229
Mutation Research, 6 5 ( 1 9 7 9 ) 2 2 9 - - 2 5 9 © Elsevier/North-Holland
Biomedical Press
MUTAGENIC, CANCEROGENIC AND TERATOGENIC EFFECTS OF ALCOHOL
GUNTER
OBE and HANSJURGEN
RISTOW
Institut fiir Genetik, Arnimallee 5-7, D-IO00 Berlin 33 (Germany) (Received 12 January 1979) (Revision received 29 March 1979) (Accepted 5 April 1979)
Summary Alcohol is mutagenic, cancerogenic and teratogenic in man. Ethanol is mutagenic via its first metabolite, acetaldehyde. This is substantiated by the findings that acetaldehyde induces chromosomal aberrations, sister-chromatid exchanges and cross-links between DNA strands. Methanol, a contaminant of many alcoholic beverages, is also mutagenic via its metabolite, formaldehyde. In addition, different indirect pathways may lead to mutations by alcohol. The cancerogenic activity of alcohol remains unverified by m o d e m standard carcinogenicity tests. Ethanol and other alcohols, as well as aldehydes, inhibit RNA synthesis in cells and in cell-free transcriptional systems. A reduction of cellular RNA synthesis may play an important role in the mutagenic, carcinogenic and teratogenic activity of alcohol.
Contents A. B. C. D.
Introduction ............................................... Teratogenicity .............................................. Cancerogenicity ............................................. Mutagenicity ................. . ............................. I. P l a n t s a n d f u n g i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. A n i m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. P o i n t m u t a t i o n s ( i n c l u d i n g r e s u l t s w i t h b a c t e r i a ) . . . . . . . . . . . . . . b. D o m i n a n t l e t h a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. C h r o m o s o m a l a b e r r a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. A n i m a l s in v i v o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. H u m a n cells in v i t r o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. S i s t e r - c h r o m a t i d e x c h a n g e s ( S C E ) . . . . . . . . . . . . . . . . . . . . . . . 1. A l c o h o l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. A l d e h y d e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. C h r o m o s o m a l a b e r r a t i o n s in lymphocytes f r o m a l c o h o l i c s . . . . . . . . . . .
230 230 233 234 234 238 238 239 240 240 242 243 243 243 245
230 E . A l c o h o l a n d t h e testis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Biochemical aspects .......................................... a. I n d u c t i o n o f c r o s s - l i n k s b y a c e t a l d e h y d e ....................... b. Effect of alcohols and aldehydes on RNA synthesis ................ G. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
247 248 248 248 251 252 252
/
A. Introduction Alcoholic beverages are complex mixtures of different types of alcohols, aldehydes and esters (e.g. fusel oils) and a large number of different organic (e.g. flavorings) and inorganic (e.g. trace elements)compounds [38,74,86,157, 180]. In this review, attention will be focused on ethanol and its first metabolic product acetaldehyde. Ethanol is ingested voluntarily by man in almost all parts of the world. It leads to a variety of severe physiological, clinical and behavioral consequences [34,43,46,52--55,64,75--79,97,98,103,116,175,200,201]. The consumption o f alcohol is rising and more and more younger people and also females are becoming addicted to alcohol [5,14,121,170,176,178,203]. In West Germany •t~e consumption of pure ethanol in 1977 was 12.25 liters per person, which is sg:~e 4 times more than in 1950 [170] (Fig. 1). Acetaldehyde is the first meta:boli c product of ethanol, formed especially in the liver, but also in other tis~§~es~Acetaldehyde is mainly formed by alcohol dehydrogenase (ADH) in the c y t g ~ ! , by the oxygen-dependent ethanol-oxidizing system (MEOS) and by catalase, whereby the ADH-mediated pathway is the main one [35,36,46,73, 8 8 , t 0 4 , 1 0 5 , 1 4 1 , 1 5 6 , 1 8 1 , 1 8 7 , 1 9 9 ]. For a recent review on the metabolism o f acetaldehyde and its possible role in the actions of alcohol see Lindros [97]. B: .Teratogenicity Ethanol i s a teratogenic agent in man, and is known to induce the fetal alcohol syndrome (FAS) or alcohol embryopathy (AE). Up to now some 400
12 t. o
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8
/ ° 1- . . . . .
/ .....
~.//
....
/"
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6
/'/
UJ o.Y
1950
1955
1960
1965 ;
:;
1970
1975 1977
Year Fig. 1, Consumption, Per person, of pure ethanol (in Hters) in the Federal RepubUc of Germany and Berlin (West) from 1950
to 1977.
Data,are
fr6m
ref.!~?~.~!
:
231
Fig. 2. Girl w i t h t h e severe f o r m o f t h e f e t a l a l c o h o l s y n d r o m e ( A E III) a g e d 1 5 m o n t h s , M i c r o c e p h a l y , e p i c a n t h i c f o l d s , s h o r t u p t u r n e d n o s e , s h o r t n a s o l a b i a l f u r r o w s a n d s m a l l v e r m i l l i o n b o r d e r o f t h e lips. T h e c h i l d s u f f e r s f r o m a c o n g e n i t a l h e a r t f a i l u r e . ( P i c t u r e k i n d l y s u p p l i e d b y P r o f . D r . F. M a j e w s k i , D i l s s e l d o r f.)
children suffering from this teratogenic damage have been described [25,25a, 58,67--69,89,106--112,133,164,179,186]. AE is characterized b y a variety o f developmental and mental defects. In 95 probands with AE the following main defects were found [133]: intrauterine and post-natal growth retardation (89%), microcephaly (84%), physical and mental retardation (88%), hyperactivity (70%), typical anomalies of the face, i.e. epicanthic folds, short upturned nose, small vermilion border of lips and hypoplasia o f the mandible (53--74%). Heart failure, anomalies of the genitalia, joints and palmar creases are rather frequent (Fig. 2). A b o u t 25 different anomalies have been described in children with AE [107,108]. The expression of AE is highly variable, ranging from very mild forms (AE I) to moderate (AE II) and severe forms (AE III) [109,133]. The frequency o f children with AE born to mothers w h o were in the chronic phase of alcoholism is assumed to be in the range of a b o u t 40% [68,111]. Many authors feel that this observation would justify an interruption of the pregnancy [112]. In a prospective study o f the births during two years (1975 and 1976) in the city of ttoubaix, France, Samaille-Villette and Samaille [164] found a frequency of children born with the severe form o f AE of 1 ~ and o f children with all forms of AE of 3 ~ . These findings indicate that AE is nowadays the most frequent teratogenic damage in man. Transposing these data to the situation in the western part o f Germany, some 600 children with the severe form of the AE and some 1800 with all forms of AE are expected to be born per year [112].
232 A teratogenic activity of ethanol has been experimentally induced in chickens [165], mice [24,84,147] and rats [166,188]. The anomalies found were comparable to those in children with AE. Randall et al. [147] fed pregnant C 57 BL/6H mice on a liquid diet containing 25 or 30% of total calories as ethanol from days 5--10 of gestation. They found significantly higher numbers of resorbed and malformed fetuses on gestation day 19, the day on which the pregnant mice were killed. In the malformed fetuses skeletal, ophthalmic, cardiovascular, urogenital and limb anomalies, hydrocephalus and other damage was observed. Other experiments with rats revealed no teratogenic activities of ethanol [ 139]. Up to n o w the etiological factors that induce AE are unknown. Ethanol itself is known to pass the placenta and to be distributed in the fetus [9,19,32,42,113,174,202,206]. The elimination rate of alcohol is slower in the fetus as compared with the mother [174,206]. The latter effect may partly be the result of a very low activity of alcohol dehydrogenase in rodent and human livers, which in man is only 3--4% of the activity in adults [142--146]. In a perfused human fetal liver the capacity to oxidize acetaldehyde is likewise very low [ 142]. Nothing is known a b o u t the distribution of acetaldehyde in human fetuses. The relative inability of the fetus to metabolize ethanol indicates that the acetaldehyde level in the fetus may be low compared with the level in the mother. Kes~iniemi and Sippel [72] treated pregnant rats (Sprague-Dawley) 4 days before term b y intraperitoneal injections with 2 g of ethanol (10% w/v) per kg. After 25 min the concentrations of ethanol and acetaldehyde were determined in the placentas, the embryos and the maternal blood. The ethanol content of the maternal aortic blood, the placental tissue and the fetal tissue was in the same range of 50 pmol per ml blood or per g tissue, indicating that ethanol is distributed freely between maternal and fetal tissues. The acetaldehyde concentration i'n the placenta was only a b o u t 25% of that in the maternal aortic blood, and in the fetal tissue no acetaldehyde could be detected. These results indicate that, at least in rats, acetaldehyde formed in the maternal metabolism is quickly oxidized in the placental tissue, a mechanism which protects the fetus against the highly toxic acetaldehyde. The milk o f lactating w o m e n who consumed alcohol contains ethanol, but no acetaldehyde [71,140]. Randall et al. [147] determined the acetaldehyde content in fetuses of mice after feeding pregnant females with liquid diets, containing 25% of their calories as ethanol, from gestation days 5--10 and 12--18. These authors found on gestation days 11 or 19 an acetaldehyde concentration in the fetal tissue which was 18% (6.7 nmoles/g) and 40% (20.7 nmoles/g), respectively, of that found in the maternal blood. These experiments with mice show that acetaldehyde can be f o u n d in the fetus, b u t clearly on a lower level than in the maternal blood. The authors speculate that the acetaldehyde in the fetus derives from the m o t h e r because of the low fetal capacity to metabolize alcohol. The low level o f acetaldehyde in the fetus indicates a protective barrier of the placenta. These findings indicate that AE m a y result from the action of alcohol rather than that of acetaldehyde. In cytogenetic analyses no elevation o f exchange-type aberrations was found in 48-h blood cultures from 23 children with AE [133].
233 In a reply to a letter in the " N e w England Journal of Medicine" stating that 'Alcohol abuse may have a role in the "fetal alcohol s y n d r o m e " , b u t the syndrome may eventually prove to be a . . . "polydrug-abuse-nutritional-deficitstress-induced fetal s y n d r o m e " ' [ 123 ], Clarren and Smith stated: "Nutritional deprivation, cigarette smoking, drugs and other factors may be shown in the future to potentiate this condition, but, to our knowledge, no complete fetalalcohol-syndrome p h e n o c o p y has been reported in a human being with a negative maternal history of ethanol use. The specific teratogenicity of ethyl alcohol in several animal species lends further credibility to this p o s i t i o n " [26]. C. Cancerogenicity Epidemiological analyses have demonstrated a correlation between alcohol consumed and cancer of the mouth, pharynx, larynx and esophagus [7,29,30, 4 0 , 6 1 , 8 0 , 9 9 , 1 1 5 , 1 2 1 , 1 7 1 , 1 9 5 , 2 0 5 , 2 0 7 ] . The ratios of actual to expected death b y cancer o f the upper digestive tract and the upper respiratory tract in alcoholics and heavy drinkers is estimated to be 2.8 up to 7.2 [171]. From the epidemiological analyses it is n o t entirely clear whether ethanol acts directly or indirectly as a cancerogenic agent. Kissin and Kaley [80] discussed some mechanisms which may be involved in alcohol-mediated carcinogenesis, some o f which will be discussed in the following. (I) Ethanol may change the reactivity of the tissues b y toxic effects, b y malnutrition or b y liver cirrhosis. Toxic effects o f ethanol are well known and m a y have different causes which cannot be discussed here [48,93]. Alcoholics often show nutritional deficiencies [62,194], and their mineral metabolism is often imbalanced [8,45]. For liver cirrhosis, which is frequently associated with alcoholism, see [39,93]. (II) Carcinogenic substances m a y be dissolved in alcoholic beverages. We have already mentioned that alcoholic beverages are highly complex mixtures of different organic and inorganic compounds. One group o f organic constituents, the fusel otis, consists of alcohols other than ethanol and of aldehydes, and is present in most alcoholic beverages [157]. Gibel et al. [50] treated rats 3 times a week orally (0.5 ml) or subcutaneously (0.25 ml) with fusel oil of the following composition: ethanol (0.8%), amyl alcohol (75%), iso-butyl alcohol (15%), n-propyl alcohol (3--4%), fatty acids and esters (0.5%) and furfurol (positive test for this substance). After some weeks of treatment they found precancerous and cancerous effects in the stomach and esophagus. Ethanol alone had no effect in these experiments. In some areas of Central Africa a very high incidence of esophageal cancer has been observed. In these regions (e.g. Eastern Zambia), people are used to drink home-made distilled alcoholic beverages, made from maize (Kachasu), which have been found to contain nitrosamine4ike c o m p o u n d s [118,119]. Other authors, using more sensitive analytical methods, could n o t confirm this result [27]. Nevertheless, a correlation has been found between regions with high esophageal cancer and the consumption of beer brewed from maize [28], indicating that substances m a y be present in this beer and in the Kachasu which, together with the alcohol, lead to the induction o f esophageal cancer. (III) Alcohol m a y increase the diffusion o f carcinogenic substances into the
234 cells. In experiments with dogs ethanol acted as a "barrier breaker" in the esophagus in the sense that it enhanced the permeability of the esophageal mucosa to H ÷ ions. Such a barrier-breaking activity may lead to the exposure of the underlying cells to carcinogenic substances [ 11,163]. (IV) Alcohol inhibits salivation [128], and this m a y lead to a higher concentration of carcinogens from tobacco smoke in the m o u t h region. This is of importance because most alcoholics are smokers as well. The epidemiological evidence that smoking is related to different carcinomas is overwhelming [ 1,33, 51,57,63]. Cigarette-smoke condensates are cancerogenic in the mouse-skin test and in mammalian and h u m a n cells in vitro [10,46a,65,85,208]. For cancer of the m o u t h and the pharynx it has been shown that both alcohol and tobacco smoking add to the risk [207]. McMichael [121] has shown that the trends of laryngeal and esophageal cancer mortalities are unlike that from mortality due to lung cancer, indicating t h a t alcohol rather than tobacco smoke is the determining risk factor for these cancers [80]. In addition to these factors, others may be of importance for the cancerogenic activity of ethanol. Lieber and his collaborators, especially, have shown t h a t chronic alcohol consumption leads to an elevation of the activity of the P-450-dependent microsomal detoxifying enzymes in the small intestine and the liver of man and rats, an effect associated with a h y p e r t r o p h y of the smooth endoplasmic reticulum and an increased clearance of drugs from the circulation, and which m a y explain the interactions between alcohol and different drugs [17,60,70,74,91,92,94,95,125,158,159,160--162,173,177]. In rats the activity of the microsomal benzpyrene hydroxylase is significantly increased, after consumption of alcohol, in the liver [161] and in the upper intestinal mucosa [173]. Seitz et al. [173] came to the conclusion that the "increased incidence of cancer seen among alcoholics may be due, at least in part, to the enhanced capacity of these individuals to activate procarcinogens in the intestine". The same may be true for the enhanced drug-metabolizing capacity of the liver in alcoholics. D. Mutagenicity
L Plants and fungi In some plants, ethanol induces chromosomal breaks, a p h e n o m e n o n comparable to the action of typical mutagens such as alkylating agents. In Vicia faba the chromosome-breaking activity of ethanol has been studied in detail by Rieger and his coworkers in a series of investigations. Their most important results are as follows. (I) Treatment of root tips of Vicia faba seedlings with ethanol leads to chromosomal aberrations of the chromatid type (open breaks and different types of reunions), indicating that ethanol acts in the S-phase of the cell cycle [149]. (II) The frequencies of aberrations are dependent on the alcohol concentration in the range between 10 -3 and 5 × 10 -1 M (Fig. 3). Concentrations higher than 5 X 10 -1 M are lethal, lower than 10 -3 M are w i t h o u t visible chromosome
Mutation Research, 6 5 ( 1 9 7 9 ) 2 2 9 - - 2 5 9 © Elsevier/North-Holland
Biomedical Press
MUTAGENIC, CANCEROGENIC AND TERATOGENIC EFFECTS OF ALCOHOL
GUNTER
OBE and HANSJURGEN
RISTOW
Institut fiir Genetik, Arnimallee 5-7, D-IO00 Berlin 33 (Germany) (Received 12 January 1979) (Revision received 29 March 1979) (Accepted 5 April 1979)
Summary Alcohol is mutagenic, cancerogenic and teratogenic in man. Ethanol is mutagenic via its first metabolite, acetaldehyde. This is substantiated by the findings that acetaldehyde induces chromosomal aberrations, sister-chromatid exchanges and cross-links between DNA strands. Methanol, a contaminant of many alcoholic beverages, is also mutagenic via its metabolite, formaldehyde. In addition, different indirect pathways may lead to mutations by alcohol. The cancerogenic activity of alcohol remains unverified by m o d e m standard carcinogenicity tests. Ethanol and other alcohols, as well as aldehydes, inhibit RNA synthesis in cells and in cell-free transcriptional systems. A reduction of cellular RNA synthesis may play an important role in the mutagenic, carcinogenic and teratogenic activity of alcohol.
Contents A. B. C. D.
Introduction ............................................... Teratogenicity .............................................. Cancerogenicity ............................................. Mutagenicity ................. . ............................. I. P l a n t s a n d f u n g i . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. A n i m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. P o i n t m u t a t i o n s ( i n c l u d i n g r e s u l t s w i t h b a c t e r i a ) . . . . . . . . . . . . . . b. D o m i n a n t l e t h a l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. C h r o m o s o m a l a b e r r a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. A n i m a l s in v i v o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. H u m a n cells in v i t r o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d. S i s t e r - c h r o m a t i d e x c h a n g e s ( S C E ) . . . . . . . . . . . . . . . . . . . . . . . 1. A l c o h o l s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. A l d e h y d e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. C h r o m o s o m a l a b e r r a t i o n s in lymphocytes f r o m a l c o h o l i c s . . . . . . . . . . .
230 230 233 234 234 238 238 239 240 240 242 243 243 243 245
230 E . A l c o h o l a n d t h e testis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Biochemical aspects .......................................... a. I n d u c t i o n o f c r o s s - l i n k s b y a c e t a l d e h y d e ....................... b. Effect of alcohols and aldehydes on RNA synthesis ................ G. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
247 248 248 248 251 252 252
/
A. Introduction Alcoholic beverages are complex mixtures of different types of alcohols, aldehydes and esters (e.g. fusel oils) and a large number of different organic (e.g. flavorings) and inorganic (e.g. trace elements)compounds [38,74,86,157, 180]. In this review, attention will be focused on ethanol and its first metabolic product acetaldehyde. Ethanol is ingested voluntarily by man in almost all parts of the world. It leads to a variety of severe physiological, clinical and behavioral consequences [34,43,46,52--55,64,75--79,97,98,103,116,175,200,201]. The consumption o f alcohol is rising and more and more younger people and also females are becoming addicted to alcohol [5,14,121,170,176,178,203]. In West Germany •t~e consumption of pure ethanol in 1977 was 12.25 liters per person, which is sg:~e 4 times more than in 1950 [170] (Fig. 1). Acetaldehyde is the first meta:boli c product of ethanol, formed especially in the liver, but also in other tis~§~es~Acetaldehyde is mainly formed by alcohol dehydrogenase (ADH) in the c y t g ~ ! , by the oxygen-dependent ethanol-oxidizing system (MEOS) and by catalase, whereby the ADH-mediated pathway is the main one [35,36,46,73, 8 8 , t 0 4 , 1 0 5 , 1 4 1 , 1 5 6 , 1 8 1 , 1 8 7 , 1 9 9 ]. For a recent review on the metabolism o f acetaldehyde and its possible role in the actions of alcohol see Lindros [97]. B: .Teratogenicity Ethanol i s a teratogenic agent in man, and is known to induce the fetal alcohol syndrome (FAS) or alcohol embryopathy (AE). Up to now some 400
12 t. o
/°
10 /.o
.J t¢g e-
8
/ ° 1- . . . . .
/ .....
~.//
....
/"
/o/O~ °/°
6
/'/
UJ o.Y
1950
1955
1960
1965 ;
:;
1970
1975 1977
Year Fig. 1, Consumption, Per person, of pure ethanol (in Hters) in the Federal RepubUc of Germany and Berlin (West) from 1950
to 1977.
Data,are
fr6m
ref.!~?~.~!
:
231
Fig. 2. Girl w i t h t h e severe f o r m o f t h e f e t a l a l c o h o l s y n d r o m e ( A E III) a g e d 1 5 m o n t h s , M i c r o c e p h a l y , e p i c a n t h i c f o l d s , s h o r t u p t u r n e d n o s e , s h o r t n a s o l a b i a l f u r r o w s a n d s m a l l v e r m i l l i o n b o r d e r o f t h e lips. T h e c h i l d s u f f e r s f r o m a c o n g e n i t a l h e a r t f a i l u r e . ( P i c t u r e k i n d l y s u p p l i e d b y P r o f . D r . F. M a j e w s k i , D i l s s e l d o r f.)
children suffering from this teratogenic damage have been described [25,25a, 58,67--69,89,106--112,133,164,179,186]. AE is characterized b y a variety o f developmental and mental defects. In 95 probands with AE the following main defects were found [133]: intrauterine and post-natal growth retardation (89%), microcephaly (84%), physical and mental retardation (88%), hyperactivity (70%), typical anomalies of the face, i.e. epicanthic folds, short upturned nose, small vermilion border of lips and hypoplasia o f the mandible (53--74%). Heart failure, anomalies of the genitalia, joints and palmar creases are rather frequent (Fig. 2). A b o u t 25 different anomalies have been described in children with AE [107,108]. The expression of AE is highly variable, ranging from very mild forms (AE I) to moderate (AE II) and severe forms (AE III) [109,133]. The frequency o f children with AE born to mothers w h o were in the chronic phase of alcoholism is assumed to be in the range of a b o u t 40% [68,111]. Many authors feel that this observation would justify an interruption of the pregnancy [112]. In a prospective study o f the births during two years (1975 and 1976) in the city of ttoubaix, France, Samaille-Villette and Samaille [164] found a frequency of children born with the severe form o f AE of 1 ~ and o f children with all forms of AE of 3 ~ . These findings indicate that AE is nowadays the most frequent teratogenic damage in man. Transposing these data to the situation in the western part o f Germany, some 600 children with the severe form of the AE and some 1800 with all forms of AE are expected to be born per year [112].
232 A teratogenic activity of ethanol has been experimentally induced in chickens [165], mice [24,84,147] and rats [166,188]. The anomalies found were comparable to those in children with AE. Randall et al. [147] fed pregnant C 57 BL/6H mice on a liquid diet containing 25 or 30% of total calories as ethanol from days 5--10 of gestation. They found significantly higher numbers of resorbed and malformed fetuses on gestation day 19, the day on which the pregnant mice were killed. In the malformed fetuses skeletal, ophthalmic, cardiovascular, urogenital and limb anomalies, hydrocephalus and other damage was observed. Other experiments with rats revealed no teratogenic activities of ethanol [ 139]. Up to n o w the etiological factors that induce AE are unknown. Ethanol itself is known to pass the placenta and to be distributed in the fetus [9,19,32,42,113,174,202,206]. The elimination rate of alcohol is slower in the fetus as compared with the mother [174,206]. The latter effect may partly be the result of a very low activity of alcohol dehydrogenase in rodent and human livers, which in man is only 3--4% of the activity in adults [142--146]. In a perfused human fetal liver the capacity to oxidize acetaldehyde is likewise very low [ 142]. Nothing is known a b o u t the distribution of acetaldehyde in human fetuses. The relative inability of the fetus to metabolize ethanol indicates that the acetaldehyde level in the fetus may be low compared with the level in the mother. Kes~iniemi and Sippel [72] treated pregnant rats (Sprague-Dawley) 4 days before term b y intraperitoneal injections with 2 g of ethanol (10% w/v) per kg. After 25 min the concentrations of ethanol and acetaldehyde were determined in the placentas, the embryos and the maternal blood. The ethanol content of the maternal aortic blood, the placental tissue and the fetal tissue was in the same range of 50 pmol per ml blood or per g tissue, indicating that ethanol is distributed freely between maternal and fetal tissues. The acetaldehyde concentration i'n the placenta was only a b o u t 25% of that in the maternal aortic blood, and in the fetal tissue no acetaldehyde could be detected. These results indicate that, at least in rats, acetaldehyde formed in the maternal metabolism is quickly oxidized in the placental tissue, a mechanism which protects the fetus against the highly toxic acetaldehyde. The milk o f lactating w o m e n who consumed alcohol contains ethanol, but no acetaldehyde [71,140]. Randall et al. [147] determined the acetaldehyde content in fetuses of mice after feeding pregnant females with liquid diets, containing 25% of their calories as ethanol, from gestation days 5--10 and 12--18. These authors found on gestation days 11 or 19 an acetaldehyde concentration in the fetal tissue which was 18% (6.7 nmoles/g) and 40% (20.7 nmoles/g), respectively, of that found in the maternal blood. These experiments with mice show that acetaldehyde can be f o u n d in the fetus, b u t clearly on a lower level than in the maternal blood. The authors speculate that the acetaldehyde in the fetus derives from the m o t h e r because of the low fetal capacity to metabolize alcohol. The low level o f acetaldehyde in the fetus indicates a protective barrier of the placenta. These findings indicate that AE m a y result from the action of alcohol rather than that of acetaldehyde. In cytogenetic analyses no elevation o f exchange-type aberrations was found in 48-h blood cultures from 23 children with AE [133].
233 In a reply to a letter in the " N e w England Journal of Medicine" stating that 'Alcohol abuse may have a role in the "fetal alcohol s y n d r o m e " , b u t the syndrome may eventually prove to be a . . . "polydrug-abuse-nutritional-deficitstress-induced fetal s y n d r o m e " ' [ 123 ], Clarren and Smith stated: "Nutritional deprivation, cigarette smoking, drugs and other factors may be shown in the future to potentiate this condition, but, to our knowledge, no complete fetalalcohol-syndrome p h e n o c o p y has been reported in a human being with a negative maternal history of ethanol use. The specific teratogenicity of ethyl alcohol in several animal species lends further credibility to this p o s i t i o n " [26]. C. Cancerogenicity Epidemiological analyses have demonstrated a correlation between alcohol consumed and cancer of the mouth, pharynx, larynx and esophagus [7,29,30, 4 0 , 6 1 , 8 0 , 9 9 , 1 1 5 , 1 2 1 , 1 7 1 , 1 9 5 , 2 0 5 , 2 0 7 ] . The ratios of actual to expected death b y cancer o f the upper digestive tract and the upper respiratory tract in alcoholics and heavy drinkers is estimated to be 2.8 up to 7.2 [171]. From the epidemiological analyses it is n o t entirely clear whether ethanol acts directly or indirectly as a cancerogenic agent. Kissin and Kaley [80] discussed some mechanisms which may be involved in alcohol-mediated carcinogenesis, some o f which will be discussed in the following. (I) Ethanol may change the reactivity of the tissues b y toxic effects, b y malnutrition or b y liver cirrhosis. Toxic effects o f ethanol are well known and m a y have different causes which cannot be discussed here [48,93]. Alcoholics often show nutritional deficiencies [62,194], and their mineral metabolism is often imbalanced [8,45]. For liver cirrhosis, which is frequently associated with alcoholism, see [39,93]. (II) Carcinogenic substances m a y be dissolved in alcoholic beverages. We have already mentioned that alcoholic beverages are highly complex mixtures of different organic and inorganic compounds. One group o f organic constituents, the fusel otis, consists of alcohols other than ethanol and of aldehydes, and is present in most alcoholic beverages [157]. Gibel et al. [50] treated rats 3 times a week orally (0.5 ml) or subcutaneously (0.25 ml) with fusel oil of the following composition: ethanol (0.8%), amyl alcohol (75%), iso-butyl alcohol (15%), n-propyl alcohol (3--4%), fatty acids and esters (0.5%) and furfurol (positive test for this substance). After some weeks of treatment they found precancerous and cancerous effects in the stomach and esophagus. Ethanol alone had no effect in these experiments. In some areas of Central Africa a very high incidence of esophageal cancer has been observed. In these regions (e.g. Eastern Zambia), people are used to drink home-made distilled alcoholic beverages, made from maize (Kachasu), which have been found to contain nitrosamine4ike c o m p o u n d s [118,119]. Other authors, using more sensitive analytical methods, could n o t confirm this result [27]. Nevertheless, a correlation has been found between regions with high esophageal cancer and the consumption of beer brewed from maize [28], indicating that substances m a y be present in this beer and in the Kachasu which, together with the alcohol, lead to the induction o f esophageal cancer. (III) Alcohol m a y increase the diffusion o f carcinogenic substances into the
234 cells. In experiments with dogs ethanol acted as a "barrier breaker" in the esophagus in the sense that it enhanced the permeability of the esophageal mucosa to H ÷ ions. Such a barrier-breaking activity may lead to the exposure of the underlying cells to carcinogenic substances [ 11,163]. (IV) Alcohol inhibits salivation [128], and this m a y lead to a higher concentration of carcinogens from tobacco smoke in the m o u t h region. This is of importance because most alcoholics are smokers as well. The epidemiological evidence that smoking is related to different carcinomas is overwhelming [ 1,33, 51,57,63]. Cigarette-smoke condensates are cancerogenic in the mouse-skin test and in mammalian and h u m a n cells in vitro [10,46a,65,85,208]. For cancer of the m o u t h and the pharynx it has been shown that both alcohol and tobacco smoking add to the risk [207]. McMichael [121] has shown that the trends of laryngeal and esophageal cancer mortalities are unlike that from mortality due to lung cancer, indicating t h a t alcohol rather than tobacco smoke is the determining risk factor for these cancers [80]. In addition to these factors, others may be of importance for the cancerogenic activity of ethanol. Lieber and his collaborators, especially, have shown t h a t chronic alcohol consumption leads to an elevation of the activity of the P-450-dependent microsomal detoxifying enzymes in the small intestine and the liver of man and rats, an effect associated with a h y p e r t r o p h y of the smooth endoplasmic reticulum and an increased clearance of drugs from the circulation, and which m a y explain the interactions between alcohol and different drugs [17,60,70,74,91,92,94,95,125,158,159,160--162,173,177]. In rats the activity of the microsomal benzpyrene hydroxylase is significantly increased, after consumption of alcohol, in the liver [161] and in the upper intestinal mucosa [173]. Seitz et al. [173] came to the conclusion that the "increased incidence of cancer seen among alcoholics may be due, at least in part, to the enhanced capacity of these individuals to activate procarcinogens in the intestine". The same may be true for the enhanced drug-metabolizing capacity of the liver in alcoholics. D. Mutagenicity
L Plants and fungi In some plants, ethanol induces chromosomal breaks, a p h e n o m e n o n comparable to the action of typical mutagens such as alkylating agents. In Vicia faba the chromosome-breaking activity of ethanol has been studied in detail by Rieger and his coworkers in a series of investigations. Their most important results are as follows. (I) Treatment of root tips of Vicia faba seedlings with ethanol leads to chromosomal aberrations of the chromatid type (open breaks and different types of reunions), indicating that ethanol acts in the S-phase of the cell cycle [149]. (II) The frequencies of aberrations are dependent on the alcohol concentration in the range between 10 -3 and 5 × 10 -1 M (Fig. 3). Concentrations higher than 5 X 10 -1 M are lethal, lower than 10 -3 M are w i t h o u t visible chromosome
