Hepatic Cytochrome P450 Reductase Research Articles

UHPLC-MS-Based Lipidomic and Metabonomic Investigation of the Metabolic Phenotypes of Wild Type and Hepatic CYP Reductase Null (HRN) Mice

Hepatic cytochrome P450 reductase (EC 1.6.2.4, POR) deficient mice provide a useful means of investigating liver-related CYP450 drug metabolism. However, the organ-wide inactivation of CYP450s has wide ranging effects on liver physiology. Untargeted UHPLC-MS metabolic and lipid profiling of aqueous and organic solvent extracts has been employed to compare the metabolic phenotypes of livers obtained from either wild type (C57Bl6) or hepatic P450 reductase null (HRNTM) mice. The metabolic phenotyping of polar aqueous extracts revealed differences between wild type and HRNTM mice for bile acids with taurochendeoxycholic acid, and tauroursodeoxycholic acid increased in proportion in the latter and taurocholic acid reduced. Lipidomic profiling demonstrated that there were numerous differences in the lipidome, particularly relating to phospholipid synthesis with significant changes in the relative amounts of phosphatidylcholines (PC) and phosphatidylethanolamines (PE). These results illustrate the wide ranging disruptive effects on the normal hepatic phenotype that result from POR-deficiency in the the HRNTM animals.

Improved hepatic physiology in hepatic cytochrome P450 reductase null (HRN™) mice dosed orally with fenclozic acid.

Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit no functional expression of hepatic cytochrome P450 (P450) when compared to wild type (WT) mice, but have normal hepatic and extrahepatic expression of other biotransformation enzymes. We have assessed the utility of HRN™ mice for investigation of the role of metabolic bioactivation in liver toxicity caused by the nonsteroidal anti-inflammatory drug (NSAID) fenclozic acid. In vitro studies revealed significant NADPH-dependent (i.e. P450-mediated) covalent binding of [14C]-fenclozic acid to liver microsomes from WT mice and HRN™ mice, whereas no in vitro covalent binding was observed in the presence of the UDP-glucuronyltransferase cofactor UDPGA. Oral fenclozic acid administration did not alter the liver histopathology or elevate the plasma liver enzyme activities of WT mice, or affect their hepatic miRNA contents. Livers from HRN™ mice exhibited abnormal liver histopathology (enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration, necrosis, inflammatory cell infiltration) and plasma clinical chemistry (elevated alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities). Modest apparent improvements in these abnormalities were observed when HRN™ mice were dosed orally with fenclozic acid for 7 days at 100 mg kg-1 day-1. Previously we observed more marked effects on liver histopathology and integrity in HRN™ mice dosed orally with the NSAID diclofenac for 7 days at 30 mg kg-1 day-1. We conclude that HRN™ mice are valuable for assessing P450-related hepatic drug biotransformation, but not for drug toxicity studies due to underlying liver dysfunction. Nonetheless, HRN™ mice may provide novel insights into the role of inflammation in liver injury, thereby aiding its treatment.

Inhibition of hepatic cytochrome P450 enzymes and sodium/bile acid cotransporter exacerbates leflunomide-induced hepatotoxicity

Leflunomide is an immunosuppressive agent marketed as a disease-modifying antirheumatic drug. But it causes severe side effects, including fatal hepatitis and liver failure. In this study we investigated the contributions of hepatic metabolism and transport of leflunomide and its major metabolite teriflunomide to leflunomide induced hepatotoxicity in vitro and in vivo. The metabolism and toxicity of leflunomide and teriflunomide were evaluated in primary rat hepatocytes in vitro. Hepatic cytochrome P450 reductase null (HRN) mice were used to examine the PK profiling and hepatotoxicity of leflunomide in vivo. The expression and function of sodium/bile acid cotransporter (NTCP) were assessed in rat and human hepatocytes and NTCP-transfected HEK293 cells. After Male Sprague-Dawley (SD) rats were administered teriflunomide (1,6, 12 mg · kg(-1) · d(-1), ig) for 4 weeks, their blood samples were analyzed. A nonspecific CYPs inhibitor aminobenzotriazole (ABT, 1 mmol/L) decreased the IC50 value of leflunomide in rat hepatocytes from 409 to 216 μmol/L, whereas another nonspecific CYPs inhibitor proadifen (SKF, 30 μmol/L) increased the cellular accumulation of leflunomide to 3.68-fold at 4 h. After oral dosing (15 mg/kg), the plasma exposure (AUC0-t) of leflunomide increased to 3-fold in HRN mice compared with wild type mice. Administration of leflunomide (25 mg·kg(-1) · d(-1)) for 7 d significantly increased serum ALT and AST levels in HRN mice; when the dose was increased to 50 mg·kg(-1) · d(-1), all HRN mice died on d 6. Teriflunomide significantly decreased the expression of NTCP in human hepatocytes, as well as the function of NTCP in rat hepatocytes and NTCP-transfected HEK293 cells. Four-week administration of teriflunomide significantly increased serum total bilirubin and direct bilirubin levels in female rats, but not in male rats. Hepatic CYPs play a critical role in detoxification process of leflunomide, whereas the major metabolite teriflunomide suppresses the expression and function of NTCP, leading to potential cholestasis.

The usage of a three-compartment model to investigate the metabolic differences between hepatic reductase null and wild-type mice.

The Cytochrome P450 (CYP) system is involved in 90% of the human body's interactions with xenobiotics and due to this, it has become an area of avid research including the creation of transgenic mice. This paper proposes a three-compartment model which is used to explain the drug metabolism in the Hepatic Reductase Null (HRN) mouse developed by the University of Dundee (Henderson, C. J., Otto, D. M. E., Carrie, D., Magnuson, M. A., McLaren, A. W., Rosewell, I. and Wolf, C. R. (2003) Inactivation of the hepatic cytochrome p450 system by conditional deletion of hepatic cytochrome p450 reductase. J. Biol. Chem. , 13480-13486). The model is compared with a two-compartment model using experimental data from studies using wild-type and HRN mice. This comparison allowed for metabolic differences between the two types of mice to be isolated. The three sets of drug data (Gefitinib, Midazolam and Thalidomide) showed that the transgenic mouse has a decreased rate of metabolism.

Hepatic effects of repeated oral administration of diclofenac to hepatic cytochrome P450 reductase null (HRN™) and wild-type mice.

Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit normal hepatic and extrahepatic biotransformation enzyme activities when compared to wild-type (WT) mice, but express no functional hepatic cytochrome P450 activities. When incubated in vitro with [(14)C]-diclofenac, liver microsomes from WT mice exhibited extensive biotransformation to oxidative and glucuronide metabolites and covalent binding to proteins was also observed. In contrast, whereas glucuronide conjugates and a quinone-imine metabolite were formed when [(14)C]-diclofenac was incubated with HRN™ mouse liver, only small quantities of P450-derived oxidative metabolites were produced in these samples and covalent binding to proteins was not observed. Livers from vehicle-treated HRN™ mice exhibited enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration and necrosis and inflammatory cell infiltration, which were not present in livers from WT mice. Elevated liver-derived alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities were also observed in plasma from HRN™ mice. When treated orally with diclofenac for 7 days, at 30 mg/kg/day, the severities of the abnormal liver histopathology and plasma liver enzyme findings in HRN™ mice were reduced markedly. Oral diclofenac administration did not alter the liver histopathology or elevate plasma enzyme activities of WT mice. These findings indicate that HRN™ mice are valuable for exploration of the role played by hepatic P450s in drug biotransformation, but poorly suited to investigations of drug-induced liver toxicity. Nevertheless, studies in HRN™ mice could provide novel insights into the role played by inflammation in liver injury and may aid the evaluation of new strategies for its treatment.

An Enhanced In Vivo Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Model for Quantification of Drug Metabolism Enzymes *

Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug–drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of drug pharmacokinetics, pharmacodynamics, and of chemically treated and genetically modified mouse models.

The anticancer drug ellipticine activated with cytochrome P450 mediates DNA damage determining its pharmacological efficiencies: studies with rats, Hepatic Cytochrome P450 Reductase Null (HRN™) mice and pure enzymes.

Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b5 alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b5, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models.

A comparison of the metabolism of midazolam in C57BL/6J and hepatic reductase null (HRN) mice

The hepatic cytochrome P450 reductase null (HRN) mouse, which has no functional hepatic Cyp P450s, may represent a useful model for examining extra-hepatic P450-related oxidative metabolism. Here the pharmacokinetics and metabolic fate of midazolam, a drug known to undergo such extra-hepatic metabolism, have been investigated in the HRN mouse and compared with a phenotypically normal strain (C57BL/6J). In addition, the effects of co-administration of the pan-P450 inhibitor 1′-aminobenzotriazole (ABT) on the metabolic profile have been compared in both strains. Significant pharmacokinetic differences for midazolam were observed between the two strains of mice with the HRN mice showing lower circulating concentrations of 1′-hydroxymidazolam but higher concentrations of 1′-hydroxymidazolam-O-glucuronide. A significant increase in midazolam exposure was seen upon ABT exposure for both strains of mice, but no differences in the area under the concentration time curves (AUC) for the monitored metabolites were observed. Although oxidative metabolism of midazolam was not abolished, significant decreases in 1′-hydroxymidazolam formation ratios were observed for both strains of mice exposed to ABT. Metabolite profiling of blood and bile showed a number of qualitative and quantitative differences between HRN and normal mice. These differences in midazolam metabolism between the two strains of mice clearly demonstrate the role that liver P450 enzymes play in the murine metabolism of midazolam. The fate of the compound in the HRN mice shows the importance of extrahepatic metabolism and also showed that these mice appear to be more capable of forming circulating phase II glucuronides than the normal strain.

Utility of the HRN™ (hepatic cytochrome P450 reductase null) mice for investigating mechanisms of liver toxicity of carboxylic-acid-containing drugs

Many carboxylic-acid-containing drugs cause liver injury in humans. Examples include fenclozic acid (FA), which was withdrawn due to jaundice observed in clinical trials, and diclofenac (DFC) which remains widely prescribed despite being associated with liver damage. To explore whether these toxicities could be due to metabolic bioactivation mediated by cytochrome P450 (CYP) or conjugative enzymes, covalent binding (CVB) assays were done using liver microsomal incubations from wild-type and hepatic cytochrome P450 reductase null (HRN™) mice, which are deficient in CYP activity. High levels of CYP-mediated CVB of [14C]-FA and [14C]-DFC were observed in wild-type microsomes, but not in HRN™ microsomes. No UDPGAmediated CVB was detected in microsomes incubated with [14C]-FA. Wild-type and HRN™ mice were orally administered DFC or FA orally for 7days. At 100 mg/kg, FA caused a significant (p<0.05) time-dependent increase in plasma alanine amino transferase (ALT) in wild-type but not HRN™ mice. Aberrant liver histopathology and liver clinical chemistry were evident in HRNTM mice and treatment with DFC and FA “normalised” the elevated ALT levels. These data demonstrate that FA undergoes CYP mediated bioactivation and that HRNTM mice are well suited to investigations of metabolism, but not of liver toxicity, due to impaired liver function.

FXR-dependent reduction of hepatic steatosis in a bile salt deficient mouse model

It has been established that bile salts play a role in the regulation of hepatic lipid metabolism. Accordingly, overt signs of steatosis have been observed in mice with reduced bile salt synthesis. The aim of this study was to identify the mechanism of hepatic steatosis in mice with bile salt deficiency due to a liver specific disruption of cytochrome P450 reductase.In this study mice lacking hepatic cytochrome P450 reductase (Hrn) or wild type (WT) mice were fed a diet supplemented with or without either 0.1% cholic acid (CA) or 0.025% obeticholic acid, a specific FXR-agonist.Feeding a CA-supplemented diet resulted in a significant decrease of plasma ALT in Hrn mice. Histologically, hepatic steatosis ameliorated after CA feeding and this was confirmed by reduced hepatic triglyceride content (115.5±7.3mg/g liver and 47.9±4.6mg/g liver in control- and CA-fed Hrn mice, respectively). The target genes of FXR-signaling were restored to normal levels in Hrn mice when fed cholic acid. VLDL secretion in both control and CA-fed Hrn mice was reduced by 25% compared to that in WT mice. In order to gain insight in the mechanism behind these bile salt effects, the FXR agonist also was administered for 3weeks. This resulted in a similar decrease in liver triglycerides, indicating that the effect seen in bile salt fed Hrn animals is FXR dependent.In conclusion, steatosis in Hrn mice is ameliorated when mice are fed bile salts. This effect is FXR dependent. Triglyceride accumulation in Hrn liver may partly involve impaired VLDL secretion.

Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling

Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kgbody weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b5 in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b5 may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por.

Modification of the metabolism and toxicity of styrene and styrene oxide in hepatic cytochrome P450 reductase deficient mice and CYP2F2 deficient mice

Styrene causes toxicity in both the lung and the liver. The study of the relationship of this toxicity to the metabolism of styrene has been aided by the use of knockout mice for both bioactivation and detoxification pathways. It has been hypothesized that CYP2E1 is primarily responsible for styrene bioactivation in mouse liver and CYP2F2 in mouse lung. Two knockout strains were used in the current studies. Mice deficient in hepatic cytochrome P450 reductase had much less hepatic metabolism of styrene to styrene oxide. Styrene (600mg/kg, i.p.) caused significant hepatotoxicity, as determined by serum sorbitol dehydrogenase and glutathione levels, in the wild-type but not in the knockout mice. It caused lung toxicity, as determined by protein levels, cell number, and lactate dehydrogenase activity in the bronchioalveolar lavage fluid of wild-type mice, but this effect was less in the knockout mice. In CYP2F2 knockout mice there was only a small decrease in the hepatic metabolism of styrene but a very large decrease in pulmonary metabolism. As expected the CYP2F2 knockout and wild-type mice were equally susceptible to styrene-induced hepatotoxicity, but the knockout mice were less susceptible to styrene-induced pneumotoxicity. Although the results are inconsistent with the simple hypothesis that styrene pneumotoxicity is due to the bioactivation of styrene to styrene oxide by CYYP2F2, they demonstrate the importance of both liver and lung in the metabolism of styrene, but additional pharmacokinetic studies are needed to help clarify the relationship between target organ metabolism and susceptibility.

7-Dehydrocholesterol reductase activity is independent of cytochrome P450 reductase

7-Dehydrocholesterol reductase (DHCR7) catalyzes the final step in cholesterol synthesis. The enzyme utilizes NADPH as a source of electrons and has been reported to require NADPH-cytochrome P450 reductase (POR) as its redox partner. To test this hypothesis, microsomes were prepared from the livers of mice in which hepatic cytochrome P450 reductase expression was extinguished during maturation. These microsomes contained negligible levels of POR but had 2.5-fold greater DHCR7 activity than did microsomes from wild-type mice. Consistent with this greater activity, immunoblot analysis of DHCR7 expression indicated that DHCR7 protein levels were elevated 2-fold in POR-null microsomes. Addition of POR to these microsomes provided no stimulation of DHCR7 activity, confirming the lack of a role for POR in DHCR7 activity. Because the original observation that POR was necessary for DHCR7 activity was based, in part, on antibody inhibition studies with POR antibody, the ability of an antibody to the full-length POR protein to inhibit DHCR7 activity and cytochrome c reductase activity was tested; the antibody had no effect on DHCR7 activity but decreased cytochrome c reductase activity (a POR-catalyzed reaction) by 50%. Immunoblot analysis further demonstrated no cross-reactivity between POR and DHCR7 with antibodies to either protein. We conclude that cytochrome P450 reductase is not involved in 7-dehydrocholesterol reductase activity.

Effect of cytochrome P450 and aldo-keto reductase inhibitors on progesterone inactivation in primary bovine hepatic cell cultures

Progesterone is required for maintenance of pregnancy, and peripheral concentrations of progesterone are affected by both production and inactivation. Hepatic cytochrome P450 (EC 1.14.14.1) and aldo-keto reductase (EC 1.1.1.145–151) enzymes play a pivotal role in the first step of steroid inactivation, which involves the addition of hydroxyl groups to various sites of the cyclopentanoperhydrophenanthrene nucleus. The current objective was to discern the proportional involvement of hepatic progesterone inactivating enzymes on progesterone decay using specific enzyme inhibitors. Ticlopidine, diltiazem, curcumin, dicumarol, and naproxen were used because of their selective inhibition of cytochrome P450s, aldo-keto reductases, and glucuronosyltransferases. Liver biopsies were collected from 6 lactating Holstein dairy cows, and cells were dissociated using a nonperfusion technique. Confluent wells were preincubated for 4h with enzyme inhibitor and then challenged with progesterone for 1h. Cell viability was unaffected by inhibitor treatment and averaged 84±1%. In control wells, 50% of the progesterone had been inactivated after a 1-h challenge with 5ng/mL of progesterone. Preincubation with curcumin, ticlopidine, or naproxen caused the greatest reduction in progesterone inactivation compared with controls and averaged 77, 39, or 37%, respectively. Hydroxylation of 4-nitrophenol to 4-nitrocatechol in intact cells was inhibited by approximately 65% after treatment with curcumin or ticlopidine. Glucuronidation of phenol red or 4-nitrocatechol in intact cells was inhibited by treatment with curcumin, dicumarol, or naproxen. In cytoplasmic preparations, aldo-keto reductase 1C activity was inhibited by curcumin, dicumarol, or naproxen treatment. Microsomal cytochrome P450 2C activity was inhibited by treatment with curcumin or ticlopidine, whereas cytochrome P450 3A activity was inhibited by treatment with curcumin or diltiazem. The contribution of cytochrome P450 2C and cytochrome P450 3A enzymes to progesterone inactivation in bovine hepatic cell cultures was 40 and 15%, respectively. Depending on the inhibitor used, it would appear that the aldo-keto reductase enzymes contribute approximately 40% to the observed progesterone inactivation, although a portion of this inactivation may be attributed to the loss of glucuronosyltransferase activity. Future work focusing on decreasing the activity of these enzymes in vivo could lead to an increase in the bioavailability of progesterone.

The adaptive response to 3‐methylcholanthrene is altered in the liver of adrenalectomized rats

The aryl hydrocarbon receptor (AHR) is a ligand‐activated transcription factor that mediates most effects of aromatic hydrocarbons, such as 3‐methylcholanthrene (MC). The in vivo regulation of hepatic AHR and whether changes in AHR levels affect the response to agonists are not fully understood. We discovered that the level of hepatic AHR protein was decreased in male adrenalectomized (ADX) rats 4 days after surgery. We studied the consequences of this AHR depletion by measuring expression of aromatic hydrocarbon‐responsive genes in the liver of MC‐treated ADX and SHAM‐operated rats at 6 and 54 h post‐MC. Hepatic AHR protein was depleted in ADX rats at 4 days, but not 6 days, post‐surgery. At this later time point, MC increased hepatic AHR protein in ADX but not SHAM rats. Hepatic cytochrome P450 1A1 (CYP1A1), AHR and P450 reductase (CYPRED) mRNA was induced by MC to a greater extent in SHAM versus ADX rats at 6 h. At 54 h post‐MC, induction by MC was greater in ADX versus SHAM rats for CYP1A1 and CYPRED mRNA. CYP1A1 protein levels corresponded to its mRNA. These results suggest that the adaptive response to MC in liver of ADX rats is impaired at 6 h but sustained and enhanced at 54 h relative to SHAM. Changes in the level of hepatic AHR caused by ADX affect the magnitude and duration of the response to aromatic hydrocarbons in vivo in a gene‐dependent manner. [Support: CIHR; OGS; U of Toronto; Peterborough K.M. Hunter Studentship]

Metabolic activation of benzo[a]pyrene in vitro by hepatic cytochrome P450 contrasts with detoxification in vivo: experiments with hepatic cytochrome P450 reductase null mice

Many studies using mammalian cellular and subcellular systems have demonstrated that polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are metabolically activated by cytochrome P450s (CYPs). In order to evaluate the role of hepatic versus extra-hepatic metabolism of BaP and its pharmacokinetics, we used the hepatic cytochrome P450 reductase null (HRN) mouse model, in which cytochrome P450 oxidoreductase, the unique electron donor to CYPs, is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated intraperitoneally (i.p.) with 125 mg/kg body wt BaP daily for up to 5 days. Clearance of BaP from blood was analysed by high-performance liquid chromatography with fluorescence detection. DNA adduct levels were measured by (32)P-post-labelling analysis with structural confirmation of the formation of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene by liquid chromatography-tandem mass spectrometry analysis. Hepatic microsomes isolated from BaP-treated and untreated mice were also incubated with BaP and DNA in vitro. BaP-DNA adduct formation was up to 7-fold lower with the microsomes from HRN mice than with that from WT mice. Most of the hepatic microsomal activation of BaP in vitro was attributable to CYP1A. Pharmacokinetic analysis of BaP in blood revealed no significant differences between HRN and WT mice. BaP-DNA adduct levels were higher in the livers (up to 13-fold) and elevated in several extra-hepatic tissues of HRN mice (by 1.7- to 2.6-fold) relative to WT mice. These data reveal an apparent paradox, whereby hepatic CYP enzymes appear to be more important for detoxification of BaP in vivo, despite being involved in its metabolic activation in vitro.

Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: Studies with the hepatic NADPH:Cytochrome P450 reductase null mouse

Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN ( Hepatic Cytochrome P450 Reductase Null) mouse model, in which cytochrome P450 oxidoreductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine–DNA adducts detected by 32P-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine–DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine–DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine–DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study.

The Disruption of Hepatic Cytochrome P450 Reductase Alters Mouse Lipid Metabolism

To elucidate the role of hepatic cytochrome P450 oxidoreductase (POR) in lipid metabolism, we characterized perturbations in lipid homeostasis in a mouse model deficient in liver POR. Using an integrative approach in which transcriptomics, lipidomics, and various bioinformatic algorithms were employed, a disruption in liver lipid mobilization, oxidation, and electron transport functions were identified. Analyzing the promoters of genes in these biological processes identified common binding motifs for nuclear receptors sensitive to lipid status, while Srebp-1c binding sites were only identified in genes involved in lipid metabolism. POR-null mice had drastic increases in hepatic lipid content (diacylglycerols, triacylglycerols, phosphatidylcholine, and cholesterol esters) and a specific enrichment in n-7 and n-9 monounsaturated fatty acids (FAs). It was found that while transporters involved in peroxisomal FA oxidation were induced, mitochondrial oxidation appeared to be more tightly controlled, supporting the increase in monounsaturated FAs. Genes coding for hepatic transporters were differentially expressed, where lipid uptake was induced and efflux repressed, indicating that in the absence of hepatic POR the liver serves as a lipid reservoir. Furthermore, while significant changes in intestinal gene expression were found in POR-deficient mice, only minor changes to plasma and intestinal lipid content were observed. Thus, while liver POR plays an important role regulating gene expression and lipid metabolism locally, the hepatic deficiency of this enzyme reverberates throughout the biological system and produces a coordinated response to the low levels of circulating cholesterol and bile.

Effect of Perinatal Low Protein Diets on the Ontogeny of Select Hepatic Cytochrome P450 Enzymes and Cytochrome P450 Reductase in the Rat

In the present study, we administered two low protein diets (LPDs) to rats during pregnancy and lactation and determined their effect on the ontogeny of select hepatic cytochrome P450 (P450) isoforms in their offspring. The L93 and LM76 LPDs were derived from the American Society of Nutrition recommended AIN93G and a modified version of the AIN76A purified control diets, respectively. The LPDs contained 8% crude protein in the form of casein, whereas the purified control diets contained 19% casein. A regular cereal-based diet (NP) was also included, and, therefore, a total of five groups were tested. Pups in all five groups were weaned onto a regular NP diet on postnatal day 28. Perinatal LPD altered the activities of a number of P450 isoforms in 28-day-old male and female offspring. However, nutritional rehabilitation abolished most of these changes as evidenced by lack of differences between the five groups in the activities of P450 isoforms in either 65- or 150-day-old offspring. Interestingly, 58-day-old female offspring in the LM76 group but not those in the L93 group exhibited shorter hexobarbital sleep time than the purified control group. However, hexobarbital hydroxylase activity and the amount of CYP2C12 protein, an important P450 isoform involved in hexobarbital metabolism in females, were unchanged. This suggests that the decrease in hexobarbital sleep time in this group is not due to an increase in the activity of hexobarbital-metabolizing enzymes. In summary, perinatal LPDs produced transient alterations in activities of select hepatic P450s and resulted in a gender- and diet-dependent long-term alteration in hexobarbital pharmacodynamics.

The hepatic cytochrome P450 reductase null mouse as a tool to identify a successful candidate entity

Cytochrome P450s (CYP) play a pivotal role in the metabolism of drugs and xenobiotics, and have been intensively studied over many years. Much of the work carried out on the role of hepatic cytochrome P450s in drug metabolism and disposition has been done in vitro, and has yielded vital information on P450 regulation and function. However, additional factors such as route of administration, absorption, drug transporters, renal clearance and extra-hepatic P450s, make it difficult to extrapolate from in vitro data to in vivo pharmacokinetics. A number of cytochrome P450s knockout mice have been generated, although many have been of limited usefulness due to either embryonic/perinatal lethality, or the functional redundancy inevitably found in a large family of isoenzymes. We have developed a mouse line (HRN) in which cytochrome P450 oxidoreductase (POR), the unique electron donor to cytochrome P450s is deleted specifically in the liver, resulting in the loss of essentially all hepatic P450 function. The HRN mouse, although having disturbances in lipid and bile acid homeostasis develops and breeds normally. We have used the HRN mouse as a model to establish the role of hepatic versus extra-hepatic metabolism in drug metabolism and disposition, and also to investigate the relationship between drug toxicokinetics and therapeutic effect, initially with the chemotherapeutic prodrug cyclophosphamide (CPA).