Pharmacology & Toxicology 1991, 69, 34-37.

Tissue Distribution and Urinary Excretion of 14C-Ethion in Goats

Resto D. Mosha

Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, 13, Biilowsvej, DK-1870 Frederiksberg C, Denmark

(Received January 7, 1991, Accepted February 7, 1991)

Abstract: Tissue concentration and elimination and urinary excretion were followed after intravenous injection of I4C- ethion in 12 goats. Tissue levels were determined in two goats after 8 hr and on each of days 1, 3, 7, 14 and 28 after exposure. During the four-week period plasma and all tissues examined (liver, kidney, fat, muscle, lung, heart and brain) had detectable I4C-residues, the highest values being found in liver, kidney and fat. Elimination of I4C-residues was faster in the first 3 days than in the later part of the experiment, where the elimination half-life for most tissues was approximately 2 weeks. During the first two weeks after exposure, 77% of the administered dose was eliminated in urine (55%) and faeces (22%). TLC of urine collected over the first 15 hr after exposure showed at least 8 bands of metabolites, five of which accounted for about two thirds of the dose excreted in urine.

Ethion, O,O,O',O'-tetraethyl-S,S'-methylene-bis-phosphoro- dithioate, is an organophosphorus (OP) compound used both as an insecticide and as an acaricide for tick control in livestock. During dipping or spraying ethion might be absorbed either through oral uptake, or more likely by the percutaneous route. It would therefore be of interest to know how ethion is distributed between the different tissues of the body, and especially to know how fast it is eliminated from these tissues once it is there.

The distribution of ethion in tissues has been studied in rats and goats after oral administration (Selim 1985a; Job- sis & Zietlow 1986). However, in goats and probably other ruminants, ethion administered orally is effectively hydro- lysed by the ruminal microflora after which the remaining ethion is subjected to first-pass extraction by the liver (O'Brien 1960). As a result less than 5% of orally adminis- tered ethion is absorbed unchanged in goats (Mosha et al. 1990a).

After dermal exposure of goats ethion is mainly absorbed unchanged, but the absorption is slow and very prolonged - approximately 20% of the dose is absorbed during the first two weeks and the absorption seems to continue for at least a month (Mosha et al. 1990a & 1991).

As the distribution pattern for the lipophilic ethion must be expected to differ from that of the more polar metabo- lites, and as the tissue elimination rate can be studied only after the process of absorption is completed, it was decided to use the intravenous route of administration for the pres- ent investigation. The purpose of the investigation was to examine tissue distribution and elimination and also to study urinary excretion of ethion in goats.

Materials and Methods Animal experiments. Twelve adult goats (5 females and 7 males) weighing between 20 and 32 kg were used in the study. Each goat was confined in a metabolism cage, where it was fed hay and concentrate and had free access to water.

(14C-methylene)-Ethion (1 1.9 mCi/mmole) with a radiochemical purity of more than 96% was purchased from Amersham, United Kingdom. This I4C-ethion was mixed with unlabelled ethion to a specific activity of 2 pCi/mg. The goats were administered 14C- ethion intravenously dissolved in glycerol formal at a dose of 2 mg/ kg, which in previous studies had been without toxic effects to goats (Mosha & Gyrd-Hansen 1990).

Sampling. Urine and faeces were collected quantitatively during the first 2 weeks of the experiment. In order to get pure samples urine was obtained from one of the female goats by means of a balloon catheter (Riisch No. 12, 5-15 ml) at I , 2, 4, 6, 8, 10, 12 and 15 hr after dosing. Subsequent samples and all urine samples from the other eleven goats were collected from the metabolism cage. All urine samples were stored at -20" until analysed.

Exhaled air from three of the goats was directed through a 12% ethanolamine solution in methanol for periods of 30 min. each in the 1st. 2nd and 3rd hr after dosing in order to see whether any significant amount of the I4C administered was excreted as I4C- carbon dioxide.

Two goats were stunned and exsanguinated at 8 hr and 1, 3, 7, 14 and 28 days after dosing. From each goat liver, kidney, heart, lungs, brain and bile were collected and weighed before samples were taken from each of them. Other tissues sampled were skeletal muscle (longissimus dorsi) and abdominal fat. Blood samples were collected just before the animals were killed and plasma obtained by centrifugation. All samples were stored at -20".

Analytical methods. Thin-layer chromatography (TLC). Silica gel 60 F254 plates, 0.25 mm (Merck) were used with the following solvent system: butanol- 1 +acetic acid + water (8 + 1 + 1 by volume) for sep- aration of metabolites in urine samples. Radioactive components were located by autoradiography (Hyperfilm-pmax, Amersham). The silica gel was scraped off the plates and the amount of 14C- ethion metabolites quantified by liquid scintillation counting.

Liquid scintillation counting (LSC) . Liquid scintillation counting was performed using an LKB Wallac 1217 Rackbeta scintillation counter with automatic quench correction by the external standards method. Solutions of ethanolamine (12%) in methanol (0.5 ml) were blended with OptiScint T (FSA Laboratory Supplies) liquid scintilation mixture (3 ml) in plastic vials and counted directly. Samples of urine and plasma, 100 pI of each, were mixed with OptiPhase HiSafe (FSA Laboratory Supplies) liquid scintillation cocktail (3 ml) and counted directly. Silica gel samples from TLC

REST0 D. MOSHA 35

plates were mixed with 300 p1 of water and then placed in an ultra- sonic bath for 30 min. before mixing with the OptiPhase HiSafe liquid scintillation cocktail (3 ml) and direct counting. Samples of tissues (approximately 100 mg), dried faeces (approximately 20 mg) and bile (100 pl) were digested by incubation with 1 ml Soluene- 350 (Packard) at 50" in glass vials until dissolution was complete. The coloured samples were bleached by addition of 0.5 ml isopro- panol and 0.2 ml hydrogen peroxide (30-35%). After 1&15 min. at room temperature the samples were incubated at 40" for one hr. Ten ml of Hionic-Fluor (Packard) liquid scintillation mixture were added to each sample, which were left to stand for at least 24 hr before counting in order to reduce the chemiluminescence.

Calculations. All concentrations of total I4C are based on the radio- activities measured and calculated as if these activities represent I4C- ethion.

Results

Tissue distribution. Tissue concentrations of total I4C from radioactive ethion (ethion plus metabolites) are shown in table 1, from which it is seen that great differences in contents were found be- tween the tissues examined. All tisues had 14C levels similar to or higher than the corresponding concentration in plasma with the highest contents being found in liver, kidney and fat. While all other tissues showed the highest levels of total I4C 8 hr after dosing, the level in fat peaked at 24 hr. Elimination of I4C from both plasma and tissues was rela- tively fast during the first three days after dosing, but be- came gradually slower during the remaining part of the 4- week experimental period (fig. 1). After four weeks 14C was still detectable in plasma and present in all tissues at the level of 0.1 p.p.m. or higher. The liver which contained, 7-10% of the dose after 8 hr remained with only 0.3% of the dose at four weeks.

Concentrations of total I4C were also measured in bile and found to be as high as 3.4 and 12.9 p.p.m. after 8 hr and 1.4 and 1.9 p.p.m. after 24 hr.

Excretion. Fig. 2 shows the cumulative excretion of total I4C in urine and faeces together with the total amount recovered. About

Table I . Tissue concentrations of total I4C in goats after intravenous admin- istration of 2 mg/kg I4C-ethion.

Concentration (p.p.m.) Goat Time No. (davsl Plasma Liver KidnevMuscle Fat Heart Lung Brain

1 0.33 1 . 1 7.8 6.1 1.1 0.85 1.7 1.8 0.83 2 0.33 1.7 8.5 10.1 1.5 2.6 2.2 2.8 1.6 3 1 0.87 5.9 3.0 0.73 5.7 1.3 1.6 0.87 4 1 0.90 7.4 5.1 0.72 1.6 1.3 1.7 0.90 5 3 0.24 2.8 1.4 0.48 1.8 0.72 0.82 0.50 6 3 0.37 2.6 1.6 0.40 1.6 0.59 0.74 0.48 7 7 0.22 1.5 1 . 1 0.24 1.3 0.40 0.64 0.24 8 7 0.21 2.2 1.2 0.21 0.63 0.39 0.81 0.25 9 14 0.09 0.57 0.47 0.13 0.46 0.20 0.31 0.11

10 14 0.06 0.71 0.42 0.13 0.64 0.18 0.32 0.13 1 1 28 0.03 0.30 0.27 0.13 0.21 0.20 0.19 0.08 12 28 0.03 0.31 0.19 0.11 0.26 0.13 0.17 0.09

10.00

h 2 1.00 h

z v

; I

0.10 r

0.01 0 7 14 21 28

DAYS Fig. 1. Concentration of total I4C from radioactive ethion in tissues from 12 goats after intravenous administration of 2 mg/kg b.wt. Plasma 0; Liver f; Kidney A ; Fat 0 ; Skeletal muscle 0. Each point represents the average of two animals.

one third of the dose was excreted in urine within the first 24 hr and at the end of the two-week collection period a total of 55% had been recovered in the urine. Another 22% was found in faeces bringing the average total recovery up to 77% of the dose administered.

Urinary metabolies. Fig. 3 shows a TLC chromatogram of eight urine samples collected over the first 15 hr after administration of I4C- ethion. The chromatogram shows that (1) ethion is exten- sively metabolised, (2) a considerable number of metabolites

100

80 w

z

i 6o 4 40 7-

8 20 8

0 0 2 4 6 8 1 0 1 2 1 4

DAYS Fig. 2. Cumulative excretion of total I4C liom ~.uciiouc~r\c c ~ l u ~ u i from goats after intravenous administration of 2 mg/kg b.wt. (8 hr, n = 12; 1 day, n = 10; 2-3 days, n = 8; 4 7 days, n = 6; 8-14 days, n=4). Urine 0; Faeces A; Total 0. MeanfS.D.

36 ETHION IN GOATS

Table 2. Major metabolites of I4C-ethion in urine from goats after intra- venous administration of 2 mg/kg.

Table 3. Exhaled I4CO, from goats after intravenous administration of 2 mg/ kg 14C-ethion.

Concentration (YO of IT-excreted)

Collection periods (hr) Band No. Rfvalue 0-2 2-4 4-6 6-10 10-15

1 0.07 8.8 13.6 15.1 18.0 21.8 2 0.10 0.0 7.3 10.7 14.3 17.5 3 0.13 17.0 18.7 22.3 24.3 24.3 4 0.35 25.0 11.2 8.0 7.1 6.6 5 0.50 13.5 13.3 9.8 4.9 2.1

14C02 exhaled (YO of dose) Collection period (min. after dosing) goat 1 goat 2 goat 3

30-60 ND 0.01 0.05 90-120 ND 0.001 0.003

150-180 ND ND ND

ND =Not detectable.

are formed and (3) unchanged ethion is not excreted in urine. It is also seen that there is a change with time from more lipophilic to more polar metabolites. The urine samples contained five major bands of metabolites, which together made up more than two thirds of the total amount excreted (table 2). Each band may contain more than one metabolite as only one-dimensional chromatography was performed.

Exhaled I4C-carbon dioxide. Collection of exhaled carbon dioxide during the first three hours after dosing revealed that only very small amounts of I4C-ethion were transformed into I4CO2 and exhaled. The largest amounts were collected in the first period (table 3).

Discussion

The plasma levels of total I4C from radioactive ethion meas- ured in the present experiments are similar to those observed

in a previous study on goats (Mosha et al. 1990a) in which the plasma concentrations of unchanged ethion were found to be more than a 100 times lower than the corresponding total 14C concentrations, and to disappear from plasma after only 24 hr. In the same study the volume of distribution (V,,,J for unchanged ethion was 9.4 l/kg indicating that ethion is present in tissues in much higher concentrations than in plasma. The tissue concentrations found in the present study show that to some extent the same is the case for total I4C.

In the present experiment the highest levels of I4C were found in liver, kidney and fat. After oral administration of I4C-ethion to goats for 7 days, Jobsis & Zietlow (1986), 4 hr after the last dosing, measured the highest I4C concen- trations in liver and kidney and the lowest in fat. This difference in distribution to fat is most likely due to the fact that 95% of orally administered ethion in goats is metabolised into much less lipophilic metabolites before distribution (Mosha et al. 1990a).

Fig. 3. Chromatogram of metabolites of 14C-ethion in 8 urine samples collected from a goat over the first 15 hr after intravenous administration of 2 mg/kg b.wt. The five dark areas at the top represent ethion standards run parallel with the urine samples. The numbers indicate collection periods in hours after administration.

REST0 D. MOSHA 37

A tissue distribution pattern similar to that in the present study was obtained in rats after oral administration of I4C- ethion (Selim 1985a), while in the hen the highest levels were observed in the liver and kidney and the lowest in skeletal muscle and fat during the first week after oral dosing (Bodden & Zietlow 1986; Mosha et al. 1990b).

Seven days after dermal application of another organ- ophosphorus compound, ''C-coumaphos, to goats, Kon- ar & Ivie (1988) found the highest residue levels in adipose tissue followed by liver and kidney.

Tissue elimination was relatively fast the first 3 days after dosing, but slow during the last two weeks of the observation period. Here the half-life was around 2 weeks for most tissues and thus similar to what Palmer et al. (1977) found for adipose tissue after dipping cattle in ethion or dioxathion. Crookshank & Smalley (1970) after administer- ing another organophosphorus compound, ronnel, orally to sheep found residues in the omental fat up to day 14 after exposure, while on day 21 ronnel was no longer detectable.

Seventy-seven% of the dose administered was recovered after two weeks in urine (55%) and faeces (22%). In an earlier study on lactating goats the recovery percentage for total I4C from radioactive ethion was similar i.e. 78%, but with 66% in urine, only 8% in faeces and 4% in milk (Mosha et al. 1990a). The difference in amounts excreted in urine and faeces can be ascribed to the use of different techniques for collection of urine. In the previous study all urine was collected by means of a balloon catheter during the first 48 hr after dosing, whereas in the present study urine was obtained from the metabolism cage where it had been in contact with faeces.

TLC analysis of urine samples revealed at least five major metabolites. Similar findings were made by Selim (1985b), who by means of HPLC found 4-6 water-soluble metabo- lites in the urine of rats treated orally with 14C-ethion. As in the present study unchanged ethion could not be detected, indicating a complete metabolism of ethion.

Only insignificant amounts of I4C were recovered as I4CO2 in exhaled air from the goats. However, after oral adminis- tration of I4C-ethion to rats 3 4 % of the dose was excreted as I4CO2 in 3 days (Selim 1985a).

FAOjWHO (1985) has recommended for goat meat and edible offal an MRL (Maximum Residue Limit) of only 0.2 p.p.m. for ethion, including parent compound, all metabo- lites and drug-based products (FAO/WHO 1989). When using labelled compounds for residue studies it is possible that the labelled group may be transferred to normal tissue components and thus stay in the tissues for a very long time. The kinetics of total I4C from radioactive ethion in goats could indicate that such a transfer takes place (Mosha et al. 1990a).

The amounts of ethion that a goat may swallow or absorb through the skin in connection with dipping or spraying

may probably only lead to blood levels 5-10 times lower than those observed in the present study (Mosha et al. 1990a) and to correspondingly lower tissue concentrations. But even then it would last several weeks for the tissue residues to get below the MRL.

Acknowledgements The study was supported by the Danish International

Development Agency (DANIDA). Technical ethion was generously supplied by Cheminova A/S, Denmark. I thank Drs. Nils Gyrd-Hansen and Poul Nielsen for assistance in the preparation of the manuscript.

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