Rhodamine 123 inhibits bioenergetic function in isolated rat liver mitochondria

Vol. 118, No. 3, 1984 February 14, 1984

RHODAMINE 123 INHIBITS BIOENERGETIC Josephine

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 717-723

BIOCHEMICAL

FUNCTION IN ISOLATED RAT LIVER MITOCHONDRIA Michael J.,Weiss,' R. AprilleI

S. Modica-Napolitano,' and June

Lan Bo Chen,2

' Dept. of Biology Tufts University Medford, 2 Dana-Farber Cancer Instiiute, Harvard MediZal School,

MA 02155 Boston MA

02115

Received December 30, 1983

Rhodamine 123 accumulates in the mitochondria of living cells and ex i its selective anticarcinoma activity. The biochemical basis of toxicity was investigated by testing the effect of the dye on isolated rat liver mitochondria. Much lower concentrations of rhodamine 123 were required to inhibit ADP-stimulated respiration and ATP synthesis in well-coupled energized mitochondria than were required to inhibit uncoupled respiration and uncoupler-stimulated ATP hydrolysis. The amount of rhodamine 123 associated with the mitochondria was several-fold greater under energized as compared to non-energized conditions, which may explain why coupled functions appeared to be more sensitive than uncoupled functions to inhibition at low concentrations of rhodamine 123. It was concluded that the site of rhodamine 123 inhibition is most likely the FOFIATPase complex and possibly electron transfer reactions as well.

YT-F*

Rhodamine anticarcinoma feature

123 is

a cationic

activity

of this

at certain

both

compound

doses

lipophilic

--in vitro

leaving

this

selective

toxicity

has yet

Rhodamine

123 is

accumulated

and retention

drial

membrane

electrical the

gradient

electrochemical with

synthesis,

potentiates

the

* Author

mitochondrion

the

is

the

to whom correspondence

mitochondria

addition

lies

promising in its

tumorigenic a biochemical

of ionophores which

mitochondrial

primary should

of the target

cells upon the

ability cells

basis

for

prevent

the

for

rhodamine

The

mitochon-

This

the

establishment

fluorescence

an inhibitor

dye (2,3).

(4,5).

known to dissipate

specific

2-Deoxyglucose, effect

of living

to be dependent

inhibitors

diminish

toxic

The most

agent

However,

123 appears

123 (6). the

viable.

in the

and respiratory

rhodamine

(3).

exhibits

to be determined.

since

gradient

vivo

many epithelial-derived

cells

of rhodamine

potential,

prestained

that

epithelial

and --in

dye which

chemotherapeutic

kill

while

uptake

normal

(1,2)

as a potential

to selectively

fluorescent

in cells

of glycolytic evidence

123 cytotoxicity.

of

ATP

suggests The

be addressed 0006-291X/84 717

$1.50

Copyright 0 1984 by Academic Press. Inc. All rights of’ reproduction in anv form resenwl.

BIOCHEMICAL

Vol. 118, No. 3, 1984 purpose

of this

study

was to investigate

rhodamine

123 on mitochondrial

MATERIALS

AND METHODS

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

possible

inhibitory

effect

of

functions.

Isolation of Mitochondria. Rat liver mitochondria were isolated by differential centrifugation as previously described (7). Protein was determined by the method of Lowry (8). Respiration. Oxygen consumption was measured polarographically in a 1 ml volume as described previously (9). The order of additions to respiratory assay medium (9) was: mitochondhia (.4-.5 mg protein); an oxidizable substrate, either glutamate + malate (5 mM each) or succinate (5 mM t 2 ug/ml rotenone); rhodamine 123 (O-20 ug/ml); ADP (120 nmol). The ADP-stimulated respiratory rate was defined as the rate after ADP minus the rate after rhodamine 123. To determine uncoupled rates, 2,4-dinitrophenol (f.c. 40 uM) was added during State 4. Measurement of Rhodamine 123 Associated with Mitochondria. Mitochondria (.4-.5 mg protein) were incubated at room temperature for 2 minutes in 1 ml respiratory medium (see above) in the presence or absence of 2,4-dinitrophenol (f.c. 40 uM), oxidizable substrate, ADP, and rhodamine 123 (5-20 ug/ml). The mixture was then layered over silicone oil layered over 12% PCA and centrifuged at 11,000 g for 1 min as described previously (7). The amount of rhodamine 123 in the supernatant was determined by absorbance at 502 nm by comparison with standards. The amount of rhodamine 123 associated with the mitochondrial pellet was determined by subtracting the amount measured in the supernatant from the amount of dye originally added. ATP Synthesis. ATP synthesis was inferred from oligomycin-sensitive Pi disappearance in the presence of 1 mM ADP and oxidisable substrate in respiratory medium (as above except 1 mM K HP0 - KH P04) at 30 C. Pi was assayed 8 et ATP synthesis calorimetrically (10) in PCA e$tra&s. was calculated from the difference in Pi present in each sample relative to a paired oligomycin (2 ug/ml) blank. 5 mM ATP and 40 uM 2,4-dinitrophenol, Identical incubations (blankgj containing 2 ug/ml oligomycin were run in parallel. Pi was assayed in PCA extracts as described above for ATP synthesis. Adenine Nucleotide Translocase Activity. This was assayed on ice by forward :~QJ;;;; ;;)0.23 M sucrose, 0.9 mM Tris, 2.5 mM each glutamate t malate, and 20 uM . RESULTS The Effect of rhodamine

of Rhodamine 123 on respiratory

on ADP-stimulated

max.

evidence consumption obtained This

at 7 ug/ml

uncoupling

using

suggested

glutamate that

several

(Fig.

Function. presence rates.

by rhodamine and >30 ug/ml

by rhodamine

was stimulated

in the

respiratory

to inhibition

inhibition for

rates

and uncoupled

much more sensitive (l/2

123 on Respiratory

123;

1).

t malate possible

718

alone,

ADP-stimulated

presence

similar

sites

There

instead

was

was some

shown

of succinate

of inhibition

as

respiration

of substrate

to those

as well

respiration

was uncoupled

respectively).

as the substrate specific

1 shows the effect

of substrate

123 than

in the

Results

Figure

alone

O2

in Fig.

1 were

(not

shown).

by rhodamine

123

BIOCHEMICAL

Vol. 118, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Rhodamine P3 Added /up/mll P e order follows: 123 (A); defined experiments.

Effect of of additions respiration ADP-stimulated under Materials

seemed unlikely, complexes

including:

I and II;

Results

like

ADP-stimulated

those

presented

respiration respiration)

being

affects

tested

transport.

concentrations

site

in Fig.

are usually some reaction However,

I of the

1 (i.e.

in intact

of uncoupler

unique

to

to the

uncoupled

at least

cells

induces

prestained

dose-response

curves

be attributable

with

obtained

to differences

mitochondria

in uncoupled

a rapid the

release

and coupled

rhodamine that

of rhodamine This

rather

than

at high

of ADP-stimulated

transport.

and absence

states.

effect

the compound pathway

inhibition

of

little

was inhibited

suggesting

amount

that

phosphorylation

chain.

of

123 having

of electron

presence

in the

inhibition

respiration

respiratory

transport

indicate

some of the

dye (6),

in the

electron

of rhodamine

may have been due to an inhibition

addition

dehydrogenases;

severe

interpreted

because 123,

substrate

transport;

at concentrations

of rhodamine

respiration the

substrate

and phosphorylation

on uncoupled

electron

rhodamine 123 on respiratory rates with succinate as substrate. is given under Materials and Methods. Symbols are as in the presence of substrate after the addition of rhodamine respiration (0) and uncoupler-stimulated respiration (0) as and Methods. Points are averages of two separate

Furthermore,

123 from mitochondria the different

of uncoupler

in Fig.

123 associated

possibility

with

1 may the

was investigated

next. The Amount Uncoupled quantitated removal

of Rhodamine

Conditions.

The association

by measuring of the

123 Associated

mitochondria

the

amount

with

Mitochondria

of rhodamine of dye left

as described

under 719

Under

123 with

in the Materials

Coupled

mitochondria

incubation

was

medium

and Methods.

and

after

Under

rapid both

Vol. 118, No. 3, 1984 coupled

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and uncoupled

conditions

the

mitochondria

varied

as a function

For added

concentrations

of

dye was associated conditions uptake

(Table with

the most

prominent

represented

detected

in whole

This

phenomenon

could

cells

with

absorbance

this

obtained

by the

first

123 up to 20 ug/ml,

of dye added

to the

medium.

much more of the to non-energized

was not adequate

upon interaction

in the

to uncouplers,

to study

under

method

in the difference indicator

(Figure

method

of rhodamine

2) were

(Table

1).

That

is,

for

more dye associated conditions.

to uncoupled

ug/ml,

there

was no further

increase

Table 1. The amount of rhodamine and non-energized conditions. Rhodamine 123 Added (w/ml 1

in the

amount between

with

of dye taken

with mitochondria

5

22.3

1.2

10

48.2

2.5

20

63.1

8.0

under Materials using succinate + malate as

720

the

The results with

data

of rhodamine the mitochondria above 30

up under

energized

under energized

and Methods. The as the substrate. the substrate.

so

Xmax and 580 nm

Rhodamine 123 associated with mitochondria (nmol/mg protein) Succinate + Succinate 40 uM 2,4 dinitrophenol

were determined as described are from a typical experiment were obtained using glutamate

1).

of dye added

At concentrations

amount

123 associated

comparing

concentrations

was 5-7-fold

as compared

for

agreement

there

coupled

Table

123 uptake.

in qualitative

mitochondria

and uncoupled

of the spectrum

it

(ref.

method

coupled

502 nm to

that

123 with

mitochondria

at 580 nm was independent

measured

suggesting

as an alternative

the mitochondria

mitochondria,

maximum from

of rhodamine

and in isolated

further

with

absorption

association

under

Values shown results

shift

a change

(6)

be used as a quantitative using

up by the

as compared

method

was sensitive

was investigated

obtained

123 taken

5 and 20 ug/ml,

energized

particular

of specific

The absorbance net

under

this

being

of dye associated

the

concentration

123 between

a spectral

feature

same kind

is

of rhodamine

> 20 ug/ml.

The peak shift

that

that

However

the

conditions.

rhodamine

123 exhibited

512 nm.

amount

1).

of the

mitochondria

concentrations

Rhodamine

about

with

amount

results Similar

BIOCHEMICAL

Vol. 118, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1.5-

2

-

3 9

l.Os

c

-

8 s p

0.5-

0

1

L

I 20

I

I 40

1

I I 60

Rhodamine 123 Added /up/m// The amount of rhodamine 123 associated with mitochondria under coupled -pled conditions. Absorbance (indicative of uptake) was determined from difference spectrum obtained for rhodamine 123 in the presence and absence of mitochondria using the wavelength pair X -580 nm as described under Results. Points are from one typical experiment w!% 5 mM succinate in respiratory assay medium with (0) or without (of 40 uM 2,4-dinitrophenol.

conditions

(Fig.

2).

sites

or to uncoupling

would

inhibit

and Fig.

that

ADP-stimulated

more sensitive

than

because

phosphorylation

mitochondria

point

rhodamine

123 which

of binding

a condition

to be derived

respiration,

to

amount

under

that

from

appeared the

greater

rhodamine

th s assay than

the

is

is total

(Figure

3).

than This

ATP synthesis 3).

respiration

oxygen

Table

1

inhibit

of dye

coupled

as compared

dye were

apparent

needed

needed

to

difference 721

by 9D% in appeared

of coupled

The reverse inhibited enzyme

to inhibit inhibit

Translocase

to be even

by rhodamine

measurement

on this

Nucleotide

reaction

to inhibition

was also

effect

were

This

consumption.

activity,

dye had a direct of the

and on Adenine

123 inhibited

a more direct

FOFIATPase

of an uncoupler (Fig.

123 on F,FIATPase

ADP-stimulated

concentrations

presence

important

uncoupled

zed mitochondria

uncoupler-stimulated

higher

not

concentrations,

was several-fold

of Rhodamine

energ

that

of

due to saturation

conditions.

well-coupled,

showing

The most

but

have been

123 at high

at concentrations

At 5 ug/ml

possibly

might

the mitochondria

The Effect Activities.

uptake.

respiration with

to uncoupled

plateau

by rhodamine

further

2 is

associated

This

the

oxidative

reaction, by rhodamine

complex.

ATP synthesis

123

However

ATP hydrolysis

in sensitivity

123,

much

in the in energized

of the

FDFIATPase

to

Vol. 118, No. 3, 1984

BIOCHEMICAL

I

I

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

0

I

10

I

I,

I

20

"

40

RhodomiM t2.3 A&fed fig/m//

w-

Effect of rhodamine 123 on mitochondrial phosphorylation reactions. Va ues are plotted relative to a control (no rhodamine 123) for ATP synthesis and ATP hydrolysis (0). Reaction conditions were as described under Materials Methods.

rhodamine

123 inhibition

readily

explained

energized

this

adenine

fact

whether

the

nucleotide

transporter.

that

non-energized

more dye associates

rhodamine

by only 123.

inhibit

inhibition

mitochondria with

of FOFIATPase

translocase,

In energized

was inhibited

not

by the

versus

is most

the mitochondria

under

conditions.

To determine of the

in energized

(e) and

mitochondria

43% even

In the

(not

the

adenine

at a relatively

presence

translocase

we measured

was secondary

high

to inhibition

effect

of rhodamine

nucleotide

translocase

concentration

of 40 uM 2,4-dinitrophenol,

123 on activi

(20 ug/ml)

20 ug/ml

tY

of

of the dye di' d

shown).

DISCUSSION A structural its

effects

this

sort

for

123.

possible

of FOFIATPase

described;

a spectral Our results

We conclude liver inhibition

that

mitochondria. of electron

123,

functions

rhodamine

Inhibition

noted.

of rhodamine

on bioenergetic for

investigated

analog

(ll-13),

and uncoupling upon

activity

interaction

rhodamine

123 inhibits

transfer

occurs reactions

report

those

findings

inhibition

may contribute 722

are

no detailed

(14),

the

for

studies

of

dye was potentials.

concentrations

dye with

oxidative via

has been investigated

membrane

at high of the

with

probably

brief

there

mitochondrial

are consistent

This

66,

but

In one recent

use in measuring

shift

rhodamine

were

mitochondria

was also

(14). phosphorylation

in intact

of FoFIATPase, as well.

although

Adenine

rat

BIOCHEMICAL

Vol. 118, No. 3, 1984 nucleotide

translocase,

respiratory

complexes

interpreting

future

finding

that

it

123 binds remains

transport

is

lipophilic

of the

effect

activity

on certain

potential

biochemical

specifically basis

be kept

in mind.

It

of rhodamine

or is

of nonspecific

clear

the

to

at present

sequestered

whether

in the matrix.

of FDFIATPase membrane

In

123 toxicity,

as compared

is not

inhibition

and

involved.

energized

membrane

whether

dehydrogenases,

and electron

perturbation

due to the

dye. 123 inhibited

energy-linked

many interesting

at understanding

use of the

not

up under

exhibited

tumor

probably

mitochondrial

rhodamine poses

substrate

taken

or a result

that

aimed

dye is

to the

mitochondria

studies,

are

to be determined

nature

anticarcinoma

transport,

on the

must

specific

The fact liver

of the

conditions

rhodamine Also,

I and II studies

more

non-energized

substrate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

questions

functions about

by the dye --in vitro the mechanism

mitochondria

in the

dye as a chemotherapeutic

(1,2)

by which intact

the

cell,

in normal

basis

for

and --in vivo

rhodamine will

rat

selective (3).

123 exerts be important

Further a toxic to the

agent.

Acknowledgements. This study will be submitted by J.S. Modica-Napolitano in partial fulfillment of the requirement for a Ph.D. in Biology at Tufts University. We thank Caroline A. Brown for technical assistance and Suzanne T. Dobson for preparing the manuscript. The work was supported by NIH grants NS14936, HD16936, and CA22427. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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