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.     

Hypersensitivity of xeroderma pigmentosum cells to dietary carcinogens

Xeroderma pigmentosum patients, in addition to ultraviolet-induced skin cancers, have an increased prevalence of neoplasms occurring in sites shielded from ultraviolet radiation. We postulated that these internal neoplasms might be related to ingestion of dietary carcinogens. As model dietary carcinogens, we studied the tryptophan pyrolysis products, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). These dietary compounds bind to DNA and are highly mutagenic and carcinogenic. Cytotoxicity of these compounds was examined in cultured lymphoblastoid cell lines from xeroderma pigmentosum patients in complementation groups A, B, C, D and E and the variant form and from normal donors. All xeroderma pigmentosum lymphoblastoid cell lines showed a greater reduction in viable cell concentration than the 2 normal lymphoblastoid cell lines following addition of Trp-P-1 or Trp-P-2 (5 micrograms/ml) to the culture medium. Possible differences in cellular activation of these compounds were overcome by treating the cells with rat-liver microsome-activated Trp-P-2. There was a greater reduction in viable cell concentration in the xeroderma pigmentosum group A and D cells than in the normal lymphoblastoid cell lines after treatment with activated Trp-P-2. These data suggest that the xeroderma pigmentosum DNA-repair system is defective in repairing Trp-P-1 and Trp-P-2 induced DNA damage in addition to being defective in repairing ultraviolet-induced DNA damage. Thus xeroderma pigmentosum patients may be at increased risk of toxicity from some dietary carcinogens.     

Repair of 4-nitroquinoline-1-oxide-induced DNA damage in normal human cells and cells from classical and variant xeroderma pigmentosum

The effect of 4-nitroquinoline-1-oxide (4NQO) upon 3 fibroblast cell lines derived from normal and xeroderma pigmentosum subjects have been compared. Excision-deficient XP cells (XP2BI), complementation group G, are nearly 200-fold more sensitive than normal cells to the lethal effect of 4NQO while XP variants (XP7TA), are 2-fold more sensitive. This cytotoxicity correlates with the levels of unscheduled DNA synthesis performed by the 3 cell lines. 4NQO causes a dose-related inhibition of DNA replication in all cell lines. However, newly replicated DNA synthesised immediately after treatment of cells with 4NQO is slightly smaller in XP7TA variant cells than in normal cells receiving the same dose of 4NQO, but DNA fragments in excision-deficient XP2BI are 50% smaller. It is likely that replicon elongation and joining together of newly replicated DNA fragments is dependent upon the excision of certain 4NQO-induced lesions, possibly normally repaired by a 'short-patch' repair process defective in XP2BI.     

An improved method for the detection of differential survival between normal and xeroderma pigmentosum lymphoblastoid cell lines in culture with 4-nitroquinoline-1-oxide

The effects of the UV-mimetic chemical 4-nitroquinoline-1-oxide (4-NQO) upon cell lines heterozygous or homozygous for the recessive mutant xeroderma pigmentosum (XP) were investigated. Human lymphoblastoid cell lines, which were established from 4 XP homozygote patients (XPL15, XPL17, XPL19 and XPL20). 2 XP heterozygote individuals (XPPL17 and XPML17) and 58 normal individuals, were cultured in the presence of 4-NQO at doses of 0, 2, 4 and 8 x 10(-6) M. Then the total cell number was counted and the viability of the cells was measured by the dye exclusion method using trypan blue and a newly devised fluorometric method with fluorescein diacetate. Results showed that 4-NQO affected, in increasing order of impairment, the cell lines: normal less than XP heterozygote less than XP homozygote.     

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.     

Number of chromosome aberrations induced by chemical carcinogens in the cells of patients with 2 forms of xeroderma pigmentosum

A study was made of the effects of a chemical mutagen of the "gamma-type"--methylmethansulfonate (MMS) and of mutagen of the "UV-type"--4-nitroquinolin-1-oxide (NQO) and 7-brommethylbenz(alpha)antracen (BMBA) exerted on chromosome aberration frequency in lymphocytes of patients with classical Xeroderma pigmentosum and with a so-called form II of the disease on different stages of the cell cycle. Mutagens were added to PHA stimulated lymphocyte cultures every 3 hours, simultaneously with pulse 3H-thymidine labelling, to fix the stage of the cell cycle at the moment of treatment. NQO and BMBA treatments were found to increase the frequency of chromosome aberrations in classical XP cells, whereas MMS was not found to. In the XP II cells, defective in repair of both UV and gamma damaged DNA, chromosome aberrations yield is higher than in normal cells after all the three mutagens treatment. The data obtained show the correlation between DNA repair and chromosome aberrations yield.     

Interferon as a protector in human cells treated with 4-nitroquinoline-1-oxide

Interferon having the activity 25-37 and 375 IU/ml decreases the frequency of chromosome aberrations induced with the cancerogen 4-nitroquinoline-1-oxide in diploid human cells. The protective effect of interferon is not connected with stimulation or inhibition of cell division. It has been revealed that interferon with the activity 25-37 IU/ml has a stimulating effect on mitotic activity of cells, while interferon with the activity 375 IU/ml inhibits cell division.     

Chromosomal hypersensitivity in mutant M10 and Q31 mouse cells exposed to ultraviolet radiation (UV) and 4-nitroquinoline-1-oxide (4NQO)

2 mutant mouse cells M10 and Q31 were examined for chromosomal aberrations induced by ultraviolet radiation (UV) and 4-nitroquinoline-1-oxide (4NQO), as compared with mouse lymphoma L5178Y cells. Q31 cells are UV- and 4NQO-sensitive cells isolated from L5178Y cells. M10 cells are similar but are sensitive to ionizing radiation and 4NQO. After treatment with UV or 4NQO, chromatid-type aberrations in these cell strains were induced more frequently in the first mitotic cells, at late fixation times. After UV exposure (2.4 J/m2), the maximal frequencies of chromatid-type breaks in Q31 cells were about 5 times higher than in L5178Y cells. In M10 cells such breaks were only as frequent as in L5178Y cells. After 4NQO treatment (50 ng/ml) the frequencies of chromatid-type breaks in M10 and Q31 cells were significantly higher than in L5178Y cells. From these results and those of previous studies (Takahashi et al., 1982), M10 cells may be considered hypersensitive to gamma-rays and 4NQO, but not to UV, and thus react similarly to L5178Y cells. The hypersensitivity of M10 cells to 4NQO may result from a defect in the ionizing-radiation repair mechanism as has been suggested to occur in ataxia telangiectasia (AT) cells. Q31 cells are hypersensitive to UV and 4NQO, but not to gamma-rays. Q31 cells may be considered to be deficient in a UV-like repair pathway. In conclusion, characteristics of murine M10 and Q31 cells are compared with those of human AT and xeroderma pigmentosum (XP) cells.     

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.     

DNA damage, DNA repair and chromosome aberrations of xeroderma pigmentosum cells and controls following exposure to nitrosation products of methylguanidine

Nitrosation of methylguanidine (MG) led to products that caused DNA fragmentation (shift in sedimentation profiles of velocity centrifugation through alkaline sucrose gradients), a DNA repair synthesis (unscheduled uptake of (³H]TdR), chromosome aberrations and a lethal effect of cultured human fibroblasts. The response of repair-deficient xeroderma pigmentosum cells did not differ from that of controls. The nitrosation of MG must be carried out at a pH level below 3, in order to obtain products that react with cellular DNA. The results show that a DNA repair synthesis of human fibroblasts appear to be a sensitive assay for carcinogenic and mutagenic nitrosation products which may be formed within an organism from non-carcinogenic compounds.     

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.     

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.