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CC SEp 28m D D T: Current Theories on its Mechanism of Poisoning by F. Matsumura DDT is perhaps our most potent insecticide, but many questions about the mechanism of its action remain unanswered. How can an "inert" chemical be so toxic to biolgical systems? As many people already know DDT, which was once a miracle chemical, has recently become a problem child of our society. Perhaps DDT has saved more human lives than any other chemical in our human history, and yet, because of its long-lasting effects upon non-targiet ecosystems, its usefulness turned into a headache. The paradox of DDT action is that DDT itself is a chemically unreactive com pound as attested by the fact that DDT persists so long in various environments. It is quite puzzling that such an "inert" chemical could he so toxic to many bio logical systems. Efforts to find the mechanisms of DDTpoisoning actually started as soon as this insecticide was first commercially used. Unfortunately, despite the efforts by many researchers for more than 20 years, the actual poisoning mechanism of DDT in animals remains as a mystery. The only facts we know for sure are that it initially attacks the nervous systems of animals and that the end result of DDT-poisoning is disruption of the ion-transport mech anism (Narahashi and Haas, 1967; Mat sumura and O'Brien, 1966a). This dis ruption may ultimately cause death through respiratory failure or secondary poisoning by neurotoxins which are pro duced by the animals themselves under DDT poisoning (Sternburg. 1963). The key question then is how DDT upsets the function of nervous system to cause such effects. It appears to be the intact DDT mole cule itself that reacts with the nervous system, for researchers have recovered only unchanged DDT from the nervous system of poisoned insects (Matsumura and O'Brien, 1966b; Eaton and Sternburg, 1967). Besides being unreactive. DDT and other chlorinated hydrocarbon insecticides have another interesting characteristic in common. That is, towards electrons they have high affinities, which become evident when researchers began adopting an elec tron-capturing device for detecting organic compounds in the eflluent gas from the gas-chromatographic systems (e.g., elec tron-capture detectors). They discovered that chlorinated hydrocarbon insecticides had outstanding susceptibilities toward this detection method allowing them to study the insecticidal residues at 1 picogram, or parts per billion ranges in various biological materials. The general mole cular structure of DDT (Figure 1) is ar ranged so that electrons are forced to flow from both the phenyl groups toward the -CCla position creating two electro negative centers at both p,p' positions. Close examination of other active chlori- nated hydrocarbon insecticides also in dicates the presence of such electronega tive centers for each molecule positioned opposite to each other across the line of symmetry (Soloway, 1965). As the reader probably noticed already, perhaps the most likely way by which a stable compound such as DDT can react with biological systems is to directly bind with some biologically vital -iles and to form a tight complex. The result of such a complex formation would be a loss of normal cell function. F. Matsumura is an Associate Professor in the Department of Entomology. I niversity of it Csronsin, Madison, lie re ceived his Ph.D. degree in 1961 from the University of W estern Ontario. He did his postdoctoral training in Cornell University before he joined Wisconsin in 1961. His main area of interest is on the chemistry and biology of chlorinated hydrocarbon in secticides. His research team members who contributed to this work are Drs. H. Brunnert, K. C. Patil, ]. N. T elford and Mr. T. A. Bratkowski. The research is supported by a grant (CC-00252) from the National Communicable Disease Cen ter. PAGE 6 DSW 255197 YALE SCIENTIFIC MAGAZINE STLCOPCB4060888 Because of llic cxcromc lipophilicity of DDT, early workers suspected the lipid components of the cell to lie the target substance for DDT. More recent studies, however, indicate that proteins take part in selective binding of DDT (Gunther et al., 1954; Matsumura and O'Brien, 1966 a, b; Holan, 1969). The net result of DDT-protein binding should be that Na+ and K+ ions are prevented from pas sing through the cell membranes. Then how does this complex formation cause such an end result? The question can only be partially answered because our knowledge of the mechanisms of ion trans port as well as of the ultrastructures of cell membranes themselves are limited. The purpose of this article is to de scribe the various experimental approaches made mainly in our laboratory and to report the current status of our under standing on this complex problem. Our initial venture was an attempt to show that DDT can be bound to nerve components. In view of the lipophilic nature of DDT it was suspected from the first that a large quantity of DDT would be simply absorbed by non-vital lipid com ponents. To distinguish the specific bind ing from non-specific absorption, we de cided to always compare the pattern of DDT-absorption to the pattern of its noninsecticidal analog DDE. which is almost as lipophilic at DDT itself. To study first the general absorption pattern of DDT by various subcellular components of the nervous system, the rat brain was homogenized. The homoge nate was then incubated with 10`5 M C14-DDT or C14-DDE for 10 minutes. The centrifugal technique adopted to separate subcellular components was that of Marchbanks et al. (1966) who modified the method of Whittaker (1959). The frac:ions were carefully separated a..d all particulate portions were washed once. The Table 1 is a brief summary of the results from matched DDT-DDE absorp:ion tests under the identical experimental conditions. It became evident from the data that the amount of DDT binding to he supernatant fraction was no more than hat of DDE, while more DDT than DDE was found in the nerve ending fraction. The above experiment revealed two im portant facts: first, soluble fraction does rot contain the target substance, and ;econd, DDT is preferentially absorbed >y the material (s) that is present in the Taction containing largely the nerve endng particles (NEP). NOVEMBER, 1969 Table 1. Distribution of C14 DDT and DDE among the subcellular fractions from the rat brain: the data are expressed in % of given (5 nanomole DDT or DDE. Fractions Supernatant (Sp) Absorption DDT DDE %% 3.96 4.63 Difference (DDT-DDE) o/o* --0.67 Cell Membrane (CM) 2.63 2.79 --0.16 Myeline Fragment (My) 20.03 19.88 0.15 Nerve Ending Particles (NEP) 37.78 33.65 4.13 Mitochondria (Mt) 1.26 1.53 --0.27 Nucleus (N) 34.38 37.63 --3.25 Figure 1 (right). The molecular structure oj DDT and its pattern of electron dis tribution. Note that two electronega tive centers are formed at p,p'positions. Figure 2 (below). Schematic representa tion of the synaptic complex and the possible site of DDT attack which is related to DDT-poisoning. Ax: axon, em: ending membrane, ssw: subsynaptic web, sm: synaptic membrane, sv: synaptic vesicle, mt: mitochondrion. 4 No No + No Cl u No ATPose --Mg-ATP K+ Normal dSNN255A98 DDT-inhibited PAGE 7 | rt \ i t fi' l i f t\lffc i i f t : 5 * t rL f j[ t t STLCOPCB4060889 snrn \ w>s. h- - /' w-c *.,V Figure 3. Electron microscopic view of synaptic membranes of the rat brain, hole that the synaptic vesicles as well as the ending membrane have been removed by the treatments of first osmotic shock and second Triton X-100 (De Robertis, 1967). It was found that DDT had the highest affinity toward this portion of the nervous system. Figure 4. Radioautographic recording of H3 DDT-binding to the axonic membrane of the abdominal nerve cord of the American cockroach. The dark filamentation rep resents the DDT molecule. Axp: axoplasm. Axm: axonic membrane. The next question was the location of DDT binding within the of NEP fraction. The tno-t helpful techno logical assistance came from the recent development of an ultra-centrifugal meth od hy E. De Roberti- and hi- associates (ref. De RoIktiD. 1%7 i to -eparate var ious neural component-: of the brain. Figure 2 schematically ilhi-trate- the ac tual sites of specific DDT-binding within the junctional complex of the rat brain. All the experimental results have in dicated that the post -ynaptic regions of the nervous y-lem play an important role in DDT binding and that the differ ence in the amounts of DDT and DDE binding does increa-e with treatment' which attempt to purify the po-t synaptic region. Electron microscopic observations of the Triton treated nerve fraction (Figure 31 indeed contained fragmented junctional complexes containing the sub-ynaptic webs and synaptic membranes joined by the intersynaptic filaments. A radioautogTaphic technique to actually locate the cite= of DDT attack has also been developed to facilitate such .studies (Figure 4i. The foregoing experiment- were helpful in establishing the order of the relative im portance in specific DDT binding among the nerve components. The experimental re sults thus indicate the position of the pri mary target within the nervous system. This way of approach does not. however, give an answer to the question of why sucli for mations of DDT-complex lead' to disrup tion of normal cell function'. To relate the action of DDT to inhibi tion of a certain system which normally performs an important role in cell function, at least three requirements were considered to l>e important: (1) the system mu-t be concerned with the mechanisms of inn transport in the nervous system. (2i it must be DDT s|>ecific and it should be susceptible to DDT at low DDT concen trations, and (3) it should be abb- to form a complex with DDT itself. Systematic search of such a sy-tem in our laboratory led to a finding that there is a certain nerve ATPase (adenosine tri phosphatase) which satisfies all the above requirements. The nerve ATPase' have been known to hydrolyze ATP fa high energy phosphate) to utilize it' energy to regulate the flow of ions across the nerve membrane. Although there are a number of ATPases in tbe nervous system, not all of them showed a special susceptibility toward DDT. One ATPa se system we PAGE 8 DSW 255199 YALE SCIENTIFIC MAGAZINE STLCOPCB4060890 found in the synapse? of the rat hrain (exactly the same synaptic fraction as lining described above) showed 1000 times more sensitivity toward DDT as compared to DDE. Moreover the concentration range of DDT to cause nerve disruption was in the same order as that required to in hibit the nerve ATPase. The close correlation of the overall phe nomenon of ATPase inhibition by DDT and nerve symptoms of DDT poisoning suggests that the site of DDT attack is very much similar to the nerve ATPase, if not the ATPase being the actual target system. The whole mechanisms by which these nerve ATPases facilitate ion tra , .>rt are still unknown. Riochemica! evirr `-'-w- ever, indicate that some ATPa-i s r- .lire Na + , K+ and Mg++ in a ccrta - pro portion for their maximum activity: im plying that the ATPases control thp rela tive concentrations of Na+ and K r by the aid of Mg++ and ATP. That is. when the sodium ion rushes in and the potas sium ion leaks out of the membrane (ex citation) thiATPase system gets acti vated by the change in the ion composi tion, and thereby acts as the brake (torn off mechanism) for the continuing inflow of Na+ ion, or as the triggering device (activation mechanism) for outflow of K+ ion. In addition, this mechanism or Tclated mechanisms also can act as the "pump" to expell unwanted Na+ ions from inside of the nerve membrane (Fig. 5). By the virtue of the rigid -spacial re quirements for the insecticidal activity of the DDT-like compounds, the apjooxi- mate shape of the target site on the nerve membrane has long been speculated by many researchers (e.g. Holan, 1969). Fig. 5 illustrates the size requirements for the DDT target and its possible relationship to the ATPase molecule. DDT by its par ticular shape and the pattern of electron distribution hinds with the ATPase. and thus blocks the channels for specific ion passage. Except lor the established fact that DDT does inhibit the nerve ATPase in vitro, the bulk of the above "ATPase" theory has to he yet confirmed hv actual experimental evidence. One factor which does not favor the above theory is that the extent of inhibition of ATPases is not too great in the nervous systems of actually DDT-poisoncd animals (in vivo). That is to say, we could not observe high degrees of ATPase inhibition even in in sects which were already in the state of complete paralysis as the result of PPT- (continued on page ?,l} NOVEMBER, 1969 DDT Figure 5 Schematic diagram explaining our current thinking on DDT-blockage oj the. '-'7 trap.-port mechanism across the nerve membrane. When the external ions /'mainly Va"L) start flowing into (i.c. excitation) the cell through the ionic channel (channel) the ATP system becomes activated by the increase oj \a~ and the decrease oj ions. It is not known whether there are two channels (one jor Nax an1 another jor K +) or the same chanel is used jor both ions. The picture leJ' ' ' to illustrate the hypothetical sodium channel only. The DDT molecule ear, , tardy ft into the potassium channel which should be bigger than that for the sodium ion. and thus blocks the outflow process oj K~ ion I inhibition oj K~ -- r-'-n mechanism). DDT also attacks ATP-systems involving \a~ channel in the sr:,,,. 'earner as it does to K3 ATP-systems except that the channel jor i\a~ is sn n't,., and the -C(Cls) end oj DDT cannot plug the hole. Thus Xa~ turn off r.i.sm. Tin DDT molecule is draw here in the. manner that both p.p'positions of phenyl ring arc attached to the ATP system and the -C(Cls) end is fitted to n-.erbnnUn. The DDT molecule is drawn here in the manner that both p.p'positions the K+ channel. Picture 6. I'f>T also weakens the egg shells of many birds, thus endangering the survival oj these species. DSW 255200 PAGE 9 STLCOPCB4060891 DDT continued jrom page 9 poisoning. However a close parallelism of 1 he elcctrophysiological symptoms of DDT-poisoning and Iowr degrees of ATPase inhibition was always observed in the nervous system of poisoned animals (un published data). This problem of "low degree inhibition" of ATPase in vivo (in live animals! has been interpreted by our group to suggest two alternative possibili ties: (1) the actual portion of the specific ATPascs that arc involved in the process of DDT-poisoning is very small in com parison to other non-target ATPases, or (2) ATPases are not the actual site of DDT attack but there is a very similar, /functionally analogous system (e.g. an ATP receptor protein) that is susceptible to DDT. The latter possibility is quite at tractive since all the data obtained in vitro (at enzyme-levels) strongly indicate that some ATP requiring system should be involved in DDT-poisoning. Such ex amples of two analogous target systems can le found in nature; e.g. the case of the acetylcholine receptor proteins vs. aceeylcholineslerases in the synaptic complex: e.g. nicotine specifically attacks the former because of its molecular similarity to acetylcholine while it does not inhibit the acetylcholinesterase in vitro. Whatever the outcome of the future, ex amination of such hypotheses, two things are aleready certain at this stage: first, DDT attacks membrane ATPases, and sec ond, it forms a tight complex with certain membrane components to alter the rates of ion exchange processes across the mem brane. The distribution of membrane ATPases is wide among various parts of any animal system. Furthermore enough evidence has accumulated to -show that various biolo gical membranes do possess similar, basic structures and properties. Recent experi mental work in our laboratory, for in stance. indieat'-s that di'-ldrin. amolcr po tent chlorinated hydrocarbon in- T-i,! upsets the ion trail-port me. hanl-m- i:i the liver cell- (Wang and Mat-umura. 1969). The experimental re-ults from an other research group al-o indicate that various chlorinated hydrocarbon in-ect- icides can inhibit ATI'a-e- i Koch. I960: Koch cl nl., 1969l. It i- entirely po--ilde that DDT and related in-orii< ide- inhibit the ATPascs and/or ATP-n-quiring sys tems in various biological membranes through complex formation ami cause the state of ion inbalance which in turn re sults in unexpected side effects in those systems. Studies on the action mechanism of DDT. therefore, could shed a light upon the various biological effects of other chlorinated hydrocarbon insecticides which are the major contaminants of our en vironment. " (References will be sent on request.) NATURE, ART and MATHEMATICS contin ued jrom page 24 somewhat at random. For a smaller error, however, the arrangement will be seen to be limited to a more regular appearance. Approximation, as recognized by Galileo, involves the choice and number of para meters; it is of paramount importance in using mathematical descriptions of natural phenomena. Ptolemy's successful description of observed planetary paths was made possible by virtually an approx imation series involving epicycles. By ap propriate choices of radii and of fre quencies, indeed, one can produce even a square orbit. The role of mathematics in art. I be lieve. is similar to that in science: in both instances it is a convenient servant of man--not an autocratic queen of exper ience. Under no circumstances should we over look the fundamental role of nature it self. The same phenomenon can be re garded from both scientific and artistic aspects, which converge in the very com monness of the single exper. nee. Is it not possible that underlying relations, due to substratum forces, may emerge out of our similar methodology? Wonder as to the ever-increasing mystery of nature may be more inspiring than beauty itself--the appeal of the unknown greater than that of the known! Suggestive of the possibility of some such deeper understanding are some pic tures exhibited about ten years ago in Basel. In each case a famous painting was set along side a scientific photograph. Two comparisons are shown here: Honeg ger's "Composition" (Picture 2a) and a hexiacontrain crystal (Picture 2h). Tau ber's "Circular Movements" (Picture 3a) and rubber latex (Picture 3b). What is the basic explanation for such remarkable similarities? (In most instances the work of art predated the scientific discovery.) Evidently the artist is unconsciously seeing something of scientific significance, the scientist something of artistic worth. What may emerge out of such convergences? Our artificial two cultures are Januslike aspects of one world. In conclusion, let us bear in mind the significance of man himself--more hu manly distinctive. I believe, in his con scious search for reality than in the fum bling grasping of surrealism. And yet. efficient reason, though necessary, is it self not sufficient: the totality of humanity requires due regard for man's own per sonality, for his spirituality. Man needs to discover--and rediscover--experiential relations to nature, to man. and to God! Otherwise we may be merely performing meaningless antics. Recall the picture showing the hero Hercules fighting the giant Antaeus. Hercules bad been advised that his opponent could be debuted only by being strangled in midair. The frantic struggles ("antics") of the giant would then be of no avail: only when he touched the ground woold his strength be in stantly renewed. To ine. this is a parable of the scientist. As long as he speculates in the rare atmosphere of isolated theory, he may merely be performing intellectual antics; Ills scientific strength is renewed only when he touches the bedrock of ex perience. Do not be hypnotized by the strangeness of the Cheshire cat's grin-- it. too, finally vanishes. NOVEMBER, 1969 OS\N 255201 PAGE 31 STLCOPCB4060892 neporx i of a Secretary's Commission on Pesticides and Their Relationship to Enyironmental Health U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE December 1969 For sale by the Superintendent o( DocumouU, U.S. Government Printing Office Washington; D.C. 20401 - Price 43.00 ft*1 t> STLCOPCB4060893 -;:rc' iircur, opportunities to main*'meam ngful observations of health . .'.:;ri`r!V:OP -kould not be overlooked. ExtrA:ions from occupationally exposed populations to the f.-onerai population always require qualification. The most significant difference between the two groups is that the general population in cludes large numbers of very young, very o!u, ill and disabled, while .-.uca persons are found rarely or r.ot at all among the occupationally exposed. These individuals arc often least resistant to the elfcots of j ,.)xic aaents: it is therefore apparent that dose-response relationships I :C1 on studies of i'ormulators or other occupational groups may be OXT rduied to cite general population only with great caution. ; i i h/t/Ciil co-in-plcxlties An appreciation of some of the more important analytic and in terpretive problems peculiar to the nrganochlorines is essential for perspective in this area. A recently-discovered source of.error in analysis of DDT and re lated materials by gas chromatography is the presence of poly chlorinated biphenyls (PCI!).-Since 19:20, polychlorinated biphenyl liquids, resins, or solids have'been spread...widely in our environment in oils, hydraulic fluids, adhesives, plastics, building materials, fuels, fire retardants, heat transfer agents, electrical equipment, paper, and many other industries. The presence of PCB has caused serious analytical errors in the nonspecific gas chromatographic analysis of the chlorinated hydrocarbons (Jensen, 1966; Richardson* 1969; Ticymolds, 1909). It is clearly necessary to confirm qualitatively the determination of DDT residues and not to be confused by gas Chroma tographic peaks that overlap DDT but represent totally different materials (Schechter, 1968). Such false peaks have been reported in gas chromatograms from some wildlife samples, along with organ- ochlorinc pesticides (Roburn, 1965). Cod liver oil from Norway gave rise to gas chromatographic peaks in the region expected for DDT, DDE, and TDE, but paper chromatography indicated the presence of halogcnated compounds which were not known pesticides at all (Eidelman, 1963). In samples for the Nature Conservancy in Britain, compounds were detected interfering with detection of p,p"DDT and p,p'TDE (Harrison, 1966). Since 1966, the presence of PCB has also been noted in the British (Holmes,'et al. 1967; Holden and Marsden, 1967; Robinson, undated reference cited in Richardson, 1969), Dutch (Koeman, et al. 19Q9), and North American (Risebrough, et al. 1968) environments. . ' Polychlorobiphenyls have been identified in fish, wildlife, and the environment as cited above, but no attempt appears to have been made to look for them in human tissues or excreta. Pooled samples of lI j 257 DSW 255203 \ `dorado. Spru'.';" i'_r!ir imwYhUlal smi . 1 J were po.'ii :v,; ;V]$ (Ki^obmu^rli, lOfiOj P:;sr l-\\ is or ouiorinarv.I hyclro- .. '/>?' might- thus have h.rn too high as a result of the Pi'.*: A further source or .mror might be introduced by in plastic contain s f'C*viinby, 19Cfi). ^ - CjX n ' of environmental s-impa-s be supported by qualita- .antitative confirmation o' ii:e. nsuits. Too many papers published that incorpomro no su?h quality controls; nor . % ...d sources of error been eons'-dc'-'ccl. Especially when the rp .'"porred as positive near r'.e lover limits of detection of i (' omatoerraphic method (which is often the case), the results isolutely misleading. The mass o:i material in question is insufficient for qualitative and quantitative corroboration of : ;u* :V\ by other techniques of analysis. ' CITED REFERENCE!* E:d:-:l.\:an. M.: Determination of micro quantities of some chlorinated organic pesticide residues in edible fats and oils. .1 of the Assoc Offlc Agricul Chem. -id: 1x2-6, 1363. Harrison. R. B.: ,T. Sci. Fd. Agric. 17:10.19GG. TLm.ukn, A. V., Marsden, K.: Organochlorine pesticides in seals and porpoises. Nature. 2H>: 1274-0, 30 Decemlicr 1007. . JTui.mem. I), c., Simmons, J. II., Tatton, J. O'G.: Chlorinated hydrocarlxms In British wildlife. Nature. 210 : 227-20, 21 October J007. Jensen, S.: New .Scientist. 32 : 012,1900/ Koh.man, J. II., Tex Xoever De Brauw, M. C., Ee Vos, R. H.: Chlorinated hi* phenyls in'fish, mussels and birds from the River Rhine and the Netherlands Coastal Area. Nature. 221:1126-28,22 March 19C9. ' Reynolds, I. M.: Polychlorobiphenyls (PCB's) and their interference with pesticide residue analysis. Bull Environ Contamin Toxicol. 4:128-43, 1969. Richardson, A.: Organochlorine compounds other than insecticides in the en vironment. "'Shell" Research Limited, 'funstnll Laboratory, Sittlngbourne, Kent, England. February 1969. ` Risebroi'cjh, R. W,, HrcGETT, R. J., Griffin, J. J., Goldberg, E. D.: Pesticides: Transatlantic movements in the northeast trades. Science, 159:1233-36, 15 March 1968. .f Robinson, J.: Undated reference. "Shell" Research Limited. Tnnstall Labora tory, Sittlngbourne, Kent, England. - .. Roburn. J: A simple concentration-cell technique for determining small amounts of halide ions and its use in the determination of residues of organochlorine pesticides. Analyst 90:487-75,1965. ` Sciiechter, M. S.: The need for confirmation (Editorial). Pesticide Monitoring Journal. June 1968^ Gom'plexitiea of Terminology , A large body of information has been published regarding the health consequences of human exposure to pesticides. Review of these publi cations reveals that the scientific terminology is often imprecise and . 258 DSW 255204