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Hydroxyurea induces site-specific DNA damage via formation of hydrogen peroxide and nitric oxide.

ABSTRACT: Hydroxyurea is a chemotherapeutic agent used for the treatment of myeloproliferative disorders (MPD) and solid tumors. The mutagenic and carcinogenic potential of hydroxyurea has not been established, although hydroxyurea has been associated with an increased risk of leukemia in MPD patients. To clarify whether hydroxyurea has potential carcinogenicity, we examined site-specific DNA damage induced by hydroxyurea using (32)P-5'-end-labeled DNA fragments obtained from the human p53 and p16 tumor suppressor genes and the c-Ha-ras-1 protooncogene. Hydroxyurea caused Cu(II)-mediated DNA damage especially at thymine and cytosine residues. NADH efficiently enhanced hydroxyurea-induced DNA damage. The DNA damage was almost entirely inhibited by catalase and bathocuproine, a Cu(I)-specific chelator, suggesting the involvement of hydrogen peroxide (H(2)O(2)) and Cu(I). Typical free hydroxyl radical scavengers did not inhibit DNA damage by hydroxyurea, but methional did. These results suggest that crypto-hydroxyl radicals such as Cu(I)-hydroperoxo complex (Cu(I)-OOH) cause DNA damage. Formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was induced by hydroxyurea in the presence of Cu(II). An electron spin resonance spectroscopic study using N-(dithiocarboxy)sarcosine as a nitric oxide (NO)-trapping reagent demonstrated that NO was generated from hydroxyurea in the presence and absence of catalase. In addition, the generation of formamide was detected by both gas chromatography-mass spectrometry (GC-MS) and time-of-flight-mass spectrometry (TOF-MS). A high concentration of hydroxyurea induced depurination at DNA bases in an H(2)O(2)-independent manner, and endonuclease IV treatment led to chain cleavages. These results suggest that hydroxyurea could induce base oxidation as the major pathway of DNA modification and depurination as a minor pathway. Therefore, it is considered that DNA damage by hydroxyurea participates in not only anti-cancer activity, but also carcinogenesis.

SUBMITTER: Sakano K

PROVIDER: S-EPMC5926660 | biostudies-other | 2001 Nov

REPOSITORIES: biostudies-other

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Hydroxyurea induces site-specific DNA damage via formation of hydrogen peroxide and nitric oxide.

Sakano K K Oikawa S S Hasegawa K K Kawanishi S S

Japanese journal of cancer research : Gann 20011101 11

Hydroxyurea is a chemotherapeutic agent used for the treatment of myeloproliferative disorders (MPD) and solid tumors. The mutagenic and carcinogenic potential of hydroxyurea has not been established, although hydroxyurea has been associated with an increased risk of leukemia in MPD patients. To clarify whether hydroxyurea has potential carcinogenicity, we examined site-specific DNA damage induced by hydroxyurea using (32)P-5'-end-labeled DNA fragments obtained from the human p53 and p16 tumor s ...[more]

PMID: 11714440

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Project description:Hydrogen peroxide (H2O2) and nitric oxide (NO·) are toxic metabolites that immune cells use to attack pathogens. These antimicrobials can be present at the same time in phagosomes, and it remains unclear how bacteria deal with these insults when simultaneously present. Here, using Escherichia coli, we observed that simultaneous exposure to H2O2 and NO· leads to prioritized detoxification, where enzymatic removal of NO· is impeded until H2O2 has been eliminated. This phenomenon is reminiscent of carbon catabolite repression (CCR), where preferred carbon sources are catabolized prior to less desirable substrates; however, H2O2 and NO· are toxic, growth-inhibitory compounds rather than growth-promoting nutrients. To understand how NO· detoxification is delayed by H2O2 whereas H2O2 detoxification proceeds unimpeded, we confirmed that the effect depended on Hmp, which is the main NO· detoxification enzyme, and used an approach that integrated computational modeling and experimentation to delineate and test potential mechanisms. Plausible interactions included H2O2-dependent inhibition of hmp transcription and translation, direct inhibition of Hmp catalysis, and competition for reducing equivalents between Hmp and H2O2-degrading enzymes. Experiments illustrated that Hmp catalysis and NAD(P)H supply were not impaired by H2O2, whereas hmp transcription and translation were diminished. A dependence of this phenomenon on transcriptional regulation parallels CCR, and we found it to involve the transcriptional repressor NsrR. Collectively, these data suggest that bacterial regulation of growth inhibitor detoxification has similarities to the regulation of growth substrate consumption, which could have ramifications for infectious disease, bioremediation, and biocatalysis from inhibitor-containing feedstocks.IMPORTANCE Bacteria can be exposed to H2O2 and NO· concurrently within phagosomes. In such multistress situations, bacteria could have evolved to simultaneously degrade both toxic metabolites or preferentially detoxify one over the other. Here, we found that simultaneous exposure to H2O2 and NO· leads to prioritized detoxification, where detoxification of NO· is hampered until H2O2 has been eliminated. This phenomenon resembles CCR, where bacteria consume one substrate over others in carbon source mixtures. Further experimentation revealed a central role for transcriptional regulation in the prioritization of H2O2 over NO·, which is also important to CCR. This study suggests that regulatory scenarios observed in bacterial consumption of growth-promoting compound mixtures can be conserved in bacterial detoxification of toxic metabolite mixtures.

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Hydroxyurea induces site-specific DNA damage via formation of hydrogen peroxide and nitric oxide.

Publications

Hydroxyurea induces site-specific DNA damage via formation of hydrogen peroxide and nitric oxide.

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