Nikolas J. Hodges

Interindividual Variability in Response to Sodium Dichromate–Induced Oxidative DNA Damage: Role of the Ser326Cys Polymorphism in the DNA-Repair Protein of 8-Oxo-7,8-Dihydro-2′-Deoxyguanosine DNA Glycosylase 1

Although the genotoxic mechanism(s) of hexavalent chromium (CrVI) carcinogenicity remain to be fully elucidated, intracellular reduction of CrVI and concomitant generation of reactive intermediates including reactive oxygen species and subsequent oxidative damage to DNA is believed to contribute to the process of carcinogenesis. In the current study, substantial interindividual variation (7.19-25.84% and 8.79-34.72% tail DNA as assessed by conventional and FPG-modified comet assay, respectively) in levels of DNA strand breaks after in vitro treatment of WBC with sodium dichromate (100 μmol/L, 1 hour) was shown within a group of healthy adult volunteers (n = 72) as assessed by both comet and formamidopyrimidine glycosylase–modified comet assays. No statistically significant correlation between glutathione S-transferases M1 or T1, NADPH quinone oxidoreductase 1 (codon 187) and X-ray repair cross complementation factor 1 (codon 194) genotypes and individual levels of DNA damage were observed. However, individuals homozygous for the Cys326 8-oxo 7,8-dihydro-2′-deoxyguanosine glycosylase 1 (OGG1) polymorphism had a statistically significant elevation of formamidopyrimidine glycosylase–dependent oxidative DNA damage after treatment with sodium dichromate when compared with either Ser326/Ser326 or Ser326/Cys326 individuals (P = 0.008 and P = 0.003, respectively). In contrast, no effect of OGG1 genotype on background levels of oxidative DNA damage was observed. When individuals were divided on the basis of OGG1 genotype, Cys326/Cys326 individuals had a statistically significant (P < 0.05, one-way ANOVA followed by Tukey test) higher ratio of oxidative DNA damage to plasma antioxidant capacity than either Ser326/Ser326 or Ser326/Cys326 individuals. The results of this study suggest that the Cys326/Cys326 OGG1 genotype may represent a phenotype that is deficient in the repair of 8-oxo-7,8-dihydro-2′-deoxyguanosine, but only under conditions of cellular oxidative stress. We hypothesize that this may be due to oxidation of the Cys326 residue. In conclusion, the homozygous Cys326 genotype may represent a biomarker of individual susceptibility of lung cancer risk in individuals that are occupationally exposed to CrVI.

Supramolecular Iron Cylinder with Unprecedented DNA Binding Is a Potent Cytostatic and Apoptotic Agent without Exhibiting Genotoxicity

The supramolecular iron cylinder, [Fe(2)L(3)]Cl(4) (L = C(25)H(20)N(4)), shows unprecedented DNA binding in vitro, inducing intramolecular DNA coiling and also targeting Y-shaped DNA junctions. We investigated its effects on proliferation and survival in both tumor and normal cell lines. Iron cylinder reduced mitochondrial activity of cultures with potency similar to cisplatin, inhibited the cell cycle, and increased cell death by apoptosis. Associated with this, we observed a lowering of the association of propidium iodide with cellular DNA consistent with an observed competitive displacement of PI from naked DNA by cylinders. Importantly, and in contrast to existing anticancer drugs such as cisplatin, the iron cylinder [Fe(2)L(3)](4+) was not genotoxic. In summary, the design of metal complexes such as [Fe(2)L(3)](4+) with potential anticancer properties in the absence of genotoxicity may represent a significant step toward therapeutic advancement.

Noncovalent DNA‐Binding Metallo‐Supramolecular Cylinders Prevent DNA Transactions in vitro

Processing of DNA information, through replication or transcription, is mediated by proteins which bind mainly to the DNA major groove through specific subunits such as helix-turn-helix, zinc fingers and leucine zippers. There have been many efforts to synthesize artificial molecules which could interact with DNA to interfere with or control biological processes. DNA replication, when unregulated or uncontrolled, results in many pathological states such as cancer, and is also inherent in viral and bacterial infections. Thus many compounds used in anticancer and antiviral therapy are able to inhibit or block DNA replication, such as platinum drugs and nucleoside analogues. In this context we have recently developed supramolecular tetracationic cylinders consisting of three bis(pyridylimine) ligand (L) strands wrapped in a helical fashion about two iron(II) or ruthenium(II) centers (Figure 1). Their size and shape are very similar to those of protein zinc fingers and the cylinders have proven to be remarkable agents with unique DNA recognition properties: They can not only bind strongly and noncovalently in the major groove of DNA, inducing substantial and unprecedented intramolecular DNA coiling in natural polymeric DNAs, but still more excitingly we have also shown that they can bind at the heart of Yshaped DNA 3-way junctions (3WJ). Such a mode of DNA recognition is without precedent and quite distinct from the classical modes of DNA interaction. 7] Yet what is particularly tantalizing is that the DNA replication fork is a form of Y-shaped junction. Indeed whenever the DNA duplex is opened to process the genetic information (replication or transcription) a Y-shaped junction is created. Gel studies have confirmed that different Y-shaped junctions can be recognized (including biologically relevant ones like splayed duplexes, as the replication fork) as well as the perfectly paired 3-way junctions. Agents that recognize this particular type of “unusual” DNA structure (by shape-fit structural specificity as opposed to traditional sequence specificity) and interfere with replication or transcription processes could be a very powerful tool not only in the fundamental study of the processes such as replication but also in the modulation of cell cycle control. These metallo-supramolecular cylinders induce cytostasis (at low micromolar concentrations) and, at slightly higher concentrations, apoptosis in various cell lines. They are not only taken up but seem to concentrate in cells, with their killing potential unusually related to amount of compound added not to concentration. We hypothesize that these biological effects are a consequence of cylinders binding to Y-shaped junctions such as DNA replication forks and preventing DNA processing. To provide further support for this we wished to probe whether the cylinders would affect DNA transactions in an in vitro experiment. While there is no simple way to study DNA-replication in vitro, polymerase chain reaction (PCR) provides a similar set of DNA transactions, is a DNA replication process involving a DNA polymerase, and is readily accessible. Herein we report the effects of the cylinders on the PCR reaction and demonstrate that they do indeed interfere with the binding of the polymerase to the DNA highlighting the potential for inhibition of DNA replication. In the first experiment we adopted a PCR-based assay to evaluate whether the ruthenium cylinder [Ru2L3] 4+ would interfere with the amplification of DNA. The DNA transaction is Taq DNA polymerase (a thermostable DNA polymerase) recognizing and elongating a primer-DNA double strand. The ruthenium(II) complex was selected as Figure 1. Bis(pyridylimine) ligand L and the structure of the tetracationic M2L3 cylinders formed from that ligand. [5e, 9a, 12]

Repair of oxidative DNA damage is delayed in the Ser326Cys polymorphic variant of the base excision repair protein OGG1

Gene-environment interactions influence an individuals risk of disease development. A common human 8-oxoguanine DNA glycosylase 1 (OGG1) variant, Cys326-hOGG1, has been associated with increased cancer risk. Evidence suggests that this is due to reduced repair ability, particularly under oxidising conditions but the underlying mechanism is poorly understood. Oxidising conditions may arise due to internal cellular processes, such as inflammation or external chemical or radiation exposure. To investigate wild-type and variant OGG1 regulation and activity under oxidising conditions, we generated mOgg1 (-/-) null mouse embryonic fibroblasts cells stably expressing Ser326- and Cys326-hOGG1 and measured activity, gene expression, protein expression and localisation following treatment with the glutathione-depleting compound L-buthionine-S-sulfoximine (BSO). Assessment of OGG1 activity using a 7,8-dihydro-8-oxodeoxyguanine (8-oxo dG) containing molecular beacon demonstrated that the activity of both Ser326- and Cys326-hOGG1 was increased following oxidative treatment but with different kinetics. Peak activity of Ser326-hOGG1 occurred 12 h prior to that of Cys326-hOGG1. In both variants, the increased activity was not associated with any gene expression or protein increase or change in protein localisation. These findings suggest that up-regulation of OGG1 activity in response to BSO-induced oxidative stress is via post-transcriptional regulation and provide further evidence for impaired Cys326-hOGG1 repair ability under conditions of oxidative stress. This may have important implications for increased mutation frequency resulting from increased oxidative stress in individuals homozygous for the Cys326 hOGG1 allele.

Use of a molecular beacon to track the activity of base excision repair protein OGG1 in live cells.

An abundant form of DNA damage caused by reactive oxygen species is 8-oxo-7,8-dihydroguanine for which the base excision repair protein 8-oxoguanine-DNA glycosylase 1 (OGG1) is a major repair enzyme. To assess the location and intracellular activity of the OGG1 protein in response to oxidative stress, we have utilised a fluorescence-quench molecular beacon switch containing a 8-oxo-dG:C base pair and a fluorescent and quencher molecule at opposite ends of a hairpin oligonucleotide. Oxidative stress was induced by treatment with potassium bromate. Flow cytometry demonstrated a concentration-dependent increase in the activity of OGG1 that was detected by the fluorescence produced when the oligonucleotide was cleaved in the cells treated with potassium bromate. This signal is highly specific and not detectable in OGG1 knock out cells. Induction of OGG1 activity is not a result of induction of OGG1 gene expression as assessed by qPCR suggesting a role for protein stabilisation or increased OGG1 catalytic activity. High resolution confocal microscopy pinpointed the location of the fluorescent molecular beacon in live cells to perinuclear regions that were identified as mitochondria by co-staining with mitotracker dye. There is no evidence of cut beacon within the nuclear compartment of the cell. Control experiments with a positive control beacon (G:C base pair and lacking the DAB quencher) did not result in mitochondrial localisation of fluorescence signal indicating that the dye does not accumulate in mitochondria independent of OGG1 activity. Furthermore, faint nuclear staining was apparent confirming that the beacon structure is able to enter the nucleus. In conclusion, these data indicate that the mitochondria are the major site for OGG1 repair activity under conditions of oxidative stress.

Organometallic nucleoside analogues with ferrocenyl linker groups: synthesis and cancer cell line studies

Examples of organometallic compounds as nucleoside analogues are rare within the field of medicinal bioorganometallic chemistry. We report on the synthesis and properties of two chiral ferrocene derivatives containing a nucleobase and a hydroxyalkyl group. These so-called ferronucleosides show promising anticancer activity, with cytostatic studies on five different cancer cell lines indicating that both functional groups are required for optimal activity.

Protection from intracellular oxidative stress by cytoglobin in normal and cancerous oesophageal cells.

Cytoglobin is an intracellular globin of unknown function that is expressed mostly in cells of a myofibroblast lineage. Possible functions of cytoglobin include buffering of intracellular oxygen and detoxification of reactive oxygen species. Previous work in our laboratory has demonstrated that cytoglobin affords protection from oxidant-induced DNA damage when over expressed in vitro, but the importance of this in more physiologically relevant models of disease is unknown. Cytoglobin is a candidate for the tylosis with oesophageal cancer gene, and its expression is strongly down-regulated in non-cancerous oesophageal biopsies from patients with TOC compared with normal biopsies. Therefore, oesophageal cells provide an ideal experimental model to test our hypothesis that downregulation of cytoglobin expression sensitises cells to the damaging effects of reactive oxygen species, particularly oxidative DNA damage, and that this could potentially contribute to the TOC phenotype. In the current study, we tested this hypothesis by manipulating cytoglobin expression in both normal and oesophageal cancer cell lines, which have normal physiological and no expression of cytoglobin respectively. Our results show that, in agreement with previous findings, over expression of cytoglobin in cancer cell lines afforded protection from chemically-induced oxidative stress but this was only observed at non-physiological concentrations of cytoglobin. In addition, down regulation of cytoglobin in normal oesophageal cells had no effect on their sensitivity to oxidative stress as assessed by a number of end points. We therefore conclude that normal physiological concentrations of cytoglobin do not offer cytoprotection from reactive oxygen species, at least in the current experimental model.

Modified comet assay as a biomarker of sodium dichromate-induced oxidative DNA damage: Optimization and reproducibility

Hexavalent chromium (Cr[VI]) is a genotoxic carcinogen that has been associated with an increased risk of nasal and respiratory tract cancers following occupational exposure. Although the precise mechanism(s) remain to be elucidated, there is evidence for a role of oxidative DNA damage in the genotoxicity of Cr(VI). In the current study, human white blood cells were treated in vitro with non-cytotoxic concentrations of sodium dichromate (1–100 μM) for 1 h. Analysis by immunocytochemistry indicated the presence of elevated levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine at concentrations of sodium dichromate greater than 10 μM. In contrast, the lowest concentration of dichromate that resulted in a statistically significant increase in levels of formamidopyrimidine DNA glycosylase (FPG)-dependent DNA strand breaks was 100 nM (p<0.05). In addition, levels of both control and dichromate-induced FPG-dependent strand breaks from blood samples taken from the same individuals over 10 months proved remarkably reproducible in the individuals studied. The coefficients of variation over three different times of the year in control and dichromate-induced oxidative DNA damage for the four individuals were 54, 1, 37 and 4, and 45, 6, 21 and 18%, respectively. In summary, these results indicate that physiologically relevant, nanomolar concentrations of sodium dichromate cause DNA base oxidation in human white blood cells in vitro as assessed by the FPG-modified comet assay. Furthermore, comet assay data from an individual are reproducible over an extended period. This consistency is sufficient to suggest that the modified comet assay might prove to be a useful and sensitive biomonitoring tool for individuals occupationally exposed to hexavalent chromium.

Direct visualization of repair of oxidative damage by OGG1 in the nuclei of live cells

Oxidative DNA damage caused by intracellular reactive oxygen species (ROS) is widely considered to be important in the pathology of a range of human diseases including cancer as well as in the aging process. A frequently occurring mutagenic base lesion produced by ROS is 8‐oxo deoxyguanine (8‐oxo dG) and the major enzyme for repair of 8‐oxo dG is 8‐oxoguanine‐DNA glycosylase 1 (OGG1). There is now substantial evidence from bulk biochemical studies that a common human polymorphic variant of OGG1 (Ser326Cys) is repair deficient, and this has been linked to individual risk of pathologies related to oxidative stress. In the current study, we have used the technique of multiphoton microscopy to induce highly localized oxidative DNA damage in discrete regions of the nucleus of live cells. Cells transfected with GFP‐tagged OGG1 proteins demonstrated rapid (<2 min) accumulation of OGG1 at sites of laser‐induced damage as indicated by accumulation of GFP‐fluorescence. This was followed by repair as evidenced by loss of the localized fluorescence over time. Quantification of the rate of repair confirmed that the Cys326 variant of OGG1 is repair deficient and that the initial repair rate of damage by Cys326 OGG1 was 3 to 4 fold slower than that observed for Ser326 OGG1. These values are in good agreement with kinetic data comparing the Ser326 and Cys326 proteins obtained by biochemical studies.

Anomalous genotoxic responses induced in mouse lymphoma L5178Y cells by potassium bromate.

Potassium bromate (KBrO3) is a well-established rodent kidney carcinogen and its oxidising activity is considered to be a significant factor in its mechanism of action. Although it has also been shown to be clearly genotoxic in a range of in vivo and in vitro test systems, surprisingly, it is not readily detected in several cell lines using the standard alkaline Comet assay. However, previous results from this laboratory demonstrated huge increases in tail intensity by modifying the method to include incubation with either human 8-oxodeoxyguanosine DNA glycosylase-1 (hOGG1) or bacterial formamidopyrimidine DNA glycosylase (FPG) indicating that, as expected, significant amounts of 8-oxodeoxyguanosine (8-OHdG) were induced. The purpose of this work, therefore, was to investigate why KBrO3, in contrast to other oxidising agents, gives a relatively poor response in the standard Comet assay. Results confirmed that it is a potent genotoxin in mouse lymphoma L5178Y cells inducing micronuclei and mutation at the tk and hprt loci at relatively non-cytotoxic concentrations. Subsequent time-course studies demonstrated that substantial amounts of 8-OHdG appear to remain in cells 24h after treatment with KBrO3 but result in no increase in frank stand breaks (FSB) even though phosphorylated histone H2AX (gamma-H2AX) antibody labelling confirmed the presence of double-strand breaks. Using bromodeoxyuracil (BrdU) incorporation together with measured increases in cell numbers, L5178Y cells also appeared to go through the cell cycle with unrepaired hOGG1-recognisable damage. Since unrepaired 8-OHdG can give rise to point mutations through G:C-->T:A transversions, it was also surprising that mutation could not be detected at the Na+/K+ATPase locus as determined by ouabain resistance. Some increases in strand breakage could be seen in the Comet assay by increasing the unwinding time, but only at highly toxic concentrations and to a much smaller extent than would be expected from the magnitude of the other genotoxic responses. It was considered unlikely that these anomalous observations were due to the inability of L5178Y cells to recognise 8-OHdG because these cells were shown to express mOGG1 and have functional cleavage activity at the adducted site. It appears that the responses of L5178Y cells to KBrO3 are complex and differ from those induced by other oxidising agents.