'- road-‘ --——-—-— —— -—— ’1 -—-¢'~-‘ —.-.\‘.\-_,...-¢, n..---._-ch--.- -7 .—~‘4...--<_ .--.--c.-4.~“~-’.--~-o--- <- . -g-.-.-_----“-_.-- ‘ ‘-4 I. '5 ~ . .. misousm 9:: 40:215. .1 . _ [2- 4,5-mmm-1,3-Dzoxo- : Lm-z-YL) 1-PHENYL * METHYLCARBAMATE. 3v RATS. HOUSE mas AND BEAN PLAN 3 ff _ Thesis for the Degree of M. S. met-mm STATE umvmsm CHACHAWAL WONGPHYAT . ' 1971 - .r ’ . ’ . . " l .— on ’ . ‘ ‘ V . . o , . . . . ‘ . .1 " n . 0 ~ . . I " ' ' - .I' - v ' . .- .v. . -/ _ ' r . . , . , o-x ‘ ' " u . v' . ‘ an ' ‘ '. . ' . -- ’ . 1 V l 9 A" . 'b . . . , . . A . . ‘ . - ‘ , . O, . ‘I . —'.‘ . 0 n' . , _ 'I .a . .1 > . .: - . ,. - ., 9 ‘ » ‘ ‘ _ . ‘ . . ' ‘~ . o ' . . .0 4 I a ' -l p; I . I '. .l'. ‘ , . ,. ‘ _ . . . . . , .,. . a Q ~ ~ ‘ ' . ‘ I , . . I . '0 . ‘ .n. ‘ ' . , u -. . 0 '3. u . . . .n .. ’ I - . . . . . .‘ . ...-o o- ‘| . , . -... . .. - ‘ . 0. . .. ‘ ' , I l ‘ '1 ~05. .y- . . . .. . . n . -- ,._ .. . o' .9 5- - - '.. . _ u 0.. ~ - ‘ ’ , , .. . .. c 1 .. , . . . . .. . ,. . . - . I ’...'-’-v"’-'-v.ov'- .. .~~.Mv—~.Mo-od‘. \ 1 ‘ o . - o . r . . . . .. . . c . . - _,. - o ’-.- ' ' . . -’ . . . V . .- . ~ , .~ 4 — . , - -_, , . r v ' ‘ ’ . _, ..'- .. .. _ ~.. ‘ . . . _ ,. . . ‘ o .. . . '- 0 I. . , ._ . . a- — , - _—‘ r v— .' .. . A . . , ' ' - ' ‘ s . , . - V . . ,.—. . . ' » . ' .-“’ ‘ l o- r ‘ . . ‘ r , . ~o r ‘ 'ar .- -. -o'o- - - ' - ~ ‘ u - .~ .. — .‘u. u .o- o ' OI ' l ’ v n r. ' 5-! 0' - '. ' <. o..' . .< ’ v ~- .v . a. _ .F- - a. ' a "I’-‘ . - — ' ‘ " —’ . ,... ¢ - '., , - .7 co *r-s ' .,. no. '. v’ .o. .... o . - -.'r‘ ’1 ...f"‘ U U a . .1 '1. I . .,;af . . l_ . ..|... . ,. .;-'-. - 4’0. . — ~.- -. - r > ‘ . . - ar-n - «v ‘ to o ,",.' - r - - c , I '0' c . a. ‘.- v- - — 0- -u. g-a a - . -¢’-- ' ... .—. o" c-, -.o-'a-O-- ., -- o o-- - 0.0‘ o 4-. O'- ‘y‘l" ‘1' -... -~.¢ V'. ,.. -'. .._ - .- -. . . -- -..~‘I . . -.lr. 'o -- -uou '.--rv ” '4' ~ . - , .— --... ..v .H-1 0 - --c... v —> '0' ~ ,,. ..-.a.,, r- - '0‘ rQ-od ‘--nl‘--t—----__-‘---.n- .-..- .n‘A m 3 3‘3““ A4 g‘r LIBRARY Michigan 5mm University I I. I 3.: t ‘ I amomc av : SflNS' I BMW M i! LIBRARY EH“ 5 puma; F! ABSTRACT METABOLISM OF C-lOOlS, [2-(4,5-DIMETHYL-l,3-DIOXO- LANE-2-YL)]-PHENYL METHYLCARBAMATE, BY RATS, HOUSE FLIES AND BEAN PLANTS BY Chachawal Wongphyat Metabolism of C-lOOlS [2-(4,5-dimethyl—l,3-dioxo- land-Z-ylfl-phenyl methylcarbamate by rat liver microsomes fortified with reduced nicotinamide-adenine dinucleotide phosphate and by house flies and bean plants was examined. C-lOOlS was metabolized i2.!l££2 to yield at least three non-hydrolytic products. Two of these metabolites were tentatively prOposed to be formed by enzymatic cleavage of the dioxolane ring to aldehyde and/or hydroxylation. It was apparent that decarbamylation was an important route for in vivo metabolism of C-lOOlS in house flies and bean plants. METABOLISM OF C-lOOlS,[2-(4,5-DIMETHYL-l,3-DIOXO- LANE-2-YL)]-PHENYL METHYLCARBAMATE, BY RATS, HOUSE FLIES AND BEAN PLANTS BY Chachawal Wongphyat A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1971 ACKNOWLEDGMENTS I wish to express my since gratitude to Dr. Norman C. Leeling, my professor, for his tireless assistance ren- dered during this research. I also wish to express my sincere thanks to Dr. Matthew Zabik for assisting me in determing the structures of metabolites from mass-spectrometry. To Dr. Ronald E. Monroe goes my appreciation for making available to me the tissue combustion equipment used for part of my research. Furthermore, I wish to convey my sincere thanks and appreciation to Dr. W. B. Drew, Dr. Jeffrey Granett, Mrs. Sandy Granett and Mrs. Karen Christlieb, who have in one way or another helped me and made my graduate study at Michigan State University a pleasant one. I wish to acknowledge the partial financial support extended me through an assistantship from the Department of Entomology, Michigan State University. My mother receives my deepest gratitude for her support and enthusiastic encouragement of my studies at Michigan State University. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INT RODUCT ION O O O O O O O O O O I O O O O O O O O I. II. III. Carbamate Insecticides And Their Mode Of Action. 0 O O O O O O O O O O O O O O O O A. B. Carbamates As Insecticides. . . . . . . Correlation of Structure and Activity . C. Mode of Action of Carbamate Insecticides. Methylcarbamate Insecticide Metabolism . . . A. B. C. Metabolism In Mammals . . . . . . . . . Metabolism In Plants. . . . . . . . . . Metabolism In Insects . . . . . . . . . Biological Oxidation As A Detoxification MeChaniSm. O O O O O O O O O O O O O I O O O A. B. C. D. Oxidation And Hydroxylation Of Insecti— cides . . . . . . . . . . . . . . . . . General Nomenclature And Distribution Of Oxygenases . . . . . . . . . . . . . Role of Oxygenases in Biological Oxidation . . . . . . . . . . . . . . . Hydroxylation Reactions . . . . . . . . l. Mechansims of Single Hydroxylation 2. Mechansim of Double Hydroxylation. Ring Hydroxylation . . . . . . . . Oxidative Demethylation. . . . . . N-Hydroxylation. . . . . . . . . . U'Ibw o o 0 Major Factors Controlling Oxygenase And Oxidase Activities. . . . . . . . . . . iii Page vi \qu U1 WNH I—‘ 11 12 13 13 14 15 17 18 Page MATERIAI‘S AND METHODS O O O O 0 0 O O O O O O Q 0 O O O 2 0 Synthesis of C-lOOlS-Carbonyl-14C, 2- (4,5- dimethyl-l,3-dioxolane- 2-yl) -phenyl methylcarbamate-carbonyl- . . . . . . . . 20 Preparation of Liver Fraction And Enzyme Incubation of c-10015 Carbonyl-14C . . . . . . . . . 21 Analysis of Metabolites. . . . . . . . . . . . . . . 22 Characterization of C-lOOlS Metabolites From The Rat Liver Microsome System. . . . . . . . . 23 Metabolism of C-10015 Carbonyl- -14C in Bean Plants. . . . . . . . . . . . . . . . . . . . 24 House Fly In Vivo And In Vitro Metabolism of 14C- 10015- -carbonly-IZC . . . . . . . . . . . . . 25 Procedures For In Vivo House Fly Metabolism Studies . . . . . . . . . . . . . . . . . 26 Procedure For In Vitro House Fly Metabolism Studies . . . . . . . . . . . . . . . . . 28 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 30 Metabolism of C-lOOlS by Rat Liver Microsomes. . . . 30 Chemical Nature Of The Carbamate Metabolites of C-IOOlS From Liver Microsomes . . . . . . . . . . 37 Metabolism of C-10015 carbonyl-14C in Bean Plants. . 40 Metabolism of C-lOOlS by the NADPHz-dependent Enzyme System from the House Fly . . . . . . . . . . 41 Identification of Organosoluble Metabolites Produced by the NADPHZ-dependent Enzyme System from House Flies. . . . . . . . . . . . . . . 43 In vivo Metabolism of C-lOOlS by House Flies . . . . 43 SUMRY O O O O O O O O O 0 O O O O O O O O O O O O O O 4 5 LIST OF REFERENCES . . . . . . . . . . . . . . . . . . 47 iv Table 1. LIST OF TABLES Page Metabolism Of C-lOOlS Carbonyl-14C by Rat Liver Microsomes. . . . . . . . . . . . . . 31 Fractions Recovered From Growing Bean Plants Injected With C-lOOlS-carbohyl—14C at Intervals After Treatment. . . . . . . . . . 42 Fractions Recovered From House Flies After Topical Application Of 0.2 - 0.3 ug of 14c-carbony1 labeled c-10015. . . . . . . . . . 44 Figure 1. LIST OF FIGURES Page Autoradiogram of one-dimensional TLC of C-lOOlS-carbonyl-14C metabolism by rat-liver microsomes. Development of TLC was with chloroform:acetonitrile, 2:1. . . . . . . . . . . . . . . . . . . . . . . 34 Autoradiogram of two-dimensional TLC of c-10015 carbonyl-14c metabolism by house fly microsomes. Development of TLC was with ether:hexane:ethanol, 77:20:3 and then chloroform:acetonitri1e, 2:1. . . . . . . . . . . . . . . . . . . . . . . 36 Autoradiogram of two-dimensional TLC of C-lOOlS-carbony1-14C metabolism by rat-liver microsomes. DevelOpment of TLC was with ether:hexane:ethanol, 77:20:3 and then chloroformzacetonitrile, 2:1. 0 O O O O O O O O O O O O O I O O O O O O O 36 The mass spectra of C-lOOlS and Metabolites A and B. O O O O O O O O C O O O O O O O O O O O 39 vi INTRODUCTION I. Carbamate Insecticides And Their Mode Of Action Carbamate insecticides have been widely used within the last decade, and have prompted numerous studies on their metabolic fate and their mode of action. Carbamate insecti- cides and many other organic insecticide chemicals are apolar and have adequate lipoid solubility to penetrate the lipoid nerve sheath and approach the site of action (Casida, 1969). Certain carbamates have a structural configuration resem- bling acetylcholine. Their inhibitory activity results from competition with acetylcholine for the active sites of the enzyme. The extent of inhibition is determined by the de- gree of stability of the carbamate insecticide to hydrolysis by the enzyme. A. Carbamates As Insecticides Development of carbamates as insecticides began with the work of H. Gysin of J. R. Geigy AG, of Switzerland, in 1947. Several well known, highly insecticidal N,Nfdimethyl— carbamates such as Isolan ® (l-isoprOpyl-3-methyl-5-pyrazolyl dimethylcarbamate) , Pyrolan ® (l-phenyl-3-methyl-5-pyrazolyl 2 dimethylcarbamate) , Dimetan ® (5 ,5-dimethyl-3-oxo-l-cyclo- hexane-l-yl dimethylcarbamate) , Dimetilan ® [1— (dimethyl- carbamoyl)-S-methyl-3-pyrazolyl dimethylcarbamate] and Pyromat ® (2-propy1-4-methyl-6-pyrimidyl dimethylcarbamate) were found. About 1950 work on meethylcarbamates was ini- tiated by Metcalf in California (Kolbezen eE_§l., 1954). It is now known that the N-methyl compounds are generally more potent insecticides than the NJNfdimethyl analogues. The best known of the present meethylcarbamate insecticides is carbaryl (l-naphthyl methylcarbamate) or Sevin® insecti- cide (O'Brien, 1967). Most of the subsequent carbamates were aromatic and phenolic rather than naphthalic. In addi- tion to carbaryl, some recent meethylcarbamate insecticides are Zectran ® (4-dimethylamino-3,5-xy1yl methylcarbamate) , propoxur (g-isopropoxyphenyl methylcarbamate) , Mesurol ® [4-(methy1thio)-3,5-xyly1 methylcarbamate] and Matacil ® (4-dimethy1amino-mftolyl methylcarbamate). B. Correlation of Structure and Activity The general formula of the current carbamate insec- ticides is RleNC(O)OX. For the present methylcarbamate insecticides, R1 is methyl, R2 is hydrogen and X is generally a substituted phenol. In present dimethylcarbamate insecti- cides x is a N-heterocyclic or hydroaromatic enol and R1 and R2 are both methyl groups. Other toxic compounds are known which contain thio- and thionocarbamate and which have various aliphatic, alicyclic and aromatic substituents on the nitrogen or which have fluorine or various aliphatic alcohols or enols for X (Casida, 1963). Kolbezen gtygl, (1954) reported an investigation of 49 substituted phenyl meethylcarbamates of which the 97 and mfalkylated phenyl derivatives were found to be the most potent insecticides. C. Mode of Action of Carbamate Insecticides Most, if not all, carbamates are inhibitors of cholinesterases because of an ability to compete with ace- tylcholine for the active sites of cholinesterase. These compounds have generally been considered to be reversible competitive inhibitors (Myers, 1956; Wilson et_al., 1960; Casida gt_§l., 1960). It was first thought that the mech- anism of inhibition of cholinesterase by carbamoyl fluoride was the formation of carbamoyl derivatives of cholinesterase at the esteratic site and that it was analogous to the phosphorylation reaction by dialkyl phosphate esters at the enzyme active centers (Myers, 1956; Myers and Kemp, 1954). From the available evidence it was shown, however, that the mechanism was competitive inhibition rather than carbamoyla- tion (Myers gE_§1., 1957). The inhibition is a time-depen- dent reaction as shown by the greater inhibition obtained when the inhibitor was added to the enzyme before the sub— strate than when the inhibitor and substrate were added at .lul'l'l’y’lllllu Ill"! the same time. In either case, as time progressed, an in- ‘ termediate "equilibrium" value was approached (Augustinsson and Nachmansohn, 1949; Goldstein, 1951; Stedman and Stedman, 1931; Wilson et_al., 1961; Wilson gt_§1., 1960; Casida gt_§l., 1960). It is now known that the mechanism of cholinesterase inhibition by carbamates is analogous to the reaction of organOphosphates with acetylcholine (O'Brien, 1967). He described the mechanism of cholinesterase inhibition by using the following equation: k1 k2 k3 EOH + AX ;=:-—_: EOH-AX 33-7-9 EOA —-—> EOH + A+ + OH— \ k-1 x’ +.H+ ' H20 ' Reversible complex A is a symbol for either the acetyl group, the dialkyl phosphoryl group, or the methylcarbamyl group, EOH for cholinesterase and X for choline in acetylcholine, p—nitrophenol in paraoxon or l-naphthol in carbaryl. The above reaction can be explained as follows: first, there is a complex formation between enzyme and substrate; second, acetylation, phosphorylation or carbamylation of the enzyme (at the serine hydroxyl site); third, hydrolysis, that is, deacetylation, dephosphorylation or decarbamylation. For carbamates, k is very slow, k 2 3 (k_l/kl) is exceptionally low. These values indicate that is even slower, but ka the EOHaAX complex is present at low levels and the carbamyl— ated enzyme (EOA) is present at high levels. Carbamate inhibition of cholinesterase thus can be explained as some of the inhibited enzyme being in the reversible form (EOH-AX) and some of it being in the carbamylated form (EOA). II. Methylcarbamate Insecticide Metabolism The metabolism of a compound is an important clue to understanding the toxicology and residue aspects of an in- secticide chemical. Metabolism of methylcarbamate insecti- cides was carried out by a mechanism at a site other than initial hydrolysis at the carbamic ester site or tox0phoric grouping (Dorough gt_§1., 1963). It is known that microsomal mixed-function oxidases play an important role in insecti- cide metabolism. The microsomal enzymes attacked at least two sites of the methylcarbamate insecticides, the aryl ring and associated structures and the mono or dialkylated atoms (Terriere, 1968). There was also evidence of cleaving of the carbamate molecule at the ester and amide linkage (Plapp et_§1., 1964). Casida and his colleagues have done extensive work on carbamate metabolism by mammals, insects and plants (Casida, 1963; Hodgson and Casida, 1960; 1961; Dorough and Casida, 1964; Krishna and Casida, 1966; Leeling and Casida, 1966; Oonnithan and Casida, 1968; Tsukamoto and Casida, 1967; Abdel-Wahab et a1., 1966; Kuhr and Casida, 1967). A. Metabolism In Mammals Mammalian liver microsome enzymes metabolize methyl- carbamate insecticides in many ways such as aromatic hydroxt ylation,a1iphatic hydroxylation, Nfdealkylation, Qfdealkyla- tion and sulfoxidation (Williams, 1959; Gillette, 1963). Study of the in vivo fate of the radiocarbon from ten var- iously labeled methyl- and dimethylcarbamate-14C insecti- cides in rats showed that decarbamylation or hydrolytic re- moval of the -OC(O)NHCH3 group was an important route of 14C appeared as 14CO2 (Krishna metabolism since much of the and Casida, 1966). There were five groupings of the carba- mate insecticides known to be susceptible to oxidation or hydroxylation by rat liver microsomal enzymes: Nfalkyl, Efformamide, Qfalkyl, §fa1ky1 and the aromatic ring groups (Oonnithan and Casida, 1968). Rabbits treated with carbaryl excreted eleven meta- bolites in the urine and six of these were identified as l-naphthyl thydroxymethylcarbamate; 4-hydroxy-1-naphthyl meethylcarbamate; 5-hydroxy-l-naphthy1 meethylcarbamate; 5,6-dihydro-5,6-dihydroxy-1-naphthyl meethylcarbamate; l-hydroxy-5,6—dihydro-5,6-dihydroxynaphtha1ene; and l-naphthol (Leeling and Casida, 1966). In the study of in Vitro metabolism of carbaryl by rat liver microsomes with added NADPH thin-layer chromatography showed seven 2! metabolites. Four of these were identified as: 1-naphthy1- thydroxymethylcarbamate, 4-hydroxy-1-naphthyl methylcarbamate, 5,6-dihydro-5,6-dihydroxy-l-naphthyl methylcarbamate and l-naphthol (Leeling and Casida, 1966). B. Metabolism In Plants Enzymes in bean plants metabolized meethylcarba- mate insecticides into organosoluble products, water solu- ble products and insoluble residues via many reactions such as N-methyl hydroxylation, ring hydroxylation and thioether oxidation (Abdel-Wahab gt_al., 1966). The hydroxylation products or metabolites produced in bean plants from methyl- carbamate insecticides were similar to those formed in the liver microsome-NADPH2 systems, but in plants these hydrox— ylated carbamates were rapidly conjugated as glycosides. These glycosides were quite persistent and, in many cases, yielded anticholinesterase agents on hydrolysis by B-glu- cosidase (Kuhr and Casida, 1967). Carbonyl-14C labeled aryl methylcarbamates such as Zectran® and Matacil ® in- jected into the bean plants yielded several metabolites including the 4-methylamino, 4-amino, 4-methy1formamido and 4-formamido analogs. Mesurol was oxidized in beans to the sulfoxide and sulfone analogs (Abdel-Wahab eE_§1., 1966). C. Metabolism In Insects In vivo, insect microsomal enzymes metabolized carbamate insecticides and yielded products similar to those obtained by in vitro incubation of these insecticides with the NADPHz-dependent system of liver microsomes (Dorough and Casida, 1964). It has been shown that under in vitro conditions, insect enzyme preparations had lower activity than that of liver microsomes (Brodie and Maickel, 1962). Microsomes prepared from house fly abdomen homogenates were more active in metabolizing methylcarbamate insecticides in the presence of NADPH2 than were homogenates of the head, thorax or whole house fly (Tsukamoto and Casida, 1967). It was also found that NADPH2 was a more specific and effective cofactor for microsomal enzyme activity than NADHZ. Strong inhibitors appeared in the nuclei and debris fraction from head and thorax homogenates (Casida, 1969). Insect microsomes metabolized methylcarbamate insec- ticides through a variety of chemical reactions such as aromatic hydroxylation, Ordealkylation, Nfdealkylation and sulfoxidation (Tsukamoto and Casida, 1967). III. Biological Oxidation As A Detoxificatibn Mechanism The chemical reactions involved in detoxification mechanisms could be classified mainly as syntheses, oxida- tions, reductions and hydrolyses (Williams, 1959). Most insecticide chemicals were susceptible to biological oxida- tion, which resulted in either activation (conversion to their derivatives of increased toxicity) or detoxification to form products of less toxicity (Casida, 1969). Insects and mammals in many cases had the same detoxification mech- anisms and most differences were quantitative rather than qualitative (Smith, 1962). Many different enzyme systems seemed to be involved in the detoxification of Nealkyl and N,Nfdia1kylcarbamates (Casida gt_§l., 1960). Esterase enzymes such as cholinesterases were able to hydrolyze cer- tain carbamates, but these enzymes could not be regarded as an important detoxification mechanism because the turnover number was very low (Goldstein and Hamlisch, 1952; Myers, 1956). Oxidation as a detoxification mechanism in mammals has been associated with enzyme systems in liver microsomes. This system required NADPH2 and oxygen for its action and could be blocked by certain inhibitors (Dorough and Casida, 1964; Leeling and Casida, 1966; Oonnithan and Casida, 1968). Similar enzyme systems from homogenates of house fly abdomens also required NADPH and oxygen for the oxidation of methyl- 2 carbamates (Tsukamoto and Casida, 1967; Casida, 1969; Dorough and Casida, 1964). A. Oxidation And Hydroxylation Of Insecticides Three majors classes of synthetic organic insecti- cides, the carbamates, the chlorinated hydrocarbons and the organophosphates undergo oxidation reactions. Among the commercial carbamate insecticides which undergo biological oxidation are carbaryl, Mesurol, Matacil and Zectran. The 10 oxidation of these compounds was detailed in the section on Metabolism of Carbamates. Among the chlorinated hydrocarbon insecticides which undergo biological oxidation are DDT, 1,1,1-trich- loro-2,2-bis(p—chlorophenyl) ethane and several cyclodiene compounds including aldrin.1,2,3,4,10,10-hexachloro-l,4, 4a,5,8,8a-hexahydro-l,4-endo-exo-5,8-dimethanonaphtha1ene, isodrin, the 1,4-endo-endo form of aldrin, and heptachlor, l,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-4,7- methanoindene. Tsukamoto (1959) first showed that DDT was oxidized to dicofol, 4,4'-dichloro-a-(trichloromethyl) benzhydrol, in the domestic fruit fly Drosophila melano- qaster. Gianotti gt_al. (1956) showed qualitatively that aldrin was converted to the 6,7-epoxy form, dieldrin, in the American cockroach. Isodrin was converted to the 6,7- epoxy form, endrin, in the house fly (Winteringham and Lewis, 1959) and heptachlor was converted to the 2,3-epoxy form, heptachlor epoxide, in the house fly (Perry et_§l., 1958). Four types of organophosphates are oxidized in zitrg: thionates, dialkylphosphoramidates, compounds with a thio- group in a side chain and a diethylaminoethyl phosphorothiolate (Heath, 1961). Phosphoramidates, X2P(O)N(CH3)2, and phosphorothiolates, (RO)2P(S)OX, were usually poor anticholinesterase agents in vitro (O'Brien, 1960). They are activated, that is converted into potent ll anticholinesterases, in Kilo in insects and mammals, in yitgg by liver slices or several insect whole-tissue pre- parations and in yitrg by specially fortified liver homo- genates. The activity of liver is principally due to the microsomes, which also catalyze a great variety of other oxidation reactions. The product of phosphoramidate acti- vation is the hydroxyalkyl derivative, X2P(O)N(CH3)CH20H, and the product of phosphorothionate oxidation is the phosphate, (RO)2P(O)OX. The thionophosphate of demeton, mixture of 9,97diethyl §f(and Q)—2-[(ethylthio)ethy1] phosphorothionate, can be oxidized to its sulfoxide and sulfone in several insect tissues and in mammalian liver (March gt_al., 1955). Liver homogenates prepared from various mammalian species possessed an enzyme system capable of catalyzing the destruction of 9,97diethy1-Sf2-diethyl- aminoethyl phosphorothiolate (DSDP) via the oxidative mechanism (Scaife and Campbell, 1959). B. General Nomenclature And Distribu- tion Of Oxygenases The term "oxygenase," in a broad sense has been applied to any enzyme capable of catalyzing the activation of oxygen and the subsequent addition of either two atoms of oxygen to a substrate or one atom of oxygen while re- ducing the other to water. In a narrow sense, the term oxygenase was applied to those enzymes which catalyze the addition of two atoms of oxygen to a substrate and the term 12 hydorxylase to those enzymes which catalyzed the addition of one atom of oxygen to a substrate while the other atom is simultaneously reduced to water (Hayaishi, 1962). Oxygenases were first described in mushrooms (Mason gt_al., 1955) and pseudomonads (Hayaishi, §E_al., 1955) and were found to be ubiquitously distributed in animals, plants and microorganisms. They are distributed in various cellular fractions such as microsomes, mitochon- dria and the soluble fraction of liver cells (Knox and Edwards, 1955; Mehler, 1956; Iaccarino gt_al., 1961; Saito gt_al., 1957; Halkerston, gt_al., 1961; Mitoma, et al., 1956). C. Role Of Oxygenases In Biological Oxidation Oxygenases play an important role in the metabolism of various aromatic and aliphatic compounds. The oxygena- tion reaction was primarily involved, in yizg, in detoxica- tion of foreign compounds through the formation of various metabolites (Hayaishi, 1962). Oxygenases and hydroxylases also play a regulatory role in energy distribution in the cell by competing with the conventional electron transport system of cytochromes for reduced nicotinamide nucleotides and for oxygen. Oxygenase activity could be inhibited in the presence of the cytochrome system, because the conven- tional electron transport system via flavoprotein and 13 cytochromes was much more active than oxygenase under normal physiological conditions (Hayaishi, 1962). D. Hydroxylation ggactions 1. Mechanism of single hydroxylation In 1955, when the use of 18O2 and H2180 was made possible, it was discovered that the oxygen atom incorpo- rated into 3,4-dimethylphenol yielding 4,5-dimethylcatechol was derived from an oxygen molecule rather than water (Mason, §£;21,, 1955). The overall reaction was represented by the following equation: S + i O SO 2 2 -—-—--—9 This type of reaction always needs an electron donor so that one atom of oxygen could be incorporated into the substrate molecu1e(s) while the other was reduced to H20 (Hayaishi, 1962) as shown by the following equation: S+02+AH2 sso+H20+A In most hydroxylation reactions, reduced nicotin- amide nucleotides appear to be specific electron donors. The reactions discussed so far involve the fixation of osygen into a substrate molecule in the presence of an appropriate external electron donor. The substrate itself can also serve as an internal electron donor for certain 18 18 reactions. Using 02 and H2 0, the reaction catalyzed by a lactic oxidative decarboxylase which converted l4 l-lactate to acetate plus CO could be shown by the follow- 2 ing equation (Hayaishi and Sutton, 1957): °CHOH:COOH + 1802 _, CH3°C0180H + co + H 18 CH 2 2 3 O l-Lactic acid accepted one atom of oxygen and simul- taneously furnished the electrons to reduce another oxygen atom. 2. Mechanism Of Double Hydroxylation It has been shown that the oxidation of nicotinic acid to 6-hydroxynicotinic acid involves hydration of a double bond followed by dehydrogenation, the oxygen atom thus coming from water (Hughes, gt_al., 1960). It has been proposed that the nitrogen atom in the ring of purines, pteridines (Forrest, §E_al., 1956) and nicotine (Hochstein and Rittenberg, 1959) produced sufficient polarization, with the presence of a lone pair of electrons, that hydration of the double bond was facilitated in such molecules. Another possible mechansim involved the enzymatic hydroxylation of kynurenic acid to 7,8-dihydroxykynurenic acid in the dehydration of dihydrodiol compounds (Kuno, gt_al., 1961). The available evidence showed that in the presence of NADH2 or NADPH2 and oxygen, kynurenic acid was converted enzymatically to the 7,8-dihydrodiol of kynurenic acid, which was then dehydrogenated to 7,8-dihydroxykynurenic acid by a NAD-linked dehydrogenase. 15 3. Ring Hydroxylation One very common metabolic pathway in animals is the hydroxylation of the aromatic ring of compounds foreign to the body. An aromatic hydroxylating system has been found in liver microsomes which required NADPH2 and oxygen for its activity as in the formation of l-naphthol and 1,2- dihydro-1,2-dihydroxy naphthalene from naphthalene (Mitoma, gt_al., 1956). The mechanism of this system involved the formation of l,2-dihydro-l,2-epoxy naphthalene as an inter- mediate which could then react either chemically or enzy- matically with water yielding 1,2-dihydro-l,2-dihydroxy naphthalene or with reduced glutathion to give a mercapturic acid which was then excreted (Booth, g£_al., 1960). The available evidence suggested that with some cyclic compounds an epoxide may be an intermediate metabolite in ring hydrox- ylated (Boyland and Sims, 1960), but it has not yet been synthesized or isolated, so epoxide formation remained hypo- thetical. A different hydroxylation mechanism is illustrated by the oxidation of nicotine, g-l-methyl-Z-(3-pyridy1)- pyrrolidine, in the presence of the liver microsome -NADPH2‘ oxygen system. The first reaction is hydroxylation at the a-position of the pyrrolidine ring by which nicotine is con- verted to hydroxynicotine. This hydroxylation was similar to the conversion of quinoline to 2-hydroxyquinoline (Knox, 16 1946) and it has been prOposed that a similar mechanism may be involved in the oxidation of purines by xanthine oxidase (Bergmann and Dikstein, 1956). 4. Oxidative Demethylation Oxidation of the NJNfdialkylcarbamates by rat liver microsomes takes place at only one of the two alkyl groups, preferentially attacking the shorter of the two if different radicals are present. Methylcarbamates incubated with rat liver microsomes in the presence of NADPH2 and oxygen give formaldehyde-yielding products (Hodgson and Casida, 1960; 1961). Thus, there is in yiE£g_evidence for the first step in oxidative demethylation in which a methyl group is oxi— dized to a hydroxymethyl group. The in yiyg_§7demethyla- tion of certain drugs such as firmethylbarbital and meethyl- phenobarbital has also been illustrated in rat livers (Kuroiwa, 1963). Many other meethylated drugs are known to be demethylated in the body. The two optical forms of N- methylglutathimide, meethyl-a-ethyl-a-phenylglutarimide, for example, are oxidized at the meethyl group to the cor- responding thydroxymethyl form.which was excreted as a glu- curonide of a new type, that of a thydroxymethyl group (Williams and Parke, 1964). The formation of a thydroxy- methyl glucuronide supports the view that Nfdemethylation proceeds by oxidation of the methyl group to hydroxymethyl, which can be removed as formaldehyde if not conjugated. Menzer and Casida (1965) reported the successive meethyl 17 hydroxyiation and subsequent demethylation of the organo- phosphate insecticide Bidrin, ® 3-hydroxy-N,N-dimethyl-g’g- crotonamide, dimethyl phosphate to 3-(dimethoxyphosphinyloxy)- meethyl-N-hydroxymethyl-gi§fcrotonamide and 3-(dimethoxy- phosphinyloxy)-N-methyl-gi§fcrotonamide, by house flies, bean plants and mammals. The same metabolites were formed in hen eggs with Bidrin injected into the yolk sac prior to incubation (Roger, et al., 1964). 5. N-hydroxylation Another type of hydroxylation reaction has been dem- onstrated in vivo with the isolation of the aryl hydroxyl- amine,thydroxy—Z-acetylaminofluorine, from the urine of rats fed 2-acety1aminofluorine. This metabolite appeared in the urine as a conjugate, probably an ether-type N-O glucuronide (N-O-C linkage) (Cramer, gt_21., 1960). Inves- tigation of the metabolism of the hydroxylamine showed that this compound and the parent amine yielded the same urinary metabolites. The excretion of larger amounts of 1—hydroxy-2-acety1aminofluorene than 3-, 5-, and 7-hydroxy derivatives after injection of the hydroxylamine instead of acetylaminofluonine suggested that the hydroxylamine may be an intermediate in the formation of the QEEE27 hydroxylation product (Miller, g£_§1., 1960). Administra- tion of a mixture of 2-acetylaminofluorine-9-l4C and un- labeled hydroxylamine showed N-hydroxyacetylaminofluorine to be a direct precursor of the acetylaminOphenol (Miller 18 and Miller, 1960). The in zizg conversion of an arylhdrox- ylamine to an aminophenol indicated that an enzyme system was present which could carry out this process. Although it has been suggested that Q- and P- hydroxylations of aromatic amines are catalyzed by different enzymes, because of the different ratios which occurred in different species (Parke and Williams, 1956; Weisburger, g£_31., 1957; Parke, 1960), it was probable that grthgfhydroxylation of aromatic amines occurred by rearrangement of thydroxy de- rivatives, probably through a quinolimide intermediate (Miller, gt_§1., 1960; Miller and Miller, 1960). That N- hydroxylation was a general reaction of aromatic amines (Miller, gt_al., 1960) was supported by a further example of this type of reaction, the identification of thydroxy- 4-acetylaminobiphenyl in the B-glucuronidase treated urine of rats treated with 4-acetylaminobiphenyl (Wyatt, §E_al., 1961). In addition to these acetyl derivatives, N-hydroxyl- ation has been demonstrated with primary amines such as 2-naphthylamine (Walpole and Williams, 1958; Boyland and Manson, 1962). E. Major Factors Controlling Oxygenase And Oxidase Activities One of the major factors controlling oxygenase and oxidase activities was their affinity for oxygen and re- duced nicotinamide nucleotides (Hayaishi, 1962). It has been shown that cytochrome oxidase (electron carrier of the 19 conventional electron transport pathway) exhibited a 10-100 times higher affinity for oxygen than do the oxy- genases, hydroxylases and other oxidases and that the affinity towards reduced nicotinamide nucleotides Was of about the same order between the electron transport sys- tem and these enzymes (Hayaishi, 1962). MATERIALS AND METHODS Synthesis Of C—lOOlS-Carbonyl-14C, 2-(4,5-dimethy1-l,3-dI6xolane-2:yl)- phenyl methylcarbamate-carbonyl-L4C Authentic non-labeled C-10015 (analytical grade) was obtained from CIBA Agrochemical Corp., Vero Beach, Florida. C-lOOlS-carbonyl-14C was obtained by reacting acetyl—1-14C chloride (New England Nuclear, Boston, Mass.) with sodium azide to yield methyl isocyanate-14C which was then reacted with appropriate phenol (prepared by alkaline hydrolysis of C-10015). The reaction tube utilized con- sisted of two compartments separated by a break seal. This apparatus provided a separated chamber for preparation of methyl isocyanate which could be brought into contact with the phenol in the other chamber by rupture of the break-seal with the glass slug. The product consisted of two isomers and was purified by liquid chromatography on a FlorisilCD column (Krishna, gt_gl., 1962). The purity was ascertained by thin-layer chromatography (TLC) on Silica Gel G (Brinkmann Instruments Inc., Westbury, New York) using 20 x 20 cm plates with a gel thickness of 0.25 mm. The plate was devel- oped with chloroformacetonitrile (2:1). More than 99% of the radioactivity co-chromatographed with authentic non- labeled C-lOOlS, using radioautography to detect the 20 21 carbon-14 material and a 10% sodium hydroxide spray followed by Gibb's (N!2,6-trichloro-pfbenzoquinoneimine) reagent (Block, gt_§1., 1958) for detection of the authentic non- labeled compound. The Rf value of the first isomer was 0.55 and that of the second was 0.45. It was later found that the isomer of lower R value was more stable than the f higher. Therefore, only the isomer with the lower Rf value was used for the subsequent metabolism studies. Preparation of Liver Fraction And 14 Enzyme Incubation of C-lOOlS—Carbonyl- C Methods used for liver microsome preparation and enzyme incubation studies were previously described by Leeling and Casida (1966) and Oonnithan and Casida (1968). Male albino rats (Wistar strain) were killed by cerebral concussion and the livers removed as quickly as possible and immersed in 0.5 M phosphate (NazHPO4-KH2PO4) buffer (pH 7.4) at 0° C. After removing any adhering tissues, each liver was rinsed one more time with chilled 0.5 M phosphate buffer, cut into pieces and homogenized in 0.5 M phosphate buffer for l min at 0° C with a glass-Teflon® homogenizer (Potter-Elvehjem type) to yield a 20% (w/v) homogenate, on a fresh liver basis. The homogenate was centrifuged at 0° for 25 minutes at 10,0009 resulting in sedimentation of the cell debris (undisrupted cells, nuclei, mitochondria, erythrocytes, etc.) The supernatant, or microsome plus soluble fraction, was further centrifuged 22 at 105,5369 for 60 minutes at 0° C to sediment the micro- some fraction from which the 'soluble fraction' was decanted. The microsomal pellet was washed twice by resuspension in 0.5 M phosphate buffer and recentrifugation. An aliquot containing 100,000 cpm of l4C-carbonyl- C-10015 (about 16 ug) was added to the bottom of a 25 m1 Erlenmeyer flask in 10 ul of ether and the solvent was al- lowed to evaporate at room temperature. To the flask was then added 0.5 ml containing each of the following compo- nents: liver microsomes (equivalent to 200 mg of liver); 2 umoles of reduced pyridine nucleotide cofactor (NADPHZ); 16 umoles of nicotinamide; 2 umoles of barium chloride in 0.5 M phosphate buffer, thus each flask contained a total volume of 2 m1. In each experiment, fresh enzyme prepara- tions and fresh cofactor solutions were used. The flasks were shaken aerobically at 37° C for three hrs. on a Dubnoff metabolic shaking incubator. After incubation the incubation mixtures were immediately either extracted or frozen. Frozen samples were stored at -15° C for not more than 24 hrs. before extraction. Analysis Of Metabolites The incubation mixtures were extracted with three 5 m1 portions of anhydrous ethyl ether. The combined ether extract was dried over anhydrous sodium sulfate and evapor- ated under reduced pressure in a rotary evaporator to Illlll‘ll’llllll 23 approximately 0.5 ml, quantitatively transferred to a grad uated centrifuge tube and reduced to 0.2 ml with a fine stream of air. One hundred ul of the ether extract was added to 15 ml of liquid Scintillation fluor for counting. The fluor consisted of toluene-methylcellosolve (2:1), 5.5 g PPO/z and 0.1 g POPOP/l. The remainder was spotted on a Silica Gel G chromatOplate, of 0.25 mm thick, for reso- lution and quantitation of radioactive components. The TLC plates were developed with chloroform:acetonitrile (2:1). Detection of resolved radioactive compounds on the plates was accomplished by radioautography using no- screen medical X-ray film (Eastman Kodak Company, Rochester, N.Y.) The radioactive regions on the plate corresponding to darkened areas on the film were scraped into scintil- lation vials and counted. This procedure for radioassay of resolved metabolites by counting the scraped regions gave an average 14C-recovery of 92% of that originally spotted on the plates. The counts for each metabolite component were related, on a percentage basis, to the total radiocarbon recovered from scraping TLC plates. Characterization of C~10015 Metabolites From The Rat Liver MiErosome System Larger quantities of C-10015 metabolites needed for characterization studies were obtained with slight changes of the procedures described above. Two-dimensional 24 TLC was used and the plates were developed first with the ether:hexane:ethanol (77:20:3) mixture and then with chloroformzacetonitrile (2:1). Each metabolite scraped from TLC plates was extracted with 7-8 ml of acetone and centrifuged in a graduated centrifuge tube. The acetone was transferred by Pasteur pipet to another graduated centrifuge tube and reduced to approximately 100 ul with a fine stream of air. The concentrated acetone extract was transferred to a melting point tube using a 50 ul syringe. The extract was evaporated to dryness in a 1y0philizer for 1-2 hours. The metabolite in each melting point tube was analyzed by direct probe mass spectrometry (Du Pont mass spectrometer 21-490). All mass spectra were obtained at an ionizing voltage of 70 eV. Metabolism of C-lOOlS-Carbony1-14C in Bean Plants The methods and materials used for the study of 14 metabolism of r C-10015 -carbony1- C by bean plants (Phaseolus nulgaris, var. Bountiful) were the same as the applicable ones described by Abdel-Wahab, et_al. (1966), with certain exceptions and changes as noted in the follow- ing procedures. About 100,000 cpm (about 16 ug) of C-10015 - carbonyl-14C were individually injected into the stems of 14 day-old, growing bean plants (Abdel-Wahab, gt_al., 1966). After injection, the growing plants were held in 25 the greenhouse for 0, 1/3, 1, 2, 3, 4 and 6 days, respec- tively. Each replicate of three identically treated plants was cut, stored at -15° C until analyzed 1-4 weeks later and extracted with acetone once and chloroform twice (each time with 30 m1) using the described procedure. This di— vided the components into three fractions: the acetone- chloroform or organic phase, the acetone-water or aqueous phase and the insoluble residue. The total radioactivity of the organic and aqueous fractions was determined using previously published procedures (Abdel-Wahab, gt_§l., 1966). The total radioactivity of the insoluble residue fraction was analyzed by placing 100 mg samples into bags prepared from 2°54 cm dialysis casing and combusting them to 14CO2 in a combustion flask according to the technique previously reported by Hopkins and Lofgren (1968). The 14CO2 was trapped in 10 m1 monoethanolamine:ethylene glycol mono- methylether (1:2 v/v) and a 3.0 m1 portion of the liquid was transferred to a scintillation vial for radioassay. House Fly In_yi Q And ¥g EEEEQ Metabolism O C- 0 15-carbony - C Methods used for the study of house fly metabolism of methylcarbamate insecticides in_vivo and in_vitro were previously described by Tsukamoto and Casida (1967) and Shrivastava, et a1. (1969). 26 Procedures For In Vivo House Fly Metabolism Studies To a group of 30-40 female flies (Musca domestica L.), 3-5 days old, 1 ul of acetone containing 0.2 - 0.3 ug l4C-carbonyl labeled C-10015 was applied topically on the ventral side of each abdomen. Groups of treated flies were placed in the metabolism chamber which was connected to a series of collecting traps. The collecting traps consisted of a chamber that was kept as acetone-dry ice temperature for trapping volatile products and two 14 The 14CO2 respired was bubbled through 5 ml of monoethanol- CO2 trapping tubes. aminezethylene glycol monomethylether (1:2 v/v) and a 1.0 ml portion of the liquid was transferred to a scintillation vial for radioactive assay. Any l4CO2 that escaped from this tube was trapped in the scrubber tube consisting of a scintered glass tube bubbling in 30 m1 of monoethanolamine: ethylene glycol monomethylether (1:2 v/v). Air was pulled through the respirometer with a vaccuum pump. The 14CO2 and the volatile products were collected at appropriate time intervals and the trapping solutions were replaced with fresh solutions. At the end of the experimental per- iods the flies in the chamber were either immediately ana- lyzed or frozen at -15° C for not longer than 24 hrs. before analysis. For analysis, the flies were transferred to another tube and surface-washed over the body with 10 ml of cold 27 acetone. The resulting acetone solution was used to wash the metabolism chamber and to dissolve any excreta in it. This procedure of washing the metabolism chamber was re- peated 3 more times, using 10 ml portions of acetone, and the four wash solutions were combined to form the acetone- soluble fraction. Acetone extracts of each group of 30-40 flies were prepared by homogenizing the flies in 10 ml of acetone at 5° C, using a Pyrex ® tissue grinder in a 50 ml tube. The homogenizing pestle was rinsed with 2-3 ml of acetone, the homogenate centrifuged in a graduated centrifuge tube and the supernatant removed with a Pasteur pipet. The sedi- ment was transferred back to the homogenizing tube and the acetone extraction was repeated in an identical manner two additional times, so that the final acetone extract (36-40 m1) represented the combination of three acetone extrac- tions. The extract was evaporated under reduced pressure in a rotary evaporator to approximately 0.3 ml and quanti- tatively transferred to a graduated centrifuge tube (final volume 0.5 ml). Fifty ul of the concentrated extract was added to 15 ml of liquid scintillation fluor for counting. The combined excrement extract was concentrated and counted in an identical manner. The dried sediment samples were analyzed by combus- 14 ting them to CO2 in a combustion flask as previously des- cribed (Hopkins and Lofgren, 1968). 28 Procedure For In Vitro House Fly Metabolism Studies Enzyme preparation consisted of homogenization of fly abdomens in 0.25 M sucrose and 0.15 M phosphate buffer ® (pH 7.4) in an ice bath using a glass-Teflon homogenizer. In each experiment, bovine serum albumin (BSA) was used at 1.5% (w/v) in both the homogenization and incubation mix- tures. Fractions of homogenates of house fly abdomens were obtained by differential centrifugation, as follows: nuclei, cell debris and mitochondria at 10,0009 for 25 min. and microsomes at 105,536g for 60 minutes. All dif- ferential sedimentation was at 0° C. A typical incubation mixture, each in a 25 m1 Erlenmeyer flask, consisted of 100,000 cpm (about 16 ug) of C-lOOlS-carbony1-14C; 5 umoles of NADPH2 and an amount of enzyme preparation equivalent to 15 fly abdomens in a 2 ml final incubation volume. The substrate-enzyme system was shaken aerobically for two hrs. at 30° C on a Dubnoff metabolic shaking incubator. After incubation, the mixtures were either held at -15° C for subsequent ex- traction or were immediately successively extracted with three 5 ml portions of ether. The combined ether extracts were evaporated under reduced pressure in a rotary evapor- ator and spotted on a TLC plate. Two dimensional TLC analy- ses were performed, the solvent being ether:hexane:ethanol (77:20:3) and chloroformzacetonitrile (2:1). 29 Quantitation studies of metabolites were accom- plished by radioautography and scraping the TLC plates as previously described. RESULTS AND DISCUSSION “Metabolism of C-10015 by Rat 'Liver'Microsomes Metabolism of C-10015 by rat liver microsomes pro- duces at least three non-hydrolytic products. These three metabolites were designated as A, B and D based on auto- radiograms of the TLC resolution of labeled components in incubation mixtures as indicated in Figure 3. Unchanged C-10015 appeared as spot C. The Rf values for these com— pounds (A to D) in ether:hexane:ethanol (77:20:3) were 0.15, 0.21,0.30 and 0.42, respectively. In chloroform: acetonitrile (2:1), the Rf values were 0.22, 0.33, 0.45 and 0.53, respectively. Reactions involved in this metab— olism include hydroxylation (metabolites A and B) and en- zyme cleavage of the dioxolane ring to the aldehyde (me- tabolite A) as will be discussed later. The pH optimum for non-hydrolytic metabolism was 7.4 for rat liver microsomes when fortified with NADPH2 (Table l-A). The Optimum incubation time for liver microsome metabolism of C-10015 was three hrs. as shown in Table l-B. The effect of selected divalent cations at 2 mmoles per flask on metabolism was ascertained (Table 1-C). The 30 31 m.H H.mm o.hN m.m N v m.H N.mm m.Nm v.m N m N.H w.vm m.mN v.m N N o m.hm m.MN m.h N H e ~.He e.H~ o.e m m.e A.mnsv poflum> mafia GONWMQSUsH suw3 v.5 mm as +Nem + amenaz + assemonoaz .m H.N m.mm H.mm H.m N m.h m.N m.Nm «.mm N.m N m.n m.m m.hv 5.5m m.oH N ¢.n m.N 5.5m m.om h.m N N.w m.N N.Nm m.mN m.m N o.w m.N «.mh m.mH v.m N m.m empowum> aminuwz +Nmm + Namafiz + moEOmouoflz .¢ a O m 4 moumowammm Annmanmwnm> can unwefluomxm uomnuxm umbum as ucosomfioulu ad 30mm m0 w O>HHMHOM moEOmOHon Ho>flq umm ma 0 «a ueseObnnoumHooauo mo snaeobebmz .H wands 32 .mmOHOSm z mN.o CH pounmonm ouw3 mosomonows Hm>fla umu usofiwuomxm was» Mom .Emummm “mumps mumnmmonm on» How pmflum> mm3 mm one An .mnn m How 0 ohm um poumnsosfl muo3 mousuxfla sowpomou HAN .Auo>fla ma ooN ou usoam>flsvov Hommsn oumnmmonm v.5 mm 2 m.o mo HE m.o cw moEOmouoHE Ho>HH umu .opHHoHso Eafluwn mmHoEE o.N .wpflfimawuoowc moaoas ma .Nmmadz assess o.~ .nmmesb scammsueoammnz 4.5 me 2 m.o .oaauaseonnsoumaeoauo as be ”unflsoaaom map pocfimucoo mnsuxfifi sowuomon sumo .mmfisuonpo pouos muons umooxm Am H.H m.om H.w m.N N opaswsflyoows + +Ncm m.H N.hn m.ma m.m N opHEwsfluooH: + +Nnm m.a ¢.mh m.ma N.m N oUHEmcHuOOHs + +ch m.H m.wo N.mN a.m N opHEmcHuOOHG + +Nmz H.H m.bm v.m N.m N opflamcHuOONs + +Nmo w.N N.Nm «.mN m.oa N opflfimcfluoowc + +Nmm >.m m.>m m.mN m.m N AoUHEMGHBOOHs osv +Nmm o.a o.Nm H.NH m.v N cowuwo ucoam>flp oz xmmam mom mmaofis ma um opflfimcHHOONG can moHOEEN um mcoflumo usoam>flp mo poommm .O a U m d mmHMONHmom moanmwum> was ucmfiflnmmxm uomupxm Honum a“ ucosomEOOIUvH comm no N m>aumaom emssebeoo .H wanes 33 Figure 1. Autoradiogram of one-dimensional TLC of C-lOOlS-carbonylél4c metabolism by rat-liver microsomes. Development of TLC was with chloroform:acetonitrile, 2:1. 34 Figure l. 35 Figure 2. Autoradiogram of two-dimensional TLC of C-10015- carbonyl-14C metabolism by house fly microsomes. Development of TLC was with ether:hexane:ethanol, 77:20:3 and then chloroform:actonitrile, 2:1. Figure 3. Autoradiogram of two-dimensional TLC of C-10015- carbonyl-14C metabolism by rat-liver microsomes. Development of TLC was with ether:hexane:ethanol, 77:20:3 and then chloroform:acetonitrile, 2:1. 36 . Origin 37 2 2+ 2+ effect was Ba2+>Mg +>Pb2+>Mn >control>Ca >Sn2+, with each of the first four increasing the total metabolism. The amount of ether-extractable metabolites was greatest with Ba2+. Chemical Nature Of The Carbamate Metabolites of C-10015 From Liver Microsomes The mass spectrum of metabolite A (Figure 4) indi- cated that the starting material (C-10015) had been modi- fied by the addition of one hydroxyl group and that an aldehyde was obtained by enzymatic cleavage of the dioxo- lane ring, based on the molecular ion peak at m/e 195. Mass spectrometry of metabolite B showed a molec- ular ion peak at m/e 283 indicating that the C-10015 had two hydroxyl groups added. The ion at m/e 255 could be obtained by elimination of CO from the parent peak at m/e 283 (Figure 4). Metabolite D was not analyzed by mass spectrometry because a sufficient quantity of material could not be re- covered. To determine the precise structure of metabolites, sufficient quantity of metabolites and authentic deriva- tives of C-lOOlS are needed for analysis by mass spectrom- etry, infrared spectrometry and nuclear magnetic resonance spectrometry. 38 Figure 4. The mass spectra of C-10015 and Metabolites A and B. 39 Figure 4. C-lOOlS .- n r\ J I I‘d-[r I T ‘ I I I I I T T 140 180 220 mhl 1007 ! Metabolite B 80f ! 60~ 40— 20-1 J I LIL J.illilll In ii 1 I r T F I r“ I I I I I l’ I 60 100 140 180 220 260 mks Metabolite A 40 Metabolism of C-lOOlS-carbonyl-14C IfiiBean Plants Table 2 lists the recovery results obtained when l4C-carbonyl labeled C-10015 was injected into the stem of l4-day-old growing bean plants. The percentage Of injected radioactivity recovered in each fraction was calculated and that not accounted for was tabulated as lost. Six days after injection into the stem, loss of radioactivity was 100%. It is apparent that C—10015 disappeared rapidly from the leaf; volatilization of the chemical or loss as 14CO2 is probably involved. The rapid disappearance of C-10015 from bean plants is comparable to that of Mesurol, of which 64% was lost by six days after injection into the stem of bean plants (Abdel-Wahab, gE_§l., 1966). Table 2 also shows that C-10015 was metabolized into organosoluble pro- ducts, water-soluble products and insoluble residues fol- lowing injection into the stem of growing bean plants. The water soluble products of C-10015 presented in Table 2 are rather small when compared with the water sol- 3uble products of other meethylcarbamate insecticides such as carbaryl, propoxur, UC 10854 and Banol® (Abdel-Wahab, gt_al., 1966). Percent recovery of water-soluble products of l4C-carbonyllabeled carbaryl, propoxur, UC 10854 and . Banol ®were 35.2, 59.0, 64.4 and 45.8, respectively, at the third day after injection into the stem of growing bean plants (Abdel-Wahab, et al., 1966). The recovery of the 41 water-soluble products of C-10015 carbonyl-14C was 8% at the third day after injection into the stem of the bean plants. Differences in the chemical nature of the substi- tuted groups of each phenyl methylcarbamate are probably involved. Metabolism of C-10015 by the NADPH,: dependent Enzyme System from the “ House Fly_ TLC autoradiograms (Figure 2) of organosoluble metabolites of C-lOOlS-carbonyl-14C plus house fly micro- somes, NADPH2 and BSA showed that at least three metabo- lites have the same two dimensional TLC Rf's as organo- soluble C-10015 metabolites produced by rat liver micro- somes. The metabolites were designated as A, B and D, respectively, (Figure 2) as previously described for the metabolism by rat liver microsomes. The quantity of metabolites produced by house fly microsome-NADPH2 systems was as follow (from A to D): 1.8%, 7.6% and 1.5%, respectively. The original compound, C-10015-carbonyl-14C (spot C), constituted 89.1% of the radioactivity. Therefore, under in vitro conditions, the house fly preparation showed very little metabolism in comparison to that of liver microsomes. Brodie and Maickel (1962) also reported that insect enzyme preparations had low activity in comparison to that of liver microsomes. 42 .Emp ooo.mNH mm3 “smam smwn comm ousw pmuommcfi 8mm Hmuoe Am ooa e.em m.Hm m.ee H.me e.mm e.m~ m smog u s.~ m.m N.OH b.e o.e e.m N nmamammn mansaonsH I I o.m m.v o.¢ h.H N.o N mmmnm msomsqm u . o.e m.m m.aH e.~m m.eb m waste oasemno b a m N a m\H o mmueoaammm macauobum How UGSOM b.n»bb ca .msau sentences ufl>fluomoapmu mmuowncfl mo usmoumm ImHOOHIU usmfiumona Hmumm mHm>HmusH um Ovalamsonumo snag mmuomncH mbsmam cmom mcfl3ouo Eoum Umum>ooom mcowuomum .N magma 43 Identification of Organosolublefi Metabolites ‘Produced by the NADPH -dependentfi Enzyme System from House F An insufficient quantity of these metabolites was obtained for analysis by mass spectrometry, however, all the spots obtained from TLC have the same two dimensional TLC Rf's as organosoluble metabolites produced by rat-liver microsomes. Therefore, they may have molecular structures identical to the metabolites produced by liver microsomes. In vivo Metabolism of C-10015 by House Flies Preliminary studies involving topical application 14C labeled C-10015 to the ventral surface of of carbonyl- the abdomen and subsequent metabolism were made. Radio- carbon recoveries in each fraction are presented in Table 3. 44 .maco Ausofiumouu Hmumm .mus wNv unmafiummxm may mo mam map um pmuwamcm Am mam .Am.Ao.Ab .osHm> sOQHmOOHUmH wooa on» now owns mmz sofl£3 amp mmonh mmz mmflam mnp ouso SHHmONQOD powammm Emu Hmuoa Am va.h AGOQHmUOHUmH wv mmoq m>.Nm mmum>ooou conHmOOHUmH w Hmuoa w.a v.H I l I I Amman» Hobbsuom may ca pommmnu NOO¢H «.mv «.mv I I I I Ammmflam :H musmsomfiou m.oa m.oa I I I I Aomucmsomeoo mmuouoxm v.m ¢.m I I I I Anospwmmu mansaomcH mm.o no.0 no.0 mo.o mH.o I muUDUOHm OHflHMHO> HOSUO e.m~ a.a o.m o.a b.eH ~.a «oneH coaumfifism vN ma m a H soaumoowmmn mcwcwmusoo cowuomum Am.psmaummnu Ho panomfioo pmnm>oomm Hmumm Amusonv mam>umbsw mswmnm> um coauomnm 30mm cw poum>ooou conumoowpmu Hmuou mo usmouom maoeano smashes Hanobneouoaa no as m.o : «.0 mo coflumONHmmd Hmowmoa Hmumm mmflam mmsom Eoum mmum>oomm chHuomHm .m magma SUMMARY The investigation of C-lOOlS-carbonyl-14C metabo— lism by rat-liver microsomes, house flies and bean plants has been conducted. Therefore, in the qualitative and quantitative analytical measurements of radioactivity, only products containing this carbon atom in their struc- ture were found. Thus, certain hydrolysis products, such as the phenol and its derivatives were not detected. Evidence from in vitro experiments suggested that hydroxylation and enzymatic cleavage of the dioxolane ring to an aldehyde were the major detoxication mechanisms for C-lOOlS. The rat-liver mecrosome fraction was selected for a detailed study of in vitro metabolism. The amount of metabolism was related to the presence of certain divalent cations, the optimum pH was 7.4 and the Optimum incubation time was three hrs. In vitro experiments suggested that house flies pro- duced the same metabolic products of C-lOOlS as did the liver microsomes. When C-lOOlS was applied topically onto the fly abdomens, about 30% of the C-lOOlS was degraded and expired 14 as CO2 within 24 hrs. 45 46 Six days after injection of C-lOOlS into growing bean plants, all of the C-10015 was lost from the plants. Rapid degradation of C-lOOlS with expiration of CO was 2 probably involved. LIST OF REFERENCES LIST OF REFERENCES Abdel-Wahabi A. M.; Kuhr, R. J.; and Casida, J. E. "Fate of '4C-carbony1-labe1ed aryl methylcarbamate insec- ticide chemicals in and on bean plants." J. Agr. Food Chem. 14 (1966) 290-298. Augustinsson, K. B., and Nachmansohn, D. "Studies on cholinesterase. VI. Kinetics of the inhibition of acetylcholinesterase." J. Biol. Chem. 179 (1949) 543-549. Bergmann, F., and S. Dikstein. "Studies on uric acid and related compounds. III. Observations on the spec- ificity of mammalian xanthine oxidases." J. Biol. Chem. 223 (1956) 765-780. Block, R. J.; Durrum, E. L.; and Zweig, G. A manual of paper chromatography and paper electrophoresis. Academic Press, New York, (19587fp. 305. Booth, J.; Boyland, E.; and Sims, P. "Metabolism of poly- cyclic compounds. 15. The conversion of naphthalene into a derivative of glutathione by rat-liver slices.“ Biochem J. 74 (1960) 117-122. Boyland, E., and Sims, P. "Metabolism of polycycyclic compounds. 16. The metabolism of 1:2-dihydronaph- thalene and 1:2-epoxy-lz2:3:4-tetrahydronaphthalene." Biochem. J. 77 (1960) 175-181. Boyland, E., and Manson D. "The reaction of phenylhydrox- ylamine and 2-naphthy1hydroxylamine with thiols." J. Chem. Soc. Unpublished work, cited in Boyland, E.; Manson, D.; and Nery, R. 1962. Brodie, B. B., and Maickel, R. P. Comparative biochemistry of drug metabolism. Proc. Intern. Pharmacol. Meeting, lst. 6 (1962) 299-329. Casida, J. E. Mode of action of carbamates. Ann. Rev. Entomol. 8 (1963) 39-58. 47 48 Casida, J. E. Insect microsomes and insecticide chemical oxidationsL_:h Microsomes and Drug Oxidations. Edited by J. R. Gillette, et a1. Academic Press, New York, 1969. Casida, J. E.; Augustinsson, K. B.; and Jonsson, G. "Sta- bility, toxicity, and reaction mechanism with ester- ases of certain carbamate insecticides." J. Econ. Entomol. 53 (1960) 205-212. Cramer, J. W.; Miller, J. A.; and Miller, E. C. "N-hydro- xylation: a new metabolic reaction observed in the rat with the carcinogen 2-acetylaminofluorene." J. Biol. Chem. 235 (1960) 885-888. Dorough, H. W.; Leeling, N. C.; and Casida, J. E. "Non- hydrolytic pathway in metabolism of N-methylcarba— mate insecticides." Science, 140 (1963) 170-171. Forrest, H. 8.; Glassman, E.; and Mitchell, H. K. "Conver- sion of 2-amino-4-hydroxypteridine to isoxanthOpterin in D. melanogaster." Science, 124 (1956) 725-726. Giannotti, 0.; Metcalf, R. L,; and March, R. B. The mode of action of aldrin and dieldrin in Periplaneta americana (L). Ann. Entomol. Soc. Am. 49 (1956) SSS-592. Gillette, J. R. Metabolism of drugs and other foreign compounds by enzymatic mechanisms. Prog. in Drug Res. 6 (1963) 11-73. Goldstein, A. Properties and behavior of purified human plasma cholinesterase. III. Competitive inhibi- tion by physostigmine and other alkaloids with special reference to differences in kinetic be- havior. Arch. Biochem. Biopays. 34 (1951). Goldstein, A. and Hamlisch, R. E. Properties and behavior of purified human plasma cholinesterase. IV. Enzymatic destruction of the inhibitors prostigmine and phySOstigmine. Arch. Biochem. Biophys. 35 (1952) 12-22. Halkerston, I. D. K.; Eichhorn, J.; and Hechter, O. "A requirement for reduced triphosphOpyridine nucleotide for cholesterol side-chain cleavage by mitochondrial fractions of bovine adrenal cortex." J. Biol. Chem. 236 (1961) 374-380. 49 Hayaishi, 0. Biological oxidation. A§n&_3§x‘_fiigghgm, 31 (1962) 25-46. Hayaishi, 0.; Katagari, M.; and Rothberg, S. "Mechanism of pyrocatechase reaction." J. Am. Chem. Soc. 77 (1955) 5450-5451. Hayaishi, O. and Sutton, W. B. "Enzymatic oxygen fixation into acetate concomitant with the enzymatic decar- boxylation of L-lactate." J. Am. Chem. Soc. 79 (1957) 4809-4810. Heath, D. F. Organophosphorus Poisons. Pergamon Press, New York, 1961, 403 pp. Hochstein, L. I., and Rittenberg, S. C. "The bacterial oxidation of nicotine. I. Nicotine oxidation by cell-free preparations." J. Biol. Chem. 234 (1959) 151-155. Hodgson, E., and Casida, J. E. Biological oxidation of N;Nrdialkyl carbamates. Biochem. BiOphys. Acta 42: (1960) 184-186. Hodgson, E., and Casida, J. E. "Metabolism of N;N-dia1kyl- carbamates and related compounds by rat-liver." Biochem. Pharmacol. 8 (1961) 179-191. HOpkins, T. L. and Lofgren, P. A. "Adenine metabolism and urate storage in the cockroach, Leuco haea maderae." J. Insect Physiol. 14 (1968) 1803-1814. Hughes, D. E.; Hunt, A. L.; Rodgers, A.; and Lowenstein, J. M. The hydroxylation of nicotinic acid by a cell-free system from Esssésmgna§_f1n2rs§2sn§. Proc. Intern- Congr. Biochem., 4th Congr. Vienna, 1958, CoIquuia I3 (1960) 189-193. Iaccarino, M.; Boeri, E.; and Scardi, V. "Preparation of purified 3-hydroxyanthrani1ic acid oxidase from rat and ox liver." Biochem. J. 78 (1961) 65-69. Knox, W. E. "The quinine-oxidizing enzyme and liver alde- hyde oxidase." J. Biol. Chem. 163 (1946) 699-711. Knox, W. E., and Edwards, S. W. "Homogentisate oxidase of liver." J. Biol. Chem. 216 (1955) 479-487. Kolbezen, M. J.; Metcalf, R. L.; and Fukuto, T. R. "Insec- ticidal activity of carbamate cholinesterase in- hibitors." J. Agr. Food Chem. 2 (1954) 864-970. 50 Krishna, J. G., and Casida, J. E. "Fate in rats of the radiocarbon from ten variously labeled methyl-and dimethylcarbamate-Cl4 insecticide chemicals and their hydrolysis products." J. Agr. Food Chem. 14 (1966) 98-105. Krishna, J. G.; Dorough, H. W.; and Casida, J. E. "Synthe- sis of N-methylcarbamates via methyl isocyanate-Cl and chromatographic purification." J. Agr. Food Chem. 10 (1962) 462-466. Kuhr, R. J., and Casida, J. E. "Persistent glycosides of metabolites of methylcarbamate insecticide chemicals formed by hydorxylation in bean plants." J. Agr. Food Chem. 15 (1967) 814-824. Kuno, S.; Tashiro, M.; Taniuchi, H.; Horibata, K.; Hayaishi, 0.; Seno, S.; Touyama, T.; and Sakan, T. Enzymatic degradation of kynurenic acid. Abstract in Fed. Proc. 20 (1961) 3. Kuroiwa, Y. "Studies on the metabolic N-demethylation. I. The metabolic fate of hexobarbital by rabbit liver slices." Chem. Pharm. Bull. 11 (1963) 160-163. Leeling, N. C., and Casida, J. E. "Metabolites of carbaryl (1-naphthyl methylcarbamate) in mammals and enzymatic systems for their formation." J. Agr. Food Chem. 14 (1966) 281-290. March, R. B.; Metcalf, R. L,; Fukuto, T. R.; and Maxon, M. G. “Metabolism of systox in the white mouse and american cockroach." J. Econ. Entomol. 48 (1955) 355-363. Mason, H. S.; Fowlks, W. L.; and Peterson, E. W. "Oxygen transfer and electron transport by the phenolase comples." J. Am. Chem. Soc. 77 (1955) 2914-2915. Mehler, A. H. "Formation of picolinic and quinolinic acids following enzymatic oxidation of 3-hydroxyanthrani- lic acid." J. Biol. Chem. 218 (1956) 241-254. Menzer, R. E., and Casida, J. E. "Nature of toxic metabolites formed in mammals, insects and plants from e-(dimethoxyphosphinyloxy) N,N-dimethy1 cis-croton- amide and its N-methyl analog." J. Agr. Food Chem. 13 (1965) 102-112. 51 Miller, J. A.; Cramer, J. W.; and Miller, E. C. The N- and ring-hydroxylation of 2-acety1aminofluorene during carcinogenesis in the rat. Cancer Research. 20 (1960) 950-962. Miller, E. C., and Miller, J. A. "A mechanism of orthol- hydroxylation of aromatic amimes in vivg." Biochem. Biophys. Acta 40 (1960) 380-382. Mitoma, D.; Posner, H. S.; Reitz, H. C.; and Udenfriend, S. "Enzymatic hydroxylation of aromatic compounds." Arch. Biochem. Biophys. 61 (1956) 431-441. Myers, D. K. "Studies on cholinesterase. 10. Return of cholinesterase activity in the rat after inhibition by carbamoyl fluorides." Biochem. J. 62 (1956) 556-563. Myers, D. K., and Kemp, A. Jr. "Inhibition of esterases by the fluorides of organic acids." Nature 173 (1954) 33-34. Myers, D. K.; Kemp, A. Jr.; Tol, J. W.; and de Jonge, M. H. T. "Studies on aliesterases. 6. Selective inhibitors of the esterases of brain and Saprophytic mycobacteria. Biochem. J. 65 (1957) 232-241. O'Brien, R. D. Toxic Phosphorus Esters. Academic Press, New York, New York, 1960, 434 pp. O'Brien, R. D. Insecticide-Action and Metabolism. Academic Press, New York, New York, 1967, 332 pp. Oonnithan, E. S., and Casida, J. E. "Oxidation of methyl- and dimethylcarbamate insecticide chemicals by microsomal enzymes and anticholinesterase activity of the metabolites." J. Agr. Food Chem. 16 (1968) 28-44. Parke, D. V. "Studies in detoxication. 85. The metabolism of [14C] aniline in the rabbit and other animals." Biochem. J. 77 (1960) 493-503. Parke, D. V., and Williams, R. T. "Species differences in the 9- and p-hydroxylation of aniline." Biochem. J. 63 (1956) 12p. Perry, A. S.; Mattson, A. M.; and Buckner, A. J. "The metabolism of heptachlor by resistant and suscep- tible house flies." J. Econ. Entomol. 51 (1958) 346-351. 52 Plapp, F. W.; Chapman, G. A.; and Bigley, W. S. "A mechanism of resistance to Isolan in the House fly." J. Econ. Entol. 57 (1964) 692-698. Roger, J. C.; Chambers, H.; and Casida, J. E. "Nicotinic acid analogs: Effects on response of chick embryos and hens to organOphospahte toxicants." Science 144 (1964) 539-540. Saito, Y.; Hayaishi, 0.‘ and Rothberg, S. "Studies on oxy- genases: Enzymatic formation of 3-hydroxy-l-kynuro- _nine from 1-kinuronine." J. Biol. Chem. 229 (1957) 921-934. Scaife, J. F., and Campbell, D. H. "The destruction of 0,0- diethyl-s-2-diethy1aminoethyl phosphorothiolate by liver microsomes." Canad. J. Biochem. Physiol. 37 (1959) 297-305. Shrivastava, S. P.; Tsukamoto, M.; and Casida, J. E. "Oxidative metabolism of C14-1abeled Baygon by living house flies and by house fly enzyme prepara- tions.“ J. Econ. Entomol. 62 (1969) 483-498. Smith, J. N. "Detoxication mechansims." Ann. Rev. Entomol. 7 (1962) 465-480. Stedman, E., and Stedman, E. "Studies on the relationship between chemical constitution and physiological action. III. The inhibitory action of certain synthetic urethanes on the activity of liver esterase." Biochem. J. 25 (1931) 1147-1167. Terriere, L. C. "Insecticide-cytoplasmic interactions in insects and vertebrates." Ann. Rev. Entomol. 13 (1968) 75-98. Tsukamoto, M. Metabolic fate of DDT in Drosophila melan- ogaster. I. Identification of a non-DDE metabolite. Botyu-Kagaku. 24 (1959) 141-151. Tsukamoto, M., and Casida, J. E. "Metabolism of methyl- carbamate insecticides by the NADPHz-requiring enzyme system from houseflies. Nature 213 (1967) 49-51. Walpole, A. L., and Williams, M. H. C. "Aromatic amines as carcinogens in industry." British Med. Bull. 14 (1958) 141-145. 53 Weisburger, J. H.; Weisburger, E. K.; and Morris, H. P. "Orientation of biochemical hydroxylation in aromatic compounds." Science 125 (1957) 503. Williams, R. T. Detoxication Mechanisms. 2nd Edition. John Wiley and Sons, Inc., New York, 1959, 796 p. Williams, R. T., and Parke, D. V. "The metabolic fate of drugs." Ann. Rev. Pharmacol. 4 (1964) 83-114. Wilson, I. 3.; Harrison, M. A.; and Ginsburg, S. "Carbamyl derivatives of acetylcholinesterases." J. Biol. Chem. 236 (1961) 1498-1500. Wilson, I. B.; Hatch, M. A.; and Ginsburg, S. "Carbamyla- tion of acetylcholinesterase." J. Biol. Chem. 235 (1960) 2312-2315. Winteringham, F. P. W., and Lewis, S. E. "On the mode of action of insecticides." Ann. Rev. Entomol. 4 (1959) 303-318. Wyatt, C. S.; Miller, J. A.; and Miller, E. C. "The N- hydroxylation of 4-acetylaminobipheny1 in the rat. Proc. Am. Assoc. Cancer Research 3, 1961, 279. ”'IIIIIIIIIIIIIIIIIIIIIIIIIIEIIIIIIIIIIIIIIII“