SUMMARY - repository civitas UGM · as in Indonesia, which is a tropical country (Marasas and...
Transcript of SUMMARY - repository civitas UGM · as in Indonesia, which is a tropical country (Marasas and...
STUDY OF TERATOGENESIS, BRAIN DEVELOPMENT AND POSTNATAL BEHAVIOR OF FETAL MICE (Mus musculus L.) Swiss Webster
INDUCED BY OCHRATOXIN A
SUMMARY
by :
Arum Setiawan 07/259516/SBI/504
FACULTY OF BIOLOGY UNIVERSITY OF GADJAH MADA
YOGYAKARTA 2012
Abstract
Ochratoxin A (OTA) is the most toxic of the ochratoxins. Ochratoxin A is structurally similar to the amino acid phenylalanine (Phe), it has an inhibitory effect on a number of enzymes that use Phe as a substrate. Its toxicity has been associated with inhibition of protein synthesis, DNA and RNA synthesis, mitochondrial dysfunction, formation of DNA adducts, disruption of calcium homeostasis, the generation of reactive oxygen species and stimulated lipid peroxidation. This experiment was performed to examine the effects of Ochratoxin A in pregnant mice at the organogenesis period on growth, development of embryos and foetuses, brain development and postnatal behavior of post weaning mice.
This research was conducted in two stages, using 30 pregnant mice in each stage. Thirthty pregnant mice in each stage were divided randomly into 5 groups of 12. Ochratoxin A was dissolved in sodium bicarbonate and administrated orally on seventh to fourteenth days of gestation. Ochratoxin A was given at the dosage of 0,5; 1,0 and 1,5 mg/kg body weight, respectively. The remaining animals were used as an untreated control, and placebo were given by sodium bicarbonate. At 18th days of gestations, thirty pregnant mice were sacrificed and caesarian sectioned to remove the foetuses. Observation covered the number of foetuses, the number of live foetuses, the number of intrauterine death, fetal morphometric observation, brain morphometric, brain protein content and protein profile of the brain. The other Dams were maintained until delivery. At 21st days of age, the offspring were performed behavioral test using swimming test and olfactory avoidance tests After behavioral tests, the offsprings were sacrificed and taken its brain. Observation covered the brain morphometry, brain protein content and brain protein profile. The cerebellum subsequently prepared by parafin method and stained using Haematoxylin-Eosin staining and Immunohistochemistry technique to detect the Inosithol 1,4,5 triphosphate receptor (IP3R) and the number of cerebellum Purkinje cells. Data Analysis using one way ANOVA and DMRT for the significance difference. Result of these study indicated that OTA given to the pregnant mice at the organogenesis period caused inhibit foetuses growth and development, intrauterine death, foetuses malformation such as haemorraghe, curved body, open eye and anotia, decreased of brain weight, the length and width of cerebrum and cerebellum, the wall thickness of cerebrum, decreased of foetuses brain protein content, decreased thickness of bands protein profile, decreased of IP3R, impaired growth of Purkinje cells of mice treated with the more marked decline in the number of Purkinje cells compared with control and placebo. In the behavioral test, OTA caused a decrease in the value of test parameters on swimming test and an increase in the percentage of failures to avoid the smell of ammonia in olfactory avoidance test. So, it can be concluded that OTA is not a specific teratogen because OTA affects on several organ in embryo and foetuses development. Ochratoxin A is neurotoxic and as a behavioral teratogen that affect the behavior of the offspring with decreased in the number of Purkinje cells and IP3R.
Key words : Ochratoxin A, mice, teratogenesis, behavior, Purkinje cells, protein, IP3R
A. Introduction
Micotoxyn is a secondary metabolite of fungi that are harmful to animals and
humans. Among various famous micotoxyn is Ochratoxin A (OTA). Ochratoxin A is
produced primarily by the fungus Aspergillus ochraceus Wilhelm and Penicillium
verrucosum. This fungus thrives in a variety of food agricultural commodities and
livestock and processed products. Contamination of agricultural commodities and
livestock by toxin-producing fungus is a problem that makes it difficult post-harvest
around the world. This is caused by the fungus Aspergillus spp and Penicillium spp.
growing fastly, especially in areas with high temperature and high relative humidity such
as in Indonesia, which is a tropical country (Marasas and Nelson, 1987).
Ochratoxin A is the main mycotoxin of ochratoxin groups that are toxic.
Ochratoxin A contains an isocoumarin moiety linked by a peptide bond to phenylalanine
(Phe) and it is generally found in cereals, oleaginous seeds, green coffee, pulses, wine and
poultry meat. Ochratoxin A production depends on both environmental and processing
conditions (climatic conditions, abnormally long storage, transportation, wet or dry
milling, roasting procedures, fermentation etc.) (Miraglia and Brera, 2002). Ochratoxin A
structure is similar to the structure of the amino acid Phe and OTA can inhibit enzymes
such as Phe-tRNA synthetase. This leads to inhibition of protein synthesis, in addition to
stimulating lipid peroxidation (Marti, 2006).
Ochratoxin A has been reports to be toxic to a number of species and produced
nephropathy, lymphoid necrosis, enteritis, and liver damage. Ochratoxin A is also known
to induce a variety of malformations in mice, rats, hamsters and chickens. Ochratoxin A
has not only renal toxicity, carcinogenicity, immunotoxicity, hepatic toxicity and
neurotoxicity, but also teratogenicity (Brown et al., 1976; Hood et al., 1978; Mayura et al.,
1982; Wei and Sulik , 1996; Wangikar et al., 2004 a,b). More recently, it was reported that
microcephaly was induced with high frequency in mice by prenatal treatment with OTA.
These malformations, including microcephaly, were also induced with nearly the same
frequency by oral intake of OTA. Ochratoxin A is also known to induce neural tube defects
(NTDs) in rodent embryos (Fukui et al., 1992; Wei and Sulik, 1996; Wangikar et al., 2004b;
Ohta et al., 2006 ; Ueta et al., 2009). It has not yet been reported that OTA induced NTDs in
humans, however OTA has been detected in the umbilical blood and maternal milk of
humans (Jonyn et al., 1995; Skaug et al., 1998; Postupolski et al., 2006).
Investigation of the effects of acute and chronic exposure to OTA on the nervous
system has been carried out, because development of nervous tissue appears to be very
susceptible to the deleterious effects of OTA (Hayes et al., 1974; Wangikar et al., 2004b;
Sava et al., 2006a). Ochratoxin A has been reported to induce teratogenic effects in neonates
rats exposed in utero, characterized by microcephaly and modification on the brain levels of
free amino acids (Belmadani et al., 1998b). Ochratoxin A was also reported to be neurotoxic
to adult male rats that were fed with OTA-containing diet. Neurotoxicity, indicated by
concentration of lactic dehydrogenase released from the dissected brain tissue, was more
pronounced in the ventral mesencephalon, hippocampus and striatum than in the
cerebellum. The bioconcentration of OTA in these brain regions did not correlate with
toxicity (Belmadani et al., 1998a).
In the peripheral nervous system of young adult rats, OTA reduces K+ channel
conductance and could interfere with cellular proliferation and the regulation of cellular
processes during myelinogenesis (Chiu and Wilson, 1989; Dubois and Dubois, 1991; Carratu
et al., 1998). Animals may be exposed in utero and obviously during the development of the
postnatal brain as well as in adulthood, since OTA is a food contaminant which is found in
the blood of humans and animals all over the world (Kane et al., 1986; Kuiper-Goodman and
Scott, 1989; Breitholtz et al., 1991; Creppy et al., 1991). Fukui et al. (1987) reported that
intracisternal injection of OTA in neonatal period caused developmental abnormalities of
mouse cerebellum such as disarranged cortical structure while layered structure of the vermis
was well preserved. Ochratoxin A may contribute to the pathogenesis of neurodegenerative
disease (e.g. Alzheimer’s and Parkinson’s disease) in which apoptotic processes are centrally
involved (Sava et al., 2006b; Zhang et al., 2009).
Half-life of OTA in humans approximately 35 days (Hagelberg et al., 1989; Studer-
Rohr et al., 1995) and is still detectable in the blood after a few weeks later. Half-life of
OTA in rats about 3 days, the pigs 3-5 days, monkeys 19-21 days, whereas in mice about
24 hours (Galtier et al., 1981; Hagelberg et al., 1989; Stander et al., 2001). Ochratoxin A
exposure with a dose of 290 µg/kgbw orally every 48 hours for 1-6 weeks, showing the
occurrence of weight loss as tired as 4 weeks in rats, but food and water consumption did
not show significant differences compared to controls. Ochratoxin A accumulates in the
brain depending on the timing of approximately 100 ng/g of brain after 6 weeks. This toxin
causes the change in concentration of amino acids tyrosine and phenanthrene and damage
to hippocampal tissue (Belmadani et al., 1998b). Research with female mice fed orally
with OTA dose 120 mg/kgbw per day for 10, 20 or 35 days, indicating the occurrence of a
significant increase of activity of the enzyme gamma-glutamyl transferase in three parts of
the brain was observed (Zanic-Grubisic et al., 1996).
Giving orally in rats at a dose of 0,12 to 12,29 mg/kgbw/day for 1-6 weeks, the
results observed in vitro and in vivo showed neurotoxic properties of the OTA (Mally et
al., 2006). In cultured rat brain cells, 10-20 nM OTA showed increased expression of genes
that cause inflammation of the brain (mRNA of Peroxisome-proliferator-activated receptor,
haem oxygenase-1 and led to the synthesis of nitric oxide) and lower expression of glial
fibrillary acidic protein, which part of filament astrocyt intermedia (Zurich et al., 2005). In
the cells of embryonic rat midbrain and a dose of 0.5 mg/ml OTA led to a reduction in the
number of living cells, and induces transcription factor activator protein-1 (AP-1) and
nuclear factor-kappa B (NF-κB) activates neurite outgrowth at high concentrations (Hong
et al., 2002).
This study aims to determine and assess the effect of OTA administration at a dose
of 0.5, 1.0 and 1.5 mg/kgbw per day during organogenesis period on fetal growth and
development of mice fetuses (Mus musculus L.) Swiss Webster that includes morphometry
(fetal weight and length) and developmental abnormalities that occur, the number of live
fetuses, numbers of pups born alive, the frequency of intrauterine death and developmental
abnormalities, mice fetal brain development covering the brain morphometry (brain
weight, length and width of cerebrum, length and width of cerebellum, and the wall
thickness of cerebrum), totally protein content of brain and brain protein profiles,
histological structure of cerebellum, profile of Inositol 1,4,5-Triphosphate receptor (IP3R),
the number of Purkinje cells and behavior changes in mice (Mus musculus L.) Swiss
Webster age 21st days (post weaning) in swimming test and olvactory avoidance test.
B. Literature Study and Theoritical Backgorund
1. Ochratoxin A
Ochratoxin A is a toxic secondary metabolites produced by the fungus Penicillium
verrucosum and Aspergillus sp. such as some types of A. ochraceus, A. carbonarius and A.
niger. Ochratoxin A is a main mycotoxin in ochratoxin groups that have toxic effects.
Ochratoxin A is a dihydro-isocoumarin derivatives bound peptides with phenylalanine and
is found in wheat, vegetable oil, coffee, wine and poultry meat (Miraglia and Brera, 2002).
The chemical name of OTA is (R)-N-[(5chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-
2-benzopyran-7-yl)-carbonyl]-L-phenylalanine.
The molecular weight of OTA (C20H18ClNO6) is 403.8da. The structure of OTA is
very similarly to the amino acid phenylalanine (Phe), so it is a competitive inhibitor of
some enzymes that use substrates such as Phe-tRNA synthetase, which in turn will inhibit
protein synthesis (Marti, 2006). OTA-shaped colorless crystals that dissolve in organic
solvents, are optically active and fluorescent blue under ultraviolet light. Ochratoxin A is a
mycotoxin that is very stable in several different solvents. Ochratoxin A in methanol under
the storage temperature of -20° C can last up to several years (Valenta, 1998).
The biosynthetic pathway of OTA biosynthetic pathway is not fully known (Ringot
et al., 2006). Research with a carbon marker (14C- and 13C-), indicating that the binding
of phenylalanine derived from the shikimate pathway and the dihydroisocoumarin bond
comes from the pentaketide pathway. The first step of the synthesis of polyketide
isocoumarin in a solution consisting of an acetate unit (Acetyl-CoA) into four malonate
units. This step requires a polyketide synthase enzyme (Callaghan et al., 2003). Once
formed, polyketide chain is converted via the formation of a lactone ring (mellein
synthesis) and the addition of the C1 carboxyl group such as S-methylmethione and
sodium formate (OT-β synthesis). Further chlorine atom comes from the action of
chloroperoxidase (OT-α synthesis). Finally, OTA synthetase catalyzes OT-α into
phenylalanine (OTA synthesis) (Harris and Mantle, 2001).
Biotransformation of OTA can not be explained in detail. The main metabolic
pathways of OTA is the process of hydrolysis to less toxic compounds (OT-α) through a
break of peptide bonds. Carboxypeptidase A, trypsin, α-chymotripsin role in this
hydrolysis process (Ringot et al., 2006). The main metabolite of OTA is hydroxylation
derivative 4(R)-, 4(S)-, and 10-OH-OTA and OT-α with Phe- bond. The metabolite of
4(R)-OH-OTA was formed after the exposure has been found in human and rat liver
microsom. In vivo studies indicate that the peptide bonds split of OTA to OT-α occurs in
the pancreas and small intestine homogenates, not in the liver. This proteolytic activity by
the enzyme α-chrymotripsine and carboxypeptidase (Pfohl-Lezkowics and Manderville,
2007).
Rahimtula et al.(1988), states that OTA leads to an increase lipid peroxidation.
Ochratoxin A stimulates both NADPH bound and ascorbate bound lipid peroxidation in
microsom, with iron ions (Fe3+) as cofactor. The bond of OTA-Fe3+ causes a decrease of
iron ions in the system NADPH-CytP450 reductase. It bonds would be generate hydroxyl
radicals (H*), which in turn play a role in membrane lipid peroxidation. Khan et al.(1989),
increase of lipid peroxidation affects the plasma membrane permeability to Ca2+ ions that
interfere with cell calcium homeostasis, increased Ca2+ ions entering the cell, the release of
stored Ca ions intracellular and affect the sensitivity of the Ca channel. Intracellular Ca2+
accumulation associated with OTA toxicity due to impaired Ca2+ regulatory mechanism is
the beginning of cell injury. Hydroxyl radical production is the result of the disruption of
calcium homeostasis due to the formation of OTA-Fe3+ bond (Hoehler et al., 1997).
Ochratoxin A toxicity involves multiple mechanisms. Ochratoxin A inhibits protein
synthesis by competition with phenylalanine aminoacylation reaction, the reaction
catalyzed by Phe-tRNA synthase. This leads to inhibition of protein synthesis, DNA and
RNA synthesis. Ochratoxin A also interfere with the liver microsomal calcium homeostasis
in the endoplasmic reticulum membrane damage through lipid peroxidation (Fung and
Clark, 2004).
Bennour et al. (2009), OTA induces apoptosis by activating mitochondrial and
trakskripsi p53 with target by some genes transcription (Bax, Bak, PUMA and p21). OTA
inhibited cell proliferation at the transcriptional level. In addition, OTA also lowered
mitochondrial membrane potential, release c cytokrom and activation of caspase 9 and
caspase 3.
2. Mice embryos development
Mice embryonic development begins with fertilization, the fusion between sperm
and egg nucleus form a zygote. Fertilization is the fusion events of the male sex cell nuclei
and female sex cells. The whole event took place in the ampulla oviduct for about 15
hours (Rugh, 1968). After fertilization the egg will have cell proliferation, cell
differentiation, cell migration and organogenesis (Lu, 2009).
According Ngatidjan (2006), the growth and development are including stages of
proliferation, differentiation, cell migration and organogenesis. During embryogenesis,
these processes occur uniformly, respectively, are related to another and controlled by a
series of commands or code that contains coded information called DNA.
Development of the zygote in the pellucida zone, and then became blastocist
attached to the endometrium to a stage of life freely, because the zygote of living in the
uterine fluid. The first proliferation started about 24 hours after fertilization and lasts for 2
to 3 days, even to move and the embryo move to the uterus. Zygote proliferation has
formed blastomeres which then developed into morula. After 3-4 days, morula enters the
uterine and develop into blastocysts. The pellucid zone started to disappear in preparation
for implantation. In mice, implantation occurs about 4-5 days after fertilization (Rugh,
1968). The second proliferation (stage 4 cells) occurred 37 hours after fertilization and the
third proliferation of the blastula stage blastomeres (8 cells) occurs approximately 47
hours after fertilization. Subsequently, the embryos will experience compression
(compaction), with a very strong bond formation between blastomeres, so that the embryo
at the age of pregnancy 2 days in the morula stage is incompressible. In the 3,5 days of
pregnancy embryos are at an advanced blastocyst stage has a small number of cells making
up approximately 32-64 cells (Rugh, 1968).
Embryonic stage is the stage where the cells have intensified differentiation,
mobilization and organogenesis, as a result of the embryo is very susceptible to the effects
of teratogens. This period usually ends on a day 10-14 days to the pregnancy in rodents,
and at week-14 in humans (Lu, 2009). The next phase of the embryo into a fetus. This
phase is characterized by a refinement of the organs, the maturation of the function, as
well as the rapid growth of the body. Morphological development of organs in conjunction
with an increase in functional activity (Tuchman-Duplessis, 1975).
3. Brain development
Brain development in mice initiated the formation of neural plate and neural groove
that occurs in pregnancy days-7th and on days-14th whole brain is shaped like the
anchestor. At the days-7¾ of pregnancy, neural plate on the right and left had elevated
neural fold that will be closed on the 8th days, while the neural crest will move toward the
ventrolateral and form the nerve ganglia V, VII, VIII, IX and X. On day-8½ of pregnancy
neuroporus anterior closures and the establishment of the sulcus opticus and the brain is
divided into three major sections. On days-9½, neuroporus posterior closures and 10 days
of pregnancy telencephalon evagination. Infundibulum began to separate from the
diencephalon at days-11th . At this stage the formation of metencephalon (cerebellum). On
days-2½, cerebellum seems very thick and growing rapidly. Oculomotoris nucleus formed
on days-13th, there is also the development of cerebral hemispherium. At this stage the
formation of one and two ventricles, corpora striata, epiphysis, optic thalamus, all of which
will grow until the 15th days. On the 18th days, the braid had fully formed (Rugh, 1968).
The rodent cerebellum offers a model to study the effects of various agents on
neurogenesis of the central nervous system because of its structural simplicity and postnatal
development. Brain development in mice initiates as the formation of the neural plate and
neural groove occurs on pregnant days-7. In the days-14 whole brain is shaped like that in
adults. On days-13 of pregnancy the cerebrum development occurrs very rapidly, while
cerebellum starts its growing fastly from 14-17 pregnant days. Precursors of Purkinje cells
are generated in the neuroepithelium of the rhombic lip in the early stages of neurogenesis,
which is about 12-15 days of pregnancy (Rugh 1968). During the late pregnancy,
microneuron precursors, namely granulosa cells, form a layer on the outside (external
granular layer/EGL) and these cells actively divide and spread to the entire surface of the
cerebellum (Darmanto et al. 2000). Precursors of Purkinje cells migrate from the
ventricular area radially towards the outer surface of the cerebellum (cortex), and at a
certain position they differentiate into Purkinje cells. In normal cerebellum Purkinje cells
shape a monolayer just beneath EGL and branching dendrites growing in the molecular
layer (ML). On the other hand, granular cells located in the cortex divided by mitosis and
synthesize of Reelin, then migrate to the inside via ML and Purkinje cell layer (PCL) to
form the internal granular layer (IGL). Reelin is a protein secreted by external granular
cells during the early migration and functions as a matrix adhesion molecule that helps
positioning and patterning arrangement of nerve cells (Altman and Bayer, 1978; Goffinet,
1984; Yuasa et al., 1993; Darmanto, 2005).
Purkinje cells is one of the largest cell neurons found in vertebrate brains. Because
of its large size, these neurons have been extensively studied by experimental methods.
Purkinje cells located in the cerebellar cortex. Purkinje cell layer consists of a single
Purkinje cell layer which is the most important cells in the cerebellum. In the field of cross
of folium, Purkinje cell dendrites clearly visible passing into the molecular layer and
having a lot of branching. Each cell has thousands of dendrites, often to have 10,000
synapses and the axon (Snell, 1987). Each Purkinje cell is in a position to receive input
from a large number of parallel fibers and parallel fibers each may come into contact with
many Purkinje cells (Purves et al., 2001).
The inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular calcium release
channel, is found in virtually all cells and is abundant in the cerebellum (Striggow and
Erlich 1996). A Ca2+ flux through IP3R from the lumen of the endoplasmic reticulum to
the cytosol constitutes an important step in intracellular Ca2+ signaling. This signaling
cascade is stimulated by the binding of an agonist to its receptor on the outer surface of the
plasma membrane followed by the activation of a phospholipase C and the formation of
diacylglycerol and inositol 1,4,5-trisphosphate (IP3) (Berridge, 1993; Clapham, 1995). The
density of the IP3R is highest in the cerebellum, specifically in the Purkinje cells
(Supattapone et al., 1988) and the majority of biochemical, molecular, and biophysical
studies of this channel have used the cerebellar receptor. Models have been proposed and
experimental evidence is accumulating suggesting that the IP3R is involved in intercellular
signaling between neuronal cells (Charles, 1994; Sneyd et al., 1995) and long term
potentiation (Malenka, 1994).
4. Behavioral teratology
Behavioral test is useful in evaluating the behavior of neonatal phase due to
prenatal exposure on various external factors on the behavior, because the behavior is an
indicator of functional integrative processes of the peripheral nervous system, both sensory
and motor. Deviation of behavior can be an early indicator for the presence of toxic and
teratogenic effects of a chemical compound, because the change can already be known
before the clinical symptoms and structural abnormalities (Spyker and Avery, 1977).
Deviation of the behavior based on biochemical processes in the brain, especially
regarding the role of neurotransmitters, particularly acetylcholine, norepinephrine,
dopamine and serotonin (Johanson, 1999). Learning and memory settings and a variety of
muscular activity is influenced by serotonin and dopamine (Kapur dan Lecrubier, 2003).
5. Theoritical background
With the widespread of contamination of the genera Penicillium and Aspergillus
fungi in food and animal feed because the content of OTA then it needs be more aware of
the danger of the side effects, as well as the target organism and the non target organism.
One of the OTA effect in organisms in their infancy, which would also impact directly on
the parent as an organism that direct contact. Both of the fungal genera can grow in a wide
temperature range, and is able to grow in the low temperature in such storage in the
refrigerator.
Organogenesis is the one stage of development was very sensitive to toxic
substances, which in mice in the lasts from day 6th -14th days of gestation (Rugh, 1968).
During the period of organogenesis, the provisionof teratogen/toxic substances (e.g. OTA)
will be able to cause abnormalities of fetal growth and development (Tuchmann-Duplessis,
1975). Giving OTA on parent will cause metabolic disturbances in the dams and the
embryos because the substance with a molecular weight of 403.8 daltons can pass the
placental barrier.
Investigation of the effects of acute and chronic exposure to OTA on the nervous
system have been carried out, because development of nervous tissue appears to be very
susceptible to the deleterious effects of OTA (Hayes et al., 1974; Wangikar et al., 2004b;
Sava et al., 2006a). It has been reported to induce teratogenic effects in neonates (rats and
mice) exposed in utero, characterized by microcephaly and modification on the brain levels
of free amino acids (Belmadani et al., 1998b). Ochratoxin A was also reported to be
neurotoxic to adult male rats that were fed with OTA containing diet. Neurotoxicity,
indicated by concentration of lactic dehydrogenase released from the dissected brain tissue,
was more pronounced in the ventral mesencephalon, hippocampus, and striatum than in the
cerebellum. The bioconcentration of OTA in these brain regions did not correlate with
toxicity (Belmadani et al., 1998a). Animals may be exposed in utero and obviously during
the development of the postnatal brain as well as in adulthood, since OTA is a food
contaminant which is found in the blood of humans and animals all over the world (Creppy
et al., 1991). Fukui et al. (1987) reported that intracisternal injection of OTA in neonatal
period caused developmental abnormalities of mouse cerebellum such as disarranged cortical
structure while layered structure of the vermis was well preserved and may contribute to the
pathogenesis of neurodegenerative disease (e.g. Alzheimer’s and Parkinson’s disease) in
which apoptotic processes are centrally involved (Sava et al., 2006b; Zhang et al., 2009).
The similarity of OTA structure with the structure of the amino acids Phe, led to
OTA may inhibit an enzyme that uses Phe such as Phe-tRNA synthetase. This leads to
inhibition of protein synthesis, as well as stimulate lipid peroxidation (Marti, 2006).
Ochratoxin A increases the incidence of ROS in the cells (Ringot et al., 2006). High levels
of ROS that would cause the oxidation of lipid and protein, so it can change the cell
structure (Dukan et al., 2000).
C. Material and Methods
This study conducted in two stages, each stage of treatment used 30 pregnant mice.
Dams in stage I kept until 18th days of gestation, while mice in stage II maintained until
delivery. The offsprings of stage II maintained until the age of 21st days. Thirty dams in
each stage were randomly divided into five treatment groups each of 6 replicates.
Ochratoxin A (from Sigma Co.) has been dissolved in sodium bicarbonate administered
orally on 7th to 14th days of gestation. Rating dose of treatment was 0.5 mg/kgbw, 1.0
mg/kgbw and 1.5 mg/kgbw, whereas the comparison group was the control (untreated)
and the placebo group (given sodium bicarbonate). On 18th days of gestation, 30 dams
from stage I, were sacrificed and dissected for fetal collection by cesarean.
Observations included the totally number of fetuses, the number of life fetuses, the
number of intrauterine death, fetal morphometry (weight and length of fetuses), and
observations of fetal brain growth and development which included the observation of fetal
brain morphometry (brain weigth, length and width of cerebrum, length and width of
cerebellum, and cerebrum wall thickness), the totally protein content, and fetal brain
protein profiles. The another 30 dams kept until birth. The offsprings were kept until age
21st days (after weaning). In the age on 21 days, the offspring treated behavioral tests
which including swimming test and olvactory avoidance test. After behavioral tests, mice
were sacrificed and then taken its brain to do the preparation and observation that includes
the brain morphometry (brain weigth, length and width of cerebrum, length and width of
cerebellum and cerebrum wall thickness), totally protein content, protein profile,
histological structure of the cerebellum with Haematoxylin-eosin staining and
immunohistochemical technique to determine the IP3R and counting the number of
cerebellar Purkinje cells.
The number of total fetuses, fetal weight, fetal length, brain morphometry, total
protein content of the brain, the number of cerebellar Purkinje cells,and behavioral data
were analyzed variant (ANOVA) pattern in one direction using a completely randomized
design (CRD) at the 5% level of confidence. If ANOVA showed significant results,
statistical test followed by Duncan's Multiple Range Test (DMRT) for the significance.
The datas of the number of intrauterine deaths, the number of fetal malformations were
analyzed with Chi-square test at 5% level. For the data of brain protein profiles and the
IP3R profiles were analyzed by qualitative descriptive analysis.
D. Results and Discussion
The results of the study of teratogenesis, brain development and postnatal behavior
of fetal mice (Mus musculus L.) Swiss Webster due to OTA treatment during
organogenesis period were shown in Table S1 and S2.
Table S1. Frequency and Percentage of External Abnormal Malformation
Frequency and Percentage of External Abnormal Malformation. (%) Dosage (mg/kgbw)
Control Placebo 0,05 1,0 1,5 Fetuses total number 59 57 52 46 39 Offsprings total number 58 55 46 37 26 Development abnormalities 0
(0%) 0
(0%) 5
(9,61%) 9
(19,57%) 16
(41,03%) Craniofacial - open eyelids
- anotia
- microtia
0
(0%) 0
(0%) 0
(0%)
0
(0%) 0
(0%) 0
(0%)
2
(3,85%) 1
(1,92%) 1
(1,92%)
2
(4,35%) 3
(6,52%) 2
(4,35%)
3
(7,69%) 3
(7,69%) 3
(7,69%) Body Malformation - Flexy
- Skin abnormality
0
(0%) 0
(0%)
0
(0%) 0
(0%)
0
(0%) 1
(1,92%)
1
(2,17%) 1
(2,17%)
1
(2,56%) 2
(5,13%) Limbs defects - Micromelia
- Phocomelia - Crossed legs
0
(0%) 0
(0%) 0
(0%)
0
(0%) 0
(0%) 0
(0%)
1
(1,92%) 2
(3,85%) 0
(0%)
2
(4,35%) 3
(6,52%) 0
(0%)
2
(5,13%) 3
(7,69%) 2
(5,13%) Cardiovascular - Hemorraghe
0
(0%)
0
(0%)
3
(5,77%)
3
(6,52%)
6
(15,38%) tail - Crooked tail
- Kinky tail
0
(0%) 0
(0%)
0
(0%) 0
(0%)
1
(1,92%) 0
(0%)
1
(2,17%) 1
(2,17%)
2
(5,13%) 1
(2,56%) Other - impaired growth hair (on the mice age 21 days)
0
(0%)
0
(0%)
1
(2,17%)
2
(5,41%)
2
(7,69%)
Tabel S2. Recapitulation of morphometry fetuses development, brain development and behavioral test
Dosage (mg/kgbw) Control Placebo 0,05 1,0 1,5
Fetuses morphometry and development - Mean of length (mm) - Mean of weight (g) - Total foetus - Mean of foetus/litter
- Total the offspring - Mean of offspring/litterpercentage
of live foetus - percentage of death foetus - percentage of resorb - percentage of malformation
27,56 ± 1,03 a 1,61 ± 0,07 a
59 9,83 ± 1,86 a
58
9,67 ± 1,63 a 59 (100%)
0 (0%) 0 (0%) 0 (0%)
27,33 ± 0,85 a 1,58 ± 0,04 a
57 9,50 ± 1,05 a
55
9,17 ± 0,75 a 57 (100%)
0 (0%) 0 (0%) 0 (0%)
25,70 ± 0,80 b 1,40 ± 0,04 b
52 8,67 ± 1,03 ab
46
7,67 ± 1,37 b 52 (86,67%)
4 (6,67%) 4 (6,67%) 5 (9,61)
22,91 ± 1,03 c 1,29 ± 0,03 c
49 7,67 ± 1,21 bc
37
6,17 ± 1,21 c 49 (77,97%) 6 (10,17%) 7 (11,86%) 9 (19,57%)
17,03 ± 1,39 d 1,13 ± 0,04 d
36 6,50 ± 0,84 c
26
4,33 ± 0,82 d 36 (66,10%) 9 (15,25%) 11(18,64%) 16 (41,03)
Brain morphometry and development (age 18 pregnancy) - mean of brain weight (g) - mean of cerebrum length (mm) - mean of cerebrum width (mm) - mean of cerebellum length (mm) - mean of cerebellum width (mm) - mean of cerebrum wall thickness (mm) - mean of total protein content (μg/ml) Brain morphometry and development (offspring 21st days /postweaning) - mean of brain weight (g) - mean of cerebrum length (mm) - mean of cerebrum width (mm) - mean of cerebellum length (mm) - mean of cerebellum width (mm) - mean of cerebrum wall thickness (mm) - mean of total protein content (μg/ml) - mean of number Purkinje cells)
0,067±0,016 a 4,213±0,090 a 5,033±0,176 a 2,723±0,121a 3,463±0,236 a 1,425±0,086 a
58,101 ± 5,912 a
0,444±0,036 a 7,950±0,363 a 9,448±0,265 a 3,477±0,410 a 7,399±0,297 a 2,850±0,172 a
84,087 ± 1,739 a 383,9 ± 2,77 a
0,066±0,011 a 4,195±0,096 a 5,021±0,179 a 2,719±0,085 a 3,437±0,169 a 1,423±0,056 a
57,624 ± 4,126 a
0,426±0,037 a 7,925±0,355 a 9,421±0,119 a 3,410±0,298 a 7,381±0,332 a 2,845±0,112 a
83,904 ± 2,289 a 383,7 ± 3,34 a
0,059±0,006 ab 4,040±0,084 b 4,899±0,031 ab 2,595±0,109 b 3,189±0,217 b 1,371±0,025 b
49,431 ± 7,001 b
0,352±0,030 b 7,600±0,269 b 9,284±0,349 ab 3,040±0,384 b 6,990±0,254 b 2,666±0,088 b
72,305 ± 6,723 b 370,4 ± 4,12 b
0,056±0,007 bc 3,885±0,120 bc 4,789±0,063 bc 2,545±0,103 b 2,910±0,380 c 1,346±0,036 bc
40,305 ± 3,884 c
0,337±0,034 b 7,456±0,119 b 9,124±0,086 b 2,911±0,222 b 6,898±0,206 b 2,573±0,098 b
63,495 ± 5,767 c 361,0 ± 3,16 c
0,049±0,012 c 3,787±0,323 c 4,639±0,375 c 2,429±0,146 c 2,868±0,282 c 1,316±0,044 c
31, 947 ± 9,758 d
0,303±0,030 c 7,375±0,159 b 8,862±0,353 c 2,555±0,160 c 6,546±0,462 c 2,438±0,181 c
54,230 ± 6,824 d 352,1 ± 3,96 d
Behavioral Test Swimming test - swim direction - swin corner - use of limbs Olfactory avoidance test - percentage of avoid the smell of
ammonia
3,000 ± 0,000 a 4,000 ± 0,000 a 3,000 ± 0,000 a
100 %
3,000 ± 0,000 a 4,000 ± 0,000 a 3,000 ± 0,000 a
100 %
2,750 ± 0,463 ab 3,750 ± 0,463 ab 2,750 ± 0,463 ab
62,50 %
2,500 ± 0,535 b 3,375 ± 0,518 b 2,375 ± 0,744 bc
37,50 %
2,125 ± 0,353 b 3,250 ± 0,886 b 2,000 ± 0,756 c
25 %
Note: different letters in the same row indicate real difference at a significant level of 95%.
From Table S1 can be seen that the OTA is given to pregnant mice during
organogenesis periods caused malformations in the fetus, such of open eyelids, no ears
(anotia), small ears (microtia), humpbacked body (flexy) , the skin shriveled, stunted limbs
(micromelia), dwarf forelimb (phocomelia), hemorrhage and the tail wrapped around the
curved tail. In the offspring age 21st days, found abnormalities in the extremities and the
back of the hair growth. This incident is an anomaly, since it is usually not found
abnormalities in mice that were born. Malformation that occurs in a significant treatment at
doses of 1.5 mg/kgbw. Abnormalities are commonly found in individuals with small body
weight (decreased) compared to normal individuals and in some fetals was found more
than one malformation (multiple congenital malformations). Some agents teratogen may
cause visceral and skeletal abnormalities without showing any abnormalities of the
external morphology (Price and Wilson, 1984).
In Table S2. can be seen clearly that OTA leads to an increase in intrauterine
mortality, reduction in total protein content of mouse brain, as well as the decrease in
protein concentration in the observation of brain protein profiles are characterized by the
thinness of the bands protein and decrease the color intensity of these bands. Ochratoxin A
also resulted in increased damage and decreased histologically structure of cerebellum dan
the color intensity of cerebellar Purkinje cells that showed a decrease in the number of IP3
receptors and causes a decrease in the number of cerebellar Purkinje cells of the brain of
mice age 21st days. Intrauterine mortality increased with higher doses of OTA, and
significant at the dose of 1.0 mg/kgbw and 1.5 mg /kgbw. Decrease the size of a number of
parameters such as fetal morphometry in 18th days of gestations, fetal brain morphometry
in fetal and the offspring ages 21st days, decreasing the number of Purkinje cells and
IP3R in cerebellum of pups showed that in addition to causing malformations, OTA also
inhibits the growth and development of the fetuses and mice are born alive. Decrease in
this parameter in line with the increasing dose of OTA treatment.
In the behavioral test, OTA causes a decreased in the value parameter in a
swimming test which showed a decreased ability of the derivative neuromotoris of pups. In
the olfactory avoidance test, OTA led to an increase in the inability to avoid the smell of
ammonia. This result indicates that the olfactory organs of mice are impaired sensitivity.
The impaired growth and development of the embryo as indicated by the small
weight and the length of the embryo, can occur if an toxic agent affects cell proliferation,
cell interaction, or a reduction in the rate sinthesis nucleic acid, protein or
mucopolysaccharide during the period of embryogenesis. Inhibited cell proliferation will
result in fetal growth is also inhibited. Developmental disorders in the uterus will cause
abnormalities include a decrease in body weight that is not normal (Wilson, 1973).
One cause of resistance is cell proliferation due to OTA structure is similar to the
structure of the amino acids Phe, led to OTA may inhibit an enzyme that uses Phe such as
Phe-tRNA synthetase. This leads to inhibition of protein synthesis, as well as stimulate
lipid peroxidation (Marti, 2006). Ochratoxin A stimulates both NADPH bound and bound
ascorbate lipid peroxidation in microsom, with iron ions (Fe3+) as cofactor. The bond of
OTA-Fe3+ causes a decrease of iron ions in the CytP450 NADPH-reductase. OTA-Fe2+
bond would generate hydroxyl radicals (H•), which in turn play a role in membrane lipid
peroxidation. Khan et al. (1989), states increased lipid peroxidation affects the plasma
membrane permeability to Ca2+ ions that interfere with cell calcium homeostasis, increased
Ca2+ ions entering the cell, the release of stored Ca ions in the cell and affect the sensitivity
of the Ca channel. Intracellular Ca2+ accumulation associated with OTA toxicity due to
impaired Ca2+ regulatory mechanism is the beginning of cell injury. Hydroxyl radical
production is the result of the disruption of calcium homeostasis due to the formation of
OTA-Fe3+ bond (Hoehler et al., 1997).
The decreased levels of protein in the brain is optimal regardless of whether or not
the blood-brain barrier function of the compounds into the brain. Blood-brain barrier
function is influenced by extracellular fluid consisting of Na+ and C+ ions, and intracellular
fluid of the brain that consists of K+ ions. Ochratoxin A is a small molecular weight allows
to pass through the blood-brain barrier so it can quickly be taken up by brain tissue (Raddel
and MacLeod, 1999). If this situation continues over time will increase the levels of OTA
in the cell. Ochratoxin A accumulation in the cells causes a decrease in intracellular K+
ions influx so that disrupt the mechanism of protein phosphorylation in neuronal cells.
Schunack et al. (1990), the unstable phosphorylation mechanism that would inhibit
phosphodiesterase in activating the hydrolysis of c-AMP and c-GMP. Furthermore Lemke
and Williams (2007), stating that the inhibition of the hydrolysis of c-AMP and c-GMP in
the brain increased the level of cyclic-nucleotide so that neural activity is also increased.
Increased neural activity will affect the biosynthesis of proteins to the lowest level. This
situation will inhibit the rate of formation of a neurotransmitter receptor protein in the post
synaptic membrane, while the protein is kept always synthesized each day. If this goes on
an ongoing basis, the reserves of protein in the brain is reduced.
Mechanisms that can cause a decrease in the number of Purkinje cells is cell death
(apoptosis). Apoptosis can be detected through changes in morphological characteristics
such as chromatin aggregation and creation of apoptotic bodies, DNA fragmentation,
expression of proteins important for apoptosis, apoptosis-specific proteolysis substrates or
phosphatydidle serine exposure on the outer side of the cell membrane (Petrik et al., 2003).
There are several factors that can lead to apoptosis, including oxidative stress, need
for blood in the brain is not adequate, mitochondrial dysfunction, and disruption of calcium
concentration in cells. This oxidative stress can cause damage to cellular components, such
as membranes, DNA and protein (Zhang et al., 2009; Jankowski et al., 2009). OTA can
lead to oxidative stress through various mechanisms. Ochratoxin A metabolism pathway
may lead to the formation of reactive oxygen species (ROS) that can reduce levels of
antioxidants. Ochratoxin A exposure may lead to decreased levels of glutahion, an increase
in catalase and increased superokside dismutase. Several OTA metabolites in microsomes
induces the formation of ROS that stimulate the formation of hydrogen peroxide which is a
cofactor for enzymatic activity of LOX (lypooxygenase) enzymes such as COX
(cyclooxygenase) enzymes with peroxidase activity (Hoehler et al., 1997).
Levels of ROS formation induced by OTA can also caused cell damage and lead to
death by affecting mitochondrial function (Marti, 2006). A side from being a producer of
energy, mitochondria also store calcium and regulate calcium levels in cells, which is
required for the process of chemical communication between neurons. When mitochondria
are not functioning, they will undergo a process called mitochondrial permeability
transition (MPT). During this process, mitochondrial membrane channel opens, and
through the channel of mitochondrial release of c-cytochrome and calcium. Both of its
were a caspase activator, which plays a role in the process of apoptosis.
Ochratoxin A also affects the activity of growth factors that regulate cell
proliferation and survival. A number of growth factors required for normal cell division,
including two-factor called insulin-like growth factors (IGF) I and II. Both are useful to
bind to a protein molecule called IGF-I receptor on the cell surface (Purves et al, 2001).
OTA influence over the activities of IGF-I receptor, so that although IGF-I receptor but
still bind to the receptor function for signaling is inhibited, and cell division does not
occur. This indicates that the OTA can prevent the production of the normally central
nervous system cells by affecting growth factor (Zhang et al., 2009).
A teratogen exposure at the stage of organogenesis may affect the offspring is a
central nervous system dysfunction indicated by a deviation include the offspring behavior,
since the formation of the central nervous system includes the process of neural tube
formation and the initial distribution of brain regions (Jacobsen et al., 1987; Vorhess,
1997).
The behavior deviation is closely associated with the physical and chemical
changes in brain tissue. The brain is an organ that serves as a central organization and
processing, as well as a place of mental processes that include learning and memory
(Scanlon and Sanders, 2007). Deviations of behavior in line with changes in different
regions of the brain neurotransmitter concentrations. Decreased in the ability of swimming
activity in mice along with the decreased concentration of acetylcholine, serotonin, and nor
epinephrine in the striatum; serotonin in the cortex; occipital, nor epinephrine and
dopamine in the dorsal hippocampus and ventral hippocampus nor epinephrine (Stemmelin
et al., 2000).
The concentrations of brain neurotransmitters decreased will affect the ability of
the nervous system to digest and convey impulses. This is caused by a disturbance in the
function of neurotransmitters. Neurotransmitters function as chemical nerve impulse
conductor from one neuron to the others, with a reduced concentration of neurotransmitters
or disturbance of the brain neurotransmitters activity affect the offspring lack of ability
respond to the stimulus (Nelson et al., 1984). Neurotranmitters concentrations decreased in
the brain closely associated with the occurrence of behavioral aberrations. Behavioral
learning and memory makes it possible to respond to stimuli that come from outside such
as escape or avoidance of a situation and approach the object, so it plays an important role
because it has adaptive value for organisms (Stemmelin et al., 2000).
Bangalore (2007), suggesting that the acetylcholine receptor is responsible for the
distribution of nerve impulses to the muscular contraction. Giving thought to disrupt the
distribution of OTA nerve impulses to the muscular contraction that will affect brain
function and behavior of mice in response to an impulse. The developing brain have more
synapses than the adult brain, which was established by the stimulation received during the
developmental period. If an increase in neural activity, namely the presence of OTA, then
the process can be inhibited synaptic development and are permanent to the anatomy of
synapses and brain function. This has led to the decline in learning and behavior of mice is
characterized by decreasing values of the activity of several parameters such as the swim
direction, swim corner and use of the limbs on OTA treatment when compared with
control and placebo.
The motor coordination decreased after administration of OTA is also associated
with a decreased number of cerebellar Purkinje cells. Purkinje cells are the major cell
cerebellum and is the sole output cell cerebellar cortex. Purkinje cells receive input from
Mossy fibers excitation (via granule cells and parallel fibers) and from the nucleus neurons
Olivarius inferior (via climbing fibers). Each Purkinje cell receives input from about
100,000 parallel fibers, but only one of the climbing fibers. Stellate cell interneurons,
basket cells and Golgi cells also receive input from parallel fibers. Stellate cells and basket
cells will cause inhibition of Purkinje cells when the Golgi cells Golgi inhibition of
granule cells. Input from parallel fibers and inhibitory interneurons results in the release
impulse of Purkinje cells is known as the simple spikes, otherwise climbing fibers produce
impulses which extended release, sometimes in the form of oscillations, known as complex
spikes (Purves et al., 2001).
The decrease in the percentage of success to avoid the smell of ammonia due to the
disruption of the thalamus functions as a center to integrate and process sensory
information thus inhibiting sensory neurons that carry impulses from receptors to the
cortex cerebrum resulting sensory nerve cells tend to decrease its ability to receive a
stimulus in this case ammonia odor response. Disruption of the process of histogenesis of
nerve cells that cause a reduction in the ability of nerve cells to receive or convey
excitatory stimulus. Presumably the reduced ability of the nerve cells of mice treated group
of the offspring to receive and convey excitatory stimulus causes the offspring decreased
ability to respond to the smell, the smell test in terms of avoiding the smell of ammonia.
According to Campbell et al. (2008), in addition to the main integration center, the
thalamus is also the center of the main sensory input information to the cerebrum and an
output center for motor information leaving the cerebrum. Information coming from all the
senses are selected in the thalamus and sent to the upper brain centers for interpretation and
further integration.
The Decrease in the success of avoiding the smell of ammonia is also caused by
OTA inhibited the growth of nerve cells in the olfactory bulbus. The bulbus olfactory
function to zoom, enlarge the sensitivity of odor detection, odor filter and to detect an odor.
Brain nerve (cranial nerve) is a peripheral nerve to the brain stem and the stem serves as
the nerve as a sensory, motor and special. Special functions are functions which are the
five senses, like smell, sight, taste, hearing and balance. Olfactory nerve connects the brain
stem to the olfactory bulbus (Langman, 2009). Ochratoxin A inhibited the growth of
peripheral nerve cells with the same mechanism through inhibition of phe-tRNA
synthetase in the process of formation of protein (Belmadani et al., 1999). This led to
inhibit the process of formation bulbus olfactorius so that in the treated mice olfactory
bulbus could not function perfectly as the olfactory nerve. In mid-organogenesis is at days
9.5 to 12.5 in mice is highly active phase in the process of neurogenesis to the formation of
a visual area, cerebral cortex, basal ganglia and forebrain as well as for hipothalamus and
limbic regions (Rodier, 1980, Kihara et al., 2001).
E. Conclussion
From the observations, data analysis and discussion, some conclusions can be
drawn that the OTA is given to pregnant mice during organogenesis period :
1. inhibited growth and embryonic development are characterized by decreased fetal
weight and length are significantly different in significant dose of 0.5 mg/kgbw, 1.0
mg/k bw and 1.5 mg/kgbw. OTA reduced the percentage of fetal life, the number of
litters born alive, increased the percentage of intrauterine death and increased the
percentage of fetal malformations, in line with the increasing dose of treatment.
2. caused a decrease in brain weight, cerebrum length and width, cerebellum length
and width, cerebrum wall thickness in the fetus at 18th days of pregnancy and the
offspring in 21 days old were significantly at a dose of 0.5 mg/kgbw, 1.0 mg/kgbw
and 1.5 mg/kgbw. OTA causes a decreased in totally protein content of the brain
and decreased brain protein concentration, increased histologically damage of
cerebellum, decreasing the amount of IP3 receptor in Purkinje cells and cerebellar
Purkinje cell numbers decline.
3. caused a decrease in coordination neuromotoris swimming behavior by lowering
the value of neuromotoris test, and the test leads to increased frequency
neurosensoris inability to avoid the smell of ammonia on olfactory avoidance test
significant at doses of 0.5 mg/kgbw, 1.0 mg/kgbw and 1.5 mg/kgbw.
4. OTA is not a specific teratogen because it causes developmental disorders in
several organs. OTA is also a behavioral teratogen because it causes impairment of
the offsprings behavior. Some of the fetus had multiple congenital malformation
which is frequency increased with the increasing doses of OTA treatment.
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