57
Chapter 6
Toxicological Properties
of Persistent Organic Pollutants
and Related Health Effects
of Concern for the Arctic Populations
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
Eva Cecilie Bonefeld-J๘rgensen and Pierre Ayotte
contaminants. Epidemiological studies have been con-
Summary
ducted in which clinical endpoints, such as psychomet-
Human exposure to environmental contaminants is
rics, neurophysiological parameters, infection incidence,
ubiquitous and not only limited to individuals living
bone density, and sexual maturation among others, were
close to the sources of contaminants. Everyone carries a
the main focus. However, in order to detect the early bio-
burden of persistent organic pollutants (POPs) in their
logical changes preceding disease, knowledge about the
body. The burden of POPs in Arctic peoples has been
mechanism of action of toxicants is required. Thus, bio-
monitored for some years, however, it is only recently
markers of effect need to be validated and used. Effect
that a programme for measuring the potential biological
biomarkers are early biological responses of the organ-
effects of these contaminants has been established: the
ism to an external toxic stress. Since the overall weight
AMAP Human Health Effects Monitoring Programme.
of evidence at the epidemiological level for adverse en-
Body burden data alone are not enough to allow the
docrine-related human health effects is not strong, fur-
health risks associated with exposure to environmental
ther studies including validated biomarkers in epidemio-
contaminants in Arctic peoples to be assessed. Further-
logical studies may help in identifying the possible rele-
more, laboratory studies on the effects of single chemi-
vant associations between exposure to contaminants
cals or chemical mixtures in laboratory animals and cell
and detrimental health effects in Arctic populations.
cultures cannot fully elucidate the human health risks.
The Canadian Arctic Contaminants Assessment Re-
Integration of epidemiological and biomarker studies
port (Jensen et al., 1997) identified areas with informa-
on humans from exposed populations in the Arctic is
tion gaps toward which Arctic contaminant research
needed in order to obtain information about the real
programmes should be oriented; effect biomarkers were,
health risks resulting from exposure to the accumulated
however, not considered. They were, however, adopted
mixtures of contaminants in the Arctic.
for use in the AMAP Phase II Human Health Effects
The broad category of human health effects that are
Monitoring Programme (see section 6.2).
suspected to result from exposure to environmental con-
In this chapter, the main focus is on effect biomarkers
taminants include cancer, birth defects, effects on the re-
of early biological responses to evaluate neurobehav-
productive and the neuro-endocrine-immune systems,
ioral, immunological, and reproductive organ status of
altered metabolism, and specific organ dysfunction. This
newborns in the Arctic. It therefore, concentrates on bi-
chapter gives an introduction to these various health ef-
ological effects related to reproductive and developmen-
fects and presents possible biomarkers that may be use-
tal effects, the neurological and immune system func-
ful to include in epidemiological studies. It also discusses
tion, and oxidative stress. Discussions are mainly re-
the connection between traditional toxicological studies
stricted to cover effects due to POPs, including meth-
and new methods designed to study the potential of
ylmercury (MeHg). Other contaminants, such as cad-
chemicals to interfere with the normal homeostasis by
mium (Cd) and lead (Pb), are now considered of lower
exerting endocrine-disrupting effects.
priority. Their major sources, relevant to human health
in the Arctic, are smoking (for Cd) and lead shot (for Pb)
and information on their toxicity and on biomarkers of
6.1. Overview
effects is already available, validated and widely used
The AMAP Phase I assessment report (AMAP, 1998) in-
(e.g., urinary 2-microglobulin level, blood -aminole-
cluded an overview of the classical toxicology of conta-
vulinic dehydratase activity). Furthermore, benchmark
minants. In 1997, the Alta Declaration extended AMAP's
doses as well as biological guidelines for Cd and Pb have
mandate to cover assessment of the combined effects of
been adopted by international health organizations. In
environmental stressors. This chapter presents a com-
contrast, health risk assessments related to the presence
prehensive and detailed description of the rationale for
of MeHg and POPs in the Arctic food chain carry much
conducting effects studies. There is some overlap with
more uncertainty.
that part of chapter 9 dealing with epidemiological stud-
ies, but this is necessary to discuss the evaluation of ef-
6.1.1. The emergence of endocrine disruption
fects parameters. Several studies considered below were
conducted outside the Arctic as only a few investigations
Human exposure to environmental contaminants is
have been carried out in this region to date.
ubiquitous. Exposure is not restricted to individuals who
Over the past decade most efforts have been focused
live next to industries or waste disposal sites, or those
on the characterization of exposure of Arctic peoples to
who reside in inner cities or third-world countries

58
AMAP Assessment 2002: Human Health in the Arctic
where, e.g., insecticides are widely used. Everyone car-
pharmaceutically from the late 1940s to the early 1970s
ries a burden of POPs and heavy metals in their body.
to prevent miscarriages and pregnancy complications in
Persistent organochlorine (OC) compounds, such as di-
women. However, it was removed from the market in
oxins/furans, polychlorinated biphenyls (PCBs) and cer-
the 1970s when studies found DES exposure to correlate
tain pesticides, e.g., toxaphene and DDT/DDE, accumu-
with increases in abortions, neonatal death and prema-
late in body fat; Hg accumulates in organs, Pb in bones,
ture birth, and an increase in the incidence of vaginal
etc. Environmental contamination is a global issue and
adenocarcinoma in young women who were exposed in
POPs are transported to the Arctic by atmospheric and
utero (Herbst et al., 1971). A study of men exposed in
oceanic currents. Because of the lipophilic and persistent
utero showed 31.5% had abnormalities of the reproduc-
nature of POPs, bioaccumulation and biomagnification
tive tract compared with 7.8% of controls (Gill et al.,
occur in the Arctic marine food web and in some fresh-
1979). The abnormalities included cryptorchidism and
water predatory fish and piscivorous birds. Although far
hypospadias, and reduced sperm concentration and
distant from the major pollution sources, some popula-
quality, although reduced fertility was not observed in
tions living in regions north of the Arctic Circle display a
these men (Wilcox et al., 1995). Exposure of mice in
greater body burden of POPs than people living in in-
utero induced very similar effects to those seen in hu-
dustrialized regions, largely due to their reliance on a
mans (McLachlan, 1981). Not all the effects of DES are
traditional diet that includes species high up in the ma-
ascribed to its binding to the estrogen receptor and re-
rine food chain (Asplund et al., 1994; Dewailly et al.,
cent studies have shown that several endocrine-disrupt-
1992, 1994b, 1999; Jensen and Clausen, 1979). Use of
ing compounds induce their effects via different recep-
several OCs, such as DDTs and PCBs, was restricted or
tors and signaling pathways (Andersen et al., 2002;
banned in most countries in the 1970s. Even though
Bonefeld-J๘rgensen et al., 2001a).
their concentrations in the environment have been
The convergence of several lines of inquiry was cru-
slowly declining over the past 30 years, these com-
cial for the rapid growth of interest in the issue of en-
pounds are still the most abundant persistent OCs found
docrine disruption in the 1990s. A number of worrying
in wildlife and in human tissue and milk samples in the
trends related to human male reproductive health had
Arctic region (Safe, 2000).
been reported globally, including decline in semen qual-
While concentrations of several OCs are decreasing,
ity parameters and increases in the incidence of testicu-
the continual introduction of `new compounds', such as
lar cancer, hypospadias and cryptorchidism. At the same
brominated flame retardants, into the environment has
time, adverse trends in the reproductive health of wild-
generated new concerns (Rahman et al., 2001). In addi-
life in some regions outside the Arctic had also been noted
tion, attention has recently shifted from chronic diseases
and correlated with exposure to environmental contam-
and reproductive endpoints to effects that are induced
inants, and in some cases specific chemicals were impli-
following exposure during the sensitive period of in
cated. Evidence was also emerging from a variety of ex-
utero development. Of particular concern are effects on
perimental studies that many widely used chemicals, dis-
development resulting from prenatal exposure to en-
tributed extensively in the environment, had the ability
docrine-disrupting compounds.
to bind and activate estrogen receptors. Although their
To date, no clear-cut evidence for adverse endocrine-
affinity for the receptor was weak compared with either
related human health effects has been obtained at the in-
the natural ligand or DES, their activity was regarded as
dividual or population level. However, data from studies
sufficient to support a working hypothesis that environ-
on wildlife species, studies on laboratory animals, and
mental chemicals might be damaging the reproductive
biomarker studies in vitro have strengthened the need
health of human and wildlife populations by interfering
for further research to address the uncertainty and alle-
with sex hormone activities. Behind this concern was the
viate concerns. Taking a precautionary approach, the
suspicion that chemicals acting through hormone recep-
weight of evidence would suggest that exposure levels
tors might mimic the natural hormones and have pro-
seen in the Arctic have some potential for adverse effects
found effects at very low concentrations. The conjunc-
on human health.
tion of threat both to human and wildlife populations
Studies on wildlife populations have documented ad-
led to responses from international organizations (in-
verse effects that correlate with exposure to one or more
cluding AMAP), governments, and the chemical indus-
putative endocrine-modulating chemicals (Safe, 2000).
try. The following general needs were identified.
Adverse developmental and reproductive effects have
ท Further research to confirm the existence of effects
been primarily linked to POPs and alkylphenols derived
from environmental exposure on reproductive health
from alkylphenol ethoxylate surfactants used in indus-
of humans and wildlife.
trial detergents. In many instances, it has been difficult
ท In cases where an adverse effect was confirmed, estab-
to assign causality because of the complexity of environ-
lishment of the causative link to exposure to an envi-
mental contaminant mixtures and the level of expo-
ronmental chemical.
sure during critical developmental windows. However,
ท Development of reliable methods, and possibly new
lower concentrations of POPs in the Great Lakes region
methods, for detecting chemicals with potential to
were correlated with dramatic improvements in repro-
cause adverse effects (monitoring).
ductive success and significant increases in an array of
ท Ranking of known and suspected endocrine-disrupt-
predatory birds in the Great Lakes basin (Tremblay and
ing compounds for possible regulatory action (prioriti-
Gilman, 1995).
zation).
The range of toxicological effects that estrogenic
ท Possible action to limit release of certain chemicals to
chemicals can produce is illustrated by work on the syn-
the environment.
thetic estrogen diethylstilbestrol (DES). DES was used

Chapter 6 ท Toxicological Properties of Persistent Organic Pollutants and Related Health Effects
59
In order to establish consensus on the scope of this
ท Rodent 5-7 day Hershberger assay: change in weight
issue, to facilitate the identification of active chemicals,
of prostate and seminal vesicles in castrated rats.
and to underpin future regulatory control it is essential
ท Frog metamorphosis assay: rate of tail resorption in
to agree on a precise definition of an endocrine-disrupt-
Xenopus laevis.
ing compound. In 1998, the International Programme
ท Fish gonadal recrudescence assay: effects on light and
on Chemical Safety and the U.S. EPA's Endocrine Dis-
temperature sensitive sexual maturation.
rupter Screening and Testing Advisory Committee (ED-
STAC) proposed the following working definition:
Tier 2 testing
(Intended to determine and characterize the effects of the
An endocrine disrupter is an exogenous chemical sub-
chemical on the endocrine system.)
stance or mixture that alters the structure or function(s)
of the endocrine system and causes adverse effects at the
ท Two-generation mammalian reproductive toxicity
level of the organism, its progeny, populations, or sub-
study or a less comprehensive test.
populations of organisms, based on scientific principles,
ท Avian reproduction test.
data, weight-of-evidence, and the precautionary principle.
ท Fish life-cycle test.
ท Mysid (shrimp) life-cycle test.
Thus, the term `endocrine disrupters' covers all kinds of
ท Amphibian development and reproduction test.
exogenous interfering chemicals; including synthetic
chemicals and synthetic and naturally occurring hor-
mones. Exposure can occur via dairy products and food
6.1.2. Single compound
intake, drinking water, and pharmaceuticals, etc. The
and chemical mixture exposures
ability of a chemical to affect humans or wildlife de-
pends on factors such as structure and concentration,
There are a number of factors that complicate the toxi-
bioavailability, degradation/metabolism, and uptake, etc.
cological evaluation of mixtures. First, it is important to
The observed potency of a chemical is, therefore, very
remember that no test can evaluate all possible end-
dependent on concentration and the system applied for
points. However, existing methods in general include nu-
testing. This can result in several classifications for any
merous endpoints that are sensitive to both strong and
one single chemical, for example as carcinogenic, terato-
weak xenoestrogens such as the reproductive and devel-
genic, toxic, and endocrine disrupter, depending on
opmental effects in humans and rodents of DES (Gill et
which characteristic of the chemical is studied.
al., 1979; Herbst et al., 1971; McLachlan, 1981; Wil-
Many of the compounds suspected of endocrine-dis-
cox et al., 1995) and DDT or chlordecone (Daston et al.,
rupting activity are known to be toxic (in some cases
1997). These endpoints, obtained by multi-generation
acutely toxic) at higher concentrations. They were there-
studies in rodents, are sufficient to indicate a hazard.
fore banned or controlled in some countries, either on
Subsequent decisions to further characterize the cellular
this basis or because of their persistence and capacity to
and molecular steps in the hazard evaluation require
bioaccumulate in biota. Chronic low dose exposure and
mechanistic research for risk assessment, taking into ac-
the subsequent bioaccumulation of lipophilic POPs with
count the possibility that the observed adverse effects
long biological half-lives is of special concern. These
may not be the most sensitive manifestation of toxicity.
POPs may over time bioaccumulate to a critical level ca-
Second, two or more compounds may have additive ef-
pable of eliciting an effect. Moreover the `life-long'
fects as a result of acting via the same mechanism in con-
duration of exposure, together with increasing environ-
cert. They may also elicit antagonistic (less then addi-
mental levels, may maximize the likelihood of induction
tive) or synergistic (greater than additive) effects. Some
of effects. Such factors must be taken into account
studies have suggested synergistic responses of steroidal
in sub-chronic, chronic and/or multi-generation tests.
estrogens in vitro (yeast) and in vivo (turtle) (Arnold et
Because of the complexity of the endocrine system, and
al., 1997a,b). However, estrogenic tests with mixtures of
the complex nature of human epidemiological studies,
dieldrin and toxaphene in human breast cancer MCF-7
animal studies and in vitro screening methods are widely
cells, yeast-based human estrogen receptor assays, and
used for toxicity evaluation and risk assessment.
mouse uterus tests showed no apparent synergism (Ra-
The U.S. EDSTAC has recently developed a strategy
mamoorthy et al., 1997).
for testing chemicals for endocrine modulating activity
There are several other complications that must be
including an initial sorting of chemicals (based on exist-
taken into account when generalizing about what is
ing data), priority setting (based on knowledge of expo-
known concerning the toxicity of single compounds
sure), and tier 1 screening and tier 2 testing, comprising:
and/or mixtures. A compound may have multiple sites
of action and its toxicity may be mediated by different
Tier 1 screening
mechanisms. Many substances are biotransformed to
metabolites (e.g., hydroxylated PCB metabolites) that
In vitro assays
may have a different biological activity than that of the
ท Estrogen receptor binding and reporter gene assays.
parent compound. In addition, a single environmental
ท Androgen receptor binding and reporter gene assays.
contaminant may induce different effects depending on
ท Steroidogenesis assay with minced testis.
the organism's age and reproductive state at the time of
In vivo assays
exposure. Lead is an example of a contaminant having
ท Rodent 3-day uterotrophic assay: increase in uterine
little effect on neurobehavioral function in adults but ir-
weight in ovariectomized rat.
reversible effects on intelligence quotient (IQ) and be-
ท Rodent 20-day pubertal female with thyroid: age of
havior when exposure occurs in utero during the devel-
rats at time of vaginal opening.
opment of the nervous system (Carpenter et al., 1998).

60
AMAP Assessment 2002: Human Health in the Arctic
It is known that developmental toxicity is dependent on
equivalents (TEQs) for mixtures comprising dioxin-like
highly susceptible periods of organogenesis, as demon-
compounds. TEQs are calculated by multiplying the
strated by prenatal exposure to, e.g., DES and thalido-
concentration of each dioxin-like compound by its Toxic
mide, and postnatal exposure to Pb, pesticides and radi-
Equivalency Factor (TEF), which corresponds to the rel-
ation (Selevan et al., 2000).
ative potency of the specific compound in generating an
Toxicity scales have been developed for compounds
AhR-mediated effect, in relation to that of TCDD, the
that share a common mechanism of action. This concept
most potent dioxin-like compound. Consequently, the
was applied to mixtures of dioxin-like compounds that
classical TEQ/TEF risk assessment only accounts for po-
bind the aryl hydrocarbon receptor (AhR). The AhR is
tential dioxin-like properties of a mixture and not other
an intracellular ligand-dependent transcription factor
relevant toxicological endpoints such as effects mediated
expressed in most tissues of mammals. Dioxins and fu-
via other receptors and biochemical pathways (e.g.,
rans (polychlorinated dibenzo-p-dioxins, PCDDs; poly-
interference with the sex hormones and thyroid hor-
chlorinated dibenzofurans, PCDFs) as well as non- or
mone systems). For example, ortho-substituted PCBs are
mono-ortho chloro-substituted PCBs are ligands to the
either weak ligands or do not bind at all to the AhR,
AhR (Birnbaum, 1995; Brouwer et al., 1999; Carpenter
therefore either very low or no TEF values are given for
et al., 1998). The activated ligand-receptor complex trig-
these compounds. Recently, however, it was reported
gers the expression of enzymes including P4501A1,
that the three most highly bioaccumulated di-ortho sub-
P4501A2, P4501B1, glutathione S-transferase, glucuro-
stituted PCBs (CB138, CB153, and CB180) elicit the po-
nyl transferase, - aminolevulinate synthethase, epider-
tential, in vitro, to interfere with cell proliferation as
mal transglutaminase, NAD(P)H:quinone oxidoreduc-
well as the function of the androgen and estrogen recep-
tase and aldehyde-3-dehydrogenase, which are involved
tors (Figures 6ท1 and 6ท2) (Bonefeld-J๘rgensen et al.,
in metabolism and detoxification of many POPs (Hahn,
2001a). These results emphasize that a full assessment of
1998; Safe and Krishnan, 1995).
the toxicological potential of a chemical mixture is
A common practice in risk assessment is to calculate
much more complex than can be deduced by the use of
the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic
TEQ values alone.
Luminiscense units (0.1 R1881 = 100%)
120
Figure 6ท1. Effects of PCB con-
geners on androgen receptor (AR)
trans-activity in Chinese hamster
100
ovary cells (CHO cells). The re-
*
sponse of PCBs and the AR ago-
nist methyltrienolone (R1881, po-
80
sitive control) were obtained by
* *
*
transiently co-transfection with
60
the pMMTV-LUC reporter plas-
mid and the pSVAR0 expression
plasmid encoding the human AR.
40
*
Asterisks indicates statistically
significant (P 0.05) decrease rel-
ative to cells treated with 0.1 nM
20
*
R1881 (which is set to 100%).
Source: Bonefeld-J๘rgensen et al.
0
(2001a).
0
1
3
5
9 18
0
1
3
5
9 18
0
1
3
5
9 18
0 3+3+3
uM
CB138
CB153
CB180
CBmix
CAT activity (10 nM E2 = 100%)
Solvent + PCB
10 nM E2 + PCB
100
* *
*
80
*
* *
*
*
*
60
40
*
20
*
0
3
5
9
3
5
9
3
5
9
3+3+3
0.1 uM
0.01
3
5
9
3
5
9
3
5
9
3+3+3
0.1 uM
ETOH
CB138
CB153
CB180
CBmix
ICI
E 2
CB138
CB153
CB180
CBmix
ICI
Figure 6ท2. Effects of PCB congeners on estrogen receptor trans-activity in human breast cancer MCF-7 cells. The response of the PCBs and
the ER agonist 17 -estradiol (E2, positive control) on transactivation of the reporter plasmid pERE-tk-cat in MCF-7 cells. Diamonds indicate
a statistically significant (P 0.05) difference from the solvent (ETOH) and asteriks from the 10 nM E2 treated cells, respectively. Source:
Bonefeld-J๘rgensen et al. (2001a).

Chapter 6 ท Toxicological Properties of Persistent Organic Pollutants and Related Health Effects
61
Table 6ท1. The AMAP Human Health Effect Monitoring Programme.
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
Bio-Physical Indicators
Epidemiological Effect Markers
Molecular/Genetic Effect Markers
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
Health Statistics
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
E
Morbidity/Mortality data
R
Cancer incidence
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
Genetic susceptibility studies
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
Gene polymorfisms
R
Gene polymorfisms
R
Genotypes
Gene expression (mRNA)
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
Fertility studies
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
Time to pregnancy
R
Time or number of menstrual cycles
R
Receptor/hormone toxicology (estrogenic-
it takes a couple to conceive from
and androgenic-like activities are performed
discontinuation of contraception
in steroid hormone cleared serum samples).
R
Estrogenic- and androgenic-like activities.
Semen quality and quantity
R
Sperm count/volume
Dioxin-like activities in blood serum.
Sperm quality/mobility
In vitro hormone receptor bindings.
Sex hormones (in blood from
R
Sex hormones
R
FSH, Inhibin-B, LH, testosterone,
women and from their male partners
estradiol, sex hormone binding globulin,
providing semen samples)
osteocalcin, pyridolins
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
Pregnancy outcome
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
General indices
E
Abortion (spontaneous)
R
Estrogenic-, androgenic- and dioxin-like
activities
Gestational age
R
Cytochrome P450 modulations,
Birth weight/length
DNA adducts
Sex (single/multiple)
Placenta weight
Developmental anomalies
R
Maldescent testis
Hypospadias
Epispadias
Ano-genital distance and other indices
Developmental effects
R
Breast milk (POPs, fatty acids)
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
Immunological effects
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
R
Hospitalization
R
Vaccination response
R
Antibody (HIB)
R
Vitamin A and cytokines,
complement system
R
Dioxin-like activities
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
Neurological effects
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
R
Milestones + age
R
Thyroid hormone
R
Pre-school tests:
R
Estrogenic- and dioxin-like activities
neurophysiological tests
GSHRd, GSHPx,
neuropsychological tests
Ubiquinol 10/Ubiquinone 10, Ox-LDL,
audiogram and visual tests
F2-isoprostanes
ญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญญ
E: Essential; R: Recommended.
The AMAP Human Health Effect Monitoring Programme is designed to examine the effects on reproduction and development of dietary expo-
sure to POPs and heavy metals within the Arctic. One requirement is that a suite of studies are carried out in parallel, including dietary question-
naires and indicators, and contaminant measurements carried out by laboratories with documented QA/QC.
Expert Group meetings held in Ottawa, Canada, Sep-
6.2. The AMAP Human Health Effects
tember 1999; Rovaniemi, Finland, January 2000; and
Monitoring Programme
T๓rshavn, Faroe Islands, October 2000, a Human Health
The broad categories of human health effects that may
Effects Monitoring Programme was recommended to
be linked to exposure to environmental contaminants in-
the eight Arctic nations.
clude: cancer, birth defects, decreased fertility, altered
The AMAP Human Health Effects Monitoring Pro-
sex hormone balance, immune system defects, neurolog-
gramme, as shown in Table 6ท1, includes several molecu-
ical effects such as reduced IQ and behavioral abnormal-
lar biomarker endpoints for use in Arctic environmental
ities, altered metabolism, and specific organ dysfunc-
health studies. Substances commonly found in the envi-
tions (Carpenter et al., 1998). At AMAP Human Health
ronment may have the potential to affect several organ

62
AMAP Assessment 2002: Human Health in the Arctic
systems. The diseases listed are identified on the basis of
mended measurements for inclusion within AMAP mon-
studies of both humans and animals, and in most cases
itoring implementation plans. The biophysical indica-
these investigations were focused on a single contami-
tors and epidemiological and molecular/genetic effect
nant. Several of these diseases, when found in a given
markers included in Table 6ท1 are, as far as possible,
individual, are difficult to ascribe to a particular expo-
linked to the different studies. In addition, dietary ques-
sure (Sharpe, 1993; Sharpe and Skakkebaek, 1993). This
tionnaires, relevant markers of a seafood based diet
is generally the case for cancer, reproductive effects
(e.g., n-3 fatty acid content in plasma phospholipids),
(such as infertility, early birth, etc.), many of the en-
and biomarkers of exposure to POPs should also be in-
docrine modulators and nervous system actions. Others
cluded in the studies. Laboratories performing POPs,
are clearly attributable to particular exposures, such as
heavy metal, and lipid analyses must have documented
kidney disease following Cd exposure or the loss of par-
quality assurance / quality control (QA/QC).
ticular neurons following MeHg exposure (Carpenter
Sections 6.3 to 6.8 describe the background and ra-
et al., 1998).
tionale for conducting the health effects studies in the
The main objective of the AMAP Human Health
Arctic that are listed in the Human Health Effects Mon-
Effects Monitoring Programme is to characterize the
itoring Programme (Table 6ท1). Some studies have al-
impact of dietary exposure to POPs by monitoring bio-
ready been initiated in some parts of the Arctic, while
physical indicators, and epidemiological and molecu-
others are still at the planning phase.
lar/genetic effect markers (see Table 6ท1). These effects
studies are directed towards examining the hypothesis
6.3. Genetic considerations
of xenobiotic interference with homeostasis of hor-
mone functions, with a special focus on circumpolar
Central to many of the influences on the biological
populations. A number of persistent OCs exhibit estro-
system are effects that occur at the gene level. Genes reg-
genic (and anti-estrogenic), androgenic (and anti-
ulate almost everything, including many aspects of hor-
androgenic) and dioxin-like activities. Some bind to
monal production and the reproductive system, brain
the estrogen receptor (e.g., DDT, toxaphene, CB138,
development and function, immune system balances,
CB153, CB180) (Bolger et al., 1998; Bonefeld-J๘r-
and organ physiology. A genetic disruption can, there-
gensen et al., 1997, 2001a), and some bind to the an-
fore, affect different organ systems, as a result of the
drogen receptor (e.g., DDE, vinclozolin) (Bonefeld-J๘r-
extensive interactions between these systems; i.e., effects
gensen et al., 2001a; Crisp et al., 1998; Kelce et al.,
on one organ system may influence the function of other
1997; Kelce and Wilson, 1997) or bind to both recep-
organs. During normal development, genes are activated
tors (e.g., metabolites of methoxychlor, CB138). Diox-
and deactivated at different stages, often under the con-
ins have been characterized as anti-estrogenic due to
trol of growth factors and hormones. Environmental
their AhR-mediated interference with estrogen receptor
factors interfere with these biologically balanced pro-
activities (Kharat and Saatcioglu, 1996; Safe and Krish-
cesses and may result in genetic dysfunction. Mutations
nan, 1995). Because of the wide variety of endocrine-
in genes, inherited or induced by environmental fac-
disrupting effects possibly induced by mixtures of per-
tors, may thus result in reproductive effects, birth de-
sistent OCs, there is a need to develop markers that inte-
fects, and cancer.
grate the effects of several chemicals on specific hor-
Gene polymorphism is known to exist between dif-
monal pathways and to combine them with epidemio-
ferent ethnic groups, which can result in differences in
logical studies that are also part of the AMAP Human
tolerance, e.g., to food components such as lactose
Health Effects Monitoring Programme.
(Harvey et al., 1998; Nei and Saitou, 1986). In addi-
Sonnenschein and colleagues have devised a method
tion, gene polymorphism in metabolizing enzymes is
for estimating human exposure to a complex mixture of
suspected to influence susceptibility to environmental
xenohormones (environmental compounds with hor-
carcinogens, affecting the risk of cancer (Autrup, 2000;
mone-like activities) (Sonnenschein et al., 1995). First,
Coughlin and Piper, 1999; Morabia et al., 2000). Ge-
endogenous steroids are separated from persistent OCs
netic polymorphism and breast cancer risk has been ex-
in human serum samples by high-performance liquid
tensively analyzed, and significant differences in geno-
chromatography (HPLC) and the resulting fractions are
type frequencies between cases and controls have been
tested for estrogenic activity using a proliferation assay
found, including the aromatase cytochrome P450
with MCF-7 cells. Other investigators have further de-
(CYP19) gene which catalyses the conversion of andro-
veloped and applied the HPLC fractionation of human
gens to estrogens (Dunning et al., 1999). Recently,
serum for separating endogenous hormones from xeno-
a study suggested an association between PCB concen-
hormones to obtain integrative measurements of estro-
trations and CYP1A1 gene polymorphism in women
genic, androgenic, and dioxin-like effects of compounds
breast cancer patients compared to control groups
using reporter-gene cell assays. Recent data, using Inuit
(Moysich et al., 1999).
serum samples from Greenland, have indicated that the
Serum concentrations of p,p'-DDT, p,p'-DDE and
concerted action of accumulated persistent OCs in the
CB138, CB153, and CB180 were found to be signifi-
human samples exerts an inhibitory effect on the estro-
cantly associated to K-ras mutations in exocrine pancre-
gen receptor function in human cells, whereas an in-
atic cancer (Porta et al., 1999). These results suggest
crease in AhR activity was observed (Bonefeld-J๘r-
new roles for OCs in the development of several cancers
gensen, in prep.).
in human beings.
The morbidity/mortality data and pregnancy out-
Mitochondrial DNA (mtDNA), which is inherited
comes are considered essential effect markers, whereas
maternally via the oocyte, has been mapped completely
the other biomarkers listed in Table 6ท1 are recom-
(Thrasher, 2000). The variation of mtDNA can be used

Chapter 6 ท Toxicological Properties of Persistent Organic Pollutants and Related Health Effects
63
to determine the ancestry of a population. For example,
PCB levels higher than the control group (Moysich et
using restriction fragment length polymorphism and
al., 1999). Thus, the importance of environmental
DNA sequence technology, the present Inuit from
xenohormones in the etiology of breast cancer remains
Greenland were shown to be descended essentially from
controversial.
Alaskan Neo-Eskimos; European mtDNA types were
Accumulation in the fatty breast tissue and the po-
not found in the Inuit samples in this study (Saillard et
tential of many persistent OCs to exert estrogenic/an-
al., 2000). Because of inadequate DNA repair mecha-
drogenic- or anti-estrogenic/anti-androgenic-like effects
nisms, relatively high rates of mutation are accumulated
are hypothesized to promote the cancer process through
in the mtDNA. Mitochondrial DNA mutations are re-
the modulation of the estrogen receptor regulated re-
sponsible for several mitochondrial syndromes. A recent
sponses (Wolff and Toniolo, 1995). Therefore, to reject
study presented a theory of possible linkages between
or verify the hypothesis future studies must include ญ in
mitochondrial defects and a possible global developmen-
addition to the epidemiological investigation and burden
tal delay in some children with maternal exposure to en-
of POPs ญ information on genetic polymorphisms and
vironmental OCs. The author suggests that investigators
biomarkers related to the total impact of components
conduct further research into the cause of maternal
with estrogenic (or anti-estrogenic), androgenic (or anti-
mtDNA mutations following exposure to mutagenic
androgenic), and dioxin-like activities. Currently, a pilot
xenobiotic compounds (Thrasher, 2000).
study including these endpoints is being carried out in
Greenland (Bonefeld-J๘rgensen, pers. comm., 2002).
PCBs and dioxin are well known for their ability to
6.4. Breast cancer
induce certain iso-enzymes of P450 in mammalian liver
The incidence of breast cancer has increased steadily
via the AhR. Some of these enzymes; P4501A1,
over the past few decades in women from a number of
P4501A2, and P4501B1, are involved in estradiol me-
countries including Finland, Denmark, the United
tabolism and might disrupt hormone levels (Spink et al.,
States, and the UK (Hakulinen et al., 1986; Quinn and
1992a,b, 1994, 1998). In vitro, several persistent OCs
Allen, 1995). The upward trend is estimated at about
have been shown to increase the 16 -OHE1:2-OHE1
1% per year since 1940. In the Arctic, the prevalence of
estradiol metabolite ratio; 16 -OHE1 is regarded as
breast cancer in Greenland Inuit is much lower than in
highly estrogenic while 2-OHE1 is a weak anti-estrogen
the general population of the Western Hemisphere; how-
(Bradlow et al., 1995). Some studies have reported
ever, during the 1990s an increasing incidence was also
higher levels of the 16 -OHE1 metabolite in urine of
observed in this population (Nielsen, 2000). The estab-
breast cancer patients (Bradlow et al., 1995; Safe, 2000),
lished risk factors, such as genetic inheritance and fac-
whereas other studies did not observe this association
tors resulting in an increased total lifetime exposure to
(McDougal and Safe, 1998; Ursin et al., 1997). Thus, in-
biologically active estrogens, can explain only about a
conclusive results exist and await further research.
third of the cases (Davis and Bradlow, 1995). Estrogens
In animal studies the anti-estrogenic capacity of
have a prominent role in the pathogenesis of breast can-
TCDD was suggested to be responsible for a decrease in
cer (Lippman and Dickson, 1989), and it has been hy-
the incidence of mammary tumors observed in female
pothesized that xenoestrogens may contribute to the
rats (Kociba et al., 1978). In vitro bioassays have sug-
total estrogenic burden.
gested that hydroxy metabolites of several PCB con-
Several studies on DDE, PCB and hexachloroben-
geners also possess anti-estrogenic properties (Moore
zene (HCB) concentrations in breast cancer patients ver-
et al., 1997).
sus controls have been carried out in Europe, Asia, and
North and South America. Overall these studies do not
6.5. The reproductive system
support a major role for exposure to persistent OCs as
and fertility studies
a risk factor (Laden and Hunter, 1998; Safe, 1997).
However, the results are inconclusive, particularly for
The development and maintenance of reproductive
high-level exposure. Six studies reported elevated levels
tissues is to a large extent controlled by steroidal hor-
of DDT or DDE among women with breast cancer.
mones. Studies in vitro or in whole animal model
Seven studies found no differences in DDT or DDE
systems have demonstrated that some environmental
level between cases and controls, and one study re-
chemicals either mimic and/or antagonize natural hor-
ported higher DDE levels in serum samples among
mone activities. Studies dating back to the late 1960s
women with breast cancer (Romieu et al., 2000). The
identified 1-[2-chlorophenyl]-1-[4-chlorophenyl]-2,2,2-
limitation of these studies has been discussed with re-
trichloroethane (o,p'-DDT), a minor constituent of tech-
gard to information on duration of lactation, other po-
nical DDT, as a weak estrogenic compound capable of
tential sources of estrogens, and replacement estrogen
causing an augmentation of rat uterine weight in the
therapy. The lack of controlling for other sources of es-
classic immature female rat model (Bitman and Cecil,
trogens or potentially confounding factors may have
1970). This compound and a few others that share es-
masked a real association between OC exposure and
trogenic properties have been implicated in abnormal
breast cancer (Romieu et al., 2000). In Denmark, H๘y-
sexual development in reptiles (Gaido et al., 1992; Guil-
er et al. (1998) reported a significant dose-related asso-
lette et al., 1994, 1995), birds (Fahrig, 1993; Fry, 1995;
ciation between accumulation of the pesticide dieldrin
Fry and Toone, 1981) as well as feminized responses in
and the risk of breast cancer. As previously mentioned,
male fish (Jobling et al., 1995).
a case-control study in western New York State found
Male reproductive disorders may be mediated by the
an increased risk of breast cancer to be associated with
estrogen receptor; however, they are also consistent with
the polymorphism of CYP1A1 among women with
inhibition of androgen receptor-mediated events. Kelce

64
AMAP Assessment 2002: Human Health in the Arctic
et al. (1995) identified the major and persistent DDT
not reveal delayed conception among consumers of fish
metabolite, 1,1-bis[4-chlorophenyl]-2,2-dichloroethylene
from Lake Ontario (Buck et al., 1997), but the duration
(p,p'-DDE), as a potent anti-androgenic agent in male
of exposure was rather short and not quantified by
rats. In addition to inhibiting androgen binding to the
measurements. Results of a Scandinavian time-to-preg-
androgen receptor, this compound, when administered
nancy study indicate delayed conception related to per-
to pregnant dams, also induced characteristic anti-an-
sistent OC body burdens in smokers but not in non-
drogenic effects in male pups (reduced anogenital dis-
smokers (Axmon et al., 2000).
tance; presence of thoracic nipples). Treatment with
p,p'-DDE at weaning delayed the onset of puberty, while
6.5.2. Semen quality and quantity
treatment of adult rats resulted in reduced seminal vesi-
cle and ventral prostate weights.
A study of sperm counts conducted worldwide suggested
TCDD is yet another OC which has been shown to
that an annual fall of 0.8% had occurred between 1938
alter sexual development in male rats (Mably et al.,
and 1990 (Carlsen et al., 1992). Since then, falling
1992). Decreases in epididymis and cauda epididymis
sperm count and quality have been reported in a number
weights, decreases in daily sperm production and cauda
of countries (Auger et al., 1995; de Mouzon et al., 1996;
epididymal sperm number were observed at day 120 and
Irvine et al., 1996; Van Waeleghem et al., 1996) and a
at most earlier times, when a dose as low as 64 ng/kg
study of testicular morphology in Finland (Pajarinen et
was administered to dams on day 15 of gestation.
al., 1997) suggested a reduction in spermatogenesis be-
During the differentiation of reproductive organs,
tween 1981 and 1991. In contrast, no evidence for a de-
hormones, growth factors, and other endogenous medi-
cline in sperm counts or quality has been found in a
ators regulate gene expression and direct differentiation.
number of locations studied within the United States
The marked difference between exposure to chemicals
(Fisch et al., 1996), although considerable geographical
including endocrine-disrupting compounds during criti-
variation in sperm counts was observed.
cal periods in development versus during adulthood is
Sperm production is controlled by the sex hormones
the irreversibility of an effect during development. Evi-
(Sharpe, 1993; Sharpe and Skakkebaek, 1993), and
dence indicates that changes in concentrations of andro-
may therefore be influenced by sex-hormone-mimicking
gen and estrogen result in permanent changes in cell
compounds. Certain chemicals lower sperm count in
function. For example, the higher level of testosterone in
animals and in exposed workers. Dioxins and the pesti-
male mouse fetuses relative to female fetuses results in
cide endosulfan are known to lower testosterone levels
the differentiation to prostate tissue as opposed to vagi-
and produce testicular atrophy in male rats, and dioxin,
nal tissue. In addition, a small increase in total circulat-
kepone, 2,4-dichlorophenoxyacetic acid (2,4-D), and
ing estradiol (50 pg/mL) permanently altered prostate
dibromochloropropane have been suggested to reduce
size in mice (Bigsby et al., 1999). Thus it is plausible that
sperm count in men (Paigen, 1999). A number of other
disruption of the action of estrogen or androgen during
possible factors may affect sperm production, including
critical periods can lead to permanent alterations in the
changes in lifestyle. Intake of selenium (Se) is reported
development of reproductive organs and other tissues
to be falling in the UK and Europe (Rayman, 1997) and
with receptors for these hormones.
dietary deficiency of Se has been suggested as a caus-
Animal experiments indicate reproductive toxicity
ative agent of lowered sperm production since selenoen-
following low-level exposure to persistent OCs. In labo-
zymes play a role in the maintenance of normal sperm
ratory animals it has been shown that prenatal exposure
motility, testicular morphology and testosterone metab-
to PCBs, PCDD or the DDT-metabolite p,p'-DDE is as-
olism. There is general consensus that, in some coun-
sociated with reduced male fertility (Kelce et al., 1995;
tries at least, semen quality and counts have declined.
Peterson et al., 1993; Sager et al., 1987).
However, taking into consideration the influence of bias
In wildlife studies on Baltic grey (Halichoerus gry-
in recruitment of study subjects and uncertainty in
pus) and ringed seals (Phoca hispida) and on Wadden
methodologies (Bromwich et al., 1994; Lerchl and Ni-
Sea harbor seals (Phoca vitulina) there is strong evidence
eschlag, 1996) it is not known to what extent semen
that PCBs in the food chain had impaired reproductive
quality reflects incidence of infertility or sub-fertility of
function resulting in population declines (SCTEE, 1999).
males as such.
Most recently, high levels of POPs have been found in
The only known study on semen quality in the Arctic
Arctic polar bears (Ursus maritimus) in Svalbard (Skaare
took place in Iceland. A study of 73 men including 27
et al., 2000), and a possible association between the re-
men with normal semen, who came to the fertility center
ported incidence of pseudohermaphroditism in polar
because of their wives' fertility problems (52% were of
bears and environmental chemicals has been discussed
proven fertility), 20 men with idiopathic sterility, and 26
(Wiig et al., 1998).
men with poor semen quality, showed no correlation be-
More information about effects of POPs on Arctic
tween fertility and persistent OC levels. There was a di-
animals can be found in the AMAP 2002 assessment on
rect relationship between OC levels in plasma and in
POPs in the Arctic (AMAP, 2003c).
semen; with concentrations approximately 20 to 50
times lower in semen. Prevalence of obesity (body mass
index (BMI) > 30 kg/m2) was more than three times
6.5.1. Time to pregnancy
higher in the group of men with semen problems, and
Time to pregnancy is a measure of the joint reproductive
sperm density correlated to BMI (P = 0.003), raising the
performance of the parents. Temporal changes in human
possibility that increased prevalence of obesity may
fertility in relation to body levels of OCs have not been
partly be involved in the decline in male fertility (Mag-
extensively investigated. A time-to-pregnancy study did
n๚sdottir et al., 2002).

Chapter 6 ท Toxicological Properties of Persistent Organic Pollutants and Related Health Effects
65
are believed to be associated with in utero exposure, i.e.,
HYPOTHALAMUS
fetal development, and may therefore have common eti-
ology (Sharpe, 1993; Sharpe and Skakkebaek, 1993).
A variety of epidemiological data suggest that the
hormonal environment of the fetus may be involved in
Pituitary
gland
the development of testicular cancer and congenital mal-
Environmental
formations. The main evidence, which suggests that pre-
estrogens
Luteinizing hormone
natal exposure to estrogens has an influence on the de-
velopment of the male reproductive tract, originates
Follicle stimulating
hormone
from the treatment of pregnant women with DES. Alter-
ation in estrogen exposure may be influenced by endoge-
ญ
Environmental
nous sources such as a change in diet (low-fiber, high fat,
estrogens
and increase in dairy products) and increase in body fat.
Intake and increase of synthetic estrogens such as DES
and ethinyl estradiol may occur through the use of oral
+
ญ
contraceptive pills, components of which are recycled
into drinking water, and also by intake of non-persistent
Environmental
phytoestrogens from plants. Finally, persistent OCs such
estrogens
Sertoli cells
Estradiol
as PCBs and dioxins are suspected to play a role because
of their potential to interfere with hormone homeostasis.
+
Aromatase
In animal studies, TCDD caused changes in both male
+
and female gonadal development and male reproduction
Environmental
Leydig cells
(Peterson et al., 1993).
estrogens
Figure 6ท3 summarizes the major hormones which
ญ
Testosterone
are involved in growth and function of the fetal testis
and illustrates how exogenous environmental estrogens
may disrupt the normal hormone homeostasis. The basis
+
for susceptibility to adverse effects of estrogen on devel-
opment of the male reproductive tract centers on the
normal hormonal control of the fetal testis.
FETAL TESTIS
Sertoli cells play an essential role in spermatogenesis.
Figure 6ท3. Means by which environmental estrogens are thought to
The production of estrogen and M�llerian inhibiting
disrupt the major hormonal mechanisms involved in growth and
substance by the Sertoli cells are thought to co-ordinate
function of fetal/neonatal testis. Green (+) and red (ญ)arrows repre-
sent positive and negative feedback mechanisms, respectively. For
the processes relating to testicular development and
explanation see the text to the right.
masculinization. Sertoli cells are also thought to regulate
the differentiation and multiplication of the early germ
cells and fetal Leydig cells and their production of
6.5.3. Male reproduction,
testosterone, which is converted to the more potent an-
testicular and prostate cancer
drogen 5 -dihydrotestosterone. The follicle stimulating
The incidence of testicular cancer has increased quite
hormone (FSH) and luteinizing hormone (LH) from the
dramatically in many countries having cancer registries,
pituitary gland control Sertoli cell multiplication and
including Scandinavian countries, the countries around
maintain testosterone production by fetal Leydig cells,
the Baltic Sea, Germany, the UK, the United States, and
respectively. A negative feedback system operates in
New Zealand (Adami et al., 1994; Brown et al., 1986;
which steroid hormones produced by the testis, testos-
Hakulinen et al., 1986; HMSO, 1992; Wilkinson et al.,
terone and estrogen, act to regulate FSH and LH and is
1992). Interestingly, the increasing incidence of testicu-
the basis for the so-called estrogen hypothesis (George
lar cancer in Denmark is several times higher than in
and Wilson, 1994; Sharpe, 1993, 1994; Sharpe and
Finland and further study of this geographical gradient
Skakkebaek, 1993). Estrogen seems to be necessary in
may give important clues on etiology. Prostate cancer in-
male development and function. In addition, immature
cidence has also increased in many countries (Boyle et
Sertoli cells, Leydig cells, and germ cells possess the en-
al., 1995; Merrill and Brawley, 1997). The incidence of
zyme aromatase, which is responsible for the synthesis
congenital malformations such as cryptorchidism (mal-
of estradiol from testosterone. Estrogens, including envi-
descent testis) and hypospadias (malformation of the
ronmental estrogens, have the potential to act via vari-
penis) are thought to be increasing in the Western coun-
ous points within this feedback loop to alter hormone
tries (Berkowitz et al., 1993; Kallen et al., 1986). Testic-
level and testicular function.
ular cancer has been related to cryptorchidism (Giwerc-
Sertoli cells produce inhibin-B in a feedback loop,
man et al., 1993, 1987) and the testicular maldescent
which regulates FSH secretion. Neonatal secretion of in-
has also been linked to hypospadias (Kallen et al., 1986;
hibin-B can be used as a measure of Sertoli cell number
Moller et al., 1996; Prener et al., 1996). Moreover it is
and a sensitive marker for Sertoli cell toxicants. In in
widely accepted that men with testicular cancer have a
vitro responses of immature rat Sertoli cells, estrogens,
higher incidence of impaired spermatogenesis in both
bisphenol-A and the pesticide lindane were shown to in-
cancerous as well as contra lateral testis (Petersen et al.,
crease the production of inhibin-B, whereas mercury(II)
1998). The increases in the incidence of testicular cancer,
and platinum(II) markedly decreased inhibin-B levels
hypospadias, cryptorchidism, and reduced sperm count
(Monsees et al., 2000).

66
AMAP Assessment 2002: Human Health in the Arctic
The extreme sensitivity of the fetus to its hormonal
complex feedback mechanisms involving hormone ac-
environment is illustrated by studies in mice demonstrat-
tion. Some chemicals are shown to interfere with this
ing that intrauterine fetal position can influence male
regulation. For example, TCDD can act to decrease
sexual behavior and androgen responsiveness (Nonne-
or increase the expression of the ER (Romkes et al.,
man et al., 1992). It has been known for some years that
1987), and compounds which bind to the ER (e.g., ICI
DDT has both estrogenic (o,p'-DDT) and anti-andro-
182,780, toxaphene, CB138 and several pesticides) in-
genic (p,p'-DDE) isomers (Kelce et al., 1995). However,
fluence receptor functions as well as the cellular level of
the range of synthetic chemicals with potential estro-
ER mRNA (Andersen et al., 2002; Bonefeld-J๘rgensen et
genic activities includes other OC pesticides (Andersen
al., 2001a; Jensen et al., 1999).
et al.; 2002, Kelce et al., 1995; Soto et al., 1994), PCBs
(Connor et al., 1997), alkylphenolic compounds (Rout-
6.5.5. Effects on synthesis, storage, release,
ledge et al., 1998), phthalate esters (Harris et al., 1997)
transport, and clearance of hormones
and bisphenol-A (Ben-Jonathan and Steinmetz, 1998;
Gould et al., 1998). All chemicals identified as having
Hormones, including sex steroids, thyroid hormones
estrogenic activity are approximately 103- to 106-fold
and glucocorticoids, are transported bound to carrier
less potent than estradiol and recent data suggest that
proteins and their effects are to some extent influenced
they have similar binding affinity for the ER- and ER-
by the level of these proteins in the blood. Sex hormone-
estrogen receptors (ER) (Kuiper et al., 1998). Moreover,
binding globulin (SHBG) is a plasma glycoprotein that
new data suggest that a number of phthalates and
binds certain estrogens and androgens with high affinity.
alkylphenol chemicals, which act as weak ER agonists in
Hormones bound to SHBG are not bioavailable for
vitro, also elicit weak androgen receptor antagonism in
transport into effector cells. In mammals, estrogen in-
vitro (Sohoni and Sumpter, 1998). This complicates the
creases the concentration of SHBG in plasma, whereas it
prediction of in vivo activity of the chemicals. There is
is decreased by androgens. Plant estrogens (phytoestro-
some in vivo evidence that high exposure of rats to
gens) also stimulate SHBG synthesis (Murkies et al.,
dibutyl-phthalate during gestation and lactation causes
1998). A positive correlation was recently reported be-
abnormalities of the male reproductive tract (Mylchreest
tween p,p'-DDE concentrations in plasma lipids and
et al., 1999). Processes regulated by testosterone were
plasma concentrations of SHBG in young men exposed
also affected by dibutyl-phthalate exposure resulting in
to DDT during anti-malarial campaigns in Chiapas,
reduction of anogenital distance, hypospadias testicular
Mexico. p,p'-DDE levels were also negatively correlated
maldescent, atrophy and underdevelopment of seminal
with bioavailable testosterone concentration, semen vol-
vesicles and prostate. Almost no effect was observed on
ume and total sperm count (Ayotte et al., 2001).
the female offspring. These observations are similar to
Xenobiotic ligands such as hydroxy-PCBs, phthalate
effects of prenatal exposure to the anti-androgen flu-
esters and chlorinated pesticides in general either do not
tamide and the anti-androgenic fungicide vinclozolin
bind to SHBG or show low affinity compared to 17 -
(Gray et al., 1994; Kelce et al., 1994; Mylchreest et al.,
estradiol (Jury et al., 2000). Although these compounds
1999). Also, the herbicide Linuron (3-[3,4-dichloro-
bind to SHBG with much lower affinity than endoge-
phenyl]-1-methoxy-1-methylurea) was shown to impair
nous sex steroids, these interactions may be physiologi-
testosterone-mediated reproductive development in rats
cally relevant in situations where SHBG levels are high
(McIntyre et al., 2000).
and endogenous hormones are low, such as prepubes-
The strongest evidence, which implicates exposure
cent children and women taking contraceptives. More-
to synthetic chemicals as a factor in reproductive tract
over, the lack of binding of xenobiotic ligands to SHBG
abnormalities, comes from wildlife. The best-known
might cause biologically relevant concentrations in spite
endocrine-disrupting compound causing serious repro-
of their relatively low concentration compared to en-
ductive abnormalities is tributyltin (TBT). TBT acts as
dogenous hormones.
an anti-estrogen since it inhibits aromatase causing an
increase in androgens and masculinization of female
6.5.6. Hormone profiles
marine gastropods (Yamabe et al., 2000). In human
prostate cancer cells TBT and triphenyltin (TPT) are
To obtain a steroid profile of androgens for both precur-
shown in vitro to stimulate cell proliferation and an-
sors and metabolites of dihydrotestosterone (DHT) a se-
drogen receptor transcription such as prostate specific
ries of steroids, including DHT, could be measured:
antigen (Yamabe et al., 2000). Moreover, a number of
dehydroepiandrosterone (DHEA), androst-5-ene-3 , 17 -
polycyclic aromatic hydrocarbons (PAHs) were shown,
diol, androstenedione, testosterone, estrone, estradiol,
in vitro, to possess anti-androgenic activities (Ving-
DHEA sulfate, androstane-3 , 17 -diol glucuronide,
gaard et al., 2000).
and androsterone glucuronide. Information on these ste-
In summary, more studies are required to support the
roid levels enables a better understanding of any alter-
involvement of environmental chemicals as a risk factor
ation in steroidogenic enzymes in classical steroidogenic
in the proposed decline in male fertility.
tissues, such as adrenal glands and the testis, and for
steroidogenic transforming enzymes localized in periph-
eral tissues such as the prostate and skin.
6.5.4. Effect on hormone receptor numbers
In the Arctic, no full studies have yet been conducted
The responsiveness of a tissue to a hormone depends on
on hormone profiles and contaminant exposure. De-
the density of receptors within its component cells. The
wailly and colleagues recently carried out a pilot study
number of receptors is determined by their rate of syn-
in Greenland (n=48 males) and the following male hor-
thesis and catabolism, which is in turn controlled by
mones were measured: DHEA, 5-diol, 4, testosterone,

Chapter 6 ท Toxicological Properties of Persistent Organic Pollutants and Related Health Effects
67
DHT, E1 and E2. These hormone levels will be corre-
be an important confounding factor, but OC exposure
lated with persistent OC levels after adjustment for age,
was significantly associated with EROD activity and
BMI and smoking (Dewailly, pers. comm., 2001).
DNA-adduct levels after stratifying for self-declared
smoking status. It was concluded that CYP1A1 induc-
tion and DNA adducts in placental tissue could consti-
6.6. Osteoporosis
tute useful biomarkers of early effects induced by envi-
Persistent OCs have recently been associated with an in-
ronmental exposure to OCs (Lagueux et al., 1999).
creased risk of osteoporosis in humans (Beard et al.,
A second study was conducted to determine whether
2000). The relationship between DDE and bone mineral
environmental exposure to PCBs induces placental
density was recently examined in 68 sedentary women
CYP1A1 in Inuit women. This more recent study was
who reported adequate dietary intake of calcium. Re-
designed to better control the confounding effect of
duced bone mineral density was significantly correlated
smoking. Cotinine concentration in meconium and Cd
with age (r = ญ 0.36, P = 0.004), as well as with increases
concentrations in placenta had previously been validated
in the log of DDE levels in serum (r = ญ 0.27, P = 0.03).
as markers of prenatal exposure to tobacco smoke
These results suggest that past community exposures to
(Pereg et al., 2002). Placenta, cord blood, and meco-
DDT may be associated with reduced bone mineral den-
nium samples were obtained from 35 Inuit women from
sity in women. As a potent androgen receptor antago-
Nunavik and 30 women from Sept-ฮles (reference popu-
nist, DDE may reduce the inhibitory effect on cytokines
lation). Efforts were made to sample more smokers in
and result in the inappropriate turnover of osteoclasts or
the Sept-ฮles population and more non-smokers in the
inadequate production of osteoblasts within bone mar-
Nunavik population in order to balance the smoker and
row, thus leading to reduced bone density (Beard et al.,
non-smoker groups. Smoking status was ascertained on
2000). Bone metabolism markers that could be used are:
the basis of cotinine concentration in the meconium and,
serum markers of bone formation (osteocalcin), and cal-
when necessary, individuals were re-assigned to the
cium resorption (urinary pyridinolines). A study of fac-
proper smoking category based on this marker. PCB
tors associated with osteoporosis in Greenland women is
concentrations were measured in cord plasma and
underway and results will be available in the near future
EROD was assessed in placenta. Despite the higher PCB
(Dewailly, pers. comm., 2001).
exposure of the Inuit population, both groups showed
similar EROD activities when the data were stratified
according to the smoking status ascertained by the coti-
6.7. Pregnancy outcome
nine concentration. In the Nunavik population, EROD
6.7.1. Developmental anomalies
activity was correlated with 2,2',4,4',5,5'-hexachlorobi-
Developmental anomalies related to OC burden are dis-
phenyl (CB153) plasma concentration (a marker of expo-
cussed in section 6.1.1. There are relatively few reports
sure to the environmental PCB mixture). However, coti-
of possible effects of OCs in humans on abortion, gesta-
nine concentrations in meconium were also significantly
tional age, and birth weight and length, and in general
correlated with CB153 plasma concentrations and multi-
this issue remains to be elucidated.
variate analyses failed to demonstrate a significant contri-
bution of PCB exposure to placental CYP1A1 activity
when tobacco smoking (as estimated by cotinine concen-
6.7.2. Placental biomarkers of in utero development
tration in the meconium) was included in the analysis.
Cytochrome P450 1A1 (CYP1A1) is a phase I biotrans-
In summary, the results from this study do not sup-
formation enzyme expressed in extra-hepatic tissues in
port the hypothesis that low-level environmental expo-
humans, and its regulation is mediated by the AhR (Safe
sure to PCBs induces an increase in CYP1A1 activity in
and Krishnan, 1995).
the placenta, and leaves tobacco smoking as the major
Hydroxylation of toxicants by P450s leads to the
modulating factor (Pereg et al., 2002).
formation of reactive intermediates that may damage
DNA. PCBs were shown to form such reactive inter-
6.7.3. Birth weight
mediates in vitro and to form DNA adducts following
their bio-activation in hepatic microsomal systems con-
CYP1A1 was reported to have been induced in the pla-
taining high levels of P450s (McLean et al., 1996; Oak-
centa of women who smoked during pregnancy and
ley et al., 1996).
lower birth weights of newborns were observed for
CYP1A1 induction and DNA adducts were investi-
smokers with high placental aryl hydrocarbon hydroxy-
gated as possible markers of early biological effects re-
lase (AHH) activity compared to smokers with lower
lated to OC exposure in Inuit women from Nunavik.
AHH activity (Pelkonen et al., 1979). Placental homo-
CYP1A1-dependent ethoxyresorufin-O-deethylase ac-
genates from Taiwanese mothers who developed Yu-
tivity (EROD) and DNA adducts were measured in pla-
Cheng disease (and thus had been highly exposed to
centa samples obtained from 22 Inuit women from
PCBs and PCDFs; see section 6.8.1.5.) had 100-fold
Nunavik. These biomarkers were also measured in 30
greater CYP1A1-related AHH activity levels than those
women from a Quebec urban center (Sept-ฮles) as a ref-
measured in homogenates from non-exposed mothers
erence group. Prenatal OC exposure was determined by
(Wong et al., 1985). This enzyme induction was signifi-
measuring these compounds in umbilical cord plasma.
cantly associated with low birth weights (Lucier et al.,
Placental EROD activity and the amount of DNA
1987). A study of Swedish fishermen's wives and 1501
adducts thought to be induced by OC exposure were sig-
children supported an association between a high con-
nificantly higher in the Nunavik group than in the refer-
sumption of POP-contaminated fish from the Baltic Sea
ence group. For both biomarkers, smoking was found to
and an increased risk of low birth weight. The women

68
AMAP Assessment 2002: Human Health in the Arctic
interviewed from the east- and west coast cohorts ate lo-
substances produced by the immune system (Marchetti
cally caught fish more than twice as often as their refer-
et al., 1995). The major focus of interest in endocrine
ents. Compared with the regional population, the
disruption has been reproduction and sexual differentia-
women in the east coast (Baltic Sea) cohort gave birth to
tion and development, with most attention to effects as-
an increased number of infants with low birth weights
sociated with steroid hormones, and to a much lesser ex-
(< 3000 g), whereas the opposite was seen in the west
tent, the thyroid hormones.
coast cohort. Infants in the east coast cohort had signifi-
Gonads primarily produce sex hormones, androgens
cantly lower birth weights than infants from the west
(male) and estrogens (female), which play vital roles in
coast cohort (median 3530 g versus 3610 g, P < 0.001)
the control of reproductive functions. The glucocorti-
(Rylander et al., 1995). Later studies have strengthened
coid hormones, such as cortisol, are produced by adre-
the hypothesized association between exposure to per-
nal glands and have fundamental effects on metabolism,
sistent OCs during childhood and adolescence and an
as well as influencing the immune and reproductive sys-
increased risk of having an infant with low birth weight
tems (Vacchio et al., 1998). Thymulin, a polypeptide
(Rylander et al., 1998, 2000). A significant relationship
hormone, is found mainly in the cortex and medulla of
between lower birth weight and PCB accumulation was
the thymus and is thought to be involved in the educa-
reported in a study of newborns of mothers who had
tion and maturation of the immune system's T-cells.
eaten fish with high PCB contents from Lake Michigan
Interference with production of this hormone affects
(Fein et al., 1984). However, at background levels of
the ability of the thymus to produce mature T-cells.
PCBs no correlation between PCB concentration and
There are thought to be feedback mechanisms linking
birth weight has been observed (Rogan et al., 1988).
thymulin to testosterone, estrogen, cortisol and thyroid
The association of maternal smoking and blood Hg
hormone balance (Marchetti et al., 1995; Marsh and
concentration with birth weight was studied in 1106 live
Scanes, 1994).
born singletons from Greenland with a gestational pe-
riod of 37+ weeks (Bjerregaard and Hansen, 1996).
6.8.1. Immune system functions
Smoking was significantly associated with low birth
weight while consumption of marine mammals, and ma-
The immune system includes a complex network of
ternal or cord blood Hg concentration were not. In West
cells. Tissue cells involve macrophages, mast cells and
Greenlanders a weak association was found between Hg
dendritic cells. The lymphocytes are divided into T- and
and low birth weight. High concentration of Hg and OC
B-cells, these are subdivided into T-helper cells (Th,
substances is suspected to reduce birth weight; however,
CD4) and T-cytotoxic cells (Tc, CD8), plasma cells
a positive influence of marine diet on birth weight due to
(CD20) and natural killer cells (NK). The immune sys-
n-3 fatty acids has been reported and may counteract the
tem cells communicate via highly regulated interleukine
effect of POPs (Bjerregaard and Hansen, 1996).
expressions. Macrophages initiate a non-specific im-
mune response by phagoctytoses of allergens and then
represent the antigen on their surface for the Th-cells.
6.7.4. Sex ratios
The Th-cells are then activated and secrete cytokines,
There have been suggestions of alteration in sex ratios
which activates other Th-, Tc-, and B-cells. Thus the Th-
following an accidental environmental exposure to
cells play a central role in the immune system.
dioxin in Seveso (Italy) in 1976. Of 74 births in the most
The extensive interaction between the immune and
heavily contaminated zone there was an excess of fe-
nervous systems involves common use of messengers
males; 26 males and 48 females were born (Mocarelli et
such as neurotransmitters and cytokines (Carpenter et
al., 1996). A similar occurrence has been noted for some
al., 1998).
other high occupational exposures to chemicals, e.g., di-
bromochloropropane (Safe, 2000). There is evidence
6.8.1.1. Effects of POPs and metals
from several countries that in the past few decades there
on the immune system
has been a small but significant decrease in the male to
female sex ratios (Safe, 2000). However, the potential
Dioxins, coplanar PCBs, and PAHs suppress the immune
role of male/female sex ratios as an indicator of environ-
system (Carpenter et al., 1998). Lead, however, affects
mental exposure to hormone-disrupting chemicals is still
the immune system differently promoting hypersensitiv-
controversial and awaits further research.
ity, rashes, and auto-immunity. Investigations suggest
that the dominance of different populations of Th-lym-
phocytes is a major factor in an individual's immune re-
6.8. The neuro-endocrine-immune system
sponsiveness. Th1-lymphocytes predominate and pro-
In vertebrate species, the neuro-endocrine-immune sys-
duce a particular profile of cytokines in individuals with
tem is responsible for many complex, inter-related phys-
normal immunity, whereas individuals with hyper-im-
iological processes including homeostatic, reproductive
munity (asthma, skin rashes, and auto-immunity) have
and immune functions. There are four main types of
predominately Th2-lymphocytes producing different cy-
hormones: polypeptides, eicosanoids, steroids, and thy-
tokines (Carpenter et al., 1998). Environmental expo-
roid hormones. The inter-dependency of the neuro-en-
sure to Pb and Hg was shown to alter the balance be-
docrine and immune systems is reflected by the produc-
tween the Th1- and Th2-lymphocytes, and contaminant
tion of hormones, neuropeptides and other neurotrans-
exposure early in life is suspected to cause prolonged ab-
mitters by some immune cells. These hormones play a
normalities in immune function. Moreover, children ex-
role in regulation of the immune system, while en-
posed prenatally to DES showed an altered immune
docrine and nervous tissues express receptors for many
function (Carpenter et al., 1998).

Chapter 6 ท Toxicological Properties of Persistent Organic Pollutants and Related Health Effects
69
Studies have shown that both synthetic and natural
non-specific defense mechanisms, such as inhibition of
estrogens suppress the immune system (Kendall et al.,
NK cell activity in rats and mice. It also decreases the ex-
1992; Osterhaus et al., 1995), and that during preg-
pression of certain activation markers of T-cells (HLA-
nancy, the female immune system is naturally suppressed
Dr, IL-2R) (NRC, 1992). Moreover, it has been well
with a decrease in thymulin and an increase in estrogen
demonstrated that MeHg can affect the functions of B-
levels. The presence of estrogen receptors on thymulin
cells and therefore reduce the humoral mediated re-
producing cells indicates coordination between these
sponse (Daum et al., 1993). Exposure to inorganic Hg
two hormones (Kendall et al., 1992). Increased concen-
induces allergies and autoimmune problems in hypersen-
tration of testosterone has a positive, enhancing effect
sitive individuals.
on the immune system, whereas increase or decrease in
thyroid hormones results in negative effects.
6.8.1.5. Effects of POPs on the human fetal
and neonatal immune system
6.8.1.2. Effects of POPs on the immune system
Few data exist regarding the potential immunotoxic ef-
of laboratory animals
fects of in utero and lactation exposure to PCBs and
Several OCs elicit immunotoxic effects in laboratory an-
dioxins/furans. In 1979, a poisoning from ingestion of
imals and humans, the most potent being substances
rice oil contaminated with PCBs and PCDFs occurred
structurally related to TCDD such as non- and mono-
in Yu-Cheng, Taiwan. In utero exposed children of
ortho chloro-substituted PCBs as well as 2,3,7,8-chloro-
highly exposed women were shown to have a higher in-
substituted PCDD/Fs. In almost all animal species tested,
cidence of respiratory symptoms during their first six
including primates, PCDD/Fs and PCBs produce myelo-
months of life (Rogan et al., 1988). An increase in the
suppression, immunosuppression, thymic atrophy, and
frequency of pulmonary diseases was suspected to re-
inhibition of immune complement system components
sult from a generalized immune disorder induced by
(NRC, 1992). Exposure to TCDD during pre- and/or
transplacental or breast milk exposure to dioxin-like
postnatal life results in more severe effects than if the
compounds, most likely PCDFs (Rogan et al., 1988). In
chemical is administered during adult life and in some
children and young adults accidentally exposed to
species it may be a prerequisite for immunosuppression
PCBs and PCDFs (`Yu-Cheng disease'), serum IgA and
(Hoffman et al., 1986; Vos and Luster, 1989). In fact,
IgM concentrations as well as percentages of total T-
available evidence in laboratory animals suggests that
cells, active T-cells and suppressor T-cells were de-
the maturation of the immune system is especially vul-
creased compared to values of age- and sex-matched
nerable to the adverse effects of dioxin-like compounds,
controls (Chang et al., 1981). In addition to a higher
chlordane, HCB, PAHs and possibly other endocrine-
frequency of middle-ear diseases among 8- to 14-year
disrupting compounds such as DDT and kepone (Bar-
old children born to Yu-Cheng mothers (Chao et al.,
nett et al., 1987; Holladay and Luster, 1996).
1997), the investigation of delayed type hypersensitiv-
ity responses further indicated that cell-mediated im-
mune system dysfunction was more frequent among
6.8.1.3. Effects of POPs on the immune system
patients than controls.
of wild mammals
Studies of 207 Dutch infants (105 breast fed and 102
Persistent organic pollutants, such as PCBs and dioxins,
bottle fed) have suggested that background levels of
can cause a broad range of immunotoxic effects in Arc-
PCB/dioxin exposure were associated with lower mono-
tic wildlife. More comprehensive information on this
cyte and granulocyte counts at three months of age and
subject can be found in the AMAP 2002 assessment on
thus influence the fetal and neonatal immune system
POPs in the Arctic (AMAP, 2003c).
(Weisglas-Kuperus et al., 1995). Follow-up studies of the
Exposure to POPs has been associated with effects
children showed that perinatal background exposure
on thyroid hormones and even low levels of PCB sup-
to PCBs and dioxins persists into childhood and might
press thyroid hormone in grey seal pups in the (non-Arc-
be associated with a greater susceptibility to infectious
tic) Baltic Sea (Jenssen et al., 1996). High body levels of
diseases such as recurrent middle-ear infections and
PCBs and other OCs have been associated with suppres-
chicken pox, and a lower prevalence of allergic reactions
sion of the immune system (Reijnders, 1986), and it has
(Weisglas-Kuperus et al., 2000).
been suggested that these compounds may be implicated
In Nunavik, an epidemiological study investigated
in mass mortality among sea mammals (e.g., seals, por-
whether OC exposure is associated with the incidence
poises, dolphins) (Osterhaus et al., 1995) following in-
of infectious diseases in Inuit infants and with immune
fection with the phocine distemper virus, morbillivirus.
system dysfunction. The number of infectious disease
In otters, environmentally exposed to PCBs, a strong
episodes in 98 breast-fed and 73 bottle-fed infants was
negative correlation was observed between vitamin A
compiled during their first year of life. Concentrations
and PCB concentrations, and a high incidence of infec-
of OCs were measured in early breast milk samples and
tious diseases was apparent in contaminated animals
used as surrogates for prenatal exposure levels. Bio-
(Murk et al., 1998).
markers of immune system function (lymphocyte sub-
sets, plasma immunoglobulins) were determined in ve-
nous blood samples collected from infants at 3, 7 and
6.8.1.4. Immunotoxic effects of mercury
12 months of age. Otitis media was the most frequent
Organic and inorganic Hg possess cytotoxic activities
disease with 80% of breast-fed and 81.3% of bottle-fed
for cellular components of the immune system in several
infants experiencing at least one episode during their
species of rodent. MeHg, a form of organic Hg, can alter
first year of life. During the second follow-up period,

70
AMAP Assessment 2002: Human Health in the Arctic
the risk of otitis media increased with prenatal exposure
vaccination is administered using two doses and that ex-
to p,p'-DDE, HCB and dieldrin. The relative risk (RR)
posure to the natural antigen is used in Nunavik. It is
for 4- to 7-month old infants in the highest tertile of
then important to note precisely when the last dose was
p,p'-DDE exposure as compared to infants in the lowest
given prior to the blood puncture (antibodies measure-
was 1.87 (95% confidence interval (CI), 1.07ญ3.26).
ment) (Raby et al., 1996).
The relative risk of otitis media over the entire first year
of life also increased with prenatal exposure to p,p'-
6.8.1.7. Complement system
DDE (RR, 1.52; 95% CI, 1.05ญ2.22) and HCB (RR,
1.49; 95% CI, 1.10ญ2.03). Furthermore, the relative
The complement (C') system plays an important role in
risk of recurrent otitis media (3 episodes) increased with
natural immunity against infectious agents. It is particu-
prenatal exposure to these compounds. No clinically
larly important in young children for whom the ac-
relevant differences were noted between breast-fed and
quired immune system is not yet fully developed. Defi-
bottle-fed infants with regard to biomarkers of immune
ciency of many of the C' components is associated with
function and immunological parameters. It was con-
increased susceptibility to infections, generally of the
cluded that prenatal OC exposure can be a risk factor
upper respiratory tract. In a murine model, exposure to
for acute otitis media in Inuit infants (Dewailly et al.,
OCs increased susceptibility to Streptococcus pneumo-
2000b).
niae infections, decreased C3 levels and lowered total C'
In 1997, an international symposium was held
hemolytic activity (White et al., 1986).
in Bilthoven (the Netherlands) to discuss the most ap-
propriate effect biomarkers of immunotoxicity that
6.8.1.8. Effects of POPs
could be used in epidemiological studies (Van Loveren
on cytokine production by Th1/Th2 cells
et al., 1999). Among the conclusions, one of the
stronger statements was to use antibody responses to
High levels of OCs and metal ions in blood and tissues
vaccination with an antigen to which no prior expo-
are frequently related to fish intake. Fish and seal oil-
sure occurred. In the scope of the ongoing cohort
supplemented diets (rich in n-3 (also called omega-3)
study on neurodevelopmental effects of Hg and POPs
fatty acids) have generally been shown to reduce plasma
(Muckle et al., 2001a), an immune component was
levels of some cytokines (Bonefeld-J๘rgensen et al.,
added in 1998. The immune function biomarkers de-
2001b). Most human studies have shown decreased
scribed in sections 6.8.1.6 to 6.8.1.9. were selected
plasma levels or diminished production of IL-1 and
based in part on the conclusions of the Bilthoven sym-
TNF , while n-3 fatty acids increased the production of
posium report.
these cytokines in mice (Blok et al., 1996). Both contam-
inants and n-3 fatty acids alter the balance between Th1-
and Th2-type cytokines, and could impair host resist-
6.8.1.6. Antibody response following vaccination
ance to infections. IL-1, IL-2, IL-4, IL-6, TNF
and
Acquired immunity produces a very specific response to
IFN
have been repeatedly associated with these
a particular microorganism or other type of challenge. It
changes and need to be measured in Arctic populations
mainly involves the activation of lymphocytes and pro-
consuming seafood products. IFN
and TNF
are
duction of antibodies. Environmental toxins may affect
known anti-viral cytokines (Zinkernagel, 1993); IL-4
acquired immunity and a broad evaluation of the com-
enhances IgG1 and IgE, but reduces IgM production
petence of the response provides a better picture of their
(Ada, 1993); IL-10 down-regulates Th1-cytokines and
effect. The development of disease represents the ulti-
inhibits IFN production (Fiorentino et al., 1989).
mate endpoint in evaluation of immune suppression.
One of the principal limitations of using cytokines
Antibody response to vaccination is an intermediate
is their high variability due to minor (usually non-de-
marker of the competence of the adaptive immunity to
tected) infections. The extremely high incidence of
infections. Vaccination programmes include essentially
minor infections in the Arctic strongly limits the use of
three types of products: killed vaccine (influenza, whole
cytokines in epidemiological studies.
cell pertussis, inactivated polio), protein-conjugated or
Organochlorine compounds and metal ions can
protein-based vaccine (Hemophilus influenza type b
modulate the production of Th1/Th2-type cytokines.
(Hib), diphtheria, tetanus, acellular pertussis vaccine,
Two studies using murine leukocytes exposed to metal
hepatitis B), and attenuated live virus (measles-mumps-
ions in vivo demonstrated that Hg inhibits the in vitro
rubella, varicella, and the tuberculosis vaccine BCG).
production of IFN , IFN and TNF by macrophages
Antibody response to conjugated Hib is of great interest.
and induces a dose- and time-dependent increase in
Hib vaccine is important in Inuit children because, prior
IL-1 activity (Ellermann-Eriksen et al., 1994; Zdolsek
to immunization, Hib was the most frequent cause of
et al., 1994). High blood levels of IL-4 and IgE and
bacterial meningitis in Inuit children, and was five to
low levels of IFN have been observed in animal stud-
ten times more frequent than in Caucasian children
ies involving treatment with Hg (Heo et al., 1996).
(Ward et al., 1986).
In humans, occupational exposure to inorganic Hg
To evaluate humoral response to Hib-conjugated
did not result in a significant variation of the immune
vaccine, two threshold values of anti-polyribosylribitol
response in terms of in vitro production of IL-1 and
phosphate (anti-PRP), a capsule polysaccharide, have
TNF , whereas a prolonged low-level exposure de-
been set. An antibody titer of 0.15 ตg/mL is indicative of
creased TNF
concentrations (Langworth et al.,
immediate protection against Hib, whereas a titer
1993). Along with their effects on Th1/Th2-type cy-
greater than 1.0 ตg/mL was found to protect for longer
tokines, OCs and metal ions are known to alter B-cell
terms (Ward et al., 1994). It should be noted that Hib
activity and to impair host resistance to several

Chapter 6 ท Toxicological Properties of Persistent Organic Pollutants and Related Health Effects
71
bacterial and viral infections (Heo et al., 1996). In
6.8.2. Neurological system
animals, Hg increased by 100-fold the virus titers fol-
lowing infection with herpes simplex virus 2 (HSV-2)
6.8.2.1. Thyroid hormone disruption
and neural development
and increased by 2-fold the number of macrophages in
the heart of mice infected with myocardial coxsack-
Thyroid hormones regulate neuronal proliferation, cell
ievirus B3 (CB3) 994 (Ellermann-Eriksen et al., 1994;
migration and differentiation including control of when
Ilback et al., 1996). Plasma levels of IFN in exposed
differentiation begins and when cell proliferation ends
animals were higher than in infected non-Hg-treated
(Hamburgh, 1969).
mice. In a recent preliminary in vitro human whole
A number of organic and inorganic compounds
blood study of induced release of the inflammatory cy-
cause toxic action in the nervous system such as abnor-
tokines IL-1
and TNF
upon incubation with vari-
malities of peripheral sensory or motor nerves, resulting
ous OC substances, 2,3,7,8-TCDD, toxaphene, and
in either abnormal or loss of sensation, or muscle weak-
CB180 elicited the potential to increase the level of the
ness (Carpenter et al., 1998). In 1979 it was shown that
two cytokines in plasma (Bonefeld-J๘rgensen, pers.
Pb at very low concentration can cause a decrease in IQ
comm., 2002).
and behavioral problems in children exposed prenatally
and in the early postnatal years. Recent studies have sug-
gested that these actions are irreversible. Moreover, sev-
6.8.1.9. Vitamin A status
eral studies suggest that PCBs may have similar effects,
Vitamin A influences the expression of over 300 genes
with prenatal exposure resulting in decreased cognitive
and thus plays a major role in cellular differentiation,
function and behavior that appears irreversible (re-
including that of cells related to immune response
viewed by Carpenter et al., 1998).
(Semba, 1994; Sommer and West, 1996). Results from
Although many theories exist as to how PCBs affect
different animal and human studies vary; however,
neurodevelopment, the main hypothesis involves PCB
almost all studies revealed that lymphopoiesis and/or
impact on thyroid hormone homeostasis (Porterfield
maturation of lymphocytes are altered (generally re-
and Hendry, 1998).
duced) in connection with vitamin A deficiency (Olson,
In addition to direct effects on neurons the thyroid
1994; Semba et al., 1993; Sommer and West, 1996).
system is important to nervous system function. In
Vitamin A deficiency could increase frequency, severity,
adults, the thyroid controls the rate of metabolism and
and duration of infections. Diseases in the lower respira-
neurological functions (Colorado State University, 2000;
tory compartment were associated with vitamin A defi-
DeVito et al., 1999; Oppenheimer et al., 1995). In the
ciency in many cross-sectional clinics and population
developing fetus and neonate, the thyroid is essential to
based studies. Also, otitis media was among the first
organ (e.g., brain) development, the development of the
infections to be associated with vitamin A deficiency
central nervous system, and cell differentiation and
in humans (Bloem et al., 1990; Semba, 1994; Sommer
growth (Porterfield, 2000; Rodier, 1994). Congenital
and West, 1996).
hypothyroidism results in minimal brain dysfunction,
Vitamin A clinical deficiency has never been docu-
even if treated after birth (Carpenter et al., 1998). PCBs
mented in Canadian Arctic populations. However, a
and dioxins and their hydroxylated metabolites have
recent report suggests that the daily vitamin A intake
some similar steric features to the thyroid hormones,
in Nunavik falls below the recommended intake
which enables these compounds to interfere with normal
(Blanchet et al., 2000). Alteration of vitamin A home-
thyroid function. The mechanisms by which PCBs can
ostasis has been associated with PCB exposure in labo-
affect thyroid hormone function, and so influence devel-
ratory animals (Ndayibagira and Spear, 1999). Fur-
opment, include increase in thyroid stimulating hor-
thermore, POPs such as OCs have been shown to alter
mone from the pituitary gland, altered structure and in-
the vitamin A homeostasis in many species, including
creased weight of the thyroid gland, decrease in thyroid
primates (Zile, 1992). It is thus important to better
hormone, thyroxine (T4) and triiodothyronine (T3), and
understand the relationships between vitamin A, OC
blockage of binding of thyroid hormone to transport
levels, and infectious disease incidence in Arctic po-
proteins and thyroid receptors (Carpenter et al., 1998).
pulations.
In a pilot study, retinol concentration was measured
6.8.2.2. Effects of POPs
in umbilical cord plasma of newborns and the vitamin A
on fetal and neonatal neurological capabilities
status was assessed in four populations. The study in-
cluded 55 First Nations newborns and 56 Caucasian
There is mounting evidence that environmental back-
newborns from the middle and the lower north shore of
ground exposure to PCBs, dioxins and furans is sufficient
the St. Lawrence River, 135 Inuit newborns from Arctic
to affect thyroid homeostasis and neurological capability.
Quebec, and 22 newborns from the general population
Studies of Japanese breast-fed neonates (Nagayama et al.,
of southern Quebec. Mean retinol concentrations in
1998) and Dutch children (Koopman-Esseboom et al.,
ng/mL were 175.2, 159.5, 148.2, and 242.8, respectively
1994b) have shown that these compounds in maternal
(Dewailly, pers. comm., 2001). These preliminary results
milk affect the thyroid hormone status in children. Esti-
may suggest an inverse relationship between retinol con-
mated TEQ was significantly and negatively correlated
centrations and the POPs burden. The difficulty of using
with the levels of T4 and T3 in the blood of breast-fed ba-
vitamin A as an effect biomarker of PCB exposure is re-
bies. A follow-up study of the same group of Dutch chil-
lated to (1) the variability of vitamin A intake among in-
dren found that exposure to high levels of PCBs and diox-
dividuals, and (2) non-systematic supplementation pro-
ins in utero and in maternal milk correlated negatively
grammes in infants.
with the cognitive score of the children (Patandin et al.,

72
AMAP Assessment 2002: Human Health in the Arctic
1997). This is in accordance with the findings of intellec-
al., 1999; Hussain et al., 2000). In animal experiments,
tual impairment of children prenatally exposed to PCBs
several chemicals such as PCBs, flame retardants, pesti-
through their mother's intake of polluted fish from Lake
cides, phthalates, and dioxins have been shown to have
Michigan (Jacobson and Jacobson, 1996). Longnecker et
the capacity to lower T4 levels in blood (Brucker-Davis,
al. (2000) did not find a significant association between in
1998; Fowles et al., 1994).
utero PCB exposure among 160 North Carolina children
and serum thyroid measured in umbilical cord sera. Nor
6.8.2.4. Thyroid effect studies
did a study of 182 children from the Faroe Islands, where
of Inuit populations exposed to POPs
marine food includes pilot whales, find any correlation
between intellectual function and PCB levels. Maternal
In Nunavik, a cord blood monitoring programme took
serum, hair, milk and umbilical cord blood were analyzed
place between 1993 and 1996. Measurements of thyroid
for contaminants. Levels of essential fatty acids, Se, and
hormones were performed on 466 Inuit newborn umbil-
thyroid hormones were determined in cord blood. The
ical cord blood samples. Free T4, total T3, thyroxine-
neurological optimality score of each infant was deter-
binding globulin (TBG) and thyroid stimulating hor-
mined at 2 weeks of age adjusted for gestational age, and
mone (TSH) were measured. Hydroxylated metabolites
predictors were assessed by regression analysis. Thyroid
of PCBs (OH-PCBs) and other phenolic compounds
function was found to be normal and not associated with
were also measured in a sub-sample (n=10). As ex-
PCB exposure (Steuerwald et al., 2000). However, a mild
pected, birth weight was positively associated with thy-
decrease in neuropsychological test scores for children 7
roid hormones (T4, TBG). For this reason, further
years of age was correlated with prenatal MeHg exposure
analyses were adjusted on birth weight. After adjust-
deduced from maternal hair (10ญ20 ตg/g) compared to
ment, TBG and TSH were significantly and negatively
controls (3 ตg/g) (Grandjean et al., 1997). A later study
associated with PCB congener levels (Dewailly, pers.
by Grandjean et al. (2001) reported neurobehavioral
comm., 2001).
deficits associated with MeHg in 7-year old children pre-
The main mechanism for the transport of thyroid
natally exposed to seafood neurotoxicants. This study in-
hormones to the brain requires them to pass through the
volved analyses of cord blood from 435 children from a
blood brain barrier via a thyroid hormone transport
Faroese birth cohort. A possible interaction between
protein called transthyretin (TTR) (Chanoine and Bra-
PCBs and MeHg was noted in this study (Grandjean et
verman, 1992). Although PCBs show some binding
al., 2001). In addition, MeHg neurotoxicity in Amazon-
affinity for TTR (Chauhan et al., 2000), OH-PCBs have
ian children living downstream from gold mining activi-
much higher in vitro binding affinities that can be as
ties has been reported (Grandjean et al., 1999b). More-
high as 12 times the binding affinity of the natural lig-
over, a cross-sectional and prospective dataset from the
and, thyroxine (T4) (Brouwer, 1991; Cheek et al., 1999;
Maastricht Aging Study suggests that exposure to pesti-
Lans et al., 1994). Binding to TTR is not limited to OH-
cides, but not metals and organic solvents, was associated
PCBs. Other chlorinated phenolic compounds such as
with increased risk of mild cognitive dysfunction in adults
pentachlorophenol (PeCP), halogenated phenols, and
(Bosma et al., 2000). A developmental study involving a
brominated flame retardants (Meerts et al., 2000; van
cohort of 7000 children carried out in the Seychelles
den Berg, 1990; van den Berg et al., 1991) also have
found no clear evidence for consistent adverse effects on
strong affinities for TTR. Recently, PeCP was found to
six developmental outcomes of pre- and postnatal study
be the dominant phenolic compound determined in Inuit
exposure to MeHg (Axtell et al., 2000).
whole blood (Sandau et al., 2000a). Thus, other halo-
In a recent study, involving 171 healthy German
genated phenolic compounds may also be important
motherญinfant pairs, the effect of prenatal and perinatal
contaminants in plasma as they have been found to ex-
exposure to PCBs (estimated as the sum of CB138,
hibit similar toxicological properties to OH-PCBs
CB153, and CB180) on prospectively measured psy-
(Schuur et al., 1998; van den Berg et al., 1991).
chodevelopment in newborn infants at age 7, 18, 30, and
PCBs have previously been measured in umbilical
42 months was estimated (Walkowiak et al., 2001). In
cord plasma; however, few studies have examined levels
summary, the findings showed that the PCB concentra-
of hydroxylated metabolites in blood, especially in hu-
tion in serum samples at 42 months increased markedly
mans. OH-PCBs have recently been quantified in whole
with duration of breast feeding, up to five times higher
blood of Inuit from northern Quebec, Canada (Sandau
than in the group of non-breastfed children. Moreover,
et al., 2000a), and Swedish and Latvian fish eaters
prenatal and postnatal exposure to European background
(Sjodin et al., 2000). One study examined chlorinated
PCB levels (1.22 ตg/L serum at age 42 months) was asso-
phenolic compounds in umbilical cord plasma to deter-
ciated with a decrease in mental and motor development
mine possible differences among three human popula-
up until 42 months of age. In comparison, the mean of the
tions with different PCB exposures due to cultural dif-
sum of these three congeners in the blood of women of
ferences in dietary habits (Sandau et al., 2002). Retinol
child-bearing age from Greenland is 5.0 ตg/L plasma
and thyroid hormone status (triiodothyronine (T3)), free
(with a range of 1.55 to 9.4 ตg/L, depending on the re-
T4, TSH, and TBG were determined in most samples.
gion, see Table 5ท2).
An inverse association was found (r = ญ 0.62; P = 0.003)
between log-normalized free T4 and log-normalized
total phenolic compounds (sum of PeCP and OH-PCBs).
6.8.2.3. Animal studies
Total chlorinated phenolic compounds were also nega-
The finding of intellectual impairment in humans is sup-
tively associated with T3 (r = ญ 0.48, P = 0.03) (Sandau
ported by animal studies on exposure to PCBs (Brouwer
et al., 2002). The results indicate that PCBs, OH-PCBs
et al., 1998; Eriksson and Fredriksson, 1998; Hany et
and PeCP affect thyroid hormone status.

Chapter 6 ท Toxicological Properties of Persistent Organic Pollutants and Related Health Effects
73
6.8.2.5. Oxidative stress induced to the nervous system
6.9. Conclusions and unanswered questions
by methylmercury
There is increasing evidence of adverse trends in human
Methylmercury is a highly toxic environmental neuro-
reproductive health, most notably testicular cancer and
toxin that can cause irreparable damage to the central
female breast cancer, whereas the decrease in sperm
nervous system (Choi, 1989; Clarkson, 1993, 1997). Al-
counts apparent from some studies is still being dis-
though the underlying biochemical and molecular mech-
cussed. However, causal links between effects and expo-
anisms that lead to impaired cell function and nerve cell
sure to environmental chemicals have still not been
degeneration are not well understood, there is abundant
firmly established.
evidence supporting the hypothesis that a major mecha-
Environmental chemicals have been focused on be-
nism of MeHg neurotoxicity involves an oxidative stress
cause of their capacity to interfere with hormone activi-
(Sarafian and Verity, 1991; Yee and Choi, 1996). Mer-
ties and hence their possible relation to trends in hor-
cury increases production of reactive oxygen species via
mone related health effects. In wildlife, there is more
deregulation of mitochondrial electron transport as well
convincing evidence of links between environmental ex-
as through glutathione (GSH) depletion (Lund et al.,
posure and endocrine disruption. This strengthens the
1993). The oxidative stress hypothesis is clearly sup-
concerns about endocrine modulation by environmental
ported by the finding that MeHg neurotoxicity can be
chemicals in humans. Because the developing fetus is
inhibited by various anti-oxidants including Se (Park et
particularly susceptible to exposure to environmental
al., 1996) and N-acetyl-L-cysteine, a precursor of GSH
chemicals, and because there are many different effect
(Ornaghi et al., 1993).
targets, evaluation in terms of both lifetime effects (gen-
Glutathione peroxidase (GSHPx) and glutathione
erations) and effects on organs (time to dysfunction) is
reductase (GSHRd) activities were measured in blood
complicated. Much research and monitoring are still re-
samples from 142 Inuit from Sallummiu, Canada (Mi-
quired, and there is a need to develop, refine, and vali-
rault and Dewailly, pers. comm., 2001). Activities of en-
date test methods that can accurately predict the effects
zymes involved in detoxification of free radicals were
of chemicals on human health.
measured in order to investigate relationships between
In this context, risk assessment must include interac-
Hg, Se and oxidative stress. It was observed that Hg was
tion between chemicals, because in general humans are
negatively correlated with GSHRd activity; an NADPH-
exposed to chemical mixtures. Finally, and most impor-
dependent enzyme that regenerates glutathione from
tantly, is the question of whether the available evidence
glutathione disulfide. In contrast, plasma Se concentra-
is strong enough to warrant regulation of the chemicals
tion was positively correlated with GSHPx activity; a se-
concerned: Which test methods should be included for
lenoenzyme that catalyses the conversion of hydrogen
the definition of an endocrine disrupter? Is demonstrat-
peroxides to water. Hence, Hg exposure may diminish
ing detrimental effects in wildlife sufficient to require
defense mechanisms against oxidative stress by limiting
regulation of a chemical? International controversy will
the availability of glutathione, while Se may afford pro-
continue until commonly accepted grounds for regula-
tection by favoring the destruction of hydrogen peroxide
tion of potential hormone-disturbing chemicals are es-
(Mirault and Dewailly, pers. comm., 2001).
tablished.
Biochemical assessment of oxidative stress markers
After reviewing what has been done, and what is
also includes three other indices. Firstly, the ratio of the
possible and desirable to do in order to obtain a better
reduced form of coenzyme Q10 (ubiquinol-10) to the oxi-
assessment of the human health impact related to the
dized coenzyme Q10 (ubiquinone-10) in plasma, which
contamination of the Arctic food web, implementation
is now considered as one of the most reliable and sensi-
of a circumpolar biomarker monitoring and research
tive indices of an oxidative stress in vivo (Finckh et al.,
programme (based on Table 6ท1) has been recommended
1995; Lagendijk et al., 1996; Yamashita and Yamamoto,
by the AMAP Human Health Expert Group. This set of
1997). In contrast to the total level of coenzyme Q10,
biomarkers will produce the information necessary to
which is reported to be associated with multiple factors
complement and support the determination of causality
including gender, age, and cholesterol and triglyceride
in relationships found in epidemiological studies. Some
levels (Kaikkonen et al., 1999), the ubiquinol-10/ubi-
of the included biomarkers are also likely to be used in
quinone-10 ratio index is apparently independent of
the future to improve risk assessments and to establish
these variables and thus represents the oxidative stress
guidelines which, at present, are still largely based on
index of choice. Secondly, an increased level of specific
experimental studies conducted on laboratory animals.
F2-isoprostanes (direct oxidation metabolites of arachi-
Finally, such a programme will effectively meet AMAP
donic acid) in plasma and/or urine is another index re-
requirements for monitoring human health impacts of
cently used to demonstrate oxidative stress in several
contamination in the Arctic.
pathological conditions involving oxygen free-radical for-
At the same time, it is important to be aware that the
mation (Patrono and FitzGerald, 1997; Pratico, 1999).
use of biomarkers of effects presents new and difficult
The most easily measurable and frequently used F2-iso-
challenges in relation to the communication of results to
prostane species as a marker of oxidative stress in vivo is
local people. The concept of biomarkers is usually diffi-
8-isoprostaglandin F2- (Patrono and FitzGerald, 1997;
cult to understand for the lay people, and results can be
Pratico, 1999). Thus the levels of 8-isoprostaglandin F2-
hard to describe and communicate in an unambiguous
in plasma samples will be measured. Finally, the level
manner. The fact that biomarkers are indices of subtle
of plasmatic low-density lipoprotein (LDL) oxidation
deleterious effects, with variable sensitivity and speci-
could also be assessed as a potential marker of oxidative
ficity to disease, makes their interpretation even more
stress (Dewailly, pers. comm., 2001).
difficult. An analogy with the effects of alcohol con-

74
AMAP Assessment 2002: Human Health in the Arctic
sumption over time might be useful in helping to illus-
Although receptor-based assays can be very useful, it
trate the role that biomarkers of effects can play in de-
is imperative to recognize that endocrine disruptive ef-
scribing the development of health impacts (i.e., the se-
fects can also be mediated through interactions at other
quence from alcohol intake, liver enzyme increases, to
levels (e.g., co-activator or repressor levels, enzymes in-
development of cirrhosis).
volved in hormone biosynthesis or degradation, etc.).
There are still major gaps and deficiencies in our un-
It will be important to design assays that can be directed
derstanding of health effects of food chain contaminants
toward improved understanding of the mechanism(s) in-
in the Arctic. Some of these are highlighted as follows.
volved, further helping in the interpretation of the re-
Exposure during the developmental period is impor-
sults and future prevention.
tant because it represents the most sensitive period for
It is important to recognize that genetic variability
several health effects. However, other critical life stages
may affect the susceptibility of individuals or popula-
such as puberty and aging can be highly relevant for ef-
tions to the effects of POPs and MeHg.
fects on reproduction, the immune system, or the nerv-
Studies on environmentally relevant mixtures (at rel-
ous system.
evant concentrations) are required to investigate possi-
Efforts should be made to relate molecular or biologi-
ble interactions between components of the mixture.
cal markers to adverse health endpoints at the individual
Understanding the relationship between subtle bio-
and population level. Biomarkers of effect should be inte-
logical effects and chronic diseases may prove to be the
grated in epidemiological studies in order to fill knowl-
greatest challenge. Many potential mechanisms of action
edge gaps between exposure and overt clinical effects.
have yet to be discovered and researched.