The priority objective of occupational and environmental toxicology is to improve the prevention or substantial limitation of health effects of exposure to hazardous agents in the general and occupational environments. To this end systems have been developed for quantitative risk assessment related to a given exposure (see the section “Regulatory toxicology”).

The effects of a chemical on particular systems and organs are related to the magnitude of exposure and whether exposure is acute or chronic. In view of the diversity of toxic effects even within one system or organ, a uniform philosophy concerning the critical organ and critical effect has been proposed for the purpose of risk assessment and development of health-based recommended concentration limits of toxic substances in different environmental media.

From the point of view of preventive medicine, it is of particular importance to identify early adverse effects, based on the general assumption that preventing or limiting early effects may prevent more severe health effects from developing.

Such an approach has been applied to heavy metals. Although heavy metals, such as lead, cadmium and mercury, belong to a specific group of toxic substances where the chronic effect of activity is dependent on their accumulation in the organs, the definitions presented below were published by the Task Group on Metal Toxicity (Nordberg 1976).

The definition of the critical organ as proposed by the Task Group on Metal Toxicity has been adopted with a slight modification: the word metal has been replaced with the expression potentially toxic substance (Duffus 1993).

Whether a given organ or system is regarded as critical depends not only on the toxicomechanics of the hazardous agent but also on the route of absorption and the exposed population.

• Critical concentration for a cell: the concentration at which adverse functional changes, reversible or irreversible, occur in the cell.
• Critical organ concentration: the mean concentration in the organ at the time at which the most sensitive type of cells in the organ reach critical concentration.
• Critical organ: that particular organ which first attains the critical concentration of metal under specified circumstances of exposure and for a given population.
• Critical effect: defined point in the relationship between dose and effect in the individual, namely the point at which an adverse effect occurs in cellular function of the critical organ. At an exposure level lower than that giving a critical concentration of metal in the critical organ, some effects may occur that do not impair cellular function per se, yet are detectable by means of biochemical and other tests. Such effects are defined as sub- critical effects.

The biological meaning of subcritical effect is sometimes not known; it may stand for exposure biomarker, adaptation index or a critical effect precursor (see “Toxicology test methods: Biomarkers”). The latter possibility can be particularly significant in view of prophylactic activities.

Table 1 displays examples of critical organs and effects for different chemicals. In chronic environmental exposure to cadmium, where the route of absorption is of minor importance (cadmium air concentrations range from 10 to 20μg/m³ in the urban and 1 to 2 μg/m³ in the rural areas), the critical organ is the kidney. In the occupational setting where the TLV reaches 50μg/m³ and inhalation constitutes the main route of exposure, two organs, lung and kidney, are regarded as critical.

Table 1. Examples of critical organs and critical effects

For lead, the critical organs in adults are the haemopoietic and peripheral nervous systems, where the critical effects (e.g., elevated free erythrocyte protoporphyrin concentration (FEP), increased excretion of delta-aminolevulinic acid in urine, or impaired peripheral nerve conduction) manifest when the blood lead level (an index of lead absorption in the system) approaches 200 to 300μg/l. In small children the critical organ is the central nervous system (CNS), and the symptoms of dysfunction detected with the use of a psychological test battery have been found to appear in the examined populations even at concentrations in the range of about 100μg/l Pb in blood.

A number of other definitions have been formulated which may better reflect the meaning of the notion. According to WHO (1989), the critical effect has been defined as “the first adverse effect which appears when the threshold (critical) concentration or dose is reached in the critical organ. Adverse effects, such as cancer, with no defined threshold concentration are often regarded as critical. Decision on whether an effect is critical is a matter of expert judgement.” In the International Programme on Chemical Safety (IPCS) guidelines for developing Environmental Health Criteria Documents, the critical effect is described as “the adverse effect judged to be most appropriate for determining the tolerable intake”. The latter definition has been formulated directly for the purpose of evaluating the health-based exposure limits in the general environment. In this context the most essential seems to be determining which effect can be regarded as an adverse effect. Following current terminology, the adverse effect is the “change in morphology, physiology, growth, development or lifespan of an organism which results in impairment of the capacity to compensate for additional stress or increase in susceptibility to the harmful effects of other environmental influences. Decision on whether or not any effect is adverse requires expert judgement.”

Figure 1 displays hypothetical dose-response curves for different effects. In the case of exposure to lead, A can represent a subcritical effect (inhibition of erythrocyte ALA-dehydratase), B the critical effect (an increase in erythrocyte zinc protoporphyrin or increase in the excretion of delta-aminolevulinic acid, C the clinical effect (anaemia) and D the fatal effect (death). For lead exposure there is abundant evidence illustrating how particular effects of exposure are dependent on lead concentration in blood (practical counterpart of the dose), either in the form of the dose-response relationship or in relation to different variables (sex, age, etc.). Determining the critical effects and the dose-response relationship for such effects in humans makes it possible to predict the frequency of a given effect for a given dose or its counterpart (concentration in biological material) in a certain population.

Figure 1. Hypothetical dose-response curves for various effects

The critical effects can be of two types: those considered to have a threshold and those for which there may be some risk at any exposure level (non-threshold, genotoxic carcinogens and germ mutagens). Whenever possible, appropriate human data should be used as a basis for the risk assessment. In order to determine the threshold effects for the general population, assumptions concerning the exposure level (tolerable intake, biomarkers of exposure) have to be made such that the frequency of the critical effect in the population exposed to a given hazardous agent corresponds to the frequency of that effect in the general population. In lead exposure, the maximum recommended blood lead concentration for the general population (200μg/l, median below 100μg/l) (WHO 1987) is practically below the threshold value for the assumed critical effect—the elevated free erythrocyte protoporphyrin level, although it is not below the level associated with effects on the CNS in children or blood pressure in adults. In general, if data from well-conducted human population studies defining a no observed adverse effect level are the basis for safety evaluation, then the uncertainty factor of ten has been considered appropriate. In the case of occupational exposure the critical effects may refer to a certain part of the population (e.g. 10%). Accordingly, in occupational lead exposure the recommended health-based concentration of blood lead has been adopted to be 400mg/l in men where a 10% response level for ALA-U of 5mg/l occurred at PbB concentrations of about 300 to 400mg/l. For the occupational exposure to cadmium (assuming the increased urinary excretion of low-weight proteins to be the critical effect), the level of 200ppm cadmium in renal cortex has been regarded as the admissible value, for this effect has been observed in 10% of the exposed population. Both these values are under consideration for lowering, in many countries, at the present time (i.e.,1996).

There is no clear consensus on appropriate methodology for the risk assessment of chemicals for which the critical effect may not have a threshold, such as genotoxic carcinogens. A number of approaches based largely on characterization of the dose- response relationship have been adopted for the assessment of such effects. Owing to the lack of socio-political acceptance of health risk caused by carcinogens in such documents as the Air Quality Guidelines for Europe (WHO 1987), only the values such as the unit lifetime risk (i.e., the risk associated with lifetime exposure to 1μg/m³ of the hazardous agent) are presented for non-threshold effects (see “Regulatory toxicology”).

Presently, the basic step in undertaking activities for risk assessment is determining the critical organ and critical effects. The definitions of both the critical and adverse effect reflect the responsibility of deciding which of the effects within a given organ or system should be regarded as critical, and this is directly related to the subsequent determination of recommended values for a given chemical in the general environment—for example, Air Quality Guidelines for Europe (WHO 1987) or health-based limits in occupational exposure (WHO 1980). Determining the critical effect from within the range of subcritical effects may lead to a situation where the recommended limits on toxic chemicals concentration in the general or occupational environment may be in practice impossible to maintain. Regarding as critical an effect that may overlap the early clinical effects may bring about the adoption of the values for which adverse effects may develop in some part of the population. The decision whether or not a given effect should be considered critical remains the responsibility of expert groups who specialize in toxicity and risk assessment.

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There are often large differences among humans in the intensity of response to toxic chemicals, and variations in susceptibility of an individual over a lifetime. These can be attributed to a variety of factors capable of influencing absorption rate, distribution in the body, biotransformation and/or excretion rate of a particular chemical. Apart from the known hereditary factors which have been clearly demonstrated to be linked with increased susceptibility to chemical toxicity in humans (see “Genetic determinants of toxic response”), other factors include: constitutional characteristics related to age and sex; pre-existing disease states or a reduction in organ function (non-hereditary, i.e., acquired); dietary habits, smoking, alcohol consumption and use of medications; concomitant exposure to biotoxins (various micro- organisms) and physical factors (radiation, humidity, extremely low or high temperatures or barometric pressures particularly relevant to the partial pressure of a gas), as well as concomitant physical exercise or psychological stress situations; previous occupational and/or environmental exposure to a particular chemical, and in particular concomitant exposure to other chemicals, not necessarily toxic (e.g., essential metals). The possible contributions of the aforementioned factors in either increasing or decreasing susceptibility to adverse health effects, as well as the mechanisms of their action, are specific for a particular chemical. Therefore only the most common factors, basic mechanisms and a few characteristic examples will be presented here, whereas specific information concerning each particular chemical can be found in elsewhere in this Encyclopaedia.

According to the stage at which these factors act (absorption, distribution, biotransformation or excretion of a particular chemical), the mechanisms can be roughly categorized according to two basic consequences of interaction: (1) a change in the quantity of the chemical in a target organ, that is, at the site(s) of its effect in the organism (toxicokinetic interactions), or (2) a change in the intensity of a specific response to the quantity of the chemical in a target organ (toxicodynamic interactions). The most common mechanisms of either type of interaction are related to competition with other chemical(s) for binding to the same compounds involved in their transport in the organism (e.g., specific serum proteins) and/or for the same biotransformation pathway (e.g., specific enzymes) resulting in a change in the speed or sequence between initial reaction and final adverse health effect. However, both toxicokinetic and toxicodynamic interactions may influence individual susceptibility to a particular chemical. The influence of several concomitant factors can result in either: (a) additive effects—the intensity of the combined effect is equal to the sum of the effects produced by each factor separately, (b) synergistic effects—the intensity of the combined effect is greater than the sum of the effects produced by each factor separately, or (c) antagonistic effects—the intensity of the combined effect is smaller than the sum of the effects produced by each factor separately.

The quantity of a particular toxic chemical or characteristic metabolite at the site(s) of its effect in the human body can be more or less assessed by biological monitoring, that is, by choosing the correct biological specimen and optimal timing of specimen sampling, taking into account biological half-lives for a particular chemical in both the critical organ and in the measured biological compartment. However, reliable information concerning other possible factors that might influence individual susceptibility in humans is generally lacking, and consequently the majority of knowledge regarding the influence of various factors is based on experimental animal data.

It should be stressed that in some cases relatively large differences exist between humans and other mammals in the intensity of response to an equivalent level and/or duration of exposure to many toxic chemicals; for example, humans appear to be considerably more sensitive to the adverse health effects of several toxic metals than are rats (commonly used in experimental animal studies). Some of these differences can be attributed to the fact that the transportation, distribution and biotransformation pathways of various chemicals are greatly dependent on subtle changes in the tissue pH and the redox equilibrium in the organism (as are the activities of various enzymes), and that the redox system of the human differs considerably from that of the rat.

This is obviously the case regarding important antioxidants such as vitamin C and glutathione (GSH), which are essential for maintaining redox equilibrium and which have a protective role against the adverse effects of the oxygen- or xenobiotic-derived free radicals which are involved in a variety of pathological conditions (Kehrer 1993). Humans cannot auto-synthesize vitamin C, contrary to the rat, and levels as well as the turnover rate of erythrocyte GSH in humans are considerably lower than that in the rat. Humans also lack some of the protective antioxidant enzymes, compared to the rat or other mammals (e.g., GSH- peroxidase is considered to be poorly active in human sperm). These examples illustrate the potentially greater vulnerability to oxidative stress in humans (particularly in sensitive cells, e.g., apparently greater vulnerability of the human sperm to toxic influences than that of the rat), which can result in different response or greater susceptibility to the influence of various factors in humans compared to other mammals (Telišman 1995).

Influence of Age

Compared to adults, very young children are often more susceptible to chemical toxicity because of their relatively greater inhalation volumes and gastrointestinal absorption rate due to greater permeability of the intestinal epithelium, and because of immature detoxification enzyme systems and a relatively smaller excretion rate of toxic chemicals. The central nervous system appears to be particularly susceptible at the early stage of development with regard to neurotoxicity of various chemicals, for example, lead and methylmercury. On the other hand, the elderly may be susceptible because of chemical exposure history and increased body stores of some xenobiotics, or pre-existing compromised function of target organs and/or relevant enzymes resulting in lowered detoxification and excretion rate. Each of these factors can contribute to weakening of the body’s defences—a decrease in reserve capacity, causing increased susceptibility to subsequent exposure to other hazards. For example, the cytochrome P450 enzymes (involved in the biotransformation pathways of almost all toxic chemicals) can be either induced or have lowered activity because of the influence of various factors over a lifetime (including dietary habits, smoking, alcohol, use of medications and exposure to environmental xenobiotics).

Influence of Sex

Gender-related differences in susceptibility have been described for a large number of toxic chemicals (approximately 200), and such differences are found in many mammalian species. It appears that males are generally more susceptible to renal toxins and females to liver toxins. The causes of the different response between males and females have been related to differences in a variety of physiological processes (e.g., females are capable of additional excretion of some toxic chemicals through menstrual blood loss, breast milk and/or transfer to the foetus, but they experience additional stress during pregnancy, delivery and lactation), enzyme activities, genetic repair mechanisms, hormonal factors, or the presence of relatively larger fat depots in females, resulting in greater accumulation of some lipophilic toxic chemicals, such as organic solvents and some medications.

Influence of Dietary Habits

Dietary habits have an important influence on susceptibility to chemical toxicity, mostly because adequate nutrition is essential for the functioning of the body’s chemical defence system in maintaining good health. Adequate intake of essential metals (including metalloids) and proteins, especially the sulphur-containing amino acids, is necessary for the biosynthesis of various detoxificating enzymes and the provision of glycine and glutathione for conjugation reactions with endogenous and exogenous compounds. Lipids, especially phospholipids, and lipotropes (methyl group donors) are necessary for the synthesis of biological membranes. Carbohydrates provide the energy required for various detoxification processes and provide glucuronic acid for conjugation of toxic chemicals and their metabolites. Selenium (an essential metalloid), glutathione, and vitamins such as vitamin C (water soluble), vitamin E and vitamin A (lipid soluble), have an important role as antioxidants (e.g., in controlling lipid peroxidation and maintaining integrity of cellular membranes) and free-radical scavengers for protection against toxic chemicals. In addition, various dietary constituents (protein and fibre content, minerals, phosphates, citric acid, etc.) as well as the amount of food consumed can greatly influence the gastrointestinal absorption rate of many toxic chemicals (e.g., the average absorption rate of soluble lead salts taken with meals is approximately eight per cent, as opposed to approximately 60% in fasting subjects). However, diet itself can be an additional source of individual exposure to various toxic chemicals (e.g., considerably increased daily intakes and accumulation of arsenic, mercury, cadmium and/or lead in subjects who consume contaminated seafood).

Influence of Smoking

The habit of smoking can influence individual susceptibility to many toxic chemicals because of the variety of possible interactions involving the great number of compounds present in cigarette smoke (especially polycyclic aromatic hydrocarbons, carbon monoxide, benzene, nicotine, acrolein, some pesticides, cadmium, and, to a lesser extent, lead and other toxic metals, etc.), some of which are capable of accumulating in the human body over a lifetime, including pre-natal life (e.g., lead and cadmium). The interactions occur mainly because various toxic chemicals compete for the same binding site(s) for transport and distribution in the organism and/or for the same biotransformation pathway involving particular enzymes. For example, several cigarette smoke constituents can induce cytochrome P450 enzymes, whereas others can depress their activity, and thus influence the common biotransformation pathways of many other toxic chemicals, such as organic solvents and some medications. Heavy cigarette smoking over a long period can considerably reduce the body’s defence mechanisms by decreasing reserve capacity to cope with the adverse influence of other life-style factors.

Influence of Alcohol

Consumption of alcohol (ethanol) can influence susceptibility to many toxic chemicals in several ways. It can influence the absorption rate and distribution of certain chemicals in the body—for example, increase the gastrointestinal absorption rate of lead, or decrease the pulmonary absorption rate of mercury vapour by inhibiting oxidation which is necessary for retention of inhaled mercury vapour. Ethanol can also influence susceptibility to various chemicals through short-term changes in tissue pH and increase in the redox potential resulting from ethanol metabolism, as both ethanol oxidizing to acetaldehyde and acetaldehyde oxidizing to acetate produce an equivalent of reduced nicotinamide adenine dinucleotide (NADH) and hydrogen (H⁺). Because the affinity of both essential and toxic metals and metalloids for binding to various compounds and tissues is influenced by pH and changes in the redox potential (Telišman 1995), even a moderate intake of ethanol may result in a series of consequences such as: (1) redistribution of long-term accumulated lead in the human organism in favour of a biologically active lead fraction, (2) replacement of essential zinc by lead in zinc-containing enzyme(s), thus affecting enzyme activity, or influence of mobil- ized lead on the distribution of other essential metals and metalloids in the organism such as calcium, iron, copper and selenium, (3) increased urinary excretion of zinc and so on. The effect of possible aforementioned events can be augmented due to the fact that alcoholic beverages can contain an appreciable amount of lead from vessels or processing (Prpic-Majic et al. 1984; Telišman et al. 1984; 1993).

Another common reason for ethanol-related changes in susceptibility is that many toxic chemicals, for example, various organic solvents, share the same biotransformation pathway involving the cytochrome P450 enzymes. Depending on the intensity of exposure to organic solvents as well as the quantity and frequency of ethanol ingestion (i.e., acute or chronic alcohol consumption), ethanol can either decrease or increase biotransformation rates of various organic solvents and thus influence their toxicity (Sato 1991).

Influence of Medications

The common use of various medications can influence susceptibility to toxic chemicals mainly because many drugs bind to serum proteins and thus influence the transport, distribution or excretion rate of various toxic chemicals, or because many drugs are capable of inducing relevant detoxifying enzymes or depressing their activity (e.g., the cytochrome P450 enzymes), thus affecting the toxicity of chemicals with the same biotransformation pathway. Characteristic for either of the mechanisms is increased urinary excretion of trichloroacetic acid (the metabolite of several chlorinated hydrocarbons) when using salicylate, sulphonamide or phenylbutazone, and an increased hepato-nephrotoxicity of carbon tetrachloride when using phenobarbital. In addition, some medications contain a considerable amount of a potentially toxic chemical, for example, the aluminium-containing antacids or preparations used for therapeutic management of the hyperphosphataemia arising in chronic renal failure.

Influence of Concomitant Exposure to Other Chemicals

The changes in susceptibility to adverse health effects due to interaction of various chemicals (i.e., possible additive, synergistic or antagonistic effects) have been studied almost exclusively in experimental animals, mostly in the rat. Relevant epidemiological and clinical studies are lacking. This is of concern particularly considering the relatively greater intensity of response or the variety of adverse health effects of several toxic chemicals in humans compared to the rat and other mammals. Apart from published data in the field of pharmacology, most data are related only to combinations of two different chemicals within specific groups, such as various pesticides, organic solvents, or essential and/or toxic metals and metalloids.

Combined exposure to various organic solvents can result in various additive, synergistic or antagonistic effects (depending on the combination of certain organic solvents, their intensity and duration of exposure), mainly due to the capability of influencing each other’s biotransformation (Sato 1991).

Another characteristic example are the interactions of both essential and/or toxic metals and metalloids, as these are involved in the possible influence of age (e.g., a lifetime body accumulation of environmental lead and cadmium), sex (e.g., common iron deficiency in women), dietary habits (e.g., increased dietary intake of toxic metals and metalloids and/or deficient dietary intake of essential metals and metalloids), smoking habit and alcohol consumption (e.g., additional exposure to cadmium, lead and other toxic metals), and use of medications (e.g., a single dose of antacid can result in a 50-fold increase in the average daily intake of aluminium through food). The possibility of various additive, synergistic or antagonistic effects of exposure to various metals and metalloids in humans can be illustrated by basic examples related to the main toxic elements (see table 1), apart from which further interactions may occur because essential elements can also influence one another (e.g., the well-known antagonistic effect of copper on the gastrointestinal absorption rate as well as the metabolism of zinc, and vice versa). The main cause of all these interactions is the competition of various metals and metalloids for the same binding site (especially the sulphhydryl group, -SH) in various enzymes, metalloproteins (especially metallothionein) and tissues (e.g., cell membranes and organ barriers). These interactions may have a relevant role in the development of several chronic diseases which are mediated through the action of free radicals and oxidative stress (Telišman 1995).

Table 1. Basic effects of possible multiple interactions concerning the main toxic and/or essential metals and matalloids in mammals

Note: Data are mostly related to experimental studies in the rat, whereas relevant clinical and epidemiological data (particularly regarding quantitative dose-response relationships) are generally lacking (Elsenhans et al. 1991; Fergusson 1990; Telišman et al. 1993).

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It has long been recognized that each person’s response to environmental chemicals is different. The recent explosion in molecular biology and genetics has brought a clearer understanding about the molecular basis of such variability. Major determinants of individual response to chemicals include important differences among more than a dozen superfamilies of enzymes, collectively termed xenobiotic- (foreign to the body) or drug-metabolizing enzymes. Although the role of these enzymes has classically been regarded as detoxification, these same enzymes also convert a number of inert compounds to highly toxic intermediates. Recently, many subtle as well as gross differences in the genes encoding these enzymes have been identified, which have been shown to result in marked variations in enzyme activity. It is now clear that each individual possesses a distinct complement of xenobiotic-metabolizing enzyme activities; this diversity might be thought of as a “metabolic fingerprint”. It is the complex interplay of these many different enzyme superfamilies which ultimately determines not only the fate and the potential for toxicity of a chemical in any given individual, but also assessment of exposure. In this article we have chosen to use the cytochrome P450 enzyme superfamily to illustrate the remarkable progress made in understanding individual response to chemicals. The development of relatively simple DNA-based tests designed to identify specific gene alterations in these enzymes, is now providing more accurate predictions of individual response to chemical exposure. We hope the result will be preventive toxicology. In other words, each individual might learn about those chemicals to which he or she is particularly sensitive, thereby avoiding previously unpredictable toxicity or cancer.

Although it is not generally appreciated, human beings are exposed daily to a barrage of innumerable diverse chemicals. Many of these chemicals are highly toxic, and they are derived from a wide variety of environmental and dietary sources. The relationship between such exposures and human health has been, and continues to be, a major focus of biomedical research efforts worldwide.

What are some examples of this chemical bombardment? More than 400 chemicals from red wine have been isolated and characterized. At least 1,000 chemicals are estimated to be produced by a lighted cigarette. There are countless chemicals in cosmetics and perfumed soaps. Another major source of chemical exposure is agriculture: in the United States alone, farmlands receive more than 75,000 chemicals each year in the form of pesticides, herbicides and fertilizing agents; after uptake by plants and grazing animals, as well as fish in nearby waterways, humans (at the end of the food chain) ingest these chemicals. Two other sources of large concentrations of chemicals taken into the body include (a) drugs taken chronically and (b) exposure to hazardous substances in the workplace over a lifetime of employment.

It is now well established that chemical exposure may adversely affect many aspects of human health, causing chronic diseases and the development of many cancers. In the last decade or so, the molecular basis of many of these relationships has begun to be unravelled. In addition, the realization has emerged that humans differ markedly in their susceptibility to the harmful effects of chemical exposure.

Current efforts to predict human response to chemical exposure combine two fundamental approaches (figure 1): monitoring the extent of human exposure through biological markers (biomarkers), and predicting the likely response of an individual to a given level of exposure. Although both of these approaches are extremely important, it should be emphasized that the two are distinctly different from one another. This article will focus on the genetic factors underlying individual susceptibility to any particular chemical exposure. This field of research is broadly termed ecogenetics, or pharmacogenetics (see Kalow 1962 and 1992). Many of the recent advances in determining individual susceptibility to chemical toxicity have evolved from a greater appreciation of the processes by which humans and other mammals detoxify chemicals, and the remarkable complexity of the enzyme systems involved.

Figure 1. The interrelationships among exposure assessment, ethnic differences, age, diet, nutrition and genetic susceptibility assessment - all of which play a role in the individual risk of toxicity and cancer

We will first describe the variability of toxic responses in humans. We will then introduce some of the enzymes responsible for such variation in response, due to differences in the metabolism of foreign chemicals. Next, the history and nomenclature of the cytochrome P450 superfamily will be detailed. Five human P450 polymorphisms as well as several non-P450 polymorphisms will be briefly described; these are responsible for human differences in toxic response. We will then discuss an example to emphasize the point that genetic differences in individuals can influence exposure assessment, as determined by environmental monitoring. Lastly, we will discuss the role of these xenobiotic-metabolizing enzymes in critical life functions.

Variation in Toxic Response Among the Human Population

Toxicologists and pharmacologists commonly speak about the average lethal dose for 50% of the population (LD₅₀), the average maximal tolerated dose for 50% of the population (MTD₅₀), and the average effective dose of a particular drug for 50% of the population (ED₅₀). However, how do these doses affect each of us on an individual basis? In other words, a highly sensitive individual may be 500 times more affected or 500 times more likely to be affected than the most resistant individual in a population; for these people, the LD₅₀ (and MTD₅₀ and ED₅₀) values would have little meaning. LD₅₀, MTD₅₀ and ED₅₀ values are only relevant when referring to the population as a whole.

Figure 2 illustrates a hypothetical dose-response relationship for a toxic response by individuals in any given population. This generic diagram might represent bronchogenic carcinoma in response to the number of cigarettes smoked, chloracne as a function of dioxin levels in the workplace, asthma as a function of air concentrations of ozone or aldehyde, sunburn in response to ultraviolet light, decreased clotting time as a function of aspirin intake, or gastrointestinal distress in response to the number of jalapeño peppers consumed. Generally, in each of these instances, the greater the exposure, the greater the toxic response. Most of the population will exhibit the mean and standard deviation of toxic response as a function of dose. The “resistant outlier” (lower right in figure 2) is an individual having less of a response at higher doses or exposures. A “sensitive outlier” (upper left) is an individual having an exaggerated response to a relatively small dose or exposure. These outliers, with extreme differences in response compared to the majority of individuals in the population, may represent important genetic variants that can help scientists in attempting to understand the underlying molecular mechanisms of a toxic response. 

Figure 2. Generic relationship between any toxic response and the dose of any environmental, chemical or physical agent

Using these outliers in family studies, scientists in a number of laboratories have begun to appreciate the importance of Mendelian inheritance for a given toxic response. Subsequently, one can then turn to molecular biology and genetic studies to pinpoint the underlying mechanism at the gene level (genotype) responsible for the environmentally caused disease (phenotype).

Xenobiotic- or Drug-metabolizing Enzymes

How does the body respond to the myriad of exogenous chemicals to which we are exposed? Humans and other mammals have evolved highly complex metabolic enzyme systems comprising more than a dozen distinct superfamilies of enzymes. Almost every chemical to which humans are exposed will be modified by these enzymes, in order to facilitate removal of the foreign substance from the body. Collectively, these enzymes are frequently referred to as drug-metabolizing enzymes or xenobiotic-metabolizing enzymes. Actually, both terms are misnomers. First, many of these enzymes not only metabolize drugs but hundreds of thousands of environmental and dietary chemicals. Second, all of these enzymes also have normal body compounds as substrates; none of these enzymes metabolizes only foreign chemicals.

For more than four decades, the metabolic processes mediated by these enzymes have commonly been classified as either Phase I or Phase II reactions (figure 3). Phase I (“functionalization”) reactions generally involve relatively minor structural modifications of the parent chemical via oxidation, reduction or hydrolysis in order to produce a more water-soluble metabolite. Frequently, Phase I reactions provide a “handle” for further modification of a compound by subsequent Phase II reactions. Phase I reactions are primarily mediated by a superfamily of highly versatile enzymes, collectively termed cytochromes P450, although other enzyme superfamilies can also be involved (figure 4).

Figure 3. The classical designation of Phase I and Phase II xenobiotic- or drug-metabolizing enzymes

Figure 4. Examples of drug-metabolizing enzymes

Phase II reactions involve the coupling of a water-soluble endogenous molecule to a chemical (parent chemical or Phase I metabolite) in order to facilitate excretion. Phase II reactions are frequently termed “conjugation” or “derivatization” reactions. The enzyme superfamilies catalyzing Phase II reactions are generally named according to the endogenous conjugating moiety involved: for example, acetylation by the N-acetyltransferases, sulphation by the sulphotransferases, glutathione conjugation by the glutathione transferases, and glucuronidation by the UDP glucuronosyltransferases (figure 4). Although the major organ of drug metabolism is the liver, the levels of some drug- metabolizing enzymes are quite high in the gastrointestinal tract, gonads, lung, brain and kidney, and such enzymes are undoubtedly present to some extent in every living cell.

Xenobiotic-metabolizing Enzymes Represent Double-edged Swords

As we learn more about the biological and chemical processes leading to human health aberrations, it has become increasingly evident that drug-metabolizing enzymes function in an ambivalent manner (figure 3). In the majority of cases, lipid-soluble chemicals are converted to more readily excreted water-soluble metabolites. However, it is clear that on many occasions the same enzymes are capable of transforming other inert chemicals into highly reactive molecules. These intermediates can then interact with cellular macromolecules such as proteins and DNA. Thus, for each chemical to which humans are exposed, there exists the potential for the competing pathways of metabolic activation and detoxification.

Brief Review of Genetics

In human genetics, each gene (locus) is located on one of the 23 pairs of chromosomes. The two alleles (one present on each chromosome of the pair) can be the same, or they can be different from one another. For example, the B and b alleles, in which B (brown eyes) is dominant over b (blue eyes): individuals of the brown-eyed phenotype can have either the BB or Bb genotypes, whereas individuals of the blue-eyed phenotype can only have the bb genotype.

A polymorphism is defined as two or more stably inherited phenotypes (traits)—derived from the same gene(s)—that are maintained in the population, often for reasons not necessarily obvious. For a gene to be polymorphic, the gene product must not be essential for development, reproductive vigour or other critical life processes. In fact, a “balanced polymorphism,” wherein the heterozygote has a distinct survival advantage over either homozygote (e.g., resistance to malaria, and the sickle-cell haemoglobin allele) is a common explanation for maintaining an allele in the population at otherwise unexplained high frequencies (see Gonzalez and Nebert 1990).

Human Polymorphisms of Xenobiotic-metabolizing Enzymes

Genetic differences in the metabolism of various drugs and environmental chemicals have been known for more than four decades (Kalow 1962 and 1992). These differences are frequently referred to as pharmacogenetic or, more broadly, ecogenetic polymorphisms. These polymorphisms represent variant alleles that occur at a relatively high frequency in the population and are generally associated with aberrations in enzyme expression or function. Historically, polymorphisms were usually identified following unexpected responses to therapeutic agents. More recently, recombinant DNA technology has enabled scientists to identify the precise alterations in genes that are responsible for some of these polymorphisms. Polymorphisms have now been characterized in many drug-metabolizing enzymes—including both Phase I and Phase II enzymes. As more and more polymorphisms are identified, it is becoming increasingly apparent that each individual may possess a distinct complement of drug-metabolizing enzymes. This diversity might be described as a “metabolic fingerprint”. It is the complex interplay of the various drug- metabolizing enzyme superfamilies within any individual that will ultimately determine his or her particular response to a given chemical (Kalow 1962 and 1992; Nebert 1988; Gonzalez and Nebert 1990; Nebert and Weber 1990).

Expressing Human Xenobiotic-metabolizingEnzymes in Cell Culture

How might we develop better predictors of human toxic responses to chemicals? Advances in defining the multiplicity of drug-metabolizing enzymes must be accompanied by precise knowledge as to which enzymes determine the metabolic fate of individual chemicals. Data gleaned from laboratory rodent studies have certainly provided useful information. However, significant interspecies differences in xenobiotic-metabolizing enzymes necessitate caution in extrapolating data to human populations. To overcome this difficulty, many laboratories have developed systems in which various cell lines in culture can be engineered to produce functional human enzymes that are stable and in high concentrations (Gonzalez, Crespi and Gelboin 1991). Successful production of human enzymes has been achieved in a variety of diverse cell lines from sources including bacteria, yeast, insects and mammals.

In order to define the metabolism of chemicals even more accurately, multiple enzymes have also been successfully produced in a single cell line (Gonzalez, Crespi and Gelboin 1991). Such cell lines provide valuable insights into the precise enzymes involved in the metabolic processing of any given compound and likely toxic metabolites. If this information can then be combined with knowledge regarding the presence and level of an enzyme in human tissues, these data should provide valuable predictors of response.

Cytochrome P450

History and nomenclature

The cytochrome P450 superfamily is one of the most studied drug-metabolizing enzyme superfamilies, having a great deal of individual variability in response to chemicals. Cytochrome P450 is a convenient generic term used to describe a large superfamily of enzymes pivotal in the metabolism of innumerable endogenous and exogenous substrates. The term cytochrome P450 was first coined in 1962 to describe an unknown pigment in cells which, when reduced and bound with carbon monoxide, produced a characteristic absorption peak at 450 nm. Since the early 1980s, cDNA cloning technology has resulted in remarkable insights into the multiplicity of cytochrome P450 enzymes. To date, more than 400 distinct cytochrome P450 genes have been identified in animals, plants, bacteria and yeast. It has been estimated that any one mammalian species, such as humans, may possess 60 or more distinct P450 genes (Nebert and Nelson 1991). The multiplicity of P450 genes has necessitated the development of a standardized nomenclature system (Nebert et al. 1987; Nelson et al. 1993). First proposed in 1987 and updated on a biannual basis, the nomenclature system is based on divergent evolution of amino acid sequence comparisons between P450 proteins. The P450 genes are divided into families and subfamilies: enzymes within a family display greater than 40% amino acid similarity, and those within the same subfamily display 55% similarity. P450 genes are named with the root symbol CYP followed by an arabic numeral designating the P450 family, a letter denoting the subfamily, and a further arabic numeral designating the individual gene (Nelson et al. 1993; Nebert et al. 1991). Thus, CYP1A1 represents P450 gene 1 in family 1 and subfamily A.

As of February 1995, there are 403 CYP genes in the database, composed of 59 families and 105 sub- families. These include eight lower eukaryotic families, 15 plant families, and 19 bacterial families. The 15 human P450 gene families comprise 26 subfamilies, 22 of which have been mapped to chromosomal locations throughout most of the genome. Some sequences are clearly orthologous across many species—for example, only one CYP17 (steroid 17α-hydroxylase) gene has been found in all vertebrates examined to date; other sequences within a subfamily are highly duplicated, making the identification of orthologous pairs impossible (e.g., the CYP2C subfamily). Interestingly, human and yeast share an orthologous gene in the CYP51 family. Numerous comprehensive reviews are available for readers seeking further information on the P450 superfamily (Nelson et al. 1993; Nebert et al. 1991; Nebert and McKinnon 1994; Guengerich 1993; Gonzalez 1992).

The success of the P450 nomenclature system has resulted in similar terminology systems being developed for the UDP glucuronosyltransferases (Burchell et al. 1991) and flavin-containing mono-oxygenases (Lawton et al. 1994). Similar nomenclature systems based on divergent evolution are also under development for several other drug-metabolizing enzyme superfamilies (e.g., sulphotransferases, epoxide hydrolases and aldehyde dehydrogenases).

Recently, we divided the mammalian P450 gene superfamily into three groups (Nebert and McKinnon 1994)—those involved principally with foreign chemical metabolism, those involved in the synthesis of various steroid hormones, and those participating in other important endogenous functions. It is the xenobiotic-metabolizing P450 enzymes that assume the most significance for prediction of toxicity.

Xenobiotic-metabolizing P450 enzymes

P450 enzymes involved in the metabolism of foreign compounds and drugs are almost always found within families CYP1, CYP2, CYP3 and CYP4. These P450 enzymes catalyze a wide variety of metabolic reactions, with a single P450 often capable of meta-bolizing many different compounds. In addition, multiple P450 enzymes may metabolize a single compound at different sites. Also, a compound may be metabolized at the same, single site by several P450s, although at varying rates.

A most important property of the drug-metabolizing P450 enzymes is that many of these genes are inducible by the very substances which serve as their substrates. On the other hand, other P450 genes are induced by nonsubstrates. This phenomenon of enzyme induction underlies many drug-drug interactions of therapeutic importance.

Although present in many tissues, these particular P450 enzymes are found in relatively high levels in the liver, the primary site of drug metabolism. Some of the xenobiotic-metabolizing P450 enzymes exhibit activity toward certain endogenous substrates (e.g., arachidonic acid). However, it is generally believed that most of these xenobiotic-metabolizing P450 enzymes do not play important physiological roles—although this has not been established experimentally as yet. The selective homozygous disruption, or “knock-out,” of individual xenobiotic-metabolizing P450 genes by means of gene targeting methodologies in mice is likely to provide unequivocal information soon with regard to physiological roles of the xenobiotic-metabolizing P450s (for a review of gene targeting, see Capecchi 1994).

In contrast to P450 families encoding enzymes involved primarily in physiological processes, families encoding xenobiotic-metabolizing P450 enzymes display marked species specificity and frequently contain many active genes per subfamily (Nelson et al. 1993; Nebert et al. 1991). Given the apparent lack of physiological substrates, it is possible that P450 enzymes in families CYP1, CYP2, CYP3 and CYP4 that have appeared in the past several hundred million years have evolved as a means of detoxifying foreign chemicals encountered in the environment and diet. Clearly, evolution of the xenobiotic-metabolizing P450s would have occurred over a time period which far precedes the synthesis of most of the synthetic chemicals to which humans are now exposed. The genes in these four gene families may have evolved and diverged in animals due to their exposure to plant metabolites during the last 1.2 billion years—a process descriptively termed “animal-plant warfare” (Gonzalez and Nebert 1990). Animal-plant warfare is the phenomenon in which plants developed new chemicals (phytoalexins) as a defence mechanism in order to prevent ingestion by animals, and animals, in turn, responded by developing new P450 genes to accommodate the diversifying substrates. Providing further impetus to this proposal are the recently described examples of plant-insect and plant-fungus chemical warfare involving P450 detoxification of toxic substrates (Nebert 1994).

The following is a brief introduction to several of the human xenobiotic-metabolizing P450 enzyme polymorphisms in which genetic determinants of toxic response are believed to be of high significance. Until recently, P450 polymorphisms were generally suggested by unexpected variance in patient response to administered therapeutic agents. Several P450 polymorphisms are indeed named according to the drug with which the polymorphism was first identified. More recently, research efforts have focused on identification of the precise P450 enzymes involved in the metabolism of chemicals for which variance is observed and the precise characterization of the P450 genes involved. As described earlier, the measurable activity of a P450 enzyme towards a model chemical can be called the phenotype. Allelic differences in a P450 gene for each individual is termed the P450 genotype. As more and more scrutiny is applied to the analysis of P450 genes, the precise molecular basis of previously documented phenotypic variance is becoming clearer.

The CYP1A subfamily

The CYP1A subfamily comprises two enzymes in humans and all other mammals: these are designated CYP1A1 and CYP1A2 under standard P450 nomenclature. These enzymes are of considerable interest, because they are involved in the metabolic activation of many procarcinogens and are also induced by several compounds of toxicological concern, including dioxin. For example, CYP1A1 metabolically activates many compounds found in cigarette smoke. CYP1A2 metabolically activates many arylamines—associated with urinary bladder cancer—found in the chemical dye industry. CYP1A2 also metabolically activates 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-derived nitrosamine. CYP1A1 and CYP1A2 are also found at higher levels in the lungs of cigarette smokers, due to induction by polycyclic hydrocarbons present in the smoke. The levels of CYP1A1 and CYP1A2 activity are therefore considered to be important determinants of individual response to many potentially toxic chemicals.

Toxicological interest in the CYP1A subfamily was greatly intensified by a 1973 report correlating the level of CYP1A1 inducibility in cigarette smokers with individual susceptibility to lung cancer (Kellermann, Shaw and Luyten-Kellermann 1973). The molecular basis of CYP1A1 and CYP1A2 induction has been a major focus of numerous laboratories. The induction process is mediated by a protein termed the Ah receptor to which dioxins and structurally related chemicals bind. The name Ah is derived from the aryl hydrocarbon nature of many CYP1A inducers. Interestingly, differences in the gene encoding the Ah receptor between strains of mice result in marked differences in chemical response and toxicity. A polymorphism in the Ah receptor gene also appears to occur in humans: approximately one-tenth of the population displays high induction of CYP1A1 and may be at greater risk than the other nine-tenths of the population for development of certain chemically induced cancers. The role of the Ah receptor in the control of enzymes in the CYP1A subfamily, and its role as a determinant of human response to chemical exposure, has been the subject of several recent reviews (Nebert, Petersen and Puga 1991; Nebert, Puga and Vasiliou 1993).

Are there other polymorphisms that might control the level of CYP1A proteins in a cell? A polymorphism in the CYP1A1 gene has also been identified, and this appears to influence lung cancer risk amongst Japanese cigarette smokers, although this same polymorphism does not appear to influence risk in other ethnic groups (Nebert and McKinnon 1994).

CYP2C19

Variations in the rate at which individuals metabolize the anticonvulsant drug (S)-mephenytoin have been well documented for many years (Guengerich 1989). Between 2% and 5% of Caucasians and as many as 25% of Asians are deficient in this activity and may be at greater risk of toxicity from the drug. This enzyme defect has long been known to involve a member of the human CYP2C subfamily, but the precise molecular basis of this deficiency has been the subject of considerable controversy. The major reason for this difficulty was the six or more genes in the human CYP2C subfamily. It was recently demonstrated, however, that a single-base mutation in the CYP2C19 gene is the primary cause of this deficiency (Goldstein and de Morais 1994). A simple DNA test, based on the polymerase chain reaction (PCR), has also been developed to identify this mutation rapidly in human populations (Goldstein and de Morais 1994).

CYP2D6

Perhaps the most extensively characterized variation in a P450 gene is that involving the CYP2D6 gene. More than a dozen examples of mutations, rearrangements and deletions affecting this gene have been described (Meyer 1994). This polymorphism was first suggested 20 years ago by clinical variability in patients’ response to the antihypertensive agent debrisoquine. Alterations in the CYP2D6 gene giving rise to altered enzyme activity are therefore collectively termed the debrisoquine polymorphism.

Prior to the advent of DNA-based studies, individuals had been classified as poor or extensive metabolizers (PMs, EMs) of debrisoquine based on metabolite concentrations in urine samples. It is now clear that alterations in the CYP2D6 gene may result in individuals displaying not only poor or extensive debrisoquine metabolism, but also ultrarapid metabolism. Most alterations in the CYP2D6 gene are associated with partial or total deficiency of enzyme function; however, individuals in two families have recently been described who possess multiple functional copies of the CYP2D6 gene, giving rise to ultrarapid metabolism of CYP2D6 substrates (Meyer 1994). This remarkable observation provides new insights into the wide spectrum of CYP2D6 activity previously observed in population studies. Alterations in CYP2D6 function are of particular significance, given the more than 30 commonly prescribed drugs metabolized by this enzyme. An individual’s CYP2D6 function is therefore a major determinant of both therapeutic and toxic response to administered therapy. Indeed, it has recently been argued that consideration of a patient’s CYP2D6 status is necessary for the safe use of both psychiatric and cardiovascular drugs.

The role of the CYP2D6 polymorphism as a determinant of individual susceptibility to human diseases such as lung cancer and Parkinson’s disease has also been the subject of intense study (Nebert and McKinnon 1994; Meyer 1994). While conclusions are difficult to define given the diverse nature of the study protocols utilized, the majority of studies appear to indicate an association between extensive metabolizers of debrisoquine (EM phenotype) and lung cancer. The reasons for such an association are presently unclear. However, the CYP2D6 enzyme has been shown to metabolize NNK, a tobacco-derived nitrosamine.

As DNA-based assays improve—enabling even more accurate assessment of CYP2D6 status—it is anticipated that the precise relationship of CYP2D6 to disease risk will be clarified. Whereas the extensive metabolizer may be linked with susceptibility to lung cancer, the poor metabolizer (PM phenotype) appears to be associated with Parkinson’s disease of unknown cause. Whereas these studies are also difficult to compare, it appears that PM individuals having a diminished capacity to metabolize CYP2D6 substrates (e.g., debrisoquine) have a 2- to 2.5-fold increase in risk of developing Parkinson’s disease.

CYP2E1

The CYP2E1 gene encodes an enzyme that metabolizes many chemicals, including drugs and many low-molecular-weight carcinogens. This enzyme is also of interest because it is highly inducible by alcohol and may play a role in liver injury induced by chemicals such as chloroform, vinyl chloride and carbon tetrachloride. The enzyme is primarily found in the liver, and the level of enzyme varies markedly between individuals. Close scrutiny of the CYP2E1 gene has resulted in the identification of several polymorphisms (Nebert and McKinnon 1994). A relationship has been reported between the presence of certain structural variations in the CYP2E1 gene and apparent lowered lung cancer risk in some studies; however, there are clear interethnic differences which require clarification of this possible relationship.

The CYP3A subfamily

In humans, four enzymes have been identified as members of the CYP3A subfamily due to their similarity in amino acid sequence. The CYP3A enzymes metabolize many commonly prescribed drugs such as erythromycin and cyclosporin. The carcinogenic food contaminant aflatoxin B₁ is also a CYP3A substrate. One member of the human CYP3A subfamily, designated CYP3A4, is the principal P450 in human liver as well as being present in the gastrointestinal tract. As is true for many other P450 enzymes, the level of CYP3A4 is highly variable between individuals. A second enzyme, designated CYP3A5, is found in only approximately 25% of livers; the genetic basis of this finding has not been elucidated. The importance of CYP3A4 or CYP3A5 variability as a factor in genetic determinants of toxic response has not yet been established (Nebert and McKinnon 1994).

Non-P450 Polymorphisms

Numerous polymorphisms also exist within other xenobiotic-metabolizing enzyme superfamilies (e.g., glutathione transferases, UDP glucuronosyltransferases, para-oxonases, dehydrogenases, N-acetyltransferases and flavin-containing mono-oxygenases). Because the ultimate toxicity of any P450-generated intermediate is dependent on the efficiency of subsequent Phase II detoxification reactions, the combined role of multiple enzyme polymorphisms is important in determining susceptibility to chemically induced diseases. The metabolic balance between Phase I and Phase II reactions (figure 3) is therefore likely to be a major factor in chemically induced human diseases and genetic determinants of toxic response.

The GSTM1 gene polymorphism

A well studied example of a polymorphism in a Phase II enzyme is that involving a member of the glutathione S-transferase enzyme superfamily, designated GST mu or GSTM1. This particular enzyme is of considerable toxicological interest because it appears to be involved in the subsequent detoxification of toxic metabolites produced from chemicals in cigarette smoke by the CYP1A1 enzyme. The identified polymorphism in this glutathione transferase gene involves a total absence of functional enzyme in as many as half of all Caucasians studied. This lack of a Phase II enzyme appears to be associated with increased susceptibility to lung cancer. By grouping individuals on the basis of both variant CYP1A1 genes and the deletion or presence of a functional GSTM1 gene, it has been demonstrated that the risk of developing smoking-induced lung cancer varies significantly (Kawajiri, Watanabe and Hayashi 1994). In particular, individuals displaying one rare CYP1A1 gene alteration, in combination with an absence of the GSTM1 gene, were at higher risk (as much as ninefold) of developing lung cancer when exposed to a relatively low level of cigarette smoke. Interestingly, there appear to be interethnic differences in the significance of variant genes which necessitate further study in order to elucidate the precise role of such alterations in susceptibility to disease (Kalow 1962; Nebert and McKinnon 1994; Kawajiri, Watanabe and Hayashi 1994).

Synergistic effect of two or more polymorphisms on the toxic response

A toxic response to an environmental agent may be greatly exaggerated by the combination of two pharmacogenetic defects in the same individual, for example, the combined effects of the N-acetyltransferase (NAT2) polymorphism and the glucose-6-phosphate dehydrogenase (G6PD) polymorphism.

Occupational exposure to arylamines constitutes a grave risk of urinary bladder cancer. Since the elegant studies of Cartwright in 1954, it has become clear that the N-acetylator status is a determinant of azo-dye-induced bladder cancer. There is a highly significant correlation between the slow-acetylator phenotype and the occurrence of bladder cancer, as well as the degree of invasiveness of this cancer in the bladder wall. On the contrary, there is a significant association between the rapid-acetylator phenotype and the incidence of colorectal carcinoma. The N-acetyltransferase (NAT1, NAT2) genes have been cloned and sequenced, and DNA-based assays are now able to detect the more than a dozen allelic variants which account for the slow-acetylator phenotype. The NAT2 gene is polymorphic and responsible for most of the variability in toxic response to environmental chemicals (Weber 1987; Grant 1993).

Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme critical in the generation and maintenance of NADPH. Low or absent G6PD activity can lead to severe drug- or xenobiotic-induced haemolysis, due to the absence of normal levels of reduced glutathione (GSH) in the red blood cell. G6PD deficiency affects at least 300 million people worldwide. More than 10% of African-American males exhibit the less severe phenotype, while certain Sardinian communities exhibit the more severe “Mediterranean type” at frequencies as high as one in every three persons. The G6PD gene has been cloned and localized to the X chromosome, and numerous diverse point mutations account for the large degree of phenotypic heterogeneity seen in G6PD-deficient individuals (Beutler 1992).

Thiozalsulphone, an arylamine sulpha drug, was found to cause a bimodal distribution of haemolytic anaemia in the treated population. When treated with certain drugs, individuals with the combination of G6PD deficiency plus the slow-acetylator phenotype are more affected than those with the G6PD deficiency alone or the slow-acetylator phenotype alone. G6PD-deficient slow acetylators are at least 40 times more susceptible than normal-G6PD rapid acetylators to thiozalsulphone-induced haemolysis.

Effect of genetic polymorphisms on exposure assessment

Exposure assessment and biomonitoring (figure 1) also requires information on the genetic make-up of each individual. Given identical exposure to a hazardous chemical, the level of haemoglobin adducts (or other biomarkers) might vary by two or three orders of magnitude among individuals, depending upon each person’s metabolic fingerprint.

The same combined pharmacogenetics has been studied in chemical factory workers in Germany (table 1). Haemoglobin adducts among workers exposed to aniline and acetanilide are by far the highest in G6PD-deficient slow acetylators, as compared with the other possible combined pharmacogenetic phenotypes. This study has important implications for exposure assessment. These data demonstrate that, although two individuals might be exposed to the same ambient level of hazardous chemical in the work place, the amount of exposure (via biomarkers such as haemoglobin adducts) might be estimated to be two or more orders of magnitude less, due to the underlying genetic predisposition of the individual. Likewise, the resulting risk of an adverse health effect may vary by two or more orders of magnitude.

Table 1: Haemoglobin adducts in workers exposed to aniline and acetanilide

Source: Adapted from Lewalter and Korallus 1985.

Genetic differences in binding as well as metabolism

It should be emphasized that the same case made here for meta-bolism can also be made for binding. Heritable differences in the binding of environmental agents will greatly affect the toxic response. For example, differences in the mouse cdm gene can profoundly affect individual sensitivity to cadmium-induced testicular necrosis (Taylor, Heiniger and Meier 1973). Differences in the binding affinity of the Ah receptor are likely affect dioxin-induced toxicity and cancer (Nebert, Petersen and Puga 1991; Nebert, Puga and Vasiliou 1993).

Figure 5 summarizes the role of metabolism and binding in toxicity and cancer. Toxic agents, as they exist in the environment or following metabolism or binding, elicit their effects by either a genotoxic pathway (in which damage to DNA occurs) or a non-genotoxic pathway (in which DNA damage and mutagenesis need not occur). Interestingly, it has recently become clear that “classical” DNA-damaging agents can operate via a reduced glutathione (GSH)-dependent nongenotoxic signal transduction pathway, which is initiated on or near the cell surface in the absence of DNA and outside the cell nucleus (Devary et al. 1993). Genetic differences in metabolism and binding remain, however, as the major determinants in controlling different individual toxic responses.

Figure 5. The general means by which toxicity occurs

Role of Drug-metabolizing Enzymesin Cellular Function

Genetically based variation in drug-metabolizing enzyme function is of major importance in determining individual response to chemicals. These enzymes are pivotal in determining the fate and time course of a foreign chemical following exposure.

As illustrated in figure 5, the importance of drug-metabolizing enzymes in individual susceptibility to chemical exposure may in fact present a far more complex issue than is evident from this simple discussion of xenobiotic metabolism. In other words, during the past two decades, genotoxic mechanisms (measurements of DNA adducts and protein adducts) have been greatly emphasized. However, what if nongenotoxic mechanisms are at least as important as genotoxic mechanisms in causing toxic responses?

As mentioned earlier, the physiological roles of many drug-metabolizing enzymes involved in xenobiotic metabolism have not been accurately defined. Nebert (1994) has proposed that, because of their presence on this planet for more than 3.5 billion years, drug-metabolizing enzymes were originally (and are now still primarily) responsible for regulating the cellular levels of many nonpeptide ligands important in the transcriptional activation of genes affecting growth, differentiation, apoptosis, homeostasis and neuroendocrine functions. Furthermore, the toxicity of most, if not all, environmental agents occurs by means of agonist or antagonist action on these signal transduction pathways (Nebert 1994). Based on this hypothesis, genetic variability in drug-metabolizing enzymes may have quite dramatic effects on many critical biochemical processes within the cell, thereby leading to important differences in toxic response. It is indeed possible that such a scenario may also underlie many idiosyncratic adverse reactions encountered in patients using commonly prescribed drugs.

Conclusions

The past decade has seen remarkable progress in our understanding of the genetic basis of differential response to chemicals in drugs, foods and environmental pollutants. Drug-metabolizing enzymes have a profound influence on the way humans respond to chemicals. As our awareness of drug-metabolizing enzyme multiplicity continues to evolve, we are increasingly able to make improved assessments of toxic risk for many drugs and environmental chemicals. This is perhaps most clearly illustrated in the case of the CYP2D6 cytochrome P450 enzyme. Using relatively simple DNA-based tests, it is possible to predict the likely response of any drug predominantly metabolized by this enzyme; this prediction will ensure the safer use of valuable, yet potentially toxic, medication.

The future will no doubt see an explosion in the identification of further polymorphisms (phenotypes) involving drug-metabolizing enzymes. This information will be accompanied by improved, minimally invasive DNA-based tests to identify genotypes in human populations.

Such studies should be particularly informative in evaluating the role of chemicals in the many environmental diseases of presently unknown origin. The consideration of multiple drug-metabolizing enzyme polymorphisms, in combination (e.g., table 1), is also likely to represent a particularly fertile research area. Such studies will clarify the role of chemicals in the causation of cancers. Collectively, this information should enable the formulation of increasingly individualized advice on avoidance of chemicals likely to be of individual concern. This is the field of preventive toxicology. Such advice will no doubt greatly assist all individuals in coping with the ever increasing chemical burden to which we are exposed.

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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.

Exposure

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.

Figure 1. A simplified pharmacokinetic model

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).

Figure 2. Reresentation of mechanisms of 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.

Conclusion

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.

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