Abstract
A cDNA isolated from a C57BL/6 mouse liver cDNA library had the identical nucleotide sequence in coding region with the mouse CYP3A11, and the NH2-terminal sequence was also identical to that of cytochrome P450 (P450) MDX-B, a microsomal alcohol oxygenase. The COS-7 cells transfected with the CYP3A11 expression vector formed 7-oxo-Δ8-tetrahydrocannabinol (7-oxo-Δ8-THC) from 7α- and 7β-hydroxy-Δ8-THC. An immunologically related protein with P450 MDX-B was expressed in the COS-7 cell microsomes. The cell microsomes expressed CYP3A11; COS-3A11 catalyzed the oxidation of 7-hydroxy-Δ8-THC and 8-hydroxy-Δ9-THC to 7-oxo-Δ8-THC and 8-oxo-Δ9-THC, respectively, in a reconstituted system. 18O derived from atmospheric oxygen was incorporated into about 30% of the corresponding ketones formed from 7α-hydroxy-Δ8-THC and 8β-hydroxy-Δ9-THC by mouse hepatic microsomes, P450 MDX-B, and COS-3A11, although incorporation of the stable isotope into the oxidized metabolites from 7β-hydroxy-Δ8-THC and 8α-hydroxy-Δ9-THC was negligible. 18O, however, was not incorporated into 7-oxo-Δ8-THC formed from 7α-hydroxy-Δ8-THC by using cumene hydroperoxide instead of NADPH under 18O2. When18O-labeled 7α-hydroxy-Δ8-THC and 8β-hydroxy-Δ9-THC were incubated with above enzymes under air, about 30% of the ketones formed released 18O from a hydroxy group at the 7 and 8 positions in the course of the oxidation. These results suggest that 7α-hydroxy-Δ8-THC and 8β-hydroxy-Δ9-THC may be oxidized to the corresponding ketones by CYP3A11 via a gem-diol pathway. 7β-Hydroxy-Δ8-THC and 8α-hydroxy-Δ9-THC may be also converted to the ketones through a stereoselective dehydration of an enzyme-bound gem-diol rather than through a direct hydrogen extraction as a peroxy form of the enzyme.
Footnotes
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This work was partially supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and by the Special Research Fund of Hokuriku University.
- Abbreviations used are::
- THC
- tetrahydrocannabinol
- MALCO
- microsomal alcohol oxygenase
- P450
- cytochrome P450
- GC/MS
- gas chromatography/mass spectrometry
- PCR
- polymerase chain reaction
- CuOOH
- cumene hydroperoxide
- Received April 30, 2001.
- Accepted August 8, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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