Biological relevance of CuFeO2 nanoparticles: Antibacterial and anti-inflammatory activity, genotoxicity, DNA and protein interactions.

Heterometal oxide nanoparticles of bioessential metals are shedding new light to nanoparticle-inspired bioapplications. Pairing bioreactive elements like copper and iron can affect the redox dynamic and biological profile of the nanomaterial. Given the complexity of physicochemical properties, biological activity and toxicity concerns, extensive exploration is demanded, especially when active and less active oxidation states participate as in case of cuprous-ferric delafossite CuFeO2 (copper(I)-iron(III)), a less widespread nanomaterial. In that vein, CuFeO2 nanoparticles were synthesized and biological profile was evaluated in comparison with cuprous oxide (Cu2O NPs) counterpart, an already established antimicrobial agent. Interactions with bacteria, proteins and DNA were examined. Cu2O NPs exhibited stronger antibacterial activity (IC50 < 25 µg/ml) than CuFeO2 NPs (IC50 > 100 µg/ml). In vitro exposure of nanoparticles on plasmid DNA unveiled toxicity in the form of DNA damage for Cu2O and enhanced biocompatibility for CuFeO2 NPs. Genotoxicity estimated by the frequency of sister chromatid exchanges, cytostaticity based on the proliferating rate indices and cytotoxicity based on the mitotic indices at human peripheral lymphocyte cultures were all significantly lower in the case of CuFeO2 NPs. Furthermore, through in vitro albumin denaturation assay, CuFeO2 NPs showed better performance in protein denaturation protection, correlating in superior anti-inflammatory activity than Cu2O and similar to acetylsalicylic acid. Synergy of copper(I)-iron(III) in nanoscale is apparent and gives rise to fruitful bioapplications and perspectives.

Elucidation of one step synthesis of PEGylated CuFe bimetallic nanoparticles. Antimicrobial activity of CuFe@PEG vs Cu@PEG.

There is a growing field of research on the physicochemical properties of bimetallic nanoparticles (BMNPs) and their potential use in different applications. Meanwhile, their antimicrobial activity is scarcely reported, although BMNPs can potentially achieve unique chemical transformations and synergetic effects can be presented. Towards this direction a reproducible simple hybrid polyol process under moderate temperature solvothermal conditions has been applied for the isolation of non-oxide contaminated bimetallic CuFe nanoparticles (NPs). 1,2-propylene glycol (PG), tetraethylene glycol (TEG) and polyethylene glycol (PEG 8000), that exhibit different physicochemical properties, have been utilized to regulate the size, structure, composition and the surface chemistry of NPs. The BMNPs were found to be of small crystalline size, 30-45nm, and high hydrophilicity, different wt% percentage of organic coating and variable hydrodynamic size and surface charge. The antimicrobial activity of the BMNPs was evaluated against the bacterial strains B. subtilis, E. coli and fungus S. cerevisiae. The IC50 values for CuFe NPs were found significantly lower compared with Cu NPs of the same size, revealing an enhancement in the antimicrobial activity when iron and copper coexist in the crystal structure. The reactive oxygen species (ROS) production was measured intracellularly and extracellularly by the nitroblue tetrazolium assay in the fungal cultures. No extracellular ROS were measured suggesting that both CuFe and Cu NPs enter the fungal cells during the incubation, also verified by optical imaging of the fungal cells in the presence of NPs. Higher ROS concentrations were generated intracellularly for CuFe NPs supporting different red/ox reaction mechanisms.

Sex-hormone binding globulin from sheep serum: purification and effects of pregnancy and treatment with exogenous estradiol.

Sex-hormone binding globulin (SHBG) is a protein that binds sex steroids in the serum of many species. SHBG binds androgens and estrogens in humans and primates with high affinity, but behaves as an androgen binding protein in other species. Here we purified SHBG from ewe and ram sera to homogeneity, by a simple and rapid method. The K(D) of the purified protein was found to be 3.63 nM for testosterone and around 600 nM for estradiol. We also studied the effect of pregnancy on SHBG levels in ewes and the effect of exogenous estradiol administration either orally or parenterally on SHBG levels in rams. Basal levels of SHBG in sheep are not affected by pregnancy or exposure to exogenous estradiol. It is concluded that SHBG regulation of expression in ewes and rams differs from that in humans in that it is not affected by estrogen and possibly is species specific.

Chemopreventive activity of very low dose dietary tannic acid administration in hepatoma bearing C3H male mice.

Tannins are plant polyphenols comprising a heterogeneous group of compounds. Tannic acid is a common tannin found in tea, coffee, immature fruits, etc. and it has also been used as a food additive. An increasing body of experimental evidence supports the hypothesis that tannins exert anticarcinogenic activity in chemically induced cancers in animal models. In the present study, tannic acid was administered in very low doses in the drinking water of C3H male mice divided into three groups (75 mg/l, 150 mg/l and 300 mg/l). These animals carry a genetic defect and show a high incidence of spontaneous liver tumors (> 50%) at an age older than 12 months. The results showed a decrease in the overall incidence of hepatic neoplasms (adenomas plus carcinomas): 53.3% of animals in the control group developed hepatic neoplasms versus 33.3% in the group given a low dose of tannic acid, 26.6% in the group given a medium dose and 13.3% in the high dosage group. The difference was more pronounced in the animals with carcinomas: 4.44% of mice who received tannic acid developed carcinomas versus 33.3% of those in the control group. Tannic acid administration did not affect the PCNA labeling index of normal hepatocytes. It is concluded that tannic acid dietary intake in low doses can exert a strong dose-dependent chemoprotective activity against spontaneous hepatic neoplasm development in C3H male mice, most probably through antipromoting mechanisms.

Albumin possesses intrinsic enolase activity towards dihydrotestosterone which can differentiate benign from malignant breast tumors.

Serum albumin was found to possess enolase activity towards the dihydrotestesterone (DHT) molecule, converting it from its 3-keto to 3-enol form. This activity was accompanied by albumin during all stages of purification, as well as following various treatments, a fact indicating that the enzymatic activity was an intrinsic property of albumin molecule and did not represent an impurity of the preparation. Enolase activity was decreased in parallel with the quantity of intact albumin molecules when proteolytic enzymes were used for their degradation. The activity was strongly inhibited by Ni (II) and Cu (II) ions, which bind to 3-histidine of the albumin molecule, as well as by oleic acid and cholesterol. It was also inhibited, in a reversible manner by surface-active agents. Enolase activity was found in all mammalian species studied, the specific activity however was very low in the serum of dogs. The administration of DHT to mice did not influence the albumin or enolase levels in their serum. The optimum pH of enolase was at 9.2, with a carbonate buffer solution. In addition to the serum enolase activity was found to be a feature of intracellular albumin. The two albumins exhibited the same specific activity and the same Km for DHT. The study of cytosolic albumin, obtained from human mammary gland tissue, revealed that benign and malignant tumors of this gland differed substantially with respect to their percentage of albumin. Significant differences were also observed in enolase activity, a consequence of the existence of a fraction of albumin in the malignant tissue in a polymeric form. This form exhibited a decreased enzymatic activity, compared to its monomeric form, exclusively encountered in benign breast specimens. The last observation, along with the quantitative differences of albumin in the two tissues, offers a possibility of reliable differentiation between benign and malignant breast tumors.

Sex hormone binding globulin inhibits strongly the uptake of estradiol by human breast carcinoma cells via a deprivative mechanism.

A controversy exists for many years about the role of sex hormone binding globulin (SHBG) in the uptake of estradiol by the cells. Using the estradiol-sensitive human breast carcinoma cell line MCF-7 and SHBG isolated from human serum by a new method, we observed a strong inhibition of estradiol uptake. The inhibition was higher when the concentration of the hormone was low. On the other hand, there seemed to be a lag period in inhibition when the concentrations of SHBG were very low, followed by an exponential increase, when the concentration exceeded a critical value. The inhibitory activity was higher when SHBG was added before or along with estradiol in the cell culture, as well as when the incubation period was elongated, while was dramatically minimized by the presence of dihydrotestosterone. Despite the inhibition of estradiol uptake caused by SHBG, the distribution of the hormone in various cell components remained practically the same. In conclusion, all indications from experimental data seem to suggest a simple deprivative mechanism being responsible for the inhibitory activity of SHBG on estradiol uptake by MCF-7 cells in culture.

Nonlinear binding of sex steroids to albumin and sex hormone binding globulin.

We have studied the binding of sex steroids to albumin and sex hormone binding globulin (SHBG) using gel filtration chromatography for the separation of the bound from the free fraction of the steroid. It was found that estradiol binds to the globulin and albumin in a nonlinear manner: a lag period of binding was observed at low concentrations of the proteins, followed by an exponential increase of the bound hormone as the protein concentration increased. The same was observed with dihydrotestosterone (DHT) and albumin but not with globulin. In the presence of a constant concentration of albumin, the increase of SHBG concentrations resulted in a rapid transfer of estradiol from albumin to globulin while the transfer of DHT was moderate. When whole serum was used, the increase of its amount again resulted in the transfer of estradiol from albumin to globulin. Our study showed that a substantial increase of globulin-bound hormone can occur, following small variations of the protein. This offers obvious advantages to the organism, by saving energy, material and time and plays a basic role in estradiol transfer from albumin to the much more biologically active globulin.

Ribonucleases protect RNA from acid precipitation.

Four, widely used, ribonucleases were found to protect their substrates from acid precipitation by causing, evidently, a modification of their physicochemical properties. The protection was dependent on the kind of substrate while the ratio of protective to nucleolytic activity varied widely between the four enzymes. The protection was enhanced by some nucleotides like UMP, CMP and IMP and decreased in the presence of several bivalent ions like Zn++, Co++ and Cu++. It was completely abolished when the substrates were hybridized with their complementary ribohomopolymers. In the case of bovine pancreatic ribonuclease, the part of the molecule which was responsible for the protective activity was localized on the enzyme domain characterized as S-protein, which lacks nucleolytic activity. The observed property of ribonucleases could lead to false data when the measurement of TCA-soluble material is the method used to follow the purification of ribonucleases or to study their activity. It was also found that ribonuclease S-protein enhances the catalytic activity of B. Cereus RNAse. S-protein could potentiate other RNAses activity like onconase, which has recently been used as an anticancer agent.

Enzymatic conversion of dihydrotestosterone from 3-keto to 3-enol form in the rat prostate.

Dihydrotestosterone (DHT) is the active androgen, as well as a strong tumor promoter in the prostate, where several enzymes are essential for the regulation of its activity. We localized four enzymes promoting the enolization of the 3-keto group of DHT in rat prostate. The enzymes were purified by ion-exchange chromatography, acetone fractionation and gel filtration to homogeneity, and found to have molecular sizes of 19.5, 22.0, 44.5 and 21.5 kDa. A partial characterization of the four enzymes revealed that their structure consisted of a common chain of 14.5 kDa with various subunits which differentiate the four enzymes from each other. All the enzymes exerted their activity only on 5-dihydro 3-keto steroids. The total enzymatic activity was strongly influenced by animal age, being very low before sexual maturation, as well as after castration. In the latter case the level of total activity fell to about 8% control animals. Activity was also estimated in human, pork, ram and bovine prostate and it was found that all these species have 20-25 times lower enzyme levels than rat. These results, in combination with the practically exclusive localization of the enzymes in the prostate, suggest a role relating to the bioavailability of DHT in this gland.

Characterization of the protein protecting RNA from Acid precipitation and its evaluation as tumor-marker in gastrointestinal and gynecological cancer.

In a previous study, we observed increased protective activity against acid precipitation of poly (U) in the serum of tumor bearing mice and hepatoma patients. In the present study we purified and partially characterized the protein responsible for this phenomenon and measured the activity in the serum of patients with various cancers. Evidence is presented that this protein forms a complex with serum ribonucleases which results in the inactivation of enzymes and protection of RNA from acid precipitation. The serum protective activity against acid precipitation of poly (U) was measured in 37 patients with gastrointestinal cancer and 79 with gynecological cancer, along with 15 male subjects with nonmalignant diseases of the gastrointestinal system, 15 with uterine leiomyoma, 19 healthy men and 30 healthy women from the hospital personnel. The mean values found in patients with various tumors were about three times higher than those found in healthy individuals, while no significant overlapping of values was observed between cancer patients and controls. On the other hand, the activity was not increased in patients with nonmalignant diseases of gastrointestinal system and uterus.

Value of serum protective activity against acid precipitation of poly (U) in the differentiation between cirrhotic patients with and without hepatocellular carcinoma.

The serum protective activity against acid precipitation of poly (U) and a-fetoprotein levels were compared in 39 cirrhotic patients with hepatocellular carcinoma (HCC) and in 33 patients with chronic liver disease (CLD) alone, in order to differentiate malignant and nonmalignant chronic liver disease. All but one (97.4%) patients with HCC were found to have serum protective activity levels of greater than or equal to 21 micrograms/ml, whereas all but one (97%) patients with CLD had serum protective activity levels of less than or equal to 20 micrograms/ml. Mean serum protective activity levels were significantly higher in the HCC group than in those with CLD (p less than 0.0001). Serum a-fetoprotein concentrations of over 500 ng/ml, suggesting malignancy, were observed in 54% of patients with HCC and in 15% of patients with CLD. Application of the best discriminating values for protective activity (greater than 21 micrograms/ml) and for a-fetoprotein (greater than 500 ng/ml) to 72 patients with or without HCC revealed an efficiency of 97.2% for protective activity and only 68.1% for a-fetoprotein.

Increased protective activity against acid precipitation of poly(U) in the serum of tumor-bearing mice.

Transplantation of leukemia L1210 cells into DBA/2 mice and of Ehrlich ascites tumor cells into BALB/C mice resulted in a significant increase of protective activity against acid precipitation of poly (U) in the serum. The increase was observed as early as one day after the tumor transplantation and seems to be connected with cancer growth, since inoculation of L1210 cells into BALB/C mice did not affect the protective activity, evidently as a result of their well established inability to cause cancer in this strain. Furthermore, no increase of activity was observed when bacteria were inoculated into mice, or when the latter were partially hepatectomized. The results suggest that the protective activity against acid precipitation of poly (U) could prove to be a tumor marker for the early detection of cancer growth.

Sialyltransferase and nucleoside diphosphatase as markers for tumor monitoring.

In this study we report serum sialyltransferase and nucleoside diphosphatase activities of patients with malignant tumors of various primary sites and extent, prior to and during chemotherapy. Enzyme levels were compared to clinical and laboratory parameters. The sialyltransferase and uridine diphosphatase (UDPase) activities in samples of 43 patients with advanced ovarian cancer was four to ten fold above the normal mean value (sialyltransferase 85.1 +/- 58 pmol/hr/ml and UDPase 26.6 +/- 7.2 nmol/hr/ml). After effective chemotherapy with adriamycin and cisplatin, the enzyme activity decreased markedly. In cases of complete remission, enzyme activity decreased to the normal range. In three cases after initial response for several months a rise of both enzymes was observed before any other biochemical finding of the forthcoming relapse. Similar patterns were observed in testicular cancer (6 cases). Clinical correlation is also obvious in other tumors except malignant lymphomas. Our findings show that the activities of these enzymes correlated with the clinical course, and therefore they can be the basis for clinical application for tumor monitoring, especially during chemotherapy.

Studies on the effect of homo-aza-steroidal ester of p-bis (2-chloroethyl) amino phenyl acetic acid in Ehrlich ascites tumor cells.

The effect of a homo-aza-steroidal ester (ASE) on the incorporation of radioactive precursor to DNA of Ehrlich ascites tumor (EAT) cells has been investigated. We found that treatment of cells with 80 micrograms/ml of ASE for 2 hours causes an inhibition of the incorporation of 3H-thymidine to DNA by 71%. This is partly because ASE affects the radioactive thymidine pool in the cell. The DNA from EAT cells after centrifugation in CsCl is shifted to higher densities when ASE is present throughout the experiment. This density shift was not observed when ASE was incubated only in the growth medium of the cells.

Poly(A) polymerases of rat liver nuclei. Purification and specificity.

Two poly(A) polymerases were isolated from rat liver nuclei and purified more than one thousand times by ion exchange chromatography on DEAE-Sephadex and phosphocellulose columns as well as affinity chromatography on a chromosomal RNA-Sepharose column. One of the two enzymes is bound to chromatin and uses as primer chromosomal RNA, while the second one is localized in the nucleoplasm and uses as primer poly(A) and hnRNA isolated from chromatin. The two enzymes seem to participate in the polyadenylation of chromosomal RNA in vitro, by a coupled mechanism. According to this mechanism, the chromatin bound enzyme adds 120-130 adenosine nucleotides to chromosomal RNA and consequently the nucleoplasmic enzyme completes the poly-adenylation by adding 80-90 more AMP units to the polyadenylated end of chromosomal RNA.