Luís Miguel Torres Cadilhe

Protective activity of plant extracts

against genotoxicity of nitric oxide

Universidade do Minho

Escola de Ciências

Março de 2017

Luís

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2017

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Luís Miguel Torres Cadilhe

Protective activity of plant extracts

against genotoxicity of nitric oxide

Universidade do Minho

Escola de Ciências

Março de 2017

Tese de Mestrado

Mestrado de Genética Molecular

Trabalho efetuado sob a orientação do

Professor Doutor Rui Pedro Soares Oliveira

iii

Acknowledgements

To Prof. Rui Pedro Soares de Oliveira, for the opportunity and pleasure to work under his supervision

in something that incites my complete interest. Thank you for being a concerned supervisor, for your

readiness to answer to all my doubts and curiosities, for all the knowledge I was able to gather

throughout this work and most importantly for the thoughtfulness and the chances to expose my work.

To Prof. Pier Parpot, for his help and expertise regarding the chemical analysis of the extracts. The

insights about the method itself were also an important input.

To all my laboratory partners, for creating such a pleasant environment to work in, for all the moments

of friendship, for all your help and specially, for always sharing your material in the most desperate

times. In other words, thank you for making this the most enjoyable experience so far.

To my friends, for sharing successes, failures and the same concerns as me, for constantly reminding

me of what is at stake and to keep my focus and for all the relaxing moments in our short coffee

breaks.

To my family, for making the physical distance that separates us feel irrelevant, for all your support, not

only during this last year, but also in all the moments of my life, in the good and the less good. Thank

you for your concern, for all the motivation, for all the help and for contributing so much to what I am

today.

v

Protective activity of plant extracts against genotoxicity of nitric oxide

Abstract

A good reputation regarding the use of plant extracts has been growing consistently over the last years,

the potential benefits of their use being heavily supported by the literature. However, as most studies

aspire very little by investigating only their antioxidant activities, a lot of work remains to be done on other

activities, such as antigenotoxicity. Excessive production of oxidative species, such as reactive oxygen

species (ROS) and reactive nitrogen species (RNS), can reveal to be harmful to the cell. Amongst various

targets, these molecules can affect DNA and may lead to the loss of its stability and integrity, imposing a

very dangerous threat to survival. Base excision repair (BER) and homologous recombination (HR) are

two major DNA repair mechanisms of the cell, and are responsible for the repair of several forms of DNA

damage, including base modification and strand breakage.

SNP is a NO-releasing agent and can be used to simulate an excessive increase of NO production

inside the cell. NO may oxidize in many different compounds, depending on the molecules it encounters

within the cell. The most damaging oxidation products include peroxynitrite and dinitrogen trioxide, a very

powerful oxidant and a very strong nitrosating agent, respectively, both being able to directly interact and

modify DNA.

This project purposed to investigate the protective properties of extracts of Dittrichia viscosa (DVE) and

Ginkgo biloba (GBE) against NO-mediated genotoxicity and in what ways protection occurs by using the

Schizosaccharomyces pombe model. In the end, the chemical profiles of DVE and GBE agreed with many

published others, as most of the detected molecules were also detected in those studies. In vitro assays

revealed a relatively (to standard) low IC50 values of DVE in DPPH reduction and iron chelating activity and

GBE in DPPH reduction and NO scavenging. DVE did not provide viability protection against NO-mediated

damage. GBE, however, was able to protect the studied BER and HR Sch. pombe mutants. Additionally,

treatment with GBE alone significantly induced a quicker progression of cell cycle and slightly attenuated

the delay produced by SNP treatment. Both extracts appear to activate oxidative-stress response through

Pap1, although the prevention of oxidative stress by SNP was not achieved in either cases. In conclusion,

GBE protection appears to be independent of BER and it may result from a combination of effects

including the scavenging of NO, the generated ROS and RNS by flavonoids and the activation of oxidative-

stress response proteins.

vii

Actividade protectora de extractos vegetais contra a genotoxicidade do óxido nítrico

Resumo

Nos últimos anos, tem-se vindo a construir uma boa reputação no que toca ao uso de extractos

vegetais, sendo os seus potenciais benefícios fortemente suportados pela literatura. No entanto, a maioria

dos estudos aspira a muito pouco ao investigar apenas as suas actividades antioxidantes, pelo que há

muito trabalho a ser feito em relação a outras actividades, como a antigenotoxicidade. A produção

excessiva de espécies oxidativas, como as espécies reativas de oxigénio (ROS) e de nitrogénio (RNS),

podem revelar-se prejudicial à célula. Entre os seus vários alvos, estas moléculas podem afectar o DNA,

podendo levar à perda da sua estabilidade e integridade, impondo assim uma grave ameaça à

sobrevivência. A reparação por excisão de bases (BER) e a recombinação homóloga (HR) são dois dos

principais mecanismos de reparação de DNA da célula e são responáveis pela reparação de vários tipos

de danos no DNA, incluindo modificações de bases e quebras de cadeia.

O nitroprussiato de sódio é um agente que liberta NO e pode ser utilizado para simular um aumento

excessivo da produção de NO por parte da célula. O NO é capaz de oxidar em muitos compostos

diferentes, dependendo das moléculas que encontra dentro da célula. Os produtos de oxidação mais

prejudiciais são o peroxinitrito e o trióxido de dinitrogénio, um poderoso oxidante e um forte agente

nitrosante, respectivamente, sendo ambos capazes de interagir e modificar directamente o DNA.

Este projecto pretendeu investigar as propriedades protectoras de extractos de Dittrichia viscosa (DVE)

e Ginkgo biloba (GBE) contra a genotoxicidade do NO e de que forma essa protecção ocorreria,

recorrendo ao modelo de Schizosaccharomyces pombe. No fim, os perfis químicos de DVE e GBE foram

semelhantes a muitos outros publicados, pelo que a maioria das moléculas detectadas foram também

detectadas nesses estudos. Os ensaios in vitro revelaram valores de IC50 relativamente (ao padrão) baixos

para DVE na reducção do DPPH e na actividade quelante do ferro e para GBE na reducção do DPPH e

na eliminação do NO. DVE não proporcionou protecção da viabilidade contra danos induzidos pelo NO.

GBE, no entanto, foi capaz de proteger estirpes de Sch. pombe mutantes na BER e HR. Ainda, o

tratamento apenas com GBE induziu uma progressão significativamente mais rápida do ciclo celular e

atenuou ligeiramente o atraso produzido pelo tratamento com SNP. Ambos os extractos pareceram

activar a resposta a stress oxidativo através da Pap1, embora a prevenção do stresse oxidativo induzido

pelo SNP não tenha acontecido em nenhum dos casos. Concluindo, a protecção pelo GBE parece ser

independente da BER e deve resultar de uma combinação de efeitos, incluindo a eliminação do NO, dos

ROS e dos RNS gerados por parte dos flavonóides e a activação de proteínas de resposta a stress

oxidativo.

ix

Table of Contents

Acknowledgements ................................................................................................................. iii

Abstract ................................................................................................................................ v

Resumo .............................................................................................................................. vii

Abbreviations and Acronyms ..................................................................................................... xi

List of figures ...................................................................................................................... xiii

List of tables ...................................................................................................................... xvii

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

1.1. DNA damage and repair ............................................................................................. 1

1.2. Nitric oxide biochemistry ............................................................................................. 7

1.3. Dittrichia viscosa (L.) Greuter and Ginkgo biloba (L.) .........................................................10

1.4. Schizosaccharomyces pombe .....................................................................................12

1.5. Scientific problem and objectives .................................................................................14

2. Materials and Methods .................................................................................................15

2.1. Yeast strains, media, and growth conditions ...................................................................15

2.2. Plant material and extracts preparation .........................................................................15

2.3. Electrospray Ionisation-Tandem Mass Spectrometry (ESI-MS/MS) ........................................16

2.4. In vitro assays .........................................................................................................16

2.5. Viability assays ........................................................................................................18

2.6. Cell cycle ...............................................................................................................19

2.7. Fluorescence microscopy ...........................................................................................20

3. Results .....................................................................................................................21

3.1. The chemical composition of DVE and GBE is similar to that of other described extracts ............21

3.2. DVE and GBE showed different in vitro activities ..............................................................24

3.3. GBE significantly protected viability loss caused by SNP ....................................................24

3.4. GBE alone induces quicker progression in cell cycle and slightly attenuates SNP-induced delay ...29

3.5. Both extracts induce relocalization of oxidative-stress response proteins ................................32

4. Discussion .................................................................................................................37

5. References ................................................................................................................43

6. Appendix ...................................................................................................................61

xi

Abbreviations and Acronyms

3’-dRP 3’-deoxyribose phosphate

3’-P 3'-phosphate

5’-dRP 5’-deoxyribose phosphate

5’-P 5'-phosphate

6-4PP 6-4 photoproducts

AP Apyrimidinic/apurinic

ATP Adenosine 5'-triphosphate

BER Base excision repair

CDK Cyclin-dependent kinases

CPD Cyclobutane pyrimidine dimer

DEA-NO Diethylamine NONOate

DNA Deoxyribonucleic acid

DPPH 2,2-diphenyl-1-picrylhydrazyl

DSB Double-strand break

DSBR Double-strand break repair

DVE Dittrichia viscosa extract

EDTA Ethylenediaminetetraacetic acid

EMM Edinburgh minimal medium

eNOS Endothelial nitric oxide synthase

GBE Ginkgo biloba leaf extract

GG-NER Global-genome NER

HJ Hollidayjunction

HR Homologous recombination

HU Hydroxyurea

IC50 Inhibition concentration 50

ICA Iron chelating activity

iNOS Inducible nitric oxide synthase

IUCN International Union for Conservation of Nature

MMR Mismatch repair

NED N-1-napthylethylenediamine dihydrochloride

xii

NER Nucleotide excision repair

NHEJ Non-homologous end joining

NMMA NG-monomethyl-L-arginine

nNOS Neuronal nitric oxide synthase

NOS Nitric oxide synthase

OD Optical density

PCNA Proliferating cell nuclear antigen

PI Propidium iodide

RFC Replication factor C

RNA Ribonucleic acid

RNS Reactive nitrogen species

ROS Reactive oxygen species

SDSA Synthesis-dependent strand annealing

SLE Systemic lupus erythematosus

SNP Sodium nitroprusside

SPER-NO Spermine NONOate

SSB Single-strand break

TC-NER Transcription-coupled NER

UV Ultraviolet

UVB Ultraviolet-B

UVER Endonuclease-dependent excision repair

YD Yeast Extract Dextrose

YES Yeast Extract with Supplements

xiii

List of figures

Figure 1 – DNA damage and respective repair pathways in Sch. pombe. Mismatched bases (except C/C) and

loops that occur during replication and recombination are mainly repaired by mismatch repair pathway. Nucleotide

excision repair and endonuclease-dependent excision repair pathways are assembled to remove bulky DNA adducts

caused by exposure to UV radiation; these two pathways have also been associated with other types of DNA damage

(dashed arrows). Base excision repair is the most competent pathway to repair non-bulky modifications that occur

from base modifications or incorporation of modified bases. Finally, homologous recombination and non-

homologous end joining repair double-strand breaks. From Egel, 2004. ................................................... 1

Figure 2 – Base excision repair (BER) pathway in Sch. pombe. Apyrimidinic/apurinic sites are primarily repaired

through short-patch BER (thicker arrows): Nth1 initiates repair by incising the AP site, which results in the formation

of 3 -dRP and 5 -P ends; the blocked 3’-end is converted in a 3’-OH by Apn2; DNA polymerase and DNA ligase fill

the gap and seal the nick, respectively. In long-patch BER, Apn2 is the initiator and incises the AP site, leaving a

5’-dRP end; DNA polymerase synthesizes 2-8 new nucleotides and displaces the damage-containing

oligonucleotide, which is cleaved by Rad2; DNA ligase completes repair by sealing the nick. Besides synthesizing

DNA, Pol4 is able to cleave 5’-dRP, only needing DNA polymerase and DNA ligase to complete the process. The

AP endonuclease independent repair pathway is initiated by Nth1 and followed by Tdp1, which cleaves 3’-dRP,

leaving a 3’-P to be processed by Pnk1 and completed by DNA polymerase and DNA ligase. From Nilsen et al.,

2012. ...................................................................................................................................... 3

Figure 3 – HR comprises SDSA, DSBR and Mus81-dependent pathways. In SDSA, the distension of the D-loop

follows strand invasion, allowing DNA synthesis. The new DNA strand is displaced from the template and returned

to the damaged molecule. DSBR pathway, proposed by Szostak et al. (1983), also involves D-loop distention,

leading to the capture of a second 3’ overhang and formation of a double Holliday junction (HJ). The resolution of

HJ results in gene conversion, with or without associated crossovers. In the last pathway, Mus81 cleaves the

invaded DNA strand disrupting the D-loop. The second 3’ overhang is captured and DNA is synthesized. Lastly,

Mus81 cleaves the strand of DNA keeping the homologs together, resulting in crossover of the strands. Edited from

Raji and Hartsuiker, 2006. ............................................................................................................ 5

Figure 4 – DNA damage checkpoint in Sch. pombe. Upon sensing of DNA damage and signal transduction,

Chk1 indirectly induces the delay of cell cycle by phosphorylating Wee1 and Cdc25. Wee1 and Mik1 phosphorylate

Y15 of Cdc2, resulting in its inactivation. Cdc25, on the other hand, dephosphorylates Cdc2 to activate it and

subsequently, induce mitosis. From Baber-Furnari et al., 2000. ............................................................. 6

xiv

Figure 5 – Chemical structure of quercetin. The three main features required for the antioxidant activity of

flavonoids are the ortho-dihydroxy structure in the B ring which facilitates electron delocalization (A), 2,3-double

bond in conjugation with the 4-oxo(=O) function in the C ring to allow electron delocalization from the B ring (B)

and the hydroxyl groups at positions 3 and 5, which allow binding of hydrogen to the oxo group (C). From

Procházková et al., 2011. ............................................................................................................11

Figure 6 – Viability of 972, Δchk1 and Δrhp51 Sch. pombe cells when cultured on solid medium incorporated

with (from left to right): water (negative control), 750 µg/mL DVE, 1 mM SNP and both 750 µg/mL DVE and 1

mM SNP. Cells were diluted up to 10–4 before pipetting onto the plates (indicated at the top of the photos). The

plates were incubated for 4 days at 30 °C. The photographs are representative of three independent experiments.

.............................................................................................................................................25

Figure 7 – Viability of 972 and Δchk1 Sch. pombe cells when incubated for 0, 30, 60 and 90 min in liquid

medium containing (from left to right): water (negative control), 750 µg/mL DVE, 300 mM SNP and both 750

µg/mL DVE and 300 mM SNP. Cells were diluted up to 10–4 before pipetting onto the plates (indicated at the top

of the photos). The plates were incubated for 4 days at 30 °C. The photographs are representative of three

independent experiments. ............................................................................................................26

Figure 8 – Viability of 972, Δsty1 and Δpap1 Sch. pombe cells when pre-incubated in 750 µg/mL DVE-

containing liquid medium and cultured in medium incorporated with (from left to right): water (negative control), 1

mM SNP and both 750 µg/mL DVE and 1 mM SNP. Cells subjected to pre-treatment without DVE are signalized

by (-) and with DVE by (+). Cells were diluted up to 10–4 before pipetting onto the plates (indicated at the top of the

photos). The plates were incubated for 4 days at 30 °C. The photographs are representative of three independent

experiments. ............................................................................................................................27

Figure 9 – Viability of 972 Sch. pombe cells when incubated for 0, 30, 60, 90 and 120 min in liquid medium

containing (from left to right): ethanol (negative control), 300 mM SNP and 1000 µg/mL GBE and 300 mM SNP.

Cells were diluted up to 10–4 before pipetting onto the plates (indicated at the top of the photos). The plates were

incubated for 4 days at 30 °C. The photographs are representative of three independent experiments. ..........27

Figure 10 – Viability of FO656, Δmag1, Δrhp51, Δnth1 and Δrhp55 Sch. pombe cells when cultured on solid

medium incorporated with (from left to right): ethanol (negative control), 1000 µg/mL GBE, 1 mM SNP and both

1000 µg/mL GBE and 1 mM SNP. Cells were diluted up to 10–4 before pipetting onto the plates (indicated at the

top of the photos). The plates were incubated for 4 days at 30 °C. The photographs are representative of three

independent experiments. ............................................................................................................28

xv

Figure 11 – Viability of 972, Δchk1, Δsty1 and Δpap1 Sch. pombe cells when cultured in solid medium

incorporated with (from left to right): ethanol (negative control), 1000 µg/mL GBE, 1 mM SNP and both 1000

µg/mL GBE and 1 mM SNP. Cells were diluted up to 10–4 before pipetting onto the plates (indicated at the top of

the photos). The plates were incubated for 4 days at 30 °C. The photographs are representative of three

independent experiments. ............................................................................................................29

Figure 12 – Cell cycle progression analysis of Sch. pombe cells under the different treatments. Before analysis,

the cells were treated with hydroxyurea (HU) in order to synchronize their cell cycle and were compared with an

asynchronous culture (top left). After washing HU, the cells were submitted to their respective treatments: water

(negative control), 750 µg/mL DVE, 1 mM SNP and both 750 µg/mL DVE and 1 mM SNP. Every 30 min, a sample

of each treatment was taken and processed for analysis. During the treatments, the cells were incubated at the

standard conditions of 30 °C, 200 rpm. ..........................................................................................31

Figure 13 – Microphotographs of fluorescence microscopy of sty1::GFP transformed Sch. pombe after 0, 10

and 60 min of incubation in liquid minimal medium containing (from left to right): water (negative control), 750

µg/mL DVE, 4 mM SNP and both 750 µg/mL DVE and 4 mM SNP. The photographs are representative of three

independent experiments. Magnification: 1000x. ...............................................................................33

Figure 14 – Microphotographs of fluorescence microscopy of of pap1::GFP transformed Sch. pombe after 0,

10 and 60 min of incubation in liquid minimal medium containing (from left to right): water (negative control), 750

µg/mL DVE, 4 mM SNP and both 750 µg/mL DVE and 4 mM SNP. The photographs are representative of three

independent experiments. Magnification: 1000x. ...............................................................................34

Figure 15 – Microphotographs of fluorescence microscopy of of pap1::GFP transformed Sch. pombe after 0,

10 and 60 min of incubation in liquid medium containing (from left to right): water (negative control), 1000 µg/mL

GBE, 4 mM SNP and both 1000 µg/mL GBE and 4 mM SNP. The photographs are representative of three

independent experiments. Magnification: 1000x. ...............................................................................35

xvii

List of tables

Table 1 – Genotypes of the strains used in this study. ....................................................................15

Table 2 – Complete list of the tentatively identified compounds for DVE. .............................................21

Table 3 – Complete list of the tentatively identified compounds for GBE. Unidentified compounds that were also

detected in other studies have (?) as their identification. ......................................................................22

Table 4 – Values of IC50 (µg/mL) determined for DVE, GBE and standards in DPPH reduction, ICA, and NO

scavenging assays. ....................................................................................................................24

1

1. Introduction

1.1. DNA damage and repair

DNA damage is any chemical or physical alteration in DNA, such as base modification, helix breakage,

adduct formation, among others. Any chemical agent able to cause any sort of DNA damage is termed

as genotoxic. However, DNA damage does not arise exclusively from this kind of agents, since it may also

be spontaneously generated within the cell during normal metabolism (Roger et al., 2006). The

consequences that arise from DNA damage range between point mutations in the DNA sequence and

chromosomal breakage, which might easily result in cell death. In case the existence of the several DNA

repair pathways is not enough to prove the importance of this molecule, many researchers have

extensively studied and documented the harmful effects of DNA repair impairments, proving that the

maintenance of genome stability is crucial to survival. There are five major DNA repair pathways (Figure

1): base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous

end joining (NHEJ) and homologous recombination (HR); each one being more adequate than the others

to repair a specific type of DNA damage. These repair pathways are highly conserved among eukaryotes,

as their central components have been identified in many organisms, including Schizosaccharomyces

pombe. In contrast, only a few organisms possess UV-damaged DNA endonuclease-dependent excision

repair (UVER), including Sch. pombe, which, on the other hand, lacks the ability to directly reverse UV

lesions by photolyase (Richard. Egel, 2004).

Figure 1 – DNA damage and respective repair pathways in Sch. pombe. Mismatched bases (except C/C) and loops

that occur during replication and recombination are mainly repaired by mismatch repair pathway. Nucleotide

excision repair and endonuclease-dependent excision repair pathways are assembled to remove bulky DNA adducts

caused by exposure to UV radiation; these two pathways have also been associated with other types of DNA damage

(dashed arrows). Base excision repair is the most competent pathway to repair non-bulky modifications that occur

2

from base modifications or incorporation of modified bases. Finally, homologous recombination and non-

homologous end joining repair double-strand breaks. From Egel, 2004.

The BER pathway (Figure 2) is mainly directed at oxidized and alkylated bases, as well as some types

of mismatched bases that result during replication or through deamination (Kanamitsu & Ikeda, 2010).

BER is initiated by a DNA glycosylase, which recognizes and nicks the N-glycosylic bond between the

damaged base and deoxyribose to release the damaged base, creating an apyrimidinic/apurinic (AP) site

in the process (H E Krokan, Standal, & Slupphaug, 1997; Hans E Krokan & Bjørås, 2013).

Apyrimidinic/apurinic sites can also occur by spontaneous hydrolysis, representing a DNA lesion as well

(Richard. Egel, 2004). AP sites are resolved by cleaving the 5’ phosphodiester bond of the lesion, by

activity of AP endonucleases or by bifunctional DNA glycosylases (both DNA glycosylase and AP lyase

activities). Then, a DNA polymerase β fills the gap and the process is concluded after ligation of the

remaining nick by a DNA ligase; this process comprises the short-patch BER. In long-patch BER, between

2 and 8 nucleotides 3’ of the lesion are removed instead and polymerase or , through DNA synthesis,

create a flap structure, which is processed by flap endonuclease Rad2. Repair by long-patch BER is

completed by DNA ligase (Richard. Egel, 2004). There is also a proposed repair pathway, independent of

AP endonucleases, which is initiated by bifunctional DNA glycosylase nth1p, followed by tyrosyl

phosphodiesterase Tdp1 and concluded by activity of DNA polymerase and DNA ligase (Nilsen, Forstrøm,

Bjørås, & Alseth, 2012).

So far, there is knowledge of about six genes coding for BER glycosylases: ung1, thp1, mag1, mag2,

myh1 and nth1, the latter being the only bifunctional DNA glycosylase in Sch. pombe; and two AP

endonucleases: apn1 and apn2 (Richard. Egel, 2004). Ung1 is suggested to remove misincorporated

uracil, while Thp1 is suggested to repair uracil residues resulting from deamination of cytosine (Richard.

Egel, 2004). Mag1 is directed to repair alkylated bases (Memisoglu & Samson, 1996). Its paralogous,

Mag2 presents high sequence and active site similarity to mag1, however, it was suggested that, alone,

it might stall at the damaged site without tackling the lesion (Adhikary, Cato, McGary, Rokas, & Eichman,

2013). On the other hand, Mag2 appears to contribute in the resistance to MMS-induced damage as

much as Mag1 (Kanamitsu, Tanihigashi, Tanita, Inatani, & Ikeda, 2007). Myh1 is suggested to prevent

mutations by removal of misincorporated adenines from G/A, C/A and 8-oxoG/A mismatches (Lu &

Fawcett, 1998). Lastly, Apn1, is the major AP endonuclease in Saccharomyces cerevisiae but not in Sch.

pombe nor humans, where it serves as back-up for Apn2, the exact opposite of what happens in the

budding yeast model (Ribar, Izumi, & Mitra, 2004).

3

Figure 2 – Base excision repair (BER) pathway in Sch. pombe. Apyrimidinic/apurinic sites are primarily repaired

through short-patch BER (thicker arrows): Nth1 initiates repair by incising the AP site, which results in the formation

of 3 -dRP and 5 -P ends; the blocked 3’-end is converted in a 3’-OH by Apn2; DNA polymerase and DNA ligase fill

the gap and seal the nick, respectively. In long-patch BER, Apn2 is the initiator and incises the AP site, leaving a

5’-dRP end; DNA polymerase synthesizes 2-8 new nucleotides and displaces the damage-containing

oligonucleotide, which is cleaved by Rad2; DNA ligase completes repair by sealing the nick. Besides synthesizing

DNA, Pol4 is able to cleave 5’-dRP, only needing DNA polymerase and DNA ligase to complete the process. The

AP endonuclease independent repair pathway is initiated by Nth1 and followed by Tdp1, which cleaves 3’-dRP,

leaving a 3’-P to be processed by Pnk1 and completed by DNA polymerase and DNA ligase. From Nilsen et al.,

2012.

MMR in not very well understood in Sch. pombe and consequently, its functioning will be resumed for

eukaryotes in general. Repair is initiated by MutS (or MutS ) binding to base-base mismatches and

small loops, following binding of MutL . This ternary complex suffers a conformational switch and

unbounds the sliding clamp from the mismatch site. The clamp may slide upstream or downstream from

the mismatch site and accordingly, specific factors direct repair. EXO1 is recruited to degrade the

mismatch-containing DNA strand and MutL delimits the extension of EXO1 activity. Pol and DNA ligase

I conclude the repair process (Jiricny, 2006).

The NER pathway is specialized on the removal of larger lesions capable of impairing DNA replication

and transcription, and is particularly important in the prevention of damage by UV radiation. UV irradiation

results in cyclobutane pyrimidine dimers (CPDs) and the 6-4 photoproducts (6-4 PPs) (Balajee, 1999;

Cadet, Sage, & Douki, 2005; Roger et al., 2006), the latter being the most mutagenic (Roger et al., 2006).

4

Impairments in this repair pathway in humans is associated to some genetic disorders, including

xeroderma pigmentosum. This disease is characterized by an extreme sensitivity to sunlight and by cancer

predisposition (Roger et al., 2006).

The two sub-pathways that comprise NER are global-genome NER (GG-NER) and transcription-

coupled NER (TC-NER). GG-NER is independent of transcription status and chromatin structure and it

repairs the non-transcribed strand of genes and non-coding sequences in a very slow reaction. TC-NER,

on the other hand, is responsible for the fast damage removal from active genes and is triggered when a

lesion in the transcribed strand blocks RNA polymerase II (Boiteux & Jinks-Robertson, 2013; Richard.

Egel, 2004). Several Sch. pombe NER factors required for damage repair have known homologs in other

organisms, indicating this pathway is similar between eukaryotes (Richard. Egel, 2004).

UV-induced DNA damage may also be repaired through a NER-independent pathway, UVER, in which

Uve1 is the key factor (Bowman et al., 1994; Yonemasu et al., 1997). Uve1 has endonuclease activity,

being able to incise 5’ of CPDs, 6-4PP, AP sites, and less efficiently 1,2 GpG (cisplatin-induced diadduct

between two adjacent guanines) intrastrand cross-links (Avery et al., 1999; Bowman et al., 1994; Yajima

et al., 1995). Some reports state that some base modifications, base-base mismatches and loops are

also substrates of Uve1 (Avery et al., 1999; Kaur & Doetsch, 2000; Kaur, Fraser, Freyer, Davey, &

Doetsch, 1999). After Uve1 incision, Rad2 (partaker in BER) excises the damaged bases, leaving a gap

to be filled in by DNA polymerase, with aid by accessory factors proliferating cell nuclear antigen (PCNA)

and replication factor C (RFC), and sealed by DNA ligase (Richard. Egel, 2004).

DNA-damaging agents may also induce the indirect formation of double-strand breaks (DSB); as BER

removes damaged bases, it creates single-strand breaks (SSB) that if close enough from each other may

turn into a DSB. In addition, if SSB repair fails, a DSB will be produced when encountered by a replication

fork. DSB may be harmful to the cell, as they can lead to the loss of large chromosome fragments and

destabilize genome integrity due to these large-scale deletions (Raji & Hartsuiker, 2006).

Repair of DSB is accomplished through one of two strategies. DSB ends can simply be joint together,

which conduces to incorrect repair in case loss of base pairs have occurred. This strategy corresponds

to NHEJ pathway, and due to this likelihood, it is considered error-prone. The second strategy corresponds

to HR pathway, which really comprises several repair mechanisms, and it involves invasion of the

homologous DNA strand, differing only in the processing of the intermediate structure that forms

afterwards. Strand invasion occurs after nucleolytic resection of DNA, by action of Mre11-Rad50-Nbs1

(MRN) complex, Rad32-Rad50-Nbs1 in Sch. pombe, and Exo1, from which results a 3’ single-stranded

overhang. Rhp51 is able to bind these DNA overhangs to form a nucleoprotein filament and promotes

5

strand exchange with the homologous double-stranded DNA, serving as a primer for DNA synthesis (Raji

& Hartsuiker, 2006). From that point on, HR divides in synthesis-dependent strand annealing (SDSA),

double-strand break repair (DSBR) and mus81p-dependent pathways, each one with a specific

mechanism of repair (see Figure 3 for more details).

Figure 3 – HR comprises SDSA, DSBR and Mus81-dependent pathways. In SDSA, the distension of the D-loop

follows strand invasion, allowing DNA synthesis. The new DNA strand is displaced from the template and returned

to the damaged molecule. DSBR pathway, proposed by Szostak et al. (1983), also involves D-loop distention,

leading to the capture of a second 3’ overhang and formation of a double Holliday junction (HJ). The resolution of

HJ results in gene conversion, with or without associated crossovers. In the last pathway, Mus81 cleaves the

invaded DNA strand disrupting the D-loop. The second 3’ overhang is captured and DNA is synthesized. Lastly,

Mus81 cleaves the strand of DNA keeping the homologs together, resulting in crossover of the strands. Edited from

Raji and Hartsuiker, 2006.

It is noteworthy that Sch. pombe has a very long G2, during which, a sister chromatid is available to

use as a repair template; that is, in an asynchronous culture, HR is the predominant repair pathway

(Manolis et al., 2001; Raji & Hartsuiker, 2006). Being a haploid organism, Sch. pombe, during G1, lacks

6

a repair template, thus HR cannot be performed in this cycle phase (Prudden et al., 2003; Raji &

Hartsuiker, 2006).

The coordination of cell cycle progression plays an important part on the response to DNA damage.

Cell cycle checkpoint pathways monitor the integrity of DNA, preventing the cell from advancing before

completing the previous stage of cell cycle or without repairing damaged DNA (Lehmann, 1996). In

eukaryotes, cell cycle progression is induced by activity of cyclin-dependent kinases (CDKs) (Pines, 1995).

Wee1 and Mik1 negatively regulate cdc2p, the CDK controlling cell progression into mitosis, by

phosphorylating it at tyrosine 15 and, in turn, Cdc25 phosphatase dephosphorylates cdc2p, activating it

(Figure 4) (Lundgren et al., 1991; MacNeill & Nurse, 1997; Millar, McGowan, Lenaers, Jones, & Russell,

1991; Tang, Coleman, & Dunphy, 1993). Upon DNA damage, Wee1 and Mik1 maintain Cdc2

phosphorylation, which led researchers to suggest them to be targets of the DNA damage checkpoint

pathway (Capasso et al., 2002; Kharbanda et al., 1994; O’Connell, Raleigh, Verkade, & Nurse, 1997;

Rhind, Furnari, & Russell, 1997). Chk1 is an essential kinase in the transduction of a delay signal to the

cell cycle machinery upon DNA damage detection (Capasso et al., 2002). Chk1 phosphorylates Wee1and

Cdc25, indirectly blocking entrance in mitosis until DNA repair is completed, which improves survival

following DNA damage (al-Khodairy et al., 1994; O’Connell, Walworth, & Carr, 2000). It has been

demonstrated that cells lacking chk1, besides failing to delay cell cycle progression, have increased

sensitivity to DNA damaging agents (al-Khodairy et al., 1994; Walworth et al., 1993). Thereby, the intricate

association between the mechanisms of DNA repair and cell cycle progression represent a well-organized

system dedicated to preserve the DNA molecule against damaging effects.

Figure 4 – DNA damage checkpoint in Sch. pombe. Upon sensing of DNA damage and signal transduction, Chk1

indirectly induces the delay of cell cycle by phosphorylating Wee1 and Cdc25. Wee1 and Mik1 phosphorylate Y15

of Cdc2, resulting in its inactivation. Cdc25, on the other hand, dephosphorylates Cdc2 to activate it and

subsequently, induce mitosis. From Baber-Furnari et al., 2000.

7

1.2. Nitric oxide biochemistry

NO is a free radical and a signalling messenger (Dawson, Dawson, & Snyder, 1992), having

cytoprotective as well as tumour promoting activity (Habib & Ali, 2011). NO is produced within the cells

through conversion of L-arginine to L-citrulline by nitric oxide synthase (NOS) enzymes. Sch. pombe was

reported for possessing NOS-like activity, however the genes encoding for that function remain unclear

(Astuti, Nasuno, & Takagi, 2016; Kig & Temizkan, 2009). There are three known isoforms of NOS in

mammals: NOS1 or neuronal nitric oxide synthase (nNOS), NOS2 or inducible nitric oxide synthase (iNOS)

and NOS3 or endothelial nitric oxide synthase (eNOS) (Habib & Ali; 2011).

Both nNOS and eNOS are Ca2+ dependent and constitutively expressed, whereas iNOS is Ca2+

independent and is activated by cytokine exposure (Moncada, Palmer, & Higgs, 1991). nNOS is expressed

in the neurons, more precisely, in its post synaptic terminals. It is activated by the influx of Ca2+ upon

binding of the neurotransmitter glutamate to receptors in the membrane and by membrane depolarization

through the opening of Ca2+ channels (Pfeiffer, Mayer, & Hemmens, 1999). eNOS is expressed in the

endothelium of blood vessels. After synthesis, NO diffuses into smooth muscle cells of blood vessels and

induces smooth muscle relaxation resulting in increased blood flow (Habib & Ali, 2011). The last one,

iNOS, is mainly expressed in macrophages and is induced by inflammatory stimuli, e.g. cytokines (Gordge,

1998), and has a very important role in organism defence.

Phagocytic cells, such as macrophages and neutrophils, are the leading peacekeepers regarding

organism immunity. Upon recognition and internalization of a potential pathogen, these cells become

activated and give rise to an antimicrobial response through the synthesis of both reactive oxygen species

(ROS) and reactive nitrogen species (RNS), the latter being synthesized by the inducible nitric oxide

synthase iNOS (Collette, Zhou, & Lorenz, 2014). NO is implicated in various health conditions, such as

ulcerative colitis (Middleton, Shorthouse, & Hunter, 1993), psoriasis (Kolb-Bachofen, Fehsel, Michel, &

Ruzicka, 1994), arthritis and systemic lupus erythematosus (SLE) (Belmont et al., 1997). The latter

disorder is characterized by an immune activated state where iNOS level is increased in macrophages,

splenic and renal tissues, and consequently, NO production is also increased (Weinberg et al., 1994).

The immune activated state promotes induction of iNOS in response to cytokines, which are responsible

for the regulation of immune and inflammatory responses, and the control of the amplitude and duration

of the response (Van der Meide & Schellekens, 1996).

The reactivity of NO mostly relies on its physicochemical properties, such as its small size,

lipophilicity and high diffusion rate (Habib & Ali, 2011). Now, the reactive behaviour of the NO molecule,

due to its unpaired electron, allows it to react with other free radicals, transition metal ions and some

8

biological molecules, since most of the biological molecules have completely filled orbitals making NO

unreactive towards them (Padmaja & Huie, 1993).

NO chemistry involves multiple pathways and processes, such as inhibition of DNA synthesis,

damage to mitochondria, loss of cell membrane integrity, apoptosis, changes in cell cycle and occurrence

of DNA strand breaks (Burney, Caulfield, Niles, Wishnok, & Tannenbaum, 1999). After trespassing the

cell membrane by diffusion, NO can react with non-heme iron or quench tyrosyl radical of ribonucleotide

reductase which may lead to inhibition of DNA synthesis (Kwon, Stuehr, & Nathan, 1991; Roy, Lepoivre,

Henry, & Fontecave, 1995).

NO effects in the mitochondria involve irreversible inactivation of enzymes, reversible inhibition of

respiration and induction of mitochondrial permeability (Murphy, 1999; Sarti et al., 2003). By reversibly

inhibiting the complex IV of the mitochondria, cytochrome c oxidase, leakage of superoxide (O2•) from the

electron transport chain may occur (Cleeter, Cooper, Darley-Usmar, Moncada, & Schapira, 1994).

Other important proteins, such as p53, can be affected by this molecule and initiate programmed

cell death. p53, also referred to as the guardian of the genome, also takes part in the maintenance of

genome stability as exposure of DNA damaging agents cause the rapid increase and accumulation of p53

in the cell (Nicotera, Bonfoco, & Brüne, 1995). The accumulation of p53 precedes DNA fragmentation

and later apoptosis. NO inhibitors such as NMMA (NG-monomethyl-L-arginine) prevent both p53

accumulation and inducible NO generation thus avoiding to apoptosis (Nicotera et al., 1995).

As mentioned previously, the toxic effects of NO mostly involve its oxidation products. The reaction

of NO with superoxide results in peroxynitrite (ONOO-). This molecule may be easily formed in the

mitochondria where superoxide continuously result as a product of respiration. It is a potent oxidant and

can react with and oxidize almost all biological molecules (Pfeiffer et al., 1999). ONOO– inhibits

mitochondrial enzymes, such as complex I, complex II, cytochrome c oxidase, the ATP synthase,

aconitase, Mn-SOD, creatine kinase, and probably many other proteins (Brown, 1999; Cassina & Radi,

1996; Murphy, 1999; Wolosker, Panizzutti, & Engelender, 1996).

The conjugate acid form (ONOOH) can diffuse through membranes and therefore cause damage at

a distance (Marla, Lee, & Groves, 1997). The formation of ONOO– is controlled by the relative amounts of

NO and superoxide, and in the excess of either one of these compounds ONOO– is converted to nitrogen

dioxide (NO2), a stable product (Habib & Ali, 2011).

NO also reacts with molecular oxygen (O2) either in gas or in aqueous phase, leading respectively, to

production of nitrogen dioxide or nitroxyl anion (NO-) and nitrate (NO3-) (Martin N Hughes, 2008).

Ultimately, NO–may still react with O2 to form ONOO– (M. N. Hughes, Nicklin, & Sackrule, 1971).

9

More complex reactions involve the oxidation of catechol-estrogens adducts to quinones which are

able to reduce oxygen to superoxide, or in the presence of NO-releasing compounds, such as sodium

nitroprusside (SNP), diethylamine NONOate (DEA-NO) or spermine NONOate (SPER-NO), lead to

production of ONOO– (Yoshie & Ohshima, 1998). In a similar way, polyhydroxy aromatic compounds,

easily autooxidize into semiquinone radicals to react with dioxygen and generate superoxide, which results

in ONOO– if in combination with a NO-releasing compound (Yoshie & Ohshima, 1997).

Additionally, it has been reported that the quinone derivative of catechol-estrogen, produced by NO-

mediated oxidation, may form adducts with nucleophilic groups of DNA (Dwivedy, Devanesan, Cremonesi,

Rogan, & Cavalieri, 1992). The human uterus and breast are sites of production of catechol-estrogens

(Yager & Liehr, 1996), and consequently, it was suggested a possible mechanism of hormonal

carcinogenesis associated with these organs (Khan, Alam, & Moinuddin, 2007; Liehr et al., 1995; Liehr

& Ricci, 1996). It has been shown that incubation with both NO-donor compound and polyhydroxy

aromatic compounds resulted in strand breakage of DNA (Habib & Ali, 2011).

As it can be noticed already by the examples provided above, NO chemical network involves a

surprisingly wide range of molecules and diverse direct and indirect reactions, evidencing its potential to

disturb the functioning of the cell. The adverse effects of NO do not end here however, as it even affects

DNA stability and its mechanisms of repair. NO exposure leads to deamination of DNA bases, causing

conversion of cytosine to uracil, guanine to xanthine and oxanosine, adenine to hypoxanthine and 5-

methylcytosine to thymine. The deamination process (T. Nguyen et al., 1992; D A Wink et al., 1991) is

caused by N2O3, a powerful nitrosating agent, formed by reaction of NO and molecular oxygen (Lee &

Pfeifer, 2007). Evidence indicates that xanthine is able to base-pair with all four natural DNA bases (Eritja

et al., 1986; Kamiya, Shimizu, Suzuki, Inoue, & Ohtsuka, 1992). Hypoxanthine, on the other hand, is

indicated to preferentially base-pair with cytosine (Vilaivan et al., 2013).

All the mispairing might lead to the transcription of mutant or aberrant proteins and affect the

metabolism of the cell. Furthermore, the instability of both xanthine and hypoxhantine leads to their

depurination and, consequently, single-strand breakage (Lindahl & Andersson, 1972). Amongst all of the

compounds oxidized by ONOO-, DNA bases may too suffer modification, preferentially guanine, producing

8-nitro-2’-deoxyguanosine (8-nitro-dG), which can depurinate and create an AP site; 8-oxo-7,8-dihydro-2’-

deoxyguanosine (8-oxo-dG), 5-guanidino-4-nitroimidazole, and 2,2-diamino-4-[(2-deoxy- -D-erythro-

pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone) (Dedon & Barth, 2007). 8-oxo-dG efficiently

undergoes further oxidation originating several other products with potential to cause G→T transversions,

similarly to 8-nitro-dG, oxazolone and 8-oxo-dG itself (Yermilov, Rubio, & Ohshima, 1995).

10

RNS, in general, have high affinity to amino acids with thiol groups, making them capable of inhibiting

enzymes having these residues (David A. Wink et al., 1994). DNA-alkyltransferase, which repairs O6-

alkylguanine back to guanine, is inhibited by nitrosation of its thiol residue (Ling-Ling et al., 1992; Zak,

Kleibl, & Laval, 1994), and so does, formamidopyrimidine glycosylase, which ends with the removal of

Zn from its zinc finger (D A Wink & Laval, 1994). Another DNA repair enzyme inhibited by RNS is DNA

ligase by nitrosation of lysine (D. Wink, 1998). DNA repair mechanisms may themselves create breaks in

DNA after the removal of the damaged base, when AP endonucleases cleave DNA at the AP site (Eastman

& Barry, 1992), creating single-strand breaks with 3’- or 5’- blocked ends that prevent DNA polymerase

or DNA ligase to perform their activities (H E Krokan et al., 1997). With DNA repair mechanisms inhibited,

single-strand breaks will eventually turn into double-strands breaks, further bringing the cell closer to

death (Tamir, deRojas-Walker, Wishnok, & Tannenbaum, 1996).

It is important to recall the cytoprotective role of NO in the cell when present in physiological

concentrations, as NO is capable of dealing with intracellular pathogens and block viral replication. NO is

also reported to protect DNA against oxidative stress and can actually prevent ROS-mediated DNA damage

by inhibiting Fenton reaction of hydrogen peroxide, which would lead to generation of the radical •OH.

In turn, this extremely reactive molecule would be available to react with almost all cell components,

including DNA (David A. Wink & Ford, 1995).

1.3. Dittrichia viscosa (L.) Greuter and Ginkgo biloba (L.)

D. viscosa and G. biloba, like many others, belong to a very interesting group of plants with

fascinating properties. Altogether, they have grown a considerable reputation due to their potential

benefits to human health. Plants with such properties are most of the times referred to as medicinal

plants. The evidence gathered among all of them suggests various possible applications, regarding, for

example, diabetes (Gushiken et al., 2016), gastro-duodenal disorders (Parolin, Ion Scotta, & Bresch,

2014) and age-related diseases (Apetz, Munch, Govindaraghavan, & Gyengesi, 2014; Bittencourt et al.,

2013; Mahdy et al., 2012), making them interesting, but mostly useful, research targets.

D. viscosa is an herbaceous plant species, mostly common in Mediterranean areas. Due to its high

resistance to adverse conditions and environmental stresses, it frequently occurs in poor or degraded

soils, such as roadsides, abandoned fields and dry riverbeds (Parolin et al., 2014). D. viscosa has been

used for many years in traditional medicine to treat wounds, bruises, and intestinal disorders (De

Laurentis, Losacco, Milillo, & Lai, 2002). A wide range of activities has been reported for D. viscosa, which

include anti-inflammatory, antidiabetic, antipyretic, antiseptic, balsamic, anti-viral, anti-fungal and anti-

11

bacterial (Barrero, Herrador, Arteaga, & Catalán, 2008; Lauro & Rolih, 1990). This species is mostly rich

in sesquiterpenes and flavonoids, the latter being associated with its antioxidant properties, through

which, D. viscosa is suggested to inhibit lipid peroxidation and also to scavenge superoxide radical

(Schinella, Tournier, Prieto, de Buschiazzo, & Rı́ os, 2002). The antioxidant activity of flavonoids (Figure

5) is dependent on the number as well as the arrangement of their hydroxyl groups (OH) (Heim,

Tagliaferro, & Bobilya, 2002). Flavonoid polymers such as proanthocyanidins, were demonstrated to be

powerful antioxidants in vitro, due to their high number of hydroxyl groups (Lotito et al., 2000). Some

structural features such as ortho-dihydroxy structure in the B ring, 2,3-double bonds in conjugation with

the 4-oxo group and hydroxyl groups at positions 3 and 5 were indicated to grant efficient radical

scavenging activities (Procházková, Boušová, & Wilhelmová, 2011).

Figure 5 – Chemical structure of quercetin. The three main features required for the antioxidant activity of flavonoids

are the ortho-dihydroxy structure in the B ring which facilitates electron delocalization (A), 2,3-double bond in

conjugation with the 4-oxo(=O) function in the C ring to allow electron delocalization from the B ring (B) and the

hydroxyl groups at positions 3 and 5, which allow binding of hydrogen to the oxo group (C). From Procházková et

al., 2011.

Research involving D. viscosa has also revealed some less desirable effects. A study on root

meristem cells of Allium cepa showed chromosomal aberrations and micronuclei formation upon

treatment with D. viscosa extract. This extract also induced cell death, membrane damage, and the

formation of binucleated cells and ghost cells (Aşkin Celik & Aslantürk, 2010). It is known that D. viscosa

is capable of inhibiting seed germination and root growth of other plant species as a competitive

advantage (Levizou, Karageorgou, Psaras, & Manetas, 2002; Parolin et al., 2014), which may be related

to the cytotoxic and genotoxic activities referred before. By contrast, during the investigation of the

epicuticular material of leaves of D. viscosa, it was determined that this material had strong absorbance

in the ultraviolet and that its removal and subsequent leaf exposure to ultraviolet-B (UVB) radiation did

not affect stomata function nor caused epidermal browning, suggesting resistance to UVB-induced

damage (Stephanou, Manetas, Stephanou, & Manetas, 1995).

G. biloba, one of the few living fossils and the only survivor of the Ginkgophyta division, is popularly

known for being among the few surviving species of the atomic bombings of Hiroshima and Nagasaki.

12

This species is currently classified as an endangered species by the IUCN Red List due to the uncertainty

of its persistence in the wild and rarity. The plant itself is known to be highly resistant to environmental

stresses and microbial diseases (Isah, 2015). Scientific research has produced an overwhelmingly large

amount of reports describing its properties and medicinal uses. G. biloba extract (GBE), made from the

leaves, is one of the most sold herbal remedies in the current market (Kressmann, Müller, & Blume,

2002), being usually prescribed to treat blood disorders, such as cerebral insufficiency and peripheral

vascular disease, and to improve memory function (Abad, Bedoya, & Bermejo, 2010; Isah, 2015). The

demand of G. biloba pharmaceutical formulations led to the large-scale cultivation of this tree in countries

such as the United States of America, China and France (Isah, 2015).

Due to its many active compounds, G. biloba has attracted the interest of many researchers. G.

biloba is stated to protect against free radicals, delaying the progress of diabetes and dementia, improve

the cognitive function of Alzheimer’s disease patients and to benefit the treatment of Parkinson’s disease

(Cheng, Xu, & Wang, 2009; M.-S. Kim, Lee, Lee, & Kim, 2004; Weinmann, Roll, Schwarzbach, Vauth, &

Willich, 2010; Yang, Wang, Sun, Zhang, & Liu, 2016; L. Zhou, Meng, Qian, & Yang, 2011). It is also

suggested that it has some beneficial effects on patients suffering angina pectoris but there are also a

number of reports defending that more clinical trials are needed to provide more conclusive evidence

(Sun, Wang, & Xu, 2015). The antihypertensive effect of GBE has been verified in vitro and in vivo (Kudolo,

2001), which suggested it as a new alternative to the treatment of hypertension, although, its

administration in humans did not reduce the incidence of hypertension (Brinkley et al., 2010).

Fairly recently, GBE was shown to protect S. cerevisiae against oxidant and genotoxic agent H2O2

(Marques, Azevedo, Johansson, & Oliveira, 2011), to decrease chromosomal damage induced by

radiofrequency radiation (Esmekaya et al., 2011), and lastly, to be a potential preventive cancer agent in

BRCA1-mutant ovarian epithelial cells by regulating cell proliferation, tumor suppression, and DNA

damage repair pathways (Jiang et al., 2011).

1.4. Schizosaccharomyces pombe

Schizosaccharomyces pombe was originally isolated from millet beer in eastern Africa and first

described by Paul Lindner in 1893 as a single-celled rod-shaped yeast. Lindner was able to point that

Sch. pombe division precedes the appearance of a wall transversal to the cell, which leads to a

constriction that completely separates the two parts. Additionally, because of this new yeast high variability

in size, Lindner named this species Schizosaccharomyces in order to express the morphological

13

difference relative to Saccharomyces as well as its mean of division (fission) and their common features,

such as spore formation and fermentation capacity (Lindner, 1893).

However, as one could possibly assume, the standard strains of today are not related to the strain

isolated by Lindner. In 1924, A. Osterwalder isolated and described what he understood to be a new

species, which he named Schizosaccharomyces liquifaciens. Later on, it ended up being renamed

Schizosaccharomyces pombe, strain liquifaciens (Osterwalder). From the collection of this strain, Urs

Leopold was able to isolate two homothallic clones, of which only one survived. This strain formed roughly

90% spores in pure culture and so, it was named h90. In the same year, 1950, Leopold also isolated two

heterothallic clones of opposite mating types, named h+ and h- (Munz, Wolf, Kohli, & Leupold, 1989).

In Sch. pombe, the mating-type of a cell is determined by the expression of the P (plus) or M (minus)

allele (Richard Egel, 1989). In short, a cluster of genes comprising an expressing locus mat1, and two

silent loci mat2-P and mat3-M control mating-type. The transposition of a copy of mat2-P or mat3-M, into

the expressing mat1, results in the production of P or M pheromone, determining its mating-type (R Egel,

Beach, & Klar, 1984; Hayles & Nurse, 2016). Through this mechanism h90 cells are capable of switching

mating-types and so, a population of this strain comprises about an half of h+ and another half of h- cells,

meaning there is always an available partner to mate (Munz et al., 1989). On the other hand, cells of the

heterothallic strains h+ and h- require the complementary pheromone in order to conjugate, otherwise

vegetative growth is the only mean of propagation (Hayles & Nurse, 2016; Munz et al., 1989). The inability

of h+ and h- strains to switch mating-types is acknowledged to have resulted from single-step events from

a h90 strain, subsequently stabilizing mat1 due to aberrant recombination (Richard Egel, 1989; Munz et

al., 1989). Of a matter of curiosity, despite the cell considering DNA breaks as DNA damage, mating-type

switching involves a site-specific DNA break in the right border of mat1. (Nielsen & Egel, 1989).

The majority of the mutants used nowadays in genetics and molecular biology research are derived

from these strains of close origin, h90, h+ and h-, and are known as 968, 975 and 972, respectively (Munz

et al., 1989). Some of Sch. pombe most appealing characteristics include its ease of culture, short

generation time (2-4 hours), easy manipulation and safe work methods (Giga‐Hama & Kumagai, 1999).

The genome sequencing of Sch. pombe was a fundamental step to encourage research concerning

this yeast due to the determination of highly conserved genes important for eukaryotic cell organization,

regarding the cytoskeleton, compartmentation, cell-cycle control, protein phosphorylation and RNA

splicing, etc. In fact, 67% of the annotated 5059 protein-coding genes are conserved in humans. Fifty of

those genes are significantly similar to human-disease related genes, almost half being involved in DNA

damage and repair, checkpoint controls and the cell cycle; all processes that highly contribute to maintain

14

genome stability (Wood et al., 2002). Sch. pombe chromosomal sequences did not reveal evidence of

large-scale genome duplications in any of its three chromosomes (Wood et al., 2002). Additionally, the

telomeres, centromeres and origins of replication of Sch. pombe are more similar to higher eukaryotes

than S. cerevisiae, the other most used yeast in research (Hayles & Nurse, 2016; Wood et al., 2002).

Throughout the years, Sch. pombe has proven itself as a remarkable model organism for the studies

of the eukaryotic cell cycle and its regulation (Moser & Russell, 2000; Yamamoto, Imai, & Watanabe,

1997), and DNA repair (Elledge & Zhou, 2000) and recombination (Wixon, 2002; Wood et al., 2002).

Sch. pombe has specially contributed to our understanding in microtubule formation (I. M. Hagan &

Petersen, 2000), meiotic differentiation (Yamamoto et al., 1997), cellular morphogenesis (Brunner &

Nurse, 2000) and polarity (Bähler & Peter, 2000) and stress response mechanisms (Toone & Jones,

1998).

1.5. Scientific problem and objectives

DNA is under constant attack from damaging agents, them being by-products of the cell metabolism

or from exogenous origin (Helleday, Eshtad, & Nik-Zainal, 2014). If it were not for the development of

DNA repair mechanisms, accumulation of DNA damage would quickly lead to serious cell dysfunction

and death. DNA damage checkpoint proteins also play an important role in DNA repair by stalling cell

cycle progression, which allows the operation of repair mechanisms. NO, when excessively produced,

causes indirect DNA damage and inhibition of proteins involved in its repair. The fission yeast model have

provided important insights in many cellular processes, regarding cell cycle and DNA repair, thus being,

the righteous subject for this study.

Plant extracts have grown a significant reputation for their potential benefits in human health (Apetz

et al., 2014; Bittencourt et al., 2013; Mahdy et al., 2012). Many plant extracts have been studied already

and a good number of recent reports provide indications of their favourable effects on numerous

conditions and diseases (Esfandiary et al., 2014; Hritcu et al., 2014; Ozarowski et al., 2013; Sutalangka,

Wattanathorn, Muchimapura, & Thukham-mee, 2013). The accumulation of DNA damage throughout life

may induce alterations in the biological functions of organisms. By changing the expression patterns of

proteins, such as transcriptional factors, an abnormal regulation of signalling pathways may be induced

resulting in different pathological processes (Sáez, 2016). Unfortunately, not much work have been

addressed to the antigenotoxic potential of such extracts (Vilar, Leite, & Chen Chen, 2009). Therefore,

the aim of this work, is to investigate if DVE and GBE confer protection against DNA damage upon

exposure to NO, and, if possible, also clarify in which way DNA damage protection occurs.

15

2. Materials and Methods

2.1. Yeast strains, media, and growth conditions

In this study, Schizosaccharomyces pombe wild-type haploid strains 972 and FO656 were used;

including DNA repair, oxidative stress-response and DNA damage checkpoint mutants (Table 1). Strains

FO656, FO763, RHP502 and FO767 were kindly provided by Ingrun Alseth (Department of Microbiology,

Oslo University Hospital, Oslo, Norway); SF869 and SF315 by Stefania Francesconi (Genome Dynamics

Unit, Pasteur Institute, Paris, France); and FO661 AV18, AV25, EHH5 and EHH14 provided by Elena

Hidalgo (Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain).

Strain stocks were kept in 50% glycerol at -80 °C and were cultured every week on solid YD medium

(1% w/v yeast extract (Acros Organics), 3% w/v glucose, and 2% w/v agar) at 30 °C and stored at 4 °C

after 3 days. All solid media contains 2% w/v agar. Media and growth conditions of each experiment is

explained to the detail in the respective subsection.

Table 1 – Genotypes of the strains used in this study.

Strain Genotype Source

972 h–

FO656 h+ ura4-D18 leu1-32 his3-D1 arg3-D4 (Alseth et al., 2004)

FO763 h+ nth1::ura4+ ura4-D18 leu1-32 his3-D1 arg3-D1 (Alseth et al., 2004)

RHP502 apn2::KanMX ura4-D18 leu1-32 his3-D1 arg3-D4+ Magnar Bjørås

FO767 mag1::ura4+ ura4-D18 leu1-32 his3-D1 arg3-D4+ Magnar Bjørås

FO661 h– rhp55Δ::arg3+ 4ura4-D18 leu1-32 his3-D1 arg3-D4+ (Alseth et al., 2004)

SF869 h+ rhp51::ura4 ura4-D18 leu1-32 Stefania Francesconi

SF315 h+ chk1::ura4 leu1-32 ade ura4-D18 Stefania Francesconi

AV18 h– sty1::kanMX6 (Zuin et al., 2005)

AV25 h– pap1::kanMX6 (Zuin et al., 2005)

EHH5 h– leu1 sty1::GFP::kanMX6 (Zuin et al., 2005)

EHH14 h+ his2 ura4 pap1::ura4– leu1 nmt::GFP-pap1::leu1 (Zuin et al., 2005)

2.2. Plant material and extracts preparation

This study relied in the use of extracts prepared from the leaves of D. viscosa and G. biloba. D.

viscosa specimens were identified by Özkan Eren (Department of Biology; University of Adnan Menderes)

and gathered from the surrounding areas of Söke, Kuşadası and Aydın (Turkey) during flowering season

16

(November 2007). D. viscosa leaf extract was prepared as described by Çelik T. and Aslantürk O. (2010).

The leaves were rinsed with water, dried in a ventilated oven at 55 °C for 24h and then reduced to a fine

powder using a blender. The powder was stored at room temperature in the dark. The extract stock

solution was prepared to a concentration of 50 mg/mL by boiling the powder with deionized water for 5

min and letting the solution cool down at room temperature for 10 min, followed by filtering with a 0.22

µm syringe filter unit. DVE stock solution was stored at -20 °C until use.

G. biloba leaves were collected at the University of Minho campus (Braga, Portugal) by the end of

summer (September 2015) and left to dry out in the dark at room temperature. The leaves were

considered dry when they became stiff and broke easily when bent. From that point on, approximately 5

g of leaves (with petiole) were triturated using a blender and put in a cellulose extraction thimble. The

powder-containing thimble was immersed in approximately 100 mL of absolute ethanol inside a 250 mL

laboratory bottle and left to incubate for 7 days at 30 °C, 200 rpm, in the dark. At the end of the incubation

period, the thimble was removed and the ethanolic solution was filtered, followed by evaporation using a

rotovapor (RE121+461, Buchi) at 40 °C, 40 rpm in the dark. Afterwards, a small amount of deionized

water was added to the evaporated extract to collect any remaining residue and facilitate freezing at

-80 °C. After freezing, the extract was put in a freeze dryer (Christ Alpha 2-4, B. Braun) for 4 days in the

dark. The resulting powder was kept at -20 °C. GBE stock solution was prepared with absolute ethanol

to a concentration of 50 mg/mL and stored at -20 °C until use.

2.3. Electrospray Ionisation-Tandem Mass Spectrometry (ESI-MS/MS)

Both GBE and DVE (100 ppm) were dissolved in 20% methanol and injected in a ion trap mass

spectrometer (LXQ Linear, Thermo) equipped with a syringe pump at a flow rate of 5 µL/min and a

vaccum of 5x10–5 Torr. The analyses were performed in the negative selective ion (see Appendix 3 for

more details). The obtained [M-H]– masses were used in the tentative identification of the detected

compounds by literature comparison. The data from the chemical analyses were processed by Xcalibur

software.

2.4. In vitro assays

Both DVE and GBE were tested for 2,2-diphenyl-1-picrylhydrazyl (DDPH) reducing, iron-chelating

(ICA) and NO scavenging activities. Each extract was tested for five concentrations. The samples were

read in a microplate reader (SpectraMaxPlus 284, Molecular Devices). All the assays were performed in

triplicate.

17

2.4.1. DPPH assay

DVE and GBE were tested for anti-radical activity. DDPH solution was prepared just before the assay

to a concentration of 0.004% (w/v) and kept protected from light at all times. Ten µL of DVE/GBE was

added to DPPH-containing wells (reactional volume: 150 µL) and the plate was left in the dark for 60 min

before reading at 515 nm. Gallic acid was used as standard. For the blank samples, water and ethanol

were used in substitution to DVE and GBE, respectively. The percentage of DPPH reduction was

calculated as follows:

% = � − � � ×

Ab – Blank sample absorbance (DPPH)

As – Sample absorbance (DPPH and extract)

DPPH reduction percentages were represented in a linear progression with the corresponding extract

concentration and IC50 was calculated. IC50 corresponds to the concentration at which DPPH is reduced by

50%.

2.4.2. Iron-chelating activity

The ICA assay evaluates the capacity to chelate Fe2+. In reaction with 3-(2-pyridyl)-5,6.diphenyl-1,2,4-

triazine-4´,4’-disulphonic acid sodium salt (ferrozine), Fe2+ forms a complex that can be quantified by

spectrophotometry. Ferrozine and FeSO4 solutions were prepared right before the experiment and kept

protected from light. Fifty µL of 0.3 mM ferrozine and 50 µL of extract were added to the plate. In order

to prevent bias due to reaction with the extract and ferrozine, Fe2+ is added in last place: 50 µL of 0.06

mM FeSO4 (reactional volume: 150 µL). Ethylenediaminetetraacetic acid (EDTA) was used as standard.

For the blank samples, water and ethanol were used in substitution to DVE and GBE, respectively. The

samples were then incubated in the dark at room temperature for 10 min and read at 562 nm. Iron-

chelating activity was calculated as follows:

� % = � − � − �� ×

Ac – Control absorbance (ferrozine, iron)

Ab – Blank sample absorbance (extract)

As – Sample absorbance (ferrozine, iron and extract)

18

ICA values were represented in a linear progression with the corresponding extract concentration

and IC50 was calculated. IC50 corresponds to the concentration at which formation of ferrozine-Fe2+ is

reduced by 50%.

2.4.3. NO scavenging

NO scavenging activity can be measured through Griess reaction: in acidic conditions, nitrite reacts

with sulfanilamide and N-1-napthylethylenediamine dihydrochloride (NED) to form an azo compound

detectable by spectrophotometry. All the solutions were prepared in the same day the assay was

performed and were kept protected from light. Griess reagent was prepared by mixing 1% w/v

sulfanilamide (dissolved in 5% H3PO4 wt. %) and 0.1% w/v NED SNP in a 1:1 ratio. One hundred µL of

SNP (20 mM) and 100 µL of extract diluted in phosphate buffer KH2PO4 19 mM pH 7.4 were added to

the plate and left under the light for 60 min. One hundred µL of Griess reagent was then added to the

samples (reactional volume: 300 µL) and incubated at room temperature in the dark for 10 min and

absorbance read at 562 nm. For the blank samples, 2% H3PO4 was used in substitution to DVE and GBE,

respectively. NO scavenging activity was calculated as follows:

% = � − � − �� ×

Ac – Control absorbance (buffer, SNP and Griess reagent)

Ab – Blank sample absorbance (extract, SNP and H3PO4)

As – Sample absorbance (extract, SNP and Griess reagent)

NO scavenging values were represented in a linear progression according to the corresponding

extract concentration and IC50 was calculated. IC50 corresponds to the concentration at which Griess

reaction is reduced by 50%.

2.5. Viability assays

Cells were grown on YES liquid medium (0.5% yeast extract, 3% glucose and 225 mg/L supplements

– adenine, histidine, leucine, lysine hydrochloride and uracil) and incubated at 30 °C, 200 rpm to an

OD600 of 0.4. Cells were submitted to four treatments, consisting in a negative control, extract control,

positive control and co-incubation. Treatments involved incorporation of the compounds in the cooling

solid medium, addition to the cellular suspensions or cells pre-treatment with extract and pipetting onto

treated medium. The extracts concentration was the same 750 µg/mL for D. viscosa and 1000 µg/mL

for G. biloba in all viability assays. For the medium incorporation and pre-treatment assays, 1 mM of SNP

19

was incorporated in the medium and 300 mM was used in the suspension assays. Cell cultures were

serially diluted up to 10–4 before pipetting on the plate. Deionized water was used to replace D. viscosa

extract and SNP, and absolute ethanol was used to replace G. biloba extract, in the corresponding

controls.

2.6. Cell cycle

Cells were grown overnight in YES liquid medium to a concentration of 3–4x106 cells/mL. A sample

of 500 µL was taken from the inoculum, for control purposes, and centrifuged (14,100 rcf), following the

discard of the supernatant and fixation of the cells in 500 µL of cold (4 °C) ethanol 70 %. The sample

was then stored at 4 °C in the dark until further processing. Every sample in this experiment was

processed has previously described.

Afterwards, the cell culture was synchronized in S phase by addition of hydroxyurea (HU) (Sigma) to

a final concentration of 12 mM and let to incubate at 30°C, 200 rpm for 4 hours. At the end of incubation

time, another control sample was taken. The culture was then centrifuged and washed twice with sterile

deionized water to remove HU and another sample was taken for control. After HU removal, the culture

was separated in four aliquots and prepared for the four described treatments (GBE at 1000 µg/mL and

SNP at 1 mM). Samples of 500 µL were taken from all the treatments every 30 min, including time 0,

up to 120 min. After collecting, the samples were washed twice with 50 mM sodium citrate pH 7.5 (4°C).

In this step, centrifuging was performed at 4 °C, 2600 rpm for 4 min. The samples remained in 50 mM

sodium citrate pH 7.5 and RNAse A (Macherey Nagel Bioanalysis) was added to a final concentration of

0.1 mg/mL, following overnight incubation at 37 °C. Before storage at 4 °C, the samples were sonicated

twice for 10 seconds at medium intensity.

Just before analysis, propidium iodide (PI) (Acros Organic, Fisher) was added to a final concentration

of 4 µg/mL. In an orderly manner, the three samples taken before the beginning of the treatments served

as references for an asynchronous culture, synchronized culture and hydroxyurea-free culture. In addition,

a nitrogen starved cell culture was also prepared as a control. Cell cycle analysis was performed in a

cytometer (Epics XL, Beckman Coulter) (300 events/s). The results of the experiment were processed

using Flowing Software 2.5.1.

20

2.7. Fluorescence microscopy

Cells were grown in EMM (3 g/L potassium hydrogen phthalate, 2.2 g/L Na2HPO4, 5 g/L NH4Cl,

glucose 2% w/v, 52.5 g/L MgCl2.6H2O, 0.735 g/L CaCl2.2H2O, 50 g/L KCl, 2 g/L Na2SO4, 1 g/L

pantothenic acid, 10 g/L nicotinic acid, 10 g/L inositol, 10 mg/L biotin, 5 g/L boric acid, 4 g/L MnSO4,

4 g/L ZnSO4.7H2O, 2 g/L FeCl2.6H2O, 0.4 g/L molybdic acid, 1 g/L KI, 0.4 g/L CuSO4.5H2O and 10 g/L

citric acid) medium to OD600 of 0.4. Before applying treatments, a sample was taken from the inoculum

and prepared for observation in the fluorescence microscope (DM5000B+CTR5000+ebq100, Leica) for

control purposes. The cell suspension was divided into 4 aliquots and each was given a different

treatment. During the experiment, the four aliquots were incubated at 30 °C, 200 rpm. Samples were

taken at 10 and 60 min and immediately observed in the fluorescence microscope.

21

3. Results

3.1. The chemical composition of DVE and GBE is similar to that of other described extracts

Chemical analysis of both DVE and GBE provided elucidation about their composition, which could

additionally allow a projection of their effects. Compounds identification relied entirely on information

found in literature regarding chemical analysis of similar D. viscosa (Mahmoudi et al., 2015; Mamoci et

al., 2011; Trimech et al., 2014) and G. biloba (Borges, Volmer, & Eberlin, 2013; S. Ding et al., 2008;

He, Bernart, Nolan, Lin, & Lindenmaier, 2000; Lin, Chen, Ozcan, & Harnly, 2008; Ma et al., 2016;

Pandey, Chandra, Arya, & Kumar, 2014; Yao et al., 2014; H. Zhou, Tang, Zeng, & Tang, 2014) extracts.

The chemical composition of D. viscosa extracts is poorly known. Most studies dedicated to the

determination of its compounds aim exclusively at the study of essential oils and consequently, only 5

peaks have been identified out of the 31 detected during mass spectrometry. Among the compounds

detected are 2 flavonoids, 2 sesquiterpenes and 5 caffeoylquinic acids (Table 2). Additionally, no mass

spectrum for D. viscosa was found for comparison.

Table 2 – Complete list of the tentatively identified compounds for DVE.

m/z Tentative identification

251 3- -hydroxycostic acid

ilicic acid

317 unknown dihydroflavonol

353 chlorogenic acid

477 isorhamnetin-O-hexoside

515 1,3-dicaffeoylquinic acid 3,4-dicaffeoylquinic acid 3,5-dicaffeoylquinic acid 4,5-dicaffeoylquinic acid

For GBE, 22 peaks were identified (of 48) corresponding, possibly, to 68 compounds (Table 3).

Unsurprisingly, among these molecules are mostly flavonoids (flavone O-glycosides, flavonoid aglycones,

biflavones, etc) and terpene lactones. Some characteristic G. biloba compounds were identified, including

ginkgolic acid I and II, bilobetin, ginkgolide A (dimer), sequoiaflavone, quercetin, rutin, syringetin,

ginkgetin, isoginkgetin. Additionally, numerous modified molecules derived from quercetin, isoharmnetin

and kaempferol were identified as well. The spectrum of GBE (Appendix 2) was compared to five other

spectra obtained in a study by (Borges et al., 2013). GBE (from this work) and those five extracts

(altogether), share 19 peaks (including unidentified peaks) out of 56, presenting a reasonable similarity

to the very few published chemical profiles of G. biloba.

22

Table 3 – Complete list of the tentatively identified compounds for GBE. Unidentified compounds that were also

detected in other studies have (?) as their identification.

m/z Tentative identification

283 dihydroxymethoxyflavone

301 pentahydroxyflavone

quercetin

345 ginkgolic acid I

syringetin

tetrahydroxydimethoxyflavone

373 ginkgolic acid II

389 ?

447 kaempferol-7-O-glucoside

quercetin-3-O-rhamnoside

quercetin-3-rhamnoside

463 quercetin 3-O-glucoside

477 isorhamnetin-3-O-glucoside

kaempferol-3-O-glucoside

kaempferol-O-hexoside

luteolin-3 -O-glucoside

quercitrin

479 myricetin-3-O-glucoside

551 bilobetin

sequoiaflavone

565 ginkgetin

isoginkgetin

577 ?

593 bisrhamnosyl–glucosylkaempferol

kaempferol-3-O-2 -glucosylrhamnoside

kaempferol-3-O-neohesperidoside

kaempferol-3-O-rhamnoside-glucoside

kaempferol-3-O-rutinoside

kaempferol-3-rutinoside

kaempferol-7-O-rutinoside

(Continues in the next page)

23

m/z Tentative identification

609 bisrhamnosyl–glucosylquercetin

quercetin-2 -glucosylrhamnoside

quercetin-3-O-rhamnoside-glucoside

quercetin-3-O-rutinoside

rutin

623 isorhamnetin 3-O-2 -glucosylrhamnoside

isorhamnetin 3-O-neohesperidoside

isorhamnetin 3-O-rutinoside

639 3’-methoxymyricetin-3-O-rutinoside

laricitrin-3-rutinoside

patuletin 3-O-neohesperidoside

patuletin 3-O-rutinoside

653 syringetin 3-O-2 -glucosylrhamnoside

syringetin-3-O-rutinoside

739 kaempferol 3-O-2 -(6 -p-coumaroyl)glucosylrhamnoside

kaempferol 3-O-2 , 6 -dirhamnosideglucoside

kaempferol 3-O-p-coumaroyldiglycoside

kaempferol 3-O-p-coumaroylrutinoside

kaempferol p-coumaroyldiglycoside

kaempferol-3-O-rhamnoside-glucoside-coumaroyl

755 kaempferol 3-O-2 -glucosyl-6 -rhamnosyl-glucoside

kaempferol 3-O-rhamnosylhexoside-7-O-glucoside

quercetin 3-O-2 , 6 -dirhamnosideglucoside

quercetin 3-O-2 , 6 -dirhamnosylglucoside

quercetin 3-O-2 -(6 -p-coumaroyl)glucosylrhamnoside

quercetin 3-O-p-coumaroyldiglycoside

quercetin p-coumaroyldiglycoside

quercetin-3-O-rhamnoside-glucoside-coumaroyl

769 isorhamnetin 3-O-2 ,6 -dirhamnosylglucoside

771 quercetin 3-O-rhamnosylhexoside-7-O-glucoside

isorhamnetin 3-O-2 , 6 -dirhamnosideglucoside

815 ginkgolide A dimer

24

3.2. DVE and GBE showed different in vitro activities

The IC50 for the activities of DVE and GBE were determined, in vitro, through DPPH, ICA and NO

scavenging assays (Table 4). DVE showed a considerably higher DPPH reduction activity than GBE. On

the other hand, GBE exhibited an equally higher NO scavenging activity than DVE. Lastly, DVE was

determined to overcome GBE regarding iron-chelating activity. Accordingly, DVE exhibited a significantly

lower IC50 than GBE in DPPH assay, of 96.9±4.2 µg/mL and 360.6±18.5 µg/mL, respectively. In ICA

assay, DVE was determined to inhibit Fe2+–ferrozine formation by 50% at 155.9±11.3 µg/mL where the

IC50 of GBE was determined to be 270.0±98.3 µg/mL. For the NO scavenging assay, DVE demonstrated

a high IC50 of 889.4±59.8 µg/mL whereas GBE was 241.2±2.2 µg/mL. The determined IC50 for all the

standard compounds, gallic acid, EDTA and curcumin, was as low as expected; 4.7±0.2 µg/mL, 2.8±0.3

µg/mL and 7.1±0.8 µg/mL, respectively, were needed to produce 50% of the expected effect.

Table 4 – Values of IC50 (µg/mL) determined for DVE, GBE and standards in DPPH reduction, ICA, and NO

scavenging assays.

DPPH reduction Iron-chelating activity NO scavenging

DVE 96.9±4.2 155.9±11.3 889.4±59.8

GBE 360.6±18.5 270.0±98.3 241.2±2.2

Standard 4.7±0.2 2.8±0.3 7.1±0.8

3.3. GBE significantly protected viability loss caused by SNP

Viability protection by DVE and GBE was examined through evaluation of numerous Sch. pombe

strains cell viability after exposure to SNP. The mutants used in these experiments are implicated in DNA

damage checkpoint, BER pathway and oxidative-stress response.

DVE protecting effect was firstly investigated by culturing 972, Δchk1 and Δrhp51 cells in treatment-

incorporated medium. As observed in Figure 6, Δrhp51 strain cells exhibited little less viability and colony

growth than 972 and Δchk1 cells in negative control, which implies a difficulty in growth for the HR-

affected mutant (Δrhp51). Medium incorporation of 750 µg/mL of DVE did not cause any loss of viability

or growth impairment comparing to the negative control, indicating DVE is not cytotoxic at the tested

concentration. The incorporation of SNP virtually caused complete loss of viability in the 3 tested strains,

which, for Δchk1 and Δrhp51, was not rescued by the presence of DVE. Co-incorporation of DVE and

SNP resulted in increased viability for 972 cells comparing to SNP treatment, however, only by an

insignificant extent.

25

Figure 6 – Viability of 972, Δchk1 and Δrhp51 Sch. pombe cells when cultured on solid medium incorporated with

(from left to right): water (negative control), 750 µg/mL DVE, 1 mM SNP and both 750 µg/mL DVE and 1 mM

SNP. Cells were diluted up to 10–4 before pipetting onto the plates (indicated at the top of the photos). The plates

were incubated for 4 days at 30 °C. The photographs are representative of three independent experiments.

The extracts were also tested for antigenotoxicity upon transient exposure of cells to SNP (Figure 7).

Cells were taken from liquid media, containing the treatments, after 30, 60 and 90 min from the

respective treatment addition and pipetted onto rich media to continue growth; this way, the effects of

SNP and GBE would be monitored more closely. In negative control, Δchk1 cells displayed a slightly

reduced viability comparing to 972 wild-type cells, as indicated by the relative number of colonies. Once

again, DVE appears not to be cytotoxic towards Sch. pombe cells, as their viability was not affected,

relatively to untreated cells, in both strains. Transient exposure to SNP decreased 972 cells viability,

indicated by the reduced colonies density especially at the more diluted drops (10–3 and 10–4). On the

contrary, 1-3 Δchk1 colonies from 10–3 and 10–4 dilutions managed to grow after incubation with SNP,

however this information should be considered irrelevant due to the smallness of their number.

The co-incubation of 972 cells with SNP and GBE did appear to produce a slight increase in viability

of 972 cells, whereas Δchk1 cells presented a significantly increased viability relative to SNP treatment,

which could be related with the role of chk1 in cell cycle progression.

26

Figure 7 – Viability of 972 and Δchk1 Sch. pombe cells when incubated for 0, 30, 60 and 90 min in liquid medium

containing (from left to right): water (negative control), 750 µg/mL DVE, 300 mM SNP and both 750 µg/mL DVE

and 300 mM SNP. Cells were diluted up to 10–4 before pipetting onto the plates (indicated at the top of the photos).

The plates were incubated for 4 days at 30 °C. The photographs are representative of three independent

experiments.

In order to investigate whether oxidative stress-response proteins are involved in the response to NO

increase and consequent nitrosative stress, Δsty1 and Δpap1 cells were submitted to treatment with DVE

prior to culture in treated medium (Figure 8). As a result, all the tested strains 972, Δsty1 and Δpap1

cells appear to maintain viability relatively to negative control however, with significant differences in

colony size; 972 appears to present some behaviour variability as can be seen by the reduced colonies

instead of loss of viability as it was observed in the first viability assay (Figure 6). Co-incorporation with

DVE and SNP following DVE pre-treatment did not rescue cell viability in any of the tested strains, which

suggests DVE is not effective against the genotoxicity of NO.

27

Figure 8 – Viability of 972, Δsty1 and Δpap1 Sch. pombe cells when pre-incubated in 750 µg/mL DVE-containing

liquid medium and cultured in medium incorporated with (from left to right): water (negative control), 1 mM SNP

and both 750 µg/mL DVE and 1 mM SNP. Cells subjected to pre-treatment without DVE are signalized by (-) and

with DVE by (+). Cells were diluted up to 10–4 before pipetting onto the plates (indicated at the top of the photos).

The plates were incubated for 4 days at 30 °C. The photographs are representative of three independent

experiments.

The antigenotoxicity of GBE was also investigated by transiently exposing cells to SNP (Figure 9).

972 cells exhibited little loss of viability after SNP treatment in comparison with the negative control

(Figure 7), also presenting a similar behaviour to what was observed in Figures Additionally, cells co-

incubation with GBE and SNP did not result in any significant differences in viability relatively to SNP-

treated cells thus, a protective effect of GBE cannot be considered.

Figure 9 – Viability of 972 Sch. pombe cells when incubated for 0, 30, 60, 90 and 120 min in liquid medium

containing (from left to right): ethanol (negative control), 300 mM SNP and 1000 µg/mL GBE and 300 mM SNP.

Cells were diluted up to 10–4 before pipetting onto the plates (indicated at the top of the photos). The plates were

incubated for 4 days at 30 °C. The photographs are representative of three independent experiments.

28

GBE was additionally tested using DNA-repair mutants affected in BER (Δmag1, Δapn2, Δnth1) and

HR (Δrhp55). As it can be observed in Figure 10, GBE did not affect the viability of any of the tested

strains relatively to the negative control, which indicates it is not cytotoxic. SNP treatment induced loss of

viability in all the strains with exception to mag1, which was only affected in colony size. Consequently,

Δmag1 viability remains unchanged when cell were in co-incubation with GBE and SNP. The observed

viability decrease in nth1 and rhp55 appears greater than that in Δmag1 and Δapn2 cells, which may

indicate that the corresponding proteins may have a more important role in repairing DNA damage

induced by NO. Additionally, the strains FO656, Δapn2, Δnth1 and Δrhp55 viability is rescued in co-

incubation. In this medium (GBE+SNP), the mutants Δnth1 and Δrhp55 presented less viability than the

wild-type FO656, which would suggest that the affected genes have a role in GBE antigenotoxicity. Based

in this assay, the protective effect of GBE is independent of the repair pathways BER and HR.

Figure 10 – Viability of FO656, Δmag1, Δrhp51, Δnth1 and Δrhp55 Sch. pombe cells when cultured on solid

medium incorporated with (from left to right): ethanol (negative control), 1000 µg/mL GBE, 1 mM SNP and both

1000 µg/mL GBE and 1 mM SNP. Cells were diluted up to 10–4 before pipetting onto the plates (indicated at the

top of the photos). The plates were incubated for 4 days at 30 °C. The photographs are representative of three

independent experiments.

Ultimately, Δchk1, Δsty1 and Δpap1 viability under SNP exposure was explored (Figure 11). Similarly

to the previous experiment, GBE has not appeared to diminish cell viability in any of the tested strains.

Growth on SNP-incorporated medium decreased viability in general, however it was more noticeable in

the mutant strains Δchk1, Δsty1 and Δpap1. The fact that the mutant strains showed less viability than

the wild-type cells when exposed to SNP, suggests that Δchk1, Δsty1 and Δpap1 are necessary to

attenuate the effects caused by NO. Lastly, by comparing SNP treatment to co-treatment with both GBE

and SNP it can be stated that viability has not changed in any of the tested strains. This observation

29

indicates that GBE does not protect agains NO, however, this could be due to differences in the genetic

background of the used strains.

Figure 11 – Viability of 972, Δchk1, Δsty1 and Δpap1 Sch. pombe cells when cultured in solid medium

incorporated with (from left to right): ethanol (negative control), 1000 µg/mL GBE, 1 mM SNP and both 1000 µg/mL GBE and 1 mM SNP. Cells were diluted up to 10–4 before pipetting onto the plates (indicated at the top of

the photos). The plates were incubated for 4 days at 30 °C. The photographs are representative of three

independent experiments.

3.4. GBE alone induces quicker progression in cell cycle and slightly attenuates SNP-induced delay

Sch. pombe spends, relatively most of the time in G2 phase. During G1 phase, protein synthesis

and accumulation take place and DNA replication is primed as preparation for S phase. Entrance in S

phase is accompanied by DNA replication initiation and ends as replication is completed. G2 phase follows

and cells grow in size as required for M phase (mitosis). During mitosis, chromosomes segregation

occurs into the dividing cells. Cytokinesis is the last step of cell cycle and separates the newly formed

cells. A very particular trait about cell division in the fission yeast is that the cytoplasmic and nuclear

divisions are somewhat out of synch, with cytokinesis being only completed when cells have already

progressed into S (Moser & Russell, 2000; Zhu et al., 2015). And therefore, during cell cycle analysis, a

G1 cell with two 1C nuclei and a G2 cell with a single 2C nucleus will be detected as equals in the

cytometer and both G1 and G2 cells will be represented in the 2C peak (see Appendix 4 for more details).

The protection effect of GBE was investigated by analysing the DNA content of wt 972 cells during

incubation in the presence of GBE, SNP and both simultaneously (Figure 12). At the beginning of the

experiment, the cells were synchronized in S-phase at the time of release from HU. 30 min later, cells of

all treatments were still in S-phase with completed cytokinesis (2C DNA content), with little difference in

progression between treatments with GBE (GBE and GBE+SNP) and without GBE (negative control and

SNP). As cells continued further into S-phase, at 60 min, treatment with GBE alone produced greater

progression in cell cycle than the remaining treatments; SNP treated cells lagged behind negative control

30

cells by very little, while cells treated with GBE+SNP continued to perform better than both negative control

and SNP treatment.

Impressively, at 90 min, most GBE-treated cells have already passed M and G1 phases and entered

S phase without completing cytokinesis (4C DNA content), while untreated cells still exhibited peaks at

both 2C and 4C DNA content, which correspond to G1-phase and S phase (before cytokinesis),

respectively. At this time-point, GBE and SNP co-incubated cells are in the transition between G1 and S

whereas, SNP-treated cells continue to be the most delayed as they are just beginning to enter G1. Finally,

at 120 min, most untreated and GBE-treated cells have 2C DNA content, indicating that cytokinesis has

been completed. Regarding GBE+SNP and SNP treatments, cells achieved to enter S phase, however it

is noteworthy that the proportion of the GBE+SNP-treated cells in the 2C and 4C peaks are roughly the

same, suggesting that half of the population has not completed S phase (2C) and the other half has

completed S phase before cytokinesis (4C), while most of the SNP-treated cells are still in the 2C peak,

which indicates that they did not complete S phase. These results indicate that GBE is abrogating the cell

cycle delay effect of SNP, which is compatible with a protecting activity against SNP genotoxicity. In

addition, it is remarkable that GBE treatment appears to cause cells to progress more rapidly in cell cycle

than untreated cells, at least in the initial stages when cells resume the cell cycle after removal of HU.

31

Figure 12 – Cell cycle progression analysis of Sch. pombe cells under the different treatments. Before analysis,

the cells were treated with hydroxyurea (HU) in order to synchronize their cell cycle and were compared with an

asynchronous culture (top left). After washing HU, the cells were submitted to their respective treatments: water

(negative control), 750 µg/mL DVE, 1 mM SNP and both 750 µg/mL DVE and 1 mM SNP. Every 30 min, a sample

of each treatment was taken and processed for analysis. During the treatments, the cells were incubated at the

standard conditions of 30 °C, 200 rpm.

32

3.5. Both extracts induce relocalization of oxidative-stress response proteins

Considering the anti-radical and NO scavenging of the extracts, the reaction of proteins involved in

oxidative-stress response Sty1 and Pap1 was investigated by fluorescence microscopy. Sty1 belongs to a

family of stress-activated protein kinases (SAPKs), a subfamily of the mitogen-activated protein kinase

(MAPKs) (A. N. Nguyen, Lee, Place, & Shiozaki, 2000), and is activated by a wide range of stress-inducing

factors such as, osmotic stress, heat shock, UV, superoxide, peroxide and certain DNA damaging agents

(Egel, 2004). Studies around Sty1 have implied not only that it is necessary to cellular response to stress

but also it controls other proteins. When stress is sensed and the MAPK cascade triggered, Sty1 relocates

into the nucleus, where it regulates other proteins, such as Atf1 and Pap1. Pap1 is a transcription factor

that accumulates in the nucleus during stress and is implicated in the expression of antioxidant-related

genes catalase, thioredoxin, and superoxide dismutase following oxidative stress (Hong, Roze, & Linz,

2013).

To test the involvement of Sty1 and Pap1, two Sch. pombe strains, in which sty1 and pap1 were

genetically fused with GFP, were used to verify if oxidative-stress response was activated by SNP treatment

and to investigate if DVE and GBE could prevent nuclear accumulation of these proteins.

Cells were incubated in the usual treatments except for SNP, which concentration was increased to

4 mM. As expected, untreated sty1::GFP cells did not exhibit nuclear accumulation throughout the entire

experiment (Figure 13). DVE treatment, however, induced accumulation of Sty1 at 10 min and 60 min of

incubation, indicating that DVE causes activation of Sty1. As expected, SNP treatment induced Sty1

translocation into the nucleus, which seems to decrease after 60 min. Co-incubation of cells in SNP+DVE

also resulted in Sty1 accumulation, although this effect is attenuated at 60 min of experiment.

SNP-induced Sty1 accumulation should most probably have been caused by the generation of RNS

and consequent nitrosative stress. At 10 min of experiment, the three treatments induced Sty1

relocalization which is decreased only in SNP incubation at 60 min, as if DVE maintained Sty1 in an

activated state.

33

Figure 13 – Microphotographs of fluorescence microscopy of sty1::GFP transformed Sch. pombe after 0, 10 and

60 min of incubation in liquid minimal medium containing (from left to right): water (negative control), 750 µg/mL

DVE, 4 mM SNP and both 750 µg/mL DVE and 4 mM SNP. The photographs are representative of three

independent experiments. Magnification: 1000x.

Following Pap1 monitoring, the untreated cells did not exhibit nuclear accumulation of Pap1 (Figure

14). All three treatments resulted in Pap1 accumulation only observable at 60 min, representing a delay

relatively to the previous experiment, which could be expected since Pap1 is downstream of Sty1 in the

signalling pathway. Cells incubated with DVE presented the lowest fluorescence in the nucleus, whereas

co-incubated cells exhibited the highest; this could indicate that DVE, in fact, induces the oxidative-stress

response and that SNP, naturally, induces a more intense reaction from that mechanism. Additionally,

treatment with DVE+SNP resulted in an intermediate nuclear fluorescence between DVE and SNP

treatments, suggesting DVE could be attenuating the effect of NO, although that effect was too tenuous

to be considered in the viability assays.

34

Figure 14 – Microphotographs of fluorescence microscopy of of pap1::GFP transformed Sch. pombe after 0, 10

and 60 min of incubation in liquid minimal medium containing (from left to right): water (negative control), 750

µg/mL DVE, 4 mM SNP and both 750 µg/mL DVE and 4 mM SNP. The photographs are representative of three

independent experiments. Magnification: 1000x.

Lastly, the effect of GBE on NO-induced Pap1 nuclear accumulation was evaluated (Figure 15).

Unexpectedly, untreated cells presented fluorescence accumulation at 60 min. All the three treatments

induced Pap1 relocalization, although co-incubated cells exhibited what appears to be the cumulative

effect of GBE and SNP. Between 10 and 60 min from the beginning of the experiment, fluorescence in

the nucleus increased, similarly to the previous assay, which involved Pap1 and DVE. Again, GBE+SNP

treatment induced an increased Pap1 nuclear accumulation, apparently resulting from the cumulative

effects of GBE and SNP.

35

Figure 15 – Microphotographs of fluorescence microscopy of of pap1::GFP transformed Sch. pombe after 0, 10

and 60 min of incubation in liquid medium containing (from left to right): water (negative control), 1000 µg/mL

GBE, 4 mM SNP and both 1000 µg/mL GBE and 4 mM SNP. The photographs are representative of three

independent experiments. Magnification: 1000x.

37

4. Discussion

The chemical analysis of the extracts, complemented by literature research, allowed some

elucidation about their composition. None of the identified DVE compounds have been associated with

DNA damage protection, however, it is worthy to highlight the remarkably high antioxidant activities of

1,3-dicaffeoylquinic (1,3-diCQA) and chlorogenic acids (3-caffeoylquinic acid) (Danino, Gottlieb,

Grossman, & Bergman, 2009; Gökbulut et al., 2013). Among the molecules identified for DVE, 3 classes

of compounds are represented, namely flavonoids, sesquiterpene acids and caffeoylquinic acids (Table

2). Sesquiterpene and caffeoylquinic acids were not very explored so far; still, considering the highly

regarded antioxidant activity of flavonoids added to the activities of the caffeoylquinic acids mentioned

above, it is expected from DVE to act mainly through a strong antioxidant activity.

GBE, on the other hand, has been subjected to numerous studies involving the activities of its

individual compounds. With regard to DNA damage protection, only myricetin was found to exhibit those

effects, more specifically, by inhibition of peroxynitrite-mediated damage (Chen et al., 2011). Some of the

remaining compounds were also studied with several flavonoids determined to interfere in cell

proliferation and to induce apoptosis in several cell lines (Klimaszewska-Wiśniewska et al., 2017; Qin,

Cui, Yang, & Tong, 2016). A small group of compounds, ginkgolic acids, are considered the downfall of

G. biloba extracts for their possible cytotoxic, mutagenic, carcinogenic and genotoxic effects, being

currently restricted to less than 5 ppm in commercial extracts (Ude, Schubert-Zsilavecz, & Wurglics,

2013); however, a research group still studied and came to demonstrate the ability of ginkgolic acids to

inhibit HIV protease and infection in vitro (Lü et al., 2012).

The analysis of GBE indicated the presence of flavonoids, most being glycosylated derivatives (Table

3). Flavonoids are best described for their antioxidant activities, which may involve direct scavenging of

ROS (Procházková et al., 2011), activation of antioxidant enzymes (Nijveldt et al., 2001), metal chelating

activity (Ferrali et al., 1997), mitigation of oxidative stress caused by nitric oxide (Vanacker, Tromp,

Haenen, Vandervijgh, & Bast, 1995), among others. In general, glycosylated flavonoids have less in vitro

antioxidant activity than the aglycon from which they are derived (Procházková et al., 2011). A study

determined that the antioxidant contribution of G. biloba flavonoid glycosides was actually higher than

that of aglycons (Ding et al., 2009), which would suggest that G. biloba is much more rich in glycosides.

In accordance, an overwhelming number of flavonoid glycosides were identified in GBE most being derived

from aglycons quercetin and kaempferol, which agrees with the indication that flavonoid glycosides are

38

more abundant in G. biloba than aglycons. In conclusion, GBE may be able to exert some protection

through a single or a combination of the mechanisms referred above.

The extracts were then tested in vitro (Table 4) to better understand which activities they may employ

in vivo. DVE showed an impressive antioxidant activity, considering the comparison of a complex mixture

to a pure compound. This activity could probably be due to the presence of caffeoylquinic acids, namely

1,3-diCQA, which exhibited higher antioxidant activity than some known antioxidants (Danino et al.,

2009). DVE also presented decent iron-chelating activity, however, this could not be directly linked to any

of its characteristic compounds nor it was found in the literature any report concerning this activity and

therefore, remains the supposition that the responsible molecules are yet to be identified. In what

concerns to NO scavenging, DVE performed very poorly, requiring more than 100-fold the concentration

needed for curcumin to scavenge 50% of NO.

On the contrary, GBE showed lesser antioxidant and iron-chelating activities. Taking into account the

vast number of the detected flavonoids and the reports concerning some of GBE compounds and their

DPPH reduction ability (Guo et al., 2015), a considerably high activity was expected, which did not occur.

The lack of insight about compound concentrations could partially explain this result as a more profound

chemical analysis might reveal the low presence of those antioxidants. With regard to iron-chelation,

literature research did not provide any information concerning G. biloba and similarly to DVE, this ability

may reside in a less investigated component of the extract.

The extracts were then put to test against genotoxicity of NO by using the fission yeast Sch. pombe.

DVE was shown not to be cytotoxic to cells in the three performed viability procedures. The use of SNP

as a NO-releasing agent was proven effective at the given concentration as it reduced the viability of yeast

cells in the incubation and suspension assays. During pre-treatment assays however, cells were able to

maintain viability being only affected in colony growth, indicating some variability in their behaviour.

Strains 972, Δchk1 and Δrhp51 were not protected in treatment-incorporated medium (Figure 6). In case

the long exposure to SNP could be too severe and overwhelming to the cells and thus, not allowing any

possible action window to the extract, cell viability upon transient SNP exposure was also evaluated. 972

cells displayed little protection whereas Δchk1 was protected to a higher degree (Figure 7). As referred

previously, Chk1 indirectly prevents cell cycle progression by inhibition of Cdc25, which happens to be

one of the proteins inhibited by NO (Majumdar, Biswas, Subhra Sarkar, Maiti, & Ghosh, 2012) and thus,

the survival of Δchk1 mutants may have relayed in the delay of cell cycle and the operation of DNA repair.

Δchk1 mutant cells are able to bypass cell cycle arrest induced by DNA damage and attempt mitosis

producing a variety of chromosomal abnormalities (Walworth et al., 1993) and so, survival through simple

39

progression of cell cycle is very unlikely, as DNA damage accumulation should be fatal. This same viability

assay could be repeated to ensure results reliability and, most importantly, cell cycle analysis must be

performed, using the same treatments, in order to investigate if Δchk1 cell cycle is effectively delayed by

NO exposure and to what extent, as it would be interesting to see if a more severe delay in cell cycle could

be behind the increased survival.

The inspection of oxidative stress-response Δsty1 and Δpap1 and wild-type 972 strains in the pre-

treatment assay demonstrated that DVE could not protect cellular viability (Figure 8). As referred above,

SNP resulted in reduced colony growth instead of loss of viability and, as strange as that, the expectable

sensitivity of, at least, Δpap1 was not observed. Other reports demonstrated that pap1 mRNA level

increases upon SNP treatment and that the protein is implicated in NO elimination (H.-J. Kim, Jung, &

Lim, 2008), and also that its absence results in increased sensitivity, indicating the involvement in the

protection against nitrosative stress (Sahoo et al., 2006).

On the other side, GBE exhibited protection towards NO exposure of some strains. The transient

exposure of 972 cells to NO did not result in protection by GBE (Figure 9), however, in that same assay,

SNP was unable to significantly reduce cell viability, contrary to what was observed while studying DVE

(Figure 7). This difference in the response to SNP treatments could also be related to the chemical stability

of SNP. The decrease of the effect of SNP could have masked the true effectiveness of GBE due to the

inexistent loss of viability in SNP treatment, which sustains the comparison between treatments.

The DNA repair mutants Δapn2, Δnth1, Δrhp55 and the respective wild-type strain FO 656 exhibited

increased viability in co-incubation of SNP and GBE (Figure 10), which indicates that the extract protects

cells independently of the impaired repair mechanisms. Even though the mutants used reasonably cover

the BER pathway and consequently provide strong evidence of GBE BER-independent mechanism of

action, the inclusion of a double Δapn2 Δnth1 mutant should be investigated as it completely abolishes

DNA repair through this pathway. The addition of HR mutants is also a suggestion as only two mutants

were involved in this study.

The strain Δmag1 was the only one to maintain viability when treated with SNP (Figure 10). The

activity of Mag1 was associated with a mutator phenotype, in which reduced levels of Mag1 activity

decreased this phenotype and high levels increased it (Glassner, Rasmussen, Najarian, Posnick, &

Samson, 1998). Based on this information, the benefit of the lack of Mag1 activity and consequent viability

maintenance of Δmag1 cells is comprehensible. On the other side, Mag1 activity might have revealed

harmful to Δapn2 and Δnth1 cells, as their impairment did not allow them to cope with the formation of

AP sites, which should have remained unrepaired in these mutants. Relatively to the mutant strains

40

Δchk1, Δsty1 and Δpap1, the protective effect of GBE was not observed (Figure 11). Since SNP did not

cause an efficient viability loss, additional assays should be performed as the best approach when looking

for this kind of protective effect is to work in cytotoxic conditions.

The evaluation of cell cycle progression provided some interesting information regarding the

influence of GBE in cellular viability. GBE treatment caused cells to progress faster in cell cycle (Figure

12), which would allow them to complete cell division more often and, consequently, a relative increase

in colony size could have been observable in co-incubation treatment. However, it is noteworthy that the

strain used in flow cytometry was 972, which was not protected by GBE, and, logically, it would be of

major interest to proceed the same test using FO656 strain in order to find out if the same effect occurs.

In addition, the mutant strain Δchk1 should be tested as well to verify if faster cell cycle advance still

occurs or not, which would clarify if GBE acts through Chk1.

It could be observed that GBE appears to take effect almost immediately, as the treatments with

GBE alone and GBE+SNP allowed enhanced cell cycle progression relatively to SNP treatment and even

the negative control at 30 min. The delaying effect of the slow-releasing agent SNP was only observable

after 60 min into the experiment, in which SNP-treated cells lagged behind until the end of the test. Co-

incubated cells started to slow down at 60 min, being ultimately overtaken by negative control cells at 90

min. Overall, the effectiveness of GBE in counteracting the SNP-induced mitotic delay was far from

impressive. Majumdar et al. (2012) reported a mitotic delay caused by NO donor detaNONOate and

investigated the origin of that delay. The group came to demonstrate that Chk1 was not involved in the

replication slowdown during nitrosative stress, although still considered it should play a role in its

maintenance. They also determined that the major protein responsible for replication slowdown was

wee1p, which phosphorylates Tyr 15 of Cdc2, inactivating it. In addition to the study of Δchk1 cell cycle,

it would also be relevant to investigate wee1 for the matter of GBE protection mechanism.

Lastly, the study of oxidative-stress response proteins Sty1 and Pap1 cellular localization revealed

that co-incubation of cells with SNP and the extracts did not prevent protein nuclear accumulation (Figure

13, 14, 15), as if nitrosative stress was not prevented. Both extracts also induced nuclear relocalization

of Sty1 (not tested with GBE) and Pap1, which suggests oxidative-stress response mechanisms were

triggered. In their work, Maeta et al. (2007) concluded that tea polyphenols could act as prooxidants and

induce a response to oxidative stress in yeasts S. cerevisiae and Sch. pombe. Similarly to DVE and GBE

of this study, green tea extract caused Yap1 (Pap1 in Sch. pombe) and Pap1 to relocate to the nucleus.

After treatment with the extract, Yap1 returned to its native localization (cytoplasm) after 60 min, which

did not occur with neither DVE nor GBE. This particular difference between both DVE and GBE relatively

41

to the green tea extract is completely understandable as different extracts should not act exactly alike,

but it is positive enough that DVE and GBE also appear to activate a response to oxidative-stress. In the

case of DVE, however, activation of Sty1 and Pap1 does not appear to have a practical effect since the

effectiveness of DVE in protecting cellular viability upon SNP exposure was not considerable.

In conclusion, despite the significant DPPH reduction and iron chelating activities of DVE and its

induction of Sty1 and Pap1-mediated oxidative stress response, no protection against DNA-damaging

agents was observed. Relatively to GBE, its DNA protection mechanism appears to result from a

combination of effects. GBE exhibited reasonable DPPH reduction and NO scavenging activities. DNA

protection seems to be independent of, at least, the BER pathway. Additionally, treatment with GBE

induced oxidative-stress response following Pap1 relocalization to the nucleus. These effects probably

result from a high flavonoid content of GBE, considering that their reported activities include scavenging

of ROS, activation of antioxidant-related enzymes, and mitigation of nitrosative stress.

43

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6. Appendix

Appendix 1 – Mass spectrum of DVE.

Appendix 2 – Mass spectrum of GBE.

62

Appendix 3 – Specific parameters regarding the negative mode of ESI-MS/MS used in the analyses of the extracts.

Sheat gas flow rate 20 (arbitrary)

Aux. gas flow rate 5 (arbitrary)

Sweep gas flow rate 5 (arbitrary)

Spray voltage 5 kV

Capillary temperature 250 °C

Capillary voltage -11 V

Tube lens -55.7 V

Appendix 4 – Cell cycle in Sch. pombe. The four phases of the cell cycle are represented as G1, S, G2 and M. The

black segmented circle represents the relative duration of each cell cycle phase. A representation of the cells and

nuclei can also be seen next to their respective stage in cell cycle. An exponentially growing G1 cell is binucleated

and each nucleus has 1C DNA content. During S phase, DNA replication occurs. Cytokinesis is only completed at

this stage; resulting in binucleated cells with 2C DNA content each. By reaching G2 phase, cells have accomplished

cytoplasmatic division and hold a single 2C nucleus. The nuclei separate in the M phase and septation occurs,

bringing the cells to G1 again. Edited from Knutsen et al., 2011.