Liver RNA Samples Research Articles

Examining effects of a novel estrogenic perfluoro-alcohol, 1H,1H,8H,8H-Perfluorooctane-1,8-diol (FC8-diol), using the fathead minnow EcoToxChip.

In a previous in vivo study, adult male fathead minnows (Pimephales promelas) were exposed via water for 4 days to 1H,1H,8H,8H-perfluorooctane-1,8-diol (FC8-diol). The present study expands on the evaluation of molecular responses to this perfluoro-alcohol by analyzing 26 male fathead minnow liver RNA samples from that study (five from each test concentration: 0, 0.018, 0.051, 0.171, and 0.463 mg FC8-diol/L) using fathead minnow EcoToxChips Ver. 1.0. EcoToxChips are a quantitative polymerase chain reaction array that allows for simultaneous measurement of >375 species-specific genes of toxicological interest. Data were analyzed with the online tool EcoToxXplorer. Among the genes analyzed, 62 and 96 were significantly up- and downregulated, respectively, by one or more FC8-diol treatments. Gene expression results from the previous study were validated, showing an upregulation of vitellogenin mRNA (vtg) and downregulation of insulin-like growth factor 1 mRNA (igf1). Additional genes related to estrogen receptor activation including esr2a (estrogen receptor 2a) and esrrb (estrogen related receptor beta) were also affected, providing further confirmation of the estrogenic nature of FC8-diol. Furthermore, genes involved in biological pathways related to lipid and carbohydrate metabolism, innate immune response, endocrine reproduction, and endocrine thyroid were significantly affected. These results both add confidence in the use of the EcoToxChip tool for inferring chemical mode(s) of action and provide further insights into the possible biological effects of FC8-diol. Environ Toxicol Chem 2024;00:1-9. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Dose-dependent effects on rat liver miRNAs 200a/b and 429: potential early biomarkers of liver carcinogenesis.

An increased incidence of liver tumours in the long term rodent bioassay is not an uncommon finding, invariably as a result of a non-genotoxic mode of action. Non-genotoxic liver carcinogenesis has been found to involve activation of certain nuclear hormone receptors (NHR) including the constitutive androstane receptor (CAR), peroxisome proliferator activated receptor alpha (PPARalpha) and arylhydrocarbon receptor (AHR) and more recently the induction of specific microRNAs (miRs), has also been demonstrated following CAR activation in studies up to 90 days (Koufaris et al., 2012). The stable induction of these tissue specific miRs, namely miR200a, 200b and 429, by liver non-genotoxic carcinogens may serve as early predictors (biomarkers) of heptocarcinogenic potential. To test this hypothesis we used RT-PCR to measure the levels of these miRs in the livers from Wistar rats treated with two rat hepatocarcinogenic and one non hepatocarcinogenic pyrazole carboxamide succinate dehydrogenase inhibitors, Isopyrazam, Sedaxane and Benzovindiflupyr, respectively. The miRs were quantified by RT-PCR in liver RNA samples from three 90 day repeat dose toxicity studies performed at the low, mid and high doses relative to control. In Isopyrazam treated rats a statistically significant (p < 0.01) dose-dependent increase in miR 200a, 220b and 429 in both males and females was observed, whilst for Sedaxane a significant (p < 0.05) increase in miR200b in males and females at the high dose was seen. Benzovindiflupyr treatment did not cause any dose related changes in miR 200a, 200b and 429 relative to control. Our results suggest that assessment of miR 200a/200b/429 levels has potential as a biomarker of the perturbation of pathways involved in hepatocarcinogenesis in Wistar rats. Further work is required to establish the possible relationship between miR200 cluster induction and CAR-mediated hepatocarcinogenesis in a more diverse range of compounds.

Oxidative stress/reactive metabolite gene expression signature in rat liver detects idiosyncratic hepatotoxicants

Previously we reported a gene expression signature in rat liver for detecting a specific type of oxidative stress (OS) related to reactive metabolites (RM). High doses of the drugs disulfiram, ethinyl estradiol and nimesulide were used with another dozen paradigm OS/RM compounds, and three other drugs flutamide, phenacetin and sulindac were identified by this signature. In a second study, antiepileptic drugs were compared for covalent binding and their effects on OS/RM; felbamate, carbamazepine, and phenobarbital produced robust OS/RM gene expression. In the present study, liver RNA samples from drug-treated rats from more recent experiments were examined for statistical fit to the OS/RM signature. Of all 97 drugs examined, in addition to the nine drugs noted above, 19 more were identified as OS/RM-producing compounds—chlorpromazine, clozapine, cyproterone acetate, dantrolene, dipyridamole, glibenclamide, isoniazid, ketoconazole, methapyrilene, naltrexone, nifedipine, sulfamethoxazole, tamoxifen, coumarin, ritonavir, amitriptyline, valproic acid, enalapril, and chloramphenicol. Importantly, all of the OS/RM drugs listed above have been linked to idiosyncratic hepatotoxicity, excepting chloramphenicol, which does not have a package label for hepatotoxicity, but does have a black box warning for idiosyncratic bone marrow suppression. Most of these drugs are not acutely toxic in the rat. The OS/RM signature should be useful to avoid idiosyncratic hepatotoxicity of drug candidates.

Thyroid-specific gene expression in chondrocytes

Previously, we demonstrated that Runx2 (Cbfa1/AML3), a chondrocyte-specific transcription factor, is expressed in thyroid glands of mice, where it stimulates expression of the thyroglobulin (Tg) gene. Here, we reverse transcribed thyroid transcription factor-1 (TTF-1), Pax-8, Tg, thyroid peroxidase (TPO) and Na+/I− symporter (NIS) cDNAs from mouse trachea and bronchus RNA samples, but were unable to recover these cDNAs from mouse liver RNA samples. Tg mRNA levels in trachea and bronchus were about 5.1% and 2.1% of those in thyroid glands. ATDC-5 cells, cultured chondrocytes, expressed about 30-fold more Tg mRNA than undifferentiated cells. Gel shift and Tg gene reporter assay revealed that TTF-1 stimulated Tg gene expression in these cells. These results indicate that chondrocytes turn on some aspects of the thyroid gene expression program and that TTF-1 plays important roles in Tg gene expression in chondrocyte.

Sex-specific and lineage-specific alternative splicing in primates

Comparative studies of gene regulation suggest an important role for natural selection in shaping gene expression patterns within and between species. Most of these studies, however, estimated gene expression levels using microarray probes designed to hybridize to only a small proportion of each gene. Here, we used recently developed RNA sequencing protocols, which sidestep this limitation, to assess intra- and interspecies variation in gene regulatory processes in considerably more detail than was previously possible. Specifically, we used RNA-seq to study transcript levels in humans, chimpanzees, and rhesus macaques, using liver RNA samples from three males and three females from each species. Our approach allowed us to identify a large number of genes whose expression levels likely evolve under natural selection in primates. These include a subset of genes with conserved sexually dimorphic expression patterns across the three species, which we found to be enriched for genes involved in lipid metabolism. Our data also suggest that while alternative splicing is tightly regulated within and between species, sex-specific and lineage-specific changes in the expression of different splice forms are also frequent. Intriguingly, among genes in which a change in exon usage occurred exclusively in the human lineage, we found an enrichment of genes involved in anatomical structure and morphogenesis, raising the possibility that differences in the regulation of alternative splicing have been an important force in human evolution.

Embrace diversity! Systems genetics-enabled discovery of disease networks

identify robust and strain-, sex-, and strain*sex-dependent responses to the diet. Global analysis of the expression data by ANOVA revealed extensive impact of both strain and sex on dietary responses in liver. The main effect of diet was apparent in the differential expression of almost 57% of genes on the arrays. Among the top co-regulated of these, the authors showed using Gene Ontology (GO) enrichment analyses that the expected effects of diet-mediated suppression of cholesterol biosynthesis occurred in all strains and that other specific biological systems functional involvements were also strongly implicated. Valuable data emerge from the ability to identify sets of genes that are reflective of strain-, sex-, and strain*sexdependent effects of diet, attributable in part from the statistical power of the study design in which the underlying genetic variations imparted systematic effects at the level of biological pathways and processes. When strain is taken into account, the majority of significantly enriched GO categories reflected a powerful correlation between effects of diet on immune function, such as antigen processing and presentation. The authors suggest that these changes may be related to diet-induced liver damage, which was indicated by changes expression of damage-related genes and by an increase in circulating levels of glutamate dehydrogenase. However, these changes may also broadly reflect genetic correlation between pathways that mediate immune responses and the development of diet-induced atherogenic plaques, the formation and propagation of which are driven by immune responses. Integrating the expression data with information about the incidence, severity, and phenotypic details of atherosclerotic lesions in this same set of strains should shed additional light on this possibility. Other important follow-on studies may also determine if strainspecific patterns of immune gene expression in liver are paralleled by qualitatively similar effects on lymphocyte populations, atherogenic lesions, and insulin pathway-associated tissues. Importantly, the authors have also provided a publicly accessible database that can be mined in a number of ways. For example, this resource could be used to select strains with divergent responses to diet (e.g., sensitive and resistant to liver damage) or to identify coexpression networks centered around a gene(s) of specific interest. It could also be used to extract supportive evidence for genes implicated in processes related

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.

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.

RNA-seq: An assessment of technical reproducibility and comparison with gene expression arrays

Ultra-high-throughput sequencing is emerging as an attractive alternative to microarrays for genotyping, analysis of methylation patterns, and identification of transcription factor binding sites. Here, we describe an application of the Illumina sequencing (formerly Solexa sequencing) platform to study mRNA expression levels. Our goals were to estimate technical variance associated with Illumina sequencing in this context and to compare its ability to identify differentially expressed genes with existing array technologies. To do so, we estimated gene expression differences between liver and kidney RNA samples using multiple sequencing replicates, and compared the sequencing data to results obtained from Affymetrix arrays using the same RNA samples. We find that the Illumina sequencing data are highly replicable, with relatively little technical variation, and thus, for many purposes, it may suffice to sequence each mRNA sample only once (i.e., using one lane). The information in a single lane of Illumina sequencing data appears comparable to that in a single array in enabling identification of differentially expressed genes, while allowing for additional analyses such as detection of low-expressed genes, alternative splice variants, and novel transcripts. Based on our observations, we propose an empirical protocol and a statistical framework for the analysis of gene expression using ultra-high-throughput sequencing technology.

Exon and junction microarrays detect widespread mouse strain- and sex-bias expression differences

BackgroundStudies have shown that genetic and sex differences strongly influence gene expression in mice. Given the diversity and complexity of transcripts produced by alternative splicing, we sought to use microarrays to establish the extent of variation found in mouse strains and genders. Here, we surveyed the effect of strain and sex on liver gene and exon expression using male and female mice from three different inbred strains.Results71 liver RNA samples from three mouse strains – DBA/2J, C57BL/6J and C3H/HeJ – were profiled using a custom-designed microarray monitoring exon and exon-junction expression of 1,020 genes representing 9,406 exons. Gene expression was calculated via two different methods, using the 3'-most exon probe ("3' gene expression profiling") and using all probes associated with the gene ("whole-transcript gene expression profiling"), while exon expression was determined using exon probes and flanking junction probes that spanned across the neighboring exons ("exon expression profiling"). Widespread strain and sex influences were detected using a two-way Analysis of Variance (ANOVA) regardless of the profiling method used. However, over 90% of the genes identified in 3' gene expression profiling or whole transcript profiling were identified in exon profiling, along with 75% and 38% more genes, respectively, showing evidence of differential isoform expression. Overall, 55% and 32% of genes, respectively, exhibited strain- and sex-bias differential gene or exon expression.ConclusionExon expression profiling identifies significantly more variation than both 3' gene expression profiling and whole-transcript gene expression profiling. A large percentage of genes that are not differentially expressed at the gene level demonstrate exon expression variation suggesting an influence of strain and sex on alternative splicing and a need to profile expression changes at sub-gene resolution.

Housekeeping Gene Expression in Bovine Liver is Affected by Physiological State, Feed Intake, and Dietary Treatment

Selection of appropriate housekeeping genes (HKG) for normalization of quantitative PCR data for genes of interest is critical for interpretation of results. Ideally, copy number of the chosen HKG mRNA will not vary with experimental treatments or physiological state in the tissue studied, which improves accuracy in detecting changes in genes of interest. Because of the liver's dynamic role in metabolism, physiological state or dietary treatments could alter mRNA expression of commonly used HKG. Therefore, the objective of this study was to evaluate stability of mRNA expression for a number of candidate HKG in bovine liver across different physiological and dietary experimental conditions during the periparturient period. A publicly available program (geNorm) was used to evaluate expression stability of 8 HKG (β-actin, glyceraldehyde 3-phosphate dehydrogenase, β-glucuronidase, peptidylprolyl isomerase A, polyubiquitin, ribosomal protein S9, ribosomal protein L32, and 18S ribosomal RNA) in 91 liver RNA samples. Screened samples included liver from cows in 3 groups: 1) cows receiving a dietary supplement pre- and postpartum (n=10); 2) cows with clinical or subclinical ketosis (n=7); and 3) cows consuming different amounts of energy prepartum (n=74). In group 3, samples from d −65, −30, −14, 1, 14, 28, and 49 relative to parturition were included to enable characterization of HKG mRNA expression across different physiological states. Initial analyses indicated that mRNA for ribosomal protein S9 (RPS9) was one of the most stably expressed across different experiment types. To determine the best gene, 200 bootstrap replications of the original data set were performed to determine if the ranking of RPS9 was superior to the other 7 genes evaluated. Average ranks and estimated standard errors for the top 3 genes were 1.64±0.06, 3.27±0.10, and 3.71±0.12 for RPS9, GAPDH, and β-actin, respectively. Ribosomal protein S9 was ranked first 59% of the time and was never ranked lower than fifth. The lowest-ranked gene was polyubiquitin, ranked last 46.5% of the time (average rank = 6.85±0.10). In this study, physiological state, amount of intake, or dietary treatment influenced the mRNA expression of commonly used HKG in bovine liver. Ideally, expression stability should be tested before collection of data in all experiments; however, we have shown that RPS9 mRNA is stable across several physiological and diet-related experimental conditions for dairy cows, making it a good HKG in liver quantitative PCR experiments.

IFN Regulatory Factor-2 Regulates Macrophage Apoptosis through a STAT1/3- and Caspase-1-Dependent Mechanism

IFN regulatory factor (IRF)-2(-/-) mice are significantly more resistant to LPS challenge than wild-type littermates, and this was correlated with increased numbers of apoptotic Kupffer cells. To assess the generality of this observation, and to understand the role of IRF-2 in apoptosis, responses of peritoneal macrophages from IRF-2(+/+) and IRF-2(-/-) mice to apoptotic stimuli, including the fungal metabolite, gliotoxin, were compared. IRF-2(-/-) macrophages exhibited a consistently higher incidence of apoptosis that failed to correlate with caspase-3/7 activity. Using microarray gene expression profiling of liver RNA samples derived from IRF-2(+/+) and IRF-2(-/-) mice treated with saline or LPS, we identified >40 genes that were significantly down-regulated in IRF-2(-/-) mice, including Stat3, which has been reported to regulate apoptosis. Compared with IRF-2(+/+) macrophages, STAT3alpha mRNA was up-regulated constitutively or after gliotoxin treatment of IRF-2(-/-) macrophages, whereas STAT3beta mRNA was down-regulated. Phospho-Y705-STAT3, phospho-S727-STAT1, and phospho-p38 protein levels were also significantly higher in IRF-2(-/-) than control macrophages. Activation of the STAT signaling pathway has been shown to elicit expression of CASP1 and apoptosis. IRF-2(-/-) macrophages exhibited increased basal and gliotoxin-induced caspase-1 mRNA expression and enhanced caspase-1 activity. Pharmacologic inhibition of STAT3 and caspase-1 abolished gliotoxin-induced apoptosis in IRF-2(-/-) macrophages. A novel IFN-stimulated response element, identified within the murine promoter of Casp1, was determined to be functional by EMSA and supershift analysis. Collectively, these data support the hypothesis that IRF-2 acts as a transcriptional repressor of Casp1, and that the absence of IRF-2 renders macrophages more sensitive to apoptotic stimuli in a caspase-1-dependent process.

High Copper Selectively Alters Lipid Metabolism and Cell Cycle Machinery in the Mouse Model of Wilson Disease

Copper is essential for human physiology, but in excess it causes the severe metabolic disorder Wilson disease. Elevated copper is thought to induce pathological changes in tissues by stimulating the production of reactive oxygen species that damage multiple cell targets. To better understand the molecular basis of this disease, we performed genome-wide mRNA profiling as well as protein and metabolite analysis for Atp7b-/- mice, an animal model of Wilson disease. We found that at the presymptomatic stages of the disease, copper-induced changes are inconsistent with widespread radical-mediated damage, which is likely due to the sequestration of cytosolic copper by metallothioneins that are markedly up-regulated in Atp7b-/- livers. Instead, copper selectively up-regulates molecular machinery associated with the cell cycle and chromatin structure and down-regulates lipid metabolism, particularly cholesterol biosynthesis. Specific changes in the transcriptome are accompanied by distinct metabolic changes. Biochemical and mass spectroscopy measurements revealed a 3.6-fold decrease of very low density lipoprotein cholesterol in serum and a 33% decrease of liver cholesterol, indicative of a marked decrease in cholesterol biosynthesis. Consistent with low cholesterol levels, the amount of activated sterol regulatory-binding protein 2 (SREBP-2) is increased in Atp7b-/- nuclei. However, the SREBP-2 target genes are dysregulated suggesting that elevated copper alters SREBP-2 function rather than its processing or re-localization. Thus, in Atp7b-/- mice elevated copper affects specific cellular targets at the transcription and/or translation levels and has distinct effects on liver metabolic function, prior to appearance of histopathological changes. The identification of the network of specific copper-responsive targets facilitates further mechanistic analysis of human disorders of copper misbalance.

Cloning, mapping and association studies of the ovine ABCG2 gene with facial eczema disease in sheep

Facial eczema (FE) is a hepatogenous mycotoxicosis in sheep caused by the fungal toxin sporidesmin. Resistance to FE is a multigenic trait. To identify QTL associated with this trait, a scan of ovine chromosomes was implemented. In addition, ABCG2 was investigated as a possible positional candidate gene because of its sequence homology to the yeast PDR5 protein and its functional role as a xenobiotic transporter. The sequence of ovine ABCG2 cDNA was obtained from liver mRNA by RT-PCR and 5' and 3' RACE. The predicted protein sequence shares >80% identity with other mammalian ABCG2 proteins. SNPs were identified within exon 6, exon 9 and intron 4. The intron 4 SNP was used to map ABCG2 to ovine chromosome 6 (OAR6), about 2 cM distal to microsatellite marker OarAE101. Interestingly, this chromosomal region contains weak evidence for a FE QTL detected in a previous genome-scan experiment. To further investigate the association of ABCG2 with FE, allele frequencies for the three SNPs plus three neighbouring microsatellite markers were tested for differences in sheep selected for and against FE. Significant differences were detected in the allele frequencies of the intronic SNP marker among the resistant, susceptible and control lines. No difference in the levels of ABCG2 expression between the resistant and susceptible animals was detected by Northern hybridisation of liver RNA samples. However, significantly higher expression was observed in sporidesmin-dosed sheep compared with naïve animals. Our inference is that the ABCG2 gene may play a minor role in FE sensitivity in sheep, at least within these selection lines.

Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA.

BackgroundThe interpretability of microarray data can be affected by sample quality. To systematically explore how RNA quality affects microarray assay performance, a set of rat liver RNA samples with a progressive change in RNA integrity was generated by thawing frozen tissue or by ex vivo incubation of fresh tissue over a time course.ResultsIncubation of tissue at 37°C for several hours had little effect on RNA integrity, but did induce changes in the transcript levels of stress response genes and immune cell markers. In contrast, thawing of tissue led to a rapid loss of RNA integrity. Probe sets identified as most sensitive to RNA degradation tended to be located more than 1000 nucleotides upstream of their transcription termini, similar to the positioning of control probe sets used to assess sample quality on Affymetrix GeneChip® arrays. Samples with RNA integrity numbers less than or equal to 7 showed a significant increase in false positives relative to undegraded liver RNA and a reduction in the detection of true positives among probe sets most sensitive to sample integrity for in silico modeled changes of 1.5-, 2-, and 4-fold.ConclusionAlthough moderate levels of RNA degradation are tolerated by microarrays with 3'-biased probe selection designs, in this study we identify a threshold beyond which decreased specificity and sensitivity can be observed that closely correlates with average target length. These results highlight the value of annotating microarray data with metrics that capture important aspects of sample quality.

Inter-individual variation of several cytochrome P450 2D6 splice variants in human liver

To examine the possibility that inter-individual differences in splicing partially explain the observed differences in CYP2D6 activity, we amplified its full-length cDNA in 96 human liver RNA samples and discovered five splice variants: intron 5 retention, intron 6 retention, intron 5 and intron 6 double retention, exon 3 skipping, and partial intron 1 retention. All of the CYP2D6 splice variants we identified are probably nonfunctional transcripts. Substantial inter-individual variation in the proportions of the CYP2D6 transcript represented by splice variants, measured by real-time PCR, suggests that the presence of these splice variants contributes to the population variation in CYP2D6 activity. Relatively high levels of intron 6 retention were not correlated with the newly discovered single nucleotide polymorphism 2988G > A in intron 6 ( CYP2D6∗ 41) but did correlate with the more common CYP2D6∗ 34 allele. Our study prompts further investigations to explore the effect of these splice variants on drug metabolism.

IMPACT OF TRANSCRIPTION FACTOR PROFILE AND CHROMATIN CONFORMATION ON HUMAN HEPATOCYTE CYP3A GENE EXPRESSION

Recent data have made it increasingly clear that the gene expression profile of a cell system, and its alteration in response to external stimuli, is highly dependent on both the higher order chromatin structure of the genome and the interaction of gene products in interpreting stimuli. To further explore this phenomenon, we have examined the role of both of these factors in controlling xenobiotic-mediated gene expression changes in primary and transformed human hepatocytes (HuH7). Using quantitative polymerase chain reaction, expression levels of several transcription factors implicated in the liver-specific regulation of the CYP3A gene family were examined in human adult and fetal liver RNA samples. These expression profiles were then compared with those obtained from both primary and transformed human hepatocytes, showing that, in general, cultured cells exhibit a distinct profile compared with either the fetal or adult samples. Transcriptome profiles before and after exposure to the CYP3A transcriptional activators rifampicin, dexamethasone, pregnane-16alpha-carbonitrile, and phenobarbital were subsequently examined. Whereas exposure to these compounds elicited a dose-dependent increase in CYP3A transcription in primary hepatocytes, no alteration in expression levels was observed for the hepatoma cell line HuH7. Alteration in the expression levels of pregnane X receptor and chicken ovalbumin upstream promoter transcription factor I, and the disruption of higher order chromatin within HuH7 cells altered CYP3A expression and/or activation by xenobiotics toward that observed in primary hepatocytes. These data provide potential roles for these two processes in regulating CYP3A expression in vivo.

A 3'-flanking NF-kappaB site mediates developmental silencing of the human zeta-globin gene.

The central developmental event in the human (h)alpha-globin gene cluster is selective silencing of the zeta-globin gene as erythropoiesis shifts from primitive erythroblasts in the embryonic yolk sac to definitive erythroblasts in the fetal liver. Previous studies have demonstrated that full developmental silencing of the hzeta-globin gene in transgenic mice requires the proximal 2.1 kb of its 3'-flanking region. In the current report, we localize this silencing activity to a 108 bp segment located 1.2 kb 3' to the zeta-globin gene. Protein(s) in nuclear extracts from cell lines representing the fetal/adult erythroid stage bind specifically to an NF-kappaB motif located at this site. In contrast, this binding activity is lacking in the nuclear extract of an embryonic-stage erythroid line expressing zeta-globin. This complex is quantitatively recognized by antisera to the NF-kappaB p50 and to a lesser extent to p65 subunits. A two-base substitution that disrupts NF-kappaB site protein binding in vitro also results in the loss of the developmental silencing activity in vivo. The data suggest that NF-kappaB complex formation is a crucial component of hzeta-globin gene silencing. This finding expands the roles of this widely distributed transcriptional complex to include negative regulation in mammalian development.

Expression of growth hormone gene in the head kidney of gilthead sea bream (Sparus aurata)

The presence of growth hormone (GH) mRNA in the head kidney of gilthead sea bream (Sparus aurata) was assessed by reverse transcription and polymerase chain reaction (RT-PCR). Reverse transcription was made with oligo-dT primers, and a single cDNA band (435 bp) corresponding in length to the distance between two specific GH primers was obtained. No corresponding product was seen using liver and brain RNA samples. The amplified DNA hybridized specifically with a pituitary GH probe after Southern transfer to nylon membranes. The identity of this DNA fragment was further confirmed by sequencing a nested PCR product (343 bp). This finding supports the hypothesis that the immune system in both higher and lower vertebrates is an extrapituitary site for the expression of GH, which may serve as an autocrine/paracrine factor in immunomodulation. J. Exp. Zool. 283:326–330, 1999. © 1999 Wiley-Liss, Inc.

34 DIFFERENTIAL: REGULATION OF GROWTH HORMONE RECEPTOR EXPRESSION BY ESTRADIOL AND TESTOSTERONE

Estradiol (E2) and testosterone (T) can stimulate growth by increasing GH secretion. Sex steriods can also regulate growth affecting GH receptor (GHR) expression in target tissues. In order to stydy the effect of physiological concentrations of E2 and T on GHR expression, we measured liver GHR mRNA by solution hybridization-RNase protection assay in rabbits treated with E2 or T. Three to five immature rabbits in each group received a s.c. pellet containing placebo, E2 (0.1; 0.5; 1.5; or 5.0 mg) or T(15, 50, or 100 mg). Liver RNA samples were hybridized to a 32 P labelled GHR antisense riboprobe and digested with RNase. After gel electrophoresis a specific 370 base long protected band, corresponding to GHR mRNA, was quantitated by autoradiography and densitometry. Liver GHR mRNA levels were significantly lower than that in controls in animals treated with 1.5 (62%; p < 0.025) and 5.0 mg of E2 (13%; p<0.003), and significantly higher in those treated with 100 mg of T (230%; p<0.01). From these findings we conclude that E2 and T, at physiological concentrations, have an opposite effect on GHR expression. The decrease on GHR expression by E2 might explain the inhibitory effect of moderate dose of estrogens on growth.