effects of nine n-nitroso compounds on the specific ... · uniform with respect to protein ... i...

[CANCER RESEARCH 37, 1564-1570, May 1977]

as a means of predicting organ-specific carcinogenicity.Such an effect might be the reduction by carcinogens of thespecific radioactivity of total tnichloroacetic acid-precipitable proteins, after the injection of radioactive amino acids.This effect was shown in the liven by many hepatocamcinogens, e.g. , aflatoxin (22), N-hydmoxy-2-fluorenylacetamide(21), and carbon tetnachlonide (12) [but umethan, also ahepatocancinogen, increased the specific radioactivity(ii)].N-Nitmoso-compoundsarea usefulgroupforourpurpose,

since more than 100 are known carcinogens (9, 16). Inprevious studies, specific radioactivity of the liver proteinsafter injection of radioactive amino acids into matswas decreased by the hepatocarcinogens DMN4 (15, 16), DEN (3,10), and N-nitmosomompholine (26), and in matsand mice itwas decreased by MNU, which is not normally a liver carcinogen (1, i3).

We report here mainly on the effects of 9 N-nitroso cornpounds on the â€œspecificradioactivity of the tnichlonoaceticacid-precipitable liver proteins, from matskilled 1 hr after aninjection of [14C]leucine.â€•The effect of the N-nitroso cornpounds was also examined on the free leucine pool in theliver and on the â€œconnectedâ€•specific radioactivity, in whichan attempt is made to correct specific radioactivity forchanges in leucine pool size. The results were correlatedwith potency of the N-nitmoso compounds in causing livernecrosis and liver cancer.

MATERIALS AND METHODS

Chemicals. N-nitnoso compounds were synthesized orpurchased as in Ref. 18, except for N-nitnososancosine andi-nitroso-5,6-dihydrounacil, which were synthesized (9, 17).Uniformly labeled [14C]Ieucine (300 mCi/mmole) and[â€˜4C]cycloleucine(9 mCi/mmole) were obtained from NewEngland Nuclear, Boston, Mass.

Animal Treatment. Male Wistar matsweighing 80 to 100 gwere used. They were given Wayne Lab Blox (Allied Mills,Chicago, Ill.) and tap water ad libitum until the [14C]leucineinjection. The rats were gavaged with the N-nitroso cornpounds dissolved in 10 ml water/kg body weight/dose orsubdose, except for the poorly soluble i-nitmoso-5,6-dihydroumacil (with 30 ml water/kg/subdose). When the compound was given as 2 subdoses, these were gavaged 3 hr

4 The abbreviations used are: DMN, dimethylnitrosamine; DEN, diethylni

trosamine; MNU, 1-methyl-1-nitrosourea; LD@, dose lethal to 50% of thepopulation.

1564 CANCERRESEARCHVOL. 37

Effects of Nine N-Nitroso Compounds on the SpecificRadioactivity of Liver Proteins after Injection of(14C]Leucineinto Rats

CecilIa Chu2 and Sidney S. Mirvish3

EppleyInstitute for Researchin Cancerand Departmentof Biochemistry,Universityof NebraskaMedicalCenter,Omaha,Nebraska68105

SUMMARY

We compared the effect of nine N-nitroso compounds,given by gavage to adult rats, on specific radioactivity of thetnichlomoacetic acid-precipitable liven proteins, 1 hr after theinjection of [14C]Ieucine. The specific radioactivity wasdecreased by dirnethylnitrosamine , diethylnitnosamine,methyl-n-butylnitmosamine, and nitnosomorpholine 5 to 10hr after their administration; was increased by nitnosopiperidine, dinitmosopiperazine, and methylnitnosoumea 5 to 24 hrafter gavage; and was unaffected by nitnososarcosine andnitnosodihydmoumacil.With dimethylnitnosamine, specific madioactivity was decreased by 10 but not 5 mg/kg. In controlrats and rats given injections of either of two nitmosamines,protein specific radioactivity at 60 mm after the [â€˜4Cjleucineinjection was 76 to 87% of that at 30 mm, indicating somedegradation of the proteins at 60 mm. The livem:blood ratioof [â€˜4C]cycloleucineconcentration was unaffected by fournitnosamines, indicating no effect on leucine transport. Theeffect of the nine compounds was examined on total poolsize of free leucine in the liver, at times close to those for themaximum specific radioactivity effect. For these data, wecalculated â€œcorrectedspecific radioactivity,â€•adjusted forchanges in pool size. This adjustment is only a first approximation since, for example, the free leucine pool is notuniform with respect to protein synthesis. The four N-nitroso compounds that decreased specific radioactivity alsodecreased corrected specific radioactivity, even thoughthey enlarged the leucine pool. Of the remaining cornpounds,two enlargedtheleucinepooland threeincreasedcorrected specific radioactivity. For all nine compounds,the decrease in specific and corrected specific radioactivitywas strongly correlated with the ability to cause acute livernecrosis. When nitmosodihydnouracil was excluded, the decrease in specific and corrected specific radioactivity wassignificantly correlated with the reported liver carcinogenicity.

INTRODUCTION

A current aim of our research is to examine the acuteeffects of carcinogens on variOus biochemical parameters,

I This investigation was supported by USPHS Contract NOl CP33278 fromthe National Cancer Institute.

2 Some of this project was conducted in partial fulfillment of the requirements for an MS. degree.

3 To whom requests for reprints should be addressed.

Received February 19, 1976; accepted February 15, 1977.

on April 3, 2017. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

J::SE.I AbA â€ AAA Iâ€˜ SAS

N-Nitroso Compounds and Protein Radioactivity

apart. Times were measured from that of the N-nitrosocompound dose or the 1st subdose. All matswere killed bydecapitation within 30 mm, at 3 to 5 p.m.

Specific Radioactivity. [â€˜4C]Leucine(2 pCi/mI 0.01 N HCI)was injected i.p. at a dose of 10 MCi/kg. One hr later, theratswere killed.The liverswere removed,frozeninliquidnitrogen,and storedat â€”15Â°.Proteinswere isolatedfromindividual livers by tnichloroacetic acid precipitation (19).Tamed20-mg protein samples were dissolved in 1 ml NCStissuesolubilizemat60Â°,2 dropsof30% H2O2were added,the samples were reheated for 30 mm, and the volume wasbrought to 2 ml with toluene. Duplicate 0.5-mI aliquots werecounted for 30 mm after adding 12 ml toluene scintillationcocktail, with an efficiency of about 90%. Each experimentof 12 to i 5 matscontained groups of 3 to 4 rats receivingdifferenttreatments,includinga controlgroup of 4 ratsgiven [14C]leucine alone. These results (in cpm/mg protein)and others were mostly expressed as a percentage of themean value for controls in the same experiment and thencombined from several experiments.

[â€˜4C@CycIoleucineTransport. [14C]Cycloleucine was injected i.p. as a 2-MCi/mI solution in 0.01 N HCI (dose, iOj.&i/kg) 2 hr before sacrifice. Radioactivity of the liver andblood (collected from the heart and hepaninized) was measured as described previously (4).

Pool Size of Free Leucine. [â€˜4C]Leucinewas injected asbefore, 1 hr before sacrifice. Each liver was minced withscissors, and samples were used to measure free leucine,specific radioactivity, and water content. For free leucine,the tissue was homogenized, and the proteins were precipitated with picnic acid. The supemnatant was freed of excesspicnic acid with an anion-exchange column and analyzedfor amino acids with a Beckman Model 1206 amino acidanalyzer (23). For water content, 0.5 to 1.0 g liver tissue wasweighed, wrapped in aluminum foil, and dried (vacuumoven,730 mm, 130Â°)toconstantweight.

Acute Hepatotoxiclty. The rats were kept for 48 hr aftergavage of the N-nitmoso compound and then killed. Piecesof 2 liver lobes were sectioned, examined histologically,and graded from 0 to 4 for liven cell necrosis (5).

50

25

0cr ooF-z00 @5

4 500,

25

0

Chart 1. Specific radioactivity (SA) at varioustimes after gavage of the N-nitroso compound.Each point gives the results for 3 to 16 rats (mean,6). At the standard times (Table 1), the results for 6to 16 rats (mean, 10) are shown. In all charts: â€¢,data significantly different from the controls (p <0.05); 0, data not significantlydifferent from thecontrols (p < 0.05); vertical bars, SE. MBN,methyl-n-butylnitrosamine; DNP, 1,4-dinitropiperazine; NPP, N-nitrosopiperidlne; NM, N-nitrosomorpholine; NB, N-nitrososarcosine; NDU, 1-nitroso5,6-dihydrouracil.

25

â€œ@ -@yY vYY yvy

0 5 0 24 0 5 10 24 48 0 5 0 24

1565MAY 1977

RESULTS

Specific Radioactivity. The 9 N-nitmosocompounds weregavaged, and their effects on specific radioactivity wereexamined (Chart i). The rats were fed ad libitum because, ina preliminary experiment, 140 mg DEN per kg decreasedspecific radioactivity more strongly in fed rats than in ratsfasted for 24 hr before and during the experiment. Theoriginal aim was to give the same dose of 1.37 mmoles/kgfor all compounds, corresponding in the case of DEN to 140mg/kg (one-half the LD5O).However, the doses of DMN andi ,4-dinitnosopiperazine were lowered because of their hightoxicity, and those of N-nitrososamcosine and 1-nitmoso-5,6-dihydmouracil were raised because of their low toxicity (seeTable 1 for the LD50values). The LD50for N-nitrosomonpholine, N-nitnosopipemidine, and 1,4-dinitmosopipemazine wasi .6 to 3.3 times greater by gavage than by injection (8).Since the required doses of these compounds would be tootoxic if injected i.p., and the p.o. route was used for mostcarcinogenesis tests, all compounds were gavaged. Because the doses of N-nitnosopipemidine, i ,4-dinitmosopipenazine, and i-nitmoso-5,6-dihydmounacil were close to theLD50, these compounds were given as 2 subdoses. Theâ€œstandardâ€•doses and times after gavage of the N-nitrosocompounds (when the specific radioactivity change wasgenerally near a maximum) are given in Table 1. The liverproteins from 87 matstreated with [14C]leucine alone contamed 127 Â±2 cpm/mg (all results are given as mean Â±S.E.).

At various times@ 24 hr after the gavage, specific radioactivity was significantly decreased by DMN, DEN, methyln-butylnitmosamine, and N-nitnosomorpholine; was increased by N-nitnosopipenidine, 1,4-dinitrosopipemazine,and MNU; and was not significantly affected by N-nitnososarcosine and i -nitroso-5,6-dihydnoumacil (the increase byi-nitroso-5,6-dihydroumacil at 8 hr was not significant)(Chart 1). With regard to the 4 compounds decreasing specific radioactivity, the maximum effectoccummed at 3 to 5 hrfor DMN, at 8 hr for DEN and methyl-n-butylnitmosamine,and at iO to 24 hr for N-nitrosomompholine. After these

TIME (HR)



Standard doses and timesused, biological activity, and summaryofresultsStandard

doseInductionLiver

carcinogeni

Summary of biochemical results (as% controlvalues)eSAdData

from Table3Leu

of liver ne cit?(%fromcineStandardLD@(mg/crosismciChartpoolCompoundmg/kgmmoles/kgtime

(hr)â€•kg)(grade)dence)1SA sizeCSADMN200.2754@Y3555656

13173DEN1401.378280â€•293706113985MBN1591.378lOOt4.U625511362NM1591.3810320@3100625912373NPP2x78h2x0.695200'@235122118100115DNP2

x 75h2 x 0.525160â€•128133133 93122NSC125010.655000â€•040102103132136MNU2

x 75h2 x 0.735110â€•00136135 123165NDU2x 300h2 x 2.105850k0989296 122 116

C. Chu and S. S. Mirvish

Table 1

aTimefromthe 1stN-nitrosocompoundgavageto sacrifice.b Highest recorded incidence in a test by chronic p.o. administration. The data are from those listed before (18), except for 1-nitroso

5,6-dihydrouracil (17).I. That is, of results obtained with the standard doses and times.

d The abbreviations used are: SA, specific radioactivity; CSA, corrected specific radioactivity; MBN, methyl-n-butylnitrosamine; NM, N-nitrosomorpholine; NPP, N-nitrosopiperidine; DNP, 1 ,4-dinitrosopiperazine; NSC, N-nitrososarcosine; NDU, 1-nitroso-5,6-dihydrouracil.

V. By the p.o. route (9).

I Determined by us, after gavage of an aqueous solution to 200-g male rats.p Methyl-n-butylnitrosamine caused liver tumors, but the incidence was not given (16).

h Gavaged as 2 doses, 3 hr apart.

I By the p. route (17).

times, specific radioactivity returned toward the normallevel or, in the case of N-nitnosomorpholine where matswerealso examined at 48 hm,was increased above normal. Theincrease of specific radioactivity caused by N-nitrosopipenidine, 1,4-dinitnosopiperazine, and MNU was relatively longlasting since it occurred at 5, 8, and 24 hr. The matstreatedwith N-nitmosopipenidine and 1,4-dinitrosopiperazine hadconvulsions soon after treatment and were sluggish beforesacrifice.

The effect of varying the DMN dose was examined, withthe matsbeing killed at 3 hr (Chart 2A). Since the shortertime might not allow full absorption to occur from thegastrointestinal tract, the DMN was injected i.p. The decreaseinspecificradioactivitycausedby20 mg DMN perkgwas similar to that for the same dose given by gavage, and itremained significant for 10 but not 5 mg DMN pen kg. Theincrease due to 2 subdoses of 1,4-dinitmosopipenazine,given by gavage, diminished when each subdose was lowened from 75 to 37.5 mg/kg, but these effects were notsignificant at the 5% level in this experiment (Chart 2B).Also, i.p. injection of MNU (75 mg/kg) increased on did notaffect specific radioactivity after 1, 2, and 3 hr (Chart 2C). Inother experiments, no effect on specific radioactivity wasobserved i , 2, and 3 hr after 300 mg i-nitroso-5,6-dihydrouracil per kg were injected i .p. and 5 hr after 5000 mg N-nitrososarcosine per kg were gavaged.

In all these experiments, [â€˜4C]leucine was injected 1 hrbefore the rats were killed. To check that this period wassuitable, untreated matsor rats treated with standard dosesof DMN or 1,4-dinitrosopipenazine were given injections of[â€˜4C]leucine30, 60, or 90 mm before sacrifice. For all 3treatment groups, specific radioactivity at 60 mm was 76 to

-j0a:z00

C/)

3755 75DOSE OF DMN DOSE OF DNP TIME (HR)AFTER

(mg/kg) (mg/kg) 75mg MNU/kg

Chart 2. Specific radioactivity (SA) for various doses of DMN and 14-dinitrosopiperazine (DNP) at the standard times (A, B) and for differenttimes after gavage of 75 mg MNU per kg (C). Each point gives the results for 3to 8 rats (mean, 4).

87% of that at 30 mm, and specific radioactivity at 90 mmwas 77 to 103% of that at 60 mm (Chart 3).

[1 4CjCycloleucine Transport. [14C]Cycloleucmne is meta

bolically inert but transported similarly to leucine (6).Hence, to check the possible effect of amino acid transporton specific radioactivity, we examined the uptake by theliver of [14C]cycloleucine (Table 2). The effect of 4 nitmosamines was examined, of which 2 inhibited and 2 enhancedspecific radioactivity. The standard doses and times wereused. Livers of the control group had about 13,000 cpm/gwet weight. For 3 of these nitmosamines,[14C]cycloleucmneconcentration in the liver was slightly but significantlygreater than that for the controls. However, the livem:blood




Effect of 4 compounds on(â€˜4CJcycloleucineconcentration, usingthe standard doses andtimesCompoundNo.of

ratsLiver % controlp<Blood % controlp<Liver:blood

ratio1'cpm/g

cpm/mIp<None

DMNMBNNPPDNP6

3333100Â±2

91 Â±9112 Â±2117 Â±5134 Â±21NSb

0.010.010.05100Â±2

95 Â±3106 Â±2103 Â±1

89 Â±3NS

NSNSNS0.96Â±0.01

0.89 Â±0.03 NS0.98 Â±0.12 NS1.06 Â±0.03 NS1.38 Â±0.12 NS


ratio for [â€˜4C]cycloleucmneconcentration did not differ significantly between the control and nitrosamine-treatedgroups. Hence changes in specific radioactivity were probably not due to an altered leucine transport into the liver.

Leucine Pool Size and Corrected Specific Radioactivity.Pool size and specific radioactivity were determined foreach N-nitroso compound, using the standard dose andtime. The specific radioactivity results (Table 3) were similarto those reported in Chart 1. Significant decreases wereobserved for the same compounds in the 2 sets of specificradioactivity results (compared in Table 1), except for N-nitrosopipemidine where the effect was significant for theChart 1 data only. However, the specific radioactivity datafrom Chart 1, which contain 90 results at the standardtimes, are more reliable than those in Table 3, which compniseonly 36 results at the standard time and are included inthe results of Chart 1.

Leucine pool size for the 15 control rats was 0.30 Â±0.01j@moleleucine peng liven. The leucine pool was significantlyenlarged by DMN, DEN, methyl-n-butylnitrosamine, and N-nitmosomompholine, which decreased specific radioactivity,and by N-nitmososarcosine and 1-njtmoso-5,6-dihyd mouracil,which did not affect specific radioactivity. Pool size wasunaffectedby N-nitmosopipenidine,1,4-dinitrosopiperazine,

zwIâ€”0

a-

DI

E

a-0

00 30 60 90

TIME (MIN) AFTER [â€˜4C]LEUCINE

INJECT ION

Chart 3. Variation of the time between [â€˜4C]leucineinjection and sacrifice:effect on protein specific radioactivity for rats receiving the standard doses of1,4-dlnitrosopiperazine (DNP) (given as 2 subdoses 3 hr apart) and DMN, andfor control rats untreated with N-nitroso compounds. 1,4-Dinitrosopiperazine and DMN were gavaged at (standard time minus 1 hr) before the[â€œC)leucineinjection. Each point shows the results for 4 rats.

and MNU. The water content (69 to 70% by weight) and totalliver weight (3.3 Â±0.1 g for the 15 controls) were notsignificantly changed in any of the treated groups, so thatthe alterations in pool size were not due to changes in thetotal amount of water in each liver.

To obtain corrected specific radioactivity, we used theequation:

Corrected specific radioactivity

â€” Specific radioactivity x pool size (A)

100

where each parameter was expressed as a percentage ofthe control value. The specific and corrected specific radioactivity results in Table 3 were similar. Thus DMN, DEN,methyl-n-butyln itrosamine , and N-nitrosomorpholine decreased both specific and corrected specific radioactivity,although the corrected specific radioactivity effect was notsignificant for the DEN group (with only 2 mats).For all 4 ofthese compounds, the decrease of corrected specific radioactivity was less than that of specific radioactivity, due tothe enlargement of the leucine pool. The remaining 5 compounds (which increased or did not affect specific madioactivity) tended to increase corrected specific radioactivity,although this effect was significant only for 1,4-dinitnosopiperazine, 1-nitroso-5,6-dihydrouracil, and MNU. Forthe lastcompound, the 35% increase in specific radioactivity wasaugmented by the leucine pool enlargement, so that conrected specific radioactivity was increased by 65%.

Summary of Biochemical Results. The results in Table 3and (for the standard times) Chart 1 are summarized inTable 1 and can be separated into 2 groups: (A) specific andcorrected specific radioactivity were decreased by DMN,DEN, methyl-n-butylnitrosamine, and N-nitnosomorpholine,and (B) specific and connected specific radioactivity andleucine pool size were increased under certain circumstances. Thus (a) all 9 compounds increased at least 1 of the3 parameters, (b) leucine pool size. was increased by 6compounds, (c) specific radioactivity was increased 48 hrafter N-nitrosomonpholine was gavaged (Chart 1), and (d)specific and/or corrected specific radioactivities were increased by 1,4-dinitrosopiperazine , N-nitnososamcosine,MNU, and 1-nitroso-5,6-dihydmouracil.

Biological Activity. To relate the biochemical data to biological activity, we assigned grades of 1 to 4 to the acuteliver necrosis in rats killed 48 hr after receiving a standard

Table 2

aMeanof theconcentrationratioscalculatedseparatelyfor eachrat.b The abbreviations used are: NS, not significant; MBN, methyl-n-butylnitrosamine; NPP, N-

nitrosopipenidine; DNP, 1 ,4-dinitrosopiperazine.

1567MAY 1977



CornpoundNo.

ofrats.

.Specific rad.

. .loactlvlty. Leucine p.ool sizeCorrected

specific radioactivity%controlp<%controlp<%control

p<None1'15100Â±

2100Â± 2100Â±4None15100Â± 4100 Â± 4100 Â±7DMN556Â± 50.001131 Â±110.0173 Â± 60.01DEN261Â± 10.02139 Â± 30.00185 Â± 1NSCMBN555Â± 20.001113 Â± 70.0562 Â± 50.001NM359Â± 40.001123 Â± 50.0273 Â± 20.05NPP3118Â±16NS100Â±13NS115Â±

6NSDNP6133Â± 80.00193 Â± 6NS122 Â± 50.01NSC3103Â± 6NS132 Â± 40.001136 Â±100.05MNU3135Â± 30.01123 Â±10NS165 Â±140.01NDU696Â± 5NS122 Â± 90.02116 Â± 4 NS


Table 3

Effects on specific radioactivity, leucine pool size, and corrected specific radioactivity, for thestandard doses and times

aAll 15controlsarereferredto here.bThree experiments were performed. These are mean values for the 3 control groups. For the

statistics, the experimental and control data of the individual experiment were compared.cThe abbreviations used are: NS, not significant, MBN, rnethyl-n-butylnitrosamine; NM, N-

nitrosomorpholine; NPP, N-nitrosopiperidine; DNP, 1 ,4-dinitrosopiperazine; NSC, N-nitrososarcosine; NDU , 1-nitroso-5,6-dihydrouracil.

dose of N-nitroso compound (Table 1). Each grade is basedon the results for 4 rats. Also, we listed the highest recordedincidence of liver tumors after chronic p.o. administrationof the N-nitnoso compound to rats (Table 1).

DISCUSSION

Most of the results describe effects of 9 N-nitroso compounds on the specific radioactivity of tnichloroacetic acidprecipitable liver proteins measured 1 hr after [â€˜4C]Ieucmneinjection (specific radioactivity). Even if only protein synthesis had been involved (and not protein degradation), wewould have measured the extent and not the initial rate ofthis synthesis. Also, the incorporation probably showedlarge variations between individual proteins. The results inChart 3 suggest that a 30-mm period after the [â€˜4C]leucineinjection would have been more suitable than the 60-mmperiod used, since protein specific radioactivity decreased13 to 24% from 30 to 60 mm. This decrease representsprotein degradation and indicates that at 60 mm we wereexamining effects on both the synthesis and degradation ofproteins and not, as we should have preferred , effects onsynthesis alone. However, at least for DMN and 1,4-dinitrosopipenazine at the standard times after their gavage, thepercentage of disappearance of specific radioactivity from30 to 60 mm was not much affected by the N-nitnoso compound treatment, and the increase caused by DMN anddecrease caused by 1,4-dinitrosopipenazine occurred atboth times (Chart 3). Hence, provided that the limitations ofthe specific radioactivity measurement are borne in mind,the results at 60 mm appear adequate for our purpose,which was to compare the action of various N-nitnoso compounds.

The effects of the 9 N-nitroso compounds on the total freeleucine pool in the liver were studied at a single time afterthe N-nitmoso compound gavage. When the free leucine

pool increased in size and other factors remained unchanged, a given dose of [14C]Ieucine would become morediluted in the pool, and [14C]leucine incorporation woulddecrease. Hence, Equation A was used to derive the conrected specific radioactivity for those data where pool sizewas measured. This connection is approximate at best, since(a) the entire leucine pool is not available for protein synthesis, i.e., the pool is not homogeneous (2); and (b) specificradioactivity involves protein degradation (where free leucine is not involved), as well as protein synthesis. Hencemore attention should perhaps be paid to the simpler butmore extensive specific radioactivity data than to the derived corrected specific radioactivity data.

Our most important findings were that 4 nitnosaminesdecreased specific and connected specific radioactivity andthat various N-nitroso compounds increased specific andconnected specific radioactivity and/or leucine pool size.With respect to the 1st effect, 3 of the nitnosamines decreasing specific radioactivity (DMN, DEN, and N-nitrosomonpholine) were previously reported to show this effect (seeâ€œIntroductionâ€•).The observation that 20 mg DMN per kgcaused an initial decrease of specific radioactivity, followedby a recovery to normal at 10 hr (Chart 1), is consistent withthat by Mukherjee et al. (20), where specific radioactivityrose above normal levels at 20 hr after the same DMN dosewas injected. The maximum specific radioactivity decreaseoccurred 5 hr after gavage of DMN, but 24 hr after gavage ofN-nitnosomompholine (Chart 1). This may be due to theslower rate of N-nitnosomonpholine metabolism, e.g., theblood nitrosamine level fell by 70% 6 hr after an injection ofDMN (16) and 14 hr after an injection of N-nitrosomorpholine (estimated from Ref. 27). MNU increased specific radioactivity in our studies but decreased specific radioactivity inprevious studies on mice and rats (1, 13), possibly becauseof differences in the species or dose. The specific nadioactivity increase caused by MNU did not appear to follow aninitial decrease (Chart 2C). A short-term decrease of specific radioactivity by 1-nitroso-5,6-dihydnoumacil had ap




No. of corn

Values of coefficientrCorrectedpoundsSpecific

ra specific ra r for p =r for p=ParametercomparedNecrosisdioactivity1'dioactivity0.050.01Specific

radioactivity1'9+0.89Â±0.63Â±0.76Necrosis9â€”0.71â€”0.90Â±0.63Â±0.76Carcinogenicityâ€•8+0.17â€”0.70â€”0.64Â±0.71Â±0.83Carcmnogenicityc7+0.58â€”0.85â€”0.78Â±0.75Â±0.87


Table 4Correlations between the data in Table 1

There were no significant correlations with leucine pool size.

a Specific radioactivity data are from Chart 1.b For all compounds except methyl-n-butylnitrosamine.C For all compounds except methyl-n-butylnitrosamine and 1-nitroso-5,6-dihydrouracil.

peared possible, since 1-nitnoso-5,6-dihydrounacil causedchanges in the liver DNA after 30 mm but not after 4 hr (26)(see below). However, 1-nitroso-5,6-dihydrouracil did notdecrease specific radioactivity 1 to 3 hr after i.p. injection(seeâ€œResultsâ€•).

With respect to the increases in specific and correctedspecific radioactivity and/on pool size, the increased poolsizecaused by DMN (20mg/kg) isincontrastto the meported lack of change in total amino acid pool size of the ratliver, 3 hr after administration of 50 mg DMN per kg (15).The reason for this difference is unknown. Related to ourresults, protein synthesis by postmitochondmial supemnatantwas inhibited when the livers of rats given 20 mg DMN perkg, 2 hr previously, were used, but was enhanced in supernatants from rats given DMN 20 to 30 hr previously (20). Thisenhancement was attributed to a stress reaction, mediatedby glucocorticoids. These hormones are known to increasespecific radioactivity (14), amino acid pool size (28), andglycogen deposition (14) in the matliver or cell-free systemsisolated therefrom. In the experiment by Mukhenjee et al.(20), liver glycogen was elevated 10-fold 20 hr after the DMNinjection. Hence the observed increase in specific radioactivity and leucine pool size could be due to an increasedsecretion of g!ucocorticoids. With regard to N-nitrosopipenidine, 1,4-dinitrosopiperazine, and MNU, a stress meaction such as glucocorticoid secretion might be related tothe high standard dose relative to the LD50(Table 1).

The biochemical results were compared with activity forthe induction of liver necrosis and liven tumors (Table 1).DMN , DEN , methyl-n-butylnitnosamine , and N-nitrosomorpholine decreased specific and corrected specific nadioactivity, had gradings of 2 to 4 for the induction of livennecrosis, and showed at least moderate liven carcinogenicity, except for methyl-n-butylnitmosamine, which was madequately tested as a carcinogen (16). N-Nitmosopipenidineand 1,4-dinitrosopipemazine, which slightly increased conmectedspecific radioactivity, were moderately toxic and carcinogenic for the liven. N-Nitnososancosine and MNU, whichincreased specific and corrected specific radioactivity, werenormally not toxic or carcinogenic for the liver, althoughMNU did induce liven tumors in partially hepatectomizedrats (7). The nitnosoumea 1-nitmoso-5,6-dihydnoumacil was astrong liver carcinogen but was nontoxic for this organ anddid not affect specific radioactivity. Thus, except for 1-

nitnoso-5,6-dihydrounacil, acute toxicity and hepatocancinogenicity appeared to be associated with a decrease in specific and connected specific radioactivity. In confirmation,Table 4 demonstrates a close correlation of effect on specific radioactivity with that on connected specific madioactivity, close correlations of necrosis with a decrease inspecific radioactivity and (especially) connected specificradioactivity, and a lack of significant correlation of camcinogenicity with a decrease in specific and connectedspecific radioactivity, when all compounds of known cancinogenicity (i.e. , excluding methyl-n-butylnitmosarnine) wereconsidered. However, theme was a significant (p < 0.05)correlation of carcinogenicity with a decrease in specificand corrected specific radioactivity when 1-nitmoso-5,6-dihydnouracil was also excluded.

From the evidence presented , we do not know whether adecrease in specific radioactivity was a cause or a resultof the events leading to liven necrosis at 48 hr, on an unmelated side-effect. The short-term decrease in specific radioactivity caused by DMN, DEN, methyl-n-butylnitmosamine,and N-nitmosomompholine was probably not a consequenceof irreversible changes leading to cell death at 48 hn, because specific radioactivity recovered completely or almostcompletely after 8 to 24 hr (Chart 1).

The doses were generally higher than those found byFarber and his colleagues to cause breaks in liven DNA;thus single-stranded DNA breaks were observed 4 hr afterthe injection (per kg) into rats of 1 mg DMN, 1 mg N-nitrosomompholine, 10 mg N-nitrosopipenidine, 10 mg 1,4-dinitmosopipemazine, and 40 mg MNU (8, 24). Doublestranded DNA breaks were observed 30 mm after the injection of 1 mg 1-nitroso-5,6-dihydrounacil pen kg (25). Forcomparison, 5 mg DMN pen kg did not affect specific radioactivity (Chart 2A). For the strong hepatocancinogen 1-nitnoso-5,6-dihydnoumacil, the potency in causing DNA breakscontrasts strikingly with the lack of decrease in specificradioactivity and the lack of necrogenic activity.

In conclusion, a decrease in specific and corrected specific radioactivity was correlated with liver cancinogenicitywhen 1-nitroso-5,6-dihydrouracil was excluded . However,DNA breakage could be more closely linked than the decrease in specific radioactivity to liven carcinogenicity.Also, the decrease in specific radioactivity was morestrongly correlated with necrosis than with carcinogenicity.

1569MAY 1977




13. Kleihues, P., and Magee, P. N. Inhibition of Protein Synthesis by N-Methyl-N-nitrosourea In Vivo. Biochem. J., 136: 303-309, 1973.

14. Leon, H. A., Arrhenius, E., and Hultin, T. Effects of GlucocorticoidAdministration on the Incorporation of Labelled Amino Acids Into Protein by Cell-Free Rat-Liver Systems. Biochim. Biophys. Acta, 63: 423-433, 1962.

15. Magee, P. N. Toxic Liver Injury. Inhibition of Protein Synthesis in RatLiver by Dimethylnitrosamine In vivo. Biochem. J. , 70: 606-61 1, 1958.

16. Magee, P. N. , and Barnes, J. M. Carcinogenic Nitroso Compounds.Advan. Cancer Res., 10: 163â€”246,1967.

17. Mirvish, S. S., and Garcia, H. 1-Nitroso-5,6-dihydrouracil: Induction ofLiver Cell Carcinomas and Kidney Adenomas in the Rat. Z. Krebsforsch.,79:304-308, 1973.

18. Mirvish, S. 5., Issenberg, P., and Sornson, H. C. Air-Water and EtherWater Distribution of N-Nitroso Compounds: Implications for LaboratorySafety, Analytical Methodology, and Carcinogenicity for the Rat Esophagus, Nose, and Liver. J. NatI. Cancer Inst., 56: 1125-1129, 1976.

19. Mirvlsh, 5. S., and Sidransky, H. Labeling In vlvo of Rat Liver Treatmentwith 3-Methylcholanthrene, Phenobarbitone, Dimethylformamide, Diethylformamide, Aminoacetonitrile, Ethionine and Carbon Tetrachloride. Biochem. Pharmacol., 20: 3493-3500, 1971.

20. Mukherjee, T., Gustafsson, R. G., Afzelius, B. A., and Arrhenlus, E.Effects of Carcinogenic Amines on Amino Acid Incorporation by LiverSystems. II. A Morphological and Biochemical Study on the Effect ofDimethylnitrosamine. Cancer Res., 23: 944-953, 1963.

21. Popp, J. A., and Shinozuka, H. Inhibition of Protein Synthesis andInduction of Helical Polysomes in Rat Liver by N-Hydroxy-2-fluorenylacetamide. Chem.-BioI. Interactions, 9: 37-43, 1974.

22. Sarasin, A., and Moule, V. Inhibition of In vitro Protein Synthesis byAflatoxin B, Derivatives. Federation European Biochem. Soc. Letters,32: 347-350, 1973.

23. Spackman, D. H. Model 120B Amino Acid Analyzer Instruction Manual.Fullerton, Calif.: Beckman Instruments, Inc., 1962.

24. Stewart, B. W., and Farber, E. Strand Breakage in Rat Liver DNA and ItsRepair following Administration of Cyclic Nitrosamines. Cancer Res. .33:3209-3215, 1973.

25. Stewart, B. W., Farber, E., and Mirvish, S. S. Introduction by a HepaticCarcinogen, 1-Nitroso-5,6-dihydrouracil, of Single and Double StrandBreaks of Liver DNA with Rapid Repair. Biochem. Biophys. Res. Commun., 53: 773-779, 1973.

26. Stewart, B. W., and Magee, P. N. Metabolism and Some BiochemicalEffects of N-Nitrosomorpholine. Biochem. J., 126: 21P-22P, 1972.

27. Stewart, B. W., Swann, P. F., Holsman, J. W., and Magee, P. N. CellularInjury and Carcinogenesis. Evidence for the Alkylatlon of Rat LiverNucleic Acids In Wvo by N-Nltrosomorpholine. Z. Krebsforsch., 82: 1-12,1974.

28. Weber, G., Srlvastava, 5. K., and Singhal, R. L. Role of Enzymes inHomeostasis. VII. Early Effects of Corticosteroid Hormones on HepaticGluconeogenic Enzymes, Ribonucleic Acid Metabolism, and Amino AcidLevel. J. Biol. Chem., 240: 740-756, 1965.


ACKNOWLEDGMENTS

We thank Dr. C. Heidelberger (McArdle Laboratories, Madison, Wis.), Dr.H. Sidransky (Pathology Department, University of Florida, Tampa, Fla.), andDr. J. Hofert, Dr. J. Johnson, and Dr. A. Barak (Biochemistry Department,this campus) for valuable advice; Dr. A. Cardesa (Eppley Institute) for thehistological examinations; and H. Sornson for the amino acid analyses.

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1977;37:1564-1570. Cancer Res Cecilia Chu and Sidney S. Mirvish into Rats

C]Leucine14Radioactivity of Liver Proteins after Injection of [-Nitroso Compounds on the SpecificNEffects of Nine

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