The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells.

The frequency of ethyl methanesulfonate (EMS)-induced mutations to 6-thioguanine resistance in a Chinese hamster ovary cells clone K1-BH4 was studied at many EMS doses including the minimally lethal range (0-100 microng/ml) as well as the exponential killing portion (100-800 microng/ml) of the survival curve. The mutation frequency increases approximately in proportion with increasing EMS concentration at a fixed treatment time. The pooled data for the observed mutation frequency, f(X), as a function of EMS dose X, is adequately described by a linear function f(X)=10(-6)(8.73+3.45 X), where 0 less than or equal to X less than or equal to 800 microng/ml. One interpretation of the linear dose-response is that, as a result of EMS treatment, ethylation of cellular constituents occurs, which is directly responsible for the mutation. Biochemical analyses demonstrate that most of the randomly isolated 6-thioguanine-resistant variants possess a highly reduced or undetectable level of HGPRT activity suggesting that the EMS-induced mutations to 6-thioguanine resistance affect primarily, if not exclusively, the HGPRT locus.     

Plasmid mediated mutagenesis of a cellular gene in transfected eukaryotic cells.

NIH3T3 cells are widely used in transformation assays and readily take up transfected DNA. A system has been devised using NIH3T3 cells to measure the mutagenic effect of transfected DNA on recipient cell genes. NIH3T3 cells can be mutated to 6-thioguanine resistance at a frequency which suggests that at least a portion of the cells have only one functional copy of the HGPRT gene. They have a low spontaneous background mutation frequency (approximately 1 X 10(-7)). Transfection of three different plasmids into NIH3T3 cells induced 6-thioguanine resistant mutants at frequencies ranging from 3 to 11 fold above background. The mutant phenotype is stable and reversion frequencies of several mutants are less than or equal to 1 X 10(-7). Southern blot analysis of the HGPRT gene in several mutants showed that 4 of 26 mutants (15.4%) had detectable alterations in the structure of the HGPRT gene. Interestingly 3 of the 4 mutants showing rearrangements were obtained by transfection of the HSV-2 morphological transforming region.     

Characterization of ultraviolet light-induced ouabain-resistant mutations in Chinese hamster cells.

Ouabain-resistant mutations in Chinese hamster cells have been quantitatively characterized. The mutation frequencies were found to be induced curvilinearly with treatments of increasing doses of ultraviolet light (UV). For the range of UV doses tested (5--20 J/m(2)), the observed frequency, Y, as a function of UV dose X, follows a curvilinear function, Y = (-28 + 13.37 X--1.52X(2) + 0.08X(3)) . 10(-6). The frequencies of UV-induced mutations were directly correlated with cell survival, indicating a similar causal relationship between cell killing and mutation induction. Under the same experimental conditions, X-rays induced 6--thioguanine-, but not ouabain-, resistant mutations. UV-induced ouabain-resistant (ouar) mutants exhibit a selection disadvantage. Their phenotypic expressions are modifiable by various agents. Wild type and 16 ouar mutants were compared with respect to their sensitivity to ouabain inhibition of 86Rb uptake by whole cells. All the ouar mutants assayed are less sensitive to the drug than are wild-type cells. In the absence of ouabain, the Na+--K+--ATPase activities can be significantly higher or lower than that of the wild-type cells.     

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.     

Relationship Between Radiation Response and the Deoxyribonucleic Acid Replication Cycle in Bacteria: Dependence on the Excision-Repair System

Prestarvation of Escherichia coli for required amino acids results in a marked enhancement in both ultraviolet light (UV) or X-ray resistance for selective strains. Preventing protein synthesis by starvation for required amino acids results in completion of the cycle of chromosomal replication then underway. We have investigated the relationship between starvation-induced resistance enhancement (SIRE) and the excision-repair (Hcr) system in several E. coli strains including E. coli B/r hcr(+) and its isogenic mutant E. coli B/r hcr(-). The following observations were made. (i) The Hcr system is the major component of SIRE in UV-irradiated strain B/r. By using the Hcr(+) strain, SIRE increases the 10% survival dose from approximately 400 ergs to approximately 1,200 ergs/mm(2). With the Hcr cells, the increase is from approximately 45 ergs to 60 ergs/mm(2). (ii) Although prestarvation leads to a moderate enhancement of resistance to X irradiation, this effect is not dependent on the Hcr system. (iii) The double mutant, E. coli B(s-1) (hcr(-)exr(-)) is completely unable to express SIRE whether studied with UV or X irradiation. It is concluded that the Hcr system is the major system responsible for SIRE in UV-treated cells, whereas Exr (resistance to X rays) may be involved to a minor extent. The Exr character appears to be required for SIRE expression in X-ray exposed cells.     

Kinetics of Mutation Induction by Ultraviolet Light in Excision-Deficient Yeast

We have measured the frequency of UV-induced reversions (locus plus suppressor) for the ochre alleles ade2-1 and lys2-1 and forward mutations (ade2 adex double auxotrophs) in an excision-deficient strain of Saccharomyces cerevisiae (rad2-20). For very low UV doses, both mutational systems exhibit linear induction kinetics. However, as the dose increases, a strikingly different response is observed: in the selective reversion system a transition to higher order induction kinetics occurs near 9 ergs/mm2 (25% survival), whereas in the nonselective forward system the mutation frequency passes through a maximum near 14 ergs/mm2 (4.4% survival) and then declines. This contrast in kinetics cannot be explained in any straightforward way by current models of induced mutagenesis, which have been developed primarily on the basis of bacterial data. The bacterial models are designed to accommodate the quadratic induction kinetics that are frequently observed in these systems. We have derived a mathematical expression for mutation frequency that enables us to fit both the forward and reversion data on the assumptions that mutagenesis is basically a "single event" Poisson process, and that mutation and killing are not necessarily independent of one another. In particular, the dose-response relations are consistent with the idea that the sensitivity of the revertants is about 25% less than that of the original cell population, whereas the sensitivity of the forward mutants is about 29% greater than the population average. We argue that this relatively small differential sensitivity of mutant and nonmutant cells is associated with events that take place during mutation expression and clonal growth.     

Resistance of Spores of Clostridium botulinum 33A to Combinations of Ultraviolet and Gamma Rays

Spores of Clostridium botulinum 33A exhibit a sigmoidal survival curve if subjected to gamma radiation. The present investigation was concerned with two questions: (i) what is the form of an ultraviolet (UV)-survival curve and (ii) what is the combined effect of UV- and gamma radiation? The UV-survival curve was found to be of sigmoidal type with a "shoulder" width of 675 ergs/mm(2) and a D(10) (exp) of 2,950 ergs/mm(2). To test the combination effect, spores were subjected to UV doses of 225, 450, 675, and 900 ergs/mm(2) followed by a series of increasing doses of gamma rays from 200 to 2,000 krad in 200-krad steps. The gamma ray-survival curves showed that increasing UV pretreatment caused a gradual loss of the "Prodiginine" yielding straight line exponential survival curves after preirradiation with UV doses of 675 ergs/mm(2) and above. Simultaneously the D(10) value for gamma-ray irradiation was reduced, e.g. UV preirradiation with 900 ergs/mm(2) reduced the D(10) by 40%. This observation emphasizes the potential practical advantage of combining UV and gamma rays for sterilization of heat-sensitive commodities.     

Resistance and recovery studies on ultraviolet-irradiated spores of Bacillus pumilus.

A spore suspension model and a procedure for recovering ultraviolet (UV)-irradiated spores of Bacillus pumilus were investigated. A most-probable-number tube dilution method using double-strength Trypticase soy broth was found to be superior to the agar plate method for recovering optimal numbers of spores irradiated with sublethal doses of UV energy. Aqueous suspensions of B. pumilus survived UV doses up to 108,000 ergs/mm2 as determined by a most-probable-number recovery and estimation procedure. Resistance and stability data were consistent and reproducible, indicating the dependability of this method for recovering UV-damaged spores. The procedures used to collect information concerning resistance characteristics for two strains of B. pumilus are discussed.     

Isolation of UV-sensitive variants of CHO-K1 by nylon cloth replica plating.

Techinques are described which permit the identification and isolation of UV-sensitive variants from mutagenized populations of Chinese hamster ovary (CHO) cells. Identification is based on the observation that within two days after receiving a dose of approximately 240 ergs/mm2 of UV irradiation most of the cells in a colony of CHO detach from the surface of a plastic tissue culture dish. At a lower dose of UV, which does not kill or detach a significant number of parental cells, UV-sensitive colonies are killed and become detached. Thus a clear plaque is produced in a lawn of unirradiated parental cells, marking the site occupied by a sensitive colony. Live cells from such sensitive colonies have been recovered from a nylon cloth replica prepared prior to irradiation and characterized. One UV-sensitive variant (CHO-UV-1) is indistinguishable from parental cells in X-ray resistance, chromosome number, generation time, and duration of the phases of the cell cycle. For UV irradiation the hit number (-n), shoulder width (Dq), and mean lethal dose (Do) for the variant are 2.8, 21 ergs/mm2, and 21 ergs/mm2, respectively, as compared to 2.6, 36 ergs/mm2, and 45 ergs/mm2 for CHO-K1 cells. These values have not changed for a period of eight months in culture.     

In vitro morphologic transformation of Syrian hamster cells by U.V.-irradiation is enhanced by X-irridation and unaffected by chemical carcinogens.

U.V.-exposure of foetal hamster cells (secondary or tertiary sub-cultures) seeded for colony formation resulted in the induction of transformation. The U.V.-effect on colony-forming ability resulted in a D37 of 58 ergs/mm2. The observed transformation frequency induced by U.V.-increases approximately in proportion to increased doses in the range of 7-5-60-8 ergs/mm2 examined. When cells seeded for colony formation were treated with U.V. and later with either benzo(a)pyrene or N-acetoxy-fluorenyl-acetamide, commencing 6 min after exposure to U.V., there was neither an additive nor a synergistic enhancement of the transformation frequency ordinarily observed with either chemical agent alone. In mass cultures that were X-irradiated with 250 R, seeded for colony formation and treated with either 15 or 30 ergs/mm2 of U.V. 48 hours later, the enhancement of transformation was approximately 12- and 6-fold on a colony basis and 6- and 3-fold on a dish basis, respectively. No transformation occurred after X-irradiation only. The enhancement obtained by the pre-treatment with X-ray is imilar to that reported for the combination of X-ray and chemical carcinogens.     

Radiation-induced specific locus mutations in human mammary epithelial cells

A specific locus mutagenesis assay using primary cultures of human mammary epithelial cells has been developed. A mutation frequency at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus of approximately 2 X 10(-5) mutations per surviving cell per gray of ionizing radiation was estimated in these cells.     

Interactions of nitrilotriacetic acid (NTA) with Cr(VI) compounds in the induction of gene mutations in cultured mammalian cells

We used the V79 Chinese hamster cell line to detect the induction by NTA of 6-thioguanine resistance, due to mutation at the HGPRT locus, with direct and indirect mutagens as positive controls. NTA was tested within the 10(-4)-1.5 X 10(-2) M concentration range: although it was cytotoxic above the 10(-2) M dose, it did not increase the frequency of mutations at any of the tested concentrations, independently of metabolic activation (rat-liver S9 fraction). NTA is known to dissolve heavy metals and therefore to increase their genotoxicity. We found that an insoluble Cr(VI) compound, lead chromate (PbCrO4), was not cytotoxic nor mutagenic on V79 cells, probably because it is taken up by the cells very slowly, whereas the presence of NTA (2.5 X 10(-3) M in water) elicited a direct cytotoxicity and mutagenicity, which was dose-dependent from 5 X 10(-5) M to 10(-4) M PbCrO4. This effect was due to solubilization of the chromate anion by NTA, as determined by comparing spectrophotometric determinations of Cr(VI) in PbCrO4 treatment solutions with a mutagenicity titration curve obtained with a completely soluble Cr(VI) salt (potassium dichromate, K2Cr2O7).     

The genotoxicity of alpha particles in human embryonic skin fibroblasts

Cell inactivation and induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus have been measured in cultured human fibroblasts (GM10) exposed to alpha particles from 238Pu (LET at the cell surface was 100 keV/microns) and 250 kVp X rays. The survival curves resulting from exposure to alpha particles are exponential. The mean lethal dose, D0, is approximately 1.3 Gy for X rays and 0.25 Gy for alpha particles. As a function of radiation dose, mutation induction at the HGPRT locus was linear for alpha particles whereas the X-ray-induced mutation data were better fitted by a quadratic function. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in cells exposed to alpha particles than to X rays.     

Cellular uptake, cytotoxic and mutagenic effects of insoluble chromic oxide in V79 Chinese hamster cells

The cellular uptake, the cytotoxicity and the induction of resistance to 6-thioguanine (6-TG) in Chinese hamster V79 cells exposed to insoluble crystalline trivalent chromium [Cr(III)], Cr2O3, were investigated. Intracytoplasmic Cr2O3 crystalline particle-containing vacuoles were observed by electron microscopy. Concentrations of 50-200 micrograms/ml did not have a marked killing effect but did show a predominantly concentration-dependent inhibitory effect on cell cycle progression with accumulation of cells in G2 phase. Exposure for 18 h to Cr2O3 induced a statistically significant (p less than 0.001) increase in the mutation frequency of up to 10-fold over the controls. Expression time was 6 days for the lowest concentration and 9 days for the highest. Culture of 6-TGr clones in selective media indicated that they were mutants at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. Examination of growth patterns of Cr2O3-induced mutants showed that, after a delay in reinitiating cell growth, they had varying growth kinetics. The results indicate the ability of a particulate (Cr(III) compound to induce mutation in a mammalian cell system and the usefulness of such systems for detecting genotoxic insoluble metal compounds.     

An effect of cell-cycle position on ultraviolet-light-induced mutagenesis in Chinese hamster ovary cells

Using synchronous populations obtained by selectively detaching mitotic cells from cultures grown in monolayer, we demonstrate here that Chinese hamster ovary (CHO) cells exhibit a differential sensitivity to mutation induction by UV as a function of position in the cell cycle. When mutation induction to 6-thioguanine (TG) resistance is monitored, several maxima and minima are displayed during cell-cycle traverse, with a major maximum occurring in early S phase. Although cells in S phase are more sensitive to UV-mediated cell lethality than those in G₁ or G₂/M phases, there is not a strict correlation with induced mutation frequency. Fluence-response curves obtained at several times during the cell cycle yield D_q values approximating 6 J/m². The primary survival characteristic which varies with cell cycle position is D₀, ranging from 2.5 J/m² at 6 h after mitotic selection to 5.5 J/m² at 11 h afterward. Based on studies with asynchronous, logarithmically growing populations, as well as those mitotically selected to be synchronous, the optimum phenotypic expression time for induced TG resistance is 7–9 days and is essentially independent of both UV fluence and position in the cell cycle. All isolated mutants have altered hypozanthine—guanine phosphoribosyl transferase (HGPRT) activity, and no difference in the residual level of activity was detected among isolated clones receiving UV radiation during G₁, S, or late S/G₂ phases of the cell cycle. Changes in cellular morphology during cell-cycle traverse do not contribute to the differential susceptibility to UV-induced mutagenesis.     

Repair of ultraviolet light-induced damage in Micrococcus radiophilus, an extremely resistant microorganism.

Repair of ultraviolet radiation damage was examined in an extremely radioresistant organism, Micrococcus radiophilus. Measurement of the number of thymine-containing dimers formed as a function of ultraviolet dose suggests that the ability of this organism to withstand high doses of ultraviolet radiation (20,000 ergs/mm2) is not related to protective screening by pigments. M. radiophilus carries out a rapid excision of thymine dimers at doses of ultraviolet light up to 10,000 ergs/mm2. Synthesis of deoxyribonucleic acid is reduced after irradiation, but after removal of photodamage the rate approaches that in unirradiated cells. A comparison is drawn with Micrococcus luteus and M. radiodurans. We conclude that the extremely high resistance to ultraviolet irradiation in M. radiophilus is at least partly due to the presence of an efficient excision repair system.     

The relationship between DNA damage and mutation frequency in mammalian cell lines treated with N-nitroso-N-2-fluorenylacetamide

The induction of DNA single-strand breaks in C3H10T1/2 mouse fibroblasts and Chinese hamster ovary (CHO) cells by N-nitroso-N-2-fluorenylacetamide (N-NO-2-FAA) was demonstrated by the alkaline elution technique. Without metabolic activating system (i.e., rat liver S9 fraction), N-NO-2-FAA exhibits more direct and strong damaging effects on DNA than its parent compound, 2-FAA, at equal concentration in both cell lines. To compare the DNA-damaging potency of N-NO-2-FAA with other well-known carcinogens, such as benzo[a]pyrene, 2-nitrofluorene, and N-methyl-N'-nitrosoguanidine (MNNG), the order of potency is as follows: MNNG (5 microM) greater than N-NO-2-FAA (150 microM) greater than benzo[a]pyrene (20 microM) at equitoxic concentrations, LD37, in the same cell system. Another parallel experiment indicated that N-NO-2-FAA could disrupt the superhelicity of circular plasmid DNA (pBR 322) at a dose range of 0.1-50 mM; however, a complete conversion to form III linear DNA was found at the highest concentration (50 mM). After treatment with various concentrations of N-NO-2-FAA, ouabain resistance (ouar) was induced in C3H10T1/2 cells, while both ouar and 6-thioguanine resistance (6-TGr) were induced in CHO cells. The mutation frequency in the Na+/K+-ATPase locus in CHO cells (1.5 X 10(-6) mutants/microM) is higher than that in C3H10T1/2 cells (1.0 X 10(-6) mutants/microM). The maximal mutation frequency at the Na+/K+-ATPase gene locus was attained with 30 min of exposure in C3H10T1/2 cells, whereas the mutation frequency in CHO cells continued to increase up to 80 min of treatment. Similarly, the maximal mutation frequency at the HPRT locus also continued to increase up to 80 min of treatment. Finally, a linear plot of alkali-labile lesions versus 6-TGr mutations was obtained; but the same relationship was not observed in the case of ouar mutation.     

Effects of growth inhibitors and ultraviolet irradiation on F pili

The effects of chloramphenicol, nalidixic acid, mitomycin C, NaCN, and ultraviolet irradiation at 253.7 nm on F pili production by Escherichia coli cells was studied by electron microscopy. The results show that cells contain pools of pili protein, and that assembly does not require synthesis of protein or deoxyribonucleic acid (DNA). NaCN (2 x 10(-2)m) prevents the reappearance of pili and causes existing pili to disappear quickly from the cell surface. This suggests that energy is used in the assembly of pili and to retain pili on the cell. Cells irradiated with high doses (10(4)ergs/mm(2)) of 253. 7 nm light produce fewer pili, and these are shorter than normal. Dose-response curves for number of pili per cell and length of pili resemble single hit kinetics, showing 37% survival at 10(4) ergs/mm(2) and 2 x 10(4) ergs/mm(2), respectively. This suggests that DNA is at the site where pili are produced, and that it may be involved in the assembly of pili.     

Mutation induction in a mouse lymphoma cell mutant sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation

The mutant mouse lymphoma cell Q31, which is sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation (UV), was compared with the parental L5178Y cell for the effect of caffeine and mutation induction after UV irradiation. Caffeine potentiated the lethal effect of UV in both cell strains to a similar extent, indicating that the defective process in Q31 cells was caffeine-insensitive. UV-induced mutation to 6-thioguanine resistance was determined in L5178Y and Q31 cells. The maximal yield of mutants was obtained 7 days post-irradiation in L5178Y cells and 14 days in Q31 cells for higher UV doses. It appears that a much longer time is required for the mutant cells than for the parental cells for full expression of the resistance phenotype even at equitoxic UV doses. A substantially higher frequency in induced mutations was observed in Q31 cells than in L5178Y cells at a given dose of UV. A plot of induced mutation frequency as a function of logarithm of surviving fraction again indicates hypermutability of Q31 cells as compared with the parental strain. In contrast, X-rays induced a similar frequency of mutations to 6-thioguanine resistance in L5178Y and Q31 cells.     

Unscheduled incorporation of thymidine in ultraviolet-irradiated human lymphocytes

The nature, degree, and kinetics of unscheduled thymidine incorporation previously shown to occur in 90 % of irradiated lymphocytes was stud-incorporation was sever ely depressed i n t h e presence of 10(-4) M acriflavine and by low temperature, but was unaffected by 10(-3) M hydroxyurea or caffeine. Over a dose range of 25 to 400 ergs/mm2, the uptake of thymidine was increased by a factor of only 1.6, although the survival of lymphocytes, measured 5 days after irradiation, decreased by almost two orders of magnitude. (The survival curve suggests that 90% of the lymphocytes have a D0 of 35 ergs/mm(2) and 10 % have a D0 of 250 ergs/mm(2).) After exposure to 25 ergs/mm(2), over 70 % of the cells survived for 5 days in culture; moreover, cells which had been stimulated by this dose to incorporate thymidine transformed and divided after exposure to phytohema-glutinin. The final uptake of thymidine was significantly greater when a total dose of 75 ergs/mm(2) was fractionated into three doses of 25 ergs/mm(2) given at six hourly intervals than when it was given as a single dose. The degree of thymidine incorporation and the fraction of leukemic cells labeled were not significantly different from those in normal lymphocytes.