Drug Metabolism In Liver Research Articles

Liver Disease and Drug Metabolism

Conventional wisdom holds that clinicians should give low doses of medications metabolized by the liver (i.e., most psychotropic drugs other than

Torsades de pointes induced by antibiotics

Background Several frequently used antibiotics are associated with an arrhythmia called “torsades de pointes” (TdP). This potentially fatal arrhythmia is considered unpredictable. Methods In order to investigate the prevalence of risk factors for TdP prior to initiation of antibiotic therapy, we conducted a literature search for all published reports on TdP induced by antibiotics and we asked pharmaceutical companies for additional unpublished reports. Results We studied 61 reports on 78 patients with TdP induced by antibiotics. Female gender was the most common risk factor for TdP: 66.7% ( n = 52) of all patients were women. Advanced heart disease and concomitant use of a QT interval-prolonging agent or an inhibitor of liver drug metabolism were also frequently present (59% and 48.7%, respectively). Most patients had at least one and 58 patients (74.3%) had two risk factors or more for TdP prior to initiation of antibiotic therapy. Conclusion Contrary to common belief, TdP induced by antibiotics may be predictable by simple history-taking and by obtaining a baseline electrocardiogram. We wish to draw attention to risk factors for TdP prior to the initiation of antibiotic therapy.

Compendium of gene expression profiles comprising a baseline model of the human liver drug metabolism transcriptome

Oligonucleotide microarrays were used to study the variability of pharmacokinetics and drug metabolism (PKDM)-related gene expression in 75 normal human livers. The objective was to define and use absorption, distribution, metabolism and excretion (ADME) gene expression variability to discern co-regulated genes and potential surrogate biomarkers of inducible gene expression. RNA was prepared from donor tissue and hybridized on Agilent microarrays against an RNA mass balanced pool from all donors. Clustering of PKDM gene sets revealed donors with distinct patterns of gene expression that grouped genes known to be regulated by the nuclear receptor, pregnane X-receptor (PXR). Fold range metrics and frequency distributions from the heterogeneous human population were used to define the variability of individual PKDM genes in the 75 human livers and were placed in context by comparing expression data with basal ADME gene expression variability in an inbred and diet/environment controlled population of 27 Rhesus livers. The most variable genes in the hepatic transcriptome were mainly related to drug metabolism, intermediary metabolism, inflammation and cell cycle control. Unique patterns of expression across 75 individuals of inducible ADME gene expression allowed their expression to be correlated with the expression of many other genes. Correlated genes for AhR, CAR and PXR responsive genes (CYP1A2, CYP2B6 and CYP3A4) were identified that may be co-regulated and, therefore, provide clues to the identity of surrogate gene or protein markers for CYP induction. In conclusion, microarrays were used to define the variable expression of hepatic ADME genes in a diverse human population, the expression variability of ADME genes was compared with the expression variability in an inbred population of Rhesus monkeys, and genes were defined that may be co-regulated with important inducible CYP genes.

The role of the cytochrome P450 3A5 enzyme for blood pressure regulation in the general Caucasian population

Cytochrome P450 3A (CYP3A) enzymes are important for drug metabolism in gut and liver. The CYP3A5 isoenzyme is also expressed in the kidney and has been implicated in renal sodium reabsorption and blood pressure regulation. Its expression and activity is strongly linked to a polymorphism (i.e. 6986G > A). Thus, appreciable expression is found in carriers of the CYP3A5*1 (6986A) but not in homozygous carriers of the CYP3A5*3 (6986G) allele. We tested whether the presence of CYP3A5*1 affects blood pressure in Caucasian individuals who were enrolled in the Prevention of REnal and Vascular ENd stage Disease (PREVEND) study. In addition, we evaluated whether the genetic effect of CYP3A5*1 on blood pressure is modulated by sodium intake. CYP3A5*1 was found in 13.3% (901 individuals) of the cohort (6777 individuals). Diastolic blood pressure was not affected by CYP3A5*1. Overall, systolic and pulse pressure were significantly lower in carriers of CYP3A5*1, both after univariate analysis adjusted for age (P = 0.012 and P = 0.008) and in logistic regression analysis (P = 0.015 and P = 0.012). The effect on systolic blood pressure was significantly modulated by sodium intake (P = 0.038). In separate analysis according to gender, CYP3A5*1 accounted for a significant age adjusted decrease in systolic blood pressure (-1.6 mmHg, P = 0.04) and pulse pressure (-1.2 mmHg, P = 0.04) in females but not in men. The present study demonstrates that the CYP3A5*1 allele affects systolic blood pressure and pulse pressure in the general population. Its role in hypertensive disease and potential gender differences should be investigated in further studies.

Metabolism of Aminoguanidine, Diaminoguanidine, and NG-Amino-l-arginine by Neuronal NO-Synthase and Covalent Alteration of the Heme Prosthetic Group

It is established that aminoguanidine (AG), diaminoguanidine (DAG), and NG-amino-l-arginine (NAA) are metabolism-based inactivators of the three major isoforms of nitric oxide synthase (NOS). In the case of neuronal NOS (nNOS), heme alteration is known to be a major cause of inactivation, although the exact mechanism by which this occurs is not well-understood. We show here by the use of LC/MS/MS techniques that AG, DAG, and NAA are metabolized by nNOS to products with corresponding mass ions at m/z of 45.2, 60.2, and 160.0, respectively. These results are consistent with the loss of a hydrazine moiety from each inactivator. These findings are confirmed by exact mass measurements and comparison to authentic standards in the case of the products for NAA and AG, respectively. Moreover, the major dissociable heme product that was formed during inactivation of nNOS by AG, DAG, and NAA had molecular ions at m/z 660.2, 675.2, and 775.3, respectively. These results are consistent with an adduct of heme and inactivator minus a hydrazine moiety. In support of this, MS/MS studies reveal a fragment ion of heme in each case. With the use of 14C-labeled heme, we also show that in the case of AG, the dissociable heme adduct accounts for approximately one-half of the heme that is altered. In addition, we employ a software-based differential metabolic profiling method by subtracting LC/MS data sets derived from samples that contained nNOS from those that did not contain the enzyme to search for products and substrates in complex reaction mixtures. The metabolic profiling method established in this study can be used as a general tool to search for substrates and products of enzyme systems, including the drug-metabolizing liver microsomal P450 cytochromes. We propose that the metabolism-based inactivation of nNOS by AG, DAG, and NAA occurs through oxidative removal of the hydrazine group and the formation of a radical intermediate that forms stable products after H-atom abstraction or reacts with the heme prosthetic moiety and inactivates nNOS.

Morphological and biochemical characterization of a human liver in a uPA‐SCID mouse chimera†‡

A small animal model harboring a functional human liver cell xenograft would be a useful tool to study human liver cell biology, drug metabolism, and infections with hepatotropic viruses. Here we describe the repopulation, organization, and function of human hepatocytes in a mouse recipient and the infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) of the transplanted cells. Homozygous urokinase plasminogen activator (uPA)-SCID mice underwent transplantation with primary human hepatocytes, and at different times animals were bled and sacrificed to analyze plasma and liver tissue, respectively. The plasma of mice that were successfully transplanted contained albumin and an additional 21 human proteins. Liver histology showed progressive and massive replacement of diseased mouse tissue by human hepatocytes. These cells were accumulating glycogen but appeared otherwise normal and showed no signs of damage or death. They formed functional bile canaliculi that connected to mouse canaliculi. Besides mature hepatocytes, human hepatic progenitor cells that were differentiating into mature hepatocytes could be identified within liver parenchyma. Infection of chimeric mice with HBV or HCV resulted in an active infection that did not alter the liver function and architecture. Electron microscopy showed the presence of viral and subviral structures in HBV infected hepatocytes. In conclusion, human hepatocytes repopulate the uPA(+/+)-SCID mouse liver in a very organized fashion with preservation of normal cell function. The presence of human hepatic progenitor cells in these chimeric animals necessitates a critical review of the observations and conclusions made in experiments with isolated "mature" hepatocytes. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Induction of CYP3As in HepG2 cells by several drugs. Association between induction of CYP3A4 and expression of glucocorticoid receptor.

The cytochrome P-450 3A (CYP3A) enzyme family is responsible for most of the drug metabolism in the human liver. In this study, we demonstrated the inductive effects of phenobarbital, rifampicin, carbamazepine, phenytoin, prednisolone, ciclosporin and clotrimazole on CYP3A4, CYP3A5 and CYP3A7 mRNA expression, and established the relationship between the expression of human glucocorticoid receptor alpha (hGR) mRNA and the induction of CYP3A4 mRNA in cultured HepG2 cells by reverse transcription polymerase chain reaction (RT-PCR). Treatment with prednisolone, rifampicin and carbamazepine rapidly induced the level of CYP3A4 mRNA expression by 3- to 6-fold. However, phenytoin and phenobarbital gradually induced CYP3A4 mRNA level by 3 to 4-fold. The induction of CYP3A4 mRNA expression by clotrimazole and ciclosporin was negligible. Treatment with phenytoin, rifampicin, carbamazepine and ciclosporin induced approximately 2-fold increases in the expression of CYP3A5 mRNA, although prednisolone, phenytoin and clotrimazole had no effect. Treatment with rifampicin, phenytoin, clotrimazole and ciclosporin resulted in approximately a 2-fold induction of the CYP3A7 mRNA level. Treatment with rifampicin and ciclosporin induced the expression of hGRalpha mRNA significantly in comparison with controls, although the induction of hGRalpha mRNA following treatment with other drugs was negligible. In cluster analysis, the induced level of CYP3A4, CYP3A5, CYP3A7 and hGRalpha mRNA by these drugs could be classified into four major clusters. This suggested that each cluster might be associated with different mechanism(s) of induction by these drugs. Furthermore, we studied the associations between the expression of hGRalpha mRNA and the induced level of CYP3A4 mRNA by prednisolone and ciclosporin. Treatment with both prednisolone and ciclosporin showed synergistic effects on induction of CYP3A4 mRNA and, following treatment with both drugs, the expression level of CYP3A4 mRNA was 2-fold greater compared with prednisolone alone after the fifth day. Positive correlations were observed between the levels of hGRalpha mRNA expression and those of CYP3A4 mRNA. This observation shows that the regulation of CYP3A4 gene expression was hGRalpha-dependent and that ciclosporin may function as a regulator of expression via hGRalpha.

Alcohol use and HIV pharmacotherapy.

Alcohol consumption by individuals infected with HIV is an important medical management issue with significant implications for the effectiveness of antiretroviral therapy as well as an important evolving field of HIV research. Alcohol consumption is a risk factor for poor medication adherence and can modify liver drug metabolism, both of which can lead to the emergence of drug-resistant virus. Research indicates that alcohol consumption greater than 50 g/day (four or five drinks) is a risk factor for liver disease progression among patients with HIV/HCV coinfection. In addition, alcohol-induced cirrhosis can result in changes in drug metabolism in the liver through compromised liver function. More research studies are needed to elucidate the biological and molecular basis of the clinical changes induced by alcohol consumption in HIV-infected individuals and on the relationship of these changes to the effectiveness of HIV pharmacotherapy. Specifically, research areas that are of particular importance are (1) determining alcohol consumption levels and patterns and its impact on antiretroviral medication adherence, efficacy, and physician prescribing practices; (2) identifying behavioral interventions to enhance adherence to HIV medications and reduce alcohol consumption; (3) clarifying the relationships and interactions among alcohol metabolism, HIV drug metabolism, and pharmacogenetics; (4) elucidating the extent of liver toxicity due to antiretroviral therapy and drug-drug interactions in individuals who consume alcohol; and (5) delineating the contribution of alcohol consumption to end-stage organ damage, particularly in HIV/HCV coinfection.

Propranolol as an inhibitor of some cytochrome P450-dependent monooxygenase activities in native and induced rat liver microsomes.

The in vitro effect of propranolol (10(-3) M and 10(-4) M), a nonselective and extensively metabolized beta-adrenergic blocking agent, on rat liver drug metabolism in native and induced (with phenobarbital and beta-naphthoflavone [beta-NF]) microsomes was studied. The type of inhibition and the inhibitory constants of some cytochrome P450-dependent microsomal enzyme reactions (hexobarbital oxidation [HBO], ethylmorphine-N-demethylation [EMND], aniline hydroxylation [AH], ethoxycoumarin-O-deethylation [ECOD], ethoxyresorufin-O-dealkylation [EROD] and penthoxyresorufin-O-dealkylation [PROD]) were estimated. The results showed that propranolol competitively inhibited AH activity in native microsomes. The type of inhibition was changed from competitive to noncompetitive in all other enzyme activities studied. This inhibition was more pronounced after phenobarbital induction in PROD (Ki = 0.11 +/- 0.01 mM), ECOD (Ki = 0.40 +/- 0.09 mM) and EMND (Ki = 0.59 +/- 0.1 mM), and after beta-NF induction in AH (Ki = 0.28 +/- 0.05 mM) and in HBO (Ki = 0.35 +/- 0.1 mM) in native microsomes. It was assumed that the noncompetitive type of inhibition is due to the covalent binding of reactive metabolites derived from propranolol to hepatic microsomal proteins. The competitive type of inhibition of AH suggested a common P450 isoenzyme in the metabolism of propranolol and aniline. Thus, in this study, propranolol has been found to be not only a selective inhibitor of CYP2D6 isoenzyme-dependent reactions, but also a nonspecific inhibitor of other cytochrome P450 isoenzymes.

Steroid hormone progesterone induces cell proliferation and abnormal mitotic processes in rat liver.

The aim of this study was to evaluate the acute hepatic effects exerted by the steroid hormone progesterone (PR) in the rat. Although the liver is not a target tissue for this hormone, a number of hepatic actions of PR have been described, and, furthermore, a specific binding site for PR (PBS) exists in rat liver microsomes. Immature male rats were treated intraperitoneally with 60 mg/kg PR per day for 1, 5 or 10 days, and different parameters were evaluated in order to detect possible alterations in liver cells. Morphological study of the livers did not present images of cytotoxicity in any group of animals. The presence of a clear hyperplasia of smooth endoplasmic reticulum (SER) was noteworthy, mainly seen in perilobular hepatocytes. Despite this SER increase, the levels of cytochrome P450 (Cyt P450) significantly decreased after 10 days of PR administration. Similarly, the concentration of PBS was significantly decreased after 10 days of treatment with PR. On the other hand, these studies revealed a clear increase of mitotic activity and Ki-67 labelling index in the livers of animals treated with PR; furthermore, livers of PR-treated animals showed an increased percentage of binucleate hepatocytes. Flow cytometry analysis showed that although ploidy status of liver cells was not modified in any case the percentage of diploid nuclei in S-phase decreased during treatment with PR. The most relevant finding was the presence of abnormal mitosis and c-mitosis in livers from animals from all PR-treated groups. This study demonstrates that PR (a) does not induce cytotoxicity although it can induce cell proliferation and spindle disturbances in liver cells, (b) may also modulate the drug-metabolizing liver enzyme function, and (c) downregulates the expression of its own microsomal specific binding site.

Cholestasis and Regulation of Genes Related to Drug Metabolism and Biliary Transport in Rat Liver Following Treatment with Cyclosporine A and Sirolimus (Rapamycin)

Abstract: Cyclosporine A and sirolimus are used alone or in combination as immunosuppressants in organ transplantation. To elucidate hepatic side effects, we examined hepatic mRNA of proteins involved in biliary and hepatocellular transport of drugs, formation of glutathione (GSH) and drug metabolising cytochrome P‐450 enzymes (CYPs) in rats treated orally for 2 weeks with cyclosporine A (15 mg/kg/day), sirolimus (0.4 mg/kg/day), their combination (same doses), or vehicle. Liver function tests (alanine aminotransferase, alkaline phosphatase, γ‐glutamyl transferase and bilirubin) in blood were then analysed as were hepatic mRNA levels of canalicular transport proteins (Mrp2, Bsep, Mdr1b and Mdr2), sinusoidal transport proteins (Ntcp, Oatp1 and Oatp2), GSH related enzymes (γ‐glutamylcysteine synthetase light (GCSlc) and heavy (GCShc) chain subunits and glutathione‐S‐transferase) and CYPs (CYP3A9, CYP1A2, CYP2E1 and CYP2BI/II). Cyclosporine A caused moderate cholestatic changes in liver enzymes, which was synergistically exacerbated by sirolimus. The data suggest that the underlying mechanisms behind cholestasis were not totally identical in the different treatment regimens. Cholestasis secondary to cyclosporine A could be related to reduction in mRNA expression of GSH synthesising enzymes and Mrp2, leading to reduced protection against oxidative stress and reduced bile acid‐independent bile flow. After sirolimus treatment, Mrp2 mRNA was also reduced together with reduced levels of most CYPs and increased Oatp2, possibly leading to accumulation of toxic metabolites in the hepatocytes. The enhanced cholestatic effect of the combination treatment could be related to reduced GSH synthesising enzymes and even more pronounced reduction in Mrp2 mRNA and increase of Oatp2 mRNA.

Cholestasis and regulation of genes related to drug metabolism and biliary transport in rat liver following treatment with cyclosporine A and sirolimus (Rapamycin).

Cyclosporine A and sirolimus are used alone or in combination as immunosuppressants in organ transplantation. To elucidate hepatic side effects, we examined hepatic mRNA of proteins involved in biliary and hepatocellular transport of drugs, formation of glutathione (GSH) and drug metabolising cytochrome P-450 enzymes (CYPs) in rats treated orally for 2 weeks with cyclosporine A (15 mg/kg/day), sirolimus (0.4 mg/kg/day), their combination (same doses), or vehicle. Liver function tests (alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase and bilirubin) in blood were then analysed as were hepatic mRNA levels of canalicular transport proteins (Mrp2, Bsep, Mdr1b and Mdr2), sinusoidal transport proteins (Ntcp, Oatp1 and Oatp2), GSH related enzymes (gamma-glutamylcysteine synthetase light (GCSlc) and heavy (GCShc) chain subunits and glutathione-S-transferase) and CYPs (CYP3A9, CYP1A2, CYP2E1 and CYP2BI/II). Cyclosporine A caused moderate cholestatic changes in liver enzymes, which was synergistically exacerbated by sirolimus. The data suggest that the underlying mechanisms behind cholestasis were not totally identical in the different treatment regimens. Cholestasis secondary to cyclosporine A could be related to reduction in mRNA expression of GSH synthesising enzymes and Mrp2, leading to reduced protection against oxidative stress and reduced bile acid-independent bile flow. After sirolimus treatment, Mrp2 mRNA was also reduced together with reduced levels of most CYPs and increased Oatp2, possibly leading to accumulation of toxic metabolites in the hepatocytes. The enhanced cholestatic effect of the combination treatment could be related to reduced GSH synthesising enzymes and even more pronounced reduction in Mrp2 mRNA and increase of Oatp2 mRNA.

Effect of troglitazone on the liver of a Gunn rat model of genetic enzyme polymorphism.

There is a possibility that serious liver dysfunction rarely observed in diabetic patients given troglitazone is attributable to idiosyncratic abnormalities in liver drug-metabolism. In addition, the results of blood biochemical examinations in serious cases of liver dysfunction showed a tendency for a high level of total bilirubin (T-Bil) over a long period compared with other indicators of liver dysfunction. Thus, we focused on genetic variation of UDP-glucuronosyltransferases (UGTs) that are involved in the conjugation of troglitazone and bilirubin. In this study, Gunn rats, which are hereditarily deficient in the UGT1 family of UGT isozymes, and Wistar rats, the parent strain of Gunn rats, were treated with troglitazone for 3 months at dose levels of 0, 100 or 400 mg/kg to investigate two possibilities: first, whether the genetic deficiency in UGT1s induces an alteration of the metabolic profile of troglitazone followed by liver dysfunction, and second, whether the dosing of troglitazone to Gunn rats which show hyperbilirubinemia result in liver dysfunction. As a result, the metabolic profile of troglitazone in Gunn rats was much the same as that of Wistar rats, suggesting that genetic deficiencies in UGT1s did not influence the metabolic profile of troglitazone. Moreover, no elevation of blood biochemical parameters, such as asparate aminotransferase (AST) and alanine aminotransferase (ALT), or histopathological liver injuries, such as hepatocellular degeneration and necrosis, were observed in either strain of rats, and hyperbilirubinemia in Gunn rats was not aggravated by the dosing of troglitazone. These results strongly suggest that troglitazone was not metabolized by UGT1s but by other UGT isozyme (s) in rats, and that glucuronidation of troglitazone did not compete with glucuronidation of bilirubin in vivo. Thus, it is suggested that high levels of total bilirubin in patients with liver dysfunction induced by troglitazone are attributable to hypofunction due to hepatocellular injury, not to metabolic competition of bilirubin with troglitazone. Moreover, it is also suggested that the deficiency in the UGT1 family of UGT isozymes itself may not be the cause of liver dysfunction associated with troglitazone treatment.

Captan impairs CYP-catalyzed drug metabolism in the mouse

To investigate whether the fungicide captan impairs CYP-catalyzed drug metabolism in murine liver, kidney and lung, the modulation of the regio- and stereo-selective hydroxylation of testosterone, including 6β-(CYP3A), 6α-(CYP2A1 and CYP2B1) and 16α-(CYP2B9) oxidations was studied. Specific substrates as probes for different CYP isoforms such as p-nitrophenol (CYP2E1), pentoxyresorufin (CYP2B1), ethoxyresorufin (CYP1A1), aminopyrine (CYP3A), phenacetin and methoxyresorufin (CYP1A2), and ethoxycoumarin (mixed) were also considered. Daily doses of captan (7.5 or 15 mg/kg b.w., i.p.) were administered to different groups of Swiss Albino CD1 mice of both sexes for 1 or 3 consecutive days. While a single dose of this fungicide did not affect CYP-machinery, repeated treatment significantly impaired the microsomal metabolism; in the liver, for example, a general inactivating effect was observed, with the sole exception of testosterone 2α-hydroxylase activity which was induced up to 8.6-fold in males. In vitro studies showed that the mechanism-based inhibition was related to captan metabolites rather than the parental compound. In the kidney, both CYP3A- and CYP1A2-linked monooxygenases were significantly induced (∼2-fold) by this pesticide. Accelerated phenacetin and methoxyresorufin metabolism (CYP1A2) was also observed in the lung. Data on CYP3A (kidney) and CYP1A2 (kidney and lung) induction were corroborated by Western immunoblotting using rabbit polyclonal anti-CYP3A1/2 and CYP1A1/2 antibodies. By means of electron spin resonance (EPR) spectrometry coupled to a spin-trapping technique, it was found that the recorded induction generates a large amounts of the anion radical superoxide (O 2 −) either in kidney or lung microsomes. These findings suggest that alterations in CYP-associated activities by captan exposure may result in impaired (endogenous) metabolism as well as of coadministered drugs with significant implications for their disposition. The adverse outcomes associated to CYP changes (e.g. cotoxicity, comutagenicity and promotion) may also have harmful consequences.

Organ Slices as an In Vitro Test System for Drug Metabolism in Human Liver, Lung and Kidney

Metabolism of xenobiotics occurs mainly in the liver, but in addition, the lungs and kidneys may contribute considerably. The choice of the animal species during drug development as a predictive model for the human condition is often inadequate due to large interspecies differences. Therefore, a universal method for the preparation and incubation of human and animal liver, lung and kidney tissue is being developed for drug metabolism and toxicity testing using precision-cut organ slices. Human tissue was obtained from surgical waste material. Slices were made from rat and human liver, kidney and agar-filled (1.5%, w/v) lung tissue using a Krumdieck tissue slicer and incubated in six-well plates. The morphology and the ATP content show that viability is maintained during 3 hours of incubation. These organ slices show a variety of phase I (hydroxylation, oxidation and O- and N-deethylation) and phase II (glucuronidation and sulfation) metabolic routes using lidocaine, testosterone, 7-ethoxycoumarin and 7-hydroxycoumarin as substrates. The metabolic patterns and rates were found to be different for the various organs and species studied. The use of human tissue slices will enable us to collect more human-specific data on drug metabolism and toxicity. This may lead to a more adequate choice of animal species used during drug development and will result in a considerable reduction in the use of experimental animals.

Inhibition of enzymes of drug metabolism in rat liver by ultrasound and correction by heparin

After 6 days following the local effect (during operation) of ultrasound (2 Wt/cm2, 1 min) the microsomal fraction showed decreased total content of cytochromes P-450 (P-450), rate of NADPH oxidation, activity of NADPH-cytochrome P450 reductase and P450 IIE1 (aniline as substrate) by 40, 28, 16 and 42 %, respectively. In addition, after 12 days the activities of P450 IIIA1 (ethylmorphine as substrate) and cytosolic sulphobromophthalein glutathione transferase (SBPh-GT) were decreased by 59 and 26 %. The administration of heparin (intramuscularly, 250 ED/kg, in a day, 3 and 6 times) exerted a normalizing effect. The P450 concentration, NADPH oxidation rate and P450 IIB1 activity (amidopyrine as substrate), IIE1 and IIIA1, SBPh-GT and 1-chloro-2,4-dinitrobenzene-GT in microsomes and cytosol exceeded the corresponding values in untreated animals by 31, 40, 68, 224, 68, 42, 24 and 36 %. The administration of heparin to control animals (intramuscularly, 250 and 500 mg/kg, in a day, 5 times) essentially unaffected both the monooxygenase, glucuro- and glutathione-conjugating systems and the elimination of antipyrine (substrate of preferably P-450 IA2) and SBPh (substrate of GT) from rat blood plasma. The experimental results provide evidence for a possible role of endogenous heparin in maintaing the optimal level of the activities of the enzyme systems of xenobiotics microsomal oxidation and conjugation in liver injury. One of the most important functions of the liver is its ability to execute biotransformation of a wide range of xenobiotics and some endogenous substances [1]. The activities of the enzyme systems catalyzing these reactions are under a sophisticated regulatory control. Among the natural factors capable of changing the function of enzymes involved in the xenobiotic biotransformation are vitamins [2], phospholipids [3], hormones [4] and many others. We studied the effect of heparin on the activities of the monooxygenase, glucuro- and glutathione transferase systems of the intact and ultrasound-treated rat liver. The significance of this study consists in the elucidation of a putative participation of heparin in the control of the activities of the enzyme system of xenobiotic biotransformation in the intact liver and under membranous pathology of the organ.

Effects of experimental dicrocoeliosis on oxidative drug metabolism in hamster liver

The purpose of this investigation was to determine the effects of experimental dicrocoeliosis on the hepatic oxidative drug-metabolizing system in hamsters. Studies were carried out 80 and 120 days after infestation with an oral dose of 40 metacercariae of Dicrocoelium dendriticum. The parasitic pathology was ascertained by detection of the fluke eggs in faeces, increased serum alanine aminotransferase and aspartate aminotransferase activities, and postmortem and histological findings. Cytochrome P-450 concentration, aniline hydroxylase activity and ethoxycoumarin O-deethylase activity were significantly decreased in both groups of infected animals. Aminopyrine N-demethylase activity and erythromycin N-demethylase activity were only reduced 120 days after infection. Effects on drug metabolizing enzymes were unrelated to changes in the physical state of the microsomal membrane, as assessed by measurement of fluorescence polarization. The results of this study indicate that the capacity of the liver for handling drugs and xenobiotics may be impaired as a consequence of dicrocoeliosis.

Expression of liver-specific functions in rat hepatocytes following sublethal and lethal acetaminophen poisoning

Aim: In order to study the short-term effect of moderate and severe reduction of liver function by acetaminophen poisoning of different severity on gene expression for liver-specific functions, rats were given 3.75 and 7.5 g per kg body weight acetaminophen intragastrically. The lower dose is associated with low mortality; after the higher dose, most rats die at between 12 and 24 h. Methods: In the morning 1 1 2 ,3,6,9, and 12 h after the injection, the rats were killed and RNA was extracted from liver tissue. By slot-blot hybridization mRNA steady-state levels were determined for enzymes involved in metabolic liver functions, i.e. unreagenesis, gluconeogensis, and drug metabolism, for acute phase proteins, “house-keeping” proteins, and for proteins related to liver regeneration. Results were expressed as per cent of the level in similarly fasted, untreated rats of the same stock Results: After the smaller dose of acetaminophen, most of the examined mRNA levels were increasing during the experimental period, being two- to four-fold elevated in relation to control after 6 to 12 h. Rats receiving the lethal dose either showed no or a later and smaller increase, and in several cases a fall towards the end of the experiment. The greatest differences were seen for mRNA of arginase, β-fibrinogne, α1-acid glycoprotein, α-tubulin, histone 3, TGFβ, and cyclin d, i.e. proteins associated with acute phase response and liver cell replication and maintenance. Conclusions: It is concluded that reversible intoxication with acetaminophen induces an adaptive modulation of mRNA expression of liver functions and regeneration which is lacking after severe intoxication. This adaptation, with emphasis on acute phase response and regeneration, may be crucial for recovery after acetaminophen intoxication. If this also applies to the intoxication in man, estimates of the corresponding variables may be clues to the prognosis of acetaminophen-induced fulminant hepatic failure.

Testosterone hydroxylase in evaluating induction and suppression of murine CYP isoenzymes by fenarimol.

This study aimed to investigate the effect of single and repeated administration of fenarimol on murine liver, kidney and lung microsomal CYP-catalyzed drug metabolism. The modulation of the regio- and stereo-selective hydroxylation of testosterone by fenarimol was considered in evaluating cocarcinogenic properties. Induction or suppression of different CYPs was recorded after a single dose of the fungicide. For example, in liver, 6 beta-(mainly associated with CYP3A), 7 alpha- and 2 beta-testosterone hydroxylase (TH) activities were induced up to 4.8-fold (7 alpha-TH) in female mice, at a dose of 150 mg/kg. In contrast, at 150 and 300 mg/kg, 16 alpha-TH (CYP2B9), 17-TH (female) and 6 alpha-TH (CYP2A1 and 2B1, male) activities were appreciably reduced. In extrahepatic tissues, the CYP modulation pattern was different, 16 alpha-TH being the only metabolite decreased (lung, male). In kidney, 16 beta-TH and 17-TH activities were increased up to 5.8-fold in female mice (lowest dose), while in lung 6 alpha-TH and 7 alpha-TH activities were induced up to 6- and 7-fold, respectively (both doses). Repeated treatment (150 mg/kg for 3 days) was able markedly to induce all steroid hydroxylations, up to 78-fold in 2 alpha-TH activity (male liver). In conclusion, fenarimol has a complex pattern of CYP induction or suppression in various tissues of both sexes, suggesting the possible toxic, cotoxic/cocarcinogenic and promoting potential of this fungicide.

Dose-related effect of aflatoxin B1 on liver drug metabolizing enzymes in rabbit

The effects of chronic administration of aflatoxin B1 (AFB1) on liver drug metabolism enzymes were measured in New Zealand rabbits divided into three groups of 5 animals, each receiving over 5 days either arabic gum or AFB1 in arabic gum at a daily oral dose of 0.05 or 0.10 mg/kg. These treatments did not lead to any lethality in any of the treated groups but the body weight gain was altered. Biochemical exploration of plasma components revealed a dose-dependent hepatotoxicity characterized by cytolysis and cholestasis. At 0.10 mg/kg/day of AFB1, significant decreases were observed in total liver microsomal cytochrome P450, several P450-dependent monooxygenase activities, all individual P450 isoenzymes levels analysed by Western-blotting and glutathione S-transferase activities. By contrast, at 0.05 mg/kg/day of AFB1, even though total cytochrome P450 was decreased by 30%, only P450 1A1 and 3A6 isoenzymes, and aniline hydroxylation, pentoxyresorufin O-depentylation, aminopyrine, erythromycin, ethylmorphine and dimethylnitrosamine N-demethylations were affected. In the same animal group, the only glutathione S-transferase accepting CDNB (1-chloro-2,4-dinitrobenzene) as substrate was decreased by 22%. UDP-glucuronyltransferase accepting p-nitrophenol as substrate was increased in both groups of animals (33–62%). The mechanisms that could contribute to the observed changes in drug metabolizing enzymes are discussed.