Spontaneous development of fatty liver in ferrets in a toxicology study.

Ferrets were maintained for 12 months on different diets (A, meat and biscuit; B, all meat; C, meat and fish; D, high fibre) to ascertain the cause of spontaneous development of fatty liver. High hepatic triglyceride contents resulted on diets B = C > D; whereas ferrets on diet A (control) showed no accumulation of lipid in liver. Serum triglyceride and total cholesterol were unchanged by diet. These ferrets (F0 generation) were mated with ferrets on the same diet and the offspring (F1 generation), maintained on the same diets as the parents, were killed at 12 months and the livers studied similarly. Histology showed that hepatic lipid accumulation in the F1 generation was identical with that in the same dietary groups of the F0 generation; liver glutathione (GSH) reductase and thiobarbituric acid-reacting substances (an index of lipid peroxidation) were increased in ferrets maintained on diets B, C and D, liver GSH concentration and GSH peroxidase activities were unchanged. Other ferrets fed a high-fat diet (diet A plus 20% w/w beef suet) for 18 days exhibited hepatic lipid accumulation and decreased hepatic cyanide-insensitive palmitoyl CoA oxidation (-30%), but hepatic lauric acid hydroxylation and carnitine acyl transferase activities were unchanged. These data indicate that ferrets on high-fat diets show no increased rates of liver fatty acid oxidation, as seen in rats, but instead accumulate triglyceride in the liver with some degree of lipid peroxidation.

Effect of high fat diet on liver microsomal oxygenations in ferret.

1. Ferret on a high fat diet accumulated large amounts of fat in its liver and had blood acetoacetate and beta-hydroxybutyrate concentrations 250 and 375% of those in control animals. 2. The high fat diet alone increased ferret liver microsomal 7-ethoxyresorufin O-deethylase (EROD) activity by 90%, but had no effect on 7-methoxy-, 7-pentoxy-, or 7-benzyloxy-resorufin, O-dealkylase activities. Administration of 3-methylcholanthrene (MC) increased only liver EROD activity, by 5- to 6-fold, in ferret on both high fat and control diets. Induction of EROD, but not MROD activity, in ferret on the high fat diet indicates that P4501A1, but not P4501A2, is induced. 3. Activation of 3H-paracetamol, measured by covalent tissue binding to ferret liver microsomal fractions, was increased three-fold in ferret on the high fat diet, nine-fold by MC administered to ferret on a control diet, and 13-fold by MC given to ferret on the high fat diet. Similar results were obtained with activation of the cooked-food amine, Glu-P-1, by ferret liver microsomes. 4. Western blots with antibodies to rat liver P450s showed that ferret liver contains proteins orthologous with rat liver P4504A1 and bifunctional protein. However, whereas clofibrate, similar to high fat diets, induced these two proteins in rat liver, no increase of these proteins occurred in liver of ferret fed a high fat diet. Western blots also showed that ferret liver contains no P4501A1 or 1A2, and although these two proteins were induced by MC, no induction occurred when ferret was fed the high fat diet alone. Ferret liver microsomes also contain a protein recognized by rat anti-P4502E1 but of a lower molecular weight. 5. Immunosorbent (ELISA) analyses of ferret liver for P4501A1 and 4A1 showed that the high fat diet increased a protein orthologous to rat P4501A1 but did not increase any protein orthologous to rat P4504A1. 6. These findings indicate that the high fat diet does not induce ferret liver bifunctional protein or P4504A1 enzyme protein, but may enhance liver P4501A1 and 1A2 activities through the hyperketonaemia resulting from the high dietary fat. The conflicting P450 results, namely Glu-P-1 activation but no MROD activity for P4501A2, high EROD activity and ELISA quantification of P4501A1, but no positive Western blot, are probably due to differences in substrate specificity and immunological characteristics between rat and ferret enzymes.

Effects of ether anaesthesia and fasting on various cytochromes P450 of rat liver and kidney.

Fed and fasted, male, Wistar albino rats exposed to light ether anaesthesia and killed immediately or after 30 or 120 min recovery were compared with non-anaesthetized rats for changes in liver and kidney cytochrome P450 (CYP) activities. In fed rats, liver total CYP (nmol/mg protein) decreased by 30% immediately after ether, but was restored to normal levels after 30 min recovery; in fasted rats, liver total CYP increased by 20% by fasting alone, then decreased by 65% immediately after ether, and recovered to only 70% of control at 2 hr after ether. Rat liver cytochrome P4501A (CYP1A; 7-ethoxyresorufin O-deethylase or EROD activity) and cytochrome P4502B (CYP2B; 7-pentoxyresorufin O-dealkylase or PROD activity) were decreased after ether anaesthesia, similar to those for total CYP. In contrast, rat liver cytochrome P4502E1 (CYP2E1), determined by p-nitrophenol hydroxylation, increased by 40% by ether anaesthesia alone, 70% by fasting alone and 140% by ether plus fasting; these increases were confirmed by the CYP2E1-mediated activation of nitrosopyrrolidine and by immunoblot analysis using antibody to CYP2E1. In rat kidney, losses of total CYP, CYP1A and CYP2B, and increases of CYP2E1, induced by ether anaesthesia, were much more marked in fasted (90% loss in total CYP, 30% increase in CYP2E1) than in fed rats (slight loss in total cytochrome P450, 30% increase in CYP2E1). As maximum losses of total CYP in liver of fasted rats exposed to ether occurred at the time of maximum increase of CYP2E1 and maximum rate of generation of reactive oxygen species (ROS), it is suggested that the increase of CYP2E1, resulting from its stabilization by fasting and ether, leads to generation of ROS, increase in lipid peroxidation and consequent loss of total CYP, associated with the hepatic and renal necrosis seen in ether intoxication and surgical trauma.

Hepatic mixed-function oxidases of ferret.

1. Ferret liver mixed-function oxidase enzymes have been quantified using a variety of substrates and the activities have been compared with those found in rat liver. 2. Ferret liver total cytochrome P-450 is only 30% of that of rat liver and exhibits higher 7-ethoxyresorufin O-deethylase (EROD) activity, and lower lauric acid hydroxylase activity than rat liver; other mixed-function oxidases are at similar levels of activity in both species. 3. Induction with 3-methylcholanthrene (MC), similar to MC-induction in rat, increases the total P-450 of ferret liver by 140%, but does not increase P-450 reductase or microsomal protein. EROD specific activity (pmol/min per mg protein) is increased 20-fold by MC treatment. 4. Turnover number of EROD for control liver microsomes of ferret, hamster, mouse, guinea pig and rat were 460, 69, 44, 36 and 35 pmol/min per nmol P-450, respectively, indicating the much higher value for ferret than for any of the rodent species studied. 5. Ferret liver EROD activity is inhibited by the P4501A1 inhibitor, alpha-naphthoflavone. Use of monospecific antibodies in ELISA, Western blot and enzyme-inhibition techniques has shown that EROD activity in ferret liver is attributable to two enzyme proteins orthologous with rat liver cytochromes P4501A1 and 1A2, with the former predominating. MC induces both P4501A enzyme proteins in ferret liver, as in rat liver, with P4501A1 activity predominating.