Structure-activity relationships imply different mechanisms of action for ochratoxin A-mediated cytotoxicity and genotoxicity

Ochratoxin A (OTA) is a fungal toxin that is classified as a possible human carcinogen based on sufficient evidence for carcinogenicity in animal studies. The toxin is known to promote oxidative DNA damage through production of reactive oxygen species (ROS). The toxin also generates covalent DNA adducts, and it has been difficult to separate the biological effects caused by DNA adduction from that of ROS generation. In the current study, we have derived structure-activity relationships (SAR) for the role of the C5 substituent of OTA (C5-X = Cl) by first comparing the ability of OTA, OTBr (C5-X = Br), OTB (C5-X = H), and OTHQ (C5-X = OH) to photochemically react with GSH and 2'-deoxyguanosine (dG). OTA, OTBr, and OTHQ react covalently with GSH and dG following photoirradiation, while the nonchlorinated OTB does not react photochemically with GSH and dG. These findings correlate with their ability to generate covalent DNA adducts (direct genotoxicity) in human bronchial epithelial cells (WI26) and human kidney (HK2) cells, as evidenced by the (32)P-postlabeling technique. OTB lacks direct genotoxicity, while OTA, OTBr, and OTHQ act as direct genotoxins. In contrast, their cytotoxicity in opossum kidney epithelial cells (OK) and WI26 cells did not show a correlation with photoreactivity. In OK and WI26 cells, OTA, OTBr, and OTB are cytotoxic, while the hydroquinone OTHQ failed to exhibit cytotoxicity. Overall, our data show that the C5-Cl atom of OTA is critical for direct genotoxicity but plays a lesser role in OTA-mediated cytotoxicity. These SARs suggest different mechanisms of action (MOA) for OTA genotoxicity and cytotoxicity and are consistent with recent findings showing OTA mutagenicity to stem from direct genotoxicity, while cytotoxicity is derived from oxidative DNA damage.     

Evidence for covalent DNA adduction by ochratoxin A following chronic exposure to rat and subacute exposure to pig

Ochratoxin A (OTA) is a nephrotoxic mycotoxin that is a potent renal carcinogen in male rats and is suspected of being the etiological agent of Balkan endemic nephropathy (BEN) and its associated urinary tract cancers. Conflicting results have been obtained regarding the genotoxicity of OTA and its ability to react directly with DNA upon oxidative bioactivation to yield covalent DNA adducts. To characterize DNA adduction by OTA, the present study utilizes the photooxidative properties of the toxin to generate authentic C8 OTA-3'-monophosphate-deoxyguanosine (3'-dGMP) adducts for use as cochromatographic standards for (32)P-postlabeling detection of OTA-mediated DNA adduction in the kidney of rat and pig. Our results show evidence for the photooxidation of OTA to yield carbon (C)- and oxygen (O)-bonded C8-3'-dGMP adducts (C-C8 and O-C8) that have been isolated and characterized by LC/MS with in-line UV and electrospray negative ionization (ES(-)) detection. A comparison to previously published work on related C8-dG adducts supports C8 attachment by OTA. The C-C8 OTA-3'-dGMP adduct standard is shown by (32)P-postlabeling to comigrate with the major lesion detected in the kidney of rat following chronic exposure to OTA and with one of four adducts detected in the kidney of pig following subacute exposure to the toxin. The O-C8 OTA-3'-dGMP adduct standard is also shown to coelute with a lesion detected in rat kidney. These findings suggest a role for the OTA phenoxyl radical in OTA-mediated DNA adduction in vivo, provide a rationale for the tumorigenesis of OTA, and strengthen the OTA hypothesis in the etiology of BEN and the associated urinary tract tumors.     

Ochratoxin A: lack of formation of covalent DNA adducts

The mycotoxin ochratoxin A (OTA) is a potent nephrotoxin and renal carcinogen in rodents. However, the mechanism of OTA-induced tumor formation is unknown and conflicting results have been obtained regarding the potential of OTA to bind to DNA. OTA is poorly metabolized, and no reactive intermediates capable of interacting with DNA have been detected in vitro or in vivo. Recently, a hydroquinone/quinone redox couple and a carbon-bonded OTA-deoxyguanosine (OTA-dG) adduct formed by electrochemical oxidation and photoreaction of OTA have been reported and suggested to be involved in OTA carcinogenicity. This study was designed to characterize the role of DNA binding and to determine if formation of these derivatives occurs in vivo and in relevant activation systems in vitro using specific and sensitive methods. Horseradish peroxidase activation of OTA and its dechlorinated analogue ochratoxin B (OTB) yielded ochratoxin A-hydroquinone (OTHQ), but the postulated OTA-dG adduct was not detectable using LC-MS/MS. In support of this, no OTA-related DNA adducts were observed by 32P-postlabeling. In vivo, only traces of OTHQ were found in the urine of male F344 rats treated with high doses of OTA (2 mg/kg body wt) for 2 weeks, suggesting that this metabolite is not formed to a relevant extent. In agreement with the in vitro data, OTA-dG was not detected by LC-MS/MS in liver and kidney DNA extracted from treated animals. In addition, DNA binding of OTA and OTB was assessed in male rats given a single dose of 14C-OTA or 14C-OTB using accelerator mass spectrometry, a highly sensitive method for quantifying extremely low concentrations of radiocarbon. The 14C content in liver and kidney DNA from treated animals was not significantly different from controls, indicating that OTA does not form covalent DNA adducts in high yields. In summary, the results presented here demonstrate that DNA binding of OTA is not detectable with sensitive analytical methods and is unlikely to represent a mechanism for OTA-induced tumor formation.     

Ochratoxin A: In Utero Exposure in Mice Induces Adducts in Testicular DNA

Ochratoxin A (OTA) is a nephrotoxin and carcinogen that is associated with Balkan endemic nephropathy and urinary tract tumors. OTA crosses the placenta and causes adducts in the liver and kidney DNA of newborns. Because the testis and kidney develop from the same embryonic tissue, we reasoned that OTA also may cause adducts transplacentally in the testis. We tested the hypothesis that acute exposure to OTA, via food and via exposure in utero, causes adducts in testicular DNA and that these lesions are identical to those that can be produced in the kidney and testis by the consumption of OTA. Adult mice received a single dose of OTA (from 0–1,056 µg/kg) by gavage. Pregnant mice received a single i.p. injection of OTA (2.5 mg/kg) at gestation day 17. DNA adducts were determined by ³²P-postlabeling. Gavage-fed animals sacrificed after 48 hours accumulated OTA in kidney and testis and showed DNA adducts in kidney and testis. Some OTA metabolites isolated from the tissues were similar in both organs (kidney and testis). The litters of mice exposed prenatally to OTA showed no signs of overt toxicity. However, newborn and 1-month old males had DNA adducts in kidney and testis that were chromatographically similar to DNA adducts observed in the kidney and testis of gavage-fed adults. One adduct was identified previously as C8-dG-OTA adduct by LC MS/MS. No adducts were observed in males from dams not exposed to OTA. Our findings that in utero exposure to OTA causes adducts in the testicular DNA of male offspring support a possible role for OTA in testicular cancer.     

Genotoxicity of the hydroquinone metabolite of ochratoxin A: structure-activity relationships for covalent DNA adduction

Ochratoxin A (OTA) is a mycotoxin that shows potent nephrotoxicity and renal carcinogenicity in rodents. One hypothesis for OTA-induced tumor formation is based on its genotoxic properties that are promoted by oxidative metabolism. Like other chlorinated phenols, OTA undergoes an oxidative dechlorination process to generate a quinone (OTQ)/hydroquinone (OTHQ) redox couple that may play a role in OTA-mediated genotoxicity. To determine whether the OTQ/OTHQ redox couple of OTA contributes to genotoxicity, the DNA adduction properties, as evidenced by the (32)P-postlabeling technique, of the hydroquinone analogue (OTHQ) have been compared to OTA in the absence and presence of metabolic activation (pig kidney microsomes) and within human bronchial epithelial (WI26) and human kidney (HK2) cells. Our experiments show that OTHQ generates DNA adduct spots in the absence of metabolic activation. These adducts are ascribed to covalent DNA adduction by OTQ generated through autoxidation of the hydroquinone precursor, OTHQ. Although OTA does not interact with DNA in the absence of metabolism, the OTQ-mediated DNA adduct spots noted with OTHQ are also observed with OTA following treatment with pig kidney microsomes and NADPH, suggesting that OTA undergoes oxidative activation to OTQ by cytochrome P450 or enzymes with peroxidase activity. Comparison of DNA adduction by OTHQ and OTA in human cell lines shows that OTQ-mediated adduct spots form in a dose- and time-dependent manner. The adduct spots form at a faster rate with OTHQ, which is consistent with more facile generation of OTQ from its hydroquinone precursor. These results establish structure-activity relationships for OTA-mediated DNA adduction and provide new evidence for the potential role of the OTQ/OTHQ redox couple in OTA-induced genotoxicity.     

In vitro cytogenetic results supporting a DNA nonreactive mechanism for ochratoxin A, potentially relevant for its carcinogenicity

Ochratoxin A (OTA) is a widespread mycotoxin of cereals and many agricultural products and causes high incidences of renal tumors in rodents. Although its carcinogenic properties have been known since the eighties, the precise mechanism of action is still relatively undefined. At present, increasing evidence suggests that OTA does not act with a direct genotoxic mechanism, opposed to other previous evidence where the formation of DNA adducts by 32P-postlabeling was observed. The genotoxic activity of OTA assessed in a variety of in vitro and in vivo studies was very low if genotoxic at all. In this study, we clearly show that OTA does not bear any clastogenic or aneugenic activity based on the absence of the induction of chromosome aberrations, sister chromatid exchanges, and micronuclei in human lymphocytes and V79 cells in vitro in both the absence and the presence of S9 metabolism. Alternatively, cytogenetic analyses evidenced significant increases in endoreduplicated cells and highly condensed metaphases with separated chromatids. This implies that OTA or its possible metabolites do not covalently bind DNA through the formation of adducts since structural chromosome aberrations are a very sensitive end points to detect chemical carcinogens with electrophilic substituents. Alternatively, induction of endoreduplication and chromatid separation provides strong evidence for a DNA nonreactive mechanism of OTA carcinogenicity involving the disruption of mitosis by interfering with key regulators of chromosome separation and progression of mitosis. This causes a temporary arrest of mitoses and premature exit from it (mitotic slippage) to generate endoreduplication and polyploidy accompanied by increased risk of aneuploidy and subsequent tumor formation.     

Metabolism of ochratoxin A: absence of formation of genotoxic derivatives by human and rat enzymes

Ochratoxin A (OTA) is a potent renal carcinogen in male rats, although its mode of carcinogenicity is not known. The metabolism and covalent binding of OTA to DNA were investigated in vitro with cytochromes P450, glutathione S-transferases, prostaglandin H-synthase, and horseradish peroxidase. Incubation of OTA with rat or human liver microsomes fortified with NADPH resulted in formation of 4-(R)-hydroxyochratoxin A at low rates [10-25 pmol min(-1) (mg of protein)(-1)]. There was no evidence of OTA metabolism and glutathione conjugate formation with rat, mouse, or human kidney microsomes or postmitochondrial supernatants (S-9) [<5 pmol min(-1) (mg of protein)(-1)]. Recombinant human cytochromes P450 (P450) 1A1 and 3A4 formed 4-(R)-hydroxyochratoxin A at low rates [0.08 and 0.06 pmol min(-1) (pmol of P450)(-1), respectively]; no oxidation products of OTA were detected with recombinant human P450 1A2 or 2E1 or rat P450 1A2 or 2C11 [<0.02 pmol min(-1) (pmol of P450)(-1)]. Prostaglandin H-synthase produced small amounts of an apolar product [33 pmol min(-1) (mg of protein)(-1)], and OTA products were not formed with horseradish peroxidase. There was no evidence of DNA adduct formation when [(3)H]OTA was incubated with these enzyme systems in the presence of calf thymus DNA (<20 adducts/10(9) DNA bases); however, these enzymes catalyzed DNA adduct formation with the genotoxins aflatoxin B(1), 2-amino-3-methylimidazo[4,5-f]quinoline, benzo[a]pyrene, and pentachlorophenol. There was also no detectable [(3)H]OTA bound in vivo to kidney DNA of male Fischer-344 rats treated orally with [(3)H]OTA (1 mg/kg, 100 mCi/mmol, 24 h exposure, <2.7 adducts/10(9) DNA bases), based upon liquid scintillation counting. However, (32)P-postlabeling experiments did show evidence of DNA lesions with total adduct levels ranging from 31 to 71 adducts/10(9) DNA bases, while adducts in untreated rat kidney ranged from 6 to 24 adducts/10(9) DNA bases. These results do not support the premise that OTA or metabolically activated species covalently bind to DNA and suggest that the (32)P-postlabeled lesions are due to products derived from OTA-mediated cytotoxicity.     

8-Oxoguanine DNA glycosylase and MutY homolog are involved in the incision of arsenite-induced DNA adducts.

Since arsenite is known to induce oxidative DNA damage in human cells, we asked if it induces other types of DNA damage and how the DNA damage is repaired. Treatment of human promyelocytic leukemia NB4 cells with 0.5muM As(2)O(3) for 30 min induced no DNA breaks, as analyzed by a standard comet assay. However, breaks were detected if these cells were then digested with endonuclease III (EnIII), formamidopyrimidine-DNA glycosylase (Fpg), or a nuclear extract (NE) of NB4 cells. Using either H(2)O(2)-Fe-treated nuclei or As(2)O(3)-treated cells, digestion with either NE or EnIII + Fpg generated the same amount of breaks, and subsequent treatment with EnIII + Fpg resulted in no increase in breaks in NE-digested cells and vice versa. The human cell lines, defective in nucleotide excision protein, such as xeroderma pigmentosum (XP) A, XPD, and XPG, excised Ultraviolet C-induced adducts less rapidly than normal fibroblasts, but excised As(2)O(3) adducts at the same rate as the normal cells. Immunodepletion of the NE with antibody against 8-oxoguanine DNA glycosylase (OGG1) or MutY homolog (MYH) decreased the incision of As(2)O(3)-induced adducts, while antibodies against XPA, XPB, XPD, XPF, or XPG, did not. These results suggest that As(2)O(3) induces the formation of only oxidative DNA adducts and that OGG1 and MYH are involved in this incision process.     

Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2.

Microcystins are naturally occurring hepatotoxins produced by strains of Microcystis aeruginosa. They are involved in promoting primary liver tumours and a previous study showed that they might also be tumour initiators. In this study we demonstrate that microcystin-LR (MCLR) at doses that were not cytotoxic (0.01-1 microg/ml), induced dose and time dependent DNA strand breaks in human hepatoma cell line HepG2. These DNA strand breaks were transient, reaching a maximum level after 4h of exposure and declining with further exposure. In the presence of the DNA repair inhibitors cytosine arabinoside (AraC) and hydroxyurea (HU), together with MCLR, DNA strand breaks accumulated after prolonged exposure. These results suggest that DNA strand breaks are intermediates, produced during the cellular repair of MCLR induced DNA damage. Digestion of DNA with purified, oxidative DNA damage specific enyzmes, endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg) markedly increased DNA strand breaks in MCLR treated cells, providing evidence that a substantial portion of the MCLR induced DNA strand breaks originate from excision of oxidative DNA adducts. A hydroxyl radical scavenger (DMSO) significantly reduced MCLR induced DNA damage. From these results we conclude that MCLR induces formation of reactive oxygen species that cause DNA damage, and that MCLR may act as an initiator of liver cancer.     

DNA damage associated with PCBs in the whole blood cells of Inuit

Lower chlorinated PCBs can damage DNA directly or via free radical mechanisms. In order to assess the DNA-damaging potential of PCBs in humans, blood samples were collected from Inuit population from Salluit, Northern Canada. Their diet comprises blubber from sea mammals and fatty fish, which accumulate non-biodegradable PCBs at varying levels. The 103 samples thus collected were categorized into low-, medium- and high-PCB exposure groups. A comprehensive (32)P-postlabeling adductomics technology, which allows measure differences in DNA adduct profiles of polar and lipophilic adducts between control and exposure groups, was applied to these samples to assess the effect of PCB on DNA damage. The adduct patterns obtained were qualitatively similar to other human tissues studied previously. A range of highly polar to lipophilic subgroups of adducts were detected. The known oxidative lesion, 8-oxodG was predominant. While some individual adducts appear to accumulate with increasing PCB levels, a definitive association could not be made. A possible confounder effect of selenium is discussed.     

Horseradish peroxidase mediates DNA and deoxyguanosine 3'-monophosphate adduct formation in the presence of ochratoxin A

Ochratoxin A (OTA) gives rise to DNA and deoxyguanosine-3'-monophosphate (dGMP) adducts in vitro using mice kidney microsomes in the presence of arachidonic acid. This result points to the involvement of prostaglandin H synthases, which are present at high levels in the kidney, urinary bladder and seminal vesicles, and/or of lipoxygenases in the metabolic activation of OTA to genotoxic compounds. These enzymes have peroxidase activities. Incubation of OTA with DNA in the presence of horseradish peroxidase (HRP) and cumene hydroperoxide at pH 7.4 led to the formation of one major and three minor adducts with a total adduct level of 42 per 10(9) nucleotides. Incubation with dGMP gave a total adduct level of 159 per 10(9) nucleotides. In the presence of H2O2 instead of cumene hydroperoxide, a lower level of adducts was obtained, both with DNA and dGMP. The concentrations of HRP and co-substrate used in this paper were higher than those used by other authors who obtained negative results when they sought DNA adducts of OTA in the presence of HRP and H2O2. The main adduct we obtained with DNA incubated with HRP and OTA had the same chromatographic behaviour as that obtained when DNA or dGMP were incubated with OTA, arachidonic acid and mice kidney microsomes. However, the main adduct obtained with dGMP incubated with HRP and OTA behaved differently. These results show that OTA can be metabolized by a peroxidase to metabolically activated species that bind covalently to DNA and dGMP; however, the main adduct obtained in vitro with HRP and dGMP cannot serve as a model for one of the adducts formed by OTA with DNA because it behaves differently in two chromatographic systems.     

Antiestrogens and the formation of DNA damage in rats: a comparison

Tamoxifen (TAM) has been used as an agent for the treatment and prevention of breast cancer. However, long-term treatment of TAM in women increases the risk of developing endometrial cancer. The secondary cancer may be due to the genotoxicity of TAM. To find safer alternatives, four selective estrogen receptor modulators (SERMs), 4-hydroxytamoxifen (4-OHTAM), toremifene (TOR), raloxifene (RAL), and ICI 182,780, were administered to rats with an equimolar dose of TAM [54 micromol/kg (20 mg/kg)/day, p.o. for 7 days]. To evaluate the genotoxicity of each SERM, the presence of bulky DNA adducts was determined by (32)P-postlabeling/polyacrylamide gel electrophoresis and (32)P-postlabeling/high-performance liquid chromatography. The formation of 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) was analyzed as a marker of typical oxidative damage, using liquid chromatography electrospray tandem mass spectrometry. Among the SERMs, bulky DNA adducts were detected in the livers of rats treated with TAM; the total amount of TAM-DNA adducts was 26.1 adducts/10(7) nucleotides. However, with a detection limit of approximately 2 adducts/10(9) nucleotides, no bulky DNA adducts were observed with 4-OHTAM, TOR, RAL, or ICI 182,780. In addition, no significant increase of hepatic 8-oxodG lesions was detected in rats treated with any of the antiestrogens. Therefore, TOR, RAL, and ICI 182,780 are likely to be less genotoxic than TAM.     

Protection by indomethacin and aspirin against genotoxicity of ochratoxin A,particularly in the urinary bladder and kidney

Ochratoxin A (OTA) is a ubiquitous nephrotoxic mycotoxin which was shown to be carcinogenic to laboratory animals and may be responsible for kidney pelvis, ureter and urinary bladder tumors associated with Balkan endemic nephropathy in man. Previous evidence from this laboratory demonstrated that OTA exposure results in adduct formation on kidney, testicles, liver and spleen DNA. We show in this study that after a single oral administration of OTA to mice (2 mg/kg body weight) a high level of DNA adducts (150 per 10⁹ nucleotides) is also detected in the urinary bladder. The metabolic pathway of OTA leading to genotoxic compounds is not yet known. We demonstrate here that two inhibitors of the prostaglandin H synthase, indomethacin and aspirin, administered to mice before OTA treatment, dramatically reduce the amounts of DNA adducts, particularly in the urinary bladder and kidney. This suggests a role of protaglandin H synthase in the metabolism of OTA leading to active metabolites which react with DNA.     

Bioenergetics and DNA alteration of normal human fibroblasts by hexavalent chromium

The effects of hexavalent chromium on mitochondria of normal human fibroblasts were investigated through the measurement of oxygen consumption, and its genotoxic effect through the analysis of chromium DNA adducts and oxidative DNA lesions. ROS production was also quantified. Chromium diminished oxygen consumption by cells in a concentration-dependent manner (IC(50)=66±8μM). This effect can be attributed to an alteration in mitochondrial functions, leading to defective glucose catabolism. The Comet assay, performed with and without the lesion-specific enzyme formamidopyrimidine-DNA glycosylase (Fpg), highlighted the extent of oxidative DNA base damage. DNA base damage was induced with low concentrations (0.5-3μM) of Cr(VI), whereas bioenergetic disturbance was only observed at higher concentrations (20-500μM).     

The protective role of ferulic acid on sepsis-induced oxidative damage in Wistar albino rats

Oxidative stress has an important role in the development of sepsis-induced multiorgan failure. Ferulic acid (FA), a well-established natural antioxidant, has several pharmacological activities including anti-inflammatory, anticancer and hepatoprotective. This study aimed to investigate the effects of FA on sepsis-induced oxidative damage in Wistar albino rats. Sepsis-induced DNA damage in the lymphocytes, liver and kidney cells of rats were evaluated by comet assay with and without formamidopyrimidine DNA glycosylase (Fpg). The oxidative stress parameters such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and total glutathione (GSH) and malondialdehyde (MDA) levels were also measured. It is found that DNA damage in sepsis + FA-treated group was significantly lower than the sepsis group. FA treatment also decreased the MDA levels and increased the GSH levels and SOD and GSH-Px activities in the sepsis-induced rats. It seems that FA might have ameliorative effects against sepsis-induced oxidative damage.     

Lung DNA adducts detected in human smokers are unrelated to typical polyaromatic carcinogens

Several studies have reported the presence of DNA adducts derived from benzo(a)pyrene and other polyaromatics by 32P-postlabeling/TLC by measuring diagonal radioactive zones (DRZs) in lung tissues of human smokers. However, our experimental studies in rodent models, which used modified chromatographic conditions to obtain distinct adduct spots, suggested that cigarette smoke-related lipophilic DNA adducts may not be derived from polycyclic aromatic hydrocarbons (PAHs) or aromatic amines. In the present study, we have performed similar analysis of human lung tissues to study the chemical nature of DNA adducts. Fifty human lung tissues from cancer patients (ages 42-83 years) with active, ex-, or never-smoking status were analyzed for highly lipophilic DNA adducts by nuclease P1- and n-butanol enrichment-mediated 32P-postlabeling assay. All DNA samples yielded low to highly intense adduct DRZs when adducts were resolved by PEI-cellulose TLC in standard high-salt, high-urea solvents. Adduct burden ranged from 6.6 to 2930 per 10(10) nucleotides. However, when adducts were resolved in a different solvent system comprising of high-salt, high-urea in direction 3 and dilute ammonium hydroxide in direction 4, which retained adducts derived from PAHs and aromatic amines on the chromatograms, this yielded no detectable adducts from human lung DNAs. Furthermore, analysis of human lung DNAs mixed with reference adducted DNAs in multisolvent systems confirmed an absence of PAH- and aromatic amine-derived adducts in human smoker lung DNA. To determine the origin of cigarette smoke-associated DNA adducts, calf thymus DNA was incubated with formaldehyde and acetaldehyde, which are known to be present in cigarette smoke in significant quantities. Analysis of purified DNAs by 32P-postlabeling resulted in adduct DRZs in the aldehyde-modified DNAs when adducts were resolved in standard urea-containing solvents, but no adducts were detected when the ammonium hydroxide-based solvent was used, suggesting that even nonpolyaromatic electrophiles can result in adduct DRZs on the chromatograms similar to those from PAH metabolites. Taken together, our data demonstrate that cigarette smoke-associated lung DNA adducts appear on chromatograms as DRZs, consistent with the literature, but they are not related to PAHs and aromatic amines.     

Formation and persistence of DNA adducts of anticancer drug ellipticine in rats

Ellipticine is an antineoplastic agent, whose mode of antitumor and/or toxic side effects is based on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochromes P450 and peroxidases. We investigated the formation and persistence of DNA adducts generated in rat, the animal model mimicking the bioactivation of ellipticine in human. Using (32)P-postlabeling, ellipticine-DNA adducts were found in liver, kidney, lung, spleen, heart and brain of female and male rats exposed to ellipticine (4, 40 and 80 mg/kg body weight, i.p.). The two major adducts were identical to the deoxyguanosine adducts generated in DNA by 13-hydroxy- and 12-hydroxyellipticine in vitro as confirmed by HPLC of the isolated adducts. At four post-treatment times (2 days, 2, 10 and 32 weeks) DNA adducts in rats treated with 80 mg/kg of ellipticine were analyzed in each tissue to study their long-term persistence. In all organs maximal adduct levels were found 2 days after administration. At all time points highest total adduct levels were in liver (402 adducts/10(8) nucleotides after 2 days and 3.6 adducts/10(8) nucleotides after 32 weeks), kidney and lung followed by spleen, heart and brain. Total adduct levels decreased over time to 0.8-8.3% of the initial levels till the latest time point and showed a biphasic profile, a rapid loss during the first 2 weeks was followed by a much slower decline till 32 weeks. These results, the first characterization of persistence of ellipticine-DNA adducts in vivo, are necessary to evaluate genotoxic side effects of ellipticine.     

DNA adducts in carp exposed to artificial diesel-2 oil slicks

In attempts to mimic field exposure, oil slicks prepared from diesel-2 oil/water emulsions were poured onto the surface of water in tanks prepared fresh every day and liver DNA adducts were analyzed by ³²P-postlabeling in carp free-swimming in these tanks. ‘Clusters’ of lipophilic DNA adducts were detected, with five major and numerus minor adducts. Essentially a similar adduct pattern was found in the liver DNA of carp exposed to crude oil-polluted water. Diesel-2 adduct induction was observed slowly with a steady increase to > 3000 amol/μg DNA at day 12. After this time fish were transferred to clean water. Adduct levels continued to increase through day 17 (≈ 10,000 amol/μg DNA) despite the cessation of exposure, but a 30% and 80% decline was evident at day 22 and day 27, respectively. All major adducts were distinct from the known benzo[a]pyrene diolepoxide-dG. These results indicate that diesel-2 oil can cause extensive DNA damage in carp in vivo and the damage accumulates proportionately with time of exposure.     

Protective effect of vitamin C towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay.

The aim of this study was to investigate the protective effect of vitamin C towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay. None of the vitamin C concentrations tested (1-10 microM) in presence or absence of formamidopyrimidine-DNA glycosylase (Fpg enzyme) caused DNA damage per se. HepG2 cells simultaneously treated with vitamin C and N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), N-nitrosodibutylamine (NDBA) or N-nitrosopiperidine (NPIP) reduced the genotoxic effects of the N-nitrosamines in a dose-dependent manner. At concentrations of 1-5 microM vitamin C, the protective effect was higher towards NPYR-induced oxidative DNA damage (78-79%) than against NDMA (39-55%), NDBA (12-14%) and NPIP (3-55%), in presence of Fpg enzyme. However, a concentration of 10 microM vitamin C led to a maximum reduction in NDBA (94%), NPYR (81%), NPIP (80%) and NDMA (61%)-induced oxidative DNA damage, in presence of Fpg enzyme. The greatest protective effect of vitamin C (10 microM) was higher towards NDBA-induced oxidative DNA damage. One feasible mechanism by which vitamin C exerted its protective effect is that may interact with the enzyme systems catalyzing the metabolic activation of the N-nitrosamines, blocking the production of genotoxic intermediates. Vitamin C (10 microM) strongly reduced the coumarin hydroxylase (82%) activity. However, the p-nitrophenol hydroxylase and the ethoxyresorufine O-deethylation activities were slightly and weakly reduced (32-19%), respectively.