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Availability during human consumption of the arsenic in tissues of chicks fed arsanilic-74As acid
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
Availability
during Tissues B.
Laboratory College,
27-30
9:
Human of
Consumption
Chicks
Fed
A.
CALESNICK,
of Human
Pharmacology Philadelphia, Pennsylvania Research Division, Abbott
(1966)
WASE,
of the
Arsanilic-‘“As AND
L. R.
Arsenic
in
Acid OVERBY
and Department of Biochemistry, Hahnemann Vedical 19143, and Biochemistry Research Department, Laboratories, North Chicago, Illinois 60043
Received
August
2, 1945
Arsanilic acid is used to improve the health, appearance, production, efficiency, and survival of poultry and swine. It is, therefore, important to determine if traces of arsenical, remaining in the edible tissues of the animals consuming arsanilic acidmedicated feed, pose an immediate or potential problem in human toxicology. This study was designed to obtain information on the absorption, excretion, and retention of orally administered pure radioactive arsanilic-74As acid, and also as a residue in poultry used as food. METHODS
Arsanilic-74As acid was prepared according to the method of Fredrickson et al. (1965). It was found to be chromatographically pure and had an initial activity of 35 yc per milligram of arsenic. In the first phase of this study, 4 healthy, adult male human volunteers received single oral dose of between 1.3 and 3.0 mg of arsenic as pure arsanilic-?“As acid1 in solution. The activity was equivalent to 16 and 4.5 l~c, respectively. The material was administered in the morning, during the fasting state, and serial blood samples were obtained at 0, 2, 4, 6, 8, 12, 24, 48, 72, and 96 hours. Pooled 24-hour urines and feces were collected for 6 days. Plasma and urine samples were assayed for radioactivity without any special preparation. The total 24-hour feces, however, were first thoroughly mixed and emulsified in a Waring blendor, weighed, and then placed into 2-inch disposable plastic petri dishes for radioassay. In the second phase, tissue-residue studies were performed in 3 healthy, adult, male volunteers. White Leghorn chicks of about 2 pounds weight were fed a commercial ration and administered arsanilic-‘+As acid twice daily, orally, with a syringe for a period of 4 days. The daily dose of arsanilic acid was about 100 mg/kg/day. The chicks were then continued on the commercial ration for three additional days to allow the easily mobile arsanilic acid to clear from the tissues. The chicks were then sacrificed and the breast, legs, thighs, and liver excised. The tissues were heated in a closed jar at 100°C for 1 hour, allowed to cool, and then shipped to Philadelphia” for further preparation and consumption by the test subjects. The 74As in the total edible tissue from one fowl represented about l/3000 of the total arsenical fed. All 1 Prepared by Abbott Laboratories. 2 The feeding of the chicks was carried
out at Abbott 27
Laboratories.
28
B.
CALESNICK,
A.
WASE,
AND
L.
R.
OVERBY
the meat was removed from the bones, mixed with the liver, and homogenized in a Waring blendor in order to obtain a representative sampling of the radioarsenictissue. Salt and pepper were added for seasoning. This pat& was pressed into 2-inch plastic, disposable petri dishes for weighing and radioassay, before and after being served to the subjects. Each individual was in a fasting state and received between 162 and 210 g of this food as a single meal. Serial blood samples were obtained at the same intervals as in the first phase of the study, when the arsanilic-74As acid solutions were used, and pooled 24-hour urines and feces were also collected for periods up to 6 days. Plasma, urine, and feces were prepared as in the first phase and radioassayed. A standard well-type scintillation detector was used for the radioassays.
URINARY
AND
FECAL
ARSANILIC
Subject
(md
61
1.3 3.0 3.0
-
6 5 4
19.5 8.7 22.7
66
2.1
-
6
18.1 lf.3b
J.M. F. R. J.U. H. B. Mean
50 100
w. c. F. R. P. G. Mean
84
a b C d
100 71
AS
Recovery
Amount of pat6 (9)
Weight (kg)
Dose of arsenic
TABLE 1 RECOVERY OF 74As ADMINISTERED Acm OR TISSUE RESIDUE Collection period (days)
Urine (%I”
-
210 163
.5 5
22.7
-
162
6
19.8 20.7”
Total (%P 102.6
83.1 so.2 63.2
88.9 86.0
71.2
-+ 6.0
74.4Ck
89.3 9.0
1.7
64.lc
7.4
82.1
59.2 &
91.7d-c93.3
70.6 62.5
19.6
As percentage of amount administered. Difference between these means not significant, Difference between these means not significant, Difference between these means not significant,
of 74As Feces (%)a
C 5.9
79.0 84.8d
-I- 7.5
P > 0.4. P > 0.3. P > 0.5.
RESULTS
A statistical analysis of the data revealed no significant differences between the total recovery of 74As following the oral administration of either the free arsanilic acid or tissue-residue material (Table 1). This was also reflected by the urinary excretion patterns (Fig. l), except for subject F. R., who developed loose bowel movements. His urinary recovery was only approximately 8.7%. The urinary recovery rates were maximal within 48 hours and then gradually tapered off. The rates thereafter were probably due to recycling of the arsenic. Fecal recovery rates ranged between approximately 60% and 80% of the total radiological dose administered, and were maximal during the first 3 days. The effective half-life ( Te) was observed to be between 0.4 and 1.7 days. Calculation of the biological (Tb) half-life from the following relationship indicated a range of 0.4 to 1.8 days ( Tp is the physical half-life of 74A~, namely, 17.5 days) : T = b
Te X TP T,---T,
ABSORPTION
OF
ARSANILIC-AS”
29
ACID
Since this was a single dose, it was estimated that on the basis of the maximum biological half-life, only 0.09% would remain in the subject after 18 days. The average life (T,, = 1.443 X Tb) of the absorbed arsanilic acid was estimated to be 2.6 days, which is equivalent to the replacement time.
0 II
0 P
0 0
f
X 0
1
X= TISSUE RESIDUE (As-741 l = ARSANILIC (As-741 ACID
2-4
48
i2
i6
Ii0
144
HOURS FIG. 1. Cumulative tissue residue (X).
urinary
recoveries
of arsenic-74
administered
as arsanilic
acid
(a)
or as
In general, extremely low levels of plasma radioactivity were detected in these subjects. However, one subject (J. M.) exhibited a significant peak plasma concentration at the sixth hour, which was estimated to approximate 2.72 X 10P3% of the administered dose of arsanilic-74As acid. DISCUSSION
It has been shown that there is a marked difference in toxicity between organic and inorganic arsenic compounds, as reflected by the arsenic retained by animals (Hogan and Eagle, 1944). There are also differences in the tolerance of various
30
B.
CALESNICK,
A.
WASE,
AND
L.
R.
OVERBY
animal species to the organic arsenicals used in feeds (Frost et al., 1955). Absorption and excretion patterns also differ for the various forms of arsenicals. For example, only a small quantity of arsenic in shrimp was retained by rats (Coulson et al., 1935). The biological action of arsenicals may be due to the combination of the arsenic with protein sulfhydryl groups. When arsenite was injected intravenously (Mealey et al., 1959), it was recovered in the urine as a mixture of arsenite and arsenate. This suggests that there is a biological mechanism for oxidizing the more toxic trivalent arsenite to arsenate. A review of the earlier literature revealed some inconclusive evidence of slow release of inorganic arsenic from arsanilic acid. Recently, however, it was shown by Overby and Straube (1965) that there was no detectable rupture of the carbon-arsenic bond when a doubly labeled molecule of arsanilic-l-14C-74As acid was fed orally to chickens. Furthermore, arsanilic acid was found to be excreted more rapidly than arsenate in chickens (Overby et al., 1965). There was also no evidence of degradative arsanilic acid metabolism. In the present studies, extremely low plasma 74As levels were detected with the tissue-residue material as with pure arsanilic acid, indicating a rapid clearance or poor absorption of both materials. This srrggests that the arsenical in tissues of chickens fed arsanilic acid is absorbed and excreted by humans in very much the same manner as is authentic arsanilic acid. SUMMARY When tissues of chicks fed arsanilic-74As acid were consumed rapid fecal excretion of the arsenic-74. There were no statistical and fecal recoveries of tissue 74As and pure arsanilic-74As acid.
by human differences
subjects, between
there was a the urinary
REFERENCES J., REMINGTON, R. E., and LYNCII, K. M. (1935). Metabolism in the rat of the naturally occurring arsenic of shrimp as compared with arsenic trioxide. J. Nutr. 10, 255-270. FREDRICKSON, R. L., BOCCHIERI, S. F., GLENN, H. J., and OVERBY, L. R. (1965). Synthesis of arsanilic-As74 acid and arsanilic-l-C14 acid. J. Assoc. Ofic. Agr. Chemists 48, 10-17. FROST, D. V., OVERBY, L. R., and SPRUTII, H. C. (1955). Studies with arsanilic acid and related compounds. J. Agr. Food Chem. 3, 235-243. HOGAN, R. B., and EAGLE, H. J. (1944). Pharmacologic basis for the widely varying toxicity of arsenicals. J. Pharmacol. Exptl. Therap. 80, 93-113. MEALEY, J., JR., BROWNELL, G. L., and SWEET, W. H. (1959). Radioarsenic in plasma, urine, normal tissues and intracranial neoplasms. Distribution and turnover after intravenous injection in man. A.M.A. Arch. Neural. Psychiat. 81, 310-320. OVERBY, L. R., and STRAUBE, L. (1965). Metabolism of arsanilic acid. I. Metabolic stability of doubly labeled arsanilic acid in chickens. Toricol. Appl. Pharmacol. 7, 850-854. OVERBY, L. R., BOCCHIERI, S. F., and FREDRICKSON, R. L. (1965). Chromatographic, electrophoretic, and ion exchange identification of radioactive organic and inorganic arsenicals. J. Assoc. Ofic. Agr. Chemists 48, 17-22. COULSON,
E.
AND
APPLIED
PHARMACOLOGY
Availability
during Tissues B.
Laboratory College,
27-30
9:
Human of
Consumption
Chicks
Fed
A.
CALESNICK,
of Human
Pharmacology Philadelphia, Pennsylvania Research Division, Abbott
(1966)
WASE,
of the
Arsanilic-‘“As AND
L. R.
Arsenic
in
Acid OVERBY
and Department of Biochemistry, Hahnemann Vedical 19143, and Biochemistry Research Department, Laboratories, North Chicago, Illinois 60043
Received
August
2, 1945
Arsanilic acid is used to improve the health, appearance, production, efficiency, and survival of poultry and swine. It is, therefore, important to determine if traces of arsenical, remaining in the edible tissues of the animals consuming arsanilic acidmedicated feed, pose an immediate or potential problem in human toxicology. This study was designed to obtain information on the absorption, excretion, and retention of orally administered pure radioactive arsanilic-74As acid, and also as a residue in poultry used as food. METHODS
Arsanilic-74As acid was prepared according to the method of Fredrickson et al. (1965). It was found to be chromatographically pure and had an initial activity of 35 yc per milligram of arsenic. In the first phase of this study, 4 healthy, adult male human volunteers received single oral dose of between 1.3 and 3.0 mg of arsenic as pure arsanilic-?“As acid1 in solution. The activity was equivalent to 16 and 4.5 l~c, respectively. The material was administered in the morning, during the fasting state, and serial blood samples were obtained at 0, 2, 4, 6, 8, 12, 24, 48, 72, and 96 hours. Pooled 24-hour urines and feces were collected for 6 days. Plasma and urine samples were assayed for radioactivity without any special preparation. The total 24-hour feces, however, were first thoroughly mixed and emulsified in a Waring blendor, weighed, and then placed into 2-inch disposable plastic petri dishes for radioassay. In the second phase, tissue-residue studies were performed in 3 healthy, adult, male volunteers. White Leghorn chicks of about 2 pounds weight were fed a commercial ration and administered arsanilic-‘+As acid twice daily, orally, with a syringe for a period of 4 days. The daily dose of arsanilic acid was about 100 mg/kg/day. The chicks were then continued on the commercial ration for three additional days to allow the easily mobile arsanilic acid to clear from the tissues. The chicks were then sacrificed and the breast, legs, thighs, and liver excised. The tissues were heated in a closed jar at 100°C for 1 hour, allowed to cool, and then shipped to Philadelphia” for further preparation and consumption by the test subjects. The 74As in the total edible tissue from one fowl represented about l/3000 of the total arsenical fed. All 1 Prepared by Abbott Laboratories. 2 The feeding of the chicks was carried
out at Abbott 27
Laboratories.
28
B.
CALESNICK,
A.
WASE,
AND
L.
R.
OVERBY
the meat was removed from the bones, mixed with the liver, and homogenized in a Waring blendor in order to obtain a representative sampling of the radioarsenictissue. Salt and pepper were added for seasoning. This pat& was pressed into 2-inch plastic, disposable petri dishes for weighing and radioassay, before and after being served to the subjects. Each individual was in a fasting state and received between 162 and 210 g of this food as a single meal. Serial blood samples were obtained at the same intervals as in the first phase of the study, when the arsanilic-74As acid solutions were used, and pooled 24-hour urines and feces were also collected for periods up to 6 days. Plasma, urine, and feces were prepared as in the first phase and radioassayed. A standard well-type scintillation detector was used for the radioassays.
URINARY
AND
FECAL
ARSANILIC
Subject
(md
61
1.3 3.0 3.0
-
6 5 4
19.5 8.7 22.7
66
2.1
-
6
18.1 lf.3b
J.M. F. R. J.U. H. B. Mean
50 100
w. c. F. R. P. G. Mean
84
a b C d
100 71
AS
Recovery
Amount of pat6 (9)
Weight (kg)
Dose of arsenic
TABLE 1 RECOVERY OF 74As ADMINISTERED Acm OR TISSUE RESIDUE Collection period (days)
Urine (%I”
-
210 163
.5 5
22.7
-
162
6
19.8 20.7”
Total (%P 102.6
83.1 so.2 63.2
88.9 86.0
71.2
-+ 6.0
74.4Ck
89.3 9.0
1.7
64.lc
7.4
82.1
59.2 &
91.7d-c93.3
70.6 62.5
19.6
As percentage of amount administered. Difference between these means not significant, Difference between these means not significant, Difference between these means not significant,
of 74As Feces (%)a
C 5.9
79.0 84.8d
-I- 7.5
P > 0.4. P > 0.3. P > 0.5.
RESULTS
A statistical analysis of the data revealed no significant differences between the total recovery of 74As following the oral administration of either the free arsanilic acid or tissue-residue material (Table 1). This was also reflected by the urinary excretion patterns (Fig. l), except for subject F. R., who developed loose bowel movements. His urinary recovery was only approximately 8.7%. The urinary recovery rates were maximal within 48 hours and then gradually tapered off. The rates thereafter were probably due to recycling of the arsenic. Fecal recovery rates ranged between approximately 60% and 80% of the total radiological dose administered, and were maximal during the first 3 days. The effective half-life ( Te) was observed to be between 0.4 and 1.7 days. Calculation of the biological (Tb) half-life from the following relationship indicated a range of 0.4 to 1.8 days ( Tp is the physical half-life of 74A~, namely, 17.5 days) : T = b
Te X TP T,---T,
ABSORPTION
OF
ARSANILIC-AS”
29
ACID
Since this was a single dose, it was estimated that on the basis of the maximum biological half-life, only 0.09% would remain in the subject after 18 days. The average life (T,, = 1.443 X Tb) of the absorbed arsanilic acid was estimated to be 2.6 days, which is equivalent to the replacement time.
0 II
0 P
0 0
f
X 0
1
X= TISSUE RESIDUE (As-741 l = ARSANILIC (As-741 ACID
2-4
48
i2
i6
Ii0
144
HOURS FIG. 1. Cumulative tissue residue (X).
urinary
recoveries
of arsenic-74
administered
as arsanilic
acid
(a)
or as
In general, extremely low levels of plasma radioactivity were detected in these subjects. However, one subject (J. M.) exhibited a significant peak plasma concentration at the sixth hour, which was estimated to approximate 2.72 X 10P3% of the administered dose of arsanilic-74As acid. DISCUSSION
It has been shown that there is a marked difference in toxicity between organic and inorganic arsenic compounds, as reflected by the arsenic retained by animals (Hogan and Eagle, 1944). There are also differences in the tolerance of various
30
B.
CALESNICK,
A.
WASE,
AND
L.
R.
OVERBY
animal species to the organic arsenicals used in feeds (Frost et al., 1955). Absorption and excretion patterns also differ for the various forms of arsenicals. For example, only a small quantity of arsenic in shrimp was retained by rats (Coulson et al., 1935). The biological action of arsenicals may be due to the combination of the arsenic with protein sulfhydryl groups. When arsenite was injected intravenously (Mealey et al., 1959), it was recovered in the urine as a mixture of arsenite and arsenate. This suggests that there is a biological mechanism for oxidizing the more toxic trivalent arsenite to arsenate. A review of the earlier literature revealed some inconclusive evidence of slow release of inorganic arsenic from arsanilic acid. Recently, however, it was shown by Overby and Straube (1965) that there was no detectable rupture of the carbon-arsenic bond when a doubly labeled molecule of arsanilic-l-14C-74As acid was fed orally to chickens. Furthermore, arsanilic acid was found to be excreted more rapidly than arsenate in chickens (Overby et al., 1965). There was also no evidence of degradative arsanilic acid metabolism. In the present studies, extremely low plasma 74As levels were detected with the tissue-residue material as with pure arsanilic acid, indicating a rapid clearance or poor absorption of both materials. This srrggests that the arsenical in tissues of chickens fed arsanilic acid is absorbed and excreted by humans in very much the same manner as is authentic arsanilic acid. SUMMARY When tissues of chicks fed arsanilic-74As acid were consumed rapid fecal excretion of the arsenic-74. There were no statistical and fecal recoveries of tissue 74As and pure arsanilic-74As acid.
by human differences
subjects, between
there was a the urinary
REFERENCES J., REMINGTON, R. E., and LYNCII, K. M. (1935). Metabolism in the rat of the naturally occurring arsenic of shrimp as compared with arsenic trioxide. J. Nutr. 10, 255-270. FREDRICKSON, R. L., BOCCHIERI, S. F., GLENN, H. J., and OVERBY, L. R. (1965). Synthesis of arsanilic-As74 acid and arsanilic-l-C14 acid. J. Assoc. Ofic. Agr. Chemists 48, 10-17. FROST, D. V., OVERBY, L. R., and SPRUTII, H. C. (1955). Studies with arsanilic acid and related compounds. J. Agr. Food Chem. 3, 235-243. HOGAN, R. B., and EAGLE, H. J. (1944). Pharmacologic basis for the widely varying toxicity of arsenicals. J. Pharmacol. Exptl. Therap. 80, 93-113. MEALEY, J., JR., BROWNELL, G. L., and SWEET, W. H. (1959). Radioarsenic in plasma, urine, normal tissues and intracranial neoplasms. Distribution and turnover after intravenous injection in man. A.M.A. Arch. Neural. Psychiat. 81, 310-320. OVERBY, L. R., and STRAUBE, L. (1965). Metabolism of arsanilic acid. I. Metabolic stability of doubly labeled arsanilic acid in chickens. Toricol. Appl. Pharmacol. 7, 850-854. OVERBY, L. R., BOCCHIERI, S. F., and FREDRICKSON, R. L. (1965). Chromatographic, electrophoretic, and ion exchange identification of radioactive organic and inorganic arsenicals. J. Assoc. Ofic. Agr. Chemists 48, 17-22. COULSON,
E.