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Enhancement of tumorigenesis by N-nitrosodiethylamine, N-nitrosopyrrolidine and N6(methylnitroso)-adenosine by ethanol
61
Cancer Letters, 68 (1993) 61- 66 Elsevier Scientific Publishers Ireland Ltd.
Enhancement of tumorigenesis by IV-nitrosodiethylamine, IV-nitrosopyrrolidine and N6(methylnitroso) -adenosine by ethanol Lucy M. Anderson”, John P. Carterb, Craig L. Driverb, Daniel L. Logsdonb, Robert M. Kovatch” and Alfred0 Giner-Sorollad OLoboratoyof Comparutiue Carcinogenesis, National Cancer Institute, bPRl/DynCorp., NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702, ‘Puthoiogy Associates, Inc., Frederick, MD 21701 and dUniuersity of Florida College of Medicine, Tampa, FL 33612 (USA) (Received 23 October 1992) (Accepted 27 October 1992)
Summary
Introduction
Inclusion of 10% ethanol with 6.8 ppm Nnitrosodiethylamine in the drinking water of strain A male mice resulted in a 4-fold enhancement of multiplicity of lung tumors and a 16-fold increase in incidence of forestomach tumors, compared with carcinogen alone. Giuen with 40 ppm N-nitrosopyrrolidine, ethanol caused a 5.5-fold increase in lung tumor multiplicity. The inclusion of 15 % ethanol with N6-(methylnitroso)adenosine, gioen orally to Swiss female mice, led to reduced body weights and shortened survival time related to hemangiosarcoma occurrence or increased incidence of thymic lymphoma, depending on dose of carcinogen. The data provide additional support for the proposal that co-administered ethanol increases the tumorigenicity of nitrosamines by blocking hepatic first-pass clearance.
Ethanol increases the risk of human cancer at several organ sites without itself being genotoxic or carcinogenic in most assays and strongly synergizes with tobacco use [l], leading to speculation that its mechanism(s) of action includes enhancement of the effects of genotoxic environmental carcinogens [Z] . Concurrent administration of ethanol with low molecular weight nitrosamines has consistently resulted in increased tumorigenesis in various target organs [3,4]. The data support the interpretation that ethanol competitively inhibits first-pass hepatic (and/or intestinal) clearance of the nitrosamines, especially by cytochrome P-450 2El [5], leading to increased exposure of more distal tissues [3,6]. Direct relevance of animal studies to the human is likely, in light of the similar activity of human P-450 2El [7] and the inhibitory effect of ethanol on nitrosamine clearance in humans [8,9] and a nonhuman primate [lo]. Although much of the work has focussed on N-nitrosodimethylamine (NDMA) [ 1 1 - 161, similar effects have also been indicated for Nnitrosodiethylamine (NDEA) in mice [17] and rats [HI, N-nitrosodipropylamine in mice [17] and N-nitrosopyrrolidine (NPyr) in hamsters [19]. In the present effort, we have extended
Keywords: nitrosamines; N-nitrosodiethylamine; N-nitrosopyrrolidine; N6-(methylnitrose)-adenosine; ethanol; lung tumors; stomach tumors; thymic lymphomas Correspondence to: L.M. Anderson, Frederick Cancer Research and Development Center, Building 538, Ft. Detrick, Frederick, MD 21702, USA.
03043835/92,‘$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
Ireland Ltd.
62
the observations on ethanol’s effect on tumorigenesis by NDEA and NPyr, since these are known environmental carcinogens. In addition, we have included N6(methyInitroso)adenosine (MNAR) , a carcinogenic nitrosamine [ZO] with the unusual property of causing mammary and uterine tumors when given chronically to adult mice [Zl]. Since ethanol has been implicated as a risk factor for mammary cancer in some epidemiological studies [22], possible potentiation by ethanol of mammary tumorigenesis due to MNAR would be of interest. Materials and Methods
Strain A/JNCr male and Swiss (NIH:Cr(S)) female mice, of 4 weeks of age, were obtained from the Animal Production Area of the Frederick Cancer Research and Development Center and were housed 5 per cage, on hardwood shavings as bedding, at a temperature of 24 f 2OC and humidity 40- 6046, with a fluorescent light/dark cycle of 12/12 h. They were fed NIH 31 Open Formula autoclavable mouse chow and given acidified water. NDEA and NPyr from Sigma Chemical Co. were stored cold and dark. MNAR was synthesized as described previously [23]. Ethanol was reagent grade. NDEA (6.8 ppm) or NPyr (6.8 or 40 ppm) were administered to strain A male mice in sterilized distilled drinking water with or without 10% ethanol for 4 weeks. The 6.8 ppm doses were chosen, for purposes of comparison, as equimolar to the 5 ppm dose of NDMA used in previous studies [4,14]. A higher dose of NPyr was also utilized in view of the expected lower tumorigenicity of this compound. The solutions were prepared twice weekly and protected from light by aluminum foil. The mice were held without further treatment for 32 weeks. MNAR was given as 3 i.g. doses per week of 60 or 120 mg/kg, with or without 15% ethanol, to Swiss female mice for 12 weeks. Thereafter the mice were killed when ill or at 18 months of age.
At kill a complete necropsy was performed and H and E-stained sections prepared of all masses and lesions for diagnosis. Lungs were fixed in Bouin’s solution and examined under a dissecting microscope for quantitative enumeration of all lesions [4,14]. We have demonstrated that tumors are accurately detected by this procedure. Representative tumors, which were primary alveologenic adenomas, were examined histologically. Statistical tests included, for incidence data, the x2 with Yates’ correction as appropriate, or the Fisher exact test as necessary; and the Student’s t-test for multiplicity data.
NDEA and NPyr
Incidence of lung tumors in strain A male mice given 10% ethanol in drinking water for 4 weeks and killed 32 weeks later was 6046, slightly but not significantly greater than that in untreated controls, 38% (Table I). Treatment with 6.8 ppm NDEA for 4 weeks resulted in a significant increase in numbers of lung tumors, to an incidence of 84% (Table I). When 10% ethanol was included with the NDEA, 100% of the mice were tumor-bearers and the multiplicity was increased 3.9-fold compared with those given NDEA alone (P < 0.01). Ethanol also strongly potentiated the tumorigenic effect of NDEA in forestomach, from l/50 with NDEA to 16/50 for NDEA plus ethanol. NPyr alone did not cause a significant number of tumors at either dose (Table I). The inclusion of ethanol with the 6.8 ppm dose increased incidence from 41 to 67% and average multiplicity from 0.5 to 1.2. These differences were statistically significant. However, the values for the NPyr/ethanol-treated mice were not significantly greater than those for mice given ethanol only, so definitive interpretation is not possible. With the higher NPyr dose, inclusion of ethanol resulted in 98% of the mice with lung tumor and a 5.5-fold increase in multiplicity compared with NPyr alone (P < 0.01). These values also differed
63
Table
1.
Treatment
Tumors after treatment Average wt. a (g zt S.D.)
with NDEA or NPyr, with or without ethanol. Average waterb (ml/mouse
Lung tumors
Other neoplasms
per day)
Incidence
Av. No. zt S.D.
NDEA, 6.8 ppm
23.1 zt 2.7
4.2 zt 0.5
42/50’
1.5 f 1.2ds”
+ EtOH
23.6 zt 2.3
4.6 zt 0.3
50/50
5.8 zt 2.2d
6.8 ppm
24.6 zt 2.3
4.4 f 0.4
20/49g
0.5 f 0.8h
+ EtOH
24.3 f 2.1
4.0 f 0.3
33/49s
1.2 f 1.2h
4.4 f 0.5
22/49’
0.6 f 0.8’
47/48ivk
3.3 f 1.7’J
15/25k 9/24’
0.8 +z 0.8’ 0.5 f 0.7”
1 liver adenoma, 1 forestomach CA’ 16 forestomach tumors (14 CA)’
Nb, 1 lymphoblastic lymphoma
Wry l
40 ppm
25.5
2.0
+ EtOH
24.7 zt 1.9
4.1
EtOH only None
22.1 zt 2.4 23.6 f 2.4
3.5 l 0.5 3.5 f 0.2
l
0.4
1 lymphoblastic lymphoma 2 lymphomas: 1 plasma cell, 1 lymphoblastic
CA, carcinoma. aWeight at the end of the 4-week treatment period. bAverage amount of water consumed per mouse per day. f S.D., ‘-’ Values with matched superscripts are significantly different, measured twice during the first week of treatment. P < 0.01.
significantly from those of the ethanol controls. Average body weights and water consumption values were generally similar among all groups (Table I), so the observed effects cannot be ascribed to general toxicity or dosing differences .
MNAR MNAR proved highly carcinogenic at the doses employed, with effects of statistical significance on lung tumors, lymphomas especially of thymus, sarcomas and hemangiosarcomas especially of the uterus and spleen and kidney carcinomas. Augmentation of MNAR’s effectiveness by ethanol was demonstrable, though not as pronounced as for NDEA and NPyr. Average body weights of both MNAR/ethanol groups at 6 months were
slightly but significantly lower than those of either control group (P< 0.01). At the lower dose, coexposure to ethanol significantly reduced survival time. Since cause of death with this dose was most commonly hemangiosarcoma, the ethanol appeared to have reduced the latency or hastened the progression of this cancer. Survival time was also shortened somewhat by inclusion of ethanol with the higher dose of MNAR, with a significant increase in number of mice with thymic lymphomas .
D&cassion The data from these experiments confirm the potentiative effect of ethanol on tumorigenesis when given concurrently with nitro-
64 Table II.
Tumors occurring
N Average age (months) at death zt SD. Body weights, 6 months (mean * S.D., g) Tumors Lung, average no. zt S.D. Lymphomas Thymic Uterus, soft tissue Hemangiosarcomas, spleen Sarcomas Kidney tubular cell CA
in significant numbers
MNAR 60 mg/kg”
MNAR
50 16.2 f 3.9e
l
after treatment
with MNAR with or without 15% ethanol.
MNAR 180 mg/kg’
MNAR 180 mg/kg + ethanold
Ethanol Only’
Saline Control’
49 13.4 f 4.6s
49 9.5 f 3.0
50 8.5 zt 3.0
49 24.1 + 5.5
48 23.9 zt 5.9
2.2
23.4 ztz 2.2
25.7 zt 3.0
22.7 + 2.6
26.1 f 2.4
27.0 zt 2.9
7.3 zt 3.8
8.0 + 6.6
5.1 f 4.7
3.2 + 4.2
1.4 f 1.4
1.1
36 32h 1’ 6
5 1 4” 0
6 1 1” 0
0 2
2’ 0
1’ 0
25.7
60 mg/kg + ethanolb
10 8 10’ 20
11 8 8’ 19
27 21h lk 9
6” 6
2p 1
lq 3
l
1.1
CA, carcinoma; AD, adenoma. “Other tumors: 3 mammary (2 adenomyoma, 1 adenosquamous CA); 2 ovary (1 thecoma, 1 granulosa-thecal cell); 1 mesothelioma; 1 schwannoma; 2 pancreas (1 acinar cell CA, 1 islet cell AD); 2 Harderian gland; 1 metastatic CA. bAlso: 2 mammary (1 adenomyoma, 1 adenoepithelioma) ; 2 ovary (1 teratoma, 1 hemangioma) ; 3 Harderian gland; 3 adrenal (2 pheochromocytomas, 1 cortical); 1 thyroid follicle. ‘Also: 1 mammary adenomyoma; 1 Harderian gland. dAlso: 1 mammary myoepithelioma; 1 thyroid follicle. ‘Also: 1 mammary CA; 4 ovary (1 thecoma, 1 tubulostromal, 1 malignant stromal, 1 hemangiosarcoma); 4 Harderian gland; 1 pituitary AD. ‘Also: 1 mammary CA; 1 ovarian thecoma; 1 Harderian gland; 1 adrenal pheochromocytoma; 1 pituitary AD; 1 mesothelioma. g,hValues with matched gP < 0.01. hP < 0.05. ‘3 Hemangiosarcomas, 1 leiomyosarcoma, 3 superscripts are significantly different, leiomyomas, 1 endometrial stromal sarcoma, 2 hemangiomas. ‘3 Hemangiosarcomas, 1 leiomyomas, 3 endometrial stromal sarcomas, 1 schwannoma. kLeiomyoma. ‘Endometrial stromal sarcoma. “Hemangiosarcomas, 2 leiomyomas. “Leiomyoma. “2 Histiocytic; 2 osteo-; 1 rhabdomyo-; 1 fibrosarcoma. p1 Osteo-; 1 fibrosarcoma. qWndifferentiated sarcomas. “1 Rhabdomyosarcoma.
samines. Ethanol at 10% in the drinking water increased multiplicity of lung tumors initiated by NDEA by about 4-fold, to the same extent as observed for an equimolar (5 mM) concentration of NDMA given under similar experimental conditions [4,14]. These results are again consistent with inhibition by ethanol of hepatic degradative metabolism of the nitrosamine, resulting in increased exposure of the
lung. Thus, while NDEA may be partially metabolized by hepatic cytochromes P-450 other than 2El [24 - 271, ethanol may be as effective in blocking its clearance as for that of NDMA. The findings also bear out the prediction made by increased alkylation of lung and esophageal DNA in rats given ethanol concurrently with NDEA [6]. The striking increase in forestomach tumors
65
with NDEA plus ethanol confirms a similar finding in C57BL/6 mice [17]. This phenomenon is of particular human relevance, because of the similarity of forestomach epithelium to that of esophagus, an important alcohol-related cancer target organ in humans. In the current and the previous [17] study, the nitrosamine and ethanol in the drinking water would have contacted this epithelium directly as well as systemically, so the role of inhibition of hepatic first pass clearance as a mechanistic component is not clear at present, but is being pursued in further experiments. NPyr at 6.8 ppm was minimally carcinogenic even in the presence of ethanol, confirming its lower effectiveness relative to NDMA and NDEA. However, at 40 ppm, ethanol caused a 5-fold increase in multiplicity of lung tumors, equivalent to its effect on NDMA and NDEA. This finding is consistent with participation of P-450 2El in the metabolism of NPyr [28]. Other relevant literature on NPyr contains a complexity of findings. Pretreatment of rodents with ethanol resulted in increased capacity of tissues to activate NPyr to a mutagen in vitro [29 - 311, but similar treatment led to reduced genotoxicity in vivo [31]. Two tests of effect of ethanol on tumorigenicity of i.p.-administered NPyr in Syrian golden hamsters gave different results, which together are consistent with our findings and hypothesis. When ethanol was given in liquid diet, which would give continuously high blood levels, it potentiated tumorigenesis by NPyr in nasal cavity and trachea [19]. When given in the drinking water, ethanol caused an increase in liver neoplastic nodules due to NPyr, but not in respiratory tract tumors [32]; this dosing protocol provides only transitory and limited increase in blood ethanol not likely to inhibit hepatic clearance of NPyr. MNAR, in a previous study of a l-mM dose administered in the drinking water to Swiss CD-l mice (-300 mg/kg per week total dose), caused primarily lung tumors and mammary carcinomas, with a 20% incidence of uterine adenocarcinomas and 1 ovarian carcinoma [21]. In the experiment reported here,
with a different Swiss mouse strain and an acute i.g. treatment mode, 180 or 540 mg MNAR/kg per week again caused lung tumors, but the predominant malignant tumors were lymphomas of thymic origin and various types of sarcomas and other soft tissue neoplasms, especially of spleen and uterus. It is of interest that organotropy for uterus was seen in both studies, though with different target tissues within the organ. A few tumors developed from mammary epithelium and a small number of assorted ovarian tumors were seen. Possible effects of ethanol on tumorigenicity of MNAR in the epithelia of these tissue must be studied with other protocols. It was however clear in the present effort that ethanol had a small potentiative effect on MNAR, as reflected in body weights, survival time and possibly sarcoma development with the lower dose and thymic lymphoma incidence with the higher dose. Although the nature of the cytochrome P-450 acting on MNAR in liver is not known, the results are consistent with some inhibition of clearance of MNAR by ethanol. This may become more apparent with drinking-water dosing. Acknowledgements
The expert technical assistance of Areitha Smith, Darlene Reever, and Gwen Magruder have been appreciated. References IARC (1988) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 44, Alcohol Drinking, Lyon. Driver, H.E. and Swann, P.F. (1987) Alcohol and human cancer. Anticancer Res., 7, 309-320. Anderson, L.M. (1992) Modulation of nitrosamine metabolism by ethanol: implications for cancer risk. In: Alcohol and Cancer, Editor: R. Watson, pp. 17 - 54. CRC Press, Boca Raton Anderson, L.M., Carter, J.P., Logsdon, D.L., Driver, C.L. and Kovatch, R.M. (1992) Characterization of tumorigenesis by Nethanol’s enhancement of nitrosodimethylamine in mice. Carcinogenesis, in press. Yang, C.S., Yoo, J.H., Ishizaki, H. and Hong, J. (1990) Cytochrome P45011El: roles in nitrosamine metabolism
66
6
7
8
9
10
11
12
13
14
15
16
17
18
and mechanisms of regulation. Drug Metab. Rev., 22, 147- 159. Swann, P.F., Coe, A.M. and Mace, R. (1984) Ethanol and dimethylnitrosamine and diethylnitrosamine metabolism and disposition in the rat. Possible relevance to the influence of ethanol on human cancer incidence. Carcinogenesis, 5, 1337 - 1343. Guengerich, F.P., Kim, D. and Iwasaki, M. (1991) Role of human cytochrome P-450 IIEI in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol., 4, 168- 179. Spiegelhalder, B. and Preussmann, R. (1985) In vivo nitrosation of amidopyrine in humans: use of ‘ethanol effect for biological monitoring of N-nitrosodimethylamine in urine. Carcinogenesis, 6, 545-548. Dunn, S.R., Simenhoff, M.L., Lele, P.S., Goyal, S., Pensabene, J.W. and Fiddler, W. (1990) N-Nitrosodimethylamine blood levels in patients with chronic renal failure: Modulation of levels by ethanol and ascorbic acid. J. Natl. Cancer Inst., 82, 783-787. Anderson, L.M., Koseniauskas, R., Burak, ES., Moskal, T.J., Gombar, C.T., Phillips, J.M., Sansone, E.B., Keimig, S., Magee, P.N., Rice, J.M. and Harrington, G.W. (1992) Reduced blood clearance and increased urinary excretion of N-nitrosodimethylamine in patas monkeys exposed to ethanol or a isopropyl alcohol. Cancer Res., 52, 1463- 1468. Peng, R., Tu, Y.Y. and Yang, C.S. (1982) The induction and competitive inhibition of a high affinity microsomal nitrosodimethylamine demethylase by ethanol. Carcinogenesis, 3, 1457 - 1461. Levin, W., Thomas, P.E., Oldfield, N. and Ryan, D.E. (1986) N-Demethylation of N-nitrosodimethylamine catalyzed by purified rat hepatic microsomal P-450: isozyme specificity and role of cytochrome b,. Archiv. Biochem. Biophys., 248, 158 - 165. Anderson, L.M., Harrington, G.W,, Pylypiw, H.M., Hagiwara, A. and Magee, P.N. (1986) Tissue levels and biological effects of N-nitrosodimethylamine in mice during chronic low or high dose exposure with or without ethanol. Drug. Metab. Dispos., 14, 733-739. Anderson, L.M. (1988) Increased numbers of Nnitrosodimethylamine-initiated lung tumors in mice by chronic co-administration of ethanol. Carcinogenesis, 9, 1717 - 1719. Harrington, G.W., Magee, P.N., Pylypiw, H.M., Kozeniauskas, R., Bevill, R.F., Nelson, D.R. and Thurmon, J.C. (1990) The formation, disposition and hepatic metabolism of dimethylnitrosamine in the pig. Drug Metab. Dispos., 18, 626-631. Griciute, L., Castegnaro, M. and Bereziat, J.C. (1981) influence of ethyl alcohol on carcinogenesis with Nnitrosodimethylamine. Cancer Lett., 13, 345 - 352. Griciute, L., Castegnaro, M. and Bereziat, J.C. (1987) Influence of ethyl alcohol on carcinogenesis induced by volatile N-nitrosamines detected in alcoholic beverages. IARC Sci. Publ., 84, 264-265. Gibel, V.W. (1967) Experimentelle Untersuchungen ZUT Synkarzinogenese beim Osophaguskarzinom. Arch. Geschwulstforsch. 30, 181- 189.
19
20
21
22 23
24
25
26
27
28
29
30
31
32
McCoy, G.D., Hecht, S.S., Katayama, S. and Wynder, E.L. (1981) Differential effect of chronic ethanol consumption on the carcinogenic&y of N-nitrosopyrrolidine and N ‘nitrosonornicotine in male Syrian golden hamsters. Cancer Res., 41, 2849-2854. Giner-Sorolla, A., Greenbaum, J.H., Last-Barney, K., Anderson, L.M. and Budinger, J.M. (1979) N6(Methylnitroso)adenosine: a carcinogenic nitrosamine derived from a naturally-occurring nucleoside. Cancer Lett., 6, 79-82. Anderson, L.M., Giner-Sorolla, A., Greebaum, J.N., Last-Barney, K. and Budinger, J.M. (1979) induction of reproductive system tumors in mice by N6(methylnitroso)-adenosine and a tumorigenic effect of its combined precursors. Int. J. Cancer, 24, 319-322. Graham, S. (1987) Alcohol and breast cancer. N. Engl. J. Med., 316, 1211- 1212. Giner-Sorolla, A., Longley-Cook, J., McCravey, M., Brown, G.B. and Burchenal, J.H. (1973) Nitrosoaminopurines and nucleosides, synthesis and biological activity. J. Med. Chem., 16, 365- 369. Yoo, J.H., Ishiiaki, H. and Yang, C.S. (1990) Roles of cytochrome P-45011El in the dealkylation and denitrosation of N-nitrosodimethylamine and N-nitrosodiethylamine in rat liver microsomes. Carcinogenesis, 11, 2239 - 2243. Puccini, P, Fioro, R., Longo, V. and Gervasi, P.G. (1989) High affinity diethylnitrosamine-deethylase in liver microsomes from acetone-induced rats. Carcinogenesis, 10, 1629- 1634. Longo, V., Citti, L. and Gervasi, P.G. (1986) Metabolism of diethylnitrosamine by nasal mucosa and hepatic microsomes from hamster and rat: species specificity of nasal mucosa. Carcinogenesis, 7, 1323- 1328. Gorsky, L.D. and Hollenberg, P.F. (1989) Metabolism of N-nitrosodimethyl and N-nitrosodiethylamine by rat hepatocytes: effects of pretreatment with ethanol. Chem. Res. Toxicol., 2, 436 -441. McCoy, G.D. and Koop, D.R. (1988) Reconstitution of rabbit liver microsomal N-nitrosopyrrolidine cY-hydroxylase activity. Cancer Res., 48, 3987 - 3992. McCoy, G.D., Chen, C.B., Hecht, S.S. and McCoy, E.C. (1979) Enhanced metabolism and mutagenesis of nitrosopyrrolidine in liver fractions isolated from chronic ethanol-consuming hamster. Cancer Res., 39, 793 - 796. Farianti, F., Zhou, Z., Bellah, J., Lieber, C.S. and Garro, A.J. (1986) Effect of chronic ethanol consumption on activation of nitrosopynolidine to a mutagen by rat upper alimentary tract, lung and hepatic tissue. Drug Metabl. Disp., 13, 210 - 214. Gold, B. and Brunk, G. (1988) The effect of pyrazole, phenobarbital, ethanol and 3-methylcholanthrene pretreatment on the in vivo and in vitro genotoxicity of Nnitrosopyrrolidine. Carcinogenesis, 9, 1001- 1005. McCoy, G.D., Hecht, S.S. and Furuya, K. (1986) The effect of chronic ethanol consumption on the tumorigenicity of N-nitrosopyrrolidine in male Syrian golden hamsters. Cancer Lett., 33, 151- 159.
Cancer Letters, 68 (1993) 61- 66 Elsevier Scientific Publishers Ireland Ltd.
Enhancement of tumorigenesis by IV-nitrosodiethylamine, IV-nitrosopyrrolidine and N6(methylnitroso) -adenosine by ethanol Lucy M. Anderson”, John P. Carterb, Craig L. Driverb, Daniel L. Logsdonb, Robert M. Kovatch” and Alfred0 Giner-Sorollad OLoboratoyof Comparutiue Carcinogenesis, National Cancer Institute, bPRl/DynCorp., NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702, ‘Puthoiogy Associates, Inc., Frederick, MD 21701 and dUniuersity of Florida College of Medicine, Tampa, FL 33612 (USA) (Received 23 October 1992) (Accepted 27 October 1992)
Summary
Introduction
Inclusion of 10% ethanol with 6.8 ppm Nnitrosodiethylamine in the drinking water of strain A male mice resulted in a 4-fold enhancement of multiplicity of lung tumors and a 16-fold increase in incidence of forestomach tumors, compared with carcinogen alone. Giuen with 40 ppm N-nitrosopyrrolidine, ethanol caused a 5.5-fold increase in lung tumor multiplicity. The inclusion of 15 % ethanol with N6-(methylnitroso)adenosine, gioen orally to Swiss female mice, led to reduced body weights and shortened survival time related to hemangiosarcoma occurrence or increased incidence of thymic lymphoma, depending on dose of carcinogen. The data provide additional support for the proposal that co-administered ethanol increases the tumorigenicity of nitrosamines by blocking hepatic first-pass clearance.
Ethanol increases the risk of human cancer at several organ sites without itself being genotoxic or carcinogenic in most assays and strongly synergizes with tobacco use [l], leading to speculation that its mechanism(s) of action includes enhancement of the effects of genotoxic environmental carcinogens [Z] . Concurrent administration of ethanol with low molecular weight nitrosamines has consistently resulted in increased tumorigenesis in various target organs [3,4]. The data support the interpretation that ethanol competitively inhibits first-pass hepatic (and/or intestinal) clearance of the nitrosamines, especially by cytochrome P-450 2El [5], leading to increased exposure of more distal tissues [3,6]. Direct relevance of animal studies to the human is likely, in light of the similar activity of human P-450 2El [7] and the inhibitory effect of ethanol on nitrosamine clearance in humans [8,9] and a nonhuman primate [lo]. Although much of the work has focussed on N-nitrosodimethylamine (NDMA) [ 1 1 - 161, similar effects have also been indicated for Nnitrosodiethylamine (NDEA) in mice [17] and rats [HI, N-nitrosodipropylamine in mice [17] and N-nitrosopyrrolidine (NPyr) in hamsters [19]. In the present effort, we have extended
Keywords: nitrosamines; N-nitrosodiethylamine; N-nitrosopyrrolidine; N6-(methylnitrose)-adenosine; ethanol; lung tumors; stomach tumors; thymic lymphomas Correspondence to: L.M. Anderson, Frederick Cancer Research and Development Center, Building 538, Ft. Detrick, Frederick, MD 21702, USA.
03043835/92,‘$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
Ireland Ltd.
62
the observations on ethanol’s effect on tumorigenesis by NDEA and NPyr, since these are known environmental carcinogens. In addition, we have included N6(methyInitroso)adenosine (MNAR) , a carcinogenic nitrosamine [ZO] with the unusual property of causing mammary and uterine tumors when given chronically to adult mice [Zl]. Since ethanol has been implicated as a risk factor for mammary cancer in some epidemiological studies [22], possible potentiation by ethanol of mammary tumorigenesis due to MNAR would be of interest. Materials and Methods
Strain A/JNCr male and Swiss (NIH:Cr(S)) female mice, of 4 weeks of age, were obtained from the Animal Production Area of the Frederick Cancer Research and Development Center and were housed 5 per cage, on hardwood shavings as bedding, at a temperature of 24 f 2OC and humidity 40- 6046, with a fluorescent light/dark cycle of 12/12 h. They were fed NIH 31 Open Formula autoclavable mouse chow and given acidified water. NDEA and NPyr from Sigma Chemical Co. were stored cold and dark. MNAR was synthesized as described previously [23]. Ethanol was reagent grade. NDEA (6.8 ppm) or NPyr (6.8 or 40 ppm) were administered to strain A male mice in sterilized distilled drinking water with or without 10% ethanol for 4 weeks. The 6.8 ppm doses were chosen, for purposes of comparison, as equimolar to the 5 ppm dose of NDMA used in previous studies [4,14]. A higher dose of NPyr was also utilized in view of the expected lower tumorigenicity of this compound. The solutions were prepared twice weekly and protected from light by aluminum foil. The mice were held without further treatment for 32 weeks. MNAR was given as 3 i.g. doses per week of 60 or 120 mg/kg, with or without 15% ethanol, to Swiss female mice for 12 weeks. Thereafter the mice were killed when ill or at 18 months of age.
At kill a complete necropsy was performed and H and E-stained sections prepared of all masses and lesions for diagnosis. Lungs were fixed in Bouin’s solution and examined under a dissecting microscope for quantitative enumeration of all lesions [4,14]. We have demonstrated that tumors are accurately detected by this procedure. Representative tumors, which were primary alveologenic adenomas, were examined histologically. Statistical tests included, for incidence data, the x2 with Yates’ correction as appropriate, or the Fisher exact test as necessary; and the Student’s t-test for multiplicity data.
NDEA and NPyr
Incidence of lung tumors in strain A male mice given 10% ethanol in drinking water for 4 weeks and killed 32 weeks later was 6046, slightly but not significantly greater than that in untreated controls, 38% (Table I). Treatment with 6.8 ppm NDEA for 4 weeks resulted in a significant increase in numbers of lung tumors, to an incidence of 84% (Table I). When 10% ethanol was included with the NDEA, 100% of the mice were tumor-bearers and the multiplicity was increased 3.9-fold compared with those given NDEA alone (P < 0.01). Ethanol also strongly potentiated the tumorigenic effect of NDEA in forestomach, from l/50 with NDEA to 16/50 for NDEA plus ethanol. NPyr alone did not cause a significant number of tumors at either dose (Table I). The inclusion of ethanol with the 6.8 ppm dose increased incidence from 41 to 67% and average multiplicity from 0.5 to 1.2. These differences were statistically significant. However, the values for the NPyr/ethanol-treated mice were not significantly greater than those for mice given ethanol only, so definitive interpretation is not possible. With the higher NPyr dose, inclusion of ethanol resulted in 98% of the mice with lung tumor and a 5.5-fold increase in multiplicity compared with NPyr alone (P < 0.01). These values also differed
63
Table
1.
Treatment
Tumors after treatment Average wt. a (g zt S.D.)
with NDEA or NPyr, with or without ethanol. Average waterb (ml/mouse
Lung tumors
Other neoplasms
per day)
Incidence
Av. No. zt S.D.
NDEA, 6.8 ppm
23.1 zt 2.7
4.2 zt 0.5
42/50’
1.5 f 1.2ds”
+ EtOH
23.6 zt 2.3
4.6 zt 0.3
50/50
5.8 zt 2.2d
6.8 ppm
24.6 zt 2.3
4.4 f 0.4
20/49g
0.5 f 0.8h
+ EtOH
24.3 f 2.1
4.0 f 0.3
33/49s
1.2 f 1.2h
4.4 f 0.5
22/49’
0.6 f 0.8’
47/48ivk
3.3 f 1.7’J
15/25k 9/24’
0.8 +z 0.8’ 0.5 f 0.7”
1 liver adenoma, 1 forestomach CA’ 16 forestomach tumors (14 CA)’
Nb, 1 lymphoblastic lymphoma
Wry l
40 ppm
25.5
2.0
+ EtOH
24.7 zt 1.9
4.1
EtOH only None
22.1 zt 2.4 23.6 f 2.4
3.5 l 0.5 3.5 f 0.2
l
0.4
1 lymphoblastic lymphoma 2 lymphomas: 1 plasma cell, 1 lymphoblastic
CA, carcinoma. aWeight at the end of the 4-week treatment period. bAverage amount of water consumed per mouse per day. f S.D., ‘-’ Values with matched superscripts are significantly different, measured twice during the first week of treatment. P < 0.01.
significantly from those of the ethanol controls. Average body weights and water consumption values were generally similar among all groups (Table I), so the observed effects cannot be ascribed to general toxicity or dosing differences .
MNAR MNAR proved highly carcinogenic at the doses employed, with effects of statistical significance on lung tumors, lymphomas especially of thymus, sarcomas and hemangiosarcomas especially of the uterus and spleen and kidney carcinomas. Augmentation of MNAR’s effectiveness by ethanol was demonstrable, though not as pronounced as for NDEA and NPyr. Average body weights of both MNAR/ethanol groups at 6 months were
slightly but significantly lower than those of either control group (P< 0.01). At the lower dose, coexposure to ethanol significantly reduced survival time. Since cause of death with this dose was most commonly hemangiosarcoma, the ethanol appeared to have reduced the latency or hastened the progression of this cancer. Survival time was also shortened somewhat by inclusion of ethanol with the higher dose of MNAR, with a significant increase in number of mice with thymic lymphomas .
D&cassion The data from these experiments confirm the potentiative effect of ethanol on tumorigenesis when given concurrently with nitro-
64 Table II.
Tumors occurring
N Average age (months) at death zt SD. Body weights, 6 months (mean * S.D., g) Tumors Lung, average no. zt S.D. Lymphomas Thymic Uterus, soft tissue Hemangiosarcomas, spleen Sarcomas Kidney tubular cell CA
in significant numbers
MNAR 60 mg/kg”
MNAR
50 16.2 f 3.9e
l
after treatment
with MNAR with or without 15% ethanol.
MNAR 180 mg/kg’
MNAR 180 mg/kg + ethanold
Ethanol Only’
Saline Control’
49 13.4 f 4.6s
49 9.5 f 3.0
50 8.5 zt 3.0
49 24.1 + 5.5
48 23.9 zt 5.9
2.2
23.4 ztz 2.2
25.7 zt 3.0
22.7 + 2.6
26.1 f 2.4
27.0 zt 2.9
7.3 zt 3.8
8.0 + 6.6
5.1 f 4.7
3.2 + 4.2
1.4 f 1.4
1.1
36 32h 1’ 6
5 1 4” 0
6 1 1” 0
0 2
2’ 0
1’ 0
25.7
60 mg/kg + ethanolb
10 8 10’ 20
11 8 8’ 19
27 21h lk 9
6” 6
2p 1
lq 3
l
1.1
CA, carcinoma; AD, adenoma. “Other tumors: 3 mammary (2 adenomyoma, 1 adenosquamous CA); 2 ovary (1 thecoma, 1 granulosa-thecal cell); 1 mesothelioma; 1 schwannoma; 2 pancreas (1 acinar cell CA, 1 islet cell AD); 2 Harderian gland; 1 metastatic CA. bAlso: 2 mammary (1 adenomyoma, 1 adenoepithelioma) ; 2 ovary (1 teratoma, 1 hemangioma) ; 3 Harderian gland; 3 adrenal (2 pheochromocytomas, 1 cortical); 1 thyroid follicle. ‘Also: 1 mammary adenomyoma; 1 Harderian gland. dAlso: 1 mammary myoepithelioma; 1 thyroid follicle. ‘Also: 1 mammary CA; 4 ovary (1 thecoma, 1 tubulostromal, 1 malignant stromal, 1 hemangiosarcoma); 4 Harderian gland; 1 pituitary AD. ‘Also: 1 mammary CA; 1 ovarian thecoma; 1 Harderian gland; 1 adrenal pheochromocytoma; 1 pituitary AD; 1 mesothelioma. g,hValues with matched gP < 0.01. hP < 0.05. ‘3 Hemangiosarcomas, 1 leiomyosarcoma, 3 superscripts are significantly different, leiomyomas, 1 endometrial stromal sarcoma, 2 hemangiomas. ‘3 Hemangiosarcomas, 1 leiomyomas, 3 endometrial stromal sarcomas, 1 schwannoma. kLeiomyoma. ‘Endometrial stromal sarcoma. “Hemangiosarcomas, 2 leiomyomas. “Leiomyoma. “2 Histiocytic; 2 osteo-; 1 rhabdomyo-; 1 fibrosarcoma. p1 Osteo-; 1 fibrosarcoma. qWndifferentiated sarcomas. “1 Rhabdomyosarcoma.
samines. Ethanol at 10% in the drinking water increased multiplicity of lung tumors initiated by NDEA by about 4-fold, to the same extent as observed for an equimolar (5 mM) concentration of NDMA given under similar experimental conditions [4,14]. These results are again consistent with inhibition by ethanol of hepatic degradative metabolism of the nitrosamine, resulting in increased exposure of the
lung. Thus, while NDEA may be partially metabolized by hepatic cytochromes P-450 other than 2El [24 - 271, ethanol may be as effective in blocking its clearance as for that of NDMA. The findings also bear out the prediction made by increased alkylation of lung and esophageal DNA in rats given ethanol concurrently with NDEA [6]. The striking increase in forestomach tumors
65
with NDEA plus ethanol confirms a similar finding in C57BL/6 mice [17]. This phenomenon is of particular human relevance, because of the similarity of forestomach epithelium to that of esophagus, an important alcohol-related cancer target organ in humans. In the current and the previous [17] study, the nitrosamine and ethanol in the drinking water would have contacted this epithelium directly as well as systemically, so the role of inhibition of hepatic first pass clearance as a mechanistic component is not clear at present, but is being pursued in further experiments. NPyr at 6.8 ppm was minimally carcinogenic even in the presence of ethanol, confirming its lower effectiveness relative to NDMA and NDEA. However, at 40 ppm, ethanol caused a 5-fold increase in multiplicity of lung tumors, equivalent to its effect on NDMA and NDEA. This finding is consistent with participation of P-450 2El in the metabolism of NPyr [28]. Other relevant literature on NPyr contains a complexity of findings. Pretreatment of rodents with ethanol resulted in increased capacity of tissues to activate NPyr to a mutagen in vitro [29 - 311, but similar treatment led to reduced genotoxicity in vivo [31]. Two tests of effect of ethanol on tumorigenicity of i.p.-administered NPyr in Syrian golden hamsters gave different results, which together are consistent with our findings and hypothesis. When ethanol was given in liquid diet, which would give continuously high blood levels, it potentiated tumorigenesis by NPyr in nasal cavity and trachea [19]. When given in the drinking water, ethanol caused an increase in liver neoplastic nodules due to NPyr, but not in respiratory tract tumors [32]; this dosing protocol provides only transitory and limited increase in blood ethanol not likely to inhibit hepatic clearance of NPyr. MNAR, in a previous study of a l-mM dose administered in the drinking water to Swiss CD-l mice (-300 mg/kg per week total dose), caused primarily lung tumors and mammary carcinomas, with a 20% incidence of uterine adenocarcinomas and 1 ovarian carcinoma [21]. In the experiment reported here,
with a different Swiss mouse strain and an acute i.g. treatment mode, 180 or 540 mg MNAR/kg per week again caused lung tumors, but the predominant malignant tumors were lymphomas of thymic origin and various types of sarcomas and other soft tissue neoplasms, especially of spleen and uterus. It is of interest that organotropy for uterus was seen in both studies, though with different target tissues within the organ. A few tumors developed from mammary epithelium and a small number of assorted ovarian tumors were seen. Possible effects of ethanol on tumorigenicity of MNAR in the epithelia of these tissue must be studied with other protocols. It was however clear in the present effort that ethanol had a small potentiative effect on MNAR, as reflected in body weights, survival time and possibly sarcoma development with the lower dose and thymic lymphoma incidence with the higher dose. Although the nature of the cytochrome P-450 acting on MNAR in liver is not known, the results are consistent with some inhibition of clearance of MNAR by ethanol. This may become more apparent with drinking-water dosing. Acknowledgements
The expert technical assistance of Areitha Smith, Darlene Reever, and Gwen Magruder have been appreciated. References IARC (1988) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 44, Alcohol Drinking, Lyon. Driver, H.E. and Swann, P.F. (1987) Alcohol and human cancer. Anticancer Res., 7, 309-320. Anderson, L.M. (1992) Modulation of nitrosamine metabolism by ethanol: implications for cancer risk. In: Alcohol and Cancer, Editor: R. Watson, pp. 17 - 54. CRC Press, Boca Raton Anderson, L.M., Carter, J.P., Logsdon, D.L., Driver, C.L. and Kovatch, R.M. (1992) Characterization of tumorigenesis by Nethanol’s enhancement of nitrosodimethylamine in mice. Carcinogenesis, in press. Yang, C.S., Yoo, J.H., Ishizaki, H. and Hong, J. (1990) Cytochrome P45011El: roles in nitrosamine metabolism
66
6
7
8
9
10
11
12
13
14
15
16
17
18
and mechanisms of regulation. Drug Metab. Rev., 22, 147- 159. Swann, P.F., Coe, A.M. and Mace, R. (1984) Ethanol and dimethylnitrosamine and diethylnitrosamine metabolism and disposition in the rat. Possible relevance to the influence of ethanol on human cancer incidence. Carcinogenesis, 5, 1337 - 1343. Guengerich, F.P., Kim, D. and Iwasaki, M. (1991) Role of human cytochrome P-450 IIEI in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol., 4, 168- 179. Spiegelhalder, B. and Preussmann, R. (1985) In vivo nitrosation of amidopyrine in humans: use of ‘ethanol effect for biological monitoring of N-nitrosodimethylamine in urine. Carcinogenesis, 6, 545-548. Dunn, S.R., Simenhoff, M.L., Lele, P.S., Goyal, S., Pensabene, J.W. and Fiddler, W. (1990) N-Nitrosodimethylamine blood levels in patients with chronic renal failure: Modulation of levels by ethanol and ascorbic acid. J. Natl. Cancer Inst., 82, 783-787. Anderson, L.M., Koseniauskas, R., Burak, ES., Moskal, T.J., Gombar, C.T., Phillips, J.M., Sansone, E.B., Keimig, S., Magee, P.N., Rice, J.M. and Harrington, G.W. (1992) Reduced blood clearance and increased urinary excretion of N-nitrosodimethylamine in patas monkeys exposed to ethanol or a isopropyl alcohol. Cancer Res., 52, 1463- 1468. Peng, R., Tu, Y.Y. and Yang, C.S. (1982) The induction and competitive inhibition of a high affinity microsomal nitrosodimethylamine demethylase by ethanol. Carcinogenesis, 3, 1457 - 1461. Levin, W., Thomas, P.E., Oldfield, N. and Ryan, D.E. (1986) N-Demethylation of N-nitrosodimethylamine catalyzed by purified rat hepatic microsomal P-450: isozyme specificity and role of cytochrome b,. Archiv. Biochem. Biophys., 248, 158 - 165. Anderson, L.M., Harrington, G.W,, Pylypiw, H.M., Hagiwara, A. and Magee, P.N. (1986) Tissue levels and biological effects of N-nitrosodimethylamine in mice during chronic low or high dose exposure with or without ethanol. Drug. Metab. Dispos., 14, 733-739. Anderson, L.M. (1988) Increased numbers of Nnitrosodimethylamine-initiated lung tumors in mice by chronic co-administration of ethanol. Carcinogenesis, 9, 1717 - 1719. Harrington, G.W., Magee, P.N., Pylypiw, H.M., Kozeniauskas, R., Bevill, R.F., Nelson, D.R. and Thurmon, J.C. (1990) The formation, disposition and hepatic metabolism of dimethylnitrosamine in the pig. Drug Metab. Dispos., 18, 626-631. Griciute, L., Castegnaro, M. and Bereziat, J.C. (1981) influence of ethyl alcohol on carcinogenesis with Nnitrosodimethylamine. Cancer Lett., 13, 345 - 352. Griciute, L., Castegnaro, M. and Bereziat, J.C. (1987) Influence of ethyl alcohol on carcinogenesis induced by volatile N-nitrosamines detected in alcoholic beverages. IARC Sci. Publ., 84, 264-265. Gibel, V.W. (1967) Experimentelle Untersuchungen ZUT Synkarzinogenese beim Osophaguskarzinom. Arch. Geschwulstforsch. 30, 181- 189.
19
20
21
22 23
24
25
26
27
28
29
30
31
32
McCoy, G.D., Hecht, S.S., Katayama, S. and Wynder, E.L. (1981) Differential effect of chronic ethanol consumption on the carcinogenic&y of N-nitrosopyrrolidine and N ‘nitrosonornicotine in male Syrian golden hamsters. Cancer Res., 41, 2849-2854. Giner-Sorolla, A., Greenbaum, J.H., Last-Barney, K., Anderson, L.M. and Budinger, J.M. (1979) N6(Methylnitroso)adenosine: a carcinogenic nitrosamine derived from a naturally-occurring nucleoside. Cancer Lett., 6, 79-82. Anderson, L.M., Giner-Sorolla, A., Greebaum, J.N., Last-Barney, K. and Budinger, J.M. (1979) induction of reproductive system tumors in mice by N6(methylnitroso)-adenosine and a tumorigenic effect of its combined precursors. Int. J. Cancer, 24, 319-322. Graham, S. (1987) Alcohol and breast cancer. N. Engl. J. Med., 316, 1211- 1212. Giner-Sorolla, A., Longley-Cook, J., McCravey, M., Brown, G.B. and Burchenal, J.H. (1973) Nitrosoaminopurines and nucleosides, synthesis and biological activity. J. Med. Chem., 16, 365- 369. Yoo, J.H., Ishiiaki, H. and Yang, C.S. (1990) Roles of cytochrome P-45011El in the dealkylation and denitrosation of N-nitrosodimethylamine and N-nitrosodiethylamine in rat liver microsomes. Carcinogenesis, 11, 2239 - 2243. Puccini, P, Fioro, R., Longo, V. and Gervasi, P.G. (1989) High affinity diethylnitrosamine-deethylase in liver microsomes from acetone-induced rats. Carcinogenesis, 10, 1629- 1634. Longo, V., Citti, L. and Gervasi, P.G. (1986) Metabolism of diethylnitrosamine by nasal mucosa and hepatic microsomes from hamster and rat: species specificity of nasal mucosa. Carcinogenesis, 7, 1323- 1328. Gorsky, L.D. and Hollenberg, P.F. (1989) Metabolism of N-nitrosodimethyl and N-nitrosodiethylamine by rat hepatocytes: effects of pretreatment with ethanol. Chem. Res. Toxicol., 2, 436 -441. McCoy, G.D. and Koop, D.R. (1988) Reconstitution of rabbit liver microsomal N-nitrosopyrrolidine cY-hydroxylase activity. Cancer Res., 48, 3987 - 3992. McCoy, G.D., Chen, C.B., Hecht, S.S. and McCoy, E.C. (1979) Enhanced metabolism and mutagenesis of nitrosopyrrolidine in liver fractions isolated from chronic ethanol-consuming hamster. Cancer Res., 39, 793 - 796. Farianti, F., Zhou, Z., Bellah, J., Lieber, C.S. and Garro, A.J. (1986) Effect of chronic ethanol consumption on activation of nitrosopynolidine to a mutagen by rat upper alimentary tract, lung and hepatic tissue. Drug Metabl. Disp., 13, 210 - 214. Gold, B. and Brunk, G. (1988) The effect of pyrazole, phenobarbital, ethanol and 3-methylcholanthrene pretreatment on the in vivo and in vitro genotoxicity of Nnitrosopyrrolidine. Carcinogenesis, 9, 1001- 1005. McCoy, G.D., Hecht, S.S. and Furuya, K. (1986) The effect of chronic ethanol consumption on the tumorigenicity of N-nitrosopyrrolidine in male Syrian golden hamsters. Cancer Lett., 33, 151- 159.