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A Comparative FTIR Spectroscopic Analysis of Rainbow Trout Liver Exposed to Nonylphenol and Estradiol

GULGUN CAKMAK, INCI TOGAN AND FERIDE SEVERCAN Department of Biological Sciences Middle East Technical University 06531, Ankara, TURKEY

Abstract: - Anthropogenic chemicals are present in the environment as pollutants and interact with the endocrine systems of animals, including fish. Many of these chemicals interfere with estrogen receptor-mediated physiological responses. Such compounds are called endocrine disrupters and either evoke estrogenic responses by mimicking, or by inhibiting the action of natural estrogen, 17-Estradiol (E2). In the present study the effect of an estrogenic endocrine disrupter, nonylphenol (NP) was examined, in comparison with the action of E2 on juvenile rainbow trout (Oncorhynchus mykiss) liver at molecular level using Fourier Transform Infrared (FTIR) Spectroscopy. NP is an alkylphenol (AP) which is a degradation product of alkylphenol polyethoxylates (APEs). APEs are non-ionic surfactants used in detergents, herbicides, pesticides, paints and cosmetics and they are found in rather high concentrations in the aquatic environment. It was found that NP and E2 induced significant alterations in the liver tissues and the NP-treated fish liver spectra were found to be quite similar to those of E2- treated fish. For example; the glycogen level and the protein concentration decreased in NP and E2-treated liver. The level of hepatic lipids, especially, triglycerides and the relative content of nucleic acids increased in both of the treatment group. In addition, a decrease in the membrane fluidity and an increase lipid order was observed in the cells of NP and E2 treated samples. These results confirmed that NP mimics the effects of E2 in immature rainbow trout.

Key-Words: - Estradiol, Nonylphenol, Rainbow trout, Oncorhynchus mykiss, Fourier Transform Infrared, FTIR Spectroscopy, Liver.

1 Introduction A large number of anthropogenic chemicals which are shown to stimulate the synthesis of vitellogenin in the present in the environment as pollutants are capable of livers of male and immature rainbow trout and inhibit disrupting the endocrine system of animals including spermatogenesis in adult rainbow trout [5]. NP has fish. These chemicals are referred to as endocrine also carcinogenic and toxic effects on animals. For disrupters because they affect the endocrine systems example, it has been shown to cause testicular and in various organisms. It has been found that many of ovarian cancer in mice and induce anemia as a these endocrine disrupters have estrogenic activity and consequence of the interaction with erythrocyte can disrupt normal functions of sex steroids [1]. A membrane [8, 9]. well known group of environmental endocrine Rainbow trout is one of the most commonly disrupters is nonylphenolethoxylates (NPEs) cultured and consumed fresh-water fish in the world. belonging to the alkylphenolethoxylates (APEs) The liver is the target tissue for estrogens where group. NPEs account for approximately 80% of the estrogens stimulate vitellogenin synthesis and has an world’s production of APEs [2]. Nonylphenol (NP) is important role in detoxification of toxic substances. the final biodegradation product of NPEs in the For these reasons, this study was conducted to environment [3]. Concentrations of NP in the aquatic compare the changes of NP and E2 on rainbow trout environment from various countries range from liver. The effects of NP and E2 on rainbow trout liver micrograms per liter to milligrams per liter [4]. were investigated at molecular level comparatively Many in vivo and in vitro studies demonstrated using Fourier Transform Infrared (FTIR) that NP affects the endocrine systems in various Spectroscopy. This technique which is a non- organisms through weak estrogen-like properties destructive, rapid, sensitive and highly selective for [5,6]. In fish as well as in mammals and avian cells, structural and functional studies, together with NP mimics the 17-estradiol (E2) by binding the infrared microscopy, is becoming an increasingly estrogen receptors [7]. E2 induces the production of important technique to study the cellular changes at hepatic vitellogenin, which is a precursor of egg-yolk molecular level in various biological samples due to protein, in mature oviparous female. NP has been its power and convenience [10-13].

1 2 Materials and Methods 157 FTIR Spectrometer (The Michelson Series, Bomem, Inc. Quebec, Canada) equipped with a deuterated triglycine sulfate (DTGS) detector. The 2.1 Chemicals spectrometer was continuously purged by dry air to eliminate atmospheric water vapour. The spectra of Nonylphenol (NP) and 17-Estradiol (E2) were -1 purchased from Sigma and Aldrich Company, liver samples were recorded in the 4000-400 cm region at room temperature. 200 scans were taken respectively. Potassium Bromide (KBr) was -1 purchased from Merck. for each interferogram at 4 cm resolution. The spectra were normalized in specific regions for data analysis by using the Win Bomem Easy software 2.2 Fish (Galactic Industries Corporation). Averages of the spectra belonging to the same experimental group Eight-month-old juvenile rainbow trout were used. and baseline correction were performed by using The fish were transferred to the laboratory in plastic GRAMS/32 programme. sacs with aeration and held in the laboratory in 35-l glass aquariums. They were acclimated, in exposure conditions, for 4 days prior the start of 2.5 Statistical Test: experimentation. During the whole experiment the fish were fed with trout chow purchased from The results were expressed as ‘mean ± standard Çağatay-Yem Company (İzmir, Turkey). deviation’. The difference in the means of the treated and control fish were compared by means of Mann- Whitney-U test. The p value of less than 0.05 was 2.3 Experimentation considered statistically significant. Experiments were conducted in a semistatic aquaria system. Charcoal filter was used to eliminate chlorine, suspended solids and metals found in water. Prior to use, these media were let to rest for 24 hours with 3 Results aerator. The fish were treated with 0 (control) (n=8) The FTIR spectrum of rainbow trout liver is a and 220 g/l NP (n=6) for three weeks in aquariums. complex spectrum consisting of several bands arises A group of fish was also exposed to 22 g/l E2 (n=6) from the functional groups belong to lipids, for three weeks. NP and E2 were dissolved in absolute carbohydrates, proteins and nucleic acids. The ethanol and then introduced into the aquaria. The detailed spectral analyses were performed in three same amount of ethanol without chemicals was also distinct frequency ranges. Figure 1A, B and C show added into the tank of control group. Five liters of the average infrared spectra of control, 220 g/l NP water siphoned and replaced with the same volume of and 22 g/l E2-treated rainbow trout liver in the 3600- water and NP and E2 concentrations were adjusted in 3030 cm-1, 3030-2800 cm-1 (C-H stretching region) accordance with the replacement everyday. and 1800-800 cm-1 (fingerprint regions), respectively, Temperature was 9 ± 1 oC in each aquarium. Livers of after three weeks treatment. the sampled fish were removed for FTIR study and The numerical comparisons of the wavenumbers were stored at –22 oC. The FTIR experiments were and the band intensities of infrared bands were listed performed in two weeks time after the removal of the in Table 1. The means and the standard deviations of liver. the wavenumbers, the band intensities and directions of the variations with respect to control were obtained for treated and untreated groups. This table illustrated 2.4 FTIR Studies the presence of significant changes in the spectra of The liver samples were dried in a HETO-MAXI dry both 220 g/l NP and 22 g/l E2 treated fish. As seen lyo freeze drier for 12 hours to remove water. The from table 1, in 4 cases for both of the treatment samples then were ground in agate mortar containing groups (NP and E2) the shift of the wavenumbers liquid nitrogen to obtain liver powder. The liver changed significantly in the same directions. For the powder was mixed with potassium bromide (at the other cases, although some of them are not significant, ratio of 1/100) and dried again in lyo freeze drier for the variation in the wavenumbers for control and 6 hours to remove water completely. The mixture treated groups are also in the same directions. As then was subjected to a pressure of ~1100kg/cm2 in shown in this table, in 4 cases both of the treatments an evacuated die to obtain KBr pellet for use in were significantly different from those of the control FTIR spectrometer. in terms of the band intensities. For these cases, all of the infrared bands were changed in the same Infrared spectra were obtained using a Bomem directions. As can be seen from this table, both E2 and

2 NP treatments generated significant changes in the The intensity of the band at 3015 cm-1, which is spectra and most of the time these changes were due to CH stretching mode of the HC=CH groups, parallel to each other. could be used as a measure of unsaturation in the acyl chains [10]. As seen from figure 1B and table 1, the intensity of this band increased in both of the treatment groups. The maximum of this band also shifted slightly to lower frequency values in the treatment groups. It was also observed that as a consequence of NP and E2 treatment, the absorption

intensity of the CH3 asymmetric stretching vibration at 2959 cm-1 slightly decreased. The band at 2876 cm-1,

which is assigned as CH3 symmetric stretching, almost diminished in the treated liver spectra. Moreover, the

intensity of CH2 symmetric stretching vibration band at 2857 cm-1 significantly increased in the NP and E2- treated livers (p<0.05) and the shape of this band was more symmetric compared to that of control liver. As seen from the figure and table 1, the frequency of this band shifted slightly to lower values. Other frequency range under consideration was that of 1800-800 cm-1 region (Figure 1C). The band centered at 1741 cm-1 is mainly assigned to C=O ester stretching vibration in triglycerides [14]. As illustrated in Figure 1C and table 1, the intensity of this band increased dramatically in the spectra of NP and E2- treated liver (p<0.05). In addition to this intensity increase, the frequency of the maximum absorption of this band shifted to higher values. The bands at 1657 and 1541 cm-1 are attributable to amide I and II vibrations of structural proteins [15]. The bandwidth of the amide I band broadened significantly (p<0.05) in the NP-treated liver (from 0.853  0.081 to 1.133  0.152) and E2-treated liver (from 0.853  0.081 to 0.933  0.057). The frequency of this band shifted slightly to higher values in both of the treatment groups. The spectra in figure 1C clearly showed that the relative intensities of both the amide I and amide II bands decreased in the treated tissues. An increase in the intensity was observed at 1452 cm-1 band. This

band is due to bending vibration of the CH2 in the lipids and proteins [16]. The band at 1395 cm-1 is due to COO- symmetric stretching vibration of amino acid Fig.1. The average infrared spectra of rainbow trout livers side chains and fatty acids. An increase was also with 0 (control) (n=8) and 220 g/l NP (n=6) and 22 g/l observed in the intensity of this band. The relatively E2 (n=6) for three weeks A) 3600-3030 cm-1 region B) strong, bands at 1236 and 1080 cm-1 are mainly due to -1 3030-2800 cm region (C-H stretching region) C) 1800- asymmetric and symmetric stretching modes of -1 800 cm region (finger print region). The spectra were phosphodiester groups in nucleic acids rather than in normalized with respect to the CH2 asymmetric stretching phospholipids [17, 18]. However, the symmetric band (figure A and B) and to the amide I band (figure C) phosphate stretching band might be masked by spectral contributions of glycogen, which exhibits As could be seen from Figure 1A and table 1, in three strong peaks at 1042, 1080, and 1152 cm-1 [19]. the NP and E2-treated liver spectra there was a As shown in Figure 1B, the treated tissue spectrum dramatic reduction (p<0.05) in the intensity of the displayed an increase in the intensity of phosphate band at 3308 cm-1, which contains strong absorbtions asymmetric stretching band and a decrease in the arising from N-H and O-H stretching modes of intensity of symmetric phosphate stretching band proteins and polysaccharides [10]. (p<0.05). As seen from table 1, the phosphate asymmetric and symmetric bands shifted to higher

3 Table 1. Numerical comparisons of the wavenumbers and band intensities of infrared bands for control, NP and E2-treated rainbow trout after three weeks. The values are the mean  standard deviation for each group. Comparisons were done by Mann-Whitney-U test. Arrows to the left indicate a decrease and to the right indicate an increase with respect to the control. The degree of significance was denoted as: *p<0.05, **p<0.01, ***p<0.001. Wavenumbers (cm-1) Band Intensities Control 220 g/l NP 22 g/l E2 Control 220 g/l NP 22 g/l E2     3308.400  0.918 3306.767  1.209* 3305.400  0.529* 2.126  0.206 1.720  0.075* 1.534  0.165* treated     liver 3015.990  0.826 3015.3  0.173 3014.833  0.057 0.140  0.010 0.168  0.006 0.155  0.018 spectra     (figure 2959.967  0.531 2960.83  0.152* 2960.367  0.838 0.695  0.026 0.689  0.031 0.651  0.060 1C).     2857.283  1.144 2856.433  1.078 2855.967  0.680 0.032  0.030 0.136  0.033* 0.172  0.048* This     was 1741,233  1.297 1742.200  0.866 1744.133  1.450* 0.285  0.059 0.5  0.014* 0.417  0.050* also   1657.300  0.774 1658.8 0.866 1658.3  1.732 1 1 1     1395.250  0.821 1393.5  0.866 1393.1  0.173* 0.334  0.018 0.387  0.015* 0.388  0. 071     1236.467  1.347 1239.56  1.674* 1240.000  0.933* 0.396  0.026 0.428  0.008* 0.415  0.032     1152.967  0.571 1162.1  1.5* 1161.233  4.361* 0.312  0.033 0.232  0.014* 0.248  0.074     1080.333  0.216 1083.1  0.866* 1083.133  1.674* 0.489  0.030 0.087  0.014* 0.393  0.040* frequency values (p<0.05). The band at 1042 cm-1 is confirmed by a decrease in the intensity of the assigned as mainly to C-O stretching vibrations in asymmetric CO-O-C stretching vibration at 1152 cm-1 polysaccharides [20]. As seen from Figure 1C, the peak band which is mainly due to glycogen in tissues (figure of this band was almost lost in treated liver spectra 1C). These results revealed that glycogen levels indicating a change in the polysaccharides in the treated decreased in the NP and E2-treated tissues. tissues. The band centered at 969 cm-1 is generally The band at 3015 cm-1 is due to the CH stretching assigned to symmetric stretching mode of dianionic mode of HC=CH groups. As seen from figure 1B and phosphat monoester of cellular nucleic acids, especially table 1, the intensity of this band increased in NP and for DNA [11]. As could be seen from the figure, this E2 treated liver. This indicates that the population of band was stronger in the NP and E2-treated liver unsaturated lipids increased [12]. The increase in the spectra than that in the control group spectrum. intensity and the shift in the frequency values of this band (table 1) are presumably the results of a change in lipid metabolism. This conclusion is supported by the intensity increase of the lipid C=O stretching vibration 4 Discussion (at 1741 cm-1) band which suggested an increased concentration of the ester groups belonging to In the present study, to compare the imposed changes of triglycerides within NP and E2 exposed livers (Figure NP and E2, the spectra of liver tissues exposed to these 1C) [14]. In addition, the dramatic shifting of this band chemicals were studied. It has been reported that the to higher frequency values (table 1) indicate a potency in estrogenic activity of NP is 100-1000 times difference in packing of ester groups within the tissue less than that of E2 [5]. For this reason, the [16]. The difference in packing might be the result of an concentration of NP was chosen at 10 fold more than increased proportion of unsaturated acyl chains in that of E2. triglycerides in the treated liver as also suggested by As can be seen from figure 1A, there was a Nara et al. [14]. This was also confirmed by the dramatic reduction in the intensities of the band at 3308 increased intensity of the olefinic=CH stretching cm-1 which arises from both the N-H and O-H stretching absorption at 3015 cm-1. Scwaiger et al., demonstrated modes in proteins and polysaccharides, in the NP and that alkylphenolic compounds exert highly toxic effects E2-treated liver spectra. The explanation for the on the organisms [9]. Since the liver is the site of dramatic reduction in the intensities of this band might synthesis, storage and release of lipids, it stands to be due to a reduced contribution from glycogen OH reason that interference with these processes by a toxic absorption [10]. This is strongly supported by the compound could lead to an accumulation of fats in the disappearance of the glycogen band at 1042 cm-1 in liver itself. The exposure of toxic compounds can block

4 the secretion of triglycerides. Another interesting division and perhaps decreased DNA condensation observation derived from figure 1B was the decreased between control and treated cells. absorbtion intensity of the CH3 asymmetric stretching vibration at 2959 cm-1 as a consequence of NP and E2 treatment. This decrease indicates a change in the composition of the acyl chains [12]. The symmetric 5 Conclusion -1 CH2 stretching band at 2857 cm shifted slightly to lower values after NP and E2 treatment. The The results of the present study indicated that when the frequencies of the CH2 stretching bands of the acyl rainbow trout were treated with 220g/l NP and 22 g/l chains depend on the degree of conformational disorder E2 for three weeks, the FTIR spectra revealed dramatic [13]. In the present work, the shift of the peak position differences between the treated and control tissues. of the symmetric CH2 stretching band to lower values Since the E2 spectral bands related to lipids, indicates that lipid order increases and acyl chain carbohydrates, proteins and nucleic acids exhibited flexibility decreases [13]. In addition, the intensity of parallel changes to those observed in the NP spectra, it the symmetric CH2 stretching band increased can be said that exposure of immature rainbow trout to significantly in treated tissues. This result suggested an NP seemed to mimic the effect of E2 in liver. The increased proportion of the CH2 groups in the treated results of the present study also indicated that FTIR liver tissues. Since the dominant mode for protein side -1 spectroscopy is an excellent technique for the chains around 1600 cm was not observed in our case, investigation of structural, compositional and metabolic the main contribution to the 1395 cm-1 band may come - changes in biological tissues at the molecular level after from the COO symmetric stretching of fatty acids [21]. in vivo exposure to toxic chemicals. We observed an increase in the intensity of this band. Consequently, the increase in the absorbtion intensities -1 of 3015, 2857, 1741, 1391 cm is mainly the result of References: the accumulation of triglycerides in the NP and E2- [1] Miura, C., Takahashi, N., Michino, F., Miura, T., treated liver tissues. The Effect of para-nonylphenol on Japanese eel There was a dramatic broadening in the (Anguilla Japonica) Spermatogenesis in Vitro, bandwidth of the amide I band and a shifting in the Aquatic Toxicology, Vol.71, 2005, pp.133-141. frequency of this band to higher values in the treatment [2] Pedersen, S. N., Christiansen L. B., Pedersen K. L., group. As the position of amide I absorbtions was Korsgaard, B., Bjerregaard P., In vivo estrogenic sensitive to protein conformation, this shifting and activity of branched and linear alkylphenols in broadening implied conformational changes in tissue rainbow trout (Onchoryncus mykiss). The Science proteins [16]. The intensities of amide I and the amide of the Total Environment, Vol.233, 1999, pp. 89- II bands decreased slightly in NP-treated liver (Figure 96. 1C). These results indicate a decrease in protein [3] Giger, W., Brunner, P. H., Schaffer C., 4- concentration in the NP and E2-treated tissues. This Nonylphenol in Sewage Sludge: Accumulation of decrease was consistent with the decrease in amide A -1 Toxic Metabolites from Nonionic Surfactants. band (at 3308 cm ) and CH3 symmetric stretching band -1 Science (Washington D. C.), Vol.225, No.4662, (at 2876 cm ) which are also due to proteins. 1984, pp. 623-625. The asymmetric and symmetric phosphate -1 [4] Uguz, C., Iscan, M., Ergüven, A., Isgor, B., Togan, stretching bands at 1236 and 1080 cm respectively are I., The Bioaccumulation of Nonylphenol and its originated mainly due to the phosphodiester backbone Adverse Effect on the Liver of Rainbow Trout of cellular nucleic acids [18]. The contribution to these (Oncorhynchus Mykiss), Environmental Research, bands from phosphate residues which are present in Vol.92, 2003, pp.262-270. membrane lipids was negligible [18,19]. As seen from [5] Jobling S., Sheahan D., Osborne J. 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