Strand breaks of mammalian mitochondrial DNA induced by carcinogens.

Closed circular mitochondrial DNA in mammalian cells was degradated to the open circular form by exposure of the cells to the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline 1-oxide (4NQO). MNNG caused more strand scission of mitochondrial DNA than 4NQO at the same concentration. The action of the carcinogens on mitochondrial DNA did not parallel that with nuclear DNA which was damaged by 4NQO more markedly than by MNNG. Mitochondrial DNA damaged by carcinogens was not repaired during 4-20 h of post-treatment incubation of the cells. Incorporation of labeled thymidine into the closed circular mitochondrial DNA, decreased by the treatment of cells with carcinogens, recovered during post-treatment incubation.     

A UV-resistant mutant without an increased repair synthesis activity, established from a UV-sensitive human clonal cell line

When cells of a human clonal cell line, RSa, with high sensitivity to UV lethality, were treated with the mutagen, ethyl methanesulfonate, a variant cell strain, UVr-1, was established as a mutant resistant to 254-nm far-ultraviolet radiation (UV). Cell proliferation studies showed that UVr-1 cells survived and actively proliferated at doses of UV-irradiation that greatly suppressed the proliferation of RSa cells. Colony-formation assays also confirmed the increased resistance of UVr-1 cells to UV. The recovery from a UV-induced inhibition in DNA synthesis, as [methyl-3H]thymidine uptake into cellular DNA, was more pronounced in UVr-1 cells than in RSa cells. Nevertheless, there was no significant difference in the activity of UV-induced DNA repair synthesis in either cell line, as estimated by the extent of unscheduled DNA synthesis and DNA repair replication. UVr-1 cells were also more refractory to 4-nitroquinoline 1-oxide (4NQO), but the activity of DNA repair synthesis induced by 4NQO in UVr-1 cells was much the same as in the RSa cells. Both N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) sensitivity and MNNG-induced DNA repair synthesis activity in UVr-1 cells were similar to that of RSa cells. These characteristics of UVr-1 cells are discussed in the light of a previously reported UV-resistant variant, UVr-10, which had an increased DNA repair synthesis activity.     

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.     

High sensitivity but normal DNA-repair activity after UV irradiation in Epstein--Barr virus-transformed lymphoblastoid cell lines from Chediak--Higashi syndrome

We established lymphoblastoid cell lines from 2 children with Chediak--Higashi syndrome (CHS), 2 xeroderma pigmentosum (XP) patients and control donors after transformation of peripheral lymphocytes by Epstein--Barr virus (EBV). We used these lymphoblastoid cell lines to investigate repair activity after ultraviolet irradiation. Cell survival of both CHS lymphoblastoid cell lines after irradiation by UV and treatment by 4-nitroquinoline 1-oxide (4NQO) fell between those of the XP and control cell lines. Unscheduled DNA synthesis of CHS cells after UV irradiation occurred at rates similar to those of control cells.     

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.     

Endoreduplication in cultured mammalian cells treated with 4-nitroquinoline I-oxide

Chinese hamster cells treated with 4-nitroquinoline 1-oxide (4NQO) developed diplochromosomes, indicating the induction of endoreduplication. The maximum ratio of diplochromosomes, about 3% of total mitoses, was reached 27 h after treatment with a concentration of 0.5 μg/ml (2.6·10^?6M) for 6 h. Various chromosomal aberrations other than changes in ploidy were observed in diplochromosomes. Spiral structures observed in diplochromosomes and the binding of 4NQO to proteins are discussed here.     

Characterization of a UV-resistant strain,UVr-10, established from a human clonal cell line,RSb, with high sensitivity to UV, 4NQO,MNNG and interferon

Characterization was performed of a UV-resistant variant strain, UV^r-10, derived from a human clonal cell line, RSb, with high sensitivity not only to the lethal effect of 254-nm far-ultraviolet (UV) irradiation but also to the effects of 4-nitroquinoline 1-oxide (4NQO) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and to the cell proliferation inhibition (CPI) effect of human leukocyte interferon (HuIFN-α) preparations.Colony-formation assays confirmed the increased resistance of UV^r-10 cells to both UV and 4NQO, but no increased resistance to MNNG. The marked recovery from the inhibition of the total cellular DNA synthesis of UV^r-10 cells, estimated by [methyl-³H]thymidine ([³H]dThd) uptake into the cellular DNA materials, was seen during 6 h after irradiation or 4NQO treatment even under the conditions without the recovery uptake into those of the parent RSb cells, but not during 6 h after MNNG treatment. Comparative studies on the activity of DNA repair synthesis between UV^r-10 and RSb cells, by measuring the extent of UV-, 4NQO- or MNNG-induced unscheduled DNA synthesis (UDS) and DNA repair replication, revealed an increased activity of UV^r-10 cells to UV and 4NQO but no significant increase of the activity to MNNG. These results suggest that increased DNA repair activities of a UV^r-10 cell line may account for its becoming resistant to the lethal effect of UV and 4NQO.Concerning the CPI effect of HuIFN-α, UV^r-10 cells showed increased resistance. Further, the DNA synthesis activity of UV^r-10 cells was not so inhibited by HuIFN-α exposure as that of RSb cells. However, HuIFN-α-exposed UV^r-10 cells showed more enhanced levels of activity of pppA(2′p5′A)_n synthetase (2–5A synthetase) than the exposed RSb, thus suggesting that HuIFN-α could exert enough intracellular effect even in UV^r-10 cells.The implication of the increased resistance of UV^r-10 cells to the effects of UV, 4NQO and HuIFN-α, but not to those of MNNG, is discussed.     

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.     

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.     

Comparative studies of the effects of carcinogenic and antitumor agents on the DNA replication of cultured mammalian cells

Cultured mouse L₅₁₇8Y cells were exposed to several carcinogenic and antitumor agents. After exposure to one of the agents, the cells were label with [³H]-thymidine for 20 min, and the DNA was subjected to alkaline sucrose gradient centrifugation immediately or after a chase period. This led us to classify the agents into 3 groups: (1) UV, 4-nitroquinoline-1-oxide (4NQO), N-methyl-N′-nitrosoguanidine (MNNG), nitrogen mustard and Mitomycin C. These were characterized by 20-min DNA labeling patterns showing the formation of small DNA and by the slowing down of their subsequent elongation. Replicated DNA strands would have gaps where “damage” was present on the parental strands. Subsequently, gap-filling replication would occur with or without repairing damage. (2) γ-rays. The 20-min DNA labeling profile displayed a larger size of DNA pieces and the subsequent elongation of this DNA was slightly affected. This probably due to a preferential depression of initiation DNA replication. (3) Methyl methanesulfonate (MMS) and low temperature (28°). The 20-min DNA labeling patterns were qualitatively similar to, but quantitatively different from those of non-irradiated control. The rate of DNA elongation was slightly retarded.     

Toxicity of 4-Nitroquinoline 1-Oxide for Crithidia fasciculata

Growth inhibition of Crithidia fasciculata by 4-nitroquinoline 1-oxide (NQO) was observed in defined and complex media at 28 C. Aromatic amino acids, cysteine, and nicotinic acid, among several other substances, were ineffective in overcoming NQO toxicity. Dicoumarol and bovine albumin reversed NQO inhibition. While bovine albumin probably acted by the extra-cellular binding of NQO, dicoumarol inhibited the activity of DT-diaphorase, which reduces NQO to 4-hydroxyaminonitroquinoline 1-oxide (HAQO). The DT-diaphorase from C. fasciculata had the same characteristics as the enzyme from rat liver. The specific protection by dicoumarol against NQO inhibition suggests that HAQO is the active toxic substance for C. fasciculata.     

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.     

Absence of DNA repair replication after chemical mutagen damage in Vicia faba

Exposure of human (Hela) cells to the mutagens 4-nitroquinoline-1-oxide (4NQO) and N-methyl-N′-nitro-nitrosoguanidine (MNNG) produces damage in DNA that is repaired by a mechanism involving the insertion of new bases into DNA (repair replication). Vicia faba root tips, either from soaked seeds containing non-proliferating cells or from growing roots, do not perform detectable amounts of repair replication even though the mutagens inhibit DNA synthesis and cause chromosome aberrations. In view of similar failures to resolve excision in Chlamydomonas, Haplopappus, and Nicotiana after irradiation with UV light and in Vicia faba after X-irradiation it appears that plants in general might lack this repair process.     

The combined action of chemical carcinogens on DNA repair in human cells

Summary Excision repair was studied in normal human and ataxia telangiectasia (AT) cells proficient in repair of UV and its mimetic chemicals, and in xeroderma pigmentosum group C (XP C) cells (deficient in repair of UV and its mimetics), after treatment with several combinations of chemical carcinogens, by the photolysis of bromodeoxyuridine incorporated into parental DNA during repair. Results indicate that repair was additive in AT, and XP C cells treated with N-acetoxy-2-acetylaminofluorene (AAAF) plus ethyl methanesulfonate (EMS) or methyl methanesulfonate (MMS) indicating that there are different rate limiting steps for removal of both types of damage. Data on the combinations of 4-nitroquinoline 1-oxide (4NQO) plus MMS or EMS are difficult to interpret, but they do not indicate inhibition of DNA repair.Research carried out under the auspices of the U.S. Dept. of Energy     

Further evaluation of an in vivo micronucleus test on rat and mouse skin: results with five skin carcinogens

In a previous paper, we presented a practical in vivo micronucleus (MN) test that used rat skin as the target organ. To evaluate the test, as well as to determine the reproducibility and applicability of the method to mice, we used it to test the effect of five skin carcinogens (N-ethyl-N′-nitro-N-nitrosoguanidine (ENNG), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4NQO), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (B[a]P)) on rat and mouse skin. All five compounds significantly and dose-dependently increased the MN frequencies in the basal cells of the chemical-treated skin. These results indicated the reproducibility of the test results and also the applicability of the test to mice as well as rats.     

Characteristics of DNA repair of a UV-sensitive mutant (radC) of Dictyostelium discoideum

Summary A radiation-sensitive mutant, TW8(radC), of Dictyostelium discoideum is more sensitive to ultraviolet light (UV) killing than the parental wild strain NC4(RAD ⁺), but is resistant to 4-nitroquinoline 1-oxide (4NQO) at almost the same level as NC4. In TW8 amoebae, single-strand breaks of DNA molecules were hardly detectable immediately after UV irradiation, and the removal of pyrimidine dimers was depressed during the postirradiation incubation when compared with that of NC4 amoebae. After treatment with 4NQO, however, single-strand breaks were detected in TW8 amoebae. The almost complete rejoining of these breaks was also detected after the removal of 4HAQO-adducts. The TW8 amoebae have an efficient repair capacity against DNA damage caused by 4NQO, MMS, MMC and MNNG but not UV.Abbreviations 4NQO 4-nitroquinoline 1-oxide - MMS methyl methanesulphonate - MMC mitomycin C - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

A comparison of the DNA binding,cytotoxicity and repair synthesis induced in human fibroblasts by reactive derivatives of aromatic amide carcinogens

The cytotoxicity of three structurally-related direct-acting carcinogens, N-acetoxy-2-acetylaminofluorene, N-acetoxy-2-acetylaminophenanthrene and N-acetoxy-4-acetylaminobiphenyl, was compared in normal cells and in excision repair deficient xeroderma pigmentosum cells (XP12BE). All three proved significantly more cytotoxic to the XP cells than to the normal cells. At equicytoxic levels, substantially more residues were initially bound to the DNA of the normal cells than to the XP cells, suggesting that the former are able to remove a large percentage of the DNA bound residues before these can result in cell death. The ability of these cell strains to remove bound residues from DNA, to incorporate thymidine into parental strands of DNA during repair replication, and to recover from potentially lethal damage if held in the non-replicating, density-inhibited G_o state was compared as a function of dose and time. The XP12BE cells proved virtually incapable of excision repair of DNA damage induced by these carcinogens and of recovery. In contrast, normal cells recovered from the potentially lethal effects of these three compounds and did so at a rate comparable to their rate of removal of bound residues and of repair synthesis. In the excision-deficient XP12BE cells, DNA adducts induced by N-acetoxy-2-acetylaminophenanthrene proved 3- to 6-fold more cytotoxic than adducts induced by the other two carcinogens.     

Carcinogens on Regeneration

A microcrystal (ca 5 μg) of N -methyl- N '-nitro- N -nitrosoguanidine (MNNG) or 4-nitroquinoline-1-oxide (4NQO) was directly administered to the regeneration blastema on day 7 after amputation of a forelimb in the newt in order to analyze the effect of such potent carcinogenic substances on regeneration cells. Although neither MNNG nor 4NQO arrested regeneration completely, they caused great retardation of the regeneration cone formation followed by various abnormalities in the bony structures. Abnormal regenerants could be classified into the following four categories; (1) complete absence of both ulna and radius; (2) subregeneration or superregeneration of carpals and digits; (3) multiple disorganization of skeletal elements; (4) arrest of regeneration at the stage of regeneration cone. The polarity of regenerants developed after application of MNNG or 4NQO was very often shifted, during which the regeneration cone was always formed from the site where a microcrystal of the carcinogens was administered. The secondary regeneration initiated by reamputation of the regenerating limb, which had received the carcinogens at the early blastema stage, proceeded in the same way as observed in the case of a simple amputation. This suggested local and temporal effects of the carcinogens applied. Nevertheless, tumor formation has not induced in the newt limb so far. We can learn from these data that both MNNG and 4NQO only alter behaviour of the newt regeneration cells without excerting their carcinogenic effects on them, and that the newt cells are highly resistant and stable against the above-mentioned carcinogens.     

Identification of 4-acetoxyaminoquinoline from the hydrolysis of 1-acetoxy-4-acetoxyimino-1,4-dihydroquinoline,in vitro and in vivo properties

4-Acetoxyaminoquinoline (Ac-4-HAQ) (1) was identified as a hydrolysis product of 1-acetoxy-4-acetoxyimino-1,4-dihydroquinoline (diAc-4-HAQO). The reaction allowing the obtention of (1) obeys to a reduction mechanism implying the N₁-O cleavage. The carcinogenic properties of (1) observed by Sato et al. (Japan J. Exp. Med., 40 (1970) 475) in mice were studied in rats with the in vivo system we used previously with 4-nitroquinoline-1-oxide (4-NQO) and 4-hydroxyaminoquinoline-1-oxide (4-HAQO). In rats (1) does not covalently bind DNA. It was, therefore, possible to propose an interpretation of the results obtained by Enomoto et al. (Proc. Soc. Exp. Biol. Med., 136 (1971) 1206) who injected diAc-4-HAQO s.c. to mice and rats. Compound 1 could be responsible for the carcinogenic effects observed through the following pathway: (1) should be formed by hydrolysis of diAc-4-HAQO and reactivated by an enzymatic system to N-oxide derivative, the 4-acetoxyaminoquinoline-1-oxide (Ac-4-HAQO), which constitutes an ultimate carcinogen model of 4-NQO.     

Organ-specific DNA damage induced in mice by the organotropic carcinogens 4-nitroquinoline 1-oxide and dimethylnitrosamine.

The extent of DNA fragmentation induced in lung, kidney, and liver of mice injected with the chemical carcinogens 4-nitroquinoline 1-oxide (4NQO), dimethylnitrosamine (DMN) and the noncarcinogenic 4-aminoquinoline 1-oxide (4AQO) was estimated by the alkaline sucrose gradient technique. A floating of minced lung tissue pieces in the alkaline lysing solution on top of the gradients afforded a gentle method of lung DNA extraction. This technique minimized mechanical shearing of lung DNA and permitted comparisons to be made with liver and kidney DNA sedimentation patterns. The extent of DNA damage induced by 4NQO followed the order: lung, kidney, liver, while that induced by DMN followed the order: liver, kidney, lung. The sites of greatest DNA damage appeared to correlate with sites of high levels of DNA repair synthesis and the sites of tumor induction. No DNA damage was induced by the noncarcinogenic 4-aminoquinoline 1-oxide (4AQO).