Abstract
Benoxaprofen (BNX), a nonsteroidal anti-inflammatory drug (NSAID) that was withdrawn because of hepatotoxicity, is more toxic than its structural analog flunoxaprofen (FLX) in humans and rats. Acyl glucuronides have been hypothesized to be reactive metabolites and may be associated with toxicity. Both time- and concentration-dependent glucuronidation and covalent binding of BNX, FLX, and ibuprofen (IBP) were determined by exposing sandwich-cultured rat hepatocytes to each NSAID. The levels of glucuronide and covalent protein adduct measured in cells followed the order BNX > FLX > IBP. These results indicate that 1) BNX-glucuronide (G) is more reactive than FLX-G, and 2) IBP-G is the least reactive metabolite, which support previous in vivo studies in rats. The proportional increases of protein adduct formation for BNX, FLX, and IBP as acyl glucuronidation increased also support the hypothesis that part of the covalent binding of all three NSAIDs to hepatic proteins is acyl glucuronide-dependent. Moreover, theses studies confirmed the feasibility of using sandwich-cultured rat hepatocytes for studying glucuronidation and covalent binding to hepatocellular proteins. These studies also showed that these in vitro methods can be applied using human tissues for the study of acyl glucuronide reactivity. More BNX-protein adduct was formed in sandwich-cultured human hepatocytes than FLX-protein adduct, which not only agreed with its relative toxicity in humans but also was consistent with the in vitro findings using rat hepatocyte cultures. These data support the use of sandwich-cultured human hepatocytes as an in vitro screening model of acyl glucuronide exposure and reactivity.
Footnotes
This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant GM61188].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.028944
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- UGT
- UDP glucuronosyltransferase
- BNX
- benoxaprofen
- FLX
- flunoxaprofen
- IBP
- ibuprofen
- HPLC
- high-performance liquid chromatography
- PBS
- phosphate-buffered saline
- SKF-525A
- proadifen hydrochloride
- LDH
- lactate dehydrogenase
- ABT
- 1-aminobenzotriazole
- DMEM
- Dulbecco's modified Eagle's medium
- MCM
- modified Chee's medium
- PMSF
- phenylmethylsulfonyl fluoride
- PH
- 1,7-phenanthroline
- BNX-G
- benoxaprofen glucuronide
- FLX-G
- flunoxaprofen glucuronide
- IBP-G
- ibuprofen glucuronide
- P450
- cytochrome P450
- NSAID
- nonsteroidal anti-inflammatory drug.
- Received June 15, 2009.
- Accepted September 17, 2009.
- Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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