[CANCER RESEARCH 51. 3143-3147, June 15. 1991]

Comparison of Oxidative Damage to Rat Liver DNA and RNA by Primary

Nitroalkanes, Secondary Nitroalkanes, Cyclopentanone Oxime,and Related Compounds1

C. Clifford Conaway, Guo Nie, Nalband S. Hussain, and Emerich S. Fiala2

American Health Foundation, Valhalla, New York 10595

ABSTRACT

The hepatocarcinogen 2-nitropropane causes oxidative damage to liverDNA and RNA after administration to rats; increases in 8-hydroxydeox-yguanosine and formation of an unknown moiety (DM ) in DNA, plusincreases in 8-hydroxyguanosine and the appearance of two unidentifiedpeaks (KM and R\2) in RNA were observed by high-performance liquidchromatography of nucleosides from 2-nitropropane-treated rats usingelectrochemical detection (E. S. Fiala et al.. Cancer Res., 49:5518-5522,1989). In the present study, damage to Sprague-Dawley rat liver RNAand DNA was assessed to determine whether the characteristic patternof oxidative nucleic acid damage caused by 2-nitropropane also occurredafter i.p. administration of primary nitroalkanes, other secondary nitroal-kanes, 2-methyl-2-nitropropane (a tertiary nitroalkane), and cyclopentan-one oxime. All of the secondary nitroalkanes and cyclopentanone oximesignificantly increased levels of 8-hydroxyguanine in both DNA and RNAand caused the appearance of DX1, RX1, and RX2. The primary nitroalkanes and the tertiary nitroalkane 2-methyl-2-nitropropane did not causea similar pattern of nucleic acid damage. The rates of reprotonation ofnitronates of the secondary nitroalkanes to the respective un-ionizedneutral forms at pH 7.7 were more than 20-fold less than the rates ofreprotonation of primary nitroalkane nitronates, suggesting that theanionic nitronates, rather than neutral compounds, are more immediatelyresponsible for the DNA and RNA damage observed in vivo. Since 8-hydroxyguanine is a miscoding lesion in DNA, these results suggest thepossibility, still to be rigorously tested, that hepatocarcinogenicity maybe associated not only with 2-nitropropane but also with other secondarynitroalkanes as well as with those ketoximes that are capable of beingconverted to secondary nitroalkanes in vivo.

INTRODUCTION

As discussed in detail in a review by Nielsen (1), nitroalkanesare perceptibly acidic compounds; the dissociation of a protonfrom a nitroalkane produces the nitroalkane anión,or nitronate,with chemical and physical properties quite different from thoseof the parent nitroalkane. We previously found that the nitron-ate form of 2-NP,3 a potent hepatocarcinogen in Sprague-

Dawley rats (2, 3), is a much more powerful mutagen inSalmonella strains TA 100 and TA 102 than the neutral parentcompound (4), an observation recently confirmed by others (5).Furthermore, administration of 2-NP to rats significantly increased the levels of 8-OH-Gua in hydrolysates of liver DNAand RNA and induced the appearance of electrochemicallyactive unknown species we designate as DX1 in DNA and RX1and RX2 in RNA (6). 8-OH-Gua, a product of hydroxyl radicalor singlet oxygen attack on guanine (7-10), has been reportedto cause misreading of DNA templates not only at the site of

Received 1/18/91: accepted 4/8/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This investigation was supported by National Institute of Environmental

Health Services Grant ES03257.2To whom requests for reprints should be addressed.3The abbretiations used are: 2-NP. 2-nitropropane: 1-NP, 1-nitropropane: 8-

OH-Gua. 8-hydroxyguanine; HPLC/EC, high-performance liquid chromatography with electrochemical detection: 8-OH-Guo. 8-h\dro\yguanosine; 8-OH-dGuo. 8-hydroxy-2'-deo\yguanosine.

8-OH-Gua but also at adjacent sites, suggesting that it may beinvolved in mutation and carcinogenesis (11). These findings,together with the results of others (12-14) which indicated thatone-electron oxidation of 2-NP nitronate to the 2-NP radicaloccurs with an accompanying uptake of oxygen and productionof Superoxide and H2O: in in vitro models, prompted us topropose a mechanism of genotoxicity of 2-NP based on production of free radicals or DNA-damaging reactive oxygenspecies in vivo (4, 6).

We also recently reported that acetoxime, a hepatocarcinogenin male rats (15), produces increased levels of 8-OH-Gua inliver DNA and RNA, as well as a characteristic pattern ofnucleoside alterations, detectable by HPLC/EC, qualitativelyidentical to that produced by 2-NP (16, 17). The effects ofacetoxime on liver nucleic acids are compatible with a suggestion made previously (15) that the compound is partially converted to 2-NP [or more accurately, metabolized by ¿V-oxidationto the ad form, which rapidly dissociates to the nitronate formof 2-NP (1, 16) in vivo]. Taken altogether, our earlier resultsraise the question of whether secondary nitroalkanes other than2-NP and ketoximes other than acetoxime would produce asimilar pattern of oxidative damage in the nucleic acids of ratliver. In this communication, we report that five other secondarynitroalkanes and cyclopentanone oxime indeed produce increased levels of 8-OH-Gua in rat liver DNA and RNA and aspecific pattern of nucleic acid alterations, detectable by HPLC/EC, identical to that previously observed following the administration of 2-NP or acetoxime. The very slow rate of reprotonation of the nitronate forms of secondary nitroalkanes to respective neutral compounds at pH 7.7 correlates with the abilityto cause oxidative damage; nitronates of primary nitroalkanesand nitrocarbinols, which do not cause oxidative damage ///vivo, are much more rapidly reprotonated at pH 7.7. These datasuggest that the nitronate forms of 2-NP and other secondarynitroalkanes are biologically active, probably by further conversion to free radicals with accompanying production of reactiveoxygen species.

MATERIALS AND METHODS

Chemicals and Standards. 2-Bromoheptane, 3-bromopentane, 2-io-dobutane, 2-methyl-2-nitropropane (b.p. 124-126°C), 1-nitrobutane(b.p. 152-153°C), nitrocyclopentane (b.p. 180°C).2-nitrooctane (b.p.74-76°C/10 mm Hg), 1-nitropentane (b.p. 75-76°C), and 2-NP (b.p.120°C)were purchased from Aldrich Chemical (Milwaukee, WI); 1-NP(b.p. 131-132°C)was obtained from Eastman Kodak (Rochester, NY).

3-Nitropentane was prepared by reaction of 3-bromopentane with sodium nitrite in dimethylformamide ( 18). The crude product was distilledunder reduced pressure (40 mm Hg) and collected at 70-72°C; 2-nitrobutane (b.p. 135-137°C)and 2-nitroheptane (b.p. 65-67°C/45 mm

Hg) were prepared by similar methods using 2-iodobutane and 2-bromoheptane, respectively, as reactants. All compounds were redistilled and stored at 4"C; the purity of each compound was verified by

gas chromatography and was found in all instances to be 99-100%,except for 3-nitropentane, which was 97.4% pure.

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OXIDATIVE DAMAGE TO RAT LIVER DNA AND RNA BY NITROALKANES

For use as Chromatographie standards, 8-OH-Guo and 8-OH-dGuowere prepared by the method of Kasai and Nishimura (19); the compounds were purified by HPLC as previously described (6). Sources ofother chemicals used have been reported (6).

Animals. Male Sprague-Dawley rats (240-305 g) were purchasedfrom Charles River Laboratories, Inc. (Kingston, NY). The rats weremaintained on NIH-07 diet and tap water ad libitum. The animals weretreated i.p. with 1.12, 1.68, 2.24, or 3.36 mmol/kg of the chemicalsinvestigated, depending on the toxicity and magnitude of responsesobserved. The chemicals were dissolved in Emulphor- 620:HiO (1:4, v/v), 0.28-0.84 mmol/ml. Control rats received Emulphor 620:H2O (1:4,v/v), 4 ml/kg. The rats were sacrificed by decapitation 6 h aftertreatment; the livers and kidneys were immediately excised, rinsed in0.15 M NaCI-0.015 M sodium citrate, pH 7.0, quick frozen in liquidnitrogen, and stored at -70°C(6. 16).

Isolation and Hydrolysis of Liver DNA and RNA. Previously described methods (6) for the isolation of DNA and RNA were utilized.For the isolation of DNA, a modification of the Marmur method (20)was used; the RNA isolation procedure was a slight modification ofthat described by Chomczynski and Sacchi (21). DNA or RNA inportions of approximately 100 \i%were hydrolyzed to the nucleosidelevel for analysis by HPLC/EC, using successive incubations withnuclease I' and alkaline phosphatase (6, 8, 16).

HPLC/Electrochemical Analysis. Analyses of DNA or RNA hydrol-ysates were performed using a 0.46- x 25-cm Ultrasphere ODS columnprotected by a 0.46- x 4.6-cm Ultrasphere ODS guard column (Beck-man Instruments, Inc., Berkeley, CA). The eluant was 5% aqueousmethanol containing 12.5 mM citric acid, 25 mM sodium acetate, 30niM NaOH, and 10 mM acetic acid, pH 5.10, at a flow rate of 1 ml/min. The effluent was routed through a Waters Model 990+ photodiodearray detector for the quantitation of 2'-deoxyguanosine and guanosineand a thermostatted (35°C)amperometric detector (model LC-4B/CC-

4; Bioanalytical Systems, West Lafayette, IN) with settings at 0.5-5.0nA (+600 mV) for quantitation of the electrochemically active species(6, 7). HPLC/EC analyses were conducted within 6 h after hydrolysisof nucleic acids. Standard curves were constructed for 8-OH-dGuo and8-OH-Guo by integration of the respective amperometric responses;standard curves were prepared for 2'-deoxyguanosine and guanosine

using the integrated UV responses at 253 nm. The location of the 8-OH-dGuo and 8-OH-Guo peaks in HPLC/EC profiles was confirmedby coinjection of purified standards.

Kinetics of Reprotonation of Nitronates. For studies on the kineticsof reprotonation of nitronates to respective neutral nitroalkanes, ni-tronates were prepared by incubating 1 mmol of the respective nitroalkanes with an equimolar amount (1 ml) of l N KOH with rapid stirringin darkness for 90 min. These solutions were then diluted to a finalconcentration of 0.1 mM nitronate by dilution with 0.1 M potassiumphosphate buffer, pH 7.7. The solutions were scanned immediatelyafter preparation in the wavelength range of 200-280 nm and thereafterat 5- or 90-min intervals (25°C)using a Beckman DU-7 spectrophotom-

eter (Beckman Instruments, Inc., Fullerton, CA). The log (A/A0) (whereAn = initial absorbance and A = absorbance at various times afterdilution of the nitronates with pH 7.7 buffer) was plotted versus timeto determine the rate of reprotonation of nitronates. The pH of 7.7 wasselected on the basis of the pKaN'"°of 2-NP, which is reported as 7.68

(1). Aqueous nitronate solutions have strong absorbance maxima in therange of 223-235 nm (22) which disappear upon reprotonation to therespective neutral nitroalkanes.

RESULTS

Of the compounds evaluated, in addition to 2-NP (6, 16, 17),2-nitrobutane, 3-nitropentane, nitrocyclopentane, 2-nitrohep-tane, 2-nitrooctane, and cyclopentanone oxime caused highlysignificant increases (P < 0.01 by Student's t test assuming

pooled variance) in 8-OH-dGuo in HPLC/EC profiles of nucleoside hydrolysates of rat liver DNA when compared withcorresponding hydrolysates prepared from Emulphor-treated

Table 1 Changes detected by HPLC ¡ECin liver DNA of Sprague-Dawley rats 6h after i.p. administration of nitroalkanes or related compounds

Compound1-Nitropropane1-Nitrobutane1-Nitropentane2-Nitro-l

-propanol.VNitro-2-butanol2-Methyl-2-nitropropane2-Nitropropane2-Nitrobutane3-Nitropentane2-Nitroheplane2-NitrooctaneNitrocyclopentaneCyclopentanone

oximeEmulphor-H2ODose

8-OH-dGuo/dGuo DXl/dGuoN" (mmol/kg) (x]05)*(area/area)'3333331035333381.122.242.243.362.243.36.51

±0.39.12±0.14.30

+0.16.70±0.29.35±0.78.53

±0.161.122.86±0.37'2.24

2.94±0.11/1.122.87 ±0.3(/2.24

2.71±O.I6/2.24:.96±0.19'1.12

2.83±0.47/3.362.58±0.18'.21

±0.45NDrfNDNDNDNDND0.20

+0.10012±0.010.08

±0.030.04±0.020.06±0.01017+0.120.07±0.02ND

°Number of animals (separate determinations) used.* Mean ±SD.' Area of EC peak (0.5 nA full scalej/area of UV peak (0.6 AU full scale at

253 nm); mean ±SD.**ND, noi detectable.' P< 0.05 compared with results from Emulphor-treated animals.f P < 0.01 compared with results from Emulphor-treated animals.

control rats (Table 1). The peak for 8-OH-dGuo appeared at28-32 ml in the HPLC/EC profiles (Fig. 1); variations in theretention times occurred for 8-OH-dGuo when different columns were utilized and with variations in ambient laboratorytemperature. Results are expressed as mol 8-OH-dGuo/IO5 mol2'-deoxyguanosine. Induction of DX1, appearing at approxi

mately 16 ml, was observed in chromatograms of deoxynucleo-sides from livers of rats treated with the secondary nitroalkanesand cyclopentanone oxime (Table 1). The primary nitroalkanes,the nitrocarbinols, and 2-methyl-2-nitropropane did not causesignificant increases in 8-OH-dGuo, and DX1 was not observed.

The 8-OH-Guo expressed as mol/105 mol guanosine in

HPLC/EC profiles of the hydrolysates of total RNA from liversof rats treated with all of the secondary nitroalkanes tested wassignificantly increased (P < 0.05) compared with correspondingvalues for liver RNA hydrolysates from Emulphor-treated rats(Table 2). Cyclopentanone oxime also caused a significantincrease (P < 0.05) in 8-OH-Guo content of liver RNA. Furthermore, all of the secondary nitroalkanes and cyclopentanoneoxime induced the formation of the unknowns RX1, which

dC

0.1 nAor

0.14 AU

NITROCYCLOPENTANE

dT 8-OH-dGdG

CYCLOPENTANONEOXIME

8-OH-dG

0.1 n«or

0.14 AU

20 30 40 0

I-NITROPENTANE 2-METHYL-2NP

10 20 30 40 10

ELUTION VOLUME, ml

Fig. 1. Representative HPLC/EC profiles of liver DNA deoxy-nucleosidesfrom male Sprague-Dawley rats treated i.p. 6 h prior to sacrifice with nitrocyclopentane. 1.12 mmol/kg; cyclopentanone oxime, 3.36 mmol/kg; 1-nitropentane,2.24 mmol/kg; and 2-methyl-2-nitropropane, 2.24 mmol/kg. Top, electrochemicaldetector response; bottom, UV absorption at 253 nm. dC, 2'-deoxycytidine; dT,

thymidine; dG, deoxyguanosine; DXI, unidentified presumed deoxynucleoside.2'-Deoxyadenosine (not shown) élûtesat 52 ml. For clarity, the electrochemicaldetector response prior to ~8 ml elution volume is not shown.

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Table 2 Changes detected by HPLC/EC in liver RNA of Sprague-Dawley rats 6 h after ¡.p.administration of nitroalkanes or related compounds

Compound1-Nitropropane1-Nitrobutane1-Nitropentane2-Nitro-

1-propanol3-Nitro-2-butanol2-Methyl-2-nitropropane2-Nitropropane2-Nitrobutane3-Nitropentane2-Nitroheptane2-NitrooctaneNitrocyclopentaneCyclopentanone

oximeEmulphor-H2ON°i33333103533336Dose(mmol/kg)1.122.242.243.362.243.361.122.241.122.242.241.123.368-OH-Guo/Guo(xl

O5)*1.63

+0.431.14±0.021.23

+0.380.97±0.231.13

±0.491.25±0.0610.84±1.59'2.54

±0.14?5.32±0.66^1.70

±0.04'2.26±0.36^6.53±l.l/2.66±l.O^1.08±0.23RXl/Guo

(area/area)c0.03

+0.01ND¿ND0.01

±0.01NDND1.02

+0.100.13+0.010.40±0.030.06±0.020.10+0.010.42+0.120.03±0.01NDRX2/GUO

(area/area)20.29

±0.02NDND0.03

±0.04NDND13.24

±1.291.99+0.256.45+0.241.10+0.241.62±0.148.58±0.060.53+0.11ND

°Number of animals (separate determinations) used.* Mean ±SD.f Area of EC peak (0.5 nA full scalei/area of UV peak (0.6 AU full scale at 253 nm): mean + SD.d ND. not detectable.' P < 0.01 compared with results from Emulphor-treated animals.*f < 0.05 compared with results from Emulphor-treated animals.

eluted at approximately 12 ml, and RX2, which occurred at19-20 ml under the experimental conditions used. The effectsof 2-nitro-l-propanol treatment on the formation of RX1 and

RX2 were equivocal, as the responses observed were very nearlyat the analytical limit of EC detection.

Typical traces of UV absorbance and amperometric responsefrom HPLC/EC chromatograms of deoxynucleosides of DNAand nucleosides of RNA from livers of male rats treated withnitrocyclopentane (2.24 mmol/kg), cyclopentanone oxime (3.36mmol/kg), 1-nitropentane (2.24 mmol/kg), and 2-methyl-2-

nitropropane (3.36 mmol/kg) are presented in Figs. 1 and 2,respectively. The appearance of peaks DX1 in DNA and RX1and RX2 in RNA was qualitatively similar to that observedafter 2-NP administration (6, 16, 17) in the instance of nitrocyclopentane and cyclopentanone oxime, but amounts of 8-OH-Guo nearly identical to those observed in liver RNA ofEmulphor-treated control rats, with no detectable RX1 or RX2,were observed after administration of 1-nitropentane and 2-methyl-2-nitropropane. HPLC/EC traces of nucleic acid hy-

drolysates for other secondary nitroalkanes resembled thoseobtained after treatment of rats with nitrocyclopentane andpossessed definite RX1 and RX2 peaks, while HPLC/EC tracesfor the primary nitroalkanes and the a-nitrocarbinols resembledthose of 1-nitropentane; no RX1 or RX2 was detectable.

The kinetics of reprotonation of secondary nitroalkanes andprimary nitroalkanes was confirmed to be first order (23),because linear curves were obtained after plotting \og(A/A0) atthe absorbance maximum for each nitronate species versus time(Fig. 3). The rates of reprotonation of the secondary nitroalkanenitronates at pH 7.7 (half-lives ranging from 15 to 151 h) weremuch slower than the reprotonation rates of the primary nitroalkane nitronates (half-lives ranging from 0.26 to 0.36 h)and the nitrocarbinols (half-lives from 0.54 to 1.23 h) studied(fable 3).

DISCUSSION

The induction of 8-OH-dGuo and DX1 in liver DNA and 8-OH-Guo, RX1, and RX2 in liver RNA after administration of2-NP has been reported to occur in both Sprague-Dawley andF344 rats (6, 17). The effect shows a typical dose-response for8-OH-dGuo, with a maximum occurring 6-18 h after i.p.

O.I nAor

0.14 AU

CRUR

RX1

NITROCYCLOPENTANE

RX2

GR CYCLOPENTANONE

8-OH-GR

CR

0.1 n A

0.14 AU

Â¥

io

GR

UR8-OH-GR

.,.URRX1OXIMERX2L8-OH-GR20 30 40 O

1-NITROPENTANE

CR

10

GR

UR

20 30 40

2-METHYL-2NP

8-OH-GR

10 20 30 40 0 10

ELUTION VOLUME, ml

20 30 40

Fig. 2. Representative HPLC/EC profiles of liver RNA nucleosides from maleSprague-Dawley rats treated i.p. 6 h prior to sacrifice with nitrocyclopentane,1.12 mmol/kg; cyclopentanone oxime, 3.36 mmol/kg; 1-nitropentane, 2.24mmol/kg; 2-methyl-2-nitropropane, 2.24 mmol/kg. Top, electrochemical detectorresponse; bottom. UV absorption at 253 nm. CR, cytidine; UR. uridine; RXI,RX2, unidentified presumed modified nucleosides; 2-methyt-2NP, 2-methyl-2-nitropropane. Adenosine (not shown) élûtesat ~41 ml. The response of theelectrochemical detector prior to ~8 ml elution volume is not shown.

administration of approximately 1.68 mmol/kg to male rats;4the amount of 8-OH-dGuo formed in DNA declines when largerdoses of 2-NP are administered, presumably because of increasing hepatotoxicity. Similar dose-response curves have beenshown to occur for 8-OH-Guo, RXI, and RX2 in RNA.4 Theformation of 8-OH-Gua from guanine has been attributed tothe action of reactive oxygen species such as hydroxyl radicalsor singlet oxygen (7-10), although other mechanisms have alsobeen proposed (24).

The identity of the peaks in HPLC/EC traces of chromatograms, labeled DX1 in hydrolysates of DNA, and RXI andRX2 in hydrolysates of RNA is currently unknown. Preliminaryresults indicate that RX2 is stable at acid pH but rapidlydegrades at alkaline pH; further experiments for the characterization of RX2 are in progress in our laboratory.

Acetoxime has been proposed to be converted by JV-oxidationto the aci form of 2-NP and hence to the nitronate in vivo (15,

4E. S. Fiala, C. C. Conaway, W. W. Asaad, and G. Nie. unpublished

observations.

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OXIDATIVE DAMAGE TO RAT LIVER DNA AND RNA BY NITROALKANES

16). Similar reactions for metabolic conversion of cyclopentan-one oxime to the aci form of nitrocyclopentane with subsequentdissociation to the nitronate would explain the results in thepresent work. Other ketoximes, including 2-butanone oxime,cyclohexanone oxime, 3-pentanone oxime, and 4-heptanoneoxime, have also been shown to cause varying amounts ofoxidative damage to DNA and RNA, with the appearance ofHPLC/EC traces of hydrolysates of nucleic acids qualitativelysimilar to those observed with acetoxime and 2-NP (25).

The effect of 2-NP may be substantially organ specific; 2-NPtreatment did not significantly increase the amounts of oxida-

tively damaged nucleic acids, with the exception of smallamounts of RX2 in RNA isolated from the kidneys of F344rats ( 17). Preliminary results (not shown) indicate that oxidativedamage to rat kidney DNA after administration of the othersecondary nitroalkanes studied in this report is not detectableusing the methods described. At the present time, however,organs other than liver and kidney have not been examined foroxidative nucleic acid damage due to treatment with 2-NP orother secondary nitroalkanes or ketoximes. Female rats, whichshow a substantially reduced response to 2-NP in terms ofcarcinogenicity (3) and oxidative damage (17), also were notinvestigated in the present study.

As noted previously (6), the appearance of small amounts ofRX1 and RX2 in RNA of rats treated with 1-NP cannot bereadily attributed to 2-NP present in the 1-NP as an impurity,since capillary gas Chromatographie analysis of the 1-NP usedin our experiments did not reveal detectable amounts of 2-NPor other secondary nitroalkanes.

The slow rate of reprotonation of the carbon bearing thenitro group of nitronates of secondary nitroalkanes has beenexplained (a) on the basis of stabilization of the nitronate byhyperconjugation, which is greater in secondary than in primarynitroalkanes; (b) by a positive inductive effect caused by twoalkyl moieties; and (c) possibly by poor solvation about thenitronate carbon (1). The data presented here demonstrate astrong correlation between the persistence of the nitronate formas measured in vitro and the occurrence of oxidative damage.The mechanism of free radical production by one-electron

-0.00

Table 3 Kinetics of reprotonation of nitronates of nitro compounds at pH 7,7(25'C)

-0.50-

100 200 300

Minutes

400 500

Fig. 3. Kinetics of nitronate protonation at pH 7.7 (25°C).The normalized logabsorbance at respective UV absorption maxima for freshly prepared 0.1-IHMsolutions of nitronates placed in O.I M potassium phosphate buffer. pH 7.7, wasplotted versus time. •.2-nitropropane. 223 nm; •2-nitrobutane. 226 nm: A. 3-nitropentane. 230 nm: *. nitrocyclopentane. 226 nm; O, 2-nitroheptane. 225.5nm; O. 1-nitropropane, 232 nm; D, 1-nitrobutane, 232 nm; A. 1-nitropentane.23l.5nm.

Compound2-Nitropropane2-Nitrobutane3-NitropentaneNitrocyclopentane2-Nitroheptane1-Nitropropane1-Nitrobutane1-Nitropentane2-Nitro-

1-propanol3-Nitro-2-butanol3-Nitro-

1-pentanolAbsorbance

maximum(nm)223.0226.0230.0226.0225.5232.0232.0231.5230.5229.5233.0Half-life(h)25.817.415.0151.030.40.260.330.360.541.230.75

oxidation of the nitronate form of 2-NP previously presented(4, 6) may thus be a more generalized pathway common toseveral of the stable secondary aliphatic and alicyclic nitronatesas well as to those ketoximes for which conversion to aci formsof corresponding secondary nitroalkanes is metabolicallyfeasible.

On the basis of the oxidative damage to DNA and RNAobserved after i.p. administration, it is possible that the secondary nitroalkanes and cyclopentanone oxime, observed to causedamage to DNA and RNA in this study, may also be hepato-carcinogenic. The secondary nitroalkanes 2-NP (26-28), 2-nitrobutane, 3-nitropentane, and nitrocyclopentane, but notprimary nitroalkanes, have been shown to be mutagenic in theAmes/Salmonella microsome assay using strains TA 100 andTA102,5 providing further evidence for their genotoxicity. In

accordance with suggestions by Löfroth which were based onvery little evidence (29), the nitronates of the secondary nitroalkanes tested were considerably more mutagenic than the respective neutral forms, both with and without microsomalactivation (4, 5).5 Bioassays to test the hypothesis regarding the

carcinogenicity of secondary nitroalkanes are presently underway in this laboratory.

ACKNOWLEDGMENTS

Capillary gas chromatography of nitro compounds was performedfor purity verification by Jonathan Cox. Some of the preliminaryexperiments for this work were performed with the assistance of Dr.Wagdy W. Asaad. We thank Dr. Ock Soon Sohn for reading thismanuscript and for her helpful comments.

REFERENCES

1. Nielsen, A. T. Nitronic acids and esters. In: H. Feurer (ed.), The Chemistryof the Nitro and Nitroso Groups, Part I, pp. 349-386. Huntington, NY:Robert E. Krieger Publishing Co., 1981.

2. Lewis, T. R., Ulrich. C. C., and Busey, W. M. Subchronic inhalation toxicityof nitromethane and 2-nitropropane. J. Environ. Pathol. Toxicol., 2: 233-249. 1979.

3. Fiala, E. S., Czerniak, R., Castonguay, A., Conaway, C. C., and Rivenson,A. Assay of 1-nitropropane, 2-nitropropane, 1-azoxypropane, and 2-axozyp-ropane for carcinogenicity by gavage in Sprague-Dawley rats. Carcinogenesis(Lond.). 8: 1947-1949, 1987.

4. Fiala, E. S., Conaway, C. C., Biles, W. T., and Johnson, B. T. Enhancedmutagenicity of 2-nitropropane nitronate with respect to 2-nitropropane—possible involvement of free radical species. Mutât.Res., 179: 15-22, 1987.

5. Dayal, R.. Gescher, A., Harpur, E. S.. Pratt, I., and Chipman, J. K. Comparison of the hepatotoxicity in mice and the mutagenicity of three nitroalkanes. Fundam. Appi. Toxicol.. 13: 341-348. 1989.

6. Fiala, E. S., Conaway, C. C, and Mathis, J. E. Oxidative DNA and RNA

5C. C. Conaway, N. S. Hussain. B. M. Way, and E. S. Fiala. Evaluation of

secondary nitroalkanes, their nitronates. primary nitroalkanes, nitrocarbinols,and other aliphatic nitro compounds in the Ames Salmonella assay, submittedfor publication.

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OXIDATIVE DAMAGE TO RAT LIVER DNA AND RNA BY N1TROALKANES

damage in the livers of Sprague-Dawley rats treated with the hepatocarcino-gen 2-nitropropane. Cancer Res., 49: 5518-5522, 1989.

7. Floyd, R. A., Watson, J. J., Wong, P. K., Altmiller, D. H., and Rickard. R.C. Hydroxyl free radical adduct of deoxyguanosine: sensitive detection andmechanisms of formation. Free Radical Res. Commun.. /: 163-172, 1986.

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1991;51:3143-3147. Cancer Res   C. Clifford Conaway, Guo Nie, Nalband S. Hussain, et al.   Oxime, and Related CompoundsPrimary Nitroalkanes, Secondary Nitroalkanes, Cyclopentanone Comparison of Oxidative Damage to Rat Liver DNA and RNA by

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