elevated formation of nitrate and w-nitrosodimethylamine ... · merville, nj; ketamine (ketaset)...

(CANCER RESEARCH 51, 3925-3929. Augusl 1. 1991]

Elevated Formation of Nitrate and W-Nitrosodimethylamine in Woodchucks

(Marmota monax) Associated with Chronic Woodchuck Hepatitis VirusInfection1

Rui Hai Liu, Betty Baldwin, Bud C. Tennant, and Joseph H. Hotchkiss2

Institute of Comparative and Environmental Toxicology, Stocking Hall [R. H. L., J. H. H.J, and College of Veterinary Medicine Â¡B.B., B. C. TJ,Cornell University, Ithaca, New York 14853

ABSTRACT

Nitrate balance and N-nitrosodimethylamine (NDMA) excretion werestudied in woodchucks chronically infected with woodchuck hepatitisvirus (WHY). Twenty-four-h urinary recovery of a bolus dose of|l5N]nitrate was 54 Â±12% in woodchucks. VVHV-infectedanimals formed3-fold more nitrate endogenously than did control animals (/' < 0.01).Treatment of WHV-infected animals with EscherÃ¬chiacoli lipopolysac-charide increased nitrate excretion 15-fold, while uninfected animalsincreased nitrate excretion 4-fold. The endogenous formation of NDMAwas higher in WHV-infected woodchucks than in uninfected controls.After administration of i,-|"N2]arginine, |15Nlnitrate, and |I5N|NDMAwere detected in urine indicating that arginine is a precursor of biosyn-thesized nitrate and the hepatocarcinogen NDMA. NDMA probablyresults from the formation of nitrosating agents during the oxidation ofarginine to oxides of nitrogen and citrulline. Woodchucks chronicallyinfected with WHY develop hepatocellular carcinomas with high frequency. Our observations suggest an additional mechanism that may beinvolved in the pathogenesis of hepatocellular carcinoma associated withchronic WHY infection.

INTRODUCTION

Nitrate exposure may be a risk factor in cancer due to thereduction of nitrate to nitrite with subsequent formation ofcarcinogenic yV-nitroso compounds within the body (1,2). Humans are exposed to nitrate from exogenous sources and fromendogenous formation. Tannenbaum et al. (3) showed thatnitrate excretion exceeded intake when humans were placed ona low-nitrate diet for several weeks. Germfree rats endogenouslyformed nitrate indicating that excess urinary nitrate is notproduced by gut microflora (4). Nonspecific diarrheal diseasein humans significantly increased urinary nitrate excretion (5).Experiments in animals treated with LPS1 from Escherichia

coli suggested that endogenous synthesis of nitrate was relatedto immunostimulation (6). LPS-stimulated macrophages produced large amounts of nitrate and nitrite in culture (7). Macrophages convert the guanidino nitrogen of L-arginine intonitric oxide (NO), with further oxidation to nitrite (NOÃ¯)andnitrate (NOj) in vitro (8, 9). Leaf et al. (IO, 11) confirmed thatL-arginine is the source of the nitrogen for nitrate formed invivo in humans, rats, and ferrets. Immunostimulated macrophages also nitrosate amines such as morpholine, thioproline,and proline in culture (12). LPS caused an increase in urinary/V-nitrosothioproline excretion in ascorbic acid-deficient rats(13). These findings suggest that immunostimulation resulted

Received 3/13/91; accepted 5/23/91.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 research was supported in part by USPHS Grant CA-37264 to B. C. T.2To whom requests for reprints should be addressed.'The abbreviations used are: LPS, lipopolysaccharide; 4-MP, 4-methylpyra-

zole, NDMA, A'-nitrosodimethylamine; NDEA, A'-nitrosodiethylamine; NMOR,jV-nitrosomorpholine, NDPA, /V-nitrosodi-n-propylamine; WHY, woodchuckhepatitis virus; GC, gas chromatography; MS, mass spectrometry; TEA, thermalenergy analyzer; DCM, dichloromethane; HPLC, high-pressure liquid chromatography; SIM, selected ion monitoring.

in increased nitrate and perhaps /V-nitroso compound formationin vivo and, thus, may represent an increased cancer risk (14).

Woodchucks chronically infected with WHY, which is closelyrelated to human hepatitis B virus, develop hepatocellular carcinoma with high frequency (15, 16). WHV-infected wood-chucks also develop hepatocellular carcinoma when exposed toNDEA (17).

Our objective was to determine if endogenous nitrate formation was elevated as a result of the chronic hepatitis associated with WHV infection. We wanted to determine if carcinogenic nitroso compounds, such as the hepatocarcinogenNDMA, were formed engenously because of chronic WHVinfection. Previous studies monitored excretion of noncarcino-genic nitrosamino acids. We also wanted to know if L-argininecould serve as a precursor for the /V-nitroso group in endoge

nously synthesized nitroso compounds.

MATERIALS AND METHODS

Reagents. NMOR, morpholine, and 4-MP were purchased fromAldrich Chemical Co. (Milwaukee, WI). LPS (E. coli, serotype0.127:B8, phenol extract), NDMA, and NDPA were purchased fromSigma Chemical Co. (St. Louis, MO). Isotopically labeled Na'5NO3(99% atom purity) and [''Nijguanido-L-arginine (99%) were purchased

from Cambridge Isotope Laboratories, Woburn, MA. Furosemide (5%,Lasix) was purchased from Hoechst-Roussel Agri-Vet Company, So-merville, NJ; Ketamine (Ketaset) was from Aveco. Ft. Dodge, IA; andxylazine (Gemini) was from Butler Co., Columbus, OH. All water wasdouble distilled in glass. NDMA and nitrate were not detected in thedistilled water, glucose, or reagent blanks.

Nitrate Balance and NDMA Excretion. Eight normal (3.3 Â±0.9 kg)and eight chronically WHV-infected 1-yr-old male captivity-born wood-chucks (3.4 Â±0.9 kg) were housed individually in stainless steel metabolic isolation chambers. WHV infection was achieved by s.c. injectionat 3 days of age with 100 ^\ of diluted woodchuck serum containingapproximately 5 x IO6 woodchuck infection dose5o of WHV. WHV

carrier became woodchuck hepatitis surface antigen positive at 2 to 3mo of age, and antigenemia persisted thereafter. The animals consumeda low-nitrate diet (3.2 mg/kg) prior to and during 8-day nitrate balanceexperiments and were given access to double distilled water (<0.05 mgof nitrate/liter) ad libitum. Food and water intakes were measured.Urine was collected every 8 h in bottles containing 25 mg of phenyl-mercuric acetate and 40 mg of sodium azide as bacteria and nitrosationinhibitors. Nitrate balance was measured during Days 1 to 5. On thesixth day the animals were given 32.6 ^mol of [15N]nitrate in 0.5 ml of

water by stomach tube followed by 5 ml of water. Animals wereanesthetized during the tube feeding by i.m. injection of 45 mg/kg ofketamine and 45 mg/kg of xylazine. The total amount of nitrate formedendogenously was calculated by correcting urinary nitrate excretion,less the dietary nitrate, for catabolic loss of ['5N]nitrate (18). On the

seventh and eighth days the animals were given 35 mg/kg of bodyweight 4-MP in 1 ml of 0.9% sterile saline solution by i.m. injectiontwice daily, and urine was collected for NDMA analysis.

Incorporation of L-|'5N2|Arginine into Nitrate and NDMA. TwoWHV-infected male woodchucks (4.2 Â±0.1 kg) and one uninfected

male woodchuck (3.9 kg) were housed as described above, and urine3925

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HEPATITIS AND IN VIVO NITRATE AND NOMA FORMATION

Table 1 Nilrate balance in control and WHV-infected woodchucks (umol/24 h)

Control (n = 8) Infected (n = 8)

DayIngested1

2.922.3932.242.885

3.29Ave.

2 73 + 044Excreted1.742.623.242.923.81287+ 077*Endogenous"0.322.463.82.533.772.58+1.4''Ingested0.771.782.442.953.312.25+1.0Excreted3.62.877.526.266.65.17+ 2.02*Endogenous"6.03.5311.498.648.917.71+ 3.0f

Â°Endogenously synthesized = (excreted/0.54) â€”ingested.* Statistically diffÃ©rent(P < 0.05).' Statistically different (P< 0.01).

was collected. The animals were given only 10% glucose in water adlibitum for 7 days, and nitrate excretion was monitored. Animals weredosed as follows: Day 3, 333 mg of L-['5N2]arginine and 35 mg/kg ofbody weight 4-MP in 1 ml of 0.9% sterile saline, both by i.m. injection;day 5, LPS (1 mg/kg of body weight) in 0.5 ml of 0.9% sterile salinesolution by i.m. injection, followed 6 h later by i.-['5N2]arginine and 4-MP as described above; Days 3 and 5, 8 h after L-C'Njjarginine and 4-

MP administration, furosemide (5 mg/kg of body weight) was giveni.m.; and Day 6, 4-MP only was given as described above. Furosemide,a diuretic, was given to increase the amount of NDMA excreted byincreasing urine volume. This was necessary so that sufficient NDMAcould be collected for analysis. 4-MP was given to inhibit NDMAmetabolism for the same reason.

Nitrate and |"N]Nitrate, and NDMA Analyses. Nitrate was quantifiedas described previously (19). ['5N]Nitrate was measured as [15N]nitro-benzene by GC-MS (20). A 25-ml aliquot of urine was analyzed forNDMA using a GC-TEA (Hewlett-Packard Model 5890 gas Chromatograph; Model 543 TEA) (21). Recovery of NDMA ( 10 ng) and NDPA(internal standard, 44 ng) from spiked urine averaged 88.4% and 99%,respectively. The limit of detection for NDMA was 80 ng/liter. Addition of MOR (IO Mg/ml) to several samples did not result in theformation of NMOR during storage or analysis.

GC-MS Analysis for |15N|NDMA. Urine samples were steam distilled

as previous described (21), and the distillate was washed 3 times with30 ml of pentane. Pooled pentane was back-washed twice with 15 mlof water. The pooled aqueous portions were saturated with sodiumchloride and extracted with 3 x 40 ml of DCM. The DCM was driedover anhydrous sodium sulfate, concentrated to 4 ml in a Kuderna-Danish concentrator, and slowly concentrated to 2 ml with N2. Theextract was filtered and chromatographed (500-fil injection volume; 25-cm x 4.6-mm CN 10-^m HPLC column; Alltech Associates; mobilephase, DCM, 1.0 ml/min). Fractions were collected at 30-s intervals,and each was analyzed for NDMA. Fractions containing NDMA werepooled, slowly evaporated to 400 p\, and rechromatographed on a 15-cm x 0.46-mm Dupont silica HPLC column (5 Mm;Alltech Associates)with DCM ( 1.0 ml/min). Fractions were again collected at 30-s intervalsand analyzed by GC-TEA. Fractions containing NDMA were pooledand concentrated (N2) to 5 to 10 n\ for GC-MS analysis. Mass spectrawere obtained on a Hewlett-Packard Model 5970 GC-mass selectivedetector. Separation of NDMA was achieved on a 25-m x 0.32-mmCarbowax 20 M capillary column with helium. Two to 3 n\ of concentrated extract were injected in the splitless mode. Conditions were asfollows: injector temperature, 180Â°C;purge delay, 30 s; oven temperature programmed (initial, 40Â°C;3.5-min hold; rate, 10'C/min to 100Â°C;

hold for 10 min). Scanning was over a mass range m/z 25-100: scanthreshold. 100; solvent delay, 3.5 min. Isotopie enrichment of NDMAwas determined by SIM at m/z 30, 31, 74, and 75. The ion ratio of mlz 75 to m/z 74 in the sample was used to calculate the M*1 enrichment(i.e., excess ("Njnitrogen) of NDMA after correlation for the m/z IS/

74 ratio from unlabeled NDMA extracted from spiked urine using theprocedures of Biemann (22).

RESULTS

Nitrate Balance. All animals excreted more nitrate than theytook in (Table 1). The average nitrate ingestion (diet + water)

3926

for infected and uninfected animals was 2.25 Â±1.0 (SD) and2.73 Â±0.44 Â¿Â¿mot/day,respectively, and was not significantlydifferent (P> 0.05) between WHV-infected and control groups.Infected woodchucks excreted 5.37 Â±2.02 ^mol/day, whileuninfected animals excreted 2.87 Â±0.77 Â¿Â¿mol/day(P < 0.05).The mean 24-h urinary recovery of the 32.6-f/mol bolus dose of[15N]nitrate was 48.7 Â±16.4%, and 57.5 Â±8.22% (SD, n = 6)for control and WHV-infected animals, respectively (combinedmean = 54.0 Â±12.5%, n = 12). The percentage of recovery of[l5N]nitrate was not different between WHV-infected and controls (P > 0.05). When corrected for ['"Njnitrate loss due to

catabolism (46%) and for dietary nitrate intake (18), infectedanimals endogenously formed nearly 3-fold more nitrate thandid control animals (P < 0.01), which resulted in 1.9-foldgreater nitrate excretion in the WHV-infected group comparedwith the control group (P < 0.05; Table 1).

NDMA Excretion. Both normal and WHV-infected wood-chucks excreted only trace (undetectable to 0.05 nmol/day)amounts of NDMA. After 4-MP treatment, WHV-infectedanimals excreted an average of 0.28 nmol/day, while controlsexcreted only trace amounts. The structure of excreted NDMAwas confirmed by GC-MS (Fig. 1).

Incorporation of i.-|"N2|Arginine into NO.T. L-[lfiN2]Arginine,

when given to the two groups along with the diuretic furosemideand 4-MP (Day 3), resulted in a 5- to 6-fold increase in nitrate

excretion over Day 2 with no treatment (Table 2). LPS inconjunction with L-["N2]arginine, furosemide, and 4-MP (Day5) increased 24-h nitrate excretion 4- and 15-fold over baselinelevels (Day 2) in normal and WHV-infected animals, respectively (Table 2).

Following combined L-['5N2]arginine, furosemide, and 4-MP

treatment (Day 3; Table 3), controls excreted 379 nmol/24 hof urinary [15N]nitrate, which represented 6.8% of total urinarynitrate and 0.045% of the ['5N]nitrogen available from the L-['5N2]arginine (Table 3), assuming that only one labeled guanidonitrogen per t-arginine molecule could be incorporated (23).['5N]Nitrogen incorporation into urinary nitrate increased to

8.3% when LPS was added to the regimen (Day 5; Table 3) incontrols. WHV-infected woodchucks excreted 500 nmol/24 hof urinary [l?N]nitrate during the same period (Day 3; Table 3),

(0

ui

UJ

4274 m+

30

B

30 50 70 90

M/E

Fig. 1. Mass spectra of NDMA standard (A) and NDMA isolated fromwoodchuck urine (B).




Table 2 Nitrate excretion for woodchucks given Â¡.-["N-Jargblbte, 4-MP, and furosemide with or without LPS

Day

AnimalA

(control)B (infected)C(infected)Mean

(B andC)Treatment110.3

1.240.710.98None21.07

1.181.971.58None35.56

8.918.958.93u-["N]Arginine,

4-MP. furosemide40.18

1.531.21.37None54.21

25.5620.8723.2LPS,

u-['5N]argi-nine, 4-MP, furosemide611.42

20.734.4212.584-MP714.93

4.4510.957.7None

Table 3 f'''ft'Â¡Nitrogenin urinary nitrate

Control Infected

Day2356TreatmentNonet.-("N]Arginine,4-MP,

furosemideLPS,

L-["N]argi-nine.4-MP, fu

rosemide4-MPNO3

excretedOimol)Â°1.075.564.2111.4"N1.66.88.34.5"NOT(nmol)16.7379351517"N

fromL-["N)arginine0.00110.0240.020.03Corrected0.0020.0450.0410.056NOTexcreted(jllllllll8.9323.212.6"N5.66.81.9"NOT

(nmol)5001580242"N

fromL-["N]arginine0.030.10.015Corrected0.0560.190.028

" Uncorrected for catabolic loss.* Assuming only one labeled guanido nitrogen per arginine molecule could be incorporated.f Corrected for catabolic loss.

Table 4 NMDA excretion for woodchucks given Â¡.-Â¡"NJargininewith or without

LPS (nmol/day)

InfectedDay3567MeanTotalTreatmentL-["N]Arginine,

4-

MP,furosemideLPS,[.-["NJargi-

nine, 4-MP, furosemide4-MPNo

treatmentNDMA

excretionControl,A0.130.200.230.220.20.8B0.210.670.310.180.34C0.730.410.150.250.39Mean(B

andC)0.470.540.230.220.371.46

which represented 5.6% of the total urinary nitrate and 0.056%of the [15N]nitrogen from the L-['5N2]arginine dose. Addition ofLPS to the L-['5N2]arginine, furosemide, and 4-MP treatments(Day 5; Table 3) increased urinary [15N]nitrate excretion to

1580 nmol/24 h, which was 6.8% of total urinary nitrate and0.19% of the available [15N]nitrogen from the L-["N2]argininedose (Table 3). The incorporation of [15N]nitrogen from L-[15N2]arginine is 3.5-fold higher than the value without LPS

treatment.Incorporation of i.-|'5N2|Arginine into NDMA. NDMA was

detected in the 24-h urine samples in both groups after thecoadministration of 4-MP, diuretic, and L-['5N2]arginine, butWHV-infected woodchucks excreted 4-fold more NDMA (Day3; Table 4). Treatment with LPS (Day 5; Table 4) resulted inincreased NDMA excretion in both groups, but infected wood-chucks excreted 2.7-fold more NDMA than did untreated control animals.

Total ion counts for m/z 75 (M+l for NDMA) when analyzedby GC-MS-SIM, were 12.1% of m/z 74 (M+ for NDMA) forinfected animals treated with LPS, L-["N2]arginine, and furo

semide. Unlabeled NDMA extracted from woodchuck urineand NDMA standards both gave m/z abundances that were 2.7and 2.5% of the m/z 74 (M+), respectively. Using the method

of Biemann (22), the excess m/z 75 was calculated to be 8.6%of the total urinary NDMA excreted.

DISCUSSION

The 24-h urinary recovery of a bolus dose of [15N]nitrate was

54 Â±12% in woodchucks, which is similar to the 53% recoveryof p.o. [15N]nitrate measured in humans (24). This suggests that

the catabolism of nitrate is quantitatively similar in humansand woodchucks.

WHV-infected woodchucks endogenously formed 3-foldmore nitrate than did control animals (P < 0.01) without anadded stimulus such as LPS. Previous studies have shown thatendogenous nitrate synthesis is elevated during immunostimu-lation (5). Tricker et al. (25, 26) reported that bilharzia patientswith parasitic Schistosoma haematobium infection and patientswith urinary diversions excreted more nitrite, nitrate, and N-nitrosoamino acids than did controls. However, they suggestedthat bacterial infection by nitrate-reducing bacteria and notimmunostimulation were responsible for the higher urinarylevels. Our data indicate that WHV infection, which in ouranimals produced no overt clinical symptoms, resulted in increased nitrate formation.

Increased nitrate formation in WHV-infected animals hasbeen related to chronic hepatitis, although portal and parenchy-mal hepatitis would be expected to be mild in 1-yr-old WHVcarriers (16, 27). The liver has a large population of tissuemacrophages or Kupffer cells, which have been shown to proliferate in vivo in inflammatory states (28). There is also markedinfiltration by other inflammatory cells into the liver in wood-chucks chronically infected with WHV (16, 29). Kupffer cellsand hepatocytes as well as other macrophages have been shownin culture to metabolize L-arginine to citrulline, nitric oxide,nitrite, and nitrate after LPS stimulation (9, 30-32). Nitricoxide is not directly a nitrosating species but can be readilyconverted to compounds that nitrosate at physiological pH byreacting with oxygen.

3927




The combination of L-["N2]arginine, furosemide, and 4-MPincreased nitrate excretion in WHV-infected woodchucks 6-fold, suggesting that L-arginine may have been a limiting factorin nitrate synthesis when animals were given only glucose water.Treatment of WHV-infected animals with LPS increased nitrate excretion 15-fold over untreated WHV-infected animals.Leaf el al. (11) did not see an increase in nitrate excretion withL-arginine alone in rats fed laboratory chow but did find a 10-fold increase with a combination of LPS and L-arginine. Ouranimals may have responded to L-arginine alone because theydid not have any dietary source of L-arginine, while those ofLeaf et al. (11) consumed low-nitrate laboratory chow. Thediuretic may have also been responsible for a portion of theincrease in nitrate excretion due to the higher amount of waterlost.

The finding that guanidino nitrogen from L-arginine is incorporated into urinary nitrate in woodchucks is similar to theresults of Leaf et al. (Il) in humans, rats, and ferrets. LPSinduced an increase in incorporation of I5N from L-['5N2]argi-

nine into nitrate in rats, but not in ferrets. Infected woodchuckshad increased incorporation of I5N from L-['5N2]arginine into

nitrate compared with uninfected controls, especially after LPStreatment. Animals received 10% glucose only in order todecrease the dilution of L-['5N2]arginine by dietary L-arginine.The body pool of L-arginine is, however, large and likelyresponsible for the relatively low rate (approximately 8%) of[15N]nitrogen incorporation into nitrate and NDMA.

Two problems limit the detection of NDMA in urine, (a)NDMA is rapidly metabolized and would not be expected toappear in the urine (33, 34). (*) NDMA equilibrates quicklywith the body water because of its miscibility and may notconcentrate appreciably in urine because of its ability to crosslipid membranes (35). NDMA excretion would, therefore, notbe expected to be much greater than the proportion of bodywater excreted. In order to overcome these limitations and toprovide sufficient NDMA for GC-MS analysis, we dosed animals with 4-MP, which is a short-term inhibitor of NDMAmetabolism (21). We also dosed animals with the diureticfurosemide, which increased the urine volume approximately4-fold. The result of this was an increase in NDMA excretion.This NDMA had to come from endogenous nitrosation, because animals were consuming only glucose in water which hadtested negative for NDMA.

For the reasons given above, data on the endogenous formation of carcinogenic /V-nitroso compounds are limited. Thepresent data are the first to suggest that a chronic infection canincrease the endogenous formation of a carcinogenic Â¿V-nitrosocompound in vivo. The 3-fold increase in the M+1 ion (and m/

z 30, 31) in the mass spectrum of NDMA from infected animalscompared with unlabeled NDMA extracted from control wood-chuck urine indicates that a portion of the yV-nitroso group inthe NDMA contained [15N]nitrogen from L-[15N2]arginine. Theincorporation of [15N]nitrogen from L-['5N2]arginine indicatesthat L-arginine is a precursor of the nitroso group in NDMA.Miwa et al. (36) have shown that immunostimulated macrophages nitrosate morpholine in vivo. Nitrosation does not, inthis case, appear to be acid catalyzed via nitrite (9, 36) but ismediated via nitric oxide, which is an intermediate in the L-arginine to nitrate/nitrite pathway (32). Nitric oxide reactsrapidly with dissolved O2 to yield NO2, which exits in equilibrium with the effective nitrosating agents N2O.i and N2O4, bothof which are capable of nitrosating dialkylamines in neutralaqueous solution to form nitrosamines or reacting with water

to yield nitrate and nitrite (23). Assuming that NDMA isequally distributed in the body water, which is approximately55% of body weight (37), and that NDMA is not metabolizedafter administration of 4-MP, infected woodchucks endoge-nously formed 5.4 nmol of NDMA/day/per animal while control animals formed 1.8 nmol/day/animal.

The development of primary hepatic neoplasms occurs withhigh frequency in woodchucks chronically infected with WHV(15, 16, 27). Tumors in woodchucks develop during persistentantigenemia, active viral replication, and in the presence ofactive hepatic inflammation (27). A characteristic influx ofinflammatory cells appears in and around the tumor (necroin-flammation) at the time of hepatocellular carcinoma development, and a tumor-promoting role for these inflammatory cellshas been postulated (16). Long-term, severe, hepatic inflammation may stimulate macrophages (Kupffer cells) in the liverto produce nitric oxide, nitrate, nitrite, NDMA, and possiblyother carcinogenic /V-nitroso compounds. Baldwin et al. (17)demonstrated that NDEA causes hepatocellular carcinomasthat are similar to those observed in WHV-infected wood-chucks. NDMA is also a well-known liver carcinogen in avariety of animals (33).

There is irrefutable evidence that nitrate is endogenouslysynthesized in mammals (3-5, 24, 38). Studies have shown thatmacrophages stimulated with LPS can nitrosate secondaryamines in vitro (36). Leaf et al. (39) recently reported theendogenous nitrosation of morpholine in rats treated with LPS.Our data indicate that woodchucks chronically infected withhepatitis virus endogenously synthesize more nitrate than dononinfected animals and confirm that L-arginine is a precursorof nitrate. Our data also show that the endogenous formationof NDMA is higher in infected animals and that at least aportion of the NDMA results from the process of formingnitrogen oxides from the oxidation of L-arginine in vivo. Thismeans that the process of forming nitric oxide in response toimmunostimulation also results in the formation of a potenthepatocarcinogen. This provides an additional mechanism forchronic hepatitis to influence the risk of liver carcinoma butdoes not prove that endogenous nitrosation is a causative factorin liver cancer.

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1991;51:3925-3929. Cancer Res Rui Hai Liu, Betty Baldwin, Bud C. Tennant, et al. Woodchuck Hepatitis Virus Infection

) Associated with ChronicMarmota monaxWoodchucks (-Nitrosodimethylamine inNElevated Formation of Nitrate and

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