Effects of inhibitors of DNA synthesis on spontaneous and ultraviolet light-induced sister-chromatid exchanges in Chinese hamster cells

Effects of inhibitors of DNA synthesis on spontaneous and ultraviolet light (UV)-induced sister-chromatid exchanges (SCE) were examined in a Chinese hamster cell line, V79 B-1. The inhibitors used were hydroxyurea (HU), 1-beta-D-arabinofuranosylcytosine (ara-C), aphidicolin (APC), 2',3'-dideoxythymidine triphosphate (ddTTP), neocarzinostatin (NCS), novobiocin (NB) and cycloheximide (CHX). HU, ara-C, and APC increased spontaneous SCE frequency, and had a synergistic effect on UV-induced SCE frequency. DdTTP, NCS and NB failed to show any statistically significant effect on either spontaneous or UV-induced SCE frequencies, though NCS and NB did slightly increase both spontaneous and UV-induced SCE frequencies. On the contrary, CHX decreased spontaneous SCE frequency, and more drastically, also UV-induced SCE frequency. These results are interpreted with respect to the replicating fork of DNA, a structure postulated to be involved in the formation of spontaneous and UV-induced SCE. A new model for SCE formation is proposed.     

Effect of prior X-irradiation on ultraviolet light-induced sister-chromatid exchange

Induction of sister-chromatid exchanges (SCEs) by ultraviolet light at 2.6 J/m2 in Chinese hamster ovary cells was significantly inhibited by pretreatment with 3 and 5 Gy of X-rays. No inhibition was observed when cells were treated first with ultraviolet light and subsequently X-irradiated with 5 Gy.     

Independent induction of sister-chromatid exchanges by 3-aminobenzamide and ultraviolet radiation in HeLa cells

The effect of 3-aminobenzamide in HeLa cells was examined in several aspects. 10(-3) M 3-aminobenzamide inhibited poly(ADP-ribose) polymerase activity to more than 95% in disrupted nuclei of HeLa cells. The mode of inhibition of the enzyme was competitive inhibition with NAD at a Ki value of 2.5 microM 3-aminobenzamide. The combined treatment of HeLa cells with 3-aminobenzamide and ultraviolet radiation revealed the independent induction of SCEs by these 2 agents.     

Influence of Neurospora endonuclease on trenimon-induced structural chromosomal aberrations and sister-chromatid exchanges in human peripheral lymphocytes and Chinese hamster ovary cells

Human peripheral lymphocytes and Chinese hamster ovary cells were treated in the G1 phase of the cell cycle with the trifunctional alkylating agent trenimon (TRN) and post-treated with a single-strand specific endonuclease from Neurospora crassa (NE). TRN induces chromosomal aberrations of the chromatid type (CA) and sister-chromatid exchanges (SCE). NE post-treatment leads to an elevation of the frequencies of CA but not of SCEs. This indicates that TRN induced CA are the result of DNA double-strand breaks and that the SCEs originate from other types of lesions, most probably base damage.     

Effect of copper coordination complexes on sister-chromatid exchanges in plant cells

The effectiveness of several copper coordination complexes in the induction of sister-chromatid exchanges (SCE) in meristematic cells of Allium cepa roots was studied. A significant increase in the frequencies of SCE was observed when cells were exposed to different concentrations of copper complexes for 3 h. This effect, which is concentration dependent, seems to be related to the possibility of intercalative binding modes between the planar copper complexes and DNA.     

3-Aminobenzamide synergistically increases sister-chromatid exchanges in cells exposed to methyl methanesulfonate but not to ultraviolet light

3-Aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, increased baseline sister-chromatid exchange (SCE) frequencies and acted synergistically with the alkylating agent methyl methanesulfonate to induce exchanges in Chinese hamster ovary and SV40-transformed human (GM637) cells. In contrast, 3-aminobenzamide did not affect the frequency of ultraviolet light-induced SCEs. Our data suggest that, in these 2 cell types, synthesis of poly(ADP-ribose) is more important in damage and repair after exposure to an alkylating agent than after exposure to ultraviolet light.     

Characterization of the enhancing effect of caffeine on sister-chromatid exchanges induced by ultraviolet radiation in excision-proficient xeroderma pigmentosum lymphoblastoid cells

Cells of some excision-proficient xeroderma pigmentosum (XP) cell lines are highly sensitive to post-UV caffeine treatment in terms of sister-chromatid exchange (SCE) induction as well as cell lethality. In the present study, we conducted a detailed investigation of the enhancing effect of caffeine on SCE frequency induced by UV in excision-proficient XP cells, and obtained the following results. (1) Continuous post-UV treatment with 1 mM caffeine markedly enhances UV-induced SCEs and such enhanced SCEs occur with similar frequency during either the 1st or the 2nd cell cycle in the presence of caffeine and 5-bromodeoxyuridine (BrdUrd). (2) The high sensitivity of the cells to post-UV caffeine treatment persists for at least 2 days after UV when irradiated cells are held in either the proliferating or the nonproliferating state prior to the addition of BrdUrd. (3) Caffeine exerts its effect on cells in S phase. (4) Neither BrdUrd in the medium nor the incorporated 5-bromodeoxyuridine monophosphate (BrdUMP) in DNA plays an appreciable role in the expression of the enhancing effect of caffeine. The most likely explanation for our findings is as follows. In excision-proficient XP cells, the cause of SCE formation such as UV-induced lesions or resulting perturbations of DNA replication persists until the 2nd round or more of post-UV DNA replication. If caffeine is given as post-UV treatment, such abnormalities may be amplified, resulting in a synergistic increase in SCE frequency.     

The ultraviolet endonuclease of bacteriophage T4. Further characterization.

The T4 ultraviolet endonuclease was previously shown to produce strand incisions (nicks) in ultraviolet-irradiated DNA on the 5' side of thymine dimers. The present studies demonstrate that the purified endonuclease creates 3'-OH and 5'-P termini at the sites of nicking. Photoreactivation of ultraviolet-sensitive sites, thereby demonstrating directly endonucleause has a molecular weight of approximately 18,000 and attacks ultraviolet-irradiated single-stranded Escherichia coli and M-13 DNA.     

Studies on the influence of photoreactivation on the frequencies of UV-induced chromosomal aberrations, sister-chromatid exchanges and pyrimidine dimers in chicken embryonic fibroblasts

Chicken embryonic fibroblasts, which possess photoreactivating enzyme were used to study the influence of photoreactivating light on the induction of pyrimidine dimers, sister-chromatid exchanges (SCEs) and chromosomal aberrations by 254 nm UV. While photoreactivation (PR) efficiently removed most of the induced dimers (75-95%), the frequencies of SCEs and chromosomal aberrations were reduced only by about 30-65%, in parallel experiments. Since pyrimidine dimers are the only photoreactivable photolesions known, the reduction in the frequencies of SCEs and chromosomal aberrations on PR has been interpreted as due to disappearance of pyrimidine dimers, implying that these lesions are the primary events responsible for the induction of the biological end points studied. The possible reasons for the lack of quantitative relationship between the frequencies of dimers and the frequencies of SCEs and chromosomal aberrations are discussed.     

Effect of sodium azide on sister-chromatid exchanges in human lymphocytes and Chinese hamster cells

Previous reports from this laboratory and others indicate that sodium azide is a unique mutagen. It is highly mutagenic in S. typhimurium TA1530 as well as in barley, rice, peas, yeast and Chinese hamster V79 cells. However, azide apparently does not produce chromosome breaks in barley, Vicia or human lymphocytes. Therefore, a study of the effects of azide on sister-chromatid exchanges (SCE) appeared warranted.Human whole blood and Chinese hamster K1 cell line were exposed for 4 and 2 h resp. to various concentrations of sodium azide ranging from 10⁻³ to 10⁻⁷ M. Cells were harvested and chromosomes stained by the FPG technique. In human lumphocytes, concentrations above 10⁻⁴ induced lethality whereas the K1 cell line was sensitive to concentrations above 10⁻⁵ M. The lower concentrations of azide produced no significant increase in SCE frequency above controls. Concurrent mitomycin C treatments produced significant increases in SCE levels.This apparent lack of induction of SCEs above background combined with previous data demonstrating negative clastogenic but very positive mutagenic activity of azide confirms the uniqueness of this mutagen. It would appear that azide is one of the few known potent mutagens that does not increase SCEs and/or break chromosomes.     

Effect of recombinant interferon-alpha on streptonigrin-induced chromosome aberrations and sister-chromatid exchanges in hamster cells

The effect of recombinant interferon-alpha-2a (rIFN-alpha-2a) on the induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) by the radiomimetic antibiotic streptonigrin (SN, 250 ng/ml, 20 min, 37 degrees C) in Chinese hamster ovary (CHO) cells was investigated. Recombinant IFN-alpha-2a (4500-45,000 IU/ml) was added to the cell cultures 30 min before SN and left in the culture medium until the end of SN treatment or until cell harvesting. A statistically significant increase in the frequency of CAs and SCEs was observed following treatment with SN (P < 0.05), whereas treatments with rIFN-alpha-2a alone did not produce any significant increase of CAs and SCEs over control values. Low rIFN-alpha-2a doses produced a reduction in the frequency of CAs and an increase in the yield of SCEs induced by SN, while high doses of the cytokine caused an increase in the yield of CAs and a reduction in the frequency of SCEs induced by the antibiotic. In addition, rIFN-alpha-2a caused a marked inhibition (around 50%) on the yield of SN-induced chromatid-type aberrations in the G(2) phase of the cell cycle. It is suggested that the inhibitory effect of rIFN-alpha-2a on the SN-induced chromosome damage is due to the stimulation of DNA synthesis and repair by the cytokine. On the other hand, our results give further support to our previous hypothesis that the induction of CAs and SCEs by SN is based on different mechanisms.     

Induction of chromosome aberrations and sister-chromatid exchanges in CHO-K1 cells by o-phenylphenol

o-Phenylphenol (OPP), is used in Japan as a fungicide in food additives for citrus fruits. The induction of chromosome aberrations and sister-chromatid exchanges (SCEs) by OPP in cultured Chinese hamster ovary (CHO-K1) cells was studied. Cells were exposed to various concentrations of OPP ranging from 50 to 175 micrograms/ml for 3 h, and further incubated for 27 and 42 h. These incubation periods are almost equal to 2 and 3 cell cycles. SCEs and chromosome aberrations were induced by OPP at concentrations of 100, 125 and 150 micrograms/ml after the incubation for 27 h. For chromosome aberrations, chromatid breaks and exchanges there was a dose-dependent increase. Diplochromosomes due to endoreduplication were also caused by the same concentrations of OPP in a dose-dependent manner. After incubation for 42 h, chromosome aberrations were also increased by OPP at concentrations of 100 and 125 micrograms/ml, but the frequencies of SCEs were not significantly different from those of the control. These results suggest that OPP has a cytogenetic toxicity, and that the DNA damage resulting in SCEs induced by OPP is relatively short-lived and can be repaired during the longer incubation time.     

Influence of beta-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells

The influence of beta-myrcene (MC) on sister-chromatid exchanges (SCE) in V79 cells induced by 4 S9 mix-activated indirect mutagens was studied. The mutagens used were cyclophosphamide (CP), benzo[a]pyrene (BP), aflatoxin B1 (AFB) and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). MC effectively inhibited SCEs induced by CP and AFB in a dose-dependent manner, but it had no effect on SCE induction by BP and DMBA. MC also reduced CP-induced SCE frequencies in a hepatic tumor cell line (HTC). These cells are metabolically competent and activate CP into its biologically active metabolites. Our results support the suggestion that MC modulates the genotoxicity of indirect-acting mutagens by inhibiting certain forms of the cytochrome P-450 enzymes required for activation of premutagens like CP and AFB.     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.