http://www.unc.edu/courses/2009spring/envr/740/001 overhead 37Begin 04/09/09The direct acting carcinogens that form covalent adducts are alkylating agents- that is, they react with DNA in a biological medium to add an alkyl group at one of the nucleophilic sites of the nucleobase. The following overhead shows the structures of a number of common alkylating agents:[OH: direct acting carcinogens]N-Methyl-N-nitrosourea (MNU) is a synthetic methylating agent, which finds use both in synthetic organic reactions and in model carcinogenesis systems. The overhead shows the reaction pathway in aqueous solution, which yields a methyl carbonium ion. Attack of hydroxideor water gives carbamic acid and monomethylnitrosamine, which is the same proximate carcinogen you saw in the activation scheme for DMN via C-hydroxylation by cytochrome P450.As in the DMN scheme, the monomethylnitrosamine yields a methyl carbonium ion non-enzymatically. The second compound on the list, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), is also a methylating agent. The pathway leading to methyl carbonium ion is the sameas for NMU, except that in this case, nitrourea is eliminated by the hydrolysis reaction, rather than carbamic acid.Both MNU and MNNG are powerful methylating agents and in addition to use in synthetic chemistry, they are used as positive controls in bioassays and as model alkylating agents in studies of tumorigenesis.Methylmethansulfonate, referred to by the acronym MMS, is a methyl donor in much the same manner as the corresponding methanesulfonic acid is a proton donor, although the direct release of H3C+ does not occur, but rather by displacement of the sulfonate from the methyl group by the target nucleophile. Dimethyl sulfate, for which the acronym is DMS, is the dimethyl ester of strong mineral acid sulfuric acid. DMS serves as a methyl donor in exactly the same manner as MMS. These compounds also find dual use as a synthetic intermediates in industry and as 1positive controls in bioassay.Ethylene oxide and propylene oxide, which are used as disinfectants, act as we have described for the metabolically generated epoxides of the PAH and aflatoxin B1, by nucleophilic opening ofthe epoxide ring, which is highly strained and also contains positively polarized carbons.Strained lactones, such as β-propiolactone, are alkylating agents through nucleophilic attack on the strained 4-membered oxygen-containing ring, in the same manner as for epoxides. Lactones are starting components in many commercial polymers and exposures occur in industry. Also, a number of antineoplastic drugs are also carcinogenic. An example is cyclophosphamide, which, as the structure suggests, forms cross-links by nucleophilic displacement of chlorines.Finally, a word about adduct formation. There are some rules of thumb regarding preferential sites of adduction on the nucleobases, although it is important to keep in mind that for each individual electrophile, preferred reaction sites must ultimately be confirmed by experiment. Alkylating agents that react by displacement or substitution, which is called an SN2 mechanism; and does not involve actually generating a discrete alkyl carbonium ion (the chemical term is hard cation), tend to add at N7 of purines. For example, MMS, DMS, which we have just described, and AFB1 oxide are examples of such alkylating agents. However, an important exception which we already have discussed is the selectivity of PAH oxides and diolepoxides, which add at the exocyclic amino groups of purines – N2 of dGuo or N6 of dAdo. Alkylating agents generating hard alkylcarbonium ions, such as MNNG, MNU preferentially alkylate O6 of dGuo, or exocyclic amino groups, and because the methyl carbonium ion is so reactive that N1 and N3 of dGuo and dAdo are also methylated to a minor extent. Aromatic nitrenium ions add atC8 of dGuo, dAdo and less commonly at N2 of dGuo via the tautomer that places (+) on the aromatic ring as we saw in the case of 2-AF.OXIDATIVE STRESSA major source of DNA damage arises from the presence of reactive oxy radicals collectively referred to as reactive oxygen species, with the acronym, ROS. The generation and subsequent 2reactions of ROS collectively comprise “oxidative stress”. The sources of ROS are several. We have already described futile cycling of cytochrome P450 through uncoupling of the oxyferrous intermediate in the P450 cycle, as shown on the next overhead, with the uncoupling reaction in the red frame.[OH: catalytic cycle of cyt. P450, futile cycling] The product of uncoupling is the reactive oxygen species, superoxide anion, written O2 ∙/-, as shown on the overhead, or often written simply as O2-, which is technically not correct. Redox cycling is another important source of superoxide anion. Redox cycling is a process in which a compound continually cycles between oxidized and reduced forms. In oxidative stress, reduced forms become re-oxidized by transferring an electron to dioxygen to generate the superoxide anion. The archetypical redox cyclers are ortho-quinones, an example of which is the benzo[a]pyrene-7,8-quinone that we described as part of the metabolic pathway of BP. The redoxcycling of a quinone is shown on the next slide. The catechol, which is at the fully reduced end of the cycle, can be generated from dihydrodiols by the action of the enzyme dihydrodiol dehydrogenase.[OH: redox cycling of a quinone]The catechol reduces dioxygen to superoxide, and is oxidized to a semiquinone, which can eitherbe reduced by NADH back to the catechol or can reduce a second dioxygen molecule to superoxide and be fully oxidized to an ortho quinone. The quinone can in turn be reduced by NADH to a semi-quinone, which can be further reduced to the catechol or can be re-oxidized with the production of another superoxide anion. The full cycle generates 2 superoxide anions. As we will discuss in another minute, the superoxide anion it not necessarily an end point, but the start of a cascade of ROS.Polychlorinated biphenyls or PCBs are also hypothesized to be genotoxic via redox cycling. The next overhead summarizes the proposed pathway: epoxidation by cytochromes P450 followed by3hydration to dihydrodiols and dehydrogenation to catechols is the most likely activation pathway.[OH; metabolic activation of PCBs]The herbicide paraquat is an example of a non-quinonoid chemical that undergoes redox cycling as shown on the next overhead. The bis-methylpyridinium