Cytochrome P450c17 Research Articles

Polymorphism in CYP17, GSTM1 and the progesterone receptor genes and its relationship with mammographic density

Radiologic breast density is one of the predictive factors for breast cancer and the extent of the density is directly related to postmenopause. However, some patients have dense breasts even during postmenopause. This condition may be explained by the genes that codify for the proteins involved in the biosynthesis, as well as the activity and metabolism of steroid hormones. They are polymorphic, which could explain the variations of individual hormones and, consequently, breast density. The constant need to find markers that may assist in the primary prevention of breast cancer as well as in selecting high risk patients motived this study. We determined the influence of genetic polymorphism of CYP17 (cytochrome P450c17, the gene involved in steroid hormone biosynthesis), GSTM1 (glutathione S-transferase M1, an enzyme involved in estrogen metabolism) and PROGINS (progesterone receptor), for association with high breast density. One hundred and twenty-three postmenopausal patients who were not on hormone therapy and had no clinical or mammographic breast alterations were included in the present study. The results of this study reveal that there was no association between dense breasts and CYP17 or GSTM1. There was a trend, which was not statistically significant (P = 0.084), towards the association between PROGINS polymorphism and dense breasts. However, multivariate logistic regression showed that wild-type PROGINS and mutated CYP17, taken together, resulted in a 4.87 times higher chance of having dense breasts (P = 0.030). In conclusion, in the present study, we were able to identify an association among polymorphisms, involved in estradiol biosyntheses as well as progesterone response, and radiological mammary density.

Are Atlantic Cod in Store Lungegårdsvann, a Seawater Recipient in Bergen, Affected by Environmental Contaminants? A qRT-PCR Survey

The aim of this study was to examine the transcriptional levels of selected genes in liver and head kidney of Atlantic cod Gadus morhua sampled in Store Lungegårdsvann, a seawater recipient situated in the middle of the city of Bergen, Norway, for effects of contaminants released from municipal sewage effluents and former dump sites. Five males and six females were caught with fish traps in Store Lungegårdsvann in 2006. Cod from a location near Jondal in the Hardanger Fjord were used as controls (five males and four females). The following 12 genes were picked as potential markers of contaminant exposure: cytochrome P-450 1A (CYP1A), cytochrome P-450 2C33-like (CYP2C33-like), cytochrome P-450 3C (CYP3C), glutathione S-transcriptase π (GST) (detoxification and biotransformation), Mn superoxide dismutase (Mn SOD), glutathione reductase (GR), heat-shock protein 70 (HSP70) (oxidative stress), vitellogenin A (VtgA), vitellogenin B (VtgB), zona pellucida 2 (ZP2) (effects of estrogen disruptors), B-cell lymphoma 2 (Bcl-2), and cyclin-dependent kinase inhibitor 1A (CDKN1A) (radiation). The results showed that two males caught in Store Lungegårdsvann possessed high transcriptional levels of VtgA, VtgB, and ZP2 mRNA in the liver. In addition, CYP1A was 4.9-fold higher expressed in males from Store Lungegårdsvann compared to males from the reference population. CYP2C33-like mRNA expression was significantly higher (1.8-fold) in females from Store Lungegårdsvann than in females from the reference population. CYP1A was significantly lower (4.7-fold) expressed in head kidney of females from Store Lungegårdsvann than in females from Hardanger Fjord. In a follow-up examination with sexually mature cod sampled in Store Lungegårdsvann in 2007, the livers were shown to contain high levels of polychlorinated biphenyls (PCB) and dioxin-like PCB. In conclusion, fish inhabiting Store Lungegårdsvann are exposed not only to endocrine disruptors but also to other contaminants that affect the transcription of phase I biotransformation genes.

Identification of HumanCYP2C8as a Retinoid-Related Orphan Nuclear Receptor Target Gene

Retinoid-related orphan nuclear receptors (RORs) alpha and gamma (NR1F1, -3) are highly expressed in liver, adipose tissue, thymus, and brain and are involved in many physiological processes, such as circadian rhythm and immune function. Enzymes in the cytochrome P450 2C subfamily metabolize many clinically important drugs and endogenous compounds, such as the anticancer drug paclitaxel and arachidonic acid, and are highly expressed in liver. Here, we present the first evidence that RORs regulate the transcription of human CYP2C8. Overexpression of RORalpha and RORgamma in HepG2 cells significantly enhanced the activity of the CYP2C8 promoter but not that of the CYP2C9 or CYP2C19 promoters. Computer analyses, promoter deletion studies, gel shift assays, and mutational analysis identified an essential ROR-responsive element at -2045 base pairs in the CYP2C8 promoter that mediates ROR transactivation. Adenoviral overexpression of RORalpha and -gamma significantly induced endogenous CYP2C8 transcripts in both HepG2 cells and human primary hepatocytes. Knockdown of endogenous RORalpha and -gamma expression in HepG2 cells by RNA interference decreased the expression of endogenous CYP2C8 mRNA by approximately 50%. These data indicate that RORs transcriptionally up-regulate CYP2C8 in human liver and, therefore, may be important modulators of the metabolism of drugs and physiologically active endogenous compounds by this enzyme in liver and possibly extrahepatic tissues where RORs are expressed.

Ginseng Extracts Facilitate Cytochrome P450 Xenobiotic Metabolism in Primary Cultures of Rat Hepatocytes

A 70% methanol extract from red ginseng (steamed and dried roots of Panax ginseng C. A. Meyer, a kind of Ginseng Radix) has been shown to have various actions on physiological functions. We investigated whether the ginseng extract (Ext.) affected the mRNA expression of cytochrome P450 (CYP) CYP1A1, 2B1, 2C11, 2D1, 3A1, and 3A2 in rat primary hepatocytes. After treatment with ginseng extract, the levels of CYP3A1 and 1A1 mRNA were significantly increased compared with those of the control. The increased protein levels of CYP3A1 were also observed after treatment with Ext. The mRNA levels of other examined CYPs exhibited little change. The mRNA levels of the pregnane X receptor, but not the constitutive androstane receptor, both transcription factors for CYPs, also significantly increased after treatment with ginseng extract. These results raise the possibility that ginseng Ext. promotes xenobiotic metabolism via an increase in CYP3A1 and 1A1 expression.

TGF-β Inhibits CYP17 Transcription in H295R Cells Acting via Activin Receptor-Like Kinase 5

Objective. Transforming growth factor β (TGF-β) is a potent inhibitor of 17α-hydroxylase/17,20 lyase activity and CYP17 gene expression. We investigated the mechanism how CYP17 is inhibited by TGF-β in adrenocortical cells. Methods. H295R cells were culture and incubated with TGF-β, transcription inhibitor (DRB), activin receptor-like kinase 5 ALK5 (TβRII) inhibitor (SB431542), mitogen activated kinases inhibitors (PD98059 and SB203580), subsequently using reverse transcription and quantitative PCR (RT-qPCR) we determined CYP17 expression. Results. TGF-β significantly decreased the level of cytochrome P450c17 mRNA and this inhibitory effect of TGF-β on CYP17 expression required activin receptor-like kinase 5 (ALK5) and on-going transcription. Mitogen activated kinases MEK1 and p38 MAPK are not involved it the inhibitory effect of TGF-β on CYP17 expression. Conclusion. We concluded that the TGF-β-dependent decrease of 17α-hydroxylase/17,20 lyase activity in the H295R cells is caused by inhibition of CYP17 transcription and is mediated by the ALK5 receptor.

Alteration of testicular steroidogenesis and histopathology of reproductive system in male rats treated with triclosan

Triclosan (TCS), a chlorophenol, is widely used as a preservative in different types of commercial preparations. The reports on TCS-mediated endocrine disruption are controversial and the present study aimed to elucidate the probable mode of action of TCS as an antiandrogenic compound using a robust study design. Male albino rats, Rattus norvegicus, were treated with three doses of triclosan for a period of 60 days followed by the analysis of various biochemical parameters. RT-PCR analysis demonstrated a significant decrease in mRNA levels for testicular steroidogenic acute regulatory (StAR) protein, cytochrome P450 SCC, cytochrome P450 C17, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) and androgen receptor (AR) in TCS treated rats ( p < 0.05). TCS also induced a perturbed translation of testicular StAR, and AR proteins as shown by Western blot analysis in treated groups of rats. A reduced level of StAR was further indicated by immunohistochemistry in testicular Leydig cells. Further, there was a significant decrease ( p < 0.05) in the level of serum lutenizing hormone (LH), follicle stimulating hormone (FSH), cholesterol, pregnenolone, and testosterone. In vitro assays demonstrated more than 30% decrease in testicular 3β-HSD and 17β-HSD enzyme activities in treated group of animals. Extensive histopathological malformations were observed in the testis and sex accessory tissues of the treated rats. Overall this study showed that TCS decreased the synthesis of androgens followed by reduced sperm production in treated male rats which could be mediated by a decreased synthesis of LH and FSH thus involving hypothalamo–pituitary–gonadal axis.

The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities

Male pigs are routinely castrated to prevent the accumulation of testicular 16-androstene steroids, in particular 5α-androst-16-en-3-one (5α-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-β synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17α-hydroxylase and C17,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and CYB5B, in both the andien-β synthase as well as the 17α-hydroxylase and C17,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17α-hydroxylase ( p < 0.013), a transient increase in C17,20 lyase, and an increase in andien-β synthase activity ( p < 0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17α-hydroxylase, but did not affect the andien-β synthase activity; however, the C17,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-β synthase activity of CYP17A1.

The Developmental Increase in Adrenocortical 17,20-Lyase Activity (Biochemical Adrenarche) Is Driven Primarily by Increasing Cytochrome b5 in Neonatal Rhesus Macaques

Adrenarche is thought to be experienced only by humans and some Old World primates despite observed regression of an adrenal fetal zone and establishment of a functional zona reticularis (ZR) in other species like rhesus macaques. Adrenal differentiation remains poorly defined biochemically in nonhuman primates. The present studies defined ZR development in the neonatal rhesus by examining androgen synthetic capacity and factors affecting it in rhesus and marmoset adrenals. Western immunoblots examined expression of 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), cytochrome b5 (b5), and 3beta-hydroxysteroid dehydrogenase (3betaHSD), among other key enzymes. 17,20-lyase activity was quantified in adrenal microsomes, as was the contribution of b5 to 17,20-lyase activity in microsomes and cell transfection experiments with rhesus and marmoset P450c17. Expression of b5 increased from birth to 3 months, and was positively correlated with age and 17,20-lyase activity in the rhesus. Recombinant b5 addition stimulated 17,20-lyase activity to an extent inversely proportional to endogenous levels in adrenal microsomes. Although 3betaHSD expression also increased with age, P450c17, 21-hydroxylase cytochrome P450, and the redox partner, reduced nicotinamide adenine dinucleotide phosphate-cytochrome P450 oxidoreductase, did not; nor did recombinant cytochrome P450 oxidoreductase augment 17,20-lyase activity. Cotransfection with b5 induced a dose-dependent increase in dehydroepiandrosterone synthesis by both nonhuman primate P450c17 enzymes. We conclude that the increase in 17,20-lyase activity characteristic of an adrenarche in rhesus macaques is driven primarily by increased b5 expression, without the need for a decrease in 3betaHSD, as suggested from human studies. The rhesus macaque is a relevant and accessible model for human ZR development and adrenal function.

Species-specific toxicity of diclofenac and troglitazone in primary human and rat hepatocytes

Troglitazone was withdrawn from the market shortly after approval for diabetes type II therapy because of strong hepatotoxic effects in man that could not be predicted from regulatory animal or in vitro studies. Another pharmaceutical that is regularly associated with adverse effects on the liver, sometimes leading to acute liver failure, is the widely used non-steroidal anti-inflammatory drug (NSAID) diclofenac. Since the underlying molecular mechanisms are not yet fully known, we treated primary rat and human hepatocyte monolayer cultures for 24 h with different doses of troglitazone and diclofenac to analyze species differences related to toxicity in vitro. Metformin an antidiabetic drug which does not cause severe adverse reactions served as negative control. Human hepatocytes showed a higher sensitivity to troglitazone than rat hepatocytes, while diclofenac-induced cytotoxicity at fairly similar concentrations. By co-treatment with specific inhibitors for cytochrome P450 (CYP) 2C and CYP3A – the major phase I enzymes involved in liver xenobiotic metabolism – we could confirm the prominent role of CYP3A in the bioactivation of troglitazone as well as the role of CYP3A and CYP2C in the activation of diclofenac. Inhibition of these enzymes increased the viability of treated cells in both species. Furthermore, we were able to demonstrate marked species differences in gene expression patterns of troglitazone treated rat and human hepatocytes. In contrast to rat hepatocytes, human cells showed distinct upregulation of various CYPs, regulators of xenobiotic metabolism and marker genes for oxidative stress. In contrast, gene expression alterations in rat and human hepatocytes treated with Diclofenac were rather similar. Altogether our study showed that species-specific effects as well as indications for the mode of action of compounds can be addressed by the use of primary hepatocyte cultures from various species in combination with gene expression profiling.

Analyses of CYP2C in Porcine Microsomes

The cytochrome P450 2C (CYP2C) subfamily in human beings includes four different isoenzymes that metabolize different substrates although with some cross reactivity. Some of these substrates (e.g. diclofenac and tolbutamide), have been investigated in porcine microsomes, but without identifying the specific CYP catalysing the reactions. The objective of this study was therefore to test some CYP2C substrates and identify the porcine CYP2C responsible for the reaction. Three substrates, paclitaxel, tolbutamide and omeprazole, were chosen, as they are metabolized by three different CYP2C isoenzymes in human beings. Microsomes, isolated from 20 different pigs, 12 conventional, and 8 minipigs, were incubated with these compounds, and correlations between the metabolism rates of these three substrates were found indicating that the reactions are catalysed by the same enzyme. Male minipigs tend to have higher average activity than female minipigs, which is in contrast to the gender-dependent expression seen for other CYP isoenzymes. The metabolic activities correlated with the protein level determined in Western blotting, using anti-human CYP2C9, indicating that this enzyme is responsible for the reaction. The expression of the CYP2C enzymes was analysed by real-time polymerase chain reaction, using a primer set that could amplify CYP2C8, CYP2C9 and CYP2C19. The melting curves (peaks) revealed that all three genes were present, showing very different expression levels in the various types of pigs. The area of one of the peaks, however, correlated with the CYP2C9-like enzyme concentration, suggesting that this peak represents CYP2C9. Among paclitaxel, tolbutamide and omeprazole, omeprazole is the best probe of CYP2C9-like enzyme in the pig.

Classification of drug molecules considering their IC50 values using mixed-integer linear programming based hyper-boxes method

BackgroundA priori analysis of the activity of drugs on the target protein by computational approaches can be useful in narrowing down drug candidates for further experimental tests. Currently, there are a large number of computational methods that predict the activity of drugs on proteins. In this study, we approach the activity prediction problem as a classification problem and, we aim to improve the classification accuracy by introducing an algorithm that combines partial least squares regression with mixed-integer programming based hyper-boxes classification method, where drug molecules are classified as low active or high active regarding their binding activity (IC50 values) on target proteins. We also aim to determine the most significant molecular descriptors for the drug molecules.ResultsWe first apply our approach by analyzing the activities of widely known inhibitor datasets including Acetylcholinesterase (ACHE), Benzodiazepine Receptor (BZR), Dihydrofolate Reductase (DHFR), Cyclooxygenase-2 (COX-2) with known IC50 values. The results at this stage proved that our approach consistently gives better classification accuracies compared to 63 other reported classification methods such as SVM, Naïve Bayes, where we were able to predict the experimentally determined IC50 values with a worst case accuracy of 96%. To further test applicability of this approach we first created dataset for Cytochrome P450 C17 inhibitors and then predicted their activities with 100% accuracy.ConclusionOur results indicate that this approach can be utilized to predict the inhibitory effects of inhibitors based on their molecular descriptors. This approach will not only enhance drug discovery process, but also save time and resources committed.

Failure to upregulate the adenosine2Areceptor-epoxyeicosatrienoic acid pathway contributes to the development of hypertension in Dahl salt-sensitive rats

Adenosine-activated renovascular dilatation in Sprague-Dawley (SD) rats is mediated by stimulating adenosine(2A) receptors (A(2A)R), which is linked to epoxyeicosatrienoic acid (EET) synthesis. The A(2A)R-EET pathway is upregulated by high salt (HS) intake in normotensive SD rats. Because this pathway is antipressor, we examined the role of the A(2A)R-EET pathway in Dahl salt-sensitive (SS) rats. Male Dahl salt-resistant (SR) and SS rats were fed either HS (8.0% NaCl) or normal salt (NS; 0.4% NaCl) diet for 7 days. On day 8, isolated kidneys were perfused with Krebs-Henseleit buffer containing indomethacin and N(G)-nitro-l-arginine methyl ester and preconstricted with phenylephrine. Bolus injections of the stable adenosine analog 2-chloroadenosine (2-CA; 0.1-20 microg) elicited dose-dependent dilation in both Dahl SR and SS rats. Dahl SR rats fed a HS diet demonstrated a greater renal vasodilator response to 10 microg of 2-CA, as measured by the reduction in renal perfusion pressure, than that of Dahl SR rats fed a NS diet (-104 +/- 6 vs. -77 +/- 7 mmHg, respectively; P < 0.05). In contrast, Dahl SS rats did not exhibit a difference in the vasodilator response to 2-CA whether fed NS or HS diet (96 +/- 6 vs. 104 +/- 13 mmHg in NS- and HS-fed rats, respectively). In Dahl SR but not Dahl SS rats, HS intake significantly increased purine flux, augmented the protein expression of A(2A)R and the cytochrome P-450 2C23 and 2C11 epoxygenases, and elevated the renal efflux of EETs. Thus the Dahl SR rat is able to respond to HS intake by recruiting EET formation, whereas the Dahl SS rat appears to have exhausted its ability to increase EET synthesis above the levels observed on NS intake, and this inability of Dahl SS rats to upregulate the A(2A)R-EET pathway in response to salt loading may contribute to the development of salt-sensitive hypertension.

Bisphosphonate-related osteonecrosis of the jaw is associated with polymorphisms of the cytochrome P450 CYP2C8 in multiple myeloma: a genome-wide single nucleotide polymorphism analysis

AbstractWe have explored the potential role of genetics in the development of osteonecrosis of the jaw (ONJ) in multiple myeloma (MM) patients under bisphosphonate therapy. A genome-wide association study was performed using 500 568 single nucleotide polymorphisms (SNPs) in 2 series of homogeneously treated MM patients, one with ONJ (22 MM cases) and another without ONJ (65 matched MM controls). Four SNPs (rs1934951, rs1934980, rs1341162, and rs17110453) mapped within the cytochrome P450-2C gene (CYP2C8) showed a different distribution between cases and controls with statistically significant differences (P = 1.07 × 10−6, P = 4.231 × 10−6, P = 6.22 × 10−6, and P = 2.15 × 10−6, respectively). SNP rs1934951 was significantly associated with a higher risk of ONJ development even after Bonferroni correction (P corrected value = .02). Genotyping results displayed an overrepresentation of the T allele in cases compared with controls (48% vs 12%). Thus, individuals homozygous for the T allele had an increased likelihood of developing ONJ (odds ratio 12.75, 95% confidence interval 3.7-43.5).

High performance liquid chromatography determination of cytochrome P450 1A and 2C activities in bovine liver microsomes

This study reports fluorescence high performance liquid chromatography (HPLC) and UV-Vis HPLC methods for the determination of 7-ethoxyresorufin O-deethylase (EROD) and tolbutamide methylhydroxylase (TMH) activities, respectively, using bovine liver microsomes. The detection limits were 0.022 and 5.5 pmol on the column, respectively; intra-day and inter-day precisions (expressed as relative standard deviation) were <10%. Both methods showed enough sensitivity to allow for an accurate determination of enzyme kinetic parameters according to Michaelis-Menten plots and the results were: K(m)=0.23+/-0.051 microM, V(max)=0.488+/-0.035 nmol/min/mg protein for EROD activity, and K(m)=1010+/-155.7 microM, V(max)=0.089+/-0.006 nmol/min/mg protein for TMH activity. An Eadie-Hofstee plot analysis showed that in bovine liver microsomes, EROD and TMH activities followed a monophasic kinetic pattern. alpha-Naphthoflavone, a cytochrome P450 1A1/2 (CYP1A1/2) inhibitor, and sulfaphenazole, a cytochrome P450 2C9 (CYP2C9) inhibitor, decreased EROD and TMH activities, respectively. The sensitivity of the methods allowed the use of microsomes with low enzyme activity, such as those from veal calf liver. Thus, EROD and TMH activities may be adopted as markers for the evaluation of CYP1A and CYP2C9-like activities in liver microsomes from veal and beef cattle.

Spearmint induced hypothalamic oxidative stress and testicular anti-androgenicity in male rats – altered levels of gene expression, enzymes and hormones

Mentha spicata Labiatae, commonly known as spearmint, can be used for various kinds of illnesses in herbal medicines and food industries. One of the prominent functions of this plant extract is its anti-androgenic activity. The present study investigated the probable correlation between oxidative stress in hypothalamic region and anti-androgenic action of this plant’s aqueous extract on rats. Decreased activities of enzymes like superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase in hypothalamus of treated rats indicated spearmint induced oxidative stress. Further RT-PCR and immunoblot analysis demonstrated the decreased expression of some of the steroidogenic enzymes, cytochrome P450scc, cytochrome P450C17, 3β-Hydroxysteroid dehydrogenase (3β-HSD), 17β-Hydroxysteroid dehydrogenase (17β-HSD) and other related proteins like, steroidogenic acute regulatory protein, androgen receptor and scavenger receptor class B-1. Further, in vitro enzyme assays demonstrated depressed activities of testicular 3β-HSD and 17β-HSD enzymes. Histopathology indicated a decreased sperm density in cauda epididymis and degeneration of ductus deference. Our study suggested that spearmint probably induced oxidative stress in hypothalamus resulting in decreased synthesis of LH and FSH which in turn down-regulated the production of testicular testosterone through the disruption of a number of intermediate cascades.

Proteasome inhibition compromises direct retention of cytochrome P450 2C2 in the endoplasmic reticulum

To determine whether protein degradation plays a role in the endoplasmic reticulum (ER) retention of cytochromes P450, the effects of proteasomal inhibitors on the expression and distribution of green fluorescent protein chimeras of CYP2C2 and related proteins was examined. In transfected cells, expression levels of chimeras of full-length CYP2C2 and its cytosolic domain, but not its N-terminal transmembrane sequence, were increased by proteasomal inhibition. Redistribution of all three chimeras from the reticular ER into a perinuclear compartment and, in a subset of cells, also to the cell surface was observed after proteasomal inhibition. Redistribution was blocked by the microtubular inhibitor, nocodazole, suggesting that redistribution to the cell surface followed the conventional vesicular transport pathway. Similar redistributions were detected for BAP31, a CYP2C2 binding chaperone; CYP2E1 and CYP3A4, which are also degraded by the proteasomal pathway; and for cytochrome P450 reductase, which does not undergo proteasomal degradation; but not for the ER membrane proteins, sec61 and calnexin. Redistribution does not result from saturation of an ER retention “receptor” since in some cases protein levels were unaffected. Proteasomal inhibition may, therefore, alter ER retention by affecting a protein critical for ER retention, either directly, or indirectly by affecting the composition of the ER membranes.

Cytochrome p450 2C Enzymes Contribute to Peritransplant Ischemic Injury and Cardiac Allograft Vasculopathy

Peritransplant ischemia and reperfusion (I/R) injury contributes to posttransplant vascular dysfunction and cardiac allograft vasculopathy (CAV). We have previously shown that cytochrome p450 (CYP) 2C inhibition significantly reduces I/R-induced myocardial infarction and postischemic vascular dysfunction. In the latter study, pretreatment with sulfaphenazole (SP), a specific inhibitor of CYP 2C, restored postischemic NO-mediated, endothelium-dependent vasodilation and reduced vascular superoxide production. Given the association between I/R injury, early vascular dysfunction and CAV, we hypothesized that CYP 2C may also contribute to the onset of CAV. Lewis-to-Fisher rat heterotopic heart transplants were performed. Donors and recipients were treated with 5 mg/kg SP or vehicle control 1 h prior to surgery. SP did not affect posttransplant morbidity, mortality or weight gain. Coronary blood vessels from rats treated with SP exhibited significantly reduced luminal narrowing and demonstrated a corresponding decrease in smooth muscle cell (SMC) proliferation compared to controls. SP did not reduce diffuse, focal, epicardial, endocardial or perivascular immune infiltration nor did it significantly alter TUNEL positivity in myocardial, endothelial or SMC populations. In conclusion, CYP 2C contributes to SMC proliferation CAV without affecting general immune infiltration.

Clinical versatility of porcine hepatocytes in the light of interspecies differences in cytochrome P450 regulation and expression

In fulminant hepatic failure, the clinical use of bioartifical liver support with porcine hepatocytes is the subject of a controversial debate. Cytochrome P450 (CYP) metabolic functions have relevant implications for drug metabolism and detoxification. In this study, we investigate interspecies differences in CYP gene expression between human and porcine primary hepatocytes and the impact of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) exposition mimicking cytokine release in fulminant hepatic failure. Primary hepatocyte cultures were isolated from human resection specimens and from German landrace pigs. Cell cultures (single and co-cultures) were exposed to porcine vs. human IL-6 and TNF-alpha, respectively. Changes in quantitative CYP gene expression were investigated by semi-quantitative RT-PCR. Significant differences in species-specific CYP gene expression by human and porcine hepatocytes were found after exposure to species-identical IL-6 (10 ng/ml) for CYP 1A1, CYP 2C, CYP 3A (P = 0.002, 0.022, 0.017, respectively) or species-identical TNF-alpha (30 ng/ml) for CYP 1A2 and CYP 2A (P = 0.037, 0.023, respectively). In single vs. co-culture, human hepatocytes demonstrated stronger repression of CYP 1A1, 2C8 and 3A4 expression after dosage with human IL-6 (10 ng/ml) (P = 0.022, 0.031, 0.014, respectively). Our findings demonstrate significant species-specific differences in CYP gene expression and regulation when high doses of IL-6 and TNF-alpha are used (10 and 30 ng/ml, respectively). These findings may point to species-specific physiological incompatibilities of porcine hepatocytes and thus limit their clinical versatility.

Cytochrome P450 2C11 5′-Flanking Region and Promoter Mediate in Vivo Suppression by 3-Methylcholanthrene

Aromatic hydrocarbons such as 3-methylcholanthrene (MC) elicit toxic and adaptive responses through the aryl hydrocarbon receptor (AHR). Aromatic hydrocarbons act via an unknown mechanism to suppress the transcription of CYP2C11, a growth hormone-regulated gene encoding the male-specific rat hepatic cytochrome P450 2C11. We hypothesize that suppression of CYP2C11 by aromatic hydrocarbons is mediated by the gene's promoter and 5'-flank. Using hydrodynamics-based injections to deliver plasmid DNA to the liver of live rats, we studied the MC responsiveness of luciferase constructs containing 10.1, 5.6, and 2.4 kilobases (kb) of the CYP2C11 5'-flank. MC suppressed CYP2C11-luciferase activity of the 10.1- and 5.6-kb constructs to less than 50% of vehicle levels by 24 and 72 h. Luciferase activity of the 2.4-kb CYP2C11 construct was decreased to 63% of vehicle levels 24 h after MC treatment, but no suppression was detected by 72 h. Negative regulatory element(s) responsible for CYP2C11 reporter suppression by MC exist in the proximal 2.4 kb of the 5'-flank; however, additional cis-acting elements located between -5.6 and -2.4 kb mediate persistent reporter suppression. As a positive control for AHR activation, MC dramatically induced the luciferase activity of a Cyp1a1-driven luciferase plasmid under AHR control. Modulation of reporter gene activity by MC was accompanied by induction of endogenous CYP1A1 and suppression of endogenous CYP2C11 mRNA/protein. This is the first demonstration of aromatic hydrocarbon-mediated suppression of a CYP2C11-luciferase construct, and this finding suggests that the 5'-flanking region and promoter mediate down-regulation of this gene in the intact rat.

Antiangiogenic Effect of Inhibitors of Cytochrome P450 on Rats with Glioblastoma Multiforme

Cytochrome P450 epoxygenase catalyzes 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) from arachidonic acid (AA). In 1996, our group identified the expression of the cytochrome P450 2C11 epoxygenase (CYP epoxygenase) gene in astrocytes. Because of our finding an array of physiological functions have been attributed to EETs in the brain, one of the actions of EETs involves a predominant role in brain angiogenesis. Blockade of EETs formation with different epoxygenase inhibitors decreases endothelial tube formation in cocultures of astrocytes and capillary endothelial cells. The intent of this investigation was to determine if pharmacologic inhibition of formation of EETs is effective in reducing capillary formation in glioblastoma multiforme with a concomitant reduction in tumor volume and increase in animal survival time. Two mechanistically different inhibitors of CYP epoxygenase, 17-octadecynoic acid (17-ODYA) and miconazole, significantly reduced capillary formation and tumor size in glial tumors formed by injection of rat glioma 2 (RG2) cells, also resulting in an increased animal survival time. However, we observed that 17-ODYA and miconazole did not inhibit the formation of EETs in tumor tissue. This implies that 17-ODYA and miconazole appear to exert their antitumorogenic function by a different mechanism that needs to be explored.