In this issue GSTsandClozapineToxicity Clozapine (CLZ) is an atypical antipsychotic drug that is used to treat schizophrenic patients who do not respond to conventional therapies. A disadvantage to CLZ is its capacity to induce idiosyncratic drug reactions, leading to agranulocytosis in 1-2% of patients and hepatotoxicity in 37% of patients, 0.06% of whom progress to liver failure. The major routes of CLZ metabolism are P450-mediated N-demethylation and N-oxidation. Minor pathways include hydroxylation of the chlorinated ring followed by glucuronidation or sulfation. However, P450or peroxidase-mediated oxidation can also generate reactive metabolites that are a likely cause of CLZ’s idiosyncratic toxicity. In vitro incubation of CLZ with rat or human liver microsomes in the presence of glutathione (GSH) leads to conjugation primarily at the 6 (CG-1) or 9 (CG-3) positions of the chlorinated ring, likely via the generation of a nitrenium ion intermediate. Two additional metabolites have been identified from in vivo studies in rats and mice. These are generated by GSH substitution of the chlorine atom (CG-6) and GSH addition to the nonchlorinated ring (CG-5). A dechlorinated metabolite in the urine of patients taking CLZ suggests that these metabolites may also be generated in humans. GSH conjugation of CLZ suggests that this is an important mechanism of re-
active intermediate detoxication. However, we do not know if these reactions are nonenzymatic in vivo or if glutathione S-transferases (GSTs) play a role. Dragovic et al. (p 1467) suggest that genetic GST polymorphisms could explain the susceptibility of some people to idiosyncratic CLZ toxicity. Here, they present data that support this hypothesis.
The bacterial P450 BM3 mutant (P450 102A M11H) produces all of the CLZ metabolites made by rat or human liver microsomes, but in higher concentrations. Dragovic et al. showed that incubation of 500 µM CLZ with P450 BM3 in the presence of 5 mM GSH resulted in the conversion of 30% of the compound to metabolites, 4 of which were GSH conjugates. GSH conjugate formation increased linearly with GSH concentration up to 100 µM and then leveled off, reaching saturation at 38 µM conjugates formed. Dragovic
Published online 09/20/2010 • DOI: 10.1021/tx100254p © 2010 American Chemical Society
et al. concluded that this level of nonenzymatic conjugate formation correlated with 100% capture of reactive intermediates generated by P450 BM3. They conducted the remainder of their experiments with 100 µM GSH, which resulted in the nonenzymatic capture of about 18% of the reactive intermediates. Incubation of CLZ in the presence of P450 BM3, GSH, and human GST P1-1 resulted in a 3.7-fold increase in total GSH conjugates and the appearance of 4 new metabolites. Mass spectrometric analysis identified the major new metabolites as CG-6 and CG-5. Under similar conditions, GST A1-1 produced only a 52% increase in GSH conjugate formation, resulting primarily from increased production of the major nonenzymatic conjugate CG-1 and some synthesis of CG-6. GST M1-1 similarly favored the formation of these two metabolites but increased total conjugate formation by 2.7fold. This enzyme also produced increased amounts of CG-4, which mass spectral studies identified as a conjugate at the C7 position or on the nonchlorinated ring. In contrast, GST T1-1 had no effect on GST conjugate levels. Dragovic et al. showed that the increase in GSH conjugate formation was linear with GST concentration for the active enzymes. They also extended their findings to include human and rat liver microsomes as the source of P450, showing a similar pattern of GSH Vol. 23,
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conjugate formation with each of the three active GSTs. The results indicate that GSTs catalyze the formation of GSH conjugates of CLZ that have only been found in vivo. Dragovic et al. propose that a nitrenium ion intermediate could serve as the precursor for all of these metabolites. The fact that CG-6 and CG-4 are present in the bile of CLZ-exposed rats and mice at levels comparable to those of CG-1 suggests that GSTs play a role in CLZ detoxication. The existence of polymorphisms in both GST P1-1 and GST M1-1 further supports the hypothesis that failure of GST-mediated detoxication in patients bearing poorly active versions of these enzymes could contribute to at least some CLZ-dependent idiosyncratic drug reactions. DTDiaphorasetotheRescue Parkinson’s disease is characterized by the selective loss of neuromelanin-containing dopaminergic neurons in specific regions of the brain. It has been proposed that dopamine oxidation to aminochrome plays a role in the degenerative process. Aminochrome is the precursor to neuromelanin but can also participate in two neurotoxic reactions. These include protein adduction leading to the formation of toxic protofibers of R-synuclein or inactivation of mitochondrial respiration and one-electron reduction to form an o-semiquinone radical, which undergoes reCHEMICAL RESEARCH IN TOXICOLOGY 1429
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In this issue dox cycling to generate reactive oxygen species.
DT-diaphorase (NAD(P)H: quinone oxidoreductase) is a flavoprotein that catalyzes the two-electron reduction of quinones to yield a hydroquinone. Prior work has indicated that DT-diaphorase protects neurons from aminochrome-mediated toxicity by preventing its ability to participate in neurotoxic reactions. However, these studies were carried out by using dicoumeral as a DT-diaphorase inhibitor, so it is impossible to know if the presence of the drug affected the outcome. To address this question, Lozano et al. (p 1492) tested the effect of RNAi knockdown of DTdiaphoraseonaminochromemediated neurotoxicity. Lozano et al. designed two siRNA duplexes (Nq6 and Nq7) targeting sites that are conserved in the human, mouse, and rat DTdiaphorase genes. Transfection of rat RCSN-3 catacholaminergic cells with retroviral particles containing these siRNAs yielded stable cell lines expressing each siRNA. The Nq6 and Nq7 siRNAs resulted in a 73% and 67% reduction of DTdiaphorase protein expression and 70% and 55% reduction in enzyme activity, respectively.
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Incubation of wild-type RCSN-3 cells with 100 µM aminochrome resulted in 21% cell death after 48 h. Cytotoxicity increased to 87% and 78% in cells expressing the Nq6 and Nq7 siRNAs, respectively. A marked change in cellular morphology from elongated to rounded accompanied aminochrome treatment in DT-diaphorasedepleted cells. These results confirm a role for DT-diaphorase in protecting cells from aminochrome-mediated cytotoxicity. Searching the Adductome When oxidative stress occurs, reactive oxidants are produced at a rate that exceeds a cell’s ability to remove them, and damage to cellular constituents results. One outcome of this process is lipid peroxidation, which produces highly electrophilic R,β-unsaturated aldehydes, including 4-oxo2(E)-nonenal (4-ONE), and 4-oxo-2(E)-hexenal (4-OHE). These aldehydes result from the peroxidation of ω-6 and ω-3 polyunsaturated fatty acids, respectively. 4-ONE and 4-OHE form exocyclic adducts with dC, dG, and dA bases in DNA, yielding heptanone-etheno adducts and butanone-etheno adducts, respectively. Although these adducts have been well-characterized structurally, their presence in human tissue DNA has notbeenfullyexplored.Now, Chou et al. (p 1442) address this question.
CHEMICAL RESEARCH IN TOXICOLOGY
Chou et al. used DNA adductomics in an attempt to comprehensively identify all adducts in the DNA isolated from 4 human lung autopsy specimens. The approach relies on mass spectrometric detection of the neutral loss of deoxyribose from positively charged DNA bases. The results revealed substantial quantities of 4ONE- and 4-OHE-derived adducts in the DNA from one lung sample, and the clear presence of these adducts, though in lesser amounts, in the other three samples. Particularly notable were the dC adducts of 4-ONE- and 4-OHE, and HεdCandBεdC,respectively. These results led Chou et al. to carry out more extensive quantification of 14 different DNA adducts in 68 autopsy specimens from multiple tissues of 26 different individuals. The results showed that adduct levels varied widely between individuals and between tissues from a given individual. The single most prevalent (found in 100% of specimens) and abundant ad-
duct (median level of 93/ 108 bases) was the oxidation product 8-oxo-dG. However, the 4-ONE-derived adducts of dC, dG, and dA were all detected, with a prevalence of 69-97% and median levels from 8.6 to 15/108 bases. The 4-OHEderived bases were also found but at levels approximately one-seventh those of the 4-ONE levels. BεdC was the most prevalent of these adducts (41% of samples), but its median level was below the limit of detection of the assay. For comparison, etheno-deoxyadenosine (εdA) was present in 93% of samples with a median level of (4.8/108 bases).
Levels of HεdC correlated with those of BεdC and εdA but not with those of 8-oxodG, consistent with the hypothesis that the etheno adducts are all derived from lipid peroxidation while 8oxo-dG is not. Together, the results confirm the presence of 4-ONE- and 4-OHE-derived adducts in human DNA and suggest that these adducts may be a contributor to mutagenesis-based disease in humans. TX100254P
Published online 09/20/2010 • DOI: 10.1021/tx100254p © 2010 American Chemical Society
