[CANCER RESEARCH 45, 2020-2024, May 1985]

Effect of Phénobarbitaland Other Liver Monooxygenase Modifiers onDimethylnitrosamine-induced Alkylation of Rat Liver Macromolecules1

Michael Schwarz,2 Albrecht Buchmann, Helmut Klormann, Dieter Schrenk, and Werner Kunz

Institute of Biochemistry, German Cancer Research Centre, Im Neuenheimer Feld 280, Heidelberg, Federal Republic of Germany

ABSTRACT

The effects of phénobarbital(PB) and other liver monooxygen-ase modifiers on dimethylnitrosamine (DMN)-induced alkylation

of rat liver DNA and protein were investigated at different carcinogen doses. In rats given single injections of radioactively labeledDMN, pretreatment with PB (80 mg/kg body weight, administered for 5 days) resulted in a small but significant decrease inthe formation of 7-methylguanine and O6-methylguanine per moleof guanine in liver DNA associated with a decrease in the 06/N7-

methylguanine ratio. The specific radioactivity of liver protein wasalso lowered in PB-pretreated rats. The degree of PB interference

was independent of DMN dose within a carcinogen dose rangeof 0.5 u.g to 10 mg/kg body weight. In parallel experiments, theeffects of pretreatment with PB, Aroclor 1254, pregnenolone-16a-carbonitrile, butylated hydroxytoluene, /3-naphthoflavone,and ethanol on DMN-induced alkylation of liver DNA were studied

at a DMN dose of 5 //g/kg body weight. In general, pretreatmentwith these modifiers of liver monooxygenase resulted in a decrease in specific alkylation of DNA and in the ratio of 7-methylguanine to guanine. If, however, 7-methylguanine levels were

related to total liver DNA, these differences in DNA alkylationbetween controls and pretreated rats became substantiallysmaller, partially being negligible, since these inducers led to anincrease in relative liver weight with concomitant increase in thecontent of liver DNA. Thus, when expressed per total liver, nosignificant changes in the overall extent of metabolic activationof DMN were evident. These findings are not consistent with theresults of in vitro studies on DMN metabolism in microsomalsystems which favored the hypothesis that changes in the metabolism of hepatocarcinogens are responsible for the reductionof liver tumor response in animals treated simultaneously withinducers of the liver monooxygenase system and hepatocarcinogens. Our findings suggest that these effects might rather berelated to drug-mediated changes on the cellular level.

INTRODUCTION

The modifying effects of phénobarbitaland other inducers ofthe monooxygenase system on chemical carcinogenesis in liverare well known. Simultaneous treatment of rats and mice withPB3 and a liver carcinogen such as diethylnitrosamine, 2-ace-

tylaminofluorene, 4-dimethylaminoazobenzene, or aflatoxin Bi

results in a decrease in tumor response together with prolongedtumor manifestation times (15, 20, 21, 26, 31, 38). In contrast,exposure of rats to PB following termination of carcinogen

1This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 302 "Kontrollfaktoren der Tumorentstehung."

2To whom requests for reprints should be addressed.3The abbreviations used are: PB, phénobarbital;DMN, W-nitrosodimethylamine;

BHT, butylated hydroxytoluene.Received 6/8/84; revised 1/21/85; accepted 1/29/85.

treatment leads to an enhancement of preneoplastic and neo-plastic response in liver (17, 21, 31, 32). This latter effect of PBwas first interpreted in terms of its tumor-promoting activity by

Peraino ef al. (31). It is generally assumed that the inhibitoryeffects of PB and of other monooxygenase modifiers such as 3-methylcholanthrene and polychlorinated biphenyls result fromtheir metabolic interference with carcinogen activation.

In experiments with liver microsomes from untreated animals,the extent of DMN demethylation was found to correlate wellwith the amounts of alkylation products formed in DNA (16).Similar analyses with liver microsomes obtained from animalspretreated with PB or other inducing drugs revealed divergenteffects, depending on the concentration of nitrosamine. At highDMN concentrations, there was always an increase in microsomal carcinogen metabolism, DNA alkylation, and subsequentmutagenicity in vitro (2, 4, 5, 7, 9). In contrast, the metabolicactivation and mutagenicity of DMN were found to decreaseprogressively when the same preparations were incubated withdescending carcinogen concentrations (2, 4, 5, 21). This differential metabolic behavior has been attributed to the existence ofDMN-demethylases with differing substrate affinities and reac

tion velocities which operate preferentially at high or low carcinogen concentrations (5). Data from our laboratory suggest thatthe paradoxical effect could be due to preferential induction ofmicrosomal nitrosamine denitrosation (3, 21). This alternativemetabolic pathway has been shown to prevail for a variety ofnitrosamines (23, 33, 34) and is assumed to result in the detox-ication of the carcinogen.

Information concerning the relevance of these in vitro resultsto the situation prevailing in vivo is inadequate. A lowering ofDNA alkylation in liver has been reported in mice which receiveda single 10-mg/kg dose of DMN (1) as a result of pretreatmentwith PB. In the present study, we therefore analyzed DMN-induced alkylation of liver DNA and protein at different carcinogendose levels both in controls and in animals pretreated withvarious liver monooxygenase modifiers in order to assess theinitial subcellular effectiveness of the carcinogen and its modification by these compounds.

MATERIALS AND METHODS

Chemicals. ["CJDimethylnitrosamine (57 mCi/mmol) was synthesizedfrom [14C]dimethylamine (Amersham Buchler, Braunschweig, Federal

Republic of Germany) according to the data of Dutton and Heath (11).[3H]Dimethylnitrosamine (6 Ci/mmol) was also obtained from Amersham

Buchler. Unlabeled DMN was purchased from Merck-Schuchardt,Darmstadt, Federal Republic of Germany. Phénobarbital-sodium was

from Serva, Heidelberg, Federal Republic of Germany. Aroclor 1254 fromAntechneka, Karlsruhe, Federal Republic of Germany; butylated hydroxytoluene from Fluka, Buchs, Switzerland; ethanol from Baker Chemicals,Deventer, Netherlands; /3-naphthoflavone from Ega Chemie, Steinheim,Federal Republic of Germany; and pregnenolone-16-a-carbonitrile from

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MODIFICATION OF DMN ACTIVATION IN RAT LIVER

Searle Laboratories, Chicago, IL.Treatment of Rats. Male Wistar rats were obtained from the Zentral

institut fuer Versuchstierkunde (Hannover, Federal Republic of Germany),and were kept on a standard diet (Altromin pellets, Altromin; Lage, Lippe,Federal Republic of Germany) and water, ad libitum. Animals of 120 to150 g body weight at the beginning of the experiment were randomlyassigned to the experimental groups. The number of rats in the varioustreatment groups is given in the legend to Chart 1 and in Tables 1 to 3.Groups were pretreated with either phénobarbital, Aroclor, pregneno-lone-16-a-carbonitrile, butylated hydroxytoluene, /3-naphthoflavone, or

ethanol for 5 consecutive days, while controls received solvent alone.Dose levels, routes of administration, and solvents are given in Table 3.3H- or 14C-labeled DMN [in 0.9% NaCI solution (saline), 5 ml/kg body

weight] was injected i.p. 24 h after the last treatment, and the rats werekilled 4 h later. The undiluted stock solution of [14C]DMN (57 mCi/rnmol)

was used for alkylation measurements at the dose levels of 0.25 and 0.5mg/kg body weight. At the higher carcinogen doses, different amountsof unlabeled DMN were added. Measurements at a dose of 5 pg/kgbody weight were performed using [3H]DMN (6 Ci/mmol).

Analysis of DNA Alkylation and Specific Radioactivity of Protein.After the rats were decapitated, the livers were weighed, frozen in liquidnitrogen, and stored at -70°C. DNA was extracted by the phenol method

described by Margison er al. (25), and purine bases were released bytreating dried DNA samples with 0.1 N HCI for 30 min at 70°C (22).

Separation of the DNA hydrolysates by Aminex A-6 (Bio-Rad, Richmond,VA) ion-exchange chromatography and determination of radioactivity

were performed as described previously (36). Amounts of adenine andguanine were calculated by determining the absórbanos of column eluatefractions at 260 nm. Molar extinction coefficients of 7,530 for guanine(at pH 4.65) and of 13,300 (liter/mo! x cm) for adenine (at pH 7) wereassumed. No variation of the molar ratio of adenine to guanine (1.4 ±0.085) was observed for controls or pretreated animals. The amounts of7-methylguanine and O6-methylguanine as well as incorporation into

guanine were calculated from the level of radioactivity in the differentfractions and were expressed as a function of guanine values. Specificradioactivities of liver protein remaining in the phenolic phase of DNAextraction were analyzed as described previously (1).

Determination of Cytochrome P-450 and Liver DNA. Liver micro-

somes were prepared from controls and pretreated rats as describedrecently (34), and the content of cytochrome P-450 was analyzed ac

cording to the method of Omura and Sato (27). The quantity of DNA inliver was determined according to the method of Burton (8) as describedpreviously (1).

Statistical Evaluation. The difference between 2 treatment groupswas analyzed by means of the Wilcoxon signed-rank test (39). Bivariate

linear regression was calculated with the method of the least squares.

RESULTS

The major alkylation product in DNA following administrationof DMN is 7-methylguanine. The amounts of this base presentin liver DNA 4-h after a single injection of various doses of [14C]-

DMN into rats are shown in Chart 1. The formation of 7-

methylguanine was found to be directly proportional to the actualdose of carcinogen, indicating a linear increase in metabolicactivation. This result is in accordance with data from othergroups (30).

Pretreatment of rats with PB (5 injections of 80 mg/kg) resultedin a significant decrease, with a mean of 12% (P = 0.02), in the7-methylguanine/guanine ratio in rat liver DNA. This decrease

was constant throughout the entire DMN dose range of 0.25 to10 mg/kg body weight as demonstrated by the parallelism of thecorresponding regression lines which were calculated from adouble logarithmic plot for better comparison (Table 1).

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dose of DMN (mg/kg)Chart 1. Effect of phénobarbitalpretreatment on 7-methylguanine/guanine (/t),

O'-metriytguanine/guanine (B), and 0'/7-methy1guanine (C) ratios in liver DNA of

mate rats treated with various dose levels of dimethylnitrosamine. PB was administered on 5 consecutive days (80 mg/kg body weight, i.p.). [14C]DMN was injected

24 h after the last PB administration, and rats were killed 4 h later. DNA wasisolated and hydrolyzed, and base analysis was performed by Aminex A-6 ion-exchange column chromatography. Each point represents the value of one individual rat treated with (0) DMN only (n = 13) or (A) DMN and PB (n = 13).

Linear increase in 06-methylguanine levels in liver DNA was

only obtained at DMN doses higher than 2 mg/kg (Chart 16).This effect reflects 06-methylguanine-speciflc repair processes,

which are overloaded at high carcinogen dose levels (13, 18,28). O6-Methylguanine/7-methylguanine ratios therefore increase

with increasing DMN dose (Chart 1c). PB treatment significantlyreduced the 06-methylguanine/guanine ratio in rat liver by about19% (P = 0.002). Moreover, the O6-methylguanine/A/7-methyl-

guanine ratio in rat liver was found to be significantly reduced inliver DNA from animals which had been pretreated with PB (P =0.03).

Radioactivity from [14C]DMN can be incorporated into purine

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MODIFICATION OF DMN ACTIVATION IN RAT LIVER

Table 1Dependence of 7-methylguanine formation and specific radioactivity of protein in

rat liver on DMN dose: double-logarithmic regression analysis

Regres-Regres- sion Correla-

No. of sion con- coeffi- tion coef-Treatment7-Methylguanine

(¿/mol/molguanine)DMN* onlyPB + DMN*' "rats13 13slant2.392.33cient1.011.02ficient1.001.00

Specific radioactivity of protein(dpm/mg)

DMN* only 13 3.24PB + DMN*'" 13 3.16

0.970.97

1.001.00

' The ["CJDMN dose level ranged from 0.25 to 10 mg/kg body weight.6 PB pretreatment for 5 consecutive days (daily injections of 80 mg/kg body

weight).

Table 2Effecf of phénobarbitalpretreatment on "C incorporation into guanine of liver

DNA 4 h after administration of p'CJDMN

dpm/fimol guanine

Dose of DMN(mg/kg)0.25

0.51

25

10DMN

only*195.3

353.7695.3

114711721315PB

+ OWN*'*247.8

591.2741.4975.2

14191828

* Values represent the mean of estimations from 2 rats.6 PB pretreatment for 5 consecutive days (daily injections of 80 mg/kg body

weight).

bases via the Ci-pool of the Å“il after metabolic activation of the

carcinogen. Determination of the amount of labeled guanineincorporated into DNA allows a rough estimate of the precedingrate of DNA synthesis. With increasing DMN dose, saturationkinetics of incorporation was observed (Table 2). The curveobtained for these kinetics does not reflect a decrease in therate of DMN metabolism at higher carcinogen doses, since therewas no obvious deviation from linearity of 7-methylguanine formation in DNA from controls and PB-pretreated rats, even at the

highest DMN dose administered (see Chart 1a). It thereforeappears that this is due to a progressive inhibition of DNAsynthesis at DMN doses higher than about 2 mg/kg. Pretreatment of rats with PB increased incorporation values in guanineof liver DNA by about 26% (P = 0.03) throughout the entire DMN

dose range.A linear increase in the amount of radioactivity covalently

bound to cellular protein with DMN-dose was observed in liver.

PB pretreatment of rats significantly reduced specific radioactivity levels of liver protein by a mean of 17% (P = 0.006). This

effect of PB was independent of DMN dose, as demonstratedby the parallelism of the log-log regression lines calculated from

values of controls and pretreated rats (Table 1).In parallel experiments, alkylation of DNA and protein was

measured at the very low DMN dose of 5 /¿g/kgbody weight.The relevance of this carcinogen dose may be underlined by thefact that it is not far from the estimated human exposure level(12). (Human exposure to DMN has been estimated to be about1 /¿g/capita.Smokers and occupationally exposed individuals,however, may reach much higher values.) Pretreatment of ratswith 6 different monooxygenase modifiers resulted in a reductionto varying degrees of both the 7-methylguanine/guanine ratioand the specific radioactivities of protein in [3H]DMN-treated rats

(Table 3). With PB, an approximate 10% reduction in the valuesof both parameters was observed at the DMN dose of 5 /jg/kgbody weight, corresponding to that seen at the higher carcinogendoses (06-methylguanine levels could not be determined at this

low carcinogen dose).With the exception of ethanol, all compounds led to increases

in relative liver weight, DNA content, and microsomal cytochromeP-450 level of liver. The degree of these changes was dependent

on the modifier applied (Table 3). There is no evidence thatdifferent amounts of total cytochrome P-450 observed in rats

pretreated with the various compounds have any effect on thebinding of DMN metabolites to cellular macromolecules in liverwhen measured 4 h after carcinogen administration. This isclearly demonstrated by the comparison of data obtained withAroclor and BHT. Pretreatment of rats with Aroclor results in180% increase of microsomal cytochrome P-450 levels over

controls, in contrast to BHT where only a 10% increase isobtained. However, with both compounds, the 7-methylguanine/

guanine ratios and the specific radioactivity levels of protein in[3H]DMN-treated rats are similarly reduced by about 30%. The

decrease in 7-methylguanine/guanine ratios and specific radio

activity levels of liver protein observed in rats pretreated with thevarious modulators (Table 3) does not necessarily reflectchanges in the extent of carcinogen activation, since drug-induced alterations in pool size are disregarded. Taking themodifier-mediated increases in relative liver weight and DNA

content of liver into account, the differences between controlsand pretreated rats in the overall alkyation rate of cellular macromolecules in liver are significantly smaller, becoming negligiblein some cases (Table 4).

DISCUSSION

The present investigation was undertaken to study the influences of PB and other modifiers of the liver monooxygenasesystem on the initial subcellular effects of DMN in rat liver underin vivo administration. Determination of DMN-induced alkylationof liver DNA provides evidence for a decrease in the 7-methyl-and 06-methylguanine/guanine ratios in inducer pretreated rats.

With the model compound PB, a lowering of these ratios isobserved at various DMN dose levels and the effect is independent of carcinogen dose. There is no indication of any increase inPB effectiveness even at the extremely low DMN dose of 5 ^g/kg body weight. An identical effect was obtained with PB withregard to its influence on the specific radioactivity levels of liverprotein in rats treated with radioactively labeled DMN. A smallbut significant reduction of both the 7-methylguanine/guanine

ratio and the specific radioactivity levels of protein was alsoobserved in kidney of PB pretreated animals (data not shown).The dose levels of DMN used in the present investigation(0.00007 to 0.14 mmol/kg body weight) might not be equivalentto the carcinogen concentrations commonly used in microsomalincubations in vitro (0.1 to 50 mw in the assay). However, thefact that PB interference appears to be entirely independent ofDMN dose in vivo sharply contrasts the effects of this cytochrome P-450 inducer seen in vitro (2, 4, 5, 7, 9).

PB causes an increase of radioactivity in liver DNA due to theuse of purine bases labeled via d-pool. This effect could beexplained by enhanced de novo synthesis of liver DNA in PB-treated rats, leading to an augmentation in the total amount of

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MODIFICATION OF DMN ACTIVATION IN RAT LIVER

TabteSEffect of different monooxygenasemodifiers on organ growth, cytochrome P-450 content, and fHJUMN activation in rat liver

Inducer*NonePBAro'BHTEth/3-NFPCNDose(mg/kg)805050020005050Route

ofadminis

trationP.O.P.O.P.O.P.O.P.O.P.O.P.O.SolventSaline

or oil orwaterSalineOilOilWaterOilWaterNo.

ofrats9333333Relativeliverwt(g/kgbody

wt)44.0

±5.6"53.6

±4.969.4±4.467.3±5.847.6±3.248.3

±1.954.2±5.4DNAcontent"(mg/liver)23.6

±1.626.1

±0.524.8±2.235.2±3.624.3±3.023.9±1.126.6±1.8Cyto

chromeP-450Ccontent(nmol/mg)0.651.451.840.690.640.820.977-Methyl-guanine/guanineGimol/mol)0.95

±0.090.83

±0.070.67±0.090.62±0.020.86±0.030.81±0.070.73±0.03Specific

radioactivityofprotein(dpm/mg)905

±73.1769

±48.7589±58.2578±32.7820±129738±66.2811±30.7

8 Inducerswere given on 5 consecutive days." Standardized for a rat of 200-g body weight.c Mean of duplicate estimations from 3 pooled livers." [3H]DMNdosewas5 Mg/kgbodyweight.' Mean ±SD.'Aro, Arador 1254; Eth, ethanol; 0-NF, (j-naphthoflavone;PCN, pregnenolone-16a-carbonitrile.

Table 4Changesin the 7-methylguaninecontent and radioactivity of protein in liver of[*H]DMN-treated rats pretreated with different cytochrome P-450 modifiers

7-Methylguanine in liverDMA" 3HRadioactivity in liver protein8

InducerNone

PBAro"

BHTEth/3-NFPCNpmd/liver"13.58

13.1310.0713.2312.6611.7311.77%

of control(100

96.774.297.493.386.486.7ipm/liver*,274,000

,319,000,308,000,245,000,249,000,141,000,407,000%

ofcontrol100

103.5102.797.798.089.6

110.4* Standardizedfor a rat of 200-g body weight."Calculated from the values presented in Table 3 by assuming a guanine

content of 0.606 ^mol/mg DNA.c Calculatedfrom the valuespresented in Table 3 by assuminga protein content

of 160 mg/g liver." For definition of abbreviations,see Table 3.

DNA in this organ (Ref. 19; see also Table 3). It should also beconsidered that a PB-mediated stimulation of DNA repair might

contribute to the intensified incorporation of labeled purines. Infavor of this is the finding that 06-/A/7-methylguanine ratios are

significantly lowered in liver DNA of PB pretreated animals,indicating an increase in 06-methylguanine-specific repair activity

(13,18,29).The ratios of 7-methylguanine/guanine in liver DNA of rats

exposed to a single DMN dose of 5 ^g/kg body weight arelowered to varying extents by pretreatment with all of the monooxygenase modifiers tested (Table 3). These compounds exhibit differential effects on hepatocarcinogenesis when administered simultaneously with liver carcinogens. In general, simultaneous treatment with hepatocarcinogens and inducers of themonooxygenase leads to a decrease in liver tumor response.Using different carcingens, such interference has been reportedfor PB (15, 20,21, 26,31,38), BHT (10, 37), and polychlorinatedbiphenyls (24). A differential effect has been observed withrespect to p'-naphthoflavone and pregnenolone-16a-carbonitrile,

whereby the former enhances while the latter inhibits DMN-

induced hepatocarcinogenesis in rat liver (6). Ethanol may eitherinhibit or enhance hepatocarcinogenesis, depending on the alcohol dose level and treatment schedule used (14, 35). Thus,the effects of the various xenobiotics on DMN-induced DNA

alkylation do not seem to correlate with the changes in tumorresponse induced by these compounds, nor is there any obviouscorrelation with the total amount of cytochrome P-450 deter

mined in liver microsomal fractions from rats of different treatment groups.

Treatment of rats with the compounds listed in Table 3 resultsin liver enlargement with concomitant increase in all cellularmacromolecules, including DNA. Assuming identical degrees ofDNA alkylation in livers of controls and rats pretreated with thevarious inducers, this increase in total liver DNA should necessarily lead to a decrease in the levels of specific alkylationproducts when related to moles of guanine or unit liver DNA.Similar considerations apply for liver protein alkylation. Takingthis change in target size into account, no significant differencesbetween PB-pretreated animals and controls are evident in theoverall extent of DNA alkylation. Comparison of inducer-media-

ted increase in liver DNA content and the concomitant decreasein the ratios of 7-methylguanine/guanine indicates that the same

may be valid to a more or less degree for the other xenobioticcompounds investigated. These results led to some generalconsiderations. Since the extent of DMN-mediated DNA alkyla

tion, when expressed per total liver, was found to be only slightlyaffected by pretreatment with different monooxygenase modifiers, significant changes in either activation or inactivation pathways of this carcinogen in vivo appear to be unlikely. Thus,preferential induction of cytochrome P-450 species leading to

detoxification of this nitrosamine by metabolic denitrosation, asobserved in studies on DMN metabolism in microsomal in vitrosystems (3, 23), is only of minor importance under in vivoconditions. This discrepancy may be explained by a differentialintralobular distribution of the particular drug-induced cytochrome P-450 species, as described by Wolf ef a/. (40). Thus,

due to the direction of blood flow and the related substrategradient across the liver lobules, the differentially located mon-

ooxygenases are exposed to unequal substrate concentrationsin vivo, while in microsomal systems in vitro, the metabolicconditions are completely uniform.

The decrease in liver tumor response observed in animalstreated simultaneously with hepatocarcinogens and inducers ofliver enlargement, such as PB, might therefore be related tochanges on the cellular level rather than to alterations in carcin-

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MODIFICATION OF DMN ACTIVATION IN RAT LIVER

ogen metabolism. The slight increase in 06-methylguanine-spe-

cific DNA repair activity observed in liver of PB-pretreated rats

may play some role but cannot by itself be taken as an explanation.

The present findings suggest that the significant decrease inspecific alkylation density of the cellular genome after pretreatment with PB and other liver-enlarging drugs might be of critical

importance. Such events should lead to a decrease in celltransformation risk which could be further intensified by theprotective effect of the drug-mediated increase in cell ploidy

status.

ACKNOWLEDGMENTS

The authors thank J. Hummel for excellent technical assistance, J. Fabermannfor textual corrections, K. Helm for typing the manuscript.

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CANCER RESEARCH VOL. 45 MAY 1985

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