by Dr. Jorge Herkovits

SUMMARY

Due to their high toxicity and persistence, POPs produce cancer and even death at such low concentrations that they have been identified as the most toxic substances produced by man. In sublethal concentrations, they produce severe adverse effects, especially in terms of reproduction, immune response, neuropsychological alterations, and endocrine disorders, particularly in the case of perinatal exposure. Although the main mechanisms for the action of these substances are probably mediated by the Ah receptor, there are probably other mechanisms, for example, those related to alterations in dopamines. In order to estimate the risk of adverse effects from these substances on human health and the ecosystem in general, studies should be conducted on the basis of biomarkers for exposure, impact, and susceptibility in order to simply, rapidly, and economically recognize potential adverse effects before the symptoms appear. Assessment of the risks from POPs to human health and the ecosystem in the MERCOSUR region would be an essential step in arriving at decisions to promote sustainable development. Bearing in mind the serious and frequently irreversible adverse impact of POPs on human health and ecosystems and following the adage that "an ounce of prevention is worth a pound of cure," it is advisable to institute all measures conducive to limiting the production/emission of these chemical substances as much as possible.

INTRODUCTION

Persistent Organic Pollutants (POPs) are substances that produce very adverse effects on both the ecosystem and human health due to: i) their very high toxicity, expressed in numerous pathologies ranging from behavioral changes to malignant neoplasms; ii) their prolonged persistence in contaminated ecosystems and in individuals because of their extremely limited biodegradability and iii) their deep penetration in even the remotest areas such as the Arctic. It is very likely that the main reason why organochlorine residues, such as DDT, continue to persist in the environment and even to increase, as seen in the Arctic ecosystem, is that the worldwide use of many of these products is more widespread now than in 1970 when they were banned in the United States due to their severe impact on human health and ecosystems.

Among the persistent organic pollutants, special attention must be given to dioxin-like compounds (PCDDs and PCDFs). These substances do not exist in nature and are not intentionally produced by man but are the result of activities such as the manufacture of other substances (e.g., PCBs and chlorinated phenols), the incineration of the waste produced by cities, hospitals, and industry; toxic waste; the combustion of petroleum derivatives, etc. Due to their severe toxic effects, these substances constitute a very high risk, particularly for those who live or work near where they are produced or ultimately concentrate, and such places may be as far removed from where they are emitted as the Arctic region.

The toxicity of these substances depends on the species, sex and age of the organisms affected. Notable adverse effects include loss of bodyweight, hepatotoxicity, gastric disorders, porphyria, carcinogenesis, immunotoxic effects, and changes in development and the reproductive function and in the nervous and endocrine systems. Although the reference value in the human population has been found to be only 20 ng/kg of bodyweight (in bodyfat), it should be noted that in cases that are exceptional for the time being the level of these substances in organisms has exceeded 50 ng/kg. 1 The fact that only 1 ng/kg a day can have an adverse impact on reproduction, while concentrations ranging from 0.01 to 0.1 ug/kg of bodyweight can produce hepatocellular carcinoma gives us an idea of the toxicity of these compounds. An additional example of the diversity of toxic effects depending on the chemical structure of each substance is the case of Aroclor 1254, where the exposure of pregnant women to 25 ug/kg resulted in a 15% reduction in the birthweight of their babies, which subsequently lost more weight, while other changes were reported in the fingers and nails and in the structure of the kidney and pancreas. The toxicity of these products varies markedly by species: 0.6 mg/kg of bodyweight of TCDD represents an LD₅₀ for a guinea-pig while a similar effect in a hamster requires 5.051mg/kg. However, it must be pointed out that the toxic effects on embryos occur with similar maternal doses regardless of species. 2

The biochemical effects of TCDD can be divided into three classes: i) Metabolic alterations due to enzymatic changes; ii) Alterations in homeostasis resulting from changes in hormones and their receptors; and iii) Alterations in growth and differentiation as a result of changes in growth factors and their receptors. 3 The current paradigm is that the toxic effects of the dioxin-like compounds (DLCs and TCDD) are primarily produced through the Ah receptor but other mechanisms such as binding with hormones, enzymes, and changes in the concentration of biological amines have also been reported. A working hypothesis could be that TCDDs and DLCs act as endocrine disrupters or environmental hormones; this seems to be consistent with the multiple adverse effects they produce during the perinatal period, especially in the immune, endocrine, and reproductive systems and in neurological development. Acting as hormones or endocrine disrupters, these substances may also produce neoplasia during the developmental period or in adults.

Embryonic development represents one of the most complex processes in the life cycle and is exceptionally sensitive to toxic substances. In addition, each stage depends largely on the normal completion of the previous stage. Chemical substances interfere with normal processes through mechanisms such as interactions with genetic material, with the components of cell membranes, enzymes, receptors, etc. Due to the differential susceptibility to toxic substances at the various stages of embryonic development, 4 when exposure to these toxic substances coincides with the most sensitive periods of development, adverse effects can be exceptionally severe and may even interrupt basic vital functions, resulting in the death of the embryo, or produce sublethal effects consisting of malformations and functional alterations. It is important to point out that, as with other substance that produce teratogenic effects, all these effects occur at significantly lower concentrations than those that are toxic for the mother. 5, 6 On the other hand, it should be noted that the developing embryo has an exceptional ability to regulate and regenerate, as has been demonstrated, for example, in the ability of embryos affected by exposure to lead to regulate shape and to recover. 7, 8 For this reason, malformations or functional alterations reported in newborns should be regarded as the damage remaining from a much more compromised situation suffered by the embryo due to chemical stress, something that happened without our knowledge.

Although POPs are stored primarily in bodyfat, it is important to note that they pass through the placenta and are excreted in breast milk, increasing exposure to these substances throughout the breast-feeding period. Children, in addition to requiring more calories per kg of weight, are also at greater risk of being affected than adults, due to a lower barrier to absorption through the skin, gastrointestinal tract, and lungs. Thus, it can be estimated that children absorb approximately 100 times more of these substances than adults and also have lower levels of detoxifying enzymes. 9

Based on the information above, it would seem that all organisms in the ecosystem including humans have absorbed dioxin-like compounds at levels that could be assumed to represent some risk to their own health or that of their progeny. Bearing in mind the high susceptibility to toxic substances during embryonic development and the perinatal period, this report will emphasize the adverse effects of this type of compound during these periods, particularly on the nervous, reproductive, immune and endocrine systems. It will also consider the need to anticipate potential adverse effects on human health and life in general through studies based on environmental monitoring and biomarkers that will allow us to obtain information on exposure levels and adverse effects. Finally, the need to conduct studies in the MERCOSUR region assessing the risks to the ecosystem and human health associated with POPs is part of the objective of achieving sustainable development.

EFFECTS ON REPRODUCTION OF PERINATAL EXPOSURE TO DIOXIN-LIKE COMPOUNDS

The adverse effects of dioxin-like compounds on the development of the reproductive system in both men and women have been widely documented. 10, 11 For example, very limited development and even the failure to reach sexual maturity was demonstrated in fish, birds, and mammals exposed to these substances. A mere 1 ug of TCDD on the 15^th day of gestation produces the de-masculinization of rats, including a reduction in the number of sperm cells and anomalies in sexual behavior after puberty. 12 Adverse effects on reproductive development have been reported in females as well, for example, malformations of the clitoris and the absence of a vaginal opening, suggesting that these malformations can be considered a result of estrogen effects. 13 The exposure of animals after birth also results in alterations such reduced size and weight of the testes and spermatogenesis and fertility disorders,10 while reduced fertility and a higher incidence of miscarriages were detected in females. Females were more severely affected in their reproductive capacity, due apparently to the presence of clinical symptoms of endometriosis. 14 Effects in humans similar to those described for other mammals have been reported, and it is important to note out that in utero exposure can lead to delays in puberty, in the development of secondary sexual characters and in growth. This is even more the case after puberty, where we may see reduced fertility, abnormal menstrual cycles, and premature menopause. In males, changes were also detected in the quality of semen. There are some pertinent epidemiological studies, such as the case of the Inuit (Eskimos) who live in the arctic region of Quebec, have a diet consisting mainly of marine mammals, and among whom the concentration of dioxin-like compounds (and PCBs) in breast milk was 7 times higher than among women living in the south of the same province. 15

EFFECTS ON NERVOUS SYSTEM DEVELOPMENT AND BEHAVIOR

Perinatal administration of PCBs (e.g., compounds 18, 28, 118 and 153) results in behavioral alterations similar to those produced by injuries to the prefrontal cortex and are apparently mediated by a dopamine deficiency. Perinatal exposure in rats and monkeys to PCBs produces deficits in memory and learning capacity. 9 The behavioral evaluation of children exposed to PCBs during gestation whose mothers had eaten contaminated fish from Lake Michigan showed reduced precision in intellectual processes. Another study conducted in Taiwan revealed that PCB and PCDF produced deficiencies in the central nervous system, demonstrated in a difference of from 4 to 8 points in intelligence tests compared to the controls. 16 These adverse effects were not reversed in subsequent years and were also verified in younger siblings. For the dioxin-like compounds specifically, we should note the case of contamination in Times Beach, Missouri, where from 1971 to 1983 when the area was evacuated, numerous cases of nervous system changes were recognized that were apparently associated with injuries to the frontal and prefrontal areas.

The neurological changes most frequently recognized in adults exposed to these substances include sexual dysfunction (lack of libido and impotence), headache, neuropathies, alterations in vision and visual memory, the sense of taste and smell, and psychiatric effects such as changes in sleep, depression, loss of vitality, and anxiety. Changes in human behavior may result from very complex interactions between the neuropsychological and neuroendocroine systems. For example, some PCB compounds can reduce dopamines in the cerebral cortex of adults as well as the T4 levels in individuals born to women exposed to these substances. 17

ENDOCRINE EFFECTS

The studies conducted in animals exposed to TCDD detected reduced libido, reduced testicular size, and alterations in male hormones; these alterations were also confirmed in humans. Perinatal exposure to TCDD may cause malformations in the reproductive system, and concentrations as low as 0.064 to 1 mg/kg administered on the 15^th day can cause a later reduction in spermatogenesis and alterations in sexual behavior depending on the dose. 1 The effect of TCDD also extends to differentiation of the central nervous system due to alterations in testosterone in the intrauterine period, reducing masculinization. Regarding thyroid function, the administration of PCB compounds during the perinatal period reduces the level of thyroid hormones but it should be noted that some PCB compounds (e.g., 118, 128, 153 and 180) and dioxin-like compounds do not always significantly affect the level of thyroid hormone. 18

In summary, perinatal exposure to dioxin-like compounds may produce endocrine effects in the absence of signs of toxicity in the mother. Adverse effects include 1) deficiency in the development the depends on male hormones and in neurological development, and 2) alterations in homeostasis of the thyroid function. These effects may be interrelated. Epidemiological studies indicate that exposure in utero or through breast-feeding may be associated with a reduction in neurological and physical development. 18

IMMUNOLOGICAL EFFECTS

The immune system is a network of cells and substances that participate in combating and eliminating all elements foreign to the organism or pathological (from viruses to tumor cells). Alterations in the immune response may result, in the case of immunosupression, in an increase in infectious diseases and tumors, while an increase in the activation or reduction of suppression that normally exists may result in allergic reactions, hypersensitivity and autoimmune diseases. The immunotoxic effects of TCDD in the perinatal period on the thymus and particularly on the immune response mediated by the T cells and macrophages are severe, while the B cells seem to be less sensitive. 19 The effects of dioxin-like substances can modify the maturation of cells in the thymus and also affect the migration of cells from the bone marrow to the thymus. Other notable adverse effects include: i) alterations in the ratio between the thymic "helper" and "suppressor" CD4/CD8 lymphocytes (2=normal; <1 compromised); and ii) effects on the CD29 thymic lymphocytes associated with memory when activated by antigens. The Inuit people in the Canadian arctic represent one of the better known cases of the adverse effects of dioxin-like compounds on the immune system. Among the Inuit, the incidence of infections such as meningitis, and otitis is 20 times higher during the first year of life than for individuals removed from the foci of contamination. Because these substances pass to the breast milk, breast-feeding has an aggravating effect on appropriate immune response in the perinatal period. 20

BIOMARKERS OF EXPOSURE TO AND EFFECTS OF DIOXIN-LIKE COMPOUNDS

A biomarker is defined as a biochemical, morphological, or functional change that indicates exposure, response, or potential susceptibility to an environmental substance or agent. Biomarkers are generally used to increase sensitivity, specificity, or response capacity, the objective being to establish a causal relationship between a chemical agent and its effect on an organism. When assessing exposure to dioxin-like compounds, their highly lipophilic nature permits these compounds to be measured in bodyfat and milk. It also allows us to recognize bioamplification phenomena from these compounds in organisms at increasingly higher levels of the food chain and to assess the level of residuals of these compounds in the organism. Biomarkers of effect represent a rapid and economical way to recognize pathological processes before clinical symptoms become apparent. This information is very important for public health. particularly in terms of diseases with extended latent periods that reduce life expectancy (e.g., cancer) or limit the productive period, such as changes in reproduction, increased neurological and immunological disorders, or the delayed effects of POPs. Significant biomarkers of effect include the sperm cell count for reproduction, the CD4-CD8 ratio for immunological factors, the T4 level for endocrine factors, dopamine for neuroendocrinological factors and tests of cognitive and motor skills for neurological behavior. At the molecular level, studies are based primarily on the high affinity between the Ah receptor, a cytoplasmic protein, and dioxin-like compounds and other persistent organic pollutants such as coplanar PCBs. The binding of the dioxin-like compounds with the Ah receptor seems fundamental to a broad spectrum of effects such as teratogenesis, carcinogenesis, immunotoxicity and other biochemical changes such as detoxifying enzymes and growth factors. The receptor-dioxin complex produces an increase in the transcription of a group of genes that includes CYP1A1 that would seem to represent one of the mechanisms for the action of these substances. 21 It should be noted that in a bioassay developed recently on the basis of the expression of the luciferase gene (the CALUX test) the limit of detection of TCDD expressed as a toxic equivalency (TEQ) is less than 0.1 fmol TEQ. 22 However, none of these biomarkers of effect and susceptibility allow us to indisputably conclude that the cause of a given alteration is dioxin-like compounds, and it is therefore important to correlate this information with data on exposure and epidemiological studies. There are precedents for this type of research such as the relationship between the metabolism of the thyroid hormone in infants whose mothers have different levels of PCBs and PCDD in their milk, 23 the study of Inuit children (Canadian Arctic) measuring dioxin-like compounds in breast milk, serum and placenta, enzymatic induction mediated by the Ah receptor, stress proteins, the ratio of CD4-CD8 lymphocytes, sister chromatid exchanges and T3, T4 and TCH hormonal levels. 15

RISK ASSESSMENT

Risk assessment is a process that allows us to scientifically determine the likelihood that a given event will occur, in this case adverse effects on the ecosystem or human health due to exposure to POPs. The assessment is divided into various stages: i) formulation of the problem; ii) characterization of the exposure; iii) characterization of the effects; and iv) the risk assessment as such. Formulation of the problem means establishing goals, scope, and objectives. This stage considers the physical/chemical nature of the compounds, the ecosystems, the populations potentially affected, and the effects observed or anticipated and identifies the ultimate objectives of the evaluation and the method for taking measurements. For this purpose, parameters are selected that are significant for the final evaluation, e.g., the objective of protecting wildlife, agricultural and livestock assets, or other assets with high economic value, and human health if the risk assessment targeted this objective. By characterizing the exposure, the interaction between the POPs selected and the organisms in the environment of interest for the study is evaluated. The assessment process involves a profile of exposure, the amount and the spatial and temporal distribution of the substance(s) being studied. Exposure depends on the concentrations of the substances in the environment, their bioavailability, and the characteristics of the organism. For humans, the greatest source of exposure is food, with average values of 0.3 pg of TCDD/kg/day and 3 to 10 pg/kg/day of PCB. 1 Estimating exposure involves analyzing the movement of substances in the environment and biota and their degradation. To characterize effects, it is important to consider three variables: the concentration of exposure, the time of exposure, and the response. Given that dioxins and related compounds are usually present in complex combinations of substances with different levels of toxicity, the concept of a toxic equivalency factor has been developed. This is a relative measure of each one or the combination as a whole in terms of TCDD toxicity. To measure the effect of a toxic substance, a fundamental relationship is the dose-response curve. Although several studies seem to indicate a dose-response ratio for POPs, quantitative PCR techniques having been used to detect sensitivity 100 times greater to such substances than that recorded through enzymatic, immunological and embryonic development effects tests, it seems irresponsible to suggest a threshold concentration for adverse effects of these substances. 24

Risk assessment as such is the final stage in the procedure. Its objective is to determine the likelihood that adverse effects will occur as a result of exposure to the POPs being studied. The profile of effects and the data on exposure are the fundamental information for this risk assessment. During this process, it is also important to analyze the evidence underlying the exposure data and the information on effects compiled in the earlier phases. Assessing risk consists of comparing the profile of exposure and the profile of effects as well as the uncertainties associated with these profiles. Based on the available data regarding exposure to this type of chemical substance and the effects produced in populations, we can anticipate that during the development and perinatal periods, living organisms, including humans, are particularly at risk of suffering adverse effects from POPs. Another at-risk population is workers and the population living close to where these substances are produced, released and/or amassed and those who eat what they catch. 3, 25, 26, 27 The magnitude of the potential adverse effect of these substances on health is revealed by the fact that, as environmental hormones alone, they may be implicated in three of the most common cancers in women in the United States. 28 Risk assessment may also extend to recognition of the potential adverse effects of the spread of POPs in ecosystems and their effects on wildlife. 29, 30 Studies assessing risk from POPs in the MERCOSUR countries would represent significant progress toward the objectives of caring for the environment and the health of the population, especially given the very high levels of toxicity in our natural resources in regions with high population density. 31, 32

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