Bioinspired Construction of a Nanozyme-Based H2O2 Homeostasis Disruptor for Intensive Chemodynamic Therapy.

The insufficient intracellular H2O2 level in tumor cells is closely associated with the limited efficacy of chemodynamic therapy (CDT). Despite tremendous efforts, engineering CDT agents with a straightforward and secure H2O2 supplying ability remains a great challenge. Inspired by the balance of H2O2 generation and elimination in cancer cells, herein, a nanozyme-based H2O2 homeostasis disruptor is fabricated to elevate the intracellular H2O2 level through facilitating H2O2 production and restraining H2O2 elimination for enhanced CDT. In the formulation, the disruptor with superoxide dismutase-mimicking activity can convert O2•- to H2O2, promoting the production of H2O2. Simultaneously, the suppression of catalase activity and depletion of glutathione by the disruptor weaken the transformation of H2O2 to H2O. Thus, the well-defined system could perturb the H2O2 balance and give rise to the accumulation of H2O2 in cancer cells. The raised H2O2 level would ultimately amplify the Fenton-like reaction-based CDT efficiency. Our work not only paves a way to engineer alternative CDT agents with a H2O2 supplying ability for intensive CDT but also provides new insights into the construction of bioinspired materials.

Silenced rice in both cytosolic ascorbate peroxidases displays pre-acclimation to cope with oxidative stress induced by 3-aminotriazole-inhibited catalase.

The maintenance of H2O2 homeostasis and signaling mechanisms in plant subcellular compartments is greatly dependent on cytosolic ascorbate peroxidases (APX1 and APX2) and peroxisomal catalase (CAT) activities. APX1/2 knockdown plants were utilized in this study to clarify the role of increased cytosolic H2O2 levels as a signal to trigger the antioxidant defense system against oxidative stress generated in peroxisomes after 3-aminotriazole-inhibited catalase (CAT). Before supplying 3-AT, silenced APX1/2 plants showed marked changes in their oxidative and antioxidant profiles in comparison to NT plants. After supplying 3-AT, APX1/2 plants triggered up-expression of genes belonging to APX (OsAPX7 and OsAPX8) and GPX families (OsGPX1, OsGPX2, OsGPX3 and OsGPX5), but to a lower extent than in NT plants. In addition, APX1/2 exhibited lower glycolate oxidase (GO) activity, higher CO2 assimilation, higher cellular integrity and higher oxidation of GSH, whereas the H2O2 and lipid peroxidation levels remained unchanged. This evidence indicates that redox pre-acclimation displayed by silenced rice contributed to coping with oxidative stress generated by 3-AT. We suggest that APX1/2 plants were able to trigger alternative oxidative and antioxidant mechanisms involving signaling by H2O2, allowing these plants to display effective physiological responses for protection against oxidative damage generated by 3-AT, compared to non-transformed plants.

[The change of cancer-related genes expression profile in Nthy-ori-3-1 cell induced by the pesticide amitrole].

OBJECTIVE: To explore the possible mechanism of amitrole causing thyroid tumor in Nthy-ori-3-1 cell by differential expression microarray analysis. METHODS: After the Nthy-ori-3-1cells were treated with 1 ~ 100 g / m L amitrole for 24 h, and the effect of amitrole on the proliferation of the cells was detected by MTT assay. Then cells were treated with 100 g / m L amitrole for 24 h, and the differential expression microarray was tested. The microarray results was analyzed by GO analysis and pathway analysis. The microarray results were verified by real-time quantitative PCR. RESULTS: MTT results showed that amitole had no significant effect on the proliferation of Nthy-ori-3-1 cells. Microarray results showed that 90( 55 up-regulated, 35 down regulated) genes were significantly changed. GO analysis showed that 43( 37 up-regulated, 6 down-regulated) of the 90 changed genes were related to biological processes, and 42( 37 up-regulated, 5down-regulated) were related to molecular function, and 44( 38 up-regulated, 6 downregulated) were related to cell components. Pathway results showed that 44 signalingpathways were influenced by the differentially expressed genes, and 10 of them were closely related to tumor. The qRT-PCR results were consistent with microarray results. wnt5 b, arnt2 and bmp2 genes were significantly related with multiple tumor-associated pathways. CONCLUSION: Amitrole may cause thyroid tumor by multiple signaling pathways, and bmp2, arnt2 and wnt5 b may beits major target genes.

Benzothiazole aniline tetra(ethylene glycol) and 3-amino-1,2,4-triazole inhibit neuroprotection against amyloid peptides by catalase overexpression in vitro.

Alzheimer's disease, Familial British dementia, Familial Danish dementia, Type 2 diabetes mellitus, plus Creutzfeldt-Jakob disease are associated with amyloid fibril deposition and oxidative stress. The antioxidant enzyme catalase is a neuroprotective amyloid binding protein. Herein the effects of catalase overexpression in SH-SY5Y neuronal cells on the toxicity of amyloid-ß (Aß), amyloid-Bri (ABri), amyloid-Dan (ADan), amylin (IAPP), and prion protein (PrP) peptides were determined. Results showed catalase overexpression was neuroprotective against Aß, ABri, ADan, IAPP, and PrP peptides. The catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and catalase-amyloid interaction inhibitor benzothiazole aniline tetra(ethylene glycol) (BTA-EG4) significantly enhanced neurotoxicity of amyloid peptides in catalase overexpressing neuronal cells. This suggests catalase neuroprotection involves breakdown of hydrogen peroxide (H2O2) plus a direct binding interaction between catalase and the Aß, ABri, ADan, IAPP, and PrP peptides. Kisspeptin 45-50 had additive neuroprotective actions against the Aß peptide in catalase overexpressing cells. The effects of 3-AT had an intracellular site of action, while catalase-amyloid interactions had an extracellular component. These results suggest that the 3-AT and BTA-EG4 compounds may be able to inhibit endogenous catalase mediated neuroprotection. Use of BTA-EG4, or compounds that inhibit catalase binding to amyloid peptides, as potential therapeutics for Neurodegenerative diseases may therefore result in unwanted effects.

A metal mixture induces transformation upon antioxidant depletion in a hepatic cell line.

INTRODUCTION: Metals are ubiquitous soil, air, and water pollutants. A mixture of arsenic cadmium and lead, in particular, has commonly been found in the vicinity of smelter areas. The mixture of As-Cd-Pb has been shown to be carcinogenic, and transforming potential and oxidative stress have been proposed as principal mechanisms involved in this process. The aim of this work was to explore the role of the antioxidant barrier in the establishment of cell transformation upon chronic exposure to a metal mixture containing 2 µM NaAsO(2), 2 µM. CdCl(2), and 5 µM Pb(C(2)H(3)O(2))(2)∙3H(2)O in WRL-68 cells-a non-transformed human hepatic cell line. MATERIAL AND METHODS: In this study, we used a WRL-68 cell model of human embryonic hepatic origin treated with antioxidant inhibitors (L-Buthionine-sulfoxamine and aminotriazole) to test the role of the antioxidant barrier in the establishment of cell transformation upon chronic exposure to a metal mixture of As-Cd-Pb (2 µM NaAsO(2), 2 µM CdCl(2) and 5 µM Pb(C(2)H(3)O(2))(2)∙3H(2)O). We evaluated oxidative damage markers, including reactive oxygen species, lipid peroxidation, and genotoxicity, as well as antioxidant response markers, including glutathione concentration, catalase activity, and superoxide dismutase activity, which promote morphological transformation, which can be quantified by foci formation. RESULTS: As expected, we found an increase in the intracellular concentration of the metals after treatment with the metal mixture. In addition, treatment with the metal mixture in addition to inhibitors resulted in a large increase in the intracellular concentration of cadmium and lead. Our results describe the generation of reactive oxygen species, cytotoxicity, genotoxicity, and oxidative damage to macromolecules that occurred exclusively in cells that were morphologically transformed upon exposure to a metal mixture and antioxidant barrier inhibition. CONCLUSION: Our results show the importance of the antioxidant barrier role in the protection of cellular integrity and the transformation potential of this metal mixture via free radicals.

[Mechanism of the effects of amitrole on the thyroglobulin in Fischer rat thyroid follicle-5 cell].

OBJECTIVE: The mechanisms of the effects of amitrole on the thyroglobulin (TG) was investigated in Fischer rat thyroid follicle-5 cell (FRTL-5 cells) and in the medium. METHODS: FRTL-5 cells were treated with 1, 10 and 100 microg/ml amitrole, and cytotoxicity was tested by 3H-TdR. The effects of amitrole on TG and thyroid transcription factor 1(TTF-1) in FRTL-5 cells were analyzed by RIA and ICC. And the TSHR in FRTL-5 cells was examined by Immunofluorescence analysis. RESULTS: 1,10 and 100 microg/ml amitrole had no significant cytotoxicity to the FRTL-5 cells (P > 0.05). The concentration of TG in the culture was decreased by 10 and 100 microg/ml amitrole (P < 0.05), and the concentration of TG in the cells was decreased by 100 microg/ml (P < 0.05) but the TTF-1 in the cells were not obviously changed (P > 0.05). The TSHR in the surface of FRTL-5 cells was significantly decreased by all doses of amitrole (P < 0.01). CONCLUSION: The influence of amitrole on TG of FRTL-5 cells may be related to significantly reduce TSHR in the surface of FRTL-5 cells.

Role of NKX2-1 in N-bis(2-hydroxypropyl)-nitrosamine-induced thyroid adenoma in mice.

NKX2-1 is a homeodomain transcription factor that is critical for genesis of the thyroid and transcription of the thyroid-specific genes. Nkx2-1-thyroid-conditional hypomorphic mice were previously developed in which Nkx2-1 gene expression is lost in 50% of the thyroid cells. Using this mouse line as compared with wild-type and Nkx2-1 heterozygous mice, a thyroid carcinogenesis study was carried out using the genotoxic carcinogen N-bis(2-hydroxypropyl)-nitrosamine (DHPN), followed by sulfadimethoxine (SDM) or the non-genotoxic carcinogen amitrole (3-amino-1,2,4-triazole). A significantly higher incidence of adenomas was obtained in Nkx2-1-thyroid-conditional hypomorphic mice as compared with the other two groups of mice only when they were treated with DHPN + SDM, but not amitrole. A bromodeoxyuridine incorporation study revealed that thyroids of the Nkx2-1-thyroid-conditional hypomorphic mice had >2-fold higher constitutive cell proliferation rate than the other two groups of mice, suggesting that this may be at least partially responsible for the increased incidence of adenoma in this mouse line after genotoxic carcinogen exposure. Thus, NKX2-1 may function to control the proliferation of thyroid follicular cells following damage by a genotoxic carcinogen.

[Effects of amitrole on thyroid hormone-associated gene transcription in FRTL-5 cells].

OBJECTIVE: To observe the effects of amitrole on the transcription of thyroglobulin (tg), thyroid peroxidase (tpo), Na(+)/I- symporter (nis), Na(+)/I- symporter (nis), thyroid-stimulating hormone receptor (tshr), thyroid transcription factor 1 (ttf-1) and paired-domain protein-8 (pax-8) genes in FRTL-5 cells and investigate the mechanism of amitrole for intervening in thyroid hormone activity. METHODS: FRTL-5 cells were treated with amitrole at 0.001, 0.01 and 0.1 mg/ml for 24 h, respectively, after which the cells were collected for extraction of the total RNA. RT-PCR was used to examine the effects of amitrole on the transcription of tg, tpo, nis, tshr, pax-8 and ttf-1 genes in FRTL-5 cells. RESULTS: Amitrole significantly induced tg gene transcription at all the doses, but produced no obvious effects on tpo and nis gene transcription. At the concentration of 0.1 mg/ml, amitrole significantly reduced pax-8 and tshr gene transcription but increased ttf-1 gene transcription. CONCLUSION: The effects of amitrole on thyroid hormone activity may be related with its actions on tg, ttf-1, tshr and pax-8 gene transcription.

Indomethacin induces small intestinal damage and inhibits amitrole-associated thyroid carcinogenesis in rats initiated with N-bis(2-hydroxypropyl)nitrosamine.

Effects of intestinal damage on thyroid carcinogenesis due to amitrole (AT) were examined in F344 male rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). In experiment 1, rats were provided with diet containing 0.03% AT for 20 weeks after a single subcutaneous injection of DHPN (2800 mg/kg body weight), and concomitantly received 0.01% indomethacin (IM) in the diet to cause small intestinal damage or 1% dextran sodium sulfate (DSS) in the drinking water for induction of colitis following a schedule of intermittent 1-week administration and 1-week withdrawal for a total of 10 times. Groups without AT- and/or IM or DSS treatment were also included. Histopathological examination revealed significant reduction in the incidence and multiplicity of follicular cell adenomas and adenocarcinomas in the group concomitantly treated with IM, but no change in the DSS group, as compared with the AT alone group. In experiment 2, rats were similarly fed diet containing AT for 3 weeks with concomitant IM or DSS treatment after a DHPN initiation, and serum thyroid stimulating hormone levels were found to be significantly elevated only in the IM case. The increase in thyroid follicular cell proliferation due to AT was also clearly suppressed in the group concomitantly treated with IM. From these findings, IM-induced intestinal damage may inhibit thyroid carcinogeneisis in rats, although contributions of other factors, such as a direct inhibitory effect of IM to thyroid follicular cell proliferation cannot be ruled out.

Lack of combination hepatocarcinogenicity of harman, norharman and amitrole when given with NaNO2 in the rat.

N-nitrosocompounds, which induce cancers in various organs, may be formed endogenously with intake of amino compounds such as secondary amines and sodium nitrite (NaNO(2)) in combination. The present study was performed to investigate whether three amino compounds, 1-methyl-9H-pyrido[3,4-b]indole (harman), 9H-pyrido[3,4-b]indole (norharman) and 2-amino-1,3,4-triazole (amitrole), might be converted in vivo to compounds capable of promoting hepatocarcinogenesis when given with NaNO(2). However, in an 8-week model, no modifying potential was evident in terms of numbers and areas of putative preneoplastic glutathione S-transferase placental form (GST-P)-positive foci in any of the groups receiving paired treatments. These results demonstrate that combinations of harman, norharman and amitrole with NaNO(2) lack promoting effects for liver carcinogenesis in our medium-term bioassay system.

Inhibition of catalase activity with 3-amino-triazole enhances the cytotoxicity of the Alzheimer's amyloid-beta peptide.

Amyloid-beta, (Abeta) is a cytotoxic peptide implicated in the pathology of Alzheimers disease. The antioxidant enzyme catalase has been suggested to protect against Abeta cytotoxicity in both neuronal and non-neuronal cell types. Inhibition of endogenous catalase using 3-amino-1,2,4-triazole (3AT) in neuronal (NT-2) and myeloma (SP2/0-Ag-14) cell lines increases Abeta toxicity, suggesting that any protective role for endogenous catalase requires active enzyme. In Abeta treated mveloma cells there was a significant decrease in the total cell catalase activity and immunoreactivity. However, when the surviving live cell population was isolated following Abeta treatment the levels of catalase were significantly increased. The surviving live cell population from groups treated with both 3AT and Abeta contain elevated immunoreactive catalase levels suggesting that the protective role for endogenous catalase may have a component independent of the antioxidant activity, possibly by acting as an Abeta binding protein. Amyloid-beta (Abeta) cytotoxicity can be prevented by Vitamin E treatment or an anti-Abeta monoclonal antibody (ALIOI), both of which also prevent Abeta cytotoxicity in cells treated with 3AT These observations suggest that Abeta mediated cell death in both neuronal and non-neuronal cells is mediated in part by actions to increase hydrogen peroxide. Catalase has a protective role, as a hydrogen peroxide-degrading enzyme and catalase inhibition by Abeta is not the direct cause of cytotoxicity.

Glutathione peroxidase and catalase modulate the genotoxicity of arsenite.

The X-ray hypersensitive Chinese hamster ovary (CHO) cells, xrs-5, are also more sensitive to sodium arsenite in terms of cell growth and micronucleus induction than CHO-K1 cells. Since reactive oxygen species are suggested to be involved in arsenic toxicity, we have measured antioxidant mechanisms in xrs-5 as well as CHO-K1 cells. There were no apparent differences in the activities of superoxide dismutase, glutathione S-transferase, glutathione reductase, and the levels of glutathione between xrs-5 and CHO-K1 cells. However, the activities of glutathione peroxidase and catalase were 5.4- and 5.8-fold lower, respectively, in xrs-5 cells. The addition of catalase or glutathione peroxidase to cultures reduced the arsenite-induced micronuclei in xrs-5 cells. Whereas, simultaneous treatment with mercaptosuccinate, an inhibitor of glutathione peroxidase, and 3-aminotriazole, an inhibitor of catalase, synergistically increased the arsenite-induced micronuclei. These results suggest that both catalase and glutathione peroxidase are involved in defense against arsenite genotoxicity. The xrs-6 cells, another line of x-ray hypersensitive CHO cells, which had 1.6-fold higher catalase activity and 2.5-fold higher glutathione peroxidase activity than xrs-5 cells, were also more sensitive than CHO-K1 cells but were less sensitive than xrs-5 cells to cell growth inhibition of arsenite. Moreover, a 1.6-fold increase of glutathione peroxidase activity by selenite adaptation effectively removed the arsenite-induced micronuclei in CHO-K1 cells. These results suggest that glutathione peroxidase is more important than catalase in defending against arsenite toxicity. Our results also suggest that increasing the intracellular antioxidant level may have preventive or therapeutic effects in arsenic poisoning.

Primary and immortalized (BEAS 2B) human bronchial epithelial cells have significant antioxidative capacity in vitro.

Antioxidant enzymes located in the bronchial epithelium can be expected to be important in protecting these cells against both endogenous and exogenous oxidants. In this study, human bronchial epithelial cells were isolated and cultured from specimens obtained from donors for lung transplantation. The levels and relative importance of different antioxidant enzymes were also assessed using an immortalized human bronchial epithelial cell line (BEAS 2B cells). Immunocytochemical studies showed a similar pattern of intracellular localization with the moderate degrees of labeling for Mn superoxide dismutase (SOD), CuZn SOD, and catalase in freshly isolated bronchial epithelial cells, bronchial epithelial cells in primary culture, and BEAS 2B cells. CuZn SOD and catalase decreased in labeling density whereas Mn SOD was unchanged when bronchial epithelial cells were placed in primary cultures. In contrast, Mn SOD and catalase were decreased in BEAS 2B cells compared with primary cultures. Although Mn SOD was low in BEAS 2B cells, it could be significantly induced by tumor necrosis factor treatment. Biochemical analysis showed remarkably similar catalase and glutathione reductase activities in primary cultured epithelial cells and BEAS 2B cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mechanism of the carcinogenic activity of amitrole.

Amitrole, a widely used herbicide found to produce thyroid and liver tumors in rodents and classified as possibly carcinogenic to humans, was investigated to acquire further information about its mechanism of action. A 20-hr exposure to amitrole concentrations ranging from 5.6 to 18 mM did not induce DNA fragmentation, as measured by the alkaline elution technique, in primary cultures of human thyroid follicular cells and of human liver cells. Under the same experimental conditions a minimal frequency of DNA breaks was detected in primary cultures of rat hepatocytes, but this event was presumably the unspecific consequence of a cytotoxic effect. In rats given amitrole with drinking water for 12 successive days at a daily dose of approximately 200 mg/kg, plasma levels of triiodothyronine and thyroxine displayed a progressive reduction, and a concurrent increase of both the mitotic index and frequency of S-phase cells revealing a clear-cut follicular cell hyperplasia was observed. In a group of these rats euthanized after 8 days of treatment any evidence of DNA fragmentation was absent in both thyroid and liver cells. Taken as a whole these results provide further evidence that the mechanism of amitrole carcinogenic activity is most likely nongenotoxic but due to hormone imbalance.

Cytotoxicity of oxidants and asbestos fibers in cultured human mesothelial cells.

The authors investigated the mechanisms caused by oxidants (superoxide and hydrogen peroxide) and asbestos (amosite) fibers in human mesothelial cells. Immortalized human pleural mesothelial cells (MET 5A) were exposed in vitro to one of the following: hypoxanthine (100-200 microM) plus xanthine oxidase (10-20 mU/ml) as a superoxide-generating system, H2O2 (50 microM-5 mM); or amosite (1-100 micrograms/cm2). Cellular adenine nucleotide depletion, DNA single strand breaks, extracellular release of nucleotides, and their catabolites and lactate dehydrogenase (LDH) were assessed as markers of cell damage after 4-6 h exposure to the oxidants or fibers. The effect of intracellular antioxidant enzymes and exogenous antioxidants on cell damage were investigated during oxidant and amosite exposure. Superoxide radical and H2O2 exposure resulted in the depletion of adenine nucleotides, accumulation of the products of nucleotide catabolism, induction of DNA single strand breaks, and extracellular LDH release. Amosite exposure did not cause nucleotide depletion or induction of DNA single strand breaks. Inactivation of the intracellular antioxidant enzymes glutathione reductase or catalase augmented cell damage during H2O2 exposure but not during amosite exposure.

Caffeic acid causes metal-dependent damage to cellular and isolated DNA through H2O2 formation.

Pulsed field gel electrophoresis showed that caffeic acid induced DNA strand breaks in cultured human cells in the presence of Mn(II). With alkali treatment, DNA single-strand breaks were observed. The strand breakage was increased by the treatment of buthionine sulphoximine (a GSH synthesis inhibitor) and 3-aminotriazol (a catalase inhibitor) and decreased by catalase, indicating the involvement of H2O2. The DNA damage was decreased by o-phenanthroline, indicating the involvement of transition metal ion. Damage to isolated DNA from c-Ha-ras-1 protooncogene was investigated by a DNA sequencing technique. Caffeic acid caused DNA damage in the presence of Cu(II) but not in the presence of either Mn(II) or Fe(III). Caffeic acid plus Cu(II) induced piperidine-labile sites frequently at thymine residues, especially of the 5'-GTC-3' and 5'-CTG-3' sequences. Typical OH scavengers showed no inhibitory effects. The inhibitory effects of bathocuproine and catalase on Cu(II)-mediated DNA damage suggest that Cu(I) and H2O2 have important roles in the production of active species causing DNA damage. The Cu(II)-mediated DNA damage was enhanced by pre-incubation of caffeic acid with Mn(II). Mn(II)- or Cu(II)-catalyzed autoxidation of caffeic acid produced H2O2 with efficiency of Mn(II) greater than Cu(II). These results suggest that in the presence of Mn(II) or Cu(II), caffeic acid produces H2O2, which is activated by transition metals to cause damage to DNA in vitro and probably in cultured cells.

Genetic toxicity of six carcinogens and six non-carcinogens in the Drosophila wing spot test.

Six rodent carcinogens, 5 of which are also human carcinogens, and 6 compounds recognized as non-carcinogens were tested for their genotoxic activity in the Drosophila melanogaster wing spot test. 72-h-old larvae trans-heterozygous for the recessive wing cell markers 'multiple wing hairs' (mwh) and 'flare' (flr3) were fed various concentrations of the test compounds for a period of 48 h. With amitrole and 4-aminobiphenyl, larvae of the same age were also given an acute treatment of 6 h with higher concentrations, and, in addition, 48-h-old larvae were fed for a longer period of 72 h. Repeats of all experiments document the good reproducibility of the results in the wing spot test. Amitrole and 4-aminobiphenyl were genotoxic after both 48-h and 72-h treatments, but their activity could not be detected following acute exposure of only 6 h. Chlorambucil and melphalan were clearly genotoxic. The carcinogens sodium arsenite and sodium arsenate, however, which are highly toxic to Drosophila, could only be tested at low exposure levels and were negative under these treatment conditions. The 6 non-carcinogens (ascorbic acid, 2-aminobiphenyl, mannitol, piperonyl butoxide, stannous chloride and titanium dioxide) were all definitely non-genotoxic in the Drosophila wing spot test. The data for the non-carcinogens demonstrate that non-genotoxic compounds can be identified in the wing spot test with a reasonable experimental effort.

Evaluation of the isobologram method for the assessment of mixtures of chemicals. Combination effect studies with pesticides in algal biotests.

The aim of this paper is an evaluation of isobolograms, a method proposed for the assessment of combined effects of chemicals. In order to examine potentials and shortcomings of this approach for ecotoxicological purposes, algal biotests with selected pesticidal compounds were performed. Additivity, as defined by the model, is demonstrated for the combination of atrazine and metribuzin for different combination ratios, response levels, and parameters. Subadditivity is shown for amitrole and glufosinate-ammonium. The results and inherent biometrical features are discussed in terms of criteria considered suitable for comparative evaluation of biometrical models for the assessment of mixtures of chemicals.