Vitamin B6 catabolism and lung cancer risk: results from the Lung Cancer Cohort Consortium (LC3).

BACKGROUND: Increased vitamin B6 catabolism related to inflammation, as measured by the PAr index (the ratio of 4-pyridoxic acid over the sum of pyridoxal and pyridoxal-5'-phosphate), has been positively associated with lung cancer risk in two prospective European studies. However, the extent to which this association translates to more diverse populations is not known. MATERIALS AND METHODS: For this study, we included 5323 incident lung cancer cases and 5323 controls individually matched by age, sex, and smoking status within each of 20 prospective cohorts from the Lung Cancer Cohort Consortium. Cohort-specific odds ratios (ORs) and 95% confidence intervals (CIs) for the association between PAr and lung cancer risk were calculated using conditional logistic regression and pooled using random-effects models. RESULTS: PAr was positively associated with lung cancer risk in a dose-response fashion. Comparing the fourth versus first quartiles of PAr resulted in an OR of 1.38 (95% CI: 1.19-1.59) for overall lung cancer risk. The association between PAr and lung cancer risk was most prominent in former smokers (OR: 1.69, 95% CI: 1.36-2.10), men (OR: 1.60, 95% CI: 1.28-2.00), and for cancers diagnosed within 3 years of blood draw (OR: 1.73, 95% CI: 1.34-2.23). CONCLUSION: Based on pre-diagnostic data from 20 cohorts across 4 continents, this study confirms that increased vitamin B6 catabolism related to inflammation and immune activation is associated with a higher risk of developing lung cancer. Moreover, PAr may be a pre-diagnostic marker of lung cancer rather than a causal factor.

No association between circulating concentrations of vitamin D and risk of lung cancer: an analysis in 20 prospective studies in the Lung Cancer Cohort Consortium (LC3).

Background: There is observational evidence suggesting that high vitamin D concentrations may protect against lung cancer. To investigate this hypothesis in detail, we measured circulating vitamin D concentrations in prediagnostic blood from 20 cohorts participating in the Lung Cancer Cohort Consortium (LC3). Patients and methods: The study included 5313 lung cancer cases and 5313 controls. Blood samples for the cases were collected, on average, 5 years before lung cancer diagnosis. Controls were individually matched to the cases by cohort, sex, age, race/ethnicity, date of blood collection, and smoking status in five categories. Liquid chromatography coupled with tandem mass spectrometry was used to separately analyze 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] and their concentrations were combined to give an overall measure of 25(OH)D. We used conditional logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for 25(OH)D as both continuous and categorical variables. Results: Overall, no apparent association between 25(OH)D and risk of lung cancer was observed (multivariable adjusted OR for a doubling in concentration: 0.98, 95% CI: 0.91, 1.06). Similarly, we found no clear evidence of interaction by cohort, sex, age, smoking status, or histology. Conclusion: This study did not support an association between vitamin D concentrations and lung cancer risk.

Anti-Mullerian hormone and risk of invasive serous ovarian cancer.

PURPOSE: Epithelial ovarian cancers either arise directly from Mullerian-type epithelium or acquire Mullerian characteristics in the course of neoplastic transformation. The anti-Mullerian hormone (AMH) causes regression of Mullerian structures during fetal development in males and has been shown to inhibit the growth of epithelial ovarian cancer. Therefore, we hypothesized that pre-diagnostic serum concentrations of AMH are inversely associated with risk of invasive serous ovarian cancer. METHODS: A case-control study (107 cases, 208 controls) was nested within the population-based Finnish Maternity Cohort (1986-2007). The sample donated during the first trimester of the last pregnancy preceding cancer diagnosis of the case subjects was selected for the study. For each case, two controls, matched on age and date at sampling, as well as parity at sampling and at cancer diagnosis were selected. AMH was measured by a second-generation AMH ELISA. Conditional logistic regression was used to compute odds ratios (OR) and 95 % confidence intervals (CI) for invasive serous ovarian cancer associated with AMH concentrations. RESULTS: Overall AMH concentrations were not associated with risk of invasive serous ovarian cancer (OR 0.93; 95 % CI 0.49-1.77 for top vs. bottom tertile, P trend=0.83). In women older than the median age at sampling (32.7 years), a doubling of AMH was associated with decreased risk (OR 0.69; 95 % CI 0.49-0.96), whereas an increased risk (OR 1.64; 95 % CI 1.06-2.54) was observed in younger women, P homogeneity = 0.002. CONCLUSIONS: In this first prospective investigation, risk of invasive serous ovarian cancer was not associated with pre-diagnostic AMH concentrations overall; however, the association may depend on age at AMH measurement.

Changes in pre-diagnostic serum C-reactive protein concentrations and ovarian cancer risk: a longitudinal study.

BACKGROUND: Evidence suggests that inflammation may be associated with increased risk of ovarian cancer but there is paucity of studies investigating this association, especially using over-time changes in inflammatory biomarkers. MATERIALS AND METHODS: We conducted a prospective population-based case-control study nested within the Finnish Maternity Cohort (FMC). Within the FMC, 170 women with ovarian cancer who had donated serum samples to the cohort twice, ≥1 year apart, before cancer diagnoses were identified. One control per case was matched for age, parity and sampling date. RESULTS: Comparing the highest with lowest tertiles, the odds ratio (OR) of ovarian cancer using the first set of serum samples (mean lag time to cancer diagnosis 9.0 years) was 1.62 [95% confidence interval (CI) 0.93-2.83]. However, analysis conducted using the second set of serum samples donated closer to cancer diagnosis (mean lag time 6.4 years) revealed a significantly increased risk of ovarian cancer comparing extreme tertiles of C-reactive protein (CRP) concentrations; OR 1.96 (95% CI 1.11-3.4). Over time, increases in individuals' CRP concentrations were also associated with increased risk; OR 1.90 (95% CI 1.12-3.23). CONCLUSION: The results suggest that inflammation may precede ovarian cancer since increasing CRP concentrations, both across tertiles and longitudinally at the individual level, were associated with increased risk.

Induction of apoptosis by intracellular potassium ion depletion: using the fluorescent dye PBFI in a 96-well plate method in cultured lung cancer cells.

Depletion of intracellular potassium ions (K+) is necessary for cells to shrink, activate caspases and induce DNA fragmentation, events which are features of apoptosis. Here we describe a 96-well plate method using the cell permeable form of K+ binding benzofuran isophtalate (PBFI-AM) to measure intracellular K+ content in relation to untreated control. Cultured human pulmonary mesothelioma cells (P31) and small-cell lung cancer cells (U1690) were treated with K+ flux modulators in order to deprive the cells of intracellular K+. The combination of K+ influx inhibition with 10 micromol/L bumetanide plus 10 micromol/L ouabain and K+ efflux stimulation with 3 mg/L amphotericin B or 5 micromol/L nigericin efficiently reduced the intracellular K+ content after 3 h. Manipulation of K+ fluxes with subsequent intracellular K+ depletion induced apoptosis of lung cancer cells, as detected by caspase-3 activity after 3 h K+ depletion followed by 24 h proliferation and TUNEL positive staining after 48 h proliferation. We concluded that the PBFI-AM assay was a useful tool to determine intracellular K+ content in relation to untreated control, and that intracellular K+ depletion of lung cancer cells by clinically used drugs of relevant concentrations induced apoptosis. These findings may lead to novel therapeutic strategies in the treatment of lung cancer.

Acquired neonatal thyroid disease due to TSH receptor antibodies in breast milk.

INTRODUCTION: We investigated whether thyroid receptor antibodies (TRAb) could result in transient neonatal thyroid disease by transfer through milk from mothers treated for thyrotoxicosis. AIM: To analyse whether breast milk content of TRAb in euthyroid mothers with treated thyrotoxicosis resulted in neonatal thyroid disease and whether extended breastfeeding prolonged the neonatal disease. PATIENTS: We tested three TRAb-positive mothers and the course, treatment and outcome for their offspring with neonatal thyrotoxicosis, and six healthy and two TRAb-negative euthyroid mothers with treated thyrotoxicosis during breastfeeding. METHOD: TRAb was analysed in serum and breast milk by a radioreceptor assay. RESULTS: TRAb in serum was detectable in all treated mothers, in one mother during her four pregnancies, resulting in all neonates requiring treatment for thyrotoxicosis. Serum TRAb concentration in neonates decreased continuously with time after birth. Breast milk TRAb was detectable in all cases but not in the controls or in TRAb-negative mothers treated for thyrotoxicosis. The calculated half-life for offspring serum and breast milk TRAb was calculated as approx. 3 weeks and 2 months, respectively. CONCLUSION: Euthyroid TRAb-positive mothers may cause transient neonatal thyroid disease which seems to be worse and more prolonged during breastfeeding as a consequence of TRAb in breast milk.

Vascular endothelial growth factor is of high prognostic value in node-negative breast carcinoma.

PURPOSE: The prognostic value of vascular endothelial growth factor (VEGF) protein, known to stimulate endothelial growth and angiogenesis, was evaluated in node-negative breast carcinoma (NNBC) and compared with established prognostic factors. PATIENTS AND METHODS: In 525 consecutive patients with primary invasive NNBC (T1-2N0M0; tumor, node, metastasis stage), of whom 500 patients did not receive any systemic therapy, the cytosolic levels of VEGF165 were measured by using a quantitative enzyme-linked immunosorbent assay. The median follow-up was 46 months. Univariate and multivariate analyses were performed. RESULTS: VEGF level was significantly inversely correlated with estrogen receptor (ER) positivity but positively associated with tumor size and histologic grade. Patients with VEGF levels above the median value (2.40 pg/microg of DNA) showed a significantly shorter survival time (P=.0012) than patients with levels less than the median value, also when analyzed as a continuous variable (P=.0277). Tumor size, grade, and ER expression were all statistically significant for overall survival in univariate analyses (P=.0069, P=.014, and P < .001, respectively). Multivariate analysis showed that VEGF level was the strongest predictor of overall survival (P=.0199). Histologic grade was also an independent predictor of survival (P=.0477). Among the 381 patients with ER-positive tumors, a group in general considered to have a good prognosis, we found a significant reduction in survival for those with levels of VEGF greater than the median value (P=.0009). CONCLUSION: The results suggest that the level of VEGF165 protein is an independent, strong prognostic factor for survival in patients with NNBC, especially in the subgroup of patients with ER positivity. Thus, cytosolic VEGF165 might be useful to select patients for adjuvant systemic therapy.

Correlation of vascular endothelial growth factor content with recurrences, survival, and first relapse site in primary node-positive breast carcinoma after adjuvant treatment.

PURPOSE: To determine the predictive value of vascular endothelial growth factor (VEGF) for relapse-free survival (RFS) and overall survival (OS) in primary node-positive breast cancer (NPBC) after adjuvant endocrine treatment or adjuvant chemotherapy. MATERIALS AND METHODS: VEGF was quantitatively measured in tumor cytosols from 362 consecutive patients with primary NPBC using an enzyme immunoassay for human VEGF(165). Adjuvant treatment was given to all patients, either as endocrine therapy (n = 250) or chemotherapy (n = 112). The median follow-up time was 56 months. RESULTS: Univariate analysis showed VEGF to be a significant predictor of RFS (P =.0289) and OS (P =.0004) in the total patient population and in patients who received adjuvant endocrine treatment (RFS, P =.0238; OS, P =.0121). In the group of patients who received adjuvant chemotherapy, no significant difference was seen in RFS, but a difference was seen in OS (P =.0235). Patients with bone recurrences tended to have lower VEGF expression (median, 2.17 pg/microg DNA) than patients with visceral metastasis (4.41 pg/microg), brain metastasis (8.29 pg/microg), or soft tissue recurrences (3.16 pg/microg). Multivariate analysis showed nodal status (P =.0004), estrogen receptor (ER) status (P <.0001), and tumor size (P =.0085) to be independent predictors of RFS. VEGF was found to be an independent predictor of OS (P =.0170; relative risk [RR] = 1.82), as were ER (P <.0001; RR = 5.19) and nodal status (P =.0002; RR = 2.58). For patients receiving adjuvant endocrine treatment, multivariate analysis showed VEGF content to be an independent predictor of OS (P =.0420; RR = 1.90) but not of RFS. CONCLUSION: The results suggest that VEGF(165) content in tumor cytosols is a predictor of RFS and OS in primary NPBC. VEGF content might also predict outcome after adjuvant endocrine treatment, but further studies in a prospective setting with homologous treatments are required.

Ascorbate-induced free radical toxicity to isolated islet cells.

Ascorbate is known to be cytotoxic by generating free oxygen radicals, a property shared with the diabetogenic drug alloxan. Some reports indicate that also ascorbate may be diabetogenic. In this study the cytotoxicity of ascorbate on isolated mouse islet cells has been investigated. Ascorbate (0.5-2.0 mmol/l) induced a concentration-dependent increase of trypan blue uptake by the cells, indicating an increase of membrane permeability to the dye. Trypan blue uptake induced by 2.0 mmol/l ascorbate was inhibited by concomitant incubation of the cells with 200 mg/l superoxide dismutase, 200 mg/l catalase, 3.0 mmol/l cytochrome-c or 50 mumol diethylenetriaminepentacetic acid (DTPA), but not by 50 mmol/l D-mannitol. The results indicate that ascorbate is cytotoxic to islet cells by metal-catalysed free radical generation.

Serum dehydroepiandrosterone sulfate in Alzheimer's disease and in multi-infarct dementia.

Circulating levels of dehydroepiandrosterone sulfate (DHEAS) and cortisol were studied in 86 patients with dementia; 45 with Alzheimer's disease and 41 with multi-infarct dementia. Compared to an elderly control group, after adjustment for age and sex, patients with Alzheimer's disease were found to have lower serum levels of DHEAS. We found a covariation between serum albumin and DHEAS levels, which may be of importance regarding peripheral hormone concentration in patients with dementia. These findings may provide evidence for a role of DHEAS in amnestic disorder in humans, either reflecting or contributing to the course of dementing diseases.

Serum interleukin-6 in relation to acute-phase reactants and survival in patients with renal cell carcinoma.

Patients with malignancies often present with signs of inflammatory reactions such as elevated erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Since interleukin-6 (IL-6) is a possible regulator of these reactions and has been proposed as a predictor of prognosis, the aim of the study was to analyse its clinical significance in patients with renal cell carcinoma. Serum samples were collected from 196 patients before any treatment. IL-6 was analysed by an enzyme-linked immunoassay and compared with tumour grade, stage, acute-phase reactants and survival. Patients with renal cell carcinoma had significantly higher IL-6 levels (mean 28.1 +/- 63.4 ng/l; median 8.3 ng/l) compared with controls (mean 1.7 +/- 2.6 ng/l; median 0.5 ng/l; P < 0.001). Serum IL-6 levels in patients with distant metastases were significantly higher than for patients with tumours confined to the kidney (P = 0.02). This difference was more pronounced when serum IL-6 levels in patients with poorly differentiated tumours were compared with well-differentiated tumours (P < 0.001). A significant correlation between the acute-phase reactants CRP, ESR and IL-6 levels was found. Survival time was significantly shorter (P = 0.001) for patients with IL-6 levels above the median serum level compared with patients with lower levels. Similar significant prognostic results were obtained in the group of patients with metastatic disease, but not in group of patients with stage I-III. Serum levels of IL-6 correlated to tumour stage, grade and acute-phase reactants. Increased levels were related to the presence of metastases and adverse survival. Serum IL-6 proved univariate prognostic information but this prognostic significance was lost using a multivariate analysis.

Early morphological detection of estramustine cytotoxicity measured as alteration in cell size and shape by a new technique of microperifusion.

The present study describes a new microscopic perifusion technique for detecting momentary alterations in cell volume and shape. The method has been applied for evaluating early signs of cytotoxicity following chemotherapeutic treatments. The effects of estramustine phosphate (EMP) have been evaluated. EMP is a complex between oestradiol-17 beta and the alkylating agent nor-nitrogen mustard and has recently demonstrated a marked cytotoxicity against malignant glioma cells. The results showed a concentration-dependent increase in cell size and a concomitant decrease in shape factor following EMP-treatment of glioma cells. These changes correlated with cytotoxicity evaluated as cell proliferation and cell membrane alterations shown by 86Rb fluxes and ultrastructural visible membrane damage. The colon cancer line HT-29 displayed no reactions at all following EMP treatment. It is suggested that acute alterations in cell morphology and shape display a strong correlation to the cytotoxicity of EMP encountered by traditional cell culture systems. The findings are discussed with respect to cell membrane disturbances caused by EMP and its potential role as an early test of cytotoxicity.

Impact of serum basic fibroblast growth factor on prognosis in human renal cell carcinoma.

Renal cell carcinoma is often characterised by extensive vascularity and angiogenic factors may be of importance for disease progression. Using a sandwich enzyme immunoassay, basic fibroblast growth factor (bFGF) was analysed in the sera from 206 patients with renal cell carcinoma before the initiation of therapy. The median bFGF level was 3.0 pg/ml (range <1.0-70.9 pg/ml). The serum levels were significantly correlated to tumour stage and nuclear grade. Patients with tumour thrombus to the renal or the inferior caval vein had significantly higher serum bFGF levels compared with those with non-invading tumours (P=0.007). Patients with serum bFGF levels above 3.0 pg/ml had a worse prognosis, compared with those with lower levels (P=0.001). Furthermore, patients with tumours with vein invasion had a worse prognosis compared with those without invasion. After multivariate analysis, only tumour stage and grade remained as independent prognostic factors.

Perivascular cell protection in vivo and increased cell survival in vitro by the antihypertensive agent carvedilol following radiation.

Carvedilol, an antihypertensive drug with activity on adrenoceptors as well as on calcium channel activity, has recently been introduced. In the present study we investigated whether carvedilol interacts with the cytotoxicity induced by irradiation in vitro as well as in vivo. A daily injection of carvedilol in clinically relevant concentrations (3 mg/kg subcutaneously), 4 days before and 3 days after a single radiation dose of 20 Gy significantly decreased the inflammatory reaction in the rat lung, evaluated as number of inflammatory cells in the perivascular area. The density of mast cells was also slightly reduced. In vitro studies revealed that carvedilol caused different radio-protective effects, dependent on dose (1-7 Gy) used and cell line studied. The effects were especially pronounced in a malignant mesothelioma cell line (P-31), and somewhat less evident in a prostatic carcinoma cell line (PC-3). No significant effect was seen in a highly radiosensitive small cell lung cancer cell line (U-1690). Thus, carvedilol may under some circumstances interact with radiation-induced tissue reactions, most probably by a direct interaction at the cellular level. The specific explanation to the differences in sensitivity to carvedilol remains to be evaluated, but the known antioxidative properties and/or scavenging of free radicals of carvedilol may be a plausible mechanism of action. Secondary induced alterations in inflammatory response may also be considered. It is suggested that a potential interaction between drugs such as carvedilol and irradiation should be considered for clinical practice.

Identification of potassium flux pathways and their role in the cytotoxicity of estramustine in human malignant glioma, prostatic carcinoma and pulmonary carcinoma cell lines.

Clinically-used drugs such as furosemide, bumetanide and cardiac glycosides, are modulators of transmembrane fluxes of cations. Recently, it has been suggested that the regulation of intracellular cation concentrations could be a primary target for anti-neoplastic drugs, and that the cytotoxic activity may be altered by inhibitors of cation fluxes at the level of the plasma membrane. Therefore, we investigated the mechanisms by which cations are translocated across the plasma membrane of malignant glioma (U251 MG), prostatic carcinoma (PC3) and pulmonary carcinoma (P31) cell lines. The interactions between cation flux inhibitors and the cytotoxicity of estramustine were also evaluated. Ouabain, the classical inhibitor of Na+, K+ATPase, markedly reduced 86Rb (K+) influx in all three lines, indicating that this ion transport system is present in the cells. Furosemide and especially bumetanide inhibited the 86Rb influx, indicating the presence of the Na+, K+, Cl- co-transport system. The potassium channel blocker, tetraethylammonium, but not apamin reduced the influx of 86Rb showing that high conductance K+ channels are present, but that channels of low conductance probably do not exist in these cell lines. The Na+, K+, Cl- co-transport inhibitors furosemide and bumetanide significantly reduced cytotoxicity of estramustine in P31 cells, whereas no interaction between other K+ flux inhibitors and the anti-neoplastic drugs were detected in any of the cell lines investigated. Thus, the data show that Na+, K+, ATPase and NA+, K+, Cl- co-transport systems and K+ channels of high conductance are present in malignant glioma (U251 MG), prostatic carcinoma (PC3) and pulmonary carcinoma (P31) cell lines, and that inhibition of the Na+, K+, Cl- co-transport system in P31 is associated with reduced cytotoxicity of estramustine. The results justify further studies evaluating the role of these cation flux pathways in terms of targets for anti-neoplastic therapy.

Serotoninergic modulation of cell volume response to estramustine: an image-analysis study on perifused individual glioma cells.

A technique of microscopy with computerised detection of early morphological changes during continuous perifusion was used to monitor the geometry changes of cultured glioma cells (MG-251) when exposed to 40 mg/L estramustine phosphate (EMP) alone or in combination with granisetron (0.1 mumol/L), ondansetron (0.1 mumol/L), or serotonin (1 mumol/L). When the cells were exposed to EMP, cell volume measured as projected cell area (PCA) rapidly increased. Serotonin and ondansetron, but not granisetron, prevented the acute EMP response (PCA). Serotonin, but none of the 5-HT3 receptor antagonists, protected against the cytotoxicity of EMP to the glioma cells as measured by a fluorometric microculture assay. Our results demonstrate hitherto unknown differences between selective 5-HT3 receptor antagonist on the cellular response to EMP and shows the necessity to study the receptor antagonists from viewpoint of interference with the antitumour drug effects on malignant cells. The perifusion technique could be used to study the effects of serotoninergic agonists and antagonists on cell volume regulation of cells exposed to anticancer drugs.

Overexpression of c-erbB-2 is related to a higher expression of vascular endothelial growth factor (VEGF) and constitutes an independent prognostic factor in primary node-positive breast cancer after adjuvant systemic treatment.

The aim of this study was to investigate possible associations between the expression of c-erbB-2 and the angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), p53 status, routine breast cancer prognostic factors and survival. Expression of c-erbB-2, VEGF, bFGF, and p53 protein was determined with an enzyme-linked immunosorbent assay (ELISA) in 656 patients with primary breast cancer (median follow-up time of 83 months). In 60 cases, we also used immunohistochemistry (IHC) for c-erbB-2 evaluation, to be used as a reference for the ELISA. Overexpression of c-erbB-2 was significantly related to a higher expression of VEGF, lower bFGF content, negative steroid receptor status, and a high S-phase fraction. In multivariate analysis, c-erbB-2 was an independent prognostic factor for relapse-free survival (RFS) and overall survival (OS) in all patients, and in node-positive patients, irrespective of the adjuvant systemic therapy. Combined survival analyses regarding c-erbB-2 and VEGF yielded additional prognostic information.

Growth hormone deficiency impairs blood clotting and reduces factor VII coagulant activity in rat.

To investigate pituitary effects on the vitamin K-dependent coagulation factors, female rats were hypophysectomized (hypox) and treated with growth hormone (GH), cortisone, thyroxine, vitamin K, or saline. After 11 days of treatment, the prothrombin time, platelet count, and factors II, VII, IX, and X were determined. The prothrombin time was 52.9 +/- 1.2% for control rats and 39.1 +/- 0.8% for hypox rats (mean +/- SEM; p < 0.001). All factors decreased after hypophysectomy, reaching significance for factor VII (from 264 +/- 23% to 131 +/- 9%; p < 0.001) and factor IX (from 28.4 +/- 2.2% to 17.1 +/- 2.5%; p < 0.01) while the platelet count was unaffected. When hypox rats were treated with GH, the prothrombin time increased to 50.9 +/- 1.0% (p < 0.001) and factor VII to 299 +/- 10% (p < 0.001). Factor II, IX, and X were slightly increased after GH substitution but not after cortisone, thyroxine, or vitamin K treatment. To summarize, GH is of importance for normal hemostasis in the female rat.

Protective effect of iron chelators on epirubicin-induced fibroblast toxicity.

Free oxygen radicals generated by anthracycline/iron complexes have been implicated in anthracycline cytotoxicity. We therefore tested whether enzymatic scavengers of free radicals or metal chelators were able to inhibit anthracycline toxicity. The survival of Chinese hamster fibroblasts was reduced when the cells were exposed to 0.1-1.0 mg/l 4'-epidoxorubicin (epirubicin). Superoxide dismutase (SOD) (250 mg/l), or catalase (250 mg/l) did not affect the clonogenic survival of the fibroblasts. The metal-chelators, diethylenetriamine-pentaacetic acid (DTPA) (100 mumol/l), EDTA (100 mumol/l), and desferrioxamine (100 mumol/l) all protected against epirubicin-induced clonogenic survival. The protection of chelators against epirubicin toxicity implies that chelators might also be able to modulate anthracycline toxicity in vivo.

Amiloride but not bumetanide protects against the cytotoxic effects of estramustine and bleomycin in cultured fibroblasts.

The effects of the clinically used diuretics amiloride (an inhibitor of Na+/H+ exchange) and bumetanide (an inhibitor of Na+, K+, Cl- co-transport) were tested on the cytotoxicity of estramustine and bleomycin in cultured fibroblasts. Both estramustine (50 micrograms/ml) and bleomycin (10 micrograms/ml) reduced the number of surviving clones. Amiloride (100 microM) but not bumetanide (100 microM) partly protected against the cytotoxic effect of both estramustine and bleomycin. The protective effect of the combination of amiloride and bumetanide was not stronger than the effect of amiloride alone. Amiloride or bumetanide alone did not affect the clonal survival. It is suggested that the protective effect of amiloride is not mediated by an effect of the molecular mechanism of cytotoxicity but may rather be due to changes in cellular metabolism and/or drug handling.