Mechanistic toxicology is the study of how chemical or physical agents interact with living organisms to cause toxicity. Knowledge of the mechanism of toxicity of a substance enhances the ability to prevent toxicity and design more desirable chemicals; it constitutes the basis for therapy upon overexposure, and frequently enables a further understanding of fundamental biological processes. For purposes of this Encyclopaedia the emphasis will be placed on animals to predict human toxicity. Different areas of toxicology include mechanistic, descriptive, regulatory, forensic and environmental toxicology (Klaassen, Amdur and Doull 1991). All of these benefit from understanding the fundamental mechanisms of toxicity.
Why Understand Mechanisms of Toxicity?
Understanding the mechanism by which a substance causes toxicity enhances different areas of toxicology in different ways. Mechanistic understanding helps the governmental regulator to establish legally binding safe limits for human exposure. It helps toxicologists in recommending courses of action regarding clean-up or remediation of contaminated sites and, along with physical and chemical properties of the substance or mixture, can be used to select the degree of protective equipment required. Mechanistic knowledge is also useful in forming the basis for therapy and the design of new drugs for treatment of human disease. For the forensic toxicologist the mechanism of toxicity often provides insight as to how a chemical or physical agent can cause death or incapacitation.
If the mechanism of toxicity is understood, descriptive toxicology becomes useful in predicting the toxic effects of related chemicals. It is important to understand, however, that a lack of mechanistic information does not deter health professionals from protecting human health. Prudent decisions based on animal studies and human experience are used to establish safe exposure levels. Traditionally, a margin of safety was established by using the “no adverse effect level” or a “lowest adverse effect level” from animal studies (using repeated-exposure designs) and dividing that level by a factor of 100 for occupational exposure or 1,000 for other human environmental exposure. The success of this process is evident from the few incidents of adverse health effects attributed to chemical exposure in workers where appropriate exposure limits had been set and adhered to in the past. In addition, the human lifespan continues to increase, as does the quality of life. Overall the use of toxicity data has led to effective regulatory and voluntary control. Detailed knowledge of toxic mechanisms will enhance the predictability of newer risk models currently being developed and will result in continuous improvement.
Understanding environmental mechanisms is complex and presumes a knowledge of ecosystem disruption and homeostasis (balance). While not discussed in this article, an enhanced understanding of toxic mechanisms and their ultimate consequences in an ecosystem would help scientists to make prudent decisions regarding the handling of municipal and industrial waste material. Waste management is a growing area of research and will continue to be very important in the future.
Techniques for Studying Mechanisms of Toxicity
The majority of mechanistic studies start with a descriptive toxicological study in animals or clinical observations in humans. Ideally, animal studies include careful behavioural and clinical observations, careful biochemical examination of elements of the blood and urine for signs of adverse function of major biological systems in the body, and a post-mortem evaluation of all organ systems by microscopic examination to check for injury (see OECD test guidelines; EC directives on chemical evaluation; US EPA test rules; Japan chemicals regulations). This is analogous to a thorough human physical examination that would take place in a hospital over a two- to three-day time period except for the post-mortem examination.
Understanding mechanisms of toxicity is the art and science of observation, creativity in the selection of techniques to test various hypotheses, and innovative integration of signs and symptoms into a causal relationship. Mechanistic studies start with exposure, follow the time-related distribution and fate in the body (pharmacokinetics), and measure the resulting toxic effect at some level of the system and at some dose level. Different substances can act at different levels of the biological system in causing toxicity.
The route of exposure in mechanistic studies is usually the same as for human exposure. Route is important because there can be effects that occur locally at the site of exposure in addition to systemic effects after the chemical has been absorbed into the blood and distributed throughout the body. A simple yet cogent example of a local effect would be irritation and eventual corrosion of the skin following application of strong acid or alkaline solutions designed for cleaning hard surfaces. Similarly, irritation and cellular death can occur in cells lining the nose and/or lungs following exposure to irritant vapours or gases such as oxides of nitrogen or ozone. (Both are constituents of air pollution, or smog). Following absorption of a chemical into blood through the skin, lungs or gastrointestinal tract, the concentration in any organ or tissue is controlled by many factors which determine the pharmacokinetics of the chemical in the body. The body has the ability to activate as well as detoxify various chemicals as noted below.
Role of Pharmacokinetics in Toxicity
Pharmacokinetics describes the time relationships for chemical absorption, distribution, metabolism (biochemical alterations in the body) and elimination or excretion from the body. Relative to mechanisms of toxicity, these pharmacokinetic variables can be very important and in some instances determine whether toxicity will or will not occur. For instance, if a material is not absorbed in a sufficient amount, systemic toxicity (inside the body) will not occur. Conversely, a highly reactive chemical that is detoxified quickly (seconds or minutes) by digestive or liver enzymes may not have the time to cause toxicity. Some polycyclic halogenated substances and mixtures as well as certain metals like lead would not cause significant toxicity if excretion were rapid; but accumulation to sufficiently high levels determines their toxicity since excretion is not rapid (sometimes measured in years). Fortunately, most chemicals do not have such long retention in the body. Accumulation of an innocuous material still would not induce toxicity. The rate of elimination from the body and detoxication is frequently referred to as the half-life of the chemical, which is the time for 50% of the chemical to be excreted or altered to a non-toxic form.
However, if a chemical accumulates in a particular cell or organ, that may signal a reason to further examine its potential toxicity in that organ. More recently, mathematical models have been developed to extrapolate pharmacokinetic variables from animals to humans. These pharmacokinetic models are extremely useful in generating hypotheses and testing whether the experimental animal may be a good representation for humans. Numerous chapters and texts have been written on this subject (Gehring et al. 1976; Reitz et al. 1987; Nolan et al. 1995). A simplified example of a physiological model is depicted in figure 1.
Different Levels and Systems Can Be Adversely Affected
Toxicity can be described at different biological levels. Injury can be evaluated in the whole person (or animal), the organ system, the cell or the molecule. Organ systems include the immune, respiratory, cardiovascular, renal, endocrine, digestive, muscolo-skeletal, blood, reproductive and central nervous systems. Some key organs include the liver, kidney, lung, brain, skin, eyes, heart, testes or ovaries, and other major organs. At the cellular/biochemical level, adverse effects include interference with normal protein function, endocrine receptor function, metabolic energy inhibition, or xenobiotic (foreign substance) enzyme inhibition or induction. Adverse effects at the molecular level include alteration of the normal function of DNA-RNA transcription, of specific cytoplasmic and nuclear receptor binding, and of genes or gene products. Ultimately, dysfunction in a major organ system is likely caused by a molecular alteration in a particular target cell within that organ. However, it is not always possible to trace a mechanism back to a molecular origin of causation, nor is it necessary. Intervention and therapy can be designed without a complete understanding of the molecular target. However, knowledge about the specific mechanism of toxicity increases the predictive value and accuracy of extrapolation to other chemicals. Figure 2 is a diagrammatic representation of the various levels where interference of normal physiological processes can be detected. The arrows indicate that the consequences to an individual can be determined from top down (exposure, pharmaco- kinetics to system/organ toxicity) or from bottom up (molecular change, cellular/biochemical effect to system/organ toxicity).
Examples of Mechanisms of Toxicity
Mechanisms of toxicity can be straightforward or very complex. Frequently, there is a difference among the type of toxicity, the mechanism of toxicity, and the level of effect, related to whether the adverse effects are due to a single, acute high dose (like an accidental poisoning), or a lower-dose repeated exposure (from occupational or environmental exposure). Classically, for testing purposes, an acute, single high dose is given by direct intubation into the stomach of a rodent or exposure to an atmosphere of a gas or vapour for two to four hours, whichever best resembles the human exposure. The animals are observed over a two-week period following exposure and then the major external and internal organs are examined for injury. Repeated-dose testing ranges from months to years. For rodent species, two years is considered a chronic (lifetime) study sufficient to evaluate toxicity and carcinogenicity, whereas for non-human primates, two years would be considered a subchronic (less than lifetime) study to evaluate repeated dose toxicity. Following exposure a complete examination of all tissues, organs and fluids is conducted to determine any adverse effects.
Acute Toxicity Mechanisms
The following examples are specific to high-dose, acute effects which can lead to death or severe incapacitation. However, in some cases, intervention will result in transient and fully reversible effects. The dose or severity of exposure will determine the result.
Simple asphyxiants. The mechanism of toxicity for inert gases and some other non-reactive substances is lack of oxygen (anoxia). These chemicals, which cause deprivation of oxygen to the central nervous system (CNS), are termed simple asphyxiants. If a person enters a closed space that contains nitrogen without sufficient oxygen, immediate oxygen depletion occurs in the brain and leads to unconsciousness and eventual death if the person is not rapidly removed. In extreme cases (near zero oxygen) unconsciousness can occur in a few seconds. Rescue depends on rapid removal to an oxygenated environment. Survival with irreversible brain damage can occur from delayed rescue, due to the death of neurons, which cannot regenerate.
Chemical asphyxiants. Carbon monoxide (CO) competes with oxygen for binding to haemoglobin (in red blood cells) and therefore deprives tissues of oxygen for energy metabolism; cellular death can result. Intervention includes removal from the source of CO and treatment with oxygen. The direct use of oxygen is based on the toxic action of CO. Another potent chemical asphyxiant is cyanide. The cyanide ion interferes with cellular metabolism and utilization of oxygen for energy. Treatment with sodium nitrite causes a change in haemoglobin in red blood cells to methaemoglobin. Methaemoglobin has a greater binding affinity to the cyanide ion than does the cellular target of cyanide. Consequently, the methaemoglobin binds the cyanide and keeps the cyanide away from the target cells. This forms the basis for antidotal therapy.
Central nervous system (CNS) depressants. Acute toxicity is characterized by sedation or unconsciousness for a number of materials like solvents which are not reactive or which are transformed to reactive intermediates. It is hypothesized that sedation/anaesthesia is due to an interaction of the solvent with the membranes of cells in the CNS, which impairs their ability to transmit electrical and chemical signals. While sedation may seem a mild form of toxicity and was the basis for development of the early anaesthetics, “the dose still makes the poison”. If sufficient dose is administered by ingestion or inhalation the animal can die due to respiratory arrest. If anaesthetic death does not occur, this type of toxicity is usually readily reversible when the subject is removed from the environment or the chemical is redistributed or eliminated from the body.
Skin effects. Adverse effects to the skin can range from irritation to corrosion, depending on the substance encountered. Strong acids and alkaline solutions are incompatible with living tissue and are corrosive, causing chemical burns and possible scarring. Scarring is due to death of the dermal, deep skin cells responsible for regeneration. Lower concentrations may just cause irritation of the first layer of skin.
Another specific toxic mechanism of skin is that of chemical sensitization. As an example, sensitization occurs when 2,4-dinitrochlorobenzene binds with natural proteins in the skin and the immune system recognizes the altered protein-bound complex as a foreign material. In responding to this foreign material, the immune system activates special cells to eliminate the foreign substance by release of mediators (cytokines) which cause a rash or dermatitis (see “Immunotoxicology”). This is the same reaction of the immune system when exposure to poison ivy occurs. Immune sensitization is very specific to the particular chemical and takes at least two exposures before a response is elicited. The first exposure sensitizes (sets up the cells to recognize the chemical), and subsequent exposures trigger the immune system response. Removal from contact and symptomatic therapy with steroid-containing anti-inflammatory creams are usually effective in treating sensitized individuals. In serious or refractory cases a systemic acting immunosuppresant like prednisone is used in conjunction with topical treatment.
Lung sensitization. An immune sensitization response is elicited by toluene diisocyanate (TDI), but the target site is the lungs. TDI over-exposure in susceptible individuals causes lung oedema (fluid build-up), bronchial constriction and impaired breathing. This is a serious condition and requires removing the individual from potential subsequent exposures. Treatment is primarily symptomatic. Skin and lung sensitization follow a dose response. Exceeding the level set for occupational exposure can cause adverse effects.
Eye effects. Injury to the eye ranges from reddening of the outer layer (swimming-pool redness) to cataract formation of the cornea to damage to the iris (coloured part of the eye). Eye irritation tests are conducted when it is believed serious injury will not occur. Many of the mechanisms causing skin corrosion can also cause injury to the eyes. Materials corrosive to the skin, like strong acids (pH less than 2) and alkali (pH greater than 11.5), are not tested in the eyes of animals because most will cause corrosion and blindness due to a mechanism similar to that which causes skin corrosion. In addition, surface active agents like detergents and surfactants can cause eye injury ranging from irritation to corrosion. A group of materials that requires caution is the positively charged (cationic) surfactants, which can cause burns, permanent opacity of the cornea and vascularization (formation of blood vessels). Another chemical, dinitrophenol, has a specific effect of cataract formation. This appears to be related to concentration of this chemical in the eye, which is an example of pharmacokinetic distributional specificity.
While the listing above is far from exhaustive, it is designed to give the reader an appreciation for various acute toxicity mechanisms.
Subchronic and Chronic Toxicity Mechanisms
When given as a single high dose, some chemicals do not have the same mechanism of toxicity as when given repeatedly as a lower but still toxic dose. When a single high dose is given, there is always the possibility of exceeding the person’s ability to detoxify or excrete the chemical, and this can lead to a different toxic response than when lower repetitive doses are given. Alcohol is a good example. High doses of alcohol lead to primary central nervous system effects, while lower repetitive doses result in liver injury.
Anticholinesterase inhibition. Most organophosphate pesticides, for example, have little mammalian toxicity until they are metabolically activated, primarily in the liver. The primary mechanism of action of organophosphates is the inhibition of acetylcholinesterase (AChE) in the brain and peripheral nervous system. AChE is the normal enzyme that terminates the stimulation of the neurotransmitter acetylcholine. Slight inhibition of AChE over an extended period has not been associated with adverse effects. At high levels of exposure, inability to terminate this neuronal stimulation results in overstimulation of the cholinergic nervous system. Cholinergic overstimulation ultimately results in a host of symptoms, including respiratory arrest, followed by death if not treated. The primary treatment is the administration of atropine, which blocks the effects of acetylcholine, and the administration of pralidoxime chloride, which reactivates the inhibited AChE. Therefore, both the cause and the treatment of organophosphate toxicity are addressed by understanding the biochemical basis of toxicity.
Metabolic activation. Many chemicals, including carbon tetrachloride, chloroform, acetylaminofluorene, nitrosamines, and paraquat are metabolically activated to free radicals or other reactive intermediates which inhibit and interfere with normal cellular function. At high levels of exposure this results in cell death (see “Cellular injury and cellular death”). While the specific interactions and cellular targets remain unknown, the organ systems which have the capability to activate these chemicals, like the liver, kidney and lung, are all potential targets for injury. Specifically, particular cells within an organ have a greater or lesser capacity to activate or detoxify these intermediates, and this capacity determines the intracellular susceptibility within an organ. Metabolism is one reason why an understanding of pharmacokinetics, which describes these types of transformations and the distribution and elimination of these intermediates, is important in recognizing the mechanism of action of these chemicals.
Cancer mechanisms. Cancer is a multiplicity of diseases, and while the understanding of certain types of cancer is increasing rapidly due to the many molecular biological techniques that have been developed since 1980, there is still much to learn. However, it is clear that cancer development is a multi-stage process, and critical genes are key to different types of cancer. Alterations in DNA (somatic mutations) in a number of these critical genes can cause increased susceptibility or cancerous lesions (see “Genetic toxic- ology”). Exposure to natural chemicals (in cooked foods like beef and fish) or synthetic chemicals (like benzidine, used as a dye) or physical agents (ultraviolet light from the sun, radon from soil, gamma radiation from medical procedures or industrial activity) are all contributors to somatic gene mutations. However, there are natural and synthetic substances (such as anti-oxidants) and DNA repair processes which are protective and maintain homeostasis. It is clear that genetics is an important factor in cancer, since genetic disease syndromes such as xeroderma pigmentosum, where there is a lack of normal DNA repair, dramatically increase susceptibility to skin cancer from exposure to ultraviolet light from the sun.
Reproductive mechanisms. Similar to cancer, many mechanisms of reproductive and/or developmental toxicity are known, but much is to be learned. It is known that certain viruses (such as rubella), bacterial infections and drugs (such as thalidomide and vitamin A) will adversely affect development. Recently, work by Khera (1991), reviewed by Carney (1994), show good evidence that the abnormal developmental effects in animal tests with ethylene glycol are attributable to maternal metabolic acidic metabolites. This occurs when ethylene glycol is metabolized to acid metabolites including glycolic and oxalic acid. The subsequent effects on the placenta and foetus appear to be due to this metabolic toxication process.
The intent of this article is to give a perspective on several known mechanisms of toxicity and the need for future study. It is important to understand that mechanistic knowledge is not absolutely necessary to protect human or environmental health. This knowledge will enhance the professional’s ability to better predict and manage toxicity. The actual techniques used in elucidating any particular mechanism depend upon the collective knowledge of the scientists and the thinking of those who make decisions regarding human health.