Total P450 Research Articles

Congenital lipoid adrenal hyperplasia: the human gene knockout for the steroidogenic acute regulatory protein.

Lipoid CAH was first described in detail as an inherited endocrine disorder by Prader and colleagues (Prader & Gurtner 1955, Prader & Siebenmann 1957, Prader & Anders 1962), although at least four autopsy cases appeared earlier in the pathology literature (Tilp 1913, Brutschy 1920, Zahn 1948, Sandison 1955). Prader’s group described male pseudohermaphroditism, an apparent lack of adrenal steroids and accumulation of lipid deposits in the adrenal, inherited in an autosomal recessive fashion. Written in German, Prader’s work was not widely recognized until Camacho et al. (1968) reported an apparently milder case, with the onset of clinically apparent salt loss at 8 months of age, and postulated that the disorder was in one of the enzymes involved in the conversion of cholesterol to pregnenolone. However, the nature of the defect was unclear, in part because the enzymology of the conversion of cholesterol to pregnenolone was unclear. At that time it was thought that the conversion of cholesterol to pregnenolone required at least three enzymes, a 20a-hydroxylase, a 22-hydroxylase, and a 20,22 desmolase (Shimizu et al. 1961), and that lipoid CAH was due to a defect in one of these enzymes. Degenhart et al. (1972) were the first to study lipoid CAH in vitro, by comparing the production of pregnenolone by mitochondria from the adrenals of affected and unaffected infants. Mitochondria from affected tissue could not convert cholesterol to pregnenolone (although normal mitochondria could), but affected mitochondria could convert 20a-hydroxycholesterol to pregnenolone. They logically concluded that the defect was in a specific 20a-hydroxylase; this conclusion was incorrect, but the experimental design was prescient: 23 years later we used a similar experiment to prove that lipoid CAH was due to a mutation in a non-enzymatic protein (Lin et al. 1995). Koizumi et al. (1977) also found that affected mitochondria failed to convert cholesterol to pregnenolone but were unable to account for Degenhart’s findings. By this time it was clear that the conversion of cholesterol to pregnenolone was catalyzed by a mitochondrial cytochrome P450 enzyme, termed P450scc, (where scc denotes side chain cleavage), which functions as the terminal oxidase in a mitochondrial electron-transfer chain where NADPH donated electrons to a flavoprotein (adrenodoxin reductase) which then transferred them to an iron-sulfur protein (adrenodoxin) which in turn donated them to P450scc (Kimura & Suzuki 1965, Omura et al. 1966, Shikita & Hall 1973) (for review see Miller 1988). Thus Koizumi examined the total P450 in the affected adrenal mitochondria in the only fashion then available, by carbon monoxide-induced difference spectra, and concluded that affected mitochondria had roughly half of the normal amount of total cytochrome P450. At that time it was known that steroid 11â-hydroxylase and 18-hydroxylase activities were also catalyzed by a mitochondrial P450, and as the affected mitochondria had normal 11â-/18-hydroxylase activity but failed to convert cholesterol to pregnenolone, Koizumi et al. (1977) logically concluded that the 227

Effects of Aroclor 1254 on the Thyroid Gland, Immune Function, and Hepatic Cytochrome P450 Activity in Mallards

Adult male mallards were exposed to 0, 4, 20, 100, 250, and 500 mg/kg Aroclor 1254 by gavage twice per week for 5 weeks. Immunotoxic effects, as measured by antibody titers to sheep erythrocytes, natural killer cell activity and lymphocyte mitogenesis to phytohemagglutinin, were not detected as a consequence of polychlorinated biphenyl (PCB) exposure. Hepatic cytochrome P450 activities were measured as microsomal dealkylations of ethoxy-resorufin (EROD) and pentoxyresorufin (PROD). Significant elevations in EROD and PROD were noted at 20 mg/kg and peaked in birds treated with 100 mg/kg. Total P450 was induced beginning at 100 mg/kg and peaked at 250 mg/kg. Relative liver weights were dose-dependently increased following treatment with 100 mg/kg or more. Thyroid weights were significantly increased in PCB-treated birds treated with 100 mg/kg or greater, but no significant histological abnormalities were observed, except at the highest dose. Plasma total triiodothyronine (T3) was decreased in a dose-dependent manner, with a significant lowest-observed-adverse-effect level (LOAEL) of 20 mg/kg. T3 was decreased following 7 days treatment with 100 mg/kg. The no-observed-adverse-effect level (NOAEL) was 4 mg/kg for decreased T3. Plasma glucose levels were decreased on days 28 and 35 in mallards treated with 500 mg/kg, while other clinical plasma biochemistry parameters were unaltered by PCB treatment. Plasma corticosterone levels were unchanged by PCB treatment. These results indicate that thyroid hormone levels and P450 activity in mallards are sensitive to subchronic PCB exposure in the absence of gross toxic effects and immunotoxicity.

Do Endogenous Volatile Organic Chemicals Measured in Breath Reflect and Maintain CYP2E1 Levelsin Vivo?

The effect oftrans-1,2-dichloroethylene (DCE), an inhibitor of cytochrome P450 (P450) 2E1 (CYP2E1), on the composition and quantity of volatile organic chemicals (VOCs) expired in the breath of male F-344 rats was determined in parallel with hepatic P450 activity and content. Hepatic microsomes were prepared from groups of rats prior to dosing and at 2, 5, 12, and 24 hr postdosing with DCE (100 mg/kg ip), and total P450 content and the activity of CYP2E1 was determined. Breath was collected from parallel groups of rats predose and at several intervals that encompassed the time points for rats euthanized for microsome preparation. Over 100 breath components were identified by GC/MS and quantitated by GC/FID. The overall change in the profile of breath VOCs resulting from administration of DCE was striking. An increase of approximately 1000% was measured in the mass of non-DCE-derived VOCs exhaled 4–6 hr after dosing, but there was no increase in hepatic lipid peroxidation. In addition to hexane, short-chain methyl ketones were particularly affected, and percentage increases in response to inhibition were inversely related to chain length, with acetone and 2-butanone > 2-pentanone ⪢ 2-hexanone > 2-heptanone. There were no statistically significant decreases in total content of P450, but the activity of CYP2E1 was diminished about 65% at 2 and 5 hr after DCE treatment. However, 24 hr after inhibitor administration the total mass of VOCs expired was only marginally elevated above baseline and CYP2E1 activity was not significantly different from that of untreated rats. The compounds most markedly increased upon inhibition of CYP2E1 are also excellent inducers of that isozyme, and this finding is consistent with the hypothesis that these chemicals are important to the normal homeostasis of CYP2E1. The increase in breath components observed following inhibition of CYP2E1 suggests that VOCs in breath can reflect the activity of that isozymein vivo.

In vivo and in vitro effects of deltamethrin on cytochrome p450 monooxygenase activity in carp (cyprinus carpio L.) liver

The in vivo and in vitro effects of the insecticide deltamethrin (DM) on hepatic cytochrome P450 (Cyt P450) monooxygenase were examined in adult carp. The in vivo experiments were carried out with 0.2 μg/l DM at 20 °C. The changes in the hepatic microsomal Cyt P450 content and the Cyt P450‐dependent monooxygenase activities were studied in DM‐treated fish. Although there were no changes in the Cyt P450 content during the exposure time, after treatment for 24 h all the investigated isoenzyme activities (para‐nitrophenetole‐O‐deethylase, p‐NPOD; aminopyrene‐N‐demethylase, APND; ethylmorphine‐N‐demethylase, EMND; 7‐ethoxycoumarin‐O‐deethyiase, ECOD; and ethoxyresorufin‐O‐deethylase, EROD) were significantly inhibited. After 72 h, all the activities were still lower than in the control animals. In vitro incubation of liver microsomes with DM led to a concentration‐dependent decrease in total microsomal Cyt P450 content. A complete loss of Cyt P450 occurred after a 5‐min incubation with 60 μM DM. The maximum in the difference spectra of microsomes was shifted to higher wavelength, showing the strong interaction of DM with Cyt P450. EROD and ECOD activities were inhibited by DM. The in vitro kinetic results on ECOD revealed that the inhibition was of non‐competitive type, with K1 = 9.8 ± 2.3 μM. This study indicates important biochemical effects of DM in fish liver, and suggests that exposure to DM may cause loss of the Cyt P450‐dependent metabolism in fish.

Gadolinium chloride reduces cytochrome P450: Relevance to chemical-induced hepatotoxicity

The Kupffer cell inhibitor, gadolinium chloride (GdCl3), protects the liver from a number of toxicants that require biotransformation to elicit toxicity (i.e. 1,2-dichlorobenzene and CCl4), as well as compounds that do not (i.e. cadmium chloride and beryllium sulfate). The mechanism of this protection is thought to result from reduced secretion of inflammatory and cytotoxic products from Kupffer cells (KC). However, since other lanthanides have been shown to decrease cytochrome P450 (P450) activity, the following studies were designed to determine if GdCl3 pretreatment alters hepatic P450 levels or activity. The toxicological relevance of GdCl3-mediated alterations in P450 activity was also estimated by determining the effect of GdCl3 pretreatment on the susceptibility of primary cultured hepatocytes to CCl4 and cadmium chloride (CdCl2). Male and female Sprague-Dawley rats were given GdCl3 (i.v., 10 mg/kg). Twenty-four hours later, livers were either processed for preparation of microsomes or for primary cultures of hepatocytes. Gadolinium chloride treatment reduced total hepatic microsomal P450 as well as aniline hydroxylase activity by approximately 30% in males and 20% in females. In hepatocytes isolated from rats pretreated with GdCl3, the toxicity caused by CCl4, but not CdCl2 was reduced. Interestingly, when GdCl3 was administered in vitro to microsomes, there was no effect on either the microsomal P450 difference spectra or p-hydroxylation of aniline. However, when GdCl3 was incubated with isolated hepatocytes, the cytotoxicity of CCl4 (but not CdCl2) was partially attenuated. These results suggest that, in addition to its inhibitory effects on KC, GdCl3 produces other effects which may alter the susceptibility of hepatocytes to toxicity caused by certain chemicals.

Down-regulation of the hepatic cytochrome P450 by an acute inflammatory reaction: implication of mediators in human and animal serum and in the liver.

1. Infection and inflammation trigger a cascade of mediators that eventually will down-regulate the hepatic cytochrome P450 (P450). The present study aimed to characterize the mediators contained in the serum of rabbits with an acute inflammatory reaction (AIR) induced by the s.c. injection of turpentine (5 ml), and in the serum of humans with an acute upper respiratory tract viral infection. 2. Hepatocytes from control (H(CONT)) rabbits and rabbits with an AIR (H(INFLA)) were isolated and cultured. Compared with H(CONT) in H(INFLA) the production of theophylline metabolites, 3-methylxanthine (3MX), 1-methyluric acid (1MU), and 1,3-dimethyluric acid (1,3DMU) was reduced as was the amount of total P450, while lipid peroxidation was increased. Incubation of H(INFLA) with serum of rabbits with an AIR (RS(INFLA)) for 4 h further reduced the formation of the metabolites of theophylline as well as the amount of P450, and enhanced the lipid peroxidation. RS(INFLA) obtained 6, 12 and 24 h after the injection of turpentine showed the same ability to down-regulate hepatic P450 as the serum obtained at 48 h. 3. The efficacy (Emax) of RS(INFLA) to inhibit the formation of theophylline metabolites differed, i.e. 1,3DMU > 1MU > 3MX, and the potency of serum mediators (IC50) was similar for 3MX and 1MU, but lower for 1,3DMU. 4. Incubation of serum of human volunteers (HS(INFLA)) with a viral infection with H(CONT) or H(INFLA) reduced the production of theophylline metabolites, as well as the amount of P450, and increased the lipid peroxidation. HS(INFLA) depressed 1,3DMU more efficiently than 3MX and 1MU. HS(INFLA) reduced 3MX with greater efficacy than did RS(INFLA). Potency was very variable but not different from rabbits. 5. It is concluded that the serum of rabbits with an AIR or of humans with a viral infection contain several mediators that inhibit noncompetitively various isoenzymes of the hepatic P450. The decrease in P450 induced by HS(INFLA) or RS(INFLA) is closely associated with the increase in lipid peroxidation (r2= 0.8870) suggesting that lipid peroxidation could directly or indirectly be involved in the P450 down-regulation.

Characterization of the P450 system in Göttingen minipigs.

It is essential to establish the activity and regulation of the cytochrome P450 system of species selected for toxicological and pharmacological studies. The minipig has become a popular substitute for the traditional non-rodent species although little information is available on its P450 system. The total P450 and the enzyme activity of the most important drug-metabolizing isoenzymes: CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 were measured in liver microsomes from 4 minipigs and 8 conventional pigs of both sexes. Immunochemical levels were determined for 4 of teh isoenzymes. The total P450 activity was slightly higher in minipigs compared to conventional pigs but no sex difference was detected. CYP1A2 activity (7-ethoxyresorufin) was 4 times higher in female minipigs than in male minipigs. The activity in male minipigs was almost identical to the activity in conventional pigs. The activity of CYP2E1 (chlorzoxazone) was 4 times higher in female than in male minipigs and 2 times higher in female than in male conventional pigs. No activity of CYP2D6 (debrisoquine) and CYP2C19 (mephenytoin) could be detected. The CYP3A4 activity (testosterone) detected in minipigs was higher than the activity in conventional pigs. A weak sex difference was seen in both strains. Western blotting using anti-human CYP2E1 and CYP3A4 confirmed the results obtained in the enzyme activity assays, while only CYP1A2 correlated with the activity in the conventional strain. The total P450 enzyme activity was close to the levels reported for human beings, as were the activities of CYP2E1 and CYP3A4.

Purification and characterization of three constitutive cytochrome P-450 isoforms from bovine olfactory epithelium.

Three constitutive forms of cytochrome P-450 (P-450s) were isolated from olfactory microsomes of cattle. The purified P-450s, designated P-450bov1, P-450bov2 and P-450bov3, were electrophoretically nearly homogeneous by SDS/PAGE and their apparent relative molecular masses were estimated to be 50000, 53000 and 51000 respectively. As indicated by several criteria including the N-terminal sequence and absorption spectra, the three olfactory forms of P-450 were distinct from each other and from all the other P-450s currently known in cattle. P-450bov1 and P-450bov2 were purified in the low-spin state, whereas P-450bov3 was in the high-spin state. Studies to evaluate, by Western blot analysis, the reactivity of these purified P-450s with antibodies raised against rat hepatic P-450 2E1, 2B, 1A and 3A and rabbit olfactory P-450NMa and P-450NMb showed that P-450bov3 strongly cross-reacted with anti-P-450NMb IgG, and P-450bov1 moderately with anti-P-450NMa IgG. As determined by immunoblots, P-450bov1 and P-450bov3 represented a great portion of the total olfactory P-450. In a reconstituted system with NADPH:cytochrome P-450 reductase and phospholipids, P-450bov1 was more active in the metabolism of xenobiotic compounds (i.e. O-de-ethylation of ethoxycoumarin and N-demethylation of hexamethylphosphoramide) than towards endogenous substrates (testosterone and progesterone). Conversely, P-450bov3 metabolized the xenobiotics at lower rates but exhibited total oxidation rates of the above sex hormones higher than those of P-450bov1. From the comparison of the catalytic, immunochemical and structural properties, it was inferred that P-450bov1 and P-450bov3 are the bovine orthologues of P-450NMa (2A) and P-450NMb (2G1) respectively, the only two olfactory P-450s previously purified from rabbit. P-450bov2, which showed low activity toward some exogenous and endogenous compounds, represents a novel purified olfactory hemoprotein possibly belonging to the 3A subfamily. These results are consistent with a specific presence of catalytically and structurally similar P-450s, at least for the major ones, in the olfactory mucosa of mammals.

Cytochromes P450 (CYP) in Tropical Fishes: Catalytic Activities, Expression of Multiple CYP Proteins and High Levels of Microsomal P450 in Liver of Fishes From Bermuda

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b 5 content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23–2.1 nmol/min/mg and 0.5–11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major ( Abudefduf saxatilis) and Bermuda chub ( Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with β-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6 β- and 16 β-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.

Effects of arsenite pretreatment on the acute toxicity of parathion

Parathion (PA) is a phosphorotioate pesticide requiring P-450-mediated oxidations to become activated to paraoxon, or to be metabolised to its less toxic metabolites. On the other hand, sodium arsenite [As(III)] markedly decreases total hepatic P-450 content and dependent monoxygenase activities. Our aim was to determine the effects of As(III) pretreatment on the acute toxicity of PA and its possible relationship with the effects of As(III) on P-450-dependent monooxygenase activities. Adult male Wistar rats were pretreated with As(III) (5.6 mg As(III)/kg s.c.), and 24 h later given PA (5 to 20 mg/kg, per os). As(III) pretreatment increased the acute toxicity of PA, reducing 38% its median lethal dose (LD50) from 11.68 to 7.21 mg PA/kg. In addition, As(III) pretreatment further decreased the inhibitory effect of PA on brain acetylcholinesterase activity, reducing 33% the median inhibitory dose (ID50) from 3.44 to 2.31 mg PA/kg, whereas As(III) alone had no significant effects. As(III) decreased the P-450 content to 87% of control values, reduced EROD activity to 74% and BROD activity to 41%; PA produced no significant effects on these parameters, whereas the joint administration of As(III) + PA produced effects similar to those of As(III). PROD activity was reduced to 36% of control value by PA, whereas As(III) alone produced no significant effects. However, As(III) pretreatment apparently protected against the inhibition of CYP2B1-mediated PROD activity produced by PA, since PROD values were similar to those of control animals. Our results also indicated that the increase in PA toxicity caused by As(III) pretreatment, could also be related to the CYP2B2 isoform, since decreases in CYP2B2-dependent BROD activity were observed in both As(III) and As(III) + PA groups, but not in PA-treated animals, suggesting that CYP2B2 is involved in PA detoxification

Separation of three P450 isozymes from liver microsomes of gilthead seabream treated with ß‐NF and partial purification of cytochrome P4501A1

Three distinct cytochrome P450 isozymes, P4501A1, P4502B and an unidentified P450 isozyme were separated and isolated from beta-NF-treated gilthead seabream liver microsomes. Cytochrome P4501A1 was partially purified from beta-NF-treated gilthead seabream liver microsomes in the presence of detergents Emulgen 913 and cholate and protease inhibitors using two DEAE-cellulose, Porapak Q and two hydroxylapatite column chromatographies. The overall yield of purified P4501A1 was 1.2% with respect to microsomal total P450 with a specific content of 3 nmol/mg protein. The purified P4501A1 was characterized with respect to spectral, electrophoretic, biocatalytic and immunochemical properties, which are found to be similar to P4501A1s purified from other teleost species such as trout, scup, cod and perch suggesting that the P450 we have purified belongs to CYP1A1.

Lanosterol 14-demethylase activity expressed in rat brain microsomes.

The occurrence of sterol 14-demethylase in rat brain microsomes was confirmed. The brain microsomes from adult rats converted lanosterol into its 14-demethylated products, 4,4-dimethylcholesta-8, 24-dienol, and 4,4-dimethylcholesta-8,14,24-trienol, in the presence of NADPH and molecular oxygen. This metabolism of lanosterol was inhibited by carbon monoxide and ketoconazole, a potent inhibitor of sterol 14-demethylase P450 (P45014DM or CYP51). These facts indicated the occurrence of lanosterol 14-demethylation in rat brain microsomes and its dependency on P45014DM. A representative value of the lanosterol demethylase activity of the brain microsomes was 8.4 pmol/min/mg protein or 640 pmol/ min/nmol of total P450. The former was about one-thirteenth of the corresponding value observed with liver microsomes from the same rats, while the latter was 4-times higher than the corresponding value obtained with the liver microsomes. This fact suggested that the ratio of P45014DM to other P450 species was higher in brain than in liver. Lanosterol 14-demethylation is situated at the root of the sterol-biosynthetic branch of the mevalonic acid pathway. Therefore, the present finding enzymologically supports the existence of the sterol biosynthetic pathway in brain.

Differential induction of carcinogen metabolizing enzymes in a transgenic mouse model of fulminant hepatitis.

The objective of this work is to examine the possible modulation of carcinogen metabolism (activation by cytochrome P450s and detoxification by conjugation via glutathione S-transferases [GST]) in relation to hepatitis B virus (HBV)-associated liver injury. In HBV transgenic mouse lineage 107.5, the hepatitis B surface antigen (HBsAg) is expressed at noncytopathic concentrations but after injection of an HBsAg-specific, major histocompatibility complex (MHC) class I restricted cytotoxic T-lymphocyte (CTL) clone, the mice develop a severe acute necroinflammatory liver disease that reaches maximum severity within 3 days and gradually subsides during the next 2 to 3 weeks. In this model, using immunohistochemical analysis, we observed an increase of P450s (CYP1A and 2A5), both involved in aflatoxin B1, metabolism, but minor changes or no changes for others (2B, 2C, 2E, 3A). There was a fivefold decrease in the total liver P450 microsomal content 3 days' post-CTL injection with the result that the relative proportion of CYP2A5 and 1A compared with other P450s is increased. Individual microsomal P450 enzyme contents estimated by Western blotting; Northern blot analysis of liver CYP messenger RNA (mRNA) levels as well as in vitro metabolism of specific substrates for different P450 isoenzymes were consistent with the immunohistochemical data. Immunohistochemical staining with antibodies to cytosolic pi class GST was increased 1 and 3 days postinjection followed by a progressive decrease at later time points (the same phenomenon was observed to a lesser extent for GST alpha). The activity of hepatic cytosols toward substrates specific for different subclasses of GST (mu, pi, alpha) showed that while GST mu was not changed in the CTL-injected HBV transgenic mice, GST pi and, to a lesser extent, alpha were increased as compared with controls. These results suggest that liver cell injury induced by a process of acute fulminant-like hepatitis can lead to the induction of some carcinogen metabolizing enzymes notably, Cyp 1A, 2A5 and GST pi in the mouse.

Differential induction of carcinogen metabolizing enzymes in a transgenic mouse model of fulminant hepatitis

The objective of this work is to examine the possible modulation of carcinogen metabolism (activation by cytochrome P450s and detoxification by conjugation via glutathione S-transferases [GST]) in relation to hepatitis B virus (HBV)-associated liver injury. In HBV transgenic mouse lineage 107.5, the hepatitis B surface antigen (HBsAg) is expressed at noncytopathic concentrations but after injection of an HBsAg-specific, major histocompatibility complex (MHC) class I restricted cytotoxic T-lymphocyte (CTL) clone, the mice develop a severe acute necroinflammatory liver disease that reaches maximum severity within 3 days and gradually subsides during the next 2 to 3 weeks. In this model, using immunohistochemical analysis, we observed an increase of P450s (CYP1A and 2A5), both involved in aflatoxin B1, metabolism, but minor changes or no changes for others (2B, 2C, 2E, 3A). There was a fivefold decrease in the total liver P450 microsomal content 3 days' post-CTL injection with the result that the relative proportion of CYP2A5 and 1A compared with other P450s is increased. Individual microsomal P450 enzyme contents estimated by Western blotting; Northern blot analysis of liver CYP messenger RNA (mRNA) levels as well as in vitro metabolism of specific substrates for different P450 isoenzymes were consistent with the immunohistochemical data. Immunohistochemical staining with antibodies to cytosolic pi class GST was increased 1 and 3 days postinjection followed by a progressive decrease at later time points (the same phenomenon was observed to a lesser extent for GST alpha). The activity of hepatic cytosols toward substrates specific for different subclasses of GST (mu, pi, alpha) showed that while GST mu was not changed in the CTL-injected HBV transgenic mice, GST pi and, to a lesser extent, alpha were increased as compared with controls. These results suggest that liver cell injury induced by a process of acute fulminant-like hepatitis can lead to the induction of some carcinogen metabolizing enzymes notably, Cyp 1A, 2A5 and GST pi in the mouse. (Hepatology 1996 Sep;24(3):649-56)

Purification and characterization of constituent androstenedione 15α-hydroxylase (Cytochrome P450 15αAD) from mouse liver : Sex- and tissue-dependent expression

Hepatic microsomal androstenedione 15α-hydroxylase (i.e. cytochrome P450 15αAD) was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-Sephacel, and hydroxylapatite columns at heme absorbing 417 nm, by cytochrome P450 content, reactivity to monoclonal antibody against female-specific rat cytochrome P450 2C12, and androstenedione 15α-hydroxylase activity. The catalytic activity for androgens of the purified cytochrome P450 15αAD, exhibiting a high degree of regioselectivity and stereospecificity, was restricted to the 7α- and 15α-hydroxylation of androstenedione, representing, respectively, >5% and >93% of the total metabolites. Polyclonal antibodies against cytochrome P450 15αAD exhibited a concentration-dependent and very selective inhibition of hepatic microsomal androstenedione 7α and 15α-hydroxylation and a 60% inhibition of benzphetamine demethylation, the latter drug appearing to be a much more effective substrate than androgens. Cytochrome P450 15αAD accounted for about 3% of the total P450 in female mouse liver microsomes. The apparent subunit molecular weight of P450 15αAD was 53,000, and the protein appeared as a single band on sodium dodecyl sulfate-polyacrylamide gels. The isoform was intensely expressed in both liver and lung of CD-1 female mice and was female-predominant in the livers of five or eight strains examined; it was sex-independent in the remaining three strains. Amino-terminal sequence analysis indicates that cytochrome P450 15αAD is a member of the murine cytochrome P450 2c subfamily.

Cytochrome P-450 expression in sudden infant death syndrome

In the human liver, the major rise of the cytochrome P-450 isoform content occurs during the first months following birth (e.g., the high vulnerability period to sudden infant death syndrome (SIDS), a syndrome frequently associated with viral infection and drug hypersensitivity. We examined the expression of individual P-450 isoforms in liver samples collected postmortem from SIDS infants and compared values with those of control adults and children of the same age suffering from various pathologies. Hepatic microsomes were prepared and examined for their content in total P-450, the level of individual isoforms (CYP1A2, CYP2E1, CYP4A, CYP3A, and CYP2C) determined with specific antibodies and for their enzymatic activities. Total RNA was extracted and probed with several CYP cDNAs and oligomers. The overall hepatic P-450 content was not modified in SIDS infants. Among cytochrome P-450 isoforms, only CYP2C was markedly increased. This rise resulted from an accumulation of RNA encoding CYP2C and was associated with a stimulation of diazepam demethylation. The precocious expression of CYP2C in SIDS could result in a higher production of epoxye-icosatrienoic acids in the neonate, believed to act as relaxant of pulmonary smooth muscles. Its consequence might be the induction of fatal apnea in SIDS.

Nitric oxide mediates hepatic cytochrome P450 dysfunction induced by endotoxin.

Animals subjected to immunostimulatory conditions (sepsis) exhibit decreased total cytochrome P450 content and decreased P450-dependent drug metabolism. Cytochrome P450 function is of clinical significance because it mediates the metabolism of some opioid and hypnotic drugs. The authors tested the hypothesis that reduced P450 function and decreased drug metabolism in sepsis are mediated by endotoxin-enhanced synthesis of nitric oxide. Hepatic microsomes were prepared from male Sprague-Dawley rats in nontreated rats, rats pretreated with phenobarbital and rats receiving aminoguanidine or NG-L-monomethyl-arginine alone. Nitric oxide synthesis was augmented for 12 h with a single injection of bacterial lipopolysaccharides. Nitric oxide synthase was inhibited with aminoguanidine or N(G)-L-monomethyl-arginine during the 12 h of endotoxemia in some animals. Plasma nitrite and nitrate concentrations were measured in vivo, and total microsomal P450 content, and metabolism of ethylmorphine and midazolam in vitro. Administration of endotoxin increased plasma nitrite and nitrate concentrations, decreased total cytochrome P450 content, and decreased metabolism of ethylmorphine and midazolam. Inhibition of nitric oxide formation by aminoguanidine or N(G)-L-monomethyl-arginine partially prevented the endotoxin-induced effects in the nontreated and phenobarbital-treated groups. Aminoguanidine or N(G)-L-monomethyl-arginine alone did not have an effect on either total cytochrome P450 content or P450-dependent drug metabolism. Plasma nitrite and nitrate concentrations correlated significantly negatively with P450 content (nontreated r = -0.88, phenobarbital r = -0.91), concentrations of formed formaldehyde (nontreated r = -0.87, phenobarbital r = -0.95), and concentrations of midazolam metabolites (4-OH midazolam nontreated r = -0.88, phenobarbital r = -0.93, and 1'-OH midazolam nontreated r = -0.88, phenobarbital r = -0.97). Altered hepatic microsomal ethylmorphine and midazolam metabolism during sepsis is mediated in large part by nitric oxide.

Liver Injury and Expression of Cytochromes P450: Evidence That Regulation of CYP2A5 Is Different from That of Other Major Xenobiotic Metabolizing CYP Enzymes

The purpose of this study was to find out how liver injury caused by two well-known hepatotoxins, chloroform and thioacetamide, alters the expression of hepatic xenobiotic metabolizing cytochrome P450 (CYP) enzymes of DBA/2N mice. Dose-dependent toxic effects of the two hepatotoxins were verified by histological examination. Along with the toxicity, intense staining of immunoreactive material was detected in the centrilobular zone, with anti-CYP2A5 antibody in hepatic tissue. This apparent increase in the expression ofCyp2a-5was verified by Northern blot and Western blot analyses and by determining the enzymatic activity, coumarin 7-hydroxylase, in hepatic tissue. The results suggest that liver injury due to these hepatotoxins increases the expression ofCyp2a-5and that the expression is pretranslationally regulated. The increased expression ofCyp2a-5is in contrast with that of other xenobiotic metabolizing CYPs because a dose-dependent decrease of the total hepatic P450 content and either a decrease or no change in the levels of CYP1A, 2B, 2C, 2E1, and 3A4 were observed. The results suggest that essential differences exist in the regulation of CYP2A5 and other major xenobiotic metabolizing CYP enzymes and that in a damaged liver CYP2A5 may be a major catalyst of xenobiotic metabolism.

Multiple forms of human P450 expressed in Saccharomyces cerevisiae: Systematic characterization and comparison with those of the rat

We systematically characterized the levels and substrate specificity of P450s from humans and rats to extrapolate drug metabolism data from experimental animals to humans. Human P450s (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, and 3A4) were expressed in Saccharomyces cerevisiae and purified. Rat P450s were purified from hepatic microsomes of rats. We investigated the catalytic activities of purified P450s in a reconstituted system. Human CYP2B6 and rat CYP2B1 had high lidocaine N-deethylation activity. Human and rat CYP2D forms had high debrisoquine 4-hydroxylation activity. Human CYP3A4 and rat CYP3A2 had high testosterone 2β- and 6β-hydroxylation activities in a modified reconstituted system with a lipid mixture. The hydroxylation site of testosterone by CYP2B6 (16α- and 16β-positions) agreed with that by rat CYP2B1. Human CYP2E1 had the highest lauric acid (ω-1)-hydroxylation activity and also had catalytic properties similar to those of rat CYP2E1. Human CYP2A and 2C forms had catalytic properties in testosterone metabolism different from those of rats. Antibodies raised against purified P450s were used to measure the levels of hepatic P450s. The level of CYP3A4 was the highest in human hepatic microsomes, comprising 30–40% of the total P450. CYP2C9 comprised 10–20% of the total. The levels of CYP1A2, 2A6, 2C8, 2D6, and 2E1 were moderate (5–15% of total P450). CYP2B6 content was very low. The information of this study is useful for drug metabolism and toxicological studies.

Role of cytochrome P-450 in the stimulation of microsomal production of reactive oxygen species by ferritin

Microsomes can remove iron from ferritin by a superoxide-dependent reaction. The released iron can then catalyze formation of a variety of reactive oxygen species. Experiments were carried out to evaluate the role of cytochrome P-450 in the release of iron from ferritin, and whether induction of certain P-450 isoforms alters ferritin-dependent reactive oxygen radical production. Rats were treated with phenobarbital, 3-methylcholanthrene, 4-methylpyrazole, or saline to produce microsomes with varying P-450 content and composition. Oxidations of 2.7′-dichlorofluorescein diacetate to a fluorescent product and chemiluminescence were used as indices of production of reactive oxygen species. The extreme sensitivity of these reactions to trolox, a potent chain-breaking oxidant, indicates the involvement of lipid peroxidation products in these reactions. In the absence of ferritin, formation of reactive oxygen species was higher in microsomes from the treated rats compared to saline controls when results were expressed on a per mg protein basis but not per nmol P-450, suggesting that the increased content of total P-450 (2-fold increases) rather than the population of isoforms was responsible for ·h, increase. Superoxide dismutase had no effect on the non-ferritin catalyzed reactions. Ferritin increased production of reactive oxygen species with all the microsomal preparations; the increase by ferritin was completely prevented by superoxide dismutase. The net increase by ferritin was higher in microsomes from the treated rats compared to saline controls, but this, again, largely reflected the increased content, rather than the type of isoforms of P-450 present. Similar results were obtained with either NADPH or NADH as microsomal reductants, although NADPH was much more effective in supporting ferritin-dependent reactive oxygen formation. In microsomes from phenobarbital-treated rats, anti-CYP2B1/B2 IgG completely prevented the NADPH- and NADH-dependent increases in reactive oxygen formation produced by ferritin. Anti-cytochrome b 5 IgG produced partial inhibition of the ferritin-stimulation. These results indicate that P-450, and to a lesser extent, cytochrome b 5, play a role in the ferritin-dependent increase in formaion of reactive oxygen species with either NADPH or NADH, most likely reflecting the requirement of these enzymes for microsomal production of superoxide anion.