Inhibition of chromosome repair by caffeine or isonicotinic acid hydrazide on chromosome damage induced by mitomycin C in human lymphocytes

The frequency of interchromatic exchanges induced by mitomycin C in cultured human lymphocytes was markedly lowered in the presence of caffeine or isonicotinic acid hydrazide (INH) during a post-treatment period. The autoradiographic experiment showed that the decrease in the exchange frequency did not result from delaying or cell-killing effects by the post-treatment with caffeine or INH. Therefore, it may deduced that the exchange formation closely related to a process sensitive to caffeine or INH.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

Inhibition of post-replication repair of alkylated DNA by caffeine in Chinese hamster cells but not HeLa cells

Caffeine has been found to potentiate the lethal effects of sulphur mustard (SM) and N-methyl-N-nitrosourea (MNU) in a line of Chinese hamster cells but not in a line of HeLa cells. The sensitization of SM-treated cells by caffeine was S phase specific, and persisted for up to 24 h after alkylation of asynchronous cell cultures. The sensitization of MNU-treated cells, however, was not S phase specific but persisted for up to 50 h after the initial alkylation. Possible explanations for this difference between these two types of alkylating agent were discussed. Previously, evidence was presented which suggested that the alkylation-induced delay in the time of the peak rate of DNA synthesis in Chinese hamster cells was associated with the operation of post-DNA replication repair mechanism in these cells. Caffeine has now been found to reverse this alkylation-induced delay of DNA synthesis in both SM- and MNU-alkylated Chinese hamster cells. It is therefore proposed that caffeine sensitizes alkylated cells by inhibition of a post-replication DNA repair mechanism. No support was obtained for the alternative possibility that caffeine inhibits alkylation-induced excision repair of damaged DNA. The role of DNA repair in the production of the lethal mutagenic and cytological effects of alkylating agents is discussed.     

Binding versus biological activity of Clostridium perfringens enterotoxin in Vero cells.

Cells resistant to $\text{Clostridium perfringens}$ enterotoxin were selected from cultures of highly sensitive Vero (African green monkey kidney) cells. Studies were done with the sensitive and resistant cells to determine the relationship between binding and biological activity. Binding studies using ¹²⁵I-enterotoxin revealed the apparent existence of high and low affinity binding sites for the enterotoxin on both cell types. The binding site density on resistant cells was found to be $\text{1}\text{10}$ that of sensitive cells. It was found that, even with high doses of enterotoxin, only partial affect upon DNA synthesis, membrane permeability, and plating efficiency was noted in resistant cells. It is concluded that without specific binding there is little or no ability of the enterotoxin to effect biological activity in cells.     

Effect of hemin and isonicotinic acid hydrazide on the uptake of iron from transferrin by isolated rat liver mitochonodria

Isolated rat liver mitochondria accumulate iron from the suspending medium when [⁵⁹Fe] transferrin is used as a model compound. The accumulation proceeds by two different mechanisms, i.e. by an energy-dependent and an energy-independent mechanism. The energy-dependent uptake of iron from transferrin is inhibited by hemin and stimulated by isonicotinic acid hydrazide. The energy-independent uptake of [⁵⁹Fe] transferrin is influenced neither by hemin nor by isonicotinic acid hydrazide.     

Investigation of styrene in the liver perfusion/cell culture system. No indication of styrene-7,8-oxide as the principal mutagenic metabolite produced by the intact rat liver

Mutagenic effect of styrene and styrene-7,8-oxide was studied with the isolated perfused rat liver as metabolizing system and Chinese hamster V79 cells as genetic target cells. Styrene-7,8-oxide which is mutagenic per se was rapidly metabolized by the perfused rat liver. Thus no mutagenic effect was detected neither in the perfusion medium nor in the bile. However when styrene was added to the perfusion system, an increase in V79 mutants was observed regardless of where in the circulating perfusion medium the V79 cells were placed: the same effect was obtained with V79 cells close to the liver as well as at a distance from the liver. No mutagenic effect was observed in the bile. Simultaneous analysis of the styrene-7,8-oxide concentration in the perfusion medium, suggest that this metabolite is not the cause of the mutagenic effect observed during perfusion with styrene.The effect of the two test compounds on some liver functions was also studied. Both styrene and styrene-7,8-oxide changed the bile flow without affecting bile acid secretion: styrene caused a reduction in bile flow as compared to control perfusions and styrene-7,8-oxide increased the bile flow. Styrene, but not styrene-7,8-oxide, reduced gluconeogenesis from lactate. Styrene had no effect on the liver's capacity to incorporate amino acids into plasma proteins, whereas styrene-7,8-oxide reduced the amino acid incorporation. The microsomal cytochrome P-450 content was not affected by the two test compounds. No alteration in microsomal N- and C-oxygenation of N, N-dimethylaniline (DMA) was observed with styrene-7,8-oxide or the lower styrene dose used (240 μmol), whereas the higher styrene concentration (480 μmol) reduced N-oxygenation and thus also the total DMA metabolism.It is suggested that the results on styrene and styrene-7,8-oxide found here using the liver perfusion/cell culture system mimic the metabolism expected to be found in the intact animal, thus indicating that styrene-7,8-oxide is not the principal mutagenic metabolite of styrene in vivo.     

The influence of the wavelength of ultraviolet radiation on survival,mutation induction and DNA repair in irradiated chinese hamster cells

Chinese hamster ovary cells were used to compare the cytotoxicity and mutagenicity of far-UV radiation emitted by a low-pressure mercury, germicidal lamp (wavelength predominantly 254 nm) with that of near-UV radiation emitted by a fluorescent lamp with a continuous spectrum (Westinghouse “Sun Lamp”), of which only the radiation with wavelengths greater than 290 nm or greater than 310 nm was transmitted to the cells. The radiation effects were compared on the basis of an equal number of pyrimidine dimers, the predominant lesion induced in DNA by far-UV, for the induction of which much more energy is needed with near-UV than with 254-nm radiation.The numbers of dimers induced were determined by a biochemical method detecting UV-endonuclease-susceptible sites. The equivalence of these sites with pyrimidine dimers was established, qualitatively and quantitatively, in studies with enzymic photoreactivation in vitro and chromatographic analysis of dimers.On the basis of induced dimers, more cells were killed by >310-nm UV than by >290-nm UV; both forms of radiation were more cytotoxic than 254-nm UV when equal numbers of dimers were induced. Moreover, 5–6 times as many mutants were induced per dimer by >310-nm UV than by >290-nm UV; the latter appeared approximately as mutagenic as 254-nm UV. The differences in lethality and mutagenicity were not caused by differences in repair of dimers: cells with an equal number of dimers induced by either 254-nm or near-UV showed the same removal of sites susceptible to a UV endonuclease specific for dimers, as well as an identical amount of repair replication.The results indicate that near-UV induces, besides pyrimidine dimers, other lesions that appear to be of high biological significance.     

Inhibition of protein and DNA synthesis in tissue culture cells by a derivative of methyl glyoxal and ascorbate

The inhibitory effect of a methyl glyoxal-ascorbate (MGA) adduct (NFCR 278021) on protein and DNA synthesis in monolayer cultures of GPK epithelial cells has been compared with the inhibitory action of methyl glyoxal (MG). GPK cells exhibited an ID₅₀ of 0.98 μM MG for both protein and DNA synthesis compared with an ID₅₀ of 0.92 mM for the adduct. Hill plots demonstrate that the characteristics of the receptor saturation are the same for MG and MGA, suggesting that the action of the two agents is mediated through the MG moiety which is modified by the presence of the ascorbate portion of the molecule in MGA. It is shown that MGA undergoes spontaneous oxidation in solution and is a substrate for ascorbate oxidase, but that no additional MG activity is released by total enzymic oxidation of MGA, and oxidised MGA possesses the same inhibitory characteristics as MGA. Inhibition of protein synthesis by ascorbate or dehydroascorbate were not demonstrated in the dose range employed for MGA. The inhibitory effect of the adduct on protein synthesis was found to be diminished in the presence of glutathione and glyoxalase I (Glo I) and II (Glo II).     

Repair of damage by ultraviolet radiation in xeroderma pigmentosum cell strains of complementation groups E and F

The xeroderma pigmentosum fibroblast strains XP2RO, complementation group E, and XP23OS, group F, were compared with normal human primary fibroblasts with regard to repair of damage induced by 254-nm UV. In XP2RO cells, repair DNA synthesis, measured by autoradiography (unscheduled DNA synthesis = UDS), was about 50% of the value found in normal human cells. In these cells also the removal of UV-induced sites recognized by a specific UV-endonuclease proceeds at a reduced rate. By having BUdR incorporated into the repaired regions, followed by the induction of breaks in these patches by 313-nm UV, it was shown that the reduced repair synthesis is not caused by a shorter length of the repair regions in XP2RO, but is solely due to a reduction in the number of sites removed by excision repair. In XP23OS a discrepancy was observed between the level of UDS, which was about 10% of the normal value, and other repair-dependent properties such as UV survival, host-cell reactivation and removal of UV-endonuclease-susceptible sites, which were less reduced than could be expected from the UDS level. However, when UDS was followed over a longer period than the 2 or 3 h normally used in UDS analysis, it appeared that in XP23OS cells, the rate of UDS remained constant whereas the rate decreased in normal control cells. Consequently, the residual level of UDS varies with the period over which it is studied.     

The scintillometric evaluation of DNA repair synthesis can be distorted by changes of thymidine pool radioactivity

The induction of DNA repair synthesis by UV radiation and methylmethane sulphonate (MMS) in mammalian cell lines of human (EUE, HeLa, FT, KB) and hamster (CHO, BHK) origin has been evaluated by means of autoradiography and the scintillometric procedure which implied the use of hydroxyurea (HU) to suppress DNA replication.While with UV radiation both methods produce concordant positive results, in the case of MMS the evidence of DNA repair synthesis obtained from the autoradiograms is occasionally accompanied by a lack of increase of DNA radioactivity in the treated cultures, as detected by scintillation counting. In such instances MMS is shown to reverse the enhancement of pool radioactivity in the cultures incubated with HU and even to reduce the radioactivity of thymidine pool below control values. By normalizing DNA radioactivities on the basis of pool variations, the discrepancy between autoradiography and scintillation counting is solved.The chromatographic analysis of thymidine pool components justifies the normalization procedure as it demonstrates that also in cultures treated with MMS or MMS + HU pool variations closely parallel the variations of thymidine triphosphate (dTTP) level.The normalization of DNA radioactivities based on the overall pool radioactivities gives an improved evaluation of the actual rate of DNA synthesis. It can be recommended for screening studies of DNA repair inducers because it allows one to correct false negative results without producing false positive data. Compared with the dTTP levels, overall pool radioactivities used as normalizing factors still produce an underestimate of DNA repair when high doses of MMS are applied to hamster cell cultures.     

Evidence against involvement of aquaporin-4 in cell-cell adhesion

Aquaporin-4 (AQP4) water channels are expressed strongly in glial cells, where they play a role in brain water balance, neuroexcitation, and glial cell migration. Here, we investigated a proposed new role of AQP4 in facilitating cell-cell adhesion. Measurements were made in differentiated primary glial cell cultures from wild-type versus AQP4 knockout mice as well as in null versus AQP4-transfected L-cells, a cell type lacking endogenous adhesion molecules, and in null versus AQP4-transfected Chinese hamster ovary (CHO)-K1 cells and Fisher rat thyroid cells. Using established assays of cell-cell adhesion, we found no significant effect of AQP4 expression on adhesion in each of the cell types. As a positive control, transfection with E-cadherin greatly increased cell-cell adhesion. High-level AQP4 expression also did not affect aggregation of plasma membrane vesicles in a sensitive quasi-elastic light-scattering assay. Further, we found no specific AQP4 binding of a fluorescently labeled oligopeptide containing the putative adhesion sequence in the second extracellular loop of AQP4. These data provide evidence against involvement of AQP4 in cell-cell adhesion.     

Evidence of DNA repair in the guinea pig pancreas, in vivo and in vitro, following exposure to N-methyl-N-nitrosourethane

The nature of DNA damage induced by N-methyl-N-nitrosourethane (NMUT) in the guinea pig pancreas, both in vitro and in vivo, and subsequent repair was investigated by alkaline sucrose density gradient analysis, using a non-radioactive fluorimetric procedure for DNA determination in gradient fractions. In vitro exposure of pancreatic slices to 20 mM NMUT for 30 min damaged DNA to less than 2.24 . 10(6) dalton fragments. However, incubation of NMUT-treated slices for 3 h in a fresh medium resulted in the repair of most of DNA damage, as indicated by the conversion of low molecular weight DNA fragments into heavy DNA of molecular weight comparable to DNA from control slices. Additionally, a single administration of NMUT (30 mg/kg, i.p.) to guinea pigs induced extensive DNA damage, to less than 2.24 . 10(6) dalton fragments in the pancreas within 4 h; similar DNA damage was observed in the liver. However, in the pancreas and liver of guinea pigs sacrificed at increasing intervals after NMUT administration, there was a gradual conversion of shortened DNA fragments to heavy high molecular weight DNA, indicating repair of DNA damage. It appears that most of DNA damage in the pancreas and liver was repaired by 14 and 7 days, respectively, following NMUT administration.     

The recovery of mammalian cells treated with methyl methanesolfonate, nitrogen mustard or UV light. II. The importance of DNA repair prior to the initiation of S phase.

CHO cells were synchronized 2 G1 phase and treated with UV light or HN2. These treatments resulted in a dose-dependent reduction in the rate of DNA replication and cell survival. Holding UV-irradiated cells in G1 phase (in HU medium) for an additional 10 h prior to their release into S phase did not assist recovery as measured by either of these criteria. The survival of cells treated with HN2 was also not enhanced by this recovery period. However, following 2 X 10(-5) M HN2 the rate of DNA replication increased from 30% to 70% of the control level when the period in HU medium was extended to 14 h. The induction of cross-links following HN2 treatment of asynchronous cells was shown to be dose dependent. Subsequent incubation in fresh medium resulted in complete recovery within 20 h at concentrations of HN2 up to 10(-5) M, and at 2 X 10(-5) M HN2, 75% of the cross-links were removed at 14 h post treatment.     

A study of triflupromazine in dominant-lethal, cytogenetic and host-mediated assays

The phenothiazine psychotherapeutant, triflupromazine (TFP), was studied for mutagenic potential in dominant-lethal, in vivo and in vitro cytogenetic and host-mediated assay procedures. No evidence of gross chromosomal aberrations or point mutations was detected in these assays even at dosage regimens which produced substantial lethality. The effect of the drug on body temperature was measured at the same doses used for mutagenicity testing. A marked and sustained temperature reduction occurs shortly after administration of as little as 10 mg/kg. Due to the pronounced physiological effects at these levels, the validity of mutagenicity studies conducted at the same levels may be seriously questioned.     

The relationship between DNA and chromosome damage after bleomycin treatment: dose-response measurements

The molecular basis for chromosome aberration formation has been studied using the sensitive techniques of premature chromosome condensation and DNA alkaline elution. The dose response of Chinese hamster ovary cells to bleomycin treatment at the DNA and chromosome levels was compared. Each DNA elution curve showed a 2-component profile, with a more sensitive component apparent at low doses. The chromosome aberration curves also exhibited a 2-component profile when determined in G2-PCC; however, this phenomenon was less apparent when chromosome damage was enumerated in mitotic figures. These results suggest that differential sensitivity to bleomycin exists within the cellular chromatin. The effect of dose rate on aberration formation was examined by administering bleomycin at 2 concentrations that, with different treatment times, yielded equivalent amounts of DNA damage. The chromatid exchange rate was independent of dose rate, suggesting that rapidly repaired DNA lesions are not involved in the formation of exchanges.     

Effect of externally added carnitine on the synthesis of acetylcholine in rat cerebral cortex cells

Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 μM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of β-oxidation might be correlated with the lower level of acetylcholine synthesis.     

Characterization of the Interaction between Diferric Transferrin and Transferrin Receptor 2 by Functional Assays and Atomic Force Microscopy

Transferrin receptor 2 (TfR2), a homologue of the classical transferrin receptor 1 (TfR1), is found in two isoforms, α and β. Like TfR1, TfR2α is a type II membrane protein, but the β form lacks transmembrane portions and therefore is likely to be an intracellular protein. To investigate the functional properties of TfR2α, we expressed the protein with FLAG tagging in transferrin-receptor-deficient Chinese hamster ovary cells. The association constant for the binding of diferric transferrin (Tf) to TfR2α is 5.6 × 10⁶ M^− 1, which is about 50 times lower than that for the binding of Tf to TfR1, with correspondingly reduced rates of iron uptake. Evidence for Tf internalization and recycling via TfR2α without degradation, as in the TfR1 pathway, was also found. The interaction of TfR2α with Tf was further investigated using atomic force microscopy, a powerful tool used for investigating the interaction between a ligand and its receptor at the single-molecule level on the living cell surface. Dynamic force microscopy reveals a difference in the interactions of Tf with TfR2α and TfR1, with Tf-TfR1 unbinding characterized by two energy barriers, while only one is present for Tf-TfR2. We speculate that this difference may reflect Tf binding to TfR2α by a single lobe, whereas two lobes of Tf participate in binding to TfR1. The difference in the binding properties of Tf to TfR1 and TfR2α may help account for the different physiological roles of the two receptors.     

Algal oxidation of aromatic hydrocarbons: formation of 1-naphthol from naphthalene by Agmenellum quadruplicatum, strain PR-6.

Hydroxyurea induces repair replication in human lymphoblastoid NC37 BaEV cells during incubation with liver microsomes and NADPH. Catalase reduces hydroxyurea-induced repair by more than 95%, suggesting that hydrogen peroxide derived from hydroxyurea in the presence of the metabolic activation system is involved in the DNA damage.     

Direct analysis of radiation-induced chromosome fragments and rings in unstimulated human peripheral blood lymphocytes by means of the premature chromosome condensation technique

Development of the procedure to stimulate peripheral blood lymphocytes has greatly facilitated the understanding of chromosome aberration formation and repair mechanisms in human cells. Yet, because radiation induces far more initial chromosome breaks than are observed as aberrations in metaphase, it has not been possible to examine the kinetics of primary chromosome breakage and rejoining with this procedure. An improved method to induce premature chromosome condensation in unstimulated lymphocytes has been used to study primary chromosome breakage, rejoining, and ring formation at various times after irradiation with up to 800 rad of X-rays. The dose-response relations for chromosome fragments analyzed immediately or 1, 2, or 24 h after exposure were found to be linear. Rapid rejoining of chromosome fragments, which takes place in the first 3 h after X-ray exposure, was not correlated with a simultaneous increase in the formation of rings. The yield of rings per cell scored 24 h after irradiation, however, increased significantly and fit a linear quadratic equation. Both chromosome fragment rejoining and ring formation were completed about 6 h after irradiation. The frequency distributions of rings among cells followed a Poisson distribution, whereas chromosome fragments were overdispersed.