Characterization of Brain and Liver Aromatase of Gilthead Seabream (Sparus aurata L., 1758) using Dibenzylfluorescein

Öz

Aromatase catalyzes the conversion of androgens into estrogens. Data on native aromatase enzyme kinetics and thus actual catalytic activity are scarce in fish, impeding comparison of aromatase activity (AA) from different organs within and between species. In the present study, fluorescence aromatase assay was optimized to measure AA in the gilthead seabream (Sparus aurata L., 1758) using dibenzylfluorescein (DBF) as a fluorometric substrate in brain and liver microsomes. Brain and liver AA have showed linearity until 20 and 40 µg protein concentration throughout 30 minutes, respectively. In brain and liver optimum pH of the enzyme was found to be 6.50 and 8.25, respectively and optimum temperature was found to be 30°C for both tissues. It has been observed that brain and liver AA have saturated at and above 2 µM DBF concentrations. Determined Vmax and Km values of brain and liver aromatase using LineweaverBurk graph have calculated 8,054±0,550 and 8,389±0,543 pmol/min/mg protein and 0,840±0,161 µM and 0,959±0,152 µM, respectively. Testosterone appeared to competitively inhibit the Sparus aurata brain and liver aromatase. In conclusion, the parameters of this assay that are reported for brain and liver aromatase in gilthead seabream could be useful to measure AA in other species

Anahtar Kelimeler

• Aromatase
• Gilthead seabream
• Brain
• Liver
• Dibenzylfluorescein

Çipura (Sparus aurata L., 1758) Beyin ve Karaciğer Aromataz Enziminin Dibenzilfloresein Kullanılarak Karakterize Edilmesi

Öz

Aromataz, androjenlerin ösrojenlere dönüşümünü katalizlemektedir. Doğal aromatazın katalitik aktivitesi ve enzim kinetikleri hakkında bilgi balıklarda çok azdır ve bu tür içi ve türler arasında farklı organlardan elde edilen aromataz aktivitesinin (AA) karşılaştırılmasını engellemektedir. Bu çalışmada, floresans aromataz metodu, florometrik bir substrat olan dibenzilfloresein (DBF)
kullanılarak çipura (Sparus aurata L., 1758) beyin ve karaciğer mikrozomlarında AA’ni ölçmek için karakterize edilmiştir. Beyin ve karaciğer AA 30 dak reaksiyon zamanı boyunca sırasıyla 20 μg ve 40 μg protein miktarına kadar doğrusallık göstermiştir. Beyin ve karaciğerde enzimin optimum pH’sı sırasıyla 6,50 ve 8,25 olarak ve optimum sıcaklığı her iki doku için 30 ˚C olarak bulunmuştur. Beyin ve karaciğer AA’inin 2 µM DBF konsantrasyonu üzerinde doyuma ulaştığı gözlenmiştir. Lineweaver-Burk grafiği kullanılarak yapılan saptamalarda beyin ve karaciğer aromataz enzimi için Vmax ve Km değerleri belirlemişi ve sırasıyla 8,054±0,550 pmol/dak/mg protein, 8,389±0,543 pmol/dak/mg protein ve 0,840±0,161 µM, 0,959±0,152 µM olarak hesaplanmıştır. Testosteronoun Sparus aurata beyin ve karaciğer aromataz enzimini yarışmalı bir şekilde inhibe ettiği gözlenmiştir. Çipurada beyin ve karaciğer aromatazı için ilk defa rapor edilmiş bu metodun parametreleri diğer türlerde de AA’ni ölçmek için faydalı olabilecektir.

Anahtar Kelimeler

• Aromataz
• Çipura
• Beyin
• Karaciğer
• Dibenzilfloresein

Kaynakça

1. Adlercreutz, H., Bannwart, C., Wahala, K., Makela, T., Brunow, G., Hase, T., Arosemena, P.J., Kellis Jr., J.T., Vickery, L.E. (1994). Inhibition of human aromatase by
2. phytoestrogens. The Journal of Steroid Biochemistry and Molecular Biology, 44, 147–153.
3. isoflavonoid Balthazart, J., Ball, G.F. (1998). New insights into the regulation and function of brain estrogen synthase (aromatase). Trends in Neurosciences, 21, 243–249.
4. Blazquez, M., Piferrer, F. (2004). Cloning, sequence analysis, tissue distribution, and sex-specific expression of the neural form of P450 aromatase in juvenile sea bass (Dicentrarchus labrax). Molecular and Cellular Endocrinology, 219, 83–94.
5. Borg, B. (1994). Androgens in teleost fishes. Comparative Biochemistry and Physiology, 109, 219– 245.
6. Callard, G.V., Petro, Z., Ryan, K.J. (1977). Identification of aromatase in the reptilian brain. Endocrinology, 100, 1214–1218.
7. Callard, G.V., Petro, Z., Ryan, K.J., Clairbone, J.B. (1981). Estrogen synthesis in vitro and in vivo in the brain of a marine teleost (Myoxocephalus). General and Comparative Endocrinology, 43, 243–255.
8. Callard, G.V. (1992). Autocrine and paracrine role of steroids during spermatogenesis: studies in Squalus acanthias and Necturus maculosus. Journal of Experimental Zoology, 261, 132–142.

9. Cheshenko, K., Pakdel, F., Segner, H., Kah, O., Eggen, R.I.L. (2008). Interference of endocrine disrupting chemicals with aromatase CYP19 expression or activity, and consequences for reproduction of teleost fish. General and Comparative Endocrinology, 155, 31-62.
10. Choi, I., Troyer, D.L., Cornwell, D.L., Kirby-Dobbels, K.R., Collante, W.R., Simmen, F.A. (1997). Closely related genes encode developmental and tissue isoforms of porcine cytochrome P450 aromatase. DNA and Cell Biology, 16, 769–777.
11. Choi, J.Y., Park, J.G., Jeong, H.B., Lee, Y.D., Takemura, A., Kim, S.J. (2005). Molecular cloning of cytochrome P450 aromatases in the protogynous wrasse, Halichoeres tenuispinis. Comparative Biochemistry and Physiology, 141, 49–59.
12. Evans, C.T., Ledesma, D.B., Schulz, T.Z., Simpson, E.R., Mendelson,
13. characterization of a complementary DNA specific for (1986).
14. human aromatase-system cytochrome P-450 mRNA. Proceedings of National Academy of Sciences, 83, 6387–6391. Isolation
15. and Fukada, S., Tanaka, M., Matsuyama, M., Kobayashi, D., Nagahama, Y. (1996). Isolation, characterization, and expression of cDNAs encoding the medaka (Oryzias latipes) ovarian follicle cytochrome P-450 aromatase. Molecular Reproduction and Development, 45, 285– 290.
16. Gelinas, D., Pitoc, G.A., Callard, G.V. (1998). Isolation of a goldfish brain cytochrome P450 aromatase cDNA: mRNA expression during the seasonal cycle and after steroid treatment. Molecular and Cellular Endocrinology, 138, 81–93.
17. Gonzalez, F.J. (1992). Control of constitutively- expressed developmentally- activated rat hepatic cytochrome P450 genes. The Keio Journal of Medicine, 41, 68–75.
18. González, A., Piferrer, F. (2002). Characterization of aromatase activity in the sea bass: effects of temperature and different catalytic properties of brain and ovarian homogenates and microsomes. Journal of Experimental Zoology, 293, 500–510.
19. Greytak, S.R., Champlin, D., Callard, G.V. (2005). Isolation and characterization of two cytochrome P450 aromatase forms in killifish (Fundulus heteroclitus): differential expression in fish from polluted and unpolluted environments. Aquatic Toxicology, 71, 371–389.
20. Harada, N., Utsumi, T., Takagi, Y. (1993). Tissue- specific expression of the human aromatase cytochrome-P-450 gene by alternative use of multiple exons-1 and promoters, and switching of tissue- specific exons-1 in carcinogenesis. Proceedings of National Academy of Sciences, 90, 11312–11316.
21. Harada, N., Sasano, H., Murakami, H., Ohkuma, T., Nagura, H., Takagi, Y. (1999). Localized expression of aromatase in human vascular tissues. Circulation Research, 84, 1285–1291.
22. Hinfray, N., Porcher, J.M., Brion, F. (2006). Inhibition of rainbow trout (Oncorhynchus mykiss) P450 aromatase activities in brain and ovarian microsomes by various environmental substances. Comparative Biochemistry Physiology-Part C Toxicology and Pharmacology, 144, 252-262.
23. Katagiri, M., Tatsuta, K., Imaoka, S., Funae, Y., Honma, K., Matsuo, N., Yokoi, H., Ishimura, K., Ishibashi, F., Kagawa, N. (1998). Evidence that immature rat liver is capable of participating in steroidogenesis by expressing 17a-hydroxylase/17,20-lyase P450cl7. Journal of Steroid Biochemistry and Molecular Biology, 64, 121–128.
24. Kellis, J.T.J., Vickery, L. E. (1984). Inhibition of human estrogen synthetase (aromatase) by flavones. Science, 225, 1032–1034.
25. Kishida, M., Callard, G.V. (2001). Distinct cytochrome P450 aromatase isoforms in zebrafish (Danio rerio) brain and ovary are differentially programmed and estrogen regulated during early development. Endocrinology, 142, 740–750.
26. Kitano, T., Takamune, K., Kobayashi, T., Nagahama, Y., Abe, S.I. (1999). Suppression of P450 aromatase gene expression in sex-reversed males produced by rearing genetically female larvae at a high water temperature during a period of sex differentiation in the Japanese flounder (Paralichthys olivaceus). Journal of Molecular Endocrinology, 23, 167–176.
27. Kobayashi, Y., Kobayashi, T., Nakamura, M., Sunobe, T., Morrey, C. E., Suzuki, N., Nagahama, Y. (2004). Characterization of two types of cytochrome P450 aromatase in the serial-sex changing gobiid fish, Trimma okinawae. Zoological Science, 21, 417–425.
28. Kwon, J.Y., McAndrew, B.J., Penman, D.J. (2001). Cloning of brain aromatase gene and expression of brain and ovarian aromatase genes during sexual differentiation in genetic male and female Nile tilapia Oreochromis niloticus. Molecular Reproduction and Development, 59, 359–370.
29. Lee, Y.-M., Seo, J.S., Kim, I.-C., Yoon, Y.-D., Lee, J.-S. (2006). Endocrine disrupting chemicals (bisphenol A, 4-nonylphenol,
30. expression of two distinct cytochrome P450 aromatase genes differently in gender types of the hermaphroditic fish Rivulus marmoratus. Biochemical and Biophysical Research Communications, 345, 894–903.
31. modulate Lephart, E.D. (1996). A review of brain aromatase cytochrome P450. Brain Research Reviews, 22, 1–26. Liu, X., Liang, B., Zhang, S. (2004). Sequence and expression of cytochrome P450 aromatase and FTZ- F1 genes in the protandrous black porgy (Acanthopagrus schlegeli). General and Comparative Endocrinology, 138, 247–254.
32. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randal, R.J. (1951). Protein Measurement with the Folin Phenol Reagent. The Journal of Biological Chemistry, 193, 265-275.
33. Luckenbach, J.A., Early, L.W., Rowe, A.H., Boski, R.J., Daniels, H.V., Godwin, J. (2005). Aromatase cytochrome P450: Cloning, intron variation, and ontogeny of gene expression in Southern flounder (Paralichthys lethostigma). Journal of Experimental Zoology, 303, 643-656.
34. Melo, A.C., Ramsdell, J.S. (2001). Sexual dimorphism of brain aromatase activity in medaka: Induction of a female phenotype by estradiol. Environmental Health Perspectives, 109, 257–264.
35. Noaksson, E., Tjarnlund, U., Bosveld, A. T., Balk, L. (2001). Evidence for endocrine disruption in perch (Perca fluviatilis) and roach (Rutilus rutilus) in a remote Swedish Lake in the vicinity of a public refuse dump. Toxicology and Applied Pharmacology, 174, 160–176.
36. Noaksson, E., Linderoth, M., Bosveld, A.T.C., Balk, L. (2003). Altered steroid metabolism in several teleost species exposed to endocrine disrupting substances in refuse dump leachate. General and Comparative Endocrinology, 134, 273–284.
37. Orlando, E.F., Davis, W.P., Guillette, L.J.J. (2002). Aromatase activity in the ovary and brain of the eastern mosquitofish (Gambusia holbrooki) exposed to paper mill effluent. Environmental Health Perspectives, 110, 429–433.
38. Pasmanik, M., Callard, G.V. (1988). A high abundance androgen receptor in goldfish brain: characteristics and seasonal changes. Endocrinology, 123, 1162– 1171.
39. Price, T., Aitken, J., Simpson, E.R. (1992). Relative expression of aromatase cytochrome P450 in human fetal tissues as determined by competitive polymerase chain reaction amplification. The Journal of Clinical Endocrinology and Metabolism, 74, 879– 883.
40. Schenkman, J.B., Cinti, D.L. (1978). Preparation of microsomes with calsium. Methods in Enzimology, 52, 83-89.
41. Silverin, B., Baillien, M., Foidart, A., Balthazart, J. (2000). Distribution of aromatase activity in the brain and peripheral tissues of passerine and nonpasserine avian
42. Endocrinolology, 117, 34–53. and
43. Comparative Simpson, E.R., Mahendroo, M.S., Means, G.D., Kilgore, M.W., Hinshelwood, M.M., Grahamlorence, S., Amarneh, B., Ito, Y.J., Fisher, C.R., Michael, M.D., Mendelson, C.R., Bulun, S.E. (1994). Aromatase cytochrome-P450, the enzyme responsible for estrogen biosynthesis. Endocrinology Reviews, 15, 342–355.
44. Strobl-Mazzulla, P.H., Moncaut, N.P., Lopez, G.C., Miranda, L.A., Canario, A.V.M., Somoza, G.M. (2005). Brain aromatase from pejerrey fish (Odontesthes bonariensis): cDNA cloning, tissue expression, and immunohistochemical localization. General and Comparative Endocrinolology, 143, 21–32.
45. Tchoudakova, A., Callard, G.V. (1998). Identification of multiple cyp19 genes encoding different cytochrome P450 aromatase isozymes in brain and ovary. Endocrinology, 139, 2179–2189.
46. Trant, J. (1994). Isolation and characterization of the cDNA encoding the channel catfish (Ictalurus punctatus) form of cytochrome P450 arom. General and Comparative Endocrinolology, 95, 155–168.
47. Weisz, J. (1982). In vitro assays of aromatase and their role in studies of estrogen formation in target tissues. Cancer Research, 42, 8, 3295-3298.
48. Wilson, J.Y., McArthur, A.G., Stegeman, J.J. (2005). Characterization of a cetacean aromatase (CYP19) and the phylogeny and functional conservation of vertebrate aromatase. General and Comparative Endocrinolology, 140, 74–83.
49. Ziegler, R.G. (2004). Phytoestrogens and breast cancer. The American Journal of Clinical Nutrition, 79, 183–184.