Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation.

Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

Promotion of experimental thrombus formation by the procoagulant activity of breast cancer cells.

The routine observation of tumor emboli in the peripheral blood of patients with carcinomas raises questions about the clinical relevance of these circulating tumor cells. Thrombosis is a common clinical manifestation of cancer, and circulating tumor cells may play a pathogenetic role in this process. The presence of coagulation-associated molecules on cancer cells has been described, but the mechanisms by which circulating tumor cells augment or alter coagulation remains unclear. In this study we utilized suspensions of a metastatic adenocarcinoma cell line, MDA-MB-231, and a non-metastatic breast epithelial cell line, MCF-10A, as models of circulating tumor cells to determine the thrombogenic activity of these blood-foreign cells. In human plasma, both metastatic MDA-MB-231 cells and non-metastatic MCF-10A cells significantly enhanced clotting kinetics. The effect of MDA-MB-231 and MCF-10A cells on clotting times was cell number-dependent and inhibited by a neutralizing antibody to tissue factor (TF) as well as inhibitors of activated factor X and thrombin. Using fluorescence microscopy, we found that both MDA-MB-231 and MCF-10A cells supported the binding of fluorescently labeled thrombin. Furthermore, in a model of thrombus formation under pressure-driven flow, MDA-MB-231 and MCF-10A cells significantly decreased the time to occlusion. Our findings indicate that the presence of breast epithelial cells in blood can stimulate coagulation in a TF-dependent manner, suggesting that tumor cells that enter the circulation may promote the formation of occlusive thrombi under shear flow conditions.

Action of aldosterone on frog skin in the presence and absence of in vitro molting effects.

The molting which occurs in frog skin following exposure to high concentrations of aldosterone interferes with the interpretation of physiological measurements. Exposure of skins from frogs maintained in standard smooth tanks to 5 - 10(-7) M aldosterone caused within a few hours erratic responses in short-circuit current Io and conductance K followed by sustained stimulation of Io and K; 10(-8) M aldosterone caused only stimulation of Io and K. Storage of frogs in "rojgh tanks" eliminated in vitro molting on exposure to 5 - 10(-7) M aldosterone. IO and K were then superimposable for 3 h, after which Io increased far more rapidly than K. These results are consistent with an early effect on permeability of the active pathway and later effects on metabolism, either a direct effect on the pump or enhanced interaction between transport and metabolism.

Sodium transport and oxygen consumption in toad bladder. A thermodynamic approach.

The relationship between active sodium transport and oxygen consumption was investigated in toad urinary bladder exposed to identical sodium-Ringer's solution at each surface, while controlling the transepithelial electrical potential difference delta phi. Rates of sodium transport and oxygen consumption were measured simultaneously, both in the short-circuited state (delta phi = 0) and when delta phi was varied. Under short-circuit conditions, when the rates of active sodium transport changed spontaneously or were depressed with amiloride, the ratio of active sodium transport to the estimated suprabasal oxygen consumption Na/O2 was constant for each tissue, but varied among different tissues. Only when delta phi was varied did the ratio Na+/O2 change with the rate of active sodium transport; under these circumstances dNa+/dO2 was constant but exceeded the ratio measured at short-circuit [(Na+/O2)delta phi = 0[. This suggests that coupling between transport and metabolism is incomplete. The results are analyzed according to the principles of nonequilibrium thermodynamics, and intepreted in terms of a simple model of the transepithelial sodium transport system.

Effects of antidiuretic hormone on kinetic and energetic determinants of active sodium transport in frog skin.

The effects of antidiuretic hormone (ADH) on the rate of transepithelial active Na transport JaNa and the rate of suprabasal O2 consumption of Jsbr were studied in paired hemiskins of frog. Within some 30 min following administration of ADH both JaNa and Jsbr increased to near-maximal levels and then remained stable for at least an hour. On symmetric perturbation of the transepithelial electrical potential delta psi at 6-min intervals, the dependence of JaNa and Jsbr on delta psi was near-linear, both in control and experimental hemi-skins. The stability and near-linearity of the system permitted systematic analysis of the parameters of linear non-equilibrium thermodynamic (NET) and electrical equivalent circuit (EC) formulations. ADH (100 mU/ml) stimulated two of the three NET phenomenological L coefficients, as well as A, the affinity (negative Gibbs free energy) of a metabolic reaction driving transport. Observations at partially depressed levels of transport indicated that the effects of kinetic and energetic factors are to some extent discrete. EC analysis showed stimulation of the amiloride-sensitive conductance Ka, but not of the apparent electromitive force of Na transport 'ENa'. Similar effects were produced by 10 mU/ml of ADH or by 10 mM dibutyryl cyclic AMP, although less marked effects on the L coefficients were noted with the lower concentration of hormone. It is suggested that, in contrast to EC analysis, the NET formulation distinguishes between kinetic and energetic determinants of transport, supporting a dual mechanism of action of ADH.

Evaluation of the rate of basal oxygen consumption in the isolated frog skin and toad bladder.

In the study of active transport it is important to distinguish between oxygen consumption sustaining transepithelial transport and that responsible for other tissue functions (basal metabolism). Since amiloride blocks transepithelial active sodium transport and the associated oxygen consumption in the frog skin and toad bladder, we and others have employed this agent to evaluate the rate of basal metabolism. This technique has recently been criticized in a report that amiloride (and ouabain) increased oxygen consumption when no sodium was available for transport. We have been unable to corroborate these observations. With magnesium-Ringer as external bathing solutions, amiloride and ouabain failed to stimulate oxygen consumption. With sodium-Ringer as external bathing solution amiloride reduced oxygen consumption about 30%, to a level indistinguishable from that found on external substitution of magnesium-Ringer for sodium-Ringer. We conclude that the use of amiloride permits evaluation of the rate of basal metabolism with acceptable accuracy; a possible slight depressant effect of ouabain on basal metabolism remains to be investigated.

Highly homologous cytochromes P-450 and b5: a model to study protein-protein interactions in a reconstituted monooxygenase system.

Cytochrome b5 from mouse and rat liver formed a type I spectral complex with two murine cytochrome P-450 isozymes, the P450Coh and P450PBI. Mouse b5 stimulated the reactions catalyzed by reconstituted P450Coh and an equimolar amount of b5 to P450Coh was needed for maximal effect. In contrast, rat b5 inhibited P450Coh-mediated reactions progressively starting from 1:1 ratio of b5 to P-450. Neither b5 had any effect on reactions catalyzed by P45015 alpha, an isozyme highly homologous to P450Coh, but with a point mutation (Arg-129----Ser) at site considered important for P-450-b5 interactions. In case of P450PBI, neither b5 protein had any effect on the associated activities at b5: P-450 ratios below 1, and a progressive inhibition occurred when b5: P-450 ratio was above 1. The results were similar with either rat or mouse liver NADPH-cytochrome P-450 reductase used in reconstitution demonstrating that the critical differences take place in P-450-b5 interactions. Kinetic and spectral experiments revealed that the stimulatory and inhibitory effects of b5 on the enzymatic reactions were due to corresponding changes in the reaction velocity, and that b5 does not compete with the flavoprotein nor with the substrate for binding to P-450. These results indicate that the high spin shift of P-450 does not necessarily correlate with enhanced reaction rates. Also, the increase in the coupling efficiency of P450PBI may result from the increased affinity for substrate in the presence of b5. Sequenation of mouse b5 peptides generated with proteinases revealed three amino acid changes between the mouse and rat b5, two of which appeared at the hydrophobic domain necessary for the P-450-b5 interaction. This could explain the species specificity of b5 proteins in supporting the P-450-mediated reactions. This is the first time functionally important differences in the interaction of highly homologous cytochromes P-450 and b5 have been demonstrated. Isozymes P45015 alpha and P450Coh, and mouse and rat b5 could serve as an excellent model for further studies on the nature and significance of P-450-b5 interactions.

Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex.

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.

Volume control by muscle fibers of the blue crab. Volume readjustment in hypotonic salines.

Single isolated muscle fibers from the walking legs of the blue crab, Callinectes sapidus act as Boyle-van't Hoff osmometers with an osmotically inactive volume of 33 %. Fibers in hypotonic salines undergo a spontaneous volume readjustment toward the initial volumes of the cells found in isotonic salines. The volume readjustment is initiated by the increase in cell volume in hypotonic salines and appears to be dependent on the duration of exposure of the fiber to external sodium, the sodium concentration, and the pH of the external medium. The volume-readjusted cells continue to behave as osmometers, but with an increased relative osmotically inactive volume and a decreased internal resistivity. The decreases in cell volumes appear to be, in large part, due to losses of osmotically active nonelectrolytes from the cells.

Dopamine D2-Receptor Antagonists Down-Regulate CYP1A1/2 and CYP1B1 in the Rat Liver.

Dopaminergic systems regulate the release of several hormones including growth hormone (GH), thyroid hormones, insulin, glucocorticoids and prolactin (PRL) that play significant roles in the regulation of various Cytochrome P450 (CYP) enzymes. The present study investigated the role of dopamine D2-receptor-linked pathways in the regulation of CYP1A1, CYP1A2 and CYP1B1 that belong to a battery of genes controlled by the Aryl Hydrocarbon Receptor (AhR) and play a crucial role in the metabolism and toxicity of numerous environmental toxicants. Inhibition of dopamine D2-receptors with sulpiride (SULP) significantly repressed the constitutive and benzo[a]pyrene (B[a]P)-induced CYP1A1, CYP1A2 and CYP1B expression in the rat liver. The expression of AhR, heat shock protein 90 (HSP90) and AhR nuclear translocator (ARNT) was suppressed by SULP in B[a]P-treated livers, whereas the AhRR expression was increased by the drug suggesting that the SULP-mediated repression of the CYP1 inducibility is due to inactivation of the AhR regulatory system. At signal transduction level, the D2-mediated down-regulation of constitutive CYP1A1/2 and CYP1B1 expression appears to be mediated by activation of the insulin/PI3K/AKT pathway. PRL-linked pathways exerting a negative control on various CYPs, and inactivation of the glucocorticoid-linked pathways that positively control the AhR-regulated CYP1 genes, may also participate in the SULP-mediated repression of both, the constitutive and induced CYP1 expression. The present findings indicate that drugs acting as D2-dopamine receptor antagonists can modify several hormone systems that regulate the expression of CYP1A1, CYP1A2 and CYP1B1, and may affect the toxicity and carcinogenicity outcome of numerous toxicants and pre-carcinogenic substances. Therefore, these drugs could be considered as a part of the strategy to reduce the risk of exposure to environmental pollutants and pre-carcinogens.

Do immature platelet levels in chest pain patients presenting to the emergency department aid in the diagnosis of acute coronary syndrome?

INTRODUCTION: Early and accurate identification of acute coronary syndrome (ACS) vs. noncardiac chest pain in patients presenting to the emergency department (ED) is problematic and new diagnostic markers are needed. Previous studies reported that elevated mean platelet volume (MPV) is associated with ACS and predictive of cardiovascular risk. MPV is closely related to the immature platelet fraction (IPF), and recent studies have suggested that IPF may be a more sensitive marker of ACS than MPV. The objective of the present study was to determine whether the measurement of IPF assists in the diagnosis of ACS in patients presenting to the ED with chest pain. METHODS: In this single-center, prospective, cross-sectional study, adult patients presenting to the ED with chest pain and/or suspected ACS were considered for enrollment. Blood samples from 236 ACS-negative and 44 ACS-positive patients were analyzed in a Sysmex XE-2100 for platelet count, MPV, IPF, and the absolute count of immature platelets (IPC). RESULTS: Total platelet counts, MPV, IPF, and IPC were not statistically different between ACS-negative and ACS-positive patients. The IPF was 4.6 ± 2.7% and 5.0 ± 2.8% (mean ± SD, P = 0.24), and the IPC was 10.0 ± 4.6 and 11.5 ± 7.5 × 10(3) /µL (P = 0.27) for ACS-negative and ACS-positive patients, respectively. CONCLUSION: In 280 patients presenting to the ED with chest pain and/or suspected ACS, no differences in IPF, IPC or MPV were observed in ACS-negative vs. ACS-positive patients, suggesting that these parameters do not assist in the diagnosis of ACS.

Characterisation and PCR-based detection of a CYP2A6 gene deletion found at a high frequency in a Chinese population.

Cytochrome P450 2A6 is an important human hepatic P450 which activates pre-carcinogens, oxidises some drugs and constitutes the major nicotine C-oxidase. In fact, results have been presented in the literature which suggested a relationship between the distribution of defective CYP2A6 alleles and smoking behaviour as well as cigarette consumption. In the present report, we describe the structure of a novel CYP2A locus where the whole CYP2A6 gene has been deleted, resulting in an abolished cytochrome P450 2A6-dependent metabolism. The origin of this locus is apparently due to an unequal crossover event between the 3'-flanking region of the CYP2A6 and CYP2A7 genes. A rapid PCR-based method for the detection of the CYP2A6del allele was developed and the allele frequency was 15.1% among 96 Chinese subjects, but only 1.0% in Finns (n=100) and 0.5% in Spaniards (n=100). In the Chinese population, we did not detect any CYP2A6*2 alleles using an improved genotyping procedure, in contrast to the 11-20% previously reported. It is concluded that genotyping for the CYP2A6del allele is of great importance in studies correlating, for example, smoking behaviour, pre-carcinogen activation or drug metabolism to the CYP2A6 genotype, in particular when oriental populations are investigated.

Induction of cytochrome P450 2A6 expression in humans by the carcinogenic parasite infection, opisthorchiasis viverrini.

The purpose of this study was to examine in vivo the activity of cytochrome P450 (CYP) 2A6, an enzyme capable of activating carcinogens, including N-nitrosodimethylamine, in humans with the carcinogenic liver fluke infection, opisthorchiasis viverrini, before and after treatment with the antiparasitic agent, praziquantel. Coumarin hydroxylase activity of CYP 2A6 was assessed by administering a probe drug, coumarin, and measuring its metabolite, 7-hydroxycoumarin, in urines collected between 0-2 h and 2-4 h of 106 people with varying intensities of Opisthorchis viverrini infection. Five individuals who did not excrete any detectable 7-hydroxy coumarin (and have a genetic defect probably leading to an absence of catalytic activity of the CYP 2A6 protein) were excluded from analysis. Infected people excreted an average of 22.7 mumol of 7-hydroxycoumarin in the first 2 h after taking the drug, whereas the mean of the uninfected group was 19.4 mumol; this difference did not reach statistical significance (P = 0.10). However, a highly significant increase in CYP 2A6-related activity was observed in infected individuals who also had radiological evidence of biliary fibrosis (28.1 mumol) compared to those without (19.4 mumol; P = 0.01). Reassessments of coumarin hydroxylase activity of CYP 2A6 made 2 months after praziquantel treatment showed highly significant reductions in the amount of 7-hydroxycoumarin excreted among the infected groups but no difference in the uninfected group. These results suggest that expression of CYP 2A6 is induced among chronically infected people who also have fibrosis of the intrahepatic bile duct. As already demonstrated in an animal model and now observed in humans for the first time, this increase in CYP 2A6-related enzyme activity may represent an important mechanistic link between inflammatory products of chronic liver fluke infection (e.g., DNA alkylation damage from endogenously formed N-nitrosamines) and the high risk of cholangiocarcinoma faced by infected individuals.

Pyrazole as a modifier of liver microsomal monooxygenase in DBA/2N and AKR/J mice.

Effects of pyrazole on liver microsomal monooxygenase was studied in two inbred strains of mice, DBA/2N (D2) and AKR/J (AKR). A selective effect on microsomal monooxygenase was found. In the D2 mouse pyrazole strongly increases the coumarin 7-hydroxylase (CoH) and 7-ethoxycoumarin O-deethylase (ECDE) activities while on the total cytochrome P-450 (P-450) content and ethylmorphine N-demethylase (EMDM) and benzo(a)pyrene hydroxylase (AHH) activities the effect is biphasic (increased with lower doses and decreased with higher). For AKR the effect of pyrazole is different from the D2. The increase of CoH and ECDE is weaker and no biphasic effect for the other three parameters can be seen. Instead only a decrease takes place. The optimal dose of pyrazole for the induction of CoH in the D2 mice is 200 mg/kg once a day during three days. The effect of pyrazole is strongest in animals (D2) of 4-10 weeks old. For young animals (2 weeks old) no effect except of a weak decrease in AHH can be seen. Also for old animals the effect is weak. Recovery of the monooxygenase after pyrazole induction takes place in about 120 hr except for the total P-450 content which is still below normal. No sex dependence in the effect of pyrazole on CoH was found.

Distinct induction profiles of three phenobarbital-responsive mouse liver cytochrome P450 isozymes.

The dose- and time-responses of three liver cytochrome P450 (P450) isozymes to 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and phenobarbital (PB) were studied in DBA/2 mice at activity, protein and mRNA levels. We found that the maximal induction ranged from about 3-fold (P4502a-4/5) and 5-fold (P4502c-x) to more than 50-fold (P4502b-10). Only P4502a-4/5 and associated mRNA displayed a biphasic time-response after TCPOBOP induction: a transient increase occurring 3-8 hr after administration with a subsequent decline at 24 hr before the maximal induction at 72 hr. The changes in P450 isozyme content reflected those in mRNA levels suggesting that the induction by TCPOBOP and PB is controlled largely at pretranslational stages. The isozyme P4502c-x and associated immunoinhibited benzphetamine N-demethylase and testosterone 16 beta-hydroxylase activities were induced half-maximally by 6-30 times smaller doses of TCPOBOP and by three to four times smaller doses of PB than isozymes P4502a-4/5, P4502b-10 or related activities. Furthermore, larger doses of TCPOBOP decreased the expression of P4502c-x to sub maximal levels. Our data show that the three isozymes, although all inducible by TCPOBOP and PB, have distinct dose dependencies and different time-responses to induction. This indicates that the induction by TCPOBOP and PB of P450s belonging even to the same subfamily may proceed by different mechanisms.

Comparison of hamster and mouse reveals interspecies differences in the regulation of hepatic CYP2A isozymes.

Three CYP2A-related activities [coumarin 7-hydroxylase (COH), testosterone 7 alpha- (test7 alpha) and 15 alpha-hydroxylases (test15 alpha)], identified in hamster liver and analysed by immunoinhibition, and western and northern blotting, were found to be similar to mouse and human CYP2As. In the microsomal fractions, anti-mouse CYP2A5 antibody recognised three bands of about 48, 49 and 52 kDa, suggesting the presence of at least three proteins immunologically similar to mouse CYP2A5. The 49 kDa band migrated close to mouse CYP2A5 and changes in its expression followed COH and test15 alpha activities. Test7 alpha activity did not associate with any of the individual bands detected on western blots despite its strong inhibition by the antibody. Despite the immunological and catalytic similarities between mouse and hamster CYP2A enzymes, their regulation is different. In mice, the enzyme activities are higher in females than males, are induced by pyrazole (PY) and phenobarbital (Pb), and are not affected by 3-methylcholanthrene (MC). In hamsters, activities are not higher in females, induced by MC and reduced by PY. MC and PY appear to regulate expression at the mRNA level, while Pb seems to act post-transcriptionally by increasing either the synthesis or the stability of the protein. Our data indicate that the modes of expression and regulation of CYP2A-related enzymes make the hamster different from mice and humans with respect to the mechanism of metabolism of certain drugs and carcinogens.

Comparative studies on coumarin and testosterone metabolism in mouse and human livers. Differential inhibitions by the anti-P450Coh antibody and metyrapone.

We have studied coumarin 7-hydroxylase (COH) and testosterone 15 alpha-hydroxylase (15 alpha OH) activities in human liver microsomes and compared them with corresponding activities catalysed by members of the P450IIA sub-family in DBA/2N mouse liver microsomes. Human liver contained low levels of 15 alpha OH (about 5-30 pmol/min/mg protein) when compared with control mouse liver microsomes (about 200 pmol/min/mg protein). The anti-P450Coh antibody efficiently inhibited mouse liver 15 alpha OH, also 7 alpha OH (which is a member of the P450IIA sub-family), but it did not inhibit human 15 alpha OH or other testosterone hydroxylases. In mouse liver microsomes, metyrapone preferentially inhibited 15 alpha OH, but in human liver microsomes it inhibited all testosterone hydroxylations measured, including 15 alpha OH (IC50 = 2.0-5.0 microM). Metyrapone clearly inhibited COH in mouse liver microsomes, but interestingly it had no effect on COH activity in human liver microsomes, although these two isozymes have earlier been shown to be immunologically similar. On the basis of available evidence human and mouse P450Coh isozymes seem to be orthologous enzymes whereas the present results indicate that the human 15 alpha OH is different from the mouse P45015 alpha.

Activation of misonidazole by rat liver microsomes and purified NADPH-cytochrome c reductase.

Rat liver microsomes and purified NADPH-cytochrome c reductase metabolized [14C]misonidazole anaerobically to a reactive intermediate that covalently binds to tissue macromolecules. Air strongly inhibited the binding whereas carbon monoxide had no effect, indicating that misonidazole is activated via reduction and not by cytochrome P-450-dependent oxidation. Both systems showed an absolute requirement for NADPH and were stimulated by flavine (FAD) and paraquat. The apparent Km for misonidazole binding to microsomal protein was 0.74 mM the apparent Vmax was 0.64 nmole 14C bound . mg-1 . min-1. At a single substrate concentration, nitrofurantoin, nitrofurazone and desmethylmisonidazole inhibited the covalent binding of misonidazole to microsomal protein by 47, 26, and 38% respectively. The effect of nitrofurantoin on the kinetics of misonidazole binding gave a complex interaction indicative of uncompetitive inhibition. Glutathione reduced the binding of misonidazole to microsomal protein below the level observed for boiled microsomes while ascorbic acid had no effect. Compared to nitrofurantoin and paraquat, misonidazole was a poor stimulator of superoxide production as measured by adrenochrome formation.

Mouse hepatic cytochrome P-450 isozyme induction by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, pyrazole, and phenobarbital.

The effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and pyrazole on mouse hepatic cytochrome P-450 isozyme expression were compared to the P-450 induction pattern elicited by phenobarbital. TCPOBOP and PB administration caused a similar induction profile by increasing microsomal protein and cytochrome P-450 content and the catalytic activities of several monooxygenases in DBA/2N and AKR/J mice. There were, however, several quantitative and some qualitative differences in the induction profile caused by phenobarbital and TCPOBOP. A few strain-related differences were also observed. Immunoblot analysis with polyclonal anti-coumarin hydroxylase (P-450Coh) antibody and epitope-specific monoclonal antibodies 1-7-1 and 2-66-3 showed that both phenobarbital and TCPOBOP increase the amount of P450IIB and P-450Coh. TCPOBOP caused a more pronounced increase in the amount of P-450IIB than phenobarbital, and TCPOBOP also caused an increase in the amount of P-450IA2. These data suggest that in the mouse, TCPOBOP increases mainly the expression of P-450 isozymes responsive to phenobarbital. The effects of pyrazole differed greatly from those caused by TCPOBOP and phenobarbital. In the DBA/2N mice, pyrazole increased coumarin 7-hydroxylation 9.4-fold, whereas in the AKR/J mice the activity was induced only to a level equivalent to the DBA/2N basal level. In immunoblot experiments with anti-P-450Coh antibody, the amount of P-450Coh was considerably higher in DBA/2N mice treated with phenobarbital, TCPOBOP, or pyrazole in comparison with the AKR/J mice, indicating a strain specificity in the inducibility of coumarin 7-hydroxylase by pyrazole.

Immunochemical and catalytical studies on hepatic coumarin 7-hydroxylase in man, rat, and mouse.

The cytochrome P-450-mediated coumarin 7-hydroxylase (COH) was studied in microsomal preparations from Wistar rat, DBA/2N mouse, and human liver. Human liver contained the highest constitutive COH activity of up to about 500 pmol/mg microsomal protein/min. The rat liver contained low levels of COH (about 3-5 pmol/mg protein/min) which could be demonstrated only with high substrate concentrations. Rabbit polyclonal antibody generated against P-450Coh (a P-450 isozyme purified from pyrazole-treated DBA/2N mouse liver showing high activity for coumarin 7-hydroxylation) inhibited COH activity by almost 100% in human liver microsomes and 86-99% in mouse liver microsomes. Also the deethylation of 7-ethoxycoumarin was inhibited somewhat by the antibody, whereas no inhibition was obtained in ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities. None of these enzyme activities was affected by the antibody in the rat liver microsomes. In Ouchterlony immunodiffusion analysis precipitin lines were obtained with human, mouse and rat liver microsomes. Complex coalescence patterns were obtained suggesting full identity between human and pyrazole-treated mouse antigens, partial identity between mouse and rat antigens, and no identity between human and rat antigens. Western blot analysis with the anti-P-450Coh antibody revealed a distinct 48-kDa protein in all four human samples tested. A 50-kDa protein comigrating exactly with P-450Coh was observed in microsomes from PB and pyrazole-treated mouse liver microsomes. No distinct protein bands appeared in rat liver samples. These data suggest that despite slightly differing molecular masses, the human and mouse P-450s supporting COH are structurally conserved at their active centers. The corresponding rat P-450 appears to differ from that of mouse and man.