Expression Of Cytochrome P450 Enzymes Research Articles

β-Catenin as a multilayer modulator of zonal cytochrome P450 expression in mouse liver

The liver is the major organ for metabolism of drugs and other xenobiotics. Expression of many drug-metabolizing enzymes is not equally distributed throughout the liver: under normal conditions, many of them, including the most relevant members of the cytochrome P450 superfamily, are exclusively expressed in a hepatocyte subpopulation located near branches of the efferent central vein. Activation of different ligand-dependent transcription factors by exogenous compounds stimulates high expression of certain cytochrome P450 isoforms. This process also occurs preferentially in perivenous hepatocytes. The mechanisms, however, which determine the zone-specificity of basal and xenobiotic-induced expression of cytochrome P450 enzymes, have remained largely unknown for decades. Very recently, signaling through the Wnt/beta-catenin pathway has been implicated in the regulation of zonal gene expression in mouse liver. In this review, current knowledge of cytochrome P450 regulation by beta-catenin-dependent transcription is summarized and underlying molecular mechanisms are discussed.

Metabolism of 17α-Hydroxyprogesterone Caproate, an Agent for Preventing Preterm Birth, by Fetal Hepatocytes

Preterm delivery (i.e., delivery before 37 completed weeks of gestation) is a major determinant of neonatal morbidity and mortality. Until recently, no effective therapies for prevention of preterm birth existed. In a recent multicentered trial, 17alpha-hydroxyprogesterone caproate (17-OHPC) was shown to reduce the rate of preterm birth by 33% in a group of high-risk women. Limited pharmacologic data exist for this drug. Previous studies have shown that CYP3A is involved in the metabolism of 17-OHPC. In this study, we evaluated the metabolism of 17-OHPC in adult and fetal human hepatocytes and in expressed cytochrome P450 enzymes. 17-OHPC was metabolized by expressed CYP3A7 and by fetal hepatocytes. The metabolite profile was qualitatively different between expressed CYP3A4 and CYP3A7. Expressed CYP3A4 demonstrated a significantly higher (>10 times) capacity to metabolize 17-OHPC than CYP3A7. Based on retention times, two unique metabolites were observed in the fetal and adult hepatocyte systems along with one common metabolite. The intrinsic clearance of 17-OHPC by fetal hepatocytes was observed to be one-half of that in adults. In summary, this study demonstrates that fetal hepatocytes and, in particular, the fetal form of CYP3A (i.e., CYP3A7) can metabolize 17-OHPC.

Estrogen receptor-dependent regulation of CYP2B6 in human breast cancer cells

Estrogen receptor α (ERα) mediates the biological actions of estrogens and also contributes to the development and progression of breast cancer. To gain a more comprehensive understanding of ERα-mediated transcription, we used chromatin immunoprecipitation and promoter focused microarrays (ChIP-chip) to identify ERα binding sites in T-47D human breast cancer cells. Transcription factor binding site analysis revealed that the estrogen response element (ERE) was significantly over-represented and was found in 50% of the 243 ERα-bound regions identified. Interestingly, multiple ERα-bound regions were detected in the upstream regulatory sequences of the CYP2B gene cluster. Because ERα has been reported to regulate the expression of other cytochrome P450 enzymes and CYP2B6 is highly expressed in ERα-positive breast tumors, we focused on characterizing the ERα-dependent regulation of CYP2B6. Reporter gene assays revealed that ERα and ERβ increased C YP2B6-regulated gene expression through a functional ERE located at −1669 to −1657 in the upstream regulatory region of CYP2B6. E2 increased ERα and nuclear receptor coactivator 3 (NCoA3) recruitment to the 5′-flanking region of CYP2B6, and increased CYP2B6 mRNA levels in T-47D but not in MCF-7 human breast cancer cells. RNAi-mediated knockdown of ERα in the T-47D cells resulted in a significant decrease in CYP2B6 mRNA levels. Taken together, our study provides evidence for cell-type specific transcriptional regulation of the CYP2B6 gene by ERs.

180. THE CONSEQUENCES OF ACRYLAMIDE EXPOSURE ON THE MALE GERMLINE

Acrylamide is a common industrial compound that has recently been identified in cooked, carbohydrate-rich foods such as potato chips, breads and cereals. Acrylamide has been found to be a reproductive toxin in rodents, eliciting male infertility and transgenerational toxicity through the male germline; thus dietary exposure to the compound may have consequences for male fertility and reproduction in humans. The aim of this project was to elucidate the mechanisms of acrylamide toxicity in male germ cells of mice. Freshly isolated early male germ cells were exposed to acrylamide and assessed for cell viability and aberrant morphology. DNA damage in these cells was also investigated using a modified version of the Comet Assay, which allows for adduct specificity. Significant increases in cell death or aberrant morphology were not observed following acrylamide exposure (1 µM, 18 hours). However, a significant increase in DNA damage (125% increase in mean tail DNA assessed by Comet) was identified; which may originate from either the metabolic conversion of acrylamide to glycidamide, leading to glycidamide adducts, or from oxidative stress. Additionally, the regulation of cytochrome P450 gene expression was measured using real time PCR and early male germ cells were found to upregulate gene expression of cytochrome P450 enzymes in response to acrylamide exposure. Collectively, these results support a genotoxic mode of action of acrylamide toxicity, in addition to potential oxidative damage in male germ cells. However, the mechanism by which acrylamide elicits toxicity in germ cells requires further investigation. Future outcomes of this research may provide insight into factors necessary for the healthy development of offspring and aid in the risk assessment of dietary acrylamide exposure in humans.

Manipulating cytochrome P450 enzymes: new perspectives for cancer treatment

Manipulating cytochrome P450 enzymes: new perspectives for cancer treatment

Update information on drug metabolism systems--2009, part II: summary of information on the effects of diseases and environmental factors on human cytochrome P450 (CYP) enzymes and transporters.

The present paper is an update of the data on the effects of diseases and environmental factors on the expression and/or activity of human cytochrome P450 (CYP) enzymes and transporters. The data are presented in tabular form (Tables 1 and 2) and are a continuation of previously published summaries on the effects of drugs and other chemicals on CYP enzymes (Rendic, S.; Di Carlo, F. Drug Metab. Rev., 1997, 29(1-2), 413-580., Rendic, S. Drug Metab. Rev., 2002, 34(1-2), 83-448.). The collected information presented here is as stated by the cited author(s), and in cases when several references are cited the latest published information is included. Inconsistent results and conclusions obtained by different authors are highlighted, followed by discussion of the major findings. The searchable database is available as an Excel file, for information about file availability contact the corresponding author.

Evaluation of liver and thyroid toxicity in Sprague-Dawley rats after exposure to polybrominated diphenyl ether BDE-209

Our goal in the present study was to evaluate whether decabromodiphenyl ether (BDE-209), which is the most abundant polybrominated diphenyl ether (PBDE) found in human samples, affects against target organs. Sprague-Dawley male rats were exposed to vehicle or BDE-209 (100, 300, or 600 mg/kg body weight, daily) from postnatal day (PND) 10 to PND 42. There was no significant difference in body and male reproductive organ weight changes compared with controls. However, liver, thyroid and adrenal gland weights were significantly increased in the high-dose of BDE-209 group. BDE-209 significantly induced the expression of cytochrome P450 (CYP1A2, CYP3A1, and CYP2B1) enzymes in the liver. Furthermore, constitutive androstane receptor (CAR) and pregnane xenobiotic receptor (PXR) expression levels were also increased in a dose-dependent manner. Total serum triiodothyronine (T3) concentration was significantly reduced in a dose-dependent manner, whereas the level of thyroid-stimulating hormone was significantly increased with BDE-209 treatment. In the histological findings, multiple areas of degenerated follicular epithelium and slight attenuation of the follicular epithelium were observed in the thyroid glands by high doses (300 and 600 mg/kg) of BDE-209 treatment. The presence of hepatocytic fatty degeneration and inflammatory foci were also observed in the 300 and 600 mg/kg of BDE-209 group. These findings demonstrate that BDE-209 induces hyperthyroidism and hepatotoxicity. In the future, further research is needed to determine the relationship between target organ toxicity and blood concentrations of BDE-209.

Modulation of carcinogen-metabolising cytochromes P450 in human liver by the chemopreventive phytochemical phenethyl isothiocyanate, a constituent of cruciferous vegetables

On the basis of studies conducted in animals, it has been established that isothiocyanates suppress cytochrome P450 activity, leading to impairment of the bioactivation of carcinogens, this being a principal mechanism of their chemopreventive activity. However, no studies have been carried out in human tissue to ascertain whether hepatic cytochrome P450 composition is similarly modulated, and this is the objective of the present studies. Precision-cut liver slices from four donors were incubated with a range of concentrations of phenethyl isothiocyanate (PEITC) for 24 h, and the expression and activity of cytochrome P450 enzymes were determined; similar studies were performed in rat slices for comparison. PEITC suppressed the O-dealkylation of methoxyresorufin in all human livers and this was accompanied by a parallel drop in CYP1A2 apoprotein levels; the same effect was noted in rat liver slices. The O-dealkylation of ethoxyresorufin was also impaired in the human livers, despite a rise in CYP1A1 apoprotein levels. The CYP3A-mediated benzyloxyquinoline dealkylation was inhibited by PEITC in only two of the four human donors, whereas a rise in CYP3A4 apoprotein levels was noted in all human livers, albeit to different extent. It is concluded that: (a) PEITC can modulate cytochrome P450 composition in human liver, and (b) PEITC, at concentrations that can be achieved by dietary intake, can antagonise the carcinogenicity of chemicals which rely on the CYP1 family for their bioactivation such as heterocyclic amines and polycyclic aromatic hydrocarbons, and this is likely to be a major contributory mechanism to its chemopreventive activity.

Expression, Function and Regulation of Mouse Cytochrome P450 Enzymes: Comparison With Human Cytochrome P450 Enzymes

The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

Endogenous Functions and Expression of Cytochrome P450 Enzymes in Teleost Fish: A Review

Environmental monitoring programs use biological tools in the form of pollutant-sensitive organisms called sentinel species and sub-organismic indicators or biomarkers to assess impacts to ecosystems by pollution. In this work, we review one of the more widely used groups of sentinel organisms by monitoring programs, the teleost fishes, and the best known biomarker of exposure, the cytochrome P450 superfamily of enzymes. We found that at least one P450 enzyme has been reported in 57 species of fish. Based on comparisons between enzymatic induction and endogenous function of P450, we discuss the specificity and sensibility of fish P450 enzymes as biomarkers in environmental monitoring. Studies in recent years show that the endogenous functions of several P450 enzymes in fishes are unknown, and, contrary to belief, the mechanisms of induction of the P450 enzymes in mammals and fishes are often different, and, thus, substrate-specific inductions are not comparable. It becomes clear that it is necessary to advance our understanding of P450 endogenous functions if we wish to correctly interpret the meaning of catalytic activity data of these mono-oxygenases for the purpose of environmental monitoring programs.

Expression of xenobiotic and steroid hormone metabolizing enzymes in hepatocellular tumors of the non-cirrhotic liver

Hepatocellular adenomas (HCA) and some hepatocellular carcinomas (HCC) arise in the non-cirrhotic liver. Although the liver is involved in the metabolism of a huge number of exogenous and endogenous substances, little is known about the role of metabolic enzymes in the development of liver tumors in the absence of cirrhosis. We analyzed the expression of glutathione S-transferases (GST) and cytochrome P450 enzymes (CYP) in 23 HCA, 20 HCC, and 22 focal nodular hyperplasias (FNH) using immunohistochemistry. The liver tissue revealed consistent specific staining for GST alpha, CYP1A1, 1A2, 2E1, and 3A4. In HCA and HCC, GST alpha expression was significantly reduced ( p<0.001 and 0.043). Reduced GST alpha expression was significantly associated with steatosis in HCA and HCC ( n=12, p=0.006), but not in non-neoplastic liver tissue. CYP3A4 expression was also reduced in HCA and HCC ( p=0.03 and 0.02), and this was correlated with diabetes mellitus type 2 ( p=0.02). In conclusion, HCA and HCC revealed changes in the expression of certain metabolic enzymes as compared with the non-neoplastic liver tissue or FNH. Therefore, reduced expression of GST alpha and CYP3A4 may indicate specific metabolic defects in the tumor tissue characterizing subgroups of HCA and HCC.

Expert Panel report on a study of Splenda in male rats

A recent study in rats investigated the retail sweetener product, Granulated SPLENDA ® No Calorie Sweetener (Splenda) (Abou-Donia et al., 2008. Splenda alters gut microflora and increases intestinal P-glycoprotein and cytochrome P-450 in male rats. J. Toxicol. Environ. Health A, 71, 1415–1429), which is composed of (by dry weight) maltodextrin (∼99%) and sucralose (∼1%). The investigators reported that Splenda increased body weight, decreased beneficial intestinal bacteria, and increased the expression of certain cytochrome P450 (CYP450) enzymes and the transporter protein, P-glycoprotein (P-gp), the latter of which was considered evidence that Splenda or sucralose might interfere with the absorption of nutrients and drugs. The investigators indicated that the reported changes were attributable to the sucralose present in the product tested. An Expert Panel conducted a rigorous evaluation of this study. In arriving at its conclusions, the Expert Panel considered the design and conduct of the study, its outcomes and the outcomes reported in other data available publicly. The Expert Panel found that the study was deficient in several critical areas and that its results cannot be interpreted as evidence that either Splenda, or sucralose, produced adverse effects in male rats, including effects on gastrointestinal microflora, body weight, CYP450 and P-gp activity, and nutrient and drug absorption. The study conclusions are not consistent with published literature and not supported by the data presented.

Hepatic and vascular mRNA expression in adult zebrafish ( Danio rerio) following exposure to benzo-a-pyrene and 2,3,7,8-tetrachlorodibenzo- p-dioxin

Developmental exposure to aryl hydrocarbon receptor (AhR) agonists in fish causes severe defects in the cardiovascular system. However, the effects of acute AhR agonist exposure on the adult fish cardiovascular system are not clear. We hypothesized that AhR-mediated changes in adult vascular tissue gene expression would differ from that of hepatic tissue. Therefore, zebrafish ( Danio rerio) were intraperitoneally injected with the AhR agonists benzo-a-pyrene (BaP; 1 mg/kg) or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD; 20 μg/kg) alone and in combination with the AhR antagonists resveratrol (Res; 10 mg/kg) or α-naphthoflavone (ANF; 50 mg/kg). Hepatic and mesenteric artery cytochrome P450 enzyme (subtypes 1A, 1B1, 1C1, and 1C2) and cyclooxygenase enzyme (subtypes 1, 2a, and 2b) mRNA expression was quantified using real-time reverse transcriptase PCR. TCDD exposure significantly increased ( p ≤ 0.05 in Tukey's posteriori test after 1-way ANOVA; n = 4–6/group) CYP1A, CYP1C1, and COX-2b mRNA expression in hepatic tissue (105 ± 21, 12 ± 2, and 2 ± 0.3 fold-increase, mean ± SEM respectively). TCDD also increased CYP1A, CYP1B1, CYP1C1, CYP1C2, and COX-1 mRNA expression in mesenteric artery (121 ± 23, 5 ± 1, 28 ± 6, 7 ± 1, and 3 ± 0.3, respectively). Importantly, while BaP exposure elicited no significant alterations in hepatic CYP mRNA expression, it increased COX-1 and COX-2b in liver tissues (3 ± 1 and 2 ± 0.1, respectively), as well as CYP1A, CYP1B1, CYP1C1, CYP1C2, and COX-1 expression in mesenteric artery (2 ± 0.3, 4 ± 0.3, 5 ± 1, 5 ± 1, and 2 ± 0.3, respectively). Resveratrol was able to antagonize TCDD-induced CYP1C2 in mesenteric artery but was without effect in all other treatments in both liver and mesenteric artery. In contrast, ANF antagonized TCDD and BaP-induced COX-2b and TCDD-induced CYP1C1 expression increases, as well as reduced baseline CYP1B1 and COX-2a expression in liver, while failing to affect BaP and TCDD-induced hepatic CYP1A increases. However, in mesenteric artery, ANF alone acted instead as an agonist to increase expression of CYP1A, CYP1B1, CYP1C1, CYP1C2, COX-2a and COX-2b. Thus, there are important differences in response to both AhR agonists and antagonists between liver and mesenteric artery in adult zebrafish. The vascular-specific changes in gene expression will be linked to future studies examining alterations in cardiovascular function produced by acute AhR agonist exposure in adult fish.

Expression of Cytochrome P450 (CYP) Enzymes in Human Nonpigmented Ciliary Epithelial Cells: Induction of CYP1B1 Expression by TCDD

Cytochrome P450 (CYP) enzymes metabolize endogenous compounds such as steroid hormones, fatty acids, and xenobiotics, including drugs and carcinogens. Expression of CYP enzymes in ocular tissues is poorly known. However, mutations in the CYP1B1 gene have been linked to congenital glaucoma. The aim of the present study was to investigate the expression and regulation of cytochrome P450 enzymes in a human nonpigmented ciliary epithelial cell line. Expression of mRNAs for major xenobiotic metabolizing CYPs in families 1-3 and regulatory factors involved in the induction of CYPs was studied using reverse transcriptase-polymerase chain reaction. For induction studies, the cells were treated with dexamethasone or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 hours. RNA and immunoblotting analysis were used to study CYP induction. Transcriptional regulation of CYP1B1 gene was studied by transient transfection of reporter gene constructs. mRNAs of CYP1A1, CYP1B1, and CYP2D6 and of the regulatory factors aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator, and glucocorticoid receptor were expressed in the human nonpigmented ciliary epithelial cell line. CYP1B1 mRNA was strongly and dose dependently induced by TCDD. CYP1B1 protein was detected only after TCDD treatment of the human nonpigmented ciliary epithelial cells. CYP1B1 promoter was activated by TCDD. The major drug-metabolizing enzymes CYP1A2, CYP2Cs, and CYP3As were not detected in these cells, and dexamethasone treatment had no effect on CYP expression. TCDD potently induces CYP1B1 mRNA in human nonpigmented ciliary epithelial cells, suggesting the involvement of an AHR-mediated pathway in the regulation of ciliary CYP1B1 expression.

Cyclic AMP-dependent Protein Kinase Signaling Modulates Pregnane x Receptor Activity in a Species-specific Manner

Pregnane x receptor is a ligand-activated transcription factor that regulates drug-inducible expression of several key cytochrome P450 enzymes and drug transporter proteins in liver and intestine in a species-specific manner. Activation of this receptor modulates several key biochemical pathways, including gluconeogenesis, beta-oxidation of fatty acids, fatty acid uptake, cholesterol homeostasis, and lipogenesis. It is of current interest to determine whether the interaction between pregnane x receptor and these key biochemical pathways is evolutionarily conserved. We show here that activation of the cyclic AMP-dependent protein kinase signaling pathway synergizes with pregnane x receptor-mediated gene activation in mouse hepatocytes. Conversely, cyclic AMP-dependent protein kinase signaling has a repressive effect upon pregnane x receptor-mediated gene activation in rat and human hepatocytes. We show that the human pregnane x receptor protein can serve as an effective substrate for catalytically active cyclic AMP-dependent protein kinase in vitro. Metabolic labeling of the protein in vivo indicates that human pregnane x receptor exists as a phosphoprotein and that activation of cyclic AMP-dependent protein kinase signaling modulates the phosphorylation status of pregnane x receptor. Activation of cyclic AMP-dependent protein kinase signaling also modulates the interactions of pregnane x receptor with protein cofactors. Our results define the species-specific impact of cyclic AMP-dependent protein kinase signaling on pregnane x receptor and provide a molecular explanation of cyclic AMP-dependent protein kinase-mediated repression of human pregnane x receptor activity. Taken together, our results identify a novel mode of regulation of pregnane x receptor activity and highlight prominent functional differences in the process across species.

Cytochrome P450 and the Biological Clock in Mammals

Cytochrome P450 (CYP) is a diverse superfamily of hemoproteins found in a large number of organisms. Most cytochrome P450 enzymes have monooxygenase activity and are involved in detoxification and hormone and lipid metabolism. In drug metabolism, the chemical modification or degradation of chemicals including exogenous and endogenous compounds, cytochrome P450 is probably the most important element of oxidative metabolism. The present understanding of the mechanisms of induction of cytochrome P450 enzymes and their regulation has made considerable progress during the last few years. However, it remains the subject of intense scientific research to better understand how the expression of cytochrome P450 enzymes is regulated at the molecular level. It has been known for three decades that the expression of cytochrome P450 gene family members is regulated by the biological clock. In addition, hepatic P450 monooxygenases metabolize melatonin, the pineal hormone whose expression is controlled by the biological clock and, in turn, resets the biological clock. This review will summarize our present understanding on how the biological clock regulates the expression and activity of cytochrome P450 enzymes to affect pharmacokinetics and detoxification and hormone and lipid metabolism and how melatonin metabolism and cytochrome P450 enzyme activity can affect circadian rhythms in mammals.

Reduced acetaminophen-induced liver injury in mice by genetic disruption of IL-1 receptor antagonist

Acetaminophen (APAP) induced increases in intrahepatic expression of interleukin (IL)-1α, IL-1β, and IL-1 receptor antagonist (IL-1ra), when administered intraperitoneally. These observations prompted us to define the pathophysiological roles of IL-1ra in APAP-induced liver injury. Compared with wild-type (WT) mouse-derived hepatocytes, IL-1ra-deficient (IL-1ra KO)-derived hepatocytes exhibited more resistance against APAP but not APAP-derived major toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Moreover, the amounts of a major APAP adduct (selenium-binding protein), an indicator of NAPQI generation from APAP, was significantly lower in IL-1ra KO mice than WT mice with depressed intrahepatic expression of CYP1A2, CYP2E1, and CYP3A11, the enzymes crucially involved in NAPQI generation from APAP. These observations would indicate that IL-1ra deficiency impaired APAP metabolism. IL-1α and IL-1β were expressed to similar extents in livers of untreated IL-1ra KO and WT mice. By contrast, the intranuclear amount of p65 of NF-κB, which can suppress the gene expression of CYP1A2, CYP2E1, and CYP3A11, was higher in untreated IL-1ra KO than WT mice. Moreover, when mice were intraperitoneally administered APAP (200 mg/kg), IL-1ra KO mice exhibited attenuated APAP-induced liver injury as evidenced by reductions in serum alanine transferase levels and histopathological changes such as centrilobular necrosis, hemorrhages, and leukocyte infiltration. Finally, when given 12 h before APAP challenge, IL-1α repressed the intrahepatic expression of CYP1A2, CYP2E1, and CYP3A11, eventually reducing APAP-induced liver injury, along with reduction in APAP adducts. Collectively, NF-κB was activated without any stimuli by the genetic disruption of IL-1ra, and suppressed cytochrome P450 enzyme expression, thereby reducing APAP-induced liver injury.

Effects of prior oral contraceptive use and soy isoflavonoids on estrogen-metabolizing cytochrome P450 enzymes

Estrogen exposure and metabolism may play an important role in the development of estrogen-sensitive cancers in postmenopausal women. In this study we investigated whether past oral contraceptive (OC) administration or current dietary isoflavonoids (IF) affected expression and/or activity of steroid hormone-metabolizing cytochrome P450 (CYP) enzymes using complementary primate and cell culture models. One-hundred-eighty-one female cynomolgus macaques were randomized to receive OC or nothing for 26 months premenopausally, then ovariectomized and randomized to one of three diets for 36 months: an IF-depleted soy protein isolate (Soy−) diet, a Soy diet with IF (Soy+), or a Soy− diet supplemented with conjugated equine estrogens (CEE). Prior OC-treatment significantly reduced CYP gene expression in the mammary gland (≤60% of OC-). Dietary IFs had no effect on CYP expression, while CEE-treatment decreased CYP1A1 and increased CYP3A4 mRNA in a tissue-specific manner. For in vitro studies, we measured effects of the isoflavonoids genistein, daidzein and equol on CYP activity using intact V79 cells stably transfected to express CYP1A1, CYP1B1, or CYP3A4. All three IFs significantly altered CYP activity in a dose-dependent and isoform-specific manner (20–95% inhibition versus controls). These results suggest potential mechanisms for prior OC and dietary IF effects on cancer risk in estrogen-responsive tissues.

Covariation of Human Microsomal Protein Per Gram of Liver with Age: Absence of Influence of Operator and Sample Storage May Justify Interlaboratory Data Pooling

Scaling of metabolic clearance values from liver microsomal data or recombinantly expressed cytochrome P450 enzymes to predict human hepatic clearance requires knowledge of the amount of microsomal protein per gram of liver (MPPGL). Identification of physiological covariates of MPPGL requires analysis of values from large diverse populations, which necessitates pooling of data from numerous sources. To ensure compatibility between results obtained within and between studies, the impact of interoperator differences and sample storage on values of MPPGL was investigated. With use of triplicate samples from one liver (HL86), no statistically significant difference was detected between values of MPPGL prepared from samples stored at -80 degrees C (23.5 +/- 1.2 mg g(-1)) and those determined using fresh tissue (21.9 +/- 0.3 mg g(-1)). Although there was a significant difference in the yield of microsomal protein obtained from another liver sample (HL43) by three different operators (17 +/- 1, 19 +/- 2, and 24 +/- 1 mg g(-1); p = 0.004, analysis of variance), no difference was observed in the estimated MPPGL after application of appropriate correction factors for each operator (28 +/- 1, 30 +/- 5, and 31 +/- 4 mg g(-1)). The result provided justification for pooling reported values of MPPGL for use in covariate analysis. Investigation of the relationship between age and MPPGL provided preliminary evidence that MPPGL values increase from birth to a maximum of 40 mg g(-1) [95% confidence interval for the geometric mean (95% CI mean(geo)): 37-43 mg g(-1) at approximately 28 years followed by a gradual decrease in older age (mean of 29 mg g(-1) at 65 years; 95% CI mean(geo): 27-32 mg g(-1)). Accordingly, appropriate age-adjusted scaling factors should be used in extrapolating in vitro clearance values to clinical studies.

Expression and Function of Cytochrome P450-Dependent Enzymes in Human Skin Cells

Scientific interest in defining the human body's ability to limit the effects of administered drugs and xenobiotics dates from the mid-19th century when developing knowledge and techniques in the field of organic chemistry first made such studies possible. The first experimental evidence documenting the existence of cytochrome p450 (CYP) dates to the year 1955, when an enzyme system capable of oxidizing xenobiotic compounds was identified in the endoplasmic reticulum of liver homogenates. From these days on several studies analyzed the expression and function of metabolizing phase I enzymes in liver cells. Due to the unique structural features of human skin, little was known about the expression and function of CYP enzymes in this tissue and their role in uptake, metabolism and elimination of xenobiotics as well as endogenous substrates. Lasting recent years it has become clear that human skin cells express various CYP enzymes, including CYP26AI which is responsible for the metabolism of retinoic acid in skin cells. It has been also shown that CYP enzyme expression patterns are cell type and tissue specific and that in skin cells this differs significantly from its expression in other environmental interfaces such as the liver, lung and gastrointestinal tract. Therefore knowledge of skin-specific CYP expression and function is a prerequisite for pharmacological studies of the skin.