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.

感染症学雑誌　第73巻　第10号

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



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



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).



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

1) Wilk IJ, Altmann RS, Berg JD: Antimicrobial activity of electrolyzed saline solutions . Sci Total Environ 1987; 63: 191-197.

2) Tanaka H, Hirakata Y, Kaku M et al.: Antimicrobial activity of superoxidized water . J Hospital Infect 1996; 34: 43-49. 3) Hotta K: Antimicrobial activity of electrolyzed NaCl solution: effect on the growth of Streptomyses spp . Actinomyce-

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.



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.

7) Shiba A, Shiba K: "Handbook of electrolyzed strong acidic water. " 1995. Igakujyoho-sha, Tokyo. 8) Okamura M, Ona H, Higuma K, Kobayashi K, Nanba K: Clinical use of electrolyzed strong acid water (the 3rd report).

J Niigata Soc Hospital Pharm 1995 ; 91: 59-62. 9) Sekiya 5, Ohmori K, Harii K: Treatment of infectious skin defects or ulcers with electrolyzed strong acid aqueous solu-

tion. Artificial Organs 1996 ; 21: 32-38. 10) Inoue Y, Endo 5, Kondo K, Ito H, Omori H, Saito K: Trial of electrolyzed strong acid aqueous solution lavage in the

treatment of peritonitis and intraperitoneal abscess. Artificial Organs 1996 ; 21: 28-31. 11) Nakae H, Endo S, Yamada Y: Two cases of the infected total hip arthroplasty for which oxidizing water was effective. J

Joint Surgery 1996 ; 15: 767-770. 12) Hayashi H, Kumon K, Yahagi N, et a!.: Successful treatment of mediastinitis after cardiovascular surgery using electro-

lyzed strong acid aqueous solution. Artifical Organs 1996 ; 21: 39-42. 13) Okubo K, Urakami H, Tamura A: Comparison of bacteriocidal activities of acidic electrolyzed water and acidic sodium

hopochlorite. J Japan Soc Environmental Infect 1998 ; 13: 184-188. 14) Iwasawa A, Nakamura Y: Bactricidal effect of acidic electrolyzed water-comparison of chemical acidic sodium hypo-

chloride (NaOCl) solution. J Japan Assoc Infect Dis 1996 ; 70: 915-922.

15) Iwasawa A, Nakamura Y: Cytotoxicity of aqua ozidation water. J Japan Soc Environ Infect 1994 ; 9: 12-18.

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.


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