Limits...
Bioactivation of nevirapine to a reactive quinone methide: implications for liver injury.

Sharma AM, Li Y, Novalen M, Hayes MA, Uetrecht J - Chem. Res. Toxicol. (2012)

Bottom Line: An analogue of NVP in which the methyl hydrogens were replaced by deuterium also produced less covalent binding than NVP.These data provide strong evidence that covalent binding of NVP in the liver is due to a quinone methide formed by oxidation of the methyl group.Liver histology in these animals showed focal areas of complete necrosis, while most of the liver appeared normal.

View Article: PubMed Central - PubMed

Affiliation: Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 3M2.

ABSTRACT
Nevirapine (NVP) treatment is associated with a significant incidence of liver injury. We developed an anti-NVP antiserum to determine the presence and pattern of covalent binding of NVP to mouse, rat, and human hepatic tissues. Covalent binding to hepatic microsomes from male C57BL/6 mice and male Brown Norway rats was detected on Western blots; the major protein had a mass of ~55 kDa. Incubation of NVP with rat CYP3A1 and 2C11 or human CYP3A4 also led to covalent binding. Treatment of female Brown Norway rats or C57BL/6 mice with NVP led to extensive covalent binding to a wide range of proteins. Co-treatment with 1-aminobenzotriazole dramatically changed the pattern of binding. The covalent binding of 12-hydroxy-NVP, the pathway that leads to a skin rash, was much less than that of NVP, both in vitro and in vivo. An analogue of NVP in which the methyl hydrogens were replaced by deuterium also produced less covalent binding than NVP. These data provide strong evidence that covalent binding of NVP in the liver is due to a quinone methide formed by oxidation of the methyl group. Attempts were made to develop an animal model of NVP-induced liver injury in mice. There was a small increase in ALT in some NVP-treated male C57BL/6 mice at 3 weeks that resolved despite continued treatment. Male Cbl-b(-/-) mice dosed with NVP had an increase in ALT of >200 U/L, which also resolved despite continued treatment. Liver histology in these animals showed focal areas of complete necrosis, while most of the liver appeared normal. This is a different pattern from the histology of NVP-induced liver injury in humans. This is the first study to report hepatic covalent binding of NVP and also liver injury in mice. It is likely that the quinone methide metabolite is responsible for NVP-induced liver injury.

Show MeSH

Related in: MedlinePlus

H&E stainingof livers from Cbl-b–/– mice treated withNVP for 2 weeks. (A) Untreated control liver with normal ALT; (B)the liver from a NVP-treated mouse with gross necrosis and an ALTof 271 U/L, and the (C) liver from another NVP-treated mouse withgross necrosis and ALT of 313 U/L. Areas of massive hepatocyte necrosissurrounded by viable hepatocytes are shown in B and C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3475366&req=5

fig10: H&E stainingof livers from Cbl-b–/– mice treated withNVP for 2 weeks. (A) Untreated control liver with normal ALT; (B)the liver from a NVP-treated mouse with gross necrosis and an ALTof 271 U/L, and the (C) liver from another NVP-treated mouse withgross necrosis and ALT of 313 U/L. Areas of massive hepatocyte necrosissurrounded by viable hepatocytes are shown in B and C.

Mentions: Liver histology of Cbl-b–/– mice sacrificed after 2 weeks of NVP treatmentat the time of maximal ALT elevation is shown in Figure 10. The presence of gross necrosis, which was visible on thesurface of the liver as white areas was observed in 4 of 7 treatedanimals. Three NVP-treated males with gross liver necrosis had ALTvalues ≥200 U/L. Two of 8 NVP-treated female mice with minorliver necrosis had ALT values of 286 and 80 U/L. Histology in femalemice did not demonstrate as much injury as in males (data not shown).Histology of the livers of affected males showed the presence of focalsubcapsular areas of massive liver necrosis (Figure 10B,C) sharply demarcated from the adjacent viable liver. Necroticareas were surrounded by and infiltrated by mononuclear cells, macrophages,and neutrophils. This pattern of liver necrosis suggests an ischemicinjury, but no evidence of thrombi or vasculitis was observed. Multifocalnecro-inflammatory hepatitis with neutrophil-rich inflammatory responsewas observed in the absence gross necrotic lesions in male Cbl-b–/– mouse livers. Lower doses of NVP were alsotested with Cbl-b–/– mice,but no injury was seen (data not shown). In contrast, hepatic histologyof C57BL/6 mice treated with NVP and sacrificed at 4 weeks displayedhepatocyte death on the edge of the lobe in one animal, as well assmall focal areas of necrosis (Figure 11B).Induction of smooth endoplasmic reticulum (Figure 11C), presumably including P450 induction, was present in thehistology of all mice strains tested but was most prominent for Cbl-b–/– male mice. This marked induction may haveled to greater reactive metabolite formation contributing to the greatertoxicity in this strain, and this appeared to be the case althoughthe difference is subtle (Figure 12).


Bioactivation of nevirapine to a reactive quinone methide: implications for liver injury.

Sharma AM, Li Y, Novalen M, Hayes MA, Uetrecht J - Chem. Res. Toxicol. (2012)

H&E stainingof livers from Cbl-b–/– mice treated withNVP for 2 weeks. (A) Untreated control liver with normal ALT; (B)the liver from a NVP-treated mouse with gross necrosis and an ALTof 271 U/L, and the (C) liver from another NVP-treated mouse withgross necrosis and ALT of 313 U/L. Areas of massive hepatocyte necrosissurrounded by viable hepatocytes are shown in B and C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475366&req=5

fig10: H&E stainingof livers from Cbl-b–/– mice treated withNVP for 2 weeks. (A) Untreated control liver with normal ALT; (B)the liver from a NVP-treated mouse with gross necrosis and an ALTof 271 U/L, and the (C) liver from another NVP-treated mouse withgross necrosis and ALT of 313 U/L. Areas of massive hepatocyte necrosissurrounded by viable hepatocytes are shown in B and C.
Mentions: Liver histology of Cbl-b–/– mice sacrificed after 2 weeks of NVP treatmentat the time of maximal ALT elevation is shown in Figure 10. The presence of gross necrosis, which was visible on thesurface of the liver as white areas was observed in 4 of 7 treatedanimals. Three NVP-treated males with gross liver necrosis had ALTvalues ≥200 U/L. Two of 8 NVP-treated female mice with minorliver necrosis had ALT values of 286 and 80 U/L. Histology in femalemice did not demonstrate as much injury as in males (data not shown).Histology of the livers of affected males showed the presence of focalsubcapsular areas of massive liver necrosis (Figure 10B,C) sharply demarcated from the adjacent viable liver. Necroticareas were surrounded by and infiltrated by mononuclear cells, macrophages,and neutrophils. This pattern of liver necrosis suggests an ischemicinjury, but no evidence of thrombi or vasculitis was observed. Multifocalnecro-inflammatory hepatitis with neutrophil-rich inflammatory responsewas observed in the absence gross necrotic lesions in male Cbl-b–/– mouse livers. Lower doses of NVP were alsotested with Cbl-b–/– mice,but no injury was seen (data not shown). In contrast, hepatic histologyof C57BL/6 mice treated with NVP and sacrificed at 4 weeks displayedhepatocyte death on the edge of the lobe in one animal, as well assmall focal areas of necrosis (Figure 11B).Induction of smooth endoplasmic reticulum (Figure 11C), presumably including P450 induction, was present in thehistology of all mice strains tested but was most prominent for Cbl-b–/– male mice. This marked induction may haveled to greater reactive metabolite formation contributing to the greatertoxicity in this strain, and this appeared to be the case althoughthe difference is subtle (Figure 12).

Bottom Line: An analogue of NVP in which the methyl hydrogens were replaced by deuterium also produced less covalent binding than NVP.These data provide strong evidence that covalent binding of NVP in the liver is due to a quinone methide formed by oxidation of the methyl group.Liver histology in these animals showed focal areas of complete necrosis, while most of the liver appeared normal.

View Article: PubMed Central - PubMed

Affiliation: Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 3M2.

ABSTRACT
Nevirapine (NVP) treatment is associated with a significant incidence of liver injury. We developed an anti-NVP antiserum to determine the presence and pattern of covalent binding of NVP to mouse, rat, and human hepatic tissues. Covalent binding to hepatic microsomes from male C57BL/6 mice and male Brown Norway rats was detected on Western blots; the major protein had a mass of ~55 kDa. Incubation of NVP with rat CYP3A1 and 2C11 or human CYP3A4 also led to covalent binding. Treatment of female Brown Norway rats or C57BL/6 mice with NVP led to extensive covalent binding to a wide range of proteins. Co-treatment with 1-aminobenzotriazole dramatically changed the pattern of binding. The covalent binding of 12-hydroxy-NVP, the pathway that leads to a skin rash, was much less than that of NVP, both in vitro and in vivo. An analogue of NVP in which the methyl hydrogens were replaced by deuterium also produced less covalent binding than NVP. These data provide strong evidence that covalent binding of NVP in the liver is due to a quinone methide formed by oxidation of the methyl group. Attempts were made to develop an animal model of NVP-induced liver injury in mice. There was a small increase in ALT in some NVP-treated male C57BL/6 mice at 3 weeks that resolved despite continued treatment. Male Cbl-b(-/-) mice dosed with NVP had an increase in ALT of >200 U/L, which also resolved despite continued treatment. Liver histology in these animals showed focal areas of complete necrosis, while most of the liver appeared normal. This is a different pattern from the histology of NVP-induced liver injury in humans. This is the first study to report hepatic covalent binding of NVP and also liver injury in mice. It is likely that the quinone methide metabolite is responsible for NVP-induced liver injury.

Show MeSH
Related in: MedlinePlus