Cytotoxicity and Microbicidal Activity of Electrolyzed...
Transcript of Cytotoxicity and Microbicidal Activity of Electrolyzed...
1025
Cytotoxicity and Microbicidal Activity of Electrolyzed Strong Acid Water
and Acidic Hypochlorite Solution under Isotonic Conditions
Koji OKUBO1), Hiroshi URAKAMI" & Akira TAMURA2)1) Department of Pharmacy, Koseiren Murakami General Hospital,
2-17 Tabata-cho, Murakami City 958-0854, Japan2) Department
of Microbiology, Niigata College of Pharmacy,
5-13-2 Kamishin'ei-cho, Niigata950-2081, Japan
(Received:April26,1999)
(Accepted:July8,1999)
Key words: acidic electrolyzed water, acidic hypochlorite solution, cytotoxity, mi-
crobicidal activity
Abstract
The cytotoxic effects of electrolyzed strong acid water and acidic hypochlorite solution, as well as
these solutions after isotonization, against cultivated L cells were compared along with their microbicidal
activities. Isotonization was accompanied by a reduction in the cytotoxic effects of these solutions against
L cells. Microbicidal activity was also reduced somewhat but was still retained after isotonization. No dif-
ference was observed in these properties between these antiseptic solutions. The results obtained indi-
cate that acidic hypochlorite solution may be useful as well as acidic electrolyzed water.
〔J. J. A. Inf. D. 73: 1025-1031, 1999〕
Electrolyzed strong acid water prepared by electrolyzing salt-containing water is known to have mi-
crobicidal activity1•`6) and is used not only for the disinfection of hands, medical instruments and the work-
ing environment, but also for the treatment of atopic dermatitis7), burned or scalded skin7), decubitue ,
and contaminated wounds9). Furthermore, recent reports have demonstrated beneficial effects of use of
electolyzed water in surgical treatment for peritonitis and intraperitoneal abscess10), total hip artro-
plasty11), and mediastinitis12). We also reported previously that severe decubitus was improved by treat-
ment with electrolyzed strong acid water8).
Acidic electrolyzed water was effective microbicidal activity when its pH is lower than 2.7, residual
chlorine content is in the range of 10 to 50 ppm, and redox potential higher than +1,100mV5,10). We re-
ported previously that acidic sodium hypochlorite solution, which has similar pH, chlorine content and re-
dox potential to electrolyzed water, showed microbicidal activities identical to those of electrolyzed
water13). This acidic hypochlorite solution can be prepared more easily and economically than electrolyzed
water14). Therefore, acidic hypochlorite solution is considered to be valuable for use in place of acidic elec-
Correspondence to: Koji OKUBO Department of Pharmacy, Kaetsu Hospital, 1-23 Nakazawa-cho, Niitsu City, Niigata 956-0831, Japan
平 成11年10月20日
1026 Koji OKUBO et al
trolyzed water.
The toxic effects of electolyzed water and acidic hypochlorite solution on eukaryotic cells have not
been investigated well7,15). As both antiseptic solutions are highly hypotonic, isotonization is considered to
be necessary to reduce their toxic effects. In this study, the toxic effects of the antiseptic solutions against
cultivated L cells, as well as their microbicidal activities, were compared before and after isotonization.
Materials and Methods
Preparation of disinfectants. Acidic electrolyzed water was prepared by electrolysis of 0 .017 mol/
ml NaCl solution in city tap water or in distilled water for injection (DWI) with a Suntron MWB 2: 2.5L
(Koshin Co., Ltd., Kyoto) . Acidic hypochlorite solution was prepared by dissolving 82 tl of 1.1 w/v% so-
dium hypochlorite (Milton, P & G, Hyogo, Japan) and 35 ƒÊl of 11.6N hydrochloric acid in 100 ml of DWI. Iso-
tonic solutions of both disinfectants were prepared by adding NaCl at 0.9 w/v% final concentration. pH,
residual chlorine concentration and redox potential in the antiseptic solution were determined by the
methods described previously13).
Assay of cytotoxicity. L-929 cells derived from mouse fibroblasts were cultivated in Eagle's mini-
mum essential medium (MEM; Nissui, Tokyo) containing 10% bovine serum (BS; the mixed medium is
abbreviated as 10%BS-MEM). Aliquots of 100 ƒÊl of the cell suspension in 10%BS-MEM at the concentra-
tion of 1•~106 cells/ml were inoculated into each well of 96-Well Cell Cluster plates (Costar Co., USA) and
after 24-hrs incubation at 37•Ž under a 5% CO, atmosphere, the cell layers formed were used to assay
the cytotoxic effects of the disinfectants.
Electrolyzed water and hypochlorite solution, and their isotonic solutions were diluted serially 2-old
with DWI or 0.9 w/v% NaCl. L cell layers in each well were washed 3 times with 150 ƒÊl of 10% BS-MEM ,
and inoculated with 100 ƒÊl of each serially diluted disinfectant solution. After 20-min incubation at room
temperature (23•`25•Ž), 100ƒÊl of 2-fold concentrated MEM containing 20% BS was added to wells
treated with hypotonic disinfectant solution, and 100 ƒÊl of 20%BS-MEM was added to wells treated with
isotonic disinfectant solution. Then, cells were stained with neutral red or trypan blue for determination
of viable and dead cell ratios as follows.
For staining with neutral red, each cell layer was washed twice with 100 ƒÊl of 10%BS-MEM and incu-
bated for 24 hrs at 37•Ž in 100 ƒÊl of 10% BS-MEM containing 0.03% neutral red. Then, the cell layer was
washed twice with 150 ƒÊl of phosphate buffered saline (PBS), all cells in one well were killed by incubation
with 100 ƒÊl of undiluted hypotonic electrolyzed water for 20 min at room temperature, and absorbance at
405nm due to the released neutral red was measured with a Bio-kinetics reader (Bio-Tek Instrument, Wi-
nooski, VT). As a control, cells incubated with 10%BS-MEM in place of disinfectant solution were stained
with neutral red by the same method as described above. The viability after treatment with disinfectant
was expressed as the ratio of absorbance of each culture against that of the control culture .
For dead cell staining, each cell layer was washed twice with 100 III of PBS and stained with 100 ƒÊl of
0.1% trypan blue in PBS for 1—min at room temperature. Then, the cell layer was washed twice with 150
μl of PBS, and color released by lysis of cells with 0.025N sodium hydroxide for 5-min were assayed at 630
nm with an ELISA plate reader. As a control, all cells in one well were killed by incubation with 100 ƒÊl of
undiluted acidic electrolyzed water for 20-min, and stained with trypan blue by the same methods . The
ratios of absorbance of each well against that of the control culture were calculated.
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Isotonic Electrolyzed Water and Acidic Hypochlorite Solution 1027
Assay of microbicidal activity. Microorganisms used were Escherichia coli ATCC 23216, Staphylococ-
cus aureus ATCC 12600, Pseudomonas aeruginosa ATCC 10145, methicillin-resistant Staphylococcus aureus
(MRSA) isolated from a clinical specimen in Murakami General Hospital, Bacillus subtilis ATCC 6051, and
Candida albicans NCTC 8143. C. albicans was cultivated in Sabouraud dextrose broth (Difco Lab., USA) for
3 days, and other bacteria were cultivated in heart infusion broth (Eiken Chem. Co. Ltd., Tokyo) for 2 days
except B. subtilis which was cultivated for 4 days. Endospore suspensions of B. subtilis were prepared by
heating B. subtilis suspension in PBS at 65t for 30 min. Numbers of microorganisms in samples were de-
termined by colony counting methods as described previously13).
Microbicidal activities of disinfectants were assayed by two methods as described previously13) .
Briefly, in one method, 10 ƒÊl of microbial suspension was mixed with 200 IA of undiluted disinfectant solu-
tion, 20 Ill samples were harvested at 10, 30, 60, 180 and 600 sec after incubation at room temperature ,
mixed with 100ƒÊl of nutrient broth containing 3% sodium thiosulfate to neutralize the effects of disinfec-
tants, and growth of surviving microorganisms was observed after 24 hr of incubation at 35•Ž by observ-
ing turbidity with the naked eye (after 72 hr incubation at 25•Ž in the case of C. albicans) . In another ex-
periment, aliquots of 10 ill of microbial suspensions were mixed with 100 il of serially 2-fold diluted disin-
fectant solutions in wells of 98-well multiplates, kept for 5 min at room temperature then 100 Ill of 2-fold
concentrated nutrient broth containing sodium thiosulfate was added to each well, and survival of the mi-
croorganisms in each well was checked after 24-hr incubation at 35•Ž
Results
1. Characterization of disinfectants.
The pH, residual chlorine contents and redox potential of disinfectants were almost identical not only
between acidic electrolyzed water and acidic hypochlorite solution but also between before and after iso-
tonization (Table 1).
2. Cytotoxicity of disinfectants before and after isotonization.
L cells were treated for 20 min with serially diluted disinfectant solutions, and ratios of surviving and
dead cells after treatment to those in control culltures were determined as described in Materials and
Methods. Fig. 1A shows the percentages of surviving cells estimated by neutral red staining , At all dilu-
tions of disinfectants examined, isotonic solutions showed higher ratios of surviving cells than those be-
fore isotonization, indicating reduction of toxicity by isotonization. Isotonic solution showed toxic effects
at dilutions of less than 2-fold, and near maximum values of 60 to 80% were reached at more than 4-fold
dilutions. In the case of non-isotonized solutions, constant surviving cell percentages were seen at dilu-
tions of 16-to 32-fold but the values were limited to 30 to 40%. This may have been due to cell damage
on exposure to hypotonic solution.
Table 1 Characterization of disinfectants used
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1028 Koji OKUBO et al
Fig. 1 Percentages of surviving (A) and dead cells
(B) after treatment with serially diluted disinfec-
tant solutions. Acidic electrolyzed water before (•¡)
and after (• ) isotonization; acidic hypochlorite so-
lution before (•œ) and after (•›) isotonization.
A
B
Fig. 1B shows dead cell ratios determined by staining with trypan blue after treatment of cells with dis-
infectant solutions. Dead cells were observed at dilutions of less than 8-fold but dead cell ratios were
much lower in cultures treated with isotonic than hypotonic solution.
From the results shown in Figs. 1A and B, we concluded that although there was no difference be-
tween electrolyzed acid water and acidic hypochlorite solutions, cytotoxic effects of both disinfectants
against L cells at lower dilutions (less than 8-fold dilution) , were clearly reduced by isotonization of the
antiseptic solutions.
3. Microbicidal activity.
Viable counts of bacteria and a fungus used here were as follows, (in 10 lil suspensions) : E. coli, 2.6•~
105; S. aureus, 3.9•~105; P. aeruginosa, 2.5•~105; MRSA, 5.8•~105; B. subtilis, 0.4•~105; and C. albicans, 1.0•~
105. When 10 IA of the suspension was mixed with 200 ƒÊl of acidic electrolyzed water or with acidic hypo-
chlorite solution, all the microorganisms except B. subtilis lost viability after 10 sec of incubation at room
temperature (upper part of Table 2) . Some bacteria survived after 10 sec of incubation with isotonic disin-
fectant solutions but none was seen after 30 sec, except in the case of B. subtilis (lower part of Table 2).
In another experiment, 10 IA of microbial suspension was mixed with 100 ill of serially 2-fold diluted
disinfectants and survival of the microorganisms after 5-min incubation at room temperature was exam-
ined. In the case of non-isotonic disinfectants, microbicidal activities were detected at 4-to 8-fold dilutions
against E. coli, S. aureus, and C. albicans, and at 1-(original) or 2-fold dilutions against P. aeruginosa and
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Isotonic Electrolyzed Water and Acidic Hypochlorite Solution 1029
Table 2 Survival tests of microorganisms after contact with disinfectants for different periods
1) and 2) microorganisms were incubated wtih DWI1) and 0.9%NaCI2) instead of disinfectants
3) Growth of microorganisms is shown as follows: +, growth; -, no growth; •}, growth in one of two experiments
Table 3 Survival tests of microorganisms treated with 2-fold serially diluted disinfectants.
1) and 2) microorganisms were contact with DWI1) and 0.9%NaCl2) instead of disinfectants
3) Growth of microorganisms is shown as follows: +, growth; -, no growth; •}, growth in one of two experiments
MRSA (upper part of Table 3). After isotonization of these antiseptic solutions, microbicidal activities
were reduced to 1-to 2-fold dilutions against E. coli, S. aureus and C. albicans, and to 1-fold dilutions again-
st P. aeruginosa and MRSA (lower part of Table 3).
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1030 Koji OKUBO et al
In summary, microbicidal activities were reduced by isotonization, but the undiluted solutions still re-
tained their activities, and no clear difference was seen between acidic electrolyzed water and acidic hy-
pochlorite solution. However, these antiseptic solutions showed no effect against bacterial endospores of
B. subtilis.
Discussion
We reported previously that acidic hypochlorite solution showed microbicidal activity identical to
that of electrolyzed strong acid water, and this hypochlorite solution was more easily prepared , cost less,
and required no special facilities for manufacture of sterilized preparations13) . Electrolyzed strong acid
water is used not only for disinfection of the hands and environment, but also for extermination of infec-
tious microorganisms from contaminated skin or the peritoneal cavity , and for prevention of bacterial
contamination in surgical operations10)•`12). For clinical application of this antiseptic solution, its safety
should be carefully examined.
The cytocidal effects of acidic electrolyzed water, as well as acidic hypochlorite solution , against eu-
karyotic cells have not been well investigated. Isotonization of these highly hypotonic antiseptic solutions
seems to be desirable for reduction of their cytocidal effects and alleviation of pain in clinical use . In this
study, we examined the cytotoxic effects of acidic electrolyzed water and acidic hypochlorite solution
against cultured L cells under hypotonic and isotonic conditions. We observed a reduction of the cytotoxic
effect of these antiseptic solutions to L cells by isotonization, although the pH, residual chlorine content
and redox potential were not altered by isotonization. The microbicidal activities were also decreased by
isotonization, but the undiluted solutions still retained their microbicidal effects. No difference was ob-
served between these two disinfectants, indicating that acidic hypochlorite solution or acidic electrolyzed
water could be used for the same purposes.
Although a small number of dead cells (5 to 10%) stained positively with trypan blue were seen after
treatment with highly diluted disinfectants (Fig. 1B), plateau levels of surviving cell percentage were ob-
served in this high dilution range (Fig. 1A); 60 to 80% after treatment with isotonic solution and 30 to 40
%with hypotonic solution. This discrepancy between dead cell and surviving cell analyses may be due to
the differences in assay methods used. Dead cell percentage was determined shortly after treatment , but surviving cell percentage was determined after 1-day incubation in neutral red-containing medium . Cells
treated with disinfectant solutions, especially with hypotonic solution, may by damaged and may lose
their ability to phagocytose neutral red during this 1-day incubation period .
References
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tologica 1994; 8: 51-56. 4) Kouda M, Satoh Y, Haraguchi Y, Hoshino M, Matsuzaki H: Bacteriocidal effect of function water (electrolyzed strong
acid solution). Jpn Pharmacol Ther 1995; 23: 843-847. 5) Iwasawa A, Nakamura Y: Antimirobiological effect and application of acidic electrolyzed waater . J Japan Soc Environ
Infect 1996; 11: 193-202.
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6) Hitomi S, Baba 5, Yano H, Morisawa Y, Kimura S: Antimicrobial effects of electrolytic products of sodium chloride-comparative evaluation with sodium hypochlorite solution and efficacy in hand washing. J Japan Assoc infect Dis 1998 ; 72: 1176-1181.
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強酸性水 と酸性次亜塩素酸水の等張条件下での細胞毒性 と殺菌効果
1)新潟県厚生連村上総合病 院薬剤部
2)新潟薬科大学微生物学教室
大 久 保 耕 嗣1)浦 上 弘2)多 村 憲2)
要 旨
食塩水の電気分解で調製した強酸性水,お よび
塩酸と次亜塩素酸ナトリウムで調製した酸性次亜
塩素酸水,及 び両消毒水を塩化ナトリウムで等張
化した場合の培養細胞に対する毒性を比較検討し
た.
その結果,両 消毒水とも等張化することにより
細胞毒性は減弱した.等 張化により殺菌効果も減
退したが,な おその活性 を保持 していた.こ うし
た現象に関 しては,強 酸性水 と酸性次亜塩素酸水
との間に全 く差異がな く,酸 性次亜塩素酸水は強
酸性水 と同様に消毒水 としての効果 を持つ ことを
確かめた.こ の結果,酸 性次亜塩素酸水は等張化
した条件下で も強酸性水 と同等の利用価値がある
ことが判明 した.
平成11年10月20日