Metabolism of arsanilic acid

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

850-854

7,

Metabolism I.

Metabolic

Stability

of

L. R. Biochemistry

Research

of Arsanilic

Doubly

Labeled

Abbott

Received

Acid Arsanilic

AND LILIAN

OVERBY

Department,

(1965)

in Chickens’

STRAUBE

Laboratories,

A’ovember

Acid

Sorth

Chicago,

Illinois

60064

25, 1964

Arsanilic acid (6aminophenylarsonic acid) was the first arsenical used for treating trypanosomiasis. The mechanism of action was paradoxical because the compound was inactive against parasites in vitro, and in vivo required a latent period during which there was no parasiticidal action. One explanation proposed was that the slow releaseof inorganic arsenic accounted for the in viva activity. Some literature in the early 1900’s tended to support this idea. For example, Igersheimer and Rothmann (1909) studied the excretion products in rabbits after injection of “Atoxyl” (sodium arsanilate). Their criterion for ‘(inorganic arsenic” was the arsenic in the insoluble residue after extraction of dried excreta with methyl alcohol. The injected material “Atoxyl,” was easily soluble in methyl alcohol. Breinl and Nierenstein (1909) injected arsanilic acid into a horse and reported that a “large amount of ‘free arsenic’ was excreted.” Williams (1959) has reviewed the scanty and inconclusive studies on the metabolism of organic arsenicals. Arsanilic acid is now widely used in medicated animal feeds. It improves health and production efficiency, but the mechanismof action is unknown. A small residue remains in body tissues unless the arsenical is removed from the ration before slaughter. It is of interest to know whether animals consuming an arsanilic acidmedicated feed, or humans who consume the edible tissues, are exposed to endogenously produced inorganic arsenic to any measurable extent. Despite the lack of modern evidence and the equivocal nature of the early work it is still widely believed that organic arsenicals are degraded to inorganic arsenic. The idea gains stature by reiteration. Actually, the most striking fact about the early German literature on arsenical metabolism is that almost all of an administered dose of arsanilic acid was excreted unchanged. Often, however, the total arsenic excreted was higher than that accounted for as diazotizable amine. The main objective of the present series of studies was to determine the nature of the arsenic retained by chickens after oral arsanilic acid administration. An attempt was first made to establish whether there was a measurable rupture of the carbon-arsenic bond in the retained arsenical. The change of isotope ratio in a doubly labeled molecule appeared suitable for this type of study. The desired compound was arsanilicl-C14-As74 acid as shown in Fig. 1. Complete cleavage of the C-As bond would result in differential metabolism of arsenate-As?”and aniline-C14.A small, partial cleav1 Presented in part at the 142nd City, New Jersey, September 9-14,

National 1962.

Meeting 850

of the .i\merican

Chemical

Society,

Atlantic

METABOLIC

STABILITY

H

N 2\_/;

/

OF ARSANILIC

\,,,-;s74

ACID

851

: ()

-P

H FIG.

1.

Structure

of doubly

labeled

arsanilic

acid

(4-aminophenyl-l-C14-arsonic-As’4

acid).

age would result in differential metabolism of a large quantity of doubly labeled arsanilic acid and small quantities of singly labeled aniline and arsenate. Extensive investigation of many animals in replicate was not possible because the acid was limited by difficulty of preparation, the amount of arsanilic-l-C14-As74 short half-life of Asi4, and the necessity for exceptional radiochemical purity. The available labeled compound was sufficient for metabolic studies in six birds. TO ensure that the methods were precise enough to detect differential retention of arsanilic acid and arsenate, singly labeled arsanilic acid and doubly labeled arsanilic acid were mixed with arsenate-As74 and studied in one animal each. Isotope ratios were then studied in four chicks given the doubly labeled compound. METHODS

Arsanilic-l-Cl4 acid was prepared from 4-nitroaniline-l-Cl4 and arsenite, and arsanilic-AsT4 acid from 4-nitroaniline and arsenite-As74 as described by Fredrickson et al. (1965). Initial specific activities were 1 mC/mM as Cl4 and 3 mC/mM as ASPS. The labeled arsanilic acids were mixed in proportions to give counting rate ratios of about 1-2 for C?“:As7”. A dose of the mixtures, calculated to give 20-30 mg/kg of arsenic, was administered orally by syringe to white Leghorn chickens of the weights shown in Table 1. After 24 hours, a second identical dose was administered. Exact replication of dose in terms of arsenic was not possible because of the short half-life of ASPS. The birds were sacrificed by withdrawing blood by heart puncture 24 hours after the second dose. Feces collections were made at 24-hour intervals. Samples of organs and tissues were freeze dried, and the levels of Cl4 and As74 were determined. The isotopes were separated by the wet combustion method of Van Slyke et al. ( 1940). Carbon dioxide was collected as the carbonate by the procedure of Jeffay and Alvarez (1961), and counted in a liquid scintillation spectrometer. Arsenic-74 remained in the acid combustion mixture and was counted in a sodium iodide well with a gamma ray spectrometer. The analytical procedure was performed in duplicate on 150 mg of dry tissue or 750 mg of wet tissue. In control experiments, normal dried liver tissue was burned with tracer amounts of arsanilic-l-Cl4 acid and arsanilicAs7” acid. Recoveries were 98-100s for As74 and 94-98y0 for Cl*. Overall counting efficiency, including the oxidation procedure, was 58-.62y0 for Asi and 30-33% for Ct4. All samples and background were counted to a 5% error or less. The minimum actual net counting rate observed among all the tissue samples was 84 cpm. Internal standards were added to each sample, and corrections made for quenching. RESULTS

The dosing schedule and the various isotope ratios are shown in Table 1. Chick 2 received a dose composed of 99.9570 arsanilic-l-C14 acid and 0.05% arsenate-As’“.

852

L.

R.

OVERBY

AND

LILIAN

TABLE

THE RATIO

OF

EXCRETA

Cl* AND

TO

As74 COUNTING

TISSUES

OF CHICKS

STRAUBE

1

RATES IN THE ADMINISTERED GIVEN

Chick Parameter Body weight (g) Arsenic per dose (mg/kg) Composition of dose Arsanilic-l-C14 acid (s) Arsenate-AsT4 (‘j&O, Arsanilic-As74 acid (%)

2

6

1

158

106 16.0

282 27.5

17.5

IN

number 3 198 31.7

5

109 31.4

78 18.0

65.00 0.00

0.00

46.00

35.00

0.00 44.00

6

9

5

6

7

6.31 6.05

0.86 0.72

2.25 2.07

1.36 1.23

0.94

7 1.81

0.87

1.69

6.49

0.99

2.42

1.45

0.98

1.91

2.40

0.76 0.57

2.32

1.23

0.96

1.57

3.15

1.07

1.06

0.62 0.92

0.70 0.53

2.02 -

3.54 -

0.54

1.02

0.53

-

0.96 0.97 -

1.7s 1.48

-

1.21 1.01 1.17

1.04

1.67

0.72

-

-

-

0.71 0.58

1.06 -

1.77 1.74 1.71

2.13

0.63

2.17

1.20

1.02

1.65

9.75

0.84

4.46 26.46 -

0.86 0.86

1.38 1.44

1.83 1.64

1.25

2.29 2.38 -

6.57 6.02

1.04 1 .oo

10.65

1.14

-

56.00

4h

53 .oo 1 .oo

Ratio of Cl4 to AsT4 in tissues Blood Muscle Heart Lung Spleen Feathers Gizzard Bone Intestinal wall Mean ratio

Change of mean ratio mean ratio in dose Excretory Body tissues

AND

Acma

99.95 0.05

Ratio of Cl4 to As7” in dose Number of determinations Mean ratio Lowest ratio Highest ratio

Ratio of 04 to As74 in excretory organs and excreta Liver Kidney Bile Intestinal contents O-24 hour excreta 2448 hour excreta Mean ratio

DOSE

ARSANIL.IC-~-C~~-AS~~

46.00 0.00

72.00 0.00

54.00

28.00

1.4s -

1.08

1.80

2.48 2.31

1.12 1.32 1.36

1.76 1.86 1.81

2.36

1.36

1.78

from (%) +59 -68

0 Two equal doses were given at 24-hour the second dose. b Died from suffocation 12-18 hours after

-t32 -27

intervals, the second

+j -4

and the birds

0 -12

were

sacrificed

24 hours

after

dose.

This dose was selected to test the precision of the methods to detect differential retention and excretion of arsanilic acid and a trace quantity of inorganic arsenate. The results indicated a large difference in affinity of the various tissues of this bird for the two compounds. The administered dose had a C14:As74 counting ratio of 6.31. The radioactivity retained in tissues had a reduced ratio, particularly in the heart and lung, indicating higher retention of arsenate. Liver, kidney, and bile also had altered ratios. The 4-fold increase in the bile ratio showed the preferential

METABOLIC

STABILITY

OF ARSANILIC

ACID

853

excretion of arsanilic acid via the liver. The lower ratio in kidney indicated an increased level of arsenate. The isotope ratios in excreta were similar to the dose ratios. A large change would not be expected in excreta because almost all the administered dose of ,both compounds was excreted, and small differences in retention would not show up here. The average change was +59ojo for excretory organs and excreta, and -68% for body tissues. acid and arsanilic-As74 acid. In Chick 6 received a mixture of arsanilic-l-Cl4 addition, 170 of arsenate-Asr4 was added. The specific activity of the added arsenate was adjusted to 46% of the arsanilic-As71 acid arsenic to simulate the comparison of a mixture formed if 1 y0 of 53 :46 mixture of doubly labeled arsanilic acid was degraded endogenously. In the experiment the mean ratio in body tissues decreased from 0.86 to 0.63. The ratio increased to 1.14 in the excreta and excretory organs. This suggested that if as little as 170 of an administered dose of doubly labeled arsanilic acid were degraded, changed isotope ratios could be detected in body tissues and excreta by the methods employed. Four chicks were given the doubly labeled arsanilic acids without added arsenate as shown in Table I. The values for Chick 1 are not significantly different from the dose. The 12% lowering in the body tissues for Chick 3 was significant at the 5% level of confidence, but there were no increased ratios. The Chick 4 ratios were not significantly changed. Only the ratios for heart and lung were significantly different from the dose in Chick 5. DISCUSSION

The present experiments with mixed isotopes revealed no large-scale rupture of the carbon-arsenic bond of orally administered arsanilic acid by chickens. In one bird, a dose composed of 99% doubly labeled arsanilic acid and 1% of arsenate-As74 was administered and significant changes in isotope ratios were observed in body tissues and excreta. In four chicks given arsanilic acid alone, there were no comparable changes in isotope ratios. Therefore, if there was degradative metabolism of the arsanilic acid it was less than 1% of the dose. The results with Chick 2 showed large differences in the retention and excretion of arsanilic acid and arsenate. Here the dose was 99.9570 arsanilic acid. Retention by tissues is a function of dose level. The differential retention probably would have been much larger with a larger percentage of arsenate in the dose. The limiting factor in the mixed isotope method is the purity of the radioactive arsanilic acid. For example, a trace impurity of arsenate-As?+ in the arsanilic acid preparation would be amplified severalfold in tissues by its differential retention, as shown in the Chick 2 test. The lowered isotope ratio found in heart, lung and spleen of Chicks 3 and 5 could be accounted for by having only about 99.97‘ isotopically pure arsanilic-As?’ acid. The preparations used in these experiments were purified on Dowex-SO@ columns, which separate arsanilic acid from arsenate (Overby et al., 1965). The compounds showed single radioactive peaks on chromatograms, but all methods investigated were too insensitive to detect fractional percentages of impurities. The double isotope labeling technique, as used in these experiments, can demonstrate best the occurrence of degradative metabolism. Proof of the absolute absence of degradation is more difficult because the limiting factor becomes the proof of

8.54

L.

R.

OVERBY

AND

LILIAN

STRAUBE

absolute purity of the isotopically labeled compounds under study. The present experiments strongly suggest that chickens, receiving arsanilic acid orally, do not cleave the C-As bond to the extent of lch of the administered dose. Subsequent to the completion of these studies, Moody and Williams (1964) reported that arsanilic acid was excreted unchanged in hens after either oral administration or intramuscular injection. The excreta did not contain any metabolic products in amounts detectable by the methods used. SUMMARY The stability of the C”:As74 isotope ratio was studied in the tissues, organs, and excreta of chicks given doubly labeled arsanilic-I-C 14-As74 acid. The method is sensitive enough to detect metabolic degradation of the compound to inorganic arsenic because (1) if as little as 1% of arsenate-As74 is added to the dose, significant increases and decreases in isotope ratios in tissues occur, and (2) trace quantities of orally administered inorganic arsenate are selectively retained in tissues. In four chicks no consistent changes were observed at 24 hours after the last oral dose of arsanilic-l-Cr4-As74 acid. If arsanilic acid was metabolically degraded, it was affected to less than 1% of the administered dose. ACKNOWLEDGMENT The authors thank R. L. Fredrickson pounds, and B. T. Main for collecting

and S. F. Bocchieri for the animal tissues.

synthesis

of the radioactive

com-

REFERENCES A., and NIERENSTEIN, M. (1909). Zum Mechanismus der Atoxylwirkung. Z. Immunitaetsforsch. 1, 620-632. FREDRICKSON, R. L., BOCCHIERI, S. F., GLENN, H. J., JR., and OVERBY, L. R. (1965). Synthesis of arsanilic-As74 acid and arsanilic-l-Cl4 acid. J. Assoc. Ofic. Agr. Chemists 48, 10-17. IGERSHEIMER, J., and ROTHMANN, A. (1909). tfber das Verhalten des Atoxyls im Organismus. Z. Physiol. Chem. 59, 256-280. JEFFAY, H., and ALVAREZ, J. (1961). Liquid scintillation counting of carbon-14. Use of ethanolamine-ethylene glycol monoethyl ether-toluene. Anal. Chem. 33, 612-615. MOODY, J. P., and WILLIAMS, R. T. (1964). The fate of arsanilic acid and acetyl arsanilic acid in hens. Food Cosmet. Toxicol. 2, 687-693. OVERBY, L. R., BOCCRIERI, 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. VAN SLYXE, D. D., FOLCH, J., and PLAZIN, J. (1940). Manometric carbon determination. J. Biol. Chem. 136, 509-541. WILLIAMS, R. T. (1959). Detoxication Mechanisms, 2nd ed., p, 703. Wiley, New York, BREINL,