Diminished amyloid-ß uptake by mouse microglia upon treatment with quantum dots, silver or cerium oxide nanoparticles: Nanoparticles and amyloid-ß uptake by microglia.

Alzheimer's disease (AD) is a chronic neurodegenerative disease leading to progressive dementia in elderly people. The disease is characterized, among others, by formation of amyloid-ß (Aß) polypeptide plaques in the brain. Although etiology of the disease is not fully understood, recent research suggest that nanomaterials may affect AD development. Here, we described the consequences of exposure of mouse BV-2 microglia to silver nanoparticles (AgNPs, 50 µg/mL), cerium oxide nanoparticles (CeO2NPs, 100 µg/mL), and cadmium telluride quantum dots (CdTeQDs, 3 or 10 µg/mL) in the context of its ability to clear Aß plaques. The brain microglial cells play an important role in removing Aß plaques from the brain. Cell viability and cycle progression were assessed by trypan blue test and propidium iodide binding, respectively. The uptake of Aß and NPs was measured by flow cytometry. Secretion of proinflammatory cytokines was measured with the use of cytometric bead array. Aß (0.1 µM) did not affect viability, whereas NPs decreased microglia growth by arresting the cells in G1 phase (CdTeQDs) or in S phase (AgNPs and CeO2NPs) of cell cycle. The uptake of Aß was significantly reduced in the presence of AgNPs and CeO2NPs. In addition, the least toxic CeO2NPs induced the release of proinflammatory cytokine, tumor necrosis factor α. In summary, each of the NPs tested affected either the microglia phagocytic activity (AgNPs and CeO2NPs) and/or its viability (AgNPs and CdTeQDs) that may favor the occurrence of AD and accelerate its development.

Dihydropyridines decrease X-ray-induced DNA base damage in mammalian cells.

Compounds with the structural motif of 1,4-dihydropyridine display a broad spectrum of biological activities, often defined as bioprotective. Among them are L-type calcium channel blockers, however, also derivatives which do not block calcium channels exert various effects at the cellular and organismal levels. We examined the effect of sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate (denoted here as DHP and previously also as AV-153) on X-ray-induced DNA damage and mutation frequency at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in Chinese hamster ovary CHO-K1 cells. Using formamido-pyrimidine glycosylase (FPG) comet assay, we found that 1-h DHP (10nM) treatment before X-irradiation considerably reduced the initial level of FPG-recognized DNA base damage, which was consistent with decreased 8-oxo-7,8-dihydro-2'-deoxyguanosine content and mutation frequency lowered by about 40%. No effect on single strand break rejoining or on cell survival was observed. Similar base damage-protective effect was observed for two calcium channel blockers: nifedipine (structurally similar to DHP) or verapamil (structurally unrelated). So far, the specificity of the DHP-caused reduction in DNA damage - practically limited to base damage - has no satisfactory explanation.

Differential DNA double strand break fixation dependence on poly(ADP-ribosylation) in L5178Y and CHO cells.

PURPOSE: To investigate the role of poly(ADP-ribosylation) in DNA double-strand break repair and fixation in murine lymphoma L5178Y (LY) sublines, LY-R and LY-S, and a pair of Chinese hamster ovary lines: wild-type and mutant xrs6 cells, that have differences in repair competence and degree of radiosensitization with poly(ADP-ribosylation) inhibitors. MATERIALS AND METHODS: Cells (asynchronous, logarithmic phase) were pre-incubated with 2 mM aminobenzamide at 37 or 25 degrees C, X-irradiated with 10 Gy and allowed to repair DNA breaks for 15, 60 and 120 min at 37 or 25 degrees C. The remaining double-strand break were estimated by the neutral comet assay. RESULTS: At 37 degrees C, no effect of AB treatment on the repair kinetics was observed either in xrs6 or Chinese hamster ovary (wild-type) cells. In contrast, aminobenzamide decreased the repair of double-strand break in the LY-S line but not the LY-R line, in agreement with the previously observed radiosensitization of LY cells by poly(ADP-ribosylation) inhibition. However, double-strand break rejoining in the repair competent cell lines, Chinese hamster ovary and LY-R, also was affected by aminobenzamide when the post-irradiation incubation was carried out at 25 degrees C. Analysis of these results together with earlier data on LY-S cells have been interpreted in terms of Radford's model of radiation damage fixation. CONCLUSION: The reported results indicate that poly(ADP-ribosylation) can be an important modulator of the conversion of DNA damage to lethal events.

Differential induction of apoptosis in x-irradiated L5178Y sublines bearing p53 mutation.

We examined apoptosis and expression of p53, E2F-1, bax, bclx(L) and bc12 proteins in two L5178Y (LY) murine lymphoma sublines, LY-R and LY-S, which differ in radiosensitivity and double-strand break (DSB) repair. Both sublines are heterozygous for a p53 mutation in codon 170 that precludes the transactivation function. Accordingly, there is no G1/S arrest after irradiation. We found that there is no change in expression of E2F-1, bax, bclx(L) or bc12 proteins in both LY sublines after x-irradiation. LY-R cells do not constitutively express bc12, whereas both sublines show high bax content. Radiation induces delayed apoptosis to a greater extent in LY-S than in LY-R cells. The apoptosis can be seen 24 h after irradiation (2 Gy) of LY-S cells, with a maximum at 48 h. LY-R cells need 5 Gy and 72 h post-irradiation incubation to show marked apoptosis (identified by the TUNEL method). The reported observations support the assumption that differential radiosensitivity of LY sublines is associated with the induction of apoptosis that is not related to transactivation by p53 and is primarily related to differential DNA repair ability.

Intracellular iron status as a hallmark of mammalian cell susceptibility to oxidative stress: a study of L5178Y mouse lymphoma cell lines differentially sensitive to H(2)O(2).

The redox properties of iron make this metal a key participant in oxygen-mediated toxicity. Accordingly, L5178Y (LY) mouse lymphoma cell lines, which display a unique inverse cross-sensitivity to ionizing radiation (IR) and hydrogen peroxide (H(2)O(2)), are a suitable model for the study of possible differences in the constitutive control of intracellular iron availability. We report here that the level of iron in the cytosolic labile iron pool (LIP), ie, potentially active in the Fenton reaction, is more than 3-fold higher in IR-resistant, H(2)O(2)-sensitive (LY-R) cells than in IR-sensitive, H(2)O(2)-resistant (LY-S) cells. This difference is associated with markedly greater content of ferritin H-subunits (H-Ft) in LY-S than in LY-R cells. Our results show that different expression of H-Ft in LY cells is a consequence of an up-regulation of H-Ft mRNA in the LY-S mutant cell line. In contrast, posttranscriptional control of iron metabolism mediated by iron-responsive element-iron regulatory proteins (IRPs) interaction is similar in the 2 cell lines, although IRP1 protein levels in iron-rich LY-R cells are twice those in iron-deficient LY-S cells. In showing that LY cell lines exhibit 2 different patterns of intracellular iron regulation, our results highlight both the role of high LIP in the establishment of pro-oxidant status in mammalian cells and the antioxidant role of ferritin. (Blood. 2000;95:2960-2966)

The response of L5178Y lymphoma sublines to oxidative stress: antioxidant defence, iron content and nuclear translocation of the p65 subunit of NF-kappaB.

We examined the response to hydrogen peroxide of two L5178Y (LY) sublines which are inversely cross-sensitive to hydrogen peroxide and X-rays: LY-R cells are radio-resistant and hydrogen peroxide-sensitive, whereas LY-S cells are radiosensitive and hydrogen peroxide-resistant. Higher initial DNA breaks and higher iron content (potentially active in the Fenton reaction) were found in the hydrogen peroxide sensitive LY-R cells than in the hydrogen peroxide resistant LY-S cells, whereas the antioxidant defence of LY-R cells was weaker. In particular, catalase activity is twofold higher in LY-S than in LY-R cells. The content of monobromobimane-reactive thiols is 54% higher in LY-S than in LY-R cells. In contrast, the activity of glutathione peroxidase (GPx) is about two times higher in LY-R than in LY-S cells; however, upon induction with selenium the activity increases 15.6-fold in LY-R cells and 50.3-fold in LY-S cells. Altogether, the sensitivity difference is related to the iron content, the amount of the initial DNA damage, as well as to the efficiency of the antioxidant defence system. Differential nuclear translocation of p65-NF-kappaB in LY sublines is due to the more efficient antioxidant defence in LY-S than in LY-R cells.

Nuclear translocation of the p65 subunit of NF-kappaB in L5178Y sublines differing in antioxidant defense.

We examined the induction of nuclear translocation of the p65 subunit of NF-kappaB in L5178Y (LY) cells. We used two LY sublines which are inversely cross-sensitive to hydrogen peroxide and x-rays: LY-R cells are radioresistant and oxidant-sensitive, whereas LY-S cells are radiosensitive and oxidant-resistant. Hydrogen peroxide, phorbol ester and x-rays caused a marked translocation of p65-NF-kappaB in LY-R cells and a weak translocation in LY-S cells. By manipulating the antioxidant defense status, we obtained an alteration in the p65-NF-kappaB translocation induction in LY-R cells. A similar effect was achieved with lovastatin pretreatment (25 microM, 24 h, 37 degrees C). The response of LY-S cells under all these conditions was considerably weaker. We conclude that differential nuclear translocation of p65-NF-kappaB in LY sublines is not related to the lethal effect of the activating, damaging agent; rather it is due to the more efficient antioxidant defense in LY-S than in LY-R cells.

Antioxidant defense system in differentially hydrogen peroxide sensitive L5178Y sublines.

Two sublines of L5178Y (LY) murine lymphoma, differing in sensitivity to hydrogen peroxide, served as a cellular model for examination of the antioxidant defense system. The contribution of catalase, glutathione peroxidase (G-Px) and glutathione were evaluated. Sensitivity to 3-amino-1,2,4-triazole (AMT), inhibitor of catalase, was higher in LY-R (hydrogen peroxide sensitive) than in LY-S (hydrogen peroxide resistant) cells. Accordingly, activity of catalase was twofold lower in LY-R than in LY-S cells. G-Px activity was about two times higher in LY-R than in LY-S cells. After induction with selenium it increased 15.6 times in LY-R cells and 50.3 times in LY-S cells. Reduced glutathione (GSH) content (and possibly other monobromobimane-reactive thiols) were determined fluorimetrically with monobromobimane and fluorescence found 54% higher in LY-S than in LY-R cells. Inhibition of catalase caused GSH decrease in LY-S cells; this decrease was abrogated by inducing G-Px by selenium treatment. On the contrary, in LY-R cells inhibition of catalase decreased GSH content only slightly and selenium treatment did not further change the GSH level. DNA damage (estimated by "comet" assay) was the same in hydrogen peroxide-treated cells in the presence or absence of AMT; however, after induction of G-Px by selenium, DNA damage was considerably lowered. This sparing effect of selenium was accompanied by decreased growth inhibition in selenium pretreated, hydrogen peroxide-treated cell cultures.

Arachidonic acid metabolism in murine lymphoma cell sublines differing in radiation sensitivity.

14C arachidonic acid incorporation and 14C radioactivity release as well as prostaglandin (PG) and 5-hydroxyeicosatetraenoic acid (5-HETE) synthesis were measured in the pair of murine lymphoma L5178Y (LY) cell sublines differing in radiation sensitivity. Both LY sublines, LY-R (resistant) and LY-S (sensitive), incorporated exogenous arachidonic acid and released it from membrane phospholipids. Ca2+ ionophores (ionomycin and A23187) but not PMA stimulated the liberation of 14C arachidonic acid in LY cells. PMA did not potentiate the 14C arachidonic acid release both in the presence or in the absence of A23187; this observation suggests that protein kinase C activation is not essential for the regulation of arachidonic acid release by LY-R and LY-S cells. X-irradiation (5 Gy) did not change the uptake of 14C arachidonic acid into LY-R and LY-S cells and did not potentiate the release of its total radioactivity from the cells. PG synthesis was stimulated in irradiated LY-R cells but not in LY-S cells. The susceptibility of eicosanoid metabolism to A23187 and H2O2 was altered in irradiated LY-R cells. A23187 stimulated only PG and 5-HETE synthesis in irradiated LY-R cells. H2O2 did not stimulate the synthesis of PG from exogenous arachidonic acid in irradiated LY-R and LY-S cells and 5-HETE synthesis in LY-R cells. An implication of the increased PG synthesis in LY-R cells in the protection against radiation is discussed.

Calcium antagonist, TMB-8, prevents the induction of adaptive response by hydrogen peroxide or X-rays in human lymphocytes.

Treatment of human lymphocytes with hydrogen peroxide (10 microM, 30 min, 37 degrees C in PBS) or with 1 cGy X-rays evoked about a 30% decrease in the frequency of micronuclei upon subsequent X-irradiation (1.5 Gy). In addition to a lower micronuclei frequency, we also found an increase in the sedimentation distance of the nucleoids, when measured 90 min (duration of the isolation procedure carried out at 4 degrees C) after the adaptive dose (hydrogen peroxide or X-rays) and preceding the challenge dose. To test whether Ca2+ is involved in the induction of the adaptive response pathway, we treated cells with the calcium chelator, EGTA. When EGTA was given at the same time as the adaptive dose, it prevented the development of the adaptive response. In addition, the calcium antagonist, TMB-8, also prevented the development of the adaptive response as it prevented the reduction of both micronuclei and increased nucleoid sedimentation. Cellular treatment with TMB-8 increased the free [Ca2+] by 40%, when given together with hydrogen peroxide. The faster sedimenting nucleoids from adapted cells were also examined by ethidium bromide titration; there was no indication of any change in supercoil density or loop size. Psi-tectorigenin, an inhibitor of phosphatidylinositol turnover, did not modify the adaptive response, indicating that inositol (1,4,5)-trisphosphate is not involved in the induction of the adaptive response, but free Ca2+ ions are.

PKA controls a level of topoisomerase I mRNA in mouse L5178Y lymphoma cells treated with db-cAMP.

The level of topoisomerase I mRNA was measured in cells of two mouse lymphoma (LY) sublines treated with db-cAMP. A transient increase of the level was observed to be of about 60% of the basic level and to have maximum after the 3 h treatment of LY-S cells. The increase in LY-R subline was two-fold lower. The activity of PKA in a cytosol fraction of LY-S cells was 1.75 times higher than that in LY-R cells. The activity of PKA in membranes and nuclear fraction did not differ significantly in both cell types. When the activity of PKA in LY-S cells was inhibited with H8, no increase of the level of topoisomerase I mRNA was observed upon db-cAMP treatment of cells. We suggest that the activity of PKA in the cytosol controls the expression of topoisomerase I gene in LY cells at high concentration of cAMP.

Thromboxane increase in irradiated animals is caused by stimulation of cyclooxygenase activity.

The irradiation of whole body of rabbits with a dose of 6.0 Gy causes an increase in thromboxane synthesis from exogenous arachidonic acid. The uptake of [14C]arachidonic acid and the total amount of radioactivity released during collagen stimulated aggregation of platelets are not changed following the exposure of animals. The irradiation changes the relation between released arachidonic acid and synthesized thromboxane. The amount of 12-hydroxyeicosatetraenoic acid remains unchanged. The results indicate that the increase in thromboxane synthesis is not associated with the activation of phospholipase but is caused by stimulation of cyclooxygenase activity.

Hydrogen peroxide induced reproductive and interphase death in two strains of L5178Y murine lymphoma differing in radiation sensitivity.

L5178Y-R (LY-R) and L5178Y-S (LY-S) cells, differing in radiation sensitivity and susceptibility to the radiosensitizing effect of benzamide (Bz) were examined for susceptibility to hydrogen peroxide. Survival and chromatid aberration frequency indicated that LY-R cells were considerably more sensitive to H2O2 than LY-S cells. So, LY strains were found to be inversely cross-sensitive to X/gamma rays and H2O2. The relative resistance to H2O2 corresponded with the previously found twofold difference in catalase activity (Jaworska et al. 1987). At higher concentrations H2O2 treatment caused interphase death, that was delayed by benzamide (Bz, 2mM), an inhibitor of poly(ADP-ribosylation), to a lesser extent in the more resistant cell subline (LY-S). From the examination of the H2O2 induced increase in the free Ca2+ concentration (with or without 2 mM Bz treatment) with the use of Fura-2 it followed, that the cells responded to the oxidative stress by Ca2+ release. The Ca2+ concentration increase was neither directly related to the killing effect of H2O2 treatment, nor did it correspond with the twofold difference in catalase activity in LY strains.

Ca2+ mobilization is related to the lethal effect of X-irradiation in L5178Y cells.

Radiation-resistant L5178Y-R (LY-R) and radiation-sensitive L5178Y-S (LY-S) murine lymphoma cells were X-irradiated and their free Ca2+ concentration was examined with Fura-2 in Ca2(+)-free salt solution. The release of free Ca2+ from intracellular stores was linear between 10 and 60 min after irradiation (1-5 Gy X-rays) and was higher in LY-S than in LY-R cells. Pre-treatment with 2 mM benzamide (Bz) further increased the concentration of free Ca2+ in LY-S cells but not in LY-R cells. In contrast with LY-R cells, LY-S cells had previously been found to be radiosensitized by continuous 2 mM Bz treatment (Szumiel et al. 1984b). Thus, there was a parallel effect of Bz on survival and on the increase in free Ca2+ concentration in LY cells. Moreover, the rates of Ca2+ release after irradiation with 1-5 Gy of X-rays with or without Bz treatment were inversely related to the respective surviving fractions of LY cells.

Irradiation of rats abolishes susceptibility of PGI2 synthesis in blood vessels to peroxidative agents.

Thoracic and abdominal aortas were obtained from rats after irradiation and used for the estimation of the synthesis of prostacyclin (PGI2) determined as 6-keto PGF1 alpha. Twenty four h after exposure to 7.0 Gy an increase was noted in the amount of PGI2 released, and 4 weeks later its level significantly decreased. The 24 h value did not increase with the further radiation dose increment (9, 12.5, 15 Gy). Cysteine or H2O2 intensified prostacyclin synthesis in control vessels but decreased it in vessels from the animals irradiated 24 h earlier. Later after the exposure cysteine or H2O2 were no longer effective.

Increased thromboxane production after whole body irradiation.

Irradiation causes an increase in serum and plasma level of thromboxane in rabbits and an increase in thromboxane synthesis in blood platelets stimulated by thrombin. Thromboxane release from thoracic aorta is also increased upon irradiation of animals. H2O2 which stimulates thromboxane release from thoracic control aorta is without effect in the irradiated one.

Photosynthetic apparatus in chilling-sensitive plants : V. Changes in protein fractions of leaves and isolated chloroplasts following cod and dark storage and illumination of tomato leaves.

Proteins of fresh, cold and dark-stored and illuminated tomato leaves were fractionated by SDS electrophoresis. The total soluble proteins extracted from fresh leaves were separated into 5 main fractions with MWs of 54,000, 45,000, 32,000, 23,000 and 14,000. The cold and dark storage of the leaves causes a marked reduction mainly in the fraction with MW of 45,000 which increased with the illumination of the cold and dark-storaged leaves. The polypeptides with MWs of 54,000 and 14,000 (probably large and small subunits of ribulose, bisphosphate carboxylase) were stable under these conditions. In contrast, the polypeptides with MWs of 54,000 and 14,000 are decreased following the storage of tomato leaves in the dark at room temperature. Chloroplast soluble proteins were seperated by SDS electrophoresis into fractions with MWs of 64,000, 54,000, 20,000 and 14,000. The same fractions in similar proportions were observed in soluble-chloroplast proteins from fresh as well as coold and dark-stored and illuminated leaves. No drastic changes in structural polypeptides were observed following cold and dark-storage and illumination of the leaves. The results indicated that the main protein fraction, which degradated following cold and dark storage of tomato leaves and synthetized during illumination, is the fraction of cytoplasmic protein which in SDS electrophoresis gives polypeptides of about 45,000 MW. The fractions of chloroplast proteins were stable under such conditions.

Photosynthetic apparatus in chilling-sensitive plants : IV. Changes in ATP and protein levels in cold and dark stored and illuminated tomato leaves in relation to Hill reaction activity.

Changes in the levels of both ATP and protein in relation to Hill reaction activity following cold and dark storage and illumination of leaves of Lycopersicon esculentum Mill. were studied. Loss of Hill reaction activity observed during cold and dark storage of leaves for 3-4 days was accompanied by about 50% decrease of both ATP and protein levels while the content of chlorophyll was not affected Illumination of cold and dark stored leaves (8000 lx for 2 h) resulted in almost a complete restoration of both ATP and protein levels as well as Hill reaction activity. The latter process proceeded, however with different kinetics than the former ones. The rate of Hill reaction activity increase very rapidly from the beginning of illumination while the ATP level diminished during the first hour of illumination. In addition there was a lag in the increases in protein content. By about two hours of illumination all these processes reached the maximum values. Following illumination of leaf dises stored in the cold and dark in the presence of either cycloheximide or DCMU, both ATP and proteins levels as well as Hill reaction activity were greatly diminished. These data seem to suggest that the lack of reactivation of Hill reaction activity in the presence of these two inhibitors is due to inhibition of ATP synthesis required primarily for manganese reincorporation into the thylakoid membrane and theraby restoration of Hill reaction activity (Kaniuga, Zabek and Sochanowicz, Planta 1978b). Contribution of cytoplasmic protein synthesis in this process appears to be of secondary importance, although the inactivation and reactivation of electron transport are accompanied by a large loss (as high as 50%) and the restoration of the initial protein content in leaves following illumination.

Photosynthetic apparatus in chilling-sensitive plants : III. Contribution of loosely bound manganese to the mechanism of reversible inactivation of hill reaction activity following cold and dark storage and illumination of Leaves.

The levels of both tightly and loosely bound Mn in chloroplasts from fresh, cold and dark stored as well as illuminated leaves of Lycopersicon esculentum Mill. were studied in relation to Hill reaction activity. The tightly bound Mn pool represents one third of the total Mn content in chloroplasts isolated from the fresh leaves, and its level does not change following cold storage and illumination of leaves. Upon cold and dark storage of leaves the loss from the chloroplasts of about 40%-50% of the total amount of Mn is accompanied by an almost complete inactivation of the Hill reactions, as studied with water as an electron donor, as well as by the appearance of an EPR signal characteristic of free Mn(2+) ions. Following illumination of such leaves, the restroration of Hill reaction activity is accompanied by an increase in the total Mn content in chloroplasts of up to 70%-80% of the Mn level measured in the fresh leaves and by disappearance of the EPR signal. In contrast, aging in the cold of isolated chloroplasts does not affect their Mn content. The addition of manganese does not result in the restoration of Hill reaction activity in chloroplasts from cold stored leaves but causes a restoration of this activity inhibited by linolenic acid. The data suggest that the loosely bound Mn pool (extractable with Tris) can be differentiated into two fractions: (1) one functionally inactive in electron transport and (2) one responsible for restoration of Hill reaction activity. Mn of the latter fraction (about 45% of the total Mn content) probably originates from the free Mn ions present in the interior of the chloroplasts following the cold and dark storage of leaves and from Mn reincorporated into chloroplasts from the cytoplasm. Incorporation of Mn from both these sources into thylakoid membrane to form a functionally active, loosely bound Mn pool proceeds during the illumination of leaves and results in the restoration of Hill reaction activity inhibited following the storage of leaves in dark and cold.