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W G Thilly

Publications and source records attributed to W G Thilly.

At least 109 records · Page 6Linked to original sources
PubMed1986

An internal survival standard for Salmonella typhimurium forward mutation assays.

A novel S. typhimurium forward mutation assay which avoids plating density artifacts is described. The new method uses a pair of multiply drug-resistant substrains of TM677, a bacterial strain used in previous forward mutation studies. This technique permits the measurement of cell survival following mutagen treatment by plating the culture on specially supplemented plates at the same cell concentration used to measure mutant yield. Thus this method is both technically easier and theoretically superior to our previous method.

Azaguanine↗
PubMed1986

Optimization of environment for high density Vero cell culture: effect of dissolved oxygen and nutrient supply on cell growth and changes in metabolites.

This study was initiated for optimization of the environment of a technologically useful mammalian cell line for high density production. Cultures of Vero cells on microcarriers were perfused with 100%, 50%, 25% and 12.5% modified L15 media (galactose was replaced with 10 mM-fructose, with 4 mM-glutamine and 5% foetal bovine serum) in phosphate-buffered saline at either 4 or 8 vol. day-1. Cell growth, pH, dissolved oxygen, and changes in the metabolites, lactate to pyruvate and lactate to ammonia indices, demonstrated that under the conditions used in the present study, perfusion of cultures with 50% L15 medium in PBS at 8 vol. day-1 provided the optimum microenvironment for Vero cell growth. The highest cell density in the perfused cultures was 3 X 10(7) cells ml-1, which at these conditions was ten times higher than the maximum cell density (3 X 10(6) cells ml-1) obtained in a batch culture. Nutrient supply and conditioning factors were the most probable growth-limiting factors in cultures that were perfused with 12.5% and 25% L15 media, while multilayering, limitation of available oxygen, and accumulation of metabolic end products in the cellular microenvironment were the most probable causes of a density-dependent inhibition of cell growth observed under the optimized and overfed (supply of 100% L15 medium at the rate of 8 vol. day-1) culture conditions. Under the optimized environmental condition, the major source of energy was probably glutamine during the first week. However, significant utilization of fructose became evident at higher cell densities during the second week, when lactate production dramatically declined and reached an almost undetectable level, while respiration progressively assumed the predominant role in energy production. It is postulated that 'available' oxygen in the multicell-layered microenvironment of the optimized cultures was higher than in the overfed culture due to the greater utilization rate of oxygen for oxidation of excess nutrients in the overfed culture.

Ammonia↗
PubMed1985

Mutational and pseudomutational effects of 5-bromodeoxyuridine in human lymphoblasts.

We have studied the effects of 5-bromodeoxyuridine (BrdUrd) at two genetic loci in diploid human lymphoblast cells. In thymidine kinase heterozygotes (tk +/-), a 2-h dose of BrdUrd induced a transient, non-heritable resistance to the thymidine analogue, trifluorothymidine (F3TdR). We have called this phenomenon pseudomutation and have shown that affected cells acquire the ability to survive in the presence of F3TdR and then, after degradation of F3TdR in the medium, return to an apparently normal wild-type state. Our data suggest that BrdUrd incorporation into DNA as a thymidine analogue is responsible for the effect, which we interpret as a temporary loss of thymidine kinase activity. This effect is not seen in tk +/+ homozygotes. In contrast, at the hypoxanthine-guanine phosphoribosyl transferase locus in tk +/- heterozygotes, BrdUrd did not induce a permanent, heritable resistance to 6-thioguanine (gene locus mutation). We detected such mutations only in the tk +/+ homozygote and only at external BrdUrd concentrations considerably higher than those which saturate the uptake of BrdUrd into DNA as a thymidine analogue. We postulate that the reduced TK enzyme levels (30%) in the heterozygote prevent the build-up of a sufficiently high intracellular BrdUrd triphosphate pool to promote the misincorporations as deoxycytidine triphosphate which may be responsible for gene locus mutation.

Aminopterin↗
PubMed1985

A human cell line sensitive to mutation by particle-borne chemicals.

A human lymphoblastoid cell line with ability to perform oxidative metabolism of various chemicals is mutated by the direct addition of an intact particulate soot. This experiment demonstrates that materials associated with combustion-generated particulates are biologically available and able to cause genetic changes in metabolically competent human cells.

Air Pollutants↗
PubMed1985

High density mammalian cell growth in Leibovitz bicarbonate-free medium: effects of fructose and galactose on culture biochemistry.

The most commonly used buffering system for mammalian cell cultures is a bicarbonate/CO2 system, which requires CO2 regulators and incubators to supply a constant level of CO2. As a replacement, Leibovitz developed a bicarbonate-free medium, L15, with relatively high levels of certain amino acids in the free base form. We found that a modified form of L15, containing 10 mM-fructose instead of galactose, supported high density growth of Vero and MDCK cells, with maintenance of a stable pH and lactate/pyruvate ratio. We report here investigations of Vero and MDCK cell growth and culture biochemistry at different concentrations of the two carbohydrates. The initial fructose concentration in the medium affected the eventual pH of the medium, the rate of production of lactic acid and ammonia, and the fructose utilization rate. The initial galactose concentration affected the growth rate but did not affect eventual culture pH, the rates of lactate and ammonia production, or the rate of its own utilization. Thus, Leibovitz' formula, modified to contain 10 mM-fructose, appears to yield satisfactory stability of culture pH and the lactate/pyruvate ratio. At all concentrations of galactose tested, the lactate/pyruvate ratio drifted out of the physiological range.

Ammonia↗
PubMed1984

Mutation of xeroderma pigmentosum lymphoblasts by far-ultraviolet light.

Survival and mutation were measured after UV irradiation in a human diploid B-lymphoblastoid line, XPA3, derived from a xeroderma pigmentosum patient of complementation group C. Relative to a normal human lymphoblastoid line, the XPA3 cells were more sensitive to killing as evidenced by a diminished shoulder in the survival curve and a steeper slope in the log-linear portion of the survival curve. While XPA3 cells were also more sensitive to mutation than normal cells, the data are complex. We interpret them to show a diminished threshold and a greater slope in the mutation curve at fluences which are not appreciably toxic. Examination of the 3H-TdR labeling index and rate of DNA synthesis after irradiation indicated that, at low equitoxic UV fluences (S greater than or equal to 0.85), XPA3 cells in S phase failed to slow their rate of DNA synthesis to the same extent as normal cells. Thus, fewer XPA3 than normal cells accumulated in S phase at relatively non-toxic fluences. However, at fluences which were toxic to XPA3 but not to normal cells, the early effects on DNA synthesis for both cell types were virtually indistinguishable. Our observations suggest that XPA3 cells have a reduced repair capacity for UV damage both because of a reduced rate of DNA repair and because uninterrupted passage through S phase reduced the time available for repair before fixation of both lethal and mutational damage. Furthermore, we interpret the relatively greater slopes in survival and mutation curves as an indication that, in XPA3 cells, there is a greater probability that an unrepaired lesion will result in cell death or mutation.

Cell Survival↗
PubMed1984

Assay for gene mutation in a human lymphoblast line, AHH-1, competent for xenobiotic metabolism.

A novel quantitative gene-locus mutation assay has been developed using a line of human lymphoblast cells, designated AHH-1, competent in oxidative xenobiotic metabolism. AHH-1 cells are sensitive to the mutagenic action of both chemically reactive mutagens and mutagens which require oxidative metabolism to exert their mutagenicity. These cells are readily mutated by direct exposure to ethyl methanesulfonate, ICR-191, 2-acetoaminofluorene, aflatoxin B1, benzo[a]pyrene (BP), cyclopenta[c, d]pyrene, dimethylnitrosamine, lasiocarpine, and 1-methylphenanthrene.

Aryl Hydrocarbon Hydroxylases↗
PubMed1983

Rate of induced forward mutation at 3 genetic loci in Salmonella typhimurium.

The rate of forward mutation to 8-azaguanine, 5-fluorouracil and azetidine carboxylic acid resistance was measured in Salmonella typhimurium following treatment with 18 chemical mutagens. Although the absolute frequency of both spontaneous and chemically-induced mutation was different at the markers scored, the 3 selection systems were found to be equisensitive: each system produced a statistically significant response at approximately the same mutagen concentration. This result suggests that in Salmonella the target size present in a given forward mutation assay is sufficiently large to approximate the level of mutation occurring elsewhere in the chromosome in sections of DNA of similar size.

DNA, Bacterial↗
PubMed1983

Mutation of human lymphoblasts exposed to low concentrations of chemical mutagens for long periods of time.

Methylnitrosourea (MNU), ethyl methanesulfonate (EMS), and 4-nitroquinoline-N-oxide (4NQO) induce mutation to 6-thioguanine resistance and trifluorothymidine resistance in diploid human lymphoblasts (TK6). In single exposure experiments in which greater than 10% of treated cells survive, mutation as a function of concentration is linear for MNU, accelerates for EMS and appears to reach a plateau for 4NQO. In order to probe the bases of these concentration dependencies, human lymphoblasts were exposed for 20 days to each of the three mutagens. Each individual exposure chosen was in itself insufficient to induce statistically significant mutation and each resulted in a cellular survival of greater than 95%. Under this regimen, induced mutation as a function of the number of exposures was linear for all three mutagens. Prior exposure to low concentrations of mutagens was found to have no significant effect on the amount of mutation induced in subsequent exposures. Thus, no biological evidence was found for the induction of repair of misrepair systems.

4-Nitroquinoline-1-oxide↗
PubMed1983

Ultraviolet light-induced mutation of diploid human lymphoblasts.

Ultraviolet irradiation (254 nm) of immortal diploid human lymphoblasts killed cells, caused mutation at three genetic loci studied, and transiently inhibited 3H-TdR uptake into DNA. A shoulder of about 6 J/m2 and a D0 of 6 J/m2 was observed for survival. Mutation rose in a monotonic non-linear fashion through 6 J/m2; above 6 J/m2, complex behavior approximating a plateau in induced mutation was observed. Irradiation at 4.4 J/m2 caused a transient increase in the number of cells synthesizing DNA and a decrease in the rate of DNA synthesis relative to mock-irradiated controls. The parameter of rate of DNA synthesis per cell in DNA synthetic phase showed a rapid recovery toward control values between 2 and 4 h after irradiation and a slower recovery to control values by 22 h post-irradiation. Fractionated dose schedules were used to measure the effects of allowing a time interval between doses at nontoxic fluences (2.2 J/m2), moderately toxic fluences (8.8 J/m2) and toxic fluences (17.6 J/m2). These measurements indicate that in the non-toxic range of fluences common to human exposure, mutational response is mediated by a post-irradiation process which seems to show an enhanced ability to protect against mutation induced by subsequent irradiation. However, at moderately toxic fluences there was little effect of dose fractionation, and at toxic fluences, a time-dependent increase in mutation fraction was observed at separation times greater than 7 h. We suggest that these latter observations arise primarily from cell cycle heterogeneity with regard to sensitivity to UV killing and mutation.

Cell Line↗
PubMed1983

Toxic and mutagenic effects of formaldehyde in Salmonella typhimurium.

Toxic and mutagenic activities of formaldehyde were studied in Salmonella typhimurium strain TM677, using forward mutation to 8-azaguanine (8-AG) resistance both in the absence and in the presence of Aroclor-induced rat-liver postmitochondrial supernatant (PMS). The results showed that formaldehyde was toxic and mutagenic to the bacteria in both systems, but toxicity and mutagenicity were reduced in the presence of PMS. The minimum concentration required to induce toxicity and mutagenicity was 0.17 mM in the absence of PMS and 0.33 mM in the presence of PMS.

Animals↗