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Journal of Toxicology and Environmental Health, Part A: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh20
Toxicity of 7-Ketocholesterol on Lethality, Growth, Reproduction, and Germline Apoptosis in the Nematode Caenorhabditis elegans a
a
b
a
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Yunfeng Zong , Junlan Gao , Huiyun Feng , Beijiu Cheng & Xin Zhang a
School of Life Sciences, Anhui Agricultural University, Hefei, P. R. China
b
Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, P. R. China Published online: 01 May 2014.
To cite this article: Yunfeng Zong, Junlan Gao, Huiyun Feng, Beijiu Cheng & Xin Zhang (2014) Toxicity of 7-Ketocholesterol on Lethality, Growth, Reproduction, and Germline Apoptosis in the Nematode Caenorhabditis elegans, Journal of Toxicology and Environmental Health, Part A: Current Issues, 77:12, 716-723, DOI: 10.1080/15287394.2014.888693 To link to this article: http://dx.doi.org/10.1080/15287394.2014.888693
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Toxicology and Environmental Health, Part A, 77:716–723, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287394.2014.888693
TOXICITY OF 7-KETOCHOLESTEROL ON LETHALITY, GROWTH, REPRODUCTION, AND GERMLINE APOPTOSIS IN THE NEMATODE Caenorhabditis elegans Yunfeng Zong1, Junlan Gao1, Huiyun Feng2, Beijiu Cheng1, Xin Zhang1 1
School of Life Sciences, Anhui Agricultural University, Hefei, P. R. China Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, P. R. China
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7-Ketocholesterol is one of the most abundant cholesterol oxides, and is known to be cytotoxic to various types of cultured mammalian cells; however, little is known regarding its effects in vivo. With the use of the nematode Caenorhabditis elegans as model organism, in vivo toxicity of 7-ketocholesterol was investigated. The aim of the study was to examine the effects on life span, as well as short-term effects on reproduction, thermotolerance, germline apoptosis, and reactive oxygen species (ROS) generation resulting from C. elegans exposure to 7-ketocholesterol at concentrations ranging from 0 to 200 µg/ml. Results indicated that 7-ketocholesterol reduced reproductive capacity, shortened the life span in a concentration-dependent manner, and impaired thermotolerance of the adult nematode. 7-Ketocholesterol also induced germline apoptotic cell death and increased ROS generation in adult worms. Thus, the model organism C. elegans is recommended for assessment of the safety and bioactivity of cholesterol oxides.
biological effects attributed to 7-ketocholesterol (Leonarduzzi et al., 2006). Although 7-ketocholesterol displays pleiotropic effects in many in vitro systems, the paucity of available data based on in vivo systems makes it difficult to assess the health risks of 7-ketocholesterol to humans and animals. Limited data from mammalian models thus far demonstrated adverse effects of 7ketocholesterol. Erickson et al. (1977) reported a decrease in liver and body weights in rats. Granulomatous angitis was observed in the rabbit lung (Santillan et al., 1980), and angiogenic and inflammatory reactions were found when 7-ketocholesterol was implanted into the anterior chamber of the rat eye (Amaral et al., 2013). Clearly, more in vivo investigations are still required to elucidate the mechanisms underlying 7-ketocholesterol-induced toxicity.
Cholesterol oxides are oxygenated products of cholesterol and appear to be involved in the physiopathology of various diseases, such as atherosclerosis, Parkinson’s, Alzheimer’s, and other age-related diseases (Sottero et al., 2009). Many foods have been analyzed to determine cholesterol content and exposure to conditions including heat, exposure to light, and prolonged storage known to promote oxidation of cholesterol (Savage et al., 2002). 7Ketocholesterol is one of the most abundant and one of the most studied cholesterol oxides. In vitro studies demonstrated 7-ketocholesterol to produce inflammatory reactions and induce apoptosis in the tumor cell lines as well as normal cells, including Caco-2 cells (Alemany et al., 2012), macrophages (Palozza et al., 2007), and vascular endothelial cells (Lizard et al., 1997; Li et al., 2011). There is evidence that reactive oxygen species (ROS) mediate the
Received 25 Januaury 2014; accepted 26 January 2014. Address correspondence to Xin Zhang, Professor of School of Life Sciences, Anhui Agricultural University, 130 West Changjiang Road, Hefei 230036, P. R. China. E-mail: [email protected] 716
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TOXICITY OF 7-KETOCHOLESTEROL IN C. elegans
Caenorhabditis elegans is a multicellular animal that is widely used as a biomedical and genetic model organism (Rajini et al., 2008; Leung et al., 2008; Ruan et al., 2009; Sese et al., 2009). As an in vivo model, C. elegans provides several characteristics that complement in vitro or cellular models for exploring sterol functions (Chitwood et al., 1984; Kurzchalia and Ward, 2003) and toxicology (Rajini et al., 2008; Leung et al., 2008; Sese et al., 2009). In recent years, several oxysterol-binding proteins (OSBP)-related genes were identified in C. elegans (Kobuna et al., 2004). In the present study, C. elegans was thus selected to investigate the biological effects induced by 7-ketocholesterol exposure, including lethality, reproduction, life span, thermotolerance, germline apoptosis, and production of reactive oxygen species (ROS).
MATERIALS AND METHODS Caenorhabditis elegans and 7-Ketocholesterol Stock Solutions The wild-type Bristol N2 strain was cultured and maintained at 20◦ C on nematode growth medium (NGM) seeded with Escherichia coli strain OP50 as described by Brenner (1974). To obtain age synchronized worms, gravid hermaphrodites were lysed in an alkaline hypochlorite solution (8% sodium hypochlorite, 5 M NaOH), and eggs were collected followed by hatching at 20◦ C overnight. 7-Ketocholesterol was purchased from Sigma-Aldrich Company (St. Louis, MO) and dissolved at 10 mg/ml in 100% dimethyl sulfoxide (DMSO). Before each experiment, this stock solution was diluted to final concentrations of 0, 25, 50, 100, and 200 μg/ml in overnight cultures of OP50 suspension in L Broth at 37◦ C. If necessary, additional DMSO was added to maintain the final concentration of DMSO at 2% (v/v) for all treatments. One hundred microliters of each working solution was separately dropped onto NGM agar plates. The plates were kept at room temperature for several days before using.
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Acute Lethality To determine whether 7-ketocholesterol affected the lethality of C. elegans, acute toxicity tests were performed in Costar 24well plates. Twenty synchronized young adult hermaphrodites were transferred into each well containing 1 ml of M9 with or without 7-ketocholesterol. Plates were incubated at 20◦ C and counts were taken after 96 h. All adult hermaphrodites survived during the experiments (data not shown). No significant changes before and after acute treatment suggest that lethality assay cannot directly reflect the adverse effects of 7-ketocholesterol to adult worms. This might be due to the impermeability of C. elegans, cuticle or undefined uptake by the oral route. Moreover, aqueous solubility of 7-ketocholesterol is limited, so testing concentration ranges exerted no significant effect on acute worm lethality.
Life-Span Measurements Twenty-five synchronized worms at L1 stage were transferred onto each of 4 fresh NGM plus OP50 plates with or without 7ketocholesterol, and this was recorded as “d 0” of the experiment. Except control, worms were maintained on plates with 7-ketocholesterol during the whole period of assays. All of the worms were inspected on the next day, “d 1” of the life-span experiment. When worms reached the gravid stage, they were transferred each day onto new plates to avoid confusing parents with progeny. After the gravid stage, worms were transferred to new plates every 5 d to ensure that 7-ketocholesterol levels remained constant and that there was sufficient food on the plates. Every other day, worms were identified and counted as dead when they were no longer responding to being touched with a platinum wire pick. Lost animals were calculated as censored.
Thermotolerance Thermotolerance assays were performed on d 5, after the majority of egg-laying ceased
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(Wilson et al., 2006). Worm plates were incubated at 35 ± 0.5◦ C for 10 h before being returned to 20◦ C, and worms were inspected on the next day to determine live or dead. Unrecovered worms were counted as dead in the computation of percent mortality. This treatment was repeated five times.
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Brood Size Determination Synchronized L1 worms were transferred individually to an E. coli layer on separate NGM plates with or without 7-ketocholesterol. During the egg-laying period, worms were transferred every other day to new plates and then eggs and freshly hatched L1 were counted. Counted plates were retained and when progeny missing from the first counting reached L4 stage, they were selected and counted to obtain L4 numbers. Apoptosis Assay Twenty synchronized young-adult hermaphrodites were picked and transferred into each well of a Costar 24-well plate containing M9 with or without 7-ketocholesterol. Worms in each well were incubated at 20◦ C with OP50 in M9 at a final optical density OD550 = 0.5, and removed after 24 h of exposure. Germline apoptosis assay with fluorescent dye acridine orange (AO; SigmaAldrich Company, St. Louis, MO) was adapted from the procedure developed by Kelly et al. (2000). Apoptotic cells were quantified in the pachytene region of one gonad arm with an Olympus I × 71 fluorescent microscope. Only worms that stained brightly were scored. Apoptotic nuclei appeared yellow-green, while intact nuclei were uniformly green in color. The test was repeated four times. ROS Determination To quantify whether 7-ketocholesterol treatment increased ROS levels in C. elegans, worms were transferred to 1 ml M9 buffer containing 10 μM 2,7-dichlorodihydrofluorescein diacetate (H2 DCFDA; Sigma-Aldrich Company, St. Louis, MO) in the Costar 24-well plate
and cultured for 2.5 h at 20◦ C. Then animals were transferred into 7-ketocholesterol solutions and cultured for 30 min. Worms were then mounted on 2% agar pads in 60 μg/ml levamisole and examined with a laser scanning confocal microscope (Olympus I × 81, Fluoview FV1000, Japan). The relative fluorescence intensities at 500 μm body length away from mouth tip were semiquantified using the Image J software. The experiment was repeated four times. Data Analysis All values were expressed as means ± standard error; the statistical significances of differences between the control and treated groups were determined by one-way analysis of variance (ANOVA) followed by Tukey’s test. To prepare survival curves, mortality data were subjected to Kaplan-Meier survival analysis. Log-rank test was used to compare the statistical significance of the mean lifespan between 7-ketocholesterol treated populations and untreated controls. The ROS levels were calculated as the relative fluorescence intensity of each worm using Image J software. All statistical analyses were conducted using Statistical Packages for the Origin Version 8.0.
RESULTS AND DISCUSSION Chronic or delayed effects of chemical compounds often are difficult to evaluate in mammalian animal models because of long life cycles. Caenorhabditis elegans, however, is a good model organism for rapid preliminary toxicity studies because of its short life cycle of 30 d. The resultant survival curves of C. elegans are shown in Figure 1A, and mean life spans in each group with standard errors are shown in Figure 1B. Caenorhabditis elegans without 7-ketocholesterol treatment survived up to d 34, while animals exposed to 200 μg/ml 7ketocholesterol treatment lived for 29 d maximum. Worms in 25 μg/ml 7-ketocholesterol treatment had a median life span similar to the control, whereas the life spans were
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FIGURE 2. Effects of 7-ketocholesterol treatments on thermotolerance. Caenorhabditis elegans were incubated at 20◦ C in the presence of 7-ketocholesterol from L1 larvae and the lethality rate was calculated after exposed to 35◦ C for 10 h on d 5. Data are expressed as means ± SE, n = 310–356 (double asterisk represents significant difference at p < .01 compared with control).
FIGURE 1. Effects of 7-ketocholesterol on lifespan of C. elegans: (A) life-span curves; (B) mean life span. Data are expressed as means ± SE (double asterisk represents significant difference at p < .01, compared with control).
significantly shortened in a concentrationdependent manner at doses above 50 μg/ml. Results indicated that 7-ketocholesterol exposure affected the life span of C. elegans at higher tested concentrations. In several studies, decreased life span was also closely associated with increased mortality rate under conditions of heat (Lithgow et al., 1994), oxidative stress (Halliwell and Whiteman, 2004), or compound treatment (Zarse and Ristow, 2008). In the present study, the intrinsic stress resistance of nematodes
was assessed by recording the lethality of C. elegans at 35◦ C after pretreatment with 7ketocholesterol for 5 d (Figure 2). The results showed that exposure to 7-ketocholesterol significantly impaired thermotolerance in a dosedependent manner. In contrast to acute toxicity tests, 7ketocholesterol treatment at concentrations above 50 μg/ml reduced the brood size of C. elegans. As shown in Figure 3, exposing C. elegans to 25 μg/ml 7-ketocholesterol produced no significant changes in brood size, indicating that fertility was not apparently sensitive to low levels of 7-ketocholesterol. However, at doses of 7-ketocholesterol above 50 μg/ml, brood size of C. elegans decreased significantly as compared to control (268 ± 5.6 at 50 μg/ml, n = 18; 256 ± 6.6 at 100 μg/ml, n = 18, and 253 ± 6.1 at 200 μg/ml, n = 18), suggesting potential genotoxic effects of 7-ketocholesterol to the germline of C. elegans. Germline apoptosis in C. elegans occurs during oogenesis where regulation of the programmed cell death is noted via different pathways (Gumienny et al., 1999). DNA damageand stress-induced apoptosis also activate multiple signaling pathways (Gartner et al., 2008).
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FIGURE 3. Brood size of C. elegans exposed to different doses of 7-ketocholesterol. All values are represented by means ± SE; n > 15 (asterisk represents significant difference at p < .05, double asterisk represents significant difference at p < .01 compared with control).
Germline apoptosis was assayed by staining worms with fluorescent dye AO, which preferentially labels highly condensed DNA characteristically present in apoptotic cells.
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In the present study, AO-stained nuclei per gonad arm were observed and the number of apoptotic nuclei were counted after 24 h of 7-ketocholesterol exposure; hydrogen peroxide (H2 O2 ) was used as positive control. As illustrated in Figures 4A–4C, similar fluorescent staining phenomena were found in C. elegans with H2 O2 and 200 μg/ml 7-ketocholesterol treatment. Compared with negative control, 25 μg/ml 7-ketocholesterol exposure for 24 h induced a 55% increment in apoptotic nuclei number per gonad arm, whereas significant ≥2-fold increase in apoptotic nuclei numbers were observed when concentration of 7-ketocholesterol was raised to 100 or 200 μg/ml (Figure 4D). Apoptotic level induced by 100 μg/ml 7-ketocholesterol was comparable to that induced by 1 mM H2 O2 . Data suggested that exposure to graded doses of 7ketocholesterol resulted in a dose-dependent rise in germline apoptosis in C. elegans. Many investigators reported a good correlation between level of oxidative damage
FIGURE 4. Representative C. elegans germline apoptosis pictures stained by AO induced by (A) 2% DMSO (control), (B) 1 mM H2 O2 , (positive control), and (C) 200 μg/ml 7-ketocholesterol, respectively. The apoptotic cells appeared as bright yellow-green spots. (D) Exposure of C. elegans to graded doses of 7-ketocholesterol caused germline apoptosis. All values are represented by means ± SE, expressed as germ cell corpses per gonad arm. About 33 to 37 worms were quantified for each dose. (Double asterisk represents significant difference at p < .01 compared with control). Scale bar = 100 μm.
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FIGURE 5. Effects of 7-ketocholesterol on ROS accumulation in C. elegans. Shown are DIC images (A–B) and ROS accumulation (a– b): (A and a) control; (B and b) 200 μg/ml 7-ketocholesterol. (C) The intensity of the ROS fluorescence of worms was determined by densitometric analysis using the Image J software. Data are expressed as means ± SE, n = 15–25 (asterisk represents significant difference at p < .05, double asterisk represents significant difference at p < .01 compared with control). Scale bar = 100 μm.
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and rate of aging of cells (Halliwell and Whiteman, 2004), tissues (Janssen et al., 1993), and individuals (Finkel and Holbrook, 2000). In organisms, ROS are produced at the mitochondria respiratory chain by the incomplete reduction of oxygen and as side products of cellular reactions. This process may also be trigged by a number of external agents like drugs, radiation, heat, and environmental toxins (Finkel and Holbrook, 2000). To determine whether increased ROS might have contributed to the adverse effects of 7-ketocholesterol, ROS production was measured using H2 DCFDA. The strongest fluorescent signals were observed in the intestine at a dose of 200 μg/ml (Figure 5b). The semiquantified ROS were expressed as arbitrary relative fluorescent units (RFU) (Figure 5C). In comparison with control, RFU in the intestine increased 3.46-fold following treatment with 50 μg/ml 7-ketocholesterol. It has been reported that 20–125 μM (equivalent to 8–50 μg/ml) 7-ketocholesterol induced apoptosis and a significant increase of intracellular ROS production in different mammalian cells during in vitro studies (Lizard et al., 1998; Berthier et al., 2005). Evidence indicates that 7-ketocholesterol enhanced ROS production in C. elegans, and this finding might be attributed to the cytotoxicity of 7-ketocholesterol in vivo.
CONCLUSIONS Higher concentrations of 7-ketocholesterol (50–200 μg/ml) treatment affected C. elegans, reproduction and life span, induced germline cell apoptosis, and increased generation of ROS, but had no significant effects on lethality rate. Our findings are relevant with a view to determining the safety of 7-ketocholesterol, and this in vivo model might aid us to better understand the safety and bioactivity of cholesterol oxides.
FUNDING The authors thank the National Natural Science Foundation of China (number 21072002), for financial support.
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Kelly, K. O., Dernburg, A. F., Stanfield, G. M., and Villeneuve, A. M. 2000. Caenorhabditis elegans msh-5 is required for both normal and radiation-induced meiotic crossing over but not for completion of meiosis. Genetics 156: 617–630. Kobuna, H., Inoue, T., and Arai, H. 2004. Analysis of oxysterol-binding protein (OSBP)related genes in C. elegans. East Asia C. elegans Meeting, June, Awaji Island, Japan. Kurzchalia, T. V., and Ward, S. 2003. Why do worms need cholesterol? Nat. Cell Biol. 5: 684–688. Leonarduzzi, G., Vizio, B., Sottero, B., Verde, V., Gamba, P., Mascia, C., Chiarpotto, E., Poli, G., and Biasi, F. 2006. Early involvement of ROS overproduction in apoptosis induced by 7-ketocholesterol. Antioxid. Redox. Signal. 8: 375–380. Leung, M. C. K., Williams, P. L., Benedetto, A., Au, C., Helmcke, K. J., Aschner, M., and Meyer, J. N. 2008. Caenorhabditis elegans: An emerging model in biomedical and environmental toxicology. Toxicol. Sci. 106: 5–28. Li, W., Ghosh, M., Eftekhari, S., and Yuan, X. M. 2011. Lipid accumulation and lysosomal pathways contribute to dysfunction and apoptosis of human endothelial cells caused by 7-oxysterols. Biochem. Biophys. Res. Commun. 409: 711–716. Lithgow, G. J., White, T. M., Hinerfeld, D. A., and Johnson, T. E. 1994. Thermotolerance of a long-lived mutant of Caenorhabditis elegans. J. Gerontol. 49: B270–B276. Lizard, G., Moisant, M., Cordelet, C., Monier, S., Gambert, P., and Lagrost, L. 1997. Induction of similar features of apoptosis in human and bovine vascular endothelial cells treated by 7-ketocholesterol. J. Pathol. 183: 330–338. Lizard, G., Gueldry, S., Sordet, O., Monier, S., Athias, A., Miguet, C., Bessede, G., Lemaire, S., Solary, E., and Gambert, P. 1998. Glutathione is implied in the control of 7ketocholesterol-induced apoptosis, which is associated with radical oxygen species production. FASEB J. 12: 1651–1663.
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Palozza, P., Serini, S., Verdecchia, S., Ameruso, M., Trombino, S., Picci, N., and Ranelletti, F. O. 2007. Redox regulation of 7-ketocholesterol-induced apoptosis by β-carotene in human macrophages. Free Radical Biol. Med. 42: 1579–1590. Rajini, P. S., Melstrom, P., and Williams, P. L. 2008. A comparative study on the relationship between various toxicological endpoints in Caenorhabditis elegans exposed to organophosphorus insecticides. J. Toxicol. Environ. Health A 71: 1043–1050. Ruan, Q. L., Ju, J. J., Li, Y. H., Liu, R., Pu, Y. P., Yin, L. H., and Wang, D. Y. 2009. Evaluation of pesticide toxicities with differing mechanisms using Caenorhabditis elegans. J. Toxicol. Environ. Health A 72: 746–751. Santillan, G., Sarma, J. S. M., Pawlik, G., Rackl, A., Grenier, A., and Bing, R. J. 1980. Toxicity, pharmacokinetics, and cholesterol-inhibitory effect of 7-ketocholesterol. Atherosclerosis 35: 1–10. Savage, G. P., Dutta, P. C., and RodriguezEstrada, M. T. 2002. Cholesterol oxides: Their occurrence and methods to prevent their generation in foods. Asia Pac. J. Clin. Nutr. 11: 72–78. Sese, B. T., Grant, A., and Reid, B. J. 2009. Toxicity of polycyclic aromatic hydrocarbons to the nematode Caenorhabditis elegans. J. Toxicol. Environ. Health A 72: 1168–1180. Sottero, B., Gamba, P., Gargiulo, S., Leonarduzzi, G., and Poli, G. 2009. Cholesterol oxidation products and disease: An emerging topic of interest in medicinal chemistry. Curr. Med. Chem. 16: 685–705. Wilson, M. A., Shukitt-Hale, B., Kalt, W., Ingram, D. K., Joseph, J. A., and Wolkow, C. A. 2006. Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans. Aging Cell. 5: 59–68. Zarse, K., and Ristow, M. 2008. Antidepressants of the serotonin-antagonist type increase body fat and decrease lifespan of adult Caenorhabditis elegans. PLoS One 3: e4062.
Journal of Toxicology and Environmental Health, Part A: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh20
Toxicity of 7-Ketocholesterol on Lethality, Growth, Reproduction, and Germline Apoptosis in the Nematode Caenorhabditis elegans a
a
b
a
a
Yunfeng Zong , Junlan Gao , Huiyun Feng , Beijiu Cheng & Xin Zhang a
School of Life Sciences, Anhui Agricultural University, Hefei, P. R. China
b
Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, P. R. China Published online: 01 May 2014.
To cite this article: Yunfeng Zong, Junlan Gao, Huiyun Feng, Beijiu Cheng & Xin Zhang (2014) Toxicity of 7-Ketocholesterol on Lethality, Growth, Reproduction, and Germline Apoptosis in the Nematode Caenorhabditis elegans, Journal of Toxicology and Environmental Health, Part A: Current Issues, 77:12, 716-723, DOI: 10.1080/15287394.2014.888693 To link to this article: http://dx.doi.org/10.1080/15287394.2014.888693
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Toxicology and Environmental Health, Part A, 77:716–723, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287394.2014.888693
TOXICITY OF 7-KETOCHOLESTEROL ON LETHALITY, GROWTH, REPRODUCTION, AND GERMLINE APOPTOSIS IN THE NEMATODE Caenorhabditis elegans Yunfeng Zong1, Junlan Gao1, Huiyun Feng2, Beijiu Cheng1, Xin Zhang1 1
School of Life Sciences, Anhui Agricultural University, Hefei, P. R. China Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, P. R. China
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2
7-Ketocholesterol is one of the most abundant cholesterol oxides, and is known to be cytotoxic to various types of cultured mammalian cells; however, little is known regarding its effects in vivo. With the use of the nematode Caenorhabditis elegans as model organism, in vivo toxicity of 7-ketocholesterol was investigated. The aim of the study was to examine the effects on life span, as well as short-term effects on reproduction, thermotolerance, germline apoptosis, and reactive oxygen species (ROS) generation resulting from C. elegans exposure to 7-ketocholesterol at concentrations ranging from 0 to 200 µg/ml. Results indicated that 7-ketocholesterol reduced reproductive capacity, shortened the life span in a concentration-dependent manner, and impaired thermotolerance of the adult nematode. 7-Ketocholesterol also induced germline apoptotic cell death and increased ROS generation in adult worms. Thus, the model organism C. elegans is recommended for assessment of the safety and bioactivity of cholesterol oxides.
biological effects attributed to 7-ketocholesterol (Leonarduzzi et al., 2006). Although 7-ketocholesterol displays pleiotropic effects in many in vitro systems, the paucity of available data based on in vivo systems makes it difficult to assess the health risks of 7-ketocholesterol to humans and animals. Limited data from mammalian models thus far demonstrated adverse effects of 7ketocholesterol. Erickson et al. (1977) reported a decrease in liver and body weights in rats. Granulomatous angitis was observed in the rabbit lung (Santillan et al., 1980), and angiogenic and inflammatory reactions were found when 7-ketocholesterol was implanted into the anterior chamber of the rat eye (Amaral et al., 2013). Clearly, more in vivo investigations are still required to elucidate the mechanisms underlying 7-ketocholesterol-induced toxicity.
Cholesterol oxides are oxygenated products of cholesterol and appear to be involved in the physiopathology of various diseases, such as atherosclerosis, Parkinson’s, Alzheimer’s, and other age-related diseases (Sottero et al., 2009). Many foods have been analyzed to determine cholesterol content and exposure to conditions including heat, exposure to light, and prolonged storage known to promote oxidation of cholesterol (Savage et al., 2002). 7Ketocholesterol is one of the most abundant and one of the most studied cholesterol oxides. In vitro studies demonstrated 7-ketocholesterol to produce inflammatory reactions and induce apoptosis in the tumor cell lines as well as normal cells, including Caco-2 cells (Alemany et al., 2012), macrophages (Palozza et al., 2007), and vascular endothelial cells (Lizard et al., 1997; Li et al., 2011). There is evidence that reactive oxygen species (ROS) mediate the
Received 25 Januaury 2014; accepted 26 January 2014. Address correspondence to Xin Zhang, Professor of School of Life Sciences, Anhui Agricultural University, 130 West Changjiang Road, Hefei 230036, P. R. China. E-mail: [email protected] 716
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TOXICITY OF 7-KETOCHOLESTEROL IN C. elegans
Caenorhabditis elegans is a multicellular animal that is widely used as a biomedical and genetic model organism (Rajini et al., 2008; Leung et al., 2008; Ruan et al., 2009; Sese et al., 2009). As an in vivo model, C. elegans provides several characteristics that complement in vitro or cellular models for exploring sterol functions (Chitwood et al., 1984; Kurzchalia and Ward, 2003) and toxicology (Rajini et al., 2008; Leung et al., 2008; Sese et al., 2009). In recent years, several oxysterol-binding proteins (OSBP)-related genes were identified in C. elegans (Kobuna et al., 2004). In the present study, C. elegans was thus selected to investigate the biological effects induced by 7-ketocholesterol exposure, including lethality, reproduction, life span, thermotolerance, germline apoptosis, and production of reactive oxygen species (ROS).
MATERIALS AND METHODS Caenorhabditis elegans and 7-Ketocholesterol Stock Solutions The wild-type Bristol N2 strain was cultured and maintained at 20◦ C on nematode growth medium (NGM) seeded with Escherichia coli strain OP50 as described by Brenner (1974). To obtain age synchronized worms, gravid hermaphrodites were lysed in an alkaline hypochlorite solution (8% sodium hypochlorite, 5 M NaOH), and eggs were collected followed by hatching at 20◦ C overnight. 7-Ketocholesterol was purchased from Sigma-Aldrich Company (St. Louis, MO) and dissolved at 10 mg/ml in 100% dimethyl sulfoxide (DMSO). Before each experiment, this stock solution was diluted to final concentrations of 0, 25, 50, 100, and 200 μg/ml in overnight cultures of OP50 suspension in L Broth at 37◦ C. If necessary, additional DMSO was added to maintain the final concentration of DMSO at 2% (v/v) for all treatments. One hundred microliters of each working solution was separately dropped onto NGM agar plates. The plates were kept at room temperature for several days before using.
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Acute Lethality To determine whether 7-ketocholesterol affected the lethality of C. elegans, acute toxicity tests were performed in Costar 24well plates. Twenty synchronized young adult hermaphrodites were transferred into each well containing 1 ml of M9 with or without 7-ketocholesterol. Plates were incubated at 20◦ C and counts were taken after 96 h. All adult hermaphrodites survived during the experiments (data not shown). No significant changes before and after acute treatment suggest that lethality assay cannot directly reflect the adverse effects of 7-ketocholesterol to adult worms. This might be due to the impermeability of C. elegans, cuticle or undefined uptake by the oral route. Moreover, aqueous solubility of 7-ketocholesterol is limited, so testing concentration ranges exerted no significant effect on acute worm lethality.
Life-Span Measurements Twenty-five synchronized worms at L1 stage were transferred onto each of 4 fresh NGM plus OP50 plates with or without 7ketocholesterol, and this was recorded as “d 0” of the experiment. Except control, worms were maintained on plates with 7-ketocholesterol during the whole period of assays. All of the worms were inspected on the next day, “d 1” of the life-span experiment. When worms reached the gravid stage, they were transferred each day onto new plates to avoid confusing parents with progeny. After the gravid stage, worms were transferred to new plates every 5 d to ensure that 7-ketocholesterol levels remained constant and that there was sufficient food on the plates. Every other day, worms were identified and counted as dead when they were no longer responding to being touched with a platinum wire pick. Lost animals were calculated as censored.
Thermotolerance Thermotolerance assays were performed on d 5, after the majority of egg-laying ceased
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(Wilson et al., 2006). Worm plates were incubated at 35 ± 0.5◦ C for 10 h before being returned to 20◦ C, and worms were inspected on the next day to determine live or dead. Unrecovered worms were counted as dead in the computation of percent mortality. This treatment was repeated five times.
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Brood Size Determination Synchronized L1 worms were transferred individually to an E. coli layer on separate NGM plates with or without 7-ketocholesterol. During the egg-laying period, worms were transferred every other day to new plates and then eggs and freshly hatched L1 were counted. Counted plates were retained and when progeny missing from the first counting reached L4 stage, they were selected and counted to obtain L4 numbers. Apoptosis Assay Twenty synchronized young-adult hermaphrodites were picked and transferred into each well of a Costar 24-well plate containing M9 with or without 7-ketocholesterol. Worms in each well were incubated at 20◦ C with OP50 in M9 at a final optical density OD550 = 0.5, and removed after 24 h of exposure. Germline apoptosis assay with fluorescent dye acridine orange (AO; SigmaAldrich Company, St. Louis, MO) was adapted from the procedure developed by Kelly et al. (2000). Apoptotic cells were quantified in the pachytene region of one gonad arm with an Olympus I × 71 fluorescent microscope. Only worms that stained brightly were scored. Apoptotic nuclei appeared yellow-green, while intact nuclei were uniformly green in color. The test was repeated four times. ROS Determination To quantify whether 7-ketocholesterol treatment increased ROS levels in C. elegans, worms were transferred to 1 ml M9 buffer containing 10 μM 2,7-dichlorodihydrofluorescein diacetate (H2 DCFDA; Sigma-Aldrich Company, St. Louis, MO) in the Costar 24-well plate
and cultured for 2.5 h at 20◦ C. Then animals were transferred into 7-ketocholesterol solutions and cultured for 30 min. Worms were then mounted on 2% agar pads in 60 μg/ml levamisole and examined with a laser scanning confocal microscope (Olympus I × 81, Fluoview FV1000, Japan). The relative fluorescence intensities at 500 μm body length away from mouth tip were semiquantified using the Image J software. The experiment was repeated four times. Data Analysis All values were expressed as means ± standard error; the statistical significances of differences between the control and treated groups were determined by one-way analysis of variance (ANOVA) followed by Tukey’s test. To prepare survival curves, mortality data were subjected to Kaplan-Meier survival analysis. Log-rank test was used to compare the statistical significance of the mean lifespan between 7-ketocholesterol treated populations and untreated controls. The ROS levels were calculated as the relative fluorescence intensity of each worm using Image J software. All statistical analyses were conducted using Statistical Packages for the Origin Version 8.0.
RESULTS AND DISCUSSION Chronic or delayed effects of chemical compounds often are difficult to evaluate in mammalian animal models because of long life cycles. Caenorhabditis elegans, however, is a good model organism for rapid preliminary toxicity studies because of its short life cycle of 30 d. The resultant survival curves of C. elegans are shown in Figure 1A, and mean life spans in each group with standard errors are shown in Figure 1B. Caenorhabditis elegans without 7-ketocholesterol treatment survived up to d 34, while animals exposed to 200 μg/ml 7ketocholesterol treatment lived for 29 d maximum. Worms in 25 μg/ml 7-ketocholesterol treatment had a median life span similar to the control, whereas the life spans were
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FIGURE 2. Effects of 7-ketocholesterol treatments on thermotolerance. Caenorhabditis elegans were incubated at 20◦ C in the presence of 7-ketocholesterol from L1 larvae and the lethality rate was calculated after exposed to 35◦ C for 10 h on d 5. Data are expressed as means ± SE, n = 310–356 (double asterisk represents significant difference at p < .01 compared with control).
FIGURE 1. Effects of 7-ketocholesterol on lifespan of C. elegans: (A) life-span curves; (B) mean life span. Data are expressed as means ± SE (double asterisk represents significant difference at p < .01, compared with control).
significantly shortened in a concentrationdependent manner at doses above 50 μg/ml. Results indicated that 7-ketocholesterol exposure affected the life span of C. elegans at higher tested concentrations. In several studies, decreased life span was also closely associated with increased mortality rate under conditions of heat (Lithgow et al., 1994), oxidative stress (Halliwell and Whiteman, 2004), or compound treatment (Zarse and Ristow, 2008). In the present study, the intrinsic stress resistance of nematodes
was assessed by recording the lethality of C. elegans at 35◦ C after pretreatment with 7ketocholesterol for 5 d (Figure 2). The results showed that exposure to 7-ketocholesterol significantly impaired thermotolerance in a dosedependent manner. In contrast to acute toxicity tests, 7ketocholesterol treatment at concentrations above 50 μg/ml reduced the brood size of C. elegans. As shown in Figure 3, exposing C. elegans to 25 μg/ml 7-ketocholesterol produced no significant changes in brood size, indicating that fertility was not apparently sensitive to low levels of 7-ketocholesterol. However, at doses of 7-ketocholesterol above 50 μg/ml, brood size of C. elegans decreased significantly as compared to control (268 ± 5.6 at 50 μg/ml, n = 18; 256 ± 6.6 at 100 μg/ml, n = 18, and 253 ± 6.1 at 200 μg/ml, n = 18), suggesting potential genotoxic effects of 7-ketocholesterol to the germline of C. elegans. Germline apoptosis in C. elegans occurs during oogenesis where regulation of the programmed cell death is noted via different pathways (Gumienny et al., 1999). DNA damageand stress-induced apoptosis also activate multiple signaling pathways (Gartner et al., 2008).
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FIGURE 3. Brood size of C. elegans exposed to different doses of 7-ketocholesterol. All values are represented by means ± SE; n > 15 (asterisk represents significant difference at p < .05, double asterisk represents significant difference at p < .01 compared with control).
Germline apoptosis was assayed by staining worms with fluorescent dye AO, which preferentially labels highly condensed DNA characteristically present in apoptotic cells.
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In the present study, AO-stained nuclei per gonad arm were observed and the number of apoptotic nuclei were counted after 24 h of 7-ketocholesterol exposure; hydrogen peroxide (H2 O2 ) was used as positive control. As illustrated in Figures 4A–4C, similar fluorescent staining phenomena were found in C. elegans with H2 O2 and 200 μg/ml 7-ketocholesterol treatment. Compared with negative control, 25 μg/ml 7-ketocholesterol exposure for 24 h induced a 55% increment in apoptotic nuclei number per gonad arm, whereas significant ≥2-fold increase in apoptotic nuclei numbers were observed when concentration of 7-ketocholesterol was raised to 100 or 200 μg/ml (Figure 4D). Apoptotic level induced by 100 μg/ml 7-ketocholesterol was comparable to that induced by 1 mM H2 O2 . Data suggested that exposure to graded doses of 7ketocholesterol resulted in a dose-dependent rise in germline apoptosis in C. elegans. Many investigators reported a good correlation between level of oxidative damage
FIGURE 4. Representative C. elegans germline apoptosis pictures stained by AO induced by (A) 2% DMSO (control), (B) 1 mM H2 O2 , (positive control), and (C) 200 μg/ml 7-ketocholesterol, respectively. The apoptotic cells appeared as bright yellow-green spots. (D) Exposure of C. elegans to graded doses of 7-ketocholesterol caused germline apoptosis. All values are represented by means ± SE, expressed as germ cell corpses per gonad arm. About 33 to 37 worms were quantified for each dose. (Double asterisk represents significant difference at p < .01 compared with control). Scale bar = 100 μm.
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FIGURE 5. Effects of 7-ketocholesterol on ROS accumulation in C. elegans. Shown are DIC images (A–B) and ROS accumulation (a– b): (A and a) control; (B and b) 200 μg/ml 7-ketocholesterol. (C) The intensity of the ROS fluorescence of worms was determined by densitometric analysis using the Image J software. Data are expressed as means ± SE, n = 15–25 (asterisk represents significant difference at p < .05, double asterisk represents significant difference at p < .01 compared with control). Scale bar = 100 μm.
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and rate of aging of cells (Halliwell and Whiteman, 2004), tissues (Janssen et al., 1993), and individuals (Finkel and Holbrook, 2000). In organisms, ROS are produced at the mitochondria respiratory chain by the incomplete reduction of oxygen and as side products of cellular reactions. This process may also be trigged by a number of external agents like drugs, radiation, heat, and environmental toxins (Finkel and Holbrook, 2000). To determine whether increased ROS might have contributed to the adverse effects of 7-ketocholesterol, ROS production was measured using H2 DCFDA. The strongest fluorescent signals were observed in the intestine at a dose of 200 μg/ml (Figure 5b). The semiquantified ROS were expressed as arbitrary relative fluorescent units (RFU) (Figure 5C). In comparison with control, RFU in the intestine increased 3.46-fold following treatment with 50 μg/ml 7-ketocholesterol. It has been reported that 20–125 μM (equivalent to 8–50 μg/ml) 7-ketocholesterol induced apoptosis and a significant increase of intracellular ROS production in different mammalian cells during in vitro studies (Lizard et al., 1998; Berthier et al., 2005). Evidence indicates that 7-ketocholesterol enhanced ROS production in C. elegans, and this finding might be attributed to the cytotoxicity of 7-ketocholesterol in vivo.
CONCLUSIONS Higher concentrations of 7-ketocholesterol (50–200 μg/ml) treatment affected C. elegans, reproduction and life span, induced germline cell apoptosis, and increased generation of ROS, but had no significant effects on lethality rate. Our findings are relevant with a view to determining the safety of 7-ketocholesterol, and this in vivo model might aid us to better understand the safety and bioactivity of cholesterol oxides.
FUNDING The authors thank the National Natural Science Foundation of China (number 21072002), for financial support.
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