J. Biol. Macromol., 5(3), 53-60 (2005)

ArticleEffect of electrolyzed water on the orientation of cortical microtubules in Spirogyra cells
Kazuyoshi Iwata and Yoh Sano
Faculty of Pharmaceutical Sciences, Setsunan University
Nagaotoge-cho, Hirakata, Osaka, 573-01, Japan
Received May 24, 2005, accepted August 19, 2005.
Effect of electrolyzed water on the orientation of cortical microtubules (MTs) in

Spirogyra cells was studied. Acidic electrolyzed water, alkaline electrolyzed water, andtap water affected the orientation of cortical MTs in different ways from each other.
About 90% of cells treated in acidic electrolyzed water had random or not repolymerized
MTs. All cells treated in alkaline electrolyzed water had transverse MTs. In tap water,
many cells were observed to have longitudinal MTs. The orientation of cortical MTs
repolymerized in tap water, pH of which was adjusted at about 4.5, was not so different
from that in tap water (pH 7.4), which indicates that this pH-adjusted tap water andacidic electrolyzed water affect MT orientation differently. Available chlorine
concentration and pH of this pH-adjusted tap water and acidic electrolyzed water werealmost similar.

Because low concentration of salts changed the effect of available

chlorine (NaClO) on MT orientation, these results imply that composition of ions might
be important for effect of acidic electrolyzed water on MT orientation in Spirogyra cells.
Key words: Spirogyra, cortical microtubules, electrolyzed water, available chlorine
concentration
Many kinds of functional water are

Spirogyra (a species of algae) cells (2).

known (1), and are often used without

To further investigate utility of thissufficient scientific evidence. We alreadymethod, it is necessary that effects ofreported that one kind of functional

other functional water on the orientationwater treated by far-infrared light couldof cortical MTs in Spirogyra cells should

affect the orientation of cortical MTs in

be studied.
Electrolyzed water, which is made by

The abbreviations used are: APM,electrolysis of tap water, is used in wide

Amiprophos-methyl; APW, Artificial

fields (1).

pond water; MT, microtubule.

anode and the cathode is as follows.

Water electrolysis on the54

Cortical microtubules in Spirogyra cellsPlant materials and treatments with2H2O Æ O2 + 4H+ + 4e- (anode)
2H2O + 2e- Æ H2 + 2OH- (cathode)solutions
The species used (Spirogyra fluviatilisHilse) and the culture method were theOn the anode side, acidic electrolyzed

same as described elsewhere (2).

Cellswater containing generated H+ with awere used for the experiments within 2 tobactericidal effect is made.

7 days (growing stage) after transferring

Hypochlorous acid (HClO) is a major

them from the stock culture to the freshchemical having a bactericidal activity.

Ichimura medium (4).

Alkaline electrolyzed water on theArtificial pond water (APW) (0.1mM

cathode side containing generated OH-

each of KCl, NaCl, CaCl2 and 5mM

has beneficial effect on disorders in

Hepes-Tris [pH7.5]) was used as thedigestive tract.

experimental medium when cortical MTs

Dissolved hydrogen isthought to be one of the effectivein Spirogyra cells were depolymerized.

elements now (3 for review).

Amiprophos-methyl (APM) was dissolved

It seems

important to investigate the effect ofin DMSO to a concentration of 5mg/ml aselectrolyzed water on the orientation ofa stock solution. Cells were treated withcortical MTs in Spirogyra cells because

3μg/ml APM in order to depolymerize

electrolyzed water is most studied in

the cortical MTs for 1 h. After the APM

functional water.

was removed by washing the cells quickly

First, we investigated the effects ofthree times with distilled water, cortical(acid and alkaline) electrolyzed water andMTs were reorganized in tap water,

tap water on the orientation of corticalelectrolyzed water or some concentration

MTs.

of NaClO solution with or without someIn addition, the effect of available

chlorine, pH or salts on the orientation ofcortical MTs was studied.

salts for 1.5 h.

In this paper,

the effects of acidic electrolyzed water,
alkaline electrolyzed water and tap waterPreparation of test solution
Tap water (Hirakata Osaka, Japan)

on the orientation of cortical MTs are

was used or was electrolyzed by DX-7000

clearly different, and these differences(Altech).

coincide with the tendency which is

NaClO solution was prepared bygenerally accepted as effects ofdiluting NaClO with distilled water.

electrolyzed water on living organisms.

of tap water was adjusted at about 4.5 by
10% HCl.

Materials and Methods
ImmunostainingpHK. Iwata and Y. Sano

MTs were observed using the method of55

According to JIS K 0101 Methods for

Hogetsu (5) as described in the previous

Examining Industrial Water (6), DPD

paper (2).

method was employed.

Cells were fixed in formalinsolution, cut with a razor blade to allow
entry of antibodies and treated with

Results and Discussion

detergent, primary antibody, andsecondary antibody.

Then, MTs wereAfter depolymerization of cortical MTs

observed with an epifluorescencewith APM, they were repolymerized in

microscopy.

tap water and acidic electrolyzed water.

The orientation of MTs was classified

In tap water, many cells had longitudinal

into three categories based on MT angles

MTs (Fig. 1).

to the cell axis, longitudinal (L) for 00-100,water, 90% of cells had random or nooblique (O) for 100-800, transverse (T) for

repolymerized MTs (Fig. 2).

800-900.

that, in distilled water or APW, all theCells having other MTs thanIn acidic electrolyzed
It is known

transverse, oblique or longitudinalcells have transverse MTs in this(random) or no repolymerized MTs were

Spirogyra species (7, 8). These results

indicated as others.

mean that a certain element(s) in tap

Response of cells is slightly different inwater affect(s) the orientation of corticaleach experiment. MT orientation of cells

MTs and that cell treated in acidic

treated with control water and sample

electrolyzed water could be damaged.

water was observed at the same time in

HClO in acidic electrolyzed water has

each experiment.

strong oxidation potential and may
damage cells in this case.

Assay of available chlorine concentration

Fr e qu e n c y(% )100t r an sve r seo bliqu elo n git u din alothers

80
60
40２０ｘ５200t ap wat e r

ac idic
e le c t r o lyze d wat e r

Fig. 1 The orientation of cortical MTs repolymerized in tap water and acidic
electrolyzed water. In one experiment, 20 cells were examined and 5 experiments
were repeated.

56

Cortical microtubules in Spirogyra cells

condition, Spirogyra cells have transverse
MTs (9).

This result implies that

Spirogyra cells in alkaline electrolyzed
water might be nearer to normal
condition than those in tap water, which
may be related to the fact that alkaline
electrolyzed water is known as functional
water good for human health, forFig. 2 Photograph of Spirogyra cells

example, beneficial effect on disorders inand immunofluorescence images ofdigestive tract (10 for review).

random cortical MTs in Spirogyra
cell.

Spirogyra cells was sensitively changed

Bar=30μm.

The orientation of cortical MTs
repolymerized in tap water and alkalineelectrolyzed water was also examined.
Apparent differentiation of MTorientation was observed between in tap
water and alkaline electrolyzed water.
In alkaline electrolyzed water, all thecells had transverse MTs.

On the otherhand, about 50% of cells had transverse
MTs in tap water (Fig. 3).

As mentioned above, MT orientation in

In normal

in tap water, acidic electrolyzed waterand alkaline electrolyzed water.

In

addition, physiological effects of water oncells could be inferred from theorientation of cortical MTs.
pH and available chlorine
concentration were also measured
because these factors are important forfunction of electrolyzed water (table 1).
Although available chlorine
concentration of tap water was only

Fr e qu e n c y(% )100t r an sve r seo bliqu elo n git u din alothers

80
60
40

２０ｘ５

200t ap wat e r

alkalin e e le c t r o lyze d wat e r

Fig. 3 The orientation of cortical MTs repolymerized in tap water and alkaline
electrolyzed water. In one experiment, 20 cells were examined and 5 experiments
were repeated.

K. Iwata and Y. Sano57slightly higher than that of acidic

Available chlorine concentration ofelectrolyzed water, cells in acidic

alkaline electrolyzed water was quite low

electrolyzed water seemed to be damaged

compared to tap water or acidic

worse than in tap water.

electrolyzed water.

It is known

It is probable thatthat pH controls the ratio of HClO in

difference of MT orientation between

available chlorine concentration (11) andalkaline electrolyzed water and tap waterthat low pH increases the ratio of HClO

might be caused by difference of available

and effects of available chlorine (1).

chlorine concentration.

These are not contradictory to anavailable chlorine on MT orientation wasinference from MT orientation.

examined.

So, effect of100
tr an sve r seo bliqu elo n gitu din alothe rsF requency ( %)806040２０ｘ５20001510Concentra ti on of N a Cl O i n D W-4( x 10 %( V /V ) )Fig. 4 The orientation of cortical MTs repolymerized in NaClO
solution. In one experiment, 20 cells were examined and 5experiments were repeated.

58

Cortical microtubules in Spirogyra cells

NaClO was used as available chlorine.

active element in alkaline electrolyzed

The orientation of cortical MTs waswater (3), affects the orientation ofclearly affected by NaClO (Fig. 4).

cortical MTs in Spirogyra cells.
MT orientation repolymerized in tap

Because effect of NaClO on MT
orientation differed quite from that ofwater and in tap water, pH of which wasNaCl (8), effect of NaClO was thought toadjusted at 4.5, was also examined.

be caused by ClO-.

addition, available chlorine concentration

As NaClO

concentration increased, ratio of cells

was also assayed.

having transverse MTs decreased.

There was no apparent difference (Fig.

Effect of alkaline electrolyzed water on

5, Table 2).

MT orientation could be explained atis lowered, the ratio of HClO andleast partially by low level of available

bactericidal effects increase (11).

chlorine concentration.

However, when pH was lowered at theIt is interestingthat how dissolved hydrogen, putative

As already known, when pH

level of acidic electrolyzed water, MT

100

Frequency (%)

80transverse
obliquelongitudinalothers

60
40
20

２０ｘ３0tap waterInacidic waterFig. 5 The orientation of cortical MTs repolymerized in tap water (pH7.4) andacidic water (pH-adjusted tap water (pH4.5)). In one experiment, 20 cells wereexamined and 3 experiments were repeated.

K. Iwata and Y. Sano59

orientation was not so affected in tap

difference of composition of salts in tap

water.

water.

These results suggest thatsomething other than available chlorine

Compared with tap water, amount ofin tap water affect MT orientation.

cation decreases and that of anion

These also imply that something in acidic

increases in acidic electrolyzed water. Inelectrolyzed water might be important foralkaline electrolyzed water, reverse

its effects.

changes occur (10).

As mentioned above,F requency of Cel l s H a v i ng
Tra ns v ers e MTs ( %)60504030
20
10000 .5

11 .5

2Concentra ti on of E a ch Mi nera l ( mM)

Fig. 6 Frequency of cells having transverse MTs. Cortical MTs repolymerized in 10-4%(V/V) NaClO solution containing the same concentration of NaCl, KCl, CaCl2 andMgCl2. In one experiment, 20 cells were examined and 5 experiments were repeated.
To investigate effects of salts on

effects of pH-adjusted tap water andavailable chlorine, MTs wereacidic electrolyzed water on MT

repolymerized in 10-4% NaClO solution

orientation were quite different, thoughcontaining the same concentration ofavailable chlorine concentration and pH

salts (NaCl, KCl, CaCl2 and MgCl2).

Aswere similar.

Change of amount of ionsconcentration of salts increased,in acidic electrolyzed water might befrequency of cells having transverse MTs

responsible for its effects.

increased (Fig. 6).

Some salts surely

In this paper, we described that effectaffect the effect of available chlorine onof electrolyzed water on living organisms

MT orientation at quite low concentration

could be detected using Spirogyra cells.

(1 mM).

This result suggests that salts

in acidic electrolyzed water might beimportant for its effects.

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Communicated by Yoshihiko Amano