RELEASE FROM THE CRABTREE EFFECT BY HYPOXIC CELL RADIOSENSITIZERS

The Crabtree effect can be observed when the 02 consumption of tumour cells or of mammalian cells grown in culture is measured in physiological medium containing glucose. The effect of 2 hypoxic cell radiosensitizers, misonidazole and NDPP, on the O2 consumption of Ehrlich ascites tumour cells was compared in media with and without glucose. A stimulatory effect on 02 consumption was found for 5-20mM misonidazole as well as for 0 5mM NDPP, both in media containing 10-2M glucose. Thus glucose induced a Crabtree effect in Ehrlich tumour cells, expressed as 38-45 o inhibition of 02 consumption relative to that in the same medium without glucose. The stimulatory effect of misonidazole and NDPP on 02 utilization in medium with glucose undoubtedly appeared as a release from the Crabtree effect. IN A RECENT PAPER we reported that misonidazole (MIS) significantly increases the 02 consumption and decreases the respiratorv-control ratio of guinea-pig liver mitochondria (Mustea et at., 1978). The respiratory-control ratio is a measure of the extent of coupling between respiration and phosphorylation. Chance (1959) has defined it as the ratio of "'the respiratory rate in the presence of added ADP to the rate obtained following its (ADP) expenditure". WN'e regarded this behaviour of MIS as rcather similar to that of the oxidative-phosphorylation uncouplers. In this work we present additional new arguments supporting our hypothesis. Starting from the findings that oxidative-phosphorylation uncouplers such as 2,4-linitrophenol (DNP) and dicumarol release from the Crabtree effect (Loomis & Ram et al., 1963), we attempted to use this test in investigating 2 hypoxic-cell radio-sensitizers, MIS acnd NDPP, r eported to have a stimulatory effect on 02 consumption (Durand et al., 1 976; Mustea et al., 21) 1978). We preferred these 2 radiosensi-tizers mainly because of their good solu-bility in water. It is known that the Crabtree effect concerns the in vitro inhibition of the 02 consumption of tumours by addition of glucose (Crabtree, 1929) but some exceptions were also reported. Thus, in some experimental cancers such as the Walker carcinoma 256 and the DBA murine ascitic thymoma, as well as in some human neoplasms, there was no Crabtree effect On the other hand, the Crabtree effect has been observed in some normal tissues (retina. cartilage) and in a series of facultative anaerobes and yeasts

IN A RECENT PAPER we reported that misonidazole (MIS) significantly increases the 02 consumption and decreases the respiratorv-control ratio of guinea-pig liver mitochondria (Mustea et at., 1978). The respiratory-control ratio is a measure of the extent of coupling between respiration and phosphorylation. Chance (1959) has defined it as the ratio of "'the respiratory r-ate in the presence of added ADP to the rate obtained following its (ADP) expenditure". WN'e regarded this behaviour of MIS as rcather similar to that of the oxidative-phosphorylation uncouplers.
It is known that the Crabtree effect concerns the in vitro inhibition of the 02 consumption of tumours by addition of glucose (Crabtree, 1929) but some exceptions were also reported. Thus, in some experimental cancers such as the Walker carcinoma 256 and the DBA murine ascitic thymoma, as well as in some human neoplasms, there was no Crabtree effect (Elliott & Baker, 1935;Levy et al., 1953;Kiricuta et al., 1965;Mustea, 1974). On the other hand, the Crabtree effect has been observed in some normal tissues (retina. cartilage) and in a series of facultative anaerobes and yeasts (Locker & Spitzy, 1956;Cohen, 1957;Noell, 1958;de Deken, 1966;Mustea & Muresian, 1967a, I 967b).

MATERIALS AND METHODS
All experiments were performed on Ehrlich ascites tumour cells, ELD hyperdiploid strain, propagated by routine inoculation of 4 x 106 cells into NMRI mice. The cells were har-vested 10-15 days later, when they were in the plateau phase of growth.
The 02 consumption was measured polarographically using an 02 Clark electrode housed in a 3-8ml closed reaction vessel and maintained at 37°C. The potential at the 02 electrode was -0-6 V. The cells were suspended in Tyrode medium free of Ca2+ and glucose, Krebs-Ringer phosphate medium, and in the same media wiith 10-2M glucose added.
The effect of the above-mentioned chemicals on the 02 consumption of Ehrlich ascites tumour cells was expressed as a relative 02 utilization ratio (OUR) obtained by dividing the rate of 02 consumption after addition of the chemical by the initial rate measured in its absence. When the effect of glucose on the rate of 02 consumption was determined, OUR was calculated as:  Fig. 2.

RESULTS
In glucose-free media, MIS and NDPP produced an inhibition of the rate of 02 consumption of Ehrlich ascites tumour cells (OUR < 1, Table I).
Glucose in a concentration of 10-2M induced a Crabtree effect measured as a pronounced decrease of the rate of 02 consumption. The OURs were 0 55 in Tyrode medium (inhibition 45%o) and 0-62 in Krebs-Ringer phosphate medium (inhibition 38%) respectively (Figs. 1 and 2).
The inhibition of 02 consumption by glucose was partially reversed by both radiosensitizers, and also by DNP. In all cases the OURs were > 1 (Fig. 1 0-92 + 0-03 (10) 1-19 + 0 047 (9) 0-81 + 0-086 (7) 1-2 + 0 037 (4) 20 1*28 + 0-061 (10) NDPP 0(5 0-96 + 0-023 (4) 1-26 + 0-082 (4) 0-88 + 0-043 (6) 1-25 + 0 095 (4) DNP 0.1 -l8 + 0-106 (6) 168 + 0-078 (9) 0-85 + 0-05 (7) 1*13 + 0-046 ( in the presence of glucose and radiosensitizer was related to the initial rate of 02 consumption in media without glucose and radiosensitizer (endogenous rate), we obtained the OURs listed in Table II. These data show that the rate of 02 consumption in the presence of glucose and radiosensitizers in this study was below the endogenous rate (Fig. 2). The data on the release from the Crabtree effect by MIS, NDPP and DNP are included in Table II. In addition correlation between drug concentration and magnitude of release was also obtained for MIS (Table II). Endogenous Irate of (2 consumption in Tyrode me(lium free of glucose= 1-0. piration may contribute to their effectiveness, in addition to electron-affinity and cytotoxicity. Chemical compounds which inhibit 02 utilization could enhance radiation response by increasing oxygenation of the tumours. On the other hand, compounds stimulating 02 consumption may alter their effectiveness by enlarging the hypoxic regions of the tumours (Biaglow & Durand, 1976;Durand et al., 1976Durand et al., , 1978Haynes & Inch, 1976). According to these criteria it appears to be of practical  importance to know the effect of hypoxic cell radio sensitizers on 02 utilization. The recent investigations permit us to divide radiosensitizers in 2 groups: respectively with inhibitory, and with stimulatory effects on 02 utilization. It is pertinent to mention that the stimulatory effects for most of these compounds was only seen when glucose was present in the reaction medium (Biaglow et al., 1975(Biaglow et al., , 1978Mustea et al., 1978). The requirement for glucose in stimulating 02 utilization was interpreted as being due to exhaustion of endogenous substrates necessary for respiration (Biaglow et al., 1978).
Our results indicate another possible mechanism and interpretation. Glucose induces in the majority of tumours and in some cell cultures a Crabtree effect expressed as an inhibition of 02 consumption (see Ibsen, 1961 for a review; Biaglow et al., 1969). For Ehrlich ascites tumour cells this inhibition was reported to be 25-57% of endogenous 02 consumption, depending upon experimental conditions (Ibsen, 1961;Mustea, 1974). The uncoupling agents of oxidative phosphorylation reverse the inhibition of 02 consumption bv glucose. MIS and NDPP have proved to have similar properties to the uncouplers regarding release from the Crabtree effect. Therefore their stimulating of 02 consumption in media containing glucose produces a marked release from the Crabtree effect. Taking our results into consideration and the attempts of various authors to find a correlation between the effectiveness of hypoxic sensitizers and their effects on oxygen utilization, we consider it necessary to re-examine the stimulatory effects on 02 consumption observed in media containing glucose.
The Crabtree effect is regarded as a regulatory mechanism between the glycolytic and respiratory compartments, and is dependent on the level of adenine nucleotides (Belitzer, 1936;Chance & Hess, 1959). The mechanism of action of hypoxic radiosensitizers on the Crabtree effect ought to be investigated, taking into account the action of these drugs on adenine factors limiting respiration (NAD+, ATP), as well as the activity of the enzymes reported as having a predominant part in release from the Crabtree effect (Yamada, 1968;Asami & Yamada, 1969).
In connection with the latter aspect, determinations of the effect of lbypoxic radiosensitizers on ATP-ase activity are now being carried out in our laboratory.
This work wvas partly supported by the Atomic Energy Agency, Vienna, Austria, Contract 1691 -RB.
We are grateful to AMr C. Donoganv for his valuiable technical assistance.