Hepatic toxicological responses of SiO2 nanoparticle on Oreochromis mossambicus.

The present study was carried out to investigate the impact of various concentrations of SiO2 nanoparticles (SiO2 NP) on the commonly available freshwater fish Oreochromis mossambicus. The 96 h median lethal concentration (LC50) of SiO2 NP was found to be between 270-280 ppm. This novel study has demonstrated histological alterations in the hepatic tissues and a dose-dependent depletion of tissue protein content and an elevated transaminases activity in the treated fish, which has facilitated understanding of the impact of SiO2 NP in O. mossambicus.

Haemato-immunological studies in ZnO and TiO2 nanoparticles exposed euryhaline fish, Oreochromis mossambicus.

The present study was aimed to analyze the effect of ZnO and TiO2 nanoparticles (NPs) on Haemato-immunological parameters in adult Tilapia, Oreochromis mossambicus. The nanoparticles size found as 47 nm and 30 nm for ZnO and TiO2 respectively. The acute toxicity (96 h) of ZnO (LC50: 100-110 ppm) and TiO2 (LC50: 80-90 ppm) NPs were identified by using probit analysis. RBC, Hb and HCT levels decreased in nanoparticles exposed groups resulted in decreased oxygen carrying capacity of RBC and other erythrocyte indices (MCH, MCV, MCHC). Increased WBC, neutrophils & monocytes and decreased lymphocyte levels were observed as increased concentration of the nanoparticles. The results were found as statistically significant (p < 0.05). In conclusion, the present study depicts that ZnO NPs exhibits more toxicity than TiO2 NPs. Nanoparticles presence even in low concentration (ppm) cause damage to the connective tissues of fish, so the existing permissible levels of these nanoparticles in water are need to be revised.

Toxicological effect of Al2O3 nanoparticles on histoarchitecture of the freshwater fish Oreochromis mossambicus.

In the present study, freshwater fish Oreochromis mossambicus were exposed to sub lethal concentrations (120, 150 and 180 ppm) of Aluminium oxide nanoparticles (Al2O3 NPs) for 96 h. Histological abnormalities were not observed in the organs of control fishes whereas severe damages and extensive architectural loss was found in the brain, gill, intestine, kidney and muscle tissues of treated fishes with more pronounced effects in 180 ppm. The results showed that the acute exposure to Al2O3NPs altered the histoarchitecture in various fish tissues.

Histological alterations in the hepatic tissues of Al2O3 nanoparticles exposed freshwater fish Oreochromis mossambicus.

Adverse effects of nanoparticles on aquatic environment and organisms have drawn much special attention to many researches. Aluminium oxide nanoparticles (Al2O3-NPs) have potential uses in varied fields and are seen entering into the ecosystem. Their potential toxicity to the freshwater fish is not much studied. Hence this study was framed to investigate the effect Al2O3 NPs on freshwater fish Oreochromis mossambicus in terms of sub lethal toxicity, histological changes and hepato somatic index (HSI) under laboratory conditions. Fishes were exposed to varying concentrations of Al2O3 NPs for 96hr. LC50 value was found to be in between 235 and 245ppm. The findings of the present work showed that the NPs were accumulated in the fish liver and caused major histological anomalies such as structural alterations in the portal vein, necrotic hepatocytes, vacuolation, aggregation of blood cells and melanomacrophages. Significant histological alterations were observed in the highest concentration. Our results evidenced that the Al2O3 NPs in the aquatic environment affects the health condition of the fishes.

Optical trapping in an absorbing medium: from optical tweezing to thermal tweezing.

We report on optical trapping in a weakly absorbing medium, hemin, an iron-containing porphyrin that is an important component of hemoglobin. By altering the hemin concentration we are able to control the amount of optical energy that is absorbed; changing the hemin concentration from <12 mg/ml to >45 mg/ml enables the onset of thermal trapping to be observed. By estimating the trap strength using two different methods we are readily able to differentiate between the optical trapping and thermal trapping regimes. We also deduce the rise in temperature that occurs within the laser focal volume: temperature changes of 5-24 K are observed for laser power values of 10-90 mW for hemin concentrations of 0-50 mg/ml.

Optical-tweezer-induced microbubbles as scavengers of carbon nanotubes.

A modified optical tweezers set-up has been used to generate microbubbles in flowing, biologically relevant fluids and human whole blood that contains carbon nanotubes (CNTs) using low power (< or =5 mW), infrared (1064 nm wavelength), continuous wave laser light. Temperature driven effects at the tweezers' focal point help to optically trap these microbubbles. It is observed that proximate CNTs are driven towards the focal spot where, on encountering the microbubble, they adhere to it. Such CNT-loaded microbubbles can be transported both along and against the flow of surrounding fluid, and can also be exploded to cause fragmentation of the bundles. Thus, microbubbles may be used for scavenging, transporting and dispersal of potentially toxic CNTs in biologically relevant environments.

Conformationally restricted analogues of 1N,14N-bisethylhomospermine (BE-4-4-4): synthesis and growth inhibitory effects on human prostate cancer cells.

Twelve analogues of 1N,14N-bisethylhomospermine (BE-4-4-4) with restricted conformations were synthesized in the search for cancer chemotherapeutic agents with higher cytotoxic activities and lower systemic toxicities than BE-4-4-4. The central butane segment of BE-4-4-4 was replaced with a 1,2-substituted cyclopropane ring, a 1,2-substituted cyclobutane ring, and a 2-butene residue. In each case, the cis/trans-isomeric pair was synthesized. Cis-monounsaturation(s) was also introduced at the outer butane segment(s) of BE-4-4-4. The two possible cis-dienes and a cis-triene formally derived from the tetraazaeicosane skeleton of BE-4-4-4 were also prepared. Four cultured human prostate cancer cell lines (LnCap, DU145, DuPro, and PC-3) were treated with the new tetramines to examine their effects on cell growth with a MTT assay. One representative cell line (DuPro) was selected to further study the cellular uptake of the novel tetramines, their effects on intracellular polyamine pools, and their cytotoxicity. All tetramines entered the cells, reduced cellular putrescine and spermidine pools while exerting only a small effect on the spermine pool, inhibited cell growth, and killed 2-3 logs of cells after 6 days of treatment at 10 microM. Four new tetramines, the two cyclopropyl isomers, the trans-cyclobutyl isomer, and the (5Z)-tetraazaeicosene, were more cytotoxic than their saturated counterpart (BE-4-4-4). Their cytotoxicity, however, could not be correlated either with their cellular uptake or with their ability to deplete intracellular polyamine pools. We attribute their cytotoxicity to their specific molecular structures. The cytotoxicity was markedly reduced when the central butane segment was deprived of its rotational freedom by replacing it with a double bond. Introduction of a triple bond or a benzene-1,2-dimethyl residue at the central segment of the polyamine chain, led to complete loss of biological activity. The conformationally restricted alicyclic derivatives were not only more cytotoxic than was the freely rotating BE-4-4-4 by several orders of magnitude but also had much lower systemic toxicities than the latter. Thus, we obtained new tetramines with a wider therapeutic window than BE-4-4-4.

cis-Unsaturated analogues of 3,8,13,18,23-pentaazapentacosane (BE-4-4-4-4): synthesis and growth inhibitory effects on human prostate cancer cell lines.

From the results of our previous physicochemical studies of polyamine-nucleic acid interactions, we concluded that polyamine analogues in cisoidal conformation are capable of wrapping around the major groove of the double helix, of displacing natural polyamines from their nucleic acid binding sites, and of inhibiting cell division. On the basis of this hypothesis, nine unsaturated pentamines, formally derived from the cytotoxic pentamine 3,8,13,18,23-pentaazapentacosane (BE-4-4-4-4), were prepared in an attempt to increase antineoplastic activity. Cis-double bonds were introduced in all possible sites in the saturated pentaazapentacosane structure of BE-4-4-4-4 to yield two pentacosenes, four pentacosadienes, two pentacosatrienes, and one pentacosatetraene. Cis-double bonds should also provide good targets for mixed-function oxidases that might eliminate the accumulation of unsaturated pentamines in serum, thereby reducing systemic toxicity in animals. We determined the ability of these new pentamines to inhibit growth in four cultured human prostate cancer cell lines (LnCap, DU145, PC-3, and DuPro) using a MTT assay. LnCap and DU145 cells were very sensitive, PC-3 cells were relatively resistant, and DuPro cells were intermediate in sensitivity to most of these synthetic pentamines. In all cell lines, pentamines that had unsaturation(s) at the end of the chain showed the highest cell growth inhibitory effects. The cellular uptake, effects on cellular polyamine levels, and cytotoxicity of these pentamines on one representative prostate cancer cell line (DuPro) were further examined with a colony-forming efficiency (CFE) assay. The pentamines with unsaturation(s) at the end of the chain were once again the most cytotoxic among both the saturated (BE-4-4-4-4) and unsaturated analogues. Appreciable amounts of all pentamines entered DuPro cells and depleted cellular polyamine pools by day 6 of treatment. For most pentamines, however, cell growth inhibitory and cytotoxic effects could not be directly correlated either with their cellular uptake or with their ability to deplete cellular polyamine pools. The position of the double bonds in the aliphatic backbone seems to be the most important determinant of cytotoxicity. For some pentamines, however, depletion of cellular polyamines may add to their efficacy.

Polyamine analog bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) enhances simian virus 40 late gene expression.

PURPOSE: The polyamine analog bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) depletes cellular polyamines and inhibits malignant cell growth. We have previously shown that BE-4-4-4-4 inhibits nucleosome condensation on supercoiled DNA in a cell-free system. Here we sought to determine whether BE-4-4-4-4 inhibits nucleosome condensation in cells, and whether that effect alters the expression of specific genes. METHODS: We used the simian virus 40 (SV-40) minichromosome as a model system and studied the expression of the viral late genes. It is known that the SV-40 late genes are regulated by the steroid receptor elements that, in turn, control gene expression by altering nucleosomal organization. RESULTS: We observed a more than six fold increase in SV-40 late gene expression in cells pretreated with BE-4-4-4-4 for 18 h. The polyamine analog bisethyl norspermine (BE-3-3-3), that does not affect nucleosomal condensation in cell free systems and has little effect on chromatin structure in cultured human tumor cells, had a negligible effect on SV-40 late gene expression under treatment conditions identical to those used with BE-4-4-4-4. CONCLUSION: Similar to the findings in the cell-free system, the polyamine analog BE-4-4-4-4 inhibited nucleosome formation and, thereby, altered the expression of specific genes in a cellular system.

Conformationally restricted analogues of 1N,12N-bisethylspermine: synthesis and growth inhibitory effects on human tumor cell lines.

Eight analogues of 1N,12N-bisethylspermine (BES) with restricted conformations were synthesized in the search for new spermine mimetics with cytotoxic activities. By replacing the central butane segment of BES with a 1,2-disubstituted cyclopropane ring, a pair of cis/trans-isomers was obtained that introduced a spatial constraint in the otherwise freely mobile butane chain. An analogous pair of isomers was obtained when the butane segment was replaced with a 1, 2-disubstituted cyclobutane ring or with a 2-butene residue. The six new BES analogues thus obtained (three pairs of cis/trans-isomers) were growth inhibitory at low-micromolar concentrations against four human tumor cell lines (A549, HT-29, U251MG, and DU145) but were less growth inhibitory against two other human tumor cell lines (PC-3 and MCF7). 1N,12N-Bisethylspermyne, where the central butane segment of BES was replaced by the rigid 2-butyne segment, was devoid of growth inhibitory activity against five of the six human cell lines studied (DU145 being the only exception), a clear indication of the importance of conformational mobility at the 4N, 9N-butane segment of BES for its biological activity. When the butane segment was replaced by a benzene-1,2-dimethyl residue, the resulting BES analogue was devoid of growth inhibitory activity despite its cisoid conformation. The cytotoxicity of the analogues does not seem to be directly related to their uptake by the cells or to their effects on cellular polyamine levels. BES analogues with restricted conformations but which contained the equivalent of a two-carbon unit, rather than the natural four-carbon unit, at the central segment, such as 1,2-diaminocyclopropyl or 1, 2-diaminocyclobutyl derivatives, were devoid of growth inhibitory effects at the concentrations studied. The development of conformationally restricted polyamine analogues appears to show promise in the further quest for polyamine-related therapeutic agents with specificity of action.

The mechanism of polyamine analog-induced enhancement of cisplatin cytotoxicity in the U-251 MG human malignant glioma cell line.

PURPOSE: During the last decade, several polyamine analogs have been developed as antineoplastic agents that replace intracellular polyamines but cannot mimic the biological functions of polyamines related to cell growth. It has been shown that pretreatment of several human brain tumor cell lines with some of these polyamine analogs increases the cytotoxicity of cis-diamminedichloroplatinum (CDDP). It has also been established that some of these polyamine analogs affect chromatin organization. In the study reported here we attempted to elucidate the mechanism by which polyamine analog-induced changes in DNA and chromatin structure may increase CDDP cytotoxicity. METHODS: We studied the micrococcal nuclease sensitivity of the nuclei and measured the amount of platinum incorporated into the nucleosomal and linker regions of chromatin isolated from CDDP-treated U-251 MG human malignant brain tumor cells with or without pretreatment with two cytotoxic polyamine analogs 1,11-bis(ethylamino)-4,8-diazaundecane (BE-3-3-3) and 1,19-bis(ethylamino)-5,10,15-diazanonadecane (BE-4-4-4-4). RESULTS: The pretreatment with the polyamine analogs decreased the MNase sensitivity and increased the incorporation of CDDP preferentially into the linker region of the chromatin. CONCLUSIONS: Pretreatment of cells with polyamine analogs probably alters the structure and/or the organization of the linker region such that more CDDP incorporates into the linker DNA. This is probably the reason for the observed enhancement of CDDP cytotoxicity in the polyamine analog-pretreated cells.

Effects of the polyamine analogues BE-4-4-4-4, BE-3-7-3, and BE-3-3-3 on the proliferation of three prostate cancer cell lines.

PURPOSE: Polyamines are biologic cations necessary for normal cell growth. Polyamine analogues have been shown to be effective inhibitors of tumor growth. We tested the effect of the polyamine analogues 1,1 9-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), N1,N11-bis(ethyl)norspermine (BE-3-3-3) and 1,15-bis(ethylamino)-4,12-diazapentadecane (BE-3-7-3) on the growth of the prostate cancer cell lines DU145, LNCaP and PC-3 in vitro. We also tested the effect of BE-4-4-4-4 on androgen-independent DU145 cells in vivo via a nude mouse xenograft model. METHODS: In vitro, cell proliferation was measured using a DNA assay or a colony-formation assay. In vivo, mice were given saline or BE-4-4-4-4 3 mg/kg or 5 mg/kg intraperitoneally twice daily on days 7-10 and 14-17 (cycle 1), days 49-52 and 56-59 (cycle 2) and days 91-94 and 98-101 (cycle 3). RESULTS: The proliferation of DU145, LNCaP and PC-3 prostate cancer cell lines was inhibited in a dose-dependent manner by BE-4-4-4-4. Intracellular putrescine, spermidine and spermine levels in all three cell lines declined after only 24 h exposure to BE-4-4-4-4 in vitro. Animals receiving BE-4-4-4-4 showed inhibition of tumor growth which continued throughout the experiment with 74% (3 mg/kg) and 81% (5 mg/kg) growth inhibition seen on day 101. No overt toxic reactions besides weight loss were observed in BE-4-4-4-4-treated animals. Tumor tissue from animals treated with BE-4-4-4-4 showed a dose-dependent decrease in spermidine and spermine levels but no decline in putrescine levels as compared with control. BE-4-4-4-4 levels were highest in tumors on day 63 with levels reaching 0.33 and 1.45 nmol/mg protein from animals treated at the 3 mg/kg and 5 mg/kg doses, respectively. CONCLUSION: These results show the polyamine analogues BE-4-4-4-4, BE-3-3-3 and BE-3-7-3 to be effective inhibitors of prostate cancer cell growth in vitro and BE-4-4-4-4 to be an effective inhibitor of DU145 cells in vivo with minimal toxicity.

Effects of spermine and its cytotoxic analogs on nucleosome formation on topologically stressed DNA in vitro.

We investigated the effects of the polyamine spermine and two of its cytotoxic analogs 1,11-bis(ethylamino)-4,8-diazaundecane (BE-3-3-3) and 1,19-bis(ethylamino)-5,10,15-tirazanonadecane (BE-4-4-4-4) on the formation of nucleosomes on negatively and positively supercoiled DNA in vitro. Histones H2A, H2B, H3 and H4 were reconstituted onto DNA to form nucleosomes and the polyamines were added either before or after histone addition. The structural state of the nucleosome was monitored by analyzing the DNA topoisomers that were present after topoisomerase I treatment. Although polyamines induced DNA aggregation to various degrees. high concentrations of topoisomerase I were able to relax the aggregated DNA and the helical pitch was found to be unaltered in the aggregates. When histones were associated with negatively coiled DNA, the polyamine-induced aggregation did not alter nucleosome structure. The induced aggregate did inhibit nucleosomal transitions when examined on positively coiled DNA. BE-4-4-4-4 was most effective and BE-3-3-3 least effective. These analogs were also extremely effective in inhibiting histone deposition onto DNA. A potential mechanism for the action of these analogs is both to inhibit histone deposition during DNA replication and also disrupt nucleosomal dynamics due to aberrant chromatin condensation. These results also suggest that BE-4-4-4-4 and BE-3-3-3 may produce their cytotoxic effect through slightly different mechanisms.

The ability of polyamine analogues to induce Z-DNA structure in synthetic polynucleotides in vitro inversely correlates with their effects on cytotoxicity of cis-diaminedichloroplatinum (II) (CDDP) in human brain tumor cell lines.

We studied the effects of 72 h pretreatment with five polyamine analogues on the cytotoxicity of cis-diaminedichloroplatinum (II) (CDDP) in U-251 MG and SF-188 human brain tumor cells. A colony forming efficiency assay showed that the pretreatment with clinically important analogues 1,11-bis(ethylamino)-4, 8-diazaundecane (BE-3-3-3), 1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4), and 1,l9-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) increased the cytotoxicity of CDDP by 1.3 to 2.3-fold; 1,19-diamino-5,10, 15-triazanonadecane (4-4-4-4) did not affect CDDP cytotoxicity, and 1,11-diamino-4,8-diazaundecane (3-3-3) protected cells from the cytotoxic effects of CDDP. An alkaline elution assay detected a small increase in DNA interstrand crosslinks accompanying the enhancement of CDDP cytotoxicity only in cells pretreated with BE-3-3-3. This study is the first to show that the Z-DNA inducing abilities of the polyamine analogues in synthetic polynucleotides in vitro correlates inversely with their effects on CDDP cytotoxicity in human tumor cells in culture.

A new model for disruption of the ornithine decarboxylase gene, SPE1, in Saccharomyces cerevisiae exhibits growth arrest and genetic instability at the MAT locus.

Ornithine decarboxylase (ODC) is a rate-determining enzyme of the polyamine-biosynthetic pathway. We sought to produce cells with impaired ODC function in order to study the biological functions of polyamines. Saccharomyces cerevisiae strains were obtained by one-step gene replacement of a 900 bp fragment of the yeast ODC gene (SPE1) with the yeast URA3 gene. Spores derived from SPE1/spe1 cells germinated at reduced efficiency relative to SPE1/SPE1. Sustained growth of spe1 haploid mutants in polyamine-free medium led to intracellular polyamine depletion, reduction in budding index, G1 arrest and cessation of growth, and cells that were large and misshapen. All of these effects were completely reversed by adding polyamines to the medium, even after 5 days of polyamine starvation. A diploid yeast strain bearing two copies of disrupted spe1 lost heterozygosity at the mating-type locus more often when grown in the absence of polyamines than when grown in their presence, indicating that polyamine deficiency leads to either chromosome loss or to mitotic recombination.

Spermine induces haemoglobin synthesis in murine erythroleukaemia cells.

The naturally occurring polyamine spermine induces haemoglobin synthesis in murine erythroleukaemia (MEL) cells. Haemoglobin production was accompanied by accumulation of cytoplasmic beta-globin mRNA and growth inhibition, but not by cell-cycle block or changes in cell volume. Hexamethylene-bisacetamide (HMBA), a well known differentiating agent, also induces haemoglobin production, but causes a G1 block and decreases cell volume. These findings indicate that HMBA and spermine affect MEL cells differently, even though both induce haemoglobin production.

The structure of polyamine analogues determines haemoglobin production and cytotoxicity in murine erythroleukaemia cells.

The naturally occurring polyamine spermine induces haemoglobin synthesis in murine erythroleukaemia (MEL) cells. We have studied the ability of various polyamine analogues to inhibit cell growth and induce haemoglobin production. Polyamine analogues with free terminal amino groups were good inducers of haemoglobin production in MEL cells. Haemoglobin levels correlated with the number of positive charges: pentamines (five positive charges) were stronger inducers than tetramines (four positive charges). Compounds ethylated at their terminal amines were poor inducers of haemoglobin production but good inhibitors of MEL cell growth. These results provide evidence that polyamine analogues support specific biological functions of polyamines in MEL cells and suggest relationships between polyamine structure and function.

Slowing proliferation in head and neck tumors: in vitro growth inhibitory effects of the polyamine analog BE-4-4-4-4 in human squamous cell carcinomas.

PURPOSE: These preclinical studies were carried out to examine the potential of the antiproliferative polyamine analog 1,19-bis-(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) to serve as a therapy adjuvant to radiation for patients with rapidly dividing tumors of the head and neck (H&N). METHODS AND MATERIALS: Cytostatic and cytotoxic effects of this polyamine analog were investigated in three squamous cell carcinoma (SCC) cell lines derived from human H&N tumors. RESULTS: Growth inhibition was achieved in all cell lines within 3-4 days of continuous 10 microM drug exposure, and inhibition of cell cycle proliferation kinetics was confirmed via flow cytometry. Cytotoxicity was pronounced (3-4 log cell kill) in the SCC-38 and SCC-4Y cell lines with continuous 10 microM analog exposure over 5 days, and was minimal in the SCC-13Y cell line. No demonstrable effect of BE-4-4-4-4 on single dose radiation survival was identified in any SCC cell line. Ornithine decarboxylase (ODC) activity was rapidly inhibited (1-2 h) following 10 microM BE-4-4-4-4 exposure in all SCC cell lines (approximately 90%), whereas identical exposure to 10 microM difluoromethylornithine (DFMO) induced animal ODC inhibition (approximately 10%). Dose-dependent depletion of endogenous polyamines (putrescine, spermidine, spermine) was achieved in all SCC cell lines following 1 microM and 10 microM BE-4-4-4-4 exposures. Difluoromethylornithine was significantly less potent than BE-4-4-4-4 in its capacity to deplete endogenous polyamines, with no measureable depletion of spermine pools even with 5 mM x 48 h DFMO exposures. CONCLUSIONS: These data evaluate cytostatic and cytotoxic properties of the polyamine analog BE-4-4-4-4 in human SCCs, and suggest a role for investigation of such agents as an adjuvant to radiation in the therapeutic approach to rapidly dividing human tumors such as those that occur in the H&N.

Radiation-induced changes in nucleoid halo diameters of aerobic and hypoxic SF-126 human brain tumor cells.

Nucleoid halo diameters were measured to assay changes in DNA supercoiling in human brain tumor cell line SF-126 after irradiation under aerobic or hypoxic conditions. In unirradiated aerobic cells, a typical propidium iodide titration curve showed that with increasing concentrations of propidium iodide, the halo diameter increased and then decreased with the unwinding and subsequent rewinding of DNA supercoils. In irradiated cells, the rewinding of DNA supercoils was inhibited, resulting in an increased halo diameter, in a radiation dose-dependent manner. To produce equal increases in halo diameter required about a threefold higher radiation dose in hypoxic cells than in aerobic cells. Quantitatively similar differences in the radiation sensitivities of hypoxic and aerobic cells were demonstrated by a colony-forming efficiency assay. These findings suggest that the nucleoid halo assay may be used as a rapid measure of the inherent radiation sensitivity of human tumors.