Metabolism of the Photo-decarboxylated Derivative

Transcription

1 J. Pesticide Sci. 10, (1985) Original Article Metabolism of the Photo-decarboxylated Derivative of Fenvalerate in Rats Nobuyoshi MIKAMI, Jun YOSHIMURA, Toshiyuki KATAGI, Hlrohiko YAMADA and Junshi MIYAMOTO Laboratory of Biochemistry and Toxicology, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Takatsukasa, Takarazuka 665, Japan (Received January 14, 1985) Upon a single oral dose of the photo-decarboxylated derivative of fenvalerate (decarboxyfenvalerate), [2-(3-phenoxyphenyl)-3-(4-chlorophenyl)-4-methylpentanenitrile] labeled with 14C in the chlorophenyl and benzyl rings to both male and female rats at the rate of 4 mg/ kg, the radiocarbon was readily absorbed, metabolized and almost completely eliminated mainly in the feces within a few days. The radiocarbon was mainly located in the liver and gastrointestinal contents, and hardly translocated to other tissues, as evidenced by the whole-body autoradiogram taken at 1, 3, 6, 9 and 24 hr after oral administration of the 14C-labeled preparations. The 14C residues in tissues 7 days after treatment were generally quite low. Decarboxy-fenvalerate was metabolized in rats via hydroxylation at the methyl group with subsequent oxidation to the carboxylic acid, and hydroxylation at the 2'-, 3'-, 4'- or 6-position of the 3-phenoxybenzyl moiety, and at the benzyl methine carbon bearing the cyano group. The resultant oxidation products were excreted in bile mainly as the glucuronide conjugates, and hardly excreted in the urine. The glucuronide conjugates underwent significant enterohepatic circulation, during which the biliary metabolites were deconjugated by gut microflora and excreted into the feces. There was no substantial difference in the nature and amounts of the metabolites, and in the patterns of the 14C excretion and tissue residues between male and female rats, and between the two 14C-labeled compounds. INTRODUCTION Fenvalerate [Sumicidin(R), a-cyano-3-phenoxybenzyl 2- (4-chlorophenyl) -3-methylbutyrate] is a synthetic pyrethroid insecticide currently used for agriculture. The insecticide was readily subjected to various types of biotic and abiotic transformation to give a large number of degradation products in the field. Decarboxy-fenvalerate [2-(3-phenoxyphenyl)-3-(4- chlorophenyl)-4-methylpentanenitrile] (I) was one of the major degradation products, and formed by photochemical reactions in water and on plant foliage. 1-4) The residue analysis of a variety of crops suggested that the photoproduct might comprise up to 10% of the total residues, particularly in forage crops which had been exposed to prolonged sunlight and drying conditions. However, the compound had no tendency to persist in tissues of hens and cows if consumed as a contaminant of feed for a long period of time, and the residue levels in egg, milk and meat were quite low. 5) Since I was not included among the metabolic pathways of fenvalerate in mammals, the toxicological studies of fenvalerate in mammals preclude the evaluation of this compound. In view of such circumstances, the mammalian metabolic study has been initiated as an attempt to provide necessary information for clarifying the toxicological profile of I. This paper deals with the absorption, distribution, excretion and biodegradability of I in rats following a single oral administration of the 14C-labeled preparations.

2 MATERIALS AND METHODS 1. Radiolabeled Compounds The following 14C preparations were used: Decarboxy-fenvalerate (I) separately labeled uniformly in the chlorophenyl ring [referred to as chlorophenyl-14c] and in the ring of the benzyl group [benzyl-14c]. These labeled compounds, which were racemic mixtures of the four optical isomers, were prepared by UVirradiation of 14C-f envalerate (1 mmol) in hexane (30 ml) for 2 hr, with subsequent purification by preparative silica gel thin-layer chromatography (TLC) in benzene saturated with formic acid/diethyl ether (10/3). The specific activity was 15.6 mci/mmol for the chlorophenyl-14c preparation, and 34.1 mci/ mmol for the benzyl-14c preparation. Both of the 14C preparations had radiochemical purities of more than 99%, as determined by TLC in hexane/toluene/acetic acid (3/15/2) (Rf=0.56). 2. Unlabeled Compounds The unlabeled compounds, shown in Fig. 4, were synthesized for reference purposes. The spectrometric properties of these compounds are summarized in Table 1. The synthetic procedures will be published elsewhere. 6) fl-glucuronidase (Type B-1, from bovine liver), sulfatase (Type IV, from limpets) and saccharo-l,4-lactone monohydrate were purchased from Sigma Chemical Co., St. Louis, Mo. 3. Radioassay Liquid scintillation counting (LSC), combustion analysis and autoradiography were carried out according to the methods reported previously. 3) 4. Thin-layer Chromatography (TLC) Precoated silica gel 60F254 chromatoplates (20 X 20 cm, 0.25 mm layer thickness, E. Merck) were used for analytical and preparative purposes. The Rf values of I and the derivatives by TLC are listed in Table 2. The solvent systems for two-dimensional development are illustrated, for example, as follows: (Ax 2, C) indicates development in the first direction twice with solvent system A, and in the second direction with solvent system C. The radioactive areas on TLC plates were detected by autoradiography, and unlabeled standard chemicals were detected by UV light. 5. Spectroscopy Nuclear magnetic resonance (NMR) were determined in deuterochloroform, using tetramethylsilane as an internal standard, with Hitachi model R-40 spectrometer at 90 MHz. Infrared (IR) spectra were recorded on Hitachi model 285 spectrometer, as a liquid thin film on NaCI crystals or KBr disks. Electron impact-mass (EI-MS) was determined at 12 and 72 ev, with Shimadzu-LKB 9000 mass spectrometer, using a direct insertion probe. Field desorption-mass (FD-MS) spectra were recorded on Hitachi M-80 spectrometer equipped with model M-003 data system. 6. Animal Experiments 6.1 Excretion balance studies The six-week old Sprague-Dawley (SD) rats weighing approximately 200 g (male) and 160 g (female) were purchased from Shizuoka Agricultural Co-operative Association for Laboratory Animals, Shizuoka, Japan. Four male and female rats were each given a single oral dose of 14C-I in 10% Tween 80 solution (1 ml/rat) at a rate of 4 mg/kg. The treated rats were individually housed in glass metabolism cages, and allowed free access to food and water. The urine and feces were separately collected for up to 7 days, and then the treated rats were sacrificed to dissect out more than 20 tissues including blood, brain, fat and liver for 14C analysis. 6.2 Time course studies of 14C tissue residues A total of ten male rats were given a single oral dose of the benzyl-14c preparation at 4 mg/kg. The tissue samples ( mg) recovered on sacrifice at 1, 3, 6, 12 and 24 hr after administration were dried over P205 in a vacuum desiccator, and then analyzed for the total radioactivity by combustion and LSC. 6.3 Studies on whole-body autoradiography The whole-body autoradiography was performed according to the method of Ullberg, 7) after male rats had been given a single oral dose of the benzyl-14c preparation (381 Ci) at 4 mg/kg. The frozen rats were mounted onto the cold stage of a microtome at -15C in a

3 Journal of Pesticide Science 10 (2), May Table 1 The analytical data for decarboxy-fenvalerate and the derivatives. PMV 450 MP cryo-microtome (PMV AB, Stockholm, Sweden). The sagittal sections, 20-micron thick, were cut at several levels to include the significant organs, and held under the X,-ray SB film (Eastman Kodak Company) at 4C for 28 days. 6.4 Bile studies During surgery, the common bile duct was

4 Table 2 The Rf values of decarboxy-fenvalerate and the derivatives. a) A, hexane/diethyl ether (20/1); B, hexane/toluene/acetic acid (3/15/2); C, benzene/chloroform (1/1); D, hexane/acetone (20/1); E, benzene saturated with formic acid/diethyl ether (10/3); F, toluene/acetic acid (7/1). cannulated in situ with a length (30 cm) of PE 50 Intramedic polyethylene tubing (0.58 mm i.d., mm o.d., Cray Adams, USA). Four male SD rats (10-week old) were given a single oral dose of the 14C-labeled preparation (I) at 4 mg/kg. The treated rats were housed individually for the duration of the experiment (48 hr) at 25C in restraining cages, which were designed to enable the separate collection of the bile, urine and feces. The bile was collected at 1, 3, 6, 9, 24 and 48 hr after administration. Animals receiving the bile (1 ml/rat) containing the radioactive metabolites (4.4 uci) also had a second cannula inserted into the duodenum via the bile duct, connected to a dosing syringe (infusion rate, 1 ml/hr). The 1-3 hr bile samples were given to other bile duct-cannulated rats via their intraduodenal cannulae. The bile from rats dosed in this manner was collected as described previously. The radiocarbon in the bile samples was taken as a direct evidence of the enterohepatic circulation. 6.5 Analysis of metabolites The 0-24 hr urine was lyophilized to dryness. Little radiocarbon was lost during the lyophilization. The resultant residues were extracted three times with 30 ml of methanol, and the extracts were combined, concentrated and analyzed by TLC. The 0-24 hr feces from male and female rats were separately extracted by homogenization twice in a Waring blender (Nikon Seiki Co., Tokyo, Japan) with four-fold volumes of methanol/water (9/1) to afford more than 80% of the fecal radioactivity. The extracts were combined, filtered through a filter paper, concentrated and analyzed by TLC. Samples of blood and liver were extracted three times in four-fold volumes of methanol/water (9/1) by homogenization twice with a polytron (Type PT 10-35, Kinematica Gmb H, Luzern, Switzerland). The homogenates were centrifuged at 5000 rpm for 10 min, and the supernatant phases were combined, concentrated and analyzed by TLC. The bile samples were directly analyzed by TLC. 6.6 Characterization of metabolites Metabolites in the bile, feces and rat tissues were identified by two-dimensional TLC cochromatography with authentic standards before or after enzymatic and/or chemical hydrolysis. The enzymatic and chemical reactions were carried out under the following conditions. 1) /3-Glucuronidase or sulfatase (2 mg) in 1 ml of 1/15 M acetate buffer (ph 4.5) incubated for 12 hr at 37 C. In the case of the sulfate conjugates, sulfatase was used together with saccharo-l,4-lactone (25 mm) to inhibit /3- glucuronidase.

5 Journal of Pesticide Science 10 (2), May Table 3 Radioactivity excreted in urine and feces following a single oral administration of 14C-decarboxy-fenvalerate to rats at the rate of 4 mg/kg. 2) Some conjugated metabolites were hydrolyzed in 3 N HCl (5 ml) at 100 C for 1 hr, or in 0.5 N NaOH (5 ml) at 100 C for 2 hr. The released aglycones were extracted three times with two-fold volumes of ethyl acetate after acidification to ph 1. RESULTS 1. Excretion Balance Studies Upon a single oral administration of the chlorophenyl- and benzyl-14c labels of I to both male and female rats, excretion of the radiocarbon was rapid and almost complete within 7 days (Table 3). The vast majority of the excreted material was observed in the feces ( %) with the urine containing less than 1.2% of the administered dose. There was no substantial difference in the overall rates of excretion between male and female rats, and between the two 14C-labeled preparations. 2. Whole-body Autoradiography The whole-body autoradiogram was taken at 1, 3, 6, 9 and 24 hr after oral administration of the benzyl-14c label to male rats at 4 mg/kg. Typical examples are shown in Fig. 1. During the period of experiments the radiocarbon located mainly in the liver, and contents of the stomach and intestines, and hardly translocated to other tissues and organs like brain, spleen, muscle, kidney and testis. At 9 and 24 hr after treatment, large amounts of the radiocarbon were found in the feces. This evidence strongly suggests that enterohepatic circulation significantly occurs, during which the radiocarbon is excreted mainly in the feces. 3. Biological Half-life of 14C in Rat Tissues Figure 2 shows the time course of 14C levels in the blood and various tissues of male rats following a single oral dose of the benzyl-14c preparation. The 14C residues in tissues reached a maximum at 1 hr (blood, heart, pancreas, spleen), 3 hr (muscle), 6 hr (liver, kidney) and 12 hr (fat) after administration, but rapidly decreased thereafter. The maximum 14C residues in the fat and liver were 1.7 and 3.8 ppm Eq., respectively, whereas the radiocarbon contents in other tissues did not exceed 0.7 ppm Eq. Based upon the data, the biological half-life (T172) of 14C was determined using the following equation ; In Y=A -BX, T112= 0.693/B. The results along with the coefficient of the correlation are summarized in Table 4. The initial biological half-life of 14C was approximately 10 hr in the pancreas and less than 7 hr in other tissues. Although it was impossible to calculate the biological half-life of the radiocarbon in the adipose tissue, it was estimated to be less than 24 hr based upon the

6 278日 本 農 薬 学 会誌 第10巻 第2号 昭和60年5月 1hr 9hr 24 hr Fig. 1 The whole-body 14C-decarboxy-fenvalerate Table 4 Half-lives of male rats after decarboxy-fenvalerate. r2: coefficient of the resultsshowninfig.2. autoradiogram at 4 mg/kg. of male rats after a single oral administration (T12) for the 14C in tissues oral administration of 14C- correlation. of 4. Tissue Residues Table 5 shows the "C residue levels in 22 tissues of male and female rats 7 days after a single oral administration. The levels of radioactivity in tissues were generally quite low. The exception was the adipose tissue which contained to ppm equivalent of "C-I. The TLC analysis showed that I amounted to 46-68% of the "C in the adipose tissue. Somewhat higher radioactivity was detected in the gastrointestinal contents. 5. Metabolites in Urine and Feces from Rats Dosed with 14C-Decarboxy fenvalerate Thin-layer chromatography showed that most of the radiocarbon in the urine remained at the origin of the TLC plates in solvent sys-

7 Journal of Pesticide Science 10 (2), May Table 5 The 14C residue levels in tissues of male and female rats 7 days after single oral administration of 14C-decarboxy-fenvalerate (I) at 4 mg/kg. Fig. 2 The 14C residues in tissues of male rats after a single oral administration of 14C-decarboxy-f envalerate. Data are expressed as the mean + range of two rats.

8 tern (B x 2). It is likely, therefore, that the urinary metabolites are present as conjugates. However, no further characterization was carried out because of the small quantity of the radiocarbon. In contrast, the extract of the feces contained more than 15 metabolites, all of which were detected with both the chlorophenyl- and benzyl-14c preparations. Each metabolite was separated on preparative TLC in solvent system (B x 2), and then identified by two dimensional TLC co-chromatography with the synthesized compounds in the following solvent systems : I, II, III, IV and V (B, C), VI (B x 2, F), VII (B, F), VIII, IX, XI and XII (B x 2,E), and X and XIII (B, E). The conjugated fecal metabolites located at the origin of the TLC plates in solvent system (B x 2) were scraped, eluted with methanol/ water (1/1) and then developed again on silica gel TLC plates in ethyl acetate/ethanol/water (4/2/1). The individual conjugates were subjected to enzymatic or chemical hydrolysis, and the released aglycones were identified by two dimensional TLC co-chromatography as described above. Table 6 shows the amounts of metabolites in the feces from rats dosed with 14C-I. The major metabolites were oxidation products hydroxylated at the 2'-position (II) and the 4'- position (IV) of the 3-phenoxybenzyl moiety. Both products were present mainly in a free form. Further, somewhat higher amounts of the metabolites oxidized at the isopropyl and Table 6 Amounts of metabolites in the feces of rats one day after a single oral administration of 14C-decarboxy-fenvalerate at 4 mg/kg. glu, glucuronide; sul, sulfate; conj, conjugated metabolites which were resistant to enzymatic and alkaline hydrolysis but were cleaved by acid hydrolysis.

9 Journal of Pesticide Science 10 (2), May phenoxybenzyl groups like IX, X, XII and XIII were found. In addition, small amounts of the corresponding glucuronide and sulfate conjugates were present, along with different types of the conjugates which were resistant toward enzymatic and alkaline hydrolysis but were cleaved by acid hydrolysis. Identification of the glycone moiety has yet to be carried out. Approximately 10-17% of the dose were not identified. However, each of the unidentified products did not exceed 3 % of the administered dose. 6. Excretion of Radiocarbon in Bile after Treatment with 14C-Decarboxy f envalerate and the Biliary Metabolites Following a single oral dose of 14C-I, approximately 64-75% of the dose was eliminated in the bile, whereas excretion of 14C in the urine and feces was in total less than 10% Treatment with 14Cdecarboxy-fenvalerate Fig. 3 The excretion of 14C into the bile after oral administration of 14C-decarboxy-fenvalerate and the biliary metabolites. Treatment with 14Cbiliary metabolites within 48 hr (Fig. 3). Nearly the same excretion profiles were observed after intraduodenal injection of the 1-3 hr bile samples to the bile duct-cannulated (B.D.C.) rats. These evidences indicate that the biliary metabolites of I undergo significant enterohepatic circulation. Table 7 Relative amounts of metabolites in the blood, liver and bile of male rats 3 hr after treatment with 14C-decarboxy-fenvalerate (oral) and the biliary metabolites (i. d. a). Values are average per cent of each metabolite on TLC plates. a) id., intraduodenal; b) B.D.C., bile duct-cannulated; glu, glucuronide; sul, sulfate.

10 During the experiment smaller amounts of feces were excreted from the B.D.C. rats than from the intact rats. This may relate to the lower recovery of radiocarbon (ca % of dose within 48 hr) from the B.D.C. rats dosed with 14C-I, as compared with the intact rats (Table 3). 7. Metabolites in Blood, Liver and Bile As shown in Table 7, the metabolites of I in the blood and liver were qualitatively similar to those in the feces. In contrast, the metabolic patterns in the bile were markedly different from those in the feces. The major biliary metabolites were glucuronide conjugates of II and IV, which amounted in total to approximately 61-72% of the radiocarbon in the bile from rats treated with 14C-I and the biliary metabolites. The enterohepatic circulation of the biliary metabolites resulted in a slight increase in amounts of the more polar products such as IX, X, XII and XIII, which also occurred as glucuronide conjugates. However, these biliary metabolites were of minor importance in the feces. DISCUSSION When 14C-decarboxy-fenvalerate was orally administered to both male and female rats, the radiocarbon was readily absorbed, metabolized and almost completely excreted mainly in the feces. In accordance with the excretion data, the 14C residues in tissues were generally quite low. In common with other chemicals, however, the adipose tissue retained small quantities of the radiocarbon in concentrations higher than that noted for other tissues. More than 15 metabolites were detected in the feces. Based upon the identified products, the proposed metabolic pathways for I in rats are shown in Fig. 4. The photoproduct underwent hydroxylation at the methyl group with subsequent oxidation to the carboxylic acid, and hydroxylation at the 2'-, 3'-, 4'- or 6-position of the 3-phenoxybenzyl moiety, and at the benzyl methine carbon bearing the cyano group. Hydroxylation at the methine carbon resulted in the formation of cyanohydrin, which was unstable under the physiological conditions and spontaneously converted to VI with a release of hydrogen cyanide. On the other hand, hydroxylation at the benzyl carbon bearing the isopropyl group resulted in the formation of XIV, a portion of which was nonenzymatically transformed to XV under acidic conditions, such as on silica gel TLC plates through pinacol-pinacolone rearrangement as shown below. Nevertheless, neither of these products was detected in the feces. Fig. 4 The proposed metabolic pathways of decarboxy-fenvalerate in rats.

11 Journal of Pesticide Science 10 (2), May were of very minor importance in the feces, and the corresponding free aglycones were predominant. It is likely, therefore, that the deconjugated biliary metabolites are excreted in the feces during the enterohepatic circulation. The excretion profiles for metabolites of I in rats are summarized in Fig. 5. Thus, although I appears to be more chemically stable than fenvalerate, it is quite biodegradable and has no tendency to persist in rat tissues for a long period of time. Fig. 5 The excretion profiles for metabolites of decarboxy-f envalerate. The oxidation products were excreted mainly in the bile as the glucuronide conjugates and to a smaller degree as the sulfate. This was not unexpected, considering that rats had a molecular weight threshold of 325±50 for the biliary excretion of such anions, 8' and the molecular weights of the conjugated metabolites were greater than 440. However, it was somewhat surprising that the metabolites were eliminated in the urine to only a limited extent. This might be due to small tendency of the metabolites to translocate to other tissues including the kidney, as evidenced by the whole-body autoradiogram (Fig. 1) as well as by radioactivity determination of the blood and tissues (Fig. 2). The biliary metabolites were glucuronide conjugates of II and IV, indicating that I was metabolized in rats primarily via hydroxylation at the 2'- and 4'- positions of the 3-phenoxybenzyl group. The biliary metabolites underwent significant enterohepatic circulation. The glucuronide conjugates might be cleaved by gut microflora and then absorbed from the intestine in free forms, because it has been reported that the gut flora were capable of cleaving the conjugates, and the deconjugation was a prerequisite for reabsorption. 9 ' 10' Following reabsorption, the hydroxylated derivatives appear to be metabolized further by direct conjugation with glucuronic acid or by oxidation at the methyl group with subsequent glucuronidation. Again, the glucuronide conjugates were excreted in the bile. However, these biliary metabolites REFERENCES 1) R. L. Holmstead & D. G. Fuller: J. Agric. Food Chem. 25, 56 (1977) 2) R. L. Holmstead, D. G. Fuller & L. 0. Ruzo: J. Agric. Food Chem. 26, 954 (1978) 3) N. Mikami, N. Takahashi, K. Hayashi & J. Miyamoto: J. Pesticide Sci. 5, 225 (1980) 4) N. Mikami, N. Takahashi, H. Yamada & J. Miyamoto: Pestic. Sci. 16, 101 (1985) 5) G. A. van Gelder: private communication 6) T. Katagi, N. Mikami & J. Miyamoto: unpublished observation 7) S. Ullberg: Proc. 2nd UNInt. Conf. Peaceful Uses of Atomic Energy, Vol. 24, p. 248, ) P. Millburn, R. L. Smith & R. T. Williams: Biochem. J. 105, 1275 (1967) 9) K. R. Huckle, J. K. Chipman, D. H. Hutson & P. Millburn: Drug Metab. Dispos. 9, 360 (1981) 10) N Mikami, J. Yoshimura, H. Kaneko, H. Yamada & J. Miyamoto: Pestic. Sci. 16, 33 (1985)

12