Evaluation of the Hemocompatibility and Anticancer Potential of Poly(ε-Caprolactone) and Poly(3-Hydroxybutyrate) Microcarriers with Encapsulated Chrysin.

In this work, novel chrysin-loaded poly(ε-caprolactone) and poly(3-hydroxybutyrate) microcarriers were synthesized according to a modified oil-in-water single emulsion/solvent evaporation method, utilizing poly(vinyl alcohol) surfactant as stabilizer and dispersing agent for the emulsification, and were evaluated for their physico-chemical and morphological properties, loading capacity and entrapment efficiency and in vitro release of their load. The findings suggest that the novel micro-formulations possess a spherical and relatively wrinkled structure with sizes ranging between 2.4 and 24.7 µm and a highly negative surface charge with z-potential values between (-18.1)-(-14.1) mV. The entrapment efficiency of chrysin in the poly(ε-caprolactone) and poly(3-hydroxybutyrate) microcarriers was estimated to be 58.10% and 43.63%, whereas the loading capacity was found to be 3.79% and 15.85%, respectively. The average release percentage of chrysin was estimated to be 23.10% and 18.01%, respectively. The novel micromaterials were further biologically evaluated for their hemolytic activity through hemocompatibility studies over a range of hematological parameters and cytoxicity against the epithelial human breast cancer cell line MDA-MB 231. The poly(ε-caprolactone) and poly(3-hydroxybutyrate) microcarriers reached an IC50 value with an encapsulated chrysin content of 149.19 µM and 312.18 µM, respectively, and showed sufficient blood compatibility displaying significantly low (up to 2%) hemolytic percentages at concentrations between 5 and 500 µg·mL-1.

In vitro cellular toxicity induced by extractable organic fractions of particles exhausted from urban combustion sources - Role of PAHs.

The bioactivity of the extractable organic matter (EOM) of particulate matter (PM) exhausted from major urban combustion sources, including residential heating installations (wood-burning fireplace and oil-fired boiler) and vehicular exhaust from gasoline and diesel cars), was investigated in vitro by employing multiple complementary cellular and bacterial assays. Cytotoxic responses were investigated by applying the MTT ((3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide)) bioassay and the lactate dehydrogenase (LDH) release bioassay on human lung cells (MRC-5). Sister Chromatids Exchange (SCE) genotoxicity was measured on human peripheral lymphocytes. Lipid peroxidation potential via reactive oxygen species (ROS) was evaluated on E. coli bacterial cells by measuring the malondialdehyde (MDA) end product. Furthermore, the DNA damage induced by the organic PM fractions was evaluated by the reporter (ß-galactosidase) gene expression assay in the bacterial cells, and, by examining the fragmentation of chromosomal DNA on agarose gel electrophoresis. The correlations between the source PM-induced biological endpoints and the PM content in polycyclic aromatic hydrocarbons (PAHs), as typical molecular markers of combustion, were investigated. Fireplace wood smoke particles exhibited by far the highest content in total and carcinogenic PAHs followed by oil boilers, diesel and gasoline emissions. However, in all bioassays, the total EOM-induced toxicity, normalized to PM mass, was highest for diesel cars equipped with Diesel Particle Filter (DPF). No correlation between the toxicological endpoints and the PAHs content was observed suggesting that cytotoxicity and genotoxicity are probably driven by other extractable organic compounds than the commonly measured unsubstituted PAHs. Clearly, further research is needed to elucidate the role of PAHs in the biological effects induced by both, combustion emissions, and ambient air particles.

Toxic organic substances and marker compounds in size-segregated urban particulate matter - Implications for involvement in the in vitro bioactivity of the extractable organic matter.

Toxic organic substances and polar organic marker compounds, i.e. polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), polybrominated diphenyl ethers (PBDEs), polycyclic aromatic hydrocarbons (PAHs) and their nitro-derivatives (N-PAHs), as well as dicarboxylic acids (DCAs) and sugars/sugar anhydrites (S/SAs) were analyzed in size-segregated PM samples (<0.49, 0.49-0.97, 0.97-3 and >3 µm) collected at two urban sites (urban traffic and urban background) during the cold and the warm season. The potential associations between the organic PM determinants and the adverse cellular effects (i.e. cytotoxicity, genotoxicity, DNA damage, oxidative DNA adduct formation, and inflammatory response) induced by the extractable organic matter (EOM) of PM, previously measured in Velali et al. (2016b), were investigated by bivariate correlations and Principal Component Analysis (PCA). Partial Least Square regression analysis (PLS) was also employed in order to identify the chemical classes mainly involved in the EOM-induced toxicological endpoints in the various particle size fractions. Results indicated that particle size range <0.49 µm was the major carrier of PM mass and organic compounds at both sites. All toxic organic compounds exhibited higher concentrations at the urban traffic site, except PCBs and OCPs that did not exhibit intra-urban variations. Conversely, wintertime levels of levoglucosan were significantly higher at the urban background site as a result of residential biomass burning. The PLS regression analysis allowed quite good prediction of the EOM-induced cytotoxicity and genotoxicity based on the determined organic chemical classes, particularly for the finest size fraction of PM. Nevertheless, it is expected that other chemical constituents, not determined here, also contribute to the measured toxicological responses.

Genotoxic effect of tocolytic drug ritodrine in combination with smoking during pregnancy.

OBJECTIVE: Tocolytic drugs are used widely in order to prevent preterm birth. Ritodrine, is the only food and drug administration (FDA) approved drug for tocolytic use. We estimated the cytogenetic effect of ritodrine administered as maternal therapy, alone or in combination with smoking, in women and their neonates. METHODS: Lymphocyte and fibroblasts cultures were evaluated and three indices were analyzed; sister chromatid exchanges (SCEs), proliferation rate index (PRI) and mitotic index (MI) as well as average generation time (AGT) and population doubling time (PDT). Campothacin (CPT-11) was used as a positive control. RESULTS: Administration of ritodrine up to a month revealed significant reduction of SCEs/cell in neonates in the presence or absence of the mutagenic agent. A statistical significant increase on SCEs, for mothers and neonates, was noticed in neonate's lymphocytes when tocolytic therapy was over a month. Ritodrine revealed a cytoprotective action against smoking when the two factors were combined, but the synergistic action of ritodrine with smoking increased genotoxicity, cytostaticity and cytotoxicity of neonates after long administration (1-3 months). CONCLUSIONS: The time-depended genotoxic, cytostatic and cytotoxic action of ritodrine alone or in combination with smoking suggests that its administration should not exceed the time period of a month.

Effect of maternal smoking during pregnancy on fetus: a cytogenetic perspective.

OBJECTIVE: The examination of the genotoxic, cytostatic and cytotoxic effects of smoking during pregnancy. METHOD: Lymphocyte cultures of peripheral blood were received from 20 women who smoked during pregnancy as well as umbilical cord blood of their newborns. Fluorescence Plus Giemsa staining technique was used in order to perform cytogenetic analyses for three indices, Sister Chromatid Exchanges (SCEs), Proliferation Rate Index (PRI) and Mitotic Index (MI). To reveal any underlying chromosome instability, CPT-11 was used as a positive control. RESULTS: Newborns whose mothers smoke during pregnancy had increased SCEs levels on their lymphocytes when they were exposed to the mutagenic agent CPT-11 (p < 0.01) compared with newborns lymphocytes exposed to the same agent with non-smoking mothers. Also, mothers smoking during pregnancy had increased SCE levels when their lymphocytes were exposed to CPT-11 (p < 0.01) compared with non smoking mothers whose lymphocytes were exposed to the same agent. In both groups newborns appeared as having decreased (p < 0.01) spontaneous SCEs levels compared with the corresponding SCE rates of their mothers. Decreases of PRIs and MIs are observed in mothers compared to their newborns. CONCLUSION: Smoking during pregnancy can promote cytogenetic damage in newborn's DNA, causing chromosome instability. The clinical importance of this indirect damage lies in the fact that this type of damage can act synergistically with other environmental and/or chemical mutagenic substances possibly leading to carcinogenicity.

Cytogenetic evaluation of pre-pregnancy smoking in maternal and newborn lymphocytes.

OBJECTIVE: To study cytogenetic damage in order to estimate the effect of pre-pregnancy smoking on pregnant women and their foetuses. STUDY DESIGN: Lymphocyte cultures were obtained from peripheral blood of 20 women who quit smoking during pregnancy, and umbilical cord blood of their newborns at delivery. Cytogenetic analyses were performed for sister chromatid exchanges (SCEs), proliferation rate index (PRI) and mitotic index (MI) using the Fluorescence Plus Giemsa staining technique. Twenty non-smoking women and their newborns were evaluated as controls. CPT-11, a known antineoplastic, was used as a positive genotoxic agent in order to correlate non-smoking women with smoking women and reveal any underlying chromosome instability. Statistical evaluation of SCE frequencies, PRI and MI was based on independent samples t-test in order to estimate the effect of pre-pregnancy smoking on mothers and their newborns. RESULTS: SCEs were induced in the cord blood lymphocytes of newborns whose mothers smoked before pregnancy when they were exposed to the mutagenic agent CPT-11 (p<0.01). A similar increase in SCEs was observed in both non-smoking and smoking mothers exposed to CPT-11. Newborns in both groups had significantly lower SCE levels than their mothers (p<0.01). CONCLUSION: Pre-pregnancy smoking results in cytogenetic damage for both mothers and newborns, and is an important risk factor for cancer and/or other genetic-related diseases. Smoking cessation needs to occur well before conception in order to avoid the strong cytogenetic association between pre-pregnancy smoking by mothers and their newborns.

Synergistic cytogenetic and antineoplastic effects by the combined action of esteric steroidal derivatives of nitrogen mustards.

We studied the effect of five newly synthesized steroidal derivatives of nitrogen mustards. These derivatives have as alkylators either P-N, N-bis(2-chloroethyl)aminophenyl-butyrate (CHL) or P-N, N-bis(2-chloroethyl)aminophenyl-acetate (PHE) groups esterified with different modified steroidal nuclei. We examined them alone or in combination, on sister chromatid exchange rates and on human lymphocyte proliferation kinetics. The antitumor activity of these compounds, alone or in combination, was also tested on Leukemia P388-bearing mice. A pronounced cytogenetic and antineoplastic action was demonstrated by the compounds that contain either PHE or CHL as alkylators and are esterified with a steroidal nucleus having added a cholestene group in the 17 position of the D-ring. The exocyclical insertion of an -NHCO- group in the D-ring of the steroidal nucleus esterified with PHE (amide ester of PHE) yielded a compound demonstrating a distinct cytogenetic and antineoplastic effect. In contrast, the ketone group in the D-ring being inserted endocyclically in the steroidal nucleus (androstene) esterified with either CHL or with PHE gave negative cytogenetic and antineoplastic effects. However, the combined action of cholestene esterified with either CHL or with PHE in combination with either the androstene ester of PHE or with the androstene ester of CHL, respectively, gave synergistic cytogenetic and antineoplastic effects. Also the amide ester of PHE in combination with the androstene ester of CHL gave distinct cytogenetic and antineoplastic effects in a synergistic manner.

Expression of 6 common antigenic markers in invasive ductal breast carcinoma: potential clinical implications.

Expression of estrogen (ER) and progesterone receptors, c-erbB-2 oncogene, mutant p53 antioncogene (mp53), e-cadherin adhesion, and apoptotic caspase-8 antigens in tumor relative to matched normal tissue specimens from 102 unselected patients with primary ductal breast carcinoma of various tumor grades was assessed by immunohistochemistry and correlated with patient's biologic and clinical features, such as age, menstrual status, age of menarche, tumor grade and diameter, the presence or absence of metastases, and number of infiltrated lymph nodes. We observed association of e-cadherin adhesion, ER and progesterone antigen marker expression with low histologic grade tumors and limited number of lymph node metastases and of c-erbB-2, mp53, and casp-8 antigen marker expression with high histologic grade tumors and increased number of lymph node metastases. We also observed strong correlation (P<0.05) between 4 of the 6 biomarkers and 4 of the 7 patient/tumor parameters examined. Our findings support the hypothesis of independent expression of these 4 strong biomarkers and reveal that nearly 40% of all breast tumor cases studied express similar proportions of 2 major phenotypic combinations [ER/c-erbB-2/mp53/casp-8: +/+/-/+ (19.6%) & +/-/-/+ (17.8%)]. We conclude that, in agreement with earlier reports, our findings support the diagnostic and potential prognostic value of these markers in the clinical assessment of breast cancer.

Cytogenetic evaluation and DNA interaction studies of the food colorants amaranth, erythrosine and tartrazine.

Food coloring agents, amaranth, erythrosine and tartrazine have been tested at 0.02-8mM in human peripheral blood cells in vitro, in order to investigate their genotoxic, cytotoxic and cytostatic potential. Amaranth at the highest concentration (8mM) demonstrates high genotoxicity, cytostaticity and cytotoxicity. The frequency of SCEs/cell was increased 1.7 times over the control level. Additionally, erythrosine at 8, 4 and 2mM shows a high cytotoxicity and cytostaticity. Finally, tartrazine seems to be toxic at 8 and 4mM. No signs of genotoxicity were observed. Reversely, tartrazine showed cytotoxicity at 1 and 2mM. Furthermore, spectroscopic titration studies for the interaction of these food additives with DNA showed that these dyes bind to calf thymus DNA and distinct isosbestic points are observed clearly suggesting binding of the dyes to DNA. Additionally DNA electrophoretic mobility experiments showed that these colorants are obviously capable for strong binding to linear dsDNA causing its degradation. PCR amplification of all DNA fragments (which previously were pre-treated with three different concentrations of the colorants, extracted from agarose gel after separation and then purified), seems to be attenuated with a manner dye concentration-dependent reflecting in a delayed electrophoretic mobility due to the possible binding of some molecules of the dyes. Evaluation of the data and curves were obtained after quantitative and qualitative analysis of the lanes of the gel by an analyzer computer program. Our results indicate that these food colorants had a toxic potential to human lymphocytes in vitro and it seems that they bind directly to DNA.

Cytogenetic damage after ischemia and reperfusion.

Tourniquets are often used to provide a bloodless operating field. However, they carry the risk of adverse effects caused by DNA damage from the free radicals generated during postischemic reperfusion of the blood. The aim of this study was to evaluate the cytogenetic damage caused by postischemic reperfusion on peripheral lymphocytes of five women and six men undergoing total knee arthroplasty "bloodless" operation using samples received before, during, immediately, and 1 h after the operations. The sister chromatid exchange assay was applied to peripheral blood lymphocyte cultures and the levels of sister chromatid exchanges were analyzed as a quantitative index of genotoxicity, along with the values of mitotic index and proliferation rate index as qualitative indices of cytotoxicity and cytostaticity, respectively. We observed that postischemic reperfusion induced cytogenetic damages specifically through reperfusion. DNA effects were most pronounced after tourniquet release and declined afterward without returning to preischemic baseline values. Our findings suggest the presence of a functional association between postischemic reperfusion and cytogenetic damage that may have important clinical implications.

Chromosome instability on children with asthma.

BACKGROUND: Asthma is a complex disease with multiple interactions between genetic and environmental factors. OBJECTIVE: The aim of our study was to investigate the possible genetic instability in asthmatic patients (AP) with asthma in human cultured peripheral blood lymphocytes. Furthermore, the presence of either cytostaticity or cytotoxicity was demonstrated. METHODS: Human peripheral blood lymphocytes were cultured from 18 admitted children to the Pediatric Clinic of the University Hospital of Alexandroupolis (average age 7.2 years), and 9 healthy blood donors were used as control subjects (average age 6.5 years), none of whom was receiving drugs for medical or other reasons. RESULTS: A significant (p < 0.05) increase in spontaneous sister chromatid exchanges (SCEs) frequency in asthmatic patients compared with control subjects was observed. No statistically significant modification in the spontaneous proliferation rate index (PRI) in AP compared with the controls was demonstrated. Finally, MMC induced a statistically significant increase in SCEs frequency both to controls and to AP, with the MMC-induced SCEs rates in AP being statistically (p < 0.01) higher compared to the MMC-induced SCEs in controls. CONCLUSION: We try to improve a new diagnostic process of possible genetic instability by a combination of genotoxic, cytostatic and cytotoxic effects of asthma on human peripheral lymphocytes.

Antineoplastic and cytogenetic effects of chlorpromazine on human lymphocytes in vitro and on Ehrlich ascites tumor cells in vivo.

The inhibitory effect of phenothiazines in tumor growth and cancer cell proliferation in vitro and in vivo has been established. These reports motivated us to investigate the genotoxic, cytotoxic, and cytostatic potential of chlorpromazine, alone or in combination with mitomycin C, in vitro and in vivo. Sister chromatid exchange levels were assessed providing a quantitative index of genotoxicity. In-vitro studies were performed on human lymphocyte cultures and in-vivo studies involved Ehrilch ascites tumor (EAT) cells. An antitumour study was also conducted on the survival time and the ascitic volume in EAT-bearing Balb/C mice. The combination of chlorpromazine plus caffeine and mitomycin C exerted cytostatic and cytotoxic actions in human lymphocytes. The combination of chlorpromazine plus mitomycin C exerted cytostatic and cytotoxic actions in EAT cells, significantly increased the survival span of the mice inoculated with EAT cells, and suppressed the expected tumor growth increase. The findings of this basic study illustrate that high chlorpromazine concentrations increase chemotherapeutic effectiveness of mitomycin C. Chlorpromazine concentrations within the observed human plasma concentration range need to be tested along with antineoplastic agents in vitro for its synergistic action so as to evaluate a potential clinical application. Further investigation including other phenothiazines, biological systems, and cancer models is required.

Supplementation of melatonin protects human lymphocytes in vitro from the genotoxic activity of melphalan.

Melatonin (MLT) is a natural oncostatic factor of the human body as well as an antioxidant thus protects the nuclear DNA from oxidative damage. It also has the ability to reduce the side effects of various drugs when used as a combination therapy. The anti-neoplastic agent melphalan (MEL), which encompasses a number of side effects, is a strong alkylating agent and a potent inducer of sister chromatid exchanges (SCEs). The aim of the current in vitro study was to investigate the ability of MLT to reduce the genotoxic effect of MEL on normal human cultured peripheral lymphocytes. Cells were treated with both agents at various concentrations (MLT 100, 200 and 400 microM and MEL 330, 490 and 650 nM) and incubated for 72 h prior harvesting. The levels of cytostaticity, cytotoxicity and genotoxicity were qualitatively evaluated using the proliferation rate index, the mitotic index and the SCE methodology, respectively. Our results demonstrated the protective effect of MLT on cells treated with MEL in vitro. The greatest protective effect of MLT at 100 and 400 microM was illustrated against 330 nM of MEL in comparison with all other doses of MEL. These observations imply that MLT may be proved useful in reducing some of the toxic effects associated with certain classes of chemotherapeutic agents and other chemical and physical mutagens and carcinogens, acting both as an antioxidant-radical scavenger and a protective mechanism against cellular damage due to exposure to free radical-producing agents. It is essential to investigate substances with protective properties which are normally produced from the human body.

DNA damage and chromosomal aberrations in various types of male factor infertility.

OBJECTIVE: To understand and delineate the nature and level of DNA damage in association to semen parameters in infertile men. DESIGN: A prospective experimental study. SETTING: Alexandroupolis University General Hospital. PATIENT(S): Eleven fertile and 27 infertile men with various types of infertility. INTERVENTION(S): DNA damage was induced by addition of mitomycin C and caffeine to lymphocyte cultures. MAIN OUTCOME MEASURE(S): Sister chromatid exchange (SCE) levels were assessed in cultures providing a quantitative index of genotoxicity and chromosomal analysis was performed using G-banding and C-banding techniques. RESULT(S): Karyotyping analysis indicated chromosomal fragility, trisomic lines, and marker chromosomes in some infertile men. Double minute chromosomes were noticed in 11 infertile men and were positively correlated with elevated SCE levels. Necrospermia and varicocele, irrespectively of the degree of severity, were positively correlated with elevated SCE levels. CONCLUSION(S): Infertile men are prone to have DNA damage; the nature and level of DNA damage varies and is associated with semen parameters. The presence of double minute chromosomes alone is associated with increased double-stranded breaks and abnormal sperm concentration. This study could provide the basis to establish whether and through which process double minute chromosomes could be related to poor semen parameters and regulation of DNA repair.

Evidence of increased chromosomal instability in infertile males after exposure to mitomycin C and caffeine.

AIM: To evaluate the genetic instability of 11 fertile and 25 infertile men. METHODS: The methodology of sister chromatid exchanges (SCEs) was applied to cultures of peripheral blood lymphocytes, and the levels of SCEss were analyzed as a quantitative index of genotoxicity, along with the values of the mitotic index (MI) and the proliferation rate index (PRI) as qualitative indices of cytotoxicity and cytostaticity, respectively. The genotoxic and antineoplastic agent, mitomycin C (MMC), and caffeine (CAF)--both well-known inhibitors of DNA repair mechanism--were used in an attempt to induce chromosomal instability in infertile men, so as to more easily detect the probable underlying damage on DNA. RESULTS: Our experiments illustrated that infertile men, compared with fertile ones, demonstrated a statistically significant DNA instability in peripheral blood lymphocytes after being exposed simultaneously to MMC and CAF. CONCLUSION: The current study showed vividly that there was genetic instability in infertile men which probably contributes to the development of an impaired reproductive capacity.