Induction Of Hepatic Cytochrome P-450 Research Articles

Exploring the Use of Serum-Derived Small Extracellular Vesicles as Liquid Biopsy to Study the Induction of Hepatic Cytochromes P450 and Organic Anion Transporting Polypeptides.

Liver-derived small extracellular vesicles (sEVs), prepared from small sets of banked serum samples using a novel two-step protocol, were deployed as liquid biopsy to study the induction of cytochromes P450 (CYP3A4, CYP3A5, and CYP2D6) and organic anion transporting polypeptides (OATP1B1 and OATP1B3) during pregnancy (nonpregnant (T0), first, second, and third (T3) trimester women; N=3 each) and after administration of rifampicin (RIF) to healthy male subjects. Proteomic analysis revealed induction (mean fold-increase, 90% confidence interval) of sEV CYP3A4 after RIF 300mg×7days (3.5, 95% CI = 2.5-4.5, N=4, P=0.029) and 600mg×14days (3.7, 95% CI = 2.1-6.0, N=5, P=0.018) consistent with the mean oral midazolam area under the plasma concentration time curve (AUC) ratio in the same subjects (0.28, 95% CI = 0.22-0.34, P<0.0001; and 0.17, 95% CI = 0.13-0.22, P<0.0001). Compared with CYP3A4, liver sEV CYP3A5 protein (subjects genotyped CYP3A5*1/*3) was weakly induced (≤1.5-fold). It was also possible to measure liver sEV-catalyzed dextromethorphan (DEX) O-demethylation to dextrorphan (DXO), correlated with sEV CYP2D6 expression (r=0.917, P=0.0001; N=10) and 3-hour plasma DXO-to-DEX concentration ratio (r=0.843, P=0.002, N=10), and show that CYP2D6 was not induced by RIF. Nonparametric analysis of liver sEV revealed significantly higher CYP3A4 (3.2-fold, P=0.003) and CYP2D6 (3.7-fold, P=0.03) protein expression in T3 vs. T0 women. In contrast, expression of both OATPs in liver sEV was unaltered by RIF administration and pregnancy.

Pathway-Based Approaches for Assessing Biological Hazards of Complex Mixtures of Contaminants: A Case Study in the Maumee River.

Assessment of ecological risks of chemicals in the field usually involves complex mixtures of known and unknown compounds. We describe the use of pathway-based chemical and biological approaches to assess the risk of chemical mixtures in the Maumee River (OH, USA), which receives a variety of agricultural and urban inputs. Fathead minnows (Pimephales promelas) were deployed in cages for 4 d at a gradient of sites along the river and adjoining tributaries in 2012 and during 2 periods (April and June) in 2016, in conjunction with an automated system to collect composite water samples. More than 100 industrial chemicals, pharmaceuticals, and pesticides were detected in water at some of the study sites, with the greatest number typically found near domestic wastewater treatment plants. In 2016, there was an increase in concentrations of several herbicides from April to June at upstream agricultural sites. A comparison of chemical concentrations in site water with single chemical data from vitro high-throughput screening (HTS) assays suggested the potential for perturbation of multiple biological pathways, including several associated with induction or inhibition of different cytochrome P450 (CYP) isozymes. This was consistent with direct effects of water extracts in an HTS assay and induction of hepatic CYPs in caged fish. Targeted in vitro assays and measurements in the caged fish suggested minimal effects on endocrine function (e.g., estrogenicity). A nontargeted mass spectroscopy-based analysis suggested that hepatic endogenous metabolite profiles in caged fish covaried strongly with the occurrence of pesticides and pesticide degradates. These studies demonstrate the application of an integrated suite of measurements to help understand the effects of complex chemical mixtures in the field. Environ Toxicol Chem 2021;40:1098-1122. © 2020 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Microplastic-associated trophic transfer of benzo(k)fluoranthene in a limnic food web: Effects in two freshwater invertebrates (Daphnia magna, Chironomus riparius) and zebrafish (Danio rerio)

The continuously growing plastic production and incomplete recycling processes open manifold entry routes for microplastic particles (MPs) into the environment. Since knowledge on trophic transfer of contaminants sorbed to MPs is still insufficient for freshwater systems, the transfer of the model pollutant benzo(k)fluoranthene (BkF) sorbed to polymethyl methacrylate (PMMA) particles in a limnic food web was investigated: Two freshwater invertebrates (Daphnia magna and Chironomus riparius larvae) were selected and either left untreated, exposed to pristine PMMA, PMMA-associated BkF, or exposed to dissolved BkF (BkFaq). As second-level consumers, zebrafish (Danio rerio) were fed twice daily with pre-treated invertebrates over two days. Induction of hepatic cytochrome P450 by BkF was determined as 7-ethoxy-O-resorufin deethylase (EROD) activity. Both invertebrate species readily ingested PMMA particles, tracked via fluorescence microscopy and accumulated BkFaq, measured via GC–MS. Fluorescence signals in gastrointestinal tracts of zebrafish were quantified with confocal laser scanning microscopy (CLSM). The fluorescence signal in gastrointestinal tracts of zebrafish was not altered, whereas, EROD activity was significantly induced when zebrafish were fed with Chironomus riparius, pre-exposed to BkFaq. Trophic exposure scenarios with BkF sorbed to PMMA did not result in any alterations of investigated endpoints in both invertebrate species and zebrafish compared to controls. Given that BkF amounts were in the low ng-range, as detected by GC–MS, the transport of MP-sorbed BkF to zebrafish was less effective than direct exposure to waterborne BkFaq, and the potential threat of trophic transfer of substances such as BkF in limnic food webs may have been overestimated.

Identification of Compounds in Coleus forskohlii Extract Involved in the Induction of Hepatic CYP and Fatty Liver in Mice.

Coleus forskohlii extract (CFE), a popular weight-loss herbal product, induces hepatic cytochrome P450 (CYP) and fatty liver in mice; however, its main bioactive ingredient, forskolin, does not show such effects. To ensure the safety of CFE as a dietary supplement, identification of the compounds implicated in the induction of hepatic CYP and fatty liver is required. In this study, we separated a crude CFE extract into 5 fractions (Fr.) by column chromatography and administered the fractions to mice for one week to assess their ability to induce CYP and fatty liver. CYP induction was detected for all fractions, indicating that many compounds may be involved in CYP induction, while fatty liver was only detected for Fr. 2. Further isolation and purification of Fr. 2 by column chromatography identified 14-deoxycoleon U as a major compound and crocetin dialdehyde as a pigment compound. An in vivo mouse study revealed that crocetin dialdehyde had no effect on the liver and, as 14-deoxycoleon U was the major compound in Fr. 2, it is likely that the active compound inducing fatty liver in CFE is 14-deoxycoleon U. These findings will facilitate the preparation of standardized safe CFE ingredients for dietary supplements.

Effects of duration of phenytoin administration on mRNA expression of cytochrome P450 and P-glycoprotein in the liver and small intestine of rats

Phenytoin (5,5-diphenylhydantoin; DPH) induces expression of cytochromes P450 (CYPs). Interactions between DPH and tacrolimus suggested that the persistence of CYP induction after discontinuation of DPH is dependent on the history of administration and dosing period of DPH. However, the relationship between the duration of DPH administration and expression of CYPs in the liver and small intestine of rats is not known. Alterations in levels of P-glycoprotein (P-gp; MDR1; ABCB1) as well as CYPs cause drug interactions in the small intestine. We examined the effects of the duration of DPH administration on expression of CYPs and P-gp in the liver and small intestine of rats. Rats were treated with DPH (100 mg/kg, peroral (p.o.) twice a day (b.d.)) for 2, 4, 8, and 16 d. mRNA levels of CYPs and P-gp were examined using the total RNA extracted from the liver and duodenum 2 h and 24 h after the final administration of DPH. CYP3A activities were determined using microsomes. DPH administration for 2 d and 4 d markedly increased mRNA levels of CYPs such as CYP3A1, CYP3A2, CYP2B1, and CYP2B2 in the liver. A relatively long duration of DPH administration (8 d and 16 d) resulted in abolition of the induction of hepatic CYP but increased CYP3A activities were maintained. These results suggest that the duration of DPH administration could be an important determinant of hepatic CYP induction.

Influence of Dietary Macronutrients on Induction of Hepatic Drug Metabolizing Enzymes by Coleus forskohlii Extract in Mice

From studies in mice, we have reported that Coleus forskohlii extract (CFE), a popular herbal weight-loss ingredient, markedly induced hepatic drug metabolizing enzymes, especially cytochrome P450 (CYP), and interacted with co-administered drugs. This study was designed to examine how the induction of drug metabolizing enzymes by CFE was influenced by different levels of macronutrients in the diet. Mice were fed a non-purified diet or semi-purified diet with and without CFE (0.3-0.5%) for 14-18 d, and changes in the ratio of liver weight to body weight, an indicator of hepatic CYP induction, and hepatic drug metabolizing enzymes were analyzed. The ratio of liver weight to body weight, content and activities of CYPs, and activity of glutathione S-transferase were higher in a semi-purified standard diet (AIN93G formula) group than in high sucrose (62.9%) and high fat (29.9%) diet groups. Different levels of protein (7%, 20%, and 33%) in the diets did not influence CFE-induced CYP induction or increase the ratio of liver weight to body weight. The effect of CFE on the ratio of liver weight to body weight was higher with a semi-purified diet than with a non-purified diet, and was similar between dietary administration and intragastric gavage when the CFE dose and the diet were the same. There was a positive correlation between CFE-induced CYP induction and the content of starch in the diets, suggesting that dietary starch potentiates CFE-induced CYP induction in mice. The mechanism of enhanced CYP induction remains unclear.

Altered expression of GADD45 genes during the development of chemical-mediated liver hypertrophy and liver tumor promotion in rats

The purpose of our study was to examine the altered gene expression associated with nongenotoxic chemical-mediated liver hypertrophy and successive liver tumor promotion. Five-week-old male rats were fed a basal diet or a diet containing phenobarbital (PB) or clofibrate (CF) for 3 days, 4 weeks, and 13 weeks. Hepatic expression profiling of cell growth- and stress-related genes, as well as those involved in xenobiotic metabolism, was performed by DNA microarray and/or real time quantitative reverse transcription-polymerase chain reaction. The induction of liver hypertrophy and hepatic cytochrome P450 (CYP) isoforms (CYP2B1/2B2 for PB and CYP4A1 for CF) by PB and CF were clearly observed at all the treatment periods examined. Genes encoding DNA damage-inducible 45 (GADD45) family proteins, in particular GADD45g (GADD45 gamma) were down-regulated by treatment with either PB or CF for 4 and 13 weeks. The chemical-mediated development of liver hypertrophy, induction of hepatic CYPs, and suppression of hepatic GADD45g gene at week 13 disappeared at 4 weeks following cessation of the chemical treatment. Additionally, DNA microarray data indicated that cell cycle-related genes such as cyclins CCNB1 and CCNA2 and cyclin-dependent kinase inhibitor CDKN3 were also down-regulated by treatment with either PB or CF at 13 weeks. Since GADD45 functions as a chemical and radiation stress sensor by interacting with cyclins and cyclin-dependent kinase inhibitors, the decrease in the gene expression of GADD45g mRNA observed in this study, may be associated with nongenotoxic chemical-induced tumor promotion of hepatocarcinogenesis rather than liver hypertrophy.

Bilobalide in ginkgo biloba extract is a major substance inducing hepatic CYPs

In a search for substances related to the marked induction of hepatic cytochrome P450 (CYP) by ginkgo biloba extract (GBE), mice were given either GBE (1000 mg kg(-1)) or fractions of GBE for 5 days. The content and activity of CYPs were induced markedly by a bilobalide-rich fraction, but not by flavonoid-rich fractions. The level of induction by the bilobalide-rich fraction was almost the same as that induced by the unfractionated GBE, suggesting that bilobalide is largely responsible for the CYPs induction. To confirm these findings, mice were given various doses of bilobalide (10.5, 21 and 42 mg kg(-1)), or GBE (1000 mg kg(-1), containing bilobalide at 42 mg kg(-1)). Treatment with bilobalide induced CYPs markedly and in a dose-dependent manner, and the level of induction was quite similar between bilobalide (42 mg kg(-1)) and GBE. Treatment with GBE and with bilobalide greatly induced pentoxyresorufin O-dealkylase activity. These findings indicate that bilobalide is the major substance in GBE that induces hepatic CYPs.

Time-Dependent Induction of Hepatic Cytochrome P450 Enzyme Activity and mRNA Expression by Bilobalide in Rats

A single dose by gavage of bilobalide (30 mg/kg) was found to produce a time-dependent induction of hepatic cytochrome P450 (CYP) enzyme activity and protein expression in rats. An RT-PCR study further showed that mRNA expression of CYP2B was maximal at 6 h. Plasma and liver bilobalide concentration in rats following administration of Ginkgo biloba extract equivalent to bilobalide of approximately 40 mg/kg showed a similar response to that exhibited by mRNA expression. These findings suggest that bilobalide markedly induced hepatic CYPs, but the induction could be mitigated due to rapid elimination from the liver.

Sterol regulatory element binding protein 1 interacts with pregnane X receptor and constitutive androstane receptor and represses their target genes

Sterol regulatory element binding protein 1 (SREBP-1) is a lipogenic transcription factor of the basic helix-loop-helix family. SREBP-1 binds to sterol regulatory elements (SREs) in the promoter of lipogenic genes and induces fatty acid and triglyceride synthesis. Decreased drug clearance has been observed in obese and other dyslipidemic rodents as well as in diabetic, obese or overfed rodents. A hallmark of these conditions is increased expression of SREBP-1 in the liver. We therefore searched for a possible link between regulation of cytochromes P450 (CYPs) and SREBP-1. We combined gene expression analysis, lipid analysis, effects of high levels of SREBP-1 in hepatocyte cultures to characterize the effects and protein interaction and chromatin immunoprecipitation assays to define the underlying mechanism. Finally, mice were fed a diet enriched in cholesterol to demonstrate the relevance of our data in vivo. By analyzing gene expression and lipids in cholesterol-fed mice or transfection of recombinant SREBP-1 in hepatocyte cultures the effect on CYPs was characterized. By use of protein interaction assays and chromatin immunoprecipitation the underlying mechanism was defined. We observed that SREBP-1 represses drug-mediated induction of hepatic CYPs, mainly members of the 2B and the 3A subfamilies. These drugs induce transcription of CYPs and other drug metabolizing enzymes via activation of the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Here we report that the activation of SREBP-1 by insulin or cholesterol in mouse liver and primary human hepatocytes inhibits the transcriptional effects in PXR and CAR. Our results suggest that SREBP-1 functions as a non-DNA binding inhibitor and blocks the interaction of PXR and CAR with cofactors such as steroid receptor coactivator 1. Consequently, mRNA induction of CYPs by drugs and other xenochemicals is impaired. We conclude that PXR and CAR respond to lipid accumulation by direct interaction with SREBP-1 and show that drug metabolism and lipid metabolism are interconnected within a complex network of transcriptional regulators.

Effects of Helicobacter infection on developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Holtzman rats

To obtain accurate results from experiments in animal models, all potential confounding variables must be identified and controlled. The authors examined the effects of Helicobacter infection on developmental toxicity resulting from exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent and ubiquitous environmental contaminant. They administered different doses of TCDD to timed pregnant Helicobacter-positive and Helicobacter-negative Holtzman rats and evaluated fetal and neonatal viability. They also assessed hepatic cytochrome P450 induction and activity of the gene Cyp1a1, which are classic indicators of TCDD exposure. All rats were affected by TCDD, and Helicobacter infection seemed to have little influence on rats' susceptibility to the compound.

Persistent induction of hepatic and pulmonary phase II enzymes by 3-methylcholanthrene in rats.

We reported earlier that exposure of rats to 3-methylcholanthrene (MC) causes sustained induction of hepatic cytochrome P450 (CYP)1A expression for up to 45 days by mechanisms other than persistence of the parent MC (Moorthy, J. 2000. Pharmacology. Exp. Ther. 294, 313-322). The CYP1A genes are members of the Ah gene battery that also encode CYP1B1 and phase II enzymes such as glutathione S-transferase (GST-alpha), UDP glucuronyl transferase (UGT)1A, NAD(P)H (nicotinamide adenine dinucleotide phosphate, reduced):quinone oxidoreductase I (NQO1), aldehyde dehydrogenase (ALDH), etc. Therefore, in this investigation, we tested the hypothesis that MC elicits persistent induction of CYP1B1 and phase II genes, which are in part regulated by the Ah receptor (AHR). Female Sprague-Dawley rats were treated with MC (100 mumol/kg), ip, once daily for 4 days, and expression of CYP1B1 and several phase II (e.g., GST-alpha, NQO1) genes and their corresponding proteins were determined in lung and liver. The major finding was that MC persistently induced (3- to 10-fold) the expression of several phase II enzymes, including GST-alpha, NQO1, UGT1A1, ALDH, and epoxide hydrolase in both tissues for up to 28 days. However, MC did not elicit sustained induction of CYP1B1. Our results thus support the hypothesis that MC elicits coordinated and sustained induction of phase II genes presumably via persistent activation of the AHR, a phenomenon that may have implications for chemical-induced carcinogenesis and chemopreventive strategies in humans.

Undernutrition enhances alcohol-induced hepatocyte proliferation in the liver of rats fed via total enteral nutrition

To assess the relative contributions of undernutrition and ethanol (EtOH) exposure to alcohol-induced hepatotoxicity, female Sprague-Dawley rats were intragastrically infused liquid diets containing 187 or 154 kcal.kg(-3/4).day(-1) with or without 11 g.kg(-1).day(-1) EtOH. EtOH clearance was impaired in the 154 kcal.kg(-3/4).day(-1) EtOH group (P < or = 0.05). A combination of undernutrition and EtOH also increased the induction of hepatic cytochrome P-450 (CYP)2E1 and CYP4A1 mRNA, apoprotein, and activities (P < or = 0.05). This was accompanied by increased oxidative stress (P < or = 0.05). The severity of liver steatosis, macrophage infiltration, and focal necrosis was comparable in both EtOH groups. Alanine aminotransferase levels were elevated (P < or = 0.05) but did not significantly differ between the two EtOH groups. TUNEL analysis also demonstrated a comparable increase in apoptosis in the two EtOH groups (P < or = 0.05). The development of alcohol-induced liver pathology was accompanied by little change in fatty acid (FA) synthesis or degradation at 187 kcal.kg(-3/4).day(-1) but at 154 kcal.kg(-3/4).day(-1) was accompanied by decreased expression of FA synthesis genes and increased expression of peroxisome proliferator-activated receptor-alpha (PPAR-alpha)-regulated FA degradation pathways (P < or = 0.05). In addition, 154 kcal.kg(-3/4).day(-1) EtOH group livers exhibited greater hepatocyte proliferation (P < or = 0.05). We conclude that undernutrition does not exacerbate alcoholic steatohepatitis despite additional oxidative stress produced by an increased induction of CYP2E1 and CYP4A1. However, enhanced ethanol-induced cellular proliferation, perhaps as a result of enhanced PPAR-alpha signaling, may contribute to an increased risk of hepatocellular carcinoma in undernourished alcoholics.

The pharmacokinetics of nelfinavir and M8 during pregnancy and post partum

The objective of this study was to explore the pharmacokinetics of nelfinavir and its active metabolite hydroxy-t-butylamidenelfinavir (M8) during pregnancy and post partum. Eleven human immunodeficiency virus type 1-infected pregnant women receiving 1250 mg nelfinavir twice daily were enrolled. Pharmacokinetics of nelfinavir and M8 were assessed over a 12-hour period during pregnancy (median, 32 weeks' gestation; range, 31-36 weeks) and post partum (median, 8 weeks post partum; range, 6-15 weeks). Drug concentrations were analyzed by HPLC coupled to tandem mass spectroscopy, and pharmacokinetic parameters were calculated by use of noncompartmental methods. The median area under the plasma concentration-time curve from 0 to 12 hours (AUC 0-12), the maximal plasma concentration (C max), and the concentration at the end of the dosing interval (C 12) for nelfinavir post partum were 33.5 h . microg/mL, 5.80 microg/mL, and 1.40 microg/mL, respectively. The values for the geometric mean ratio (GMR) (third trimester/post partum) for the nelfinavir AUC 0-12 , C max , and C 12 were 0.76 (90% confidence interval [CI], 0.54-1.06), 0.81 (90% CI, 0.57-1.15), and 0.43 (90% CI, 0.25-0.76), respectively. The GMR values for the M8 AUC 0-12 , C max , and C 12 were 0.32 (90% CI, 0.18-0.55), 0.31 (90% CI, 0.19-0.51), and 0.30 (90% CI, 0.14-0.64), respectively. The median ratio values of the AUC 0-12 of M8 and nelfinavir (M8/nelfinavir) during the third trimester and post partum were 11% and 27%, respectively (GMR, 0.42 [90% CI, 0.33-0.53]). Nelfinavir exposure was reduced during pregnancy, and the reduction was statistically significant for C 12 . M8 concentrations were about 70% lower during pregnancy compared with post partum, suggesting either induction of hepatic cytochrome P450 (CYP) 3A4 or inhibition of CYP2C19, or both, during pregnancy. Because 8 of 11 women had subtherapeutic nelfinavir trough concentrations during pregnancy, the safety and efficacy of therapeutic drug monitoring should be investigated.

Blarina brevicauda as a biological monitor of polychlorinated biphenyls: evaluation of hepatic cytochrome P450 induction.

We assessed the value of short-tailed shrews (Blarina brevicauda) as a possible biomonitor for polychlorinated biphenyl pollution through measurement of the induction of hepatic cytochrome P450 and associated enzyme activities. First, we checked the inducibility of four monooxygenases (benzyloxyresorufin-O-dealkylase [BROD], ethoxyresorufin-O-dealkylase [EROD], methoxyresorufin-O-dealkylase [MROD], and pentoxyresorufin-O-dealkylase [PROD]) by measuring the activity of these enzymes in hepatic microsomes prepared from shrews injected with beta-naphthoflavone (betaNF) or phenobarbital (PB), typical inducers of cytochrome P4501A (CYP1A) and CYP2B enzyme families, respectively. Enzyme activity was induced in shrews that received betaNF but not in shrews that received PB; PROD was not induced by either exposure. Later, shrews were exposed to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1242:1254, in 1:2 ratio) at 0.6, 9.6, and 150 ppm in food, for 31 d. Induction in these shrews was measured by specific enzyme activity (BROD, EROD, and MROD) in hepatic microsomes, by western blotting of solubilized microsomes against antibodies to CYP1A or CYP2B, and by duration of sodium pentobarbital-induced sleep. These three CYP enzymes were induced in shrews by PCBs at similar levels of exposure as in cotton rat (Sigmodon hispidus). Neither sleep time nor the amount of CYP2B family protein were affected by PCB exposure. Blarina brevicauda can be a useful biomonitor of PCBs that induce CYP1A, especially in habitats where they are the abundant small mammal.

Accumulation of tributyltin in olive flounder, Paralichthys olivaceus: its effect on hepatic cytochrome P450.

Accumulation of tributyltin (TBT) was determined in liver of olive flounder exposed to TBT (3.65, 36.5, 365, 3,650, and 7,300 ng Sn/L) for 10 or 30 days, followed by 60 days depuration. Effect of TBT on hepatic cytochrome P450 content was also measured in liver of olive flounder. TBT was highly accumulated in liver of fish during the 10- to 30-day exposures, and hepatic cytochrome P450 content decreased with increasing TBT concentration. Hepatic cytochrome P450 contents were affected in olive flounder exposed to even environmentally relevant TBT concentrations, such as 3.65 ng Sn/L. In addition, the liver TBT levels demonstrated strong negative correlation to the hepatic cytochrome P450 content. The effects started to appear from 20 ng Sn/g dry weight of TBT in liver. Tributyltin concentrations and hepatic cytochrome P450 were also determined in feral fine-spotted flounder. The relationship between other organic pollutants known as cytochrome P450 inducers (e.g., polychlorinated biphenyls) as well as TBT and hepatic cytochrome P450 in the feral fish implied that TBT even at ppt level could impose antagonistic effects on hepatic cytochrome P450 induction.

Reduced urinary 6β-hydroxycortisol to cortisol ratios in patients with liver cirrhosis

While the urinary concentration ratio of 6β-hydroxycortisol (6β-HC) to cortisol (C) in 24-h urine samples is an established parameter for the induction of hepatic cytochrome P450 (CYP) 3A activity, it remains unclear whether it would be reduced in association with chronic liver damages. To clarify this issue we measured this parameter in 12 patients with chronic hepatitis, 15 patients with liver cirrhosis and 12 healthy subjects. Urinary 6β-HC and C were assayed with high-performance liquid chromatography with ultraviolet absorption. Multiple regression analysis was performed to search for which biochemical or demographic parameter(s) would be associated with the overall variability of urinary 6β-HC/C ratios. Results showed that the mean (±S.D.) 6β-HC/C ratio obtained from the patients with liver cirrhosis (4.3±2.6), but not that from patients with chronic hepatitis (7.4±7.2), was significantly ( P<0.05) lower than that obtained from the healthy controls (11.8±8.3). It was also revealed that among the clinical parameters examined (e.g. serum transaminases concentrations) only serum albumin concentrations were correlated significantly ( R=0.61, P<0.05) with the urinary 6β-HC/C ratio. In conclusion, the present study implies that that the reduced urinary 6β-HC/C ratios in patients with liver cirrhosis may be associated with attenuated hepatic CYP3A activity in these patients.

Environmental polychlorinated biphenyl exposure and cytochromes P450 in raccoons (Procyon lotor)

An investigation involving raccoons as a sentinel species at the Paducah Gaseous Diffusion Plant (PGDP) and Ballard Wildlife Management Area in western Kentucky (USA) delineated the extent of exposure to polychlorinated biphenyls (PCBs). Three separate measures of hepatic cytochrome P450 (CYP) induction were used to evaluate raccoon physiological responses to PCB exposure. Hepatic CYP induction was estimated via determination of total CYP, dealkylase activities, and immunoreactive proteins. There were no differences in raccoon biomarker responses between study sites. Significant relationships between and among PCB residues and biomarkers indicated that hepatic CYP induction had occurred in response to PCB exposure. Pentoxyresorufin O-deethylase (PROD) activity, CYP1A1, and CYP1A2 were biomarkers most closely associated with PCB exposure. The rank order of responses was CYP1A1 > CYP1A2 > PROD > ethoxyresorufin O-deethylase (EROD) as related to raccoon liver PCB concentrations, whereas the order was CYP1A1 > PROD > EROD > CYP1A2 when regressed with total PCB concentrations in abdominal fat.

Population pharmacokinetic meta-analysis with efavirenz.

A population-based pharmacokinetic (PK) model has been developed for efavirenz based on 16 phase I studies. The combined data set consisted of 334 healthy volunteers, 2,907 efavirenz dose administrations and 9,342 measured plasma concentrations across a range of doses from 100-600 mg. The pharmacokinetic structural model was a 2-compartment model with first-order absorption with differentiation between single- and multiple-dose exposure to account for known hepatic cytochrome P450 induction of efavirenz metabolism. Model-building was performed on the index data set (66% of the total database), as a data-splitting technique was used to validate the final model using NONMEM. The final model confirmed the appropriateness of separate clearance terms for single and multiple dose administration (2.65 versus 10.2 l/h, respectively). Clearance increased with dose and frequency of administration. A lower clearance was predicted in Asians and Blacks relative to Caucasians. A slightly lower clearance was observed in females relative to males (9.08 compared to 10.2 l/h in males) and interactions on clearance due to co-administration of fluconazole, ritonavir, rifampin, indinavir and azithromycin were identified. The magnitudes of these effects were small and did not suggest dose adjustment in the various subpopulations. With little exception, these results agree with the findings from the non-compartmental analyses. The residual variability was 21% CV and the intersubject variation in CL/F and V/F was 48 and 85%, respectively. The phase I meta-analysis was able to substantiate the pharmacokinetic characteristics of efavirenz derived from the composite of individual well-defined studies. The model was deemed adequate for subsequent evaluation in HIV-infected patients. Covariates and outlier classes identified in this phase I meta-analysis were similarly identified in subsequent analyses of patient data.

Conservation of signaling pathways of xenobiotic-sensing orphan nuclear receptors, chicken xenobiotic receptor, constitutive androstane receptor, and pregnane X receptor, from birds to humans.

Chicken xenobiotic receptor, pregnane X receptor, and constitutive androstane receptor are orphan nuclear receptors that have recently been discovered to regulate drug- and steroid-mediated induction of hepatic cytochromes P450 (CYP). This induction is part of an adaptive response involving numerous genes to exposure to drugs and chemicals and has major clinical and toxicological implications. Here we report experiments in the chicken hepatoma cell line LMH that suggest evolutionary conservation of the signaling pathways triggered by pregnane X receptor, constitutive androstane receptor, and chicken xenobiotic receptor. Thus, the phenobarbital-inducible enhancer units of the mouse Cyp2b10, rat CYP2B2, and human CYP2B6 genes were activated in reporter gene assays by the same compounds that activate the chicken CYP2H1 phenobarbital-inducible enhancer units. Chicken xenobiotic receptor, pregnane X receptor, and constitutive androstane receptor all bound to the CYP2H1 phenobarbital-inducible enhancer units in gel-shift experiments. In CV-1 cell transactivation assays, mammalian pregnane X receptors activate the chicken phenobarbital-inducible enhancer units to the same extent as does chicken xenobiotic receptor, each receptor maintaining its species-specific ligand spectrum. To assess the reported role of protein phosphorylation in drug-mediated induction, we treated LMH cells with okadaic acid and observed increased mRNA of delta-aminolevulinate synthase and CYP2H1 whereas expression of CYP3A37 was decreased. The effects of okadaic acid and other modifiers of protein phosphorylation in LMH cells are comparable to those seen on CYP2Bs and CYP3As in mammalian primary hepatocyte cultures. These results indicate that closely related nuclear receptors, transcription factors, and signaling pathways are mediating the transcriptional activation of multiple genes by xenobiotics in chicken, rodents, and man.