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Metabolism of busulfan in the boll weevil (Anthonomus grandis Boheman)
PESTICIDE
BIOCHEYIYTitY
.\Sl)
Metabolism
I’lIl-sIOI.OGY
1,
of Busulfan
418-423
in the
grads GLENN Entomology
Research
Division
Weevil
(Anthonomus
Boheman)
WIYGUL
, dgricullural State
Boll
AND
NORMAN
Research Service, College, Mississippi
~IITLIS 7:nitetJ 39762
States
Department
of
Agriulltllre
AND
,4. C. Pesticide Received
December
THOMPSOX
Chemicals
Research
7, 1971;
accepted
Branch December
8, 1971
One-day-old male boll weevils, Anthonomus grandis Boheman, that had been forcefed 3H, and IYXabeled busulfan were held for 24 and 72 hr, respectively. The resulting metabolites from the feces and extracts of the weevils were analyzed by thin-layer chromatography, gas-liquid chromatography, and mass spectroscopy. At 72 hr postingestion, 34.32Tc of the total radioactivity was recovered from the feces, about 1.09ci; as busulfan. Most metabolism of busulfan appeared t,o t)ake place within 24 hr postingestion. Organic acids, amino acids, busulfan, 1,4- and 2,3-butanediols, and sldfolane were identified as metabolites. Methanesulfonic acid was det,ermined as a metabolite by using unlabeled busulfan.
reared by the methods of Gast (3). Busulfan labclcd with 3H at the 1,4-positions of the butanediol moiety was purchased from Schwarz Biorescarch Inc., Orangrburg, NY as a 1’ ; ethanol solution with a specific activity of 8000 ;\ICi/mM. When tlio compound was chromatographcd in thrw thinlayer systems, it gave only one spot. Busulfan labeled with 14C on the 1,4-positions of the butancdiol moiety was supplied by the Radiation and Metabolism Laboratory, Agr. Rrs. Serv., USDA, I’argo, KD; it had a and also specific activity of 2 MCi/mR’I gave only onr spot when it was chromatographed on three t,hin-layer systems.
INTRODUCTION
Busulfan (Myleran); (1-4-butanediol dimethanesulfonatc) has only recently been found to be an effective chemosterilant for the male boll weevil (Anthonomus granclis Boheman) (1) though it has been used for some time in the treatment of myelocytic leukemia (3). However, the compound is the chemosterilant of choice for the pilot eradication program against thcl boll wwvil currcntly underway. It, was thcreforc necessary to determine the fate of tht> compound and to identify the metabolitcs resulting when it was fed to the boll wwvil. MATERIALS
AND
METHODS
Treatment
Insects ancl Reagents
The test insects were force-fed aliquots of the original solution of the 3H or 14C-labeled busulfan by the method of McLaughlin et al.
The insects used were one-day-old males from our laboratory culture that had been 418
METABOLISM
OF
BUSTJLFAK
(4) as modified by Wiygul et al. (5). Then they wrc held in an incubator at 33°C for 24 or 72 hr with cotton squares (buds) as food. After the> requisite t)ime, the wewils were ground in a Potter-Elvejhem homogcnizcr, and the homogenate was extracted twice wit,11 10 YI acetone-water and once with acctonc,. (In preliminary tests, no radioactive metabolitcs were found in a nonpolar extract of bhc wrwils.) The acetonc c>xtract was then taken to a known volume by evaporation at, room temperature wit’h nitrogen. Also, the feces were collected from thr containers where the insects had been held a,nd ext,ractcd in the same way. lletemiriatio?r.
0-f c:lllabeled Metabolites
In addition, a brei of ground boll weevils wit,11 enough insect saline solution to give it a liquid consistency was incubated at 37°C for 24 hr with busulfan added in the amount’ of 100 pg;jinscct’ used to make the brci. After incubation, the brei was extracted as described, spotted on TLC plates, and chromatographcd. The ext’ract was used to: (1) drtcrmine the presence of 1,4-butanediol by GLC and (2) t,o determine that methanesulfonic acid was a metabolitc of busulfan in the boll wwvil.
Thin-layw chromat,ograph\(TLC), gasliquid chromatography (GLC), and mass spectroscopy were used for quantitative separation and identification of the radioactiw mt+abolitw. All analyses were rcplicatc>d five timc>s.
TLC Separations Trams’ carlior work with rats (6) in which 1~0uwd paper chromatography supplied us with a pattwn for idcnt’ification of 1,4butancldiol, sulfolane, and busulfan. Howwwr, in the TLC system wc developed to scparatch thcsc mrtabolitcs WCused act’ivated silica gr,l G and chloroform-methyl et’hyl kc+orw (1: 1) in :I saturat,ed atmosphere;
Ipi
THE
BOLL
lYEEVIL
419
maximum separation was achieved by developing the plates in both directions. The organic acids and wlatrd compounds wrr separated by using II -butanol-awtic acid-wat’er, (120 : 30: 50) in a sat,uratcd atmosphere in OIW direction (7) and ethanol-ammonia-water (160: 10: 30) (8) in a saturated atmosphrre in the other dircction with activated silica gel CTas the sub&rate. The methods of Fox et al. (Y) were used to separate met~harw sulfonic acid by TLC, but me used activated silica gel G as a substrate rather than papw, and the solvent systems were 1~-butanol-dioxane-~ N ammonia, (4: 1:5) and n-butanol-2 N acetic acid (1: 1). The amino acids and related compounds were separated by the TLC method of Jonts and Hrathcot’c (10). After t,he mctabolitrs n-w> located, the spots were scraped from the TLC plates into vials containing O’Brien’s scintillation fluid (ll), and a thixotropic gel was added (Cab-O-Sil) to suspend the> TLC particlcs. Tht> radioactive samples were counted in a Packard model X320 liquid scintillation counter (Packard Instrument Co., Des Plainrs, IL). Results were first calculated as disintegrations prr minute (dpm’s) and then convcrtcld to ptlrccntagc of material force-fed. Also, unlabeled standards of all suspected metabolites were added to the cxt’ract of weevils to facilitate idcntificat’ion on the TLC platw. Husulfan \vas idcntificd by modifying the procedure dcvelopcd by Sawicki et al. (12). The TLC plate was sprayed with 4-(p-nitrobenzyl)p):ridinc (1 ‘,; w/v acetone sol), heated to 120°C for 10 min, and thPn sprayed with a %.5“;, cyclohcxylaminr solution ; t,hc busulfan then devrlopcd a purple color. Vanallin-sulfuric acid (13) was used to locate the 1,4 and 2,3 butancdiols. Sulfolane \vas visible at the concent,rations used without treatmclnt. The organic acids n-crc visualizt>d by using TJ(dimethylamino) bcnzaldchgdc in acet,ic anhydride as a spray and than spraying wit,11
420
WIYGUL,
MITLIN,
AND
bromcresol green reagent (7). The amino acids and related compounds were detected by using ninhydrin as a spray reagent.
Xass
The quantitative determinations of the presence of 1,4 butanediol as a metabolite of busulfan were made on a GLC equipped with a flame ionization detector with a 6-ft, s in. o.d. stainless steel column packed with 10.5% DEGA on 60/80 mesh HNDStreated Gas Chrom-P. The injector and detector temperatures were 175’ and 19O”C, respectively; the column temperature was 140°C. Standards consisted of the isomeric butanediols and sulfolane. Sulfolane and 2,3 butanediol were apparently present in such minor quantities that’ n-e were not able to det’ermine their presence by our GLC methods though we were able to determine their presence by mass spectral analysis and, in the case of sulfolanc, by TLC done with radioisotopes.
from
the boll weevil
at 24 and
‘,L, Boll weevil
y. recovered
in extract
DISCUSSION
1
of
YZ hr postingestion extract resultsC
24 hr postfeeding (%A
Total
AND
The results (Table 1) indicated that the butanediol port’ion of the busulfan molecule enters into a number of metabolic reactions which would be expected since the 4 carbon structure of 1,4-butanediol would seem to allow easy conversion to acids of the citric acid cycle. (Trams et al. (6) fed 14C-labeled 1,4-butanediol to rats and found several labeled organic acids in the urine.) At 24 hr postingestion, 1.5 5 of the radioactivity fed the insects was recovered as identifiable
and %On
1.03% 3. 042d 0.2% 0.1 0.29 0.27 0.58 0.31 1.44 6.52 1.71d 6.61 37.68%
a Expressed as percentage of busulfan force-fed. * Replicated three times unless otherwise noted. c No differences could be determined at the 5y0 level tract by using lkmcan’s new multiple range test. d Replicated five times.
3H- and
W-lateled
busulfan”,
feces Extract (%‘I
72 hr postfeeding (‘G 1
f
(%I
0.53 1.73 0.85 0.62 0.50 0.48 0.85 2.01 0.98 4.46 1.64
1.09 2.40 0.85 0.01 4.92 1.54 0.01 0.05 4.09 0.73 4.03
0.77 1.55 0.80 0.0 2.13 0.87 0.01 0.01 3.20 0.61 0.78
25.77”;
34.32:4,
5.35
of significance
b
test 72-hr
Metabolite
Busulfan 1,4- and 2,3-butanediol Sulfolane Citric acid Malic and malonic acids Succinic acid Fumaric acid (Y Ketoglutaric acid Amine Aldehyde Amino acids co2
Analysis
RESULTS
TABLE recovered
Spectral
For the mass spectral analyses, radioactive spots from samples of metabolites were located on TLC plates by using a thinlayer radio chromatogram scanner and elutcd and subjected t’o mass spectral analysis for confirmation of busulfan, 1,4and 2,3-butanediols, and methane sulfonic acid.
GLC
Metabolites
THOMPSON
between
24 and
72 hr weevil
ex-
METABOLISM OF BUSULFAN IX THE BOLL B:EEVIL organic acids. Also, another 6.52 5 was recovered as a compound or compounds that gave a positive aldehyde test with 2,4dinitrophcnylhydrazine (14). We spcculatc that’ thcsc aldehydcs arc intermediates brtown 1,4-butarwdiol and the di- and tricarboxylic acids. 1,4-Butanediol itself appearcld at 24 hr postingestion as 3.045 of tht> chcmostcrilant originally fed. Thcrefort>, bccausr hydrol\-tic cleavage of the busulfan molecule would yield 1,4-butanediol, a busulfan * 1,4-butanediol -+ dicarboxylic acid mct,abolic path would seem to b(l ow route by which this compound is metabolized. Also, the results appear to indicate t’hat most metabolism takes place in the first 24 hr posting&ion. After the busulfan molecules entered into citric acid cycle reactions, fragments would bc, expected to cntcr into a number of metabolic reactions. WC, therefore determincxd t,hc percentage of radi0act’ivit.y in the amino acid fraction of the extract and found that 1.71 c;, of thr radioactivit,y was present thcrc at 24 hr postingestion. ,4lso, a compound that had an Rf value of 74 in the butanol-acetic acid-water TLC system (7) and an Rf value of 42 in t’he ethanol-ammania-water TLC system (8) gave a positive reaction for amincs with p-(dimethylamino) bcnzaldehyde-hydrochloric acid (15) but. a mgative ninhydrin test, which indicated that then> were no free amino groups. Moreover, in an ethyl ethw, 0.1 N HCl extraction of this compound from TLC plates, the radioactivit’) wmaincd in the HCl, further evidenw that an amine was present. If t,his spot, consists of tertiary amines as the evidence indicates, it may be nitrogen-coupled mctabolites that we have already identified. At 72 hr postingestion of the 3H-labelcd busulfan, about 4.46 ’ ( of the radioactivity was recovered as citric acid cycle acids, an amount that appears to be somewhat, larger than thr amounts recovcrcd at 24 hr. Howwcr, the values were not significantly differrnt at thr 5 ‘; lcvc~l of confidence. Also,
421
6.Gl ‘,c of the chemosterilant appeared as 14C02 at 24 hr postingrstion. (It was not possible to determine 14COs _ at 72 hr because of the difficulty in keeping n-cevils alive and vigorous in the CO, tc‘st apparatus for t,hat length of time.) AIalic and malonic acids formed a large prrccwtagr of the organic acids (4.92’; and 2.13”;), but the standard deviat’ion \vas largcl, so error may be the reason for t,hc disproportionatcly high value; ~v:chave no othrr explanation. Of the total, 4.91 ‘is appcbared as amino acids, and :L complete array appeawd to bc labeled. About 1.09 5‘; of the force-fed busulfan was excret,ed in the feces as busulfan at] 72 hr postingestion. Also, 2.40 and (i.53 5 appeared in t,ht: feces as 1,4- and 2, %butancdiols and organic acids, rcspectivcly. In our laboratory, a boll xveevil allowed to feed on u 0.1 ‘A concentration of busulfan in its diet will ingest, about 3 pg of that’ chemosterilant each day. Then about 0.03 pg of busulfan is apparently cxcrrted in a 72-hr period for each day that t’hc insect feeds on the treated dirt. Of the radioactivity ingested, 34.32 “; was recovered in t’he frees at 72 hr postfrcding. The brci of homogenized boll n-wvils and busulfan gave spots in two TLC systems which showed acid positive with bromcresol green and had the same R, value ax the methanesulfonic acid standard. These spots wrc not present in the control, an ext,ract of boll weevils with no busulfan added to the broi. Trams et al. (6) used 35S-labelcd busulfan and found that mt%hane-sulfonic acid was a major urinary met’abolitc when he injccted the compound int,ravenously int,o rats. The mass spwtrum of busulfan indicat,c>s a high mass peak at w/e 175 (20). The compound had the following fragmentation n?/e values intensities: 771/e 119(13), 97(14), 79(80), 71(100), 55(39), 54(3S), 43(2(i), 42(53), and 41(29). In this spectrum therefore, rearrangement. occurs and no molecular
422
W’IPGUL,
MITLIN,
ion corresponding to a molecular weight of 246 is observed. However, the high m/e peak evidently involves a skeletal rearrangement since it contains two sulfur atoms as dctermined by the ratio of the intensity of m/e 175 to m/e 177 (20:2). This rearrangement resulted in a methyl pyrosulfate 0
0
[ cH3-~-o-;-ol+ J+ 0 0 m/e
*
THOMPSO?i
45 corresponding to those of 1,4- and 2,3butanediol, respectively, but t>hcra was no base peak at m/e 43 for 1,3-butanediol, which corresponds to the GLC dat,a. Spot 2 had a mass peak at m/e 120 which was t’he most significant high mass and was attributed to sulfolane; further, the base peak at m/e 41 (1I-CH&02)+ plus other major fragment-ion peaks at m/e 55 (RI-HSOz)+ and 56 (XSOz)+ arc characteristic of sulfolane. The compounds present in thinlayer spot 3 had such a complex nature that we could not determine wi-ith certainty that busulfan was present.
175
The base peak of m/e 71, AI-CH&06 was thus formed by the cyclic rearrangement of busulfan. Figure 1 shows the two-dimensional TLC separation of the labeled metabolites from boll weevils that had ingested labeled busulfan 24 hr previously. The mass spectral examination of the spots indicated that they contained a complex mixture of material. Thus spot 1 had base peaks at m/e 42 and
03
0
AND
2
0
CONCLUSIOSS
Busulfan, 1,4- and 2, X-butanediols, sulfolane, citric acid cycle acids, and amino acids were found as metabolites of busulfan labeled with 3H on the but,anediol portion of the molecule. These metabolites were found in weevils at 24 and 72 hr postingest’ion and in frees collected at, 72 hr postingestion. 14C0, was found as a metabolite by using 14C-labeled busulfan. (Methanesulfonic acid was determined as a metabolitr by using unlabeled active busulfan.) Most metabolism of t’he chemosterilant took place within 24 hr postingestion. Unmetabolized busulfan (1.03 and 0.53 ‘Q) was present in the weevil at 24 and 72 hr postingestion, respectively; 1.09’; appeared in the feces at 72 hr postingestion. A total of 34.32 ( ; of the radioactivity appeared in the frees at 72 hr postingest’ion.
1
1st
FIG. 1. The two-dimensional thin-layer chromatographic separation of 3H-labeled metabolites of busulfan fed to boll weevils. The plate was developed in both direct,ions in chloroform-methyl ethyl ketone (l:l).
ACKNOWLEDGMENTS
The authors are indebted to M. C. Kirk, Southern Research Institute, Birmingham, AL for mass spect,ra of the metabolites of busulfan, and tjo H. W. Chambers, Entomology Department, Mississippi State University, for advice concerning experimental procedure. REFERENCES
1. W.
Klassen and sterility induced
N. W. Earle, in boll weevils
Permanent with busul-
METABOLISM
2.
3. 4.
5.
6.
OF
BUSULFAh-
fan without reducing production of pheromone, J. Econ. Entomol. 3, 1195 (1970). W. Dameshek, II. B. Granville, and F. Rubio, Therapy of the myeloproliferative disorders with myleran, Ann. K.I-. Acad. Sci. 68, 1001 (1958). 11. Gast, Oviposition and fecundity of boll weevils in mass-rearing cultures, J. Econ. Entomol. 59, 173 (1966). R. E. McLaughlin, M. It. Bell, and 1~. J. Daum, Suspension of microorganisms in a thixotropic solution, J. Invertebr. Pathol. 9, 35 (1967). G. Wiygul, N. Mitlin, J. N. Love, and G. J. Lusk, The effect of irradiat,ion on absorption and metabolism of glycine-U14C in the boll weevil Anthonomus grandis Boheman, Comp. Biochem. Physiol. 33, 475 (1970). E. G. Trams, M. V. Nadkarni, V. DeQuattro, G. 1). Maengwyn-Davies, and P. K. Smith, Myleran. Preliminary st,udies on distribut,ion and metabolic fate in the rat, Biochem. Pharmacol.
2, 7 (1959).
7. J. Nordmann and R. Nordmann, in “Chromatographic and Electrophoretic Tech(I. Smith, Ed.), John Wiley & niques” Sons, New York, 1969. 8. R. I. Cheftel, R. Munier, and M. Macheboeuf, Partition microchromatography on paper of nonvolatile water-soluble aliphatic acids, Bull. Sot. Chim. Biol. 33, 840 (1951).
IX
THE
BOLL
IVEEVIL
423
9. B. W. Fox, A. W. Craig, and H. Jackson, Comparative met,abolism of myleran-3% in the rat, mouse, and rabbit, Biochem. Pharmacol. 3, 27 (1960). 10. K. Jones and J. G. Heathcot,e, Rapid resolution of naturally occurring amino acids by thin-layer chromatography, 1. Chromatogr. 24, 106 (1966). 11. R. D. O’Brien, Nitric acid digestion of tissues for liquid scintillation counting, Anal. Biochem. 7, 251 (1964). 12. E. Sawicki, D. F. Bender, T. R. Hauser, R. M. Wilson, Jr., and J. E. Meeker, Five new methodsfor t>he spectrophotometric determination of alkylating agents including some ext,remely sensitive autocatalytic methods, Anal. Chem. 35, 1479 (1963). 13. J. S. Matthews, Color reagent for steroids in t,hin-layer chromatography, Biochim. Biophys. Acta 69, 163 (1963). 14. A. Mehlitz, K. Gierschner, and T. Minas, Thin-layer chromatographic separation of 2,4-dinitrophenyl hydraxones. Differentiation of 2,4-dinitrophenyl-hydrozones of saturated aldehydes and ketones as well as of unsaturated carbonyl compounds by means of a color reaction with potassium ferricyanide, Chern. Ztg. 8i, 573 (1963). 15. R. A. Heacock and M. E. Mahon, The color reactions of the hydroxy skatoles, J. Chromatogr. 17, 338 (1965).
BIOCHEYIYTitY
.\Sl)
Metabolism
I’lIl-sIOI.OGY
1,
of Busulfan
418-423
in the
grads GLENN Entomology
Research
Division
Weevil
(Anthonomus
Boheman)
WIYGUL
, dgricullural State
Boll
AND
NORMAN
Research Service, College, Mississippi
~IITLIS 7:nitetJ 39762
States
Department
of
Agriulltllre
AND
,4. C. Pesticide Received
December
THOMPSOX
Chemicals
Research
7, 1971;
accepted
Branch December
8, 1971
One-day-old male boll weevils, Anthonomus grandis Boheman, that had been forcefed 3H, and IYXabeled busulfan were held for 24 and 72 hr, respectively. The resulting metabolites from the feces and extracts of the weevils were analyzed by thin-layer chromatography, gas-liquid chromatography, and mass spectroscopy. At 72 hr postingestion, 34.32Tc of the total radioactivity was recovered from the feces, about 1.09ci; as busulfan. Most metabolism of busulfan appeared t,o t)ake place within 24 hr postingestion. Organic acids, amino acids, busulfan, 1,4- and 2,3-butanediols, and sldfolane were identified as metabolites. Methanesulfonic acid was det,ermined as a metabolite by using unlabeled busulfan.
reared by the methods of Gast (3). Busulfan labclcd with 3H at the 1,4-positions of the butanediol moiety was purchased from Schwarz Biorescarch Inc., Orangrburg, NY as a 1’ ; ethanol solution with a specific activity of 8000 ;\ICi/mM. When tlio compound was chromatographcd in thrw thinlayer systems, it gave only one spot. Busulfan labeled with 14C on the 1,4-positions of the butancdiol moiety was supplied by the Radiation and Metabolism Laboratory, Agr. Rrs. Serv., USDA, I’argo, KD; it had a and also specific activity of 2 MCi/mR’I gave only onr spot when it was chromatographed on three t,hin-layer systems.
INTRODUCTION
Busulfan (Myleran); (1-4-butanediol dimethanesulfonatc) has only recently been found to be an effective chemosterilant for the male boll weevil (Anthonomus granclis Boheman) (1) though it has been used for some time in the treatment of myelocytic leukemia (3). However, the compound is the chemosterilant of choice for the pilot eradication program against thcl boll wwvil currcntly underway. It, was thcreforc necessary to determine the fate of tht> compound and to identify the metabolitcs resulting when it was fed to the boll wwvil. MATERIALS
AND
METHODS
Treatment
Insects ancl Reagents
The test insects were force-fed aliquots of the original solution of the 3H or 14C-labeled busulfan by the method of McLaughlin et al.
The insects used were one-day-old males from our laboratory culture that had been 418
METABOLISM
OF
BUSTJLFAK
(4) as modified by Wiygul et al. (5). Then they wrc held in an incubator at 33°C for 24 or 72 hr with cotton squares (buds) as food. After the> requisite t)ime, the wewils were ground in a Potter-Elvejhem homogcnizcr, and the homogenate was extracted twice wit,11 10 YI acetone-water and once with acctonc,. (In preliminary tests, no radioactive metabolitcs were found in a nonpolar extract of bhc wrwils.) The acetonc c>xtract was then taken to a known volume by evaporation at, room temperature wit’h nitrogen. Also, the feces were collected from thr containers where the insects had been held a,nd ext,ractcd in the same way. lletemiriatio?r.
0-f c:lllabeled Metabolites
In addition, a brei of ground boll weevils wit,11 enough insect saline solution to give it a liquid consistency was incubated at 37°C for 24 hr with busulfan added in the amount’ of 100 pg;jinscct’ used to make the brci. After incubation, the brei was extracted as described, spotted on TLC plates, and chromatographcd. The ext’ract was used to: (1) drtcrmine the presence of 1,4-butanediol by GLC and (2) t,o determine that methanesulfonic acid was a metabolitc of busulfan in the boll wwvil.
Thin-layw chromat,ograph\(TLC), gasliquid chromatography (GLC), and mass spectroscopy were used for quantitative separation and identification of the radioactiw mt+abolitw. All analyses were rcplicatc>d five timc>s.
TLC Separations Trams’ carlior work with rats (6) in which 1~0uwd paper chromatography supplied us with a pattwn for idcnt’ification of 1,4butancldiol, sulfolane, and busulfan. Howwwr, in the TLC system wc developed to scparatch thcsc mrtabolitcs WCused act’ivated silica gr,l G and chloroform-methyl et’hyl kc+orw (1: 1) in :I saturat,ed atmosphere;
Ipi
THE
BOLL
lYEEVIL
419
maximum separation was achieved by developing the plates in both directions. The organic acids and wlatrd compounds wrr separated by using II -butanol-awtic acid-wat’er, (120 : 30: 50) in a sat,uratcd atmosphere in OIW direction (7) and ethanol-ammonia-water (160: 10: 30) (8) in a saturated atmosphrre in the other dircction with activated silica gel CTas the sub&rate. The methods of Fox et al. (Y) were used to separate met~harw sulfonic acid by TLC, but me used activated silica gel G as a substrate rather than papw, and the solvent systems were 1~-butanol-dioxane-~ N ammonia, (4: 1:5) and n-butanol-2 N acetic acid (1: 1). The amino acids and related compounds were separated by the TLC method of Jonts and Hrathcot’c (10). After t,he mctabolitrs n-w> located, the spots were scraped from the TLC plates into vials containing O’Brien’s scintillation fluid (ll), and a thixotropic gel was added (Cab-O-Sil) to suspend the> TLC particlcs. Tht> radioactive samples were counted in a Packard model X320 liquid scintillation counter (Packard Instrument Co., Des Plainrs, IL). Results were first calculated as disintegrations prr minute (dpm’s) and then convcrtcld to ptlrccntagc of material force-fed. Also, unlabeled standards of all suspected metabolites were added to the cxt’ract of weevils to facilitate idcntificat’ion on the TLC platw. Husulfan \vas idcntificd by modifying the procedure dcvelopcd by Sawicki et al. (12). The TLC plate was sprayed with 4-(p-nitrobenzyl)p):ridinc (1 ‘,; w/v acetone sol), heated to 120°C for 10 min, and thPn sprayed with a %.5“;, cyclohcxylaminr solution ; t,hc busulfan then devrlopcd a purple color. Vanallin-sulfuric acid (13) was used to locate the 1,4 and 2,3 butancdiols. Sulfolane \vas visible at the concent,rations used without treatmclnt. The organic acids n-crc visualizt>d by using TJ(dimethylamino) bcnzaldchgdc in acet,ic anhydride as a spray and than spraying wit,11
420
WIYGUL,
MITLIN,
AND
bromcresol green reagent (7). The amino acids and related compounds were detected by using ninhydrin as a spray reagent.
Xass
The quantitative determinations of the presence of 1,4 butanediol as a metabolite of busulfan were made on a GLC equipped with a flame ionization detector with a 6-ft, s in. o.d. stainless steel column packed with 10.5% DEGA on 60/80 mesh HNDStreated Gas Chrom-P. The injector and detector temperatures were 175’ and 19O”C, respectively; the column temperature was 140°C. Standards consisted of the isomeric butanediols and sulfolane. Sulfolane and 2,3 butanediol were apparently present in such minor quantities that’ n-e were not able to det’ermine their presence by our GLC methods though we were able to determine their presence by mass spectral analysis and, in the case of sulfolanc, by TLC done with radioisotopes.
from
the boll weevil
at 24 and
‘,L, Boll weevil
y. recovered
in extract
DISCUSSION
1
of
YZ hr postingestion extract resultsC
24 hr postfeeding (%A
Total
AND
The results (Table 1) indicated that the butanediol port’ion of the busulfan molecule enters into a number of metabolic reactions which would be expected since the 4 carbon structure of 1,4-butanediol would seem to allow easy conversion to acids of the citric acid cycle. (Trams et al. (6) fed 14C-labeled 1,4-butanediol to rats and found several labeled organic acids in the urine.) At 24 hr postingestion, 1.5 5 of the radioactivity fed the insects was recovered as identifiable
and %On
1.03% 3. 042d 0.2% 0.1 0.29 0.27 0.58 0.31 1.44 6.52 1.71d 6.61 37.68%
a Expressed as percentage of busulfan force-fed. * Replicated three times unless otherwise noted. c No differences could be determined at the 5y0 level tract by using lkmcan’s new multiple range test. d Replicated five times.
3H- and
W-lateled
busulfan”,
feces Extract (%‘I
72 hr postfeeding (‘G 1
f
(%I
0.53 1.73 0.85 0.62 0.50 0.48 0.85 2.01 0.98 4.46 1.64
1.09 2.40 0.85 0.01 4.92 1.54 0.01 0.05 4.09 0.73 4.03
0.77 1.55 0.80 0.0 2.13 0.87 0.01 0.01 3.20 0.61 0.78
25.77”;
34.32:4,
5.35
of significance
b
test 72-hr
Metabolite
Busulfan 1,4- and 2,3-butanediol Sulfolane Citric acid Malic and malonic acids Succinic acid Fumaric acid (Y Ketoglutaric acid Amine Aldehyde Amino acids co2
Analysis
RESULTS
TABLE recovered
Spectral
For the mass spectral analyses, radioactive spots from samples of metabolites were located on TLC plates by using a thinlayer radio chromatogram scanner and elutcd and subjected t’o mass spectral analysis for confirmation of busulfan, 1,4and 2,3-butanediols, and methane sulfonic acid.
GLC
Metabolites
THOMPSON
between
24 and
72 hr weevil
ex-
METABOLISM OF BUSULFAN IX THE BOLL B:EEVIL organic acids. Also, another 6.52 5 was recovered as a compound or compounds that gave a positive aldehyde test with 2,4dinitrophcnylhydrazine (14). We spcculatc that’ thcsc aldehydcs arc intermediates brtown 1,4-butarwdiol and the di- and tricarboxylic acids. 1,4-Butanediol itself appearcld at 24 hr postingestion as 3.045 of tht> chcmostcrilant originally fed. Thcrefort>, bccausr hydrol\-tic cleavage of the busulfan molecule would yield 1,4-butanediol, a busulfan * 1,4-butanediol -+ dicarboxylic acid mct,abolic path would seem to b(l ow route by which this compound is metabolized. Also, the results appear to indicate t’hat most metabolism takes place in the first 24 hr posting&ion. After the busulfan molecules entered into citric acid cycle reactions, fragments would bc, expected to cntcr into a number of metabolic reactions. WC, therefore determincxd t,hc percentage of radi0act’ivit.y in the amino acid fraction of the extract and found that 1.71 c;, of thr radioactivit,y was present thcrc at 24 hr postingestion. ,4lso, a compound that had an Rf value of 74 in the butanol-acetic acid-water TLC system (7) and an Rf value of 42 in t’he ethanol-ammania-water TLC system (8) gave a positive reaction for amincs with p-(dimethylamino) bcnzaldehyde-hydrochloric acid (15) but. a mgative ninhydrin test, which indicated that then> were no free amino groups. Moreover, in an ethyl ethw, 0.1 N HCl extraction of this compound from TLC plates, the radioactivit’) wmaincd in the HCl, further evidenw that an amine was present. If t,his spot, consists of tertiary amines as the evidence indicates, it may be nitrogen-coupled mctabolites that we have already identified. At 72 hr postingestion of the 3H-labelcd busulfan, about 4.46 ’ ( of the radioactivity was recovered as citric acid cycle acids, an amount that appears to be somewhat, larger than thr amounts recovcrcd at 24 hr. Howwcr, the values were not significantly differrnt at thr 5 ‘; lcvc~l of confidence. Also,
421
6.Gl ‘,c of the chemosterilant appeared as 14C02 at 24 hr postingrstion. (It was not possible to determine 14COs _ at 72 hr because of the difficulty in keeping n-cevils alive and vigorous in the CO, tc‘st apparatus for t,hat length of time.) AIalic and malonic acids formed a large prrccwtagr of the organic acids (4.92’; and 2.13”;), but the standard deviat’ion \vas largcl, so error may be the reason for t,hc disproportionatcly high value; ~v:chave no othrr explanation. Of the total, 4.91 ‘is appcbared as amino acids, and :L complete array appeawd to bc labeled. About 1.09 5‘; of the force-fed busulfan was excret,ed in the feces as busulfan at] 72 hr postingestion. Also, 2.40 and (i.53 5 appeared in t,ht: feces as 1,4- and 2, %butancdiols and organic acids, rcspectivcly. In our laboratory, a boll xveevil allowed to feed on u 0.1 ‘A concentration of busulfan in its diet will ingest, about 3 pg of that’ chemosterilant each day. Then about 0.03 pg of busulfan is apparently cxcrrted in a 72-hr period for each day that t’hc insect feeds on the treated dirt. Of the radioactivity ingested, 34.32 “; was recovered in t’he frees at 72 hr postfrcding. The brci of homogenized boll n-wvils and busulfan gave spots in two TLC systems which showed acid positive with bromcresol green and had the same R, value ax the methanesulfonic acid standard. These spots wrc not present in the control, an ext,ract of boll weevils with no busulfan added to the broi. Trams et al. (6) used 35S-labelcd busulfan and found that mt%hane-sulfonic acid was a major urinary met’abolitc when he injccted the compound int,ravenously int,o rats. The mass spwtrum of busulfan indicat,c>s a high mass peak at w/e 175 (20). The compound had the following fragmentation n?/e values intensities: 771/e 119(13), 97(14), 79(80), 71(100), 55(39), 54(3S), 43(2(i), 42(53), and 41(29). In this spectrum therefore, rearrangement. occurs and no molecular
422
W’IPGUL,
MITLIN,
ion corresponding to a molecular weight of 246 is observed. However, the high m/e peak evidently involves a skeletal rearrangement since it contains two sulfur atoms as dctermined by the ratio of the intensity of m/e 175 to m/e 177 (20:2). This rearrangement resulted in a methyl pyrosulfate 0
0
[ cH3-~-o-;-ol+ J+ 0 0 m/e
*
THOMPSO?i
45 corresponding to those of 1,4- and 2,3butanediol, respectively, but t>hcra was no base peak at m/e 43 for 1,3-butanediol, which corresponds to the GLC dat,a. Spot 2 had a mass peak at m/e 120 which was t’he most significant high mass and was attributed to sulfolane; further, the base peak at m/e 41 (1I-CH&02)+ plus other major fragment-ion peaks at m/e 55 (RI-HSOz)+ and 56 (XSOz)+ arc characteristic of sulfolane. The compounds present in thinlayer spot 3 had such a complex nature that we could not determine wi-ith certainty that busulfan was present.
175
The base peak of m/e 71, AI-CH&06 was thus formed by the cyclic rearrangement of busulfan. Figure 1 shows the two-dimensional TLC separation of the labeled metabolites from boll weevils that had ingested labeled busulfan 24 hr previously. The mass spectral examination of the spots indicated that they contained a complex mixture of material. Thus spot 1 had base peaks at m/e 42 and
03
0
AND
2
0
CONCLUSIOSS
Busulfan, 1,4- and 2, X-butanediols, sulfolane, citric acid cycle acids, and amino acids were found as metabolites of busulfan labeled with 3H on the but,anediol portion of the molecule. These metabolites were found in weevils at 24 and 72 hr postingest’ion and in frees collected at, 72 hr postingestion. 14C0, was found as a metabolite by using 14C-labeled busulfan. (Methanesulfonic acid was determined as a metabolitr by using unlabeled active busulfan.) Most metabolism of t’he chemosterilant took place within 24 hr postingestion. Unmetabolized busulfan (1.03 and 0.53 ‘Q) was present in the weevil at 24 and 72 hr postingestion, respectively; 1.09’; appeared in the feces at 72 hr postingestion. A total of 34.32 ( ; of the radioactivity appeared in the frees at 72 hr postingest’ion.
1
1st
FIG. 1. The two-dimensional thin-layer chromatographic separation of 3H-labeled metabolites of busulfan fed to boll weevils. The plate was developed in both direct,ions in chloroform-methyl ethyl ketone (l:l).
ACKNOWLEDGMENTS
The authors are indebted to M. C. Kirk, Southern Research Institute, Birmingham, AL for mass spect,ra of the metabolites of busulfan, and tjo H. W. Chambers, Entomology Department, Mississippi State University, for advice concerning experimental procedure. REFERENCES
1. W.
Klassen and sterility induced
N. W. Earle, in boll weevils
Permanent with busul-
METABOLISM
2.
3. 4.
5.
6.
OF
BUSULFAh-
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THE
BOLL
IVEEVIL
423
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