Observation of hepatotoxic effects of 2-n-pentylaminoacetamide (Milacemide) in rat liver by a combined in vivo/in vitro approach.
Milacemide or 2-n-pentylaminoacetamide hydrochloride, a new glycine derivative, was found to cause elevations of plasma transaminases in patients suffering from severe depression and Alzheimer's disease. However, no signs of liver toxicity were observed during the course of earlier conducted subchronic and chronic in vivo studies in rodents and cynomolgus monkeys. In this study an in vivo/in vitro approach has been proposed to detect early alterations in key metabolic and functional liver capacities. Milacemide was administered by continuous i.v. infusion for 7 days to male Sprague-Dawley rats using subcutaneously implanted osmotic pumps. Doses were given of 0, 250 and 500 mg/kg per day. Body weight and food intake were recorded and at day 7 of exposure, Milacemide concentration, glucose, urea, triglycerides and cholesterol levels and alanine (ALT) and aspartate aminotransferase (AST) activities were measured in plasma. Non-esterified fatty acids were determined in serum. On day 8, after overnight fasting, hepatocytes were isolated. A portion of the cells derived from untreated animals (no osmotic pumps) were cultured in a primary monolayer and exposed in vitro to different Milacemide concentrations. The xenobiotic biotransformation capacity of the isolated hepatocytes was studied by measuring the cytochrome P450 content, ethoxycoumarin-O-deethylase (ECOD), pentoxyresorufin-O-deethylase (PROD), ethoxyresorufin-O-deethylase (EROD), aldrin epoxidase (AE), epoxide hydrolase (EH) and glutathione S-transferase (GST) enzyme activities. Triglycerides, cholesterol and phospholipid contents were measured on the isolated cells. At plasma concentrations of 43 and 130 microM Milacemide, the ALT activity was unchanged or significantly decreased, whereas the AST activity was increased in both cases. Other clinical chemistry parameters remained unchanged. Weight gain was significantly lower in rats treated with the high Milacemide dose. In addition, decreased food consumption was observed in all treated animals leading to significantly lower food efficiency factors for the rats treated with the high dose. Milacemide had a specific inhibitory effect on xenobiotic biotransformation: ECOD activity decreased to 60% of the control value for both Milacemide doses, PROD activity remained unaffected whereas EROD activity decreased to 65% of the control value. A decrease was also observed at the highest drug concentration for AE (to 41%), EH (to 65%), cytochrome P450 content (to 80%) and GST (to 85%). At 500 mg Milacemide kg/day, hepatocyte triglycerides levels increased 3.1-fold while cholesterol and phospholipid levels remained unaffected. Electron and light microscopy on total liver and isolated hepatocytes indicated a concentration-dependent accumulation of lipid droplets, the occurrence of numerous vacuoles in the cytoplasm and other structural abnormalities. When the cultured hepatocytes of control animals (without osmotic pumps) were exposed to Milacemide, the appearance of vacuoles and myeloid bodies could be confirmed in vitro. The results of this study using an in vivo/in vitro approach clearly show potential hepatotoxic properties of Milacemide, an effect not observed in conventional toxicity studies.
Rogiers
V
V
Department of Toxicology, Vrije Universiteit Brussel, Belgium.
Vandenberghe
Y
Y
Vanhaecke
T
T
Geerts
A
A
Callaerts
A
A
Carleer
J
J
Roba
J
J
Vercruysse
A
A
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000081:Acetamides / Q000097:blood / Q000633:toxicity*; D000818:Animals; D000927:Anticonvulsants / Q000097:blood / Q000633:toxicity*; D001835:Body Weight / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism; D004435:Eating / Q000187:drug effects; D004851:Epoxide Hydrolases / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D050356:Lipid Metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D008297:Male; D006899:Mixed Function Oxygenases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D000081:Acetamides; D000927:Anticonvulsants; C039332:milacemide; D006899:Mixed Function Oxygenases; C019647:aldrin epoxidase; D015660:7-Alkoxycoumarin O-Dealkylase; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; D005982:Glutathione Transferase; D004851:Epoxide Hydrolases