We have learned much about the metabolic activation of unsymmetric dialkylnitrosamines such as N-nitrosomethylamylamine (NMAA), which induce esophageal cancer in rats and could induce this cancer in humans. The following new studies are proposed. 1. Microsomal metabolism of NMAA: We will improve our method of isolating rat esophageal microsomes, study NMAA and N-nitrosodimethylamine metabolism by microsomes from various rat and human tissues to establish the tissue specificity, survey the microsomal metabolism in 20 human esophagi, study formaldehyde (CH20) formation from [3Hmethyl]NMAA, and test non-P450 cofactors and effects of monoclonal antibodies that inhibit specific P450s in the rat esophageal metabolism of NMAA. We will search for chemicals that inhibit the esophageal metabolism, including isothiocyanates, diallyl sulfide and ellagic acid. 2. DNA alkylation by NMAA: DNA from esophagus and liver of NMAA-treated rats contained O6- and 7-MeG, but not O6- and 7-pentylguanine. After confirming the absence of pentylguanines, 7- and O6-pentylguanine in esophageal and liver DNA will be followed after injecting pentylnitrosourea. To elucidate the fate of active NMAA intermediates, [14C-pentyl]NMAA will be incubated with microsomes and the label will be followed in tissue fractions. 1-Pentene formation from NMAA will also be studied. Alkylation of rat esophageal DNA by NMAA will be examined after treatment with chemical inhibitors. 3. Metabolism of other nitrosamines: We will study the metabolism of 5 methylalkyl and 3 cyclic nitrosamines that induce esophageal cancer in rats or people are exposed to. Effects of inhibitory antibodies and of chemical inhibitors will be studied on NMAA metabolism by esophageal and liver microsomes and tissue slices. 4. NMAA metabolism by vaccinia viruses with P450s: We will examine NMAA metabolism by 5 rat and 12 human liver P450 isozymes incorporated into vaccinia virus, to ensure that rat and human esophageal P450 (P450-es) are not known P450s. 5. Identification of the esophageal P450:. We will attempt to identify the rat P450-es that catalyzes NMAA metabolism and is believed critical for cancer induction. We will isolate fragments of P450. CDNA using polymerase chain reaction technology applied to rat esophagus mRNA, with primers corresponding (initially) to portions of P450 2El cDNA. To isolate full-length clones, the cDNA fragments will be used as primers on esophageal mRNA or on a rat esophagus cDNA library. The clones will be expressed in vaccinia virus or COS-1 cells and their metabolism of nitrosamines used to identity P450-es. We will study how levels of P450-es, and its mRNA vary under conditions that affect NMAA metabolism.
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