Human exposure to metals. Pathways of exposure, biomarkers of effect, and host factors

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Ecotoxicology and Environmental Safety 56 (2003) 93–103

Human exposure to metals. Pathways of exposure, biomarkers of effect, and host factors Jaqueline Caldero´n, Deogracias Ortiz-Pe´rez, Leticia Ya´n˜ez, and Fernando Dı´ az-Barriga
Laboratorio de Toxicologı´a Ambiental, Facultad de Medicina, Universidad Auto´noma de San Luis Potosı´, Avenida Venustiano Carranza No. 2405, Col. Lomas los Filtros, CP 78210, San Luis Potosi, SLP, Mexico Received 20 March 2003; accepted 20 March 2003

1. Introduction

1.1. Sources and environmental media

The Agency for Toxic Substances and Disease Registry (ATSDR, 1992) has defined an exposure pathway as the process by which an individual is exposed to contaminants that originate from a specific source. An exposure pathway consists of the following five elements:

Metals are widely used, and their sources are numerous. Major sources are smelters, mining activities, hazardous waste sites, and even natural sources. Pyrometallurgical nonferrous metal production is the major global source of airborne arsenic, cadmium, copper, zinc, and lead (Nriagu and Pacyna, 1988). The metallurgicals are also primary sources of cadmium, nickel, and lead for aquatic ecosystems, whereas for soil the most important sources of metals worldwide are mine tailings, smelter wastes, and atmospheric fallout (Nriagu and Pacyna, 1988). As a result, metal concentration in air, water, soil, and food (fish, grains, etc.), may be higher than background levels in areas located in the vicinity of smelters and mines. Arsenic, cadmium, and lead contamination have been reported in smelter and mining areas located in different countries, among them Poland (Dunnette et al., 1994), Russia (Bustueva et al., 1994), the United States (Hwang et al., 1997), Mexico (Dı´ az-Barriga et al., 1993a; Benin et al., 1999), Bolivia (Dı´ az-Barriga et al., 1997a), Chile (Rivara et al., 1997), Peru (Ramı´ rez, 1986), Brazil (Malm, 1998), the Phillippines (Appleton et al., 1999), and Zimbabwe and Tanzania (Van Straaten, 2000). Hazardous waste sites are also important sources of metals. For example, in the United States, inorganic compounds were reported to be present at 65% of the hazardous waste sites assessed by ATSDR through 1992 (ATSDR, 1993). At those sites, inorganic compounds were most often reported to be present in groundwater (77% of the sites) and in soils (58%). There were fewer reports of their presence in surface water (39%), air (6%), and biota (6%). Natural sources are very important for groundwater contamination. Aquifers polluted with arsenic have been reported in several countries (United Nations, 2001), including Argentina, Chile, Mexico, Bangladesh, India, China, and Taiwan. Additionally, high levels of fluoride

1. Source of contaminant release into the environment. 2. Environmental media (including groundwater, surface water, air, soil, sediment, household dust, biota) serve to move contaminants from the source to points where human exposure can occur. 3. Point of exposure (a location where humans contact a contaminated medium, such as a playground, water body, well water, and food services). 4. Route of exposure (means by which the contaminant actually enters or contacts the body, such as ingestion, inhalation, and dermal absorption). 5. Receptor population (persons who are exposed to the contaminants of concern at a point of exposure). It should be noted that an exposure pathway is not simply an environmental medium (e.g., air, soil, water) or a route of exposure. Rather, an exposure pathway includes all the elements that link a contaminant source to a receptor population. However, for exposure to be measured, variables such as intensity, frequency, and duration of the contact with the polluted media are crucial. Furthermore, the metal of concern must be bioavailable. As a result of the exposure, and depending on both the amount of chemical that actually enters the body, and host influences (e.g., five nutritional status), a biological effect may become manifest in the exposed population.


Corresponding author. Fax: +52-444-8262354. E-mail address: [email protected] (F. Dı´ az-Barriga).

0147-6513/03/$ - see front matter r 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0147-6513(03)00053-8

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in groundwater have been reported in some nations (UNICEF, 2001); also, very high concentrations have been identified in aquifers located in regions of Mexico (Dı´ az-Barriga et al., 1997b), India (Choubisa, 2001), and China (Teng et al., 1996). Smelters, mining activities, hazardous waste sites, and polluted natural aquifers may be major sources of metals; still, in some countries, folk medicines, traditional dyes, lead-glazed ceramics, etc., are sources for a vast number of individuals. 1.2. Metal mixtures at the exposure points Around smelters and mining areas, metals are commonly present as a mixture. For example, in Mexico, in the vicinity of smelters (Dı´ az-Barriga et al., 1993a, 1997c) and mining areas (Mejia et al., 1999), lead and arsenic are often present as a mixture in soil, air, and household dust. Similar conditions have been observed in hazardous waste sites. For example, in the United States, among the most frequent combinations of contaminants in soil, the mixture of cadmium, chromium, and lead, was identified in 12% of the sites assessed by ATSDR (1993). The mixture of arsenic, cadmium, and lead was identified in soil samples in 11% of the sites, whereas this same mixture was identified in water in 10% of the sites. Regarding natural sources, mixtures such as fluoride and arsenic are not uncommon in some regions of the world (Del Razo et al., 1993). Mixtures can influence expected adverse health effects because their components can individually attack the same organs or, together, overwhelm a particular mechanism the body uses to defend itself against toxic substances. Thus, metal mixtures can interact in the body in such a way that the combined toxicity is more serious than the individual toxicity of each metal alone. In this way, low doses that might not individually cause health effects, in concert may become a public health issue. 1.3. Receptor populations In view of the diverse environmental sources of metals, multiple scenarios of exposure can be expected. In each of them, high-risk populations can be described. However, children and pregnant women deserve special considerations—children because of their unique physiology and behavior, and pregnant women because of the inherent susceptibility of the fetus arising from transplacental transfer of metals in maternal blood. Human exposure to metals has been reported in children and women living in the vicinity of smelters and mining areas (Morse et al., 1979; Caldero´n-Salinas et al., 1996; Dı´ az-Barriga et al., 1993a, 1997a, c; Wasserman et al., 1997; Cikrt et al., 1997; Mejia et al., 1999; Hilts et al., 1998), in individuals working in hazardous waste

sites (Dı´ az-Barriga et al., 1993b), and in populations exposed to polluted groundwater (Grimaldo et al., 1995; Smith et al., 2000b). Depending on the level of exposure, metal-induced biological effects can be anticipated in the exposed population. Different biological effects for specific metals have been extensively described in the literature; however, less is known about the effects related to exposure to mixtures. This observation is relevant, because human exposure to metals is rarely limited to a single element. In response to the issues just discussed, we focus our analysis on some biological effects that have been described in the literature and that could be applied in the study of individuals exposed to metal mixtures. However, the objective of the paper is not an extensive review of the literature.

2. DNA damage and apoptosis Health risk assessment in areas polluted with metals can be improved by using biomarkers of effect. The development of biomarkers has given rise to the field of molecular epidemiology, which uses these laboratory measurements, rather than disease, to assess biological effects related to environmental exposure. In this regard, DNA damage, an effect induced by a variety of metals, can be used as a biomarker in sites polluted with mixtures. Chromium, copper, cobalt, nickel, cadmium, and arsenic are among the metals that may induce DNA damage. In the past few decades, new methodologies to assess DNA damage have been developed. Among them, the ‘‘single-cell gel electrophoresis technique’’ (comet assay) has not only become a very useful test for genotoxicity, but is also an invaluable tool for investigating the fundamental aspects of DNA damage and resulting cellular responses. It is being used successfully to monitor DNA damage in exposed human populations. The comet assay is capable of detecting DNA singlestrand breaks (SSBs), alkali-labile sites, DNA–DNA or DNA–protein crosslinking, oxidative DNA adducts, and SSB associated with incomplete excision repair sites (Tice et al., 2000). Relative to other genotoxicity tests, the advantages of the comet assay include its demonstrated sensitivity for detecting slight DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study (Tice et al., 2000). Using the comet assay, metals such as chromium (Blasiak and Kowalik, 2000), mercury (Ben-Ozer et al., 2000), sodium arsenite, and cadmium (Hartmann and Speit, 1994) have shown in vitro genotoxicity, and positive results were observed in animals treated with copper (Hayashi et al., 2000), lead nitrate (Devi et al., 2000), arsenic trioxide (Banu et al., 2001), or cadmium

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(Valverde et al., 2000). Furthermore, we have demonstrated that mice subjected to cadmium inhalation had DNA damage in several organs, with a distribution from high to low: on brain4bone marrow4 nasal cells4lungs4leukocytes4testicles4liver4kidneys (Valverde et al., 2000). In mice treated for different durations of exposure (1–4 weeks; inhalations were performed for 60 min twice a week—Monday and Wednesday), there was a positive correlation between DNA damage and cadmium concentration in lungs, liver, and kidneys (Valverde et al., 2000). DNA damage can result from a direct DNA–metal interaction. However, it has been shown that lead and cadmium were not able to induce DNA strand breaks in the plasmid pUSE amp+ or when incubated with naked DNA (Valverde et al., 2001). In this regard, the case of arsenic is more interesting. It has been reported that methylated trivalent arsenicals were able to nick and/or completely degrade phiX174 DNA in vitro (Mass et al., 2001), whereas sodium arsenite, sodium arsenate, and the methylated pentavalent arsenicals did not. Furthermore, using the comet assay in human lymphocytes, methylated trivalent arsenicals were found to be much more potent than any other arsenicals tested (Mass et al., 2001). Because methylated trivalent arsenicals were the only arsenic compounds that were observed to damage naked DNA and required no exogenously added enzymatic or chemical activation systems, they are considered to be direct-acting forms of arsenic that are genotoxic, although they are not necessarily the only genotoxic species of arsenic (Mass et al., 2001). For example, DNA damage was observed on stimulated human lymphocytes treated in vitro with sodium arsenite (Sordo et al., 2001). Thus, in addition to a direct interaction, DNA damage can arise by other indirect mechanisms such as DNA repair inhibition, induction of DNA–protein crosslinks, and oxidative damage. DNA repair is a system of defenses designed to protect the integrity of the genome; it has been suggested that deficiencies in this system probably lead to carcinogenesis (Berwick and Vineis, 2000). Current evidence suggests that DNA repair systems are very sensitive targets for nickel, cadmium, cobalt, and arsenic(III) (Hartmann and Speit, 1996; Hartwig et al., 1997; Hartwig, 1998), and although the mechanism could depend on the ability of toxic metal ions to displace zinc ions in zinc-finger structures of DNA repair enzymes (Hartwig, 1998), inhibition of DNA repair by arsenic is probably not the result of direct enzyme inhibition. It has been shown that purified human DNA repair enzymes are insensitive to arsenic; however, treatment of human cells in culture with arsenic produces a significant dose-dependent decrease in DNA ligase activity in nuclear extracts from the treated cells (Hu et al., 1998). The authors stated that

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this inhibition may be an indirect effect caused by arsenic-induced changes in cellular redox levels or alterations in signal transduction pathways (Hu et al., 1998). In contrast, inhibition of oxidative DNA repair was reported in cadmium-adapted alveolar epithelial cells, in which thiol-containing antioxidants such as metallothionein and glutathione were increased (Potts et al., 2001). Well-known and widely distributed DNA–protein crosslinkers are metal compounds such as arsenic (As2O3), chromate (chromium(VI)), nickel, cadmium, cobalt, and platinum (Wedrychowski et al., 1986; Merk and Speit, 1999). Two mechanisms have been suggested to explain this effect: a direct participation of the metal in complexing DNA with proteins (Miller et al., 1991) and an indirect participation through the induction of reactive oxygen species (ROS) (Zhuang et al., 1994; Chakrabarti et al., 2001). Regardless of the mechanism, actin (Miller et al., 1991) and cytokeratins (Ramirez et al., 2000) are among those proteins that have been identified in the formation of DNA–protein crosslinks. Oxidative damage implies the development of ROS, which in their turn modify the structure of proteins, lipids, and nucleic acids by interacting with them. It has been shown that some cytotoxic properties of metals involve ROS induction. For example, arsenic and cadmium increase lipid peroxidation (Ramos et al., 1995; Shaikh et al., 1999), whereas protein oxidation was not observed in nickel-treated cells (Zhuang et al., 1994), and oxidative DNA adducts were reported in mammalian cells exposed to arsenite (Wang et al., 2001). One possible pathway for oxidative damage induction in metal-treated cells could involve the release of iron from ferritin and the iron-dependent formation of ROS (Ahmad et al., 2000). Other mechanisms that have been described, include the activation of NADH oxidase to produce superoxide (Lynn et al., 2000), and a coppermediated Fenton reaction that catalyzes the production of hydroxyl radicals (Wang et al., 1996). Accumulating evidence now suggests that ROS may act as signaling molecules for the initiation and execution of the apoptotic death program in many, if not all, current models of apoptotic cell death (Carmody and Cotter, 2001). Therefore, it is not unusual to find reports relating the exposure to metals with apoptosis. Involvement of ROS in metal-induced apoptosis have been reported for arsenite (Chen et al., 1998), cadmium (Bagchi et al., 2000), and chromium(VI) (as sodium dichromate) (Bagchi et al., 2000). Nevertheless, the upregulation of p53 (Jiang et al., 2001) and induction of several key G2/mitosis regulatory proteins (Park et al., 2001) are other mechanisms involved in the induction of apoptosis by arsenic. Interestingly, genes involved in cell-cycle regulation, apoptosis, and stress response were among those aberrantly expressed in arsenic-exposed cells (Chen et al., 2001) or in arsenic-exposed mice (Liu

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et al., 2001). In the case of mercury, different species have been described to share common features in the apoptotic process, but at the same time, profound differences exist in a number of key steps in the pathway (Shenker et al., 2000). For example, cells treated with Methyl HgCl or HgCl2 affect mitochondrial activity by inducing the development of a membrane-permeability transition. However, whereas methyl HgCl caused a significant increase in cytosolic cytochrome c; HgCl2 did not. Yet, regardless of whether cytochrome c is released from the mitochondria, both mercurial species were capable of activating the caspase cascade (Shenker et al., 2000). As stated earlier, human monitoring may lead to identification of potentially hazardous exposures before adverse health effects appear. In this regard, DNA damage has been used as a biomarker of effect for the study of populations exposed to metals. For example, a positive comet assay was observed in humans exposed to high concentrations of natural arsenic in drinking water (Valverde et al., 1999), or exposed to arsenic and lead in mining areas (Dı´ az-Barriga et al., manuscript submitted), whereas in workers exposed to lead, the metal seems to sensitize the cells to damage induced by other genotoxicants (Restrepo et al., 2000). Analysis of oxidative DNA adducts revealed increased oxidative damage in cases of arsenic-related skin neoplasms when compared with arsenic-unrelated Bowen’s disease (Matsui et al., 1999). Furthermore, a positive association between nickel and the rate of oxidative DNA lesions was observed in an urban population (Merzenich et al., 2001). In relation to cell death, an important percentage of apoptosis was found in buccal epithelial cells of persons chronically exposed to arsenic in China (Feng et al., 2001). The importance of analyzing DNA damage as a biomarker of effect in humans exposed to metals was confirmed, using the human cancer cDNA expression array to profile aberrant gene expression. For example, in samples obtained by liver needle biopsy from an arsenic-exposed population in China, among the aberrantly expressed genes were those involved in cell-cycle regulation, apoptosis, and DNA damage response (Lu et al., 2001). In a different direction, it is clear that DNA damage can serve as a measure of the effectiveness of remediation programs. A reduction in bladder micronuclei prevalence with reduction in arsenic intake was reported in Chile (Moore et al., 1997). The preceding results lead us to three conclusions: (1) DNA damage can be used for the monitoring of humans exposed to metal mixtures, because several metals can induce this effect; (2) as illustrated by arsenic and mercury, some of the effects are species specific; and (3) data obtained in humans have to take into account that many chemicals are also able to induce DNA damage (Gonsebatt et al., 1995); thus, this effect is not specific to the exposure to metals.

3. Neurological effects There is conclusive evidence from experimental and epidemiological investigations that lead, mercury, manganese, and arsenic are neurotoxic agents (ATSDR, 1998, 1999b, c, 2000). Lead, mercury, and manganese neurotoxicity is mainly associated with central nervous system (CNS) dysfunction, whereas arsenic is often associated with peripheral nervous system alterations (Gerr et al., 2000). Recently, CNS effects of arsenic or arsenic mixtures have been reported in human and experimental models (Mejia et al., 1997; Rodriguez et al., 1998, 2001; Delgado et al., 2000; Calderon et al., 2001). Although for the pollutants just mentioned there is enough evidence and an increased interest in testing their neurotoxicity, there are other pollutants that have received less or no attention regarding their potential for damage to the nervous system. One example is fluoride. Recently, some data from studies conducted in endemic fluorosis areas, reported intelligence quotient (IQ) score reduction in children exposed to fluoride, when compared with children not exposed. In all these investigations, a flattening of the normal IQ distribution and a shift of the curve toward the lower end of it in the exposed population was observed (Li et al., 1995; Zhao et al., 1996; Lu et al., 2000, Machado et al., 2003). In adults, effects on concentration and memory have been reported (Spittle, 1994). Furthermore, at the experimental level there are data to support the hypothesis that fluoride is associated with adverse CNS events (Mullenix et al., 1995; Varner et al., 1998). Whereas neurotoxicity is tested or reported from one single pollutant, human populations are usually exposed to mixtures of compounds, increase the risk of illness. As was illustrated earlier, examples include mining and smelter areas (Mejia et al., 1999; Calderon et al., 2001). In Table 1, the reduction in the proportionate reductions in IQ in children exposed to different combinations of neurotoxic agents is summarized. There are several validated instruments to measure CNS function in humans (ATSDR, 1995). One of the most widely used is the Wechsler Intelligence Scale for Children Revised (WISC-R) test to evaluate cognitive function (Wasserman et al., 1997; Calderon et al., 2001). Additional screening batteries include the continuous performance tests, the digit symbol and coding test, the Benton visual retention test, the Rey–Osterreith complex figure, the motor-free visual perceptual test, the developmental test of visual motor integration, and drawing the human figure (ATSDR, 1995; Guillette et al., 1998). Neurobehavioral screening test batteries can help to identify subclinical behavioral or neurological changes at early stages, offering the opportunity to identify important disease processes and persons affected by

ARTICLE IN PRESS J. Caldero´n et al. / Ecotoxicology and Environmental Safety 56 (2003) 93–103 Table 1 Proportionate distribution across WISC-RM categories in children exposed to various neurotoxic agentsa IQ

Proportion of IQ across categories (%) o89

90–110

4110

Expected

Full

25

50

25

Fluoride (n ¼ 61)

Full

26

59

15

Verbal Performance

38 21

49 54

13 25

Full

33

62

5

Verbal Performance

46 36

49 56

5 8

Full

22

66

12

Verbal Performance

32 12

51 61

17 27

Fluoride+lead (n ¼ 39)

Lead+arsenic (n ¼ 41)

a

All children were aged 6–9 years. Children in the fluoride group were exposed to 1.5–3.0 mg/L of fluoride in drinking water and the mean blood lead level was 6.2 mg/dL. Children in the fluoride+lead group were exposed to 4.0 mg/L of fluoride in drinking water and the mean lead level in blood was 9.7 mg/dL. In the lead+arsenic group the mean blood lead level was 8.9 mg/dL and the mean urinary arsenic was 62.0 mg/g creatinine. Data were obtained using WISC-RM. Based on Calderon et al. (2001), Machado et al. (2003).

exposures at stages that offer more effective opportunities for intervention.

4. Endocrine effects in the male reproductive system Evidence is accumulating that links chemicals exposure with potential interference in endocrine function and resultant effects on the male reproductive system. These chemicals may affect the reproductive system by acting in any of the three components of the hypothalamo-pituitary-testicular axis (HPT axis): (1) gonadotropin-releasing hormone neurons from the hypothalamus, (2) gonadotropes in the anterior pituitary gland, and (3) the somatic cells of the testis (Leydig and Sertoli cells). Gonadotropes secrete the gonadotropin, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which then act on their respective target cells in the testis: LH on Leydig cells and FSH on Sertoli cells. As a consequence, testosterone is secreted by the Leydig cells and inhibin B by the Sertoli cells. These hormones, in turn, provide negative feedback on LH and FSH secretion, by acting in the hypothalamus and pituitary gonadotropes. The Leydig and the Sertoli cell products are among the elements that are needed for spermatogenesis.

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Considering all the elements involved in the HPT axis, it seems reasonable to study the effect of xenobiotics on the male reproductive endocrine function. Any alteration in the hormonal levels in serum may be used as a biomarker for individuals exposed to reproductive toxicants. Consequences, of high circulating blood lead levels on the reproductive system, have been reported (ATSDR, 1999b); therefore, it would be relevant to study gonadotropin serum concentrations in exposed populations, to determine whether this endpoint can be a useful biomarker of effect. Although only a few studies have addressed this item, their results are provocative. For example, in male adolescents in whom a substantial negative relationship was found between blood lead levels and stature, a negative correlation was found between lead in blood and LH and FSH serum concentrations; the correlation with the gonadotropins was evident only with lead levels 49.0 mg/dL (Vivoli et al., 1993). In workers with abnormal semen quality and lead levels o40 mg/dL, no significant lead-related influence was found on FSH or LH serum levels; yet, an increase in serum testosterone was found (Telisman et al., 2000). Finally, in a group of 90 males who were occupationally exposed to inorganic lead, an increase in FSH was observed related to blood lead levels 447 mg/ dL (McGregor and Mason, 1990). Additional evidence about the lead-induced effects in the reproductive endocrine system came from experimental studies. For example, Sertoli cells revealed injuries in the cynomolgus monkey with circulating lead concentrations of 35 mg/dL (Foster et al., 1998), whereas prenatal lead exposure in rats, linked with long-term behavioral, physiological, and anatomical effects associated with reproduction, revealed irregular release patterns of both FSH and LH in some exposed rats (McGivern et al., 1991). The alterations of FSH and LH serum levels in humans exposed to lead or in lead-treated animals may be indicative of a lead-induced effect in the cellular components of the HPT axis. Therefore, it is worth mentioning that cytotoxic effects were observed in a rat Sertoli–germ cell coculture exposed to this metal. The addition of lead to the culture medium caused progressive detachment of germ cells from the Sertoli cell monolayer (Adhikari et al., 2000). Cadmium is also a well-known toxicant of the reproductive system (ATSDR, 1999a); thus, this metal could also affect the HPT axis. In workers exposed to cadmium, a cadmium-induced effect was not observed on serum levels of testosterone, LH or FSH (Mason, 1990). However, LH and testosterone plasma levels decreased whereas FSH was increased in adult rats treated with cadmium (Lafuente et al., 2001). Taking into account that in this model both LH and testosterone decreased, it is important to mention that cadmium accumulation increased in the hypothalamus, pituitary

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gland, and testes in the cadmium-treated rats (Lafuente et al., 2000, 2001).Therefore, cadmium may act at all three levels, although in vivo (Boscolo et al., 1985; Lymberopoulos et al., 2000) and in vitro (Syed et al., 1997; Chung and Cheng, 2001) studies have shown that Sertoli cells are unusually susceptible to cadmium. In workers exposed to elemental mercury, FSH and LH levels were not different from those found in a referent group (Erfurth et al., 1990). However, in adult rats treated with mercuric chloride or methyl mercuric chloride, mercury accumulation was found in Sertoli cells (Ernst et al., 1991), whereas in vitro studies with Hg(II) revealed mercury-induced cytotoxic effects in Sertoli cells (Monsees et al., 2000). All of the preceding in vivo studies assessed HPT axis by analyzing testosterone, FSH, and LH concentrations, but none of them studied inhibin B levels. As previously stated, inhibin B generated by the Sertoli cells provides negative feedback on FSH secretion, and therefore, it has been postulated as a marker of exocrine testicular function (Hipler et al., 2001). Inhibin B concentrations in serum could be used as a biomarker of effect for Sertoli cell toxicants. Fluoride-induced effects on the reproductive system have been reported in human populations (Tokar and Savchenko, 1977; Susheela and Jethanandani, 1996). Therefore, in workers exposed to this element, FSH, LH, testosterone, and inhibin B serum concentrations were analyzed (D. Ortı´ z-Pe´rez et al., 2003). When compared to a control population, a substantial increase in FSH, as well as a reduction of free testosterone and inhibin B in serum, were observed in the exposed workers. No abnormalities were found in the semen parameters in these workers. The results obtained showed that exposure to fluoride caused a subclinical effect on the reproductive system that can be explained by a fluoride-induced toxic effect on Sertoli cells. In fact, low concentrations of inhibin B in the presence of high FSH levels has been considered a biomarker of cellular damage for Sertoli cells (Nachtigall et al., 1996; Pierik et al., 1998). Children may be a susceptible population for Sertoli cell damage. It has been reported that inhibin B peaked at 3 months of age and remained elevated up to at least the age of 15 months (Byrd et al., 1998; Andersson et al., 1998). Thus, the neonatal period may be a developmental window important for Sertoli cell proliferation and maturation. During this period, the gonads may be potentially vulnerable to exogenous endocrine interference, for example, from the exposure to xenobiotics during this period of life (Andersson et al., 1998). Measurement of serum levels of inhibin B in infants may give clinical clues about developmental deficiencies in the testis that otherwise become apparent only around puberty or later in life (Andersson et al., 1998).

In conclusion, more studies are needed to define biomarkers for the assessment of the HPT axis in individuals exposed to metals. However, initial evidence, illustrated in the studies, just outlined, is giving positive results. Furthermore, it would be interesting to analyze effects on the female reproductive system. It has been shown, for example, that arsenic may affect the reproductive axis in female rats (Chattopadhyay et al., 1999).

5. Host factors Development of biological effects in humans exposed to metals is a result of interactions between metal toxicity and host factors involved in detoxification. Among them, some micronutrients and selective genes may modify the toxicokinetics of metals. In regard to micronutrients, the effect of calcium on lead exposure is one of the best examples of an interaction between dietary components and environmental toxicants. Among children, an inverse relationship has been observed between blood lead levels and daily calcium intake (Lacasana et al., 2000). Furthermore, higher milk intake during pregnancy has been shown to be associated with lower maternal and umbilical cord lead concentrations in postpartum women (Hernandez-Avila et al., 1997). At the experimental level, an increase in dietary calcium during pregnancy can reduce fetal lead accumulation but cannot prevent lead-induced decreases in birth weight and length (Han et al., 2000). All these results suggest that diet may influence metal toxicity; however, caution is needed before the results can be generalized. For example, in male rats selenium reduced cadmiuminduced DNA damage; in contrast, however, selenium alone induces DNA damage in female rats, (Forrester et al., 2000). Studies with vitamins lead to similar conclusions. For example, folic acid is a protective agent for fibroblasts exposed to arsenic (Ruan et al., 2000), whereas ascorbic acid potentiates arsenic-induced cytotoxic effects in vitro, by decreasing glutathione (Grad et al., 2001). In agreement with this study, it has been recently reported that vitamin C may mediate the formation of genotoxins from lipid hydroperoxides (S.H. Lee et al., 2001). Regarding human populations, contrasting information also has been reported. For example, no differences in the prevalence of arsenicinduced skin lesions were observed in natives with good nutrition, when compared to that reported with corresponding arsenic drinking water concentrations in Taiwan and West Bengal, India (Smith et al., 2000a). By contrast, in an area where fluoride concentration in drinking water was higher than normal, and considering only children with the same level of exposure (similar concentrations of urinary fluoride), dental fluorosis was

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higher in children living in a low-income area (Dı´ azBarriga et al., 1997b). In its turn, one of the best examples of the association between genotype and susceptibility to metals is the case of d-aminolevulinic acid dehydratase (ALAD). This enzyme is a component of the catalytic pathway for heme synthesis and is polymorphic. The genetic polymorphism produces two alleles, ALAD-1 and ALAD-2. ALAD-2 has been associated with high blood lead levels and thus may increase the risk of lead toxicity. ALAD-2 generates a protein with a higher affinity for lead than the ALAD-1 protein. However, at the same time ALAD2 may confer resistance by making lead less bioavailable. Individuals who are homozygous for ALAD-1 have higher bone lead levels, this implies that they may have a greater body lead burden and may be at higher risk for the long-term effects of lead (Kelada et al., 2001). Another protein known to modify the toxicokinetics of lead is the vitamin D receptor. Three genotypes commonly termed bb, Bb, and BB have been identified. It has been shown that subjects with B allele had higher concentrations of lead in blood and bone as well as more chelatable lead (Schwartz et al., 2000). It is relevant that lead workers having the B allele also had a higher prevalence of hypertension compared with lead workers having the bb genotype (B.K. Lee et al., 2001). Genotype influence is less clear in the case of other metals. For example, it is well known that biomethylation is a major detoxification pathway for inorganic arsenicals. According to the metabolic scheme, methylation of inorganic arsenic yields methylated metabolites in which arsenic is present in both pentavalent and trivalent forms. It has been published that trivalent monomethylated species are the most cytotoxic, whereas the dimethylated arsenicals were at least as cytotoxic as trivalent inorganic arsenic (Styblo et al., 2000). Pentavalent arsenicals are less cytotoxic than their trivalent analogs. In the context of these results, it is of note that methylation capacity has been shown in human hepatocytes but not in human bladder cells (Styblo et al., 2000). Furthermore, there seems to be a genetic polymorphism in the biomethylation of arsenic, because recent studies have identified groups with unusually low or high urinary excretion of methylated arsenicals (Vahter, 1999). In conclusion, it is becoming evident that health risk assessments have to consider environmental issues, but also that host variables such as the nutritional status of the population and the genotype of those individuals at risk may influence the development of health outcomes.

6. Conclusions 1. Exposure and effect assessments in communities exposed to metals have to consider the issue of mixtures into consideration.

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2. To identify high-risk populations early enough to institute risk reduction programs in sites polluted with metals, health assessments would be improved by analyzing biomarkers of noncarcinogenic effects. 3. Furthermore, in sites contaminated with metals, it would be useful to have biological tools for the detection of susceptible populations; therefore, the gene–environment interaction must be further investigated.

Acknowledgments This work was supported by grants from the Consejo Nacional de Ciencia y Tecnologı´ a, Sistema Miguel Hidalgo, Mexico (20000206021).

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