BELLE Newsletter Vol. 5, No. 1, June 1996

Toxicological defense mechanisms and how they may affect the nature of
dose-response relationships

Hart RW and Frame L
National Center for Toxicological Research, Jefferson, Arkansas, USA
Tel: 501-543-7000, Fax: 501-543-7332, E-mail rhart@nctr.fda.gov

I. Introduction and basic principles:
Historically, physiologically high doses of chemical agents have been used in toxicological studies to minimize cost and increase the probability of obtaining positive dose-response relationships. Often, lethality is measured since it is precise, quantal, and unequivocol, though this response may not always be relevant in the range of normal human exposure levels. To make information from standard bioassays more useful for risk assessment, mathematical models are often employed to extrapolate high-dose toxicity and carcinogenicity data to the low-dose situation (Kreski et al, 1991). In general, they do not take into account the diverse toxicological defense mechanisms that enhance survival in the face of minor environmental adversity. The significance of these strategies in the physiological range of environmental stressors is the emphasis of this review.

Since organisms are dependent on their immediate surroundings for basic needs, they must be able to gauge changes that take place in the external environment. Survival of the individual and species often depends on making the correct systemic adjustment (biochemical, physiological, or behavioral) when food is scarce, when noxious stimuli are encountered, or when injury occurs. Sensory proteins (in bacteria) or sensory systems (in higher organisms) receive a wide range of signals that help orient the organism properly to its surroundings. Low-level exposure/ sensitivity/susceptibility to noxious compounds and physical agents may be another important aspect of environmental monitoring and conditioning. In higher eukaryotes, most of the burden of risk assessment falls to cell-types that are highly specialized for surveillance and defense, but this normally poses little immediate risk to the organism as a whole. Cells of the immune system, for instance, are well-designed to act as "interpreters" of the outside world. They freely traverse the extracellular space, and have access to cell/environment interfaces as well as internal organs. They are responsive both to environmental stimuli and endogenous factors/hormones, have a high rate of proliferation and inducible DNA-repair, and readily undergo apoptosis in response to disruption of homeostasis or severe DNA damage (Janeway and Bottomly, 1994; Anderson, 1992; Buttke and Sandstrom, 1994). Every species has an arsenal of such defense mechanisms to cope with an anticipated range of noxious environmental stimuli, while evolutionary pressures continually reinforce or redefine the limits of that range (Poran et al, 1987; Guila, 1991).

Interestingly, low-level exposure to certain stressors can often lead to net benefits, including in some instances, decreases in age-associated disease or increased lifespan in laboratory models (Luckey, 1986; Sagan, 1987; Stebbing, 1987; Bauxenbaum, 1992;). A classic example of this may be dietary restriction, which while apparently inducing a stress response (Aly et al, 1994) does not induce any noticeable form of biochemical damage in rodent models (Haley-Zitlin and Richardson, 1994; Turturro and Hart, 1991). Understandably, there is much interest in determining the underlying mechanisms of this and similar responses since intervention strategies may be able to maximize potential health benefits for humans. It is our contention that, under certain conditions, physiological stress may stimulate endogenous defense mechanisms, and thus modulate many observed low-dose effects.

II. Toxicological defense mechanisms:
Organisms that are sensitive to environmental change and have the capacity to mount an appropriate and timely response have a selective advantage in evolution. For single-celled organisms, information about the environment is readily accessible, but defense options are relatively limited. The organization of cells into multicellular organisms afforded a more sophisticated defense strategy, but simultaneously created new challenges for dissemination of information and coordination of response. This may in part account for the differences that are observed between mechanisms of toxicity in mammals and simple organisms.

Table 1.Toxicological defense mechanisms operate on a number of levels to minimize the potential for exposure and/or permanent damage.

• 1. Exposure can be minimized by (or toxicity neutralized by):
  • a. cellular barriers
  • b. metabolism/detoxication
  • c. avoidance behavior

• 2. If damage has occurred, reproductive potential may be normalized by:
  • a. stereotypical biochemical changes that enhance short-term survival, including
    • i. stress-protein responses
    • ii. DNA repair mechanisms
    • iii. apoptosis (particularly as a response in the immune system)
    • iv. cell-cycle check points
  • b. biochemical/physiological/behavioral changes that lead to
    (long-term,stressor specific) adaptation
  • c. cell or tissue repair mechanisms that re-establish host integrity.

Table 2.Enzymes important in biotransformation of toxicants:

• Phase I:
  
  • cytochrome P-450
  • amine oxidase
  • epoxide hydrolase
  • esterases and amidases
  • alcohol, aldehyde, ketone oxidation-reduction systems

• Phase II:
  • glucuronosyltransferases
  • sulfotransferases
  • methylating enzymes (catechol-o-methyltransferase, N-methyltransferases, S methyltransferases)
  • N-acetyltransferses
  • amino acid conjugation enzymes
  • glutathione S-transferases

While prokaryotes and eukaryotes utilize many of the same mechanisms for preserving cell integrity in an oxidizing atmosphere, many higher organisms have evolved with the ability to harness the destructive potential of radical oxygen species for beneficial purposes. However, this is an imperfect balancing act. For instance, the production of superoxide radical by activated polymorphonuclear leukocytes and other phagocytes serves an important role by destroying and engulfing host pathogens. At the same time, this action may contribute to tissue damage associated with inflammation. Under normal conditions, superoxide production may stimulate cell division, but it may also contribute to malignancy under a different set of circumstances. Oxygen free radicals have been implicated in the development or exacerbation of many human diseases, including ischemia reperfusion injury in heart attacks, organ transplantation, stroke, cancer, emphysema, inflammatory immune and neurodegerative disorders, as well as aging (McCord, 1995; Spitzer, 1995). Very low-level oxidant stress, however, may be necessary for optimal function (James et al, 1991).

Another common approach for limiting exposure is avoidance. In single-celled organisms, this response is mediated by proteins that are directly linked to motile elements. However, in higher organisms, avoidance is a complex learned behavior. While it is beyond the scope of this article to discuss behavioral mechanisms, suffice it to say that there are many components, and the history of exposure is one of the more critical variables that impact on the dose-response. Depending on the particular stressor and the physiological state of the organism, prior exposure can either be sensitizing or desensitizing (Johnson et al, 1992; Basso et al, 1994).

Minimizing the potential for permanent damage:
Cell and DNA damage is not entirely avoidable, even under ideal conditions. Fortunately, there are a number of defense strategies that minimize the risk of permanent damage. Since these processes protect both germ and soma, they are highly conserved in evolution.

When environmental conditions change rapidly, the "stress-protein response" is often stimulated. On the cellular level, this response is fairly stereotypical in organisms as diverse as plants, bacteria, and mammals, and involves stimulation of DNA repair, cell-cycle changes, and programmed cell death to eliminate damaged cells (Smith-Sonneborn, 1992).

Stress responses:
The two major families of stress proteins are the heat-shock proteins (Hsps) which are induced when cells are exposed to nonphysiological heat, and the glucose-regulated stress proteins which are stimulated when cells are deprived of glucose or oxygen or when calcium homeostasis has been altered (Smith-Sonneborn, 1992). Heat-shock proteins, such as Hsp 70 and the ubiquitins, appear in response to minor DNA damage. They also appear in response to caloric restriction (Aly et al, 1994) without an increase in DNA damage (Haley-Zitlin and Richardson, 1994). The stress response is often associated with increases in specific proteases, metallothione, DNA repair enzymes, oncogenes, cell cycle mediators (p53), and chromatin changes. The topological changes in chromatin structure correlate with increased synthesis of poly (ADP-ribose), changes in histone methylation, and dependence on the presence of ubiquitin-histone conjugants. Changes in chromatin are assumed to facilitate stress induced gene expression and repression and/or provide access to DNA for repair. Abnormal proteins that arise as a consequence of environmental stress are able to catalyze the ATP-driven refolding which activates ubiquitin-mediated increases in protein degradation (Smith-Sonneborn, 1992).

In higher organisms, an integral part of the stress response involves stimulation of the brain hypothalamic-pituitary-adrenal axis (Pagliacci et al, 1993). Immune-derived factors, like interleukin-1, which are released in response to injury or inflammation, signal hypothalamic corticotropin-releasing hormone neurons to activate the pituitary-adrenal axis (thus counteracting the effects of inflammation); these events also set in motion a coordinated series of responses thought to be important for immunologic and behavioral homeostasis/adaptation (Castanon and Mormede, 1994). When cell-cell communication is perturbed, and adaptation fails, chronic stress may be a critical factor in the development of inflammatory disease, such as rheumatoid arthritis. Poor neurochemical adaptation in response to stress may also be the basis for certain affective disorders, such as major depression (Sternberg et al, 1992; Anisman and Zacharko, 1992; Johnson et al, 1992). The link between neurobehavioral and immune functions is also evident in the adaptation to low-level intermittent restraint stress which protects the heart from a wide spectrum of harmful factors (Meerson et al, 1992). Restraint stress is thought to attenuate the immunosuppressive response induced by novel aversive stimuli (Basso et al, 1994).

Because of this link between the neurochemical, immune, and behavioral systems, alterations in any one system will initiate adjustments in the others; this is how an overall adaptive "strategy" is thought to develop in response to environmental change. As previously hypothesized in a series of evolving papers, many of the beneficial aspects of adaptation to caloric restriction appear to operate through this mechanism. Under this scenario, food deprivation is hypothesized to induce stress in the absence of macromolecular damage. This triggers the hypothalamic-pituitary-adrenal axis and the series of events associated with adaptation to decreased caloric intake: changes in drug metabolism, increases in free-radical protective mechanisms, increases in DNA repair and fidelity, enhancement of cell replacement mechanisms (apoptosis) and enhancement of selective immune functions. When food again becomes available, and the stress is relieved, these systems are down-regulated (probably as a pleiotropic response to an internal signal) and reproduction is again enhanced (Turturro and Hart, 1991; Leakey et al, 1994; Aly et al, 1994; James and Muskhelishvili, 1994).

It follows that breeding practices which select for certain behavioral traits will often inadvertently impact on patterns of disease susceptibility (Newman, 1994). A relevant example for this particular discussion is the inbred rodent animal model. Bred for high reproductive capacity and fast growth, most rodents are well-adapted for the stresses of captivity. However, compared to their predecessors, they are maladaptive in the sense that they show increased susceptibility to the adverse effects of environmental and endogenous toxins (Rao et al, 1990). Interestingly, the high background rate of chronic disease in these animals can be reversed when the animals are calorically restricted (Hart et al, 1995). Again, caloric restriction appears to result in "caloric stress" (i.e. increased expression of stress proteins), which induces a physiological response characterized by both neurochemical and immune changes. These global physiological changes enhance a number of specific toxicological defense mechanisms, which may in part account for the decrease in degenerative disease. It has been suggested that glucocorticoids may play an important role in coordinating these responses (Leakey et al, 1994; Sabatino et al, 1991).

DNA repair:
DNA repair mechanisms are one aspect of the stress response, and represent the best characterized of the adaptive responses. This process is essential to protect DNA from the consequences of damage caused by internal or external agents, as well as errors that may occur during replication. The SOS system in Escherichia coli, for instance, is induced by UV-irradiation and a large number of DNA damaging chemicals (including alkylating agents). Any block to DNA replication caused by DNA damage produces a signal (thought to be an excess of single-stranded DNA) that induces an increase in specific gene transcription. There are at least 17 genes under the coordinate control of the recA and lexA genes in the SOS regulatory network. Collectively, they are involved in the repair of many types of DNA lesions.

Another DNA-repair system is induced specifically by exposure to alkylating (primarily methylating) agents, and is sometimes referred to simply as the "adaptive response". It was so coined because of the observation that low level exposure to methylating and ethylating agents improved the resistance of E.coli to subsequent (higher) challenge doses of these powerful mutagenic and toxic compounds. There are at least 4 genes (ada, alkA, alkB, and aidB) involved in the induction of this particular DNA repair system. Interestingly, the Ada protein acts both as a DNA repair enzyme (O6-meG-DNA methyltransferase I) and as a transcriptional activator of the (ada) gene. The enzyme first demethylates O6-meG, O4-methylthymine, and the S-diastereoisomer of methylphosphotriesters present in damaged DNA. Then, by transferring the methyl adducts from these lesions to the enzyme, the ada protein is converted to a strong transcriptional activator of the four inducible genes (Takahashi et al, 1988; Defais, 1985).

E. coli also has a low level of a constitutive O6-meG-DNA methyltransferase II activity (encoded by the ogt gene) which accounts for about 94% of uninduced activity. Since this repair enzyme is easily inactivated by self-methylation, its repair capacity is limited by the amount of enzyme which is immediately present. Consequently, this enzyme is only useful to counteract low levels of cellular alkylation damage. When cells are exposed to higher levels of exogenous alkylating agents, it is the Ada protein activity which is induced; subsequently, it becomes the major O6-meG-DNA methyltransferase activity (Defais, 1985; Nakabeppu et al, 1985).

Other enzymes that participate in DNA replication and repair (i.e.DNA-glycolases, DNA polymerases, topoisomerases etc) may also be stimulated as part of the adaptive response (Srivastava et al, 1993; Skov et al, 1994).

Cells of higher organisms also show the adaptive response. In 1984, Olivieri et.al. showed that lymphocytes had fewer chromatid aberrations induced by high doses of ionizing radiation if they were first exposed to a low dose of ionizing radiation. Subsequently, it was found that many toxic chemicals (bleomycin, hydrogen peroxide) produced similar effects in mammalian cells, or could substitute for either the high or low dose radiation challenge (Wolff et al, 1988; Dominguez et al, 1993; Vijayalaxmi, 1989; Zhou et al, 1993). While the molecular events have not been fully defined, induced DNA-repair has been proposed to account for much of the complexity of the dose-response relationship in the low-dose range (Laval, 1985; Joiner, 1994; Hall et al, 1988; Frankenberg-Schwager and Frankenberg, 1994; Lambin et al, 1994; Ruiz de Almodovar et al, 1994; Cai and Liu, 1992; Kelsey et al, 1991). The cytogenetic adaptive response is also evident in mammalian germ cells (Cai et al, 1993).

Despite success with in vitro modeling in tumor cell lines, results with primary human-derived cells have been highly variable, leading many to question the general significance of hormesis for the human population. (Shadley, 1994). Olivieri and Bosi (1989) have suggested that occasional failure to show adaptive responses in human cells may be a consequence of the physiological state of the cells at the time of the low-dose exposure. Since then, it has been found that endogenous levels of lymphokines at the time of exposure appear to correlate with the magnitude of the adaptive response (Shadley, 1994).

In the context of dose-response relationships, it is significant that individual DNA-repair pathways can be:

1.	influenced (qualitatively and quantitatively) by the history, timing, and conditions of exposure
2.	optimally induced at relatively low exposures to DNA-damage (where inducible)
3.	saturable and self-limiting, depending on the lesion and the enzyme
4.	moderately redundant with other DNA-repair pathways
5.	variable between species, cell-types, and cell cycle (Tang et al, 1994), and influenced by physiological state

These features are reflected in the dose-response relationships that relate to chemical carcinogenesis. Strongly signmoidal shapes are observed for dose-tumor incidence relationships in animal bioassays, supporting the idea that DNA repair mechanisms are effective in the low-dose range (Fig 1, as published in Lutz, 1991).

Fig 1.

From Lutz (1991) The relationship between carcinogen dose and DNA adduct formation (A) is usually described in terms of induction and saturation of bioactivation pathways. The influence of toxicological defense mechanisms, however, is also important. Within the sublinear phase, for example, saturation of inactivation mechanisms and saturation of DNA repair may occur. Within the superlinear phase, induction of DNA repair mechanisms and inactivation mechanisms are important. Collectively, these influences impact on the mutation rate (B), which shows a steep increase, concomitant with the stimulation of DNA replication and compensatory cell proliferation (often subsequent to inflammation). Collectively, the slopes of A plus B, as represented by the linear lines in fig 1C, can result in an exponential increase in tumor incidence, as represented in fig 1C.

A superlinear shape for DNA adduct formation, often seen at highest doses, are assumed to relate to enzymatic saturation or inactivation.

Apoptosis:
Apoptosis is another strategy for minimizing damage which is particularly important in the physiological range of exposure (Steller, 1995). As with some forms of DNA-repair, this process can be stimulated in some cell-types as a response to cellular stress. Unfortunately, this process has not been as well-studied in the context of toxicology because bioassays have traditionally focused on the high-dose range, where cellular necrosis is the common pathological feature. Since necrotic cells stimulate inflammation, much of the damage observed in bioassays can often be attributed to the host's own defense system. At doses that are actually relevant to human exposure levels, inflammatory processes may not be indicated. At low exposures, damaged cells may normally be eliminated by apoptosis (or "programmed cell death" ) (Collins and Lopez Rivas, 1993; Barr and Tomei, 1994; Kerr et al, 1994).

Apoptosis is a highly regulated and energy-consuming process which effectively removes damaged, effete, or preneoplastic cells from the cell population. Balanced against factors that regulate proliferation, apoptosis is also essential for keeping cell numbers constant in tissues. This process is not only important for defense, but for maintaining the social order (Kerr et al, 1994; Williams, 1991).

As may be expected, the presence of two modes of cell death (apoptosis and necrosis) in toxicity studies can contribute significantly to the complexity of the dose-response continuum. Each process has a distinctive time course, an optimal dose range, a unique set of morphological criteria, and very different functional consequences for the organism. Yet for the individual cell, the end-result is the same. Apoptotic cell death can be stimulated by small to moderate doses of ionizing radiation, cancer chemotherapeutic agents, hyperthermia, hormone withdrawal or addition, antibodies to the APO-1 or Fas antigen, cytotoxic lymphocyte action (Kerr et al, 1994), and caloric restriction (James and Muskhelishvili, 1994). In its role as a defense mechanism, this form of cell death represents a beneficial response, and it clearly operates within the range that is consistent with homeostasis. In other words, it can be considered both a sign of cell damage and a sign of a healthy cellular response. Apoptosis is particularly prevalent in the immune system where it performs a number of important functions, including thymic selection, cell-mediated cytotoxicity, and activation-induced death (Kerr et al, 1994). At the high doses generally used in toxicity assays, the failure of apoptosis may seem an insignificant aspect of the host response. However, for understanding low-dose effects and the etiology of environmental disease, the regulation, function, and failure of these (and other homeostatic) processes is a critical area of research.

Cell cycle check points and regulation:
DNA fidelity can be facilitated by alterations in cell cycle progression. For instance, there is good evidence that the tumor-suppressor gene p53 can participate in the repair of damaged DNA by blocking cells in the G1 phase of the cell cycle. This allows extra time for repair prior to division, and appears to be accompanied by increased production of free radical scavengers. This safeguards the organism and the genome. If irreparable DNA damage has occurred, however, the cell defaults to the apoptosis pathway and is deleted from the system (Levine et al, 1991).

The proto-oncogene c-Myc can induce both proliferation and apoptosis, depending on the presence of growth factors or other survival stimuli. Like p53, the response is cell type and stimulus specific. Other proteins are important in the regulation of the apoptotic mechanism, but more indirectly. The Bcl-2 protein and its family members, for instance, can repress apoptosis that is normally induced by p53, TNF, Fas/Apo1, or phorbol esters. Mechanistic studies indicate that Bcl-2 protein may function in an antioxidant pathway, although its exact mechanism is unknown (Williams and Smith, 1993; Collins and LopezRivas, 1993).

Cell damage can also initiate other adaptive strategies, such as compensatory cell proliferation, which further complicate the dose-response relationship (Fabrikant, 1987).

Physiological adaptation:
Species thrive and reproduce under a wide range of environmental conditions due to their ability to adapt. For instance, adaptative changes (or tolerance) occur when organisms are chronically exposed to acid (Foster, 1991), oxidative stress (Lu et al, 1993), food deprivation (Balam and Gurri, 1994), radiation (Macklis and Beresford, 1991), DNA-damaging agents, excessive heat (Baracos et al, 1987), stress damage (Prendergast and Taylor, 1994), physical exercise (Higuchi et al, 1985), and other sources of adversity. While these stressors appear to be very different, they all have the ability to threaten reproductive potential. Throughout evolution, the ability to normalize reproductive potential, in spite of environmental uncertainty, has provided some species a selective advantage. As previously mentioned, the stress response is important for dealing with immediate dangers to individual cells and the information content of living organisms. However, other (more stressor- and species-specific) adaptative changes often occur over the longer term. These processes ultimately alter behavior, reproduction, disease susceptibility, and lifespan.

Many aspects of cellular adaptation are counter-intuitive from anything but an evolutionary point of view. For instance, stressor-induced biochemical changes that correlate with early carcinogenesis may be transiently advantageous. As well, the physiological changes that accompany food deprivation may result in decreases in age-degenerative disease and increased lifespan. These processes (discussed below) buy time for the organism, permitting the individual (as the host of the germ cell) to survive periods of stress until reproduction becomes the more evolutionarily desirable option.

Adaptation in Cancer:
The multistep hypothesis of chemical carcinogenesis stipulates that a carcinogen (or its bioactivated metabolite) initiates a primary lesion (mutation, strand break etc.). Cells that are able to sustain this lesion arise from a phenotypically diverse initial population (Ogawa et al, 1980; Baron et al, 1986), and under the appropriate conditions, proliferate and expand into focal lesions. In the liver, the most widely studied model of chemical carcinogenesis, these focal lesions can develop further into hepatocellular nodules. One of the essential characteristics of primary lesions appears to involve the initiated cells acquiring resistance to the cytotoxic actions of carcinogens and hepatotoxins. This resistance is essential for the development of nodules; by overriding the mitoinhibitory effects of the carcinogen, the new phenotype allows the initiated cells to respond to mitogenic stimuli (Farber and Cameron, 1980).

Alterations of drug-metabolizing enzyme activities in experimentally-induced rat hepatocyte nodules suggest that only certain of these activities may contribute to the resistant phenotype. Certain cytochrome P-450 dependent monooxygenase activities are severely reduced (Tsuda et al, 1980; Cameron et al, 1976), whereas many of the Phase 2 enzyme activities are significantly increased. For example, UGT activity toward 1-naphthol is increased 5-fold in hepatocyte nodules compared to control tissue, but other isozyme-specific activies (e.g. bilirubin and 4-hydroxybiphenyl glucuronidation) are unchanged or only slightly elevated (Bock et al, 1982; 1990). The rat Pi class form of glutathione S transferase is strongly expressed in hepatic foci and hepatomas and also in initiated cells (Tsuchida and Sato, 1992). Other metabolizing enzymes that appear to be differentially expressed during chemical carcinogenesis include epoxide hydrolase (Kuhlmann et al, 1981), DT-diaphorase (Pickett et al, 1984), class 3 aldehyde dehydrogenase (Lindahl, 1992), and aryl sulfotransferase IV (Yerokun et al, 1994) . Though there is considerable variability in the pattern of phenotypic expression between cancer models, the source of selection pressure probably varies widely depending on the initiating agent and the tissue. It has been suggested that the development "preneoplastic" changes represents a normal aspect of carcinogen adaptation (Anilkumar et al, 1995; Farber and Cameron, 1980; Farber, 1992,1993). Increased cell survival as a consequence of transformation has also been documented using in vitro culture techniques (Boothman, 1994).

With chronic exposure to toxic compounds, many cell-types also have the capacity to alter their rates of uptake, excretion, or permeability as a means of lowering the dose to critical cell components. This adaptive response is observed after chronic exposure to chemical carcinogens and chemotherapeutic agents, and often leads to decreased toxicity (effectiveness) over time. This process is also thought to contribute to the multidrug resistance phenotype (Simon and Schindler, 1994).

Most of these cell changes that occur with carcinogen exposure are reversible. In other words, when the stress is removed, the cell reverts back to its original phenotype. Even fully-transformed (genotypically-altered) cells, however, may have the potential to revert back to their original (non transformed) phenotypes by a process known as "adaptive", "directed", or "selection-induced" mutation. When populations of microorganisms or NIH 3T3 cells are subjected to certain nonlethal selections, for example, only "useful" mutants arise among the nongrowing cells, and "useless" mutants do not (Foster, 1994) . The model that most readily explains the evidence is that cells under stress produce genetic variants continuously and at random, but these variants are immortalized as mutations only if they are permissive for cell growth (Foster, 1994). While controversial, a role for adaptive mutation in chronic disease processes of higher organisms is beginning to gain experimental support (Chuang et al, 1993; Ruiz de Almodovar et al, 1994).

At present, the significance of carcinogen adaptation is unknown for the etiology of the disease. However, there is an immediate relevance for the dose-response relationship: a history of low exposure (to carcinogens or other toxic agents) can be transiently advantageous for the organism if further exposure is inevitable. Adaptations notwithstanding, carcinogens do increase the likelihood of cancer over the longer timecourse, but the dose may vary depending on the history and consequences of prior exposure. Therefore, for the time being, cancer susceptibility should be considered in the context of multiple possible causes which may be additive.

Adaptation during food deprivation:
To overcome the problem of long-term or seasonal adversity, more global physiological changes are required to normalize reproductive potential. Some species are able to lower body temperature and basal rates of metabolism (as in hibernation or torpor). Delay of sexual maturation, with a delay in degenerative disease, appears to be another adaptive mechanism, and has validation in several animal models.

A regimen of 30% caloric restriction in rats, for instance, significantly increases the time to onset of sexual maturity, the length of the reproductive period, and the average lifespan. Furthermore, in the absence of malnutrition in general, the lower the body weight the greater is the ability of an animal to cope with chemical or agent exposure (Hart et al, 1995). Though the mechanism of action is unknown, this form of adaptation can have a significant impact on the dose-response relationship which is so critical to regulatory decision-making. At doses of stress that result in a decrease in body weight, significant decreases in age-degenerative disease can often be observed. Consequently, a common observation at the end of the chronic bioassay is increased survival in test animals compared to their (larger) controls. Failure to account for body-weight differences in toxicity testing can significantly increase variability, and in some cases can provide misleading information. Recently, dietary control has been proposed as a means to improve the bioassay and strengthen the regulatory utility of its results (Turturro and Hart, 1994).

Cell and Tissue Repair:
Many toxicological defense mechanisms mentioned above impact on cell and tissue repair mechanisms and consequently shape the dose-response continuum. In particular, Mehendale and colleagues have shown a pivotal role for hepatocellular division, adaptation, and tissue healing processes in the mechanism of autoprotection against many toxicants. Interactive toxicity of chemicals can be greatly amplified, for instance, if the mechanism of action involves interference with the timing or effectiveness of these essential processes (Mehendale, 1992, 1994; Mangipudi et al, 1995).

III. Response vs responsiveness
Having outlined the types of responses that are important in host defense, it may be useful to distinguish the concept of "host response" from "host responsiveness". While these terms are obviously related, "responsiveness" may be a more appropriate term with which to discuss some of the more intriguing aspects of low-dose exposure.

The physiological state of an organism affects how responsive it is to environmental input; in turn, environmental components are important factors for setting up the physiological state. While teleologically, this cross-talk between the organism and the environment makes sense, it is not well understood on the mechanistic level. Nevertheless, "responsiveness" should be in the forefront of variables to consider in the dose-response relationship. The term "responsiveness" implies that there may be an optimal level of stimulation, and possibly even a desired level of stimulation of toxicological defense mechanisms. Furthermore, unlike the term "response", it is not tethered to the expectations of a single causative agent. Therefore it may serve as a unifying concept for further discussion of biochemical mechanisms. Numerous host factors (including the history of exposure) can affect the responsiveness of an organism, but may bear little similarity to the spectrum of effects that occur with higher doses.

In that context, it is particularly interesting to consider the diversity of agents/conditions that cause toxicity at high doses but show net "beneficial effects" at very low doses. So-called "hormetic agents" include heavy metals, polychlorinated biphenyls, insecticides, alcohol, oxygen poisoning, cyanide, antibiotics, ionizing radiation, cosmic or gamma radiation, electromagnetic radiation, ultraviolet plus photoreactivation (Smith-Sonneborn, 1992) and caloric restriction (James and Muskhelishvili, 1994). The mechanistic challenge is to link this diversity with the stereotypical responses of the cell, and still consider the larger driving forces of the organism in its environment.

IV. Common mechanisms:
Smith-Sonneborn(1992) previously reported on the similarity, and possible connection, between the actions of hormetic agents and features of the stress response. Such mechanistic explanations have traditionally focused on "downstream" events, in an effort to analyze the link between the stress and the biological effect. For instance, it has been suggested that "hormetic" benefits may be derived as part of an "overcorrection" response, since "step" increases are associated with induction and signal amplification processes. Alternatively (or in addition), net benefits may be a consequence of improved repair of age-associated cell damage, or improved resistance to other environmental toxins that operate through a common mechanism.

Other mechanistic studies have focused on "upstream" events, to help resolve how agents as diverse as radiation and caloric restriction can initiate similar responses. A number of investigators feel that oxidative stress may play an important role in determination of cell fate, since agents that cause redox imbalances are also able to initiate the chain of events leading to apoptosis in immune cells (Buttke and Sandstrom, 1994). Acting through stimulation of the stress response, low-level oxidant exposure can stimulate signal transduction , compensatory cell proliferation, antioxidant defense, and DNA repair. Acting directly on the membrane components, oxygen radicals can also sensitize receptors to specific effectors (Kuzin et al, 1991).

Laboratory evidence from a number of overlapping disciplines now appear to converge on a proposed role for enhanced immune functioning as a response to low-level stress. James and Makinodan (1988), for instance, found that prolonged exposure of mice to low doses of radiation and/or caloric restriction results in augmentation of the proliferative response of effector T cells. Low dose radiation may also inhibit suppressor T cells (Kondo, 1988). The significantly lower spontaneous liver tumor rates in calorically restricted animals also correlate well with higher basal rates of apoptosis (James and Muskhelishvili, 1994).

Focusing on the various mechanisms by which cell lesions are eliminated in higher organisms (DNA repair, apoptosis, and immune response), Yakovlev et al (1993) recently developed a "stochastic" model of hormesis. The model provides a format to interpret experimental findings, and has successfully been used to analyze published data on the hormetic effects of prolonged irradiation and of procaine on animal longevity.

V. Guidelines for modeling low-dose effects:
The shape(s) of dose-response curves that demonstrate hormesis have been reviewed by Calabrese and others (Calabrese and Baldwin, 1993). A feature they share is complexity (biphasic or multiphasic) in the low-dose range, and responses that are highly dependent on the history of exposure and rate of dosing (Ruiz de Almodovar et al, 1994). All of this is consistent with the idea that responses in the low dose range are homeostatic in nature. In order to understand defense strategies that operate in the low dose range, however, it is useful to have multiple model systems for contrast and comparison. In toxicological models that are mechanism-based, it is important to consider the following:

Multiple processes occur concurrently.The "threshold dose", observed in whole animal studies, is often described pictorially rather than mechanistically: "the dose below which there is no effect." This is unfortunate. The subthreshold and threshold dose range is relevant for most human exposure situations, and many toxicological defense mechanisms operate optimally in this range. Furthermore, a number of chronic disease processes (including cancer and aging) appear to be associated with significant changes in the expression or effectiveness of homeostatic mechanisms.

When repair and replacement mechanisms are able to keep pace with the damage and death of cells, it is true that there may be very little net change in certain responses. However, multiple and opposing processes are balanced to achieve this steady state, and "beneficial" effects will always coincide with "detrimental" effects (Mehendale, 1992). For instance, in a population of eukaryotic cells continuously exposed to a moderate dose of a noxious compound, induction of DNA-repair, apoptosis, and necrosis may occur simultaneously. While increases in DNA repair and apoptosis may be a sign that mild cellular damage has occurred, they also have to be considered signs of a healthy cell response. Necrosis, on the other hand, is usually indicative of high exposure, more severe damage (or more susceptible cells), and stressor-induced loss of major cell functions that cannot be efficiently repaired on the local-cell level. From a mechanistic standpoint, therefore, simple measurement of cell number may not be particularly informative; apoptosis and necrosis both lead to transient reductions in cell number, and yet each has a distinctive time course and optimal range. More importantly, they appear to have different functional consequences in the intact organism. Regulation of apoptosis is handled primarily on the local tissue level, and represents an efficient means of eliminating delinquent, damaged, or aging cells. Necrosis, on the other hand, is not regulated. However, in the aftermath of exposure, the products of necrotic cells will stimulate other processes which are regulated, including inflammation, DNA repair, and/or cell replacement. The occurrence of multiple processes dictates a need for precise cell phenotyping and temporal monitoring in order to adequately interpret multiple consequences of (particularly) low-dose exposure. As well, relevant in vitro and mathematical models are needed to better understand the superpositioning of multiple dose-responses which characterize survival curves in eukaryotic cells.

Zukhbaya and Smirnova (1991) have developed a mathematical model of lymphopoiesis which has predictive value for the shape of the dose-response curve, and illustrates the concepts above (Fig 2). The model is based upon several biologically- plausible assumptions about cellular responses in complex tissues, including: a) heterogeneous cell populations, b) differential susceptibility, c) different degrees of damage leading to different cellular consequences, d) different rates of cell proliferation between cell types, e) developmental transitions and migration between compartments, and f) adaptive mechanisms that, in time, re-establish a steady-state.

Fig 2.

From Zukhbaya and Smirnova (1991) Health Physics 61:87-95. Lymphopoiesis dynamics under continuous irradiation at a dose N=0.4 Gy d -1. Experimental data are mean values and standard deviations of unitless concentrations of the lymphoid cells in blood (A) and in bone marrow (B) of rats. In panel A, the numerical calculations are presented by curves describing the unitless concentrations of proliferating cells (I), non proliferating cells (II) and mature lymphocytes (III). In panel B: the dimensionless total concentration of proliferating plus non-proliferating cells, and, in panel C, the mitotic index M (%) of the proliferating cells.

Not all processes are induced directly by the environmental agent. Compensatory cell proliferation, for instance, appears to be induced primarily by cell-derived factors (Ward, 1994). In other words, this process is stimulated as a consequence of cell damage, as opposed to exposure per se. Particularly in complex organisms, therefore, toxicological defense strategies should be considered a sequence of events.

Enzyme induction, hormone (paracrine or pheremone) stimulation, apoptosis, inflammation, cell proliferation, and cell differentiation are regulated processes that take a prescribed length of time, and "complicate" the dose-response relationship. In higher organisms, the distance between cells and the time-to-response are important variables to consider. Not surprinsingly, multiphasic dose-response relationships are not uncommon, as defense processes proceed through various phases. Despite some flexibility in the regulated response, there may be physiological factors which are more or less desirable for optimal functioning. Increased body weight or disease, for example, may be factors which interfere with the cell communication necessary to enforce interdependence between specialized cell types.

Each species may be optimally responsive and/or susceptible to a different set of environmental stimuli. A fundamental tenet of evolutionary theory states that every species occupies a unique environmental niche. Each species is therefore responsive to a unique and limited set of possible stressors. Since sources of stress are not universal, defense systems reflect both the evolutionary history of a species and reigning evnvironmental conditions. In other words, to some extent, defense mechanisms may be "made-to-order". They may be inducible, expressed constitutively at a high rate, or missing altogether and still be appropriate.

Within species as well, there may be considerable interindividual variability, depending on numerous environmental or host-specific factors (i.e. age, sex, diet, disease, etc). In describing dose-response relationships in a highly diverse population, most of the variability is generally assumed to derive from genetic differences. However, the inbred rodent model has highlighted the critical role of other factors, particularly dietary intake, on the susceptibility to degenerative and environmental disease. In toxicological studies, failure to control or stabilize body weight, for instance, may cause a significant increase in background pathologies and (in many cases) the risk of toxicity. Thus, the impact of body weight differences on the induction of chemical or agent toxicity can be as significant as the test agent dose (Hart et al, 1995 ; Turturro and Hart, 1994). For the human population as well, lifestyle choices are thought to lead the list of risk factors for the development of cancer (Ames et al, 1995).

Within the human population, many drug-metabolizing enzyme families express polymorphisms, which are thought to correlate with susceptibility to acute toxicity or environmental disease (Vineis and Ronco, 1992; Ketterer et al, 1992).

Individual cell types may be optimally responsive and/or susceptible to a different set of environmental stimuli. Environmental monitoring and defense strategies in higher organisms are the responsibility of individual specialized cell types. Because of this organization, cells of higher eukaryotes may be differentially responsive and/or susceptible to damage. Susceptibility may also depend on the stage of the cell cycle or the proliferative rate (Tang et al, 1994; Gorczyca et al, 1993) of the tissue. While standard biochemical assays may be useful for characterizing the metabolic competence of an organ system, they generally lack the specificity necessary to characterize subtle changes in cell phenotype or tissue organization that may occur subsequent to toxin exposure.

Both drug-metabolizing enzymes (Baron et al, 1986) and stress-proteins (Vamvakopoulus, 1993), for instance, are differentially expressed and induced in multiple tissue-types. In some cases (as in the UDP-glucuronosyltransferase UGT1 family) there are independent 5'-upstream regulatory regions and transcriptional initiation sites; because of this organization, expression levels and regulation can differentiate further within the same organism to accommodate local tissue requirements and environmental challenges (Munzel et al, 1994).

One fact that is rarely taken into account in the design and interpretation of in vitro models, is the long term influence a cell may have in modulating its own extracellular microenvironment and vice versa. Cells which are organized in tissues usually share (sometimes unequally) in this responsibility, but the strategy pays off in relative stability for the organism. While toxicity testing of individual cell types in culture may give some indication of potential susceptibility, it does not adequately model this in vivo dependence. Despite these limitations (and largely because of them), in vitro methods can be very useful for characterizing cell-mediated detoxication processes that operate in the the subthreshold range. By restricting the focus to single tissues or cell types, however, only a portion of the homeostatic mechanism may be characterized, and this should be taken into account in interpreting in vitro-derived data.

When cells are taken from an organism and then transferred to short-term cell culture, the in vivo history of those cells is often sustained as a significant factor in the response. For example, the dietary history of an animal is often reflected in the rate of in vitro cell transformation, oncogene expression, or DNA repair (Hass et al, 1993). As well, senescent cells may show decreased responsiveness to environmental challenges (Wang, 1995).

Toxicological defense mechanisms may themselves be targets for toxicity.Triggering of normal toxicological defense mechanisms may be one of the more sensitive indicators of exposure to a noxious agent. Due to their placement and function within the host organism, cells that specialize in surveillance and/or defense functions are generally more responsive to environmental agents in the low-dose range. However, as with other functions of the organism, these defense mechanisms may be inactivated or saturated at higher doses, or lose effectiveness with age. Catalase and superoxide dismutase, for instance, are inducible by low level ozone, but capable of inactivation at high doses (Whiteside and Hassan, 1987). The tissue repair response in liver, also inducible at low doses, can be delayed and attenuated at higher doses, leading to a threshold effect for toxicity (Mangipudy et al, 1995).

VI. Conclusions:
The concept of hormesis is both biologically and socially complex. It is reasonable to assume that organisms able to cope with intermittent stress would have a reproductive advantage over those unable to do so. The question is whether low-dose exposure to environmental stress improves the responsiveness of the organism, and whether this, in some cases, translates to increased survival. On the surface, this would appear unreasonable; however, a number of carefully documented studies suggest exactly this.

From a molecular or biochemical perspective, the induction of protective mechanisms (e.g. DNA repair, apoptosis etc.) may provide one explanation for the dose-response relationship. Many biochemical processes (particularly those involved in cell-cell communication) are induced in a step fashion, as a consequence of signal amplification. In some instances, therefore, the ability of the organism to deal with the stress might theoretically be greater than the damage induced by the stress. When the stressor initiates very little direct damage, or enhances mechanisms for selective recognition of damage (as it does in the immune system), then the stressor might be deemed as beneficial to survival and protection of the genome. As the potential of the stressor to induce harm increases and the ability of the organism to respond decreases, linearity would ensue. Whether this does or does not serve as a mechanism for hormesis is yet to be determined experimentally. The difficulty in doing so is as it has always been- the dose range within which such studies must be conducted. Supportive of this concept, however, are the extensive studies reported recently on the response of higher organisms to "caloric stress" (ILSIE). If, as suggested in this and previous papers (Turturro and Hart, 1991; 1994) caloric restriction does induce such an all-encompassing organismic stress response in the absence of macromolecular/biochemical/metabolic/cellular and organ system damge, this mechanism may also provide a reasonable explanation of how organisms protect themselves in the face of a hostile environment until such times that the opportunity for reproduction is optimized.

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