Stannous chloride mediates single strand breaks in plasmid DNA through reactive oxygen species formation

Stannous ion (Sn) has been employed in nuclear medicine and in food industry. We described that Stannous Chloride (SnCl₂) inactivation effect in Escherichia coli is mediated by a Fenton-like reaction. The effect of SnCl₂ was studied through: (i) the alteration of plasmid topology in neutral and acidic pH by gel electrophoresis; and (ii) the transformation efficiency of an wild type E. coli strain. Treatment of plasmid DNA pUC 9.1 with SnCl₂, at pH 7.4, results in DNA single-strand breaks (SSB), in a dose-dependent manner. Addition of sodium benzoate partly inhibited the DNA damage, while EDTA completely abolishes DNA-SSB. Furthermore, the ability of the plasmid to transform E. coli was reduced. At pH 1.3, SnCl₂ exerts a protective effect on plasmid against HCl depurination. Our results suggest the generation of ROS, such as √OH by a Fenton-like reaction, close to the site of the lesions due to a possible complexation of stannous ion to DNA.     

Photoactivated uranyl ion produces single strand breaks in plasmid DNA

Uranium is an important emerging toxicant whose use has outpaced the rate at which we are learning about its health effects. One unexplored pathway for uranium toxicity involves the photoactivation of uranyl ion by UV light to produce U(5+) and oxygen radicals. The purpose of this study was to provide proof of principle data by testing the hypothesis that coexposures of DNA to uranyl acetate and UVB irradiation should produce more DNA strand breaks than individual exposures. Results supported the hypothesis and suggest that investigations of uranium toxicity be expanded to include skin as a potential target organ for carcinogenesis, especially in populations with high uranium and high UV radiation exposures.     

Induction of DNA strand breaks in peripheral lymphocytes by soluble chromium compounds.

1. Incubation of human lymphocytes with sodium dichromate (CrVI) at 37 degrees C for 3 h resulted in a dose-dependent increase in DNA strand breaks without concurrent cytotoxicity. In contrast, chromium acetate hydroxide (CrIII) failed to induce DNA strand breaks at sub-cytotoxic concentrations. 2. DNA strand breaks were also detected in the peripheral lymphocytes of Wistar rats, 24 h after intratracheal instillation of sodium dichromate (1.3 and 2.5 mg kg-1). Instillation of chromium acetate hydroxide (up to 21.8 mg kg-1) failed to induce DNA strand breaks in peripheral lymphocytes. In accord with previous studies, hexavalent chromium was found to be more readily absorbed from the lungs into the peripheral blood than chromium in its trivalent form. 3. The results of this study indicate that fluorometric analysis of DNA unwinding (FADU) in peripheral lymphocytes might be a convenient method of measuring an important biological effect of chromium in occupationally-exposed workers.     

DNA strand breaks and apoptosis induced by oxaliplatin in cancer cells

Platinum anticancer drugs, such as cisplatin, are thought to exert their activity by DNA damage. Oxaliplatin, a clinically active diaminocyclohexane platinum compound, however, requires fewer DNA-Pt adducts than cisplatin to achieve cell growth inhibition. Here we investigated whether secondary DNA damage and apoptotic responses to oxaliplatin compensate for the reduced formation of DNA adducts. Oxaliplatin treatment of leukemic CEM and ovarian A2780 cancer cells resulted in early (4 hr) induction of DNA single-strand breaks measured by nucleoid sedimentation. These infrequent early lesions progress with time into massive double-stranded DNA fragmentation (fragments >50k bp) paralleled by characteristic apoptotic changes revealed by cell morphology and multivariate flow cytometry. Profound oxaliplatin-induced apoptotic DNA fragmentation was detectable following a 24 hr treatment of A2780 and CEM cells with 2 and 10 microM oxaliplatin, respectively. This DNA fragmentation was inhibited completely by the broad-spectrum caspase inhibitor Z-VAD-fmk. Cisplatin, which forms markedly more DNA-Pt adducts in CEM and A2780 cells than equimolar oxaliplatin, was similarly potent as oxaliplatin in terms of early strand breaks and later apoptotic responses. Oxaliplatin was also profoundly apoptotic in several other tumor cell lines of prostate origin but had only a marginal effect in normal prostate PrEC cells. Collectively, the results demonstrate that, relative to the magnitude of the primary DNA-Pt lesions, oxaliplatin is disproportionately more potent than cisplatin in the induction of apoptosis. Apoptosis induction, possibly enhanced by a contribution of targets other than DNA, seems to be an important factor in the mechanism of action of oxaliplatin.     

DNA strand breaks induced by hydrogen peroxide in isolated rat hepatocytes

It has been proposed that increased rates of hepatic hydrogen peroxide (H2O2) production may initiate or promote the liver tumors that appear following chronic exposure of rodents to chemicals that cause peroxisome proliferation. However, the effect of H2O2 on the structural integrity of DNA in parenchymal hepatocytes, the target cells of peroxisome proliferator-induced carcinogenesis, is largely uncharacterized. Furthermore, oxidant-induced cellular damage has been invoked as causal of a number of hepatotoxic effects associated with exposure to chemicals other than peroxisome proliferators. For these reasons, alkaline elution analysis was used to study the action of H2O2, added exogenously, on DNA of intact, isolated rat hepatocytes. Addition of a bolus of H2O2 (0.01-1.0 mM) to monolayer cultures of hepatocytes caused concentration-dependent increases in single-strand DNA breaks (SSDB), which were maximal within 30 min of exposure. Cytotoxicity, as measured by lactate dehydrogenase (LDH) release, was minimal during a 1-h exposure to H2O2 concentrations less than 1 mM, but the efflux of oxidized glutathione was increased. Formation of SSDB was nearly linear with respect to H2O2 concentration in the range 0.1-1.0 mM. No double-strand DNA breaks or DNA-protein crosslinks were identified at H2O2 concentrations of 1 mM or less. Repair of SSDB in H2O2-free medium occurred in a rapid, linear manner only for the first 15 min, resulting in disappearance of 65% of the SSDB. A second, slower phase of SSDB rejoining occurred between 20 and 60 min of incubation in H2O2-free media; at 60 min rejoining was maximal (80% repair). These results define a specific type of DNA damage associated with H2O2 exposure of hepatocytes and suggest that primary cultures of rat hepatocytes are a suitable model for characterizing the potential genotoxic effects of oxidants, particularly excess H2O2 that may occur in the livers of animals exposed chronically to peroxisome proliferators.     

菲啰啉对不同氧化剂和多柔比星所致CHL细胞DNA链断裂的影响

目的 为了研究菲啰啉对2种氧化剂和抗癌药多柔比星诱发细胞DNA损伤的影响, 并初步探讨其损伤机制。方法 用不同浓度菲啰啉预处理CHL细胞30 min, 再分别加入3种不同染毒受试物,共同培养一定时间(0.3 mmol·L^-1重铬酸钾：105 min； 0.5 μmol·L^-1多柔比星：5 min； 0.4 mmol·L^-1过氧化氢(H₂O₂)：25 min)后, 用碱性单细胞凝胶电泳方法(ASCGE)测定DNA链断裂情况, 并同时以菲啰啉与二甲亚砜(DMSO，0.33 mol·L^-1)比较对H₂O₂致DNA损伤中·OH的产生和清除。结果 3种染毒受试物均可明显引起CHL细胞DNA链断裂；而当3 μmol·L^-1菲啰啉预处理后, 可使重铬酸钾、H₂O₂所致DNA迁移长度和细胞拖尾率明显降低, 并超过DMSO降低H₂O₂的损伤作用, 当菲啰林浓度升至12 μmol·L^-1时, 可完全消除这两种因素所致的DNA链断裂损伤；10 μmol·L^-1菲啰啉可抑制多柔比星所致DNA损伤, 但浓度直至60 μmol·L^-1仍不能完全消除多柔比星的损伤作用。结论 菲啰啉对2种氧化剂和多柔比星所致DNA损伤均有不同程度的防护作用,同时提示重铬酸钾和H₂O₂所致的DNA损伤主要与需过渡金属离子参与的·OH产生有关, 而多柔比星所致损伤仅部分与此有关。     

菲啰啉对不同氧化剂和多柔比星所致CHL细胞DNA链断裂的影响

目的　为了研究菲口罗啉对 2种氧化剂和抗癌药多柔比星诱发细胞DNA损伤的影响 ,并初步探讨其损伤机制。方法　用不同浓度菲口罗啉预处理CHL细胞 30min ,再分别加入 3种不同染毒受试物 ,共同培养一定时间 (0 .3mmol·L- 1重铬酸钾 :10 5min ;0 .5μmol·L- 1多柔比星 :75min ;0 .4mmol·L- 1过氧化氢(H2 O2 ) :2 5min)后 ,用碱性单细胞凝胶电泳方法 (AS CGE)测定DNA链断裂情况 ,并同时以菲口罗啉与二甲亚砜 (DMSO ,0 .33mol·L- 1)比较对H2 O2 致DNA损伤中·OH的产生和清除。结果　 3种染毒受试物均可明显引起CHL细胞DNA链断裂 ;而当 3μmol·L- 1菲口罗啉预处理后 ,可使重铬酸钾、H2 O2 所致DNA迁移长度和细胞拖尾率明显降低 ,并超过DMSO降低H2 O2 的损伤作用 ,当菲口罗林浓度升至 12 μmol·L- 1时 ,可完全消除这两种因素所致的DNA链断裂损伤 ;10 μmol·L- 1菲口罗啉可抑制多柔比星所致DNA损伤 ,但浓度直至 60 μmol·L- 1仍不能完全消除多柔比星的损伤作用。结论　菲口罗啉对 2种氧化剂和多柔比星所致DNA损伤均有不同程度的防护作用 ,同时提示重铬酸钾和H2 O2 所致的DNA损伤主要与需过渡金属离子参与的·OH产生有关 ,而多柔比星所致损伤仅部分与此有关     

DNA single strand breaks in peripheral blood lymphocytes induced by three nitroimidazole derivatives

Tinidazole (TNZ), ornidazole (ONZ) and metronidazole (MTZ) are antiparasitic drugs (nitroimidazole derivatives) that have proven to be effective against Trichomonas vaginalis, Entoamoeba histolytica, Giardia lamblia and Helicobacter pylori. The reduction of the nitro group and the generation of short-lived reactive intermediates are the basis of its parasiticidal activity. This reduction is associated with its mutagenic activity in bacteria, although in mammalian cells DNA damage seems to be related to the production of reactive oxygen species (ROS). Using alkaline single cell electrophoresis, a significant increase in single strand breaks and alkali labile sites in human peripheral blood lymphocytes (PBL) exposed to MTZ, ONZ and TNZ at 10, 100 and 500 microg/ml is observed. MTZ causes less damage, especially at higher concentrations, when compared with TNZ, the most harmful of the drugs tested. These findings suggest that primary damage is induced under aerobic conditions and confirms that these nitroimidazoles are DNA damaging agents.     

Photosensitization of DNA strand breaks by three phenothiazine derivatives

The interaction and the photosensitizing activity of three phenothiazine derivatives, fluphenazine hydrochloride (FP), thioridazine hydrochloride (TR), and perphenazine (PP), toward DNA were studied. Evidences obtained from various spectroscopic studies such as fluorimetric and linear dichroism measurements indicate that these derivatives bind to the DNA at least in two ways: intercalation and external stacking on the DNA helix, depending on their relative concentrations. Irradiation of supercoiled plasmid DNA in the presence of these phenothiazines leads to single strand breaks. The DNA photocleavage appears to be due to externally bound molecules rather than to those intercalated. The highest photocleavage activity was observed with PP and TR whereas FP was less efficient. The efficiency of the photocleavage in aerated and deaerated solutions does not change thus indicating that an involvement of singlet oxygen can be excluded. Primer extension analysis of plasmid DNA irradiated in the presence of phenothiazines indicates that photocleavage of DNA occurs predominantly at Gua and Cyt residues. Laser flash experiments carried out in the presence of 2'-deoxyguanosine 5'-monophosphate reveal an efficient electron transfer between the nucleotide and the radical cations produced by photoionization of the phenothiazines. In the presence of DNA, an electron transfer process takes place within the laser pulse from the lowest singlet state of phenothiazines to the DNA bases; the time-resolved measurements showed that the back-electron transfer is a negligible decay pathway for the charged species.     

Synergistic cytotoxicity and DNA strand breaks in cells and plasmid DNA exposed to uranyl acetate and ultraviolet radiation

Depleted uranium (DU) has a chemical toxicity that is independent of its radioactivity. The purpose of this study was to explore the photoactivation of uranyl ion by ultraviolet (UV) radiation as a chemical mechanism of uranium genotoxicity. The ability of UVB (302 nm) and UVA (368 nm) radiation to photoactivate uranyl ion to produce single strand breaks was measured in pBR322 plasmid DNA, and the presence of adducts and apurinic/apyrimidinic sites that could be converted to single strand breaks by heat and piperidine was analyzed. Results showed that DNA lesions in plasmid DNA exposed to UVB‐ or UVA‐activated DU were only slightly heat reactive, but were piperidine sensitive. The cytotoxicity of UVB‐activated uranyl ion was measured in repair‐proficient and repair‐deficient Chinese hamster ovary cells and human keratinocyte HaCaT cells. The cytotoxicity of co‐exposures of uranyl ion and UVB radiation was dependent on the order of exposure and was greater than co‐exposures of arsenite and UVB radiation. Uranyl ion and UVB radiation were synergistically cytotoxic in cells, and cells exposed to photoactivated DU required different DNA repair pathways than cells exposed to non‐photoactivated DU. This study contributes to our understanding of the DNA lesions formed by DU, as well as their repair. Results suggest that excitation of uranyl ion by UV radiation can provide a pathway for uranyl ion to be chemically genotoxic in populations with dermal exposures to uranium and UV radiation, which would make skin an overlooked target organ for uranium exposures. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of benzene on DNA strand breaks in vivo versus benzene metabolite-induced DNA strand breaks in vitro in mouse bone marrow cells

Previously, we identified p-benzoquinone (BQ) and 1,2,4-benzenetriol (BT) as toxic metabolites of benzene on the basis of their inhibitory effect on DNA synthesis. In the present study, the capability of benzene and the two metabolites to induce DNA strand breaks was investigated in either the in vivo or the in vitro system by comparing the DNA elution rate on a fine membrane filter at alkaline pH. In the in vitro system were bone marrow cells were reacted with test chemicals for 60 min, both BQ and BT induced a dose-related increase in alkali-labile DNA single-strand breaks (SSBs) of bone marrow cells. However, when glutathione (350 micrograms/ml) was added to the same reaction system, the DNA damaging effect of BQ (24 microM) and BT (24 microM) was blocked by 100 and 53%, respectively. Catalase (130 units/ml) completely blocked the DNA damaging effect of BT, while no protection was afforded with BQ. Consistent with these observations, no induction of alkali-labile DNA SSBs was observed in the in vivo system by an anesthetic dose of benzene (1760 mg/kg, ip or po) at 1, 24, and 36 hr postadministration in both male and female ICR mice. These results suggest that benzene exposure would not induce direct DNA strand breaks in vivo under realistic work-related or accidental exposure conditions and also indicate that caution should be exercised in the interpretation of in vitro data for whole-body toxicity evaluation.     

The preparation of poly (dT)-5'-transferrin conjugates and hybridisation studies with poly (dA)-tailed linearised pBR322 plasmid DNA

The formation of transferrin-DNA complexes intended for ligand-directed transfection studies has been achieved through a hybridisation technique involving complementary homodeoxypolynucleotide chains attached to the participating protein and DNA species. Oligothymidylate residues (pT)n obtained by dicyclohexylcarbodiimide (CDI) polymerisation of thymidine-5'-monophosphate (5'-TMP) were activated to the 5'-imidazolides which on incubation with transferrin yielded the 5'linked phosphoramidates (pT)n-5'-transferrin. Homopolymeric chain extension of (pT)5-5'-transferrin by terminal transferase and dTTP at 30 degrees for 30 min yielded (pT) 300-5'-transferrin. Cleavage of the phosphoramide link in the polymer modified transferrin at 37 degrees was pronounced after 30 min although at 25 degrees hydrolysis was less than 5% after 4 hr. Poly(dT)-5'-transferrin readily hybridised with [3H]poly(dA)-tailed Pst 1 linearised pBR322 DNA. Resultant complexes were demonstrated by nitrocellulose filter binding and immunoprecipitation with anti-transferrin antibody. In contrast with poly(dT)-5'-transferrin, poly(dT)-5'-transferrin-poly(dA)-tailed pBR322 DNA complexes were stable at 37 degrees suggesting that annealing is followed by further stabilising interactions between the DNA and protein components.     

Cell alterations but no DNA strand breaks induced in vitro by cylindrospermopsin in CHO K1 cells

Cylindrospermopsin has been involved in some cyanobacterial blooms associated with animal and human intoxications in different countries. Liver is the main target organ even though thymus and kidney are also affected. Its toxic effect has been shown to be induced by protein synthesis inhibition. However, further research about its toxicological potential is required, as revealed by the U.S. Unregulated Contaminant Monitoring Regulation (US UCMR) meeting in 2001. Induction of DNA damage by cylindrospermopsin has been reported by some authors either by a direct effect on DNA or by an indirect effect on associated macromolecules. This study focused on evaluating its in vitro genotoxic potential using the comet assay coupled to various cell alteration measurements. No DNA damage was detected by the alkaline comet assay on Chinese hamster ovary (CHO) K1 cells after 24 h of treatment with cylindrospermopsin concentrations of 0.5 and 1 microg mL(-1). However, inhibition of cell growth was noticed as well as cell blebbing and rounding. These morphological effects were linked to cytoskeletal reorganization (mainly microfilaments) but not to apoptosis. This study concluded that cylindrospermopsin does not obviously react directly with DNA in CHO K1 cells. But the hypothesis of its metabolization into a genotoxic product must be explored further.     

Cadmium/zinc-metallothionein induces DNA strand breaks in vitro

The in vitro DNA strand breaking activity of metallothionein (MT) containing Cd²⁺ and Zn²⁺ in a molar ratio of 52 is described. Studies with radical scavengers and electron paramagnetic resonance spectroscopy indicate that the DNA damage might be caused by a radical species formed by the native protein (i.e., MT) charged with the heavy metal ions. No DNA strand breaks are detectable with the heat-denatured MT or with Cd²⁺ or Zn²⁺ alone. Inhibition studies using EDTA as a metal ion chelator orN-ethylmaleimide to alkylate sulfhydryl groups suggest that both the bound heavy metal ions as well as the SH groups of the various cysteine residues of MT may be involved in the MT-dependent DNA cleavage. Further characterization showed that the DNA cleavage is more likely random than sequence- or base-specific. These observations may provide a clue in the search for initial events in Cd-related carcinogenicity.     

Application of alkaline unwinding to analysis of strand breaks induced by bleomycin in hamster lung DNA in vivo.

Strand breaks in hamster lung DNA were analyzed following in vivo exposure to bleomycin. An alkaline unwinding procedure involving separation of single-strand from double-strand DNA by hydroxylapatite chromatography followed by fluorescence detection of DNA with bisbenzimide was adapted for these studies. Procedures were developed that allowed preparation of DNA from lungs of control animals with a relatively low amount of single-strand DNA relative to double-strand DNA. Time dependence of unwinding was demonstrated using samples that were damaged deliberately by brief probe sonication. To verify that residual bleomycin remaining in lungs at the time of sacrifice did not cause strand breaks during sample preparation, bleomycin was added to minced lung in vitro. Under these conditions, the ratio of single-strand to double-strand DNA was not increased significantly. Substantial strand breaks were produced in vivo at 15 min and 1 h following intratracheal instillation of bleomycin into hamsters, as evidenced by a 5-6-fold increase in the ratio of single-strand to double-strand DNA relative to controls. The DNA damage appeared to be repaired within 1 day following exposure.     

Induction of DNA strand breaks by reduced nitroimidazoles. Implications for DNA base damage

Radiation-reduced 2-nitroimidazoles (misonidazole, RSU-1137, Ro.03-8799 and Ro.03-8800) incubated in air with plasmid DNA (pH 7.0, 310K) induce DNA strand breakage, as revealed following subsequent heat or alkali treatment. Only RSU-1137 resulted in the binding of a [2-14C] fragment and significant yields of heat-labile strand breaks (greater than 20% loss of type-I DNA after 48 hr incubation). RSU-1137 was shown to be greater than 6 times more effective than misonidazole at producing alkali-labile breaks. In fact, the efficiency of alkali-induced strand break production is in the order: misonidazole less than Ro.03-8799 approximately Ro.03-8800 less than RSU-1137. Reaction of these reduced 2-nitroimidazoles with 2'-deoxyguanosine (dG) also results in the formation of a common glyoxal-dG product, with its yield and rate of production being dependent upon the 2-nitroimidazole used. It has been demonstrated that these variations are influenced by the N-1 side-chain of the 2-nitroimidazole. Product yields are approximately 5-6 times greater with misonidazole than with RSU-1137. From the evidence presented, it is apparent that formation of glyoxal (or a glyoxal-like product) is not responsible for the DNA strand breakage seen. It is inferred that these breaks are induced by a nitro-reduction product(s) which remains unidentified.     

Detection of dichromate (VI)-induced DNA strand breaks and formation of paramagnetic chromium in multiple mouse organs

DNA single-strand breaks (and/or alkali-labile sites) induced by Cr(VI) were evaluated with the alkaline single cell gel electrophoresis (SCG) (Comet) assay in five organs (liver, kidney, spleen, lung, and brain) of male mice dosed with K(2)Cr(2)O(7) (20 mg Cr/kg) by a single ip injection in vivo, and the formation of paramagnetic Cr(V) in these organs was investigated by electron spin resonance (ESR) spectrometry. Furthermore, the in vivo effects of deferoxamine (DFO), an iron chelator, and dimethylthiourea (DMTU), a hydroxyl radical scavenger, on the formation of Cr(V) and DNA strand breaks induced by the metal in the liver and kidney were examined. SCG assay detected DNA strand breaks were detected in the liver and kidney at 15 min and showed that they were being repaired at 3 h after Cr(VI) injection. The ESR spectra of paramagnetic Cr(V) were also observed in the liver and kidney for 15 min to 24 h after Cr(VI) injection. In contrast, there were no significant levels of DNA strand breaks and Cr(V) in the spleen, lung, or brain. The pretreatment of mice with DFO reduced the formation of Cr(VI)-induced DNA strand breaks and Cr(V) complexes as well as the total contents of Cr in the liver and kidney at 15 min after the metal injection. In the case of the pretreatment with DMTU, DNA strand breaks induced by Cr(VI) were suppressed in the liver and kidney at 15 min, without any influence on the levels of Cr(V) complexes and total Cr contents in the organs. The in vitro study showed that DFO decreased the levels of Cr(V)-GSH complexes and Cr(V)-mediated hydroxyl radicals, while DMTU reduced only the levels of Cr(V)-mediated hydroxyl radicals without affecting the formation of Cr(V)-GSH complexes. These results demonstrated that the SCG assay may be useful for detecting DNA strand breaks and/or alkali-labile sites caused by Cr(VI) in vivo. The results also indicated that the in vivo formation of hydroxyl radicals during the reduction of Cr(VI) may play an important role in the induction of the DNA strand breaks caused by this metal and implied that the levels of Cr(V) inside the cells may not always be related to the induction of DNA strand breaks.     

Basal levels of DNA strand breaks in human leukocytes determined by comet assay

In order to determine background levels of DNA strand breaks, we examined 80 healthy individuals by comet assay considering age, sex, and smoking as confounding factors. Only age was found to have a significant effect on basal levels. One thousand cells of each donor were graded by eye into 5 categories according to the amount of DNA in the tail: classification group A (no damage) <5%, B (low damage) 5-20%, C (medium damage) 20-40%, D (high damage) 40-95%, and group E (total damage) >95%. The interpretation of the comet assay was modified to achieve a tail factor, which represents the DNA damage of 1000 scored cells as a single number, without the need of an image analysis software package. Hydrogen peroxide and bleomycin used for in vitro exposure of lymphocytes, produced clear dose-related responses in the comet assay. Our data encourage the application of the used classification model for a sensitive and fast quantification of DNA damage. Results in this study are in agreement with most of the earlier investigations.     

DNA strand breaks in embryonic mouse limbs exposed to methylnitrosourea

The alkaline elution assay was used to monitor DNA single strand breaks in hindlimbs of embryonic mice exposed to methylnitrosourea (MNU) on day 13 of gestation, a stage of development at which the embryotoxicity of MNU was minimal. DNA breakage was maximal by 1 h after exposure, and rapidly declined to control levels by 8 h. There was a reduction of fetal weights in the MNU-treated litters, but no increase in embryomortality. No malformations of the limbs were observed. The data are discussed in relation to an earlier study, where the same dose of MNU administered on day 11 of gestation was highly teratogenic and produced a substantially different pattern of DNA breakage in hindlimbs.     

DNA strand breaks photosensitized by benoxaprofen and other non steroidal antiinflammatory agents

Benoxaprofen, a non steroidal antiinflammatory drug is known to be highly phototoxic. Upon irradiation at 300 nm, benoxaprofen is shown to enhance the cleavage of phi X 174 DNA in buffered aqueous solution (pH 7.4). A linear relationship between the number of single strand breaks and the irradiation time is found. In deaerated solutions, these breaks are three times greater in the presence than in the absence of benoxaprofen. In both cases the rate of cleavage decreases in the presence of air. The rate of DNA damage increases with the drug per base pair ratio up to approximatively 0.2 and then decreases at higher ratios. Other NSAIDs, naproxen, ketoprofen, diflunisal, sulindac and indomethacin have been tested as photocleavers of DNA by using the same experimental conditions. A comparison of the efficiency of cleavage of all these drugs (including BNP) was obtained at drug concentrations such that the light absorbance was the same. Benoxaprofen, naproxen, ketoprofen and diflunisal induce single strand breaks. Sulindac and indomethacin do not cause breaks, and they can in some conditions even act as screening agents. The most efficient of the series are naproxen and ketoprofen. In the presence of oxygen, at the same concentrations as above, the efficiency of benoxaprofen, ketoprofen and diflunisal is decreased while that of naproxen is increased. This suggests that all these compounds do not interact with DNA by the same mechanism. In the case of BNP, the mechanism of photoinduced DNA cleavage is discussed in detail. It is shown that the photoactive agent is the decarboxylated derivative of benoxaprofen, as the photodecarboxylation of benoxaprofen is much faster than the photocleavage of DNA.