Flumazenil (Ro15-1788) does not affect ethanol tolerance and dependence.

There are conflicting reports concerning whether flumazenil (Ro15-1788) can antagonize the central effects of ethanol and ethanol withdrawal reactions. C57BL/6J mice were treated chronically with an ethanol liquid diet. Control mice were pair fed an isocaloric diet containing no ethanol. These mice were injected with either Ro15-1788 (25 mg/kg) or vehicle immediately before, 14 h or 24 h before ethanol withdrawal. Under these conditions, no attenuation of the severity of handling-induced withdrawal seizures or of withdrawal hypothermia was observed in the ethanol-dependent mice injected with Ro15-1788. Likewise, there was no abolition or attenuation of tolerance to the ataxic effects (sleep time and horizontal dowel tests) and hypothermic effects of ethanol by Ro15-1788 when the mice were tested on day 3 of ethanol withdrawal. It is concluded that Ro15-1788 (25 mg/kg) has no effect on ethanol tolerance and dependence.

The ability of chlordiazepoxide to maintain ethanol tolerance and dependence.

Two criteria need to be satisfied in the demonstration of cross-dependence to chlordiazepoxide (CDP) in ethanol-dependent mice. These are the ability of CDP to suppress ethanol withdrawal and to maintain the dependent state. In this study, mice which had been fed chronically an ethanol diet followed by two days of CDP diet treatment had more severe CDP withdrawal signs induced by Ro15-1788 than drug-naive mice which were similarly exposed to the CDP diet treatment. The data indicate that CDP can maintain the dependent state acquired from the prior ethanol treatment. Alternatively, the prior ethanol treatment would have facilitated the development of CDP dependence, but it was not deemed likely. Three behavioral tests, namely, runway, sleep time, and drug-induced hypothermia, were used to test whether CDP could maintain the ethanol tolerance acquired from the prior ethanol treatment. The runway test showed that CDP could maintain the previously acquired ethanol tolerance. However, interpretations of the data from the sleep time and hypothermia tests are more equivocal because of factors such as peak tolerance, differential rates of development and dissipation of ethanol (or CDP) tolerance as determined by different behavioral tests.

Chronic treatment with ethanol or chlordiazepoxide alters the metabolism of chlordiazepoxide.

Although chronic ethanol administration in C57BL/6J mice did not cause an induction of ethanol metabolism, it altered the metabolism of chlordiazepoxide (CDP). Significantly lower blood levels of CDP, but higher levels of N-desmethyl CDP (NDCDP), were observed in ethanol-dependent mice compared to pair-fed controls during the first hour after CDP injection. Mice treated chronically with CDP showed significantly lower blood levels of CDP and NDCDP than pair-fed controls after a test dose of CDP. In response to an injection of ethanol, the CDP-dependent mice had lower blood alcohol levels (BAL) than the pair-fed controls, but the rate of fall of BAL was not different in the two groups. Thus, chronic CDP treatment affected the absorption and distribution of ethanol. These results provide a metabolic basis for the manifestations of CDP tolerance and ethanol cross-tolerance that have been reported in CDP-dependent mice.

Partial cross-dependence on ethanol in mice dependent on chlordiazepoxide.

Mice which had been fed chronically a liquid diet containing chlordiazepoxide (CDP) showed spontaneous and Ro15-1788-induced withdrawal signs upon CDP withdrawal. Ethanol (1.5 g/kg) injected 5 min before Ro15-1788 injection almost completely suppressed the withdrawal signs induced by the benzodiazepine receptor antagonist. However, neither ethanol injection nor ethanol diet administration could prevent the loss of appetite and weight loss on day 1 of CDP withdrawal. Likewise, the addition of saccharin in the ethanol diets did not prevent the loss of appetite. Mice which had been fed the CDP diet followed by 9 days of ethanol treatment (CDP/ethanol) showed more severe hypothermia during ethanol withdrawal compared to mice which had been fed the control/ethanol diets. The CDP/ethanol mice also retained the increase in runway activity attained from the prior CDP treatment. The data indicate that CDP-dependent mice showed partial rather than full cross-dependence on ethanol.

A liquid diet model of chlordiazepoxide dependence in mice.

Mice fed chronically (3 to 4 weeks) a liquid diet containing chlordiazepoxide (CDP) became physically dependent on the drug as demonstrated by the occurrence of withdrawal signs precipitated by injection of the benzodiazepine antagonist Ro15-1788 (5 to 25 mg/kg) or by omitting CDP from the diet (spontaneous withdrawal). Very low blood concentrations of CDP, but medium to high levels of the active metabolites N-desmethyl CDP and demoxepam were found during the period of CDP administration. The Ro15-1788-induced withdrawal signs appeared within 1 min after the injection of the antagonist and lasted for at least 10 min. Quantifiable withdrawal signs included tail lift, tremor, impaired movement and handling-induced seizures. Mice undergoing spontaneous withdrawal had milder withdrawal signs such as weight loss, in appetite and suppression of runway and head-dipping activities on day 1 or day 2 of withdrawal. These signs were also present in Ro15-1788-induced withdrawal. A long-lasting rebound increase in runway and head-dipping activities occurred several days after CDP withdrawal.

Transferrin and mitochondrial aspartate aminotransferase in young adult alcoholics.

Two recently proposed biochemical markers of alcoholism, namely, quantitation of plasma transferrin variant (Tf5.7) and the ratio of plasma mitochondrial aspartate aminotransferase (m-AspAT) to total AspAT (t-AspAT), were tested for their ability to detect young adult alcoholics. Another commonly used biochemical test, namely, activity of plasma gamma glutamyltransferase (GGT) was included as a comparison. Although mean values of GGT, TF5.7, total transferrin (Tftot), m-AspAT and t-AspAT in alcoholics were significantly higher than those in controls, there were too many overlapping values in each test between alcoholics and controls to render any of these tests suitable as a marker for young adult alcoholics. Depending on cut-off limits, the sensitivity of each test ranged from 0-52% and the specificity ranged from 80-97%. Moreover, the m-AspAT/t-AspAT and Tf5.7/Tftot ratios were not significantly different between alcoholics and controls. A stepwise linear discriminant function analysis of all the variables resulted in a slight increase in classification sensitivity (66%) but a decrease in specificity (77%). The relatively short duration (mean = 5.6 years) of heavy alcohol intake and the time elapsed (mean = 5.8 days) since the alcoholics last consumed alcohol very likely contributed to the low sensitivity. Young adults might also be more resilient with regard to the damaging biochemical effects of ethanol. Abnormal biochemical values might reverse to normal values much more quickly in young adult alcoholics than in those who are older and have more years of alcohol abuse.

Does chronic ethanol intake confer full cross-tolerance to chlordiazepoxide?

Four behavioral tests, namely, hypothermia, horizontal dowel, runway and head-dipping, were used to assess tolerance to ethanol and cross-tolerance to chlordiazepoxide (CDP) in mice chronically treated with an ethanol diet for 15 days. Mice were tested on day 3 of ethanol withdrawal, with some being retested on day 8. In terms of hypothermia and the horizontal dowel test, ethanol tolerance conferred full cross-tolerance to CDP, but the conclusion based on results of the latter test may be equivocal. Partial cross-tolerance to CDP was observed in the runway test, while no cross-tolerance to CDP was detected in the head-dipping test. For these latter two tests ethanol tolerance was present in the mice. Thus, the degree of equivalence between tolerance to ethanol and cross-tolerance to CDP in ethanol-dependent mice varied with the behavioral tests to assess tolerance. Possible mechanisms are discussed.

Differential effects of Ro15-1788 in actions of chlordiazepoxide and ethanol.

Three behavioral tests, namely, runway activity, horizontal dowel test and hypothermia, were used to compare the effects of Ro15-1788, a specific benzodiazepine antagonist, on the common neuropharmacological actions of chlordiazepoxide (CDP) and ethanol in C57BL/6J mice. Ro15-1788 completely reversed the CDP-induced inhibition of runway activity and incoordination on a horizontal dowel, but only partially antagonized the hypothermic effects of CDP. The latter phenomenon was likely to be due to the rapid elimination of Ro15-1788, but could also be due to the fact that hypothermia might not be a specific action of CDP. The sedative actions of ethanol were not antagonized at all by Ro15-1788. In fact, Ro15-1788 potentiated the incoordinating effect of ethanol as determined by the horizontal dowel test such that mice injected with Ro15-1788/ethanol had lower brain ethanol levels than mice injected with vehicle/ethanol when they fell off the dowel. In contrast, mice injected with Ro15-1788/CDP took longer to fall off and had significantly higher CDP levels at fall-off than mice injected with vehicle/CDP. The stimulatory effect of a low dose of ethanol on runway activity was reversed by Ro15-1788. These data are discussed in terms of the possible mechanisms of actions for CDP and ethanol.

Substitution of chlordiazepoxide for ethanol in alcohol-dependent mice.

Alcohol dependence was induced in C57BL/6J mice by administration of a liquid diet containing ethanol. These mice showed alcohol withdrawal signs when the alcohol diet was withdrawn. However, when the alcohol diet was substituted with three liquid diets containing different amounts of chlordiazepoxide (CDP; 0.4, 1 and 2 mg/ml), the alcohol withdrawal signs were fully suppressed by CDP. The CDP diet administration was continued for 14-24 days. At termination of the diet treatment, the mice showed CDP withdrawal signs. Similar signs, but much more short-lived, can be precipitated by injection of the benzodiazepine receptor antagonist, RO-15-1788. The results are consistent with the hypothesis that alcohol-dependent mice are cross-dependent on CDP. This report constitutes the first experimental demonstration of cross-dependence between ethanol and CDP. The reverse phenomenon, namely, CDP-dependent mice being cross-dependent on ethanol, remains to be investigated.

Cross-tolerance between ethanol and chlordiazepoxide.

Drug-induced hypothermia was used to investigate drug tolerance and cross-tolerance. C57BL/6J mice, which were injected with a single dose of chlordiazepoxide (CDP; 30 mg/kg) one day before and reinjected with an equivalent dose of CDP the next day, did not develop tolerance to the drug. However, ethanol-pretreated (3.5 g/kg, 24 hr earlier) mice, when injected with CDP (30 mg/kg), showed cross-tolerance to CDP. The cross-tolerance was short-lived (less than 48 hr). On the other hand, CDP-pretreated mice (30 mg/kg, 24 hr earlier) did not show cross-tolerance to ethanol. The lack of a reciprocating effect of CDP-pretreatment was not likely to be due to the difference in initial dosage between ethanol and CDP. It may be due to different rates of tolerance development or different mechanisms of actions between CDP and ethanol. Mice chronically treated with ethanol also showed a similar degree of cross-tolerance to CDP compared to those exposed to an acute dose of ethanol.

Long-lasting reduction in ethanol selection after involuntary intake of ethanol/chlordiazepoxide.

C57BL/6J mice, after having been exposed to a free-choice condition between water and aqueous chlordiazepoxide (CDP, 25 mg/100 ml) or between water and ethanol/CDP, showed a significant trend for decreased preference for ethanol when tested 2 weeks later. Similarly, mice previously exposed to a no-choice intake of ethanol showed a significant decrease in ethanol preference when tested subsequently. A long-lasting (greater than 20 weeks) reduction in ethanol selection developed after mice were previously exposed to ethanol/CDP in a no-choice condition. This was also accompanied by a decrease in the subsequent selection of ethanol/CDP, but not CDP. The exact mechanisms for the long-lasting decrease in ethanol selection was unknown, but it was not due to the development of fluid aversion. It is suggested that the combined central effects of ethanol/CDP might be partially responsible.

Influence of chlordiazepoxide on alcohol consumption in mice.

In a free-choice situation, chlordiazepoxide (CDP; 12.5 or 25 mg/100 ml; groups B or C), when incorporated in ethanol solutions (2 to 20%, v/v), caused a significant decrease in ethanol preference index (P.I.). This was probably due to the combined CNS effects of both drugs rather than a taste effect, since the mice did not discriminate between aqueous CDP solutions and water. However, when the mice had prior exposure to ethanol and CDP was incorporated intermittently, no significant decreases in P.I. resulted. In a no-choice situation, ethanol intake was increased only on the first day of each intermittent incorporation of CDP (3 days for each 6-day cycle), being more persistent in group B (2 to 15% ethanol) than in group C (2 to 6.5%). Ethanol intake decreased in group C when alcohol concentrations exceeded 10%. The "first-day" CDP effect also occurred in the no-choice situation of an ethanolic liquid diet. Possible factors for this effect are discussed. Thus the effects of CDP on alcohol consumption in non-deprived mice vary with experimental designs.

Dissociation of tolerance and physical dependence after ethanol/chlordiazepoxide intake.

The previously-observed attenuation of withdrawal reactions in mice (group B) fed an ethanol diet containing chlordiazepoxide (CDP) was not due to a difference in the rate of disappearance of blood ethanol levels during chronic diet treatment in group B compared to mice which received only the ethanol diet (group A). Injection of group A mice with CDP or N-demethyl CDP (10 mg/kg) at the time of diet withdrawal did not result in any significant attenuation of withdrawal scores. Injection of the lactam metabolite of CDP (LCDP; 10 mg/kg) resulted in significantly attenuated withdrawal scores at 4 and 6 hr only, but the pattern of withdrawal scores were different from that for group B mice. Moreover, blood LCDP level, in mice injected with LCDP, at 4 hr was at least five times higher than that attained in group B mice (from diet containing CDP). These results support our previous conclusion that the presence of major metabolites of CDP during withdrawal could only account for a minor contribution to the protective effect. Mice in A and B did not differ in the degree of functional tolerance which developed as a result of ethanol intake. Thus, there was an apparent dissociation between tolerance and physical dependence in the mice which had consumed the CDP/ethanol diet. The magnitude of decrease of GABA levels in the cerebellum and cerebral cortex at 4 hr after withdrawal also did not differ between the two groups, suggesting the reduction in GABA levels could not be correlated with the intensity of withdrawal signs.

Alcohol withdrawal reactions after chronic intake of chlordiazepoxide and ethanol.

Withdrawal reactions were compared in C57BL/6J mice, which had been fed an ethanolic liquid diet containing chloridazepoxide (CDP, 3.2 or 6.4 mg/100 ml, group B or C, respectively) with those which had been administered an ethanol diet alone (group A) for 15 days. Group A showed a significantly more pronounced decrease in rectal temperature (4 to 10 hr) and a higher withdrawal score (4 to 14 hr) than mice in groups B and C. The differences in withdrawal signs still persisted even after mice were fed an ethanol diet without CDP for one extra day before withdrawal. The presence of metabolites of CDP in the blood during withdrawal could only account for a minor contribution to the protective effect. Our data are more suggestive of an increased rate of ethanol metabolism leading to lower blood alcohol levels during diet intake period as being the major factor. However we cannot rule out the alternative possibility that CDP or its metabolites might interfere with the development of tolerance to and physical dependence on alcohol.