Effects of cigarette smoke and disulfiram on tumorigenicity and clastogenicity of ethyl carbamate in mice

ELSEVIER

CANCER LETTERS

Cancer Letters 94 (1995) 91-95

Effects of cigarette smoke and disulfiram on tumorigenicity clastogenicity of ethyl carbamate in mice

and

Roumen M. Balansky National

Centre

of Oncology,

Sofa-1

7.56, Bulgaria

Received 10 January 1995; revision received 9 March 1995; accepted 27 April 1995

Abstract

Exposure of male Balb/C mice to mainstream cigarette smoke for 4 months, starting 10 or 30 days before the administration of ethyl carbamate (0.3% in drinking water for 3 weeks), resulted in an up to 57.6% (P < 0.05) decrease of lung adenoma multiplicity. However, the number of ethyl carbamate-induced lung tumors was not significantly affected by exposure to cigarette smoke when ethyl carbamate was injected i.p. in single doses of 0.5 or 1.0 g/kg, irrespective of the different treatment schedules used, i.e. (a) 10 days before and 4 days after the ethyl carbamate injection; (b) throughout the experiment starting 10 days before the ethyl carbamate injection, and (c) until the end of the experiment, starting 30 days after the ethyl carbamate injection. Disulfiram (500 mg/kg), given by gavage 24 h and 1 h before the ethyl carbamate injection, decreased by 88.5% (PC 0.001) the multiplicity of lung adenomas but had no effect on tumorigenesis when administered after the carcinogen injection. Proadifen (SKF-525 A, 50 mg/kg) injected i.p. 24 h and I h before and 24 h and 48 h after the injection with ethyl carbamate tended to decrease the multiplicity of lung adenomas, but not to a significant extent. Furthermore, disulfiram given 24 h and 1 h before the i.p. administration of ethyl carbamate completely prevented its clastogenicity in mouse bone marrow. On the other hand, cigarette smoke, which was per se a weak clastogen in bone marrow erythroblasts, synergistically potentiated the clastogenic response to ethyl carbamate in a more than additive fashion. Keywords:

Ethyl carbamate (urethane); Cigarette smoke; Disulfiram;

1. Introduction Cigarette smoke (CS) has been recognized as a major risk factor involved in human lung carcinogenesis.It contains more than 3800 identified chemi-

cal compounds, many of them being mutagensor/and co-mutagens as well as potent tumor initiators or/and promoters [l]. The role of CS in carcinogenesis is

rather complex, since it may trigger or affect different stages of neoplastic transformation and its carcinogenic

activity

could be considered

as a result of

Proadifen; Mice; Lung tumors; Micronuclei

the combined effects on target cells of different CS constituents [I]. Although the epidemiological evidence for CS carcinogenicity in humans is quite convincing, the spectrum and peculiarities of the possible tumor responseto CS in experimental animals, including rodents have not been fully clarified [l]. In fact, the data from 6 chronic

experiments

carried

out in mice

exposed to CS throughout their life did not reveal significant differences in tumor frequency compared to untreated controls. Furthermore, CS had no strik-

0304-3835/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3835(95)03829-L

mg effects on carcinogenesis induced by benzofalpyrene, 7,12-dimethylbenz[a]anthracene or 4-(methyln$rosamino)- 1-(3-pyridyl)- 1-butanone in rats and hamsters [l]. On the other hand, the few data availcable indicate that CS can promote respiratory tract carcinogenesis in hamsters treated with N-diethylnilrosamine ]2] or to potentiate the lung carcinogenesis induced in mice by benzo[a]pyrene or ethyl ::nrbamate (EC. urethane) [3,4]. In any case, the combined exposure of experimental animals to CS plus known carcinogen(s) has not received the .attenrion that it merits as an approach to providing valu;rble information concerning the effects of CS on different stages of carcinogen&is as well as the putative mechanisms of CS involvement in this process. The present investigation was designed in order to study the possible influence of CS on initiation and/or promotion phases of lung carcinogenesis induced by EC in mice as well as on the ciastogenic ,ictivity of’ this compound in mouse bone marrow. Since this carcinogen requires a metabolic activation io exert its genotoxicity, parallel experiments were carried out in order to study the-effects of two known inhibitors otmetabolism, namely disulfiram @SF) and proadifen (SKF-525 A) on EC tumorigenicity
and methods

2 i, Animals

A total of 450 male and 30 female Balb/C mice [Animal Laboratory, Nationai Center of Oncology, Sof’ta, Bulgaria). aged 8-12 weeks and weighing 2226 g, were housed in plastic cages on sawdustbedding and maintained on standardrodent chow and tap water ad libitum. ‘_ : 2. Chemicals All chemicals used were purchased from Sigma ChemicaI Co. (St Louis, MO’). -2.1. Experimental

protocol

All animals included in carcinogenic&y experiments (see Table 1) were treated with EC either by addition to drinking water (3 g/l) for 3 weeks (Experiments 1 and 2) or by its injection i.p-., dissolved in 0‘9% NaCl, in single doses of 0.5 g/kg or 1.Og/kg (Experiments 3-6).

Some of the animals were also whole-body exposed (60 mitt/day) to mainstream CS by placing each group of mice in a sealed 14 1 glass chamber, which was filled by meansof a 50 ml plastic syringe with mainstreamCS (600 ml) generatedby commercially available filter-tipped cigarettes, containing 31.5 mg tar and 1.6 mg nicotine as describedearlier [S-7]. After 15 min, the chamber was opened and, after a 2-min interval to renew the air, filled again with fresh CS for three additional times, thereby accounting for a cumulative daily exposure to the CS produced by three cigarettes in 60 min. Control mice were sham-exposed.i.e. kept inside the samechamber for the sameperiods of time, but in the absence of CL The following treatment time schedules were used: (a) throughout the experiments, starting 30 days (Experiments 2 and 3) or 10 days (Experiments I, 3 and 4) before EC treatment; (b) for a total of 14 days, starting 10days before EC injection (Experiment 4), and (c) throughout the experiment, starting.30 days after EC treatment (Experiment 4). Other groups of animals were treated with DSF (500 trig/kg, dissolved. in corn oil) by gavage 24 h and I h before EC injection or during 4 consecutive days, starting on day 4 after the carcinogen injection (Experiment 5). In Experiment 6, besides EC, the mice were also treated with SKF-525 A (50 mg/kg) injected i.p. 24 h and 1 h before and 24 h and 48 h after EC administration. Four months after exposure to the carcinogen mice were killed by diethyl ether, the lungs were fixed in 10% buffered formalin, the number of superficial adenomaswas determined macroscopically, and verified histopathologically after samples of lungs had further been processed by routine histological techniques for microscopic examination. The practice of other investigators [8] as well as our preliminary microscopic investigations of serial lung sectionshave indicated that EC-induced tumors were mostly localized beneaththe pleura. Thus, the macroscopic count of adenomasprovides precise enough data to calculate the frequency of tumor-bearing mice and the tumor multiplicity (mean number of lung tumors per mouse). In clastogenicity experiments (see Table 2>, groups-of mice (10 animals/group) were injected i.p; with a single dose of EC (0.5 g/kg) or/and received

R. M. Balansky

I Cancer

Letters

an additional treatment either with CS (840 ml CS in a 14 1 chamber, 90 mm/day) for 4 days, starting 3 days before EC injection or DSF (500 mg/kg) given by gavage 24 h and 1 h before carcinogen injection. Mice were killed by cervical dislocation 24 h after EC administration, and bone marrow smears were prepared, air-dried and stained with the MayGrunwald-Giemsa technique for analyzing the frequency of micronucleated (MN) polychromatic erythrocytes (PCE) [9]. At least 1000 PCE per mouse were scored. The data obtained were statistically analyzed according to Student’s t-test. 3. Results and discussion The data summarized in Table 1 indicate that, after 4 months, treatment of mice with EC induced Table

91-95

93

multiple lung tumors being mostly diagnosed as alveolar and papillary adenomas. The well-known carcinogenic effect of EC in mouse lung [lo] was significantly inhibited by the additional treatment of animals with CS throughout the experiments starting 10 or 30 days before carcinogen exposure, but only in mice treated with EC per OS (Experiments 1 and 2). No effect of CS on EC-induced lung carcinogenesis was conversely detected when the carcinogen was injected i.p., irrespective of the different treatment schedules used, aimed at evaluating the possible influence of CS on initiation and/or promotion phases of EC carcinogenesis. These data might indirectly suggest that the inhibition of EC-induced adenomagenesis observed when the carcinogen was applied per OS was not related to a possible non-specific irritant effect of CS. Thus, CS appears to inhibit EC carcinogenesis

I

Modulation Exp. no

94 (1995)

of lung carcinogenesis

induced

Treatment

EC”, p.o., 3 g/l, EC + CSb EC, p.o., 3 g/l,3 EC + CSc EC, sin le, i.p., EC+CS fi EC+CSC EC, single i.p., EC + CSd EC + CSb EC+ CSe EC, single, i.p., EC + DSF, p.o., EC + DSF, p.o., EC, single, i.p., EC+SKF-525A,

3 weeks weeks 0.5 g/kg

1 g/kg

1 g/kg 500 mg/kgf 500 mg/kgg 0.5 g/kg i.p., 50 mg/kgh

by ethyl carbamate

in Balb/c

mice by cigarette

smoke, disulftram

No. of mice initial/survivors

% of mice with tumors

No. of tumors mouse (mean f SE)

2.5116 25113 30129 30125 25112 25/18 25117 25120 25117 25123 25122 25115 25116 25117 25/18 2512 1

100 84.6 100 92.0 91.7 100 88.2 90.0 100 100 100 100 62.5 94.1 94.4 90.5

10.5 f 1.03 5.1 f 1.29* 9.9 2 0.58 4.2 f 0.68*** 4.6 + 0.99 4.7 + 1.16 3.4 k 0.73 12.3 f 2.33 15.8 f 3.37 11.0+2.40 9.4 * 1.75 15.7 f 2.71 1.8 f 0.63*** 12.7 f 3.03 4.3 + 0.83 3.1 f 0.71

a EC, ethyl carbamate. b CS, cigarette smoke, 600 ml in a 14 1, 60 mitt/day (4 exposures of 15 min each with 2 min intervals 10 days before EC administration and until the end of the experiment. ’ CS, starting 30 days before EC administration and until the end of the experiment. d CS treatment started 10 days before EC injection and lasted 4 days after that. e CS, started 30 days after EC injection and lasted until the end of experiment. f DSF, disulfiram was administered 24 hand 1 h before EC injection. 6 DSF was administered once a day for 4 days starting on day 4 after EC injection. h SKF-525 A (proadifen) was injected 24 h and 1 h before EC exposure and 24 h and 48 h after that. * P < 0.05; ***P < 0.001, compared to the corresponding EC data, as assessed by Student’s f-test.

and SKF-525 per

A Modulation of carcinogenesis (%)

-51.4 -57.6 i2.2 -26.1 +28.5 -10.6 -23.6 -88.5 -19.1 -27.9

for renewing

the air),

starting

only when the carcinogen was administered at low doses in drinking water but not when it was given in *t single massive dose i-p., probably due to changes in its metabolic activaticm. It is likely that only under the former conditions was CS successful in modulating EC metabolism. In a previous study, treatment with EC p.o. for 3 weeks, accompanied and followed by exposure of animals to CS for a total of 8 months. had been found to enhance lung carcinogenesis in BalbiC, but not in AKR or C57Bl mice [4]. The observed inhibition by CS of EC lung carcinogenesis, when the carcinogen was applied in drinking water, is likely to depend on changes in its metabolic activation. EC is metabolically activated by dehydrogenation to form vinyl carbamate (proximate electrophilic metabolite), which is further OXIdized to form vinyl carbamate epoxide (ultimate clectrophilic metabolite), both reactions being cata.Iy~ed by P45O 2E I [I 1,171. In order to support this suggestion, the effects of two known inhibitors ot xenobiotic metabolism on EC lung carcinogenesis were investigated. One of thetn, i.e. DSF was shown to block specifically the activity of cytochrome P450 2E t [ 111, while the other, i.e. SKF-525 A. inhibited P4502Hl [i2]. The data obtained revealed that the administration of’ DSF, 24 h and 1 h before the injection of EC, significantly inhibited the induction of lung adenomas (Table 1, Experiment 5). However, no effect of DSF in carcinogenic activity of EC occurred when the inhibitor was administered for 4 consecutive days. starting 4 days after the carcinogen injection. These data indirectly confirm that the alteration of EC me. tabolism by inhibiting the cytochrome P4SO 2E1 activity is crucial for the inhibitory effect of DSF on iung carcinogenesis. Similarly, diethyldithiocarbainate, which is a proximate metabolite of DSE:, also inhibited EC carcinogenicity in B6C3F1 mice, but in young PIIJax mice it. failecl to influence EC-induced lung carcinogenesis [ 131. In three series of experiments, one of which IS :;hown in Table 1 (Experiment 6), the other inhibitor of metabolism tested, namely SKF-525 A, tended to diminish the multiplicity of lung adenotnas induced by EC, although not to a significant extent. Previously, SKF-525 A had been shown to be ineffective in influencing EC carcinogenicity in mouse lung but lnhihited the carcinogemc activity of N-hydroxy-EC

[ 14-161. Thus. it appears that the metabolic activation of EC is not significantly altered by inhibiting the P450 231 activity with SKF-525 A. However, some marginal involvement of the latter izoenzyme in N-hydroxy-EC metabolism, probably in its reduction back to EC, might be suggested [IO]. The combined genotoxic effects of EC co-administered with CS or DSF were further investigated in vivo by means of the micronucleus test in mouse bone marrow (Table 2). The data obtained indicated that DSF given 24 h and 1 h before the EC injection, completely prevented the clastogenicity of this carcinogen (Experiment 1). On the contrary, the 3-day pretreatment of mice with CS significantly enhanced their clastogenic response to EC, compared to mice treated with the carcinogen only (Experiment 2). Thus, for the first time it was demonstrated that CS can act as a strong syn-clastogen in vivo. The data presented might suggest that CS could modulate the activity of P450 2El or additional isoenzymes of cytochrome P450 that might also be involved in the metabolic activation of EC. It is well known that CS may alter the metabolism of xenobiotits, and in particular may enhance the microsomal Table 2 Modulation by cigarette smoke and disulfiram clastogenicity in hone marrow of female (Exp. &lb/c mice ---___1__ Exp. no

of ethyl carbamate 1) or male (Exp. 2)

Treatment

No. of MN PCE per 1000 PCE

Untreated control EC, single, I+., 0.5 g/kg EC + DSFh. p,o.. SO0 mg/kg lintrented control CSc,4 days, 90 min/day EC, single. i.p.. 0.5 g/kg EC + cs

2.5 + 0.48” 19.8 c 1.87*** 2.2 -r 0.36”“” 2.4 j, 0.78 5.7 It 0.90* 47.3 5 3.86 69.4 + 3.71””

“ Mean f SE. Average data from IO mice, 1000 PCE scored per mouse. h DSF, disulfiram, administered 24 h and 1 h hefore the EC injection. i CS, cigarette smoke, 840 ml in 14 I glass chamber, 9 exposures of 10 min each with 2 min interval between themfor renewing the air, carried out for 3 days before EC as well as 30 min before the EC injection. * P < 0.05; ***P < 0.001, compared to untreated controls, as assessed by Student’s f-test. onP < 0.01; “““P < 0.001, compared to EC-treated mice.

R. hf. Balansky

I Cancer Letters

monooxygenase activity in rodent and human tissues, probably due to induction of P450 1Al and P450 lA2 isoenzymes [ 1,l I]. On the other hand, recently it was found that cigarette smoke condensate strongly inhibits the activation of some procarcinogens and particularly the reactions catalyzed by P450 lA2, thus demonstrating that CS contains inhibitors of P450 enzymes [ 181. The possibility of modulating the P450 2El activity by CS warrants further consideration as this izoenzyme plays a key role in the metabolic activation of some human carcinogens [I 1,191. In conclusion, this study evaluated the frequency of micronuclei in bone marrow as an intermediate biomarker and the yield of lung tumors as a final end-point in EC-treated mice. Disulfiram, a specific inhibitor of cytochrome P450 2E1, strongly inhibited both end-points. On the other hand, exposure of mice to CS, which is a particularly complex mixture, significantly inhibited EC tumorigenicity when the carcinogen was given in drinking water. When the carcinogen was given i.p. in single massive doses, exposure of mice to CS significantly enhanced EC clastogenicity in bone marrow without affecting its pulmonary tumorigenicity. Acknowledgments This study was supported by the Bulgarian Ministry of Science and Education. References [I]

[2]

[3]

[4]

[S]

IARC (1986) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 38, Tobacco Smoking. IARC, Lyon. Takahashi, M., Imaida, K., Mitsumori, K., Okamiya, H., Shinoda, K., Yoshimura, H., Furukawa, F. and Hayashi, Y. (1992) Promoting effects of cigarette smoke on the respiratory tract carcinogenesis of Syrian golden hamsters treated with diethylnitrosamine. Carcinogenesis, 13, 569-572. Gupta, M.P., Khanduja, K.L., Koul, LB. and Sharma, R.R. (1990) Effect of cigarette smoke inhalation on benzo(a)pyrene-induced lung carcinogenesis in vitamin A deficiency in the rat. Cancer Len., 55, 83-88. Keast, D., Tam, N., Sheppard, N. and Papadimitriou, .I. (1985) The role of tobacco smoke, iron ore mine dust, viruses and chemicals in experimental cancer. Arch. Environ. Health, 40, 296-300. Balansky, R.M., Blagoeva, P.M. and Mircheva, Z.I. (1987) Investigation of the mutagenic activity of tobacco smoke. Mutat. Res., 188, 13-19.

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