Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2001-12-31
2004-10-12
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S029000
Reexamination Certificate
active
06803206
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for identifying an endocrine disruptor using a cell line having aromatase activity, particularly, a human granulosa-like tumor cell line; and a kit for carrying out the method.
BACKGROUND OF THE INVENTION
The use of tributyltin (TBT) as a biocide in antifouling paints and wood preservatives leads to the contamination of the marine and freshwater environment with TBT. TBT has been shown to be highly toxic to a number of aquatic animals. Especially in marine prosobranch snails, TBT induces reproductive abnormalities and sterilization in female animals. This phenomenon, which has been called either pseudohermaphroditism or imposex, is characterized by the development of male sex organs (penis and/or vas deferens and prostate tissue) on females (Bryan et al,
J. Mar. Biol. Ass. UK,
66:611-640 (1986); and Gibbs et al,
J. Mar. Biol. Ass. UK,
66:767-777 (1986)). Not only TBT, but also triphenyltin (TPT) has been shown to have a strong effect on the development of imposex in the rock shell,
Thais clavigera
(Horiguchi et al,
Environ. Pollution,
95:85-91 (1997)). However, the detailed biochemical mechanism of this phenomenon remains obscure. The involvement of sex steroids in the expression of imposex in marine neogastropods has been suggested based on the fact that pure female displays the lowest testosterone content, whereas advanced imposex stages have the highest testosterone content (Bettin et al,
Helgolander Meeresunters,
50:299-317 (1996)), as well as the fact that the TBT-induced imposex is completely suppressed by antiandrogen cyproterone acetate, which is a competitive inhibitor of androgen receptors (Bettin et al, supra). TBT-induced imposex is mediated by an increasing androgen levels relative to estrogen levels, thus suggesting a decreased conversion of androgens to estrogens, i.e., aromatization (Bettin et al, supra; and Spooner et al,
Mar. Environ. Res.,
32:37-49 (1991)). This has been further supported by the fact that a specific aromatase inhibitor, SH489, exhibited the same imposex-inducing effect in neogatropods as did TBT-exposure (Bettin et al, supra). On the other hand, there is a controversial report which demonstrated no decrease in the activity of aromatase in gastropods, which were contaminated and exhibited clear evidence of imposex (Morcillo et al,
Environ. Res.,
81:349-354 (1999)). There is yet no clear direct in vitro evidence demonstrating that TBT compounds truly affect aromatase activity in any species, including marine species, as well as in humans.
A low dose of Dioxin or PCB, is known to cause problems in sperm formation in males that affects rates of hypospadias and cryptorchism. An orchioncus rise results which is passed to the next generation. Further, it is known that the pesticide Benomyl, can cause man sterility.
Thus, long-term and excessive exposure to endocrine disruptors in humans causes clinical problems, which relate to excessive androgens and/or reduced estrogens. Clinical problems in women include ovarian dysfunction, osteoporosis and hirsutism. Clinical problems in men include sterility. Hence, it is important to be able to identify endocrine disruptors so that exposure to the same can be eliminated.
A steroidogenic human ovarian granulosa-like tumor cell line, KGN, from a patient with invasive granulosa cell carcinoma has been established (Nishi et al,
Endocrinol.,
142:437-445 (2001); which is incorporated by reference herein in its entirety). The cell line possesses properties very similar to normal ovarian granulosa cells, including the expression of functional FSH receptor and a relatively high aromatase activity.
The KGN cell line was found in the present invention to be a useful model for investigating the in vitro effects of various compounds on aromatase activity in the mammalian system, and thus useful in identifying compounds which are effective as endocrine disruptors.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a cell line useful for investigating the effects of compounds on aromatic activity.
Another object of the present invention is to provide a method for identifying an endocrine disruptor.
Still another object of the present invention is to provide a kit for carrying out said method.
These and other objects of the present invention, which will be apparent from the detailed description of the present invention provided hereinafter, have been met in one embodiment by a method for identifying an endocrine disruptor comprising the steps of:
(a) incubating a cell line having aromatase activity with a test compound;
(b) assaying for inhibition or activation of aromatase activity due to said test compound so as to identify an endocrine disruptor.
REFERENCES:
Kitawaki et al., “Growth Suppression of MCF-7 Human Breast Cancer Cells by Aromatase Inhibitors: A New System for Aromatase Inhibitor Screening” (1993) J. Steroid Biochem. Molec. Biol., 44(4-6), 667-670.*
Powlin et al., “Ex Vivo and In Vitro Testis and Ovary Explants: Utility for Identifying Steroid Biosynthesis Inhibitors and Comparison to a Tier I Screening Battery” (1998) Toxicological Sciences, vol. 46, 61-74, Abstract.*
Stresser et al., “A High-Throughput Screen to Identify Inhibitors of Aromatase (CYP19)” (Sep. 10, 2000) Anal. Biochem. 284(2), 427-430.*
Okubo et al., “Down-Regulation of Promoter 1.3 Activity of the Human Aromatase Gene in Breast Tissue by Zinc-finger Protein, Snail (SnaH)” (Feb. 15, 2001) Cancer Research, 61, 1338-1346.*
Yang et al., “Two Organochlorine Pesticides, Toxaphene and Chlordane, Are Antagonists for Estrogen-related Receptor-1 Orphan Receptor” (Sep. 15, 1999) Cancer Research, 59, 4519-4524.*
Almstrup et al. “Dual Effects of Phytoestrogens Result in U-Shaped Dose-Response Curves” (Jun. 2002) Environ. Health Perspect., vol. 110, pp. 743-748*
Zacharewski, T. “Identification and Assessment of Endocrine Disruptors: Limitations of in Vivo and in Vitro Assays” (Apr. 1998) Environ. Health Perspect. 106(Suppl 2), 577-582.*
Mak et al., “A Yeast Screen System for Aromatase Inhibitors and Ligands for Androgen Receptor: Yeast Cells Transformed with Aromatase and Androgen Receptor” (1999) Environ. Health Perspect., 107, 855-860.*
Chen et al., “Breast Tumor Aromatase: Functional Role and Transcriptional Regulation” (1999) Endocrine-Related Cancer, 6, 149-156.*
Saitoh et al,Biochem. Biophys. Res. Comm., 289:198-204 (2001).
Nishi et al,Endocrinology , 142(1):437-445 (2001).
KGN Cell Line (http://www.ric.riken.go.jp/cgi-bin/CELL/cell.p1? DATA=314).
RGN Animal Cell Line Catalog, General Catalog No. 5-1, p. 158 (May 2001).
Mu et al,Mol. Cell. Endrocrin., 181:239-248 (2001).
Mu et al,Endocrinology, 142(8):3590-3597 (2001).
Mu et al,Biochem. Biophys. Res. Comm., 271:710-713 (2000).
Nawata et al,J. Steroid Biochem. Molec. Biol., 53(1-6):165-174 (1995).
Rainey et al,J. of Clin. Endocrin. Metabol., 78(3):705-710 (1994).
Nawata et al,76thAnnual Meeting Program and Abstracts of the Annual Meeting-Endocrine Society, “Aromatase Activity in Human Osteoblast-Like Osteosarcoma Cell”, No. 1195 (1994) title only.
Tanaka et al,Calcif Tissue Int., 52:107-109 (1993).
Steinkampf et al,Mol. Endocrinology, 1(7):465-471 (1987).
Simms et al,Cancer Res., 40:4356-4363 (1980).
Nawata, “Effects of Endocrine Disruptors on Aromatase Activity in the Human Ovary Granulosa-like Cell Line KGN”, Endocrine Disruptors The First Symposium on Each Category, Lecture Summary (Sep. 19, 2002), partial english transl.
Nawata Hajime
Yanase Toshihiko
Otsuka Pharmaceutical Co. Ltd.
Weber Jon P.
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