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Endocrine-disrupting effects of nonylphenol in the newt, Triturus carnifex (Amphibia, Urodela)
Comparative Biochemistry and Physiology, Part C 155 (2012) 352–358
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Endocrine-disrupting effects of nonylphenol in the newt, Triturus carnifex (Amphibia, Urodela) Anna Capaldo a,⁎, Flaminia Gay a, Salvatore Valiante a, Maria De Falco a, Rosaria Sciarrillo b, Massimo Maddaloni a, Vincenza Laforgia a a b
Department of Biological Sciences, Section of Evolutive and Comparative Biology, University Federico II, Via Mezzocannone 8, 80134 Naples, Italy Department of Biological and Environmental Sciences, University of Sannio, Benevento, Italy
a r t i c l e
i n f o
Article history: Received 31 August 2011 Received in revised form 14 October 2011 Accepted 14 October 2011 Available online 25 October 2011 Keywords: Adrenal gland disruption Amphibian endocrine disruption Environmental contaminants Nonylphenol Triturus carnifex Water pollution
a b s t r a c t The aim of our study was to verify whether environmental concentrations of nonylphenol influenced the adrenal gland of Triturus carnifex. Newts were exposed to 19 μg/L nominal concentration of nonylphenol throughout the periods of December–January and March–April, corresponding to different stages of the chromaffin cell functional cycle. The morphological features of the steroidogenic and chromaffin tissues, and the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine were evaluated. Nonylphenol did not influence ACTH serum levels. During the two periods examined, the steroidogenic tissue had the same reaction: the quantity of cytoplasmic lipids, and the corticosteroid serum levels, decreased, suggesting the inhibition of synthesis and release of corticosteroids. During the two periods examined, the chromaffin tissue reacted differently to nonylphenol. During December–January, the numeric ratio of norepinephrine granules to epinephrine granules, and the epinephrine serum levels, increased, suggesting the stimulation of epinephrine release. During March–April, the numeric ratio of norepinephrine granules to epinephrine granules did not change, and the norepinephrine serum levels decreased, suggesting the inhibition of norepinephrine release. Our results show that nonylphenol influences the activity of the newt adrenal gland; considering the physiological role of this gland, our results suggest that nonylphenol may contribute to amphibian decline. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Nonylphenol ethoxylates (NPEs) are surfactants used worldwide, found in many types of products including detergents, plastics, emulsifiers, pesticides, and industrial and consumer cleaning products. As a consequence of their use in a variety of products, they are quite common in rivers and other aquatic environments that receive sewage discharges (Arukwe et al., 2000). Nonylphenol ethoxylates are transformed in the environment by microorganisms to form more toxic compounds, such as nonylphenol (NP) and short-chain nonylphenol ethoxylates (Vazquez-Duhalt et al., 2005). Because of the enhanced resistance towards biodegradation, toxicity, estrogenic effects, and ability to bioaccumulate in aquatic organisms of nonylphenol, it has been regarded as the most critical metabolite of NPE (Arukwe et al., 2000). Endocrine disruptors, such as nonylphenol, are considered a contributing factor in the global decline of amphibians (Renner, 2002); indeed, due to their highly permeable skin and their lifecycle which goes through both aquatic and terrestrial stages, amphibians are very
⁎ Corresponding author. Tel.: + 39 081 2535037; fax: + 39 081 2535035. E-mail addresses: [email protected], [email protected] (A. Capaldo), [email protected] (F. Gay), [email protected] (S. Valiante), [email protected] (M. De Falco), [email protected] (R. Sciarrillo), [email protected] (M. Maddaloni), [email protected] (V. Laforgia). 1532-0456/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2011.10.004
sensitive to waterborne chemicals (Kloas, 2002; Bogi et al., 2003; Levy et al., 2004; Polzonetti-Magni et al., 2004; Steinberg et al., 2004; Lutz et al., 2005) and can be considered excellent bioindicators of the general health of the environment (Noriega and Hayes, 2000). The greater part of studies evaluating nonylphenol effects on amphibian physiology found alterations in the reproductive biology. Environmental concentrations of nonylphenol caused malformations of tail flexure in Rana nigromaculata tadpoles during metamorphosis (Yang et al., 2005) and altered gonadal differentiation in Rana pipiens and R. sylvatica (Mackenzie et al., 2003). Higher doses of nonylphenol inhibited the rate of bullfrog (Rana catesbeiana) tadpole metamorphic progression and tail resorption (Christensen et al., 2005) and induced abnormalities in developing Xenopus laevis embryos (Sone et al., 2004). Moreover, intraperitoneal injections of nonylphenol induced hepatic vitellogenin mRNA in male Bombina orientalis (Kang et al., 2006). Considering the physiological importance of the adrenal gland of amphibians,it is surprising that no studies have been undertaken that regard the effects of nonylphenol on this gland. Indeed, the adrenal gland plays a key role in the stress response, which includes the release of both corticosteroids and catecholamines. Corticosterone and, to a lesser extent, aldosterone, are gluconeogenic and hyperglycaemic, but the primary role of these steroids appears to be the regulation of ion and water balance. Epinephrine is a potent stimulator of lypolysis in fat bodies and of glycogenolysis in both the liver and muscle and is
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therefore hyperglycaemic. These hormones induce changes in metabolism and/or ionic regulation that work to combat physiological factors and to eliminate or to neutralize the stressful stimulus, allowing the organism to adapt to its environment, and to survive (Norris, 2007). Moreover, the adrenal gland is involved in the amphibian reproductive processes (Moore and Miller, 1984; Moore and Zoeller, 1985; Moore and Jessop, 2003) and, in T. carnifex, its activity is correlated with some events of the reproductive cycle (Gay et al., 2008). Therefore, the aim of our research was to verify whether nonylphenol was able to influence the amphibian adrenal gland. To this aim, we chose a urodele amphibian, the newt Triturus carnifex, for the experimental model. Urodele amphibians spend a great part of their life in the aquatic environment, and therefore can easily adsorb waterborne chemicals through their permeable skin. In addition, T. carnifex has the features of an ideal bioindicator, due to its high sensitivity to chemicals (Kloas, 2002; Capaldo et al., 2006). The studies investigating the effects of nonylphenol on this species examined the gonads and the pituitary gland, showing the induction of plasma vitellogenin in the male T. carnifex (Mosconi et al., 2002; Polzonetti-Magni et al., 2004), the change in secondary sex characteristics, and the increase in the androgen hormone levels in plasma. Moreover, the inhibition of gonadotropin and prolactin secretion by pituitary, and the increase in the number of pituitary prolactin cells, were found (Mosconi et al., 2002). As far as we know, there are no data published concerning nonylphenol effects on the newt adrenal gland. 2. Materials and methods 2.1. Reagents Nonylphenol (analytical grade, 98%) and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). A stock solution of nonylphenol was prepared in DMSO at a concentration of 1 mg/mL (Mariager et al., 2005; Hirano et al., 2009).
was changed every four days in all the tanks. The test was carried out in triplicate. One month after the beginning of the experiment (January in the first experiment; April in the second experiment), the newts were anaesthetized by hypothermia, chilling them in chipped ice, within 5 min after removal from tank. Blood was immediately collected by heart puncture, between 11:00 h and 14:00 h, centrifuged for 15 min at 2000 g and serum was collected and stored, together with standards to correct for degradation, at −22 °C until assayed, as previously described (Capaldo et al., 2006). The experiment was carried out in accordance with the European Directive 2010/63/EU for animal experiments, and authorized by the National Committee of the Italian Ministry of Health for in vivo experimentation (Dept. for Veterinary Public Health, Nutrition and Food Safety). 2.4. Transmission electron microscopy The animals were killed by decapitation immediately after collection of blood samples. The adrenals were fixed in 2.5% glutaraldehyde in Millonig's phosphate buffer at pH 7.4 at 4 °C, rinsed in buffer, and postfixed in 1% OsO4 (2 h, 4 °C), dehydrated in ethanol, cleared in propylene oxide, embedded in epoxy resin, and polymerized. Ultrathin sections (300 nm) were cut with glass knives on a Reichert– Jung ultracut ultramicrotome (SUPER NOVA), collected on formvarcoated copper grids, stained with solutions of uranyl acetate and lead citrate, and observed with a Philips EM 301 transmission electron microscope at the Interdepartmental Center of Services for Electron Microscopy (C.I.S.M.E.) (Naples). For each specimen from each group (ten newts per group), ten low-power micrographs of the steroidogenic tissue and ten of the chromaffin tissue, each containing at least four cells, were subject to morphometric investigation by a computerized image analysis system (KS 300 for Windows 98, Zeiss). The area occupied by the lipid droplets in the steroidogenic cells was calculated using the following formula:
2.2. Animals Adult male specimens of T. carnifex (mean mass 8.0 g.), captured in the field around Naples, were kept in 50 L glass tanks (10 newts per tank, with a loading rate of 1.6 g newt/L water), under a natural photoperiod, in dechlorinated, well-aerated tap water, at seasonal temperature (12 °C mean temperature during December–January period; 20 °C mean temperature during March–April period). The animals were fed minced cow liver and used after an acclimation period of two weeks. 2.3. Experimental design Adult male newts were exposed to a nominal, environmental concentration of nonylphenol, included in the range internationally reported for surface water (between 5 ng/L and 180 μg/L) (Mariager et al., 2005). The concentration used (19 μg/L) and the exposure period (one month) were selected on the basis of the results of preliminary dose–response and time-course tests. At the beginning of December and at the beginning of March, corresponding to different stages of the chromaffin cell functional cycle (Gay et al., 2010), thirty adult male newts were collected (sixty animals in all) and kept under the previously described conditions. In both months, after an acclimation period of two weeks, the newts were divided into three groups (treated, carrier and control), each containing ten specimens. Each group was kept in 50 L glass tank (loading rate of 1.6 g newt/L water). Treated newts were exposed to a nominal nonylphenol concentration of 19 μg/L. Since the stock solution was prepared in DMSO, ten newts were kept in a glass tank and exposed to the same concentration of DMSO as the treated newts (0.019 mL/L); ten untreated newts were kept in a tank with tap water only. The water
353
Lipid=cytoplasm ratio ¼
Mean total lipid area μm2 Mean total cytoplasmic area μm2
:
In the chromaffin cells, the mean total number of chromaffin granules/μm 2; the mean number of norepinephrine and epinephrine granules/μm 2; the numeric ratio of norepinephrine granules to epinephrine granules (NE/E ratio); the mean number of intermediate granules/μm 2, i.e. the secretory granules in intermediate stage of synthesis, were calculated. The sampling criteria to selectively discriminate between norepinephrine, epinephrine and intermediate granules were the following: granules recognized as norepinephrine granules were of variable shape, with a very electron-dense and compact core filling the granule; the core was separated from the limiting membrane. Granules identified as epinephrine granules were roundish, homogeneous, with a finely granular core of medium electron density, separated from the limiting membrane by a narrow electron-lucent space. All the intermediate forms of granules, not showing these distinctive features, were considered intermediate granules (Laforgia and Capaldo, 1991). 2.5. Hormone assay Aldosterone and corticosterone serum levels were determined by radioimmunoassay (RIA) as previously described (Capaldo et al., 2006). Briefly, non hemolyzed serum samples (80 μL for aldosterone and 30 to 40 μL for corticosterone) were incubated for 30 min at 37 °C with known amounts of radioactive steroids ( 3H-aldosterone, and 3 H-corticosterone from Bio-Rad, Hercules, CA, USA) in 0.06 M Naphosphate buffer containing 0.01 M EDTA disodium salt and 0.1%
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BSA pH 7.4. Samples were applied to an extraction column (Sep-Pak C18, Waters, Milford, MA, USA) and washed with 500 μL of pure methanol. Methanol extracts were dried at 37 °C under vacuum and redissolved in 1400 μL of PBS. An aliquot was taken to determine the labeled hormone recovery and on two other aliquots aldosterone and corticosterone were assayed by RIA. After incubation with rabbit antiserum (Biogenesis, Poole, UK) for 30 min at 37 °C and for another 2 h in an ice bath, dextran-coated charcoal was used to separate free from bound steroids. After immersion for 10 min in an ice bath and centrifugation (600 g), a supernatant aliquot was counted with a liquid scintillation spectrometer (Tri-Carb Packard, GMI, Albertville, MN, USA). Extraction yields ranged from 80% to 90% for both hormones. Data were obtained through a standard calibration curve linearized with a log–logit method and corrected for individual extraction yield. Sensitivity was 5 pg/tube for aldosterone and corticosterone. Intra-assay coefficient of variation was 10%, and inter-assay coefficient of variation was 12% for both steroids. ACTH concentrations were measured in 100 μL of plasma by a two-site immunoradiometric assay using mouse monoclonal antibodies (Diagnostic Products Corp.) as previously described (De Falco et al., 2004). Cross-reactivities of the ACTH antiserum as determined by the kit manufacturer were 0.03% for a-MSH, 0.01% for β-MSH, and 0.02 for β-endorphin. Sensitivity was 0.1 pg.mL − 1 as determined by the kit manufacturer, and the inter- and intra-assay coefficients of variation were 10% and 6%, respectively. Norepinephrine and epinephrine levels were determined in 150 μL serum. For catecholamine extraction, 50 μL of dihydroxybenzylamine was added as an internal standard. Ten milligrams activated aluminum oxide (Sigma) was used as adsorbent for catecholamines and the internal standard. After 15 min shaking and centrifugation, the supernatant was removed and the aluminum oxide containing the adsorbed catecholamines and the internal standard was washed three times with 1 mL distilled water by shaking, centrifuging, and discarding the supernatant-extracted samples using high performance liquid chromatography (HPLC), with electrochemical detection, according to the method previously used in T. carnifex (Capaldo et al., 2006). Electrochemical HPLC detection was carried out using an acid eluant;
NE and E levels were calculated in comparison to the internal standard (dihydroxybenzylamine). The detection limit for NE and E was around 20 pg. 2.6. Statistical analysis All data were expressed as mean± standard error of mean (SEM). Each experimental group consisted of ten animals of the same weight and sex. The numeric data were obtained considering, for each experimental group, an equal number of micrographs at the same magnification (100 micrographs), and examining only the cytoplasmic areas, excluding the nucleus. The control and experimental data of all the groups were normalized and then tested together for significance using one-way analysis of variance (ANOVA), followed by Duncan's test for multigroup comparison and Student's t test for between group comparison. Differences were considered significant when P b 0.05. 3. Results 3.1. Steroidogenic tissue The “adrenal gland” of urodeles includes numerous discrete bodies scattered on the ventral surface of the functional opistonephros kidney, close to its medial margin. The bodies contain tightly intermingled steroidogenic and chromaffin cells. The steroidogenic cells are characterized by the presence of many mitochondria, a smooth endoplasmic reticulum arranged in tubules and vesicles, and a large quantity of lipid droplets (Hanke, 1978). The cells of December–January (Fig. 1A) and March–April (Fig. 1B) control newts, showed a similar morphology: a cytoplasm rich in lipid droplets and numerous mitochondria with tubular cristae. The lipid/cytoplasm ratio was 0.45 in December–January and 0.40 in March–April (Table 1). The exposure to nonylphenol always strongly reduced the quantity of lipid droplets (Fig. 1C, D); the lipid/cytoplasm ratio decreased (Table 1). The steroidogenic cells mainly produce the corticosteroids aldosterone and corticosterone, according to an annual pattern. Corticosterone levels are low from October to November, peak in January,
Fig. 1. Electron micrographs of steroidogenic cells of Triturus carnifex adrenal gland. (A) December–January and (B) March–April control specimens. The cells show many lipid droplets (L) and mitochondria (M) filling the cytoplasm. (C) December–January and (D) March–April treated specimens. The cytoplasm shows a strong decrease in lipid (L) droplets. Scale bar: a: 2 μm; b: 1.8 μm; c: 3 μm; d: 2.3 μm.
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Table 1 Mean ± SE of the different parameters evaluated in control, carrier (DMSO at 0.019 mL/L) and treated (nonylphenol at 19 μg/L) specimens. Chromaffin g/μm2
Intermediate g/μm2
NE g/μm2
E g/μm2
NE/E ratio
Lip/cyt.ratio
December–January Control Carrier Treated
7.59 ± 2.01 7.56 ± 1.97 6.15 ± 1.01
0.003 ± 0.0009 0.002 ± 0.0004 2.23 ± 1.51◊
7.38 ± 2.13 7.37 ± 1.89 3.90 ± 1.02◊
0.21 ± 0.001 0.19 ± 0.005 0.02 ± 0.003◊
35.1/1 38.7/1 195/1
0.45 0.44 0.15
March–April Control Carrier Treated
7.72 ± 2.41 7.74 ± 2.26 5.38 ± 1.16a
0.07 ± 0.001 0.08 ± 0.005 3.24 ± 1.03b
3.89 ± 1.61 3.85 ± 1.46 1.05 ± 0.03b
3.83 ± 1.13 3.89 ± 1.58 1.09 ± 0.03b
1.0/1 1.0/1 0.9/1
0.40 0.39 0.25
a b
Significantly (P b 0.05) different from the control values. Significantly (P b 0.001) different from the control values.
fall in February and remain low until March, after which they peak in July (Zerani and Gobbetti, 1993). Aldosterone levels are low from September to November, then rise from December up to a maximum in April, decrease in May, with values similar to those found during December–March period, and remain steady until July (Gay et al., 2010). During both periods, December–January and March–April, nonylphenol reduced aldosterone and corticosterone serum levels (Fig. 2). During both periods, nonylphenol did not influence ACTH serum levels (Fig. 3).
A
6000
Control Carrier
Corticosteroids (pg/ml)
5000
Treated
4000
3000
2000
1000
**
**
0
Aldosterone
Corticosterone
B
6000
Corticosteroids (pg/ml)
December-January
5000
3.2. Chromaffin tissue The chromaffin tissue of T. carnifex is composed of a single cell type that has an annual cycle in which the quantities of norepinephrine and epinephrine change during the year. During the December– February and May–August periods, large quantities of norepinephrine and small quantities of epinephrine are present in the chromaffin cells. During the March–April and September–November periods, the production of epinephrine increases, becoming similar to norepinephrine. During both periods, a small quantity of intermediate granules is present (Laforgia and Capaldo, 1991; Gay et al., 2010). In December– January control cells (Fig. 4A), almost exclusively norepinephrine granules were present. In March–April control cells (Fig. 4B), norepinephrine and epinephrine granules were both present in almost equal quantities. Moreover, a small quantity of intermediate granules was always present (Table 1). In December–January treated cells (Figs. 4C, D), the cytoplasm showed many chromaffin granules; the rough endoplasmic reticulum appeared increased in size (Fig. 4D). As it can be seen in Table 1, the total number of chromaffin granules did not change, but there was a strong decrease in the number of norepinephrine and epinephrine granules, and an increase in the number of intermediate granules, and in the numeric ratio of norepinephrine granules to epinephrine granules. In March–April treated cells (Fig. 4E, F), the cytoplasm did not show many chromaffin granules; most of them were intermediate granules (Fig. 4F). As it can be seen in Table 1, there was a decrease in the total number of chromaffin granules, and in the number of norepinephrine and epinephrine granules. The number of intermediate granules increased, whereas the numeric ratio of norepinephrine granules to epinephrine granules did not change. During the December–January period (Fig. 5A), nonylphenol slightly reduced norepinephrine serum levels and significantly increased epinephrine serum levels, whereas, during the March–April period
Control Carrier Treated
700
December-January March-April
600
ACTH (pg/ml)
4000 3000 2000 1000
Aldosterone
400 300 200
**
**
0
500
100 Corticosterone
March-April Fig. 2. Aldosterone and corticosterone serum levels in (A) December–January and (B) March–April control, carrier (DMSO at 0.019 mL/L) and treated (nonylphenol at 19 μg/L) specimens. Values are mean ± SE of the mean. **Values significantly (P b 0.001) different from the control values.
0
Control
Carrier
Treated
Fig. 3. ACTH serum levels in control, carrier (DMSO at 0.019 mL/L) and treated (nonylphenol at 19 μg/L) specimens. Values are mean ± SE of the mean. Values in the treated groups do not significantly differ from the control values.
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Fig. 4. Electron micrographs of the chromaffin cells of Triturus carnifex adrenal gland. (A) December–January control specimens: the cytoplasm prevalently contains norepinephrine (NE), very electron-dense, granules (B) March–April control specimens: the cytoplasm contains norepinephrine (NE) and epinephrine (E), of medium electron density, granules, in almost equal quantities. (C, D) December–January and (E, F) March–April treated specimens. The chromaffin cells contain few norepinephrine and epinephrine granules, and many intermediate (i) granules. In March–April treated specimens, an increase in rough endoplasmic reticulum (RER) size was found. Scale bar: a, b: 1 μm; c: 3 μm; d: 1 μm; e: 3,7 μm; f: 1.2 μm.
(Fig. 5B), nonylphenol strongly reduced norepinephrine serum level and slightly reduced epinephrine serum level. 4. Discussion The results of our study show that nonylphenol is able to influence the adrenal gland of the newt, T. carnifex. Since the effects of nonylphenol on ACTH serum levels were negligible, a direct effect on the adrenal gland may be supposed. Both the steroidogenic and the chromaffin tissues were affected by nonylphenol. The steroidogenic tissue had a similar reaction to nonylphenol during the two periods examined. The lipid/cytoplasm ratio, and the corticosteroid serum levels, decreased; this suggests that nonylphenol inhibited the synthesis and the release of both corticosteroids. Previous research, performed in vitro on rat zona fasciculatareticularis cells (Chang et al., 2010) has shown that nonylphenol directly stimulates corticosterone release. Our results confirm a direct action on the steroidogenic tissue, but opposite to what found in mammals, even if it is possible that such a difference may be due to the different type of exposure (acute and in vitro in rats vs. chronic and in vivo in newts). In contrast, the reaction of the newt
steroidogenic tissue to nonylphenol is quite similar to the reaction of the same tissue to a fungicide, thiophanate methyl, that decreased the steroidogenic activity in this species (Capaldo et al., 2006). The chromaffin tissue reacted differently to nonylphenol during the two periods examined. During the December–January period, the increase in the numeric ratio of norepinephrine granules to epinephrine granules and the increase in the epinephrine serum levels, suggest that nonylphenol stimulated the release of epinephrine. Since the total number of chromaffin granules did not change and the number of intermediate granules, i.e. the secretory granules in intermediate stage of synthesis, increased, an increase in the biosynthetic activity of the chromaffin cells may be supposed, as suggested by the increased size of the rough endoplasmic reticulum. During the March–April period, the unchanged numeric ratio of norepinephrine granules to epinephrine granules, and the decrease in the norepinephrine serum levels, suggest that nonylphenol inhibited the release of norepinephrine. Since norepinephrine is a precursor to epinephrine, and epinephrine serum levels only slightly reduced, probably the biosynthesis of catecholamines was shifted towards epinephrine, as suggested by the increase in the number of intermediate granules.
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A
1500
Control Carrier Treated
Catecholamines (pg/ml)
1200
900
600
*
300
0 NE
E
December-January
B
1500 Control Carrier
Catecholamines (pg/ml)
1200
Treated
900
357
high activity level, and the chromaffin cells produce large quantities of epinephrine (Laforgia and Capaldo, 1991; Gay et al., 2010). Therefore, probably, during this period, the PNMT is not sensitive to further stimulation by nonylphenol. However, further studies are needed to verify the mechanism of action of nonylphenol on the adrenal gland, and the reason for the varied reaction of the chromaffin tissue during the adrenal cycle periods. The results of our study show that, at environmental concentrations, nonylphenol changes the activity of the adrenal gland. Up to now, nonylphenol was shown to influence the gonads and the pituitary gland of T. carnifex (Mosconi et al., 2002; Polzonetti-Magni et al., 2004). However, considering the physiological role of the adrenal gland, not less important are the effects on this gland, found in our study. Indeed, corticosteroids regulate ion and water balance and, together with epinephrine, are involved in glucose metabolism. The adrenal hormones allow the organism to adapt to the environment, and to survive (Norris, 2007). Moreover, the adrenal gland is involved in the amphibian reproductive processes (Moore and Miller, 1984; Moore and Zoeller, 1985; Moore and Jessop, 2003; Gay et al., 2008). Since nonylphenol, at environmental concentrations, was able to alter both corticosteroid and catecholamine serum levels, it could be assumed that it interferes with the processes regulated by these hormones. Considering that nonylphenol is routinely found at microgram per liter levels around the world in rivers and lakes (Hirano et al., 2009) where amphibians, closely linked to the aquatic environment, live, it could be assumed that this endocrine disruptor may contribute to amphibian decline.
5. Conclusions
600
300
** 0 NE
E
In conclusion, our results show that nonylphenol, at environmental concentrations, influences the morphology and the activity of both the steroidogenic and the chromaffin tissues of the newt adrenal gland. Considering the wide diffusion of nonylphenol in the aquatic environment, and the physiological importance of this gland in the amphibian physiology, these results suggest that nonylphenol may contribute to amphibian decline.
March- April Fig. 5. Norepinephrine and epinephrine serum levels in (A) December–January and (B) March–April control, carrier (nonylphenol at 19 μg/L) and treated (nonylphenol at 19 μg/L) specimens. Values are mean ± SE of the mean. *Values significantly (Pb 0.05) different from the control values. **Values significantly (P b 0.001) different from the control values.
Few data are present that regard the effect of nonylphenol on the chromaffin tissue. Yanagihara et al. (2005) showed that pnonylphenol stimulates catecholamine synthesis from tyrosine, and tyrosine hydroxylase activity, in bovine adrenal medullary cells. Liu et al. (2008) found that nonylphenol suppressed the Ca(2 +) signaling coupled with nicotinic acetylcholine receptors and voltage operated Ca(2 +) channels, in bovine adrenal chromaffin cells. Our results, regarding the stimulatory role played by nonylphenol on epinephrine release, during the December–January period, agree with the results regarding the stimulation of catecholamine synthesis by p-nonylphenol (Yanagihara et al., 2005). The reason of the varied reaction of the chromaffin cells to nonylphenol during the two periods examined may be the different activity level of the enzyme phenyletanolamine-N-methyl transferase (PNMT), methylating norepinephrine into epinephrine. During the December–January period, the newt PNMT has a low activity level, and the chromaffin cells prevalently produce norepinephrine (Laforgia and Capaldo, 1991; Gay et al., 2010). Therefore, probably, during this period, the PNMT is sensitive to stimulation by nonylphenol, increasing the release of epinephrine. During the March–April period, the PNMT has a
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Kang, H.S., Noh, J.S., Gye, M.C., 2006. Effect of nonylphenol on the expression of hepatic vitellogenin mRNA in male Bombina orientalis (Boulenger). Bull. Environ. Contam. Toxicol. 77, 15–20. Kloas, W., 2002. Amphibians as a model for the study of endocrine disruptors. Int. Rev. Cytol. 216, 1–57. Laforgia, V., Capaldo, A., 1991. Annual cycle of the chromaffin cells of Triturus cristatus. J. Morphol. 208, 83–90. Levy, G., Lutz, I., Kruger, A., Kloas, W., 2004. Bisphenol A induces feminization in Xenopus laevis tadpoles. Environ. Res. 94, 102–111. Liu, P.S., Liu, G.H., Chao, W.L., 2008. Effects of nonylphenol on the calcium signal and catecholamine secretion coupled with nicotinic acetylcholine receptors in bovine adrenal chromaffin cells. Toxicology 244, 77–85. Lutz, I., Jie, Z., Opitz, R., Kloas, W., Ying, X., Menzel, R., Steinberg, C.E., 2005. Environmental signals: synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis. Chemosphere 61, 1183–1188. Mackenzie, C.A., Berrill, M., Metcalfe, C., Pauli, B.D., 2003. Gonadal differentiation in frogs exposed to estrogenic and antiestrogenic compounds. Environ. Toxicol. Chem. 22, 2466–2475. Mariager, N.M., Falkenberg Hansen, S., Dahl, M.P.J., 2005. Effects of 4-n-nonylphenol on the Prosobranch Snail Marisa cornuarietis. RUDAR (Roskilde University Digital Archive)http://hdl.handle.net/1800/1020. Moore, I.T., Jessop, T.S., 2003. Stress, reproduction, and adrenocortical modulation in amphibians and reptiles. Horm. Behav. 43, 39–47. Moore, F.L., Miller, L.J., 1984. Stress-induced inhibition of sexual behaviour: corticosterone inhibits courtship behaviours of a male amphibian (Taricha granulosa). Horm. Behav. 18, 400–410. Moore, F.L., Zoeller, R.T., 1985. Stress-induced inhibition of reproduction: evidence of suppressed secretion of LH–RH in an amphibian. Gen. Comp. Endocrinol. 60, 252–258.
Mosconi, G., Carnevali, O., Franzoni, M.F., Cottone, E., Lutz, I., Kloas, W., Yamamoto, K., Kikuyama, S., Polzonetti-Magni, A.M., 2002. Environmental estrogens and reproductive biology in amphibians. Gen. Comp. Endocrinol. 126, 125–129. Noriega, C.N., Hayes, T.B., 2000. DDT congener effects on secondary sex coloration in the reed frog Hyperolius argus: a partial evaluation of the Hyperolius argus endocrine screen. Comp. Biochem. Physiol. B 126, 231–237. Norris, D.O., 2007. Vertebrate Endocrinology, 4th ed. Academic Press, Amsterdam. Polzonetti-Magni, A.M., Mosconi, G., Palermo, F., Cottone, E., Forno, S., Franzoni, M.F., 2004. Amphibians as a model for studying xenoestrogen-dependent neuroendocrine disruption. Ital. J. Zool. 71, 95–100. Renner, R., 2002. Amphibian declines. Conflict brewing over herbicide's link to frog deformities. Science 298, 938–939. Sone, K., Hinago, M., Kitatama, A., Morokuma, J., Ueno, N., Watanabe, H., Iguchi, T., 2004. Effects of 17β-estradiol, nonylphenol, and bisphenol-A on developing Xenopus laevis embryos. Gen. Comp. Endocrinol. 138, 228–236. Steinberg, C.E., Hoss, S., Kloas, W., Lutz, I., Meinelt, T., Pflugmacher, S., Wiegand, C., 2004. Hormonelike effects of humic substances on fish, amphibians, and invertebrates. Environ. Toxicol. 19, 409–411. Vazquez-Duhalt, R., Marquez-Rocha, F., Ponce, E., Licea, A.F., Viana, M.T., 2005. Nonylphenol, an integrated vision of a pollutant. Scientific review. Appl. Ecol. Environ. Res. 4 (1), 1–25. Yanagihara, N., Toyohira, Y., Ueno, S., Tsutsui, M., Utsunomiya, K., Liu, M., Tanaka, K., 2005. Stimulation of catecholamine synthesis by environmental estrogenic pollutants. Endocrinology 146, 265–272. Yang, F.X., Xu, Y., Wen, S., 2005. Endocrine-disrupting effects of nonylphenol, bisphenol A, and p, p -DDE on Rana nigromaculata tadpoles. Bull. Environ. Contam. Toxicol. 75, 1168–1175. Zerani, M., Gobbetti, A., 1993. Corticosterone during the annual reproductive cycle and in sexual behaviour in the crested newt, Triturus carnifex. Horm. Behav. 27, 29–37.
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Comparative Biochemistry and Physiology, Part C journal homepage: www.elsevier.com/locate/cbpc
Endocrine-disrupting effects of nonylphenol in the newt, Triturus carnifex (Amphibia, Urodela) Anna Capaldo a,⁎, Flaminia Gay a, Salvatore Valiante a, Maria De Falco a, Rosaria Sciarrillo b, Massimo Maddaloni a, Vincenza Laforgia a a b
Department of Biological Sciences, Section of Evolutive and Comparative Biology, University Federico II, Via Mezzocannone 8, 80134 Naples, Italy Department of Biological and Environmental Sciences, University of Sannio, Benevento, Italy
a r t i c l e
i n f o
Article history: Received 31 August 2011 Received in revised form 14 October 2011 Accepted 14 October 2011 Available online 25 October 2011 Keywords: Adrenal gland disruption Amphibian endocrine disruption Environmental contaminants Nonylphenol Triturus carnifex Water pollution
a b s t r a c t The aim of our study was to verify whether environmental concentrations of nonylphenol influenced the adrenal gland of Triturus carnifex. Newts were exposed to 19 μg/L nominal concentration of nonylphenol throughout the periods of December–January and March–April, corresponding to different stages of the chromaffin cell functional cycle. The morphological features of the steroidogenic and chromaffin tissues, and the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine were evaluated. Nonylphenol did not influence ACTH serum levels. During the two periods examined, the steroidogenic tissue had the same reaction: the quantity of cytoplasmic lipids, and the corticosteroid serum levels, decreased, suggesting the inhibition of synthesis and release of corticosteroids. During the two periods examined, the chromaffin tissue reacted differently to nonylphenol. During December–January, the numeric ratio of norepinephrine granules to epinephrine granules, and the epinephrine serum levels, increased, suggesting the stimulation of epinephrine release. During March–April, the numeric ratio of norepinephrine granules to epinephrine granules did not change, and the norepinephrine serum levels decreased, suggesting the inhibition of norepinephrine release. Our results show that nonylphenol influences the activity of the newt adrenal gland; considering the physiological role of this gland, our results suggest that nonylphenol may contribute to amphibian decline. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Nonylphenol ethoxylates (NPEs) are surfactants used worldwide, found in many types of products including detergents, plastics, emulsifiers, pesticides, and industrial and consumer cleaning products. As a consequence of their use in a variety of products, they are quite common in rivers and other aquatic environments that receive sewage discharges (Arukwe et al., 2000). Nonylphenol ethoxylates are transformed in the environment by microorganisms to form more toxic compounds, such as nonylphenol (NP) and short-chain nonylphenol ethoxylates (Vazquez-Duhalt et al., 2005). Because of the enhanced resistance towards biodegradation, toxicity, estrogenic effects, and ability to bioaccumulate in aquatic organisms of nonylphenol, it has been regarded as the most critical metabolite of NPE (Arukwe et al., 2000). Endocrine disruptors, such as nonylphenol, are considered a contributing factor in the global decline of amphibians (Renner, 2002); indeed, due to their highly permeable skin and their lifecycle which goes through both aquatic and terrestrial stages, amphibians are very
⁎ Corresponding author. Tel.: + 39 081 2535037; fax: + 39 081 2535035. E-mail addresses: [email protected], [email protected] (A. Capaldo), [email protected] (F. Gay), [email protected] (S. Valiante), [email protected] (M. De Falco), [email protected] (R. Sciarrillo), [email protected] (M. Maddaloni), [email protected] (V. Laforgia). 1532-0456/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2011.10.004
sensitive to waterborne chemicals (Kloas, 2002; Bogi et al., 2003; Levy et al., 2004; Polzonetti-Magni et al., 2004; Steinberg et al., 2004; Lutz et al., 2005) and can be considered excellent bioindicators of the general health of the environment (Noriega and Hayes, 2000). The greater part of studies evaluating nonylphenol effects on amphibian physiology found alterations in the reproductive biology. Environmental concentrations of nonylphenol caused malformations of tail flexure in Rana nigromaculata tadpoles during metamorphosis (Yang et al., 2005) and altered gonadal differentiation in Rana pipiens and R. sylvatica (Mackenzie et al., 2003). Higher doses of nonylphenol inhibited the rate of bullfrog (Rana catesbeiana) tadpole metamorphic progression and tail resorption (Christensen et al., 2005) and induced abnormalities in developing Xenopus laevis embryos (Sone et al., 2004). Moreover, intraperitoneal injections of nonylphenol induced hepatic vitellogenin mRNA in male Bombina orientalis (Kang et al., 2006). Considering the physiological importance of the adrenal gland of amphibians,it is surprising that no studies have been undertaken that regard the effects of nonylphenol on this gland. Indeed, the adrenal gland plays a key role in the stress response, which includes the release of both corticosteroids and catecholamines. Corticosterone and, to a lesser extent, aldosterone, are gluconeogenic and hyperglycaemic, but the primary role of these steroids appears to be the regulation of ion and water balance. Epinephrine is a potent stimulator of lypolysis in fat bodies and of glycogenolysis in both the liver and muscle and is
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therefore hyperglycaemic. These hormones induce changes in metabolism and/or ionic regulation that work to combat physiological factors and to eliminate or to neutralize the stressful stimulus, allowing the organism to adapt to its environment, and to survive (Norris, 2007). Moreover, the adrenal gland is involved in the amphibian reproductive processes (Moore and Miller, 1984; Moore and Zoeller, 1985; Moore and Jessop, 2003) and, in T. carnifex, its activity is correlated with some events of the reproductive cycle (Gay et al., 2008). Therefore, the aim of our research was to verify whether nonylphenol was able to influence the amphibian adrenal gland. To this aim, we chose a urodele amphibian, the newt Triturus carnifex, for the experimental model. Urodele amphibians spend a great part of their life in the aquatic environment, and therefore can easily adsorb waterborne chemicals through their permeable skin. In addition, T. carnifex has the features of an ideal bioindicator, due to its high sensitivity to chemicals (Kloas, 2002; Capaldo et al., 2006). The studies investigating the effects of nonylphenol on this species examined the gonads and the pituitary gland, showing the induction of plasma vitellogenin in the male T. carnifex (Mosconi et al., 2002; Polzonetti-Magni et al., 2004), the change in secondary sex characteristics, and the increase in the androgen hormone levels in plasma. Moreover, the inhibition of gonadotropin and prolactin secretion by pituitary, and the increase in the number of pituitary prolactin cells, were found (Mosconi et al., 2002). As far as we know, there are no data published concerning nonylphenol effects on the newt adrenal gland. 2. Materials and methods 2.1. Reagents Nonylphenol (analytical grade, 98%) and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). A stock solution of nonylphenol was prepared in DMSO at a concentration of 1 mg/mL (Mariager et al., 2005; Hirano et al., 2009).
was changed every four days in all the tanks. The test was carried out in triplicate. One month after the beginning of the experiment (January in the first experiment; April in the second experiment), the newts were anaesthetized by hypothermia, chilling them in chipped ice, within 5 min after removal from tank. Blood was immediately collected by heart puncture, between 11:00 h and 14:00 h, centrifuged for 15 min at 2000 g and serum was collected and stored, together with standards to correct for degradation, at −22 °C until assayed, as previously described (Capaldo et al., 2006). The experiment was carried out in accordance with the European Directive 2010/63/EU for animal experiments, and authorized by the National Committee of the Italian Ministry of Health for in vivo experimentation (Dept. for Veterinary Public Health, Nutrition and Food Safety). 2.4. Transmission electron microscopy The animals were killed by decapitation immediately after collection of blood samples. The adrenals were fixed in 2.5% glutaraldehyde in Millonig's phosphate buffer at pH 7.4 at 4 °C, rinsed in buffer, and postfixed in 1% OsO4 (2 h, 4 °C), dehydrated in ethanol, cleared in propylene oxide, embedded in epoxy resin, and polymerized. Ultrathin sections (300 nm) were cut with glass knives on a Reichert– Jung ultracut ultramicrotome (SUPER NOVA), collected on formvarcoated copper grids, stained with solutions of uranyl acetate and lead citrate, and observed with a Philips EM 301 transmission electron microscope at the Interdepartmental Center of Services for Electron Microscopy (C.I.S.M.E.) (Naples). For each specimen from each group (ten newts per group), ten low-power micrographs of the steroidogenic tissue and ten of the chromaffin tissue, each containing at least four cells, were subject to morphometric investigation by a computerized image analysis system (KS 300 for Windows 98, Zeiss). The area occupied by the lipid droplets in the steroidogenic cells was calculated using the following formula:
2.2. Animals Adult male specimens of T. carnifex (mean mass 8.0 g.), captured in the field around Naples, were kept in 50 L glass tanks (10 newts per tank, with a loading rate of 1.6 g newt/L water), under a natural photoperiod, in dechlorinated, well-aerated tap water, at seasonal temperature (12 °C mean temperature during December–January period; 20 °C mean temperature during March–April period). The animals were fed minced cow liver and used after an acclimation period of two weeks. 2.3. Experimental design Adult male newts were exposed to a nominal, environmental concentration of nonylphenol, included in the range internationally reported for surface water (between 5 ng/L and 180 μg/L) (Mariager et al., 2005). The concentration used (19 μg/L) and the exposure period (one month) were selected on the basis of the results of preliminary dose–response and time-course tests. At the beginning of December and at the beginning of March, corresponding to different stages of the chromaffin cell functional cycle (Gay et al., 2010), thirty adult male newts were collected (sixty animals in all) and kept under the previously described conditions. In both months, after an acclimation period of two weeks, the newts were divided into three groups (treated, carrier and control), each containing ten specimens. Each group was kept in 50 L glass tank (loading rate of 1.6 g newt/L water). Treated newts were exposed to a nominal nonylphenol concentration of 19 μg/L. Since the stock solution was prepared in DMSO, ten newts were kept in a glass tank and exposed to the same concentration of DMSO as the treated newts (0.019 mL/L); ten untreated newts were kept in a tank with tap water only. The water
353
Lipid=cytoplasm ratio ¼
Mean total lipid area μm2 Mean total cytoplasmic area μm2
:
In the chromaffin cells, the mean total number of chromaffin granules/μm 2; the mean number of norepinephrine and epinephrine granules/μm 2; the numeric ratio of norepinephrine granules to epinephrine granules (NE/E ratio); the mean number of intermediate granules/μm 2, i.e. the secretory granules in intermediate stage of synthesis, were calculated. The sampling criteria to selectively discriminate between norepinephrine, epinephrine and intermediate granules were the following: granules recognized as norepinephrine granules were of variable shape, with a very electron-dense and compact core filling the granule; the core was separated from the limiting membrane. Granules identified as epinephrine granules were roundish, homogeneous, with a finely granular core of medium electron density, separated from the limiting membrane by a narrow electron-lucent space. All the intermediate forms of granules, not showing these distinctive features, were considered intermediate granules (Laforgia and Capaldo, 1991). 2.5. Hormone assay Aldosterone and corticosterone serum levels were determined by radioimmunoassay (RIA) as previously described (Capaldo et al., 2006). Briefly, non hemolyzed serum samples (80 μL for aldosterone and 30 to 40 μL for corticosterone) were incubated for 30 min at 37 °C with known amounts of radioactive steroids ( 3H-aldosterone, and 3 H-corticosterone from Bio-Rad, Hercules, CA, USA) in 0.06 M Naphosphate buffer containing 0.01 M EDTA disodium salt and 0.1%
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BSA pH 7.4. Samples were applied to an extraction column (Sep-Pak C18, Waters, Milford, MA, USA) and washed with 500 μL of pure methanol. Methanol extracts were dried at 37 °C under vacuum and redissolved in 1400 μL of PBS. An aliquot was taken to determine the labeled hormone recovery and on two other aliquots aldosterone and corticosterone were assayed by RIA. After incubation with rabbit antiserum (Biogenesis, Poole, UK) for 30 min at 37 °C and for another 2 h in an ice bath, dextran-coated charcoal was used to separate free from bound steroids. After immersion for 10 min in an ice bath and centrifugation (600 g), a supernatant aliquot was counted with a liquid scintillation spectrometer (Tri-Carb Packard, GMI, Albertville, MN, USA). Extraction yields ranged from 80% to 90% for both hormones. Data were obtained through a standard calibration curve linearized with a log–logit method and corrected for individual extraction yield. Sensitivity was 5 pg/tube for aldosterone and corticosterone. Intra-assay coefficient of variation was 10%, and inter-assay coefficient of variation was 12% for both steroids. ACTH concentrations were measured in 100 μL of plasma by a two-site immunoradiometric assay using mouse monoclonal antibodies (Diagnostic Products Corp.) as previously described (De Falco et al., 2004). Cross-reactivities of the ACTH antiserum as determined by the kit manufacturer were 0.03% for a-MSH, 0.01% for β-MSH, and 0.02 for β-endorphin. Sensitivity was 0.1 pg.mL − 1 as determined by the kit manufacturer, and the inter- and intra-assay coefficients of variation were 10% and 6%, respectively. Norepinephrine and epinephrine levels were determined in 150 μL serum. For catecholamine extraction, 50 μL of dihydroxybenzylamine was added as an internal standard. Ten milligrams activated aluminum oxide (Sigma) was used as adsorbent for catecholamines and the internal standard. After 15 min shaking and centrifugation, the supernatant was removed and the aluminum oxide containing the adsorbed catecholamines and the internal standard was washed three times with 1 mL distilled water by shaking, centrifuging, and discarding the supernatant-extracted samples using high performance liquid chromatography (HPLC), with electrochemical detection, according to the method previously used in T. carnifex (Capaldo et al., 2006). Electrochemical HPLC detection was carried out using an acid eluant;
NE and E levels were calculated in comparison to the internal standard (dihydroxybenzylamine). The detection limit for NE and E was around 20 pg. 2.6. Statistical analysis All data were expressed as mean± standard error of mean (SEM). Each experimental group consisted of ten animals of the same weight and sex. The numeric data were obtained considering, for each experimental group, an equal number of micrographs at the same magnification (100 micrographs), and examining only the cytoplasmic areas, excluding the nucleus. The control and experimental data of all the groups were normalized and then tested together for significance using one-way analysis of variance (ANOVA), followed by Duncan's test for multigroup comparison and Student's t test for between group comparison. Differences were considered significant when P b 0.05. 3. Results 3.1. Steroidogenic tissue The “adrenal gland” of urodeles includes numerous discrete bodies scattered on the ventral surface of the functional opistonephros kidney, close to its medial margin. The bodies contain tightly intermingled steroidogenic and chromaffin cells. The steroidogenic cells are characterized by the presence of many mitochondria, a smooth endoplasmic reticulum arranged in tubules and vesicles, and a large quantity of lipid droplets (Hanke, 1978). The cells of December–January (Fig. 1A) and March–April (Fig. 1B) control newts, showed a similar morphology: a cytoplasm rich in lipid droplets and numerous mitochondria with tubular cristae. The lipid/cytoplasm ratio was 0.45 in December–January and 0.40 in March–April (Table 1). The exposure to nonylphenol always strongly reduced the quantity of lipid droplets (Fig. 1C, D); the lipid/cytoplasm ratio decreased (Table 1). The steroidogenic cells mainly produce the corticosteroids aldosterone and corticosterone, according to an annual pattern. Corticosterone levels are low from October to November, peak in January,
Fig. 1. Electron micrographs of steroidogenic cells of Triturus carnifex adrenal gland. (A) December–January and (B) March–April control specimens. The cells show many lipid droplets (L) and mitochondria (M) filling the cytoplasm. (C) December–January and (D) March–April treated specimens. The cytoplasm shows a strong decrease in lipid (L) droplets. Scale bar: a: 2 μm; b: 1.8 μm; c: 3 μm; d: 2.3 μm.
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355
Table 1 Mean ± SE of the different parameters evaluated in control, carrier (DMSO at 0.019 mL/L) and treated (nonylphenol at 19 μg/L) specimens. Chromaffin g/μm2
Intermediate g/μm2
NE g/μm2
E g/μm2
NE/E ratio
Lip/cyt.ratio
December–January Control Carrier Treated
7.59 ± 2.01 7.56 ± 1.97 6.15 ± 1.01
0.003 ± 0.0009 0.002 ± 0.0004 2.23 ± 1.51◊
7.38 ± 2.13 7.37 ± 1.89 3.90 ± 1.02◊
0.21 ± 0.001 0.19 ± 0.005 0.02 ± 0.003◊
35.1/1 38.7/1 195/1
0.45 0.44 0.15
March–April Control Carrier Treated
7.72 ± 2.41 7.74 ± 2.26 5.38 ± 1.16a
0.07 ± 0.001 0.08 ± 0.005 3.24 ± 1.03b
3.89 ± 1.61 3.85 ± 1.46 1.05 ± 0.03b
3.83 ± 1.13 3.89 ± 1.58 1.09 ± 0.03b
1.0/1 1.0/1 0.9/1
0.40 0.39 0.25
a b
Significantly (P b 0.05) different from the control values. Significantly (P b 0.001) different from the control values.
fall in February and remain low until March, after which they peak in July (Zerani and Gobbetti, 1993). Aldosterone levels are low from September to November, then rise from December up to a maximum in April, decrease in May, with values similar to those found during December–March period, and remain steady until July (Gay et al., 2010). During both periods, December–January and March–April, nonylphenol reduced aldosterone and corticosterone serum levels (Fig. 2). During both periods, nonylphenol did not influence ACTH serum levels (Fig. 3).
A
6000
Control Carrier
Corticosteroids (pg/ml)
5000
Treated
4000
3000
2000
1000
**
**
0
Aldosterone
Corticosterone
B
6000
Corticosteroids (pg/ml)
December-January
5000
3.2. Chromaffin tissue The chromaffin tissue of T. carnifex is composed of a single cell type that has an annual cycle in which the quantities of norepinephrine and epinephrine change during the year. During the December– February and May–August periods, large quantities of norepinephrine and small quantities of epinephrine are present in the chromaffin cells. During the March–April and September–November periods, the production of epinephrine increases, becoming similar to norepinephrine. During both periods, a small quantity of intermediate granules is present (Laforgia and Capaldo, 1991; Gay et al., 2010). In December– January control cells (Fig. 4A), almost exclusively norepinephrine granules were present. In March–April control cells (Fig. 4B), norepinephrine and epinephrine granules were both present in almost equal quantities. Moreover, a small quantity of intermediate granules was always present (Table 1). In December–January treated cells (Figs. 4C, D), the cytoplasm showed many chromaffin granules; the rough endoplasmic reticulum appeared increased in size (Fig. 4D). As it can be seen in Table 1, the total number of chromaffin granules did not change, but there was a strong decrease in the number of norepinephrine and epinephrine granules, and an increase in the number of intermediate granules, and in the numeric ratio of norepinephrine granules to epinephrine granules. In March–April treated cells (Fig. 4E, F), the cytoplasm did not show many chromaffin granules; most of them were intermediate granules (Fig. 4F). As it can be seen in Table 1, there was a decrease in the total number of chromaffin granules, and in the number of norepinephrine and epinephrine granules. The number of intermediate granules increased, whereas the numeric ratio of norepinephrine granules to epinephrine granules did not change. During the December–January period (Fig. 5A), nonylphenol slightly reduced norepinephrine serum levels and significantly increased epinephrine serum levels, whereas, during the March–April period
Control Carrier Treated
700
December-January March-April
600
ACTH (pg/ml)
4000 3000 2000 1000
Aldosterone
400 300 200
**
**
0
500
100 Corticosterone
March-April Fig. 2. Aldosterone and corticosterone serum levels in (A) December–January and (B) March–April control, carrier (DMSO at 0.019 mL/L) and treated (nonylphenol at 19 μg/L) specimens. Values are mean ± SE of the mean. **Values significantly (P b 0.001) different from the control values.
0
Control
Carrier
Treated
Fig. 3. ACTH serum levels in control, carrier (DMSO at 0.019 mL/L) and treated (nonylphenol at 19 μg/L) specimens. Values are mean ± SE of the mean. Values in the treated groups do not significantly differ from the control values.
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Fig. 4. Electron micrographs of the chromaffin cells of Triturus carnifex adrenal gland. (A) December–January control specimens: the cytoplasm prevalently contains norepinephrine (NE), very electron-dense, granules (B) March–April control specimens: the cytoplasm contains norepinephrine (NE) and epinephrine (E), of medium electron density, granules, in almost equal quantities. (C, D) December–January and (E, F) March–April treated specimens. The chromaffin cells contain few norepinephrine and epinephrine granules, and many intermediate (i) granules. In March–April treated specimens, an increase in rough endoplasmic reticulum (RER) size was found. Scale bar: a, b: 1 μm; c: 3 μm; d: 1 μm; e: 3,7 μm; f: 1.2 μm.
(Fig. 5B), nonylphenol strongly reduced norepinephrine serum level and slightly reduced epinephrine serum level. 4. Discussion The results of our study show that nonylphenol is able to influence the adrenal gland of the newt, T. carnifex. Since the effects of nonylphenol on ACTH serum levels were negligible, a direct effect on the adrenal gland may be supposed. Both the steroidogenic and the chromaffin tissues were affected by nonylphenol. The steroidogenic tissue had a similar reaction to nonylphenol during the two periods examined. The lipid/cytoplasm ratio, and the corticosteroid serum levels, decreased; this suggests that nonylphenol inhibited the synthesis and the release of both corticosteroids. Previous research, performed in vitro on rat zona fasciculatareticularis cells (Chang et al., 2010) has shown that nonylphenol directly stimulates corticosterone release. Our results confirm a direct action on the steroidogenic tissue, but opposite to what found in mammals, even if it is possible that such a difference may be due to the different type of exposure (acute and in vitro in rats vs. chronic and in vivo in newts). In contrast, the reaction of the newt
steroidogenic tissue to nonylphenol is quite similar to the reaction of the same tissue to a fungicide, thiophanate methyl, that decreased the steroidogenic activity in this species (Capaldo et al., 2006). The chromaffin tissue reacted differently to nonylphenol during the two periods examined. During the December–January period, the increase in the numeric ratio of norepinephrine granules to epinephrine granules and the increase in the epinephrine serum levels, suggest that nonylphenol stimulated the release of epinephrine. Since the total number of chromaffin granules did not change and the number of intermediate granules, i.e. the secretory granules in intermediate stage of synthesis, increased, an increase in the biosynthetic activity of the chromaffin cells may be supposed, as suggested by the increased size of the rough endoplasmic reticulum. During the March–April period, the unchanged numeric ratio of norepinephrine granules to epinephrine granules, and the decrease in the norepinephrine serum levels, suggest that nonylphenol inhibited the release of norepinephrine. Since norepinephrine is a precursor to epinephrine, and epinephrine serum levels only slightly reduced, probably the biosynthesis of catecholamines was shifted towards epinephrine, as suggested by the increase in the number of intermediate granules.
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A
1500
Control Carrier Treated
Catecholamines (pg/ml)
1200
900
600
*
300
0 NE
E
December-January
B
1500 Control Carrier
Catecholamines (pg/ml)
1200
Treated
900
357
high activity level, and the chromaffin cells produce large quantities of epinephrine (Laforgia and Capaldo, 1991; Gay et al., 2010). Therefore, probably, during this period, the PNMT is not sensitive to further stimulation by nonylphenol. However, further studies are needed to verify the mechanism of action of nonylphenol on the adrenal gland, and the reason for the varied reaction of the chromaffin tissue during the adrenal cycle periods. The results of our study show that, at environmental concentrations, nonylphenol changes the activity of the adrenal gland. Up to now, nonylphenol was shown to influence the gonads and the pituitary gland of T. carnifex (Mosconi et al., 2002; Polzonetti-Magni et al., 2004). However, considering the physiological role of the adrenal gland, not less important are the effects on this gland, found in our study. Indeed, corticosteroids regulate ion and water balance and, together with epinephrine, are involved in glucose metabolism. The adrenal hormones allow the organism to adapt to the environment, and to survive (Norris, 2007). Moreover, the adrenal gland is involved in the amphibian reproductive processes (Moore and Miller, 1984; Moore and Zoeller, 1985; Moore and Jessop, 2003; Gay et al., 2008). Since nonylphenol, at environmental concentrations, was able to alter both corticosteroid and catecholamine serum levels, it could be assumed that it interferes with the processes regulated by these hormones. Considering that nonylphenol is routinely found at microgram per liter levels around the world in rivers and lakes (Hirano et al., 2009) where amphibians, closely linked to the aquatic environment, live, it could be assumed that this endocrine disruptor may contribute to amphibian decline.
5. Conclusions
600
300
** 0 NE
E
In conclusion, our results show that nonylphenol, at environmental concentrations, influences the morphology and the activity of both the steroidogenic and the chromaffin tissues of the newt adrenal gland. Considering the wide diffusion of nonylphenol in the aquatic environment, and the physiological importance of this gland in the amphibian physiology, these results suggest that nonylphenol may contribute to amphibian decline.
March- April Fig. 5. Norepinephrine and epinephrine serum levels in (A) December–January and (B) March–April control, carrier (nonylphenol at 19 μg/L) and treated (nonylphenol at 19 μg/L) specimens. Values are mean ± SE of the mean. *Values significantly (Pb 0.05) different from the control values. **Values significantly (P b 0.001) different from the control values.
Few data are present that regard the effect of nonylphenol on the chromaffin tissue. Yanagihara et al. (2005) showed that pnonylphenol stimulates catecholamine synthesis from tyrosine, and tyrosine hydroxylase activity, in bovine adrenal medullary cells. Liu et al. (2008) found that nonylphenol suppressed the Ca(2 +) signaling coupled with nicotinic acetylcholine receptors and voltage operated Ca(2 +) channels, in bovine adrenal chromaffin cells. Our results, regarding the stimulatory role played by nonylphenol on epinephrine release, during the December–January period, agree with the results regarding the stimulation of catecholamine synthesis by p-nonylphenol (Yanagihara et al., 2005). The reason of the varied reaction of the chromaffin cells to nonylphenol during the two periods examined may be the different activity level of the enzyme phenyletanolamine-N-methyl transferase (PNMT), methylating norepinephrine into epinephrine. During the December–January period, the newt PNMT has a low activity level, and the chromaffin cells prevalently produce norepinephrine (Laforgia and Capaldo, 1991; Gay et al., 2010). Therefore, probably, during this period, the PNMT is sensitive to stimulation by nonylphenol, increasing the release of epinephrine. During the March–April period, the PNMT has a
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