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.

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.

A low-dimensional deformation model for cancer cells in flow.

A low-dimensional parametric deformation model of a cancer cell under shear flow is developed. The model is built around an experiment in which MDA-MB-231 adherent cells are subjected to flow with increasing shear. The cell surface deformation is imaged using differential interference contrast microscopy imaging techniques until the cell releases into the flow. We post-process the time sequence of images using an active shape model from which we obtain the principal components of deformation. These principal components are then used to obtain the parameters in an empirical constitutive equation determining the cell deformations as a function of the fluid normal and shear forces imparted. The cell surface is modeled as a 2D Gaussian interface which can be deformed with three active parameters: H (height), σ(x) (x-width), and σ(y) (y-width). Fluid forces are calculated on the cell surface by discretizing the surface with regularized Stokeslets, and the flow is driven by a stochastically fluctuating pressure gradient. The Stokeslet strengths are obtained so that viscous boundary conditions are enforced on the surface of the cell and the surrounding plate. We show that the low-dimensional model is able to capture the principal deformations of the cell reasonably well and argue that active shape models can be exploited further as a useful tool to bridge the gap between experiments, models, and numerical simulations in this biological setting.

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.

In vivo and mechanistic evidence of nuclear receptor CAR induction by artemisinin.

Artemisinin (a sesquiterpene lactone endoperoxide) has become important in multi-drug treatment of malaria. There is evidence that artemisinin induces drug metabolism which could result in drug-drug interactions. The objective of this study was to characterize the inductive properties of artemisinin on drug-metabolizing cytochrome P450 (CYP450) enzymes. The possibility of artemisinin to induce CYP450 was studied in artemisinin-treated (orally for four days) and vehicle-treated rats using reverse transcriptase polymerase chain reaction (RT-PCR). The effect on enzymatic activities in mouse microsomes from multiple artemisinin administration (intraperitonally) to mice were also studied as well as the effect on the expression in mouse primary hepatocytes and HEK293 cells. Increased CYP2B1 mRNA levels in rats could be seen after artemisinin treatment as well as a weak but reproducible increase in the intensity of CYP1A2. Administration of artemisinin to mice up-regulated hepatic CYP2B10-dependent, and to a lesser extent, CYP2A5-dependent enzyme activities. In primary hepatocyte culture, artemisinin significantly increased the CYP2B10 mRNA levels whereas the CYP2A5 mRNA levels were increased to a lesser extent. No significant changes were seen in the levels of other CYP enzymes. Artemisinin was an activator of constitutive androstane receptor (CAR) but not pregnane X receptor (PXR) in HEK293 cells. The results demonstrate that the drug exerts its effects on drug metabolism via the CAR receptor that results in up-regulation of genes such as the Cyp2b. The weaker up-regulation of CYP2A5 might also be CAR-dependent or alternatively, a consequence of artemisinin toxicity. The results of this study are of importance when predicting potential drug-drug interactions in multi-drug therapies with artemisinin.

Variable expression of CYP and Pgp genes in the human small intestine.

BACKGROUND: The small intestine is receiving increased attention for its importance in drug metabolism. However, knowledge of the intervariability and regulation of the enzymes involved, cytochrome p450 and P-Glycoproteins (CYP and Pgp), is poor when compared with the corresponding hepatic enzymes. METHODS: The expression of eight different CYP genes and the Pgp were determined by reverse transcription polymerase chain reaction (RT-PCR) in 51 human duodenum biopsies. And the variability and correlation of expression was analyzed. RESULTS: Extensive interindividual variability was found in the expression of most of the genes. Only CYP2C9, CYP3A4 and Pgp were found in all samples. CYP1A2, CYP2A6 and CYP2E1 exhibited the highest interindividual variability. No strong correlation of expression existed between the genes. But a highly significant correlation was found between CYP2D6/1A2, 2D6/2E1, 1A2/2E1 and 2B6/2C9. Acetylsalicylic acid and omeprazole significantly increased the expression of CYPs 2A6, 2E1 and 3A4, respectively. CONCLUSIONS: Extensive interindividual variability is characteristic for the expression of drug-metabolizing CYP and Pgp genes in human duodenum, and external factors such as drugs may further increase the variability. It is possible that the large interindividual variability may lead to variable bioavailability of orally used drugs and hence complicate optimal drug therapy, especially for drugs with a small therapeutic window. Elucidation of factors contributing to clinically important variances warrants further investigation.

Rat oesophageal cytochrome P450 (CYP) monooxygenase system: comparison to the liver and relevance in N-nitrosodiethylamine carcinogenesis.

N-nitrosodiethylamine (NDEA) is able to induce tumours in the rat oesophagus. It has been suggested that this could be due to tissue specific expression of NDEA activating cytochrome P450 enzymes. We investigated this by characterizing the oesophageal monooxygenase complex of male Wistar rats and comparing it with that of the liver. Total amount of cytochrome P450, NADPH P450 reductase, cytochrome b5 and cytochrome b5 reductase of the oesophageal mucosa was approximately 7% of what was found in the liver. In addition, major differences were found in the cytochrome P450 isoenzyme composition between these organs: CYP 2B1/2B2 and CYP3A were found only in the liver, whereas CYP1A1 was constitutively expressed only in the oesophagus. Of the two well-known nitrosamine metabolizing enzymes, CYP2A3 was found only in the oesophagus whereas CYP2E1 was exclusively expressed in the liver. Catalytic studies, western blotting and RT-PCR analyses confirmed the expression of CYP2A3 in the oesophagus. CYP2A enzymes are known to be good catalysts of NDEA metabolism. Oesophageal microsomes had a K(m) for NDEA metabolism, which was about one-third of that of hepatic microsomes, but they showed similar activities when compared per nmol of total P450. NDEA activity in the oesophagus was significantly increased by coumarin (CO), which also induced oesophageal CYP2A3. Immunoinhibition of the microsomal NDEA activity showed that up to 70% of this reaction is catalysed by CYP2A3 in the oesophagus, whereas no inhibition of the hepatic NDEA activity could be achieved by the anti-CYP2A5 antibody. NDEA, but not N-nitrosodimethylamine (NDMA) inhibited the oesophageal metabolism of CO. The results of the present investigation show major differences in the enzyme composition of the oesophageal and hepatic monooxygenase complexes, and are in accordance with the hypothesis that the NDEA organotropism could, to a large extent, be due to the tissue specific expression of the activating enzymes.

Predicting retention in a residential drug treatment alternative to prison program.

One hundred and fifty felony drug offenders diverted from prison to community-based, residential drug treatment alternative to prison program completed a comprehensive interview as part of a longitudinal study. Treatment completion predictors were sought examining intake data (demographics, family, social, employment, medical, psychological, criminal, sexual behavior, drug use and treatment histories). Logistic regression results found completers had more social conformity and close friends, and less need for employment counseling, felony drug convictions, drug dealing income, and unprotected sex than dropouts. Completers were also less likely to encounter recent problems with significant other, have a psychiatric history, experience gunshot or stabbing, and commenced heroin use at older ages than dropouts. However, completers reported higher alcohol use than counterparts. Further analyses explored subcategory models: "life choice" (substance use, criminal and sexual behavior), static (background and dispositional), and dynamic situational influences (employment, psychological state, recent and past encounters). Treatment implications considering findings are discussed.

Stress modulates the enzymatic inducibility by benzo[alpha]pyrene in the rat liver.

The role of stress on the inducibility by benzo[alpha]pyrene (B[alpha]P), a representative polycyclic aromatic hydrocarbon, of several drug-metabolizing enzymes was investigated in rats, using restraint stress and mild unpredictable stress as models of psychological stress. Restraint stress was found to significantly suppress basal ethoxyresorufin 7-dealkylase (EROD) and pentoxyresorufin 7-dealkylase (PROD) activities (two-fold). In contrast, mild unpredictable stress markedly increased basal EROD activity, while PROD activity was not affected. In addition, both types of stress resulted in a significant reduction of basal p -nitrophenol hydroxylation (PNP). It is worth noting that restraint stress greatly enhanced the inducibility of EROD, methoxyresorufin 7-dealkylase (MROD) and to a lesser extent PROD activities by B[alpha]P, while mild unpredictable stress had no, or only a mild effect on the inducibility of cytochrome P450s (CYPs) by B[alpha]P. In conclusion, psychological stress may modulate different enzymatic systems which are vital elements of the detoxification mechanisms of the body. The two distinct types of psychological stress used in this study appear to affect the enzymatic systems under investigation in a stress-specific manner at the basal level and at the induced state by B[alpha]P.

Noradrenaline, dopamine, serotonin: different effects of psychological stress on brain biogenic amines in mice and rats.

The effect of restraint stress on central neurotransmission was evaluated in mice and rats. Noradrenaline (NA), dopamine (DA) and serotonin (5-HT) levels and their primary metabolites were measured in discrete brain regions following exposure to stress. Mice and rats demonstrated a similar response to stress in some brain regions. Both species responded to stress with lower NA and 5-HT in the locus coeruleus compared to non-stressed controls. Dopaminergic activity, assessed by DA turnover, was elevated in the hypothalamus. While DA turnover was suppressed in the amygdala, 5-HT turnover was similarly elevated in both species. In most cases, however, there were differences in biogenic neurotransmission between mice and rats in response to stress. In particular, NA levels were suppressed by stress in the dorsal cortex of mice, but in the rats NA levels were decreased in the hypothalamus. While stress produced lower DA levels in the hypothalamus, DA levels demonstrated a marked increase in the amygdala of mice. Stress was also associated with a decrease in DA levels in the rat striatum and with an increase of DA turnover in the locus coeruleus of mice. On the other hand, 5-HT was suppressed in the mouse striatum and in the rat hypothalamus and amygdala, while 5-HT turnover was markedly decreased in the hippocampus and dorsal cortex of rats alone. In conclusion, the changes in the central neurotransmission which are evoked by stress appear to be species-specific in most cases, a fact which may trigger discrete alterations in homeostatic mechanisms.

Clinicians' and clients' perspectives on the impact of assertive community treatment.

OBJECTIVE: Clients in an assertive community treatment program and their clinicians were asked to rate clients' current difficulties in 13 quality-of-life areas to determine whether improvement in any area predicted reductions in hospitalization and incarceration. METHODS: A peer counselor interviewed 45 clients about psychiatric symptoms, substance use and abuse, medical issues, medication compliance, primary supports, social supports, vocational and occupational issues, housing, daily living skills, economic issues and entitlements, legal involvement, behavioral issues, and treatment involvement. The clients' clinicians rated the clients in these same areas. Ratings of clients' difficulties in these areas at program entry were based on combined ratings made at intake and after a review of clients' charts. Data on hospitalization and incarceration were obtained from medical and police records. Logistic regression analyses were used to seek predictors of declines in admissions to hospitals and jails (referred to as institutional admissions). RESULTS: Institutional admissions decreased after program entry; decreases were larger among clients admitted in recent years. Clients improved significantly in all 13 quality-of-life areas based on comparisons of both clinicians' and clients' ratings and baseline ratings; however, clients rated themselves as having less difficulty than their clinicians thought they had in the areas of substance abuse, medication compliance, primary supports, social supports, daily living skills, and treatment involvement. Based on clinicians' ratings, improvement in substance abuse issues predicted declines in institutionalized admissions. Based on clients' ratings, improvement in social support and economic issues predicted declines. CONCLUSIONS: These findings emphasize the importance of clients' perspectives in treatment planning and suggest that clinicians may overlook the smaller incremental steps toward improvement that are valued by clients.

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.

Analysis of RNA-protein interactions of mouse liver cytochrome P4502A5 mRNA.

In our previous studies we have identified a 37/39 kDa, pyrazole-inducible, cytochrome P4502A5 (CYP2A5) mRNA binding protein and provided evidence that it may play a role in the stabilization and processing of the RNA [Geneste, Rafalli and Lang (1996) Biochem. J. 313, 1029-1037; Thulke-Gross, Hergenhahn, Tilloy-Ellul, Lang and Bartsch (1998) Biochem. J. 331, 473-481]. Details of the RNA-protein interactions are, however, not known. In this report we have performed an analysis of the interaction between the CYP2A5 mRNA and the 37/39 kDa protein. With UV-cross linking experiments, using RNA probes corresponding to various parts of the CYP2A5 mRNA, and with antisense oligonucleotides complementary to certain areas of the 3'-untranslated region (3'UTR), we could map the primary binding site to the tip of a 71 nt hair-pin loop at the 3'-UTR. This analysis also showed that the protein may have more than one site of interaction with the RNA and/or that, within the binding region, there could be more than one protein molecule binding to the RNA. Analysis of the probable conformations of the various probes used in the UV cross-linking experiments, in combination with the estimated binding affinities of the protein to the different probes, suggests that important factors in the high-affinity binding are the UAG triplet flanked by GA-rich sequences at the tip of the hair-pin loop, in addition to the conformation of the loop itself. Within the binding region, similarities with known binding sites of heterogeneous nuclear ribonucleoprotein (hnRNP) A1 in other RNA molecules were revealed by sequence alignment analysis. Moreover, competition experiments with an oligoribonucleotide corresponding to a known high-affinity binding site of hnRNP A1, and immunoprecipitation of the UV cross-linked 37/39 kDa complex showed that the protein binding to the CYP2A5 mRNA could be hnRNP A1 or its close analogue. It was also shown that the 37/39 kDa protein binds with less affinity to CYP2A4 mRNA than to CYP2A5 mRNA. This is in accordance with experiments characterizing the binding site, since these two otherwise highly homologous genes are kown to have a three nucleotide difference within the region important for the high binding affinity. Since the response of CYP2A4 to pyrazole is known to be weak, as compared with CYP2A5, this observation provides further evidence for a regulatory role of the 37/39 kDa protein in CYP2A5 mRNA metabolism.

Expression of cytochrome P450 2A5 in preneoplastic and neoplastic mouse liver lesions.

Cytochrome P450 (CYP) 2A5 is involved in the metabolism of carcinogens like aflatoxin B1 and N-nitrosodiethylamine (NDEA), and CYP2A5 levels are increased in some pathological states of the liver (e.g., infectious hepatitis and porphyria). We analyzed the expression of CYP2A5 during experimental liver carcinogenesis in three different mouse strains (C3H/He, C57BL/6J, and B6C3F1) with immunohistochemical techniques and in situ hybridization. In normal liver, CYP2A5 protein and mRNA were detected in centrilobular hepatocytes only. Phenobarbital treatment increased the number of CYP2A5-positive centrilobular hepatocytes and the CYP2A5-positive areas were extended into the middle zone in all strains, but periportal hepatocytes remained negative. Fifty percent of the spontaneous foci in untreated mice, over 90% of the foci in mice treated with NDEA or phenobarbital and all of the hepatocellular adenomas and carcinomas displayed positive immunostaining and a strong CYP2A5 mRNA signal by in situ hybridization. In the liver tumors metastasized to the lung, expression of CYP2A5 had largely disappeared. CYP2A5 expression in neoplastic and putative preneoplastic lesions, although sometimes heterogeneous, was apparently independent of the typical zonal expression pattern in normal tissue. As expected, the C57BL/6J mice developed fewer foci and tumors than the C3H/He and B6C3F1 mice, but the phenotype of CYP2A5 overexpression was similar in all the strains. Our data suggest that the increased expression of CYP2A5 may play an important role in the development of liver cancer in mice and may be used as a novel marker for spontaneous and NDEA-induced mouse liver foci.

The role of stress in the regulation of drug metabolizing enzymes in mice.

The role of stress in the regulation of several enzymatic systems which are involved in the biotransformation of xenobiotics in the liver was investigated in this study using restraint stress as a stress model. The results demonstrated that stress suppressed total basal P450 content (35%) and basal ethoxyresorufin 7-dealkylase (EROD) activity (33%), while slightly increasing basal methoxyresorufin 7-dealkylase (MROD) activity (20%). Basal pentoxyresorufin 7- dealkylase (PROD) and coumarin 7-hydroxylase (COH) activities were not affected. On the other hand, restraint stress increased total P450 content in 1,4-bis[2-(3,5- dichloropyridyloxy)]benzene (TCPOBOP)-treated mice (35%), while slightly suppressing PROD activity (26%). In addition, CYP2E1 dependent p-nitrophenol hydroxylation (PNP), was suppressed (40%) by stress in TCPOBOP-treated animals and cytosolic aldehyde dehydrogenases were not affected. Although stress had no effect on basal P4502A5 activity, the inducibility of this hepatic activity increased 2-fold after stress exposure. A pronounced suppression (7-fold) in glutathione content was observed in lungs of TCPOBOP treated mice after stress, whereas basal levels remained unaffected. In addition, only a slight suppression (20%) in liver glutathione content was found in both treatment groups. Northern blot analysis revealed that restraint stress had a relatively suppressive effect on control CYP1A2 expression in the liver. In contrast, stress markedly enhanced the expression of liver CYP2A5 in TCPOBOP-treated mice, but did so to a lesser extent in controls. Stress also increased CYP2A5 mRNA in TCPOBOP-treated mice to a greater degree than the activity of the corresponding cytochrome. On the other hand, liver P4502A5 activity was found to be induced by TCPOBOP by about 2.5-fold. However, the drug does not appear to be involved in the expression of CYP2A5. Finally, although the activity of liver P4502A5 cytochrome was found to be increased 3, 8 and 27 h after stress, after which it gradually declined up to 75 h, CYP2A5 liver expression appeared to be suppressed 3, 8, 27 and 51 h after stress, while 75 h later it apparently reached normal levels. In conclusion, the results of this study showed that restraint stress significantly alters several enzymatic systems differently at a basal level than under conditions of TCPOBOP induction. In addition, stress was found to significantly interfere with the expression processes of CYP1A2 and CYP2A5.

Evidence of alpha2-adrenoceptor involvement in B[alpha]P induction processes of drug-metabolizing enzymes: the effect of stress.

Central to the appropriate regulation of behavioral and physiological changes induced by stress are the noradrenergic neuronal systems which have been implicated in a large number of stress-induced pathophysiological states. Endoplasmic reticulum-bound cytochromes (CYPs) play a crucial role in drug metabolism, resulting in deactivation or formation of reactive derivatives. In turn, these products may be responsible for the chemotherapeutic, mutagenic or carcinogenic properties of the parent compound. The present study assesses the effect of a specific alpha2- adrenoceptor agonist, dexmedetomidine (DEXT), on stress-induced modification of cytochrome activity in rats using a restraint stress model. The results indicated that activation of the alpha2-adrenoceptor with DEXT did not alter basal hepatic methoxyresorufin 7-dealkylase (MROD). On the other hand, it appeared to enhance MROD in benzo[alpha]pyrene (B[alpha]P) treated animals. Of interest was the finding that stress blocked DEXT-induced MROD enhancement in B[alpha]P- treated rats. In addition, DEXT had no effect on basal hepatic pentoxyresorufin 7-dealkylase (PROD), while it further enhanced the strong induction by B[alpha]P. Stress was also found to block this effect. Hepatic ethoxyresorufin 7-dealkylase (EROD) activity was strongly increased by B[alpha]P; this effect was enhanced by DEXT. In contrast, the DEXT enhanced induction was further strengthened by stress. These findings suggest that alpha2-adrenoceptors may modulate the induction of cytochromes CYP1A1, 1A2 and 2B1 by B[alpha]P in rats and that stress may modify this process. In particular, stress may regulate the inducibility of P4501A1 activity by B[alpha]P via mechanisms related to alpha2-adrenoceptors.

Characterization of two nuclear proteins that interact with cytochrome P-450 1A2 mRNA. Regulation of RNA binding and possible role in the expression of the Cyp1a2 gene.

Regulation of the expression of the cytochrome P-450 la2 gene (cyp1a2) occurs mainly at the transcriptional level, but the molecular events involved in the induction process are partly unknown. Some reports have proposed involvement of post-transcriptional mechanisms [Adesnik, M. & Atchison, M. (1986) Crit. Rev. Biochem. 19, 247-305; Silver, G. & Krauter, K. S. (1990) Mol. Cell. Biol. 10, 6765-6768]. Here we report the identification of two proteins in the nuclear fraction of mouse liver, with specific binding characteristics towards CYP1A2 mRNA. The proteins have apparent molecular masses of 37 kDa and 46 kDa and exhibit a high affinity for a poly(U) motif in the 3' untranslated region of CYP1A2 mRNA. This motif seems to be important for their specific and apparently competitive binding to CYP1A2 mRNA. Treatment of mice with an inducer of CYP1A2, 3-methylcholanthrene, increases the binding of the 46-kDa protein and decreases the binding of the 37-kDa protein to the mRNA, suggesting that changes in the binding of the proteins to the mRNA could play a role in the upregulation of CYP1A2 mRNA by 3-methylcholanthrene. Phosphorylation of the 46-kDa protein, or of an intermediary factor, may play a role in its binding activity. Furthermore, the 46-kDa but not the 37-kDa protein is recognized by a monoclonal antibody against the heterogeneous nuclear ribonucleoprotein C, a nuclear protein probably involved in pre-mRNA processing. While more work is needed to understand the function of the proteins that bind to the 3' untranslated region of CYP1A2, it is possible that the 37-kDa protein has a role in the maintenance of uninduced levels of CYP1A2 mRNA, while the 46-kDa protein could be important in the maturation of elevated levels of CYP1A2 pre-mRNA, during induction.

Interaction of aflatoxin B1 with cytochrome P450 2A5 and its mutants: correlation with metabolic activation and toxicity.

Among members of the mouse cytochrome P450 2A family, P450 2A5 is the best catalyst of aflatoxin B1 (AFB1) oxidation to its 8,9-epoxide (Pelkonen, P., Lang, M., Wild, C. P., Negishi, M., and Juvonen, R. O. (1994) Eur. J. Pharmacol., Environ. Toxicol. Pharmacol. Sect. 292, 67-73). Here we studied the role of amino acid residues 209 and 365 of the P450 2A5 in the metabolism and toxicity of AFB1 using recombinant yeasts. The two sites have previously been shown to be essential in the interaction of coumarin and steroids with the P450 2A5. Reducing the size of the amino acid at position 209 or introducing a negatively charged residue at this site increased the 8,9-epoxidation of AFB1 compared to the wild type. In addition, replacing the hydrophobic amino acid at the 365 position with a positively charged lysine residue strongly decreased the metabolism of AFB1. These mutations changed the KM values generally less than the Vmax values. The changes in AFB1 metabolism contrast with the changes in coumarin 7-hydroxylation caused by these amino acid substitutions, since reducing the size of the 209 residue strongly reduced coumarin metabolism and increased the K(M) values. On the other hand, the results with AFB1 are similar to those obtained with steroid hydroxylation. This suggests that the size of the substrate is important when interacting with the residue 209 of the protein. The catalytic parameters of AFB1 correlated generally with its toxicity to the recombinant yeasts expressing the activating enzyme and with the binding of AFB1 to yeast DNA. Furthermore high affinity substrates and inhibitors (e.g., methoxsalen, metyrapone, coumarin 311, 7-methylcoumarin, coumarin, and pilocarpine) of P450 2A5 could efficiently block the toxicity of AFB1. It is suggested that the recombinant yeasts expressing engineered P450 enzymes are a useful model to understand the substrate protein interactions, to study the relationship of metabolic parameters to toxicity, and to test potential inhibitors of metabolism based toxicity.