Effects of pH in the in vitro chromosomal aberration test

The relation between the pH of the medium and clastogenic activity was studied in Chinese hamster ovary (CHO) K1 cells in vitro. The pH was adjusted with NaOH, KOH, HCl or H2SO4. No clastogenic activity was observed over the initial pH range of 7.3-10.9 without S9 mix, but a few chromosomal aberrations were induced at pH 10.4 with S9 mix. The frequency of aberrations increased with the increase in amount of S9. At acidic pH, many chromatid breaks were induced at initiatial pH 5.5 or below without S9 mix, and aberrations such as chromatid breaks and chromatid exchanges were induced at initial pH 6.2 or below with S9 mix. Using MES and Bis-Tris as buffers instead of sodium bicarbonate, we observed that aberrations of the chromatid break type were inducible at pH 6.2 or below. These results show that the combination of strong alkalinity and S9 is clastogenic to CHO-K1 cells, and also that weakly acidic media are genetically active. The results indicate that incubations at non-physiological pH might give false-positive responses.     

cis-diamminedichloroplatinum(II)-induced sister-chromatid exchanges and chromosome aberration formation in cultured human lymphocytes and their inhibition by sodium thiosulfate

cis-Diamminedichloroplatinum(II) (cis-DDP)-induced sister-chromatid exchanges (SCEs) and chromosome aberration formation were studied in human lymphocytes. The mitotic index decreased abruptly at 2 X 10(-6) M cis-DDP and the frequency of SCEs was dose-related; a marked increase was recorded at 10(-6) M cis-DDP. A dose-dependent effect was also found for chromosome aberration formation at concentrations between 10(-11) and 4 X 10(-6) M. The aberrations observed were primarily chromatid breaks and gaps. We also examined the inhibition of these genotoxicities by treating the cells with sodium thiosulfate (STS). Simultaneous treatment with 10(-4)-10(-3) M STS (100-1000-fold molar ratio to cis-DDP) significantly reduced the frequency of SCEs induced by 10(-6) M cis-DDP. Furthermore, a 3-h delay in treating with STS significantly reduced cis-DDP-induced SCEs, but not chromosome aberration formation.     

The effects of radon daughter alpha-particle irradiation in K1 and xrs-5 CHO cell lines

We investigated the radiobiological effects of the radon daughter bismuth-212 (212Bi) in Chinese hamster ovary (CHO) K1 cells and in xrs-5 cells, which are X-ray sensitive and deficient in the ability to rejoin DNA double-strand breaks. The cells were exposed to 250 kVp X-rays or to 212Bi chelated to diethylene triamine pentaacetic acid (DTPA); chelation of 212Bi to DTPA prevented its attachment to or entry into the cells. Cytotoxic, clastogenic, and mutagenic responses of the cells were measured and RBEs (D10, 2 chromatid aberrations/cell and 10 induced 6-thioguanine-resistant mutants) were calculated to be 3.8, 3.5, and 3.9, respectively for K1, and 1.4, 0.8, and 5.1, respectively, for xrs-5. With the exception of the RBE of less than 1 for alpha-induced aberrations in xrs-5, the results are consistent with the following conclusions: (1) alpha-particles are in general more effective cytotoxic, clastogenic and mutagenic agents than X-rays; (2) the primary lethal and clastogenic lesion induced by both X-rays and alpha-particles is probably a DNA double-strand break; (3) DNA double-strand breaks induced by alpha-radiation are less well repaired than those induced by X-rays, although a portion of alpha-induced damage is repairable; and (4) deficiencies in rejoining DNA double-strand breaks affect the clastogenic and cytotoxic effects of X-rays and alpha-radiation, not their mutagenic effects. The RBE of 0.8 for aberration induction in xrs-5 cells could reflect a deficiency in the ability of these cells to convert alpha-induced damage to chromosome aberrations. Alternatively, the RBE of less than 1 might reflect an unusual sensitivity of xrs-5 cells to alpha-induced G2 delays.     

Induction by chemical clastogens of aberrations in prematurely condensed interphase chromatin of Chinese hamster ovary cells

The clastogenic activities of diepoxybutane and bleomycin were comparatively studied on prematurely condensed interphase chromatin and metaphase chromosomes of Chinese hamster ovary cells. The yield of chromosomal aberrations was distinctly higher in G2-premature chromosome condensation as compared to metaphase. Most notably, the clastogenic activity of bleomycin was visible in premature chromosome condensation after application of much lower final concentrations than necessary for induction of chromosome aberrations in metaphase. In addition, the different mechanisms of action of both clastogens were reflected by the aberration yield in GI and G2 immediately after exposure. While bleomycin induced aberrations throughout all stages of interphase, diepoxybutane did not induce aberrations in GI or G2. Though certainly not a routine system for genotoxicity testing, premature chromosome condensation analyses provide a powerful opportunity to demonstrate relationships between DNA damage and repair, and the production of chromosomal changes at the site of their formation.Abbreviations BM bleomycin - BrdUrd bromodeoxyuridine - CHO Chinese hamster ovary - DEB diepoxybutane - DMSO dimethylsulfoxide - FCS fetal calf serum - PCC premature chromosome condensation, prematurely condensed chromosomes - PEG polyethylene glycol     

Clastogenic factors in plasma of HIV-1 infected patients

The objective of this study was to investigate the clastogenic activity of plasma ultrafiltrates from HIV-I infected patients. Clastogenic factors are chromosome-damaging agents with low molecular weight (<10,000 daltons) which cause chromosome aberrations, sister chromatid exchanges, DNA strand breakage, and gene mutation. They have first been described in the plasma of irradiated persons, but they are also found in hereditary breakage syndromes and chronic inflammatory diseases with autoimmune reactions. Their formation and their clastogenic effects are modulated by superoxide anion radicals. We analyzed a total of 22 HIV-1 positive patients in comparison to 20 reference plasma samples from healthy HIV negative blood donors of similar age. The plasma ultrafiltrates (filter cutoff 10,000 daltons) from patients induced a statistically significant increase in chromosomal breakage in the cytogenetic test system (20.5 ± 6.8 aberrations per 100 cells), while no increase was observed in test cultures exposed to plasma ultrafiltrates from healthy blood donors (6.3 ± 2.9 aberrations per 100 cells). The breakage values were slightly, but not significantly, lower in the 10 patients with more than 200 T-helper cells/ml (18 ± 4 aberrations per 100 cells), than in the 12 patients with less than 200 T-helper cells/ml (22.3 ± 7.9 aberrations per 100 cells). HIV patients with high clastogenic activity (induction of more than 20 aberrations per 100 cells, range 20 to 39) showed higher plasma levels for malondialdehyde than those with lower clastogenic activity (less than 20 aberrations per 100 cells, range 12 to 18). However, the difference was statistically not significant. Another lipid peroxidation product, 4-hydroxynonenal, was increased equally in both groups. There were no significant differences in water- and lipid-soluble plasma antioxidants between the low- and high-breakage group. In agreement with previous findings, the clastogenic effects of plasma ultrafiltrates in the test cultures were reduced by the antioxidant enzyme superoxide dismutase. The presence of clastogenic factors in the plasma of HIV patients is further evidence for a prooxidant state in these persons. Since clastogenic factor formation appears to occur at an early stage of the disease, it may be significant for virus release or activation, because of the superoxide anion stimulating effects of clastogenic factors. From a practical standpoint, clastogenic factors may be useful for evaluation of promising drugs.     

Chromosomal aberrations and sister-chromatid exchanges induced by N-nitroso-2-acetylaminofluorene and their modifications by arsenite and selenite in Chinese hamster ovary cells.

The frequencies of chromosomal aberrations (CA) and sister-chromatid exchanges (SCE) in Chinese hamster cells were significantly increased by the direct-acting mutagen N-nitroso-2-acetylaminofluorene (N-NO-AAF) at the concentration of 0.1 mM. N-NO-AAF was prepared by nitrosation of the protohepatocarcinogen 2-acetylaminofluorene. The induced CA, which included chromatid breaks, chromatid exchanges, chromosome breaks, and chromosome ring formation were significantly potentiated by the presence of sodium arsenite (10 microM), but not by hydroxyurea (20 mM) or cytosine arabinoside (25 microM). On the other hand, the clastogenic effect of N-NO-AAF was effectively inhibited by sodium selenite (100 microM). Arsenite (10 microM) was shown to be moderately active in CA induction which was partially blocked by the presence of selenite (10 nM). N-Nitroso compounds such as N-nitroso-N-methylurea, N-nitroso-N-ethylurea and N-methyl-N'-nitro-N-nitrosoguanidine were equally or more active in the induction of CA and SCE in CHO cells when compared with N-NO-AAF. The cell cycle was significantly delayed by the intervention of N-NO-AAF.     

Cytogenetic effects of 2-methoxyethanol and its metabolite,methoxyacetaldehyde, in mammalian cells in vitro

Glycol ethers such as 2-methoxyethanol (2-ME) are reproductive toxins. The genotoxicity of 2-ME, especially its metabolites: methoxyacetaldehyde (MALD) and methoxyacetic acid (MAA), is not adequately investigated yet. We have shown previously that MALD induced mutation in the bacterial gpt gene which is inserted in an autosome of CHO-AS52 cell line but not in the hprt gene on the X chromosome of CHO-K1-BH4 cell line. These data suggest that MALD induces major deletion-type mutation. If this prediction is correct we would expect to observe that MALD is an efficient inducer of chromosome aberrations in both CHO cell lines. We have conducted a cytogenetic study using both CHO cell lines and human lymphocytes to investigate this phenomenon. Our results show that human lymphocytes treated with 10–30 mM MALD for 1 h or 0.05–0.5 mM MALD for 24 h induced significant dose-dependent increase of sister-chromatid exchanges (SCE) (p < 0.05). It also induced significant dose-dependent increase (p < 0.05) of chromosome aberrations in human lymphocytes (10–40 mM treated for 1 h, or 0.05–2.5 mM for 24 h) and in both CHO cell lines (1.25–20 mM for 3 h). Treatment of these cells with the parent compound, 2-ME did not induce chromosome aberrations nor SCE unless very high doses of the chemical were used. In conclusion, these results indicate that MALD is clastogenic to different cell types therefore it is potentially carcinogenic. The genotoxic effects of 2-ME in humans will be dependent upon the metabolic capability of individuals to bioactivate 2-ME to MALD.     

Genotoxicity of 2-amino-6-N-hydroxyadenine (AHA) to mouse lymphoma and CHO cells.

2-Amino-6-N-hydroxyadenine (AHA) treated L5178Y/TK (+/-)-3.7.2C mouse lymphoma cells were evaluated for mutations at the tk, hgprt, and Na+/K+ ATPase loci, as well as for gross chromosome aberrations and induction of micronuclei. In addition, AHA was evaluated for its ability to induce HGPRT mutants in CHO cells. AHA was found to induce mutations at all evaluated loci and in both cell types. The TK mutants were primarily large colonies although a few small colonies were also induced, particularly at the higher concentrations. Preliminary cytogenetic analysis of AHA-treated mouse lymphoma cells indicated that some gross aberrations but not micronuclei were induced. The 20 small-colony TK mutants evaluated by banded karyotype indicate that only a small fraction (2 of 20) showed chromosome 11 abnormalities. From these studies, it appears that AHA may be one of a very few chemicals that is capable of inducing multi-locus point mutations, with only slight clastogenic activity. Particularly at the higher concentrations, some of the mutants may contain multi-locus point mutations that result in slow growth.     

Chromosome damage in rat pulmonary alveolar macrophages following ozone inhalation

To determine whether ozone is clastogenic at environmentally relevant exposure levels, rats were exposed for 6 h to 0.0, 0.12, 0.27, or 0.80 ppm ozone. The alveolar macrophages were isolated from animals sacrificed 28 h after the end of the exposure. The mitotic index and frequency of chromosome aberrations were determined. No change in the mitotic index was detected following 0.12 ppm ozone exposure. A significant decrease in mitotic index was observed after exposure to 0.27 ppm ozone; a significant (4-fold) increase in the frequency of dividing macrophages was detected following exposure to 0.8 ppm ozone. Only chromatid-type aberrations were observed. There was a significant increase in the frequency of cells with chromatid gaps and in the frequency of cells with chromatid deletions. Animals exposed to 0.27 ppm ozone had the highest proportion of cells with chromatid deletions (0.172) relative to background level (0.028). No exchanges or chromosome-type aberrations were detected in any of the animals. These data suggest that ozone, at relatively low levels, is clastogenic in macrophages from exposed rats.     

Genotoxicity studies of di(2-ethylhexyl)phthalate and its metabolites in CHO cells

The widely used plasticizer di(2-ethylhexyl)phthalate (DEHP), its hydrolysis products mono(2-ethylhexyl)phthalate (MEHP) and 2-ethylhexanol, and also phthalic acid have been tested for clastogenic activity in cultured Chinese hamster ovary (CHO) cells. Only MEHP was found to cause chromosome damage. MEHP was without effect in the SCE and HGPRT mutation test in CHO cells. The clastogenicity of MEHP suggests a role for this compound in the observed carcinogenicity of DEHP and its positive effect in the dominant lethal assay.     

Effects of G2 treatments with inhibitors of DNA synthesis and repair on chromosome damage induced by X-rays and chemical clastogens in root tips of Vicia faba. Comparison with corresponding effects in cultured human lymphocytes

The frequencies of chromatid aberrations produced in roots of Vicia faba by clastogenic (chromosome-damaging) agents were strongly enhanced by exposing the root-tip cells to inhibitors of DNA synthesis during the G2 phase. Chromosome damage produced by both S-dependent (maleic hydrazide, methyl methanesulfonate, thio-TEPA) and S-independent (X-rays, streptonigrin) mechanisms was enhanced by the inhibitor treatments. The types of aberrations affected by the inhibitors were mainly chromatid gaps and breaks and isochromatid breaks of the non-union type. Most effective among the inhibitors tested were hydroxyurea (HU) and 5-fluorodeoxyuridine (FdUrd). Post-treatments with caffeine were effective in enhancing clastogen-induced chromosome damage when given during the S phase. All types of aberrations, exchanges as well as breaks, were enhanced by the post-treatments. When given during the G2 phase, caffeine enhanced only the frequency of chromatid aberrations produced by X-rays. The enhancement was slight and obtained only when the cells were irradiated in the G2 phase and immediately post-treated with caffeine. Clastogen-treated cultures of human lymphocytes responded to post-treatments with inhibitors of DNA synthesis in very much the same way as clastogen-treated root-tip cells of Vicia faba. Thus, the frequencies of chromatid gaps and breaks and isochromatid breaks of the non-union type were strongly enhanced by exposing clastogen-treated lymphocytes to inhibitors of DNA synthesis during the G2 phase. The efficiency of the inhibitors, however, varied considerably in the two materials. On the whole, the number of inhibitors capable of enhancing induced chromosome damage was much larger in lymphocytes than in bean root tips. Only HU was equally effective in both materials. The most striking difference between the two materials was found when caffeine was given as a post-treatment. Thus, in human lymphocytes the frequencies of chromatid aberrations induced by most clastogenic agents were strongly enhanced when caffeine was given during the G2 phase, but little affected by post-treatments with caffeine during the S phase.     

Mutagenic activity of anticancer agent cis-dichlorodiammine platinum-II.

cis-Dichlorodiamminoplatinum-II (cis-DDP) has been widely used as an anticancer chemotherapeutic agent. The mutagenicity of cis-DDP was investigated in vitro and in vivo using sister-chromatid exchange analysis and the analysis of chromosomal aberrations. Parallel human lymphocyte cultures were incubated with and without the addition of BrdU at 4 concentrations of cis-DDP. Significant increases in SCE rate were observed at 0.25 micrograms/ml and higher, showing a clear dose-response relation between SCE rate and cis-DDP concentration. A significant increase in chromosome breakage and tetraradial figures was observed in BrdU free cultures treated with cis-DDP again showing a dose dependency. Analysis of the distribution of cells in the first, second and third division in cis-DDP treated cultures demonstrated the depressing effect of the drug on mitotic activity. In vivo analysis of SCE and chromosome aberrations in mouse showed that 13.85 mg/kg i.p. of cis-DDP produces significant increases in the rate of SCE and chromosome aberrations in bone-marrow cells.     

XRCC1 protects cells from chromate-induced chromosome damage, but does not affect cytotoxicity

Hexavalent chromium Cr(VI) is a well known human carcinogen. This genotoxic metal induces DNA strand breaks and chromosome damage. However, the relationship between these lesions is uncertain. Our study focused on examining the role of XRCC1 in sodium chromate-induced cytotoxicity and chromosomal aberrations in Chinese Hamster Ovary (CHO) cells. Three different cell lines were used: AA8 (parental), EM9 (XRCC1 mutant) and H9T3 (EM9 complemented with human XRCC1 gene). Results show that concentration-dependent decreases in relative survival are similar in all three cell lines, indicating that XRCC1 is not crucial for protecting cells from sodium chromate-induced cytotoxicity. Similarly the frequency of damaged metaphase cells was not affected by XRCC1 deficiency. However, the total number of Cr(VI)-induced chromosome aberrations was exacerbated by XRCC1 deficiency and the spectrum of chromosome damage changed dramatically. Specifically, chromatid and isochromatid lesions were the most prominent aberrations induced in the cell lines and XRCC1 was essential to reduce the formation of chromatid lesions. In addition, XRCC1 deficiency caused a dramatic increase in the number of chromatid exchanges indicating that it is involved in protection from Cr(VI)-induced chromosome instability.     

XRCC1 protects cells from chromate-induced chromosome damage, but does not affect cytotoxicity

Hexavalent chromium Cr(VI) is a well known human carcinogen. This genotoxic metal induces DNA strand breaks and chromosome damage. However, the relationship between these lesions is uncertain. Our study focused on examining the role of XRCC1 in sodium chromate-induced cytotoxicity and chromosomal aberrations in Chinese Hamster Ovary (CHO) cells. Three different cell lines were used: AA8 (parental), EM9 (XRCC1 mutant) and H9T3 (EM9 complemented with human XRCC1 gene). Results show that concentration-dependent decreases in relative survival are similar in all three cell lines, indicating that XRCC1 is not crucial for protecting cells from sodium chromate-induced cytotoxicity. Similarly the frequency of damaged metaphase cells was not affected by XRCC1 deficiency. However, the total number of Cr(VI)-induced chromosome aberrations was exacerbated by XRCC1 deficiency and the spectrum of chromosome damage changed dramatically. Specifically, chromatid and isochromatid lesions were the most prominent aberrations induced in the cell lines and XRCC1 was essential to reduce the formation of chromatid lesions. In addition, XRCC1 deficiency caused a dramatic increase in the number of chromatid exchanges indicating that it is involved in protection from Cr(VI)-induced chromosome instability.     

Bloom's syndrome and EM9 cells in BrdU-containing medium exhibit similarly elevated frequencies of sister chromatid exchange but dissimilar amounts of cellular proliferation and chromosome disruption

Bloom's syndrome (BS) and EM9 cells both display elevated frequencies of sister chromatid exchange (SCE) following growth for two rounds of DNA replication in bromodeoxyuridine (BrdU)-containing medium. To learn whether hyperresponsiveness to BrdU itself might play a role in causing the SCE elevation, the effects of BrdU on two other parameters, cellular proliferation and chromosome disruption, were examined, comparing the responses of BS and normal lymphoblastoid cells and of EM9 and CHO cells. BS and normal cells responded similarly with respect to growth for 4 days in BrdU-containing medium (0, 1, 3, and 5 g/ml). Chromosome aberrrations were increased only slightly in the BS and normal cells after 2 days in BrdU. CHO cells responded to growth in BrdU-containing medium like BS and normal cells; however, little growth of EM9 was detected at any of the BrdU concentrations employed. CHO and EM9 cells also exhibited strikingly different amounts of chromosome damage following growth in BrdU. After 2 days in 1, 3, and 5 g/ml BrdU 21%, 46%, and 50%, respectively, of the CHO cells had chromosome aberrations in contrast to 92%, 96%, and 98% of the EM9 cells. Most of the aberrations in the BrdU-treated CHO cells consisted of what appeared to be polycentric and ring chromosomes or chromosomes exhibiting telomere association. Acentric fragments were absent from most cells with polycentric and ring chromosomes, indicating either that the abnormal chromosomes were formed during an earlier cell cycle or that the abnormal chromosomes represent a form of association in which the telomeres are apposed so tightly that the juncture between chromosomes cannot be identified microscopically. EM9 cells treated with BrdU exhibited many chromatid and isochromatid gaps and breaks as well as numerous quadriradial, triradial, and complex interchange configurations. In addition, the types of aberrations present in CHO cells also were increased greatly in number. The different responses of BS and EM9 cells to growth in BrdU suggest that the molecular defects in the two cell types are different.     

Molecular mechanisms involved in the production of cromosomal aberrations II. Utilization of neurospora endonuclease for the study of aberration production by X-rays in G1 and G2 stages of the cell cycle

Chinese hamster ovary cells (CHO) were X-irradiated in G₁ and G₂ stages of the cell cycle and subsequently Neurospora endonuclease (NE) (E.C.3.1.4), an enzyme which is specific in cleaving single-stranded DNA, was introduced into the cells, after making the cells permeable by treatment with inactivated Sendai virus. With this treatment all classes of X-ray-induced chromatid aberrations increased in G₂ cells, whereas in G₁ cells an increase in cromosome type of aberrations was found, associated with a profound induction of chromatid type of aberrations as well. Duration of the availability of single-strand gaps for the action of NE has been studied in G₂ cells following X-irradiation and the influence of different parts of the G₂ stage on the type and frequencies of chromatid aberrations was discerned. While the increase in chromosome type of aberrations by NE in X-irradiated G₁ cells has been interpreted as due to the conversion of DNA single-strand breaks or gaps to double-strand breaks by NE, the induction of chromatid aberrations in G₁ has been assumed to be due to conversion of some of the damaged bases strand breaks by NE. Biochemical evidence is presented for the conversion by NE of DNA single-strand breaks induced by X-rays into double-strand breaks using neutral sucrose gradient centrifugation.     

Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP

Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man. The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-[Pt(AF)2Cl2]) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation. It was shown that cis-[Pt(AF)2Cl2] acts directly and is mutagenic for three strains of S. typhimurium: TA97a, TA98 and TA100. In comparison with cis-DDP this compound showed a weaker genotoxicity. Contrary to cis-DDP it has not shown toxic properties in the tester bacteria. The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro. The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared. In all test systems applied, cis-[Pt(AF)2Cl2] was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation. cis-[Pt(AF)2Cl2] has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound. These results provide important data on the genotoxicity of cis-[Pt(AF)2Cl2] and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.     

Effects of arginine deprivation upon chromosome aberrations, sces and survival of cho cells treated with mutagenic agents.

Arginine deprivation sensitizes CHO cells to the clastogenic activity of the mutagenic agents UV light, N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C and 4-nitroquinoline-1-oxide. Cells were allowed to undergo proliferative arrest by deprivation of the amino acid arginine, treated with mutagenic agent and refed with complete medium. The resulting mitotic cells displayed more chromosome aberrations than did mitotic cells in proliferating cell cultures which had been treated similarly. This effect was observed at each dose tested (representing a 300-fold range in concentration). Survival of arginine-deprived cells exposed to UV light was also markedly reduced in comparison to the response of proliferating cells. Sister-chromatid exchange levels induced by MNNG, in contrast, were similar in arginine-deprived and proliferating cells.     

Clastogenicity, photo-clastogenicity or pseudo-photo-clastogenicity: Genotoxic effects of zinc oxide in the dark, in pre-irradiated or simultaneously irradiated Chinese hamster ovary cells

Zinc oxide (ZnO), a widely used ingredient in dermatological preparations and sunscreens, is clastogenic in vitro, but not in vivo. Given that ZnO has an approximately four-fold greater clastogenic potency in the presence of UV light when compared with that in the dark, it has been suggested to be photo-clastogenic. In order to clarify whether this increased potency is a genuine photo-genotoxic effect, we investigated the clastogenicity of ZnO (mean particle size, 100 nm) in Chinese hamster ovary (CHO) cells in the dark (D), in pre-irradiated (PI, i.e. UV irradiation of cells followed by treatment with ZnO) and in simultaneously irradiated (SI, i.e. ZnO treatment concurrent with UV irradiation) CHO cells at UV doses of 350 and 700 mJ/cm(2). The cytotoxicity of ZnO to CHO cells under the different irradiation conditions was as follows: SI>PI>D. In the dark, ZnO produced a concentration-related increase in chromosome aberrations (CA). In PI or SI CHO cells, ZnO was clastogenic at significantly lower concentrations (approximately two- to four-fold) when compared with effective concentrations in the dark, indicating an increased susceptibility of CHO cells to ZnO-mediated clastogenic effects due to UV irradiation per se. The incidence of CA in SI or PI cells was generally higher than that in the dark. At similar ZnO concentrations, SI conditions generally produced higher CA incidence than PI conditions. However, when ZnO concentrations producing similar cytotoxicity were compared, CA incidences under PI or SI conditions were nearly identical. The modest increase in the clastogenic potency of ZnO following UV irradiation contrasts with the results observed with genuine photo-clastogenic agents, such as 8-MOP, which may produce an increase in clastogenic potency of >15,000-fold under SI conditions. Our results provide evidence that, under conditions of in vitro photo-clastogenicity tests, UV irradiation of the cellular test system per se may produce a slight increase in the genotoxic potency of compounds that are clastogenic in the dark. In conclusion, our data suggest that minor increases in clastogenic potency under conditions of photo-genotoxicity testing do not necessarily represent a photo-genotoxic effect, but may occur due to an increased sensitivity of the test system subsequent to UV irradiation.     

Further studies on toxic and radiosensitizing properties of ruthenium complexes of 4-nitroimidazoles

Transition metal complexes containing nitroimidazole ligands have been shown previously to act as radiosensitizers of hypoxic cells in vitro. As part of our study on metal-radiosensitizer complexes, we were encouraged by a ruthenium (Ru) sensitizer, RuCl2(DMSO)2(4(5)-nitroimidazole)2, 1, which showed better radiosensitizing properties and lower toxicity than the free ligand. In this study, we have extended our investigation to include the various other substituted 4-nitroimidazoles as ligands. The new Ru complexes, analogues of 1, were synthesized, identified and characterized and their toxicity and radiosensitizing abilities examined in vitro using Chinese hamster ovary (CHO) cells. Like 1, each of these ruthenium complexes has lower CHO hypoxic toxicity than the free ligands alone at equimolar concentration. These newer complexes gave sensitizing enhancement ratio (SER) values of 1.1 to 1.3 at 1.0 X 10(-4) mol dm-3 compared with 1.6 for 1. Unlike complex 1, the new complexes do not bind to plasmid DNA (assessed by inhibition of restriction endonuclease activity), possibly because the chloride (Cl-) ligand does not dissociate. In addition, the redox potential of the coordinated imidazole ligands is relatively unchanged compared to that of the free ligand. These factors may explain the more favourable properties of 1 compared with those of the new 4-nitroimidazole complexes of Ru.