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Phenobarbital (PB) therapy is frequently associated with elevated serum alanine aminotransferase (ALT) and alkaline phosphatase (AP) activities in dogs without clinical signs of liver disease. The goal of this study was to determine if increased serum ALT and AP activities in clinically healthy PB-treated epileptic dogs are due to hepatic enzyme induction or to subclinical liver injury. Liver biopsies were obtained from 12 PB-treated dogs without clinical signs of liver disease but with elevated serum ALT and/or AP activities or both. Liver biopsies were obtained from eight healthy control dogs not receiving PB. Biopsies were evaluated histopathologically (all dogs) and liver homogenates were assayed for ALT (all dogs) and AP (six treated dogs, all controls) activities. As a positive control, liver cytochrome P4502B, an enzyme known to be induced by PB, was measured by benzyloxyresorufin-O-dealkylase activity and immunoblotting (five treated dogs, all controls). Serum AP isoenzyme analyses were performed. Results showed that ALT and AP activities in liver homogenates were not increased in treated dogs compared with controls, whereas the positive control for induction, CYP2B, was dramatically increased in treated dogs. Histopathological examination of liver biopsies revealed more severe and frequent abnormalities in treated dogs compared to controls, but similar types of abnormalities were found in both groups. Serum AP isoenzyme analyses in treated dogs demonstrated increased corticosteroid-induced and liver isoenzyme activities compared to controls. Results do not support induction of ALT or AP in the liver as the cause of elevated serum activities of these enzymes due to PB.

The aim of this study was to determine the amount of excess dietary copper (Cu) necessary to experimentally induce liver lesions characteristic of Cu-associated disease in Fischer 344 rats. Male weanling Fischer 344 rats of uniform age were divided into 6 groups (n = 5) and fed a rodent diet containing 18 (control), 750, 1000, 1250, 1500, and 2000 microg/g Cu added as CuSO4. Rats were euthanized after 3 months on the experimental diets and their livers processed for histology, histochemistry, Cu analysis (by atomic absorption spectrophotometry), and quantification of malondialdehyde (MDA) by the thiobarbituric acid reaction. Hepatic Cu levels were significantly higher (P < 0.01) in rats receiving over 1000 microg/g Cu compared to the controls (means for each diet: control = 4.8 microg/g, 750 microg/g Cu = 39.6 microg/g, 1000 microg/g Cu = 111.2 microg/g, 1250 microg/g Cu = 389 microg/g, 1500 microg/g Cu = 509.4 microg/g, and 2000 microg/g Cu = 766 microg/g). Histological lesions increased gradually according to the level of dietary Cu. Significant morphologic changes (necrosis, portal inflammation, hyaline remnants) and reduced growth rate occurred in rats receiving over 1250 microg/g Cu. However, no significant differences were found for MDA levels between groups. The present study demonstrates that compared to other species, very high levels of excess dietary Cu are needed to induce significant liver injury in Fischer 344 rats. Increased MDA content was not detected in rats with morphologic evidence of liver damage, suggesting that lipid peroxidation may not play a major role in this model of Cu toxicity.

The purpose of this study was to determine the molecular basis for a relative deficiency in the cat of cytosolic arylamine N-acetyltransferase (NAT), an enzyme family that is important in the metabolism of xenobiotics and that normally consists of at least two related enzymes, NAT1 and NAT2. N-acetyltransferase in feline liver showed high affinity (mean Km = 2.1 microM) for p-aminobenzoic acid, an NAT1 selective substrate in humans and rabbits, but showed a very poor affinity (mean Km > 10 mM) for sulfamethazine, an NAT2 selective substrate in humans and rabbits. Immunoreactive N-acetyltransferase was detected in feline liver, bladder and colon using an NAT1-specific antipeptide antibody, but was not detected in any tissues using an NAT2-specific antibody. Southern blot analysis of genomic DNA demonstrated a single band in domestic cats using each of six restriction digests; single bands were also found on Southern blot analysis of six wild felids. The deduced amino acid sequence of the central portion of feline N-acetyltransferase, obtained by polymerase chain reaction amplification in both domestic cats and seven wild felids (lion, tiger, lynx, snow leopard, bobcat, Asian leopard cat and cheetah), contained three residues, Phe125, Arg127, and Tyr129, which determine NAT1-like substrate specificity in humans. These results support the conclusion that cytosolic arylamine N-acetylation activity is low in the cat because of the presence of a single N-acetyltransferase that has substrate specificity, immunogenicity and sequence characteristics similar to human NAT1, and that the unusual presence of only a single N-acetyltransferase gene appears to be a family wide trait shared by other felids.

The purpose of this study was to determine the molecular basis in the dog for an unusual and absolute deficiency in the activity of cytosolic N-acetyltransferase (NAT), an enzyme important for the metabolism of arylamine and hydrazine compounds. NAT activity towards two NAT substrates, p-aminobenzoic acid and sulfamethazine, was undetectable in dog liver cytosol, despite substrate concentrations ranging from 10 microM to 4 mM and a wide range of incubation times. Similarly, no protein immunoreactive to NAT antibody was evident on western blot analysis of canine liver cytosol. Southern blot analysis of genomic DNA from a total of twenty-five purebred and mixed bred dogs, and eight wild canids, probed with a full-length human NAT2 cDNA, suggested an absence of NAT sequences in all canids. Polymerase chain reaction amplification of genomic DNA using degenerate primers designed to mammalian NAT1 and NAT2 consensus sequences generated products of the expected size in human, mouse, rabbit, and cat DNA, but no NAT products in any dog or wild canids. These results support the conclusion that cytosolic NAT deficiency in the domestic dog is due to a complete absence of NAT genes, and that this defect is shared by other canids.

Potentially serious idiosyncratic reactions associated with sulfamethoxazole (SMX) include systemic hypersensitivity reactions and hepatotoxicity. Covalent binding of SMX to proteins subsequent to its N-hydroxylation to form N4-hydroxysulfamethoxazole (SMX-HA) is thought to be involved in the pathogenesis of these reactions. A polyclonal antibody was elicited in rabbits against a SMX--keyhole limpet hemocyanin conjugate that recognized covalent protein adducts of SMX in microsomal protein and was used to characterize the covalent binding of SMX and its putative reactive metabolites to hepatic protein in vivo and in vitro. In vitro covalent binding of SMX to rat and human liver microsomal protein was NADPH-dependent, while binding of SMX-HA was not dependent on NADPH. SMX and SMX-HA produced similar patterns of covalent binding, with major protein targets in the region of 150, 100 (two bands), 70 (two bands), and 45-55 kDa. The pattern of covalent binding to human and rat liver microsomal protein was similar. Binding of SMX-HA was completely eliminated by GSH or by addition of cytosolic fractions and acetylcoenzyme A. The acetoxy metabolite of SMX also led to covalent binding, but it was primarily attributable to the formation of SMX-HA from acetoxySMX. In vivo exposure of rats to SMX did not result in detectable covalent binding by the methods employed. When rat liver slices were incubated with 2 mM SMX or 500 microM SMX-HA, no toxicity was observed and yet covalent binding of SMX-HA to 130, 100, 70, and 55 kDa proteins could be detected. These results confirm that covalent binding of SMX occurs via the formation of SMX-HA and that covalent binding of SMX-HA in vitro results from its conversion to the more reactive nitroso metabolite. Acetylation of SMX-HA protected against its covalent binding. Further studies are required to determine how this in vitro covalent binding relates to in vivo covalent binding in humans and to either direct or immune-mediated cytotoxicity in SMX idiosyncratic drug reactions.

Interferon and interferon inducers are well known to depress hepatic cytochrome P-450 (P-450). Previous reports have suggested that all constitutive members of the P-450 family of proteins are affected in this manner, whereas inducible P-450s--including cytochrome P-4503A1 (CYP3A1)--are resistant to the effects of interferons. We examined the effect of interferon [produced in response to polyinosinic acid-polycytidylic acid (polyIC; 10 mg/kg) administration] on the induction of CYP3A1 in the female rat by the macrolide antibiotic troleandomycin (TAO; 200 mg/kg), and the antiglucocorticoid pregnenolone-16 alpha-carbonitrile (PCN; 300 mg/kg). The induction of CYP3A1 was characterized by erythromycin N-demethylation, Western blotting, and mRNA quantitation with a specific oligonucleotide cDNA probe. PCN-mediated induction of erythromycin metabolism was depressed by 85% following polyIC administration. PolyIC depressed the induction of CYP3A1 apoprotein by TAO (84%) and PCN (73%). The depression of enzyme activity and protein were accompanied by a corresponding decrease in hepatic CYP3A1 mRNA. It is concluded that CYP3A1 is sensitive to the depressant effects of interferon, and that interferon appears to act at a pretranslation step that is independent of the induction process per se.