This research will address the mechanisms of toxicity of the alkylating anticancer drugs cyclophosphamide, procarbazine, dacarbazine, and the nitrosoureas (carmustine, lomustine and semustine). As an indicator of the abilities of these drugs alkylate enzymes, we will assess the capabilities of these agents to destroy hepatic cytochrome P-450 enzymes, which are intimately involved as the enzymatic ativators of the drugs in question. We will elucidate the in vitro inactivating capabilities of these drugs with the final objective to extend this work to in vivo applications of the treatment of melanomas. Additionally, we will determine the capabilities of cysteine and other sulfur containing molecules to protect cytochrome P-450 from inactivation, and we will correlate this protection with the in vivo protection against the toxicities of these drugs in tumor-bearing (B16 melanoma) mice while maintaining the anticancer properties of the alylating agents. We will use rabbit microsomes and 10,000 x g supernatants to initially evaluate the extent of cytochrome P-450 inactivation by each drug by measuring P-450 content and enzymatic activity. We will then study the mechansims by which each drug destroys cytochrome P-450. The abilities of the sulfur containing molecules cysteine, cysteamine, and N-acetylcysteine to decrease cytochrome p-450 destruction will be determined. We will characterize the nature of the alkylating species of the activated drugs by trapping the electrophilic intermediates with thiols or other nucleophiles, followed by isolation and identification of the products. These studies will provide valuable information concerning the nature of the reactive moities and the therapeutic use of protective agents. The destruction of Cytochrome P-450 by these drugs is not only important in relationship to the toxicities of the drugs, but also in regard to the metabolic activation and/or deactivation of coadministered drugs. Our goals in this project are to learn more about the molecular toxicities of these alkylating agents and to apply this knowledge to the more effective use of the drugs.
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