Local chemical sympathectomy of rat bone marrow and its effect on marrow cell composition.

Existing experimental studies of the effect of sympathetic nerve fibers on bone marrow cells are based on the systemic administration of neurotoxic 6-hydroxydopamine. The method of global chemical sympathectomy has some serious disadvantages and could lead to questionable results. We describe a new method of local chemical sympathectomy of rat femoral bone marrow using guanethidine (Ismelin) delivery using an osmotic mini pump. Local guanethidine treatment for 14days led to complete elimination of sympathetic fibers in femoral bone marrow in contrast to bone marrow of contralateral or naïve femurs. Ablation of sympathetic fibers was associated with a loss of rat endothelial cell marker (RECA) indicating immunophenotype changes in blood vessel endothelial cells, but no significant effect of guanethidine was found on the survival of endothelial cells and mesenchymal stem cells in vitro. Moreover, local guanethidine treatment also elicited a significant reduction of Nestin+/SDF1+ mesenchymal stem cells and c-Kit+/CD90+ hematopoietic stem cells in femoral bone marrow. Tissue-specific chemical sympathectomy of rat bone marrow by guanethidine overcomes some of the drawbacks of systemic administration of neurotoxic compounds like 6-hydroxydopamine and delivers unequivocal evidence on the effects of sympathetic innervation on the cell content of bone marrow.

Detecting minimal residual disease in patients with chronic lymphocytic leukemia using 8-color flow cytometry protocol in routine hematological practice.

INTRODUCTION: Minimal residual disease (MRD) detection has become increasingly important for the assessment of therapy response in chronic lymphocytic leukemia (CLL). However, current MRD analysis methods, both molecular genetic and flow cytometric, are time-consuming and require experienced laboratory staff. METHODS: To reduce the demands of flow cytometric MRD detection in CLL, we have introduced a novel flow cytometric 8-color protocol. The MRD analysis results using this protocol were then compared with the commonly employed 4-color protocol and the molecular genetic (real-time quantitative allele-specific oligonucleotide IGH polymerase chain reaction; RQ-ASO IGH PCR) approach. RESULTS: Forty-two CLL patient samples were repeatedly analyzed after allogeneic stem cell transplantation (n = 20) or after fludarabine-based therapy (n = 22), and 100% concordance was found using both flow cytometric protocols. Furthermore, there was a strong correlation (r = 0.94) between flow cytometric and RQ-ASO IGH PCR results in MRD detection. CONCLUSION: Flow cytometry is less time-consuming, less financially demanding, and moreover, MRD assessment using our novel 8-color protocol is less complicated than the 4-color approach and molecular methods.

[Our experience in treatment of multicentric plasma-cell Castleman disease associated with vasculitis manifestations - case report and literature review]. / Nase zkusenosti s lécbou multicentrické plazmocelulární Castlemanovy choroby s projevy vaskulitidy - popis prípadu a prehled literatury.

Castleman disease is a rare idiopathic non-neoplastic lymphoproliferative disorder with 2 clinical (unicentric and multicentric) and 3 histomorphological (hyaline-vascular, plasma-cell and mixed) forms identified. The case report given here describes the 3-year experience with therapy in a patient, male born 1961, diagnosed with multicentric plasma-cell Castleman disease (HIV and HHV-8 negative) with the finding of generalized lymphadenopathy and splenomegaly. During first line treatment (R-CHOP: rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, 3 cycles in total, 12/2008-2/2009) the development of bilateral upper and lower limb edemas with clinical manifestation of vasculitis occurred and a restaging computed tomography (CT) examination revealed a stable finding of the lymphadenomegaly. Greater success was achieved with thalidomide regimen (CTD: cyclophosphamide, thalidomide, dexamethasone, 10 cycles, 3/2009-1/2010) leading to reduction in the size of the hypervascularized lymph nodes (almost by 50%) as well as their radiopharmaceutical (fluorodeoxyglucose) uptake as seen on a combined positron emission tomography and computed tomography (PET/CT) scan imaging. Thalidomide was given daily at doses between 100 and 200 mg. We returned to the CTD regimen again in April 2010 after a short period of monoclonal antibody tocilizumab treatment (400 mg intravenous in 2-week intervals with 50% dose reduction due to a limited supply of the drug, 5 doses in total) during which edemas reoccurred with a CT scan finding of stable lymphadenomegaly. However, the renewed regimen with thalidomide was stopped after 2.5 cycles due to adverse effects of thalidomide (neuropathy) and corticoids (Cushing syndrome). In September 2010, after enrollment in the Celgenes Compassionate Use Program we were able to start treating the patient with the derivative of thalidomide, lenalidomide, at a dosage of 25 mg on days 1-21 in a 28-day cycle, 15 cycles in total (10/2010-12/2011). The monotherapy with lenalidomide was very well tolerated by the patient without any effects of myelotoxicity, thromboembolism or relapses of edemas and vasculitis, additionally now with apparent improvement of fatic disorder and the patients motor abilities. Thus, lenalidomide represents an attractive alternative agent for patients with Castleman disease after rituximab and cytostatics failures. It has a favourable safety profile and could be therefore considered for administering in first line treatment.

[Schnitzler syndrome: case report, the experience with glucocorticoid and anakinra (Kineret) therapies and monitoring of systemic cytokine response]. / Schnitzler-syndrom: popis prípadu, zkusenosti s lécbou glukokortikoidy a preparátem anakinra (Kineret) a sledování cytokinové odpovedi organizmu.

Schnitzler syndrome is a rare idiopathic disease characterized by chronic urtica, presence of monoclonal IgM immunoglobuline and further, less common symptoms. This case report describes another case of this disease affecting a male adult born in 1963. The first symptoms, eruptions of non-pruritic urticarial rash, appeared in this patient at the age of 43. In addition, bone pains (mainly tibias) and joint pains (mainly knees) were present. Later on however, severe attacks of fever, chills and shaking together with bone and joint pains were added to during which new urticarial eruptions appeared. Primarily, the man was followed up without any substantial therapeutic results at a department of dermatovenerology, subsequently, due to a finding of monoclonal IgM kappa immunoglobulin (serum concentration 1.9 g/l) he was referred to our department for the reason of gammopathy being a differential diagnosis. On a CT scan hyperostosis in claviculae and pelvic bones was identified. Also on the CT, an increase in cortical thickness was described in the long bones of the lower extremities, where areas of technetium pyrophosphate accumulation were identified on a bone scintigraphy. These areas were found in the chest and sacral regions as well. From the blood exams, the proinflammatory status of the organism was apparent (CRP 35.9 mg/l, erythrocyte sedimentation rate 92 mm/h, leukocytes 12.4 x 10(9)/l). After excluding other differential diagnoses, the patient was diagnosed with Schnitzler syndrome. As regards therapy, we made initial use of the effect of corticoids which abated the symptoms, however, these were causing serious adverse reactions in the form of iatrogenous Cushing's syndrome. The therapy took a turn only after biologic therapy with anakinra (interleukin-1 receptor antagonist) had started, which minimized the Schnitzler symptoms with very good drug tolerance. In the work we measured serum levels of interleukins for disease activity monitoring. The most sensitive were interleukins IL-6 and especially IL-18 the levels of which were the highest at the time of clinical exacerbation of the disease, whereas the levels of IL-1beta and TNF-alpha (tumour necrosis factor) were during all measurements below the limit of detection. Concerning the growing numbers of the reports on successful biological therapy with anakinra and our positive experience, we propose that the therapeutic response to anakinra should be included within the diagnostic criteria of Schnitzler syndrome, which is significant above all in differential diagnosis thereof.

Administration of isothiocyanate (E-4IB) and cisplatin leads to altered signalling and lysosomal export in human ovarian carcinoma sensitive- and cisplatin-resistant cells.

The aim of this study was to compare the effect of a new synthetic isothiocyanate derivative, ethyl 4-isothiocyanatobutanoate (E-4IB) and cisplatin (CDDP) in CDDP-sensitive human ovarian carcinoma cell line (A2780) and its resistant subline (A2780/CP). In parental cells, in comparison to untreated cells, sequential administration of both compounds led to higher exosomal dye (LysoTracker Green DND-26) retention and to alterations of mitogen-activated protein kinases (MAPKs), JNK, ERK and p38, or Akt kinase accompanied by changes in several anti- and pro-apoptotic molecules and lysosomal protein LAMP-1, as detected by Western blotting. On the contrary, variant A2780/CP cells were resistant to CDDP- or to combined sensitizer (E-4IB)/inducer (CDDP)-related apoptosis induction and exerted minor changes in the levels of these molecules.

Expression of new prognostic markers, peripheral-type benzodiazepine receptor and carbonic anhydrase IX, in human breast and ovarian carcinoma cell lines.

Peripheral benzodiazepine receptor (PBR), a mitochondrial protein involved in cell proliferation and differentiation, and carbonic anhydrase IX (CA IX), an intrinsic marker of hypoxia, have been studied in the panel of human breast (MCF-7, BT- 20, MDA-MB-453, MDA-MB-231) and ovarian (A2780, A2780/CP, A2780/ADR, CH1, SKOV-3) carcinoma cell lines that differ by malignant progression. The expression of both antigens was detected by staining with the PBR-specific 8D7 and CA IX-specific M75 monoclonal antibodies and quantitated by flow cytometry. PBR was related to mitochondrial mass and CA IX to the cell density. Breast carcinoma cell lines showed higher relative fluorescence intensity of PBR expression than ovarian cell lines, with the exception of A2780/CP cisplatin-resistant subline that was comparable to highly invasive MDA-MB-231 breast line. Among the breast cell lines, PBR expression increased with their invasive potential. The ovarian cell lines showed greater variability in fluorescence intensities and the expression of PBR did not correlate with the amount of mitochondria. Mitochondrial PBR density disclosed significant difference between cisplatin-sensitive (low PBR density) and -resistant (high PBR density) ovarian cell lines. MTT test showed higher sensitivity of 2 breast cell lines MCF-7 and MDA-MB-231 (IC50 < 75 microM) to PBR ligand PK 11195 than all examined ovarian cell lines (IC50 > 90 microM, in chemo- and radio- resistant lines IC50 > 110 microM). Growth inhibitory effect of PK 11195 did not correlate with the amount of PBR and was mediated probably by another, PBRindependent mechanisms. The expression of CA IX was only marginal in majority of tested cell lines in subconfluent conditions and was inducible by high cell density. More than 5% of positive cells in sparse culture have been found in MDA-MB-231 and MDA-MB-453 breast cell lines while more than 15% of A2780/ADR adriamycin-resistant ovarian cells were positive for CA IX expression under the same conditions. Our data indicate that PBR expression in breast and ovarian carcinoma cell lines is not proportional to the amount of mitochondria and should be expressed relatively to the cell mitochondrial mass. This assessment allows establishing high PBR density as a measure of aggressiveness (invasion in breast and resistance in ovarian cancer). Observation of relatively high CA IX expression in A2780/ADR cells evokes the assumption that multidrug resistance might be connected with selection advantage towards CA IX expressing cells.

Multiple organochlorine pollution and the thyroid.

OBJECTIVE: Although the biological effects of major persistent organochlorinated pollutants (POPs) appear to be essentially similar, some effects which would be specific for certain substance cannot be excluded. We attempted to study the thyroid volume and thyrotropin level in the population living in the area with multiple pollution by polychlorinated biphenyls (PCBs) and pesticides (DDE and hexachlorobenzene - HCB). METHODS: A total of 454 adults was examined within the pilot field survey in 1998. Among them were 237 males (age range 19-78 years, median 47) and 227 females (age range 19-78 years, median 48). Fifteen environmentally prevalent congeners of polychlorinated biphenyls and also p,p-DDE (2,2-bis(4-chlorophenyl)-1,1-dichloroethylene), p,p-DDT (2,2-bis(4-chlorophenyl)- 1,1,1-trichloro-ethane), hexachlorobenzene (HCB) as well as alpha-, beta- and gamma-hexachlorocyclohexane (HCH) were determined in serum by high resolution gas chromatography using microelectron capture detector and microcapillary column. Thyroid volume (ThV) was measured by real time sonography using the ellipsoid method with the aid of sonographic instrument Sonoline SI-400 (Siemens, Germany). The level of TSH was estimated by supersensitive immunoradiometric method using commercial kits by Immunotech (Marseille, France). Pearsons correlation coefficients after logarithmic transformation of values and Spearmans correlation coefficients were used for statistical evaluation. RESULTS: Significant positive association (p<0.01) was found between DDE and PCB, DDE and HCB, while that between PCB and HCB was not significant. Similar positive association (p<0.01) was also found between each individual organochlorine and their sum. Significant negative association (p<0.01) was found between ThV and TSH. When using categorical PCB values either >2000 (N=208) or >3000 (N=127) ng/g lipid, significant positive association (p<0.05 and p<0.01, respectively) was found between the sum of all organochlorines (PCB+DDE+HCB) and ThV, while that between PCB and ThV (p<0.01) was found only at the PCB levels >3000 ng/g lipid. When using Spearmans correlation coefficients, significant negative association appeared between PCB and TSH (p<0.05), sum of organochlorines and TSH (p<0.05) and ThV and TSH (p<0.01). CONCLUSIONS: Although several significant positive and negative associations were found, this study, like several others, could not exactly define the participation level of individual POPs in their common toxic effects, but possibly contributed to the recognition and elucidation of some problems related to this task.

Enhanced sensitivity of human ovarian carcinoma cell lines A2780 and A2780/CP to the combination of cisplatin and synthetic isothiocyanate ethyl 4-isothiocyanatobutanoate.

Naturally occurring and synthetic isothiocyanates (ITCs) are known as chemopreventive agents. The present study shows a new synthetic ITC derivate ethyl 4-isothiocyanatobutanoate (E-4IB) as an effective modulator of cellular proliferation and inducer of apoptosis with potential utility as an anticancer drug, as well as a sensitizer to routinely used chemotherapeutic agent cisplatin (cis-Pt). Evaluation of the growth inhibitory effects of E-4IB in the human ovarian carcinoma cell line A2780 and its cisplatin-resistant variant A2780/CP using MTT-test and its apoptosis-inducing properties by flow cytometry was performed. Effect of E-4IB was assessed both alone and in paired combination with cisplatin. Combination index (CI) values from Calcusyn software were used to characterize the interactions as synergistic, additive, or antagonistic. Significant synergistic effect in growth inhibition of E-4IB (0.5-5 microM) with cis-Pt (2.5-10 microM) on A2780 parental cell line (CI from 0.39 to 0.75) was also observed on A2780/CP resistant subline, although to a lesser extent (CI from 0.43 to 0.86) for cis-Pt concentrations 5-25 microM and the same concentrations of E-4IB. Synergy in growth inhibition correlated with the potential of E-4IB to stimulate apoptosis induced by cis-Pt (from 9.5% to 24.7% at 24 hours) while E-4IB alone induced 3.6% of apoptotic cells in A2780 cell line. We conclude that E-4IB may be worth of further studies assessing its value in the ovarian carcinoma treatment, in combination with the other chemotherapeutic agents.

Human thyroid in the population exposed to high environmental pollution by organochlorinated pollutants for several decades.

OBJECTIVE: To study possible effects of long-time exposure of chemical factory employees and population of surrounding polluted area to polychlorinated biphenyls and pesticides on the thyroid volume and function as compared to the population from the area of background pollution. METHODS: A total of 461 adults consisting of 239 men and 222 women was examined and divided into four groups according to their permanent domicile as related to the level of environmental pollution, e.g. SR (area of background pollution, n = 207), SI (slightly polluted area, n = 59), MI (polluted city of Michalovce, n = 94) and CH (employees of chemical factory subjected to high PCB exposure, n = 101), combined first three groups being also called LPA (less polluted areas, n = 360). Thyroid volume (ThV) and echogenicity were measured by real time sonography. The level of polychlorinated biphenyls (PCB) and pesticides (hexachlorbenzene--HCB, DDE (2,2'-2-bis(4-chlorobiphenyl)- 1,1-dichloroethylene), p,p'-DDT (2,2'-bis(4-chlorophenyl)- 1,1,1-trichloroethane) and alpha-, beta- and gamma-hexachlorcyclohexane--HCH) was estimated by congener specific analysis using HP 5890 gas chromatograph with a 63Ni electron capture detector. Serum levels of thyrotropin (TSH) and thyroid peroxidase antibodies (anti-TPO) were measured by specific sensitive immunoassays. RESULTS: The association of very high PCB level (e.g. 7300 +/- 871 ng/g lipid; mean +/- S.E.) with increased ThV (e.g. 16.3 +/- 0.73 ml) in CH has been found, the values being significantly higher than these of 360 subjects in LPA (e.g. 2045 +/- 147 ng/g, p < 0.001 for PCB and 14.0 +/- 0.32 ml, p < 0.001 for ThV). In 23 subjects from CH with PCB level > 10000 ng/g the ThV was 18.7 +/- 2.32 ml, while that in 251 subjects from LPA with PCB level of < 2000 ng/g was 13.8 +/- 0.35 ml (p < 0.05). In addition, ThV as well as PCB levels were strikingly increasing with age. In parallel with PCB levels, also the levels of other organochlorines estimated (namely these of DDE) were increasing. Although the participation of these substances in the development of adverse effects cannot yet be defined, it cannot be excluded. The association of increased levels of episodic congener PCB 101 with increased ThV appeared to be more pronounced than that of stable congeners PCB 153 and 180. Finally, significant increase in the frequency of thyroid hypoechogenicity by ultrasound, ThV > 20.0 ml and thyroperoxidase antibodies in CH area was observed as compared to LPA. CONCLUSIONS: Several associations of high PCB and pesticides level with characteristics of thyroid disorders (e.g. increased thyroid volume, frequency of hypoechogenicity and frequency of positive thyroperoxidase antibodies level in blood) were observed in the area with heavy industrial pollution by PCB.

Environmental contamination with polychlorinated biphenyls in the area of their former manufacture in Slovakia.

Evidently increased environmental pollution as a consequence of the 25-year manufacture of polychlorinated biphenyls (PCBs) in eastern Slovakia was observed. PCB levels determined in ambient air, soil, surface water, bottom sediment, wildlife (fish and game) samples collected in a potentially contaminated area of about 250 km2 (a part of the Michalovce district) were compared with those determined in a control area (Stropkov district). Up to 1700 ng/m3 were found in ambient air in a village close to a manufacturer's dumping site and a highly contaminated manufacturer's effluent canal whereas PCB concentrations in ambient air samples taken in villages in the control area were about 80 ng/m3 only. While soil samples taken from the agricultural fields of the polluted area contained PCBs at levels comparable with soil samples from the control area (about 0.008 mg/kg) much higher values (from 0.4 to 53,000 mg/kg) were determined in soil taken in the vicinity of manufacturer's landfill and storage sites and especially plants preparing asphalted gravel using formerly PCBs in their heat-exchanging systems. The contamination of the Laborec river and large Zemplinska Sirava reservoir is caused by the manufacturer's effluent canal since PCB levels in the canal sediment are still to be found about 3000 mg/kg. While PCB levels in sediment samples from Michalovce watercourses ranged between 1.7 and 6 mg/kg, sediment samples from the control Stropkov district ranged between 0.007 and 0.052 mg/kg only. Fish living in contaminated Michalovce waters contained about hundred times higher PCB levels than those caught in Stropkov ones. Similarly, game animals shot in Michalovce forests contained several times higher levels than those shot in Stropkov ones.

Radiation-induced apoptosis and cell cycle alterations in human carcinoma cell lines with different radiosensitivities.

Radiosensitivity of examined human neoplastic cell lines was assessed with the aid of MTT assay. Differences between radiosensitive and radioresistant human neoplastic cell lines were as follow: a) radiation-induced apoptosis detected by flow cytometry was apparent in the most radiosensitive (i.e. CH-1 ovarian carcinoma cell line), but not in the radioresistant (i.e. SKOV-3 ovarian carcinoma) cell lines, b) radiation-induced G2/M arrest appeared early after irradiation (6 hours) in both the radioresistant SKOV-3 cells and in the radiosensitive CH-1 human ovarian carcinoma cell line, but a different pattern was observed 24 hours after irradiation with 2 Gy dose with G2/M arrest only in radiosensitive cell line. The radiosensitivity and resistance to radiation-induced apoptosis in the radioresistant human breast carcinoma MDA-MB-231 cell line were similar to those observed in SKOV-3 cells. These data suggest that radiation-induced apoptosis and cell cycle alterations can predict radiosensitivity at least in some examined human malignant cells in vitro.

Detection of apoptosis in a heterogenous cell population using flow cytometry.

Apoptosis induced in human leukemic cells (promyelocytic human leukemic cells HL-60, multidrug-resistant subline HL-60/VCR) and human ovarian carcinoma cells (A2780 and multidrug-resistant subline A2780/ADR) in vitro was detected by flow cytometric analysis or DNA electrophoresis. The cytofluorometric techniques utilized, i. e. detection of phosphatidylserine exposed at the outer surface of the plasma membrane, identification of cells with "sub-G0" DNA content or increased light side scatter (cell internal structure) correlated with the electrophoretic determination of DNA fragmentation ("DNA ladder"). Detection of the 34 kDa mitochondrial protein recognized by the monoclonal antibody Apo2.7 yielded elevated percentages of apoptotic cells, suggesting that this technique detecting both early and late apoptosis in digitonin-fixed cells might not be restricted to the specific detection of programmed cell death.

Angiogenesis inhibitor TNP-470: cytotoxic effects on human neoplastic cell lines.

Angiostatic substance TNP-470 displayed moderate cytotoxicity towards human leukemia HL-60, HL-60/ADR, HL-60/VCR and myeloma ARH77 cell lines with IC50 in the range 5-10 microM of concentrations and slightly higher IC50 for myeloma cell line U266. IC50 for ovarian CH-1, A2780 and A2780/ADR cell lines was in the range 10-15 microM with the exception of platinum-resistant SKOV3 cell line (more than 40 microM ). The IC50 values for MDA-MB-231 and MCF-7 breast carcinoma cell lines were 15 and 25 microM, respectively. In human hemopoietic neoplastic cell lines examined, TNP-470 induced the appearance of subpopulation with sub-G0 DNA content, suggesting the apoptosis-inducing potential of TNP-470 in these cells. No TNP-470-induced drug uptake modulation in drug-resistant leukemia cell line HL-60/VCR was observed. TNP-470 induced accumulation of cells in G0/G1 phase of cell cycle. There was no TNP-470-induced inhibition of MMP collagenase activity or MMP (MMP2 and MMP9) production in the human fibrosarcoma cells HT 1080 in vitro.

Synthesis, antimicrobial activity and bleaching effect of some reaction products of 4-oxo-4H-benzopyran-3-carboxaldehydes with aminobenzothiazoles and hydrazides.

The synthesis of the biologically active novel systems derived from reaction of 3-formylchromones with three types of amino derivatives, 6-R2-2-aminobenzothiazoles, 6-amino-2-R3-thiobenzothiazoles and hydrazide derivatives (derived from cyanoacetic, isonicotine, salicylic and gallic acids) was carried out. The structures of the prepared compounds have been proved by elemental analysis, 1H NMR and IR spectra. Antimicrobial activity was studied against the following microorganisms--bacteria G+ (Staphylococcus aureus 29/58, Bacillus subtilis 18/66), G- (Escherichia coli 326/71, Pseudomonas aeruginosa); yeasts: Candida albicans, Saccharomyces cerevisiae; moulds: Microsporum gypseum, Aspergillus niger, Scopulariopsis brevicaulis; and against typical and atypical mycobacteria: Mycobacterium tuberculosis (H37Rv), Mycobacterium kansasii (PFG 8), Mycobacterium avium (My 80/72), Mycobacterium fortuitum (1021). The hereditary bleaching effect on the plastid system of Euglena gracilis, a unique phenomenon of the biological activity of chromone derivatives, is reported. The bleaching test on E. gracilis is used for detecting extranuclear mutations.

Human multidrug-resistant (MRP,p190) myeloid leukemia HL-60/ADR cells in vitro: resistance to the mevalonate pathway inhibitor lovastatin.

Mevalonate pathway inhibitor lovastatin inhibited proliferation of human multidrug-resistant promyelocytic leukemia HL-60/ADR cells in vitro, with MRP-gene coded p190 mediated drug resistance, to a markedly lesser extent than that of the parental drug sensitive HL-60 cells and also that of the other human multidrug resistant (MDR-1, P-glycoprotein) myeloid leukemia cell line HL-60/VCR. The sensitivity of the examined human leukemia cell lines to the cytostatic activity of lovastatin correlated approximately with the potential of lovastatin to induce the characteristic cell cycle alteration (i.e. the accumulation of lovastatin-treated cells in the G0/G1 phase of the cell cycle). The P-glycoprotein positive HL-60/VCR cells and the parental drug sensitive HL-60 cells were more sensitive to this cell cycle alteration than the HL-60/ADR multidrug resistant leukemia cells with MRP drug resistance. Lovastatin (72 hours, 20 micromol) induced apoptosis and cell necrosis in HL-60 cells, apoptosis but not cell necrosis in HL-60/VCR cells and neither apoptosis nor necrosis in HL-60/ADR cells.

TOCOEN project.

The basic goals of the TOCOEN project (Toxic Organic COmpounds in the ENvironment) are to detect and understand the fate of selected organic pollutants (PAHs, PCBs and PCDDs/Fs mainly) in the environment. The TOCOEN project was established in 1988 as a free association of Czech and Slovak environmental chemists, ecototoxicologists and other environmentalists.

Levels of PCBs and some organochlorine pesticides in the human population of selected areas of the Slovak Republic. I. Blood.

Fifty samples of human blood collected in 1992 from the general human population living in five selected areas of the Slovak Republic (the Michalovce, Velký Krtís and Nitra District, Myjava area and Bratislava) were analysed for polychlorinated biphenyls (IUPAC Nos. 28, 52, 77, 101, 105, 114, 118, 123, 126, 138 (+163), 153, 156, 157, 167, 169, 180 and 189) and some organochlorine pesticides (HCB, gamma-HCH, p,p'-DDE and p,p'-DDT). PCB levels (the sum of the congeners analysed) in serum lipids averaged for all the samples analysed were 1.79 micrograms.g-1 (range: 0.53-9.20 micrograms.g-1, median: 1.33 micrograms.g-1), HCB 5.38 micrograms.g-1 (0.16-23.20 micrograms.g-1, 4.27 micrograms.g-1), gamma-HCH 0.012 microgram.g-1 (< 0.01-0.18 microgram.g-1), p,p'-DDE 6.05 micrograms.g-1 (1.30-34.80 micrograms.g-1, 4.39 micrograms.g-1) and p,p'-DDT 0.27 microgram.g-1 (< 0.01-0.79 microgram.g-1, 0.23 microgram.g-1). About three times higher levels of PCBs were found in the samples from the district where PCB formulations had been produced (the Michalovce District).

Determination of ethylenethiourea in grapes and wine.

Residues of ethylenethiourea (ETU) in grapes and wine were determined by capillary gas chromatography and paper chromatography, without a cleanup step, and after derivatization to S-benzyl-ETU. The detection limit was 0.0002 mg/kg for flame ionization detection, 0.008 mg/kg for paper chromatography with photodensitometric evaluation of the detected spot. Results were compared with a generally used GC method specifying electron capture detection of trifluoroacetylated S-benzyl-ETU. The recoveries of ETU in grapes and wine at different concentration levels were determined. ETU residues were determined in treated grapes but no residues were detected in wine.