IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1) 2011 Toxicological and Physiological Effects of DDT on Caenorhabditis elegans S.M. Mahmood Department of Biology, College of Education Ibn Al-Haitham,University of Baghdad Reseived in ,30 ,Nov,2010 Accepted, 2,Feb,2011 Abstract Toxicity assays were used in this study to test how DDT affects lethality and brood size of Caenorhabditis elegans (C.elegans) by exposing them to various concentrations of this agent. These nematodes have provided a very informative system that was utilized to study the behavioural and physiological processes. The results showed that DDT affected the lethality in a dose-dependent manner, but 100% kill was not achieved with concentration tested. Whereas, sodium azide, positive control does have an effect on C. elegans and significantly inhibiting lethality (LC50 0.01mM). Similarly, DDT led to a pronounced effect in brood size of C.elegans compared to the mean brood size recorded for the control (0.1% DM SO). Sodium azide results showed a greater difference in brood size compared to DDT. Both agents, DDT and sodium azide caused a remarkable inhibitory effect on C.elegans pharyngeal pumping rate. It can be concluded that the target site of DDT in C.elegans might not be the same target in insect. Key words : DDT – S odium azide - Caenorhabditis elegans Introduction The long term use of many insecticides is continually threatened by the ability of insect to evolve resistance mechanism that renders the chemical ineffective [1] DDT has had a significant impact in the control of malaria and other insect diseases, and was extensively used as an agricultural insecticide after the Second World War . This agent was found to have a chronic effect on the nervous system, liver, kidney, and immune system in experimental animals . [2] Caenorhabditis elegans (C.elegans) has been extensively utilized as a model animal for the study of nematode behavioural and physiological processes [3]. C.elegans has also proven itself a model of biological studies relevant to higher animals in such areas as neuroscience, genetics, aging, and development. Thus, toxicological results in C.elegans are likely to higher animals [4]. However, one tissue that has proven more amenable to physiological analysis is the pharynx. This is a muscular pump which sucks bacteria into its lumen, grinds them and then passes them to the gut [5]. This provided insight into the mode of action of toxins and IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1) 2011 how C.elegans responds to protect itself from toxin. This has contributed and will continue to contribute to our biological understanding of all animals including human. In the present work an attempt has been made to investigate the effect and the mode of action of DDT in order to probe the toxicity and the accumulation of this pesticide. Until recently little is known about the pharmacological effect of this insecticide and how it works on C. elegans which is used as model organism in this study. Materials and Methods C.elegans were cultured on agar and fed with E.coli (OP50) throughout this study. Worms were grown at 15 0 C on NGM plates containing (NaCl 3g, Agar 17g, Peptone 2.5g, and 1ml Cholesterol (5mg/ml ethanol) in 1L). For the lethality assay 50 L4 C.elegans N2 worms per dose group were placed in 400ul of liquid medium (S-basal) supplemented with 40ul E.coli (O.D 0.7 600nm) with varying concentrations of DDT or sodium-azide (NaN3) to each well 0f 24-well plate [6] . The 24-well plate was then sealed with a laboratory film (to prevent evaporation). Wrapped in a damp paper towel (to provide humidity) and placed in an air tight plastic container and incubated for 3 days. After the incubation period, the percentage of live worms were determined by counting the number of viable and dead worms in each well based on movement. To measure the effects of DDT on brood size, the assay was set up in 48 well-plates, with 4 well for each concentration. An L4 worm was p laced in each well with 40ul of bacteria culture, the toxin and s-basal to make a total volume of 400ul.The 48 well-plate was then sealed and placed in an air tight plastic container and incubated at 150C for 3 days. After 3 days the number of offspring in each well was counted under a stereo microscope. An identical assay was set up and run alongside with sodium azide (as positive control). Pharyngeal pumping assay was set up the same as the lethality bioassays. Briefly, the worms were exposed to various DDT or sodium-azide concentrations for an hour, before the worms transferred to NGM plates and left for another an hour to settle down before the pharyngeal pumping rate was counted. Reagents: All solutions were made up to 1 litre with UHP water and sterilized by autoclaving storage was at room temperature. DDT stock solution made up to 10 mg/ml in DM SO (Dimethyl sulfoxide) this was serially diluted to make 1 ml of each concentration used in this investigation. Results C. elegans has been used as a model organism in order to probe toxicity and the accumulation of pesticides as well as various metal ions. In this study C.elegans was used as a system to gauge the toxicological effects of DDT and sodium azide. - Lethality assay 50 L4 larvas were exposed up to 5ug/ml for 3 days. The experimental results showed that DDT had marked inhibitory effect at 5ug/ml, but little effect at 1ug/ml (fig.1). Lethality was affected in a dose-dependent manner, but 100% kill was not achieved with concentration tested. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1) 2011 Sodium-azide was also employed as positive control due to its well known toxic effect [7]. The results obtained demonstrated that exposure of C.elegans to the respiratory inhibitor sodium azide, this agent caused a remarkable inhibitory effect on C.elegans lethality and the LC50 was 0.01mM (fig.2). The graph represents a single experiment with each concentration tested in triplicate to produce standard error (represented by bar) and mean percentage of viable worms (presented by dots) - Brood size assay Individual L4 C.elegans were placed in 400ul of liquid medium with varying concentrations of DDT, incubated for 3 days at 15 0 C the effect was determined by counting the offspring in each well. It was found that exposure of the worms to (0.001ug/ml-1.0ug/ml) DDT led to a pronounced effect in the brood size of C.elegans (fig.3). The mean brood size recorded (41.6) for the control (0.1% DM SO). In parallel with DDT sodium azaide was tested as a positive control, the results showed that sodium-azide has an effect on C.elegans and inhibits brood size. The maximum brood size recorded was 30.5 and the greatest inhibition was at 0.01mM. There was significant difference in brood size seen for the concentration of sodium azide tested (fig.4) -Pharyngeal pumping assay The pharynx of C.elegans has several features that make it suitable for cellular and molecular studies of behaviour. This model system has been studied by observing its behaviour in normal worms and treated worms. Pharyngeal pumping is the coordinated and regulated process by which the pharynx mediates intake and forces the food and waste products through the worm gut [8] .The results p resented here showed that DDT does have an inhibitory effect on C.elegans by decreasing the pumping rate in a dose-dependent manner. These results summarized graphically in fig.5. The pharyngeal pumping is also reduced in response to sodium azide; however, the higher concentration of sodium azide stopped the pharynx activity in some worms (fig.6). Discussion The exposure of animals to exogenous agents has been of considerable value in analysing C.elegans response. Most of pharmacological agents that have been used in C.elegans affect various aspects, such as brood size, lethal concentration, and growth measurement. DDT is an orgaochlorine insecticide used mainly to control insect typhus and malaria vectors. This agent has been banned for use in different countries, although it is still used in other. There is evidence that DDT causes reproductive effects in test animals [2] and this seems to agree with results obtained in this study because DDT had a dose-dependent inhibitory effect on the brood size of C.elegans. Insecticides may act on membrane proteins (receptors, channels) increasing the ability of the insect to detoxify the insecticide or by changes in the target protein with which the insecticide interacts [1]. DDT is an insect neurotoxin that interferes with ion flow regulation across the sodium ion channels [9,10] increasing the flow of sodium ions via sodium channels, thus, the channels are kept open and there is prolonged inward conductance of sodium causing repetitive nerve firing which can lead to paralysis and death of the insect [11,10]. This may not be the story in C.elegans because patch-clamp experiments on C.elegans neurones [12] and body wall muscle cells[13,14] showed that no IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (1) 2011 sodium current was observed, furthermore, intracellular recordings from pharyngeal muscle cells have shown a dependence sp ike generation on external sodium [15]. Moreover, the muscle cell’s of the pharynx communicate with each other via a network of gap-junction made up of innexins this confers a high degree of electrical connectivity which likely to play a central role in coordinating waves of myogenic excitation and the response to neuronal modulation [5]. [16] found that, although the pharyngeal pumping rate is under the control of the neurones, the pharynx continues to pump even when the pharyngeal neurones have been ablated, providing evidence that the pharynx may be myogenic. Further investigation (intracellular recording) might be needed to find out the target site of DDT which cannot be the same target as in insects. References 1- Davies T, Field L, Usherwood P and Williams M. (2007). DDT, pyrethrins, pyrethroids and insect sodium channels. IUBMB Life 59: 151-162 2-ATSDR (1994) . Agency for Toxic Substance and Diseases Registry.(ATSDR)/US Public Health serves, Toxicological profile for 4-4’DDT, 4-4’DDE, 4-4’DDD(update)ATSDR Atlanta GA. 3- de Bono Maricq (2005). Neuronal substances of complex behaviours in C. elegans. Neurosci 28: 457-501. 4- Strange K (2006).C.elegans M ethods and Application. Human press. 5- Franks CJ, Holden-Dye L, Bull K, Luedtke S and Walker RJ(2006). Anatomy, Physiology and Pharmacology of Caenorhabditis elegans pharynx: a model of define gene function in a simple neural system. Invert neuroscience 6: 105-122. 6- Bischof L, Huffman L, and AROIAN R V (2006). C. elegans: Methods and application. Methods in molecular Biology. K. Strange pp.139 Human press. 7- Szabadas T, Dul C, Majtenyi K, Hargital J, Penzes Z, and Urbnics R (2004). A chronic Alzheimer’s model evoked by mitochondrial poison sodium azide for pharmacological investigations. Behav Brain Res. 154: 31-40 . 8- Avery L, and Thomas JH (1997). Feeding and defecation in: Riddle D, Blumenthal T, Meyer B, Pries J, editors. C.elegans II.cold Springs Harbor: Cold Spring Harbor Laboratory Press. P 679-716. 9- Bloomquist JR (1996). Ion channels as target for insecticides. Annu.Rev.Entomol. 41: 163- 190. 10- Balkew M, Ibrahim M, Koekemoer L, Brook BD, Engers H, Aseffa A, Gebre-Michael, and Elhassan I. (2010). Insecticide resistance in anopheles arabiensis (Diptera: culicidae) from villages in central, northern and south west Ethiopia and detection of kdn mutation. Parasites & vectors 3:40-45. 11- Ranson H, Jansen B, Vulule JM , Wang X, Hemingway J, and Collins FH (2000). Identification of a point mutation in the voltage-gated sodium channel gene of Kenyan Anopheles gambiae associated with resistance to DDT and pyrethroids. Insect Mol Biol 9: 491-497. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1) 2011 12- Goodman MB, Hill DH, Avery L, and Locker SR(1998). Active regulate sensitivity and dynamic range in C. elegans neurones. Neurone 20: 763- 772. 13- Richmond JE,and Jorgenson EM (1999). One GABA and two acety lcholine receptors function at C.elegans neuromuscular junction. Nat. Neurosci. 2: 791-797. 14- Jospin M, Jacquemond V, Mariol MC, Segalat L and Allard B(200). The L-type voltage- dependent Ca channels EGL-19 controls body wall mauscle function in Caenorhabditis. J. cell Biol.159: 337-348. 15- Franks CJ, Pemberton D, Vinogradova I, Cook A, Walker RJ and Holden-dye L(2002). The ionic basis of the resting potential and action potential in the pharyngeal muscle of the Caenorhabditis elegans. J. Neurophysiol. 87 : 954-961. 16- Avery L, and Horvitz (1989). Pharyngeal pumping continues after laser killing of pharyngeal nervous system of C.elegans Neuron 3: 473-485. Acknowledgments The author is indebted to Dr.D.Bell, School of Biology, Nottingham University , UK for his kind invitation to do this work. Other thanks is extended to D.Brady for providing expert technical advice. -1 0 50 100 150 0 1 2 3 4 5 6 Control DDT ug/ml % A liv e Fig.( 1) Lethal concentration assay. This graph depicts a single LC50 in which the number of worms live after 3 day of exposure to dose DDT (0.1ug/ml–5.0 ug/ml) or vehicle control (0.1% DMSO) L4 C.elegans. The graph represents a single experiment with each concentration tested in triplicate where n=50, t=15 0 C. This experiment was repeated with similar results. -4 0 50 100 150 -3 -2 -1 0Control [Log 10] Na-Azide (mM) %A live Fig.( 2) Lethal concentrations assay of sodium azide (0.0001mM-1.0mM). This graph was set up as described in figure: 1 as appositive control. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VO L.24 (1) 2011 -4 0 10 20 30 40 50 -3 -2 -1 0Control [Log 10] DDT ug/ml Bro od siz e Fig.( 3) Effect of various concentrations of DDT (0.001ug/ml-1.0ug/ml) or vehicle control (0.1% DMSO) on brood size of L4 C.elegans N2 worms, where n=4, t=3days at 150 C. This experiment was repeated giving similar results. -4 0 10 20 30 40 -3 -2 -1 0Control [Log 10] Na-Azide (mM) Br oo d si ze Fig.( 4) Effect of various concentrations of sodium azide (0.0001mM-0.1mM) on brood size. This experiment was set up as in figure 3 as a positive control. -1 0 50 100 150 0 1 2 3 4 5 6Control DDT ug/ml ph ar yn ge al p um pin g Fig.( 5) Effect of various concentrations of DDT (1ug/ml-5ug/ml) or vehicle control (0.1%DMSO) on pharyngeal pumping rate after one hour of exposure where n=6. -5 0 50 100 150 200 -4 -3 -2 -1 0Control [Log 10] Na-Azide (Mm) Ph ar yn ge al pu m pi ng Fig. (6) Effect of various concentrations of sodium azide(0.0001mM-1.0mM).This experiment was set up as describe in figure 5 where n=6. 2011) 1( 24مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد Caenorhabditis elegans في DDT التاثیرات السمیة والفسیولوجیة لل سعدي محمد محمود ابن الھیثم ، جامعة بغداد -كلیة التربیة ، قسم علوم الحیاة 2010، تشرین الثاني ،30،ستلم في أ 2011،شباط ،8قبل البحث في الخالصة في تحدید الجرعة القاتلة وحجم الحضنة الواحدة DDTـ فة مدى تاثیر الت اختبارات السمیة في ھذه الدراسة لمعرعملاست Caenorhabditis elegansفي دیدان )C.elegans ( وذلك من خالل تعریضھا لتراكیز مختلفة من ھذا المبید. .ان ھذه الدیدان وفرت معلومات قیمة استخدمت في دراسة العملیات الفسیولوجیة والسلوكیة المختلفة اً على التركیز في تحدید الجرعة القاتلة ولكن دون الوصول دكان معتم DDT ـوقد اظھرت نتائج ھذه الدراسة ان تاثیر ال LC50كان الـ أذاالذي كان لھ تاثیراً معنویاً Na – azideة على عكس لمعغي جمیع التراكیز المست% 100الى تاثیر قاتل .ملي مول 0.01عند التركیز 0.1( مقارنة بمجموعة السیطرة C.elegansفي حجم الحضنة الواحدة لدیدان الـ DDTمشابھة فقد اثر الـ وبطریقة DMSO ( وكان التاثیرNa- azide اكثر وضوحا من الـDDT .واضحا ً في معدل ضخ العضالت البلعومیة سببا تثبیطاً Na- azideو DDTكال المركبین ال یمكن ان یكون ھو الموقع ) C.elegans( في DDTن االستنتاج ان الموقع الذي یستھدفھ الـ ومن خالل ھذه النتائج یمك .الذي یستھدفھ في الحشرات نفسة Caenorhabditis elegans ،صودیوم أزاید ،دي دي تي: الكلمات المفتاحیھ