Liver RNA Research Articles

Induced expression of drug metabolizing enzymes by preventive agents: Role of the antioxidant response element

Identifying agents that block tumor initiation is a goal of cancer prevention. The ability of a chemically varied group of agents to induce various drug metabolizing genes in livers of rats was examined. Sprague-Dawley rats were treated for 7 days with various agents in the diet or by gavage. The agents examined, which might be expected to respond via specific nuclear receptors (CAR, AhR) as well as antioxidant response elements (AREs), included Phase I/II inducers [5,6-benzoflavone (BF, 5000 mg/kg diet), diallyl sulfide (DAS, 500 mg/kg BW/day), ethoxyquin (EXO, 300 mg/kg BW/day) and phenobarbital (PB, 500 mg/kg diet)] or pure Phase II inducers [1,2-dithiol-3-thione (DTT, 500 mg/kg diet), and cyclopentadithiolthione (CPDTT, 175 mg/kg BW/day)]. Liver RNA expression was analyzed employing oligonucleotide microarrays. The agents yielded unique expression profiles. In genes with known AREs, the induction ratios (Levels Treated/Levels Controls) were: quinone oxidoreductase (BF, 8:1; DTT, 3.2:1; CPDTT, 3:1; DAS, 1.8:1; Exo, 1.7:1), glutatione transferase Pi (DTT, 36:1; CPDTT, 34:1; EXO, 8:1; DAS, 5:1; BF, 2.5:1), and aldehyde keto reductase 7A3 (AFAR) (DTT and CPDTT, 14:1; DAS, 6:1; EXO, 4:1; PB, 1.5:1). When the search included a wider variety of Phase II drug metabolizing enzymes, no clear pattern was observed. Agent induced gene expression and preventive activity in published carcinogen induced tumor models showed limited correlation; questioning whether measuring the induction of one or two genes (e.g., quinone reductase) is a surrogate for overall Phase II inducing (antioxidant) and potential anti-tumor activity.

Expression of ECM proteins fibulin-1 and -2 in acute and chronic liver disease and in cultured rat liver cells

Fibulin-2 has previously been considered as a marker to distinguish rat liver myofibroblasts from hepatic stellate cells. The function of other fibulins in acute or chronic liver damage has not yet been investigated. The aim of this study has been to evaluate the expression of fibulin-1 and -2 in models of rat liver injury and in human liver cirrhosis. Their cellular sources have also been investigated. In normal rat liver, fibulin-1 and -2 were both mainly present in the portal field. Fibulin-1-coding transcripts were detected in total RNA of normal rat liver, whereas fibulin-2 mRNA was only detected by sensitive, real-time quantitative polymerase chain reaction. In acute liver injury, the expression of fibulin-1 was significantly increased (17.23-fold after 48 h), whereas that of fibulin-2 was not modified. The expression of both fibulin-1 and -2 was increased in experimental rat liver cirrhosis (19.16- and 26.47-fold, respectively). At the cellular level, fibulin-1 was detectable in hepatocytes, “activated” hepatic stellate cells, and liver myofibroblasts (2.71-, 122.65-, and 469.48-fold over the expression in normal rat liver), whereas fibulin-2 was restricted to liver myofibroblasts and was regulated by transforming growth factor beta-1 (TGF-β1) in 2-day-old hepatocyte cultures and in liver myofibroblasts. Thus, fibulin-1 and -2 respond differentially to single and repeated damaging noxae, and their expression is differently present in liver cells. Expression of the fibulin-2 gene is regulated by TGF-β1 in liver myofibroblasts.

Effects of Bovine Growth Hormone in Infant Rats from Reduced Litters

The objective of this study was to determine the effects of growth hormone on body growth and the content of RNA, DNA and protein in liver, brain and heart muscle in rat pups from reduced nests. Suckling rats raised in litters of 4 youngs were daily injected from 3rd to 28th day of life with 1 microgram of bGH/g body weight. Our results show that GH administration evoked higher body weight gain and increase of tail length after 23rd day of life. There was significant increase of liver content of nucleic acids and protein. This GH-responsivity of pups from reduced nests might be related to differences in their metabolic profile and accelerated somatic and psychosocial development in comparison to normal youngs.

Tissue tropism of nervous necrosis virus (NNV) in Atlantic cod, Gadus morhua L., after intraperitoneal challenge with a virus isolate from diseased Atlantic halibut, Hippoglossus hippoglossus (L.)

Atlantic cod, Gadus morhua, averaging 100 g, were experimentally challenged by intraperitoneal injection of nervous necrosis virus (NNV) originating from Atlantic halibut. Cod tissues, including blood, gill, pectoral fin, barbel, ventricle, atrium, spleen, liver, lateral line (including muscle tissue), eye (retina) and brain, were sampled at day 25 and 130 and investigated by real-time RT-PCR for the presence of NNV. Relative quantifications at day 130 were calculated using the 2(-DeltaDeltaCt) method. Immunosuppression by injection of prednisolone-acetate was introduced for a 30-day period, and tissue sampled at day 180 and relative quantification estimated. No mortality or clinical signs of disease were observed in the challenged group. The challenge resulted in detection of NNV in blood, spleen, kidney, liver, heart atrium and heart ventricle at day 25, and by the end of the experiment NNV showed a clear increase in brain and retina, suggesting these to be the primary tissues for viral replication. There was no increase in the relative amount of NNV in blood, atrium, ventricle, spleen, liver and kidney. Corticosteroid implants resulted in a weak increase in virus RNA in spleen, kidney, liver and brain. These findings suggest that Atlantic cod is susceptible to infection with NNV from halibut. The observed tissue tropism patterns suggest an initial viraemic phase, followed by neurotrophy. Head-kidney is the best tissue identified for possible NNV detection by non-lethal biopsy, but detection was not possible in all injected fish.

Effects of Ovine Prolactin in Infant Rats

The objective of this study was to determine the effects of ovine prolactin (oPRL) on body growth and the content of RNA, DNA and protein in liver, brain and heart muscle in rat pups. Suckling rats were daily injected from 3rd to 28th day of life with 1 microgram of oPRL/g body weight. Our results show that oPRL administration evoked higher body weight gain up to 8th day of life (p less than 0.05). There was significant (p less than 0.05) increase of DNA liver content and significant decrease (p less than 0.001) of RNA/DNA ratio in the hepatocytes. The findings suggest that prolactin influences growth rate of rats in the early postnatal period of life.

Structure, Genetic Mapping, and Function of the Cytochrome P450 3A37 Gene in the Turkey (Meleagris gallopavo)

Cytochromes P450 (P450 for protein; CYP for gene) are a superfamily of membrane-bound hemoproteins that oxidize a large number of endogenous and exogenous compounds. Through oxidation reactions, these enzymes are often responsible for the toxic and carcinogenic effects of natural food-borne toxicants, such as the mycotoxin aflatoxin B₁ (AFB₁). Previous studies in our laboratory have shown that the extreme sensitivity of turkeys to AFB₁ is in part explained by efficient hepatic P450-mediated epoxidation to the toxic and reactive metabolite the exo-AFB₁-8,9-epoxide (AFBO). Using 3′-5′-rapid amplification of cDNA ends (RACE), we amplified CYP3A37 from turkey liver RNA, the E. coli-expressed protein which efficiently epoxidates AFB₁. Turkey CYP3A37 has an ORF of 1512 bp, and the protein is predicted to be 504 amino acids with 97% homology to chicken CYP3A37. The turkey gene is organized into 13 exons and 12 introns. A single nucleotide polymorphism in the 11th intron was used to assign CYP3A37 to turkey linkage group 10 (corresponding to chicken chromosome 14, GGA14). Because of the important role of P450s in the extreme sensitivity of turkeys to the toxic effects of AFB₁, this study will contribute to the identifying allelic variants of this important gene in poultry.

The myth of occult hepatitis C infection #

We read with interest the review by Welker and Zeuzem on occult hepatitis C virus (HCV) infection and also the reply by Carreno et al.1, 2 The latter reported in their answer to Welker and Zeuzem that HCV can replicate in patients with occult HCV infection. Because this conclusion would have substantial consequences for patients considered cured of their HCV infection, as well as for the health care system, the data must be interpreted most carefully. They reported two studies that “unconvincingly failed to detect occult HCV infection in cryptogenic hepatitis because of several concerns with the methodology employed.”3, 4 We would like to correct Carreno et al. on these two important points. In the first study published by Tanari et al., it was shown that the ability for HCV RNA detection is more sensitive in whole blood instead of peripheral blood mononuclear cells (PBMCs), due to circulation of the infectious virus in patient sera in the form of triglyceride-rich particles which is present in the whole blood and not in PBMCs.5 In the second study, we looked for the so-called “occult hepatitis C”, in a different population of patients and controls, including patients with long-term normal alanine aminotransferase level, by using an original ultrasensitive validated real-time polymerase chain reaction (PCR) method. We failed to find any occult hepatitis C.4 Moreover, we would like to underscore that for a long time, Carreno et al. reported the same results on the same cohort of patients.6 The method proposed used PBMCs from a patient chronically infected with HCV for RNA isolation and HCV RNA detection. Serial dilutions of the isolated RNA cannot be a gold standard due to the limit of detection of HCV RNA by an in-house reverse transcription PCR method. PBMCs from our patients were subjected to HCV RNA transcription-mediated amplification assays without any HCV RNA detectable using this high sensitivity assay.6 The second issue addressed is related to the concept of the presence of HCV RNA in the liver in the absence of HCV RNA detected in PBMCs. Carreno et al. suggested that negativity for HCV-RNA in PBMC does not exclude the existence of occult HCV infection because the gold standard method to identify this occult infection is by detection of viral RNA in liver cells. To address this question, Maylin et al. recently assessed the presence of residual HCV RNA in serum, liver, and PBMCs using TMA (sensitivity <9.6 IU/mL) in a long-term follow-up study of patients infected with chronic hepatitis C who achieved a sustained virological response after interferon-based treatment.6 A total of 114 patients had a posttreatment liver tissue and 156 had PBMCs. Serum HCV RNA remained undetectable (1300 samples), indicating that none of the patients had a relapse. HCV RNA was detectable in 2 of 114 (1.7%) liver specimens, and in none of 156 PBMC specimens. These authors strongly suggest that SVR may be considered to show eradication of HCV infection.7 This latest observation was confirmed by another recent study with a complete clearance of HCV RNA from the PBMCs of blood donors who spontaneously or therapeutically control their plasma viremia.8 In conclusion, the excellent review performed by Welker and Zeuzem on occult HCV infection confirm our data and those of two other studies using sensitive assays and liver/PBMC assessment, and reinforce the absence of evidence of occult HCV infection. Philippe Halfon M.D., Ph.D.*, Michele Martinot-Peignoux Ph.D. , Patrice Cacoub M.D., Ph.D. , * Département Virologie Laboratoire Alphabio, Service Maladie Infectieuses Hôpital Ambroise Paré, Marseille, France, Université Paris VII, Hôpital Beaujon, Clichy, France; Service de Microbiologie, Hôpital Beaujon, Clichy, France; Institut National de la Santé et de la Recherche Médicale U-773, Centre de Recherche Biomédicale Bichat-Beaujon CRB3, Hôpital Beaujon, Clichy, France, Université Pierre et Marie Curie-Paris 6, Centre National de la Recherche Scientifique, UMR 7087, Paris, France; Hôpital Pitié-Salpêtrière, Service de Médecine Interne, Assistance Publique–Hôpitaux de Paris, Paris, France.

Effect of saponins from Helicteres isora on lipid and glucose metabolism regulating genes expression

Ethnopharmacological relevance We characterized saponins as active constituents from traditionally used antidiabetic plant Helicteres isora. Aim of the study To evaluate the changes in the gene expression of the glucose and lipid metabolism regulating genes in C57BL/KsJ- db/ db mice. Materials and methods C57BL/KsJ- db/ db mice were divided into four different groups; one diabetic control, the mice in other three groups were treated with methanol extract (100 mg/kg), saponins (100 mg/kg) and pioglitazone (30 mg/kg) for 14 days. After completion of the treatment period biochemical parameters and the expression levels of adipsin, adiponectin, glucose transporter 4 (Glut4), peroxisome proliferator activated receptor gamma (PPARγ), fatty acid binding protein 4 (FABP4), lipoprotein lipase (LPL) in adipose tissue and for liver RNA samples glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), glucose transporter 2 (Glut2) and acyl-co-enzyme A oxidase (ACOX) were determined by quantitative real time PCR and angiopoeitin like 3 (ANGPTL3), angiopoeitin like 4 (ANGPTL4) and peroxisome proliferator activated receptor alpha (PPARα) by semiquantitative reverse transcription PCR. Results Treatment caused a significant reduction in the serum lipid and glucose levels and increased the expression of adipsin, PPARγ and Glut4 while reduced expression of FABP4 and G6Pase, whereas there was no effect on the expression levels of adiponectin, LPL, PEPCK, ACOX, Glut2, ANGPTL3, ANGPTL4 and PPARα. Conclusions Saponins are beneficial for improving hyperlipidemia and hyperglycemia by increasing the gene expression of adipsin, Glut4 and PPARγ and reducing the gene expression of the enzyme G6Pase and FABP4 in C57BL/KsJ- db/ db mice.

Selective Up-Regulation of Intact, but Not Defective env RNAs of Endogenous Modified Polytropic Retrovirus by the Sgp3 Locus of Lupus-Prone Mice

Endogenous retroviruses are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Because four different classes of endogenous retroviruses, i.e., ecotropic, xenotropic, polytropic, or modified polytropic (mPT), are expressed in mice, we investigated the possibility that a particular class of endogenous retroviruses is associated with the development of murine SLE. We observed >15-fold increased expression of mPT env (envelope) RNA in livers of all four lupus-prone mice, as compared with those of nine nonautoimmune strains of mice. This was not the case for the three other classes of retroviruses. Furthermore, we found that in addition to intact mPT transcripts, many strains of mice expressed two defective mPT env transcripts which carry a deletion in the env sequence of the 3' portion of the gp70 surface protein and the 5' portion of the p15E transmembrane protein, respectively. Remarkably, in contrast to nonautoimmune strains of mice, all four lupus-prone mice expressed abundant levels of intact mPT env transcripts, but only low or nondetectable levels of the mutant env transcripts. The Sgp3 (serum gp70 production 3) locus derived from lupus-prone mice was responsible for the selective up-regulation of the intact mPT env RNA. Finally, we observed that single-stranded RNA-specific TLR7 played a critical role in the production of anti-gp70 autoantibodies. These data suggest that lupus-prone mice may possess a unique genetic mechanism responsible for the expression of mPT retroviruses, which could act as a triggering factor through activating TLR7 for the development of autoimmune responses in mice predisposed to SLE.

Hepatitis C virus replication in patients with occult hepatitis C virus infection†

We read with interest the review by Welker and Zeuzem1 on occult hepatitis C virus (HCV) infection. In this review, the authors claim that viral replication has not been proven in occult HCV infection. However, the presence of the antigenomic HCV RNA strand has been demonstrated in liver and in peripheral blood mononuclear cells (PBMCs) of patients with occult HCV who are negative for anti-HCV and serum HCV RNA2 (and references in Carreño et al.2). There is no doubt that detection of the antigenomic strand is an indicator of viral replication as recognized by Welker and Zeuzem in their review.1 Furthermore, ultracentrifugation of serum samples before performance of real-time reverse transcription polymerase chain reaction (RT-PCR) detects very low levels of viremia in patients with occult HCV infection, and the density of the circulating HCV particles was identical to those found in the serum of patients with chronic hepatitis C.2 In addition, family members of patients with occult HCV infection show serological markers of HCV infection even more frequently than those of patients with chronic hepatitis C.3 Other authors have reported that low-level persisting HCV remains infectious.4 Finally, HCV-specific CD4+ T cell proliferative responses to nonstructural 3 and/or 4 proteins have been detected among patients infected with occult HCV,2 which may not have occurred unless there was ongoing HCV replication, either within the liver or in immune-privileged sites. So, taking all these data into account, one may convincingly assume that HCV replication takes place in patients with occult HCV infection who no doubt are potentially infectious. However, Welker and Zeuzem1 review two studies that unconvincingly failed to detect occult HCV infection in cryptogenic hepatitis,5, 6 because of several concerns with the methodology employed. Thus, one study5 used whole blood to assess occult HCV infection, but it was demonstrated in patients with occult hepatitis C that the sensitivity of HCV RNA detection substantially decreased when using whole blood instead of PBMCs.2 Another study6 included a very small and heterogeneous population, and these authors did not perform detection of HCV RNA either in liver or in serum by PCR after ultracentrifugation. Thus, their results do not exclude the existence of occult HCV infection. On the other hand, an anti-HCV core antibody assay has been developed recently, which adds another diagnostic tool because it has been proposed as a potential new marker of the serologically silent form of occult HCV infection.7 In contrast, other studies have confirmed the existence of occult HCV infection by detection of HCV RNA in liver.8, 9 Furthermore, contrary to what is concluded by Welker and Zeuzem, HCV proteins have been detected in liver10 and in lymph nodes11 of patients who are negative for anti-HCV and serum HCV RNA. In summary, considering the data available, there is cumulative evidence strongly supporting the idea that HCV replication takes place in patients with occult HCV infection. Therefore, in order to diagnose occult HCV infection among these patients, it is mandatory to perform appropriate molecular and immunoserological tests in serum, PBMCs, and liver biopsies. Vicente Carreño*, Javier Bartolomé*, Inmaculada Castillo*, Juan Antonio Quiroga*, * Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain.

Regulator of sex-limitation KRAB zinc finger proteins modulate sex-dependent and -independent liver metabolism

Krüppel-related zinc finger proteins (KRAB-zfps) comprise the largest mammalian transcription factor family, but their specific functions are largely unknown. Two KRAB-zfps, regulator of sex-limitation (Rsl) 1 and Rsl2, repress expression of the mouse sex-limited protein (Slp) gene, the hallmark of Rsl activity, as well as some other male-predominant liver genes. This phenotype suggests Rsl modifies sex-specific transcription. The scope of Rsl control was determined by expression profiling of liver RNA from wild-type (wt), rsl, and transgenic mice with hepatic overexpression of Rsl1 or Rsl2. About 7.5% of the liver transcriptome was Rsl-responsive. More genes in males than females were affected by the loss of Rsl (e.g., in rsl mice), whereas Rsl overexpression altered more transcripts in females than males. Rsl dramatically repressed some female-predominant genes, but most were modestly (1.25- to 2-fold) influenced. In males, most Rsl-responsive genes unexpectedly expressed at lower levels in rsl than wt, suggesting not all are direct targets of Rsl repression. Gene Ontology analysis showed Rsl targets enriched in pathways of cholesterol, steroid, and lipid metabolism, linking Rsl to energy balance. In accord with this, blood glucose levels were less in male rsl than wt mice, and less responsive to fasting and refeeding. rsl mice were also leaner than wt, consistent with their hepatic regulation of phosphoenolpyruvate carboxykinase 1 and stearoyl-Coenzyme A desaturase 1. Altogether, Rsl's effect on sexually dimorphic and metabolically sensitive liver gene expression suggests a role for KRAB-zfps as broad genetic modulators of individual adaptation.

Genomic and Proteomic Analysis of Hepatocellular Carcinogenesis in HBV Transgenic Mice (44.14)

Abstract Hepatitis B virus (HBV) transgenic mice invariably developed macroscopically evident tumors within the 20th month of life. Thus HBV transgenic mouse can be used as a natural model of hepatocellular carcinoma (HCC). We confirmed this phenomenon by observation of BALB/c mice harboring HBV complete genome (subtype ayw). To identify molecular changes involved in HCC, we performed both microarray profiling and proteomic analysis between HBV transgenic mice and their syngeneic controls at 6, 12, and 20-month-old. The total liver RNA was hybridized on an oligonucleotide chip containing 35,000 Mus musculus genes. The total liver protein was used for proteomic analysis. Out of all genes and proteins compared and screened, the number of differentially expressed transcripts and proteins was increased along with the growth of mice. We concluded a molecular portrait in different stages of HCC. These molecules were involved in important cellular pathways, including cell growth regulation, apoptosis, immune response, metabolisms, stress, cell adhesion, et al. The dynamics of molecular profiling during hepatocellular carcinogenesis would better elucidate the molecular mechanisms of HCC and help for the discovery of novel therapeutic targets. Correspondence to Wensheng Sun: wsw@sdu.edu.cn Supported by the National Nature Science Foundation of China, Grant No. 30440040, 30772031, and 30801036.

Analysis of hepatic genes involved in the metabolism of fatty acids and iron in nonalcoholic fatty liver disease

Hepatic steatosis and iron cause oxidative stress, thereby progressing steatosis to steatohepatitis. We quantified the expression of genes involved in the metabolism of fatty acids and iron in patients with nonalcoholic fatty liver disease (NAFLD). The levels of transcripts for the following genes were quantified from biopsy specimens of 74 patients with NAFLD: thioredoxin (Trx), fatty acid transport protein 5 (FATP5), sterol regulatory element-binding protein 1c (SREBP1c), fatty acid synthase (FASN), acetyl-coenzyme A carboxylase (ACAC), peroxisome proliferative activated receptor alpha (PPARalpha), cytochrome P-450 2E1 (CYP2E1), acyl-coenzyme A dehydrogenase (ACADM), acyl-coenzyme A oxidase (ACOX), microsomal triglyceride transfer protein (MTP), transferrin receptor 1 (TfR1), transferrin receptor 2 (TfR2) and hepcidin. Twelve samples of human liver RNA were used as controls. Histological evaluation followed the methods of Brunt. The levels of all genes were significantly higher in the NAFLD patients than in controls. The Trx level increased as the stage progressed. The levels of FATP5, SREBP1c, ACAC, PPARalpha, CYP2E1, ACADM and MTP significantly decreased as the stage and grade progressed (P < 0.05). Hepatic iron score (HIS) increased as the stage progressed. The TfR1 level significantly increased as the stage progressed (P < 0.05), whereas TfR2 level significantly decreased (P < 0.05). The ratio of hepcidin mRNA/ferritin (P < 0.001) or hepcidin mRNA/HIS (P < 0.01) was significantly lower in NASH patients than simple steatosis patients. Steatosis-related metabolism is attenuated as NAFLD progresses, whereas iron-related metabolism is exacerbated. Appropriate therapies should be considered on the basis of metabolic changes.

Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes

Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV-H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.

Identification and characterization of phospholipase A 2 inhibitors from the serum of the Japanese rat snake, Elaphe climacophora

Two distinct phospholipase A 2 (PLA 2) inhibitory proteins (PLIs) were purified from the serum of the Japanese rat snake, Elaphe climacophora. The 150-kDa inhibitor, a trimer of a 50-kDa subunit, specifically inhibited the basic PLA 2 purified from the venom of Gloydius brevicaudus, whereas the 120-kDa one composed of two distinct 25-kDa subunits, A and B, inhibited both the acidic and basic PLA 2s of G. brevicaudus. On the basis of their amino acid sequences, these inhibitors were assigned as PLIβ and PLIγ, respectively. A PLIα homolog (PLIα-like protein; PLIα-LP) having an apparent molecular weight of 50-kDa and composed of 15-kDa subunits was also purified from the E. climacophora serum. This homolog was immunoreactive with antibody raised against the G. brevicaudus PLIα, but lacked in the inhibitory activity toward the acidic and basic PLA 2s. The cDNAs encoding PLIα-LP, PLIβ, PLIγ-A, and PLIγ-B were cloned from liver RNA, and their nucleotide sequences were compared with those of other venomous and non-venomous snakes.

Changes in transcriptome after in vivo exposure to ionising radiation reveal a highly specialised liver response

Purpose: To identify transcriptional gene-networks involved in the early in vivo response of liver cells to radiation exposure and improve our understanding of the molecular processes responsible for tissue radiosensitivity.Materials and methods: Transcriptome variations of liver RNA samples were measured 3 hours post-irradiation using microarray technology. The results were confirmed and extended using real-time polymerase-chain-reaction (RT-PCR).Results: We identified quantitative changes in the expression of 126 genes, most of which were observed for the first time. We show that some modifications, such as the upregulation of the cyclin-dependent kinase inhibitor 1A (Cdkn1A) gene, persisted for at least two months after the initial exposure. Other genes regulated by the transformation-related protein 53 (Trp53/p53) such as Bcl2-associated X protein (Bax) or etoposide-induced-2.4 (Ei24/PIG8) were not upregulated. Grouping differentially expressed genes into functional categories revealed that the primary response of liver cells to radiation exposure was the enhancement of oxidoreductase activity and inhibition of cell proliferation, involving cell cycle progression and apoptosis-related genes.Conclusions: The data provides evidence of gene expression modifications associated with the hepatic response to radiation exposure. One of the main differences observed with radiation-sensitive tissues such as the spleen was cell proliferation. The comparison of our data with transcriptome modifications in different biological models enabled the identification of networks of genes that might be co-regulated. Overall, our expression data revealed genes and cellular pathways that might help to improve our understanding of the molecular basis underlying tissue radiosensitivity and to identify possible targets for novel therapeutic strategies.

Purification, molecular cloning, heterologous expression and characterization of pig CYP1A2

Porcine cytochrome P450 (CYP) 1A2 was purified to electrophoretic homogeneity from the hepatic microsomes of β-naphthoflavone-treated male pigs. In a reconstituted system, this enzyme showed a good catalytic activity towards caffeine, acetanilide, and methoxyresorufin, all known markers of mammalian CYP1A2. Using 3′- and 5′-rapid amplification of coding DNA (cDNA) ends (RACE), we amplified from the liver RNA of control pigs a full-length 1827 bp cDNA containing an open reading frame of 1548 bp which encoded a putative CYP1A2 protein of 516 amino acids and an estimated Mr of 58 380 Da. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that the messenger RNA (mRNA) of CYP1A2 was expressed in liver, heart and nasal mucosa but not in lung, small intestine, kidney and brain. Using the pCW vector containing a N-terminal modified cDNA, pig CYP1A2 was expressed in Escherichia coli. 3-[(3-Chloroamidopropyl)dimethylmmonio]-1-propane-sulfonate (CHAPS)-solubilized E. coli preparations expressing CYP1A2 produced a functionally isoform which, in a reconstituted system, was catalytically active toward ethoxyresorufin and methoxyresorufin showing Km's similar to those obtained with CYP1A2 purified from pig liver or human recombinant CYP1A2. Taken together, these results demonstrate that domestic pigs have a functionally active CYP1A2 gene well expressed in the liver with biochemical properties quite similar to those corresponding to the human enzyme.

Antisense Oligonucletide Mediated Depletion of Factor VII Provides Protection from Ferric Chloride Induced Thrombosis without Increased Bleeding Risk in Mice

The Tissue Factor-Factor VIIa complex is the initiating protease in the extrinsic pathway of the coagulation cascade. Knockout of Factor VII in mice results in embryonic lethality. However, only humans with severe Factor VII deficiency exhibit any coagulopathy. In order to determine the potential of Factor VII as an anticoagulant target and the utility of antisense oligonucleotides (ASOs) as Factor VII inhibitors, ASOs were designed to promote RNaseH mediated degradation of Factor VII RNA and evaluated in mice. Factor VII is predominantly produced in the liver, a tissue that is tractable for antisense mediated target depletion. Following three weeks of treatment, the Factor VII ASO was well tolerated and supported significant reduction of Factor VII RNA and protein in mouse liver (ED50 of 3.5 mg/kg for RNA and 2.8 mg/kg for protein). Greater than 95% reduction in Factor VII RNA levels was observed at a dose of 25 mg/kg, administered twice weekly. Following three weeks exposure to 100 mg/kg/wk, prothrombin times (PT) from platelet poor mouse plasma was more than doubled, compared to untreated animals, while activated partial thromboplastin times (aPTT) was only increased 16%. Antithrombotic activity of Factor VII ASO was determined following ferric chloride injury to the inferior vena cava. ED50 for inhibition of thrombosis was 24 mg/kg/wk. The effects of Factor VII depletion on hemostatic control were also determined using a mouse tail nick model. No increase in blood loss was detected, compared to untreated animals, up to a weekly exposure of 80 mg/kg, the highest dose utilized in the study. Compared to warfarin, ASO mediated depletion of Factor VII promoted equivalent protection from thrombosis following ferric chloride injury while maintaining better hemostatic control. These findings support the development of an ASO drug targeting Factor VII as an anticoagulant.

Hepatic function is preserved in the absence of mature microRNAs #

MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression through partial or complete complementarity with target messenger RNAs. The function of miRNAs in normal liver physiology is largely unknown. We address the role of Dicer1 in the differentiated liver. We derived mice lacking Dicer1 function in hepatocytes and assessed the loss of mature miRNA via quantitative polymerase chain reaction. Gene expression microarray analysis was performed on liver RNA from mutant and control mice. Liver sections from mutant and control mice were examined and liver function tests were performed. Mice lacking Dicer1 function in hepatocytes appeared and behaved normally. Despite the loss of mature miRNAs, hepatic function was maintained, as reflected by normal blood glucose, albumin, cholesterol, and bilirubin. However, mutant mice between 2 and 4 months of age exhibited progressive hepatocyte damage with elevated serum alanine aminotransferase and aspartate aminotransferase. Liver mass was increased in mutant mice, as were cellular markers of both proliferation and apoptosis. Microarray analysis indicated large-scale changes in gene expression, with increased expression of many miRNA targets, particularly imprinted genes. Loss of miRNA processing in the liver at late gestation has a remarkably mild phenotype, suggesting that miRNAs do not play an essential role in hepatic function. However, miRNA deficiency results in hepatocyte apoptosis, hepatocyte regeneration, and portal inflammation. Finally, microarray analysis of gene expression in the mutant liver supports a previously hypothesized role for Dicer1 in the repression of imprinted genes.

Gene expression profiling in hepatic tissue of newly weaned pigs fed pharmacological zinc and phytase supplemented diets

BackgroundZinc (Zn) is an essential trace element. However, Zn bioavailability from commonly consumed plants may be reduced due to phytic acid. Zn supplementation has been used to treat diarrheal disease in children, and in the U.S. swine industry at pharmacological levels to promote growth and fecal consistency, but underlying mechanisms explaining these beneficial effects remain unknown. Moreover, adding supplemental phytase improves Zn bioavailability. Thus, we hypothesized that benefits of pharmacological Zn supplementation result from changes in gene expression that could be further affected by supplemental phytase. The goal of this study was to investigate the effects of feeding newly weaned pigs dietary Zn (150, 1,000, or 2,000 mg Zn/kg) as Zn oxide with or without phytase [500 phytase units (FTU)/kg] for 14 d on hepatic gene expression. Liver RNA from pigs fed 150, 1,000, or 2,000 mg Zn/kg, or 1,000 mg Zn/kg with phytase (n = 4 per treatment) was reverse transcribed and examined using the differential display reverse transcription polymerase chain reaction technique. Liver RNA from pigs fed 150 or 2,000 mg Zn/kg (n = 4 per treatment) was also evaluated using a 70-mer oligonucleotide microarray.ResultsExpressed sequence tags for 61 putatively differentially expressed transcripts were cloned and sequenced. In addition, interrogation of a 13,297 element oligonucleotide microarray revealed 650 annotated transcripts (FDR ≤ 0.05) affected by pharmacological Zn supplementation. Seven transcripts exhibiting differential expression in pigs fed pharmacological Zn with sequence similarities to genes encoding GLO1, PRDX4, ACY1, ORM1, CPB2, GSTM4, and HSP70.2 were selected for confirmation. Relative hepatic GLO1 (P < 0.0007), PRDX4 (P < 0.009) and ACY1 (P < 0.01) mRNA abundances were confirmed to be greater in pigs fed 1,000 (n = 8) and 2,000 (n = 8) mg Zn/kg than in pigs fed 150 (n = 7) mg Zn/kg. Relative hepatic HSP70.2 (P < 0.002) mRNA abundance was confirmed to be lower in pigs fed 2,000 mg Zn/kg than in pigs fed 150 or 1,000 mg Zn/kg.ConclusionResults suggest that feeding pharmacological Zn (1,000 or 2,000 mg Zn/kg) affects genes involved in reducing oxidative stress and in amino acid metabolism, which are essential for cell detoxification and proper cell function.