Ecotoxicology and Environmental Safety 101 (2014) 103–106
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Ionizing radiation effects on sex steroid hormone levels in serum and milt of freshwater fish Oreochromis mossambicus M. Saiyad Musthafa a,n, A. Jawahar Ali a, T.H. Mohamed Ahadhu Shareef b, S. Vijayakumar a, K. Iyanar c, K. Thangaraj c a
P.G. & Research Department of Zoology, The New College, Chennai 600014, Tamil Nadu, India Department of Chemistry, The New College, Chennai 600014, Tamil Nadu, India c Centre for Plant Breeding & Genetics, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 7 June 2013 Received in revised form 15 December 2013 Accepted 20 December 2013 Available online 11 January 2014
Effects of gamma rays on the sex steroid hormone levels [testosterone (T), 11-ketotestosterone (11-KT) and 17β-estradiol (E2)] were studied in the freshwater fish Oreochromis mossambicus. Gamma radiation induced effects on hormone levels reported here for the first time in the fish. Since radionuclides released accidentally or during a nuclear disaster can contaminate inland water bodies, biomonitoring methods are required for assessing the impacts of certain dose levels of radiation that may ultimately result in ionizing radiation exposure to both humans and non-human biota. Three groups of (n ¼15 in each group) fishes were irradiated with a single dose of 60Co 10 Gy, 15 Gy and 20 Gy with a duration of .33, .50 and .66 min. Significant decrease of the hormone levels was seen at higher doses of 15 Gy and 20 Gy. The sex steroid hormone levels in the fishes are vital for sperm production, development, differential functions related to the physiology and reproductive behavior. This study serves as biomonitoring tool to assess the ionizing radiation effects on reproductive behavior of aquatic biota. & 2013 Elsevier Inc. All rights reserved.
Keywords: Gamma radiation Freshwater fish Sex-steroids Milt Serum
1. Introduction All living organisms are exposed to a certain amount of ionizing radiation during their life span. Sources of radioactivity in the aquatic environment include natural radionuclides, fallout from atmospheric runoff, watersheds that have received atmospheric deposition and radioactive effluents from medical, industrial and nuclear facilities released either accidentally or intentionally. Moreover, the growing dependence in a number of developing countries on nuclear fission as a source of power for the generation of electricity causes concern on the release of nuclear radiation through radioactive waste disposal and reprocessing. Besides, Chernobyl nuclear accident and the recent Fukushima disaster are wake-up calls for radiation protection of the humans and the environment (Anbumani and Mohankumar, 2012). Ionizing radiation damages biological tissues by exciting or ionizing their atoms and molecules. Additional indirect damage is caused by the formation of free radicals in water, which makes up 75–80 percent of the mass of living systems. The primary products of water radiolysis are the hydroxyl radical, hydrogen radical (hydrogen atom), and hydrated electron; hydroperoxy radicals and hydrogen peroxide are also formed in the presence of oxygen. The production of lysosomal enzymes and biological mediators, such as histamine and prostaglandins, is another biological response to radiation exposure (Bacg and Alexander, 1966; Snyder, 1977; Donlon and Walden, 1988). The risks of ionizing radiation to non-human biota (biota) are of considerable n
Corresponding author. E-mail address: [email protected] (M. Saiyad Musthafa).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.12.015
current interest, and both the International Commission on Radiological Protection (ICRP) and the International Atomic Energy Agency (IAEA), among others, have ongoing activities in this area (Chambers et al., 2006). Reproduction in fish is under hormonal regulation by gonadal steroids. In teleosts, testosterone (T), 11-ketotestosterone (11-KT) and 17β-estradiol (E2) regulate a number of reproductive processes (Fostier et al., 1983; Kime, 1993). T levels increase in both females and males during gonadal development, while 11-KT is considered to be a dominant androgen in males (Kime, 1993; Borg, 1994). On the other hand, E2 has been considered to be the main hormone of female fish; however, recent studies have suggested that estrogens are “essential” for normal male reproduction. For example, E2 regulates spermatogonial proliferation and sertoli cell physiology in teleost males. (Miura et al., 1991) The present study is an attempt to meet the lacuna over the scientific data on the effect of ionizing radiation on non-human species. Fish is one of the abundant population of an aquatic environment. Hence, commonly available fish Oreochromis mossambicus was selected and subjected to assess the ionizing radiation effects on sex steroid hormone levels in the serum and milt. 2. Materials and methods 2.1. Experimental fish specimens Matured adult male O. mossambicus weighing between 150 and 180 g and of standard length 13 72.0 cm were procured from a commercial fish farm and
104
M. Saiyad Musthafa et al. / Ecotoxicology and Environmental Safety 101 (2014) 103–106
transported to the laboratory in oxygenated bags and released into 150 L fiber tank (fifteen fishes per tank) filled with dechlorinated tap water. They were then acclimatized for 21 days under laboratory conditions with natural photoperiod and fed with commercial feed. The fecal matter and other waste materials were siphoned off daily to reduce ammonia content in water that was renewed once in two days with dechlorinated tap water. The water quality parameters were analyzed and maintained within the normal range (pH – 7.5, dissolved oxygen – 8.2 mg/L, temp. – 257 1 1C and hardness-in terms of CaCO3 was 220 mg/L).
space in the tubes and maintain the temperature of the collected milt at 4 1C until further analysis (Billard et al., 1995).
2.4. Collection of blood After 72 h of gamma irradiation, the blood samples were drawn from the caudal artery of O. mossambicus by sterilized syringes. They were centrifuged at 2000g (10 min, at 4 1C).The serum was stored at 20 1C for hormone assay.
2.2. Irradiation process The freshwater fish O. mossambicus were irradiated to three different dose levels of gamma radiation 10 Gy, 15 Gy and 20 Gy with duration of .33, .50 and .66 min, respectively using the facility of Atomic Energy Regulatory Board, Govt. of India recognized 60Co gamma chamber source (GAMMA CHAMBER 1200; Authorization no. AERB/RSD/AUTH/GIC/TN-06/2013/5850) at Plant Breeding & Genetics Department, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. 2.3. Collection of milt After 72 h of gamma irradiation, the milt samples were obtained from two year matured O. mossambicus male fishes by stripping the abdomen of each fish. The initial male ejaculate was discarded and the external urogenital pore was wiped dry with paper towel to avoid contamination from seawater, urine fecal matter and blood and to provide enough oxygenation to the sperm by keeping enough head
Table 1 Gamma radiation (60Co) induced sex steroid hormone levels in the serum and milt of freshwater fish Oreochromis mossambicus. Dose rate
11-Ketotestosterone Testosterone (T) 17 β-Estradiol (E2) (11-KT) (ng/ml) (ng/ml) (ng/ml)
Serum samples Control 23.60 7.75 10 Gy irradiated 18.58 7.53 15 Gy irradiated 11.32 7.84 20 Gy irradiated 3.12 7.37
10.50 7.58 8.357.58 7.53 7.35 1.80 7.33
2.95 7 .34 1.707 .28 .92 7 .10 .067 .03
Milt samples Control 10 Gy irradiated 15 Gy irradiated 20 irradiated
15.20 7.39 11.90 7.53 8.60 7.20 6.50 7.23
11.357 .80 6.78 7 .45 6.50 7 .14 1.82 7 .15
42.13 71.01 35.77 7.84 14.50 7.32 5.25 7.50
‘7 ’ Standard deviation, No. of analyzed samples: 06.
2.5. Hormone analysis Radio-immunoassay (RIA) was used to measure testosterone (T), 11ketotestosterone (11-KT) and 17β-estradiol (E2) in the serum and milt samples of freshwater fish O. mossambicus. The concentrations of the steroid hormones were determined using appropriate FRANSA radioimmunoassay kits (Cat nos. Testosterone: CM-TESTO; Estradiol 17-b: SB-ESTR; CM-PROG supplied by FRANSA) and plasma was analyzed. All FRANSA RIA test kits made use of 125I labeled hormones, which are intended for use with human samples. All readings of radioactivity were taken using a Beckman Gamma 8500 Microprocessor Counter.
3. Results and discussion Results of the sex-steroid hormone levels in the serum and milt of the freshwater fish O. mossambicus, irradiated and non-irradiated with gamma (60Co) radiation experiments are shown in Tables 1–3. In irradiated fish groups the sex steroid hormone levels were decreased with increasing dosage of gamma ray indicating the effect of radiation in the sex-specific hormones, 11-KT, T and E2. At lower dose, 10 Gy did not show a significant decrease of these hormones compared with control groups. Significant decreases of these hormones were seen at higher doses of 15 Gy and 20 Gy. 11-KT, T and E2 levels in serum samples decreases about 86, 82 and 97 percent, respectively at 20 Gy irradiated group. Gamma irradiation significantly affected the sex steroid hormone levels in the milt samples of the freshwater fish O. mossambicus. In tune the sex-specific steroid hormones such as 11-KT, T and E2 decrease with increasing dose levels of gamma irradiation. In response to gamma irradiation stress at higher dose of 20 Gy, these three hormone levels were decreased to about 86, 57 and 83 percent correspondingly.
Table 2 Spearman0 s correlation between non-irradiated and irradiated with gamma radiation (60Co) induced sex steroid hormone levels in the serum of freshwater fish Oreochromis mossambicus. Control 11-KT Control 11-KT T E2
10 Gy irradiated T
E2
11-KT
T
15 Gy irradiated E2
11-KT
T
.314 .257
.371
10 Gy irradiated 11-KT T E2
.086 .543 .087
.143 .086 .406
.200 .257 .464
.314 .783
.058
15 Gy irradiated 11-KT T E2
.429 .029 .657
.371 .464 .257
.143 .928nn .029
.657 .319 .429
.771 .551 .829n
.232 .500 .058
.319 .943nn
.232
20 Gy irradiated 11-KT T E2
.471 .147 .086
.500 .265 .086
.412 .029 .543
.471 .441 .543
.294 .588 .029
.134 .746 .319
.000 .294 .086
.388 .090 .348
Bold letters of table 2 and table 3 states that the hormone levels are significantly decreased with the respective dose level. n
Correlation is significant at the .01 level (2-tailed). Correlation is significant at the .05 level (2-tailed).
nn
20 Gy irradiated E2
11-KT
T
.235 .441 .029
.348 .794
.412
E2
M. Saiyad Musthafa et al. / Ecotoxicology and Environmental Safety 101 (2014) 103–106
105
Table 3 Spearman0 s correlation between non-irradiated and irradiated with gamma radiation (60Co) induced sex steroid hormone levels in the milt of freshwater fish Oreochromis mossambicus. Control 1-KT Control 11-KT T E2
.493 .371
10 Gy irradiated T
E2
11-KT
15 Gy irradiated
T
E2
11-KT
T
20 Gy irradiated E2
11-KT
T
.574 .132 .956nn
.500 .676
.015
E2
.522
10 Gy irradiated 11-KT .429 T .543 E2 .943nn
.232 .348 .406
.257 .486 .543
.943nn .600
15 Gy irradiated 11-KT .1.000nn T .406 E2 .203
.493 .750 .118
.371 .899n .232
.429 .493 .232
.543 .638 .058
.943nn .551 .116
.406 .203
.015
20 Gy irradiated 11-KT .406 T .348 E2 .377
.309 .132 .221
.348 .551 .232
.174 .058 .319
.087 .029 .029
.116 .493 .232
.406 .348 .377
.279 .279 .015
.714
Bold letters of table 2 and table 3 states that the hormone levels are significantly decreased with the respective dose level. n
Correlation is significant at the .01 level (2-tailed). Correlation is significant at the .05 level (2-tailed).
nn
Sex steroid hormones play important roles in many physiological processes, particularly in the reproduction of vertebrates. In many species of teleost, three sex steroid hormones, 11-KT, T and E2 are abundantly produced in gonadal tissues under the control of pituitary gonadotropins (GTH), and are essential for critical steps of gametogenesis (Wallace, 1985; Agahama and Yamashita, 2008; Miura et al., 1991). The measurement of sex steroid hormone concentrations is a reproductive parameter useful to assess the reproductive cycle of the fish. The endocrine changes that accompany the reproductive process have been described for many species of teleosts (Kobayashi et al., 1988; Prat et al., 1990; Pavlidis et al., 2000; Tricas et al., 2000). 11-KT can be the main circulating androgen in stimulating spermatogenesis and/or spermiogenesis in male sea bass. These results are supported by the work of Schulz and Goos (1999) in African catfish, suggesting that a balanced production of 11-oxygenated 11-KT and of aromatizable androgens T is crucial to the activation of the brain–pituitary–gonad (BPG) axis during puberty. Similarly, a direct stimulatory effect of 11-KT on spermatogenesis has been reported in male Japanese eel (Anguilla japonica, Miura et al., 1991) and in African catfish (Cavaco et al., 1998), indicating that this androgen is an important endocrine signal to promote spermatogenesis in this species. This is in contrast with findings reported for mammals, in which the most important androgen in stimulating spermatogenesis is T, instead of 11-KT (McLachlan et al., 1996). Lee (2000) reported that exposure of juvenile male freshwater prawn Macrobrachium rosenbergii to 10 Gy and 20 Gy of gamma (60Co) radiations reduced their total sperm count by 55 and 100 percent respectively. Furthermore, those irradiated male M. rosenbergii which had sperm were unable to fertilize, oocytes of non-irradiated females. Lee (2000) also reported complete sterility in adult size females M. rosenbergii when exposed to juveniles to 15 Gy. According to Sellars et al. (2005), gamma irradiation impaired the reproductive performance of male and female Penaues japonicus. Males were sensitive to irradiation being reproductively impaired at 10 Gy as compared to 20 Gy for females. Testis is one of the most important radiosensitive tissues because even very low doses of radiation deteriorate testicular functions. Leydig cells reside in the interstitium of the testes and contribute
about 75 percent of the total testosterone produced by normal adult male to support spermatogenesis and to maintain masculinity. Therefore, any defect in Leydig cell functions is expected to have a significant effect upon the quality of life manifested by sexual and psychosocial problems. Various clinical and experimental reports reveal the adverse effects of radiation on Leydig cell function (Izard, 1995). Decreased response of Leydig cells to human chorionic gonadotropin (hCG) was also reported in lymphoblastic leukemia patients treated with 20 Gy of testicular irradiation for leukemic infiltration (Carrascosa et al., 1984). In healthy adult men, a single dose of 600 rad of radiation directed to the testis decreased testosterone levels and raised LH levels in serum due to Leydig cell dysfunction (Rowley et al., 1974). Radiotherapy for testicular seminoma, unilateral germ cell cancer, and carcinoma in situ of the test showed decreased serum testosterone with a concomitant increase in LH (Petersen et al., 2002, 2003; Tsatsoulis et al., 1990). In accordance with the present study, radiation has been shown to induce both acute and chronic damage to Leydig cells of prepubertal and adult rats, as decreased testosterone secretion and increased gonadotropin release are reported (Delic et al., 1985, 1986a, 1986b). The clinical and experimental studies revealed the adverse effects of radiation on testicular steroidogenesis, diverse opinion exists on the sensitivity of Leydig cells to radiation. Studies on the molecular mechanisms behind radiation-induced Leydig cell dysfunction would be of great interest to adopt therapeutic or preventive measures to preserve Leydig cell function in postirradiated state. None of the previous reports describe the plausible molecular mechanism involved in radiation-induced Leydig cell dysfunction. Based on the existing information, it is hypothesized that gamma radiation impairs Leydig cell steroidogenesis by affecting LH receptor expression and its signal transduction (Ramadoss Sivakumar et al., 2006).
4. Conclusion To our knowledge, this is the first study focusing on effects of sexspecific steroid hormone levels in the serum and milt samples of freshwater fish O. mossambicus exposed to three dose levels of gamma (60Co) radiation under laboratory conditions. This
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investigation has identified significant decrease of the sex steroid hormones, 11-KT, T and E2 at higher dose 20 Gy of gamma radiation. The present study suggests that, the endocrine hormones were tremendously decreased with high doses and it leads to infertile condition in male fishes. This biomonitoring study is going to be useful to assess the ionizing radiation effects on reproductive behavior of aquatic biota. Acknowledgments Authors are thankful to U. Mohamed Khalilullah, Chairman, A. Mohamed Ashraf, Hon. Secretary and Correspondent, Dr. S. Abdul Maliq, Principal and Dr. M. Asrar Sheriff, Head, P.G. & Research Department of Zoology, The New College, Chennai for institutional support. We also thank the Director, Centre for Plant Breeding & Genetics, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu for technical support. References Agahama, Y.N., Yamashita, M., 2008. Regulation of oocyte maturation in fish. Dev. Growth Differ. 50, 195–219. Anbumani, S., Mohankumar, M.N., 2012. Gamma radiation induced micronuclei and erythrocyte cellular abnormalities in the fish Catla catla. Aquat. Toxicol. 122–123, 125–132. Bacg, Z.M., Alexander, P., 1966. 2nd ed. Fundamentals of Radiobiology, 45–62. Pergamon Press, Oxford, pp. 122–156. Borg, B., 1994. Androgens in teleost ¢shes. Comp. Biochem. Physiol. 109 C, 219–245. Billard, R., Cosson, J., Crim, L., Suquet, M., 1995. Sperm physiology and quality. In: Bromage, N.R., Roberts, R.J. (Eds.), Broodstock Management and Egg and Larval Quality. Institute of Aquaculture, Stirling and Blackwell Science, UK, pp. 25–52. Carrascosa, A., Audi, L., Ortega, J.J., Javier, G., Toran, N., 1984. Hypothalamohypophyseal-testicular function in prepubertal boys with acute lymphoblastic leukemia following chemotherapy and testicular radiotherapy. Acta Paediatr. Scand. 73, 364–371. Cavaco, J.E.B., Vilrokx, C., Trudeau, V.L., Schulz, R.W., Goos, H.J.T., 1998. Sex steroids and the initiation of puberty in male African catfish Clarias gariepinus. Am. J. Physiol.: Regul. Integr. Comp. Physiol. 275 (44), R1793–R1802. Chambers, D.B., Osborne, R.V., Garva, A.L., 2006. Choosing an alpha radiation weighting factor for doses to non-human biota. J. Environ. Radioact. 87, 1–14. Delic, J.I., Hendry, J.H., Morris, I.D., Shalet, S.M., 1985. Dose and time related responses of the irradiated prepubertal rat testis. I. Leydig cell function. Int. J. Androl. 8, 459–471. Delic, J.I., Hendry, J.H., Morris, I.D., Shalet, S.M., 1986a. Leydig cell function in the pubertal rat following local testicular irradiation. Radiother. Oncol. 5, 29–37. Delic, J.I., Hendry, J.H., Morris, I.D., Shalet, S.M., 1986b. Dose and time relationships in the endocrine response of the irradiated adult rat testis. J. Androl. 7, 32–41. Donlon, M.A., Walden Jr., T.L., 1988. The release of biological mediators in response to acute radiation injury. Comment. Toxicol. 2, 205–216.
Fostier, A., Jalabert, B., Billard, R., Breton, B., 1983. The gonadal steroids. In: Hoar, W.S., Randall, D.J., Donaldson, E.M. (Eds.), Fish Physiology, Vol. IXA. Academic, New York, pp. 277–372. Izard, M.A., 1995. Leydig cell function and radiation: a review of the literature. Radiother. Oncol. 34, 1–8. Kime, D.E., 1993. ‘Classical’ and ‘non-classical’ reproductive steroids in fish. Rev. Fish Biol. Fish. 3, 160–180. Kobayashi, M., Aida, K., Hanyu, I., 1988. Hormone changes during the ovulatory cycle in gold fish. Gen. Comp. Endocrinol. 69, 301–307. Lee, N., 2000. Effects of Cobalt-60 Gamma Irradiation on the Malaysian Prawn Macrobrachium rosenbergii (Ph.D. thesis). Louisiana State University, Louisiana, USA. McLachlan, R.I., Wreford, N.G., O’Donell, L., de Kretser, D.M., Robertson, D.M., 1996. The endocrine regulation of spermatogenesis: independent roles for testosterone and FSH. J. Endocrinol. 148, 1–9. Miura, T., Yamauchi, K., Takahashi, H., Nagahama, Y., 1991. Hormonal induction in all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica). Proc. Natl. Acad. Sci. USA 88, 5774–5778. Pavlidis, M., Greenwood, L., Mourot, B., Kokkari, C., Le Menn, F., Divanach, P., Scott, A.P., 2000. Seasonal variations and maturity stages in relation to differences in serum levels of gonadal steroids, vitellogenin, and thyroid hormones in the Common Dentex (Dentex dentex). Gen. Comp. Endocrinol. 118, 14–25. Petersen, P.M., Daugaard, G., Rorth, M., Skakkebaek, N.E., 2003. Endocrine function in patients treated for carcinoma in situ in the testis with irradiation. Acta Pathol. Microbiol. Immunol. Scand. 111, 93–99. Petersen, P.M., Giwercman, A., Daugaard, G., Rorth, M., Petersen, J.H., Skakkeaek, N.E., Hansen, S.W., von der Maase, H., 2002. Effect of graded testicular doses of radiotherapy in patients treated for carcinoma in situ in the testis. J. Clin. Oncol. 20, 1537–1543. Prat, F., Zanuy, S., Carrillo, M., De Mones, A., Fostier, A., 1990. Seasonal changes in plasma levels of gonadal steroids of sea bass, Dicentrarchus labrax L. Gen. Comp. Endocrinol. 78, 361–373. Sivakumar, Ramadoss, Sivaraman, Puliyangudi Balakrishnan, Mohan-Babu, Narayanasami, Jainul-Abideen, Ibrahim Mohamed, Kalliyappan, Perumal, Balasubramanian, Karundevi, 2006. Radiation exposure impairs luteinizing hormone signal transduction and steroidogenesis in cultured human Leydig cells. Toxcol. Sci. 91 (2), 550–556. Rowley, M.J., Leach, D.R., Warner, G.A., Heller, C.G., 1974. Effect of graded doses of ionizing radiation on the human testis. Radiat. Res. 59, 665–678. Schulz, R.W., Goos, H.J.Th., 1999. Puberty in male fish: concepts and recent developments with special reference to the African catfish Clarias gariepinus. Aquaculture 177, 5–12. Sellars, M.J., Degnan, B.M., Carrington, L.F., Preston, N.P., 2005. The effects of ionoizing radiation on the reproductive capacity of adult Penaeus (Marsupenaeus) japonicus (Bate). Aquaculture 250, 194–200. Snyder, S.L., 1977. Radiation-induced alterations in serum and splenic lysosomal hydrolases of the rat. Radiat. Res. 69, 306–316. Tricas, T.C., Maruska, K.P., Rasmussen, L.E.L., 2000. Annual cycles of steroid hormone production, gonad development, and reproductive behaviour in the Atlantic stingray. Gen. Comp. Endocrinol. 118, 209–225. Tsatsoulis, A., Shalet, S.M., Morris, I.D., de Kretser, D.M., 1990. Immunoactive inhibin as a marker of sertoli cell function following cytotoxic damage to the human testis. Horm. Res. 34, 254–259. Wallace, R.A., 1985. Vitellogenesis and oocyte growth in non-mammalion vertebrates. In: Browder, L.W. (Ed.), Developmental Biology: A Comprehensive Synthesis. Plenum Press, New York, pp. 127–177.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Ionizing radiation effects on sex steroid hormone levels in serum and milt of freshwater fish Oreochromis mossambicus M. Saiyad Musthafa a,n, A. Jawahar Ali a, T.H. Mohamed Ahadhu Shareef b, S. Vijayakumar a, K. Iyanar c, K. Thangaraj c a
P.G. & Research Department of Zoology, The New College, Chennai 600014, Tamil Nadu, India Department of Chemistry, The New College, Chennai 600014, Tamil Nadu, India c Centre for Plant Breeding & Genetics, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 7 June 2013 Received in revised form 15 December 2013 Accepted 20 December 2013 Available online 11 January 2014
Effects of gamma rays on the sex steroid hormone levels [testosterone (T), 11-ketotestosterone (11-KT) and 17β-estradiol (E2)] were studied in the freshwater fish Oreochromis mossambicus. Gamma radiation induced effects on hormone levels reported here for the first time in the fish. Since radionuclides released accidentally or during a nuclear disaster can contaminate inland water bodies, biomonitoring methods are required for assessing the impacts of certain dose levels of radiation that may ultimately result in ionizing radiation exposure to both humans and non-human biota. Three groups of (n ¼15 in each group) fishes were irradiated with a single dose of 60Co 10 Gy, 15 Gy and 20 Gy with a duration of .33, .50 and .66 min. Significant decrease of the hormone levels was seen at higher doses of 15 Gy and 20 Gy. The sex steroid hormone levels in the fishes are vital for sperm production, development, differential functions related to the physiology and reproductive behavior. This study serves as biomonitoring tool to assess the ionizing radiation effects on reproductive behavior of aquatic biota. & 2013 Elsevier Inc. All rights reserved.
Keywords: Gamma radiation Freshwater fish Sex-steroids Milt Serum
1. Introduction All living organisms are exposed to a certain amount of ionizing radiation during their life span. Sources of radioactivity in the aquatic environment include natural radionuclides, fallout from atmospheric runoff, watersheds that have received atmospheric deposition and radioactive effluents from medical, industrial and nuclear facilities released either accidentally or intentionally. Moreover, the growing dependence in a number of developing countries on nuclear fission as a source of power for the generation of electricity causes concern on the release of nuclear radiation through radioactive waste disposal and reprocessing. Besides, Chernobyl nuclear accident and the recent Fukushima disaster are wake-up calls for radiation protection of the humans and the environment (Anbumani and Mohankumar, 2012). Ionizing radiation damages biological tissues by exciting or ionizing their atoms and molecules. Additional indirect damage is caused by the formation of free radicals in water, which makes up 75–80 percent of the mass of living systems. The primary products of water radiolysis are the hydroxyl radical, hydrogen radical (hydrogen atom), and hydrated electron; hydroperoxy radicals and hydrogen peroxide are also formed in the presence of oxygen. The production of lysosomal enzymes and biological mediators, such as histamine and prostaglandins, is another biological response to radiation exposure (Bacg and Alexander, 1966; Snyder, 1977; Donlon and Walden, 1988). The risks of ionizing radiation to non-human biota (biota) are of considerable n
Corresponding author. E-mail address: [email protected] (M. Saiyad Musthafa).
0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.12.015
current interest, and both the International Commission on Radiological Protection (ICRP) and the International Atomic Energy Agency (IAEA), among others, have ongoing activities in this area (Chambers et al., 2006). Reproduction in fish is under hormonal regulation by gonadal steroids. In teleosts, testosterone (T), 11-ketotestosterone (11-KT) and 17β-estradiol (E2) regulate a number of reproductive processes (Fostier et al., 1983; Kime, 1993). T levels increase in both females and males during gonadal development, while 11-KT is considered to be a dominant androgen in males (Kime, 1993; Borg, 1994). On the other hand, E2 has been considered to be the main hormone of female fish; however, recent studies have suggested that estrogens are “essential” for normal male reproduction. For example, E2 regulates spermatogonial proliferation and sertoli cell physiology in teleost males. (Miura et al., 1991) The present study is an attempt to meet the lacuna over the scientific data on the effect of ionizing radiation on non-human species. Fish is one of the abundant population of an aquatic environment. Hence, commonly available fish Oreochromis mossambicus was selected and subjected to assess the ionizing radiation effects on sex steroid hormone levels in the serum and milt. 2. Materials and methods 2.1. Experimental fish specimens Matured adult male O. mossambicus weighing between 150 and 180 g and of standard length 13 72.0 cm were procured from a commercial fish farm and
104
M. Saiyad Musthafa et al. / Ecotoxicology and Environmental Safety 101 (2014) 103–106
transported to the laboratory in oxygenated bags and released into 150 L fiber tank (fifteen fishes per tank) filled with dechlorinated tap water. They were then acclimatized for 21 days under laboratory conditions with natural photoperiod and fed with commercial feed. The fecal matter and other waste materials were siphoned off daily to reduce ammonia content in water that was renewed once in two days with dechlorinated tap water. The water quality parameters were analyzed and maintained within the normal range (pH – 7.5, dissolved oxygen – 8.2 mg/L, temp. – 257 1 1C and hardness-in terms of CaCO3 was 220 mg/L).
space in the tubes and maintain the temperature of the collected milt at 4 1C until further analysis (Billard et al., 1995).
2.4. Collection of blood After 72 h of gamma irradiation, the blood samples were drawn from the caudal artery of O. mossambicus by sterilized syringes. They were centrifuged at 2000g (10 min, at 4 1C).The serum was stored at 20 1C for hormone assay.
2.2. Irradiation process The freshwater fish O. mossambicus were irradiated to three different dose levels of gamma radiation 10 Gy, 15 Gy and 20 Gy with duration of .33, .50 and .66 min, respectively using the facility of Atomic Energy Regulatory Board, Govt. of India recognized 60Co gamma chamber source (GAMMA CHAMBER 1200; Authorization no. AERB/RSD/AUTH/GIC/TN-06/2013/5850) at Plant Breeding & Genetics Department, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. 2.3. Collection of milt After 72 h of gamma irradiation, the milt samples were obtained from two year matured O. mossambicus male fishes by stripping the abdomen of each fish. The initial male ejaculate was discarded and the external urogenital pore was wiped dry with paper towel to avoid contamination from seawater, urine fecal matter and blood and to provide enough oxygenation to the sperm by keeping enough head
Table 1 Gamma radiation (60Co) induced sex steroid hormone levels in the serum and milt of freshwater fish Oreochromis mossambicus. Dose rate
11-Ketotestosterone Testosterone (T) 17 β-Estradiol (E2) (11-KT) (ng/ml) (ng/ml) (ng/ml)
Serum samples Control 23.60 7.75 10 Gy irradiated 18.58 7.53 15 Gy irradiated 11.32 7.84 20 Gy irradiated 3.12 7.37
10.50 7.58 8.357.58 7.53 7.35 1.80 7.33
2.95 7 .34 1.707 .28 .92 7 .10 .067 .03
Milt samples Control 10 Gy irradiated 15 Gy irradiated 20 irradiated
15.20 7.39 11.90 7.53 8.60 7.20 6.50 7.23
11.357 .80 6.78 7 .45 6.50 7 .14 1.82 7 .15
42.13 71.01 35.77 7.84 14.50 7.32 5.25 7.50
‘7 ’ Standard deviation, No. of analyzed samples: 06.
2.5. Hormone analysis Radio-immunoassay (RIA) was used to measure testosterone (T), 11ketotestosterone (11-KT) and 17β-estradiol (E2) in the serum and milt samples of freshwater fish O. mossambicus. The concentrations of the steroid hormones were determined using appropriate FRANSA radioimmunoassay kits (Cat nos. Testosterone: CM-TESTO; Estradiol 17-b: SB-ESTR; CM-PROG supplied by FRANSA) and plasma was analyzed. All FRANSA RIA test kits made use of 125I labeled hormones, which are intended for use with human samples. All readings of radioactivity were taken using a Beckman Gamma 8500 Microprocessor Counter.
3. Results and discussion Results of the sex-steroid hormone levels in the serum and milt of the freshwater fish O. mossambicus, irradiated and non-irradiated with gamma (60Co) radiation experiments are shown in Tables 1–3. In irradiated fish groups the sex steroid hormone levels were decreased with increasing dosage of gamma ray indicating the effect of radiation in the sex-specific hormones, 11-KT, T and E2. At lower dose, 10 Gy did not show a significant decrease of these hormones compared with control groups. Significant decreases of these hormones were seen at higher doses of 15 Gy and 20 Gy. 11-KT, T and E2 levels in serum samples decreases about 86, 82 and 97 percent, respectively at 20 Gy irradiated group. Gamma irradiation significantly affected the sex steroid hormone levels in the milt samples of the freshwater fish O. mossambicus. In tune the sex-specific steroid hormones such as 11-KT, T and E2 decrease with increasing dose levels of gamma irradiation. In response to gamma irradiation stress at higher dose of 20 Gy, these three hormone levels were decreased to about 86, 57 and 83 percent correspondingly.
Table 2 Spearman0 s correlation between non-irradiated and irradiated with gamma radiation (60Co) induced sex steroid hormone levels in the serum of freshwater fish Oreochromis mossambicus. Control 11-KT Control 11-KT T E2
10 Gy irradiated T
E2
11-KT
T
15 Gy irradiated E2
11-KT
T
.314 .257
.371
10 Gy irradiated 11-KT T E2
.086 .543 .087
.143 .086 .406
.200 .257 .464
.314 .783
.058
15 Gy irradiated 11-KT T E2
.429 .029 .657
.371 .464 .257
.143 .928nn .029
.657 .319 .429
.771 .551 .829n
.232 .500 .058
.319 .943nn
.232
20 Gy irradiated 11-KT T E2
.471 .147 .086
.500 .265 .086
.412 .029 .543
.471 .441 .543
.294 .588 .029
.134 .746 .319
.000 .294 .086
.388 .090 .348
Bold letters of table 2 and table 3 states that the hormone levels are significantly decreased with the respective dose level. n
Correlation is significant at the .01 level (2-tailed). Correlation is significant at the .05 level (2-tailed).
nn
20 Gy irradiated E2
11-KT
T
.235 .441 .029
.348 .794
.412
E2
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105
Table 3 Spearman0 s correlation between non-irradiated and irradiated with gamma radiation (60Co) induced sex steroid hormone levels in the milt of freshwater fish Oreochromis mossambicus. Control 1-KT Control 11-KT T E2
.493 .371
10 Gy irradiated T
E2
11-KT
15 Gy irradiated
T
E2
11-KT
T
20 Gy irradiated E2
11-KT
T
.574 .132 .956nn
.500 .676
.015
E2
.522
10 Gy irradiated 11-KT .429 T .543 E2 .943nn
.232 .348 .406
.257 .486 .543
.943nn .600
15 Gy irradiated 11-KT .1.000nn T .406 E2 .203
.493 .750 .118
.371 .899n .232
.429 .493 .232
.543 .638 .058
.943nn .551 .116
.406 .203
.015
20 Gy irradiated 11-KT .406 T .348 E2 .377
.309 .132 .221
.348 .551 .232
.174 .058 .319
.087 .029 .029
.116 .493 .232
.406 .348 .377
.279 .279 .015
.714
Bold letters of table 2 and table 3 states that the hormone levels are significantly decreased with the respective dose level. n
Correlation is significant at the .01 level (2-tailed). Correlation is significant at the .05 level (2-tailed).
nn
Sex steroid hormones play important roles in many physiological processes, particularly in the reproduction of vertebrates. In many species of teleost, three sex steroid hormones, 11-KT, T and E2 are abundantly produced in gonadal tissues under the control of pituitary gonadotropins (GTH), and are essential for critical steps of gametogenesis (Wallace, 1985; Agahama and Yamashita, 2008; Miura et al., 1991). The measurement of sex steroid hormone concentrations is a reproductive parameter useful to assess the reproductive cycle of the fish. The endocrine changes that accompany the reproductive process have been described for many species of teleosts (Kobayashi et al., 1988; Prat et al., 1990; Pavlidis et al., 2000; Tricas et al., 2000). 11-KT can be the main circulating androgen in stimulating spermatogenesis and/or spermiogenesis in male sea bass. These results are supported by the work of Schulz and Goos (1999) in African catfish, suggesting that a balanced production of 11-oxygenated 11-KT and of aromatizable androgens T is crucial to the activation of the brain–pituitary–gonad (BPG) axis during puberty. Similarly, a direct stimulatory effect of 11-KT on spermatogenesis has been reported in male Japanese eel (Anguilla japonica, Miura et al., 1991) and in African catfish (Cavaco et al., 1998), indicating that this androgen is an important endocrine signal to promote spermatogenesis in this species. This is in contrast with findings reported for mammals, in which the most important androgen in stimulating spermatogenesis is T, instead of 11-KT (McLachlan et al., 1996). Lee (2000) reported that exposure of juvenile male freshwater prawn Macrobrachium rosenbergii to 10 Gy and 20 Gy of gamma (60Co) radiations reduced their total sperm count by 55 and 100 percent respectively. Furthermore, those irradiated male M. rosenbergii which had sperm were unable to fertilize, oocytes of non-irradiated females. Lee (2000) also reported complete sterility in adult size females M. rosenbergii when exposed to juveniles to 15 Gy. According to Sellars et al. (2005), gamma irradiation impaired the reproductive performance of male and female Penaues japonicus. Males were sensitive to irradiation being reproductively impaired at 10 Gy as compared to 20 Gy for females. Testis is one of the most important radiosensitive tissues because even very low doses of radiation deteriorate testicular functions. Leydig cells reside in the interstitium of the testes and contribute
about 75 percent of the total testosterone produced by normal adult male to support spermatogenesis and to maintain masculinity. Therefore, any defect in Leydig cell functions is expected to have a significant effect upon the quality of life manifested by sexual and psychosocial problems. Various clinical and experimental reports reveal the adverse effects of radiation on Leydig cell function (Izard, 1995). Decreased response of Leydig cells to human chorionic gonadotropin (hCG) was also reported in lymphoblastic leukemia patients treated with 20 Gy of testicular irradiation for leukemic infiltration (Carrascosa et al., 1984). In healthy adult men, a single dose of 600 rad of radiation directed to the testis decreased testosterone levels and raised LH levels in serum due to Leydig cell dysfunction (Rowley et al., 1974). Radiotherapy for testicular seminoma, unilateral germ cell cancer, and carcinoma in situ of the test showed decreased serum testosterone with a concomitant increase in LH (Petersen et al., 2002, 2003; Tsatsoulis et al., 1990). In accordance with the present study, radiation has been shown to induce both acute and chronic damage to Leydig cells of prepubertal and adult rats, as decreased testosterone secretion and increased gonadotropin release are reported (Delic et al., 1985, 1986a, 1986b). The clinical and experimental studies revealed the adverse effects of radiation on testicular steroidogenesis, diverse opinion exists on the sensitivity of Leydig cells to radiation. Studies on the molecular mechanisms behind radiation-induced Leydig cell dysfunction would be of great interest to adopt therapeutic or preventive measures to preserve Leydig cell function in postirradiated state. None of the previous reports describe the plausible molecular mechanism involved in radiation-induced Leydig cell dysfunction. Based on the existing information, it is hypothesized that gamma radiation impairs Leydig cell steroidogenesis by affecting LH receptor expression and its signal transduction (Ramadoss Sivakumar et al., 2006).
4. Conclusion To our knowledge, this is the first study focusing on effects of sexspecific steroid hormone levels in the serum and milt samples of freshwater fish O. mossambicus exposed to three dose levels of gamma (60Co) radiation under laboratory conditions. This
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investigation has identified significant decrease of the sex steroid hormones, 11-KT, T and E2 at higher dose 20 Gy of gamma radiation. The present study suggests that, the endocrine hormones were tremendously decreased with high doses and it leads to infertile condition in male fishes. This biomonitoring study is going to be useful to assess the ionizing radiation effects on reproductive behavior of aquatic biota. Acknowledgments Authors are thankful to U. Mohamed Khalilullah, Chairman, A. Mohamed Ashraf, Hon. Secretary and Correspondent, Dr. S. Abdul Maliq, Principal and Dr. M. Asrar Sheriff, Head, P.G. & Research Department of Zoology, The New College, Chennai for institutional support. We also thank the Director, Centre for Plant Breeding & Genetics, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu for technical support. References Agahama, Y.N., Yamashita, M., 2008. Regulation of oocyte maturation in fish. Dev. Growth Differ. 50, 195–219. Anbumani, S., Mohankumar, M.N., 2012. Gamma radiation induced micronuclei and erythrocyte cellular abnormalities in the fish Catla catla. Aquat. Toxicol. 122–123, 125–132. Bacg, Z.M., Alexander, P., 1966. 2nd ed. Fundamentals of Radiobiology, 45–62. Pergamon Press, Oxford, pp. 122–156. Borg, B., 1994. Androgens in teleost ¢shes. Comp. Biochem. Physiol. 109 C, 219–245. Billard, R., Cosson, J., Crim, L., Suquet, M., 1995. Sperm physiology and quality. In: Bromage, N.R., Roberts, R.J. (Eds.), Broodstock Management and Egg and Larval Quality. Institute of Aquaculture, Stirling and Blackwell Science, UK, pp. 25–52. Carrascosa, A., Audi, L., Ortega, J.J., Javier, G., Toran, N., 1984. Hypothalamohypophyseal-testicular function in prepubertal boys with acute lymphoblastic leukemia following chemotherapy and testicular radiotherapy. Acta Paediatr. Scand. 73, 364–371. Cavaco, J.E.B., Vilrokx, C., Trudeau, V.L., Schulz, R.W., Goos, H.J.T., 1998. Sex steroids and the initiation of puberty in male African catfish Clarias gariepinus. Am. J. Physiol.: Regul. Integr. Comp. Physiol. 275 (44), R1793–R1802. Chambers, D.B., Osborne, R.V., Garva, A.L., 2006. Choosing an alpha radiation weighting factor for doses to non-human biota. J. Environ. Radioact. 87, 1–14. Delic, J.I., Hendry, J.H., Morris, I.D., Shalet, S.M., 1985. Dose and time related responses of the irradiated prepubertal rat testis. I. Leydig cell function. Int. J. Androl. 8, 459–471. Delic, J.I., Hendry, J.H., Morris, I.D., Shalet, S.M., 1986a. Leydig cell function in the pubertal rat following local testicular irradiation. Radiother. Oncol. 5, 29–37. Delic, J.I., Hendry, J.H., Morris, I.D., Shalet, S.M., 1986b. Dose and time relationships in the endocrine response of the irradiated adult rat testis. J. Androl. 7, 32–41. Donlon, M.A., Walden Jr., T.L., 1988. The release of biological mediators in response to acute radiation injury. Comment. Toxicol. 2, 205–216.
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