Archives of
Arch. Toxicol. 36, 159--162 (1976)
TOXICOLOGY 9 by Springer-Verlag 1976
Short Communications Biotransformation of As(llI) to As(V) and Arsenic Tolerance V. Bencko, B. BeneL and M. Cikrt Medical Faculty of Hygiene, Charles University, Prague Institute of Hygiene and Epidemiology 100 42 Prague 10, ~robfirova48, Czechoslovakia
Abstract. The paper deals with the excretion of As(III) and As(V) in the urine of mice exposed to a toxic concentration of As(III) (250 mg/1) in drinking water. After the exposures of 2, 6, and 8 days, the mice were given i.m. a solution of 74As(III) labeled sodium arsenite in a dose of 1.3 mg As(III)/kg b.w. and an activity of 3 ~Ci/1 ml. After the sacrifice of the animal, the urine was drawn off directly from the bladder. The urine samples were subjected to separation of As(III) from As(V) by using a paper radiochromatography technique. The results showed that unexposed control mice excreted one-half of the administered As(III) in the form of As(V). Mice exposed to arsenic in drinking water exceted already, after 2 days of exposure, four-fifths of the applied As(III) in the form of As(V), after 4 days of exposure the proportion of As(V) was more than 95%, and after 8 days of exposure only traces of As(III) were present. The authors discussed the possibility of participation of the biotransformation of As(III) to As(V) on arsenic tolerance. Key words: Arsenic -- Biotransformation -- Tolerance.
Zusammenfassung. Die Arbeit behandelt die Ausscheidung von As(III) und As(V) im Urin von M/~usen, die einer toxischen Konzentration von As(III) im Trinkwasser (250 mg/1) ausgesetzt waren. Nach der Exposition von 2, 6 und 8 Tagen erhielten die M/iuse eine i.m. Injektion yon 74As-markiertem Natriumarsenit (3 ~Ci/ml) in einer Dosis von 1,3 mg As(III)/kg K6rpergewicht. Nach TStung der Tiere wurde der Urin direkt der Blase entnommen. In den Urinproben wurden As(III) und As(V) unter Verwendung der Radio-Papierchromatographie aufgetrennt. Die Ergebnisse zeigen, dal3 nicht exponierte Kontrollm/iuse die H/ilfte des am Ende der Expositionszeit gegebenen As(III) als As(V) ausscheiden. M/iuse, die Arsen im Trinkwasser erhielten, scheiden bereits nach 2 Tagen Exposition 4/5 des injizierten As(III) als As(V) aus. Nach 4 Tagen Exposition lagen mehr als 95% als As(V) vor, und nach 8 Tagen waren lediglich Spuren von As(Ill) vorhanden. Die Autoren diskutieren die M6glichkeit, dab die Biotransformation von As(Ill) zu As(V) zur Arsentoleranz beitr/igt.
160
v. Bencko et al.
A n interconversion of arsenite and arsenate ions in vivo has been known for a long time (Crawford, 1947). Regarding this fact we extended the series of our experiments, by the study presented here, dealing with the hygienic and toxicologic problems of arsenic (Bencko and Symon, 1969, 1969a; Bencko and N~merkova, 1971; Bencko, 1972; Bencko et al., 1975), including its kinetics in the organism (Bencko et al., 1973; Cikrt and Bencko, 1974). The objective was to find out whether there occurs a change in the As(III)/As(V) ratio in the urine of mice exposed to a toxic dose of trivalent arsenic given in drinking water in the course of exposure.
Material and Methods From the number of techniques recommended for the separation of trivalent and pentavalent forms of arsenic we have selected paper chromatography (Miket'ukovfi, 1968), which was modified by using radioarsenic 74As. Adult ICR-SPF male mice, weighing25-30 g, were used in the experiment after 3 weeks of adaptation to experimental conditions. Arsenic was administered in drinking water in a concentration of 250 mg/l, in the form of sodium arsenite. No arsenic was added to the drinking water given to the control group. The mice were fed with a pelleted Larsen's diet. The intake of drinking water was calculated per weight of the experimental animal. The amount of water and feed was given ad lib. From the total daily intake of drinking water it was possible to estimate the average dose of As(Ill), which was approx. 12 mg/kg b.w./day. After 2, 4, and 8 days of exposure, the mice were injected i.m. into the left hind limb a solution of 74As(llI) labeled sodium arsenite in a dose of 1.3 mg As3+/kg b.w. and approx, specific activity 3 ~Ci/1 ml. From experimental and control groups 6-12 animals, were sacrificed by decapitation 1, 3, 5, 6, 12, and 24 h after the i.m. administration of arsenic. The above-mentionedintervals were chosen for technical reasons. The animals' abdominal cavities were opened and urine drawn off directly from the bladder with an injection syringe. The well-type scintillation counter NZQ 717 Tesla was used to measure urine activity. The paper chromatography technique was then used to separate As(III) from As(V) in the urine samples (Mikethkovh, 1968). For the purpose of chromatographic separation, 50 p~l of each sample was applied. The activity of urine samples was adjusted by adding redistilled water to 0.01 ~Ci/10 ~xlof urine. Concurrently with the urine sample, reference standards were applied (20 ~1 of mixture of As(IlI) and As(V) in a ratio of 1 : 1, 5 : 1, and 1 : 5, respectively).The mixture was labeled by 74As(III) and 74As(V) in the same ratio. The chromatograms were measured using the registration apparatus Radiochromatograph NOQ 611 Tesla. The comparison of sample readings with the standards revealed an agreement between individualmaxims of separated ions. By comparing the area of the peaks with the total activity applied, final results were received in percent of total activity.
Results and Discussion Under the described experimental conditions, a total of 32 urine samples, with the a m o u n t of urine ranging from 0 . 3 - 1 . 2 ml at different time exposures, was obtained and analyzed. No difference in the arsenite to arsenate ratio was found among samples of Urine taken from animals of the same exposure group in spite of the fact that they were sacrificed at different time after i.m. administration of labeled doses of As(III) ranging from 1 . 5 - 2 4 h. The relative percentage of excreted 74As(III) and 74As(V) in relation to total activity is presented in Figure 1. Figure 1 shows that in the control group about one-half of the arsenite dose that was administered was excreted in the form of arsenate ions, which is in agreement to the findings by Crawford (1974). The oxidation of As(III) to As(V) has also been
Biotransformation of As(III) to As(V) and Arsenic Tolerance
161
(g) fJ r /
80
~
/J
f~ r~
As(V)
F~
fj
// ,// // // // //
70.
9 s
fi // i J
f
/ /
r.,
~.
/ /
"f j "
//
/ /
/'/ /-j // /j
40
f/ 9 30
Fig. 1. Ratio of As(III) and As(V) excreted in urine of mice during their exposure to arsenic in drinking water in concentration of 250 mg As(III)/1 in the form of sodium arsenite. The ratio expressed in per cent of total 74As(III) and 74As(V) activity after radiochromatographic separation of both ions
/
F/
/j fJ 20
fJ r j r'/
10
,~ / f/ f J r
c
// //
f~
;;
z
""
@
2
//
4
/ / / / j-
/
8 (DAYS)
described in some bacteria (Mandel and Mayerdak, 1962), even though the increased tolerance to arsenic may be due, in this case, to other detoxicating mechanisms, e.g., to reduced permeability of the cellular membrane (Arima and Beppu, 1964). Mice, exposed to arsenic in drinking water, excreted already after 2 days of exposure, four-fifths of the trivalent arsenic administered in the form of arsenate ions. After 4 days of exposure the share of As(V) reached more than 95% and after 8 days of exposure almost exclusively, pentavalent arsenic was found in the urine. Trivalent arsenic was present only in traces. Since arsenate ions are less toxic than arsenite ions, then theoretically the tolerance to arsenic may be enhanced by accelerating the biotransformation of arsenite to arsenate ions with its subsequent excretion from the organism. We have encountered the phenomenon of increased tolerance to arsenic in experiments with mice exposed for 3 months to arsenic in a concentration of 50 mg As(III) in the form of sodium arsenite in drinking water. The phenomenon was further encountered when we studied the metabolic oxygen consumption (Bencko, 1972) and dehydrogenase activity of the liver parenchyma of mice (Bencko et al., 1975) under the same experimental conditions. In the preliminary communication presented here we report on the results we have obtained with a toxic concentration of arsenic. In our next experiment we intend to study this phenomenon at a concentration ensuring the intake of near threshold doses (5 mg/1) and at an already tested concentration 50 mg As(III)/1 in drinking water to inducing the tolerance until it is suitable to arsenic. A c k n o w l e d g e m e n t s . The authors are grateful to Miss St. Jechov/t and Mr. K. Hradeck~, for their careful and untiring technical assistance.
162
V. Bencko et al.
References Arima, K., Beppu, M.: Induction and mechanism of arsenite resistence on psedomonas pseudomaUei. J. Bact. 88, 141 (1964) Bencko, V., Symon, K.: Suitability of hairless mice for experimental work and their sensitivity to arsenic. J. Hyg. Epidem. (Praha) 13, 1--6 (1969) Bencko, V., N~me~kovfi, H.: Oxygen consumption by mouse liver homogenate during drinking water arsenic exposure. J. Hyg. Epidem. (Praha) 15, 104 (1971) Bencko, V.: Oxygen Consumption by mouse liver homogenate during drinking water arsenic exposure. J. Hyg. Epidem. (Praha) 16, 42--46 (1972) Bencko, V., Rtssner, P., Mokr~,, M.: Dehydrogenase activity of liver parenchyma in mice exposed to arsenic in drinking water. J. Hyg. Epidem. (Praha) 19, 17--21 (1975) Bencko, V., Symon, K.: Dynamics of cumulation of arsenic in hairless mice after its application in drinking water. J. Hyg. Epidem. (Praha) 13, 245-253 (1969) Bencko, V., Dvo~fik, V., Symon, K.: Organ retention of parenterally administered arsenic (labelled with 74As)in mice preliminarily exposed to the element in drinking water. A study in arsenic tolerance. J. Hyg. Epidem. (Praha) 17, 165-168 (1973) Cikrt, M., Bencko, V.: Fate of arsenic after parenterai administration to rats with particular reference to excretion via bile. J. Hyg. Epidem. (Praba) 18, 129-136 (1974) Crawford, T. B.: Changes underdone by phenylarsenious acid and phenylarsenic acid in the animal body. Biochem. J. 41, 333 (1947) Miket'ukov~., V." Separation of arsenite and arsenate ions by paper chromatography. Study of methanol-ammonia/water system. J. Chromatogr. 34, 284-288 (1968) Mandel, H. G., Mayerdak, J. S.: The metabolisms and action of arsenite in microorganism. Fed. Proc. 21, 179 (1962) Received June 14, 1976
Arch. Toxicol. 36, 159--162 (1976)
TOXICOLOGY 9 by Springer-Verlag 1976
Short Communications Biotransformation of As(llI) to As(V) and Arsenic Tolerance V. Bencko, B. BeneL and M. Cikrt Medical Faculty of Hygiene, Charles University, Prague Institute of Hygiene and Epidemiology 100 42 Prague 10, ~robfirova48, Czechoslovakia
Abstract. The paper deals with the excretion of As(III) and As(V) in the urine of mice exposed to a toxic concentration of As(III) (250 mg/1) in drinking water. After the exposures of 2, 6, and 8 days, the mice were given i.m. a solution of 74As(III) labeled sodium arsenite in a dose of 1.3 mg As(III)/kg b.w. and an activity of 3 ~Ci/1 ml. After the sacrifice of the animal, the urine was drawn off directly from the bladder. The urine samples were subjected to separation of As(III) from As(V) by using a paper radiochromatography technique. The results showed that unexposed control mice excreted one-half of the administered As(III) in the form of As(V). Mice exposed to arsenic in drinking water exceted already, after 2 days of exposure, four-fifths of the applied As(III) in the form of As(V), after 4 days of exposure the proportion of As(V) was more than 95%, and after 8 days of exposure only traces of As(III) were present. The authors discussed the possibility of participation of the biotransformation of As(III) to As(V) on arsenic tolerance. Key words: Arsenic -- Biotransformation -- Tolerance.
Zusammenfassung. Die Arbeit behandelt die Ausscheidung von As(III) und As(V) im Urin von M/~usen, die einer toxischen Konzentration von As(III) im Trinkwasser (250 mg/1) ausgesetzt waren. Nach der Exposition von 2, 6 und 8 Tagen erhielten die M/iuse eine i.m. Injektion yon 74As-markiertem Natriumarsenit (3 ~Ci/ml) in einer Dosis von 1,3 mg As(III)/kg K6rpergewicht. Nach TStung der Tiere wurde der Urin direkt der Blase entnommen. In den Urinproben wurden As(III) und As(V) unter Verwendung der Radio-Papierchromatographie aufgetrennt. Die Ergebnisse zeigen, dal3 nicht exponierte Kontrollm/iuse die H/ilfte des am Ende der Expositionszeit gegebenen As(III) als As(V) ausscheiden. M/iuse, die Arsen im Trinkwasser erhielten, scheiden bereits nach 2 Tagen Exposition 4/5 des injizierten As(III) als As(V) aus. Nach 4 Tagen Exposition lagen mehr als 95% als As(V) vor, und nach 8 Tagen waren lediglich Spuren von As(Ill) vorhanden. Die Autoren diskutieren die M6glichkeit, dab die Biotransformation von As(Ill) zu As(V) zur Arsentoleranz beitr/igt.
160
v. Bencko et al.
A n interconversion of arsenite and arsenate ions in vivo has been known for a long time (Crawford, 1947). Regarding this fact we extended the series of our experiments, by the study presented here, dealing with the hygienic and toxicologic problems of arsenic (Bencko and Symon, 1969, 1969a; Bencko and N~merkova, 1971; Bencko, 1972; Bencko et al., 1975), including its kinetics in the organism (Bencko et al., 1973; Cikrt and Bencko, 1974). The objective was to find out whether there occurs a change in the As(III)/As(V) ratio in the urine of mice exposed to a toxic dose of trivalent arsenic given in drinking water in the course of exposure.
Material and Methods From the number of techniques recommended for the separation of trivalent and pentavalent forms of arsenic we have selected paper chromatography (Miket'ukovfi, 1968), which was modified by using radioarsenic 74As. Adult ICR-SPF male mice, weighing25-30 g, were used in the experiment after 3 weeks of adaptation to experimental conditions. Arsenic was administered in drinking water in a concentration of 250 mg/l, in the form of sodium arsenite. No arsenic was added to the drinking water given to the control group. The mice were fed with a pelleted Larsen's diet. The intake of drinking water was calculated per weight of the experimental animal. The amount of water and feed was given ad lib. From the total daily intake of drinking water it was possible to estimate the average dose of As(Ill), which was approx. 12 mg/kg b.w./day. After 2, 4, and 8 days of exposure, the mice were injected i.m. into the left hind limb a solution of 74As(llI) labeled sodium arsenite in a dose of 1.3 mg As3+/kg b.w. and approx, specific activity 3 ~Ci/1 ml. From experimental and control groups 6-12 animals, were sacrificed by decapitation 1, 3, 5, 6, 12, and 24 h after the i.m. administration of arsenic. The above-mentionedintervals were chosen for technical reasons. The animals' abdominal cavities were opened and urine drawn off directly from the bladder with an injection syringe. The well-type scintillation counter NZQ 717 Tesla was used to measure urine activity. The paper chromatography technique was then used to separate As(III) from As(V) in the urine samples (Mikethkovh, 1968). For the purpose of chromatographic separation, 50 p~l of each sample was applied. The activity of urine samples was adjusted by adding redistilled water to 0.01 ~Ci/10 ~xlof urine. Concurrently with the urine sample, reference standards were applied (20 ~1 of mixture of As(IlI) and As(V) in a ratio of 1 : 1, 5 : 1, and 1 : 5, respectively).The mixture was labeled by 74As(III) and 74As(V) in the same ratio. The chromatograms were measured using the registration apparatus Radiochromatograph NOQ 611 Tesla. The comparison of sample readings with the standards revealed an agreement between individualmaxims of separated ions. By comparing the area of the peaks with the total activity applied, final results were received in percent of total activity.
Results and Discussion Under the described experimental conditions, a total of 32 urine samples, with the a m o u n t of urine ranging from 0 . 3 - 1 . 2 ml at different time exposures, was obtained and analyzed. No difference in the arsenite to arsenate ratio was found among samples of Urine taken from animals of the same exposure group in spite of the fact that they were sacrificed at different time after i.m. administration of labeled doses of As(III) ranging from 1 . 5 - 2 4 h. The relative percentage of excreted 74As(III) and 74As(V) in relation to total activity is presented in Figure 1. Figure 1 shows that in the control group about one-half of the arsenite dose that was administered was excreted in the form of arsenate ions, which is in agreement to the findings by Crawford (1974). The oxidation of As(III) to As(V) has also been
Biotransformation of As(III) to As(V) and Arsenic Tolerance
161
(g) fJ r /
80
~
/J
f~ r~
As(V)
F~
fj
// ,// // // // //
70.
9 s
fi // i J
f
/ /
r.,
~.
/ /
"f j "
//
/ /
/'/ /-j // /j
40
f/ 9 30
Fig. 1. Ratio of As(III) and As(V) excreted in urine of mice during their exposure to arsenic in drinking water in concentration of 250 mg As(III)/1 in the form of sodium arsenite. The ratio expressed in per cent of total 74As(III) and 74As(V) activity after radiochromatographic separation of both ions
/
F/
/j fJ 20
fJ r j r'/
10
,~ / f/ f J r
c
// //
f~
;;
z
""
@
2
//
4
/ / / / j-
/
8 (DAYS)
described in some bacteria (Mandel and Mayerdak, 1962), even though the increased tolerance to arsenic may be due, in this case, to other detoxicating mechanisms, e.g., to reduced permeability of the cellular membrane (Arima and Beppu, 1964). Mice, exposed to arsenic in drinking water, excreted already after 2 days of exposure, four-fifths of the trivalent arsenic administered in the form of arsenate ions. After 4 days of exposure the share of As(V) reached more than 95% and after 8 days of exposure almost exclusively, pentavalent arsenic was found in the urine. Trivalent arsenic was present only in traces. Since arsenate ions are less toxic than arsenite ions, then theoretically the tolerance to arsenic may be enhanced by accelerating the biotransformation of arsenite to arsenate ions with its subsequent excretion from the organism. We have encountered the phenomenon of increased tolerance to arsenic in experiments with mice exposed for 3 months to arsenic in a concentration of 50 mg As(III) in the form of sodium arsenite in drinking water. The phenomenon was further encountered when we studied the metabolic oxygen consumption (Bencko, 1972) and dehydrogenase activity of the liver parenchyma of mice (Bencko et al., 1975) under the same experimental conditions. In the preliminary communication presented here we report on the results we have obtained with a toxic concentration of arsenic. In our next experiment we intend to study this phenomenon at a concentration ensuring the intake of near threshold doses (5 mg/1) and at an already tested concentration 50 mg As(III)/1 in drinking water to inducing the tolerance until it is suitable to arsenic. A c k n o w l e d g e m e n t s . The authors are grateful to Miss St. Jechov/t and Mr. K. Hradeck~, for their careful and untiring technical assistance.
162
V. Bencko et al.
References Arima, K., Beppu, M.: Induction and mechanism of arsenite resistence on psedomonas pseudomaUei. J. Bact. 88, 141 (1964) Bencko, V., Symon, K.: Suitability of hairless mice for experimental work and their sensitivity to arsenic. J. Hyg. Epidem. (Praha) 13, 1--6 (1969) Bencko, V., N~me~kovfi, H.: Oxygen consumption by mouse liver homogenate during drinking water arsenic exposure. J. Hyg. Epidem. (Praha) 15, 104 (1971) Bencko, V.: Oxygen Consumption by mouse liver homogenate during drinking water arsenic exposure. J. Hyg. Epidem. (Praha) 16, 42--46 (1972) Bencko, V., Rtssner, P., Mokr~,, M.: Dehydrogenase activity of liver parenchyma in mice exposed to arsenic in drinking water. J. Hyg. Epidem. (Praha) 19, 17--21 (1975) Bencko, V., Symon, K.: Dynamics of cumulation of arsenic in hairless mice after its application in drinking water. J. Hyg. Epidem. (Praha) 13, 245-253 (1969) Bencko, V., Dvo~fik, V., Symon, K.: Organ retention of parenterally administered arsenic (labelled with 74As)in mice preliminarily exposed to the element in drinking water. A study in arsenic tolerance. J. Hyg. Epidem. (Praha) 17, 165-168 (1973) Cikrt, M., Bencko, V.: Fate of arsenic after parenterai administration to rats with particular reference to excretion via bile. J. Hyg. Epidem. (Praba) 18, 129-136 (1974) Crawford, T. B.: Changes underdone by phenylarsenious acid and phenylarsenic acid in the animal body. Biochem. J. 41, 333 (1947) Miket'ukov~., V." Separation of arsenite and arsenate ions by paper chromatography. Study of methanol-ammonia/water system. J. Chromatogr. 34, 284-288 (1968) Mandel, H. G., Mayerdak, J. S.: The metabolisms and action of arsenite in microorganism. Fed. Proc. 21, 179 (1962) Received June 14, 1976
