The mechanism of acetaminophen-induced methemoglobinemia in dogs and cats

Acetaminophen (APAP) overdose in most species is associated with hepatotoxicity due to covalent binding of the reactive metabolite N-acetyl- p-benzoquinoneimine (NAPQI) to hepatocellular proteins. Dogs and cats are unique in that acetaminophen overdose primarily causes methemoglobinemia and hemolysis. It has been proposed that NAPQI is the responsible reactive intermediate in dogs and cats but it does not have the chemical or pharmacokinetic characteristics that would favor methemoglobin formation. Our hypothesis is that the deficiency of N-acetyltransferase (NAT) activity in dogs and cats allows accumulation of another acetaminophen metabolite, para-aminophenol (PAP) that then induces methemoglobinemia. We have demonstrated that dogs, cats, mice and rats can all deacetylate APAP to PAP in vitro with no significant difference between species. Dogs were unable to acetylate PAP to APAP in vitro while feline acetylation of PAP in vitro was significantly less than rats and mice (feline Vmax=0.088 ± 0.004 nmol/mg prot/min, apparent rat Vmax=0.63 ± 0.027 nmol/mg prot/min and apparent wildtype mouse Vmax=1.42 ± 0.1 nmol/mg prot/min; P<0.001).

Erythrocytes of healthy dogs, cats and rats were exposed in vitro to APAP, NAPQI and PAP. The only compound that caused significant methemoglobin formation at physiologically relevant concentrations was PAP. The 500 μM PAP-induction of methemoglobin at 60 minutes was greater in feline and canine erythrocytes than in rat and mouse erythrocytes (60.9 ± 2.0%, 67.3 ± 1.9%, 27.1 ± 2.5% and 28.5 ± 1.1% respectively, n=4 in each species) (P<0.01). Methemoglobin induction was significantly higher in erythrocytes from NAT1/NAT2 double knockout mice than wildtype C57BL/6 mice (35.8 ± 1.2% versus 28.5 ± 1.1%; P<0.05). There was significantly more methemoglobin in the lysed erythrocytes of C57BL/6 mice and rats, most likely due to the loss of methemoglobin reductase activity. In dogs and cats, there was significantly more methemoglobin in the intact erythrocytes, likely reflecting prolonged PAP and oxyhemoglobin redox cycling due to lower NAT activity in these species. The NAT1/NAT2 double knockout mice showed no significant difference in methemoglobin production between intact and lysed erythrocytes, possibly due to their high methemoglobin reductase activity but minimal acetylation. These results support our hypothesis that PAP contributes to APAP-induced methemoglobinemia and demonstrates a species difference in the sensitivity to methemoglobin induction. They also support that the decreased ability to re-acetylate PAP to APAP in dogs and cats is one contributing factor to the species sensitivity to APAP-induced methemoglobinemia.

The in vivo response of C57BL/6 wildtype and C57BL/6 NAT1/NAT2 double knockout mice to hepatotoxic doses of APAP was compared. Both groups had markedly elevated liver enzymes and evidence of centrilobular hepatic necrosis. There were no significant changes in the erythrocyte morphology. One group of knockout mice treated with 250 mg/kg APAP intra-peritoneally (ip) did have a significantly lower hematocrit at 48 hours than wildtype mice treated with the same dose of APAP. However, this was not a consistent change across multiple experiments. These results suggest that deficient N-acetylation alone is usually insufficient to create susceptibility to methemoglobinemia and hemolytic anemia and that it must be accompanied by either a relative deficiency in the ability to reverse methemoglobinemia or another defect.

There was significantly higher methemoglobin induction in knockout mice compared to wildtype mice treated with 400 mg/kg PAP (P=0.008). This was due to the significantly higher methemoglobinemia in female knockout mice compared to female wildtype mice (P=0.0047). There was no significant difference in methemoglobinemia in male knockout and wildtype mice. This again supports that a deficiency in N-acetylation would contribute to differing species sensitivity towards methemoglobin induction by the APAP metabolite PAP.

The H2 blocker cimetidine has been proposed as a treatment for feline and canine APAP toxicity. However, it has been shown that cimetidine inhibits N-acetylation in rats. We examined the in vitro effect of cimetidine on feline APAP N-acetylation. There was a significant inhibition of acetylation activity, suggesting the possibility that cimetidine could increase the risk of methemoglobinemia.

Our findings support that the hypothesis that PAP is the metabolite responsible for APAP hematotoxicity and that deficient N-acetylation in dogs and cats is a contributing factor to their high species sensitivity to PAP-induced methemoglobinemia. Furthermore, we have shown that cimetidine inhibits feline N-acetylation of PAP and therefore should not be used in the therapy of feline APAP toxicity.