Reductive metabolism and DNA binding of misonidazole

TOXICOLOGY

AND

APPLIED

Reductive

PHARMACOLOGY

Metabolism

101,47-54

(1989)

and DNA Binding of Misonidazole’ ZORADJURIC

Department of Obstetrics and Gynecology, Wayne State University, 275 E. Hancock, Detroit, Michigan 48201

Received January 25,1989: accepted June 19.1989 Reductive Metabolism and DNA Binding of Misonidazole. DJURIC, Z. (1989). Toxicol. Appl. Pharmacol. 101, 47-54. Misonidazole (I-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol) is an experimental anticancer drug. Reductive metabolism is thought to be important for the cytotoxicity of misonidazole. In this study, the DNA binding of misonidazole was examined after chemical and enzymatic reduction. Under anaerobic conditions, both rat liver microsomes and cytosol catalyzed the reductive metabolism and DNA binding of misonidazole. The misonidazole utilized in these studies was radiolabeled on the side chain. The adduct formed was too unstable for structural analysis. Little or no metabolism of misonidazole was detected in aerobic incubations. Likewise, very little DNA binding occurred in the presence of oxygen. Xanthine oxidase, a model nitroreductase, also was capable of catalyzing the DNA binding of misonidazole. However, unlike the xanthine oxidase catalyzed DNA binding of carcinogenic nitropolycyclic aromatic hydrocarbons, the DNA binding of misonidazole was not increased at slightly acidic pH. The putative reactive intermediate, the N-hydroxylamine, was synthesized by zinc reduction of misonidazole. The DNA binding ofthe N-hydroxylamine derivative increased with increasing pH. The observed pH dependence of the reactions with DNA is similar to other heterocyclic N-hydroxylamines, but is in contrast to the reactivity of a number of aromatic Nhydroxylamines. 0 1989 academic Press, Inc.

Misonidazole (Fig. 1) is an experimental anticancer drug that has been shown to be selectively cytotoxic toward hypoxic tumor cells. The mechanism of cytotoxicity by misonidazole and other 2-nitroimidazole drugs is believed to be due to more efficient nitroreduction under hypoxic conditions (reviewed in Adams and Stratford, 1986). The N-hydroxylamine derivative of misonidazole is formed by four electron reduction. This derivative is unstable and can rearrange followed by fragmentation of the nitroimidazole ring to form numerous products, including glyoxal, which has been shown to bind to DNA (Varghese ’ This study was supported in part by a developmental grant from the Meyer L. Prentis Comprehensive Cancer Center of Metropolitan Detroit. A portion of this work was presented at the 28th meeting ofthe American Association for Cancer Research, 1987.

and Whitmore, 1983). However, this fragmentation pathway does not account for the DNA binding of misonidazole in numerous systems, including in tumor cells, where it appears that the side chain has not cleaved from the molecule (reviewed in Whitmore and Varghese, 1986). Reduction of 2-nitroimidazole to the N-hydroxylamine, which can subsequently form a reactive nitrenium ion, is considered to be an activation pathway that also could result in binding to biological nucleophiles (McClelland et aE., 1985). Other heterocyclic nitro compounds can react with DNA after nitroreduction without cleavage of the molecule (Ludlum et al., 1988; Snyderwine et al., 1988). Therefore, the DNA binding of misonidazole was investigated after chemical and enzymatic nitroreduction. To examine the reactivity of the N-hydroxylamine derivative of misonidazole, the effects 47

0041-008X/89$3.00 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

48

ZORA DJURIC

of changes in pH on binding were investigated.

to DNA also

METHODS [3H]Misonidazole (I-(2-nitro-1-imidazolyl)-3-methoxy-[2-3H]-2-propanol, 1.3 Ci/mmol) was obtained from Chemsyn (Lenexa, KS). Unlabeled misonidazole was obtained from Hoffmann LaRoche (Nutley, NJ) and the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute. I-[4,.5,9, lo3H]Nitropyrene was a gift from F. A. Beland (National Center for Toxicological Research). Biochemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Male Sprague-Dawley rats were from Charles River Laboratories (Portage, MI). The amine derivative of misonidazole was prepared under argon by reducing 5 mg misonidazole in 10 ml ethanol with 1 mg 10% palladium on activated carbon and 100 ~1 hydrazine hydrate (Heflich ef al., 1985). After 1 hr at room temperature, the yield of amine was essentially quantitative, as determined by uv/vis, ms, and nmr spectroscopy. The uv and mass spectra were in agreement with published spectra of the compound (Varghese and Whitmore, 198 1). The proton chemical shifts relative to TMS in methanol-& for I-(2-amino- 1-imidazolyl)-3-methoxy-2-propanol were 6 6.7 1 (1 H, d, H-4 or H5, J4,5 = 1.9 Hz), 6.63 (lH, d, H-4 or H-5), 4.00 (IH, m, 2-CH), 3.96 (2H, dd, I-CH2, J= 3.53 Hz and 14.7 Hz), 3.84 (2H, dd, 3-CHz, J = 7.06 Hz and 14.7 Hz), 3.39 (3H, s, CH2). Azo and azoxy derivatives of misonidazole were prepared by zinc reduction of 1 mg/ml misonidazole in 2 mg/ml ammonium chloride using 20 mg/ml zinc (Varghese and Whitmore, 1981). After vigorous shaking of the mixture in air at room temperature for 2 hr, the solution became bright yellow and the products were isolated by high-performance liquid chromatography (HPLC). The reduction products were identified from uv/vis and mass spectra by comparison to published spectra (Varghese and Whitmore, 198 1). Rat liver fractions were obtained from male SpragueDawley rats (200-250 g) and used fresh. Homogenates (25%) were prepared in 50 mM Tris, pH 7.4, 250 mM sucrose, and 1 mM EDTA and were centrifuged at 15,OOOgfor 15 min. Cytosol was prepared by centrifugation of the 15,OOOgsupematant at 105,OOOgfor 1 hr. The pellet was resuspended in 1.15% KC1 and microsomes were prepared after centrifugation once again at 105,OOOgfor 1 hr. Incubations with the rat liver fractions and [3H]misonidazole were conducted as described in the legend to Table 1. Reactions were terminated by addition of 2 vol of ice-cold methanol followed by centrifugation at 13,OOOgfor 1 min. Metabolite formation was determined by HPLC analyses on the same day. A large-scale incubation (100 ml) was conducted with cytosol and unlabeled

NOz

MISONIDAZOLE

FIG. 1. Structure of misonidazole. [3H]Misonidazole was labeled on the hydrogen indicated by a star. misonidazole to allow for analysis of metabolites by uv/ vis and mass spectroscopy. Metabolites were separated by HPLC with a Waters C18 pBondapack column eluted with 100% water for 5 min followed by a linear gradient to 35% methanol over 25 min. Radioactivity associated with metabolites was detected with anon-line Radiomatic FLO-ONE HS detector. Glyoxal eluted at 2.5 min, the N-hydroxylamine at 3.5 mitt, the amine at 5 min, misonidazole at 16 mitt, and the azo and azoxy derivatives at 18 and 24 min, respectively (Fig. 2). DNA binding of [3H]misonidazole was quantified in incubations containing 2 mg/ml calf thymus DNA and rat liver fractions or xanthine oxidase as described in the legends to Tables 2 and 3. These incubations were stopped by addition of 2 vol of ice-cold ethanol and $5 vol of 5 M NaCI. The DNA was collected by spooling onto glass pipets, dissolved in 1.5 mM sodium citrate, 15 mM sodium chloride buffer, and treated with RNases A and Tl for 10 min at 37°C followed by treatment with proteinase K for an additional 10 min (Djuric er al., 1986). The DNA was extracted three times with 2 vol of n-butanol and three times with 2 vol of ether. The DNA was precipitated two additional times with 0.5 M NaCl and 2 vol of ethanol and DNA was redissolved in 5 mM Bis-Tris, 0. I mM EDTA, pH 7.1. For the incubations with I-nitropyrene, the reactions were stopped with phenol and the DNA was extracted as described previously (Djuric ef al., 1986). The recovered DNA was quantified by its absorbance at 260 nm, and the binding levels were determined by scintillation counting after digestion with DNase. Further precipitations and/or extractions did not change the observed binding levels. DNA binding also was quantified after zinc reduction of [3H]misonidazole in dilute solution, which yields the IV-hydroxylamine derivative (Varghese and Whitmore, 198 1). The reduction was carried out under argon purge for 1 hr on ice using 100 fiM [3H]misonidazole, 0.2 mg/ ml ammonium chloride, and 1 mg/ml zinc. The pH of the solution was slightly acidic with a pH value of 5. At

DNA BINDING zl

MISONIDAZOLE ----

0

400

SYNTHETIC

STANDARDS

“Ill 0.m

CYTOSDLIC

5

AND

49

230nm

ammtl

-

OF MISONIDAZOLE

METABOLITES

IO

OF [“I-] MISONIDAZOLE

15 Time

20

25

30

(minutes)

FIG. 2. HPLC separation of misonidazole and its reduced derivatives. (A) Chromatogram of synthetic standards detected by uv/vis absorption at the wavelengths indicated and (B) radioactivity associated with cytosolic metabolites. Experimental details are under Methods.

this pH, the half-life of the N-hydroxylamine is about 1 (McClelland et al., 1984). Presence of the N-hydroxylamine was determined by its uv absorption at 230-240 nm, which distinguishes it from misonidazole that absorbs at 325 nm (Varghese and Whitmore, 1981). Furthermore, the HPLC retention time of the zinc-reduced product is about 1 min earlier than that of the amine. As judged by HPLC, yields of N-hydroxylamine were typically 80-90%. Aliquots ofthe remaining mixture (250 ~1) were immediately added to an equal volume of an argonpurged solution of 4 mg/ml DNA. The DNA was buffered with 100 mM of the following buffers: potassium phthalate at pH 3 and 4, sodium citrate at pH 5, potasday

sium phosphate at pH 6, 7, and 8, and sodium borate at pH 9 and Il. After the addition of reduced misonidazole in 0.2 mg/ml ammonium chloride, the pH values were lowered and are shown in Fig. 3. The mixtures were purged with argon and kept at 0°C for 4 hr. The DNA was extracted by the method described above for enzymatic incubations, except that the DNA was not treated with protease. The DNA modified by misonidazole after chemical or enzymatic reduction was hydrolyzed and analyzed by HPLC for the presence of covalent adducts. Enzymatic hydrolysis of the DNA to nucleosides involved incubation with DNase, nuclease PI, and phosphatases at neu-

ZORA DJURIC

RESULTS

FH FIG. 3. DNA binding of zinc-reduced [‘Hlmisonidazole. Incubations were conducted with [‘H]misonidazole which had been reduced with zinc at 0°C under argon. A solution of 100 PM reduced [3H]misonidazole was added to an equal volume of 4 mg/ml DNA buffered at various pH values. The reaction was allowed to proceed under argon for 4 hr at 0°C. Binding levels were determined by liquid scintillation counting after purification of the DNA by multiple extractions and precipitations. The recovery of DNA was estimated by its absorbance at 260 nm. See Methods for detailed procedures.

tral pH overnight (Djuric et al., 1986). Hydrolysisat 70°C for I hr with trifluoroacetic acid (Tang and Lieberman, 1983) to yield bases was also performed. HPLC analyses were conducted with a Waters PBondapack column and a water/methanol gradient (Waters No. 2) of 20% methanol to 65% methanol over 20 min followed by 100% methanol for 5 min. The flow rate was 2 ml/mitt. Binding of [3H]misonidazole to bovine serum albumin (BSA) was quantified after zinc reduction in ammonium chloride as described above for DNA binding. Aliquots of reduced [3H]misonidazole (250 1rl,80 PM) were added to equal vclumes of 20 mg/ml BSA buffered at pH 5, 6, 7, and 8, using the same buffers as for the DNA reactions above. The reactions were allowed to proceed at 0°C under argon for 3 hr. Reactions were extracted sequentially three times with each of the following: ether, ethanol, methanol, and ether again (Krauss et a/., 1987), after which no more radioactivity could be extracted. The protein was redissolved in I N NaOH by incubation at 37°C overnight. The protein content was determined by the Lowry method (Lowry et a/., 1951), and binding levels were calculated by scintillation counting of small aliquots of the solution.

Rat liver fractions reduced [3H]misonidazole to the amine, N-hydroxylamine, and other unidentified reduction products in the presence of NADPH and FMN (Fig. 2). These polar reduction products may consist of glyoxal and various degradation products of the nitroimidazole ring, as has been reported previously for metabolism of misonidazole in Chinese hamster ovary cells (Varghese and Whitmore, 198 1). These same compounds also were detected by HPLC analyses after zinc reduction of [3H]misonidazole, with the N-hydroxylamine being the main product (80-90%). In agreement with published results (Josephy et al., 198 1), the major product of xanthine oxidase reduction was the N-hydroxylamine. Metabolite formation was very low in the presence of air. Similar to the iV-hydroxylamine, the amine was unstable with a half-life of l-2 days at 0°C in a slightly acidic solution. Azo and azoxy derivatives were not detected after either enzymatic or chemical reduction. Microsomes metabolized [3H]misonidazole about twofold faster than cytosol (Table 1). Likewise, the DNA binding of [3H]misonidazole was twofold higher in microsomal incubations than in cytosolic incubations (Table 2). Under aerobic conditions or without FMN, both the formation of metabolites and the DNA binding of [3H]misonidazole was greatly decreased. O-Acetylation is an important metabolic pathway in the activation of a number of aromatic N-hydroxylamines (Beland and Kadlubar, 1985) as well as of the heterocyclic N-hydroxylamine, N-hydroxy-2-amino-3-methylimidazolo[4,5-fl-quinoline (Snyderwine et al., 1988) to DNA reactive nitrenium ions; therefore, the importance of this pathway for misonidazole was examined. [3H]Misonidazole was apparently a poor substrate for Oacetylation as indicated by DNA binding (Table 2). When aerobic microsomal incubations were conducted with [3H]amino-misonidazole, DNA, and NADPH, DNA bind-

DNA BINDING

OF MISONIDAZOLE

TABLE 1 METABOLISM OF MISONIDAZOLE BY RAT LIVER FRACTIONS Incubation” Microsomes Aerobic -FMN Cytosol Aerobic -F-MN

Total metabolism (pmol/mg protein/min)h 530-+ 10 so*20 4Ok 0 220 r 20 o+ 0 02 0

“Incubations were conducted under argon at 37°C with 20 FM [3H]misonidazole, 1 mg/ml protein, 1 mM NADPH, 0.1 mM FMN, 100 mM potassium phosphate, pH 7.4, 1 mM EDTA, and an oxygen-scavenging system of glucose, glucose oxidase, and catalase (Djuric et al., 1986). Aerobic incubations were conducted in air without the oxygen-scavenging system. Reactions were stopped after 10 min by addition of methanol. * Total metabolism was calculated from HPLC analysis of substrate disappearance. Data are the average of three incubations + the standard deviation.

ing was not detected (co.2 pmol/mg DNA, data not shown). Xanthine oxidase, a model nitroreductase (Howard and Beland, 1982; Colvert and Fu, 1986), also was capable of catalyzing the DNA binding of [3H]misonidazole (Table 3). The level of binding was decreased by the xanthine oxidase inhibitor, allopurinol. The binding of misonidazole was lower than that of the carcinogen I-nitropyrene, and unlike with I-nitropyrene, the DNA binding of [3H]misonidazole was higher at pH 7.4 than at pH 6. When metabolism was examined in identical incubations without DNA (using the same methods as for the cytosolic incubations), there was more metabolism of misonidazole to the N-hydroxylamine at pH 7.4 than at pH 6 (100 pmol/min/ml vs 32 pmol/ min/ml). Likewise, an examination of l-nitropyrene reduction in cytosol (using the methods of Djuric et al. (1986)) indicated more amine formation at pH 7.4 than at pH 6 (160 pmol/min/ml vs 40 pmol/min/ml). Interestingly, the N-hydroxylamine deriva-

51

tive of misonidazole can be detected by HPLC while the direct observation of this intermediate from I-nitropyrene has not been reported in the literature and was not accomplished here. Zinc reduction of [3H]misonidazole, which is known to form the N-hydroxylamine (Varghese and Whitmore, 198 1), also resulted in DNA binding (Fig. 2). Similar to the incubations with xanthine oxidase, the binding was higher with increasing pH. Incubations could not be performed above pH 11 or below pH 3 due to instability of the DNA. At 0°C the binding was typically 5- to IO-fold higher than at 37°C. After a 4-hr reaction at 0°C the yields of adducts were about lo-fold higher than after a 16-hr reaction at 0°C. These re-

TABLE 2 DNA BINDING OF MISONIDAZOLE CATALYZED BY RAT LIVER FRACTIONS Incubation“ 20 PM misonidazole Microsomes 15,OOOgsupernatant Cytosol 40 pM misonidazole, cytosol Complete Complete +air Complete -FMN Complete +DTT Complete +DTT, +S-acetyl CoA

DNA binding (pmol/mg DNA/ 15 min)h 13.9 + 1.4 4.3 + 0.6 7.0& 1.1 15.2+0.1 0.3 f 0.0 0.2 f 0.2 5.7 f 0.7 3.6 + 0.5

“Incubations were conducted under argon at 37°C with 1 mg/ml protein, 2 mg/ml DNA, I mM NADPH, 0.1 mM FMN. 100 mM potassium phosphate, pH 7.4, 1 mM EDTA, and an oxygen-scavenging system of glucose oxidase, glucose, and catalase (Djuric et al., 1986). DNA was precipitated and extracted after 15 min reaction as described under Methods. In complete anaerobic incubations conducted with boiled cytosol, 15.OOOgsupematant, or microsomes, 2.0 * 0.3 pm01 misonidazole/mg DNA/15 min was bound. Some cytosolic incubations were conducted with added 1 mM dithiothreitol (DTT) and 1 mM S-acetyl CoA. ’ Data are the mean of triplicate incubations + the standard deviation.

52

ZORA DJURIC TABLE 3 DNA BINDING

XANTHINE~XIDASE-CATALYZED

Compound“

pH

Misonidazole

6.0 6.0 7.4 6.0 6.0 7.4

1-Nitropyrene

Allopurinol + + -

DNA binding (pmol/w DNA/30 min)’ 5.6? 0.1 0.6k 0.2 7.0* 1.4 162 k20 41 f 8 57 + 9

a Incubations were conducted under argon with 20 PM tritiated compound, 500 pg/ml hypoxanthine, 2 mg/ml DNA, 0.1 U/ml xanthine oxidase. 100 mkt potassium phosphate, pH 6.0 or pH 7.4, and I tIIM EDTA at 37°C for 30 min. Some incubations also contained I mM allopurinol. Reactions were stopped after 30 min by addition of ethanol, and DNA was extracted as described under Methods. ’ Data are the mean of duplicate incubations f the range.

sults are consistent with formation of an unstable adduct. DNA binding of [3H]misonidazole without prior reduction was below 0.2 pmol/mg DNA. The DNA from incubations with either enzymatically or chemically reduced [3H]misonidazole was analyzed by HPLC after hydrolysis. Enzymatic hydrolysis of the DNA overnight at neutral pH failed to yield any HPLC peaks of radioactivity other than the void volume or methanol wash. Acid hydrolysis of the DNA, however, did yield a HPLC peak at about 12 min if the HPLC analysis was conducted immediately after hydrolysis. Typically ZO-50% of the radioactivity was associated with this peak, with the remainder being present in the void volume and methanol wash. The relative amount of the 12-min peak decreased to undetectable levels after a day. Attempts to isolate and characterize this adduct were unsuccessful due to its instability. Binding of zinc-reduced [3H]misonidazole to BSA also was quantified. Similar to the results with DNA, binding increased with increasing pH (19 f 2 pmol/mg BSA at pH 5

vs 97 + 5 pmol/mg BSA at pH 8). Below pH 5, the BSA did not stay in solution and above pH 8, the BSA could not be recovered by precipitation. DISCUSSION Rat liver fractions catalyzed the reductive metabolism and DNA binding of [3H]misonidazole (Tables 1 and 2). Little metabolism of [3H]misonidazole was detected in aerobic incubations, and likewise very little DNA binding occurred in the presence of oxygen (Table 2). These results indicate that oxygen prevents formation of DNA reactive metabolites from [3H]misonidazole. Previous studies have demonstrated that the nitroanion radical of misonidazole is not detected in the presence of oxygen (Moreno et al., 1983). Although this could be due to the rapid reaction of the nitro-anion radical with oxygen, it has been proposed that oxygen may compete with misonidazole for reducing equivalents at the enzyme active site (Moreno et al., 1983). The latter proposal appears likely since misonidazole failed to stimulate NADPH consumption in aerobic microsomal incubations, as measured by HPLC (Z. Djuric, unpublished data). An increase in NADPH utilization would be expected if the nitro-anion radical were redox cycling with oxygen. Xanthine oxidase, a model nitroreductase (Howard and Beland, 1982), also was capable of catalyzing the DNA binding of [3H]misonidazole. However, unlike the xanthine oxidase catalyzed DNA binding of aromatic nitro compounds (Howard and Beland, 1982; Colvert and Fu, 1986), the DNA binding of [3H]misonidazole was not facilitated at slightly acidic pH (Table 3). When zinc-reduced [3H]misonidazole was reacted with DNA, the binding also increased with increasing pH (Fig. 2). Since the misonidazole utilized in these studies was tritiated on the side chain, this indicates that misonidazole can bind covalently to DNA by a mechanism

DNA

BINDING

OF

other than the previously characterized binding via fragmentation of the imidazole ring to form glyoxal (Varghese and Whitmore, 1983). There are at least two mechanistic explanations for the increased DNA binding of misonidazole at higher pH values. Unlike aromatic N-hydroxylamines, 2-(N-hydroxylamino)imidazole can form a reactive nitrenium ion without acid catalysis (McClelland et al., 1985). Second, 2-(N-hydroxylamino)-imidazole exhibits a pK, of 7 (McClelland et al., 1985). Thus, at lower pH values the N-l is protonated to form an oxime, which would interfere with nitrenium ion formation. This type of mechanism also may explain the observed pH dependence of the binding of other heterocyclic N-hydroxylamines to DNA (Snyderwine et al., 1988; Mita et al., 1982). Reaction of misonidazole with DNA is not like that with glutathione, which results in formation of relatively stable adducts that are formed in lesser amounts under both acidic and basic conditions (Chacon et al., 1988). The increased amounts of covalent DNA binding by misonidazole with increases in pH are in contrast to DNA damage elicited by electrolytically reduced misonidazole (Edwards et al., 1986). However, in the latter case, DNA damage was estimated by measuring the viscosity of the DNA (Edwards et al., 1986), and this type of damage may be the result of a different type of lesion. The binding of reduced misonidazole to BSA was typically lower than the binding to DNA (see Results), which is similar to results obtained upon in vivo administration of [2“Clmisonidazole (Varghese and Whitmore, 1980). Similar to the pH dependence of DNA binding, the binding of misonidazole to BSA also was higher at higher pH values (see Results). However, reactions with BSA are more difficult to interpret since changes in pH will presumably change protonation of reactive sulfhydryls as well as of the N-hydroxylamine. For aromatic N-hydroxylamines, reaction with BSA has not been influenced by

53

MISONIDAZOLE

pH in a consistent manner (Martin et al., 1982; Kadlubar et al., 1978, 1980). In summary, the results presented here indicate that [3H]misonidazole can bind to DNA after reductive activation by rat liver enzymes and that the binding does involve retention of tritium from the side chain of misonidazole. Unlike DNA adducts formed by aromatic N-hydroxylamines (Beland and Kadlubar, 1985), the misonidazole DNA adduct(s) is unstable and binding to DNA is increased under slightly basic conditions. REFERENCES ADAMS, G. E.. AND STRATFORD, I. J. (1986). Hypoxiamediated nitroheterocyclic drugs in the radio- and chemotherapy of cancer. Biochem. Pharmacof. 35, 71-76.

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typhimurium

TA1538.

Mutat.

Res. 149, 25-

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JOSEPHY, P. D., PALCIC, B., AND SKARSGARD, L. D. (1981). Reduction of misonidazole and its derivatives by xanthine oxidase. Biochem. Pharmacol. 30, 849853.

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MITA, S., YAMAZOE, Y., KAMATAKI, T., AND KATO, R. (1982). Effects of ascorbic acid on the nonenzymatic binding to DNA and the mutagenicity of N-hydroxylated metabolite of a tryptophanpyrolysis product. Biothem. Biophys. Rex Commun. 105,1396140 1. MORENO, S. N. J., MASON, R. P., MUNIZ, R. P. A., CRUZ, F. S., AND DOCAMPO, R. (1983). Generation of free radicals from metronidazole and other nitroimidazoles by Tritrichomonas foetus. J. Biol. Chem. 258, 405 l-4054. SNYDERWINE, E. Cl., ROLLER, P. P., ADAMSON, R. H.. SATA, S., AND THORGEIRSSON. S. S. (1988). Reaction of the N-hydroxylamine and N-acetoxy derivatives of 2-amino-3-methylimidazolo [4,5-f] quinoline with DNA: Synthesis and identification of N-(deoxyquanosin-8-yl)-IQ. Carcinogenesis 9, 106 1- 1065. TANG, M. S.. AND LIEBERMAN, M. W. (1983). Quantification of adducts formed in DNA treated with Nacetoxy-2-acetylaminofluorene or N-hydroxy-2-aminofluorene: Comparison of trifluoroacetic acid and enzymatic degradation. Carcinogenesis 4, 100 I- 1006. VARGHESE, A. J., AND WHITMORE, G. F. (1980). Binding to cellular macromolecules as a possible mechanism for the cytotoxicity of misonidazole. Cancer Res. 40,2165-2169. VARGHESE, A. J., AND WHITMORE, G. F. (198 I). Cellular and chemical reduction products of misonidazole. Chem. Biol. Interact. 36, 14 1- 15 1. VARGHESE, A. J., AND WHITMORE, G. F. (1983). Modification ofguanine derivatives by reduced 2-nitroimidazoles. Cancer Rex 43,78-82. WHITMORE, G. F., AND VARGHESE, A. J. (1986). The biological properties of reduced nitroheterocyclics and possible underlying biochemical mechanisms. Biothem. Pharmacol. 35,97-10X