S-XVII: Role of oxidative damage, mutagens and anumutagens in relation to nutrition S167

Acquired resistance to oxygen free radicals represents I SIgnificant problemin the treatment of cancer patients with specific antitumour drugs and maybave other important implications since oxygen free radicals arc increasinglyimplicated IS mediators of cell injury under I variely of pathologicalcircumstances. Recent worle performed in our laboratory has demonstratedthat some cell lines exposed to nontoxic dose of H202 became less sensitiveto the cytotoxic effect induced by a high doses of adriamycin. We undertookthe present investigation with the goal of exarmnmg whether a relationshipexists between the hydrogen peroxide pretreatment and delays in transitionsbetween phases of cell cycle in cell lines used before: human melanomacells (ME18), human embryo cells (CLV98), Chinese hamster ovary cells(CHO), African Green monkey kidney cells (Vl) and human melanomaadriamycin-resistant (MEI8IR) and Chinese hamster ovary adriamycin­resistant (CHOIR) sublines. The DNA content in the phases of cell cyclewas examined using flowcytometty. We have obtained influence of the H202pretreatment at various concentrations on phases of cell cycle in CHO andCHOIR cells and MEIS and MEISIR cells. The chromosome analysis hasconfirmed the data received.

Keyword(s): Hydrogen peroxide; Cell cycle

Ip XVII A.61 Development of a postlabelln!: assay as a blomonl­tor of oxidative DNA dama!:e In humans

George Jones, Neil Bennett, Ian Podmore, Michael Routledge. C.UH.T.,Uniuers lry ofLeicester. Leicester LEI 9HN, UK

Evaluating a role for oxidative effects in human disease and judgement ofnutritional antioxidant vitamins, in their potential role to prevent of suchdisorders, necessitates the accurate measure of oxidative damage in uilJO.We arc currently developing/evaluating the Weinfeld SVPD-based l2 P-DNApostlabeling procedure (l) as a bioassay of oxidative DNA damage. The pro­tocol has the advantage of allowing for the exclusive l2 P-labelling/deteClionof several oxidative DNA base and sugar lesions, most notably thymineglycols and phosphoglycolates, at femtomole levels. Development has al­lowed for detection limits as low as 1.8 fmol of damagcll!g of DNA assayed(5.7 damagcsllO' normal nucleotides) and has permitted an assessment ofbasal levels of damage in both calf-thymus and human peripheral bloodlymphocyte DNAs. For phosphoglycolates, values ofca. 20-25 fmoVl!g DNAhave been determined for both DNA samples whilst for thymine glycolsvalues of ca. 10-12 fmoVl!g DNA are ROIed. The assay is being furthervalidatcd by the study of damage-coruaining standards and by comparisonswith Ge-MS/SIM and a plasmid DNA strand break assay. Preliminaryresults indicate that the assay is underdetermining damage levels by CD.

2-5-fold. This research is supported by MAFF (AN0426) and the MRC(G9527655MA).

Keyword(s): Oxidative DNA damage; 32p-postlabeling

(I) Weinfeld & Soderlind, (1991), Biochemistry, 3D,1091-1097.

Ip XVII A.71 Steady-slate levels of oxidative DNA base modifica­tions In mammalian cells

Michael Pflaum" , Olaf wui', Andre-Patrick Arrig02, Bernd Epel. / Institute

of Pharmacy. Uniuerslry of Mainz, D-JJ099 Mainz, Germany; JClalJdeBernard Uniuersity. Lyon, Frana

Various repair endonucleases (Fpg protein, endonuclease Ill, exonucleaseIll, T4-endonuclease V) in combination with the alkaline elution techniquewere used to quantify steady-state (background) levels of oxidative basemodifications in various types of mammalian cells. The number of basemodifications sensitive to Fpg protein, which include S-hydroxyguanine, was0.25 ± 0.05 per 106 base pain in human Iymphocytcs and only 0.07 ± 0.02per 106 bp in HeLa cells. The numbers of sites sensitive to the other repairendonucleases were below the detection limit (0.1 per 106 base pairs). Ina direct comparison, the background level of Fpg-sensitive modificationsdetermined by alkaline elution was much lower than the background level

of 8-hydroxydesoxyguanosine (8-oxodG) determined after enzymatic DNAhydrolysis by HPLC and electrochemical detection. However, the enzymicassay does not systematically underestimate the number of lesions since thenumber of additional Fpg-sensitive modifications induced by a photosensi­tizer plus light was similar to the additional number of 8-oxodG residuesdetermined by HPLC. The level of Fpg-sensitive sites was elevated in cellscultured at 41"C. In contrast, the constitutive expression of the heat-shockprotein hsp27 in L929 fibroblasts decreased the steady-stale level by approx.20%. Treatment with various thiols increased the DNA damage or had noeffect.

Kcyword(s): 8-hydroxyguanine; endogenous DNA damage

Ip XVII A.sl 8-oxoguanlne: Mutagenei!s and repair

Florence Le Page, Alain Genti!, Alain Sarasin. Laboratory of MolecularGenet ics, UPR42-IFCl/CNRS-Villeiui/. France

The aerobic metabolism is accompanied by the formation of reactive freeradicals of oxygen that can damage cellular components and DNA. Thecase with which the 8-oxoguanine is formed by numerous oxidizing agentssuggests that this lesion could make a significant contribution to mutagenesis.In both bacteria and mammalian cells, repair enzymes have been discoveredwhich possess activities towards 8-oxoguanine (Fpg, OGGI) . The generalrepair systems that recognize diverse lesions, such as the uvrABC systemIn E. coli or NER (nucleotide excision repair) in S. cereoisiae may beimplicated. The objective of this work was to investigate the mutationfrequency induced by a unique 8-oxoguanine in different human cell linesnormal or deficient in NER in order to approach a possible intervention of theNER system in the removal of 8-oxoguanine. Results show that replicationof the modified base in I single-stranded plasmid in repair proficient humancells and repair deficient cells from xeroderma pigmentosum patients ofgroup C or D induces a similar mutation frequency of about 3-5% .

In the other hand the base mispairing GO:C as well as the base mispairingGO:A have also been shown as being Intermediates of replication of thealtered base. Studies concerning the biological consequences of these basepairing show the absence of efficient repair of GO:A in human cells underour experimental conditions. This may explain the mutational potency of 8­oxoguanine and also the observed mutation spectrum (G to T transversionsin 95% of mutants).

Keyword(s): S-oxoguanine; DNA vector; normal human cells