The toxicity of organophosphorus compounds to mammals

SESSION VI TOXIC EFFECTS PRODUCED IN VERTEBRATES BY ANTICHOLINESTERASES Bull. Org. mond. Santl 1971, 44, 233-240Bull. Wid Hith Org. The Toxicity of Organophosphorus Compounds to Mammals KENNETH P. DuBOIS 1 The acute toxicity of most of the commonly used organophosphorus insecticides is essentially the same. A few compounds with low toxicity, such as malathion, have been developed but further efforts in that direction are needed. Most of the organophosphorus insecticides exert a generalized cholinergic action by inhibiting central and peripheral cholinesterases. The phosphoramides are an exception in that they do not gain access to the cholinesterase of the central nervous system in vivo and consequently atropine is a more effective antidote for them than for organophosphorus compounds. Young animals are more susceptible to the organophosphorus compounds than are adults. Enzyme-inducing agents decrease the toxicity of the phosphorothioates and phosphorodithioates. All organo- phosphorus insecticides can inhibit esterases that catalyse the detoxification of some insecticides of this class and ester-type drugs. The toxicology of the organophosphorus insecti- cides has received a tremendous amount of attention during the past 25 years because of the established value and widespread use of these compounds as agricultural insecticides. The use of organophos- phorus compounds as insecticides has prompted extensive investigation of their toxicity to various laboratory animals as a means of evaluating the possible hazards to the health of those engaged in their manufacture and use and those who may consume food containing small residues of these agents. Any consideration of the available data on the toxicity of organophosphorus insecticides must take into account the fact that studies of this type are generally conducted in connexion with some aspect of their practical use, because toxicological studies are costly and time consuming. As a result, each group of investigators conducts toxicity tests in which the types of experiment and the procedures used are selected with primary consideration being given to the practical problems at hand. Thus, toxicity studies are frequently carried out on technical materi- als of the same purity as those used for insecticidal purposes rather than on highly purified materials. The solvents that are used to dissolve the compounds for experimental studies or that are present in vari- 1 Director, Toxicity Laboratory, and Professor of Phar- macology, University of Chicago, Chicago, Ill., USA. ous formulations sometimes influence the toxicity of the organophosphorus compounds. Nevertheless, sufficient toxicity data are available to permit rela- tively accurate comparisons of the toxicity of various members of the group. At present, the toxicological evaluation of organo- phosphorus insecticides usually consists of a series of experiments that include the measurement of (a) acute toxicity by several routes of administra- tion, (b) subacute toxicity by repeated injection or by feeding the compounds, and (c) chronic toxicity by feeding various low levels of the compounds in the diet for long periods of time. With this particular group of compounds measurement of the anticholin- esterase action has become an established part of toxicity evaluations. This paper discusses the toxi- city of a number of organophosphorus insecticides and considers their mode of action and the way in which they are metabolized, in so far as these factors are related to and govern the toxicity of these chem- ical agents. Acute toxicity is the problem of greatest impor- tance for the organophosphorus insecticides. Acute intoxication by every member of this group always results in at least some symptoms characteristic of stimulation of the parasympathetic nervous system (muscarinic effects). These symptoms, which con- sist of bronchoconstriction, sweating, salivation and other increased glandular secretions, anorexia, nau- sea, abdominal cramps, vomiting, diarrhoea, invol- 2633 - 233 - K. P. DUBOIS untary defaecation, and increased x of paramount importance because all phorus insecticides produce some c muscarinic actions of acetylcholine. 1 organophosphorus compounds on st (nicotinic effects) include muscular tx cular fasciculation, increased liability t muscular weakness (which also involv of the respiratory system). Most ol phosphorus compounds are able to E the central nervous system, where their in anxiety, restlessness, impairment speech defects, convulsions, and corr and symptoms produced by anticholimn in different mammalian species are esse quantitatively, suggesting similar tissu patterns. However, the doses requ toxic effects in different species vary probably owing largely to differences i extent of detoxification of the compc PYROPHOSPHORIC ACID DERIV) In the early studies on the toxicolo phosphorus insecticides a great deal was given to the derivatives of pyroph and particularly to tetraethyl pyrophos, Interest in this compound led to the ir other pyrophosphates containing variot (DuBois & Coon, 1952). Table 1 sh( peritoneal toxicity of five derivatives phoric acid for mice and their anti activity in vitro. The results of these s- strated the influence of various alkyl Table 1 Toxicity and anticholinesterase of alkyl pyrophosphates Compound Intra- peritoneal LDso (mg/kg) tetramethyl pyrophosphate tetraethyl pyrophosphate diethyl dimethyl pyrophosphate (asymmetrical) diisopropyl dimethyl pyro- phosphate (asymmetrical) tetraisopropyl pyrophosphate 1.7 0.85 1.1 2.5 16.0 irination, are Iorganophos- )r all of the[he actions of ,eletal muscle iitching, mus- o fatigue, and es the muscles the organo- gain access to action results of memory, la. The signs toxicity and anticholinesterase activity of organo- phosphorus compounds and the correlation between in vitro anticholinesterase activity and toxicity. Since the toxicity of pyrophosphates can be varied over a considerable range by appropriate alkyl substitu- tion, and since the hydrolysis products are simple nontoxic derivatives of phosphoric acid, it seems that these organophosphorus compounds might be worthy of further consideration in efforts to develop insecticides with low persistence and nontoxic degra- dation products. Schradan -sterase agents Further studies of pyrophosphates in the early tntially similar 1950s led to the finding that metabolic changes in e distribution the parent molecule can result in the transformation ired to elicit of an inactive compound into an anticholinesterase considerably, agent. The first observation along this line was n the rate and made with octamethyl pyrophosphoramide (schra-)unds. dan), the analogue of tetraethyl pyrophosphate in which dimethylamino groups replace the ethoxy kTIVES groups of TEPP. The toxicity of this compound is not strikingly different from that of the alkoxy pyro- gy of organo- phosphates, since the LDro values for several species *ofrattention by the parenteral route are 5-20 mg/kg. However, osphortce cipd the replacement of alkoxy groups with amide groupsphate (TEPP). resulted in a marked difference in the inherent ivestigation of biological activity of the compound and in its siteis alkyl groups of action. The ineffectiveness of schradan as an ows the intra- inhibitor of cholinesterase in vitro, in contrast to its of pyrophos- strong anticholinesterase action in vivo (DuBois ,cholinesterase et al., 1950), is well known. The early demonstra-tudies demon- tion that schradan is converted by an oxidative reac- groups on the tion in the liver to a metabolite with strong anticholin- esterase activity (DuBois et al., 1950; Gardiner & Kilby, 1950) is also well known. It is unnecessary to describe the details of experiments on the oxida- action tive activation of phosphoramides by the enzyme system that is now known to be located in the microsomal fraction of the liver. Knowledge that 150 for ChE schradan must undergo biochemical transformation in vitro (M) to exert an anticholinesterase action provided the first clear evidence that the potency of organophos- phorus insecticides as inhibitors of cholinesterase 1.8 x 10-8 cannot always be ascertained by conducting in vitro 4.0 x 10-9 tests. The other unique property of schradan that has 8.0 X 10-9 not been intentionally exploited in the development of new insecticides is its selective action. Schradan 2.0 x 10-7 produces all the muscarinic effects of organophos- 1.4 X 10-6 phorus insecticides and also the typical stimulant action on skeletal muscle, but it has no action on 234 TOXICITY OF OP COMPOUNDS FOR MAMMALS Table 2 Selective anticholinesterase action of phosphoramides Maximum inhibition Compound (mg/kg) o ChE xi%) Ban Submaxillary ________________________________________________ _______ _Brain__ gland octamethyl pyrophosphoramide 5.0 0 88 diethyl bisdimethyl pyrophosphordiamide (symmetrical) 6.25 3 82 diethyl bisdimethyl pyrophosphordiamide (asymmetrical) 1.95 5 78 tetramethyl phosphorodiamidic fluoride 3.1 25 85 a Each dose represents 5/8 of the acute intraperitoneal LDso. the central nervous system. Small doses (5 mg/kg) given intraperitoneally cause marked inhibition of the cholinesterase of peripheral tissues but no inhi- bition of brain cholinesterase, which is consistent with the absence of signs caused by central nervous system stimulation. Further studies (DuBois et al., 1953) have demonstrated that an amide linkage in other pyrophosphates as well as in phosphoro- thioates also imparts a selective peripheral action. Table 2 gives some examples of organophosphorus compounds that exert a selective action on the cholin- esterase of peripheral tissues. From the standpoint of mammalian toxicology the advantage of a selec- tive peripheral action is that atropine is much more effective than it is in treating poisoning by com- pounds that also inhibit the cholinesterase activity of the brain. PHOSPHOROTHIOATES Of all the organophosphorus compounds, the phosphorothioates and phosphorodithioates have received by far the greatest amount of attention from toxicologists. The widespread use of these agents and the continued development of new com- pounds in these classes have served to maintain interest in their toxicology. In addition, a number of interesting features of the toxicity of these com- pounds have attracted the attention of many investi- gators interested in the reasons for age and species differences in susceptibility, especially in relation to the metabolism of the compounds. It can probably be safely stated that more is known about the details of the factors governing the toxicity of organo- phosphorus insecticides than is true for any other group of insecticides. However, efforts to utilize this information for the development of new agents have been somewhat limited. It is probable that experience with parathion, the first member of this series to be widely used as an insecticide, served to indicate that organophosphorus compounds with high acute toxicity could be em- ployed without large numbers of accidental poison- ing incidents. On the other hand, the incidence of acute poisoning by parathion and other organo- phosphorus compounds with similar toxicity has been high enough to demonstrate that greater efforts should be made to replace the most toxic compounds of this class. Complete data on the toxicity of many phosphorothioates and phosphorodithioates have been published in many original articles and in reviews. Figures for the acute oral toxicity of a few common compounds of these classes are presented in Table 3. The acute toxicity of the common phosphoro- thioates and phosphorodithioates is well known and only brief comments on certain aspects of this subject need be made. All these compounds show essentially the same pattern of toxicity, although their oxygen analogues show appreciable differences in potency as cholinesterase inhibitors and there are differences in the rate and type of metabolic degradation. The sex difference in susceptibility is largely confined to rats and is the result of a higher rate of metabolism catalysed by oxidative micro- somal enzymes. The toxicity of all agents of this class when administered by the intraperitoneal route is approximately twice their toxicity when adminis- tered orally. However, members of this group show greater variation in toxicity when admin- istered dermally than they do when administered 235 K. P. DUBOIS Table 3 Acute oral toxicity of several organophosphorus insecticides for male and female rats Compound parathion parathion-methyl EPN t demeton azinphos-methyl fenthion malathion LD5o (mg/kg) for: females males 4.0 4.5 7.0 2.5 10.0 310.0 1 000.0 7.0 9.7 32.0 6.0 18.0 190.0 1 375.0 by other routes, and the overall structure of a compound is apparently more important in deter- mining absorption by the dermal route than is the case for other routes. Species differences in the toxicity of organophos- phorus compounds for the common experimental animals are generally small. Age differences in susceptibility have not been routinely investigated. However, Brodeur & DuBois (1963) have com- pared the toxicity of 15 organophosphorus insecti- cides for weanling and adult male rats, and some of the results are given in Table 4. Table 4 Acute toxicity of organophosphorus insecticides for weanling and adult male rats Compound parathion parathion- methyl EPN t demeton carbophenothion ethion azinphos- methyl malathion schradan dioxathion Intraperitoneal LDso (mg/kg) for: weanlings adults ___ 1.5 3.5 8.0 2.5 5.4 100.0 3.4 340.0 49.0 49.0 3.6 5.8 33.0 3.8 9.4 128.0 4.9 750.0 10.0 94.0 Weanling male rats (23 days old) are more suscep- tible to most of the phosphorothioates and phos- phorodithioates than are adult rats, because the detoxification enzymes in the former have not yet developed to the adult level. The degree of age difference varies from one compound to another, and probably depends on the extent to which a given compound is detoxified by means of oxidation processes catalysed by microsomal enzymes. The detoxification of phosphorothioates and phosphoro- dithioates seems to be more dependent upon micro- somal oxidation than that of the phosphates and phosphonates. It was clear from this sttudy that age differences in susceptibility must be considered separ- ately for each organophosphorus compound. It appears that conversion to the oxygen analogues requires only a small amount of the total microsomal oxidase activity in adult liver. Thus, the rate of de- toxification would tend to have a marked influence on the toxicity of the compounds. Further studies should be carried out on young animals of various species. INTERACTIONS WITH OTHER CHEMICALS One important aspect of the toxicity of organo- phosphorus insecticides that has received relatively little attention is the increase or decrease in toxicity that may result from interactions with other chemi- cals or drugs to which the subject may be exposed simultaneously. The use of toxicity tests as a gen- eral procedure for detecting such interactions would not be practicable, owing to the numerous combina- tions of chemicals that would have to be tested. A more practical procedure is to consider the bio- chemical mechanisms that might be responsible for the increases and decreases in the toxicity of organo- phosphorus insecticides resulting from interaction with other chemical agents. Enzyme induction One interaction mechanism of obvious importance for the organophosphorus insecticides is interference by another chemical agent with the activity of the hepatic microsomal enzymes that activate and degrade these insecticides. It would be impractical to test the effects of enzyme-inducing agents in combination with each of the organophosphorus insecticides. For this reason DuBois & Kinoshita (1968) attempted to develop a procedure that could be employed generally to ascertain whether an enzyme-inducing agent affects the toxicity of any 236 TOXICITY OF OP COMPOUNDS FOR MAMMALS 237 Table 5 Influence of phenobarbital treatment on the acute toxicity of organophosphorus insecticides for rats and mice LD5o (mg/kg) for- Compound rats mice control phenobarbital- control phenobarbital-treated treated parathion 2.45 7.3 8.1 14.2 parathion-methyl 7.0 8.0 9.3 14.3 EPN t 7.3 75.0 23.7 64.4 demeton 2.1 16.9 6.7 16.3 carbophenothion 10.1 70.5 27.0 44.0 ethion 25.9 302.6 34.5 40.8 azinphos-methyl 8.7 11.4 4.0 4.9 malathion 619.4 949.9 193.0 234.0 schradan 28.7 14.5 9.6 9.9 dioxathion 17.2 118.7 33.2 87.0 organophosphorus insecticide. The procedure used in these experiments consisted of the daily intraperi- toneal administration of 50 mg of phenobarbital sodium per kg of body weight for 5 days. On the day following the fifth treatment with this broad- spectrum enzyme-inducing agent the acute toxicity of a number oforganophosphorus insecticides was meas- ured, and some of the results are given in Table 5. The results of this study demonstrated that the treatment of rats and mice with phenobarbital for a period long enough to cause marked induction of hepatic microsomal enzymes decreases the acute toxi- city for them of several common organophosphorus insecticides. A marked decrease in the toxicity of some compounds, including disulfoton, dioxathion, ethion, carbophenothion, and EPN t was observed in the phenobarbital-treated animals. Smaller de- creases in the toxicity of some other compounds were noted, but the significant finding was that the induction of hepatic microsomal enzymes did not increase the toxicity of any of the compounds except schradan, which is activated but not detoxified by hepatic microsomal enzymes. Since phenobar- bital treatment decreased the toxicity of most of the compounds it is apparent that the rate of micro- somal detoxification is more responsive to enzyme- inducing agents than is the activation reaction. t Names against which this symbol appears are identified in the Glossary on pages 445-446. The demonstration that exposure to an enzyme- inducing agent does not increase the toxicity of organophosphorus insecticides is of considerable importance with respect to the combined toxicity of such insecticides and other chemicals. Any chemical agent capable of producing enzyme induction would be expected to influence the toxicity of organophos- phorus insecticides in the same way as phenobar- bital, or to a smaller extent if it induced fewer micro- somal enzymes. From the mechanistic point of view, therefore, it is not necessary to test the effects of all potential enzyme-inducing agents to ascertain their possible effects on the toxicity of organo- phosphorus insecticides. Many pesticides do not fall within a chemical class such as the organophosphorus compounds or the chlorinated hydrocarbons, and the possibility of interactions between such agents and other pesti- cides and drugs should also be studied. Measure- ment of the toxicity of these agents in combination with each important drug or pesticide is the most certain method of detecting potentiation or anta- gonism, but practical considerations limit its use. Since the alteration of detoxification enzymes plays a prominent role in causing interactions, our labora- tory is attempting to elucidate the structural features of chemical agents that cause such alterations. We have developed a procedure for detecting the ability of various chemical agents to induce or inhibit K. P. DUBOIS hepatic microsomal detoxification enzymes. An essential requirement for such a procedure is the availability of quantitative assay methods. Previous studies in this laboratory (Kinoshita et al., 1966) have resulted in the development of quantitative pro- cedures for measuring the levels of oxidative micro- somal enzymes. Our procedure for detecting the induction of enzymes by pesticides and other chemi- cals consists of two types of experiment. Initially, the acute toxicity of the pesticide is determined in mice and rats. Groups of 4 or 5 animals of each species are then treated daily for 5 days with 1/5 or 1/10 of the acute LD50, and microsomal enzyme activity is measured on the sixth day. If enzyme induction does not occur under these conditions, further studies are considered to be unnecessary. When enzyme induction is observed, it is considered essential to carry out further studies to determine whether the effect occurs at a practical dose level. With pesticidal chemicals this is done by feeding various dietary levels in the vicinity of the estab- lished or proposed tolerance levels to rats under essentially the same experimental conditions as are used in the conventional subacute and chronic toxicity tests. The procedure described above was applied to a number of pesticidal chemicals, few of which were observed to cause enzyme induction, and it was con- cluded that these agents would not alter the toxicity of other chemicals by changing their rates of meta- bolism. Occasionally, however, new classes of indu- cing agents are found; for example, Kinoshita & DuBois (1970) discovered that the substituted- urea herbicides produce this effect. When informa- tion of this type is available for a number of classes of insecticides, drugs, and other chemicals it should be possible to utilize it to predict whether or not interactions would occur as a result of changes in levels of microsomal enzymes, thus avoiding the necessity for conducting large numbers of toxicity tests. Esterase inhibition Another type of interaction for which a bio- chemical approach is now preferable to toxicity tests is the potentiation of the toxicity of various esters as a result of the inhibition of esterases (car- boxylic ester hydrolases, B-esterases) by organo- phosphorus insecticides. Since Frawley et al. (1957) discovered the ability of EPN t to potentiate the toxicity of malathion, the toxicity of each new anticholinesterase agent under development as an insecticide has been measured in combination with each agent of the same class for which tolerance levels have been established. The increase in the number of anticholinesterase insecticides has resulted in a great increase in the number of toxicity measure- ments that must be made to ascertain whether any combinations cause potentiation. Furthermore, it has become apparent that the simultaneous admin- istration of two compounds may not reveal the cap- ability of one of them to potentiate the toxicity of the other. From the point of view of the mechanism involved, it is more logical to study the combined action of organophosphorus insecticides by measur- ing their potency as inhibitors of esterases than to measure the toxicity of every possible combination of compounds. It has become apparent from bio- chemical studies of the mechanism of potentiation that only one implication of esterase inhibition has been considered during the past 10 years-the pos- sibility that one organophosphorus insecticide will potentiate the toxicity of another by inhibiting its detoxification. However, any compound, such as EPN, that can inhibit esterases would be expected to inhibit not only the detoxification of another organophosphorus insecticide but also the hydro- lytic detoxification of any drug or other type of chemical agent that is normally detoxified by ester- ases. It follows, therefore, that the procedure of testing the toxicity of different combinations of organophosphorus insecticides is too restricted, even if it involves a large number of tests, and to us it seemed more logical to devise quantitative methods of measuring esterase inhibition for determining the ability of these insecticides to potentiate the toxicity of other esters. Recent studies in our laboratory (DuBois et al., 1968) resulted in the development of a procedure for measuring the potency of these insecticides as esterase inhibitors using diethyl succinate and tri- butyrin as substrates for liver and serum esterases. Different levels of a number of organophosphorus insecticides were fed in the diet for several weeks and assays were conducted periodically. It was found that, at a given level, maximum inhibition occurred when the diet was fed for only 1 week. This infor- mation was then applied to a study of 18 organo- phosphorus insecticides, in which different levels were fed in the diet for 1 week and the degree of inhibition of carboxylic ester hydrolase and cholines- terase activity. The dietary levels of each compound that would cause 50% inhibition of the enzyme activity were calculated from the results obtained. 238 TOXICITY OF OP COMPOUNDS FOR MAMMALS Table 6 Dietary levels of organophosphorus insecticides that produce 50 % inhibition of hydrolysis of tributyrin and diethyl succinate by rat liver and serum Compound parathion parathion- methyl EPN t demeton dioxathion ethion coumaphos fenchlorphos Folex t Dietary level (ppm) for 50 % inhibition of hydrolysis of: diethyl succinate liver serum 4.0 8.8 4.5 2.5 4.8 12.0 4.7 21.0 1.7 6.7 > 25.0 7.5 12.3 16.0 108.0 14.5 38.0 2.6 tributyrin liver serum 1.5 2.5 3.9 0.7 2.2 13.0 1.8 8.0 1.6 7.2 > 25.0 8.5 12.0 14.0 > 25.0 9.5 37.0 5.0 Some of the results of these measurements are presented in Tables 6 and 7. The results showed that all the organophosphorus insecticides inhibit the hydrolysis of diethyl succinate and tributyrin by liver and serum. Comparisons indicated that there is no consistent relationship between the potency of the compounds as inhibitors of carboxylic ester hydrolases and their potency as cholinesterase inhibitors. All the insecticides inhi- Table 7 Dietary levels of organophosphorus insecticides that produce 50 % inhibition of cholinesterase activity of the tissues of rats Compound parathion parathion-methyl EPN t demeton dioxathion ethion coumaphos fenchlorphos Folex t Dietary level (ppm) for 50 % inhibition of ChE in: brain liver 12.5 14.5 40.0 5.4 54.0 108.0 80.0 280.0 130.0 17.0 25.0 > 100.0 15.0 48.0 70.0 50.0 90.0 50.0 bited one or both of the carboxylic ester hydrolases at lower dietary levels than were necessary to pro- duce an equivalent degree of cholinesterase inhibi- tion. Differences exceeding 10-fold were observed between the inhibition of cholinesterase and that of carboxylic ester hydrolases produced by several insecticides, including EPN,t dioxathion, ethion, coumaphos, fenchlorphos, and Folex.t If informa- tion on dietary levels that inhibit esterase activity were to be used in establishing tolerances for organo- phosphorus insecticides, adherence to such toler- ances would insure that interactions through this mechanism would not occur. CONCLUSIONS The toxicity of the organophosphorus insecticides has been extensively studied over the past 25 years, but-apart from a few new compounds-the amount of information on the subject has not increased appreciably since it was reviewed by DuBois (1963). The organophosphorus insecticides differ quantita- tively in their toxicity for mammals, but most of the widely used ones are highly toxic. The pharma- cological effects of different compounds are essen- tially the same in that they inhibit cholinesterase of the central and peripheral nervous systems and thus produce generalized cholinergic effects. However, octamethyl pyrophosphoramide and related com- pounds having phosphoramide linkages form an exception: these agents do not inhibit brain cholin- esterase in vivo because they are unable to gain access to brain cholinesterase. Their selective action on peripheral tissues makes atropine a more effective antidote for them than for compounds that affect the central nervous system. This factor, together with the lack of biological activity of their hydrolysis products, may render pyrophosphates worthy of consideration for more extensive development as insecticides. The phosphorothioates and phosphorodithioates are still the most important organophosphorus insecticides. These agents, of which parathion may be considered to be the parent compound, usually have high toxicity for mammals and exert a gener- alized cholinergic action. Only malathion, fenchlor- phos, and a few other compounds have low toxicity for mammals. It seems worth while to make further efforts to develop organophosphorus compounds with low toxicity for mammals, especially if they could be used generally as substitutes for the chlor- inated hydrocarbons for non-agricultural uses. 17 239 240 K. P. DUBOIS REFERENCES Brodeur, J. & DuBois, K. P. (1963) Proc. Soc. exp. Biol. (N.Y.), 114, 509 DuBois, K. P. (1963) In: Handbuch der experimentellen Pharmakologie, Suppi. XV, Cholinesterases and anti- cholinesterase agents, pp. 833-859 DuBois, K. P. & Coon, J. M. (1952) Arch. indust. Hyg., 6,9 DuBois, K. P., Doull, J., Okinaka, A. J. & Coon, J. M., (1953) J. Pharmacol. exp. Ther., 107, 464 DuBois, K. P., Doull, J., Salerno, P. R. & Coon, J. M. (1950) J. Pharmacol. exp. Ther., 95, 79 DuBois, K. P. & Kinoshita, F. K. (1968) Proc. Soc. exp. Biol. (N. Y.), 129, 699 DuBois, K. P., Kinoshita, F. K. & Frawley, J. P. (1968) Toxicol. appl. Pharmacol., 12, 273 Frawley, J. P., Fuyat, H. N., Hagan, E. C., Blake, J. R. & Fitzhugh, 0. G. (1957) J. Pharmacol. exp. Ther., 121, 96 Gardiner, J. E. & Kilby, B. A. (1950) Biochem. J., 46, 78 Kinoshita, F. K. & DuBois, K. P. (1970) Toxicol. appl. Pharmacol., 17, 406 Kinoshita, F. K., Frawley, J. P. & DuBois, K. P. (1966) Toxicol. appl. Pharmacol., 9, 505 DISCUSSION HOLLINGWORTH: Would Dr DuBois comment on the suggestion that we concentrate too much of our atten- tion on the liver as a metabolic tissue in mammals? This organ is highly active in both toxifying and detoxi- fying phosphorothioates, but I recall that some years ago Dr Gage reported that parathion is just as toxic to hepatectomized rats as to those with a liver. We know that many other tissues are also able to activate phos- phorothioates. What is the relative importance of these other tissues compared with that of the liver, how does this change with age and sex, and what is the effect of dosage with inducing agents such as phenobarbital? DuBois: Quantitative measurements of the rates of acti- vation and degradation of phosphorothioates in tissues other than the liver have been attempted. In the rat, the liver is by far the most active organ for both processes, although the contribution of other tissues should be studied further. It is clear from studies that have been carried out on liver with a variety of phosphorothioates that we should not generalize on the basis of work on one compound, such as parathion. To obtain the ans- wers to the questions asked by Dr Hollingworth, we shall have to consider all possible sites of activation and detoxification for every important compound.