Inhibition of the rat liver mitochondrial low Km ALDH activity by acute and chronic administration of kynurenine metabolites
Acute time-course (Fig. a) and dose–response (Fig. b) experiments were performed. Initially, we found that the high Km ALDH activity (assayed in the presence of 5 mM acetaldehyde) was not influenced by kynurenine metabolites. Consequently all results reported in this section concern the low Km activity. As shown in Fig. a, the mitochondrial low Km ALDH activity was inhibited by all three kynurenine metabolites after administration of a 10 mg/kg body wt dose. At 0.5 h after administration, ALDH activity was inhibited by 3-HK and KA by 45 and 37%, respectively (P = 0.0267–0.0024). Inhibition by all three metabolites was then maintained at 40–51% at 1 h. Thereafter, inhibition by KA remained at this latter level until 4 h, whereas that by 3-HAA continued to strengthen, reaching 84% at 4 h (P = 0.0000). With 3-HK, ALDH activity began to recover at 2 h, but remained significantly inhibited at 2 and 3 h (by 30 and 17%, respectively; P = 0.0128–0.0431).
As shown in Fig. b, significant inhibition of 32–56% (P = 0.0398–0.0008) of ALDH activity was observed at 1 h after administration of a 1 mg/kg body wt dose of kynurenine metabolites. Maximum inhibition at 1 h was observed with a 2.5 mg/kg dose of KA (86%) and with a 7.5 mg/kg dose of 3-HK (59%) and 3-HAA (70%) (P = 0.0035–0.0001).
ALDH activity was also inhibited when a 10 mg/kg body wt dose of the above 3 kynurenine metabolites was administered daily for 8 days (data not shown). At 2 h after the (final) injection on the 8th day, inhibition by KA, 3-HK and 3-HAA was 37%, 39% and 64% respectively (P = 0.0292–0.0007). Although this inhibition could very well be due to the acute effect of kynurenine metabolites, it suggests that no tolerance develops towards it after chronic treatment.
Inhibition of ALDH activity in vivo by acute administration of kynurenine metabolites
ALDH activity in vivo was determined by measuring the accumulation of acetaldehyde in blood following acute ethanol administration. The results in Fig. show blood-ethanol (a) and acetaldehyde (b) concentrations after intraperitoneal administration of a 2 g/kg body wt dose of ethanol. In saline-pretreated control rats, ethanol concentration rose to 35.8 mM at 1 h and to 37.1 mM at 2 h before declining to 26.0 mM at 3 h. None of the three kynurenine metabolites exerted a significant effect on ethanol concentration at 1 h (P > 0.1). With 3-HAA, ethanol concentration resembled that in saline-treated controls at 2 and 3 h. By contrast, ethanol concentration at 2 and 3 h after ethanol administration was significantly decreased by pretreatment of rats with KA and 3-HK, by 24–27% (P = 0.05–0.0175).
Blood acetaldehyde concentration following ethanol administration (Fig. b) to saline-pretreated control rats remained at a constant level of 45–47 μM, suggesting a constant rate of ethanol and acetaldehyde oxidation over the 1–3 h observation period. Pretreatment of rats with KA increased acetaldehyde concentration by 109, 125 and 115%, respectively (P = 0.0104–0.0029). 3-HK induced a stronger elevation of acetaldehyde concentration, of 180, 174 and 202%, respectively at 1–3 h after ethanol administration (P = 0.0017–0.0000), whereas with 3-HAA, the elevation of acetaldehyde concentration was modest (42, 34 and 56%, respectively), and not significant (P > 0.1). The elevation of blood acetaldehyde concentration by 3-HK was significantly greater than that by KA at 1–3 h (P = 0.05–0.0095).
Demonstration of aversion to alcohol after administration of kynurenine metabolites and disulfiram
In the aversion model of Garver et al. (2000)
, rats treated with the classical ALDH inhibitor disulfiram consumed equal amounts of the ethanol drinking solution as control animals during the first hour of the test. Thereafter, alcohol consumption by disulfiram-treated rats remained static, unlike that by controls, which continued to increase cumulatively up to the fifth hour. The results in Fig. a, show that disulfiram actually inhibited alcohol consumption significantly and maximally during the first hour (by 51%; P
0.0351, paired t
-test), thereafter the animals continued to drink the ethanol solution, but to a lesser degree than controls. Thus, the inhibition of ethanol consumption by disulfiram was maintained for two more hours, as 45–46% (P
= 0.0158–0.0138), but, at 4 h, the 37% decrease was not significant. The results in Fig. b show that kynurenine metabolites also inhibited alcohol consumption in this model significantly (P
= 0.05–0.005) over the first 3 h. Thus, as was the case with disulfiram, inhibition of alcohol consumption was strongest at 1 h after administration of KA, 3-HK and 3-HAA (by 28, 26 and 50%, respectively). Inhibition remained significant at 2 and 3 h, but, by 4 h, only that by KA was still significant.
Fig. 4. Inhibition of alcohol consumption by disulfiram (a) and kynurenine metabolites (b) in a rat alcohol aversion model Experimental details are as described in the ‘Materials and Methods’ section. Ethanol consumption was monitored hourly for (more ...)
It will be noted from the data in Fig. that alcohol consumption by control rats in the disulfiram experiment (Fig. a) is lower than that of the control animals in the kynurenine metabolite experiment (Fig. b). This is almost certain to be due to the use of dimethylformamide, along with saline, to dissolve disulfiram, rather than to variations among different batches of animals, because, as will be seen in the accompanying paper (Badawy et al., 2011
), the control data obtained in animals given saline only in two different experiments were broadly similar to those in the present experiment with kynurenines.
Alcohol consumption by C57 mice
As no attempt was made to enhance alcohol preference by acclimatizing the mice to increasing ethanol concentrations, there were wide variations in levels of consumption of the 10% (v/v) ethanol solution, with ~37–50% of the mice in each group consuming >10 g/kg/day, and showing >50% preference, with the remainder consuming up to 7 g/kg/day with a level of preference <32%. To establish comparability between group at baseline (Day 0), six mice from each group were selected whose results are shown in Fig. . As shown, the % preference (Fig. a) and absolute ethanol intake in g/kg body wt (Fig. b) were broadly similar across groups at baseline, with no significant differences (P > 0.1). In the saline-treated control group, preference remained stable for 4 days and the decrease on Day 5 did not reach statistical significance (P = 0.095). By contrast, preference decreased in mice receiving the three kynurenine metabolites, with 3-HAA causing the largest decrease. Compared with baseline, the decrease in the % preference with 3-HAA (23–46%) was significant on all days (P = 0.043–0.009), except Day 4. With 3-HK and KA, only the decreases on Days 4 and/or 5 (19–39%) were significant (P = 0.05–0.011). When the % preference values with kynurenine metabolites were compared with those of the control group, significant decreases were also observed with 3-HK (37%) on Day 4, with KA (26–30%) on Days 1 and 4 and with 3-HAA (26–36%) on Days 1–5 (P = 0.05–0.0075).
Fig. 5. Effects of repeated administration of kynurenine metabolites 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 ...)
When alcohol consumption was expressed in absolute amounts (g/kg body wt) (Fig. b), a broadly similar pattern emerged, with 3-HAA causing the greatest decrease in alcohol intake. However, the only significant differences were those compared with the saline controls for KA on Days 1 and 4 and for 3-HAA on Days 1 and 5 (31–45%; P = 0.021–0.001).
Hepatic concentrations of kynurenine metabolites after their administration
Hepatic concentrations of KA, 3-HK and 3-HAA were determined after acute administration of each individual compound in the same rats in which ALDH activity was assessed. Although concentrations of other kynurenine metabolites and of Trp were also determined, these are not reported here, as their relevance largely falls outside the scope of the present paper. When a 10 mg/kg body wt dose of the three kynurenine metabolites was administered (Fig. a), maximum increases (of 4.2–7.3-fold) in their concentrations were observed at 1 h (P = 0.0002) and were maintained for 3–4 h. Dose-dependent increases in liver kynurenine metabolite concentrations were observed at 1 h (Fig. b) in the 1–10 mg/kg dose-range with KA and 3-HK. However, with 3-HAA, the maximum increase was observed with the 5 mg/kg dose. Baseline [3-HAA] in the time-course experiment was lower than that in the dose–response one. This is most likely due to variations across different animal batches, as the relative increases in this metabolite concentration at 1 h after administration of the 10 mg/kg dose were similar in both experiments (2.67 and 2.97-fold, respectively).
Fig. 6. Hepatic kynurenine metabolite concentrations after their acute administration Kynurenine metabolite concentrations were determined as described in the ‘Materials and Methods’ section in livers of the same rats undergoing the time-course (more ...)
Animal body weights during repeated administration of kynurenine metabolites and disulfiram
Body wt was measured in chronic experiments, not only to determine daily dose levels, but also as a measure of animal welfare and safety of administered compounds. In the alcohol aversion study in which compounds were injected once daily for 4 days, both rats treated with kynurenine metabolites and their saline-treated controls gained weight at the normal rate during the first 3 days. However, small losses of 6–9% were observed in both control and test rats on the morning of the final (fourth) day, compared with body weights the day before, almost certainly due to introducing a water-deprivation regimen during the preceding 18 h. In the aversion experiments with disulfiram and its control rats receiving the vehicle (saline: dimethylformamide, 1:1), both control and test rats showed a small wt loss (2.8 and 4.2%) on Day 3, compared with Day 1, presumably due to this solvent, in addition to a 3–6% wt loss following the water-deprivation period, as was the case with kynurenine metabolites and their pure saline control.
In the chronic study of changes in rat liver ALDH activity in which kynurenine metabolites were administered daily for 8 days, changes in body weights were also recorded. As shown in Fig. a, all groups gained wt significantly (P = 0.001), with the gains by control rats reaching 26% on Day 8. Rats receiving KA and 3-HK also gained wt at a rate close to that of controls (respectively 21 and 24% on Day 8). 3-HAA-treated rats, however, gained wt less strongly, achieving only a 10% wt gain on Days 6–8. All animals appeared to tolerate kynurenine metabolites and showed no adverse reactions or unusual behaviours.
Fig. 7. Body weights of rats and mice during chronic treatment with kynurenine metabolites on Day 1 (rats) or Day 0 (mice), animals received single daily intraperitoneal injections of saline or kynurenine metabolites (10 mg/kg each) for 8 (rats) or 5 (more ...)
Body wt was also recorded for mice during the preference study. The body weights of all mice (n = 32) at the start of the study (mean ± SEM in g) (26.83 ± 0.25) rose by 5% during the 21-day free choice period to reach 28.09 ± 0.23 g (P = 0.003) on Day 0 of the 5-day drug administration experiment. As shown in Fig. b, body weights remained stable over the 5-day period and none of the changes in the control and test groups was significant when compared with the zero day value (P > 0.9). This is reflected in the small (not exceeding 2.5%), but insignificant, gains observed in the control group and those receiving 3-HK and KA and in the small (2%) and insignificant loss in mice of the 3-HAA group. As was the case with rats, mice appeared healthy and showed no adverse reactions or unusual behaviors.