Chem.-Biol. Interactions, 16 (1977) 223--233 223 © Elsevier/North-Holland Scientific Publishers, Ltd.

C Y T O T O X I C I T Y A N D D N A D A M A G E TO M A M M A L I A N C E L L S B Y N I T R O F U R A N S *

P.L. OLIVE ** and D.R. McCALLA

Department of Biochemistry, McMaster University, Hamilton, Ontario (Canada)

(Received May 19th, 1976) (Revision received November 5th, 1976) (Accepted November l l t h , 1976)

SUMMARY

Nitrofurazone, ni trofurantoin, furaz olidone, furaltadone and N- [ 4-(5-nitro- 2-furyl)-2-thiazolyt]formamide (FANFT) were toxic to cultured mouse L cells. The extent of toxicity and the rate of reduction of nitrofurazone increased markedly as the oxygen content of the incubation medium was lowered. The toxic effect of nitrofurans was decreased by addition of serum and was much greater in phosphate-buffered saline containing glucose (PSG) than in medium.

Damage to L cell DNA by nitrofurans increased as the oxygen concentra- tion decreased from 21% to 0%. The concentration of nitrofurazone and duration of exposure also determined the number of DNA single-strand breaks. It is suggested that toxicity and DNA damage may result from the actions of toxic intermediates in the metabolic reduction of nitrofurans.

INTRODUCTION

Humans are exposed to nitrofuran derivatives used as antibacterial agents, as additives to livestock feed and, until recently, as additives to human food. Most nitrofurans tested are mutagenic and carcinogenic [1--4]. The nitro

* This work was supported by a grant from the National Cancer Insti tute of Canada. ** Present address: Division of Clinical Oncology, Unversity of Wisconsin Center for Health Sciences, 420 N. Charter, Madison, Wisconsin 53706 (U.S.A.). Abbreviations: AF-2, 2-(2-furyl)-2-(5-nitro-2-furyl)acrylic acid; DMSO, dimethylsulph- oxide; FANFT, N-[ 4-(5-nitro-2-furyl )-2-thiazolyl ] formamide; FCS, fetal calf serum; MEM, minimal essential medium, NFTA, N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide; PBS, phosphate-buffered saline; PSG, phosphate-buffered saline with 0 .1% glucose, SDS, sodi- um dodecyl sulphate.

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group appears to be essential fo r these ac~vities, since the furan analogues that have been tested are neither mutagenic nor carcinogenic [3,4]. Work with E. coli has demonstrated that enzymatic reduction of the nitro group is required before mutat ions [1] or DNA single-strand breaks are induced [5]. The identity of the ultimate mutagenic agent has not been established, but it appears to be a highly reactive compound intermediate in oxidation level between the nitro compound and the amine [5,6].

Similar reductive activation can take place in animal cells and tissues under some conditions. At least five mammalian enzymes can reduce nitro- furans in vitro (see ref. 7 for references); all are inhibited by oxygen although some may have residual activity in air [8]. Cultured cells (mouse L-929, Ehrlich ascites, hamster BHK-21 and human KB) have been shown to metabolize nitrofurans at slow but measurable rates in nitrogen [9]. No activity was detected in air. We have also reported that DNA of cultured cells is damaged when cells are incubated with nitrofurazone in nitrogen but no t in air [9]. These observations suggest that compounds derived from nitro- furazone may be responsible for the breakage of mammalian DNA.

Work from several laboratories has documented the toxici ty and mutagen- icity of several nitrofurans to cultured cell lines under aerobic conditions [10--13]. However, with the exception of studies reported by Mohindra and Rauth [14], the effects of ambient oxygen concentrat ion have not been systematically studied.

In this paper, we report that nitrofurantoin and nitrofurazone are more toxic to cultured cells under hypoxic conditions than in air. We also present data confirming our earlier report [9] on nitrofuran reduction and DNA breakage and show that the rate of reduction and the number of DNA strand breaks increase as the oxygen concentration is lowered.

MATERIALS AND METHODS

Nitrofurans. Nitrofurazone, nitrofurantoin, furaltadone, and furazolidone were obtained from Norwich Pharmacal Co., Norwich, N.Y. FANFT was supplied by Abbo t t Laboratories, Chicago, Ill. Stock solutions of nitrofurans in DMSO (10 mg/ml) were prepared before use and were diluted in medium or buffer to the appropriate nitrofuran concentration. The final concentra- tions of DMSO was less than 1%, and no effects of this concentrat ion of DMSO were detected. Nitrofurazone reduction in intact L cells was measured spectrophotometrical ly by observing a decrease in absorbance at 375 nm [9].

Ceils. Mouse L ceils were purchased from GIBCO, Grand Island, N.Y. Cells were maintained in suspension culture in Joklik-modified minimal essential medium with 10% FCS from ABS, Buffalo, N.Y. For isotopic labelling, cells in exponential growth were transferred to plastic tissue culture dishes in medium containing 10% FCS and 0.4 #Ci/ml [3H]thymidine (specific activ- ity 18 Ci/mmole) from New England Nuclear. After 2 0 h , radioactive medium was replaced with fresh medium for 1 h. Although the amount of

225

[aH]thymidine incorporated in these cells affected survival, preliminary experiments indicated that highly labelled cells (more than 2 cpm/cell) were no more sensitive to nitrofurans than cells of low specific radioactivity (less than 0.1 cpm/cell).

Measurement o f nitrofuran toxicity. Approx. 2 . 1 0 s exponentially growing cells/ml were suspended in 20 ml of medium or PBS (2.68 mM KC1, 1.47 mM KH2PO4, 0.137 M NaC1 and 6.46 M Na2HPO4 • 2H20) with nitro- furan in glass-stirring culture vessels (Bellco) at 37°C. In some experiments, 0.1% glucose was added to the buffer (PSG). At specified times, a sample of cells was removed, washed, resuspended at a suitable density, and approx. 600 surviving cells were plated in a 100-mm plastic tissue culture dish. All cell counts were performed using an electronic cell counter (Coulter Elec- tronics, Hialea, Fla.). Normally, duplicates of 2 dilutions were plated in MEM with 15% FCS and antibiotics. After 10 days, colonies were stained with methylene blue and counted. The number of colonies divided by the cells plated gave the plating efficiency.

When toxici ty was measured under anaerobic conditions, medium (with or wi thout bicarbonate) or buffer-containing nitrofuran was equilibrated with humidified oxygen-free nitrogen (Canada Liquid Air, Hamilton, Ontario) with or wi thout 3% CO2 for 1 h before addition of cells. The pH of the medium was maintained between 7.0 and 7.4 throughout the experiment. Gas flow was adjusted through 6 vessels in parallel at a rabe of 1.5 1/min. After cells were added (about l 0 s cells/ml), the gas flow continued over the surface of the stirred suspension during the experiment. Samples equilibrated with nitrogen for 1 h were considered to contain 0% oxygen. Gas mixtures containing 2% or 5% oxygen in nitrogen (Certified Standard) were obtained from Matheson of Canada, Toronto, Canada.

Alkaline sucrose gradients. Approx. 104 intact cells in 0.02 ml PBS were lysed directly on top of an alkaline sucrose gradient for DNA analysis, using a method modified from that of McGrath and Williams [15]. Lysing solution (0.2 ml of 0.5 M NaOH, 0.4 M NaCI and 0.01 M disodium EDTA, pH 13) was layered onto a 4.6-ml gradient (5 to 20% sucrose, formed by gravity) con- raining 0.9 M NaOH, 0.1 M NaC1 and 0.003 M disodium EDTA (pH 12.8). Cells were lysed for 4 h at room temperature in the dark, then centrifuged at 18 000 rpm for 4 h at 20°C using a Beckman SW 50.1 rotor in an L2-65B ultracentrifuge. T w e n t y fractions were collected from the top using an Isco model 260 density gradient fractionator. Fractions were dissolved in xylene- Triton X-114 fluor [16] and counted in a Nuclear Chicago Liquid Scintilla- tion Counter.

Weight average molecular weights of DNA were calculated using the equa- tions given by Palcic and Skarsgard [17]. AD-2 phage (35.2S) was used as a marker for molecular weight determinations. The molecular weight repre- sented by the DNA in the peak fraction was calculated, as well as the weight average molecular weight. The peak molecular weight is independent of the shape of the rest of the profile, so that molecular weights of DNA samples near the top of the gradient are not influenced by molecules having higher

226

molecular weight. The distribution of control DNA over the gradient was tested and found to be random, so the number of single-strand breaks was proport ional to weight average as well as number average molecular weight [17].

Hydroxylapatite chromatography. The percent of DNA in double stranded form was determined according to the method of AhnstrSm and Edvardsson [18]. Approx. 104 cells in 0.05 ml were lysed in alkaline solution (0.02 M NaOH, 0.98 M NaC1) for 45 min at 0°C in the dark. Neutralization with 1.0 ml of NaH2PO4 was followed by 15 sec of sonication using a Bronwill sonicator with a microtip. Then 0.05 ml of 10% SDS was added and each sample was poured onto a hydroxylapat i te column containing 150 mg Bio-Rad DNA grade hydroxylapat i te at 60°C. After washing with 3 ml 0.012 M sodium phosphate buffer, single-stranded DNA was eluted with 3 ml of 0.12 M sodium phosphate buffer, pH 6.8, containing 0.4% SDS. Double- stranded DNA was eluted with 3 ml of 0.4 M buffer. Radioactivity in each sample was determined by dissolving 0.2 ml in xylene-Triton X-114 fluor and counting in a liquid scintillation spectrometer.

R E S U L T S

Nitrofuran reduction and toxicity Nitrofurazone reduction by intact L cells was dependent on the oxygen

concentrat ion during incubation (Fig. 1). Maximum reduction occurred under anaerobic conditions with a rate of approx. 2.6 pM/h/lO s cells. Partial reduction occurred when solutions were gassed with 2% and 5% oxygen in

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Fig. 1. R e d u c t i o n o f n i t r o f u r a z o n e by L cells as a f u n c t i o n o f oxygen tens ion . Approx . 0.8 • 108 L cells were s u s p e n d e d in 5 ml PSG plus 250 /~M n i t r o f u r a z o n e u n d e r 0, 2, 5 or 21% oxygen in n i t r ogen a t 37°C. A t specif ied t imes, a 0 .5-ml sample was r emoved , pre- c ip i t a t ed in 3 ml co ld e t h a n o l and cen t r i fuged . The a b s o r b a n c e (A) of the clear super- n a t a n t was read a t 375 n m (A 0.1 = 1.05 p g / m l n i t r o f u r a z o n e reduced) .

227

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TIME (HI Fig. 2. Killing of L cells by nitrofurans in MEM (wi thout bicarbonate) under anaerobic condit ions . Cells were incubated with 500 p_M nitrofurazone, 334 paM furaltadone, 43 pM FANFT and 444 #M furazol idone. There was no decrease in survival w h e n cells were incubated with the same concentrations o f ni trofurazone in air.

Fig. 3. Ef fect of oxygen concentrat ion on killing o f L cells by nitrofurazone. PSG con- taining 250 pM nitrofurazone was equil ibrated wi th 0, 2, 5 or 21% oxygen in nitrogen for 1 h before addit ion o f L cells. Control (untreated) cells were equil ibrated wi th air (C,21) or nitrogen (C,0).

nitrogen, resulting in reduction rates of 1.4 and 0.4 #M/h/108 cells, respec- tively. No measurable reduction occurred in air during the first 20 min of incubation.

In medium saturated with nitrogen, extensive killing of L cells by five nitrofurans was observed (Fig. 2). When the medium was saturated with air, or when incubation was carried out at 0°C under anaerobic conditions, there was no detectable cell killing by 750 pM nitrofurazone or 430/~M nitro- furantoin (data not shown).

L cells incubated in glucose-containing buffer remained viable in air, although slight toxicity was noted in nitrogen (Fig. 3). Nitrofurans added to buffer plus glucose were toxic to L cells even in air. However, the extent of killing was dramatically increased as the oxygen content of the gas phase decreased (Fig. 3). Thus, after 1.5 h, 100% of untreated cells incubated in air or nitrogen survived, while only 30% of cells incubated with nitrofurazone in air survived. This value decreased to 10%, 0.8% and 0.003% when the gas phase consisted of 5%, 2% and 0% oxygen in nitrogen, respectively.

Serum reduced the toxicity of nitrofurantoin (Fig. 4) and nitrofurazone (Fig. 2) both in medium and in buffer. Conversely, a large increase in toxic-

228

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TIME (H) Fig. 4. Effect of medium of incubation on the toxicity of nitrofurantoin. L cells were incubated with 430 ~ nitrofurantoin under anaerobic conditions in the solutions indi- cated. Cells incubated in MEM were equilibrated with nitrogen containing approx. 3% CO2 which maintained the pH at 7.4. Numbers in brackets indicate the plating efficiency of the corresponding drug-free controls after 6 h. Initial plating efficiency was 0.67-+ 0.04.

i ty of nitrofurantoin was observed in PSG relative to that found in PBS (Fig. 4). Nitrofurantoin was considerably less toxic in medium than in buffer containing an equal concentrat ion of glucose.

Single-strand breaks in DNA In contrast to toxici ty, 500 pM nitrofurazone produced nearly equal

numbers of single,strand breaks when L cells were incubated for I h in MEM, PBS or PSG under nitrogen. The extent of breakage of L cell DNA by nitro- furazone and nitzofurantoin decreased progressively as the oxygen concen- tzafion of the incubation medium was increased (Figs. 5, 6). No breaks were detec ted in air or at 0°C using alkaline sucrose gradient sedimentation. However, measurable damage did occur in air when strand breaks were assayed using hydroxylapat i te chromatography (Fig. 6), perhaps reflecting the greater sensitivity of this procedure [18]. The amount of DNA damage increased with the time of incubation and with the concentrat ion of drug used (Fig. 7).

229

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FRACTION NUMBER FROM MENISCUS

Fig. 5. Effect of oxygen concentration on the formation of single-strand breaks in L cell DNA by nitrofurazone and nitrofurantoin. [3H]Thymidine-labelled L cells were incu- bated with 500/~M nitrofurazone or 430 ~M nitrofurantoin in PSG equilibrated with 0, 2, 5 or 21% oxygen in nitrogen. After 2 h, 104 cells were lysed on top of alkaline sucrose gradients and spun at 18 000 rpm for 4 h. The peak position of DNA from cells treated without drug was the same as for cells treated with drug in air.

DISCUSSION

Our results indicate that nitrofurazone, nitrofurantoin, furazolidone, furaltadone, and FANFT are toxic to cultured L cells even in air. Similarly, Kuroda [10] and Umeda et al. [11] have shown that another nitrofuran derivative, AF-2 (furylfuramide), is both toxic and mutagenic to mammalian cells in air. At present, it is no t known whether these effects are caused by the nitrofurans themselves, by activated derivatives formed by partial reduc- tion (or some other process), or by the production of superoxide radical

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Fig. 6. Hydroxylapatite chromatography of DNA from L cells treated with nitrofurazone. Cells in exponential growth were incubated for 90 min in PSG. The buffer was equi- librated with 0, 2, 5 or 21% oxygen in nitrogen before addition of cells. The dotted line indicates L cells in plateau phase growth incubated with nitrofurazone in buffer equilib- rated with 2% oxygen in nitrogen.

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(%) (pM) (H)

Fig. 7. Summary of DNA single-strand breakage data showing the effect of oxygen con- centration, nitrofurazone concentration and incubation time. See text for further details. o o, peak molecular weight; X X, weight average molecular weight.

231

anion via the auto-oxidation of partially reduced nitrofumns [19,20]. Our work also shows that hypoxic conditions enhance the toxicity of the

nitrofurans tested, which is in agreement with results reported by Mohindra and Rauth [10]. It should be noted, however, that these researchers used different experimental conditions and found little increase in toxicity when CHO cells were exposed to nitrofurazone in complete medium equilibrated with 1% oxygen in nitrogen, but considerable increase with 0.1% oxygen. We found a dramatic increase in toxicity with 2% oxygen in nitrogen when L cells were incubated in buffer with glucose. It appears that some components of the medium (perhaps thiols and amino acids) exert a protective effect. Serum also conferred a small additional protective effect which may have been due to absorption of nitrofurans onto serum albumin [21], thus lowering the effective concentration. The enhanced toxicity of nitro- furantoin in the presence of glucose is probably due to the greater capacity of cells Which are metabolizing glucose to reduce nitrofurans [22]. Small changes in pH might also have an important effect, as preliminary experi- ments suggest that the toxicity of nitrofurantoin and furazolidone increases as the pH decreases.

It seems likely that the enhancement of toxicity and DNA breakage of mammalian cells under hypoxic conditions reflect an increase in the rate of enzymatic reduction of nitrofurans under these conditions. It is of interest that in the presence of 2% oxygen, both the rate of reduction and toxicity of nitrofurazone are inhibited by about 50%, and only half as many DNA breaks are observed compared to use of pure nitrogen. It has been estab- lished that the activated compounds formed during the reduction of nitro- furans by enzymes in E. coli are toxic, bind to protein, cause breaks in DNA, and induce mutations (see 5 for references). Cell-free extracts of E. coli also reduce nitrofurans to compounds which bind to protein and break DNA. While there has been no systematic study of the "activation" of nitrofurans by mammalian cells, there is some evidence that cytotoxic reduction prod- ucts are formed. Reduction of nitrofurazone by rat liver xanthine oxidase yields a compound which binds to protein [5]. Likewise, NFTA binds to protein after activation by either xanthine oxidase or by cytochrome c reductase [23].

It may also be relevant that such tissues as liver and kidney, which have high levels of nitrofuran-reducing enzymes, show marked changes when animals are fed large amounts of nitrofurazone, while tissues having lower reductase levels do not [24]. Such tissues also bind [14C]NFTA more readily than tissues lacking high levels of nitrofuran reductase [23], and they also lose radioactivity associated with DNA more rapidly as a result of nitro- furazone feeding [25]. The rate of nitrofuran reduction in tissue will obviously depend on several factors, including the enzyme complement of the tissue and its oxygen tension.

Nitrofurans and other nitroheterocyclic compounds are currently under investigation as radiosensitizers which may inprove the efficacy of the radio- therapy of solid tumors containing hypoxic cells [12,13,26]. In addition to

232

acting as radiosensitizers, our results suggest that compounds of this type may be reductively activated to a much greater extent in hypoxic areas of tumors than in the better oxygenated normal tissues, thus contributing a "chemotherapeutic" component to preferentially kill hypoxic cells.

ACKNOWLEDGEMENT

The authors are indebted to Dr. Colin Arlett for reading the manuscript and making helpful suggestions.

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14 J.K. Mohindra and A.M. Rauth, Increased cell killing by metronidazole and nitro- furazone of hypoxic compared to aerobic mammalian cells, Cancer Res., 36 (1976) 930.

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