ToxSci Advance Access originally published online on April 7, 2008
Toxicological Sciences 2008 104(1):100-106; doi:10.1093/toxsci/kfn071
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Evidence that the Anticarcinogenic Effect of Caffeic Acid Phenethyl Ester in the Resistant Hepatocyte Model Involves Modifications of Cytochrome P450
* Departamento de Biología Celular
Sección Externa de Toxicología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV), Avenida IPN No. 2508 Colonia San Pedro Zacatenco, México 14, DF, CP 07360, México
1 To whom correspondence should be addressed at Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV), Avenida IPN No. 2508 Colonia San Pedro Zacatenco, México 14, DF, CP 07360, México. Fax: (52) 55 57473393. E-mail: svilla{at}cell.cinvestav.mx.
Received October 3, 2007; accepted March 28, 2008
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Caffeic acid phenethyl ester (CAPE), a natural component of propolis, shows anticarcinogenic properties in the modified resistant hepatocyte model when administered before initiation or promotion of hepatocarcinogenesis process; however, information about the mechanism underlying this chemoprotection is limited. The aim of this work was to characterize the effect of CAPE on cytochrome P450 (CYP), which is involved in diethylnitrosamine (DEN) metabolism during the initiation stage of chemical hepatocarcinogenesis. Male Fischer-344 rats were treated as in the modified resistant hepatocyte model. Liver samples were obtained at four different times: at 12 h after pretreatment with CAPE and at 12 and 24 h and 25 days after DEN administration. Liver damage was determined by histology with hematoxylin and eosin, measurement of total CYP levels and enzyme activity, and
-glutamyl transpeptidase–positive (GGT+) staining of hepatocyte foci. CAPE administration prevented DEN-induced necrosis at 24 h. It also decreased O-dealkylation of 7-ethoxy-resorufin (EROD), O-dealkylation of 7-methoxyresorufin (MROD), and 7-pentoxy-resorufin activities at 12 h after its administration and EROD and MROD activities at 12 h after administration of DEN. CAPE treatment decreased GGT+ foci by 59% on day 25. Our results suggest that CAPE modifies the enzymatic activity of CYP isoforms involved in the activation of DEN, such as CYP1A1/2 and CYP2B1/2. These findings describe an alternative mechanism for understanding the ability of CAPE to protect against chemical hepatocarcinogenesis. Key Words: chemoprevention; cytochrome P450; DEN metabolism; hepatocarcinogenesis.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Liver cancer is the fifth most frequent cancer and the third most frequent cause of cancer death worldwide (Parikh and Hyman, 2007
). Although millions of people live with cancer or have received medical treatment for it, prevention remains the best option. Chemoprevention is based on the hypothesis that disruption of the biological events involved in any stage of carcinogenesis can decrease cancer incidence. There are three principal stages in carcinogenesis: initiation, when mutations occur and cells are initiated; promotion, when clonal expansion of initiated cells takes place and forms preneoplastic lesions; and progression, when preneoplastic lesion becomes tumors through an increase in genetic and metabolic changes. These stages constitute a lengthy process during which chemoprevention can be applied (Klaunig and Kamendulis, 2004).
Caffeic acid phenethyl ester (CAPE), a natural component of propolis collected from honeybee (Apis mellifera) hives, has been studied since 1987 (Bankova et al., 1987). It shows a broad spectrum of biological properties, including activity as an anti-inflammatory (Michaluart et al., 1999), antioxidant (Oktem et al., 2005; Song et al., 2002; Sud`ina et al., 1993), and anticarcinogen (Carrasco-Legleu et al., 2004, 2006; McEleny et al., 2004; Na et al., 2000).
The anticarcinogenic properties of CAPE have been studied in the modified resistant hepatocyte model. Its effects have been tested when it was administered before the initiation and promotion stages. Administering CAPE in several doses during promotion caused a 90% decrease in the induction of -glutamyl transpeptidase–positive (GGT+) foci on day 25; decreases in markers of preneoplastic lesions, GGT activity, and the amount of glutathione-S-transferase class Pi (GST-p) protein were also observed. CAPE administration 12 h before exposure to diethylnitrosamine (DEN) caused an 85% decrease in the translocation of the p65 subunit of nuclear factor kappa B into the nucleus; it reduced GGT+ foci by 84% and GST-p levels by 90% on day 25. In addition, in primary hepatocyte culture, it lowered liver thiobarbituric reactive species and it prevented DNA damage. The protective effects of CAPE on initiation have been attributed to its antigenotoxic, antioxidative, and free radical scavenging activities, though it may also be due to an ability to interfere with DEN bioactivation (Carrasco-Legleu et al., 2004, 2006).
It is not clear which of several isoforms of cytochrome P450 (CYP) participate in DEN metabolism. Isoforms proposed to be the principal bioactivators are CYP2E1 (Amelizad et al., 1988; Sook-Jeong et al., 1990; Verna et al., 1996; Yamazaki et al., 1992a,b) or CYP2B1/2 (Bellec et al., 1996; Yamazaki et al., 1992b). It is also possible that all or any of these three isoforms can bioactivate DEN (Espinosa-Aguirre et al., 1997). In fact, evidence suggests that CYP1A1/2 plays an important role in this process (Bellec et al., 1996; Yamazaki et al., 1992b). In summary, CYP2E1, CYP1A1/2, and CYP2B1/2 may be DEN bioactivators.
Experimental evidence concerning the effect of CAPE on liver CYP regulation is limited. An alcoholic extract of propolis, which contains CAPE, has been shown to reduce the enzyme activity associated with the isoforms CYP1A1/2, thereby reducing the bioactivation of carcinogens like benzo[a]pyrene. Therefore, both these CYP isoforms have been proposed as DEN bioactivators (Jen et al., 2000).
The aim of the present study was to analyze the protective effect of CAPE on liver CYP isoforms involved in DEN metabolism when administered before cancer induction in a resistant hepatocyte model of chemically induced hepatocarcinogenesis. The effects of CAPE were assessed during the first 24 h post-administration of DEN and included assays of tissue damage, total CYP levels, enzyme activity, and induction of GGT+ foci on day 25 in Fischer-344 male rats.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Animals
Male Fischer-344 rats (180–200 g) were obtained from the Unit for Production of Experimental Laboratory Animals (UPEAL-Cinvestav, México City, México). Animals had free access to food (PMI Nutrition International, Richmond, Indiana, Feeds, Inc., Laboratories Diet) and water at all times; each rat consumed approximately 12–15 g of food and 10–15 ml of water per day. After treatment, the animals were transferred to the holding room and kept under controlled conditions of 12 h light/12 h dark, 50% relative humidity, and 21°C. Animal care followed institutional guidelines for the use of laboratory animals.
Animal Treatments
Animals were subjected to three different treatments to test chemoprevention, to confirm the chemoprotective effect when DEN or 2-acethyl aminofluorine (2-AAF) is absent, and to perform microsomal assays.
Chemoprotective effect of CAPE on GGT+ foci.
Animals were treated according to the modified resistant hepatocyte model of Semple-Robert (Carrasco-Legleu et al., 2004, 2006). In the first group, rats were administered 200 mg/kg DEN ip on day 0. On days 7, 8, and 9, 20 mg/kg of 2-AAF suspended in carboxymethyl cellulose and dimethylsulfoxide (DMSO) were administered by gavage before partial hepatectomy (PH) on day 10 (n = 8). A second group was treated with a single 20 mg/kg ip dose of CAPE (kindly provided by Dr Javier Hernández-Martínez, CIAD, Hermosillo, México, with 99% of purity, obtained according to Grunberger et al., 1988) using DMSO as vehicle and a third group was treated using DMSO 12 h before administration of DEN (n = 8 for CAPE pretreatment and n = 8 for DMSO control group). All groups were sacrificed by exsanguination under ether anesthesia on day 25 after DEN administration. Livers were excised, washed in saline solution, frozen in 2-methyl butane, and stored at –80°C. Other liver sections were formalin-fixed and paraffin-embedded for histology (weight of the rats, 220–230 g; liver weight, 5–7 g).
Effect of DEN and 2-AAF on GGT+ foci.
One group received only DEN treatment (200 mg/kg) (n = 6) and the other received only 2-AAF treatment (20 mg/kg) (n = 6). Both received a PH on day 10 and were sacrificed by exsanguination under ether anesthesia on day 25. Livers were excised, washed in saline solution, frozen in 2-methyl butane, and stored at –80°C. Other liver sections were formalin-fixed and paraffin-embedded for histology (weight of the rats, 220–230 g; liver weight, 5–7 g).
Microsomal assays.
Two groups received only DEN treatment (200 mg/kg) and were sacrificed 12 and 24 h after its administration (n = 4). Another group received CAPE alone (20 mg/kg) in DMSO vehicle (n = 4) and was sacrificed 12 h after its administration. Finally, two more groups received DEN together with CAPE and were sacrificed 12 and 24 h after DEN administration (n = 4). Animals were sacrificed under ether anesthesia by perfusion with physiological solution via the portal vein and processed immediately to obtain microsomes (Mayer et al., 1990). The treatment did not affect the weight of the rats or the weight of their livers.
Histochemistry of GGT and Hematoxylin and Eosin
Liver sections of 20 µm thickness were stained for GGT activity (Rutenburg et al., 1969). Images of the GGT+ foci were captured with a digital camera (Color View 12, Soft Imaging System GmbH, Germany) and quantified with AnalySIS software (AnalySIS, Soft Imaging System GmbH). Only GGT+ areas larger than 0.01 mm2 were registered to avoid detecting background. In addition, paraffin-fixed sections of 5 µm thickness were processed for routine histological examination by staining with hematoxylin and eosin (H&E) (n = 5).
Total CYP Levels and Enzyme Activities
Total liver CYP content was measured according to Omura and Sato (1964). To detect enzyme activity of CYP1A1, microsomal O-dealkylation of 7-ethoxy-resorufin (EROD) was assayed. In contrast, O-dealkylation of 7-methoxyresorufin (MROD) was used for CYP1A2 and O-dealkylation of 7-pentoxy-resorufin (PROD) for CYP2B1/2. All these reactions were followed fluorimetrically at 37°C with excitation at 530 nm and emission at 585 nm (Burke et al., 1985; Lubet et al., 1985; Nerurkar et al., 1993). The enzyme activity of CYP2E1 was measured by p-nitrophenol hydroxylase (PNPH) assay, which detects the formation of 4-nitrocatechol by colorimetric assay at 510 nm (Reinke and Moyer, 1985) (n = 4). Protein was measured with the Bio-Rad DC Protein Assay Kit (Richmond, CA) (Lowry et al., 1951; Peterson, 1979).
Statistical Analysis
The data were analyzed using one-way ANOVA and further analyzed using Bonferroni's pairwise multiple comparison post hoc test (Klockars et al., 1995). Analyses were made using SigmaStat 3.1.1 software (Systat Software, Inc., Point Richmond, CA). In all tests, the level of significance was p < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
CAPE Treatment Reduces GGT+ Foci
GGT+ foci were considered to be preneoplastic lesions, and their number reached a maximum approximately 25 days after initiation of the carcinogenic treatment (Pérez-Carreón et al., 2006
). CAPE has been shown to reduce GGT+ foci in Wistar rats with a precise end point, which allows measurement of the chemoprotective effect in a few days (Carrasco-Legleu et al., 2004, 2006). These results have since been confirmed in male Fischer-344 rats, where CAPE reduces the induction of GGT+ foci. The only significant difference in the experimental animals, compared to the vehicle-only controls, was GGT+ area (59% in the experimental animals; Fig. 1A), but the number of foci per square centimeter was reduced too (40%; Fig. 1B). These results show that a single dose of the chemoprotector CAPE, prior to DEN administration, reduced the preneoplastic lesions in our model, probably by interfering with the effects of DEN at the initiation stage.
|
DEN Is Required to Produce GGT+ Foci
To determine whether CAPE interferes with the effects of DEN in the modified resistant hepatocyte model, the effect of omitting DEN during induction of chemical hepatocarcinogenesis was tested as in previous experiments (Tsuda et al., 1980
). We observed that administering 2-AAF alone caused 98% and 98.4% inhibition of the total number of foci induced per square centimeter and the GGT+ area, respectively, once treatment was complete. These values were higher than the corresponding results (87.4% and 89.7%) obtained when only DEN was administered (Fig. 2). The results of omitting DEN were therefore similar to the results seen when CAPE was administered before initiation. Therefore, we cannot exclude that CAPE may be blocking DEN activation.
|
CAPE Treatment Prevents Necrosis Induced by DEN
Liver cell necrosis induced by DEN plays an important role in the early stages of experimental hepatocarcinogenesis leading to the formation of preneoplastic lesions such as GGT+ foci (Ying et al., 1981
). Generalized necrosis of the liver was observed by H&E staining at 24 h after the DEN administration (Fig. 3A). With DEN + CAPE, however, the focal necrosis areas were minimal and the livers resembled those of untreated rats (Fig. 3B). These results indicate that CAPE administration reduces the toxicity of DEN—very likely by affecting its metabolism—and consequently alters DEN's activity at the initiation stage in the resistant hepatocyte model.
|
Modulation of CYP Liver Concentration and Enzyme Activities
Our first approach to analyzing the metabolism of DEN was to measure total liver CYP content; the results show that the total content was not altered by any treatment (Table 1). Next, several enzyme activities associated with the isoforms CYP1A1, CYP1A2, CYP2B1/2, and CYP2E1 were determined to assess the effect of CAPE on specific CYP isoforms involved in DEN bioactivation. Vehicle alone (DMSO) increased EROD and MROD activities by 39% and 62%, respectively, and it decreased PNPH activity by 27% compared to untreated rats. CAPE treatment, however, showed the opposite effect with respect to DMSO. By 12 h after CAPE treatment, the EROD, MROD, and PROD activities had decreased by 54%, 60%, and 67%, respectively; in contrast, CYP2E1 activity increased by 89%. Pretreatment with CAPE 12 h before DEN administration reduced EROD, MROD, and PROD activities by 64%, 65%, and 22%, respectively (Table 2). Similar results were obtained when these activities were assayed 24 h after DEN administration (data not shown). These results show that CAPE administration modifies the enzyme activity related to CYP isoforms proposed to function as DEN bioactivators, which suggests that these events are involved in CAPE chemoprotection in resistant hepatocyte model.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
During the last 20 years, the chemoprotective properties of CAPE have been studied intensively in several kinds of cancer models and several mechanisms of action have been proposed to explain its various properties (Carrasco-Legleu et al., 2004
, 2006; McEleny et al., 2004; Na et al., 2000). In the present study, we examined the chemopreventive effect of CAPE on the initiation stage in the modified resistant hepatocyte model when the compound was administered prior to DEN treatment. The main findings of this study are that CAPE reduced the induction of GGT+ foci by 59%, preventing the tissue damage that is characteristic of DEN treatment, and it modified the enzyme activity associated with CYP1A1, CYP1A2, CYP2B1/2, and CYP2E1 in a time-dependent manner.
The metabolites produced during bioactivation of DEN induce mutations in the DNA and increase oxidative stress that triggers several signaling pathways, ultimately leading to the production of initiated cells. DEN requires metabolic activation by CYP to give 
-hydroxynitrosamines, and these intermediate compounds decompose spontaneously to give acetaldehyde and mono-N-ethyl-nitrosamines, followed by ethyl-diazohydroxides and nitrogen-separated pairs. The ethyl-diazohydroxides and nitrogen-separated pairs can lead to the formation of diazoalkanes or carbocations and ultimately to the alkylation of nucleophiles, and these very reactive species are known to induce cancer in mammals. Nearly all the DEN is biotransformed within the first 12 h after its administration (Verna et al., 1996). In our modified resistant hepatocyte model, every GGT+ focus is assumed to originate from a single initiated cell that underwent neoplastic conversion. Thus, because CAPE treatment reduces the occurrence of GGT+ foci, it is likely to interfere with the initiating activity of DEN. Since both carcinogens, DEN and 2-AAF, are required to induce liver cancer, the absence of one of them results in a drastic reduction in the occurrence of GGT+ foci.
Detection of tissue damage produced by the carcinogen 24 h after its administration confirmed that CAPE interferes with cell initiation. CAPE protected the liver from necrosis, suggesting that it blocks DEN activity at the initiation stage. CAPE has previously been proposed to act as an active antioxidant scavenger of reactive species produced during bioactivation of DEN (Carrasco-Legleu et al., 2004, 2006). Several mechanisms of action have been proposed, and CAPE could in fact be acting like garlic powder, which blocks the bioactivation of carcinogens (Park et al., 2002).
CYP1A, CYP2B, and CYP2E subfamilies constitute the main groups of enzymes involved in the activation of environmental mutagens/carcinogens. These isoforms represent 11% of total CYP protein in rat liver, and all of them are involved in the metabolism of N-nitroso-dialkylamines (Bellec et al., 1996; Escobar-García et al., 2001). CYP1A1/2, CYP2B1/2, and CYP2E1 have been shown to participate in DEN bioactivation in rats (Amelizad et al., 1988; Bellec et al., 1996; Espinosa-Aguirre et al., 1997; Sook-Jeong et al., 1990; Verna et al., 1996; Yamazaki et al., 1992a,b). Our results show that CAPE decreased the enzyme activity associated with CYP1A1/2 and CYP2B1/2 12 h after administration of CAPE. The level of activity for CYP1A1 and CYP1A2 remained low until 12 h after DEN administration. Together, these results suggest that CAPE can modulate the activity of CYP isoforms involved in DEN biotransformation when it is administered both on its own and in the presence of DEN. CYPs are well-known to have distinct but often overlapping substrate specificity, and more than one CYP isoform may be involved in the metabolism of a given chemical. Therefore, if different CYPs are involved in the biotransformation of DEN, any change in the expression of CYP isoforms due to CAPE exposure may both modify the proportions of primary metabolites and alter the bioactivation of DEN and its downstream effects. Further studies are needed to characterize the hepatic CYP isoforms involved in the bioactivation of DEN.
Experimental evidence about the effect of CAPE on the regulation of liver CYP isoforms is very limited. The present study is the first report of the effects of CAPE treatment on the regulation of CYP1A1/2, CYP2B1/2, and CYP2E1 in the liver. An alcoholic extract of propolis containing a very high level of CAPE has been reported to decrease the enzyme activity of CYP1A1 and CYP1A2 and, consequently, reduce carcinogen bioactivation (Jen et al., 2000). In the same way, dietary garlic powder has been shown to act as an anticarcinogen by modifying the expression of CYP2E1 (Park et al., 2002). Bicyclol has been shown to act as an anticarcinogen by increasing CYP2B1 enzyme activity and, in particular, enhancing the denitrosation of DEN (Zhu et al., 2006). Therefore, the ability of CAPE to modify the expression of several CYP isoforms evidences an additional mechanism for explaining the chemopreventive activity of CAPE in the initiation stage of chemical carcinogenesis.
It is an oversimplification to hypothesize that only one CYP isoform participates in a given reaction in vivo. DEN is a carcinogen used in several models, but it is not clear which of several CYP isoforms participate in its bioactivation in our model in particular. The measurements of enzymatic activity reported here are insufficient to conclude definitively that the chemoprotective activity of CAPE is related to CYP. Nevertheless, our results strongly suggest that CAPE acts through the inhibition of carcinogen metabolism, and they warrant further study in search of a definitive answer.
In conclusion, CAPE may modify the enzyme activity of CYP isoforms involved in DEN activation. The modification of CYP-dependent metabolism in the liver may constitute an alternative mechanism for understanding CAPE's chemoprotective effect in a hepatocarcinogenesis model.
|
FUNDING |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
CONACYT (39525-M); a fellowship from CONACYT (OBR 185604).
|
ACKNOWLEDGMENTS |
|---|
We would like to acknowledge the excellent animal technical support of UPEAL-Cinvestav from M.V.Z María Antonieta López-López, Rafael Leyva-Muñoz, Manuel Flores-Cano, Ricardo Gaxiola-Centeno, and UPEAL Chairman Dr. Jorge Fernández. We are deeply grateful to Dr Javier Henández-Martínez (CIAD, Hermosillo, México) for providing CAPE and to Dr Arnulfo Albores-Medina in his laboratory for providing valuable technical support. Part of this work was presented orally in a lecture entitled "Modulation of carcinogenesis by naturally occurring polyphenolic compounds" during the plenary session of the 45th SOT Annual Meeting and ToxExpo in San Diego, CA, USA (5–9 March 2006).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Amelizad Z, Appel KE, Oesch F, Hildebrandt AG. Effect of antibodies against cytochromes P-450 on demethylation and denitrosation of N-nitrosodimethylamine and N-nitrosodimethylaniline. J. Cancer Res. Clin. Oncol. (1988) 144:380–384.
Bankova V, Dyulgerov A, Popov S, Marekov N. A GC/MS study of the propolis phenolic constituents. Z. Naturforsch. C. (1987) 42:147–151.
Bellec G, Goasduff T, Dreano Y, Menez JM, Berthou F. Effect of the length of alkyl chain on the cytochrome P450 dependent metabolism of N-dialkylnitrosamines. Cancer Lett. (1996) 100:115–123.[CrossRef][Web of Science][Medline]
Burke MD, Thompson S, Elcombe CR, Halpert J, Haaparanta T, Mayer RT. Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochem. Pharmacol. (1985) 34:3337–3345.[CrossRef][Web of Science][Medline]
Carrasco-Legleu CE, Márquez-Rosado L, Fattel-Fazenda S, Arce-Popoca E, Pérez-Carreón JI, Villa-Treviño S. Chemoprotective effect of caffeic acid phenethyl ester on promotion in a medium-term rat hepatocarcinogenesis assay. Int. J. Cancer (2004) 108:488–492.[CrossRef][Web of Science][Medline]
Carrasco-Legleu CE, Sánchez-Pérez Y, Márquez-Rosado L, Fattel-Fazenda S, Arce-Popoca E, Hernández-García S, Villa-Treviño S. A single dose of caffeic acid phenethyl ester prevents initiation in a medium-term rat hepatocarcinogenesis model. World J. Gastroenterol. (2006) 12:6779–6785.[Web of Science][Medline]
Escobar-García D, Camacho-Carranza R, Pérez I, Dorado V, Arriaga-Alba M, Espinosa-Aguirre JJ. S9 induction by combined treatment with cyclohexanol and albendazole. Mutagenesis (2001) 16:523–528.
Espinosa-Aguirre JJ, Rubio J, López I, Nosti R, Asteinza J. Characterization of the CYP isozyme profile induced by cyclohexanol. Mutagenesis (1997) 12:159–162.
Grunberger D, Naberjee R, Eisinger K, Oltz EM, Efros L, Caldwell M, Estevez V, Nakanisgi K. Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis. Experientia (1988) 44:230–232.[CrossRef][Web of Science][Medline]
Jen SN, Shih MK, Kao CM, Liu TZ, Chen SC. Antimutagenicity of ethanol extracts of bee glue against environmental mutagens. Food Chem. Toxicol. (2000) 38:893–897.[CrossRef][Web of Science][Medline]
Klaunig JE, Kamendulis LM. The role of oxidative stress in carcinogenesis. Annu. Rev. Pharmacol. Toxicol. (2004) 44:239–267.[CrossRef][Web of Science][Medline]
Klockars AJ, Hancock GR, McAweeney MJ. Power of unweighted and weighted versions of simultaneous and sequential multiple comparison procedures. Psychol. Bull. (1995) 118:300–307.[CrossRef][Web of Science]
Lowry OH, Rosebroug NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem. (1951) 193:265–275.
Lubet RA, Mayer RT, Cameron JW, Nims RW, Burke MD, Wolff T, Guengerich FP. Dealkylation of pentoxyresorufin: a rapid and sensitive assay for measuring induction of cytochromes(s) P-450 by phenobarbital and other xenobiotics in the rat. Arch. Biochem. Biophys. (1985) 238:43–48.[CrossRef][Web of Science][Medline]
Mayer RT, Netter KJ, Heubel F, Hahnemann B, Buchheister A, Mayer GK, Burke MD. 7-Alkoxyquinolines: new fluorescent substrates for cytochrome P450 monooxygenases. Biochem. Pharmacol. (1990) 40:1645–1655.[CrossRef][Web of Science][Medline]
McEleny K, Coffey R, Morrissey C, Fitzpatrick JM, Watson RWG. Caffeic acid phenethyl ester-induced PC-3 cell apoptosis is caspase-dependent and mediated through the loss of inhibitors of apoptosis proteins. BJU Int. (2004) 94:402–406.[CrossRef][Web of Science][Medline]
Michaluart P, Masferrer JL, Carothers AM, Subbaramaiah K, Zweifel BS, Koboldt C, Mestre JR, Grunberger D, Sacks PG, Tanabe T, et al. Inhibitory effects of caffeic acid phenethyl ester on the activity and expression of cyclooxygenase-2 in human oral epithelial cells and in a rat model of inflammation. Cancer Res. (1999) 59:2347–2352.
Na H, Wilson MR, Kangb K, Changa C, Grunbergerc D, Trosko JE. Restoration of gap junctional intercellular communication by caffeic acid phenethyl ester (CAPE) in a ras-transformed rat liver epithelial cell line. Cancer Lett. (2000) 157:31–38.[CrossRef][Web of Science][Medline]
Nerurkar PV, Park SS, Thomas PE, Nims RW, Lubet RA. Methoxyresorufin and Benzyloxy substrates preferentially metabolized by cytochromes P4501A2 and 2B respectively in the rat and mouse. Biochem. Pharmacol. (1993) 46:933–943.[CrossRef][Web of Science][Medline]
Oktem F, Ozguner F, Sulak O, Olgar S, Akturk O, Yilmaz HR, Altuntas I. Lithium-induced renal toxicity in rats: protection by a novel antioxidant caffeic acid phenethyl ester. Mol. Cell Biochem. (2005) 277:109–115.[CrossRef][Web of Science][Medline]
Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes. J. Biol. Chem. (1964) 239:2370–2378.
Parikh S, Hyman D. Hepatocellular cancer: a guide for the internist. Am. J. Med. (2007) 120:194–202.[CrossRef][Web of Science][Medline]
Park KA, Kweon S, Choi H. Anticarcinogenic effect and modification of cytochrome P450 2E1 by dietary garlic powder in diethylnitrosamine-initiated rat hepatocarcinogenesis. J. Biochem. Mol Biol. (2002) 35:615–622.[Web of Science][Medline]
Pérez-Carreón JI, López-García C, Fattel-Fazenda S, Arce-Popoca E, Alemán-Lazarini L, Hernández-García S, Le Berre V, Sokol S, Francois JM, Villa-Treviño S. Gene expression profile related to the progression of preneoplastic nodules toward hepatocellular carcinoma in rats. Neoplasia (2006) 8:373–383.[CrossRef][Web of Science][Medline]
Peterson GL. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal. Biochem. (1979) 100:201–220.[CrossRef][Web of Science][Medline]
Reinke LA, Moyer MJ. p-Nitrophenol hydroxylation. A microsomal oxidation which is highly inducible by ethanol. Drug Metab. Dispos. (1985) 13:548–552.[Abstract]
Rutenburg AM, Kim H, Fischbein JW, Hanker JS, Waserkrug HL, Seligman AM. Histochemical and ultrastructural demonstration of gamma-glutamyl transpeptidase activity. J. Histochem. Cytochem. (1969) 17:517–526.[Abstract]
Song YS, Park EH, Hur GM, Ryu YS, Lee YS, Lee JY, Kim YM, Jin C. Caffeic acid phenethyl ester inhibits nitric oxide synthase gene expression and enzyme activity. Cancer Lett. (2002) 175:53–61.[CrossRef][Web of Science][Medline]
Sook-Jeong HY, Ishizaki H, Yang CS. Roles of cytochrome P450IIE1 in the dealkylation and denitrosation of N-nitrosodimethylamine in rat liver microsomes. Carcinogenesis (1990) 11:2239–2243.
Sud`ina GF, Mirzoeva OK, Pushkareva MA, Korshunova GA, Sumbatyan NV, Varfolomeev SD. Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett. (1993) 329:21–24.[CrossRef][Web of Science][Medline]
Tsuda H, Lee G, Farber E. Induction of resistant hepatocytes as a new principle for a possible short-term in vivo test for carcinogens. Cancer Res. (1980) 40:1157–1164.
Verna L, Whysner J, Williams M. N-Nitrosodiethylamine mechanistic data and risk assessment: bioactivation, DNA-adduct formation, mutagenicity, and tumor initiation. Pharmacol. Ther. (1996) 71:57–81.[CrossRef][Web of Science][Medline]
Yamazaki H, Inui Y, Guenguerich FP, Shimada T. Cytochrome P450 2E1 and 2A6 enzymes as a major catalyst for metabolic activation of N-nitrosodialquilamines and tobacco-related nitrosamines in human liver microsomes. Carcinogenesis (1992a) 13:1789–1794.
Yamazaki H, Oda Y, Funae Y, Imaoka S, Inui Y, Guenguerich FP, Shimada T. Participation of rat liver cytochrome P450 2E1 in the activation of N-nitrosodimethylamine and N-nitrosodiethylamine to products genotoxic in an acetyltransferase-overexpressing Salmonella thyphimurium strain (NM2009). Carcinogenesis (1992) 13:979–985.
Ying TS, Sarma DSR, Farber E. Role of acute hepatic necrosis in the induction of early steps in liver carcinogenesis by diethylnitrosamine. Cancer Res. (1981) 41:2096–2102.
Zhu B, Liu GT, Wu RS, Strada SJ. Chemoprevention of bicyclol against hepatic preneoplastic lesions. Cancer Biol. Ther. (2006) 5:1665–1673.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||