Absence Of Phenobarbital Research Articles

Drug–drug interaction comparison between tacrolimus and phenobarbital in different formulations for paediatrics and adults

To compare drug–drug interaction (DDI) between tacrolimus and different formulations of phenobarbital in paediatrics and adults. Physiologically based pharmacokinetics (PBPK) models were used to evaluate DDI between phenobarbital (oral (p.o.) and intravenous (i.v.) formulations) and tacrolimus in paediatrics and adults. All dosing regimens were maintained for 7 days. Compared to i.v. phenobarbital, p.o. phenobarbital could decrease pharmacokinetic (PK) parameters of tacrolimus much more in both paediatrics and adults. On day 7, the results showed that the ratio of Cmax for tacrolimus in the presence and absence of phenobarbital were 0.13 (p.o.) and 0.48 (i.v.), respectively, in paediatrics, while 0.54 (p.o.) and 0.73 (i.v.) in adults, respectively. The ratios of the area under the concentration–time curve (AUC) were 0.06 (p.o.) and 0.18 (i.v.) in paediatrics, while 0.46 (p.o.) and 0.53 (i.v.) in adults, respectively. PK parameters of tacrolimus decreased more significantly in paediatrics compared to adults. In paediatric, phenobarbital had a greater impact on PK of tacrolimus than that in adults. P.o. phenobarbital reduced PK parameters of tacrolimus even more than i.v. administration. In clinical practice, the concentration monitoring and dosage adjustment of tacrolimus should be emphasised when co-administrated with phenobarbital, especially in paediatric or in p.o. formulation. Key points The results indicated that p.o. and i.v. phenobarbital both had a significant DDI with tacrolimus in paediatrics and adults. Phenobarbital had a greater impact on the PK of tacrolimus over time in paediatrics. P.o. administration of phenobarbital can reduce the PK parameters of tacrolimus more.

Latent carcinogenicity of early-life exposure to dichloroacetic acid in mice.

Environmental exposures occurring early in life may have an important influence on cancer risk later in life. Here, we investigated carryover effects of dichloroacetic acid (DCA), a small molecule analog of pyruvate with metabolic programming properties, on age-related incidence of liver cancer. The study followed a stop-exposure/promotion design in which 4-week-old male and female B6C3F1 mice received the following treatments: deionized water alone (dH2O, control); dH2O with 0.06% phenobarbital (PB), a mouse liver tumor promoter; or DCA (1.0, 2.0 or 3.5g/l) for 10 weeks followed by dH2O or PB (n = 20-30/group/sex). Pathology and molecular assessments were performed at 98 weeks of age. In the absence of PB, early-life exposure to DCA increased the incidence and number of hepatocellular tumors in male and female mice compared with controls. Significant dose trends were observed in both sexes. At the high dose level, 10 weeks of prior DCA treatment induced comparable effects (≥85% tumor incidence and number) to those seen after continuous lifetime exposure. Prior DCA treatment did not enhance or inhibit the carcinogenic effects of PB, induce persistent liver cytotoxicity or preneoplastic changes on histopathology or alter DNA sequence variant profiles within liver tumors compared with controls. Distinct changes in liver messenger RNA and micro RNA profiles associated with prior DCA treatment were not apparent at 98 weeks. Our findings demonstrate that early-life exposure to DCA may be as carcinogenic as life-long exposures, potentially via epigenetic-mediated effects related to cellular metabolism.

The connection of β-catenin and phenobarbital in murine hepatocarcinogenesis: a critical discussion of Awuah et al., PLoS ONE 7(6):e39771, 2012

Phenobarbital (PB) is frequently used as a promoter of rodent liver tumors. The mechanisms by which PB exerts its tumorpromoting activity are still not fully understood, but the constitutive androstane receptor (CAR) and b-catenin seem to be essentially involved: Experiments with Car knockout (KO) mice have proven that the presence of CAR is mandatory for PB-mediated tumor promotion (Yamamoto et al. 2004). In mice, PB selects for the outgrowth of hepatocellular tumors with activating mutations in the Ctnnb1 gene, which encodes the transcription factor b-catenin (Aydinlik et al. 2001). Similar observations were made with the PB-like tumor promoter PCB153 (Strathmann et al. 2006). Furthermore, CAR and b-catenin interact in the regulation of enzyme induction and hepatocyte proliferation triggered by PB (Braeuning et al. 2009; Braeuning et al. 2011). In the absence of PB, two groups have studied hepatocarcinogenesis induced by diethylnitrosamine (DEN), a potent tumor initiator, in b-catenin-deficient livers from transgenic mice with conditional hepatocyte-specific KO of Ctnnb1. The unexpected finding of both studies was an increased number of tumors in Ctnnb1 KO mice, which contradicts the classic view of b-catenin as an oncogenic driver of tumor formation (Rignall et al. 2011; Zhang et al. 2010). Possible explanations for this finding are increased inflammation and oxidative stress in the KO mice (Zhang et al. 2010), as well as the increased number of hepatocytes with high levels of O-ethylguanine after DEN injection (Rignall et al. 2011). Subsequent administration of PB promoted tumor formation in livers of wild-type (WT) C3H mice, whereas a lack of tumor promotion was observed in Ctnnb1 KO mice in the same experiment (Rignall et al. 2011). This can be attributed to the fact that the target cells of PB-mediated tumor promotion, namely Ctnnb1-mutated hepatoma cells, are largely absent from Ctnnb1 KO livers. The group of Dr. Monga has recently reported an increase in liver tumor burden of DEN/PB-treated Ctnnb1 KO mice, as compared to WT controls (Awuah et al. 2012). This seemingly contradicts previous findings (Rignall et al. 2011) and thus questions the established connection of PB and b-catenin, at least for readers who are not highly specialized in the field of tumor promotion. In their recent study, the authors treated 2-week-old C57BL/6 mice with a single dose of DEN, followed by chronic exposure to PB (Awuah et al. 2012). The age of mice at DEN injection is highly relevant since, in C3H mice, it has been shown that PB inhibits hepatocarcinogenesis when the initiator DEN is given to 2-week-old mice, whereas it promotes tumorigenesis after DEN injection at 6 weeks of age (Moennikes et al. 2000). Comparably, PB inhibits tumor development in DENinduced (2 weeks) mice from strain B6C3F1, a crossbreed of C3H and C57BL/6 (Diwan et al. 1984; Klaunig et al. 1987, 1988; Lee et al. 1998). The paradoxical phenomenon of tumor inhibition by PB has been reviewed by Lee (2000). Using CD1 mice, however, Tamano et al. (1994) have demonstrated tumor promotion by PB in animals treated with DEN at 2 weeks of age. Therefore, it cannot be excluded that PB has acted as a tumor promoter in the study by Awuah and colleagues. However, the majority of available studies argue against a tumor-promoting activity of PB when using the 2-week DEN treatment regimen, especially since the C57BL/6 strain is rather refractory to liver tumor formation and promotion (Maronpot 2009). A. Braeuning (&) Department of Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Tubingen, Wilhelmstr. 56, 72074 Tubingen, Germany e-mail: albert.braeuning@uni-tuebingen.de

Hormone receptor-like in 96 and Broad-Complex modulate phenobarbital induced transcription of cytochrome P450 CYP6D1 in Drosophila S2 cells

Phenobarbital (PB) is a prototypical inducer for studies of xenobiotic responses in animals. In mammals, the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) have been identified as key transcription factors regulating PB induced transcription of xenobiotic responsive genes. In insects, much less is known about the transcription factors involved in regulating PB induced transcription, although CAR and PXR have a single orthologue hormone receptor-like in 96 (HR96) in Drosophila melanogaster. Using dual luciferase reporter assays in Drosophila Schneider (S2) cells, constructs containing variable lengths of the promoter of the PB inducible cytochrome P450 CYP6D1 were evaluated in the presence and absence of PB. The promoter region between -330 and -280 (relative to the position of transcription start site, +1) was found to be critical for PB induction. Putative binding sites for Drosophila Broad-Complex (BR-C) and deformed (Dfd) were identified within this promoter region using TFsearch. RNA interference (RNAi) treatment of S2 cells in conjunction with CYP6D1 promoter assays showed that suppression of Drosophila HR96 and BR-C transcription in S2 cells resulted in a significant decrease and increase, respectively, of PB induction. Effects of HR96 and BR-C in mediating PB induction were PB specific and PB dependent. This represents new functional evidence that Drosophila HR96 and BR-C can act as an activator and repressor, respectively, in regulating PB induced transcription in insects.

Comparative Study on the Mode of Action of Chlorinated Ethylenes on the Expression of Rat CYP Forms.

Many environmental chemicals (xenobiotics), including polychlorinated ethylenes (CEs), are known to be metabolically intoxicated via the formation of hazardous intermediates (referred to as biological activation), while the expression of metabolic enzymes might be interfered with by xenobiotics at various stages in a system to minimize their adverse biological effects on the host animals. Three CEs, i.e., tetrachloroethylene (PCE), trichloroethylene (TCE) and 1, 1-dichloroethylene (1, 1-DCE), were comparatively studied for their effects on the in vivo expression of CYP forms, which are responsible for their metabolic activation, as well as organospecificities between the liver and lung. Furthermore, their effects on the expression of CYP forms were studied in the animals simultaneously administered with phenobarbital (PB), known as an inducer of CYP2B. Individual CEs were administered intraperitoneally at 0.5 g/kg alone or simultaneously with PB (80 mg/kg/d) to 7-week-old male Wistar rats weighing about 200 g. The testosterone hydroxylase activities, CYP2B- and 2E1-mRNA, and CYP2B- and 2E1-proteins were measured 24 hr after the treatment. Testosterone 2β-hydroxylase (2βTSH) and 16βTSH activities are attributable to the functions of CYP3A2, and CYP2B plus CYP3A2, respectively. The induction of hepatic CYP2B in the PB-treated animals might be masked by the suppressive effect of CYP3A2 in terms of the 16βTSH activity, while the pulmonary 16βTSH activity was confined to the function of CYP2B, which was insensitive to the inducing effect of PB. The inhibition of hepatic 16βTSH activity was observed exclusively in the presence of 1, 1-DCE, especially in PB-coadministered animals, suggesting the preferable suppression of CYP2B. In the lung, however, PCE suppressed the 16βTSH activity in the absence of PB. The expression levels of hepatic CYP2B mRNA and protein were significantly lowered by 1, 1-DCE in the presence of PB. Concerning hepatic CYP2E1, no alternation was observed in the levels of mRNA and protein by any of the CEs in the absence or presence of PB, except for a marked decrease in the amount of mRNA when the rats were treated with 1, 1-DCE in combination with PB. In the lung, both mRNA and protein were not detected under the given assay conditions, as in our previous studies. Based on these results, it is suggested that 1, 1-DCE suppresses the induction of hepatic CYP2B and 2E1 in advance of the transcriptional stage. The expression of pulmonary CYP2B was obstructed by PCE posttranslationally in the absence of PB.

TGF-alpha sustains clonal expansion by promoter-dependent, chemically initiated rat hepatocytes.

A series of promoting and non-promoting barbiturates and hydantoins were examined for their ability to sustain the growth of a phenobarbital (PB)-dependent hepatocyte line in cell culture. The effective liver tumor promoters, pentobarbital, allobarbital and 5-ethyl-5-phenylhydantoin, replaced PB and supported 6/27C1 hepatocyte colony formation in vitro at 52-87% of the level induced by PB. The weak promoters secobarbital and amobarbital supported colony formation at only 11-19% of the PB control. A significant correlation was observed for in vivo and in vitro promotion activities of barbiturates and hydantoins, indicating that clonal expansion by 6/27C1 hepatocytes was promoter-dependent. Cell density also appeared to influence hepatocyte growth in vitro. Hepatocyte colonies acquired the ability to grow in the absence of PB, such that after 10 days incubation with PB, approximately 50% of colonies continued to grow in the absence of promoter. This phenomenon of clone-size-dependent hepatocyte growth suggested the operation of an autocrine growth factor pathway. Addition of the hepatocyte mitogen and autocrine growth factor, transforming growth factor-alpha (TGF-alpha), to culture medium lacking PB induced a dose-dependent increase in 6/27C1 hepatocyte colony formation. At the optimal concentration of 3 ng/ml, TGF-alpha sustained hepatocyte clonal expansion at 84% of the level induced by 2 mM PB. Individual 6/27C1 colonies that grew from single cells in the presence of TGF-alpha were tested for promoter-dependent colony formation. Either PB or TGF-alpha supported colony formation by these cells at similar levels and when combined at optimal concentrations, the response appeared to be saturated. When these factors were tested in combination at suboptimal concentrations, the two compounds were additive for supporting colony formation by the parental 6/27C1 line. The ability of TGF-alpha to replace PB and sustain hepatocyte clonal expansion was confirmed with the tumorigenic 6/15 hepatocyte line. These results suggest that TGF-alpha and PB may promote hepatocarcinogenesis by stimulating a common signal transduction pathway.

Phenobarbital modulates the type of cell death by rat hepatocytes during deprivation of serum in vitro

An immortal line of chemically altered rat hepatocytes was used to study the effects of the liver tumor promoter, phenobarbital (PB), on hepatocyte growth and viability in vitro. When the serum concentration in medium was changed from 10% to 0.5%, cell proliferation decreased and hepatocytes died. Death of the hepatocytes occurred after 2 days in low-serum medium. PB appeared to control the type of cell death that occurred. In the absence of PB in low-serum medium, most dead cells had morphological changes that are characteristic of necrosis as determined by both light and electron microscopy. In the presence of PB, the dead cells had alterations typical of apoptosis. Biochemical features of cell death in low-serum medium were also analyzed. DNA isolated from cells in low serum with PB showed nucleosome-length fragments after gel electrophoresis, whereas DNA from cells in low serum without PB appeared as randomly degraded fragments. Although proliferation of hepatocytes in low-serum decreased by 75%, the appearance of apoptosis in the presence of PB was associated with increased expression of the c-myc gene. Based on these observations, we conclude that PB can modulate the type of cell death that occurs after serum deprivation in this line of immortal rat hepatocytes. PB seemed to prevent necrotic cell death in low serum, and cells died through a gene-directed pathway of apoptosis.

Phenobarbital modulates the type of cell death by rat hepatocytes during deprivation of serum in vitro

An immortal line of chemically altered rat hepatocytes was used to study the effects of the liver tumor promoter, phenobarbital (PB), on hepatocyte growth and viability in vitro. When the serum concentration in medium was changed from 10% to 0.5%, cell proliferation decreased and hepatocytes died. Death of the hepatocytes occurred after 2 days in low-serum medium. PB appeared to control the type of cell death that occurred. In the absence of PB in low-serum medium, most dead cells had morphological changes that are characteristic of necrosis as determined by both light and electron microscopy. In the presence of PB, the dead cells had alterations typical of apoptosis. Biochemical features of cell death in lowserum medium were also analyzed. DNA isolated from cells in low serum with PB showed nucleosome-length fragments after gel electrophoresis, whereas DNA from cells in low serum without PB appeared as randomly degraded fragments. Although proliferation of hepatocytes in low-serum decreased by 75%, the appearance of apoptosis in the presence of PB was associated with increased expression of the c-myc gene. Based on these observations, we conclude that PB can modulate the type of cell death that occurs after serum deprivation in this line of immortal rat hepatocytes. PB seemed to prevent necrotic cell death in low serum, and cells died through a gene-directed pathway of apoptosis.

Effect of Rat Developmental Stage at Initiation on the Expression of Biochemical Markers during Liver Tumor Promotion

The phenotypic response of rat liver to a carcinogenic protocol involving initiation/selection and promotion with and without phenobarbital (PB) feeding was studied in pubertal and adult male rats. Considering the early presence of preneoplastic nodular areas, it appeared that pubertal rats, initiated at 6-7 weeks, presented a higher susceptibility to the protocol than adult rats initiated at 9-10 weeks. Altered liver phenotype was characterized by: (1) gamma-glutamyl-transpeptidase (GGT) and glutathione S-transferase (GST) activities; (2) the expression of two forms of cytochrome P-450; de novo PB-inducible P-450 II B 1,2 and P-450 II C 7 normally expressed in 45-day-old rats and PB-inducible, and (3) the expression of albumin and alpha-fetoprotein cDNAs. In the absence of PB, the susceptibility of pubertal rat liver to hepatocarcinogenesis was related to a special metabolic phenotype enriched in GGT and GST activities by comparison with the quasi-normal expression of both P-450s. Adult rat liver presented a less altered pattern closer to that of noninitiated rat liver. During PB promotion, the loss of PB inducibility of P-450 II C 7 in pubertal rat liver suggested that the hormonal status of the animals could interact with initiation to modulate specific gene expression. The late phase of PB promotion revealed the loss of highly differentiated functions (P-450s, albumin), whereas enzymatic markers associated with preneoplastic foci showed a persistent high expression.

Phenobarbital-dependent proliferation of putative initiated rat hepatocytes.

The mitogenic effects of phenobarbital (PB) were examined using cultures of putative initiated hepatocytes that proliferate and form colonies under conditions in which normal hepatocytes senesce and die. The frequencies of colony-forming hepatocytes in primary cultures isolated 2 weeks after initiation with methyl(acetoxymethyl)nitrosamine or benzo[a]pyrene-7,8-diol-9,10-epoxide(anti) were in the range of 2-38 per million in the presence of PB. Colony-formation frequencies were 0.1 per million in the absence of PB. Proliferative hepatocyte colonies were not observed in cultures grown in serum-free medium containing PB, epidermal growth factor, nor-epinephrine and insulin. The requirement for PB was characterized further using secondary cultures of hepatocytes that had been isolated from a liver 5 weeks after initiation. The colony-forming efficiency of these hepatocytes was about 10% in the presence of 2 mM PB and less than 0.2% in its absence. Colony formation displayed a linear response to concentrations of PB in the range of 0.5-2 mM and a decline above the optimal 2 mM concentration. Autoradiography was used to determine the percentages of hepatocytes in secondary cultures that synthesized DNA in the presence or absence of PB. By the third day after seeding as single cells, hepatocytes exhibited a labeling index of about 50% and this level of labeling was preserved for up to 2 weeks after seeding. Very few hepatocytes were found to synthesize DNA in the absence of PB and most senesced. A small fraction of the colony-forming hepatocytes continued to proliferate in the absence of PB and formed colonies with a high labeling index. These results suggest that the proliferation of initiated hepatocytes in vivo may be conditional upon the presence of the hepatic tumor promoter, PB.

Influence of phenobarbital on the hepatic handling of [3H]vitamin D3 in the dog.

The effect of phenobarbital (PB) on the hepatic handling of vitamin D3 (D3) and its metabolism to 25-hydroxyvitamin D3 [25(OH)D3] was studied in eight mongrel dogs. The hepatic uptake and clearance of [3H]D3 were evaluated by the multiple indicator-dilution curve technique, and the formation of [3H]25(OH)D3 was evaluated by sampling the hepatic effluent. The hepatic enzyme induction was assessed in six dogs by the 14CO2 breath excretion test. The results show that the hepatic uptake of [3H]D3 was not significantly affected but that its hepatic clearance was significantly increased during PB treatment. The [3H]25(OH)D3 production was increased during PB administration by a factor of 3-5 times over the pre- or post-PB period. Evaluation of the enzyme induction produced by PB revealed that two of the dogs studied had a blunted response to PB; furthermore, these dogs were the only animals that showed no increase in [3H]25(OH)D3 production during PB treatment and that in the presence of similar serum PB, endogenous 25(OH)D3 and 1,25-dihydroxyvitamin D3 pools, and hepatic uptake and clearance of [3H]D3. Strong positive correlation coefficients were observed between the breath excretion of 14CO2 and the [3H]25(OH)D3 production during PB treatment, whereas no correlation was present in the absence of PB. These observations show that, in most animals, PB is accompanied by an increased hepatic clearance of [3H]D3 and by an increased production of [3H]25(OH)D3. The data obtained during the present study also show that the response to PB is heterogeneous and that some animals escaped PB-mediated enzyme induction.(ABSTRACT TRUNCATED AT 250 WORDS)

The phenotypic stability of altered hepatic foci: effect of the short-term withdrawal of phenobarbital and of the long-term feeding of purified diets after the withdrawal of phenobarbital.

The effects of the short-term withdrawal of phenobarbital (PB) and of the feeding of purified diets during the long-term withdrawal of dietary PB on the stability of altered hepatic foci (AHF) were studied. In both experiments female CD rats initially received an intragastric dose of diethylnitrosamine (10 mg/kg) 20 h after being subjected to partial hepatectomy. In the short-term study rats were fed 0.05% PB in a cereal-based diet for 6 months; at this time half of the rats were killed, whereas the other half were withdrawn from PB for 10 days before sacrifice. Withdrawing PB for 10 days resulted in a decrease in the number and volume of AHF, particularly those which stained positively for gamma-glutamyltranspeptidase (GGT). In the long-term experiment rats were fed 0.05% phenobarbital in a cereal-based diet for 3 months; they were then withdrawn from the cereal-based diet containing PB and fed either a low-fat or a high-fat purified diet without PB for 8 months. At this time the number and volume of AHF were much less than that seen at the time of PB withdrawal. In addition, the distribution of phenotypes was altered: the percentage of foci containing GGT as a marker decreased dramatically. These results indicate that certain phenotypic characteristics of AHF, specifically their observable number and total volume, rapidly decrease after the withdrawal of PB from rats fed a cereal-based diet and that the feeding of purified diets after such PB withdrawal does not result in the reappearance of AHF. These studies suggest that the phenotype, volume and observable number of AHF may change in the absence of PB as compared with its presence in the diet and that some cereal-based diets may contain unspecified agents which 'stabilize' or enhance the phenotypic appearance of AHF.

The effect of barbiturates on microtubular assembly

Purified tubulin was prepared from fresh pig brain. Purity was determined by gel electrophoresis. Polymerization and depolymerization of microtubules were studied as a function of the presence (10 −3 M ) and absence of phenobarbital. The drug markedly depressed tubular assembly. Dialysis of presumably protein bound phenobarbital affected recovery of polymerization properties favorably, but only to 65% of control levels. Protein assembly was determined by biophysical methodology as incubation of tubulin proceeded at 37°C. Phenobarbital added to assembly medium interrupted polymerization and reduced both viscosity and turbidity. The various preparations were monitored by transmission electron microscopy. The lessened production of tubules in presence of drug was characterized by the presence of tubules of only 10 μ m in length. The significance of this drug effect on CNS development is discussed.

Induction by phenobarbital of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) cytochrome c reductase in regenerating rat liver

The administration of a single dose of phenobarbital (100 mg/kg) to rats resulted in a 60 per cent increase in the activity of NADPH cytochrome c reductase within 48 hr after the inducer. The imposition of the competitive stress of partial hepatectomy of animals 1 hr after phenobarbital treatment prematurely terminated the inductive process and reduction of NADPH cytochrome c reductase activity ensued thereafter, reaching control non-induced levels by 49 hr. Partial hepatectomy lowered the basal level of cytochrome b 5 by 30 per cent at 24–72 hr after the operation, but no alteration in the level of cytochrome b 5 was produced by phenobarbital in either sham-operated or partially hepatectomized rats. The rate of incorporation of [4,5- 3H] l-leucine into trypsin-solubilized microsomal protein at 2, 4, 6 and 9 hr after treatment with phenobarbital was equivalent to that of untreated sham-operated rats ; whereas, at 25 hr after the inducer a significant increase in the rate of incorporation of leucine into the trypsin-solubilized microsomal protein of sham-operated animals was obtained. No significant change in the incorporation of leucine into microsomal protein was observed in the livers of partially hepatectomized rats in either the presence or absence of phenobarbital. Measurement of the loss of microsomal radioactivity following labeling with [ 14C-guanidino] l-arginine revealed a similar rate of decay at 25–73 hr after administration of phenobarbital in the livers of sham-operated and partially hepatectomized animals.