Q 184 anng 12/99: transcription of typed text dated 4/17/81 The following are some draft notes that I gave at CIIT on March 10th and at Queens College on April 16th on the theme of "Contribution of Toxicological Study to Basic Science". This is of course modeled on Beecher’s 1960 complilation. Reference to my own experiences with toxicology starting with colchicine. Looking up the questions of colchicine resistance in a hamster and in colchicum for comparative toxicology got me thinking more seriously about this. Attached are some notes on my own experiences. The preliminaries had to do with the public policy implications of our ignorance about standards, for example whether one PPB of chloroform was really worth spending very much effort to try to avoid. Recollection of Avogadro’s number and (where did I first hear this?) we must be inhaling at least one molecule of Julius Caesar’s dying breath "Et Tu Brute" with every inhalation. (That was years ago and it may go back to Jaffe’s "Crucibles", probably not in a pollution context.) The academic environment for toxicology. I was asked later at Queens whether there were any examples of undergraduate toxicology programs in letters at science and could hardly answer but certainly some of the environmentally oriented schools must do that. That may be a confusion of environment with toxicology I don’t know whether appropriate for chemistry department. Whitaker College and the Human Biology Program at Stanford should be examples. I would be surprised if there were not some similar things at, say, Davis and perhaps at Santa Cruz. Biographically recall also my letter to Fieser about 1939 or 1940 asking about Kellaway and the chemistry of carcinogenic hydrocarbons. Some deep questions that toxicology still poses: like Mithridates as purported adaptation to arsenic. Recall Roger Stanier in re: Mithridates. Could teach most of biochemistry as an interpretation of toxic actions and this may not be a bad undergraduate approach. Interpretation of "arsenic and old lace": arsenic stands both for the image of toxicities since Roman times (homicide and other pharmacological purposes’ as cited in Schumberg) and also very specific toxicology. Old lace is not the topology of knots or the enzymology of Retting of linen but just an attribution to traditions that we might wish to overcome. Natural toxins have evolved special selectivity that make them more and more interesting for the dissection of very complex processes. Anticipating later wrapup keep in mind that inhibitors were the main tools for the interruption of pathways and the accumulation of intermediates prior to the development of radioisotope tracer methodology, chromatographic sensitive methods of analysis, and genetic tools for interruption as well as the far more sophisticated apparatus we have today for the actual isolation of specific enzymes in its intermediary metabolism. Slide showing the use of toxins in the dissection of intermediary metabolism. DNP was discovered for producing fever in dogs in 1885. Malonate was probably first looked at as a homolog of oxalate proving to be not toxic but Thunberg in 1909 had found that both oxalate and malonate do inhibit muscle respiration and succinate stimulates it. This may have been forgotten. This is all quoted in Webb, Vol. 2, 1966. Emphasize the centrality of the models of competitive inhibition and how they fit into the Paul Ehrlich and Karl Landsteiner tradition of understanding of steric relationships in biology. Quastel 1927 and 1930 elaborated this into a formal theory which has been the basis of almost all rational drug designs ever since. I still have only scattered notes on the history of discovery of various competitive inhibitors but Webb is probably the best back source for that. Arsenic is quoted out of Metzler, and Schumberg and Spencer. The explanations are not very convincing. Why not ketoglutarate instead of pyruvate dehydrogenase as the target for a lipoic acid block? Neurotoxins the examples out of the White and Handler book. DFP was picked up in 1932. Schrader had already noted that miticides which were acid fluorides and the idea was to produce analogs of these in the fluorophosphates. 1940 the antagonism of PAB and sulfanilamide was a new chapter in biochemistry and launched antimetabolite concepts for chemotherapy. How explain the differential toxicity of sulfanilamide: it inhibits the endogenous synthesis of folic acid. Animal cells which acquire a folic acid from the environment must have more potent means of doing this from very low concentrations but I do not know whether this has really been worked out. A long list of antibiotics involved in RNA and DNA metabolism from Stryer and from Kornberg. Go into the history of cholera and cholera toxin. The wrongheaded hypothesis started from Koch and the numerous elaborations both practically and as a research tool. Colchicine E. W. Taylor in 1967. Stryer also comments on cytochalasin which binds the end of actin chains and phalloidin which prevents the depolimerization of actin, tubulin? Table of miscellaneous cases which have given both extraordinary new insights and many practical uses. The specificity of carcinogenicity. Fluorouracil almost unique in an effective rationally designed product. P-450: need to go into the initial history of its discovery. What was the first substrate of it? The need to use more rational approaches in carcinogenesis assay: since we know that initiation and promotion are at least two distinct steps so we should do tests for initiation on fully promoted cells and vice versa instead of relying on our present crude methods that look for complete carcinogens. A note on evolving methodology out of my Recordex. Toxicity study tends to be compartmentalized. Many observations of pharmacological effect are thrown away if found in the toxicology rather than the pharmacology laboratory and then some more notes on dubious success of antimetabolite design. Evolving method - Natural product isolation and identification -- leads to +++ chromatography, mass spectrometry and NMR. in vitro reactions inhibitors to isolate reaction steps [enzyme isolation] +++ Tracer methods Comparative biochemistry Genetic interruption Toxics: more complex systems -- discovering receptors integrating: for example carcinogenesis and therapeutics From Potts to DNA activation - mutation vs. cancer critical DNA not yet sorted out ... as skin irritant? not sorted out on account of binary system (check Berenblum) Futility of testing without using theoretical base Cell toxicity Promotion Accessible DNA (specific genes) Ubiquity of SARC Hormone dependence progression Immunology and Macrophage activation factors “Initiators" must be tested on fully "promoted" animals/cells Toxicity compartmentalized. Today’s toxins are tomorrow’s medicines. But today’s toxics are viewed as substantive and regulatory threats. That is business risks much more than {technological opportunities}. In many case, outside CIIT, toxicity is not pursued re: mechanism. Most data not published. Dubious success of anti-metabolite design. Discovery vs. invention. Folding enigma. Inhibition as natural regulator. < {interest in mechanism of toxicity competition of other methods of pathway analysis - pragmatics of perseverance} “more complex systems - organogenesis, cancer, specificity moving toward sequences Notes dated April 16, 1981 User: jlab Host: j 1 notes Class: jl-pce2 _ Job: OBR Chistory with toxic agents Colchicine and toxicology of mitosis (and embryology) 1941-2 Clear chromosomes!! << Tjio and Levan (I didn’t know 2n=48 was problematical) Later Taylor --> --> tubulin Penicillin - as selective agent (--> Japanese industry!) - mode of action Drug resistance markers in genetics Mutagens ~ 1951 periodate S’R as suppressor (ribosome action) toxic glucosides: chloroacetate Arsenic - mucous membrane irritation: keratosis 1. Peripheral neuropathy of arsenic: no good animal models mimics beri-beri thiamin pyrophosphate is co factor for pyruvate dehydrogenase lipoic acid Why vulnerability of peripheral nerves? Guess at transport; binding; competition of other - SH ligands. 2. Arsenic as carcinogen (skin, lungs) unique to human? Promoter via keratosis 3. Differential toxicity of organic arsenicals passage blood brain barrier Arsenic/old lace a conversation intimate personal account Many discussions about toxicology Why my personal resonance with it? colchicine 1941 - not quite prehistoric later... uses of toxins Notes dated March 1, 1981 Woolley - a study of anti-metabolites Michaelis 1910-1914 product inhibition Quastel & Wooldrige 1927 malonic/succinic dehydrogenase enzyme... how discovered Landsteiner - hapten (inhibitor) 1938 Dyer ethionine as anti methionine 1932-7 acetylcholine vs atropine quarternary-ammonium AJ Clark vs antagonists and pharmacology 1938 B-acetylpyridine, pyridine sulfonic ac. toxic in nic-def dogs 1940 Woods pab vs SA predicted pab as vitamin, folic acid Fildes: design anti-metabolites By 1947 most vitamins had no antagonists but need selectivity for chemoth. action Lethal synthesis. Fluroacetate yields fluorcitric errors - e.g. Vit K/bacteria ? specific adsorb refs? ? recent RR’s 111 paradoxes stimulation mutual inhibition 116 effects as crystal habit errors phenylalanine vs chloramphenicol methionine sulfoximine from bleached flour Toxicity --> practical uses curare--> neurotransmitter In most cases enlarged our pharmacological and biochemical knowledge opioids Carcinogenesis: mechanism? ion antagonisms K vs Na vs Ca Heavy metal mechanisms Spoiled sweet clover 3, 3 prime methylene bis (4 HO-courmarin) --> blindness Link: antagonized by vitmain K --> dicumarol 1943 pyrithiamine --> B, def. in mice 1980 today, competition complicated permeation synthesis activity incorporation into lethal product Multiple functions of a metabolite - glutamine anti-metabolite - FU Accidental confusions e.g. thiouracil => thyroid not Uracil antagonist Competition non competitive: e.g. independent addition colchicine - EW Taylor 1967 cytochalasin - binds ends of actin chain phalloidin - prevents depolymerization no clear rationale for mutagenesis vs. cancer ?? base analogues relatively non toxic; non carcinogenic No tests on fully promoted cells! Cholera toxin - wrongheadiness general implications for toxicology Antibacterials 1940 PAB vs sulfa new chapter in biochemistry launched anti metabolite concepts Antibiotics - *RNA, DNA feedback controls Neurotoxins DFP 1932 Schrader miticides from sulfonyl fluorides Opioids Competitive inhibitors Arsenic DNP 1885 fever in dogs Lardy and Elvejhem Green 1945-49 oligomycin inhibits ATP formation/does not hydrolyze ATP... Quastel 1963 - mechanism? Intermediary metabolism HCN CO DNP malonate fluoride Iodoacetate arsenate phlorizin alloxan Malonate - most studied re: other biological effects Hazards of interpretation of loci of action! accumulation of succinate relief by fumarate ~ specific symptoms chelaters. calcium teratogens? Sprott on CNS differentiation occurs in green plants. Malonate Webb 2 1966 1889 Heymans - frog oxalate toxic malonate not. Quastel tested series. 1896 Pohl m. enhances oxalate excretion 1909 Thunberg m. inhibits muscle respirations succinate stimulates muscle respiration forgotten 1925 Quastel Cook 1930 competitive with succinate prior notes Tox Bio Lecture 4/81 Mercury, known since antiquity (mad hatters) 1900-1940’s antisepsis Koch 1881, about 1930 an SH reagent Arsenate - iodoacetate and iodine Heinz - iodobenzoate from iodine 1899 1874 - bromoacetate kills frogs 1930 - Iodoacetate better Lundsgaard - muscle rigor: lactic acid depletion 10 Fluoride blocks enolase -magnesium ++ dependent Iodoacetate :=> reduction of pyruvate 3 - phosphoglyceraldehyde dehydronase 1933 - problems of selectivity Embden and Myerhof 1948 Cori Webb 3 page 4 - Fluoride, iodoacetate perhaps the most important tools to work out glycolytic pathway Could teach most of biochemistry as interpretation of toxic actions and physiology and pathology as well. Natural toxins have evolved specific selectivity. Not a toxicologist - perhaps that why I am much more interested. ? credentials 40 years ago as a college freshman, I was injecting colchicine into mice trying to understand spindle disruption, organ-specific effects, chain of consequence of chromosome aberration in spermatogenesis. And also use respiratory poisons - the idea of physiology of mitosis in the root tip. Genetics instead. My letter to Fieser ~ 1940. 25 years ago - mode of action of penicillin toxicology poses deep questions Mithridates Repair Specificity and threshold Message to promulgate to academic as well as to industrial research community - contributions of toxicology to basic science - Beecher 1960 as prototype cf Irving Page, I think that’s NEJM (New England Jrnl of Medicine).