Noninvasive fluorescence imaging of small animals.

Noninvasive in vivo fluorescence imaging of small animals as a method in preclinical research has developed considerably in recent years, and is used widely across a variety of disciplines such as oncology and infectious disease research. It provides a means of detecting a fluorescent signal within a living animal reflecting specific, mostly disease-related, processes, such as parts of the host immune response, inflammation, cancer growth or presence of pathogens. As well as offering many advantages as a standalone technique, it can also be highly complementary to other imaging modalities. This review discusses aspects of light distribution in animal tissue and the implications on in vivo imaging; the most widely used imaging techniques including planar and tomographic imaging; advantages and challenges of the techniques; fluorescent contrast agents and some examples of applications. Rather than in detail reviewing studies using in vivo fluorescence imaging, we focus on the principles and practicalities of the method itself, so that the reader can apply these to their own research question.

Phase I pharmacokinetic and pharmacodynamic study of the bioreductive drug RH1.

BACKGROUND: This trial describes a first-in-man evaluation of RH1, a novel bioreductive drug activated by DT-diaphorase (DTD), an enzyme overexpressed in many tumours. PATIENTS AND METHODS: A dose-escalation phase I trial of RH1 was carried out. The primary objective was to establish the maximum tolerated dose (MTD) of RH1. Secondary objectives were assessment of toxicity, pharmacokinetic determination of RH1 and pharmacodynamic assessment of drug effect through measurement of DNA cross linking in peripheral blood mononuclear cells (PBMCs) and tumour, DTD activity in tumour and NAD(P)H:quinone oxidoreductase 1 (NQO1) polymorphism status. RESULTS: Eighteen patients of World Health Organization performance status of zero to one with advanced refractory solid malignancies were enrolled. MTD was 1430 µg/m(2)/day with reversible bone marrow suppression being dose limiting. Plasma pharmacokinetic analysis showed RH1 is rapidly cleared from blood (t(1/2) = 12.3 min), with AUC increasing proportionately with dose. The comet-X assay demonstrated dose-related increases in DNA cross linking in PBMCs. DNA cross linking was demonstrated in tumours, even with low levels of DTD. Only one patient was homozygous for NQO1 polymorphism precluding any conclusion of its effect. CONCLUSIONS: RH1 was well tolerated with predictable and manageable toxicity. The MTD of 1430 µg/m(2)/day is the dose recommended for phase II trials. The biomarkers of DNA cross linking, DTD activity and NQO1 status have been validated and clinically developed.

Maintenance of Golgi structure and function depends on the integrity of ER export.

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.

Quantitative multiplexed quantum dot immunohistochemistry.

Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.

Biomarker method validation in anticancer drug development.

Over recent years the role of biomarkers in anticancer drug development has expanded across a spectrum of applications ranging from research tool during early discovery to surrogate endpoint in the clinic. However, in Europe when biomarker measurements are performed on samples collected from subjects entered into clinical trials of new investigational agents, laboratories conducting these analyses become subject to the Clinical Trials Regulations. While these regulations are not specific in their requirements of research laboratories, quality assurance and in particular assay validation are essential. This review, therefore, focuses on a discussion of current thinking in biomarker assay validation. Five categories define the majority of biomarker assays from 'absolute quantitation' to 'categorical'. Validation must therefore take account of both the position of the biomarker in the spectrum towards clinical end point and the level of quantitation inherent in the methodology. Biomarker assay validation should be performed ideally in stages on 'a fit for purpose' basis avoiding unnecessarily dogmatic adherence to rigid guidelines but with careful monitoring of progress at the end of each stage. These principles are illustrated with two specific examples: (a) absolute quantitation of protein biomarkers by mass spectrometry and (b) the M30 and M65 ELISA assays as surrogate end points of cell death.

Validation of the comet-X assay as a pharmacodynamic assay for measuring DNA cross-linking produced by the novel anticancer agent RH1 during a phase I clinical trial.

PURPOSE: RH1 is a novel anticancer agent with potent DNA-cross linking activity. RH1 has the potential to be activated within tumors over expressing NQO1, giving maximal antitumour activity with reduced toxicity in normal tissues. RH1 has recently completed a Cancer Research UK sponsored phase I clinical trial at two different centers in the United Kingdom. The comet-X assay was a secondary endpoint in this trial and assay validation was necessary. We describe here this validation process. Whilst it is impossible to cover all variations/conditions of a pharmacodynamic assay, we have strived to evaluate and demonstrate that this assay conforms to the three R's of validation, that is robustness, reliability and reproducibility. METHODS: K562 and peripheral blood mononuclear cells were treated with either radiation alone, or with a combination of radiation and drug. These samples were then embedded in low melting point agarose and subjected to a modified version of the alkaline single cell gel electrophoresis (Comet) assay, described here as the comet-X assay. Variations in the preparation, electrophoresis, storage and scoring of these samples was investigated. In addition radiation and drug dose response curves were constructed. Finally stability of QC standards was investigated over a 30-month period. RESULTS: We have demonstrated a linear radiation-dose response in cells up to 20 Gy and drug induced DNA cross-linking up to 50 nM. From the radiation dose response curves we were able to show that the relative inaccuracy measured against a global mean value was less than 25% and the relative (within day) imprecision was less than 30% over all doses. Between day runs produced an intra assay imprecision of 21.2%. Variables involved in the electrophoresis process showed the voltage across all slides in the tank ranged from 3.1 to -2.0 (mV) whilst the current ranged from 0.8-5.5 mA. QC standards were prepared from PBMCs of healthy donors and frozen at -80 degrees C. The stability of these frozen QC standards was measured over a 30-month period. No significant deterioration in any of the control, irradiated or drug treated samples was observed. CONCLUSIONS: The comet-X assay has been shown to be a robust, reliable and reproducible assay. It is ideally suited for the evaluation of the pharmacodynamic effects of DNA cross-linking agents undergoing early clinical trials. Furthermore, this assay may provide valuable data, in conjunction with pharmacokinetics, when measuring toxicity and efficacy as part of the RH1 phase I clinical trial.

Comparison of the structural and cytotoxic activity of novel 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone analogues.

Eight analogues of 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone have been synthesized and tested for cytotoxicity against four different leukemic and lymphomic cell lines. For K562 and BSM cells, the toxicity could be correlated with the ease of reduction of the compounds as determined by the one-electron reduction potentials and the electron spin resonance detection of the reduced compounds produced by the cells. The cell toxicity could also be correlated with the efficiency of the compounds to form cross-links in DNA. However, no such correlations could be observed for the L1210 and Raji cells, although the activity of the NADPH dependent reducing enzymes in these cells was similar to that in the others. It is believed that for the L1210 and Raji cells, the influx/efflux of the different compounds may be more important to the cytotoxicity than their reduction or alkylation.

DT-diaphorase: a target for new anticancer drugs.

DT-diaphorase (DTD) is an obligate two-electron reductase which bioactivates chemotherapeutic quinones. DTD levels are elevated in a number of tumour types, including non-small cell lung carcinoma, colorectal carcinoma, liver cancers and breast carcinomas, when compared to the surrounding normal tissue. The differential in DTD between tumour and normal tissue should allow targeted activation of chemotherapeutic quinones in the tumour whilst minimising normal tissue toxicity. The prototypical bioreductive drug is Mitomycin C (MMC) which is widely used in clinical practice. However, MMC is actually a relatively poor substrate for DTD and its metabolism is pH-dependent. Other bioreductive drugs have failed because of poor solubility and inability to surpass other agents in use. RH1, a novel diaziridinylbenzoquinone, is a more efficient substrate for DTD. It has been demonstrated to have anti-tumour effects both in vitro and in vivo and demonstrates a relationship between DTD expression levels and drug response. RH1 has recently entered a phase I clinical trial in solid tumours under the auspices of Cancer Research UK. Recent work has demonstrated that DTD is present in the nucleus and is associated with both p53 and the heat shock protein, HSP-70. Furthermore, DTD is inducible by several non-toxic compounds and therefore much interest has focussed on increasing the differential in DTD levels between tumour and normal tissues.

Cytotoxicity of the bioreductive agent RH1 and its lack of interaction with radiation.

BACKGROUND AND PURPOSE: RH1 is a new bioreductive agent that was developed as a cytotoxic agent with selectivity for tumour cells expressing high levels of the enzyme DT-diaphorase (DTD). The aim of the present study was to investigate the cytotoxicity of RH1 in relation to cellular levels of reducing enzymes and any interaction of RH1 with ionizing radiation under oxic and hypoxic conditions. PATIENTS AND METHODS: The MB-MDA231 human breast cancer cell line (WT) and WT cells transfected with the NQO1 gene encoding DTD (the D7 cell line) were used to examine the dependency of RH1's cytotoxicity on cellular DTD activity. The role of the 1-electron reducing enzyme P450 reductase was also studied using a P450 reductase-transfected isogenic cell line (R4). A clonogenic assay was used to investigate the cytotoxicity of RH1 with and without irradiation in air and in nitrogen. In all cases drug exposure was for 3 h. RESULTS: DTD levels were around 300-fold higher in D7 compared to WT and R4 cells. RH1 was cytotoxic at nanomolar concentrations to all the cell lines, and was 2-3 times more toxic in the D7 cells with high DTD than in the other two cell lines. Doses of RH1 was around 2-fold more effective in hypoxic than in oxic WT cells, but not by as much in D7 cells. RH1 did not radiosensitise the cells but showed an additive effect when combined with irradiation under oxic and hypoxic conditions. CONCLUSIONS: RH1 shows high clonogenic cytotoxicity to MDA231 cells with high DTD activity but its selectivity based on the presence of DTD is much less than as shown in previous reports. RH1 showed an additive cell killing effect when combined with irradiation under both oxic and hypoxic conditions.

The alkylation of DNA in vitro by 2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ)--I.

Cell toxicity by BZQ could not be explained by free radical formation and thus further work has been undertaken to elucidate a possible mechanism of cell killing. By using radiolabelled BZQ, in vitro DNA-drug binding has been investigated. The effect of salt, buffer and drug concentrations was determined in the pH range 4.0 to 8.0. The influence of in situ oxidation and reduction on BZQ binding was also studied as a function of pH. In an effort to ascertain any base specificity of BZQ binding the homopolymers, Poly[dG]. Poly[dC] and Poly[dA]. Poly[dT] were treated with radiolabelled BZQ in the pH range 4.0 to 8.0. A fluorescence assay was used to demonstrate the possible involvement of DNA cross-linking in cellular activity. From this work, it was concluded that BZQ functions as a bifunctional alkylating agent by an acid-assisted aziridine ring-opening mechanism and that other factors including oxidation or reduction are much less important.

Comet assay studies on the activation of two diaziridinylbenzoquinones in K562 cells.

Two versions of the comet assay have been used to identify the difference in the modes of action of AZQ (2,5-diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone) and BZQ (2,5-diaziridinyl-3,6-bis(ethanolamino)-1,4-benzoquinone) in human leukaemia K562 cells and a K562-derived resistant cell line, BZQR. Using the standard alkaline assay, AZQ produced dose-dependent changes in the mean comet moments from K562 cells, consistent with the formation of strand breaks. This damage was repaired over a period of 6 hr after removal of the drug. The resistant cell line, BZQR, showed much smaller changes in comet moment under identical conditions. In contrast to AZQ, BZQ did not produce any measurable strand breaks in the K562 or BZQR cells. However, the comet radiation/crosslinking assay and a fluorescence-based assay revealed that BZQ extensively cross-links DNA in K562 cells. The extent of cross-linking is greatly reduced in the resistant cell line.

The lack of correlation between toxicity and free radical formation of two diaziridinyl benzoquinones.

L1210 and K562 leukaemic cells have been used to study the relationship between cytotoxicity and free radical production by two aziridinyl benzoquinones, 2,5-bis(carboethoxyamino)3,6-diaziridinyl-1,4-benzoquinone (AZQ) and 2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ). BZQ showed a high level of toxicity in both cell lines, but no ESR signal was detectable, while AZQ readily produced an ESR signal but much lower cytotoxicity was observed, particularly in L1210 cells. The rate of superoxide formation was measured for each drug. The results demonstrate that cell killing and free radical production do not necessarily concur.

Cross-resistance studies on two K562 sublines resistant to diaziridinylbenzoquinones.

Two resistant K562 sublines have been developed by treatment with AZQ (2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone) and BZQ (2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone). The ID50 values of for AZQ on K562, the AZQ-resistant sublines (AZQR) and the BZQ-resistant sublines (BZQR) were 0.063, 1.47 and 0.244 microM, respectively. The relative ID50 values for BZQ on the same cell lines were 0.2, 0.67 and 0.83 microM, respectively. Although it is generally believed that these two quinones function by different mechanisms, the two sublines have similar decreased levels of cytochrome P-450 reductase and DT-diaphorase and increased levels of glutathione and superoxide dismutase, compared to the parent cell line. The sublines are also cross-resistant to adriamycin, mitozolamide, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and mitomycin C. This work indicates the potential multifactorial mechanisms by which drug resistance can be induced in cell lines in the absence of conventional 'P'-glycoprotein multidrug resistance.

Childhood lead poisoning and vinyl miniblind exposure.

OBJECTIVE: To determine the contribution of vinyl miniblinds to childhood lead poisoning. DESIGN: A descriptive investigation was undertaken to estimate attributable risk among all reported childhood lead poisoning cases in North Carolina for which home environmental sampling was conducted between March and August 1996. PARTICIPANTS: Ninety-two children, aged 6 to 72 months, identified through a statewide screening program were included. Blood lead and environmental sampling test results were obtained from routine surveillance data collected for all lead-poisoned children. RESULTS: Exposure to vinyl miniblinds with dust lead levels of 100 micrograms/ft2 or more occurred for 44 (48%) of the lead-poisoned children; 25 (27%) of the children were exposed to levels of 500 micrograms/ft2 or more. Vinyl miniblinds were the predominant source (ie, other major sources of lead were not identified) for 8 (9%) of the children. Overall, the median dust lead level for vinyl miniblind field samples was 590 micrograms/ft2, and the highest level reported was 73,302 micrograms/ft2. Even new vinyl miniblinds manufactured before July 1996 contained dust lead levels that on average exceeded 100 micrograms/ft2. The levels for recently available nonleaded vinyl miniblinds were below the limits of detection. CONCLUSIONS: Vinyl miniblinds, introduced into this country 10 years ago, with sales estimated at 30 million sets a year, include brands containing lead. Although new formulations with no lead added are available, millions of children may still be at risk because a product recall has not been issued (ie, lead-contaminated vinyl miniblinds are still in general use). In addition, the risk assessment evaluations proposed in lieu of universal blood lead screening for low-risk communities could overlook children with exposure to this source.

Comparative studies of the uptake of daunorubicin in sensitive and resistant P388 cell lines by flow cytometry and biochemical extraction procedures.

The intracellular accumulation of daunorubicin as determined by flow cytometry correlates well with that as determined by extraction of the drug from cell homogenates. Two P388 mouse leukaemia cell lines showing differential sensitivity to the drug have been used to investigate the transport changes associated with resistance. Resistance to daunorubicin in these cell lines occurs through an alteration in the intracellular accumulation of the drug, resulting from the increased efflux of the anthracycline from the resistant cells. The effect of temperature, drug concentration, pH, and metabolic inhibitors on this process have been investigated. Uptake by a carrier-mediated process of the un-ionised form of the drug (pK = 8.25), coupled with an energy-dependent efflux process, is proposed as the mechanism of cellular accumulation in the case of the resistant cell line.

DNA damage and repair in Gorlin syndrome and normal fibroblasts after aminolevulinic acid photodynamic therapy: a comet assay study.

Using normal, untransformed, human fibroblasts, the effectiveness of aminolevulinic (ALA)-mediated photodynamic therapy (PDT) was investigated in terms of both clonogenic survival and DNA damage. The response of normal fibroblasts was then compared with Gorlin syndrome-derived fibroblasts (basal cell nevus syndrome [BCNS]). In terms of clonogenic survival, no significant differences were observed between the two groups of cells. Using the alkaline comet assay, initial DNA damage after PDT was measured. Some DNA damage was detected at higher doses, but this was fully repaired within 24 h of treatment. The BCNS-derived cells showed levels of initial damage that did not differ significantly from normal lines.

Influence of extracellular pH on the cytotoxicity and DNA damage of a series of indolequinone compounds.

BACKGROUND: The existence of an acidic extracellular pH (pHe) within solid tumours is regarded as a potential target for drug development. The indolequinone EO9 has a complex mechanism of action which includes enhanced potency under acidic pHe conditions in vitro. In order to identify compounds which have a simpler mechanism of action where activation under acidic pHe is the predominant mechanism of toxicity, this study has determined the cytotoxic properties of a series of analogues of EO9 under both physiological and acidic pHe conditions. MATERIALS AND METHODS: H460 human NSCLC cells were exposed to EO compounds under acidic (pH 6.04) and physiological (pH 7.24) pHe conditions for one hour and chemosensitivity assessed 4 days later using the MTT assay. For compounds of interest, DNA damage (both single strand breaks and cross links) in H460 cells was determined using the comet assay. RESULTS: All the compounds tested were more potent under acidic pHe conditions although a broad range of enhancement ratios (defined as the IC50 at pHe 7.24/IC50 at pHe 6.04) were obtained ranging from 3.25 to 116.53. The activity of EO72 was significantly enhanced under acidic conditions and activity was associated with a pH dependent increase in DNA cross linking in H460 cells. As EO72 is a poor substrate for purified human DT-diaphorase, pHe conditions appear to be a major factor determining cell kill. CONCLUSIONS: This study has identified several compounds whose cytotoxic properties in vitro are pHe dependent with EO72 emerging as the lead compound on the basis of the magnitude of the pH dependent chemosensitivity and the fact that it is a poor substrate for DT-diaphorase. Further studies are required to determine whether or not EO72 has suitable pharmacokinetic properties to allow it to reach regions of low pHe within solid tumours.

Technical report: SYBR Green I and the improved sensitivity of the single-cell electrophoresis assay.

The single-cell electrophoresis (comet) assay is an established method for measuring radiation-induced strand breaks in DNA. The extent of damage is quantified in individual cells by assessing the intensity and distribution of a fluorescent DNA-binding dye using image analysis software. Using the Kinetic Imaging Komet3 system, it is demonstrated that the fluorochrome SYBR Green I improves the resolution and sensitivity of the comet assay, particularly for measuring radiation-induced double strand breaks.

Ethanol enhances the formation of endogenously and exogenously derived adducts in rat hepatic DNA.

To investigate the role of ethanol in chemically-induced carcinogenesis, we exposed Wistar rats to ethanol, either as an acute dose or for prolonged periods in a liquid diet and looked for effects on endogenously and exogenously derived DNA adducts. Changes in the cytochrome P450 protein (CYP 2E1) and its catalytic demethylase activity were also followed in order to provide a sequence of relatively well understood changes that are associated with free radical production and, therefore, potentially capable of affecting DNA. The exocyclic DNA adducts, ethenodeoxyadenosine (varepsilondA) and ethenodeoxycytidine (varepsilondC), known to arise from oxidative stress and lipid peroxidation (LPO) sources, were detected in the liver DNA of Wistar rats at background concentrations of 4-6 (varepsilondA) and 25-35 (varepsilondC) adducts per 10(9) parent bases. When rats were given either an acute dose of ethanol (5g/kg, i.g.) or exposed for 1 week to ethanol in a liquid diet (5%, w/v), etheno adduct levels were increased approximately 2-fold and this was statistically significant for varepsilondC (P<0.05 and P<0.02, respectively) for the two separate treatments.In N-nitrosodimethylamine (NDMA)-treated rats, acute ethanol treatment significantly increased the level of O(6)-methylguanine (O(6)-MeG) in hepatic DNA and this was paralleled by a decrease in O(6)-alkylguanine DNA alkyltransferase (ATase) activity; immunohistochemistry confirmed this increase of O(6)-MeG in both hepatic and renal nuclei. When rats were given ethanol in the diet and treated with NDMA, O(6)-MeG levels in hepatic DNA increased at 1 week which coincided with the peak of CYP 2E1-dependent NDMA-demethylase activity. Single cell gel electrophoresis of liver cells showed that after 1 week of exposure to ethanol, there was a small but significant increase in the frequency of DNA strand breaks induced by NDMA (P<0.05); after 4 weeks the increase was 1.4-fold (P<0.01). Our results indicate that exposures to ethanol, which resulted in blood ethanol concentrations similar to those seen in chronic alcoholics and increased levels of expression of the CYP 2E1 protein can exacerbate the DNA damaging effects of endogenous and exogenous alkylating agents. These observations provide indications of possible mechanisms for the carcinogenic or co-carcinogenic action of ethanol.

Measurement of drug-induced DNA interstrand crosslinking using the single-cell gel electrophoresis (comet) assay.

DNA damaging agents have been widely used in cancer chemotherapy for many years and have proved successful in the treatment of both solid tissue and haematological malignancies. Many commonly used clinical agents, such as members of the nitrogen mustard, chloroethylnitrosourea, dimethane-sulphonate and platinum classes, are bifunctional. DNA interstrand crosslinks (ISC) formed in cells are clearly critical cytotoxic lesions and the formation of DNA ISC has been shown to correlate with cytotoxicity in vitro (1-5). Acquired resistance in vitro to such agents can occur by a number of mechanisms, for example altered drug transport (6), intracellular detoxification via enhanced glutathione and glutathione-S-transferase activity (7), but enhanced DNA repair capacity can also play an important role (3). Clinically the mechanisms of acquired resistance to DNA damaging agents are less clear but enhanced repair of ISC has been suggested to play a role in the acquired resistance of some cancers, e.g., chronic lymphocytic leukaemia to nitrogen mustards (8). In addition, the inherent sensitivity (and curability) of some tumors, e.g., testicular cancer, to DNA damaging agents may result in part from their inability to repair critical DNA lesions (9).