The effect of the antioxidant sesamol on ultraviolet radiation induced damage in Drosophila melanogaster: observing fertility and larval development

The effect of the antioxidant sesamol on ultraviolet radiation induced damage in Drosophila melanogaster: observing fertility and larval development ...
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The effect of the antioxidant sesamol on ultraviolet radiation induced damage in Drosophila melanogaster: observing fertility and larval development

Simona Erikkilä Helsingin Suomalainen Yhteiskoulu Biology

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Abstract

The role of ultraviolet radiation in the etiology of cancer has been well established. Its capacity to generate reactive oxygen species (ROS) within cells and induce oxidative stress, causing damage to vital macromolecules, essentially contributes to the classification of ultraviolet radiation as an environmental carcinogen. The highly reactive oxygen radicals, characterized by an instable open shell configuration, initiate biochemical cascades causing damage to nucleic acids, lipids, and proteins. Organisms have their own defense mechanisms such as endogenous antioxidants, which prevent or slow down the process of oxidation by pairing with the single paired electrons in the outer shell of free radicals. However, exposure to ultraviolet radiation can significantly increase the level of ROS production, causing oxidative stress to the extent where the cell’s own defense mechanisms cannot sufficiently cope with the damages. Thus, antioxidant supplementation may play a vital role in providing protection against oxidative stress, induced by such exogenous sources as UV. The aim of this investigation was to assess the radioprotective efficacy of an antioxidant called sesamol – a phytochemical derived from sesame seed lignans. Sesamol’s radioprotective potential has been evaluated to some extent only very recently and thus sufficient in-vivo experimentation is still lacking. This research was conducted on Drosophila melanogaster. The effect of sesamol on fertility and larval development following UV-C (254 nm) exposure was assessed. The results of the experiment indicate that following UV irradiance, there is no statistically significant difference in the length of larval development and rate of eclosion for D. melanogaster grown in sesamol and control growth mediums. Also, no significant difference in fertility of the sesamol and control parent generation was observed. Several suggestions to improve the investigation were made to enable more extensive analysis of future results.

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Tiivistelmä

Ultraviolettisäteilyn on todistettu edistävän syövän syntyä ja näin ollen se on luokiteltu ympäristökarsinogeeneihin.

Altistuminen

UV-säteilylle

saattaa

johtaa

reaktiivisten

happimolekyylien (ROS) tuotantoon soluissa, mikä voi puolestaan aiheuttaa vaurioita elintärkeille makromolekyyleille.

Epävakaan

avoimen

elektronikuorikonfiguraation

vuoksi

reaktiiviset

happimolekyylit aiheuttavat biokemiallisia reaktioita, vahingoittaen esimerkiksi nukleiinihappoja, lipidejä ja proteiineja. Soluilla on omia puolustusmekanismeja, esimerkiksi endogeenisia antioksidantteja, jotka ehkäisevät tai hidastavat hapettumista sitoutumalla vapaiden radikaalien parittomien elektronien kanssa atomiorbitaalissa. Säteilyaltistus saattaa kuitenkin nostaa merkittävästi ROS-tuotantoa, joka voi johtaa oksidatiiviseen stressiin, jolloin solun omat puolustusmekanismit eivät riitä ehkäisemään säteilyn tuottamaa vahinkoa. Näin ollen antioksidantit voivat mahdollisesti tarjota suojaa oksidatiivista stressiä vastaan, joka voi syntyä sellaisista eksogeenisista lähteistä niin kuin UV-säteily. Tämän tutkimuksen tavoite oli arvioida tietyn antioksidantin, sesamolin, vaikutusta UV-säteilyn aiheuttamien vaurioiden syntyyn Drosophila melanogasterissa. Sesamol on seesamsiemenistä saatu fytokemikaali, jonka mahdollisuutta tarjota säteilysuojaa on arvioitu osin vasta äskettäin ja näin ollen oleellista tietoa sen toiminnasta, varsinkin in-vivo –olosuhteissa, vielä puuttuu. Sesamolin vaikutus D. melanogasterin hedelmällisyyteen ja toukan kehitykseen arvioitiin UV-C (254 nm) säteilytyksen jälkeen. Tulokset osoittavat, että toukkien kehityksen pituudessa ja kuoriutumisessa ei ollut tilastollisesti merkittävää eroa sesamol- ja kontrollibanaanikärpästen välillä. Tämän lisäksi myöskään hedelmällisyydessä ei havaittu merkittävää eroa. Lopuksi työssä ehdotettiin useita parannuksia, joiden avulla voidaan mahdollistaa tulevien tulosten laajempi analyysi.

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Acknowledgments

I wish to thank the following people for their valuable help and contribution to this investigation. Hanna-Leena Nikkinen for her supervision, support and guidance. Elina Näsäkkälä for the time she dedicated to help me find the necessary UV lamp and for acquiring the chemical studied in this investigation. Prof. Reija Jokela from the Helsinki University of Technology for letting me use their UV-C lamp.

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Table of Contents

1. Introduction......................................................................................................................

1

1.1. Metabolism of oxidative stress and reactive oxygen species.....................................

2

1.2. Role of antioxidants in oxidative stress......................................................................

4

1.3. Effect of ultraviolet light and oxidative stress on reproduction and development.....

4

1.4. Aim of the current investigation.................................................................................

5

2. Materials and methods....................................................................................................

6

2.1. The fruit fly Drosophila melanogaster.......................................................................

6

2.2. Antioxidant sesamol...................................................................................................

7

2.3. Experimental setup.....................................................................................................

8

3. Results...............................................................................................................................

9

3.1. Fertility.......................................................................................................................

10

3.2. Larval development....................................................................................................

11

4. Discussion.........................................................................................................................

14

5. Conclusion........................................................................................................................

16

6. Bibliography..................................................................................................................... 18 7. Appendix A 8. Appendix B 
 


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1 1. Introduction

Reactive oxygen species (ROS) are naturally produced in low quantities as a by-product of aerobic metabolism in mitochondria.1 As a result of an open shell configuration, these molecules are unstable and readily take part in chemical reactions – oxidizing nucleic acids, proteins, and lipids.2 The damage caused by these chemical intermediates are constantly repaired by the cell and endogenous antioxidants are used as a defense mechanism to keep the ROS level at a minimum.3 However, this balance can be disrupted by a number of factors, such as heat4, chemical agents5, and radiation6, inducing oxidative stress in an organism. An imbalance is created between the production of ROS and the cell’s ability to neutralize the chemical intermediates. Prolonged exposure to high levels of oxidative stress may lead to severe cellular damage and mutagenesis7. Ultraviolet radiation is an established environmental carcinogen due to the resulting oxidative damage. It has been generally accepted that of the ultraviolet radiation that reaches the earth’s atmosphere, UV-B (280-320 nm) imposes the greatest health hazard, while UV-C (200-280 nm), which is filtered by the ozone layer8, represents the most damaging part of the UV spectrum. Both UV-B and UV-C are capable of inducing mutagenesis due to their potential to directly oxidize DNA9 and cause indirect damage through the production of exogenous ROS in cells. The free oxygen radicals are effectively generated by such mechanisms as radiolysis of water, which can initiate chains of biochemical reactions, most notably causing lipid peroxidation, protein oxidation or further DNA damage.

























































 1

Thiele, J. and Elsner P. (2001), 117.

2

Karbowik, M. and Reiter, R. J. (2000), 9. Sen, C. K. et al. (2000), 614. 4 Zhang, H. J. et al. (2003), 2293. 5 Shi, Y. et al. (2008), 19. 6 Ibid. 7 Rhodes, C. J. (2000), 328. 8 However, extensive depletion of the stratospheric ozone layer, may present with a potential threat where UV-C could also reach the earth’s surface (Väkevä, L. 2006). 9 Pastila, R. (2006) 3

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2 Growth abnormalities have been reported in various species, particularly in arthropods and amphibians, exposed to high levels of ambient UV. 10 11 The changes are thought to be the result of the UV generated free oxygen radicals interacting with DNA regulation, or directly with DNA and proteins.12 While only limited research has been conducted to assess the effect of narrow-spectrum UV on fertility, it has been, nevertheless, established that direct irradiation of cells of the germ line may decrease the rate of fertilization.13 Therefore, further research for finding means by which UVinduced damage could be prevented is of great value and importance.

1.1.

Metabolism of oxidative stress and reactive oxygen species

In aerobic organisms, metabolism can occasionally produce reactive oxygen species (ROS) as a byproduct. Such ROS are produced inside the mitochondria and can be released into the cytosol14, causing damage to the mitochondrial membrane and proteins. Persistently elevated levels of ROS can lead to oxidative stress, meaning a rise in cellular reduction potential, which in turn can cause further damage to important cellular macromolecules. Ultraviolet radiation can significantly increase the ROS production, causing oxidative stress in an organism. This in turn can initiate a cascade of biochemical reactions. One of the common mechanisms by which the ROS are produced in cells by ultraviolet radiation is through the radiolysis of water,15 resulting in a self-perpetuating reaction where the main end products are a proton (H•) and a hydroxyl radical (OH•). These radicals will readily react with other molecules, however they can also react with each other giving rise to new oxygen radicals. While the two protons will combine to form harmless hydrogen gas, the addition reaction of two OH• will generate hydrogen peroxide (H2O2), a molecule that can induce further cellular damage.16

























































 10 11

Ovaska K. et al. (1997) Veteli, T. (2003)

12

Ibid. Seaver, R. W. et al. (2009) 14 Han, D. (2001), 411. 13

15 16

Karbowik, M. and Reiter, R. J. (2000), 10. Toivonen, H. et al. (1988)

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3 The free oxygen radicals target most biological molecules and react with them at a very high rate.17 However DNA damage is most significant as it imposes a higher risk of a malignant transformation of a cell, which essentially contributes to the formation of cancer.18 ROS, which interact with DNA, have been shown to play a significant role in both initiation and promotion of multistage carcinogenesis.19 There are three main ways by which OH•, generated by UV, can cause indirect DNA damage: 1) DNA single and double strand breaks can be initiated by the abstraction of a deoxyribose hydrogen atom;20 2) purine or pyrimidine bases of DNA can be oxidized, for example thymidine can be transformed into a hydroxy-peroxide;21 3) abasic sites, meaning the loss of a nucleotide base, arise from the breakage of N-glycosyl bonds. These DNA damages primarily occur through the interaction of the free oxygen radical with DNA bases and a lesser extent with DNA sugars.22 However, apart from DNA, lipids and proteins can also be targeted by free oxygen radicals with extensive consequences. The oxidation of lipids, known as lipid peroxidation, often initiates a chain reaction that leads to the formation of a variety of degradation products. The radicals of lipid breakdown can react with other lipids in biological membranes, degrading membrane fluidity23 and subsequently altering plasma membrane permeability. Other consequences of lipid peroxidation may include carcinogenesis if the end products react with DNA.24 ROS induced damage to proteins usually result in the formation of altered side-chains. The consequences of protein oxidation may include altered gene regulation and expression as well as promotion of apoptosis or necrosis.25 While UV can effectively generate ROS and induce irreversible changes to cellular components, oxidative stress can also cause the depletion of endogenous antioxidants and enzymes26 such as superoxide dismutase, which plays a significant role in cell’s defense mechanism against ROS. If the cell’s ability to defend against free radicals and capacity to repair DNA becomes inefficient, the 























































 17

Ibid.

18

Rhodes, C. J. (2000), 328. 19 Ibid. 20 Balasubramanian, B. et al. (1998), 9738. 21 Acendaño, C. and Menéndex, J. C. (2008), 100. 22 Karbowik, M. and Reiter, R. J. (2000), 10. 23 Arivazhagan, P. et al. (2003) 24 Marnett, L. (1999) 25 26

Davies, M. J. (2003) Vayalil, P. K. et al. (2003)

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4 damages can accumulate, exacerbated by the likelihood that the damaged DNA strands will be replicated. This can alter or eliminate the cell’s ability to transcribe the genes that the damaged DNA encodes. If DNA repair is not facilitated, other consequences may include an irreversible state of dormancy, apoptosis or unregulated cell division, otherwise known as carcinogenesis.

1.2. Role of antioxidants in oxidative stress Reactive oxygen species (ROS) can start complex chain reactions involving the oxidation of important macromolecules. The process of oxidation can be prevented or slowed down by antioxidants. They react with free radicals by pairing with the single paired electrons in the outer shell and neutralize these chemical intermediates. Organisms have their own defense systems such as endogenous antioxidants to keep the ROS level at minimum. However, exposure to ultraviolet radiation can significantly increase the level of ROS production and can cause oxidative stress to the extent where the cell’s own defense mechanisms are not able to cope with the damages. Thus, as antioxidants can readily neutralize ROS generated by such exogenous source as radiation, antioxidant supplementation may play a vital role in providing protection against oxidative stress.

1.3. Effect of ultraviolet light and oxidative stress on reproduction and development The effect of ultraviolet radiation on various species at different stages of development has been closely studied for the past few decades, particularly due to the increasing ambient levels of UV-B radiation in the atmosphere27. It has been generally shown that UV-B exposure causes physiological abnormalities in amphibians and reduces the survival of larva.28

29

In arthropods, exposure to UV

has been also associated with delayed embryonic and larval development.30

























































 27 28 29 30

Blaustein, A. R. (2004) Ovaska K. et al. (1997) Blaustein, A. R. (2003) Veteli, T. (2003)

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5 During embryonic or larval stage, the cells are particularly susceptible to UV induced damage31 and thus various implications may rise, collectively contributing to delayed development. Cells, which exhibit rapid cell division, such as nerve cells are particularly radiosensitive at initial stages of organism’s development.32 The role of oxidative stress in the inhibition of for example neural precursor cell33 growth has been established.34 This may play a crucial role in the formation of the nervous system during the embryonic stage. Additionally, an investigation evaluating the effect of UV-B irradiance on amphibians concluded that exposing fertilized eggs to UV induces changes to the neural induction system that constitutes the initial step in the generation of a vertebrate nervous system.35 UV irradiation can also suppress the functioning of the immune system by damaging lymphocytes and decreasing hematocrit, plasma protein, and plasma immunoglobulin levels.36 All these factors may essentially contribute to delayed development. With respect to reproduction, limited research has been conducted to assess the effect of radiation, specifically UV, on fertility. It has been, however, established that germ cells are particularly sensitive to radiation exposure, and if subjected to direct irradiance, consequences may include mutagenesis or apoptosis.37 Additionally, a very recent investigation concluded that eggs and sperm of zebra mussels are highly sensitive to UV-B and sperm irradiation decreases the rate of fertilization.38

1.4. Aim of the current investigation The aim of this investigation is to assess to what extent UV induced damage could be prevented with sesamol supplementation, a phytochemical derived from sesame seed lignans. Sesamol’s radioprotective potential has been evaluated to some extent only very recently and therefore 























































 31

Toivonen, Harri. et al. (1988): 408. Ibid. 33 Precursor cells are stem cells, which have developed to the stage where they are committed to form into a specific type of cell. 32

34

Limoli, C. L. et al. (2006) Youn, B. W. and Malacinski, G. M. (1980) 36 Salo, H. M. et al. (2000) 35

37 38

Toivonen, H. et al. (1988) Seaver, R. W. et al. (2009)

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6 sufficient in-vivo experimentation is yet to be done. The research is to be conducted on Drosophila melanogaster – an organism commonly used in comparative experimental research. The effect of sesamol on D. melanogaster fertility following UV-C (254 nm) exposure will be assessed. Whether radiation given to parent generation of D. melanogaster may affect the level of oxidative stress during larval development and whether this stress can be compensated for with the antioxidant sesamol will also be evaluated.

2. Materials and methods

2.1. The fruit fly, Drosophila melanogaster The fruit fly, Drosophila melanogaster, is one of the most commonly used genetic model organisms in biology and has been extensively used in comparative experimental research39. D. melanogaster has a relatively short lifespan and can be easily cultured in a laboratory. The stages in the life cycle of a D. melanogaster involve metamorphosis, the four distinct phases being egg, larva, pupa and adult. As an ectothermic species its life cycle and development time varies with temperature conditions. At 18°C the development takes 4 – 5 weeks.40 Such temperature is often used to maintain stocks. Under ideal conditions at 25°C, which is the optimum temperature for breeding D. melanogaster, the development time takes 8.5 days.41 Generally, the maturation times decrease with increasing temperature, until reaching an optimum. At temperature conditions exceeding 30°C, the development times begin to increase due to heat stress. Other factors that may influence the development and growth of D. melanogaster include the composition of the growth medium, crowding in the culture bottle and humidity. The growth medium is essentially made of malt and semolina, and also includes yeast, agar and antimicrobial agents42.

























































 39 40 41 42

Markow, A. T. and O’Grady, P. M. (2005) Ashburner, M. et al. (2000), 590. Ashburner, M. et al. (2005), 162-4. For the exact composition of the growth medium used in this investigation, see Table B-1 (Appendix B).

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7 2.2. Antioxidant sesamol Sesamol (3,4-methylenedioxyphenol)43 is a natural organic compound that is derived from sesame seed lignans and is a constituent of sesame oil. This white crystalline is a phenol derivative due to its phenyl ring and hydroxyl group. Its capacity to resist oxidative rancidity and ability to prevent the spoilage of oils44 has been ascribed to its antioxidant activity, which may also provide a cell with radioprotection against oxidative damage. However no extensive research, particularly in vivo, has been yet performed to assess the latter. A very recent in-vitro investigation evaluated the photoprotective effect of sesamol using human blood lymphocytes, in which oxidative stress was induced by ultraviolet radiation. The sesamol pretreated lymphocytes showed significantly reduced lipid peroxidation and an increase in natural antioxidant defenses.45 Cancer chemopreventive potential of sesamol has been also assessed to a certain extent. In a research conducted in 2002, sesamol was shown to reduce papillomas in mice skin by 50%, affirming its chemopreventive activity. In this same investigation sesamol’s profound free radical scavenging activity was confirmed.46 Toxicological investigations have shown that sesamol is a nonirritant to the skin and does not cause skin sensitization.47 The physiological activity of the phytochemical has been also evaluated. For instance long-term feeding of sesamol to rats showed no effect on growth, mortality, or blood morphology.48 The sesamol concentration used in the growth medium of Drosophila melanogaster in this investigation is 1.5 mM.

























































 43

For the chemical structure, see Figure B-1 (Appendix B).

44

Kim, J. Y. et al. (2003) Prasad, N. R. et al. (2004) 46 Kapadia, G. J. et al. (2002) 45

47 48

Ambrose, A. M. et al. (1958), 600. Ibid.

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8 2.3. Experimental setup The flies were divided into two groups – ones having sesamol in their growth medium and ones forming the control group. Each group comprised of six culture bottles containing four females and five males. Larval development of the F1 generation and the fertility of the parent generation was observed and compared following the UV-C irradiance of the parent generation. Larval development was assessed by calculating the average larval development time and rate of eclosion, while fertility was evaluated by calculating the average total number of offspring in the growth bottles. Prior to the UV treatment the fruit flies were kept for four days in the corresponding growth bottles to ensure that the group of flies with sesamol in their growth medium had sufficient amount of time to first consume the chemical. During this period and the UV treatment which was to follow, the male and female flies were kept in separate bottles to avoid early mating. Altogether four bottles were exposed to UV. 49 Constrained by time, the female flies were exposed to UV-C (254 nm)50 continuously for three hours, while the males were irradiated for four hours. For the UV treatment the flies were removed from their culture bottles and placed into smaller vials without their growth medium. This was done due to height restrictions imposed by the UV lamp. Two bottles with flies were placed under the lamp at a time and subjected to radiation.51 The openings of the bottles were covered with gauze, thus assuring that the flies were exposed to direct UV. The temperature was periodically monitored to avoid overheating. Following the radiation treatment, the flies were moved to new growth bottles for mating. Sesamol was kept in the diet. After two days the parent generation was removed.

























































 49

The first contained sesamol group males; the second, sesamol group females; the third, control males; and the fourth control group females. 50 51

The source of radiation was a Desaga MinUVIS ultraviolet radiation lamp. For visualization of the set up, see Figure B-2 (Appendix B).


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9 3. Results

The raw data, representing the total number of offspring produced among sesamol and control growth bottles against time in hours after the distribution of irradiated parent flies into corresponding growth bottles for mating, is presented in Figure 3.1. For the tabulated form of the

Number of offspring

raw data, see Table A-1 (Appendix A).

Time in hours

Figure 3.1. The number of offspring produced in the sesamol (circle) and control (square) growth bottles against time – hours after the distribution of irradiated D. melanogaster for mating.

As it can be seen from Figure 3.1., the fluctuation in the number of “sesamol” and “control” offspring emerging from pupa against time follows a similar pattern, illustrating that D. melanogaster raised in sesamol growth medium show the same rate of development as D. melanogaster raised in control growth medium.

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10 3.1. Fertility The fertility of the sesamol and control parent generation Drosophila melanogaster was assessed and compared by calculating the mean number of offspring produced per growth bottle. The results are presented in Table 3.1.1. Table 3.1.1. The mean and standard deviation for the total number of offspring recorded per growth bottle, a comparison between sesamol and control growth mediums.

Total number of offspring Sesamol

Control

Mean

47.8

49.2

Standard deviation

9.78

9.18

Figure 3.1.1. The mean and standard deviation for the total number of offspring recorded in the growth bottles with either sesamol or control growth medium. Error bars represent ± 1 standard deviation.

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11 As the values representing the mean number of offspring produced in the sesamol and control growth bottles are quite close to each other and the standard deviations overlap, illustrating that both sesamol and control flies showed similar variation in their response to the UV treatment, it can be concluded that there is no statistically significant difference between the fertility of D. melanogaster grown in sesamol and control growth mediums.

3.2. Larval development The larval development of Drosophila melanogaster, cultured in sesamol and control growth mediums, was assessed by calculating the mean larval development time and the rate of eclosion, meaning the rate at which D. melanogaster hatched from pupa. While the mean length of larval development is presented in hours, determined with an error of ± 3 h, the rate of eclosion is given by the number of flies emerging from pupa per day. Table 3.2.1. The mean larval development time (hours) and standard deviation according to the growth medium, determined with an error of ± 3 h.

Larval development time (h) ± 3 Sesamol

Control

Mean (h)

249.35

248.06

Standard deviation

17.90

21.10

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12

Figure 3.2.1. The mean and standard deviation for larval development time, a comparison between sesamol and control growth mediums. Error bars represent ± 1 standard deviation.

As it can be seen from Figure 3.2.1, the difference between the mean larval development time (h) for D. melanogaster cultured in sesamol and control growth mediums is small and as the standard deviations once again overlap, it can be concluded that here is no statistically significant difference between the length of larval development of D. melanogaster grown in sesamol and control growth mediums. The mean rate of eclosion, another criterion based on which larval development was assessed, is presented in Table 3.2.2.

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13 Table 3.2.2. The mean and standard deviation for the rate of D. melanogaster eclosion (flies/day), a comparison between sesamol and control growth mediums.

Rate of eclosion (flies/day) Sesamol

Control

Mean

14.90

16.88

Standard deviation

3.05

2.86

Figure 3.2.2. The mean and standard deviation for the rate of eclosion (flies/day), a comparison between sesamol and control growth mediums. Error bars represent ± 1 standard deviation.

Once again it can be seen that there is no statistically significant difference between the D. melanogaster offspring grown in sesamol and control mediums as the mean rate of eclosion for sesamol and control subjects are close to each other and the standard deviations overlap, illustrating a similar response in sesamol and control flies to the UV exposure.

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14 4. Discussion

Based on our data analysis in the section 3.1, it is evident there was no statistically significant difference between the fertility of UV-irradiated Drosophila melanogaster administered with a sesamol included diet and those administered with a sesamol-free, control, diet. Likewise, no considerable difference was apparent in the length of larval development and rate of eclosion for D. melanogaster cultured in sesamol and control growth mediums. As illustrated by Figure 3.1., representing the total number of offspring produced against time (hours), bottles with control growth medium essentially showed a similar pattern of fluctuation in the numbers of total offspring emerging from pupa, as growth bottles with sesamol, indicating that in general sesamol and control D. melanogaster developed at the same rate with no contrasting delays. Considering the results of this investigation, it appears that sesamol does not provide protection against UV radiation induced damage in D. melanogaster since sesamol and control subjects show nearly same kind of variation. However, there are other implications that should be taken into consideration. One of the possible sources of error lies in the method of acquiring the new generation of D. melanogaster for this investigation. The process of collecting flies, soon after eclosion, extended to a few days as not enough flies could be obtained at once. Therefore, some of the flies of the parent generation are older than others, the age difference ranging from of 1 – 3 days. As the flies were also kept for an additional few days in their growth bottles before the UV treatment due to complications with the UV lamp, accordingly the flies became even older. Therefore, particularly those flies who were three days older than others, could have displayed declined interest in courtship as the likelihood that flies will mate decreases with age52. As the mean age distribution of parent flies varied among different bottles, the probability and frequency of mating may also have varied and therefore could have caused certain growth bottles to exhibit to a certain extent a larger offspring population than others. However, it should be noted that as the critical age at which fertility or interest in courtship begins to decline is unclear, it may as well be that the age difference was not significant enough to contribute to a varied rate of offspring production. Nevertheless, as the initial method of collecting flies may present a possible source of error, in further investigations

























































 52

Markow, A. T. and O’Grady, P. M. (2005)

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15 it should be ensured that there are many more growth bottles available at once, with developing D. melanogaster, to collect the flies for the investigation. Another possible source of error could have occurred when the flies were distributed into the appropriate bottles for mating following the UV exposure. Four females and five males were placed into each growth bottle. If it was noticed on the same day that certain flies failed to wake up from the etherization, they were replaced. However on the following day, possibly due to other implications, in several bottles it was observed that one or two male flies had died. Thus, this may have influenced the success of mating to a certain extent as in some bottles there was in the end more females than males. Subsequently, this may have affected the results related to fertility, with certain growth bottles exhibiting a larger offspring population than others. As the imbalanced ratio of males to females may have been a source of possible error, in further similar investigations it would be important to have many more males in relation to females, while keeping the female count equal among the growth bottles to ensure that each growth bottle in theory will produce an offspring population equal in size. However, as there is an equal chance that a female fly might die as well, resulting in the growth bottles having an unequal proportion of females, the experiment should comprise of more growth bottles to have the possibility to discard them in case certain bottles end up having less parent flies than initially intended. With respect to the radiation treatment, it would be important that the female and male flies are given the same radiation dosage. Due to time constraints, the female flies were exposed to UV for only three hours, while the male flies were irradiated for four hours. Thus, the effect of irradiance cannot be equally assessed here. It would also be an asset if a higher radiation dosage would be given to the flies to ensure that sufficiently elevated oxidative stress is induced to cause enough damage with such repercussions as declined fertility and delayed development. Without sufficient UV damage being induced, there would be an imprecision in the evaluation of antioxidant supplementation as the radioprotective potential of the chemical is essentially assessed, not at a molecular level, but indirectly by examining fertility and larval development. In further investigations it would be also essential to expose the larva to direct UV radiation, an approach that this investigation lacked due to time constraints. This is therefore a major source of flaw when assessing the radioprotective efficacy of sesamol during the larval development of D. melanogaster. In addition to carrying out direct irradiation of the larva, it would be vital to expose

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16 D. melanogaster to UV at an early stage of its life cycle – during the embryo or first-instar larva stage when the organism is most vulnerable to radiation exposure. This would ensure that the flies are induced to sufficiently elevated oxidative stress, the importance of which was previously discussed. In the case of fertility, another criterion based on which the radioprotective efficacy of a phytochemical is evaluated, it would be important to expose young female and particularly young male flies to radiation before cuticle darkening in order to acquire more accurate results. The reason for this being that as UV does not penetrate far enough into the tissues53, by exposing D. melanogaster at a stage when the tissues have not yet thickened there is a higher chance that the damage is inflicted to the gonads. Thus we would be able to evaluate more accurately whether sesamol, as a result of its antioxidant activity, may provide protection against UV induced damage to fertility.

5. Conclusion

With the gradual depletion of the stratospheric ozone layer over the years, there has become an increased concern over the implications of elevated levels of ambient ultraviolet radiation as the role of UV in the etiology of carcinogenesis has become well established. A considerable amount of attention has been paid to find various means by which a certain degree of protection against UVinduced damage could be provided. The assessment of the radioprotective potential of antioxidant supplementation has, particularly, become an area of great interest. This investigation aimed at assessing the effect of an antioxidant called sesamol on UV-induced damage in Drosophila melanogaster. The results of the experiment conducted indicate that there is no statistically significant difference between the fertility of the sesamol and control parent generation treated with UV radiation. According to the results the mean number of offspring produced in the growth bottles with sesamol was 47.8, while for the control growth bottles the value was 49.2. The difference is small and taking into account the overlapping standard deviation, 























































 53

Browning, L. S. and Altenburg, E. (1963)

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17 illustrating a similar response in sesamol and control flies to the UV treatment, the difference is negligible. This suggests that with the experimental conditions present in this study, referring to the flaws in the method of irradiance, for instance, that have been discussed, further investigations should be conducted to assess with certainty whether sesamol may provide radioprotection in D. melanogaster. Likewise, no significant difference in the length of larval development and the rate of eclosion for D. melanogaster grown in sesamol and control growth mediums was observed, suggesting that radiation given to the parent generation of D. melanogaster most likely does not affect the level of oxidative stress during larval development of the offspring and therefore further studies where the larva are directly exposed to UV radiation should be conducted in order to conclude what effect sesamol may have on UV-induced damage during larval development of D. melanogaster.

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18 6. Bibliography

Acendaño, Carmen and Menéndex, J. Carlos. Medicinal chemistry of anticancer drugs. Elsevier: 2008. Ambrose, Anthony M. et al. “Antioxidant Toxicity, Toxicological Studies on Sesamol.” Journal of agricultural and food chemistry, vol. 6, Aug. 1958: 600. Arivazhagan, Palaniyappan et al. “Effect of DL-α-Lipoic Acid on the Status of Lipid Peroxidation and Lipids in Aged Rats.” The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, vol. 58, 2003: B788B791. Ashburner, Michael et al. Drosophila protocols. CSHL Press: 2000. Ashburner, Michael et al. Drosophila: A Laboratory Handbook. 2nd ed. Cold Spring Harbor Laboratory Press: 2005. Balasubramanian, Bhavani et al. “DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone.” Proceedings in National Academy of Sciences USA, vol. 95, Aug. 1998: 9738-9743. Blaustein, Andrew R. et al. “Ultraviolet radiation, toxic chemicals and amphibian population declines.” Diversity & Distribution, vol. 9, March 2003: 123-140. Blaustein, Andrew R. “UV-B Radiation.” AmphibiaWeb, 23 March 2004. The Regents of the University of California. 13 Oct. 2009 Browning, Luolin S. and Altenburg, Edgar. “Studies in ultraviolet mutagenesis in Drosophila involving treatment of inseminated females.” Genetics, vol. 50, Oct. 1964: 695-699. “Chemical structure of sesamol.” Illustration. Pubchem database, National Center for Biotechnology Information. 29 Oct. 2009 . Davies, Michael J. “Protein oxidation: concepts, mechanisms and new insights.” The Heart Research Institute, Sydney. 2003. Han, Derick et al. “Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space.” Biochemical Journal, vol. 353, 15 Jan. 2001: 411-416.

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19 Kapadia, G. J. et al. “Chemopreventive effect of resveratrol, sesamol, sesame oil and sunflower oil in the epstein-barr virus early antigen activation assay and the mouse skin two-stage carcinogenesis.” Pharmacological Research, vol. 45, June 2002: 499-505. Karbowik, Malgorzata and Reiter, Russel J. “Antioxidative effects of melatonin in protection against cellular damage caused by ionizing radiation.” Experimental Biology and Medicine, vol. 225, 2000: 9-22. Kim, Joo Yeon et al. “Antiphoto-oxidative Activity of Sesamol in Methylene Blue- and Chlorophyll-Sensitized Photooxidation of Oil.” Journal of agricultural and food chemistry, vol. 51, 2003: 3460-3465. Limoli, C. L. et al. “Altered growth and radiosensitivity in neural precursor cells subjected to oxidative stress” The Journal of Radiation Biology, vol. 82, 2006: 640-647. Markow, Therese Ann and O’Grady, Patrick M. Drosophila: a guide to species identification and use. Academic Press: 2005. Marnett, LJ. “Lipid peroxidation-DNA damage by malondialdehyde.” Mutation research, vol. 424, 8 March 1999: 8395. Ovaska, K. et al. “Hatching success and larval survival of the frogs Hyla regilla and Rana aurora under ambient and artificially enhanced solar ultraviolet radiation.” Canadian Journal of Zoology, vol. 75, 1997: 1081-1088. Prasad, Nagarajan Rajendra et al. “Photoprotective effect of sesamol on UVB-radiation induced oxidative stress in human blood lymphocytes in vitro”. Environmental Toxicology and Pharmacology, vol. 20, July 2005: 1-5. Pastila, Riikka. Effect of long-wave UV radiation on mouse melanoma: an in vitro and in vivo study. Diss. U. of Helsinki, 2006. Rhodes, Christopher J. Toxicology of the human environment: the critical role of free radicals. CRC Press, 2000. Salo, H. M. et al. “Comparative effects of UVA and UVB irradiation on the immune system of fish.” The Journal of Photochemistry and Photobiology B: Biology, vol. 56, July 2000: 154-162. Seaver, R. W. et al. “Effects of ultraviolet radiation on gametic function during fertilization in zebra mussels (Dreissena polymorpha).” The Journal of Shellfish Research, 1 Aug 2009. Sen, Chandan K. et al. Handbook of oxidants and antioxidants in exercise. Elsevier: 2000. Shi, Yimin et al. “Fast repair of oxidative DNA damage by phenylpropanoid glycosides and their analogues.” Mutagenesis, vol. 23, Jan. 2008: 19-26.

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20 Thiele, Jens and Elsner, Peter. Oxidants and antioxidants in cutaneous biology. Karger Publishers: 2001. Toivonen, Harri et al. Säteily ja turvallisuus. Säteilyturvakeskus, Valtion painatuskeskus, Helsinki: 1988. Veteli, Timo. Global Atmospheric Change and Herbivory. Effects of elevated levels of UV-B radiation, atmospheric CO2 and temperature on boreal woody plants and their herbivores. Diss. U. of Joensuu, 2003. Vayalil, P. K. et al. “Treatment of green tea polyphenols in hydrophilic cream prevents UVB-induced oxidation of lipids and proteins, depletion of antioxidant enzymes and phosphorylation of MAPK proteins in SKH-1 hairless mouse skin.” Carcinogenesis, vol. 24, May 2003: 927-936. Väkevä, Liisa. The risk of cancer associated with immunosuppressive therapy for skin diseases. Diss. U. of Helsinki, 2006. Youn, B. W. and Malacinski, G. M. “Action spectrum for ultraviolet irradiation inactivation of a cytoplasmic component(s) required for neutral induction in the amphibian egg.” The Journal of Experimental Zoology, vol. 211, 1980: 369-377. Zhang, H. J. et al. “Heat-induced liver injury in old rats is associated with exaggerated oxidative stress and altered transcription factor activation.” The Journal of the Federation of American Societies for Experimental Biology, vol. 17, 18 Sep. 2003: 2293-2295.

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7. Appendix A

The raw data collected from this investigation is presented in Table A-1. The time represents hours after the parent flies, following the UV treatment, were distributed into corresponding growth bottles for mating. Table A-1. The number of offspring emerging from the pupa in each growth bottle (Nr. 1 – 12) every four hours. A comparison between D. melanogaster grown in sesamol and control growth mediums. The length of the development period of each fly is determined with an error of ± 3 hours.

Number of offspring in the growth bottle Sesamol

Control

Time in

Nr.1

Nr.3

Nr.4

Nr.5

Nr.6

217

3

-

-

-

2

221

4

2

-

2

Total

Nr.7

Nr.8

Nr.10

Nr.11

Nr.12

Total

5

3

2

5

5

-

15

2

10

7

3

9

5

2

26

hours

225

6

-

1

1

1

9

-

-

2

-

-

2

229

-

1

2

-

-

3

-

-

3

-

-

3

233

12

4

13

4

11

44

4

7

13

11

6

41

237

-

-

1

-

6

7

2

1

4

-

1

8

241

5

9

2

2

9

27

7

-

7

3

7

24

245

2

7

2

4

-

15

4

3

-

6

-

13

249

-

8

2

1

-

11

2

2

2

2

10

18

253

-

-

-

1

4

5

-

2

1

-

1

4

257

2

5

3

12

5

27

5

5

2

4

2

18

261

2

4

4

4

9

23

4

3

2

4

3

16

4

4

4

2

15

1

3

1

-

-

5

265

1

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269

-

2

2

3

-

7

-

1

-

4

-

5

273

1

-

1

-

2

4

1

-

1

1

2

5

277

2

3

1

1

4

11

3

1

5

-

7

16

281

1

1

1

-

3

6

-

2

3

1

3

9

285

4

-

-

1

1

6

1

1

3

3

2

10

289

1

1

-

-

2

4

-

2

-

-

3

5

293

-

-

-

-

-

-

1

-

-

2

-

3

Total

46

51

39

40

63

239

45

38

63

51

49

246

Only those bottles which produced more than 30 flies or less than 70 were taken into account. Others were discarded since overcrowding can affect the rate of development and bottles with too few individuals may have been affected by a poor condition of the growth medium. This allows us to assume, with a reasonable degree of certainty that all the flies developed under similar conditions. As it can be seen from the table above, of the twelve growth bottles, only ten were taken into account.

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8. Appendix B

Table B-1. The composition of D. melanogaster growth medium, a recipe for 100 culture bottles.

Ingredient

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Amount

Water

1700 ml

Agar

10 g

Semolina

50 g

Malt

100 g

Yeast

15 g

Propionic acid

11 ml

Nipagin

39 ml

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Figure B-1. The chemical structure of sesamol. Retrieved from the NCBI’s (National Center for Biotechnology Information) Pubchem database. 29 Oct. 2009. .

Figure B-2. UV irradiation set up. The equipment was covered with aluminium foil for personal protection from the UV light. Photograph by the candidate. 14 October 2009.



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