Microsoft word - anschubprojekt_2004.doc

Projektskizze

1. Projektbezeichnung

Evaluation of phytochemicals for CYP1B1 inhibition and their application 2. Kurztitel CYP1B1 Inhibition Screening
3. Zusammenfassung
Beside CYP1A1, CYP1B1 is the major human CYP enzyme involved in the metabolic activation of environmental pollutants, such as polycyclic aromatic hydrocarbons including benzo[a]pyrene, as well as endogenous substrates, such as estrogens, to ultimate carcinogens. CYP1B1 displays the highest level of expression in breast tissues and is there the main estradiol hydroxylase. Because metabolic activation of estrogens to their 4-hydroxy form (catechol estrogens) by CYP1B1 has been postulated to be a major factor in mammary carcinogenesis, selective inhibition of CYP1B1 may be an attractive mechanism for cancer prevention. We plan to investigate inhibition by a number of phytochemicals of recombinant CYP1B1 in vitro using microsomes prepared from insect cells expressing both CYP1B1 and NADPH-cytochrome P450 reductase. To identify and evaluate potent and selective inhibition of CYP1B1, IC50 values for inhibition of CYP1B1-catalyzed estradiol and benzo[a]pyrene activation will be determined for a series of about 30 natural plant-derived polyphenols (components of diet and phytopharmaka). Finally, to examine whether inherited variants of CYP1B1 differ from wild-type CYP1B1 in carcinogen activation activity, polymorphic variants of CYP1B1 will be expressed and functionally characterized. For most potent inhibitors the role of CYP1B1 polymorphisms will be determined to explain possible interindividual differences in (breast) cancer risk. 4. Stand der Forschung und eigene Vorarbeiten
Many chemical carcinogens (both endogenous and xenobiotic) are unreactive and require metabolic activation by drug-metabolizing enzymes, such as cytochrome P450 and epoxide hydrolase, to exert their carcinogenic or mutagenic effects. The most important CYPs for the activation of procarcinogens are CYP1A1, 1A2, 1B1, 2C9/19, 2E1, and 3A4 with CYP1A1 and CYP1B1 showing the highest activities (Shimada et al., 2001). Both latter enzymes are mainly extrahepatically expressed and they can be induced by environmental toxicants (e.g. polychlo-rinated biphenyls such as 2,3,7,8-tetrachlorodibenzo-p-dioxin). Whereas CYP1A1 is predomi-nantly expressed in the lung, gastro-intestinal tract, placenta, and brain, CYP1B1 is mainly found in steroidogenic tissues such as breast, uterus, and prostate. The latter is the major estradiol hydroxylase and activation of estrogens has been postulated to be a major factor in mammary carcinogenesis (Guengerich et al., 2003). In most cases, estrogens are hydroxylated in several positions, but 4-hydroxylation is the major metabolic path leading to tumorigenesis. Several reports suggested that the 4-OH catechol estrogens are most important because they are oxidized to quinones which represent electrophilic metabolites and are capable of forming DNA adducts (Han and Liehr, 1994); it was also shown that they are able to induce endometrial adeno-carcinoma in mice (Newbold and Liehr, 2000). Because of the postulated significant role of CYP1B1 in the carcinogenicity of estradiol, selective and potent inhibition of CYP1B1 may prevent formation of mammary tumors. Several agents have been already studied to determine a possible role as a cancer chemo-
preventive agent, such as α-naphthoflavone, pyrene, homoeriodictyol, hydroxystilbenes obtained
from natural sources (resveratrol, rhapontigenin, ) and synthetic derivatives of stilbenes such as
2,4,3´,5´-tetramethoxystilbene (Chun et al., 2001). However, except the latter stilbene derivative,
they did not show selectivity for inhibiting of CYP1B1 compared to CYP1A1 or CYP1A2.
Work in the applicants lab (within a 5-years DFG-supported project, RO1287/1-3) led to the
establishment of an efficient expression system for polymorphic human CYP enzymes which we
hope can be successfully applied to the expression of CYP1B1, too (Chernogolov et al., 2003).
Based on this, we developed a novel in vitro system to evaluate the potency of CYP1A1
inhibitors for their application in chemopreventive cancer strategies (Schwarz et al., 2003;
Biochem. Biophys. Res. Commun.). Recently, we found that St. John´s Wort, a herbal extract
widely used in depression, and several of its major constituents are extraordinary strong
inhibitors of carcinogen activation (Schwarz et al., 2003 [Cancer Res.]). These discoveries led to
a patent application suggesting use of St. John´s wort extracts, hypericin and other constituents
in chemoprevention strategies and as components in functional food. Lastly, we first time could
demonstrate genotype-dependent inhibition of carcinogen activation by CYP1A1, with the
consequence that carriers of the alleles CYP1A1*2 and *4 may be at higher risk of developing
lung cancer (Schwarz et al., 2003 [J. Natl. Cancer Inst.]). Based on this expertise, the planned
project is aimed to continue these studies by inclusion of CYP1B1 as target for inhibition in
anticarcinogenesis strategies. Studies will be performed for a number of dietary and medicinal
constituents to evaluate their mechanism of action and chemopreventive potential.
The following recent publications relevant to the project prove the expertise of the applicant:
- A. CHERNOGOLOV, J. BEHLKE, W.-H. SCHUNCK, I. ROOTS, D. SCHWARZ (2003)
Human CYP1A1 allelic variants: Baculovirus expression and purification, hydrodynamic,
spectral and catalytical properties and their potency in the formation of all-trans-retinoic acid.
Prot. Expr. Purif. 28, 259-269.
- D. SCHWARZ, I. ROOTS (2003) In vitro assessment of inhibition by natural polyphenols of
metabolic activation of procarcinogens by human CYP1A1. BBRC 303, 902-907.
- D. SCHWARZ, P. KISSELEV, I. ROOTS (2003) St. John´s wort extracts and some of their
constituents potently inhibit ultimate carcinogen formation from benzo[a]pyrene-7,8-dihydrodiol
by human CYP1A1. Cancer Res. 63, 8062-8068.
- D. SCHWARZ, P. KISSELEV, I. ROOTS (2003) CYP1A1 genotype-selective inhibition of
benzo[a]pyrene activation by quercetin. J. Natl. Cancer Inst. (submitted).
Further preparatory work and preliminary studies related to the project include:
- Installation and validation of assays for the analysis of estradiol and estrogen metabolism by
reversed-phase HPLC
- Development and optimization of coexpression methods to prepare enzymatically active CYP
systems by coinfection of insect cells with both recombinant CYP and P450 reductase viruses
(Schwarz et al., 2001 [Xenobiotica]; Schwarz et al., 2001 [Carcinogenesis])
5.1 Bisherige bzw. beantragte Förderung des Projektes bzw. von themenverwandten
Teilprojekten im Rahmen der haushaltsfinanzierten Forschungsförderung

Projektkurztitel
Gesamtfördersumme Förderzeitraum CYP1B1 Inhibition Screening Dr. Schwarz, Dieter
5.2 Drittmittelperspektive

Angestrebte Drittmittelförderung: DFG
Vorgesehener Termin Drittmittelantragstellung: 11/2004
6. Ziele

The aim of the project is the assessment of the inhibitory capacity of a number of phyto-
chemicals in the metabolic activation of estrogens by human CYP1B1 to their carcinogenic
metabolites and the analysis of a possible role of polymorphisms of the CYP1B1 gene in the
inhibition process. Therefore, it will be necessary
(i) to develop an efficient expression system for human CYP1B1 and its polymorphic variants
based on baculovirus/insect cells,
(ii) to characterize catalytic activity of CYP1B1 in cancer-related hydroxylation reactions using
the prototype estrogen estradiol as substrate,
(iii) to determine IC50 values for a quantitative evaluation of the inhibitory capacity of a number
of
phytochemicals, and
(iv) to characterize the isozyme selectivity of inhibition by examination of inhibition of
CYP1A1-
and CYP1A2-catalyzed reactions.
Finally, for selected phytochemicals with highest inhibitory capacity the role of CYP1B1
polymorphisms in the inhibition will be characterized with the aim to contribute to a better
understanding of interindividual differences in cancer risk and to develop individualized
prevention strategies.
7. Arbeitsprogramm
Permanent work: Insect cell culture for the expression of CYP1B1 and P450 reductase.
Based on microsomes prepared from insect cells coexpressing CYP1B1 and P450 reductase the
influence of a number of phytochemicals on the CYP1B1-catalyzed epoxidation of 7,8-
dihydrodiol-BaP and hydroxylation of 17ß-estradiol will be studied. We use enzymatic assays
based on HPLC analysis already established in the lab: RP-HPLC with UV/VIS detection of
metabolites for 7,8-diol-BaP epoxidation and RP-HPLC with radioactive monitoring of
metabolites for estradiol hydroxylation using 14C-labeled estradiol (Schwarz et al., 2001).
Besides determining IC50 values of CYP1B1 inhibition for all phytochemicals, an analysis of the
role of polymorphisms in inhibition is planned for selected (most potent) inhibitors.
In detail, the program includes the following work:
1. Expression of CYP1B1 in Sf9 insect cells
- Plasmid construction, preparation of recombinant baculoviruses, expression of CYP1B1
and its characterization by CO difference spectroscopy and immunoblotting
- Coexpression of CYP1B1 and P450 reductase and its characterization by CO difference
spectroscopy, protein determination, and immunoblotting
- Preparation and characterization of catalytically active microsomal systems (optimization
of CYP/reductase stoichiometry, cytochrome b5 addition, protein concentration, etc.)
2. Measurements of the catalytic activity of CYP1B1 in dependence on the polyphenol
(inhibitor)
concentration:
- Hydroxylation of 17ß-estradiol by CYP1B1 and determination of IC50 values for the
selected phytochemicals listed below (see enclosure) (this is a preliminary list which will
be
actualized according to progress in this rapidly developing area of research)
- Depending on the data obtained, investigation of inhibition of epoxidation of 7,8-diol-BaP
by CYP1B1 for most potent inhibitors
- Analysis of selectivity of inhibition for selected phytochemicals by comparison of activity
in
CYP1A1- and CYP1A2-catalyzed reactions
3. Role of polymorphisms of the CYP1B1 gene:
- Construction of plasmids by site-directed mutagenesis for the generation of variant
CYP1B1 cDNAs encoding the substitutions Arg48Gly/Ala119Ser (CYP1B1.2), Leu432Val
(CYP1B1.3), and Asn453Val (CYP1B1.4)
- Expression of the polymorphic variants of CYP1B1
- Coexpression of the variants with P450 reductase and functional characterization of
"coexpressed microsomes"
- Analysis of the role of CYP1B1 polymorphisms in the inhibition by investigation of
estradiol activation by the different CYP1B1 variants
8. Voraussetzungen für die Durchführung des Vorhabens

Zusammenarbeit mit anderen Wissenschaftlern
- Dr. W.-H. Schunck (Max-Delbrück-Center for Molecular medicine, Berlin-Buch (MDC)):
Cooperation in enzymatic assays using radioactive substrates (existing successful cooperation
over the last years)
- Prof. Dr. P. Kisselev (Institute Bioorganic Chemistry, Academy of Sciences, Minsk,
Weißrußland): Cooperation in protein preparation and enzymatic analyses
Apparative Voraussetzungen und investive Mittel
All equipment and methods for insect cell culture, microsome preparation, enzymatic
measurements and analysis are established at the Institute of Clinical Pharmacology, Charite-
University Medicine Berlin and/or at MDC (AG Dr. Schunck).
Investment costs are not planned.
9. Förderdauer Regelförderzeit: 2004 (ein Jahr)
10. Beantragte Mittel und Begründung Gesamt: 11.500,- €.
Die Begründung für die einzelnen Positionen Verbrauchsmaterial, Reisekosten und sonstige
Kosten geht aus der Spezifikation im Abschnitt III.7 des Deckblattes hervor.
Literatur:
Chun, Y.-J., Kim, S., Kim, D., Lee, S.-K., and Guengerich, F.P. (2001) A new selective and potent inhibitor of human cytochrome P450 1B1 and its application to antimutagenesis. Cancer Res. 61, 8164-8170. Guengerich, F.P., Chun, Y.-J., Kim, D., Gillam, E.M.J., and Shimada, T. (2003) Cytochrome P450 1B1: a target for inhibition in anticarcinogenesis strategies. Mutation Res. 523-524, 173-182. Han, X., and Liehr, J.G. (1994) DNA single-strand breaks in kidneys of Syrian hamsters treated with steroidal estrogens: hormone-induced free radical damage preceding renal malignancy.
Carcinogenesis 15, 997-1000)
Newbold, R.R., and Liehr, J.G. (2000) Induction of uterine adenocarcinoma in CD-1 mice by catechol
estrogens. Cancer Res. 60, 235-237.
Schwarz, D., Kisselev, P., Cascorbi, I., Schunck, W.-H., und Roots, I. (2001) Differential metabolism of
benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 variants. Carcinogenesis 22
453-459.
Schwarz, D., Kisselev, P., Honeck, H., Cascorbi, I., Schunck, W.-H., and Roots, I. (2001) Coexpression
of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase
in the baculovirus/insect cell system. Xenobiotica 31, 345-356.
Shimada, T., Oda, Y., Gillam, E.M.J., Guengerich, F.P., and Inoue, K. (2001) Metabolic activation of
polycyclic aromatic hydrocarbons and other procarcinogens by cytochrome P450 1A1 and P450 1B1
allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009. Drug
Metab. Dispos. 29, 1176-1182.



I. Gentechnische Sicherheit

Die Genehmigung für die Durchführung gentechnischer Arbeiten (S1) liegt vor:
II. Kopie einer eigenen Publikation
D. SCHWARZ, P. KISSELEV, I. ROOTS (2003)
St. John´s wort extracts and some of their constituents potently inhibit ultimate carcinogen
formation from benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1.
Cancer Res. 63, 8062-8068.

III. List of selected phytochemicals planned to be studied



III. List of selected phytochemicals planned to be studied
COMPOUND
CONSTITUENT OF
Hydroxystilbenes
herbal extracts, Rheum undulatum Linne Flavonoles
ubiquitous in all plants (fruits, herbs, vegetables, Polyphenoles
Carotenoids
(St. John´s Wort, Johanniskraut-Extrakt) Hyperforin Phloroglucinoles I3,II8-biapigenin Biflavones Hyperoside Flavonoids Chlorogenic acid Phenolic acids
Isoflavones
soy beans, soy products, Leguminosae (Phytoestrogenes)
Brassinosteroids
28-homobrassinol. Benzyl isothiocyan. Isothiocyanates Indol-3-carbinol

Source: http://forschungsberichte.charite.de/FOB_2003-2005/english/PJ/PJ15051Anschubprojekt_2004.pdf