Influence of heavy metals and 4-nonylphenol on reproductive function in fish

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1 Vol. 6, Suppl Influence of heavy metals and 4-nonylphenol on reproductive function in fish Włodzimierz Popek 1,2, Grzegorz Dietrich 3, Jan Glogowski 3,4, Krystyna Demska-Zakęś 4, Ewa Drąg-Kozak 2, Jan Sionkowski 2, Ewa Łuszczek-Trojan 2, Piotr Epler 2, Wiesław Demianowicz 3, Beata Sarosiek 3, Radosław Kowalski 3, Małgorzata Jankun 4, Zdzisław Zakęś 5, Jarosław Król 4, Stanisław Czerniak 5, Mirosław Szczepkowski 5 2 Department of Ichthyobiology and Fisheries, Agriculture University of Cracow; 3 Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn; 4 Chair of Ichthyology, University of Warmia and Mazury in Olsztyn, 5 Department of Aquaculture, Inland Fisheries Institute, Olsztyn, Poland SUMMARY Many industrial and agricultural chemicals (including heavy metals and alkylphenols) present in the environment have adverse effects on the reproductive function in fish. Three studies were conducted to assess toxicity of these chemicals towards reproduction of freshwater fish. It was shown that heavy metals added to the diets accumulate in brain tissue of carp, and this accumulation results in inhibition of the secretion of noradrenaline and stimulation of the secretion of dopamine in the hypothalamus. These processes results in a disturbance of hormonal equilibrium of the hypothalamo-pituitary system, which can unfavorably influence the efficiency of artificial spawning in fish. Quality of salmonid and sturgeon sperm was impaired after in vitro exposure to heavy metals. The degree of this toxic effect was species-specific. 1 Corresponding author: Departmen of Ichthyobiology and Fisheries, Agricultural University of Cracow, ul. Prof. Spiczakowa 6, Kraków-Mydlniki, Poland; rzpopek@cyf-kr.edu.pl Copyright 2006 by the Society for Biology of Reproduction

2 176 Xenobiotics and fish reproduction It was demonstrated that sperm motility parameters appeared to be good indicators of adverse effects of heavy metals fish sperm. The protection role of seminal plasma against toxic effects of heavy metals was suggested for salmonid fish. Oral application of 4-nonylphenol (NP) disrupted reproduction in pikeperch. In juvenile fish a decrease in the percentage of males and an increase of intersex fish was observed in relation to dose of NP and time of exposure to this alkylphenol. Exposure of adult males to the NP led to the reduction in fecundity, milt quality and fertility. Reproductive Biology Suppl. 1: Key words: fish, heavy metals, sperm quality, CASA, 4-nonylphenol, sex differentiation, brain, reproduction INTRODUCTION Due to increasing pollution, concentrations of various chemical compounds have increased in aquatic environment. The toxic effects of pollutants may affect behavior, cellular metabolism, endocrine regulations and reproduction of aquatic organisms [28]. Disrupted reproduction may result in limited propagation of species and eventually can lead to their extinction. A serious threat towards reproduction is posed by heavy metal compounds (mainly heavy metal salts), which are resorbed by organisms living in contaminated environments. Heavy metals are known to be cumulated in fish tissues, reaching concentrations of up to fold higher than in surrounding water environment. It has been reported that heavy metals affect both quality and quantity of the gametes as well as the endocrine system, disrupting the gametogenesis [16]. Mechanisms of these harmful effects depend on the type of metal and the degree of environmental pollution. For example, it is known that lead s toxic effects are linked with the disturbance of LH secretion [1]. Cadmium disrupts reproductive function in female fish by reducing the weight of ovaries and arresting their development [33]. Although zinc and copper are essential for fish at physiological concentrations, these heavy metals may become dangerous at higher concentrations. Disruption of

3 Popek et al. 177 reproductive function by zinc has been described at different stages of fish development [21, 22, 24] and it has been shown that copper is detrimental to the development of fish ovaries. The toxic effects of copper can be neutralized by metallothionins [27]. Action of heavy metals such as mercury and cadmium on spermatozoa may include disturbances in trajectory of sperm movement, inhibition of the enzymes or fragmentation of DNA [7, 10, 16]. As a result of these disturbances, fertilization rate and hatchability are often impaired [7, 28]. Natural sexual steroid hormones play a major role in controlling sex differentiation and reproduction in fish. Additionally, physiological concentrations of these hormons are essential for the maintenance of cell proliferation, growth and several other biological activities. Imbalance in hormonal status caused by higher level of endogenous hormones as well as by exposure to exogenous steroid-like compounds is known to produce adverse effects on reproductive processes [19]. Alkylphenols, including 4-nonylphenol, are good examples of such compounds. The current study was aimed to evaluate the effects of heavy metals and 4-nonylphenol on selected aspects of reproduction and sex differentiation in fish. EFFECT OF HEAVY METALS ON THE HYPOTHALAMIC CONTROL OF SEXUAL MATURATION AND THE EFFICIENCY OF ARTIFICIAL SPAWNING IN FEMALE CARP Heavy metals are especially harmful for Cyprinidae species that feed and live at the bottom of water bodies and therefore are more prone to contamination than predatory fish [23]. No data are available on the effects of accumulation of the heavy metals in the central nervous system on fish reproduction. It was shown that heavy metals added to the fodder are accumulated in brain tissue of carp and this results in inhibition of noradrenaline (NA) secretion and stimulation of the dopamine (DA) secretion in the hypothalamus. These processes result in the disturbance of hormonal equilibrium of the hypothalamo-pituitary system, which can unfavorably influence the efficiency of artificial spawning in fish.

4 178 Xenobiotics and fish reproduction Hypothalamus a strategic organ, mediates between seasonal environmental changes and the endocrine system and is involved in the control of maturation and reproduction. Neurohormones produced in the hypothalamus can either stimulate [gonadoliberin (GnRH)], or inhibit (dopamine) pituitary LH secretion. Both neurohormones control the gonadotropic function of the pituitary [20], while DA is additionally involved in the inhibition of GnRH secretion [38]. Stimulation of LH secretion in females of some fish species may be modulated by NA [38]. Noradrenalin acting through α1 receptors can stimulate the release of GnRH in the preoptic area of the brain and LH secretion by the pituitary [2]. The aim of the study was to test whether heavy metals (Pb, Cd, Zn and Cu) present in the diet may accumulate in the nervous system of mature female carp and whether the accumulation of heavy metals in fish brain may influence the hypothalamic concentrations of NA and DA. The ultimate goal of the research was to test whether the continuous presence of these heavy metals in the diet may affect the success of artificial spawning in female carp. Accumulation of heavy metals in fish nervous system A total of 150 mature female carp (Cyprinus carpio L.) were divided into five groups and stocked in five carp ponds of similar area and depth. Fish from the control group received a standard feed (carp pellets). The experimental fish received pellets supplemented with lead acetate, cadmium sulphate, zinc sulphate or copper sulphate. The concentration of particular heavy metals in pellets was 5-7 times of that in the control feed. After nine months of feeding, ten fish from each group were sacrificed and brains and the spinal cords were collected. After mineralization, samples were analysed for the concentrations of Pb, Cd, Zn and Cu using atomic absorption spectrometry. Average concentrations of all studied elements in the brain and the spinal cord of experimental carps were significantly higher than that of control carps [11, 12]. Therefore, it can be concluded that even a slight increase in the concentrations of heavy metals in the feed, that is within permitted values [34], leads to their significant accumulation in the nervous tissue.

5 Popek et al. 179 The influence of Pb, Cd, Zn and Cu on hypothalamic concentration of NA and DA During the spawning period (following nine months of feeding), ten females from each group were sacrificed in order to collect the hypothalamic sections of the brain (for determination of NA and DA concentrations by radioimmunoassay). Average hypothalamic concentration of NA in fish fed diets contaminated with Cu salts was significantly higher than that of control fish. The average hypothalamic concentration of DAin experimental fish was significantly lower than that of control fish. These data suggest that Cd, Cu and Pb reduce secretion of the stimulating hormone and increase the secretion of the inhibiting hormone involved in the regulation of the gonadotropic activity of the pituitary. Effects of heavy metals on efficiency of artificial spawning in female carp Ten females from each group were collected during the spawning period (following nine months of feeding). Half of the females from each group were hypophysed (injected with carp pituitary homogenate). The other females were intraperitoneally injected with Ovopel (a commercial preparation used for stimulation of the ovulation in fish). The use of classical hypophysation (stimulation of ovaries with exogenous LH) and Ovopel injections (pituitary stimulation with LHRH analog and anti-da) induced ovulation in 80% of the control females. The ovulation in the experimental females did not occur or the ovulation rate was markedly decreased. The poorest results of stimulation were obtained in fish receiving a feed supplemented with Cd [25] and Cu salts. It is alarming that results occurred relatively quickly after only nine months of feeding. Thus, even the low (within permitted values) dietary levels of heavy metals may disrupt many vital functions when present in feed for a prolonged period. This threat also concerns predatory fish [26]. The results obtained under pond breeding conditions correspond with natural conditions. There are evidences that Pb, Cd, Zn and Cu salts pose a threat to many fish species by impairing reproduction. This may result in the

6 180 Xenobiotics and fish reproduction disappearance of many commercially valuable species of fish. Additional studies also showed that feeding fish feeds containing higher levels of heavy metals caused undesirable changes in the environment, such as significant increases in the Cd concentration in bottom mud [30] and aquatic vegetation [31] as well as Zn concentration in pond water [32]. The results obtained in the presented studies are also of practical importance since the complete knowledge of the effects of heavy metals on the neurohormonal brain function should provide new insights into their potentially dangerous influence on the hypothalamus-pituitary-gonadal axis. DNA INTEGRITY, SPERM MOTILITY AND FERTILIZING CAPACITY OF SALMONID AND STURGEON MILT EXPOSED MERCURY AND CADMIUM The male reproductive system of fish is especially sensitive towards adverse effects of heavy metals. It has been reported that high levels of mercury ions affect gametogenesis and fertilization success and cadmium ions induce necrosis of the testis. Prolonged bioaccumulation of heavy metals in gonads is related to decreased quality of gametes, including sperm motility, plasmalemma integrity or DNA fragmentation [10, 16, 28]. Recent studies have shown that sperm motility characteristics are sensitive and reliable bioindicators of aquatic pollution [17, 28]. The most common method for assessment of the motility of fish sperm is microscopic estimation of the percentage of motile cells. However, this technique is much less sensitive than objective evaluation of sperm motility using computer-assisted sperm analysis (CASA). By using CASA it is possible to distinguish sublethal effects of pollutants on spermatozoa including changes in sperm velocity and trajectory of sperm movement. Moreover, it has been proven that CASA parameters are well correlated with fertilization [7, 28]. The aims of the present study were to investigate the effects of short-time in vitro exposure (0-24 h) of rainbow trout and sturgeon sperm to mercury and cadmium ions (0-100 mg/l) on sperm quality, including motility parameters, plasmalemma integrity, DNA fragmentation and fertilizing capacity.

7 Popek et al. 181 The influence of mercury and cadmium ions on sperm motility parameters We investigated the effects of short-term exposure (0, 4, 24 h) of rainbow trout, Siberian sturgeon and sterlet milt to mercury and cadmium ions (1, 10, 100 mg/l) on sperm motility characteristics measured using CASA [9]. Mercury and cadmium ions significantly affected sperm motility parameters. Total inhibition of sperm motility by Hg 2+ and Cd 2+ (at concentrations of 100 mg/l) was observed at all exposure times [7, 8]. Cadmium ions decreased percentages of motile sperm and sperm velocities at a dose and time dependent manner. Immediately after dilution of milt with heavy metals, percentages of motile sperm were decreased at concentration of 10 mg/l of Hg 2+ and Cd 2+, whereas decreased values of straight-line velocity (VSL) were observed at a concentration as low as 1 mg Hg 2+ /l. A similar pattern was observed after 4 h of milt exposure to heavy metals. Interestingly, there were no significant differences in rainbow trout sperm motility parameters between control and 10 mg Hg 2+ / l treated milt after prolonged (24 hours) incubation. This phenomenon may be probably explained by protective action of fish seminal plasma used as a incubation medium. These results are contrary to the results of other experiments where artificial buffers were used [28]. Our results suggest a protective role of rainbow trout seminal plasma components, such as albumin, against toxic effects of mercury ions. However, contrary to rainbow trout, this effect was not observed for sturgeon where both mercury and cadmium significantly decreased percentages of motile sperm and sperm velocities at dose and time dependent manner. These results suggest that spermatozoa of sturgeon and salmonid fishes have different sensitivity to negative impact of heavy metals. The influence of mercury and cadmium ions on sperm membrane integrity and DNA fragmentation The aim of the study was to determine the harmful effects of short-time exposure of rainbow trout and sturgeon milt to mercury and cadmium ions

8 182 Xenobiotics and fish reproduction on plasmalemma integrity. Sperm viability was assessed by supravital staining by applying a combination of the fluorophores SYBR-14 and propidium iodide. This method rapidly distinguishes live and dead cells. Deterioration effects of 24 h incubation on rainbow trout spermatozoa integrity were observed at a concentration of 100 mg Hg 2+ /l and 10 mg Cd 2+ /l, whereas Siberian sturgeon spermatozoa integrity were affected at 10 mg Hg 2+ /l and 100 mg Cd 2+ /l. Our results indicate that the negative impact of mercury and cadmium ions on plasmalemma integrity was observed at the higher concentrations and longer exposure time compared to effects of these heavy metals on CASA parameters. In addition we tested whether mercury and cadmium ions may led to DNA fragmentation in salmonid and sturgeon sperm. We used alkaline comet assay protocol which has recently been successfully used for the evaluation of DNA fragmentation in rainbow trout spermatozoa [10]. Incubation of sterlet spermatozoa for 24 h with mercury ions at a concentration of 10 mg Hg 2+ /l or higher resulted in increased DNA fragmentation. Our results indicate that the adverse effects of mercury and cadmium ions on DNA integrity of rainbow trout and sturgeon sperm are relatively minor in comparison to their effects on CASA parameters and plasmalemma integrity. There were species-specific differences in results of comet assay analysis of salmonid and Siberian sturgeon sperm. The latter was characterized by very high DNA fragmentation even in nontreated group (72-78% of DNA in comet tail). Fertilizing capacity of salmonid and sturgeon sperm after 4 h exposure of milt to mercury and cadmium The aim of the study was to examine the effects of short-term (4 h) exposure of rainbow trout milt to mercury and cadmium ions (0, 1, 10, 100, 1000 mg/l) on the fertilizing ability of salmonid (fresh milt) and sturgeon (cryopreserved milt) spermatozoa. The percentages of hatched rainbow trout embryos were significantly decreased at 10 mg/l or higher concentrations of both heavy metals [6, 7]. The percentages of hatched sterlet embryos were decreased at concentrations of 10 mg Hg 2+ /l and 100

9 Popek et al. 183 mg Cd 2+ /l or higher [13]. Although similar results of adverse effects of mercury ions were observed on Siberian sturgeon embryos, cadmium ions affected hatchability only at a very high concentration (1000 mg/l). The percentages of hatched Siberian sturgeon embryos were highly correlated with VSL and percentages of motile sperm [13]. Our results indicate that there are species-specific differences in sensitivity of sperm to adverse effects of heavy metals on sperm fertilizing capacity. Parameters of CASA may be successfully used as predictors of the hatching success. EFFECT OF ORAL ADMINISTRATION OF 4-NONYLPHENOL ON REPRODUCTIVE FUNCTION IN FISH There is an increasing concern about long-chain phenolic compounds, such as 4-nonylphenol (NP), because of their estrogenic properties [19]. Such compounds are hardly biodegradable but can easily accumulate; therefore they are a serious threat to populations of aquatic animals and other organisms [14, 18, 19]. To our knowledge, evaluation of this threat in terms of the effects produced by NP as a nutritional factor has not been studied. The study focused on the assessment of 4-nonylphenol effects on sexual differentiation, sex ratio and quality of milt in the pikeperch, Sander lucioperca L. The transgenerational effects of NP on the F 1 offspring were also analyzed. Effect of 4-nonylphenol on sex differentiation The embryonic gonad in fish is bipotential and can either develop into an ovary or a testis [4]. In fish, gonadal sex is determined not only genetically but also environmentally. Therefore, exposure to exogenous sex hormones or chemicals that mimic or block endogenous hormones during sensitive developmental period can disrupt sexual differentiation in fish. A few studies demonstrated that chronic exposure to aqueous solution of 4-nonylphenol significantly increased the number of female and/or intersex fish produced [14, 35]. The present data have demonstrated that oral

10 184 Xenobiotics and fish reproduction administration of 4-nonylphenol (0.1, 1, 10 and 100 ppm) disrupted the sexual differentiation in juvenile pikeperch without affecting the survival and growth rate. Morphological and histological studies revealed that administration of NP produced intersex fish. Intersex gonads had morphological features of testes or ovaries, but contained both female and male germ cells. In addition, we found that NP induced testis-ova formation in fish when the exposure occurred during sex differentiation [4]. The increase in NP dose and exposure time reduced the percentage of males and increased the percentage of intersex fish. However, the permanent feminizing effect was not observed. It is possible that gonad sensitivity to 4-nonylphenol administration depended not only on dose and exposure time but also on fish species and the method of NP application [3]. Effect of 4-nonylphenol on milt quality Several studies have been performed to evaluate the potential risk of 4-nonylphenol exposure to the reproductive system of adult fish. Disruptions to fish reproduction caused by NP include general effects on testes morphology, rates of spermatogenesis, and reproductive behavior [18, 36]. Additionally, nonylphenol is able to induce vitellogenin production in male individuals [18]. We investigated the effects of long-term exposure of NP ( ppm) on pikeperch milt quality. The examined endpoints included sperm concentration (spectrophotometric method), sperm motility parameters (computer-assisted sperm analysis), DNA fragmentation (comet assay) and fertilizing capacity [5]. We found that oral administration of NP resulted in decreases in milt quality and hatching success [3] without affecting the survival of the spawners. The negative impact of NP on milt quality was observed in all treated groups and was characterized by decreased spermatozoa concentration and increased level of DNA fragmentation. The data on the effect of alkylphenols on milt quality are scarce [3], but corresponding morphological effects including inhibition of testicular growth and spermatogenesis, and pathological changes of germ and Sertoli cells (e.g. fibrosis, vacuolation and atrophy) were reported [18, 36].

11 Popek et al. 185 Our results indicate that nonylphenol significantly affected sperm motility parameters. An increase in dose and time of exposure to NP resulted in an elevation in percentages of motile sperm and sperm velocities. For example, straight-line spermatozoa velocity ranged from 14 to 16.8 µm/s for the fish receiving a diet with NP for nine months vs 10.7 µm/s for untreated fish. A few studies showed that percentage of hatched embryos correlate with percentages of sperm motility and straight-line spermatozoa velocity (VSL). This correlation was not observed in the current study, because the percentages of fertilized eggs and hatched embryos in NPtreated groups were lower than those in control groups. However, no significant differences in survival and growth rate, gonad morphology and in sex ratio of the offspring were observed between control and NP-treated group. Genotoxicity of 4-nonylphenol The extent of DNA damage can serve as a useful indicator of toxicity of xenobiotics [29, 37]. Genotoxic substances can create adducts or cause breaks in a single and/or double DNA strand. As a result of DNA strand breaks chromosomal aberrations appear, which can be monitored as micronuclei in interphase cells or as fractures of chromatids in dividing chromosomes. The results of single cell gel electrophoresis (comet assay) [29, 37], chromosome aberration [15] and micronucleus tests suggest that 4-nonylphenol neither cause DNA breaks nor disturb cell divisions. Even long-term exposure (above 63 days) did not significantly increase DNA fragmentation; DNA damage ranged from 0 to 33% for the fish receiving feed with NP vs 0 to 23% for the control fish. Offspring of NP-treated males DNA were characterized by low DNA fragmentation. Altogether, these data suggest that NP is a nongenotoxic chemicals with estrogenic activity. The chronic exposure to NP can lead to negative changes in the sexual structure, milt quality, and, consequently, may cause the disappearance of natural fish populations.

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