2-Nitropropane (2-NP), a secondary nitroalkane, has been extensively used in industry as a solvent for extractions and as a component of paints, inks, and varnishes. The World Health Organization estimates that approximately 4000 U.S. workers may have had significant occupational exposure to this chemical. 2-NP is a bacterial mutagen, produces unscheduled DNA synthesis in rat hepatocytes, and is a strong hepatocarcinogen in male Sprague-Dawley rats. The overall goal of this project is to elucidate the mechanism of the genotoxicity and carcinogenicity of 2-NP, thereby obtaining information for predicting whether or not the compound could also cause neoplasms in man. Previous work by this laboratory has shown that 2-NP produces specific and unique base modifications in rat liver DNA and RNA, namely the appearance of 8- aminoguanine, increases in 8-oxoguanine, and the appearance of thus far unidentified """"""""X1"""""""" adducts in RNA and DNA. The first Specific Aim of this proposal is to structurally identify these X1 adducts. Since the formation of 8-aminoguanine and X1 in DNA may represent tumor initiating events, the second Specific Aim is to determine the mutagenic potency of these lesions in bacterial and mammalian cells using a site-specifically modified single stranded shuttle vector. Metabolic conversion of 2-NP to a species capable of aminating guanine residues at C8 is mediated through aryl sulfotransferase (AST), probably isozyme IV.
Specific Aim 3 is to test the proposed central role of AST in 2-NP-induced hepatocarcinogenesis by a bioassay of 2-NP in control and pentachlorophenol-pretreated 12-day old B6C3F1 mice and in 12-day old normal and brachymorphic mice. For the AST-catalyzed conversion of 2-NP to a species capable of aminating guanine at C8 a mechanism has been proposed involving acetoxime-O-sulfonic acid and hydroxylamine-O-sulfonic acid as intermediates.
In Specific Aim 4, the mutagenicity of these intermediates will be determined and 35S-labeled PAPS will be used in attempts to detect these intermediates in the in vitro AST reactions using 2-NP nitronate as substrate. Since the AST-catalysed activation of 2-NP nitronate is a completely novel biochemical reaction, its kinetic parameters will be determined using purified rat liver ASTIV, (Specific Aim 5). The genotoxicity of secondary nitroalkanes, including 2-NP, depends on the dissociation of the a-hydrogen to yield stable nitronate anions.
In Specific Aim 6, primary nitroalkanes with acidic a-hydrogens will be synthesized and tested for mutagenicity and ability to serve as substrates in the AST-catalysed activation. The 7th Specific Aim is to determine the extents of metabolic activation and detoxication of 2-NP in various rodent species of both sexes and correlate these with 2-NP- induced rat liver nucleic acid modifications and susceptibility to 2-NP hepatocarcinogenesis. Since acute exposure to 2-NP can result in severe hepatotoxicity, it is proposed to establish whether this toxicity is also mediated through the activation of 2-NP by AST, and whether its severity can be reduced by inhibitors of AST (Specific Aim 8). Having obtained information on the extents of the activation and detoxication pathways of 2-NP in rodent species of varying susceptibility to its carcinogenicity, the last Specific Aim (9) is to determine the ability of normal human liver to activate and detoxify 2-NP in order to assess its potential human carcinogenicity.
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