IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 Toxicological and Physiological Effects of DDT o n Caenorhabditis elegans S.M. Mahmood Departme nt of Biology, College of Education I bn Al-Haitham,Unive rsity of Baghdad Reseived in ,30 ,Nov,2010 Accepted, 2, Feb,2011 Abstract Toxicity assay s were used in this st udy to test how DDT affects lethality and brood size of Caenorhabditis elegans (C.elegans) by exp osing them to various con centrations of t his agent. These nematodes have p rovided a very informative sy st em that was utilized to st udy the behavioural and p hy siological p rocesses. The results showed that DDT affected the lethality in a dose-dependent manner, but 100% kill was not ach ieved with concentration tested. Whereas, sodium azide, p ositive control does have an effect on C. elegans and significantly inhibitin g lethality (LC50 0.01mM ). Similar ly, DDT led to a pronounced effect in brood size of C.elegans comp ared to the mean brood size record ed for the control (0.1% DM SO). Sodium azide r esults showed a greater difference in brood size compared to DDT. Both agents, DDT and sodium azide caused a r emarkable inhibitory effect on C.elegans p haryngeal 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 - Caenorhabdi tis 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 ineff ective [1] DDT has had a significant imp act in the control of malaria and other insect diseases, and was extensively used as an agr icultural insecticide after the Second World War . This agent was found to have a chronic effect on the nervous sy stem, liver, kidney , and immune sy stem in exp erimental animals . [2] Caenorhabditis elegans (C.elegans) has been extensively utilized as a model animal for the study of nematode behavioural and phy siological processes [3]. C.elegans has also p roven itself a model of biological st udies relevant to higher animals in such areas as neuroscience, genetics, aging, and dev elop ment. Thus, t oxicological results in C.elegans are likely to high er animals [4]. However, one tissue that has p roven more amenable to p hysiological analy sis is the p hary nx. This is a muscular p ump which sucks bacteria into its lumen, grinds them and then passes t hem to the gut [5]. This p rovided insight into t he mode of action of toxins and IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 how C.elegans resp onds t o p rotect itself from toxin. This has contributed and will continue to contribute to our biological underst andin g of all animals including human. In the present work an attempt has been made to invest igate the effect and the mode of action of DDT in order to p robe the toxicity and the accumulation of this p esticide. Unt il recently little is known about the p harmacological effect of this insecticide and how it works on C. elegans which is used as mod el or ganism in this st udy . Materials and Methods C.elegans were cultured on agar and f ed with E.coli (OP50) throughout this st udy . Worms were grown at 15 0 C on NGM p lates containing (NaC l 3 g, Agar 17 g, Pep tone 2.5g, and 1ml Cholest erol (5mg/ml ethanol) in 1 L). For t he lethality assay 50 L4 C.elegans N2 worms p er dose group were placed in 400ul of liquid medium ( S-basal) sup p lemented with 40ul E.coli (O.D 0.7 600nm) with varying concentrations of DDT or sodium-azide (NaN3) to each well 0f 24-well p late [6] . The 24-well p late was then sealed with a laboratory film (to p revent evap oration). Wrapp ed in a damp p ap er towel (to p rovide humidity ) and p laced in an air tight p last ic container and incubated for 3 days. After the incubation p eriod, the p ercentage of live worms were determin ed 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-p lates, 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-p late was then sealed and p laced in an air tight p last ic container and incub ated at 15 0 C for 3 days. Aft er 3 days the number of offsp ring in each we ll was counted under a st ereo microscop e. An identical assay was set up and run alongsid e with sodium azide (as p ositive control). Phary ngeal pumping assay was set up the same as t he lethality bioassays. Briefly , the worms were exp osed to various DDT or sodium-azide concentrations for an hour, before the worms transferred to NGM p lates and left for another an hour to settle down before the p hary ngeal pumping rate was counted. Reagents: All solutions were made up to 1 litre with UHP water and st erilized by autoclaving st orage was at room temp erature. DDT stock solution made up to 10 mg/ml in DM SO (Dimethy l sulfoxide) this was ser ially diluted to make 1 ml of each concentration used in this invest igation. Results C. elegans h as been used as a model or ganism in ord er to p robe toxicity and the accumu lation of pesticides as well as v arious metal ions. In this st udy C.elegans was used as a system to gauge the toxicological effects of DDT and sodium azide. - Lethal ity assay 50 L4 larv as were exp osed up to 5ug/ml for 3 d ays. The exp erimental r esults 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 p ositive control due to its well known toxic effect [7]. The results obtained demonst rated that exp osure of C.elegans to the resp iratory inhibitor sodium azide, this agent caused a remarkab le inhibitory effect on C.elegans lethality and the LC50 was 0.01mM (fig.2). The graph represents a single exp eriment with each concentration tested in trip licate to p roduce standard error (represented by bar) and mean p ercentage of viable worms (p resented by dots) - Brood si ze assay Individual L4 C.elegans were p laced in 400ul of liquid med ium with varying concentrations of DDT, incubated for 3 days at 15 0 C the effect was determined by counting the offsp ring in each well. It was found t hat e xp osure of the worms to (0.001u g/ml-1.0u g/ml) DDT led to a p ronounced 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 p arallel with DDT sodium azaide was tested as a p ositive control, the results showed that sodium-azide has an effect on C.ele gans 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 p hary nx of C.elegans has several features that make it suitable for cellular and molecu lar st udies of behaviour. This model sy st em has been st udied by observing its behaviour in normal worms and treated worms. Phary ngeal p umping is the coordinated and regu lated p rocess by which the p harynx mediates intake and forces the food and wast e p roducts t hrough the worm gut [8] .The results p resented here showed that DDT does have an inhibitory effect on C.elegans by decreasin g the p umping r ate in a dose-dependent mann er. These results summarized graphically in fig.5. The p hary ngeal p umping is also reduced in resp onse to sodium azide; however, the higher concentration of sodium azide st opp ed the p hary nx activity in some worms (fig.6). Discussion The exp osure of animals to exo genous agents has been of considerable valu e in analysin g C.elegans resp onse. M ost of p harmacological agents that have been used in C.elegans affect various asp ects, such as brood size, lethal concentration, and growt h measurement. DDT is an orgaochlorine inse cticide used mainly to control insect typhus and malar ia vectors. This agent has been banned for use in differ ent countries, although it is st ill used in other. There is evidence that DDT causes rep roductive effects in test animals [2] and this seems to agr ee with results obtained in this st udy because DDT had a dose-dependent inhibitory effect on the brood size of C.elegans. Insecticides may act on membrane p roteins (receptors, channels) increasin g the ability of the insect to detoxify the insecticide or by chan ges in the target p rotein with which the insecticide interacts [1]. DDT is an insect neurotoxin that interferes with ion flow re gulation across the sodium ion chann els [9,10] increasing the flow of sodium ions via sodium chann els, thus, the channels are kept op en and there is p rolonged inward conductance of sodium causin g repetitive nerve firin g whi ch can lead to p aralysis and death of the insect [11,10]. This may not be the st ory in C.elegans because p atch-clamp exp eriments on C.elegans neurones [12] and body wall muscle cells[13,14] showed that no IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (1) 2011 sodium current was observ ed, furthermore, intracellular recordin gs from p haryngeal muscle cells h ave shown a d ependence sp ike generation on external sodium [15]. M oreover, the muscle cell’s of the p harynx communicate with each other via a n etwork of gap-junction made up of innexins this confers a high degr ee of electrical connectivity which likely to p lay a central role in coordin ating wav es of myogenic excitation and the resp onse to neuronal modulation [5]. [16] found that, a lthough the pharyngeal p umping r ate is und er the control of the neurones, the phary nx continues to p ump even when the p hary ngeal neurones have been ablated, p rovidin g evidence that the p harynx may be myogenic. Furt her invest igation (intracellu lar record ing) might be need ed to find out the target site of DDT which cannot be the same target as in insects. Re ferences 1- Davies T, Field L, Usherwood P and Williams M . (2007). DDT, py rethrins, py rethroids and insect sodium chann els. IUBM B Life 59: 151-162 2-AT SDR (1994) . Agency for Toxic Subst ance and Diseases Registry .(AT SDR)/US Public Health serves, Toxicological p rofile for 4-4’DDT, 4-4’DDE, 4-4’DDD(up date)ATSDR At lanta GA. 3- de Bono M aricq (2005). Neuronal substances of comp lex b ehaviours in C. ele gans. Neurosci 28: 457-501. 4- Strange K (2006).C.elegans M ethods and App lication. Human p ress. 5- Franks CJ, Holden-Dy e L, Bu ll K, Luedtke S and Walker RJ(2006). Anatomy, Phy siology and Pharmacology of Caenorhabditis elegans p harynx: a model of defin e gene function in a simple neural sy st em. Invert neuroscience 6: 105-122. 6- Bischof L, Huffm an L, and AROIAN R V (2006). C. elegans: M ethods and app lication. M ethods in molecular B iolo gy . K. Strange pp .139 Human press. 7- Szabadas T, Dul C, M ajtenyi K, Hargital J, Penzes Z, and Urbnics R (2004). A chronic Alzheimer’s model evoked by mitochondrial p oison sodium azide for p harmacological invest igations. Behav Brain Res. 154: 31-40 . 8- Avery L, and Thomas JH (1997). Feeding and defecation in: R iddle D, Blumenthal T, M ey er B, Pries J, editors. C.elegans II.cold Sp rings Harbor: Cold Sp ring Harbor Laboratory Press. P 679-716. 9- Bloomquist JR (1996). Ion channels as t arget for insecticides. Annu.Rev.Entomol. 41: 163- 190. 10- Balkew M , Ibrahim M , Koekemoer L, Brook BD, Engers H, Aseffa A, Gebre-M ichael, and Elhassan I. (2010). Insecticide resist ance in anop heles arab iensis (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, Vu lule JM , Wang X, Hemin gway J, and Collins FH (2000). Identification of a p oint mutation in the voltage-gated sodium chann el gene of Keny an Anop heles gambiae associated with resistance to DDT and py rethroids. Insect M ol Biol 9: 491-497. IBN AL- HAITHAM J. FO R PURE & APPL. SC I. VO L.24 (1 ) 2011 12- Goodman M B, Hill DH, Avery L, and Locker SR(1998). Active regulate sensitivity and dy namic range in C. elegans neurones. Neurone 20: 763- 772. 13- Richmond JE, and Jorgenson EM (1999). One GABA and two acety lcholine recep tors function at C.elegans neuromuscu lar junction. Nat. Neurosci. 2: 791-797. 14- Jospin M, Jacquemond V, M ariol M C, Segalat L and Allard B(200). The L-type voltage- dependent Ca channels EGL-19 controls body wall mauscle fun ction in Caenorhabditis. J. cell Biol.159: 337-348. 15- Franks CJ, Pemberton D, Vinogradova I, Cook A, Walker RJ and Hold en-dye L(2002). The ionic basis of the r esting p otential and action p otential in the p haryngeal muscle of the Caenorhabditis elegans. J. Neurophy siol. 87 : 954-961. 16- Avery L, and Horvitz (1989). Phary ngeal p umping continues after laser killin g of p hary ngeal nervous sy stem of C.elegans Neuron 3: 473-485. Acknowle dgments 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 p roviding exp ert 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. Th e graph represents a single experiment with each concentration tested in triplicate where n=50, t=15 0 C. This ex periment was repeated with similar results. -4 0 50 100 150 -3 -2 -1 0Control [Log 10] Na-Az ide (mM) %A liv e Fig.( 2) Lethal concentrations assay of sodium azide (0.0001mM-1.0mM). Th is graph was set up as described in figure: 1 as appositive control. IBN AL- HAITHAM J . FOR PURE & APPL. SC I. VO L.24 (1) 2011 -4 0 10 20 30 40 50 -3 -2 -1 0Control [ Log 10] DDT ug/ml Br oo d 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 15 0 C. This ex periment was repeated givin g similar results. -4 0 10 20 30 40 -3 -2 -1 0Control [Log 10] Na-Azide (mM) B ro od s iz e Fig.( 4) Effect of various concentrations of sodium azide (0.0001mM-0.1mM) on b rood size. Th is experiment was set u p as in figure 3 as a positive control. -1 0 50 100 150 0 1 2 3 4 5 6 Control DDT ug/ml ph ar yn ge al p um pi ng 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 ex posure where n=6. -5 0 50 100 150 200 -4 -3 -2 -1 0Control [ Log 10] Na-Az ide (Mm) Ph ar yn ge al p um pi ng Fig. (6) Effect of various concentrations of sodium azide(0.0001mM-1.0mM).Th is experiment was set u p as d escribe 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 راسة لمعرعملاست في تحدید الجرعة القاتلة وحجم الحضنة الواحدة DDTـ فة مدى تاثیر الت اختبارات السمیة في ھذه الد Caenorhabditis elegansفي دیدان )C.elegans ( وذلك من خالل تعریضھا لتراكیز مختلفة من ھذا المبید. تلفة .ان ھذه الدیدان وفرت معلومات قیمة استخدمت في دراسة العملیات الفسیولوجیة والسلوكیة المخ 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 دان الـ DDTمشابھة فقد اثر الـ وبطریقة دة لدی 0.1( مقارنة بمجموعة السیطرة C.elegansفي حجم الحضنة الواح DM SO ( 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 رات نفسة .الذي یستھدفھ في الحش Caenorhabditis elegans ،صودیوم أزاید ،دي دي تي: الكلمات المفتاحیھ