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THE 2-METHOXYETHANOL TOXICITY TOWARDS THE STRUCTURES OF PLASMA MEMBRANE AND MITOCHONDRIA OF THE RAT’S SPERMATOZOA Alfiah Hayati1, Soesanto Mangkoewidjojo2, Aucky Hinting3, Sukarti Moeljopawiro4 1 Department of Reproductive Biology, Airlangga University School of Mathematics and Natural Sciences 2 Department of Clinical Pathology, Gadjah Mada University School of Veterinary Medicine 3 Department of Medical Biology, Airlangga University School of Medicine 4 Department of Biochemistry, Gadjah Mada University School of Biology ABSTRACT 2-Methoxyethanol (2-ME) is characterised as a toxic and teratogenic compound in which the exposure to it can lead to increased incidences of embryonic death or damage, teratogenesis, or growth retardation. Epidemiologic studies have been conducted on a certain group of populations where it was shown that 2-ME can result abnormalities from olygospermia to azoospermia. The main reason for this research was to understand how this compound could affect the ultrastructures of spermatozoa membrane and mitochondria. Therefore, an experiment has been performed on 10 samples of male rats where the first five were injected with 0.2ml of 200 mg/kg of 2-ME, 6 days per week within 2 weeks. The rest were taken as a control group and were injected with 0.2ml of physiological electrolytes for the same duration as the first group. After the experiment was over, the samples were anesthetised with chloroform to perform a necropsy where the spermatozoa were taken from the caudal epididimis and vas deferens by using a flushing method. Spermatozoa were fixed with glutaraldehyde so that the structures could be examined thoroughly and it was observed by using an electron microscope transmission. The results showed that there were differences of the ultra structures between the tested rats and the control group. The exposure could damage the plasma membrane of the head and also the middle piece and also resulted in vacuolated mitochondria at the middle piece. The conclusion is that exposure of 2-ME can cause damage in the plasma membrane and mitochondria of the rats’ spermatozoa. Keywords: 2-Methoxyethanol (2-ME), ultra structures, spermatozoa. Correspondence : Alfiah Hayati, Department of Reproductive Biology, Airlangga University School of Mathematics and Natural Sciences, Jl. Mulyorejo, Surabaya, phone 62-31-5992785 INTRODUCTION 2-methoxyethanol is used as a solvent for many purposes: cellulose esters, dyes, resins, lacquers, varnishes, and stain; and as a perfume fixative and jet fuel educing additive. This compound is a stable, colourless substance, and highly inflammable, with a slight scent of aromatic ether. It is also water soluble substance with a major constituent of organic solvent and it tastes bitter and sweet. 2ME can enter the body through inhalation or just by touching the compound. It is rapidly distributed to the liver, vesicle urine, bone marrow, epididymis, and throughout the whole tissues of the body except the adipose tissues (Johanson 2000). 2ME is converted to metoxy asetate acid (MAA) in a reaction catalysed by alcohol dehydrogenase and aldehyde dehidrogenase enzymes. The MAA is known as a toxic and teratogenic compound (Moslen et al. 1995). The epidemiologic studies that have been conducted on painters, who paint ships, have shown that 2ME can result in abnormalities from oligospermia to azoospermia since 2ME is used as solvent in the making of paints (Li et al. 1996; Li et al. 1997). This compound

is highly toxicable for male reproduction system. Besides being harmful on the system, it can also cause other effects such as the decreasing diameter of the seminiferous tubules, degenerations of the spermatogenic cells especially the pachytene spermatocytes and the decreasing numbers of the spermatozoa (Hayati et al. 2003). These effects appeared after 2ME were given within 21 days with 200mg/kg. However, according to the research that had been conducted by Hayati et al. (2004), there is a regeneration process (gradually within 4 weeks) of spermatogenic cells and spermatid cells where it could be found in the third week. Besides that, the regeneration process could also occur in spermatozoa morphology and motility back to normal (Hayati et al. 2005). 2ME is a high intense oxidant where it induces oxidation stress to the spermatozoa (Millar 1983). Hydroxyl radical, a reactive oxygen compound, involves in a certain pathological process. If the hydroxyl radical reacts with the cell membrane, the unsaturated fatty acid, which is the main component of

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phospholipids that coated the spermatozoa’s cell membrane, will be easily damaged. The spermatozoa membrane is mostly consisted of unsaturated fatty acid, thus, making it very sensitive to free radical. Lipid peroxidization is a chain reaction that occurs when hydroxyl radicals react with unsaturated fatty acids. In the end, the spermatozoa membranes will be damaged due to the breakdown of the unsaturated fatty acid chains (Sanocka et al. 2004). There is a significant positive correlation between the damaged structures and the functions of plasma membranes (Zaneveld 1985). This study should be conducted because the damages upon the organelles structures can be observed closely. Based on the background above, a study on the 2ME toxicities on plasma membrane and mitochondria of the samples’ spermatozoa (considering that it might relate to decreasing spermatozoa qualities) should be performed. MATERIALS AND METHODS Ten white male rats (Rattus norvegicus) of Wistar strains were used for this experiment. The tested animals were 3 months old, 125 – 135 gram and in a healthy condition. The rats were placed in a cage under the temperature of 250c – 260c. The rats were given drinks from tap water and they were fed everyday with Hi-Pro-Vite 524-2 in a pellet form produced by PT Chaeron Pokphand Indonesia, Surabaya. Reagent that has been used for this experiment was the 2-methoxyethanol produced by Wako Company, Japan. 0.2ml of the 200mg/kg 2ME was injected subcutaneously at the dorsal neck. The rats were injected with 2ME for 6 days in a week within 2 weeks and the control group were injected with only physiological electrolytes. This experiment was done at the same time for both groups. After the experiment was over, the samples were anesthetised with chloroform to perform a necropsy where the spermatozoa were taken from the caudal epididymis and vas deferens. The spermatozoa were collected by using methods modified from Haila (2001) and Goyal et al (2001). The caudal epididymis and vas deferen were taken from each group from the testes. Subsequently, the spermatozoa were placed in Petri dishes containing 4ml of physiological substances. The sperms were collected by flushing method using stereo

microscope, and syringe consisting of 1ml physiologies substances was injected into the hole of vas deferens. Later, the syringe was injected slowly until the substance is able to push the sperms, so that the spermatozoa suspension was ready to be fixed. An electron microscope transmission was used to observe the ultrastructures. The suspension containing the spermatozoa was fixed with glutaraldehyde in a buffer (0.2 M Na3PO4, pH 7.4). The second fixation was applied with osmium tetra oxide 1% in a buffer. The dehydration and cleansing stages were done to get rid of the remaining osmium tetraoxide by soaking the preparations with ethanol 30%, 50%, 70%, 80% and 90% for 10 minutes in a room temperature and left them overnight. After the agar of 1 – 2% was heated under the temperature of 4000c - 5000c, the spermatozoa were concealed into the agar. Afterwards, the agar was cut and processed again by dehydrating it 2-3 times with ethanol 30%, 50%, 70%, 80% and 90%. Impregnation (in water-free propylene oxide) was done twice for 10 minutes in a room temperature and it was done again in a propylene oxide : eponmix = 1:1, where it was left overnight or for 16 hours in a room temperature. The following procedure was block embedding process. After the process was accomplished, the block was sliced by using ultra microtome, 1µm, and it was observed with an inverted microscope. The slice had to be as thick as 90nm, and toluidine blue was applied to ensure the thickness. The process was then followed by a grading stage. After the grading has been dried off, then the samples were observed with an electron microscope transmission (Hoediasmoro et al. 1985). RESULTS The 2ME exposures caused differences in ultrastructures between the tested rats and the control group. Under the magnification of 25000 times, the spermatozoa plasma membrane of the posterior head appeared to be in good physical shape for the control group (figure 1A). However, for the tested group, the middle head of the plasma membrane seemed to be damaged, where it appeared lysis and discontinuous (figure 1B). The horizontal and vertical views at the midpiece of plasma membrane and mitochondria for the control group appeared to be in perfect structures (figure 2.I.A and 2.II.A), whereas for the tested group, the plasma membrane seemed to be swollen and the mitochondria was vacuolated (Figure 2.I.B and 2.II.B).

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Figure 1. The rats’ spermatozoa morphology and the horizontal view of the electron microscope transmission from the

head of the rats’ spermatozoa. A) control group, the posterior head of spermatozoa, perfect shape of plasma membrane. B) tested group, the middle piece of the spermatozoa head, lysis and discontinuous plasma membrane; mp, plasma membrane; n, nucleous; thickness of 60nm; uranil acetat stain; magnification 25000 x.

DISCUSSION Spermatozoa, unlike other cells, have specific structures and functions and it is also sensitive to the lipid peroxidization. This process, besides damaging the plasma membrane, it can also trigger the destruction of DNA and protein. It can also induce oxidization towards the SH group of the protein, causing changes of the spermatozoa structures and functions. In this research, it was shown that there were damaged structures of the spermatozoa plasma membrane and mitochondria due to the length of the exposure, which was 6 days per week for 2 weeks. The horizontal and longitudinal views of the head or the middle piece showed impairments of the plasma membrane clearly. This damage was due to the 2ME exposure that could cause stress oxidization of the spermatozoa, affecting the lipid peroxidization to increase. This caused the unsaturated fatty acids chains that coated the cell membrane to break up, thus, producing a toxic

compound (MDA) (WHO, 2002). This reaction affects the functions and structures of the spermatozoa, as a result, changing the membranes’ permeability (Timbrell 1994). The increased permeability of the plasma membrane caused the Ca2+ influx to increase and blocked the efflux. Consequently, the free Ca2+ increases in cytosol. This will lead to cytoskeleton changes and activate several enzymes, thus, causing an intense damage of the plasma membrane. Besides that, at the middle piece, vacuolated mitochondria could be seen. Vacuolisation occurred due to the space that filled in the mitochondria. One of the factors that cause vacuolisation is as a result of the damaging mitochondria membrane due to lipid peroxidization by 2-ME. Therefore, the membranes’ permeability was decreasing, causing molecules from the cytoplasm to penetrate into the mitochondria. This condition could probably be the reason of the injured organelles and the deteriorating mitochondria activities. There was a positive correlation between the mitochondria activities and the spermatozoa motility (Pesini et al. 1998).

200 nm

200 nm

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A mp

mp

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Figure 2. The rats’ spermatozoa morphology and I) horizontal view; II) vertical view from the electron microscope

transmission of the rats’ spermatozoa midpiece. IA and IIA) control group, undamaged plasma membrane, normal mitochondria and microtubules; IB and IIB) tested group, bloated plasma membrane and vacuolated mitochondria; mp, plasma membrane; m, mitochondria; df, thick fiber; 60 nm; uranil acetate stain; magnification 25000 x.

CONCLUSION In conclusion, 2ME exposure could damage the plasma membrane and mitochondria of the rats’ spermatozoa, resulting in decrease of its quality.

ACKNOWLEDGMENT First and foremost, we would like to thank the Third Batch of the Committee DUE-Like Project of Airlangga University for the generous contribution to support this research. Also, for the students (Jean, Mustaeja, Ratri, Ulul, Ergina, Yuyun, and Pepe) who have dedicated their time and energy to make this research a success.

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