Ecotoxicology Ecotoxicological study is a multi-step

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Ecotoxicology

• Ecotoxicological study is a multi-step process,
  involving
• The entry, distribution and fate of pollutants
  within the environment
• The entry and fate of pollutants in living
  (biota) organisms within an ecosystem and
• The harmful effects of the chemical pollutants on
  the constituents (biotic abiotic) of ecosystems
  (which include man).

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Beyond our toxin trailIs the grave deeper than
we thought?
Metabolized and/or stored
Reaches an organ
molecular mechanism
Adverse health effect
Physiological chain of events
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• Ecotoxicology
• Bioaccumulation
• Bioconcentration (in water)
• Biomagnification
• Never single effects
• Movement between media (air, water)

• Toxicology
• Host defense mechanisms
• Individual susceptibility states
• Single effects
• Cumulative exposure

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Ecological bases of Ecotoxicology

• The basis for determining the effects of
  contaminants on ecosystem is at organism level
• At organism level, response can be
• Acute toxicity causing mortality
• Chronically accumulating damage ultimately
  causing death
• Sublethal impairment of various aspects of
  physiology and morphology
• Sublethal behavioral effects
• Measurable biochemical changes

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• At population level, response can be
• Size and dynamics (based on birth rates, death
  rates, gains, from immigration and losses from
  emigration)
• Cause a reduction or an increase in the natural
  flowchart of numbers, in the biomass, sex ratio,
  etc.
• At community level, response can be
• species diversity
• predator prey relationship, etc
• Change in ecosystem
• nutrient cycling rates, patterns of nutrient
  flow,
• physical-chemical conditions etc.

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Assessment of Structural Changes
    changes in species / population  structure
                  -  appearance/disappearance of
an indicator species                   - 
number of individuals of a species
                  -  biomass of a species
                  -  presence or absence of a
species Biomass-a quantitative estimate of the
total mass of living plant or animal materials
       
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changes in community/ecosystem structure
                        -  biomass
                        -  abundance
                        -  biotic indices (e.g.
trophic types)                         - 
species richness / diversity                     
    -  dominance                         -  food
chain length/complexity
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Chemicals of ecotoxicological interest

• They are toxic and in many cases their
  metabolites are also harmful e.g. DDT DDE
  (metabolite of DDT)
• They are very stable both chemically and
  environmentally
• Their stability has lead to their persistence and
  ubiquitous nature in the environment
• Almost all chemicals of ecotoxicologigal interest
  are bioavailable and in most cases undergo
  bioaccumluation and biomagnification (food chain)

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Bioavailabiltiy The fraction of a chemical that
is in an available form to an organism e.g. fish
food, absorption from water
Bioconcentration - where the chemical
concentration in an organism exceeds the
concentration in the surrounding media (i.e.
aquatic environment) as a result of exposure
through the respiratory surfaces (i.e.
gills/dermal surfaces) - not food! Bioconcentrati
on Factor conc. in organism conc. in
ambient medium (usually water)
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• Bioaccumulation - where the chemical
  concentration in an organism achieves a level
  that exceeds that in the water/media as a result
  of chemical uptake through all routes of
  exposure.
• Bioaccumulation factor Conc. in organism
• Conc. in
  food
• (or
  ingested water)
• Bio-accumulation of Cd is higher than most metals
  as it is assimilated rapidly and excreted slowly
• depends on the rate of excretion

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Biomagnification - where the chemical
concentration in an organism achieves a level
that exceeds that in the organisms diet due to
dietary absorption. i.e. higher trophic levels
accumulate more chemical
Biomagnification Factor Conc. in
predator Conc. in
prey Factors that influence bioaccumulation -Envir
onmental persistence -Lipophilicity -Biotransforma
tion
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Toxic Effects

• The biochemical (molecular in nature) or
  physiological (observed at organ and whole
  organism levels) changes which adversely affect
  individual organisms birth, growth or mortality
  rates.
• Both biochemical and physiological changes could
  lead to behavioral (whole organism level) changes.

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• Toxicant binding
• Reversible vs. Irreversible binding
• Irreversible binding (covalent) causes harmful
  effects.
• Types of bonding
• Covalent gt ionic gt Hydrogen binding gt Vanderwaals
  gt hydrophilic
• Biochemical responses
• Biochemical response could be protective or
  non-protective (may or may not cause harmful
  effect).
• Non-protective biochemical responses have
  carcinogenic, mutagenic, teratogenic and
  neurotoxic potentials.

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• Protective biochemical responses
• Monoxygenase (OCs and PAHs)
• Induction and binding to metalothionein (Cu, Cd,
  Zn and Hg)
• Binding to blood plasma, bones and hair (Metals
  and xenobiotics)
• Dissolving in fat (organics- e.g. OCs)
• Mineralization ( e.g. MeHg to Hg 2)
• Demineralization (As to MeAs)

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Protective biochemical response

• Heavy metals for example can be stored and
  detoxified by organisms either by binding to
  specific proteins e.g. metallothioneins (-SH
  proteins)
• In some cases it is mineralized to inorganic
  form, which is less toxic e.g. Hg bound to Se is
  a mineralized Hg (detoxified Hg MeHg to Hg). On
  the other hand, the inorganic form, which is more
  toxic can be methylated to a less toxic form e.g.
  As.

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Protective biochemical response

• PHASE 1 REACTION.
• Organic pollutants could also be metabolized and
  detoxified by Cytochrome P450 enzymes (Microsomal
  Monoxygenase MMO).
• PHASE 2 REACTION
• The metabolites undergo conjugation with
  endogenous molecules e.g. GSH.
• For some chemicals the metabolites/conjugated
  form are more toxic than the parent compound and
  can lead to cancer formation.

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Non-protective response

• Binding to DNA (DNA adduct)
• DNA structural damage (strands break) induced by
  genotoxic compounds
• Binding to SH-Protein (Protein adduct) enzymes
  and proteins
• Neurotoxicity prolongation of K and Na flow and
  inhibition of AChE activity in the brain

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Non-protective response

• Mitochondrial Poison (lost of proton gradient)
• Inhibition of vitamin K cycle (competition with
  vit K binding site)
• Inhibition of Thyroxine (competition with
  thyrosine binding site)
• Inhibition of ATPase (enzymes for transport of
  ions e.g. K, Na, Ca)

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Non-protective response

• Environmental estrogens (eg DDT) and androgens
  (tributhyl Tin)
• Endocrine disrupters (binding to endocrine
  receptors)
• Photosystems of Plants (interruption of electron
  flow)
• Plant growth regulation

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Physiological changes

• Non-protective biochemical responses lead to
  Physiological changes which could be observed at
  organ and organism levels
• Organ level
• accumulation of Cd in kidney, which could cause
  cell death (cytotoxicity), resulting in
  dysfunction of the kidney
• PAHs and Lung cancer
• Organism level
• decrease in production (growth and reproduction)
• changes in gene frequency
• decrease in resources acquisition and uptake

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Behavioral Changes

• Either or both physiological and biochemical
  effects could lead to behavioral effects at
  organism level-
• migration,
• intraspecific attraction,
• aggregation,
• aggression,
• predation,
• vulnerability,
• mating
• caring for young ones and avoidance of predator.
• .

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Population Changes

• Changes in population may come about as a result
  of direct changes in numbers of individual
  organism and gene frequency
• By indirect means (decrease in population of
  predators due to toxic chemicals could lead to
  increase in numbers of its prey).

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Diclofenac residues as the cause of vulture
population decline in Pakistan.Nature. 2004 Feb
12427(6975)630-3.

• Diclofenac causes kidney damage, increased serum
  uric acid concentrations, visceral gout, and
  death.

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• Changes in community structure
• change in pyhtoplankton assemblage due to
  eutrophication
• acid rain affecting microorganisms in the soil,
• aquatic life
• Changes in Ecosystem level (earth as an
  ecosystem)
• carbon dioxide increase
• ozone depletion

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What is an Endocrine Disruptor ?
An exogenous agent that interferes with the
synthesis, secretion, transport, binding, action,
or elimination of natural hormones in the body
that are responsible for the maintenance of
homeostasis, reproduction, development and/or
behavior.
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Mechanisms of endocrine disrupting compounds 1)
Binding and activating the estrogen receptor 2)
Binding but not activating the estrogen receptor
(therefore acting as an anti-estrogen) 3) Binding
other receptors 4) Modifying the metabolism of
natural hormones 5) Modifying the number of
hormone receptors in a cell 6) Modifying the
production of natural hormones
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Hormone regulation and feedback control Estrogen
levels depend on Estrodiol sulfate Estrodiol
serum-binding proteins ?-fetoprotein
(AFP) Testosterone-estradiol binding
globulin Xenoestrogens (ex. DES) 100-fold lower
affinity than E2 to these binding
protein Bioavailability increased
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Non-genomic mechanisms of ED action

• Compounds of the azole type, such as ketoconazole
  and the fungicide fenarimol, inhibit CYP isoforms
  and consequently can also affect steroid
  synthesis while the now-banned anti-fouling agent
  tributyltin and its metabolites, which have
  strong ED potential, are thought to act by the
  same mechanism, probably by inhibition of
  aromatase.

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Genomic mechanisms of ED action

• bind to oestrogen receptors and so act as
  pseudoestrogens in vivo, giving feminizing
  effects
• tamoxifen and diethylstilbestrol and industrial
  chemicals (e.g. octylphenol and bisphenol-A
• fungicide vinclozolin binds competitively to the
  androgen receptor, blocking the cellular actions
  of testosterone on androgen-dependent tissue
  growth and behaviour patterns
• chlordecone, inhibit binding to the oestrogen and
  progesterone receptors, whereas bisphenol-A can
  block ligand binding to the thyroid receptor

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Timing, duration, and amount of exposure.
Organization vs. activation Timing, duration, and
amount of exposure are each important
determinants of the outcome. There are windows of
vulnerability during fetal development in which
small exposures to endocrine disruptors may have
profound effects not observed in adults. Studies
of the intrauterine position of mice during fetal
development show that slight fluctuations of
steroid hormone levels influence genital
morphology, timing of puberty, sexual
attractiveness, sexual behavior, aggressiveness,
and activity level of offspring.
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Various Classes of EDCs

• Flame Retardants
• Fungicides
• Herbicides
• Insecticides
• Metals
• Pharmaceuticals
• Phenols
• Plasticizers
• Polyaromatic Hydrocarbons
• Soy Products
• Surfactants

Polybrominated diphenyl ether Vinclozolin Atrazine
Methoxychlor Tributyltin Ethynyl
Estradiol Bisphenol A Phthalates PCBs,
dioxins Genistein Alkylphenol Ethoxylates
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PBDEs(??????)

• Polybrominated diphenyl ethers (PBDEs) are a
  class of recalcitrant and bioaccumulative
  halogenated compounds that have emerged as a
  major environmental pollutant. PBDEs are used as
  a flame-retardant and are found in consumer goods
  such as electrical equipment, construction
  materials, coatings, textiles and polyurethane
  foam (furniture padding).

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Bioavailability of PBDEs

• Found in animals
• Increase in fish
• Increase in whales
• Sewage sludge
• PCBs Found in Lake Washington Fish (PBDEs next?)
• Found in human (breast milk)

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PBDEs Breast Milk - Sweden
(Norén and Mieronyté, 1998)
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Health Effects of PBDEs

• Similar to PCBs (Polychlorinated biphenyls)
• Persistent Bioaccumulative Toxicant
• No human data
• Animals studies indicate
• Changes in thyroid hormone levels
• Neurobehavioral toxicity
• Development effects- alters Behavior
• Impairs memory and learning
• Delays sexual development

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Vinclozolin

• Vinclozolin is a fungicide that has been shown to
  cause Leydig cell tumors and atrophy of the
  accessory sex glands in adult rodents. In
  addition, exposure of rats during pregnancy
  causes a pattern of malformations in the male
  urogenital tract .
• Androgen receptor antagonist

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Atrazine

• A chlorotriazine herbicide, is used to control
  annual grasses and broadleaf weeds.
• Suppression of the luteinizing hormone surge
  during the estrus cycle by atrazine leads to the
  maintenance of elevated blood levels of
  17beta-estradiol (E2) and prolactin.
• The mechanism for tumor development may include
  one or more of the following the induction of
  aromatase (CYP19) and/or other P450 oxygenases,
  an antagonist action at the estrogen feedback
  receptor in the hypothalamus, an agonist action
  at the mammary gland estrogen receptor or an
  effect on adrenergic neurons in the
  hypothalamic-pituitary pathway.

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??A Bisphenol-A
BPA is used in the manufacture of polycarbonate
plastics and epoxy resins from which food and
beverage containers and dental materials are
made. Perinatal exposure to environmentally
relevant BPA doses results in morphological and
functional alterations of the male and female
genital tract and mammary glands that may
predispose the tissue to earlier onset of
disease, reduced fertility and mammary and
prostate cancer.
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????(PVC)????????????????????????????????????????
??????PVC??,?????????????(phthalates)?????????????
???????????????,????????????????????PE(???)???
??? alkylphenol(???) ???????????????(???????)??
??????????????????(polystyren)?????,???PS,????????
??????????,??????,????????????alkylphenol(???)????
??????????,?????????????????
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• ??????????
• ????????????????????????
• ??????(??????????)
• ?????(phthalates)
• ???(alkylphenol)

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Phthalates??????

• ??????????????????? ?
• ????????????????(???)?
• ????????????,???????PVC?PVDC???PVC?????
• male infertility
• Interfere with cholesterol uptake and androgen
  biosynthesis

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Tributyltin (TBT) ?????????????,??????????,?????
????????,????????,????????????
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?????????????????,?????????????,??????????????????
,?????????????????
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TBT
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Yucheng (??) of Taiwan, 1979

• 2000 people exposed to PCB-contaminated rice oil
• with chloracne, fatigue, skin and nail
  pigmentation, polyneuropathy, and abnormal liver
  function
• Long-term follow-up study was established for 1st
  and 2nd generation (prenatal exposed)

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• Likely Kanechlor 500 (a Japanese PCB mixture)

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Semen Analysis40 Yucheng men (37-50 yr old)
compared with 28 controls

• Volume and count no difference
• Oligospermia 23 vs. 4
• Morphology 28 vs. 23 abnormal (18 increase)
• Motile sperm no difference
• Speed by CASA no difference
• Chinese hamster oocyte penetration after one
  re-thaw cycle 16 vs. 32 (50 drop)

(Hsu et al., JAMA 2003)
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Children Prenatally Exposed

• More stillbirth (Yu et al., 2000)
• Called Yucheng children
• Born with darker skin, nail pigmentation and
  deformity, and developmental delay (Rogan et al.,
  1988)
• Reduced neurocognitive functioning up to ages of
  12 years (Chen et al., 1992)
• Behavioral problems (Lai et al., 2002)

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Semen Analysis

• Young men prenatally exposed to PCBs/PCDFs as
  well as their neighborhood-matched controls were
  recruited for semen analysis
• Volume, counts, morphology, motility, oocyte
  penetration
• Aged 16-21, consent obtained from young men and
  their parents

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Penile length (cm) in Yucheng and Control Boys
by Age
Measured by Blinded physicians
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Hormones in prenatal PCB/PCDF-exposed boys and
unexposed controls after puberty
gt
lt
gt
lt
gt
lt
No difference luteinizing hormone
prolactin
(Hsu et al., JTEH 2005)
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(?)?????????????26?, (?)?????9?(?13)? (?)?????
9?(?13) (?)????????9?,(?13)????????????????????
(?)????????????????6?? (?)??????????????????????
3?,????????????????????????????????????? (?)??????
??2?,??????,????,??????,???????,?????? (?)???????
???????????????????????????????? (?)?????2?,????
????????????? (?)???????????,?????????????
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EDSTAC Tier 1 Assays Concerned with detecting

• Receptor binding assays (ER and AhR)
• Uterotrophic
• Hershberger
• Pubertal female
• Steroidogenesis
• Frog metamorphosis
• Fish reproductive screen

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EDSTAC Tier 2 dose-response relationship

• Mammal development and reproduction
• Bird development and reproduction
• Mysid shrimp life cycle
• Fish reproduction and development
• Amphibian development and reproduction

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Species-dependent sex determination Mammal XY/XX
synthesis of testosterone/functional androgen
receptors estrogen receptor in the brain Birds
WZ/WW The ability to synthesize and recognize
17?-estradiol is necessary for female CNS and
gonadal sexual development to occur Reptile temper
ature-dependent sex determination (aromatase
related)
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Temperature-dependent sex determination
thermosensitive period (TSP)
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Temperature determines their sex. A nest
temperature of 73.5 degrees would develop males.
If it heats up to 83.5, hormones would trigger
changes causing the embryonic cells to
differentiate as females.
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