Br. J. exp. Path. (1977) 58, 606
HEPATIC TRIGLYCERIDE ACCUMULATION AND THE ETHANOL PHYSICAL WITHDRAWAL SYNDROME IN MICE C. ABU MURAD, S. J. BEGG, P. J. GRIFFITHS* AND J. M. LITTLETON Fromn the Department of Pharnmacology, King's College, Strand, London WC2R 2LS Received for publication April 1, 1977
Summary.-Physical dependence on ethanol was induced in TO strain mice by chronic administration of ethanol by inhalation. The severity of the behavioural syndrome of withdrawal from ethanol was quantified by a subjective scoring method. During the chronic administration of ethanol, triglycerides accumulated in livers of male or female mice with a time course similar to that of the induction of physical dependence. When ethanol was withdrawn from adult or weanling dependent mice, a relationship was observed between the decline of triglyceride concentrations in liver and the duration of the ethanol withdrawal syndrome. The addition of DL-carnitine (7%0 w/w) to diet during the administration of ethanol markedly inhibited the accumulation of triglycerides, and significantly reduced the intensity of the ethanol withdrawal syndrome. Administration of carbon tetrachloride ((1 .3 ml/kg i.p.), however, although augmenting hepatic triglyceride accumulation, had no significant effect on the withdrawal syndrome. The results are interpreted as suggesting either that ethanol-induced liver dysfunction plays a part in dependence, or, more likely, that triglyceride accumulation reflects an ethanol -induced metabolic disorder which is itself related to the induction of dependence. THE use of animal models to elucidate the mechanism of ethanol dependence has concentrated on ethanol-induced changes in central neurotransmitter metabolism (Littleton, 1975). A variety of changes have been reported, some of which seem to apply to most models, whereas others are reported comparatively rarely (e.g. Pohorecky, Jaffe and Berkeley, 1974; Hunt and Majchrowicz, 1974). It now seems very likely that some of the observed changes in neurotransmitter metabolism are important in determining the expression of the ethanol withdrawal syndrome (Littleton, 1975). However, the wide variety of neurotransmitter changes reported in ethanol-dependent animals suggests that it may be futile to pursue this approach, and that some more fundamental alteration of brain metabolism may be of primary importance in the induction of physical dependence. We have chosen *
to examine the possibility that an ethanolinduced alteration in hepatic function might alter brain metabolism in such a way as to predispose to physical depend-
ence.
The recognition of an association between liver damage and alcoholism is by no means new, and fatty change in liver has long been recognized as a sign of excessive ethanol consumption in man (Lieber and Rubin, 1968). It is not, however, clear what part these pathological changes may play in the development of alcoholism, and whether they contribute in any way to the establishment of the addicted state. Such hypotheses would be very difficult to test in clinical alcoholism, where presentation is normally delayed until the condition is established. In animal models,however,the
development of dependence and of ethanolinduced liver dysfunction can be studied together. For example, preliminary find-
Present address: Battelle Institute, 7 rue de Drize, 1227 Carouge, Geneva, Switzerland.
ETHANOL WITHDRAWAL AND HEPATIC TRIGLYCERIDE ACCUMULATION
ings (Abu Murad, Griffiths and Littleton, 1976) suggested that, in mice exposed to ethanol vapour, the time course of accumulation of triglycerides in liver (fatty change) was similar to the time course of induction of physical dependence. This paper explores this relationship further and demonstrates that, in many situations, there is an association between the degree of dependence (as shown by the severity of the ethanol withdrawal syndrome) and the amount of triglyceride accumulated by liver. It should be emphasized that this relationship applies only to the triglyceride accumulation produced by chronic administration of ethanol. The triglyceride accumulation in liver induced by single large doses of ethanol seems to be mediated by a different mechanism (Kalant et al., 1975) and is unlikely to have any relationship to dependence. MATERIALS AND METHODS
Induction of ethanol dependence.-Ethanol dependence was induced in groups of mice by administration of ethanol by inhalation (Griffiths, Littleton and Ortiz, 1973, 1974). Ethanol withdrawal severity was assessed by a subjective procedure iri which the major signs of withdrawal (convulsions, tremor, piloerection and tail erection) are scored 0 to 4 for severity
(Griffiths et al., 1974). In most animals the withdrawal syndrome lasts about 10 h. This period has therefore been used for assessment of the syndrome, unless stated otherwise in the text. Measurement of ethanol concentrations.-Ethanol concentrations in inspired air were measured by taking 1 ml of air from the inhalation chamber into a gas-tight syringe and injecting this sample directly on to the gas liquid chromatograph (glc) column described previously (Griffiths et ai., 1974). Blood ethanol concentrations were measured in the air of the head space above a protein-precipitated, centrifuged blood sample of mixed arterio-venous blood obtained after decapitation (Griffiths et al., 1974). It has been found possible to obtain an accurate estimate of the blood ethanol concentration in the living mouse by measuring the concentration of ethanol in the expired air. This concentration was measured by forcing individual mice to rebreathe air in a 5-ml chamber for 30 sec. At the end of this period a 1-ml sample of air was withdrawn and injected on to the glc column. Several experiments, in which estimated blood
607
ethanol concentrations of individual mice were verified by subsequent killing and direct measurement, established the validity and reproducibility of this method. It has been used routinely in this study. Meassurement of hepatic triglyceride concentration. Mice were killed by whole-body immersion in liquid nitrogen. Livers were dissected in the cold, weighed and taken for triglyceride estimation. Total triglyceride content of each liver was measured by a modification of the method of van Handel and Zilversmit (1957) after extraction of lipids by the method of Folch, Lees and Sloane Stanley (1957). Experimental design.-To investigate the relationship between the time course of accumulation of triglycerides in liver and the induction of ethanol dependence, 100 male TO mice, 2025 g, were exposed to ethanol vapour in the usual way. On each day, 10 mice were removed from the inhalation chamber. Five were killed for estimation of hepatic triglycerides and 5 were taken for assessment of the severity of the ethanol withdrawal syndrome. To further investigate any relationship between the concentration of triglycerides in liver and the severity of ethanol withdrawal a direct comparison was made between male and female mice. It has previously been reported that, when exposed to the same concentration of ethanol vapour, female mice show a significantly less severe ethanol withdrawal syndrome than males (Goldstein, 1973). It was therefore decided to compare also the time course of accumulation of triglycerides in liver with the time course of induction of ethanol dependence in female mice. This experiment was carried out at the same time and in exactly the same way as that described above. The possibility that some relationship might exist between the time course of the fall of hepatic triglyceride concentrations after the withdrawal of ethanol and the time course of the withdrawal syndrome was also considered. This was tested by comparing the fall in triglyceride concentrations and the withdrawal syndrome in adult and weanling mice. It had previously been noted (unpublished) that when adult and weanling mice were exposed to the same ethanol concentration weanling mice showed a significantly shorter duration of the ethanol withdrawal syndrome, although severity at the peak of withdrawal was very similar. In the experiments reported here 50 male TO mice, 30-35 g, were compared with 50 male TO mice, 8-10 g. Each group was exposed to ethanol vapour in the same chamber for 10 days. Blood ethanol concentrations were estimated frequently by the expired air method. After 10 days, 5 mice of each group were removed from the inhalation chamber and immediately killed for hepatic triglyceride measurement. The remainder were used
608
C. ABU MURAD, S. J. BEGG, P. J. GRIFFITHS AND J. M. LITTLETON
to assess the time course and severity of the withdrawal syndrome. After 12 h 5 mice of each group were killed for estimation of hepatic triglyceride concentrations. Testing for withdrawal signs in the remaining animals continued until no evidence of ethanol withdrawal was apparent. Some experiments were made to investigate the effects of drug-induced alteration of hepatic triglyceride accumulation on the ethanol withdrawal syndrome. The administration of DL-carnitine has been reported previously to inhibit ethanol-induced triglyceride accumulation (Hosein and Bexton, 1975). We therefore compared two groups of 30 male TO mice, 20-25 g, in an experiment in which one group received DL-carnitine (7% w/w) in diet, while the other group received the normal laboratory diet. Both groups were exposed to ethaniol vapour in the same inhalation chamber for 10 days. Ten mice of each group were killed for estimation of hepatic triglyceride concentration immediately before ethanol withdrawal. The remainder were used to assess the severity of the ethanol withdrawal syndrome. Frequent estimations of blood ethanol concentration were made throughout the period of the experiment. The i.p. injection of carbon tetrachloride has been reported to potentiate ethanol-induced triglyceride accumulation in liver (Strubelt, BuettnerandSiegers, 1975). Theeffectsofpotentiating triglyceride accumulation on the induction of ethanol dependence were therefore tested in experiments in which carbon tetrachloride (1.3 ml/kg i.p.) was administered to one group of mice (30 male TO, 20-25 g) and arachis oil, the vehicle for carbon tetrachloride, was administered to a similar group during the exposure of both groups to the same concentration of ethanol vapour. This experiment was performed in two ways: in one, the additional treatments were administered on the first day of ethanol administration; in the other, the additional treatments were given on the penultimate day of ethanol exposure. In both cases frequent estimations of blood ethanol concentrations were made. Ten animals from each group were killed for hepatic triglyceride measurement before ethanol withdrawal, and the remainder used for assessment of the withdrawal syndrome. In all these experiments comparison of biochemical and behavioural parameters has been made between the experimental groups and control animals exposed to the same conditions, except for the absence of ethanol from the
All reagents used for biochemical analysis
were Analar grade when these were commercially available. Ethanol (99-8 %, A.R. grade) was
obtained from James Burroughs Ltd, DL carnitine HCI (99 %) was obtained from Aldrich Chemical Co Ltd, carbon tetrachloride (S.L.R. grade) from Fisons, and arachis oil (special for injections) from Boots Ltd. RESULTS
Time course of accumulation of triglycerides in livers of male and female TO mice: relation to withdrawal syndrome The results, shown in Figs la and Ib, indicate a very similar time course for the ethanol-induced accumulation of triglycerides in liver and the onset of a recognizable ethanol withdrawal syndrome. In male mice significant triglyceride accumulation does not occur until after 5 days of ethanol administration (Fig. la). The onset of a withdrawal syndrome follows closely so that, after 6 days of administration, a recognizable syndrome can be observed in some male animals. The appearance of significant changes in these parameters is slightly delayed in female mice, but, once again, there is close agreement between the time courses (Fig. lb). The intensity of the withdrawal syndrome after 10 days confirms that female mice suffer a less severe withdrawal syndrome than males exposed to the same concentration of ethanol vapour. It is important to note that in this experiment no attempt was made to maintain the blood ethanol concentration at the same level in the two sexes. Female mice do, in fact, show significantly lower blood ethanol concentrations than males throughout this regime of ethanol administration.
Time course of reversal of ethanol-induced triglyceride accumulation in adult and weanling mice: relation to withdrawal syndrome inspired air. When weanling and mature adult mice Animals and materials.-TO strain white mice were obtained from Animal Suppliers Ltd, were exposed to the same concentration of Welwyn. Mice were maintained on Spillers ethanol vapour, similar blood ethanol Labsure 41B diet. Food and drinking water were concentrations were obtained. Both groups freely available throughout every experiment. showed accumulation of triglycerides in
609
ETHANOL WITHDRAWAL AND HEPATIC TRIGLYCERIDE ACCUMULATION
liver after 10 days (Table I). The accumulation of triglycerides in liver of weanling animals is greater than that in adult animals; this difference is significant at the P
HEPATIC TRIGLYCERIDE ACCUMULATION AND THE ETHANOL PHYSICAL WITHDRAWAL SYNDROME IN MICE C. ABU MURAD, S. J. BEGG, P. J. GRIFFITHS* AND J. M. LITTLETON Fromn the Department of Pharnmacology, King's College, Strand, London WC2R 2LS Received for publication April 1, 1977
Summary.-Physical dependence on ethanol was induced in TO strain mice by chronic administration of ethanol by inhalation. The severity of the behavioural syndrome of withdrawal from ethanol was quantified by a subjective scoring method. During the chronic administration of ethanol, triglycerides accumulated in livers of male or female mice with a time course similar to that of the induction of physical dependence. When ethanol was withdrawn from adult or weanling dependent mice, a relationship was observed between the decline of triglyceride concentrations in liver and the duration of the ethanol withdrawal syndrome. The addition of DL-carnitine (7%0 w/w) to diet during the administration of ethanol markedly inhibited the accumulation of triglycerides, and significantly reduced the intensity of the ethanol withdrawal syndrome. Administration of carbon tetrachloride ((1 .3 ml/kg i.p.), however, although augmenting hepatic triglyceride accumulation, had no significant effect on the withdrawal syndrome. The results are interpreted as suggesting either that ethanol-induced liver dysfunction plays a part in dependence, or, more likely, that triglyceride accumulation reflects an ethanol -induced metabolic disorder which is itself related to the induction of dependence. THE use of animal models to elucidate the mechanism of ethanol dependence has concentrated on ethanol-induced changes in central neurotransmitter metabolism (Littleton, 1975). A variety of changes have been reported, some of which seem to apply to most models, whereas others are reported comparatively rarely (e.g. Pohorecky, Jaffe and Berkeley, 1974; Hunt and Majchrowicz, 1974). It now seems very likely that some of the observed changes in neurotransmitter metabolism are important in determining the expression of the ethanol withdrawal syndrome (Littleton, 1975). However, the wide variety of neurotransmitter changes reported in ethanol-dependent animals suggests that it may be futile to pursue this approach, and that some more fundamental alteration of brain metabolism may be of primary importance in the induction of physical dependence. We have chosen *
to examine the possibility that an ethanolinduced alteration in hepatic function might alter brain metabolism in such a way as to predispose to physical depend-
ence.
The recognition of an association between liver damage and alcoholism is by no means new, and fatty change in liver has long been recognized as a sign of excessive ethanol consumption in man (Lieber and Rubin, 1968). It is not, however, clear what part these pathological changes may play in the development of alcoholism, and whether they contribute in any way to the establishment of the addicted state. Such hypotheses would be very difficult to test in clinical alcoholism, where presentation is normally delayed until the condition is established. In animal models,however,the
development of dependence and of ethanolinduced liver dysfunction can be studied together. For example, preliminary find-
Present address: Battelle Institute, 7 rue de Drize, 1227 Carouge, Geneva, Switzerland.
ETHANOL WITHDRAWAL AND HEPATIC TRIGLYCERIDE ACCUMULATION
ings (Abu Murad, Griffiths and Littleton, 1976) suggested that, in mice exposed to ethanol vapour, the time course of accumulation of triglycerides in liver (fatty change) was similar to the time course of induction of physical dependence. This paper explores this relationship further and demonstrates that, in many situations, there is an association between the degree of dependence (as shown by the severity of the ethanol withdrawal syndrome) and the amount of triglyceride accumulated by liver. It should be emphasized that this relationship applies only to the triglyceride accumulation produced by chronic administration of ethanol. The triglyceride accumulation in liver induced by single large doses of ethanol seems to be mediated by a different mechanism (Kalant et al., 1975) and is unlikely to have any relationship to dependence. MATERIALS AND METHODS
Induction of ethanol dependence.-Ethanol dependence was induced in groups of mice by administration of ethanol by inhalation (Griffiths, Littleton and Ortiz, 1973, 1974). Ethanol withdrawal severity was assessed by a subjective procedure iri which the major signs of withdrawal (convulsions, tremor, piloerection and tail erection) are scored 0 to 4 for severity
(Griffiths et al., 1974). In most animals the withdrawal syndrome lasts about 10 h. This period has therefore been used for assessment of the syndrome, unless stated otherwise in the text. Measurement of ethanol concentrations.-Ethanol concentrations in inspired air were measured by taking 1 ml of air from the inhalation chamber into a gas-tight syringe and injecting this sample directly on to the gas liquid chromatograph (glc) column described previously (Griffiths et ai., 1974). Blood ethanol concentrations were measured in the air of the head space above a protein-precipitated, centrifuged blood sample of mixed arterio-venous blood obtained after decapitation (Griffiths et al., 1974). It has been found possible to obtain an accurate estimate of the blood ethanol concentration in the living mouse by measuring the concentration of ethanol in the expired air. This concentration was measured by forcing individual mice to rebreathe air in a 5-ml chamber for 30 sec. At the end of this period a 1-ml sample of air was withdrawn and injected on to the glc column. Several experiments, in which estimated blood
607
ethanol concentrations of individual mice were verified by subsequent killing and direct measurement, established the validity and reproducibility of this method. It has been used routinely in this study. Meassurement of hepatic triglyceride concentration. Mice were killed by whole-body immersion in liquid nitrogen. Livers were dissected in the cold, weighed and taken for triglyceride estimation. Total triglyceride content of each liver was measured by a modification of the method of van Handel and Zilversmit (1957) after extraction of lipids by the method of Folch, Lees and Sloane Stanley (1957). Experimental design.-To investigate the relationship between the time course of accumulation of triglycerides in liver and the induction of ethanol dependence, 100 male TO mice, 2025 g, were exposed to ethanol vapour in the usual way. On each day, 10 mice were removed from the inhalation chamber. Five were killed for estimation of hepatic triglycerides and 5 were taken for assessment of the severity of the ethanol withdrawal syndrome. To further investigate any relationship between the concentration of triglycerides in liver and the severity of ethanol withdrawal a direct comparison was made between male and female mice. It has previously been reported that, when exposed to the same concentration of ethanol vapour, female mice show a significantly less severe ethanol withdrawal syndrome than males (Goldstein, 1973). It was therefore decided to compare also the time course of accumulation of triglycerides in liver with the time course of induction of ethanol dependence in female mice. This experiment was carried out at the same time and in exactly the same way as that described above. The possibility that some relationship might exist between the time course of the fall of hepatic triglyceride concentrations after the withdrawal of ethanol and the time course of the withdrawal syndrome was also considered. This was tested by comparing the fall in triglyceride concentrations and the withdrawal syndrome in adult and weanling mice. It had previously been noted (unpublished) that when adult and weanling mice were exposed to the same ethanol concentration weanling mice showed a significantly shorter duration of the ethanol withdrawal syndrome, although severity at the peak of withdrawal was very similar. In the experiments reported here 50 male TO mice, 30-35 g, were compared with 50 male TO mice, 8-10 g. Each group was exposed to ethanol vapour in the same chamber for 10 days. Blood ethanol concentrations were estimated frequently by the expired air method. After 10 days, 5 mice of each group were removed from the inhalation chamber and immediately killed for hepatic triglyceride measurement. The remainder were used
608
C. ABU MURAD, S. J. BEGG, P. J. GRIFFITHS AND J. M. LITTLETON
to assess the time course and severity of the withdrawal syndrome. After 12 h 5 mice of each group were killed for estimation of hepatic triglyceride concentrations. Testing for withdrawal signs in the remaining animals continued until no evidence of ethanol withdrawal was apparent. Some experiments were made to investigate the effects of drug-induced alteration of hepatic triglyceride accumulation on the ethanol withdrawal syndrome. The administration of DL-carnitine has been reported previously to inhibit ethanol-induced triglyceride accumulation (Hosein and Bexton, 1975). We therefore compared two groups of 30 male TO mice, 20-25 g, in an experiment in which one group received DL-carnitine (7% w/w) in diet, while the other group received the normal laboratory diet. Both groups were exposed to ethaniol vapour in the same inhalation chamber for 10 days. Ten mice of each group were killed for estimation of hepatic triglyceride concentration immediately before ethanol withdrawal. The remainder were used to assess the severity of the ethanol withdrawal syndrome. Frequent estimations of blood ethanol concentration were made throughout the period of the experiment. The i.p. injection of carbon tetrachloride has been reported to potentiate ethanol-induced triglyceride accumulation in liver (Strubelt, BuettnerandSiegers, 1975). Theeffectsofpotentiating triglyceride accumulation on the induction of ethanol dependence were therefore tested in experiments in which carbon tetrachloride (1.3 ml/kg i.p.) was administered to one group of mice (30 male TO, 20-25 g) and arachis oil, the vehicle for carbon tetrachloride, was administered to a similar group during the exposure of both groups to the same concentration of ethanol vapour. This experiment was performed in two ways: in one, the additional treatments were administered on the first day of ethanol administration; in the other, the additional treatments were given on the penultimate day of ethanol exposure. In both cases frequent estimations of blood ethanol concentrations were made. Ten animals from each group were killed for hepatic triglyceride measurement before ethanol withdrawal, and the remainder used for assessment of the withdrawal syndrome. In all these experiments comparison of biochemical and behavioural parameters has been made between the experimental groups and control animals exposed to the same conditions, except for the absence of ethanol from the
All reagents used for biochemical analysis
were Analar grade when these were commercially available. Ethanol (99-8 %, A.R. grade) was
obtained from James Burroughs Ltd, DL carnitine HCI (99 %) was obtained from Aldrich Chemical Co Ltd, carbon tetrachloride (S.L.R. grade) from Fisons, and arachis oil (special for injections) from Boots Ltd. RESULTS
Time course of accumulation of triglycerides in livers of male and female TO mice: relation to withdrawal syndrome The results, shown in Figs la and Ib, indicate a very similar time course for the ethanol-induced accumulation of triglycerides in liver and the onset of a recognizable ethanol withdrawal syndrome. In male mice significant triglyceride accumulation does not occur until after 5 days of ethanol administration (Fig. la). The onset of a withdrawal syndrome follows closely so that, after 6 days of administration, a recognizable syndrome can be observed in some male animals. The appearance of significant changes in these parameters is slightly delayed in female mice, but, once again, there is close agreement between the time courses (Fig. lb). The intensity of the withdrawal syndrome after 10 days confirms that female mice suffer a less severe withdrawal syndrome than males exposed to the same concentration of ethanol vapour. It is important to note that in this experiment no attempt was made to maintain the blood ethanol concentration at the same level in the two sexes. Female mice do, in fact, show significantly lower blood ethanol concentrations than males throughout this regime of ethanol administration.
Time course of reversal of ethanol-induced triglyceride accumulation in adult and weanling mice: relation to withdrawal syndrome inspired air. When weanling and mature adult mice Animals and materials.-TO strain white mice were obtained from Animal Suppliers Ltd, were exposed to the same concentration of Welwyn. Mice were maintained on Spillers ethanol vapour, similar blood ethanol Labsure 41B diet. Food and drinking water were concentrations were obtained. Both groups freely available throughout every experiment. showed accumulation of triglycerides in
609
ETHANOL WITHDRAWAL AND HEPATIC TRIGLYCERIDE ACCUMULATION
liver after 10 days (Table I). The accumulation of triglycerides in liver of weanling animals is greater than that in adult animals; this difference is significant at the P
