Megabase pair deletions in mutant mammalian cells following exposure to amsacrine, an inhibitor of DNA topoisomerase II.

Amsacrine, [4'-(9-acridinylamino)-methanesulfon-m-auisidide], belongs to the class of cancer chemotherapeutic agents that target DNA topoisomerase II. We show that, over its cytotoxic range, amsacrine is a potent mutagen of the S1 phenotype in the AL (human x hamster) hybrid cell line. By contrast, amsacrine induction of the HPRT- phenotype in AL cells is at least two decades less frequent and is not concentration dependent. Such differential mutation frequencies are hypothesized to reflect the concomitant loss of essential genes neighboring the hprt locus. It may be that some amsacrine cytotoxicity is due to the inactivation of essential genes by large deletions. The AL mutation system is well suited for the detection and mapping of mutations which are large deletions because its MIC1 locus, which controls the expression of the selectable cell surface antigen S1, is on a single human chromosome. This human chromosome 11 is in addition to the genome of the Chinese hamster ovary cell and is basically nonessential. Since there are no sister human chromosomes in AL cells, deletions which extend beyond the MIC1 locus may be conveniently and unambiguously mapped. We have detected the presence or absence of 9 different chromosome 11 markers in 48 S1- mutants cloned from amsacrine-treated cultures. We find that almost all (92%) of the mutants have deletions of at least 1.5-2 megabase pairs in length. The distribution of marker loss frequencies flanking the MIC1 locus does not appear symmetric with respect to distance from that locus. We speculate that amsacrine-induced deletions are mediated by a series of subunit exchanges between overlapping topoisomerase II dimers at the bases of replicons or larger chromosomal structures such as replicon clusters or chromosome minibands.

Induction and repair of strand breaks and 3'-hydroxy terminals in the DNA of mammalian cells in culture following gamma-ray irradiation.

DNA was isolated in a fairly pure and intact state from cultured mouse leukaemia cells (L5178Y) after gamma-ray irradiation using a hydroxyapatite column chromatography method, and analysed further by sucrose gradient centrifugation or DNA polymerase (EC 2.7.7.7, enzyme A of Klenow from Escherichia coli) assay. Irradiation of the cells induced single- and double-strand breaks in the DNA with an efficiency of 100 eV/break and 1300 eV/break, respecitvely. Approximately 50% of the single-strand breaks were estimated to be those arising from allali-labile lesions. A linear, dose-dependent increase was found in the template activity of the DNA, indicating the induction of 3'-OH terminals by gamma-irradiation. Post-irradiation incubation of the cells in serum-free medium allowed the majority of the breaks to rejoin within a few hours. Repair of the alkali-labile lesions was, however, found to be much slower than that of "actual" single-strand breaks. A slight increase of the DNA template activity was found during the period of post-irradiation incubation. The reason for the increase is discussed.

Cell killing and mutation to 6-thioguanine resistance after exposure to tritiated amino acids and tritiated thymidine in cultured mammalian cells (L5178Y).

Cell killing and mutation to 6-thioguanine resistance were studied in growing mouse leukemia cells in culture after exposure to tritiated amino acids and tritiated thymidine. These effects varied widely among the tritiated compounds tested, being greatest for tritiated thymidine followed by tritiated arginine and tritiated lysine, in that order, for a given concentration of 3H expressed in kBq/ml of 3H in the medium. The differences between each tritiated amino acid disappeared almost totally when the effects were compared on the basis of the absorbed dose to the cells. The effects of tritiated thymidine, however, remained more than twofold greater compared to other tritiated compounds. These results indicate the importance of determining the absorbed dose for assessment of the radiotoxicity of tritiated organic compounds. For an exceptional case (tritiated thymidine), contribution of a mechanism(s) other than beta irradiation should also be taken into account.

Mutation induction by very low dose rate gamma rays in cultured mouse leukemia cells L5178Y.

Induction of cell killing and mutation to 6-thioguanine resistance was studied in growing mouse leukemia cells in culture following gamma rays at dose rates of 30 Gy/h, 20 cGy/h, and 6.3 mGy/h, i.e., acute, low dose rate, and very low dose rate irradiation. A marked increase was observed in the cell survival with decreasing dose rate; no reduction in the surviving fraction was detected after irradiation at 6.3 mGy/h until a total dose of 4 Gy. Similarly, the induced mutation frequency decreased after low dose rate irradiation compared to acute irradiation. However, the frequency after irradiation at 6.3 mGy/h was unexpectedly high and remained at a level which was intermediate between acute and low dose rate irradiation. No appreciable changes were observed in the responses to acute gamma rays (in terms of cell killing and mutation induction) in the cells which had experienced very low dose rate irradiation.

Inhibition of gamma-ray dose-rate effects by D2O and inhibitors of poly(ADP-ribose) synthetase in cultured mammalian L5178Y cells.

Effects of deuterium oxide (D2O) and 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, on cell proliferation and survival were studied in cultured mammalian L5178Y cells under growing conditions and after acute and low-dose-rate irradiation at about 0.1 to 0.4 Gy/hr of gamma rays. Growth of irradiated and unirradiated cells was inhibited by 45% D2O but not by 3-aminobenzamide at 10 mM, except for treatments longer than 30 hr. The presence of these agents either alone or in combination during irradiation at low dose rates suppressed almost totally the decrease in cell killing due to the decrease in dose rate. The D2O did not inhibit the radiation-induced increase in poly(ADP-ribose) synthesis as measured by the incorporation of [14C]NAD into the acid insoluble fraction, contrary to 3-aminobenzamide. Among other inhibitors tested, theobromine and theophylline were found to be effective in eliminating the dose-rate effects of gamma rays. Possible mechanisms underlying the inhibition are discussed.

Mutant quantity and quality in mammalian cells (AL) exposed to cesium-137 gamma radiation: effect of caffeine.

We examined the effect of caffeine (1,3,7-trimethylxanthine) on the quantity and quality of mutations in cultured mammalian AL human-hamster hybrid cells exposed to 137Cs gamma radiation. At a dose (1.5 mg/ml for 16 h) that reduced the plating efficiency (PE) by 20%, caffeine was not itself a significant mutagen, but it increased by approximately twofold the slope of the dose-response curve for induction of S1- mutants by 137Cs gamma radiation. Molecular analysis of 235 S1- mutants using a series of DNA probes mapped to the human chromosome 11 in the AL hybrid cells revealed that 73 to 85% of the mutations in unexposed cells and in cells treated with caffeine alone, 137Cs gamma rays alone or 137Cs gamma rays plus caffeine were large deletions involving millions of base pairs of DNA. Most of these deletions were contiguous with the region of the MIC1 gene at 11p13 that encodes the S1 cell surface antigen. In other mutants that had suffered multiple marker loss, the deletions were intermittent along chromosome 11. These "complex" mutations were rare for 137Cs gamma irradiation (1/63 = 1.5%) but relatively prevalent (23-50%) for other exposure conditions. Thus caffeine appears to alter both the quantity and quality of mutations induced by 137Cs gamma irradiation.

A low, adaptive dose of gamma-rays reduced the number and altered the spectrum of S1- mutants in human-hamster hybrid AL cells.

We examined the effects of a low, adaptive dose of 137Cs-gamma-irradiation (0.04 Gy) on the number and kinds of mutants induced in AL human-hamster hybrid cells by a later challenge dose of 4 Gy. The yield of S1- mutants was significantly less (by 53%) after exposure to both the adaptive and challenge doses compared to the challenge dose alone. The yield of hprt- mutants was similarly decreased. Incubation with cycloheximide (CX) or 3-aminobenzamide largely negated the decrease in mutant yield. The adaptive dose did not perturb the cell cycle, was not cytotoxic, and did not of itself increase the mutant yield above background. The adaptive dose did, however, alter the spectrum of S1- mutants from populations exposed only to the adaptive dose, as well as affecting the spectrum of S1- mutants generated by the challenge dose. The major change in both cases was a significant increase in the proportion of complex mutations compared to small mutations and simple deletions.

Mutant yields and mutational spectra of the heterocyclic amines MeIQ and PhIP at the S1 locus of human-hamster AL cells with activation by chick embryo liver (CELC) co-cultures.

Cooking meat and fish at high temperature creates heterocyclic amines (HA) including 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Several HA are mutagens in the Ames' S9/Salmonella assay. While PhIP is a substantial Ames' test mutagen, it is 1000-fold less active than the extraordinarily potent MeIQ. In contrast, MeIQ is significantly less mutagenic than PhIP in several mammalian cell assays, especially in repair-deficient Chinese hamster ovary (CHO) cells. HA are suspect human carcinogens on the basis of (i) epidemiological evidence, (ii) induction of tumors in rodents and monkeys, (iii) DNA adduct formation and (iv) mutagenic capacity. In this study, MeIQ and PhIP were significant mutagens at the S1 locus of co-cultivated human/hamster hybrid AL cells following metabolic activation by beta-napthoflavone (betaNF)-induced chick embryonic liver cultures (CELC). MeIQ was more mutagenic than PhIP in the CELC+AL cell assay. The mutant response curves increase with dose and then plateau (PhIP), or decrease (MeIQ). The inflections in these response curves coincide with dose-dependent decreases in cytochrome CYP1A1 activity. Molecular analysis of S1- mutants indicates that a substantial fraction, >65%, of the mutations induced by PhIP are deletions of 4.2 to 133 (Mbp); half are larger than 21 Mbp. Mutations induced by MeIQ were smaller, most (56%) being less than 5.7 Mbp. When appropriate metabolic activation is combined with a target locus, which can detect both small and large chromosomal mutations, both MeIQ and PhIP are significant mutagens and clastogens in repair proficient mammalian cells.

Differential response of human and rodent cell lines to chemical inhibition of the repair of potentially lethal damage.

We have examined the effects of several classes of metabolic inhibitors on the repair of potentially lethal damage in density-inhibited cultures of two rodent and two human cell systems which differ in their growth characteristics. Aphidicolin, 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea showed no effect on PLD repair, whereas the effects of 9-beta-D-arabinofuranosyladenine (ara-A) and 3-aminobenzamide (3-AB) were cell line dependent. For example, 3-AB suppressed PLD repair almost completely in CHO cells, but showed no inhibitory effects in human diploid fibroblasts. These results indicate that inhibitors of DNA replication and poly(ADP-ribose) synthesis are not efficient inhibitors of cellular recovery in irradiated cells and, moreover, that such effects may be cell line dependent.

Vanillin (3-methoxy-4-hydroxybenzaldehyde) inhibits mutation induced by hydrogen peroxide, N-methyl-N-nitrosoguanidine and mitomycin C but not (137)Cs gamma-radiation at the CD59 locus in human-hamster hybrid A(L) cells.

We have investigated the ability of the naturally occurring plant essence vanillin (3-methoxy-4-hydroxybenzaldehyde) to inhibit mutation at the CD59 locus on human chromosome 11 by hydrogen peroxide, N-methyl-N-nitrosoguanidine, mitomycin C and (137)Cs gamma-radiation in human-hamster hybrid A(L) cells. Previous studies using vanillin have suggested that it can inhibit chromosome aberrations induced by hydrogen peroxide and mitomycin C, as well as inhibiting X-ray- and UV-induced mutations at the hprt locus. Other studies with vanillin have shown that it can increase both the toxicity and mutagenicity of ethyl methane sulfonate and increase the induction of sister chromatid exchange by mitomycin C and a variety of other mutagens. The increased sensitivity of the A(L) assay, which is due in part to its ability to detect both small (single locus) and large (multilocus) genetic damage, allows us to measure the effect of vanillin at low doses of mutagen. Vanillin is shown, in these studies, to inhibit mutation induced by hydrogen peroxide, N-methyl-N-nitrosoguanidine and mitomycin C, as well as to enhance the toxicity of these agents. Vanillin had no effect on either toxicity or mutation induced by (137)Cs gamma-radiation. The vanillin-induced potentiation of H(2)O(2) toxicity is shown not to involve inhibition of catalase or glutathione peroxidase. These results show that vanillin is able to inhibit mutation at the CD59 locus and modify toxicity in a mutagen-specific manner. Possible mechanisms to explain the action of vanillin include inhibition of a DNA repair process that leads to the death of potential mutants or enhancement of DNA repair pathways that protect from mutation but create lethal DNA lesions during the repair process.