serum protein, glucose, and ion levels or porphyrin profiles; and measuring increases in toxicant-inducible protein levels or activities. [8][9][10] This last group of molecular biomarkers, toxi-Stress proteins are produced as part of cellular responses to cant-inducible proteins, appears to have good potential as molecular damage by physical agents such as heat, chemicals biomarkers because of their direct links to cellular stress. including pharmaceuticals and environmental pollutants, They protect cells either by removing, binding or otherwise and pathogens including viruses, bacteria, and eukaryotic detoxifying toxicants before they cause damage or by reparasites. 1 Proteins known historically as heat shock proteins pairing or removing the toxicant-induced damage so that cel-(hsps) particularly members of the 70 kilodalton protein famlular function is not impaired. These cellular responses to ily are among the most intensively studied stress proteins.
toxicants can thus be separated into two major categories Heat shock genes have provided valuable models for tightly that can be used to classify biomarkers: 1) adaptive responses regulated systems of eukaryotic gene expression. Structural to prevent damage that are useful as biomarkers of exposure biologists and biochemists have now provided molecular to toxicants; or 2) responses to repair or remove toxicantstructures and detailed enzymology for several of these proinduced damage that are useful as biomarkers of effect. 11 teins. Cell biologists have begun to fit these proteins into Thus it is necessary to distinguish into which category a cellular metabolism and in the process, they have revealed stress protein response falls, so its potential use as a bioentirely new functions, the molecular chaperoning of proteins marker can be more accurately determined. 12,13 into folding pathways. 2 In contrast, the induced states of cel-
The following group of toxicant-inducible proteins, whose lular protection such as acquired thermotolerance that accellular functions are to remove, bind or detoxify potentially company the production of hsps, remain poorly characterized.
harmful toxicants or their by-products, appears to belong to However, sufficient interest has been generated to stimulate the first category of adaptive cellular responses. These proexploration of the roles of these responses in animal physiolteins include phase I and phase II conjugating enzymes and ogy and pathology. 3 their associated proteins, such as cytochrome P450 enzymes More recently, toxicologists have become interested in (mixed function oxidases), glutathione transferases, and these inducible cellular defenses and the stress proteins that UDP-glucouronosyltransferases that attempt to biotranscontribute to them. 4,5 What is the attraction of cellular stress form toxicants into forms that are more readily excreted or responses for this very practical discipline? Undoubtedly, it less toxic 9-14 ; metallothioneins and other metal-binding prois an interesting appreciation that these defenses can be trigteins that sequester some heavy metals 15,16 ; P-glycoproteins gered by macromolecular damage caused by chemicals in the and other proteins that function as cellular efflux pumps 17 ; cellular environment. For example, the heat shock response and antioxidant proteins such as superoxide dismutases, catis triggered by chemicals that share a capacity to damage alases, peroxidases, and glutathione-related enzymes that atcellular proteins, and the induced cellular defenses fretempt to prevent oxidative stress. 16-18 quently offer cross-protection against toxic concentrations of There have been a considerable number of studies pubstructurally unrelated chemicals. In my laboratory, we refer lished evaluating these toxicant-inducible proteins as molecto the heat shock response as the proteotoxic response, 6 the ular biomarkers, many of which have focused on cytochrome protein version of the more familiar consequences of DNA P450 proteins. 19 The consensus from these studies is that, damage known as genotoxicity and inducible DNA repair. In although the induction of cytochrome P450 proteins is a good the parlance of toxicology, protein damage is an adverse effect indicator of a biological response to specific classes of toxiof a toxicant that can be detected by cells and to which they cants, their induction has not been directly linked with respond by inducing stress proteins. Thus, measurements of adverse cellular and environmental effects caused by these hsps in cells are molecular biomarkers of effect. The retoxicants. The same appears to be true for the other toxicantsponding cells themselves are biosensors that can be configinducible proteins listed above. Thus these inducible proteins ured into in vitro testing formats for cytotoxicity assays. 7 are potentially useful as biomarkers of exposure that show Biological indicators or ''biomarkers'' are defined as ''bioonly the presence of toxicants rather than their adverse efchemical, physiological, or histological indicators of either fects. In contrast, at least one stress protein Hsp70 is linked exposure to, or effects of, xenobiotic chemicals at the suborby both its induction pathway and its function directly to ganismal or organismal level.'' 8 Many measurements of celluprotein damage, i.e., its presence in cells is a biomarker of lar molecules or their activities have been proposed or used as effect. How do cells detect protein damage? Recent studies biomarkers. These include, but are not limited to, measuring suggest that heat shock transcription factor (Hsf) is held in inhibition of enzymes such as acetylcholinesterases for studyan inactive form in cells as a complex with Hsc70, the Hsp70 ing pesticide exposures, or inhibition of aminolevulinic acid dehydratase activity as an indicator of lead exposure; de-family member normally present in cells. 20,21 When a toxicant termining energy levels and reserves by measuring mitochoncauses protein damage, Hsc70 is recruited to the damaged drial activity, adenosine triphosphate, glycogen or lipid levproteins to prevent them from aggregating irreversibly. The els, and cellular redox or oxygen:nitrogen ratios; blood equilibrium is shifted in favor of unbound Hsf, which then forms trimers capable of binding to the regulatory region of heat shock genes. The activated genes produce new messenger RNA that is quickly translated into hsps. Hsp70 aug-Abbreviations: hsps, heat shock proteins; Hsf, heat shock transcription factor.