Roles of lead-binding proteins in mediating lead bioavailability.

The intracellular bioavailability of lead (Pb) at low dosage levels in major target organs such as the kidney and brain appears to be largely determined by complexation with a group of low molecular weight proteins. These proteins are rich in aspartic and glutamic dicarboxyl amino acids. The proteins are chemically similar but not identical across all species examined to date and the brain protein appears to be different from that found in the kidney. These proteins possess dissociation constant values for Pb on the order of 10(-8) M and appear to normally bind zinc. In rats, these proteins attenuate the Pb inhibition of the heme pathway enzyme delta-aminolevulinic acid dehydratase by a mechanism involving both Pb chelation and zinc donation to this highly Pb-sensitive zinc-dependent enzyme. Other studies in rats have shown that the kidney protein facilitates the intranuclear movement of Pb in vitro followed by chromatin binding, suggesting that this protein may be involved in alterations of the pathognomonic Pb intranuclear inclusion bodies in renal gene expression associated with the mitogenic effects of Pb in the kidney.

All models for predicting blood lead values from soil or dust exposures require the movement of lead (Pb) in a soluble form between the gastrointestinal tract, lungs, blood, major organs, and urinary or fecal routes of excretion. The models also assume that blood Pb values are useful predictors of Pb toxicity for all segments of the population. The movement of Pb in blood and its transport into hard and soft tissues implies binding to soluble carriers such as metal-binding proteins. It has been known for some time that other metals such as iron are transported in blood via proteins such as transferrin and that cadmium and zinc are transported bound to metallothionein. Soluble Pb-binding proteins (PbBPs) in blood (1,2) and target tissues (3)(4)(5)(6)(7)(8)  toxic potential of Pb. Protein-mediated mechanisms of toxicity for metals such as calmodulin and calcium have been studied extensively (9,10), and research from this laboratory has shown that PbBPs from the rat are capable of both attenuating the effects of Pb on 6-aminolevulinic acid dehydratase (6-ALAD) (11)(12)(13) and mediating the intranuclear movement and chromatin binding (14,15) of Pb in target cell nuclei from the kidney. These latter effects appear to be quite important in regulating alterations in renal gene expression patterns (16,17) and possibly Pb-induced renal cancer (18). PbBPs may also be involved in the formation of Pb-containing intranuclear inclusion bodies in target tissues (19)(20)(21)(22)(23), as they appear to undergo aggregation after addition of Pb in vitro (24).
The following discussion reviews the literature on these molecules and examines how they influence individual susceptibility to Pb toxicity in humans and other species.

Lead-Binding Proteins Rats
Kidney. Studies by Oskarsson et al. (4) demonstrated that the rat kidney possessed several low molecular weight protein peaks from rat kidney cytosol. One of these proteins had an estimated molecular mass of 63,000 daltons, which may be a tetramer of the lower molecular weight peak, and showed stable binding of 203Pb on sodium dodecyl sulfate (SDS) gels, indicating a high degree of stability. Subsequent studies (11)(12)(13) demonstrated that both molecular forms were capable of attenuating the inhibitory effects of Pb2+ on 6-ALAD activity by a mechanism involving both Pb chelation and donation of zinc to this zinc-activated enzyme. Pb-binding studies demonstrated that the low molecular weight protein had an apparent dissociation constant (Kd) for Pb of 10-8 M, with extremely stable binding. Cell-free nuclear translocation studies (14,15) demonstrated time-and temperature-dependent nuclear uptake of 203Pb-labeled PbBP with subsequent chromatin binding. In vitro metal competition studies (15) demonstrated that cadmium and zinc were the most effective competitors for inhibiting Pb binding to the PbBP. These data are of interest, as they are consistent with the results of an in vivo lead x cadmium x arsenic feeding study (25) that demonstrated that concomitant exposure to cadmium abolished formation of Pb intranuclear inclusion bodies. Biochemical characterization studies (5) identified the rat renal PbBP as a2,-globulin, which is a member of the retinol-binding protein supergene family. These studies also confirmed the apparent KI value of 10-8 M Pb for binding to the purified protein. Other studies (24) also showed that addition of Pb to the purified protein in vitro also resulted in an apparent aggregation phenomenon, indicated by the appearance of higher molecular weight bands on SDS gels as a function of metal and incubation time. These findings suggest the soluble PbBP in rat kidney cytosol may be involved in the formation of pathognomonic Pb intranuclear inclusion bodies (18)(19)(20)(21)(22)(23). These inclusions (20,(22)(23) contain acidic proteins with estimated molecular masses of 30,000 daltons.
Brain. The other major target organ in the rat also showed two protein peaks with higher molecular masses than those found in the kidney (3); Western blot analysis studies using polyclonal antibodies to the rat renal PbBP did not cross-react with the brain PbBP. Preliminary characterization studies (3) showed that, like the kidney, brain PbBP was an acidic protein with a high content of glutamic and aspartic amino acids and ion exchange chromatographic

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characteristics similar to those of renal PbBP. The exact identity of this protein is currently unknown. Humans Blood. Ragavan and Gonick (2) reported earlier the increased presence of low molecular weight PbBPs in blood of PbBPexposed workers. Subsequent studies by Lolin and O'Gorman (1) also showed the presence of soluble proteins in workers with moderate Pb exposure. The identity of these proteins is currently unknown. Kidney. Recent studies by Smith and co-workers (7,8) have shown that there are two PbBPs in human kidney and that these are identified as diazepam-binding inhibitor and thymosin f4. These proteins were found to exhibit Kd values of approximately 10-8 M, which is similar to that reported for the rat (15).
Brain. Studies by Quintanilla-Vega et al. (6) showed that human brain also contained Pb endogenously bound to thymosin 4. 203Pb-binding studies also showed an apparent Kd value of 10-8 M.

Other Species
Preliminary studies in monkeys (26) showed that this species has kidney and brain PbBPs proteins with chromatographic characteristics similar to those of humans. The major bands on the gels were enriched in glutamic and aspartic amino acids, but the exact identity of these proteins is currently unknown. Studies on the PbBP from catfish liver (27) also showed similar highly anionic characteristics and the capacity to attenuate the direct inhibitory effects of Pb on 6-ALAD (28). The chemical and functional similarities between the PbBPs in different species appear to be highly conserved, although the proteins seem to be of different molecular masses. This could suggest that there are conserved binding regions of these various proteins across species that act in a similar manner.

Relevance of Molecular
Handling of Lead to Kinetic Modeling of Lead in Vivo Clearly, intracellular partitioning of Pb in target organs may be involved in mediating the low dose effects of Pb. These proteins are chemically similar but vary between species and target organs, with Kd values for Pb on the order of 10-8 M. In vitro studies suggest that the binding of Pb to these proteins is highly stable and that they represent an important Pb pool in the cytosolic compartment. Thus, they appear to be important molecular factors for regulating the bioavailability of Pb to sensitive molecular processes such as 6-ALAD activity in kidney and brain, and gene regulation in the kidney. From the perspective of kinetic modeling, they probably represent a highly stable intracellular compartment that mediates the toxic potential of Pb to the important processes noted above. It is hypothesized that differences in tissue levels of these proteins play a major role in determining individual variation in intracellular Pb bioavailabilty and hence susceptibility to toxicity from this metal. A diagram of this concept is presented in Figure 1, which shows the hypothesized regulation of Pb movement between the target tissue-specific Pb-binding proteins, sensitive effector molecules such as calmodulin, 6-ALAD, or chromatin-binding sites, and (at higher dose levels) the Pb intranuclear inclusion bodies. This last point is central to improving the predictive value of kinetic modeling for Pb toxicity. As noted above, accurate prediction of blood values is only a surrogate approach for estimating toxic potential of Pb; individuals vary greatly in their susceptibilty to Pb toxicity for equal blood Pb values. It is our hypothesis that the soluble Pb-binding proteins in major target tissues such as the brain and kidney are involved in mediating these individual differences in susceptibility, particularly at low-dose exposures. Further research is needed to test this hypothesis and to delineate how differences in tissue levels of these molecules influence the kinetics of Pb retention and bioavailability to other sensitive molecular processes.