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Metabolism and binding of cyclophosphamide and its metabolite acrolein to rat hepatic microsomal cytochrome P-450.
Cancer Res. 1984 Oct; 44(10):4615-21.CR

Abstract

The hepatic cytochrome P-450-mediated metabolism and metabolic activation of [chloroethyl-3H]cyclophosphamide [(chloroethyl-3H]CP) and [4-14C]cyclophosphamide [(4-14C]CP) were investigated in vitro in the reconstituted system containing cytochrome P-450 isolated from phenobarbital-treated rats. In addition, hepatic microsomal binding and the hepatic microsome-mediated metabolism of [14C]acrolein, a metabolite of [4-14C]CP, were also investigated. The metabolism of [chloroethyl-3H]CP and [4-14C]CP to polar metabolites was found to depend on the presence of NADPH and showed concentration dependence with respect to cytochrome P-450 and NADPH:cytochrome P-450 reductase. Km and Vmax values were essentially similar (Km, 0.44 and 0.42 mM; Vmax, 4.8 and 7.0 nmol of polar metabolites formed/min/nmol of cytochrome P-450 for [4-14C]CP and [chloroethyl-3H]CP, respectively). The patterns of inhibition by microsomal mixed-function oxidase inhibitors, anti-cytochrome P-450 antibody, and heat denaturation of the cytochrome P-450 were essentially similar, with subtle differences between [4-14C]CP and [chloroethyl-3H]CP metabolism. The order of inhibition by various mixed-function oxidase inhibitors was SKF greater than alpha- and beta-naphthoflavones greater than metyrapone. The in vitro metabolic activation of CP in the reconstituted system demonstrated predominant binding of [chloroethyl-3H]CP to nucleic acids and almost exclusive binding of [4-14C]CP to proteins. Gel electrophoresis-fluorography of the proteins in the reconstituted system treated with [4-14C]CP demonstrated localization of the 14C label in the cytochrome P-450 region. To examine this association further, hepatic microsomes were modified with [14C]acrolein in the presence and the absence of NADPH. The results confirmed covalent association between [14C]acrolein and cytochrome P-450 in the microsomes and also demonstrated further metabolism of [14C]acrolein, apparently to an epoxide, which is capable of binding covalently to proteins. The results of these investigations not only confirm the significance of primary metabolism but also emphasize the potential role of the secondary metabolism of cyclophosphamide in some of its toxic manifestations.

Authors

No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

6380709

Citation

Marinello, A J., et al. "Metabolism and Binding of Cyclophosphamide and Its Metabolite Acrolein to Rat Hepatic Microsomal Cytochrome P-450." Cancer Research, vol. 44, no. 10, 1984, pp. 4615-21.
Marinello AJ, Bansal SK, Paul B, et al. Metabolism and binding of cyclophosphamide and its metabolite acrolein to rat hepatic microsomal cytochrome P-450. Cancer Res. 1984;44(10):4615-21.
Marinello, A. J., Bansal, S. K., Paul, B., Koser, P. L., Love, J., Struck, R. F., & Gurtoo, H. L. (1984). Metabolism and binding of cyclophosphamide and its metabolite acrolein to rat hepatic microsomal cytochrome P-450. Cancer Research, 44(10), 4615-21.
Marinello AJ, et al. Metabolism and Binding of Cyclophosphamide and Its Metabolite Acrolein to Rat Hepatic Microsomal Cytochrome P-450. Cancer Res. 1984;44(10):4615-21. PubMed PMID: 6380709.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Metabolism and binding of cyclophosphamide and its metabolite acrolein to rat hepatic microsomal cytochrome P-450. AU - Marinello,A J, AU - Bansal,S K, AU - Paul,B, AU - Koser,P L, AU - Love,J, AU - Struck,R F, AU - Gurtoo,H L, PY - 1984/10/1/pubmed PY - 1984/10/1/medline PY - 1984/10/1/entrez SP - 4615 EP - 21 JF - Cancer research JO - Cancer Res VL - 44 IS - 10 N2 - The hepatic cytochrome P-450-mediated metabolism and metabolic activation of [chloroethyl-3H]cyclophosphamide [(chloroethyl-3H]CP) and [4-14C]cyclophosphamide [(4-14C]CP) were investigated in vitro in the reconstituted system containing cytochrome P-450 isolated from phenobarbital-treated rats. In addition, hepatic microsomal binding and the hepatic microsome-mediated metabolism of [14C]acrolein, a metabolite of [4-14C]CP, were also investigated. The metabolism of [chloroethyl-3H]CP and [4-14C]CP to polar metabolites was found to depend on the presence of NADPH and showed concentration dependence with respect to cytochrome P-450 and NADPH:cytochrome P-450 reductase. Km and Vmax values were essentially similar (Km, 0.44 and 0.42 mM; Vmax, 4.8 and 7.0 nmol of polar metabolites formed/min/nmol of cytochrome P-450 for [4-14C]CP and [chloroethyl-3H]CP, respectively). The patterns of inhibition by microsomal mixed-function oxidase inhibitors, anti-cytochrome P-450 antibody, and heat denaturation of the cytochrome P-450 were essentially similar, with subtle differences between [4-14C]CP and [chloroethyl-3H]CP metabolism. The order of inhibition by various mixed-function oxidase inhibitors was SKF greater than alpha- and beta-naphthoflavones greater than metyrapone. The in vitro metabolic activation of CP in the reconstituted system demonstrated predominant binding of [chloroethyl-3H]CP to nucleic acids and almost exclusive binding of [4-14C]CP to proteins. Gel electrophoresis-fluorography of the proteins in the reconstituted system treated with [4-14C]CP demonstrated localization of the 14C label in the cytochrome P-450 region. To examine this association further, hepatic microsomes were modified with [14C]acrolein in the presence and the absence of NADPH. The results confirmed covalent association between [14C]acrolein and cytochrome P-450 in the microsomes and also demonstrated further metabolism of [14C]acrolein, apparently to an epoxide, which is capable of binding covalently to proteins. The results of these investigations not only confirm the significance of primary metabolism but also emphasize the potential role of the secondary metabolism of cyclophosphamide in some of its toxic manifestations. SN - 0008-5472 UR - https://www.unboundmedicine.com/medline/citation/6380709/Metabolism_and_binding_of_cyclophosphamide_and_its_metabolite_acrolein_to_rat_hepatic_microsomal_cytochrome_P_450_ DB - PRIME DP - Unbound Medicine ER -