After several successful animal experiments with radioactive digitoxin, Geiling and his colleagues were prepared to use the new technology in tests with humans. In the first human experiments, Tox Lab scientists returned to the question of renal excretion. Their original study with dogs demonstrated that the animals excreted up to 46 percent of a single dose of the drug (a glycoside) in the urine. This finding sharply contradicted early experiments using only bioassay techniques without the benefit of radioisotopes. For the human tests, Tox Lab researchers administered radioactive digitoxin to three patients suffering from arteriosclerotic heart disease with congestive failure of varying degrees of severity. The subjects excreted radioactive digitoxin over a span of thirty-one to forty-two days, suggesting the drug’s considerable level of persistence. Nevertheless, the level of digitoxin dropped off quickly during the first day (10 percent). By the end of the third day, each subject had excreted an additional 10 percent of the digitoxin. Between the seventh and eighth days, excretion leveled off. From a comparison between “unchanged” digitoxin and its metabolites, the researchers concluded that the major route of excretion of digitoxin in cardiac patients was through the kidneys.43
In a slightly larger study of eight patients with cardiac failure, Tox Lab researchers measured the disappearance of unchanged digitoxin from the blood and discovered two rate constants. One component showed a half-life of twenty-five to thirty minutes and another had a half-life of forty-eight to fifty-four hours. The first half-life may have represented the rate at which digitoxin in the blood was equilibrating with the various body tissues, and the slower rate could have represented the rate at which “loosely” bound glycoside was being liberated from the body tissues.44
Yet another study explored the metabolic fate of radioactive digitoxin. Using three terminal subjects, Tox Lab researchers administered multiple doses of biosynthetically labeled carbon-14 digitoxin intravenously. Tissue analysis revealed where digitoxin and its metabolites concentrated in the body. Most interestingly, the researchers found that the myocardium did not particularly attract digitoxin, whereas the kidney, gall bladder, jejunum, ileum, and colon all showed the highest concentration of unchanged digitoxin. Metabolites of digitoxin pooled in the gall bladder contents, jejunum contents, and spleen in the highest concentrations. The liver contained the largest amount of both digitoxin and its metabolites, suggesting to scientists that the liver was the major organ involved in the detoxification of the drug and confirming earlier findings that the kidney was the major organ involved in the ultimate removal of digitoxin and its metabolic products.45
Clearly, radioactive digitoxin allowed a new level of sophistication in scientists’ ability to monitor the metabolism of chemicals in living systems, including humans. In time, however, pharmacologists developed new methods to synthesize radioactive drugs, which resulted in much higher concentrations of radioisotopes. Chemically synthesized radioactive drugs facilitated experiments that were much more precise than those conducted by Geiling and Tox Lab researchers.46 Nevertheless, the Tox Lab’s research demonstrated the importance of radioactive technologies after World War II. Moreover, the spirit of the research, tracing minute doses within systems, became very important conceptually as toxicologists evaluated the risks posed by increasingly toxic insecticides at ever diminishing exposures.
In a related project after the Air Force took over the Tox Lab contract in 1951, John Doull along with Vivian Plzak and Mildred Root established a screening program for radio protective elements. For each chemical analyzed at the Tox Lab, the scientists determined the LD50 in male mice. This critical piece of information represented the only toxicological information for many of the agents analyzed. The resulting database of LD50s was more valuable than the few radio protective elements identified.47
Under the parochial leadership of E. M. K. Geiling, the Toxicity Laboratory at the University of Chicago emerged as one of the leading centers for research in toxicology. Over the course of World War II, up to sixty scientists became employed in its toxicological research. Many more studied for graduate degrees and advanced training in the evolving discipline. The search for antimalarial drug therapies, the study of nitrogen mustards, research using radioisotopes in plants, and the search for radio protective elements yielded valuable data and new approaches and methodologies in the study of toxicology. Specifically, the antimalarial studies added to an understanding of the important toxicological concepts of persistence and synergism in chemicals as well as the critical theoretical issues of joint toxicity and drug/chemical resistance. This research transcended antimalarial drugs and also found application in pesticides. In the process of preliminary screening during the war, Tox Lab scientists dismissed nitrogen mustards as “nontoxic,” but their effect on white blood cells suggested promise as chemotherapeutic agents. In addition, radioisotopes facilitated tracking minute, subtherapeutic doses of a drug. In short, Geiling and the growing group of scientists at the Tox Lab explored a broad range of studies with implications for pharmacology and, more important, for toxicology as a distinct discipline. Moreover, such research clarified toxicological methodologies as well as the toxicity of numerous chemical agents.
John Doull inoculating a hibernating gopher with tagged digitoxin. Courtesy of the University of Chicago Photographic Archive apf 1–05858. Special Collections Research Center, University of Chicago Library.
CHAPTER 4
The Toxicity of Organophosphate Chemicals
In the preceding chapters we have followed several episodes in the development of a notion of environmental risk. Along with other early cases, the Elixir Sulfanilamide tragedy refined scientific methodology with an analytical technique for deriving LD50s and prompted passage of the Food, Drug, and Cosmetics Act of 1938. With the advent of World War II there was renewed interest in insecticides that could control the spread of malaria and other insect-borne diseases. DDT was the most promising of these, and its potential effects on target organisms, lab animals, wildlife, and humans underwent extensive analysis. Much of the interest in DDT was concentrated in governmental organizations—the PHS, the FWS, the USDA, and the U.S. armed forces. Such scrutiny demonstrated that DDT had opened a new era in insect control and toxicology. No other insecticide killed such a broad spectrum of insects without damaging the crops it was protecting. No other insecticide inspired such extensive investigation. For DDT, scientists extended the scope of toxicology to include effects on wildlife populations.
The wartime pursuit of an effective insecticide against malaria-carrying mosquitoes was just part of the fight against malaria (one of DDT’s important uses). Scientists in the Toxicity Laboratory at the University of Chicago also sought antimalarial drug therapies and contributed new techniques to measure the joint toxicity of drugs and drug resistance. In addition to antimalarial drug therapies, Tox Lab scientists developed methods to trace minute quantities of drugs like bufagin and digitoxin by rendering them radioactive. Equally important, the Tox Lab laid the foundation for an independent discipline of toxicology by training graduate students and supporting research. Through his paternalistic direction, E. M. K. Geiling inspired these and other developments.
Despite the extensive publicity focused on it, DDT was only one of many insecticides that scientists developed during World War II.1 Governmental organizations exhaustively tested DDT, but the task of evaluating other pesticides fell mainly to a young scientist named Kenneth DuBois (1917–73) who was working in the Tox Lab. Like many other scientists, DuBois joined the war effort shortly after completing his Ph.D. in physiology and biochemistry at the University of Wisconsin. At the Tox Lab, DuBois’s research revealed several strong commitments. First, DuBois pursued the biochemical aspects of toxicology and stressed the importance of in vivo confirmation of the effects of toxic agents observed in in vitro enzyme studies. Second, he endeavored to develop methods to measure these effects quantitatively.2
Like DDT and other chlorinated hydrocarbons, the organic phosphate
insecticides (later, “organophosphates” or “OPs”) were first examined by German chemists as potential nerve gases to be used in combat.3 Organic phosphate compounds link many phosphorous atoms to oxygen atoms (termed esters of polyphosphoric acids). Among the compounds investigated was diisopropyl fluorophosphate (DFP), which contained only one phosphorous atom. The Germans eventually discarded DFP as a nerve gas, but their experiments indicated that it inhibited cholinesterase, a critically important enzyme needed for the proper functioning of the nervous systems of humans, other vertebrates, and insects. It was the quality of cholinesterase inhibition that convinced physicians to use DFP to treat glaucoma, by reducing the abnormally high tension of the eyeball, and also myasthenia gravis, an autoimmune neuromuscular disease, for which it was more effective than treatment with eserine.4 The first organic phosphate, HETP (hexaethyl tetra phosphate), emerged from Gerhard Schrader’s laboratory at Farbenfabriken, Germany, in the early 1940s. Schrader discovered the insecticidal properties of the organic phosphates during the war, and the chemicals reached America in 1945, when the British and American Technical Intelligence Committee interrogated German chemists in the immediate aftermath of the war. One of the first groups to gain access to the organic phosphates was the Tox Lab, where DuBois and his associates recognized cholinergic symptoms (i.e., changes in the action of the neurotransmitter acetylcholine) produced by the new chemicals and found that atropine would be an effective antidote.5
Kenneth Dubois, studying the effect of radioactive digitoxin on beating mammalian heart. Courtesy of the University of Chicago Photographic Archive apf 1–05876, Special Collections Research Center, University of Chicago Library.
In the U.S., there was considerable interest in the in the new insecticides because the organic phosphates could allegedly control aphids, against which DDT was proving ineffective. The Tox Lab assumed the responsibility for testing the toxicity of the new chemicals largely because the University of Chicago was near one of the major labs, Chemagro, where organic phosphates were synthesized. HETP (C12H30P4O13) was one of the organophosphorous chemicals that German chemists had developed, and under interrogation they compared this new compound to nicotine for its action in destroying aphids. Tox lab researchers could not locate any references to HETP’s mechanism of action other than these possible nicotinic effects. Several researchers at the lab noted during routine testing, however, that animals showed symptoms similar to those produced by DFP. Such symptoms included muscular twitching, tonic and tonic-clonic convulsions, involuntary defecation, micturition (urination), and salivation. The case for parallel actions of the two chemicals was reinforced when researchers produced miosis by placing a dilute solution of HETP in the eyes of rabbits. In the case of HETP, the dilation effect lasted for five to twelve hours compared to three days in response to DFP. The similarities in the action of the two chemicals prompted DuBois to consider their possible effects on cholinesterase.
Through a series of in vitro and in vivo tests, DuBois and George Mangun (also a researcher at the Tox Lab and its director from 1946 to 1953) investigated the effect of HETP on cholinesterase. In the in vitro experiments, they measured HETP’s effect on the cholinesterase of rat and cockroach tissue by adding solutions of the inhibitor dissolved in the buffer to the test system, which facilitated manometric measurement of the cholinesterase activity. The final concentration of 1 × 10−7 M HETP inhibited cholinesterase by 47 percent in the brain tissue of rats, by 53 percent in the submaxillary, 60 percent in serum, 45 percent in erythrocytes, and 58 percent in cockroach tissue. In comparisons of HETP with two recognized cholinesterase inhibitors (DFP and carbamic acid ester), DuBois and Mangun found HETP to be the most effective. For the in vivo experiments, the researchers administered HETP intraperitoneally (directly into the body peritoneum or body cavity) to rats and then measured the cholinesterase activity of the brain, submaxillary glands, and serum with the manometric test system. The results of the in vivo experiments verified the in vitro experiments, revealing cholinesterase inhibition in all of the tissues. Thus DuBois and Mangun concluded: “Hexaethyl tetraphosphate exerts a strong inhibitory effect on mammalian and insect cholinesterase in vitro and in vivo. This finding, in conjunction with its gross effects on animals, suggests that its physiological effects may be at least in part due to its inhibition of this enzyme.”6 This research connected the new insecticides with cholinesterase inhibition. The comparison with carbamic ester anticipated by nearly a decade the development of carbamate insecticides (see below).
DuBois and other researchers at the Tox Lab examined the toxicity of other organic phosphate insecticides as well. For much of this research, DuBois was joined by John Doull. For his doctoral dissertation, Doull used radioisotopes to evaluate cardiotoxic and other effects of bufagin. He received his Ph.D. in 1950 and his M.D. in 1953. Later Doull recalled that he had initially contributed to the analysis of the toxicity of organic phosphate chemicals.7
One of the most important chemicals investigated at the Tox Lab was a new insecticide called parathion. Parathion appeared to be particularly effective against plant insects, and its potential use stimulated researchers to examine its toxicity and pharmacologic action in mammals. Their approach to the analysis of parathion shared many similarities with the toxicological analyses of DDT. Along with Paul R. Salerno and Julius M. Coon, DuBois and Doull evaluated the acute and subacute toxicity of parathion as well as its inhibitory action on cholinesterase. They determined that LD50s were low (less than 20 mg/kg) in all species (rats, mice, cats, and dogs), whether parathion was administered intraperitoneally or orally. Recall that a low LD50 corresponds to a very high toxicity. When sublethal doses of parathion were administered daily, its toxic action was cumulative. DuBois and his team also noted that the symptoms produced by parathion were similar in all species tested. These symptoms were typical of parasympathomimetic drugs (i.e., cholinergic drugs or those that mimic acetylcholine and inhibit cholinesterase). Like HETP, the Tox Lab researchers showed parathion to be a strong inhibitor of cholinesterase. In vitro a final concentration of 1.2 × 10−6 M inhibited by 50 percent rat brain cholinesterase. Finally, they explored possible antidotes to the lethal effects of the drug.8 A picture of consistency within the class of organic phosphate insecticides gradually emerged from toxicological assessments like this one.
DuBois and his team also conducted the first toxicological evaluation of OMPA or Pestox III (Octamethyl Pyrophosphoramide). Like other organic phosphates, OMPA was first synthesized by Schrader, who demonstrated its insecticidal properties. He also noted that plants absorbed OMPA from the soil, which rendered them insecticidal. In fact, one group of researchers showed that OMPA had limited value as a contact insecticide, though plants grown in soil containing the chemical became highly toxic to insects for several weeks. This same group of researchers claimed that OMPA was toxic to mammals when mixed with food and administered orally.9 On the whole, DuBois and his colleagues found the toxicity of OMPA similar to that for parathion. LD50 values for several species were virtually identical to those for parathion. All of the species exhibited symptoms typical of parasympathomimetic drugs except symptoms linked to the stimulation of the central nervous system. In regards to OMPA’s potential as a systemic insecticide, DuBois’s group noted that plants grown in soil containing OMPA contained an anticholinesterase agent. This finding indicated that plants converted OMPA much like the mammalian liver.10
In the Tox Lab, DuBois mainly studied the toxicity of organophosphates to animals, while other labs assessed the risks posed to humans from information gained through occupational accidents. David Grob and other researchers at the Johns Hopkins University School of Medicine reviewed the toxic effects of parathion in thirty-two men and eight women following accidental exposure. The research at Johns Hopkins, like that at the Tox Lab, was supported by the Medical Division of the Chemical Corps of the U.S. Army. Grob and his colleagues identified a disturbing characteristic of parathion: it could be readily absor
bed through the skin, respiratory tract, conjunctivae, gastrointestinal tract, or following injection, most likely due to its high solubility in lipids (fats). Moreover, parathion did not produce inflammation in the skin so absorption could remain undetected.11 That parathion could be absorbed through the skin contrasted with DDT, which had a low rate of dermal absorption, a real advantage in the eyes of economic entomologists. This basic difference between the two chemicals accounted for parathion’s much higher level of toxicity. More troubling than dermal absorption was the absence of an inflammatory reaction, which suggested that an individual could suffer a toxic exposure without being aware of it. Still, the Johns Hopkins researchers were able to determine several general warning symptoms—intermittent nausea, vomiting, giddiness, weakness, drowsiness, and fasciculations (muscle twitches) of the eyelids—which appeared for one to seven days before the more severe manifestations developed.
Grob and his colleagues also addressed symptoms that were particular to organic phosphate chemicals and specifically to parathion. They classified the symptoms with respect to two classes of action on the sympathetic nervous system: muscarine-like symptoms (anorexia and nausea, vomiting, abdominal cramps, excessive sweating, and salivation) and nicotine-like symptoms (nausea and vomiting, muscular fasciculations or twitches in the eyelids and tongue, followed by fasciculations in the muscles of the face and neck, in the extra-ocular muscles, and finally generalized fasciculations and weakness). Although categorizing symptoms may seem esoteric, the symptoms suggested that the organophosphates affected both the muscarinic receptors and the nicotinic receptors in the nervous system. The term “muscarine” tied the organophosphates to the long history of poisons. First isolated from a mushroom, Amanita muscaria, in 1869, muscarine was the first parasympathomimetic substance ever studied; it causes profound activation of the peripheral parasympathetic nervous system that may end in convulsions and death. More troubling still, the subjects developed all these symptoms and some of them died, despite the fact that most of them had worn carbon filter respirators, rubber gauntlets, and protective coveralls during their exposure to parathion. Some of the subjects had even worn hip-length rubber boots and rubber aprons. The Johns Hopkins researchers suspected that breaks in these safety procedures had occurred. Moreover, they discovered that the face piece respirators did not fully protect the workers from inhaling organophosphates as aerosols, dusts, or sprays.
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