Development of an XTT tetrazolium salt-based assay for detection of specific hyperthermia sensitizers in a high-flux screening programme.

It is now possible to search for new drugs using high-throughput screening of chemical libraries accumulated over the past few years. To detect potential new hyperthermia sensitizers, we are screening for chemical inhibitors of thermotolerance. For the screening of a large chemical library, a rapid and simple assay based on the XTT-tetrazolium salt with the addition of intermediate electron acceptor, phenazine methosulphate (PMS) as a promoter, was developed. It was found that the sensitivity of the XTT/PMS assay is sufficient for assessing thermal cell killing and thermotolerance, although it was highly dependent on cell number and type. When the formazan assay system was challenged with the bioflavonoid drug quercetin (up to 25mm) and validated against the clonogenic cell survival assay, significant decreases in thermotolerant cell viability were observed, directly reflecting inhibition of thermotolerance. Although short-term assays can, in some instances, underestimate overall cell killing, the dose dependency of inhibition of thermotolerance by quercetin recorded in this study by clonogenic and XTT/PMS assays was similar. Application of the XTT/PMS assay in chemical library screening was highly effective in differentiating potential thermotolerance inhibitors from both chemicals with lack of efficacy and from toxic compounds. Taken together, these results show that the XTT/PMS assay, when carried out under careful conditions, is well suited for primary high-flux screen of many thousands of compounds, thus opening up new areas for discovery of hyperthermia sensitizers.

Cyclooxygenase inhibitors are potent sensitizers of prostate tumours to hyperthermia and radiation.

It has previously been demonstrated that hyperthermia can activate prostaglandin synthesis and that prostaglandins are protective against hyperthermia. This study examined the use of inhibitors of prostaglandin synthesis on the response of prostate tumours to hyperthermia. The non-steroidal anti-inflammatory drugs (NSAID) ibuprofen and sulindac, known cyclooxygenase inhibitors that inhibit prostaglandin production, were effective hyperthermia sensitizers and augmented growth delay of DU-145 and PC-3 prostate tumours to combined radiation and hyperthermia treatment protocols. Pre-treatment of mice with ibuprofen and sulindac at hyperthermia sensitizing doses resulted in significant (p < 0.01) inhibition of hyperthemia-induced serum prostaglandin E2. These findings indicate that NSAID may have both sensitizing effects on prostate tumour growth and may function by inhibiting prostaglandin synthesis.

First beneficial results of the implementation of Croatian new law on salt iodination.

Presented pilot-study on urinary iodine excretion among schoolchildren in main geographic regions of Croatia indicates a significant improvement in iodine excretion only 12 months after introduction of a new mandatory salt iodination with 25 mg KI/kg of salt. Measurements of urine iodine excretion in 663 schoolchildren of both sexes, aged 9-16 years, from Zagreb, Rude, Bednja, Osijek, Dakovo, Rijeka, Pazin and Dubrovnik showed that from 77.9% to 94.8% of subjects have had urinary iodine above 5 microg/dl. Also from 41.2% to 70.8% of children have had urinary iodine excretion above 10 microg/dl. Before introduction of a new law mostly borderline values of urinary iodine excretion were recorded; therefore recent values present significant positive shift and actually they are the best signs of almost adequate iodine intake in Croatian youth. Nevertheless, further measures for improvement of iodine intake are recommendable because still 31% of controlled table-salt samples from households and 15.7% of imported salt samples have had KI level below 20 mg/kg of NaCl. Salt produced in Croatia was (Pag and Nin salt plants) completely in accordance with iodination regulations. On the other hand, as recent increase in urinary iodine excretion could not be simultaneously followed by reduction in goiter prevalence, in the following years further epidemiological surveys of iodine deficiency disorders shall be warranted.

The cluster of basic amino acids in vitronectin contributes to its binding of plasminogen activator inhibitor-1: evidence from thrombin-, elastase- and plasmin-cleaved vitronectins and anti-peptide antibodies.

Derivatives of vitronectin obtained by specific cleavage at its cluster of basic amino acids with thrombin, elastase and plasmin are shown to have a decreased ability to bind plasminogen activator inhibitor-1 (PAI-1). The identification and localization of the segment involved in the binding of PAI-1 (Lys348-Arg379) were carried out by purification of these cleaved vitronectins and their subsequent structural characterization (sequence analysis, phosphorylation of Ser378 with cAMP-dependent protein kinase and immunostaining with peptide-specific antibodies), then measurement of the vitronectin-PAI-1 interaction by (a) a two-phase system (ELISA); (b) co-precipitation of the vitronectin-PAI-1 complex out of solution, and (c) analysis of the stereospecific interaction between the active conformation of PAI-1 and a peptide derived from the above-mentioned cluster; this interaction occurs when the peptide is composed of all-l-amino acids but not when it is composed of all-d-amino acids. Our results explain why workers who have used immobilized vitronectin to study this interaction could not have observed the involvement of the cluster of basic amino acids in PAI-1 binding, since the immobilization of vitronectin is shown to render this cluster inaccessible for interaction. We propose that vitronectin binds active PAI-1 by interaction via amino acid residues that originate from distal locations in the N- and C-termini of vitronectin.

Successful struggle against iodine deficiency disorders (I.D.D.) in Croatia.

In Croatia a law requiring obligatory iodination of all salt with 10 mg KI per kg of NaCl was proclaimed in 1953. After first ten years of implementation a threefold reduction of goiter prevalence was recorded. Presented study indicates that, four decades later, mild iodine deficiency still persists, keeping goiter prevalence among schoolchildren between 8% and 35%. In continental parts of Croatia 69% to 86% of children had urinary iodine excretion above 5 micrograms/dL, while only 17% to 34% had it above 10 micrograms/dL. In Zagreb, 5% to 13.2% of children had thyroid volumes above the upper limits for their age. Control of salt produced in Pag revealed the average KI value of 8.3 mg/kg. On the basis of these results Croatian Ministry of Health proclaimed, on October 9, 1996, a new regulation requiring 25 mg KI in each kg of NaCl.

Cancer consequences of the Chernobyl accident in Europe outside the former USSR: a review.

The accident which occurred during the night of April 25-26, 1986 in reactor 4 of the Chernobyl nuclear power plant in the Ukraine released considerable amounts of radioactive substances into the environment. Outside the former USSR, the highest levels of contamination were recorded in Bulgaria, Austria, Greece and Romania, followed by other countries of Central, Southeast and Northern Europe. Studies of the health consequences of the accident have been carried out in these countries, as well as in other countries in Europe. This report presents the results of a critical review of cancer studies of the exposed population in Europe, carried out on the occasion of the 10th anniversary of the Chernobyl accident. Overall, three is no evidence to date of a major public health impact of the Chernobyl accident in the field of cancer in countries of Europe outside the former USSR.

Endogenously labeled thyroid hormones (131I-T3/T4) in sera of patients with differentiated thyroid carcinoma.

Patients with differentiated thyroid carcinoma (DTC), who have undergone thyroidectomy and radioablation with iodine (131I), are usually monitored with ultrasonography (US) of the neck, 131I whole body scan (WBS), and determination of thyroglobulin (Tg) concentrations in serum. A chromatographic method (CHROM), designed for detection of in vivo labeled thyroid hormones (131I-T3/T4) that circulate in the body after administration of 131I for a WBS, may be used in monitoring of these patients. This study included 35 patients with DTC. Ultrasonography, WBS, Tg, and CHROM method were performed for each of them. One patient was followed-up 6 times, 11 were monitored twice, and the remaining group of 23 patients was examined only once (51 diagnostic tests). We found CHROM results to be in disagreement with the WBS and/or Tg findings in three patients out of 35 at the time of the first visit. In one of them WBS was negative, while Tg and CHROM findings were positive. In this patient the local metastases were proven by US with fine needle biopsy (FNB). In another patient (without signs of thyroid tissue remnant, determined by WBS, Tg, and US) only CHROM was positive. Finally, in the third patient, a thyroid remnant was proven positive on WBS and US (negative CHROM and Tg). We registered an additional peak (peak 4) in chromatograms of 12 out of 35 patients. This component could be tentatively characterized as 3,3'-diiodo-L-thyronine (131I-3,3'-T2). Although this preliminary study included a small number of patients, we show that the CHROM method can be useful as an additional test in monitoring of patients with DTC, especially those with discordant WBS and Tg results.

[Endemic goiter in Croatia]. / Endemska gusavost u Hrvatskoj.

In the early nineties, 40 years after the introduction of iodine prophylaxis in Croatia (10 mg KI/kg salt), a nationwide study was initiated with the aim to determine the real prevalence of goiter in the country. A total of 2436 schoolchildren of both sexes, aged 7-15 years, were included in the study. Investigations were designed in a way to cover most of geographical regions in Croatia and subjects were randomly selected. The prevalence of goiter in schoolchildren was assessed by palpation and in part by ultrasonography of the neck. At the same time urinary iodine excretion was measured and potassium iodide content in salt samples was determined. The results revealed the persistence of mild endemic goiter in the inland parts of Croatia with the prevalence of 6-29% in the age group 7-11 years and 10-43% among the age group 12-15 years. The overall goiter prevalence in schoolchildren in Croatia fluctuates from 8% to 35%. The urinary iodine excretion in Zagreb varied from 1.6 to 24.4 micrograms/dL with a median of 9.0, while in 14% it was below 5 micrograms/dL. The urinary iodine excretion in Samobor varied between 0.5 and 19 micrograms/dL with a median of 7.4 micrograms/dL, while in 30% it was below 5 micrograms/dL. Examination of salt from all three salt plants in Croatia showed iodine contents between 0.0 and 12.73 mg/kg with the average value of 5.39 mg/kg. Such prevalence, most probably due to less than optimum iodine intake, is unlikely to change until potassium iodide content of salt is increased from its present level of 10 mg of KI per kg of salt, with concomitant stricter observance of legal regulations.

Current status of endemic goiter in Croatia: the results of a nationwide study (1995).

In the beginning of the nineties, 40 years after introduction of iodine prophylaxis in Croatia, on a basis of a frequent reports coming from general practitioners about the presence of a rather high prevalence of goiter among schoolchildren, a nationwide study was initiated with the aim to determine the real prevalence of goiter in the country. A total of 2856 schoolchildren of both sexes, aged 7-15 years, were included into the study. Investigations were designed in a way to cover most of geographical regions in Croatia and subjects were randomly selected. The prevalence of goiter in schoolchildren was assessed by palpation and in part by ultrasonography of the neck. At the same time urinary iodine excretion was measured and iodine content in salt samples was determined. The results have revealed the persistence of mild endemic goiter in inland parts of Croatia with the prevalence of 6-29% in the age group 7-11 years and those of 10-43% among the age group 12-15 years. The overall goiter prevalence in schoolchildren in Croatia fluctuates from 8% to 35%. Such prevalence, most probably due to less than optimum iodine intake, is unlikely to change until iodine content of the salt is increased from its present level of 10 mg of Kl per kg of salt.