differential effect of chronic ethanol consumption on the … · [cancer research 41, 2849-2854,...

[CANCER RESEARCH 41, 2849-2854, July 1981]0008-5472/81 70041-OOOOS02.00

Differential Effect of Chronic Ethanol Consumption on the Carcinogenicityof /V-Nitrosopyrrolidine and A/'-Nitrosonornicotine in Male SyrianGolden Hamsters1

G. David McCoy,2 Stephen S. Hecht, Shoichi Katayama, and Ernst L. Wynder

Way/or Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York 10595

ABSTRACT

The effect of chronic ethanol consumption on the carcino-genicity of A/-nitrosopyrrolidine (NPYR) and W-nitrosonornic-

otine (NNN) in male Syrian golden hamsters has been investigated. Groups of hamsters were maintained on either ethanol-

containing or control liquid diets for 4 weeks prior to and duringcarcinogen treatment. NPYR or NNN was administered toethanol-consuming or control hamsters by i.p. injection over a25-week period in total doses of either 1 or 2 mmol. In the

group treated with 1 mmol of NPYR and maintained on a controldiet, we observed 1 of 20 hamsters with a nasal cavity tumorand 4 of 20 hamsters with trachÃ©altumors. In the group treatedwith 1 mmol of NPYR and maintained on the ethanol-containingdiet, we observed 8 of 18 hamsters with nasal cavity tumorsand 9 of 18 hamsters with trachÃ©altumors. The correspondingresults in hamsters given 2 mmol of NPYR were: nasal cavitytumors, 14 of 21 (control) and 16 of 17 (ethanol); trachÃ©altumors, 8 of 21 (control) and 11 of 17 (ethanol). These resultsdemonstrate that the carcinogenicity of NPYR is enhanced inethanol-treated Syrian golden hamsters, particularly at the

lower dose. In the groups treated with 1 mmol of NNN and acontrol diet, we observed 1 of 21 hamsters with a nasal cavitytumor and 4 of 21 with trachÃ©altumors. In the correspondingethanol-treated groups, we observed 1 of 17 hamsters with a

nasal cavity tumor and 5 of 17 with trachÃ©al tumors. In thehamsters given 2 mmol of NNN, the results were: nasal cavitytumors, 5 of 21 (control) and 4 of 21 (ethanol); trachÃ©altumors,9 of 21 (control) and 7 of 21 (ethanol). Thus, the carcinogenicityof NNN in the Syrian golden hamster was not affected byethanol treatment. These results suggest that the metabolicactivation of NPYR, but not that of NNN, may be enhanced bychronic ethanol consumption in the Syrian golden hamster.

INTRODUCTION

There is now an extensive body of epidemiological evidenceindicating that the combination of both chronic alcohol andtobacco consumption are major risk factors for cancers of thehead and neck. Positive association of these risk factors forcancers of the oral cavity, esophagus, and larynx have beennoted by numerous investigators (see Ref. 26 for a recentreview). However, the possible role that each plays in theetiology of the disease has remained elusive. In order to studythe mechanism(s) by which alcohol and tobacco might increasethe risk for cancer, our working hypothesis assumes that

1This work was supported by National Cancer Institute Grants CA 12376 and

CA 23901.2 To whom requests for reprints should be addressed.

Received December 15, 1980; accepted March 24, 1981.

tobacco and/or tobacco smoke are the source of the carcinogenic stimuli and that ethanol facilitates the activation oftobacco-associated carcinogens.

Although tobacco and, more specifically, tobacco smokecontain numerous potential carcinogens and cocarcinogens,we have focused our attention on the structurally related cyclicnitrosamines, NPYR3 and NNN (Chart 1). NPYR is found in the

gas phase of mainstream and sidestream tobacco smoke andis also present in cooked bacon and other processed meats(4, 15). NNN is one of the major tobacco-specific carcinogens,

occurring in tobacco and the particulate phase of tobaccosmoke (16, 18).

a-Hydroxylation as a likely activation mechanism for nitros

amines was first suggested by Druckrey ef al. (9). The role ofo-hydroxylation in the microsomal activation of cyclic nitros

amines, such as NPYR and NNN, has been supported bystructure-activity studies in which decreased carcinogenicity

was observed when the a positions are either deuterated orsubstituted with methyl groups (20, 21) and by the high muta-genicity without activation of a-acetoxy cyclic nitrosamines and

related model compounds (3, 6). Our previous studies haveshown that chronic (28-day) ethanol consumption by Syrian

golden hamsters causes a marked increase in liver microsomala-hydroxylation of NPYR and NNN as well as in the conversion

of NPYR to a mutagen by postmicrosomal supernatants (22,24).

Since the in vitro data suggested that ethanol consumptionby hamsters could increase the metabolic activation of NPYRand NNN, we studied the effect of chronic ethanol consumptionby hamsters on the carcinogenicity of these 2 tobacco-asso

ciated carcinogens.

MATERIALS AND METHODS

NPYR was obtained from Aldrich Chemical Company, Milwaukee, Wis., and NNN was synthesized as described previously (19). NIH-07 chow was from Ziegler Brothers, Gordeners,Pa.; liquid diets were obtained from Bio-Serv, Frenchtown, N.

J.; ethanol (95%) was from Publicker Chemical Corporation,Cranford, N.J. and IMC Chemical Group, Newark, N. J.

Two hundred ten outbred male Syrian golden hamsters (3weeks old) from Bio-Research (Telaco), Cambridge, Mass.,were housed 3/cage and allowed free access to NIH-07 laboratory chow and water. Cages were solid-bottomed polycarbonate and contained hardwood chip bedding. The laboratorieswere maintained at 21 Â±1Â° (S.D.) and 50 Â± 10% relative

humidity, respectively. Animals were kept on light-dark cycles

3 The abbreviations used are: NPYR, W-nitrosopyrrolidine; NNN, W-nitroso-

nornicotine.

JULY 1981 2849

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G. D. McCoy et al.

JÃ®N-NITROSOPYRROLIDINE

(NPYR)

N =0

N'-NITROSONORNICOTINE

(NNN)

Chart 1. Structures of NPYR and NNN. Metabolic activation of these 2 nitros-amines is believed to occur by hydroxylation of positions 2 or 5 of NPYR andposition 2' or 5' of NNN.

of 12-hr duration starting at 7 a.m. At 8 weeks of age, all

animals were placed on control liquid diet (Diet 71 1 supplemented with yS-cellulose, 4.14 g/liter). At 9 weeks of age,animals were randomized by weight and one-half (1 05 animals)

was continued on control liquid diet. The remaining animalswere placed on an ethanol-containing liquid diet. Both control

and ethanol diets were as originally described by DeCarli andLieber (7). In these diets, ethanol was added as an isocaloricreplacement for carbohydrate such that 35% of the total caloriccontent was as ethanol. The percentage of total calories asprotein, fat, and carbohydrate was 18, 35, and 47%, respectively. The ethanol concentration in the diet was 6% (w/v). At13 weeks of age, each group was again randomized by weightand further subdivided into 5 treatment groups of 21 animalseach.

Animals received 0.5-ml i.p. injections 3 times weekly for 25

weeks of: 1.33 mg NPYR (total dose, 1 mmol) (Groups 1 and2); 2.67 mg NPYR (total dose, 2 mmol) (Groups 3 and 4); 2.37mg NNN (total dose, 1 mmol) (Groups 5 and 6); 4.75 mg NNN(total dose, 2 mmol) (Groups 7 and 8); and 0.9% NaCI solutionvehicle (Groups 9 and 10).

Odd-numbered groups received control liquid diets, andeven-numbered groups received ethanol-containing liquid

diets. Diet consumption and weight gain were measured onceweekly, and consumption by animals on control diet was variedto equal the consumption of the animals on the ethanol-con

taining diet.After 16 weeks of injections, a decrease in the weight of all

groups on liquid diets was observed. Since the weight lossappeared to be diet related and enhanced by both ethanol andcarcinogen, liquid diet consumption and carcinogen administration were suspended for 1 month. All groups were givenNIH-07 laboratory chow and water ad libitum. The weights of

all groups increased promptly, and the weight differentialamong groups was abolished. Control and ethanol liquid dietswere reinstated, and the carcinogen injection schedule wasresumed 1 week later and continued without further interruption. At the end of the 25-week carcinogen administrationperiod, all animals were returned to NIH-07 laboratory chow

and water. Animals were sacrificed when moribund. SurvivingNPYR animals (Groups 1 to 4) were sacrificed 15 months afterthe beginning of carcinogen administration, and surviving NNN-

treated animals and controls (Groups 5 to 10) were sacrificed18 months after the beginning of carcinogen administration. All

grossly observable lesions were submitted for histological evaluation. Longitudinal sections of all tracheas were also submitted for histopathological examination. Heads were decalcifiedfor 48 hr, cut in at least 3 frontal sections, embedded inparaffin, and stained with hematoxylin and eosin. The Fisherexact probability test was used for statistical analysis.

RESULTS

The mortality rates of control and ethanol-consuming ham

sters treated with NPYR and NNN are shown in Charts 2 and3, respectively. Ethanol-consuming animals receiving either 1

or 2 mmol of NPYR exhibited increased mortality comparedwith NPYR-treated animals on control liquid diet. In contrast,little difference was seen in the mortality of control and ethanol-

consuming animals treated with NNN.The effect of chronic ethanol consumption on the tumor yield

in NPYR- and NNN-treated animals is presented in Table 1.

With both carcinogens, tumors were observed most frequentlyin the nasal cavity and trachea. Ethanol-consuming animals

receiving NPYR had a higher incidence of both nasal cavityand trachÃ©al tumors compared to NPYR-treated animals on

control diet. This difference was greater in animals receiving 1mmol NPYR than for those receiving 2 mmol NPYR. Seventy% of the nasal cavity tumors were observed in animals sacrificed prior to the termination of the study. The first nasal cavitytumor was observed in an animal sacrificed in ExperimentalMonth 10, but the majority of nasal cavity tumors was observedin animals sacrificed in Experimental Months 14 and 15. Thefirst trachÃ©altumor was also seen in Experimental Month 10,and approximately one-half of the total trachÃ©al tumors was

observed in animals sacrificed prior to the termination of theexperiment.

The NNN-treated animals developed more trachÃ©althan did

nasal cavity tumors, and no increased tumor incidence wasobserved in the ethanol-consuming groups. In these animals,

approximately 60% of the total nasal cavity tumors and 15% ofthe trachÃ©altumors were found prior to the termination of theexperiment. The first nasal cavity and trachÃ©al tumors wereobserved in animals sacrificed in Experimental Months 14 and15, respectively.

Almost one-half of the animals treated with either NPYR or

NNN had multiple trachÃ©altumors, but no difference due to

S20

NPYR [ Im mol ) t

NPYR( 2 m mol } â€¢¿�'9 D ETHANOL + NPYR ( I m mol } O

0 ETHANOL+ NPYR l2mmol)O

24 68 10 12 14 16

EXPERIMENTAL MONTHS

Chart 2. Mortality of control and ethanol-consuming hamsters treated withNPYR. Carcinogen administration began in Experimental Month 1 and wasterminated in Experimental Month 8. A pause in carcinogen administrationoccurred in Experimental Month 5.

2850 CANCER RESEARCH VOL. 41



Effect of Ethanol on NPYR and NNN Carcinogenesis

120-

Ã•

10-

vo---o--o--o a..

2 4 6 8 10 12 14 16 18 20EXPERIMENTALMONTHS

Chart 3. Mortality of control and ethanol-consuming hamsters treated withNNN. Carcinogen treatment period was described in legend of Chart 2.

ethanol consumption was observed. In a few animals, thetrachÃ©alneoplasms almost totally obstructed the trachÃ©al lumen. Histological examination showed squamous papillomas,but no malignant transformation was seen. Neoplastic livernodules were observed only in animals treated with carcinogenand more frequently in NPYR-treated animals. The morphology

of these lesions was similar to those described by Stewart efal. (32) for rats.

Benign and malignant tumors of the adrenal gland are amongthe more common spontaneous tumors found in Syrian goldenhamsters (see Ref. 33 for a review). In this study, adrenaltumors were found in both carcinogen-treated and control

groups. The incidence of adrenal tumors generally was higherin Groups 5 through 10, which were sacrificed 18 months afterthe beginning of carcinogen treatment. Fewer adrenal tumorswere seen in Groups 1 through 4, which were sacrificed 15months after the beginning of carcinogen administration. Therewere marked differences in the number of animals in Groups 1to 4 which survived until termination (Chart 2). It is possiblethat the suppression of adrenal tumors (Groups 2 versus 1) isa reflection of animal age at the time of sacrifice.

A detailed examination of the nasal cavity lesions is presented in Table 2. Areas of focal epithelial hyperplasia wereobserved, with the respiratory epithelia being the major site.However, all of the tumors appeared to arise from the olfactorymucosa (Fig. 1) and ranged from small localized tumors, withina single turbinate, to large invasive neoplasms (Fig. 2). In theadvanced stages, the tumors almost completely filled the nasalcavity, destroying bone and infiltrating other tissue (Fig. 3).More than one-half of the tumors in the NPYR-treated animals

were malignant invading bone, brain, and surrounding tissuesand giving mÃ©tastases to the cervical lymph nodes. In somecases, tumors appeared to be of multicentric origin. When thetumor was still localized, it was easy to identify its origin in theolfactory mucosa. However, the origin of the more advancedtumors was difficult to determine due to the destruction ofnormal adjacent structures. Nevertheless, since the tumor morphology of the advanced tumors was nearly identical to that ofthe more localized lesions, a reasonable presumption of theolfactory origin could be made.

Several tumors displayed a glandular arrangement of relatively small cells (Fig. 4); however, a medullary type image ofrelatively large tumor cells was found more frequently (Fig. 5).In a few incidences, compact growth in cords of tumor cellswith hyperchromatic nuclei was observed (Fig. 6). Some tumors

displayed intermingling of different cellular components andpatterns. Tumors with a similar morphology were found inhamsters treated with A/'-nitrosopiperidine by Hilfrich ef al.

(17). It was not possible to further classify these tumors asolfactory neuroepitheliomas, olfactory neurocytomas, or olfactory neuroblastomas in the absence of any demonstrable Bod-ian's staining for neurofibers and Grimelius staining for cyto-

plasmic argyrophilic granules.

DISCUSSION

The results presented here indicate that chronic ethanolconsumption can increase the incidence of nasal cavity andtrachÃ©altumors in hamsters exposed to NPYR. The fact thatthe enhancing effect of ethanol is more evident at the lowerlevel of NPYR suggests that ethanol consumption in somemanner increases the susceptibility of the nasal and trachÃ©almucosa to NPYR. Our previous studies have shown that ethanolconsumption increased liver microsomal metabolism and mu-

tagenicity of NPYR (24). The present observations could be theresult of similar changes occurring in the target tissues.

Ethanol by itself does not increase tumor incidence orchange the organotropism of either NPYR or NNN, indicatingthat in this system ethanol is not a carcinogen and that theincreased tumor incidence in the target tissues is not causedby decreased incidence at some other site. The observationthat chronic ethanol consumption increased the carcinogenic-ity of NPYR but had no effect with NNN suggests that themechanism is cocarcinogenic rather than promotional, sincethe latter mechanism should have resulted in the enhancedcarcinogenicity of both nitrosamines.

In view of the structural similarity of NPYR and NNN (Chart1), the lack of an effect of ethanol consumption on the carcinogenicity of NNN indicates that enhanced metabolism per seis not sufficient to explain increased carcinogenicity. Previousstudies have shown that like NPYR, hamster liver microsomalmetabolism of NNN is increased by ethanol consumption (22).However, unlike NPYR, the 2 sites of a-hydroxylation of NNNare not equivalent. Although the major site of in vitro hydrox-ylation (5') is the site which is increased by ethanol, it ispossible that 2'-hydroxylation is involved in carcinogenesis andthat 5'-hydroxylation is a detoxification pathway. There arespecies differences in the response of 5'- and 2'-hydroxylationof NNN to induction. In the hamster liver, it is 5'-hydroxylationwhich is inducible, while in the rat liver only 2'-hydroxylation isinducible (23). If 2'-hydroxylation is the pathway involved in

metabolic activation, the rat may be a more suitable model inwhich to examine the influences of chronic ethanol consumption on the carcinogenicity of NNN.

The high incidence of nasal cavity and trachÃ©al tumors aswell as the increased mortality of NPYR-treated hamstersshows that NPYR is a more potent carcinogen than NNN inhamsters. Except for the brief report by Dontenwill (8), thecarcinogenicity of NPYR in hamsters has not been previouslyreported. Administration (p.o.) of NPYR has been shown tocause hepatocellular carcinomas in several strains of rats (9,14, 20). The effects of other routes of carcinogen administration have not been examined. The data presented here indicatethat the trachÃ©al and nasal cavity mucosa are major targetsites, while only early neoplastic changes were found in theliver. Whether these differences are due to species suscepti-

JULY 1981 2851



G. D. McCoy et al.

Table 1

Influence of chronic alcohol consumption on the carcinogenicity of NPYR and NNN in male Syrian golden hamsters

ExperimentalconditionsCarcinogenGroup12345e78910TypeNPYRNPYRNPYRNPYRNNNNNNNNNNNNNoneNoneDose(mmol)1122112200DietControlEthanolControlEthanolControlEthanolControlEthanolControlEthanolEffectiveanimals320182117211721212119AnimalswithtumorsUpper

respiratorytractNasalcavity18e1416Â°115400Trachea49Â°811459700Bothsites14710001100Eithersite413C1517e56131000Liverneoplas-

tic nodules3425002200Adrenal81348111171110Othertumors1600^d6"5'4"2"5'e'

Animals that survived the carcinogen administration period.0 Pancreatic islet cell tumor.c Significantly different (p s 0.05) from control." Parathyroid adenoma.e Forestomach papilloma, follicular adenoma of the thyroid, lymphoma of the mesenteric lymph node, pancreatic

islet cell tumor, parathyroid adenoma, and kidney papillary adenoma.Pancreatic islet cell tumor (3 animals), papillary adenoma of the thyroid (2 animals).

9 Pancreatic islet cell tumor, parathyroid adenoma, papillary adenoma of the thyroid, adenosquamous cell carcinoma

of the lung." Pancreatic islet cell tumor, lung adenoma.' Pancreatic islet cell tumor, lymphoma, hemangioma of the spleen, forestomach papilloma, s.c. sarcoma.' Lymphoma, hemangioendothelioma of the liver, follicular adenoma of the thyroid, parathyroid adenoma, pancreatic

acinar cell adenoma, pancreatic islet cell tumor.

Table 2

Detail of neoplastic changes in the nasal cavity mucosa

Experimental conditionsOlfactory epithelial

tumorsFocal epithelial

hyperplasia

Carcinogen

Group12345678910TypeNPYRNPYRNPYRNPYRNNNNNNNNNNNNNoneNoneDose(mmol)1122112200DietControlEthanolControlEthanolControlEthanolControlEthanolControlEthanolEffectiveanimal820182117211721212119Total181416115400Invasive0e89102100uiiac-

torymucosa0221000200nespi-

ratorymucosa047e41se00

Animals that survived the carcinogen administration period.

bility, administration routes, or total dose administered cannotbe determined in the absence of strictly comparable studies inthe 2 species. The tumor incidence in hamsters treated withNNN at the 2-mmol dose level reported here is similar to the

data presented by Hilfrich ef al. (17), who administered similarquantities s.c.

Beginning with the investigations by Protzel ef al. (27), several studies have shown that ethanol consumption can increasethe tumor incidence in animals exposed to benzo(a)pyrene (5,31 ) or 7,12-dimethylbenz(a)anthracene (10-12). The role that

ethanol plays in increased tumor incidence in polynucleararomatic hydrocarbon-treated animals is not known. However, the enhanced 3-hydroxylation and mutagenicity of

benzo(a)pyrene by subcellular fractions of small intestinal mucosa from ethanol-consuming rats indicates that ethanol can

increase microsomal metabolism in at least one extra hepatictissue (30). Two studies on the effect of alcohol on the carcinogenicity of nitrosamines have been reported. In 1965, Gibel(13) presented evidence that simultaneous administration of

ethanol and diethylnitrosamine by gastric intubation increasedthe incidence of esophageal tumors in rats. Both control andethanol-treated rats had 100% incidence of liver tumors. Ani

mals treated with dinitrosopiperazine showed no increase dueto ethanol. More recently, SchmÃ¤hl (28) has shown that thecarcinogenicity of A/-nitrosomethylphenylamine administeredeither p.o. or s.c. to male and female Sprague-Dawley rats wasnot increased by ethanol consumption. In none of these studieswas there any indication of an increased tumor incidence duesolely to ethanol consumption in the absence of carcinogen. Arecent study by Schrauzer et al. (29) has shown, however, thatchronic ethanol consumption can decrease the latent periodand increase the tumor volume of spontaneously occurringmammary adenocarcinoma in female C3H/St mice.

The hamster, in contrast to most commonly used laboratoryspecies, will preferentially consume up to 90% of its total liquidfrom ethanol-water mixtures (1, 2). However, we have observed

(25) that as caloric intake from ethanol increased, the caloricintake from diet decreased, making this method of ethanol

2852 CANCER RESEARCH VOL. 41



Effect of Ethanol on NPYR and NNN Carcinogenesis

delivery impractical for comparative carcinogenicity studies.The use of liquid diet preparations in which ethanol isocalori-

cally replaces carbohydrate (7) prevents this disparity in nutrient intake. The effect of consumption of liquid diets per se onthe carcinogenicity of NPYR and NNN cannot be determined inthe absence of carcinogen-treated chow-fed animals. However,

it should be noted that, relative to the usual laboratory chowformulation, these liquid diets have a high fat content.

The data presented here as well as the previous studies onthe effect of ethanol on the carcinogenicity of nitrosaminesindicate the importance of both the dose and the carcinogenused, since ethanol administration clearly does not increasethe carcinogenicity of all nitrosamines. Procedures in whichcarcinogen administration is decreased with respect to bothtime and number of injections need to be devised so that thecritical time of ethanol consumption can be defined. Furtherstudies are needed to establish whether enhanced metabolicactivation is the reason for increased carcinogenicity of NPYRin hamsters. Nevertheless, insight into this aspect as well asother possible mechanisms by which ethanol consumption mayincrease the risk for cancer can be studied in this modelsystem.

ACKNOWLEDGMENTS

The authors would like to thank Maria Nicolais for her excellent technicalassistance.

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7. DeCarli, L. M., and Lieber, C. S. Fatty liver in the rat after prolonged intakeof ethanol with a nutritionally adequate new liquid diet. J. Nutr., 91: 331-336, 1976.

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Fig. 1. Early tumor arising in the nasoturbinate in a hamster treated with 2 mmol NPYR on ethanol diet. H & E, x 6.

Fig. 2. Fully developed nasal cavity tumor in a hamster treated with 2 mmol NPYR on ethanol diet. The cavity is completely filled by the tumor. H & E, x 6.

Fig. 3. Advanced nasal cavity tumor in a hamster treated with 2 mmol NPYR on control diet. Note invasion and overgrowth of tumor. H & E, x 6.

Fig. 4. One of the representative cellular components and structural pattern of nasal cavity tumors in hamsters. Note glandular pattern of relatively small tumorcells. A hamster treated with 1 mmol NPYR on ethanol diet. H & E, x 100.

Fig. 5. Most frequent nasal cavity tumor morphology in this study. Note the presence of relatively large anaplastic tumor cells. Taken from the tumor shown in Fig.3. H & E, x 100.

Fig. 6. Less frequently encountered nasal cavity tumor morphology in the present study. Note the streaming-like pattern of hyperchromatic small tumor cells in ahamster treated with 2 mmol NPYR on control diet. H & E, x 100.

JULY 1981 2853



G. D. McCoy ef al.

r

Sv .;

1981;41:2849-2854. Cancer Res G. David McCoy, Stephen S. Hecht, Shoichi Katayama, et al. -Nitrosonornicotine in Male Syrian Golden Hamsters

′N-Nitrosopyrrolidine and NCarcinogenicity of Differential Effect of Chronic Ethanol Consumption on the

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differential effect of chronic ethanol consumption on the … · [cancer research 41, 2849-2854,...

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