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.     

Synergistic DNA damaging effects of 4-nitroquinoline-1-oxide and non-effective concentrations of methyl methanesulfonate in human fibroblasts

DNA damage and DNA repair in human fibroblasts induced by the combination mixture of the genotoxic agents methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4-NQO) were studied using the comet assay and the unscheduled DNA synthesis (UDS), respectively. Cells were simultaneously treated for 1h with the no observed effect concentration (noec) of MMS and increasing concentrations of 4-NQO or vice versa. Different results were obtained with the two types of mixtures. When the noec of 4-NQO was combined with increasing concentrations of MMS, no combination effects were observed. However, in experiments with increasing concentrations of 4-NQO and the noec of MMS, an increase in DNA damage and repair (and an enhancement of cytotoxicity) was demonstrated. Quantitative analysis of the effects by the isobologram method confirmed synergistic responses in both tests. We are proposing interactive actions between 4-NQO and MMS, whereby 4-NQO facilitates the attack of MMS on the DNA bases.     

Mutagenicity of the oral carcinogen 4-nitroquinoline-1-oxide in cultured BigBlue rat tongue epithelial cells and fibroblasts

Environmental carcinogen exposures contribute to the development of oral cancer and improved test systems for the analysis of such carcinogens are needed. We have previously isolated and characterized an epithelial cell line from the tongue of a BigBlue rat. Now, we have established an immortalized fibroblast cell line from the same organ. We exposed these cells to 4-nitroquinoline-1-oxide (NQO), a well-known experimental oral carcinogen in the rat and other species, and measured its cytotoxic and genotoxic (cII transgene mutagenesis) effects. Both cell lines were very sensitive to NQO toxicity and showed dose-dependent mutant frequency responses. At the highest NQO dose tested, 70 ng/ml, the mutant frequency was elevated more than eight-fold above background for the epithelial cells and more than 25-fold for the fibroblast cells. We examined cellular parameters which could affect glutathione-dependent detoxication of mutagens. Glutathione (GSH) contents of the two cell lines were similar. Glutathione transferase (GST) activities were measured with several substrates and were generally higher in the epithelial cells. Although multiple biochemical and biological characteristics of individual cell lines are likely to determine responses to mutagens, the greater sensitivity of the fibroblast cells to NQO mutagenicity is in accord with the lower GST activity and the lower DNA content of these cells. These new cell lines are suitable for in vitro testing of chemicals as possible oral mutagens and for studies of their biochemical mechanisms of action.     

Repair of DNA damage after exposure to 4-nitroquinoline-1-oxide in heterokaryons derived from xeroderma pigmentosum cells

Xeroderma pigmentosum (XP) cells are dificient in the repair of damage induced by ultraviolet irradiation. Excision-repair-deficient XP cell strains have been classified into 7 distinct complementation groups, according to results of studies on cell fusion and UV irradiation. XP cells are not only abnormally sensitive to UV, but also to a variety of chemical carcinogens, including 4-nitroquinoline-1-oxide (4NQO). Complementation analysis with XP strains from 4 different complementation groups with respect to the repair of 4NQO-induced DNA damage revealed that the classification of the strains into complementation groups with respect to 4NQO-induced repair coincides with the classification based on the repair of UV damage.     

4-nitroquinoline-1-oxide: A good mutagen for Aspergillus nidulans

Conditions for using 4-nitroquinoline-1-oxide as a mutagen for Aspergillus nidulans were established. High frequencies of mutations (about 0.5%) and a broad spectrum of mutants were obtained. The stability of NQO in solutions and safety precautions are described.     

Reversion of nonsense mutants induced by 4-nitroquinoline-1-oxide in Schizosaccharomyces pombe.

We have studied the reversion of 8 nonsense alleles located in 7 different genes of Schizosaccharomyces pombe using 4-nitroquinoline-1-oxide (NQO) as a mutagenic agent. The nonsense mutants of S. pombe have been classified according to their suppressibility by defined opal and ochre suppressors into a class of efficiently suppressed opal and a class of inefficiency suppressed ochre mutants. The UGA alleles tested all revert consistently with NQO, in agreement with the high specificity of this mutagen for G-residues reported for bacteria and yeast. The UAA alleles show a lack or a low level of reversion with NQO. This low level of reversion is due to the low level of non-G-specific transversions at A sites of the UAA triplet. Within each class of nonsense mutants the extent of induction is site-dependent. We conclude that NQO acts predominantly on G-residues in S. pombe.     

Application of alkaline sucrose gradient sedimentation to the study of DNA damage and its repair in mammalian cells treated with methylmethanesulfonate and 4-nitroquinoline-1-oxide.

KB cells and L cells were treated with methylmethanesulfonate (MMS) or 4-nitroquinoline-1-oxide (4 NQO) and the resulting damage to DNA and its repair were examined by sedimentation in an alkaline sucrose gradient. The sedimentation profiles obtained were found to be the resultant of a complex interrelationship between drug dosage, duration of the lysis period and the repair capacity of the cells. A systematic study of these variables was made which led to a plausible and useful interpretation of the sedimentation profiles. Both drugs produce two kinds of DNA modifications which show up as a single-strand breaks but affect the sedimentation profile in characteristic ways. One of these modifications which is quite alkali-labile can be studied using a 30-min lysis period. The other modification is less alkali-labile and can be studied using a long lysis period. Both KB cells and L cells can repair the former type of damage but only KB cells can repair the latter type of damage.     

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.     

Infection with adeno-associated virus type 5 inhibits mutagenicity of herpes simplex virus type 1 or 4-nitroquinoline-1-oxide

Infection with adeno-associated virus type 5 (AAV-5) reduced the number of mutants arising in the hypoxanthine phosphoribosyltransferase locus of human RD 176 cells after infection with herpes simplex virus type 1 (HSV-1; partially inactivated) or 4-nitroquinoline-1-oxide (4-NQO). The mutation frequency was reduced by AAV-5 infection from 11.4 to 1.8 after mutation with HSV-1 and from 3.2 to 2.5 when mutation was induced by 4-NQO. This was analyzed by determination of the number of cells resistant to 8-azaguanine when infected with AAV-5 prior to induction of mutations with HSV-1 or 4-NQO.     

Malignant transformation of rat bone marrow-derived mesenchymal stem cells treated with 4-nitroquinoline 1-oxide

Recent studies indicated that extensive culture of bone marrow-derived mesenchymal stem cells (BMSCs) can lead to malignant transformation, supporting the concept that tumor may originate from adult stem cells. Also, neoplastic transformation of BMSCs induced by virus and ionizing radiation were verified. However, the capacity for BMSCs to become mutated by chemical carcinogens and become precursors of cancer is still poorly understood. In this study, BMSCs were used to test the hypothesis that tumorigenesis can originate from the mutation of stem cells induced by chemical carcinogen. BMSCs were intermittently treated with 10^?6 M 4-nitroquinoline 1-oxide (4-NQO) from population doublings level (PDL) 3 until senescence occurred. Proliferation data demonstrated that BMSCs treated with 4-NQO bypassed the senescence phase and exhibited unlimited proliferation and anchorage independence. These cells underwent a malignant transformation that resulted in tumor formation in 12/12 immunodeficient mice that received the cells by tail vein injection. In contrast, spontaneous transformation of BMSCs was observed in 6/12 immunodeficient mice injected with BMSCs that had been cultured over PDL 30 in vitro. For both BMSCs treated with 4-NQO, and BMSCs maintained in long-term culture, their transformation into neoplastic cells was found to involve chromosomal abnormalities, increased telomerase activity, and reduced, or absent, expression of p53. Our results also indicate that BMSCs are susceptible to carcinogen-induced malignant transformation rather than spontaneous transformation. Therefore, carcinogen-induced BMSCs transformation models may be ideal for studying mechanisms associated with the promotion of tumor formation by chemical carcinogens.     

Application of a wheat seedling assay for detecting aneuploidy induced by N-ethyl-N-nitrosourea and 4-nitroquinoline-1-oxide.

N-Ethyl-N-nitrosourea (ENU) and 4-nitroquinoline-1-oxide (4NQO) were evaluated in the allohexapolyploid wheat seedling assay developed by Redei and Sandhu (1988), for its ability to induce aneuploidy and/or small chromosome deletions. The wheat strain used (Neatby's virescent) is homozygous for a pair of recessive alleles (v1) present on chromosome 3B and produces virescent seedlings grown at temperatures below 26 degrees C. When the developing embryos are treated with a test chemical, loss of chromosome 3B or its segment bearing the v1 allele in a progenitor cell produces a green sector in the leaf, whereas a gain of this chromosome induces a white sector. ENU and 4NQO induced dose-dependent increases in the frequency of leaf sectors at concentrations ranging from 0.128 to 1.280 mM and 0.052 to 0.263 mM, respectively. The assay is very simple and can be employed for evaluating the genetic potential of chemicals in a laboratory as well as for in situ hazards assessment under natural environmental conditions.     

Mechanism of increased susceptibility to 4-nitroquinoline-1-oxide in cultured skin fibroblasts from patients with familial polyposis coli

About 50% of the strains of cultured fibroblasts from patients with familial polyposis coli (FPC) exhibited increased susceptibility to cytotoxicity of 4-nitroquinoline-1-oxide (4NQO) compared with cells from normal individuals. The FPC cells that showed hyper-sensitivity to 4NQO were also hyper-sensitive to mitomycin C (MMC), but susceptibilities of these cells to UV radiation, methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were within the normal range. The extent of single-strand scission of DNA in the 4NQO-sensitive FPC cells was greater than in normal cells, and the amount of [14C]4NQO bound to DNA in the FPC cells was twice as high as in normal cells. The rate of release of [14C]4NQO from DNA by the post-culture was the same as in both FPC and normal cells. The 4NQO-sensitive FPC cells exhibited increased 4NQO-reductase activity; the level of this activity was consistent with the extent of the decrease in colony formation by 4NQO. These results suggest that the enhanced ability to activate 4NQO might be an important factor in the mechanism of susceptibility of FPC cells to 4NQO rather than the reduced ability to repair DNA.     

Guanyl-C8-arylamination of DNA by the ultimate carcinogen of 4-nitroquinoline-1-oxide: a spectrophotometric titration

Native and denatured DNAs and polynucleotides were modified by 4-acetoxyaminoquinoline-1-oxide, the ultimate carcinogen of 4-nitroquinoline-1-oxide (4 NQO). The N-( deoxyguanosin -C8-yl)-4-aminoquinoline-1-oxide adduct, the so-called "dG III," was quantified on the DNA and on poly(dG-dC) in absorption spectroscopy, by using a spectral property of dG III, i.e., the variation of the absorption spectrum as a function of the pH. Using the "free-dG III" absorption reference spectra, a simple graphic determination of the percentage of dG III was established by recording the absorption spectra of the 4-acetoxyaminoquinoline-1-oxide-modified polymers. It was found that the dG III adduct accounts for about 30% of the total modification in the case of native modified DNA and poly(dG-dC) and for about 70% in the case of denatured modified DNA.     

Lactic acid bacteria isolated from dairy products inhibit genotoxic effect of 4-nitroquinoline-1-oxide in SOS-chromotest

Antigenotoxic activity against 4-nitroquinoline-1-oxide (4-NQO) of lactic acid bacteria isolated from commercial dairy products was studied using SOS-Chromotest. The supernatants from bacteria-genotoxin co-incubations in general exhibited a strong suppression on SOS-induction produced by 4-NQO on the tester organism Escherichia coli PQ37 (sfiA::lacZ). High genotoxicity inhibition (>75%) was found for 31/67 of the examined bacteria and the maximum values of some strains within the species were as follows: Lactobacillus casei, 99.1%; L. plantarum, 93.3%; L. rhamnosus, 93.4%; L. acidophilus, 90.9%; L. delbrueckii subsp. bulgaricus, 85.7% and Bifidobacterium bifidum, 89.6%; Strains with low antigenotoxicity (5-60%) were evidenced in both L. acidophilus and L. delbrueckii subsp. bulgaricus, whereas some inactive strains were found only in L. casei and L. delbrueckii subsp. bulgaricus. Cell exposure to 100 degrees C for 15 min prevented antigenotoxicity and no effect was evidenced for cell-free spent media. The active strains survived at 0.1 mM 4-NQO exposure and generally presented some relevant functional properties, such as tolerance to bile (0.5%) or acid environment (pH 2.0) and adherence to Caco-2 enterocytes. Antigenotoxicity was always associated with modification of the 4-NQO absorbance profile.