Metabolism and pharmacokinetics of a single oral dose of O-4-bromo-2,5-dichlorophenyl O-methyl...

TOXICOLOGY AND APPLIED PHARMACOLOGY 55, 131-145 (1980)

Metabolism and Pharmacokinetics of a Single Oral Dose of 0-4-Bromo- 2,5Dichlorophenyl O-Methyl Phenylphosphonothioate

(Leptophos) in Hens

MOHAMED B. ABOU-DONIA

Department oj.Pharmacology. Duke University Medical Center, Durham. North Carolina 277/O

Received November 27, 1979: accepted April 17. 1980

Metabolism and Pharmacokinetics of a Single Oral Dose of O-4-Bromo-2.5dichlorophenyl O-Methyl Phenylphosphonothioate (Leptophos) in Hens. ABOU-DONIA, M. B. (1980). Tosicol. Appl. Pharmacol. 55, 131-145. The metabolism and pharmacokinetics of a subneurotoxic dose of leptophos were determined in laying hens following a single oral dose of 50 mgikg (0.9 &i/hen) of [phenylY]leptophos (O-4-bromo-2,Sdichlorophenyl O-methyl [‘Clphenylphosphonothioate). This study adds confirmatory evidence to the hypothesis that species selectivity for delayed neurotoxicity is related to interspecies differences in pharmacokinetics and metabolism. Oral leptophos was metabolized and excreted slowly in hens. The major portion of the radioactivity (86.5%) was excreted during the 20-day experiment. Significant amounts of the dose were deposited in egg albumen and yolk-3.4 and 2.5%. respectively. Only 1.3% was excreted in expired CO,. Radioactivity in tissues reached a peak of 14.6% of the dose 12 hr after administration; radioactivity decreased to 6.2% after 20 days (42.6% of peak value). The highest ‘“C concentration was present in the bile, followed by the gall bladder, kidney, adipose tissue, and liver. Brain, spinal cord, and sciatic nerve, which are affected by the neurotoxicity of leptophos, had smaller but constant concentrations throughout the experiment. Following the oral administration of [“Qleptophos the change in the r’C body burden with time was biex- ponential. The physiological disposition of leptophos may therefore be defined in terms of a two-compartment open-system model. Radioactivity was excreted at a slow rate, p value of 0.05 day-‘, corresponding to a half-life of 12.0 days. Leptophos was the only compound identified in nerve tissues, muscle, fat, and blood. Most of the radioactive substances in the excreta and liver were identified as unchanged leptophos with minor amounts of polar metabolites. The metabolic fate of leptophos can be explained on the basis of its physical properties of lipid solubility and tissue binding, and the predominant biliary secretion and gastrointestinal excretion of the compound.

Leptophos (O-4-bromo-25dichlorophenyl phenylphosphonothioate) causes delayed neurotoxicity in a number of species, in- cluding water buffaloes. chickens, and Mallard ducklings (Abou-Donia et al., 1974; Herin et al., 1978). This study continues investigations into the factors contributing to the species selectivity of this insecticide. Organophosphorus-induced delayed neurotoxicity was first characterized in man

(Smith et al., 1930); later other species (cats, dogs, cows, and chickens) were found to be susceptible. Rodents and some pri- mates are not susceptible. Since the adult chicken reacts like humans to these chemicals, the hen has become the test animal of choice in demonstrating and studying this effect.

Peripheral motor neuropathy of the limbs is the earliest neurological manifestation of

131 0041-008x/80/100131-15$02.00/0 Copyright 0 1980 by Academic Presr. Inc. All rights of reproduction m any form rererved.

132 MOHAMED B. ABOU-DONIA

poisoning by leptophos. The toxic effects METHODS later spread to other parts of the central nervous system: spinal cord and medulla, but not the higher brain (Abou-Donia and Chemica’s Preissig, 1976a,b). The associated histopathological changes can be characterized as Wallerian degeneration of the axons and myelin (Preissig and Abou-Donia, 1978; Abou-Donia and Graham, 1978), similarly observed after treatment with tri-O-cresyl phosphate (TOCP) (Cavanagh, 1973).

Certain physical properties of leptophos may contribute to its neurotoxic effect and further increase its human health hazard. It degrades slowly in the environment: leaves of tomato plants retained 35% of the pesticide 5 weeks after application (Aharon- son and Ben-Aziz, 1974). In addition, leptophos is highly soluble in lipid solvents. Its partition coefficient between octanol and water is 2,000,OOO (Davies et al., 1975). It also binds strongly to proteins (Abou- Donia, 1979a). All these properties enhance both penetration into the central nervous system and accumulation at the site(s) of neurotoxic action.

In an earlier limited study we suggested that species susceptible to delayed neurotoxicity have a higher accumulation rate, coupled with slower elimination of leptophos (Abou-Donia, 1976). Previously, orally administered leptophos has been shown in nonsusceptible species, e.g., mice (Holm- stead et al., 1973) and rats (Whitacre et al., 1976), to be rapidly metabolized and excreted. We later established that in susceptible species leptophos is not as com- pletely metabolized as in nonsensitive species (Abou-Donia and Ashry, 1978). Also, we have shown that topically applied leptophos in hens was persistent, with a long biologic half-life of 17 days (Abou-Donia, 1979a).

The present report was planned to study the absorption, distribution and elimination of a single oral subneurotoxic dose of uniformly phenyl-labeled [14C]leptophos in laying hens. This study also analyzes the metabolic fate of leptophos in the chicken.

Leptophos, its related compounds, and the radioactive material used were provided by Velsicol Chemical Company.’ The following analytical grade compounds were investigated: leptophos, leptophos oxon (U-4-bromo-2,5-dichlorophenyl O-methyl phenylphosphonate), MPPTA (U-methyl phenylphosphonothioic acid), MPPA (O-methyl phenylphosphonic acid), PPA (phenylphosphonic acid), desmethyl leptophos (DML, O-4-bromo-2,5-dichlorophenyl phenylphosphonothioate). and desmethyl leptophos oxon (DMLO, O-4-bromo-2,5-dichlorophenyl phenylphosphonate). Uniformly phenyl-labeled [“Clleptophos (O-4-bromo-2,5dichlorophenyl O- methyl [‘“Clphenylphosphonothioate) has a specific activity of 6.23 mCi/mmol.2

Care and Treatment of‘Birds.

Laying hens (Callus gallus domesricus), mixed breed, each 18 months old and weighing approximately I.5 kg (1.4-1.6 kg), were used.3 The birds were housed in individual metabolic cages in an air-con- ditioned room and were allowed I week to adjust to their environment before treatment was begun. After I week, 15 hens were given an oral dose of 50 mg/kg (0.9 &i/hen) of leptophos in gelatin capsules. The desired specific radioactivity was obtained by dissolu- tion of labeled and radioactive leptophos in acetone. Five similar hens given an empty gelatin capsule served as the control. The birds were returned to their cages and supplied with food and water ad /ibitum.4 Expired carbon dioxide, excrements and eggs were collected daily. One control and three treated hens were killed at each of the following time inter- vals: 0.5, 2, 4,8, and 20 days. Hens were anesthetized with an intraperitoneal injection of pentobarbital sodium solution (Nembutal),5 then killed by heart puncture. The blood was collected, and individual organs removed and weighed. The contents of the gastrointestinal tract parts, e.g., crop, proventriculus, ventriculus. small intestine, ceca, and large intestine, were separated. and each part washed with acetone. The wash of each part was added to its corresponding content and air dried.

1 Velsicol Chemical Co., Inc., Chicago, III. * Prepared by New England Nuclear, Boston, Mass. ’ SPAFAS, Norwich, Conn. ’ Layena Chicken Feed, Ralston Purina Co.,

St. Louis, MO. ’ Abbott Laboratories, North Chicago, Ill.

METABOLISM OF LEPTOPHOS 133

Determination of “C Radioactivity

i4C radioactivity was determined with a Packard Tri-Carb Model 32556 liquid scintillation spectrometer after preparation of the samples as indicated below. All data were corrected for background interference, dilution effects, quenching, and counting efficiency. Counting efficiencies were determined by correlating external standard values with a series of quenched standards. Counting efficiency was above 90%.

“CO, was trapped in two containers (100 ml each) containing a solution of ethanolamine-ethylene glycol monomethyl ether (1:2, v/v) (purified reagents).’ The scintillation medium was toluene-ethylene glycol monomethyl ether (4:l. v/v) containing 5 g of 2.5-diphenyl oxazole (PPO) and 200 mg of 1,4-bis- [2(5-phenyloxazolyl)benzene] (POPOP) per liter.

Samples from fresh tissues, body fluids, contents of gastrointestinal tract parts, and homogenized excreta were oxidized by combustion in a Packard tissue oxidizer Model 306B using 10 ml of the trapping solution Carbo-Sorb and 12 ml of the scintillation cocktail PermaAuor V.”

Extraction of Radioactivity from Tissues and Excreta

Tissue and body fluid samples selected for identihca- tion of metabolites were: brain, spinal cord, sciatic nerve, plasma, liver, fat, and bile. Samples were taken from birds sacrificed 2 days after administration. Tissue or excreta samples were homogenized using the Polytron combination sonicator-homogenize?’ with water five times their weight. The homogenized sample was excreted three times with ethyl ether followed by ethyl acetate to ensure the extraction of nonpolar and polar compounds, and the combined solvent extracts were dried over anhydrous Na,SO, and evaporated to dryness at 40°C in vacua. The residue was partitioned between n-hexane and aceto- nitrile. cleaned up from interfering materials by passing through Florisil column (Jones and Riddick, 1952; Mills, 1968) and the solvent concentrated at 40°C in vacua.

Identificurion of Metabolites

Sequential thin-layer chromatography (stlc). The concentrated extracts were subjected to stlc (Abou- Donia, 1978). In this system Gelman-type SA tic, silicic acid-impregnated glass fiber sheets9 were first developed with the primary solvent, acetronile-

6 Packard Instrument Co., Inc., Downers Grove, III. ’ Fisher Scientific Company, Raleigh, N.C. * Brinkman Instruments, Westbury, N.Y. y Gelman Instruments Company, Ann Arbor, Mich.

water-ammonia (40:9:1), for 6 cm followed by the secondary solvent n-hexane-diethyl ether (9:l) for 16 cm.

For characterization of metabolites, a mixture of leptophos and structurally related compounds was added as standard to the solvent extracts of tissues or excreta from [‘4C]leptophos-treated hens. Their chromatography pattern with the labeled unknown was determined on stlc. The standards were detected by their color in iodine vapor. Quantification of leptophos and its metabolites was achieved by cutting the sheets into 5-mm strips, placing them into scintillation vials, and vigorously mixing them with the scintillation medium mentioned above. When chromatography of one of the standards with a radioactive metabolite was indicated, the metabolite was isolated in sufficient quantity, when possible, on tic plates with the unknown only. The compounds were eluted from the sheet by repeated extractions with ether and acetone and the solvents were evaporated.

Reverse-phase high-pressure liquid chromatography (hplc). Leptophos and related compounds were separated by reverse-phase hplc” and a RP-8 column” using methanol-water gradient elution (1:99, v/v, -+ 95:5. v/v, in 30 min after a IO-min delay); quantification was performed by measuring uv absorbance at 280 nm (Lasker er al., 1980). Detection at this wave- length was very sensitive, allowing detection of lo-20 ng of leptophos and the oxygen analog; detection of phosphonic acid-related compounds was 10 times less sensitive. Retention times and peak areas were highly reproducible for all compounds.

Mass sprctrometry. Mass spectrometry was used to confirm the identity of leptophos and most of its metabolites. The spectra were recorded in triplicate at 70 eV and source temperature 150°C on an AEI MS 902 high-resolution mass spectrometer.‘”

RESULTS

Clinical and Histological Observation

None of the hens given a single oral dose of 50 mglkg leptophos showed any signs of cholinergic or delayed neurotoxic effects. During the experimental period the eating habits of treated animals did not change; treated and control hens consumed com- parable amounts of feed and water. No changes in tissues were observed when

iI’ Waters Associates Inc., Milford, Mass. I1 EM Reagents, Cincinnati, Ohio. if AEI Scientific Instruments, San Diego, Calif.


TABLE 1

CUMULATIVE PERCENTAGE RECOVERY OF ‘T RADIOACTIVITY FROM HENS GIVEN A SINGLE 50 mg/kg ORAL

(0.9 pCi/HEN) OF [‘TILEPTOPHOS

Days Expired

air” Excreta” Egg

albumen” Egg yolk” Tissues”

contents of gastrointestinal

tract” Total

0.5 0.05 -c 0.01 28.55 t 3.29 0.41 + 0.08 0.24 i 0.05 14.50 f 2.89 47.24 2 9.93 91.05 k 16.24 2 0.16 2 0.04 70.94 _f 5.78 1.24 2 0.48 0.78 -c 0.24 14.76 + 3.06 6.02 +- 1.92 93.90 t 11.52 4 0.40 + 0.15 77.16 -t 7.40 1.79 + 0.69 1.34 i- 0.37 13.94 t 4.98 0.52 r 0.18 95.15 _t 13.77 8 1.03 t 0.45 79.75 i 7.92 2.46 i 0.86 2.05 2 0.53 10.61 -c 2.92 0.28 2 0.03 96.18 t 12.70

20 1.25 2 0.52 86.47 k 9.97 3.40 2 1.19 2.54 2 0.65 6.15 k 1.63 0.11 t 0.03 99.92 2 13.99

” Each value is an average -C SE of the daily excretion values of the “C radioactivity from all the birds used (15. 12. 9. 6, and 3 birds for 0.5. 2. 4, 8, and 20 days, respectively).

’ Each value is an average ir SE of the “C radioactivity of tissues from three birds. Tissues that were analyzed are listed in Table 2.

treated and control birds were compared for weight, size, shape, and color of different tissues. Tissues which were taken from the brain, spinal cord and sciatic, tibia1 and peroneal nerves and examined histologically revealed no histologic alter- ations.

14C Radioactivity in Expired Air

During the experimental period accumulated total la’c in expired air increased steadily (Table 1). The daily rate of ‘“C radioactivity, however, reached a peak on Day 5 after administration, then generally decreased until Day 9 when it leveled off. Cumulative percentage recovery of llC in expired air throughout the 20-day experiment was 1.2% of the administered dose.

Combined Urinary-Fecal Excretion of 14C

A sharp increase was noted in the accumulated total urinary-fecal excretion of 14C through Day 4 after oral administration of [‘“Clleptophos. Radioactivity in excreta increased slowly thereafter. The daily rate of 14C excreted reached a peak on Day 1 after application of leptophos. Following this, there was a sharp decrease in the rate through Day 4 and slower decrease thereafter. By the end of the 20-day

experiment, 86.5% of the orally administered [‘“Clleptophos had been recovered in the combined urinary-fecal excreta (Table 1).

14C Radioactivity in the Egg

“C rapidly accumulated in egg albumen during the first 5 days following the oral application of [14C]leptophos (Table 1). The daily rate of “‘C reached a peak on Day 2, then decreased with time.

The yolk of eggs of hens treated orally with [“Clleptophos followed a pattern similar to that seen in albumen. A rapid increase occurred in the accumulated total Y through Day 6 in the yolk of eggs from treated hens. The daily rate of 14C showed peaks 1 and 6 days after administration. Table 1 shows that accumulated total “‘C was 2.5 and 3.4% in egg yolk and egg albumen, respectively.

Tissue Disposition of 14C Radioactivity

Table 2 shows that 12 hr after the oral administration of [14C]leptophos all tissues had taken up radioactivity. The highest concentration (dpm/g tissue) of radioactivity was in bile, followed by gall bladder, kidney, adipose tissue, liver, spinal cord, and uterus. Of all nervous tissue, the spinal


TABLE 2

CONCENTRATION OF RADIOACTIVITY IN VARIOUS TISSUES AND GASTROINTESTINAL TRACT CONTENTS OF HENS

GIVEN A SINGLE 50 mg/kg ORAL DOSE (0.9 @/HEN) OF [‘*C]LEPTOPHOS”

Tissue 0.5 Day 2 Days 4 Days 8 Days 20 Days

Brain Spinal cord Peripheral nerves Muscles RBC Plasma Urinary bladder

contents Urinary bladder Trachea Esophagus Lungs Heart Liver Spleen Pancreas Kidney Gall bladder 3ile Mesentary Adipose tissue Skin Ovary Ovary contents lsthmus Uterus Crop Proventriculus Ventriculus Ventricular lining Small intestine Ceca Large intestine Cloaca

Tissue contents Crop Proventriculus Ventriculus Small intestine Ceca Large intestine

235 f 43 411 2 71 216 f 60 247 t 54 384 k 93 300 k 35

I89 -t 49 245 -t 35 239 + 32 331 + 54 389 t 79 270 -t 52 644 2 66 275 2 22 357 + 69 665 r 167

1,023 + 147 2,270 + 272

333 t 53 662 + 344 I88 + I2 233 ” 39 177 f 33 238 + 35 405 r+ 124 550 + 250 313 f 75 161 t 43

1,458 ? 468 610 2 I54 665 k 148 562 _C I14 363 + 35

25,530 r 6,774 118,882 + 22,849 IX.483 + 62,829

18,137 i 5,537 26,948 2 5,100 30.317 2 1,743

314 2 65 420 -t 89 250 2 56 224 + 37 230 4 42 294 rt_ 53

565 2 55 507 2 72 260 2 54 207 f 22 341 2 69 202 t 12 575 k 60 217 r 51 289 i 36 347 f 51

1,768 ‘- 380 4,654 2 1,209

243 r+_ 63 409 + 87 312 ? 68 271 -c 60 285 & 40 186 f 40 146 c 41 222 + 52 221 5 31 275 5 51 523 2 67 405 2 91 449 + 69 694 r 230 469 rt 157

2,826 t 1,728 1,206 z 553 6,410 t 3,413 2,440 t 399

13,603 r 3,460 21,771 f 6,214

294 k 61 371 + 109 225 + 94 308 + 94 282 + 77 250 2 47

420 + 89 253 + 76 371 + 55 I87 k 40 326 k 105 193 2 34 746 + 290 301 2 109 274 k 43 308 +- 80

2,700 + 1,405 5,994 2 3,256

591 k 161 509 2 I51 223 + 71 320 + 80 I98 f 31 234 2 30 281 i 77 361 2 76 389 z!z 125 222 k 30 559 2 171 306 2 73 244 + 7 499 2 106

1,043 rt_ 842

30s r+_ 41 3,230 k 2,375

832 ? 324 1,167 k 434 3,800 2 1,739 1,143 AZ 162

238 rc_ 50 414 2 170 150 ‘- 64 250 + 63 324 c 38 I85 2 57

344 c 132 275 2 42 253 1 49 193 c 55 309 k 96 I81 k 20 366 IT 177 239 f 70 205 -c 70 I95 t 32 808 r 327

5,012 2 66 209 r 36 885 -+ 348 224 2 54 I98 2 38 I81 -t 44 265 ? 64 241 I 61 194 e 36 211 k 54 I41 t 23

1,099 + 476 248 2 44 266 + 49 215 i 34 512 ” 75

1,631 ” 787 348 ? II 565 + 83 371 2 58 472 2 12 636 rt 290

204 r 42 259 zt 56 113 t 62 130 t 31 121 ? 22 98 + 21

89-+ II 70 ‘- IO

292 f 77 87 + 31

114 k 63 65 ‘-’ 20

159 f 27 184 2 58

39 ” 25 I58 2 37

83 2 45 4,321 _t 74

181 r+_ 83 375 k 63 126 + 35 142 k 26

52 f 17 156 2 54 116 + 23 124 + 30 125 ? 25 106 + 67 172 2 46 130 t 29 62 k 33

189 + 18 233 r I08

I86 + 48 288 c 59 344 2 62 281 _t 72 356 f 54 429 c 89

‘I Values expressed as dpm/g fresh tissue, dpm/ml plasma and other fluids, or dpm/g dry gastrointestinal tract contents.

cord contained the highest concentration of the administered dose. As time passed, of 14C; the brain, followed closely by the there was a general decrease in the amount peripheral nerves, had the second highest of radioactivity in all tissues, as measured concentration. The total 14C in the tissues 4, 8, and 20 days after administration. The 12 hr after the administration was 14.6% total amount of 14C in all tissues had fallen


to 6.2% (41.7% of peak value) of the administered dose by Day 20 after administration.

The different tissues of the alimentary canal had varying concentrations of radioactivity, with the ventriculus lining having the highest. The same general pattern was noted in the birds that were killed at other periods. The contents of different parts of the gastrointestinal tract reached peak concentrations of 14C at 12 hr, with the contents of ventriculus having the highest concentration. The concentration of 14C in the contents of the gastrointestinal tract parts generally declined with time.

In chickens, urine and feces are voided together, and no attempts were made to separate them in this study. An approximate measure of amounts of 14C in the excreta components was made from serial analyses of radioactivity in the urinary bladder and the gall bladder. The urine plasma ratios of 14C concentration increased from 0.6 at 12 hr after administration to 1.9, 1.7, and 1.9 after 2, 4, and 8 days, respectively. This ratio decreased to 0.9, at 20 days. Bile/plasma ratio steadily increased with time and was 7.6, 15.8, 24.0, 27.1, and 44.1 after 0.5, 2, 4, 8, and 20 days, respectively. This suggested that leptophos and/or metabolites were excreted primarily in the feces via the bile.

Kinetics of the Body Tissue Pools of 14C Radioactivity

Since leptophos has a selective toxic effect on various parts of the nervous system, we determined whether there were similar differences in pool sizes and other parameters of fluxes of radioactivity among various tissues. A plot of log percentage of dose against time yielded a straight line. The tissue pool size of radioactivity is then zero-time tissue content of radioactivity. The turnover rate of tissue radioactivity is the pool size times the slope of the curve = percentage of dose/day. Turnover time is

pool size divided by turnover rate or the reciprocal of the slope. The slope can be determined from the slope of the line that equals -k/2.303, where k is the apparent first-order elimination rate constant for tissue radioactivity. The biologic or elimination half-life of radioactivity in the tissue (t& is determined from the relationship t ,,z = 0.693lk.

Pool size, turnover rate, turnover time and half-life of lJC radioactivity are based on the total 14C content in each tissue (Table 3). Pool sizes in all nerve tissues were small relative to most other body tissues. Pool size in nerves could be ranked in the following order, from lowest to highest: sciatic nerve, spinal cord, and brain. These tissues were among the tissues that showed the slowest turnover rates, largest turnover times, and the longest half-lives. Other tissues with large pool size were muscle, skin, adipose tissue, and liver, but these magnitudes were largely functions of the size of these tissues. All had high turnover rates, muscle being the highest, followed by skin, adipose tissue, and liver. The lowest turnover rate was calculated for urinary bladder, spleen, and nerve tissues. The pool size of 14C for whole body tissues was 18.6% of the administered dose, with turnover rate of 0.47% of dose per day. The turnover time was 39.9 days and half-life was 12 days. It is interesting to note that the value of the pool size of 14C for whole body tissues, which was calculated from the total 14C in the tissues, was identical to the sum of the pool size values, which were calculated for each tissue individually. Turnover time and half-life values were highest for the brain and lowest for gall bladder and ceca. The contents of gastrointestinal tract parts had very high pool sizes; however, their turnover rates were among the most rapid and except for the crop, they generally had short turnover times and half-lives. The pool size for the total contents of all gastrointestinal tract parts was 100% of the dose, with high turnover rate of 3.8% of dose per day. The


TABLE 3

POOL SIZE, TURNOVER RATE, TURNOVER TIME, AND HALF-LIFE OF RADIOACTIVITY IN VARIOUS TISSUES AND

GASIROINTESTINAL TRACT CONTENTS OF HENS GIVEN A SINGLE 50 mgikg ORAL DOSE (0.9 @/HEN) OF [TILEPTOPHOS”

Sample Pool size Turnover rate Turnover time Half-life

(% of dose x 103) (% of dose/day x 103) (days) (days)

Brain Spinal cord Sciatic nerve Muscle Red blood cells Plasma Urinary bladder contents Urinary bladder Trachea Esophagus Lungs Heart Liver Spleen Pancreas Kidney Gall bladder Bile Mesentary Adipose tissue Skin Ovary Ovary contents Isthmus Uterus Crop Proventriculus Ventriculus Ventricular lining Small intestine Ceca Large intestine Cloaca Whole body tissues Gastrointestinal tract

contents Crop Proventriculus Ventriculus Smail intestine Ceca Large intestine

Total gastrointestinal tract contents:

49 0.46 106.3 32 42 0.63 66.5 20 25 0.84 29.9 9

5,000 601.82 83.1 25 500 16.72 29.9 9 750 25.08 29.9 9 600 25.79 23.3 7

9 0.10 93.1 28 29 0.87 33.2 10 50 1.25 39.9 12

110 3.01 36.6 11 100 5.02 19.9 6

1,050 39.49 26.6 8 16 0.30 53.2 16 55 2.76 19.9 6

210 7.02 29.9 9 65 4.89 13.3 4

300 3.76 79.8 24 250 8.36 29.9 9

2,500 62.69 39.9 12 3,500 75.23 46.5 14

105 2.63 39.9 I2 550 18.39 29.9 9 500 25.08 19.9 6 450 27.08 16.6 5 200 12.04 16.6 5 100 2.31 31.1 13 300 15.05 19.9 6 125 6.27 19.9 6 625 31.34 19.9 6 200 15.05 13.3 4

60 1.13 53.2 16 12.5 3.13 39.9 12

18,550 465.16 39.88 12

40,000 752.28 52.2 16 15,000 501.52 29.9 9 36,150 1.812.99 19.9 6 7,000 351,06 19.9 6

600 60.18 10.0 3 1,250 125.38 10.0 3

100,000 3,761.40 26.59 8

” All parameters are defined in the text.


overall time was 26.6 days and the half- life was 8 days.

Pharmacokinetics of [14C]Leptophos in Hens’ Bodies

The time course of change in body contents of “C radioactivity following the oral administration of a single dose of [‘“Cl- leptophos to laying hens is presented in Fig. 1. The pharmacokinetics profile is sum- marized in Table 4. Following the oral administration of [14C]leptophos, 14C in the body declined biphasically. The early phase is referred to as the fast disposition phase; its rate constant, a, reflects the disposition and distribution of leptophos from the central compartment into the body tissues. The later phase is referred to as the slow disposition rate or “apparent” elimination rate, with the rate constant /3, and reflects the elimination of the drug after the distribution phase has been completed; cr and p are hybrid first-order constants, each of

II ’ 3 ’ 0 ’ ’ ’ ’ * ’

2 4 6 6 IO I2 14 I6 IO 20

FIG. 1. lJC-Body disappearance curve in hens given a single 50 r&kg oral dose (0.9 &i/hen) of [‘“Clleptophos.

TABLE 4

PHARMACOKINETIC PARAMETERS ASSOCIATED WITH

THE PHYSIOLOOICAL DI~PUSITION OF A SINGLE 50 mg/kg ORAL DOSE (03 &~/HEN) OF [W]LEPTOPHOS

IN HENS”

Parameters

Dose, mgihen 75 Dose, &i/hen 0.9 Weight, kg 1.50 Dose, mg/kg 50 aeneral equation:

(F), = Ae-"' + Be-"' A. % of dose B, % of dose a, day-’ ,R day-’ t,,2 of (Y phase, day t1,2 of p phase, day

V, (volume of central compartment), % of body weight

V rl(es., (total volume of distribution), % of body weight

Vdlk) (kinetic volume of distribution), % of body weight

k,2, day-’

k,,, day-’

k,,, day-’

81.45 18.55 1.39 0.06 0.50

12.00

73.00

285.00

336.00 0.87 0.30 0.27

Q rl,,,: elimination rate constant from the central compartment, and is the sum of the simultaneous process of metabolism and excretion all assumed to be first order; k,,. k,,: first-order rate constants of distribution; 01, (~t,,~, /?, PtllP: first-order rate constants and corresponding half-times of LY and p disposition phases: p reflects leptophos elimination from the body; V,: volume of distribution of the central compartment; V ,i,eq.): volume of distribution at steady state; Vdcks: volume of distribution after the distribution phase is completed.

which are influenced by all of the processes for each tissue type involved in the disposition of the organophosphorus insecticide.

The characterization of the pharmacokinetics of [14C]leptophos thus requires a minimum of a two-compartment open- system model. The individual rate constants from the models k12, kzl, and k,, are the first- order rate constants for the designated processes. The assumptions and mathe- matical solutions for this model have been previously described (Gillette, 1974). The


first-order rate constant for the removal of ‘“C from the central compartment to the peripheral compartment (k12) and the first- order rate constant of the reverse move- ment of 14C from the peripheral to the central compartments (k,,) were 0.87 and 0.30 day-‘, respectively. The elimination rate constant from the central compartment (k,,), which is the sum of the simultaneous processes of metabolism and excretion (both assumed to be first order), was 0.27 day -I. The percentage volume of the central compartment was calculated to be 73% of body weight, and the corresponding total volume of distribution was calculated to be 285% of body weight. The kinetic volume of distribution was found to be 336% of body weight. The insecticide was eliminated at a very slow rate, p value of 0.06 day-l, corresponding to a half-life of 12.00 days. The time course of 14C radioactivity in the body of hens orally treated with a single 50 mg/kg dose of [14C]leptophos is described by the following equation

(F), = 81.45e-1.3s’ + 18.55ee0.0’jt,

where (F), is the percentage of 14C radioactivity in the body at time t (in days).

Leptophos and Metabolites

In order to identify the radioactive material present in various tissues, purified extracts from the brain, spinal cord, sciatic nerve, plasma, liver, adipose tissue, and bile of birds sacrificed 2 days after administration were examined, along with urinary- fecal excreta. The stlc and hplc, in combination with scintillation counting and mass spectral analysis, were used to study the excreta and extract. Using two-solvent stlc system, all leptophos-type compounds were separated, as has been previously described (Abou-Donia, 1979a). Some of these compounds were resolved using hplc on reverse-phase RP-8 column. The retention times (min) of these compounds were:

PPA, 5.8; MPPA, 7.0; MPPTA, 10.0; 4-bromo-2,5-dichlorophenol, 26.6; leptophos oxon, 28.8; leptophos, 33.8.

The amounts of radioactivity found in these tissues expressed as equivalents of leptophos ranged from 17 pg/g tissue for sciatic nerve to 191 &g tissue for adipose tissue. These values assume that the measured amounts of radioactivity are due to the unchanged administered leptophos. These calculated leptophos equivalents were well above the lower limit of sensitivity of the hplc method for leptophos-type compounds.

Quantitative extraction of leptophos and related compounds was obtained with the solvent system above used in the absence of tissue homogenate. Extraction of fresh liver homogenate fortified with leptophos indicated some binding or entrainment of leptophos to liver homogenate (yielding an 80 to 85% recovery). Recovery of 14C from tissues of hens treated with oral doses of [llC]leptophos and killed 2 days after administration was always very low (rang- ing between 0 and 10%). No radioactivity could be extracted from sciatic nerves. Only leptophos, and no metabolites, was detected in most tissues, which had the following amounts (pg/g tissue): brain, 0.5; spinal cord, 0.5; plasma, 0.8; bile, 1.5; and adipose tissue, 19. The following metabolites were recovered from the liver &g/g tissue): leptophos, 5.9; leptophos oxon, 0.5; MPPTA, 0.1; MPPA, 0.3: and PPA. 0.4. The low recovery of leptophos or metabolites from the tissues indicate extensive tissue binding of leptophos or metabolites. It may also be attributed to loss in the cleaning up of tissue homogenate extracts required for hplc analysis, since leptophos is highly soluble in lipids and strongly binds to proteins (Abou-Donia, 1979a). Although the low recoveries may be indicative of metabolic incorporation, the overall results suggest that most of the detected radioactivity in tissues may be attributed to leptophos.


“C extracted from urinary-fecal excreta after 0.5, 2, 4, 8, and 20 days of administration of [14C]leptophos was 80, 71, 58, 30, and 35%. The decrease in extractable 14C as time passed suggests that with time leptophos is metabolized to metabolites that are bound to excreta and not extractable. Twelve hours after the oral administration of leptophos, most of the radioactivity recovered from the excreta was identified as leptophos (99.58%) with traces of leptophos oxon (1.17%) and PPA (0.25%) (Table 5). After 2 days 93.59% of the radioactivity recovered was identified as leptophos, with minimal quantities of desmethyl leptophos (1.55%), leptophos oxon (0.90%), desmethyl leptophos oxon (0.79%), MPPTA (1.17%), MPPA (0.51%), and PPA (1.4%). At other times, these same metabolites were identified along with an unknown and leptophos. The quantities of these metabolites slightly increased as time passed, at the expense of leptophos, which accounted for 92.15, 91.09, and 89.82% after 4, 8, and 20 days, respectively.

The mass spectral analysis of the recovered leptophos and metabolites gave fragmentation patterns identical to those of their authentic samples as previously described (Abou-Donia, 1979a). The finding that most of the radioactivity recovered from the tissues and excreta was unchanged leptophos was confirmed by mass spectral

analysis. Fragmentation of the molecule in hydrolytic fashion gave rise to two major ion clusters centered at m/e 172 and 240. Thus, the major radiochemical product was unequivocally identified as leptophos. Des- methyl leptophos and desmethyl leptophos oxon were tentatively identified by sequential thin-layer chromatography.

DISCUSSION

This study has demonstrated that a single nontoxic oral dose of [“Clleptophos is slowly metabolized and excreted in the hen, a species susceptible to delayed neurotoxicity. In the present investigation neither acute nor delayed neurotoxic effect was seen in treated birds. These results were expected since the dose used (50 mg/kg) was far below the reported LD50; e.g., 4700 mg/kg (Abou-Donia et al., 1974), and was half of the threshold oral dose required to cause clinical signs of delayed neurotoxicity (Abou-Donia, 1979b). The biologic half-life (12 days) of 14C determined in this study in hens given a nontoxic 50 mg/kg oral dose of [14C]leptophos is similar to that (11.55 days) found in hens given a neurotoxic oral dose of 400 mg/kg [‘“Cl- leptophos (Abou-Donia, 1976). This finding suggests that the results of the present investigation on the pharmacokinetics and metabolism of a nontoxic dose of leptophos

TABLE 5

LEPTOPHOS AND METABOLITE DISTRIBUTION IN EXCRETA OF HENS GIVEN A SINGLE 50 mgikg ORAL DOSE

(0.9 &~/HEN) OF [%]LEPTOPHOS”

Days after administration PPA MPPA MPPTA DMLO LO DML Leptophos Unknown

0.5 0.25 0.00 0.00 0.00 0.17 0.00 99.58 0.00 2 1.49 0.51 1.17 0.79 0.90 1.55 93.59 0.00 4 1.73 1.71 1.39 0.91 1.09 1.87 92.15 0.15 8 1.95 0.87 1.51 1.07 1.23 1.99 91.09 0.29

20 2.08 0.98 1.79 1.33 1.45 2.17 89.82 0.38

0 [Y]Leptophos was administered in a gelatin capsule. * See Fig. 2 for identification of metabolites.


may reflect the condition seen with a neurotoxic dose. These results show that leptophos is less absorbed from the gastrointestinal tract than from the skin. When a single oral dose was administered, the maximum amount of l*C radioactivity recovered in the tissues was 16.69%, as compared to 35.35% when the same dose was applied topically (Abou-Donia, 1979a).

The degradation of the phenyl ring in the phenylphosphonothioate moiety of leptophos molecule was not an important pathway for the detoxification and elimination of orally administered [‘Qleptophos from the hen’s body. Phenyl ring fission most likely occurs through oxygenated intermedi- ary products, analogous to that reported for aromatic xenobiotics (Abou-Donia and Dieckert, 1975; Abou-Donia and Menzel, 1976). After ring oxidation the ‘“CO, would be absorbed and excreted by respiration.

‘V (mostly as leptophos) was eliminated slowly, with combined urinary-fecal excreta being the main route of excretion. In the hen, urine and feces are voided together; indirect evidence, however, indicates that most of the excreted radioactivity was in the feces. 14C radioactivity in the urinary bladder and its contents was low throughout the experiment. The higher levels of radioactivity in the kidney relative to the urinary bladder contents may be explained by extensive binding of leptophos to plasma proteins and tissues (Abou-Donia, 1979a), and consequent low glomerular filtration of the insecticide (Weiner et al., 1960). Also, the high lipid solubility of leptophos facilitates nonionic diffusion and passive reabsorption in the renal tubules (Milne et al., 1958).

These results are in contrast to the results in metabolism of [‘“C]leptophos in the mouse (Holmstead et al., 1973) and the rat (Hassan et al., 1977). In these two species, which are not sensitive to delayed neurotoxicity induced by organophosphorus esters, orally administered leptophos was metabolized and excreted mostly

in urine, as water-soluble degradation products.

Excretion into the intestine via the bile seems to be the major pathway by which absorbed 14C is removed from the body of [14C]leptophos-treated hens. The bile/ plasma concentration ratio of 14C ranged between 7.6 and 44. These results indicate that leptophos and its metabolites are transferred from the blood to the bile. This process might be attributed to the following characteristics of leptophos: (a) high lipid solubility , (b) high apparent volume of distribution, Vdch.), (c) strong and multiple site binding to proteins, and (d) large molecular weight. The assumed high rate of bile secretion of leptophos is in harmony with the finding that compounds with high molecular weight (more than 300) and two or more aromatic rings tend to be excreted into bile (Williams et al., 1965). Leptophos has two phenyl rings and a molecular weight of 412. Because substances that bind strongly and preferentially to proteins in the plasma are transferred efficiently and rapidly to the secreting cells (Baker and Bradley, 1966) and leptophos has these binding characteristics (Abou-Donia, 1979a), the designation of bile as the domi- nant pathway is further confirmed.

Although biliary excretion seems to be the major one, other routes of entry to the gastrointestinal tract may exist. This would explain why 14C persisted in tissues and contents of the stomach, proventriculus, and ventriculus. This suggestion of extra- biliary excretion is in agreement with the proposed gastrointestinal excretion of digoxin (Harrison et al., 1966), diphenyl- hydantoin (Noach et al., 1958), and dieldrin (Williams et al., 1965).

A significant portion of radioactivity was absorbed into the blood and concentrated in the egg, the latter probably explained by protein binding of leptophos. The protein content in the egg is 16.6 and 10.6% for yolk and albumen, respectively (Sturkie, 1976).


Pharmacokinetic profile of an orally administered subneurotoxic dose of [14C]- leptophos in the hen reflects the large volume of the peripheral compartment which includes muscle, adipose tissue, and skin. This indicates a high distribution of the insecticide into the tissues. Radio- activity appeared in all tissues at 12 hr, peaked at 2 days and rapidly declined after oral dosing. The rapid decline in blood and well-perfused tissues would appear to be due to the redistribution of leptophos from the blood into tissues, a phenomenon consistent with the lipophilic nature of the compound. In support of this hypothesis, high contents of leptophos were found in the fat, skin, and muscle of hens. This pattern of distribution is similar to that found in hens treated topically with leptophos (Abou-Donia, 1979a). The massive retention of 14C in adipose and muscle tissues is striking. Even at the end of the 20-day experiment, considerable amounts of radioactivity were deposited in the muscle. Most of this reservoir is attributed to high lipid solubility and protein binding of leptophos. These results are in agreement with the report of Konno and Kinebuchi (1978).

After administration of [‘“C]leptophos. the spinal cord does accumulate l*C more avidly than the brain or peripheral nerves. This difference in the uptake plays at least some part in determining differences in the pattern of nerve damage seen after leptophos ingestion. This hypothesis is suggested by ranking parts of the nervous system according to the extent and severity of degeneration (Abou-Donia and Preissig, 1976a,b; Preissig and Abou-Donia, 1978), and comparing this sequence with the order of turnover time which they require to rid themselves from 14C. Peripheral nerves, which accumulated small amounts of 14C and had the shortest turnover time and the shortest half-life of all nerve tissue, showed early degeneration of axons and myelin, which subsequently recovered (Abou-

Donia and Graham, 1978; Abou-Donia, 1979b). Although overall nerve tissues were among tissues containing least amounts of [C, nevertheless, brain and spinal cord were among tissues having the longest turnover times and longest half-lives. These results are in harmony with the finding that in leptophos poisoning degeneration of myelin and axons in the spinal cord is the most consistent histologic change. Brain damage, however, was only seen in the medulla; the upper brain stem, cerebellum, or cerebrum are not damaged (Abou-Donia and Preissig, 1976a,b). These results suggest that besides the differences in the uptake of 14C by various cells, neurons must also vary in their susceptibility to the effect of equal quantities of leptophos. A possible explanation in the uptake of leptophos in the different parts of the nervous system could be sought in variations of the per- meability of the blood-brain barrier. Also, the functioning of the blood-brain barrier may change during poisoning by leptophos. Regional variations of the defect of the barrier could possibly explain the regional differences of the concentration of 14C.

The slow metabolism and elimination of leptophos is the most important finding in this study. Figure 2 illustrates a suggested sequence of leptophos metabolism in the hen. Most of these metabolic pathways represent detoxification mechanisms, since they enhance the excretion of leptophos from the bird’s body and render it less toxic: only leptophos oxon produces acute and delayed neurotoxic effect in hens (Abou-Donia et al., 1980).

Figure 2 presents a scheme illustrating a suggested sequence of leptophos metabolism in the hen. The reactions that have been involved in this sequence were oxidation and hydrolysis. Leptophos was oxidized to leptophos oxon, which is subsequently hydrolyzed to methanol and desmethyl leptophos oxon. Hydrolysis was also exemplified by the conversion of leptophos to methanol and desmethyl lepto-


co.2 (=J-i;ob Br

T @-Qc~B. %---+

I& Cl -%A &,,

DMLO Cl

0 0

/

LEPTOPHOS OXON

sg,bBr Y+nOC$Bl + (=&T

‘9

MPwibcH3 -%A

(-J&m

3 Cl Cl

LEPTOPHOS BDP MPPTA3y ,/f PP:”

&&Br

OH Cl

DML PPTA

FIG. 2. Suggested leptophos metabolic pathways in hens given a single 50 mg/kg oral dose (0.9 &i/hen) of j*‘VZ]leptophos. DML, desmethyl leptophos; DMLP, desmethyl leptophos oxon: BDP, 4-bromo-2,5-dichlorophenol; MPPTA, methyl phenylphosphonothioic acid: MPPA, methyl phenylphosphonic acid; PPTA, phenylphosphonothioic acid; PPA, phenylphosphonic acid.

phos. The latter compound might have been oxidized to desmethyl leptophos oxon. Leptophos was also hydrolyzed to form 4- bromo-2,Sdichlorophenol and MPPTA. Oxidation was responsible for the conversion of MPPTA to MPPA, which was hydrolyzed to PPA. Hydrolysis took place in the formation of desmethyl leptophos oxon to PPA and the phenol. 4-Bromo- 2,5-dichlorophenol was conjugated to form glucuronides, ethereal sulfates, and hy- brids.13

This study adds confirmative evidence to the hypothesis that species selectivity for delayed neurotoxicity of leptophos is related to the different profiles of the pharmacokinetics and metabolism of the insecticide (Abou-Donia, 1976, 1979a). This hypothesis is supported by the following criteria: (a) although leptophos is rapidly absorbed and excreted in nonsusceptible species (e.g., rats and mice), it is only partially absorbed and slowly excreted in susceptible species (e.g., chickens); (b) the half-life value for labeled leptophos is much longer in the hen (12.0 days) than in the

“’ M. B. Abou-Donia, unpublished results.

mouse (9 hr); (c) leptophos is metabolized in nonsusceptible species and excreted largely as water soluble conjugates in the urine. In the hen only a small fraction is metabolized. This suggests that the target is exposed for longer periods of time to leptophos in susceptible species, and may also be more sensitive to leptophos than that in nonsusceptible species.

We have previously described the clinical and histopathological anatomical manifesta- tions of leptophos poisoning in hens treated with a neurotoxic dose. Overt clinical signs of neurological disturbances begin to appear 4- 14 days after treatment. When the per- sistence of l“C in body tissues in general, and in the nervous tissues in particular, described in this article are compared with the progression of pathological anatomical malfunction described earlier, a relationship between the accumulation and stability of leptophos in sensitive species and their susceptibility can be seen. This relationship may explain the implication of leptophos in the poisoning and paralysis of farm animals (Abou-Donia et al., 1974) and of workers in a Texas factor where it was formerly manufactured and packaged


(Anonymous, 1976). This study has also demonstrated the necessity of conducting similar pharmacokinetic and metabolic studies of organophosphorus esters sus- pected of causing delayed neurotoxicity, in order to better assess the potential risk of these chemicals to humans,

ACKNOWLEDGMENTS

Acknowledgment is made to Ms. Diana Carter for her technical assistance, and to Ms. Marcine Basden for her secretarial work. Mass spectral analysis of leptophos and metabolites was carried out in the Research Triangle Institute, Research Triangle Park. North Carolina. The supply of radioactive and pure leptophos and metabolites by Velsicol Chemical Company, Inc. (Chicago, Ill.) is acknowledged. This study was supported in part with the Federal funds from the Environmental Protection Agency under Contract 68-02-2452. The content of this publication does not necessarily reflect the views or policies of the U.S. Environmental Protection Agency, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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