Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp. 245-250

Dye-mediated photodynamic inactivation of Bacillus subtilis cells: Involvement of singlet oxygen and superoxide radicals

Alok Dube*, Harsha Bansal and Pradeep K Gupta

Laser Programme, Centre for Advanced Technology, Indore 452013, Indi a

Received 7 May 1999; accepted 16 January 2000

Studies were carried out on the photodynamic inactivation of Bacillus subtilis cells by He-Ne laser irradiation in pres­ence of methylene blue and toluidine blue. Electron paramagnetic resonance (EPR) measurements show decrease in the cell membrane nuidity due to photodynamic damage of the lipophilic compartments. Study of cell damage in presence of so­dium azide, 0 20 and glutathione as well as measurements on singlet oxygen/free radical generation by EPR show that with toluidine blue, si nglet oxygen is the major reactive species leading to cell membrane damage. No free radical generation was observed in toluidine blue medi ated photodamage. However, with methylene blue our observations suggest that the photo­damage ari ses due to both si nglet oxygen mediated membrane damage and intracellular damage by superoxide radical.

Photosensitised inactivation of prokaryotic cells and eukaryotic cells mediated by phenothiazine dyes has been investigated by many groups1. In particular, methylene blue (MB) and toluidine blue (TB) in combination with low power lasers have been used for photodynamic inactivation of bacteria as an ap­proach to the treatment of localized infections2-6.

However, the primary site of photodamage and the reactive species responsible for photodynamic cell inactivation are not well understood . Although it is widely accepted that the main target for MB photo­sensitized biological action is DNA7

, evidence for membrane damage by MB plus light has been re­ported in eukaryotic system8 (rat liver microsomes) . For TB-mediated photodynamic action, cell mem­brane appears to be the primary site of photodam­age9-11. However, more information on this aspect can be obtained from studies on structural alterations in cell membrane, which has not yet been reported . As for the nature of reactive species, while several stud­ies have implicated singlet oxygen (10 2) as the pri­mary damaging species in both MB and TB mediated

h d · fC 7 12-14 h ' ' ' f p oto ynamtc e 1ects · , t e parttctpatiOn o super-oxide (02- ") and hydroxyl radicals (OH-) has also

*To whom correspondence may be addressed at the address: Laser R & D Block D, Centre for Advanced Technology, P. 0 . CAT, lndore45201 3, India. Fax: 091-731488430, E-mail : okdube@cat.ernet.in Abbrevi ations used: MB, Methylene blue; PBN, N-t-butyl-a­phenylnitrone; 5-DS, 5-doxyl stearic acid ; TEMP, 2,2,6,6-tetramethylpiperidone; TB , toluidine blue.

been demonstrated 15 . In the present communication we describe results

of our investigation on photodynamic inactivation of Bacillus subtilis. EPR measurements show decrease in the cell membrane fluidity due to photodynamic damage of the lipophilic compartments. Investiga­tions on cell damage in the presence of sodium azide, D20 and glutathione as well as measurements on 10 2 and 0 2- · generation by EPR spectrometry show that whereas in TB-mediated photodamage, 10 2 is the major reactive species, in ME-mediated photodamage both 10 2and o2-· radicals are involved .

Materials and Methods Methylene blue and toluidine blue were obtained

from Himedia, India. Bacillus subtilis (MTCC 441) was grown aerobically for 18 hr at 30°C in LB me­dium. The cells were collected by centrifugation at approx. 15,000 g for 10 min , washed twice with phosphate buffered saline (PBS), pH 7.4 and sus­pended in 1 ml of the same buffer. To 0.9 ml cell suspension (approx . 108 cell/ml) 0 . 1 ml dye was added to give a final concentration of I 00 J...Lg/ml. Af­ter incubation for 30 min in the dark, the cell suspen­sion containing dye was transferred to a quartz cu­vette and irradiated with He-Ne laser (632.8 nm, made in Russia) under continuous stirring for varying time periods. The laser power measured using a Sci­entech-372 power meter (Boulder Co., USA) was 25 mW at the front face of the cuvette. Subsequent to laser irradiation 100 J...Ll aliquots were taken, diluted

246 INDIAN J. BIOCHEM. BIOPHYS., VOL. 37, AUGUST 2000

appropriately and plated onto solidified agar medium. After overnight incubation at 30°C, resultant colonies were counted.

Spin labeling of the cell membrane The cells were collected by centrifugation, sus­

pended in PBS containing I rnM 5-doxylstearic acid (5-DS), incubated for 30 min at room temperature in dark, the cell suspension was recentrifuged and the cells were collected and washed twice with PBS to remove excess 5-DS . The cells were finally sus­

pended in PBS containing I 00 !J.g/ml dye, re­incubated for 30 min in dark and then irradiated as

described above. Each I 00 !J.I aliquot was mixed with 10 !J.I NiCI 2 (I 0 rnM) to quench the 5-DS molecules extrinsic to the cell membrane. The sample was transferred to glass capillaries and one end was sealed. EPR spectra of the labeled probe were re­corded on EPR spectrometer (Varian E-104) . The instrument settings were: field 3237 G, power I 0 mW, microwave frequency 9.01 GHz, modulation 2X I G, time constant I sec and scan time 4 min. The rotational correlation time of the labeled probe was calculated according to McRae et al. 16• The hyperfine tensor 2T11 , which is a convenient measure of the flu­idity of membranes 17 was also measured .

Spin trap studies The cells were collected by centrifugation, sus­

pended in PBS containing I 0 mM TEMP (Sigma Chemicals Co., USA) as spin trap for 10 2 or I 0 mM PBN (Sigma Chemicals Co., USA) as spin trap for

free radicals. After incubation at 30°C for 30 min ,

dye was added to a final concentration of I 00 !J.glml. The cell suspension was re-incubated for 30 min in dark, and irradiated as described above. After irradia­tion 100 !J.I aliquots were transferred to glass capil­laries and one end was sealed. The 10rTEMP ad­ducts and 0 2--PBN adducts formed during irradiation were recorded on EPR spectrometer with instrument settings as above.

Results and Discussion Fig. I shows the dose dependent decreases in per

cent cell survival of Bacillus subtilis cells following He-Ne laser irradiation for 0-6 min in the presence of MB or TB. The I 00% cell survival in this figure rep­resents the cell density at 0 time irradiation . As de­scribed in Materials and Method section, the cells were mixed with dye and incubated for 30 min in

100 6

80

"@ 60 > .E

::l VJ

......... 40 a) u ~ 0

20

0

0

r0YNyYCH3

CH -W'~S~'-N-H 3 I + I

~ CH3 II Toluidine blue

2 4 6 Irradiation time (min)

Fig. 1-Cell survival (%) of Baciflus subtilis cells following photodynamic treatment with He-Ne laser irrad iation in the pres­ence of MB, (0 ) or TB , (6). [Each data point represents mean of three independent experiments. The standard deviation was less than 5%]

dark before He-Ne laser irradiation . Both MB and TB caused a dark toxicity on the cell suspension. The dark toxicity was maximum after 15 min incubation and remained constant for further incubation in dark. For MB it was about 17% and for TB about 13% as compared to a control sample without dye (data not shown). All the data points shown in Fig. I have been normalised with respect to the cell dens ity at 0 time irradiation.

Understanding photodamage requires knowledge of the primary site of photodamage and the primary re­active species. While the possible site for photodam­age can be cell membrane, enzymes, DNA etc., the primary reactive species are expected to be singlet oxygen and/or free radicals . In the case of MB it is generally believed that the main target of photodam­age is DNA7

. The possible involvement of cell mem­brane damage in photodynamic inactivation of bacte­ria has not been investigated. In the case of TB, sev­eral experimental observations suggest that photody­namic treatment leads to cell membrane damage. For example, it has been reported that photodynamic treatment with TB leads to enhanced transport of ac­ridine orange through the membrane, which in turn leads to increase in the mutation frequency of the yeast cells9

. It has also been observed to inhibit

DUBE eta /.: DYE-MEDIATED PHOTODYNAMIC INACTIVATION OF BACILLUS SUBTILIS CELLS 247

membrane transport system in yeast cells 10. Further, the fact that osmotic stabilizers were found to protect E. coli cells against TE-induced photodamage 11 also suggests involvement of cell membrane damage. The exact nature of cell membrane damage is however not well understood. We have therefore investigated bio­physical properties of the cell membrane by EPR spectrometry. The cells were labeled with spin label 5-DS, which intercalates into the lipophilic domains of the cell membrane and the motion parameter of the label in the microenvironment was computed to un­derstand lipid mobility. The changes in the rotational correlation time ('tc) of the labeled probe following laser irradiation of cells in the presence of ME as well as TE are shown in Fig. 2a. In both the cases, a

decrease in 'tc is observed which suggests an en­hancement in the rotation of the spin label, possibly due to damage to the lipophilic compartments of the cell membrane. Decrease in rotational correlation time generally indicates increase in membrane fluid­ity. However, since the rotational correlation time also depends on the cone angle, which defines the range of the rotational motion of the probe18, a reduc­tion in cone angle brought about by membrane altera­tions may also lead to a decrease in the rotational cor­relation time. Indeed, we observed increase in the value of hyperfine tensor 2T11 following He-Ne laser irradiation in presence of ME or TE (Fig. 2b). This suggests decrease in the fluidity of the cell mem­brane. Similar observations have been reported earlier by Joshi et a!. 19, from experiments on photodynami­cally induced alterations in plasma membrane of glioblastoma cells (U-87 MG) in which, they ob­served increase in plasma membrane rigidity but de­crease in the rotational relaxation time due to a re­duction in cone angle 19. The decrease in cell mem­brane fluidity is expected to be due to damage to un­saturated lipids caused by photodynamically gener­ated reactive oxygen species (ROS). This aspect has been investigated in different systems by several in­vestigators20.

In order to understand the role of reactive oxygen species (ROS) in membrane damage, mechanistic studies employing various quenchers for ROS were carried out. In presence of sodium azide, a quencher of singlet oxygen, photodynamic treatment of cells with both ME and TE did not lead to significant change in the value of 'tc and 2T11 of the labeled probe as compared with unirradiated cell sample (Fig. 2a, b). This would suggest involvement of singlet oxy-

20

16

0

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~ u

t--4

0

60

57

t= 54 N

51

6 48~~----~------~------~

2 0 4 Irradiation time (min)

Fig. 2-(a): Rotational correlation time (1:, ) and (b): hyperfine tensor 2T11 of 5-DS following photodynamic treatment of Bacillus subtilis cells with MB, (0) or TB , (~) in the absence (open sym­bol) or presence of 20 rnM sodium azide (fi lled symbol) . [Inset : EPR spectra of the 5-DS labeled cells after ( I), 0 min ; (2), 2 min ; (3), 4 min and (4) , 6 min of laser irradiation in the presence of MB. Each data point represents mean of three independent ex­periments. The standard deviation was less than 5%]

gen in cell membrane damage. Interestingly, whereas sodium azide protected the cells (almost completely) against TE-mediated phototoxicity, its effect on cell survival was only partial in the case of ME-mediated toxicity (Table I) . Substitution of D20 for water caused increase in the cell damage, which was s ig­nificantly higher in TE-mediated photodamage as compared to ME-mediated photodamage (Table I). Glutathione (GSH), a quencher of free radicals as well as 10 2 was found to cause an increase in the %

248 INDIAN J. BIOCHEM. BIOPHYS., VOL. 37, AUGUST 2000

Table 1-Per cent cell survival of the Bacillus subtilis cell in MB and TB-mediated photodynamic action in the presence of 0 20, sodium azide and glutathione [LIR- laser irradiation for 2 min . (Mean± SO, n = 3)]

Treatment

Control LIR

LIR+ 0 20 LIR + Sod. azide, 20 mM LIR+ glutathione, 15 mM

% Cell survival MB TB

100 ± 3.5 100 ± 4.2 64 ± 3.6 60 ± 3.8 45 ± 3.2 21 ± 2.5 69 ± 4 .3 91 ± 2.8 89 ± 3.2 66 ± 3.6

cell survival in ME-mediated cell damage. However, in TE-mediated cell damage the effect of GSH was not significant (Table 1 ). Control experiments with sodium azide, D20 or glutathione revealed no effect on Bacillus subtilis in dark either in presence or ab­sence of dye. The 100% cell survival in Table I rep­resents the cell density at 0 time irradiation.

In order to check whether the difference in re­sponse of ME- and TE-mediated photodamage to­wards quenchers of ROS is due to a difference in the magnitude of singlet oxygen generation, we moni­tored generation of singlet oxygen under both condi­tions. Fig. 3 shows the EPR signal intensity of TEMPO adduct formed by the reaction between 10 2 and TEMP when cell suspension containing ME or TE was irradiated with He-Ne laser for varying time periods. The TEMPO adduct obtained was identified by its characteristic nitrogen hyperfi ne splitting (aN) of 16.4 G. No significant difference in the generation of singlet oxygen during ME and TE-mediated pho­todamage was observed (Fig. 3). Since the magnitude of singlet oxygen generation was similar for ME and TE, the observed difference in the effect of quenchers for 10 2, on the ME- and TE-mediated photodamage (Table 1) suggest a possible involvement of free radi­cals in photodamage.

In order to confirm the involvement of free radical s in photodamage, we monitored generation of free radicals by EPR spectrometry. The use of PEN allows the measurements of both 0 2- • and OH. radicals. In TB-mediated photodamage, generation of free radi­cals was not observed. In ME-mediated photodamage, there was a dose dependent increase in the o2- . gen­eration both in the absence and presence of 20 mM sodium azide (Fig. 4). The presence of 0 2- ·was con­firmed by comparing absorption line characteristics of PBN-02- adduct obtained when 0 2- • was generated artificially using a pyrogallol autooxidation system

20 -H-t- 0

SG ;:::J

15 ~ ..._.,

.£ Vl c Q)

10 ....... .s c;:j c -~ Vl

~ 5 p... ~

0 2 4 6 Irradiation time (min)

Fig. 3-EPR signal intensities of 10 r TEMP adducts formed during photodynamic treatment of Bacillus subtilis cells with MB , (0) or TB, (11) . [Inset: EPR spectra of 10rTEMP adducts, re­ceiver gain 3.2x l04 G. Hyperfine splitting (aN)= 16.4 G. Each data point represents mean of three independent experiments. The standard deviation was less than 5%]

(Fig. 4, inset). The PEN-superoxide adduct obtained was identified by its characteristic nitrogen hyperfine splitting (aN) of 14.8 G . Further, by comparing ab­sorption line characteristics of PEN-OH adduct when hydroxyl radical s were generated artificially by Fen­ton reaction, it was confirmed that hydroxyl radicals were not generated.

The fact that 0 2- . generation was observed in ME­mediated photodamage and not in TE-mediated pho­todamage may be due to difference in the ability of these dyes to penetrate the cells. This difference is important because inside the cell the prevailing re­ducing conditions wi ll favour the free radical path­wa/5'21 '22. While ME is believed to penetrate the cells , there exists several reports to the effect that TE d h 11 9- 11 ?1 I . . oes not penetrate t e ce ·--. t 1s pertment to note here that in a recent study by Paardekooper et al. 2~, it was confirmed that incubation of yeast cells with TE from even up to 16 hr in dark did not result in diffu­sion of dye inside the cell. However, a rap id diffu­sion of a small amount of dye was observed followin g photodynamic treatment of cell s23

. T hi s was indicated by nullification of blue shift of absorption spectrum of the cell bound dye after photodynamic treatment.

DUBE eta/.: DYE-MEDIATED PHOTODYNAMIC INACTIVATION OF BACILLUS SUBTILIS CELLS 249

15~------------------------. SG

12

~I .Jj .-lift- .A a II

~~ ~~~ T

9

6

3

0 2 4 6 Irrad iation time (min)

Fig. 4-EPR signal intensities or 0 2--PBN adducts formed du ri ng photodynamic treatment or Bacillus subtilis cell s with MB in the absence (open symbol) and presence of 20 mM sodi um azide (filled symbol) . [Inset: EPR spectra of 0 2--PBN adducts formed in methylene blue photodynamic act ion. (I); in pyrogall ol au tooxi­dation system, (II ). Arrows represent absorption line characteris­tics of o 2- -PB N adducts. Recei ver gain 8 X 10~ G. Hyperlinc splitting (aN) = 14.8 G. Each data point represents mean of three independent experiments. The stand ard devi ati on was less than 5%]

However, in our study on Bacillus subtilis, for the irradi ation conditions used, no such change in the absorption spectra of ce ll bound dye was observed (data not shown) . Thi s suggests that the dye does not enter the cell s even after the photodynamic alterati on of cell membrane.

The difference in the permeability of the cells to the two dyes can a lso qualitative ly explain the differ­ence in the effec t of quenchers for ROS on photody­namic damage to ce ll s (Tab le 1). For example , 0 2- •

can be generated by intrace llul ar ME direct ly via type I pathway or as secondary spec ies from dye generated 10 2 in the presence of intrace llular reductants22

. Since sodium azide cannot penetrate the ce ll s, it cannot quench 10 2 generated by intracellul ar ME. Thi s is consi stent with our observations that in ME-mediated photodamage there was no significant diffe rence be­tween the magnitude of 0 2- • generation in the pres­ence and absence of sodium azide (Fi g. 4) . Further,

the fact that sodium azide did not lead to significant reduction in ME-mediated photodamage (Table l ) would suggest that involvement of o2- . in cell dam­age is dominant. In the case of TE, whi ch does not penetrate the cell s, 10 2 generated extracellularly can be quenched by sodium azide leading to protecti on against cell damage (Table 1). Glutathione, unlike sodium azide penetrates cel ls and can quench the re­active oxygen spec ies produced during photodynamic reac tion of intracellular ME resulting in the preven­tion of cell damage.

To conclude, our studies show that outside the cell both ME and TE lead to photodynamic generation of 10 2 causing a decrease in cell membrane fluidity. Further, our results suggest that whereas TB remains outside the ce ll and does not lead to free radical gen­erati on, in ME-mediated photodynamic action , 0 2- •

generated intracellularly is the major contributor to the cell damage.

Acknowledgement Authors wish to thank ProfS Eharti, School of Life

Sc iences, D A University , Indore for providing EPR faci lity and for he lpful di scussions.

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250 INDIAN J. BIOCHEM . BIOPHYS., VOL. 37, AUGUST 2000

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23 Paardekooper M, De Bruijne A W, Van Steveninck J & Van den Broek P J A ( 1995) Photochem Photobiol 61 , 84-89