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dieldrin metabolite dihydrochlordene dicarboxylic acid-14C in rats
l’ESTI(‘IDE
BIOCHEMISTRY
Conversion
AND
PHYSIOLOGY
5, 226-232
of the Aldrin/Dieldrin Metabolite Dihydrochlordene Dicarboxylic Acid-14C in Rats1 J. P. LAY, I.
Znstitut
fiir
(107.5)
Bkoloyische Mfjnchen.
C’hemie
WEISGERBER,
AND
W.
KLEIN
der G’esellschaft fiir Strahlenund A~t.g~~stin I, Schloa Birlinghoven,
Unaweltforschung
D-5%16 St.
Received
August
Y, 1974 ; accepted
November
mbH,
Germany
13, 1974
Upon intravenous application of dihydrochlordene dicarboxylic acid-W to rats, the radioactivit,y is quickly excreted, and 44yc;, of the excreted radioactivity consists of metabolites. Nine metabolites have been isolated from feces and urine extract.s. Three metabolites could he identified by means of authentic samples by thin layer chromatography, gas chromatography, and mass spectromet.ry : two isomers of dechlorodihydrochlordene-dicarboxylic acid (metabolites I and II, total 22.5yc,) and dihydrochlordenedicarboxylic acid-dimet,hyl-est.er (mebabolite III, 11.3?:).
1NTROl)UCTION
MATERIALS
The metabolism of the cyclodiene insccticidcs aldrin and dieldrin in warm blooded organisms has been investigated in detail (2-5). There is very little knowledge about a further conversion of identified metabolites (6-8). A main metabolitc, dihydrochlordene dicarboxylic acid (P’ig. 1) is formed from aldrin and dicldrin in warmblooded animals (5-7) as ~11 as in higher plants and soil (S-10). In a former publication, we reported on the microsynthesis, application and conversion of dihydrochlordenc dicarboxylic acid-14(r to isolat,ed organclles of rat liver homogenates (11). The following investigations aimed at verifying the metabolism of dihydrochlordene dicarboxylic acid in warm-blooded organisms by means of in uirw experiments and proving this substance not to bc the final physiological degradation product of aldrin a,nd dicldrin. ’ This is paper number 87 in the Series “Contributions to Ecological Chemistry.” For number 86 see Ref. (I).
Co),yright rights
All
0 197.5 hy .4eademic of reproduction in any
I'res~, Inc. form
reserved.
.4ND
hIETHOI)S
Application.. Each of six female and six male albino rats were given a single intravenous dose of dihydrochlordenc dicarboxylic acid-14C (Fig. 1, specific activity: 1.47 mCi/mM). Each male was given 4.03 @g/g body weight, each female 4.09 pg/g body weight of dihydrochlordenc dicarboxylic acid-14C, dissolved in 1,‘L-propancdiol/cthanol 6: 1. The animals wcrc: kept in individual metabolism cages allowing separat,e collection of the excret.a. During the ow-week experiment, all rats were fed with normal grains; an enzyme induction by drug treatment brfore or after applicaCon was not made. lcurther data pertaining to application and excretion are listed in Table 1. Determirlation.
of
radioactiuity
it1
the
e.rcreta. Icrces and urine were collected daily. The radioactivity in the urine was det’ermined by direct liquid-scintillation counting. lcor determination of radioactivity in the feces, t’hey were air-dried, pulverized, and treated with methanol in a
DIHYDROCHLORDENE
DICARBOXTLIC
ACID-14C
IN
RATS
227
COOH COOH
CL
Cl DHCDS
Metabolite
I FIG.
Metabolite 1. Scheme
of
conversion
II
of dihydrochlordene
soxhlet for 48 hr. The radioactivity in the extracts was determined in a liquid scintillation counter; the unextracted radioactivity was determined by combustion and liquid scintillation counting of 14C0,. In order to determine individual radioactive compounds separately, the rcnally excreted radioactivity was recovered by 48 hr extraction of urine (natural pH) with ether in a liquid-liquid extractor. The et,her extracts of urine and the methanol extracts of feces were analyzed by thin layer chromatography (tic). Thin layer chromatography on silica gel was carried out on the concentrated extracts using the following solvents: Benzene/petrol ether (60-90)/acetic acid 1O:lO:l; 4:4:1; 2:2:1. Benzene Benzene/chloroform 1: 1 Benzenc,/hexanc 1: 1 Quantitative measurement of the separated compounds was performed by liquid scintillation counting. Determination of radioactivity i?~ the orgam. In order to determine the distribution and residues of radioactivity in the animal organisms, the rats were sacrificed one week after dosage. Their organs were
Metabolitc dicarhoxylic
acid
III in rats.
prepared, weighed, homogenized, and extracted individually in a soxhlet with methanol for 48 hr. The collected blood of the animals was extracted several times with ether in a separator-y funnel. The radioactivity in organs and blood was determined by liquid scintillation counting of definite aliquots of rxt’ract. The unext’ractable radioactive &dues were counted after combust,ion t.o r4C02. Isolation of mefabolites.Since the metabolites were qualitatively identical in feces and urine extracts, they were isolated from both extracts by the same procedure. The extracts were separated by preparative layer chromatography on silica gel with benzene/petrol ether/acetic acid 10 : 10 : 1 as solvent, and gave a zone at the origin corresponding to t,hc polar mctabolites IV and V, zones corresponding to unchanged dihydrochlordene dicarboxylic acid and the metabolites I-III, and a zone at the front corresponding to the metabolites VI-IX. For Rf-values, see section “ResultsIdentification of Conversion Products.” The zones were desorbcd with methanol and purified further. llletabolites I and II were subjected several times to preparative layer chromatography using successively the following
228
LAY,
WEISGERBER
solvents : benzene/petrol ether/acetic acid 2:2:1; 4:4:1 and 1O:lO:l. Then, they were methylated by stirring t’hem for 12 hr-in absence of light-with CHJ/ AgzO in methanol, or with freshly prepared diazomethanc. Both methods gave identical methylated products. These were purified with benzene as solvent. Metabolite III was purified by preparat,ivc layer chromatography using benzene as solvent. Metabolites IV and V were purified by using benzene/petrol ether/acetic acid 2 : 2 : 1 on preparative layer chromatography. Then, both products were hydrolyzcd by refluxing them with 6 N HCl in methanol for 24 hr. The hydrolyzed products were methylated with CH,I/ Ag,O and purified on TLC with benzene as solvent. The purification of the metabolites VI-IX was carried out by tic using benzene/ chIoroform 1: 1 and benzene/hcxane 1: 1. 8eparat’ion of these nonpolar products was not achieved on tic, but they were separated by glc. TABLE Experimental Data Dihydrochlordene
1
Dose pCi Dose mg g body weight before application g body weight after one week Dose pg/g body weight/animal Amount of excreted urine after one week (ml) cyo of renally excreted radioactivity y. of radioactivity excreted via feces Total excretion r. after one week Biological halflife (days) ea.
KLEIN
Identification,. For glc, a gaschromatograph Packard, model 873, with a splitter for separate collection of radioactivity, was used. Column packing : 1% OV1 silicon gum on chromosorb W-AW-DMCS SO/l00 mesh. detector : G3Ni EC ; column tempcrature ;SO’C; carrier gas PI;,. Second apparatus: Fractovap G I Carlo Erba; column : 30 m metal capillary column ; column packing and detector as above ; ECD Exiter model 200 Carlo Erba; column t,empernture 250°C ; carrier gas Ns. Mass spectrometry was performed with a GLC-MS-combination LKB 9000 A, LKB Produkter, Sweden; data processing with IBM calculator, model 1130, plotter model 1627. NMR spectroscopy was carried out with Varian, type A 60. Radioactive counting methods. For counting urine, extracts, and tic-zones, Packard liquid-scintillation counters, model Tri Carb 3380 and 3375, were used. 14C0,determinat,ion of unextractable residues was achieved with Oximat, Int,ertechnique. The thin layer scanner was LB 2722 from Bert’hold/Friesekc. RESULTS
of
and Results of the Application Dicarbox$ic Acid to Rats
Rats
AND
633 12.7 3.53
69 0 12.7 3.53
865.5
878.0
1,026.O
999.0
4.09
4.06
78.0
104.0
28.3
37.7
64.9
57.6
93.2
95.3
1.6
1.8
During t,he investigation period none of the animals showed any disability as a consequence of dihydrochlordene dicarboxylic acid injection. At the end of the experiment, a general increase in body weight of about 2&30 g/individuum could be detected. Excretion. The dihydrochlordene dicarboxylic acid was excreted rather quickly from the warm-blooded organism. The maximum excretion was observed the first day after application; on the sixth day,
DIHYDROCHLORDENE
DICARBOXYLIC
well as muscle fat (0.51 ppm for 8, 0.62 ppm for 9>, ventral fat (0.47 ppm for 8, 0.23 ppm for 9 ), and blood (0.13 ppm for c? and 0.22 ppm for 9 ). The absolute quantity of radioactivity, however, was not sufficient for isolation and identification of conversion products. Identification of conversion products. The following conversion products, all of which were isolated from feces and urine extracts with slight differences among the animal sexes, were identified by means of thin layer chromatographic and mass spectrometric comparison with authentic samples : two isomers of dechloro-dihydrochlordene-dicarboxylic acid (Fig. 1, I and II) = 4,5,6,7,8pentachloro-2,3,3a,4,7,7a-hexahydro-4,7methano-indene-1,3-dicarboxylic acid (22.5% of the total excretion). The dimethylestcr of dihydrochlordene dicarboxylic acid ( 11.3yo) (Fig. 1, III) : two very polar conjugates which could be hydrolyzed with methanol/HCl and, upon their methylation, formed the dimethylester of dihydrochlordene dicarboxylic acid (IV and V) (4.5% of the total excretion). Furthermore, there were other conversion products (VI-IX) (5.6% of the total excretion), but, except for glc-MS-spectrometry, no further information on their structures was obtained. Of the total
ACID-‘IC
IN
RATS
229
excreted radioactivity 46.10/, was due to unchanged dihydrochlordene dicarboxylic acid, and 10% was not extractable by the methods used. The compourlds I and II have, on tic, Rf values of 0.51 and 0.53 (solvent: benzene/ petrol ether/acetic acid 10 : 10 : 1). After methylation, the Rf values were 0.72 and 0.77 (three times developed with benzene), and the glc retention times 23.3’ and 24.1’ (experimental conditions as described in “Materials and Methods” for the second apparatus). The mass spectra of the methylated compounds I and II (Figs. 2 and 3) show differences only in peak intensities. Figure 2 shows the mass spectrum of metabolite I. A part of the mass spectrum of metabolite II has been placed above the corresponding part of I, in order to indicate the differences. Likewise, Fig. 3 shows the mass spectrum of metabolite II, and a part of I has been added. Both compounds contain only 5 chlorine atoms. Due to the characteristic Retro-Dicls-Alder fragment ions at m/e 236 and 201, a bridged molecule can be excluded. The R!I+-peak and important fragment ions with proposed gross formula are given I in Table 2. For the exact structure elucidation, the two dihydrochlordene dicarboxylic acids (Fig. 1, I and II) de-
l
Ii’
I
IX 200 5cAr4 NR. nECILURO-3I~~ORUM;ROEN-~*~~-~~~~~~~~~~~~~ FIG.
2. Mass
spectrum
of metabolite
I of dihydrochlordene
h4*420
dicarboxylic
acid in rats, after
methylation.
LAY,
FIG.
3. dlass
spectrwn
of metabolite
WEISGERBER
AND
II of dihvdrochlordene
chlorinated at, C4 were synthesized (11). Chromatographic data and mass spectra were identical to those of the metabolites. A 60 MHz NXIR spectrum (solvent: deuteroacetonc) was obtained for the synthesized substances. The signal occurring at 3.1 ppm (relating to T3IS = 10) indicates the presence of a proton at the dechlorinated C,. Unfortunately, there was not enough pure metabolitc material to obtain a N-\IR spectrum thereof t,oo. Furthermore, a dihydrochlordenc dicarboxylic acid dechlorinated at the double bond was synthesized (13) and methylated. The mass speckurn of this substance was compared to those of the mcthylated
KLEIN
dicarboxylic
acid in rats, after
,methylation.
metabolites I and II; clear diffcrenccs in intensities exclude the identity of bot,h isomers with this compound. Metabolite III, on tic, had an R,-value of 0.62 with benzene/petrol ether/acetic acid 10: 10: 1 as solvent and, on glc, a retention time of 25.2’. Table 3 contains the typical fragment ions from the mass spectrum of conversion product, III. The mass spectrum of metabolite III is shown in E’ig. 4. h reference substance was obtained by methylation of the dihydroTABLE MS-Fragnzentation Dihydrochlordene
3 oj Metabolite Dicarbo&ic
III Acid
of
-__ TABLE
2
Mass
MS-Fragmentation of the Methylated Metabolites II of Dihgdrochlordene Dicarboxylic Acid M&W 420 (M+) 389 3% 3x4 325 324 289 236 201
Fragment
ion
GrHuOXls CI,H~OO~X, ‘&H~aOaCla ‘&H120& C,zH,O&l~ ChH80#.% C,zHsO&l, CsHCl? CbHCk ~---
a R,etro-Dieb-Alder.
Fragment,
I and 454 (M+) 423 419 418 387 36s 359 358 351 323 297 270 23.5 __-.. 0 Retro-Die&Alder.
G~H,~O~Cl~
CdLOaCls ChH120&1:,
C~Hdl&l~ CdWXl, GaH,OnCls CieHnOzCl, C,zH,O&ls C,3H,O&la C,2H70rCl~ C&Clc C&lea CLC16LL __
ion
DIHYDROCHLORDENE
FIG.
4. Mass
spectrum
of metabolzte
DICARBOXYLIC
III
ACID-14C
of dikydrochlordene
chlordene dicarboxylie acid with diazomethane. Its chromatographic properties and the mass spectrum were identical to those of the metabolite. Th,e products VI-IX, on tic, were at the front with all solvents used for their purification. The treatment of the products VI-IX with methanol/HCl and CfIJ/ Ag,O, respectively, did not lead to any changes in their tic-properties, which is contrary to compounds I-V. The products VI-IX had lower molecular w-eights than dihydrochlordene dicarboxylic acid. Table 4 gives t.he characteristic dat,a of thcsc minor met,abolites. Figure 1 gives a survey on t)hc structures of isolated and idcmified conversion products of dihydrochlordene dicarboxylic acid in rats.
IN
231
RATS
dicaybozylic
acid in rats.
as reported earlier (11) with rat-live1 homogenat’es in in vitro experiments. The dechlorinabcd acids (metabolites I and II) may br regarded as the first step in the degradation of the chlorinated ring. The metabolites VI-IX have a lower molecule weight, and they seem to bc products of further degradat,ion of the parent compound. But as these metabolites were available in minor yuantit#ies, not much information could be obtained regarding their structure. However, it was not possible to detect them in in vitro experiments with liver homogenates (11). This suggest,st,hat they might. be formed in vitro in very small quant’it,ies which were beyond t,he detection limit. Alternatively, they arc formed in the living animals in some other part of the organism.
DISCUSSION
TABLE
From the present experiments it is apparent that dihydrochlordenc dicarboxylic acid is not the end product of aldrin/dieldrin metabolism. It was metabolized to a large degree within one week of the experiment; about 44% of the radioactivity excreted consisted of met’abolites. The dechlorination resulting in the metabolites I and II is the major metabolic pathway, besides esterification and conjugation resulting in the metabolites III-V. It is obvious that the dechlorination takes places in the liver,
Data on hlinor Dihydrochlordene ~Conversion product no.
Concentration in excreta in terms of % of total excretion
VI VII VIII IS u glc Conditions Second apparatus, 0.35 kp/cm*.
1.3 1.2 1.4 1.7
4
Conversion Products Dicarboxylic Acid nlc
M W
Retention time-
3.5’ 3.Y’ 3.7’ Y.0’
244 278 302 336
of Number chlorine atoms
4 5 4 6
as described in “Materials and Methods.” oven temperature 25O”C, N?-pressure:
of
LAY,
232
WEISGERBER
ACKNOWLEDGMENT
We thank W. Tomberg for the preparation of mass spectra and for kind assistance in the interpretation. REFERENCES
I. K. Sandrock, D. Bieniek, W. Klein and F. Korte, LXXXVI. Communication : Isolierung und Strukturaufklsrung von Kelevan-“CMetaboliten und Bilanz in Kartoffeln und Boden, Chemosphere 3, 199 (1974). 2. F. Korte and H. Arent, Metabolism of insecticides IX: Isolation and identification of dieldrin metabolites from urine of rabbits after oral administration of dieldrir+C, Life fki. 4, 2017 (1965). 3. F. Korte and W. Kochen, Insektizide im Stoffwechsel XII : Isolierung und Identifizierung von Metaboliten des Aldrin-lPC aus dem Urin von Kaninchen, Med. Pharmacol. Exp. 15, 409 (1966).
4. V. J. Feil, R. D. Hedde, R. G. Zaylskie, and C. H. Zachrison, Dieldrin-14C metabolism in sheep: Identification of trans-6,7-dihydroxydihydroaldrin and 9-(syn-epoxy) hydroxy1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a,~5,6,7,8,8a-octahydro-1,4-endo-&&exo-dimethanonaphthalene, J. Agr. Food Chem. 18, 120 0970). 5.
M. 6. Baldwin, J. Robinson and D. V. Parke, A comparison of the metabolism of HEOD (dieldrin) in the CFl mouse with that in the CFE rat, Food Cosmet. Toxiwl. 10,333 (1972).
AND
KLEIN
6. F. Korte, Recent results of investigations in environmental chemistry, in “Radiotracer Studies of Chemical Residues in Food and Agriculture,” Panel, Vienna, October 1971, IAEA STI/PUB 332, p. 3. 7. J. Oda and W. Miiller, Identification of a mammalian break-down product of dieldrin, Symposium on chemistry of pesticides under metabolic and environmental conditions, Bonn, Sept. 1970. Summary, Env. Qnal. safety 1, 248 (1972). 8. L. Nilzer, S. Detera, I. Weisgerber und W. Klein, BeitrSige zur iikologischen Chemie LXXVII : Verteilung und Metabolsmus des Aldrin-Dieldrin-Metaboliten Trans-4,5-dihydroxy-4,5-dihydroaldrin-14C in Salatpflanzen und Boden, Chemosphere 3, 143 (1974). 9. W. Klein, J. Kohli, I. Weisgerber, and F. Kort,e, Fate of aldrin-14C in potatoes and soil under outdoor conditions, 1. Agr. Food Chem. 21, 1.52 (1973).
10. J. Kohli, S. Zarif, I. Weisgerber, W. Klein, and F. Korte, Fate of aldrin-“C in sugar beets and soil under outdoor conditions, J. Agr. Food Chem.
21, 855 (1973).
11. J. P. Lay, W. Klein und F. Korte, BeitrLge zur tikologischen Chemie LXXXV : Mikrosynthese und in vitro Metabolismus von Dihydrochlorden-Dicarbons&ure-‘4C durch Rattenleberorganellen, Chemosphere 3, 193 (1974). 12. S. G&b, W. Klein und F. Korte, Beitrgge zur iikologischen Chemie LVI : Photoreaktionen des Aldrin/Dieldrin-Metaboliten Dihydrochlorden-1,3-Dicarbonslure, Chemusphere 2, 107 (1973).
BIOCHEMISTRY
Conversion
AND
PHYSIOLOGY
5, 226-232
of the Aldrin/Dieldrin Metabolite Dihydrochlordene Dicarboxylic Acid-14C in Rats1 J. P. LAY, I.
Znstitut
fiir
(107.5)
Bkoloyische Mfjnchen.
C’hemie
WEISGERBER,
AND
W.
KLEIN
der G’esellschaft fiir Strahlenund A~t.g~~stin I, Schloa Birlinghoven,
Unaweltforschung
D-5%16 St.
Received
August
Y, 1974 ; accepted
November
mbH,
Germany
13, 1974
Upon intravenous application of dihydrochlordene dicarboxylic acid-W to rats, the radioactivit,y is quickly excreted, and 44yc;, of the excreted radioactivity consists of metabolites. Nine metabolites have been isolated from feces and urine extract.s. Three metabolites could he identified by means of authentic samples by thin layer chromatography, gas chromatography, and mass spectromet.ry : two isomers of dechlorodihydrochlordene-dicarboxylic acid (metabolites I and II, total 22.5yc,) and dihydrochlordenedicarboxylic acid-dimet,hyl-est.er (mebabolite III, 11.3?:).
1NTROl)UCTION
MATERIALS
The metabolism of the cyclodiene insccticidcs aldrin and dieldrin in warm blooded organisms has been investigated in detail (2-5). There is very little knowledge about a further conversion of identified metabolites (6-8). A main metabolitc, dihydrochlordene dicarboxylic acid (P’ig. 1) is formed from aldrin and dicldrin in warmblooded animals (5-7) as ~11 as in higher plants and soil (S-10). In a former publication, we reported on the microsynthesis, application and conversion of dihydrochlordenc dicarboxylic acid-14(r to isolat,ed organclles of rat liver homogenates (11). The following investigations aimed at verifying the metabolism of dihydrochlordene dicarboxylic acid in warm-blooded organisms by means of in uirw experiments and proving this substance not to bc the final physiological degradation product of aldrin a,nd dicldrin. ’ This is paper number 87 in the Series “Contributions to Ecological Chemistry.” For number 86 see Ref. (I).
Co),yright rights
All
0 197.5 hy .4eademic of reproduction in any
I'res~, Inc. form
reserved.
.4ND
hIETHOI)S
Application.. Each of six female and six male albino rats were given a single intravenous dose of dihydrochlordenc dicarboxylic acid-14C (Fig. 1, specific activity: 1.47 mCi/mM). Each male was given 4.03 @g/g body weight, each female 4.09 pg/g body weight of dihydrochlordenc dicarboxylic acid-14C, dissolved in 1,‘L-propancdiol/cthanol 6: 1. The animals wcrc: kept in individual metabolism cages allowing separat,e collection of the excret.a. During the ow-week experiment, all rats were fed with normal grains; an enzyme induction by drug treatment brfore or after applicaCon was not made. lcurther data pertaining to application and excretion are listed in Table 1. Determirlation.
of
radioactiuity
it1
the
e.rcreta. Icrces and urine were collected daily. The radioactivity in the urine was det’ermined by direct liquid-scintillation counting. lcor determination of radioactivity in the feces, t’hey were air-dried, pulverized, and treated with methanol in a
DIHYDROCHLORDENE
DICARBOXTLIC
ACID-14C
IN
RATS
227
COOH COOH
CL
Cl DHCDS
Metabolite
I FIG.
Metabolite 1. Scheme
of
conversion
II
of dihydrochlordene
soxhlet for 48 hr. The radioactivity in the extracts was determined in a liquid scintillation counter; the unextracted radioactivity was determined by combustion and liquid scintillation counting of 14C0,. In order to determine individual radioactive compounds separately, the rcnally excreted radioactivity was recovered by 48 hr extraction of urine (natural pH) with ether in a liquid-liquid extractor. The et,her extracts of urine and the methanol extracts of feces were analyzed by thin layer chromatography (tic). Thin layer chromatography on silica gel was carried out on the concentrated extracts using the following solvents: Benzene/petrol ether (60-90)/acetic acid 1O:lO:l; 4:4:1; 2:2:1. Benzene Benzene/chloroform 1: 1 Benzenc,/hexanc 1: 1 Quantitative measurement of the separated compounds was performed by liquid scintillation counting. Determination of radioactivity i?~ the orgam. In order to determine the distribution and residues of radioactivity in the animal organisms, the rats were sacrificed one week after dosage. Their organs were
Metabolitc dicarhoxylic
acid
III in rats.
prepared, weighed, homogenized, and extracted individually in a soxhlet with methanol for 48 hr. The collected blood of the animals was extracted several times with ether in a separator-y funnel. The radioactivity in organs and blood was determined by liquid scintillation counting of definite aliquots of rxt’ract. The unext’ractable radioactive &dues were counted after combust,ion t.o r4C02. Isolation of mefabolites.Since the metabolites were qualitatively identical in feces and urine extracts, they were isolated from both extracts by the same procedure. The extracts were separated by preparative layer chromatography on silica gel with benzene/petrol ether/acetic acid 10 : 10 : 1 as solvent, and gave a zone at the origin corresponding to t,hc polar mctabolites IV and V, zones corresponding to unchanged dihydrochlordene dicarboxylic acid and the metabolites I-III, and a zone at the front corresponding to the metabolites VI-IX. For Rf-values, see section “ResultsIdentification of Conversion Products.” The zones were desorbcd with methanol and purified further. llletabolites I and II were subjected several times to preparative layer chromatography using successively the following
228
LAY,
WEISGERBER
solvents : benzene/petrol ether/acetic acid 2:2:1; 4:4:1 and 1O:lO:l. Then, they were methylated by stirring t’hem for 12 hr-in absence of light-with CHJ/ AgzO in methanol, or with freshly prepared diazomethanc. Both methods gave identical methylated products. These were purified with benzene as solvent. Metabolite III was purified by preparat,ivc layer chromatography using benzene as solvent. Metabolites IV and V were purified by using benzene/petrol ether/acetic acid 2 : 2 : 1 on preparative layer chromatography. Then, both products were hydrolyzcd by refluxing them with 6 N HCl in methanol for 24 hr. The hydrolyzed products were methylated with CH,I/ Ag,O and purified on TLC with benzene as solvent. The purification of the metabolites VI-IX was carried out by tic using benzene/ chIoroform 1: 1 and benzene/hcxane 1: 1. 8eparat’ion of these nonpolar products was not achieved on tic, but they were separated by glc. TABLE Experimental Data Dihydrochlordene
1
Dose pCi Dose mg g body weight before application g body weight after one week Dose pg/g body weight/animal Amount of excreted urine after one week (ml) cyo of renally excreted radioactivity y. of radioactivity excreted via feces Total excretion r. after one week Biological halflife (days) ea.
KLEIN
Identification,. For glc, a gaschromatograph Packard, model 873, with a splitter for separate collection of radioactivity, was used. Column packing : 1% OV1 silicon gum on chromosorb W-AW-DMCS SO/l00 mesh. detector : G3Ni EC ; column tempcrature ;SO’C; carrier gas PI;,. Second apparatus: Fractovap G I Carlo Erba; column : 30 m metal capillary column ; column packing and detector as above ; ECD Exiter model 200 Carlo Erba; column t,empernture 250°C ; carrier gas Ns. Mass spectrometry was performed with a GLC-MS-combination LKB 9000 A, LKB Produkter, Sweden; data processing with IBM calculator, model 1130, plotter model 1627. NMR spectroscopy was carried out with Varian, type A 60. Radioactive counting methods. For counting urine, extracts, and tic-zones, Packard liquid-scintillation counters, model Tri Carb 3380 and 3375, were used. 14C0,determinat,ion of unextractable residues was achieved with Oximat, Int,ertechnique. The thin layer scanner was LB 2722 from Bert’hold/Friesekc. RESULTS
of
and Results of the Application Dicarbox$ic Acid to Rats
Rats
AND
633 12.7 3.53
69 0 12.7 3.53
865.5
878.0
1,026.O
999.0
4.09
4.06
78.0
104.0
28.3
37.7
64.9
57.6
93.2
95.3
1.6
1.8
During t,he investigation period none of the animals showed any disability as a consequence of dihydrochlordene dicarboxylic acid injection. At the end of the experiment, a general increase in body weight of about 2&30 g/individuum could be detected. Excretion. The dihydrochlordene dicarboxylic acid was excreted rather quickly from the warm-blooded organism. The maximum excretion was observed the first day after application; on the sixth day,
DIHYDROCHLORDENE
DICARBOXYLIC
well as muscle fat (0.51 ppm for 8, 0.62 ppm for 9>, ventral fat (0.47 ppm for 8, 0.23 ppm for 9 ), and blood (0.13 ppm for c? and 0.22 ppm for 9 ). The absolute quantity of radioactivity, however, was not sufficient for isolation and identification of conversion products. Identification of conversion products. The following conversion products, all of which were isolated from feces and urine extracts with slight differences among the animal sexes, were identified by means of thin layer chromatographic and mass spectrometric comparison with authentic samples : two isomers of dechloro-dihydrochlordene-dicarboxylic acid (Fig. 1, I and II) = 4,5,6,7,8pentachloro-2,3,3a,4,7,7a-hexahydro-4,7methano-indene-1,3-dicarboxylic acid (22.5% of the total excretion). The dimethylestcr of dihydrochlordene dicarboxylic acid ( 11.3yo) (Fig. 1, III) : two very polar conjugates which could be hydrolyzed with methanol/HCl and, upon their methylation, formed the dimethylester of dihydrochlordene dicarboxylic acid (IV and V) (4.5% of the total excretion). Furthermore, there were other conversion products (VI-IX) (5.6% of the total excretion), but, except for glc-MS-spectrometry, no further information on their structures was obtained. Of the total
ACID-‘IC
IN
RATS
229
excreted radioactivity 46.10/, was due to unchanged dihydrochlordene dicarboxylic acid, and 10% was not extractable by the methods used. The compourlds I and II have, on tic, Rf values of 0.51 and 0.53 (solvent: benzene/ petrol ether/acetic acid 10 : 10 : 1). After methylation, the Rf values were 0.72 and 0.77 (three times developed with benzene), and the glc retention times 23.3’ and 24.1’ (experimental conditions as described in “Materials and Methods” for the second apparatus). The mass spectra of the methylated compounds I and II (Figs. 2 and 3) show differences only in peak intensities. Figure 2 shows the mass spectrum of metabolite I. A part of the mass spectrum of metabolite II has been placed above the corresponding part of I, in order to indicate the differences. Likewise, Fig. 3 shows the mass spectrum of metabolite II, and a part of I has been added. Both compounds contain only 5 chlorine atoms. Due to the characteristic Retro-Dicls-Alder fragment ions at m/e 236 and 201, a bridged molecule can be excluded. The R!I+-peak and important fragment ions with proposed gross formula are given I in Table 2. For the exact structure elucidation, the two dihydrochlordene dicarboxylic acids (Fig. 1, I and II) de-
l
Ii’
I
IX 200 5cAr4 NR. nECILURO-3I~~ORUM;ROEN-~*~~-~~~~~~~~~~~~~ FIG.
2. Mass
spectrum
of metabolite
I of dihydrochlordene
h4*420
dicarboxylic
acid in rats, after
methylation.
LAY,
FIG.
3. dlass
spectrwn
of metabolite
WEISGERBER
AND
II of dihvdrochlordene
chlorinated at, C4 were synthesized (11). Chromatographic data and mass spectra were identical to those of the metabolites. A 60 MHz NXIR spectrum (solvent: deuteroacetonc) was obtained for the synthesized substances. The signal occurring at 3.1 ppm (relating to T3IS = 10) indicates the presence of a proton at the dechlorinated C,. Unfortunately, there was not enough pure metabolitc material to obtain a N-\IR spectrum thereof t,oo. Furthermore, a dihydrochlordenc dicarboxylic acid dechlorinated at the double bond was synthesized (13) and methylated. The mass speckurn of this substance was compared to those of the mcthylated
KLEIN
dicarboxylic
acid in rats, after
,methylation.
metabolites I and II; clear diffcrenccs in intensities exclude the identity of bot,h isomers with this compound. Metabolite III, on tic, had an R,-value of 0.62 with benzene/petrol ether/acetic acid 10: 10: 1 as solvent and, on glc, a retention time of 25.2’. Table 3 contains the typical fragment ions from the mass spectrum of conversion product, III. The mass spectrum of metabolite III is shown in E’ig. 4. h reference substance was obtained by methylation of the dihydroTABLE MS-Fragnzentation Dihydrochlordene
3 oj Metabolite Dicarbo&ic
III Acid
of
-__ TABLE
2
Mass
MS-Fragmentation of the Methylated Metabolites II of Dihgdrochlordene Dicarboxylic Acid M&W 420 (M+) 389 3% 3x4 325 324 289 236 201
Fragment
ion
GrHuOXls CI,H~OO~X, ‘&H~aOaCla ‘&H120& C,zH,O&l~ ChH80#.% C,zHsO&l, CsHCl? CbHCk ~---
a R,etro-Dieb-Alder.
Fragment,
I and 454 (M+) 423 419 418 387 36s 359 358 351 323 297 270 23.5 __-.. 0 Retro-Die&Alder.
G~H,~O~Cl~
CdLOaCls ChH120&1:,
C~Hdl&l~ CdWXl, GaH,OnCls CieHnOzCl, C,zH,O&ls C,3H,O&la C,2H70rCl~ C&Clc C&lea CLC16LL __
ion
DIHYDROCHLORDENE
FIG.
4. Mass
spectrum
of metabolzte
DICARBOXYLIC
III
ACID-14C
of dikydrochlordene
chlordene dicarboxylie acid with diazomethane. Its chromatographic properties and the mass spectrum were identical to those of the metabolite. Th,e products VI-IX, on tic, were at the front with all solvents used for their purification. The treatment of the products VI-IX with methanol/HCl and CfIJ/ Ag,O, respectively, did not lead to any changes in their tic-properties, which is contrary to compounds I-V. The products VI-IX had lower molecular w-eights than dihydrochlordene dicarboxylic acid. Table 4 gives t.he characteristic dat,a of thcsc minor met,abolites. Figure 1 gives a survey on t)hc structures of isolated and idcmified conversion products of dihydrochlordene dicarboxylic acid in rats.
IN
231
RATS
dicaybozylic
acid in rats.
as reported earlier (11) with rat-live1 homogenat’es in in vitro experiments. The dechlorinabcd acids (metabolites I and II) may br regarded as the first step in the degradation of the chlorinated ring. The metabolites VI-IX have a lower molecule weight, and they seem to bc products of further degradat,ion of the parent compound. But as these metabolites were available in minor yuantit#ies, not much information could be obtained regarding their structure. However, it was not possible to detect them in in vitro experiments with liver homogenates (11). This suggest,st,hat they might. be formed in vitro in very small quant’it,ies which were beyond t,he detection limit. Alternatively, they arc formed in the living animals in some other part of the organism.
DISCUSSION
TABLE
From the present experiments it is apparent that dihydrochlordenc dicarboxylic acid is not the end product of aldrin/dieldrin metabolism. It was metabolized to a large degree within one week of the experiment; about 44% of the radioactivity excreted consisted of met’abolites. The dechlorination resulting in the metabolites I and II is the major metabolic pathway, besides esterification and conjugation resulting in the metabolites III-V. It is obvious that the dechlorination takes places in the liver,
Data on hlinor Dihydrochlordene ~Conversion product no.
Concentration in excreta in terms of % of total excretion
VI VII VIII IS u glc Conditions Second apparatus, 0.35 kp/cm*.
1.3 1.2 1.4 1.7
4
Conversion Products Dicarboxylic Acid nlc
M W
Retention time-
3.5’ 3.Y’ 3.7’ Y.0’
244 278 302 336
of Number chlorine atoms
4 5 4 6
as described in “Materials and Methods.” oven temperature 25O”C, N?-pressure:
of
LAY,
232
WEISGERBER
ACKNOWLEDGMENT
We thank W. Tomberg for the preparation of mass spectra and for kind assistance in the interpretation. REFERENCES
I. K. Sandrock, D. Bieniek, W. Klein and F. Korte, LXXXVI. Communication : Isolierung und Strukturaufklsrung von Kelevan-“CMetaboliten und Bilanz in Kartoffeln und Boden, Chemosphere 3, 199 (1974). 2. F. Korte and H. Arent, Metabolism of insecticides IX: Isolation and identification of dieldrin metabolites from urine of rabbits after oral administration of dieldrir+C, Life fki. 4, 2017 (1965). 3. F. Korte and W. Kochen, Insektizide im Stoffwechsel XII : Isolierung und Identifizierung von Metaboliten des Aldrin-lPC aus dem Urin von Kaninchen, Med. Pharmacol. Exp. 15, 409 (1966).
4. V. J. Feil, R. D. Hedde, R. G. Zaylskie, and C. H. Zachrison, Dieldrin-14C metabolism in sheep: Identification of trans-6,7-dihydroxydihydroaldrin and 9-(syn-epoxy) hydroxy1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a,~5,6,7,8,8a-octahydro-1,4-endo-&&exo-dimethanonaphthalene, J. Agr. Food Chem. 18, 120 0970). 5.
M. 6. Baldwin, J. Robinson and D. V. Parke, A comparison of the metabolism of HEOD (dieldrin) in the CFl mouse with that in the CFE rat, Food Cosmet. Toxiwl. 10,333 (1972).
AND
KLEIN
6. F. Korte, Recent results of investigations in environmental chemistry, in “Radiotracer Studies of Chemical Residues in Food and Agriculture,” Panel, Vienna, October 1971, IAEA STI/PUB 332, p. 3. 7. J. Oda and W. Miiller, Identification of a mammalian break-down product of dieldrin, Symposium on chemistry of pesticides under metabolic and environmental conditions, Bonn, Sept. 1970. Summary, Env. Qnal. safety 1, 248 (1972). 8. L. Nilzer, S. Detera, I. Weisgerber und W. Klein, BeitrSige zur iikologischen Chemie LXXVII : Verteilung und Metabolsmus des Aldrin-Dieldrin-Metaboliten Trans-4,5-dihydroxy-4,5-dihydroaldrin-14C in Salatpflanzen und Boden, Chemosphere 3, 143 (1974). 9. W. Klein, J. Kohli, I. Weisgerber, and F. Kort,e, Fate of aldrin-14C in potatoes and soil under outdoor conditions, 1. Agr. Food Chem. 21, 1.52 (1973).
10. J. Kohli, S. Zarif, I. Weisgerber, W. Klein, and F. Korte, Fate of aldrin-“C in sugar beets and soil under outdoor conditions, J. Agr. Food Chem.
21, 855 (1973).
11. J. P. Lay, W. Klein und F. Korte, BeitrLge zur tikologischen Chemie LXXXV : Mikrosynthese und in vitro Metabolismus von Dihydrochlorden-Dicarbons&ure-‘4C durch Rattenleberorganellen, Chemosphere 3, 193 (1974). 12. S. G&b, W. Klein und F. Korte, Beitrgge zur iikologischen Chemie LVI : Photoreaktionen des Aldrin/Dieldrin-Metaboliten Dihydrochlorden-1,3-Dicarbonslure, Chemusphere 2, 107 (1973).