Validation of a zebrafish developmental defects assay as a qualified alternative test for its regulatory use following the ICH S5(R3) guideline.

Zebrafish is a popular toxicology model and provides an ethically acceptable small-scale analysis system with the complexity of a complete organism. Our goal is to further validate this model for its regulatory use for reproductive and developmental defects by testing the compounds indicated in the "Guideline on detection of reproductive and developmental toxicity for human pharmaceuticals" (ICH S5(R3) guideline.) To determine the embryotoxic and developmental risk of the 32 reference compounds listed in the ICH S5(R3) guideline, the presence of morphological alterations in zebrafish embryos was analyzed at two different stages to calculateLC50 and EC50 values for each stage. Teratogenic Indexes were established as the ratio between LC50 and EC50 critical for the proper compound classification as teratogenic when it is ≥ 2. A total of three biological replicates have been conducted to study the reproducibility of the assay. The chemicals' concentration in the medium and internally in the zebrafish embryos was evaluated. In this study, the 3 negative compounds were properly categorized while 23 compounds out of the 29 reference ones (sensitivity of 79.31%) were classified as teratogenic in zebrafish. The 6 that had false-negative results were classified 4 as inconclusive, 1 as not toxic, and 1 compound resulted toxic for zebrafish embryos under testing conditions. After the bioavailability experiments, some of the obtained inconclusive results were refined. The developmental defects assay in zebrafish gives an accuracy of 89.66%, sensitivity of 88.46%, specificity and repeatability of 100% compared to mammals; therefore, this is a well-integrated strategy using New Alternative Methods, to minimize the use of animals in developmental toxicity studies.

Retrospective analysis of the prognostic implications of tumor spread through air spaces in lung adenocarcinoma patients treated with surgery.

BACKGROUND: Tumor spread through air spaces (STAS) in lung adenocarcinoma is a novel mechanism of invasion. STAS has been proposed as an independent predictor of poor prognosis. The aim of this study was to evaluate the correlations between STAS status and other clinicopathologic variables and to assess the prognostic implications of STAS and the distance from the edge of the tumor to the farthest STAS in patients with resected lung adenocarcinoma. MATERIAL AND METHODS: This is a single-institution retrospective observational study. We included all patients with resected lung adenocarcinoma from January 2017 to December 2018 at La Paz University Hospital. The cut-off for the distance from the edge of the tumor to the farthest STAS was 1.5 mm and was assessed by the area under the receiver operating characteristic curve. RESULTS: A total of 73 patients were included. STAS was found in 52 patients (71.2%). Histological grade 3 (P = 0.035) and absence of lepidic pattern (P = 0.022) were independently associated with the presence of STAS. The median recurrence-free survival (RFS) was 48.06 months [95% confidence interval (CI) 33.58 months to not reached]. STAS-positive patients had shorter median RFS [39.23 months (95% CI 29.34-49.12 months)] than STAS-negative patients (not reached) (P = 0.04). STAS-positive patients with a distance from the edge of the tumor to the farthest STAS ≥1.5 mm had an even shorter median RFS [37.63 months (95% CI 28.14-47.11 months)]. For every 1 mm increase in distance, the risk of mortality increased by 1.26 times (P = 0.04). CONCLUSIONS: Histological grade 3 and absence of lepidic pattern were independently associated with the presence of STAS. STAS was associated with a higher risk of recurrence. The distance from the edge of the tumor to the farthest STAS also had an impact on overall survival.

Malignant pleural mesothelioma: clinical experience and prognostic value of derived neutrophil-to-lymphocyte ratio and PD-L1 expression

Background Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor, with a poor prognosis. MPM needs to find prognostic factors of survival. We provided the management of patients with MPM and sought to determine whether pre-treatment levels of derived neutrophil-to-lymphocyte ratio (dNLR) as well as PD-L1 expression were reliable prognostic factors of survival. Methods We conducted a single-institution retrospective study, including all patients with MPM treated at La Paz University Hospital between December 2009 and March 2018. Baseline disease, demographics, clinical data, treatment characteristics and complete blood cell counts were collected. We examined dNLR at baseline and data for PD-L1 expression were analyzed in tumor cells by immunohistochemistry. Results We included 25 patients. The median overall survival (OS) was 15.7 months (95% CI 11.3–20.0). 5 patients had a dNLR greater than 3 (20%). Patients with a dNLR greater than 3 had shorter median OS (8.5 months), than patients with a dNLR less than 3 (17.0 months), with statistically significant differences (p = 0.038). Ten patients (40%) had positive PD-L1 expression (≥ 1%). Patients with positive PD-L1 expression had shorter median OS (8.5 months) than patients with negative PDL1 expression (15.7 months), but without statistically significant association (p = 0.319). Conclusion The survival data obtained in our sample are consistent with those previously reported. Pretreatment levels of dNLR greater than 3 and positive PD-L1 expression could be significant prognostic factors for poor survival in patients with MPM. Further and prospective studies are needed to explore this relationship and to derive definitive conclusions (AU)

Prognostic factors in advanced pancreatic ductal adenocarcinoma patients-receiving second-line treatment: a single institution experience

Background Second-line (2L) treatments for advanced pancreatic ductal adenocarcinoma (PDAC) achieve a modest benefit at the expense of potential toxicity. In the absence of predictive factors of response, the identification of prognostic factors could help in the therapeutic decisions-making. The purpose of this study was to assess the prognostic factors associated with shorter survival in patients with advanced PDAC who received 2L treatment. Methods We conducted a single institution retrospective study, which included all patients with advanced PDAC who received 2L treatment between September 2006 and February 2020 at La Paz University Hospital, Madrid (Spain). Significant variables in the logistic regression model were used to create a prognostic score. Results We included 108 patients. The median overall survival (OS) was 5.10 months (95%CI 4.02–6.17). In the multivariate analysis, time to progression (TTP) shorter than 4 months after first-line treatment (OR 4.53 [95%CI 1.28–16.00] p = 0.01), neutrophil-to-lymphocyte ratio (NLR) greater than 3 at the beginning of 2L (OR 9.07 [95%CI 1.82–45.16] p = 0.01) and CA-19.9 level higher than the upper limit of normal at the beginning of 2L (OR 7.83 [95%CI 1.30–49.97] p = 0.02) were independently associated with OS shorter than 3 months. The prognostic score classified patients into three prognostic groups (good, intermediate and poor) with significant differences in OS (p < 0.001). Conclusions TTP shorter than 4 months after first-line treatment, NLR greater than 3 and CA-19.9 level higher than the upper limit of normal at the beginning of 2L were associated with shorter overall survival. We developed a prognostic score that classifies patients with advanced PDAC into three prognostic groups after progression to the first-line. This score could help in the decision-making for 2L treatment (AU)

Prognostic value of neutrophil-to-lymphocyte ratio in advanced cancer patients receiving immunotherapy

Background The prognostic value of neutrophil-to-lymphocyte ratio (NLR) has been extensively studied in cancer patients. However, the performance of NLR as an early marker of efficacy of immune checkpoint inhibitors (ICI) is still understudied. We studied the utility of NLR at baseline (bNLR), before the second dose of immunotherapy (NLR2) and the NLR trend for predicting efficacy outcomes. Methods We included all patients with advanced cancer treated with ICI from June 2013 to April 2019 at La Paz University Hospital, Madrid (Spain). We examined bNLR, NLR2 and NLR trend and explored the association with progression-free survival (PFS) at 6 months, median PFS and overall survival (OS). Results We included 211 patients. PFS and OS were significantly longer in the low bNLR group than in the high bNLR group [HR 0.71 (95% CI 0.60–0.84) and HR: 0.66 (95% CI 0.55–0.79), respectively]. Regarding NLR2, patients with low NLR2 had significantly longer PFS and OS than patients with high NLR2 [HR 0.67 (95% CI 0.57–0.79) and HR: 0.60 (95% CI 0.50–0.72), respectively]. Finally, for NLR trend, PFS and OS for patients with NLR trend < 1 were significantly longer than those patients with NLR trend ≥ 1 [HR 0.59 (95% CI 0.43–0.82) and HR 0.63 (95% CI 0.44–0.90), respectively]. At the multivariate analysis for PFS and OS, bNLR, NLR2 and NLR trend were all independent prognostic factors for PFS and OS. Conclusions bNLR, NLR2 and NLR trends are independent prognostic factors for survival in patients on immunotherapy. The dynamics of NLR in patients on immunotherapy is a promising marker that needs further investigation (AU)

Incidence of venous thromboembolic events in cancer patients receiving immunotherapy: a single-institution experience

Background Cancer and cancer therapies have been associated with an increased incidence of venous thromboembolic events (VTE). However, the incidence of VTE in patients on immunotherapy has not been well characterized. The aim of this study was to assess the incidence of VTE in cancer patients receiving immunotherapy and ascertain its prognostic utility. Materials and methods We conducted a single-institution retrospective study, including all cancer patients treated with anti-Programmed cell Death 1 (PD-1), anti-Programmed cell Death Ligand-1 (PD-L1), anti-Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA4), a combination of anti-PD-1/anti-PD-L1 and anti-CTLA4 or a combination including any of these drugs with chemotherapy, antiangiogenic agents or both between June 2013 and April 2019 at La Paz University Hospital, Madrid (Spain). Results We selected 229 patients. VTE occurred in 16 of 229 patients (7%). VTE occurred more frequently in patients with lung cancer followed by melanoma. Female sex and melanoma were independently associated with an increased risk of VTE. 12 of 16 VTE (75%) were symptomatic. Progressive disease to immunotherapy [HR 31.60 (95% CI 11.44–87.22), p = 0.00], lung cancer [HR 2.55 (95% CI 1.34–4.86), p = 0.00] and melanoma [HR 2.42 (1.20–4.86), p = 0.01] were independently associated with shorter OS. VTE occurrence was not independently associated with shorter OS [HR 1.33 (95% CI 0.63–2.80), p = 0.44]. Conclusions The incidence of VTE in cancer patients receiving immunotherapy in our study appeared to be similar to the incidence previously reported in other series of cancer patients treated with systemic therapies. VTE occurrence did not correlate with the prognosis. Further and prospective studies are needed to derive definitive conclusions (AU)

Prognostic factors in advanced pancreatic ductal adenocarcinoma patients-receiving second-line treatment: a single institution experience.

BACKGROUND: Second-line (2L) treatments for advanced pancreatic ductal adenocarcinoma (PDAC) achieve a modest benefit at the expense of potential toxicity. In the absence of predictive factors of response, the identification of prognostic factors could help in the therapeutic decisions-making. The purpose of this study was to assess the prognostic factors associated with shorter survival in patients with advanced PDAC who received 2L treatment. METHODS: We conducted a single institution retrospective study, which included all patients with advanced PDAC who received 2L treatment between September 2006 and February 2020 at La Paz University Hospital, Madrid (Spain). Significant variables in the logistic regression model were used to create a prognostic score. RESULTS: We included 108 patients. The median overall survival (OS) was 5.10 months (95%CI 4.02-6.17). In the multivariate analysis, time to progression (TTP) shorter than 4 months after first-line treatment (OR 4.53 [95%CI 1.28-16.00] p = 0.01), neutrophil-to-lymphocyte ratio (NLR) greater than 3 at the beginning of 2L (OR 9.07 [95%CI 1.82-45.16] p = 0.01) and CA-19.9 level higher than the upper limit of normal at the beginning of 2L (OR 7.83 [95%CI 1.30-49.97] p = 0.02) were independently associated with OS shorter than 3 months. The prognostic score classified patients into three prognostic groups (good, intermediate and poor) with significant differences in OS (p < 0.001). CONCLUSIONS: TTP shorter than 4 months after first-line treatment, NLR greater than 3 and CA-19.9 level higher than the upper limit of normal at the beginning of 2L were associated with shorter overall survival. We developed a prognostic score that classifies patients with advanced PDAC into three prognostic groups after progression to the first-line. This score could help in the decision-making for 2L treatment.

Malignant pleural mesothelioma: clinical experience and prognostic value of derived neutrophil-to-lymphocyte ratio and PD-L1 expression.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor, with a poor prognosis. MPM needs to find prognostic factors of survival. We provided the management of patients with MPM and sought to determine whether pre-treatment levels of derived neutrophil-to-lymphocyte ratio (dNLR) as well as PD-L1 expression were reliable prognostic factors of survival. METHODS: We conducted a single-institution retrospective study, including all patients with MPM treated at La Paz University Hospital between December 2009 and March 2018. Baseline disease, demographics, clinical data, treatment characteristics and complete blood cell counts were collected. We examined dNLR at baseline and data for PD-L1 expression were analyzed in tumor cells by immunohistochemistry. RESULTS: We included 25 patients. The median overall survival (OS) was 15.7 months (95% CI 11.3-20.0). 5 patients had a dNLR greater than 3 (20%). Patients with a dNLR greater than 3 had shorter median OS (8.5 months), than patients with a dNLR less than 3 (17.0 months), with statistically significant differences (p = 0.038). Ten patients (40%) had positive PD-L1 expression (≥ 1%). Patients with positive PD-L1 expression had shorter median OS (8.5 months) than patients with negative PDL1 expression (15.7 months), but without statistically significant association (p = 0.319). CONCLUSION: The survival data obtained in our sample are consistent with those previously reported. Pretreatment levels of dNLR greater than 3 and positive PD-L1 expression could be significant prognostic factors for poor survival in patients with MPM. Further and prospective studies are needed to explore this relationship and to derive definitive conclusions.

Incidence of venous thromboembolic events in cancer patients receiving immunotherapy: a single-institution experience.

BACKGROUND: Cancer and cancer therapies have been associated with an increased incidence of venous thromboembolic events (VTE). However, the incidence of VTE in patients on immunotherapy has not been well characterized. The aim of this study was to assess the incidence of VTE in cancer patients receiving immunotherapy and ascertain its prognostic utility. MATERIALS AND METHODS: We conducted a single-institution retrospective study, including all cancer patients treated with anti-Programmed cell Death 1 (PD-1), anti-Programmed cell Death Ligand-1 (PD-L1), anti-Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA4), a combination of anti-PD-1/anti-PD-L1 and anti-CTLA4 or a combination including any of these drugs with chemotherapy, antiangiogenic agents or both between June 2013 and April 2019 at La Paz University Hospital, Madrid (Spain). RESULTS: We selected 229 patients. VTE occurred in 16 of 229 patients (7%). VTE occurred more frequently in patients with lung cancer followed by melanoma. Female sex and melanoma were independently associated with an increased risk of VTE. 12 of 16 VTE (75%) were symptomatic. Progressive disease to immunotherapy [HR 31.60 (95% CI 11.44-87.22), p = 0.00], lung cancer [HR 2.55 (95% CI 1.34-4.86), p = 0.00] and melanoma [HR 2.42 (1.20-4.86), p = 0.01] were independently associated with shorter OS. VTE occurrence was not independently associated with shorter OS [HR 1.33 (95% CI 0.63-2.80), p = 0.44]. CONCLUSIONS: The incidence of VTE in cancer patients receiving immunotherapy in our study appeared to be similar to the incidence previously reported in other series of cancer patients treated with systemic therapies. VTE occurrence did not correlate with the prognosis. Further and prospective studies are needed to derive definitive conclusions.

Prognostic value of neutrophil-to-lymphocyte ratio in advanced cancer patients receiving immunotherapy.

BACKGROUND: The prognostic value of neutrophil-to-lymphocyte ratio (NLR) has been extensively studied in cancer patients. However, the performance of NLR as an early marker of efficacy of immune checkpoint inhibitors (ICI) is still understudied. We studied the utility of NLR at baseline (bNLR), before the second dose of immunotherapy (NLR2) and the NLR trend for predicting efficacy outcomes. METHODS: We included all patients with advanced cancer treated with ICI from June 2013 to April 2019 at La Paz University Hospital, Madrid (Spain). We examined bNLR, NLR2 and NLR trend and explored the association with progression-free survival (PFS) at 6 months, median PFS and overall survival (OS). RESULTS: We included 211 patients. PFS and OS were significantly longer in the low bNLR group than in the high bNLR group [HR 0.71 (95% CI 0.60-0.84) and HR: 0.66 (95% CI 0.55-0.79), respectively]. Regarding NLR2, patients with low NLR2 had significantly longer PFS and OS than patients with high NLR2 [HR 0.67 (95% CI 0.57-0.79) and HR: 0.60 (95% CI 0.50-0.72), respectively]. Finally, for NLR trend, PFS and OS for patients with NLR trend < 1 were significantly longer than those patients with NLR trend ≥ 1 [HR 0.59 (95% CI 0.43-0.82) and HR 0.63 (95% CI 0.44-0.90), respectively]. At the multivariate analysis for PFS and OS, bNLR, NLR2 and NLR trend were all independent prognostic factors for PFS and OS. CONCLUSIONS: bNLR, NLR2 and NLR trends are independent prognostic factors for survival in patients on immunotherapy. The dynamics of NLR in patients on immunotherapy is a promising marker that needs further investigation.

Prognostic value of neutrophil-to-lymphocyte ratio and other inflammatory markers in patients with high-risk soft tissue sarcomas.

BACKGROUND: Soft tissue sarcomas (STS) have a high risk of relapse in spite of the use of (neo)adjuvant chemotherapy. In this context, looking for new prognostic biomarkers is an interesting field of research. Our aim is to analyze the prognostic impact of neutrophil-to-lymphocyte ratio (NLR) and other serum markers in patients with STS who received chemotherapy with curative intent. MATERIALS AND METHODS: This is a retrospective observational study. We included all patients with STS (primary tumor, local recurrence or resected metastatic disease) treated with high-dose ifosfamide and epirubicin with curative intent from January 2007 to December 2018. The pretreatment NLR and other serum markers were calculated, selecting the median as the cut-off value for the survival and multivariate analysis. RESULTS: Seventy-nine patients were included. Median NLR, platelet-to-lymphocyte ratio (PLR) and lymphocyte-to-monocyte ratio (LMR) were 2.83, 174.05 and 3.25, respectively. Median progression-free survival (PFS) was significantly longer in patients with low NLR [not reached (NR) vs 21, 92 months, P < 0.01]. No significant differences were found for PFS regarding PLR or LMR. For overall survival (OS), a significant survival advantage was also found for patients with low NLR (NR vs 65.45 months, P = 0.01), without differences for PLR or LMR. In multivariate analysis, NLR remains an independent prognostic factor for PFS. CONCLUSION: In our cohort, low NLR was significantly associated with a longer PFS and OS, and is consolidated as an independent prognostic factor.

Magnetoresistive immunosensor for the detection of Escherichia coli O157:H7 including a microfluidic network.

A hand held device has been designed for the immunomagnetic detection and quantification of the pathogen Escherichia coli O157:H7 in food and clinical samples. In this work, a technology to manufacture a Lab on a Chip that integrates a 3D microfluidic network with a microfabricated biosensor has been developed. With this aim, the sensing film optimization, the design of the microfluidic circuitry, the development of the biological protocols involved in the measurements and, finally, the packaging needed to carry out the assays in a safe and straightforward way have been completed. The biosensor is designed to be capable to detect and quantify small magnetic field variations caused by the presence of superparamagnetic beads bound to the antigens previously immobilized on the sensor surface via an antibody-antigen reaction. The giant magnetoresistive multilayer structure implemented as sensing film consists of 20[Cu(5.10nm)/Co(2.47 nm)] with a magnetoresistance of 3.20% at 235Oe and a sensitivity up to 0.06 Omega/Oe between 150Oe and 230Oe. Silicon nitride has been selected as optimum sensor surface coating due to its suitability for antibody immobilization. In order to guide the biological samples towards the sensing area, a microfluidic network made of SU-8 photoresist has been included. Finally, a novel packaging design has been fabricated employing 3D stereolithographic techniques. The microchannels are connected to the outside using standard tubing. Hence, this packaging allows an easy replacement of the used devices.

Ecstasy-induced toxicity in rat liver.

BACKGROUND/AIMS: The aim of the present study was to examine the effects of single and repeated administration of 3,4-methylenedioxyme-thamphetamine (MDMA, "ecstasy") on rat liver. METHODS: Animals were given an acute (20 mg/kg) and repeated (20 mg/kg, b.i.d., for 4 consecutive days) intraperitoneal dose of MDMA, and at various times after administration the hepatic and serum determinations were made. RESULTS: The effect of acute MDMA administration included increased triglyceride and cholesterol levels and an increase in all enzyme activities 6 h post administration. The toxic effect of MDMA was also observed in other hepatic processes. Glycogen content showed a marked decrease, which was accompanied by a decrease in serum glucose levels. No significant changes in lipid peroxidation and hepatic GSH content were observed. In contrast, multiple MDMA administration produced some evidence of oxidative stress, namely, increased MDA content and decreased GSH content, a small decrease in liver glycogen at 3 h recovering 6 h post dose, no effect on blood glucose and increased AST and ALP activities but no effects on ALT activity. Seven days after the last MDMA injection a tendency towards recovery was shown. CONCLUSION: Our results show that the liver toxicity caused by MDMA administration involves several mechanisms.

3,4-Methylenedioxymethamphetamine (ecstasy)-induced hepatotoxicity: effect on cytosolic calcium signals in isolated hepatocytes.

AIMS/BACKGROUND: Hepatocellular damage has been reported as a consequence of 3,4-methylenedioxymethamphetamine (MDMA) intake. However, little is known about the cellular mechanisms involved. The present study was undertaken to evaluate the effects of MDMA on cell viability as well as free calcium levels ([Ca2+]i) in short-term cultured hepatocytes. Reduced glutathione (GSH), adenosine-5'-triphosphate (ATP) and lipid peroxidation were investigated to evaluate the toxic effect of MDMA, in vitro, using freshly isolated rat hepatocytes. METHODS: In order to measure cytosolic free Ca2+ concentrations ([Ca2+]i), rat hepatocytes were loaded with the Ca2+ indicator fura-2-acetoxymethylester (fura-2-AM). RESULTS: A sustained rise of ([Ca2+]i) after incubation with MDMA was the most noteworthy finding. In Ca2+-free medium, MDMA caused a reduced increase of ([Ca2+]i). On the other hand, MDMA (0.1-5 mM) induced a concentration-dependent and time exposure-dependent GSH and ATP depletion. Although it did not reach statistical significance, GSH deficits were accompanied by a tendency to increase lipid peroxidation 3 h after MDMA incubation. CONCLUSIONS: The above data suggest that the marked rise of ([Ca2+]i) and subsequent ATP and GSH depletion can lead to a rapid decrease in cell viability.

Cloning of two genes encoding potassium transporters in Neurospora crassa and expression of the corresponding cDNAs in Saccharomyces cerevisiae.

Two Neurospora crassa genes, trk-1 and hak-1, encode K+ transporters that show sequence similarities to the TRK transporters described in Saccharomyces cerevisiae and Schizosaccharomyces pombe, and to the HAK transporters described in Schwanniomyces occidentalis and barley. The N. crassa TRK1 and HAK1 transporters expressed by the corresponding cDNAs in a trk1 delta trk2 delta mutant of S. cerevisiae exhibited a high affinity for Rb+ and K+. Northern blot analysis and comparison of the kinetic characteristics of the two transporters in the trk1 delta trk2 delta mutant with the kinetic characteristics of K+ uptake in N. crassa cells allowed TRK1 to be identified as the dominant K+ transporter and HAK1 as a transporter that is only expressed when the cells are K+ starved. The HAK1 transporter showed a high concentrative capacity and is identified as the K(+)-H+ symporter described in N. crassa, whereas TRK1 might be a K+ uniporter. Although the co-existence of K+ transporters of the TRK and HAK types in the same species had not been reported formerly, we discuss whether this co-existence may be the normal situation in soil fungi.

Hepatotoxicity of ethanol: protective effect of calcium channel blockers in isolated hepatocytes.

This study examines the effects of three calcium channel blockers (verapamil, nifedipine and diltiazem) on isolated rat hepatocytes exposed to ethanol. In the first part of our study, hepatocytes were incubated with increasing concentrations of ethanol (100, 300, 500, 1000 mM) for varying times. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) release were measured to evaluate the cytotoxic effects of ethanol. The concentration of 300 mM and time of incubation of 45 min were chosen for cytoprotection experiments in which calcium channel blockers, at two different concentrations, were added to the medium 30 min prior to the addition of ethanol. ALT, AST and LDH release as well as lipid peroxidation and cellular reduced glutathione (GSH) were measured. Nifedipine and verapamil (25 microM) reduced ALT, AST and LDH activities. The highest dose of diltiazem (50 microM) was more effective than the lowest one (25 microM). Ethanol caused a significant depletion of cellular GSH content as well as a moderate enhancement of lipid peroxidation. While none of the three calcium channel blockers was able to restore the decrease in GSH levels, diltiazem (25 microM) and nifedipine (50 microM) showed the greatest effect, significantly reducing lipid peroxidation.