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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