Intracellular Oxidation Reduction Studies

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VOL.

VIII, No. i

JANUARY,

193I

INTRACELLULAR OXIDATION-REDUCTION

STUDIES

III. PERMEABILITY OF ECHINODERM OVA TO INDICATORS

BY ROBERT CHAMBERS, BARNETT COHEN

AND HERBERT POLLACK.

(New York University, The Johns Hopkins University and the Eli Lilly Research Division, Marine Biological Laboratory, Woods Hole, Mass.)

{Received 14th May, 1930.)

IN a number of studies on the reduction potentials of living tissues and cells, the method of immersing the material under investigation in solutions of oxidation-reduction indicators has found considerable employment. Under controlled conditions and with sufficient knowledge concerning the physico-chemical charac-teristics of the indicator systems, this method is of value for determining the magnitude of extracellular reduction potentials. Its use, however, for the determin-ation of intracellular reduction potentials has been hampered by the lack, among other things, of knowledge concerning the penetrating power of the indicators into living cells. Results obtained from immersion experiments alone have proved to be inconsistent. An example of this is given by J. and D. Needham (1) who examined a series of indicators and showed that cells become coloured when immersed in solutions of indicators which should be reduced intracellularly, while they give no sign of coloration in solutions of other indicators which should remain oxidised. These authors recognised the discrepancy but did not examine it more thoroughly. The problem reduced to its simplest terms may be described as follows. When a cell is immersed in a solution of a reversible oxidation-reduction indicator the latter may or may not enter, depending on whether or not it can penetrate or be carried across the semi-permeable membrane. If it enters, the dye oxidant may be reduced by cell reductants so that the cell shows no coloration. Hence failure of the cell to become coloured in reversible dye solutions is an inconclusive test of either non-penetration or of cytoplasmic reduction. The additional informa-tion can be obtained by the micro-injecinforma-tion method, as we shall show presently. On the other hand, dye oxidant may enter the cytoplasm at a greater rate than it can be reduced by the cell mechanism and, as a result, the cell becomes stained almost at once. Superficially, it would appear that the reduction intensity of the cell is not great enough to permit reduction of the dye in question, while in reality, it is merely the reducing capacity of the cell that is inadequate. This and similar

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2 R. CHAMBERS, B. COHEN and H. POLLACK

fusion of the capacity and intensity factors of reduction is unfortunately present in much of the literature on biological oxidations.

With the above considerations in mind, we have made a study of the problem by combining the immersion method with that of micro-injection. In doing so we have been able to detect a possibly distinguishing characteristic of those indicators which do not penetrate from those which do and are reduced in the living cell. It has also been possible to find a consistent explanation for the intracellular coloration of the marine ova caused by easily reducible indicators.

MATERIAL AND METHODS.

The relatively colourless and translucent ova of the sanddollar

(Echinarach-nins partna) and starfish (Asterias rubens) were used as material. The eggs were

fertilised and then divested of their jelly and membranes by straining through fine-meshed, silk bolting-cloth, or aspirating the eggs into glass capillary tubes, the bore of which was smaller than the diameter of the fertilisation membranes and larger than that of the eggs. By this means naked eggs were obtained which could be readily washed after removal from the immersion fluids.

The naked eggs, 3 minutes after insemination, were immersed in sea-water coloured with freshly made aqueous solutions of the indicator salts, the proportions used being one to several drops of a 1 per cent, solution of the indicator in 10 c.c. sea-water.

At various intervals after immersion the eggs were removed, and rinsed several times in sea-water. If the eggs were colourless they were injected with an oxidising agent (1 per cent, solution of potassium ferricyanide) as a test for the possible presence of the indicator in the reduced form. If the eggs were coloured the experiment was repeated by immersing eggs in more dilute solutions and for shorter periods.

The relative toxicity of the indicator solutions was judged from the effects of continued immersion on the subsequent development of the eggs.

EXPERIMENTS ON PENETRATION AND TOXICITY. The accompanying table is a record of the results obtained.

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Table I. Immersion experiments on sanddollar and starfish eggs. Indicators I 2 I Phenol m-sulfonate indo-2, 6-dibromophenol

. .

.

:

m-Bromophenol indophenol

...

1 o-Chlorophenol indophenol ...

...

5 o-Bromophenol indophenol

...

7 Phenol blue chloride

...

7 2 Phenol blue sulfonate

.

. .

.

. .

.

. .

.

3 Phenol indo-2, 6-dichlorophenol

...

Phenol indo-2, 6-dibromophenol

.

. .

.

az-Cresol indophenol

. .

.

.. .

'C o-Cresol indophenol

...

L o-Cresol indo-2, 6-dichlorophenol

.

. .

.

M I-Naphthol-2-sulfonate indophenol o-sulfonatef V 111-Toluylene diamine indophenol chloride

. .

.

3 I-Naphthol-2-sulfonate indophenol

...

P I-Naphthol-2-sulfonate indo-2, 6-dichlorophenol 2 Toluylene blue chloride

...

fJ I Brilliant cresyl blue chloride

...

R Methylene blue chloride

...

... S & indigo tetrasulfonate

...

S I Ethyl Capri blue nitrate

...

.. T Ks indigo trisulfonate

...

..

U Ka indigo disulfonate (also Na3

...

..

V Neutral red iodide

. .

.

. . .

.

. .

.

Janus green

. .

.

. .

.

. . .

.

E,' at OH 7.0 (volts)

Blue Blue Blue Blue Blue

Green? Blue Blue

Purple blue Brick red Purple blue

Blue Pink _Purple Blue Blue Blue Blue

Purple

blut

Blue Blue Blue Red Blue

Of phenolic hydrion. 1 Erroneously described as the m-sulfonate in previous publications. Colour

-

'ink (after I hr.) 'ink (after 3 hr.) ?ink (after 3 hr.) Greenish

- _Blue

Greenish

Red Red _Bluish

-

Pink - - Blue Blue Blue - Blue

- -

Red Blue

Test for penetration in still colourless eggs Cytolysis by tearing

4 _-

Pink

-

Pink -

-_ -

Already coloured Already coloured Injection of ferricyanide 5

- +(Pink)

+

+(Pink) +(Pink) _+(Green) - +(Blue) +(Blue)

+

+(Red) +(Red) +(Blue) - S(Pink) - - +(Blue) +(Blue) +(Blue) -

+

+(Blue) - -

Already coloured Already coloured - Indicator in

colourless cytoplasm

6

Toxicity lmmerslon

7

None

Reduced Reduced Reduced Reduced None Reduced Reduced Reduced Reduced Reduced None Reduced None None Reduced Reduced Reduced None

Partly

reducec

None None

Not

reduced5

Not

reduced5

Non-toxic Toxic Toxic Toxic Toxic Toxic

Toxic Toxic Toxic Toxic Toxic Non-toxic Toxic

Rel.

non-toxic

Not

tested

Toxic Toxic Toxic

Non-toxic Toxic Non-toxic Non-toxic Toxic Toxic

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All the other indicators (which lack the sulfonate radical) sooner or later colour the cytoplasm of the immersed eggs. In these solutions the rate of staining varied considerably. Of the acid dyes the most easily reducible ones, C, D and E, took the longest to stain, the time varying from one to three hours; the rest of the acid dyes, except L, took from 5 to 15 minutes. Indicator L stained the eggs poorly and irregularly. The amphoteric dyes, F, N and the basic dyes, Q, Q 1, R 1, S 1 and V stained the eggs the most readily of all.

The colours assumed by the eggs immersed in the penetrating dyes were consis-tent with the cytoplasmic pH of 6'6 to 6-8 which has already been published (1, 6). Some of the indicators have pK values (acid-base dissociation constants) near this range and the colours they impart to the cytoplasm after penetration serve as a rough independent confirmation of the intracellular pH found by micro-injection of the sulfonephthalein indicators. This is shown in the table.

In this connection, it is important to bear in mind a defect in the immersion method which may lead to incorrect conclusions unless properly controlled or discounted. While the dry salts of our oxidation-reduction indicators are relatively indefinitely stable, their solutions begin to undergo decomposition almost at once. At moderate pH values and in pure solution the rate of breakdown is relatively low. But when mixed with unfiltered sea-water containing surface-active eggs as well as countless numbers of micro-organisms, and with a large surface exposed to the air, the rate of dye decomposition may be serious. By working with fresh dye solutions, by employing careful technique with repeated tests and by controls with direct micro-injection, we believe we have avoided all the major pitfalls so that the results reported are substantially correct.

The results of testing for penetration of the indicators in the still colourless eggs are given in columns 4 and 5. All visible traces of colour around the eggs were first removed by washing in several changes of sea-water. Some of the eggs were then cytolysed by tearing with a micro-needle, and others were injected with a solution of 1 per cent, potassium ferricyanide. Positive results with a return of colour of the oxidant are indicated by + signs; negative results are indicated by — signs. In a few cases (D, J and N) the induction of cytolysis by tearing the eggs with micro-needles sufficed to cause the colour of the oxidant to appear. The pink colour produced by the injection of the ferricyanide solution in eggs from solutions of indicators C, D and E is consonant with the pH virage of their oxidants in the presence of an acid of injury having a pH of 5-0 to 5-4.

In column 6 are indicated the conclusions arrived at from the tests listed in the preceding columns regarding the presence of the indicators in the cytoplasm of the eggs while still colourless. These results agree in every respect with those already published (a, 3). In the last column are indicated the relative toxicities of the indi-cators with reference to the ability of the fertilised eggs to segment and to develop into swimming larvae while immersed in solutions of the indicators in sea-water.

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Intracellular Oxidation-Reduction Studies 5

into irregular blastulae. In solutions of all the penetrating dyes the eggs coloured and died before segmentation took place. It is of interest to note that the concen-trations used in all these experiments were such that neutral red was toxic.

The most careful attempts to detect signs of reduction of neutral red iodide either aerobically or anaerobically yielded negative results. Eggs were immersed for several minutes in sea-water barely tinged with neutral red. They were then washed thoroughly in three changes of fresh sea-water and placed in a hanging drop in the nitrogen chamber. For a description of this method the reader is referred to our previous publication (2). The eggs in the drop least tinged with the dye were kept under observation in nitrogen for 15 minutes and no noticeable decoloration occurred. The eggs were then injected with ferricyanide but no sign of intensification of colour occurred. From these results we must conclude that neutral red is not reduced even anaerobically under conditions in which injected indigo sulfonates are promptly decolorised.

One lot of fertilised sanddollar eggs was stained with Janus green. After an exposure of the eggs to a nitrogen atmosphere for i hour the eggs changed from a blue to a red colour, indicating a breakdown of the Janus green to the safranin compound. The eggs, coloured red, appeared normal but did not segment.

This reaction is in accord with results already reported on sperm mother cells of the squash-bug, Anasa tristisb).

The equilibria of the Janus green : leuco-Janus green system have not yet been worked out satisfactorily. Preliminary studies at the Hygienic Laboratory have indicated that the change from the blue to the red stage is practically irreversible. Very rough measurements place this unstable equilibrium between the methylene blue and the indigo tetrasulfonate systems1. It is, however, unsafe to conclude even approximately that a system able to cause the complete conversion of Janus green to safranin has a reduction potential equal to that indicated above. No stable equili-brium state occurs between oxidant (Janus green) and reductant (leuco-Janus green); and the safranin ultimately obtained is a fragment apparently not easily reconstituted to the original Janus green molecule. Hence the system can have no appreciable true poising ability. Consequently, given sufficient time and concen-tration, the reductant of another system more electropositive but overlapping ever so slightly can completely reduce Janus green. The reduction of Janus green under such circumstances would be a very crude and uncertain indication of the reduction

potential of the active system. It becomes evident therefore, that this constitutes a

serious objection to such utilisation of irreversible indicators for reduction-potential determinations, although they may have some value for rough exploration.

1

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EXPERIMENTS ON THE EFFECT OF CYTOLYSIS ON REDUCTION. It is known that crushed tissues and tissue extracts may exhibit a decided reducing intensity. This can be shown with starfish eggs cytolysed by distilled water. A mass of the cytolysed material in a test-tube completely reduced methylene blue. The addition of potassium ferricyanide brought back the colour. This pheno-menon seems to run counter to the fact that in the case of several of the indicators

(D, J and N; see table) the reverse occurs when the eggs containing the reduced

dye are cytolysed by tearing, viz. that cytolysis results in the appearance of the colour of the oxidant.

In order to analyse the above apparent discrepancy the following experiment was performed. Eggs in a hanging drop were cytolysed both by tearing with needles and by exposure to distilled water blown against them with a micro-pipette. By such means cytolysed eggs were secured with intact contours, the protoplasm of the eggs disintegrating into irregular globules within the enveloping membranes of the egg. Oxidants of indicators A and R were injected into these eggs within a few seconds after cytolysis; In the case of A the debris took on the pink colour (colour of oxidant at/>H 5-4). The colour persisted from 10 seconds to 20 minutes. As soon as the colour faded, ferricyanide was injected with no return of colour. In the case of methylene blue, R, the cytolysed material took on a blue colour, which gradually faded and no colour returned upon injecting ferricyanide. The experiments with A and R were done both in air and in nitrogen atmosphere with identical results. The lack of return of colour by injecting an oxidising agent leads to the conclusion that the fading is due to a diffusion of the dye out of the freely permeable disintegrated remains of the eggs into the surrounding medium.

DISCUSSION.

In their immersion experiments J. and D. Needham(i) found that the indicators which they used differ in their penetrating ability into marine eggs. The experiments described in this paper show that the ova of the sanddollar and starfish are im-permeable to all the indicators tested, which possess a sulfonic acid group in their molecular constitution irrespective of their position on the scale of potential. This is in harmony with J. and D. Needham's results, for the five indicators which they list as not capable of staining are sulfonated compounds, while the four which do stain are indophenols lacking a sulfonate radical. In conformity with this may also be cited the results of Harvey (9) on the effect of immersing luminescent bacteria in solutions of the oxidants. By examining the list of indicators which he used, it is noted that all those which possess a sulfonic acid radical were apparently non-toxic to the bacteria and caused no dimming of luminescence.

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Intracellular Oxidation-Reduction Studies J

of the sulfonate group which prevents the anion, Dye-SO3, from penetrating the cell membrane ?

In considering the toxicity of the penetrating indicators one must bear in mind that the introduction of these same compounds by micro-injection was followed by toxic effects only when excessive amounts were injected. The micro-injection experiments gave results which show that the introduction of limited amounts of reversible oxidation-reduction indicators does not appreciably upset the level of intracellular reduction potential. Under aerobic conditions, the cell acts like a poised system, and this depends to a considerable degree upon the oxygen tension of the surrounding medium. The experiments reported in this paper show also that the presence of the oxidants in the medium around the cells does not necessarily have a harmful effect, unless the indicator accumulates within the cells. The injection experiments indicated quite convincingly that toxic oxidants were tolerated only if they were introduced in small enough quantities so as not to overwhelm the intrinsic reducing capacity of the cell (specific effects on the reducing mechanism are not considered here). The results obtained in the present immersion experiments, therefore, confirm our inference that the penetrating oxidant was at first reduced, and succeeding quantities then taxed the limits of cellular reducing power with consequent irreversible repression of activity.

It is noteworthy, in regard to the penetrating indicators, that eggs immersed in the easily reducible dyes took a comparatively long time to exhibit visible coloration, whereas the injection of ferricyanide demonstrated their presence within a very short time after the eggs had been immersed. On the other hand, the time difference between early detection with ferricyanide and visible coloration was very small with those dyes whose reducing potential lies close to our estimated reduction potential level for these eggs. These observations again demonstrate the variations in reducing

capacity at different levels of reducing intensity in the cell.

It is well known that mashed tissue and tissue extracts possess a high reducing power. We find, however, that individual eggs upon cytolysis lose their reducing action. This was best shown by inducing cytolysis with a return of colour in eggs which contained the indicators D, J and TV in their colourless, reduced state. It would seem, therefore, that the substance or substances which are responsible for the maintenance of a reducing intensity in the living cell may be lost to the surrounding medium upon cytolysis, while oxygen, or its equivalent, in the medium enters the cytolysed structure and re-oxidises the reduced dye as well as residual cell reductants.

SUMMARY.

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2. Under aerobic conditions all the penetrating dyes down to and including methylene blue can be completely reduced within the ova. Ultimately, however, through progressive intracellular accumulation of the oxidant, the dyes all impart a visible coloration to the cytoplasm. There is an apparent relationship between the time necessary for the ultimate visible coloration and the ease with which the dye is reduced in the cytoplasm.

3. Eggs, immersed in the penetrating indicators, eventually die without under-going segmentation. On the other hand, the eggs immersed in solutions of all but one of the non-penetrating indicators developed into swimming larvae.

4. Upon cytolysis the increased permeability of the disintegrated remains of the eggs permits the washing out of the contained indicator. The dissipation of reducing power has been shown to occur in several instances upon cytolysis by tearing. The agent or agents responsible for the reducing activity of protoplasm apparently escapes from the cytolysed cell debris into the surrounding medium in which it may still exert its reducing action.

BIBLIOGRAPHY.

(1) NEEDHAM, J. and D. M. (1926). "The hydrogen-ion concentration and oxidation-reduction potential of the cell-interior before and after fertilisation and cleavage. A micro-injection study on marine eggs." Proc. Roy. Soc. London, B, 99, 173.

(2) COHEN, B., CHAMBERS, R. and REZNIKOFF, P. (1928). " Intracellular oxidation-reduction studies.

I. Reduction potentials of Amoeba dubia by micro-injection of indicators." Journ. Gen. Physiol. " ,

585-(3) CHAMBERS, R., POLLACK, H. and COHEN, B. (1929). " Intracellular oxidation-reduction studies,

II." Brit. Journ. Exp. Biol. 6, 229.

(4) CLARK, W. M., COHEN, B. and co-workers (1928). "Studies on oxidation-reduction, I-X." (Suppl. notes, p. 349.) Hygienic Lab. Bull. No. 151, U.S. Public Health Service.

(5) HALL, W. L., PREISLER, P. W. and COHEN, B. (1928). "Studies on oxidation-reduction, XIV." U.S. Public Health Rep., Suppl. No. 71.

(6) CHAMBERS, R. and POLLACK, H. (1927). "Micrurgical studies in cell physiology. IV. Colori-metric determination of the nuclear and cytoplasmic pH in the starfish egg." Journ. Gen.

Physiol. 10, 739.

(7) KITE, G. L. and CHAMBERS, R. (1912). "Vital staining of chromosomes and the structure of the nucleus." Sc. N.S. 36, 639.

(8) RAPKINE, L., STRUYK, A. P. and WURMSER, R. (1929). " L e potentiel d'oxydo-reduction de

quelques colorants vitaux." Journ. chim. phys. 26, 340.

(9) HARVEY, E. N. (1929). " A preliminary study of the reducing intensity of luminous bacteria.