Chapter 17
The Role of Wine in Ethyl Carbamate Induced Carcinogenesis Inhibition 1
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Gilbert S. Stoewsand , J . L. Anderson , and L. Munson
2
1
Department of Food Science and Technology, Cornell University, Geneva, NY 14456 Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37901 2
A 12% (v/v) ethanol solution and, to a greater extent, wine fed to C3H male mice inhibited tumorigenesis in their liver and lungs induced by the carcinogen, ethyl carbamate. Ethyl carbamate (urethane), a water soluble carcinogen originally used for numerous commercial processes, is a contaminant of fermentation. Wine phenols used in these studies, i.e. caffeic acid, catechin hydrate, and gallic acid, without the presence of ethanol, appeared to enhance liver, but not lung, tumor incidence and frequency. Thus, outside of the presence of ethanol in wine, no other wine constituents have been identified that affords protection against cancer development of ethyl carbamate. Any cancer risk assessment of ethyl carbamate in wine should take into account this ethanol interaction.
Ethyl carbamate (EC), also known as urethane, urethan, or carbamic acid ethyl ester, was formerly used in producing amino resins, as a solvent in the manufacture of pesticides, fumigants and cosmetics and as an antineoplastic drug. These commercial purposes have ceased and the main public exposure to E C is from its presence as a natural contaminant present in alcoholic beverages and other fermented products such as soy sauce (1-3). In one of the largest surveys on wines, E C present in 261 wine samples ranged from undetected to 102 | i g / L (4). The U . S. Food and Drug Administration reached an agreement in 1988 with the American Association of Vintners and the Wine Institute that the weighted average of E C in table wines, containing 14 percent or less alcohol by volume, "is not to exceed 15 parts per billion ^ g / L ) starting with wines produced from the 1988 harvest crush" (5). Guidelines in Canada were established in 1985 limiting the content of EC to 30 μg/L (6). Carcinogenesis by E C Exposure EC, the first water soluble carcinogen discovered, can induce many types of tumors in the lungs, liver, thymus, skin and mammary tissue of laboratory animals (7). Distribution of E C after dosing, whether by oral or dermal routes (8) or intraperitoneal injection (9), is widely distributed in tissues and organs. A review of metabolism, carcinogenic effects and risks from E C exposure has been published (10). Carcinogenesis is initiated by the oxidation of E C that is probably accomplished by the
© 1997 American Chemical Society
Watkins; Wine ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
17. STOEWSAND ET A L Ethyl Carbamate Induced Carcinogenesis Inhibition
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specific co-factor, cytochrome P-450 ΠΕ1 to a vinyl carbamate, and then epoxidation to an epoxyethyl species forming etheno adducts with nucleic acids in D N A and R N A of various tissues (ll-14).These studies on carcinogenesis mechanisms have been accomplished with rodents, but malignant tumors have been observed in nonhuman primates dosed over a long period with EC (15).
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Ethanol Interaction with E C Waddell, et al (16) discovered that radioactive EC administered to mice in a 12% ethanol solution showed an inhibition of radioactivity in tissues as compared to the localization of radioactivity in liver, bone marrow, pancreas and other tissues when E C was administered in water. Further studies in this laboratory (17) showed that ethanol delayed E C metabolism with an inhibition of active metabolites. Ethanol inhibits micronuclei induction by E C in mouse bone marrow erythrocytes (18). The microsomal ethanol oxidizing system (MEOS) plays a significant role in ethanol oxidation at high ethanol intake or chronic use of alcohol. Cytochrome P450ΠΕ1 is a necessary co-factor in this MEOS pathway (19). Strain A female mice drinking 10 or 20% ethanol solutions for 12 weeks exhibited significantly reduced incidences of EC-induced pulmonary tumors (20). Studies in our laboratory showed that a 12% ethanol solution, or white or red wines containing this same level of ethanol, fed to C 3 H mice inhibited E C induced hepatocellular adenomas, hepatocellular carcinomas, as well as pulmonary tumors (21). Ethanol inhibition of E C induced carcinogenesis may be due, in part, to competitive inhibition for cytochrome Ρ-450ΠΕ1. Metabolism of ethanol, especially under continuous intake, requires this co-factor with the M E O S . The limitation of cytochrome Ρ-450ΠΕ1 for concomitant E C metabolism to effectively form the ultimate carcinogen, namely the E C epoxide metabolite, could play a role in this interactive mechanism. Wine Interaction with E C Our initial studies showed that an EC level of 20mg/kg/day in wine given to C3H male mice for 41 weeks resulted in a decreased incidence and frequency of hepatocellular adenomas and other liver tumors as compared to mice drinking water, and even somewhat to a greater extent than ethanol fed mice (21). After alcohol, acids, residual sugars, and perhaps proline, phenols are the constituents major in amount in many wines (22). The wine phenol known as caffeic acid, a nonflavonoid, is found in both red and white wines in appreciable amounts (22). Since caffeic acid has been shown to inhibit tumor development in mice (23,24), we conducted a study to determine if caffeic acid, together with either water or a 12% ethanol solution, could inhibit lung and/or liver tumorigenesis in C3H male mice induced by E C (10 mg/kg body wt/day) with ad libitum drinking. Caffeic acid was mixed into a semi-purified diet at 0, 200 or 400 mg/kg of diet and the mice were treated via a similar experimental protocol described in our initial investigation (21). The results of this study, as mean adenoma frequency (number of tumors/tumor bearing mice) in both liver and lung, are presented in Fig. 1. Tumor frequency in both mouse tissues were lower with ethanol treatments as repeated from our earlier work (21). Dietary caffeic acid at 200 mg/kg slightly lowered tumor frequency in the liver of ethanol treated animals but this lowered frequency did not continue at the highest caffeic acid level. Indeed, the highest caffeic acid dietary level with water treatment produced the highest frequency of liver tumors. About 75% of the total number of mice in this study exhibiting liver tumors were on this caffeic acid/water treatment. Caffeic acid produced no changes, in either ethanol or water treatments, on the frequency of lung tumors (Fig. 1). It appeared clear that caffeic acid did not enhance E C induced tumor inhibition and may even be responsible, at high dietary levels without ethanol, for liver tumor development.
Watkins; Wine ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
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WINE: NUTRITIONAL AND THERAPEUTIC BENEFITS
(a) 5.0
• Water Z3 12% Ethanol
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9 g
3.0
ο
2.0 1.0
τ
0.0 OCA
(b) 3.0
• 0
200 mg/kg C A 400 mg/kg C A
Water 12% Ethanol
OCA
200 mg/kg C A 400 mg/kg C A
Figure 1· Mean tumor frequency (number of tumors/tumor bearing mice) of EC fed mice, a) Liver b) Lung CA = Caffeic acid E C = Ethyl carbamate Wine Phenols. We conducted a more complete study of wine phenolics using the averaged amounts of three major phenolics (P) estimated content in red and white wines (22). Ρ was added to either a 12% ethanol (v/v) solution or water as: caffeic acid, catechin hydrate, and gallic acid at 140, 50, and 25 mg/L, respectively. The wine (Cayuga White/Seyval 50:50 blend), was adjusted to 12% ethanol. In addition, a commercial non-alcoholic (N/A) wine, actually a dealcoholized Chardonnay (.05% ethanol), and water control completed the five treatments used in this 41 week feeding study. The purified diet used in the past (21) was again fed to the mice, but only for 12 hrs during the night (-7:30 pm to 7:30 am) in order to attempt to stop diet wastage and excess body weights. The liquids were fed ad libitum with either 0 or 20 mg/kg body wt/day of EC. The incidence and frequency of the two tumor types observed in the liver by detailed pathology, hepatocellular adenomas and hemangioendotheliomas, are presented in Table I. The incidence of spontaneous adenomas, as seen in the 0 E C
Watkins; Wine ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
Watkins; Wine ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
b
0 0 20 20 20 20 20
Water
Wine
Ethanol + Ρ
N/AWine
Water + Ρ
Water
0
17.4
1
0
58.3
77.7
83.3
56.5
47.8
45.8
33.3
1.9±0.5 1.2±0.5
2
1.6±0.5
1.5±0.7
2.2±0.7
0
0
0
0
0
4
4
4
4
4
Hemangio endothelioma
3
2.4±0.4 3
3.1±0.3
3.5 ±0.33
37.5 31.8
1.510.4 »
17.4
2
1.6±0.4U
17.4
1
1.610.4 »
2
2
2
0
1
1.610.4 *
1.5 + 0.3 *
0
70.8 66.7
1
0
1.010.7 1.4 ± 0 . 5 U
1
Hepatocellular Adenoma
Frequency**
31.8
2
Hemangio endothelioma
Hepatocellular Adenoma
a
d
c
a Percentage of mice with tumors (n= 21-24). Significant (p
