Physiology & Behavior 131 (2014) 142–148
Contents lists available at ScienceDirect
Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
Glucose feeding during development aggravates the toxicity of the organophosphorus insecticide Monocrotophos in the nematode, Caenorhabditis elegans Chinnu Salim, P.S. Rajini ⁎ Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi, India Food Protectants and Infestation Control Department, CSIR-Central Food Technological Research Institute, Mysore 570020, India
H I G H L I G H T S • High glucose diet increases body size, decreases locomotion, lifespan, egg laying, and brood size in C. elegans. • Monocrotophos decreases locomotion, lifespan, egg laying, and brood size in C. elegans. • Monocrotophos significantly enhances all the adverse effects induced by high glucose diet.
a r t i c l e
i n f o
Article history: Received 24 February 2014 Accepted 8 April 2014 Available online 26 April 2014 Keywords: Caenorhabditis elegans Monocrotophos High glucose Physiology Behavior Biochemical Lifespan
a b s t r a c t Several studies have demonstrated that high glucose feeding induced oxidative stress and apoptosis thereby affecting growth, fertility, aging and lifespan in Caenorhabditis elegans. Earlier studies from our laboratory had clearly established the propensity of monocrotophos, an OPI to alter the physiological and behavioral responses of C. elegans. The present study was aimed to investigate the effect of monocrotophos (MCP) on physiological/ behavioral and biochemical responses in C. elegans that were maintained on high glucose diet. We exposed the worms through development to high glucose diet (2%) and then treated with sublethal concentrations of MCP (0.5, 0.75, 1.5 mM). We measured the behavioral responses in terms of locomotion, physiological responses in terms of egg laying, brood size, lifespan; morphological alterations; and biochemical responses including glucose content. The worms exposed from egg stage through development to high glucose diet showed enhanced toxic outcome of MCP in terms of physiological, behavioral and biochemical responses. Our studies showed that C. elegans is a good model to study glucose–OPI interactive neurotoxicity since all the responses could be studied at ease in this organism and the outcome could be well extrapolated to those that one would expect in higher animals. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Dietary habits in Asian countries and most industrialized cities tend to be characterized by high glycemic index (GI). The high-GI diets, which predominantly include processed carbohydrates or sugars, are easily metabolized to glucose and raise blood glucose level very quickly. High-GI diets have been linked to obesity, type 2 diabetes, and cardiovascular diseases [1,2]. Further, excess intake of sugars can potentially alter the pharmacological and toxicological outcome of numerous xenobiotics. Hence, regardless of the mechanism, excessive sugar intake has been suggested to alter the biotransformation and in turn activity of a ⁎ Corresponding author at: Senior Principal Scientist, Food Protectants and Infestation Control Department, CSIR-Central Food Technological Research Institute, Mysore 570 020, India. Tel.: +91 821 2513210; fax: +91 821 2517233. E-mail address: [email protected] (P.S. Rajini).
http://dx.doi.org/10.1016/j.physbeh.2014.04.022 0031-9384/© 2014 Elsevier Inc. All rights reserved.
plethora of xenobiotics [3]. Recent research has also demonstrated that the metabolic pathways downstream of raised glucose have damaging effects on neurons whose functional consequences have yet to be understood [4]. Glucose homeostasis is a critical determinant of various fundamental processes such as development, fertility, lifespan and response to infection and toxicity in diverse organisms ranging from yeast, nematodes and mammals [5]. Loss of homeostatic control of glucose is a well known causative factor for the development of diabetes. Several endogenous, as well as exogenous factors, such as pesticides, have been shown to disrupt glucose homeostasis in mammals [6]. Recent studies have clearly established that organophosphorus insecticides (OPI), one of the most widely used classes of insecticides which are neurotoxic also possess the propensity to disrupt glucose homeostasis in mammals through several mechanisms [7]. OPI elicit toxicity through inhibition of the enzyme acetylcholinesterase (AChE)
C. Salim, P.S. Rajini / Physiology & Behavior 131 (2014) 142–148
resulting in the accumulation of acetylcholine (ACh), a neurotransmitter at the cholinergic synapse leading to alterations in several physiological responses. Interestingly, studies have also demonstrated that glucose feeding can exacerbate the toxicity of some of the OPI in rats [3,8]. Several recent studies have employed the nematode, Caenorhabditis elegans as the model system to study glucose toxicity [5,9,10]. These studies have demonstrated that high glucose feeding induced oxidative stress and apoptosis thereby affecting growth, fertility, aging and lifespan in the worms. Earlier studies from our laboratory have clearly established the propensity of monocrotophos, an OPI to alter the physiological and behavioral responses of C. elegans [11]. However, there are no studies till date that have looked into the toxicity of MCP in a scenario of high glucose feeding in C. elegans. Hence, the present study was aimed to investigate the effect of MCP on physiological/behavioral and biochemical responses in C. elegans that were maintained on high glucose diet. We exposed the worms through development to high glucose diet and then treated with sublethal concentrations of MCP. We measured the behavioral responses in terms of locomotion, physiological responses in terms of egg laying, brood size, lifespan; morphological alterations; and biochemical responses including glucose content. 2. Materials and methods 2.1. Materials Monocrotophos (MCP) (technical grade, 72% pure) was a gift from Hyderabad Chemicals Ltd., (Hyderabad, India). All other chemicals used were of analytical grade. C. elegans, wild-type strain (N2) and Escherichia coli (OP50—uracil auxotroph) were obtained from the Caenorhabditis Genetics Center (CGC, Minneapolis, MN, USA), which is funded by the National Center for Research Resources (NCRR). 2.2. Worm culture and glucose treatment Worms were cultivated on nematode growth medium (NGM) plates [12] with OP50 strain of E. coli and maintained at 20 °C. Worms were synchronized by preparing the eggs from gravid adults by sodium hypochlorite treatment. The synchronous eggs were allowed to develop on NGM or NGM plates supplemented with 2% glucose (111 mM) until the L4 stage. LC 50 of MCP was determined separately for glucose-fed (GF) and control worms (CO) before starting the experiment. 2.3. Morphological responses The L4 stage worms (both CO and GF) were exposed to various sublethal concentrations of MCP (0, 0.5, 0.75 and 1.5 mM) for 24 h in multiwell plates with K-medium. After the exposure period, the worms were washed thrice with K-medium [13] and mounted on the 1% agar pad in a slide and observed under a CX 21 (Olympus) fluorescence microscope fitted with a digital camera (E-PL3, Wide Zoom, Olympus). All images were observed at 20× objective. 2.4. Analysis of behavioral and physiological responses 2.4.1. Locomotion For analysis of locomotion, after the exposure period of 24 h, both CO and GF worms were transferred to agar plates without bacterial food. The worms were allowed to adapt for 10 min and then the locomotory rate was quantified in these worms by counting the number of body bends produced by each worm in 20 s under a stereomicroscope. A body bend was considered when there was a change in the direction of movement [14,15]. Ten worms from each group were studied, and the assay was repeated three times in triplicate.
143
2.4.2. Lifespan L4 stage worms (CO and GF) were exposed to different concentrations of MCP (0, 0.5, 0.75, 1.5 mM) for 24 h at 20 °C in a 12 well microtiter plate with 1 ml K-medium in each of the well. After the exposure period, worms were washed thrice with K-medium and 20 ± 1 L4 worms were re-exposed in a 24 well plate containing K-medium with, heat killed OP50 as food and 5-fluoro-2-deoxyuridine (FudR 50 μM). The worms were maintained at 20 °C and checked for survivability daily until all the worms died [16]. 2.4.3. Egg laying Ten age-synchronized L4 stage worms were used for both CO and GF groups. They were exposed in a 24 well tissue culture plate with NGM agar with MCP incorporated at 0, 0.5, 0.75, and 1.5 mM for 4 h. The number of eggs laid by each worm during 1 h after exposure to MCP was counted [17]. This was repeated 3 times in triplicates. 2.4.4. Brood size Age synchronized (L4 stage) worms were exposed to different concentrations of MCP (0, 0.5, 0.75, 1.5 mM) for 24 h at 20 °C. After exposure, the worms were washed with K-medium and a single worm was picked and transferred to 12-well tissue culture plates containing 1 ml K-medium, which contained E. coli at a dilution of 1 OD at 550 nm. GF worms were placed in wells with dead E. coli. The plates were incubated at 20 °C for 72 h. After 72 h, the worms were washed, pelleted, and the progeny was counted under the dissecting microscope [18]. Assays were repeated 3 times in triplicates. 2.5. Biochemical responses Age synchronized worms (L4 stage) were exposed to different concentrations of MCP (0, 0.5, 0.75, 1.5 mM) in 1 ml K-medium in a 12-well tissue culture plate and incubated for 24 h at 20 °C. After the exposure period, the worms were washed thrice with K-medium and homogenized in 50 mM Tris–HCl buffer (pH 7.4). Worm homogenates were centrifuged at 10,000 rpm for 10 min, and the supernatant was used for various assays. Protein content in C. elegans homogenate was determined by Lowry's method. Bovine serum albumin (BSA) was used as the standard [19]. 2.5.1. Acetylcholinesterase (AChE) activity Supernatants of worm homogenates were used for assaying acetylcholinesterase activity Briefly, a required amount of phosphate buffer (100 mM, pH 8) was added along with acetylthiocholine iodide (ATCI, 0. 01 ml of 0. 1 M) to a mixture containing a suitable amount of worm homogenate and DTNB in phosphate buffer (100 mM, pH 8). The rate of change in absorbance was monitored over 2 min in a microplate reader at 406 nm. The amount of the enzyme causing a change of 0.001 units of absorbance per minute was considered as one unit of enzyme, and the results were expressed as units/mg protein [20,21]. 2.5.2. Glucose content Supernatant of the CO and GF worms was used to estimate the total glucose content using a commercial kit from Rapid diagnostic Private limited (New Delhi India) which was based on the Glucose Oxidase– Peroxidase (GOD–POD) method. The results were expressed as µg glucose/mg protein. 2.6. Statistical analysis Mean and standard error (SE) were determined for all parameters, and the results were expressed as mean ± SE. All the experiments were repeated at least three times with three replicate each. The data were analyzed employing analysis of variance (ANOVA) followed by the Tukey B test. Values below 0.05 were considered as significant. All
144
C. Salim, P.S. Rajini / Physiology & Behavior 131 (2014) 142–148
the calculations were carried out by Graph pad prism (computer software program) and Stat plus (statistics package). 3. Results 3.1. Morphological, behavioral and physiological responses of C. elegans Worms treated with different concentrations of MCP were observed under the microscope for morphological difference. Compared to CO worms, the GF worms were larger in size (in terms of width) (Fig. 1A & B). MCP treatment did not induce any further change in body size in these worms (Fig. 1C & D). Morphologically, CO worms exposed to
even 1.5 mM MCP showed a normal structure of nose muscles while GF worms exposed to MCP showed hyper contracted nose muscles (Fig. 1E & F). GF worms showed slower locomotory rate (18% less) compared to CO worms (Fig. 2). MCP treatment induced further slowing of locomotory rate in both CO and GF worms, and the response was concentration-dependent (43, 59 and 76% in CO vs. 50, 70 and 92% in GF worms exposed to 0.5, 0.75 and 1.5 mM MCP respectively). Interestingly, the number of body bends was much lower in GF worms compared to their CO counterparts at any given concentration of MCP. Monocrotophos decreased the life span of GF worms compared to CO worms in a concentration-dependent manner. The reduction in
Fig. 1. Effect of MCP on morphology of CO (Control) and GF (Glucose fed) worms. A—Control (CO); B—Glucose-fed (GF); C—CO + MCP (1.5 mM); D—GF + MCP (1.5 mM); E—CO + MCP (1.5 mM); F—GF + MCP (1.5 mM).
C. Salim, P.S. Rajini / Physiology & Behavior 131 (2014) 142–148
Fig. 2. Effect of MCP on locomotion in CO (control) and GF (Glucose fed) worms. Data are expressed as mean ± SEM. * difference among CO worms of various treatment groups compared to the untreated worms (p b 0.003); # difference among GF worms of various treatment groups compared to the untreated worms (p b 0.001); a,b,c,d represent difference between control and glucose fed worms (CO + GF) with and without MCP treatment.
lifespan in MCP treated worms was statistically significant (p b 0.01). Normal N2 strain survived till 25 days while GF worms survived only for 17 days (32% decrease) (Fig. 3A). GF worms exposed to MCP showed much lower survival rate compared to their CO counterparts (p b 0.001). The percent decrease in the life span of GF worms on MCP exposure compared to CO was 30, 41, and 43% at 0.5, 0.75 and 1.5 mM MCP respectively (Fig. 3B). The number of eggs laid by GF worms was significantly lower (36 %) than that laid by CO worms (Fig. 4). MCP treatment induced a concentration-dependent decrease in egg laying in both CO and GF worms. The extent of decrease in egg laying in GF worms compared to CO worms was: 38, 43 and 50% at 0.5, 0.75, and 1.5 mM MCP respectively (Fig. 4). The number of eggs laid/worm was significantly lower in GF worms compared to their CO counterparts at any given concentration of MCP. Intrinsically, GF worms showed marked reduction (30%) in brood size compared to CO worms (Fig. 5). Further decrease in brood size per worm was evident in both CO and GF worms exposed to varying concentrations of MCP. Percent decrease in brood size of GF worms compared to CO worms was: 40, 43, and 60% at 0.5, 0.75, and 1.5 mM MCP respectively.
145
Fig. 4. Effect of MCP on egg laying in CO (Control) and GF (Glucose fed) worms. Data are expressed as mean ± SEM. * difference among CO worms of various treatment groups compared to the untreated worms (p b 0.001); # difference among GF worms of various treatment groups compared to the untreated worms (p b 0.001); a,b,c,d represent difference between control and glucose fed worms (CO + GF) with and without MCP treatment.
3.2. Biochemical responses of C. elegans Marked increase (34%) in glucose content was observed in GF worms (Fig. 6). While MCP exposure only marginally increased the glucose content in CO worms, it markedly elevated the glucose content in GF worms in a concentration dependent manner compared to their CO counterparts (36, 41, and 47 % at 0.5, 0.75 and 1.5 mM MCP respectively). GF worms showed markedly lower AChE activity (29% lower) compared to that in CO worms (Fig. 7). A concentration-dependent decrease in AChE activity was evident in CO worms exposed to varying concentrations of MCP (42–76%). GF worms showed a further significant decrease (38, 47 and 48 %) in the enzyme activity compared to CO worms on exposure to MCP. 4. Discussion In the present study, we investigated whether glucose feeding during development would modify the toxicity of the organophosphorus insecticide Monocrotophos in the nematode, C. elegans. The worms
Fig. 3. Effect of MCP on lifespan in (A) CO (Control) and (B) GF (Glucose fed) worms. Data are expressed as mean ± SEM (3 replicates each from 3 different experiments).
146
C. Salim, P.S. Rajini / Physiology & Behavior 131 (2014) 142–148
Fig. 5. Effect of MCP on brood size in CO (Control) and GF (Glucose fed) worms. Data are expressed as mean ± SEM. * difference among CO worms of various treatment groups compared to the untreated worms (p b 0.001); # difference among GF worms of various treatment groups compared to the untreated worms (p b 0.001); a,b,c,d represent difference between control and glucose fed worms (CO + GF) with and without MCP treatment.
exposed from egg stage through development to high glucose diet showed enhanced toxic outcome of monocrotophos in terms of physiological, behavioral and biochemical responses. The responses of C. elegans to feeding high glucose concentrations have gained much importance in the recent years. Such an intervention has been shown to reduce the lifespan by increased reactive oxygen species formation and by apoptosis induction [10,22,23]. However, there is limited evidence to show how such an intervention would affect the toxic outcome of a neurotoxic insecticide. Glucose feeding has been shown to exacerbate the neurotoxicity of an OPI, parathion in rats [3,8]. We undertook the present study since in our earlier studies we had demonstrated unequivocally that MCP augments toxic outcome in experimentally-induced diabetic rats [24,25]. Hence, we proposed utilize C. elegans as a model to study the impact of high glucose diet on the toxic outcome of MCP. In the present study, initially, we screened various concentrations of glucose (2, 4 and 6%) for their effect on development in the worms. We observed that the eggs exposed to concentrations greater than 2% glucose in the NGM exhibited marked abnormality in development while the worms developed normally in 2% glucose supplemented NGM. Hence we selected 2% glucose supplemented NGM as “high glucose”
Fig. 6. Effect of MCP on glucose content in CO (Control) and GF (Glucose fed) worms. Data are expressed as mean ± SEM. * difference among CO worms of various treatment groups compared to the untreated worms (p b 0.003); # difference among GF worms of various treatment groups compared to the untreated worms (p b 0.001); a,b,c,d represent difference between control and glucose fed worms (CO + GF) with and without MCP treatment.
Fig. 7. Effect of MCP on AChE activity in CO (Control) and GF (Glucose fed) worms. Data are expressed as mean ± SEM. * difference among CO worms of various treatment groups compared to the untreated worms (p b 0.001); # difference among GF worms of various treatment groups compared to the untreated worms (p b 0.001); a,b,c,d represent difference between control and glucose fed worms (CO + GF) with and without MCP treatment.
diet for the worms. Earlier report [26] has shown that glucose at 32.5 mg/ml was equivalent to supported maximal population of C. elegans while 162.5 mg/ml was toxic in a chemically defined medium; 2% glucose has been commonly deployed as glucose-rich diet for C. elegans [10,22,27]. We confirmed that the worms ingested glucose by measuring the glucose content in their whole body. We found that the GF worms had 1.5 times more glucose content than CO worms. Upon MCP treatment, while CO worms showed a marginal increase in glucose content, GF worms revealed marked and concentrationdependent increase in glucose content. Our earlier studies [28] had demonstrated the potential of MCP to induce hyperglycemia in rats and also augment the glucose levels in experimentally-induced diabetic rats [24]. Based on these studies, as well as the observation of the present study, we conclude that MCP per se increases the glucose content. We observed that worms developing in glucose-supplemented diet were larger in size compared to the control worms although morphologically they appeared normal. The body width of GF worms were almost 100% greater than that of control worms. Glucopenia is reported to induce obesity in C. elegans [29]. Although, in the present study we did not measure the adipose stores in the worms, nevertheless our results concur with the earlier findings. Interestingly, MCP exposure did not affect the body size of either CO or GF worms, indicating that MCP exerts toxicity through other mechanisms. Morphologically, CO worms exposed to various concentrations of MCP appeared normal. However, GF worms exposed to the highest concentration of MCP (1.5 mM) displayed contracted nose muscle. Such nose-contraction has been earlier reported in worms exposed to the OPI, dichlorvos wherein the nose contraction response correlated with ACh accumulation as a result of AChE inhibition. This endpoint has been suggested as an easily measurable behavioral endpoint for agents inducing ACh accumulation in C. elegans [30]. Locomotion in C. elegans involves a number of discrete motor activities such as omega bends (deep bends typically on the ventral side of the body which reorient the direction of forward locomotion) and reversals (changes in the direction of the locomotion wave that cause a switch from forward to backward crawling) [31]. In the present study, we quantified the locomotory rate of the worms moving on an agar surface of a petriplate by counting the number of bends in the anterior body region during a 20 s interval [15]. We observed that GF worms
C. Salim, P.S. Rajini / Physiology & Behavior 131 (2014) 142–148
exhibited greater number of reversals and hence slower locomotion compared to the CO worms. In the present study, GF worms exhibited slower locomotion compared to the CO worms. Our observations are in accordance with the earlier reports, wherein reduced motility has been reported in worms fed high glucose diets [10]. MCP per se induced the locomotory rate in worms as has been reported for other OPI [32]. Movement has been suggested as reliable and easier tests in determining the toxicity of OPI to C. elegans [32]. GF worms exposed to MCP exhibited much slower movement compared to the CO worms exposed to same concentrations of MCP. This suggests the enhanced toxicity of MCP in GF worms. Further [33] demonstrated that C. elegans on exposure to OPI could move normally with less than 15% of normal AChE activity. In the present study, the slower movement of GF worms exposed to MCP compared to their CO counterparts could be attributed to the lower AChE activity in these worms. Lifespan reduction in C. elegans by high glucose diet is an area of contemporary research. The outcome of several studies conducted along this line has suggested that high glucose diets induce shortened lifespan accompanied with a loss of locomotion capacity [10], and also production of excess reactive oxygen species [9] by down-regulating the activities of the life-span extending proteins DAF-16/FOXO and HSF-1 [22]. In the present study, we also noted a marked reduction (32%) in lifespan in GF worms, which is in accordance with the earlier findings. MCP also reduced the lifespan in CO worms. Marked reduction in lifespan had been earlier reported from our laboratory in worms exposed to much lower concentrations of MCP (50–200 μM) although the mechanism of this action was not clear [14]. In the present study, since the GF worms exposed to MCP showed further lifespan reduction, this could be attributed to the augmented toxicity of MCP in these worms. OPI have been reported to possess the potential of significantly reducing the reproductive output in C. elegans. In reproduction assays using numerous agrochemicals, including diquat and paraquat, parathion more potently inhibited reproduction [34,35]. In the present study, MCP significantly reduced the reproductive output both in terms of eggs laying as well as brood size. Interestingly, worms developing in glucoseenriched diet (10 mM) also exhibited reduced reproductive output. This is contrary to an earlier report [5] wherein it was observed that worms exposed to glucose concentrations up to 250 mM were fully fertile, and fertility was reduced only in the presence of glucose beyond 333 mM. The threshold for glucose toxicity in lifespan assays has been found to be nearly 100-fold less than the threshold for glucose toxicity in the fertility assay [22]. However, in the present study, we observed that 2% glucose induced marked decrease in both lifespan and reproductive outcome in worms. It has been suggested that nutrient buffering systems allowed robust fertility over a range of glucose concentrations while the same glucose concentrations induce a sharp decline in lifespan [5]. Insulin signaling has been implicated in both fertility and lifespan, with fertility being regulated by insulin-like activity in the germline [36 while lifespan being regulated by activity in neurons and intestine [37]. This probably explains the robust decrease in lifespan and reproductive output in GF worms exposed to MCP, since MCP besides being neurotoxic also has an adverse impact on insulin signaling [38]. Diet and especially sugars have been reported to influence the activity of the cholinergic system in rats by modifying the microenvironment of the membrane-bound AChE or by a diminished synthesis of ACh or AChE [39]. In the present study, GF worms exhibited relatively lower AChE activity compared to the CO worms. Further, GF worms when exposed to MCP showed more inhibition of AChE activity than their CO counterparts. Perhaps this is indicative of the vulnerability of the nervous system in these worms. 5. Conclusions Our findings demonstrate for the first time that C. elegans grown in glucose-rich diet when exposed to OPI MCP exhibit greater toxic response to the insecticide in terms of physiological, behavioral and
147
biochemical responses. Our studies showed that C. elegans is a good model to study glucose–OPI interactive neurotoxicity since all the responses can be studied at ease in this organism and the outcome can be well extrapolated to those that one would expect in higher animals.
Acknowledgments The authors acknowledge the support of the Director, CSIR-CFTRI for this study and the funding from the Council of Scientific and Industrial Research (CSIR), India through the BSC0202 grant. CS acknowledges junior research fellowship from University Grants Commission, New Delhi, India. Nematode strain used in this work was provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
References [1] Aston LM. Glycaemic index and metabolic disease risk. Proc Nutr Soc 2006;65:125–34. [2] Venn BJ, Green TJ. Glycemic index and glycemic load: measurement issues and their effect on diet-disease relationships. Eur J Clin Nutr 2007;61(Suppl. 1):S122–31. [3] Olivier K, Liu J, Karanth S, Zhang H, Roane DS, Pope CN. Glucose feeding exacerbates parathion-induced neurotoxicity. J Toxicol Environ Health A 2001;63:253–71. [4] Tomlinson DR, Gardiner NJ. Glucose neurotoxicity. Nat Rev Neurosci 2008;9:36–45. [5] Mondoux MA, Love DC, Ghosh SK, Fukushige T, Bond M, Weerasinghe GR, et al. Olinked-N-acetylglucosamine cycling and insulin signaling are required for the glucose stress response in Caenorhabditis elegans. Genetics 2011;188:369–82. [6] Thayer KA, Heindel JJ, Bucher JR, Gallo MA. Role of environmental chemicals in diabetes and obesity: a National Toxicology Program workshop review. Environ Health Perspect 2012;120:779–89. [7] Joshi AKR, Rajini PS. Organophosphorus insecticides and glucose homeostasis. In: Perveen Farzana, editor. Insecticides pest engineering. InTech Publisher; 2012. p. 63–84. [8] Liu J, Gupta RC, Goad JT, Karanth S, Pope C. Modulation of parathion toxicity by glucose feeding: is nitric oxide involved? Toxicol Appl Pharmacol 2007;219:106–13. [9] Schlotterer A, Kukudov G, Bozorgmehr F, Hutter H, Du X, Oikonomou D, et al. C. elegans as model for the study of high glucose mediated life span reduction. Diabetes 2009;58:2450–6. [10] Choi SS. High glucose diets shorten lifespan of Caenorhabditis elegans via ectopic apoptosis induction. Nutr Res Pract 2011;5:214–8. [11] Leelaja BC, Rajini PS. Biochemical and physiological responses in Caenorhabditis elegans exposed to sublethal concentrations of the organophosphorus insecticide, monocrotophos. Ecotoxicol Environ Saf 2013;94:8–13. [12] Brenner S. The genetics of Caenorhabditis elegans. Genetics 1974;77:1–94. [13] Williams PL, Dusenbery DB. Aquatic toxicity testing using the nematode Caenorhabditis elegans. Environ Toxicol Chem 1990;9:1285–90. [14] Ali SJ, Rajini PS. Elicitation of dopaminergic features of Parkinson's disease in C. elegans by monocrotophos, an organophosphorous insecticide. CNS Neurol Disord Drug Targets 2012;11:993–1000. [15] Sawin ER, Ranganathan R. Horvitz HR. C elegans locomotory rate is modulated by the environment through a dopaminergic pathway and by experience through a serotonergic pathway Neuron 2000;26:619–31. [16] Keaney M, Matthijssens F, Sharpe M, Vanfleteren J, Gems D. Superoxide dismutase mimetics elevate superoxide dismutase activity in vivo but do not retard aging in the nematode Caenorhabditis elegans. Free Radic Biol Med 2004;37:239–50. [17] Bany IA, Dong MQ, Koelle MR. Genetic and cellular basis for acetylcholine inhibition of Caenorhabditis elegans egg-laying behavior. J Neurosci 2003;23:8060–9. [18] Middendorf PJ, Dusenbery DB. Fluoroacetic acid is a potent and specific inhibitor of reproduction in the nematode Caenorhabditis elegans. J Nematol 1993;25:573–7. [19] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [20] Galgani F, Bocquene G. Semi-automated colorimetric and enzymatic assays for aquatic organisms using plate readers. Water Res 1991;25:147–50. [21] Ellman GL, Courtney KD, Andres V, Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88–95. [22] Lee SJ, Murphy CT, Kenyon C. Glucose shortens the life span of C. elegans by down regulating DAF-16/FOXO activity and aquaporin gene expression. Cell Metab 2009;10:379–91. [23] Fitzenberger E, Boll M, Wenzel U. Impairment of the proteasome is crucial for glucose-induced lifespan reduction in the mev-1 mutant of Caenorhabditis elegans. Biochim Biophys Acta 2013;1832:565–73. [24] Begum K, Rajini PS. Augmentation of hepatic and renal oxidative stress and disrupted glucose homeostasis by monocrotophos in streptozotocin-induced diabetic rats. Chem Biol Interact 2011;193:240–5. [25] Vismaya, Rajini PS. Exacerbation of intestinal brush border enzyme activities and oxidative stress in streptozotocin-induced diabetic rats by monocrotophos. Chem Biol Interact 2014;211:11–9. [26] Lu NC, Goetsch. Carbohydrate requirement of Caenorhabditis elegans and the final development of a chemically defined medium. Nematologica 1993;39:303–11.
148
C. Salim, P.S. Rajini / Physiology & Behavior 131 (2014) 142–148
[27] Lavigne JP, Audibert S, Molinari N, O'Callaghan D, Keriel A. Influence of a highglucose diet on the sensitivity of Caenorhabditis elegans towards Escherichia coli and Staphylococcus aureus strains. Microbes Infect 2013;15:540–9. [28] Joshi AKR, Rajini PS. Hyperglycemic and stressogenic effects of monocrotophos in rats: evidence for the involvement of acetylcholinesterase inhibition. Exp Toxicol Pathol 2012;64:115–20. [29] Yen K, Mastitis JW, Mobbs CV. Lifespan is not determined by metabolic rate: evidence from fishes and C. elegans. Exp Gerontol 2004;39:947–9. [30] Jadhav KB, Rajini PS. Evaluation of sublethal effects of dichlorvos upon Caenorhabditis elegans based on a set of end points of toxicity. J Biochem Mol Toxicol 2009;23:9–17. [31] Huang K-M, Pamela C, Schafer W. Machine vision based detection of omega bends and reversals in C. elegans. J Neurosci Meth 2006;158:323–36. [32] Rajini PS, Melstrom P, Williams PL. A comparative study on the relationship between various toxicological endpoints in Caenorhabditis elegans exposed to organophosphorus insecticides. J Toxicol Environ Health A 2008;71:1043–50. [33] Opperman CH, Chang S. Effects of aldicarb and fenamiphos on acetycholinesterase and motility of Caenorhabditis elegans. J Nematol 1991;23:20–7.
[34] Boyd WA, McBride SJ, Freedman JH. Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay. PLoS One 2007;2:e1259. [35] Boyd WA, McBride SJ, Rice JR, Snyder DW, Freedman JH. A high-throughput method for assessing chemical toxicity using a Caenorhabditis elegans reproduction assay. Toxicol Appl Pharmacol 2010;245:153–9. [36] Michaelson D, Korta DZ, Capua Y, Hubbard EJA. Insulin signaling promotes germline proliferation in C. elegans. Development 2010;137:671–80. [37] Wolkow CA, Kimura KD, Lee MS, Ruvkun G. Regulation of C. elegans life-span by insulin like signaling in the nervous system. Science 2000;290:147–50. [38] Joshi AKR, Raju N, Rajini PS. Insights into the mechanisms mediating hyperglycemic and stressogenic outcomes in rats treated with monocrotophos, an organophosphorus insecticide. Toxicology 2012;294(1):9–16. [39] Ruano MJ, Sánchez-Martín MM, Alonso JM, Hueso P. Changes of acetylcholinesterase activity in brain areas and liver of sucrose- and ethanol-fed rats. Neurochem Res 2000;25:461–70.
Contents lists available at ScienceDirect
Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
Glucose feeding during development aggravates the toxicity of the organophosphorus insecticide Monocrotophos in the nematode, Caenorhabditis elegans Chinnu Salim, P.S. Rajini ⁎ Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, New Delhi, India Food Protectants and Infestation Control Department, CSIR-Central Food Technological Research Institute, Mysore 570020, India
H I G H L I G H T S • High glucose diet increases body size, decreases locomotion, lifespan, egg laying, and brood size in C. elegans. • Monocrotophos decreases locomotion, lifespan, egg laying, and brood size in C. elegans. • Monocrotophos significantly enhances all the adverse effects induced by high glucose diet.
a r t i c l e
i n f o
Article history: Received 24 February 2014 Accepted 8 April 2014 Available online 26 April 2014 Keywords: Caenorhabditis elegans Monocrotophos High glucose Physiology Behavior Biochemical Lifespan
a b s t r a c t Several studies have demonstrated that high glucose feeding induced oxidative stress and apoptosis thereby affecting growth, fertility, aging and lifespan in Caenorhabditis elegans. Earlier studies from our laboratory had clearly established the propensity of monocrotophos, an OPI to alter the physiological and behavioral responses of C. elegans. The present study was aimed to investigate the effect of monocrotophos (MCP) on physiological/ behavioral and biochemical responses in C. elegans that were maintained on high glucose diet. We exposed the worms through development to high glucose diet (2%) and then treated with sublethal concentrations of MCP (0.5, 0.75, 1.5 mM). We measured the behavioral responses in terms of locomotion, physiological responses in terms of egg laying, brood size, lifespan; morphological alterations; and biochemical responses including glucose content. The worms exposed from egg stage through development to high glucose diet showed enhanced toxic outcome of MCP in terms of physiological, behavioral and biochemical responses. Our studies showed that C. elegans is a good model to study glucose–OPI interactive neurotoxicity since all the responses could be studied at ease in this organism and the outcome could be well extrapolated to those that one would expect in higher animals. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Dietary habits in Asian countries and most industrialized cities tend to be characterized by high glycemic index (GI). The high-GI diets, which predominantly include processed carbohydrates or sugars, are easily metabolized to glucose and raise blood glucose level very quickly. High-GI diets have been linked to obesity, type 2 diabetes, and cardiovascular diseases [1,2]. Further, excess intake of sugars can potentially alter the pharmacological and toxicological outcome of numerous xenobiotics. Hence, regardless of the mechanism, excessive sugar intake has been suggested to alter the biotransformation and in turn activity of a ⁎ Corresponding author at: Senior Principal Scientist, Food Protectants and Infestation Control Department, CSIR-Central Food Technological Research Institute, Mysore 570 020, India. Tel.: +91 821 2513210; fax: +91 821 2517233. E-mail address: [email protected] (P.S. Rajini).
http://dx.doi.org/10.1016/j.physbeh.2014.04.022 0031-9384/© 2014 Elsevier Inc. All rights reserved.
plethora of xenobiotics [3]. Recent research has also demonstrated that the metabolic pathways downstream of raised glucose have damaging effects on neurons whose functional consequences have yet to be understood [4]. Glucose homeostasis is a critical determinant of various fundamental processes such as development, fertility, lifespan and response to infection and toxicity in diverse organisms ranging from yeast, nematodes and mammals [5]. Loss of homeostatic control of glucose is a well known causative factor for the development of diabetes. Several endogenous, as well as exogenous factors, such as pesticides, have been shown to disrupt glucose homeostasis in mammals [6]. Recent studies have clearly established that organophosphorus insecticides (OPI), one of the most widely used classes of insecticides which are neurotoxic also possess the propensity to disrupt glucose homeostasis in mammals through several mechanisms [7]. OPI elicit toxicity through inhibition of the enzyme acetylcholinesterase (AChE)
C. Salim, P.S. Rajini / Physiology & Behavior 131 (2014) 142–148
resulting in the accumulation of acetylcholine (ACh), a neurotransmitter at the cholinergic synapse leading to alterations in several physiological responses. Interestingly, studies have also demonstrated that glucose feeding can exacerbate the toxicity of some of the OPI in rats [3,8]. Several recent studies have employed the nematode, Caenorhabditis elegans as the model system to study glucose toxicity [5,9,10]. These studies have demonstrated that high glucose feeding induced oxidative stress and apoptosis thereby affecting growth, fertility, aging and lifespan in the worms. Earlier studies from our laboratory have clearly established the propensity of monocrotophos, an OPI to alter the physiological and behavioral responses of C. elegans [11]. However, there are no studies till date that have looked into the toxicity of MCP in a scenario of high glucose feeding in C. elegans. Hence, the present study was aimed to investigate the effect of MCP on physiological/behavioral and biochemical responses in C. elegans that were maintained on high glucose diet. We exposed the worms through development to high glucose diet and then treated with sublethal concentrations of MCP. We measured the behavioral responses in terms of locomotion, physiological responses in terms of egg laying, brood size, lifespan; morphological alterations; and biochemical responses including glucose content. 2. Materials and methods 2.1. Materials Monocrotophos (MCP) (technical grade, 72% pure) was a gift from Hyderabad Chemicals Ltd., (Hyderabad, India). All other chemicals used were of analytical grade. C. elegans, wild-type strain (N2) and Escherichia coli (OP50—uracil auxotroph) were obtained from the Caenorhabditis Genetics Center (CGC, Minneapolis, MN, USA), which is funded by the National Center for Research Resources (NCRR). 2.2. Worm culture and glucose treatment Worms were cultivated on nematode growth medium (NGM) plates [12] with OP50 strain of E. coli and maintained at 20 °C. Worms were synchronized by preparing the eggs from gravid adults by sodium hypochlorite treatment. The synchronous eggs were allowed to develop on NGM or NGM plates supplemented with 2% glucose (111 mM) until the L4 stage. LC 50 of MCP was determined separately for glucose-fed (GF) and control worms (CO) before starting the experiment. 2.3. Morphological responses The L4 stage worms (both CO and GF) were exposed to various sublethal concentrations of MCP (0, 0.5, 0.75 and 1.5 mM) for 24 h in multiwell plates with K-medium. After the exposure period, the worms were washed thrice with K-medium [13] and mounted on the 1% agar pad in a slide and observed under a CX 21 (Olympus) fluorescence microscope fitted with a digital camera (E-PL3, Wide Zoom, Olympus). All images were observed at 20× objective. 2.4. Analysis of behavioral and physiological responses 2.4.1. Locomotion For analysis of locomotion, after the exposure period of 24 h, both CO and GF worms were transferred to agar plates without bacterial food. The worms were allowed to adapt for 10 min and then the locomotory rate was quantified in these worms by counting the number of body bends produced by each worm in 20 s under a stereomicroscope. A body bend was considered when there was a change in the direction of movement [14,15]. Ten worms from each group were studied, and the assay was repeated three times in triplicate.
143
2.4.2. Lifespan L4 stage worms (CO and GF) were exposed to different concentrations of MCP (0, 0.5, 0.75, 1.5 mM) for 24 h at 20 °C in a 12 well microtiter plate with 1 ml K-medium in each of the well. After the exposure period, worms were washed thrice with K-medium and 20 ± 1 L4 worms were re-exposed in a 24 well plate containing K-medium with, heat killed OP50 as food and 5-fluoro-2-deoxyuridine (FudR 50 μM). The worms were maintained at 20 °C and checked for survivability daily until all the worms died [16]. 2.4.3. Egg laying Ten age-synchronized L4 stage worms were used for both CO and GF groups. They were exposed in a 24 well tissue culture plate with NGM agar with MCP incorporated at 0, 0.5, 0.75, and 1.5 mM for 4 h. The number of eggs laid by each worm during 1 h after exposure to MCP was counted [17]. This was repeated 3 times in triplicates. 2.4.4. Brood size Age synchronized (L4 stage) worms were exposed to different concentrations of MCP (0, 0.5, 0.75, 1.5 mM) for 24 h at 20 °C. After exposure, the worms were washed with K-medium and a single worm was picked and transferred to 12-well tissue culture plates containing 1 ml K-medium, which contained E. coli at a dilution of 1 OD at 550 nm. GF worms were placed in wells with dead E. coli. The plates were incubated at 20 °C for 72 h. After 72 h, the worms were washed, pelleted, and the progeny was counted under the dissecting microscope [18]. Assays were repeated 3 times in triplicates. 2.5. Biochemical responses Age synchronized worms (L4 stage) were exposed to different concentrations of MCP (0, 0.5, 0.75, 1.5 mM) in 1 ml K-medium in a 12-well tissue culture plate and incubated for 24 h at 20 °C. After the exposure period, the worms were washed thrice with K-medium and homogenized in 50 mM Tris–HCl buffer (pH 7.4). Worm homogenates were centrifuged at 10,000 rpm for 10 min, and the supernatant was used for various assays. Protein content in C. elegans homogenate was determined by Lowry's method. Bovine serum albumin (BSA) was used as the standard [19]. 2.5.1. Acetylcholinesterase (AChE) activity Supernatants of worm homogenates were used for assaying acetylcholinesterase activity Briefly, a required amount of phosphate buffer (100 mM, pH 8) was added along with acetylthiocholine iodide (ATCI, 0. 01 ml of 0. 1 M) to a mixture containing a suitable amount of worm homogenate and DTNB in phosphate buffer (100 mM, pH 8). The rate of change in absorbance was monitored over 2 min in a microplate reader at 406 nm. The amount of the enzyme causing a change of 0.001 units of absorbance per minute was considered as one unit of enzyme, and the results were expressed as units/mg protein [20,21]. 2.5.2. Glucose content Supernatant of the CO and GF worms was used to estimate the total glucose content using a commercial kit from Rapid diagnostic Private limited (New Delhi India) which was based on the Glucose Oxidase– Peroxidase (GOD–POD) method. The results were expressed as µg glucose/mg protein. 2.6. Statistical analysis Mean and standard error (SE) were determined for all parameters, and the results were expressed as mean ± SE. All the experiments were repeated at least three times with three replicate each. The data were analyzed employing analysis of variance (ANOVA) followed by the Tukey B test. Values below 0.05 were considered as significant. All
144
C. Salim, P.S. Rajini / Physiology & Behavior 131 (2014) 142–148
the calculations were carried out by Graph pad prism (computer software program) and Stat plus (statistics package). 3. Results 3.1. Morphological, behavioral and physiological responses of C. elegans Worms treated with different concentrations of MCP were observed under the microscope for morphological difference. Compared to CO worms, the GF worms were larger in size (in terms of width) (Fig. 1A & B). MCP treatment did not induce any further change in body size in these worms (Fig. 1C & D). Morphologically, CO worms exposed to
even 1.5 mM MCP showed a normal structure of nose muscles while GF worms exposed to MCP showed hyper contracted nose muscles (Fig. 1E & F). GF worms showed slower locomotory rate (18% less) compared to CO worms (Fig. 2). MCP treatment induced further slowing of locomotory rate in both CO and GF worms, and the response was concentration-dependent (43, 59 and 76% in CO vs. 50, 70 and 92% in GF worms exposed to 0.5, 0.75 and 1.5 mM MCP respectively). Interestingly, the number of body bends was much lower in GF worms compared to their CO counterparts at any given concentration of MCP. Monocrotophos decreased the life span of GF worms compared to CO worms in a concentration-dependent manner. The reduction in
Fig. 1. Effect of MCP on morphology of CO (Control) and GF (Glucose fed) worms. A—Control (CO); B—Glucose-fed (GF); C—CO + MCP (1.5 mM); D—GF + MCP (1.5 mM); E—CO + MCP (1.5 mM); F—GF + MCP (1.5 mM).
C. Salim, P.S. Rajini / Physiology & Behavior 131 (2014) 142–148
Fig. 2. Effect of MCP on locomotion in CO (control) and GF (Glucose fed) worms. Data are expressed as mean ± SEM. * difference among CO worms of various treatment groups compared to the untreated worms (p b 0.003); # difference among GF worms of various treatment groups compared to the untreated worms (p b 0.001); a,b,c,d represent difference between control and glucose fed worms (CO + GF) with and without MCP treatment.
lifespan in MCP treated worms was statistically significant (p b 0.01). Normal N2 strain survived till 25 days while GF worms survived only for 17 days (32% decrease) (Fig. 3A). GF worms exposed to MCP showed much lower survival rate compared to their CO counterparts (p b 0.001). The percent decrease in the life span of GF worms on MCP exposure compared to CO was 30, 41, and 43% at 0.5, 0.75 and 1.5 mM MCP respectively (Fig. 3B). The number of eggs laid by GF worms was significantly lower (36 %) than that laid by CO worms (Fig. 4). MCP treatment induced a concentration-dependent decrease in egg laying in both CO and GF worms. The extent of decrease in egg laying in GF worms compared to CO worms was: 38, 43 and 50% at 0.5, 0.75, and 1.5 mM MCP respectively (Fig. 4). The number of eggs laid/worm was significantly lower in GF worms compared to their CO counterparts at any given concentration of MCP. Intrinsically, GF worms showed marked reduction (30%) in brood size compared to CO worms (Fig. 5). Further decrease in brood size per worm was evident in both CO and GF worms exposed to varying concentrations of MCP. Percent decrease in brood size of GF worms compared to CO worms was: 40, 43, and 60% at 0.5, 0.75, and 1.5 mM MCP respectively.
145
Fig. 4. Effect of MCP on egg laying in CO (Control) and GF (Glucose fed) worms. Data are expressed as mean ± SEM. * difference among CO worms of various treatment groups compared to the untreated worms (p b 0.001); # difference among GF worms of various treatment groups compared to the untreated worms (p b 0.001); a,b,c,d represent difference between control and glucose fed worms (CO + GF) with and without MCP treatment.
3.2. Biochemical responses of C. elegans Marked increase (34%) in glucose content was observed in GF worms (Fig. 6). While MCP exposure only marginally increased the glucose content in CO worms, it markedly elevated the glucose content in GF worms in a concentration dependent manner compared to their CO counterparts (36, 41, and 47 % at 0.5, 0.75 and 1.5 mM MCP respectively). GF worms showed markedly lower AChE activity (29% lower) compared to that in CO worms (Fig. 7). A concentration-dependent decrease in AChE activity was evident in CO worms exposed to varying concentrations of MCP (42–76%). GF worms showed a further significant decrease (38, 47 and 48 %) in the enzyme activity compared to CO worms on exposure to MCP. 4. Discussion In the present study, we investigated whether glucose feeding during development would modify the toxicity of the organophosphorus insecticide Monocrotophos in the nematode, C. elegans. The worms
Fig. 3. Effect of MCP on lifespan in (A) CO (Control) and (B) GF (Glucose fed) worms. Data are expressed as mean ± SEM (3 replicates each from 3 different experiments).
146
C. Salim, P.S. Rajini / Physiology & Behavior 131 (2014) 142–148
Fig. 5. Effect of MCP on brood size in CO (Control) and GF (Glucose fed) worms. Data are expressed as mean ± SEM. * difference among CO worms of various treatment groups compared to the untreated worms (p b 0.001); # difference among GF worms of various treatment groups compared to the untreated worms (p b 0.001); a,b,c,d represent difference between control and glucose fed worms (CO + GF) with and without MCP treatment.
exposed from egg stage through development to high glucose diet showed enhanced toxic outcome of monocrotophos in terms of physiological, behavioral and biochemical responses. The responses of C. elegans to feeding high glucose concentrations have gained much importance in the recent years. Such an intervention has been shown to reduce the lifespan by increased reactive oxygen species formation and by apoptosis induction [10,22,23]. However, there is limited evidence to show how such an intervention would affect the toxic outcome of a neurotoxic insecticide. Glucose feeding has been shown to exacerbate the neurotoxicity of an OPI, parathion in rats [3,8]. We undertook the present study since in our earlier studies we had demonstrated unequivocally that MCP augments toxic outcome in experimentally-induced diabetic rats [24,25]. Hence, we proposed utilize C. elegans as a model to study the impact of high glucose diet on the toxic outcome of MCP. In the present study, initially, we screened various concentrations of glucose (2, 4 and 6%) for their effect on development in the worms. We observed that the eggs exposed to concentrations greater than 2% glucose in the NGM exhibited marked abnormality in development while the worms developed normally in 2% glucose supplemented NGM. Hence we selected 2% glucose supplemented NGM as “high glucose”
Fig. 6. Effect of MCP on glucose content in CO (Control) and GF (Glucose fed) worms. Data are expressed as mean ± SEM. * difference among CO worms of various treatment groups compared to the untreated worms (p b 0.003); # difference among GF worms of various treatment groups compared to the untreated worms (p b 0.001); a,b,c,d represent difference between control and glucose fed worms (CO + GF) with and without MCP treatment.
Fig. 7. Effect of MCP on AChE activity in CO (Control) and GF (Glucose fed) worms. Data are expressed as mean ± SEM. * difference among CO worms of various treatment groups compared to the untreated worms (p b 0.001); # difference among GF worms of various treatment groups compared to the untreated worms (p b 0.001); a,b,c,d represent difference between control and glucose fed worms (CO + GF) with and without MCP treatment.
diet for the worms. Earlier report [26] has shown that glucose at 32.5 mg/ml was equivalent to supported maximal population of C. elegans while 162.5 mg/ml was toxic in a chemically defined medium; 2% glucose has been commonly deployed as glucose-rich diet for C. elegans [10,22,27]. We confirmed that the worms ingested glucose by measuring the glucose content in their whole body. We found that the GF worms had 1.5 times more glucose content than CO worms. Upon MCP treatment, while CO worms showed a marginal increase in glucose content, GF worms revealed marked and concentrationdependent increase in glucose content. Our earlier studies [28] had demonstrated the potential of MCP to induce hyperglycemia in rats and also augment the glucose levels in experimentally-induced diabetic rats [24]. Based on these studies, as well as the observation of the present study, we conclude that MCP per se increases the glucose content. We observed that worms developing in glucose-supplemented diet were larger in size compared to the control worms although morphologically they appeared normal. The body width of GF worms were almost 100% greater than that of control worms. Glucopenia is reported to induce obesity in C. elegans [29]. Although, in the present study we did not measure the adipose stores in the worms, nevertheless our results concur with the earlier findings. Interestingly, MCP exposure did not affect the body size of either CO or GF worms, indicating that MCP exerts toxicity through other mechanisms. Morphologically, CO worms exposed to various concentrations of MCP appeared normal. However, GF worms exposed to the highest concentration of MCP (1.5 mM) displayed contracted nose muscle. Such nose-contraction has been earlier reported in worms exposed to the OPI, dichlorvos wherein the nose contraction response correlated with ACh accumulation as a result of AChE inhibition. This endpoint has been suggested as an easily measurable behavioral endpoint for agents inducing ACh accumulation in C. elegans [30]. Locomotion in C. elegans involves a number of discrete motor activities such as omega bends (deep bends typically on the ventral side of the body which reorient the direction of forward locomotion) and reversals (changes in the direction of the locomotion wave that cause a switch from forward to backward crawling) [31]. In the present study, we quantified the locomotory rate of the worms moving on an agar surface of a petriplate by counting the number of bends in the anterior body region during a 20 s interval [15]. We observed that GF worms
C. Salim, P.S. Rajini / Physiology & Behavior 131 (2014) 142–148
exhibited greater number of reversals and hence slower locomotion compared to the CO worms. In the present study, GF worms exhibited slower locomotion compared to the CO worms. Our observations are in accordance with the earlier reports, wherein reduced motility has been reported in worms fed high glucose diets [10]. MCP per se induced the locomotory rate in worms as has been reported for other OPI [32]. Movement has been suggested as reliable and easier tests in determining the toxicity of OPI to C. elegans [32]. GF worms exposed to MCP exhibited much slower movement compared to the CO worms exposed to same concentrations of MCP. This suggests the enhanced toxicity of MCP in GF worms. Further [33] demonstrated that C. elegans on exposure to OPI could move normally with less than 15% of normal AChE activity. In the present study, the slower movement of GF worms exposed to MCP compared to their CO counterparts could be attributed to the lower AChE activity in these worms. Lifespan reduction in C. elegans by high glucose diet is an area of contemporary research. The outcome of several studies conducted along this line has suggested that high glucose diets induce shortened lifespan accompanied with a loss of locomotion capacity [10], and also production of excess reactive oxygen species [9] by down-regulating the activities of the life-span extending proteins DAF-16/FOXO and HSF-1 [22]. In the present study, we also noted a marked reduction (32%) in lifespan in GF worms, which is in accordance with the earlier findings. MCP also reduced the lifespan in CO worms. Marked reduction in lifespan had been earlier reported from our laboratory in worms exposed to much lower concentrations of MCP (50–200 μM) although the mechanism of this action was not clear [14]. In the present study, since the GF worms exposed to MCP showed further lifespan reduction, this could be attributed to the augmented toxicity of MCP in these worms. OPI have been reported to possess the potential of significantly reducing the reproductive output in C. elegans. In reproduction assays using numerous agrochemicals, including diquat and paraquat, parathion more potently inhibited reproduction [34,35]. In the present study, MCP significantly reduced the reproductive output both in terms of eggs laying as well as brood size. Interestingly, worms developing in glucoseenriched diet (10 mM) also exhibited reduced reproductive output. This is contrary to an earlier report [5] wherein it was observed that worms exposed to glucose concentrations up to 250 mM were fully fertile, and fertility was reduced only in the presence of glucose beyond 333 mM. The threshold for glucose toxicity in lifespan assays has been found to be nearly 100-fold less than the threshold for glucose toxicity in the fertility assay [22]. However, in the present study, we observed that 2% glucose induced marked decrease in both lifespan and reproductive outcome in worms. It has been suggested that nutrient buffering systems allowed robust fertility over a range of glucose concentrations while the same glucose concentrations induce a sharp decline in lifespan [5]. Insulin signaling has been implicated in both fertility and lifespan, with fertility being regulated by insulin-like activity in the germline [36 while lifespan being regulated by activity in neurons and intestine [37]. This probably explains the robust decrease in lifespan and reproductive output in GF worms exposed to MCP, since MCP besides being neurotoxic also has an adverse impact on insulin signaling [38]. Diet and especially sugars have been reported to influence the activity of the cholinergic system in rats by modifying the microenvironment of the membrane-bound AChE or by a diminished synthesis of ACh or AChE [39]. In the present study, GF worms exhibited relatively lower AChE activity compared to the CO worms. Further, GF worms when exposed to MCP showed more inhibition of AChE activity than their CO counterparts. Perhaps this is indicative of the vulnerability of the nervous system in these worms. 5. Conclusions Our findings demonstrate for the first time that C. elegans grown in glucose-rich diet when exposed to OPI MCP exhibit greater toxic response to the insecticide in terms of physiological, behavioral and
147
biochemical responses. Our studies showed that C. elegans is a good model to study glucose–OPI interactive neurotoxicity since all the responses can be studied at ease in this organism and the outcome can be well extrapolated to those that one would expect in higher animals.
Acknowledgments The authors acknowledge the support of the Director, CSIR-CFTRI for this study and the funding from the Council of Scientific and Industrial Research (CSIR), India through the BSC0202 grant. CS acknowledges junior research fellowship from University Grants Commission, New Delhi, India. Nematode strain used in this work was provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
References [1] Aston LM. Glycaemic index and metabolic disease risk. Proc Nutr Soc 2006;65:125–34. [2] Venn BJ, Green TJ. Glycemic index and glycemic load: measurement issues and their effect on diet-disease relationships. Eur J Clin Nutr 2007;61(Suppl. 1):S122–31. [3] Olivier K, Liu J, Karanth S, Zhang H, Roane DS, Pope CN. Glucose feeding exacerbates parathion-induced neurotoxicity. J Toxicol Environ Health A 2001;63:253–71. [4] Tomlinson DR, Gardiner NJ. Glucose neurotoxicity. Nat Rev Neurosci 2008;9:36–45. [5] Mondoux MA, Love DC, Ghosh SK, Fukushige T, Bond M, Weerasinghe GR, et al. Olinked-N-acetylglucosamine cycling and insulin signaling are required for the glucose stress response in Caenorhabditis elegans. Genetics 2011;188:369–82. [6] Thayer KA, Heindel JJ, Bucher JR, Gallo MA. Role of environmental chemicals in diabetes and obesity: a National Toxicology Program workshop review. Environ Health Perspect 2012;120:779–89. [7] Joshi AKR, Rajini PS. Organophosphorus insecticides and glucose homeostasis. In: Perveen Farzana, editor. Insecticides pest engineering. InTech Publisher; 2012. p. 63–84. [8] Liu J, Gupta RC, Goad JT, Karanth S, Pope C. Modulation of parathion toxicity by glucose feeding: is nitric oxide involved? Toxicol Appl Pharmacol 2007;219:106–13. [9] Schlotterer A, Kukudov G, Bozorgmehr F, Hutter H, Du X, Oikonomou D, et al. C. elegans as model for the study of high glucose mediated life span reduction. Diabetes 2009;58:2450–6. [10] Choi SS. High glucose diets shorten lifespan of Caenorhabditis elegans via ectopic apoptosis induction. Nutr Res Pract 2011;5:214–8. [11] Leelaja BC, Rajini PS. Biochemical and physiological responses in Caenorhabditis elegans exposed to sublethal concentrations of the organophosphorus insecticide, monocrotophos. Ecotoxicol Environ Saf 2013;94:8–13. [12] Brenner S. The genetics of Caenorhabditis elegans. Genetics 1974;77:1–94. [13] Williams PL, Dusenbery DB. Aquatic toxicity testing using the nematode Caenorhabditis elegans. Environ Toxicol Chem 1990;9:1285–90. [14] Ali SJ, Rajini PS. Elicitation of dopaminergic features of Parkinson's disease in C. elegans by monocrotophos, an organophosphorous insecticide. CNS Neurol Disord Drug Targets 2012;11:993–1000. [15] Sawin ER, Ranganathan R. Horvitz HR. C elegans locomotory rate is modulated by the environment through a dopaminergic pathway and by experience through a serotonergic pathway Neuron 2000;26:619–31. [16] Keaney M, Matthijssens F, Sharpe M, Vanfleteren J, Gems D. Superoxide dismutase mimetics elevate superoxide dismutase activity in vivo but do not retard aging in the nematode Caenorhabditis elegans. Free Radic Biol Med 2004;37:239–50. [17] Bany IA, Dong MQ, Koelle MR. Genetic and cellular basis for acetylcholine inhibition of Caenorhabditis elegans egg-laying behavior. J Neurosci 2003;23:8060–9. [18] Middendorf PJ, Dusenbery DB. Fluoroacetic acid is a potent and specific inhibitor of reproduction in the nematode Caenorhabditis elegans. J Nematol 1993;25:573–7. [19] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [20] Galgani F, Bocquene G. Semi-automated colorimetric and enzymatic assays for aquatic organisms using plate readers. Water Res 1991;25:147–50. [21] Ellman GL, Courtney KD, Andres V, Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88–95. [22] Lee SJ, Murphy CT, Kenyon C. Glucose shortens the life span of C. elegans by down regulating DAF-16/FOXO activity and aquaporin gene expression. Cell Metab 2009;10:379–91. [23] Fitzenberger E, Boll M, Wenzel U. Impairment of the proteasome is crucial for glucose-induced lifespan reduction in the mev-1 mutant of Caenorhabditis elegans. Biochim Biophys Acta 2013;1832:565–73. [24] Begum K, Rajini PS. Augmentation of hepatic and renal oxidative stress and disrupted glucose homeostasis by monocrotophos in streptozotocin-induced diabetic rats. Chem Biol Interact 2011;193:240–5. [25] Vismaya, Rajini PS. Exacerbation of intestinal brush border enzyme activities and oxidative stress in streptozotocin-induced diabetic rats by monocrotophos. Chem Biol Interact 2014;211:11–9. [26] Lu NC, Goetsch. Carbohydrate requirement of Caenorhabditis elegans and the final development of a chemically defined medium. Nematologica 1993;39:303–11.
148
C. Salim, P.S. Rajini / Physiology & Behavior 131 (2014) 142–148
[27] Lavigne JP, Audibert S, Molinari N, O'Callaghan D, Keriel A. Influence of a highglucose diet on the sensitivity of Caenorhabditis elegans towards Escherichia coli and Staphylococcus aureus strains. Microbes Infect 2013;15:540–9. [28] Joshi AKR, Rajini PS. Hyperglycemic and stressogenic effects of monocrotophos in rats: evidence for the involvement of acetylcholinesterase inhibition. Exp Toxicol Pathol 2012;64:115–20. [29] Yen K, Mastitis JW, Mobbs CV. Lifespan is not determined by metabolic rate: evidence from fishes and C. elegans. Exp Gerontol 2004;39:947–9. [30] Jadhav KB, Rajini PS. Evaluation of sublethal effects of dichlorvos upon Caenorhabditis elegans based on a set of end points of toxicity. J Biochem Mol Toxicol 2009;23:9–17. [31] Huang K-M, Pamela C, Schafer W. Machine vision based detection of omega bends and reversals in C. elegans. J Neurosci Meth 2006;158:323–36. [32] Rajini PS, Melstrom P, Williams PL. A comparative study on the relationship between various toxicological endpoints in Caenorhabditis elegans exposed to organophosphorus insecticides. J Toxicol Environ Health A 2008;71:1043–50. [33] Opperman CH, Chang S. Effects of aldicarb and fenamiphos on acetycholinesterase and motility of Caenorhabditis elegans. J Nematol 1991;23:20–7.
[34] Boyd WA, McBride SJ, Freedman JH. Effects of genetic mutations and chemical exposures on Caenorhabditis elegans feeding: evaluation of a novel, high-throughput screening assay. PLoS One 2007;2:e1259. [35] Boyd WA, McBride SJ, Rice JR, Snyder DW, Freedman JH. A high-throughput method for assessing chemical toxicity using a Caenorhabditis elegans reproduction assay. Toxicol Appl Pharmacol 2010;245:153–9. [36] Michaelson D, Korta DZ, Capua Y, Hubbard EJA. Insulin signaling promotes germline proliferation in C. elegans. Development 2010;137:671–80. [37] Wolkow CA, Kimura KD, Lee MS, Ruvkun G. Regulation of C. elegans life-span by insulin like signaling in the nervous system. Science 2000;290:147–50. [38] Joshi AKR, Raju N, Rajini PS. Insights into the mechanisms mediating hyperglycemic and stressogenic outcomes in rats treated with monocrotophos, an organophosphorus insecticide. Toxicology 2012;294(1):9–16. [39] Ruano MJ, Sánchez-Martín MM, Alonso JM, Hueso P. Changes of acetylcholinesterase activity in brain areas and liver of sucrose- and ethanol-fed rats. Neurochem Res 2000;25:461–70.
