This volume contains the main papers presented at the 1997 EUROTOX Congress, Århus, Denmark, 24-28 June 1997. Diversification in toxicology is seen as the application of basic science to such diverse
The nuclear receptor superfamily, which includes receptors for steroid hormones, thyroid hormone, vitamin D, retinoic acid, peroxisome proliferators and ecdysone, consists of a surprisingly large number of genes (Tsai and O’Malley, 1994); a large number of genes having extensive sequence homology to the nuclear receptor family, but for which no ligands have yet been identified, are furthermore also known (Enmark and Gustafsson, 1996). It remains possible that, as with vitamin A and vitamin D, the natural ligands for these receptors are components of the diet. Equally likely is the possibility that, like the peroxisome proliferator activated receptor (PPAR), environmental contaminants or xenobiotics can activate some of these receptors and can thus interfere with normal physiological functions in the body. This lecture focuses on two members on the nuclear receptor super gene family, the newly discovered estrogen receptor, ERß, and the PPAR to illustrate how these receptors could be involved in receptor mediated toxicity.
Glutathione transferases (GSTs) catalyzing the conjugation of glutathione with electrophilic substrates are important enzymes in the metabolism of xenobiotics. Several isozymes exhibit polymorphisms in humans. The two deletion polymorphisms of hGSTM1 and hGSTT1 result in total loss of enzyme activity in homozygous null genotype (GSTM1*0 and GSTT1*0 respectively) individuals (Seidegård et al. 1988; Pemble et al. 1994). Individuals that are heterozygous for hGSTT1 show distinctly lower enzyme activities than individuals carrying two functional alleles of hGSTT1 (Wiebel et al. 1996). A similar effect is conceivable for the hGSTM1 polymorphism but has not been verified so far.
Bis(tri-n-butyltin)oxide (TBTO) has been shown to be immunotoxic in rodents, resulting in decreased resistance to infections. The no-effect level assessed by estimating effects on host resistance in rats has been found to lie between 0.5 and 5.0 mg TBTO/kg food (0.025 and 0.25 mg/kg body weight). For risk assessment such animal data need to be extrapolated to the human situation. In risk assessment procedures uncertainty factors are used to account for interspecies variation (extrapolation from animal to man) and for variation within the human species. For both factors a value of to is often used, based on international guidelines. Hence, exposures below 0.00025 mg/kg body weight should not pose a risk for the human population.
The potential immunotoxicity of xenobiotics has been a matter of growing concern during the past two decades. A large number of research papers showed that many industrial and environmental chemicals as well as medicinal products or food additives, can adversely influence the immune competence of mammals, including man, but can also induce hypersensitivity and autoimmune reactions. In 1982, the US Environmental Protection Agency issued guidelines for the immunotoxicity evaluation of pesticides and this was followed by a recommendation from the Council of the European Communities to pay attention to immunotoxicity during the safety evaluation of new medicinal products in 1983. Presumably because these regulatory texts were either premature (EPA) or grossly general (CEC), they have never been implemented. Surprisingly, no guidelines on immuno toxicity evaluation have been published until very recently and therefore, immunotoxicity evaluation remains largely unregulated today.
There is considerable evidence that cancer development in humans and experimental animals includes different stages that are results of interactions between target cells and various endogenous/exogenous factors. In order to protect man from chemically induced cancer, the development of suitable test systems for the detection of carcinogenic potency has been and still is an important task of applied toxicological research. Conventional cancer bioassays in which rodents are exposed to a certain chemical over two years is commonly used to investigate possible carcinogens. This test procedure is costly with respect to time and money and makes use of a large number of animals.
Allelism has been found in human glutathione S-transferase (GST) genes of the alpha, mu, theta and pi families with the best characterised examples being those in mu class GSTM1 and theta class GSTT1. Isoenzymes encoded by these genes catalyse the detoxification of various reactive toxic and mutagenic compounds including epoxides resulting from the cytochrome P450-mediated metabolism of polycyclic aromatic hydrocarbons as well as lipid and DNA products of oxidative stress (Hayes and Strange, 1995, Smith et al, 1995). Homozygosity for null alleles or those encoding low activity variants are likely therefore, to be associated with a biochemical consequence. However, while accumulating evidence suggests the importance of different GST, it remains unclear precisely which in vivo processes are influenced by these polymorphisms. In this chapter we discuss firstly, a new polymorphism in GSTM3 and secondly, the role of GST polymorphisms in determining cancer susceptibility with particular reference to allelism at GSTMI, GSTT1 and GSTM3 and their interactions with cytochrome P450 (CYP) genotypes in basal cell carcinoma of skin (BCC).
Two transgenic in vivo mutation assays are described which are based on LacZ (Muta Mouse) and LacI (Big Blue) shuttle vector systems. Their utility has already been explored by a number of investigators including our laboratory. The evaluation of data derived from these assays confirm that they offer a practical method for studying mutagenic activity and mechanism in a wide range of tissues including those of the respiratory and gastrointestinal tract. Therefore, these transgenic mutation assays are valuable tools to assess the organotropic effects of genotoxic carcinogens.
Several recent studies indicate declines in sperm production, as well as increases in the incidence of genitourinary abnormalities such as testicular cancer, cryptorchidism and hypospadias (Toppari et al., 1996). It is not known if these effects are due to exposure to chemical pollutants or if other ethiological factors are involved. Animal studies indicate that chemicals will induce such effects by various genetic, epigenetic or non-genetic mechanisms. Recently, much attention has been focused on embryonic/fetal exposure to oestrogen-mimicking chemicals (Toppari et al., 1996). However, the possibility that chemicals may cause reproductive toxicity by other mechanisms such as interactions with DNA, should not be ignored. DNA damage in germ cells may lead to the production of mutated spermatozoa, which in turn may result in spontaneous abortions, malformations and/or genetic defects in the offspring. Regarding the consequences of DNA alterations for carcinogenesis it is possible that genetic damage may occur germ cells, but the consequences are not expressed until certain genetic events occur in postnatal life. Transmission of genetic risk is best demonstrated by cancer-prone disorders such as hereditary retinoblastoma and the Li-Fraumeni syndrome. A number of experiments indicate that germ cells and proliferating cells may be particularly sensitive to DNA damaging agents compared to other cells (Masters et al., 1993; Holme et al., 1997).
The formation of covalent adducts to cellular macromolecules, including proteins, phospholipids, and DNA or RNA, is associated with the exposure of humans and animals (most probably to any given living organism) to a large number of xenobiotics, including drugs as well as occupational and environmental pollutants (Hinson and Roberts, 1992; Park and Kitteringham, 1990; Nelson and Pearson, 1990). Only few parent compounds form, based on their intrinsic chemical reactivity, such adducts spontaneously; the majority of compounds do form adducts to cellular macromolecules only after metabolic activation to reactive intermediates (Park and Kitteringham, 1990; vanWelie et al., 1992). From an immunological point of view, adducted cellular target molecules constitute “modified self” or even “non-self” structures, which might provoke immune responses against themselves (Allison 1989; deWeck 1983). In general, low molecular mass organic compounds (<l000 Da), such as most drugs, are thought to be non-immunogenic per se. However, many of these compounds (coined haptens) may become immunogenic, when covalently linked to a macromolecular carrier such as a protein. Once formed, drug-carrier conjugates might act as immunogens and elicit immune responses at the humoral level, at the cellular level, or at both levels (Allison 1989; deWeck 1983). These immune responses might be directed against at least three different types of antigenic determinants. First, haptenic epitopes may include the derivative of the xenobiotic (i.e. hapten) bound to the carrier molecule. Second, new antigenic determinants (NAD) may comprise newly created linear or conformational structures on the carrier molecule elicited upon binding of the hapten to the carrier molecule. Last, cryptic autoantigenic determinants of the carrier molecule, which normally are seen as self or are ignored, could bypass the mechanisms of immunological self-tolerance as a consequence of hapten binding (Allison, 1989; Blooksma and Schuurman, 1989; Roitt et al., 1985). An immunological basis for the development, in susceptible individuals, of a number of severe, sometimes life-threatening adverse effects after therapeutic
Variability among individuals in their responses to toxic chemicals arises from several sources, the most important of which are genetic differences, environmental influences (including maternal effects and historical factors) and measurement error. Effective risk assessment requires that estimates of toxicant response (e.g., LD50, EC50, LOEC, NOEC) are precise--that is, have narrow confidence limits-, repeatable--that is, different laboratories must obtain the same or very similar result-, and accurate--that is, they must provide a reasonable approximation of the effects of toxicants on real ecological systems. Determining which of the above-mentioned sources of variability has the greatest influence on toxicant response has implications for both the design and interpretation of ecotoxicological tests. If, for example, genetic influences are of overriding importance, controlling genotype (by using clones or inbred strains) can lead to greater precision but at the expense of accuracy when the objective is to estimate toxicant response for the species as a whole. Likewise, if environmental influences are of primary importance in controlling the response to toxicants, performing experiments under a standard temperature, light, and food regime may provide highly repeatable test results that have little relevance to the responses of populations in nature. Although there is little doubt that the development of standard ecotoxicological test guidelines (e.g., by the OECD), that control genetic and environmental sources of variability, has led to improvements in the practice of risk assessment, further advances will require a more sophisticated approach for dealing with these sources of uncertainty. There is a need for more systematic approaches for quantifying the sources of variability in toxicant response and for formally combining the error associated with each source in key risk assessment endpoints.
Excitotoxins are a special group of neurotoxic substances that excite somatic and dendritic receptors in such a way that the neurons may die. All excitotoxins are in principle agonists of glutamate receptors in the brain and are structurally related to glutamate. The excitotoxins can be administered systemically before the blood brain barrier is fully developed (at day 10 in rats) or they can be injected locally into the brain. In the latter case, they produce the so called axonsparing/dendrito somatic lesions.
Stress refers to a physiological and emotional state of man and higher animals in which the autonomic regulation is overstrained and temporarily disturbed under the impact of conflicting stimuli. Stress activates, invigorates, acts life-sustaining, and initiates and drives adaptive changes towards improved fitness. While the positive action is commonly underestimated, much attention is given to the discomfort and the strain of efforts required during coping. The label of stress as being bad and the core of suffering has been applied with particular empathy to laboratory animals, for they are kept in captivity and are exposed to experimental procedures. The husbandry conditions to which the animals are adapted are commonly standardized. This applies to procedures for subacute and chronic toxicity testing. Acute toxicity tests are the classical example of stress research in which the demands on the organism exceed the limits of its regulative capacity. Stressors are: the test compound, the procedure proper and preceeding treatment of the animal. The experimental stress contributes to model the real situation. The weighting between the stressors may modify the outcome of the test.
There are scientific, ethical and financial reasons why toxicologists seek alternatives to the use of animals in toxicity testing. The scientific drive to develop alternatives is aimed at improving the process of hazard assessment which currently relies predominantly on the results of animal tests. Ethical concern for the welfare of animals is a potent driving force for the development of alternatives. However, the protection of human health also places strong ethical demands on the toxicologist to provide the best available hazard information and currently it is believed to be that based on the results of animal experiments. Alternative testing which is cheaper and carried out in a shorter time, provided that the concern for scientific and ethical standards is met, is beneficial both to industry and to the research community
The development of the preimplantation embryo seems morphologically very simple, and embryologists previously assumed that an embryo that developed to the blastocyst stage was fully capable of normal development after transfer to the uterus of a recipient female. This complacency was disturbed by reports that exposure of early embryos to mutagens such as methylnitrosourea led to fetal abnormalities, decreased birth rates, and decreased life-span. Even more disturbing are recent reports that culture of early embryos in supposedly benign conditions can adversely affect their subsequent development. Techniques have been developed for the production of cattle and sheep embryos by in-vitro fertilization and by cloning. Such embryos must be cultured for several days before they can be transferred, and, in some cases, this has been related to abortion, very high birthweight, physical abnormalities and peri-natal mortality of the calves and lambs. This syndrome may result from an unbalanced development of the trophoblast relative to the inner-cell mass, possibly related to the presence of serum, glucose, or ammonium in the culture medium. An analogous phenomenon has been observed in human in-vitro fertilization where babies from single pregnancies have below-normal birth-weight. There is also evidence to suggest that the in-vitro environment of the gametes before fertilization can affect subsequent embryonal and fetal development. Exposure of mouse oocytes to vitrification solutions has been shown to lead to fetal malformations, and treatment of bull sperm with glutathione improves early embryo development. The common thread in these diverse observations is that development can be affected by events that occur long before any defect is apparent. Consequently, the production of a morphologically normal embryo is no guarantee that fetal development and post-natal life will be normal. This is of immediate concern in human reproductive medicine due to the increasing use of sperm injection for fertilization, and the emergence of in-vitro oocyte maturation. Further development and application of reproductive techniques would benefit from a toxicological evaluation of risk factors and exposure limits.
Evidence that a number of chemicals affect wildlife populations or individuals via interaction with endocrine systems has been increasing in recent years. Worldwide effects of tributyltin from antifouling paints on mollusc populations (Langston 1996; Oehlmann et al. 1996), effects of polychlorinated biphenyls on Baltic and Wadden Sea seals (Reijnders 1986; Brouwer et al. 1989), masculinisation of North American fish affected by pulp and paper mill effluents (Howell 1980; Munkittrick et al. 1991, 1992), feminisation of male fish in British rivers receiving effluents from waste water plants (Jobling and Sumpter 1993; Purdom et al. 1994; Harries et al. 1997), demasculinisation of alligators in Lake Apopka after a chemical spill (Guillette et al. 1994, 1995a,b, 1996) and effects on North American birds (Fry and Toone 1981; Fry 1995) are some of the most prominent and best documented examples, all attributed to chemicals exerting endocrine disrupting effects.
Many natural dietary phytochemicals found compounds found in fruits, vegetables, spices and tea have been shown in recent years to be protective against cancer in various animal models. In the light of the potential impact of these compounds on human health it is important to elucidate the mechanisms involved. We therefore developed and characterized relevant in vitro models using immortalized human epithelial cell lines derived from target tissues in carcinogenesis, such as lung, liver and colon. Assays were established, allowing the evaluation of the cytotoxic and genotoxic effects of various procarcinogens, including nitrosamines, mycotoxins and heterocyclic amines on these metabolically-competent human epithelial cell lines. These cellular models appeared to be a useful tool to study the capacity of certain food components to block the initiation stage of carcinogenesis. The ability of carnosol and carnosic acid from rosemary as well as the synthetic dithiolethione, oltipraz, to block the formation of DNA adducts, and their effects on the expression of phase I and phase II enzymes was investigated. We have observed that both rosemary extracts and oltipraz inhibited benzo(a)pyrene- or aflatoxin B,-induced DNA adduct formation by strongly inhibiting CYP45° activities and inducing the expression of glutathione S-transferase. These results in human cell models give some insight into the different mechanisms involved in the chemopreventive action of both natural and synthetic compounds in relation to phase I and phase II enzymes.
A new area of ecotoxicology, now usually called endocrine disruption, has arisen in the last few years. However, despite the present topicality of this issue, some of the best documented examples of endocrine disruption were reported a decade or more ago (see, for example, Fry, 1995). The issue is concerned with the effects of chemicals that mimic endogenous hormones on the physiology of exposed wildlife and humans. As many of these xenohormones mimic steroid hormones, especially oestrogens and androgens, most of the reported effects have involved effects on the reproductive system of exposed organisms. Many of these reported effects on wildlife have concerned aquatic, rather than terrestrial, organisms (even the well-documented effects on birds are primarily concerned with water birds, which feed predominantly on fish); the reproductive abnormalities seen in alligators living in some lakes in Florida (Guillette et al, 1994), and the oestrogenic effects on fish reported in British rivers (this example is discussed in detail below) provide good examples of the type of effects observed in aquatic organisms. This predominance of effects on aquatic organisms could reflect an unconscious bias of the interests of research scientists (are there more wildlife biologists interested in aquatic, rather than terrestrial, animals?), but is perhaps more likely a consequence of the fact that the aquatic environment is the ultimate “sink” for the intentional or unintentional disposal of much waste. Thus, this brief review is focused exclusively on the aquatic environment, and particularly on the effects on fish. However, I have attempted to emphasize the general nature of the phenomena illustrated by studies on endocrine disruption in fish, because they apply to most, if not all, examples of endocrine disruption in all wildlife.
A complex chemical mixture is defined as a mixture that consists of tens, hundreds or thousands of chemicals, and of which the composition is qualitatively and quantitatively not fully known. In contrast, a simple mixture consists of a relatively small number of chemicals, say ten or less, and the composition of which is fully known. In the present paper a number of options for hazard identification and risk assessment of complex chemical mixtures is discussed, and a scheme aimed at selecting the most appropriate approach for each (type of) complex mixture is presented. A conspicuous element of this scheme is the dichotomy of complex mixtures into mixtures that are readily available and mixtures that are virtually unavailable for testing in their entirety. Another characteristic aspect of the scheme is the inclusion of the "top-ten" and "pseudo top-ten" approaches, which in essence are ways to select the, say ten, most risky chemicals or pseudocomponents to be dealt with as a simple chemical mixture.
An essential step to protect human health from chemicals at the workplace or in the environment was to measure their concentration in the environment and to correlate them with adverse health effects. Tolerance values, like the maximum workplace concentrations (MAK-values), are established on that basis. A next step to improve exposure control was the introduction of human biomonitoring. Measuring the parent chemical or its stable metabolites in biological material, like blood or urine, allowed to assess the internal stress as compared to the external stress given by environmental monitoring. However, the biological activity of chemicals, of genotoxic carcinogens in particular, depends mostly on reactive intermediates generated in the course of metabolic activation. The biologically active dose cannot be assessed from stable metabolites usually measurable in biological metarial, but from the effects they produce. In contrast to „biomarkers of exposure“ the expression „biomarkers of response“ has been introduced. In this category two kinds of markers exist: biochemical effect markers and biological effect markers. The former markers are mainly protein and DNA adducts, the latter mutations, micronuclei, chromosomal aberrations or sister chromatid exchanges. Whereas the biochemical effects are not considered pathological per se, the biological effects can be seen one step further down to disease. However, as a dosimeter for the biologically active dose, adduct measurements are more specific and more sensitive than the biological endpoints. Moreover, they represent the strain of individuals best.
