In this issue 3-MI: A Major Lung Carcinogen The association of smoking with lung cancer is no longer in doubt; however, with at least 81 carcinogenic compounds in cigarette smoke, identification of the most important players remains an important question. It is clear that 4-(methylnitrosamino)-1-(3pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P) are major contributors to smoking-related carcinogenicity. In contrast, 3-methylindole (3-MI), which is present in considerably higher amounts in cigarette smoke, has received little attention. 3-MI is subject to cytochrome P450-dependent bioactivation to 3-methyleneindolenine (3-MEIN), an electrophilic compound that forms adducts with dA, dC, and dG bases of DNA in vitro and in vivo. Yet, 3-MI is not mutagenic in standard bioassays using human or rat liver microsomes as sources of P450 enzymes. Now Weems et al. (p 1682) report that the P450s primarily responsible for 3-MI activation are not present in the liver, but are richly expressed in the lung, indicating that a correct evaluation of the potential carcino genicity of this compound requires the use of the relevant source of microsomes.
Mutagenicity testing of NNK, B[a]P, and 3-MI using the TA98 strain of S. typhimurium in the presence of human lung microsomes (S9) yielded positive results for all three compounds, suggesting that all three are
capable of inducing frameshift mutations under these conditions. At concentrations of 1 and 10 µM, 3-MI was more mutagenic than either NNK or B[a]P, while at higher concentrations, the cytotoxicity of 3-MI led to lower levels of mutations than were seen with the other two compounds. 3-MI was not mutagenic when rat or human S9 was used as the source of P450 enzymes. None of the compounds was mutagenic in the absence of NADPH, confirming the requirement for P450-dependent bioactivation. Further confirmation of the role for P450 enzymes came from the near total inhibition of mutagenicity of all three compounds in the presence of the nonspecific P450 suicide substrate 1-aminobenzotriazole. Studies of individual P450 enzymes present in lung tissue showed that CYP1A1, CYP2A13, and CYP2F3 all induced the mutagenicity of 3-MI using the TA98 bacterial strain. Of these enzymes, B[a]P was activated only by CYP1A1, and NNK was activated only by CYP2A13. In the presence of lung S9, the selective CYP2A13 inhibitor 8-methyoxypsoralen blocked the CYP2A13-dependent mutagenicity of 3-MI by about 30%, and the mutagenicity of NNK by 90%. This finding suggests that of human lung P450s, CYP2A13 is almost totally responsible for NNK activation, but only partially responsible for 3-MI activation. Quantitative immunoblotting demonstrated that CYP1A1 is present in human lung microsomal fractions at a concentration approximately 10-fold higher than that of CYP2A13, a finding
Published online 11/15/2010 • DOI: 10.1021/tx100329j © 2010 American Chemical Society
that may explain the relatively minor role of CYP2A13 in 3-MI bioactivation. Together, the results confirm that 3-MI can be activated to a mutagenic reactive intermediate, likely 3-MEIN, in the presence of human lung microsomes. Considering its high concentration in cigarette smoke, these results show that 3-MI must be considered as a potential major toxicant in lung carcinogenesis in smokers. Unusual Nevirapine Adduct Nevirapine (NVP) is a nonnucleoside reverse transcriptase inhibitor that has become one of the mainstays for the treatment of HIV infection. NVP is a primary drug used to prevent the passage of HIV from a pregnant woman to her fetus. Although rare, NVP may cause idiosyncratic reactions including a characteristic skin rash and a potentially fatal hepatotoxicity. The fact that NVP covalently binds to liver proteins in vitro and in vivo suggests that formation of a reactive drug metabolite may be the cause of its idiosyncratic toxicity.
NVP is normally metabolized by multiple P450 enzymes leading to hydroxylation at positions 2, 3, 8, and 12. The products are subject to glucuronidation and excretion; however, sulfonation of 12-hydroxy-NVP produces the unstable intermediate 12-sulfoxy-NVP, which can decompose to yield an electrophilic quinone methide. To explore the hypothesis that this pathway is the mechanism by which NVP adducts proteins, Antunes et al. (p 1714) have characterized the adducts formed from the reaction of the 12-sulfoxy-NVP surrogate, 12-mesyloxy-NVP, with human serum albumin (HSA) and human hemoglobin (Hb).
Following overnight incubation of 12-mesyloxy-NVP with HSA or Hb, Antunes et al. used multiple ap-
Special Features Protein sulfenic acids, generated by the oxidation of cysteine thiols, are an intermediate in the formation of reversible and irreversible cysteine oxidation products. In light of the recognition of the importance of these post-translational modifications to cell signaling and toxicity, Kettenhofen and Wood (p 1633) provide an excellent overview of the chemistry of their formation and fate. Here is a great opportunity to learn all you need to know on this timely topic! Vol. 23,
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In this issue proaches to assess the extent and site of protein adduction. MALDI-TOF mass spectrometry of the whole protein indicated adduction with 3 or 4 NVP molecules to HSA exposed once or twice, respectively, to the compound at a ratio of 1:0.5 (wt/wt). Exposure of Hb to 12-mesyloxy-NVP at a ratio of 5:3 (wt/wt) produced a complex pattern of adducts suggesting the attachment of 1, 2, or 3 NVP molecules on the A and/or B chains. Total hydrolysis of 12-mesyloxy-NVP-treated HSA followed by LC-ESI-MS/MS analysis of the individual amino acids revealed adducts to histidine, cysteine, andtryptophan.Similartreatment of Hb yielded adducts of cysteine and tryptophan along with a third compound tentatively identified as a serine adduct. Direct Edman degradation of the treated Hb also revealed adducts at the Nterminal valine residues. Use of MALDI-TOF-TOF analysis of peptides obtained by tryptic digestion of 12-mesyloxy-NVP-treated HSA identified adducts at Trp-238, His-362, Lys-214, and Lys-548 or Lys-549. In the case of Hb, this approach identified adducts at Trp-38 and Ser-90 of HbB and His21 of HbA. Antunes et al. note that results from their various methods support one another with regard to the
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majority of adducts, although each approach detected at least one adduct not observed by the other methods. It is clear, however, that 12-sulfoxy-NVP reacts at typical nucleophilic sites such as cysteine, histidine, lysine, and serine. The most remarkable finding, however, was the consistent detection of tryptophan adducts in both proteins. Tryptophan is not a common site of electrophilic attack, suggesting that this is an unusual pathway of reactivity for the putative quinone methide of NVP and may prove to be a valuable proteomic biomarker for NVP exposure and toxicity in future studies. On the Alert for Isothiazoles A major cause of drug toxicity is bioactivation to a reactive intermediate that may form adducts with DNA and/or proteins, leading to damage of critical cellular macromolecules. Consequently, medicinal chemists try to avoid chemical structures for which an “alert” has been issued, indicating the potential for bioactivation. As described by Teffera et al. (p 1743), this principle guided the discovery of Compound 1 (7-methoxy-N-((6-(3-methylisothiazol5-yl)-[1,2,4]triazolo[4,3b]pyradiazin-3-yl)methyl-1,5napthyridin-4-amine). This compound shows promise as an antitumor agent as a result of its potent inhibitory activity against the recep-
CHEMICAL RESEARCH IN TOXICOLOGY
tor tyrosine kinase c-Met. There were no structural alerts on the isothiazole ring, so its inclusion in Compound 1 appeared to be safe. As an extra precaution, the chemists added a methyl group to the ring since this approach improves the safety of the related thiazole ring structure. Incubation of Compound 1 with liver microsomes from several species resulted in the formation of four metabolites. However, a portion of the molecule became bound to microsomal proteins, suggesting that, despite the chemists’ best efforts, bioactivation of Compound 1 had occurred. When glutathione (GSH) was included in the reaction mixture, protein adduction was eliminated in favor of the formation of a GSH conjugate. Excretion of this conjugate in the bile of rats and mice following the administration of Compound 1 confirmed that the bioactivation process occurred in vivo.
Careful mass spectrometric analysis defined the structure of the GSH conjugate, indicating GSH addition to the isothiazole ring. Teffera etal.proposethatthemechanism of conjugate formation involves sulfoxidation of the isothiazole sulfur atom followed by GSH adduction
and loss of water. Having identified the isothiazole ring as the site of bioactivation, the researchers sought to alter Compound 1’s structure to prevent the reaction from occurring. They first attempted modification of the napthyridine ring methoxy group to a methoxyethoxy group to provide an alternative site of metabolism. Although this change succeeded in changing the pattern of metabolite formation, it did not eliminate protein adduct formation during incubation with liver microsomes. Greater success was achieved when the isothiazole ring of Compound 1 was replaced with one of two alternative bioisosteric heterocycles lacking a sulfur atom. These compounds had similar potency against cMet and pharmacokinetic properties similar to those of Compound 1, but did not form protein or GSH adducts upon incubation with liver microsomes. Teffera et al. conclude that the isothiazole ring provides a potential site of bioactivation reactions and should,therefore,betagged with a structural alert. However, their work also offers strategies to avoid this possible toxicity by replacing the isothiazole with heterocycles of similar structure. TX100329J
Published online 11/15/2010 • DOI: 10.1021/tx100329j © 2010 American Chemical Society
