Lifespans in wild-type and mutants exposed to clenbuterol or ractopamine.

<p>Exposures were performed from L1-larvae to adult (prolonged exposure) at the concentration of 10 µg/L. Thirty nematodes were examined per treatment. clen, clenbuterol; rac, ractopamine. Bars represent mean ± S.E.M. **<i>P</i><0.01.</p

Comparison of lethality and growth in nematodes exposed to different concentrations of clenbuterol or ractopamine.

<p>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 ± S.E.M. **<i>P</i><0.01.</p

Comparison of brood size and locomotion behavior in nematodes exposed to different concentrations of clenbuterol or ractopamine.

<p>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 ± S.E.M. *<i>P</i><0.05, **<i>P</i><0.01.</p

Adverse Effects from Clenbuterol and Ractopamine on Nematode <i>Caenorhabditis elegans</i> and the Underlying Mechanism

<div><p>In the present study, we used <i>Caenorhabditis elegans</i> assay system to investigate <i>in vivo</i> 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 <i>sod-2</i> 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 <i>daf-16</i>, <i>sgk-1</i>, <i>skn-1</i>, and <i>aak-2</i> genes than clentuberol, and increased expression levels of <i>daf-2</i> and <i>age-1</i> genes at the examined concentration. Therefore, the <i>C. elegans</i> 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.</p></div

Effects of clenbuterol or ractopamine exposure on expression patterns of genes required for aging control.

<p>(A) Effects of clenbuterol or ractopamine exposure on expression patterns of genes in insulin/IGF-1 signaling pathway. (B) Effects of clenbuterol or ractopamine exposure on expression patterns of genes in TOR signaling pathway. (C) Effects of clenbuterol or ractopamine exposure on expression patterns of genes in germline signaling pathway. Exposures were performed from L1-larvae to adult (prolonged exposure) at the concentration of 10 µg/L. Bars represent mean ± S.E.M. **<i>P</i><0.01.</p

Effects of overexpression of <i>sod-2</i> gene on toxicity from clentuberol or ractopamine in <i>C. elegans</i>.

<p>(A) Effects of overexpression of <i>sod-2</i> gene on growth in clentuberol or ractopamine exposed nematodes. Twenty nematodes were examined per treatment. (B) Effects of overexpression of <i>sod-2</i> gene on brood size in clentuberol or ractopamine exposed nematodes. Twenty nematodes were examined per treatment. (C) Effects of overexpression of <i>sod-2</i> gene on locomotion behavior in clentuberol or ractopamine exposed nematodes. Fifty nematodes were examined per treatment. (D) Effects of overexpression of <i>sod-2</i> gene on intestinal autofluorescence in clentuberol or ractopamine exposed nematodes. Twenty nematodes were examined per treatment. (E) Effects of overexpression of <i>sod-2</i> gene on intestinal ROS production in clentuberol or ractopamine exposed nematodes. Twenty nematodes were examined per treatment. (F) Effects of overexpression of <i>sod-2</i> 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 µg/L. Bars represent mean ± S.E.M. **<i>P</i><0.01.</p

Comparison of intestinal autofluorescence (A) and ROS production (B) in nematodes exposed to clenbuterol or ractopamine.

<p>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. Bars represent mean ± S.E.M. **<i>P</i><0.01.</p

Comparison of lifespan in nematodes exposed to clenbuterol or ractopamine.

<p>(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 ± S.E.M. *<i>P</i><0.05, **<i>P</i><0.01.</p