Excision repair of DNA base damage in human cells treated with the chemical carcinogen 4-nitroquinoline 1-oxide.

Excision repair of DNA damage produced by 4-nitroquinoline 1-oxide (4NQO), a potent chemical carcinogen, was compared in a normal human amnion FL cell line and a xeroderma pigmentosum (XP) cell line unable to repair ultraviolet-induced pyramidine dimers. The main objective of this study was to investigate, by a direct assay of the loss of damage from DNA, whether DNA damage induced by 4NQO in human cells is repaired by the excision-repair system as in Escherichia coli cells. DNA was extracted from FL and XP cells treated with [³H]4NQO, hydrolyzed and subjected to radiochromatographic analysis in order to quantitate the initial formation of 4NQO damage and subsequent disappearance during post-incubation. Two peaks of stable 4NQO-quanine adducts appeared on the chromatogram, together with one peak of stable 4NQO-adenine adduct and a peak due to 4-aminoquinoline 1-oxide (4AQO) released from a labile fraction of 4NQO-guanine adduct during hydrolysis. The three kinds of stable 4NQO-purine adduct disappeared from DNA of the FL cells at almost the same rate of about 60% during 24-h post-incubation in culture medium, and 4AQO disappeared somewhat faster. In the XP cells, however, the stable adducts did not disappear from DNA, whereas about 40% of the 4AQO-releasing adduct disappeared from DNA. These findings at the molecular level quantitatively parallel the previous findings at the cellular level that the XP cells are several times as sensitive as normal cells to killing by 4NQO. These results lead to the conclusion that in human cells 4NQO-induced lethality is mainly due to the four kinds of 4NQO-purine adduct as it is in E. coli, and that the adducts are excisable by the same excision-repair mechanism that works on pyramidine dimers.     

Inhibition of 4-nitroquinoline 1-oxide induced unscheduled DNA synthesis in primary cultures of rat urothelial cells by dicumarol and pyrophosphate

Primary cultures of rat urothelial cells were exposed to hydroxyurea, [3H]thymidine, and 4-nitroquinoline 1-oxide (NQO) or N-hydroxy-4-aminoquinoline 1-oxide (HAQO) in a serum-free media for 2 h; unscheduled DNA synthesis (UDS) was measured by autoradiography. Both NQO and HAQO produced unscheduled DNA synthesis. Dicumarol, an inhibitor of NQO nitroreductase, inhibited the activity of NQO and, to a lesser extent, HAQO. Pyrophosphate, an inhibitor of seryl-AMP synthetase, inhibited the activity of both compounds. Neither dicumarol nor pyrophosphate, under similar experimental conditions, inhibited the activity of N-hydroxy-N-2-acetylaminofluorene (N-OH-AAF). These results support the idea that nitro-reductase and seryl-AMP synthetase may be involved in the activation of NQO.     

Oxidative DNA damage is a preliminary step during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

The aim of this study was to investigate oxidative DNA damage during 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis. For this purpose, male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12, and 20 weeks. Ten animals were used as negative control. The alkaline Comet assay modified with lesion-specific enzymes was used to detect single and double strand breaks, labile sites (SBs), and oxidised purines and pyrimidines. Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, oxidative DNA damage was detected in the ‘normal’ oral epithelium. In pre-neoplastic lesions and squamous cell carcinomas induced after 12 and 20 weeks following carcinogen exposure, respectively, oxidative DNA damage was also increased (P < 0.05) when compared to negative control. In conclusion, our results suggest that oxidative DNA damage is an early event during multistep carcinogenesis assay induced by 4NQO. This kind of approach should be considered to persons with high risk of oral cancer, such as in smokers or alcohol consumers.     

The 4-nitroquinoline 1-oxide mutational spectrum in single stranded DNA is characterized by guanine to pyrimidine transversions.

4-Nitroquinoline-1-oxide is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. In ds or ss damaged DNA the ratio C8/N2 adducts is 1:2 and 8-10:1, respectively. In bacteria and yeast 4NQO has been shown to be a base substitution mutagen acting at G residues inducing mainly G to A transitions. We determined the mutational spectrum induced by the 4NQO metabolite, acetoxy-4-aminoquinoline 1-oxide, in the M13lacZ'/E. coli lacZ delta M15 alpha complementation assay using ssDNA. Among 68 Ac-4HAQO induced mutants, G to Pyr transversion was the most frequent base substitution observed. By comparison with dsDNA based systems, our data suggest that dGuo-C8-AQO induces G to Pyr transversions. A mechanism to explain how this lesion may induce transversions is proposed.     

Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagens

The clone-forming capacity and level of DNA repair was examined on normal human cells and repair-deficient Xeroderma pigmentosum (XP) fibroblasts exposed to various chemical carcinogens and mutagens.The cultured fibroblasts were treated for 90 min with the carcinogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO), 4-hydroxyaminoquinoline 1-oxide (4HAQO), 2-methyl-4-nitroquinoline 1-oxide (2-Me-4NQO), 3-methyl-4-nitropyridine 1-oxide 3-Me-4NPO) and the non-carcinogenic 6-nitroquinoline 1-oxide (6NQO). The response of the cells to the N-oxides was compared to that induced by the mutagen and carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and UV-irradiation.The XP cells showed (1) a reduced level of DNA repair synthesis when exposed to various carcinogenic N-oxides, (2) no unscheduled DNA synthesis following 6NQO and (3) a normal degree of DNA repair synthesis after treatment with MNNG.When the clone-forming capacity was examined the XP cells exhibited (1) a higher increased sensitivity to the various carcinogenic N-oxides, (2) no reduction in the clone formation following 6NQO and (3) a sensitivity virtually comparable to that of normal cells after treatment with MNNG.The results suggest a link between extent of DNA damage, level of DNA repair and degree of sensitivity in human cells exposed to various chemical carcinogens and which induce DNA alterations that cannot be repaired by DNA repair synthesis.     

In vivo repair of rat liver DNA damaged by dimethylnitrosamine or diethylnitrosamine.

Effects of hepatocarcinogens dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) on the sedimentation pattern of rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. Both DMN (10 mg/kg body weight) and den (13.4 or 134 mg/kg) induced appreciably decreased DNA sedimentation rates at 24 h after injection. DMN at 10 mg/kg was as effective in decreasing the DNA sedimentation rate at 24 h after injection as was the higher dose of DEN (134 mg/kg). Sedimentation patterns at 1, 6 and 14 days after injection indicated that damage induced by DEN (134 mg/kg) was repaired at a substantially lower rate than DMN (10 mg/kg) induced damage. When effects of equimolar doses of DMN (10 mg/kg) and DEN (13.4 mg/kg) were compared at 1, 6 and 14 days after injection, it was observed that the more pronounced damage of rat liver DNA induced by DMN was repaired at a faster rate than was the DEN-induced damage. At the molecular level this difference in repair between damage induced by the two nitrosamines is probably related to different DNA alkylation patterns. The relatively persistent nitrosamine-induced DNA lesions (observed especially after DEN administration) are thought to represent phosphotriesters which give rise to single strand DNA breaks at strongly alkaline conditions of lysis on top of the gradient. The results are discussed in relation to the possible significance of alkylation and repair of DNA in the formation of (pre)cancerous lesions in rat liver.     

The formation and repair of single-strand breaks in DNA of cultured mammalian cells treated with UV-light, methylating agents or 4-nitroquinoline-1-oxide.

The technique of sedimentation in alkaline sucrose was used to examine the formation and repair of single-strand (SS) breaks in cultured mammalian cells that were treated with methyl methanesulfonate (MMS), methyl nitrosourea (MNUA), 4-nitroquinoline-1-oxide (4NQO) or UV-light. The SS breaks induced by MMS and 4NQO were largely repaired by HeLa cells during a 5-h post-treatment incubation. The SS breaks induced by MNUA and UV-light were not repaired by HeLa cells. L-cells were not able to repair the SS breaks induced by any of the agents, which correlates with the deficiency of these cells for repair synthesis of DNA. The following conclusions are discussed. MNUA and UV-light produce modifications in DNA which are not repaired but are translated into SS breaks in alkali. MMS produces SS breaks intracellularly but these are not derived from a simple depurination of methylated purines. 4NQO produces a modification in DNA which is translated into an SS break in alkali but which can be removed by an intracellular process.     

Repair synthesis and sedimentation analysis of DNA of human cells exposed to dimethylnitrosamine and activated dimethylnitrosamine

The capacity of dimethylnitrosamine(DMN), and of DMN activated by a NADPH-fortified mouse liver microsomal preparation, to elicit DNA alterations in cultured human fibroblasts was examined. A maximum induction of DNA repair synthesis, estimated by unscheduled incorporation of tritiated thymidine, occurred following 60-minute incubation of the human cells with DMN activated by a NADPH-fortified mouse liver microsomal preparation. A low level of DNA repair activity followed exposure to DMN alone, or to DMN mixed with the microsomal preparation without NADPH or without O₂. The extent of DNA damage, estimated by velocity sedimentation of DNA through alkaline sucrose gradients, was maximum following treatment with DMN mixed with the NADPH-fortified microsomal preparation. The combined application of $\text{in vitro}$ activation systems and estimation of DNA repair synthesis in cultured cells may be exploited in the detection of precarcinogens.     

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.     

Alkaline, Endo III and FPG modified comet assay as biomarkers for the detection of oxidative DNA damage in rats with experimentally induced diabetes

Increased production of reactive oxygen species under diabetic condition underlines the higher oxidatively damaged DNA in different tissues. However, it is practically difficult to assess the oxidatively damaged DNA in different internal organs. Therefore, the present study was aimed to evaluate the extent of oxidative stress-induced DNA damage in different organs with the progression of diabetes. Diabetic and control Sprague Dawley rats were sacrificed in time-dependent manner and the lung, liver, heart, aorta, kidney, pancreas and peripheral blood lymphocytes (PBL) were analyzed for both alkaline and modified comet assay with endonuclease-III (Endo III) and formamidopyrimidine-DNA glycosylase (FPG) (hereafter called modified comet assay) for the detection of oxidative DNA damage. The statistically significant increase in olive tail moment (OTM) was found in all the tested tissues. The extent of DNA damage was increased with the progression of diabetes as revealed by the parameter of OTM in alkaline and modified comet assay. Further, the positive correlations were observed between OTM of the lung, liver, heart, aorta, kidney and pancreas with PBL of diabetic rat in the alkaline and modified comet assay. Moreover, significant increase in the 8-oxodG positive nuclei in the lung, liver, heart, aorta, kidney and pancreas was observed in 4th and 8th week diabetic rat as compared to control. Results of the present study clearly indicated the suitability of alkaline and modified comet assay for the detection of multi-organ oxidative DNA damage in streptozotocin (STZ)-induced diabetic rat and showed that damaged DNA of PBL can be used as a suitable biomarker to assess the internal organs response to DNA damage in diabetes.     

Reduced DNA repair during differentiation of a myogenic cell line

Repair synthesis induced by 4-nitroquinoline-1-oxide (4NQO) in L6 myoblasts before and after cellular fusion was measured by [3H] thymidine incorporation into unreplicated DNA. The level of repair synthesis was reuced after the cells had fused into myotubes. The terminal addition of radioactive nucleotides into DNA strands occurred only to a minor extent, and the dilution of [3H] thymidine by intracellular nucleotide pools was shown not to be responsible for the observed difference in repair synthesis, Both the initial rate and the overall incorporation of [3H] thymidine were found to be 50% lower in the myotubes. 4NQO treatment of myoblasts and myotubes induced modifications in the DNA which were observed as single-strand breaks during alkaline sucrose sedimentation. After the myoblasts were allowed a post-treatment incubation, most of the single-strand breaks were not longer apparent. In contrast, a post-treatment incubation of myotubes did not change the extent of single-strand breakage seen. Both myoblasts and myotubes were equally effective in repairing single- strand breaks induced by X radiation. It would appear that when myoblasts fuse, a repair enzyme activity is lost, probably an endonuclease that recognizes one of the 4 NQO modifications of DNA. The result observed is a partial loss of repair synthetic ability and a complete loss of ability to remove the modification that appears as a single-strand break in alkali.     

Differential repair of 1-beta-D-arabinofuranosylcytosine-detectable sites in DNA of human fibroblasts exposed to ultraviolet light and 4-nitroquinoline 1-oxide

The extent of DNA excision repair was determined in dermal fibroblast strains from clinically normal and xeroderma pigmentosum (XP; complementation group A) human donors after single or combined exposures to 254-nm ultraviolet light and 4-nitroquinoline 1-oxide (4NQO). The repair was monitored by incubation of the treated cultures in the presence of 1-beta-D-arabinofuranosylcytosine (araC), a potent inhibitor of long-patch excision repair, followed by quantitation of araC-accumulated DNA single-strand breaks (representing repair events) by velocity sedimentation analysis in alkaline sucrose gradients. The amount of repair in normal fibroblast strains increased as a function of UV fluence and reached a plateau at 15 J/m2; strand breaks were not detected when these same cultures were irradiated with as much as 60 J/m2 UV and incubated in the absence of araC, implying that an initial (incision) step is rate-limiting in the repair of UV damage. In normal fibroblasts (i) the incidence of araC-detectable lesions removed during fixed intervals following exposure to 4NQO (4 microM; 30 min) was approximately 2.5 times greater than that seen following irradiation with repair-saturating fluences (greater than or equal to 15 J/m2) of UV-rays; and (ii) the amount of repair in cultures treated simultaneously with 4NQO (0.5-6 microM; 30 min) and a repair-saturating fluence of UV (20 J/m2) was found to approach the sum of that arising from exposure to each separately. The XP cells (XP12BE) exhibited a deficiency in the removal of araC-detectable DNA lesions following exposure to either of the carcinogens. Since araC is known to inhibit the repair of alkali-stable 4NQO-DNA adducts (i.e., lesions assumed to be removed by the UV-like excision pathway) but not that of alkali-labile sites (i.e., DNA lesions operated on by the X-ray-like repair pathway), our results strongly imply that the multistep excision-repair pathway operative on UV photoproducts in human fibroblasts differs from that responsible for removing alkali-stable (araC-detectable) 4NQO adducts by at least one step, presumably the rate-limiting incision reaction mediated by a lesion-recognizing endonuclease.     

DNA excision in repair proficient and deficient human cells treated with a combination of ultraviolet radiation and acridine mustard (ICR-170) or 4-nitroquinoline 1-oxide

Excision repair was measured in normal human and xeroderma pigmentosum group C fibroblasts treated with ultraviolet radiation and the carcinogens acridine mustard (ICR-170) or 4-nitroquinoline 1-oxide (4NQO) by the techniques of unscheduled synthesis, photolysis of bromodeoxyuridine incorporated into parental DNA during repair, and assays of sites sensitive to ultraviolet (UV)-endonuclease. Doses of ICR-170 and 4NQO, low enough not to inhibit unscheduled DNA synthesis (UDS), caused damage to DNA that was repaired by a long patch type mechanism and the rates of UDS decreased rapidly in the first 12 h after treatment. Repair after a combined action of UV plus ICR-170 or UV plus 4NQO was additive in normal cells and no inhibition of loss of endonuclease sensitive sites was detected. In xeroderma pigmentosum (XP) C cells there was less repair after UV plus ICR-170 than after each treatment separately; whereas there was an additive effect after UV plus 4NQO and no inhibition of loss of endonuclease sensitive sites. The results indicate that in normal human fibroblasts there are different rate limiting steps for removal of chemical and physical damages from DNA and that XP cells have a different repair system for ICR-170, not just a lower level, than normal cells. Possibly the same long patch repair system works on 4NQO damage in both normal and XP cells.     

The repair of large DNA adducts in mammalian cells.

This paper describes experiments involving the measurement of DNA damage and repair after treatment with 4-nitroquinoline 1-oxide (4NQO) or aflatoxin B1 (AFB1) epoxide in a number of mammalian cell cultures primarily associated with defects in the excision repair of UV-induced DNA damage. The results with transformed derivatives of XP cells belonging to different complementation groups showed that the extent of repair of 4NQO adducts at the N2 or C8 of guanosine did not correlate to the extent of repair reported by others after UV-irradiation. An examination of 4NQO repair in rodent UV-sensitive cell lines from different ERCC groups indicated that again there was little correlation between the extent of 4NQO and UV repair. However, regardless of complementation group those mutants that were defective in the repair of pyrimidine dimers and 6,4-photoproducts did exhibit a reduced ability to repair the 4NQO N2 guanosine adduct, whereas those mutants defective in pyrimidine dimer repair alone were able to repair this lesion as normal. In all of these cell lines there was a normal capacity to repair the 4NQO C8 guanosine adduct. Less extensive experiments involving AFB1 epoxide showed an XPC-transformed cell line was able to repair 40% of lesions after 6 h, whereas only 20% of repair is seen after UV. The rodent mutant V-C4 which belongs to the same ionising radiation group as irs2, was partially defective in repairing AFB1-induced damage. These experiments highlight the fact that although there are many commonalities between the repair of UV damages and lesions classed as large DNA adducts differences clearly exist, the most striking example here being the repair of the C8 guanosine 4NQO adduct which rarely correlates with a defect in UV repair.     

Protective effect of <Emphasis Type="Italic">Spirulina</Emphasis> against 4-nitroquinoline-1-oxide induced toxicity

In the present study, we focused on the protective effect of Spirulina against 4-nitroquinoline-1-oxide (4NQO) induced hepato and nephrotoxicity in the experimental rats. The 4NQO administration resulted in increased levels of hepatic and renal markers [Alanine Transaminase (ALT), Aspartate Transaminase (AST), Lactate Dehydrogenase (LDH), urea, creatinine and uric acid] in the serum of experimental animals. It also increased the oxidative stress resulting in increased levels of the lipid peroxidation with a concomitant decline in the levels of non enzymic [reduced glutathione (GSH)] and enzymic antioxidants [(Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx), and Glutathione-S-transferase (GST)] in both liver and kidney. Oral pretreatment with aqueous extract of Spirulina prevented 4NQO induced changes in the levels of hepatic and kidney diagnostic marker enzymes in the serum of experimental rats. It counteracted the 4NQO induced lipid peroxidation and maintained the hepatic and kidney antioxidant defense system at near normal in both liver and kidney. The antioxidant responsiveness mediated by Spirulina may be anticipated to have biological significance in eliminating reactive free radicals that may otherwise affect normal cell functioning and provide a scientific rationale for the use of Spirulina.     

A new method to reveal the genotoxic effects of N-nitrosodimethylamine in pregnant mice

DNA damage and repair in kidney and liver of mouse fetuses exposed to selected doses of N-nitrosodimethylamine (NDMA) (CAS No. 62.75.9) were studied using the alkaline elution technique. CD1 female mice (15 days pregnant) were treated i.p. with 2 and 10 mg/kg b.w. of NDMA; a slight increase in DNA damage was observed in their fetuses compared to untreated controls. A 2-fold higher extent of DNA damage was induced when mice were treated by intrafetal injections of a rat S9 activating fraction (S9) immediately before exposure to the same dose of NDMA by transplacental means. The DNA-strand breaks disappeared as a function of time in animals treated with NDMA alone. In contrast, a significant persistence of DNA damage was detected in the liver and lung of fetuses which were treated with S9 and NDMA in sequence. These experiments demonstrate the metabolic immaturity of unborn mice as far as the carcinogenic activation of NDMA is concerned and show the high susceptibility of fetal tissues to DNA-damaging agents. The alkaline elution applied in vivo by the transplacental route combined with the intrafetal injection of an exogenous activating microsomal fraction allow to extend our knowledge on the interaction of metabolism-dependent chemicals with fetal tissues.     

In vivo genotoxicity of heterocyclic amines detected by a modified alkaline single cell gel electrophoresis assay in a multiple organ study in the mouse

We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of 6 heterocyclic amines, Trp-P-1 (25 mg/kg), Trp-P-2 (13 mg/kg), IQ (13 mg/kg), MeIQ (13 mg/kg), MeIQx (13 mg/kg) and PhIP (40 mg/kg), in mouse liver, lung, kidney, brain, spleen, bone marrow and stomach mucosa. Mice were sacrificed 1, 3, and 24 h after intraperitoneal injection. Trp-P-2, IQ, MeIQ, and MeIQx yielded statistically significant DNA damage in the stomach, liver, kidney, lung and brain; Trp-P-1 in the stomach, liver and lung; and PhIP in the liver, kidney and brain. None of the heterocyclic amines induced DNA damage in the spleen and bone marrow. Our results suggest that the alkaline SCG assay applied to multiple organs is a good way to detect organ-specific genotoxicity of heterocyclic amines in mammals.     

The formation and stability of methyl phosphotriesters in the DNA of rat tissues after treatment with the carcinogen N,N-dimethylnitrosamine

Following the injection i.p. of N,N-dimethylnitrosamine (DMN) into Chester Beatty (CB) hooded, female rats (2 mg/kg) measurable concentrations of methyl phosphotriesters were found in the DNA of liver, lung and kidney but not in spleen, thymus or brain. In lung and kidney these lesions were stable for at least 14 days but in liver there was a steady loss (t 1/2 9-11 days). Administering the same total dose in 10 weekly injections produced the same concentration of phosphotriesters in lung and kidney DNA as the single injection but in liver only half of the concentration induced by the single injection was found. It was calculated that the half-life of methyl phosphotriesters in the liver DNA of animals given repetitive injections was of the order of 6 weeks.     

DNA fragmentation by N-nitrosodimethylamine and methyl methanesulfonate in human hepatocyte primary cultures

The ability of N-nitrosodimethylamine (DMN) and methyl methanesulfonate (MMS) to induce DNA damage in primary cultures of human hepatocytes was examined by the alkaline elution technique. Both the agents induced a dose-dependent increase in DNA elution rate, but appreciable differences in the degree of response to the procarcinogen DMN were observed among cultures obtained from the livers of four patients. A comparative analysis of DNA fragmentation indicated a substantial similarity between human and concurrently studied rat hepatocytes in their response to both DMN and MMS.     

DNA repair in human fibroblasts treated with a combination of chemicals.

Excision repair of DNA damage was measured by the photolysis of bromodeoxy-uridine incorporated during repair in normal human and xeroderma pigmentosum group C fibroblasts (XP C) treated with a combination of the carcinogens N-acetoxy-2-acetylamino-fluorene (AAAF), and 4-nitroquinoline 1-oxide (4NQO). Repair was additive in normal and XP C cells treated with AAAF plus 4NQO, indicating that there are different rate limiting steps for removal of 4NQO and AAAF lesions.