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Process Biochemistry 49 (2014) 195–202

Contents lists available at ScienceDirect

Process Biochemistry

jo ur nal home p age: www.elsev ier .com/ locate /procbio

he effect of menadione-induced oxidative stress on the in vivoeactive oxygen species and antioxidant response system ofhanerochaete chrysosporium

urcu Tongul, Leman Tarhan ∗

niversity of Dokuz Eylul, Faculty of Science, Department of Chemistry, 35160 Buca, Izmir, Turkey

r t i c l e i n f o

rticle history:eceived 4 August 2013eceived in revised form 31 October 2013ccepted 4 November 2013vailable online 13 November 2013

eywords:enadionexidative stress

a b s t r a c t

The antioxidant response system of Phanerochaete chrysosporium against menadione-induced oxidativestress was investigated in this study. The superoxide anion radical levels in tested menadione-supplemented conditions generally decreased over the incubation period. The level of hydrogen peroxideand the activities of NAD(P)H oxidase, superoxide dismutase (SOD) and catalase (CAT) were higher thanthose in the controls at all incubation times. The highest NADH and NADPH oxidase activities were deter-mined to be 4.9- and 5.0-fold higher than those in the control, respectively in cells exposed to 0.75 mMmenadione. The SOD and CAT activities increased with increasing menadione, and their highest activitieswere 5.4- and 5.1-fold higher than those in the control, respectively. In 0.1–0.5 mM menadione exposed

ntioxidant response system cells, the lipid peroxidation levels did not change significantly when compared to each other, except 8thhour of incubation (p > 0.01). Our result shows that although menadione induces the formation of reactiveoxygen species, the antioxidant response system of P. Chrysosporium is able to negate menadione-inducedoxidative stress up to relatively high menadione concentrations, as 0.75 mM. These results are importantto determine the effects of menadione, as a medicine, on the antioxidant response system of eukaryotic

level

models and the resulting

. Introduction

All living aerobic microorganisms are inevitably exposed toeactive oxygen species (ROS), including the superoxide anion radi-al (O2

−), hydrogen peroxide (H2O2) and the hydroxyl radical (•OH)1]. ROS can be generated by both endogenous and exogenousources. Potential endogenous sources include the mitochondrialespiratory chain, cytochrome P450 metabolism, peroxisomes,nd the activation of inflammatory cell [2]. NADH and NADPHxidase are also responsible for ROS production as an endoge-ous source Exogenous factors include ionising radiation, tobaccomoke, polluted air, industrial toxins and drug exposure. Exoge-ous factors, such as xenobiotics and chemotherapeutics, haveeen intensively investigated in terms of their effects on intracel-

ular ROS production and cell damages [3–9]. They catalyse thene-electron reduction of O2 leading to the production of O2

−,y transferring one electron to O2 from various reductases. Thisedox cycle explains the toxic effects of exogenous factors. Antiox-

dant response mechanism exists to regulate the cellular levels ofOS; otherwise, their reactive nature damages important macro-olecules, including DNA, protein, and lipids [1]. The antioxidant

∗ Corresponding author. Tel.: +90 0232 301 86 94; fax: +90 0232 453 41 88.E-mail address: leman.tarhan@deu.edu.tr (L. Tarhan).

359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.procbio.2013.11.004

of damage.© 2013 Elsevier Ltd. All rights reserved.

response system consists of antioxidant enzymes, such as super-oxide dismutase (SOD), and catalase (CAT), and non-enzymaticantioxidants, which participate in the action against ROS [3,4,10].

Under normal physiological conditions, the toxic effects of ROSare minimised by the antioxidant defence system. However, oxi-dant levels may increase under various stressful conditions, andoverwhelm the antioxidants, cell damage results, which are definedas oxidative stress [11]. On the other hand, it has been proven thatthe exposure of aerobic cells to oxidative stress induces the syn-thesis of the antioxidant enzymes up to a certain level [4,5,11–14].

Vitamin K derivatives, which are efficient exogenous source ofROS generation, were found to have electron carrier roles betweenelectron-donating and electron-accepting enzyme complexes ofthe mitochondrial respiratory chain [15–17]. Menadione, a vitaminK derivative, is a quinone that has been extensively used in studiesof cellular oxidative stress [1–4] as well as a therapeutic agentwith anticancer activity. It is known to generate intracellular ROSat multiple cellular sites with a concomitant decrease in NAD(P)H,consistent with redox cycling [18]. Previous studies have shownthat menadione leads to O.−

2 generation in exposed cells [19–21].In vivo, several flavoenzymes, such as NADPH cytochrome P-450

reductase, catalyse the one-electron reduction of quinones tosemiquinone radicals. These unstable radicals can be re-oxidisedby O2, giving rise to O2

−. Menadione also acts as an artificialelectron carrier in the treatment of certain mitochondrial

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96 B. Tongul, L. Tarhan / Process

yopathies [19,22]. Menadione was shown to inhibit the growthf mammalian tumour cells both in vitro and in vivo, e.g., mousend rat liver tumours, glioma, melanoma and neuroblastoma cells23,24], human glioma, hepatoma cells [25] and urologic tumours26]. Moreover, a synergistic effect has been shown when thisubstance is given in combination with other anti-tumour agents27] and potentiation of radiotherapy by vitamin K has beenevealed [23].

In this study, the activity of the antioxidant response system ofhanerochaete chrysosporium, as a eukaryotic model, against mena-ione, as an exogenous factor of ROS production, was investigated.or this purpose, the variation in the intracellular O2

− and H2O2 lev-ls, the activities of the O2

− producer NADH oxidase and NADPHxidase and the antioxidants SOD and CAT activities and the mem-rane lipid peroxidation (LPO) levels were investigated at variousenadione concentrations (0.1, 0.2, 0.3, 0.5 and 0.75 mM)and incu-

ation times (1, 2, 3, 4, 5, 6, 7 and 8 h).

. Materials and methods

.1. Microorganism and culture conditions

The DSM-1547 strain of P. chrysosporium, obtained from German Collection oficroorganisms and Cell Culture (DSMZ), was used as model microorganism for

nvestigating the antioxidant responses to menadione-induced oxidative stress.Spore suspension was generated in potato dextrose agar (PDA) medium (pH 5.6)

s described by Beever and Bollard [28], Sterilisation of the medium was carried outy autoclaving at 121 ◦C for 20 min. Inoculation was performed at 28 ◦C for 7 days

n Petri dishes.P. chrysosporium was cultured in modified Tien and Kirk liquid medium [29]

g/L); KH2PO4 (2.0), CaCl2 (0.114), MgSO4 (0.7), NH4Cl (0.12), d-glucose (2.0),hiamin-HCl (1.10−3), Tween 80 (0.05), FeSO4.7H2O (70 �g), ZnSO4.7H2O (46 �g),nSO4·2H2O (35 �g); CoCl2.6H2O (7 �g). The liquid medium was sterilised by auto-

laving at 121 ◦C for 20 min. Incubation was carried out at 28 ◦C for 10 days and withgitation at 150 rpm in the 250 mL Erlenmayer flask containing 90 mL liquid mediumnd 10 mL spore suspension (OD650; 0.800).

.2. Stress conditions

After 10 days culture, menadione (0.1–0.75 mM) was added to the growthedium that containing stationary phase P. chrysosporium in 250-mL Erlenmayer

ask. Incubation was performed at 28 ◦C with agitation at 150 rpm for 1–8 h. Controlamples were incubated without menadione. The menadione-treated and controlells were harvested and washed several times with 20 mM potassium phosphateuffer (pH 7.4) at 4 ◦C, and then the crude extracts were prepared.

.3. Preparation of crude extracts for enzyme activities

The harvested cells were resuspended in 20 mM potassium phosphate buffer (pH.4) in a volume equal to 3.0 times of the cells’ wet weight. The optimised homogeni-ation procedure was performed for 3 min at 9000 rpm with 30-seconds intervals.he cell debris in the homogenate was removed by centrifugation at 15,000 rpm andor 15 min at 4 ◦C. The crude extract was not frozen before use.

.4. Determination of ROS levels

.4.1. Measurement of the O2− level

O2− production was measured by chemiluminescence in the presence of the

hemiluminogenic probe lucigenin [30]. Lucigenin (5 �M) was added to the platehat contained 250 �l cell suspensions (OD650 = 0.300). The O2

− level was deter-ined after 1 h incubation at 37 ◦C in luminescence counter.

.4.2. Measurement of the H2O2 LevelThe H2O2 level was measured using a modification of the method described

y Barja [31]. Briefly, the cell homogenate was incubated at 30 ◦C with 1.5 mL phos-hate buffer (pH 7.4) containing 2.5 mM pyruvate/malate. After 15 min, the reactionas stopped with 0.5 mL cold stop solution (0.1 M glycine, 25 mM EDTA-NaOH, pH

2.0) and then fluorescence was determined at excitation wavelength of 312 nmnd emission wavelength of 420 nm. The H2O2 level was calculated using a standardurve of H2O2 and expressed in nmol/gww.

.5. Enzyme activity assays

.5.1. NADH oxidase activity assayThe NADH oxidase activity was determined by spectrophotometry. The proce-

ure was based on the disappearance of NADH at 340 nm [32]. The decrease in the340 value was recorded of two 5-min intervals. A millimolar extinction coefficientf 6.22 was used to calculate the NADH disappearance.

emistry 49 (2014) 195–202

2.5.2. NADPH oxidase activity assayThe NADPH oxidase activity was determined by spectrophotometry. The pro-

cedure was based on the disappearance of NADPH at 340 nm [32]. A millimolarextinction coefficient of 6.22 was used to calculate the volume activity of theenzyme.

2.5.3. SOD activity assayThe SOD enzyme activity was determined by spectrophotometry at 490 nm.

The SOD activity assay was based on the measurement of autoxidation of 6-hydroxydopamine (6-OHDA), which is inhibited by SOD [33]. One unit is the amountof SOD required to inhibit the initial rate of 6-OHDA autoxidation by 50%.

2.5.4. Catalase activity assayThe catalase enzyme activity was determined by spectrophotometry with Aebi

method [34], which depends on the decrease in the absorbance at 240 nm that occursupon the hydrolysis of H2O2 to H2O and O2.

The extinction coefficient for H2O2 at 240 nm is 43.6 M−1 cm−1. The specificactivity of catalase (U/mg protein) is the enzyme amount necessary to decreasethe H2O2 absorbance at 240 nm from 0.450 to 0.400 in 20 s.

2.6. Total protein assay

Bradford method (A595) was used for the measurement of the total proteinconcentration in the samples [35].

2.7. Determination of the LPO level

Lipid peroxidation was measured by the formation of malondialdehyde (MDA)using the thiobarbutiric acid reaction [36].

2.8. Statistical analysis

Tukey’s test, a multiple comparison test, was used to determine statistical sig-nificance. The values are the means of three separate experiments. Also comparisonwas made using Pearson’s correlation.

3. Results

In this study, the level of O2− and H2O2, the activities of the

O2− producing enzymes NADH oxidase and NADPH oxidase and

the antioxidant enzymes SOD and CAT and the levels of membraneLPO in P. chrysosporium, as a eukaryotic model, were investigatedupon supplementation with 0.1–0.75 mM menadione for variousincubation times, and the results were compared with those of non-supplemented control cells.

3.1. Variation in the intracellular O2− level

As shown in Fig. 1a, the O2− level in control cells decreased sig-

nificantly after hour 4 of the incubation (p < 0.01). The O2− levels in

all of the menadione supplementation conditions decreased fromthe beginning of investigated incubation period and were gener-ally lower than that of the control starting at hour 3 (Fig. 1b–f). Theperiod of this decrease in the O2

− levels generally shifted from 3 to5 h of incubation depending on the menadione concentration, andthe minimum levels were similar for all the samples, approximately21 RLU (p < 0.05). After the minimum levels were observed, whilethe O2

− levels of P. chrysosporium showed no significant changesat 0.1–0.3 mM menadione, they significantly increased at 0.5 and0.75 mM menadione after 7 and 6 h of incubation, respectively(p < 0.01).

3.2. Variation in the intracellular H2O2 level

The H2O2 level of P. chrysosporium incubated in menadione-freemedium did not show any significant changes until 5 h of incuba-tion and then decreased significantly up to 8 h (p < 0.05). Duringhour 1 the levels of H2O2 in P. chrysosporium incubated with mena-

dione showed significant increases in a concentration-dependentmanner (r = 0.939, p < 0.01) (Fig. 2b–f). The H2O2 levels were higherthan the control cells at all incubation times and increased unex-pectedly after hour 5 at 0.75 mM menadione. The highest H2O2

B. Tongul, L. Tarhan / Process Biochemistry 49 (2014) 195–202 197

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evels in menadione-supplemented conditions was determined as2.77 ± 0.68 nmol/gww for 0.75 mM at hour 1 which was 2.2-foldigher than that in the control cells.

.3. Variations in the NADH and NADPH oxidase activities

As shown in Fig. 3a, the NADH oxidase activity in the control

ells significantly increased between hours 4 and 5th, althoughhe values were similar before and after this periods (p < 0.01). TheADH oxidase activities of all the samples treated with menadioneere significantly higher than the control cells at all incubation

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ne supplementation; 0.1 mM (b), 0.2 mM (c), 0.3 mM (d) and 0.5 mM (e), 0.75 mMan ± SD. *Significant difference (p < 0.05) from control.

times. The maximum NADH oxidase activities in P. chrysosporiumwere positively correlated to menadione concentration (r = 0.95).The highest NADH oxidase activity value was determined as4.27 ± 0.12 U/mg at 0.75 mM menadione at hour 5 of incubation;this value was 4.9-fold higher than that in the control.

As shown in Fig. 4a–f, while the NADPH oxidase activities in thecontrol cells did not show significant changes at all incubation times

, the activities for the cells at all menadione concentrations andincubation period were higher than those of the control. NADPHoxidase activity, which depended on the menadione concentration,showed a similar trend to the NADH oxidase activity. The highest

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ne supplementation; 0.1 mM (b), 0.2 mM (c), 0.3 mM (d), 0.5 mM (e) and 0.75 mMan ± SD. *Significant difference (p < 0.05) from control.

198 B. Tongul, L. Tarhan / Process Biochemistry 49 (2014) 195–202

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ADPH oxidase activity in menadione-supplemented conditionsas determined as 7.9 ± 0.21 for 0.75 mM at hour 6, which was

-fold higher than that in the control cells.

.4. Variation in the SOD activity

As shown in Fig. 5, the activity of the antioxidant enzyme SODas higher than the control cells for all of the tested menadione

oncentrations and incubation times (p < 0.01). The SOD activity

evels showed a positive correlation with the menadione concen-ration at all incubation times (rhighest = 0.998 for 7th hour). The

aximum SOD activities depending on menadione concentrationsf P. chrysosporium also increased with increasing menadione. The

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3.5. Variation in CAT activity

The CAT activity level of the control P. chrysosporium increasedsignificantly after 6 h of incubation (p < 0.01) (Fig. 6a). Althoughthe CAT activity was similar from 0.1 to 0.3 mM menadione and

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B. Tongul, L. Tarhan / Process Biochemistry 49 (2014) 195–202 199

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8,698.2 ± 873.04 U/mg for 0 .75 mM at 6 h of incubation, whichas 5.1-fold higher than that in the control cells. The CAT activity

t 0.5 and 0.75 mM menadione was higher than all other samplest all incubation times but showed similar trends with one another.

.6. Variations in membrane LPO level

The membrane LPO level of P. chrysosporium was significantlyigher at all the menadione concentrations when compared toontrol (p < 0.01) (Fig. 7a–f). At 0.1–0.5 mM menadione, the LPOevels did not change significantly when compared to each other

menadione; control (a), and menadione supplementation; 0.1 mM (b), 0.2 mM (c),ts experiments and expressed as mean ± SD. *Significant difference (p < 0.05) from

(approximately 3.2 nmol MDA/gww) except at 8 h of incubation(p > 0.01). The membrane LPO levels of P. chrysosporium at hour 8were positively correlated with menadione concentration (r = 0.92).

The LPO level reached its highest value (5.32 ± 0.29 nmolMDA/gww) at 0.75 mM menadione and hour 4, which was 2-foldhigher than that in the control cells.

4. Discussion

In this study, menadione supplementation in growth mediaof P. chrysosporium caused an increase in O2

− for all of the

200 B. Tongul, L. Tarhan / Process Biochemistry 49 (2014) 195–202

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Fig. 7. Variation of LPO level depending on the incubation period and concentration of menadione; control (a), and menadione supplementation; 0.1 mM (b), 0.2 mM (c),0 endenc

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.3 mM (d), 0.5 mM (e) and 0.75 mM (f). The results were obtained from three indepontrol.

ested concentrations. The highest O2− level was determined as

7.22 ± 1.4 RLU at 0.3 mM menadione at 1 h of incubation, whichas 1.66-fold higher than that in the control. In a previous study,2

− levels in Fomes fomentarius and Tyromyces pubescens treatedith 1 mM menadione were found 4.28- and 8.42-fold higher than

he non-supplemented cells, respectively, at the end of the first dayf incubation period [6]. After 5 days of incubation, while the O2

−

evel in F. fomentarius fell under the control, which is similar to ouresults, it was still 2.66 fold higher in T. pubescens. In addition, forenicillium chrysogenum exposed to 0.5 mM menadione, this valueas 7-fold higher than the control at the end of hour 5, whereas

he O2− level was lower than the control for P. chrysosporium at

ll of the tested menadione concentrations when comparing theame incubation times [37].

The NADH and NADPH oxidase activities in P. chrysosporium,n which the highest activity was approximately 5-fold higher inhe menadione-supplemented conditions than in the control, con-ribute to the generation of O2

− as endogenous sources of ROS.he NADH oxidase activity of P. chrysosporium in the control waslso higher than the previously determined activities of NADH oxi-ases of other fungi [38]. While we found the NADH oxidase activityo be 0.490 U/mg protein in P. chrysosporium, the fungi Glomerellaingulata, Schizophyllum commune, Pythium debaryanum, Monoliniaructicola showed activities of 0.111, 0.100, 0.082, 0.081 U/mg in,espectively [39].

Menadione has been shown to stimulate intracellular ROS gen-ration via activation of NADPH oxidase [40–43]. The level of H2O2n A549-S cells exposed to 0.1 mM menadione decreases 8-foldfter pretreatment with diphenyleneiodonium as an inhibitor ofADPH oxidase [44]. The induction of NADH and NADPH oxidase

n P. chrysosporium by menadione treatment may be proof thathese enzymes are involved in the production of ROS. Nevertheless,he O2

− levels in P. chrysosporium treated with menadione did not

ncrease as much as the other reported species despite the increasesn the activities of NADH and NADPH oxidases. This situation cane explained by the induction of SOD against menadione-inducedxidative stress.

ts experiments and expressed as mean ± SD. *Significant difference (p < 0.05) from

The rapid decrease in O2− at the beginning of hour 1 of the

investigated incubation period was the results of the dismuta-tion reaction of SOD, which was triggered by menadione-inducedoxidative stress in P. chrysosporium. The SOD activity was 2.8-foldhigher at 0.1 mM menadione and 5.4-fold higher at 0.5 mM mena-dione than that in the control. In previous researches conducted onbacteria and white root fungi, SOD activity is generally induced bymenadione treatment at a level that is 2- to 5-fold higher than thatof control [21,45–47].

The SOD activity in P. chrysosporium was induced more stronglywhen compared with these studies, and this induction in SODresulted in the maintenance of O2

− to a level that was under thatof the control for a short period of the incubation at all investigatedmenadione concentrations. The unexpected increases in the O2

−

level at 0.5 and 0.75 mM menadione after 6 h of incubation may berelated to the inhibition of SOD by excessive ROS, such as O2

− andH2O2, in P. chrysosporium.

The H2O2 levels in P. chrysosporium exposed to menadioneincreased in a time-dependent manner and reached its maxi-mum, except for 0.5 and 0.75 mM menadione supplementation.Despite the increased CAT activity, the H2O2 level was generallyhigher than the control at all of the investigated incubation times(p < 0.01). The H2O2 level was found to be approximately 1.2-,2.4- and 1.7-fold higher than the control in menadione-exposedS. pombe, P. chrysogenum, and Human PBMC cells, respectively, inprevious studies [37,46,48]. The highest observed value of H2O2 inP. chrysosporium was also 2.2 fold higher than the control, and thisresult is similar to the findings in the published literature. On theother hand, the activity of the antioxidant enzyme CAT increasedup to 5.1-fold in response to menadione-induced oxidative stress.The highest CAT activity in A. niger is 1.2-fold higher, while it isapproximately 2.0-fold in S. pombe, F. fomentarius and Bacillussp. F26 [21,47,48]. However, the CAT activity of P. chrysogenum

exposed to 0.5 mM menadione did not show any significantvariation, and CAT activity has also been shown to decreased by44% in menadione-exposed T. pubescens [21,37]. The 5.1fold higherCAT activity of menadione-exposed P. chrysosporium reveals

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B. Tongul, L. Tarhan / Process

he strong induction of antioxidant response system againstenadione-induced oxidative stress, although it is not sufficient to

uppress the increases in H2O2 level. This condition may be relatedo the conversion of O2

− to H2O2. According to our results and othereports, the CAT activity is affected by menadione supplementationo varying degrees depending on the investigated species.

The increase in LPO level, an indicator of oxidative damages, wasot substantial in P. chrysosporium, except in the 0.75 mM mena-ione treated samples. In this study although the LPO levels inll of the menadione treated samples were approximately 1.3-foldigher than those of control, significant dose dependent differencesere not seen at 0.1–0.5 mM menadione. The significant induction

f antioxidant enzymes depending on the menadione concentra-ion could protect the cells against menadione-induced oxidativetress at a certain level, while the LPO level could not be con-rolled by antioxidant system at 0.75 mM menadione despite highernzyme activities; thus, the LPO level reached 2-fold higher thanhe control. The increase in LPO level was also observed in Bacillusp. F26 (2.3-fold higher) and S. pombe (1.25-fold higher) [46,47].

Our results show that although menadione induces the O2− pro-

ucing enzymes NADH oxidase and NADPH oxidase as well as theormation of the O2

− and H2O2 in P. chrysosporium, the antioxidantnzymes SOD and CAT, are able to suppress LPO considerably up to.75 mM menadione.

. Conclusion

Although NADH oxidase and NADPH oxidase were notably trig-ered by menadione treatment, O2

− was maintained at a levelhat was below the control through the induction of antioxidantesponse system of P. chrysosporium by menadione. On the otherand, while the CAT activity of P. chrysosporium showed a large

ncrease, the level of H2O2 was higher than that of the control buttill quite lower than the data obtained in previous studies. Fur-hermore, the induction of ROS production by menadione did notffect the LPO level until 0.75 mM menadione. Compared to previ-us studies, the large induction of the enzymes of the antioxidantesponse systems, the stability of ROS levels and the minimal effectsf menadione on the LPO levels of P. chrysosporium as a eukaryoticodel provide hope that menadione used in tumour treatment will

ot lead to significant damages in healthy cells.

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