Sex-differentiated Neurotoxicity of Metals and other Endocrine Disrupting Compounds

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>> Welcome to this lecture on

sex differentiated mechanisms
0:02
of neurotoxicity, specifically
focused on exposure to metals
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and other endocrine
disrupting chemicals.
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The recognition that sex, as
a biological variable matters
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in cellular biology,
neuroscience,
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and neurotoxicology is
unfortunately very recent.
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Over the years many
researchers have advocated
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that scientists include
females in their studies.
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In fact, multiple review
articles have been published
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highlighting that since the
1970's, here specifically
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in pharmacology and physiology,
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studies have increasingly
only used male animals
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to the exclusion of females.
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In fact, other reviews overall
on the basic scientists indicate
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that even as of 2009,
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most studies did not
consider science.
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In neuroscience specifically,
studies that focused
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on males outnumber
studies that focused
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on both sexes at a 5 to 1 ratio.
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But these demands have been
successful in the United States,
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as in 2015 the National
Institute of Health demanded
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that all grants addressed
sex as a biological variable
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in their experimental plan
and statistical methods.
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Historically, females were
removed from studies based
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on the suggestion that
estrus cyclicity created more
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variability in measured
phenotypes.
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However, a recent study
by Prendergast et al
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in 2014 supports that overall,
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females are not more
variable than male rodents.
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In fact, for many phenotypes
males show more variability
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than females.
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Secondly, other variables
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such as group housing add much
more phenotypic variability
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to analyses than sex.
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This critical need to
include females in clinical
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and basic research,
does not mean
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that every phenotype will
be sexually differentiated.
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In fact, many will not.
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Nor does it affirm a strict
categorical determination
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of sex.
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In fact, sex differentiated
phenotypes are determined
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by interactions between
chromosomal compliment,
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cellular genetic functions,
hormones, epigenetics,
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and developmental environments
including toxicant exposures.
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As such, sex differences
often occur on a spectrum.
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Further, sex and
gender are not the same.
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Gender is culturally determined.
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So rodent studies
investigating the role of sex
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in neurotoxicity should use
the word sex and not gender.
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Sex differentiation
as a spectrum
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of phenotypes is a critical
concept for neurotoxicology.
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Because there is no
whole pink or blue brain.
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There's no whole male or
whole female phenotype.
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Instead, work from a variety
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of labs supports McCarthy's
sex differentiated neural
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mosaic model.
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Each brain region has
different mechanisms
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to establish a sex
differentiated neurophenotype
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at the population level.
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However, any one individual
can vary region by region,
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where they fall on
that continuum.
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It is important to note
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that a mosaic is not a
non-differentiatable blend.
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There is still a
measurable continuum
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of sex-differentiated
phenotypes in the rodent brain,
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for both cellular,
neurochemical,
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and neuro-architectural
features.
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Taken together, interactions
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between sex-differentiated
neural development,
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early maternal influences,
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and external environmental
risk factors
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in cultural perception all
play a role in the reality
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that neurobehavioral disorders
are some of the most sex-biased
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in their prevalence rates.
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Given the important role the
environment and hormones play
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in determining
sex-differentiated
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neurophenotypes, it's not
surprising xenobiotic exposures,
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particularly endocrine
disrupting chemicals,
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have been increasingly
implicated
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in the increased incidence rates
of neurobehavioral disorders
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with sex-biased prevalence
rates.
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There are three key
parts to this lecture.
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First, we will discuss
relevant features
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of the endocrine
system important
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for neurotoxicology studies
and give a brief review.
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Secondly, we will
discuss metals exposures,
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stress axis disruption, and the
consequent sex-differentiated
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cellular and behavioral
toxicity.
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And finally, I will
give a brief review
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on some region-specific
mechanisms
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of sex-differentiated
neurodevelopment.
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And how EDC exposure may
disrupt early androgens
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that play an organizational
role.
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And to begin our discussion
of the endocrine system,
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the very origin or the word
hormone makes the first point.
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The root of the hormone means
to stimulate or to urge on.
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And often hormones act as
integrators, modulators,
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or transcription
factors to activate
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or enhance cellular pathways.
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Fundamentally, the endocrine
system is the body's chemical
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communication system and
it helps maintain stasis
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and coordination through change,
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change induced either
developmentally
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or environmentally.
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In fact, these dual roles both
coordinating developmental life
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history traits, and responding
to environmental change,
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have classically been separated
into the organizational effects
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of hormones in early development
and the activational effects,
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shifts in hormones in adulthood
needed to maintain a phenotype.
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Of course, classic dogma
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as often is the case,
has many exceptions.
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But overall alterations
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in hormone concentrations help
organize and coordinate growth,
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social attachment, sex,
secondary sex characteristics,
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and critical windows
of learning.
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Furthermore, shifts in hormone
activation are controlled
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by negative feedback loops.
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A variety of endocrine
systems work on cascades.
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Principally functioning along
a hypothalamic, pituitary,
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and distal organ axis.
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For example,
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the
hypothalamic-pituitary-gonadal
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axis,
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the
hypothalamic-pituitary-adrenal
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axis, where signals from the
hypothalamus stimulate the
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pituitary to begin
secreting systemically
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circulating hormones.
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As these systemic concentrations
increase, they each act
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as their own negative
feedback, terminating activation
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in the hypothalamic and
pituitary messengers.
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Systemic hormones in the blood
can occur in three forms.
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Either free, unconjugated
and unbound, or bioavailable,
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bound to low affinity carrier
proteins such as albumin,
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or inactive as they are bound to
high affinity binding proteins
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such as alpha-fetoprotein.
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When consider these
forms, circulating levels
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of free hormones in the
blood that are active
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at target tissues, occur at
extremely low concentrations.
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Furthermore, overall
the general function
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of each hormone is well
conserved across species.
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Although there are important
differences in the details,
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such as the rodent
reliance on corticosterone,
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and the primate reliance
on cortisol
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as the primary glucocorticoids
in the stress axis.
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These key aspects
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of the endocrine system
influence neurotoxicology.
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One, the endocrine
system communicates
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across a broad range of
physiological targets.
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Two, given the low
serum concentrations
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of active systemic hormones,
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research has increasingly
found low dose
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and non-monotonic
dose response effects.
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Three, given the
important role of hormones
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in organism development
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and sexually-differentiated
development, neurotoxicity
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of [inaudible] compounds
is often dependent
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on the critical windows
of exposure,
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and effects may be
late emerging.
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And finally, given that these
systems adapted to respond
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to other environmental
stimuli, exposure to compounds
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with endocrine disrupting
properties may be modulated
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by other non-xenobiotic
environmental stimuli.
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In fact, the concept
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of endocrine disrupting
chemicals is fairly recent.
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Many attribute its origin to the
Wingspread conference in 1991,
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where Theo Colborn and her
colleagues first coined the term
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endocrine disrupting
chemical or EDC.
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Given that the endocrine
system works on cascades
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and coordinates development
and environmental response
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across a variety
of target tissues,
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and that an endocrine
disrupting chemical is defined
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as any chemical that interferes
with hormone activity,
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and that includes production,
secretion, transportation,
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metabolism, binding
action, and/or the excretion
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of endogenous hormones.
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To me it's not surprising
that we've identified
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over 1,000 compounds
that have some form
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of endocrine disrupting
property.
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Metals, although typically not
immediately thought of as EDCs,
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have long been known to
disrupt the endocrine system.
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Lead, methylmercury, arsenic,
and cadmium have all been shown
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to disrupt serum/glucocorticoid
concentrations
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and alter the
hypothalamic-pituitary-adrenal
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axis, the HPA axis.
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This is critically
important for neurotoxicology
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because the HPA axis has
been shown to interact with
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and support the function of
mesocorticolimbic networks
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and alter neurotransmitter
concentrations critical
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for appropriate learning
and cognitive function.
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But to understand how the HPA
axis influences neurotoxicology,
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we first need to
take a deeper look
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at the stress response itself.
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For a quick review of the HPA
axis and the stress response,
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especially given that the word
stress has so many definitions,
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it is important to start by
defining stress and stressor.
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Stress, for our purposes will
be limited to activation of
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and changes
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to the
hypothalamic-pituitary-adrenal
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or HPA axis function.
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Stressors are stimuli, either
environmental or physiology
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or both, that activate
the HPA axis.
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The stress response is
a critical adaptation
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that helps animals internalize
and accommodate to changes
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in homeostatic balance.
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It is a generalized response
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that prioritizes short-term
mechanisms necessary
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for escaping from or
responding to stressors,
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primarily by shunting
sugar into the bloodstream.
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But it also shuts down
long-term repair mechanisms,
 12:55
such as digestion,
muscle repair,
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and certain immune functions.
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Taken together, activation
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of the HPA axis provides
the metabolic support
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for an organism to
preserve homeostasis
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in a variety of circumstances.
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Increasing glucocorticoids
help wake organisms
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up in the morning.
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They provide metabolic support
for life history events,
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such as the physical
demands of late pregnancy.
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And they provide metabolic
support for the flight
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or fight response, activated
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if one perhaps was
chased by an elephant.
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But despite the adaptive
mechanisms of HPA activation
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in providing the metabolic
support for behavior,
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chronic stress where an
organism constantly feels
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like an elephant is chasing
them when there is no elephant
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in the room, leads to disease.
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Given that it shuts down
long-term repair mechanisms,
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these diseases are often immune
deficiency, muscle wasting,
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gastrointestinal dysfunction,
impaired brain function,
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reproductive suppression
and growth reduction.
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Taken together, chronic or
excessive developmental stress
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and developmental
metals exposures,
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share biological targets
including the stress response,
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and mesocortiocolimbic
neurotransmitter function.
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Importantly, particularly
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in low socioeconomic
status communities,
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these developmental
risk factors co-occur.
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Metals exposure, including
lead, is a particular problem
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for children living
in these communities,
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as they typically
sustain the highest blood
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lead concentrations.
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Furthermore, resource
deprivation, both material
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and social, as well as dangerous
neighborhood conditions,
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violence and racism, all
make low socioeconomic status
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communities' potential sources
for persistent, intense,
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and chronic stressors.
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This shared co-occurrence
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and shared biological targets
provides mechanistic support
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for the neurobehavioral shifts
induced by lead, stress,
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and the potential
for them to interact.
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For the following studies on
developmental lead exposure
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and prenatal stress, dams
either rats or mice were treated
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with lead water two months prior
to breeding through weaning.
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On gestational days 15 through
18, during this critical window
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of glucocorticoid
sensitivity, dams were assigned
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to receive either restraint
stress or no exposure,
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or no stress exposure.
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Restraint stress lasts for 30
minutes, three times a day.
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For many analyses one
pup per liter, per sex,
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per behavioral endpoint was used
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to minimize liter
specific effects.
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These breeding and
exposure methods indicate
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that developmental lead
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and prenatal stress
share a biological target
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in the
hypothalamic-pituitary-adrenal
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axis.
16:17
One measurement suggesting or
corroborating that hypothesis,
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is that for rats exposed to lead
and prenatal stress, both lead,
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stress, and lead plus
stress increase serum
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corticosterone levels.
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In addition to the enhanced
disruption of the HPA axis
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by developmental lead
and prenatal stress,
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metals exposures including
methylmercury, arsenic,
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and lead have been shown
in combination with stress,
 16:46
to disrupt behavioral function
in a sex-specific faction.
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This includes behavioral
analyses using the
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fixed-interval schedule
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of controlled behavior,
the FI schedule.
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The FI schedule encompasses many
behavioral domains including
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response inhibition, motivation,
attention and temporal learning.
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It is translationally
relevant behavioral essay
17:13
because children can be placed
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on the same reward
mediated schedule
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to identify correlates
associated
17:20
with neurobehavioral
disorders in children.
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During the FI schedule,
rodents are trained
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to press a lever to
receive a reward.
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Following that training, a 60
second interval is imposed.
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That 60 second interval changes
the reward schedule such that,
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any lever presses that occurred
during the 60 second interval do
 17:46
not immediately provide
a reward.
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Only lever presses
that occur immediately
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after that 60 second interval
has passed will provide a reward
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for the rodent.
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This creates a very
characteristic rate
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of behavioral responding.
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As the animals begin
to learn that responses
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on the lever early on in the
interval do not provide any
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reward, they begin to allocate
their responding on the lever
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to the end of the interval.
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So characteristically
you will see a pause,
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and then you will see the
animal begin to respond
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and those response
rates will increase
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in frequency towards
the end of the interval.
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Then as the next lever press
produces a reward, that interval
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and the behavioral paradigm
or the behavioral pattern
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of waiting, then beginning
to respond at the end
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of the interval begins again.
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The ability of a rodent to
learn the FI schedule can be
18:50
quantitated via a variety
of different variables.
18:57
But today, we will speak mostly
19:01
about the correct
lever response rates.
19:02
For individuals interested
19:11
in understanding
behavioral toxicity,
19:12
I always say it's easiest
to understand behavior
19:14
if you're able to
watch your animals.
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So here is a wonderful
example of a female mouse
19:19
that has been trained
on the FI schedule.
19:22
You will see that
her behavior begins
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with a post-reinforcement pause.
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This video begins
approximately 20 seconds
19:31
into the 60 second interval.
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Following her pause, she will
begin pressing the lever,
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and as the end of the
interval becomes near,
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she will increase her correct
lever response rate frequency.
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[ Background Noise ]
19:47
Historically, work in Doctor
[inaudible] lab has shown sex
20:33
specific changes in overall
correct lever response rates,
20:37
following developmental exposure
to lead and prenatal stress.
20:40
Depending on the lead
dose, timing of the stress,
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and rodent species, these
effects shift slightly.
20:49
But overall, developmental
lead and stress have been shown
20:52
to combine to increase
correct lever response rates.
20:55
As mentioned, maternal arsenic
exposure has also been shown
20:59
in combination with
prenatal stress,
21:07
to alter FI performance
including correct overall
21:09
response rates.
21:14
However, for arsenic, the
effects are a bit different.
21:16
Effects are seen in
both males and females.
21:19
However in males, only animals
that had both arsenic exposure
21:23
and were exposed to
developmental prenatal stress,
21:30
showed any deficits
on the FI schedule.
21:32
And those deficits
were reductions
21:35
in overall correct
lever response rates.
21:39
For females, arsenic,
prenatal stress,
21:43
and the arsenic plus
prenatal stress group,
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all showed reductions in
correct lever response rates
21:49
on the fixed interval schedule.
21:53
Identifying the cellular
mechanisms connecting
22:00
alterations in serum
hormone concentrations,
22:02
and neurobehavioral toxicity
is critically important.
22:05
Epigenetic reprogramming
is emerging
22:09
as a clear cellular mechanism
of neuro-reprogramming
22:12
across the lifespan following
developmental exposures.
22:15
For example, many studies have
been conducted in the Meaney
 22:20
and Shift labs, investigating
how early maternal behavior
22:23
or developmental stress
influences the HPA axis
22:27
and alters brain and
behavioral development
22:31
through epigenetic
reprogramming or NR3C1,
22:34
the glucocorticoid
receptor gene.
22:37
Their studies, mostly
unspecified sex
22:40
or on male offspring, indicate
22:43
that different maternal
behaviors alter both DNA
22:45
methylation and histone
acetylation profiles
22:48
across NR3C1.
22:52
Remember classically,
reductions in DNA methylation
22:55
and increased histone
acetylation represent permissive
22:59
modifications, suggesting
increased accessibility
23:02
of the genome.
23:06
To investigate the
epigenetic consequences
23:09
of developmental lea, prenatal
stress, and the combination,
23:13
we evaluated both methylation
and H3K9 acetylation profiles
23:17
across NR3C1 in adult
postnatal day 60 mice.
23:22
We processed frontal
cortex and hippocampus
23:29
for both males and females.
23:31
For DNA methylation, we
measured methylation percentage
23:33
at 10 CPG sites along NR3C1.
23:37
Averaged together, lead had a
stronger effect on methylation
23:41
than prenatal stress
in our studies.
23:45
Lead reduced methylation
percentage in both males
23:48
and females, but the
effects were region specific.
23:51
Females showed reductions
in methylation percentages
23:55
in hippocampus, while
males showed reductions
23:58
in frontal cortex.
24:01
For H3K9 acetylation
concentrations,
24:04
42 different sites along NR3C1
were assessed and averaged
24:09
across the entire NR3C1
gene presented here.
24:14
Here again, the effects
of developmental lead
24:18
and prenatal stress
were sex specific.
24:21
In male hippocampus,
our results were similar
24:23
to previous Meaney studies,
24:26
indicating that stress
associates with increases
24:27
in H3K9 acetylation
along NR3CC1.
24:30
Lead also increased
acetylation concentrations
24:35
in male hippocampus.
24:39
However, for females,
24:41
in the hippocampus the
opposite effect was observed
24:43
with prenatal stress lowering
H3K9 acetylation concentrations.
24:47
This is a critical
example demonstrating
24:54
that if sex is not considered,
the neurotoxic effects
24:56
of lead would not be recognized.
24:59
By averaging both sex, you
would eliminate the influence
25:03
of developmental lead on H3K9
acetylation concentrations.
25:06
Further, there was no
effect of lead or stress
 25:12
in male frontal cortex but
lead increased h3K9 acetylation
25:15
concentration in female
frontal cortex, again,
25:19
supporting that the
mechanisms of developmental lead
25:22
and prenatal stress
are region specific.
25:26
Additionally, the
developmental effects of lead
25:35
and prenatal stress on global
posttranslational histone
25:37
modifications, both permissive
25:39
and restrictive modifications
change across the lifespan
25:42
with effects in female
hippocampus showing the most
25:47
dramatic divergence between
lead and stress exposed animals
25:50
and controls, late
emerging into adulthood.
25:54
In contrast, the
most significant lead
26:02
and stress-induced changes
26:05
to global posttranslational
histone modifications
26:06
in male hippocampus,
occurred shortly
26:09
after birth at postnatal day 6.
26:11
Suggesting alterations
following shifts
26:14
in critical sex specific
developmental programming.
26:17
In fact, one of the most sex
differentiated developmental
26:20
events important for central
nervous system development
26:27
in rodents and primates is the
perinatal testosterone surge.
26:30
This male specific surge in
testosterone occurs prenatally,
26:35
at the end of gestation and
peaks just hours after birth.
26:39
This surge in testosterone
occurs
26:47
in rats, mice, and humans.
26:49
And studies in rodents
have shown
26:53
that if you inject
female rodents
26:55
with testosterone during this
critical window, her behavior
26:57
in adulthood, particularly
sexual behavior will
27:01
be masculinized.
27:04
Interestingly, although
this surge
27:07
in serum testosterone
peaks hours after birth,
27:10
elevated levels of
androgens remain elevated
27:14
in the hippocampus until
postnatal day 6 or 8,
27:17
around the same time lead
27:21
and stress exposed males
showed significant differences
27:22
in posttranslational
histone modifications.
27:26
More work is needed to
investigate how metals exposures
27:30
or prenatal stress or both, may
influence androgen disruption
27:34
or directly influence
epigenetic reprogramming induced
27:38
by the perinatal
testosterone surge.
27:45
As discussed, the influence
of androgens and testosterone
27:49
in organizing the sexually
differentiated central nervous
27:55
system, has been shown to
be highly region specific.
27:58
Work by McCarthy's lab
in the hypothalamus,
28:02
has eloquently discovered that
the sex differentiated phenotype
28:06
of elongated dendritic spines
in the preoptic area of rats,
28:11
is originally facilitated by
androgen induced reductions
 28:17
in DNA methylation
profiles in this region.
28:20
This alteration in DNA
methylation results
28:25
in permissive access for immune
response genes to be expressed.
28:27
It is the expression of these
genes and the activation
28:33
of immune cells, such as
microglia and astrocytes,
28:36
that results in the
elongated dendritic spines
28:40
and the sexually differentiated
synaptic patterning
28:46
that we see in the
preoptic area.
28:50
Further work from the Forger lab
suggests that in the bed nucleus
28:55
of the stria terminalis, histone
acetylation plays a key role
29:00
in the sex differentiated
phenotype including region
29:04
volume and cell number.
29:08
Histone deacetylase inhibitors
block the androgen induced
29:13
shifts in these neurophenotypes.
29:17
Again, this work
reemphasizes that the mechanisms
29:20
of sex differentiated
neurophenotypes are extremely
 29:25
region specific.
29:30
Given the only recent commitment
29:36
to understand sex differentiated
development of the rodent brain
29:38
and how these differences
may result in unique toxicity
29:42
of EDCs by sex, there is still
a lot of work to be done.
29:45
One interesting emerging pattern
is that many of the effects
29:49
of early hormone
disruption are late emerging.
29:53
One example that we discussed
is the late emergence
29:56
into adulthood, of lead and
stress induced alterations
29:59
in global histone
modifications only in females.
30:03
But another is worked by
Ghahramani et al in 2014,
30:09
that showed that the
administration of testosterone
30:12
at birth to females resulted
in late emerging changes
30:14
in methylation patterns
in the hypothalamus,
30:18
but actually more
extremely in the striatum.
30:21
The extent of the
methylation changes induced
 30:24
by early testosterone exposure,
had become more evident
30:27
in adulthood when compared
30:31
to methylation changes
assessed immediately
30:33
after the administration of
testosterone at postnatal day 4.
30:36
Despite increasing evidence
30:42
that the perinatal testosterone
surge plays a critical role
30:45
in sex differentiated
neurodevelopment,
30:48
is there any evidence that EDCs
directly disrupt the perinatal
30:50
testosterone surge?
30:54
What we do know, is based on
work from Ear Grey, Howdeshell
30:56
and Ryder labs, on developmental
exposure to phthalate mixtures,
31:02
testosterone, and sex
differentiated reproductive
31:05
system development.
31:08
These studies have demonstrated
enhanced male reproductive
31:09
dysfunction following
developmental exposures
31:13
to combinations of
anti-androgens.
31:17
These effects occur even
 31:20
when single exposures
show no phenotypic change,
31:23
and when single exposures act
via different direct molecular
31:29
mechanism and are administered
at subthreshold doses.
31:34
These enhanced effects
of phthalate mixtures
31:39
on male specific
reproductive development
31:44
and androgen production,
leave open questions
31:47
about what's happening
following exposure
31:52
to endocrine disrupting
chemical mixtures
31:55
and the sex-differentiated
development
31:57
of the nervous system.
31:59
and to begin addressing
some of these questions,
32:02
studies from my lab have exposed
pregnant mice to a mixture
32:05
of endocrine disrupting
chemicals known
32:12
to target the central
nervous system and behavior.
32:14
Again here, we see sex
specific alterations
32:19
in developmental endocrine
disrupting chemical mixture
32:23
exposure, where the mixture
shows significant effects
32:25
where the single
compounds did not.
32:29
Some of the behavioral
alterations we saw
32:33
in only male mice included
social conditioned place
32:35
preference, where mix exposed
males seemed to prefer to be
32:39
in isolation rather than to
be in a social condition.
32:42
Whereas females both showed
a preference for sociality.
32:46
And again, on the FI schedule
32:49
where we saw male
specific increase
32:52
in correct lever response rates.
32:54
These behavioral data
left us wondering
33:01
if developmental exposure
33:03
to this endocrine disrupting
chemical mixture was partially
33:05
mediated by sex specific
disruption
33:08
of the perinatal
testosterone surge.
33:12
And sure enough, measuring serum
testosterone concentration just
33:14
hours after birth in
males and females,
33:18
from the same dams
exposed to these EDCs,
33:21
showed that male mice had
elevated serum testosterone
33:24
concentration while
females showed no change.
33:28
More research is needed to
begin to uncover how mixtures
33:35
or single exposures to endocrine
disrupting chemicals including
33:41
metals, disrupts
maternal environments
33:45
in hormone signaling
and how those changes
33:48
in early maternal environments
shift the developmental
33:53
trajectory in a sex-biased
direction.
33:56
For example, if endocrine
disrupting mixtures disrupt this
34:04
early surge in testosterone
experienced by male fetuses,
34:08
and that disruption of
testosterone is associated
34:12
with alterations in methylation
profiles, how can we then begin
34:17
to connect shifts in key
developmental programming,
34:24
alterations across
the lifespan that sort
34:30
of helped provide a
physiological or epigenetic echo
34:33
that could then change the
nervous system into adulthood,
34:38
and could help us begin
34:41
to understand the sex
differentiated origins
34:42
of neurobehavioral disorders.
34:45
And furthermore,
variations and fluctuations
34:47
in hormone concentrations across
the lifespan between males
34:51
and females, are
categorically different.
34:54
So how can we begin
to understand some
34:58
of these patterns of
hormone fluctuations
35:00
in early development and
then in late adulthood,
35:05
that might provide
different critical windows
35:07
of susceptibility for
male fetuses than females.
35:10
To fully understand
how differences
35:20
in early sex differentiated
endocrine profiles may modulate
35:21
the toxicity of EDCs
including metals,
35:25
we need to integrate
cellular mechanisms
35:29
with functional outcomes.
35:31
This is a perspective
highlighted
35:33
by Kembra Howdeshell,
35:35
to understand the role endocrine
disrupting compounds play
35:36
in reproductive disease.
35:40
And the same perspective has
relevance for neurotoxicology.
35:42
This integrative approach
is reflected in one
35:46
of my favorite quotes
from George Bartholomew,
35:52
the integrative physiologist.
35:54
Every scale in biology
receives its mechanism
35:56
from the scale below
and its significance
36:00
from the scale above.
36:03
Neurotoxicology has a great and
exciting challenge and that is
36:07
to help us integrate how
diverse cellular mechanisms
36:12
of toxicity are integrated into
organismal functional outcomes
36:18
that can help us understand
and predict disease.
36:23
For additional information,
here are the resources
36:26
and references used to
develop this lecture.
36:30
If you're interested in any
of these topics further,
36:34
feel free to contact me
and thank you for joining.
36:36