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Mechanisms of Absorption of As2O5 from Rat Small Intestine: the Effect of Different Parameters
Journal of
Trace Elements
J. Trace Elements Med. BioI. Vol. 1 L pp. 239 - 247 (1997)
In Medicine and Biology
© 1997 by Gustav Fischer Verlag
Mechanisms of Absorption of As 20 s from Rat Small Intestine: the Effect of Different Parameters MJ. GONzALEZ I , M.Y. AGUILAR and M.e. MARTINEZ Departamento de Nutrici6n y Bromatologfa, Facultad de Farmacia, Universidad de Alcala de Henares, E-28871 Alcala de Henares, Madrid, Spain (Received April/July 1997)
Summary
The main purpose of this research was to study the effects of water movement on arsenic absorption. In order to appreciate and measure the interaction between these two variables we investigated the perfusion of isotonic and hypotonic buffers (containing 3.2 !lg/ml AS 2 0) into rat small intestines. The As (V) depended on osmolarity sensitive. This implied the presence of a saturable uptake kinetic mechanism and suggested the participation of some kind of carrier-mediated transport system. An increase in arsenate absorption and its accumulation in organs was detected in the presence of an Na+ gradient. The same technique, combining isotonic buffers with different pH values and buffers containing valinomycin, was used in order to study the effects of intraluminal pH on membrane potential. A significant decrease in arsenic levels and As(V) absorption in organs was detected as a result of an increase in the pH. This influence of pH on the As (V) absorption mechanism indicated dependence on a proton (H+) gradient. An inside negative potassium potential induced by valinomycin increased significantly the uptake of arsenate, suggesting an electrogenic transport of arsenate. In conclusion, the As(V) might be absorbed from the small intestine through a carrier-mediated mechanism, which would depend on the Na+ - and H+ -gradients and the membrane potential differential from both sides of the intestinal epithelial cells.
Keywords: Arsenate, absorption, solvent drag, pH-dependency, membrane potential. Introduction
Arsenicals are well known as poisonous agents and were commonly used in homicides and suicides during the Middle Ages. Over the years, scientists have discovered more constructive and beneficial ways to use these products. For instance, arsenicals are commonly used in the agricultural and industrial fields, and they are also employed in the development of new drugs for medical use (1). Arsenical drugs are still used for treating certain tropical diseases such as African sleeping sickness and 'To whom correspondence should be addressed.
amoebic dysentery. Moreover, they are also utilized in veterinary medicine to treat parasitic diseases (2). Several studies have revealed the ubiquitous presence of arsenic in the environment. The exposure of humans and wildlife to arsenic may occur through the air (emissions from smelters, coal-fired power plants, herbicide sprays), water (mine tailings runoff, smelter wastes, natural mineralization), food (especially seafood) and food chains (additives for poultry and swine) (3, 4, 5, 6, 7, 8). Therefore, one of the most important routes of entry of arsenic into the human organism is the gastrointestinal tract.
240
M.J. Gonzalez, M.V. Aguilar and M.e. Martinez
The mechanism responsible for intestinal absorption of arsenic is still not well understood. Arsenic absorption at the intestinal level depends on the physical characteristics of the arsenical derivative, that is, its chemical form, solubility in water, and other specific parameters such as solvent drug effect, intraluminal pH, etc. (9, 10). Several studies of the absorption process of arsenic in the gastrointestinal tract have been undertaken, although, many of the results obtained have been contradictory. In 1984, Tabche et al. (11) discovered that arsenate transport through isolated rat gut membranes depends on concentration and they identified passive diffusion as the most likely mechanism for its absorption. However, Klevay (12) and Cullen et al. (13) suggested that the As (V) transport system across the small intestine was partially shared with another active phosphate transport system. Hwang and Schanker (14) found that certain organic arsenic forms such as carbasone, tryparsamide, and dimethylarsenic acid were absorbed in rat intestine at a rate directly proportional to their concentration over a 100-fold range. This finding indicated that some organic arsenicals are absorbed mainly by simple diffusion through the lipoid areas of the intestinal boundary. Solvent drag effect seems to be a mechanism of absorption of low molecular weight substances (15). Therefore, the first aim of our research was to study, using small intestine perfusion on rats, whether arsenic is absorbed in this way.
Table 1. Salt composition of hypotonic and isotonic buffers Hypotonic buffcr 8 Phosphatc buffer (pH=6.4) Na,HP0 4 KH,P°4 **NaCI
20.l mM 47.0mM 0.5 or 101 mM
Tris NaCI **Mannitol
30.0mM 60.0mM 0.63 or 126 mM
Iris NaCI LiCI KCl Mannitol Urea
30.0mM 125.0 mM 125.0mM 133.0 mM 242.0 mM 250.0 mM
., (Tris)-HCl buffer (pH= 7.4)
Isotonic buffer *(Tris)-HCl buffer (pH=7.4)
"Buffer in which one-half ofNaCI or urea was isotonically replaced by mannitol * *NaCl and mannitol being added to vary the osmolarity
The perfusion technique proved to be more effective than the everted gut sac procedure, because it maintained intestine viability (14, 16, 17) and preserved the orientation of absorptive epithelial cells to the circulatory and lymphatic system (18). In addition, three variables of the absorption pathway were quantified: 1) uptake from the intestinal lumen; 2) transport into the circulatory system; and 3) retention by the mucous membrane. On the other hand, there are several limitations in this technique such as the short duration of the study because of the possible deterioration of organs over time, the level of expertise required in setting up the perfusion experiments and, finally, despite refinement of the technique for maintaining the organ in as near a normal state as possible, the resulting data may differ from the situation in vivo. Therefore, interpretation of the results must be done with care. Nevertheless, the proposed technique was considered suitable for a first approach to the subject under study. Similarly, the effect of pH and membrane potential was studied by this technique in order to achieve a further understanding of the transport process involved in the intestinal absorption of pentavalent arsenic.
Materials and Methods
Perfusion experiment The experimental subjects were male rats of a Wistarderived strain (n=5 per experiment) with a body weight between 200 and 300 g. They were maintained in a temperature-and-humidity controlled environment and fed a standard commercial diet (Panlab, Barcelona). Food and drinking water were supplied ad libitum. The subjects were starved overnight in order to ensure an empty intestinallumen. They were anaesthetized with an intraperitoneal injection of sodium pentothal (60 mg/kg body wt.) . Then, an abdominal incision was made and the subject's small intestine was exposed. The intestinal inflow nozzle was inserted at the duodenum, distal to the pyloric sphincter, and the outflow nozzle was inserted at the ileum, close to the ileo-cecal valve. The subjects were keept under a constant temperature of 37-38" C using a heating set with a incorporated thermostat. During a previous experiment, carried out with a control glucose solution, we observed that, under these conditions, the anesthetic agent and nozzle introduction had not effect on the absorption phenomena in the small intestine.
The absorption of Asp, from small intestine
In order to investigate the solvent drag effect, we perfused the intestine with an isotonic or hypotonic buffer (see Table I) for 60 minutes at a flow rate of 50 ml/h using the single perfusion method (19). This buffer contained 3.2 mg/l of As 20, (Merck) and/or 4.0 mg/l of phenol red. The recovered buffer from the outflow nozzle was collected at fixed intervals of 5, 15, 30, 45, and 60 min. We observed a decrease in arsenic concentration and a change in the phenol red concentration. Comparing the infused volume with the effluent volume we obtained a measure of the net absorption or "intestinal retention" (understood as the amount of As retained in the intestinal lumen). Asp, and phenol red were added to the buffers just before start of the perfusion. Several authors who have performed similar experiments have confirmed that the small intestine remains viable under these conditions (20,21). We took t=O as the moment when we started the perfusion. Logically, at this moment there is no absorption and the degree of retention is considered to be 100%. At the end of the perfusion (60 min.), blood was withdrawn by a syringe from the descendant aorta and the small intestine. The liver and kidneys were also removed. Phenol red was chosen as a marker of water movement bccausc this substance is not absorbed from the rat intcstinal tract (22). To express the balance of bi-directional water flows, the concept of phenol red factor (PRF) was introduced by Koizumi et al. in 1964 (23). PRF was defined as the ratio of phenol red concentration in the buffcr aftcr thc perfusion (C f ) to that before the perfusion (C), which indicates a predominance of water absorption (> 1) or secretion (<1). The buffer recovered 60 min. after t=O was used for Cf measurement. The effect of pH was studied adding 3.2 mg/L of As (V) to two series of isotonic buffer systems. The phosphate buffers of pH 5.5, 6.4 and 7.4 used by Koizumi et al. (23) were diluted with a saline solution (system I) or an isotonic mannitol solution (system II) to minimize the difference in sodium ion concentration among the three buffers. The difference in sodium concentration can be neglected (15, 18). All buffers contained 4.0 mg/l phenol red. The effect of membrane potential on arsenic transport was studied using a 20 mM Tris-HCI buffer (pH=6.0) containing 100 mM NaCl and 100 mM mannitol, with valinomycin dissolved in ethanol added. The resulting concentrations of valinomycin and ethanol were 10 ~M and 0.5%, respectively.
241
Analytical determination Total arsenic levels in the intestinal perfusion solution and in thc different tissues were measured by means of hydride-generation Atomic Absorption Spectrometry (AAS-HG), using a Perkin-Elmer MHS-lO system combined with a Perkin-Elmer 2380 spectrophotometer. The blood samples and organs were submitted to a wet mineralization (HNO y H2 S0 4 and HCI0 4) prior to analysis (24). After extraction, the organs were washed and homogenized with an isotonic saline solution. The process of mineralization was performed once they were dried and weighed. It was necessary to remove the H 2 S0 4 from the samples by total evaporation, because this acid had a negative influence on arsine formation. The residue obtained aftcr cvaporation was dissolved in 25 ml distilled water. It was necessary to sonicate this solution to achieve complete solubilization. At the same time, we performed blank assays to detect any impurities resulting from the reagcnts, and the recovery assays were carried out. The As recovery was 100.9 ± 1,2 %. The analytical parameters of this technique were determined in parallel by means of standard aqueous solutions. The results obtained from thc rccovcry test, combined with the analytical parameters, indicated the validity of this method for measuring the total As content. The phenol red concentration was evaluated by absorbance at 550 nm, using a Perkin-Elmer Mod. Lambda 3B spectrophotometer, inmediately after the addition of an equal volume of 2.0 N NaOH. The instrumental setting used were those recommended in the manufacturer's manual and the analytical % Intestinal retention of As
110,---------------------------------------,
70
60 '-'-_'___'____'_____~____'______'_-L_'__'___'____'_____~____'______'___'____'____'__'__'____LJ o 5 15 30 45 60 Time (min) ~315 mOsm +240 mOsm *128 mOsm
Figure 1. Effect of the osmolarity on intestinal absorption of As(V). Percentage of intestinal retention after perfusion of rat small intestine with various phosphate buffers. The results are mean ± SO (n=5)
M.J. Gonzalez, M.V. Aguilar and M.e. Martinez
242
% Intestinal retention of As 110,---------------------------------------,
ures 1 and 2. In both experiments, the percentage of arsenic remaining in the intestinal perfusate decreased significantly (P<0.05) and proportionally in dependence on a decrease in osmolarity. As a result, we obtained a direct and significant correlation (P
o
5
15
30
45
60
Time (min) ~315 mOsm +240 mOsm *128 mOsm
Figure 2. Effect of the osmolarity on intestinal absorption of As(V). Percentage of intestinal retention after perfusion of rat small intestine with various Tris-(IICl) buffers. The results are mean ± SD (n=5)
These results on arsenic distribution, showed that its absorption increased simultaneously with the osmolarity decrease in both buffers.
The effect of water movement generated by isotonic solutions on arsenic absorption
parameters for As-AAS were: Detection limit, 0.00315
As hypotonic buffers appeared to damage the cellular
mg; sensitivity (1 % absorbance), 4.2 A/mg; precision (6
membrane and alter its permeability (26), isotonic buffers
measurement), 1.00; linear calibration range, 0-0.0250
combined with an appropriate composition of solute were used to alter the direction and degrec of the water move-
mg.
ment (Table 1). The results of these experiments are
Statistical analysis
shown in the Tables 3 and 4.
Statistical analysis was by the Biostatistical Package
"Sigma data base" [Horus Hardware1(25), which includ-
showed the highest significant values for the percentage of arsenic absorption (P<0.05) and also the highest levels
ed a MANOVA analysis (multiple analysis of variance).
for the As (V) in blood, liver, kidney, intestine, and PRF
Values were expressed as mean ± SD.
(Table 4). As the isotonic replacement of NaCI in the
Perfusion with the buffer containing 125 mM NaCI
buffer by LiCI or KCl resulted in a decrease in arsenic concentrations, As absorption, and PRF, sodium ion apResults
peared responsible for the maximum values obtained.
The effect of water movement generated by hypotonic solutions on arsenic absorption
tion, and the PRF resulting from perfusion with the buffer
The As (V) levels in organs, the percentage of absorpcontaining urea (250 mM) were high, although lower than those obtained with NaCI. The percentages of arsenic retention after perfusion
When we replaced one-half of the NaCI or urea by
with phosphate and Tris-HCl buffers are presented in Fig-
mannitol, it lead to a reduction in both the arsenic levels
Table 2. Arsenic concentration in organs (mg/kg wet weight) and blood (Ilg/ml) and PRF after perfusion using with phosphatelTris-HCI buffers with different osmolarity Phosphate buffer (pH 6.4) Osmolarity (mOsM)
Liver
Kidney
Intestine
Blood
PRF(C,fC)
315 240 128
0.089 ± 0.001 * 0.118 ± 0.021 * * 0.171 ± 0.051 *",
0.250 ± 0.089* 0.271 ± 4.2.10" 0.417 ± 4.8.1 O·H
0.402 ± 0.012* 0.412 ± (J.023 * 0.4 75 ± (J.087* *
1.738 ± 0.033* 2.382 ± 0.039* 2.840 ± 0.055*"
1.01 ± 0.002* 1.Of> ± (H)04 * 1.12 ± 0.007**
Tris-HCl buffer (pH 7.4) Osmolarity (mOsM)
Liver
Kidney
Intestine
Blood
PRF(C,fC)
315 240 128
O.I9h ± 0.056* 0.238 ± 0.023** 0.257 ± {Hl03***
0.471 ± 0.027* 0.507 ± 0.019* 0.697 ± O.O(]] **
1.007 ± 0.052* 1.167 ± 0.021 * 1.211 ± 0.024 * *
2.519 ± (J.029* 2.701 ± 0.032* 3.298 ± 0. lO9**
1.02 ± 0.004 * 1.06 ± (H111 * 1.I3 ± 0.017**
The results are mean ± SD for 5 experiments. Values in the same column bearing a common symbol are not significantly different (P> 0.(1); MAN OVA analysis of variance
The absorption of As 20, from small intestine
243
Table 3. Percentage of intestinal retention of As after the perfusion of rat small intestine with several pH=7A Tris-HCI buffers containing 3.2 mg/ml Asp,. 60 min
Salt composition
5 min
15 min
30 min
Tris + NaCI 30mM 125mM
54.7 ± 3.95
81.3 ± 0.64
82.2 ± 0042
84.2 ± 3042
81.1 ± 0.941c.Jd.g)
Tris+NaCI+Mannitol 30mM 62,5mM 121mM
70.5 ± 0.99
8304 ± 2.11
86.5 ± 0.10
85.9 ± 1.91
86.0±0.71")
Tris+Mannitol 30mM242mM
8604 ± 0.75
98.5 ± 2.12
95.1 ± 1.98
Tris+Urea+Mannitol 30mM 125mM 121mM
82.9 ± 0.28
84.2 ± 2.00
87.9 ± 1.62
94.2 ± 7.07.10"
88.9 ± 1.71 1'1
Tris + Urea 30mM 250mM
75.2 ± 0.95
8704 ± 2.05
86.3 ± 0.73
87.9 ± 5.16
86.5 ± 0.911"1
Tris + LiCI 30mM 125mM
79.2 ± 0.78
93.9 ± 1.91
94.7 ± 2.81
90.1 ± 4.03
92.7 ± 0.73"')
Tris + KCl 30mM 133mM
75.1 ± 4.53
91.9 ± 1.87
8804 ± 0.63
9004 ± 3.54
89.1 ± 1.601")
45 min
100
± 0.03
94.9 ± 0.05 10."
The results are mean ± S.D. for 5 experiments. The letters indicate statistically significant differences (P<0.05) from Tris-HCI buffers containing:(a)NaCI, (b)NaCI and mannitol, (c)mannitol, (d)urea and mannitol, (e)urea, (f)LiCI and (g)KCI; MANOYAanalysis of variance.
in all organs and PRE Perfusion with the buffer containing 242 mM of mannitol revealed the lowest values for arsenic absorption and PRE
The effect of pH on water movement and on arsenic absorption and distribution The interrelation between buffer system pH and arsenic retention or As (V) levels in organs and PRF after perfusion are shown in Figures 3 and 4 and Table 5.
The pH variations in buffer systems I and II did not alter the PRF value after 60 minutes of perfusion. The PRF after perfusion with buffer system I was higher than 1.0, and with buffer system II was lower than 1.0. At each pH, the arsenic absorption and levels in organs and PRF were significantly different between the two buffer systems. This fact corroborated the Na+ -gradient dependence of arsenic transport at the intestinal level. Because the PRF values were not altered by pH variation, this parameter did not affect water movement, al-
Table 4. Concentration of As in organs (/lg/g wet weight), blood (/lg/ml) and PRF after the perfusion with several pH=7A Tris-HCI buffers. Salt composition
Liver (mg/kg)
Kidney (mg/kg)
Intestine (mg/kg)
Blood (mg/kg)
PRF (C/C)
Tris + NaCI 30mM, 125 mM
0.216 ± 0.03
0.335 ± 0.03
1.281 ± 0.04
2.985 ± 0.08
1.08 ± O.()]
+ NaCI + Mannitol 62,5 mM, 121 mM
0.166 ± 0.01
0.278 ± 0.02
1.079 ± 0.03
1.947 ± 0.06
1.02 ± 0.01
+ Mannitol 242mM
0.092 ± 0.02
0.165 ± 0.03
0.796 ± 0.03
1.716 ± 0.03
0.99 ± 0.01
+ Urea + Mannitol 125 mM, 121 mM
0.160 ± 0.01
0.284 ± (l.(ll
1.806 ± 0.09
1.01 ± 0.01
+ Urea 250mM
0.190±0.01
0.303 ± 0.01
1.0l0 ± 0.11
1.846 ± 0.17
1.04 ± 0.01
+ LiCI 125 mM
0.064 ± 0.00
0.204 ± 0.02
00465 ± 0.07
1.650 ± 0.07
1.00 ±O.O
+KCL 133mM
0.134 ± 0.02
0.266 ± 0.01
0.973 ± 0.11
1.904 ± 0.07
un ±O.O
The results arc the mean ± S.D. for 5 experiments.
0.862 ± 0.06
244
M.1. Gonzalez, M.V. Aguilar and M.e. Martinez
% Intestinal retention of As % Intestinal retention of As 110----------------------------------------, 110"------------------------------------,
100
I
90
o
15
5 ~pH
=
30
Time (min) 5.5 +pH 6.4 *pH
=
45
60
::l
0
though it modified arsenic absorption: an increase in the pH resulted in a noticiable decrease in the arsenic level in organs and in As absorption (Figures 3 and 4). Furthermore, high, and negative correlation coefficients (P
The effect of membrane potential on As (V) absorption The effect of K+ diffusion potential generated by valinomycin on the uptake of As (V) was studied in the presence of an outward-directed gradient of K+ . Valinomycin is an antibiotic which behaves as a ionophore due to specific transport of K+ ions through cell membranes (27) (see Figure 5 and Table 6). In the presence of valinomycin, both the absorption of arsenic and its levels in organs were significantly enhanced.
15
30
Time (min) ~pH = 5.5 ~pH 6.4 *pH
=
= 7.4
Figure 3. Effect of pH on intestinal absorption of As(V). Percentage of intestinal retention after perfusion of rat small intestine with buffer system I. The results are mean ± SD (n=5). *=significant difference (p<0.05) with respect to pH=5.5.
5
45
60
=7.4
Figure 4. Effect of pH on intestinal absorption of As(V). Percentage of intestinal retention after perfusion of rat small intestine with buffer system II. The results are mean ± SD (n=5). *=significant difference (p<0.05) with respect to pH=5.5.
Discussion
The effect of water movement generated by hypotonic solutions on arsenic absorption The As osmolarity-sensitive uptake by the intestine implies a saturable uptake kinetics; suggesting the participation of some type of carrier-mediated transport system (28). In addition, this phenomenon suggests that the increase in arsenic absorption and levels in the intestinal tissue is accompanied by an increase in water absorption, due to the correlation between arsenic absorption and the PRF observed (the PRFs are greater than 1 in all the cases). Statistically significant differences have been observed between blood levels and concentrations in organs, as well as between percentages of intestinal retention obtained with phosphate buffers and Tris-HCI buff-
Table 5. Effect of pH of perfusion buffers on As (V) concentrations in organs (mg/kg wet weight) and blood (mg/L) System I (Na+) pH
Liver
Kidney
Intestine
Blood
PRF(C;C)
5.5 6.4 7.4
0.221 ± 0.018* 0.201 ± 0.021 * 0.175 ± 0.039*
0.482 ± 0.035* 0.437 ± 0.008* 0.353 ± 0.042**
1.398 ± 0.089* 1.199 ± 0.140** 1.006 ± 0.068 * *
3.076 ± 0.248* 2.120 ± 0.154* 1.793 ± 0.029**
1.07±0.003* 1.06±0.006* 1.05±0.005 *
pH
Liver
Kidney
Intestine
Blood
PRF(C;C)
5.5 6.5 7.4
0.165 ± 0.025* 0.131 ± 0.020** 0.126 ± (J.0l2**
0.321 ± 0.027* 0.290 ± 0.068* 0.249 ± 0.026*
0.878 ± 0.047* 0.854 ± 0.043* 0.773 ± 0.055*
2.457 ± 0.610* 2.214 ± 0.084* 1.432 ± 0.184**
O. 997±0.004 * 0.986±0.005* O.971±O.003*
System" (Mannitol)
The results are mean ± SO for 5 experiments. Values in the same column bearing the common symbol are not significantly different. Values in the same column bearing the different symbol are significantly different P< 0.001. MANOVA analysis of variance.
The absorption of Asp, from small intestine
ers. These differences may be caused by the pH, which is different in both solutions, or a possible interaction of the phosphate anion with arsenate intestinal absorption.
The effect of water movement generated by isotonic solutions on arsenic absorption Results suggest that the degree of As (V) absorption and accumulation in the intestinal tissue depend on the direction and volume of the water movement. Thus, a decrease in osmolarity and the variation of the sodium ion and urea concentration in buffer solutions cause an increase in water absorption. Consequently, this increase in water absorption also implies an increase in the levels of As (V) in blood, liver, kidney, and intestine, partially due to the solvent drag effect. A similar effect has been reported in experiments with mercury (15), magnesium (26), and certain organic compounds (29). The results of these experiments may indicate that arsenate is transported across epithelial cells of the intestinal mucous membranes by a secondary active transport. This transport system depends on an Na+ -gradient (sodium transport). A similar transport system is used for the intestinal absorption of glucose, galactose, and various aminoacids. The carrier of As (V), like those utilized by the substances mentioned above, as probably located within the intestinal brush-border membrane (30). However, this carrier does not seem to transport arsenate without the presence of sodium ions. The carrier appears to have a
245
receptor place for As (V) and Na+. In addition, this carrier does not introduce arsenate into the cell if the receptor place for As (V) and Na+ are not simultaneously occupied. The driving energy required to allow the passage of the carrier from the outer side of the membrane comes from the difference in sodium ion concentration between both sides of the membrane. When Na+ diffuses into the cells across the sodium channel, it "pulls" the carrier and the arsenate too, providing the energy necessary for transport. Inside the epithelial cells, arsenate ions diffuse in every direction, including into the portal blood. As regards the nature of the possible carrier, it may be a protein with a high molecular weight (31) and rich in SH-groups. This hypothesis is supported by Sorensen (32), who observed that As was found in the form of inclusions as a metal-protein complex in the nucleus of the cell. The Rosen (33) and San Francisco (34) teams identified and purified a protein called "Ars B protein", which is located in the membrane of Escherichia coli, and is part of a specific anion pump. This anion pump is responsible for resistance to and transport of arsenate from these cells. The amino-acid sequence for this protein proposed by these authors is: Met-Gln-Phe-Lue-Gln-Asn-Jle-ProPro-Tyr-Leu-Phe. This protein, or another with similar characteristics, may be the carrier involved in the arsenate transport at the intestinal level. The exact number of the possible carriers for arsenate will be studied by our team in the near future.
The effect of pH on water movement and on arsenic absorption an distribution % Intestinal retention 105r-----------------------------------~ I
1009S
L
90 85 80
The same effect of pH on Asps absorption revealed in our experiments was observed by Meharg and Macnair (1991)(35) in research on As (V) uptake in Holcus lanatus. These authors attributed the increase in arsenate uptake to the different chemical form this compound Table 6. Effect of valinomycin on levels of As (V) in organs (mg/kg wet weight) and blood Cmg/L)
75 70~------~-------------------------L~
o
5
15
30
45
60
Time (min) ~ Control -fr Valinomycine Figure 5. Effect of membrane potential on intestinal absorption of As (V). Percentage of intestinal retcntion after perfusion of rat small intestine with Tris-(HCl) buffer with or without valinomycin. The resulta are mean ± SD (n=5). *=significant difference with rcspect to contra.1.
Liver Treatment with Valinomycin O.361±0.030*
Kidney
Intestine
0.282±0.016* 1.798±0.1l3*
Blood
2.122±0.212*
Control 0.149±O.037* * O.234±O.003 * O.658±O.123 * * 1. 754±0.065* * The results are mean ± SD for 5 experiments. Values in the same column bearing a common symbol are not significantly different (P> 0.(1); MAN OVA analysis of variance.
246
MJ. Gonzalez, M.V. Aguilar and M.e. Martinez
assumes depending on the pH: arsenate uptake is optimal at pH=5 when HcAs04 is the dominant anion in solution; as the pH is increased to 8 and HAs0 42 becomes the dominant form, arsenate uptake decreases due to its lower liposolubility. The pH influence on arsenic absorption may indicate dependence on an W -gradient involved in the As (V) absorption mechanism. Arsenate transport across the intestinal cell might be mediated by a coupled symport with W (or antiport with the hydroxy ion). Although the H+ -gradient-dependent transport system responsible for As (V) absorption was not defined, a similar proton-gradient-dependent transport for antibacterial agents has been found, ~-lactamic antibiotics in the rat small intestine (36, 37). Furthermore, several nutrients such as a small peptide (38), folate (39), oxalate (40), Dglucose (41), lactic acid (42), and nicotinic acid (43) are transported via some proton- or hydroxy ion-gradient dependent carrier system cross the intestinal brush-border membrane. Consequently, the role of an H+-gradient across the brush-border membrane in the intestinal absorption of nutrients and drugs is as important as the role of the Na+ -gradient. The pH effect and the solvent drag effect appear to act independently because: the pH effect was observed under predominance of water absorption (system I) and water secretion (system II); the arsenic concentration in each organ and the arsenic absorption after perfusion with buffer system I were higher than with buffer system II.
The effect of membrane potential on As (V) absorption The arsenic absorption, as well as its levels in organs (except kidney), were significantly enhanced by inducing a relatively inside-negative membrane potential, suggesting an electrogenic transport of arsenate was suggested. All the characteristics of Na+-gradient, H+-gradient and membrane potential dependencies are advantageous for the arsenate absorption in the small intestine under physiological conditions; since the luminal side of the brush-border membrane is known to be Na+ -rich, to show an acidic microclimate pH (pH=6.0) and a relatively positive membrane potential compared to intraenterocytes due to the presence of sialic acid residues and sulfate groups (44). In conclusion, As (V) may be absorbed from the small intestine via a carrier-mediated mechanism depending on Na+- and H+-gradients and potential differences across
the intestinal brush-border membrane. The carrier system available for arsenate is suggested to be partially shared with the inorganic transport system. This hypothesis is based on the transport characteristics of phosphate, which show Na+- and H+-gradient dependence which quite similar to those present in arsenate transport (45,46,47,48).
References 1. IRGOLIC, K.J. (1986 )Arsenic in the Euvironment. In Frontiers in BioinorganicChemistry. Edited by Xavier AV Ed. VCH Publishers, Weinheim, 399-408 2. NASS (1977) Arsenic. Natl Acad Sci, Washington, D.e., 332. 3. GATES, M.; WILLIAMS, J.W AND ZAPP, lA. (1942)Arsenicals. In: Chemical warfare agents and related chemical problems, Summary Technical Report of Division 9, NRDC vol I, Parts I-II, Chapter 7. National Defense Research Committee, Washington, D.C., 83-114 4. PERSHAGEN, G. (1983) The Epidemiology of Human Arsenic Exposure. In Biologica and Environmental Effect of Arsenic. Edited by B.A. Fowler Ed. Vol 6. Elsevier Science Publisher, Amsterdam,199-232 5. SQUIBB, KS. AND FOWLER, B.A. (1983) The toxicity of Arsenic and its Compounds. In Biological and Environmental Effect of Arsenic. Edited by B.A. Fowler Ed. Vol 6, Elsevier Science Publisher, Amsterdam, 233-269. 6. FIELDER, R.J.; DALE, E.A. AND WILLIAMS, S.D. (1986) Inorganic Arsenic Compounds. Health and Safe Commission's Advisory Committee on Toxic Substance. Health and Safety Executive. Her Majesty's Stationery Office, London, Toxicity Review 16, 1-95. 7. IRGOLIC, KJ. (1986) Health effects of arsenic in the aqueous environment. Northeast Environmental Science 5, 86-90. 8. PER SHAG EN, G. (1986) Sources of Exposure and Biological Effects of Arsenic. In Environmental carcinogens-selected methods of analysis. Edited by I.K O'Neill, P. Schuller & L. Fishbein Eds. vol8. International Agency for Research on Cancer, World Health Organization, Lyon, 45-61 9. DIAZ DE TORANZO, E.G. (1990) Absorption of toxic substances. A basic concept in toxicology. Acta Bioquimica Clinica Latinoamericana 24 (4), 375-83. 10. REITZ, R.H. (1990) Distribution, persistence and elimination of toxic agents (pharmacokinetics). In Progress in Predictive Toxicology. Edited by D.B. Clayson, I.e. Munro, P. Shubik & Swenberg Ed. Elsevier Science Publishers B.Y., Amsterdam, 79-90. 11. TABCHE, L.M.; NAVARRETE, L.M. AND GERMAN, F.e. (1984) Effect of iron, calcium and lead on arsenic absorption in isolated rat intestine. Acta Mexicana de Ciencia y Tecnologia 2(6), 57-62. 12. KLEVAY, L.M. (1976) Pharmacology and toxicology of heavy metals: Arsenic. Pharmacological Therapeutics AI, 189-209 13. CULLEN, WR.; MCBRIDE B.e. AND PICKETT. A.W (1990) The uptake of arsenicals by Candida humicola. Applied Organometallic Chemistry 4,119-125. 14. HWANG, S.W AND SCHANKER, L.S. (1973) Absorption of organic arsenical compounds from the rat small intestine. Xenobiotica 3, 351-355. 15. ENDO, T.; NAKAYA, S. AND KIMURA, R. (1988) Mechanisms of Absorption of Inorganic Mercury from Rat Small Intestine. 1. Solvent drag Effect on Absorption of Inorganic Mercury. Pharmacology and Toxicology (Copenhagen) 65, 128-35.
Thc absorption of 16. CASTLE, SJ.; TUCKER. G.T.; WOODS, H.E; UNDERWOOD, J.C.E.; NICHOLSON, M.E.; HAVLER, C.M.; LEWIS, CJ.; FLOCKHART, 1.R. AND LLOYD-JONES, G. (1985) Assessment of an in situ rat intestine preparation with perfused vascular bed for studying the absorption and first -pass metabolism of drugs. Journal of Pharmacological Methods 14, 255-274. 17. SAKAI. K.; AKIMA M. AND HINOHARA, Y. (1980) Vasculariy perfused rat small intestine: a research model for drug absorption. Japan Journal of Pharmacology 30, 231-241. 18. OCHSENFAHRT, H. AND WINNE, D. (1979) The contribution of solvent drag to the intestinal absorption of tritiated water and urea from the jejunum of the rat. Naunyn-Schmiedebergs Archives of Pharmacology 279,133-152. 19. ENDO, T; NAKAYA, S.; KIMURA R. AND MURATA, T (1984) Gastrointestinal Absorption of Inorganic Mercuric Compounds in Vivo and in Situ. Toxicology and Applied Pharmacology 74, 223-229. 20. WINDMUELLER, H.; SPAETH, AE. AND GANOTE, C.E. (1970) Vascular perfusion of the isolated rat gut: norepinephrine and glucocorticoid requirements. American Journal of Physiology 218,197-204. 21. DOWLING, HJ.; OFFENBACHER, E.G. AND PI-SUNYER, EX. (1989) Absorption of Inorganic, Trivalent Chromium from the Vascularly Perfused Rat Small Intestine. American Institute of Nutrition 1138-1145.
Asp, from small intestine
247
33. ROSEN, B.P.; WEIGEL, U.; KARKARIA, C. AND GANGOLA P. (1988) Molccular characterization of an anion pump. The ars A gene product is an arsenite (antimonate)-stimulated ATPase. Journal of Biology and Chemistry 263, 3067-3070. 34. SAN FRANCISCO, MJ.D.; TISE, L.S. AND ROSEN, B.P. (1989) Identification of the membrane component of the anion pump encoded by the arsenical resistance operon of R-factor R773. Molecular Microbiology 3( 1), 15-21. 35. MEHARG, A. AND MACNAIR, M.R. (1990) An altered phosphate uptake system in arsenate tolerant Holcus lanatus L. New Physiology 116,29-35. 36. TSUJI, A.; TERASAKI, T; TAMAI, I. AND HIROOKA, H. (1987) H+ Gradient dependent and carrier~mediatcd transport of cefiximc, a new cephalosporin antibiotic, across brush-border membrane vesicles from rat small intestine. Journal of Pharmacology and Experimental Therapeutics 241, 594-60 I. 37. TAMAI, I.; TSUJI, A. AND KIN, Y. (1988) Carrier-mediated transport of cefixime, a new antibiotic, via an organic anion transport system in the rat renal brush-border membrane. Journal of Pharmacology and Experimental Therapeutics 246, 338344. 38. GANAPATHY, V AND LEIBACH, EH. (I985) Intestinal pep~ tide transport energized by proton gradient? American Journal of Physiology 249, GI53-GI60.
22. KITAZAWA, S.; ITO, H. AND SEZAKI, H. (1975) Transmucosal fluid movement and its effect on drug absorption. Chemical and Pharmaceutical Bull 23, 1856-1865.
39. SCHRON, C.M.; WASHINGTON, C. JRAND BLITZER, B.L. (1985) The transmembrane pH gradient drive uphill folate transport in rabbit jejunum. Direct evidence for folate hydroxyl exchange in brush-border membrane vesicles. 10urnal of Clinical Investigation 76, 2030-2033.
23. KOIZUMI, T.: ARITA, T AND KAKEMI, K. (1964) Absorption and excretion of drugs Some pharmacokinetic aspect of absorption and excretion of sulfonamides. (1). Absorption from rat stomach. Chemical and Pharmaceutical Bull 12,413-420.
40. KNICKELBEIN, R.G.; ARONSON, P.S. AND DOBBINS, J.W. (1986) Oxalate transport by anion exchange across rabbit ileal brush-border. Journal of Clinical Investigation 77, 170175.
24. HERSHEY, J.w. AND OOSTDYK. TS. (1988) Determination of arsenic and selenium in environmental and agricultural samples by hydride generation atomic absorption spectrometry. Journal of Association Official of Analysis and Chemistry 71(6), lOW).
41. HOSHI T; TAKUWA, N.; ABE, M. AND TAJIMA, A. (1986) Hydrogen ion-coupled transport of D-glucose by phlorizinesensitive sugar carrier in intestinal brush-border membranes. Biochemical and Biophysical Acta 861, 483-488.
25. FERNANDEZ-PERIS, E.; MOLINERO CASARES, C.M. AND MOERU-ABOAL, E. (1986) Sigma. Base de datos bioestadisticos para un ordenador personal. Horus Hardware. Madrid. 26. BEHAR, J. (1974) Magnesium absorption by rat ileum and colon. American Journal of Physiology 227, 334-340. 27. RAWN, J.D. (1990) Bioquimica, Inter Americana. McGrawHill, Madrid, 1037. 28. ISHIZAWA, T; TSUJI, A; TAMAI, I.; TERASAKI, T; HOSOI, K. AND FUKATSU, S. (1990) Sodium and pH Dependent Carrier-Mediated Transport of Antibiotic, Fosfomycin, in the Rat Intestinal Brush-Border Membrane. Journal of Pharmacobiology-Dyn 13, 292. 29. KARINO, A; HORIE HAYASHI, T; AWAZU, S.; MINAMI, H. AND HANANO, M. (1982) Solvent drag effect in drug intestinal absorption. 1. Studies on drug and D,O absorption clearances. Journal of Pharmaceutical Dyn 5, 410-417. 30. CHAUNCEY, B.; SCHMID, E.C. AND GOLDTEIN, L. (1988) Arsenical and mercurial inhibition of tyrosine transport by the flounder intestine. Journal of Toxicology and Environmental Health 23(2), 275-65. 31. MARAFANTE, E.; RADE, J.; PIETRA, R.; SABBIONI, E. AND BERTOLERO, E (1980) Metabolic fate of inorganic arsenic in laboratory animals. In 3 Spurenelement-Symposium, Arsen.Edited by M. Anke, HJ. Schneider and Chr. Brucker Eds. Karl-Marx Univ., Leipzip, Fried.-Schiller-Univ., Jena, 49-57. 32. SORENSEN, E.M.B. (1987) Ultrastructural cytochemical analysis of intranuclear arsenic inclusions. Journal of Toxicology and Environmental Health 22, 273-285.
42. SIMANJUNTAK, M.T; TAMAI, I.; TERASAKI, T AND TSUJI, A. (1990) Carrier-mediated uptake of nicotinic acid by rat intestinal brush-border membrane vesicles and relation to monocarboxylic acid transport. Journal of PharmacobiologyDyn 13,301-309. 43. TIRUPPATHI, c.; BALKOVETZ, D.E; GANAPATHY, V; MIYAMOTO, Y. AND LEIBACH, EH. A proton gradient, not a sodium gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles. Biochemical Journal 256, 219-223. 44. VAN HOOGDALEM, EJ.; DE BOER, A.G. AND BREIMER, D.D. (1989) Intestinal Drug Absorption Enhancement: an overview. Pharmaceutical Therapeutics 44, 407-443. 45. DANISI, G.; MURER, H. AND STRAUB, R.W. (1982) Effect of pH on phosphate transport into intestinal brush-border membrane. American Journal of Physiology 242, G533 G539. 46. BELIVEAU, R. AND STREVEY, 1. (1991) Phosphate transport in kidneys: effect of transmembrane electrical potential. American Journal of Physiology 261(4, Pt. 2), F663-F669. 47. QUAMME, G.A. AND SHAPIRO, RJ. (I986) Membrane controls of epithelial phosphate transport. Canadian Journal of Physiology and Pharmacology 65, 275-286. 48. SHIRAZI-BEECHEY, S.P.; BEECHEY, R.B.; PENNY, J.; VAYRO, S.; BUCHAN, W. AND SCOTT, D. (1991) Mechanisms of phosphate transport in shcep intestine and parotid gland: response to variation in dietary phosphate supply. Experimental Physiology 76(2), 231-241.
Trace Elements
J. Trace Elements Med. BioI. Vol. 1 L pp. 239 - 247 (1997)
In Medicine and Biology
© 1997 by Gustav Fischer Verlag
Mechanisms of Absorption of As 20 s from Rat Small Intestine: the Effect of Different Parameters MJ. GONzALEZ I , M.Y. AGUILAR and M.e. MARTINEZ Departamento de Nutrici6n y Bromatologfa, Facultad de Farmacia, Universidad de Alcala de Henares, E-28871 Alcala de Henares, Madrid, Spain (Received April/July 1997)
Summary
The main purpose of this research was to study the effects of water movement on arsenic absorption. In order to appreciate and measure the interaction between these two variables we investigated the perfusion of isotonic and hypotonic buffers (containing 3.2 !lg/ml AS 2 0) into rat small intestines. The As (V) depended on osmolarity sensitive. This implied the presence of a saturable uptake kinetic mechanism and suggested the participation of some kind of carrier-mediated transport system. An increase in arsenate absorption and its accumulation in organs was detected in the presence of an Na+ gradient. The same technique, combining isotonic buffers with different pH values and buffers containing valinomycin, was used in order to study the effects of intraluminal pH on membrane potential. A significant decrease in arsenic levels and As(V) absorption in organs was detected as a result of an increase in the pH. This influence of pH on the As (V) absorption mechanism indicated dependence on a proton (H+) gradient. An inside negative potassium potential induced by valinomycin increased significantly the uptake of arsenate, suggesting an electrogenic transport of arsenate. In conclusion, the As(V) might be absorbed from the small intestine through a carrier-mediated mechanism, which would depend on the Na+ - and H+ -gradients and the membrane potential differential from both sides of the intestinal epithelial cells.
Keywords: Arsenate, absorption, solvent drag, pH-dependency, membrane potential. Introduction
Arsenicals are well known as poisonous agents and were commonly used in homicides and suicides during the Middle Ages. Over the years, scientists have discovered more constructive and beneficial ways to use these products. For instance, arsenicals are commonly used in the agricultural and industrial fields, and they are also employed in the development of new drugs for medical use (1). Arsenical drugs are still used for treating certain tropical diseases such as African sleeping sickness and 'To whom correspondence should be addressed.
amoebic dysentery. Moreover, they are also utilized in veterinary medicine to treat parasitic diseases (2). Several studies have revealed the ubiquitous presence of arsenic in the environment. The exposure of humans and wildlife to arsenic may occur through the air (emissions from smelters, coal-fired power plants, herbicide sprays), water (mine tailings runoff, smelter wastes, natural mineralization), food (especially seafood) and food chains (additives for poultry and swine) (3, 4, 5, 6, 7, 8). Therefore, one of the most important routes of entry of arsenic into the human organism is the gastrointestinal tract.
240
M.J. Gonzalez, M.V. Aguilar and M.e. Martinez
The mechanism responsible for intestinal absorption of arsenic is still not well understood. Arsenic absorption at the intestinal level depends on the physical characteristics of the arsenical derivative, that is, its chemical form, solubility in water, and other specific parameters such as solvent drug effect, intraluminal pH, etc. (9, 10). Several studies of the absorption process of arsenic in the gastrointestinal tract have been undertaken, although, many of the results obtained have been contradictory. In 1984, Tabche et al. (11) discovered that arsenate transport through isolated rat gut membranes depends on concentration and they identified passive diffusion as the most likely mechanism for its absorption. However, Klevay (12) and Cullen et al. (13) suggested that the As (V) transport system across the small intestine was partially shared with another active phosphate transport system. Hwang and Schanker (14) found that certain organic arsenic forms such as carbasone, tryparsamide, and dimethylarsenic acid were absorbed in rat intestine at a rate directly proportional to their concentration over a 100-fold range. This finding indicated that some organic arsenicals are absorbed mainly by simple diffusion through the lipoid areas of the intestinal boundary. Solvent drag effect seems to be a mechanism of absorption of low molecular weight substances (15). Therefore, the first aim of our research was to study, using small intestine perfusion on rats, whether arsenic is absorbed in this way.
Table 1. Salt composition of hypotonic and isotonic buffers Hypotonic buffcr 8 Phosphatc buffer (pH=6.4) Na,HP0 4 KH,P°4 **NaCI
20.l mM 47.0mM 0.5 or 101 mM
Tris NaCI **Mannitol
30.0mM 60.0mM 0.63 or 126 mM
Iris NaCI LiCI KCl Mannitol Urea
30.0mM 125.0 mM 125.0mM 133.0 mM 242.0 mM 250.0 mM
., (Tris)-HCl buffer (pH= 7.4)
Isotonic buffer *(Tris)-HCl buffer (pH=7.4)
"Buffer in which one-half ofNaCI or urea was isotonically replaced by mannitol * *NaCl and mannitol being added to vary the osmolarity
The perfusion technique proved to be more effective than the everted gut sac procedure, because it maintained intestine viability (14, 16, 17) and preserved the orientation of absorptive epithelial cells to the circulatory and lymphatic system (18). In addition, three variables of the absorption pathway were quantified: 1) uptake from the intestinal lumen; 2) transport into the circulatory system; and 3) retention by the mucous membrane. On the other hand, there are several limitations in this technique such as the short duration of the study because of the possible deterioration of organs over time, the level of expertise required in setting up the perfusion experiments and, finally, despite refinement of the technique for maintaining the organ in as near a normal state as possible, the resulting data may differ from the situation in vivo. Therefore, interpretation of the results must be done with care. Nevertheless, the proposed technique was considered suitable for a first approach to the subject under study. Similarly, the effect of pH and membrane potential was studied by this technique in order to achieve a further understanding of the transport process involved in the intestinal absorption of pentavalent arsenic.
Materials and Methods
Perfusion experiment The experimental subjects were male rats of a Wistarderived strain (n=5 per experiment) with a body weight between 200 and 300 g. They were maintained in a temperature-and-humidity controlled environment and fed a standard commercial diet (Panlab, Barcelona). Food and drinking water were supplied ad libitum. The subjects were starved overnight in order to ensure an empty intestinallumen. They were anaesthetized with an intraperitoneal injection of sodium pentothal (60 mg/kg body wt.) . Then, an abdominal incision was made and the subject's small intestine was exposed. The intestinal inflow nozzle was inserted at the duodenum, distal to the pyloric sphincter, and the outflow nozzle was inserted at the ileum, close to the ileo-cecal valve. The subjects were keept under a constant temperature of 37-38" C using a heating set with a incorporated thermostat. During a previous experiment, carried out with a control glucose solution, we observed that, under these conditions, the anesthetic agent and nozzle introduction had not effect on the absorption phenomena in the small intestine.
The absorption of Asp, from small intestine
In order to investigate the solvent drag effect, we perfused the intestine with an isotonic or hypotonic buffer (see Table I) for 60 minutes at a flow rate of 50 ml/h using the single perfusion method (19). This buffer contained 3.2 mg/l of As 20, (Merck) and/or 4.0 mg/l of phenol red. The recovered buffer from the outflow nozzle was collected at fixed intervals of 5, 15, 30, 45, and 60 min. We observed a decrease in arsenic concentration and a change in the phenol red concentration. Comparing the infused volume with the effluent volume we obtained a measure of the net absorption or "intestinal retention" (understood as the amount of As retained in the intestinal lumen). Asp, and phenol red were added to the buffers just before start of the perfusion. Several authors who have performed similar experiments have confirmed that the small intestine remains viable under these conditions (20,21). We took t=O as the moment when we started the perfusion. Logically, at this moment there is no absorption and the degree of retention is considered to be 100%. At the end of the perfusion (60 min.), blood was withdrawn by a syringe from the descendant aorta and the small intestine. The liver and kidneys were also removed. Phenol red was chosen as a marker of water movement bccausc this substance is not absorbed from the rat intcstinal tract (22). To express the balance of bi-directional water flows, the concept of phenol red factor (PRF) was introduced by Koizumi et al. in 1964 (23). PRF was defined as the ratio of phenol red concentration in the buffcr aftcr thc perfusion (C f ) to that before the perfusion (C), which indicates a predominance of water absorption (> 1) or secretion (<1). The buffer recovered 60 min. after t=O was used for Cf measurement. The effect of pH was studied adding 3.2 mg/L of As (V) to two series of isotonic buffer systems. The phosphate buffers of pH 5.5, 6.4 and 7.4 used by Koizumi et al. (23) were diluted with a saline solution (system I) or an isotonic mannitol solution (system II) to minimize the difference in sodium ion concentration among the three buffers. The difference in sodium concentration can be neglected (15, 18). All buffers contained 4.0 mg/l phenol red. The effect of membrane potential on arsenic transport was studied using a 20 mM Tris-HCI buffer (pH=6.0) containing 100 mM NaCl and 100 mM mannitol, with valinomycin dissolved in ethanol added. The resulting concentrations of valinomycin and ethanol were 10 ~M and 0.5%, respectively.
241
Analytical determination Total arsenic levels in the intestinal perfusion solution and in thc different tissues were measured by means of hydride-generation Atomic Absorption Spectrometry (AAS-HG), using a Perkin-Elmer MHS-lO system combined with a Perkin-Elmer 2380 spectrophotometer. The blood samples and organs were submitted to a wet mineralization (HNO y H2 S0 4 and HCI0 4) prior to analysis (24). After extraction, the organs were washed and homogenized with an isotonic saline solution. The process of mineralization was performed once they were dried and weighed. It was necessary to remove the H 2 S0 4 from the samples by total evaporation, because this acid had a negative influence on arsine formation. The residue obtained aftcr cvaporation was dissolved in 25 ml distilled water. It was necessary to sonicate this solution to achieve complete solubilization. At the same time, we performed blank assays to detect any impurities resulting from the reagcnts, and the recovery assays were carried out. The As recovery was 100.9 ± 1,2 %. The analytical parameters of this technique were determined in parallel by means of standard aqueous solutions. The results obtained from thc rccovcry test, combined with the analytical parameters, indicated the validity of this method for measuring the total As content. The phenol red concentration was evaluated by absorbance at 550 nm, using a Perkin-Elmer Mod. Lambda 3B spectrophotometer, inmediately after the addition of an equal volume of 2.0 N NaOH. The instrumental setting used were those recommended in the manufacturer's manual and the analytical % Intestinal retention of As
110,---------------------------------------,
70
60 '-'-_'___'____'_____~____'______'_-L_'__'___'____'_____~____'______'___'____'____'__'__'____LJ o 5 15 30 45 60 Time (min) ~315 mOsm +240 mOsm *128 mOsm
Figure 1. Effect of the osmolarity on intestinal absorption of As(V). Percentage of intestinal retention after perfusion of rat small intestine with various phosphate buffers. The results are mean ± SO (n=5)
M.J. Gonzalez, M.V. Aguilar and M.e. Martinez
242
% Intestinal retention of As 110,---------------------------------------,
ures 1 and 2. In both experiments, the percentage of arsenic remaining in the intestinal perfusate decreased significantly (P<0.05) and proportionally in dependence on a decrease in osmolarity. As a result, we obtained a direct and significant correlation (P
o
5
15
30
45
60
Time (min) ~315 mOsm +240 mOsm *128 mOsm
Figure 2. Effect of the osmolarity on intestinal absorption of As(V). Percentage of intestinal retention after perfusion of rat small intestine with various Tris-(IICl) buffers. The results are mean ± SD (n=5)
These results on arsenic distribution, showed that its absorption increased simultaneously with the osmolarity decrease in both buffers.
The effect of water movement generated by isotonic solutions on arsenic absorption
parameters for As-AAS were: Detection limit, 0.00315
As hypotonic buffers appeared to damage the cellular
mg; sensitivity (1 % absorbance), 4.2 A/mg; precision (6
membrane and alter its permeability (26), isotonic buffers
measurement), 1.00; linear calibration range, 0-0.0250
combined with an appropriate composition of solute were used to alter the direction and degrec of the water move-
mg.
ment (Table 1). The results of these experiments are
Statistical analysis
shown in the Tables 3 and 4.
Statistical analysis was by the Biostatistical Package
"Sigma data base" [Horus Hardware1(25), which includ-
showed the highest significant values for the percentage of arsenic absorption (P<0.05) and also the highest levels
ed a MANOVA analysis (multiple analysis of variance).
for the As (V) in blood, liver, kidney, intestine, and PRF
Values were expressed as mean ± SD.
(Table 4). As the isotonic replacement of NaCI in the
Perfusion with the buffer containing 125 mM NaCI
buffer by LiCI or KCl resulted in a decrease in arsenic concentrations, As absorption, and PRF, sodium ion apResults
peared responsible for the maximum values obtained.
The effect of water movement generated by hypotonic solutions on arsenic absorption
tion, and the PRF resulting from perfusion with the buffer
The As (V) levels in organs, the percentage of absorpcontaining urea (250 mM) were high, although lower than those obtained with NaCI. The percentages of arsenic retention after perfusion
When we replaced one-half of the NaCI or urea by
with phosphate and Tris-HCl buffers are presented in Fig-
mannitol, it lead to a reduction in both the arsenic levels
Table 2. Arsenic concentration in organs (mg/kg wet weight) and blood (Ilg/ml) and PRF after perfusion using with phosphatelTris-HCI buffers with different osmolarity Phosphate buffer (pH 6.4) Osmolarity (mOsM)
Liver
Kidney
Intestine
Blood
PRF(C,fC)
315 240 128
0.089 ± 0.001 * 0.118 ± 0.021 * * 0.171 ± 0.051 *",
0.250 ± 0.089* 0.271 ± 4.2.10" 0.417 ± 4.8.1 O·H
0.402 ± 0.012* 0.412 ± (J.023 * 0.4 75 ± (J.087* *
1.738 ± 0.033* 2.382 ± 0.039* 2.840 ± 0.055*"
1.01 ± 0.002* 1.Of> ± (H)04 * 1.12 ± 0.007**
Tris-HCl buffer (pH 7.4) Osmolarity (mOsM)
Liver
Kidney
Intestine
Blood
PRF(C,fC)
315 240 128
O.I9h ± 0.056* 0.238 ± 0.023** 0.257 ± {Hl03***
0.471 ± 0.027* 0.507 ± 0.019* 0.697 ± O.O(]] **
1.007 ± 0.052* 1.167 ± 0.021 * 1.211 ± 0.024 * *
2.519 ± (J.029* 2.701 ± 0.032* 3.298 ± 0. lO9**
1.02 ± 0.004 * 1.06 ± (H111 * 1.I3 ± 0.017**
The results are mean ± SD for 5 experiments. Values in the same column bearing a common symbol are not significantly different (P> 0.(1); MAN OVA analysis of variance
The absorption of As 20, from small intestine
243
Table 3. Percentage of intestinal retention of As after the perfusion of rat small intestine with several pH=7A Tris-HCI buffers containing 3.2 mg/ml Asp,. 60 min
Salt composition
5 min
15 min
30 min
Tris + NaCI 30mM 125mM
54.7 ± 3.95
81.3 ± 0.64
82.2 ± 0042
84.2 ± 3042
81.1 ± 0.941c.Jd.g)
Tris+NaCI+Mannitol 30mM 62,5mM 121mM
70.5 ± 0.99
8304 ± 2.11
86.5 ± 0.10
85.9 ± 1.91
86.0±0.71")
Tris+Mannitol 30mM242mM
8604 ± 0.75
98.5 ± 2.12
95.1 ± 1.98
Tris+Urea+Mannitol 30mM 125mM 121mM
82.9 ± 0.28
84.2 ± 2.00
87.9 ± 1.62
94.2 ± 7.07.10"
88.9 ± 1.71 1'1
Tris + Urea 30mM 250mM
75.2 ± 0.95
8704 ± 2.05
86.3 ± 0.73
87.9 ± 5.16
86.5 ± 0.911"1
Tris + LiCI 30mM 125mM
79.2 ± 0.78
93.9 ± 1.91
94.7 ± 2.81
90.1 ± 4.03
92.7 ± 0.73"')
Tris + KCl 30mM 133mM
75.1 ± 4.53
91.9 ± 1.87
8804 ± 0.63
9004 ± 3.54
89.1 ± 1.601")
45 min
100
± 0.03
94.9 ± 0.05 10."
The results are mean ± S.D. for 5 experiments. The letters indicate statistically significant differences (P<0.05) from Tris-HCI buffers containing:(a)NaCI, (b)NaCI and mannitol, (c)mannitol, (d)urea and mannitol, (e)urea, (f)LiCI and (g)KCI; MANOYAanalysis of variance.
in all organs and PRE Perfusion with the buffer containing 242 mM of mannitol revealed the lowest values for arsenic absorption and PRE
The effect of pH on water movement and on arsenic absorption and distribution The interrelation between buffer system pH and arsenic retention or As (V) levels in organs and PRF after perfusion are shown in Figures 3 and 4 and Table 5.
The pH variations in buffer systems I and II did not alter the PRF value after 60 minutes of perfusion. The PRF after perfusion with buffer system I was higher than 1.0, and with buffer system II was lower than 1.0. At each pH, the arsenic absorption and levels in organs and PRF were significantly different between the two buffer systems. This fact corroborated the Na+ -gradient dependence of arsenic transport at the intestinal level. Because the PRF values were not altered by pH variation, this parameter did not affect water movement, al-
Table 4. Concentration of As in organs (/lg/g wet weight), blood (/lg/ml) and PRF after the perfusion with several pH=7A Tris-HCI buffers. Salt composition
Liver (mg/kg)
Kidney (mg/kg)
Intestine (mg/kg)
Blood (mg/kg)
PRF (C/C)
Tris + NaCI 30mM, 125 mM
0.216 ± 0.03
0.335 ± 0.03
1.281 ± 0.04
2.985 ± 0.08
1.08 ± O.()]
+ NaCI + Mannitol 62,5 mM, 121 mM
0.166 ± 0.01
0.278 ± 0.02
1.079 ± 0.03
1.947 ± 0.06
1.02 ± 0.01
+ Mannitol 242mM
0.092 ± 0.02
0.165 ± 0.03
0.796 ± 0.03
1.716 ± 0.03
0.99 ± 0.01
+ Urea + Mannitol 125 mM, 121 mM
0.160 ± 0.01
0.284 ± (l.(ll
1.806 ± 0.09
1.01 ± 0.01
+ Urea 250mM
0.190±0.01
0.303 ± 0.01
1.0l0 ± 0.11
1.846 ± 0.17
1.04 ± 0.01
+ LiCI 125 mM
0.064 ± 0.00
0.204 ± 0.02
00465 ± 0.07
1.650 ± 0.07
1.00 ±O.O
+KCL 133mM
0.134 ± 0.02
0.266 ± 0.01
0.973 ± 0.11
1.904 ± 0.07
un ±O.O
The results arc the mean ± S.D. for 5 experiments.
0.862 ± 0.06
244
M.1. Gonzalez, M.V. Aguilar and M.e. Martinez
% Intestinal retention of As % Intestinal retention of As 110----------------------------------------, 110"------------------------------------,
100
I
90
o
15
5 ~pH
=
30
Time (min) 5.5 +pH 6.4 *pH
=
45
60
::l
0
though it modified arsenic absorption: an increase in the pH resulted in a noticiable decrease in the arsenic level in organs and in As absorption (Figures 3 and 4). Furthermore, high, and negative correlation coefficients (P
The effect of membrane potential on As (V) absorption The effect of K+ diffusion potential generated by valinomycin on the uptake of As (V) was studied in the presence of an outward-directed gradient of K+ . Valinomycin is an antibiotic which behaves as a ionophore due to specific transport of K+ ions through cell membranes (27) (see Figure 5 and Table 6). In the presence of valinomycin, both the absorption of arsenic and its levels in organs were significantly enhanced.
15
30
Time (min) ~pH = 5.5 ~pH 6.4 *pH
=
= 7.4
Figure 3. Effect of pH on intestinal absorption of As(V). Percentage of intestinal retention after perfusion of rat small intestine with buffer system I. The results are mean ± SD (n=5). *=significant difference (p<0.05) with respect to pH=5.5.
5
45
60
=7.4
Figure 4. Effect of pH on intestinal absorption of As(V). Percentage of intestinal retention after perfusion of rat small intestine with buffer system II. The results are mean ± SD (n=5). *=significant difference (p<0.05) with respect to pH=5.5.
Discussion
The effect of water movement generated by hypotonic solutions on arsenic absorption The As osmolarity-sensitive uptake by the intestine implies a saturable uptake kinetics; suggesting the participation of some type of carrier-mediated transport system (28). In addition, this phenomenon suggests that the increase in arsenic absorption and levels in the intestinal tissue is accompanied by an increase in water absorption, due to the correlation between arsenic absorption and the PRF observed (the PRFs are greater than 1 in all the cases). Statistically significant differences have been observed between blood levels and concentrations in organs, as well as between percentages of intestinal retention obtained with phosphate buffers and Tris-HCI buff-
Table 5. Effect of pH of perfusion buffers on As (V) concentrations in organs (mg/kg wet weight) and blood (mg/L) System I (Na+) pH
Liver
Kidney
Intestine
Blood
PRF(C;C)
5.5 6.4 7.4
0.221 ± 0.018* 0.201 ± 0.021 * 0.175 ± 0.039*
0.482 ± 0.035* 0.437 ± 0.008* 0.353 ± 0.042**
1.398 ± 0.089* 1.199 ± 0.140** 1.006 ± 0.068 * *
3.076 ± 0.248* 2.120 ± 0.154* 1.793 ± 0.029**
1.07±0.003* 1.06±0.006* 1.05±0.005 *
pH
Liver
Kidney
Intestine
Blood
PRF(C;C)
5.5 6.5 7.4
0.165 ± 0.025* 0.131 ± 0.020** 0.126 ± (J.0l2**
0.321 ± 0.027* 0.290 ± 0.068* 0.249 ± 0.026*
0.878 ± 0.047* 0.854 ± 0.043* 0.773 ± 0.055*
2.457 ± 0.610* 2.214 ± 0.084* 1.432 ± 0.184**
O. 997±0.004 * 0.986±0.005* O.971±O.003*
System" (Mannitol)
The results are mean ± SO for 5 experiments. Values in the same column bearing the common symbol are not significantly different. Values in the same column bearing the different symbol are significantly different P< 0.001. MANOVA analysis of variance.
The absorption of Asp, from small intestine
ers. These differences may be caused by the pH, which is different in both solutions, or a possible interaction of the phosphate anion with arsenate intestinal absorption.
The effect of water movement generated by isotonic solutions on arsenic absorption Results suggest that the degree of As (V) absorption and accumulation in the intestinal tissue depend on the direction and volume of the water movement. Thus, a decrease in osmolarity and the variation of the sodium ion and urea concentration in buffer solutions cause an increase in water absorption. Consequently, this increase in water absorption also implies an increase in the levels of As (V) in blood, liver, kidney, and intestine, partially due to the solvent drag effect. A similar effect has been reported in experiments with mercury (15), magnesium (26), and certain organic compounds (29). The results of these experiments may indicate that arsenate is transported across epithelial cells of the intestinal mucous membranes by a secondary active transport. This transport system depends on an Na+ -gradient (sodium transport). A similar transport system is used for the intestinal absorption of glucose, galactose, and various aminoacids. The carrier of As (V), like those utilized by the substances mentioned above, as probably located within the intestinal brush-border membrane (30). However, this carrier does not seem to transport arsenate without the presence of sodium ions. The carrier appears to have a
245
receptor place for As (V) and Na+. In addition, this carrier does not introduce arsenate into the cell if the receptor place for As (V) and Na+ are not simultaneously occupied. The driving energy required to allow the passage of the carrier from the outer side of the membrane comes from the difference in sodium ion concentration between both sides of the membrane. When Na+ diffuses into the cells across the sodium channel, it "pulls" the carrier and the arsenate too, providing the energy necessary for transport. Inside the epithelial cells, arsenate ions diffuse in every direction, including into the portal blood. As regards the nature of the possible carrier, it may be a protein with a high molecular weight (31) and rich in SH-groups. This hypothesis is supported by Sorensen (32), who observed that As was found in the form of inclusions as a metal-protein complex in the nucleus of the cell. The Rosen (33) and San Francisco (34) teams identified and purified a protein called "Ars B protein", which is located in the membrane of Escherichia coli, and is part of a specific anion pump. This anion pump is responsible for resistance to and transport of arsenate from these cells. The amino-acid sequence for this protein proposed by these authors is: Met-Gln-Phe-Lue-Gln-Asn-Jle-ProPro-Tyr-Leu-Phe. This protein, or another with similar characteristics, may be the carrier involved in the arsenate transport at the intestinal level. The exact number of the possible carriers for arsenate will be studied by our team in the near future.
The effect of pH on water movement and on arsenic absorption an distribution % Intestinal retention 105r-----------------------------------~ I
1009S
L
90 85 80
The same effect of pH on Asps absorption revealed in our experiments was observed by Meharg and Macnair (1991)(35) in research on As (V) uptake in Holcus lanatus. These authors attributed the increase in arsenate uptake to the different chemical form this compound Table 6. Effect of valinomycin on levels of As (V) in organs (mg/kg wet weight) and blood Cmg/L)
75 70~------~-------------------------L~
o
5
15
30
45
60
Time (min) ~ Control -fr Valinomycine Figure 5. Effect of membrane potential on intestinal absorption of As (V). Percentage of intestinal retcntion after perfusion of rat small intestine with Tris-(HCl) buffer with or without valinomycin. The resulta are mean ± SD (n=5). *=significant difference with rcspect to contra.1.
Liver Treatment with Valinomycin O.361±0.030*
Kidney
Intestine
0.282±0.016* 1.798±0.1l3*
Blood
2.122±0.212*
Control 0.149±O.037* * O.234±O.003 * O.658±O.123 * * 1. 754±0.065* * The results are mean ± SD for 5 experiments. Values in the same column bearing a common symbol are not significantly different (P> 0.(1); MAN OVA analysis of variance.
246
MJ. Gonzalez, M.V. Aguilar and M.e. Martinez
assumes depending on the pH: arsenate uptake is optimal at pH=5 when HcAs04 is the dominant anion in solution; as the pH is increased to 8 and HAs0 42 becomes the dominant form, arsenate uptake decreases due to its lower liposolubility. The pH influence on arsenic absorption may indicate dependence on an W -gradient involved in the As (V) absorption mechanism. Arsenate transport across the intestinal cell might be mediated by a coupled symport with W (or antiport with the hydroxy ion). Although the H+ -gradient-dependent transport system responsible for As (V) absorption was not defined, a similar proton-gradient-dependent transport for antibacterial agents has been found, ~-lactamic antibiotics in the rat small intestine (36, 37). Furthermore, several nutrients such as a small peptide (38), folate (39), oxalate (40), Dglucose (41), lactic acid (42), and nicotinic acid (43) are transported via some proton- or hydroxy ion-gradient dependent carrier system cross the intestinal brush-border membrane. Consequently, the role of an H+-gradient across the brush-border membrane in the intestinal absorption of nutrients and drugs is as important as the role of the Na+ -gradient. The pH effect and the solvent drag effect appear to act independently because: the pH effect was observed under predominance of water absorption (system I) and water secretion (system II); the arsenic concentration in each organ and the arsenic absorption after perfusion with buffer system I were higher than with buffer system II.
The effect of membrane potential on As (V) absorption The arsenic absorption, as well as its levels in organs (except kidney), were significantly enhanced by inducing a relatively inside-negative membrane potential, suggesting an electrogenic transport of arsenate was suggested. All the characteristics of Na+-gradient, H+-gradient and membrane potential dependencies are advantageous for the arsenate absorption in the small intestine under physiological conditions; since the luminal side of the brush-border membrane is known to be Na+ -rich, to show an acidic microclimate pH (pH=6.0) and a relatively positive membrane potential compared to intraenterocytes due to the presence of sialic acid residues and sulfate groups (44). In conclusion, As (V) may be absorbed from the small intestine via a carrier-mediated mechanism depending on Na+- and H+-gradients and potential differences across
the intestinal brush-border membrane. The carrier system available for arsenate is suggested to be partially shared with the inorganic transport system. This hypothesis is based on the transport characteristics of phosphate, which show Na+- and H+-gradient dependence which quite similar to those present in arsenate transport (45,46,47,48).
References 1. IRGOLIC, K.J. (1986 )Arsenic in the Euvironment. In Frontiers in BioinorganicChemistry. Edited by Xavier AV Ed. VCH Publishers, Weinheim, 399-408 2. NASS (1977) Arsenic. Natl Acad Sci, Washington, D.e., 332. 3. GATES, M.; WILLIAMS, J.W AND ZAPP, lA. (1942)Arsenicals. In: Chemical warfare agents and related chemical problems, Summary Technical Report of Division 9, NRDC vol I, Parts I-II, Chapter 7. National Defense Research Committee, Washington, D.C., 83-114 4. PERSHAGEN, G. (1983) The Epidemiology of Human Arsenic Exposure. In Biologica and Environmental Effect of Arsenic. Edited by B.A. Fowler Ed. Vol 6. Elsevier Science Publisher, Amsterdam,199-232 5. SQUIBB, KS. AND FOWLER, B.A. (1983) The toxicity of Arsenic and its Compounds. In Biological and Environmental Effect of Arsenic. Edited by B.A. Fowler Ed. Vol 6, Elsevier Science Publisher, Amsterdam, 233-269. 6. FIELDER, R.J.; DALE, E.A. AND WILLIAMS, S.D. (1986) Inorganic Arsenic Compounds. Health and Safe Commission's Advisory Committee on Toxic Substance. Health and Safety Executive. Her Majesty's Stationery Office, London, Toxicity Review 16, 1-95. 7. IRGOLIC, KJ. (1986) Health effects of arsenic in the aqueous environment. Northeast Environmental Science 5, 86-90. 8. PER SHAG EN, G. (1986) Sources of Exposure and Biological Effects of Arsenic. In Environmental carcinogens-selected methods of analysis. Edited by I.K O'Neill, P. Schuller & L. Fishbein Eds. vol8. International Agency for Research on Cancer, World Health Organization, Lyon, 45-61 9. DIAZ DE TORANZO, E.G. (1990) Absorption of toxic substances. A basic concept in toxicology. Acta Bioquimica Clinica Latinoamericana 24 (4), 375-83. 10. REITZ, R.H. (1990) Distribution, persistence and elimination of toxic agents (pharmacokinetics). In Progress in Predictive Toxicology. Edited by D.B. Clayson, I.e. Munro, P. Shubik & Swenberg Ed. Elsevier Science Publishers B.Y., Amsterdam, 79-90. 11. TABCHE, L.M.; NAVARRETE, L.M. AND GERMAN, F.e. (1984) Effect of iron, calcium and lead on arsenic absorption in isolated rat intestine. Acta Mexicana de Ciencia y Tecnologia 2(6), 57-62. 12. KLEVAY, L.M. (1976) Pharmacology and toxicology of heavy metals: Arsenic. Pharmacological Therapeutics AI, 189-209 13. CULLEN, WR.; MCBRIDE B.e. AND PICKETT. A.W (1990) The uptake of arsenicals by Candida humicola. Applied Organometallic Chemistry 4,119-125. 14. HWANG, S.W AND SCHANKER, L.S. (1973) Absorption of organic arsenical compounds from the rat small intestine. Xenobiotica 3, 351-355. 15. ENDO, T.; NAKAYA, S. AND KIMURA, R. (1988) Mechanisms of Absorption of Inorganic Mercury from Rat Small Intestine. 1. Solvent drag Effect on Absorption of Inorganic Mercury. Pharmacology and Toxicology (Copenhagen) 65, 128-35.
Thc absorption of 16. CASTLE, SJ.; TUCKER. G.T.; WOODS, H.E; UNDERWOOD, J.C.E.; NICHOLSON, M.E.; HAVLER, C.M.; LEWIS, CJ.; FLOCKHART, 1.R. AND LLOYD-JONES, G. (1985) Assessment of an in situ rat intestine preparation with perfused vascular bed for studying the absorption and first -pass metabolism of drugs. Journal of Pharmacological Methods 14, 255-274. 17. SAKAI. K.; AKIMA M. AND HINOHARA, Y. (1980) Vasculariy perfused rat small intestine: a research model for drug absorption. Japan Journal of Pharmacology 30, 231-241. 18. OCHSENFAHRT, H. AND WINNE, D. (1979) The contribution of solvent drag to the intestinal absorption of tritiated water and urea from the jejunum of the rat. Naunyn-Schmiedebergs Archives of Pharmacology 279,133-152. 19. ENDO, T; NAKAYA, S.; KIMURA R. AND MURATA, T (1984) Gastrointestinal Absorption of Inorganic Mercuric Compounds in Vivo and in Situ. Toxicology and Applied Pharmacology 74, 223-229. 20. WINDMUELLER, H.; SPAETH, AE. AND GANOTE, C.E. (1970) Vascular perfusion of the isolated rat gut: norepinephrine and glucocorticoid requirements. American Journal of Physiology 218,197-204. 21. DOWLING, HJ.; OFFENBACHER, E.G. AND PI-SUNYER, EX. (1989) Absorption of Inorganic, Trivalent Chromium from the Vascularly Perfused Rat Small Intestine. American Institute of Nutrition 1138-1145.
Asp, from small intestine
247
33. ROSEN, B.P.; WEIGEL, U.; KARKARIA, C. AND GANGOLA P. (1988) Molccular characterization of an anion pump. The ars A gene product is an arsenite (antimonate)-stimulated ATPase. Journal of Biology and Chemistry 263, 3067-3070. 34. SAN FRANCISCO, MJ.D.; TISE, L.S. AND ROSEN, B.P. (1989) Identification of the membrane component of the anion pump encoded by the arsenical resistance operon of R-factor R773. Molecular Microbiology 3( 1), 15-21. 35. MEHARG, A. AND MACNAIR, M.R. (1990) An altered phosphate uptake system in arsenate tolerant Holcus lanatus L. New Physiology 116,29-35. 36. TSUJI, A.; TERASAKI, T; TAMAI, I. AND HIROOKA, H. (1987) H+ Gradient dependent and carrier~mediatcd transport of cefiximc, a new cephalosporin antibiotic, across brush-border membrane vesicles from rat small intestine. Journal of Pharmacology and Experimental Therapeutics 241, 594-60 I. 37. TAMAI, I.; TSUJI, A. AND KIN, Y. (1988) Carrier-mediated transport of cefixime, a new antibiotic, via an organic anion transport system in the rat renal brush-border membrane. Journal of Pharmacology and Experimental Therapeutics 246, 338344. 38. GANAPATHY, V AND LEIBACH, EH. (I985) Intestinal pep~ tide transport energized by proton gradient? American Journal of Physiology 249, GI53-GI60.
22. KITAZAWA, S.; ITO, H. AND SEZAKI, H. (1975) Transmucosal fluid movement and its effect on drug absorption. Chemical and Pharmaceutical Bull 23, 1856-1865.
39. SCHRON, C.M.; WASHINGTON, C. JRAND BLITZER, B.L. (1985) The transmembrane pH gradient drive uphill folate transport in rabbit jejunum. Direct evidence for folate hydroxyl exchange in brush-border membrane vesicles. 10urnal of Clinical Investigation 76, 2030-2033.
23. KOIZUMI, T.: ARITA, T AND KAKEMI, K. (1964) Absorption and excretion of drugs Some pharmacokinetic aspect of absorption and excretion of sulfonamides. (1). Absorption from rat stomach. Chemical and Pharmaceutical Bull 12,413-420.
40. KNICKELBEIN, R.G.; ARONSON, P.S. AND DOBBINS, J.W. (1986) Oxalate transport by anion exchange across rabbit ileal brush-border. Journal of Clinical Investigation 77, 170175.
24. HERSHEY, J.w. AND OOSTDYK. TS. (1988) Determination of arsenic and selenium in environmental and agricultural samples by hydride generation atomic absorption spectrometry. Journal of Association Official of Analysis and Chemistry 71(6), lOW).
41. HOSHI T; TAKUWA, N.; ABE, M. AND TAJIMA, A. (1986) Hydrogen ion-coupled transport of D-glucose by phlorizinesensitive sugar carrier in intestinal brush-border membranes. Biochemical and Biophysical Acta 861, 483-488.
25. FERNANDEZ-PERIS, E.; MOLINERO CASARES, C.M. AND MOERU-ABOAL, E. (1986) Sigma. Base de datos bioestadisticos para un ordenador personal. Horus Hardware. Madrid. 26. BEHAR, J. (1974) Magnesium absorption by rat ileum and colon. American Journal of Physiology 227, 334-340. 27. RAWN, J.D. (1990) Bioquimica, Inter Americana. McGrawHill, Madrid, 1037. 28. ISHIZAWA, T; TSUJI, A; TAMAI, I.; TERASAKI, T; HOSOI, K. AND FUKATSU, S. (1990) Sodium and pH Dependent Carrier-Mediated Transport of Antibiotic, Fosfomycin, in the Rat Intestinal Brush-Border Membrane. Journal of Pharmacobiology-Dyn 13, 292. 29. KARINO, A; HORIE HAYASHI, T; AWAZU, S.; MINAMI, H. AND HANANO, M. (1982) Solvent drag effect in drug intestinal absorption. 1. Studies on drug and D,O absorption clearances. Journal of Pharmaceutical Dyn 5, 410-417. 30. CHAUNCEY, B.; SCHMID, E.C. AND GOLDTEIN, L. (1988) Arsenical and mercurial inhibition of tyrosine transport by the flounder intestine. Journal of Toxicology and Environmental Health 23(2), 275-65. 31. MARAFANTE, E.; RADE, J.; PIETRA, R.; SABBIONI, E. AND BERTOLERO, E (1980) Metabolic fate of inorganic arsenic in laboratory animals. In 3 Spurenelement-Symposium, Arsen.Edited by M. Anke, HJ. Schneider and Chr. Brucker Eds. Karl-Marx Univ., Leipzip, Fried.-Schiller-Univ., Jena, 49-57. 32. SORENSEN, E.M.B. (1987) Ultrastructural cytochemical analysis of intranuclear arsenic inclusions. Journal of Toxicology and Environmental Health 22, 273-285.
42. SIMANJUNTAK, M.T; TAMAI, I.; TERASAKI, T AND TSUJI, A. (1990) Carrier-mediated uptake of nicotinic acid by rat intestinal brush-border membrane vesicles and relation to monocarboxylic acid transport. Journal of PharmacobiologyDyn 13,301-309. 43. TIRUPPATHI, c.; BALKOVETZ, D.E; GANAPATHY, V; MIYAMOTO, Y. AND LEIBACH, EH. A proton gradient, not a sodium gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles. Biochemical Journal 256, 219-223. 44. VAN HOOGDALEM, EJ.; DE BOER, A.G. AND BREIMER, D.D. (1989) Intestinal Drug Absorption Enhancement: an overview. Pharmaceutical Therapeutics 44, 407-443. 45. DANISI, G.; MURER, H. AND STRAUB, R.W. (1982) Effect of pH on phosphate transport into intestinal brush-border membrane. American Journal of Physiology 242, G533 G539. 46. BELIVEAU, R. AND STREVEY, 1. (1991) Phosphate transport in kidneys: effect of transmembrane electrical potential. American Journal of Physiology 261(4, Pt. 2), F663-F669. 47. QUAMME, G.A. AND SHAPIRO, RJ. (I986) Membrane controls of epithelial phosphate transport. Canadian Journal of Physiology and Pharmacology 65, 275-286. 48. SHIRAZI-BEECHEY, S.P.; BEECHEY, R.B.; PENNY, J.; VAYRO, S.; BUCHAN, W. AND SCOTT, D. (1991) Mechanisms of phosphate transport in shcep intestine and parotid gland: response to variation in dietary phosphate supply. Experimental Physiology 76(2), 231-241.