Ecotoxicology DOI 10.1007/s10646-015-1510-0
Transcriptional Responses in Adult Zebrafish (Danio rerio) Exposed to Propranolol and Metoprolol Liwei Sun1 • Fang Liu1 • Haigang Chen2 • Sisi Wang1 • Xia Lin1 • Jian Chi1 Qing Zhu1 • Zhengwei Fu1
•
Accepted: 8 June 2015 Springer Science+Business Media New York 2015
Abstract b-adrenergic receptor blockers (b-blockers) are widely detected in the aquatic environment; however, the effects of these pharmaceuticals on aquatic organisms remain uncertain. In this study, adult zebrafish were exposed to two different b-blockers, propranolol and metoprolol, for 96 h. After exposure, the transcriptional responses of genes encoding the b-adrenergic receptor (i.e., adrb1, adrb2a, adrb2b, adrb3a and adrb3b), genes involved in detoxification and the stress response (i.e., hsp70, tap, mt1 and mt2), and genes related to the antioxidant system (i.e., cu/zn-sod, mn-sod, cat and gpx) were examined in the brain, liver and gonad. Our results show that both propranolol and metoprolol exposure changes the mRNA level of b-adrenergic receptors, indicating clear pharmacological target engagement of the b-blockers. The transcription of genes related to antioxidant responses and detoxification process were induced, suggesting that bblocker exposure can activate the detoxification process and result in oxidative stress in fish. Moreover, the transcriptional responses displayed substantial tissue- and gender-specific effects. Considering the environmental concentrations of propranolol and metoprolol, these results
Electronic supplementary material The online version of this article (doi:10.1007/s10646-015-1510-0) contains supplementary material, which is available to authorized users. & Zhengwei Fu [email protected] 1
College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, People’s Republic of China
2
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Science, No. 231 Xingangxi Road, Guangzhou 510300, People’s Republic of China
suggest that these pharmaceuticals are unlikely to pose a risk to fish. However, the impacts in prolonged exposure, along with other possible side effects due to b-adrenergic receptor blockade, should be further assessed. Keywords Adrenergic receptor b-blockers Oxidative damages Detoxification process
Introduction The presence of numerous human pharmaceuticals in the aquatic environment is currently a worldwide concern. These pharmaceuticals are released in large amounts into the environment as parent compounds or as metabolites following absorption, distribution, metabolism and elimination (ADME) from patients (Ashton et al. 2004; Bartram et al. 2012). Additionally, it was reported that the disposal of unused and expired pharmaceuticals, either by household waste or via the sink or toilet, may be a prominent route requiring greater attention (Bound and Voulvoulis 2005). Many pharmaceuticals and their metabolites can survive conventional water-treatment processes (Santos et al. 2010); therefore, as pharmaceuticals are continuously delivered and not efficiently removed, they exhibit a different pseudo-persistence when compared with certain conventional pollutants (Daughton 2003). Due to the bioavailability and the intended target engagement of specific receptors/enzymes or signal transduction pathways that are often conserved across vertebrate families, pharmaceuticals pose a significant health risk for non-target aquatic species, especially fish (Owen et al. 2007). It has been suggested that aquatic organisms are not as efficient as mammals in detoxification processes (Contardo-Jara et al. 2010). Consequently, considerable research efforts
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over the last two decades have examined the potential detrimental effects to fish induced by pharmaceuticals (Santos et al. 2010). According to Santos et al. (2010), approximately 8 % of the therapeutic classes detected in the environment are bblockers. b-blockers or b-adrenergic receptor blockers work through the competitive inhibition of b-adrenergic receptors (b-ARs) and are prescribed for the treatment of human cardiovascular conditions, such as hypertension, heart failure, angina and arrhythmia (Massarsky et al. 2011; Owen et al. 2007). Since the discovery of propranolol in the 1960s, many b-blockers have emerged and more than 30 types are used clinically (Massarsky et al. 2011; Owen et al. 2007). Within the most commonly used b-blockers, propranolol and nadolol are non-specific antagonists that block both b1- and b2-receptors, while metoprolol and atenolol exhibit b1-receptors specificity (Fraysse and Garric 2005; Huggett et al. 2003). Due to the high incidence of cardiovascular disease, the global market value of b-blockers rises substantially each year, and over the last decade the number of prescriptions written has doubled (Massarsky et al. 2011). As a consequence of this substantial use, b-blockers have been detected in aquatic environments worldwide, such as in influents and effluents from sewage treatment plants, surface water, groundwater and drinking water (Ashton et al. 2004; Benotti et al. 2009; Kolpin et al. 2002; Santos et al. 2010). In Spain, propranolol and atenolol have been detected at concentrations up to 6.5 and 122 lg/L, respectively, in hospital effluents (Gomez et al. 2006). To date, these values are some of the highest levels detected. In China, a study found that 13 bblockers existed in water samples taken from hospitals and sewage treatment plants (Liu 2010). Among these compounds, metoprolol was detected at high levels in clinical wastewater and in the influents and effluents from sewage treatment plants at concentrations up to 340.0, 2669.7 and 1287.4 ng/L, respectively. There has been an increase in research designed to assess the toxicity of b-blockers on fish. Acute toxicity tests are quick and convenient tools that are considered to be necessary for adequate experimental designs to determine the effects of chronic drug exposure. Following a 48-h exposure to propranolol, an LC50 value of 24.3 mg/L was obtained for 3 to 4-day-old Japanese medaka; however, acute exposure to metoprolol did not affect the survival of the fish even at concentrations greater than 100 mg/L (Huggett et al. 2002). Our previous work has shown that the 96-h LC50 value for propranolol in zebrafish larvae was 2.48 mg/L, but exposure to 50 mg/L of metoprolol did not increase mortality (Sun et al. 2014). Exposure to propranolol ranging from 4 to 32 mg/L also affected the hatching and mortality rate, decreased spontaneous movement and caused morphological abnormalities of the zebrafish
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embryos (Fraysse et al. 2006). Pharmaceuticals are likely to be found in trace quantities in the aquatic environment, which poses a low risk for acute toxicity; therefore, studies examining chronic low-concentration exposures can provide information to specifically address the effects of pharmaceutical contamination (Fent et al. 2006). A previous study showed that propranolol reduced growth rates and changed plasma steroid levels in medaka after a two-week exposure (Huggett et al. 2002). In a subsequent four-week propranolol exposure, egg production was significantly decreased at concentrations as low as 0.5 lg/L, but no difference was observed at higher concentrations (Huggett et al. 2002). For metoprolol, the liver, kidney and gills of rainbow trout displayed ultrastructural changes after a 28-day exposure at concentrations ranging 1–100 lg/L (Triebskorn et al. 2007). These data highlight the idea that b-blockers can affect fish at very low, but environmentally relevant, concentrations. However, an adult reproduction study using fathead minnows found that propranolol exposure at environmental concentrations did not pose a risk to fish (Giltrow et al. 2009). Similar findings were reported using rainbow trout (Owen et al. 2009) and the fathead minnow (Lorenzi et al. 2012). Taken together, major gaps in our understanding of b-blocker toxicity to aquatic organisms remain. Research into the ecotoxicity of environmental pollutants on the level of gene showed that gene transcriptional responses represent the primary interaction site between chemical and organism and often ultimately relate with physiological processes; thus, research into the ecotoxicity of environmental pollutants on the level of gene transcription has grown significantly over the last decade (Filby et al. 2007). However, to the best of our knowledge, relatively few studies have examined the transcriptional responses in fish exposed to b-blockers. Therefore, the objective of this study was to determine the transcriptional response of genes encoding the b-ARs, the potential target of b-blockers, and genes involved in other physiological processes, including antioxidant and detoxification pathways, in adult zebrafish after exposure to the b-blockers propranolol and metoprolol. This work can improve our understanding of the impact of b-blockers on fish and will ultimately be helpful for the risk assessment of these pharmaceuticals.
Materials and Methods Test Chemicals The (R, S)-propranolol hydrochloride (purity [99 %) and (R, S)-metoprolol tartrate salt (purity [98 %) were purchased from Sigma (St. Louis, MO, USA). No carrier
Transcriptional Responses in Adult Zebrafish (Danio rerio) Exposed to Propranolol and…
solvent was required for the preparation of the stock solutions because these test substances are all readily soluble. Test Fish The zebrafish (Danio rerio) used in this study were kindly provided by the Institute of Hydrobiology of the Chinese Academy of Science (Wuhan, China). The fish were maintained in charcoal-dechlorinated tap water at a constant temperature (26 ± 1 C) with a 16:8 h (light:dark) photoperiod. The brood stock was fed three times daily, once with newly hatched brine shrimp (Artemia nauplii) and twice with commercial food. Exposure Conditions and Fish Sampling Prior to the initiation of the experiment, adult zebrafish (approximately 7 months-old) were sex-separated and acclimated for 1 week. Exposure studies were conducted in glass tanks containing 3 L of the test solution under the conditions described above. Based on the results of our previous studies, fish were exposed to propranolol and metoprolol for 96 h at 0.03, 0.3 or 3 mg/L. There were three replicate tanks of seven male fish each and another three tanks with females, for a total of 21 males and females per treatment. Dechlorinated tap water controls were used in addition to the test series. The test solutions were 80 % renewed every 24 h. The test solutions were stored at 4 C between the semi-static renewal periods. Previous analytical studies indicated that both propranolol and metoprolol were stable in water when kept at 4 C in the dark (Dzialowski et al. 2006). The fish were not fed during exposure because fecal matter and uneaten food may decrease water quality and affect the biological activity of test chemicals. The fish were observed three times daily, and no mortality was found. At the end of the exposure, the fish were sacrificed by keeping them on ice. The brain, liver and gonad were excised, flash-frozen in dry ice and stored at -80 C for subsequent analyses. Quantitative Real-Time PCR Assay For gene expression analysis, the tissue of four or five fish were homogenized as one sample and a total of four replicates from each treatment group for each gender were prepared. The total RNA was extracted using RNAiso Plus (Takara, Dalian, China) according to the manufacturer’s instructions. The quality of the total RNA was evaluated by agarose gel electrophoresis and optical density reading (260/280 ratio 1.8–2.0). First-strand cDNA synthesis was performed using the ReverTra Ace qPCR RT kit (Toyobo,
Osaka, Japan). Real-time PCR with SYBR green detection was performed on a Mastercycler ep realplex (Eppendorf, Hamburg, Germany) according to the protocols established by the manufacturer (SYBR Green Realtime PCR Master Mix, Toyobo). The transcription of the genes encoding the b-ARs and those associated with other physiological processes, including detoxification, stress response and antioxidant pathways, were selected for quantification. The oligonucleotide primers specific for the selected genes (Supplemental Table S1) were identical to those used in previous studies (Gonzalez et al. 2005; Jin et al. 2010; Wang et al. 2009). The mRNA levels were expressed relative to the transcription level of the reference gene, bactin. The relative quantification of gene expression was analyzed from the measured cycle threshold (Ct) using the 2-DDCt method (Livak and Schmittgen 2001). Data Analysis Gene transcription was calculated as the fold-change relative to the average transcription in the vehicle control. Prior to conducting statistical comparisons, data were assessed for normality and homogeneity of variances using the Kolmogorov–Smirnov one-sample test and Levene’s test, respectively. Significant differences were assessed using a one-way analysis of variance (ANOVA) followed by the Dunnett’s post hoc test. The set value for statistical significance was p \ 0.05. All statistical analyses were performed with SPSS 13.0 software (SPSS, Chicago, IL).
Results Transcriptional Responses of Target Genes in Zebrafish Following Exposure to Propranolol In the brain, exposure to 3 mg/L of propranolol significantly up-regulated the transcription of arb1 and downregulated the mRNA levels of arb2b and arb3b in females when compared with the control (Fig. 1). In addition, the mRNA levels of arb2b and arb3b decreased in a concentration-dependent manner in male zebrafish exposed to propranolol (Fig. 1). No obvious changes in arb2a and arb3a were observed in any male or female treated groups. For genes involved in the stress response and detoxification mechanisms, including hsp70, mt1, mt2 and tap, a clear concentration-dependent induction was observed following propranolol exposure, especially in male fish (Fig. 1). Similar inductions were found for genes encoding antioxidant proteins, including cu/zn-sod, mn-sod, cat and gpx. In female fish, these genes were up-regulated significantly after exposure to 3 mg/L of propranolol; however, in male fish, 0.3 mg/L propranolol was sufficient to induce
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Fig. 1 Changes in the transcription of b-AR genes and genes involved in stress, detoxification and antioxidant responses in the brain of zebrafish exposed to propranolol. The results are presented as
the mean ± SEM and are expressed as the ratio of the target gene mRNA/b-actin mRNA. The asterisks denote significant differences when compared with the control group (*p \ 0.05 and **p \ 0.01)
Fig. 2 Changes in the transcription of b-AR genes and genes involved in stress, detoxification and antioxidant responses in the liver of zebrafish exposed to propranolol. The results are presented as
the mean ± SEM and are expressed as the ratio of the target gene mRNA/b-actin mRNA. The asterisks denote significant differences when compared with the control group (*p \ 0.05 and **p \ 0.01)
statistically significant changes when compared with the control (Fig. 1). In the liver of treated fish, a significant induction of arb1 mRNA was observed in females exposed to 3 mg/L propranolol and in males exposed to both 0.3 and 3 mg/L propranolol (Fig. 2). In contrast, 3 mg/L propranolol
down-regulated the mRNA level of arb3b in both females and males. Propranolol exposure did not alter the mRNA levels of arb2a, arb2b or arb3a. For genes involved in the stress response and detoxification, propranolol exposure significantly up-regulated the transcriptional level of hsp70, mt1 and mt2 in both female and male fish, but it did
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Transcriptional Responses in Adult Zebrafish (Danio rerio) Exposed to Propranolol and…
In the brain, metoprolol exposure did not produce any obvious changes in the transcription of b-AR genes except for arb3b, which was significantly increased after exposure
to a concentration of 0.03 mg/L in both females and males (Fig. 4). No obvious changes were observed in the transcription of genes involved in the stress response, detoxification or antioxidant pathways (Fig. 4). In the liver, exposure to metoprolol significantly downregulated the transcription of arb1 in both females and males; however, no changes were observed for other bAR genes (Fig. 5). For genes involved in the stress response and detoxification pathways, only the transcription of hsp70 was induced in treated males (Fig. 5). For antioxidant genes, there was a clear concentration-dependent induction of cat that reached a 4.1- and 4.3-fold increase relative to that of the control in treated females and males, respectively. However, no changes were found in cu/zn-sod, mn-sod and gpx in either female or male fish (Fig. 5). In the gonads, exposure to metoprolol resulted in an increase of arb1 and arb3b in females and arb1, arb2a, arb2b and arb3a in males (Fig. 6). However, these changes were significant only at a concentration of 0.03 mg/L and not at higher exposures. For genes involved in the stress response and detoxification pathways, exposure to metoprolol caused no changes in transcription in females, and it up-regulated the mRNA levels of hsp70, mt1 and mt2 in males at the highest concentration tested (Fig. 6). For antioxidant genes, metoprolol exposure increased the mRNA level of mn-sod in treated males and cat in both females and males (Fig. 6).
Fig. 3 Changes in the transcription of b-AR genes and genes involved in stress, detoxification and antioxidant responses in the gonad of zebrafish exposed to propranolol. The results are presented
as the mean ± SEM and are expressed as the ratio of the target gene mRNA/b-actin mRNA. The asterisks denote significant differences when compared with the control group (*p \ 0.05 and **p \ 0.01)
not change the mRNA level of tap (Fig. 2). For genes encoding antioxidant proteins, the transcription of cat was induced after exposure to propranolol in females; however, no changes were found in any other genes (Fig. 2). In males, the mRNA levels of mn-sod, cat and gpx were upregulated after exposure to 3 mg/L of propranolol. For b-AR genes in the gonads of treated fish, no changes were observed for arb1 and arb2a in either females or males (Fig. 3). Significant changes were observed in arb2b and arb3a in males, although a trend toward a down-regulation was observed in females. Significant decreases were found in arb3b in both males and females when compared with the control (Fig. 3). Exposure to propranolol induced no obvious changes in hsp70, mt1, mt2 and tap in the gonads from either females or males (Fig. 3). For antioxidant genes, the transcriptional responses to propranolol exposure were gender-specific. No changes were observed in treated females; however, significant increases in cu/zn-sod, mn-sod and cat were observed in males when compared with the controls (Fig. 3).
Transcriptional Responses of Target Genes in Zebrafish Following Exposure to Metoprolol
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Fig. 4 Changes in the transcription of b-AR genes and genes involved in stress, detoxification and antioxidant responses in the brain of zebrafish exposed to metoprolol. The results are presented as
the mean ± SEM and are expressed as the ratio of the target gene mRNA/b-actin mRNA. The asterisks denote significant differences when compared with the control group (*p \ 0.05 and **p \ 0.01)
Fig. 5 Changes in the transcription of b-AR genes and genes involved in stress, detoxification and antioxidant responses in the liver of zebrafish exposed to metoprolol. The results are presented as
the mean ± SEM and are expressed as the ratio of the target gene mRNA/b-actin mRNA. The asterisks denote significant differences when compared with the control group (*p \ 0.05 and **p \ 0.01)
Discussion
pharmacological target of b-blockers, the transcriptional responses of b-ARs were determined in zebrafish exposed to propranolol and metoprolol. Our results showed that the mRNA tissue distribution of these genes (data not shown) was consistent with the report of Wang et al. (2009). For propranolol, with the exception of arb1, the b-AR genes showed a concentration-dependent decrease. Although the
b-ARs are members of the G-protein-coupled receptor superfamily and mediate various physiological processes (Massarsky et al. 2011; Wang et al. 2009). In zebrafish, Wang et al. (2009) identified five b-AR genes designated as adrb1, adrb2a, adrb2b, adrb3a, and adrb3b. Being the
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Transcriptional Responses in Adult Zebrafish (Danio rerio) Exposed to Propranolol and…
Fig. 6 Changes in the transcription of b-AR genes and genes involved in stress, detoxification and antioxidant responses in the gonad of zebrafish exposed to metoprolol. The results are presented as
the mean ± SEM and are expressed as the ratio of the target gene mRNA/b-actin mRNA. The asterisks denote significant differences when compared with the control group (*p \ 0.05 and **p \ 0.01)
function of b-AR genes in the zebrafish has not been fully described, the down-regulation of their transcriptional response may be due to the antagonistic activity of bblockers on adrenoceptors. For metoprolol, with the exception of arb1 in the liver, the altered b-AR genes showed an increase at low-concentration exposures, followed by a decrease to levels similar to that observed in the unexposed controls. The increase in b-AR genes induced by low concentrations of metoprolol may be related to the compensatory response for adrenoceptor blockade; however, at higher concentrations, the down-regulation of bAR genes was similar to the observed propranolol effects, indicating the therapeutic effects of b-blockers. These results also revealed that the pharmacological effect of propranolol on fish is more potent than metoprolol, which could be explained by the lipophilicity of pharmaceuticals because the octanol–water partition coefficient of propranolol is greater than metoprolol (Massarsky et al. 2011). However, the difference in transcriptional patterns of b-AR genes between the two b-blockers may be due to receptor selectivity. Propranolol binds to any b-AR, while metoprolol has a higher affinity for the b1-AR (Fraysse and Garric 2005; Huggett et al. 2003). It has been confirmed that exposure to a variety of chemical pollutants produces oxygen free radicals and other reactive oxygen species (ROS) during metabolic processes in biological systems, which can subsequently cause oxidative damage to membrane lipids, DNA, and proteins (Valavanidis et al. 2006). Over the past decades,
molecular biomarkers of oxidative stress have been widely applied to study mechanisms of toxicity in aquatic organisms exposed to chemical pollutants. Here, the transcriptional responses of antioxidant enzymes, including Cu/ZnSod, Mn-Sod, Cat, and GPx, were determined in fish exposed to propranolol and metoprolol. With the exception of the genes in the brain of fish exposed to metoprolol, concentration-dependent changes were observed in antioxidant gene transcripts in both male and female fish. Although limited data regarding oxidative stress caused by b-blockers in fish were available for comparison, our observations are in agreement with previous studies in aquatic invertebrates. For example, propranolol exposure resulted in elevated lipid peroxidation damage in the gill of the marine mussel Mytilus galloprovincialis (Sole et al. 2010), and reduced the lysosomal membrane stability of hemocytes and induced CAT activity in the digestive gland (Franzellitti et al. 2011). Liu and Williams (2007) suggested that the formation of intermediate radicals, which are highly reactive and cause oxidative stress, occurs during the degradation of propranolol. As for metoprolol, mRNA levels of the antioxidant enzymes SOD and CAT were significantly induced in the digestive gland of the freshwater mussel Dreissena polymorpha, presumably generated as a side-effect of phase I biotransformation (Contardo-Jara et al. 2010). In addition, it was found that high propranolol concentrations were associated with increased CAT activity in fluvial biofilms, indicating that exposure to b-blockers can also cause oxidative stress in
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algae and bacteria within river biofilms and the whole aquatic ecosystem (Bonnineau et al. 2010). Generally, xenobiotic exposure alters homoeostasis. HSP70 acts as an indicator for protein damage and subsequent protective processes (Contardo-Jara et al. 2010), and it is involved in the oxidative stress response (Gonzalez et al. 2005). In the present study, propranolol exposure induced the up-regulation of hsp70 mRNA in the brain and liver of both genders. Induction of hsp70 was found in the gonad and liver of males exposed to metoprolol. Similarly, Contardo-Jara et al. (2010) found a significant upregulation of hsp70 mRNA in the gill of mussels exposed to metoprolol; furthermore, this induction persisted for 1 week. Metallothionein (Mt) functions in primary metal storage, transport and detoxification, and the induction of MT mRNA can indirectly indicate the level of oxidative stress in an organism (Contardo-Jara et al. 2010; Gonzalez et al. 2005). In this study, the induction of mt1 and mt2 mRNA was observed after propranolol and metoprolol exposure. This induction, coupled with similar observations previously reported in mussels exposed to metoprolol (Contardo-Jara et al. 2010), may be due to the hydroxyl radical derived from biotransformation processes of bblockers. The gene that encodes TAP belongs to the family of ATP binding-cassette (ABC) transporters that utilize ATP as an energy source and extrude xenobiotics by active efflux (Gonzalez et al. 2005). Results of pervious studies showed that various metals, such as Hg and Cd, could induce the transcription of tap in the zebrafish (Arini et al. 2015; Gonzalez et al. 2006, 2005). However, to our knowledge, no data about the effects of b-blockers on tap mRNA changes was available. In this study, the tap gene was increased only in the brain of male zebrafish after propranolol exposure. It should be noted that the transcriptional responses of the selected genes displayed substantial tissue-specificity. For example, significant induction of antioxidant and detoxification genes were stimulated by propranolol exposure in the brain. The transcript levels of all eight genes were increased in males, and six genes were increased in females. Conversely, in the gonad, only cu/znsod, mn-sod and cat were induced by propranolol in males. The tissue-specific effects of b-blockers was found previously. CAT activities were increased in the digestive gland and reduced in the mantle/gonads in marine mussels exposed to propranolol (Franzellitti et al. 2011). For metoprolol, the mRNA levels of SOD and CAT were significantly induced in the digestive gland, but not the gill, of the freshwater mussel (Contardo-Jara et al. 2010). The specific biotransformation or detoxification process in different tissues might account for the difference in susceptibility to b-blockers. Moreover, it has been shown that propranolol can cross the blood–brain barrier in higher
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organisms due to its relatively high lipophilicity when compared with most other b-blockers. One study reported that the concentration in human brain tissue was 10 to 26 times higher than in plasma (Cruickshank and Neil-Dwyer 1985). Although no convincing data were demonstrated in fish, the relatively high concentration of propranolol in the brain likely produced the substantial changes in gene transcription. In addition to tissue-specificity, the genderspecificity of the b-blockers should be emphasized as well. For most of the genes tested, the changes observed in males were greater than those of females. Giltrow et al. (2009) also reported that the plasma concentration of propranolol in male adult fathead minnows after a 21-day exposure was greater than the levels found in females. These results demonstrate that it is extremely important to consider the gender of fish when assessing the effects of b-blockers and other pharmaceuticals. Based on the results of present study, the transcriptional changes in b-ARs genes are likely due to the expected pharmacological activities of b-blockers. Similarly, transcriptional changes in genes involved in the antioxidant response and detoxification process were found. Considering the concentrations of b-blockers found in the aquatic environment, the results of the present study, our previous studies (Sun et al. 2014) and those of other researchers (Giltrow et al. 2009; Owen et al. 2009), these pharmaceuticals seem unlikely to pose a risk to the well-being of fish. However, more specific consequences of adrenoceptor blockade should be assessed before a definitive answer can be given. However, it was reported that propranolol exposure at concentrations that produced a fish plasma concentration within the range recorded in patients also induced an effective b-adrenoceptor blockade; however, when the fish plasma concentration exceeded the human therapeutic plasma concentration, undesired toxicity of propranolol was elicited (Giltrow et al. 2009; Owen et al. 2009). Although the present study did not measure fish plasma concentrations, the effective concentrations of propranolol fall within the range of these previous reports. Thus, our data supports the ‘‘read-across’’ hypothesis from human to fish, and the existing human pharmacological or toxicological data could be useful for predicting the ecotoxicological risk of these pharmaceuticals on fish. In conclusion, our study demonstrates that both propranolol and metoprolol can change the mRNA levels of bARs, the target of b-blockers, indicating the expected pharmacological effects. Additionally, the transcription of genes related to the antioxidant response and detoxification process were induced after exposure to propranolol and metoprolol, suggesting that exposure to b-blockers can activate the detoxification process and result in oxidative stress in fish. It appears that environmentally relevant concentrations of these pharmaceuticals are unlikely
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sufficient to elicit adverse effects on fish because an effective b-adrenoceptor blockade occurred only within the therapeutic range. However, further studies with prolonged exposure should be performed and, in fact, these studies are being conducted by our research group. Acknowledgments We gratefully acknowledge the National Natural Science Foundation of China (Nos. 21377118, 20907044), Program for Changjiang Scholars and Innovative Research Team in University (IRT13096), and Key Laboratory of Fishery Ecology and Environment, Guangdong Province (LFE-2013-2) for supporting this research. Conflict of interest of interest.
The authors declare that they have no conflict
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Fraysse B, Garric J (2005) Prediction and experimental validation of acute toxicity of b-blocks in Ceriodaphnia dubia. Environ Toxicol Chem 24:2470–2476 Fraysse B, Mons R, Garric J (2006) Development of a zebrafish 4-day toxicity of embryo-larval bioassay to assess chemicals. Ecotoxicol Environ Safe 63:253–267 Giltrow E, Eccles PD, Winter MJ, McCormack PJ, Rand-Weaver M, Hutchinson TH, Sumpter JP (2009) Chronic effects assessment and plasma concentrations of the b-blocker propranolol in fathead minnows (Pimephales promelas). Aquat Toxicol 95:195–202 Gomez MJ, Petrovic M, Fernandez-Alba AR, Barcelo D (2006) Determination of pharmaceuticals of various therapeutic classes by solid-phase extraction and liquid chromatography-tandem mass spectrometry analysis in hospital effluent wastewaters. J Chromatogr A 1114:224–233 Gonzalez P, Dominique Y, Massabuau JC, Boudou A, Bourdineaud JP (2005) Comparative effects of dietary methylmercury on gene expression in liver, skeletal muscle, and brain of the zebrafish (Danio rerio). Environ Sci Technol 39:3972–3980 Gonzalez P, Baudrimont M, Boudou A, Bourdineaud JP (2006) Comparative effects of direct cadmium contamination on gene expression in gills, liver, skeletal muscles and brain of the zebrafish (Danio rerio). Biometals 19:225–235 Huggett DB, Brooks BW, Peterson B, Foran CM, Schlenk D (2002) Toxicity of select b adrenergic receptor-blocking pharmaceuticals (b-blockers) on aquatic organisms. Arch Environ Contam Toxicol 43:229–235 Huggett DB, Khan IA, Foran CM, Schlenk D (2003) Determination of b-adrenergic receptor blocking pharmaceuticals in united states wastewater effluent. Environ Pollut 121:199–205 Jin YX, Zhang XX, Shu LJ, Chen LF, Sun LW, Qian HF, Liu WP, Fu ZW (2010) Oxidative stress response and gene expression with atrazine exposure in adult female zebrafish (Danio rerio). Chemosphere 78:846–852 Kolpin DW, Furlong ET, Meyer MT, Thurman EM, Zaugg SD, Barber LB, Buxton HT (2002) Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999-2000: a national reconnaissance. Environ Sci Technol 36:1202–1211 Liu J (2010) Stereoisomer analysis of b-adrenergic receptor blockers in wasterwater samples (in Chinese with English abstract). School of Pharmaceutical Science. Shanxi Medical University, Taiyuan Liu QT, Williams HE (2007) Kinetics and degradation products for direct photolysis of b-blockers in water. Environ Sci Technol 41:803–810 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCt method. Methods 25:402–408 Lorenzi V, Mehinto AC, Denslow ND, Schlenk D (2012) Effects of exposure to the b-blocker propranolol on the reproductive behavior and gene expression of the fathead minnow, Pimephales promelas. Aquat Toxicol 116–117:8–15 Massarsky A, Trudeau VL, Moon TW (2011) b-blockers as endocrine disruptors: the potential effects of human b-blockers on aquatic organisms. J Exp Zool A 315:251–265 Owen SF, Giltrow E, Huggett DB, Hutchinson TH, Saye J, Winter MJ, Sumpter JP (2007) Comparative physiology, pharmacology and toxicology of b-blockers: mammals versus fish. Aquat Toxicol 82:145–162 Owen SF, Huggett DB, Hutchinson TH, Hetheridge MJ, Kinter LB, Ericson JF, Sumpter JP (2009) Uptake of propranolol, a cardiovascular pharmaceutical, from water into fish plasma and its effects on growth and organ biometry. Aquat Toxicol 93:217–224
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Transcriptional Responses in Adult Zebrafish (Danio rerio) Exposed to Propranolol and Metoprolol Liwei Sun1 • Fang Liu1 • Haigang Chen2 • Sisi Wang1 • Xia Lin1 • Jian Chi1 Qing Zhu1 • Zhengwei Fu1
•
Accepted: 8 June 2015 Springer Science+Business Media New York 2015
Abstract b-adrenergic receptor blockers (b-blockers) are widely detected in the aquatic environment; however, the effects of these pharmaceuticals on aquatic organisms remain uncertain. In this study, adult zebrafish were exposed to two different b-blockers, propranolol and metoprolol, for 96 h. After exposure, the transcriptional responses of genes encoding the b-adrenergic receptor (i.e., adrb1, adrb2a, adrb2b, adrb3a and adrb3b), genes involved in detoxification and the stress response (i.e., hsp70, tap, mt1 and mt2), and genes related to the antioxidant system (i.e., cu/zn-sod, mn-sod, cat and gpx) were examined in the brain, liver and gonad. Our results show that both propranolol and metoprolol exposure changes the mRNA level of b-adrenergic receptors, indicating clear pharmacological target engagement of the b-blockers. The transcription of genes related to antioxidant responses and detoxification process were induced, suggesting that bblocker exposure can activate the detoxification process and result in oxidative stress in fish. Moreover, the transcriptional responses displayed substantial tissue- and gender-specific effects. Considering the environmental concentrations of propranolol and metoprolol, these results
Electronic supplementary material The online version of this article (doi:10.1007/s10646-015-1510-0) contains supplementary material, which is available to authorized users. & Zhengwei Fu [email protected] 1
College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, People’s Republic of China
2
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Science, No. 231 Xingangxi Road, Guangzhou 510300, People’s Republic of China
suggest that these pharmaceuticals are unlikely to pose a risk to fish. However, the impacts in prolonged exposure, along with other possible side effects due to b-adrenergic receptor blockade, should be further assessed. Keywords Adrenergic receptor b-blockers Oxidative damages Detoxification process
Introduction The presence of numerous human pharmaceuticals in the aquatic environment is currently a worldwide concern. These pharmaceuticals are released in large amounts into the environment as parent compounds or as metabolites following absorption, distribution, metabolism and elimination (ADME) from patients (Ashton et al. 2004; Bartram et al. 2012). Additionally, it was reported that the disposal of unused and expired pharmaceuticals, either by household waste or via the sink or toilet, may be a prominent route requiring greater attention (Bound and Voulvoulis 2005). Many pharmaceuticals and their metabolites can survive conventional water-treatment processes (Santos et al. 2010); therefore, as pharmaceuticals are continuously delivered and not efficiently removed, they exhibit a different pseudo-persistence when compared with certain conventional pollutants (Daughton 2003). Due to the bioavailability and the intended target engagement of specific receptors/enzymes or signal transduction pathways that are often conserved across vertebrate families, pharmaceuticals pose a significant health risk for non-target aquatic species, especially fish (Owen et al. 2007). It has been suggested that aquatic organisms are not as efficient as mammals in detoxification processes (Contardo-Jara et al. 2010). Consequently, considerable research efforts
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over the last two decades have examined the potential detrimental effects to fish induced by pharmaceuticals (Santos et al. 2010). According to Santos et al. (2010), approximately 8 % of the therapeutic classes detected in the environment are bblockers. b-blockers or b-adrenergic receptor blockers work through the competitive inhibition of b-adrenergic receptors (b-ARs) and are prescribed for the treatment of human cardiovascular conditions, such as hypertension, heart failure, angina and arrhythmia (Massarsky et al. 2011; Owen et al. 2007). Since the discovery of propranolol in the 1960s, many b-blockers have emerged and more than 30 types are used clinically (Massarsky et al. 2011; Owen et al. 2007). Within the most commonly used b-blockers, propranolol and nadolol are non-specific antagonists that block both b1- and b2-receptors, while metoprolol and atenolol exhibit b1-receptors specificity (Fraysse and Garric 2005; Huggett et al. 2003). Due to the high incidence of cardiovascular disease, the global market value of b-blockers rises substantially each year, and over the last decade the number of prescriptions written has doubled (Massarsky et al. 2011). As a consequence of this substantial use, b-blockers have been detected in aquatic environments worldwide, such as in influents and effluents from sewage treatment plants, surface water, groundwater and drinking water (Ashton et al. 2004; Benotti et al. 2009; Kolpin et al. 2002; Santos et al. 2010). In Spain, propranolol and atenolol have been detected at concentrations up to 6.5 and 122 lg/L, respectively, in hospital effluents (Gomez et al. 2006). To date, these values are some of the highest levels detected. In China, a study found that 13 bblockers existed in water samples taken from hospitals and sewage treatment plants (Liu 2010). Among these compounds, metoprolol was detected at high levels in clinical wastewater and in the influents and effluents from sewage treatment plants at concentrations up to 340.0, 2669.7 and 1287.4 ng/L, respectively. There has been an increase in research designed to assess the toxicity of b-blockers on fish. Acute toxicity tests are quick and convenient tools that are considered to be necessary for adequate experimental designs to determine the effects of chronic drug exposure. Following a 48-h exposure to propranolol, an LC50 value of 24.3 mg/L was obtained for 3 to 4-day-old Japanese medaka; however, acute exposure to metoprolol did not affect the survival of the fish even at concentrations greater than 100 mg/L (Huggett et al. 2002). Our previous work has shown that the 96-h LC50 value for propranolol in zebrafish larvae was 2.48 mg/L, but exposure to 50 mg/L of metoprolol did not increase mortality (Sun et al. 2014). Exposure to propranolol ranging from 4 to 32 mg/L also affected the hatching and mortality rate, decreased spontaneous movement and caused morphological abnormalities of the zebrafish
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embryos (Fraysse et al. 2006). Pharmaceuticals are likely to be found in trace quantities in the aquatic environment, which poses a low risk for acute toxicity; therefore, studies examining chronic low-concentration exposures can provide information to specifically address the effects of pharmaceutical contamination (Fent et al. 2006). A previous study showed that propranolol reduced growth rates and changed plasma steroid levels in medaka after a two-week exposure (Huggett et al. 2002). In a subsequent four-week propranolol exposure, egg production was significantly decreased at concentrations as low as 0.5 lg/L, but no difference was observed at higher concentrations (Huggett et al. 2002). For metoprolol, the liver, kidney and gills of rainbow trout displayed ultrastructural changes after a 28-day exposure at concentrations ranging 1–100 lg/L (Triebskorn et al. 2007). These data highlight the idea that b-blockers can affect fish at very low, but environmentally relevant, concentrations. However, an adult reproduction study using fathead minnows found that propranolol exposure at environmental concentrations did not pose a risk to fish (Giltrow et al. 2009). Similar findings were reported using rainbow trout (Owen et al. 2009) and the fathead minnow (Lorenzi et al. 2012). Taken together, major gaps in our understanding of b-blocker toxicity to aquatic organisms remain. Research into the ecotoxicity of environmental pollutants on the level of gene showed that gene transcriptional responses represent the primary interaction site between chemical and organism and often ultimately relate with physiological processes; thus, research into the ecotoxicity of environmental pollutants on the level of gene transcription has grown significantly over the last decade (Filby et al. 2007). However, to the best of our knowledge, relatively few studies have examined the transcriptional responses in fish exposed to b-blockers. Therefore, the objective of this study was to determine the transcriptional response of genes encoding the b-ARs, the potential target of b-blockers, and genes involved in other physiological processes, including antioxidant and detoxification pathways, in adult zebrafish after exposure to the b-blockers propranolol and metoprolol. This work can improve our understanding of the impact of b-blockers on fish and will ultimately be helpful for the risk assessment of these pharmaceuticals.
Materials and Methods Test Chemicals The (R, S)-propranolol hydrochloride (purity [99 %) and (R, S)-metoprolol tartrate salt (purity [98 %) were purchased from Sigma (St. Louis, MO, USA). No carrier
Transcriptional Responses in Adult Zebrafish (Danio rerio) Exposed to Propranolol and…
solvent was required for the preparation of the stock solutions because these test substances are all readily soluble. Test Fish The zebrafish (Danio rerio) used in this study were kindly provided by the Institute of Hydrobiology of the Chinese Academy of Science (Wuhan, China). The fish were maintained in charcoal-dechlorinated tap water at a constant temperature (26 ± 1 C) with a 16:8 h (light:dark) photoperiod. The brood stock was fed three times daily, once with newly hatched brine shrimp (Artemia nauplii) and twice with commercial food. Exposure Conditions and Fish Sampling Prior to the initiation of the experiment, adult zebrafish (approximately 7 months-old) were sex-separated and acclimated for 1 week. Exposure studies were conducted in glass tanks containing 3 L of the test solution under the conditions described above. Based on the results of our previous studies, fish were exposed to propranolol and metoprolol for 96 h at 0.03, 0.3 or 3 mg/L. There were three replicate tanks of seven male fish each and another three tanks with females, for a total of 21 males and females per treatment. Dechlorinated tap water controls were used in addition to the test series. The test solutions were 80 % renewed every 24 h. The test solutions were stored at 4 C between the semi-static renewal periods. Previous analytical studies indicated that both propranolol and metoprolol were stable in water when kept at 4 C in the dark (Dzialowski et al. 2006). The fish were not fed during exposure because fecal matter and uneaten food may decrease water quality and affect the biological activity of test chemicals. The fish were observed three times daily, and no mortality was found. At the end of the exposure, the fish were sacrificed by keeping them on ice. The brain, liver and gonad were excised, flash-frozen in dry ice and stored at -80 C for subsequent analyses. Quantitative Real-Time PCR Assay For gene expression analysis, the tissue of four or five fish were homogenized as one sample and a total of four replicates from each treatment group for each gender were prepared. The total RNA was extracted using RNAiso Plus (Takara, Dalian, China) according to the manufacturer’s instructions. The quality of the total RNA was evaluated by agarose gel electrophoresis and optical density reading (260/280 ratio 1.8–2.0). First-strand cDNA synthesis was performed using the ReverTra Ace qPCR RT kit (Toyobo,
Osaka, Japan). Real-time PCR with SYBR green detection was performed on a Mastercycler ep realplex (Eppendorf, Hamburg, Germany) according to the protocols established by the manufacturer (SYBR Green Realtime PCR Master Mix, Toyobo). The transcription of the genes encoding the b-ARs and those associated with other physiological processes, including detoxification, stress response and antioxidant pathways, were selected for quantification. The oligonucleotide primers specific for the selected genes (Supplemental Table S1) were identical to those used in previous studies (Gonzalez et al. 2005; Jin et al. 2010; Wang et al. 2009). The mRNA levels were expressed relative to the transcription level of the reference gene, bactin. The relative quantification of gene expression was analyzed from the measured cycle threshold (Ct) using the 2-DDCt method (Livak and Schmittgen 2001). Data Analysis Gene transcription was calculated as the fold-change relative to the average transcription in the vehicle control. Prior to conducting statistical comparisons, data were assessed for normality and homogeneity of variances using the Kolmogorov–Smirnov one-sample test and Levene’s test, respectively. Significant differences were assessed using a one-way analysis of variance (ANOVA) followed by the Dunnett’s post hoc test. The set value for statistical significance was p \ 0.05. All statistical analyses were performed with SPSS 13.0 software (SPSS, Chicago, IL).
Results Transcriptional Responses of Target Genes in Zebrafish Following Exposure to Propranolol In the brain, exposure to 3 mg/L of propranolol significantly up-regulated the transcription of arb1 and downregulated the mRNA levels of arb2b and arb3b in females when compared with the control (Fig. 1). In addition, the mRNA levels of arb2b and arb3b decreased in a concentration-dependent manner in male zebrafish exposed to propranolol (Fig. 1). No obvious changes in arb2a and arb3a were observed in any male or female treated groups. For genes involved in the stress response and detoxification mechanisms, including hsp70, mt1, mt2 and tap, a clear concentration-dependent induction was observed following propranolol exposure, especially in male fish (Fig. 1). Similar inductions were found for genes encoding antioxidant proteins, including cu/zn-sod, mn-sod, cat and gpx. In female fish, these genes were up-regulated significantly after exposure to 3 mg/L of propranolol; however, in male fish, 0.3 mg/L propranolol was sufficient to induce
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Fig. 1 Changes in the transcription of b-AR genes and genes involved in stress, detoxification and antioxidant responses in the brain of zebrafish exposed to propranolol. The results are presented as
the mean ± SEM and are expressed as the ratio of the target gene mRNA/b-actin mRNA. The asterisks denote significant differences when compared with the control group (*p \ 0.05 and **p \ 0.01)
Fig. 2 Changes in the transcription of b-AR genes and genes involved in stress, detoxification and antioxidant responses in the liver of zebrafish exposed to propranolol. The results are presented as
the mean ± SEM and are expressed as the ratio of the target gene mRNA/b-actin mRNA. The asterisks denote significant differences when compared with the control group (*p \ 0.05 and **p \ 0.01)
statistically significant changes when compared with the control (Fig. 1). In the liver of treated fish, a significant induction of arb1 mRNA was observed in females exposed to 3 mg/L propranolol and in males exposed to both 0.3 and 3 mg/L propranolol (Fig. 2). In contrast, 3 mg/L propranolol
down-regulated the mRNA level of arb3b in both females and males. Propranolol exposure did not alter the mRNA levels of arb2a, arb2b or arb3a. For genes involved in the stress response and detoxification, propranolol exposure significantly up-regulated the transcriptional level of hsp70, mt1 and mt2 in both female and male fish, but it did
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Transcriptional Responses in Adult Zebrafish (Danio rerio) Exposed to Propranolol and…
In the brain, metoprolol exposure did not produce any obvious changes in the transcription of b-AR genes except for arb3b, which was significantly increased after exposure
to a concentration of 0.03 mg/L in both females and males (Fig. 4). No obvious changes were observed in the transcription of genes involved in the stress response, detoxification or antioxidant pathways (Fig. 4). In the liver, exposure to metoprolol significantly downregulated the transcription of arb1 in both females and males; however, no changes were observed for other bAR genes (Fig. 5). For genes involved in the stress response and detoxification pathways, only the transcription of hsp70 was induced in treated males (Fig. 5). For antioxidant genes, there was a clear concentration-dependent induction of cat that reached a 4.1- and 4.3-fold increase relative to that of the control in treated females and males, respectively. However, no changes were found in cu/zn-sod, mn-sod and gpx in either female or male fish (Fig. 5). In the gonads, exposure to metoprolol resulted in an increase of arb1 and arb3b in females and arb1, arb2a, arb2b and arb3a in males (Fig. 6). However, these changes were significant only at a concentration of 0.03 mg/L and not at higher exposures. For genes involved in the stress response and detoxification pathways, exposure to metoprolol caused no changes in transcription in females, and it up-regulated the mRNA levels of hsp70, mt1 and mt2 in males at the highest concentration tested (Fig. 6). For antioxidant genes, metoprolol exposure increased the mRNA level of mn-sod in treated males and cat in both females and males (Fig. 6).
Fig. 3 Changes in the transcription of b-AR genes and genes involved in stress, detoxification and antioxidant responses in the gonad of zebrafish exposed to propranolol. The results are presented
as the mean ± SEM and are expressed as the ratio of the target gene mRNA/b-actin mRNA. The asterisks denote significant differences when compared with the control group (*p \ 0.05 and **p \ 0.01)
not change the mRNA level of tap (Fig. 2). For genes encoding antioxidant proteins, the transcription of cat was induced after exposure to propranolol in females; however, no changes were found in any other genes (Fig. 2). In males, the mRNA levels of mn-sod, cat and gpx were upregulated after exposure to 3 mg/L of propranolol. For b-AR genes in the gonads of treated fish, no changes were observed for arb1 and arb2a in either females or males (Fig. 3). Significant changes were observed in arb2b and arb3a in males, although a trend toward a down-regulation was observed in females. Significant decreases were found in arb3b in both males and females when compared with the control (Fig. 3). Exposure to propranolol induced no obvious changes in hsp70, mt1, mt2 and tap in the gonads from either females or males (Fig. 3). For antioxidant genes, the transcriptional responses to propranolol exposure were gender-specific. No changes were observed in treated females; however, significant increases in cu/zn-sod, mn-sod and cat were observed in males when compared with the controls (Fig. 3).
Transcriptional Responses of Target Genes in Zebrafish Following Exposure to Metoprolol
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Fig. 4 Changes in the transcription of b-AR genes and genes involved in stress, detoxification and antioxidant responses in the brain of zebrafish exposed to metoprolol. The results are presented as
the mean ± SEM and are expressed as the ratio of the target gene mRNA/b-actin mRNA. The asterisks denote significant differences when compared with the control group (*p \ 0.05 and **p \ 0.01)
Fig. 5 Changes in the transcription of b-AR genes and genes involved in stress, detoxification and antioxidant responses in the liver of zebrafish exposed to metoprolol. The results are presented as
the mean ± SEM and are expressed as the ratio of the target gene mRNA/b-actin mRNA. The asterisks denote significant differences when compared with the control group (*p \ 0.05 and **p \ 0.01)
Discussion
pharmacological target of b-blockers, the transcriptional responses of b-ARs were determined in zebrafish exposed to propranolol and metoprolol. Our results showed that the mRNA tissue distribution of these genes (data not shown) was consistent with the report of Wang et al. (2009). For propranolol, with the exception of arb1, the b-AR genes showed a concentration-dependent decrease. Although the
b-ARs are members of the G-protein-coupled receptor superfamily and mediate various physiological processes (Massarsky et al. 2011; Wang et al. 2009). In zebrafish, Wang et al. (2009) identified five b-AR genes designated as adrb1, adrb2a, adrb2b, adrb3a, and adrb3b. Being the
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Transcriptional Responses in Adult Zebrafish (Danio rerio) Exposed to Propranolol and…
Fig. 6 Changes in the transcription of b-AR genes and genes involved in stress, detoxification and antioxidant responses in the gonad of zebrafish exposed to metoprolol. The results are presented as
the mean ± SEM and are expressed as the ratio of the target gene mRNA/b-actin mRNA. The asterisks denote significant differences when compared with the control group (*p \ 0.05 and **p \ 0.01)
function of b-AR genes in the zebrafish has not been fully described, the down-regulation of their transcriptional response may be due to the antagonistic activity of bblockers on adrenoceptors. For metoprolol, with the exception of arb1 in the liver, the altered b-AR genes showed an increase at low-concentration exposures, followed by a decrease to levels similar to that observed in the unexposed controls. The increase in b-AR genes induced by low concentrations of metoprolol may be related to the compensatory response for adrenoceptor blockade; however, at higher concentrations, the down-regulation of bAR genes was similar to the observed propranolol effects, indicating the therapeutic effects of b-blockers. These results also revealed that the pharmacological effect of propranolol on fish is more potent than metoprolol, which could be explained by the lipophilicity of pharmaceuticals because the octanol–water partition coefficient of propranolol is greater than metoprolol (Massarsky et al. 2011). However, the difference in transcriptional patterns of b-AR genes between the two b-blockers may be due to receptor selectivity. Propranolol binds to any b-AR, while metoprolol has a higher affinity for the b1-AR (Fraysse and Garric 2005; Huggett et al. 2003). It has been confirmed that exposure to a variety of chemical pollutants produces oxygen free radicals and other reactive oxygen species (ROS) during metabolic processes in biological systems, which can subsequently cause oxidative damage to membrane lipids, DNA, and proteins (Valavanidis et al. 2006). Over the past decades,
molecular biomarkers of oxidative stress have been widely applied to study mechanisms of toxicity in aquatic organisms exposed to chemical pollutants. Here, the transcriptional responses of antioxidant enzymes, including Cu/ZnSod, Mn-Sod, Cat, and GPx, were determined in fish exposed to propranolol and metoprolol. With the exception of the genes in the brain of fish exposed to metoprolol, concentration-dependent changes were observed in antioxidant gene transcripts in both male and female fish. Although limited data regarding oxidative stress caused by b-blockers in fish were available for comparison, our observations are in agreement with previous studies in aquatic invertebrates. For example, propranolol exposure resulted in elevated lipid peroxidation damage in the gill of the marine mussel Mytilus galloprovincialis (Sole et al. 2010), and reduced the lysosomal membrane stability of hemocytes and induced CAT activity in the digestive gland (Franzellitti et al. 2011). Liu and Williams (2007) suggested that the formation of intermediate radicals, which are highly reactive and cause oxidative stress, occurs during the degradation of propranolol. As for metoprolol, mRNA levels of the antioxidant enzymes SOD and CAT were significantly induced in the digestive gland of the freshwater mussel Dreissena polymorpha, presumably generated as a side-effect of phase I biotransformation (Contardo-Jara et al. 2010). In addition, it was found that high propranolol concentrations were associated with increased CAT activity in fluvial biofilms, indicating that exposure to b-blockers can also cause oxidative stress in
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algae and bacteria within river biofilms and the whole aquatic ecosystem (Bonnineau et al. 2010). Generally, xenobiotic exposure alters homoeostasis. HSP70 acts as an indicator for protein damage and subsequent protective processes (Contardo-Jara et al. 2010), and it is involved in the oxidative stress response (Gonzalez et al. 2005). In the present study, propranolol exposure induced the up-regulation of hsp70 mRNA in the brain and liver of both genders. Induction of hsp70 was found in the gonad and liver of males exposed to metoprolol. Similarly, Contardo-Jara et al. (2010) found a significant upregulation of hsp70 mRNA in the gill of mussels exposed to metoprolol; furthermore, this induction persisted for 1 week. Metallothionein (Mt) functions in primary metal storage, transport and detoxification, and the induction of MT mRNA can indirectly indicate the level of oxidative stress in an organism (Contardo-Jara et al. 2010; Gonzalez et al. 2005). In this study, the induction of mt1 and mt2 mRNA was observed after propranolol and metoprolol exposure. This induction, coupled with similar observations previously reported in mussels exposed to metoprolol (Contardo-Jara et al. 2010), may be due to the hydroxyl radical derived from biotransformation processes of bblockers. The gene that encodes TAP belongs to the family of ATP binding-cassette (ABC) transporters that utilize ATP as an energy source and extrude xenobiotics by active efflux (Gonzalez et al. 2005). Results of pervious studies showed that various metals, such as Hg and Cd, could induce the transcription of tap in the zebrafish (Arini et al. 2015; Gonzalez et al. 2006, 2005). However, to our knowledge, no data about the effects of b-blockers on tap mRNA changes was available. In this study, the tap gene was increased only in the brain of male zebrafish after propranolol exposure. It should be noted that the transcriptional responses of the selected genes displayed substantial tissue-specificity. For example, significant induction of antioxidant and detoxification genes were stimulated by propranolol exposure in the brain. The transcript levels of all eight genes were increased in males, and six genes were increased in females. Conversely, in the gonad, only cu/znsod, mn-sod and cat were induced by propranolol in males. The tissue-specific effects of b-blockers was found previously. CAT activities were increased in the digestive gland and reduced in the mantle/gonads in marine mussels exposed to propranolol (Franzellitti et al. 2011). For metoprolol, the mRNA levels of SOD and CAT were significantly induced in the digestive gland, but not the gill, of the freshwater mussel (Contardo-Jara et al. 2010). The specific biotransformation or detoxification process in different tissues might account for the difference in susceptibility to b-blockers. Moreover, it has been shown that propranolol can cross the blood–brain barrier in higher
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organisms due to its relatively high lipophilicity when compared with most other b-blockers. One study reported that the concentration in human brain tissue was 10 to 26 times higher than in plasma (Cruickshank and Neil-Dwyer 1985). Although no convincing data were demonstrated in fish, the relatively high concentration of propranolol in the brain likely produced the substantial changes in gene transcription. In addition to tissue-specificity, the genderspecificity of the b-blockers should be emphasized as well. For most of the genes tested, the changes observed in males were greater than those of females. Giltrow et al. (2009) also reported that the plasma concentration of propranolol in male adult fathead minnows after a 21-day exposure was greater than the levels found in females. These results demonstrate that it is extremely important to consider the gender of fish when assessing the effects of b-blockers and other pharmaceuticals. Based on the results of present study, the transcriptional changes in b-ARs genes are likely due to the expected pharmacological activities of b-blockers. Similarly, transcriptional changes in genes involved in the antioxidant response and detoxification process were found. Considering the concentrations of b-blockers found in the aquatic environment, the results of the present study, our previous studies (Sun et al. 2014) and those of other researchers (Giltrow et al. 2009; Owen et al. 2009), these pharmaceuticals seem unlikely to pose a risk to the well-being of fish. However, more specific consequences of adrenoceptor blockade should be assessed before a definitive answer can be given. However, it was reported that propranolol exposure at concentrations that produced a fish plasma concentration within the range recorded in patients also induced an effective b-adrenoceptor blockade; however, when the fish plasma concentration exceeded the human therapeutic plasma concentration, undesired toxicity of propranolol was elicited (Giltrow et al. 2009; Owen et al. 2009). Although the present study did not measure fish plasma concentrations, the effective concentrations of propranolol fall within the range of these previous reports. Thus, our data supports the ‘‘read-across’’ hypothesis from human to fish, and the existing human pharmacological or toxicological data could be useful for predicting the ecotoxicological risk of these pharmaceuticals on fish. In conclusion, our study demonstrates that both propranolol and metoprolol can change the mRNA levels of bARs, the target of b-blockers, indicating the expected pharmacological effects. Additionally, the transcription of genes related to the antioxidant response and detoxification process were induced after exposure to propranolol and metoprolol, suggesting that exposure to b-blockers can activate the detoxification process and result in oxidative stress in fish. It appears that environmentally relevant concentrations of these pharmaceuticals are unlikely
Transcriptional Responses in Adult Zebrafish (Danio rerio) Exposed to Propranolol and…
sufficient to elicit adverse effects on fish because an effective b-adrenoceptor blockade occurred only within the therapeutic range. However, further studies with prolonged exposure should be performed and, in fact, these studies are being conducted by our research group. Acknowledgments We gratefully acknowledge the National Natural Science Foundation of China (Nos. 21377118, 20907044), Program for Changjiang Scholars and Innovative Research Team in University (IRT13096), and Key Laboratory of Fishery Ecology and Environment, Guangdong Province (LFE-2013-2) for supporting this research. Conflict of interest of interest.
The authors declare that they have no conflict
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