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Intestinal absorption of arsenate in the chick
ENVIRONMENTAL
36, 206-217 (1985)
RESEARCH
Intestinal
Absorption
C. S. FULLMER
of Arsenate
in the Chick
AND R. H. WASSERMAN
Department of Physiology, New York State College of Veterinary Medicine, Cornell University. Ithaca, New York 14853 Accepted March 25, 1984 The intestinal absorption of arsenate(As(V)) has been investigated in the chick by means of the in situ ligated duodenal loop technique. By this procedure, it was observed that arsenate is rapidly and essentially completely absorbed @O-95%) from the lumen at As(V) concentrations up to 5 mM, declining to about 50% absorption at 50 mM. Transfer from the intestinal lumen to the mucosal cells at low As(V) concentration (0.1 IIIM) is rapid, while transfer from the mucosal cells to the body occurs more slowly. At stable As(V) concentrations greater than I IIIM, fractional mucosal cell accumulation of As(V) remains constant, while fractional transfer to the body declines. However, total mucosal accumulation of As(V) and that transferred to the body increase in a linear logarithmic fashion from 0.05 to 5 mm As(V). The results indicate that As(V) readily penetrates both the mucosal and serosal surfaces of the epithelial membrane. Furthermore, arsenate and phosphate do not appear to share a common transport pathway in the duodenum and no evidence was obtained for any interaction between the two at this level. Vitamin D, administration to rachitic chicks was effective in significantly elevating duodenal arsenate absorption, acting primarily to enhance serosal transport. 0 1985 Academic Press. Inc.
INTRODUCTION
While considerable information is available regarding the metabolism and manifestation of the toxic effects of arsenicals, less is known of the mechanisms for their intestinal absorption. Hwang and &hanker (1973) have reported on the mechanism of absorption of some organic arsenicals in rats. Solutions of carbarsone, tryparsamide, and sodium cacodylate were injected into isolated small intestinal loops of anesthetized rats and the remaining arsenic determined periodically. The results indicated that the process, in these cases, was simple diffusion and not active transport. Wasserman and Taylor (1973) examined the effects of sodium arsenate on in viva ileal phosphate absorption in chicks. In these experiments, arsenate was found to be more effective than phosphate itself in signiticantly reducing mucosal accumulation and intestinal absorption of phosphate. While no direct measurements of arsenate absorption were made, the results suggested that arsenate may act as a competitive inhibitor of phosphate absorption, although the possibility of metabolic inhibition of phosphate transport by arsenate was not excluded. Ducoff et al. (1948) injected rats, rabbits, mice, and humans with sodium arsenite and examined arsenic excretion rates and tiseue distribution patterns. Rats were found to retain most of the administered dose in the blood cells and consequently exhibited much greater body retention times than humans. Lanz et al. 0013-9351/85 $3.00 Copyright Q 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
206
INTESTINAL
ABSORPTION
OF
ARSENATE
207
(1950) observed similar results, reporting 80-90% of the arsenic bound to hemoglobin and not removable by dialysis. Complicating the understanding of arsenic metabolism are various reports relating to the biotransformation of As(II1) and As(V) to methylarsonic acid and dimethylarsinic acid in animals (Charbonneau et al., 1978, 1979; Tam et al., 1978; Vahter, 1981) and in humans (Bramen and Foreback, 1973; Crecelius, 1977; Smith et al., 1977). Significant gastrointestinal absorption of inorganic arsenic has been shown in animals (Dutkiewicz, 1977; Charbonneau et czl., 1978; Vahter and Norin, 1980) and in humans (Crecelius, 1977), although this process has not been examined in detail. The purpose of the present study was to investigate several of the parameters relating to the intestinal absorption of arsenate by the chick, as well as the relationship of phosphate to arsenate absorption, and vice-versa. MATERIALS
AND METHODS
Day-old White Leghorn cockerels were generally maintained on the appropriate diets for 3 weeks and fasted overnight prior to the absorption experiments. Measurements of arsenate absorption were performed by the in situ ligated duodenal loop procedure (Wasserman and Taylor, 1973). Chicks (-200 g) were anesthetized with ether and a laparotomy performed, exposing the duodenal segment. One end of the segment was tied securely with suture and another ligature placed loosely around the free end. A hypodermic needle was inserted through this tie, into the intestinal lumen, the ligature tightened about the needle, and 1 ml of dosing solution injected. The intestinal segment was carefully replaced in the peritoneal cavity and the incision closed with wound clips. At the termination of the recovery and absorption period, the chick was killed by intracardiac injection of sodium pentobarbital and the intact segment removed. placed in a counting vial in ice-cold saline and counted in a Beckman Gamma-300 counter for 1 min. The appropriate standard dosing solution was also counted for 1 min immediately before that of each segment to correct for counting geometry and decay. The intact segment was then removed from the vial, snipped at each end, inside the ligatures, rinsed with 50 ml saline and counted for 1 min, as before. In some cases, carcasses were counted for radioactivity in a Tobor (Nuclear Chicago) whole body counter. In these instances, a counting standard was prepared by injecting the appropriate dose into an already dead chick, this was done to correct for decay and counting geometry. 32P absorption was assessed as described below. Following the 15min absorption period, the intact ligated loop was removed, the ends were snipped, and the contents rinsed into a graduated tube with 30-35 ml of saline and diluted to 40 ml. The duodenal segment was slit lengthwise and the mucosal tissue scraped from the underlying muscle layers and placed in a counting vial with 10 ml of 1 N NaOH. The tissue was digested at room temperature for 72 hr and thoroughly dispersed by sonication. The resultant suspension was sampled (0.1 ml) and counted in a liquid scintillation counter after the addition of an appropriate counting solution. Samples were corrected for quenching and decay against the original dosing solution treated in an identical fashion. The luminal solution was vigorously mixed and 1.0-ml aliquots were dried in counting vials and counted for radioactivity as described above.
208
FULLMER
AND WASSERMAN
The following terminology is used to describe the overall process of absorption. The term “luminal efflux” refers to the amount of total dose which left the lumen during the absorption period, and is equivalent to the sum of “mucosal accumulation” and “transferred to body”. The terms “mucosal accumulation” and “percent dose in the mucosa” are equivalent to the percentage of original radioactivity in the dose which remained in the segment following rinsing, and the term “transferred to body” indicates that percentage of initial dose which left the isolated segment parenterally during the absorption period. The latter value is determined by taking the difference between the total dose injected and that remaining in the intact, unrinsed segment, or by evaluation of carcass radioactivity. 74As (an arsenic acid, 1 mCi/Fg As) and 32P (as the orthophosphate, 50 mCi/pg P) were employed as tracers and purchased from Amersham (Arlington Heights, Ill.). Statistical evaluation. The significance of all treatment differences was determined by Duncan’s New Multiple Range Test (Steel and Torrie, 1960). RESULTS
Experiment 1. Retention of 74As To determine the retention of 74As with time after a single orally administered dose of radionuclide, 3-week-old normal White Leghorn cockerels (two chicks per treatment) were dosed by gastric intubation with 1 ml of one of the following solutions: (a) 1.0 mM Na,HAsO,, 0.5 t&i 74As, in saline, pH 7.4; (b) 1.0 mM Na,HPO,, 0.5 PCi 74As, in saline, pH 7.4. The chicks were then subjected to whole body counting at various times following the dose. All values were corrected for decay and counting geometry by orally dosing and immediately killing a “zero time” control bird. Orally administered 74As was rapidly excreted from the chick regardless of whether it was administered with 1 mM stable arsenate or I mM phosphate (Fig. 1). Whole-body levels of 74As were reduced to less than 50% after 7 hr and to about 7% after 24 hr.
m-
\ a***~-.~\@
10 II Oo 2 4
I 8
I 12
1 24 POST-DOSE
----------‘------s--m-e-, 46 TIME
(hr)
FIG. 1. Retention of 74As by the chick, as a function of time following a single oral dose. Stable carrier was either I mM Na,HAsO, (-), or 1 mM Na,HPO, (- - -). Values represent the means of 2 chicks for each dosing solution.
INTESTINAL
Experiment
2. Duodenal
ABSORPTION
Absorption
209
OF ARSENATE
of 74As as a Function
of Time
Chicks were maintained on the nutritionally adequate semisynthetic diet for 3 weeks prior to the absorption experiment. Ligated duodenal segments were prepared as described and injected with 1 ml of the following solution; 150 mM NaCl, 0.1 mM Na,HAsO, 0.1 pCi 74As, pH 7.4. Following injection of the dose, absorption periods of 0 (injection of dose into a previously-sacrificed bird), 5, 10, 15, 30, and 60 min were allowed. Chicks were killed and the duodenal segments processed as described above. The results (Fig. 2) show that the luminal efflux of the administered dose of 74As occurred rapidly, plateauing at the 90% level after only 15 min. Within the first 5 min, mucosal accumulation constituted the major component of absorption, reaching a maximum at 15 min and declining thereafter. Transfer of 74As to body occurred more slowly than mucosal accumulation, indicating a time delay in transfer of 74As from the apical region of the intestinal cell to its exit across the basal lateral membrane and this is consistent with a two-step process of 74As absorption-accumulation and subsequent release of the radionuclide from the enterocyte. For subsequent studies, a 15-min absorption period was chosen since, at this time, the transfer to body is increasing, mucosal accumulation is maximal and luminal efflux has approached a plateau value. Experiment 3. Effect of Varying Levels of Stable As(V) on the Duodenal Absorption of 74As Chicks maintained on the nutritionally adequate semisynthetic diet for 3 weeks were dosed, via the ligated duodenal loop procedure, with 1 ml of solution containing 150 mM NaCl, 0.1 t&i 74As, and either 0, 0.05, 0.10, 0.15, 1 .O, 2.0, 5.0, 10.0, 20.0, or 50.0 mM Na,HAsO,. The dosing solutions were adjusted to pH 7.4; the absorption period was 15 min.
100 90 60 70 60 a
x
50
;;140 iz a@
MUCOSAL
ACCUMULATION
a----P
30 20 10 0
0
5
lo
15 ABSORPTION
30 TIME
60 (mtn)
FIG. 2. Absorption of 74As by the ligated duodenal loop procedure as a function of absorption time. Each point represents the mean -c SEM of 5 chicks. The dose contained 0.1 mM stable As(V) (as Na,HAsO,).
210
FULLMER
AND WASSERMAN
A linear representation of the effect of stable arsenate in the dosing solution on 74As absorption and mucosal accumulation is presented in Fig. 3. At the lower stable As(V) concentrations (below 1.0 mM>, a significant accumulation of 74As by the mucosal tissue was apparent. As stable As(V) was increased from 0 to 1.0 mM, a greater percentage of the administered dose was transported to the body. Above 2 mM, and certainly above 5 mM As(V) in the dosing solution, a significant reduction occurred in the percentage 74As transferred to body, while only a slight reduction was observed for mucosal accumulation, even at the 50 mM As(V) level. Logarithmic representation of the arsenate (based on 74As levels) accumulated in the mucosa and transferred to the body is shown in Fig. 4. The log of the total mucosal accumulation of As(V) appears to be a relatively linear function of the log of the arsenate concentration in the dosing solution, at least from the 0.1 to 50 mM As(V) level. Slight deviation of points from the line at lower arsenate concentrations suggest an enhanced accumulation at these levels. Total transfer to body of As(V) also appears to be a log-linear function of arsenate in the dosing solution from the lowest level (.05 mM) to between 2.0 to 5.0 mM. At this point, total transfer remains unchanged over a four-fold concentration increase (5 to 20 mM As(V)). Transfer appears to increase again at 20 to 50 mM As(V). Experiment 4. Effect of Stable Phosphate on the Duodenal Absorption of 74As The structural similarities between phosphate and arsenate suggest the possibility that they may also share similarities in their modes of intestinal absorption and metabolism. In order to examine the possible interaction between P(V) and As(V) absorption, the previous 74As absorption experiment was repeated except the effect of different levels of stable phosphate included in doses containing no added stable arsenate were studied. Rather different results were observed when phosphate replaced stable arsenate (Fig. 5). At the lower leveis of phosphate, no significant effect on the transfer of 74As to body above the zero As(V) control
0
012345
”
”
’ ARSENIC
CONCE&7ATION
(mm)
FIG. 3. Absorption of 74As as a function of stable As(V) (as Na,HAsO,). mean 2 SEM for 5 chicks. Time of absorption was 15 min.
Each
Point represents the
INTESTINAL
ABSORPTION
211
OF ARSENATE
ACCUMULATKWd
o.l.05-
.Ol .Ol
I 50
.5ol.o2.0 ARSENATE
CONCENTRATION
I la0
I 2cm
1 5on
ImMl
FIG. 4. Full logarithmic representation of data depicted in Fig. 3. based on total As(V) concentration in the loops. Experimental conditions were identical to those described in the legend to Fig. 3.
levels occurred nor were there changes in the dose accumulated in the mucosal tissue (compare 0- 1.O mM As(V) and P(V) levels in Figs. 3 and 5). The percentage dose in the mucosa did decline somewhat over the concentration range 0 to 20 mM P(V) and that transferred to body became slightly elevated, but these effects were minimal. There was a significant decline in all parameters between 20 and 50 mM stable P(V).
I
100
I
1
I
1
-
k *.w----
s-
A
_ --
8D]+--a-..
_______
T l I
pLlJMlNAL ---
------+--m z4
------_____
70ao-
----mm__ ,x ____ ~,w~OSAL ---
EFFLUX ---
---_
ACCUMULATION ---- -______ ____
zw0 z 40-
--•
T
----
--_____.
L\ I* ‘\
yTRANSFER
TO
-
BODY
I 3.
,D’y/’ Oo
-
:,\ ‘* 2k’;P
3020 -
-
n
% 1
2
I 3
I 4
I 5
10 PHOSPHOROUS
CONCENTRATION
-
26 m50 (2)
FIG. 5. Absorption of 74As as a function of stable P(V) (as Na,HPO,). Each point represents the mean ? SEM for 5 chicks. Time of absorption was 15 min and As(V) concentration was at tracer levels.
212
FULLMER
AND
WASSERMAN
Experiment 5. Effect of Varying Concentrations Arsenate on Duodenal Absorption of 74As
of Stable Phosphate
and
The absorption of 74As as a function of varying levels and concentrations of stable arsenate and phosphate in the dosing solution was examined using six chicks per treatment group. A 15min absorption period was allowed. It is apparent that stable phosphate, at the levels employed, produced no noticeable effect on 74As absorption at any of the stable arsenate levels (Table 1). On the other hand, it is evident that the elevation of arsenate in the dosing solution produced an obvious elevation in the percentage of 74As transferred to body with a concomitant decline in percentage 74As remaining in the mucosal tissue. These results corroborate the findings of the previous experiment and supply additional evidence of a differential effect of As(V) and P(V) on 74As absorption. Experiment 6. Effect of Stable Arsenate and Phosphate on Duodenal Absorption of 74As and 32P 74As and 32P absorption were determined by the ligated duodenal loop procedure as described under Materials and Methods. Several of the preceeding experiments indicated that arsenate absorption (mucosal accumulation and transfer to the body) is not significantly affected by stable phosphate in the solution. The present experiment (Table 2) again confirms those results (at a single phosphate concentration) and also suggests that stable arsenate does not influence phosphate absorption. Experiment 7. Effects of Vitamin D,, calcium, and Phosphorus on the Duodenal Absorption of 74As Day-old White Leghorn cockerels were maintained for a period of 2 weeks on a semisynthetic diet either deficient or adequate in vitamin D, (1200 IU/kg). At the end of this period the dietary treatment groups (6 chicks each) were continued for 1 week, as follows:
ABSORPTION Dose
OF 74As AS A FUNCTION
TABLE 1 OF STABLE As(V)
(mM)
P(V)
AND
IN THE DOSING
SOLUTION
74As (% dose)
As
P
0 0 0.1 0.1 1.0 1.0
0 1.0 0 1.0 0 1.0
Transferred to body 10.24 12.14 28.08 29.39 52.71 55.73
t4 2 k t +-
1.65” 0.72” 1.40” 1.29b 4.63‘ 2.42’
Mucosal accumulation 81.41 78.78 60.10 57.52 32.92 28.90
f 2 _’ 2 k f
Luminal efflux
3.71” 0.80” 1.4gh 1.69b 1.42” 0.94’
Note. Values represent means _’ SEM for 6 chicks. Values within same letter superscript are not significantly different (P 5 0.05).
91.65 90.92 87.67 86.91 85.62 84.64
the same column
2 2 t 2 ” 4
2.11” 1.21” 1.49”,b 2.15”.’ 4.24”,h 1.80” and having
the
INTESTINAL
ABSORPTION TABLE
ABSORITION
OF
14As AND
32P AS A FUNCTION
213
OF ARSENATE 2
OF STABLE
As(V)
AND
P(V)
IN THE DOSE
(% Dose) Mucosal accumulation
Luminal efflux
Treatment
Transferred to body
‘4AS 1.0 mM As 1.0 mM P
59.73 4 2.05” 12.94 rf~ 0.74b
31.38 ” 0.86” 83.26 5 1.696
91.11 2 1.27” 96.20 k 2.14a
QP 1.O mM As 1.0 mM P
38.01 2 1.60’ 37.98 k 2.94’
56.71 -r- 0.41c 57.26 2 2.99’
94.12 ‘- 1.41“ 95.24 2 1.26”
Note. Values represent the means ? SEM for 6 chicks. Values within the same column and having the same letter superscript are not significantly different (P G 0.05).
(a) semisynthetic diet adequate in vitamin D,, (1200 IU/kg), calcium (1.2%), and phosphorus (0.7%) (control diet); (6) semisynthetic diet adequate in calcium (1.2%) and phosphorus (0.7%), but containing no vitamin D, (rachitic diet); (c) as in (b) above, but chicks were dosed with 500 IU vitamin D, (im) 48 hr prior to the absorption experiment; (6) semisynthetic diet adequate in vitamin D, (1200 IU/kg) and phosphorus (0.7%) but containing 0.1% calcium (low-calcium diet); (e) semisynthetic diet adequate in vitamin D, (1200 IU/kg) and calcium (1.2%) but containing 0.2% phosphorus (low phosphorus diet). 74As absorption data was generated as previously described, following a 30min absorption period. The dosing solution (1.0 ml) contained 0.5 mM stable arsenate as Na,HAsO,. Chicks raised on diets deficient in vitamin D, exhibited considerably less transfer to body of 74As than animals maintained on the control diet (Table 3). There was, however, no difference in mucosal accumulation of 74As as a result of dietary vitamin D, supplementation. Administration of 500 IU vitamin D, (im), 48 hr prior to the absorption experiment appeared to enhance the percentage of 74As transferred to body by both loop measurements and carcass counts, but neither measurement produced statistically significant differences, owing to the large individual variation within treatment groups. Feeding diets low in calcium or phosphorus resulted in no statistically significant differences from the control groups in either component of absorption (Table 3). The effect of vitamin D, administration on 74As absorption was further investigated in chicks maintained for 3 weeks on a semisynthetic diet deficient in vitamin D,. At 24, 48, and 72 hr prior to the 74As absorption experiment, chicks were dosed with 500 IU vitamin D, (im). Absorption experiments were conducted as described previously. The dosing solution (1 .O ml) contained 0.5 mM stable arsenate as Na,HAsO,. Results of the previous experiment indicated that dietary vitamin D, status
214
FULLMER
AND WASSERMAN
TABLE 3 EFFECTOFDIETAKY VITAMIN D,, LOWCALCIUM,AND Low PHOSPHORUSONTHE ABSORPTION OF 74As 74As (% Dose) Transferred to body
Treatment Control diet Rachitic Rachitic + 500 IU D, (48 hr) Low Ca Low P
Mucosal accumulation
Luminal efflux
In carcass
49.11 _’ 1.83” 26.90 2 3.89b
40.34 2 1.17” 41.43 k 3.10”
90.09 2 1.02” 68.25 4 6.0gb
47.30 t 4.81” 28.36 t 4.70b
34.61 k 3.21b 47.11 k 3.25” 44.06 f 1.13”
41.76 k 1.88” 40.40 k 2.75” 40.22 2 1.91”
76.37 2 2.93b 87.86 2 1.23” 84.29 +- 1.44”
38.13 ? 3.6Tb 49.30 k 4.28” 48.23 _’ 1.29“
Note. Values represent the means * SEM for 6 chicks. Values within the same column and having the same letter superscript are not significantly different (P < 0.05).
influenced 74As absorption and, perhaps, a single im dose of 500 IU D,, 48 hr prior to the absorption experiment, may have also produced an effect. In the present experiment a time range of dosing from 0 to 72 hr was investigated (Table 4). Again, no statistically significant change in the components of absorption was noted at 48 hr following the dose. At 72 hr following the im dose of vitamin D,, a rather large elevation of 74As transferred to the body occurred, this in spite of a constant mucosal arsenate accumulation. DISCUSSION
The results presented herein clearly establish that arsenate (As(V)) is rapidly and extensively absorbed from the intestinal tract of the chick and, thereafter, rapidly eliminated from the body. In this latter respect, the chick appears to be a more suitable animal model for the study of arsenate absorption in humans than is the rat, where considerable arsenate is retained for an extended time period (see Ducoff et al., 1948; Lanz et al., 1950). At a relatively low concentration of stable As(V), 74As was rapidly and comTABLE 4 EFFECT OF VITAMIN D, ADMINISTRATION ON 74As ABSORPTION 74As (% Dose) Treatment Rachitic Rachitic + 500 IU D, (24 hr) Rachitic + 500 IU D, (48 hr) Rachitic + 500 IU D, (72 hr)
Transferred to body
Mucosal accumulation
Luminal efflux
29.96 rt 2.52O
42.69 I 1.53”
72.65 5 3.00”
33.73 -c I.700
38.57 k 1.80b
72.31 2 2.74O
32.92 -t 2.91a
38.23 ” 0.31b
71.16 k 3.04O
45.54 f 1.60b
37.53 2 l.Olb
83.08 k 1.40b
Note. Values represent the means ? SEM for 6 chicks. Values within the same column and having the same letter superscript are not significantly different (P G 0.05).
INTESTINAL
ABSORPTION
OF
ARSENATE
215
pletely absorbed (90% of the dose left the lumen in 15 min) from the duodenum. The overall absorption was easily separated into two components (Fig. 2); mucosal tissue accumulation constituted the rapid component, while transfer of 74As to the body occurred more slowly and apparently represents the rate-limiting phase of absorption, under these conditions. At increasing concentrations of stable luminal arsenate from 0 to 5 mM (Fig. 3), overall 74As luminal efflux remained relatively constant (80-90%), however, dramatic changes in the two absorption components occurred. 74As transferred to the body increased at the 1 mM stable As(V) level, plateaued between 1 and 5 mM, and declined thereafter. Between 0 and 1 mM As(V) there was a decline in fractional mucosal tissue accumulation of 74As from 70 to 40%, a constant value which was maintained to the 50 mM level of stable luminal As(V), and this in spite of a 50-fold increase in total accumulated arsenate (Fig. 4). These results indicate that, at the lower levels of stable As(V), 74As becomes sequestered primarily in or on the mucosal tissue and is transferred to the body only minimally during 15 min. As the stable luminal As(V) concentration is elevated to 1 mM, these sites of sequestration, or compartments, become tilled and, eventually, overloaded with concomitant movement of 74As to the body. The number of such sites must not be extensive since they appear to be saturated at or about 1 mM stable As(V), corresponding to a total As(V) level of 0.3 to 0.4 kmole over the entire segment length. Above 1 mM and extending to 50 mM stable As(V) the percentage of 74As retained by the mucosal tissue is not appreciably diminished, despite a 50-fold enhancement of in total accumulated arsenate (Fig. 4). Coincidentally, the percent of dose transferred to body declines considerably (to about IO%, Fig. 3), even though the total concentration of As(V) in the mucosal tissue has reached about 15 kmole over the segment length. Between 2 and 50 mM stable As(V), however, total arsenate @moles) transferred to body lags considerably behind macosal accumulation. This may represent saturation of serosal transport components or the effects of high arsenate concentration on metabolic processes. The maintenance of constant fractional mucosal As(V) accumulation in the face of concomitant elevation in total mucosal tissue concentration is difficult to explain. Perhaps, above the 1 mM level of accumulated arsenate, additional sets of arsenate-sequestering sites become available, which are more extensive in number or capacity than those sites sequestering arsenate below the 1 mM level. The experiments presented here do not eliminate the possibility that As(V) (above the 1 mM stable luminal level) is simply moving down a concentration gradient from the lumen to the mucosal tissue, and this may well be the case. At any rate, it is apparent that the duodenal mucosal tissue does not present a substantial limiting barrier to the movement of arsenate. On the basis of results presented here, it appears that, despite structural similarities, arsenate and phosphate do not share a common transport pathway in the duodenum. The results (Table 1) indicate that, while 74As transferred to the body is elevated by increasing stable As(V) from 0 to 1.0 mM, no such enhancement is noted for increasing the stable P(V) concentration. Whereas 1.O mM stable As(V) is effective in significantly elevating 74As transferred to the body and re-
216
FULLMER
AND WASSERMAN
ducing mucosal 74As accumulation compared to the 0 mM As(V) control values, 1.0 mM P(V) is not effective in this regard (Tables 1 and 2). Also, there was no significant change in duodenal 32P absorption in the presence of 1.O mM As(V) (Table 3). These experiments clearly establish differences in duodenal P(V) and As(V) absorption and the differential effects of these two anions are further evidenced by comparison of Figs. 3 and 5. Elevation of stable phosphate from 0 to 20 mM in the dosing solution produced little effect on the transfer of 74As to the body, whereas a modest decline in mucosal accumulation occurred (Fig. 5). Significant declines in both components of absorption occurred at 50 mM stable phosphate, possibly as the result of direct inhibition of As(V) transport or, more likely, toxicity resulting at such high P(V) levels. Wasserman and Taylor (1973) have shown that arsenate inhibits ileal phosphate absorption at concentrations as low as 2 mM and, indeed, was a more effective inhibitor than phosphate itself. This effect was more pronounced in vitamin Dreplete than rachitic chicks, suggesting that the inhibitory action is directed primarily against the vitamin D,-responsive component of ileal phosphate transport. The present studies indicate no substantial inhibitory effect of phosphate on either component of duodenal 74As absorption until between 20 and 50 mM stable luminal phosphate. The interaction between these two ions noted by Wasserman and Taylor (1973) may be confined to the ileal tissue. Duodenal arsenate absorption was significantly influenced by vitamin D status of the chicks. This effect was most clearly demonstrated by comparing vitamin D-deficient and normal animals (Table 3) where vitamin D3-depletion resulted in a 45% decline in 74As transferred to the body. There was no sign&ant alteration in mucosal accumulation of 74As. Acute administration of vitamin D, (500 IU) required 72 hr to effect a significant elevation of 74As transferred to body and, perhaps, a slight decrease in mucosal accumulation. Wasserman and Taylor (1973) reported a significant elevation in both components of phosphate transport in chick ileum only 48 hr following a similar dose of vitamin D,. The differences noted between that study and the present work may be tissue-related or may provide another example of noncorrelation between intestinal arsenate and phosphate transport. ACKNOWLEDGMENTS This work was supported by Contract EV-S-2792 from the U.S. Department of Energy. The excellent technical assistance of Steven Maxwell is acknowledged.
REFERENCES Bramen, R. S., and Foreback, C. C. (1973). Methylated forms of arsenic in the environment. Science (Washington, D.C.) 182, 1247-1249. Charbonneau, S. M., Spencer, K., Bryce, F., and Sandi, E. (1978). Arsenate excretion by monkeys dosed with arsenic-containing fish and with inorganic arsenate. Bull. Environ. Contam. Toxicol. 20, 470-477. Charbonneau, S. M., Tam, G. K. H., Bryce, F., Zawidzka, Z., and Sandi, E. (1979). Metabolism of orally administered inorganic arsenic in the dog. Toxicol. Let?. 3, 107-l 13. Crecelius, E. A. (1977). Changes in the chemical speciation of arsenic following ingestion by man. EHP, Environ Health Perspect. 19, 147- 150.
INTESTINAL
ABSORPTION
OF ARSENATE
217
Ducoff, H. S., Neal, W. B., Straube, R. L., Jackson, L. O., and Brues, A. M. (1948). Biological studies with arsenic excretion and tissue localization. Proc. Sot. Exp. Biol. Med. 69, 548-554. Dutkiewicz, T. (1977). Experimental studies on arsenic absorption routes in rats. EHP, Environ. Health Perspect. 19, 173-177. Hwang, S. W.. and Schanker, L. S. (1973). Absorption or organic arsenical compounds from the rat small intestine. Xenobiotica 3, 351-355. Lanz. H., Wallace, P. G., and Hamilton, J. G. (1950). The metabolism of arsenic in laboratory animals with As’~ as a tracer. Univ. Calif. Pub!. Pharmacol. 2, 263-282. Smith, T. J.. Crecelius, E. A., and Reading, J. C. (1977). Airborne arsenic exposure and excretion of methylated arsenic compounds. EHP, Environ. Health Perspect. 19, 89-93. Steel, R. G. D., and Torrie, J. H. (1960). “Principles and Procedures of Statistics.” McGraw-Hill, New York. Tam, K. H., Charbonneau, S. M., Bryce, F., and Lacroix, G. (1978). Separation of arsenic metabolites in dog plasma and urine following intravenous injection of 74As. Anal. Biochem. 86, 505-511. Vahter. M., and Norin, H. (1980). Metabolism of 74As-labeled trivalent and pentavalent inorganic arsenic in mice. Environ. Res. 21, 446-457. Vahter, M. (1981). Biotransformation of trivalent and pentavalent inorganic arsenic in mice and rats. Environ.
Res.
25, 286-293.
Wasserman, R. H., and Taylor, A. N. (1973). Intestinal absorption of phosphate in the chick: Effect of vitamin D and other parameters. J. Nutr. 103, 586-599.
36, 206-217 (1985)
RESEARCH
Intestinal
Absorption
C. S. FULLMER
of Arsenate
in the Chick
AND R. H. WASSERMAN
Department of Physiology, New York State College of Veterinary Medicine, Cornell University. Ithaca, New York 14853 Accepted March 25, 1984 The intestinal absorption of arsenate(As(V)) has been investigated in the chick by means of the in situ ligated duodenal loop technique. By this procedure, it was observed that arsenate is rapidly and essentially completely absorbed @O-95%) from the lumen at As(V) concentrations up to 5 mM, declining to about 50% absorption at 50 mM. Transfer from the intestinal lumen to the mucosal cells at low As(V) concentration (0.1 IIIM) is rapid, while transfer from the mucosal cells to the body occurs more slowly. At stable As(V) concentrations greater than I IIIM, fractional mucosal cell accumulation of As(V) remains constant, while fractional transfer to the body declines. However, total mucosal accumulation of As(V) and that transferred to the body increase in a linear logarithmic fashion from 0.05 to 5 mm As(V). The results indicate that As(V) readily penetrates both the mucosal and serosal surfaces of the epithelial membrane. Furthermore, arsenate and phosphate do not appear to share a common transport pathway in the duodenum and no evidence was obtained for any interaction between the two at this level. Vitamin D, administration to rachitic chicks was effective in significantly elevating duodenal arsenate absorption, acting primarily to enhance serosal transport. 0 1985 Academic Press. Inc.
INTRODUCTION
While considerable information is available regarding the metabolism and manifestation of the toxic effects of arsenicals, less is known of the mechanisms for their intestinal absorption. Hwang and &hanker (1973) have reported on the mechanism of absorption of some organic arsenicals in rats. Solutions of carbarsone, tryparsamide, and sodium cacodylate were injected into isolated small intestinal loops of anesthetized rats and the remaining arsenic determined periodically. The results indicated that the process, in these cases, was simple diffusion and not active transport. Wasserman and Taylor (1973) examined the effects of sodium arsenate on in viva ileal phosphate absorption in chicks. In these experiments, arsenate was found to be more effective than phosphate itself in signiticantly reducing mucosal accumulation and intestinal absorption of phosphate. While no direct measurements of arsenate absorption were made, the results suggested that arsenate may act as a competitive inhibitor of phosphate absorption, although the possibility of metabolic inhibition of phosphate transport by arsenate was not excluded. Ducoff et al. (1948) injected rats, rabbits, mice, and humans with sodium arsenite and examined arsenic excretion rates and tiseue distribution patterns. Rats were found to retain most of the administered dose in the blood cells and consequently exhibited much greater body retention times than humans. Lanz et al. 0013-9351/85 $3.00 Copyright Q 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
206
INTESTINAL
ABSORPTION
OF
ARSENATE
207
(1950) observed similar results, reporting 80-90% of the arsenic bound to hemoglobin and not removable by dialysis. Complicating the understanding of arsenic metabolism are various reports relating to the biotransformation of As(II1) and As(V) to methylarsonic acid and dimethylarsinic acid in animals (Charbonneau et al., 1978, 1979; Tam et al., 1978; Vahter, 1981) and in humans (Bramen and Foreback, 1973; Crecelius, 1977; Smith et al., 1977). Significant gastrointestinal absorption of inorganic arsenic has been shown in animals (Dutkiewicz, 1977; Charbonneau et czl., 1978; Vahter and Norin, 1980) and in humans (Crecelius, 1977), although this process has not been examined in detail. The purpose of the present study was to investigate several of the parameters relating to the intestinal absorption of arsenate by the chick, as well as the relationship of phosphate to arsenate absorption, and vice-versa. MATERIALS
AND METHODS
Day-old White Leghorn cockerels were generally maintained on the appropriate diets for 3 weeks and fasted overnight prior to the absorption experiments. Measurements of arsenate absorption were performed by the in situ ligated duodenal loop procedure (Wasserman and Taylor, 1973). Chicks (-200 g) were anesthetized with ether and a laparotomy performed, exposing the duodenal segment. One end of the segment was tied securely with suture and another ligature placed loosely around the free end. A hypodermic needle was inserted through this tie, into the intestinal lumen, the ligature tightened about the needle, and 1 ml of dosing solution injected. The intestinal segment was carefully replaced in the peritoneal cavity and the incision closed with wound clips. At the termination of the recovery and absorption period, the chick was killed by intracardiac injection of sodium pentobarbital and the intact segment removed. placed in a counting vial in ice-cold saline and counted in a Beckman Gamma-300 counter for 1 min. The appropriate standard dosing solution was also counted for 1 min immediately before that of each segment to correct for counting geometry and decay. The intact segment was then removed from the vial, snipped at each end, inside the ligatures, rinsed with 50 ml saline and counted for 1 min, as before. In some cases, carcasses were counted for radioactivity in a Tobor (Nuclear Chicago) whole body counter. In these instances, a counting standard was prepared by injecting the appropriate dose into an already dead chick, this was done to correct for decay and counting geometry. 32P absorption was assessed as described below. Following the 15min absorption period, the intact ligated loop was removed, the ends were snipped, and the contents rinsed into a graduated tube with 30-35 ml of saline and diluted to 40 ml. The duodenal segment was slit lengthwise and the mucosal tissue scraped from the underlying muscle layers and placed in a counting vial with 10 ml of 1 N NaOH. The tissue was digested at room temperature for 72 hr and thoroughly dispersed by sonication. The resultant suspension was sampled (0.1 ml) and counted in a liquid scintillation counter after the addition of an appropriate counting solution. Samples were corrected for quenching and decay against the original dosing solution treated in an identical fashion. The luminal solution was vigorously mixed and 1.0-ml aliquots were dried in counting vials and counted for radioactivity as described above.
208
FULLMER
AND WASSERMAN
The following terminology is used to describe the overall process of absorption. The term “luminal efflux” refers to the amount of total dose which left the lumen during the absorption period, and is equivalent to the sum of “mucosal accumulation” and “transferred to body”. The terms “mucosal accumulation” and “percent dose in the mucosa” are equivalent to the percentage of original radioactivity in the dose which remained in the segment following rinsing, and the term “transferred to body” indicates that percentage of initial dose which left the isolated segment parenterally during the absorption period. The latter value is determined by taking the difference between the total dose injected and that remaining in the intact, unrinsed segment, or by evaluation of carcass radioactivity. 74As (an arsenic acid, 1 mCi/Fg As) and 32P (as the orthophosphate, 50 mCi/pg P) were employed as tracers and purchased from Amersham (Arlington Heights, Ill.). Statistical evaluation. The significance of all treatment differences was determined by Duncan’s New Multiple Range Test (Steel and Torrie, 1960). RESULTS
Experiment 1. Retention of 74As To determine the retention of 74As with time after a single orally administered dose of radionuclide, 3-week-old normal White Leghorn cockerels (two chicks per treatment) were dosed by gastric intubation with 1 ml of one of the following solutions: (a) 1.0 mM Na,HAsO,, 0.5 t&i 74As, in saline, pH 7.4; (b) 1.0 mM Na,HPO,, 0.5 PCi 74As, in saline, pH 7.4. The chicks were then subjected to whole body counting at various times following the dose. All values were corrected for decay and counting geometry by orally dosing and immediately killing a “zero time” control bird. Orally administered 74As was rapidly excreted from the chick regardless of whether it was administered with 1 mM stable arsenate or I mM phosphate (Fig. 1). Whole-body levels of 74As were reduced to less than 50% after 7 hr and to about 7% after 24 hr.
m-
\ a***~-.~\@
10 II Oo 2 4
I 8
I 12
1 24 POST-DOSE
----------‘------s--m-e-, 46 TIME
(hr)
FIG. 1. Retention of 74As by the chick, as a function of time following a single oral dose. Stable carrier was either I mM Na,HAsO, (-), or 1 mM Na,HPO, (- - -). Values represent the means of 2 chicks for each dosing solution.
INTESTINAL
Experiment
2. Duodenal
ABSORPTION
Absorption
209
OF ARSENATE
of 74As as a Function
of Time
Chicks were maintained on the nutritionally adequate semisynthetic diet for 3 weeks prior to the absorption experiment. Ligated duodenal segments were prepared as described and injected with 1 ml of the following solution; 150 mM NaCl, 0.1 mM Na,HAsO, 0.1 pCi 74As, pH 7.4. Following injection of the dose, absorption periods of 0 (injection of dose into a previously-sacrificed bird), 5, 10, 15, 30, and 60 min were allowed. Chicks were killed and the duodenal segments processed as described above. The results (Fig. 2) show that the luminal efflux of the administered dose of 74As occurred rapidly, plateauing at the 90% level after only 15 min. Within the first 5 min, mucosal accumulation constituted the major component of absorption, reaching a maximum at 15 min and declining thereafter. Transfer of 74As to body occurred more slowly than mucosal accumulation, indicating a time delay in transfer of 74As from the apical region of the intestinal cell to its exit across the basal lateral membrane and this is consistent with a two-step process of 74As absorption-accumulation and subsequent release of the radionuclide from the enterocyte. For subsequent studies, a 15-min absorption period was chosen since, at this time, the transfer to body is increasing, mucosal accumulation is maximal and luminal efflux has approached a plateau value. Experiment 3. Effect of Varying Levels of Stable As(V) on the Duodenal Absorption of 74As Chicks maintained on the nutritionally adequate semisynthetic diet for 3 weeks were dosed, via the ligated duodenal loop procedure, with 1 ml of solution containing 150 mM NaCl, 0.1 t&i 74As, and either 0, 0.05, 0.10, 0.15, 1 .O, 2.0, 5.0, 10.0, 20.0, or 50.0 mM Na,HAsO,. The dosing solutions were adjusted to pH 7.4; the absorption period was 15 min.
100 90 60 70 60 a
x
50
;;140 iz a@
MUCOSAL
ACCUMULATION
a----P
30 20 10 0
0
5
lo
15 ABSORPTION
30 TIME
60 (mtn)
FIG. 2. Absorption of 74As by the ligated duodenal loop procedure as a function of absorption time. Each point represents the mean -c SEM of 5 chicks. The dose contained 0.1 mM stable As(V) (as Na,HAsO,).
210
FULLMER
AND WASSERMAN
A linear representation of the effect of stable arsenate in the dosing solution on 74As absorption and mucosal accumulation is presented in Fig. 3. At the lower stable As(V) concentrations (below 1.0 mM>, a significant accumulation of 74As by the mucosal tissue was apparent. As stable As(V) was increased from 0 to 1.0 mM, a greater percentage of the administered dose was transported to the body. Above 2 mM, and certainly above 5 mM As(V) in the dosing solution, a significant reduction occurred in the percentage 74As transferred to body, while only a slight reduction was observed for mucosal accumulation, even at the 50 mM As(V) level. Logarithmic representation of the arsenate (based on 74As levels) accumulated in the mucosa and transferred to the body is shown in Fig. 4. The log of the total mucosal accumulation of As(V) appears to be a relatively linear function of the log of the arsenate concentration in the dosing solution, at least from the 0.1 to 50 mM As(V) level. Slight deviation of points from the line at lower arsenate concentrations suggest an enhanced accumulation at these levels. Total transfer to body of As(V) also appears to be a log-linear function of arsenate in the dosing solution from the lowest level (.05 mM) to between 2.0 to 5.0 mM. At this point, total transfer remains unchanged over a four-fold concentration increase (5 to 20 mM As(V)). Transfer appears to increase again at 20 to 50 mM As(V). Experiment 4. Effect of Stable Phosphate on the Duodenal Absorption of 74As The structural similarities between phosphate and arsenate suggest the possibility that they may also share similarities in their modes of intestinal absorption and metabolism. In order to examine the possible interaction between P(V) and As(V) absorption, the previous 74As absorption experiment was repeated except the effect of different levels of stable phosphate included in doses containing no added stable arsenate were studied. Rather different results were observed when phosphate replaced stable arsenate (Fig. 5). At the lower leveis of phosphate, no significant effect on the transfer of 74As to body above the zero As(V) control
0
012345
”
”
’ ARSENIC
CONCE&7ATION
(mm)
FIG. 3. Absorption of 74As as a function of stable As(V) (as Na,HAsO,). mean 2 SEM for 5 chicks. Time of absorption was 15 min.
Each
Point represents the
INTESTINAL
ABSORPTION
211
OF ARSENATE
ACCUMULATKWd
o.l.05-
.Ol .Ol
I 50
.5ol.o2.0 ARSENATE
CONCENTRATION
I la0
I 2cm
1 5on
ImMl
FIG. 4. Full logarithmic representation of data depicted in Fig. 3. based on total As(V) concentration in the loops. Experimental conditions were identical to those described in the legend to Fig. 3.
levels occurred nor were there changes in the dose accumulated in the mucosal tissue (compare 0- 1.O mM As(V) and P(V) levels in Figs. 3 and 5). The percentage dose in the mucosa did decline somewhat over the concentration range 0 to 20 mM P(V) and that transferred to body became slightly elevated, but these effects were minimal. There was a significant decline in all parameters between 20 and 50 mM stable P(V).
I
100
I
1
I
1
-
k *.w----
s-
A
_ --
8D]+--a-..
_______
T l I
pLlJMlNAL ---
------+--m z4
------_____
70ao-
----mm__ ,x ____ ~,w~OSAL ---
EFFLUX ---
---_
ACCUMULATION ---- -______ ____
zw0 z 40-
--•
T
----
--_____.
L\ I* ‘\
yTRANSFER
TO
-
BODY
I 3.
,D’y/’ Oo
-
:,\ ‘* 2k’;P
3020 -
-
n
% 1
2
I 3
I 4
I 5
10 PHOSPHOROUS
CONCENTRATION
-
26 m50 (2)
FIG. 5. Absorption of 74As as a function of stable P(V) (as Na,HPO,). Each point represents the mean ? SEM for 5 chicks. Time of absorption was 15 min and As(V) concentration was at tracer levels.
212
FULLMER
AND
WASSERMAN
Experiment 5. Effect of Varying Concentrations Arsenate on Duodenal Absorption of 74As
of Stable Phosphate
and
The absorption of 74As as a function of varying levels and concentrations of stable arsenate and phosphate in the dosing solution was examined using six chicks per treatment group. A 15min absorption period was allowed. It is apparent that stable phosphate, at the levels employed, produced no noticeable effect on 74As absorption at any of the stable arsenate levels (Table 1). On the other hand, it is evident that the elevation of arsenate in the dosing solution produced an obvious elevation in the percentage of 74As transferred to body with a concomitant decline in percentage 74As remaining in the mucosal tissue. These results corroborate the findings of the previous experiment and supply additional evidence of a differential effect of As(V) and P(V) on 74As absorption. Experiment 6. Effect of Stable Arsenate and Phosphate on Duodenal Absorption of 74As and 32P 74As and 32P absorption were determined by the ligated duodenal loop procedure as described under Materials and Methods. Several of the preceeding experiments indicated that arsenate absorption (mucosal accumulation and transfer to the body) is not significantly affected by stable phosphate in the solution. The present experiment (Table 2) again confirms those results (at a single phosphate concentration) and also suggests that stable arsenate does not influence phosphate absorption. Experiment 7. Effects of Vitamin D,, calcium, and Phosphorus on the Duodenal Absorption of 74As Day-old White Leghorn cockerels were maintained for a period of 2 weeks on a semisynthetic diet either deficient or adequate in vitamin D, (1200 IU/kg). At the end of this period the dietary treatment groups (6 chicks each) were continued for 1 week, as follows:
ABSORPTION Dose
OF 74As AS A FUNCTION
TABLE 1 OF STABLE As(V)
(mM)
P(V)
AND
IN THE DOSING
SOLUTION
74As (% dose)
As
P
0 0 0.1 0.1 1.0 1.0
0 1.0 0 1.0 0 1.0
Transferred to body 10.24 12.14 28.08 29.39 52.71 55.73
t4 2 k t +-
1.65” 0.72” 1.40” 1.29b 4.63‘ 2.42’
Mucosal accumulation 81.41 78.78 60.10 57.52 32.92 28.90
f 2 _’ 2 k f
Luminal efflux
3.71” 0.80” 1.4gh 1.69b 1.42” 0.94’
Note. Values represent means _’ SEM for 6 chicks. Values within same letter superscript are not significantly different (P 5 0.05).
91.65 90.92 87.67 86.91 85.62 84.64
the same column
2 2 t 2 ” 4
2.11” 1.21” 1.49”,b 2.15”.’ 4.24”,h 1.80” and having
the
INTESTINAL
ABSORPTION TABLE
ABSORITION
OF
14As AND
32P AS A FUNCTION
213
OF ARSENATE 2
OF STABLE
As(V)
AND
P(V)
IN THE DOSE
(% Dose) Mucosal accumulation
Luminal efflux
Treatment
Transferred to body
‘4AS 1.0 mM As 1.0 mM P
59.73 4 2.05” 12.94 rf~ 0.74b
31.38 ” 0.86” 83.26 5 1.696
91.11 2 1.27” 96.20 k 2.14a
QP 1.O mM As 1.0 mM P
38.01 2 1.60’ 37.98 k 2.94’
56.71 -r- 0.41c 57.26 2 2.99’
94.12 ‘- 1.41“ 95.24 2 1.26”
Note. Values represent the means ? SEM for 6 chicks. Values within the same column and having the same letter superscript are not significantly different (P G 0.05).
(a) semisynthetic diet adequate in vitamin D,, (1200 IU/kg), calcium (1.2%), and phosphorus (0.7%) (control diet); (6) semisynthetic diet adequate in calcium (1.2%) and phosphorus (0.7%), but containing no vitamin D, (rachitic diet); (c) as in (b) above, but chicks were dosed with 500 IU vitamin D, (im) 48 hr prior to the absorption experiment; (6) semisynthetic diet adequate in vitamin D, (1200 IU/kg) and phosphorus (0.7%) but containing 0.1% calcium (low-calcium diet); (e) semisynthetic diet adequate in vitamin D, (1200 IU/kg) and calcium (1.2%) but containing 0.2% phosphorus (low phosphorus diet). 74As absorption data was generated as previously described, following a 30min absorption period. The dosing solution (1.0 ml) contained 0.5 mM stable arsenate as Na,HAsO,. Chicks raised on diets deficient in vitamin D, exhibited considerably less transfer to body of 74As than animals maintained on the control diet (Table 3). There was, however, no difference in mucosal accumulation of 74As as a result of dietary vitamin D, supplementation. Administration of 500 IU vitamin D, (im), 48 hr prior to the absorption experiment appeared to enhance the percentage of 74As transferred to body by both loop measurements and carcass counts, but neither measurement produced statistically significant differences, owing to the large individual variation within treatment groups. Feeding diets low in calcium or phosphorus resulted in no statistically significant differences from the control groups in either component of absorption (Table 3). The effect of vitamin D, administration on 74As absorption was further investigated in chicks maintained for 3 weeks on a semisynthetic diet deficient in vitamin D,. At 24, 48, and 72 hr prior to the 74As absorption experiment, chicks were dosed with 500 IU vitamin D, (im). Absorption experiments were conducted as described previously. The dosing solution (1 .O ml) contained 0.5 mM stable arsenate as Na,HAsO,. Results of the previous experiment indicated that dietary vitamin D, status
214
FULLMER
AND WASSERMAN
TABLE 3 EFFECTOFDIETAKY VITAMIN D,, LOWCALCIUM,AND Low PHOSPHORUSONTHE ABSORPTION OF 74As 74As (% Dose) Transferred to body
Treatment Control diet Rachitic Rachitic + 500 IU D, (48 hr) Low Ca Low P
Mucosal accumulation
Luminal efflux
In carcass
49.11 _’ 1.83” 26.90 2 3.89b
40.34 2 1.17” 41.43 k 3.10”
90.09 2 1.02” 68.25 4 6.0gb
47.30 t 4.81” 28.36 t 4.70b
34.61 k 3.21b 47.11 k 3.25” 44.06 f 1.13”
41.76 k 1.88” 40.40 k 2.75” 40.22 2 1.91”
76.37 2 2.93b 87.86 2 1.23” 84.29 +- 1.44”
38.13 ? 3.6Tb 49.30 k 4.28” 48.23 _’ 1.29“
Note. Values represent the means * SEM for 6 chicks. Values within the same column and having the same letter superscript are not significantly different (P < 0.05).
influenced 74As absorption and, perhaps, a single im dose of 500 IU D,, 48 hr prior to the absorption experiment, may have also produced an effect. In the present experiment a time range of dosing from 0 to 72 hr was investigated (Table 4). Again, no statistically significant change in the components of absorption was noted at 48 hr following the dose. At 72 hr following the im dose of vitamin D,, a rather large elevation of 74As transferred to the body occurred, this in spite of a constant mucosal arsenate accumulation. DISCUSSION
The results presented herein clearly establish that arsenate (As(V)) is rapidly and extensively absorbed from the intestinal tract of the chick and, thereafter, rapidly eliminated from the body. In this latter respect, the chick appears to be a more suitable animal model for the study of arsenate absorption in humans than is the rat, where considerable arsenate is retained for an extended time period (see Ducoff et al., 1948; Lanz et al., 1950). At a relatively low concentration of stable As(V), 74As was rapidly and comTABLE 4 EFFECT OF VITAMIN D, ADMINISTRATION ON 74As ABSORPTION 74As (% Dose) Treatment Rachitic Rachitic + 500 IU D, (24 hr) Rachitic + 500 IU D, (48 hr) Rachitic + 500 IU D, (72 hr)
Transferred to body
Mucosal accumulation
Luminal efflux
29.96 rt 2.52O
42.69 I 1.53”
72.65 5 3.00”
33.73 -c I.700
38.57 k 1.80b
72.31 2 2.74O
32.92 -t 2.91a
38.23 ” 0.31b
71.16 k 3.04O
45.54 f 1.60b
37.53 2 l.Olb
83.08 k 1.40b
Note. Values represent the means ? SEM for 6 chicks. Values within the same column and having the same letter superscript are not significantly different (P G 0.05).
INTESTINAL
ABSORPTION
OF
ARSENATE
215
pletely absorbed (90% of the dose left the lumen in 15 min) from the duodenum. The overall absorption was easily separated into two components (Fig. 2); mucosal tissue accumulation constituted the rapid component, while transfer of 74As to the body occurred more slowly and apparently represents the rate-limiting phase of absorption, under these conditions. At increasing concentrations of stable luminal arsenate from 0 to 5 mM (Fig. 3), overall 74As luminal efflux remained relatively constant (80-90%), however, dramatic changes in the two absorption components occurred. 74As transferred to the body increased at the 1 mM stable As(V) level, plateaued between 1 and 5 mM, and declined thereafter. Between 0 and 1 mM As(V) there was a decline in fractional mucosal tissue accumulation of 74As from 70 to 40%, a constant value which was maintained to the 50 mM level of stable luminal As(V), and this in spite of a 50-fold increase in total accumulated arsenate (Fig. 4). These results indicate that, at the lower levels of stable As(V), 74As becomes sequestered primarily in or on the mucosal tissue and is transferred to the body only minimally during 15 min. As the stable luminal As(V) concentration is elevated to 1 mM, these sites of sequestration, or compartments, become tilled and, eventually, overloaded with concomitant movement of 74As to the body. The number of such sites must not be extensive since they appear to be saturated at or about 1 mM stable As(V), corresponding to a total As(V) level of 0.3 to 0.4 kmole over the entire segment length. Above 1 mM and extending to 50 mM stable As(V) the percentage of 74As retained by the mucosal tissue is not appreciably diminished, despite a 50-fold enhancement of in total accumulated arsenate (Fig. 4). Coincidentally, the percent of dose transferred to body declines considerably (to about IO%, Fig. 3), even though the total concentration of As(V) in the mucosal tissue has reached about 15 kmole over the segment length. Between 2 and 50 mM stable As(V), however, total arsenate @moles) transferred to body lags considerably behind macosal accumulation. This may represent saturation of serosal transport components or the effects of high arsenate concentration on metabolic processes. The maintenance of constant fractional mucosal As(V) accumulation in the face of concomitant elevation in total mucosal tissue concentration is difficult to explain. Perhaps, above the 1 mM level of accumulated arsenate, additional sets of arsenate-sequestering sites become available, which are more extensive in number or capacity than those sites sequestering arsenate below the 1 mM level. The experiments presented here do not eliminate the possibility that As(V) (above the 1 mM stable luminal level) is simply moving down a concentration gradient from the lumen to the mucosal tissue, and this may well be the case. At any rate, it is apparent that the duodenal mucosal tissue does not present a substantial limiting barrier to the movement of arsenate. On the basis of results presented here, it appears that, despite structural similarities, arsenate and phosphate do not share a common transport pathway in the duodenum. The results (Table 1) indicate that, while 74As transferred to the body is elevated by increasing stable As(V) from 0 to 1.0 mM, no such enhancement is noted for increasing the stable P(V) concentration. Whereas 1.O mM stable As(V) is effective in significantly elevating 74As transferred to the body and re-
216
FULLMER
AND WASSERMAN
ducing mucosal 74As accumulation compared to the 0 mM As(V) control values, 1.0 mM P(V) is not effective in this regard (Tables 1 and 2). Also, there was no significant change in duodenal 32P absorption in the presence of 1.O mM As(V) (Table 3). These experiments clearly establish differences in duodenal P(V) and As(V) absorption and the differential effects of these two anions are further evidenced by comparison of Figs. 3 and 5. Elevation of stable phosphate from 0 to 20 mM in the dosing solution produced little effect on the transfer of 74As to the body, whereas a modest decline in mucosal accumulation occurred (Fig. 5). Significant declines in both components of absorption occurred at 50 mM stable phosphate, possibly as the result of direct inhibition of As(V) transport or, more likely, toxicity resulting at such high P(V) levels. Wasserman and Taylor (1973) have shown that arsenate inhibits ileal phosphate absorption at concentrations as low as 2 mM and, indeed, was a more effective inhibitor than phosphate itself. This effect was more pronounced in vitamin Dreplete than rachitic chicks, suggesting that the inhibitory action is directed primarily against the vitamin D,-responsive component of ileal phosphate transport. The present studies indicate no substantial inhibitory effect of phosphate on either component of duodenal 74As absorption until between 20 and 50 mM stable luminal phosphate. The interaction between these two ions noted by Wasserman and Taylor (1973) may be confined to the ileal tissue. Duodenal arsenate absorption was significantly influenced by vitamin D status of the chicks. This effect was most clearly demonstrated by comparing vitamin D-deficient and normal animals (Table 3) where vitamin D3-depletion resulted in a 45% decline in 74As transferred to the body. There was no sign&ant alteration in mucosal accumulation of 74As. Acute administration of vitamin D, (500 IU) required 72 hr to effect a significant elevation of 74As transferred to body and, perhaps, a slight decrease in mucosal accumulation. Wasserman and Taylor (1973) reported a significant elevation in both components of phosphate transport in chick ileum only 48 hr following a similar dose of vitamin D,. The differences noted between that study and the present work may be tissue-related or may provide another example of noncorrelation between intestinal arsenate and phosphate transport. ACKNOWLEDGMENTS This work was supported by Contract EV-S-2792 from the U.S. Department of Energy. The excellent technical assistance of Steven Maxwell is acknowledged.
REFERENCES Bramen, R. S., and Foreback, C. C. (1973). Methylated forms of arsenic in the environment. Science (Washington, D.C.) 182, 1247-1249. Charbonneau, S. M., Spencer, K., Bryce, F., and Sandi, E. (1978). Arsenate excretion by monkeys dosed with arsenic-containing fish and with inorganic arsenate. Bull. Environ. Contam. Toxicol. 20, 470-477. Charbonneau, S. M., Tam, G. K. H., Bryce, F., Zawidzka, Z., and Sandi, E. (1979). Metabolism of orally administered inorganic arsenic in the dog. Toxicol. Let?. 3, 107-l 13. Crecelius, E. A. (1977). Changes in the chemical speciation of arsenic following ingestion by man. EHP, Environ Health Perspect. 19, 147- 150.
INTESTINAL
ABSORPTION
OF ARSENATE
217
Ducoff, H. S., Neal, W. B., Straube, R. L., Jackson, L. O., and Brues, A. M. (1948). Biological studies with arsenic excretion and tissue localization. Proc. Sot. Exp. Biol. Med. 69, 548-554. Dutkiewicz, T. (1977). Experimental studies on arsenic absorption routes in rats. EHP, Environ. Health Perspect. 19, 173-177. Hwang, S. W.. and Schanker, L. S. (1973). Absorption or organic arsenical compounds from the rat small intestine. Xenobiotica 3, 351-355. Lanz. H., Wallace, P. G., and Hamilton, J. G. (1950). The metabolism of arsenic in laboratory animals with As’~ as a tracer. Univ. Calif. Pub!. Pharmacol. 2, 263-282. Smith, T. J.. Crecelius, E. A., and Reading, J. C. (1977). Airborne arsenic exposure and excretion of methylated arsenic compounds. EHP, Environ. Health Perspect. 19, 89-93. Steel, R. G. D., and Torrie, J. H. (1960). “Principles and Procedures of Statistics.” McGraw-Hill, New York. Tam, K. H., Charbonneau, S. M., Bryce, F., and Lacroix, G. (1978). Separation of arsenic metabolites in dog plasma and urine following intravenous injection of 74As. Anal. Biochem. 86, 505-511. Vahter. M., and Norin, H. (1980). Metabolism of 74As-labeled trivalent and pentavalent inorganic arsenic in mice. Environ. Res. 21, 446-457. Vahter, M. (1981). Biotransformation of trivalent and pentavalent inorganic arsenic in mice and rats. Environ.
Res.
25, 286-293.
Wasserman, R. H., and Taylor, A. N. (1973). Intestinal absorption of phosphate in the chick: Effect of vitamin D and other parameters. J. Nutr. 103, 586-599.