http://www.unc.edu/courses/2009spring/envr/740/001 overhead 8Begin 03/26/09There are a number CYP transformations that are toxicologically important and are obligatory to present. They were part of ENVR430, but the whole class has not seen them, and, anyway, I can hopefully add some new insights. The PAH are perhaps archetypical examples of P450 activation, and the scheme for benzo[a]pyrene is the best studied and representative. The metabolic scheme is summarized on the next overhead:[OH; B[a]P metabolism]The overhead shows the structure, numbering convention and major metabolic transformations characterized for BP. The transformations to the left were traditionally ascribed to “carbon hydroxylation”. However, they arise through initial epoxidation of an arene double bond followed by non-enzymatic rearrangement to phenols, illustrated on the next overhead. This is true even in the case of the 6-position, by analogy to the demonstrated epoxidations of other PAH[fluoranthene at a ring junction]. [OH; Schematic epoxidation → phenol rearrangement] In the case of benzene metabolism, an initial epoxidation step has been confirmed by direct observation of benzene oxide.OHHOH1Of the metabolic transformations on the scheme, those leading to phenols and dihydrodiols can be considered as detoxifying - the hydroxy group is conjugated by phase II enzymes and the metabolites are excreted. The membrane-bound cytochrome P450s in eukaryotes are topologically juxtaposed to an enzyme called epoxide hydrolase (EH) that can detoxify epoxides by catalysis of the addition of water to form a product called a dihydrodiol. The formal reaction is illustrated on the next overhead:[OH; trans hydration of cyclic epoxides by EH]Because the cyclic structure of the aromatic rings fixes the geometry of the hydroxy groups with respect to the plane of the ring, two isomers- cis and trans - are possible. A hallmark of the epoxide hydrolase-catalyzed hydrations is trans geometry of the resulting dihydrodiol. Some proportion of the 1-, 3-OH BPs, if they are not conjugated rapidly, are oxidized either enzymatically or non-enzymatically to diketones called quinones. The 6-phenol is very unstable and leads to the formation of all three quinones through rapid non-enzymatic oxidation by dissolved dioxygen. There had been some controversy over the biological activity of the quinones. Their formation was initially considered to be a detoxification pathway; however, theyare mutagenic in bacterial tester strains that are sensitive to oxidative damage (TA104 and TA102). Consistent with this result, evidence is accumulating that quinones have the potential for mutagenic activity through oxidative damage arising from redox cycling. Of the quinones, the 7,8-quinone has been directly investigated and found to undergo redox cycling. We will revisit this aspect of mutagenic activity shortly under the heading of “oxidative damage”. The epoxidations leading to dihydrodiols are also detoxifying pathways except for one- the 7,8-dhdo from the 7,8-oxide. Besides oxidation to the 7,8-quinone, this metabolite is cycled through the MFO system a second time to yield the benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide, often referred to by the acronym BPDE. BPDE is a poor substrate for EH, so in vivo it escapes the OHHOHOHHHOHHOH2smooth endoplasmic reticulum and survives for a sufficient time to form adducts with cellular nucleophiles like proteins and DNA and is in fact responsible for most of the adduct formation from B[a]P metabolism. As we have implied earlier in the course, both geometric and optical isomerism are important in determining the level of activity of the diolepoxide. To review quickly: geometric isomers of the BPDE are identified based on the orientation of the epoxide oxygen relative to the 7-OH as reference: as the next overhead shows, epoxidation on the same face as the 7-OH leads to the syn geometrical isomer and on the opposite face, leads to the anti isomer.[OH; BPDE stereoisomers]There are two enantiomeric forms of the trans-7,8-dhdo with the result that the epoxidation/hydration sequence leads to a total of four syn and anti diolepoxides. The metabolites have been designated ± based on the direction in which they rotate plane polarized light; however, the more rigorous and structurally informative R,S notation is now often used, and since you will encounter this notation in any discussion of B[a]P activity, I will briefly describe the R, S notation.[OH; R, S convention]Asymmetric carbons are assigned an R or S configuration based on a set of priorities assigned to the substituents of the asymmetric carbon. As the next overhead shows, the tetrahedral carbon is oriented so that the lowest priority substituent points away from the viewer. Then, priorities of the 3 substituents on the face towards the viewer are determined, and the configuration is definedas R if the priorities from highest to lowest are in a clockwise rotation, and S, if the rotation is counterclockwise. Priorities are assigned first on the basis of atomic weight: H<C<N<O<S<Cl, etc. When identical atoms are bonded to the asymmetric carbon, then the substituents once removed are considered, and so on. [OH; asymmetric carbon with priorities assigned]3In mammalian test systems, only one of the four BPDE isomers is highly active, and that one is the (+)-anti, or 7R, 8S, 9S, 10R isomer:.Repeat [OH; 4 isomeric benzo[a]pyrene diolepoxides]Intuitively, the result that one specific stereoisomer is the most reactive metabolite is not surprising, since DNA is chiral and so the BPDE adduct is formed by reaction of a chiral target with a chiral metabolite. However, in order to understand adduct formation with a view to learning something of predictive value, as we have seen from our discussion of lesion processing, it is necessary as a first step to understand the consequences of adduct formation withregard to changes in structure of DNA and how the adducts are processed by cellular machinery. As we have already seen from our discussion of polymerases and repair strategies, this is one area of rapid progress as a result of technological developments in spectroscopic methods. A great deal of headway has also been made regarding characterization of adduct conformations. Using anti-BPDE as an example, the next overhead shows the 2D representation of the reaction of BPDE at the exocyclic amino groups of dGuo and dAdo, which are the major BPDE