Adverse Effects from Clenbuterol and Ractopamine on Nematode Caenorhabditis elegans and the Underlying Mechanism

In the present study, we used Caenorhabditis elegans assay system to investigate in vivo toxicity from clentuberol and ractopamine and the possible underlying mechanism. Both acute and prolonged exposures to clentuberol or ractopamine decreased brood size and locomotion behavior, and induced intestinal autofluorescence and reactive oxygen species (ROS) production. Although acute exposure to the examined concentrations of clentuberol or ractopamine did not induce lethality, prolonged exposure to 10 µg/L of clentuberol and ractopamine reduced lifespan. At relatively high concentrations, ractopamine exhibited more severe toxicity than clentuberol on nematodes. Overexpression of sod-2 gene encoding a Mn-SOD to prevent induction of oxidative stress effectively inhibited toxicity from clentuberol or ractopamine. Besides oxidative stress, we found that clentuberol might reduce lifespan through influencing insulin/IGF signaling pathway; however, ractopamine might reduce lifespan through affecting both insulin/IGF signaling pathway and TOR signaling pathway. Ractopamine more severely decreased expression levels of daf-16, sgk-1, skn-1, and aak-2 genes than clentuberol, and increased expression levels of daf-2 and age-1 genes at the examined concentration. Therefore, the C. elegans assay system may be useful for assessing the possible toxicity from weight loss agents, and clentuberol and ractopamine may induce toxicity through different molecular mechanisms.


Introduction
Illegal or unsuitable use of weigh loss agents has gradually become a public health concern [1][2]. Clenbuterol, a typical weight loss agent, is a kind of b2-adrenergic agonist, and was illegally used as a feed additive to improve production performance and a carcass composition in many countries [3][4]. With the increased use of internet sales, the internet has made this even banned product to be readily accessible for the aim of weight loss or dieting addition [2]. Ractopamine, a synthetic b2-adrenoceptor agonist, is now widely used as a feed additive in the United States to promote a reduction in body fat and to enhance muscle growth in cattle, pigs, or turkeys [5].
Among the used weight loss agents, clenbuterol and ractopamine belong to products that may have health hazards upon accidental or intentional exposure and ingestion [2,6]. The in vitro study has indicated that clenbuterol exhibited potential toxicity on structure and function of trypsin, an important digestive enzyme, and stimulated guinea-pig heart rate [4,7]. Following consumption of meat or liver from clenbuterol administrated cattle, intoxication cases were described [7]. Moreover, at least the toxic effects on cardiovascular systems (such as tachycardia and hypertension) are considered to be of clinical relevance [7][8][9]. More recently, it was further reported that ractopamine administration might cause the myocardial toxicity in dogs [5].
Several toxicological studies have been performed for clenbuterol. Administration of growth-promoting doses of clenbuterol adversely affected the liver function in female pigs [10]. Dietary administration of clenbuterol decreased androgen receptor (AnR) expression in testicle, glucocorticoid receptor (GR) expression in lymphoid tissues, and b-adrenergic receptor (b-AR) expression in targeted organs of chickens [3]. Clenbuterol caused an impairment of collagen turnover by down-regulating MMP-9 activity [11]. Clenbuterol not only enhanced muscle fiber size but also increased expression of GATA-2 protein in skeletal muscle of rat uterus [12]. The preferential involvement of calpain 2 autolysis was found for clenbuterol-induced skeletal muscle remodelling in rats [13]. Overexpression of calpastatin in skeletal muscle of mice prevented clenbuterol-induced muscle hypertrophy and phenotypic shift [14]. However, so far no toxicological study on ractopamine has been performed.
In the present study, we first compared the toxicity between clentuberol and ractopamine with the aid of a series of endpoints in C. elegans. Moreover, considering the fact that we still know limited information about toxicological mechanism for the clentuberol and especially the ractopamine, we examined the underlying mechanism for toxicity from clentuberol and ractopamine. Our results here will be useful for the further understanding multiple toxicities from clentuberol and ractopamine and the underlying mechanism.

Results
Comparison of lethality and growth in clentuberol or ractopamine exposed nematodes Considering the fact that many toxicants at the low concentrations may have the adverse effects on nematodes after prolonged exposure [18,25,[44][45][46][47][48][49], we performed both the acute exposure and the prolonged exposure for clentuberol or ractopamine. Concentrations of 0.01-5 mg/L were used for acute exposure to clentuberol or ractopamine. After acute exposure from young adult stage for 24-hr, both clentuberol and ractopamine did not induce lethality and alteration of body length in nematodes (Fig. 1). Concentrations of 0.01-10 mg/L were used for prolonged exposure to clentuberol or ractopamine. After prolonged exposure from L1-larvae to the adult stage, although both clentuberol and ractopamine still did not induce lethality of nematodes, both clentuberol and ractopamine at concentrations more than 1 mg/L significantly reduced body length of nematodes (Fig. 1).
Comparison of reproduction and locomotion behavior in clentuberol or ractopamine exposed nematodes Reproductive organ and neuron may be important secondary targeted organs for toxicants in nematodes [18,44,[48][49]52]. Acute exposure to clenbuterol or ractopamine at concentrations of 0.01-0.1 mg/L and prolonged exposure to 0.01 mg/L of clenbuterol or ractopamine did not significantly alter brood size (Fig. 2). Acute exposure to 0.01 mg/L of clenbuterol or ractopamine did not significantly influence locomotion behavior of nematodes (Fig. 2). In contrast, acute exposure to clenbuterol or ractopamine at concentrations more than 1 mg/L and prolonged exposure to clenbuterol or ractopamine at concentrations more than 0.1 mg/L significantly reduced brood size (Fig. 2). Acute exposure to clenbuterol or ractopamine at concentrations more than 0.1 mg/L and prolonged exposure to clenbuterol or ractopamine at concentrations more than 0.01 mg/L significantly decreased locomotion behavior of nematodes (Fig. 2). More interestingly, we observed that acute exposure to 1-5 mg/L of ractopamine and prolonged exposure to 0.1-10 mg/L of ractopamine exhibited more severe toxicity on locomotion behavior than clenbuterol in nematodes, although ractopamine at the examined concentrations still showed the similar toxicity on brood size to clenbuterol in nematodes (Fig. 2).
Comparison of intestinal autofluorescence in clentuberol or ractopamine exposed nematodes Intestine is the primary targeted organ for toxicants in nematodes [18,41,43,48]. Acute exposure to clenbuterol or ractopamine at concentrations of 0.01-0.1 mg/L and prolonged exposure to clenbuterol or ractopamine at concentrations of 0.01-0.1 mg/L did not induce the significant intestinal autofluorescence compared with control (Fig. 3A). In contrast, acute exposure to 5 mg/L of clenbuterol or ractopamine and prolonged exposure to clenbuterol or ractopamine at concentrations of 1-10 mg/L significantly induced the intestinal autofluorescence compared with control (Fig. 3A). More interestingly, we observed that acute exposure to 5 mg/L of ractopamine and prolonged exposure to 1-10 mg/L of ractopamine induced more pronounced intestinal autofluorescence than clenbuterol in nematodes (Fig. 3A).
Comparison of intestinal reactive oxygen species (ROS) production in clentuberol or ractopamine exposed nematodes In C. elegans, toxicants usually cause damage on animals by inducing oxidative stress [18,29,53]. We further examined the intestinal ROS production in clentuberol or ractopamine exposed nematodes. After acute exposure, both clentuberol and ractopamine at the concentration of 5 mg/L induced the significant intestinal ROS production, and acute exposure to ractopamine at the concentration of 5 mg/L resulted in more severe intestinal ROS production than clentuberol (Fig. 3B). After prolonged exposure, similarly, both clentuberol and ractopamine at the concentration of 10 mg/L caused significant intestinal ROS production, and prolonged exposure to ractopamine at the concentration of 10 mg/L induced more severe intestinal ROS production than clentuberol (Fig. 3B). These data suggest that toxicity from clentuberol or ractopamine may be closely associated with the induction of oxidative stress in nematodes.

Comparison of lifespan in clentuberol or ractopamine exposed nematodes
We further investigated the effects of exposure to clentuberol and ractopamine on lifespan in nematodes. Lifespan is an important endpoint and may reflect the long-term effects of a specific toxicant in nematodes [52,54]. Acute exposure to clentuberol or ractopamine at the concentration of 5 mg/L did not significantly alter lifespan of nematodes (Figs. 4A and 4B). In contrast, prolonged exposure to clentuberol or ractopamine at the concentration of 10 mg/L significantly reduced the lifespan of nematodes (Figs. 4C and 4D). Moreover, we found that prolonged exposure to ractopamine at the concentration of 10 mg/L more severely inhibited the lifespan of nematodes than clentuberol (Figs. 4C and 4D).
Overexpression of SOD-2 prevented the toxicity of clentuberol or ractopamine on nematodes In C. elegans, sod-2 and sod-3 genes encode Mn-SODs, which function in protecting animals from the damage from oxidative stress [45,55]. Previous studies have demonstrated that overexpression of Mn-SODs inhibited the oxidative stress and prevented the toxicity from metals and nanomaterials [45,55]. To examine the role of oxidative stress in inducing the toxicity from clentuberol or ractopamine on nematodes, we investigated the effects of overexpression of sod-2 gene in all cells of nematodes (Ex(Pdpy-30-sod-2)) on toxicity from clentuberol or ractopamine. Transgenic strain of Ex(Pdpy-30-sod-2) had no deficits in body length, brood size, head thrash, and body bend, and did not have significant intestinal autofluorescence and intestinal ROS production (Figs. 5A-5E). After prolonged exposure to 10 mg/L of clentuberol or ractopamine, we did not detect the significant decreases in body length, brood size, head thrash, and body bend in nematodes overexpressing sod-2 gene compared with control (Figs. 5A-5C). Moreover, after prolonged exposure to 10 mg/L of clentuberol or ractopamine, we did not observe the significant induction of both intestinal autofluorescence and intestinal ROS production in nematodes overexpressing the sod-2 gene compared with control (Figs. 5D and 5E). Nematodes overexpressing sod-2 gene had the similar lifespan to that of wild-type N2 (data not shown). After prolonged exposure to 10 mg/L of clentuberol or ractopamine, our results further demonstrate that no significant reduction of lifespan was observed in nematodes overexpressing sod-2 gene compared with control (Fig. 5F).
To further confirm the functions of the dysregulated genes in regulating the toxicity formation from clentuberol or ractopamine, we used the corresponding mutants to investigate the lifespans of these mutants exposed to clentuberol or ractopamine. With the aid of lifespan as the endpoint, interestingly, we found that the daf-16(mu86), sgk-1(ok538), skn-1(zu67), and aak-2(ok524) mutants had the susceptible property to the toxicity of clentuberol or ractopamine (Fig. 7). In contrast, the daf-2(e1370), age-1(hx546), Figure 1. Comparison of lethality and growth in nematodes exposed to different concentrations of clenbuterol or ractopamine. Exposures were performed from the young adult for 24-hr (acute exposure) or from L1-larvae to adult (prolonged exposure). Fifty nematodes were examined per treatment for lethality assay, and twenty nematodes were examined per treatment for growth assay. Bars represent mean 6 S.E.M. **P,0.01. doi:10.1371/journal.pone.0085482.g001 daf-15(m81), and rict-1(mg360) mutants had the resistant property to the toxicity of ractopamine (Fig. 7). These data further confirm the involvement of the related signaling pathways in regulating the toxicity formation from clentuberol or ractopamine.

Discussion
In the present study, we first provide a series of evidence to indicate the usefulness of C. elegans assay system in assessing the in vivo toxicity of weight loss agents, such as clentuberol or ractopamine. With the aid of brood size, head thrash, body bend, intestinal autofluorescence, and intestinal ROS production as the endpoints, we detected the toxicity from acute exposure to clentuberol or ractopamine (Figs. 2 and 3). With the aid of body length, brood size, head thrash, body bend, intestinal autofluorescence, intestinal ROS production, and lifespan as the endpoints, we further detected the toxicity from prolonged exposure to clentuberol or ractopamine (Figs. 1-4). Using head thrash and body bend as the endpoints, we could observe the potential toxicity from acute exposure to 0.1 mg/L of clentuberol or ractopamine, and the potential toxicity from prolonged exposure to 0.01 mg/L of clentuberol or ractopamine (Fig. 2). Therefore, C. elegans may be a relatively sensitive assay system for toxicity assessment of food additives. Especially, the prolonged exposure assay system may be more suitable for detecting the potential toxicity of long-term adverse effects of weight loss agents. For example, prolonged exposure to 1-10 mg/L of clentuberol or ractopamine significantly decreased the body length of nematodes (Fig. 1). In contrast, acute exposure to all the examined concentrations of clentuberol or ractopamine did not noticeably influence the body length of nematodes (Fig. 1). Nevertheless, we did not observe the induction of lethality in nematodes exposed to clentuberol or ractopamine at the examined concentrations (Fig. 1). The careful selection of suitable and sensitive endpoints for toxicity assessment of weight loss agents should be paid attention to, since the toxicity of illegal weight loss agents may be not so severe as heavy metals [21,23,[25][26][27][28][29][30] or organic pesticides [33][34]. Comparison of brood size and locomotion behavior in nematodes exposed to different concentrations of clenbuterol or ractopamine. Locomotion behavior of nematodes was evaluated by endpoints of head thrash and body bend. Exposures were performed from the young adult for 24-hr (acute exposure) or from L1-larvae to adult (prolonged exposure). Twenty nematodes were examined per treatment for brood size assay, and fifty nematodes were examined per treatment for locomotion behavior assay. Bars represent mean 6 S.E.M. *P,0.05, **P,0.01. doi:10.1371/journal.pone.0085482.g002 Based on our data, we indicate that ractopamine may somewhat have more severe toxicity than clentuberol in nematodes. With the aid of head thrash, body bend, intestinal autofluorescence, and intestinal ROS production as the endpoints, after acute exposure, we detected the toxicity differences between clentuberol and ractopamine at concentrations of 1 and/or 5 mg/L (Figs. 2 and 3). With the aid of head thrash, body bend, intestinal autofluorescence, intestinal ROS production, and lifespan as the endpoints, after prolonged exposure, we observed the toxicity differences between clentuberol and ractopamine at relatively high concentrations  (Figs. 2-4). Considering the fact that the toxicity for toxicants in C. elegans may be similar to that observed in mammals [16,19], our data here imply the possible potential more severe toxicity of ractopamine than clentuberol in mammals.
Oxidative stress is a key mechanism for toxicants to induce the damage on nematodes [16,18]. Our data demonstrate that both clentuberol and ractopamine induced the significant intestinal ROS production (Fig. 3B). Moreover, with the aid of the transgenic strain of Ex(Pdpy-30-sod-2), we found that overexpression of sod-2 gene in all cells effectively prevented the potential toxicity from clentuberol or ractopamine on growth, reproduction, locomotion behavior, intestinal development and lifespan in nematodes (Fig. 5). Therefore, oxidative stress is an important mechanism for clentuberol and ractopamine to induce the damage on nematodes.
Moreover, we found that clentuberol and ractopamine also induce the toxicity on nematodes through other mechanisms. In C. elegans, our data demonstrate that clentuberol and ractopamine caused the reduction in lifespan of animals possibly through different molecular mechanisms. Clentuberol might reduce the lifespan of nematodes through influencing the insulin/IGF signaling pathway; however, ractopamine might reduce the lifespan of nematodes through affecting both the insulin/IGF signaling pathway and the TOR signaling pathway (Fig. 6). In C. elegans, insulin/IGF-1 receptor (DAF-2) activates its tyrosine kinase activity and initiates a cascade of phosphorylation events that activate several kinases: phosphatidiylinositol 3-kinase (PI3K/ AGE-1), 3-phosphoinositide-dependent kinase 1 (PDK-1), and serine/threonine-protein kinase (SGK-1) [56]. Ultimately, SGK-1 phosphorylates and inactivates the FOXO transcription factor DAF-16 and thereby blocks the transcription of targeted genes [57]. The catalytic subunit of AMP-activated protein kinase, AAK-2, is necessary for the long lifespan of daf-2 mutants, which explains the energy mechanism [58]. TOR exists in two complexes, TORC1 and TORC2, and TORC1 and TORC2 contain different coactivators, DAF-15/Raptor and RICT-1/ Rictor [59]. We hypothesize here that the more reduction in lifespan in ractopamine exposed nematodes than in clentuberol exposed nematodes may at least due to the induction of more severe alteration in genes required for aging control and the changes of more signaling pathways. Our data suggest that ractopamine more severely decreased the expression levels of daf-16, sgk-1, skn-1, and aak-2 genes than clentuberol (Fig. 6A). In addition, ractopamine not only decreased the expression levels of daf-16, sgk-1, skn-1, and aak-2 genes like clentuberol, but also increased the expression levels of daf-2 and age-1 genes (Fig. 6A).
Previous studies further imply the possible crosstalk between insulin/IGF signaling pathway and oxidative stress in clentuberol or ractopamine exposed nematodes. The transcription factor Skinhead (SKN-1) regulates resistance to oxidative stress and expression of detoxification genes in response to reduced isulin/ IGF-1 signaling [60]. That is, the activated insulin/IGF signaling may at least partially contribute to the induction or regulation of oxidative stress in clentuberol or ractopamine exposed nematodes.
In conclusion, we provide the evidence in the present study to demonstrate that the C. elegans assay system was useful for assessment of possible in vivo toxicity from weight loss agents such as clentuberol and ractopamine. Our data imply that ractopamine exposure might induce more severe toxicity than clentuberol exposure in nematodes. Besides the oxidative stress, our results demonstrate that both insulin/IGF-1 signaling pathway and TOR signaling pathway were involved in the regulation of toxicity Figure 4. Comparison of lifespan in nematodes exposed to clenbuterol or ractopamine. (A and C) Lifespan curves of nematodes exposed to clenbuterol or ractopamine. (B and D) Comparison of mean lifespans in nematodes exposed to clenbuterol or ractopamine. Exposures were performed from the young adult for 24-hr (acute exposure) or from L1-larvae to adult (prolonged exposure). Thirty nematodes were examined per treatment. Bars represent mean 6 S.E.M. *P,0.05, **P,0.01. doi:10.1371/journal.pone.0085482.g004 Figure 5. Effects of overexpression of sod-2 gene on toxicity from clentuberol or ractopamine in C. elegans. (A) Effects of overexpression of sod-2 gene on growth in clentuberol or ractopamine exposed nematodes. Twenty nematodes were examined per treatment. (B) Effects of overexpression of sod-2 gene on brood size in clentuberol or ractopamine exposed nematodes. Twenty nematodes were examined per treatment. (C) Effects of overexpression of sod-2 gene on locomotion behavior in clentuberol or ractopamine exposed nematodes. Fifty nematodes were examined per treatment. (D) Effects of overexpression of sod-2 gene on intestinal autofluorescence in clentuberol or ractopamine exposed nematodes. Twenty nematodes were examined per treatment. (E) Effects of overexpression of sod-2 gene on intestinal ROS production in clentuberol or ractopamine exposed nematodes. Twenty nematodes were examined per treatment. (F) Effects of overexpression of sod-2 gene on lifespan in clentuberol or ractopamine exposed nematodes. Thirty nematodes were examined per treatment. Exposures were performed from L1-larvae to adult (prolonged exposure) at the concentration of 10 mg/L. Bars represent mean 6 S.E.M. **P,0.01. doi:10.1371/journal.pone.0085482.g005 Toxicity from Clenbuterol and Ractopamine PLOS ONE | www.plosone.org induction from clentuberol or ractopamine in nematodes. Moreover, we hypothesize that clentuberol and ractopamine might induce the toxicity on nematodes through different molecular mechanisms. Our data here will be helpful for our further understanding the potential damage from illegal use of weight loss agents on health of human and animals and the future design of effective strategies against the adverse effects from weight loss agents.

Lethality and growth
Lethality was evaluated by the percentage of survival animals. Following exposure, inactive ones were scored under a dissecting microscopy and nematodes were judged to be dead if they did not respond to stimulus using a small, metal wire. Fifty nematodes were examined per treatment. Growth was assessed by the body length, which was determined by measuring the flat surface area of nematodes using the Image-ProH Express software. Twenty nematodes were examined per treatment. Three replicates were performed.

Brood size and locomotion behavior
Reproduction was assessed by the brood size of adult nematodes. To assess brood size, we counted the number of offspring at all stages. Nematodes were transferred daily to new agar plates, until the completion of the egg laying period. Hatched progeny were allowed to grow to L1/L2 stage and counted manually. Twenty nematodes were examined per treatment. Three replicates were performed.
Locomotion behaviors of nematodes were evaluated by head thrash and body bend [62]. To assay head thrash, every examined nematode was transferred into a microtiter well containing 60 mL of modified K medium on the top of agar, and head thrashes were counted for 1-min after a 1-min recovery period. A thrash was defined as a change in the direction of bending at the mid body. To assay body bend, nematodes were picked onto a second plate and scored for the number of body bends in an interval of 20 sec. A body bend was counted as a change in the direction of the part of the nematodes corresponding to the posterior bulb of the pharynx along the y axis, assuming that the nematode was traveling along the x axis. Fifty nematodes were examined per treatment. Three replicates were performed.

Intestinal autofluorescence
Intestinal autofluorescence caused by lysosomal deposits of lipofuscin can accumulate over time in aging nematodes [63][64]. Images were collected for endogenous intestinal fluorescence using a 525-nm bandpass filter and without automatic gain control in order to preserve the relative intensity of different animal's fluorescence. Observations of fluorescence were recorded and color images subjected to a common exposure time were taken for the documentation of results with MagnafireH software (Olympus, Irving, TX, USA). Lipofuscin levels were measured using ImageJ Software (NIH Image) by determining average pixel intensity in each animal's intestine. Twenty nematodes were examined per treatment. Three replicates were performed.

ROS production
To quantify whether the clenbuterol or ractopamine exposure activated the oxidative damage, ROS production was assayed. The examined nematodes were transferred to M9 buffer containing 1 mM of 5-(and-6)-chloromethyl-29, 79-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) to pre-incubate for 3-h at 20uC, and then mounted on agar pads for examination with a laser scanning confocal microscope (Leica, TCS SP2, Bensheim, Germany) at 488 nm of excitation wavelength and 510 nm of emission filter. Relative fluorescence intensities of the intestines were semi-quantified as Figure 7. Lifespans in wild-type and mutants exposed to clenbuterol or ractopamine. Exposures were performed from L1-larvae to adult (prolonged exposure) at the concentration of 10 mg/L. Thirty nematodes were examined per treatment. clen, clenbuterol; rac, ractopamine. Bars represent mean 6 S.E.M. **P,0.01. doi:10.1371/journal.pone.0085482.g007 described previously [30]. The semiquantified ROS was expressed as relative fluorescent units (RFU). Twenty nematodes were examined per treatment. Three replicates were performed.

Lifespan assay
Lifespan assay was performed basically as described [65][66]. In the test, the hermaphrodites were transferred daily for the first 4 days of adulthood. Nematodes were checked every day and would be scored as dead when they did not move even after repeated taps with a pick. Thirty nematodes were examined per treatment. For lifespan, graphs are representative of at least three trials.
Quantitative real-time polymerase chain reaction (PCR) Total RNA was extracted using RNeasy Mini Kit (Qiagen). Total nematode RNA (,1 mg) was reverse-transcribed using a cDNA Synthesis kit (Bio-Rad Laboratories). Quantitative reverse transcription PCR (RT-PCR) was run at the optimized annealing temperature of 58uC. Relative quantification of the targeted genes in comparison to the reference act-1 gene was determined, and the final results were expressed as the relative expression ratio (between targeted genes and reference gene). The designed primers for targeted genes and reference act-1 gene were shown in Table S2.

DNA construct and germline transformation
To construct plasmid of Pdpy-30-sod-2, dpy-30 gene promoter fragment (1907 bp, PstI/BamHI) was subcloned into the pPD95_75 vector, and the full length of sod-2 cDNA was inserted into the site of SmaI/KpnI of the pPD95_75 vector behind Pdpy-30 fragment. Transgenic nematodes of Ex(Pdpy-30-sod-2) were generated as described [67]. Plasmids were injected as a mix at 20 ng/mL using Pdop-1::rfp as a transgenic marker.

Statistical analysis
All data were expressed as means 6 standard error of the mean (S.E.M.). Statistical analysis was performed using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) followed by Tukey post-hoc test was used to determine the significance of differences between the groups. Probability levels of 0.05 and 0.01 were considered statistically significant. The lifespan data were statistically analyzed using a 2-tailed 2 sample t-test (Minitab Ltd., Coventry, UK).