ARTICLE IN PRESS
Ecotoxicology and Environmental Safety 56 (2003) 93–103
Human exposure to metals. Pathways of exposure, biomarkers
of effect, and host factors
Jaqueline Calderón, Deogracias Ortiz-Pérez, Leticia Yáñez, and Fernando Dı́az-Barriga
Laboratorio de Toxicologı´a Ambiental, Facultad de Medicina, Universidad Autónoma 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 (Dı́az-Barriga et al., 1993a; Benin et al., 1999),
Bolivia (Dı́az-Barriga et al., 1997a), Chile (Rivara et al.,
1997), Peru (Ramı́rez, 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. Dı́az-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
(Dı́az-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 (Dı́az-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; Calderón-Salinas et al.,
1996; Dı́az-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 (Dı́az-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
95
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 (Dı́az-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
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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.
97
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. Ortı́z-Pérez 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 (Dı́azBarriga 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.
99
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 Tecnologı́a, Sistema Miguel
Hidalgo, Mexico (20000206021).
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