Graduate Thesis Or Dissertation
 

Metabolic pathway of the nitrosoureas catalyzed by cytochrome P-450 and NADPH cytochrome P-450 reductase

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  • Three highly purified forms of cytochrome P-450 (P-450a, P-450b, P-450c) from Aroclor 1254-induced rat hepatic microsomes catalyzed the monooxygenation of 1-(2- chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) and 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) in the presence of purified NADPH cytochrome P-450 reductase, NADPH and oxygen. Cytochrome P -450- dependent monooxygenation of CCNU gave six alicyclic hydroxylation products with cis-4-hydroxy CCNU the principal metabolite of cytochrome P-450a and P-450; trans-3- hydroxy CCNU was the principal metabolite with cytochrome P-450c. MeCCNU monooxygenation catalyzed by microsomal and purified isozymes gave four alicyclic hydroxylation products, an "-hydroxylation on the 2-chloroethyl moiety and a trans-4-hydroxymethyl derivative with Cis-4-hydroxytrans- 4-methyl CCNU the major hydroxylation product. The nitrosoureas undergo denitrosation in the presence of NADPH and deoxygenated hepatic microsomes or purified NADPH cytochrome P-450 reductase to yield NO. A microsomal cytochrome P-450 ferrous·NO ligand which rapidly formed was used to quantitate nitrosourea denitrosation after establishing the ferrous·NO extinction coefficient as 36 mM⁻¹cm⁻¹ at 444-500 nm by optical difference spectroscopy. Further analysis with highly purified PB cytochrome P-450 and NADPH cytochrome P-450 reductase, reconstituted with dilauroylphosphatidylcholine, suggested that NADPH cytochrome P-450 reductase was solely responsible for denitrosation. Although PB cytochrome P-450 was not directly involved in denitrosation it stimulated NADPH cytochrome P-450 reductase catalytic activity nearly 3-fold at a concentration equal to that of the reductase. Furthermore, NADPH cytochrome P-450 reductase dependent CCNU denitrosation was also stimulated by FMN.
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