delayed pyridine nucleotide reoxidation induced by the anticancer agent vm-26 as measured in vivo...

Europ. J. Cancer Vol. 1 I, pp. 709-715. Pergamon Press 1975. Printed in Great Britain

Delayed Pyridine Nucleotide Reoxidation Induced by the Anticancer Agent VM-26 as measured In Vivo and In Situ by NADH Microfluorimetry*

MARIO GOSALVEZ,~ R. G~mcL~-C~m~ot AND HUBERT REINHOLD~ ~ Dept. of Experimental Biochemistry, Cltnica Puerta de Hierro, Facultnd de Medicina,

Universidad AutJnoma, Madrid, Spain and

The Radiobiological Institute of the Organization for Health Research, TNO, Rijswik, The Netherlands

Abstraet--Anoxia cycles have been performed in rats bearing rhabdomyosarcoma while focusing an alpha-diphosphopyridine nueleotide ( NAD H) microfluorimeter in the surface of the tumor tissue. A delayed re-oxidation of the pyridine nudeotides upon re-oxygenation of the animal was observed after the animal was injected with chemotherapeutic doses of the anticancer agent 4'-demethyl-epipodophyllotoxin thenylidene glueoside (NSC- 122819) (VM-26). These results confirm in vivo and in situ that VM-26 is a respiratory inhibitor.

I N T R O D U C T I O N

IN PREVIOUS papers, we reported that the anticancer agent VM-26 [1, 2] inhibits NADH- linked respiration at low concentrations [3] and that this respiratory inhibition seems to be somewhat more specific for certain tumour cells than for certain normal cells [4]. Conversely, we have also reported that rotenone, a classical inhibitor of NADH-linked respiration, is able to inhibit tumour growth in vivo and therefore we have postulated that an anticancer chemotherapy based on specific inhibitors of NADH- linked respiration may be worth investiga- ting [5].

We have shown that i.p. chemotherapeutic treatment with VM-26 results in respiratory inhibition of the cells [4], a fact favoring our

Accepted 30 May 1975. *This work was supported by Grant 12-913-74 of the Spanish Instituto National de Prevision and by Contract NO 1-CM-53792 from the Division of Cancer Treatment, National Cancer Institute, H.E.W. (U.S.A.)

709

proposition that the mechanism of action and/ or toxicity of VM-26 may lie in its effect on respiration. However, as the abdominal cavity of an ascites tumour might behave as an in vitro chamber if the drug is not rapidly absorbed, it will be convenient to demonstrate in vivo and in situ that V M - 2 6 inhibits the respiration of parentally-treated solid tumours, in order to ascertain the biological significance of the effects of the drug in cellular respiration.

In this report we illustrate in vivo and in situ that VM-26, injected i.p. at chemotherapeutic doses, is able to delay the reoxidation ofpyridine nucleotides of solid rat rhabdomyosarcomas growing in the skin of rats. This effect is interpreted as an inhibition of tissue respiration by VM-26 and provides additional evidence sup- porting the proposed mechanism of action and/ or toxicity of VM-26 as based on its effect on respiration. To perform in vivo and in situ the measurement of redox cycles of pyridine nucleotides, we have taken advantage of the NADH microfluorimetric technique for tissue surfaces [6-9] and the so-called "sandwich

710 Mario Gosalvez, R. Garcla-Caaero and Hubert Reinhold

tumor" technique [10]. The NADH microfluorimetry in the sandwich tumor is defined as a very suitable technique for the precise evaluation of the possibilities of an anticancer chemotherapy based on respiratory inhibitors.

MATERIAL AND METHODS

Tumor system The tumor under investigation was the

rhabdomyosarcoma BAl12 [I 1-14] growing in a "sandwich" chamber implanted in the subcutis of the back of a rat. Female WAG/Rij rats, isologues to the tumor, were used when 5 weeks old. The average weight was 120 g. The "sandwich" tumor system was a modifi- cation of the mouse-sandwich system [10]. In essence, a thin sheet of subcutis is fixed between a mica base plate and a glass cover slip. The tumor is implanted in this thin sheet ofsubcutis, next to a skin nerve, and grows as a circular sheet of transparent tumor tissue. The diameter of such a tumor is about 4 mm and its thickness ranges from 200 to 300 (/zm) [15]. Through the glass cover slip the tumor can be observed in vivo and investigated by focusing the microfluorimeter on it.

NAD H micro fluorimetry The microfluorimeter was a modified version

of the microfluorimeter for NADH surface fluorescence described by Chance [6-7], and the technique was essentially as described by us in the mouse-sandwich tumor [8, 9]. The microfluorimeter consisted o f a standard type Leitz Orthoplan microscope with M P V I . This microscope has an advantage in that the microscope stage is large enough to carry the rat in a simple holder. The lamp was an HBO 100 W, the filter system consisted of a BG 12 pre-filter, a "Ploem" Dichroic system (BG 38 /4+UGI /4 Selection filters, H-400 dichroic mirror and barrier filter K-400), a K430 u.v. filter and a 500 mm wide bad interference filter.

The photomultiplier operated on 0.8 kV, cooling to - 2 8 ° C reduced the dark current to about 0.02 nA. The signal was recorded on a chart recorder that was connected to the photomultiplier via a digital voltmeter and a low- frequency band filter ( l /2Hz) . The latter served as a noise-suppressing filter.

The objective used was a Leitz 11 × Ultropal without condensor. The measuring aperture equaled 100/am at the tissue level.

To study the sandwich tumors under the microfluorimeter, the animals were anaesthe- tized with a composite mixture consisting of the following anaesthetics: • chloralose 1 g ~ ,

urethane 1.25 g ~ , pentobarbital 0'12 g~o, tribromo ethanol 0 .5g~ , valium 0.1 g~o, to which, besides the appropriate solvents (poll thylen glycol and tert. amyl alcohol), 8.4 g % NaHCO3 was added to counteract the respiratory acidosis that may result from hypoxia cycles. This mixture was given i.v., slowly; 4ml/kg. The animal was then intubated via a larynx canula and received artificial respiration via a Starling-type respiratory pump. The respiratory rate was 60/min, the respiratory volume was adjusted for every individual animal depending on the weight of the animal, according to Guyton's formula [16]. The gas mixtures given consisted of a mixture of pure nitrogen and oxygen which was fed with an overflow into the respiratory pump. Calibrated flow meters and electro-magnetic gas valves facilitated the adjustment and changes in oxygen content when required. Rotenone and VM-26 were administered to the rat at the indicated doses, by i.p. injection, using poli ethylen glycol plus amyl alcohol, and a mixture of ricinus-oil plus ethyleneoxide [2] as solvents, respectively.

For the microfluorimetric determination of rat brain, normal animals were subjected to a trepanation of 3 mm of diameter. All other aspects of the technique were otherwise as in tumors except that the microfluorimeter was focused on the brain surface.

To ascertain that the effects of rotenone and VM-26 on the redox level of tissue pyridine nucleotides, were not due to circulation changes induced in the tumors by spurious effects of the drugs in the rat blood pressure, blood pressure was measured separately in rats treated with drugs at different doses. Blood pressure measurements were taken from the carotic artery and recorded via a pressure transducer with a Schwarzer strip-chart recorder. Rotenone, at 1-5mg/Kg, i.p., either did not affect or decreased the blood pressure by 10%, depend- on the rat. VM-26 did not affect blood pressure at any of the tested doses in any rat.

Oxygen uptake and redox measurements in mitochondria

Rat-liver, rat-rhabdomyosarcoma and rat- brain mitochondria were prepared essentially as described by Schneider [17], Devlin [18] and Basford [19], respectively. Mitochondrial oxygen uptake was measured with a Clark-type oxygen electrode at 22°C in a 3 ml chamber. The assay medium for mitochondrial respiration was composed of 225 mM sucrose, 20 m M KC1, 7 m M MgCI2, 1 0 m M Tris HC1 and 5 mM Tris-Pi, at pH 7.2. The redox states of

Delayed Pyridine Nucleotide Reoxidation Induced by the Anticancer Agent VM-26 711

mitochondrial pyridine nucleotides were measured with a dual wavelength spectro- photometer (Perkin-Elmer Hitachi 356) as described by Chance and Williams [20, 21]. Substrates, salts and co-factors were purchased from Sigma Company. Mitochondrial protein was determined by the Biuret reaction [22].

RESULTS

Figure 1 shows typical results of microfluorimetric experiments in sandwich rhabdomyosarcoma and rat brain of rats injected with VM-26 or rotenone.

RESET

) %02

#/ .q ~'/.o~

lO(O/*Oz lO0"1"Oz iO0"1o~ 100%0, ~ I0 rag,

f - \

,../.o, ~'/oO~ ~./. o, 4./.o,

,oo./~,oo./.o, ,oo'~.o, ,oo./.q,oo.1. o'~.o, ,007.o '

----t i' i ¢/.o, ¢/.q ,.'y.o, ~'/.o, ~'/.o, ~'/.o, ~./.o,

Fig. 1. Pyridine nucleotide redox changes monitored by N A D H microfluorimetr~ in vivo and in situ, performed in rats subjected to anoxia cycles during the i.p. rotenone and VM-26 treatments. The upper recording is an experiment done by focusing the microfluorimeter on an arterial region of the sandwich rhabdomyosarcoma. The middle recording is an experiment with another rhabdomyosarcoma, focusing the microfluorimeter on a venous region. The lower recording shows an experiment recorded from the rat brain surface. 100% 02: the animal was switched to breathing pure oxygen. 4% 02: the animal was switched to breathing a mixture of 4% oxygen and 96% nitrogen. 2"6 mg of Rot./ Kg LP.) i.p. injection of this dosage to the tumor-bearing rat. 10 rag. VM-26/Kg (LP.) i.p. injection of this dosage to the tumor-bearing rat (middle recording) or t , a normal rat (lower recording). The traces in the figure represent a smooth line drawn through the actual recordings; the level of electronic "noise" ranged from 3 to 8% of the changes.

The upper recording of Fig. t shows an experiment performed in a sandwich rhabdomyosarcoma. After anesthesia and intubation, the sandwich-tumor-bearing animal was placed on the stage of the microfluorimeter which was then focused on a tumor surface area free of blood vessels but near an arterial capillary. After a few minutes, allowed for the equilibra- tion of the animal, the NADH tissue fluorescence was recorded by switchingthe prismafrom

the visual-observation position to the photo- multipliel position. The recording of fluorescence (far left) starts with the animal breathing 100% oxygen. Immediately after the starting of the recording, the animal is subjected to anoxia by forcing it to breathe 4% oxygen in nitrogen. As shown in the recording, after a brief delay, this procedure induces an increase in NADH tissue fluorescence due to the reduction of mitochondrial pyridine nucleotides by intracellular substrates in the absence of intracellular oxygen [8, 9]. Two hundred seconds after the induction of anoxia, the anoxic period is terminated by switching the animal back to breathing 100% oxygen, and an immediate decrease of fluorescence is observed due to the recuperation of the aerobic-level redox state of intracellular pyridine nucleotides. The fluorescence recovery from anoxia is very rapid as the recorded tissue point lies very near to an arterial capillary. Afterwards, the recording shows a second anoxia cycle followed by the i.p. injection of rotenone, to the rat (2.6 mg/ kg of weight). Upon the injection of rotenone, there is a slight decrease in the tumor NADH fluorescence followed, after a few minutes, by a great increase in fluorescence and fluorescence oscillations, until the fluorescence is stabilized at a new, highly reduced, aerobic level. The cause of the fluorescence oscillations after the rotenone treatment is not yet clear but could be due either to reduction of cytoplasmic pyridine nucleotides, which has been reported to take place in an oscillating fashion under certain circumstances [23-25], or to small variations induced by rotenone, in the rat arterial pressure. After the fluorescence is equilibrated, a new 200-second anoxic cycle is performed and, in this case, the recovery from anoxia is considerably delayed. In this case, after inducing the respiration with 100% oxygen, the fluorescence remains at the anaerobic level for a few minutes and later on rapidly decreases to almost the aerobic level of fluorescence attained upon the rotenone injection. This delay in the recovery from anoxia is interpreted as a rotenone-induced inhibition of the tissue respiratory rate. Intracellularly, rotenone would partially block the mitochondrial respiratory chain and then the reoxidation of mitochondrial pyridine nucleotides, in the presence of oxygen, would take place slowly. The fluorescence decrease would then occur the moment pyridine nucleotides are completely re-oxidized. It is important to note that the extent of the fluorescence change of the anoxic cycle performed after rotenone, i s smaller than the fluorescence

712 Mario Gosalvez, R. Garda-Cahero and Hubert Reinhold

change of the previous control anoxic cycles. Therefore, rotenone would have induced a mitochondrial reduction of pyridine nucleotides not re-oxidable by oxygen, which is in agreement with previous reports [26]. On the other hand, the high level of fluorescence attained in aerobiosis after rotenone administration, is higher than would be expected if only the total reduction of mitochondrial pyridine nucleotides were to be involved. Thus a reduction of cytoplasmic pyridine nucleotides must collaborate in the attaining of such a highly reduced state of intracellular nucleotides, induced indirectly by rotenone. This would be supported by the increase in the glycolytic rate of tumor cells treated with rotenone as reported by us [27].

The middle recording of Fig. 1 shows an experiment performed on a sandwich rhabdomyosarcoma, but in this case the microfluorimeter was focused on a free area near a venous capillary. The recording shows first, two control anoxic cycles. Here the recovery from the anaerobic level of fluorescence takes place more slowly than in the previous experiment, due to the slower difusion of oxygen in the tissue, in venous regions. After the control cycles, the i.p. injection of VM-26 to the rat (10 mg/kg of weight) induces a slight oxidation of pyridine nucleotides (fluorescence decreases) that levels off in a few minutes. Two subsequent similar 200-second anoxic cycles performed, show a long delay in the recovery of anoxia with the administration of 100% oxygen. This delay is interpreted as a VM-26 induced inhibition of the tissue respiratory rate, by the same reasons stated in the previous experiment. The second anoxic cycle, after the VM-26injection, is not as wide as the first one; however, cycles that followed the second one, remained at similar widths (not shown). I f one could assume that the width of the cycle is proportional to the extent of the tissue respiratory inhibition, VM-26 would induce first, a strong respiratory inhibition which later stabilizes itself. The lower recording of Fig. 1, shows a microfluorimetric experiment performed on the brain surface of a rat. Here again, two control anoxic cycles recorded from a free area near a venous capillary, are shown, followed by the i.p. injection of VM-26 to the rat (10mg/kg of weight). The injection of VM-26 does not induce any change in the aerobic level of fluorescence but when perform- ing, after a few minutes, successive anoxic cycles, a progressive widening of the cycles is observed due to the delay in the recovery from anoxia after the administration of 100% oxygen. Again, the delay in the recovery is interpreted

as an inhibition of the respiratory rate of the brain tissue by VM-26; and as the widening of the rat brain cycles is less apparent than the widening of the tumor cycles, it would seem that VM-26 has less effect in brain respiration than in rhabdomyosarcoma respiration.

To provide a basis for the interpretations given to the microfluorimetric experiments, anoxia cycles were performed in isolated mitochondria, measuring the redox changes of pyridine nucleotides. Figure 2 shows two cycles in isolated mitochondria, the lower cycle being recorded in the absence of inhibitor. In both recordings the far left indicates the redox level of the mitochondrial pyridine nucleotides in the absence of substrate. In the lower cycle, with the addition of succinate and after a brief delay, the oxygen in the cell is exhausted and a great anaerobic reduction appears. This

Suc ;f--J

Oz I NAD f ~ reduction / ~ T M

~k 4 2 ~in I-

O.O.

Fig. 2. Changes in the redox state of p~ridine nucleotides of isolated mitochondria (2 mg/ml) in the presence and absence of rotenone (0.1/tM). Suc." 5 mM succinate ; 02: bubbling

oxygen.

reduced level is reoxidized by bubbling oxygen. The reoxidation is very rapid at first but slows down before attaining complete reoxidation. In the upper cycle, rotenone is added to the mitochondria prior to the addition ofsuccinate. In this case, the second and last phase of reoxidation are much slower due to the inhibition of I espiration by rotenone. This experiment in mitochondria supports the con- cept that a delay of reoxidation of pyridine nucleotides can be caused by inhibiting respiration with an inhibitor of NADH-linked substrates.

As the microfluorimetric experiments in Fig. 1 seem to indicate that rat brain tissue is less sensitive to VM-26 inhibition than is rat rhabdomyosarcoma, we compared, in Fig. 3,

Delayed Pyridine Nudeotide Reoxidation Induced by the Anticancer Agent VM- 26 713

the effect of VM-26 on the respiration of mitochondria isolated from rat rhabdomyosarcoma with both rat brain and rat liver. The concentrations of each type of mitochondria were adjusted to yield equivalent respiration [4, 28] to facilitate the comparison. Using this criteria, it appears that rat brain mitochondrial respiration is less sensitive to VM-26 than is rat rhabdomyosarcoma mitochondria.

100

o I.--.-

C~C

50 U3 Lid

25 50

~N VNI-26

Fig. 3. Effect of increasing concentrations of VM-26 on the respiration of mitochondria isolated from rat rhabdomyosarcoma, rat liver and rat brain. The concentration of the different mitochondria was adjusted to yield equivalent respira- tions in order to fadlitate the comparison (crosses: rat rhabdomyosarcoma mitochondria, 2 mg[ml; circles: rat liver mitochondria, 0.9 mg]ml ; triangles: rat brain mitochondria, 0.9

mg/ml).

DISCUSSION

Erecinska et al. [29, 30] have demonstrated that delayed reoxidation in mitochondria can be produced by addition of respiratory inhibitors which block partially the respiratory chain. This blocking acts as a bottle-neck for the reoxidation of the pool of carrier molecules behind the blocked point [29, 30]. Although the experiments of Erecinska et al. were done with terminal inhibitors, they have provided the basis for the interpretation of our experiments. Thus, the delay of the reoxidation of pyridine nucleotides found in the treatments with VM-26 and rotenone is secondary to a blocking of the mitoehondrial respiratory chain caused by the respiratory inhibitors. NADH microfluorimetry, therefore, appears to be a suitable

technique for the in vivo and in situ qualitative estimation of the effect of drugs on tissue respiratory rates. However, for the precise quantitative use of this technique it would be convenient to correlate the microfluorimetric experiments with data collected with micro-oxygen elec- trodes suitable for in vivo and in situ tissue determinations. However, presently NADH microfluorimetry offers the possibility of relative qualitative estimation of tissue respiratory rates in vivo and in situ.

From our results, it can be concluded that 10 mg/kg of VM-26 administered i .p.--a dose well within the range of the chemotherapeutic dose for rodent tumors [1]--affects considerably the respiration of the rabdomyosarcoma. Rotenone, a well known potent inhibitor of NADH-linked respiration [26], in a dose of 2"6 rag, affects the respiration of the rhabdomyosarcoma to the same extent as 10 mg of VM-26. Our results tend to confirm the proposed mechanism of action and toxicity of VM-26 as based on its effects and relative specificity for tumor respiration [3, 4]. On the other hand, we do not exclude the possibility that VM-26 might have effects in other bio- chemical parameters. VM-26 has been reported to inhibit in vitro the entering of the cell into mitosis and, in short incubation periods, is also able to arrest the cells in metaphase [1].

Within this context, it is interesting to point out that a respiratory inhibitiion would prevent the formation of the energy necessary in the premitotic period [31] and in the metaphase and anaphase transitions [32].

The application of NADH microfluorimetry to the in vivo and in situ evaluation of the mechanism of action of VM-26, provides a model study for the use of this technique in the precise determination of the possibilities of an anticancer chemotherapy based on respiratory inhibitors [5, 28].

Acknowledgements--Dr. M. Gosalvez acknowledges the personal contributions of Mr. J. Barrett (Torrance, California) and Mr. G. Blanco (Madrid), and the discussions and comments of Prof. B. Chance. Dr. H. Reinhold acknowledges the assistence of Mrs. M. Sahadat (rat preparations) and Mrs. J. Verwey, Mrs. G. H. Buisman and Miss M. Poess6 (blood pressure measurements).

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714 Mario Gosalvez, R. Garcla-Ca~ero and Hubert Reinhold

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delayed pyridine nucleotide reoxidation induced by the anticancer agent vm-26 as measured in vivo...

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