Effects of administration of BSZ or carbidopa alone or in combinations with tryptophan on the rat liver mitochondrial low Km ALDH activity
The acute effects are depicted in Fig. . Neither BSZ alone (Fig. a) nor carbidopa alone (Fig. b) exerted any significant effects on ALDH activity. Similarly, Trp administered alone or with carbidopa did not influence ALDH activity (Fig. b). By contrast, combined administration of Trp plus BSZ caused a remarkable inhibition (Fig. a). Thus, ALDH activity was decreased by this combined treatment by 46% at 1 h (P = 0.0005). Inhibition then progressed over time, reaching 92% at 3 h and remaining at this level 1 h later.
Fig. 2. Time-course of effects of acute administration of tryptophan, BSZ, carbidopa and combinations of tryptophan with BSZ or carbidopa on activity of the rat liver mitochondrial low km ALDH. Tryptophan (50 mg/kg), BSZ (100 mg/kg) or carbidopa (more ...)
Chronic experiments were also performed in which rats were treated with Trp, BSZ or a combination of the two for 7 days. In data not shown, neither Trp alone nor BSZ alone exerted any significant effect on ALDH activity, whereas the combined treatment caused a 55% inhibition (P = 0.0001) Although this could be attributed to an acute effect at 2 h after the final treatment, it suggests that no tolerance develops to the acute inhibition after repeated administration of Trp plus BSZ.
Inhibition of AlDH activity in vivo by combined administration of tryptophan and BSZ
The accumulation of acetaldehyde and ethanol in blood following acute ethanol administration was studied in rats treated with Trp, BSZ or a combination of the two (Fig. ). Blood acetaldehyde concentration (Fig. a) after pretreatment with BSZ did not differ significantly from that in saline-pretreated controls (P > 0.0980). A non-significant trend towards elevated acetaldehyde concentration after pretreatment with Trp was observed (P > 0.0971). By contrast, combined pretreatment with Trp plus BSZ caused a significant elevation of blood acetaldehyde concentration. The strongest effect was at 1 h after ethanol (145 μM), which was significantly different from all the other three groups (P = 0.0287–0.0001). At 2 h, the increase caused by Trp plus BSZ was also significant, compared with those in saline or BSZ-pretreated rats (P = 0.0029), but, at 3 h, only the difference from saline-pretreated controls was still significant (P = 0.0011). Acetaldehyde concentration in Trp plus BSZ-pretreated rats did not differ significantly from that in Trp-pretreated rats at 2 or 3 h.
Fig. 3. Effects of acute administration of BSZ, tryptophan or both on rat blood ethanol and acetaldehyde concentrations. Rats received an intraperitoneal injection of BSZ (100 mg/kg body wt) or an equal volume (1 ml/kg) of saline 1 h before (more ...)
The increase in blood ethanol concentration following ethanol administration to saline-pretreated controls was not significantly influenced by pretreatment with BSZ, Trp or both (Fig. b). Pretreatment with Trp alone showed a slightly lower blood ethanol concentrations at 3 h, though not significant (P > 0.1). The ethanol area under the curve (AUC) was similar among the saline-, BSZ- and Trp plus BSZ-pretreated groups (80.35, 80.95 and 81.85 mM, respectively) and slightly higher than that in the Trp-pretreated group (76.9 mM).
Demonstration of aversion to alcohol after administration of tryptophan in combination with BSZ, but not with carbidopa
In the preceding paper (Badawy et al., 2011
), we have successfully applied the alcohol aversion model of Garver et al. (2000)
to demonstrate aversion to alcohol by three kynurenine metabolites and to confirm that by the classical AlDH inhibitor disulfiram. In the present work, we tested the same model for possible aversion by Trp and the two kynureninase inhibitors BSZ and carbidopa either alone or in combination with Trp. As the results in Fig. show, no aversion to alcohol was observed after administration of Trp alone. BSZ alone (Fig. a) also did not inhibit alcohol consumption, except only at 2 h. This particular effect appears to be a spurious result, as it did not occur earlier nor was it maintained thereafter, and it does not fit in with other results described in this paper. Carbidopa alone (Fig. b) caused aversion to alcohol, decreasing alcohol consumption by 30–33% consistently at 2–4 h (P
= 0.0162–0.0017), in comparison with saline-treated controls.
Fig. 4. Inhibition of alcohol consumption by carbidopa alone and by combined treatment with BSZ and tryptophan in a rat alcohol aversion model. Groups of rats received single daily intraperitoneal injections for 4 days of BSZ (100 mg/kg body wt), carbidopa (more ...)
Combined administration of Trp with carbidopa (Fig. b), however, reversed the carbidopa-induced aversion. Here, alcohol consumption resembled that of saline-treated controls. By contrast, combined treatment with Trp plus BSZ (Fig. a) induced consistent inhibition of alcohol consumption, of 38–44% at 1–4 h (P = 0.0127–0.0024), compared with saline-treated controls. Alcohol consumption with this combined treatment also differed significantly from that with the Trp-treated group at all four time-intervals (P = 0.0253–0.0030) and from the BSZ-treated group at 1 and 4 h (P = 0.0274–0.0106).
Effects of tryptophan, BSZ or both on alcohol consumption and preference by C57 mice
After a 16-day period of provision of free choice between drinking water and a 10% (v/v) ethanol solution, four groups (n = 8 each) of male alcohol-preferring C57BL/6J mice were treated with single daily doses of saline, Trp, BSZ or a combination of Trp plus BSZ. Six mice from each group were matched on Day 0 for level of alcohol consumption (in g/kg body wt) and preference (%) adjusted per kg body wt, as shown in Fig. . There were no significant differences on any one day between the groups receiving saline, Trp or BSZ, either in absolute ethanol intake (Fig. a) or preference (Fig. b) (ANOVA's, P > 0.1). By contrast, combined administration of Trp + BSZ led to lower levels of alcohol consumption and preference. Alcohol intake (g/kg) in this combined group differed significantly only on Day 5 from that in the BSZ- and Trp-treated groups (P = 0.05–0.034), whereas alcohol preference (%) differed significantly from that in the saline group on Days 1and 3 (P = 0.039–0.015), the Trp group on Days 1, 3 and 5 (P = 0.044–0.003) and the BSZ group on Days 1, 3 and 5 (P = 0.05–0.009). From these data and the graphs, it is clear that, whereas the three single groups exhibited a cyclical alcohol consummatory behaviour, with a high and a lower intake on alternate days, the combined Trp + BSZ group consistently decreased its alcohol intake continuously with time. Accordingly, because of this cyclical behaviour of the single treatment groups, the absence of significant differences between the Trp + BSZ group and the other three was apparent only on days on which mice of these latter groups decreased their alcohol intake.
Fig. 5. Effects of repeated administration of BSZ, tryptophan or both on alcohol consumption and preference in male alcohol-preferring C57BL/6J mice. Experimental details are as described in the ‘Materials and Methods’ section. Alcohol consumption (more ...)
Inhibition of liver kynureninase activity by BSZ and carbidopa and the differential effect of combined treatment with tryptophan
The two most potent inhibitors of kynureninase activity in vitro
are carbidopa and BSZ, with the former being a stronger inhibitor (Bender and Smith, 1978
; Bender, 1980
). This was partially confirmed in the present work. As shown in Table , kynureninase inhibition was similar between BSZ and carbidopa at drug concentrations of 10 μM (17–22%), 25 μM (25–28%) and 100 μM (32–33%). However, at larger concentrations, the inhibition by carbidopa was almost twice as strong (61 vs 36% at 250 μM and 80 vs 44% at 500 μM).
Inhibition of liver kynureninase activity in vitro by BSZ and carbidopa
Kynurenine aminotransferase activity was also determined simultaneously with that of kynureninase by measuring the increase in KA formation. In data to be reported elsewhere, both BSZ and carbidopa inhibited kynurenine aminotransferase activity, with the inhibition by carbidopa being stronger than that by BSZ at all concentrations tested.
Kynureninase activity was also determined after the acute administration of BSZ, carbidopa, Trp and combinations of Trp with either drug. The results in Fig. a show that Trp alone did not inhibit kynureninase activity. Trp administration (30 mg/kg intraperitoneally) has previously been reported (Takeuchi and Shibata, 1984
) to enhance kynureninase activity by 15 and 21% at 2 and 3 h, respectively. As no such enhancement was observed in the present work at these time-intervals, a closer examination of a possible early increase was made. At 1.5 h after Trp administration, kynureninase activity (6.08 ± 0.49, expressed as in Fig. ) was still not significantly different (P
> 0.1) from the control zero-time value (5.78 ± 0.27). However, a significant increase of 37% was observed at 0.5 h (7.91 ± 0.26; P
Fig. 6. Time-course of effects of acute administration of tryptophan, BSZ, carbidopa or a combination of tryptophan with BSZ or carbidopa on rat liver kynureninase activity. Experimental details are as described for the ALDH experiments in Fig. . Kynureninase (more ...)
As expected, both BSZ and carbidopa inhibited kynureninase activity. As shown in Fig. b, BSZ caused a significant inhibition (43%; P = 0.0003) only at 1 h, whereas carbidopa (Fig. c) caused a similar inhibition (41%; P = 0.0013) at 2 h. The inhibition by BSZ was not undermined by combined Trp administration (Fig. b). In fact, a constant level of inhibition (of 30–35%; P = 0.0196–0.0036) was sustained over the first 3 h, and that at 4 h (23%) was still significant (P = 0.0462). As BSZ was administered 30 min before Trp, the latter could be said to have prolonged the BSZ inhibition by 3.5 h. By contrast, the kynureninase inhibition by carbidopa was totally reversed by co-administration of Trp (Fig. c).
Kynureninase activity was also determined after chronic administration of Trp, BSZ or both for 7 days. In data not shown, kynureninase activity was decreased by BSZ by 22% and co-administration of Trp did not modify this decrease. In fact, Trp induced a further decrease. Trp alone exerted no effect on kynureninase activity. Under these chronic conditions, BSZ also inhibited kynurenine aminotransferase activity, by 61% and Trp, which exerted no significant effect when given alone, caused a further significant decrease when co-administered with BSZ (data not shown).
Hepatic kynurenine metabolites after the acute administration of tryptophan, carbidopa, BSZ or various combinations
As stated above, the main effect of kynureninase inhibition (by BSZ and carbidopa) is the elevation of hepatic [3-HK]. However, because these two drugs also inhibit kynurenine aminotransferase activity, the potential increase in xanthurenic acid (due to 3-HK elevation) may be hampered by such latter inhibition. Although hepatic concentrations of Trp and six of its kynurenine metabolites were measured in the present work, only the data on [3-HK] are reported here, as details of other changes and their discussion fall outside the scope of this paper. As shown in Fig. , the 3-HK precursor Trp administered alone caused relatively large increases in liver [3-HK] at 3–4 h, with the 190% increase at 3 h being significant, compared with the zero-time control value (P = 0.016). The much smaller increases at 1 and 2 h were not significant. By contrast and as expected, both BSZ (Fig. a) and carbidopa (Fig. b) induced large increases in [3-HK] (P = 0.004–0.001), which were comparable at 1 and 2 h. Thereafter, the [3-HK] elevations by carbidopa remained close to the maximum increases, whereas those by BSZ showed a dramatic drop towards normal values.
Fig. 7. Time-course of increases in hepatic 3-HK concentration after acute administration of tryptophan, BSZ, carbidopa or a combination of tryptophan with BSZ or carbidopa. 3-HK concentration was determined in livers of the same rats undergoing the time-course (more ...)
When BSZ administration was followed 0.5 h later by that of Trp, a dramatic elevation of [3-HK] was observed at 1 h after the Trp injection, with a 1069% increase over the baseline zero-time value (P = 0.008). This increase was a 152% higher than that induced by BSZ alone at this time-interval (P = 0.008). The [3-HK] elevation by the combined BSZ plus Trp treatment continued to be much higher than that by BSZ alone at the 2–4 h time points (P = 0.05–0.001). By contrast, when carbidopa was administered 0.5 h before Trp, there was no potentiation of the carbidopa-induced elevation of [3-HK] by Trp. In fact, none of the [3-HK] values observed with this combination differed significantly from those obtained with carbidopa alone at 1–3 h (P > 0.1) and the value at 4 h was even 26% smaller (P = 0.034) after the combined treatment.
Animal body weights during repeated administration of tryptophan, BSZ or both
In the aversion experiments reported in Fig. , rats gained weight during the first 3 days of the test. However, as was the case with disulfiram and kynurenine metabolites in the preceding paper (Badawy et al., 2011
), a wt loss of 6–7% occurred in all group rats on the morning of the fourth day, following the 18 h-water-deprivation period.
Changes in body wt of rats given single daily injections of saline, Trp, BSZ or a combination of Trp plus BSZ for 7 days in the chronic experiments were also recorded. Rats of all groups gained wt. On Day 7, the wt gain over the starting wt on Day 1 was 19, 21, 21 and 17%, respectively for the above four groups. No significant differences in body wt were observed after analysis of variance in within- or between-group comparisons (P > 0.1).
Body weights of mice in the preference experiments were also recorded. All mice (n = 32) gained wt during the 16-day free choice period (from 19.44 ± 0.16 to 21.85 ± 0.21 g; P < 0.001) (means ± SEM). Body weights remained stable during the 6-day drug administration period, with small rises of 1–3%. No significant differences within- or between-groups were observed (P > 0.1). Both rats and mice appeared healthy and showed no adverse reactions or unusual behaviours.