Inr. J Radrurton Ondog? Bmi. Phg Vol Prmred I” the U.S A. Al, nghts reserved
22. pp. 79-86 Copyrrghr
0364-3016192 $5.00 + .oO 0 1991 Pergamon Press plc
0 Biology Original Contribution
PHARMACOLOGICAL MODIFICATION OF TUMOR BLOOD FLOW: LACK OF CORRELATION BETWEEN ALTERATION OF MEAN ARTERIAL BLOOD PRESSURE AND CHANGES IN TUMOR PERFUSION HELEN B. STONE, PH.D.,
ANDREW I. MINCHINTON,
DOUGLAS MENKE,
PH.D.,
MARILYN LEMMON, B. S.,
B. A. AND J. MARTIN BROWN, D.PHIL.
Department of Radiation Oncology, Division of Radiation Biology, Stanford University Medical Center, Stanford, CA 94305 The correlation between mean arterial blood pressure (MABP) and vascular perfusion in SCC-VIUSt tumors in mice was compared following administration of three vasoactive drugs: flavone acetic acid (200 mgkg), hydralazine (5 mgkg), or nicotinamide (500, 750, and 1000 mgkg). MABP was measured by the direct method in unanesthetized, unrestrained mice bearing a carotid catheter. Vascular perfusion of the tumor was measured using the 86RbCI extraction method. Body temperature was maintained at 36” to 37°C after drug administration when necessary. All three drugs reduced MABP from a control value of 125 + 2 (s.e.) mm Hg in mice without tumors. Flavone acetic acid at this dose had the least effect on blood pressure, with a minimum of 86% of control values at 10 to 20 min, and a return to control values by 1 hr. However, it produced a profound reduction in tumor perfusion that lasted more than 48 hr. Hydralazine and nicotinamide reduced blood pressure to minima between 55% and 69% of control values within 30 min, followed by a gradual return toward control values by about 8 hr. The reduction in tumor perfusion by hydralazine paralleled its effect on blood pressure. However, nicotinamide produced a transitory, although not statistically significant, increase in tumor perfusion at the highest dose given. These data demonstrate that tumor blood flow modification by drugs is not necessarily the result of changes in MABP, and blood pressure changes alone do not inevitably lead to changes in tumor perfusion. Flavone acetic acid, Hydralazine,
Nicotinamide,
Mean arterial blood pressure, Tumor perfusion,
Tumors.
blood flow by a thermal washout technique, and oxygen tension with a polarographic technique. Lymph node metastases, intramuscular, and intrahepatic tumors showed similar responses, whereas blood flow in liver, brain, bone marrow, and subcutaneous tissues remained relatively constant under these conditions. In the studies by both groups, the animals were anesthetized by pentobarbital sodium, which itself can reduce blood pressure and body temperature (12, 32, 40, 44) and can affect the response of tumors to radiotherapy, most likely by its effects on oxygenation of tumors (12, 14, 44, 50, 53). Studies on the effects of vasoactive agents on blood flow or perfusion in tumors have been reviewed by Jain (29) and by Jirtle (30). The purpose of the present study was to determine the correlation between MABP and tumor perfusion following administration of three vasoactive agents, flavone acetic acid, hydralazine, and nicotinamide, using unanesthetized, unrestrained mice whose body temperature was maintained at 36 to 37°C after drug administration.
INTRODUCTION
Many studies have investigated the interrelationship between tumor cells and the anatomy and function of a tumor’s vascular network and the importance of the vascular supply to tumor growth and response to radiotherapy, chemotherapy, hyperthermia (reviewed in (28, 30, 61)). Manipulation of a tumor’s blood supply to enhance the effectiveness of therapy is the ultimate goal of such studies. Vaupel (60) has observed a linear relationship between mean arterial blood pressure (MABP) and blood flow in tissue-isolated sarcomas in rats. Blood pressure was reduced by controlled bleeding and corrected by transfusion, and blood flow was measured by cannulating the single vein draining the tumor. Conversely, Suzuki et al. (55, 56) elevated mean arterial blood pressure in rats to 140 to 160 mm Hg using angiotensin II, and found sharp increases in blood flow and oxygen tension in subcutaneous tumors, to as much as 4 to 12 times control values. They measured Reprint requests to: Helen B. Stone, Ph.D., NIH-NCI, EPN800, 9000 Rockville Pike, Bethesda, MD 20892. Acknowledgements-We gratefully acknowledge the technical assistance of Nixy Kaul and the helpful suggestions of Dr. James
veloping the techniques for carotid catheterization of the mouse and measuring mean arterial blood pressure. Supported by grant #CA 25990 from the National Cancer Institute. Accepted for publication 14 June 1991.
Weeks, Dr. William Ebling, Dr. Hideyashi Harashima, Dr. Michael Horsman, Dr. William Wilson, and Kathy Groom in de79
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AND MATERIALS
Mice and tumor C3H/Km female mice aged 3 to 4 months were obtained from the defined flora colony of the Stanford Radiation Biology Mouse Facility. The tumor line was squamous cell carcinoma SCC-VII/St. Details of the derivation and handling of this cell line have been published previously (20). Suspensions containing 2 X lo5 cells were implanted intradermally on the backs of the mice, and grew to 8 mm diameter in approximately 2 weeks. The effects of flavone acetic acid on blood pressure were examined in mice with and without tumors. Mice used for blood pressure studies with hydralazine and nicotinamide did not have tumors.
Drugs All drugs were prepared fresh daily in sterile saline solution containing no preservatives. Flavone acetic acid* was dissolved to give a concentration of 20 mg/ml, hydralazinei 0.5 mg/ml, and nicotinamide$ 50, 75, or 100 mg/ml. The injected volume of all drugs was 0.01 ml/g body weight.
Blood pressure measurements Mean arterial blood pressure was measured by the direct method in unanesthetized, unrestrained mice bearing a carotid catheter. Mice were anesthetized with pentobarbital sodium (65 mg/kg, intraperitoneally) for catheter implantation, and were given ampicillin (100 mglkg, subcutaneously) at the completion of surgery and daily thereafter. The animals were kept warm until they recovered from the anesthesia. Between experiments, they were caged individually to prevent them from biting each other’s catheters. Fabrication and implantation of carotid catheters have been described previously (10, 18, 46, 64, 65). Briefly, the catheter was made of polyethylene tubing.§ It was filled with heparinized saline, inserted 4 to 5 mm into the carotid artery, and held in place with two sutures. After a 180” bend it passed subcutaneously behind the front leg, and after another bend, exited at the nape of the neck, pointing forward. It was plugged with a short steel wire when not connected to the transducer. A second catheter of PE- 10 tubing was implanted intraperitoneally through the muscles of the flank, and passed subcutaneously to exit at the nape of the neck. It was also plugged with a short piece of steel wire. This catheter allowed administration of drugs without picking the mouse up or puncturing the skin, thus minimizing stress, although blood pressure returned to preinjection levels within two min after release of mice injected intraperitoneally with saline using a hypodermic needle. *National Cancer Institute. tCiba-Geigy, Basel, Switzerland. *Sigma Chemical Co., St. Louis, MO. §PE-10 (ID 0.011 in, OD 0.024 in) and PE-50 (ID 0.023 in,
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Blood pressure measurements were made only after the mice had fully recovered from anesthesia, either in the afternoon of the day of surgery, or the next morning. The body temperature of the mice treated with nicotinamide was maintained after injection of drugs by placing them in a standard polycarbonate mouse cage floating in a 37°C water bath. Rectal temperatures of all mice were measured periodically during the course of each experiment with a thermocouple, and did not decrease below 36°C. A disposable transducer* was placed at the level of the mouse’s heart, and was connected to a clinical monitor-t The monitor was calibrated daily before use, rechecked periodically during experiments and did not drift significantly during use. The connections between the catheter and the transducer were made from 22 gauge disposable hypodermic needles and PE-50 tubing filled with heparinized saline, taking care to exclude air bubbles. Mean arterial blood pressures prior to administration of the drugs were 125 ? 2 (s.e.) mm Hg in mice without tumors, and 113 -+ 3 in mice with tumors. Readings were taken when the mice were sitting quietly. Animals were retested with the same drug and dose on subsequent days as long as the catheter remained open. There was no evidence that the magnitude or duration of the changes in blood pressure was altered in the mice given multiple injections, and data were pooled. Tumor perfusion Tumor perfusion was measured by extraction of “RbCl (49, 67). Briefly, mice bearing 8 mm diameter tumors were injected intraperitoneally with the appropriate vasoactive compound, and were kept warm as described above, with food and water available ad lib. At the appropriate time, 3 $Ji of s6RbCl in 0.1 ml was injected intravenously into the tail vein. Two minutes later, the mice were killed by cervical dislocation. The tumors and tails were removed immediately, and placed in double glass tubes and counted in a gamma scintillation spectrometer.* Counts from tumors were corrected for background, normalized to tumor weight, and expressed as % of control. Data from mice whose tails contained > 10% of the injected radioactivity were not included. Statistical analysis Means and standard errors were calculated for each group and time point. The significance of the difference between blood pressure measurements before and after drug administration was determined with paired-difference t-tests. Comparisons of blood pressure in mice with and without tumors and of tumor perfusion data were made using unpaired two-group t-tests. P values of 5 0.05 were considered significant, and those 5 0.01 were considered highly significant. OD 0.038 in) Intramedic, Clay Adams. *Medi-Trace, Graphic Controls, Buffalo, NY. tMode1 #78901A, Hewlett Packard, Palo Alto, CA. $Model
#5360,
Packard
Instrument
Co.,
Laguna
Hills,
CA.
Blood
pressure and tumor perfusion ??H.
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HYDRALAZINE,
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Fig. 1. Effect of 200 mg FAA/kg ip on (a) mean arterial blood pressure, as a percentage of pretreatment control values and on (b) tumor blood perfusion as measured by s6Rb extraction, as a function of time after FAA administration. 0: mice bearing SCCVII/St tumors, and 0: mice without tumors. The shaded areas represent pretreatment control values 2 1 se.; error bars are s.e. In (a), symbols with error bars were from groups with 6 to 20 mice, and in (b), symbols with error bars were from groups with 3 to 4 mice; symbols without error bars were from individual mice.
RESULTS Flavone acetic acid Flavone acetic acid at a dose of 200 mg/kg reduced the mean arterial blood pressure to a minimum of 8 1% to 86% of control in mice with and without tumors, respectively (Fig. la). The reduction was statistically significant from 5 to 30 min, but returned to control values by 1 hr. There
was no statistically significant difference between mice with and without tumors in the percentage of reduction following FAA, although MABP was 7 to 20 mm Hg lower in tumor-bearing mice both before and after treatment @ > 0.05 at 1 and 1.5hr; p < 0.01 at all other times). Tumor perfusion was reduced within 15 min after FAA, and the reduction persisted for more than 48 hr (Fig. lb). Hydralazine blood pressure was reduced to 55 to value between 5 min and 2 hr after a dose of 5 mg hydralazine/kg (Fig. 2a). The blood pressure then returned gradually to normal values within 24 hr. The mean
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Fig. 2. Effect of 5 mg hydralazine/kg ip on (a) mean arterial blood pressure, as a percentage of pretreatment control values, and on (b) tumor blood perfusion, as in Fig. 1. A: mice bearing SCC-VII/St tumors, and A: mice without tumors. Symbols with error bars were from groups with 4 to 8 (a) or 3 to 4 mice (b); symbols without error bars represent individual mice.
Tumor perfusion was reduced for at least 6 hr, although in some individual mice, no reduction was observed (Fig. 2b). Nicotinamide Mean arterial blood pressure was rapidly reduced by a dose of 500 mg nicotinamide/kg, reaching a minimum of 69% of the control value by 30 min. It gradually returned toward control values over the next several hours (Fig. 3a). Tumor perfusion was not significantly reduced during the first 4 hr after administration of this dose of nicotinamide (Fig. 3b). A similar reduction in mean arterial blood pressure and gradual return to control values were seen following nicotinamide doses of 750 (data not shown) and 1000 mg/kg (Fig. 4a). Tumor perfusion was increased over control levels at 30 min and 1 hr after a dose of 1000 mg/kg, but the increases were not statistically significant (Fig. 4b).
arterial
60% of the control
DISCUSSION The present studies show that agents that decrease mean arterial blood pressure do not necessarily cause a decrease
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1. J. Radiation Oncology 0 Biology 0 Physics NICOTINAMIDE, E
Volume 22 , Number 1, 1992 NICOTINAMIDE,
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in tumor perfusion. Of the three agents studied vone acetic acid produced the smallest reduction
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pressure, and blood pressure had returned to normal by 1 hr, but it produced a profound, prolonged reduction in tumor perfusion. Hydralazine and all three doses of nicotinamide produced similar degrees of reduction in blood pressure, followed by a slow return to nearly normal values by about 5 to 10 hr. However, hydralazine reduced tumor perfusion for several hours, whereas nicotinamide did not significantly reduce tumor perfusion, and probably increased it somewhat following a dose of 1000 mg/kg. The increase in tumor perfusion after nicotinamide in the present study was of the same order of magnitude as that reported by Horsman et al. (21). Flavone acetic acid Reduced perfusion or vascular collapse in tumors 4 to 48 hr following administration of flavone acetic acid has been observed by several investigators, using Evans blue concentrations, single labeling or mismatch of labeling fol-
Fig. 4. Effect of 1000 mg nicotinamide/kg ip on (a) mean arterial blood pressure and on (b) tumor blood perfusion, as in Fig. 1. ?? : mice bearing SCC-VII/St tumors, and 0: mice without tumors. There were 4 to I2 (a) or 3 to 4 mice/group (b).
lowing administration of the fluorescent compounds Hoechst 33342 and DiOC7(3), 86RbC1 extraction, 2H-NMR, or histological methods (3, 15, 16, 19, 51, 54, 68). Murray et al. (41) have reported a decrease in plasma clotting time 15 to 30 min after administration of 300 mg FAA/kg to tumor-bearing mice, but an increase in clotting time, thrombin time, and fibrin degradation products 4 to 6 hr after FAA, which suggested intravascular coagulation. The plasma from mice without tumors showed only the transient decrease in clotting time. The effects of FAA may vary with the site of the tumor: hepatic metastases of colon tumors showed extensive necrosis but less hemorrhage than occurred with subcutaneous tumors (51). Whether tumor sizes were comparable at the time of treatment was not reported. Vascular collapse in normal tissues was not observed histologically by Smith et al. (51). Dose-limiting hypotension occurs in patients following doses of FAA that produce no objective tumor response (38). Loss of clonogenic cells and growth delay has been observed in tumors after FAA administration in vivo (4, 19, 45, 68). Denekamp et al. (13) have shown that clamping the blood supply to murine tumors for 2 hr or longer de-
Blood pressure and tumor perfusion
lays their growth, and for 15 to 24 hr can induce local tumor control. A detailed histological study by Zwi et al. (69) showed that FAA caused necrosis and hemorrhage in vascularized but not in avascular regions of EMT6 tumors growing in the peritoneal cavity of mice. Thus, vascular collapse following FAA is probably a major cause of the cytotoxicity of FAA in viva. However, other mechanisms may contribute as well, such as direct cytotoxicity by FAA or its metabolites, production of irreparable single strand breaks in DNA, decreasing ATP levels, increasing NK cell activity, and interferon production, as pointed out by Chabot et al. (6). The similar reduction in MABP in mice with and without SCC-VII/St tumors indicates that FAA did not have an additional effect on MABP that was mediated through its effects on the tumors. Hydralazine Hydralazine is a vasodilator that relaxes the smooth muscle of the arterioles, lowering peripheral vascular resistance. It is used clinically, in conjunction with other agents, as an antihypertensive agent. At doses that do not produce a marked decrease in blood pressure, it may increase splanchnic, coronary, cerebral, and renal blood flow (48). It increases heart rate and contractility, plasma renin activity, and fluid retention (48). Decreases in tumor blood flow or perfusion have been observed in transplanted rodent tumors at intervals of 15 to 90 min after hydralazine, by the methods of radioactive microspheres, laser Doppler flowmetry, binding of 3H-misonidazole, 86RbC1 extraction, and double labeling with the fluorescent dyes Hoechst 33342 and DiOC7(3) (2, 7, 24, 37, 52, 58, 63). Changes in the magnetic resonance spectrum after hydralazine show increases in anaerobic metabolism of tumors (42, 43, 57). However, at low doses of hydralazine (0.25 kg/g), tumor blood flow was increased in a murine fibrosarcoma (37). Data suggest that in humans, an increase in tumor blood flow is produced by maximum tolerated doses of hydralazine (47). The effects on normal tissue blood flow do not appear to be consistent: increases (2, 7, 63), decreases (37, 52), and no change (37, 52) have all been observed. The magnitude of the effect depends on the normal tissue and dose of hydralazine, and perhaps the method of measurement as well. Hydralazine enhances the toxicity of agents that are preferentially toxic to hypoxic cells, including SR 4233 (5) melphalan (52), the hypoxic cell sensitizers misonidazole (ll), RSU 1069 (8), and hyperthermia (24, 25, 37). Radioprotection of both tumors (1, 11) and normal spleen cells in vivo (43) has been observed following hydralazine administration. Tozer et al. (57) found a reduction in MABP following hydralazine (5 2 mg/kg) and a simultaneous increase ininorganic phosphate, as measured in a rat fibrosarcoma by magnetic resonance spectroscopy. However, the latter peaked at about 30 min, and then decreased while MABP
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showed little change from minimal values from 10 through 150 min. They concluded that the degree of reduction in MABP was not the sole determinant of tumor energy status. Horsman et al. (24) found a correlation between the duration of reduction in MABP and tumor perfusion (86RbC1 extraction method) in murine mammary tumors following 5 mg hydralazine/kg. In the present study, perfusion was reduced in tumors for 4 to 6 hr after hydralazine. This roughly correlated with the curve of the reduction of blood pressure with time after hydralazine, although there was considerable heterogeneity in individual responses, which we cannot explain. Nicotinamide Horsman et al. have reported a reduction in mean arterial blood pressure in unanesthetized mice after 1000 mg nicotinamide/kg (26), as well as an increase in tumor perfusion in SCC-VII and EMT6 tumors, as measured by *‘RbCl extraction and by uptake of Hoechst 33342 (9, 21, 26). They also reported a decrease in binding of 14C misonidazole and an increase in radiation response of SCCVII, EMTG, and Lewis lung tumors, both of which are consistent with increased oxygenation of the tumors (9, 21, 22, 23, 26, 27, 33, 34). Although the increase in tumor perfusion 30 to 60 min after nicotinamide was not statistically significant in our study, it is consistent with the above findings. Relationship between MABP and tumor blood flow: The relationship between blood pressure and blood flow rate in a tissue is described by the equation: n Y
=
P rlz
where Q is the blood flow rate, AP is the pressure difference, q is the blood viscosity, and Z is a term representing the geometrical resistance to flow (28). Because there are many control points in the vascular tree, changes in MABP may not be transmitted to the vessels supplying the vascular bed of a given tissue, or conversely, the distribution of blood to various organs and tissues may change even in the absence of changes in MABP, as in the vascular collapse in tumors following flavone acetic acid. Although many agents are known that reduce tumor blood flow or perfusion (29), hyperthermia and several drugs can increase it: acetyl-Bmethylcholine (17)) angiotensin (3 1), nitroglycerine (39), oxytocin (59), and certain anesthetics (40). The calcium channel blockers verapamil and flunarazine have been shown to increase blood flow or perfusion in rodent tumors at doses that do not alter MABP (35, 36, 62, 66). When MABP was reduced by verapamil, however, Vaupel and Menke (62) found a reduction in tumor blood flow. Menke and Vaupel compared the effects of various anesthetics, neuroleptic, neuroleptanalgesic, and sedative agents on MABP, tumor blood flow, and tumor vascular resistance in rats bearing DS-carcinosarcoma (40).
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All of the agents studied either decreased MABP or had no effect on it. Their data show a better correlation between changes in tumor blood flow and tumor vascular resistance than between either of these parameters and reduction in MABP. Changes in cardiac output and in blood viscosity could contribute to changes in tumor blood flow and perfusion as well. A final consideration is that most studies of tumor blood flow and perfusion give data on the tumor as a whole. The
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response to therapy, however, may be governed by subpopulations of tumor cells that are resistant because of insufficient access to oxygen and nutrients or chemotherapeutic agents. Similarly, agents that modify tumor blood flow will affect vascularized and nonvascularized regions differently, as has been shown for FAA (69). Conversely, it is also possible that a large proportion of the cells in deficient environments are nonclonogenic. Therefore, studies of tumor response to manipulations of blood flow are needed.
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Int. J. Radiat. Biol. 35:277-280; 1979. Evelhoch, J. L.; Bissery, M.-C.; Chabot, G. G.; Simpson, N. E.; McCoy, C. L.; Heilbrun, L. K.; Corbett, T. H. Flavone acetic acid (NSC 347512)-induced modulation of mutine tumor physiology monitored by in vivo nuclear magnetic resonance spectroscopy. Cancer Res. 48:47494755; 1988. Finlay, G. J.; Smith, G. P.; Fray, L. M.; Baguley, B. C. Effect of flavone acetic acid on Lewis lung carcinoma: evidence for an indirect effect. JNCI 80:241-245; 1988. Gullino, P. M.; Grantham, F. Studies on the exchange of fluids between host and tumor. III. Regulation of blood flow in hepatomas and other rat tumors. JNCI 28:21 l-229; 1962. Heatley, N. G.; Weeks, J. R. Fashioning polyethylene tubing for use in physiological experiments. J. Appl. Physiol. 19:542-545; 1964. Hill, S.; Williams, K. B.; Denekamp, J. Vascular collapse after flavone acetic acid: a possible mechanism of its anti-tumour action. Eur. J. Cancer Clin. Oncol. 25:1419-1424; 1989. Hirst, D. G.; Brown, J. M.; Hazlehurst, J. L. Effect of partition coefficient on the ability of nitroimidazoles to enhance the cytotoxicity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Cancer Res. 43:1961-1965; 1983. Horsman, M. R.; Brown, J. M.; Hirst, V. K.; Lemmon, M. J.; Wood, P. J.; Dunphy, E. P.; Overgaard, J. Mechanism of action of the selective tumor radiosensitizer nicotinamide. Int. J. Radiat. Oncol. Biol. Phys. 15:685-690; 1988. Horsman, M. R.; Chaplin, D. J.; Brown, J. M. Radiosensitization by nicotinamide in vivo: a greater enhancement of tumor damage compared to that of normal tissues. Radiat. Res. 109:479489; 1987. Horsman, M. R.; Chaplin, D. J.; Brown, J. M. Tumor radiosensitization by nicotinamide: a result of improved perfusion and oxygenation. Radiat. Res. 118:139-150; 1989. Horsman, M. R.; Christensen, K. L.; Overgaard, J. Hydralazine-induced enhancement of hyperthermic damage in a C3H mammary carcinoma in vivo. Int. J. Hyperther. 5:123-126; 1989. Horsman, M. R.; Overgaard, J.; Chaplin, D. J. The interaction between RSU-1069, hydralazine and hyperthermia in a C3H mammary carcinoma as assessed by tumour growth delay. Acta Oncol. 861-862; 1988. Horsman, M. R.; Overgaard, J.; Christensen, K. L.; Trotter, M. J.; Chaplin, D. J. Mechanism for the reduction of tumour hypoxia by nicotinamide and the clinical relevance for radiotherapy. Biomed. Biochim. Acta. 48:s 251-S 254; 1989. Horsman, M. R.; Wood, P. J.; Chaplin, D. J.; Brown, J.M. ; Overgaard, J. The potentiation of radiation damage by nicotinamide in the SCCVII tumour in vivo. Radiother. Oncol. 18:49-57; 1990. Jain, R. K. Determinants of tumor blood flow: a review.
Blood pressure and tumor perfusion 0 H. B. Cancer Res. 48:2641-2658; 1988. 29. Jain, R. K.; Ward-Hartley, K. Tumor blood flow-characterization, modifications, and role in hyperthermia. IEEE Trans. Sonics Ultrasonics. SU-31504-526; 1984. 30. Jirtle, R. L. Chemical modification of tumour blood flow. Int. J. Hyperther. 4:355-371; 1988. 31. Jirtle, R. L.; Clifton, K. H.; Rankin, J. H. Effects of several vasoactive drugs on the vascular resistance of MT-W9B tumors in W/Fu rats. Cancer Res. 38:2385-2390; 1978. 32. Johnson, R.; Fowler, J. F.; Zanelli, G. D. Changes in mouse blood pressure, tumor blood flow, and core and tumor temperatures following nembutal or urethane anesthesia. Radiology 118:697-703; 1976. 33. Jonsson, G. G.; Kjellen, E.; Pero, R. W. Nicotinamide as a radiosensitizer of a C3H mouse mammary adenocarcinoma. Radiother. Oncol. 1:349-353; 1984. 34. Jonsson, G. G.; Kjellen, E.; Pero, R. W.; Cameron, R. Radiosensitization effects of nicotinamide on malignant and normal mouse tissue. Cancer Res. 45:3609-3614; 1985. 35. Kaelin, W. G. J.; Shrivastav, S.; Jirtle, R. L. Blood flow to primary tumors and lymph node metastases in SMT-2A tumor-bearing rats following intravenous flunarizine. Cancer Res. 44:896-899; 1984. 36. Kaelin, W. G. J.; Shrivastav, S.; Shand, D. G.; Jirtle, R. L. Effect of verapamil on malignant tissue blood flow in SMT-2A tumor-bearing rats. Cancer Res. 42:394&3949; 1982. 37. Kalmus, J.; Okunieff, P.; Vaupel, P. Dose-dependent effects of hydralazine on microcirculatory function and hyperthermic response of murine FSaII tumors. Cancer Res. 50: 1519; 1990. 38. Kerr, D. J.; Kaye, S. B.; Cassidy, J.; Bradley, C.; Rankin, E. M.; Adams, L.; Setanoians, A.; Young, T.; Forrest, G.; Soukop, M.; Calvel, M. Phase I and pharmacokinetic study of flavone acetic acid. Cancer Res. 47:677(X781; 1987. 39. Kruuv, J. A.; Inch, W. R.; McCredie, J. A. Blood flow and oxygenation of tumors in mice. II. Effects of vasodilator drugs. Cancer 20:60-65; 1967. 40. Menke, H.; Vaupel, P. Effect of injectable or inhalational anesthetics and of neuroleptic, neuroleptanalgesic, and sedative agents on tumor blood flow. Radiat. Res. 114:6&76; 1988. 41. Murray, J. C.; Smith, K. A.; Thurston, G. Flavone acetic acid induces a coagulopathy in mice. Brit. J. Cancer 60:729733; 1989. 42. Okunieff, P.; Kallinowski, F.; Vaupel, P.; Neuringer, L. J. Effects of hydralazine-induced vasodilation on the energy metabolism of murine tumors studied by in viva 31P-nuclear magnetic resonance spectroscopy. JNCI 80:745-750; 1988. 43. Okunieff, P.; Walsh, C. S.; Vaupel, P.; Kallinowski, F.; Hitzig, B. M.; Neuringer, L. J.; Suit, H. D. Effects of hydralazine on in vivo tumor energy metabolism, hematopoietic radiation sensitivity, and cardiovascular parameters. Int. J. Radiat. Oncol. Biol. Phys. 16:1145-l 148; 1989. 44. Pallavicini, M. G.; Hill, R. P. Effect of tumor blood flow manipulations on radiation response. Int. J. Radiat. Oncol. Biol. Phys. 9:1321-1325; 1983. 45. Plowman, J.; Narayanan, V. L.; Dykes, D.; Szarvasi, E.; Briet, P.; Yoder, 0. C.; Paull, K. D. Flavone acetic acid: a novel agent with preclinical antitumor activity against colon adenocarcinoma 38 in mice. Cancer Treat. Rep. 70:631435; 1986. 46. Popovic, P.; Sybers, H.; Popovic, V. P. Permanent cannulation of blood vessels in mice. J. Appl. Physiol. 25:626627; 1968. 47. Rowell, N. P.; Flower, M. A.; McCready, V. R.; Cronin, B.; Horwich, A. The effects of single dose oral hydralazine
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on blood flow through human lung tumors. Radiother. Oncol. 18:283-292; 1990. Rudd, P. Antihypertensive agents and the drug therapy of hypertension. In: Gilman, A. G.; Goodman, L. S.; Rall, T. W.; Murad, F., eds. Goodman and Gilman’s the pharmacological basis of therapeutics. New York: Macmillan Publ. Co.; 1985:78&805. Sapirstein, L. A. Regional blood flow by fractional distribution of indicators. Am. J. Physiol. 193:161-168; 1958. Sheldon, P. W.; Chu, A. M. The effect of anesthetics on the radiosensitivity of a murine tumor. Radiat. Res. 79:568-578; 1979. Smith, G. P.; Calveley, S. B.; Smith, M. J.; Baguley, B. C. Flavone acetic acid (NSC 3475 12) induces haemorrhagic necrosis of mouse colon 26 and 38 turnours. Eur. J. Cancer Clin. Oncol. 23:1209-1211; 1987. Stratford, I. J.; Adams, G. E.; Godden, J.; Nolan, J.; Howells, N.; Timpson, N. Potentiation of the anti-tumour effect of melphalan by the vasoactive agent, hydralazine. Brit. J. Cancer. 58:122-127; 1988. Suit, H. D.; Sedlacek, R. S.; Silver, G.; Dosoretz, D. Pentobarbital anesthesia and the response of tumor and normal tissue in the C3Hf/Sed mouse to radiation. Radiat. Res. 104: 47-65; 1985. Sun, J.; Brown, J. M. Enhancement of the antitumor effect of flavone acetic acid by the bioreductive cytotoxic drug SR 4233 in a murine carcinoma. Cancer Res. 49:5664-5670; 1989. Suzuki, M.; Hori, K.; Abe, I.; Saito, S.; Sato, H. A new approach to cancer chemotherapy: selective enhancement of tumor blood flow with angiotensin II. JNCI 67:663-669; 1981. Suzuki, M.; Hori, K.; Abe, I.; Saito, S.; Sato, H. Selective increase in tumor oxygen tension with angiotensin II. Invasion Metas. 2:33-39; 1982. Tozer, G. M.; Maxwell, R. J.; Griffiths, J. R.; Pham, P. Modification of the 3’P magnetic resonance spectra of a rat tumour using vasodilators and its relationship to hypotension. Brit. J. Cancer. 62:553-560; 1990. Trotter, M. J.; Acker, B. D.; Chaplin, D. J. Histological evidence for the nonperfused vasculature in a murine tumor following hydralazine administration. Int. J. Radiat. Oncol. Biol. Phys. 17:785-789; 1989. Tvete, S.; Gothlin, J.; Lekven, J. Effects of vasopressin, noradrenalin and oxytocin on blood flow distribution in rat kidney with neoplasm. Acta Radiol. Oncol. 20:253-260; 1981. Vaupel, P. Interrelationship between mean arterial blood pressure, blood flow, and vascular resistance in solid tumor tissue of DS-carcinosarcoma. Experientia. 3 1:587-589; 1975. Vaupel, P.; Kallinowski, F.; Okunieff, P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 49:6449-6465; 1989. Vaupel, P.; Menke, H. Effect of various calcium antagonists on blood flow and red blood cell flux in malignant tumors. Prog. Appl. Microcirc. 14:88-103; 1989. Voorhees, W. D., III; Babbs, C. F. Hydralazine-enhanced selective heating of transmissible venereal tumor implants in dogs. Eur. J. Cancer Clin. Oncol. 18:1027-1033; 1982. Weeks, J. R. Experimental narcotic addiction. Sci. Amer. 210:46-52; 1964. Weeks, J. R.; Jones, J. A. Routine direct measurement of arterial pressure in unanesthetized rats. Proc. Sot. Exp. Biol. Med. 104:64&648; 1960. Wood, P. J.; Hirst, D. G. Cinnarizine and flunarizine as ra-
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diation sensitisers in two murine tumours. Brit. J. Cancer 58:742-745; 1988. 67. Zanelli, G. D.; Fowler, J. F. The measurement of blood perfusion in experimental tumors by uptake of 86Rb. Cancer Res. 34:1451-1456; 1974. 68. Zwi, L. J.; Baguley, B. C.; Gavin, J. B.; Wilson, W. R.
Volume 22, Number 1, 1992 Blood flow failure as a major determinant in the antitumor action of flavone acetic acid. JNCI 81:1005-1013; 1989. 69. Zwi, L. J.; Baguley, B. C.; Gavin, J. B.; Wilson, W. R. The use of vascularised spheroids to investigate the action of flavone acetic acid on tumour blood vessels. Brit. J. Cancer 62:23 l-237; 1990.
22. pp. 79-86 Copyrrghr
0364-3016192 $5.00 + .oO 0 1991 Pergamon Press plc
0 Biology Original Contribution
PHARMACOLOGICAL MODIFICATION OF TUMOR BLOOD FLOW: LACK OF CORRELATION BETWEEN ALTERATION OF MEAN ARTERIAL BLOOD PRESSURE AND CHANGES IN TUMOR PERFUSION HELEN B. STONE, PH.D.,
ANDREW I. MINCHINTON,
DOUGLAS MENKE,
PH.D.,
MARILYN LEMMON, B. S.,
B. A. AND J. MARTIN BROWN, D.PHIL.
Department of Radiation Oncology, Division of Radiation Biology, Stanford University Medical Center, Stanford, CA 94305 The correlation between mean arterial blood pressure (MABP) and vascular perfusion in SCC-VIUSt tumors in mice was compared following administration of three vasoactive drugs: flavone acetic acid (200 mgkg), hydralazine (5 mgkg), or nicotinamide (500, 750, and 1000 mgkg). MABP was measured by the direct method in unanesthetized, unrestrained mice bearing a carotid catheter. Vascular perfusion of the tumor was measured using the 86RbCI extraction method. Body temperature was maintained at 36” to 37°C after drug administration when necessary. All three drugs reduced MABP from a control value of 125 + 2 (s.e.) mm Hg in mice without tumors. Flavone acetic acid at this dose had the least effect on blood pressure, with a minimum of 86% of control values at 10 to 20 min, and a return to control values by 1 hr. However, it produced a profound reduction in tumor perfusion that lasted more than 48 hr. Hydralazine and nicotinamide reduced blood pressure to minima between 55% and 69% of control values within 30 min, followed by a gradual return toward control values by about 8 hr. The reduction in tumor perfusion by hydralazine paralleled its effect on blood pressure. However, nicotinamide produced a transitory, although not statistically significant, increase in tumor perfusion at the highest dose given. These data demonstrate that tumor blood flow modification by drugs is not necessarily the result of changes in MABP, and blood pressure changes alone do not inevitably lead to changes in tumor perfusion. Flavone acetic acid, Hydralazine,
Nicotinamide,
Mean arterial blood pressure, Tumor perfusion,
Tumors.
blood flow by a thermal washout technique, and oxygen tension with a polarographic technique. Lymph node metastases, intramuscular, and intrahepatic tumors showed similar responses, whereas blood flow in liver, brain, bone marrow, and subcutaneous tissues remained relatively constant under these conditions. In the studies by both groups, the animals were anesthetized by pentobarbital sodium, which itself can reduce blood pressure and body temperature (12, 32, 40, 44) and can affect the response of tumors to radiotherapy, most likely by its effects on oxygenation of tumors (12, 14, 44, 50, 53). Studies on the effects of vasoactive agents on blood flow or perfusion in tumors have been reviewed by Jain (29) and by Jirtle (30). The purpose of the present study was to determine the correlation between MABP and tumor perfusion following administration of three vasoactive agents, flavone acetic acid, hydralazine, and nicotinamide, using unanesthetized, unrestrained mice whose body temperature was maintained at 36 to 37°C after drug administration.
INTRODUCTION
Many studies have investigated the interrelationship between tumor cells and the anatomy and function of a tumor’s vascular network and the importance of the vascular supply to tumor growth and response to radiotherapy, chemotherapy, hyperthermia (reviewed in (28, 30, 61)). Manipulation of a tumor’s blood supply to enhance the effectiveness of therapy is the ultimate goal of such studies. Vaupel (60) has observed a linear relationship between mean arterial blood pressure (MABP) and blood flow in tissue-isolated sarcomas in rats. Blood pressure was reduced by controlled bleeding and corrected by transfusion, and blood flow was measured by cannulating the single vein draining the tumor. Conversely, Suzuki et al. (55, 56) elevated mean arterial blood pressure in rats to 140 to 160 mm Hg using angiotensin II, and found sharp increases in blood flow and oxygen tension in subcutaneous tumors, to as much as 4 to 12 times control values. They measured Reprint requests to: Helen B. Stone, Ph.D., NIH-NCI, EPN800, 9000 Rockville Pike, Bethesda, MD 20892. Acknowledgements-We gratefully acknowledge the technical assistance of Nixy Kaul and the helpful suggestions of Dr. James
veloping the techniques for carotid catheterization of the mouse and measuring mean arterial blood pressure. Supported by grant #CA 25990 from the National Cancer Institute. Accepted for publication 14 June 1991.
Weeks, Dr. William Ebling, Dr. Hideyashi Harashima, Dr. Michael Horsman, Dr. William Wilson, and Kathy Groom in de79
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AND MATERIALS
Mice and tumor C3H/Km female mice aged 3 to 4 months were obtained from the defined flora colony of the Stanford Radiation Biology Mouse Facility. The tumor line was squamous cell carcinoma SCC-VII/St. Details of the derivation and handling of this cell line have been published previously (20). Suspensions containing 2 X lo5 cells were implanted intradermally on the backs of the mice, and grew to 8 mm diameter in approximately 2 weeks. The effects of flavone acetic acid on blood pressure were examined in mice with and without tumors. Mice used for blood pressure studies with hydralazine and nicotinamide did not have tumors.
Drugs All drugs were prepared fresh daily in sterile saline solution containing no preservatives. Flavone acetic acid* was dissolved to give a concentration of 20 mg/ml, hydralazinei 0.5 mg/ml, and nicotinamide$ 50, 75, or 100 mg/ml. The injected volume of all drugs was 0.01 ml/g body weight.
Blood pressure measurements Mean arterial blood pressure was measured by the direct method in unanesthetized, unrestrained mice bearing a carotid catheter. Mice were anesthetized with pentobarbital sodium (65 mg/kg, intraperitoneally) for catheter implantation, and were given ampicillin (100 mglkg, subcutaneously) at the completion of surgery and daily thereafter. The animals were kept warm until they recovered from the anesthesia. Between experiments, they were caged individually to prevent them from biting each other’s catheters. Fabrication and implantation of carotid catheters have been described previously (10, 18, 46, 64, 65). Briefly, the catheter was made of polyethylene tubing.§ It was filled with heparinized saline, inserted 4 to 5 mm into the carotid artery, and held in place with two sutures. After a 180” bend it passed subcutaneously behind the front leg, and after another bend, exited at the nape of the neck, pointing forward. It was plugged with a short steel wire when not connected to the transducer. A second catheter of PE- 10 tubing was implanted intraperitoneally through the muscles of the flank, and passed subcutaneously to exit at the nape of the neck. It was also plugged with a short piece of steel wire. This catheter allowed administration of drugs without picking the mouse up or puncturing the skin, thus minimizing stress, although blood pressure returned to preinjection levels within two min after release of mice injected intraperitoneally with saline using a hypodermic needle. *National Cancer Institute. tCiba-Geigy, Basel, Switzerland. *Sigma Chemical Co., St. Louis, MO. §PE-10 (ID 0.011 in, OD 0.024 in) and PE-50 (ID 0.023 in,
Volume 22, Number
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Blood pressure measurements were made only after the mice had fully recovered from anesthesia, either in the afternoon of the day of surgery, or the next morning. The body temperature of the mice treated with nicotinamide was maintained after injection of drugs by placing them in a standard polycarbonate mouse cage floating in a 37°C water bath. Rectal temperatures of all mice were measured periodically during the course of each experiment with a thermocouple, and did not decrease below 36°C. A disposable transducer* was placed at the level of the mouse’s heart, and was connected to a clinical monitor-t The monitor was calibrated daily before use, rechecked periodically during experiments and did not drift significantly during use. The connections between the catheter and the transducer were made from 22 gauge disposable hypodermic needles and PE-50 tubing filled with heparinized saline, taking care to exclude air bubbles. Mean arterial blood pressures prior to administration of the drugs were 125 ? 2 (s.e.) mm Hg in mice without tumors, and 113 -+ 3 in mice with tumors. Readings were taken when the mice were sitting quietly. Animals were retested with the same drug and dose on subsequent days as long as the catheter remained open. There was no evidence that the magnitude or duration of the changes in blood pressure was altered in the mice given multiple injections, and data were pooled. Tumor perfusion Tumor perfusion was measured by extraction of “RbCl (49, 67). Briefly, mice bearing 8 mm diameter tumors were injected intraperitoneally with the appropriate vasoactive compound, and were kept warm as described above, with food and water available ad lib. At the appropriate time, 3 $Ji of s6RbCl in 0.1 ml was injected intravenously into the tail vein. Two minutes later, the mice were killed by cervical dislocation. The tumors and tails were removed immediately, and placed in double glass tubes and counted in a gamma scintillation spectrometer.* Counts from tumors were corrected for background, normalized to tumor weight, and expressed as % of control. Data from mice whose tails contained > 10% of the injected radioactivity were not included. Statistical analysis Means and standard errors were calculated for each group and time point. The significance of the difference between blood pressure measurements before and after drug administration was determined with paired-difference t-tests. Comparisons of blood pressure in mice with and without tumors and of tumor perfusion data were made using unpaired two-group t-tests. P values of 5 0.05 were considered significant, and those 5 0.01 were considered highly significant. OD 0.038 in) Intramedic, Clay Adams. *Medi-Trace, Graphic Controls, Buffalo, NY. tMode1 #78901A, Hewlett Packard, Palo Alto, CA. $Model
#5360,
Packard
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pressure and tumor perfusion ??H.
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B. STONE et al
HYDRALAZINE,
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Fig. 1. Effect of 200 mg FAA/kg ip on (a) mean arterial blood pressure, as a percentage of pretreatment control values and on (b) tumor blood perfusion as measured by s6Rb extraction, as a function of time after FAA administration. 0: mice bearing SCCVII/St tumors, and 0: mice without tumors. The shaded areas represent pretreatment control values 2 1 se.; error bars are s.e. In (a), symbols with error bars were from groups with 6 to 20 mice, and in (b), symbols with error bars were from groups with 3 to 4 mice; symbols without error bars were from individual mice.
RESULTS Flavone acetic acid Flavone acetic acid at a dose of 200 mg/kg reduced the mean arterial blood pressure to a minimum of 8 1% to 86% of control in mice with and without tumors, respectively (Fig. la). The reduction was statistically significant from 5 to 30 min, but returned to control values by 1 hr. There
was no statistically significant difference between mice with and without tumors in the percentage of reduction following FAA, although MABP was 7 to 20 mm Hg lower in tumor-bearing mice both before and after treatment @ > 0.05 at 1 and 1.5hr; p < 0.01 at all other times). Tumor perfusion was reduced within 15 min after FAA, and the reduction persisted for more than 48 hr (Fig. lb). Hydralazine blood pressure was reduced to 55 to value between 5 min and 2 hr after a dose of 5 mg hydralazine/kg (Fig. 2a). The blood pressure then returned gradually to normal values within 24 hr. The mean
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Fig. 2. Effect of 5 mg hydralazine/kg ip on (a) mean arterial blood pressure, as a percentage of pretreatment control values, and on (b) tumor blood perfusion, as in Fig. 1. A: mice bearing SCC-VII/St tumors, and A: mice without tumors. Symbols with error bars were from groups with 4 to 8 (a) or 3 to 4 mice (b); symbols without error bars represent individual mice.
Tumor perfusion was reduced for at least 6 hr, although in some individual mice, no reduction was observed (Fig. 2b). Nicotinamide Mean arterial blood pressure was rapidly reduced by a dose of 500 mg nicotinamide/kg, reaching a minimum of 69% of the control value by 30 min. It gradually returned toward control values over the next several hours (Fig. 3a). Tumor perfusion was not significantly reduced during the first 4 hr after administration of this dose of nicotinamide (Fig. 3b). A similar reduction in mean arterial blood pressure and gradual return to control values were seen following nicotinamide doses of 750 (data not shown) and 1000 mg/kg (Fig. 4a). Tumor perfusion was increased over control levels at 30 min and 1 hr after a dose of 1000 mg/kg, but the increases were not statistically significant (Fig. 4b).
arterial
60% of the control
DISCUSSION The present studies show that agents that decrease mean arterial blood pressure do not necessarily cause a decrease
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1. J. Radiation Oncology 0 Biology 0 Physics NICOTINAMIDE, E
Volume 22 , Number 1, 1992 NICOTINAMIDE,
500 mglkg
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in tumor perfusion. Of the three agents studied vone acetic acid produced the smallest reduction
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pressure, and blood pressure had returned to normal by 1 hr, but it produced a profound, prolonged reduction in tumor perfusion. Hydralazine and all three doses of nicotinamide produced similar degrees of reduction in blood pressure, followed by a slow return to nearly normal values by about 5 to 10 hr. However, hydralazine reduced tumor perfusion for several hours, whereas nicotinamide did not significantly reduce tumor perfusion, and probably increased it somewhat following a dose of 1000 mg/kg. The increase in tumor perfusion after nicotinamide in the present study was of the same order of magnitude as that reported by Horsman et al. (21). Flavone acetic acid Reduced perfusion or vascular collapse in tumors 4 to 48 hr following administration of flavone acetic acid has been observed by several investigators, using Evans blue concentrations, single labeling or mismatch of labeling fol-
Fig. 4. Effect of 1000 mg nicotinamide/kg ip on (a) mean arterial blood pressure and on (b) tumor blood perfusion, as in Fig. 1. ?? : mice bearing SCC-VII/St tumors, and 0: mice without tumors. There were 4 to I2 (a) or 3 to 4 mice/group (b).
lowing administration of the fluorescent compounds Hoechst 33342 and DiOC7(3), 86RbC1 extraction, 2H-NMR, or histological methods (3, 15, 16, 19, 51, 54, 68). Murray et al. (41) have reported a decrease in plasma clotting time 15 to 30 min after administration of 300 mg FAA/kg to tumor-bearing mice, but an increase in clotting time, thrombin time, and fibrin degradation products 4 to 6 hr after FAA, which suggested intravascular coagulation. The plasma from mice without tumors showed only the transient decrease in clotting time. The effects of FAA may vary with the site of the tumor: hepatic metastases of colon tumors showed extensive necrosis but less hemorrhage than occurred with subcutaneous tumors (51). Whether tumor sizes were comparable at the time of treatment was not reported. Vascular collapse in normal tissues was not observed histologically by Smith et al. (51). Dose-limiting hypotension occurs in patients following doses of FAA that produce no objective tumor response (38). Loss of clonogenic cells and growth delay has been observed in tumors after FAA administration in vivo (4, 19, 45, 68). Denekamp et al. (13) have shown that clamping the blood supply to murine tumors for 2 hr or longer de-
Blood pressure and tumor perfusion
lays their growth, and for 15 to 24 hr can induce local tumor control. A detailed histological study by Zwi et al. (69) showed that FAA caused necrosis and hemorrhage in vascularized but not in avascular regions of EMT6 tumors growing in the peritoneal cavity of mice. Thus, vascular collapse following FAA is probably a major cause of the cytotoxicity of FAA in viva. However, other mechanisms may contribute as well, such as direct cytotoxicity by FAA or its metabolites, production of irreparable single strand breaks in DNA, decreasing ATP levels, increasing NK cell activity, and interferon production, as pointed out by Chabot et al. (6). The similar reduction in MABP in mice with and without SCC-VII/St tumors indicates that FAA did not have an additional effect on MABP that was mediated through its effects on the tumors. Hydralazine Hydralazine is a vasodilator that relaxes the smooth muscle of the arterioles, lowering peripheral vascular resistance. It is used clinically, in conjunction with other agents, as an antihypertensive agent. At doses that do not produce a marked decrease in blood pressure, it may increase splanchnic, coronary, cerebral, and renal blood flow (48). It increases heart rate and contractility, plasma renin activity, and fluid retention (48). Decreases in tumor blood flow or perfusion have been observed in transplanted rodent tumors at intervals of 15 to 90 min after hydralazine, by the methods of radioactive microspheres, laser Doppler flowmetry, binding of 3H-misonidazole, 86RbC1 extraction, and double labeling with the fluorescent dyes Hoechst 33342 and DiOC7(3) (2, 7, 24, 37, 52, 58, 63). Changes in the magnetic resonance spectrum after hydralazine show increases in anaerobic metabolism of tumors (42, 43, 57). However, at low doses of hydralazine (0.25 kg/g), tumor blood flow was increased in a murine fibrosarcoma (37). Data suggest that in humans, an increase in tumor blood flow is produced by maximum tolerated doses of hydralazine (47). The effects on normal tissue blood flow do not appear to be consistent: increases (2, 7, 63), decreases (37, 52), and no change (37, 52) have all been observed. The magnitude of the effect depends on the normal tissue and dose of hydralazine, and perhaps the method of measurement as well. Hydralazine enhances the toxicity of agents that are preferentially toxic to hypoxic cells, including SR 4233 (5) melphalan (52), the hypoxic cell sensitizers misonidazole (ll), RSU 1069 (8), and hyperthermia (24, 25, 37). Radioprotection of both tumors (1, 11) and normal spleen cells in vivo (43) has been observed following hydralazine administration. Tozer et al. (57) found a reduction in MABP following hydralazine (5 2 mg/kg) and a simultaneous increase ininorganic phosphate, as measured in a rat fibrosarcoma by magnetic resonance spectroscopy. However, the latter peaked at about 30 min, and then decreased while MABP
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showed little change from minimal values from 10 through 150 min. They concluded that the degree of reduction in MABP was not the sole determinant of tumor energy status. Horsman et al. (24) found a correlation between the duration of reduction in MABP and tumor perfusion (86RbC1 extraction method) in murine mammary tumors following 5 mg hydralazine/kg. In the present study, perfusion was reduced in tumors for 4 to 6 hr after hydralazine. This roughly correlated with the curve of the reduction of blood pressure with time after hydralazine, although there was considerable heterogeneity in individual responses, which we cannot explain. Nicotinamide Horsman et al. have reported a reduction in mean arterial blood pressure in unanesthetized mice after 1000 mg nicotinamide/kg (26), as well as an increase in tumor perfusion in SCC-VII and EMT6 tumors, as measured by *‘RbCl extraction and by uptake of Hoechst 33342 (9, 21, 26). They also reported a decrease in binding of 14C misonidazole and an increase in radiation response of SCCVII, EMTG, and Lewis lung tumors, both of which are consistent with increased oxygenation of the tumors (9, 21, 22, 23, 26, 27, 33, 34). Although the increase in tumor perfusion 30 to 60 min after nicotinamide was not statistically significant in our study, it is consistent with the above findings. Relationship between MABP and tumor blood flow: The relationship between blood pressure and blood flow rate in a tissue is described by the equation: n Y
=
P rlz
where Q is the blood flow rate, AP is the pressure difference, q is the blood viscosity, and Z is a term representing the geometrical resistance to flow (28). Because there are many control points in the vascular tree, changes in MABP may not be transmitted to the vessels supplying the vascular bed of a given tissue, or conversely, the distribution of blood to various organs and tissues may change even in the absence of changes in MABP, as in the vascular collapse in tumors following flavone acetic acid. Although many agents are known that reduce tumor blood flow or perfusion (29), hyperthermia and several drugs can increase it: acetyl-Bmethylcholine (17)) angiotensin (3 1), nitroglycerine (39), oxytocin (59), and certain anesthetics (40). The calcium channel blockers verapamil and flunarazine have been shown to increase blood flow or perfusion in rodent tumors at doses that do not alter MABP (35, 36, 62, 66). When MABP was reduced by verapamil, however, Vaupel and Menke (62) found a reduction in tumor blood flow. Menke and Vaupel compared the effects of various anesthetics, neuroleptic, neuroleptanalgesic, and sedative agents on MABP, tumor blood flow, and tumor vascular resistance in rats bearing DS-carcinosarcoma (40).
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All of the agents studied either decreased MABP or had no effect on it. Their data show a better correlation between changes in tumor blood flow and tumor vascular resistance than between either of these parameters and reduction in MABP. Changes in cardiac output and in blood viscosity could contribute to changes in tumor blood flow and perfusion as well. A final consideration is that most studies of tumor blood flow and perfusion give data on the tumor as a whole. The
Volume 22, Number
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response to therapy, however, may be governed by subpopulations of tumor cells that are resistant because of insufficient access to oxygen and nutrients or chemotherapeutic agents. Similarly, agents that modify tumor blood flow will affect vascularized and nonvascularized regions differently, as has been shown for FAA (69). Conversely, it is also possible that a large proportion of the cells in deficient environments are nonclonogenic. Therefore, studies of tumor response to manipulations of blood flow are needed.
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