http://www.unc.edu/courses/2009spring/envr/740/001 overhead 34Begin 04/07/09Another important class of environmental carcinogens undergoes activation by other metabolic pathways, in addition to cytochrome P450. These are the nitroaromatics, defined by substitution of a nitro- (NO2) group at a peripheral carbon of a polycyclic aromatic hydrocarbon and often referred to by the abbreviation nitroPAH. NitroPAH were originally discovered by bioassay-directed chemical fractionation of diesel soot and were responsible for the largest proportion of the mutagenicity in the Ames assays. Several of the nitroPAH (1,6- and 1,8-dinitropyrene), when tested in pure form, proved to be among the most potent bacterial mutagens ever observed. In addition, the mutagenicity in the Ames assay was expressed without the need for exogenous microsomal activation (meaning, an added source of MFO activation = cytochrome P450 metabolism). Remember, bacterial P450s are very restricted in their substrates, and therefore test chemicals requiring oxidative metabolism for activation - which represent the majority of compounds tested- have to be assayed in the presence of eukaryotic cytochromes P450 supplied exogenously, usually in the form of rat-liver or mouse-liver microsomes. Initially, this observation of apparent “direct” activation was misinterpreted to mean that the nitroPAH required no metabolic transformation for their biological activity to be expressed. However, it was ultimately determined that the nitroPAH did, in fact, have to be metabolized. Bacteria are particularly rich in a family of enzymes called nitroreductases, which reduce the nitroPAH to hydroxylamines, which we just identified as being the proximate carcinogenic metabolites of aromatic amines in the N-hydroxylation pathway. Mammalian cells do not contain nitroreductases and as a consequence do not efficiently reduce the nitro group. So in assays based on mammalian cells, the nitroPAH were not nearly as mutagenic and most were only weakly transforming. Nevertheless, nitroaromatics are of interest because some members of the class were demonstrated to be animal carcinogens, by routes other than nitroreduction and there is significant exposure to diesel soot both in community and industrial settings. In eukaryotic cells, the transformations of nitroPAH are complex, and there appear to be multiple activation routes and eukaryotic cells which do have some reductive capacity through other enzymes than nitroreductases. The activation pathway of the most environmentally prevalent nitroPAH, 1-nitropyrene, is summarized on the following overhead:1[OH35: metabolic transformations of 1-nitropyrene; left panel]Pathway A, outlined in red, represents the bacterial reduction pathway, which involves successive 2-electron reductions, first to the level of a nitroso compound, and then to the hydroxylamine level, where conjugation followed by elimination of an anionic leaving group results in generation of a nitrenium ion, the same electrophile involved in the activation scheme of aromatic amines. Pathway C indicates that the leaving group may be generated by acetylationof the hydroxyl group by O-acetyl transferases (acronym OAT). Pathway B, highlighted in blue, represents a cytochrome P450 mediated oxidative pathway, which involves epoxidation of an aromatic double bond. This pathway is proposed as a result of the in vitro activity of a synthetic epoxide metabolite and needs to be confirmed in vivo. Pathway D represents a mammalian pathway in which the nitroPAH is first reduced completely to the level of an aromatic amine. This may be accomplished by a non-heme system- read non-cytochrome P450 containing system- called xanthine oxidase. The resulting aromatic amine can then follow the same pathwaywe have just outlined for this class of carcinogen: N-hydroxylation by cytochrome P450 with or without N-acetylation (accomplished by N-acetyl transferase; acronym= NAT). It is important to point out that the nitro-reduction pathways in mammalian cells are not nearly as efficient as in bacteria: bacterial pathway A leads to conversion of virtually all of the nitro compound directly to a proximate carcinogenic species, while there are other outlets for the mammalian pathways.Two additional nitroPAH that may play a significant role in human exposures are 3-nitrofluoranthene and 2-nitrofluoranthene. The structures are shown in the right-hand panel of the overhead:[OH35: right panel, next to NO2-PAH activation scheme]3-Nitrofluoranthene is present in soot generated by internal combustion engines, while the 2-nitro isomer is generated photolytically in the atmosphere by the reaction of the parent fluoranthene with NOx. To date, the only adduct characterized in mammalian cells is the adduct at C8 of dGuo derived from the completely reduced metabolite 3-aminofluoranthene.2DIRECT ACTING CARCINOGENS/MUTAGENSSo far we have described compounds requiring metabolic transformation to exert genotoxic effects. There is a smaller but still important group of chemicals that are active without the necessity of initial biotransformation. These compounds are called direct acting. Direct acting compounds can exert their effects in two ways: modification of the nucleobases without formation of covalent adducts, and modification via formation of covalent adducts, in the same manner as the indirect acting agents that we have just discussed.Of the chemicals that cause DNA damage without formation of covalent adducts, the most widespread and important is nitrite ion (NO2-). Thus, nitrite is not only toxic as a reagent in nitrosamine formation when it is an additive to meat, but it is also mutagenic in its own right. In an acidic medium and somewhat more slowly at physiological pH, nitrite acts to deaminate the exocyclic amino groups of the nucleobases. The reaction name will be familiar to those of you who can call up some of your organic chemistry: the diazotization reaction. The reaction pathway is summarized on the next overhead. This isn’t to memorize, and you have it for reference, but in the interest of time, we won’t step through the pathway.[OH1: diazotization reaction]The reaction is written for the deamination of dCyd, which is the base most commonly transformed, and the brackets show the products resulting from deamination of dAdo and dGuo. [OH1: transformation of bases]dCyd is converted to dUrd, which you remember is an RNA base, that is dThyd without the