Metabolism of m-tert.-butylphenyl N-Methylcarbamate in

Transcription

1 Bichem. J. (1971) 125, Printed in Great Britain 385 Metablism f m-tert.-butylphenyl N-Methylcarbamate in Insects and Mice By P. G. C. DOUCH AND J. N. SMITH Department f Bichemistry, Victria University f Wellingtn, P.O. Bx 196, Wellingtn, New Zealand (Received 17 May 1971) The metablism f m-tert.-butylphenyl N-methylcarbamate was studied in mice and five species f insects. Bth the tert.-butyl grup and the N-methyl grup were hydrxylated. The majrphenlic metablitewasm-(,-hydrxy-tert.-butyl)phenl, which was identified by mass spectrscpy. Significant amunts f dihydrxy cmpunds were frmed at a cnstant rate frm the start f the enzymic xidatin prcess. The cnsiderable species variatin in the yields f the different types f xidatin prducts suggests that N-demethylatin and xidatin f the tert.-butyl grups were catalysed by different enzymes. A micrsmal NADPH-dependent enzyme als catalysed the splitting f the ester link in the insecticide. Carbamate insecticides have arused interest because f the frequency with which selective txicity is exhibited. One imprtant reasn fr this is the rapid degradatin f these cmpunds in the mre resistant species (Winteringham, 1969), but the interpretatin f txicity results in terms f species differences in metablism has been hampered by a lack f adequate quantitative infrmatin abut the nature and rates f frmatin f the varius metablites frmed by different insects and mammals. Oxidative rutes f metablism are f majr imprtance in the case f the carbamate grup f insecticides, and their txicity may be greatly increased by the use f synergists that act by preventing their xidatin by the micrsmal fractin (Metcalf & Fukut, 1965; Metcalf et al. 1966; Casida, 1969, 197), but whether these synergists influence a single enzyme r whether several micrsmal hydrxylases are cncerned in carbamate detxicatin is nt knwn. It was thught that an assessment f the different types f hydrxylated metablites f a single carbamate in several species might thrw sme light n this. The chice f m-tert.-butylphenyl N-methylcarbamate fr study was made because it had relatively lw txicity and because xidative detxicatin culd be presumed t be imprtant, since in enzyme assays this insecticide is a highly effective inhibitr f acetylchline esterase (Mrefield, 196; Metcalf & Fukut, 1965; Metcalf, Fukut & Wintn, 1962). A further reasn fr the study f this cmpund was the great difference in its effective txicity in 13 flies and mice (Fraser, Greenwd, Harrisn & Wells, 1967), since we have suggested that the xidatin f a selectphric alkyl grup in an insecticide mlecule might accunt fr the relatively lw txicity f sme carbamates in mammals as cmpared with insects (Hk & Smith, 1967). MATERIALS AND METHODS Cmpunds. -tert.-butylphenyl N-methylcarbamate, m.p. 95C, m-tert.-butylphenyl N-methylcarbamate, m.p. 11C, p-tert.-butylphenyl N-methylcarbamate, m.p. 143C, 3,5-di-tert.-butylphenyl N-methylcarbamate, m.p. 12 C, and -isprpxyphenyl N-methylcarbamate, m.p. 91C, were prepared frm the phenls and methyl iscyanate in the presence f triethylamine (Klbezen, Metcalf & Fukut, 1954). m-tert.-butylphenyl carbamate was prepared by the methd f Raifrd & Inman (1934) and had m.p. 168C (Fund: C, 68.2; H, 7.9; N, 7.2. C11H17NO2 requires C, 68.4; H, 7.8; N, 7.3%). m-tert.-butylphenl was tritiated by the methd f Hiltn & O'Brien (1964) and purified by t.l.c. as described belw. It was cnverted int the N-methylcarbamate by treatment with methyl iscyanate, and the [3H]m-tert.- butylphenyl N-methylcarbamate was purified by t.l.c. Stck slutins f this material were diluted with unlabelled material t give a specific radiactivity f.9ci/ ml fr experimental use. 3,5-di-tert.-Butylphenyl N- methylcarbamate (Butacarb) was similarly tritiated and purified (.1 Ci/ml). ['4C]-Isprpxy-[1,3-14C]phenyl N-methylcarbamate (1. Ci/ml) was prvided by the Vectr Cntrl Divisin, Wrld Health Organizatin, Geneva, Switzerland. Experimental animals. Huseflies (MU8ca dmestica) and blwflies (Lucilia sericata) were reared and maintained as described previusly (Jrdan & Smith, 197) and were used as 3-day-ld adults in mst experiments. Bich. 1971, 125

2 386 P. G. C. DOUCH AND J. N. SMITH 1971 Seven strains f huseflies were used in this wrk; six f these had been selected frm an riginal plymrphic strain. These were a strain with a lw resistance t DDT [1,1,1-trichlr-2,2-di-(p-ahlrphenyl)ethane] (V), strains with high DDT resistance assciated with a dehydrchlrinatin mechanism (VD and A), strains that were under pressure with carbamates and lindane respectively but had nt yet shwn increased resistance t these insecticides (VC and VL), a strain with white eyes but therwise identical with the V strain (Y), and a wild strain cllected in Wellingtn with a lw DDT resistance f abut the same degree as the V strain (M). Grass grubs (C8telytra zealandica) were cllected by the New Zealand Department f Scientific and Industrial Research Entmlgy Divisin and stred at 5C in mist earth befre use. Bees (wrkers frm an Api8 mellifera clny) were cllected frm the frnt f the hive in a net and used immediately. Meal wrms (Tenebri sp.) were reared in a dry flur-yeast medium; larvae, pupae and adults were used. Eight-week-ld male mice were used fr wrk with vertebrates. Preparatin f enzyme8. Muse liver was hmgenized at C in a Tefln-and-glass Ptter-Elvehjem hmgenizer in 6vl. f.1 M-KH2P4-Na2HPO4 buffer, ph7.4. The mixture was centrifuged at 1OOg fr 1min and the supernatant used as enzyme fr mst experiments. Muse micrsmal fractin was prepared by centrifuging the 1OOOOg supernatant at 1OOOOg in an MSE Superspeed 5 centrifuge and 421 angle rtr fr lh. All muse enzyme incubatins were at 37C and cntained enzyme equivalent t 1 g f liver in IOml f the O.1Im-phsphate buffer, ph7.4. In incubatins with loooog supernatant in a shaking water bath in air, the cfactrs added were 1mm-nictinamide, 15 mm-mgcl2,.1 mm-nad+,.1 mm-nadp+ and 4mM-glucse 6- phsphate. Enzymes frm huseflies r blwflies were prepared frm fly abdmens by the prcedure described by Jrdan & Smith (197). Incubatin mixtures cntained the same cfactrs as fr the muse enzyme except fr nictinamide and MgC12, and a cncentratin f fly enzyme equivalent t 2 abdmens/ml was used (cf. Jrdan & Smith, 197). Incubatins were at 3C and were terminated, usually after 3 min, by additin f an equal vlume f ether t extract substrates and metablites. Substrate cncentratins in all experiments were 1 mm and cntrls were used that cntained n substrate. Dsing f in8ect8. Nn-radiactive cmpunds were given tpically in acetne slutin by using calibrated 1 pl capillary tubes. In experiments with radiactive cmpunds insects were dsed tpically with.13,iu f acetne slutin cntaining O.9,ug f [3H]m-tert.-butylphenyl N-methylcarbamate frm a calibrated capillary tube and the adults were kept in grups f2-5 in 2 cm x 4 cm test tubes clsed with wire mesh. Larval stages were kept in small Petri dishes. In mst experiments the dse cntained 885±6c.p.m./insect. Mea8surement f 3H radiactivity. Radiactivity was measured in a Packard series 4 scintillatin spectrmeter. Materials sluble in rganic slvents were cunted at 25% efficiency in a sintillatin mixture cntaining 5g f 2,5-diphenylxazle and.3g f 1,4-bis-(5-p-tlylxazl-2-yl)benzene/I f tluene. Aqueus samples (1 ml) were cunted at 2% efficiency in similar slutins in which ne-third f the tluene had been replaced by Tritn X-1 (Rhm and Haas C., Philadelphia, Pa., U.S.A.) (Turner, 1968). Efficiencies in cunting f radiactivities f slutins were assessed by reference t a quench crrelatin curve by using the autmatic external standardizatin facility. Radiactivities f samples f tluene-sluble metablites n sectins f chrmatgraphy paper (2 cm x 1 cm) were cunted in 2 ml f scintillatin fluid. N difference in the c.p.m. was fund between samples whse radiactivities were cunted in this way and duplicate samples whse radiactivities were cunted after chrmatgraphic elutin and assay in full vials f scintillatin fluid. Radiactivities f slutins r suspensins (1 ml) cntaining prtein were cunted after heating at 1 with 1 ml f frmic acid. These slutins were neutralized by 2m-NaOH and their radiactivities cunted in the Tritn X-1-tluene scintillant. Extractin f metablite8. Enzyme incubatins, usually 1 ml ttal vlume, were extracted after 3 min fur times with equal vlumes f ether; this, in experiments with radiactive cmpunds, was sufficient t remve all radiactivity frm the aqueus layer. The ether was dried with anhydrus Na2SO4, evaprated in vacu at 3C and the extract disslved in.2 ml f dixan fr further examinatin. Whle insects were immbilized with C2 and then grund with an equal weight f acid-washed sand in a glass mrtar with 5 ml f ether. Fur successive prtins f ether were used t rinse ut the sand and ether; this, in experiments with radiactive cmpunds, was sufficient t remve all radiactivity frm the aqueus layer. The ether was dried with Na2SO4, evaprated in vacu at 3C and the extract disslved in.2 ml f dixan fr further examinatin. Cntainers that had held live insects were rinsed ut with 2ml f acetne-ethanl-water (1: 1:2, by vl.) and the rinsings added t the extract f grund insects. The radiactivity f a pellet f centrifuged tissue and sand remaining after centrifuging these extracts was als cunted, but this was negligible in experiments with radiactive cmpunds. Measured prtins f the aqueus fractin frm this extractin prcedure were hydrlysed enzymically t decmpse any cnjugated metablites present. An acetne-dried pwdered preparatin f Halitis au8trali8 viscera (1mg) was used as enzyme and the aqueus layers were incubated at ph 5.5 fr 12h befre extractin f metablites as described abve. This enzyme preparatin cntained,-glucsidase, fl-glucurnidase, arylsulphatase and phsphatase, but did nt hydrlyse the carbamate ester linkage in cntrl experiments with 3H-labelled m-tert.-butylphenyl N- methylcarbamate. Ttal recvery f radiactive material frm live insects in these experiments was in the range 98-12% f the applied dse. Chrmatgraphy and inphreai8. Sme t.l.c. was carried ut with.25 mm-thick layers f silica gel G (E. Merck A.-G., Darmstadt, Germany) activated at 12C and plates were usually develped fr 15cm in ether-is-ctane (4:1, v/v). Whatman silica-gel-laded paper n. SG81 was fund mre cnvenient, as it required n activatin and samples were develped by descending chrmatgraphy fr 45cm in the same slvent. lnphresis was carried ut in the Shandn high-vltage

3 Vl. 125 CARBAMATE METABOLISM 387 apparatus as previusly desribed (Jrdan, McNaught & Smith, 197) (Table 1). Clur reactin8. Chrmtrpic acid (.2% in 6M- HASW) at 1 C gave purple clurs with N-hydrxymethyl derivatives (Metcalf et al. 1968). Phenlic metablites were detected with diaztized p-nitraniline r 2,6-dichlrquinnechlrimide (Hk & Smith, 1967). Carbamate esters and phenls were detected by first spraying with 1 M-NaOH in ethanl-water (1:1, v/v) fllwed by the phenl-detecting reagents. Phenyl arbamates and phenyl N-methylcarbamates were detected by red-purple clurs given in their reactin with 2% (w/v) ninhydrin in acetne cntaining cllidine (.1%)at1OC(Gemrich,1967). Withp-dimethylaminbenzaldehyde (2%, w/v, in acetic acid) phenyl carbamates gave yellw clurs in the cld, and phenyl N- methylcarbamates gave similar clurs n heating t 1C (Feigl, 1956, p. 273). p-dimethylamincinnamaldehyde gave red t range clurs under similar cnditins. 1,3-Dihydrxyphenls gave clured and flurescent prducts when heated fr 3min at 1 C after spraying with 1% (w/v) -phthalaldehyde in cnc. H2SO4 r with 1% (w/v) -sulphbenzic anhydride in cnc. H2SO4 (Feigl, 1956, p. 382). Clurs were intensified by spraying with lom-naoh; 1,3-dihydrxyphenls als gave yellw clurs when they were sprayed with 5% (w/v) fructse in 6M-HCI and heated t 1 C. Aldehydes gave yellw clurs when sprayed with di--anisidine in acetic acid and als dark spts with ammniacal AgNO3. Gaa-liquid chrmatgraphy. A Perkin-Elmer 81 gas chrmatgraph was used with a flame inizatin detectr. Clumns (.4 cm x 4 cm lng) were packed with 5% (w/w) Carbwax 6 n 8-mesh Celite that had been treated with 2% (w/v) phsphric acid (Kllff, Breuklander & Barkley, 1963). Retentin times at 7, 85 and 125 C with N2 as carrier at 12 ml/min clumn flw are shwn in Table 1. Cmparisns f retentin times were made with injectins f bth separate and mixed cmpunds. RESULTS Enzymnic xidatin f m-tert.-butylphenyl N-methylcarbamate In mu8e liver preparatin8. Incubatins were made as described abve with enzyme frm 1g f liver in a ttal vlume f loml. After.5h the substrate and prducts were extracted with ether as described abve and samples f the extract were separated in the t.l.c. system r n Whatman n. SG81 paper. Eight clured spts were btained, and these were identified as the cmpunds listed in Table 2 by use f the clur reactins described belw. Tw, metablites e and h, were phenlic, since they gave clurs immediately with diaztized p-nitraniline; the rest were phenl esters which gave this reactin after preliminary treatment with 1 M-sdium hydrxide. Tw spts, metablites a and d, gave the purple clur with chrmtrpic acid characteristic fn-hydrxymethyl derivatives. Tw, metablites b and f, gave yellw clurs with p-dimethylaminbenzaldehyde and acetic acid in ee d._ c Ca H p C) 5 ^ Ca C4q A-. r S,) 4.anQ 1 5- )n---c S.t SS)C.n6.5 3 ), Ca81 d t ri 1 1 ~ 5Ca e e S jo.1- -a... -a C) A- -4 6,C 5 I.-, OI P P, P4 p4 pq 4'5) 1) 4 rn P4 c c l C t P4 C CO 6 c c O " t O 1 " C CO ).W NO C)"5 ; X "t 33H x.d 1)

4 388 P. G. C. DOUCH AND J. N. SMITH 1971 the cld, which is given by esters f carbamic acid, and the metablites a, c, d and g gave this 6 h reactin nly n heating and were presumed t be v N-substituted carbamates. Spts were eluted 4) chrmatgraphically frm Whatman n. SG81 paper with ether, and the cncentrated eluates were "4 hydrlysed by warming with a drp f 1 M-sdium 4) hydrxide. After acidificatin and transfer t a small vlume f ether the hydrlysis prducts were z chrmatgraphed n Whatman n. SG81 paper r the g.l.c. system and separated by inphresis in p4 each f the systems given in Table 1. N carbxylic acids culd be detected by their inphretic * Ca behaviur. Metablites a, b and c yielded n Sd CB + I I I hydrlysis a cmpund identical with the phenlic metablite e n chrmatgraphy and inphresis 4s) _ "-. p4 "l ) - 4) e4 Cs * ) OC) -4 + X4) P4 4) m Id ds 4) I + + v CeXC O e P O OO ( O 4) -4. ID -4) k 4)'1 "4 -?e,4a ff) 3X sus and by clur reactins. Metablites d, f and g yielded a hydrlysis prduct with behaviur identical with that f m-tert.-butylphenl (metablite h). Only ne phenlic prduct was fund after hydrlysis f each f the eight spts, and n diphenls were detected with ammniacal silver nitrate r clur reagents fr m-dihydrxybenzenes. N clur reactins fr aldehydes were btained. Samples f the tw phenlic hydrlysis prducts frm a number f separate experiments were pled and chrmatgraphed in ether-is-ctane n the t.l.c. system, and the apprpriate znes (RFO.5 and.8) were eluted and evaprated t yield apprx..5mg f residue in each case. These were heated separately n a biling-water bath in.5ml f aq. 5% (w/v) phsphric acid and.5ml f aq. 5% (w/v) ptassium permanganate fr 1Bmin. Each slutin was declrized with slid sdium metabisulphite and 2.5ml f frmic acid, and then extracted with ether. After being dried with sdium sulphate and evapratin, prtins f the tw ether slutins were examined in the separatin systems and cmpared with the reference cmpunds listed in Table 1. Tw cmpunds giving the clur reactins f phenls were present in each case, ne f which behaved identically with the unchanged hydrlysis prduct (metablite e r h) and the ther with m- hydrxybenzic acid. Prducts with RF values identical with that f m-hydrxybenzic acid in system B (Table 1) were als btained by similar xidatins f reference m-tert.-butylphenl, but rcinl yielded a different prduct, presumably cxresrcylic acid, having RFO.3 in slvent system B. Samples f metablite e were btained by pling 5 muse liver incubatins, hydrlysing the ether extracts with sdium hydrxide as abve and separating the liberated phenlic material in slvent system A. The phenlic metablite e, RFO.S5 Was eluted with ether and purified by micr-distillatin at 15C and 2mmHg. The clurless il was disslved in ether, and mass-spectral measurements were made by Prfessr R. Hdges with an A.E.I.

5 Vl. 125 CARBAMATE METABOLISM 389 MS92b mass spectrmeter. Majr m/e peaks were btained at 166, 135, 17, 95 and 77; the empirical frmula fr each f these peaks was determined by precise mass measurements and the pathways were shwn by metastable peaks. Mass-spectral data were cnsistent with the identificatin f metablite e as m-(,-hydrxy-tert.-butyl)phenl and shwed that breakdwn pathways were prbably as indicated in Scheme 1. In husefly and blwfly enzyme preparatins. Similar incubatins were carried ut in enzyme prepared frm husefly and blwfly abdmens with m-tert.-butylphenyl N-methylcarbamate as substrate. Each incubatin mixture cntained the equivalent f 1 flies in a ttal vlume f 5ml. Extractin and chrmatgraphy were carried ut as abve and eight spts were detected by t.l.c. with RF values identical with thse btained in parallel experiments with muse enzymes. The metablites were identified afs described abve fr the muse metablites by their clur reactins and by inphresis, chrmatgraphy and g.l.c. f hydrlysis prducts in the svstems given in Table 1. Fly metablites were identical with thse frm the muse experiments, and n ther metablites were fund. Quantitative assays. Incubatins were carried ut with muse, husefly and blwfly preparatins with.1,uci f [3H]m - tert. - butylphenyl N - methyl - carbamate. Incubatin mixtures were extracted with ether after.5h and chrmatgraphed n t.l.c. systems r n Whatman n. SG81 paper as described abve. Znes crrespnding with the eight chrmatgraphic spts were eluted and their radiactivities cunted in the scintillatin spectrmeter as described abve. Results f these experiments are listed in Table 3. Similar experiments with muse enzyme and husefly enzyme were perfrmed at OH 166 -CH,-OH 15+ CH3 sch3 i ~~~~135 -c * 17 OH H -H2 H Scheme 1. Prbable spectrmetry. breakdwn pathway f m-(fl-hydrxy-tert.-butyl)phenl (metablite e) during mass Table 3. Enzymic metablism f [3H]m-tert.-butylphenyl N-methylcarbamate The substrate (1 mm) was incubated at 37 C in loml f.1 M-phsphate buffer, ph 7.4, cntaining.1 mm- NADP+, O.lmM-NAD+, 4mM-glucse 6-phsphate and the loooog supernatant f a hmgenate f I.Og f muse liver in.1 M-phsphate buffer. Husefly and blwfly experiments were similar except that the enzyme preparatin was an hmgenate f 2 fly abdmens in.1 M-phsphate buffer, ph 7.4. Metablites were thse identified in Table 2. Results are listed as mean values with ranges in parentheses and numbers f experiments as a superscript. Rate f frmatin (,uml/h per g f fly r liver) Metablite Surce f enzyme... a b c de f A Blwfly.5 (.5)4.3 (.3)4.17 (.17)4.8 (.7-.9)4.2 (.1-.2)4.17 (.17)4.11 (.9-.13)4 Husefly, V strain.14 (.1-.32)12.14 (.8-.29)12.18 (.1-.58)12.25 ( )12.7 (.-.19)12.38 ( )12.12 (.-.36)8 Husefly, M strain.57 (.55-.6) (.22)2.34 ( )2.35 ( )2.15 (.1-.19)2.17 ( )2.83 (.8-.86)2 Muse liver.11 (.2-.17)1.4 (.1-.6)1.7 ( )1 1.6 ( )1.4 (.1-.6)1.96 ( )1.86 ( )1

6 39 P. G. C. DOUCH AND J. N. SMITH 1971 ph values between 6 and 9, and the amunt f each butylphenl frm metablites f and h and m-(,- metablite prduced at the different values was hydrxy-tert.-butyl)phenl frm metablites b and e. pltted as ph-activity curves. Fr each metablite the maximum rate f prductin ccurred between Enzymic xidatin f - and p-tert.-butylphenyl ph 7 and 7.5. Similar experiments were made with N-methylcarbamate muse enzyme and husefly enzyme at ph 7.4 in which prtins f the incubatin mixture were withdrawn and analysed chrmatgraphically fr the Incubatins were carried ut with muse enzyme and extractins made as described abve with seven metablites at 1min intervals. Rates f p-tert.-butylphenyl N-methylcarbamate, and the frmatin f each f the metablites were cnstant extracts were chrmatgraphed in the t.l.c. system as up t.5h (Fig. 1). described abve. Eight spts were again detected n these chrmatgrams with the clur reagents Enzymic xidatin f m-tert.-butylphenl and used, and these spts were nt significantly different m-tert.-butylphenyl carbamate in RF values frm the crrespnding spts fund in Muse liver incubatins were perfrmed, as experiments with the meta ismer. Each f the described abve, in which m-tert.-butylphenl was eight spts was eluted with ether and hydrlysed used as a substrate. Extractin f the incubatin with sdium hydrxide, and the liberated phenl mixture after.5h fllwed by chrmatgraphy in was subjected t inphresis at the ph values ether-is-ctane n Whatman n. SG81 paper shwed the presence f nly tw prducts. These were eluted and examined inphretically in each f the chrmatgraphic systems given in Table 1 and shwn t have prperties identical with thse f m-tert.-butylphenl and m-(p-hydrxy-te.rt.-butyl)- phenl (metablite e frm mice). Similar experiments with m-tert.-butylphenyl carbamate yielded chrmatgrams with fur spts. These had the same RF values and clur reactins as metablites b, e, f and h ftnd in the metablism f m-tert.- butylphenyl N-methylcarbamate in muse liver. On elutin with ether and hydrlysis in 1 M-sdium hydrxide each f the fur metablites gave nly ne phenlic hydrlysis prduct, namely m-tert.- f-4 C) bo I.c given in Table 1. These hydrlysed slutins frm the para ismer cntained phenlic material that culd nt be distinguished, by the methds used, frm the crrespnding phenls liberated by hydrlysis f the analgus spts frm chrmatgrams f the m-tert.-butylphenyl N-methylcarbamate. Similar incubatin mixtures carried ut with the -tert.-butylphenyl N-methylcarbamate were extracted and after chrmatgraphy in the t.l.c. system als gave rise t eight spts with RF values nt significantly different frm thse btained frm either the meta r the para ismer. Hwever, with the rth ismer the spts with RF values.5,.14,.37 and.46 (spts a, b, d and e) were nt due t single substances. Hydrlysis f each f these spts with sdium hydrxide gave rise t tw phenlic prducts that culd be distinguished by chrmatgraphy, ne f which readily reduced ammniacal silver nitrate. These metablites were nt further investigated and were presumed t arise frm hydrxylatin f the armatic ring ) I'd 1.5 [ 3 6 Time (min) Fig. 1. Frmatin f metablites f [3H]m-tert.-butylphenyl N-methylcarbamate in muse liver enzyme preparatins. Incubatin cnditins were the same as in Table 3. Prtins were withdrawn, extracted and analysed fr metablites by t.l.c. as described in the text. Metablites a-h are thse identified in Table 2. Enzymic hydrlysis f carbamates The radiactively labelled carbamate insecticides were incubated at ph 7.4 in.1 M-phsphate buffer with fly abdmen enzymes at 3C r at 37C with the 1OOg supernatant f lg f muse liver and with the cfactrs listed in Table 4. Reactin mixtures were extracted after.5h and the ether extracts chrmatgraphed n Whatman n. SG81 paper as described abve. Znes crrespnding t m-tert.-butylphenl, -isprpxyphenl and 3,5- di-tert.-butylphenl were lcated by cmparisn with reference samples and the apprpriate znes were cut ut and assayed fr radiactivity. Cnversin int the phenl was expressed as a percentage f the ttal radiactivity used (Table 4). Similar experiments with muse liver micrsmal fractin frm 1 g liver (1 OOOg sediment) shwed

7 Vl. 125 CARBAMATE METABOLISM 391 Table 4. Cfactr-dependent cleavage f sme N-methylcarbamates Incubatin mixtures were prepared as in Table 3 except that cfactrs and glucse 6-phsphate were added as shwn belw. Substrates were: A, m-tert.-butylphenyl N-methylcarbamate; B, -isprpxyphenyl N- methylcarbamate; C, 3,5-di-tert.-butylphenyl N-methylcarbamate. % f substrate cleaved Surce f enzyme... Substrate... Cfactrs Nne NAD+, glucse 6-phsphate NADP+, glucse 6-phsphate NAD+, NADP+, glucse 6- phsphate Muse liver A B C Husefly Blwfly A B A B C that n hydrlysis ccurred in unfrtified preparatins and that additin f either NADPH r a mixture f NADP+, glucse 6-phsphate and glucse 6-phsphate dehydrgenase t the incubatin mixture resulted in the liberatin f phenl equivalent t 2% f the substrate added. N hydrlysis f the three carbamates used was detectable when they were incubated with a 1 :1 dilutin f muse bld at 37C fr 3min at ph 7.4. Metablism f m-tert.-butylphenyl N-methylcarbamate in viv Grups f insects weighing in all abut 1-2g were dsed tpically with [3H]m-tert.-butylphenyl N- methylcarbamate and extracted after either 12h r 24h by grinding with sand and ether as described abve. Unextracted material was enzymically hydrlysed and the slutin re-extracted. The pled extracts were chrmatgraphed in slvent system A (Table 1) and eight spts were detected at the same RF values and having the same clur reactins as thse fund in the enzyme incubatin experiments. The quantitative values btained in these experiments with [3H]m-tert.-butylphenyl N- methylcarbamate are summarized in Table 5; the difference between the ttal extractable metablite and that extractable nly befre enzymic treatment has been assumed t be cnjugated material. Qualitative experiments were als made with larvae f the prina mth (Wiseana sp.) and the whitefringed weevil (Graphgnathus leuklma): chrmatgraphy f these extracts als shwed eight spts, which were shwn by the chrmatgraphy, inphresis and clur reactins described abve t be the same as thse frmed by mice. Mice (25g) were starved fr 24h and dsed rally r intraperitneally with 1mg f [3H]m-tert.- butylphenyl N-methylcarbamate in.25ml f aq..5% Tritn X-1 slutin. N significant differences in excretin rates were discerned after the tw methds f administratin, and in six experiments 7% (59-82%) f the radiactivity was excreted in the urine after 24h. A further 7.9% (5-12%) was excreted during the next 3 days. Each 24h cllectin f urine was extracted as described abve, befre and after treatment with the hydrlytic enzyme. In cntrast with the insect experiments, material cntaining 8.6% ( %) f the radiactivity in muse urine was nt hydrlysed by the enzymic treatment and this fractin was nt further investigated. Extracted metablites were chrmatgraphed n Whatman n. SG81 paper as described abve, and eight spts were recgnized with Rp values and clur reactins identical with thse f the metablites a-h fund in the insect and enzymic experiments. Hydrlysis f each f the metablites with 1 M-sdium hydrxide yielded phenlic materials identical in chrmatgraphic and inphretic prperties and clur reactins with m -tert. -butylphenl r m- (P-hydrxytert.-butyl)phenl, as shwn in the experiments with enzymic metablism described abve. DISCUSSION The same seven metablites fm -tert. -butylphenyl N-methylcarbamate were fund in all the species and instars examined, thugh the amunts f each metablite shwed sme species variatin (Table 5). The mst striking quantitative difference was in the amunt fund f the hydrlysed metablites, m-tert.-butylphenl and m-(fl-hydrxy-tert.-butyl)- phenl, which in uncnjugated r cnjugated frms accunted fr mst f the metablized dse in mice and meal wrms. Esterases that can hydrlyse carbamate insecticides t the crrespnding phenls have been fund in insect haemlymph and in vertebrate bld (Casida, 1963; Matsumura & Sakai, 1968), but it seems likely that these were f nly minr significance in the frmatin f the tw phenlic metablites. The lack f any hydrlysis f this insecticide in fly hmgenates has been

8 392 P. G. C. DOUCH AN] J. N. SMITH 1971 ) C4 _ ) t _ e bserved by Mrefield (196). The actin f gut cd scq 4 ' c) P -e esterases in the mice seems als t be unlikely, since ;-4-4 '.e4 CO 1 t It- O "46C61' CO -; C little difference in the pattern f excreted metablites was fund after either ral r -4 1 eq intraperitneal 4"e 1, Ca C e ~fcd Cae *Ce C Ced.CCB ~ Ca Wa 1'e, CB 41 s Ce a, u X 1 Ca11 4- Oe CB,581 H g Ce< 4) Ca O 41 w, c I- S ++ _-O _O -t * cw el I-r> = ed :L -1P O eq, H:,, -,' 1 ^'I 1 ++ O - ^ O 6 O c6 c- 4 OC1 C9 CO 4 ~~~~~~~ We c c1 CO1 C ~ ~ ~ ~ ~ ~ s41 'D Ca _ --D p 4 X^m~4111C e L.a Nt acs *-c -1 uzls m r P ~~~~ m, ) C;l -I - 1 O-SO e e 5 1,5 1 4O e c i C'Ca r~~~~~~~c COi& -.eq s.h4 4-.i e * 4- C &D administratin f the insecticide. The unfrtified liver micrsmal fractin, the liver 1 g supernatant and muse bld cntained an abundance f esterase capable f rapid hydrlysis f, fr instance, phenyl butyrate r ethyl acetate, but these had n significant actin n the insecticide (J. N. Smith & J. C. Turner, unpublished wrk). Hydrlysis f m-tert.-butylphenyl N-methylcarbamate by fly hmgenate was als negligible until NADP+ and glucse 6-phsphate were added (Table 4). Similarly the muse liver 1g supernatant gave n hydrlysis until these cfactrs were added. It seems prbable that mst f the cleavage f the carbamate insecticide in viv was brught abut by a micrsmal NADPH-dependent system similar t that bserved by Neale (1967) and by Nagatsugawa & Dahm (1969), which liberates p-nitrphenl frm parathin (OO-diethyl O-p-nitrphenyl phsphrthinate) nly in the presence f the usual hydrxylatin cfactrs fund in the micrsmal fractin. If this is s, then all the detxicatin reactins undergne by this insecticide are catalysed by the micrsmal system, thugh the species differences between the degree f hydrxylatins and the extent f ester-cleavage suggest that these tw prcesses are nt mediated by the same enzyme. N metablites frmed by hydrxylatin f the armatic ring were fund in any f the species examined, and thse xidized metablites present may be divided int tw grups. They are metablites resulting frm hydrxylatin f the tert.- butyl grup and thse frmed as a cnsequence f hydrxylatin in the N-methylcarbamyl functin. It seems reasnable t include m-tert.-butylphenyl carbamate (metablite f) as well as metablite b (Table 2) in this grup since they may be assumed t be the prducts f xidative demethylatin. Metablites were present where bth types f xidatin had taken place (metablites a and b), thugh these usually represented less than 1% f the ttal xidized metablites. On the ther hand, the grass grub, which has the lwest rate f xidatin by the micrsmal fractin f any insect examined in this labratry (Hk, Jrdan & Smith, 1968), cnverted abut half f the insecticide int dubly xidized metablites (Table 5). Frmatin f the dubly xidized prducts was nt dependent n the prir frmatin f large quantities f the singly xidized metablites, since in bth muse (Fig. 1) and fly enzyme systems the rate f frmatin f the dubly hydrxylated prducts was cnstant frm the start f the incubatin. The analytical results frm experiments with intact insects and mice suggest that the mechanisms

9 Vl. 125 CARBAMATE METABOLISM 393 that give rise t the tw types f xidized metablites may be different. Thus in blwflies the insects s far as xidatins by the micrsmal (apprx. 5mg/g), which are amng the mst active amunt f hydrxylatin f the tert.-butyl grup fractin are cncerned (Hk et al. 1968), and high greatly exceeds that f the N-methylcarbamyl functin (Table 5). If metablites c and e are taken as an index f the alkyl hydrxylatin and metablites d andf (Table 2) as an index f the xidative demethylatin pathway the rati f these is abut 5: 1 in blwflies and 4: 1 in mice cmpared with a rati f 1: 3 in the grass grub. This distributin f metablites in the intact animal des nt, hwever, reflect in the rates f hydrxylatin by the micrsmal fractin in the enzymic experiments with flies and muse liver (Table 4). In these experiments the xidative demethylatin prcess takes place at a greater rate than the xidatin f the tert.-butyl grup. This des nt exclude the pssibility that mre than ne micrsmal hydrxylase is cncerned in the detxicatin f this insecticide, since the enzyme systems were prvided in vitr with ptimum cncentratins f cfactrs and in these circumstances hydrxylatin rates (as uml/min per fly) may be much greater than in intact insects (Jrdan & Smith, 197). If mre than ne hydrxylatin system is present in the micrsmal fractin f muse liver r in insects the relative effectiveness f these systems wuld be mdified by the availability f cfactrs and differences in the affinities f several pssible systems fr them. It is nt thught that limited availability f substrate in the experiments described abve culd have affected either the results btained in viv r thse btained in vitr, since the dses and cncentratins used were sufficient t give maximum and cnstant rates f metablism (Hk & Smith, 1967; Jrdan & Smith, 197). It has been shwn with ther carbamate insecticides that the hydrxylated metablites may still pssess significant anti-chlinesterase activity and that sme metablites may exceed the effectiveness f the parent insecticide in their abilitv t inhibit this enzyme (Onithan & Casida, 1968; Drugh, 197). It is unlikely, hwever, that xidatin prcesses in the whle insect give any effective activatin f a carbamate insecticide, since the xidatin prducts are themselves substrates fr thse enzymes that detxify by cnjugatin. In the present wrk majr prprtins f all the metablites were present in a frm that was nt extractable by ether until after enzymic hydrlysis (Table 5). It seems prbable that the critical detxicatin prcess that inactivates the insecticide is the xidatin r NADPH-dependent ester-cleavage in the micrsmal fractin and the txicity f this insecticide is inversely related t the ability f the varius species t carry ut xidatin in the micrsmal fractin. The txicity is thus lw in flies in thse species, like grass grubs and bees (Metcalf & Fukut, 1965), the micrsmal fractin f which is knwn t have nly a lw xidatin ability. This wrk has been supprted by equipment grants frm the New Zealand University Grants Cmmittee, the Department f Scientific and Industrial Research, the Glden Kiwi Bard f Cntrl and the Internal Research Cmmittee f this University. P.G.C.D. was maintained in part by a grant frm Merck, Sharpe and Dhme Ltd. REFERENCES Casida, J. E. (1963). A. Rev. Ent. 8, 39. Casida, J. E. (1969). In Micrsmes and Drug Oxidatins, p Ed. by Gillette, J. R. & Futs, J. R. New Yrk: Academic Press Inc. Casida, J. E. (197). J. agric. Fd Chem. 18, 753. Drugh, H. W. (197). J. agric. Fd Chem. 18, 115. Feigl, F. (1956). Spt Tests in Organic Analysis. New Yrk: Elsevier Publishing C. Fraser, J., Greenwd, D., Harrisn, I. R. & Wells, W. H. (1967). J. Sci. Fd Agric. 18, 372. Gemrich, E. G. (1967). J. agric. Fd Chem. 15, 617. Hiltn, B. D. & O'Brien, R. D. (1964). J. agric. Fd Chem. 12, 236. Hk, G. E. R., Jrdan, T. W. & Smith, J. N. (1968). In Enzymatic Oxidatins f Txicants, p. 27. Ed. by Hdgsn, E. Raleigh: Nrth Carlina State University Press. Hk, G.E. R. & Smith, J. N. (1967). Bichem.J.12,54. Jrdan, T. W., McNaught, R. W. & Smith, J. N. (197). Bichem. J. 118, 1. Jrdan, T. W. & Smith, J. N. (197). Int. J. Bichem. 1, 139. Klbezen, M. J., Metcalf, R. L. & Fukut, T. R. (1954). J. agric. Fd Chem. 2, 864. Kllff, R. H., Breuklander, L. J. & Barkley, L. B. (1963). Analyt. Chem. 35, Matsumura, F. & Sakai, K. (1968). J. ecn. Ent. 61,598. Metcalf, R. L. & Fukut, T. R. (1965). J. agric. Fd Chem. 13, 22. Metcalf, R. L., Fukut, T. R., Cllins, C., Brck, K., Abdel Aziz, S., Munz, R. & Cassil, C. C. (1968). J. agric. Fd Chem. 16, 3. Metcalf, R. L., Fukut, T. R., Wilkinsn, C., Fahmy, M. H., El Aziz, S. A. & Metcalf, E. R. (1966). J. agric. Fd Chem. 14, 555. Metcalf, R. L., Fukut, T. R. & Wintn, M. Y. (1962). J. ecn. Ent. 55, 345. Mrefield, H. H. (196). Ent. Sc. Am. Misc. Publ. n. 2, p Nagatsugawa, T. & Dahm, P. A. (1969). Bichem. Pharmac. 18, 113. Neale, R. A. (1967). Bichem. J. 15, 289. Onithan, E. S. & Casida, J. E. (1968). J. agric. Fd Chem. 16, 28. Raifrd, W. C. & Inman, G.. (1934). J. Am. chem. Sc. 56, Turner, J. C. (1968). Int. J. appl. Radiat.Istpes,2, 499. Winteringham, F. P. W. (1969). A. Rev. Ent. 14, 49.