Differential sensitivity of Chinese hamster V79 and Chinese hamster ovary (CHO) cells in the in vitro micronucleus screening assay.

Both the V79 and CHO cell lines are routinely used in the in vitro MN screening assay for the detection of possible genotoxicants. The CHO cell line is the predominant cell line currently used in the genetic toxicology testing industry. However, some laboratories routinely utilize the V79 cell line since the in vitro MN screening assay was initially developed using V79 cells. Our laboratory has historically used the CHO cell line. Therefore, our laboratory was interested in comparing the two cell lines with regard to possible similarities or differences in MN induction sensitivity after exposure to cyclophosphamide (CPA) and mitomycin C (MMC), the two standard positive control chemicals routinely used in this assay. Three exposure conditions in the presence of CPA and MMC were examined in both cell lines. Replicate cultures of CHO cells in McCoy's 5A and V79 cells in both McCoy's 5A and E-MEM were established and treated with 5 microg CPA/ml (4h exposure with S9), 0.5 microg MMC (4h exposure without S9) and 0.5 microg MMC (24h exposure without S9). A total of 400 cytochalasin B-blocked binucleated cells and 200 consecutive cells were analyzed from each culture for MN and cell cycle kinetics, respectively. Analysis of the data demonstrated that CHO cells were up to approximately five-fold more sensitive to the induction of CPA- and MMC-induced MN than V79 cells. Both cell lines exhibited similar average generation times among identical exposure groups. Therefore, the difference in MN sensitivity cannot be attributed to possible differences in cell cycle kinetics and is possibly related to inherent cellular differences in the processing of and/or repair of CPA- and MMC-induced damage by V79 and CHO cells.     

Use of the photo-micronucleus assay in Chinese hamster V79 cells to study photochemical genotoxicity

2-Methoxyaniline (o-anisidine) is a urinary bladder carcinogen in both mice and rats. Since the urinary bladder contains substantial peroxidase activity, we investigated the metabolism of this carcinogen by prostaglandin H synthase (PHS), a prominent enzyme in the urinary bladder, and lactoperoxidase as model mammalian peroxidases. Horseradish peroxidase (HRP)-mediated oxidation of o-anisidine was also determined and compared with the reactions catalyzed by mammalian peroxidases. All three peroxidases oxidized o-anisidine via a radical mechanism. Using HPLC combined with electrospray tandem mass spectrometry, we determined that peroxidases oxidized o-anisidine to a diimine metabolite, which subsequently hydrolyzed to form a quinone imine. Two additional metabolites were identified as a dimer linked by an azo bond and another metabolite consisting of three methoxybenzene rings, which exact structure has not been identified as yet. Using [¹⁴C]-labeled o-anisidine, we observed substantial peroxidase-dependent covalent binding of o-anisidine to DNA, tRNA and polydeoxynucleotides [poly(dX)]. The ³²P-postlabeling assay (a standard procedure and enrichment of adducts by digestion with nuclease P1 or by extraction into 1-butanol prior to ³²P-labeling) was employed as the second method to detect and quantitate binding of o-anisidine to DNA. Using these versions of the ³²P-postlabeling technique we did not observe any DNA adducts derived from o-anisidine. The o-anisidine-DNA adducts became detectable only when DNA modified by o-anisidine was digested using three times higher concentrations of micrococcal nuclease and spleen phosphodiesterase (MN/SPD). We found deoxyguanosine to be the target for o-anisidine binding in DNA using poly(dX) and deoxyguanosine 3′-monophosphate (dGp). A diimine metabolite of o-anisidine is the reactive species forming adducts in dGp. The results strongly indicate that peroxidases play an important role in o-anisidine metabolism to reactive species, which might be responsible for its genotoxicity, and its carcinogenicity to the urinary bladder in rodents. The limitation of the ³²P-postlabeling technique to analyze DNA adducts derived from o-anisidine as a means to estimate its genotoxicity is discussed.     

Reproductive death of Chinese hamster V79 cells after exposure to chemical inhibitors of DNA synthesis.

1. The results of this study have contributed to the definition of three categories of chemical inhibitors of DNA replication in mammalian cells. 2. Inhibitors of replicon cluster initiation [4-nitroquinoline-N-oxide (4-NQO), etoposide (VP-16), teniposide (VM-26), amsacrine (m-AMSA), N-methyl-N'-nitro-N-nitrozoguanidine (MNNG), cis-Pt(II)diammine dichloride (cis-PDD)], which needed similar doses to produce a slow and persistent (up to 4 hr) inhibition of DNA synthesis, followed by significant cell killing. 3. Inhibitors of DNA replication by indirect action [3-aminobenzamide [correction of 3-aminobezamide] (3-AB), cycloheximide (CHX), puromycin (PRC), bisbenzimide Hoechst No. 33258 (H-33258]), that showed reduced cytotoxic effects, and caused a slow (60 min) and reversible inhibition of DNA synthesis. 4. Inhibitors of formation and/or polymerization of deoxyribonucleotides [5-aminouracil (5-AU), bisbenzimide Hoechst No. 33342 (H-33342)], which induced a fast (20 min) and reversible suppression of DNA replication, associated with limited cell killing.     

In vitro and in vivo inhibition of telomerase activity from Chinese hamster V79 cells

While studying the inhibition of telomerase activity in Chinese hamster V79 cells using polymerase chain reaction (PCR) based telomeric repeat amplification protocol (TRAP) assay, we had earlier observed that 7-deaza deoxy guanosine triphosphate (7-deaza dGTP) and oligonucleotide (TTAGGG)4 inhibited telomerase activity in vitro. In the present study, we report inhibition of telomerase activity by modified base 7-deaza deoxy adenosine triphosphate (7-deaza dATP) and phosphorothioate TTAGGG (PS-TTAGGG). Both the compounds inhibited telomerase activity in a concentration dependent manner; 8.5 microM of 7-deaza dATP and 0.1 microM of PS-TTAGGG being the concentration for 50% of the maximum inhibition. This observation supports our earlier hypothesis that incorporation of a modified nucleotide into telomere possibly interferes with the recognition of the telomerase and TTAGGG interferes with the RNA component of telomerase. We have further shown that treatment of cells with nicotinamide (NA) and benzamide (BA), well known inhibitors of poly (ADP-ribose) polymerase, reduced telomerase activity. We speculate that modification of the telomeric binding proteins or other components by poly (ADP-ribosyl)ation may be involved in such inhibition.     

The application of the micronucleus test in Chinese hamster V79 cells to detect drug-induced photogenotoxicity.

Recent reports on the photochemical carcinogenicity and photochemical genotoxicity of fluoroquinolone antibacterials led to an increasing awareness for the need of a standard approach to test for photochemical genotoxicity. In this study the micronucleus test using V79 cells was adapted to photogenotoxicity testing. Results of using different UVA/UVB relationships enabled us to identify a suitable irradiation regimen for the activation of different kinds of photosensitizers. Using this regimen, 8-methoxypsoralen and the fluoroquinolones lomefloxacin, grepafloxacin and Bay Y 3118 were identified to cause micronuclei and toxicity upon photochemical activation. Among the phenothiazines tested, chlorpromazine and 2-chlorophenothiazine, were positive for both endpoints, whereas triflupromazine was only slightly photoclastogenic in the presence of strong phototoxicity. Among the other potential human photosensitizers tested (oxytetracycline, doxycycline, metronidazole, emodin, hypericin, griseofulvin), only hypericin was slightly photogenotoxic. Photochemical toxicity in the absence of photochemical genotoxicity was noted for doxycycline and emodin. With the assay system described, it is possible to determine photochemical toxicity and photochemical genotoxicity concomitantly with sufficient reliability.     

Critical importance of microsome concentration in mutagenesis assay with V79 Chinese hamster cells.

For optimum mutagensis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1-5%) concentrations of the liver 15 000 X g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the S15 concentrations of more than 10%. Similarly, aflatoxin B1 induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic aflatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased continuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the S15. In contrast with these compounds, N-nitrosodimethylamine induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the lipid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.     

Evaluation of the genotoxicity of domoic acid in a hepatocyte-mediated assay with V79 Chinese hamster lung cells

Domoic acid, a recognized neurotoxin derived from contaminated samples of the blue mussel (Mytilus edulis L.), was analyzed for mutagenicity at 2 loci and for 2 cytogenetic parameters in a hepatocyte-mediated assay with V79 Chinese hamster lung fibroblasts. Genetic end-points measured were: mutation to 6-thioguanine resistance at the HGPRTase locus; mutation to ouabain resistance at the Na+,K+-ATPase locus; sister-chromatid exchange (SCE) and micronucleus frequency (MN). None of these genetic end-points was significantly affected by exposure to domoic acid at dose levels of 27.2 and 54.4 micrograms/ml with or without activation by freshly isolated rat liver hepatocytes. It was concluded that, within the limits of the test system employed, domoic acid was non-genotoxic to V79 cells.     

Recovery of the viability of Chinese hamster V79-4 cells after combined exposure to hyperthermia and radiation

The survival of cells overheated (42 degrees C) before gamma irradiation is increased by holding them in the growth medium at 37 degrees C before treatment with hypertonic NaCl solution. The substantial synergistic effect of hyperthermia and radiation takes place when the cells are treated with a 1.5 M NaCl solution immediately after the combined action of these inactivating factors. The synergistic effect is decreased by holding the cells in the nutrient medium at 37 degrees C for 4 hours before hypertonic treatment.     

Induction of HPRT- mutants in Chinese hamster V79 cells after heavy ion exposure

The induction of resistance to 6-thioguanine by heavy ion exposure was investigated with various accelerated ions (oxygen-uranium) up to linear energy transfer (LET) values of about 15000 keV/µm.31 y Survival curves are exponential with fluence; mutation induction shows a linear dependence. Cross-sections (_i: inactivation, _m: mutation) were derived from the respective slopes. Generally, _i rises over the whole LET range, but separateas into different declining curves for single ions with LET values above 200 keV/µm. Similar behaviour is seen for _m. The new SIS facility at GSI, Darmstadt, makes it possible to study the effects of ions with the same LET but very different energies and track structures. Experiments using nickel and oxygen ions (up to 400 MeV/u) showed that inactivation cross-sections do not depend very much on track structure, i.e. similar values are found with different ions at the same LET. This is not the case for mutation induction, where very energetic ions display considerably smaller induction cross-sections compared with low-energy ions of identical LET. Preliminary analyses using the polymerase chain reaction (PCR) demonstrate that even heavy ions cause small alterations (small deletions or base changes). The proportion of the total deletions seems to increase with LET.Submitted paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

Patulin,a further clastogenic mycotoxin,is negative in the SCE assay in Chinese hamster V79-E cells in vitro

Patulin is a potent inducer of chromatid-type aberrations in Chinese hamster V79-E cells, but loses its activity when 9000 g supernatant of rat-liver homogenate is added. The narrow dose range of patulin clastogenicity shows a quantitative relationship between absolute amount of mycotoxin applied and the number of indicator cells treated. Within a dose range permitting survival of V79-E, patulin does not induce an increase of the SCE rate. It is suggested that patulin clastogenicity is caused by interaction with chromosomal proteins and that DNA is not the virtual target of this mycotoxin.     

Cadmium-resistant Chinese hamster V79 cells with decreased accumulation of cadmium

Cells resistant to 3 x 10(-5) M CdCl2 (Cdr cells) were isolated from cultures of Chinese hamster V79 cells by a procedure that involved stepwise increase in the concentration of Cd2+ and subsequent mass selection. Cdr cells grew as fast as wild-type cells (Cds) in medium without cadmium. Cdr cells were not cross-resistant to other divalent metal ions, such as Hg2+, Ni2+, Pb2+, and Zn2+. Both Cds and Cdr cells induced similar levels of metallothioneins (MT) in response to zinc. Depletion of glutathione (GSH) did not significantly influence the sensitivity of Cdr cells to Cd2+ but markedly enhanced the sensitivity to Cd2+ of Cds cells. Furthermore, the rate of synthesis of GSH after depletion did not differ greatly between sensitive and resistant cells. The rate of uptake of 109Cd2+ by Cdr cells was only 10-15% that by Cds cells. The difference in rates of uptake between Cds and Cdr cells was observed irrespective of the presence or absence of serum in the culture medium. These results indicate that, in this system, resistance to Cd2+ is attributable neither to increased inducibility of MT nor to increases in intracellular levels of GSH, and that only a decrease in the rate of uptake of Cd2+ contributes to the acquisition of resistance to Cd2+. Uptake of Cd2+ by cells was dependent on temperature and the rate of uptake of Cd2+ by Cdr cells was lower at all temperatures examined than the rate of uptake by Cds cells. Cycloheximide did not suppress the uptake of Cd2+, suggesting that uptake does not require synthesis of cell proteins de novo. Preincubation of cells with N-ethylmaleimide suppressed the uptake of Cd2+ to some extent, a result that suggests the involvement of surface SH groups in the uptake of Cd2+ by these cells.     

Genotoxicity of 1-nitronaphthalene in Chinese hamster V79 cells

1-Nitronaphthalene (1-NN) has been identified in the U.S. National Toxicology Program as a non-carcinogen showing some evidence of in vitro genotoxicity. We tested this compound in Chinese hamster V79 cells at 20-80 micrograms/ml with two endpoints: sister-chromatid exchange (SCE) and thioguanine resistance (TGR), with 5 repeat experiments. The SCE values in the presence of rat or hamster hepatocytes were consistently above the 95% and usually the 99% upper confidence limits for the corresponding control. Without hepatocyte activation, the control upper confidence limits were not exceeded except in one experiment in which the control SCE value was unusually low. TGR was scored both as proportion of plates with mutant colonies and as number of mutant colonies per plate. In 2 of 5 experiments, these values exceeded control 95% or 99% upper confidence limits; on the other hand, these values were substantially lower than those of the positive controls, dimethylbenz[a]anthracene (2.6 micrograms/ml) with activation and ethyl methanesulfonate (155 microgram/ml), which is direct-acting. For TGR, activation of 1-NN by either rat or hamster hepatocytes produced inconsistent results. Overall we would consider this compound to be a weak genotoxin, to which a cancer bioassay would be expected to be relatively insensitive.     

Mutagenicity of hydrogen peroxide in V79 Chinese hamster cells

Hydrogen peroxide (H2O2) was investigated for its potential to induce gene mutations in V79 Chinese hamster cells. Exposure of 2-3 X 10(6) cells/100-mm dish to 0.5-4.0 mM H2O2 for 1 h resulted in a concentration-dependent increase in the frequency of 6-thioguanine-resistant clones. At 4 mM H2O2 the mutation frequency was increased about 6-fold above that in controls and survival of the cells was reduced by 50%. Cytotoxicity was markedly increased at lower cell densities. When only 100-200 cells/100-mm dish were exposed to H2O2 for 1 h, 50% were killed at an H2O2 concentration as low as 60 microM. The results show that mutagenicity of H2O2 in mammalian cells in vitro has escaped attention previously because the concentrations tested were too low, presumably because the likely toxicity of H2O2 to V79 cells treated at high cell densities was overestimated.     

Mutagenicity of 4-hydroxynonenal in V79 Chinese hamster cells

4-Hydroxynonenal (HNE), a major product of the peroxidation of liver microsomal lipids, was examined for mutagenic activity at the hypoxanthine-guanine phosphoribosyltransferase locus in V79 Chinese hamster lung cells. At concentrations ranging from 10 to 45 microM, HNE induced a dose-dependent increase in the number of mutations to 6-thioguanine resistance, which reached the level of 4.7X baseline at the highest concentration tested.     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

Recovery of thioguanine-resistant cells from Chinese hamster V79 spheroids

Multicell spheroids may prove useful in evaluting the interactions of mutagens with cells exposed in a tissue-like environment. However, direct comparisons among populations of Chinese hamster V79 spheroids of different sizes or with monolayers are complicated by the observation that as spheroids enlarge, the fraction of mutant cells resistant to 6-thioguanine (TG^r) gradually decreases from about 5 in 10⁵ to less than 1 in 10⁵. There appear to be at least 2 explanations for these observations. First, TG^r cells grow less well as spheroids than do 6-thioguanine-sensitive (TG^s) cells. Second, the clonal nature of spheroid growth means that small samples fo spheroids are likely to contain fewer pre-existing TG^r cells.     

Elimination of MNU-induced mutational lesions in V79 Chinese hamster cells

Studies were performed on the non-linear dose response for gene mutations induced by low doses of monofunctional methylating agents in V79 Chinese hamster cells. When treatment with methylnitrosourea was applied at the beginning of the S phase in synchronized cells, a linear dose-response curve was obtained, whereas application of the dose after gene replication resulted in a strong reduction of the number of induced mutations. Additional time for repair resulted in reduced dose response of MNU, indicating that an error-free repair process operates on methylated DNA in V79 Chinese hamster cells.