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Alcohol & Alcoholism. Vol. 31. No. 2. pp. 205-215. 1996

EFFECTS OF CHRONIC ADMINISTRATION AND SUBSEQUENT WITHDRAWAL OF ETHANOL-CONTAINING LIQUID DIET ON RAT LIVER TRYPTOPHAN PYRROLASE AND TRYPTOPHAN METABOLISM SAM1NA BANO, ROSSANA G. ORETT1, CHRISTOPHER J. MORGAN 1 , ABDULLA A.-B. BADAWY", PAUL R. BUCKLAND and PETER McGUFFIN Department of Psychological Medicine. University of Wales College of Medicine. Heath Park. Cardiff CF4 4XN and 'Cardiff Community Healthcare NHS Trust. Biomedical Research Laboratory. Whitchurch Hospital. Cardiff CF4 7XB. UK (Received 19 June 1995: in revised form 21 November 1995: accepted 7 December 1995)

INTRODUCTION The effects of chronic ethanol administration on the metabolism of the cerebral indolylamine 5-HT (5-hydroxytryptamine, serotonin) have been the subject of much investigation. The results, however, have been contradictory, with increases, decreases or no changes in 5-HT synthesis or turnover rate having all been reported (for recent review, see LeMarquand et al., 1994). These differences could be explained by differences in doses of ethanol, duration of its administration, methods of assessment of some of the parameters tested, e.g. turnover rate, species and/or strains of animals used and route of ethanol administration. It is generally accepted, however, that

•Author to whom correspondence should be addressed.

ethanol administration enhances 5-HT synthesis and turnover, whereas subsequent withdrawal causes the opposite effects (for references, see LeMarquand et al., 1994). We have previously demonstrated these effects (Badawy er al.. 1979a. 19806) and showed that they are caused by corresponding changes in circulating tryptophan (Trp) availability to the brain brought about respectively by inhibition and induction of activity of the major Trp-degrading enzyme, liver Trp pyrrolase (tryptophan 2,3-dioxygenase, EC 1.13.11.11) (for these latter effects, see Badawy and Evans, 1973, 1975a). In our previous studies, ethanol was administered to rats orally in drinking water. Under these conditions, very little behavioural changes were observed during the withdrawal phase (Badawy el al., 1980b). To further examine the possible role of Trp and 5-HT metabolic changes

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1996 Medical Council on Alcoholism

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Abstract — An investigation of the effects of chronic administration of ethanol by the liquid diet procedure and its subsequent withdrawal on tryptophan (Trp) metabolism and disposition was performed in rats. Treatment with the control liquid diet caused an enhancement of liver Trp pyrrolase activity and mRNA abundance. These effects are not due to the starvation associated with this feeding procedure, because they occur in rats maintained on the liquid diet ad libitum. Chronic ethanol administration in the liquid diet did not further influence the above increased expression of Trp pyrrolase mRNA but caused inhibition of pyrrolase activity in competition with the effects of the diet. The control liquid diet decreased liver Trp concentration, but exerted no significant effects on other aspects of Trp disposition. The most striking and robust finding was a highly significant elevation in both Trp pyrrolase activity and mRNA expression at 7 h following discontinuation of ethanol availability, at which time there were demonstrable behavioural signs of ethanol withdrawal. The increase in Trp pyrrolase mRNA during alcohol withdrawal may be caused by corticosteronc. whose circulating concentration was also increased. The changes in Trp pyrrolase activity during ethanol withdrawal were associated with significant alterations in Trp disposition including decreased brain Trp concentration and 5-hydroxytryptamine synthesis and turnover. These alterations may play a pivotal role in the behavioural manifestations of ethanol withdrawal including the hypcrexcitement underlying audiogenic seizures. We suggest that rat Trp pyrrolase gene regulation may be an important biological determinant of the ethanol withdrawal syndrome and requires further study, and that the use of the liquid diet procedure in Trp metabolic studies requires inclusion of adequate controls and special attention to the effects of the liquid diet itself.

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MATERIALS AND METHODS Animals and treatments Locally bred male Wistar rats, weighing 150-

200 g at the start of experiments, were housed 5 per cage under standard conditions (12 h dark: 12 h light cycle at 22 ± 1°C) and had free access to solid lab chow and drinking water. The rats were then divided into groups of 5 each and were all given an ethanol-free liquid diet ad libitum (Dyets Inc, 2508 Easton Avenue, Bethlem, Pennsylvania, PA 18017, USA), the composition of which is as described by Lieber et al. (1989), for 3 days before introducing ethanol into the diet. Ethanol was then added to the liquid diet in the proportion of either 5% or 8% (v/v), representing a calorific contribution to the diet of either 36% or 47% respectively and providing ~ 1.0-1.2 kcal/ ml. Matched control rats for each group were fed isocaloric amounts of the ethanol-free liquid diet, in which the ethanol contribution was substituted with maltose-dextrin. Freshly prepared liquid diets were provided daily between 09:00 and 10:00. All rats were housed in grid-bottomed cages to prevent coprophagia. Matched control rats, being limited in their food intake, were noted to ingest their total daily food ration more rapidly than their corresponding ethanol-fed counterparts, resulting in an altered feeding pattern and hence periods of starvation. To test the possible effect of any likely starvation, an additional experiment was performed in which a control group of rats was maintained on the control liquid diet ad libitum. Additionally in all experiments, a control group of rats maintained ad libitum on solid lab chow was included. Treatment of rats with ethanol-containing or control liquid diets was for either 14 or 28 days. For assessment of the effects of ethanol withdrawal, the ethanol-containing liquid diet was substituted with drinking water for 7h (before the rats were killed), as by this time there were demonstrable signs of withdrawal including the ability to induce seizures audiogenically and other behavioural signs (Hunter et al., 1975). Matched controls for the ethanol withdrawal group(s) were also starved for 7 h to counter any likely effects of the liquid diet itself in relation to interpretation of the effects of ethanol withdrawal. It should be noted here that, during the 7 h ethanol-withdrawal period, rats do not consume any diet and, accordingly, it was neither necessary nor desirable to provide the control liquid diet during the withdrawal phase. The withdrawal groups and the ad libitum liquid diet control group were all killed between 14:00 and 16:00.

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in alcohol dependence, a robust withdrawal syndrome was needed, which necessitated administration of ethanol by another procedure. Of the other procedures of chronic ethanol administration available, the liquid diet is one of the least stressful. We have in the past, however, avoided the use of this procedure to assess the 5-HT and related Trp-metabolic effects of ethanol on the assumption that administration to control rats of isocaloric amounts of carbohydrate may exert effects on Trp metabolism and disposition similar to those of ethanol itself (for a discussion, see Badawyefa/., 1980a). Previous studies from other laboratories of the effects of chronic ethanol administration on Trp and 5-HT metabolism using the liquid diet procedure have been limited to assessment of either cerebral 5-HT turnover (Frankel et al., 197'4; Rawat, 1974; Yamanaka and Kono, 1974) or hepatic Trp pyrrolase activity (Branchey and Lieber, 1982), and were thus not designed to address certain questions related specifically to the role or the nature of the liquid diet procedure itself or any associated physiological changes in the effects produced by, and/ or attributed to, ethanol. For example, could substitution of ethanol with an isocaloric amount of carbohydrate in matched controls cause inhibition of Trp pyrrolase activity (see Badawy et al., 1980a), or is pyrrolase activity more likely to be enhanced in control or even ethanol-treated rats secondarily to the partial starvation known to be associated with the liquid diet feeding procedure? We have accordingly performed a detailed investigation of the effects of chronic ethanol administration by the liquid diet procedure and its subsequent withdrawal on various aspects of tryptophan metabolism and disposition in the rat. We have additionally determined the effect of chronic ethanol administration by this procedure on the expression of Trp pyrrolase mRNA to examine further the suggestion by M0rland (1974) that the ethanol-induced decrease in pyrrolase activity is caused by inhibition of synthesis of the enzyme at the level of transcription. The results of these investigations form the subject of the present paper.

ALCOHOL AND TRYPTOPHAN PYRROLASE

Rats in all other groups were killed between 10:00 and 12:00. Rats were killed by decapitation under ether anaesthesia and their livers were perfused in situ with ice-cold 0.9% (w/v) NaCl and were rapidly removed and frozen in liquid nitrogen until analysis. Brains were also recovered and treated similarly. In addition, serum was isolated in some experiments. Chemicals Guanidinium thiocyanate and hydrochloride (>97% and >98% purity respectively) were from Fluka Chemicals Ltd (The Old Brickyard, New Road, Gillingham, Dorset SP8 4JL, UK). All other chemicals and reagents were from either the former source or from Sigma and/or BDH Chemicals (both of Poole, Dorset, UK) and were of the purest commercially available grades.

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Preparation and quantification of tryptophan pyrrolase mRNA Preparation of total RNA. Livers were mechanically homogenized in 20 ml of 5 M guanidinium thiocyanate, 50 mM TRIS buffer, pH 7.5, 25 mM EDTA and 8% 2-mercaptoethanol. The homogenate was centrifuged at 6000 g for 40 min at Chemical, enzymic and other determinations Blood ethanol concentration was determined 15°C. Total RNA from each liver was then prepared by repeat precipitations from 6 M guan- by an alcohol dehydrogenase (EC 1.1.1.1) based idinium HC1, 25 mM EDTA and 10 mM 2- enzymatic procedure (Badawy and Aliyu, 1984). Liver Trp pyrrolase activity was determined in mercaptoethanol (Buckland et al., 1992). Multiprobe oligonucleotide solution hybridiz- homogenates either in the absence (holoenzyme ation. Oligonucleotide probes (Genosys Ltd, 162a activity) or in the presence (total enzyme activity) Science Park, Milton Road, Cambridge CB4 of added (2 fiM) haematin. as described pre4GH, UK) were end-labelled with polynucleotide viously (Badawy and Evans, 1975ft: see also the kinase with [32P]ATP using standard procedures. fuller description by Badawy, 1981 and additional The Trp pyrrolase probe consisted of the following comments by Badawy et al.. 1983). The apo33 bases: CTAGCAGCCGGAACTGAAGA- enzyme activity was obtained by difference. The CTCTGGAAGCCTG (Haber et al., 1993). The saturation of the enzyme with its cofactor 28s ribosomal RNA probe consisted of the follow- haem was expressed as the percentage haem (100 X holoenzyme activity/total ing 42 bases: GCTTAAATTCAGCGGGTCG- saturation CCACGTCTGATCTGAGGTCGCGG (Hadji- enzyme activity). Because, as stated earlier, the olov et al., 1984). Solution hybridization was per- livers were perfused in situ before frozen storage, formed by the following procedure (O'Donovan the possibility of an artefactual increase in the et al.. 1991). Total RNA (20 ^g) and probes were haem saturation of the enzyme (through haemosuspended in 30 fA of hybridization buffer (probe globin haem) was excluded (see also Badawy et concentration 1 pmol/ml in 0.4 M NaCl, 40 mM al., 1983). Free (ultrafiltrable) serum, total PIPES, pH 6.4, 1 mM EDTA), heated at 90°C for (free + albumin-bound) serum, liver and brain 3 min and then incubated at 65°C for 2 h. Excess tryptophan concentrations were determined by a probe was removed by the addition of 300 fA of modification (Bloxam and Warren, 1974) of the Si nuclease buffer [S, nuclease, Amersham, UK method of Denckla and Dewey (1967) as 120 U/ml, 4.5 mM zinc sulphate. 50 mM sodium described previously (Badawy and Evans, 1976).

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acetate. pH 4.5. 0.3 M NaCl and 10 mg/ml singlestranded DN A] and incubation at 37°C for 15 min. A 300 fA portion of the reaction mixture was then transferred to a fresh tube, incubated at room temperature for 5 min after which the reaction was terminated by the addition of 48 fi\ of 4 M ammonium acetate, 0.1 M EDTA. Doublestranded hybrids were precipitated with ethanol, resuspended in 10 /A of formamide running buffer, denatured at 90°C for 2 min and subjected to acrylamide gel (10%) electrophoresis. Quantification of mRNA. The dried gels were analysed by autoradiography using Amersham Hyperfilm MP (Amersham UK) (exposure for 4 days) followed by scanning densitometry (Ultrascan Densitometer. LKB, UK). Liver samples were analysed individually and all bands measured on the autoradiographs were in the linear range of the film used. Values for Trp pyrrolase were normalized to the corresponding 28s values. All ratios thus obtained in the liquid diet-treated groups were related to the mean value (taken as 100%) for rats in the corresponding untreated solid chow control groups.

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Table 1. Effects of liquid diet, chronic ethanol administration (8%) and withdrawal on rat liver tryptophan pyrrolase activity and mRNA expression Exp no 1

Treatment SD LD

E (8%) 2

SD LD

E (8%) 3

SD LDa

E (8%) LDb

W (E 8%) 4

SD LD

E (5%)

Duration (days) 14 14 28 28 28 28 28 28 14 14

Holoenzyme activity

Total enzyme activity

1.7 ±0.06 3.7 ± 0 06 4.4 ±0.21*** 7.0 ±0.64"* 1.9 + 0.26*** 2.3 itO.32*** 1.6 ±0.02 3.7 st0.22 4.2 + 0.39*** 7.3 it0.38"* 1.6 ±0.07*** 2.3 dtO 20*** 1.2 + 0.05 2.3 dt0.08 4.0 ±0.25*** 4.3 itO.10*" 3.7 ±0.43 43 it 0.44 4.3 + 0.15*** 5.1 it0.34"* 11.2± 1.31** 11.3 it 135* 1.8 ± 0.17 4.0 ±0.15 6.1 ±0.21** 9.6 ± 0.29** 5.2 ± 0.24t 5.9 ±0.40**

Apoenzyme activity

Haem saturation (%)

% encoding mRNA level

2.0 + 0.05 2.6 ±0.57 0.4 + 0.13* 2.1 ±0.24 3.1 +0.38 0.7 + 0.16*** 1.1 ±0.09

46 ± 2 63 ± 5tt

100 ± 1 167 + 2 " * 162 ± 4 " 100 ± 1 196 ± 4 " * 204 ± 6 " * 100 + 3 164 ± 8 * " 285 ± 1 4 " * 100 ± 5 145 ± 14 204 ± 9 t

_ -

2.2 + 0.09 3.5 ±0.14** 0.7 ±0.19**

-

43 ± 3 57 + 4tt -

52 ifc3 93 ih 4««* 86 it 3 84 dfc6* 99 itot 4 5 :t 3 63 -t 1 " -

Concentrations of brain 5-hydroxytryptamine (5HT) and its major metabolite 5-hydroxyindol-3ylacetic acid (5-HIAA) (Curzon and Green, 1970) and of serum albumin (Doumas and Biggs, 1972), corticosterone (Glick et al., 1964), glucose (Slein, 1963) and non-esterified fatty acids (NEFA) (Mikac-Devic et al., 1973) were all determined by standard procedures. Statistical analysis of results was performed by Student's t test and, where appropriate, by one-way analysis of variance (ANOVA). RESULTS Effects of chronic administration and subsequent withdrawal of ethanol-containing liquid diets on rat liver tryptophan pyrrolase activity Changes in rat liver Trp pyrrolase activity following chronic administration of both control and ethanol-containing liquid diets and subsequent withdrawal of the diets are shown in Table 1. The

results of four separate experiments are presented in Table 1. Here ethanol was administered in the liquid diet either at the 8% level for 14 (experiment 1) or 28 days (experiments 2 and 3) or at the 5% level for 14 days (experiment 4). Body weight was monitored twice weekly during the experiments. As expected, body weight gain by ethanol-treated rats was similar to that achieved by their matched controls, both of which showed a lower gain rate than solid chow control rats (results not shown). Blood ethanol concentrations in the rats described in the first three experiments in Table 1 were as follows: 138 ± 35, 233 ± 40 and 306 ± 43 respectively (means in mg/dl ± SEM for each group of 5 rats). The corresponding values in mM were 30 ± 8, 51 ± 9 and 67 ± 9 respectively. As the results in Table 1 show, liver Trp pyrrolase activity in rats given the control liquid diet was significantly increased in comparison with control rats receiving lab chow. Thus the control liquid diet increased the holoenzyme activity by

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Experimental details are as described in the Materials and methods section. The control liquid diets were administered to matched controls for the same duration. The results obtained in liquid diet controls (LD) were compared with those in solid diet controls (SD), whereas those obtained with ethanol (E) or at 7 h after its withdrawal (W) were compared with the corresponding matched liquid diet (LD) controls, except the Trp pyrrolase mRNA, which is expressed as % relative to SD controls. The significance of the differences is indicated as follows: t/> < 0.05; t t P < 0.02; *P < 0.01; **P < 0.002; ***P < 0.001. Values are means ± SEM for each group of 4-5 rats. Tryptophan pyrrolase activity was determined in liver homogenates either in the absence (holoenzyme activity) or in the presence (total enzyme activity) of added (2 ^M) haematin, and is expressed in /rniol of kynurenine formed per h per g wet weight of liver. The apoenzyme activity was obtained by difference and the saturation of the enzyme with its cofactor haem is expressed as a percentage. Please note that in some instances neither the apoenzyme activity nor the percentage haem saturation is given; this would have been inappropriate, because in the former case the saturation of the enzyme with its cofactor haem is enhanced, whereas in the latter case the apoenzyme activity is inhibited. - Denotes not determined.

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Table 2. Effects of control liquid diets and ethanol withdrawal on rat liver tryptophan pyrrolase activity and mRNA expression Tryptophan pyrrolase activity (/imol of kynurenine formed per h per g wet wt of liver) Treatment SD LD ad libitum LD (matched) W (E 8%)

Duration (days)

Holoenzyme

Total enzyme

Apoenzyme

14 14 14

1.1 ±0.09 2.0 ±0.19* 1.9 + 0.16* 9.1 ± 1.12**

2.3 ±0.16 4.4 ±0.41* 4.1 ±0.26** 10.1 ±0.60**

1.2 ±0.14 2.4 + 0.23* 2.2 ±0.15*

Haem saturation (%)

% encoding mRNA level

48 ± 4 45 ± 1 46 ± 2 90 ± 7**

100 ± 4 143 + 5** 167 ±2**

Experimental details are as described in the Materials and methods section and also in Table 1, except that the control liquid diet was made available to rats either ad libitum or to match an ethanol-treated group which subsequently underwent withdrawal. Duration of treatment with control and ethanol-containing (8%) liquid diets was 14 days. Values are means ± SEM for each group of 4-5 rats. The values in liquid diet receiving controls (LD) are compared with those in controls maintained on the solid diet (SD). whereas those observed in the 7 h ethanol-withdrawn group (W) are compared with those in its matched liquid diet control group, and the significance of the differences is indicated as follows: *P< 0.005; **P< 0.001. - Denotes not determined.

the first two experiments (Table 1). Thus, although the holoenzyme activity was not altered by ethanol (as is the case when ethanol is administered in drinking water; see the Discussion section), activities of the total enzyme and apoenzyme were significantly decreased, by 38% (P = 0.01-0.002) and 67-80% (P = 0.002-0.001) respectively. In experiment 4 shown in Table 1, ethanol was administered in the liquid diet for 14 days at a 5% concentration. Here, all forms of the pyrrolase were significantly decreased in comparison with values in the liquid diet controls (by 15, 39 and 80% respectively), whereas in comparison with lab chow controls, only the apoenzyme activity appeared to be decreased, though this was not the case; the low free apoenzyme merely reflects the increase in its saturation with haem, and in fact, both the holoenzyme and total pyrrolase activities were increased, by 189% and 48% respectively (P= 0.01-0.001). In addition the blood ethanol levels in these rats were only 4.2 mg/dl (about 1 mM) when measured. This might indicate that these animals were in withdrawal. However, the Trp pyrrolase activities were not increased compared with non-withdrawn; cf. other data in Table 1 and those in Table 2. Overall, this indicates that a 5% ethanol concentration in the liquid diet may not be sufficient to induce strong inhibition of Trp pyrrolase activity (see also the Discussion). The results of experiment 3 in Table 1 show

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135-239%, whereas the corresponding increases in the total pyrrolase activity were 89-140%. Because the increase in the holoenzyme activity was relatively greater than in that of the total enzyme, the saturation of the pyrrolase with its cofactor haem was accordingly increased by the control liquid diet treatment, and this was reflected in the significant elevation of the percentage haem saturation in all four experiments by 33-79%. It should however be stated here that the control liquid diet does not always increase this saturation, as the results of the experiments in Table 2 show. The results in Table 1 also show that chronic ethanol administration in the liquid diet at the 8% level caused a significant inhibition of rat liver Trp pyrrolase activity, in comparison with either the liquid diet controls or solid lab chow controls. The statistical significance of the ethanol inhibition is expressed in Table 1 in relation to the corresponding liquid diet control groups. In comparison with these latter groups, the inhibition by ethanol of the holoenzyme activity varied in the first two experiments between 57% and 62%, whereas the corresponding inhibition of the total enzyme activity was 67-68%. As a result, the decrease in activity of the apoenzyme (which is calculated by difference) was 77-85%. When the values observed in ethanol-treated rats were compared to those in control animals receiving the solid diet chow, inhibition of Trp pyrrolase activity was still evident in

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TP mRNA Expression

33 BASES

W

M

Fig. 1. Expression of rat liver tryptophan pyrrolase mRNA after chronic administration and subsequent withdrawal of ethanol-containing liquid diets. Tryptophan pyrrolase mRNA expression was determined by multiprobe oligonucleotide solution hybridization as described in the Materials and methods section. The control 28s ribosomal RNA band is not shown. Abbreviations: C (solid diet controls); M (matched liquid diet controls): E (ethanol-treated group, 8% in liquid diet for 28 days); W (7 h withdrawal following ethanol treatment as in E).

Effects of chronic administration and subsequent withdrawal of ethanol containing liquid diets on rat liver tryptophan pyrrolase mRNA expression The results of a typical experiment are illustrated in Fig. 1. Trp pyrrolase mRNA expression was examined in liver samples from the majority of the rats used in the experiments described in Tables 1 and 2. For all the experimental groups described, the expression of Trp pyrrolase mRNA was significantly enhanced by the control liquid diet by 45%-96% (P = 0.05-0.001) relative to the corresponding solid lab chow control group. The addition of ethanol to the diet did not significantly alter the mRNA levels from those of matched controls. However, expression was enhanced 7 h after ethanol withdrawal by an extra 121% (P < 0.001) in comparison with the (starved) matched control group. Effects of chronic administration and subsequent withdrawal of ethanol-containing liquid diets on tryptophan metabolism and disposition and related metabolic processes These effects are shown in Table 3. The data on liver Trp pyrrolase activities of these rats are shown in experiment 3 in Table 1. As the results in Table 3 show, brain [Trp] was not significantly

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that inhibition of Trp pyrrolase activity is not always observable following ethanol administration in the liquid diet. Thus, both the holoenzyme and total enzyme activities in ethanoltreated rats were similar to those in their matched controls, both of which were two-fold higher than the corresponding activities in solid chow controls. This variability no doubt reflects the fact that two antagonistic influences (liquid diet and ethanol) are competing to influence Trp pyrrolase activity and that blood alcohol levels are bound to vary with the rats' feeding behaviour. Experiment 3 in Table 1 also shows the effect of ethanol withdrawal on rat liver Trp pyrrolase activity. As shown, the holoenzyme and total enzyme activities at 7 h after withdrawal were 122— 160% higher than the corresponding activities in matched liquid diet controls. This increase during withdrawal has been repeatedly confirmed in the present and other experiments (see also Table 3). Because the enzyme appears to be almost fully saturated with its cofactor haem, such an increase may be of the cofactor (haem)- or substrate (Trp)type activation, rather than the hormonal (glucocorticoid)-type induction mechanism (see Badawy and Evans, 19756). However, as described below, the mRNA encoding Trp pyrrolase also increases and this would be expected to increase the enzyme protein levels. Because, as stated in the Materials and Methods section, control rats receiving the normal liquid diet tended to consume their ration rapidly and thus undergo periods of food deprivation, the possibility was considered that the increase in liver Trp pyrrolase activity in these controls may be secondary to starvation. An experiment whose results are shown in Table 2 was therefore performed in which a group of rats received the control liquid diet ad libitum. Compared to the matched control group, the ad libitum fed rats consumed on average an extra 28 ml of the liquid diet per day each, and, as a consequence, gained weight at a higher rate (46% vs 28% over a 14-day period). As shown in Table 2, liver Trp pyrrolase activities in this ad libitum-fed group did not differ significantly from those in another control liquid diet group (acting as the matching control group for another ethanol-withdrawal group), both of which were 73-100% higher than the corresponding activities in a third control group receiving solid chow.

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Table 3. Effects of chronic administration and subsequent withdrawal of ethanol-containing liquid diets on rat tryptophan metabolism and disposition and serum corticosterone concentration Treatment groups Parameter Liver [Trp] Serum Trp: [Free] [Total] Free (%) Brain: [Trp] [5-HT] [5-HIAA] Serum: [corticosterone]

SD

LDa

E

LDb

W

8.25 ±0.31

6.72 ± 0.09**

5.45 ±0.02***

5 80 ± 0.07***

4.49 ±0.14***

1.20 ±0.05 11.56 ±0.41 10.38 ± 0.37

1.24 ±0.03 12.71 ±0.41* 9.76 ± 0.37

1.44 ±0.03** 12.95 ± 0.86 11.12 ±0.93

1.22 ±0.05 13.47 ±0.61* 9.06 ± 0 74

1.05 ±0.05* 10.53 ±0.32* 9.97 ±0.71

3.42 ± 0.07 0.65 ± 0.008 0.38 ± 0.007

3.29 ± 0.06 0.62 ±0.016 0.39 ±0.012

3.05 ±0.10 0.77 ± 0.020*** 0.31 ±0.015*

3.42 ± 0 13 0.63 ± 0 025 0.48 ±0.014***

2.29 ±0.06*** 0.56 ± 0.024* 0.29 ±0.007***

33.9 ± 1.7

52.0 ±2.2***

52.6 ±4.1

57.8 ±2.2***

71.7 ± 1.4***

altered by administration of either control or ethanol-containing liquid diets. Only ethanol withdrawal caused a significant decrease in brain [Trp], of 33%. Brain [5-HT] was also not significantly altered by the control liquid diet, but was increased by chronic ethanol administration (by 24%) and decreased moderately during subsequent withdrawal (by 11%, compared to the matched control, and by 27% compared to the non-withdrawn ethanol-treated group). Brain [5-HIAA] was decreased by both chronic administration and subsequent withdrawal of ethanol, by 20% and 40% respectively. These results show that ethanol withdrawal exerts clear-cut effects on brain Trp metabolism, namely inhibition of 5-HT synthesis and turnover secondarily to decreased brain [Trp], whereas the only discernible effect of chronic ethanol treatment is that of a modest inhibition of 5-HT turnover by an unknown mechanism. The results in Table 3 also show that free (ultrafiltrable) serum [Trp] was not significantly influenced by the control liquid diet, whereas that of total (free + albumin-bound) Trp was significantly elevated by 11-16%. Chronic ethanol

administration in the liquid diet caused a 16% increase in free serum [Trp]. but did not influence significantly that of total Trp. By contrast, ethanol withdrawal caused significant decreases in both free and total serum [Trp], of 14% and 22% respectively and these changes were not associated with altered Trp binding (expressed as the percentage free serum Trp). The results in Table 3 show additionally that liver [Trp] was significantly decreased by the control liquid diet by 19-30%. Chronic ethanol administration decreased liver [Trp] further by 19% and subsequent withdrawal decreased it further by 23%. Serum corticosterone concentration (Table 3) was elevated by the control liquid diet by 53-71%. but chronic ethanol administration did not cause a further increase. Subsequent withdrawal caused a marked increase in serum corticosterone of 24% over its matched control. In experiments not detailed here, we also found that the concentrations of both albumin and nonesterified fatty acids (NEFA; the physiological displacers of serum albumin-bound Trp) were not significantly altered, except for a 33% increase (P < 0.001) in NEFA induced by chronic ethanol

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Experimental details are as described in the Materials and methods section and Table 1 Liver tryptophan pyrrolase activities of the rats used here have been described in experiment 3 in Table 1. Values are means ± SEM for each group of 5 rats. The values in liquid diet receiving controls (LD) are compared with those in controls maintained on the solid diet (SD). whereas those observed in the ethanol-treated group (E) or the 7 h ethanol-withdrawn group (W) are compared with their respective matched control groups (LDa and LDb respectively). The significance of the differences is indicated as follows: *P< 0.005; ***p< 0.001. Tryptophan and 5-hydroxyindole concentrations are expressed in ug/ml of serum or per g wet wt of tissue (except the percentage free serum tryptophan). whereas serum corticosterone concentration is in /(g/l. Other abbreviations used: Trp (tryptophan); 5-HT (5-hydroxytryptamine); 5-HIAA (5-hydroxyindol-3-ylacetic acid).

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administration, which explains the above preferential increase in free serum [Trp] caused by ethanol. Similarly, serum [glucose] was not significantly altered by the control liquid diet nor by chronic ethanol administration or its subsequent withdrawal. Thus the mean values for 5 rats per group ± SEM in mg/dl for the five groups of rats used in the experiments described in Table 3 were respectively as follows: 177 ± 10, 150 ± 5, 165 ± 4 , 158 ± 11 and 141 ± 7.

The results of the present work show clearly that ethanol withdrawal produces a consistent and dramatic enhancement of rat liver Trp pyrrolase activity. We have previously demonstrated this enhancement following withdrawal of ethanol from drinking water (Badawy and Evans, 1973, 1975a) and showed that it is caused by a hormonaltype induction mechanism associated with elevation of circulating corticosterone concentration (Badawy era/., 1980b). The results presented here also show an increase in circulating corticosterone concentration (Table 3). The pyrrolase enhancement was associated with a dramatic increase in Trp pyrrolase mRNA expression (Table 1) and it follows, therefore, that this increase may be

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DISCUSSION Effects of chronic ethanol administration on rat liver tryptophan pyrrolase activity We have previously shown that chronic ethanol administration to rats in their drinking water decreases liver tryptophan pyrrolase activity (Badawy and Evans, 1973, 1975a). This decrease is specific to the apoenzyme, because it is demonstrable only with the activity of the total enzyme but not with that of the holoenzyme, and is caused by an NAD(P)H-dependent allosteric mechanism (Badawy and Evans, 1973, 1975a; Badawy et al., 19796). From these and other experiments (Badawy and Evans, 1979), we concluded that the above decrease is due to inhibition of pyrrolase activity and not synthesis, contrary to the suggestion by M0rland (1974) on the basis of the observation that induction of the enzyme activity by dexamethasone in perfused livers is impaired in rats chronically treated with ethanol. We have, however, shown previously that the cortisol induction of pyrrolase activity in intact rats is not influenced by chronic ethanol administration (Badawy and Evans, 1975a; Evans and Badawy, 1977). Glucocorticoid induction of liver Trp pyrrolase activity is known to involve increased mRNA production (DeLap and Feigelson, 1978), and in the present work, the expression of Trp pyrrolase mRNA, as determined by the technique of oligonucleotide solution hybridization, was shown not to be influenced by chronic ethanol administration (Fig. 1, Table 1), despite the observed decrease in enzyme activity. The results of these molecular experiments together with our previously published observations therefore strongly suggest that the inhibition of rat liver Trp pyrrolase activity by chronic ethanol administration does not involve altered synthesis of the enzyme, at least at the level of expression of its message.

Effects of ethanol withdrawal on rat liver tryptophan pyrrolase activity By contrast with our present and previous results and those by M0rland (1974), who also administered ethanol in drinking water, Branchey and Lieber (1982), using a 5% ethanol concentration in the liquid diet, observed an enhancement in pyrrolase activity and in urinary kynurenine excretion following oral Trp loading. One possible explanation of these opposite findings is provided in the present work, which demonstrates the rapid activation of liver Trp pyrrolase after ethanol withdrawal. Thus, the increased urinary excretion of the Trp metabolite kynurenine following Trp loading was observed by Branchey and Lieber (1982) at 7 h after withdrawal of the ethanol-containing liquid diet, i.e. at a time at which the enzyme is dramatically activated (Tables 1 and 2 of the present work). Such an increase must therefore be ascribed to ethanol withdrawal, rather than ethanol itself. Similarly, the moderate increase in pyrrolase activity reported by Branchey and Lieber (1982) and which is also attributed to ethanol (not withdrawal) may be explained by a possible withdrawal-induced activation, as these latter authors did not specify the time of death after the rats ceased to consume their ethanol-containing liquid diet; we have shown that pyrrolase activity begins to rise within 2h after withdrawal (unpublished observations). Thus, the effect of chronic ethanol administration by the liquid diet procedure will depend critically on the time of testing after cessation of ethanol intake, which is also determined in part by the rat's nocturnal eating habit.

ALCOHOL AND TRYPTOPHAN PYRROLASE

corticosterone-mediated. Although corticosterone causes a hormonal type induction of Trp pyrrolase activity, the increase in the haem-saturation of the enzyme observed during ethanol withdrawal in the present work suggests a cofactor- or substrate-type activation mechanism (for differences between these mechanisms, see Badawy and Evans, 19756); further work is therefore required to establish the possible role(s) of these three mediators of the Trp pyrrolase enhancement during alcohol withdrawal.

and probably varies at different times of the day and during the eating/resting cycle. Effects of chronic ethanol administration and subsequent withdrawal on tryptophan metabolism and disposition and related metabolic processes Cerebral 5-HT synthesis is controlled mainly by brain [Trp], because the rate-limiting enzyme of the 5-HT biosynthetic pathway, Trp hydroxylase, exists unsaturated with its Trp substrate (Fernstrom and Wurtman, 1971; Carlsson and Lindqvist, 1978; Curzon, 1979). It follows therefore that peripheral factors influencing circulating Trp availability to the brain must play important roles in cerebral 5-HT synthesis. These factors include Trp binding to albumin, extent of competition between Trp and itsfivecompetitors (Val, Leu, He, Phe and Tyr) for the same cerebral uptake mechanism, and activity of the major Trp degrading enzyme, hepatic Trp pyrrolase. We have previously shown (Badawy et al., 19806) that, as a result of the enhancement of liver Trp pyrrolase activity during ethanol withdrawal from drinking water, brain 5-HT synthesis and turnover are inhibited secondarily to a decrease in brain [Trp] brought about by a decrease in circulating Trp availability to the brain as a consequence of increased hepatic Trp degradation. The results in Table 3 of the present work confirm that withdrawal of ethanol-containing liquid diets also decreases rat brain 5-HT synthesis and turnover by the same pyrrolase-dependent mechanism. The present results demonstrate that changes in Trp disposition and 5-HT metabolism occur rapidly after withdrawal, preceding the appearance of the behavioural disturbances of the withdrawal syndrome, and suggest that the liquid diet procedure of chronic ethanol administration is a valid method for studying the Trp metabolic disturbances of, and their possible role in, the alcohol withdrawal syndrome. By contrast with these withdrawal effects, chronic ethanol administration in liquid diets caused a modest inhibition of cerebral 5-HT turnover leading to an increase in brain 5-HT (Table 3). Previous studies with ethanol-containing liquid diets have shown inconsistent effects, namely no change (Frankel et al., 1974; Rawat, 1974) or a decrease (Yamanaka and Kono, 1974) in 5-HT turnover. This is in marked contrast with changes

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Effects of the control liquid diet on rat liver tryptophan pyrrolase activity Another likely determinant of the effect of ethanol-containing liquid diets on Trp pyrrolase activity is the diet itself, which as the results in Tables 1 and 2 show, was capable of enhancing rat liver Trp pyrrolase activity and mRNA level. It is likely that the increased enzyme activity is a result of the increased mRNA levels which in turn may well be due to the raised serum corticosterone. These effects could be caused by one or more components of the liquid diet or as a consequence of the administration procedure, e.g. the associated food starvation or non-specific stress such as a diet in liquid form. The results in Table 2, which show that pyrrolase activity (and mRNA expression) are equally enhanced in control liquid diet-receiving rats irrespective of whether the diet was provided ad libitum, exclude the possible involvement of starvation. However, the fact that the control liquid diet alters Trp pyrrolase activity in the opposite direction to the established ethanol effect emphasizes the need to use solid chow control groups in addition to the usual liquid diet controls in studies of the effects of ethanol on Trp pyrrolase and related aspects of Trp metabolism. The ability of ethanol to inhibit pyrrolase activity after its administration in liquid diets is therefore likely to depend on the extent of enhancement of enzyme activity by the liquid diet itself, as well as on whether ethanol is administered in amounts and for durations sufficient to both overcome this enhancement and produce a net inhibition of the basal enzyme activity (compare experiments 1-4 in Table 1). This clearly appears from the present work to be a variable phenomenon due to the two competing effects.

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Advances in Experimental Medicine and Biology 35, 105-123. Badawy, A. A.-B. and Evans, M. (1975a) The effects of ethanol on tryptophan pyrrolase activity and their comparison with those of phenobarbitone and morphine. Advances in Experimental Medicine and Biology 59, 229-251. Badawy, A. A.-B. and Evans, M. (19756) The regulation of rat liver tryptophan pyrrolase by its cofactor haem— experiments with haematin and 5-aminolaevulinate and comparison with the substrate and hormonal mechanisms. Biochemical Journal 150, 511-520. Badawy, A. A.-B. and Evans, M. (1976) The role of free serum tryptophan in the biphasic effect of acute ethanol administration on the concentrations of rat brain tryptophan, 5-hydroxytryptamine and 5-hydroxyindol-3ylacetic acid. Biochemical Journal 160. 315-324. Badawy, A. A.-B. and Evans, M. (1979) Further evidence against inhibition of synthesis of rat liver tryptophan pyrrolase by chronic ethanol administration. British Journal on Alcohol and Alcoholism 14, 59-64. Badawy, A. A.-B., Punjani, N. F. and Evans. M. (1979a) Enhancement of rat brain tryptophan metabolism by chronic ethanol administration and possible involvement of decreased liver tryptophan pyrrolase activity. Biochemical Journal 178, 575-580. Badawy, A. A.-B.. Punjani, N. F. and Evans, M. (19796) Prevention by pyrazole of the effects of chronic ethanol administration on the redox states of the hepatic nicotinamide adeninc dinucleotide (phosphate) couples and on liver and brain tryptophan metabolism in the rat. Biochemical Journal 184, 165-168. Badawy. A. A.-B., Punjani, N. F. and Evans, M. (1980a) Unsuitability of control sucrose or glucose in studies of the effects of chronic ethanol administration on brain 5-hydroxytryptamine metabolism. Journal of Pharmacological Methods 3, 167-171. Badawy, A. A.-B., Punjani, N. F., Evans, C. M. and Evans, M. (19806) Inhibition of rat brain tryptophan metabolism by ethanol withdrawal and possible involvement of the enhanced liver tryptophan pyrrolase activity. Biochemical Journal 192, 449-455. Badawy, A. A.-B., Morgan, C. J. and Davis, N. R. (1983) Determination of tryptophan pyrrolase activity in rat liver homogenates. Biochemical Journal 215, 709-710. Bloxam, D. L. and Warren, W. H. (1974) Error in the Acknowledgements — S.B. is a British Commonwealth Scholar determination of tryptophan by the method of Denckla and R.G.O. is an MRC Training Fellow. We thank the Mental and Dewey. A revised procedure. Analytical BioHealth Foundation for additional financial support. chemistry 60, 621-625. Branchey, L. and Lieber, C. S. (1982) Activity of tryptophan pyrrolase after chronic alcohol administration. Substance and Alcohol Actions/Misuse 2, 225-229. Buckland, P. R., O'Donovan, M. C. and McGuffin, P. REFERENCES (1992) Changes in dopamine D,, D2 and D3 receptor mRNA levels in rat brain following antipsychotic treatBadawy, A. A.-B. (1981) Haem utilization by rat liver ment. Psychopharmacology 106, 479—483. tryptophan pyrrolase as a screening test for exacerbation of hepatic porphyrias by drugs. Journal of Phar- Carlsson, A. and Lindqvist, M. (1978) Dependence of 5HT and catecholamine synthesis on concentrations of macological Methods 6, 77-85. precursor amino acids in rat brain. Naunyn SchmieBadawy, A. A.-B. and Aliyu, S. U. (1984) Antagonism of deberg's Archives of Pharmacology 303, 157-164. acute alcohol intoxication by naloxone. Alcohol and Curzon, G. (1979) The relationship between plasma, CSF Alcoholism 19, 199-201. and brain tryptophan. Journal of Neural Transmission Badawy, A. A.-B. and Evans. M. (1973) Tryptophan pyr15 (Suppl.). 81-92. rolase in ethanol administration and withdrawal.

in 5-HT synthesis and turnover observed when ethanol is administered chronically by other procedures, as reported by us (see e.g. Badawy et al., 1979a,b, l980a,b) and others (for references, see LeMarquand et al., 1994). The inability of chronic ethanol administration by the liquid diet procedure to influence Trp metabolism and disposition and 5-HT synthesis in the present work (Table 3) is not surprising, as alcohol in this particular experiment failed also to inhibit liver Trp pyrrolase activity (see experiment 3 in Table 1). The present results therefore show that the liquid diet procedure is also likely to give variable results in studies of the chronic effects of ethanol on Trp metabolism and disposition and this is almost certainly determined by whether ethanol can overcome the effect of the liquid diet itself on Trp pyrrolase activity and possibly also on other aspects of Trp disposition. In this latter context, it is noteworthy that, although the control liquid diet enhanced liver Trp pyrrolase activity (Tables 1 and 2), and caused the expected decrease in liver [Trp] (Table 3), presumably because of increased hepatic Trp degradation secondarily to this enhancement, it failed to alter, as a consequence, circulating Trp availability to the brain or 5-HT synthesis. Other compensatory factors must have been involved, related to circulating Trp concentration and availability to the brain, e.g. increased Trp supply through the diet itself or an effect of the liquid diet and/or its administration procedure on aspects of body physiology altering Trp disposition in such a way as to overcome the expected decrease in circulating [Trp]. Further work is therefore required to examine these possibilities.

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