Aromatase mRNA expression in the brain of adult Xenopus laevis exposed to Lambro river water and endocrine disrupting compounds

General and Comparative Endocrinology 168 (2010) 262–268

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Aromatase mRNA expression in the brain of adult Xenopus laevis exposed to Lambro river water and endocrine disrupting compounds A. Massari a,*, R. Urbatzka c, A. Cevasco a, L. Canesi a, C. Lanza a, L. Scarabelli a, W. Kloas b, A. Mandich a,d a

Department of Biology, University of Genoa, Italy Department of Ecophysiology and Aquaculture, Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany c CIIMAR, Centre of Marine and Environmental Research, Porto, Portugal d INBB, Centro Interuniversitario, Rome, Italy b

a r t i c l e

i n f o

Article history: Received 30 November 2009 Revised 8 April 2010 Accepted 20 April 2010 Available online 22 April 2010 Keywords: Xenopus laevis Endocrine disruption RQ-RT-PCR Aromatase

a b s t r a c t Aromatase P450 (P450 arom; Cyp19) is a key enzyme for vertebrate reproduction and brain development that catalyzes the conversion of androgens to estrogens. The aim of this study was to improve the knowledge on EDC effects by analysing their potential impact on brain P450 arom in adult Xenopus laevis exposed for 4 weeks to an environmental sample, the water of the river Lambro (LAM), the most polluted tributary of the Po river in North Italy. Other groups were exposed to individual compounds 108 M tamoxifen (TAM), ethinylestradiol (EE2), flutamide (FLU) and methyldihydrotestosterone (MDHT) known for their (anti)estrogenic and (anti)androgenic modes of action. Expression of CYP19 was evaluated in brain extracts by quantitative RT-PCR, using a pair of primers located in the open reading frame (ORF) that allowed the simultaneous amplification of all transcripts (Aro-ORF) and a pair of primers specific for brain aromatase (Aro-B). Significant increase in Aro-ORF and Aro-B mRNA levels were observed in both females and males exposed to LAM. Different changes were observed for the model compounds using two pairs of primers. Aro-ORF mRNA expression was significantly increased in EE2 and MDHT exposed males and in FLU-exposed females, while it was significantly decreased in TAM exposed females. Aro-B mRNA was significantly increased in both sexes exposed to FLU and decreased in TAM exposed females. In conclusion, aromatase mRNA in the brain of X. laevis was regulated differentially in a gender specific manner by certain (anti)estrogenic and (anti)androgenic EDCs, supporting previous hypotheses that diverse compounds present in the river Lambro may induce feminization and demasculinization effects. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Aromatase cytochrome P450 (P450 arom), the product of the aromatase gene Cyp19, is the terminal enzyme in the estrogen biosynthetic pathway that catalyzes the transformation of testosterone to estradiol-17b (E2). The gonads are considered to be the major source of estrogens in the body, however, local estrogen production in other sites, especially in the brain, is also very important, since it is essential for gonad development as well as for other diverse physiological processes. In the brain, E2 regulates multiple functions like control of neuroendocrine system, sex differentiation, activation of sexual behavior and stimulation of gonadal activity by increasing gonadotropin secretion in the pituitary (Balthazart and Ball, 1998; Forlano et al., 2006; Guerriero et al., 2000; Hutchinson, 1993; Lephart, 1996, 1997; McEwen, 1997). In

* Corresponding author. Address: Department of Biology, University of Genoa, V. le Benedetto XV, 5, 16132 Genoa, Italy. Fax: +39 010 3538047. E-mail address: [email protected] (A. Massari). 0016-6480/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2010.04.012

amphibians, P450 aromatase is encoded by one CYP19 gene leading to two gonad and one brain P450 aromatase transcripts which differ in their 5-untranslated region (UTR) but contain an identical open reading frame (Iwabuchi et al., 2007). These transcripts have been detected at different expression levels in brains and gonads. Brain P450 arom is mainly expressed in the embryonic and adult brain, but also in undifferentiated gonads and in adult ovaries and testis. Gonad P450 aroms 1 and 2 are expressed in undifferentiated gonads and in adult ovaries and testis, but also weakly in the brain (Iwabuchi et al., 2007). Aromatase is considered as one of the potential targets of the many environmental chemicals, called Endocrine Disrupting Compounds (EDC) that can interfere with the endocrine system affecting the reproductive biology of aquatic vertebrates (Hogan et al., 2008; Kloas, 2002; Kloas et al., 2009; MacKenzie et al., 2003; Thibaut and Porte, 2004). Changes in aromatase expression may alter the rate of estrogen and androgen production and disturb the local and systemic levels of these steroids (Cheshenko et al., 2008; Kazeto et al., 2004; Kishida and Callard, 2001; Mann et al., 2009; Murphy et al., 2006; Sanderson et al., 2002).

A. Massari et al. / General and Comparative Endocrinology 168 (2010) 262–268

Xenopus laevis has been widely used as a sensitive model for the screening of EDCs in vitro (Kloas et al., 1999; Lutz et al., 2005) and in vivo (Bögi et al., 2002; Kloas, 2002; Levy et al., 2004; Urbatzka et al., 2006). In this study, adult X. laevis were exposed to 17a-ethinylestradiol (EE2) as estrogenic compound, tamoxifen (TAM) as anti-estrogenic compound, 17a-methyldihydrotestosterone (MDHT) as androgenic compound, and flutamide (FLU) as antiandrogenic compound, in a 4 week exposure at the concentration of 108 M. The condition of exposure where chosen in order to mimic environmental exposure conditions to both EDC mixtures and individual EDCs as previously described (Cevasco et al., 2008; Urbatzka et al., 2007a). Furthermore, adult X. laevis were exposed to Lambro river water to assess the interference of an environmental mixture. The river Lambro is a heavily polluted tributary of the river Po that drains a sub-basin densely inhabited, characterized by many industrial activities and extensive plain agriculture (Camusso et al., 1995). Recent studies have demonstrated that water and sediment of the Lambro river are mainly polluted by substances exhibiting estrogenic (Viganò et al., 2008) and anti-androgenic mode of actions (MOAs) (Urbatzka et al., 2007b). The aim of the study was to compare the possible effects of (anti)estrogenic and (anti)androgenic EDC model compounds and of Lambro river water samples on the level of aromatase mRNA in the brain of adult X. laevis. Both total (Aro-ORF) and brain (Aro-B) transcripts were evaluated by quantitative real-time PCR assays. 2. Materials and methods 2.1. In vivo exposure All exposure protocols were performed as previously described (Urbatzka et al., 2007a; Cevasco et al., 2008) within the activities of the EU-project EASYRING (Contract No. QLK4-CT-2002-02286). Adult X. laevis (3–4 years old) were taken from the breeding stock of the Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin. The frogs were fed twice a week and kept under a 12 h light:12 h dark cycle. For the exposure, adult male and female X. laevis were transferred to aerated 10 L tanks containing reconstituted tap water using distilled water supplemented with 2.5 g marine salt (Tropic Marin Meersalz, Tagis, Dreieich, Germany). X. laevis were exposed to tamoxifen (TAM), ethinylestradiol (EE2), flutamide (FLU) and methyldihydrotestosterone (MDHT), chosen as (anti)estrogenic and (anti)androgenic compounds, respectively, and to Lambro river water. Chemicals were dissolved in ethanol (Roth, Karlsruhe, Germany) and a solvent control (0.001% ethanol) was included in the experimental design of the study. The treatment lasted 4 weeks and consisted of eight females and eight males per compound equally distributed into two 10 L tanks containing four females and four males, respectively. The test concentration of the chemicals was 108 M. Rearing water and chemicals were renewed completely every Monday, Wednesday and Friday. During exposure, X. laevis were fed once a week with commercial fish diet (Fisch-Fit, Interquell, Wehringen, Germany) and water temperature was adjusted to 22 ± 1 °C.

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2.3. Sampling and RNA-isolation At the end of exposure, all X. laevis frogs were sacrificed. Whole brain (n = 6 for treatment) including the pituitary was dissected, transferred to an Eppendorf tube and snap-frozen in liquid nitrogen. Isolation of total RNA was carried out using the phenolic reagent TRIZOL (Invitrogen, Karlsruhe, Germany) according to the manufacturers instructions as described in Bögi et al. (2002). The amount of isolated total RNA ranged between 10 and 32 lg per tissue sample and was adjusted to 1 lg total RNA per 8 ll RNAse free water (diethyl pyrocarbonate-treated water). First-strand cDNA was synthesized with 0.5 lg RNA using the SuperScript first-strand synthesis system (Invitrogen, San Diego, CA) and oligo(dT) primers. The resulting cDNA was used in PCR experiments. 2.4. RT-Q-PCR The expression levels of Cyp19 gene was quantified by using a Chromo 4TM System real-time PCR apparatus (Biorad, Milan, Italy). Real-time quantitative PCR (q-PCR) reactions were performed in triplicate in a final volume of 20 ll containing 10 ng cDNA, 10 ll of iTaq SYBR Green Supermix with ROX (Biorad), and 0.25 lM of each primer pairs. The GAPDH mRNA was used as housekeeping gene to normalize the expression data. For normalization, expression of GAPDH mRNA was used since no differences were found between all treatment groups. Similar results were obtained using the EF-1a, elongation factor-1 alpha-chain (not shown). Expression of CYP19 was evaluated in brain extracts using a pair of primers located in the open reading frame (ORF) that allowed the simultaneous amplification of all transcripts (Aro-ORF) and a pair of primers specific for brain aromatase (Aro-B). Aro-ORF primers were designed in our laboratory on the basis of the sequence reported in GenBank and location is indicated in Table 1. Aro-B primers were used as designed by Iwabuchi et al. (2007). The accession numbers of the genes used in the study, the primer sequences and the product sizes are given in Table 1. The thermal protocol included an enzymatic activation step at 95 °C (3 min) and 40 cycles of 95 °C (15 s), 60 °C (30 s) and 72 °C (20 s). The melting curve of the PCR products (55–94 °C) was also recorded to check the reaction specificity. Relative expression of target genes in comparison with that of the GAPDH mRNA reference gene was conducted following the comparative Ct threshold method (Pfaffl et al., 2002) using the Biorad software tool Genex-Gene Expression Macro™ (Vandesompele et al., 2002). The normalized expression was then expressed as relative quantity of mRNA (fold induction) with respect to the control sample. 2.5. Statistical analyses Data (n = 6/treatment) are presented as the mean ± SD of analyses in triplicate. Statistical analysis was performed by using ANOVA followed by Tukey ad post hoc test (INSTAT software, GraphPad Software Inc., San Diego, CA, USA). 3. Results

2.2. Chemicals and Lambro sampling

3.1. Males

All test chemicals (EE2, TAM, MDHT, FLU) were purchased from Sigma (Taufkirchen, Germany). A composite sample (total volume 280 L) of Lambro river water was sampled using 2 L glass vessels on March 18th 2004 during the whole day, about 60 km south of Milano (Lat: 45:09:57N, Long: 9:31:55E) and transported to Germany. The Lambro water sample was kept at 4 °C and exposure started immediately after arrival at the IGB in Berlin.

The effects of different conditions of exposure on the transcription of Aro-ORF and Aro-B in male brain are reported in Fig. 1(A and B). Exposure to Lambro river water (LAM) induced a significant increase in the level of mRNA of both Aro-ORF (Fig. 1A) and Aro-B (Fig. 1B), that was apparently higher for Aro-B than for Aro-ORF (with relative expressions of 2.42 ± 0.51 and 1.67 ± 0.15, respectively). Exposure to the estro-

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Table 1 GenBank accession number, primer sequences, nucleotide position, product size and reference of the target genes. Aro-ORF oligonucleotide sequences were designed in this laboratory on the base of previously reported GenBank entries. Accession number NCBI

Primer sequence (50 –30 )

Nucleotide position

PCR product

Reference

GAPDH-F GAPDH-R

U41753

50 -CACTACAAACTGTCTGGCTC-30 50 -TGGTCTTGTGTGTATCCCA-30

450–469 815–833

384 bp

Cemerikic et al. (2007)

Aro-ORF For Aro-ORF Rev

AB 272088

50 -CCAGCTTATTGCATGGGACT-30 50 -TTTCCTCGCCATTAATCCAG-30

142–161 255–274

133 bp

Aro-B For Aro-B Rev

AB 272088

50 -AAGGAGACATAGGCGAGCAG-30 50 -TGTTGTTGTAGTAATTTGCTGCTTT-30

(40) to (20) 208–232

272 bp

relative expression of Aro-ORF mRNA

Primer name

4

A

Iwabuchi et al. (2007)

MALE

3.5

*

3

*

2.5

*

2 1.5 1 0.5 0

C

relative expression of Aro-B mRNA

4

LAM

EE2

TAM

MDHT

B

FLU

*

3.5

*

3 2.5 2 1.5 1 0.5 0

C

LAM

EE2

TAM

MDHT

FLU

Fig. 1. Relative Aro-ORF (A) and Aro-B (B) mRNA expression in male X. laevis brain in response to Lambro river water and to 17a-ethinylestradiol (EE2), tamoxifen (TAM), 17a-methyldihydrotestosterone (MDHT) and flutamide (FLU) 108 M. Significant differences were indicated by asterisks (*p < 0.05).

gen EE2 also increased both Aro-ORF and Aro-B mRNA; however, the effect was significant for Aro-ORF (2.08 ± 0.27) but not for Aro-B. The anti-estrogen TAM induced a decrease, although not significant, in the level of both transcripts. In males exposed to the androgen MDHT, about a twofold increase in both Aro-ORF and Aro-B mRNA levels was observed, although the effect was significant only for Aro-ORF (2.3 ± 0.61) (Fig. 1A). The anti-androgen FLU also induced an increase in the level of aromatase transcripts that, however, was significant only for Aro-B; in particular, relative expression of Aro-B in FLU-exposed males was about 3-fold higher than in controls (2.88 ± 0.65) (Fig. 1B).

No significant changes in either Aro-ORF or Aro-B mRNAs were observed in response to EE2 exposure, whereas the anti-estrogen TAM induced a significant decrease in both aromatase isoforms (with relative expression of 0.46 ± 0.22 and 0.34 ± 0.056, respectively, for Aro-ORF and Aro-B). The androgen MDHT induced a decrease in Aro-ORF and Aro-B mRNA, that was significant for Aro-B (0.55 ± 0.2) (Fig. 2B). In contrast, in the FLU treatment group, the level of both Aro-ORF and Aro-B transcripts was significantly increased (with relative expression of 1.55 ± 0.28 and 1.69 ± 0.33, respectively, for Aro-ORF and Aro-B) (Fig. 2A and B).

3.2. Females

3.3. Sex-related differences

The results obtained in female brain are reported in Fig. 2 (A and B). Both Aro-ORF (Fig. 2A) and Aro-B (Fig. 2B) mRNA levels were significantly increased in the brain of females exposed to Lambro river water (LAM; 1.51 ± 0.43 and 1.76 ± 0.25, respectively).

Expression of Aro-ORF and Aro-B was determined by quantitative RT-PCR in total RNA fractions of adult X. laevis brain. Fig. 3 shows the amplification curves recorded for Aro-ORF in males (A) and females (B), and for Aro-B in males (C) and females (D); aver-

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relative expression of Aro-ORF mRNA

A. Massari et al. / General and Comparative Endocrinology 168 (2010) 262–268 2.5

A

FEMALE

*

2

*

1.5

1

* 0.5

0

C

relative expression of Aro-B mRNA

2.5

LAM

EE2

TAM

MDHT

FLU

B *

*

2

1.5

1

* *

0.5

0

C

LAM

EE2

TAM

MDHT

FLU

Fig. 2. Relative Aro-ORF (A) and Aro-B (B) mRNA expression in female X. laevis brain in response to Lambro river water and to 17a-ethinylestradiol (EE2), tamoxifen (TAM), 17a-methyldihydrotestosterone (MDHT) and flutamide (FLU) 108 M. Significant differences were indicated by asterisks (*p < 0.05).

age Ct values of about 21 ± 0.12 for Aro-ORF, and of 22.5 ± 0.17 for Aro-B, were observed in both males and females, respectively, indicating no sex-related differences in basal mRNA levels of the two aromatase isoforms. When the effects of different treatments on males and females were compared, EE2 and MDHT induced significantly distinct responses in terms of changes in Aro-ORF mRNA levels (p < 0.001). Such a gender-related specificity in response was more evident for Aro-B mRNA, whose levels were statistically different in males and females exposed not only to EE2 and MDHT, but also to FLU (p < 0.001). 4. Discussion Aromatase is considered as a target for different EDCs in vertebrate system. To gain a further insight on the effects and mechanisms of action of EDCs on aromatase expression in amphibian brain, adult X. laevis were exposed to Lambro river water and the effects were compared with those of four model compounds EE2, TAM, MDHT and FLU with different MOAs. We chose to utilize the Aro-ORF pair of primers in order to amplify all aromatase transcripts present in the brain and Aro-B pair of primers to specifically study the brain aromatase variant, known to be more sensitive to EDCs (Cheshenko et al., 2008). The obtained results indicate that the levels of both Aro-ORF and Aro-B mRNAs were significantly altered in the brain of both sexes of X. laevis exposed to LAM, EE2, TAM, MDHT and FLU, with the most consistent patterns of mRNA expression observed in the LAM and FLU treatment groups. In teleosts, brain aromatase may be regulated by estrogens and androgens, interacting with estrogen response element (ERE) (Mouriec et al., 2009) or under the control of androgens, since its

promoter contains a potential androgen response element (ARE) (Tong and Chung, 2003). Recently, an ERE half site has been identified in the promoter region of the single aromatase gene in X. laevis as well as binding sites for cAMP and steroidogenic factor-1 (Akatsuka et al., 2005). This implies that natural steroids and numerous steroid-mimetic chemicals in the environment may affect aromatase expression in aquatic species living in polluted areas. The river Lambro is highly contaminated with agricultural runoff, as well as with domestic and industrial waste water. Several studies have demonstrated, in male teleosts sampled strictly downstream its confluence with the main river Po, the occurrence of intersexuality and altered sex ratio (Viganò et al., 2001) in concomitance with organic compound, heavy metal, natural estrogen and pharmaceutical loads (Barghigiani et al., 2001; Fattore et al., 2002; Viganò et al., 2003; Zuccato et al., 2007). Our results show that exposure of adult X. laevis to Lambro river water significantly increased the level of both Aro-ORF or Aro-B mRNAs in the brain of both males and females. These data are consistent with those of a recent study using adult carps, where Cyp19B, but not Cyp19A was significantly increased in females exposed to an artificial mixture of six compounds with estrogenic MOA, formulated on the base of chemical analyses of water and sediment of Lambro river (data unpublished). In X. laevis, exposure to the estrogen EE2 induced a significant increase in aromatase mRNA in males, significant for Aro-ORF mRNA, but not for Aro-B. An increase in brain aromatase expression has been demonstrated in early prometamorphic Rana pipiens tadpoles exposed to EE2 demonstrating the estrogen responsiveness of the gene (Duarte et al., 2006). The observed expression pattern of Aro-ORF in male X. laevis suggests that the effect of LAM may be caused by an estrogenic MOA.

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Fig. 3. Transcription profile of aromatase isoforms in male and female brain of X. laevis. Basal expression of Aro-ORF and Aro-B was quantified in brain tissue from control males and females by RT-PCR. Amplification curves are shown by triplicate samples with a crossing point at approximately 21 cycles for Aro-ORF in males (A) and females (B) and at approximately 22.5 cycles for Aro-B in males (C) and females (D). X-axis: amplification cycle number. Y axis: normalized fluorescence signal.

However, the more consistent pattern of increased mRNA expression of aromatase in both sexes were observed in response to the anti-androgenic model compound FLU. Exposure to FLU re-

sulted in a general increase in both Aro-ORF and Aro-B in the brain of males and females. We hypothesize that anti-androgenic compounds known to be present in the river Lambro (Urbatzka et al.,

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2007b) may significantly contribute to increasing aromatase mRNA, since the observed effects of the LAM and FLU treatment are very similar in males and in females. These results might reflect that under normal conditions androgens suppress brain-specific Aro-mRNA expression, which is abolished by addition of antiandrogenic compounds such as FLU. However, this could be only true in females, since in males both androgens and anti-androgens induce Aro-B mRNA expression. In fact exposure of adult X. laevis to the androgen MDHT provoked a significant increase in AroORF, while on the contrary, Aro-B was significantly decreased in females. Exposure to the anti-estrogen TAM induced in both sexes a decrease in mRNA of Aro-ORF and Aro-B, significant in females with both pair of primers. The observed expression pattern is likely due to inhibition of ER binding to the ERE present in the promoter of the aromatase gene. TAM is an estrogen receptor (ER) antagonist forming a relatively stable complex with the 17b-estradiol receptor (Jordan et al., 1977) but also shows estrogen agonistic properties, depending on the species and tissue (Osborne et al., 1996). Recently, it has been demonstrated in zebrafish that aromatase B transcripts and protein are increased by E2 and 5a-androstane3b,17b-diol as estrogenic metabolites due to the conversion of T and 5a-dihydrotestosterone, respectively (Mouriec et al., 2009). At the moment it is very difficult to explain all effects exerted by model compounds used in this study that possibly modulate the aromatase expression both directly, via hormonal response elements, second messengers or transcription factors and indirectly, through systemic changes in hormone levels. In fact, in the same experimental conditions of 4 weeks of exposure, significant alterations of plasma sex steroid levels were observed, probably via feed back mechanisms (Urbatzka et al., 2007a). These data were in accordance with changes in LH expression in brain previously observed (Urbatzka et al., 2006). Moreover, data on gonad histomorphology showed anti-androgenic effects of LAM and EE2 on males and anti-estrogenic effects of TAM and MDHT on females (Cevasco et al., 2008). Overall, the results support the hypothesis of a feedback mechanisms on the hypothalamus–pituitary–gonad axis and on the production of sex steroids exerted by exposure to certain EDCs Interestingly, from the results of the present study a distinct expression pattern emerges for the two primer pairs that were designed to analyse all aromatase transcripts (Aro-ORF) or specifically the brain-specific transcript (Aro-B). From the comparison of the observed results, a different regulation can be hypothesized for the gonadal and brain transcripts in response to different treatments. For example, the high and low induction of Aro-B and AroORF, respectively, due to FLU suggest a regulation of only the brain transcript. When comparing data obtained in males and females, exposure to EE2 and MDHT induced distinct effects on the level of both AroORF and Aro-B mRNAs, indicating a sex-specific regulation of aromatase expression by estrogenic and androgenic compounds; moreover, for Aro-B, a significantly different response was observed in males and females also for the anti-androgenic compound FLU. On the other hand, no sex-related differences were observed in response to LAM exposure. Overall, the results indicate that in X. leavis brain aromatase can be gender-specifically regulated by individual compounds with different MOAs. Even when considering that the endocrine-mimetic activities of Lambro river are due to a mixture of anthropogenic, industrial and agricultural pollutants, these data further support the hypothesis that this watercourse may exert both estrogenic and anti-androgenic effects in X. laevis males. Acknowledgment This work was partly funded by the European Community within the EU-project EASYRING, Contract No. QLK4-CT-2002-02286.

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