Mobilization of cellular calcium-45 and lead-210: effect of physiological stimuli.

Isolated rat hepatocytes in primary culture were used as a model system to evaluate the effects of selected hormones and culture conditions on the efflux of calcium-45 and lead-210 from cells labeled with these isotopes. Alpha-adrenergic stimuli, angiotensin, vasopressin, dibutyryl adenosine 3',5'-monophosphate, and reduced phosphate concentrations in the medium increased the efflux of calcium-45 and lead-210. Glucagon and insulin had no effect, but increased phosphate concentrations decreased the efflux of both isotopes. Experiments with hepatocytes cultured in a medium free of calcium and lead demonstrated that the increased efflux of calcium-45 and lead-210 induced by hormones was the result of mobilization of the ions from intracellular stores. The data indicate that the physiological stimuli that mobilized calcium ions also mobilized lead ions, and that the mobilized lead would be available to interact with calcium-mediated cell functions.

Problems in determination of skeletal lead burden in archaeological samples: an example from the First African Baptist Church population.

Human bone lead content has been demonstrated to be related to socioeconomic status, occupation and other social and environmental correlates. Skeletal tissue samples from 135 individuals from an early nineteenth century Philadelphia cemetery (First African Baptist Church) were studied by electrothermal atomic absorption spectrometry and X-ray fluorescence for lead content. High bone lead levels led to investigation of possible diagenetic effects. These were investigated by several different approaches including distribution of lead within bone by X-ray fluorescence, histological preservation, soil lead concentration and acidity as well as location and depth of burial. Bone lead levels were very high in children, exceeding those of the adult population that were buried in the cemetery, and also those of present day adults. The antemortem age-related increase in bone lead, reported in other studies, was not evidenced in this population. Lead was evenly deposited in areas of taphonomic bone destruction. Synchrotron X-ray fluorescence studies revealed no consistent pattern of lead microdistribution within the bone. Our conclusions are that postmortem diagenesis of lead ion has penetrated these archaeological bones to a degree that makes their original bone lead content irretrievable by any known method. Increased bone porosity is most likely responsible for the very high levels of lead found in bones of newborns and children.

Dehydroretronecine-induced skin tumors in mice.

Female Swiss mice susceptible to skin tumors received 6 sc injections and/or topical applications of dehydroretronecine, a metabolite of the pyrrolizidine alkaloid monocrotaline, and were observed for 15 months for tumor development. Of 92 animals examined, 63 had tumors at the site of application or injection; 47 of these had skin tumors, mainly basal cell and squamous cell carcinomas. These data indicate that dehydroretronecine is a proximate carcinogen capable of causing a high incidence of skin tumours in mice at the site of sc injection or topical application.

The role of cell calcium in current approaches to toxicology.

All cells contain elaborate systems for the spatial and temporal regulation of the calcium ion, [Ca2+]i, and diverse Ca2+ receptor and biochemical response systems that are regulated by these changes in [Ca2+]i. Toxicants that perturb the mobilization or homeostasis of [Ca2+]i will place the regulation of these processes outside the normal range of physiological control. Many classes of chemical toxicants, including metals, solvents, and pesticides, may have particular aspects of cell calcium as key cellular and molecular targets of toxicant action. However, experimental proof of these targets as a specific site of toxicant action is challenging and technically difficult as a result of the complexity and diversity of these processes. To fully establish and understand the target role of the calcium messenger system in toxicant action, it is necessary to distinguish between the effects of a toxicant on (a) the calcium mobilization and homeostatic processes, (b) the calcium-mediated processes, and (c) from those processes which co-regulate or counter-regulate these calcium-mediated processes. As our understanding of the calcium messenger system expands, these insights will be increasingly applied to understanding the mechanisms of action of toxic chemicals.

A nonlinear isobologram model with Box-Cox transformation to both sides for chemical mixtures.

The linear logistical isobologram is a commonly used and powerful graphical and statistical tool for analyzing the combined effects of simple chemical mixtures. In this paper a nonlinear isobologram model is proposed to analyze the joint action of chemical mixtures for quantitative dose-response relationships. This nonlinear isobologram model incorporates two additional new parameters, Ymin and Ymax, to facilitate analysis of response data that are not constrained between 0 and 1, where parameters Ymin and Ymax represent the minimal and the maximal observed toxic response. This nonlinear isobologram model for binary mixtures can be expressed as [formula: see text] In addition, a Box-Cox transformation to both sides is introduced to improve the goodness of fit and to provide a more robust model for achieving homogeneity and normality of the residuals. Finally, a confidence band is proposed for selected isobols, e.g., the median effective dose, to facilitate graphical and statistical analysis of the isobologram. The versatility of this approach is demonstrated using published data describing the toxicity of the binary mixtures of citrinin and ochratoxin as well as a new experimental data from our laboratory for mixtures of mercury and cadmium.

The ICRP age-specific biokinetic model for lead: validations, empirical comparisons, and explorations.

The objective of this manuscript is to provide a description of the International Commission for Radiation Protection (ICRP) model and a comparison to other models (the integrated exposure uptake biokinetic [IEUBK] and O'Flaherty models), including the software used with the models, and a comparison of the model predictions for selected situations. The ICRP biokinetic model for Pb is a multicompartmental model for Pb uptake and disposition in children and in adults. The model describes deposition and retention of absorbed Pb in numerous tissues, removal from tissues to plasma, and movement along various routes of excretion. Long-term skeletal behavior of Pb is described in terms of age-specific rates of restructuring of compact and trabecular bone. The ICRP model is more flexible and has wider applicability than the IEUBK model. The major disadvantages are that application of the computer model requires some basic computer skills, and the user must convert the Pb concentrations in food, air, soil, dust, paint, or other media to the amount of Pb ingested or inhaled per day. Direct comparisons between the ICRP model and the IEUBK model are provided by modeling blood Pb levels using the IEUBK v0.99d default Pb uptakes and intake values. The model is used to simulate occupational exposure cases and a controlled Pb inhalation experiment in adult humans. Finally, use of the model to explore situations with limited data is illustrated by simulating the kinetics and disposition of Pb during acute Pb poisoning and chelation therapy in a child.

Cellular and molecular toxicity of lead in bone.

To fully understand the significance of bone as a target tissue of lead toxicity, as well as a reservoir of systemic lead, it is necessary to define the effects of lead on the cellular components of bone. Skeletal development and the regulation of skeletal mass are ultimately determined by the four different types of cells: osteoblasts, lining cells, osteoclasts, and osteocytes. These cells, which line and penetrate the mineralized matrix, are responsible for matrix formation, mineralization, and bone resorption, under the control of both systemic and local factors. Systemic components of regulation include parathyroid hormone, 1,25-dihydroxyvitamin D3, and calcitonin: local regulators include numerous cytokines and growth factors. Lead intoxication directly and indirectly alters many aspects of bone cell function. First, lead may indirectly alter bone cell function through changes in the circulating levels of those hormones, particularly 1,25-dihydroxyvitamin D3, which modulate bone cell function. These hormonal changes have been well established in clinical studies, although the functional significance remains to be established. Second, lead may directly alter bone cell function by perturbing the ability of bone cells to respond to hormonal regulation. For example, the 1,25-dihydroxyvitamin D3-stimulated synthesis of osteocalcin, a calcium-binding protein synthesized by osteoblastic bone cells, is inhibited by low levels of lead. Impaired osteocalcin production may inhibit new bone formation, as well as the functional coupling of osteoblasts and osteoclasts. Third, lead may impair the ability of cells to synthesize or secrete other components of the bone matrix, such as collagen or bone sialoproteins (osteopontin). Finally, lead may directly effect or substitute for calcium in the active sites of the calcium messenger system, resulting in loss of physiological regulation. The effects of lead on the recruitment and differentiation of bone cells remains to be established. Compartmental analysis indicates that the kinetic distribution and behavior of intracellular lead in osteoblasts and osteoclasts is similar to several other cell types. Many of the toxic effects of lead on bone cell function may be produced by perturbation of the calcium and cAMP messenger systems in these cells.

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.

Meso-2,3-dimercaptosuccinic acid induces calcium transients in cultured rhesus monkey kidney cells.

The maintenance of intracellular Ca2+ homeostasis is critical to many cellular functions that rely on the calcium ion as a messenger. While attempting to characterize the effects of lead on intracellular calcium levels ([Ca2+]i) in LLC-MK2 Rhesus Monkey kidney cells, we observed that treatment with the metal chelating drug, meso-2,3-dimer-captosuccinic acid (DMSA) evoked transient increases in [Ca2+]i. Changes in [Ca2+]i were monitored using the Ca2+ indicator dye Fura-2 and a dual wavelength fluorescence imaging system. In the presence of 2 mM extracellular Ca2+, DMSA treatment caused a concentration-dependent (15-500 microM) transient increase in [Ca2+]i returning to baseline levels within 30-60 s. Pharmacologic concentrations of DMSA (30 microM) stimulated a three-fold increase in [Ca2+]i, which was spatiotemporally comparable to Ca2+ transients induced by other calcium agonists. Depletion of inositol trisphosphate (IP3)-sensitive [Ca2+]i stores with the smooth endoplasmic reticulum calcium-ATPase (SERCA) inhibitor thapsigargin did not prevent DMSA-elicited increases in [Ca2+]i, suggesting that Ca2+ mobilized by DMSA was either extracellular or from an non-IP3 releasable Ca2+ pool. Treatment with glutathione, cysteine, or 2-mercaptoethanol caused similar but not identical calcium transients. Adenosine-5'-trisphosphate (ATP) also elicited transient increases in [Ca2+]i similar to those of DMSA. No transient increases in [Ca2+]i were elicited by DMSA or ATP in the absence of extracellular calcium. These data indicate that DMSA and other sulfhydryl compounds trigger an influx of extracellular calcium, suggesting a previously unobserved and unanticipated interaction between DMSA and the Ca2+ messenger system.

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.

Effect of lead intoxication on calcium homeostasis and calcium-mediated cell function: a review.

The interaction between lead and essential metals is a complex, well recognized, but poorly understood phenomenon. Dietary deficiencies or excesses of certain essential metals may alter the absorption, elimination, or dose response of lead. Lead, in turn, may alter the homeostasis and function of essential metals. This review will be limited to the effect of lead on calcium, an essential metal with elaborate systemic and cellular homeostatic mechanisms and innumerable second messenger and coupling-factor functions. Lead may ultimately perturb calcium-regulated or calcium-mediated functions (a) directly by interfering with calcium transport or storage processes, e.g., Ca2+ transport proteins, calcium gates, etc.; (b) indirectly by altering cell functions required for calcium homeostasis, e.g., energy production, plasma membrane permeability, etc.; (c) by substitution of Pb2+ for Ca2+ at functionally important calcium binding sites, e.g., calmodulin. While any individual study may not provide adequate experimental verification of the causal role of perturbations of calcium metabolism and function as the primary toxic lesion of lead intoxication, the collective observations of many studies do strongly support lead-calcium interactions as an important functional lesion in lead intoxicated organelles, cells, tissues, and organ systems.

Potentiation of dimethylnitrosamine genotoxicity in rat hepatocytes isolated following ethanol treatment in vivo.

Unscheduled DNA synthesis (UDS), following exposure to dimethylnitrosamine (DMN), was potentiated in cultured hepatocytes isolated following treatment of rats for 14 or 28 days with 20% ethanol/5% sucrose solution. Ethanol treatment was associated with increased UDS, a concomitant increase in hepatic microsomal protein concentration and DMN N-demethylase activity. Increased aniline hydroxylase activity of hepatic microsomes from ethanol-treated rats preceded the measured increase in microsomal protein content or DMN metabolism. The increase in metabolism of DMN in vitro and potentiation of DMN-induced UDS associated with ethanol treatment may contribute to a synergistic effect of ethanol on DMN hepatotoxicity and carcinogenicity. In contrast, ethanol pretreatment did not increase the cytotoxicity of DMN as characterized by enzyme release.

The potential laboratory health hazard of 210Pb and a simple procedure for separation of 210Pb from the daughters 210Bi and 210Po.

Lead 210 (Radium D) is a naturally occurring radionuclide which is frequently used in toxicological studies due to its long half-life. The use of 210Pb in tracer studies poses two problems. First, 210Pb, along with its daughters 210Bi and 210Po, presents a significant health hazard to laboratory personnel. Second, the presence of the daughter products may interfere with the detection of 210Pb, particularly by techniques which discriminate poorly between different radioactive emissions, e.g., autoradiography. The potential laboratory health hazards of 210Pb and its daughters are briefly reviewed and a simple dithiozone extraction procedure which allows quantitative separation of 210Pb frm the daughters 210Po and 210Bi is described. The purified 210Pb may then be utilized to reduce the health hazard from the daughter products and to construct calibration curves for the quantitation of 210Pb in the presence of 210Bi and 210Po by liquid scintillation counting.

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.

A method for rapid, sensitive quantitation of short-patch DNA repair in cultured rat hepatocytes.

The sensitivity of DNA-repair detection after incubation of cultured rat hepatocytes with dimethylnitrosamine (DMN) and [Me-1',2'-3H]thymidine ([3H]TdR) was enhanced by the preparation of isolated nuclei. Nuclear isolation-liquid scintillation counting (NI-LSC) provided a rapid method for the determination of unscheduled DNA synthesis (UDS). DMN-induced UDS detected by autoradiography and NI-LSC correlated well and provided similar dose-response curves, indicating the utility of the NI-LSC method for the quantitation of short-patch DNA repair.

X-ray fluorescence with synchrotron radiation.

The use of synchrotron radiation for X-ray fluorescence has several advantages over the use of other conventional X-ray sources. The principles of synchrotron radiation and methods for applying synchrotron radiation to the X-ray fluorescence measurements of trace elements are discussed. The Brookhaven National Laboratory X-ray microprobe, facilities dedicated to X-ray fluorescence, and related analytical techniques are discussed. Some examples of trace element analyses in biological materials with synchrotron radiation are presented.

Role of nuclear analytical probe techniques in biological trace-element research.

Many biomedical experiments require the qualitative and quantitative localization of trace elements with high sensitivity and good spatial resolution. The feasibility of measuring the chemical form of the elements, the time course of trace element metabolism, and conducting experiments in living biological systems are also important requirements for biological trace element research. Nuclear analytical techniques that employ ion or photon beams have grown in importance in the past decade and have led to several new experimental approaches. Some of the important features of these methods are reviewed here along with their role in trace element research. Examples of their use are given to illustrate potential for new research directions. It is emphasized that the effective application of these methods necessitates a closely integrated multidisciplinary scientific team.

An X-ray microprobe facility using synchrotron radiation.

An X-ray microprobe for trace elemental analysis at micrometer spatial resolutions, using synchrotron radiation (SR), is under development. The facility consists of two beamlines, one including a 1:1 focusing mirror and the other an 8:1 ellipsoidal mirror. At present, "white light" is used for excitation of the characteristic X-ray fluorescence lines. Sensitivities in thin biological samples are in the range of 2-20 fg in 100 microns2 areas in 5 min irradiation times. Scanning techniques, as well as microtomography and chemical speciation, are discussed. Application to a specific biomedical study is included.

Preparation and Characterization Challenges to Understanding Environmental and Biological Impacts of Nanoparticles.

Increasingly, it is recognized that understanding and predicting nanoparticle behavior is often limited by the degree to which the particles can be reliably produced and adequately characterized. Two examples that demonstrate how sample preparation methods and processing history may significantly impact particle behavior are: 1) an examination of cerium oxide (ceria) particles reported in the literature in relation to the biological responses observed and 2) observations related that influence synthesis and aging of ceria nanoparticles. Examining data from the literature for ceria nanoparticles suggests that thermal history is one factor that has a strong influence on biological impact. Thermal processing may alter many physicochemical properties of the particles, including density, crystal structure, and the presence of surface contamination. However, these properties may not be sufficiently recorded or reported to determine the ultimate source of an observed impact. A second example shows the types of difficulties that can be encountered in efforts to apply a well-studied synthesis route to producing well-defined particles for biological studies. These examples and others further highlight the importance of characterizing particles thoroughly and recording details of particle processing and history that too often are underreported.