Microsomal Cytochrome P450 3A4 Research Articles

In Silico Prediction of the Metabolic Resistance of Vitamin D Analogs against CYP3A4 Metabolizing Enzyme.

The microsomal cytochrome P450 3A4 (CYP3A4) and mitochondrial cytochrome P450 24A1 (CYP24A1) hydroxylating enzymes both metabolize vitamin D and its analogs. The three-dimensional (3D) structure of the full-length native human CYP3A4 has been solved, but the respective structure of the main vitamin D hydroxylating CYP24A1 enzyme is unknown. The structures of recombinant CYP24A1 enzymes have been solved; however, from studies of the vitamin D receptor, the use of a truncated protein for docking studies of ligands led to incorrect results. As the structure of the native CYP3A4 protein is known, we performed rigid docking supported by molecular dynamic simulation using CYP3A4 to predict the metabolic conversion of analogs of 1,25-dihydroxyvitamin D2 (1,25D2). This is highly important to the design of novel vitamin D-based drug candidates of reasonable metabolic stability as CYP3A4 metabolizes ca. 50% of the drug substances. The use of the 3D structure data of human CYP3A4 has allowed us to explain the substantial differences in the metabolic conversion of the side-chain geometric analogs of 1,25D2. The calculated free enthalpy of the binding of an analog of 1,25D2 to CYP3A4 agreed with the experimentally observed conversion of the analog by CYP24A1. The metabolic conversion of an analog of 1,25D2 to the main vitamin D hydroxylating enzyme CYP24A1, of unknown 3D structure, can be explained by the binding strength of the analog to the known 3D structure of the CYP3A4 enzyme.

Correlations between 4β-hydroxycholesterol and hepatic and intestinal CYP3A4: protein expression, microsomal ex vivo activity, and in vivo activity in patients with a wide body weight range

PurposeVariability in cytochrome P450 3A4 (CYP3A4) metabolism is mainly caused by non-genetic factors, hence providing a need for accurate phenotype biomarkers. Although 4β-hydroxycholesterol (4βOHC) is a promising endogenous CYP3A4 biomarker, additional investigations are required to evaluate its ability to predict CYP3A4 activity. This study investigated the correlations between 4βOHC concentrations and hepatic and intestinal CYP3A4 protein expression and ex vivo microsomal activity in paired liver and jejunum samples, as well as in vivo CYP3A4 phenotyping (midazolam) in patients with a wide body weight range.MethodsThe patients (n = 96; 78 with obesity and 18 normal or overweight individuals) were included from the COCKTAIL-study (NCT02386917). Plasma samples for analysis of 4βOHC and midazolam concentrations, and liver (n = 56) and jejunal (n = 38) biopsies were obtained. The biopsies for determination of CYP3A4 protein concentration and microsomal activity were obtained during gastric bypass or cholecystectomy. In vivo CYP3A4 phenotyping was performed using semi-simultaneous oral (1.5 mg) and intravenous (1.0 mg) midazolam.Results4βOHC concentrations were positively correlated with hepatic microsomal CYP3A4 activity (ρ = 0.53, p < 0.001), and hepatic CYP3A4 concentrations (ρ = 0.30, p = 0.027), but not with intestinal CYP3A4 concentrations (ρ = 0.18, p = 0.28) or intestinal microsomal CYP3A4 activity (ρ = 0.15, p = 0.53). 4βOHC concentrations correlated weakly with midazolam absolute bioavailability (ρ = − 0.23, p = 0.027) and apparent oral clearance (ρ = 0.28, p = 0.008), but not with systemic clearance (ρ = − 0.03, p = 0.81).ConclusionThese findings suggest that 4βOHC concentrations reflect hepatic, but not intestinal, CYP3A4 activity. Further studies should investigate the potential value of 4βOHC as an endogenous biomarker for individual dose requirements of intravenously administered CYP3A4 substrate drugs.Trial registrationClinical.Trials.gov identifier: NCT02386917.

Effects of alcohol on metabolism and toxicity of cocaine in rats

As most cocaine users drink alcohol, it is interesting to understand how a non-lethal dose of alcohol affects the metabolism and toxicity of cocaine. In this study, we examined the correlation between dose-dependent toxicity and the metabolism/pharmacokinetic (PK) profile of cocaine with or without alcohol (ethanol, 1 g/kg) co-administration in rats. The cocaine toxicity in rats with or without alcohol co-administration is characterized by not only the commonly used LD50, but also the average times for the appearance of convulsion and death as well as total toxicity level (TTL) in the blood. All these data have consistently demonstrated that co-administration of alcohol increased cocaine toxicity, and that the alcohol-enhanced toxicity of cocaine is mainly attributed to the observed two additional metabolites (cocaethylene and norcocaethylene – products of chemical reactions of cocaine with alcohol catalyzed by metabolic enzymes carboxylesterase-1 and liver microsomal cytochrome P450 3A4) that are more toxic than cocaine itself. So, evaluation of the substance TTL should account for the blood levels of not only cocaine itself, but also its all toxic metabolites. In addition, for rats died of a lethal dose of cocaine (60 or 100 mg/kg) combined with 1 g/kg alcohol, we also determined the TTL at the time of death, demonstrating that death would occur once the TTL reached a threshold (~16 μM).

СASE STUDY OF HEPATO-RENAL FAILURE IN A PATIENT AFTER ORTHOTOPIC HEART TRANSPLANTATION

Introduction. Heart transplantation remains the only radical treatment for end-stage heart failure (HF). Liver and / or renal dysfunction is common in patients with HF, which is also exacerbated by the use of artificial circulation and immunosuppressive therapy, and leads to postoperative complications and mortality. Case description. Patient P., 49 years old, after orthotopic heart transplantation was admitted to the intensive care unit (ICU) with signs of multiple organ failure. Graft rejection syndrome was suspected, but was not confirmed after the detailed clinical and laboratory examinations and according to the myocardial biopsy. Because of severe renal and hepatic insufficiency, patient at the ICU started to receive hemodiaultrafiltration with a flow of 190 ml/min; ultrafiltration – 100 ml/h. The condition, that developed was due to the direct effect of tacrolimus as the patient had a critically high plasma concentration of this drug (&gt; 30 ng / ml) after the standard recommended postoperative dose (0.2 mg / kg per day). According to the literature, the elimination of the tacrolimus is provided by the liver, with microsomal cytochrome P450 3A4. Thus, the patient most likely had a failure of hepatic metabolism. Conclusion: Because of the systemic toxicity of tacrolimus, it is important to monitor its concentration after the first dose. Diagnosis of metabolic disorders at an early stage will prevent further systemic toxicity of tacrolimus. Efferent methods at ICU are the important tools for the correction of hepatic and renal insufficiency throughout toxic effects of tacrolimus.

Biocompatible Palladium Telluride Quantum Dot-Amplified Biosensor for HIV Drug

Indinavir (IDV) is a potent and well-tolerated protease inhibitor antiretroviral (ARV) drug used as a component of the highly active antiretroviral therapy (HAART) of human immunodeficiency virus (HIV). It undergoes hepatic first-pass metabolism that is catalysed by microsomal cytochrome P450-3A4 enzyme (CYP3A4), which results in pharmacokinetics that may be favourable or adverse. Therapeutic drug monitoring (TDM) of IDV during HIV treatment is therefore critical, in order to prevent the adverse effects of its first-pass metabolism and optimise an individual’s dosage regime. Biosensors are now the preferred diagnostic tools for TDM assessment at point-of-care, due to their high sensitivity and real-time response. An electrochemical biosensor for IDV was prepared by depositing a thin film of CYP3A4 (a thiolate enzyme) and thioglycolic acid-capped palladium telluride quantum dot (TGA-PdTeQD) on a cysteamine-functionalised gold disk electrode (Cyst|Au) using a combination of thiol and carbodiimide covalent bonding chemistries. The electrochemical signatures of the biosensor (CYP3A4|TGA-PdTeQD|Cyst|Au) were determined by cyclic voltammetry (CV) that was performed at a scan rate of 500 mV s−1, and the sensor responses at the characteristic reduction peak potential value of − 0.26 V were recorded. The sensitivity, linear range (LR) and limit of detection (LOD) values of the indinavir biosensor were 4.45 ± 0.11 μA nM−1 IDV, 0.5–1.0 nM IDV (i.e. 3.6 × 10−4–7.1 × 10−4 mg L−1 IDV) and 4.5 × 10−4 mg L−1 IDV, respectively. The values of the two analytical parameters (LR and LOD) of the biosensor were by up to four orders of magnitude lower than the maximum plasma concentration (Cmax) values of indinavir (0.13–8.6 mg L−1 IDV). The IDV biosensor was successfully used to detect IDV in human serum samples containing dissolved indinavir tablet. This, therefore, indicates the indinavir biosensor’s suitability for TDM applications, using samples obtained within 1–2 h of drug intake at point-of-care, for which very low levels of the drug are expected.

Continuous monitoring of Naproxen by a cytochrome P450-based electrochemical sensor

This paper reports the characterization of an electrochemical biosensor for the continuous monitoring of Naproxen based on cytochrome P450. The electrochemical biosensor is based on the drop-casting of multi-walled carbon-nanotubes (MWCNTs) and microsomal cytochrome P4501A2 (msCYP1A2) on a graphite screen-printed electrode (SPE).The proposed biosensor was employed to monitor Naproxen (NAP), a well-known anti-inflammatory compound, through cyclic voltammetry. The dynamic linear range for the amperometric detection of NAP had an upper limit of 300µM with a corresponding limit of detection (LOD) of 16±1µM (S/N=3), which is included in NAP physiological range (9–300µM). The MWCNT/msCYP1A2-SPE sensor was also calibrated for NAP detection in mouse serum that was previously extracted from mice, showing a slightly higher LOD (33±18µM).The stability of the msCYP1A2-based biosensor was assessed by longtime continuous cyclic voltammetric measurements. The ability of the sensor to monitor drug delivery was investigated by using a commercial micro-osmotic pump. Results show that the MWCNT/msCYP1A2-SPE sensor is capable of precisely monitoring the real-time delivery of NAP for 16h. This work proves that the proposed electrochemical sensor might represent an innovative point-of-care solution for the personalization of drug therapies, as well as for pharmacokinetic studies in both animals and humans.

Maternal obesity alters feto-placental cytochrome P4501A1 activity

Cytochrome P4501A1 (CYP1A1), an important drug metabolizing enzyme, is expressed in human placenta throughout gestation as well as in fetal liver. Obesity, a chronic inflammatory condition, is known to alter CYP enzyme expression in non-placental tissues. In the present study, we test the hypothesis that maternal obesity alters the distribution of CYP1A1 activity in feto-placental unit. Placentas were collected from non-obese (BMI < 30) and obese (BMI > 30) women at term. Livers were collected from gestation day 130 fetuses of non-human primates fed either control diet or high-fat diet (HFD). Cytosol and microsomes were collected using differential centrifugation, and incubated with 7-ethoxyresorufin. The CYP1A1 specific activity (pmoles of resorufin formed/min/mg of protein) was measured at excitation/emission wavelength of 530/590 nm. Placentas of obese women had significantly reduced microsomal CYP1A1 activity compared to non-obese women (0.046 vs. 0.082; p < 0.05); however no such effect was observed on cytosolic activity. Similarly, fetal liver from HFD fed mothers had significantly reduced microsomal CYP1A1 activity (0.44 ± 0.04 vs. 0.20 ± 0.10; p < 0.05), with no significant difference in cytosolic CYP1A1 activity (control, 1.23 ± 0.20; HFD, 0.80 ± 0.40). Interestingly, multiple linear regression analyses of placental efficiency indicate cytosolic CYP1A1 activity is a main effect (5.67 ± 2.32 (β ± SEM); p = 0.022) along with BMI (−0.57 ± 0.26; p = 0.037), fetal gender (1.07 ± 0.26; p < 0.001), and maternal age (0.07 ± 0.03; p = 0.011). In summary, while maternal obesity affects microsomal CYP1A1 activity alone, cytosolic activity along with maternal BMI is an important determinant of placental efficiency. Together, these data suggest that maternal lifestyle could have a significant impact on CYP1A1 activity, and hints at a possible role for CYP1A1 in feto-placental growth and thereby well-being of fetus.

Catalytic and immunochemical detection of hepatic and extrahepatic microsomal cytochrome P450 1A1 (CYP1A1) in white-sided dolphin ( Lagenorhynchus acutus)

We have characterized microsomal systems and measured the levels of microsomal cytochrome P450 1A1 (CYP1A1) and ethoxyresorufin-O-deethylase (EROD) activity in multiple internal organs of male and female white-sided dolphin ( Lagenorhynchus acutus) from the northwest Atlantic Ocean. Internal organs were sampled within 24 h of death, sometimes in a period of hours, collection times which are significantly less than usually seen for marine mammals. Tissue autolysis, as assessed by histological analysis of liver, was minimal to none in all individuals. Total P420 did not correlate with time from death to sampling, suggesting that it is a poor indicator of P450 degradation in cetacean tissues where perfusion is not practical. The total hepatic microsomal P450 content, cytochrome b5 content, and NADPH-cytochrome c (P450) reductase (CPR) activity averaged 0.29 nmol mg −1, 0.12 nmol mg −1, and 238 nmol mg −1 min −1, respectively. Microsomal CPR activity in liver was higher than that in lung and kidney, and was higher than that reported in liver of most other cetacean species. Immunodetected CYP1A1 content was low in all organs, less than 3 pmoles CYP1A equivalents mg −1. EROD activity ranged from 9 to 376 pmoles mg −1 min −1 and was greater in liver than in other tissues. Hepatic microsomal EROD activity and CYP1A1 content did not correlate. However, hepatic EROD activity, but not CYP1A1 protein content, was well correlated with both total PCB and Σmono- ortho PCB concentrations in blubber. Length, as a proxy for age, did not correlate with hepatic EROD activity or CYP1A1 protein levels, and sex did not influence the relationship between EROD and contaminant concentrations. We cannot easily control for the extent of tissue degradation in cetacean studies nor do we have a complete history of these animals. Therefore, other factors such as degradation or hormonal state may have a role in the observed relationships. Yet, as in other mammals, hepatic tissues appear to be a major site of CYP1A1 expression and probably of biotransformation of CYP1A substrates in white-sided dolphin. The expression of an EROD catalyst in liver likely reflects induction by PCBs, but the P450 enzyme catalyzing hepatic EROD activity in these whales may not be CYP1A1.

Identification of mitochondrial cytochrome P450 induced in response to polycyclic aromatic hydrocarbons in the mummichog ( Fundulus heteroclitus)

Increasing evidence suggests that polycyclic aromatic hydrocarbons (PAHs) such as benzo[ a]pyrene (BaP) are localized to the mitochondria. Because the toxic effects of many PAHs are the result of metabolism by cytochrome P4501A (CYP1A), it is important to investigate whether active forms of these enzymes can be identified in the mitochondria. In this study, we identified mitochondrial P450s with a monoclonal antibody against scup ( Stenotomus chrysops) CYP1A in the isolated mitochondrial fraction of the liver from adult male mummichog ( Fundulus heteroclitus) livers. The size of the protein in the mitochondria was similar to that of microsomal CYP1A. Fish dosed with 10 mg/kg BaP had increased EROD activity in the mitochondrial fraction compared to controls. In mummichog larvae dosed with 100 µg/L BaP and 100 µg/L benzo[ k]fluoranthene, CYP1A protein levels as well as enzyme activity were elevated. However, fish from a PAH-polluted Superfund site (Elizabeth River, Portsmouth VA) showed recalcitrant mitochondrial CYP1A protein levels and enzyme activity in a similar manner to microsomal CYP1A.

Macrolide–theophylline interactions: no role for the inhibition of cytochrome P4501A2

Macrolide–theophylline interactions: no role for the inhibition of cytochrome P4501A2

Inhibition on Human Liver Cytochrome P450 3A4 by Constituents of Fennel (Foeniculum vulgare): Identification and Characterization of a Mechanism-Based Inactivator

Fennel, a seed of Foeniculum vulgare, is used as a culinary spice and traditional medicine. The methanolic extract of fennel showed a characteristic of mechanism-based inactivation on erythromycin N-demethylation mediated by human liver microsomal cytochrome P450 3A4 (CYP3A4). The present study was conducted to identify the fennel constituent having the inhibition. Thirteen compounds have been isolated from a methanol extract of fennel and tested for their inhibition on CYP3A4. Among them, 5-methoxypsoralen (5-MOP) showed the strongest inhibition with an IC50 value of 18.3 microM and a mixed type of inhibition. In addition, with the preincubation time of 20 min only 5-MOP showed preincubation time dependency; the IC50 value decreased from 18.3 microM with a preincubation time of 0 min to 4.6 microM with a preincubation time of 20 min. Further investigation on 5-MOP showed the characteristics of time-dependent inhibition, requirement of NADPH, lack of protecting effect of nucleophiles, and recovery of CYP3A4 activity by the competitive inhibitor. This result suggests that the inhibitory activity of CYP3A4 by 5-MOP was a mechanism-based inactivation. The kinetic parameter for mechanism-based inactivation was characterized by a KI value of 15.0 microM and a kinact value of 0.098 min(-1).

Uncoupling of cytochrome P450 1A and stimulation of reactive oxygen species production by co-planar polychlorinated biphenyl congeners

The non- ortho-polychlorinated biphenyl (PCB) congener 3,3′4,4′-tetrachlorobiphenyl (PCB 77) can uncouple the catalytic cycle of fish (scup) cytochrome P4501A (CYP1A) and mammalian (rat, human) CYP1A1, stimulating release of reactive oxygen species (ROS). PCB 77 also inactivates CYP1A in an NADPH-, oxygen-, and time-dependent process, linked to uncoupling. We addressed a hypothesis that planar halogenated hydrocarbons generally will uncouple CYP1A. Thus, additional PCB congeners including non- ortho-3,3′,4,4′,5′-pentachlorobiphenyl (PCB 126) and 3,3′,4,4′,5,5′-hexachlorobiphenyl (PCB 169), mono- ortho-2,3,3′,4,4′-pentachlorobiphenyl (PCB 105) and di- ortho-2,2′,5,5′-tetrachlorobiphenyl (PCB 52), as well as the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P), were examined for their ability to stimulate microsomal ROS production and to inactivate CYP1A. Incubated without NADPH, non- ortho-PCB 126 and -PCB 169 both inhibited microsomal CYP1A activity (ethoxyresorufin O-deethylase; EROD). When NADPH was included, these congeners caused a progressive inactivation of CYP1A, in addition to the inhibition. The determined K Inact values for inactivation were 0.14 and 0.08 μM, respectively, for PCB 126 and PCB 169, similar to the 0.05 μM for PCB 77 previously reported. The mono- ortho-PCB 105 weakly inhibited and weakly inactivated CYP1A. The di- ortho-PCB 52 neither inhibited nor inactivated CYP1A. Alone, B[a]P strongly inhibited CYP1A, but when NADPH was added that inhibition was reversed, apparently by metabolic depletion of the substrate, and there was no inactivation. PCB 126 and PCB 169 stimulated release of ROS from induced liver microsomes, while B[a]P, PCB 52 and PCB 105 did not. ROS release and CYP1A inactivation stimulated by the non- ortho-PCB 126 and PCB 169 indicate an uncoupling of CYP1A like that previously shown with PCB 77. The uncoupling and release of ROS further suggest a participation of CYP1A in the oxidative stress associated with some planar halogenated aryl hydrocarbon receptor agonists.

The Effect of Mild Hypothermia on Plasma Fentanyl Concentration and Biotransformation in Juvenile Pigs

Therapeutic hypothermia may alter the required dosage of analgesics and sedatives, but no data are available on the effects of mild hypothermia on plasma fentanyl concentration during continuous, long-term administration. We therefore assessed in a porcine model the effect of prolonged hypothermia on plasma fentanyl concentration during 33 h of continuous fentanyl administration. Seven female piglets (weight: 11.8 +/- 1.1 kg) were anesthetized by IV fentanyl (15 microg . kg(-1) . h(-1)) and midazolam (1.0 mg . kg(-1) . h(-1)). After preparation and stabilization (12 h), the animals were cooled to a core temperature of 31.6 degrees +/- 0.2 degrees C for 6 h and were then rewarmed and kept normothermic at 37.7 degrees +/- 0.3 degrees C for 6 more hours. Plasma fentanyl concentrations were measured by radioimmunoassay, cardiac index by thermodilution, and blood flows of the kidney, spleen, pancreas, stomach, gut, and hepatic artery by a colored microspheres technique. Furthermore, in an additional 4 pigs, temperature dependency of hepatic microsomal cytochrome P450 3A4 (CYP3A4) was determined in vitro by ethylmorphine N-demethylation. Plasma fentanyl concentration increased by 25% +/- 11% (P < 0.05) during hypothermia and remained increased for at least 6 h after rewarming. Hypothermia reduced the cardiac index (41% +/- 15%, P < 0.05), as well as all organ blood flows except the hepatic artery. A strong temperature dependency of CYP3A4 was found (P < 0.01). Mild hypothermia induced a distribution and/or elimination-dependent increase in plasma fentanyl concentration which remained increased for several hours after rewarming. Consequently, a prolonged increase of the plasma fentanyl concentration should be anticipated for appropriate control of the analgesia/sedatives during and early after therapeutic hypothermia.

Mechanism-Based Inactivation of Human Liver Microsomal CYP3A4 by Rutaecarpine and Limonin from Evodia Fruit Extract

Evodia fruit (Evodiae Fructus) is used as a herbal medicine prepared from the matured fruit of the Evodia rutaecarpa Bentham or Evodia officinalis Dode, of the Rutaceae plant family. An extract of Evodia fruit in the presence of NADPH was shown to inhibit human liver microsomal erythromycin N-demethylation activity, mediated by cytochrome P450 3A4 (CYP3A4), in a preincubation-time dependent manner. The present study was conducted to identify components of Evodia fruit extract having preincubation-time dependent inhibitory effects on CYP3A4 by analyzing human liver microsomal erythromycin N-demethylation activity. Rutaecarpine, a major component of Evodia fruit, and limonin caused the most dramatic decrease in residual CYP3A4 activity (IC50 before and after 20 min preincubation with: rutaecarpine, >100 microM and 1.4 microM; limonin, 23.5 microM and 1.8 microM, respectively). Furthermore, rutaecarpine and limonin were identified as mechanism-based inhibitors of CYP3A4 from the following observations: 1) The inhibitory effects of rutaecarpine and limonin on CYP3A4 activity were dependent on the preincubation time, 2) The inhibition required NADPH, 3) The inhibition was depressed in the presence of the competitive CYP3A4 inhibitor, ketoconazole, 4) Dialysis resulted in no recovery of CYP3A4 activity. The kinetic parameters for inactivation k(inact) and K(I) were: 0.387 min-1 and 107.7 microM for rutaecarpine, 0.266 min-1 and 23.2 microM for limonin, respectively. These results indicate that rutaecarpine and limonin are mechanism-based inhibitors of CYP3A4.

The Structure of Human Microsomal Cytochrome P450 3A4 Determined by X-ray Crystallography to 2.05-Å Resolution

The structure of P450 3A4 was determined by x-ray crystallography to 2.05-A resolution. P450 3A4 catalyzes the metabolic clearance of a large number of clinically used drugs, and a number of adverse drug-drug interactions reflect the inhibition or induction of the enzyme. P450 3A4 exhibits a relatively large substrate-binding cavity that is consistent with its capacity to oxidize bulky substrates such as cyclosporin, statins, taxanes, and macrolide antibiotics. Family 3A P450s also exhibit unusual kinetic characteristics that suggest simultaneous occupancy by smaller substrates. Although the active site volume is similar to that of P450 2C8 (PDB code: 1PQ2), the shape of the active site cavity differs considerably due to differences in the folding and packing of portions of the protein that form the cavity. Compared with P450 2C8, the active site cavity of 3A4 is much larger near the heme iron. The lower constraints on the motions of small substrates near the site of oxygen activation may diminish the efficiency of substrate oxidation, which may, in turn, be improved by space restrictions imposed by the presence of a second substrate molecule. The structure of P450 3A4 should facilitate a better understanding of the substrate selectivity of the enzyme.

Chicken Egg Yolk as an Excellent Source of Highly Specific Antibodies Against Cytochromes P450

Using chicken antibodies IgY (purified from egg yolks) against mammalian cytochromes P450 and by means of cytochrome P450 marker substrates, we found for the first time the presence of hepatopancreatic cytochrome P450 in crayfishOrconectes limosus(an inducible cytochrome P450 2B-like enzyme) and we were able to detect and quantify cytochrome P450 1A1 in microsomes of human livers. Expression levels of cytochrome P450 1A1 in human livers constituted less than 0.6% of the total hepatic cytochrome P450 complement. The results obtained in our study are clear examples that chicken IgY are suitable for cytochrome P450 detection and quantification. Due to the evolutionary distance, chicken IgY reacts with more epitopes on a mammalian antigen, which gives an amplification of the signal. Moreover, this approach offers many advantages over common mammalian antibody production since chicken egg is an abundant source of antibodies (about 100 mg IgY/yolk) and the egg collection is a non-invasive technique. In the case of antibodies against cytochrome P450 2B4, we documented fast and steady production of highly specific immunoglobulins. Thus, chicken antibodies should be considered as a good alternative to and/or superior substitute for conventional polyclonal antibody produced in mammals.

CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes.

Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6beta-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.

Evaluating molar CYP1A level in fish hepatic microsomes by competitive ELISA using recombinant membrane-free CYP1A standard protein

Fish cytochrome P4501A (CYP1A) is a widely accepted environmental biomarker, detecting biological effects of several xenobiotic groups present in aquatic environments, when evaluated in target tissues of a biosensor species. However, appropriate utilization of its protein level as a routine environmental diagnostic tool requires evaluation of properly normalized molar levels, mitigating comparison among different laboratories, during a multi-annual time scale and over a variety of tested populations of the biosensor species. A competitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of CYP1A of the striped sea bream, Lithognathus mormyrus, using our previously described antibody raised to a trout CYP1A synthetic peptide, and a recombinant L. mormyrus CYP1A as a competitor. The L. mormyrus CYP1A-cDNA was cloned and modified by truncating its 5′ hydrophobic membrane anchor, as well as by addition of 4× histidine tag, permitting its partial purification on a nickel–NTA column. The modified cDNA was ligated into the PCWOri+ vector and heterologously produced in Escherichia coli as a cytosolic, membrane-free protein, retaining its immuno-affinity with the anti-CYP1A antibody in the presence of the detergent Triton X-100. The detergent was added to the ELISA solution during the competitive step, rendering the microsomal CYP1A more accessible to the antibody. ELISA components, including coated levels of the modified standard CYP1A, and the concentrations of the Triton X-100, CYP1A-specific antibody, and the range of dissolved CYP1A standard protein, were optimized. Hypothesized immuno-affinity differences between the microsomal and the recombinant CYP1As, and among microsomal samples, as well as assay accuracy, were examined and discussed. This ELISA can serve for more efficient utilization of fish CYP1A as a pollution biomarker, and also as a model for establishing competitive ELISAs aimed at quantification of many different microsomal P450 proteins.

Regulation of cell morphology and cytochrome P450 expression in human hepatocytes by extracellular matrix and cell-cell interactions.

The influence of extracellular matrix conditions and plating density on cell cytoarchitecture and the constitutive and chemically induced expression of cytochrome P450 3A4 (CYP3A4) was examined in primary cultures of human hepatocytes. Constitutive and drug-induced microsomal CYP3A4 expression occurred equally well in human hepatocyte cultures maintained on either a complex or simple substratum (Matrigel vs collagen, type I), or in a sandwich configuration (i.e., between two layers of extracellular matrix), despite the markedly different morphological properties exhibited by each condition. However, a density-dependent decrease in both the constitutive and induced levels of CYP3A4 was observed in hepatocytes maintained on a simple collagen substratum as plating density was reduced from 100% to 25%. Marked alterations in cell shape and cytoarchitecture were noted concomitant with decreases in the expression and localization of intercellular gap junctions and E-cadherin-mediated cell adhesions. In addition, the intracellular distribution of microtubules and microfilaments was altered substantially and the expression of immunoreactive actin and beta-tubulin increased as cell density was decreased. These effects were reversed to some extent by overlaying monolayers with extracellular matrix or by co-culturing with another cell type. Efforts to maintain normal cell shape and cytoskeletal distribution in hepatocytes at low cell density with a Matrigel substratum failed to restore normal basal levels of CYP3A4 expression or responsiveness to rifampicin (RIF). Likewise, E-cadherin and Cx-32 expression was again reduced, even though the distribution and expression of cytoskeletal elements returned to normal levels. These results suggest that cell-cell contacts, but not the extracellular matrix configuration or composition, play a critical role in determining normal responsiveness to chemical modulators in human hepatocytes.

Induction of CYP1A1. The AhR/DRE paradigm: transcription, receptor regulation, and expanding biological roles.

The CYP1A1 gene encodes microsomal cytochrome P4501A1 that catalyzes the metabolism of many xenobiotics, including the oxygenation of polycyclic aromatic hydrocarbons (PAH). Induction of CYP1A1 enhances the metabolism of PAHs, and therefore, represents an adaptive response to chemical exposure in mammalian cells. Mechanistic studies reveal an AhR/DRE paradigm for the induction, which involves activation of the aryl hydrocarbon receptor (AhR) by an agonist, dimerization of AhR with the Ah recceptor nuclear translocator (Arnt), followed by binding of the AhR/Arnt heterodimer to the dioxin-responsive enhancer (DRE) and transcription of the gene. The AhR mediated transcription is tightly regulated through, at least, two mechanisms: (a) the cytoplasmic AhR interacts with hsp90 and an immunophilin chaperone AIP for proper folding and receptivity, and (b) the agonist-activated, nuclear AhR is degraded through the ubiquitin-26S proteasome mediated protein turnover, such that the transcription by AhR is controlled at a physiologically adequate level. In addition to CYP1A1 induction, AhR mediates a broad range of biological responses to CYP1A1 inducers, typified by the environmental contaminant dioxin, via modulating gene expression. Thus, mechanistic studies of CYP1A1 induction have provided insights into P450 induction, PAH carcinogenesis, dioxin action, AhR function, and receptor-mediated mammalian gene expression.