LEAD TRANSPORT PROPERTIES OF CARBOXYLIC ACID AND SYNTHETIC IONOPHORES
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of the Ohio State University
By
Shawn A. Hamidinia, B.A.
*****
The Ohio State University 2005
Dissertation Committee: Approved by Professor Douglas Pfeiffer, Adviser
Professor Ruth Altschuld ______Adviser Professor Ross Dalbey Biophysics Program
Professor Thomas Clanton
ABSTRACT
Metal ion transport studies were performed using naturally occurring carboxylic acid ionophores, a synthetic ionophore, and phospholipid vesicles. Several compounds were identified that are highly selective for the transport of Pb2+ compared to physiological monovalent and divalent cations.
Based upon their respective second-order rate constants, the compounds nigericin and monensin were shown to be selective for the transport of Pb2+, with nigericin being slightly more selective in that regard. Plots of log rate vs. log Pb2+ or log ionophore concentration, in addition to pH dependency, indicate that the predominant transporting species are NigPbOH or MonPbOH. Agents that collapse membrane potential were not required to achieve a high transport rate, which is indicative of an electroneutral mechanism. Nigericin and monensin catalyzed transport of Pb2+ are only modestly affected by physiological concentrations of Ca2+, Mg2+, Na+, or K+. These findings led to the testing of monensin as a therapeutic agent for Pb intoxication using an intact rat model.
Monensin at a concentration of 100 ppm in the feed was given to rats exposed to
100 ppm Pb(acetate)2 in drinking water over a three week period. A reduced Pb
ii accumulation in several organs and tissues was shown. Furthermore, there was an acceleration in the excretion of Pb without depleting organs of essential trace metals such as Zn2+ and Cu2+.
An additional study showed that the co-administration of 100 ppm monensin with meso-dimercaptosuccinate after an exposure to 100 ppm Pb(acetate)2 in drinking water for three weeks significantly reduced the Pb content from femur, brain, and heart without markedly perturbing the concentrations of physiological elements. Thus, monensin may be useful for the treatment of Pb poisoning when combined with DMSA.
KTC-15-cr-5 was synthesized and found to be an effective ionophore for Pb2+ and
Cd2+ transport across a phospholipid bilayer. The results suggest that Pb2+ and Cd2+ are
primarily transported as a 1:1 complex and by an electrogenic mechanism. The high
selectivity for Pb and Cd is of possible value for Pb/Cd intoxication and furthermore to
wastewater treatment.
iii
Dedicated to my family
iv ACKNOWLEDGMENTS
I wish to thank my adviser, Douglas Pfeiffer, for his guidance,
encouragement, and support which made this thesis possible.
My deep gratitude goes to Warren Erdahl for intellectual support and
encouragement.
I also wish to thank those in the lab including, Gregory Steinbaugh, Clifford
Chapman, Jittendra Kumar, and Ron Louters who have provided me with consistent
support and a very friendly work environment.
This research was supported by grant GM 66206 from the National Institute of
General Medical Sciences, National Institutes of Health, by grant 0255017B fom
the American Heart Association, Ohio Valley Affiliate, and by Grant HR00-030
from the Oklahoma Center for the Advancement of Science and Technology.
v VITA
December 15, 1975...... Born - Bridgeport, Connecticut
1998...... B.A. Biochemistry, Case Western Reserve University
1998-present...... Graduate Teaching and Research Associate, The Ohio State University
PUBLICATIONS
Research Publication
1. Hamidinia, S.A., Tan, B., Erdahl, W.L., Chapman, C., Taylor, R.W., and Pfeiffer, D.R. The ionophore nigericin transports Pb2+ with high activity and selectivity: A comparison to monensin and ionomycin. Biochemistry. 43, 15956- 15965, 2004.
2. Hamidinia, S. A., Shimelis, O. I., Tan, B., Erdahl, W. L., Chapman, C. J., Renkes, G. D., Taylor, R. W., and Pfeiffer, D. R. Monensin mediates a rapid and selective transport of Pb2+. The Journal of Biological Chemistry. 277(41): 38111- 38120. 2002.
3. Wang, E., Erdahl, W. L, Hamidinia, S. A., Chapman, C. J., Taylor, R. W., and Pfeiffer, D. R. Transport properties of the calcium ionophore ETH-129. Biophysical Journal. 81, 3275-3284, 2001.
vi FIELDS OF STUDY
Major Field: Biophysics
vii TABLE OF CONTENTS
Page
Abstract…...... ……………. ii
Dedication...... ……………. iv
Acknowledgments...... ……………. v
Vita...... …………….. vi
List of Tables...... …....……….. xi
List of Figures...... ………..…. xii
List of Abbreviations...... ………….... xv
Chapters:
1. Introduction and Background...... …..…………... 1
1.1 Properties of ionophores………………………………………….………….1 1.2 Model membrane system…………………………………………….…….... 3 1.3 The impact of Pb toxicity……..……………………………………...……... 7 1.4 Current treatment for Pb intoxication…………...... …………….………… 8 1.5 The discovery that ionomycin is a Pb2+ ionophore………………..…………9
2. Monensin mediates a rapid and selective transport of Pb2+: Possible application of monensin for the treatment of Pb2+ intoxication….………………………….………11
2.1 Introduction...... ………………..11 2.2 Materials and Methods...... …………..14 Reagents and solvents………………………………..………………… 14 Preparation of phospholipid vesicles……………………….………….. 15 Pb2+ buffers and the determination of transport………………….……..17 Potentiometric titrations and the determination of pH in aqueous methanol……………………………………………………… 19 viii Treatment of experimental animals……………………..…………...… 21 Determination of Pb in biological samples….……………………….…21
2.3 Results…………………………………………………….……………….. 23 Monensin mediated Pb2+ transport………………...…………...……… 23 Competitive relationships between Pb2+ and other cations……………. 25 Monensin promotes the excretion of Pb2+ in rats..……..…………….... 27 2.4 Discussion………………………………………………………………….. 29 2.5 Summary…………………………………………………..………….……. 34
3. Monensin improves the effectiveness of meso-dimercaptosuccinate when used in the treatment of Pb intoxication……….……………….………….…………... 52
3.1 Introduction………………………………………………………………....52 3.2 Materials and methods………………………………………….………….. 54 Treatment of experimental animals...……………...…………...……… 54 The determination of Pb and other elements by ICP-MS….…...……… 56 3.3 Results……………………………………………………………………....57 Levels of Pb and other elements following a period of Pb administration………………………………………………………..… 57 Depletion of previously accumulated Pb……………….….…...……… 59 Effects of monensin alone on the levels of other elements...…...……... 60 Pertubations produced by monensin in DMSA treated rats..…...………61 Pertubations produced by monensin plus DMSA compared to no treatment……………………………………………….....……… 62 Effects of treatment on other parameters………………...... …...……… 63 3.4 Discussion………………………………………………………………….. 63 3.5 Summary…………………………………………………………………… 70
4. The ionophore nigericin transports Pb2+ with high activity and selectivity: A comparison to monensin and ionomycin…………………………...……………….. 91
4.1 Introduction...... …………………. 91 4.2 Materials and methods…...... …………………. 94 Reagents and solvents……………………………………...…...……… 94 Potentiometric titrations and the determination of pH in aqueous methanol.…...………………………………………………… 94 4.3 Results……………………………………………………….……………...95 4.4 Discussion…………………………………………………………..…….. 100 4.5 Summary………………………………………..……….……………...... 104
5. Selective transport of Pb2+ and Cd2+ by a Kemp’s triacid capped 15-crown-5 ether across a phospholipid bilayer membrane…………………………….……….....116
5.1 Introduction………………………………………………….………….....116 ix 5.2 Materials and Methods... ……………………………….….…………….. 117 Reagents…………………………………………………...…..………117 Synthesis of crown ether derivative………….…………...…...………118 Determination of transport………………………………...…...... ……118 Pb2+ and Cd2+ buffers……………………………………...…...……...119 5.3 Results……………………………………………………………………..119 5.4 Discussion………………………………………………………………… 122 5.5 Summary…………………………………………………….……………. 126
6. Discussion…………………………………………………………………………. 136
6.1 Conclusion...... ……….. 136 6.2 Future Perspectives...... …………………… 139
Literature Cited...... ….………... 142
x LIST OF TABLES
Table Page
2.1 Monensin effects on Pb2+ accumulation…………………...….……….. 49
2.2 Monensin effects on Pb2+ clearance…………………………………… 50
2.3 Monensin effects on trace cation levels……………………………….. 51
3.1 Effects of Pb administration on the levels of selected elements in rat tissues……………………………………………………………. 90
4.1 Equilibrium constants for selected reactions………………...……….. 111
xi LIST OF FIGURES
Figure Page
1.1 POPC vesicles observed by electron microscopy by the freeze fracture technique………………………………………….…………..... 5
1.2 Model membrane system…………………………………..…………..... 6
2.1 Structure of representative carboxylic acid ionophores…...…………....36
2.2 Equilibria between an ionized carboxylic acid ionophore (A-1), a metal cation of charge n+ (Mn+), and selected anions (A-, X-, and OH-)……………………………………………………………….. 37
2.3 Transport of Pb2+ and other divalent cations by ionomycin and monensin……………………………………………………………….. 38
2.4 Pb2+ transport dependence on the concentrations of Pb2+ and monensin……………………………………………………………….. 39
2.5 Equilibrium behavior of monensin and selected cations...…………….. 41
2.6 Effect of external pH on ionophore mediated Pb2+ transport………….. 43
2.7 Actions of physiological cations as inhibitors of Pb2+ transport………. 44
2.8 Inhibition of Pb2+ transport by Na+…………………………….………. 46
2.9 Effect of monensin on Pb excreted in urine………...………….……….47
2.10 Potential mechanisms of monensin catalyzed Pb2+ transport………….. 48
3.1 Changes in tissue Pb levels produced by selected treatments…….…… 72
3.2 Changes in tissue Ca levels occurring during selected treatments..…… 75
xii 3.3 Changes in tissue Co levels occurring during selected treatments.……. 76
3.4 Changes in tissue Cu levels occurring during selected treatments.……. 77
3.5 Changes in tissue Fe levels occurring during selected treatments.……. 78
3.6 Changes in tissue Mg levels occurring during selected treatments.…… 79
3.7 Changes in tissue Mn levels occurring during selected treatments.…… 80
3.8 Changes in tissue Mo levels occurring during selected treatments.….... 81
3.9 Changes in tissue Zn levels occurring during selected treatments.……. 82
3.10 Changes in tissue As levels occurring during selected treatments.……. 83
3.11 Changes in tissue Cr levels occurring during selected treatments.……. 84
3.12 Changes in tissue Cd levels occurring during selected treatments.……. 85
3.13 Changes in tissue Ni levels occurring during selected treatments.……. 86
3.14 Changes in tissue Sr levels occurring during selected treatments.……. 87
3.15 Effect of treatment on weight gain in Pb intoxicated rats………..……. 88
3.16 Potential relationships between the transport of Na+ and Pb2+…..……. 89
4.1 Transport of Pb and other divalent cations by nigericin, monensin, and ionomycin.……………………………………………………….. 105
4.2 Dependence of Pb2+ transport on the concentration of free Pb2+...... 107
4.3 Dependence of Pb2+ transport on the concentration of nigericin...... … 108
4.4 Dependence of Pb2+ transport on external pH………………..….….... 109
4.5 Equilibrium behavior of nigericin and Pb2+………………….....……. 110
4.6 Actions of Ca2+ and Mg2+ as inhibitors of Pb2+ transport....…....……. 112
4.7 Actions of K+ and Na+ as inhibitors of Pb2+ transport……..…………. 113
4.8 Possible involvement of electrogenic processes in ionophore- mediated transport……...………...…………………………………... 115
xiii 5.1 Synthesis of Kemp’s triacid-capped 15-crown-5 ether…...... …..……. 127
5.2 Transport of Pb2+ and other divalent cations by KTC-15-cr-5, ionomycin, and monensin...……………………………………..….... 128
5.3 Effect of ionophore concentration on the rate of Pb2+ transport…..…. 130
5.4 Effect of ionophore concentration on the rate of Cd2+ transport….…. 131
5.5 Effect of Pb2+ and Cd2+ concentration on the initial rate of transport…132
5.6 Dependence of Pb2+ transport on membrane potential……….………. 134
5.7 Influence of external pH on the rate of ionophore-mediated Pb2+ transport………...………………………..………………………….... 135
xiv LIST OF ABBREVIATIONS
BCECF 2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein CCCP carbonyl cyanide m-chlorophenylhydrazone CHES 2-(N-cyclohexylamino)ethanesulfonic acid EDTA ethylenediamine-tetraacetic acid HEPES 4-(2-hydroxyethyl)-1-piperazinethane sulfonic acid KTC 15-cr-5 ether Kemp’s triacid-capped 15-crown-5 ether ICP-MS inductively coupled plasma mass spectroscopy MES 2-(N-morpholino)ethanesulfonic acid POPC 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine ∆pH pH gradient TEA+ tetraethylammonium cation TEAP tetraethylammonium perchlorate VAL valinomycin
xv CHAPTER 1
INTRODUCTION & BACKGROUND
1.1 Properties of ionophores
The unique ion-transporting properties of ionophores offer value for a broad area of applications including manipulating cellular ion concentrations, forming the basis of ion-selective electrodes, and serving as potential therapeutic agents. Ionophores are lipophilic molecules which can move cations across membranes by binding the cation, diffusing through the hydrophobic phase, and then releasing the cation on the other side.
To accomplish this task, natural or synthetic ionophores delocalize or shield the charge on the ion being transported. Since their discovery in the 1960s by Pressman and co- workers, ~130 naturally occurring compounds have been isolated from soil bacteria in the Streptomyces genus (Prosser and Palleroni, 1982;Pressman, 1968b). They are distinguished from pore forming antibiotics such as gramicidin by the involvement of a discrete complex in the transport mechanism. Ionophores exhibit discrimination between different ions, and as such can be useful tools in cell biology because of their ability to selectively transport particular cations (Taylor et al., 1982;Painter and
Pressman, 1982). In addition to their usefulness in cell biology, many ionophores are important commercially. Monensin, for example, is widely used in agriculture as a growth promoting feed additive and as an anticoccidial agent (Ruff, 1982a). As a
1 growth promoter in ruminant animals used for meat production, monensin increases weight gain per feed consumed by ~10% (Herberg et al., 1978;Walker et al., 1980a).
This action occurs because the ionophore inhibits the growth of specific rumen microbes resulting in changes in rumen fermentation and metabolism. More specifically, production of propionic acid increases, resulting in a decrease in the acetate:propionate ratio and a reduction in the amount of energy wasted as carbon dioxide and methane
(Ruff, 1982a). Monensin is also used in the poultry industry to combat coccidiosis, a protozoan infection of chickens (Ruff, 1982a).
Ionophores are broadly divided into two groups: the electrogenic and the electroneutral ionophores (Dobler, 1981;Westley, 1982). Electrogenic ionophores, such as ETH 129 and valinomycin (Hladky et al., 1995), are neutral molecules that form cation complexes that carry a net positive charge, such that transbilayer charge movements accompany transport catalyzed by this class. Transport of divalent cations by electrogenic ionophores is a function of membrane potential, which also determines the transmembrane distribution of the transported cation at equilibrium. Electroneutral ionophores, such as the carboxylic acid ionophores monensin, nigericin, and A23187, transport cations in exchange for H+ or another cation, without net charge movement.
The rates of transport by ionophores of this type are influenced by transmembrane pH gradients, as is the equilibrium distribution of cations.
Carboxylic acid ionophores are characterized by an open chain-like structure, with a carboxyl group attached on one terminal end of the molecule and a hydroxyl or ketone group positioned on the other. The deprotonated forms of carboxylic acid ionophores are negatively charged and form complexes with cations via electrostatic and
2 coordinate covalent interactions. The complex is then stabilized by hydrogen bonding between the terminal ends (Dobler, 1981;Pfeiffer and Lardy, 1976). The previous view on carboxylic acid ionophores has been that individual compounds are able to complex/transport a small group of cations having the same charge, with transport through a single species by a mechanism that is strictly electrogenic or electroneutral.
Thus, monensin and nigericin were known as ionophores for Na+ and K+, respectively, with transport occurring via a 1:1 complex and electroneutral mechanism. However, recent studies have shown that a more accurate representation shows a broad range of cations (n=1-3), and that this 1:1 complex can react to form higher order species which involve free ionophore, hydroxide ion, and other anions(Taylor et al., 1985;Chapman et al., 1987b;Chapman et al., 1987a). Using this model depicted in Figure 2.2, the mode and rate of transport reflect the various complexes and their respective transmembrane diffusion constants. A consequence of this finding is that for a given ionophore and set of conditions, transport selectivity will depend on how these factors vary with the properties of the cation.
1.2 Model membrane system
Phopholipid bilayer vesicles or liposomes are a powerful model system used to study ionophores. Large unilamellar vesicles (LUV), prepared by freeze-thaw extrusion, have been routinely used in our laboratory to model membrane systems (Chapman et al.,
1990a;Chapman et al., 1991). Unlike biological systems, liposomes are stable to a wide range of cations. In addition, determining ionophoretic properties such as cation
3 specificities (Erdahl et al., 1996;Wang et al., 1998) and transport mechanisms (Erdahl et al., 1994;Erdahl et al., 1995;Thomas et al., 1997) are now possible because this model system is free of endogenous channels and carriers.
The freeze-thaw extrusion technique is used to make phospholipids vesicles and is described later in the Materials and Methods section of chapter 2. Once formed, the vesicles are closed, unilamellar, and have an average diameter of 71 nm. Figure 1.1 shows vesicle images gathered by freeze-fracture electron microscopy (Chapman,
Erdahl, Taylor, and Pfeiffer, 1990a). Loading a chelator/indicator, such as Quin-2, introduced by Tsien and co-workers (Tsien, 1980), inside these vesicles enables monitoring of cation transport by UV-vis spectroscopic analysis of Quin-2•cation complexes. Also, solutes of interest can be entrapped by repeated freeze-thaw cycles
(Chapman, Erdahl, Taylor, and Pfeiffer, 1990a;Chapman, Erdahl, Taylor, and Pfeiffer,
1991) between extrusions. The model membrane system is illustrated in Figure 1.2. For comparison to the size of the vesicle, the relative dimensions of the hydrated Ca2+ ion and the transporting complexes, (A23187)2Ca and Ionomycin•Ca are also shown. To make homogenous unilamellar vesicles with high solute entrapment, our vesicle preparation protocol includes alternative cycles of freeze-thaw and extrusion. The phospholipid bilayer in our system is composed of 1-palmitoyl-2-oleoyl-sn- glycerophosphatidylcholine (POPC) which constitutes a significant percentage of lipid in biological organisms. Of practical importance, POPC has a low phase transition temperature -7 C, making it possible to prepare, store, and use the vesicles in its fluid state at room temperature.
4
Figure 1.1 POPC vesicles observed by electron microscopy by the freeze fracture technique. (adapted from Chapman, et al., 1990) 5
Dimensions (to Scale) of the Transport System
Ionomycin Ca Complex
10 X 6
(A23187)2 Ca Complex
POPC Vesicle Diameter ~ 70 nm Ca2+ Ion
Figure 1.2 Model Membrane system. (adapted from Chapman, et al., 1990) 1.3 The impact of Pb toxicity
Lead poisoning is a serious health problem in the United States. There are ~6000 deaths per year due to Pb poisoning, and a much larger number of individuals who suffer from sub-lethal exposure. The U.S. Department of Health and Human Services reported that between 0.5 and 1.5 million industrial workers are exposed to lead in the workplace.(ATSDR (Agency for Toxic Substances and Disease Registry), 1999) These people include those who work in lead smelting and refining industries, brass/bronze foundries, rubber products and plastics industries, soldering, steel welding and cutting operations, battery manufacturing plants, and lead compound manufacturing industries.
Health problems arising from Pb intoxication include hypertension, anemia, hearing loss, kidney damage, peripheral neuropathies, infertility, diminished life span, and encephalopathy, listed in the order associated with increasing blood levels (ATSDR
(Agency for Toxic Substances and Disease Registry), 1999). Both adults and children can be afflicted with lead poisoning, but there is greater concern for children who are more sensitive to the effects of Pb and experience symptoms at lower blood Pb levels than do adults(Centers for Disease Control and Prevention, 1997). The Centers for
Disease Control and Prevention estimates that about 900,000 U.S. children younger than
5 years old possess blood Pb levels above the toxic threshold of 10 µg/dL. The sources of this exposure are primarily leaded paint, which was not banned in the United States until 1978, and contaminated soil (Mielke and Reagan, 1998).
Many toxic effects of Pb arise through actions exerted on components of Ca2+ based cell signaling systems (Büsselberg et al., 1994;Sun and Suszkiw, 1995;Quinn and
Harris, 1995;Cory-Slechta, 1995;Akke et al., 1995;Schanne et al., 1997;Sun and
7 Suszkiw, 1995;Quinn and Harris, 1995;Cory-Slechta, 1995;Akke, Forsén, and Chazin,
1995;Schanne, Long, and Rosen, 1997). In particular, lead interferes with the ability of the Ca2+-binding protein synaptotagmin to bind to its protein partner syntaxin; this inhibition offers a possible molecular explanation for the disruption of calcium-triggered neurotransmitter release (Bouton et al., 2001). Lead also displaces Zn2+ from proteins that normally bind Zn2+. These include: 5-aminolevulinic acid dehydratase (ALAD), human protamine 2, and TFIIIA (Bridgewater and Parkin, 2000;Payne et al.,
1999;Godwin, 2001). Each of these proteins has an important physiological role in the body, and the perturbation of their function contributes to the symptoms associated from
Pb poisoning.
1.4 Current treatment for Pb intoxication
The standard treatment for Pb intoxication has been chelation therapy using calcium disodium EDTA, which is given I.V. and secreted as the Pb·EDTA complex via the kidney (Goyer et al., 1995a). Despite its widespread use as a Pb therapeutic agent,
CaNa2EDTA has several disadvantages. It enhances lead uptake from the gastrointestinal tract, thus contraindicating usage via this route (Cory-Slechta, 1988a).
Consequently, CaNa2EDTA is administered intramuscularly and is not well tolerated by young children. Urinary elimination of essential trace metals such as copper and zinc often accompanies lead excretion during treatment (Flora et al., 1995). Recent studies also indicate that Ca disodium EDTA chelation redistributes Pb from bone and kidney into both brain and liver, raising questions of safety (Cory-Slechta et al., 1987a). Newer
8 Pb2+ chelating agents, offer some advantage over EDTA, particularly oral administration and higher selectivity against trace cations with biological roles(Goyer, Cherian, Jones, and Reigart, 1995a;Lifshitz et al., 1997).
DMSA is a chelator given orally that is commonly used to remove Pb from the blood and soft tissues. Unfortunately, DMSA cannot remove Pb from bone, where approximately 90% of the Pb taken in is deposited/stored for periods of 104 days
(Bronner, 1996). As a consequence of this, Pb continues to leach out of the bone and is redistributed throughout the body. The disadvantage to using these compounds is that chelation therapy is a repetitive process that proceeds on a time scale of months or longer. This extended time frame is particularly problematic in young children who are undergoing rapid growth and development (Roper et al., 1991;Rogan et al.,
2001;Besunder et al., 1997).
1.5 The discovery that ionomycin is a Pb2+ ionophore
Pfeiffer and co-workers (Erdahl et al., 2000a) first discovered that ionomycin, originally classified as a Ca2+ ionophore, was better described as an ionophore for Pb2+.
They demonstrated that ionomycin transports Pb2+ rapidly, and with a selectivity over
Ca2+ near 103 when both cations are present simultaneously. Ionomycin was also found to effect an efficient transport of Pb2+ into cultured cells, as well as to facilitate the depletion of Pb2+ when the cells had been previously loaded. They concluded that ionomycin should be considered primarily as an ionophore for Pb2+, rather than Ca2+, and further suggested that its Pb2+ transporting activity might be adapted to improve existing treatments for Pb2+ intoxication (Erdahl, Chapman, Taylor, and Pfeiffer, 2000a).
9 It is within the context of the above background information that the present studies were undertaken. The initial goal was to detect if other ionophores transport and are specific for Pb2+ transport. Our findings revealed that the naturally occurring carboxylic ionophores monensin and nigericin are highly selective for the transport of
Pb2+ in model membrane systems. Subsequently, our investigations using Sprague-
Dawley rats have shown that monensin mediated lead transport utilized alone or in conjunction with DMSA may aid in the treatment of lead intoxication. Another study was undertaken to study a synthetic ionophore based on a crown ether backbone and its selective properties for the heavy metals Cd2+ and Pb2+.
10 CHAPTER 2
MONENSIN MEDIATES A RAPID AND SELECTIVE TRANSPORT OF Pb2+:
POSSIBLE APPLICATION OF MONENSIN FOR THE TREATMENT OF Pb2+
INTOXICATION
2.1 Introduction
Ionophores are lipophilic chelating agents that transport cations across phospholipid bilayer membranes, such as the plasma and subcellular membranes of cells. The naturally occurring compounds (about 100) are antibiotics produced primarily by soil bacteria of the Streptomyces genus (Prosser and Palleroni, 1982). They are distinguished from pore forming antibiotics such as gramicidin by the involvement of a discrete complex in the transport mechanism. Thus, true ionophores can be highly selective for particular cations (Taylor, Kauffman, and Pfeiffer, 1982;Painter and
Pressman, 1982). The known compounds are generally divided into two groups; the so- called electrogenic and the electroneutral ionophores (Dobler, 1981;Westley, 1982).
Electrogenic ionophores are typified by the well-known compound valinomycin
(Hladky, Leung, and Fitzgerald, 1995). They are neutral molecules that form cation complexes that carry a net positive charge, such that transbilayer charge movements accompany transport catalyzed by this class. Accordingly, the rate of transport is influenced by membrane electrical potential, which also determines the transmembrane
11 distribution of the transported cation at equilibrium. Compounds such as monensin, nigericin, and A23187 (Fig. 2.1) typify electroneutral ionophores, also called carboxylic acid or polyether ionophores. The anionic forms of these compounds complex the cation, which is exchanged for H+, or another cation, without net charge movement.
Transport catalyzed by this class is therefore influenced by transmembrane pH gradients, as is the equilibrium distribution of cations.
Among the carboxylic acid ionophores it has been common to consider individual compounds as able to complex/transport a small group of cations having the same charge, with transport occurring through a single species by a mechanism that is purely electrogenic or electroneutral. Thus, monensin and nigericin were known as ionophores for Na+ and K+, respectively, with transport occurring via a 1:1 complex and electroneutral mechanism: A23187 was known as a Ca2+ ionophore transporting via a
1:2 complex and an electroneutral mechanism, and so forth. In work conducted over a period of time, we showed that the model depicted in Fig. 2.2 is a more accurate representation of the factors that establish the transport properties of a carboxylic acid ionophore (Taylor, Chapman, and Pfeiffer, 1985;Chapman, Puri, Taylor, and Pfeiffer,
1987b;Chapman, Pfeiffer, Thomas, and Taylor, 1987a;Chapman et al., 1990b;Stiles et al., 1991;Taylor et al., 1993;Erdahl, Chapman, Wang, Taylor, and Pfeiffer, 1996;Wang,
Taylor, and Pfeiffer, 1998;Erdahl, Chapman, Taylor, and Pfeiffer, 2000a;Wang et al.,
2001). The equilibrium shown at top represents formation of 1:1 complexes and emphasizes that carboxylic acid ionophores react with a broad range of cations (n = 1-3), not just those with a particular charge as often assumed. The 1:1 complexes then react to form higher-order species by competing equilibria I-III, which involve free ionophore
12 (A-), hydroxide ion (OH-) and other anions (X-), respectively. Complexes of differing stoichiometry therefore arise, which include mixed species containing OH- and X-.
Within this scheme, the mode and overall rate of transport will reflect the distribution of ionophore between these various complexes, and their respective transmembrane diffusion constants. As a corollary of this interpretation, for a given ionophore and set of conditions, transport selectivity will also depend on how these factors vary with properties of the cation. The latter include size, charge, coordination and hydration number, preferred donor atom geometry, Lewis acidity, and ease of hydrolysis.
In considering the above model, the characteristics of the donor atoms in typical ionophores, and the metal ion complexation properties of analogous synthetic polyethers
(Izatt et al., 1985), we realized that some of the antibiotic compounds might be efficient ionophores for Pb2+ and for other cations with biological toxicity. Subsequently we showed that ionomycin transports Pb2+ rapidly, and with selectivity over Ca2+ near 103 when both cations are present simultaneously. Ionomycin was also found to affect an efficient transport of Pb2+ into cultured cells, as well as to facilitate the depletion of Pb2+ when the cells had been previously loaded. We indicated that ionomycin should be considered primarily as an ionophore for Pb2+, rather than Ca2+, and suggested that its
Pb2+ transporting activity might be adapted to improve existing treatments for Pb2+ intoxication (Erdahl, Chapman, Taylor, and Pfeiffer, 2000a).
In the present report we extend the investigation of ionophore mediated Pb2+ transport by demonstrating that monensin is also effective as a Pb2+ionophore, and is more selective in that regard than is ionomycin. We also show that monensin promotes
13 the excretion of Pb2+ from rats when the cation has been previously provided in drinking water and the ionophore is administered in feed. Aspects of these data have been presented in abstract form (Hamidinia et al., 2002a).
2.2 Materials and Methods
Reagents and Solvents. High purity nitric acid (Fisher, trace metal) and perchloric acid (GFS Chemicals, double distilled) were obtained from commercial sources. Synthetic 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine (POPC)1 was obtained from Avanti Polar Lipids, Inc. Purity was confirmed by thin-layer chromatography before use. For the transport studies monensin and ionomycin were obtained from Calbiochem and used without further purification. Ionomycin stock solutions were prepared in ethanol and standardized spectrophotometrically using an extinction coefficient of Σ278 = 13,560 at 278 nm. In the case of monensin,
+ standardization was gravimetric or by titration with Me4NOH. Quin-2 (K salt) from
Sigma was purified by passage over Chelex 100 resin (100-200 Mesh) in the Cs+ form as described previously (Erdahl, Chapman, Taylor, and Pfeiffer, 1994), or in the Na+ form when Na+ loaded vesicles were employed. The nitrate and chloride salts of divalent cations were the ultrapure grade from Alfa Products. Stock solutions were standardized by titration with a primary standard EDTA solution (Vogel, 1961), or by atomic absorption spectroscopy using certified solutions (Fisher).
For solution chemical studies a mixed solvent of 80% (w/w) methanol in water was prepared gravimetrically, using distilled deionized water and reagent grade methanol (Fisher) that had been freshly distilled. The Et4NClO4 that was used to
14 maintain ionic strength in this solvent was prepared by reaction of Et4NOH (Aldrich) with 70% perchloric acid (GSH Chemicals, distilled). The salt obtained was
+ recrystallized four times from water. Solvent containing Et4NClO4 and H buffering compounds was further deionized by passage over Chelex 100. For this purpose the
+ resin was in the Et4N form, which was prepared as previously described (Chapman,
Puri, Taylor, and Pfeiffer, 1987b).
Preparation of Phospholipid Vesicles. The preparation of freeze-thaw extruded
POPC vesicles loaded with Quin-2 has also been described previously (Chapman,
Erdahl, Taylor, and Pfeiffer, 1990a;Chapman, Erdahl, Taylor, and Pfeiffer, 1991).
Briefly, 300 mg of POPC in chloroform was dried by rotation under a nitrogen stream to produce a film on the wall of a 25 x 150 mm culture tube. Residual solvent was removed under high vacuum (4 h) and the film was subsequently hydrated in 6 mL of a solution containing 6.6 mM of purified Quin-2 and 10.0 mM Hepes buffer adjusted to pH 7.00 with Chelex-treated CsOH or NaOH (Erdahl, Chapman, Taylor, and Pfeiffer,
1994), depending on the internal composition required. The mixture was vortexed and the resulting multilamellar vesicles were frozen in a dry ice-acetone bath, thawed in lukewarm water, and vortexed again. The freeze-thaw and vortexing procedures were repeated two additional times, after which the vesicles were extruded three times through two stacked 100 nm polycarbonate membrane filters. This step was followed by six additional freeze-thaw cycles coupled with additional extrusions. The resulting preparations were applied to Sephadex G-50 mini-columns (Fry et al., 1978) to remove extravesicular Quin-2. These columns were eluted by low speed centrifugation and had previously been equilibrated with a solution containing 10 mM Hepes buffer, pH 7.00.
15 A single pass over such columns effectively removes the external Quin-2 (Chapman,
Erdahl, Taylor, and Pfeiffer, 1990a;Chapman, Erdahl, Taylor, and Pfeiffer, 1991;Erdahl,
Chapman, Taylor, and Pfeiffer, 1994).
The nominal concentration of POPC in the final preparations was determined by measurement of lipid phosphorus (Bartlett, 1959) and was near 80 mM. The average diameter of these vesicles is 71 nm as determined by freeze-fracture electron microscopy
(Chapman, Erdahl, Taylor, and Pfeiffer, 1990a), and they contain entrapped solutes at the following concentrations; Quin-2, 10.5 ± 0.8 mM; Hepes, 34 ± 8 mM (pH ≈ 7.4); and Cs+/Na+, 60 ± 5 mM. Specific values for Quin-2 and Cs+/Na+ were determined for each preparation by the methods described previously (Erdahl, Chapman, Taylor, and
Pfeiffer, 1994;Erdahl, Chapman, Taylor, and Pfeiffer, 1995). Briefly, entrapped Quin-2 is determined by spectrophotometric titration with standard CaCl2 following dispersion of the vesicles in deoxycholate. The entrapped monovalent cation is determined by atomic absorption spectroscopy, following replacement of the external medium with one containing a different cation, and dispersion of the vesicles in 0.1 N HCl. When of interest, buffer entrapment is determined from the other values by calculation, using the
Henderson-Hasselbach equation, the buffer pKa, and the internal pH. When buffer entrapment is to be determined, the vesicles also contain the fluorescent pH indicator
BCECF, so that the internal pH can be ascertained. The internal pH and solute concentrations differ from those of the vesicle formation medium because of a freeze- thaw driven solute concentrating effect that operates during preparation of the vesicles
(Chapman, Erdahl, Taylor, and Pfeiffer, 1990a;Chapman, Erdahl, Taylor, and Pfeiffer,
1991).
16 Pb2+ Buffers and the Determination of Transport. A buffer system was used to control the concentration of Pb2+ available for transport into the vesicles. 15 mM or 5 mM citrate was employed to buffer the concentration of this cation, whereas 10 mM each of Hepes and Mes were present to buffer H+. Seventeen equilibria involving citrate3-, H+, Pb2+, and OH- were accounted for when calculating the free Pb2+ concentration. When Ca2+ or Mg2+ were to be buffered simultaneously, five additional equilibria involving those cations were also considered. The respective equilibrium constants were taken from literature sources (Smith and Martell, 1976;Martell and
Smith, 1977) and, when necessary, the Davies equation (Davies, 1962) was used to correct these to an ionic strength of 100 mM. The species distribution program
COMICS (Perrin and Sayce, 1967) was used to solve the applicable sets of simultaneous equations at experimental conditions of interest, and to allow the generation of standard curves. Examples of the latter were shown previously (Erdahl, Chapman, Taylor, and
Pfeiffer, 2000a).
The transport of Pb2+ and other divalent cations into Quin-2 loaded vesicles was determined by monitoring formation of the Quin-2:cation complexes spectroscopically.
Unless otherwise indicated, vesicles containing Quin-2 were present at a nominal POPC concentration of 1.0 mM in a medium that also contained 50 mM CsCl, and 10 mM each of Hepes and Mes. The medium pH ranged from 6.00 to 9.50 and was adjusted with
CsOH that had been passed over Chelex 100 columns to remove contaminating divalent cations (Erdahl, Chapman, Taylor, and Pfeiffer, 1994). In some cases, valinomycin
(VAL), (0.5 µM) and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), (5 µM) were also present to maintain internal pH at the external value and to dissipate any
17 transmembrane electrical potential that might otherwise arise (Erdahl, Chapman, Taylor, and Pfeiffer, 1995). Specific concentrations of ionophores, divalent cations, and pH values are given in the figure legends. Reactions were started by addition of the ionophore, following an initial 2 min preincubation to allow equilibration of transmembrane pH.
The formation of Quin-2:cation complexes was followed continuously by difference absorbance spectroscopy, using an Aminco DW2a spectrophotometer operated in the dual wavelength mode. An Oriel No. 59800 band pass filter was used between the cuvette and the beam scambler-photomultipler assembly to prevent detection of the fluorescent light emitted by Quin-2. The sample wavelength used for all cations was 264 nm. The reference wavelengths were at an isosbestic point in the Quin-
2/Quin-2:cation complex difference spectrum of interest. These wavelengths vary slightly from cation to cation, as previously described (Erdahl, Chapman, Wang, Taylor, and Pfeiffer, 1996). Data were collected on disk using Unkel Scope software (Unkel
Software, Inc., Lexington, MA).
To determine initial transport rates, an early portion of the progress curves was fit to Eq (1) using standard nonlinear least-squares methods.
2 AT = A0 + Bt + Ct (1)
In this expression, AT and A0 are the observed and the initial absorbance values, respectively, B is the initial rate in units of absorbance per second, C is a correction factor for nonlinearity, and t is time in seconds. The values presented are in units of µM external cation transported into the vesicles per second. B values obtained from Eq (1) were converted to the latter unit by referring to a standard curve for the cation of interest
18 that was generated by titrating the vesicles in the presence of excess ionophore, or after they had been lysed with 0.33% (w/v) of Cs+-deoxycholate (Erdahl, Chapman, Taylor, and Pfeiffer, 1994;Erdahl, Chapman, Wang, Taylor, and Pfeiffer, 1996). Transport selectivities are expressed as S values defined by Eq (2).
S = Initial Rate of Pb2+ Transport / Initial Rate of Ca2+ Transport (2)
When determining S, the concentration of the Pb2+ or Ca2+ was 20 µM and all other conditions were held constant. All transport data were obtained at 25.0° C.
Potentiometric Titrations and the Determination of pH in Aqueous Methanol.
The protonation constants and complex formation constants of monensin were measured by potentiometric methods in the mixed solvent 80% (w/w) methanol-water. For these studies, Na+ monensin was purified by column chromatography on silica gel, using ethyl acetate as the eluent. The Na+ salt was converted to the acid form by repeated back- extraction of a CHCl3 solution with 1.0 M HCl. The CHCl3 solution was then washed three times with distilled water, the solvent removed, and the product dried under vacuum. Analysis by flame atomic emission showed that less than 0.05% Na+ (w/w) remained.
Pb(ClO4)2 and Zn(ClO4)2 were prepared by reaction of the metal oxide (Aesar,
(Pb) 99.9995%, (Zn) 99.99%) with 70% HClO4 (GFS Chemicals, distilled). CaCl2⋅xH2O
(99.999%) and MgCl2⋅6H2O (Aldrich, 99.995%) were used as received. Stock solutions of Pb2+, Zn2+, Ca2+, and Mg2+ salts were standardized by titration using EDTA (Vogel,
1961). NaCl (Aldrich, 99.999%) and KCl (Aesar, 99.997%) were dried at 110°C and stock solutions of these salts were prepared gravimetrically.
19 Test solutions for potentiometric titration typically contained 0.5-1.0 mM monensic acid (HL), and where appropriate, the metal ion (M) at concentration ratios
([HL]:[M]) in the range 1.0-3.0. In the case of Mg2+ and K+, [HL]:[M] ratios of 0.2-0.25 were also used. Ionic strength was maintained at 0.050 using Et4NClO4, except in the
+ case of K where Et4NCl (Fluka, >99%) was used. The titrations were carried out using a digital burette, (Metrohm, model 665) and pH meter (Fisher 825MP) that was interfaced to a computer (Stiles, Craig, Gunnell, Pfeiffer, and Taylor, 1991). pH* measurements were made using double junction combination electrodes (Sensorex
S1021CD, Orion Ross 8175BN, Thomas 4080-B49) where the external filling solution was replaced with 0.1 M Et4NClO4 or 0.1 M Me4NCl in 20% methanol-water.
The pH meter-electrode system was calibrated in aqueous solution using standard buffers (Fisher Gram-Pac); then the electrodes were equilibrated in 80% methanol-water for at least two hours prior to measurement. The operational pH* scales developed by de Ligny et al. (de Ligny et al., 1960a;de Ligny et al., 1960b) and Gelsema et al. (Gelsema et al., 1966;Gelsema et al., 1967) were utilized to determine the value of
+ pH*. The term pH* is defined as -log aH*, where aH* is the activity of H in the mixed solvent. Accordingly, the term pH* when used in reference to a specific methanol/water mixture has the same meaning as the term pH when used in reference to an aqueous solution (see (Rorabacher et al., 1971) and references therein).
All titrations were carried out at 25.0°C using a thermostatted cell and 20 mM
Me4NOH as the titrant. A nitrogen or argon atmosphere was maintained to minimize contamination by CO2. The Me4NOH was standardized using KH2PO4 and checked for carbonate content (Gran, 1956). Typical titrations consisted of 50-100 pairs of pH*-mL
20 Me4NOH readings. The titration data were analyzed using the computer programs
PKAS for the protonation constants (Motekaitis and Martell, 1982b), and BEST for the metal ion complexation constants (Motekaitis and Martell, 1982a).
Treatment of Experimental Animals. Male rats were utilized when investigating effects of monensin on the pathophysiology of Pb2+. They were housed in AALAC approved animal facilities at the College of Medicine, Ohio State University. A 12 hr light-dark cycle, single housing in plastic cages, or in metabolic cages, and conditions of constant temperature and humidity were employed. One week was allowed for acclimation before an experimental protocol began. During this period a standard laboratory chow was employed, whereas the AIN-93M diet containing 0.5% calcium was employed thereafter. During the administration of Pb2+ and/or monensin, water and feed were provided ad libitum, and records of consumption were maintained, together with periodic measurements of body weight.
At the beginning of an experimental protocol the rats weighed 245-255g. They were divided randomly into groups of six or eight, and the administration of Pb2+ was begun. It was provided at 100 ppm in the drinking water, and was in the form of
Pb(acetate)2. The water was rendered slightly acidic with acetic acid to prevent the precipitation of PbCO2 (Bogden et al., 1995b). When utilized, monensin was provided in the feed, also at 100 ppm.
Determination of Pb in Biological Samples. Blood samples were taken periodically from the tail artery and blood Pb levels were determined by electrothermal atomic absorption spectroscopy against certified standards (Shuttler and Delves, 1986).
For the determination of Pb in urine and feces, total outputs were collected over two day
21 periods from each rat individually. The urine samples were neutralized by the careful addition of nitric acid while the samples were stirred and the pH was monitored with an electrode. Samples were then extracted with methyl isobutyl ketone in the presence of excess ammonium pyrrolidine dithiocarbamate, which quantitatively extracts Pb2+ into the organic layer, and concentrates it compared to the original concentration in urine
(Zinterhofer et al., 1971). The Pb content was thereafter determined by flame atomic absorption spectroscopy. For the determination of Pb in feces, weighed samples comprising ∼1.0 g were dispersed in 10 mL of concentrated nitric acid and allowed to stand at room temperature overnight. They were then concentrated to ∼1.5 mL by heating on a hot plate, diluted to a total volume of 10.0 mL with water, and Pb was determined by flame atomic absorption spectroscopy (Briggs-Reed and Heagler, 1997).
At completion of experimental protocols the rats were sacrificed by the injection of excess Nembutal and perfused briefly with Hepes buffered 0.9% NaCl, via the left ventricle, to remove blood from the organs. Organs and tissues of interest were then removed and stored at -20°C. For Pb analysis, the samples were thawed and weighed portions were digested with 9 volumes of nitric/percholoric acid (3:1). After reducing the volume to 1 mL by heating, the samples were diluted to a total volume of 10.0 mL with a solution containing 0.140 mM NH4OH, 29 mM NH4(H2PO4), and 3 mM EDTA.
For samples containing relatively high levels of Pb, such as bone and kidney, an initial dilution with water proceeded that step. Following all of the above steps, the Pb content was determined by electrothermal atomic absorption spectroscopy (Miller et al., 1987).
The resultant values, and other parameters relevant to rats, were examined by univariant
ANOVA to determine if the differences observed were statistically significant.
22 2.3 Results
Monensin Mediated Pb2+ Transport. Fig. 2.3 compares the efficiency of ionomycin and monensin as ionophores for Pb2+, and contrasts their selectivity for Pb2+ compared to other divalent cations. Under conditions employed, the two compounds are similarly effective from the perspective of rate, whereas monensin is much more selective. Selectivity for Pb2+ over Ca2+ for example, as defined by equation 2, is ∼100 for ionomycin and ∼3,400 for monensin2.
The stoichiometry of the complex between ionomycin and Pb2+ that is responsible for transport is 1:1, cation: ionophore, based upon plots of log rate vs. log ionomycin or log Pb2+ concentration, which both display slopes of 1.0 (Erdahl,
Chapman, Taylor, and Pfeiffer, 2000a). By the same criteria, monensin-mediated Pb2+ transport also occurs through formation of a 1:1 complex (Fig. 2.4), although the plot of log rate vs. log Pb2+ concentration progressively deviates from a slope of 1 as the free
Pb2+ concentration rises above 1 µM. This negative deviation suggests that the steady state fraction of monensin that is located at the external membrane interface approaches saturation with Pb2+ at concentrations above that value.
Ionomycin is dibasic because both the carboxylic acid function and the enolized
β diketone moiety can be ionized (Fig. 2.1) (Toepilitz et al., 1979). Accordingly ionomycin can form uncharged complexes with divalent cations, presumably including
Pb2+, and can exchange these for 2H+ in an electroneutral manner (Erdahl, Chapman,
Taylor, and Pfeiffer, 1994;Erdahl, Chapman, Taylor, and Pfeiffer, 1995). In contrast, monensin is monobasic (Fig. 1) (Pinkerton and Steinrauf, 1970b) and would form a 1:1 complex with Pb2+ having a net charge of +1. Thus the 1:1 complex between Pb2+ and 23 monensin might result in electrogenic Pb2+ transport, or it might associate with an anion as depicted in Fig. 2.2, to produce transport through a charge neutral mechanism. To examine the mode of Pb2+ transport we initially determined complex stability constants of potential transporting species. Potentiometric titration methods were employed using
80% methanol-water as the solvent. This mixed solvent provides an effective polarity similar to that experienced by ionophores at a POPC membrane interface (Pfeiffer et al.,
1983;Taylor, Pfeiffer, Chapman, Craig, and Thomas, 1993;Taylor, Chapman, and
Pfeiffer, 1985;Kauffman et al., 1982). Analogous complexation equilibria for other cations were also examined to provide insight into the basis of the high selectivity that is seen in Fig. 2.3B.
Fig. 2.5A shows examples of the primary data obtained, whereas Fig. 2.5B reports the complex stability constants. It is seen that the complex PbMon+ is relatively stable, displaying a log K value of 7.25. The uncharged complex PbMonOH that is formed upon reaction of PbMon+ with OH- is nearly 4 orders of magnitude more stable than the 1:2 complex formed from PbMon+ and a second molecule of the ionized ionophore. When these stability constants and the protonation constant of monensin are used to generate a species distribution diagram (Perrin and Sayce, 1967) (Fig. 2.5C) it is seen that significant levels of both PbMon+ and PbMonOH are expected at the membrane interface during Pb2+ transport, within the pH range of 6.5-7.5. In contrast, the species Pb(Mon)2 is negligible, (<0.003% of total monensin) throughout the broad range of pH considered (not shown). Thus, the complex stability data indicate that
24 monensin might transport Pb2+ by a mixed mode (electrogenic and electroneutral transport occurring simultaneously), and that the fraction transported by the neutral process would likely occur via the species PbMonOH.
To further examine these possibilities, the effect of external pH on the rate of
Pb2+ transport was examined, using Na+ containing vesicles with Val and CCCP excluded. These conditions limit transport to the fraction occurring by neutral mechanisms because no provision is made to collapse the membrane potential arising from electrogenic transport, which is very effective at limiting the process e.g.(Wang,
Taylor, and Pfeiffer, 1998;Wang, Erdahl, Hamidinia, Chapman, Taylor, and Pfeiffer,
2001). In addition, the use of Na+ rather than Cs+ containing vesicles allows the ionophore to exchange Pb2+ for Na+, assuring that the rate of Pb2+ transport does not become limited by the protonation of monensin at the internal membrane interface. As seen in Fig. 2.6A, monensin remains active as an ionophore for Pb2+ under these conditions, demonstrating that the electroneutral mode is indeed active.
Regarding the species PbMonOH, the data in Fig. 2.6B show that increasing external pH enhances the rate of transport, consistent with a model involving dissociation of a single proton from hydrated PbMon+ to form PbMonOH. This finding and a comparison of the transport data to the pH dependence for PbMonOH formation shown in Fig. 2.5C indicate that PbMonOH is indeed the major transporting species. In
Fig. 2.6 the half-maximal rate was seen at pH = 7.8 (pOH = 6.2) or at pPbOH = 6.9, if it is considered that that is the actual species which is transported.
Competitive Relationships Between Pb2+ and Other Cations. Given the potential use of ionophores to manipulate Pb2+ in living systems, we considered the possibility
25 that the relatively high concentrations of other cations found in blood and intracellular compartments might substantially prevent monensin from transporting Pb2+ in vivo.
Specifically, the rates of Pb2+ transport were compared when the free Pb2+ concentration was buffered at 1.0 µM alone, and when 1.0 mM free Ca2+ or Mg2+ was also present.
Only negligible differences were seen (Fig. 2.7A and B), as might be expected based upon the complex stability constants shown in Fig. 2.5B. Potential interference by K+ and Na+ was also examined. The actions of K+ were again negligible when it was present at 5.0 mM or 100 mM, approximately the levels of this cation in blood and cytoplasm, respectively (data not shown). The same concentrations of Na+ did have modest inhibitory effects (Fig. 2.7C), however these were smaller than expected based on the KML values listed in Fig. 2.5B. That is to say the ratio KPb/KNa is approximately
300, whereas at a ratio of free Na+/Pb2+ of 105 (i.e. when Na+ was present at 100 mM and Pb2+ at 1.0 µM) the rate of Pb2+ transport is reduced by only a factor of < 2.
To further examine the effectiveness of Na+ as an inhibitor of monensin mediated Pb2+ transport the Na+ concentration was varied from 0 to 100 mM, while another solute was varied in the opposite direction, so as to maintain the external ionic strength and/or the osmotic pressure at constant values. As seen in Fig. 2.8, Na+ is more effective when used to replace Cs+, compared to K+ or TEA+ and is ineffective when used to replace mannitol. These data presumably reflect modest differences in the stability of complexes between monensin and monovalent cations (Hoogerheide and
Popov, 1979;Cox et al., 1984b;Pointud et al., 1985a) together with effects of ionic
26 strength on complex stability. However of greater importance, they further indicate that concentrations of Na+ found in living systems have little effect on the efficiency of monensin as a Pb2+ ionophore.
Monensin Promotes the Excretion of Pb2+ in Rats. Given that the above data are consistent with the notion that monensin might alter the dynamics of Pb2+ in whole organisms, we determined the effect of monensin on the accumulation and disposition of
Pb in rats. In one experiment the ionophore and Pb2+ were administered simultaneously, at 100 ppm in feed and 100 ppm in drinking water, respectively, or Pb2+ was administered without the ionophore. Blood samples were taken at weekly intervals over a twenty eight-day period, and thereafter the rats were sacrificed to allow the determination of Pb in organs and tissues. Throughout the entire experimental period the average concentration of Pb in blood was lower by ∼25% in the rats that had received monensin, and this difference was significant at p = 0.05 (Table 2.1 and data not shown). Monensin also reduced the accumulation of Pb in several organs, with a particularly large effect seen in heart where it was reduced by 66% (Table 2.1). The reduced values seen in brain, muscle and bone were also significant.
In a second experiment the rats were loaded with Pb2+ over a twenty-one day period, and in the absence of monensin. Thereafter they were divided into groups and given drinking water that did not contain Pb2+. One group was given monensin at 100 ppm in feed, whereas another was given the same feed without monensin. After an additional twenty-one days the rats were sacrificed and Pb in organs and tissues was determined. This time there was no difference in blood Pb, either at the time of sacrifice
(Table 2.2) or at earlier sampling times (data not shown). However, the rats given
27 monensin had lower levels of Pb in brain, kidney, liver, and bone. The greatest effect was seen in kidney (a 55% reduction), with the effect on liver being of similar magnitude. Consistent with the transport data shown in Fig. 2.3, and the complex stability constants shown in Fig. 2.5B, the reduced levels of Pb in organs were obtained without reducing the levels of Zn or Cu (Table 2.3). Thus, the actions of monensin on
Pb are not accompanied by perturbation of these trace elements having biological roles.
During this second experiment the rats were housed in metabolic cages so that urine and feces could be collected to determine the fate of Pb released from the organs.
Amounts excreted in urine were low, and were unaffected by the presence or absence of monensin (Fig. 2.9). Determining excretion via the feces is more problematic because the gastrointestinal tract in rats rejects most Pb that is ingested, resulting in high levels of Pb in feces during the loading period [(Quarterman et al., 1976;Barltrop and Meek,
1979) and data not shown]. When Pb2+ is withdrawn, feces Pb remains high for several days while the contents of the gastrointestinal tract turn over. These large values of excreted Pb obscure smaller differences between groups, such as differences that might be produced by monensin. Nevertheless, at later times a statistically significant effect of monensin on Pb in feces was observed. That is to say, during the second and third week of monensin administration the treated rats lost Pb at a rate of ∼120 nmol/day, whereas without monensin the rate was about half that value. As further considered below, these data suggest that the Pb mobilized from organs by monensin is ultimately excreted via the feces.
28 2.4 Discussion
Monensin was among the first group of carboxylic acid ionophores to be discovered, a group that also includes dianemycin, nigericin, compound X-206, and lasalocid A [(reviewed in (Westley, 1982)]. Soon after these compounds were reported to be potent anticoccidial agents (Shumard and Callender, 1968a) and to derive this activity through a direct interaction with monovalent cations, leading to altered cation transport (Estrada-O, 1968). Among the monovalent alkali metal cations, monensin was subsequently shown to be most effective as an ionophore for Na+ and it has long been utilized as a research tool in that regard [reviewed in (Pressman and Fahim, 1982;Taylor,
Kauffman, and Pfeiffer, 1982)]. It has also been used as a feed additive in agriculture, because of the anticoccidial activity, and because it promotes the growth of several animal species that are used in that industry (Raun et al., 1976;Ruff, 1982a).
Relatively recent studies have shown that monensin also forms complexes with several divalent cations and that they are generally more stable than those formed with monovalent cations, when compared in nonaqueous solvents such as methanol or diethylformamide (Hoogerheide and Popov, 1979;Cox, van Truong, Rzeszotarska, and
Schneider, 1984b;Hebrant et al., 1992;Mimouni et al., 1993;Mimouni et al., 1994).
More limited data have furthermore shown that monensin displays some capacity to extract divalent cations from a bulk aqueous to a bulk organic phase (Hebrant, Mimouni,
Tissier, Pointud, Juillard, and Dauphin, 1992) and to convey these cations through three phase bulk solvent systems (Tsukube et al., 1994) and polyvinyl chloride membranes in ion-selective electrodes (Suzuki et al., 1987;Suzuki et al., 1988). Thus, previous studies have suggested that monensin might not be strictly an ionophore for monovalent cations,
29 but they did not reveal that it is actually a highly selective and efficient ionophore for
Pb2+ when phospholipid membranes are employed. That activity is newly discovered and is shown in the present report (Figs 2.3 and 2.4).
The stoichiometry of the complex that transports Pb2+ is clearly 1:1, ionophore: cation, based upon the relationships between log rate and log monensin or log Pb2+ concentration, which are both linear over a wide range and display slopes of 1.0 (Fig.
2.4). As illustrated in Fig. 2.10, the 1:1 complex PbMon+ may then be active as a transporting species and would function by an electrogenic mechanism. This type of mechanism may have contributed to the transport seen in Figs. 2.3 and 2.4 because VAL and CCCP were present during those experiments. VAL and CCCP, acting together, are fully effective at collapsing an induced membrane potential in the present vesicle system
(Wang, Taylor, and Pfeiffer, 1998;Wang, Erdahl, Hamidinia, Chapman, Taylor, and
Pfeiffer, 2001), which is required for sustained electrogenic transport.
No provision was made to collapse membrane potential during the experiments reported in Figs. 2.6-2.8, so only electroneutral transport was contributing. The transporting species under those conditions is PbMonOH, as shown by the relationship between rate and external pH (Fig. 2.6), and by comparing that relationship to the distribution of Pb/monensin species that arises when the pH is varied (Fig. 2.5C).
PbMonOH can form by the pathways shown by Eqs 1 or 2, as further illustrated in Fig.
2.10, and both of these pathways can be considered to involve the acid ionization of a hydrating water molecule associated with Pb2+.
Pb2+ + Mon- PbMon+ + OH- PbMonOH (1)
Pb2+ + OH- PbOH+ + Mon- PbMonOH (2)
30 The present data do not provide insight into which of the pathways may be the most significant, but that may not be a point of central interest, assuming that all equilibria shown in Fig. 2.10 remain at or near equilibrium during monensin catalyzed Pb2+ transport.
Apart from the pathway forming PbMonOH, the extent of equilibration between reactants forming the transporting species is of interest when the origin of high selectivity for Pb2+ transport is considered, compared to the transport of other divalent cations. If near equilibrium conditions are maintained selectivity is expected to arise from differing complex stabilities, together with variation in the transmembrane diffusion constants of the various metal cation/monensin complexes. Several alkaline earth and first transition series divalent cations are poorly shielded when complexed by monensin (Hebrant, Mimouni, Tissier, Pointud, Juillard, and Dauphin, 1992), a factor that reduces transmembrane diffusion constants. However, the role of differential shielding in establishing the selectivity of monensin as an ionophore for Pb2+ is uncertain because shielding in Pb2+-monensin complexes has not been investigated.
Regarding complex stabilities, the CaMon+ and the MgMon+ complexes are less stable than the PbMon+ complex by ∼4 orders of magnitude [Fig. 2.5B, references
(Hebrant, Mimouni, Tissier, Pointud, Juillard, and Dauphin, 1992) and (Mimouni,
Pointud, and Juillard, 1994)], and in addition, no interaction of these complexes with
OH- was detected during the potentiometric titrations. The latter finding was expected, given the hydrolysis properties of these cations (Baes, Jr. and Mesner, 1976), but more importantly these considerations further explain why monensin has little or no activity as an ionophore for Ca2+ (Fig. 2.3), and why neither Ca2+ nor Mg2+ are effective as
31 inhibitors of Pb2+ transport (Fig. 2.7). Likewise, Zn2+ is bound by monensin with low affinity compared to Pb2+, although the ZnMon+ and PbMon+ complexes bind OH- with similar affinities (Fig. 2.5A). From those considerations one might expect monensin to have some activity as an ionophore for Zn2+, and that was observed in the vesicle system
(Fig. 2.3). Thus relative complex stabilities clearly contribute to establishing the transport selectivity sequence seen in Fig. 2.3.
On the other hand, Na+ is much less effective as an inhibitor of monensin mediated Pb2+ transport than would be expected on the basis of complex stability constants alone, were near equilibrium maintained between monensin, OH- and the two competing cations (Figs 2.7 and 2.8). Thus, kinetic constants of the cation complexation/decomplexation reactions, transmembrane diffusion constants, or a differing tendency of Pb2+ and Na+ to accumulate at the membrane interface may contribute to the selectivity of monensin as an ionophore for Pb2+ when significant concentrations of Na+ are also present.
The current data obtained in model systems suggest that like ionomycin (Erdahl,
Chapman, Taylor, and Pfeiffer, 2000a), monensin might be useful in the treatment of Pb intoxication, however there are no studies that have tested that possibility in animals.
Accordingly, we determined if monensin alters the accumulation or distribution of Pb when the two agents are given simultaneously, and if monensin effects the disposition of
Pb that was accumulated previously. The level of Pb2+ employed and the method of administration are typical of those used when investigating Pb toxicity in rats (Cory-
Slechta, 1988a;Gruber et al., 1997;Smith et al., 1998a;Seaton et al., 1999a). The level of monensin is the same as that usually given to chickens as an agricultural practice (Ruff,
32 1982a), and is below the toxic threshold for monensin in rats (Todd et al.,
1984b;Bogden, Gertner, Kemp, McLeod, Bruening, and Chung, 1991). Monensin was administered as a component of feed because that is the practice in agriculture and because activity upon oral administration is considered to be advantageous for agents used to treat Pb intoxication.
The data show that in both types of experiment monensin reduced the level of Pb in several organs and tissues (Tables 2.1 and 2.2). The design of studies which investigate the efficiency of agents used to treat Pb intoxication vary widely, so it is difficult to compare the effectiveness of monensin to that of the traditional agents (e.g.
EDTA and dimercaptosuccinate) in a succinct manner. Nevertheless, monensin appears to be similarly effective to these better investigated compounds [e.g., compare to references (Hammond et al., 1967;Pappas et al., 1995a;Tandon et al., 1998)], even though the dose employed here, the treatment interval, etc., have not been optimized. In addition, monensin reduces Pb in bone and brain, which is not true for the traditional compounds under some circumstances (Cory-Slechta, Weiss, and Cox, 1987a;Cory-
Slechta, 1988a), and does not deplete the tissues of Zn and Cu, which can occur as an unwanted side effect with some of the traditional compounds (Perry, Jr. and Perry,
1959;Braide, 1984;Kostial et al., 2000). Thus, monensin may indeed be useful for promoting the elimination of Pb from animals.
At this early stage it is appropriate to consider the possibility than monensin acts differently than the traditional compounds as regards the mechanism by which of Pb excretion occurs. The traditional compounds are hydrophilic cation chelators that circulate in blood and form Pb2+ complexes having stability constants of ∼1015 and
33 higher. They are not membrane permeant and are excreted together with Pb2+, typically via kidney. The Pb/Mon complex is much less stable (K= 107.25 in 80% methanol-water) and has little solubility in an aqueous environment. Moreover, the complex easily crosses a phospholipid membrane, whereas Pb excretion occurs primarily via the colon.
Thus, monensin may act primarily by transporting Pb out of cells, where it eventually returns to the lumen of the gastrointestinal tract and is excreted by a normal mechanism, possibly involving the enterohepatic circulation. Since monensin is highly active as an ionophore for Na+, as well as for Pb2+, its presence will couple the gradients of these two cations across cells membranes, providing a driving force for Pb transport out of the cell.
Overall, this potential mechanism suggests that the application of monensin, together with a hydrophilic chelator, might be particularly effective at promoting the excretion of
Pb from animals. That possibility is currently under investigation.
2.5 Summary
The carboxylic acid ionophore monensin, known as an electroneutral Na+ ionophore, an anticoccidial agent, and a growth promoting feed additive in agriculture, is shown to be highly efficient as an ionophore for Pb2+, and to be highly selective for Pb2+ compared to other divalent cations. Monensin transports Pb2+ by an electroneutral mechanism in which the complex PbMonOH is the transporting species. Electrogenic transport via the species PbMon+ may also be possible. Monensin catalyzed Pb2+ transport is little affected by Ca2+, Mg2+ or K+ concentrations that are encountered in living systems. Na+ is inhibitory, but its effectiveness at 100 mM does not exceed
∼50%. The poor activity of monensin as an ionophore for divalent cations other than
34 Pb2+ is consistent with the pattern of complex formation constants observed in the mixed solvent 80% methanol-water. This pattern also explains why Ca2+, Mg2+ and K+ are ineffective as inhibitors of Pb2+ transport, but it does not fully explain the actions of Na+, where kinetic features of the transport mechanism may also be important. When given to rats at 100 ppm in feed together with Pb2+ at 100 ppm in drinking water, monensin reduces Pb accumulation in several organs and tissues. It also accelerates the excretion of Pb that was accumulated previously and produces this effect without depleting the organs of Zn or Cu. Monensin, used alone or in combination with other agents, may be useful for the treatment of Pb intoxication.
35 Me
H Me Me H H H O H OO H H Me Me N N N H O H H H OO A23187
OH Me Me Me OMe Me Et H Me Me O O OO HHH O H CH2OH CO2H Me OH
Monensin
OMe Me Me Me Me Me Me Me H Me Me O O O O O H H HHH O CH OH CO H 2 2 OH Nigericin
Me Me Me Me Me Me
O O OH OH OH OH Me Me Me Me
CO2H O Ionomycin
Figure 2.1. Structure of representative carboxylic acid ionophores.
36
n+ - M + A MAn-1 +++
A- OH- X-
I II III
n-2 n-2 MA2 MA(OH)n-1 MAX
Figure 2.2. Equilibria between an ionized carboxylic acid ionophore (A-1), a metal cation of charge n+ (Mn+), and selected anions (A-, X-, and OH-). Following formation of 1:1 complex between the ionophore and a cation (MAn−1), additional species can arise that may contribute to transport. In I, a 1:2 complex is formed by reaction with a second ionophore molecule. In II and III, mixed complexes arise by reaction with OH- or another anion (X-), respectively.
37 A. Ionomycin 20 Pb2+
Zn2+ 16 Cd2+ M µ Iono t, 12 por Mn2+ ans
Tr 8 2+ Ca2+ Me 2+ 4 Cu Co2+ Ni2+ 0 Sr2+
0 200 400 600 800
seconds
B. Monensin 20 Pb2+
16 M µ Mon
ort, 12 p s n
a 2+
r Cu
T 8 Zn2+ 2+ Cd2+ Me Mn2+ 4 Co2+ Ca2+ Ni2+ 2+ 0 Sr
0 200 400 600 800
seconds
Figure 2.3. Transport of Pb2+ and other divalent cations by ionomycin (panel A) and monensin (panel B). POPC vesicles loaded with Quin-2 (Cs+) were prepared as described in the Materials and Methods. They were incubated at 25°C and a nominal phospholipid concentration of 1.0 mM. The external medium contained 50 mM CsNO3, 10 mM each of Mes and Hepes (both Cs+), pH 7.00, 0.5 µM valinomycin, 5.0 µM carbonyl cyanide-m-chlorophenylhydrazone (CCCP), and 20 µM of the indicated divalent cation (nitrate in the case of Pb2+, chlorides for all others). After a 2 min preincubation 0.10 µM ionophore was added to initiate transport (designated as 0 seconds in the figure). The sequence of cations shown on the right corresponds to their relative rate of transport as observed directly for panel A, or on an expanded scale for panel B. The y-axis unit refers to cation concentration in the external medium. When this parameter reaches 20 µM, all of the cation that was originally present in the external medium has been transported into the vesicles and sequestered by Quin-2. 38
Figure 2.4. Pb2+ transport dependence on the concentrations of Pb2+ and monensin. Data were obtained as described in the legend to Fig. 2.3 except that the concentrations of Pb2+ and monensin were varied. In addition, the external free Pb2+ concentration was established by a 15 mM citrate-based buffer system, as described in the Materials and Methods. Panel A; the monensin concentration was 0.10 µM and the concentration of free Pb2+ was varied from 10 nM (bottom most curve) to 5.63 µM (topmost curve). Panel B; the free Pb2+ concentration was 1.0 µM and he concentration of monensin was varied from 1.17 nM (bottom most curve) to 2.40 µM (topmost curve). Panel C, the data from panels A and B are shown as log initial rate vs. log Pb2+ ({) or log monensin (z) concentration. Initial rates were obtained from the individual progress curves as described in the Materials and Methods.
39 A. Variable Monensin 24
20 M µ
t, 16 Mon or sp
n 12 a r T
2+ 8 Pb
4
0
0 200 400 600 800
seconds
2+ 20 B. Variable Pb
16 M µ
, 12 Mon ansport r 8 T 2+ Pb 4
0
0 200 400 600 800
seconds
1 C. Variable Monensin Variable Pb2+
c 0 e s M/ µ -1 te, a R -2 Initial
Log -3
-4
-10-9-8-7-6-5-4
2+ Log Pb (Monensin), M
40
Figure 2.5. Equilibrium behavior of monensin and selected cations. Panel A: potentiometric titrations of 1.0 mM monensin in an 80% methanol-water mixed solvent were conducted as described in the Materials and Methods. The temperature was 25 oC and the ionic strength was 0.1 M, maintained by the presence of TEAP. (I), monensin alone; (II), monensin in the presence of 0.5 mM Pb2+; (III), monensin in the presence of 1.0 mM Pb2+. Panel B: equilibrium constants of interest, determined from data analogous to those shown in panel A. The symbol L- refers to the monensin carboxylate anion. Panel C: the species distribution diagram for a solution containing a total of 0.10 µM monensin and with the Pb2+ concentration fixed at 0.10 µM. Individual values were calculated using the program COMICS (Perrin and Sayce, 1967), the equilibrium 2+ constants of interest from panel B, and a pKa of 7.96 for the hydrolysis of Pb in 80% methanol-water (Ivanova, 2000).
41 A. III
10
8
pH 6 III
4
2 0.0 0.5 1.0 1.5 2.0
Base Added, mL
B. + - H +L HL ; log KH =6.83 2+ - + Pb +L PbL ; log KML =7.25 + - PbL +OH PbLOH ; log KMLOH =7.10 + - PbL +L PbL2 ; log KML2 =3.35 2+ - + Ca +L CaL ; log KML =3.10 2+ - + Mg +L MgL ; log KML =3.16 2+ - + Zn +L ZnL ; log KML =3.74 + - ZnL +OH ZnLOH ; log KMLOH =6.67 + - Na +L NaL ; log KML =5.00 + - K +L KL ; log KML =3.76
C.
0.10
HMon PbMPbMonOHonOH 0.08 M µ
,
0.06 tration
0.04 + - ncen PbMon Mon o C
0.02
0.00 45678910 pH*
42 A. 8 pH 9.00
7
6 M
µ Mon
, 5
4
Transport 3 2+
Pb 2
1 pH 6.50
0 0 200 300 400 500 600
seconds
pPbOH
8.0 7.5 7.0 6.5 6.0
0.8 B. n 0.6 M/mi µ te,
a 0.4 Initial R 0.2
0.0 6.5 7.0 7.5 8.0 8.5 9.0
pH
Figure 2.6. Effect of external pH on ionophore mediated Pb2+ transport. Vesicles were prepared as described in the Materials and Methods except that Na+ rather than Cs+ was employed as the counter ion to Quin-2. The external medium contained 10 mM each of Mes, Hepes and Ches, to provide for H+ buffering over a broad range of pH, and 5 mM citrate was present to buffer free Pb2+. K+ was used as the external counter ion for citrate and the H+ buffers. The external pH was set at a value of interest and the 2+ concentration of Pb(NO3)2 required to produce a free Pb concentration of 0.10 µM was added to the cuvette. Pb2+ transport was initiated by the addition of 0.1 µM monensin. Panel A, individual progress curves covering the pH region of 6.5-9.0. The earliest portions are omitted because the initial equilibration of H+ and monovalent cation gradients perturbs the UV-Vis spectrum of Quin-2. Accordingly rates pertaining at 140 seconds were determined, rather than the initial rates. Panel B, rates obtained from panel A at the pH indicated. Values are the mean of duplicate determinations. They were fit to the Henderson-Hasselbalch equation to obtain the solid line. 43
Figure 2.7. Actions of physiological cations as inhibitors of Pb2+ transport. Vesicles were prepared as described in the Materials and Methods using Cs+ as the counter ion to + Quin-2. The external medium contained 5 mM citrate (Cs ) plus Pb(NO3)2 sufficient to produce a free Pb2+ concentration of 1.0 µM (except for traces labeled –Pb2+), and 5 mM each of Mes and Hepes (both Cs+), pH =7.0. As indicated, the medium also contained 2+ CaCl2 sufficient to produce a free Ca concentration of 1.0 mM (panel A), MgCl2 sufficient to produce a free Mg2+ concentration of 1.0 mM (panel B), or the indicated concentration of NaCl (panel C). Transport was initiated by the addition of 0.1 µM monensin.
44 A. 10 +Pb2+, +Ca2+
8
2+ 2+ M +Pb , -Ca µ Mon
rt, 6 anspo r 4 T 2+ Pb 2 -Pb2+, +Ca2+
0 0 100 200 300 400 500 600 700
seconds
B. 10 +Pb2+, -Mg2+
8
M 2+ 2+
µ +Pb , +Mg 6 Mon ansport, r 4 T 2+ Pb 2 -Pb2+, +Mg2+
0 0 100 200 300 400 500 600 700
seconds
C. 10
+Pb2+, Na+ = 0 8 +Pb2+, Na+ = 5.0 mM M µ Mon rt, 6
anspo 2+ + r +Pb , Na = 100 mM 4 T 2+ Pb 2
-Pb2+, Na+ = 100 mM 0 0 100 200 300 400 500 600 700
seconds 45
14 CsCl 12 KCl
n 10 TEAP mi Mannitol M/
µ 8 te, a 6
4 Initial R
2
0 0 20406080100
NaCl, mM
Figure 2.8. Inhibition of Pb2+ transport by Na+. Vesicles were prepared as described in the Materials and Methods using Cs+ as the counter ion to Quin-2. The external medium 2+ contained 5 mM citrate plus Pb(NO3)2 sufficient to produce a free Pb concentration of 1.0 µM, and 5 mM each of Mes and Hepes, pH =7.0. At NaCl =0 the medium also contained (z), 100 mM CsCl; ({), 100 mM KCl; (S), 100 mM TEAP; (U), 200 mM mannitol. Cs+, K+ or TEA+ was used as the counter ion for the external buffers, so as to match the cation that was added at 100 mM. In the case of mannitol, Na+ was used. As the NaCl concentration was increased, the concentration of the external cation was decreased by an equal value, or was decreased by twice that value in the case of mannitol. Monensin was used at 0.1 µM.
46
+Pb2+ -Pb2+
30
25 ay /d
20 nmol , rat
n/ 15 io
cret 10 ex 2+
Pb 5
0 0 5 10 15 20 25 30 35 40 45
days
Figure 2.9. Effect of monensin on Pb excreted in urine. Two groups of eight rats were given 100 ppm Pb2+ in drinking water, for twenty-one days, as further described in the Materials and Methods. The rats were housed in metabolic cages, allowing total urine output by each rat to be collected over two-day intervals. Pb was determined by atomic absorption spectroscopy and expressed as nmol Pb excreted per rat per day. Up to and including day twenty-one, the values plotted are means of those obtained for all sixteen rats. On day twenty-two Pb2+ was withdrawn and one group was shifted to a diet containing 100 ppm monensin, while the second was maintained without monensin. ({), Pb excreted by rats receiving monensin; (z), Pb excreted by rats not receiving monensin.
47
Mon- +
Pb2+
1
PbMon+ Electrogenic Transport + OH-
2
PbMonOH Electroneutral Transport
3
Mon- + + PbOH
Figure 2.10. Potential mechanisms of monensin catalyzed Pb2+ transport. The curved lines together represent a phospholipid bilayer, whereas the heavy arrows indicate transmembrane diffusion of a Pb/monensin complex. The various species illustrated are assumed to be present near the membrane-bulk aqueous phase interface.
48
Tissue Control Pb Pb plus monensin
Blood* 0.10 1.17 ± 0.32 0.88 ± 0.24
Heart* 0.005 0.33 ± 0.11 0.11 ±0.04
Brain# 0.050 0.71 ± 0.35 0.50 ± 0.13
Liver 0.20 3.82 ± 2.04 2.88 ± 0.65
Kidney 0.20 23.3 ± 8.73 21.0 ± 5.35
Muscle# Nil 0.20 ± 0.07 0.13 ± 0.05
Bone* Nil 264 ± 82 173 ± 29
Table 2.1. Monensin Effects on Pb2+ Accumulation Two groups of six rats were given Pb at 100 ppm in their drinking water, as further described in Experimental procedures. One group also received monensin at 100 ppm in feed, whereas the other group did not. After twenty-eight days the rats were sacrificed and Pb was determined in the indicated organs and tissues, as further described in Experimental Procedures. Muscle and bone refer to the quadriceps muscle and the femur, respectively. All values are means, ± one standard deviation, and are in units of nmoles per g wet weight, except for blood which is in units of µM. Control values (no Pb, no monensin) are from the literature (Bogden, Gertner, Kemp, McLeod, Bruening, and Chung, 1991). For tissues marked with an *, the Pb plus monensin value was lower than the Pb value at p < 0.05. For those marked #, the threshold was p < 0.10. For liver and kidney, the differences did not reach either threshold.
49
Tissue Normal values No monensin Plus monensin
Blood 0.10 0.82 ± 0.23 0.83 ± 0.27
Heart 0.005 0.13 ± 0.09 0.09 ± 0.07
Brain# 0.05 0.90 ± 0.44 0.65 ± 0.20
Liver* 0.20 1.19 ± 0.48 0.608 ± 0.46
Kidney* 0.20 16.4 ± 6.4 7.32 ± 3.3
Muscle Nil 0.033 ± 0.03 0.014 ± 0.01
Bone* Nil 310 ± 109 189 ± 78
Table 2.2. Monensin Effects on Pb2+ Clearance Two groups of eight rats were given Pb2+ 100 ppm in drinking water without the administration of monensin. After twenty- one days Pb was withdrawn and the administration of monensin at 100 ppm in feed began in one of the groups, whereas normal feed was given to the other group. After an additional twenty-one days the rats were sacrificed and Pb was determined in the indicated organs and tissues, as further described in Experimental Procedures. Muscle and bone refer to the quadriceps muscle and the femur, respectively. All values are means, ± one standard deviation, and are in units of nmoles per g wet weight, except for blood which is in units of µM. Control values (no Pb, no monensin) are from the literature (Bogden, Gertner, Kemp, McLeod, Bruening, and Chung, 1991). As in table 2.1, for tissues marked with an *, the plus monensin value was lower than the no monensin value at p < 0.05. For those marked #, the threshold was p < 0.10. For heart and muscle, the differences did not reach either threshold.
50
Tissue Zn, nmol/g wet weight Cu, nmol/g wet weight No monensin Plus monensin No monensin Plus monensin
Heart 249 ± 14 238 ± 12 61.9 ± 4.7 74.1 ± 6.9
Brain 230 ± 4.0 226 ± 13 26.8 ± 2.3 35.3 ± 2.5
Liver 428 ± 25 395 ± 48 56.7 ± 5.1 57.6 ± 6.4
Kidney 330 ± 27 319 ± 16 99.0 ± 18 97.0 ± 17
Muscle 166 ± 33 150 ± 34 11.1 ± 1.7 10.7 ± 1.5
Bone 2136 ± 112 1975 ± 66 44.6 ± 2.3 44.9 ± 3.2
Table 2.3 Monensin Effects on Trace Cation Levels Values are means, ± one standard deviation, and were determined by flame atomic absorption spectroscopy. Samples were the same ones used to obtain the data in Table 2.2. Of the small variations observed, none were statistically significant.
51 CHAPTER 3
MONENSIN IMPROVES THE EFFECTIVENESS OF MESO-
DIMERCAPTOSUCCINATE WHEN USED IN THE TREATMENT OF Pb
INTOXICATION
3.1 Introduction
Several polyether ionophores, including A23187, ionomycin, monensin, and nigericin, have been shown to transport Pb2+ across phospholipid bilayers and to form stable complexes with Pb2+ in homogeneous solution (Erdahl et al., 2000b;Hamidinia et al., 2002b;Hamidinia et al., 2004). Among this group, the order of transport activity is ionomycin > nigericin > monensin > A23187, whereas the order of selectivity, compared to other divalent cations, is nigericin > monensin > ionomycin, > A23187. Ionomycin can be used to load cultured cells and to deplete them of Pb2+ (Erdahl, Chapman, Taylor, and Pfeiffer, 2000b), whereas, effects of monensin on Pb dynamics in rats have also been investigated (Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and
Pfeiffer, 2002b). In the latter area it was found that simultaneous administration of monensin in feed and Pb2+ in drinking water lowers the prevailing concentration of Pb in blood and decreases the accumulation of Pb in several soft tissues, and in bone. Feeding monensin after a period of Pb2+ administration was furthermore effective at accelerating
52 the clearance of Pb from brain, liver, kidney and bone, with the monensin related increment being found in feces, as opposed to the Pb excreted spontaneously which included a component found in urine (Hamidinia, Shimelis, Tan, Erdahl, Chapman,
Renkes, Taylor, and Pfeiffer, 2002b).
The actions of monensin on Pb dynamics in rats suggest the possibility of utilizing ionophores in the treatment of human Pb intoxication. At present, the ongoing and wide-spread problem of Pb intoxication is treated first by removing the individual from the Pb contaminated environment, and thereafter by administering a Pb2+ chelating agent. Meso-dimercaptosuccinic acid (DMSA) is the most commonly used agent at present, in part because it can be administered orally and is well-tolerated (Graziano et al., 1985;Cory-Slechta, 1988b). DMSA is a water soluble compound that forms a strong complex with Pb2+ in blood, which is thereafter secreted via the kidney. EDTA and other chelating agents are used in a similar way to treat Pb intoxication (Llobet et al.,
1990;Goyer et al., 1995b). In all cases, several cycles of chelator administration given over a period of months are required to produce an adequate and durable reduction of Pb in blood. This is because the blood pool is equilibrated with Pb in other internal compartments, such that a blood pool is reestablished as Pb is mobilized from the other compartments following a cycle of chelator administration (Graziano, Siris, LoIacono,
Silverberg, and Turgeon, 1985;Graziano, 1986;Graziano et al., 1988;Graziano, 1993).
The repetitive treatments and extended time frames are problematic, particularly when treating children, because important aspects of Pb toxicity arise to a greater degree if Pb is present before development is completed. The propensity of Pb to lower IQ and otherwise interfere with functions of the central nervous system is perhaps the most
53 important of these aspects (Goyer, 1993). An uneven effectiveness of water soluble chelators at removing Pb from particular organs is another problem with existing protocols for the treatment of Pb intoxication, with bone (Castellino and Aloj,
1964;Smith et al., 2000;Cory-Slechta, 1988b) and brain (Cremin et al., 1999;Seaton et al., 1999b;Smith et al., 1998b) being particularly difficult to free of the accumulated cation. In addition, chelators can provoke an undesirable redistribution of Pb among soft tissues and between bone and soft tissues (Cory-Slechta et al., 1987b)
Within this context of less than ideal treatments for Pb intoxication and the recent discovery that several carboxylic acid ionophores transport Pb2+ with high specificity, we are seeking to determine if some of these compounds might be used together with the traditional chelators to improve the effectiveness of existing treatment protocols. The present report describes the effectiveness of co-administering monensin and DMSA in this regard, using rats as an experimental model.
3.2 Materials and Methods
Treatment of Experimental Animals. Male Sprague-Dawley rats were utilized throughout the study. They were housed in AALAC approved animal facilities at the
College of Medicine, Ohio State University. A 12 hr light-dark cycle, dual housing in plastic cages and conditions of constant temperature and humidity were employed. One week was allowed for acclimation before the experimental protocol began. During this period a standard laboratory chow was employed, whereas the AIN-93M diet containing
0.5% calcium (normal chow) (Harlan Techlad) was employed thereafter. During the
54 administration of Pb2+ and/or monensin and DMSA, water and feed were provided ad libitum and records of consumption were maintained together with periodic measurements of body weight.
Initially the rats weighed 245-255g. They were divided into five groups of eight, and the administration of Pb2+ was begun. It was provided at 100 ppm in the drinking water (0.48 mM), and was in the form of Pb(acetate)2. The water was rendered slightly acidic with acetic acid to prevent the precipitation of PbCO2 (Bogden et al., 1995a).
After three weeks one group of rats was sacrificed to determine the Pb content of organs at that time (see below, the Pb loaded group), whereas the remaining groups thereafter received water that did not contain Pb2+. One of these groups received the normal chow
(no treatment group), another received this chow containing 100 ppm of monensin
(monensin group), a third group received normal chow and DMSA, administered by oral gavage (DMSA group), while a final group received the chow containing monensin and
DMSA delivered by oral gavage (monensin plus DMSA group). When administered, the dose of DMSA was 50 mg/kg given every other day. The solution volume administered was varied between ~0.25 to 0.45 mL as body weight increased so as to maintain the prescribed dose. The DMSA solution was freshly prepared by dissolving the compound in 5% NaHCO3. All treatments were continued for three weeks beyond the time when the administration of Pb2+ had been discontinued. The rats were then sacrificed by the injection of excess Nembutal and were subsequently perfused briefly with Hepes buffered 0.9% NaCl, via the left ventricle, to remove blood from the organs.
Following this organs and tissues of interest were removed and stored at -20°C.
55 The determination of Pb and other elements by Inductively Coupled Plasma
Mass Spectroscopy. All aspects of the analytical procedures were conducted in laminar flow hoods within the Microscopic and Chemical Analysis Research Center at Ohio
State University. The frozen organs were first thawed and weighed, and were then digested in 5.00 ml of trace metal-grade concentrated nitric acid. One intact kidney, the entire brain, the entire heart, and the left femur from each animal were analyzed. For liver, a portion of the right lateral lobe weighing ~2 g was analyzed, whereas for muscle it was a similar sized portion of the left biceps femoris. Digestion was conducted in acid cleaned quartz vials that were contained in a teflon liner which was itself contained in a closed high pressure vessel (Milestone Inc). The teflon liners contained 10 mL of a 20%
H2O2 solution, in which the quartz vial was partially immersed, so as to minimize the rise in internal pressure that occurs as digestion proceeds. Samples were heated to
180˚C in a microwave apparatus (Ethos Plus, Milestone Inc). Temperature programming provided for a linear rise to that value over a 10 min period, a holding period of 10 min, and a 15 min cool down period. Sample blanks containing no tissue were generated in the same way, as were occasional tissue samples that were spiked with
Ga (used to estimate overall recovery from the procedure). After digestion the samples were transferred quantitatively to Nalgene HDPE Boston round bottles (Fisher), diluted to 50.0 ml with double distilled deionized water, and then stored at room temperature.
Subsequent steps were carried out within one month. A cocktail of three internal standards (Bi, Sc and Rh) made from CPI International-Peak Performance standards was added to each sample (100 µl in 10.0 ml) so as to give a 10 ppb concentration of each standard. The signals arising from these standards were used to correct data for
56 variations in instrument performance that occur while a set of samples is being analyzed.
A set of calibration standards, similar in composition to the unknown samples, was prepared for each of the six tissues and used to convert data output from the instrument into units of concentration. These were prepared using SPEX Certi Prep multielement standards with appropriate adjustments made using single element standards obtained from CPI International. The samples were analyzed using a Thermal Finnigan Magnetic
Sector Inductively Coupled Mass Spectrometer (ICP mass spectrophotometer), which is capable of resolutions greater than 0.005 amu. The data were expressed first in units of ppb and were subsequently converted to units of nmol/g wet weight of tissue. Replicate analysis of single samples showed that deviation arising from analysis per se was on the order of ±2%, whereas overall recovery of unknowns was ~98-101%.
Pb levels in the various tissues were expressed as means, ± the standard error of the mean, and thereafter by the percentage change in mean values. Comparisons between groups were made using Student’s test with differences reported as significant for p values < 0.05. For elements other than Pb we asked if mean values differed between treatment groups and the two tailed test was employed, accordingly. In the case of Pb, previous work indicated that all treatments might enhance depletion, but would not be expected to elevate the levels observed. Accordingly, when analyzing the Pb data we asked if treatment in question lowered Pb, relative to the appropriate control, and the one-tailed test was employed (Christenson and Stoop, 1986).
3.3 Results
Levels of Pb and other elements following a period of Pb administration.
57 Monensin is highly selective for the transport of Pb2+, compared to other divalent cations
(Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002b), and this is one of its characteristics which led us test this ionophore for possible use in conjunction with DMSA for the treatment of Pb intoxication. That is to say, it seemed possible that monensin might aid in the delivery of intracellular Pb2+ to the circulating chelator without greatly perturbing the intracellular level of other cations. On the other hand, the available selectivity data were obtained using a model transport system based on phospholipid vesicles and did not include all cations of possible interest. Thus, it was not clear initially to what extent the selectivity for Pb2+ would be manifest in vivo, or if cations having a physiological role, but not yet considered in terms of selectivity, might also be perturbed. Accordingly, we examined the tissue levels of several physiological elements (Ca, Co, Cu, Fe, Mn, Mg, Mo, Zn) using ICP mass spectrometry as the detection modality. We also examined levels of several elements having no physiological role (As, Cd, Cr, Ni, Sr) to determine if the ionophores actions are specific for Pb among a known group of potential toxins.
Table 3.1 compares literature values for the endogenous levels of these cations in the organs examined, as they were reported in another study (Naveh et al., 1987), to those that we found at the end of the three week Pb loading period. As expected, Pb rose markedly in all organs examined, but there were large differences in the absolute levels that were attained. Specifically, the order of Pb levels observed was femur > kidney > liver > brain > heart > skeletal muscle, with the highest value exceeding the lowest one by approximately 2.5 × 103. These findings are similar to those reported by others (Naveh, Weis, Chung, and Bogden, 1987;Bogden et al., 1992;Han et al., 1996).
58 Among the other elements considered, comparisons were possible for Ca, Cu, Fe,
Mg, Mn and Zn, but not for Co, Mo, As, Cd, Cr, Ni, and Sr, because the reference study did not include data for the latter group. Among the former group three large variations were seen, namely the level of Ca in kidney, and the levels of Cu and Mn in femur. In all three cases the values reported here are about 10% of the reference values and the differences are statistically significant. While there are several possible reasons for variations of this magnitude, as further considered in the Discussion section, we doubt that they reflect authentic effects of Pb administration and have used our current values when interpreting other aspects of the data. Among the other organs and elements considered, variations were much smaller. Reference values for Fe in kidney and Mn in skeletal muscle are higher than what we found following Pb loading, by about a factor of
2, and these differences were also statistically significant. As regards the other variations, many were not significantly different whereas others were, even when the extent of variation was quite small (Table 3.1).
Depletion of previously accumulated Pb. To express the relative effectiveness of the four treatments that were applied after the period of Pb administration, we calculated the fraction of the Pb loaded value which remained when the treatment period was complete (three weeks), and compared these values to each other and to the endogenous values. Results are shown graphically and numerically in Fig. 3.1. Two subsets are apparent among the six tissues examined, namely kidney, liver, and skeletal muscle compared to heart, brain and femur. For those in the first subset the mean Pb level fell dramatically simply in response to halting its administration, and these were fully significant declines compared to the levels that existed at the end of the loading period
59 (Fig. 3.1). For this subset, monensin alone did not alter the values significantly, compared to withdrawal alone, whereas the further decrease produced by DMSA was significant in kidney and liver. The former result might not be expected, whereas the latter was expected, based upon previous reports (Cory-Slechta, 1988b;Pappas et al.,
1995b;Jones et al., 1997;Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002b). As regards monensin used together with DMSA, the mean values in kidney and liver were lower than those produced by DMSA alone, but the p values
(0.08 in kidney, 0.14 in liver) fell short of the usual threshold that is taken to demonstrate significance (0.05).
For the other subset of tissues (heart, brain and femur) halting administration alone did not significantly lower the level of Pb during the three week test period that was employed. Compared to the values obtained by withdrawal alone, the effects of monensin alone were again not significant except in the case of heart where the ionophore did produce a lower value. As regards DMSA alone, this treatment lowered
Pb in heart and brain, but did not decrease Pb in bone, again as might be expected (Cory-
Slechta, 1988b;Jones, Singh, Kostial, Blanusa, Piasek, and Restek-Samarozija,
1997;Smith, Woolard, Luck, and Laughlin, 2000). Of greater interest, in all three tissues, monensin plus DMSA lowered mean Pb values more noticeably than was seen in the other tissues, compared to the effect of DMSA alone, and these differences were statistically significant (Fig. 3.1). Furthermore, the decrease produced by monensin plus
DMSA in bone was significant compared to withdrawal alone, and this was also true in the five other tissues (Fig. 3.1).
Effects of monensin alone on the levels of other elements. Regarding the other
60 elements, data are presented in Figs. 3.2-3.14, which are in the same format used for Fig.
3.1. Given that we are primarily interested in determining if monensin might be useful in the treatment of Pb intoxication we focus first on whether or not the ionophore given alone perturbs physiological elements. This point is of interest because such perturbations might lead to secondary forms of toxicity. Regarding Ca, monensin alone increased the level in heart by ~24%, compared to rats that received no treatment for accumulated Pb, but had no significant effect in the other tissues (Fig. 3.2). By the same comparison, Co was decreased in kidney, heart and brain, but here it is seen in the no treatment group, that Co fell during the three week period after Pb administration had ended (Fig. 3.3). Thus, the 25-30% increases in Co produced by monensin were tending to reverse that initial decline and might therefore be viewed as advantageous. Cu was increased in kidney and brain, decreased slightly in liver and skeletal muscle, while it was not changed significantly in in heart and femur (Fig. 3.4). Fe rose modestly in heart and brain but was not perturbed significantly elsewhere (Fig. 3.5). Mg was altered only in heart (Fig. 3.6), Mn in liver, heart and brain (Fig. 3.7), and these were all modest variations of around 15% or less. Changes in Mo and Zn were similarly small (Figs. 3.8 and 3.9), except for the Mo level in femur which decreased by 70% (Fig. 3.8).
Among the non-physiological elements (Figs. 3.10-3.14), the only statistically significant effect produced by monensin alone was in heart, where the trace level of As rose by ~24% (Fig. 3.10).
Perturbations produced by monensin in DMSA treated rats. Because Fig. 3.1 indicates that monensin plus DMSA is more effective at depleting Pb, than either agent used alone, it is also of interest to determine if the two agents used together perturb other
61 elements more so than does DMSA alone, and to examine perturbations produced by the combination of compounds compared to no treatment. Considering the former comparison first, and for the physiological elements, monensin altered the levels of Ca,
Co, Cu, Fe and Zn in one or more tissues, more so than did DMSA alone, whereas the levels Mg, Mn, and Mo were not effected statistically (Figs. 3.2-3.9). Among the changes of this type, all were small (on the order of 15% or less), except for the level of
Co in femur which increased by 53% (Fig. 3.3). Furthermore, the directions of these changes were such that they tended to reverse changes that arose from DMSA alone and thus might be viewed as beneficial. This is with the exception of Zn in skeletal muscle and heart (Fig. 9), which was perturbed to a greater extent by monensin plus DMSA, than it was by DMSA alone, although the effects were small (+16% and -10%, respectively).
Considering the non-physiological elements, monensin plus DMSA increased Ni substantially in liver, skeletal muscle and heart, compared to DMSA alone (Fig. 3.13), and likewise decreased Sr in kidney and heart (Fig. 3.14). The changes in Sr were in the direction that corrected a perturbation produced by DMSA alone, but this was not true with Ni.
Perturbations produced by monensin plus DMSA compared to no treatment.
Within this last area of interest, and among the physiological elements, monensin plus
DMSA produced a change in one or more tissue, in all cases except Ca. Co rose in kidney, heart, and femur, but these changes were again tending to reverse declines which otherwise occurred in rats that were not treated (Fig. 3.3). Cu fell modestly in liver,
62 heart, and brain; Fe rose in liver and femur, whereas Zn was altered in liver, skeletal muscle and femur (Figs. 3.7-3.9). All of these changes were again small, with the exception of Mo femur where the increase was 62%.
Considering the non-physiological elements (Figs. 3.10-3.14), significant changes were confined to Cr and Ni, which were both increased in liver (Figs. 3.11 and
3.13).
Effects of treatment on other parameters. As regards macroscopic effects of the treatment strategies investigated, there was no indication that the rats were differently stressed, as indicated by their behavior, activity level, general appearance, or the macroscopic appearance of internal structures seen during dissection. Records of weight gain throughout the experimental period are shown in Fig. 3.15. During the period of Pb administration, when all rats were maintained in the same way, the per animal average weight of the five groups diverged such that there was a 25g range in this parameter at the time that Pb was withdrawn. Comparing the no treatment and the monensin alone groups showed no significant difference in the subsequent rate of weight gain (Fig.
3.15). For the rats receiving DMSA, or DMSA plus monensin, weight gain lagged initially but then returned to a rate that was similar to that seen in the no treatment or the monensin alone groups. An unpleasant odor of the dimercaptide and gavage-related irritation of the G.I. tract may have contributed to these lags in weight gain by discouraging eating.
3.4 Discussion
The present results demonstrate that monensin plus DMSA is more effective than
63 DMSA alone in terms of depleting rats of previously accumulated Pb, and that the combination of agents is effective in all organs/tissues examined. Comparing the agents used in combination to DMSA alone, monensin significantly improved the outcome in heart, brain and femur, and showed a tendency to do this in kidney and liver. The improved clearance seen in heart, brain and femur is notable because withdrawal of Pb alone is not very effective at reducing Pb in these tissues, because DMSA used alone is less effective than in some other tissues, and because the actions of Pb in heart, brain and bone account for significant aspects of Pb pathophysiology. Thus in heart, Pb accumulation generates a set of effects that are analogous to those produced by various forms of human cardiac disease (Williams et al., 1983). In brain, Pb effects are manifest at low overall levels and include cognitive impairment and reduced IQ (Goyer,
1993;Goyer, Cherian, Jones, and Reigart, 1995b;Rice, 1996). Bone Pb is problematic because this pool is large, and is the main source of Pb that reestablishes an elevated blood concentration following treatment by existing methods (Cory-Slechta,
1988b;O'Flaherty, 1991;Leggett, 1993;O'Flaherty, 1995;O'Flaherty et al., 1998). Bone
Pb can furthermore be mobilized together with Ca during aging and pregnancy to produce toxic effects even when there is no ongoing exposure to Pb from the environment. Thus, it seems worthwhile to further explore the possibility of using ionophores together with the traditional agents in the clinical treatment of Pb intoxication.
Potential toxicity of the ionophore itself must be considered when contemplating a clinical application. There are no data available on the toxic actions of monensin in humans; however the compound has long been administered to a variety of animal
64 species that are used to produce food in agriculture. This practice arose because monensin is an anticoccidial agent, because it promotes growth, and because it is easily administered in feed as was done during this study (Shumard and Callender,
1968b;Walker et al., 1980b;Ruff, 1982b). The level of 100 ppm used here is typical of that used in agriculture and is well below the level of 200 ppm where toxic actions are first seen in rats (Ruff, 1982b;Todd et al., 1984a). To these points we can now add that monensin plus DMSA also produces no overt toxicity in rats, and has little effect on growth, beyond that seen with DMSA alone (Fig. 3.15).
In an attempt to further examine the potential for toxicity we determined how tissue levels of other elements changed during a period of Pb administration and during our attempts to remove it. Among the elements having a physiological role, no situation was found in which the level of an element was markedly decreased compared to that which existed when Pb administration had been completed and treatment was about to begin. The closest we saw to this type of situation was with Co, where levels tended to decline following removal of Pb from the drinking water. The various treatments used to remove Pb either had little further effect, or tended to restore Co to its pretreatment value (Fig. 3.3).
Likewise, we did not encounter examples where the levels of a physiological element rose markedly, compared to a pretreatment value, as a result of any approach taken to remove Pb. Cu, Fe, Mo, and Zn rose variously in some tissues, by as much as
50%, and there were a number of smaller changes (increases and decreases) where statistical significance could be demonstrated (Figs. 3.2-3.9). It is difficult to say if any of these are of consequence because data describing the range of values found in normal
65 rats as a function of strain, diet, age, etc., is sparse. It also is not clear to what extent a
“normal value” may be decreased or increased before symptoms of deficiency or overload toxicity arise, respectively.
Some further insight into these types of questions is obtained by examining
Table 3.1, where we compare tissue levels of physiological elements in rats that had received Pb to the levels in rats that had not been exposed. The latter data are from
Bogden and coworkers and were selected for comparison because the rats they used were of a similar age and dietary history (Naveh, Weis, Chung, and Bogden, 1987). As seen globally in the table, the size of variations that were identified are similar to those that arose during attempts to remove Pb after it had accumulated. This supports our view that the changes in physiological element levels that were seen during treatment for
Pb intoxication are of little consequence. This is with the exception of Ca levels in kidney and the levels of Cu and Mn in femur. There the argument cannot be employed because the normal values reported by Bogden and coworkers are about ten fold higher than what we found following Pb administration. These large variations may reflect methodological problems arising during calibration or data analysis. We rechecked our own methods upon seeing these large differences and note that our values are close to others reported in the literature (Oishi et al., 2000;Seaborn and Nielsen, 2002b;Seaborn and Nielsen, 2002a).
Another type of potential toxicity to consider in contemplating the use of monensin to treat Pb intoxication is the possibility that other toxic elements present in the individual might be perturbed in such way as to enhance their toxicity; even through the toxicity from Pb had been abated. Among the five toxic elements examined we
66 found scattered examples were one or more of the treatment strategies increased the level of a toxin in one or more of the tissues (Figs. 3.10-3.14). When observed, these perturbations were sometimes much larger than what was seen with physiological elements (e.g. the two to four fold rise of Cr and Ni in liver during treatment with monensin plus DMSA), supporting the prospects for increased toxicity. On the other hand, none of the non-physiological elements were being administered beyond the trace quantities presumably present in diet, water, and general environment provided to the rats. Accordingly, the levels of these found were very low (Table 3.1) even when they had increased during attempts to removed Pb. Thus, it is again difficult to judge if these changes are meaningful in terms of toxicity without conducting additional studies in which the element of interest is purposely administered. The same can be said for the scattered examples where certain treatments for Pb intoxication decreased the level of another toxin (e.g. Cr and Ni in brain; As in liver).
Interest is growing in the combined use of multiple chelating agents for the treatment of metal intoxication (Andersen and Aaseth, 2002;Wu et al., 2003;Link et al.,
2001;Kalia and Flora, 2005), although the success of this approach with Pb intoxication has been limited (Jones, Singh, Kostial, Blanusa, Piasek, and Restek-Samarozija,
1997;Kachru et al., 2005;Kostial et al., 1999). The question then arises; how does monensin improve the effectiveness of DMSA at removing Pb from various tissues to the extent that is seen here? The multiple chelator approach seems to work best when one of the agents is fully water soluble while the other has some hydrophobic character
(Andersen and Aaseth, 2002;Giardina and Grady, 2001). This has been explained by the so-called relay hypothesis, or shuttle hypothesis, which maintains that the more
67 hydrophobic agent is able to bind toxic cations that are located in compartments not accessible to the hydrophilic compound, and can thereafter facilitate their movement and transfer to the hydrophilic compound. Once this has occurred the hydrophilic compound is excreted via the kidney, together with the chelated cation. Monensin is highly hydrophobic and is in fact a highly selective and efficient ionophore for Pb2+
(Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002b). It may then be particularly effective at shuttling Pb2+ to DMSA located in blood or in the interstitial fluid compartment.
Additional factors to consider are illustrated in Fig. 3.16 and relate to the fact that monensin is an effective ionophore for Na+ in comparison to K+ (Pressman,
1968a;Henderson et al., 1969a;Kinnally et al., 1991). In addition, near a membrane
2 surface the pKa for monensin is 6.85, pKNa is 5.00, whereas the in vivo concentrations of H+ and Na+ in extracellular volumes are about 10-7 M and 10-3 M, respectively
(Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002b).
Furthermore, at physiological pH, it appears that the predominant species by which monensin transports Pb2+ is the mixed complex monensin●Pb●OH (Hamidinia,
Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002b). Thus, when acting to deliver intracellular Pb2+ to extracellular DMSA it is probable that the compound enters cells as the species monensin●Na, leaves as the mixed complex, and so in effect catalyzes an exchange of extracellular Na+ for intracellular PbOH+ (equivalent to exchanging an extracellular Na+ and a H+ for an intracellular Pb2+) (see Fig. 3.16).
Given that the external/internal Na+ concentration gradient is maintained by the Na,K
ATPase, the presence of monensin partially couples the release of intracellular Pb2+ to
68 ATP hydrolysis, giving a direction and a driving force to the process. This may also be a factor helping to explain why the presence of monensin enhances the effectiveness of
DMSA at removing Pb. However, it should also be pointed out that monensin may act in a more indirect way to enhance the action of DMSA. One possibility relates to the depletion of Pb from bone where monensin might increase the rate of bone turnover.
Were that to occur, access of Pb to DMSA would possibly be increased without a requirement for direct Pb2+ transport mediated by the ionophore.
A final point arises upon comparing an aspect of the present data to those from a previous study in which monensin alone was administered to Pb intoxicated rats
(Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002b). The earlier study showed that monensin alone accelerated the removal of Pb from several tissues, compared to no treatment, whereas in the present study this was seen only in heart (Fig. 3.1). In the present study Pb was released more efficiently under the no treatment condition, than what was seen before, even though the design of both studies was very similar. Differences related to how the rats were housed. During the earlier study they were housed individually, and in metabolic cages (i.e. standing on metal gratings), which are both considered to be stressful circumstances. In the present study they were housed in pairs, and in plastic cages containing normal bedding. When viewed collectively, these considerations suggest that the mechanism(s) which remove
Pb under no treatment conditions are less efficient in stressed rats and that monensin used alone is more efficient when organ Pb levels are higher. The later point is of interest here because it implies that monensin will be even more effective at higher
69 levels of Pb intoxication, and that other Pb ionophores having a higher affinity for Pb may also be more effective. These possibilities can be tested experimentally and are under investigation.
3.5 Summary
Among divalent cations, the carboxylic acid ionophore monensin shows high activity and selectivity for the transport of Pb2+ across phospholipid bilayer membranes
(S. A. Hamidinia et.al. (2002) J. Biol. Chem. 277, 38111-38120). When co-administered to rats (100 ppm in feed) that were receiving meso-dimercaptosuccinate (DMSA) for treatment of Pb intoxication (three week treatment periods), monensin significantly increased the amount of Pb removed from femur, brain, and heart. It showed a tendency to increase Pb removal from liver and kidney, but had no effect of this type in skeletal muscle. Tissue levels of several physiological (Ca, Co, Cu, Fe, Mg, Mn, Mo, Zn) and non-physiological (As, Cr, Cd, Ni, Sr) elements were also determined following the application of these compounds. Among the physiological elements a number of significant changes were seen; including both rising and falling values. The size of these changes was typically around 20%, compared to control values, with the largest examples seen in femur. These changes often tended to reverse those of similar size that had occurred during Pb administration. Among the non-physiological elements, which were present only in trace amounts, the significant changes were smaller in number but larger in size. None of these changes appear likely to be significant in terms of toxicity, and there were no signs of overt toxicity under any of the conditions employed.
Monensin may act by co-transporting Pb2+ and OH- ions out of cells, in exchange for
70 external Na+ ions. The net effect would be to shuttle intracellular Pb2+ to extracellular
DMSA, thereby enhancing its effectiveness. Thus, monensin may be useful for the treatment on Pb intoxication when applied in combination with a hydrophilic Pb2+ chelator.
71
Figure 3.1. Changes in tissue Pb levels produced by selected treatments. Bar graph portion: Five groups of 8 rats were given Pb(acetate)2 in their drinking water as further described in the Materials and Methods. The level was 100 ppm (0.48 mM) while the duration of exposure was 21 days. One group (the Pb loaded group) was then sacrificed and elements of interest were determined in the indicated tissues using ICP mass spectroscopy. The Pb levels observed were given numerically in Table 3.1. To construct this figure these values are defined as 1.0 (to aid in normalizing the range of values that were subsequently observed), and are represented by open bars. The remaining groups thereafter received drinking water that did not contain Pb. From left to right, one of these received no other intervention (no treatment group), another received monensin in feed at 100 ppm (monensin group), another received DMSA by repeated oral gavage (see Materials and Methods), while the last group received monensin and DMSA. After an additional 21 days all groups were sacrificed and the organs of interest were analyzed. The Pb values observed were divided by the loaded value and so represent the fractional change that was seen in that tissue. A standard error of the mean value was calculated for each condition and these are shown in the figure. Tabular portion: The same data shown in the bar graph were used to calculate the comparisons specified in the left most column, with the values shown being the percent changes in mean values. When the change was statistically significant (p < 0.05) a gray background was used for that cell. A white background indicates that the change was not statistically significant.
72 Figure 3.1:Pbdata
Fraction of Loaded 0.0 0.2 0.4 0.6 0.8 1.0 1.2
Co pl pl vs vs M M M tr tr tr tr us us DM m e e e e . vs vs vs . onens onens onens D l a a a a D D No Lo p tm tm tm tm o . . . no no no M
a ade S M M ad r
e e e e A i SA Kid s n n n n S S i i i
o ed n n n d t t t t A A n
ney , K + - - - i 75. 36. 61. - 34. dne 77
4 3 4 0 y N Liv
o Trea e r tmen + Li - - - - 83. 36. 74. 86. 17. v
e 6 5 2 3 8 r t,
M u s c l 73 e S Mu k - - - - -4 34. 34. 34. 68. Mo Ti e l s
. et 5 s c 0 0 7 0 s ne l
al e u
e
nsin, , H % eart Ch a He - - - - +6 n 70. 38. 52. 36. ge
a . 4 r 9 5 7 7 D
t
M B SA, rai n Br - - - - +2 77. 45. 58. 30. a
. i 1 6 9 6 6 n
D Femur MSA &Mon Fe + - - - - 49. 43. 10. 22. 18. m
u 6 8 6 8 5 e r
n s in
Figures 3.2-3.14. Changes in tissue levels of selected elements occurring during treatment for Pb intoxication. In addition to Pb, all other elements considered in Table 3.1 were determined in each sample. Figures 3.2-3.14 were constructed for the individual elements using the format described for Figure 3.1. The order of these figures is the same order used for Table 3.1, as follows: Figure 3.2, Ca; Figure 3.3, Co; Figure 3.4, Cu; Figure 3.5, Fe; Figure 3.6, Mg; Figure 3.7, Mn; Figure 3.8, Mo; Figure 3.9, Zn; Figure 3.10, As; Figure 3.11, Cr; Figure 3.12, Cd; Figure 3.13, Ni; Figure 3.14, Sr. In the tabular portion of these figures a gray background again indicates that the observed change was statistically significant (p < 0.05), whereas a white background was used when the change was not significant. In Figures 3.11 and 3.13, where data are missing from the bar graph portion, the element of interest was too low to be determined relative to the levels in blanks that arose as contaminants.
74 Figure 3.2CaData
Fraction of Loaded 0. 0. 0. 0. 0. 1. 1. 1. 1. 0 2 4 6 8 0 2 4 6
Co pl pl vs v M M M t t t t u u r r r r s DM m eat eat eat eat . vs vs vs . s s o o o D l D D ne ne ne No p Loa o . . . m m m m no no no M
a ad S M M ns ns ns r
e e e e A i S Ki s ed n n n n ded, S S i i i A
o n n n t t t t A A dn
n
ey Ki + + +1 +8 -8 1 1 d 0. 9.
n . . . 1 3 6 No e 2 1
y L
T i v r e eatmen r + Li - - - +3 14. 17. 10. 15. v t
. , e 5 2 1 3 5 r
M us c l M e 75 S Mu onen k +7 +7 +0 +7 +0 Ti el s
. . . . . et s c 9 3 5 3 1 s l s al
e u i
n, e
H , % ear C t h a + H +2 +6 n -3 -4 2 ear ge 3.
. . DMSA, . . 3 7 8 3
9
t
B ra in + + + Br - -2 15. 1 3 1 a 3. 4. 8.
. 0 i 6 n 7 6 6
D
Fem M SA &Monensi u r Fe +4 +4 -0 -4 -0 m
. . . . . 6 8 9 4 5 u
r
n
Loaded, No Treatment, Monensin, DMSA, DMSA & Monensin
1.4
1.2
1.0
0.8 on of Loading i 0.6 Fract
0.4
0.2
0.0 Kidney Liver Muscle Heart Brain Femur
Tissue, % Change
Comparison Kidney Liver Skeletal Heart Brain Femur Muscle No treatment -31.8 -37.7 +12.2 -22.8 -17.7 -57.3 vs. loaded Monensin vs. no +35.1 +8.3 +13.2 +26.6 +23.3 +14.5 treatment DMSA vs. no +15.1 -6.8 -22.3 +10.4 +6.1 +14.4 treatment Monensin plus DMSA +28.5 +25.4 +13.5 +17.5 +8.3 +52.7 vs. DMSA Monensin plus DMSA +48.0 +16.9 -11.8 +29.6 +14.9 +74.7 vs. no treatment
Figure 3.3 Co data
76
Loaded, No Treatment, Monensin, DMSA, DMSA & Monensin
1.8
1.6
1.4
1.2
1.0 of Loaded n tio
c 0.8 Fra 0.6
0.4
0.2
0.0 Kidney Liver Muscle Heart Brain Femur
Tissue, % Change
Comparison Kidney Liver Skeletal Heart Brain Femur Muscle No treatment +28.2 +13.2 +0.2 +10.1 -0.5 +54.4 vs. loaded Monensin vs. no +19.3 -7.0 -9.3 +1.0 +11.3 -24.7 treatment DMSA vs. no -2.0 -11.8 -10.2 -2.7 -6.6 -16.6 treatment Monensin plus DMSA -6.9 +5.9 +2.9 -13.1 -1.2 -2.5 vs. DMSA Monensin plus DMSA -8.8 -6.6 -7.6 -15.5 -7.7 -18.7 vs. no treatment
Figure 3.4 Cu data
77
Loaded, No Treatment, Monensin, DMSA, DMSA & Monensin
1.6
1.4
1.2 d
de 1.0 Loa
of 0.8 tion c a r F 0.6
0.4
0.2
0.0 Kidney Liver Muscle Heart Brain Femur
Tissue, % Change
Comparison Kidney Liver Skeletal Heart Brain Femur Muscle No treatment +26.0 +12.8 +3.2 +8.3 -5.8 +18.6 vs. loaded Monensin vs. no -3.0 -2.5 -4.4 +15.7 +17.9 +6.2 treatment DMSA vs. no +3.8 +16.4 +0.3 +13.3 +3.9 +19.1 treatment Monensin plus DMSA -10.3 +5.4 +2.1 -9.2 -5.8 +11.0 vs. DMSA Monensin plus DMSA -6.9 +22.7 +2.4 +2.8 -2.1 +32.1 vs. no treatment
Figure 3.5 Fe data
78
Loaded, No Treatment, Monensin, DMSA, DMSA & Monensin 1.2
1.0
0.8 aded
0.6 of Lo n tio Frac 0.4
0.2
0.0 Kidney Liver Muscle Heart Brain Femur
Tissue, % Change
Comparison Kidney Liver Skeletal Heart Brain Femur Muscle No treatment -6.9 +3.5 +12.4 -1.6 -5.9 0 vs. loaded Monensin vs. no +7.5 -6.2 +1.9 +9.5 +4.1 +5.5 treatment DMSA vs. no +8.1 -9.7 -0.5 +1.9 -10.3 +3.1 treatment Monensin plus DMSA +3.1 +4.2 -1.8 -2.1 -6.2 +3.7 vs. DMSA Monensin plus DMSA +11.4 -5.9 -2.2 -0.3 -15.8 +6.9 vs. no treatment
Figure 3.6 Mg data
79
Loaded, No Treatment, Monensin, DMSA, DMSA & Monensin
1.0
0.8
0.6 tion of Loaded ac r
F 0.4
0.2
0.0 Kidney Liver Muscle Heart Brain Femur
Tissue, % Change
Comparison Kidney Liver Skeletal Heart Brain Femur Muscle No treatment -4.6 +5.8 -7.6 +0.2 -2.4 -5.1 vs. loaded Monensin vs. no +4.9 -10.8 -5.0 +7.5 +6.7 treatment DMSA vs. no +4.2 -6.3 -4.0 +1.1 -2.1 -2.4 treatment Monensin plus DMSA +1.6 +5.5 +0.3 -2.8 -2.5 +3.6 vs. DMSA Monensin plus DMSA +5.8 -1.2 -3.7 -1.7 -4.6 +1.2 vs. no treatment
Figure 3.7 Mn data
80
Loaded, No treatment Monensin DMSA DMSA & Monensin
1.6
1.4
1.2 d e
d 1.0 a
of Lo 0.8 on ti ac r F 0.6
0.4
0.2
0.0 Kidney Liver Muscle Heart Brain Femur
Tissue, % Change
Comparison Kidney Liver Skeletal Heart Brain Femur Muscle No treatment +12.5 +25.9 +11.2 -3.3 -1.3 -17.6 vs. loaded Monensin vs. no +1.9 -8.4 +6.4 +10.2 +8.5 +70.7 treatment DMSA vs. no +8.1 -11.1 +13.8 +8.4 -1.0 +56.3 treatment Monensin plus DMSA -2.1 +2.8 -1.2 -2.3 +2.2 +3.5 vs. DMSA Monensin plus DMSA +5.8 -8.6 -12.5 +5.8 +1.2 +61.7 vs. no treatment
Figure 3.8 Mo data
81
Loaded, No treatment Monensin DMSA DMSA & Monensin
1.6
1.4
1.2 d e
d 1.0 a
of Lo 0.8 on ti ac r F 0.6
0.4
0.2
0.0 Kidney Liver Muscle Heart Brain Femur
Tissue, % Change
Comparison Kidney Liver Skeletal Heart Brain Femur Muscle No treatment +12.5 +25.9 +11.2 -3.3 -1.3 -17.6 vs. loaded Monensin vs. no +1.9 -8.4 +6.4 +10.2 +8.5 +70.7 treatment DMSA vs. no +8.1 -11.1 +13.8 +8.4 -1.0 +56.3 treatment Monensin plus DMSA -2.1 +2.8 -1.2 -2.3 +2.2 +3.5 vs. DMSA Monensin plus DMSA +5.8 -8.6 -12.5 +5.8 +1.2 +61.7 vs. no treatment
Figure 3.9 Zn data
82
Loaded, No Treatment, Monensin, DMSA, DMSA & Monensin 2.5
2.0
1.5 Loaded on of i 1.0 Fract
0.5
0.0 Kidney Liver Muscle Heart Brain Femur
Tissue, % Change
Comparison Kidney Liver Skeletal Heart Brain Femur Muscle No treatment -23.7 -47.0 -4.9 -14.2 +11.6 +59.1 vs. loaded Monensin vs. no +0.5 +23.6 +7.9 +23.6 -11.7 +20.3 treatment DMSA vs. no +8.3 -18.6 +7.0 +9.4 -11.7 +27.8 treatment Monensin plus DMSA -7.2 +6.2 -3.0 -8.0 -2.4 -23.4 vs. DMSA Monensin plus DMSA +0.5 -13.5 +3.8 +0.6 -2.2 -2.1 vs. no treatment
Figure 3.10 As data
83
Loaded, No Treatment, Monensin, DMSA, DMSA & Monensin 5.0
4.0
3.0 tion of Loaded
ac 2.0 r F
1.0
0.0 Kidney Liver Muscle Heart Brain Femur
Tissue, % Change
Comparison Kidney Liver Skeletal Heart Brain Femur Muscle No treatment +14.8 -21.2 +13.3 -42.5 -63.1 ND vs. loaded Monensin vs. no +40.7 -17.9 -8.2 -13.6 -33.5 ND treatment DMSA vs. no -81.4 +143 +41.0 -24.3 +370.9 ND treatment Monensin plus DMSA ND +96.9 -3.4 -4.1 -83.9 ND vs. DMSA Monensin plus DMSA ND +379 +36.2 -27.4 -24.4 ND vs. no treatment
Figure 3.11 Cr data
84
Loaded, No Treatment, Monensin, DMSA, DMSA & Monensin 3.0
2.5
2.0 ed oad L f 1.5 tion o c a r F 1.0
0.5
0.0 Kidney Liver Muscle Heart Brain Femur
Tissue, % Change
Comparison Kidney Liver Skeletal Heart Brain Femur Muscle No treatment +28.6 +17.5 +20.1 +3.6 +9.3 +14.3 vs. loaded Monensin vs. no +28.4 +22.7 -36.9 +16.3 +15.4 -18.9 treatment DMSA vs. no +0.7 -10.2 +0.1 -5.8 -1.7 +0.9 treatment Monensin plus DMSA +59.5 +5.0 +57.9 +2.0 +11.8 -13.3 vs. DMSA Monensin plus DMSA +60.6 -5.7 +58.1 -3.9 +9.9 -12.6 vs. no treatment
Figure 3.12 Cd data
85
Loaded, No Treatment, Monensin, DMSA, DMSA & Monensin 6.0
5.0
4.0 ed
3.0 of Load tion ac r F 2.0
1.0
0.0 Kidney Liver Muscle Heart Brain Femur
Tissue, % Change
Comparison Kidney Liver Skeletal Heart Brain Femur Muscle No treatment +63.3 +38.1 -5.5 +28.2 -51.1 ND vs. loaded Monensin vs. no +48.6 +168.9 -58.9 -26.8 -22.2 -42.2 treatment DMSA vs. no +37.1 -40.5 -67.0 -31.7 -35.4 -74.6 treatment Monensin plus DMSA +3.8 +458 +260 +61.9 +2.9 +515.8 vs. DMSA Monensin plus DMSA +42.3 +232 +19.0 +10.6 -33.6 +56.6 vs. no treatment
Figure 3.13 Ni data
86
Loaded, No treatment, Monensin, DMSA, DMSA & Monensin 4.0
3.5
3.0
2.5 oaded L 2.0 action of r
F 1.5
1.0
0.5
0.0 Kidney Liver Muscle Heart Brain Femur
Tissue, % Change
Comparison Kidney Liver Skeletal Heart Brain Femur Muscle No treatment +162 +31.5 -37.1 +136 -15.0 +12.7 vs. loaded Monensin vs. no 10.4 +1.7 +9.7 +21.6 +17.1 -0.3 treatment DMSA vs. no +29.2 -9.7 -10.3 +39.3 +35.6 +0.3 treatment Monensin plus DMSA -31.9 +13.5 +10.8 -49.3 -12.6 +2.0 vs. DMSA Monensin plus DMSA -12.0 +2.5 -0.6 -29.4 +18.5 +2.3 vs. no treatment
Figure 3.14 Sr data
87
440
Pb Loaded No treatment Monensin DMSA 400 DMSA & Monensin
360
Start Pb 320 Weight, grams
280 End Pb/ Start Treatment
240 0 1020304050 Day
Figure 3.15. Effect of treatment on weight gain in Pb intoxicated rats. Data are from the same rats used to obtain the other figures. The treatments applied following day 21, and other aspects of the experiment, are described in the legend to Figure 3.1.
88
2K+ A.
3Na+
+ ATP Na , + Na , ADP + Pi
Mon- Na+ B. Na+
NaMon NaMon Mon-
PbMonOH PbMonOH Mon- + Pb2+
OH-
OH- Mon- C. + 2+ Pb
Figure 3.16. Potential relationships between the transport of Na+ and Pb2+. The two parallel curved lines represent a section of plasma membrane bounding a cell that contains Pb2+. To the left and right of these lines are the extra- and intra- cellular compartments, respectively. The upper portion of the scheme (section A.) shows the formation of an electrochemical Na+ gradient across this membrane through action of the NaK ATPase. In section B. monensin transports Na+ down the Na+ gradient, whereas in section C. Pb2+ is transported out. The net result of processes occurring in B. and C. is the electroneutral exchange of an intracellular Pb2+ and an OH- for an external Na+.
89 Tissue
Element, Kidney Liver Skeletal Heart Brain Femur nmol/gm Muscle
Pb 29.8 5.77 0.073 0.163 1.19 203 0.20 0.20 ND 0.005 0.05 ND
Ca 897 574 1,318 522 2,418 4,024,813 12,775 669 1,193 903 1,327 3,220,000 Co 1.24 0.352 0.025 0.523 0.093 0.130 NR NR NR NR NR NR Cu 62.1 51.1 13.6 101 38.7 5.29 96.9 59.2 14.0 78.4 33.4 45.5 Fe 524 1,534 133 1,049 239 771 1,112 1,003 191 1,010 315 702 Mg 6,627 8,203 10,028 8,885 6,561 117,285 7,365 6,665 9,998 8,023 6,048 91,750 Mn 13.1 32.0 1.02 7.34 7.12 6.44 19.8 37.3 2.40 10.32 8.83 52.8 Mo 2.21 4.10 0.083 0.492 0.299 0.187 NR NR NR NR NR NR Zn 295 472 162 315 247 1,844 327 329 284 246 180 1,743
As 0.035 0.444 0.121 0.437 0.071 0.207 NR NR NR NR NR NR Cr 0.012 0.004 0.033 0.886 0.066 ND NR NR NR NR NR NR Cd 0.082 0.062 0.003 0.005 0.005 0.006 NR NR NR NR NR NR Ni 0.070 0.029 0.097 0.420 0.080 ND NR NR NR NR NR NR Sr 0.088 0.061 0.313 0.110 0.277 56.3 NR NR NR NR NR NR
Table 3.1. Effects of Pb administration on the levels of selected elements in rat tissues The elements are separated into three groups with the first being Pb alone, the second being those having a known physiological role, and the third being those, in addition to Pb, having no known role. Values shown in bold are from this study and were determined after the rats had been given Pb2+ for three weeks, at 100 ppm, in their drinking water (see Materials and Methods). The other values are from the literature and were determined using rats that had not been given Pb. Among the latter group, Pb values are from Hamidinia et al (Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002b), whereas the others are from Naveh et al (Naveh, Weis, Chung, and Bogden, 1987). In cells with a gray background, the two values were statistically different (p < 0.05) from each other. In cells with a white background the values were not different. NR = not reported: ND = not detected.
90 CHAPTER 4
THE IONOPHORE NIGERICIN TRANSPORTS Pb2+ WITH HIGH ACTIVITY AND
SELECTIVITY: A COMPARISON TO MONENSIN AND IONOMYCIN
4.1 Introduction
The antibiotic agent nigericin was first described in 1951 as a compound produced by a then unidentified Streptomyces species present in soil samples arising from Nigeria (Harned et al., 1951). The producing organism was later identified as
Streptomyces hydroscopicus and the compound was found to be identical to polyetherin
A and X-464, which were independently described by other investigators (Stempel et al.,
1969;Shoji et al., 1968). The actions of nigericin on mitochondria led to the discovery of its well-known activity as a K+ ionophore, and it has long been used as a research tool in that regard (Reed, 1982).
Structurally, nigericin belongs to the polyether class of antibiotics, a group which also includes the well-known ionophores monensin and ionomycin (Fig. 2.1). About
130 naturally occurring compounds belonging to this class have been described (Dutton et al., 1995). Their ion transport properties are unknown in most cases, and when they have been investigated, a limited set of the classical methods were often employed.
These include two- and three- phase bulk solvent extraction techniques, potentiometric methods based on stabilized membrane electrodes or Mueller-Rudin bilayers, and ion
91 transport studies carried out with subcellular preparations. While data obtained by such methods are of interest, they are often difficult to apply when interpreting the action of ionophores on intact biological systems. For example, bulk solvent systems do not model the potential energy or structural characteristics of a phospholipid bilayer, which is the actual environment wherein the cation complexation and decomplexation reactions associated with transport occur. This problem is not manifest when subcellular preparations are employed; however, preparations of that type are generally unstable to extremes of pH, ionic strength, and other conditions that are of interest when investigating mechanisms of transport. In addition, they contain endogenous transporters which can obscure an activity parameter of interest, and do not readily lend themselves to the investigation of toxic and trace cation transport, depending upon the cation in question and its concentration.
Several groups utilize model transport systems based upon phospholipid vesicles to circumvent the problems that are inherent with other systems. For example, these structures were employed in conjunction with temperature jump techniques to investigate factors that limit rates of Na+ and K+ transport by monensin and nigericin
(Prabhananda and Ugrankar, 1991;Prabhananda and Kombrabail, 1992). Riddell and coworkers used NMR-based methods for similar proposes (Riddell and Hayer,
1985a;Riddell et al., 1988a;Riddell and Arumugam, 1988;Riddell et al., 1988b;Riddell et al., 1990;Riddell, 1992), but that method of detection is limited to cations which are
NMR active and requires their presence at high concentrations. We have been utilizing a highly defined system in which the vesicles are loaded with a chelator/indicator, allowing transport to be monitored by UV-Vis or fluorescence spectroscopy. It is well
92 suited to the determination of polyvalent cation transport and has so far been applied to investigate the properties of A23187, 4-BrA23187, and ionomycin (Erdahl, Chapman,
Taylor, and Pfeiffer, 1994;Erdahl, Chapman, Taylor, and Pfeiffer, 1995;Erdahl,
Chapman, Wang, Taylor, and Pfeiffer, 1996;Thomas, Wang, Pfeiffer, and Taylor,
1997;Wang, Taylor, and Pfeiffer, 1998;Erdahl, Chapman, Taylor, and Pfeiffer, 2000a), properties of monensin (Hamidinia et al., 2002a), and those of a synthetic ionophore for divalent cations called ETH 129 (Wang, Erdahl, Hamidinia, Chapman, Taylor, and
Pfeiffer, 2001). Our interest, in part, is to identify ionophores that are efficient and selective for the transport of Pb2+, and then to apply these towards the development of improved treatments for Pb2+ intoxication. Among the three naturally occurring compounds that have been considered, all are ionophores for Pb2+, but marked differences are seen in terms of selectivity. A23187 is relatively unselective, ionomycin is of intermediate selectivity, and monensin is highly selective for Pb2+ transport, compared to other divalent cations. This range of selectivity suggests that we may not yet have identified the most suitable compound for the intended application and we are therefore investigating other members of the group.
The present report describes Pb2+ transport properties of nigericin and compares them to those of monensin and ionomycin. This compound was selected because it is similar to monensin in terms of structure, but shows significant differences in cation complexation chemistry and transport selectivity. More specifically, nigericin contains a pyran ring between the spiroketal group and the terminal carboxylate that is not present in monensin (Fig. 2.1). As a result, the carbon backbone chain length is increased from
26 to 30 atoms, which is associated with differing coordination modes for Na+, K+, and
93 Ag+, as seen in the solid state (SHiro and Koyama, 1970;Gedes et al., 1974;Barrans and
Alleaume, 1980;Pinkerton and Steinrauf, 1970a;Pangborn et al., 1987;Duax et al.,
1980;Ward et al., 1978). It is also associated with a reversal in the Na+/K+ complexation selectivity where monensin favors Na+, while nigericin forms stronger complexes with the larger K+ ion (Lutz et al., 1970;Pointud et al., 1983). Accordingly, it seemed possible that nigericin might be particularly selective for a larger cation like Pb2+, relative to smaller ones such as Ca2+, Mg2+ and Zn2+. That is in fact the case as shown by the present communication.
4.2 Materials and Methods
Reagents and Solvents. Nigericin was obtained from Calbiochem and used without further purification.
Preparation of Phospholipid Vesicles. The preparation of freeze-thaw extruded
POPC vesicles loaded with Quin-2 was described earlier in the Materials and Methods section 2.2.
Pb2+ Buffers and the Determination of Transport. A buffer system was used to control the concentration of Pb2+ available for transport into the vesicles. This is described earlier in section 2.2.
Potentiometric Titrations and the Determination of pH in Aqueous Methanol.
For solution chemical studies a mixed solvent of 80% (w/w) methanol in water was prepared gravimetrically, as previously discussed in section 2.2. The protonation constants and complex formation constants of nigericin were measured by potentiometric methods in the mixed solvent 80% (w/w) methanol-water. For these
94 studies, Na+ nigericin (Fluka, Fermentek) was purified by column chromatography on silica gel, using ethyl acetate as the eluent. The Na+ salt was converted to the acid form by repeated back-extraction of a CHCl3 solution with 1.0 M HCl. The CHCl3 solution was then washed three times with distilled water, the solvent removed, and the product dried under vacuum. Analysis by flame atomic emission showed that less than 0.05%
Na+ (w/w) remained. The pH meter-electrode system was calibrated and titrated as discussed in section 2.2.
4.3 Results
The activity of nigericin as an ionophore for divalent cations is compared to that of monensin (Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer,
2002a) and ionomycin (Erdahl, Chapman, Taylor, and Pfeiffer, 2000a) in Fig. 4.1.
Under the conditions employed all three compounds are effective as Pb2+ ionophores, with monensin and nigericin being particularly selective in that regard. In the past we have used S values, as defined by equation 2, to express the selectivity of ionophores for the transport of Pb2+, compared to Ca2+ transport (Hamidinia, Shimelis, Tan, Erdahl,
Chapman, Renkes, Taylor, and Pfeiffer, 2002a;Erdahl, Chapman, Taylor, and Pfeiffer,
2000a). When considered this way, the selectivity sequence for the three compounds is monensin (3340) > nigericin (2890) > ionomycin (100), with the numerical values in parenthesis being the calculated value for that compound. However, a close comparison of Figs 4.1A and 4.1B shows that the selectivity of nigericin exceeds that of monensin
95 when the total set of cations investigated is considered. That is particularly apparent when the efficiencies of Cu2+, Zn2+ and Cd2+ transport by the two compounds are compared.
As with monensin and ionomycin, the predominant complex by which nigericin transports Pb2+ has a 1:1 stoichiometry, ionophore:cation, as indicated by plots of log rate vs. log of the free Pb2+ concentration (Fig. 4.2) or log rate vs. log of the nigericin concentration (Fig. 4.3). In both cases the slopes of these plots are near to 1.0, as expected for a transporting species of that stoichiometry2. Figs. 4.2B and 4.3B also contain log rate vs. log Pb2+ or log ionophore concentration data obtained with ionomycin and monensin. A comparison of these plots shows the relative efficiency sequence of the three compounds as ionophores for Pb2+, which is ionomycin > nigericin
> monensin.
Medium pH is an important factor determining the efficiency of Pb2+ transport catalyzed by monensin, with the rate increasing as pH rises over the range of 6.5-9, and with a half maximal value obtained at pH 7.80 (Hamidinia, Shimelis, Tan, Erdahl,
Chapman, Renkes, Taylor, and Pfeiffer, 2002a). Ionomycin and nigericin display similar dependencies, with half maximal values obtained at pH 7.88 and 7.94 respectively (Fig. 4.4). In the case of monensin, the pH dependence was thought to reflect the protonation equilibrium of the carboxylic acid function, together with hydrolysis of the 1:1 species (MonPb+) to form MonPbOH, which is one of the transporting species (Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and
Pfeiffer, 2002a). Ionomycin may form the analogous transporting species HIonoPbOH, or may be ionized twice to form the neutral species IonoPb (Erdahl, Chapman, Taylor,
96 and Pfeiffer, 2000a). Given that nigericin has a single ionizable function, like monensin
(Fig. 2.1), it is also unable to form an uncharged complex of 1:1 stoichiometry without participation of a second anion. Uncharged complexes are responsible for the nigericin catalyzed Pb2+ transport seen in Fig. 4.4 because no provision was made to collapse the membrane potential that would arise if electrogenic transport were occurring (i.e. valinomycin and CCCP were not present). Based on these considerations it seemed possible that the NigPbOH complex is a major species by which nigericin transports
Pb2+, and that formation of this species accounts for the pH dependency seen in Fig. 4.4.
To determine if the above view is consistent with the complexation chemistry of
Pb2+ with nigericin, protonation constants and complex formation constants for several cations were determined by potentiometric methods using 80% methanol/water as the solvent. This medium provides a solvent polarity which is similar to that experienced by ionophores located at a POPC membrane interface and so the values obtained can be used to analyze the mechanism of transport (Kauffman, Taylor, and Pfeiffer,
1982;Pfeiffer, Chapman, Taylor, and Laing, 1983;Taylor, Chapman, and Pfeiffer,
1985;Taylor, Pfeiffer, Chapman, Craig, and Thomas, 1993). These values are shown in
Table 4.1, together with analogous values for monensin (Hamidinia, Shimelis, Tan,
Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002a), and were used to construct the species distribution diagram shown in Fig. 4.5. In Table 1 it is seen that nigericin is a slightly weaker acid than monensin, but forms a 1:1 complex (PbL+) with Pb2+ that is slightly more stable. As with monensin, this complex can hydrolyze (react with OH-) to form the charge neutral mixed complex NigPbOH, although the equilibrium constant is
97 smaller with the latter compound. These differences tend to cancel each other, such that the overall formation constants (βPbLOH) for MonPbOH and NigPbOH are effectively equal (Table 4.1).
The distribution diagram (Fig. 4.5) shows that the species NigPb+ is most prevalent near pH 7 and then declines as the pH rises, which is the opposite of what would be expected if it were the major species transporting Pb2+, based on the behavior
2+ shown in Fig. 4.4. The species (Nig)2Pb, which a priori might transport Pb in an electroneutral fashion, is not prominent at any of pH values examined and under the present concentration conditions, whereas the prevalence of the NigPbOH complex closely parallels the effect of pH on the rate of transport. The similarity is emphasized by the inserted panel, wherein the two parameters are compared side by side. Thus we conclude that NigPbOH is primarily responsible for Pb2+ transport catalyzed by that compound, at least under conditions were an electroneutral mechanism is required.
The equilibrium constants shown in Table 4.1 also provide a rationale for the high selectivity of nigericin for Pb2+ transport, compared to other divalent cations (Fig.
4.1). That is to say, the NigCa+, NigMg+, and NigZn+ complexes are less stable than
NigPb+ by approximately five orders of magnitude, with this differential being greater than that seen with monensin by about one order of magnitude (Table 4.1). The same relative relationships are seen comparing the overall stability constants (β) for the
NigPbOH and the MonPbOH complexes while the analogous complexes formed with
Ca2+ and Mg2+ could not be detected. These differences in complex stability constants
98 appear adequate to explain the selectivity of nigericin for Pb2+ transport, among divalent cations, as was the case with monensin described previously (Hamidinia, Shimelis, Tan,
Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002a).
With regards monovalent cations, the equilibrium constant for formation of
NigNa is similar to those for the analogous complexes formed with Ca2+, Mg2+ and Zn2+ and notably smaller than for the MonNa complex. This is in line with data showing that monensin is selective for Na+ transport, among monovalent cations (Henderson et al.,
1969b;Antonenko and Yaguzhinsky, 1988;Pressman, 1968b), and is the same pattern seen by others using different solvent conditions (Cox et al., 1984a;Pointud et al.,
1985b;Hoogerheide and Popov, 1978;Lutz, Wipf, and Simon, 1970;Pointud, Tissier, and
Juillard, 1983). The NigK complex is more stable than the analogous complexes with all divalent cations except Pb2+, and has the same stability as the MonK complex. All of these data are consistent with the possibility of nigericin acting as a Pb2+ ionophore in biological systems, although several physiological cations such as Ca2+, Mg2+, Na+, and
K+ would be present at much higher concentrations and might reduce the rate of transport by competition.
To examine competitive relationships more directly we determined if (near) physiological concentrations of Ca2+ and Mg2+ (1 mM) are inhibitory to Pb2+ transport at a free Pb2+ concentration of 1.0 µM. No inhibition was seen (Fig. 4.6). There were effects of K+ and Na+, however, as shown in Fig. 4.7. Na+ produced a modest and concentration-dependant stimulation of Pb2+ transport, whereas K+ had the opposite effect (Fig. 4.7A). These monovalent cations also have small effects on the rate of Pb2+ transport catalyzed by ionomycin and these are shown here (Fig. 4.7B) to complete the
99 asset of parallel data which are now available on Pb2+ transport catalyzed by both compounds and by monensin [(Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes,
Taylor, and Pfeiffer, 2002a;Erdahl, Chapman, Taylor, and Pfeiffer, 2000a) and the present communication].
4.4 Discussion
Based upon the present data, we can now add nigericin to the set of naturally occurring ionophores that transport Pb2+. In comparison to the others (A23187, ionomycin, and monensin) nigericin has the greatest overall selectivity for Pb2+ as shown in part by Fig. 4.1, and by work reported previously in the case of A23187
(Erdahl, Chapman, Taylor, and Pfeiffer, 2000a). The stability constants for ionophore-
Pb2+ complexes which are available for monensin and nigericin (Table 4.1) confirm that the relative stability of complexes seen in solution is an important factor establishing transport selectivity. This can be seen in Table 4.1, in part by comparing the constants for 1:1 complexes formed with other divalent cations to that of the analogous complex formed with Pb2+. With either compound the Pb2+ complex is the most stable, and by 4-
5 orders of magnitude.
The tendency of 1:1 complexes to hydrolyze forming a ternary complex containing OH- appears to be another factor that relates complex stability constants to the efficiency of Pb2+ transport and the transport of other divalent cations. Such complexes could not be detected for either compound interacting with Ca2+ or Mg2+ and both of these cations are transported very poorly (Fig. 4.1 and data not shown). A weak activity for Zn2+ transport was detected, as was the ternary complex ionophoreZnOH
100 formed with either compound. Assuming that Zn2+ is transported via these ternary complexes, as is Pb2+, the overall equilibrium constants for their formation (log β values) can be compared to each other and to the relative transport efficiencies. Log β for the ionophorePbOH complexes are 4-5 orders of magnitude greater than those for the analogous Zn2+ complexes, in line with the much greater activity of both compounds for
Pb2+ transport (Fig. 4.1). Comparing the two compounds to each other shows that
2+ 2+ nigericin is more selective than monensin for Pb , compared to Zn (βPbLOH/βZnLOH), and that this arises because βZnLOH for the monensin complex is greater than for the nigericin complex by ~10 fold. These differences are in line with the noticeably higher activity of monensin for Zn2+ transport, and with the apparent higher selectivity of nigericin for Pb2+ transport compared to Zn2+. Thus, one might suggest that relative complex stabilities seen in solution will predict the selectivities of other ionophores for
Pb2+ transport. However, such parallel relationships were generally not found with
A23187, 4BrA23187 and ionomycin transporting a different group of divalent cations
(Erdahl, Chapman, Wang, Taylor, and Pfeiffer, 1996), or transporting the lanthanide series trivalent cations (Wang, Taylor, and Pfeiffer, 1998). Accordingly, the patterns of complex formation constants and transport selectivities, as well as details of the transport mechanisms, must be determined for a larger group of ionophores and cations to establish the relationships among these characteristics.
The very high transport selectivity of nigericin and monensin for Pb2+ is one of the obstacles to obtaining fully quantitative expressions of a sequence which includes a more diverse set of divalent cations. This is because the range of initial rates that can be accurately determined is limited to about 5 ×103 when all conditions are held constant
101 except for the divalent cation identity, whereas the selectivity values for Pb2+ will approach or exceed this limit in several cases. It should be possible to overcome these limitations with the compounds that have been investigated to date, however, given that they all transport Pb2+ predominantly through formation of a 1:1 complex, as indicated by broad regions in plots of log rate vs. log ionophore and log Pb2+ concentrations which are linear and have slopes near 1.0 (Figs. 4.2 and 4.3). Within these regions the efficiency of transport can be expressed as a second order rate constant, as defined by equation 3.
2+ kPb = rate / [ionophore][Pb ] (3)
Presumably, analogous constants for various cations can also be obtained and these would be directly comparable even when different conditions of ionophore and cation concentration were used to obtain them. This approach may overcome the limitations associated with the range of rates that can be observed using a single set of conditions and allow a broader range of transport efficiencies to be determined and compared using the vesicle system. To initiate the use of rate constants when evaluating the transport selectivity in our system we calculated kPb for monensin, nigericin, and ionomycin using the data shown in Fig. 4.3B. The values obtained were 1.0, 2.4, and 11.9 × 105 M-1 sec-1 for the three compounds, respectively. To the best of our knowledge these are the first examples of such constants to be reported for divalent cation transport by polyether ionophores.
The above type of quantitation in analyzing ionophore transport properties requires an understanding of the overall reaction and the stoichiometry of the transporting species. Rapid transport in the absence of agents which would dissipate a
102 membrane potential shows that an efficient electroneutral mechanism is operating (Fig.
4.5), whereas ionophorePbOH complexes are the apparent species responsible, as pointed out in the Results section, referable to Figs. 4.5, 4.6, and Table 4.1. It remains to be seen if other species contribute, depending upon conditions, and if significant electrogenic transport can occur. A small electrogenic component does seem possible because Pb2+ is transported slightly faster when VAL plus CCCP are present, compared to when they are absent (see preliminary data in Fig. 4.8). This could indicate that a mixed mode arises when membrane potential is dissipated, and that this is shifted to strictly neutral when an opposing potential arises. Alternatively, CCCP may substitute for OH- to form a neutral species of the type ionophorePbCCCP, similar to the mixed complex that was previously found to arise between ETH 129, Ca2+, and the uncoupler
(Wang, Erdahl, Hamidinia, Chapman, Taylor, and Pfeiffer, 2001). Other possible explanations for what is seen in Fig. 4.8 relate to small changes in intravesicular pH that are brought about by VAL plus CCCP, and the effects these have on rate by altering the distribution of ionophore between the two sides of the vesicle membrane (Erdahl,
Chapman, Taylor, and Pfeiffer, 1995).
Monensin has already been shown to promote the excretion of previously accumulated Pb2+ from rats (Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes,
Taylor, and Pfeiffer, 2002a) and to increase the effectiveness of dimercaptosuccinate, which is a hydrophilic chelating agent that is used clinically to treat Pb intoxication (to be presented elsewhere). These actions are thought to reflect Pb2+ transport by that compound, facilitating the mobilization of Pb from membrane bounded compartments.
Given that nigericin is of higher activity as a Pb2+ ionophore (Figs. 4.2 and 4.3), is
103 apparently more selective for Pb2+ compared to other divalent cations (Fig. 4.1), and is little effected by the major physiological cations in terms of its Pb2+ transport activity
(Figs. 4.6 and 4.7) it seems possible that nigericin may be the preferable compound for those types of applications. That possibility is under further investigation, as is the potential suitability of other polyether ionophores.
4.5 Summary
The K+ ionophore nigericin is shown to be highly effective as an ionophore for
Pb2+, but not other divalent cations, including Cu2+, Zn2+, Cd2+, Mn2+, Co2+, Ca2+, Ni2+, and Sr2+. Among this group a minor activity for Cu2+ transport is seen, while for the others activity is near or below the limit of detection. The selectivity of nigericin for
Pb2+ exceeds that of ionomycin or monensin and arises, at least in part, from a high stability of nigericin-Pb2+ complexes. Plots of log rate vs. log Pb2+ or log ionophore concentration, together with the pH dependency, indicate that nigericin transports Pb2+ via the species NigPbOH, and by a mechanism that is predominately electroneutral. As with monensin and ionomycin, a minor fraction of activity may be electrogenic, based upon a stimulation of rate that is produced by agents which prevent the formation of transmembrane electrical potentials. Nigericin catalyzed Pb2+ transport is not inhibited by physiological concentrations of Ca2+ or Mg2+ and is only modestly affected by K+ and
Na+ concentrations in the range of 0-100 mM. These characteristics, together with higher selectivity and efficiency suggest that nigericin may be more useful than monensin in the treatment of Pb intoxication.
104
Figure 4.1. Transport of Pb2+ and other divalent cations by nigericin (panel A), monensin (panel B), and ionomycin (panel C). POPC vesicles loaded with Quin-2 (Cs+) were prepared as described in Materials and Methods. They were incubated at 25°C and a nominal phospholipid concentration of 1.0 mM. The external medium contained 50 + mM CsNO3, 10 mM each of Mes and Hepes (both Cs ), pH 7.00, 0.5 µM valinomycin, 0.5 µM carbonyl cyanide m-chlorophenylhydrazone, and 20 µM indicated divalent cation (nitrate in the case of Pb2+, chlorides for all others). After a 2 min preincubation 0.10 µM ionophore was added to initiate transport (designated as 0 s in the figure). The sequence of cations shown on the right corresponds to their relative rate of transport as observed directly for panel C or on an expanded scale for panels A and B. The y-axis unit refers to cation concentration in the external medium. When this parameter reaches ~ 20 µM, all of the cation that was originally present in the external medium has been transported into the vesicles and sequestered by Quin-2.
105 A. Nigericin 20 Pb2+
16 M µ Nig 12
2+
Transport, Cu 8 2+ 2+ Zn 2+
Me Cd Mn2+ 4 2+ Co Ca2+ Ni2+ 0 Sr2+
0 200 400 600 800
seconds
B. Monensin 20 2+ Pb
16 M µ
t, Mon r
o 12 p s an r 2+
T Cu 8 2+ 2+ Zn 2+
Me Cd Mn2+ 4 2+ Co Ca2+ Ni2+ 0 Sr2+
0 200 400 600 800
seconds
C. Ionomycin 20 Pb2+
Zn2+ 16 Cd2+ M µ
,
t Iono
or 12 p 2+ s Mn an
Tr 8 2+ Ca2+ Me 2+ 4 Cu Co2+ Ni2+ 0 Sr2+
0 200 400 600 800
seconds
106 16 A.
12 M µ
t, Nig r
spo 8 an r T 2+
Pb 4
0
0 200 400 600 800
seconds
0 B.
Ionomycin c
e -1 Nigericin M/s
µ Monensin , te -2 l Ra ia it In g
o -3 L
-4
-8 -7 -6 -5
2+ Log [Pb ]
Figure 4.2. Dependence of Pb2+ transport on the concentration of free Pb2+. Panel A: Data were obtained as described in the legend to Figure 4.2, except that the free Pb2+ concentration was buffered and varied as follows (beginning with the bottom most curve): 10 nM, 20.1 nM, 40.2 nM, 80.6 nM, 162 nM, 327 nM, 669 nM, 1.41 µM, 2.23 µM, 3.19 µM, 5.63 µM, and 10.0 µM (topmost curve). CsNO3 was omitted from the medium, and 15 mM citrate was present to form the Pb2+ buffer, as further described in the Materials and Methods. Panel B: Initial rate values were obtained from the progress curves shown in panel A, as described in the Materials and Methods. The log of these values is shown vs log of the free Pb2+ concentration. Panel B also shows analogous data obtained with ionomycin and monensin(Erdahl, Chapman, Taylor, and Pfeiffer, 2000a;Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002a). 107 A. 8
M 6 µ Nig
4 ansport, r T 2+
Pb 2
0
0 200 400 600 800
seconds
0 B.
Ionomycin c
e -1 s Nigericin M/ µ Monensin -2 Rate,
-3 Log Initial
-4
-8 -7 -6 -5 -4
Log [ionophore], M
Figure 4.3. Dependence of Pb2+ transport on the concentration of nigericin. Panel A: Data were obtained as described in the legend to Figure 4.3 except that the nominal concentration of POPC was 0.50 mM and the free Pb2+ concentration was held constant at 10 nM. The concentration of nigericin was varied as follows (beginning with the bottom most curve): 0, 39.1 nM, 78.1 nM, 156 nM, 313 nM, 625 nM, 1.25 µM, 2.50 µM, 5.00 µM, 10.0 µM, and 20.0 µM (topmost curve). Panel B: Initial rate values were obtained from the progress curves shown in panel A, as described in the Materials and Methods. The log of these values is shown vs log of the nigericin concentration. Panel B also shows analogous data obtained with ionomycin and monensin (Erdahl, Chapman, Taylor, and Pfeiffer, 2000a;Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002a). 108
8
Ionomycin 6 Nigericin M/min µ
4 Transport, 2+ 2 Pb
0
678910
pH
Figure 4.4. Dependence of Pb2+ transport on external pH. Data were obtained as described in Figure 4.3 except that the external medium contained 10 mM each of Mes, Ches, and Hepes to provide for buffering of H+ across the full range of pHs examined. Cs+ was used as the counterion to all buffer anions and to citrate or Quin-2 in the external and internal volumes, respectively. In addition, valinomycin and CCCP were not present, the free Pb2+ concentration was held constant at 100 nM, and the ionophores were used at this same concentration. Points shown are experimental and were fit to the Henderson-Hasselbach equation to obtain the solid lines.
109 4
3 value
2 [PbNigOH] lative
e Transport rate R 1
0 0.10 6.5 7.0 7.5 8.0 8.5 9.0 9.5
HNig pH 0.08 M µ
, PbNigOH 0.06 tration en
c 0.04 n o C
Pb(Nig)2 0.02 PbNig+
0.00
45678910
* pH
Figure 4.5. Equilibrium behavior of nigericin and Pb2+. Main panel: Individual values were calculated using the program COMICS (Perrin and Sayce, 1967) and represent the distribution expected at total Pb2+ and nigericin concentrations of 100 nM. Equilibrium 2+ constants of interest were from Table 1, and the pKa for hydrolysis of Pb in 80% methanol/water was taken to be 7.96 (Ivanova, 2000). Inserted panel: The PbNigOH values from the main panel are plotted together with the nigericin transport data from Figure 4.4.
110
Equilibrium Reaction Nigericin Monensin constant
+ - H + L HL : log KH = 7.02 6.83
2+ - + Pb + L PbL : log KML = 7.57 7.25
+ - PbL + OH PbLOH : log KMLOH = 6.69 7.10
+ - PbL + L PbL2 : logKML2 = 3.80 3.35
2+ - - Pb + L + OH PbLOH : log βPbLOH = 14.26 14.35
2+ - - Zn + L + OH ZnLOH : log βZnLOH = 9.46 10.41
2+ - + Ca + L CaL : log KML = 2.59 3.10
2+ - + Mg + L MgL : log KML = 2.46 3.16
2+ - - Zn + L ZnL : log KML = 2.99 3.74
+ - ZnL + OH ZnLOH : log KMLOH = 6.47 6.67
+ - Na + L NaL : log KML = 2.89 5.00
+ - K + L KL : log KML = 3.77 3.76
Table 4.1. Equilibrium constants for selected reactions. Equilibrium constants for the reactions shown were obtained by potentiometric titrations in an 80% methanol/water mixed solvent as described in the Materials and Methods (nigericin) or were taken from data reported previously (monensin) (Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002a;Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002a). The temperature was 25˚C, and the ionic strength was maintained constant at 0.05 by the presence of tetraethylammonium perchlorate. L- refers to the ionized form of nigericin or monensin, as indicated.
111 A. Ca2+ 10 +Pb2+, -Ca2+ +Pb2+, +Ca2+
8 M µ Nig t, 6 or sp an r
t 4 2+ Me 2
-Pb2+, +Ca2+ 0
0 100 200 300 400 500 600 700
seconds
2+ B. Mg 2+ 2+ 10 +Pb , +Mg +Pb2+, -Mg2+
8 M µ Nig t, 6 spor
Tran 4 2+ Me 2
-Pb2+, +Mg2+ 0
0 100 200 300 400 500 600 700
seconds
Figure 4.6. Actions of Ca2+ and Mg2+ as inhibitors of Pb2+ transport. Data were obtained as described in the legend to Figure 4.3 except, when present, the free Pb2+ concentration 2+ was held constant at 1.00 µM. Panel A: CaCl2 was present with or without Pb , as indicated in the figure, sufficient to give a free Ca2+ concentration of 1.00 mM. Panel B: 2+ Same as panel A except MgCl2 was used instead of CaCl2 to give a free Mg concentration of 1.00 mM. 112
Figure 4.7. Actions of K+ and Na+ as inhibitors of Pb2+ transport. Vesicles were prepared as described in the Materials and Methods using Cs+ as the counter ion to Quin-2. The external medium contained 5 mM citrate plus Pb(NO3)2 sufficient to produce a free Pb2+ concentration of 100 nM. The external pH was buffered at 7.00 by 10 mM Hepes. NaOH or KOH was used to adjust the external pH, so as to match the monovalent cation that was under examination as a potential inhibitor. NaCl or KCl was also present in the external medium at the indicated concentrations. When these concentrations were 0 the medium also contained 200 mM mannitol and this solute was decreased by twice the concentration of NaCl or KCl that was subsequently added so as to maintain a constant osmotic pressure. Nigericin (panel A) and ionomycin (panel B) were used at 100 nM and 50 nM, respectively.
113 A. Nigericin Mannitol, mM
200 160 120 80 40 0
14
12
+ NaNa+
M/min 10 µ
rt, 8 anspo r
T 6
2+ + K+K Pb 4
2
0 20406080100
MeCl, mM
B. Ionomycin Mannitol, mM
200 160 120 80 40 0
8
7
Na++
M/min 6 Na µ
t, 5
anspor ++ r KK
T 4 2+ Pb 3
2
0 20406080100
MeCl, mM
114 A. Nigericin 10
8 M µ
rt, 6 Nig + VAL, CCCP anspo r
t 4 2+ -VAL, CCCP Pb 2
0
0 100 200 300 400 500 600 700
seconds
B. Monensin 10
8 M µ t, r 6 o Mon sp + VAL, CCCP
Tran 4 2+
Pb -VAL, CCCP 2
0
0 100 200 300 400 500 600 700
seconds
C. Ionomycin 12
10 M µ t, r 8 Iono o
sp + VAL, CCCP 6
Tran -VAL, CCCP 2+ 4 Pb
2
0
0 100 200 300 400 500 600 700 seconds
Figure 4.8. Possible involvement of electrogenic processes in ionophore-mediated Pb2+ transport. Experiments were conducted as described in the Materials and Methods at a free Pb2+ concentration of 1.00 µM. Valinomycin plus CCCP were present, or not present, as indicated in the figure. Nigericin (panel A), monensin (panel B), and ionomycin (panel C) were utilized at 100 nM. 115 CHAPTER 5
SELECTIVE TRANSPORT OF Pb2+ AND Cd2+ BY A KEMP’S TRIACID-CAPPED
15-CROWN-5 ETHER ACROSS A PHOSPHOLIPID BILAYER MEMBRANE
5.1 Introduction
Highly selective liganding agents for divalent metal cations, such as those based upon the crown ethers general structure, are of potential interest in the design of novel synthetic ionophores. An added sidearm to a crown ether, forming a lariat crown ether, offers a functional approach to enhance the cation-binding ability of a given crown ether.(Vincent et al., 2000) The U-shaped structural motif of Kemp’s triacid has been used in this regard, and has resulted in the design of several supramolecular systems.
(Mathur et al., 2000;Oliver et al., 2000;Baldwin et al., 1997) However, few reports have addressed the binding of these related derivatives for the recognition of heavy metal ions,(Hüser et al., 2000;Hermesh et al., 2000) particularly, lead and cadmium. Of practical importance is to develop synthetic ionophores which can be used for the efficient removal of heavy metal ions known to have toxic effects. [ATSDR (Agency for
Toxic Substances and Disease Registry), 1999;Sakamoto et al., 2000)]
The ionophore KTC 15-cr-5 ether (Fig. 5.1) was among several derivatives synthesized by Chang and co-workers employing a 15-crown-5 ether as the molecular
116 framework.(Maravall et al., 2000) It was demonstrated that this compound selectively transports Pb2+ over a diverse set of divalent cations as well as monovalent cations including: Li+, Na+, K+, and Rb+. Chang and co-workers demonstrated that KTC 15-cr-5 transports Pb2+ by a simple three-phase system using chloroform as the organic phase.
In this paper we extend their results by reporting that KTC 15-cr-5 is a selective ionophore for the heavy metals Pb2+ and Cd2+ in a phospholipid vesicle system and by further presenting the kinetics of transport by this compound.
A variety of different membrane systems have been utilized to study carrier transport across a membrane barrier. These classical methods include bulk solvent liquid membranes, liquid emulsion, and polymer-supported liquid membrane systems.
We have been using phospholipid vesicles to more closely mimic biological cell membranes. This highly defined transport system has been widely applied by several groups on numerous studies.(Dejean et al., 2000;Kavanagh et al., 2000;Riddell and
Hayer, 1985b;Riddell et al., 1988c;Erdahl, Chapman, Taylor, and Pfeiffer, 1994;Wang,
Taylor, and Pfeiffer, 1998;Hamidinia, Shimelis, Tan, Erdahl, Chapman, Renkes, Taylor, and Pfeiffer, 2002a;Wang, Erdahl, Hamidinia, Chapman, Taylor, and Pfeiffer, 2001)
The results reported here may be of use for the development of separation processes for medicinal value and waste water treatment.
5.2 Materials and Methods
Reagents. 1H NMR spectra were obtained with a Bruker DPX 400. Synthetic 1- palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine (POPC) was obtained from Avanti
Polar Lipids, Inc. Purity was confirmed by thin layer chromatography before use. 2-
117 (aminomethyl)-15-crown-5 and propyl analogue of Kemp’s triacid were purchased from
Aldrich and did not undergo further purification. Quin-2 (K+ salt) from Calbiochem
Corp. was purified by passage over Chelex 100 resin (100-200-mesh) in the Cs+ form as described by Erdahl, et al., 1994.(Erdahl, Chapman, Taylor, and Pfeiffer, 1994)
Synthesis of Crown Ether Derivative. The Kemp’s triacid-capped 15-crown-5 ether was synthesized by procedures outlined by Kim et al., 2002.(Maravall, Mainen,
Sabatini, and Svoboda, 2000) Briefly, a mixture of 2-(aminomethyl)-15-crown-5 (1 mmol) and tripropyl Kemp’s triacid (1 mmol) was mixed and heated to 200 °C for 3 hours under nitrogen atmosphere. After cooling, the crude product was dissolved in 30 mL of CHCl3 and filtered. The crude product was purified by column chromatography
(silica gel, solvent; 50:50 hexane:chloroform and eluted with MeOH. TLC plate analysis showed possible unreacted 2-(aminomethyl) 15-crown-5 in the fraction. As a result of this impurity, a less polar solvent was chosen. Further purification was carried out by silica gel chromatography with 100% chloroform, and eluted with 2-propanol.
1 Yield: 0.054 g (10 %). The MW of the product is 555.70. NMR (D2O, ppm)
Structure was confirmed my 1NMR.
Preparation of Phospholipid Vesicles. Freeze-thaw extruded POPC vesicles loaded with Quin-2 were prepared as described in the Materials and Methods section
2.2.
Determination of Transport. The transport of Pb2+ and other divalent cations into Quin-2 loaded vesicles was determined by monitoring formation of the Quin-
2:cation complexes spectroscopically, as described in section 2.2. Transport
118 selectivities, S, were obtained from Equation 2. To determine S, an equal concentration of the cation in question (Pb or Cd) and calcium is 20 µM, with all other conditions held constant. All transport data were obtained at 25.0° C.
Pb2+ and Cd2+ Buffers. Pb2+ or Cd2+ was usually presented from a buffer system for transport into the vesicles. 15 mM citrate was employed to buffer the concentration of this cation, whereas 10 mM each of Hepes and Mes were present to buffer H+.
Seventeen equilibria involving citrate3-, H+, Pb2+, and OH- were accounted for when calculating the free Pb2+ concentration. In addition, fourteen equilibria involving citrate3-, H+, Cd2+, Cl-, and OH- were accounted for when calculating the free Cd2+ concentration. The respective equilibrium constants were taken from literature sources.(Smith and Martell, 1976;Martell and Smith, 1977) The species distribution program COMICS(Perrin and Sayce, 1967) was used to solve the applicable sets of simultaneous equations at experimental conditions of interest, and to allow the generation of standard curves.
5.3 Results
The transport activity of KTC-15-cr-5 is compared to that of ionomycin(Erdahl,
Chapman, Taylor, and Pfeiffer, 2000a) and monensin(Hamidinia, Shimelis, Tan, Erdahl,
Chapman, Renkes, Taylor, and Pfeiffer, 2002a) as depicted in Fig. 5.2. Under the conditions described, all three compounds facilitate transport of Pb2+ over several other divalent metals, with the lariat crown ether being further selective for Cd2+. Initial rates were obtained from these data (equation 1), and were used to calculate the selectivity values (equation 2). The SPb is 98 for ionomycin, 3432 for monensin, and 280 for the 119 KTC-15-cr-5 under the conditions of Fig. 5.2. The SCd is 10 for ionomycin, 2.1 for monensin, and 68 for the KTC-15-cr-5 under the conditions of this figure. A comparison of panels A, B, and C shows that the KTC-15-cr-5 is an effective Pb2+ ionophore, but also has an added selectivity for the transport of Cd2+ over several other divalent cations.
The rate of the lariat crown ether-catalyzed Pb2+ or Cd2+ transport is a function of the ionophore and the Me2+ concentration. When the ionophore concentration was varied with 1 µM free Pb2+ present (Fig 5.3), a plot of log initial rate versus log of the ionophore concentration revealed a straight line of slope 0.9, close to a value of 1 that is expected if this compound transports through a 1:1 (ionophore/cation) complex.
Similarly, when 20 µM CdCl2 was present, a slope of 1.0 was also obtained from the log initial rate versus log ionophore concentration (Fig. 5.4).
Varying the free Pb2+ or the free Cd2+ concentration both yielded log initial rate versus log Me2+ plots that display a more complex multiphasic pattern (Fig 5.5).
Analysis of the variable Pb2+ and Cd2+ data in Fig. 5.5 C shows linearity in the mid regions (b) and (d) of the multiphasic pattern with slopes of 0.8 and 1.0, respectively. In both cases the slopes of these plots are near 1. At lower Pb2+ (a) and Cd2+ (c) concentrations, however, there is an increase in the slope values to 1.4 and 3, respectively. This suggests the involvement of higher order transporting species which will further be described in the Discussion. Overall, the data indicate a predominant stoichiometry of 1:1 (ionophore:cation), and that species of higher stoichiometry are also contributing. To determine the selectivity of Pb2+ over Cd2+ for KTC-15-cr-5, a value was calculated based upon the x-axis separation of the plots within the regions where
120 slopes near 1 were observed. Within these regions, both sets of data were separated by
1.50 log units, which translates into a selectivity value for Pb2+ over Cd2+ of ~30.
KTC-15-cr-5 is monobasic in the fully ionized state, because the carboxylic acid sidearm can be ionized (Fig. 5.1). Additionally, the 15-crown-5 ether moiety can form a stable complex with Pb2+ through size selectivity from its cavity.(Saks et al., 2000)
However, a 1:1 complex containing Pb2+ could still carry a partially positive charge if the lariat crown ether does not completely neutralize the 2+ charge. Thus it seemed possible that Pb2+ is transported by an electrogenic mechanism when Val plus CCCP is present to collapse the resulting membrane potential. To investigate this possibility, the effect of an imposed potential was determined using vesicles containing a high internal
Cs+ concentration and an external medium containing Na+. In that system, when Val and CCCP are excluded, KTC-15-cr-5 remains only slightly active for Pb2+ transport.
This shows that there is a modest electroneutral mechanism operating when no potential is imposed. However, when Val was also present, but CCCP was not present, the rate of
Pb2+ transport was accelerated approximately four-fold (Fig. 5.6). This suggests that
Pb2+ is transported through a predominately electrogenic mechanism when a membrane potential of the appropriate orientation is present.
Figure 5.7 shows the effect of pH on the initial rate of Pb2+ transport by this proton-ionizable crown ether. Transport is more rapid between the ranges of pH 6 to 7 and then declines progressively at higher pH values. One possibility which explains this observation is that two hydroxide groups could associate with the Pb2+ • crown ether complex at higher pH preventing transport thru the membrane because of the negatively charged complex. The lariat crown ether is presumably a weak acid with proton
121 donating groups at the carboxyl and the amide nitrogen. Further studies, however, need to be done to determine the pka of the crown ether derivative as well as the transporting species across the pH range.
5.4 Discussion
Based on the present results, KTC-15-cr-5 can now be classified with other ionophores that transport Pb2+. A comparison of KTC-15-cr-5 to naturally occurring carboxylic ionophores such as monensin, ionomycin, and nigericin shows that this synthetic ionophore shares the common selectivity for Pb2+ over other divalent cations, with the added property for Cd2+ transport as depicted in Figure 5.2.
Pb2+ and Cd2+ transport by KTC-15-cr-5 is a first-order function of ionophore concentration, as indicated by the linear behavior and slope near 1.0 that are observed from plots of log initial rate versus log ionophore concentration (Fig. 5.3 & 5.4). The same is true with respect to varying Pb2+ and Cd2+ concentration within the central portion of the concentration range examined (Fig. 5.5). Within these regions the efficiency of transport can be expressed as a second-order rate constant, as defined by equation 3. These rate constants are useful in the evaluation of the transport selectivity for various cations in this vesicle system, regardless of the selected ionophore and cation concentration used to obtain them. Additionally, these rate constants provide a way to compare transport efficiencies even with the diverse range of rates that are observed using a single set of conditions by the vesicle transport system. From the present data,
2+ 2+ 4 -1 -1 2 -1 -1 ktrans values for Pb and Cd transport are 1.9 ·10 M s and 5.9 ·10 M s ,
122 respectively. To put these rate constants into perspective, previous reported kPb values for the carboxylic ionophores monensin and ionomycin were 1.0 ·105 and 11.9 ·105 M-1 s-1, respectively.(Van Hall, 2000)
In the regions where slopes near 1 were observed from Fig. 5.5 C, with 30 µM
KTC-15-cr-5 and an external concentration of 70 nM Pb2+ or 2 µM Cd2+ present, the initial rate of Pb2+ or Cd2+ transported was 0.0389 µM/sec and 0.03535 µM/sec, respectively. These values correspond to uptake rates of 0.0389 ·10-9 moles Pb2+/1 ·10-6 moles POPC per sec or 0.03535 ·10-9 moles Cd2+/1 ·10-6 moles POPC per sec, and, assuming a PC headgroup area of 68 Å2,(Chapman, Erdahl, Taylor, and Pfeiffer, 1990a)
Pb2+ and Cd2+ fluxes of 1.73 ·10-14 and 1.57 ·10-14 mole/cm2 per sec, respectively. When these fluxes are divided by the initial concentration of the external cation (70 ·10-12 and 2
·10-9 mole/cm3), permeability coefficients of 2.47 ·10-4 and 7.89 ·10-6 cm/sec are calculated. The permeability coefficient for Pb2+ and Cd2+ when no ionophore is present is calculated to be 4.0 ·10-7 cm/sec and 1.3 ·10-9 cm/sec, respectively. Permeability coefficients on the order of 10-12 cm/sec have been reported for K+ and Na+ in liposome systems.(D'Herde et al., 2000).
Within the concentration regions where equation 3 is applicable, the stoichiometry of the transporting complex is 1:1, ionophore to cation, based upon first- order kinetics which is demonstrated by slopes near 1.0 in log versus log plots. The 1:1 complex PbKTC+ or CdKTC+ may then be active as the transporting species and would function by an electrogenic mechanism. This mechanism likely occurred in figures 5.2-
5.5, and 5.7 because Val plus CCCP were present during these experiments. Val and
CCCP are effective at collapsing an induced membrane potential in the current vesicle
123 system(Wang, Taylor, and Pfeiffer, 1998), which is required for continued transport operating in an electrogenic mode(Wang, Erdahl, Hamidinia, Chapman, Taylor, and
Pfeiffer, 2001). Further support that the major transporting species is the 1:1 charged species PbKTC+, formed according to Equation 4, is shown by the pH dependency depicted in figure 5.7.
Pb2+ + KTC¯ PbKTC+ (4)
In addition to the predominant 1:1 complex, the data also indicate that species of higher stoichiometry are also contributing to Pb2+ and Cd2+ transport. Considering Pb2+ first, the slope of log initial rate versus log free Pb2+ exceeded 1 within the cation concentration range of 3-30 nM (Fig. 5.5C). These findings can be rationalized if more than one species transports Pb2+. More precisely, the primary transporting species would be subject to comproportionation with an increasing Pb2+ concentration, as represented by Equation 5 (charges omitted):
2+ Pb(KTC)2 + Pb 2PbKTC (5)
Thus, the fraction of transport occurring via the 1:1 complex is expected to increase as the Pb2+ concentration rises. If the 2:1 or 1:1 complexes were both capable of Pb2+ transport at a lower cation concentration, then the slope of a log rate versus log plots would be less than 2, in proportion to the particular complex and their relative efficiencies as transporting species. This can explain how a slope of 1.4 might arise under the conditions of Fig. 5.5.
Regarding Cd2+, the comproportionation equilibria of the various transporting species is as follows:
124 2+ + Cd(KTC)3 + Cd Cd(KTC)2 + CdKTC ;
2+ + Cd(KTC)2 + Cd 2CdKTC (6)
When these reactions are considered with Fig. 5.5C, the indications are that Cd(KTC)3 exists, and this is an explanation to the high slope value of 3 at Cd2+ concentration less than 0.75 µM.
The above interpretations explain the kinetics data relating the concentrations of
Pb2+ and Cd2+ with KTC-15-cr-5, however an unambiguous identification of the transporting species still awaits investigation of the complexation equilibria between this ionophore and Pb2+ or Cd2+. Explanations for the multiphasic pattern in plots of log rate versus log Pb2+ or Cd2+ also await investigation by the complexation equilibria between this ionophore and Pb2+ or Cd2+. Regarding the upper portion of the multiphasic pattern, one possibility is that it reflects an approaching saturation of the ionophore with Pb2+ or
Cd2+. However, it is also possible that it reflects the reaction of a 1:1 transporting species with a second Pb2+ or Cd2+ cation to form an impermeant complex that is inefficient for transport across membranes.
n+ MKTC + M M2KTC (7)
If higher order species were less stable than the transporting species, the effect would be less ionophore contributing to transport as the free Pb2+ or Cd2+ increased, producing the upper region of the multiphasic pattern which is observed in Fig. 5.5.
This report allows the possibility that KTC-15-cr-5 could serve as a therapeutic agent for the efficient and selective transport of the heavy metals Pb2+ and Cd2+ from biological systems. Van Zanten and co-workers have also suggested metal-sorbing
125 vesicles as a potential technology for ionophores in the removal of metal ions from a dilute aqueous solution.(Hüser, Blatter, and Sheu, 2000) An application of this technology has been employed in the selective separation and concentration of heavy metals from wastewater.(Johnson et al., 2000;Crompton, 2002;Crompton, 2000;Li et al.,
2000;Jones et al., 2000;Fernández-Salas et al., 1999;Balsinde et al., 2000)
5.5 Summary
A Kemp’s triacid-capped 15-crown-5 ether was synthesized and found to be effective as an ionophore for transporting Pb2+ and Cd2+ across a phospholipid bilayer membrane. Data from quin-2 loaded POPC vesicles obtained at pH 7.0 show that this compound transports divalent cations with the selectivity sequence Pb2+ > Cd2+ >> Zn2+
> Mn2+ > Co2+ > Ni2+ > Ca2+ > Sr2+. When Pb2+ or Cd2+ was present individually, relative rates of transport indicated selectivity for Pb2+ over Cd2+ of 4-fold to 30-fold, depending on the experimental conditions. Plots of log rate vs log Mn+ or log ionophore concentration suggest that Pb2+ and Cd2+ are transported primarily as a 1:1 complex, ionophore:Mn+, but that higher order complexes also occur. The ionophore transports
Pb2+ and Cd2+ by a predominately electrogenic mechanism, based upon enhanced rates of transport that are seen in the presence of agents that dissipate transmembrane potential potentials. The rate of transport is dependent on external medium pH; increasing at acidic to neutral pH and progressively declining at higher pH values. The high selectivity for Pb2+ and Cd2+ transport by the Kemp’s triacid-capped 15-crown-5 is of possible value for removal of these heavy metals from wastewater and in the treatment of Pb and Cd intoxication.
126
OH O O HO O O O OH
+ O O Pr Pr
O NH2 Pr
in neat 200 °C
O
O O
O O
O N O CO2H
Pr Pr
Pr
Figure 5.1. Synthesis of Kemp’s triacid-capped 15-crown-5 ether.
127
Figure 5.2. Transport of Pb2+ and other divalent cations by KTC-15-cr-5 (A), ionomycin (B), and monensin (C). POPC vesicles loaded with Quin-2 (Cs+) were prepared as described in the Materials and Methods. Vesicles were incubated at 25 °C in a medium containing 50 mM CsNO3 plus 10 mM Hepes. The nominal POPC concentration was 1.0 µM. The external pH was adjusted to pH 7.0 with CsOH, and valinomycin (0.5 µM) plus carbonyl cyanide m-chlorophenyl-hydrazone (5 µM) were present to maintain the internal pH at the external value. Divalent cations were present at 20 µM in all cases (nitrate in the case of Pb2+ and chlorides for all others). Transport was initiated at 0 seconds by the addition of 10 µM KTC-15-cr-5 or 0.1 µM for ionomycin and monensin. A 2 minute pre-incubation preceded the initiation of transport to permit the equilibration of internal and external pH. The sequence of cations shown on the right corresponds to their relative rate of transport as observed directly for A and B or on an expanded scale for C. The y-axis unit refers to cation concentration in the external medium. When this parameter reaches 13 µM as seen in A or 20 µM as seen in B and C, all of the cation that was originally present in the external medium has been transported into the vesicles and bound by Quin-2.
128 14 A. KTC-15-cr-5 2+ Pb 2+ 12 Cd M µ
10 ,
port 8 Crown ans r 6 T 2+ 4 2+
Me Zn Mn2+ 2 Co2+ Ni2+ 0 Ca2+ Cu2+ 0 200 400 600 800 Sr2+ seconds
B. Ionomycin 20 Pb2+
Zn2+ 16 2+ M Cd µ
, Iono
12 2+
sport Mn an r
T 8 2+
2+ Ca
2+ Me Cu 4 Co2+ Ni2+ 2+ 0 Sr
0 200 400 600 800
seconds
C. Monensin 20 Pb2+
16 M µ
, Mon
port 12 ans r 2+
T 8 Cu Zn2+ 2+ Cd2+ 2+ Me 4 Mn Co2+ Ca2+ Ni2+ 0 Sr2+ 0 200 400 600 800
seconds 129 A. Crown Ether Derivative, µM
30 27.5 12 25 22.5 20 10 17.5
M 15 µ 8 12.5 10
6 Crown 7.5 Transport, 4 5.0 2+ Pb 2 1.0 0 0 100 200 300 400 500
seconds
B. -0.8
-1.0 M/sec µ -1.2 te, a R -1.4
og Initial -1.6 L
-1.8 -5.4 -5.2 -5.0 -4.8 -4.6 -4.4
Log [Crown Ether], M
Figure 5.3. Effect of ionophore concentration on the rate of Pb2+ transport. (A) Conditions were the same as described in the legend to Fig. 5.2, except that the external 2+ medium contained 15 mM citrate plus Pb(NO3)2 sufficient to produce a free Pb concentration of 1 µM, and the lariat crown ether was varied as shown to the right of the individual traces. (B) Shown is the log of the initial Pb2+ transport rate as a function of log KTC-15-cr-5 concentration. Initial rate values were obtained from the progress curves shown in A. 130 A. Crown, µM
18 50 40 30 16 20 14 M 15 µ
t, 12 Crown 10 10 7.0
anspor 8 5.0 Tr 6 4.0 2+ 3.0
Cd 4
2 2.0 0 0 0 100 200 300 400 500
seconds
B. -0.5 c e -1.0 M/s µ
-1.5 Rate,
ial
-2.0 Log Init
-2.5 -5.6 -5.4 -5.2 -5.0 -4.8 -4.6 -4.4 -4.2
Log [Crown Ether Derivative], M
Figure 5.4. Effect of ionophore concentration on the rate of Cd2+ transport. (A) Conditions were the same as described in the legend to Fig. 5.2. The external medium contained 20 µM CdCl2, and the lariat crown ether was varied as shown to the right of the individual traces. (B) Shown is the log of the initial Cd transport rate as a function of log KTC-15-cr-5 concentration. Initial rate values were obtained from the progress curves shown in A. 131
Figure 5.5. Effect of Pb2+ and Cd2+ concentration on the initial rate of transport. Conditions were the same as described in the legend to Fig. 5.2, except that 30 µM ionophore was added to initiate transport and the concentration of (A) Pb2+ or (B) Cd2+ was varied by a 15 mM citrate-based buffer system, as described in the Experimental Section. Some of the progress curves from (B) have been omitted for the sake of clarity. (C) The log initial rate is shown versus log Pb2+ (○) or log Cd2+ (●) concentration.
132 A. Variable Pb2+ Free Pb2+, nM
400
10 200
100 M
µ 8 80
, 70 60 ort
p 6 50 s n Crown 40 30 Tra 4 2+ 20 Pb 2 15 10 8.0 7.0 0 6.0 4.0 0 50 100 150 200 250
seconds
B. Variable Cd2+ 18 Free Cd2+, µM
16 10 6.0 14 5.0
M 4.0 µ
3.0 , 12 t 2.5 or 10 2.0 sp Crown n 1.5 a 8
tr 1.0 0.9
2+ 6 0.8 Cd 4 0.6 2 0.5 0.4 0.25 0 0 100 200 300 400 500
seconds
C. Variable Pb2+ -1.0 Variable Cd2+
c -1.5 e
M/s b µ -2.0 d
ial Rate, -2.5
a -3.0 Log Init c
-3.5 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0
2+ Log [Me ], M
133
25
20 M
µ + val
e, 15
ak Crown
upt 10 2+ Pb 5 - val
0 0 100 200 300 400 500 600
Seconds
Figure 5.6. Dependence of Pb2+ transport on membrane potential. Vesicles were prepared as described in the Materials and Methods using Cs+ as the counter ion to Quin-2, except the external medium contained 50 mM NaCl, 10 mM Hepes (Na+), pH 7.00. The external medium also contained 15 mM citrate plus Pb(NO3)2 sufficient to produce a free Pb2+ concentration of 1 µM. Where indicated, Val was present at 0.5 µM, and was added before the indicated crown ether derivative, which was used at 10 µM. CCCP was not present during this experiment.
134
7.0
6.0
5.0 M/min
µ 4.0
3.0
2.0 Initial Rate, 1.0
0.0
6.0 6.5 7.0 7.5 8.0 8.5 9.0
pH
Figure 5.7. Influence of external pH on the rate of ionophore-mediated Pb2+ transport. Data were obtained as described in the legend to Fig. 5.2, except that the external 2+ medium contained 15 mM citrate plus Pb(NO3)2 sufficient to produce a free Pb concentration of 1 µM. CsOH was also used to adjust the external pH, as well as to match the monovalent cation used as the counter ion to Quin-2. In addition, 20 µM ionophore was added to initiate transport. Points shown are experimental and were fit to an inverse third order polynomial equation to obtain the solid line.
135 CHAPTER 6
DISCUSSION
6.1 Conclusion
After our laboratory discovered that several ionophores have lead transporting properties, our objective was to determine if the widely used ionophore in agriculture, monensin, is a potential harmful agent to humans. Monensin is commonly used in agriculture as a feed additive for weight gain in cattle and to combat coccidial infections in livestock. Our hypothesis was that a carry over of this ionophore from the consumption of beef or poultry could be dangerous for individuals exposed to lead.
However, the opposite finding was obtained. Monensin used alone was found to reduce the accumulation of lead administered in the drinking water, and to accelerate the clearance of previously accumulated lead from several organs and tissues, including brain and bone. Furthermore, unlike EDTA, the commonly given drug in the treatment of Pb poisoning, monensin was shown not to deplete rats of essential trace metals such as Cu, Mn, and Zn.
Upon completion of this study, our lab investigated whether monensin could more effectively enhance Pb removal if utilized in conjuction with a newer therapeutic chelating agent, DMSA. DMSA is a more specific chelator for Pb than EDTA, however it is inadequate in removing Pb from bone where the largest amount of Pb is deposited.
136 For these reasons, our hypothesis was if monensin and DMSA were used in a combined treatment, then a synergistic effect would result in depleting Pb more successfully. The hypothesis that a reduction of Pb will occur after a combined treatment is based on the idea that if monensin transports Pb from several target organs, especially bone, then this mobilized Pb will bind with DMSA. From the experiments undertaken, the combination of monensin and DMSA in Pb intoxicated rats was more effective in reducing lead concentration than DMSA alone. Furthermore, Pb depletion was also observed in bone and other tissues without significantly effecting endogeneous trace metals.
The finding that monensin is an effective Pb ionophore, led to an exploration of other ionophores that could transport Pb more efficiently. The ionophore nigericin and a synthetic ionophore based on a 15-crown-5 ether skeleton both showed the ability to transport Pb across a phospholipid bilayer. The latter ionophore was also shown to be selective for the transport of Cd2+ which is of possible value to waste water applications.
Most recently, a preliminary study by our lab showed that the simultaneous administration of nigericin and Pb for three weeks led to an increase in Pb content in bone, brain, kidney and liver compared to the no treatment group. However, with the exception of this preliminary study, neither ionophore has been thoroughly investigated in an animal model.
The observation that monensin reduces Pb levels in Pb intoxicated rats is noteworthy, but an understanding of how this occurs is necessary. Metal ion transport studies were undertaken on monensin, nigericin, and KTC-15-cr-5 using POPC liposomes in an attempt to clarify the mechanism of heavy metal transport in a defined model membrane system. Experiments using quin-2 loaded liposomes showed that both
137 monensin and nigericin transport Pb2+ in a 1:1 stoichiometry, ionophore:cation, as indicated by plots of log rate vs log of the free Pb2+ concentration or log rate vs log of the ionophore concentration. These plots show linearity across a wide concentration range and display slopes of 1 which is consistent with a 1:1 stoichiometry. This data taken together with an increase rate of transport at higher pH suggests that the main transporting species under the experimental conditions was PbMonOH and PbNigOH and by a mechanism that is predominately electroneutral. Further support for these species come from stability constants obtained in 80% methanol/water which show complex stabilites of ~1014 for PbMonOH and PbNigOH which is 7 orders of magnitude more stable than the PbMon+ and PbNig+.
In addition to the pathway forming PbMonOH or PbNigOH, the equilibration between reactants forming the transporting species is of interest, especially when the high selectivity for Pb2+ is compared to the transport of other divalent cations.
Regarding the selectivity of monensin, the complex stability constants for the CaMon+ and the MgMon+ species are ~4 orders of magnitude less than for the PbMon+ species.
Moreover, no interaction of these complexes with OH- was detected during the potentiometric titrations. Similarly, in the case of nigericin, the complex stability constants for the CaNig+ and the MgNig+ species was shown to be ~5 orders of magnitude less than for the PbNig+ species. Together, these findings illustrate why monensin and nigericin are selective for Pb2+ over several physiological abundant divalent cations.
The situation is more complex with the synthetic Kemp’s triacid capped 15- crown-5 ether compound. The stoichiometry of the transporting complex is the 1:1
138 PbKTC+ or CdKTC+ species. Therefore, the active transporting species functions by an electrogenic mechanism. Yet the 1:1 complex occurs at higher metal ion concentrations, whereas at lower metal ion concentrations the primary transporting species is subject to comproportionation with an increasing Pb2+ or Cd2+ concentration. Thus, the fraction of transport via the 1:1 complex is expected to increase as the Pb2+ or the Cd2+ concentration rises. Further support that the transporting species is the 1:1 PbKTC+ or the CdKTC+ complex is shown by the pH dependency and the increasing rate of transport at lower pH.
6.2 Future Perspectives
What is currently unknown is the precise mechanism of Pb depletion in the animal model after monensin is given either alone or in combination with DMSA. A plausible explanation is that monensin works by taking advantage of the Na+ gradient to gain intracellular access to Pb2+ stores. However, this concept has not been proven conclusively. Another explanation underlying the mechanism of action for monensin is that this ionophore may promote bone turnover. According to this idea, monensin could either affect the balance between the activity of osteoblasts that form bone, and osteoclasts that break it down, by an indirect cell signaling pathway. This notion can be tested experimentally by first loading the animal with a radioactive isotope, such as 45Ca, until a steady state is achieved. Then once administration of the isotope was halted, one could measure whether monensin decreases the concentration of the radioactive isotope
139 in bone further than 45Ca removal alone by a scintillation counter. Additionally, one could determine if there is an increase in the concentration of the radioactive isotope in the urine and feces following the addition of monensin.
Dose-response studies are an important set of experiments that remains to be performed to test the efficacy of the ionophore by varying concentrations of both monensin and Pb. Unfortunately, the logistics of these experiments, such as excessive man hours and high cost, are limitations for implementation. Nevertheless, these experiments would be useful in gaining insight to the level of monensin most effective for Pb reduction without causing toxicity to the animal.
As described in the materials and method section of chapters 2 & 3, rats received
100 ppm of monensin in their diet which was administered ad libitum. Yet, it is unclear what the actual concentration of monensin is in the animal and its organs. Additionally, the half-life of monensin in the rat remains uncertain. Liquid chromatography using pre- column derivitization is one approach to elucidate the active concentration of monensin in organs and blood. This method has already been applied in agriculture for safety purposes by testing the levels of monensin and other ionophores in poultry feeds using the ultraviolet compound 2,4-dinitrophenylhydrazine(Dusi and Gamba, 1999).
Ultimately, the applications of these studies could be used on Pb poisoned individuals in our society. Accordingly, human trials should be undertaken to examine the efficacy of monensin or other Pb selective ionophore in cases of Pb poisoining.
Administration of these drugs could either be alone or in combination with a traditional chelator, preferably DMSA. It is important to note that these drugs are administered in agriculture at relatively high doses, and the LD50 of these compounds in rats, rabbits,
140 mice, etc. are known and are in this high range. Thus, we expect that monensin can be safely given to humans at a dose that is effective for treatment of Pb intoxication.
A final point relates to the future for ionophore studies. A wide range of applications exist for ionophores such as: making selective electrodes, tools for cell biologists, therapeutic agents, and environmental cleanup. In each of these areas the design of more selective compounds that target a particular ion is of value. Although many ionophores used today are isolated from natural sources, synthetic derivatives can be designed for the appropriate application, and an example of this was demonstrated by the work on the Kemp’s triacid 15-crown-5 ether.
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