Influence of Extracellular Matrix Overlay on Phenobarbital-Mediated Induction of CYP2B1, 2B2, and 3A1 Genes in Primary Adult Rat Hepatocyte Culture

Sidhu, Jaspreet S.; Farin, Federico M.; Omiecinski, Curtis J.

doi:10.1006/abbi.1993.1121

Cited by 126 publications

(72 citation statements)

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“…The signaling mechanisms and DNA elements involved in phenobarbital-induced transcription of CYP2B1/2 genes have not been elucidated in detail. Important efforts have been made to develop primary hepatocyte cultures responsive to phenobarbital (Schuetz et al, 1988;Jauregui et al, 1991;Sidhu et al, 1993). Recently, a DNA sequence element conferring responsiveness to phenobarbital was shown to localize to a 163-bp fragment of the CYP2B2 promoter (Trottier et al, 1995).…”

Section: Resultsmentioning

confidence: 99%

Regulation of the Major Detoxication Functions by Phenobarbital and 3‐Methylcholanthrene in Co‐Cultures of Rat Hepatocytes and Liver Epithelial Cells

Lerche

Fautrel

Shaw³

et al. 1997

European Journal of Biochemistry

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In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CUP), including CYPlAU2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase a (GSTa), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin 0-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. CYPl A1 /2, CYP2B1/2, CYP3All2, GSTa, 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 CYPl A1/2 (13.6-fold) and GSTu (3.9-fold) mRNA expression in 3-methylcholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3A1/2 (1 1 .Zfold), GSTu (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 CYP2B 1 (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.

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Section: Resultsmentioning

confidence: 99%

Regulation of the Major Detoxication Functions by Phenobarbital and 3‐Methylcholanthrene in Co‐Cultures of Rat Hepatocytes and Liver Epithelial Cells

Lerche

Fautrel

Shaw³

et al. 1997

European Journal of Biochemistry

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“…1 Modeling this response in vitro therefore requires a welldefined system exhibiting concordant expression of a battery of other liver-specific markers and signaling pathways. 1,2,4 To assist with the study of molecular mechanisms involved in the PB induction response and expression mechanisms of other liver-specific genes, we have tested a diverse array of DNA transfection techniques and reagents in cultured primary hepatocytes. Our best results were obtained using lipofectin as a DNA delivery lipid, however, ␤-galactosidase staining experi- ments indicated that even this reagent yielded Ͻ0.1% transfection efficiency in the primary cells (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…These endpoints are highly sensitive indicators of the highly differentiated hepatocyte phenotype. [1][2][3] Attempts to rescue the baculovirus-mediated repression of PB induction or albumin expression with gadolinium chloride were without effect. Adding gadolinium chloride to adenovirus-exposed cells also resulted in negative modulation of these endpoints ( Figure 8).…”

Section: Viral Specificity and Kupffer Cell Disruptionmentioning

confidence: 99%

“…Recent advances in primary hepatocyte culture techniques using defined preparations of serum-free media coupled with extracellular matrices now allow for the in vitro preservation of differentiated hepatocyte function. [1][2][3] One parameter that has been especially difficult to model in primary hepatocytes, or in established hepatoma cell lines, is the phenobarbital (PB) gene induction response, a process that is transcriptionally regulated and results in dramatically enhanced expression of a battery of genes, including several involved in drug biotransformation. 4 A continued difficulty with current primary hepatocyte culture protocols is the inability to perform efficient DNA transfections, a procedure commonly used for investigating gene regulatory processes.…”

Section: Introductionmentioning

confidence: 99%

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Baculovirus vectors repress phenobarbital-mediated gene induction and stimulate cytokine expression in primary cultures of rat hepatocytes

2000

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Baculovirus transfection strategies have proven successful at transferring foreign DNA into hepatoma cells and primary hepatocytes. When testing the utility of these methodologies in cultured hepatocytes, we discovered that the presence of baculovirus disrupts the phenobarbital (PB) gene induction process, a potent transcriptional activation event characteristic of highly differentiated hepatocytes, and repressed expression of the albumin gene. In concert with previous reports from our laboratory demonstrating that increased cAMP levels can completely repress the induction of specific cytochrome P450 (CYP) genes, cAMP concentrations and PKA activities were measured in the primary hepatocytes subsequent to baculovirus exposure. However, neither parameter was affected by the presence of the virus. To evaluate whether immune response modulation was triggered by baculovirus exposure, RNase protection assays were performed and demonstrated that baculovirus infection activates TNF-␣, IL-1␣ and IL-1␤ expression in the primary hepatocyte cul-

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“…Consequently, DST causes a false positive result. Phenobarbital and rifanpin also accelerate dexamethasone metabolism [37], and estrogen and mitotane can increase CBG [38]. Similarly, women taking an oral contraceptive pill may produce a false positive result with DST because of increased CBG levels.…”

Section: Acknowledgmentsmentioning

confidence: 99%

Evaluation of the diagnostic criteria for Cushing’s disease in Japan [Review]

et al. 2013

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Abstract. Adrenocorticotropic hormone (ACTH)-dependent Cushing's syndrome is caused by an ACTH-producing tumor, as is the case with Cushing's disease and ectopic ACTH syndrome (EAS). Diagnosis and differential diagnosis of Cushing's disease from EAS in ACTH-dependent Cushing's syndrome are thus challenging problems in clinical endocrinology. The diagnostic criteria for Cushing's disease in Japan, established by the working group of the Japan Ministry of Health, Labour and Welfare, were originally reported in 2003 and revised in 2007 and 2010. In addition, criteria for subclinical Cushing's disease were established in Japan in 2010. In this review, we evaluate the usefulness and accuracy of the most recent diagnostic criteria. Previous data suggest that as an initial test of Cushing's syndrome, 0.5 mg dexamethasone is more sensitive than 1 mg in the overnight dexamethasone suppression test (DST). Here, we recommend 0.5 mg plus a plasma cortisol cut-off level of 3 µg/dL as a suitable low-dose overnight DST for screening of all cases of ACTH-dependent Cushing's syndrome in Japan. Recently, standardization of cortisol measurements by the ID-LC/MS/MS method using seven assay kits with standard plasma material containing synthetic hydrocortisone-d4 was carried out in Japan. The resulting relative standard deviation was within 10%. The cut-off value remains valid even after standardization of plasma cortisol measurements. Although the recent diagnostic criteria achieve higher diagnostic specificity, care should be taken since data for Cushing's disease partially overlaps with some cases of EAS. Overall, therefore, this review suggests that the accuracy of each diagnostic test should be considered.

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Influence of Extracellular Matrix Overlay on Phenobarbital-Mediated Induction of CYP2B1, 2B2, and 3A1 Genes in Primary Adult Rat Hepatocyte Culture

Cited by 126 publications

References 0 publications

Regulation of the Major Detoxication Functions by Phenobarbital and 3‐Methylcholanthrene in Co‐Cultures of Rat Hepatocytes and Liver Epithelial Cells

Regulation of the Major Detoxication Functions by Phenobarbital and 3‐Methylcholanthrene in Co‐Cultures of Rat Hepatocytes and Liver Epithelial Cells

Baculovirus vectors repress phenobarbital-mediated gene induction and stimulate cytokine expression in primary cultures of rat hepatocytes

Evaluation of the diagnostic criteria for Cushing’s disease in Japan [Review]

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