Somatic cell mutation. Detection and quantification of x-ray-induced mutation in cultured, diploid human fibroblasts

We describe a system for detecting somatic cell mutation to 8-azaguanine (8AG) resistance in cultured, diploid human fibroblasts. Hypoxanthine-guanine phosphoribosyltransferase (HG-PRT)-deficient, AG-resistant fibroblasts from boys with the X-chromosomal, Lesch-Nyhan (L-N) mutation served as one type of prototype mutant cells. Both spontaneous and X-ray-induced mutation were studied. Recovery of L-N cells was a function both of density of normal cells and of the AG concentration used for selection. Optimum recovery was achieved at an initial inoculum of 2·10⁴ normal cells per 60 mm diameter culture dish and an AG concentration of 8·10^?6M. Efficiency of recovery was between 39 and 90% and controls to determine this efficiency were included in mutagenesis experiments.Attempts to free normal cell populations of pre-existing AG-resistant mutant cells by pregrowth in HAT medium failed because, unlike L-N mutants, most spontaneous AG-resistant mutants can grow in HAT medium. Although pre-existing mutants probably caused overestimation, the average spontaneous mutation rate derived from our experiments was 4.5·10^?6 per cell generation. Eliminating one large-yieldv experiment reduced this estimate to 1.9·10^?6. Clonal survival of cultured human fibroblasts as a function of X-ray dose was studied. X-Irradiation increased the mutation rate above spontaneous background. Minimum estimates of the increases were 1.13·10^?9 per R per cell at 75 R, 7.49·10^?8 per R per cell at 125 R, 6.87·10^?8 per R per cell at 150 R and 2.16·10^?7 per R per cell at 250 R. The total mutagenic effect and the induced mutation rate appeared to be dose-dependent. Normal parental cell strains and their derived AG-resistant mutants had similar X-ray sensitivities indicating that X-rays induced mutations rather than selected for pre-existing mutants.Because of the realism of the cultured diploid, human fibroblast model vis-a-vis in vivohuman cellular events, the mutation detection system described herein is proposed as being potentially useful for environmental monitoring.     

UV-induced mutagenesis at the hypoxanthine—guanine phosphoribosyl transferase locus in two L5178Y mouse lymphoma cell strains with different UV sensitivities

Two strains of L5178Y mouse lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S), differ markedly in their sensitivity to 254 nm UV radiation (D₀ = 0.7 and 5.5 J/m²; n = 6.0 and 2.0 for LY-R and LY-S cells, respctively). In this study, the frequency o hypoxanthine-guanine-phosporibosyl-transferase-deficient mutants was determined, using 6-thioguanine (TG) as a selective agent, in populations of LY-R and LY-S cells exposed to various fluences of UV radiation. The spontaneous mutation frequency for LY-R cells was (3.7 ± 0.6) × 10^?5 TG^r mutants per viable cell, and the UV induction rate was (2.2 ± 0.8) × 10^?4 TG^r mutants per viable cell, per J/m². Both spontaneous and induced mutantion frequencies were much lower for LY-S cells. The sopntaneous mutation frequency for these cells were too low to make its measurement practicable ( < 0.0013 × 10^?5 TG^r mutants per viable cell). Mutation induction rate was (4.2 ± 2.2) × 10^?7 TG^r mutants per viable cell, per J/m². These differences in mutability do not appear to be due to gene duplication in LY-S cells, or to selective growth disadvantage of LY-S-derived TG-resistant mutants. Possible mechanisms underlying the differences in mutability of LY-R and LY-S cells are considered.     

Dilution of hypoxanthine-guanine phosphoribosyl transferase and other factors affecting the frequency of 6-thioguanine resistance in Chinese hamster lung cells.

Factors influencing the frequency of thioguanine resistant mutations were examined in Chinese hamster lung cells damaged with a carcinogen, N-acetoxy-2-acetyl aminofluorene. Factors such as inoculum density, expression time, and concentration of selective agent were found to have a profound effect on the mutation frequency.Over a range of doses, a longer expression time is required for mutant cells from a more damaged population to reach their maximum frequency. In order to investigate the elements involved in this phenomenon, the increment in the plating efficiency of treated cells as a function of expression time, spontaneous mutation rate per cell per generation, viability of mutant as well as wild type cells, and half life of HGPRTase were evaluated.There was an observed relationship between induced mutation frequency and plating efficiency of treated cells. When treated cells had recovered from effects of the treatment and arrived at the normal level of plating efficiency, they also yielded the maximum frequency of mutations.The estimated mutation rate was 5.5 × 10^?8 per cell per generation. This number is too small to account for the increment in mutation frequency with the increase in the expression time. The mutation frequency of spontaneous origin was 4 × 10^?6 and that of induction of 10^?5 M NA-AAF was 10^?4. Lower growth rates of mutant cells cannot explain this increase in the number of mutants recovered, either.Continuous diminution in the level of HGPRTase, at 35% daily, interpreted as an important factor responsible for the recovery of mutation frequency during expression time, was observed in non-dividing cells. None of a large number of mutants sampled from those isolated had HGRPT activity. This indicates that they are true mutants and are not a result of phenocopy. Only cells completely deficient in HGPRT activity are recovered in TG selection medium. It is suggested, therefore, that this cell line is suitable for mutagenicity testing in the induction of mutation at the HGPRT locus.     

Mutagenesis studies on cultured mammalian cells. The sensitivity of the asparagine-requiring phenotype to several chemical agents.

The effect of several chemical agents on the mutation frequency from asparagine dependence to asparagine independence has been studied in Jensen sarcoma cells. It was found that ethylmethanesulfonate brought about a dramatic exponential increase, while nitrosoguanidine was not lighly effective as a mutagen, causing only a modest increase in mutation frequency, and quinacrine HCl was ineffective. The results presented here are compared with those obtained in other systems and with our previous work on the effects of UV on mutation induction in the asparagine system. They suggest that the basis of the asparagine requirement of mammalian cell lines resides in a specific genetic alteration in nuclear DNA which is corrected by the mutagenic action of the agents tested here.     

Mutagenic effect of ultraviolet radiation on the non-acid-fast strain ofMycobacterium phlei

The mutability of the PN strain ofMycobacterium phlei was examined after induction of auxotrophic mutants and of STM and VM-resistant mutants, by UV irradiation. A total of 30 auxotrophic mutants were isolated, most of them amino acid-dependent five purine-dependent, and one uracil-dependent. To induce the mutants higher UV doses had to be used so that the survival of cells in the original suspension would not exceed a few per cent. For further genetic work use can be made of 8 auxotrophic mutants (PN try^?ura^?, PN arg^?ura^?, PN ileu^?val^?, PN ileu^?, PN leu^?, PN lys^?, PN lys^?-VM^r, PN val^?), these showing a low frequency of spontaneous reversions. No spontaneous auxotrophic mutants have been found. The frequency of STM and VM-resistant mutants is increased upon UV irradiation, a post-irradiation incubation in a liquid medium without the drug being essential for their phenotypic expression. The highest increase of the number of these mutants is attained after 48 h of post-irradiation incubation and it has been found that, within a certain experimental scatter, the same frequency increase is found on using a complete or a minimal liquid medium. The frequency of spontaneous STM-resistant mutants lies within 5.8×10^?6–8.8×10^?6, of those VM-resistant between 3.1×10^?5 and 4.1×10^?5. The highest frequency of induced STM-resistant mutants lies between 3.0×10^?5 and 9.3×10^?5 and of VM-resistant mutants between 1.1×10^?4 and 2.2×10^?4     

X-ray-induced mutation to 6-thioguanine resistance in cultured human diploid fibroblasts

X-ray induced mutation to 6-thioguanine (6TG)-resistance was studied in early passage cultures of human diploid fibroblasts.The appearance of phenotypic induced mutants in irradiated cell populations was linearly related to the number of post-irradiation cell doublings and to the duration of the growth period prior to mutant selection; the maximum yield of X-ray induced mutants was observed when cells surviving radiation had completed 3–4 doublings (6–7 days growth_in non-selective medium.The maximum induced mutation frequency was linearly related to X-ray dose and the mutation rate was estimated to be 3.1 · 10^?7 mutations per viable cell per rad.The data obtained for X-ray induced mutations in cultured human diploid fibroblasts were compared with (a) similar experimental data obtained with established cell cultures and (b) theoretical predictions of X-ray mutation rates in human germ cells.     

Electrokinetic properties of asparaginase sensitive or resistant mouse leukemic cells treated with L-asparaginase

The electrophoretic mobility of L5178Y cells in 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO₃, pH 7.2, at 25°C was — 1.78 μ·s^?1·V^?1·cm^?1 while that of an L-asparaginase resistant subline, L5178Y/ASN, was — 1.11 μm·s^?1·V^?1·cm^?1. Both cell lines were characterized by terminal sialic acid residues on their surfaces. Treatment of L5178Y cells for 90 min with 10 units of L-asparaginase per ml in saline decreased the electrophoretic mobility of the cells to — 1.65 μm·s^?1·V^?1·cm^?1 while treatment in Fischer's medium decreased the mobility to — 1.25 μm·s^?1·V^?1·cm^?1; neither treatment had a significant effect on the L5178Y/ASN electrophoretic mobility. The results suggest that L-asparaginase has an immediate and specific effect on synthesis of cell surface asparaginyl glycoproteins.     

CELL SURVIVAL CHARACTERISTICS AND MOLECULAR RESPONSES OF ANTARCTIC PHYTOPLANKTON TO ULTRAVIOLET-B RADIATION1

Twelve species of Antarctic diatoms were studied to assess UV sensitivity in relation to cellular and molecular aspects of DNA damage and repair. Responses of cell survival, induction of DNA damage, and DNA repair capacity were determined. There was a wide range of interspecific UV-sensitivity among diatoms. D₃₇ values (average fluence to kill one cell) ranged from 681 J · m^?2 (most sensitive) to 25,338 J · m^?2 (most resistant). Molecular analysis (by radioimmunoassay) of UV-induced DNA damage [induction of cys-syn cyclobutane dimers and pyrimidine (6-4) pyrimidone photoproducts] also revealed considerable variability among species [0.98–84 lesions · (10⁸ daltons DNA)^?1 induced by exposure to 2500 J · m^?2]. Repair of DNA damage ranged from 0.18 to 2.72 lesions removed · (10⁸ daltons DNA)^?1 in 6 h; removal represented 0.72–73.5% of initial damage. Comparison of cellular responses associated with photoenhanced repair and nucleotide excision (“dark”) repair indicated that light-mediated correction of UV damage was an important factor in cell survival. There was a relationship between the number of photoproducts induced and cell survival, but not between repair efficiency and survival. The data also indicate a general dependence of photoproduct induction and D₃₇ values on cell size and shape (expressed as the surface area: volume ratio which ranged from 0.07 to 0.66 between species) and suggest that these factors are indicators of UV sensitivity. Smaller cells with greater surface area: volume ratios sustained more damage per unit of DNA, had lower D₃₇ values, and were more sensitive to UV exposure. The wide species variations observed in molecular and cellular responses to UV exposure emphasize the ecological implications of changes in natural UV regimes. These changes can act as determinants of cell size and taxonomic structure within phytoplankton communities and have as yet unknown effects on trophic interactions within the Antarctic ecosystem.     

An electrogenic Na+/K+ pump in the choroid plexus

Intracellular electrical potential and potassium activity was measured by means of microelectrodes in the epithelial cells of choroid plexus from bullfrogs (Rana catesbeiana). Ouabain applied from the ventricular side caused an abrupt depolarisation of 10 mV but only a gradual loss of potassium from the cells. Readministration of potassium to the ventricular solution of plexuses which were previously depleted of potassium, caused a hyperpolarisation of about 4 mV. These two experiments are consistent with the notion of an electrogenic Na⁺/K⁺ pump situated at the ventricular membrane and which pumps potassium into the cell and sodium into the ventricle. The numerical values obtained suggest that 3 sodium ions are pumped for 2 potassium ions. The permeability coefficient for potassium exit from the cell is calculated to be 1.24 · 10^?5 cm^?1 · s^?1 expressed per cm² of flat epithelium.     

Ventilation and oxygen consumption in the hagfish, Myxine glutinosa L.

Ventilation was measured directly in the hagfish, Myxine glutinosa L., by means of an electro-magnetic blood flowmeter. Ventilatory flow and frequency increased from 0.86 ± 0.27 ml·min^?, and 18.2 ± 5.1·min^?, respectively, at 7°C to 1.70 ± 0.20 ml·min^?, and 70.1 ± 9.5·min^? at 15 ·C.Standard oxygen consumption,V?O₂, was measured in non-buried hagfish. V?O₂ was 0.57 ± 0.17μl O₂·g^?1·min^?1 at 7°C, and 0.85 ± 0.12μl O₂·g^?1·min^?1 at 15°C.     

Improvement of Biomethane Production from Sewage Sludge in Co-digestion with Glycerol and Waste Frying Oil,Using a Design of Experiments

A central composite design circumscribed method was used to define the experimental conditions that improve the methane production rate (k_CH4, liters of methane per kilogram of VS of waste added and per day) and the cumulative methane production (cMP, liters of methane per kilogram of VS of waste added) of the co-digestion of sewage sludge (SS) with crude glycerol (cGly) and waste frying oil (WFO). Three factors were selected, i.e., SS concentration, global co-substrate concentration, and mass fraction of cGly (x_cGly) in a mixture of cGly and WFO (in chemical oxygen demand, COD). SS digestion without co-substrate reached a cMP of (294?±?6) L·kg^?1 and a k_CH4 of (64?±?1) L·kg^?1·d^?1, at standard temperature and pressure conditions and expressed relatively to the initial volatile solids. After statistical analysis, SS and co-substrate concentrations of 4.6 g·L^?1 and 8.8 g·L^?1 (in COD), respectively, with x_cGly of 0.8, were defined to simultaneously boost cMP (91 % more) and k_CH4 (3-fold increase). Application of these conditions would yield 214 MWh more in electricity per 1000 m³ of SS digested.     

Transposition of the hobo element in Drosophila melanogaster somatic cells

Somatic mutation and recombination test on wing cells of Drosophila melanogaster showed that the recombination frequency in the somatic tissues of strains studied correlated with the presence of a full-length copy of the hobo transposable element in the genome. Transposition of hobo in somatic tissue cells at a frequency 3.5 × 10^?2 per site per X chromosome was shown by fluorescence in situ hybridization with salivary gland polytene chromosomes of larvae of one of the D. melanogaster strains having a full-length hobo copy.     

Mutagenesis in cultured mammalian cells. II. Induction of gene mutations in Chinese hamster cells

Mutations controlling the resistance to 6-mercaptopurine (6-M) and the ability to multiply in a medium with a low concentration of glucose (“glucose-independent” mutants) were induced in cultured Chinese hamster cells by N-nitrosomethylurea (NMU), 5-bromodeoxyuridine (BUdR), UV and X-rays. The chemical agents were found to be very active in induction of mutations to 6-M resistance (NMU and BUdR) and mutations of “glucose independence” (NMU). These agents increase the yield of mutations as compared to the spontaneous mutation rate by about two orders of magnitude. The induced rate of 6-M-resistant mutations by X-rays was 2.0 ? 10⁻⁷ per viable cell per roentgen. BUdR approximately equally increases the cell's sensitivity to both inactivating and mutagenic action of X-rays. The maximum induction of mutations to 6-M resistance by UV was observed at 100 erg/mm². This dose leads to 1 16-fold increase of the mutation frequency as compared to the spontaneous rate. Further increase of the UV dose up to 200 erg/mm² resulted in a lower yield of mutations per dose unit. The highest yield of mutations to 6-M resistance induced by NMU, BUdR and X-rays was observed if cells were plated in selective medium several generations after the mutagenic treatment. The maximum yield of mutations to 6-M resistance induced by UV and of glucose-independence induced by NMU was recorded if cells were transferred to selective media immediately after treatment. The kinetics of expression of mutations and the decline of their number observed after prolonged incubation of treated cells in nonselective conditions are discussed.     

Glucose uptake rate as a tool to estimate hybridoma growth in a packed bed bioreactor

The immobilisation of cells in a perfusion culture allows to obtain a high cell concentration and an efficient removal of the catabolites without cell loss. A disadvantage of this system is that the cell density cannot be directly monitored. The cellular metabolism is just followed by online measurements of pH and dissolved oxygen (DO) and off-line determinations of residual metabolites. In this article, we report a high cell density achieved by the cultivation of a hybridoma in a bubble-column bioreactor filled with hollow glass cylinders. The parameters monitored during the cultivation were pH, temperature, DO, glucose, lactate and monoclonal antibody. The glucose uptake rate was used to estimate the cell concentration along the time. The maximum cell concentration calculated for the considered cultivation time was 2.7?×?10⁷ cell?·?ml^?1. The glucose concentration in the media decreased stepwise twice, causing a decrease on the specific growth rate, while maintaining high antibody productivity levels. Maximum monoclonal antibody productivity was 503?μg?·?l^?1?·?day^?1 and specific productivity, considering calculated cell density, was 0.019?ng?·?cell^?1?·?day^?1.     

Membrane methylation in isolated rat testis interstitial cells unmasks functional luteinizing hormone receptors

Treatment of intact isolated rat testis interstitial cells with S-adenosylmethionine as methyl donor, increases substantially the number of LH human CG receptors (100–200%) without modifying the equilibrium dissociation constant. The increase in binding capacity was associated with an augmentation in the sensitivity of the rat testis interstitial cells to produce testosterone in response to LH, suggesting a functional role of the unmasked receptors. The amount of S-adenosylmethionine necessary to obtain an increase in LH binding capacity and preserve cell viability was 25–50 μg/ml per 1.6·10⁷ cells. 10 mM MgCl₂ in addition to the Mg²⁺ present in the medium was necessary to maintain cell viability. ³H-labelled methyl groups were incorporated mainly into the lipid fraction (208 fmol/10⁶ cells) when ³H-S-adenosylmethionine was incubated with the cells for 2 h at 30°C. Our results are consistent with the conclusion that early action of LH may involve an activation of methyltransferase activity, phospholipid methylation, an increase in LH binding capacity and an increase in receptor function.     

Effects of Initial Phosphorus Concentration and Light Intensity on Biomass Yield per Phosphorus and Lipid Accumulation of Scenedesmus sp. LX1

Phosphorus has been considered as one of the most important limiting resources of large-scale production of microalgal biofuel. The approaches to increase biomass yield per phosphorus, along with the lipid accumulation properties of Scenedesmus sp. LX1, were investigated in this study. It was found that practical biomass yield per phosphorous was reduced with the increase of initial phosphorus (P) concentration, but increased with light intensity. The highest biomass yield per P of 4,500 kg-biomass/kg-P was achieved at initial phosphorus concentration of 0.05 mg?·?L^?1 under the light intensity of 320 μmol photon?·?m^?2?·?s^?1. Furthermore, the lipid content per biomass and triacylglycerols (TAGs) content per lipid were found to be positively correlated to biomass yield per P. With the biomass yield increased from 2,800 kg-biomass/kg-P to 4,500 kg-biomass/kg-P, the lipid content per microalgal biomass and TAG content per lipid increased from 18.7 % to 35.0 % and from 69.5 % to 83.0 %. These results suggested a possible approach to achieve high biomass production and high lipid content simultaneously.     

Kinetic analysis of the binding of immunoglobulins IgG1 and IgG2 to bovine mammary cells

Previous reports were confirmed that specific binding sites exist on bovine mammary cells near parturition presumably involved in the transfer of immunoglobulins IgG1 and IgG2 across the mammary gland at the time of colostrum formation. Determination of the kinetic parameters of these binding sites using ¹²⁵I-labeled IgG1 and IgG2 immunoglobulins indicated the presence of sites with association constants (K_a) of about 5 · 10⁸?10 · 10⁸ M^?1 for both subclasses during normal lactation with about 9000 and 3000 sites per cell for each, respectively. The number of IgG1 sites tended to increase as the time of parturition approached. In addition, a new group of sites numbering about 5000 per cell with very strong binding of IgG1 (K_a about 45 · 10⁸ M^?1) appeared on the cells about a week before parturition. The numbers and affinity of the IgG1 and IgG2 binding sites bear a relationship to the approximate 7:1 ratio of these immunoglobulin subclasses found in colostrum and normal milk and to the time of maximum colostrum formation. The results support the premise that a highly selective transport mechanism exists in the bovine mammary epithelial cell for the transfer of IgG1 and IgG2 immunoglobulins from blood to the lacteal secretions.     

Sub-lethal effect of ultraviolet radiation on the growth, intestinal adherence ability and cholesterol removal potentials of parent cells and subsequent sub-culturing of Lactobacillus acidophilus BT 1088 under conditions that mimic the human gastrointestinal tract

The aim of this study was to evaluate the sub-lethal effect of ultraviolet radiation (UV) on the cell growth, intestinal adherence ability and cholesterol removal potential of parent cells and the possible inheritance of such effects on subsequent sub-cultures of Lactobacillus acidophilus BT 1088 cells under conditions that mimic the human gastrointestinal tract. We found that UV decreased (P?<?0.05) growth of the parent cells immediately upon treatment (0 h), although an increase (P?<?0.05) in growth was observed at 8–24 h of fermentation compared to that of the control. The intestinal adherence ability of the parent cells decreased significantly by 15.62 % (P?<?0.05) compared to that of the control. Nevertheless, UV led to increased (>26.22 %; P?<?0.05) cholesterol removal from the parent cells, accompanied by an increased incorporation of cholesterol into the cellular membrane and an increased ratio of membrane cholesterol:phospholipids (C:P; P?<?0.05; 95 % confidence interval 8.71–121.95 %) in parent cells, compared to that of the control. Incorporated cholesterol was found in the interface of apolar and polar regions, polar heads and also apolar tails of phospholipids in the cellular membrane bilayer. However, such traits were not inherited by the treated cells in subsequent sub-cultures (first, second and third sub-culture). Our data suggest that UV could be a potential physical treatment to increase the cholesterol removal ability of parent cells without inducing permanent damage to the treated cells. UV treatment did not affect the intestinal adherence functionality of the treated cells in subsequent sub-cultures.     

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.