Joel G. Pounds

Whole-Body Lifetime Occupational Lead Exposure and Risk of Parkinson’s Disease

Background Several epidemiologic studies have suggested an association between Parkinson’s disease (PD) and exposure to heavy metals using subjective exposure measurements. Objectives We investigated the association between objective chronic occupational lead exposure and the risk of PD. Methods We enrolled 121 PD patients and 414 age-, sex-, and race-, frequency-matched controls in a case–control study. As an indicator of chronic Pb exposure, we measured concentrations of tibial and calcaneal bone Pb stores using 109Cadmium excited K-series X-ray fluorescence. As an indicator of recent exposure, we measured blood Pb concentration. We collected occupational data on participants from 18 years of age until the age at enrollment, and an industrial hygienist determined the duration and intensity of environmental Pb exposure. We employed physiologically based pharmacokinetic modeling to combine these data, and we estimated whole-body lifetime Pb exposures for each individual. Logistic regression analysis produced estimates of PD risk by quartile of lifetime Pb exposure. Results Risk of PD was elevated by > 2-fold [odds ratio = 2.27 (95% confidence interval, 1.13–4.55); p = 0.021] for individuals in the highest quartile for lifetime lead exposure relative to the lowest quartile, adjusting for age, sex, race, smoking history, and coffee and alcohol consumption. The associated risk of PD for the second and third quartiles were elevated but not statistically significant at the α = 0.05 level. Conclusions These results provide an objective measure of chronic Pb exposure and confirm our earlier findings that occupational exposure to Pb is a risk factor for PD.

Chemical mixtures from a public health perspective: the importance of research for informed decision making.

When considered from a public health perspective, the central question regarding chemical mixtures is deceptively simple: Are current approaches to risk assessment for chemical mixtures affording effective (adequate) and efficient (cost-effective) protection for members of our society? Answering this question realistically depends on an understanding of the hierarchical goals of public health (i.e. prevention, intervention, treatment) and an accurate evaluation of the extent to which these goals are being achieved. To allow decision makers to make informed judgments about the health risks of chemical mixtures, adequate scientific knowledge and understanding must be available to support risk assessment activities, which are an integral part of the regulatory decision making process. Designing and implementing relevant research depends on the existence of a feedback loop between researchers and regulators, where the information needs of regulators influence the nature and direction of research and the information and understanding generated by researchers improves the scientific basis for public health decisions. A clear, consistent, commonly accepted taxonomy for describing important mixture-related phenomena is a key factor in creating and maintaining the necessary feedback loop. Ultimately, both researchers and regulators share a common goal with regard to chemical mixtures; improving the state-of-the-science so that we can make informed decisions about protecting public health. A survey of research issues and needs that are crucial to attaining this goal is presented.

Lead intoxication alters basal and parathyroid hormone-regulated cellular calcium homeostasis in rat osteosarcoma (ROS 17/2.8) cells

SummaryThe skeleton is the major reservoir of lead and calcium in humans, and plays an important role in systemic calcium regulation. Lead perturbs normal calcium transport and second messenger function, directly or indirectly, in virtually all cells studies so far. Therefore, we and others have postulated that an early and discrete toxic effect of lead is perturbation of one or more loci within the calcium messenger system. To understand further the role of lead on calcium homeostasis in bone, we undertook this study to characterize calcium homeostasis and the effect of lead on calcium homeostasis in rat osteosarcoma (ROS 17/2.8) cells, which exhibit the osteoblast phenotype. ROS cells were incubated in medium containing 45Ca for 20 hours. Monitoring the efflux of 45Ca from the cultures for 210 minutes allowed for the determination of kinetic parameters defining steady state calcium homeostasis. Three distinct intracellular kinetic calcium pools characterized 45Ca homeostasis. Treatment with either 400 ng parathyroid hormone (PTH)/ml culture medium for 1 hour or 25 μM lead for 20 hours increased total cell calcium. Treatment with PTH caused a larger increase of cell calcium in lead-intoxicated cells than either lead intoxication or PTH treatment alone. This increase suggests that lead may perturb normal calcium-mediated PTH responsiveness of the osteoblast. These experiments further establish a kinetic model for the study of calcium homeostasis in osteoblastic bone cells. The studies also advance the hypothesis that lead-induced perturbations of calcium-mediated processes represent and early effect of lead toxicity at the cellular level.

Cocaine toxicity in cultured rat hepatocytes

Cocaine hydrochloride was added to primary cultures of hepatocytes isolated from naive and phenobarbital-induced (80 mg/kg i.p. for 3 d) Sprague-Dawley rats. Cocaine was cytotoxic, as measured by lactate dehydrogenase release, to cells from naive rats in concentrations of 1 mM or greater. Phenobarbital induction greatly increased the cytotoxic potency of cocaine in vitro, with nearly complete loss of cell viability at cocaine concentrations in culture as low as 0.01 mM. The addition of 10 microM SK&F-525-A to the cultures blocked cocaine cytotoxicity in cells from both naive and phenobarbital-induced rats. These results suggest that the metabolic pathways leading to cocaine hepatotoxicity identified in the mouse also exist in the rat hepatocyte.

Lead inhibits meso-2,3-dimercaptosuccinic acid induced calcium transients in cultured rhesus monkey kidney cells

Previously we have shown that meso-2,3-dimercaptosuccinic acid (DMSA, 15-500 microM) elicits concentration-dependent increases in intracellular calcium levels ([Ca2+]i) in untreated rhesus monkey kidney cells (LLC-MK2) (Pokorski et al., 1997, unpublished results). Little is known about the restorative effects of the chelating agent 2,3-dimercaptosuccinic acid on intracellular calcium homeostasis in the presence of lead. Lead interacts at numerous sites in Ca2+ homeostasis and may mimic Ca2+ to interfere with Ca2+-mediated intracellular signaling. To examine the effects of lead on [Ca2+]i and DMSA-induced calcium transients, LLC-MK2 were plated on 35 mm coverslip dishes (10(4) cells/dish) and pre-treated with non-cytotoxic concentrations of lead (0-100 microM) for 24 h. Cells were washed, loaded with the calcium-sensitive probe Fura-2/AM, rinsed again, and examined in loading buffer in the absence of any additional lead. Intracellular calcium was measured using a dual-wavelength calcium imaging system. Basal [Ca2+]i levels did not change between Pb-exposed (0-50 microM, 24 h) and non-lead exposed cells. In cells treated with > or = 10 microM lead for 24 h, the ability of DMSA to elicit a calcium response was blocked. These results provide evidence that pre-exposure to lead blocks the entry of extracellular calcium into LLC-MK2 cells when stimulated by specific calcium mobilizing agents.

LEAD (Pb)|[ndash]|CALCIUM (Ca) INTERACTIONS IN CULTURED OSTEOCLASTIC BONE CELLS (OC)

Perturbations in intracellular Ca homeostasis, produced by Pb at low concentrations, may be an unifying hypothesis to explain Pb toxicity at the cellular level. Such perturbations may place the regulation of cellular processes out of the physiological range of normal control through changes in cytosolic free Ca concentration. The present study was undertaken to characterize more fully Ca:Pb ratios in different structural compartments of OC. Bone cells, enriched for OC, were prepared by a collagenase digestion of murine calvaria. After OC grew to confluence in 7 days, intracellular Ca:Pb ratios were calculated using kinetic analyses of dual label 45Ca and 210pb washout curves by desaturation techniques that employed a validated 3 compartment model. The ratio of Ca:Pb half-times and rate constants were <4 thereby indicating similar intracellular pathways for these two metals. However, the kinetic distribution of Ca and Pb in OC was not symmetrical; the Ca:Pb ratio for two rapidly exchanging compartments was 40:1; for the most slowly exchanging compartment, which includes mitochondrial Ca and Pb, the Ca:Pb ratio was 7:1. The Ca:Pb ratios for the steady state flux across the plasma membrane was 35:1; and the ratio was 30:1 across the mitochondrial membrane. These observations reveal both similarities and differences in the intracellular homeostasis of Ca and Pb in OC. These quantitative relationships should be considered for the evaluation of Pb effects on intracellular Ca homeostasis and Ca functions in OC, other cell types and in cell-free systems.

1249 CELLULAR METABOLISM OF LEAD: A KINETIC ANALYSIS IN CULTURED OSTEOCLASTIC BONE CELLS

Detailed characterization of the modulation of lead metabolism in bone is necessary to understand the role of skeletal lead in the expression of clinical and biochemical effects of lead. The metabolism of lead in osseous tissue is also important clinically because it is the major site of chelation by therapeutic agents, such as CaNa2EDTA and d-penicillamine. Experiments were conducted to characterize the steady-state kinetic distribution and behavior of 210pb, and to identify the biological structures or functions associated with the kinetic pools. Bone cells, derived from mouse calvaria, were enriched for osteoclasts by a sequential collagenase digestion and maintained in primary culture for 1 week. Cultures were labeled with 210pb as 5 μM lead acetate for 20 hr and the kinetic parameters were obtained by analysis of 210pb washout curves. Cellular metabolism was defined by three kinetic pools of intracellular lead containing ∼10% (Sl), ∼15% (S2) and ∼757. (S3) of total cellular lead (1.7-2.2 nmol/mg cell protein). The halftimes for isotopic exchange were 1, 40 and 1000 minutes, respectively. Less than one half of S1 was labile to chelation by EGTA and thus defined as extracellular. KCN, DNP and DBcAMP decreased S3, whereas increasing medium PO4 to 4 mM increased S3, thereby suggesting that S3 includes mitochondrial 210Pb. These data indicate that lead is readily mobilized from osteoclastic bone cells and, like soft tissues (hepatocytes), the bulk of cellular lead is associated with mitochondria.