The effect of oxidative stress induced by t-butyl hydroperoxide on the structural dynamics of membrane proteins of Chinese hamster fibroblasts

A method based on the measurement at room temperature of tryptophan phosphorescence (RTTP) gives the unique possibility to investigate the dynamic structure of membrane proteins without their isolation from cells. This method was used to study the influence of tert- butyl hydroperoxide (t -BHP) on Chinese hamster fibroblasts. The treatment of fibroblasts with t -BHP in a concentration range of 0. 5-2 m m for 60 min caused an increase of frequency and amplitude of membrane protein motions with lifetimes of hundreds miliseconds (a decrease of RTTP tau(2)). In parallel, cell viability was studied by trypan blue exclusion test and the content of thiobarbituric acid reactive substances was measured in cells. The dependences of the RTTP tau(2)and cell viability on t -BHP concentration were similar. Contrary to this, t -BHP did not induce the activation of lipid peroxidation processes in cells. This indicates that cell death is connected with the excessive increase of intramolecular dynamics of membrane proteins during t -BHP action.     

Oxidative DNA damage induced by a hydroperoxide derivative of cyclophosphamide

Interstrand DNA cross-linking has been considered to be the primary action mechanism of cyclophosphamide (CP) and its hydroperoxide derivative, 4-hydroperoxycyclophosphamide (4-HC). To clarify the mechanism of anti-tumor effects by 4-HC, we investigated DNA damage in a human leukemia cell line, HL-60, and its H(2)O(2)-resistant clone HP100. Apoptosis DNA ladder formation was detected in HL-60 cells treated with 4-HC, whereas it was not observed in HP100 cells. 4-HC significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, a marker of oxidative DNA damage, in HL-60 cells. On the other hand, CP did not significantly induce 8-oxodG formation and apoptosis in HL-60 cells under the same conditions as did 4-HC. Using (32)P-labeled DNA fragments from the human p53 tumor suppressor gene, 4-HC was found to cause Cu(II)-mediated oxidative DNA damage, but CP did not. Catalase inhibited 4-HC-induced DNA damage, including 8-oxodG formation, suggesting the involvement of H(2)O(2). The generation of H(2)O(2) during 4-HC degradation was ascertained by procedures using scopoletin and potassium iodide. We conclude that, in addition to DNA cross-linking, oxidative DNA damage through H(2)O(2) generation may participate in the anti-tumor effects of 4-HC.     

Toxicity and DNA damage induced by 1-nitropyrene and its derivatives in Chinese hamster lung fibroblasts

1-Nitropyrene and its chemically synthesised derivatives were investigated for their cytotoxicity and ability to induce DNA-strand breaks in Chinese hamster lung fibroblasts. Both 1-nitrosopyrene (0.25-60 micrograms/ml) and 1-aminopyrene (0.25-25 micrograms/ml) were cytotoxic, and induced the formation of DNA lesions, which were measured as DNA single-strand breaks after sedimentation in alkaline sucrose-density gradients. Higher doses of 1-aminopyrene (25-60 micrograms/ml) inhibited the formation of DNA single-strand breaks. 1-Nitropyrene was not toxic (0.25-60 micrograms/ml) and induced low levels of detectable DNA strand breaks, whilst N-acetyl-1-aminopyrene was inactive. The post-mitochondrial supernatant fraction of Aroclor-induced rat-liver containing 4 mM NADPH (S9 mix) did not promote the activation of 1-nitropyrene. In fact DNA strand breaks induced by either 1-nitropyrene or 1-nitrosopyrene was abolished in the presence of S9 mix. The 1-nitropyrene reduced intermediate, N-hydroxy-1-aminopyrene was synthesised by the reduction of 1-nitrosopyrene with ascorbic acid. In the presence of ascorbic acid, 1-nitrosopyrene caused a 5-fold increase in the number of DNA single-strand breaks when compared to cells treated with 1-nitrosopyrene alone. The results are discussed in terms of the metabolic activation of 1-nitropyrene and 1-aminopyrene in Chinese hamster lung cells.     

Visible chemiluminescence induced by t-butyl hydroperoxide in red blood cell suspensions

Red blood cells from Wistar rats were exposed to milimolar concentrations of t-butyl hydroperoxide. Extensive hemoglobin oxidation (methemoglobin formation), t-butyl hydroperoxide cleavage (t-butanol formation) and peroxidation (measured by oxygen consumption and thiobarbituric acid reactive substances) was observed. Significant chemiluminescence was emitted by the system. Hemoglobin oxidation and t-butanol production were independent of oxygen pressure and free radical scavengers, however, luminescence was enhanced as oxygen pressure increased and it was reduced by addition of free radical scavengers. The spectral distribution of the light emitted suggests that the luminescence detected is not due to singlet oxygen dimol emission. The results are in agreement with a lipid peroxidative mechanism initiated by t-butoxy radicals produced in the interaction of hemoglobin and t-butyl hydroperoxide.     

Polycentric chromosomes induced by X-rays in giant cells of Chinese hamster in vitro

The induction of polycentric chromosomes by X-rays supports a previous interpretation that at least some giant cells are polyploid cells that result from the repetition of endomitosis.Work supported by U.S. Atomic Energy Commission.     

DNA damage in Chinese hamster cells repeatedly exposed to low doses of X-rays

In a recent paper we reported the results of an experiment carried out by analysing chromosomal damage in Chinese hamster (CHO) cells exposed to low doses of X-rays. The present investigation was undertaken in order to validate those results using a different approach, the single cell gel electrophoresis assay (comet assay) immediately after irradiation. Cells were cultured during 14 cycles, irradiation treatment was performed once per cycle when the cells were at 90-95% of confluence. Doses of 2.5, 5.0 and 10.0 mSv were used. Sequential irradiation of CHO cells induced a decrease of cells without migration and an increase of cells showing DNA damage with the three doses employed. Significant increases of low-level damaged cells (p < 0.001) were found for the 14 exposures when compared to controls except for the first irradiations with 2.5 and 10 mSv, respectively. No significant increase of the frequency of cells with severe damage was observed in any case. These findings could be explained by assuming a complex interactive process of cell recovery, DNA damage and repair together with the induction of genomic instability, the incidence of bystander effects as well as some kind of radioadaptative response of the cells. If these phenomena are limited to the cell line employed deserves further investigation.     

Haematoporphyrin derivative photosensitization and gamma-radiation damage interaction in Chinese hamster ovary fibroblasts

The interaction of haematoporphyrin derivative (HPD) photosensitization and gamma-irradiation was studied with regard to clonogenicity of Chinese hamster ovary (CHO) fibroblasts. Exposure to either treatment alone resulted in shouldered response curves. Exposure to 4.2 Gy gamma-radiation immediately before graded doses of visible light had no effect on the shape of the visible-light survival curve; similarly, exposure to 8.75 kJ/m2 light immediately before graded doses of gamma-radiation had no effect on the shape of the gamma-radiation response curve. These data indicate that damage due to gamma-radiation and HPD photosensitization did not interact, suggesting that the mechanisms of cell killing are different.     

The role of phospholipase A2 in microsomal lipid peroxidation induced with t-butyl hydroperoxide

The role of phospholipase A2 (PlA2) in lipid peroxidation induced with t-butyl hydroperoxide was examined in rat liver microsomes. Exposure of microsomes to t-butyl hydroperoxide was associated with activation of endogenous PlA2. When PlA2 was inhibited with chlorpromazine, mepacrine, or p-bromphenacyl bromide, the accumulation of thiobarbituric acid reactive substances (TBARS) was reduced in a dose dependent manner. In contrast, the accumulation of conjugated dienes was not affected by chlorpromazine, and was slightly increased by mepacrine. When endogenous PlA2 was activated with mellitin prior to induction of peroxidation, accumulation of both TBARS and dienes was reduced. Analogously, pretreatment with exogenous PlA2 reduced both dienes and TBARS. In contrast, addition of mellitin following the induction of peroxidation did not alter either TBARS or dienes.     

Irradiation damage in chromatin isolated from V-79 Chinese hamster lung fibroblasts

The effect of chromatin structure on the extent of radiation damage induced by low doses of 100 KeV X rays was investigated using a fluorescent assay for DNA unwinding. Chromatin was isolated from V-79 Chinese hamster lung fibroblast nuclei by partial digestion with micrococcal nuclease. Gel electrophoresis of the isolated DNA showed the molecular weight of the chromatin preparation to be 10.6 X 10(6) with a size range of 6.6-21.7 X 10(6) Da while a size of 10.2 +/- 0.9 X 10(6) Da was found by sedimenting the DNA in alkaline sucrose gradients. The repeat length of V-79 chromatin was found to be 194 +/- 3 bp. The typical nucleosomal repeat structure of the isolated chromatin and that of intact nuclei was identical. Irradiation with 50 and 100 Gy of 100 KeV X rays and analysis by alkaline sucrose density centrifugation indicated that V-79 chromatin sustained 0.56 +/- 0.19 and 0.69 +/- 0.09 single-strand breaks per 10 Gy per 10(8) Da of DNA, respectively. Irradiation with doses of 0.5-3.0 Gy of 100 KeV X rays and analysis by the fluorometric assay showed that the radiation sensitivity of V-79 chromatin decreases sharply on compaction with MgCl2. Histone H1 depletion, which inhibits compaction and causes chromatin to expand by increasing the linker from 26 to 48 bp, results in a considerable increase in the radiation sensitivity. It is concluded that radiation damage sustained by DNA is greatly influenced by chromatin structure.     

Mutations induced by X-rays at the HPRT locus in cultured Chinese hamster cells are mostly large deletions

We investigated the molecular basis of 19 X-ray-induced HPRT-deficient mutants of V79 Chinese hamster cells with Southern hybridisation techniques. 12 of those mutants suffer from a big deletion (greater than 10 kb) of HPRT DNA sequences. Cytological studies of chromosome preparations of those 12 deletion mutants showed that in at least 3 of these mutants part of the long arm of the X-chromosome was lost. After correction for spontaneous arising mutations we estimate that at least 70-80% of X-ray-induced mutations are caused by large deletions.     

Metal-induced DNA damage and repair in human diploid fibroblasts and Chinese hamster ovary cells

Cloning efficiency and DNA strand breaks induction were compared in human diploid fibroblasts (HSBP) and chinese hamster ovary (CHO) cells treated with various metal salts. Cadmium (Cd2+), nickel (Ni2+) and chromate (Cr2O7) reduced the cloning efficiency of HSBP cells more than that of CHO cells whereas the reverse was true after treatment with mercury (Hg2+), manganese (Mn2+) and cobalt (Co2+). The effects on cloning efficiency did not consistently correlate with DNA strand breaking activity as all metals except Cr(VI) were more effective at producing DNA strand breaks in CHO cells than in human cells. The differential responses of the two cell types was shown to be only partially due to differences in cellular uptake of metals. DNA breaks induced in human cells by Hg2+ and Cr2O7 were shown most likely to be alkaline labile sites rather than true strand breaks since no damage was detected in a nick translation assay which measures the amount of free 3'-OH terminals. Damage induced by Mn2+ and Co2+, however, appeared to be comprised at least in part by true DNA strand breaks. DNA damage was also induced in HSBP cells following treatment with selenium but only in the presence of reduced glutathione. These studies indicate that DNA damage is not as major a consequence following some metal treatments in human cells as it appears to be in rodent cells. This suggests that rodent models for risk estimation of metal-induced tumorigenesis may not always be appropriate for extrapolation to humans.     

Changes in oxygen consumption induced by t-butyl hydroperoxide in perfused rat liver. Effect of free-radical scavengers.

The addition of t-butyl hydroperoxide to perfused rat liver elicited a biphasic effect on hepatic respiration. A rapid fall in liver oxygen consumption was initially observed, followed by a recovery phase leading to respiratory rates higher than the initial steady-state values of oxygen uptake. This overshoot in hepatic oxygen uptake was abolished by free-radical scavengers such as (+)-cyanidanol-3 or butylated hydroxyanisole at concentrations that did not alter mitochondrial respiration. (+)-Cyanidanol-3 was also able to facilitate the recovery of respiration, the diminution in the calculated rate of hydroperoxide utilization and the decrease in liver GSH content produced by two consecutive pulses of t-butyl hydroperoxide. It is suggested that the t-butyl hydroperoxide-induced overshoot in liver respiration is related to increased utilization of oxygen for lipid peroxidation as a consequence of free radicals produced in the scission of the hydroperoxide by cellular haemoproteins.     

Suppressing effect of antimutagenic flavorings on chromosome aberrations induced by UV-light or X-rays in cultured Chinese hamster cells

Chromosome aberrations induced by UV-light or X-rays were suppressed by the post-treatment with antimutagenic flavorings, such as anisaldehyde, cinnamaldehyde, coumarin, and vanillin. UV- or X-ray-irradiated surviving cells increased in the presence of each flavoring. X-ray-induced breakage-type and exchange-type chromosome aberrations were suppressed by the vanillin treatment in the G1 phase of the cell cycle and a greater decrease in the number of X-ray-induced chromosome aberrations during G1 holding was observed in the presence of vanillin. Furthermore, a greater decrease in the number of X-ray-induced DNA single-strand breaks was observed in the presence of vanillin. Treatment with vanillin in the G2 phase suppressed UV- and X-ray-induced breakage-type but not exchange-type chromosome aberrations. The suppression of breakage-type aberrations was assumed to be due to a modification of the capability of the post-replicational repair of DNA double-strand breaks. These G1- and G2-dependent anticlastogenic effects were not observed in the presence of 2',3'-dideoxythymidine, an inhibitor of DNA polymerase beta. Based on these results, the anticlastogenic effect of vanillin was considered to be due to the promotion of the DNA rejoining process in which DNA polymerase beta acts.     

Protective effect of ellagic acid on t-butyl hydroperoxide induced lipid peroxidation in isolated rat hepatocytes

Ellagic acid, a plant polyphenol, showed protective effect on isolated rat hepatocytes against destruction due to lipid peroxide formation induced by t-butyl hydroperoxide in vitro. Ellagic acid inhibited the generation of superoxide anions and hydroxyl radicals both in enzymic and non enzymic systems, thus providing protection against oxidative damage.     

Zinc suppression of free radicals induced in cultures of rat hepatocytes by iron, t-butyl hydroperoxide, and 3-methylindole

The effect of zinc on lipid peroxidation initiated by either ferric-nitrilotriacetate, t-butyl hydroperoxide, or 3-methylindole was studied using primary monolayer cultures of rat liver parenchymal cells. The malondialdehyde content of the cells and culture medium was used to estimate the extent of lipid peroxidation. As the zinc concentration of the culture medium was increased from 1 to 48 microM, peroxidation was diminished. Cellular zinc and metallothionein levels were proportionally increased by supplemental zinc. Zinc supplementation of the medium inhibited NADPH-cytochrome c reductase activity and stimulated glutathione peroxidase activity. The uptake of iron into the hepatocytes was significantly reduced as the level of zinc was raised, suggesting that zinc antagonizes uptake of chelated iron into isolated hepatocytes and in this way blocks iron-induced peroxidation. Furthermore, induction of metallothionein synthesis by zinc may contribute to the reduction in free radicals. Spectra from electron spin resonance studies, using phenylbutylnitrone as a spin-trapping reagent, demonstrated that free radical production was inversely related to the zinc concentration of the culture medium. Spin trap data suggest that metallothionein added to lysed cells in vitro decreases free radical production. Studies using the spin trap, 3,3,5,5-tetramethylpyrroline-N-oxide indicated that cumulatively the predominant radical present in the cultures was a phenyl radical with hydroperoxide or methylindole. Collectively, our data demonstrate that zinc inhibits free radical production and lipid peroxidation in cultured hepatocytes. The mode of action of zinc could occur via free radical scavenging by zinc-induced metallothionein and/or by processes related to cytochrome P-450 and glutathione peroxidase, since these were also found to be sensitive to zinc supplementation levels of the culture medium.     

A mechanism of mitochondrial damage induced by tert-butyl hydroperoxide and microsomes in vitro

Isolated potato ( Solanum tuberosum L. cv. Dansyaku) tuber mitochondria showed a significant loss in respiratory activity when treated with tert -butyl hydroperoxide (BHP), especially in the presence of microsomes. The following alterations appeared in parallel with the gradual decrease in the respiratory activity: The outer membrane became leaky, probably due to peroxidation of phospholipids. The level of sulfhydryl (SH) groups in mitochondrial proteins decreased in contrast to non-protein SH groups. A considerable amount of phospholipids was degraded and lost. A mechanism of the mitochondrial damage induced by BHP and microsomes is discussed with respect to a significant role of free radicals which may be formed at the onset of senescence or physiological disorders.     

Hypoxanthine transport by cultured Chinese hamster lung fibroblasts.

The uptake of hypoxanthine by Chinese hamster lung fibroblasts grown in tissue culture was studied in wild type clones and 8-azaguanine-resistant mutant clones devoid of hypoxanthine-guanine phosphoribosyltransferase. Wild type fibroblasts rapidly accumulate [3H]hypoxanthine from the medium and over 80% of the intracellular radioactivity is found in acid-soluble nucleotides. The phosphoribosyltransferase-deficient clones accumulate much lower levels of hypoxanthine and over 85% of the intracellular 3H label is associated with chemically unaltered hypoxanthine. The internal level of hypoxanthine in the mutant clones rapidly approaches but does not exceed that present in the medium. Wild type and phosphoribosyltransferase-deficient cells take up hypoxanthine at almost identical initial rates at external hypoxanthine levels from 2 to 300 muM. Analysis of these data reveals two transport systems that obey the Michaelis-Menten relationship. These differ markedly in affinity, yielding average Km values of 20 and 600 muM for both cell types. Hypoxanthine transport by both low and high affinity transport systems is blocked by p-chloromercuriphenylsulfonate and N-ethylmaleimide. Counter-transport of hypoxanthine was demonstrated in phosphoribosyltransferase-deficient fibroblasts. It is concluded that hypoxanthine is transported into Chinese hamster cells by means of carrier-mediated processes (facilitated diffusion) that operate independently of phosphoribosylation.     

Iron uptake by Chinese hamster fibroblasts from human transferrin

The manner of uptake or iron by Chinese hamster fibroblasts, type DON, from human transferrin was investigated by means of replacement studies, in which the cells that were incubated with ¹²⁵I-labelled human transferrin were chased with non-radioactive transferrin for only a few minutes. The results did not support the reversible endocytosis hypothesis for the uptake of iron from transferrin. The uptake of iron measured as ⁵⁹Fe during several cell divisions was found to be a function of time and cell number. It was found that the total uptake of iron in the harvests was directly proportional to the incubation, and that the uptake per 10⁶ cells levelled off in the course of time.     

Endocytosis in Chinese hamster fibroblasts : Inhibition by glucose

Endocytosis in Chinese hamster ovary fibroblasts was investigated by measuring the rate of uptake of ³H-sucrose, which is known to enter cells only by endocytosis. Serum, polyvinylpyrrolidone (PVP), adenosine triphosphate, insulin, and cyclic 3′,5′-adenosine monophosphate, all of which are known to increase the rate of endocytosis by other cell systems, had no effect on Chinese hamster fibroblasts. However, medium in which these cells had been maintained for several days, referred to as conditioned medium, had a profound effect on endocytosis. These cells endocytosed 3 to 5 times as rapidly in conditioned medium as in fresh medium. A logarithmic inhibition of this effect was observed with increasing -glucose concentrations, however, glucose-free medium did not produce as great an effect as conditioned medium. This suggests that these cells may endocytose in response to their nutritional requirements.