Transcript 2061-2008 ecotoxicology under

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).
Beyond our toxin trail
Is the grave deeper than we thought?
Transport
and fate
Toxin
emitted
Ecosystem
effects
Community
effects
Metabolized
and/or stored
Ingested
Contacts
human
Population
effects
Reaches
an organ
Physiological
chain of events
Toxicology
Ecotoxicology
• Host defense
mechanisms
• Individual
susceptibility states
• Single effects
• Cumulative
exposure
• Bioaccumulation
• Bioconcentration
(in water)
• Biomagnification
• Never single
effects
• Movement
between media (air,
water)
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
•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.
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
changes in community/ecosystem structure
-
biomass
abundance
biotic indices (e.g. trophic types)
species richness / diversity
dominance
food chain length/complexity
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)
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!
Bioconcentration Factor =
conc. in organism
conc. in ambient medium (usually water)
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
Biomagnification - where the chemical
concentration in an organism achieves a
level that exceeds that in the organism’s 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
-Environmental persistence
-Lipophilicity
-Biotransformation
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.
• Toxicant binding:
– Reversible vs. Irreversible binding
• Irreversible binding (covalent) causes harmful
effects.
– Types of bonding:
– Covalent > ionic > Hydrogen binding > Vanderwaals >
hydrophilic
• Biochemical responses:
– Biochemical response could be protective or nonprotective (may or may not cause harmful effect).
• Non-protective biochemical responses have
carcinogenic, mutagenic, teratogenic and
neurotoxic potentials.
• 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)
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.
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.
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
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)
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
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
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.
.
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).
Diclofenac residues as the cause of
vulture population decline in Pakistan.
Nature. 2004 Feb 12;427(6975):630-3.
• Diclofenac
causes kidney
damage,
increased serum
uric acid
concentrations,
visceral gout, and
death.
• 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
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. “
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
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
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 nowbanned 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.
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
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.
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
PBDEs（多溴二苯基醚）
• 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).
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)
PBDEs Breast Milk - Sweden
(Norén and Mieronyté, 1998)
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
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
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 17betaestradiol (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.
雙酚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.
聚氯乙烯（PVC）製的嬰兒固齒器、玩具
讓長牙的嬰兒咬玩的固齒器、洗澡玩的軟性玩具、價格不貴的流
行卡通玩具常常是PVC製品，在使用中可能釋出鄰苯二甲酸
（phthalates）這類有致癌性的環境荷爾蒙。
【安全替代品】仔細查看成分標示，凡是嬰幼兒可能放到口中把
玩的玩具一定選購PE（聚乙烯）製品。
苯乙烯 alkylphenol（烷基酚）
攤販、自助餐店、速食店的熱飲杯（裝湯、茶、咖啡）、泡麵的
碗麵及杯麵絕大多數都使用聚苯乙烯（polystyren）的塑膠容器，
簡稱為PS，被國人稱為保麗龍。其原料單体叫苯乙烯，是已知致
癌物，且製造過程所添加的塑化劑alkylphenol（烷基酚）也是
會干擾內分泌的環境荷爾蒙，二者在使用過程很容易溶出到食物
中。
化妝品中的環境荷爾蒙
多數的化妝品、卸妝用清潔用品含有幾類的環境
荷爾蒙：
•壬基苯酚乙烯（一種非離子界面活性劑）
•鄰苯二甲酸（phthalates）
•烷基酚（alkylphenol）
Phthalates鄰苯二甲酸酯
•廣泛存在於化粧品、兒童玩具、食品包裝中 。
•香水中可當作溶煤或香料固著劑使用(已禁用)。
•常用於塑膠類製品的塑化劑，可能遇熱溶出。
PVC或PVDC保鮮膜；PVC塑膠手套。
•male infertility
•Interfere with cholesterol uptake and
androgen biosynthesis
Tributyltin (TBT)
三丁基錫是一種有機錫化合物，常被
添加於船舶油漆中，以防止貝類及藻
類附著於船身，由於具有殺菌效果，
所以也可以作為殺菌劑使用
受到三丁基錫或三苯基錫污染的雌岩螺，因生殖孔阻塞受
精卵無法排出，堆積在生殖管道內變紅變黑形成壞死組織，
此時長出陰莖的雄化作用也同時被引發
TBT
Yucheng (油症) of Taiwan,
1979
• ~2000 people exposed to PCBcontaminated 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)
Likely
Kanechlor 500
(a Japanese
PCB mixture)
Semen Analysis
40 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)
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)
Penile length (cm)* in Yucheng and
Control Boys by Age
9
8
Yucheng
Controls
7
6
5
4
3
2
1
0
6 to 7
8 to 9
10 to 11
12 to 13
14 to 15
*Measured by “Blinded” physicians
Hormones in prenatal PCB/PCDF-exposed
boys and unexposed controls after puberty
Sex Hormones
Control
Yucheng
Testosterone (TT, ng/mL)
4.2 ± 2.2
√(TT/E2)
0.5 ± 0.2
> 3.0 ± 2.4
< 48.6 ± 53.9
> 0.3 ± 0.2
Follicle-stimulating
hormone (FSH, mIU/mL)
√(TT/FSH)
3.4 ± 0.8
<
4.6 ± 2.2
1.1 ± 0.3
0.8 ± 0.5
√(E2/FSH)
2.4 ± 0.8
>
<
Estradiol (E2, pg/mL)
21.3 ± 13.2
3.1 ± 1.4
No difference: luteinizing hormone
prolactin
(Hsu et al., JTEH 2005)
（一）殺蟲劑或其代謝中間產物：計有26種，
（二）殺菌劑：計有9種（佔13％）。
（三）除草劑：亦有9種（佔13％）
（四）塑膠之塑化劑：亦有9種，（佔13%）。禁用鄰苯二甲酸鹽
作為玩具塑膠之塑化劑。
（五）醫藥、化工原料合成之中間產品：計有6種。
（六）有機氯化物之污染副產品或菸煙中之芳香族烴計有3種，
其中以戴奧辛夫喃廣布於空氣、土壤、底泥、甚至於食品、乳製
品中最為令人憂心。
（七）熱媒及防火材料：有2種，其中多氯聯苯，惡名昭彰，雖
已禁用多年，但在環境介質中，仍時常檢出。
（八）界面活性劑之代謝分解中間產物：非離子界面活性劑廣用
於各種民生日用清潔劑、乳化劑中
（九）有機錫：計有2種，作為魚網之防腐劑及船上抗腐蝕油漆。
（十）重金屬：計鉛、鎘、汞三種，亦列為內分泌干擾之疑似物
質
EDSTAC Tier 1 Assays
Concerned with detecting
•
•
•
•
•
•
•
Receptor binding assays (ER and AhR)
Uterotrophic
Hershberger
Pubertal female
Steroidogenesis
Frog metamorphosis
Fish reproductive screen
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
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
temperature-dependent sex determination (aromatase
related)
Temperature-dependent sex determination
thermosensitive period (TSP)
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.