1973, British Journal of Radiology, 46, 623-630 AUGUST 1973

Effectiveness of nitrofuran derivatives in sensitizinghypoxic mammalian cells to X raysBy J. D. Chapman, B.E., M.Sc, Ph.D., A. P. Reuvers, A.C.I.C., and J. Borsa, B.E., M.Sc, Ph.D.

Medical Biophysics Branch, Whiteshell Nuclear Research Establishment, Atomic Energy of Canada Limited,Pinawa, Manitoba, Canada{Received February ,1973)

ABSTRACTVarious nitrofuran derivatives have been tested with

Chinese hamster cells growing in vitro to determine theirtoxicity and radiosensitizing effectiveness. All nitrofuranstested displayed a greater radiosensitizing potential than/>-nitroacetophenone for hypoxic cells at concentrationsproducing no acute toxicity. The most effective compoundtested to date is nifuraldezone which at 250 ju.m in completemedium gives the same extent of radiosensitization(ER'--'2-85) as oxygen in air-saturated medium. Additionalnitroheterocyclic compounds in current clinical usage havealso been characterized. The chemical and pharmacologicalproperties of the various compounds are discussed in rela-tion to their application in animal tumour studies. Highwater solubility appears to be an important property forradiosensitizers to be used in animal tumour studies. Apositive demonstration of radiosensitization in vivo withnitroheterocyclic drugs will depend upon whether or not asensitizing concentration of the drugs can be establishedin the hypoxic cells of a solid animal tumour for the durationof the radiation treatment.

Cells in solid tumours are inactivated by ionizingradiation according to a multi-component survivalcurve (Powers and Tolmach, 1963; Hill, Bush andYeung, 1971) which is likely to be related to a varia-tion in oxygen concentration within the tumourmass (Tannock, 1972). Certain chemicals are knownto sensitize cells to the lethal action of radiation(Bridges, 1969) and those which selectively sensitizehypoxic cells have been considered as possible ad-juvants in the radiotherapy of some cancers (Mit-chell, 1964; Berry, 1969; Adams, 1972; Emmerson1972; Chapman et al., 1972b). Adams and co-work-ers (1969, 1970, 1971) have successfully used theproperty of electron-affinity in the selection ofcompounds which subsequently have been shown tobe effective radiosensitizers selective in hypoxia.The sensitizer />-nitroacetophenone (PNAP) wasone product of their studies and the characteriza-tion of its radiosensitizing properties in mammaliancells has formed the basis of many studies in thislaboratory (Chapman, Webb, and Borsa, 1971).Studies with analogues of PNAP indicated the im-portance of the NO2 group in cellular radiosensi-tization and led to the selection of numerousnitrobenzene derivatives with good radiosensitizingproperties (Chapman et al., 1972a; Raleigh et ah,1973).

Recently, the highly electron-affinic class ofcompounds, the nitrofurans, has been shown to haveexcellent radiosensitizing effectiveness in hypoxicmammalian cells (Reuvers, Chapman and Borsa,1972; Chapman et al, 1972b). This class of com-pounds is of great interest to radiotherapists sincesome derivatives have been used clinically as anti-bacterial agents for a number of years. We reporthere on a study of various nitrofuran derivatives forthe purpose of selecting those with propertiesamenable to application in vivo. The toxicity andradiosensitizing effectiveness of the nitrofuran ana-logues have been measured in an in vitro mammaliancell system. Other nitroheterocyclic compoundswith current clinical usage have also been tested andthe data are discussed in relation to the prospec-tive application of such drugs in mammalian tumoursystems.

MATERIALS AND METHODS

Chinese hamster cell line V79-379-A was usedthroughout this study and was cultured accordingto procedures previously described (Chapman et al.,1972b). The toxicity of the various compoundstested was estimated from both the cytostatic andcytocidal effect of various concentrations of eachdrug on cells growing asynchronously at 37°C. Theprocedures for both toxicity tests have been pre-viously described (Chapman et al., 1971,1972b).

Irradiations were performed with a WestinghouseX-ray therapy unit operating at 250 kVp and 15mA. Beam filtration, dose-rate, and nitrogen gas-sing procedures were identical with those previouslydescribed (Chapman et ah, 1971). The radiationsensitivity of single cells attached to glass petridishes was determined from clonogenic survival datameasured after six days of post-irradiation incuba-tion at 37°C.

The following nitrofuran derivatives were gen-erously supplied by Norwich Pharmacal Co., Nor-wich, N.Y.: 5-nitro-2-furaldehyde semicarbazone(nitrofurazone), N-(5-nitro-2-furfurylidene)-l-am-inohydantoin (nitrofurantoin), anfr'-5-nitro-2-fural-doxime (nifuroxime), 5-nitro-2-furaldehyde acetyl

623

VOL. 46, No. 548

J. D. Chapman, A. P. Reuvers and J. Borsa

hydrazone (nihydrazone), N-(5-nitro-2-furfuryli-dene)-3-amino-5(4-Morpholinyl methyl)-2-oxazoli-dinone (furaltadone), 5-nitro-2-furaldehyde 2-(hy-droxyethyl) semicarbazone (nidroxyzone), 5-nitro-2-furaldehyde semioxamazone (nifuraldezone), 3-(5-nitrofurfurlideneamino)-2-oxazolidinone (fura-zolidone), 2(Methoxymethyl)-5-nitrofuran (nitro-furfuryl methyl ether), l-(5-nitro-2-furyl)-3-piperi-dino-1-propanone semicarbazone Hydrochloride(NF-131), 1 -(5-nitro-2-furyl)-3-piperidino-1 -pro-panone Hydrochloride (NF-132), 5-nitro-2-fural-dehyde 5-(3-diethylaminopropyl) semioxamazoneHydrochloride (NF-167), Pyridinium-acetic acid(5-nitrofurfurylidene) hydrazide chloride (NF-168),5-nitro-2-furaldehyde 4-(3-diethylaminopropyl)semicarbazone Hydrochloride (NF-185), 5-Morpho-linomethyl-3- (5-nitrofurfurylidine)amino -2-oxa-zolidinone Hydrochloride (NF-269), and 5-nitro-2-furaldehyde 2-(Morpholinoethyl) semicarbazoneHydrochloride (NF-435).

N-(2-hydroxyethyl)-a-(5-nitro-2-furyl) nitrone(nifuratrone) was supplied by Dainippon Pharma-ceutical Co. Ltd., Osaka, Japan; l-(5-nitro-2-thia-zolyl) 2-imidazolidinone (niridazole) by Ciba-Geigy,Basel, Switzerland; and 2-methyl-5-nitroimidazole-1-ethanol (metronidazole) by Poulenc Ltd., Mon-treal, Canada.

The compounds are listed in the table along withtheir chemical structures and water solubilities.Reference to the compounds in the text is by theircommon or trade name or by their numerical desig-nation.

RESULTSToxicity of nitrofuran analogues

Mammalian cells growing in vitro provide a con-venient system for the screening of potential radio-sensitizers. The effectiveness of a drug in enhancingthe lethal action of ionizing radiations can be quan-titatively measured for cells in a variety of physio-logical states, and in vitro cellular toxicity can bedetermined. Such information in conjunction with aknowledge of drug administration techniques canthen be used to select potential radiosensitizers foruse in animal studies.

The compounds tested as radiosensitizers in thisstudy were initially characterized for cytostatic tox-icity. The concentration of each compound in com-plete medium which was effective in reducing netcell proliferation to 50 per cent of that of an un-treated control (over a 24-hour growth period) islisted in the table. Some merits and limitations ofthis in vitro toxicity test have been discussed (Chap-man et al, 1972a). By the criterion of cytostatic

toxicity, the nitrofurans are more toxic than theradiosensitizer PNAP and are comparable in toxicitywith the more electron-affinic nitrobenzene deri-vatives which have good radiosensitizing properties(Raleigh et al, 1973). Structures NF-185 and NF-167, derivatives of nitrofurazone and nifuraldezone,respectively, with increased water solubilities, wereapproximately twice as toxic to cells as the parentstructures. Other water-solubilized derivatives ofnitrofurazone were found to be more toxic (com-pound NF-131) and less toxic (compound NF-435) than the parent structure.

We have found that cytostatic toxicity serves as auseful guide for quickly defining the concentrationrange over which the drug should be tested forradiosensitizing potential. The concentrations ofdrugs which inhibit cell proliferation to 50 per centof that of an untreated control are usually 10-100times lower than the drug concentration which canbe tolerated by hamster cells for the duration of theirradiation procedure (~20 minutes at 21°C) with-out altering their subsequent plating efficiency. Wehave also characterized these drugs according to theircytocidal toxicity for cells growing in vitro. A stand-ard cytocidal toxicity test was used which involvedmeasuring the effect on subsequent plating efficiencyof cell populations exposed to various concentra-tions of each drug in complete medium at 37°C forone hour. Data from such experiments are shown inFig. 1, and the maximum concentration of each drugproducing no apparent reduction in plating effi-ciency has been estimated from the plots. Cytocidaltoxicities have been determined only for the deriva-tives which have relatively high water solubilities,and these are listed in the table. The cytocidaltoxicities of nitrofurazone, nitrofurantoin, and ni-furoxime have been reported previously in detail(Chapman et ah, 1972b). It is interesting to notethat the concentrations of the drugs resulting inequivalent cytocidal toxicity for mammalian cellsare more variable than the concentrations of thesame drugs producing equivalent cytostatic toxicity.Whether or not either measure of in vitro cellulartoxicity can be related to animal toxicity remains tobe seen.

Radiosensitization by nitrofuran analoguesThe radiosensitizing effectiveness of various con-

centrations of each drug was estimated from theenhanced killing produced by the drug when hypoxiccells were irradiated with a standard dose (1,650rads). Enhancement ratios (ERs) were computedfrom single dose survival data by using the multi-target equation S/S0 = l-(l-e-D/D

0)n with a value of

624

AUGUST 1973

Ejfectiveness of nitrofuran derivatives in sensitizing hypoxic mammalian cells to X rays

TABLE ICELLULAR TOXICITY AND HYPOXIA-SELECTIVE RADIOSENSITIZING EFFECTIVENESS OF VARIOUS NITROHETEROCYCLIC COMPOUNDS

Compound

Nitrofurazone

Nitrofurantoin

Nifuroxime

Nihydrazone

Furaltadone

Nidroxyzone

Nifuraldezone

Furazolidone

NitrofurfurylMethyl Ether

NF-131

NF-132

NF-167

NF-168

NF-185

NF-269

Nifuratrone

NF-435

Niridazole

Metronidazole

Molecular structure

NO2 0

R-CH = NNHCONH2

R-CH = NN-N = 0

1 N

R-CH = NOH

R-CH = NNHCOCH3

R-CH = N N - \ = OI 0 y vl_yCH2N 0

C2H4OHR-CH = NNCONH2

R-CH = NNHCOCONH2

R-CH = NN~\ = 0

R-CH2OCH3

R-C = NNHCONH2

CH2CH2N )-HCl

R-COCH2CH2N^~)-HCI

R-CH = NNHCOCONH(CH2)3NEt2 -HC1

R-CH = NNHCOCH2N+ fo-

R-CH = NNHCONH(CH2)3NEt2 -HC1

R-CH = NN-^ = 0L ^ C H 2 N 0HC1

Ot

R-CH = NCH2CH2OH

CH2CH2N^2OHC1

R-CH = NNCONH2

fT— NJ l S II (

NO> S-N

Hn—N

NO2 N CH3

CH2CH2OH

Watersolubility

(mg/1at pH 7-0)

210

190

1,040

90

750

1,090

120

40

11,000

12,100

32,000

13,400

35,600

43,000

66,000

—8,000

10,000

~ 5 0

10,000

Cytostatictoxicity (/tm)

(Radiosensitizingenhancement

ratio)

44 (1 -4)

72(1-2)

44(1-5)

33(1-45)

68(1-4)

98(1-45)

29(1-6)

39(1-4)

220(1-3)

15(1-4)

60(1-3)

11(1-4)

56(1-2)

26(1-45)

30(1-35)

28(1-25)

60(1-45)

120(1-2)

4,000 (1 -6)

Cytocidaltoxicity (/un)

(Radiosensitizingenhancement

ratio)

—

—

—

—.

—

—

—

—

—

160(1-9)

—

40(1-7)

450(1-35)

220(1-9)

400(2-1)

270(1-8)

1,400(2-25)

>10mM (1-7)

625

VOL. 46, No. 548

J. D. Chapman, A. P. Reuvers and J. Borsa

> 120-

UJ

ou.u.UJ

o

p<

100-

8 0 -

60 -

4 0 -

20 -

0.1 1.0

CONCENTRATION OF DRUG [rnM]FIG. 1.

Cytocidal toxicity of various nitrofuran derivatives as determined by subsequent platingefficiency of Chinese hamster cells exposed to various concentrations of each drug in

complete medium at 37°C for one hour.

+ OXYGEN* NF-I3Io NF- 132• NF- 167A NF- 168A NF- 185

3 0 ^ • NF- 269• NF-435

I 10 100CONCENTRATION OF DRUG

10000

FIG. 2.The radiosensitizing enhancement ratios of various con-centrations of nitrofuran derivatives as measured inChinese hamster cells made acutely hypoxic in vitro.

n=6. The enhancement ratio (ER) is the ratio of Do(nitrogen control)/D0 (nitrogen+drug). Whole sur-vival curves constructed with all electron-affiniccompounds tested to date have shown the radio-sensitizing effect to be dose-modifying to a firstorder approximation for asynchronously growingChinese hamster cells (Chapman et al, 1971, 1972b).

The extrapolation number for the radiation survivalcurve of asynchronously growing Chinese hamstercells (V79-379-A) in over 40 determinations has been6±1- Figure 2 shows the radiosensitizing effective-ness (ERs) of various concentrations of the morewater-soluble nitrofuran derivatives tested. Theradiosensitizing effectiveness of oxygen is from thedata of Elkind et al. (1965) and has been plotted inFig. 2 for comparison. Of the various ways one couldcompare the compounds tested, we have chosen tocompare their radiosensitizing effectiveness atconcentrations producing equivalent toxicity. Theenhancement ratios measured in this study at equi-valent levels of cytostatic toxicity (as previouslydefined) are listed in the table in parenthesesfollowing the toxicity values. Likewise, the en-hancement ratios measured at equivalent cytocidaltoxicity are listed in the table in parentheses follow-ing the toxicity values. It should be pointed outthat there was no appreciable cytocidal toxicity inthe radiation survival studies described in this paper.The longest time of exposure of a drug to cells was~ 2 0 minutes at 21 °C. It is obvious from the toxicitydata in the table that some concentrations of thederivatives used in the characterization of the radio-sensitizing effect would result in reduced platingefficiencies if cells were exposed for 60 minutes at37°C.

At equivalent cytostatic toxicity the nitrofurananalogues radiosensitize with ERs near 1 -4. Compari-

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FIG. 3A-D.

Whole survival curves constructed with Chinese hamster cells irradiated under acutely hypoxic conditions and air-saturatedconditions with and without chemical sensitizers in the medium. Panels (A)-(D) shows results with 5 mM metronidazole,

500 pM NF-269, 500 /xM nifuratrone, and 250 fiM NF-167 respectively.

VOL. 46, No. 548

J. D. Chapman, A. P. Reuvers and J. Borsa

son on the basis of cytostatic toxicity shows nifural-dezone and metronidazole to be the most effectivesensitizers of hypoxic mammalian cells, and nitro-furantoin, nitrofurfuryl methyl ether, and NF-168to be somewhat less effective than the others tested.Comparison of the more water-soluble derivatives atequivalent cytocidal toxicity showed decreasingradiosensitizing ability in the order: NF-435 > N F -269>NF-185, NF-131 > nifuratrone > NF-167>NF-168. The radiosensitizing effectiveness of othernitroheterocyclic drugs in current clinical usage wasalso measured and their toxicities and sensitizingabilities are listed in the table. Niridazole, a nitro-thiazole derivative, was tested and found to have lowradiosensitizing ability. Unfortunately, its watersolubility limited its characterization to concentra-tions less than 250 /xM. The compound 2-bromo-5-nitrothiazole, on the other hand, at 500 ^M incomplete medium gave an enhancement ratio of 2-1,suggesting that the nitrothiazole structure could alsobe modified to give excellent radiosensitizing pro-perties. Metronidazole, a nitroimidazole derivative,displayed moderate radiosensitizing effectivenessbut only at relatively high concentrations comparedwith the nitrofuran derivatives. Again, these datasuggest that the nitroimidazole structure could bemodified to enhance its radiosensitizing effective-ness.

The initial screening of potential radiosensitizerswas based on single dose survival data and it is im-portant to characterize more fully the sensitizingability of compounds deemed interesting by thiscriterion. Figures 3A-3D show whole survival curvesdemonstrating the hypoxia-selective radiosensitizingability of 5 raM Metronidazole, 500 /xM NF-269,500 /xM nifuratrone, and 250 ^M NF-167. For eachof these drugs the radiosensitization is adequatelydescribed as a dose-modifying effect, and the ex-trapolation number of 6 is confirmed. Again, it isapparent that 250 /xM NF-167 completely mimics,in hypoxic medium, the radiosensitizing effectivenessof molecular oxygen in air saturated medium. Onthe basis of the radiosensitizing data here presented,NF-435 and NF-269 appear to have potential assensitizers in animal systems, because of their goodradiosensitizing ability and low cytocidal toxicity.

DISCUSSIONThis study with analogues of nitrofuran and

other nitroheterocycles has resulted in the selection ofsome extremely effective radiosensitizers of hypox-ic mammalian cells in vitro and has demonstrated awide variation in cellular toxicity for compoundstested at concentrations producing equivalent sensiti-

zation. Nifuraldezone, and a water-solubilized deri-vative of nifuraldezone, NF-167, were found to com-pletely mimic the radiosensitizing effect of molecu-lar oxygen in hypoxic mammalian cells in vitroat concentrations in tissue culture medium whichshowed no measurable acute toxicity (20 minutesexposure at 21°C). Also, NF-185, NF-269, NF-435,and nifuratrone were shown to sensitize hypoxicmammalian cells with ERs in excess of 2-0 at con-centrations displaying no acute toxicity. The maxi-mum radiosensitizing effectiveness in mammaliancells of most of the nitrofuran derivatives tested isequal to or greater than the effectiveness of NDPP,a water-solubilized derivative of PNAP (Adamset al., 1972). The only other sensitizers reported tohave effectiveness in mammalian cells with ERsgreater than 2-0 are methyl glyoxal (Ashwood-Smith et al., 1967) and some highly electron-affinicnitrobenzene derivatives (Raleigh et al., 1973).The remarkable radiosensitizing ability of nifuralde-zone is not completely understood, yet it is inter-esting to point out that within this one structurethere are two electron-affinic sites; the nitrohetero-cyclic group and the glyoxal-like group on the sidechain, both of which are known to enhance theradiosensitizing properties of molecules.

The demonstration that niridazole and metroni-dazole have intermediate radiosensitizing propertiessuggests that both the nitrothiazole and nitroimida-zole heterocycles could be modified to optimizeproperties desirable for application in the radio-therapy of tumours. Metronidazole, in fact, has thelongest half-life in the blood of mice (~80 min) ofany of the potential radiosensitizers measured todate. Unfortunately, at 500/xM in complete medium,it radiosensitizes hypoxic mammalian cells in vitrowith an enhancement ratio of only 1-3.

As regards the potential application of such sen-sitizers in the radiotherapy of tumours, the re-cent demonstration of chemical radiosensitizationwith NDPP of cells made acutely hypoxic in vivois encouraging (Denekamp and Michael, 1972).Furthermore, it has been shown that nifuroxime cancompletely mimic oxygen in eliminating the resis-tant component of the survival curve (believed toresult from hypoxia) for multicellular spheroids(Sutherland and Durand, 1972).

It has been assumed that the drug concentrationin the vicinity of hypoxic tumour cells in vivo re-quired to effect radiosensitization should be similar tothe drug concentration used with single cells in vitroto effect equivalent radiosensitization. This meansthat sensitizer concentrations of up to 100 /xM arerequired in the tumour if ERs in excess of 1-5 are

628

AUGUST 1973

Effectiveness of nitrofuran derivatives in sensitizing hypoxic mammalian cells to X rays

desired. In the absence of means for selectively con-centrating such drugs in the tumour, the requiredsensitizer concentration (100 /u,M) would have to beestablished over the whole volume of the animal. Forthe compounds characterized in this study (mole-cular weights of 150-375), drug doses of 15-38 mg/kg would be required to achieve this concentrationover the whole animal. This is a relatively largedrug dose, and water solubility becomes extremelyimportant in relation to the available means of drugadministration. Such a drug dose would have to beadministered prior to each radiation treatment (in afractionated therapy regime) with the radiationgiven at some optimum time after administration ofthe drug, since it is known that the nitrofurans aremetabolized and cleared by animals quite quickly(Paul and Paul, 1964). This radiosensitizing drugdose (100 fiM over the whole animal) is at leastten times lower than the doses given per oz. to miceto produce acute toxicity (LD50); and repeateddoses of furaltadone at 80 mg/kg/day per oz. to mon-keys over a three-month period did not result in theneurotoxic symptoms typical of some nitrofurans(Paul and Paul, 1964). Of course, these data do notsuggest that the drugs are distributed uniformlyover the whole animal after oral dosage. Preliminarydata from animal studies in this laboratory withfour different nitrofuran derivatives indicate thatsingle doses of 100-150 mg/kg given i.v. or i.p. tomice are not acutely toxic. These studies also indi-cate that concentrations in excess of 200 ^M ofvarious nitrofuran derivatives can be maintained inthe blood of mice for at least 30 minutes withoutacute toxicity to the animals. The distribution andexcretion of many of the nitrofurans have been meas-ured in various laboratory animals and these datashould prove useful in selecting derivatives for ap-plication in mammalian tumour systems (Paul et al.,1960; Buzard and Conklin, 1961). Studies are con-tinuing to determine the drug concentration in asolid mouse tumour at various times after admini-stration of various radiosensitizing drugs by differentroutes.

ACKNOWLEDGMENTSWe would like to thank our colleagues of the Medical

Biophysics Branch for their interest and helpful discussionsduring the course of this work. Special thanks go to Mr.Frank F. Ebetino, Director of the Chemistry Division ofThe Norwich Pharmacal Co., for his assistance in theselection of many of the derivatives tested in this study.

REFERENCESADAMS, G. E., 1970. Molecular mechanisms of cellular

radiosensitization and protection. In Radiation Protec-tion and Sensitization, Eds. H. L. Moroson and M.Quintiliani, pp. 3-14 (Taylor & Francis Ltd., London).

1972. Radiation chemical mechanisms in radiation bio-logy. In Advances in Radiation Chemistry, Eds. M. Burton

and J. L. Magee, Vol. 3, pp. 125-208. (Academic Press,New York).

ADAMS, G. E., ASQUITH, J. C , DEWEY, D. L., FOSTER, J. L.,and WILLSON, R. L., 1971. Electron affinic sensitizationII para-nitroacetophenone: a radiosensitizer for anoxicbacterial and mammalian cells. International Journal ofRadiation Biology, 19, 575-585.

ADAMS, G. E., ASQUITH, J. C , WATTS, M. E., and SMITHEN,C. E., 1972. Radiosensitization of hypoxic cells in vitro:a water-soluble derivative of para-nitroacetophenone.Nature, 239, 23-24.

ADAMS, G. E., and Cooke, M. S., 1969. Electron affinicsensitization I A structural basis for chemical radio-sensitizers in bacteria. International Journal of RadiationBiology, 15, 457-471.

ASHWOOD-SMITH, M. J., ROBINSON, D. M., BARNES, J. H.,and BRIDGES, B. A., 1967. Radiosensitization of bac-terial and mammalian cells by substituted glyoxals.Nature, 216, 137-139.

BERRY, R. J., 1969. Radiotherapy plus chemotherapy—Have we gained anything by combining them in thetreatment of human cancer? In Frontiers of RadiationTherapy and Oncology, Vol. 4, pp. 1-16 (Karger: Basel;New York).

BRIDGES, B. A., 1969. Sensitization of organisms to radia-tion by sulf hydryl-binding agents. In Advances in Radia-tion Biology, Vol. 3, pp. 123-176 (Academic Press, Lon-don and New York).

BUZARD, J. A., and CONKLIN, J. D., 1961. The distributionof furaltadone in the rat. Antibiotics and Chemotherapy,11, 89-96.

CHAPMAN, J. D., RALEIGH, J. A., BORSA, J., WEBB, R. G.,and WHITEHOUSE, R., 1972a. Radiosensitization ofmammalian cells by ̂ -nitroacetophenone II Effectivenessof analogues. International Journal of Radiation Biology,21, 475-482.

CHAPMAN, J. D., REUVERS, A. P., BORSA, J., PETKAU, A., andMCCALLA, D. R., 1972b. Nitrofurans as radiosensitizersof hypoxic mammalian cells. Cancer Research, 32, 2616-2624.

CHAPMAN, J. D., WEBB, R. G., and BORSA, J., 1971. Radio-sensitization of mammalian cells by ^-nitroaceto-phenone I Charterterization in asynchronous and syn-chronous populations. International Journal of RadiationBiology, 19, 561-573.

DENEKAMP, J., and MICHAEL, B. D., 1972. Preferentialsensitization of hypoxic cells to radiation in vivo. Nature,239, 21-23.

ELKIND, M. M., SWAIN, R. W., ALESCIO, T., SUTTON, H.,and MOSES, W. B., 1965. Oxygen, nitrogen, recovery andradiation therapy. In Cellular Radiation Biology, pp. 442-466 (Williams & Wilkins; Baltimore).

EMMERSON, P. T., 1972. X-ray damage in DNA and loss ofbiological function: Effect of sensitizing agents. In Ad-vances in Radiation Chemistry, Eds. M. Burton and J. L.Magee, Vol. 3,209-270.

HILL, R. P., BUSH, R. S., and YEUNG, P., 1971. The effect ofanaemia on the fraction of hypoxic cells in an experi-mental tumour. British Journal of Radiology, 44, 299-304.

MITCHELL, J. S., 1965. A review of clinical and laboratorystudies of radiosensitizers in radiotherapy. In Progress inBiochemistry and Pharmacology, Vol. 1, 335-351 (Karger,Basel/New York).

PAUL, H. E., and PAUL, M. F., 1964. The nitrofurans—Chemotherapeutic properties. In Experimental Chemo-therapy, Eds. R. J. Schnitzer and F. Hawking, Vol. 2,part 1, 307-370 (Academic Press, New York).

PAUL, M. F., PAUL, H. E., BENDER, R. C , KOPKO, F.,HARRINGTON, C. M., ELLS, V. R., and BUZARD, J. A., 1960.Studies on the distribution and excretion of certain nitro-furans. Antibiotics and Chemotherapy, 10, 287-302.

POWERS, W. E., and TOLMACH, L. J., 1963. A multicom-ponent X-ray survival curve for mouse lymphosarcomacells irradiated in vivo. Nature, 197, 710-711.

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RALEIGH, J. A., CHAPMAN, J. D., BORSA, J., KREMERS, W.,and REUVERS, A. P., 1973. Radiosensitization of mamma-lian cells by ^-nitroacetophenone III Effectiveness ofnitrobenzene analogues. International Journal of RadiationBiology (in press).

REUVERS, A. P., CHAPMAN, J. D., and BORSA, J., 1972.Potential use of Nitrofurans in radiotherapy. Nature,237,402-403.

SUTHERLAND, R. M., and DURAND, R. E., 1972. Radiosensi-tization by nifuroxime of the hypoxic cells in an in vitrotumour model. International Journal of Radiation Biology,22,613-618.

TANNOCK, I. F., 1972. Oxygen diffusion and the distributionof cellular radiosensitivity in tumours. British Journal ofRadiology, 45, 515-524.

Book reviewThe Upper Cervical Spine. Regional Anatomy, Pathology andTraumatology. A Systematic Radiologic Atlas and Textbook.By D. Von Torklus and W. Gehle, pp. 101+244 illus.,1972 (Butterworth, London), £7 00.

This short monograph, written by two orthopaedic sur-geons, deals with the development, regional anatomy andphysiological variations and pathology of the upper cervicalspine. The subject is covered in a systematic fashion in aconcise and well-written text, which is most adequatelyillustrated with diagrams, anatomical photograms and a

large number of radiographs chosen from the authors' ownmaterial,which clearly portray the lesions being described.There is only incidental consideration of the contents of thespinal canal and special neuro-radiological findings are notpart of the authors' brief.

The book brings together much detailed information ofinterest to radiologists on this complex region. It is supple-mented by extensive references and a good index.

B. KENDALL.

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