Selective protection of zidovudine-induced DNA-damage by the antioxidants WR-1065 and tempol.
The cytokinesis-block micronucleus cytome (CBMN) assay, introduced by Fenech, was used to demonstrate different types of DNA damage in MOLT-3 human lymphoblastoid cells exposed to 10 μM zidovudine (AZT). In addition, we explored the cytoprotective potential of two antioxidants, WR-1065 and Tempol, to decrease AZT-induced genotoxicity. Binucleated cells, arrested by Cytochalasin B (Cyt B), were evaluated for micronuclei (MN), caused by DNA damage or chromosomal loss, and chromatin nucleoplasmic bridges (NPBs), caused by telomere attrition. Additionally, nuclear buds (NBUDs), caused by amplified DNA, and apoptotic and necrotic (A/N) cells were scored. We hypothesized that AZT exposure would increase the frequency of genotoxic end points, and that the antioxidants Tempol and WR-1065 would protect against AZT-induced genotoxicity. MOLT-3 cells were exposed to 0 or 10 µM AZT for a total of 76 hr. After the first 24 hr, 0 or 5 µM WR-1065 and/or 0 or 200 µM Tempol were added for the remainder of the experiment. For the last 28 hr (of 76 hr), Cyt B was added to arrest replication after one cell division, leaving a predominance of binucleated cells. The nuclear division index (NDI) was similar for all treatment groups, indicating that the exposures did not alter cell viability. MOLT-3 cells exposed to AZT alone had significant (P < 0.05) increases in MN and NBs, compared to unexposed cells. Both Tempol and WR-1065 protected against AZT-induced MN formation (P < 0.003 for both), and WR-1065, but not Tempol, reduced the levels of A/N (P = 0.041). In cells exposed to AZT/Tempol there were significantly reduced levels of NBUDs, compared to cells exposed to AZT alone (P = 0.015). Cells exposed to AZT/WR-1065 showed reduced levels of NPBs, compared to cells exposed to AZT alone (P = 0.037). Thus WR-1065 and Tempol protected MOLT-3 cells against specific types of AZT-induced DNA damage.
Carcinogen-DNA Interactions Section, Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
Ofelia A
OA
Olivero
Michael O
MO
Ongele
Hannan M
HM
Braun
Ariadna
A
Marrogi
Kathyiani
K
Divi
James B
JB
Mitchell
Miriam C
MC
Poirier
D000975:Antioxidants / Q000737:chemistry*; D017209:Apoptosis; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival; D002843:Chromatin / Q000737:chemistry*; D002875:Chromosomes / Q000648:ultrastructure; D003497:Cyclic N-Oxides / Q000737:chemistry*; D003571:Cytochalasin B / Q000737:chemistry; D004249:DNA Damage; D006801:Humans; D008624:Mercaptoethylamines / Q000737:chemistry*; D015162:Micronucleus Tests; D009153:Mutagens / Q000737:chemistry; D009336:Necrosis; D011837:Radiation-Protective Agents / Q000737:chemistry; D013113:Spin Labels; D015215:Zidovudine / Q000737:chemistry*
D000975:Antioxidants; D002843:Chromatin; D003497:Cyclic N-Oxides; D008624:Mercaptoethylamines; D009153:Mutagens; D011837:Radiation-Protective Agents; D013113:Spin Labels; C020174:N-(2-mercaptoethyl)-1,3-diaminopropane; D003571:Cytochalasin B; D015215:Zidovudine; C001803:tempol
CBMN assay; MOLT-3 cells; apoptosis; chromatin neoplasmic bridges; cytome assay; micronuclei; necrosis; nuclear buds