kinetic characterization of in vitro lead transport across the rat small intestine: mechanism of...

TOXICOLOGY AND APPLIED PHARMACOLOGY 61, 39-47 ( 198 1)

Kinetic Characterization of in vitro Lead Transport across the Rat Small Intestine

Mechanism of Intestinal Lead Transport

BRUCE J. AUNGST AND HO-LEUNG FUNGI

Department of Pharmaceutics, School of Pharmacy, State University of New York at Buffalo, Amherst, New York 14260

Received February 18. 1981: accepted June 4, 1981

Mechanism of Intestinal Lead Transport. Kinetic Characterization of in Vitro Lead Trans- port across the Rat Small Intestine. AUNGST, B. J., AND FUNG, H.-L. (198 1). Toxicol. Appl. Pharmacol. 61, 39-47. Lead transport through the everted rat small intestine was used as an in vitro model to examine the kinetics and mechanism of gastrointestinal lead absorption. Mucosal-to-serosal lead flux increased nonlinearly with increasing mucosal lead concentrations (0.5-48.3 PM), and an apparent capacity-limited lead flux was observed with intestines of both adult and adolescent rats. Uptake of lead by the adult intestinal tissue, measured at the end of the flux experiments, was also nonlinearly related to mucosal lead concentration. Both intestinal uptake and mucosal-to-serosal flux were reduced by anoxia and by inhibition of glycolysis using fluoride. The data were consistent with a model of lead intestinal transport which included both a carrier-mediated component and passive diffusion. Kinetic parameters for these transport processes were obtained. The relative contribution of passive diffusion to total lead llux increased with increasing lead concentration, but was quantitatively minor (~20%) at all lead concentrations tested. The apparent capacity-limited component of lead flux was not due artifactually to reduced lead solubility in the mucosal solution at high metal concentrations, nor was it due apparently to reduced viability of the intestinal tissue, since lead did not alter 3-Gmethylglucose transport over the range of lead concentration used. When the same lead solution was placed on both sides of an everted intestine, net serosal-to- mucosal lead flux was observed at low lead concentrations. It is therefore likely that intestinal lead transport may be bidirectional, similar to calcium transport.

The identification of impaired intelligence in children with abnormally high concentrations of lead in dentine (Needleman et al., 1979) emphasizes the need for limiting exposure to environmental lead. Major sources of the body lead burden at normal levels of exposure are food (Mahaffey, 1977; Rabi- nowitz et al., 1977), including specific foods with high concentrations of lead (Settle and

’ To whom correspondence should be addressed: De- partment of Pharmaceutics, School of Pharmacy, 517 Hochstetter Hall, State University of New York at Buf- falo, Amherst, N.Y. 14260.

Patterson, 1980), and water (Thomas et al., 1979). In children, ingestion of dust and soil also contributes significantly to elevated blood lead levels (Roels et al., 1980). Thus, lead toxicity could be prevented, in principle, by inhibiting its absorption through the gastrointestinal tract. A thorough understand- ing of the mechanism of lead absorption is essential if efforts toward prevention of lead exposure via this approach are to be ratio- nally formulated.

The mechanism of gastrointestinal absorption of lead has not been clearly described. It has been postulated that lead

39 0041-008X/81/130039-09$02.00/0 Copyright 8 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

40 AUNGST AND FUNG

shares intestinal absorption sites with calcium and iron, because of the apparent changes in lead absorption when dietary calcium (Six and Goyer, 1970; Meredith et al., 1977) and iron (Six and Goyer, 1972) are manipulated. Indeed, competitive inhibition of lead binding to mucosal proteins by calcium (Barton et al., 1978a) and iron (Barton et al., 1978b) has been demonstrated. The absorption of orally administered lead, like that of calcium and iron, is highest in new- borns (Kostial et al., 1971) and decreases with age (Forbes and Reina, 1972). Thus, it appears likely that absorption of lead through the gastrointestinal tract is regulated, and perhaps limited, by a requirement of binding to transport protein(s).

The role of passive diffusion in lead absorption has not been delineated. Meredith et al. (1977) proposed that more than one mechanism may be responsible for lead absorption because calcium, even at very high concentrations in the gut, had a limited competitive effect on lead absorption. Based on the differences in absorption kinetics between normal and iron-deficient mice, Flan- agan et al. (1979) also suggested multiple mechanisms for lead absorption. No quan- titative data exist, however, which assess the relative contribution of passive diffusion vs active transport in lead absorption. In this report, we examined this aspect of lead absorption by studying the effect of lead concentration on its in vitro flux through everted intestines from both adult and adolescent rats. In vitro kinetic parameters for passive and active lead transport were determined.

METHODS

Animals. Male Sprague-Dawley rats (Holtzman Breeders, Madison, Wise.) were used for all experiments. One group consisted of adult animals weighing 200-300 g, and another group was 27- to 33-day-old rats which weighed 40-90 g. All rats were housed prior to the experiments in stainless-steel cages in a room with a 12-hr dark, 12-hr light illumination cycle. The diet was Charles River RMH 1000 (Agway). The experiments were performed following an overnight fast.

Preparation of isolated intestine. Rates of lead and 3-Qmethylglucose transfer across the rat small intestines were determined in vitro using the everted intestine preparation of Crane and Wilson (1958). Rats were decapitated and a single 12-cm-length segment of intestine was removed from each rat approximately 10 cm from the pylorus. This was rinsed with saline, everted,

and tied at one end, with the other end attached to glass tubing for removal of samples. The intestine was then put into 90 ml of an isotonic Tris buffer (pH 7.4) containing 0.2% glucose and a known concentration of lead and/or 3-O-methylglucose. The buffer was kept at 37°C and continually gassed with 95% 0,/5% CO*. A Tris buffer was used instead of the usual buffers containing carbonate or phosphate in order to avoid lead precipitation.

3-0-Methylglucoseflux. As an index of the biochem- ical viability of the intestine preparations, flux of 3-O [ “C]methyl-d-glucose (Amersham) was determined in the absence of lead and in the presence of 0.5 and 48.3 &M lead. Intestines from adult rats were prepared as described and placed in Tris buffer containing 0.2% glucose and 0.5 pCi 3-O-[‘4C]methylglucose (specific activity, 58.9 mCi/mmol). Tris buffer placed inside the intestine sacs was free of lead, glucose, and 3-Ometh- ylglucose. The solution inside the intestine sac was replaced five times during 40 min of incubation. Total radioactivity in the samples removed from the sac was then determined, and the counting efficiency was obtained with the addition of [“C]toiuene (Packard) in- ternal standard. 3-0-methylglucose flux was measured in intestines from five rats at each lead concentration.

Lead flux and intestinal lead uptake. Lead flux was measured in intestines from both adult and adolescent rats. The range of lead concentrations in the mucosal bathing medium was from 0.5 to 48.3 NM. One milliliter of a lead-free Tris buffer was placed inside the intestinal sac from the adult rats, while 0.7 ml was put into the sacs from the adolescent rats. The solution inside the sac was removed entirely four or five times during 40 min of incubation and was replaced with lead-free buffer each time. At the lowest mucosal lead concentration used (0.5 PM), only three samples, at 15-min intervals, were removed. The samples were assayed for lead as described later. Intestines from five to nine rats were used at each lead concentration. To examine the effects of anoxia, experiments also were performed in which the mucosal buffer solution was gassed with N2 instead of the Or/CO2 mixture. The effect of metabolic inhibition on lead flux was studied using an isotonic Tris buffer containing 0.02 M NaF and 0.2% glucose.

Following the 40-min incubation, each adult rat intestine was removed from the apparatus and blotted to remove excess buffer. The segment of intestinal tissue was then weighed, minced, and frozen until analysis for lead concentration as described later.

In those experiments in which the buffer within the

LEAD TRANSPORT ACROSS SMALL INTESTINE 41

intestinal sac was removed at regular intervals and replaced with lead-free buffer, the mucosal-to-serosal lead concentration gradient was maintained, creating a “sink” condition. Lead transport by the rat intestine at a nonsink condition also was investigated. Everted intestines were prepared as described, but instead of in- serting a lead-free buffer inside the sac, the same lead- containing buffer was placed on both sides of the intestine. For these experiments a single sample was taken from both the mucosal and serosal buffers after 40 min of incubation, and the lead concentration ratio was calculated. Two initial lead concentrations were used for these experiments (2.4 and 48.3 PM), with six animals in each group.

Sample analyses. All glassware was washed with 10% nitric acid prior to use to prevent lead contamination. Lead concentrations in the Tris buffer samples were determined by atomic absorption spectrophotometry (AAS) after diluting the samples with 2 or more vol of 0.1 N nitric acid. Five milliliters of an organic base tissue solubilizer (TS- I, Research Products International) was added to minced intestines. This mixture was then son- icated and the resulting solution, which was free of par- ticulate matter, was diluted to at least 10 ml with toluene and analyzed by AAS. Intestinal lead uptake was calculated by subtracting individual endogenous intestinal lead concentrations from those obtained following incubation in the lead-containing buffer.

For all analyses, a Perkin-Elmer Model 603 atomic absorption spectrophotometer equipped with an HGA- 2200 graphite furnace and deuterium arc background correction was used. Tbe analytical wavelength was 283.3 nm. The optimum dry, char, and atomization parameters were determined for both the buffer and sol- ubilized intestine matrices. The buffer samples were dried for 70 set at 135”C, charred for 15 set at 400°C. and atomized for 10 set at 2lOO“C. The optimum program for the solubilixed intestine samples was to dry at 60°C for 60 set, char 30 set at 4OO”C, and atomize 10 set at 2100°C. The graphite tube was purged with NZ, with flow interrupted for 7 set at the start of atomixa- tion. Calibration curves were made daily using blank

buffer or intestine samples spiked with a certified atomic absorption lead standard (4.8 mM lead in dilute nitric acid, Fisher). This standard was also used to prepare the mucosal buffer solutions prior to the flux experiments.

RESULTS

Chronic administration of lead to rats impaired intestinal transport mechanisms for glucose, sodium, glycine, lysine, and phe- nylalanine noncompetitively (Wapnir ef al., 1977). To determine whether short-term

lead exposure had any damaging effect on intestinal function in vitro, flux of 3-G [ “C]methyl-d-glucose was measured at lead concentrations of 0, 0.5, and 48.3 pM. Flux was calculated as the cumulative amount (dpm) of 3-O-methylglucose transferred from the mucosal solution to the serosal solution per minute of incubation. As shown in Fig. 1, lead had no effect on 3Gmethylglucose transport. Methylglucose was transported at a constant rate during the incubations, sug- gesting that intestinal viability was retained during the 40-min experiments.

Lead flux across everted rat intestines and intestinal lead uptake were measured after 40 min of incubation in biological buffers containing lead concentrations ranging from 0.5 to 48.3 pM. The flux patterns were similar at all concentrations. Plots of the mean data obtained from the intestines incubated with the highest and lowest lead concentrations are shown representatively in Fig. 2. For each experiment, the amount transferred vs. time plot was linear and the slope was identified as lead flux. The intercept values were small. Linear flux during the course of everted intestine experiments has been considered an index of maintenance of intestinal integrity (Benet et al., 197 1).

Lead flux increased with increasing medium concentrations more steeply at mu-

I I 1 I 0

10 20 30 40 Lead Concrntrotion (pM)

I 50

FIG. 1. Flux of 3-0-[‘4C]methytd-glucose across proximal rat small intestines in vitro, measured with no lead added to the bathing medium and at two lead concentrations. The mean of five experiments and the standard error are represented for each group.

42 AUNGST AND FUNG

A is the area of membrane exposed to lead, and h is the thickness of the membrane (Bates and Gibaldi, 1971). If A and h are considered constant and C,,, b C,, Eq. (1) can be simplified to

=KC,,,, (2) passive

0 15 30 45 Time (min)

where K is a constant with units of liters per minute. Flux due to a capacity-limited process can be represented by

FIG. 2. Results obtained from everted intestine ex-

periments using buffer lead concentrations of 0.5 (0) and 48.3 PM (0), as the mean + SE of six and seven

intestines, respectively. Lead flux was calculated as the regression slope of the amount transferred vs time plot

for each individual intestine.

V max *Gl = K,,, -I- C,,,

(3) capacity-limited

with V,,,,, and K,,, being Michaelis-Menten parameters for the transport system.

cosal lead concentrations below 2.4 PM than those above it. In fact, lead flux at concentrations above 4.8 PM was only marginally dependent on mucosal lead concentration (Fig. 3). In all the experiments, the lead concentrations measured in buffer from the mucosal side following 40 min of incubation and after centrifugation were within 90% of the initial concentrations. Thus, the plateau- ing of flux at higher lead concentrations was not due artifactually to precipitation of in- soluble lead salts from the medium bathing the mucosa.

The total flux contributed jointly by both passive diffusion and capacity-limited processes can then be described using

V max. Cm

tota, = Km + Cm +KC,,,. (4)

The mean data reported on Fig. 3 were fit to Eq. (4) with the nonlinear least-squares computer program NONLIN (Metzler, 1969). The estimates (mean + SD) obtained for the transport kinetic parameters were: K,,,, 0.59 f 0.13 PM; V,,,,,, 1.44 X lo-5 + 7.7 X lo-’ pmol/min; and K, 7 t- 2

The flux data were consistent with a lead transport model in which both capacity-limited and passive processes were involved. The rate of appearance of lead on the serosal side of the membrane due to passive diffusion can be expressed by

2.5-

8 x 2.0-

.f I T i

dx, c-1

= MCln - Cd tA z 0.5

dt hX 1000 ’ (1) passive

4 F I I I I I

0 lo 20 30 40 50 where X and C are amount and concentra- Maeowl Lead Concentration (FM)

tion (nM), respectively, with the subscripts s and m representing serosal and mucosal

FIG. 3. Lead flux across adult rat small intestines

levels, respectively; k is a diffusion constant (mean f SE) measured at various buffer lead concen-

with units of square centimeters per minute, trations. The line was drawn from the parameters obtained by computer fitting the data to Eq. (4).


TABLE 1

EXPERIMENTAL AND PREDICTED FLUXIS USING EQ. (4). WITH K,,, = 0.59 CM, V,, = 1.44 X 10-s cmol/min, K = 7 x lo-* liters/min AND THE PREDICTED RELATIVE CONTRIBUTIONS OF CARRIER-MEDIATED TRANSPORT

AND PASSIVE DIFFUSION

G (PM)

Measured Prexlicted flux bmol/min) flux (pmol/ AIT

min) Total (7) Passive (P) Active (A) XlOO?G

0.5 5.98 x IO+ 1.2 1.03 x lo-5 2.4 1.19 x lo-s 4.8 1.32 X lo-’

12.1 1.39 x lo-+ 24.1 1.66 x lo-5 36.2 1.60 X lo-’ 48.3 1.80 x lo+

6.50 x lo-+ 9.76 X lo+ 1.17 x 10-S 1.32 X lo-’ 1.46 X lo+ 1.58 X lo+ 1.68 x lo-’ 1.77 x 10-s

3.47 x 10-s 8.74 x 10-s 1.74 x lo-’ 3.49 x lo-’ 8.72 X lo-’ 1.74 x 10-6 2.62 x lO-6 3.49 x 10-6

6.46 X lo+ 99.4 9.68 X 10d 99.2 1.16 X 1O-5 99.1 1.28 X lO-5 97.0 1.37 x lo+ 93.8 1.40 x 10-J 88.6 1.41 x lo+ 83.9 1.42 X 10-s 80.2

X lo-* liters/min. Table 1 gives a compar- ison of the predicted flux, based on these parameters, the measured flux, and the predicted contributions of capacity-limited transport and diffusion to the total flux. Lead transport was effected almost exclu- sively through the active route at low lead concentrations, with increasing relative contribution from passive diffusion as the lead concentration was increased. However, even at the highest lead concentration studied, passive diffusion contributed less than 20% to the total flux of lead through the intestine.

Intestinal lead concentrations also were measured in the adult rats at the end of the flux experiments. The tissue concentration is expected to reflect both the buffer/tissue partition coefficient and the extent of tissue binding that might have occurred. Uptake of lead by the intestines increased with increasing buffer concentrations, but as was seen with flux, the increases in uptake were not proportional to the increases in lead concentration (Fig. 4). Because of the irregular shape of the uptake/concentration curve, these data were not suitable for computer fitting to obtain estimates for the binding parameters and partition coefficient. Within any group there was no apparent relationship between the intestinal lead content and the measured lead flux.

Because lead absorption in vivo is age dependent, the kinetics of intestinal lead transport in vitro in young rats were compared to those in adult rats. Due to technical prob- lems associated with the handling of intestines from very young rats, the youngest animals used for the in vitro work were 27 days old. Forbes and Reina (1972) have shown that in 27-day-old rats lead absorption in vivo was approximately double that of adult rats, with greater interanimal variability than adult rats; absorption also decreased rapidly after 27 days. There were similarities in lead transport between the adult and adolescent rats in vitro in that the flux of lead

Mucosal Led comantmtion (pM)

FIG. 4. Intestinal lead accumulation (mean 2 SE) determined after 40 min of incubation in buffers of various lead concentrations. These are the same preparations from which flux was determined (see Fig. 3).

44 AUNGST AND FUNG

TABLE 2

LEAD FLUX ACROSS EVERTED INTE~TINE~ 0~

TAINED FROM AD~LF.SCENT (27- to 33-Day Old) AND

ADULT (200-300 g) RATS AT VARIOUS MUCOSAL ME- DIUM LEAD CONCENTRATIONS (C,)

Adolescent Adult

Cm Flux (xl06 Flux (Xl06 (PM) N ~mol/min) N rmol/min)

0.5 7 9.5 + 2.2 6 6.0 f 0.4 1.2 7 10.3 f 2.3 2.4 6 11.9 f 2.7 4.8 6 11.8 f 1.5 9 13.2 f 2.0

12.1 6 13.9 + 1.1 24.1 4 14.6 + 1.4 9 16.6 + 2.5 36.2 6 16.0 f. 2.5 48.3 5 12.3 f 4.1 7 18.0 f 3.0

Note. The number of animals (IV) and mean + SE are given for each group.

across the adolescent intestines was also apparently saturable (Table 2). A direct com- parison of the values of flux in adult vs adolescent rat intestine is difficult, however, because different weights of intestines were used, and there were probably differences between these two groups in membrane surface area as well. A rough estimate indicated that the surface area of the adolescent intestine used was approximately 60-70s of that of the adult intestine. If this correction factor is applied to the measured flux values (Table 2), it can be seen that the flux per unit surface area across the adolescent intestine is, in most cases, greater than that observed for the adult intestine at the same mucosal lead concentration.

One characteristic common to active transport systems is the requirement for metabolic energy. The dependence of intestinal uptake and transport of lead on a metabolic energy supply were investigated using adult rat intestines. Lead flux and intestinal lead concentrations at 40 min were measured as before, using a mucosal buffer lead concentration at 4.8 PM. Anoxia was pro- duced by removing the oxygen supply and

replacing it with nitrogen. Flux was reduced by about 40% under anoxic conditions (Ta- ble 3). The intestinal uptake of lead was like- wise reduced. Similar results were observed in the presence of fluoride, an inhibitor of glycolysis; flux was reduced by about 50% and the mean intestinal concentration of lead appeared to also decrease, albeit only modestly. The dependence of intestinal uptake and flux of lead on metabolic energy suggest then that the saturable component of the flux might be an active transport system.

We also attempted to demonstrate, using adult rat intestines, active lead transport against a concentration gradient by measur- ing serosal-to-mucosal concentration ratios after 40 min of uninterrupted incubation. Lead concentrations initially were equal on both sides of the intestinal membrane. When the lead concentration was 48.3 PM, the 40- min serosal-to-mucosal lead concentration ratio (s/m) was 0.97 5 0.04 (mean + SE, n = 6). However, at a lead concentration of 2.4 PM the final s/m ratio was 0.72 + 0.07 (n = 6) which was significantly different from that at 48.3 PM using an unpaired Stu-

TABLE 3

DEPENDENCE OF INTESTINAL LEAD FLUX AND

UPTAKE ON A METABOLIC ENERGY SUPPLY~

Flux (Xl@ pmollmin)

Intestinal lead uptake

(X 10’ pmol/g)

Control Anoxia by

removal of 02 supply

In presence of 0.02 M NaF

13.2 + 2.0 9.5 + 0.9

8.4 + 0.4 4.6 + 0.4’

6.1 + l.ob 7.5 + 0.8

’ The mucosal lead concentration in each group was 4.8 PM and there were nine animals in the control group and seven in each other group. Data are expressed as mean f SE.

‘p < 0.05 significantly different from the control group using unpaired Student’s f test.


dent’s t test. Thus, it appeared that net transport of lead in the serosal-to-mucosal direc- tion was also present and this process was also concentration dependent. The intestinal transport mechanism for lead may therefore be bidirectional in nature.

DISCUSSION

In order to understand the mechanism of absorption of lead through the rat gastrointestinal tract more completely, we have eval- uated the concentration dependency of intestinal transport and accumulation of lead in vitro. Under circumstances in which in vivo absorption is influenced by many vari- ables (e.g., presence or absence of food, gas- tric emptying rate, GI motility), the everted intestine can be a valuable tool for exam- ining absorptive mechanisms. Our methods differed from the previous in vitro work using everted intestines (Blair et al., 1979) in that the buffer on the serosal side of the intestine was continually replenished with lead-free buffer in our experiments, creating a “sink” condition. The maintenance of sink conditions was essential for measurement of a true lead flux because serosal-to-mucosal back-transport was identified.

From our measurements of mucosal-to- serosal intestinal lead flux at various lead concentrations, we conclude that flux pro- ceeds by a capacity-limited process in par- allel with passive diffusion. Lead transport was reduced under conditions of anoxia or inhibition of the metabolic energy supply. Using our estimates of transport parameters obtained using adult rat intestines, the relative contributions of active vs passive transport can be assessed. At a buffer lead concentration on the mucosal side of 0.5 PM, the capacity-limited process contributed approximately 200 times more to the mucosal-to- serosal lead flux than did diffusion. At a lead concentration of 48.3 pM, diffusion still ac- counted for less than 20% of the flux ac- cording to our model.

The substantial contribution of capacity- limited intestinal lead binding and transport is consistent with evidence obtained by others implying that the mechanism of intestinal lead absorption is similar to, and possibly the same as those which transport the essential metals, calcium and iron. The ac- tivities of the calcium and iron transport systems are regulated by the requirement for maintenance of relatively constant serum concentrations of these minerals. When the diet is deficient in these metals, absorption efficiency improves. Dietary calcium and iron deficiencies have also been associated with elevated tissue lead concentrations, pre- sumably due to improved efficiency of lead absorption.

Another similarity in the way the rat intestine handles calcium and lead is its apparent ability to transport both metals in both directions. In situ experiments by Ghis- han et al. (1980) have demonstrated the ability of the immature rat intestine to transport calcium, at low mucosal calcium concentrations, in a serosal-to-mucosal di- rection. In this study, we identified net serosal-to-mucosal lead transport at low lead concentration using adult rat intestines.

The age dependency in lead absorption could be due to greater activity of the transport systems (possibly calcium and iron) or greater diffusion, or both. It has also been proposed that in suckling mice intestinal pi- nocytosis contributed significantly to lead absorption (Keller and Doherty, 1980). Al- though we could not estimate kinetic parameters with our flux data from adolescent (27- to 33-day-old) rats, it would appear that capacity-limited absorption is operative.

The in vitro data we have presented suggest that in vivo lead absorption might also be concentration dependent. However, con- flicting evidence exists in the literature re- garding this point. In the in situ work by Barton and Conrad (1978) and Conrad and Barton (1978), the percentage of lead doses absorbed was dependent on the magnitude

46 AUNGST AND FUNG

of the dose. However, Barltrop and Meek (1975) reported that tissue lead concentrations increased in proportion to increases in the magnitude of chronic oral doses. Others have reported dose-dependent lead absorption in starved (Garber and Wei, 1974), or iron-deficient (Hamilton, 1978) animals, but not in fed or iron-replete animals. Rigorous bioavailability experiments have not appeared in the literature. In a follow-up study which examined blood and tissue lead concentrations in adult and suckling rats given a broad range of oral and intravenous lead doses, we showed that lead absorption in vivo was indeed dose dependent (Aungst et al., 198 1). If lead absorption in man is simi- larly determined largely by capacity-limited transport, the primary objective in reducing absorption should be to limit the activity of the transport system(s).

ACKNOWLEDGMENT

This work was supported in part by NIH Grant ES01317.

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kinetic characterization of in vitro lead transport across the rat small intestine: mechanism of...

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