs to N-methyl formamide- and diethylnitrosamine-induced toxicity, suggesting metabolic activation by CYP2E1. Thus, RLECs may cooperate with hepatocytes to CYP2E1-mediated metabolism in the co-culture model. In addition, transfection experiments with a CYP2E1 promoter construct, in which the proximal 539 bp containing the binding site for HNF1alpha were inserted upstream of the chloramphenicol acetyl transferase gene, demonstrated a strong induction upon co-transfection with an HNF1alpha expression plasmid. Thus, RLECs provide a useful tool for studying metabolism and cytotoxicity of CYP2E1 substrates in the absence of other expressed CYPs, and for analyzing CYP2E1 promoter function.

International audience; Although carbon tetrachloride (CCl(4))-induced acute and chronic hepatotoxicity have been extensively studied, little is known about the very early in vivo effects of this organic solvent on oxidative stress and mitochondrial function. In this study, mice were treated with CCl(4) (1.5 ml/kg ie 2.38 g/kg) and parameters related to liver damage, lipid peroxidation, stress/defense and mitochondria were studied 3 h later. Some CCl(4)-intoxicated mice were also pretreated with the cytochrome P450 2E1 inhibitor diethyldithiocarbamate or the antioxidants Trolox C and dehydroepiandrosterone. CCl(4) induced a moderate elevation of aminotransferases, swelling of centrilobular hepatocytes, lipid peroxidation, reduction of cytochrome P4502E1 mRNA levels and a massive increase in mRNA expression of heme oxygenase-1 and heat shock protein 70. Moreover, CCl(4) intoxication induced a severe decrease of mitochondrial respiratory chain complex IV activity, mitochondrial DNA depletion and damage as well as ultrastructural alterations. Whereas DDTC totally or partially prevented all these hepatic toxic events, both antioxidants protected only against liver lipid peroxidation and mitochondrial damage. Taken together, our results suggest that lipid peroxidation is primarily implicated in CCl(4)-induced early mitochondrial injury. However, lipid peroxidation-independent mechanisms seem to be involved in CCl(4)-induced early hepatocyte swelling and changes in expression of stress/defense-related genes. Antioxidant therapy may not be an efficient strategy to block early liver damage after CCl(4) intoxication.

BackgroundIdiopathic pulmonary fibrosis is a devastating as yet untreatable disease. We demonstrated recently the predominant role of the NLRP3 inflammasome activation and IL-1β expression in the establishment of pulmonary inflammation and fibrosis in mice.MethodsThe contribution of IL-23 or IL-17 in pulmonary inflammation and fibrosis was assessed using the bleomycin model in deficient mice.ResultsWe show that bleomycin or IL-1β-induced lung injury leads to increased expression of early IL-23p19, and IL-17A or IL-17F expression. Early IL-23p19 and IL-17A, but not IL-17F, and IL-17RA signaling are required for inflammatory response to BLM as shown with gene deficient mice or mice treated with neutralizing antibodies. Using FACS analysis, we show a very early IL-17A and IL-17F expression by RORγt(+) γδ T cells and to a lesser extent by CD4αβ(+) T cells, but not by iNKT cells, 24 hrs after BLM administration. Moreover, IL-23p19 and IL-17A expressions or IL-17RA signaling are necessary to pulmonary TGF-β1 production, collagen deposition and evolution to fibrosis.ConclusionsOur findings demonstrate the existence of an early IL-1β-IL-23-IL-17A axis leading to pulmonary inflammation and fibrosis and identify innate IL-23 and IL-17A as interesting drug targets for IL-1β driven lung pathology.

Background/aims During iron overload of dietary origin, iron accumulates predominantly in periportal hepatocytes. A gradient in the basal and normal transcriptional control of genes involved in iron metabolism along the portocentral axis of liver lobules could explain this feature. Therefore, we aimed at characterizing, by quantitative RT-PCR, the expression of iron metabolism genes in adult C57BL/6 mouse hepatocytes regarding lobular localisation, with special emphasis to cell ploidy, considering its possible relationship with lobular zonation. Methods We used two methods to analyse separately periportal and perivenous liver cells: 1) a selective liver zonal destruction by digitonin prior to a classical collagenase dissociation, and 2) laser capture microdissection. We also developed a method to separate viable 4N and 8N polyploid hepatocytes by flow cytometer. Results Transcripts of ceruloplasmin, involved in iron efflux, were overexpressed in periportal areas and the result was confirmed by in situ hybridization study. By contrast, hepcidin 1, hemojuvelin, ferroportin, transferrin receptor 2, hfe and l -ferritin mRNAs were not differentially expressed according to either lobular zonation or polyploidisation level. Conclusions At variance with glutamine or urea metabolism, iron metabolism is not featured by a metabolic zonation lying only on a basal transcriptional control. The preferential periportal expression of ceruloplasmin raises the issue of its special role in iron overload disorders involving a defect in cellular iron export.

Little is known about the nucleotide excision repair (NER) pathway in the resistance of human hepatocellular carcinoma (HCC) to chemotherapeutics. We investigated expression of several NER genes in human HCC and matching non-tumor tissue (NT) and in normal liver.Expression of CSA, CSB, XPC, hHR23B, XPA, XPB, ERCC1 and p53 genes was analyzed by quantitative RT-PCR and immunoblotting in 26 HCC and 9 normal livers.The seven NER genes and p53 were frequently overexpressed in HCC compared to matched NT. XPA, XPC, hHR23B and ERCC1 mRNA levels were significantly increased (p0.05) in HCC arising in cirrhotic livers compared to non fibrotic tissue. Moreover, expression of ERCC1, XPA and XPC mRNA was significantly augmented in HCC, even more in tumors arising in cirrhotic liver. ERCC1, XPC ad XPA mRNA levels were highly correlated in NT and HCC. XPC and ERCC1 protein levels were also increased in HCC.Our findings strongly suggest that overexpression of two key genes involved in the early steps of the NER process, ERCC1 and XPC, is associated with liver fibrogenesis and cancer and could be related to the well recognized resistance of HCC to chemotherapeutics.

We investigated whether exposure to noise, in addition to its well-known potentiating effect on toluene-induced ototoxicity, may also exacerbate behavioral disturbances and brain neurochemical alterations produced by subchronic exposure to low toluene concentration. To test this hypothesis, we evaluated whether subchronic combined exposure (16 weeks, 104 h per week) to noise at 80 dB-A and toluene at 40 ppm potentiates the recently reported neurotoxic effects of subchronic exposure to 40 ppm toluene. Locomotor and rearing activities, sensitization to narcosis induced by acute toluene at high concentration, and tyrosine and tryptophan hydroxylase activities in the caudate-putamen and hippocampus were investigated in both male and female rats. Our results confirm that subchronic exposure to 40 ppm toluene significantly decreases rearing activity and leads to a sensitization to toluene-induced narcosis, as evaluated by loss of righting reflex, but fails to demonstrate any adverse effect of noise, alone or in combination with toluene. Given that toluene has addictive properties, the lack of potentiating behavioral and neurochemical effect of noise is discussed with regards to a recent study that has shown that methamphetamine neurotoxicity is potentiated by exposure to loud noise.

Expression of cytochromes P450 3A (CYP3A) has been reported in the lung, but its regulation has received little attention. In the present study, we assessed lung levels of Cyp3a mRNA, protein and activity in control mice and in mice treated with either dexamethasone (DEX), pregnenolone 16alpha-carbonitrile (PCN) or a mixture of DEX+PCN. Lung expression of the pregnane X receptor (PXR) was also investigated. Constitutive levels of Cyp3a mRNA were found in the lung from control mice by polymerase chain reaction after reverse transcription of total RNA (RT-PCR). These levels were significantly increased (2.0-fold, P

Phenobarbital (PB) has long been known as an inducer of drug-metabolizing enzymes in liver, but the molecular mechanism underlying this induction is still poorly understood. Using primary mouse hepatocyte culture, we have investigated the possible involvement of different regulatory pathways in PB action, by exposing PB-treated cells to various protein kinase/phosphatase modulators. Our results showed a negative role of the cAMP-dependent pathway, as treatment with cAMP-dependent protein kinase (PKA) activators (10 microM dibutyryl-cAMP and 50 microM forskolin) dramatically inhibited PB-induced Cyp2b9/10 mRNA accumulation, whereas PKA inhibitor potentiated the PB responsiveness of this gene. The cGMP-dependent protein kinase (PKG) seems to play a positive role as PKG inhibitor reduced the PB-induced level of Cyp2b9/10 mRNA. We also obtained two lines of evidence for the involvement of Ca2+ in modulating PB action. Firstly, measurements of intracellular Fura-2 fluorescence ratio in murine hepatocytes showed that long-term PB incubation (24 and 48 h) led to a significant increase of [Ca2+]i. Secondly, treatment with an intracellular Ca2+ chelator (BAPTA-AM) nearly completely abolished PB-induced Cyp2b9/10 expression. Ca2+ thus appeared to mediate PB action likely via Ca2+/calmodulin-dependent protein kinase II, as KN62, a specific inhibitor of this enzyme, also dramatically inhibited PB induction of the Cyp2b9/10 genes.

By screening Balb/c male mouse liver cDNA library with a rat CYP2B1 cDNA probe, we have isolated a 1795 bp cyp2b10-like clone, referred to as P16. Its sequence exhibited 34 base differences (98% similarity) with the cyp2b10 published sequence, together with a 97% identity at the amino acid sequence level. By RT-PCR and PCR analyses with Balb/c female and male liver RNA and genomic DNA, using a region showing 8 base differences between the P16 and the cyp2b10 sequences, we have confirmed the identity of our cloned cDNA, and failed in finding a PCR product exhibiting a sequence 100% identical with that of cyp2b10. Our results therefore suggest that the P16 sequence is the authentic cyp2b10 sequence. We have also isolated a partial clone, P21, which 1609 bp sequence overlapped with that of P16, except for a T→G transversion, giving rise to a premature TGA stop codon, indicating that it was derived from a pseudogene.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.

In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CYP), including CYP1A1/2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase alpha (GST alpha), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin O-deethylation activity was well maintained and ethoxyresorufin O-deethylation activity gradually decreased during co-culture time. In addition, we showed that phenobarbital and 3-methylcholanthrene treatments resulted in a significant increase of these activities. Two general patterns of accumulation of liver-specific mRNAs were observed. CYP1A1/2, CYP2B1/2, CYP3A1/2, GST alpha, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CYP2C6 and CYP2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CYP1A1/2 (13.6-fold) and GST alpha (3.9-fold) mRNA expression in 3-methylcholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3A1/2 (11.2-fold), GST alpha (9-fold), aldehyde dehydrogenase (6-fold) and epoxide hydrolase (5-fold) mRNA expression in phenobarbital-treated co-cultures. Furthermore, we demonstrated that liver-specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CYP2E1 which were expressed in both cell types during the co-culture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells. Finally, to investigate whether co-cultures could be used to study the molecular mechanisms regulating CYP transcription, we performed transfection of hepatocytes, before the establishment of the co-culture, with large CYP2B1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163-bp DNA sequence element reported to confer phenobarbital responsiveness. A 2-3-fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital-treated co-cultures transfected with the phenobarbital-responsive element construct, although phenobarbital had no effect on large CYP2B1 or CYP2B2 promoter fragments. Our results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.

We have isolated a novel cDNA sequence from a male mouse liver library. It consists of an open reading frame containing no stop codon of 1348 bp which share 98 % similarity with the cytochrome P450 2b10 followed by 95 nucleotides sharing 88 % identity with the mouse NADPH cytochrome P450 oxidoreductase. Proteins are encoded by the opposite DNA strands. Genomic amplification excludes a library artefact or a de novo recombination event. RT-PCR analyses revealed expression of this clone in the liver of male and female mice as well as in kidney, lung and intestine. This cDNA was not, however, induced by phenobarbital. Preliminary results indicate that this cDNA could span more than 2.5 kb. This study is the first description of a cytochrome P450 cDNA sequence encoding part of both a cytochrome P450 and the NADPH cytochrome P450 oxidoreductase in mammalian liver.

The aim of this study was to investigate the effect of the genetic background on the phenobarbital inducibility of cytochrome P-450 2b-9, cytochrome P-450 2b-10 and aldehyde dehydrogenase type 2 mRNAs in mice. We analysed the basal expression and the phenobarbital inducibility of both cytochrome P-450 mRNAs by semi-quantitative specific reverse transcription-PCR analyses in five inbred mouse strains (A/J, BALB/cByJ, C57BL/6J, DBA/2J and SWR/J). Male mice constitutively expressed cytochrome P-450 2b-9 and cytochrome P-450 2b-10 mRNAs, but a number of differences in their response to phenobarbital were observed. In all these mouse strains, phenobarbital induced cytochrome P-450 2b-10 mRNA whereas it could have either a positive or a negative effect on cytochrome P-450 2b-9 expression, depending on the strain and the sex of the mice. Specifically, phenobarbital increased cytochrome P-450 2b-9 expression in C57BL/6J males while it decreased it in DBA/2J mice. Interestingly, dexamethasone was able to mimic the phenobarbital effect on both cytochromes P-450 in these two strains. Aldehyde dehydrogenase type 2 mRNA was always induced by phenobarbital, except in the C57BL/6J strain. Genetic analysis revealed that the phenobarbital-inducible phenotype was either a semi-dominant or a recessive trait in F1 animals from a C57BL/6J×DBA/2J cross for the cytochrome P-450 2b-9 and the aldehyde dehydrogenase type 2 genes, respectively. This study suggests that the genetic basis for phenobarbital induction in mice depends on the target gene, and that more than one regulatory step would be involved in this response pathway.

A panel of four novel human hepatoma cell lines was isolated from a single tumor from a male individual. BC1, B16 and B16A2 lines were well differentiated, while cells of the B9 line were only poorly differentiated, being essentially negative for the functions analyzed. These cell lines have been surveyed for expression of a large set of plasma proteins, accumulation of liver-specific mRNAs and DNA-binding activity of ubiquitous and liver-enriched transcription factors. BC1 cells expressed the highest levels of albumin mRNA, whereas B16 and B16A2 cells accumulated the largest amounts of haptoglobin mRNA. In addition, B16 and B16A2 cells were unique in that they expressed CYP2E1 mRNA, a species absent from the available human liver cells, including HepG2 hepatoma cells, and 3-methylcholanthrene-inducible CYP1A2 mRNA. The activities of genes encoding transcription factors were evidenced in all four cell lines which expressed mRNAs for nuclear factor interleukin 6 and hepatocyte nuclear factor 1 (HNF) together with the DNA-binding activity of NFY and AP1 nuclear proteins. Strikingly, HNF-1 and HNF-4-like DNA-binding activities were restricted to BC1, B16 and B16A2 cells, supporting the idea of the potential role of these (or closely related) factors in the maintenance and/or in the establishment of the differentiated phenotype. B9 cells contained variant HNF1-like DNA-binding activity, similar to dedifferentiated rat hepatoma cells of the H5 line. CCAAT/enhancer-binding protein and HNF-3-like activities were found in all cell lines, although at a lower level and/or activity in B9 cells. Finally, transfection experiments of plasmids containing the whole hepatitis-B virus genome demonstrated that B16 cells, but not B9 cells, were able to support hepatitis-B virus replication and virion production, in agreement with the notion that HNF-1 activity is necessary for viral replication. We believe that the specific complement of transcription factors expressed in the differentiated BC1, B16 and B16A2 cells, and in the poorly differentiated B9 cells, will allow studies on the regulation of hepatic gene expression in these human lines, and will also aid the analysis of xenobiotic metabolism and the biology of hepatitis-B virus replication.

Rat livers were perfused and stored for 48 hr in cold University of Wisconsin solution before dissociation by the two-step collagenase method. At that time, glycogen content was significantly reduced, but no obvious changes in albumin, β-actin and aldolase B mRNAs and in glutathione levels were observed. Enzymatic perfusion yielded 280 ± 30 × 106 viable hepatocytes vs. 520 ± 40 × 106 viable hepatocytes from unstored organs. Cell viability determined by trypan blue exclusion was 74% and 90%, respectively. Hepatocytes from University of Wisconsin—preserved livers had a 29% reduced adenosine triphosphate content, but glutathione levels did not significantly differ from those found in unstored cells. When put into culture, hepatocytes formed typical monolayers of granular epithelial cells and did not exhibit alteration of their fine structure when compared with cells from unstored organs. After 24 and 48 hr, they showed variations in cytochrome P-450 content and ethoxyresorufin O-deethylase activity similar to those observed with unstored cells. By contrast, overall protein synthesis and albumin secretion rate were 40% and 30% lower, respectively. Hepatocytes from University of Wisconsin—preserved organs could be cryopreserved and further cultured as unstored cells. The University of Wisconsin solution was also used to preserve isolated hepatocytes. Viability of freshly isolated hepatocytes was decreased by only 10% after 48 hr of hypothermic liver storage when assayed by intracellular lactate dehydrogenase content. However, after 4 hr of storage, in contrast with hepatocytes preserved in L15 Leibovitz medium, the cells attached poorly to plastic and exhibited morphological alterations. These results show that rat hepatocytes are still viable, well functional and able to survive in culture after 48 hr University of Wisconsin preservation of the liver and suggest that isolated hepatocytes survive better in a culture medium than in this organ storage solution. (HEPATOLOGY 1990;12:1329–1336).

We have investigated the effects of interleukin (IL)-1 beta and IL6 on expression and phenobarbital (PB) induction of ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-deethylase (PROD) activities, as well as on mRNA levels of cytochromes P450 (CYP) 1A, 2B, 2C, 2E and 3A, in rat hepatocytes in primary culture. IL6 slightly antagonized PB-induced PROD activity. Strikingly, IL1 beta strongly inhibited basal EROD and PROD activities, and fully blocked their induction by PB in a dose-dependent fashion. Furthermore IL1 beta completely suppressed PB induction of all CYP mRNAs analyzed. Our results demonstrate that IL1 beta can suppress basal CYP activities, as well as PB-inducible expression of five CYP mRNAs in rat hepatocytes in primary culture.