Fyziologie působení farmak a
toxických látek
Přednáška č.5
Endokrinní disrupce u obratlovců II.
Modulace funkcí RAR/RXR, TR a PPAR –
deregulace vývoje organismu, modulace
endokrinních signálů a karcinogenní účinky;
Efekty spojené s deregulací hladiny retinoidů:
•• Funkce RA;Funkce RA;
•• Vznik konVznik konččetin;etin;
•• Vývoj nervovVývoj nervovéé soustavy;soustavy;
•• VývojovVývojovéé abnormality obojabnormality obojžživelnivelnííkkůů;;
•• NaruNaruššeneníí hladin vitaminu A;hladin vitaminu A;
Struktura aStruktura a
syntsyntééza kyselinyza kyseliny
retinovretinovéé
• Abnormalities caused by exogenous agents (certain chemicals or viruses,
radiation, or hyperthermia) are called developmental disruptions. The agents
responsible for these disruptions are called teratogens. Most teratogens
produce their effects only during certain critical periods of development. The
most critical time for any organ is when it is growing and forming its
structures. Different organs have different critical periods, but the time from
period from day 15 through day 60 of gestation is critical for many human
organs.
• Retinoic acid is important in forming the anterior-posterior axis of the
mammalian embryo and also in forming the limbs. In these instances, retinoic
acid is secreted from discrete cells and works in a small area. However, if
retinoic acid is present in large amounts, cells that normally would not receive
such high concentrations of this molecule will respond to it. Inside the
developing embryo, vitamin A and 13-cis-retinoic acid become isomerized
to the developmentally active forms of retinoic acid, all-trans-retinoic
acid and 9-cis-retinoic acid. Some of the Hox genes have retinoic acid
response elements in their promoters.
• In the early 1980s, the drug Accutane® (the trade name for isoretinoin, or
13-cis-retinoic acid) was introduced as a treatment for severe acne. Women
who took this drug during pregnancy had an increased number of spontaneous
abortions and children born with a range of birth defects.
There are discrete positions where limb fields are generated.
Researchers have precisely localized the limb fields of many
vertebrate species. Interestingly, in all land vertebrates, there are
only four limb buds per embryo, and they are always opposite each
other with respect to the midline. Although the limbs of different
vertebrates differ with respect to which somite level they arise
from, their position is constant with respect to the level of Hox
gene expression along the anterior-posterior axis. For instance,
in fishes (in which the pectoral and pelvic fins correspond to the
anterior and posterior limbs, respectively), amphibians, birds, and
mammals, the forelimb buds are found at the most anterior
expression region of Hoxc-6, the position of the first thoracic
vertebra.
Retinoic acid appears to be critical for the initiation of limb
bud outgrowth, since blocking the synthesis of retinoic acid with
certain drugs prevents limb bud initiation, suggested that a
gradient of retinoic acid along the anterior-posterior axis might
activate certain homeotic genes in particular cells and thereby
specify them to become included in the limb field.
RA reguluje vznik a vývoj konRA reguluje vznik a vývoj konččetinetin
Legs regenerating from retinoic acid-treated tadpole tail. (A) The tail
stump of a balloon frog tadpole treated with retinoic acid after
amputation will form limbs from the amputation site. (B) Normal tail
regeneration in a Rana temporaria tadpole 4 weeks after amputation. A
small neural tube can be seen above a large notochord, and the muscles
are arranged in packets. No cartilage or bone is present. (C) A retinoic
acid-treated tadpole tail makes limb buds (arrows) as well as pelvic
cartilage and bone. The cartilaginous rudiment of the femur can be seen
in the right limb bud.
The Hox complex of
an insect and the
Hox complexes of a
mammal compared
and related to body
regions.
Expression domains of Hox genes in a mouse. The photographs show
whole embryos displaying the expression domains of two genes of
the HoxB complex (blue stain). These domains can be revealed by in
situ hybridization or, as in these examples, by constructing
transgenic mice containing the control sequence of a Hox gene
coupled to a LacZ reporter gene, whose product is detected
histochemically. Each gene is expressed in a long expanse of tissue
with a sharply defined anterior limit. The earlier the position of the
gene in its chromosomal complex, the more anterior the anatomical
limit of its expression. Thus, with minor exceptions, the anatomical
domains of the successive genes form a nested set, ordered
according to the ordering of the genes in the chromosomal complex.
Teratogenesis in frogs. (A) Wild green frog (Rana clamitans) with an
eye deformity, collected in New Hampshire in 1999 by K. Babbitt. (B)
Xenopus tadpole with eye deformities caused by incubating newly
fertilized eggs in water containing methoprenic acid, a by-product of
methoprene. (C) One of several pathways by which methoprene can
decay into teratogenic compounds such as methoprenic acid. (D) An
isomer of retinoic acid showing the structural similarities to
methoprenic acid.
Top panel: At left, retinoic acid
activates gene expression in a
subset of cells in the normal
developing forebrain of a
midgestation mouse embryo (blue
areas indicate β-galactosidase
reaction product, an indicator of
gene expression in this experiment);
at right, after maternal ingestion of
a small quantity of retinoic acid
(0.00025 mg/g of maternal weight),
gene expression is ectopically
activated throughout the forebrain.
Bottom panel: At left, the brain of
a normal mouse at term; at right,
the grossly abnormal brain of a
mouse whose mother ingested this
same amount of retinoic acid at
mid-gestation.
RA reguluje vývoj CNSRA reguluje vývoj CNS
Location of some inductive signals in the
developing neural tube. Inductive signals
are provided by either the notochord, the
floorplate, the roofplate and dorsal
ectoderm, or the somites. These signals
act locally on either the ventral or dorsal
neuroepithelium of the developing spinal
cord and hindbrain to elicit distinct
patterns of gene expression and,
ultimately, differentiation of specific
classes of neurons. The peptide hormone
sonic hedgehog (shh) is the most
important ventral signal and is produced
by both the notochord and floorplate. In
addition, noggin, chordin, and retinoic acid
are produced either by the notochord or
floorplate. In contrast, a variety of
signals including dorsalin and other
members of the TGF family as well as
noggin and retinoic acid are provided by
the roofplate and dorsal ectoderm. These
signals influence the differentiation of
several dorsal cell types including the
neural crest
PHAHs a PAHs naruPHAHs a PAHs naruššujujíí funkci a strukturufunkci a strukturu ššttíítntnéé
žžlláázy a hladiny thyroidnzy a hladiny thyroidníích hormonch hormonůů a retinoida retinoidůů
PHAHs modulují hladiny retinoidůPHAHs modulují hladiny retinoidů –– mobilizace zásobmobilizace zásob
vitaminu A v játrechvitaminu A v játrech
BPA moduluje hladiny retinoidnBPA moduluje hladiny retinoidníích receptorch receptorůů
v prv průůbběěhu embryogenezehu embryogeneze -- mymyššii
Funkce thyroidních hormonů v ontogenezi a vliv
organických polutantů:
•• Funkce thyroidnFunkce thyroidníích hormonch hormonůů v metamorfv metamorfóózeze
•• Funkce thyroidnFunkce thyroidníích hormonch hormonůů ve vývoji nervovve vývoji nervovéé
soustavy;soustavy;
HypotHypotéézaza –– environmentenvironmentáálnlníí polutanty jakopolutanty jako
kauzkauzáálnlníí faktor neurologických poruchfaktor neurologických poruch
(autismus, poruchy u(autismus, poruchy uččeneníí, hyperaktivita,, hyperaktivita,
nnáádorovdorováá onemocnonemocněěnníí, juveniln, juvenilníí formyformy
diabetes);diabetes);
•• ToxickToxickéé lláátky narutky naruššujujííccíí thyroidnthyroidníí regulace;regulace;
T3 a T4 majT3 a T4 majíí zzáásadnsadníí význam pro iniciacivýznam pro iniciaci
metamorfmetamorfóózy obojzy obojžživelnivelnííkkůů
Although it has been known for a century that
hypothyroidism leads to retardation and other serious
developmental effects, the role of thyroid
hormones in brain development is still not completely
understood. It is also accepted that thyroid hormones
transferred from the mother to the embryo and fetus are
critical for normal brain development, even though the
thyroid gland of a fetus starts producing thyroid
hormones at about 10 weeks.
We now recognize that only a slightWe now recognize that only a slight
difference in the concentration of thyroiddifference in the concentration of thyroid
hormones during pregnancy can lead tohormones during pregnancy can lead to
significant changes in intelligence insignificant changes in intelligence in
children.children.
• Inhibition of active transport of inorganic iodide into the
follicular cell
• Interference with the sodium/iodide transporter system
• Inhibition of thyroid peroxidases to convert inorganic iodide into
organic iodide to couple iodinated tyrosyl moieties into thyroid
hormone
• Damage to follicular cells
• Inhibition or enhancement of thyroid hormone release into the
blood
• Inhibition or activation of the conversion of T4 to T3 by 5´monodeiodinase
at various sites in the body, for example, the fetal
brain
• Enhancement or interference of the metabolism and excretion of
thyroid hormone by liver uridine diphosphate
• Interference with transport of thyroid hormones
• Vitamin A (retinol) disturbances
• Blocking of or interfering with thyroid receptors
MoMožžnnéé mechanismy disrupce funkcemechanismy disrupce funkce
thyroidnthyroidníích hormonch hormonůů
Mechanisms of Action of ThyroidMechanisms of Action of Thyroid--Disrupting ChemicalsDisrupting Chemicals
The complexity of the development of both the neurologic and thyroid
systems offers numerous opportunities for chemicals to interfere as the
systems develop, mature, and function. Briefly, there are chemicals that
interfere with iodine uptake (the herbicides 2,4-D and mancozeb, several
PCB congeners, and thiocyanates) and peroxidation at the molecular level
(the herbicides aminotriazole and thioureas, the insecticides endosulfan and
malathion, and PCBs). They also interfere with the protein transporter that
provides a pathway for iodine to enter the cell (military and aerospace
chemicals, perchlorates). Certain antagonists (PCBs, the herbicides
aminotriazole and dimethoate, and the insecticide fenvalerate) prevent the
release of thyroid hormone from the cell and inhibit conversion of T4 to
triiodothyronine (T3). Various chemicals enhance excessive excretion of
thyroid hormones, some through activation of the cytochrome P450 system
(dioxin, hexachlorobenzene, and fenvalerate). Some PCBs, phthalates, and
other widely used chemicals compete for sites on the thyroid transport
proteins that deliver thyroid hormones throughout the body. New research
focuses on the role of chemicals as they interfere with vitamin A (retinols).
retinols, a process essential for thyroid hormone expression.
HydroxylovanHydroxylovanéé PCBPCB
During normal enzyme detoxification
of PCBs in the maternal liver, certain
PCB congeners are hydroxylated.
This metabolic step enhances the
binding affinity of the hydroxylated
PCBs to TTR. Through their highaffinity
binding the hydroxylated
congeners displace essential fT4
that must get to the fetal brain to
be converted to fT3. Hydroxylated
PCBs also interfere with the normal
excretion of thyroid hormones by
inhibiting their sulfation. PCB
hydroxylates also have estrogenic
and antithyroid properties.
ThyroidnThyroidníí disrupce u volndisrupce u volněě žžijijííccíích obratlovcch obratlovcůů::
Obojživelníci
Gutleb and co-workers did a series of exposure studies with Xenopus
laevis and Rana temporaria. They found increased incidence of
mortality in tadpoles weeks after they ceased dosing the animals. Over
an 80-day period, 47.5% of the tadpoles died. The X. laevis exposed to
7.7 pM and 0.64 nM PCB 126 exhibited swimming disorders prior to
death. Both increased mortality and reduced T4 concentrations
occurred in a dose– response manner in X. laevis. Severe eye and tail
malformations increased in the froglets in a dose–response manner
after approximately 60–68 days.
Ptáci
Thyroid hormones in birds have been investigated for their role in
migration and courtship. Preventing migrating species from breeding
out of season is especially critical for their survival. From the 1950s
through to the 1970s, fish-eating birds in the Great Lakes were
experiencing very poor reproductive success. Keith suggested that the
high embryo mortality and low chick survival in herring gulls nesting in
upper Green Bay in the mid 1960s was both the result of
a) the effects of the chemical residues from the mother on the
embryo and b) the effects of the adult’s contamination on its parental
behavior.
Ryby
Migration of salmonids is linked with THs effecting a sequence of
behaviors. In the laboratory, increases in T4 led to less display of
aggressive behavior such as territoriality. Elevated concentrations of
both T3 and T4 reduced the fishes’ preference for shade to more
open areas (phototaxis). T3 treatment caused the fish to swim with
the current rather than against the flow (rheotaxis).
Savci
PCBs and dioxins have been shown to alter thyroid function in rodents
by multiple mechanisms, including direct toxic effects on the thyroid
gland, induction of thyroid hormone metabolism via the UDPglucuronyl
transferases, and interactions with thyroid hormone
plasma transport proteins, particularly transthyretin. A number of
investigators have evaluated the effects of maternal PCB exposure
on thyroid function of rat pups. Pup serum thyroxine (T4) levels are
markedly reduced by PCB or dioxin exposure, but the levels of the
active form of the hormone, triiodothyronine (T3), are generally
unchanged, or only slightly reduced. A relationship between exposure
to dioxins and PCBs and alterations in thyroid hormones has also been
reported in human infants. Infants exposed to higher levels of PCBs
and dioxins had lower free T4 levels and higher thyroid-stimulating
hormone levels.
PPAR
•• Deregulace PPAR a reprodukceDeregulace PPAR a reprodukce
•• PPAR a karcinogenitaPPAR a karcinogenita
FtalFtalááty jako ligandy PPARty jako ligandy PPAR –– efekty na samefekty na samččíí aa
samisamiččíí reprodukreprodukččnníí systsystéémm
TDS = testicular dysgenesis syndromeTDS = testicular dysgenesis syndrome
FtalFtalááty modulujty modulujíí expresi enzymexpresi enzymůů kontrolujkontrolujííccíích syntch syntéézu azu a
odpourodpouráávváánníí steroidnsteroidníích hormonch hormonůů
Peroxisomes
 Peroxisomes: 0.5M diameter,
contribute to 20% cytoplasmic
volume.
 Enzymes for oxidation of fatty
acids
 Oxidation produces H2O2
 Increase in peroxisomal number
and volume induced by peroxisome
proliferators
Oxidation of fatty acids by peroxisomes.
Peroxisomes degrade fatty acids with more
than 12 carbon atoms by a series of
reactions similar to those used by liver
mitochondria. In peroxisomes, however, the
electrons and protons transferred to FAD
and NAD+ during the oxidation reactions are
subsequently transferred to oxygen,
forming H2O2
PPAR a karcinogeneze
Peroxisome proliferation
 Liver growth
– hypertrophy
– hyperplasia
 Induction of liver enzymes
– peroxisomal enzymes (peroxisome proliferation)
– P450 - the CYP4 genes
 Proliferation of the Endoplasmic Reticulum
and peroxisomes
 Hypolipidaemia
Oxidative Theory
Fatty acids
increase in peroxisomal -oxidation
without catalase (<2 fold)
Increase H2O2 genotoxic
DNA damage
Cancer
Úloha Kupfferových buněk
 PPAR exists in mouse, rat, guinea pig and
human
 Low hepatic levels in human and g-pig
 Human liver
– No peroxisome proliferation
– No induction of liver growth
 If PPARs cause cancer in rats, do they
cause cancer in humans? Therefore, no risk
of cancer???
Species Differences and Human Risk
Assessment