Genotoxicity evaluation of a commonly used synthetic androgen, Oxymetholone, was carried out in human peripheral blood lymphocytes in vitro. Sister chromatid exchange (SCE) was used as genetic end point. The concentrations of the drug were determined after observing its effects on the mitotic index. A wide range of concentrations, i.e., 25, 50, and 100 micro g/ml of the drug, were used to determine the genotoxic effects in the absence as well as in the presence of rat liver microsomal activation system (S9 mix). The drug did not induce any significant increase in the SCE frequency at any of the concentrations either in the presence or in the absence of S9 mix. The maximum value of SCE was observed in the absence of S9 mix at 100 microg/ml concentration (i.e., 1.38+/-0.080/cell), which was not significant statistically. Moreover, the drug was not effective in increasing the SCE frequency even in the presence of S9 mix. The maximum value of SCE (i.e., 1.78+/-0.103/cell) was observed at 50 microg/ml of concentration in the presence of S9 mix. A dose relationship was also not observed. It was concluded that Oxymetholone does not affect the genetic material in human lymphocytes at a wide range of concentrations in the SCE assay.
Inadequate doses or prolonged chemotherapy can be cytotoxic or genotoxic to cancer patients, increasing the risk for the development of a second cancer, particularly acute leukemia. The association between therapeutic and genotoxic properties of oncocalyxone A (Onco A), make cytotoxic tests (mitotic index and chromosomal aberrations) fundamental in the accompaniment of the effects of this active compound. Therefore, the aim of the present study was to determine the genotoxic action of Onco A in vitro, during different phases of the cell cycle, utilizing primary cultures of lymphocytes of healthy individuals. The results showed that Onco A is cytotoxic during the cell cycle phases G1, G1/S, and S, however, not in G2. Onco A did not demonstrate a genotoxic effect in any of the cell cycle phases at the concentration studied. It is concluded that during the period of exposure, this active substance inhibits DNA synthesis and consequently cell division. Therefore, the absence of such genotoxicity for Onco A in the tests performed in this study provides important information in regard to the therapeutic use of this agent. Further studies are necessary to better understand the molecular mechanism of action of Onco A.
The year 2001 witnessed the sequencing of 90% of the euchromatic region in the human genome but the ultimate goal to delineate the positions of all genes is yet to be achieved. Fluorescence In Situ Hybridization (FISH) is one of the methods for localizing genes on chromosomes. In the present study, diagnostic utility of single-, dual-, and multicolor FISH was evaluated for prenatal diagnosis, cancer genetics, and screening of various congenital anomalies (sex chromosomal and autosomal). Centromeric probes for chromosomes X and Y were used for screening minor aneuploid cell lines (XXY, XO, and XXX) in the cases of primary amenorrhea and suspected Klinefelter syndrome. The cases with ambiguous genitalia were analyzed using a probe specific for the sex-determining region (SRY). Suspected cases of Down syndrome were subjected to FISH using probe specific for chromosome 21. FISH was also used to study gene alterations in retinoblastoma and myeloid leukemias. Prenatal diagnosis was done to screen for aneuploidies of chromosomes 13, 18, 21, X, and Y using FISH on uncultured cells from amniotic fluid and chorionic villi sampling. The screening for common aneuploidies was extended to abortuses from spontaneous abortions. Using FISH, low-level mosaicism could be identified in some cases of primary amenorrhea and suspected Klinefelter syndrome. Submicroscopic gene rearrangements could be detected using FISH in cases of ambiguous genitalia and cancers. Further interphase FISH could provide results within 24 hours. To conclude, FISH adds to the diagnostic utility of routine cytogenetics and its use on interphase nuclei overcomes the difficulty of conventional cytogenetics, thereby reducing the time between sampling and diagnosis to 24 hr. (C) 2003 Wiley-Liss, Inc.
Selenium is an environmental metal that occurs ubiquitously and is produced throughout the world for various industrial activities. Selenium has been reported to have anticarcinogenic and preventive effects in clinical and epidemiological studies. Selenium supplements can inhibit chemically-induced tumours. From the viewpoint of genotoxicity, selenium has not been adequately studied and an IARC review concluded that there were not sufficient data to consider it a carcinogen for man. In contrast, hexavalent chromium is classified as a known respiratory carcinogen producing DNA damage through free oxygen radicals. In the present study, a collaborative study has been carried out to evaluate the genotoxicity of selenium compounds and their possible interactions with potassium dichromate and hydrogen peroxide. Thus, in laboratory 1 (U.K.), the genotoxic effects of three selenium compounds were examined. Sodium selenate, sodium selenite, and selenous acid were investigated in the Ames test using strain TA102 and in the Comet assay using human lymphocytes, and also investigated for their interaction with potassium dichromate. In the Ames test, it was shown that potassium dichromate produced a highly mutagenic response, whilst the three selenium compounds did not. In combination, sodium selenate reduced the genotoxicity of potassium dichromate, but sodium selenite and selenous acid had no effect. In the Comet assay, potassium dichromate induced DNA damage, but so did the selenium compounds. In combination with potassium dichromate, however, only sodium selenate reduced its effect, whereas sodium selenite and selenous acid exacerbated DNA damage. In laboratory 2 (Spain), in the TK6 lymphoblastoid cell line, the Comet assay showed that sodium selenite was non-genotoxic, while potassium dichromate and hydrogen peroxide induced DNA damage. It was also shown that sodium selenite did not decrease the genotoxicity of potassium dichromate or hydrogen peroxide when administered as a pre-treatment or at the same time, or when potassium dichromate and sodium selenite treatments were for different time periods. Thus, only sodium selenate has shown antigenotoxic properties against potassium dichromate in the Ames test and in human lymphocytes in the Comet assay.
The human diet contains a variety of compounds that exhibit chemopreventive effects towards an array of xenobiotics. In the present study, the antigenotoxic potential of selected dietary constituents including Diallyl sulfide (DAS), Indole-3-carbinol (I3C), Curcumin (CUR), and Black tea polyphenols (BTP) has been evaluated in the Salmonella typhimurium reverse mutation and mammalian in vivo cytogenetic assays. In addition, the anticlastogenic effect of the above dietary constituents was identified towards Benzo(a)pyrene (BaP) and cyclophosphamide- (CP) induced cytogenetic damage in mouse bone marrow cells. The induction of BaP and CP induced chromosomal aberrations, micronuclei formation, and sister chromatid exchanges (SCEs) were found to be inhibited in a dose-dependent manner by DAS, I3C, CUR, and BTP. Thus the study reveals the antimutagenic potential of these dietary compounds towards BaP- and CP-induced genotoxicity in microbial and mammalian test systems.
Microsatellite instability (MSI) is an indicator of a defective DNA mismatch repair system (MMR) that results from somatic mutations. The present work has been planned to investigate MSI and its clinical significance in human urinary bladder and thyroid cancers in Indian patients. Tumor tissues of histologically confirmed cases of urinary bladder and thyroid cancers, respectively, were obtained. Clinical data on tumor stage and histopathological grades were recorded. Corresponding matched peripheral blood was taken as a control. Genomic DNA was isolated from the tumor tissues and blood using a standard phenol-chloroform extraction method. Polymerase chain reaction was done to amplify mononucleotide microsatellite markers, BAT-26, BAT-40, TGFbetaRII, IGFIIR, hMSH3, and Bax by using specific primer sequences. For analysis of allelic patterns, the PCR products were run on 8% denaturing Polyacrylamide gel and sizing was done using a pUC18 sequencing ladder. The instability with BAT-26 and BAT-40 was found to be 20% and 45% in urinary bladder and 33% and 19% in thyroid cancers, respectively. However, no instability was observed with the other four-mononucleotide markers in either of the cancers studied. Eighty-three percent of the unstable urinary bladder cancers were found to have a high grade in a superficial group, whereas only 27% MSI+ve were muscle invasive cancers. Forty percent of unstable thyroid lesions were found to be at high risk of developing metastasis. Association of BAT-26 and BAT-40 instabilities with high grade tumors as well as risk tumors may help in choosing a more definite therapy at the outset.
India is one of the 12 mega diversity countries in the world so it has a vital stake in conservation and sustainable utilization of its biodiversity resources. Plant secondary metabolites have been of interest to man for a long time due to their pharmacological relevance. With this in view, the bark powder of Acacia auriculiformis, A. nilotica, Juglans regia, and the fruit powder of Terminalia bellerica, T. chebula, Emblica officinalis, and a combination drug "Triphala," which are known to be rich in polyphenols, were tested for their antimutagenic activities. Antimutagenic activities of the extracts were estimated by employing the plate incorporation Ames Salmonella histidine reversion assay by using the frame shift mutagen tester strain TA98 and base pair substitution strain TA100 against direct acting mutagens (NPD, sodium azide), and the S9-dependent mutagen 2-aminofluorene(2AF). Acetone extracts of all the plants exhibited significant antimutagenic activities among the other extracts tested, but an acetone extract of Acacia nilotica showed a marked anti-mutagent effect. Furthermore, it was more effective against indirect acting mutagen, 2AF, in both TA98 and TA100 tester strains of Salmonella typhimurium than against the direct acting mutagens. The results indicate that an acetone extract of bark and fruit of the medicinal plants under study harbors constituents with promising antimutagenic/anticarcinogenic potential that could be investigated further.
Brain cells are continuously exposed to reactive oxygen species generated by oxidative metabolism, and in certain pathological conditions defence mechanisms against oxygen radicals may be weakened and/or overwhelmed. DNA is a potential target for oxidative damage, and genomic damage can contribute to neuropathogenesis. It is important, therefore, to identify tools for the quantitative analysis of DNA damage in models of neurological disorders. The aim of this study was to compare the susceptibility of DNA to oxidative stress in cells freshly dissociated from the mouse brain, to that in cultured brain cells. Both primary cultures and a continuous cell line of astrocytes were considered. All cells were treated by xanthine/xanthine oxidase, a superoxide generator or hydrogen peroxide, applied alone or in the presence of the oxygen radical scavengers, superoxide dismutase, catalase, or ascorbic acid. DNA damage, quantified with the Comet assay, was consistent in all the different cell preparations exposed to oxidative stress, and was attenuated in similar ways by superoxide dismutase and catalase, scavengers of superoxide anion and hydrogen peroxide, respectively. The results with ascorbic acid were more variable, presumably because this compound may switch from anti- to pro-oxidant status depending on its concentration and other experimental conditions. Overall, similar responses were found in freshly dissociated and cultured brain cells. These results suggest that the Comet assay can be directly applied to cells freshly dissociated from the brain of rodents, including models of neurological disorders, such as stroke models and animals with targeted mutations that mimic human diseases.
Metanil yellow (MY) and malachite green (MG) are textile dyes, which, despite a ban, are used as food-coloring agents. MY and MG have promoter effects on the development of hepatic preneoplastic lesions induced by N-nitrosodiethylamine (DEN). Tumor-promoting agents are not mutagenic but may alter the expression of genes whose products are associated with hyper-proliferation, tissue remodeling, and inflammation. Cell cycle controls normally function to ensure the integrity of the genome and arrest of cells at G1/S or G2/M checkpoints until all the prerequisite events are completed. In order to understand the mechanism(s) of tumor promotion by MY and MG, we have studied the levels of PCNA, a marker of cell proliferation and cell cycle regulatory proteins, cyclin D1, and its associated kinase, cdk4, cyclin B1, and associated kinase, cdc2. Immunohistochemical staining showed an elevated level of PCNA in animals administered MY and MG subsequent to DEN treatment. Western and Northern blot hybridization showed an increased expression of both cyclin D1 and its associated kinase cdk4, and cyclin B1 and its associated kinase cdc2, in livers of rats administered MY and MG after administration of DEN as compared to untreated or DEN controls. The increased level of mRNA was due to the increased rate of transcription of these genes as studied by run-on transcription assay. These data obtained by the in vivo model of liver tumor development provide strong evidence for a link between dysregulation of the two critical checkpoints of the cell cycle as one of the possible mechanism(s) during tumor promotion by malachite green and metanil yellow.
Mutant spectra analysis was conducted with spontaneous hisG46 revertants of Salmonella typhimurium strain YG1029 and revertants induced by the plant- and mammalian S9-activation of benzidine and 4-aminobiphenyl (4-ABP). Under preincubation conditions, YG1029 cells were exposed to benizidine or 4-ABP with mammalian S9 activation or to a high molecular weight fraction that contained the plant-activated products. The induced revertants were isolated at mutagen concentrations that caused an increased mutant frequency of approximately 4- to 10-fold above background. Genomic DNA from each revertant was isolated and the hisG region was amplified using polymerase chain reaction (PCR). Using a series of specific probes and a modified version of the ECL3's-oligolabelling and detection system, each of the six possible base-pair substitution mutations at hisG46 that leads to a reversion event was determined. Of the YG1029 spontaneous revertants, transition mutations were 31.8% and transversion mutations were 68.2%. The YG1029 spontaneous mutant spectrum differed significantly from the spontaneous spectrum of TA1535 but did not significantly differ from the spontaneous TA100 mutant spectrum. The differences of the spontaneous mutant spectra among these highly related strains illustrate that the introduction of the plasmid pKM101 into S. typhimurium increased the frequency of transversions (CCC-->ACC; CCC-->CAC) and reduced site 2 (CCC-->CTC) transitions. With plant-activated benzidine, 21.1% of recovered revertants resulted from transitions and 78.9% from transversions while S9 activated-benzidine induced revertants were recovered as 14.2% from transition and 85.8% from transversion mutations. Plant-activated 4-ABP recovered 20.0% transitions and 80.0% transversions. S9-activated 4-ABP-induced 21.4% transitions and 78.6% transversions. Chi-square analysis of mutant spectra indicated that the DNA lesions that resulted in reversion at the hisG46 allele induced by plant-activated benzidine or 4-ABP were different from those generated after mammalian S9 activation of these promutagens. The plant-activated benzidine and 4-ABP induced statistically identical mutant spectra. Also, the mammalian-activated benzidine and 4-ABP induced statistically similar mutant spectra. These data show that the plant-activated and mammalian-activated aromatic amine products inflicted different types or distributions of DNA lesions that were reflected in the resulting induced mutant spectra.
Bleomycin is a radiomimetic antitumor agent with unique genotoxic properties. 1-nitropyrene is an environmental mutagen and carcinogen that undergoes both oxidative and reductive metabolism. In the present study, hepatocellular carcinomas were induced in male A/J mice by the intraperitoneal injection of bleomycin (120 mg/kg) followed by the intraperitoneal administration of 1-nitropyrene (total dose: 1,575 mg/kg). In order to understand the mechanism by which these two compounds induce hepatocellular carcinomas, the incidence and spectrum of mutations in the K-ras proto-oncogene in these hepatocellular carcinomas were analyzed. The hepatocellular carcinomas were induced by the administration of bleomycin and 1-nitropyrene were evaluated for point mutations in exon 1 and exon 2 of the K-ras gene by the polymerase chain reaction and a sequencing analysis. No mutation was found in the hotspots regions of the K-ras gene codon 12, 13, or 61. However, the codon 64 of the K-ras gene mutation was identified in 10 of 10 (100%) hepatocellular carcinomas. All mutations showed the same pattern, which was TAC-CAC transition. Codon 64 of the K-ras gene mutation may thus play an important role in the induction of hepatocellular carcinomas by bleomycin in the existence of 1-nitropyrene. As far as we know, this is the first report of a codon 64 mutation in the K-ras gene in a chemically induced tumor.
Thalassaemia is an inherited group of disorders caused by a reduction or total absence of one or more of the globin chains of the haemoglobin molecule. It has been shown that lymphocytes isolated from a sickle/beta thal double heterozygote-sickle phenotype patient showed increased sensitivity to the dietary food mutagen 3-amino-1-methyl-5H-pyridol(4,3-b)indole (Trp-P-2) when compared to the control. Furthermore, when a combination of Trp-P-2 with either quercitin or kaempferol was compared, the responses to Trp-P-2 were reduced to untreated control levels at the highest doses of quercitin and kaempferol. It has now been shown that using the food mutagens 2-amino-2-methylimidazolo(4,5-f)quinolone (IQ) and 2-amino-1-methyl-6-phenylimidazol(4,5-b)pyridine (PhIP) on lymphocytes of three different thalassaemia patients, a beta-thalassaemia major, a beta-thalassaemia/Hb E, and an alpha-thalassaemia trait with a 3.7-kb deletion, similar increased sensitivity could also be demonstrated. The present study investigated whether the modulatory effects of the flavonoids could be demonstrated in lymphocytes isolated from a beta-thalassaemia major and a beta-thalassaemia/Hb E patient. Lymphocytes from both a beta-thalassaemia major and beta-thalassaemia/Hb E patient showed increased sensitivity to PhIP when compared to the normal control. When a combination of PhIP and either quercitin or kaempferol was used, a reduction in the responses was seen, and at the highest doses of quercitin and kaempferol the responses were reduced to near untreated control levels and were significantly different when compared to PhIP alone (P < 0.05). It was concluded that lymphocytes from different thalassaemia genotypes showed increased sensitivity to different dietary food mutagens compared to normal lymphocytes and that flavonoids such as quercitin and kaempferol modulated the effects of these food mutagens in an antigenotoxic/antioxidant manner.
We have recently reported that the fluoroquinolone antibacterial DW-116 induces a significant developmental toxicity in rat. The present study was conducted to better understand the teratogenic effects of DW-116 at several developmental toxic doses in rats. DW-116 was orally administered to pregnant rats from gestational day (GD) 6 through 16 at dose levels of 0, 320, 400, and 500 mg/kg/day. All dams were subjected to caesarean section on GD 20 and their fetuses were examined for external, visceral, and skeletal abnormalities. At above 400 mg/kg, severe decreases in maternal body weight gain, food consumption, litter size, fetal weight and placental weight, and severe increases in resorption rate and fetal morphological alterations were observed. At 320 mg/kg, mild decreases in maternal body weight gain, food consumption, fetal weight and placental weight, and mild increases in fetal variations and retardations were observed. These results suggest that DW-116 is embryotoxic at above 320 mg/kg/day and is embryolethal and teratogenic at above 400 mg/kg in pregnant rats and that DW-116 is a selective developmental toxicant in rat conceptuses.
In girls of adolescent age, primary amenorrhea is a major problem and it is often suspected as Turner syndrome (TS), with complete or partial absence of one of the two X chromosomes. The girls who are unable to menstruate are primarily investigated by the gynecologists with the help of a physical examination, sonogram of the pelvis, endocrinologic tests, and ultimately cytogenetic analysis. Chromosomal analyses have been carried out in 280 such cases that were referred from different parts of the country. The standard protocol for peripheral blood lymphocyte culture was followed for metaphase chromosome preparation and conventional analysis of G-banded chromosomes. A total of 29% cases were found to have some chromosomal abnormality, including TS and testicular feminization syndrome involving sex chromosomes. Amongst those with sex chromosomal anomaly, 34% had evidence of a 46,XY karyotype in phenotypic females and 51% had pure line 45,X or mosaic with normal XX or other aberrations in X. The classification of the TS group further showed the spectrum of variant TS in Indian adolescent girls who suffered from absence or delayed menarche to correspond well with the Belgian, Danish, or Russian population. However, it has been reported that only 1% of the pure line 45,X conception is viable, indicating the necessity of mosaicism with X or Y chromosome. It has been understood that conventional banding analysis is absolutely necessary for segregating the variant nature of TS. In addition, molecular genetic or molecular cytogenetic investigations can determine the nature of mosaicism. The present study further indicated the involvement of autosomes in causing improper sexual development in girls of adolescent age.
The aim of this work was to test the cytotoxicity of newly synthesized cis-type complexes of platinum(II) and palladium(II) dichloride with methyl 3,4-diamine-2,3,4,6-tetradeoxy-alpha-L-lyxohexopyranoside, [M(C(7)H(16)N(2)O(2))Cl(2)].H(2)O, against two mouse lymphoma cell lines (L5178Y) differing in their double strand breaks and nucleotide excision repair ability. cis- Diaminedichloroplatinum (CDDP) was used as a reference compound. The toxicity of Pt(C(7)H(16)N(2)O(2))Cl(2) appeared to be similar for both cell lines: IC(50) is 8 microM for L5178Y-R cells and 12 microM for L5178Y-S cells, respectively. In contrast, the palladium complex was found to be more toxic for the LY-R cells than for the LY-S cells. The cytotoxicity of both compounds was compared with their ability to induce DNA crosslinks, as measured by the modified comet assay. CDDP caused retardation of the DNA migration induced by 2 Gy of the X-irradiation in a dose-dependent manner. The ability of Pd(C(7)H(16)N(2)O(2))Cl(2) to retard X-ray induced DNA migration was more pronounced than its platinum analogue and CDDP (see Fig. 6). However, this was not reflected in the toxicity of the compound. Such results indicate that these two compounds may cause a different type of DNA damage and/or that the DNA damage caused by the palladium(II) compound was dealt with in a different manner from that induced by the platinum(II) complex.
Many natural agents including fruits and vegetables are known to provide protection from different degenerative diseases including cancer, by preventing damage to the cellular components. The effect of two important dietary agents, alpha tocopherol, and the flavonoid quercetin, along with two commonly consumed vegetables, bitter gourd and tomato, were investigated on spontaneous and dimethylbenz(a)anthracene (DMBA)-induced DNA damage in murine lymphocytes in vitro. DNA damage was determined by single cell gel electrophoresis (comet assay). The rationale for such an approach for this study is that DNA damage can lead to genetic disorders that occur at different stages of carcinogenesis and protection from such damages may in the long run help to prevent development of cancer. Both alpha tocopherol and quercetin as single agents were found to be potent inhibitors of DNA damage (spontaneous and carcinogen induced) in a dose-dependent manner. Fresh juices of bitter gourd and tomato could also protect from DMBA-induced DNA damage but not as effectively as the single agents. The anticarcinogenic role of nutrients as well as non-nutrient dietary components need to be explored more extensively. The Comet assay is a simple, fast, and reliable method to determine the protective effect against DNA damage, one of the prerequisites for carcinogenesis.
Sperm is produced by a highly complex and poorly understood differentiation process known as spermatogenesis. Occupational exposure to high temperatures adversely affect testicular function, causing partial or complete spermatogenic arrest. Dyers, cooks, blast furnace workers, and men with varicocele are known to develop testicular hyperthermia, which leads to oligoasthenoteratozoospermia (OAT) and azoospermia. Semen analysis of 122 infertile men (and 25 fertile controls), following the WHO guidelines, 1999, showed azoospermia in 106 men and oligozoospermia in 16 men. Twenty azoospermic and fourteen oligozoospermic men had high testiculoepididymal temperatures, either due to occupational exposure to high temperature or varicocele. All the 14 oligozoospermic men showed a very high percentage of sperm with abnormal morphology, impaired motility and they were subclassified as OAT group. Observations made in this study reiterates that high intratesticular temperature causes partial or complete spermatogenic arrest and may lead to increased production of morphologically abnormal sperm with impaired motility. This inverse relationship of sperm function with elevated temperature has implications in clinical medicine both in understanding pathological states and for therapeutic measures. (C) 2003 Wiley-Liss, Inc.
8-oxo-7,8-dihydroguanine (8-oxoG) is a potent mutagenic lesion that forms at elevated levels in cellular DNA and is repaired with low efficiency in human cells. Unlike its human counterpart, the Drosophila S3 ribosomal/repair protein is endowed with a vigorous 8 oxoG repair activity that is associated to beta,delta-elimination AP lyase activity. We have recently observed that pure GST-tagged Drosophila S3 protein can significantly accelerate the in vitro repair of 8 oxoG performed by human and mouse cell extracts [Cappelli et al., unpublished data]. In this work, we have transfected Chinese hamster cells with mammalian expression vectors containing the Drosophila S3 cDNA. The cells synthesized both S3 mRNA and protein but no improved repair of 8 oxoguanine was observed. Factors important for the proper expression of Drosophila genes in mammalian cells are discussed.
In order to determine differences in repair after treatment with DNA damaging agents, normal and cancer cells were selected for analysis of single strand breaks and DNA crosslinks using the Comet assay. Normal human lymphocytes, human colorectal adenocarcinoma SW620 cells, lung carcinoma A549, and H460 cell lines were exposed to an ethylating agent (ethylmethane sulfonate [EMS]), and a cross-linking agent (mitomycin C [MMC]). Differences in repair profiles of DNA damage demonstrated using the comet assay were observed in human lymphocytes and tumour cell lines with both mutagens. Results were also indicative that MMC repair is concentration-dependent. It was also apparent that normal cells repair DNA damage more readily than tumour cells. Repair also varied between different cell lines. To investigate the mechanistic differences of these two chemicals, flow cytometry studies were undertaken in tumour cells, namely cell cycle analysis and frequency of micronuclei induction (FMN). A G2M phase block was clearly evident following treatment with EMS at all concentrations tested. With MMC, an initial arrest of cells in G2M was accompanied by a build-up in S-phase over longer exposure periods. Also, at the highest mutagen doses there were different patterns of micronuclei induction. Thus, using the mutagens with different mechanisms of action highlighted the differences in repair patterns between normal and tumour cells.
An iron chelate, ferric nitrilotriacetate (Fe-NTA), is a potent nephrotoxic agent, and induces acute and subacute renal proximal tubular necrosis, a consequence of the Fenton-like reaction that eventually leads to a high incidence of renal adenocarcinoma in rodents. In order to examine the possible mechanism for carcinogenic activity, we investigated the DNA damage with Fe-NTA in the presence of various peroxides/organic hydroperoxides. S1 nuclease hydrolysis and deoxyribose degradation assays were performed. Incubation of calf thymus DNA with ferric nitrilotriacetate (0.1 mM) in the presence of peroxides/organic hydroperoxides at a final concentration of 40 mM of each in phosphate buffer (0.1 M, pH 7.4) augmented DNA damage severalfold as compared to the damage caused by individual treatments. Fe-NTA in the presence of hydrogen peroxide caused DNA single-strand breaks and damage to its deoxyribose sugar moiety as measured, respectively, by S1 nuclease hydrolysis and deoxyribose degradation using calf thymus DNA. However, only deoxyribose degradation could be recorded in the presence of other peroxide/organic hydroperoxides. No DNA single-strand break was observed by this treatment. The observed differences in DNA damage by hydrogen peroxide and organic hydroperoxides/peroxide have been ascribed to the differential reactivity of DNA with hydroxyl and alkoxy/aryloxy free radicals produced, respectively, from these inorganic and organic peroxides. These studies suggest that Fe-NTA not only mediated the production of reactive oxygen species, but also catalysed the decomposition of these peroxides and organic hydroperoxides, which may cause a clastogenic change in DNA. This reactivity enhances the clastogenic activity in DNA. These changes in the DNA structure may ultimately be responsible, at least in part, for the induction of carcinogenesis in Fe-NTA-exposed animals.
