Meeting Report: Moving Upstream—Evaluating Adverse Upstream End Points
for Improved Risk Assessment and Decision-Making
Tracey J. Woodruff,1 Lauren Zeise,2 Daniel A. Axelrad,3 Kathryn Z. Guyton,4 Sarah Janssen,5,6 Mark Miller,2,7
Gregory G. Miller,3 Jackie M. Schwartz,1 George Alexeeff,2 Henry Anderson,8 Linda Birnbaum,9 Frederic Bois,10
Vincent James Cogliano,11 Kevin Crofton,9 Susan Y. Euling,4 Paul M.D. Foster,12 Dori R. Germolec,12 Earl Gray,9
Dale B. Hattis,13 Amy D. Kyle,14 Robert W. Luebke,9 Michael I. Luster,15 Chris Portier,12 Deborah C. Rice,16
Gina Solomon,5 John Vandenberg,4 and R. Thomas Zoeller 17
1Program on Reproductive Health and the Environment, Department of Obstetrics and Gynecology, University of California, San Francisco,San Francisco, California, USA; 2Ofﬁce of Environmental Health Hazard Assessment, California Environmental Protection Agency, Oakland,California, USA; 3Ofﬁce of Policy, Economics and Innovation, and 4National Center for Environmental Assessment, U.S. EnvironmentalProtection Agency, Washington, DC, USA; 5Department of Medicine, University of California, San Francisco, San Francisco, California,USA; 6National Resource Defense Council, San Francisco, California, USA; 7Pediatric Environmental Health Specialty Unit, University ofCalifornia, San Francisco, California, USA; 8Wisconsin Division of Public Health, Madison, Wisconsin, USA; 9National Health andEnvironmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA; 10InstitutNational de l’Environnement Industriel et des Risques, Verneuil-en-Halatte, France; 11International Agency for Research on Cancer, Lyon,France; 12National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services,Research Triangle Park, North Carolina, USA; 13George Perkins Marsh Institute, Clark University, Worcester, Massachusetts, USA; 14Schoolof Public Health, University of California, Berkeley, Berkeley, California; 15National Institute for Occupational Safety and Health, Atlanta,Georgia, USA; 16Environmental and Occupational Health Program, Maine Center for Disease Control and Prevention, Augusta, Maine, USA;17Department of Biology, University of Massachusetts, Amherst, Amherst, Massachusetts, USA
defines an adverse effect as “a biochemical
BACKGROUND: Assessing adverse effects from environmental chemical exposure is integral to public
change, functional impairment, or pathologic
health policies. Toxicology assays identifying early biological changes from chemical exposure are
lesion that affects the performance of the
increasing our ability to evaluate links between early biological disturbances and subsequent overt
whole organism, or reduces an organism’s abil-
downstream effects. A workshop was held to consider how the resulting data inform consideration
ity to respond to an additional environmental
of an “adverse effect” in the context of hazard identification and risk assessment.
challenge” (U.S. EPA 2007a) and, for exam-
OBJECTIVES: Our objective here is to review what is known about the relationships between chemical
ple, considers such end points as alterations in
exposure, early biological effects (upstream events), and later overt effects (downstream events)
circulating levels of sex hormones to be an
through three case studies (thyroid hormone disruption, antiandrogen effects, immune system disrup-
adverse effect (U.S. EPA 1996). Identifying an
tion) and to consider how to evaluate hazard and risk when early biological effect data are available.
adverse effect forms the basis for hazard identi-
DISCUSSION: Each case study presents data on the toxicity pathways linking early biological pertur-
fication and for defining the critical effect for
bations with downstream overt effects. Case studies also emphasize several factors that can influ-
ence risk of overt disease as a result from early biological perturbations, including background
chemical exposures, underlying individual biological processes, and disease susceptibility. Certain
overt disease to elucidating toxicologic path-
effects resulting from exposure during periods of sensitivity may be irreversible. A chemical can act
ways has been recognized and endorsed by the
through multiple modes of action, resulting in similar or different overt effects.
CONCLUSIONS: For certain classes of early perturbations, sufficient information on the disease
process is known, so hazard and quantitative risk assessment can proceed using information on
upstream biological perturbations. Upstream data will support improved approaches for consider-
University of California-San Francisco, Suite 1100,1330 Broadway St., Oakland, CA 94612, USA.
ing developmental stage, background exposures, disease status, and other factors important to
Telephone: (510) 986-8942; fax: (510) 986-8960.
assessing hazard and risk for the whole population.
KEY WORDS: adverse health effects, androgen antagonists, hazard identification, immunotoxicants,
This work was supported by California Environ-
risk assessment, science policy, thyroid hormone, toxicologic assessments. Environ Health Perspect
mental Protection Agency, Office of Environmental
116:1568–1575 (2008). doi:10.1289/ehp.11516 available via http://dx.doi.org/ [Online 10 July 2008]
and Health Hazard Assessment, contract OEHHA-06-S34; the U.S. Environmental Protection Agency,Office of Policy, Economics, and Innovation,
To evaluate the potential of environmental
hazard and risk assessments have often been
National Center for Environmental Economics and
chemicals to cause harm, to estimate the risks
the more overt diseases or defects, rather than
National Center for Environmental Assessment,
that chemical exposures pose to the popula-
events that occur earlier in the disease process.
contract EP07H001060; the Intramural ResearchProgram of the National Institute of Environmental
tion, and to identify opportunities for preven-
Increasingly, toxicology assays are providing
Health Sciences (NIEHS), National Institutes of
tion and intervention, the type and extent of
more information on how chemicals can inter-
Health (NIH); University of California, Berkeley,
adverse effects associated with exposure to a
fere with cellular signaling or metabolism, dis-
NIEHS Superfund Program at Berkeley, NIH grant
chemical must be elucidated. To date, hazard
P42 ES04705; and University of California,
and risk assessments have relied largely on data
expression, or otherwise play a role early in dis-
from traditional toxicologic studies, such as the
ease processes. As scientific understanding of
This report has been reviewed by the National
Institute of Occupational Safety and Health and the
2-year, chronic toxicology and carcinogenesis
the mechanisms through which chemical expo-
U.S. Environmental Protection Agency’s Office of
studies or the two-generation reproductive tox-
sures advance pathologic processes resulting in
Research and Development, and approved for publica-
icity assay. A primary goal of these studies is to
disease increases, so too does the opportunity
tion. Approval does not signify that the contents neces-
identify whether chemical exposures cause
for effective and efficient hazard identification
sarily reflect the views and policies of the agency. The
overt disease outcomes, such as birth defects
and risk assessment. A necessary step in incor-
findings and conclusions in this report are those of the
and neoplasia. These studies also provide data
porating data on early biological perturbations
authors and do not necessarily represent the views ofinstitutions affiliated with the authors.
on biological events that precede these overt
is to consider how these early events relate to
The authors declare they have no competing
disease outcomes, often referred to as precursor
the concept of “adverse effects.” The U.S.
effects. Adverse effects identified in existing
Received 27 March 2008; accepted 9 July 2008.
VOLUME 116 | NUMBER 11 | November 2008 • Environmental Health Perspectives
• Were the steps from the upstream event(s) to
deficits of TH and is diagnosed primarily by
agencies (Collins et al. 2008; National Research
the overt downstream effect(s) identified?
comparing an individual’s level of TH and TSH
Council 2007). These organizations note that
How does our understanding of these steps
to population reference ranges. Untreated con-
moving toward a focus on perturbations along
inform the use of the upstream event as a basis
genital (neonatal) hypothyroidism can have
the disease pathway should result in assays that
for risk assessment, particularly in situations
severe consequences on neurologic develop-
can test more chemicals with reduced cost and
when we only have data on upstream events?
time and fewer animals, and improve the scien-
• How does an understanding of variability in
hypothyroidism are treated with T4, and even
tific understanding of the relationships between
background biological status (e.g., susceptibil-
small doses (e.g., 2 µg/kg/day) significantly
ity or sensitivity, genetic or otherwise) affect
improve later cognitive performance, demon-
strating the sensitivity of the developing brain to
sary to consider how data on early biological
• How might each end point be influenced by
TH insufficiency (Oerbeck et al. 2003; Selva
changes relate to the concept of an adverse
effect and how these data might best be inte-
• In considering an upstream biological end
grated into hazard and risk assessment. To
point that is measured as a continuous variable
ing pregnancy can cause lasting developmental
[e.g., thyroid-stimulating hormone (TSH)],
deficits in the child. Decrements in human
what approach should be taken in deciding
maternal T4 during the early fetal period are
whether a certain degree of upstream change
and Risk Assessment, was held 16–17 May
will lead to the overt downstream outcomes
reduced IQ scores, even for small T4 deficits
that do not constitute maternal hypothyroidism
should changes in upstream indicators within
“normal” ranges be considered in evaluating
Three case studies were presented at the work-
potential for overt downstream outcomes in
shop that described available data on toxico-
by a complex interplay of dynamic processes,
logic pathways connecting chemical exposures
• Can the association between upstream events
including dietary intake of iodine (necessary
to early upstream biological events and then to
and downstream effects for chemicals consid-
for synthesis of TH); the transport of iodine
subsequent overt effects, which are considered
ered in the case study be generalized to other
into the thyroid; its synthesis and storage in
) thyroid hormone disruption
chemicals that have the same upstream effects?
the thyroid gland; its release and transport
and related toxicities; b
• What are the obstacles to the use of the
through circulation; the deiodination of T4 to
male reproductive effects; and c
upstream event in risk assessment (hazard
T3 in peripheral tissues; and the degradation
indicators of immunosuppression. To frame
identification; dose–response assessment)?
discussions in terms of implications for hazard
is regulated through a negative feedback loop
identification and risk assessment processes, the
involving the hypothalamus and pituitary.
case studies explored the following questions:
Case Study Synopsis:
• What are the precursor effects or other early
Thyroid Hormone Disruption
signals the pituitary gland to release TSH,
biological changes linked to the case study
and Related Toxicities
which stimulates the thyroid to increase TH.
Thyroid hormone overview.
The thyroid hor-
When levels rebound, the hypothalamus sig-
• Is there sufficient evidence to associate the
mones (TH) thyroxine (T4) and triiodothyrine
nals the pituitary to decrease TSH. Finally,
upstream events with the overt downstream
(T3) are essential to neurologic development,
TH action is mediated through TH receptors
skeletal growth, and normal function of the pul-
• What related information would or does
monary system, metabolism, and cardiovascular
In hazard identification and risk assess-
enhance our understanding of the relation-
system. This case study emphasized the effects of
ment, TH levels could serve as an upstream
ship between the upstream event and down-
thyroid disruption on neurologic development.
indicator of adverse effects on neurologic
Hypothyroidism is a condition of persistent
development. Table 1 provides an overview of
Classes, mechanisms of action, and effects of thyroid-disrupting chemicals on thyroid hormone homeostasis.
Decreased thyroidal synthesis of T3 and T4
Perchlorate, chlorate, bromate, nitrates, thiocyanate
Decreased thyroidal synthesis of T3 and T4
Methimazole, propylthiourea, amitrole, mancozeb,
soy isoﬂavones, benzophenone 2, 1-methyl-3-propyl-imidazole-2-thione
Increased biliary elimination of T3 and T4
Acetochlor, phenobarbital, 3-methylcholanthrene,
Increased biliary elimination of T3 and T4
TCPOBOP, pregnenolone-16α-carbonitrile, TCDD,
Hydroxlyated PCBs, triclosan, pentachlorophenol
FD&C Red dye no. 3, propylthiouracil, PCBs,
Abbreviations: AhR, aryl hydrocarbon receptor; CAR: constitutive androstane receptor; FD&C, Federal Food, Drug, and Cosmetic Act (1938); MCT, monocarboxylate transporter; OATPs,organic anion-transporting polypeptides; PXR, pregnane X receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p
-dioxin; TCPOBOP, 1,4-bis
[2-(3,5-dichloropyridyloxy)]benzene; TH, thyroidhormone; TR, thyroid receptor; TRE, thyroid hormone response elements. Data from Crofton (2008).
Environmental Health Perspectives • VOLUME 116 | NUMBER 11 | November 2008
classes, mechanisms of action, and effects for
among individuals in the level of TH that rep-
multiple mechanisms of action. There are also
chemicals that disrupt TH homeostasis and
resents homeostasis, and the normal range of
numerous uncertainties in extrapolating thy-
action. For example, perchlorate inhibits the
fluctuations for an individual is narrower than
roid data from laboratory animals to estimate
uptake of iodine, resulting in decreased synthe-
the normal range for a population (Figure 1).
effects in humans, including species differ-
sis of TH. Polychlorinated biphenyls (PCBs)
Consequently, a TH value within the popula-
ences in the expression or structure of specific
and the pesticide acetochlor activate enzymes
tion reference range is not necessarily normal
functional proteins that may affect the toxicity
in the liver that increase excretion of TH, thus
or healthy for the individual (Andersen et al.
reducing circulating levels (Crofton 2008).
TH levels influence a spectrum of health
Factors influencing effects of thyroid-
population range may therefore be associated
outcomes and symptoms, in addition to neuro-
with adverse effects for some individuals.
logic development. Lower
T4 and higher TSH
markedly with life stage. The fetus, infant,
Assessments considering thyroid-disrupting
levels are correlated with adverse effects on the
and child appear the most susceptible to neu-
chemicals (TDCs) in isolation are likely to
rologic disruption. The permanent effects of
underestimate the potential disruption of TH
1991) and cardiovascular system, including
TH insufficiency on neurologic development
levels or action by real-world chemical mixtures.
increased blood pressure and less favorable
were discussed above. Infants are likely less
blood lipid profiles (Asvold et al. 2007a,
resistant to fluctuations in TH synthesis than
2007b). A meta-analysis of 14 epidemiologic
TDCs, including dioxins, PCBs, perchlorate,
studies found an overall increase in risk of
months’ supply of TH and the half-life of T4
brominated flame retardants, bisphenol A, and
coronary heart disease of > 65% in those with
in serum is 7–10 days (Greer et al. 2002;
several pesticides (Centers for Disease Control
subclinical hypothyroidism (elevated in TSH
Vulsma et al. 1989). In contrast, the newborn
and Prevention 2008). A mixture of 18 TDCs
with normal T4) (Rodondi et al. 2006).
thyroid stores a 1-day supply of TH and the
(dioxins, dibenzofurans and PCBs) was tested
Conclusions regarding thyroid data.
half life of T4 in serum is 3 days (Vulsma
at doses comparable to human exposure levels
environmental chemicals are capable of dis-
et al. 1989). Therefore, effects on TH synthe-
for effects on serum T4 in rats. Components of
rupting thyroid hormone levels, and many of
this mixture affect T4 through two different
these have the effect of decreasing circulating
mechanisms of action: The dioxins, dibenzo-
levels of T4. Compensatory mechanisms may
Recognizing subpopulations at risk is a key
furans, and dioxin-like PCBs in the mixture
not be sufficient to counteract the potential
consideration in evaluating effects on TH levels
activate one set of liver enzymes, and the non-
downstream consequences of these T4 decre-
or action as adverse.
Hypothyroidism is preva-
dioxin-like PCBs activate a separate set of liver
ments. First, findings in both animals and
lent in the U.S. population. Between 1999 and
humans indicate that even small maternal T4
2002, 7.3% of the U.S. population ≥ 12 years
effect on T4 at environmentally relevant doses
of age reported having thyroid disease or taking
and a 2- to 3-fold greater than dose-additive
range during pregnancy can have adverse neu-
thyroid medication. Hypothyroidism is fre-
effect on T4 at higher doses (Crofton et al.
rodevelopmental consequences, such as reduc-
quently undetected and therefore untreated.
2005), demonstrating that exposures to chemi-
tion in IQ, in the developing child. Second,
This condition often persists even in those who
cals acting on different pathways can have
fetuses and infants do not have stored thyroid
cumulative effects on an upstream marker.
hormone, and thus have limited capacity to
Assessments considering single TDCs in isola-
respond to thyroid hormone decrements dur-
tion are therefore likely to underestimate the
ing critical stages of development. Third, there
is a substantial prevalence of thyroid hormone
causes an increased demand on the thyroid
exposure to chemical mixtures. It is appropri-
insufficiency in the population of U.S. women,
gland, and hypothyroidism is twice as com-
ate to presume cumulative effects unless there
indicating that compensatory processes are
mon during pregnancy (Aoki et al. 2007).
is evidence to the contrary, and it is important
already compromised for many individuals.
The current method for assessing thyroid
for risk assessments to consider real-life
Therefore, for hazard identification pur-
health may not be predictive of adverse down-
Thyroid health is evaluated pri-
Challenges for assessing risks of exposure to
TDC exposures that would result in reduced
marily by comparing an individual’s level of
Currently available mathematical mod-
T4 in a population should be considered an
TH and TSH to population reference ranges.
els may not be able to accurately predict the
adverse effect. Further-upstream disruptions
However, there is a substantial variability
that result in lowered T4 levels, such as inhi-
bition of iodine uptake, should similarly be
considered adverse when their potential con-sequences include alteration of T4 levels.
Because additivity or synergy of TDCs withdifferent modes of action has been demon-strated, and background exposure to many
TDCs is common, risk assessments shouldconsider simultaneous exposures to multiple
agents and account for these interactions.
demand on the thyroid gland, pregnant womenare likely to have heightened sensitivity to thy-
roid toxicants, particularly if they have low
dietary iodine or thyroid peroxidase antibodies.
These sensitive subpopulations are likely to be
Distribution of 12 monthly measurements of total T4 in 15 healthy men (white bars) and one indi-
vidual (black bars). The distribution in one individual is about half the width of the distribution in the group.
sizable; for example, 38% of 126 pregnant
Frequency represents number of measurements. Adapted from Andersen et al. (2002) with permission.
women in the National Health and Nutrition
VOLUME 116 | NUMBER 11 | November 2008 • Environmental Health Perspectives
Examination Survey (NHANES), 2001–2002,
antiandrogens in that they bind antagonisti-
had low iodine intake (< 100 µg/L urinary
cally to the androgen receptor (AR), interfering
semen quality in adult males (Hauser et al.
iodine) (Caldwell et al. 2005). Because thyroid
with endogenous testosterone and dihydro-
2006). The exposure levels in most subjects in
hormone levels affect cardiovascular risk fac-
these studies were comparable to the general
tors, the potential impact of TDCs on the
decreasing the expression of androgen-depen-
health of adults in the general population also
dent genes. In fetal rat studies, exposure to
Antiandrogen effects as adverse end points
is large. Therefore, effects on thyroid represent
these chemicals has been consistently associated
in hazard identification and risk assessment.
a risk to the general population and not just
with retained nipples, decreased sperm counts,
An antiandrogenic agent, therefore—one that
decreased anogenital distance, hypospadias,
and decreased size or agenesis of the accessory
action (e.g., binding to the androgen receptor,
Case Study Synopsis:
sex glands (Gray et al. 2006). The severity of
or via interference with androgen synthesis)—
can be predictably associated with a series of
adverse end points in animals and likely also
Overview of mammalian male reproductive
industrial chemicals with widespread exposure
in humans. Several additional scientific find-
Early in fetal development,
in the general population—that do not bind
ings need also be considered when consider-
male sexual differentiation is determined
ing antiandrogen effects as upstream adverse
by expression of a Y chromosome gene, the
activity by inhibiting testosterone synthesis.
sex-determining region Y (SRY). Appropriately
Specifically, phthalates with four to six carbon
Specific periods during development
timed SRY expression results in differentiation
side-chains interfere with testosterone produc-
are uniquely susceptible to perturbations.
of Sertoli cells in the testes, initiating a cascade
tion by inhibiting cholesterol uptake into the
Experiments in rats have demonstrated that
of events that result in development and differ-
mitochondria by steroidogenic acute regulatory
both the dose and the timing of exposure are
entiation of male reproductive structures and
protein and by inhibiting some, but not all, of
important for development of phthalate syn-
regression of female structures. The interstitial
drome. Gestation days 12–19 have been iden-
cells of the testes, Leydig cells, produce two
(Barlow et al. 2003). These phthalates have
tified as a sensitive period in rats, during
been most consistently shown to reduce testos-
which exposure to dibutyl phthalate induces
development: the steroid sex hormone testos-
terone production in animal research to date,
phthalate syndrome, with some irreversible
terone and the peptide hormone insulin-like-3
effects (Carruthers and Foster 2005).
(INSL-3). Testosterone is necessary for differ-
(Gray et al. 2000; Liu et al. 2005).
entiation of the Wolffian duct into the epi-
bioactive metabolites responsible for phthalate
didymis and secondary sex organs such as the
phthalate-induced decrease in testosterone
toxicity. Excretion of phthalate monoesters in
seminal vesicles. Both testosterone and INSL-3
results in a syndrome of anti-androgenic repro-
urine is enhanced by glucuronide conjugation.
are necessary for testicular descent in mam-
ductive abnormalities, referred to as the
mals: Testosterone mediates transabdominal
“phthalate syndrome,” which is characterized
curonidation pathway is immature and ineffi-
descent of the testes (Klonisch et al. 2004),
by malformations of the epididymis, vas defer-
cient during the period of susceptibility for
whereas INSL-3 is necessary for gubernacular
ens, seminal vesicles, and prostate; hypospa-
phthalate syndrome. This relatively low level of
dias, cryptorchidism, and testicular injury;
glucuronidation compared to adults could ren-
permanent changes (feminization) in the reten-
der the fetus much more susceptible to the bio-
descent of the testes into the scrotum (Wilson
tion of nipples and areolae (sexually dimorphic
logically active phthalate metabolites during a
et al. 2004). Disruption of testosterone and/or
structures in rodents); and demasculinization
critical period of sexual development. Exposure
INSL-3 production or action can result in
of the growth of the perineum, resulting in a
to rats during this period of gestation has
cryptorchidism (undescended testes), one of
reduced anogenital distance (Mylchreest et al.
demonstrated the proportion of free phthalate
the most common birth defects in humans.
2000). As with other antiandrogenic chemicals,
monoesters relative to glucuronidated phtha-
Exposure to antiandrogens during fetal
the severity of effects increases with dose.
late monoesters in amniotic fluid is much
During fetal life, there is a transient peak
The carboximide pesticides and four to six
higher than levels in the urine of the pregnant
in testosterone levels necessary for proper
side-chain carbon phthalates have different
development of male reproductive tissues.
modes of action resulting in the same spectrum
Exposure to mixtures of AR antagonists
Disruption in androgen action during this
and androgen synthesis disruptors.
critical time window results in a number of
development. In the developing male repro-
95% of the population from 6 to > 65 years of
abnormalities that consistently develop in lab-
ductive tissues, disruption of either of these
age are exposed to at least five phthalates on a
oratory animals (e.g., rats and rabbits), includ-
two parts of the androgen action pathway con-
regular basis (Silva et al. 2004). Recent studies
ing retained female structures, such as nipples
verges into a final common pathway: a decrease
show that exposure to mixtures of chemicals
in male rodents, and malformations of male
in androgen-dependent gene expression.
that interfere with androgen action results in
reproductive structures, such as hypospadias
The spectrum of effects seen in phthalate
dose-additive effects. Rats exposed to a mixture
(an abnormal location of the urethral opening)
syndrome parallels a spectrum of human dis-
of AR antagonists, vinclozolin, procymidone,
eases called “testicular dysgenesis syndrome”:
and flutamide, all acting through the same
infertility, cryptorchidism, hypospadias, and
mode of action, at doses that would not have
androgen activity, including a decrease in
testicular cancer. Several epidemiologic stud-
caused hypospadias alone, resulted in > 50% of
testosterone production or interference with
ies have shown associations between certain
the animals having hypospadias (Christiansen
phthalate metabolites and male developmen-
et al. 2008). Rider et al. (2008) found that pre-
modes of action has been well described in
tal reproductive outcomes, including short-
natal exposure to a mixture of seven phthalates
the literature for two different classes of
and pesticides with differing modes of action
chemicals with antiandrogenic effects.
prenatally exposed to phthalates (Swan et al.
(i.e., AR antagonist or inhibition of androgen
First, carboximide pesticides such as lin-
2005), changes in reproductive hormone lev-
synthesis) produced cumulative, dose-additive
uron, vinclozolin, and procymidone are classic
els in male infants exposed to phthalates in
outcomes in the androgen-dependent tissues.
Environmental Health Perspectives • VOLUME 116 | NUMBER 11 | November 2008
Exposures to phthalates can result in
mediator production, and generation of long-
Effects on the immune system during periods
adverse effects from modes of action other
lived memory cells that respond rapidly on sub-
In a review of select develop-
than disrupting testosterone synthesis.
sequent exposures to the same or closely related
mental immunotoxicology literature, Luebke
addition to inhibiting the production of testos-
antigens. The acquired immune response is
et al. (2006) concluded that exposure to envi-
terone, phthalates also interfere with pro-
mediated by two types of lymphocytes, T cells
ronmental chemicals during key developmental
duction of INSL-3, which is necessary for
and B cells. T cells are a source of soluble media-
periods can affect immune function in labora-
gubernaculum development (Wilson et al.
tors known as cytokines that stimulate other
tory animals. Developmental exposure sup-
2004) and subsequent descent of the testis into
immune system cells, including B cells, and act
presses T-cell function through adolescence in
the scrotum. Unique to phthalate exposure is
as effector cells with cytotoxic activity. B cells
mice and throughout adulthood in rats.
an absence of the gubernacular ligament in
mature into plasma cells, which serve to produce
Gestational/neonatal exposure to diazepam
males exposed in utero.
Thus, phthalates can
immunoglobulins (Ig), or antibodies, of various
(DZP) and diethylstilbestrol (DES) in rodents
act through two different modes of action to
subclasses, including IgM, IgG, IgE, IgA, and
can cause long-lasting (up to lifetime) effects on
IgD. Each of these antibody subclasses serves a
the immune system, including suppression of
Conclusion regarding antiandrogen data.
unique function in the immune response.
IgM and IgG antibody responses and nonspe-
The prenatal exposure of males to antiandro-
Factors influencing immune function.
cific lymphocyte proliferation (DES) at doses
genic chemicals illustrates several important
Numerous factors influence the outcome of
that cause only short-term immunotoxicity in
points when considering upstream indicators
an encounter with an infectious agent. Innate
adults. Developmental exposure to DZP, lead,
of adverse effects: First, it is necessary for expo-
defenses, critical in the early phase of resis-
or tributyltin oxide caused immunotoxicity at
sure to occur during the critical window, the
tance, may be overcome by large numbers of
lower doses in young than in mature animals,
period of reproductive organ development, in
and developmental effects were persistent.
order for certain developmental effects to be
pathogen may also prevent effective innate
Clinical experience indicates that adult immune
observed. Second, perturbations early in the
function typically recovers soon after therapeutic
development of the male reproductive system
microorganisms before infection ensues. Host
immunosuppressive treatment ends. However,
predictably result in a wide array of adverse
attributes such as age, sex, genotype, lifestyle,
the developmental data suggest that screening
outcomes that are permanent and irreversible.
and disease status all influence immunocom-
chemicals exclusively in adult animals may fail
Third, exposures to different chemicals with
petence, and each may influence the inci-
to detect persistent effects or those effects that
different modes of action can result in the same
occur at lower doses (Luebke et al. 1999).
outcomes due to a deficiency in androgen-
Immune function also declines with age, as
mediated gene expression. Finally, exposure to
at birth and are thus at increased risk of infec-
do the normal physiologic processes that limit
tion. Protective antibodies are transferred from
microbial invasion. Although data are limited,
action with effects on the same end point. In
mother to fetus across the placenta and to the
this suggests that relatively small changes in
newborn in breast milk, although passive pro-
cause adverse effects in upstream indicators,
tection decreases rapidly as these proteins are
exposure may have more severe consequences if
including a reduction in fetal testosterone lev-
catabolized. Average IgM and IgG antibody
combined with normal immunosenescence.
els or androgen receptor binding, which can
levels do not reach 50% of adult levels until
Several epidemiologic studies have described
increase the risk of a constellation of down-
7–12 months, and IgA reaches adult levels by
associations between early-life chemical expo-
3–5 years. A preponderance of naive T cells
sure, altered immune end points, and frequency
hypospadias, and, later in life, infertility.
of infections. Increased incidence of otitis
Case Study Synopsis: Immune
reduced cell-mediated immunity in the young.
media and respiratory infections were reported
in children exposed to PCBs (Dallaire et al.
Function, Immunotoxicity, and
advanced age; however, few studies have evalu-
2006; Dewailly et al. 2000; Nakanishi et al.
Resistance to Infection and
ated the effects of immunotoxicants in older
1985; Weisglas-Kuperus et al. 2000; Yu et al.
Neoplasia: The Downstream
1998). For example, at 3 months of age, multi-
Impacts of Unintended
ple upstream indicators of impaired immune
have established that extrinsic factors, including
function were detected in breast-fed infants
Immune system overview.
The immune system
with higher PCB exposure levels, including
consists of a complex system of tissues, cells, and
agents, and psychological factors may influence
reduced numbers of white blood cells and lym-
soluble mediators that protects the body against
the course of infection. Furthermore, un-
phocytes, and lower serum IgA levels at 7 and
foreign substances, including infectious agents
intended immunosuppression resulting from
12 months of age (Dewailly et al. 2000). These
and some types of tumor cells. Immune cells are
exposure to environmental chemicals alone or
studies also reported changes in immune system
located throughout the body, either in discrete
in combination with other intrinsic and extrin-
biomarkers, such as changes in blood cell
organs, such as the spleen, thymus, and lymph
sic factors can shift the distribution of normal
counts and T-cell subsets or decreased serum Ig
nodes, or in diffuse accumulations of lymphoid
immune response, resulting in an increase in
levels with increasing PCB exposures (Karmaus
and myeloid cells, as are found in association
individuals classified as “immunosuppressed.”
et al. 2001; Weisglas-Kuperus et al. 2000).
with the skin, lungs, and gastrointestinal tract.
Immunosuppressed individuals might express
Antibody responses to tetanus toxoid vaccine
Destruction of infectious agents relies on both
alterations of immune function such as reduced
were significantly decreased after early postnatal
innate and adaptive immune responses. Innate
antibody production, decreased immune cell
PCB exposure (Heilmann et al. 2006).
responses are rapid, do not require clonal expan-
counts, or ineffective cell signaling. Recent data
sion, and are stimulated by recognition of
also suggest that, in addition to suppression,
pathogen-associated molecules by macrophages,
developmental exposure (i.e., exposure from
natural killer cells, and granulocytes. Adaptive,
birth through puberty) to certain chemicals
quently develop opportunistic infections.
or acquired antigen-specific responses, rely on
may shift the pattern of cytokine production,
Xenobiotic agents are likely to cause consider-
antigen recognition and subsequent events that
leading to a greater incidence of allergy and
ably more subtle immunologic effects. The
culminate in cell proliferation and maturation,
interaction among host genetics, pathogen
VOLUME 116 | NUMBER 11 | November 2008 • Environmental Health Perspectives
virulence, and pathogen dose plays a role in
issues that should be addressed in existing haz-
greater than additive response (“synergism”)
determining the frequency and severity of ill-
ard identification and risk assessment practices.
are possibilities, the case studies support appli-
ness. For individuals exhibiting mild immuno-
Chemical and biological background.
cation of dose additivity as a default, as recom-
of the case studies illustrated the importance of
mended in the guidelines. Considering the
xenobiotic chemical exposure, the infectious
considering preexisting or continuous exposure
influence of multiple chemical exposures on
dose of a given pathogen may be lower than
to environmental chemicals as well as preexist-
upstream events can facilitate a more realistic
that which would cause disease in an individual
ing biological or disease susceptibilities that
characterization of the risks of downstream
contribute independently to risk of overt dis-
effects when assessing a single chemical that is
mally. Limited studies in populations with mild
ease. Preexisting exposures or biological vulner-
an additional increment to the background
forms of immunosuppression (e.g., under psy-
abilities can increase the baseline risk of the
chological stress or administered immuno-
population or enhance already initiated disease
suppressive therapies) have provided qualitative
processes (Figure 2). Consequently, slight per-
health status, as influenced by age, preexisting
turbations in upstream biological indicators are
disease, genetics, and other factors, can influ-
function can also lead to an increased incidence
more likely to increase risk of subsequent
of infectious or neoplastic diseases (Cohen et al.
downstream events given an already more acti-
upstream and downstream adverse end points.
vated state among segments of the population.
For example, the implications of a chemical’s
Conclusions regarding immunosuppression
Chemical background and exposure to
interference with iodine uptake into the thy-
In summary, exposures to environmen-
NHANES data indicate that the
roid are substantially greater for an infant than
tal contaminants have the potential to affect
entire U.S. population has measurable levels of
for an adult because of differences in thyroid
upstream immune indicators, including anti-
multiple environmental contaminants in their
hormone storage and circulating half life.
body synthesis, T-cell function, and other
Likewise, the implications are greater for the
Prevention 2008). Other studies of exposure
38% of pregnant women in the United States
result in compromised downstream resistance
pathways show the population is in constant
who are deficient in iodine (Aoki et al. 2007)
to infection. However, the impact of back-
contact with xenobiotics in air, food, water,
and who simultaneously have greater thyroid
ground levels of xenobiotics on the burden of
disease has not been clearly established. If a
Working Group 2005, 2007; National Library
development of a fetus. Individuals with pre-
positive correlation between increasing expo-
of Medicine 2008; U.S. EPA 2007b; U.S.
existing immune suppression, such as organ
sure and disease is assumed, then even small
Food and Drug Administration 2007). U.S.
transplant patients, those who are HIV posi-
changes in immune function will represent an
EPA guidelines for mixtures risk assessment
tive, and those at early or late life stages, might
increased risk for developing disease.
recommend default assumptions of dose addi-
experience disproportionately higher disease
tivity for mixtures of chemicals with similar
toxicologic activity and response additivity for
Periods of susceptibility.
Exposure to envi-
The case studies demonstrate that certain toxi-
mixtures of chemicals that act independently
ronmental chemicals during periods of suscep-
city pathways are understood well enough that
(U.S. EPA 2000a). The case studies showed
tibility can pose a unique risk of subsequent
screening assays to identify early biological
that these preexisting and concurrent exposures
downstream effects, both in the short and long
changes could be used for hazard identification
can increase the effect a given chemical expo-
term, as well as diminished capacity for recov-
and risk assessment. For example, if a chemical
sure has on disease risk, consistent with the
ery from decrements to physiological systems.
is found to suppress thyroid function, the asso-
dose-additivity default. For example, separate
For example, exposure to thyroid-disrupting
ciated data could be used to assess risk for
studies of antiandrogenic chemicals and thy-
chemicals such as perchlorate during the fetal
developmental neurotoxicity, because the latter
roid-disrupting chemicals found that mixtures
effect can be predicted on the basis of the
of chemicals acting on common systems via
adverse effects on neurologic development by
mechanism of action alone. Similarly, when a
different modes of action had dose-additive
decreasing TH levels; the same mild perturba-
chemical is found to be antiandrogenic, the
effects. Although a less-than-additive response
current science would support using the data
[referred to in the U.S. EPA mixtures guide-
adverse challenge to a healthy adult. Similarly,
to assess increased risk of developmental
lines as “antagonism” (U.S. EPA 2000a)], or a
to impair male reproductive development in
effects. Data from whole-animal tests of thedownstream outcomes would not be requiredfor risk assessment of these outcomes. The
findings of this workshop support the recentNational Academy of Sciences recommenda-
tion that toxicity testing move toward the
direct assessment of upstream events along tox-icity pathways and gradually move away from
the current whole-animal end point–based
assays (National Research Council 2007).
challenges that arise when using upstream
Percent of population
events to assess the adverse effects of chemicalexposures during hazard identification or riskassessment. Several common themes emergedfrom the case study discussions. Some of thesethemes are not unique to the use of upstream
Value of physiological parameter
indicators as the basis of hazard identification
Distribution of a typical physiological parameter within the population and how that may vary
and risk assessment. Rather, they underscore
depending on the inﬂuence of chemical and biologic background.
Environmental Health Perspectives • VOLUME 116 | NUMBER 11 | November 2008
rodents, exposure to phthalates must occur
process of considering continuous upstream
while development of appropriate principles
during gestation days 12–19. When exposure
and practices for assessing the emerging science
occurs during periods of developmental sus-
for timely public health–based decision-making
ceptibility, the dose required to produce an
resulting from chemical exposure are often
effect will typically be lower than the dose
Identifying classes of upstream events.
required to produce effects in fully developed,
reversible. For example, exposure to chemicals
case studies of thyroid perturbation, anti-
can result in immune system suppression and
androgen activity, and certain types of immune
Multiple or complex modes of action.
increased opportunity for infections. A sup-
changes each represent a class of perturbations
Exposure to a chemical can influence disease
whose links to downstream events were deter-
processes through multiple modes of action and
chemical exposure is stopped, and the risk of
mined by workshop participants to be suffi-
also increase the risk of more than one down-
infectious disease returns to original levels.
ciently characterized by the data to move
stream, overt effect. Hence, measurements that
However, for assessments of a constant chronic
toward considering issues of implementation.
focus on a particular pathway or individual
or lifetime exposure, recovery from shorter
A suggested next step is to evaluate chemicals
downstream event may provide an incomplete
duration exposures should not be considered
for their ability to cause the upstream pertur-
picture of the range of effects produced by the
when evaluating whether the observed response
bation identified in the case studies. For these
chemical. Phthalates work through multiple
is adverse. Whether a perturbation is reversible
chemicals, hazard and risk assessment could be
pathways to decrease androgen production but
can depend on intrinsic factors, such as age or
initiated with data on the relationship between
also interfere with the production of INSL-3.
disease status, and extrinsic factors, such as pre-
exposures and early perturbations. Fewer data
The resulting deficits in these hormones during
existing or co-exposures to other contaminants
are needed for subsequent downstream events,
male fetal development can increase the risk of
or the timing of exposure. For example, gesta-
given what is already known on risks linking
several different downstream effects, both man-
tional and neonatal exposure to DZP and DES
the pathway from early to late events, and
ifested early in life (e.g., cryptorchidism) and
in rodents can cause long-lasting effects on
with the presumption that downstream conse-
later in life (e.g., effects on sperm). Data inter-
immune function; in contrast, adult rodents
quences are likely to occur when the upstream
pretations therefore should consider the possi-
exposed to the same dose experience reversible
bility of a complex set of modes of action that
Participants noted other types of early bio-
produce adverse effects on the whole system.
Population variability and defining the
logical perturbations that were not discussed in
Focusing on one single aspect of these complex
Basing the definition of “nor-
the case studies but that are also likely suffi-
mal” function for a biologic parameter on
ciently characterized as to be defined as a class.
characterization of system response.
population reference ranges may not reflect
Early biological perturbations that appeared to
Continuous versus discrete events.
an individual’s normal range or variability
be sufficiently characterized include certain
downstream effects, such as cancer, birth
and may fail to detect an adverse effect. For
high affinity and persistent interactions with the
defects, and infectious illnesses are typically dis-
Ah receptor, changes in hormonal responses
crete events. In contrast, upstream indicators of
adverse decline in thyroid hormone but still
[which are identified in reproductive toxicity
adverse effects, such as changes in hormone
remain within the normal population refer-
risk assessment guidelines as an adverse event
levels or cellular markers of immune function,
ence range (Figure 1). For situations in which
(U.S. EPA 1996)], and cholinesterase inhibi-
are often measured on a continuous scale. Risk
the interindividual variability in a continuous
tion [which has been identified as an indicator
assessment practices recognize differences in
upstream indicator is high, alternative methods
of adverse effects (U.S. EPA 2000b)]. It would
frequency of discrete events as an indication of
to detect adverse changes in the biologic
also be beneficial to expand the list to include
an adverse effect. Characterization of changes
parameter will need to be developed.
biological perturbations that appear common to
in continuous outcomes in risk assessment
many environmental chemicals, such as effects
change constituting adversity. Should any
Workshop participants concluded that for the
information on upstream indicators does not
chemical-induced increase or decrease be con-
toxicity pathways represented in these case
necessarily reduce uncertainty, although it does
sidered adverse? Should there be a focus on
studies, it may be feasible to move toward
add more information about the exposure–
particular cut points in the distribution of
using direct evaluation of upstream mechanis-
disease continuum and increases the predictive
population or individual baseline levels? These
tic indicators as the basis for risk assessment
power of risk assessment and can demonstrate
questions should be considered in the context
greater variability across study subjects than
of the issues of background exposures and age-
The case studies illustrate the complexities
previously recognized. Hence, using upstream
related susceptibilities noted above. One possi-
indicators does not necessarily mean reducing
bility is to consider that deviations from
exposures to environmental contaminants.
applicable uncertainty or adjustment factors.
expected baseline (e.g., for a given individual,
Capturing and translating the complexities of
The case studies suggest
with all its characteristics, susceptibilities, age,
the science is an ongoing challenge in the regu-
sex, physiologic state) will increase the proba-
latory and policy arena. While science pursues
upstream biological markers have utility for
bility (risk) of downstream adverse effects. For
new areas of inquiry, decision making requires
hazard identification and risk assessment. The
example, changes in biological function dur-
timely answers to questions about risks and haz-
approaches to using these upstream indicators
ing susceptible periods, such as during devel-
ards to public health in order to mitigate future
should consider factors such as biological back-
or current potential harm. The regulatory con-
ground, chemical background (and the poten-
text also requires a different sufficiency of evi-
tial for dose additivity or synergism), periods of
function changes of the same magnitude at
dence whereby regulatory decisions can be
other life stages. Whether the perturbation is
made based on evidence that a chemical is likely
modes of action that can increase the risk of
reversible and whether it is possible to use
to cause a particular outcome. The value of a
subsequent downstream events. The case stud-
population reference ranges to detect adversity
parallel-tracks approach was noted, in which
ies also illustrated the need to better integrate
are two key questions that may arise in the
scientific investigation continues in one track
the nonzero baseline concept into hazard and
VOLUME 116 | NUMBER 11 | November 2008 • Environmental Health Perspectives
risk assessment—in other words, background
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Environmental Health Perspectives • VOLUME 116 | NUMBER 11 | November 2008
Mötesprotokoll HAFF Styrelsemöte Tid : Onsdagen den 11 december 2013 kl 18:00-19:30 Plats : Fritids Vindkulla Närvarande : Örjan Liebendörfer (rektor), Cia Håkansson (ordf, förälder klass 3), Pär Ekelöw (förälder klass 3 och 5 och Solvindens lekskola), Per Kristav (förälder klass 3) Elisabet Andersson (förälder klass 1), Olivia Gatte (fd elev har gått ut 9:an vt10), Kat
CURRICULUM VITAE Dott. Ing. Antonella Bogoni CNIT head of research area Via Moruzzi 1 56124 Pisa Italy Tel. +39 050 5492221 Fax. +39 050 5492194 e-mail: antonella. Bogoni@cnit.it CNIT (Consorzio Nazionale Interuniversitario per le Telecomunicazioni) head of research area (digital and microwave photonics) at the Integrated Research Center for Photonic Networks and Technologie