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.


Introduction
Intracellular Ca2" homeostasis and the messenger role of the Ca2" ion in the regulation and control of cell functions have been a very active and productive area of basic biological research during the last decade. The processes that regulate intracellular Ca2' are extraordinarily intricate, numerous, and diverse. The Ca21 messenger system has a central role in mediating the contraction of all forms of muscle, the secretion of exocrine, endocrine, and neurocrine products, the regulation of glycogenolysis and gluconeogenesis, the transport and secretion of fluids and electrolytes, and the growth and division of cells. Based upon the central role of the Ca2+ messenger system in these aspects of cell function, it is logical, if not inevitable, to examine possible disturbances in Ca2' homeostasis and Ca2"-mediated functions as underlying mechanisms of toxicant action. The Ca2' messenger system may be considered as the integrated function of several constitutive systems (Fig. 1). The Ca2+ messenger system is described in detail elsewhere and is briefly presented in this overview (1)(2)(3)(4)(5)(6)(7)(8)(9). The  the second messengers that carry the message to mobilize Ca2' from the plasma membrane to the endoplasmic reticulum and other Ca2" stores. The Ca2" homeostatic processes are those Ca2+ gates, pumps, and channels that operate to keep the free cytosolic calcium ion, [Ca21]i, within normal physiological range. The Ca2' receptor system is a family of homologous calcium-binding proteins that act as transducers for relaying changes in [Ca21]i to the appropriate receptor enzymes that transduce the Ca2' signal to the biochemical response system. The Ca2' receptor system includes calmodulin, calcimedins, parvalbumins, troponin-C, and other Ca2'-binding proteins. Calmodulin, perhaps the most versatile Ca2+ receptor, activates a multi-functional Ca2' calmodulin-dependent protein kinase and Ca2' calmodulindependent protein phosphorylases, which elicit the wide range of biochemical and physiological responses. Other second messengers, such as cyclic nucleotides, also modulate or counter regulate the calcium signal and calcium response.
Three general types of calcium-mediated functions have evolved: cell movement, including muscle contraction, ameboid movement, and cytoskeletal functions; information processing, such as nerve impulse conduction and sensory mechanisms; and the actions of hormones, including growth factors, on cell growth and differentiation. All three of these general types of Ca2'-mediated functions may be important targets for toxicant action. These diverse functions are regulated and mediated by temporal and spatial changes

Ca2+-Mediated Processes As Targets for Toxicant Action
Experimental approaches to toxicology, toxicological concepts, and toxicologists are often classified according to their emphasis on target organ systems, chemical class of toxicants, and the level of biological organization used. The potential involvement of the calcium messenger system embraces all classifications of toxicology and toxicologists. These classifications are not inclusive or independent, but are a useful framework for considering toxicology data and concepts. The following discussion is not meant to be extensive but illustrates the pervasive and ubiquitous role of cell calcium in all aspects of toxicology (Tables  1 and 2).

Metals
Many metals may substitute for Ca2' or displace Ca2' from sites of Ca2' transport, binding, and storage. Lead, cadmium, mercury, and some other metals are transported across cell membranes by Ca2+ transporters and Ca2+ gates (10)(11)(12)(13)(14)(15)(16). Furthermore, the presence of these metals on these proteins affects Ca2+ movement at these sites (10,11,13,14). For example, the calcium channel in adrenal medullary cells is about ten times as permeable to Pb2+ as to Ca2', however, the channels do not inactivate when Pb2+ passes through them in contrast to Ca2+ (14). In addition, many of those normal hormonal signals that mobilize Ca21 to increase [Ca21]i also mobilize Pb2+, although the quantitative mobilization is not yet clear (17).  (18)(19)(20). This substitution could lead to the situation where the activity of these Ca2" receptor proteins is no longer regulated by [Ca2+]i, with potentially far-reaching perturbation of cell and organ function. This hypothesis is attractive and is supported by many observations, but is difficult to verify in vivo. Metals have high affinity for many other ligands, including the sulfhydryls, phosphate, and even other carboxyl groups. It remains to be established if toxic metals are bound to calmodulin and other Ca2'-binding proteins in situ in sufficient quantity to perturb the biochemical functions regulated by these proteins.

Pesticides
Numerous aspects of the calcium messenger system may be considered or are proposed as targets of pesticide action. Egg shell thinning in birds, caused by DDT, is an example of impaired calcium utilization. Other organochorine insecticides, including heptachlor and lindane, which is a structural analog of inositol, have been shown to alter Ca2' homeostasis. The changes in nerve terminal [Ca2"]i may be responsible for the neurotoxic action of these pesticides (21)(22)(23)(24).

Tumor Promoters
Phorbol esters and some other tumor promoters substitute for diacylglycerol in the activation of protein kinase C. Protein kinase C is a Ca2" and phospholipid-dependent enzyme that is activated by the natural activator, diacylglycerol, which occurs transiently during phosphatidylinositol turnover. This enzyme occurs in a wide range of tissues and phosphorylates a broad spectrum of proteins with a crucial role in signal transduction for activating cellular functions and proliferation. Protein kinase C also appears to be the receptor protein for tumor-promoting phorbol esters and many of the pleiotropic actions of tumor promoters, such as induction of ornithine decarboxylase, alkalinization of the cytoplasm, DNA synthesis, and expression of oncogenes may be mediated by the direct and unregulated activation of this enzyme by promoters (25,26).

Halogenated Hydrocarbons, Solvents, and Other Organic Toxicants
The acute toxicity of organic toxicants has been linked to altered Ca2" homeostasis through several mechanisms (27)(28)(29)(30). In particular, the exposure of hepatocytes to CCl4 and certain other halocarbons, carbon disulfide, or thioacetamide results in the rapid loss of Ca2'-ATPase activity and the ability of the endoplasmic reticulum to sequester Ca2` (31)(32)(33). Other toxicants such as cisplatin may increase renal Ca2'-ATPase activity (34).

Maintenance of normal [Ca2+]i homeostasis is also
dependent upon the status of other cell constituents, including sulfhydryl, glutathione, and nicotinamidenucleotide redox levels of the cytoplasm and mitochondria. Agents such as t-butyl hydroperoxide that cause oxidative stress, or agents which deplete cellular glutathione through conjugation reactions will perturb Ca2" fluxes and intracellular compartmentation, resulting in deregulation of cell function and even cell death (35).

Toxicological End Point
Oncology Tumor cells show various degrees of autonomy in that they do not react to the normal physiological and hormonal control over growth, cell proliferation, cell differentiation, and glycolysis and/or respiration. the calcium messenger system has important roles in the cellular and molecular process that regulate or modulate the expression of neoplasia. In contrast to normal cells, transformed cells continue to proliferate in suboptimal concentrations of extracellular Ca2" and appear to have reduced Ca2" requirements for other Ca2"-mediated functions. Altered calcium homeostasis and cAMP metabolism and the phosphatidylinositol cycles may be related to oncogene expression. The products of several viral oncogenes affect the signal transduction mechanism involving phosphatidylinositol turnover, protein kinase C activation, and intracellular Ca2" release (25). Future advances in tumor biology will include a more complete understanding of the differences in Ca2" homeostasis between normal and transformed cells, as well as the target role of cell calcium in the initiation and promotion processes.

Developmental Toxicology
Several calcium-mediated processes may be important cellular and molecular targets for toxicants in developing systems. intercellular adhesion is mediated by a variety of molecules and intercellular junctions. In the last several years a family of adhesion proteins called cadherins has been discovered that mediates a prominent Ca2'-dependent intercellular adhesion between cells in multicellular tissues. It is thought that specific recognition between like cadherin proteins mediates the sorting out of cell types during the cellular rearrangements and morphogenetic movements that occur during embryogenesis (36).
Gap junctions are other important cellular targets; these are dynamic cell structures that regulate communication and metabolic cooperation between adjacent cells. Gap junctions are also regulated, in part, by protein kinase C and other Ca2"-dependent kinases (37). Inhibition of gap junction-mediated communication may interfere with embryonic development (38). Furthermore, the secretion and action of many growth factors is regulated by [Ca2+]i so toxicants which modify the calcium messenger system could modify the availability and response of these hormones.

Cell Injury/Cell Death
The close temporal and biochemical relationship between the regulation of ions and cell injury and cell death has been of considerable interest for many years (27,39). Deregulation of [Ca2+]i appears to be especially important in the development of irreversible cell injury. Following a biochemical injury, cells pass through a sequence of morphological and biochemical stages. The early stages, which are charac-

Target Organ Nervous System
Calcium ions are well established as playing important roles in the signal transduction, neurotransmitter secretion, and axonal transport (40), and more recently in the biosynthesis neurotransmitters (41). At least three different calmodulin-dependent protein kinases with different substrate specificities are present in rat brain cytosol (42). Both the secretions and biosynthesis of monamine neurotransmitters stimulated by Ca2' influx and Ca21 mobilization may be regulated by the calmodulin-dependent protein kinase phosphorylation of microtubule proteins and the phosphorylation of the monooxygenases that are the rate-limiting enzymes in the biosynthesis of the neurotransmitters (41).

Immune System
Cell Ca is important in both cell and humoral immune responses. Phagocytosis and cell migration are dependent upon [Ca2+]i. Many of the processes responsible for lymphocyte activation, proliferation, and differentiation are initiated by antigen-receptor and growth-factor interactions that are mediated by the calcium messenger system. Signals induced by antigens or mitogens initiate responses including gene activation for the production of hormone-like lymphocytic growth factors and their specific receptors. The physiologic mitogenesis of lymphoscytes is then regulated by interactions of these specific growth factors with receptors that transmit the signals across the plasma membrane to induce a proliferative response. The integrated transduction of these complex signals is essential to lymphocyte immunobiology, and perturbations of the calcium messenger system would be expressed as an immunodeficiency of the host (43)(44)(45).

Cardiovascular System
Calcium couples excitation to contraction in all muscle cells; however, in smooth muscle, the relationship between tension and [Ca2+]i is not fixed but varies both with the nature of the stimulant and the duration of its application. At least three Ca2' channels have been identified including the voltage-sensitive Ca2' channel, stretch-activated Ca2' channel, receptor-operated channel, and a Ca2' leak channel in vascular smooth muscle. Contraction is mediated by Ca2' binding with troponin-C. Other important calciumbinding proteins with high homology to calmodulin include the calcimedins in smooth muscle that are, however, virtually absent from cardiac and smooth muscles. The precise function of calcimedins is not known. Ca2' has other roles in regulating including mediating response to adrenoreceptor activation, lipid metabolism, ion regulation, and the secretion of hormones that regulate cardiovascular function.

Renal System
The calcium messenger system is involved in many critical renal functions, mediating the osmotic, baroand hormonal regulation of fluid and electrolytes and renal ischemic injury, intermediary metabolism, vitamin D activation, and the glomerular filtration rate. Second, in the juxtaglomerular cells, [Ca2+]i is an inhibitory second messenger in the renin secretory process (46)(47)(48). Toxicant-induced alteration of renal function may also include effects on other organ systems, such as the cardiovascular system, thus altering the pharmacokinetics of the toxicant.

Functions
Because of the complexity and diversity of Ca2+homeostatic processes and Ca2'-mediated responses, it is useful to consider the many ways in which a toxicant may act on the Ca2' messenger system. Perturbations of Ca2' homeostasis and Ca2'-mediated cell functions following toxicant exposure may each result from at least three general types of interactions: direct, indirect, and secondary. These classifications are neither mutually exclusive or absolute. The direct interactions are the most significant with respect to the mechanistic or target role of the calcium messenger system. Rather, the purpose of this classification is to give some organization to an often overwhelming number of real and potential actions of toxicants on cell calcium. This classification should also assist in defining an experimental approach, the interpretation of observations, and the formulation of new hypotheses. This range of actions of a toxicant on cell calcium is illustrated with lead, a toxicant with diverse actions on the calcium messenger system (Table 3). It is important to note that a given toxicant may conceivably, if not probably, exhibit more than one type of action depending on the dose of the toxicant, duration of toxicant exposure, time-course of the experimental observation, and level of biological organization under investigation.

Toxicant Effects on Ca2" Cellular Homeostasis
Toxicants may directly alter Ca2' homeostasis by substituting for Ca2' at specific sites of Ca2" mobilization, transport, or storage. In this sense these actions are very loosely defined as competitive with Ca2" and generally reversible at the molecular level. Direct actions are most commonly observed with other divalent metals, including lead, ruthenium, cadmium, and others, which directly compete with, or displace Ca2' at transport sites in the plasma membrane and inner mitochondrial membrane (10,14,15,19). Although the direct action of these metals on Ca2" homeostasis at these sites is well documented, the explicit correlation between these effects and the manifestations of toxicity are confounded by the concurrent actions of these metals with other aspects of the Ca2' messenger system as well as cell functions unrelated to Ca2' homeostasis. Other examples include the direct actions of the calcium channel blockers, nifedipine or verapamil, on cardiovascular function by a blockade of calcium mobilization through membrane channels in myocardial and smooth muscle cells. It must be recognized aMost of these actions of lead on cell calcium have been well established in several in vitro studies using diverse biological systems. The difficulty is separating the direct from the less direct actions and experimentally characterizing those actions as key events in chronic lead toxicity in humans, such as altered cognitive function reproductive disorders and hypertension. that a direct interaction does not necessarily result in an inhibition of a Ca2'-mediated function but may also produce an exaggerated Ca2'-mediated response if the clearance of Ca2" from the cytosol and other cellular compartments is impaired (16,49).
Toxicants may indirectly act on specific cellular and molecular constituents of Ca2'-homeostatic processes, such as Ca2+ pumps or gates, but at molecular sites that are independent or remote of Ca2', not at the Ca2+ transport or binding site. Thus the interaction is non-competitive. Adding more calcium will not alter the biochemical lesion. This type of interaction is observed with many organic toxicants. The most fully characterized example of an indirect action is the inactivation of the Ca2' transporter in smooth endoplasmic reticulum by carbon tetrachloride that leads eventually to cell death (30,50). An indirect interaction may be irreversible at the molecular level; if the injury is sublethal, the cell and organism may be able to repair and recover-in this instance, with the biosynthesis of new endoplasmic reticulum and the associated Ca2' pump. Toxicants that dissipate the Na+ gradient will secondarily alter Ca2' homeostasis in cells where the Ca/Na exchange is a component of Ca2' homeostasis. Na+ transport is affected by numerous drugs, thiol status, and energy metabolism.
Indirect actions of lead on Ca2' homeostasis would include effects on adenylate cyclase and the Na+, K+-ATPase (51).
Toxicants may secondarily alter Ca2' homeostasis through non-specific effects on cell function that are biochemically and functionally remote from the processes of Ca2' transport and storage. For example, ethanol, other aliphatic alcohols, and local anesthetics including dibucaine or tetracaine produce changes in nerve cell excitability and neurotransmitter release that are the result of physical-chemical changes in the cell membrane; increased fatty chain motion within the membrane bilayer, expansion of membrane volume, increased membrane fluidity resulting in altered Ca2' permeability and transport properties of the membrane (52). Thus toxicants may alter Ca2' homeostasis secondarily to the primary site of action of the toxicant; and although the effects on Ca2+ homeostasis may be nonspecific, the effects on Ca'mediated processes may still be significant and mechanistically important.

Toxicant Effects on Ca2"-Mediated Celi
Functions Toxicants may directly act on Ca2+-mediated cell functions, including Ca2'-receptor proteins or their substrates. These actions may occur independently of altered Ca2' homeostasis. A well-characterized example is the inhibition of Ca-calmodulin-mediated processes by the phenothiazines that bind to the hydrophobic region of calmodulin and prevents the formation of a Ca-calmodulin-receptor enzyme complex thereby inhibiting Ca2"-mediated functions. Some pharmacologic and, presumably, adverse effects of these agents are a result of this interaction with calmodulin. However, this conclusion must be tempered by observations that some calmodulin antagonists also alter membrane currents of potassium and sodium; thus, not all effects can be attributed to inhibition of calmodulin (53). Lead, aluminum, cadmium, and other divalent metals effectively compete with Ca2' for binding sites on a variety of Ca2' binding proteins including calmodulin (18). Replacement of Toxicants may indirectly alter Ca2"-mediated cell functions independently of changes in Ca2' homeostasis, independent of direct interaction with the Ca2" receptor or effector proteins. The response to a Ca2" signal is a cascade of biochemical events that often include phosphorylation, and it is subject to feedback control by cyclic nucleotides and to sensitivity and response modulation. Because many cAMP-mediated responses are antagonistic to the Ca2"-mediated response, toxicants that effect adenylate cyclase, phosphodiesterase, or other parts of the cAMP messenger system will likely alter Ca2"-mediated cell functions. Examples of indirect effects include the inhibitory effects of xanthenes on phosphodiesterase leading to pharmacologic and toxic changes in muscle contraction. Although observed effects are clearly on Ca2'-dependent processes, it is important to recognize that the site of the primary biochemical lesion is not directly related to Ca2" homeostasis or the Ca2" effectors.
Toxicants may secondarily perturb Ca2'-dependent functions as a result of toxic events remote from Cadependent processes. That is, toxicants that grossly alter cell and organelle function may impair the ability of a cell to respond to a Ca2' signal, but as a result of toxic action which has no relationship to the Ca2' messenger system. For example, methyl mercury impairs neuronal migration and cell division, but the cellular target for these actions is the neurotubule and microtubules of the cells, rather than the Ca'mediated aspects of cell movement and division. Another example might be the inhibition of ornithine decarboxylase activity by cycloheximide. This enzyme is induced by hormones that mobilize diacylglycerol and calcium, thus activating protein kinase C. The action of cycloheximide, however, is not on any aspect of the calcium messenger system, but rather on translation of RNA. Thus, many, if not all, toxicants that alter cellular energetics, protein synthesis, etc., will eventually alter the ability of cells and tissues to maintain Ca2' response systems. However, these secondary effects on Ca2'-mediated function are more an expression of toxicity than the cause of toxicity. Considerable research effort must be directed at distinguishing between the mechanistically significant direct and indirect effects, and these mechanistically inconsequential (as it relates to the Ca2' messenger system) secondary effects.

Challenges to Establishing the Target Role of Cell Calcium in Toxicant Action
There are several difficulties in experimentally identifying and characterizing the role of the calcium messenger system as the principal or most sensitive molecular target for toxicant action. The most important of these difficulties is the complexity an diversity of the calcium messenger system itself. First, toxicants may act on many components of the calcium messenger system in direct and less direct ways. The temporal phases of Ca2' homeostasis and function may be affected independently, and the [Ca2+]i signal may not be directly proportional to the Ca2'-elicited function (6,7,55). Finally, the Ca2'-mediated processes are generally under coregulation or counterregulation by other messenger systems such as cyclic nucleotides. This complexity makes it difficult to determine if changes in the calcium messenger system are a cause or a consequence of toxicity.

Component Complexity of Signal Transduction and the Ca2+ Homeostatic
Processes Intracellular Ca2' homeostasis is an extraordinarily complex process that both integrates and requires the integration of innumerable processes occurring in all structural and functional components of cells (Fig. 2). Often it is necessary to experimentally dissect cells and tissues so that the individual components and processes of Ca2' mobilization and Ca2' homeostasis can be distinguished and manipulated experimentally. However, the more an experimental system is simplified, the less it will resemble the component and process at higher levels of biological control. Thus, incomplete information is obtained by investigating Ca2' transport systems, or Ca2'-mediated processes in an experimental system free of normal physiological controls, without attempting to demonstrate similar results at higher levels of biological organization. It  Ca2'-mediated processes are generally co-regulated or counter-regulated by the cAMP messenger system and the arachidonic acid cascade. This complexity of these messenger systems in constituent parts and the diversity in regulation often necessitates the investigation of a small portion or discrete components of the system, that is, isolated cells, organelles, membrane vesicles, or enzymes systems. It is important to understand the relationship of measurements made within the isolated system to the system as a whole, in order to design and interpret the appropriate experiments and to establish the target role of the calcium messenger system.

Temporal Complexity of the Calcium Messenger System
The most important point to consider for this discussion is that there are four distinct physiological phases of the calcium messenger system that must be considered as targets for toxicant action: basal or resting state, transition to the activated state, the fully activated state, and transition to the resting state. The time scale for these processes may typically range from less than a second to a few minutes, or even hours. Just as a single measure of cAMP is inadequate to define cAMP metabolism, so too are measurements of [Ca2+]i that do not consider the dynamic aspects of [Ca2+]i. Unfortunately, when the effects of toxicants are studied during a single phase, typically the basal or activation phase, rather than all four phases, and the results extrapolated to the full cycle real or potential effects on the other phases are ignored. Thus, important effects of a toxicant on Ca2h omeostasis and function may be missed completely, or the results may be misinterpreted (49,55).

Specific (Direct) vs. NonspecifiLc Effects
The experimental distinction must be made between direct and the nonspecific toxicant effects in order to establish the calcium messenger system as a principal target of toxicant action. Unlike investigations of, for example, genetic damage, the key or target molecule(s) is generally not known. Cellular Ca2' homeostasis is dependent upon the energy state and redox state of the cell; thus, alterations in energy metabolism would eventually result in perturbations of cellular Ca2' homeostasis, which may not be important in understanding the mechanism of action of these agents. This distinction between direct and nonspecific effects is generally more difficult to establish with the Ca2" messenger system than with many other cellular targets of toxicant action, such as DNA or receptors, because of the vast number and diversity of Ca2' homeostatic and Ca2'-mediated processes.

Postulates
Establishing cell calcium as a key target for the action of a toxicant is a complex and challenging process. Four general postulates or conditions for establishing the calcium messenger system as the critical molecular or cellular target are proposed.
First, perturbation of the calcium messenger system must be demonstrated in an experimental system that provides strict experimental control and manipulation of the calcium messenger system. This demonstration will often be at the cellular and subcellular level in vitro and will define the target cell and appropriate molecular targets to be investigated at higher levels of biological organization. Second, to be considered a key molecular or cellular target, these perturbations should precede in time and dose other signs of toxicity. This condition is to establish the specificity of the action for the calcium messenger system. Third, a toxicant must reach the molecular or cellular target process at comparable concentrations both in vivo and in vitro. That is to say the demonstration of an effect in an isolated system is of little mechanistic significance if the molecular dosimetry is not appropriate. And fourth, perturbation of Ca'mediated function should be experimentally associated with the biochemical/physiological manifestation of toxicity in vivo.

Summary
Because of the central role of the calcium messenger system in diverse functions of tissues, organs, and cells, Ca2' homeostasis and Ca2'-mediated processes may prove to be critical cellular and molecular targets for a diverse range of toxicants. However, experimental proof of these targets as a specific site of toxicant action is challenging and technically difficult because of the complexity and diversity of these processes. Nevertheless, the investigation of the Ca2' messenger system and Ca2'-mediated functions will continue to be an active and productive area of basic research for several years. These insights will be increasingly applied to the understanding of the mechanisms of action of toxic agents.