Critical evaluation of the use of total reflection X-ray fluorescence spectrometry for the analysis of whole blood samples: application to patients with thyroid gland diseases.

Multielemental analysis of whole blood can provide significant information for the evaluation of nutritional status and diagnosis of certain diseases as well as for the assessment of exposure to potentially toxic metals. However, the quantification of multiple elements in whole blood is not easy partly because of the wide variation in element concentrations (from ng L-1 to g L-1) and the complex matrix. The aim of this work was to develop a fast, sustainable, and reliable analytical method, in combination with low-power TXRF, for multielemental analysis of blood samples. Firstly, a set of experiments were carried out to select the best diluent type and dilution factor using the control material SeronormTM Trace Elements Whole Blood L-1. A critical evaluation of the parameters affecting the sample deposition on the reflector was also carried out including a study of the shape and element distribution of the deposited residue on the reflector by micro X-ray fluorescence spectrometry. Using the best analytical conditions, limits of detection estimated were in the low milligrams per kilogram range and similar to those obtained using more complex sample treatments such as digestion. Accuracy and precision of the results were in most cases acceptable (recoveries 89-102%, RSD 6-8%, n = 5). Only underestimated values were obtained for light elements such as potassium. To prove the applicability of the method, several blood samples from control and thyroid disease patients were analyzed. Despite the fact that more samples need to be analyzed, it seems that Zn and Br contents in some of the patients are significantly higher compared to control samples. Graphical abstract.

The effect of apigenin on cyclophosphamide and doxorubicin genotoxicity in vitro and in vivo.

The aim of this study was to investigate the mutagenic and antigenotoxic effects of different doses of the flavonoid, apigenin, alone and in combination with the antitumor drugs, cyclophosphamide and doxorubicin, in vitro and in vivo. Using bacterial reverse mutation inhibition in vitro, with and without metabolic activation, the effect of apigenin (10-400 µg/plate) was studied on genotoxicity induced by cyclophosphamide (800 µg/plate) and by doxorubicin (0.2 µg/plate). Subsequent to a dose-finding study in vivo, CD1 mice were treated with either cyclophosphamide (40 mg/kg, i.p.) or doxorubicin (5 mg/kg, i.p.) with or without co-administration of apigenin (1-100 mg/kg, p.o.). Micronuclei were determined microscopically in blood smears and glutathione peroxidase (GPX), superoxide dismutase (SOD) and total antioxidative status (TAS) in whole blood, erythrocytes and plasma, respectively. Apigenin decreased doxorubicin-induced, but not cyclophosphamide-induced mutagenicity in vitro. In vivo, apigenin caused a statistically significant decrease in micronucleus frequency in response to cyclophosphamide, possibly due to active flavonoid metabolite formation or inhibition of cyclophosphamide metabolic activation. In animals treated with apigenin and doxorubicin, a significant decrease in micronucleus frequency was not observed, probably due to interindividual variability. No changes in GPX, SOD or TAS were observed in response to either cytotoxic agents or the flavonoid, possibly due to limited metabolic transformation of the drugs at the doses used. The results of the present study provide further evidence for the chemo-preventative properties of apigenin.

Ganglioside GT1b protects human spermatozoa from hydrogen peroxide-induced DNA and membrane damage.

We have reported previously that various gangliosides, the sialic acid containing glycosphingolipids, provide protection against sperm injury caused by reactive oxygen species (ROS). In this study, we investigated the effect of treatment of human spermatozoa with ganglioside GT1b on hydrogen peroxide (H(2)O(2))-induced DNA fragmentation and plasma membrane damage. Single-cell gel electrophoresis (Comet assay) used in the assessment of sperm DNA integrity showed that in vitro supplemented GT1b (100 microm) significantly reduced DNA damage induced by H(2)O(2) (200 microm) (p < 0.05). Measurements of Annexin V binding in combination with the propidium iodide vital dye labelling demonstrated that the spermatozoa pre-treated with GT1b exhibited a significant increase (p < 0.05) in the percentage of live cells with intact membrane and decreased phosphatidylserine translocation after exposure to H(2)O(2). Flow cytometry using the intracellular ROS-sensitive fluorescence dichlorodihydrofluorescein diacetate dye employed to investigate the transport of the extracellularly supplied H(2)O(2) into the cell interior revealed that ganglioside GT1b completely inhibited the passage of H(2)O(2) through the sperm membrane. These results suggest that ganglioside GT1b may protect human spermatozoa from H(2)O(2)-induced damage by rendering sperm membrane more hydrophobic, thus inhibiting the diffusion of H(2)O(2) across the membrane.

DNA and cytogenetic damage in white blood cells of postmenopausal breast cancer patients treated with radiotherapy.

The primary and residual genome damage and its elimination rate were evaluated in peripheral blood lymphocytes of breast cancer patients treated with adjuvant radiotherapy after surgical removal of the tumor by mastectomy or quadrantectomy. The levels of DNA/chromosome damage were estimated before, throughout, as well as after six months, respectively one year after the radiotherapy, using the alkaline comet assay, the chromosome aberration analysis and the cytokinesis-block micronucleus assay. The marked individual differences in the baseline genome damage were observed in patients, which additionally increased until the end of the radiotherapy cycle. The levels of DNA/cytogenetic damage slowly declined during post-irradiation period; although in the majority of subjects they did not return to pre-therapy levels. In addition to the well-established comet parameters, the long-tailed nuclei were also proved as a useful indicator of individual DNA damage and response to radiation. One of the most important observation was that older breast cancer patients, irradiated after mastectomy, had higher values of almost all parameters evaluated. We found positive correlations between the comet assay parameters and the cytogenetic biomarkers that confirmed their complementary value in the assessment of the radiation sensitivity/susceptibility in elderly breast cancer patients. The specific patterns of DNA damage observed in the majority of subjects after a prolonged exposure to ionizing radiation indicate the possibility of adaptive response. Such results may also be linked to the hormesis theory and support previous observations, but the underlying mechanisms should be further investigated on a much larger population.

Cytogenetic follow-up in testicular seminoma patients exposed to adjuvant radiotherapy.

Early stage testicular seminoma is a radiosensitive tumor. Its incidence has significantly increased during the last decade especially in the young population. Although the therapy for testicular seminoma gives very satisfying results, the evaluation of genome damage caused by the therapy is of a great importance in order to recognize possible related health risks. The present study was performed on ten patients diagnosed with seminoma stage I; pT1/2N0M0S0, treated with adjuvant radiotherapy (a radiation dose of 25 Gy divided in 16 fractions) after orchidectomy. To assess the possible existence of an increased baseline DNA/chromosome damage in patients we also selected the appropriate control group often healthy men. The levels of primary DNA/chromosome damage in peripheral blood lymphocytes, as well as the dynamics of their repair were studied using the alkaline comet assay, chromosome aberration and cytokinesis-block micronucleus assay. Altogether four blood samples per patient were collected in the course of the therapy: before and after receiving the first dose of radiotherapy, in the middle of the radiotherapy cycle, and after the last dose of radiotherapy. Other two follow-up blood samples were collected six and twelve months after the cessation of therapy. As observed, the administration of the first radiation dose significantly increased the levels of DNA damage in almost all patients compared to their baseline values. Specific patterns of DNA damage were recorded in samples analyzed in the middle of radiotherapy and after receiving the last dose, indicating the possibility of an adaptive response in some patients. The levels of chromosomal aberrations and the incidence of micronuclei also increased in the course of therapy but gradually declined during the follow-up period. Our results confirmed the existence of post-irradiation damage in peripheral blood lymphocytes (and possibly in other non-target cells) of cancer patients which may represent a risk for the secondary cancer development. Considering that the majority of patients with testicular cancer are of a younger age, they represent a population deserving special attention. As cytogenetic screening may detect high-risk individuals, it might be useful in regular medical monitoring of seminoma patients after the successful therapy.

A survey on the cytogenetic status of the Croatian general population by use of the cytokinesis-block micronucleus assay.

The cytokinesis-block micronucleus assay (CBMN) was used to assess the variability and determine possible influences of external and internal factors on the background levels of cytogenetic damage in peripheral blood lymphocytes (PBL) of 50 healthy volunteers selected at random from the general population of Croatia. The mean MN frequency for all subjects was 4.74+/-0.31 per 1000 cells and the mean cytokinesis-block proliferation index (CBPI) was 1.82+/-0.01. The mean frequency of nucleoplasmic bridges (NPB) for all subjects was 0.06+/-0.04 and of nuclear buds (NB) 0.12+/-0.05. The canonical correlation analyses indicate a positive non-significant correlation between the MN frequency and age, gender and smoking habits. Results of factor structure and canonical weights showed that age and gender rather than smoking habits control the incidence of MN in PBL of healthy volunteers. The lowest median value of MN was observed in subjects younger than 30 years (both smoking and non-smoking). Generally, non-smokers had lower median values of MN compared to smokers. In non-smokers, males showed lower micronucleus incidence than females. Within the non-smokers smaller differences in the median values of MN between subgroups (male and female; age subgroups) were observed. Among smokers, females had a two-fold higher median value of MN frequency than males, but this difference was not significant (p=0.2643, Mann-Whitney U test). Canonical correlation analyses indicate a strong and significant correlation between cell proliferation parameters (M1-M4 and CBPI) and age, gender and smoking habits. The most sensitive parameters were M3 and M4. Age had the strongest effect on M3, while M4 was highly influenced by smoking habits. Gender had an equal non-significant effect on both parameters. The usefulness of the new criteria for the cytokinesis-block MN assay measuring DNA damage as a sensitive biomarker in biomonitoring studies is confirmed.

T-2 toxin: incidence and toxicity in poultry.

T-2 toxin is the most toxic type A trichothecene mycotoxin. It is the secondary metabolite of the Fusarium fungi, and is common in grain and animal feed. Toxic effects have been shown both in experimental animals and in livestock. It has been implicated in several outbreaks of human mycotoxicoses. Toxic effects in poultry include inhibition of protein, DNA, and RNA synthesis, cytotoxicity, immunomodulation, cell lesions in the digestive tract, organs and skin, neural disturbances and low performance in poultry production (decreased weight gain, egg production, and hatchability). Concentrations of T-2 toxin in feed are usually low, and its immunosuppressive effects and secondary infections often make diagnosis difficult. If at the onset of the disease, a change in diet leads to health and performance improvements in animals, this may point to mycotoxin poisoning. Regular control of grain and feed samples is a valuable preventive measure, and it is accurate only if representative samples are tested. This article reviews the incidence and toxic effects of T-2 toxin in poultry.

Cytogenetic outcomes of adjuvant chemotherapy in non-target cells of breast cancer patients.

Spontaneous and chemotherapy-induced sister chromatid exchanges (SCES) and lymphocyte proliferation rate index (PRI) in cultured peripheral lymphocytes were evaluated in 30 patients with diagnosed breast cancer before and after adjuvant chemotherapy and in 30 healthy women with no known familial history of breast cancer. Before chemotherapy, the breast cancer patients had a significantly increased background level of SCE, and lowered PRI as compared with the healthy women. Marked inter-individual variations were observed in both endpoints among the patients. Significantly elevated frequency of SCE and depressed PRI were recorded in blood samples collected after the first cycle of chemotherapy, with high inter-individual variations in the responses to the chemotherapy. FAC (5-fluorouracil, adriamycin and cyclophosphamide) protocol was the most genotoxic of the protocols studied, but also AC (adriamycin, cyclophosphamide) and CMF (cyclophosphamide, methotrexate and 5-fluorouracil) clearly increased SCE. All protocols significantly retarded lymphocyte proliferation in vitro. Our findings indicate that both SCE and PRI may serve as sensitive biomarkers for the routine detection of critical lesions produced by the administration of antineoplastic drugs in the clinical setting, as well as for possible screening of high-risk individuals among patients who have successfully completed chemotherapy.

Evaluation of DNA damage in white blood cells of healthy human volunteers using the alkaline comet assay and the chromosome aberration test.

The present study was undertaken to contribute to the characterization of the degree of variability in baseline damage in white blood cells from control population, and to investigate how this variability is associated with external and internal factors. Altogether 170 healthy volunteers, randomly selected from the general population of the Republic of Croatia, participated in the study. Two sensitive tests: the alkaline comet assay and the chromosome aberration test were applied to study the background levels of DNA damage in their white blood cells. The results point to inter-individual differences, indicating different genome sensitivity. As revealed by both assays, the background levels of DNA damage were mostly influenced by smoking habit as well as medical exposure (especially to diagnostic X-rays). Sex and age of subjects did not significantly influence the values of DNA damage recorded in the white blood cells. Although higher levels of DNA damage were recorded in blood samples collected during winter and autumn, they were mostly influenced by medicinal exposure and smoking habit. Statistical evaluation of the data confirmed that a positive correlation exists between DNA migration and the number of long-tailed nuclei found with the comet assay and the total number of chromosome aberrations. The data obtained can serve as control values in forthcoming biomonitoring studies.

Safety of Radiographic Imaging in Pregnancy

There are many false assumptions, both in the general population and among physicians, regarding the influence of radiation on pregnant patients and the fetus during diagnostic procedures, in spite of scientific facts based on studies. These false assumptions are mostly based on the idea that every diagnostic procedure using ionizing radiation is a cause for serious concern and that artificial abortion as a possible solution might be considered. We analyzed data from counseling of pregnant patients exposed to ionizing radiation during diagnostic procedures in the Merkur University Hospital during a 4-year period. In this period, 26 patients presented for counseling after exposure to diagnostic ionizing radiation during pregnancy. Results showed most of these patients to have been exposed to radiation between the 2nd and 3rd week of gestation (36%), between the 4th and 5th week 32%, before the 2nd week 24%, and after the 6th week of gestation less than 8%. To provide reasonable estimate of fetal doses, Report No. 174 from the National Council on Radiation Protection and Measurements (NCRP) was used. Data from the Report include estimate of the fetal dose from direct and indirect exposures. Th e mean doses were up to 0.01 cGy in 46.2%, 0.01-0.15 cGy in 19.2%, 0.2-1 cGy in 26.9% and 1 cGy or more in 7.7% of patients. None of the counseled patients had medical indication for abortion, even though in a small percentage of patients abortion was a personal subjective decision. Considering that there are no Croatian guidelines for counseling patients exposed to ionizing radiation during pregnancy, it is recommended to use the International Commission on Radiological Protection guidelines in the management of pregnant patients exposed to ionizing radiation.

Carboplatin resistant human laryngeal carcinoma cells are cross resistant to curcumin due to reduced curcumin accumulation.

Curcumin is a natural compound that exhibits a wide range of beneficial effects, among them the anti-tumor activity. Recently it was shown that curcumin may be efficient against drug resistant tumor cells. The goal of our investigation was to examine if human laryngeal carcinoma cells resistant to carboplatin display sensitivity to curcumin, as compared to parental cells, and if this sensitivity is altered, to determine the molecular mechanisms that are responsible for it. We found that carboplatin resistant 7T cells were also cross resistant to curcumin. After the treatment with equimolar concentration of curcumin, 7T cells exhibited lower intracellular accumulation of curcumin which coincided with reduced formation of reactive oxygen species (ROS), diminished lipid and DNA damage followed by reduced induction of apoptosis and expression of heat shock protein 70 (Hsp70), as compared to parental HEp-2 cells. However, after the treatment with equitoxic concentration of curcumin, intracellular accumulation and all the explored downstream effects were similar in both cell lines suggesting that resistance of 7T cells to curcumin was based on its reduced intracellular accumulation. Since curcumin accumulates mostly in the membranes, we explored the fatty acid composition of both cell lines, but we did not find any difference between them.

Protective effect of gangliosides on DNA in human spermatozoa exposed to cryopreservation.

Gangliosides, the sialic acid-containing glycosphyngolipids, are amphiphilic compounds which in micellar form affect the properties and functions of a cellular membrane. The aim of this study was to test whether exogenous gangliosides supplied to cryopreservation media before freezing could protect sperm cells from cryopreservation-induced DNA damage assessed by Comet assay. Additionally, to investigate whether gangliosides were also able to reduce membrane integrity damage, malonaldialdehyde as a measure of lipid peroxidation and sperm-specific lactate dehydrogenase-C4 activity as an enzyme marker of sperm membrane leakage were determined. The monosialogangliosides (GM1) and trisialogangliosides (GT1b) were examined at a concentration of 100 µM, which was above their respective critical micellar concentrations. Exogenous gangliosides were not found to protect sperm membrane from lipid peroxidation. However, a freezing-/thawing-induced increase in Comet parameters was equally significantly prevented by the presence of both GM1 and GT1b (P < .05), indicating that the ceramide moiety, rather than the polar groups, is involved in the protective ability of gangliosides. The observed phenomena suggest that ganglioside micelles could modulate hydrophobic properties of the sperm membrane responsible for better tolerance to DNA fragmentation, thus protecting DNA integrity from cryopreservation-induced damage.

Evaluation of the cytogenetic status of human lymphocytes after exposure to a high concentration of bee venom in vitro.

Several studies have reported radioprotective, antimutagenic, anti-inflammatory, antinociceptive, and anticancer effects of bee venom both in the cell and the whole organism. The aim of this study was to assess the effects of a single high dose of 100 microg mL(-1) of whole bee venom in human lymphocytes in vitro over a variety of time spans (from 10 min to 24 h). After the treatment, we used the comet assay and micronucleus test to see the effect of bee venom on the cell. The comet assay confirmed that the venom damaged the DNA molecule. Tail length, tail intensity, tail moment showed a significant increase (P < 0.05). The percentage of long-tailed nuclei (LTN) with the tail length exceeding the 95th percentile also increased in a time-dependent manner. The micronucleus parameters (number of micronuclei, nucleoplasmic bridges, and nuclear buds) also showed a significant time-dependent increase (P < 0.05). This research indicates that high concentrations of bee venom can lead to cellular instability. Further research is needed to understand the mechanism of action of bee venom and its components in human cells and to see if this natural product may find application in medicine.

Assessment of genotoxic risks in Croatian health care workers occupationally exposed to cytotoxic drugs: a multi-biomarker approach.

The aim of the present study was to evaluate genome damage induced in peripheral blood lymphocytes of Croatian health care workers occupationally exposed to cytotoxic drugs. A comprehensive multi-biomarker approach using the alkaline comet assay and cytogenetic endpoints (analysis of structural chromosome aberrations, SCE assay, lymphocyte proliferation kinetics and cytokinesis-block micronucleus assay) was employed. The study included two populations of subjects: 50 health care workers occupationally exposed to cytotoxic drugs and 50 control subjects matched in age, gender and smoking habit. An investigation regarding the handling practice with cytotoxic drugs was conducted in parallel. Results obtained indicate high exposure levels at workplace that should be reduced. The values recorded among the occupationally exposed subjects were as follows: mean comet tail length: 17.46+/-0.08 microm; the incidence of long-tailed nuclei: 54.68+/-3.93%; 4.48+/-0.33 structural chromosome aberrations per 200 cells; 5.81+/-0.04 SCE per 50 cells; 29.28+/-2.21% of high-frequency cells; proliferation rate index: 1.97+/-0.12; and 16.32+/-0.85 micronuclei per 1000 binuclear cells. All these values indicated higher levels of DNA and cytogenetic damage compared to the general population. Obtained results also confirmed that the frequency of long-tailed nuclei in the alkaline comet assay represents a helpful complement to other well-established comet parameters. The age of subjects and smoking habit significantly influenced the values of both comet and cytogenetic endpoints. Overall results of this study confirmed that handling cytotoxic drugs without appropriate safety precautions involves a potential genotoxic risk for exposed subjects. Before a strict monitoring of exposure levels on each workplace becomes a standard practice in Croatian hospitals, cytogenetic surveillance of exposed workers is also recommended, at least in cases of accidental exposure.

Investigation into possible DNA damaging effects of ultrasound in occupationally exposed medical personnel--the alkaline comet assay study.

In the present paper the possible DNA damaging effects of ultrasound in occupationally exposed medical personnel were investigated using the alkaline comet assay. The extent of DNA migration in peripheral blood leucocytes was measured. Parameters of the comet assay were studied in 30 medical workers occupationally exposed to ultrasound and in 30 corresponding unexposed control subjects. It was found that the subjects who were occupationally exposed to ultrasound for various periods of time showed a highly significant increase in levels of DNA damage compared with the control. The results obtained have confirmed the usefulness of the alkaline comet assay as a sensitive biodosimetric method, reflecting the current level of DNA damage and/or repair in peripheral blood leucocytes of ultrasound-exposed subjects. In spite of their limitations, the results of the present investigation indicate that individuals occupationally exposed to ultrasound may experience an increased genotoxic risk, emphasizing the need for more research into the nature and extent of the biological consequences to medical personnel working with ultrasonic equipment.