Animal metabolism
Information was made available to the Meeting on metabolism studies in rats, lactating goats and laying hens.
Gay et al. (1992) summarized the conclusions from a series of metabolism studies on rats with [thiocarbonyl-14C]thiram. When rats were dosed orally with [14C]thiram much of the 14C (40-60%) was eliminated as volatiles in exhaled air, 25-35% was excreted in the urine and 2-5% in the faeces. After an interval of 96 hours 2-3% of the 14C remained in the tissues. Polar metabolites and conjugates were identified in the urine.
Groups of rats (5 male + 5 female) were given single doses of [14C]thiram at 125 mg/kg bw for the high-dose group and 1.9 mg/kg bw for the low-dose group (Gay, 1987). Excreta were collected for 7 days, when the animals were slaughtered for tissue collection. The total recovery of the administered 14C was low, with 21 and 29% in the urine, 3.9 and 2.8% in the tissues and 4.1 and 2.4% in the tissues (recoveries in low-dose and high-dose groups respectively). The low total recovery suggested loss in exhaled air.
Exit gases were collected for 96 hours from metabolism cages containing groups of 3 rats given single oral doses of [14C]thiram at 2.1-2.5 mg/kg bw (Norris, 1989). The majority of the volatile 14C was produced in the first 24 hours, and its rate of production peaked between 2 and 4 hours after dosing. The total 14C recovered in the expired air was 57-63% and in the urine 25-43% of the dose. The composition of the exhaled 14C was not examined in this experiment, but the trapping system would have collected CO2, CS2 and COS.
Rats (6 male + 6 female) were dosed orally for 14 days with unlabelled thiram at 2 mg/kg bw then with [14C]thiram at 2 mg/kg bw on day 15 (Nomeir and Markham, 1990). Expired air and excreta were collected for a further 96 hours and then the animals were slaughtered for tissue collection. Approximately 33-35% of the administered 14C was eliminated in the urine, 2.6-5.3% in the faeces and 47-48% in the expired air. The nature of the volatile 14C was not investigated in detail, but 75-85% of it was collected in a KOH trap suggesting CO2, or COS, with the remainder collected in a reagent for CS2.
Norris (1991) identified by HPLC the metabolites in the urine of the rats from the single dose study of Gay (1987) and the multiple dose study of Nomeir and Markham (1990). There were no sex differences in the metabolism but the proportions of some of the metabolites depended on the dosage level and the time after dosing. Table 1 lists the proportions of the metabolites as percentages of the 14C in the urine samples.
Table 1. Levels of metabolites (as % of total 14C in the sample) in urine of rats given [14C]thiram as single doses 125 mg/kg bw or multiple doses of 2 mg/kg bw/day for 15 days (Norris, 1991).
|
Metabolite |
14C, % of total in sample |
|||
|
Single dose |
Multiple dose |
|||
|
Male, 0-24 hr sample |
Female, 0-24 hr sample |
Male, 4-8 hr sample |
Female, 4-8 hr sample |
|
|
U1 |
13 |
7.8 |
1.4 |
1.7 |
|
Unidentified |
|
|
0.3 |
0.4 |
|
U2 |
5.6 |
8.7 |
12 |
11 |
|
Unidentified |
|
|
5.1 |
4.0 |
|
U3 |
37 |
42 |
40 |
36 |
|
U4 |
3.4 |
2.4 |
1.6 |
0.6 |
|
U5 |
34 |
36 |
36 |
42 |
McManus (1991) fed rats with 50 ppm unlabelled thiram in the diet for 9 weeks and then gave them single oral doses of [14C]thiram. The five urinary metabolites shown above were identified by HPLC and mass spectrometry.
Dalvi and Deoras (1986) showed that CS2 was present in expired air from rats dosed with thiram by intraperitoneal injection.
Residues in the tissues, milk and excreta were measured in lactating goats (2 goats for the low dose (x 1) and 1 for the high dose (x 10), each animal weighing approx 40-50 kg) dosed orally twice daily for 4 consecutive days by capsule with [thiocarbonyl-14C]thiram equivalent to 2.5 and 3.3 ppm (x 1) and 23 ppm (x 10) thiram in the feed (Norris, 1993b). The feed consumption was 2 kg/animal/day. The animals were milked twice daily; milk production was at least 800 ml per day. Respiration gases were collected from the low-dose animals for 10 hours after the final dose. Milk and excreta were collected throughout, and the goats were slaughtered 13 hours after the final dose for tissue collection. The 14C was distributed as shown below.
|
A |
14C, % of (low) dose |
|
|
Goat 1 |
Goat 2 |
|
|
Milk |
1.3 |
1.0 |
|
Urine |
9.2 |
9.6 |
|
Faeces |
4.6 |
5.6 |
|
Tissues |
7.2 |
7.5 |
|
Expired air |
48 |
39 |
|
Total |
70 |
63 |
A major part of the 14C was eliminated in the respired gases, with smaller amounts in the urine and faeces. Approximately 60-70% of the dose was accounted for, but the value for the expired air is an estimate based on the 10-hour collection period. The value for the tissues includes the stomach and intestine contents, which accounted for almost half of the 7.2 and 7.5% reported.
The level of 14C in the milk reached a plateau within 1.5 to 3 days of the first dose, and that in the urine by day 2 or 3, but the level in the faeces may still have been increasing at the completion of the study.
The ethanol + diethylamine traps (for CS2 and COS) on the expired air collected 9.3 and 2.8% of the dose. Most of the 14C in the expired air was present as CO2.
The metabolism of thiram was quite extensive and much of the 14C in the milk and tissues was present as very polar extractable material or bound residues. 14C was present in glycogen, amino acids, proteins, lactose and saponifiable lipids. The only xenobiotic metabolites detected were CS2 and COS. It is likely that thiram is rapidly converted to dimethyldithiocarbamate and then to dimethylamine and CS2. CS2 is converted to COS and carbonate. [14C]carbonate then enters fat, protein and carbohydrates.
Table 2. Total 14C (as thiram) in milk from goats dosed orally by capsule for 4 days with [14C]thiram equivalent to 2.5 and 3.3 ppm (x 1) and 23 ppm (x 10) in the feed (Norris, 1993b).
|
Day |
Total 14C as thiram, mg/kg |
||
|
x 1 dose |
x 10 dose |
||
|
0.5 |
0.024 |
0.027 |
0.27 |
|
1 |
0.036 |
0.040 |
0.33 |
|
1.5 |
0.051 |
0.054 |
0.47 |
|
2 |
0.044 |
0.047 |
0.36 |
|
2.5 |
0.056 |
0.064 |
0.40 |
|
3 |
0.048 |
0.060 |
.0.45 |
|
3.5 |
0.045 |
0.070 |
0.48 |
|
4 |
0.050 |
0.074 |
0.48 |
|
% of dose in milk |
1.3 |
1.0 |
1.8 |
Table 3. Distribution of radiolabel in tissues from goats dosed orally for 4 days by capsule with [14C]thiram equivalent to 2.5 and 3.3 ppm (x 1) and 23 ppm (x 10) in the feed (Norris, 1993b).
|
Sample |
Goats dosed at 2.5 and 3.3 ppm |
Goat dosed at 23 ppm |
||
|
14C as % of total administered 14C |
14C as thiram, mg/kg |
14C as % of total administered 14C |
14C as thiram, mg/kg |
|
|
Muscle |
0.81 1.1 |
0.008 0.013 |
1.1 |
0.12 |
|
Kidneys |
0.049 0.064 |
0.055 0.093 |
0.064 |
0.68 |
|
Liver |
2.4 2.4 |
0.51 0.58 |
3.4 |
7.0 |
|
Blood |
0.42 0.49 |
0.021 0.030 |
0.52 |
0.27 |
|
Fat |
0.035 0.064 |
0.005 0.013 |
0.075 |
0.12 |
Residues in the tissues, eggs and excreta were measured in laying White Leghorn hens (groups of 4 for the low dose and 6 for the high dose, each bird weighing approx 1.5 kg) dosed orally by capsule once daily for 4 days with [thiocarbonyl-14C]thiram equivalent to 0.6 and 6.0 ppm thiram in the feed (Norris, 1993a). The feed consumption was 80-110 g/bird/day. Eggs and excreta were collected throughout, and the birds were slaughtered 24 hours after the final dose for tissue collection.
Table 4 shows the distribution of the 14C. The total recovery of the administered dose was only 61-68%. The likely reason is loss as volatiles. Liver was the tissue with the highest level of 14C. A high percentage of the 14C was extractable with chloroform or aqueous methanol, or was made soluble on enzymic or acid digestion.
The residues extracted by aqueous methanol from the liver were determined by reversed-phase HPLC and the resultant three peaks, comprising 4.7% of the total 14C in the liver, were identified by mass spectrometry as dimethyldithiocarbamate ornithine, 2-thioxo-4-thiazolidinecarboxylic acid and dimethyldithiocarbamate glucuronide. Approximately 48% of the 14C in the liver was identified as incorporated into natural products such as acids, amino acids, peptides and proteins. The 14C residues in the other tissues were at quite low levels were characterized by ion-exchange chromatography also as natural products.
Thiram itself was identified in an aqueous methanolic extract of the excreta. Other metabolites in the extract appeared to be conjugates of dimethyldithiocarbamate.
Table 4. Distribution of radiolabel in tissues, eggs and excreta from hens dosed orally for 4 days with [thiocarbonyl-14C]thiram equivalent to 0.6 and 6.0 ppm thiram in the feed (Norris, 1993a).
|
Sample |
Hens dosed at 0.6 ppm |
Hens dosed at 6.0 ppm |
||
|
14C as % of total administered 14C |
14C as thiram, mg/kg |
14C as % of total administered 14C |
14C as thiram, mg/kg |
|
|
Egg white, days 1-4 |
0.09 |
0.00-0.003 |
0.069 |
0.003-0.020 |
|
Egg yolk, days 1-4 |
0.075 |
0.00-0.006 |
0.058 |
0.001-0.044 |
|
Excreta, days 1-4 |
66 |
0.14-0.18 |
59 |
1.2-1.5 |
|
Muscle, breast |
0.18 |
0.002 |
0.19 |
0.025 |
|
Muscle, thigh |
0.17 |
0.003 |
0.21 |
0.036 |
|
Liver |
1.2 |
0.11 |
1.0 |
0.89 |
|
Kidney |
0.086 |
0.041 |
0.12 |
0.51 |
|
Fat |
0.027 |
0.002 |
0.013 |
0.009 |
|
Heart |
0.017 |
0.008 |
0.025 |
0.11 |
|
Blood |
0.20 |
0.007 |
0.23 |
0.081 |
|
GI tract |
0.49 |
0.007 |
0.44 |
0.053 |
|
Gizzard |
0.035 |
0.004 |
0.040 |
0.041 |
|
Skin |
0.049 |
0.003 |
0.052 |
0.029 |
The metabolic pathways are shown in Figure 1.
