Photochemistry und Pkotohioloyy Vol. 35 , pp. 501 to 505, 1982 Printed in Great Britain. All rights reserved

003 1 -8655/82/040501-05$03.00/0 Copyright 0 1982 Pergamon Press Ltd

ENHANCING EFFECT OF ASCORBATE ON TOLUIDINE BLUE-PHOTOSENSITIZATION OF YEAST CELLS

ATSUSHI IT0 and TAKASHI ITO*

Institute of Physics, College of General Education, University of Tokyo, Meguroku, Komaba, Tokyo 153, Japan

(Received 21 September 1981 ; uccrpted 29 October 1981)

Abstract -Using toluidine blue, a potent photosensitizer with a '02 dominated mechanism in yeast cell inactivation, it was found that addition of ascorbate to the sensitizer-cell mixture during illumination enhanced the inactivation. The enhancement required the presence of oxygen in the reaction mixture. The same enhancement was observed with methylene blue and thionine but not with xanthenes (Rose Bengal and eosin Y). The consumption of O2 and ascorbate seemed coupled in the enhancement. From the observation that the presence of ascorbate for a very short time (1 s) in the reaction mixture was enough to exhibit the same enhancement, it was concluded that the ascorbate enhancement processes are probably initiated in bulk medium, not intracellularly. The ascorbate enhancement may be a com- bined consequence of the high electron-accepting property of triplet toluidine blue and the strong tendency of ascorbate to act as an electron donor. The role of oxygen was not specified whether it was directly involved in the photoinactivation of cells. Addition of N; appeared to suppress the photoinacti- vation only in the higher fluence region where ascorbate had been consumed. Thus the ascorbate enhancement seems to occur under low fluence conditions and may probably be independent of the singlet oxygen mechanism.

INTRODUCTION

Many lines of evidence indicate that the photodyna- mic inactivation of yeast cells by sensitization with toluidine blue (TB)? occurs largely from the damage inflicted on the surface layer of cells through the sing- let oxygen mechanism (Ito, 1977; Ito and Kobayashi, 1977). More recent work showed that TB underwent some interaction with cells in the dark if incubated for a sufficient period of time in the presence of TB (Ito, 1980). Other recent work on TB sensitization with Escherichia coli (Wakayama et a/., 1980) and Neuro- spora conidia (Shimizu-Takahama et a/., 1981) con- firmed cell membrane as the site of photoinduced damage.

Since an in vivo system is so complex that often straight analysis is difficult, we have been looking for agents which directly interfere with the presumed sensitization pathways. There are a number of chemi- cals which suppress the photosensitized inactivation by quenching the active intermediate '02 or scaveng- ing responsible radicals (Foote, 1976 for review). On the other hand, apart from the reaction conditions such as temperature, O2 concentration or pH (Spikes and Livingston, 1969), few agents have been known which enhance the photosensitized inactivation via di- rect interaction with reaction pathways. In the present paper we will present some observations on the en- hancement of photoinactivation by ascorbate in yeast cells. As yet we have no definite answer for the mech- anism of enhancement, but in view of the unique

~~

* To whom correspondence should be addressed. t Ahbreurations: EY, eosin Y; MB, methylene blue; RB,

Rose Bengal; TB, toluidine blue: TH, thionine.

photochemical activity of ascorbate, the ascorbate effect provides a new aspect for the analysis of TB photosensitization of cells. During the preparation of manuscript we noticed Davies et al. (1976) had reported that an ascorbate-methylene blue coupled system produced a remarkable photosensitization reaction in an in vitro system.

MATERIALS AND METHODS

Cells of Saccharomyces cerevisiae. The AD4 strain was used. Stationary cells were harvested from 48 h cultures in YEP (yeast extract and peptone) medium as usual. Cells were washed 3 times with distilled water and suspended in 0.01 M phosphate buffer. The pH was adjusted to 6.8 or 7.5 depending on the experiments. Chemicals used for prepar- ing the buffer were of reagent grade and obtained from Koso Chemical Co.

Sensitiiufion of cells. The following dyes were used as photosensitizers, toluidine blue (TB), thionine (TH), methyl- ene blue (MB), eosin Y (EY) and Rose Bengal (RB). These dyes were obtained from Tokyo Kasei Co. except for EY which was obtained from Koso Chemical Co. Dyes were used without further purification. Concentrated stock sol- utions were diluted with the 0.01 M phosphate buffer before use to give a final optical density of about 1 at maximum absorption wavelength for each sensitizer. Cells were usually incubated with sensitizers for 30 min at 30°C.

Addition of ascorbate. When necessary, L-ascorbic acid (Tokyo Kasei Co.) was added to the reaction mixture (cells plus sensitizer) immediately before illumination. Separate tests showed that the addition of 10 mM ascorbate caused the bleaching of thiazines. The concentration used in the present experiments (< 1 mM) had no effect on the above mentioned dyes as far as tested by the absorption spectra. After illumination the reaction mixture was immediately diluted by a factor of at least 1 x lo3 to avoid possible complications due to secondary reactions.

Illumination. The illumination setup was the same as de- scribed previously (Ito and Kobayashi, 1977). White light was used. The fluence rate was roughly 560W/m2 as

501

502 ATSUSHI ITO and TAKASHI ITO

measured by a calibrated photometer (IL200, International Light Co.). The density of cells was 1 x 107/m/ and the illumination was performed in an ordinary 1 cm-light path cuvette with stirring. For short illumination experiments a more intense light source providing a fluence rate of approx 20kW/m2 was used. Coupled with this short illumination a rapid mixing technique was employed which was used for investigating the time course of sensitizer-cell interaction (Ito, 1980). Depending on the experiments, NaN, (Kishida Kagaku Co., Tokyo) was added to the reaction mixture at least 20 min prior to illumination as a source of N;.

Assay for inactivation. The survival fraction was obtained by plating the treated cells, after appropriate dilution, on complete medium. Colony countings were made after 3 days of incubation at 30°C.

Measurements of O2 and ascorbate. The concentration of oxygen in the reaction mixture was measured by Beckman oxygen analyzer (Model 777, Beckman-Toshiba, Ltd.). A home-made container with an adaptor for the polaro- graphic oxygen sensor (type 39065) on top was held in the light beam in the same arrangement as the inactivation experiments. The output of the oxygen analyzer was traced by a recorder. Ascorbate, added to the reaction mixture, was monitored by the absorption at 268 nm.

RESULTS AND DISCUSSION

Effect of ascorbate on the TB-photosensitization of yeast cells

Figure 1 shows the effect of addition of ascorbate to the TBcel l mixture on the photodynamic inactiva-

t \

1 0 2 4 6 8 10 12 15 16 18

I IL u mi n a t ion (mi n)

Figure 1. Fluence-survival curves for the TB-sensitized photoinactivation of yeast cells. Experiments under N, are marked by filled symbols. (o), Control (TB plus cells); (A), presence of 0.005 mM ascorbate; (0). presence of 0.01 mM ascorbate; (V). presence of 0.1 mM ascorbate; ( x ), absence of TB but in the presence of 0.1 mM ascorbate; (Dj, under N, bubbling in the absence of ascorbate; (V), under N, bubbling (presence of TB and axorbate). Other conditions: cell density 1 x 10’/m/; concentration of TB, OD636 = 1.0; medium, 0.01 M phosphate buffer, pH 6.8; temperature 28°C; fluence rate of light 560 W/mZ. Unless

otherwise stated the reaction mixture was air saturated.

tion of yeast cells. The ascorbate was added immedi- ately prior to illumination. It can be seen that 0.1 mM ascorbate enhanced the photodynamic inactivation of the cells quite significantly. Ascorbate (0.5 mhf) plus illumination under either atmospheric or N2 con- ditions had no effect in the absence of TB on survival of cells. We also observed in other experiments that the length of preincubation (48 h) with ascorbate prior to illumination was not an important factor in the enhancement. Ascorbate alone had no damaging effect in the dark at a concentration lower than 10 mM. Therefore, the present enhancement is clearly distinguishable from a dark inactivation effect of ascorbate as reported with viruses (Murata and Kita- gawa. 1973). Figure 1 also shows that thc unhance- ment was dependent on the concentration of ascor- bate. The minimal concentration with a discernible effect was 0.005mM. The effect increased gradually up to 0.1 mM where it seemed saturated.

Requirement of O2 in the ascorhate enhancement

To see whether O2 is required for the ascorbate enhancement we replaced O2 in the reaction mixture (cells, TB and ascorbate) with N,. In thesc experi- ments N2 (Research grade) was bubbled through the reaction mixture for 5 min prior to illumination (50 m//min). The bubbling continued during illumina- tion at the same rate, The results are also shown in Fig. 1. As will be seen, inactivation was well sup- pressed under N2, although a small initial drop of survival was noticed (inverted filled triangles). Since this small drop could be ascribed to the residua{ O2 present in the system, the involvement of O2 in the enhancing reaction may be assumed. We also noticed that after the initial phase. roughly corresponding to the drop mentioned above, the bleaching of dyes pro- gressed rapidly under N,. Thus, O2 seemed to play a dual role; one concerned directly with enhancing reaction and the other with regeneration of dyes from the photo-reduced state (see below).

Speczfificity of t h i i i i i i i ~ dyes in ascorhutc enltancwnent

Similar enhancement was also observed with other dyes in the thiazine group, T H and MB. The results are shown in Fig. 2. To see if the enhancement by ascorbate is selective for thiazine dyes, we also tested RB and EY as a model for xanthene dyes. EY is known to attack the yeast cells in the presence of light (Cohn and Tseng, 1977). As shown, the photoinactiva- tion with EY as a sensitizer was not enhanced by ascorbate. Similar results were observed with RB (data not shown). These results suggest that the enhancing effect of ascorbate is associated specifically with thiazine dyes.

Consumption of O2 and ascorhate The thiazine dyes are commonly susceptible to

bleaching upon illumination, the phenomenon being ascribable to the electron or proton accepting prop- erty of the excited state of the dye. In fact, the above

Photosensitization in yeast 503

0 5 10 15 20 0 5 10 15 XI25 30 0 5 0 15 20

I l i u m i n a t i o n ( m i n )

Figurc 2. Effects of ascorbate on TH, MB, and EY sensi- tized photoinactivation of yeast cells. (0). Control (sensi- tizers plus cells); (A) illumination in the presence of ascor- bate (0.1 m M for TH and 1 mM for MB, and EY). Concen- trations of sensitizers; OD,,, = 1.1 for TH, OD,6, = 1.0 for MB, and ODsl7 = 1.1 for EY. Other conditions were

the same as in Fig. 1.

results showed a rapid bleaching under N,, suggesting that O2 acts on the semi-reduced dye to restore its original state. Oster and Watherspoon (1954) ob- served a rapid photobleaching of MB and TH in the presence of 1 mM ascorbate with subsequent restora- tion of the color by 02. In our system, it was thus of interest to see if O2 and ascorbate are consumed in the process of enhancement. The results of such measurements are shown in Table 1. In the presence of ascorbate the consumption of O2 was very much faster than in the absence of ascorbate. It should be noted, however, that the concentration of ascorbate in these measurements was considerably higher for tech- nical reasons than in the usual photodynamic experi- ments reported in this paper. The table also shows that ascorbate consumption occurred in the presence of 02, and that it was significantly faster than the

consumption of 0, as measured in the absence of cells. We could not perform the absorption measure- ment on ascorbate in the presence of cells. The stoi- chiometric relationship between the consumption of O2 and ascorbate was not established. Since only a few percent drop of 0, was observable after about 30 min illumination with the TB system in the absence of ascorbate, the observed rapid consumption of O2 is likely to be a characteristic of the ascorbate-TB sys- tem. This table also shows that TB was bleached under N2 with the consumption of ascorbate. Of course. in the absence of ascorbate the bleaching of TB was much slower under 02. A slower rate of bleaching of TB under O2 may be ascribed to the competitive restoration of TB by 0, as mentioned above. Based on these observations it seems that the presence of ascorbate is the primary factor for the bleaching of TB. Davies et al. (1976) reported the pho- todegradation of alginate, a polysaccharide, by the M B sensitization coupled with thc consumption of ascorbate. They measured the consumption of both O2 and ascorbate, and based on the kinetic consider- ations, they concluded that initiation of enhancing processes was the proton transfer from ascorbate to MB.

Reaction site of ascorbute enhancement

In order to obtain information on the location of ascorbate with respect to the cells in initiating the enhancement reaction, a fast mixing-short illumina- tion technique was applied. This technique enables us to investigate dye-cell interaction in the time range from several seconds to 1 min in the reaction system (Ito, 1980). In the present attempt ascorbate-contain- ing TB solution (kept in the dark) was rapidly mixed with cells (in 20 ms) and then illuminated immediately (less than I S ) with a short duration of the intense light. The illumination times were 8-32s. A remark- able enhancement under these conditions, as seen in

Table 1. Consumption of 0, and ascorbate in TB system upon illumination in the absence and in the presence of yeast cells. The consumption has been expressed as the light fluence (min) required to reduce the initial values of O 2 and ascorbate concen-

trations to SO?,

0, consumption

In the prescnce of ascorbate In the absence

( 1 m M ) of ascorbate

In the absence 2.9 undetectable

In the presence 3.2 undetectable3 of cells

of cells

Ascorbate consumption*

Under Oz Under N, (air saturation) bubblingt

0.9 I .s (bleach of TB)$

not measured not measured (bleach of TB)

* Initial O2 pressure was attained by air saturation. Initial concentration of ascor- bate was 0.05 mM. Illumination conditions are the same as in inactivation experiments.

t Bubbling of Nz at the flow rate of 50m//min for 5 min prior to and during illumination.

$ After less than 3 min of illurninatton bleaching became noticeable. p After long illumination (e.g. 34 min) only a few percent of decrease in O2 preaaure

was measured.

P A P 1 5 4 ,

504 ATSUSHI ITO and TAKASHI ITO

100

50

h

s - 20 .? 10

- 0

>

2 0 5 10 15 20 25 30 35

I l l u m i n a t i o n ( s )

Figure 3. Fluence-survival curves in the fast mixing-short illumination experiments. (O), Control (TB plus cells); (A), ascorbate (0.8 mM) present in the TB solution. Period of incubation before illumination was less than 1 s. Concen-

trations of TB and cells were the same as in Fig. 1.

the difference of two fluence-survival curves in Fig. 3, indicates that ascorbate was already potent immedi- ately after it was mixed with cells, suggesting that the ascorbate enhancement reaction was initiated in coupling with the formation of excited TB in such a situation that both components were presumably located in the bulk phase of the medium (outside the cell).

Effects of N ;

If the interaction of the excited triplet of TB with ascorbate is crucial in the enhancing effect, as sug- gested in the above, it would be natural to investigate how modifying agents of known characters would affect the enhancing effect of ascorbate. We tested if a singlet oxygen quencher could affect the enhanced inactivation. As cab be seen in Fig. 4a, the addition of 0.05 M N; indeed suppressed the ascorbate effect sig- nificantly over the whole range of survival tested, but more so in the high fluence region (lower survival region), where protection occurred beyond the control (TB alone). This rather strange fluence-survival re- sponse promoted further careful experiments. When a lower concentration of N; (0.01 M) was used (Fig. 4b), the above feature was more apparent. In this case, the suppression of ascorbate enhancement by N; was small until 4 min of illumination. Then, afterwards, in the high fluence region, the protection occurred beyond the control as in the former case. It seems that the time when the trend of the protection became apparent corresponds roughly to the time when the ascorbate is substantially consumed (see results in previous section). In other words, as far as ascorbate exists, the protection by N; which is usually expected for the singlet oxygen mechanism occurs only

slightly. Thus we speculate that the ascorbate en- hancement, which was conspicuous in the low fluence region as the decrease of a shoulder in the survival curve (Figs. 1 and 4), is not caused by the increased generation of '02 during that period of illumination. Some suppression at a higher concentration of N; (Fig. 4a) could partly be ascribed to the quenching of triplet TB (Kraljic and Lindqvist, 1974).

CONCLUDING REMARKS

It would be expected that the illumination of the TEascorbate system produces semi-reduced TB fol- lowing triplet formation, since TB is easily reduced in the excited state via an electron transfer reaction (Kramer and Maute, 1972; Bonneau et a/., 1974; Bon- neau and Pereyre, 1975) with ascorbate. Thus the ascorbate enhancement observed in the present study is likely to be related to the fate of semi-reduced TB. The semi-reduced TB may give O2 an electron, pro- ducing O j (Rizzuto and Spikes, 1977), for which no attempt to identify was made in the present study. Then H 2 0 2 would also be produced when 0; and ascorbate react (Epel and Neumann, 1973 ; Nishikimi, 1975); however, we have found that H,Oz exerts little inactivating effect on yeast cells even at fairly high concentrations (unpublished observation). I t is not known whether the ascorbate radical or dehydro- ascorbate have an inactivating effect on cells. O i (or HO;) could be a responsible intermediate in the en- hancement, as suggested by a recent in uitro model experiments (Muller-Runkel et a/., I981), but obviously needs confirmations.

Formation of '02 and semi-reduced TB via triplet TB may be competitive in the presence of electron

100

50 - s - 20

10

> 5

- > .- L

3 m

2

1- 0 2 4 6 8 1012 0 2 4 6 8 1012

1 1 1 u m i n a t io n ( m i n )

Figure 4. Effects of N; on the fluence-survival curves of the TB sensitized photoinactivation of yeast cells in the presence and absence of ascorbate (0.1 mM). (a): 0.05 M N;. (b): 0.01 MN;. Symbols: (0) TB plus cells; (O), TB, cells, and N; ; (A), TB. cells and ascorbate; (A), TB, cells, ascorbate and N;. Other conditions were the same as in

Fig. 1.

Photosensitization in yeast 505

donors. Our system, including cells, unfortunately is not suitable for detailed kinetic analyses. Being aware of the limitations, we propose to outline the photo- sensitized inactivation of yeast cells in the presence of TB and ascorbate in the following way. The presence of ascorbate and 0, (with TB) makes the shoulder of the fluence-survival curve smaller than in the absence of ascorbate. I t persists as long as ascorbate is present (first several minutes under the present conditions). The active intermediate responsible for this inactiva- tion enhancement is not likely to be ‘02 in view of the possible photochemical reactions and of the inef- fectiveness of N;. After the ascorbate dominated reaction is over, the survival curve appears to follow the same slope as in the absence of ascorbate. This latter phase can be explained by the usual TB-sensi- tized ‘02 mechanism since the protection by N; occurs normally.

Acknowtrdgements We thank Drs. J. D. Spikes and Y. Usui for comments and for providing invaluable literature. We are also grateful for the technical assistance of Akiko Hirose. This work has been supported by a Grant-in-aid from the Ministry of Education, Sciences and Culture, No. 348382 and 558080.

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