Physiological performance of common carp (Cyprinus carpio, L., 1758) exposed to a sublethal copper/zinc/cadmium mixture.

In a natural ecosystem, fish are subjected to a multitude of variable environmental factors. It is important to analyze the impact of combined factors to obtain a realistic understanding of the mixed stress occurring in nature. In this study, the physiological performance of juvenile common carp (Cyprinus carpio) exposed for one week to an environmentally relevant metal mixture (4.8 µg/L of copper; 2.9 µg/L of cadmium and 206.8 µg/L of zinc) and to two temperatures (10 °C and 20 °C), were evaluated. After 1, 3 and 7 days, standard (SMR) and maximum metabolic rate (MMR) were measured and aerobic scope (AS) was calculated. In addition, hematocrit, muscle lactate, histology of the gills and metal accumulation in gills were measured. While SMR, MMR and AS were elevated at the higher temperature, the metal mixture did not have a strong effect on these parameters. At 20 °C, SMR transiently increased, but no significant changes were observed for MMR and AS. During metal exposure, hematocrit levels were elevated in the 20 °C group. The bioaccumulation of Cd in the gills reflected the increased metabolic rate at the higher temperature, with more accumulation at 20 °C than at 10 °C. Anaerobic metabolism was not increased, which corresponds with the lack of significant histopathological damage in the gill tissue. These results show that common carp handled these metal exposures well, although increased temperature led to higher Cd accumulation and necessitated increased hematocrit levels to maintain aerobic performance.

Antagonistic bioaccumulation of waterborne Cu(II) and Cd(II) in common carp (Cyprinus carpio) and effects on ion-homeostasis and defensive mechanisms.

In the aquatic environment, metals are present as mixtures, therefore studies on mixture toxicity are crucial to thoroughly understand their toxic effects on aquatic organisms. Common carp (Cyprinus carpio) were used to assess the effects of short-term Cu(II) and Cd(II) mixtures, using a fixed concentration of one of the metals, representing 25 % of its individual 96h-LC50 (concentration lethal for 50 % of the population) combined with a variable concentration of the other metal corresponding to 10, 25 or 50 % of its 96h-LC50, and vice versa. Our results showed a fast Cu and Cd bioaccumulation, with the percentage of increase in the order gill > liver > carcass. An inhibitory effect of Cu on Cd uptake was observed; higher Cu concentrations at fixed Cd levels resulted in a decreased accumulation of Cd. The presence of the two metal ions resulted in losses of total Na, K and Ca. Fish tried to compensate for the Na loss through the induction of the genes coding for Na+/K+-ATPase and H+-ATPase. Additionally, a counterintuitive induction of the gene encoding the high affinity copper transporter (CTR1) occurred, while a downregulation was expected to prevent further metal ion uptake. An induction of defensive mechanisms, both metal ion binding protein and anti-oxidant defences, was observed. Despite the metal accumulation and electrolyte loss, the low mortality suggest that common carp is able to cope with these metal levels, at least during a one-week exposure.

Marine species as safe source of LC-PUFA and micronutrients: Insights in new promising marine food in Peru.

Seafood could be a promising way to supplement healthy fatty acids and trace elements to the Peruvian diet. Seafood from northern Peru was characterized with the highest relative concentrations of long-chain polyunsaturated fatty acids (LC-PUFAs), while in the center region marine species had the lowest As and Pb contents. Peruvian marine species are rich in LC-PUFAs and micro-nutrients (Cu, Fe, Mn, Zn), including species considered as potentially edible (e.g. Cycloxanthops sexdecimdentatus), but also non-edible species (e.g. Caulerpa filiformis). Nevertheless, it is crucial to consider toxic metals, e.g. As and Cd, which could pose a risk for consumers. High levels of beneficial LC-PUFAs and micro-nutrients would be taken up (up to 80% of the recommended values) when the Peruvian population would consume the estimated safe amount of seafood. Scoring species for fatty acid and metal content resulted in gastropods (e.g. Bursa ventricosa) as being the least beneficial species.

Investigating the effects of a sub-lethal metal mixture of Cu, Zn and Cd on bioaccumulation and ionoregulation in common carp, Cyprinus carpio.

The aquatic environment is continuously under threat because it is the final receptor and sink of waste streams. The development of industry, mining activities and agriculture gave rise to an increase in metal pollution in the aquatic system. Thus a wide occurrence of metal mixtures exists in the aquatic environment. The assessment of mixture stress remains a challenge considering that we can not predict the toxicity of a mixture on the basis of single compounds. Therefore the analysis of the effects of environmentally relevant waterborne mixtures is needed to improve our understanding of the impact of metal pollution in aquatic ecosystems. Our aim was to assess whether 10 % of the concentration of the 96 h LC50 (the concentration that is lethal to 50 % of the population in 96 h) of individual metal exposures can be considered as a "safe" concentration when applied in a trinomial mixture. Therefore, common carp were exposed to a sublethal mixture of Cu 0.07 ±â€¯0.001 µM (4.3 ±â€¯0.6 µg/L), Zn 2.71 ±â€¯0.81 µM (176.9 ±â€¯52.8 µg/L) and Cd 0.03 ±â€¯0.0004 µM (3.0 ±â€¯0.4 µg/L) at 20 °C for a period of one week. Parameters assessed included survival rate, bioaccumulation and physiological biomarkers related to ionoregulation and defensive mechanisms such as MT induction. Our results showed a sharp increase in Cu and Cd concentration in gills within the first day of exposure while Zn levels remained stable. The accumulation of these metals led to a Na drop in gills, liver and muscle as well as a decreased K content in the liver. Biomarkers related to Na uptake were also affected: on the first day gene expression for H+-ATPase was transiently increased while a concomitant decreased gene expression of the Na+/H+ exchanger occurred. A fivefold induction of metallothionein gene expression was reported during the entire duration of the experiment. Despite the adverse effects on ionoregulation all fish survived, indicating that common carp are able to cope with these low metal concentrations, at least during a one week exposure.

Potential health risks via consumption of six edible shellfish species collected from Piura - Peru.

Scallops and their potential predators were collected in Sechura Bay and in front of the Illescas Reserved Zone (north Peru), during El Niño-Southern Oscillation (ENSO) 2016, and analyzed for the metals chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), cadmium (Cd) and lead (Pb). This study showed that ~20% of the molluscs exceeded the maximum residual levels (MRLs) for human consumption in inorganic As, while ~30% of the crustaceans did. For Cd, around 10% and 40% of the molluscs and the crustaceans were above the MRLs, respectively. The cephalopod Octopus mimus exhibited As concentrations, but not Cd concentrations, that exceeded the MRLs. Cr, Ni, Cu, Zn and Pb in muscle exhibited generally concentrations below the MRLs. Integrated risk indices were estimated to determine if there is a health risk for consumption. Target hazard quotients (THQs) and total hazard indices (HIs) were mostly < 1, implying no human health risk. Provisional tolerable weekly intake (PTWI) for Cd was exceeded in Bursa ventricosa at Illescas Reserved Zone. Target cancer risks (TRs) for inorganic As were always higher than the threshold (1 × 10-6), therefore an actual cancer risk is present.

Gene expression profiling of giant cell tumor of bone reveals downregulation of extracellular matrix components decorin and lumican associated with lung metastasis.

Giant cell tumor of bone (GCTB) displays worrisome clinical features such as local recurrence and occasionally metastatic disease which are unpredictable by morphology. Additional routinely usable biomarkers do not exist. Gene expression profiles of six clinically defined groups of GCTB and one group of aneurysmal bone cyst (ABC) were determined by microarray (n = 33). The most promising differentially expressed genes were validated by Q-PCR as potential biomarkers in a larger patient group (n = 41). Corresponding protein expression was confirmed by immunohistochemistry. Unsupervised hierarchical clustering reveals a metastatic GCTB cluster, a heterogeneous, non-metastatic GCTB cluster, and a primary ABC cluster. Balanced score testing indicates that lumican (LUM) and decorin (DCN) are the most promising biomarkers as they have lower level of expression in the metastatic group. Expression of dermatopontin (DPT) was significantly lower in recurrent tumors. Validation of the results was performed by paired and unpaired t test in primary GCTB and corresponding metastases, which proved that the differential expression of LUM and DCN is tumor specific rather than location specific. Our findings show that several genes related to extracellular matrix integrity (LUM, DCN, and DPT) are differentially expressed and may serve as biomarkers for metastatic and recurrent GCTB.

Difluorodeoxyuridine plasma concentrations after low-dose gemcitabine during chemoradiation in head and neck cancer patients.

PURPOSE: The aim of this study was to investigate whether relevant plasma levels of dFdU could be detected during concurrent chemoradiation (CRT) with low doses of dFdC administered in patients with head and neck cancer and to assess the toxicity related to dose. METHODS: dFdC was administered at doses of 5 mg/m² twice weekly or 10, 50, or 100 mg/m² weekly. Plasma concentrations of dFdU were determined daily for 7 days after the first administration and before each administration, thereafter. A high-performance liquid chromatographic method was used. During CRT, skin and mucosal toxicity were scored weekly according to the RTOG toxicity scoring system. RESULTS: Eight patients were sampled at the 10-50 mg/m² dose and nine at the 5-100 mg/m² dose. dFdU levels were in the micromolar range, inducing RS in vitro. There was a strong correlation between the area under the curve of dFdU and the dose of dFdC (r = 0.803, P < 0.001) and a weak correlation between trough concentrations and total dose of dFdC (r = 0.408, P = 0.017). Duration of severe mucositis correlated with dFdC dose. CONCLUSIONS: During CRT with 10-100 mg/m(2) of dFdC weekly or 5 mg/m(2) twice weekly, dFdU remains detectable at potentially radiosensitizing concentrations.

Telomere biology in giant cell tumour of bone.

Giant cell tumour of bone (GCTB) is a benign bone tumour known for the unpredictable clinical behaviour of recurrences and, in rare instances, distant metastases. It consists of uniformly distributed osteoclastic giant cells in a background of mononuclear rounded and spindle-shaped cells. Cytogenetically, telomeric associations are the most common chromosomal aberrations, which, however, are normally almost exclusively found in high-grade malignancies. GCTB has often been regarded as a polyclonal tumour, but more recently a recurrent specific aberration was reported, which suggests a possible role for disturbed telomere maintenance. Here we further investigate telomere maintenance in GCTB using 19 samples from 19 patients. A combination of immunofluorescence and FISH was performed, applying antibodies directed against promyelocytic leukaemia body-related antigen and hTERT and using telomere peptide nucleic acid probes. The TRAP assay and telomere restriction fragment length analysis were performed for functional detection of telomerase activity and alternative telomere lengthening. Both osteoclastic giant cells and mononuclear cells showed positivity for hTERT and promyelocytic leukaemia body-related antigen. In most mononuclear cells, co-expression was present. The TRAP assay demonstrated heterogeneous telomerase activity, while telomere restriction fragment length analysis showed non-heterogeneous telomere lengths, indicating the absence of alternative telomere lengthening. Confocal microscopy showed stereometric co-localization of nucleolin with promyelocytic leukaemia body-related antigen in association with telomeres in the spindle-shaped cells. hTERT was more diffusely distributed throughout the nucleus. Our results show that GCTB demonstrates remarkable telomere maintenance of activated telomerase and inactivated alternative telomere lengthening in the presence of normal mean telomere restriction fragment lengths. These findings strongly suggest that these aggregates, while activating telomerase, are part of a structural telomere protective-capping mechanism rather than of a telomere-lengthening mechanism. Telomere maintenance could be considered an important key factor in the pathogenesis of GCTB.

Copper toxicity in the spiny dogfish (Squalus acanthias): urea loss contributes to the osmoregulatory disturbance.

Previous research showed that the spiny dogfish, Squalus acanthias, is much more sensitive to silver exposure than typical marine teleosts. The aim of the present study was to investigate if spiny dogfish were equally sensitive to copper exposure and whether the toxic mechanisms were the same. We exposed cannulated and non-cannulated spiny dogfish to measured concentrations of Cu (nominally 0, 500, 1000 and 1500 microg L(-1) Cu) for 72-96 h. All Cu exposures induced acidosis and lactate accumulation of either a temporary (500 microg L(-1)) or more persistent nature (1000 and 1500 microg L(-1)). At the two highest Cu concentrations, gill Na(+)/K(+)-ATPase activities were reduced by 45% (1000 microg L(-1)) and 62% (1500 microg L(-1)), and plasma Na(+) and Cl(-) concentrations increased by approximately 50 mM each. At the same time urea excretion doubled and plasma urea dropped by approximately 100 mM. Together with plasma urea, plasma TMAO levels dropped proportionally, indicating that the general impermeability of the gills was compromised. Overall plasma osmolarity did not change. Cu accumulation was limited with significant increases in plasma Cu and elevated gill and kidney Cu burdens at 1000 and 1500 microg L(-1). We conclude that Cu, like Ag, exerts toxic effect on Na(+)/K(+)-ATPase activities in the shark similar to those of teleosts, but there is an additional toxic action on elasmobranch urea retention capacities. With a 96 h LC(50) in the 800-1000 microg L(-1) range, overall sensitivity of spiny dogfish for Cu is, in contrast with its sensitivity to Ag, only slightly lower than in typical marine teleosts.

A phase I dose-finding clinical pharmacokinetic study of an oral formulation of irinotecan (CPT-11) administered for 5 days every 3 weeks in patients with advanced solid tumours.

BACKGROUND: Oral administration of irinotecan (CPT-11) should allow sustained exposure to the drug without the inconvenience of intravenous delivery and with fewer side-effects. PATIENTS AND METHODS: The present phase I trial of CPT-11, administered orally as a powder-filled capsule for 5 consecutive days every 3 weeks at doses ranging from 30 to 90 mg/m(2)/day, was conducted in 47 patients for whom a satisfactory standard treatment option was no longer available (24 males/23 females; median age 51 years, range 26-85). Tumour types included melanoma (11), colorectal (4), urinary tract (3), lung/pleura (4), thyroid (3), liver (3), gallbladder (2), cervix/uterus (3), breast (2), pancreas (2), carcinoma and other cancer types (10). RESULTS: A total of 171 cycles were administered (median 3, range 1-11). Dose limiting toxicities (DLTs) occurred during the first cycle in five of 31 patients in the dose-escalation part of the study: one patient at the 50 mg/m(2)/day dose level (diarrhoea grade 4); one patient at the 80 mg/m(2)/day dose level (prolonged neutropenia grade 4 and diarrhoea grade 3); and three patients at the 90 mg/m(2)/day dose level (diarrhoea, vomiting and neutropenia). The 80 mg/m(2)/day dose level was expanded, as a feasibility study, to include 16 additional patients, five of whom had received extensive prior pelvic irradiation. A further three patients in this cohort experienced DLTs, two of whom had received extensive prior pelvic irradiation. One patient died on study day 15 during the first cycle of oral CPT-11 following grade 3 diarrhoea, febrile neutropenia and a necrotic enterocolitis. Overall the grade 3/4 toxicities in 47 patients were asthenia (19%), anorexia (17%), neutropenia (14.9 %), diarrhoea (13%), nausea (12.7%), vomiting (8.5%) and thrombocytopenia (8.5%). Partial responses were observed in two melanoma patients and disease stabilisation was noted in 17 (36.1%) patients. Pharmacokinetic parameters were recorded for 46 patients. CONCLUSIONS: At the maximum tolerated dose, defined as 80 mg/m(2)/day for 5 days every 3 weeks, oral CPT-11 was shown to be well tolerated and safe with few of the haematological toxicities associated with the intravenous formulation.

Sensitive and specific quantification of the anticancer agent ZD1839 (Gefitinib) in plasma by on-column focusing capillary liquid chromatography-tandem mass spectrometry.

The development of an on-column focusing gradient capillary LC method coupled to tandem mass spectrometry (quadrupole-linear ion trap) for the quantitative determination of the anticancer agent ZD1839 (Gefitinib, Iressa) in blood plasma is described. Plasma samples (0.2 ml) were extracted with methyl tert-butyl ether. The analytes of interest, ZD1839 and the internal standard [(2)H8]ZD1839 (ZD1839-d8) were eluted on a 50 mm x 1 mm, 5 microm particle size, capillary ODS Hypersil column using an aqueous ammonium acetate gradient at 40 microl/min. Mass spectrometric detection was performed by a Q-Trap tandem mass spectrometer with electrospray positive ionisation, and monitored in the multiple reaction monitoring transitions 447 >128 and 455 >136, respectively. The limit of quantification of ZD18395 was 0.1 ng/ml. The method proved to be robust, allowing quantification of ZD1839 with sufficient precision, accuracy and sensitivity.

Anterograde versus retrograde isolated lung perfusion with melphalan in the WAG-Rij rat.

OBJECTIVE: Isolated lung perfusion (ILuP) is an experimental technique currently tested to increase the 5-year survival of 40% after surgical resection of pulmonary metastases from certain solid tumors. The standard technique of anterograde perfusion was compared with retrograde isolated lung perfusion in which the drug is introduced through the pulmonary veins while the effluent is collected from the pulmonary artery. Since the lung has a dual arterial circulation through the pulmonary artery and bronchial circulation, perfusion through the pulmonary veins can result in a more homogeneous distribution throughout the lung with subsequent higher melphalan concentration. METHODS: We randomized 20 rats into two groups. Group one underwent anterograde isolated left lung perfusion while group two underwent retrograde isolated left lung perfusion. A dose of 2 mg/kg melphalan (MN) was administered to the lung at a flow of 0.5 mL/min during 30 min, followed by a 5-min washout with buffered hetastarch (BHE). The final melphalan lung concentration (FMLC) was determined in the hilum, at the apex, the mid-periphery and the base of the lung. Statistical analysis was done with an unpaired student's t-test. RESULTS: Retrograde left ILuP resulted in a higher FMLC in the hilum (P<0.0001) and in the base of the lung (P=0.03), while anterograde ILuP induced a higher concentration at the apex of the lung (P=0.04). No difference was seen in the mid-peripheral area of the lung (P=0.92). CONCLUSIONS: In this experimental study, retrograde perfusion seems to increase final melphalan lung concentration in hilar and basal regions of the lung compared to anterograde perfusion.

Balloon catheter hypoxic abdominal and pelvic perfusion with tumour necrosis factor-alpha, Melphalan and Mitomycin C: a pharmacokinetic study in pigs.

BACKGROUND: Addition of tumour necrosis factor-alpha (TNF) to hypoxic abdominal perfusion (HAP) and hypoxic pelvic perfusion (HPP) with chemotherapeutic agents for treatment of un-resectable malignancies may lead to similar enhanced anti-tumour effects as are observed when TNF is added to isolated limb perfusions (ILP) with Melphalan. Here, we validate the methodology of HAP and HPP using balloon catheter techniques, and investigate the distribution of TNF, Melphalan and Mitomycin C (MMC) over the regional and systemic blood compartments when applying these techniques. MATERIALS AND METHODS: Twelve pigs underwent HAP or HPP with TNF, Melphalan and MMC for 20 min. Throughout and after the procedures blood samples were obtained from hepatic, portal and systemic blood compartments and plasma concentrations of perfused agents were determined. RESULTS: We demonstrated that HAP and HPP result in temporary loco-regional concentration advantages of all perfused agents, although from start of perfusion significant systemic leakage occurred. CONCLUSION: On basis of these results it seems that the advantage in terms of regional plasma concentration of TNF may be insufficient for TNF-mediated effects to occur, making future addition of this cytokine to these procedures in the clinical setting questionable. The observed regional concentration advantages of MMC and Melphalan, however, warrant further studies on clinical application of these agents in both settings.

Combretastatin A-4 phosphate enhances CPT-11 activity independently of the administration sequence.

We evaluated the effect of different intervals and sequences of the vascular targeting agent combretastatin A-4 disodium phosphate (CA4DP) and CPT-11 administration on tumour growth delay and intratumoral uptake of CPT-11 using a syngeneic rhabdomyosarcoma tumour model. Irrespective of the administration sequence, the combination of CA4DP and CPT-11 significantly increases tumour growth delay in comparison with both drugs alone (P<0.001). Intratumoral CPT-11 concentration generally decreased (up to 5-fold) in the combination groups, while SN-38, the active metabolite of CPT-11, increased up to 9-fold. However, the increased amount of intratumoral SN-38 trapping after CA4DP injection did not correlate with the observed tumour growth delay. In conclusion, CA4DP significantly enhances the antitumour effect of CPT-11, which is not greatly influenced by the administration sequence, and which lacks a correlation with the intratumoral trapping of CPT-11 or SN-38. Mechanisms other than trapping are likely to be involved in the chemosensitising capacity of CA4DP.

Effect of antivascular endothelial growth factor treatment on the intratumoral uptake of CPT-11.

Promising preclinical activity with agents blocking the function of vascular endothelial growth factor (VEGF) has been observed in various cancer types, especially with combination therapy. However, these drugs decrease microvessel density, and it is not known whether this reduced vessel density (VD) results in decreased delivery of concomitantly administered classical anticancer drugs. We designed an in vivo study to investigate the relation between VEGF-blocking therapy, tumoral blood vessels, and intratumoral uptake of anticancer drugs. Nude NMRI mice bearing colon adenocarcinoma (HT29) were treated with the anti-VEGFmAb A4.6.1 or placebo. After 1 week, CPT-11 was administered 1 h prior to killing the animals. In A4.6.1 treated tumours, there was a significant decrease in VD, more pronounced with potentially functional large vessels than endothelial cords. Interestingly, a trend to increased intratumoral CPT-11 concentration was observed (P=0.09). In parallel, we measured an increase in tumour perfusion, as estimated by high-performance liquid chromatography determination of intratumoural Hoechst 33342 concentration. In the growth delay study, CPT-11 was at least equally effective with or without pretreatment with A4.6.1. These data suggest that tumour vascular function and tumour uptake of anticancer drugs improve with VEGF-blocking therapy, and indicate the relevance for further investigations.

Degree of tumour vascularity correlates with drug accumulation and tumour response upon TNF-alpha-based isolated hepatic perfusion.

Isolated hepatic perfusion (IHP) with melphalan with or without tumour necrosis factor alpha (TNF-alpha) is currently performed in clinical trials in patients with hepatic metastases. Previous studies led to the hypothesis that the use of TNF-alpha in isolated limb perfusion causes specific destruction of tumour endothelial cells and thereby induces an increased permeability of tumour vasculature. However, whether TNF-alpha contributes to the therapeutic efficacy in IHP still remains unclear. In an in vivo rat liver metastases model we studied three different tumours: colon carcinoma CC531, ROS-1 osteosarcoma and BN-175 soft-tissue sarcoma which exhibit different degrees of vascularisation. IHP was performed with melphalan with or without the addition of TNF-alpha. IHP with melphalan alone resulted, in all tumour types, in a decreased growth rate. However in the BN-175 tumour addition of TNF-alpha resulted in a strong synergistic effect. In the majority of the BN-175 tumour-bearing rats, a complete response was achieved. In vitro cytoxicity studies showed no sensitivity (CC531 and BN-175) or only minor sensitivity (ROS-1) to TNF-alpha, ruling out a direct interaction of TNF-alpha with tumour cells. The response rate in BN-175 tumour-bearing rats when TNF-alpha was coadministrated with melphalan was strongly correlated with drug accumulation in tumour tissue, as only in these rats a five-fold increased melphalan concentration was observed. Secondly, immunohistochemical analysis of microvascular density (MVD) of the tumour showed a significantly higher MVD for BN-175 tumour compared to CC531 and ROS-1. These results indicate a direct relation between vascularity of the tumour and TNF-alpha mediated effects. Assessment of the tumour vasculature of liver metastases would be a way of establishing an indication for the utility of TNF-alpha in this setting.

Hyphenated techniques in anticancer drug monitoring. I. Capillary gas chromatography-mass spectrometry.

Most anticancer agents are relatively unstable substances and are subjected to intensive metabolism in vivo and radiation during sample pretreatment. Hyphenated techniques including a separation technique and, most frequently, mass spectrometry are therefore chosen to obtain insight into the in vivo behavior of anticancer agents. Once established, simpler assays can be derived from those based on hyphenation, which are less expensive. Capillary gas chromatography (cGC)-mass spectrometry (MS) is amongst the most frequently applied hyphenated analytical technologies in anticancer drug monitoring. Here a selection has been made of: (i) cGC-MS applied to the analysis of agents frequently used in clinical oncology (e.g. tamoxifen, oxazaphosphorines); (ii) cGC-MS applied to the development of new agents (Swainsonine and Brefeldin); (iii) cGC-MS applied to the analysis of agents for which comparisons with other frequently applied hyphenation technologies are possible (see Part I of this series). cGC-MS played a key role in the elucidation of the in vivo behavior of the oxazaphosphorine cyclophosphamide, historically the most frequently applied anticancer agent. cGC-MS appeared to be of special interest in the analysis of cyclophosphoramide and congeners in human erythrocytes by coupling of the hyphenated technique with a measurement of sediment technique. This resulted in the quantitative and qualitative analysis of oxaphosphorine-related mustard gas moieties in human erthrocytes for the first time.

Hyphenated techniques in anticancer drug monitoring. II. Liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry.

High-performance liquid chromatography has become the separation technique of choice for the monitoring of generally thermolabile anticancer agents. With the introduction of electrospray mass spectrometry, the coupling of liquid chromatogaphy and mass spectrometry has opened the way to widely and routinely applied anticancer drug monitoring. Real-time metabolism versus degradation can now be distinguished, since derivatization is no longer obligatory. This is important for the monitoring of the anabolic and catabolic pathways of the same agent, such as 5-fluorouracil. Detection limits almost equal to those obtained with capillary gas chromatography-mass spectrometry are realistic with the latest generation of mass spectrometers, enabling quantitative analysis of various anticancer agents and their metabolites down to the low ng/ml level. Furthermore, sample clean-up and chromatography can be downscaled markedly using the latest column technologies, such as the generally applied 10 cm x 2.8 mm I.D. RP 18 columns. The coupling of capillary electrophoresis to mass spectrometry is today far from a routine application in anticancer drug monitoring. Nevertheless, interesting applications have been reported and are selected for the present review.

The impact of drug administration sequence and pharmacokinetic interaction in a phase I study of the combination of docetaxel and gemcitabine in patients with advanced solid tumors.

Our objective was to determine the maximum tolerated dose (MTD) of two administration sequences of docetaxel and gemcitabine in cancer patients, and to describe the pharmacokinetics of both drugs. Patients were treated in a 4-weekly schedule at two dose levels: gemcitabine 800 mg/m2 on days 1, 8 and 15, and docetaxel 85 or 100 mg/m2 on day 15 (levels I and II). The protocol was amended to a 3-weekly schedule, testing gemcitabine 800 or 1000 mg/m2 on days 1 and 8, with docetaxel 85 mg/m2 on day 8 given initially (dose levels IIIa and IV). At the recommended dose, an extra cohort of patients initially received gemcitabine (dose level IIIb). Eleven patients were treated with the 4-week schedule; 29% of cycles were delayed predominantly because of hematological toxicity. Four patients developed dose-limiting toxicities (DLTs), predominantly hematological. In the 3-week schedule, 14 patients were treated. At level IV, three of four patients developed DLTs, defining the MTD. With the reverse sequence, three patients received a total of 10 cycles. Overall, nine partial remissions were observed. We conclude the recommended dose for phase II studies is gemcitabine 800 mg/m2 on days 1 and 8, combined with docetaxel 85 mg/m2 on day 8, on a 3-weekly schedule. Gemcitabine distribution is significantly altered upon docetaxel administration.

Sensitivity of the spiny dogfish (Squalus acanthias) to waterborne silver exposure.

The physiological effects of waterborne silver exposure (added as AgNO(3)) on spiny dogfish, Squalus acanthias, were evaluated at 30, 200 and 685 microg silver per l in 30 per thousand seawater. These concentrations cover the toxic range observed for freshwater teleosts, where silver is extremely toxic, to seawater teleosts which tolerate higher silver concentrations. However, these levels are considerably higher than those that occur in the normal environment. At 685 microg l(-1), dogfish died within 24 h. Causes of death were respiratory as well as osmoregulatory failure. Arterial P(a)O(2) rapidly declined below 20 Torr, and blood acidosis (both respiratory and metabolic) occurred. Urea excretion increased dramatically and plasma urea dropped from 340 to 225 mM. There were pronounced increases in plasma Na(+), Cl(-), and Mg(2+), indicative of ionoregulatory failure due to increased diffusive permeability as well as inhibited NaCl excretion. At 200 microg l(-1), fish died between 24 and 72 h of silver exposure. The same physiological events occurred with a small time delay. At 30 microg l(-1), effects were much less severe, although slight mortality (12.5%) still occurred. Respiratory alkalosis occurred, together with moderate elevations in plasma Na(+) and Cl(-) levels. Silver accumulated to the highest concentrations on gills, with only low levels in the intestine, in accord with the virtual absence of drinking. Na(+)/K(+)-ATP-ase activities of gill and rectal gland tissue were impaired at the highest silver concentration. Normal gill function was impaired due to swelling and fusion of lamellae, lamellar aneurism and lifting of the lamellar epithelium. Our results clearly indicate that this elasmobranch is much more sensitive (about 10-fold) to silver than marine teleosts, with silver's toxic action exerted on the gill rather than on the intestine, in contrast to the latter.