Neural Tube Defects (NTDs)

3rd International Conferences and Workshop on Basic & Applied Sciences 2011

ISBN: 978-979-19096-1-7

Neural Tube Defects (NTDs) of Mice Embryo As Effect of 2-Metoxyethanol Treatment E. Prihiyantoro1, I.b. Sa`id2, S.A. Husen1, W. Darmanto1 Department of Biology Airlangga University, Surabaya, Indonesia 1

STAIN Kediri, Indonesia E-mail: [email protected]

2

Abstract

dehydrogenase (ADH). Based on several studies that have been done, it was reported that the compound was the most harmful to the body cells is the result of the oxidation process of 2-ME 2methoxyacetic acid (MAA) (Moslen et al., 1995). Previous studies reported that 2-ME can cause growth failure in multiple organ systems of experimental animals (Maldonado et al., 2003), increased incidence of degeneration of brain development (Terry et al., 1996), Exencephaly (Terry et al., 1994 ), and cerebrum cortex tissue damage of embryo (Darmanto et al., 2004). Toxicity of 2-ME from the above studies reinforced with European studies report that says that there is a relationship between pregnant women exposed to glycol ether (a group of compounds of 2-ME) during the first trimester of gestation, with abnormalities of the central nervous system (Johanson , 2000). Central nervous system disorders can be caused by the disruption of embryonic development, especially neural tube formation that can trigger the occurrence of Neural Tube Defects (NTDs) (DeSesso et al., 1999). Neural Tube Defects (NTDs) are a form of structural abnormality in the development of the composition that caused by failure of neural tube closure that occurred in the prospective brain, and the prospective spinal cord (DeSesso et al., 1999; Harris & Juriloff, 2000; Harris & Juriloff , 1999; Wu et al., 1996). In 1989 in Atlanta, USA have NTDs incidence 0.6 per 1000 births (Yen et al., 1992). Research conducted by Guvenc et al. (1993) in eastern Turkey, reported that from 1000 there were 5.6 births, affected by NTDs. Refers to research conducted by Wu et al. (1996) Neural Tube Defects (NTDs) in humans appear between 1-9 per 1000 live births in the world. To study this disorder (NTDs) should be performed using animal models. The best animal model for studying neural tube defects are mice, because the disorder is easily produced in mice through a gene mutation or through toxic material that is inserted in the body (Harris & Juriloff, 2000). Based on the teratogenic properties of 2-ME above, the research was

2-Metoxyethanol (2-ME) is liquid flammable compound which extensively used as solvent in various industries production like plasticizer and paint solvent. The objective of this study was to showed the Neural Tube Defects (NTDs) on the prospective brain of mice embryo in neurulation stage as effect of 2-ME 7,5 mmol/kg body weight (BW) and were administered intraperitoneally on gestation day (GD) 7. The control groups were administered sterilized bidestilled water. The pregnant mice were killed by cervical dislocation at GD 8, 8 and 2 hours (8:02), 8:04, 8:10, 8:14, 8:19, 9:04, 9:12, and 9:14 on control group. On treatment groups were killed at GD 8:10, 8:14, 8:19, 9:04, 9:12, and 9:14. The results were analyzed descriptively. In the treatment group on GD 8:10 (that induced 2-ME), had 88,89% embryos with NTDs or 8 of 9 embryos had NTDs. On GD 8:14, 8:19, and 9:04, the percentage was 100%, it means all embryos had NTDs, which are 9 embryos on GD 8:14, 8 embryos on GD 8:19, and 7 embryos on GD 9:04. On GD 9:12, 5 of 6 embryos had NTDs (83,33%). On GD 9:14, 7 of 9 embryos had NTDs (77,78%). From the results of this study showed that 2-ME 7,5 mmol/kg BW could caused NTDs on mice BALB/c embryo. Keywords:2-Metoxyethanol,mice,neurulation, NTDs. 1

Introduction

2-Metoxyethanol (2-ME) is a glycol ether that are widely used in industry. 2- Metoxyethanol can enter the body of animals and humans through breathing (inhalation), gastrointestinal (ingested), and through direct contact with skin (Johanson, 2000). In the body, these compounds will oxidize into 2methoxyacetaldehyde (2-MALD) with a catalytic enzyme alcohol dehydrogenase (ALDH) and undergo further oxidation into 2-methoxyacetic acid (2-MAA) with the help of aldehyde

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Prihiyantoro, Said, Husen, Darmanto, Neural Tube Defects (NTDs) of Mice Embryo as Effect of 2-Metoxyethanol Treatment 3 Result and Discussion

conducted to test the effect of 2-ME to the occurrence of abnormalities in neural tube closure process/ Neural Tube Defects (NDTs) in the prospective brain of mouse embryo Gestation Day (GD) 8 days 10 hours (8:10), 8:14, 8:19, 9:04, 9:12, 9:14.

2 Methodology Animals used in this study were mice (Mus musculus L.) strain BALB / c aged 2-3 months who have never married, weighing 20 -30 g. fifteen male and female mice obtained from the Center Veterinaria Farma (PUSVETMA) Surabaya. Male and female mice were maintained separately in the animal house. Acclimation of the mice was performed for 1-2 weeks. 2.1 Treatment In the treatment group (6 pregnant mice), treated with 2-ME 7.5 mmol / kg body weight (BW) intraperitoneally on GD 7 before surgery in the GD 8 and 10 hours (8:10), 8:14, 8:19, 9:4, 9:12, and 9:14. While in the control group (9 pregnant mice) without giving 2-ME, and replaced with a sterile aqua bidestillata intraperitoneally on GD 7 days before surgery in the GD 8, 8:2, 8:4, 8:10, 8:14, 8:19, 9:4, 9:12, and 9:14. Volume that used for all control and treatment groups was 0.1 ml for every 10 grams of BW female mice pregnant. 2.2 Data Collection The whole embryo control group (9 embryos) observed normal development of embryos, while six control group embryos (GD 8:10, 8:14, 8:19, 9:04, 9:12, and 9:14) and whole embryo treatment group (GD 8:10, 8:14, 8:19, 9:04, 9:12, and 9:14) observed morphological abnormalities (NTDs) ratio of each embryos in the cephal which is a candidate for embryonic brain/ prospective brain. The disorder can be a decrease in the volume of prospective brain or late stages in the process of neural tube closure in the embryo cephal. Observations were carried out by using photomicroscope (microscope that is connected directly to computers equipped with special software). 2.3 Analysis NTDs incidence (morphology) calculated the percentage of abnormalities that occur for each subgroup, subsequently analyzed descriptively. To calculate the percentage of NTDs per sub group by summing all embryos with NTDs occurrence, the result is divided by the total embryos that have NTDs in the same group. The results of the division are multiplied by 100%.

Table 1: The percentage of mice embryo that have Neural Tube Defects (NTDs) in the prospective brain.

GD

Group

Control (aqua bidestillated) Σ embryo

8:00 8:02 8:04 8:10 8:14 8:19 9:04 9:12 9:14

5 7 9 9 8 9 9 9 9

Σ embryo with NTDs

Treatment (2-ME 7,5 mmol/kg BB)

% NTDs

Σ embryo

0 0 0 0 0 0 0 0 0

9 9 8 7 6 9

-

Σ embryo with NTDs

8 9 8 7 5 7

% NTDs

-

88,89 % 100 % 100 % 100 % 83,33 % 77,78 %

rc cnp

cnp

A

cnf

B

ng

C

D

msp msp nf

E

mtp myp G msp

F msp

mtp

mtp myp myp H

I

Figure 1: Embryonic development of mice (63X magnification) in the control group GD 8 (A), 8:02 (B), 8:04 (C), 8:10 (D), 8:14 (E), 8:19 ​ ​ (F), 9:04 (G), 9:12 (H), 9:14 (I). CNP; cephalic neural plate, CNF; cephalic neural fold, ng; neural groove, rc; rostrocephal, nf: neural fold, MSP; mesencephalon, MTP; metencephalon, MYP; myelencephalon.

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Prihiyantoro, Said, Husen, Darmanto, Neural Tube Defects (NTDs) of Mice Embryo as Effect of 2-Metoxyethanol Treatment

cp

msp

msp

mtp

mtp myp

mtp

A

A

B psp

C

psp

B

C

Figure 4: Embryonic mice (63X magnification) at GD 8:10 control group (A) and treatment (B & C). Arrow shows the difference of neural tube development. Cephal (cp) in the treatment group appear smaller.

myp

myp

cp

cp

msp

psp

cp

D

E

F

cp

Figure 2: Comparison of embryonic development of mice (63X magnification) the dorsal (A, B, C) and ventral (D, E, F) in the control group GD 9:04 (A and D), 9:12 (B and E ) and 9:14 (C and F). msp; mesencephalon, MTP; metencephalon, MYP; myelencephalon, PSP; prosencephalon.

nf

nf

A

B

nf

A

cp

cp

D cp

A B C Figure 6: Embryonic mice (63X magnification) at GD 9:04 control group (A) and treatment (B & C). Arrow shows the difference of neural tube development. Cephal (cp) in the treatment group appear smaller.

nf

nf E

C

Figure 5: Embryonic mice (63X magnification) at GD 8:19 ​ ​ control group (A) and treatment (B & C). Arrow shows the difference of neural tube development. Cephal (cp) in the treatment group appear to be smaller and abnormal.

np

C

B

cp

F

Figure 3: Comparison of embryonic mice (63X magnification) control group (A, C, E) and treatment (B, D, F) in the GD 8:10 (A & B), 8:14 (C & D), 8:19 (E & F). np; neural plate, nf: neural fold.

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Prihiyantoro, Said, Husen, Darmanto, Neural Tube Defects (NTDs) of Mice Embryo as Effect of 2-Metoxyethanol Treatment

rc

MSP

rc

A

neural groove (Figure 1) while the reason for the last surgery performed on mice with GD 9:14 (for the control and treatment group) because, according to Harris & Juriloff (1999) a clear embryos if the embryos are not promising NTD have prenatal age more than 9 days, by reason of embryos with prenatal age less than 9 days which experienced a similar incident to the nature of NTD has not been established that these embryos had NTD. These abnormalities may only delay the process of growth that would normally be back to normal at prenatal age more than 9 days or more than GD 9:12. This can be understood by looking at the results of research on embryos in GD 9:04, 9:12, 9:14 (Figure 2) which showed no differences are quite striking. 2-Metoxyethanol are undergoing a process of oxidation in the mitochondria of liver cells to MAA, after previously having a MALD oxidation causes elevated levels of Reactive Oxygen Species (ROS). Reactive Oxygen Species (ROS) are compounds that have a negative electron and will always react with other molecules that have positive electrons in order to stabilize themselves. Reactive Oxygen Species (ROS) potential forming other free radical that stronger namely hydrogen peroxide (H2O2) and hydroxyl (OH-) (Dhalluin et al., 1999; Hayati et al., 2006; Lieber, CS, et al. 2003 in Lunitasari, 2004; Niwa, 1997). High levels of ROS can oxidize lipids in cell membranes that lead to the formation of malondialdehyde (MDA) which is toxic to cells (Maslachah et al., 2003). The process of oxidation of lipids in cell membranes can lead to low permeability of cell membranes that induced influx of Ca2+ ions become excessive. With increasing of Ca2+ ions within the cell will cause the interruption of respiratory chain by inhibiting oxidative phosphorylation, so the acquisition of ATP decreases. With the loss of mitochondrial membrane potential, the apoptotic inducing factor (AIF) will be released by membrane intermitochondrial (Kluck et al., 1997 in Darmanto, 2002). The presence of AIF is to stimulate the occurrence of chromatin condensation and nucleus fragmentation (Ma `ruf, 2002). Nuclear fragmentation may result in termination of double chain nuclear DNA as a sign of cell death (apoptosis), so this cell death process called the process of cell death through apoptotic inducing factor (AIF) and cell death including apoptotic cell death (Duval & Wyllie, 1986 in Darmanto, 2002; Kimball `s, 2004), this information can be seen in figure 9 below.

B

C

MSP

MSP D

E

MSP

F

Figure 7: Comparison of embryonic mice (63X magnification) control group (A, C, E) and treatment (B, D, F) in the GD 9:04 (A & B), 9:12 (C & D), 9:14 (E & F). rc; rostrocephal, MSP; mesencephalon.

cp

cp

cp

A B C Figure 8: Comparison of embryonic mice (63X magnification) control group (A) and treatment (B & C) at GD 9:14. Arrow shows the difference of neural tube development. Cephal (cp) in the treatment group appear smaller. Giving teratogenic material in early embryo development can cause abnormal development of these embryos (Sadler, 2000). In this study used 2ME as chemicals that are teratogenic, and embryos of mice strain BALB /c as experimental animals. Gived 2-ME intraperitoneally in female mice with GD 7, meant 2-ME has become MAA in mice in GD 8 which is early neurulation (Harris & Juriloff, 1999). This is as it has been reported by Kim & Smialowicz (1997), that the MAA from the metabolism of 2-ME has been distributed into the tissues of mice embryos within 24 hours. In the control group initial surgery performed on mice with the GD 8:00, because on that GD the embryos of mice experiencing the beginning of the neurulation. In the treatment group surgery performed on mice with the GD 8:10 in order to provide the estimated time over the distribution of MAA in the embryo, but this cannot be said too late, because in the GD 8:10 is the early formation of

2-ME

MALD

MAA

ROS

APOPTOSIS

Ca2+ intra cell

ATP

Mitochondrial potential membrane

Chromatin condensation & nuclear fragmentation

Figure 9: Apoptosis caused by 2-ME

156

AIF released

Prihiyantoro, Said, Husen, Darmanto, Neural Tube Defects (NTDs) of Mice Embryo as Effect of 2-Metoxyethanol Treatment Please note that neural cell death is normal in mice embryonic development and induction of 2-ME led to increased incidence of cell death and the death of the neural tube (Ambroso et al., 1998; Harris & Juriloff, 1999). Apoptotic cell death is an excessive mortality affects four important processes in the formation of neural tube, namely the formation of nural plate, nural plate growth into neural folds, upward movement of neural folds and neural groove closure and becoming neural tube. And errors in 4 stages can create neural tube defects (Selcuki et al., 2005), if the situation occurs in part of cephal then obtained disorders are called NTDs. From the observation, there are 8 embryos in the GD 8:10 who have NTDs of total 9 embryo. There are 6 of 8 embryos still looks flat on the cephal area. This also happened on 9 embryo GD 8:14. Fifteen embryos (6 embryos at the GD 8:10 and 9 embryos in the GD 8:14) in the treatment group looks like the embryo on GD 8:04 in control group. The difference is 9 embryo on GD 8:14 appear more developed than embryos in the UK 8:10. Referring to Harris & Juriloff (1999) state is possible due to the abnormal cellular process caused by abnormal cell formation. Abnormalities allegedly originated from the disruption of methionine synthesis which causes the cells to be round/ circular, which should be columnar. The changing shape of these cells is for elevation. So disruption of metioinin synthesis can cause a cephal still be flat that should be concave. For the 7th embryo (from 8 embryos with NTDs in the GD 8:10) the cephal looks smaller, this can be caused due to the absence of retionic acid which causes a decrease in cell proliferation in the neural tube and this situation can cause a smaller cephal than normal which originated from the small neural tube formation (Wilson et al., 2003). Meanwhile, the 8th embryo looks like an embryo on GD 8:02 or 8:04 is could be happened because the embryo had stopped growing due to the effects of 2-ME that could cause embryonic death and resorption by the parent. It is like it has been reported by Scott et al. (1989). As in the GD 8:19, on GD 8:14 whole embryo (8 embryos) had NTDs. Of 8 embryos 6 embryos had been developed as an embryo in the GD 8:10 in control group, this situation may result because the recovery process on a flat cephal, causes the development of the cephal late. Harris & Juriloff Research (1999) which knockout of P300, obtained embryos show abnormalities in the form of fusion failure in the dorsal cephal as research results shown in Figure 3.F, failure of fusion in the dorsal cephal also can cause abnormalities in the form opening of the mesencephalon as happened on 5 embryos on GD 9:12 and 5 embryos on GD 9:14 (Figure 7.F. 7.D) While abnormalities in 2 others embryos at GD 8:19 can be caused by 2 things, the

first failure of communication between cells in the cephal that causes an abnormal shape of cephal (figure 5.B), the second possibility is the disrupted expression of the gene Lama5 causes basal lamina appear more narrow/ slender causing cephal shape like the Figure 5.C looks much thinner. This also occurs in embryos as shown by the GD 9:14 (Figure 8.B) (Harris & Juriloff, 1999). A hundred percent abnormalities were also still occurring in the GD 9:04. Five of 7 embryos have abnormalities in the form of the opening of the rostral cephal. With reference to the research of Juriloff and Harris (1999) using mice with knockout of Cart1 gene, that causes cell death increasing in the mesenchyme that can potentially cause abnormalities in the form of fuse failure or the opening of the rostral cephal as embryos in the treatment group GD 9:04. In 2 other embryos seem didn’t grow and is assumed an embryonic death that why their body size is much smaller compared with the control group that would eventually resorption by the parent. This situation is also seen in the embryo on GD 9:14 (Figure 8.C) who did not growth (Scott et al., 1989). From the above, can be presumed that the addition of 2-ME in mice on the possibility to interfere with the synthesis of methionine, the absence of retionic acid, expression of P300 genes, expression of Cart1 genes, expression of Lama5 genes, and the worst effect was the death of the embryo which later resorption by the parent. Induction of 2-ME suspected as the cause of decline in the number of embryos at several GD. Eight, 7, and 6 embryos in the mice on GD 8:19, 9:04, and 9:12. This is allegedly due to the effects of 2-ME could cause the death of embryos and embryonic resorption by the parent. From the observation, the decline in the quality of 2-ME toxicity of embryo deaths have occurred at 62 hours after treatment (GD 9:14). This is thought to be caused by enzyme systems in the body have been able to reduce the excessive amount of radical compounds (Niwa, 1997) that caused by 2-ME metabolic processes in the body of mice.

4

Conclusions

Based on research that has been done can be concluded that the induction/treatment of 2-ME 7.5 mmol / kg intraperitoneally to the mice in the GD 7 cause 88.89% NTDs in the embryonic prospective brain for GD 8:10, 83.33% and 77.78% for GD 9:12 and 9:14, and 100% for GD 8:14, 8:19, and 9:04.

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