Amino-functionalized hypercrosslinked polymer as sorbent for effective extraction of nitroimidazoles from water, drink and honey samples.

The three hypercrosslinked polymers (HCP) materials, designated as OPD-HCP, MPD-HCP and PPD-HCP, were synthesized by using o-phenylenediamine (OPD), m-phenylenediamine (MPD) and p-phenylenediamine (PPD) as monomers. They were characterized by infrared spectroscopy, powder X-ray diffraction, nitrogen sorption isotherms, and scanning electron microscopy. Then, the HCPs were explored as solid-phase extraction (SPE) adsorbent for the extraction of five nitroimidazoles (NDZs) (metronidazole, ronidazole, secnidazole, dimetridazole and ornidazole). Among the three HCPs, the MPD-HCP has the best adsorption performance for the NDZs. With the help of high-performance liquid chromatography with ultraviolet detection (HPLC-UV), good linear response range (0.07-40.0 ng mL-1), high method recovery (86.8%-113.3%), low limits of detection (0.02-0.15 ng mL-1) and good precision with the relative standard deviations of less than 8.1% were achieved for the determination of the NDZs in water samples. The effective determination of the NDZs in peach juice, honey tea, and honey samples were also realized by the developed method with satisfactory results. Based on both the experimental results and density functional theory calculation, the adsorption mechanism can be attributed to multiple interactions between the MPD-HCP and the NDZs, including hydrogen bonding, hydrophilic, and electrostatic interactions. The method provides a new alternative of choice for the determination of some NDZs in real samples.

[Determination of nitroimidazoles and their metabolites in animal-derived foods by liquid chromatography-tandem mass spectrometry coupled with dispersive solid phase extraction].

A method for the quantitative analysis of nitroimidazoles and their metabolites in four kinds of animal-derived foods by liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with dispersive solid phase extraction (dSPE) was established. The samples (2.0 g) were extracted with ethyl acetate. The extracts were degreased with hexane and purified with 50 mg primary-secondary amine (PSA), and then filtered through a hydrophilic polytetrafluoroethylene (PTFE) membrane. The analytes were separated on a C18 column, and detected in selected reaction monitoring (SRM) mode via positive electrospray ionization. The matrix matching and internal standard method was used. The nitroimidazoles and their metabolites showed good linearity in the range of 0.5-20.0 µg/L with correlation coefficients greater than 0.99. The limits of detection of the nitroimidazoles and their metabolites in animal-derived foods were between 0.1 and 0.5 µg/kg. The recoveries varied between 84.2% and 120.8%, with relative standard deviations (n=6) ranging from 2.0% to 16.2% at spiked levels of 1.0, 3.0, and 10.0 µg/kg. The proposed method is accurate, fast, cheap, easy, and can satisfy the requirements of monitoring nitroimidazoles and their metabolites in animal-derived foods.

Development and validation of a QuEChERS method for the analysis of 5-nitroimidazole traces in infant milk-based samples by ultra-high performance liquid chromatography-tandem mass spectrometry.

QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) methodology in combination with UHPLC-MS/MS is proposed for the determination of 5-nitroimidazole (5-NDZ) residues in infant milk-based products. Chromatographic separation was accomplished in a C18 Zorbax Eclipse Plus RRHD (50 × 2.1 mm, 1.8 µm) column under gradient elution conditions. Mobile phase consisted of 0.025% (v/v) aqueous formic acid (eluent A) and MeOH (eluent B), and was supplied at a flow rate of 0.5 mL/min. Under these conditions, eleven 5-NDZs including three metabolites were separated in less than 4 min. A novel QuEChERS method was optimized, and primary-secondary amine (PSA) sorbent was selected as clean-up agent. In addition, the proposed QuEChERS procedure was compared with other sample preparation methodologies, which are usually used in the analysis of 5-NDZs, namely solid phase extraction (SPE) using mixed cation exchange (MCX) cartridges and molecularly imprinted solid phase extraction (MISPE). Method comparison was carried out in terms of process efficiency, which includes matrix effect and extraction recovery. Higher process efficiency was generally achieved for QuEChERS and MISPE than for SPE. However, matrix effect was more significant for the non-selective QuEChERS method than for the selective MISPE. Higher extraction recoveries (63.2-94.1%) were observed for QuEChERS. The studied methods were validated in terms of linearity, detection limits (CCα), detection capabilities (CCß) and precision, observing similar performance characteristics independently of the proposed sample treatment. CCα and CCß ranged between 0.05 and 1.69 µg/L for all analytes and extraction techniques. QuEChERS-UHPLC-MS/MS method was also validated in terms of precision (relative standard deviations <10.3%), trueness (recovery>70.2%) and selectivity according to Regulation 2002/657/EC. It is proposed as a good alternative for the monitoring of 5-NDZ residues in enriched infant-milk based products and other similar milk based-products.

Integrated assessment of persistent toxic substances in sediments from Masan Bay, South Korea: Comparison between 1998 and 2014.

Complexity of anthropogenic influences on coastal ecosystems necessitates use of an integrated assessment strategy for effective interpretation and subsequent management. In this study a multiple lines of evidence (LOE) approach for sediment assessment, that combined use of chemistry, toxicity, and benthic community structure in the sediment quality triad was used to assess spatiotemporal changes and potential risks of persistent toxic substances (PTSs) in sediments of Masan Bay highlighting "long-term changes" between 1998 and 2014. Specific target objectives encompassed sedimentary PTSs (PAHs, alkylphenols (APs), and styrene oligomers), potential aryl hydrocarbon receptor (AhR; H4IIE-luc assay)- and estrogen receptor (ER; MVLN assay)-mediated activities, and finally several ecological quality (EcoQ) indices of benthic community structure. Concentrations of target PTSs in Masan Bay sediments were generally less by half in 2014 compared to those measured in 1998. Second, AhR-mediated potencies in sediments also decreased during this time interval, whereas ER-mediated potencies increased (+3790%), indicating that there has been substantial ongoing, input of ER agonists over the past 16 years. Potency balance analysis revealed that only 3% and 22% of the AhR- and ER-mediated potencies could be explained by identified known chemicals, such as PAHs and APs, respectively. This result indicated that non-targeted AhR and ER agonists had a considerable presence in the sediments over time. Third, EcoQ indices tended to reflect PTSs contamination in the region. Finally, ratio-to-mean values obtained from the aforementioned three LOEs indicated that quality of sediments from the outer region of the bay had recovery more during the period of 16-years than did the inner region. Overall, the results showed that even with the progress supported by recent efforts from the Korean governmental pollution control, PTSs remain a threat to local ecosystem, especially in the inner region of Masan Bay.

A QuEChERS-Based Sample Preparation Method for the Analysis of 5-Nitroimidazoles in Bovine Milk by HPLC-DAD.

In this study, a simple, cost-effective, and sensitive HPLC diode-array detection method was developed for the simultaneous determination of six different 5-nitroimidazoles [metronidazole, 2-hydroxymethyl-1-methyl-5-nitro-1H-imidazole, dimetridazole (DMZ), ronidazole, ornidazole, and ipronidazole] in bovine milk samples. A QuEChERS-based sample preparation procedure was optimized by evaluating different cleanup sorbents, including zirconium-based sorbents (Z-Sep and Z-Sep+), C18, and primary-secondary amine (PSA), as well as EMR-Lipid cleanup solution. Acceptable analytical performance for all analytes was observed with recoveries in the range of 45-93% and RSDs of less than 15%. Negligible matrix interference was observed for most of the analytes due to application of PSA sorbent in a dispersive solid-phase extraction cleanup step. Method LOQs (mLOQs) for five of the six investigated analytes were set at a satisfactory low food product value of 2.5 ng/mL. For DMZ only, the mLOQ was set at 10 ng/mL. The procedure was evaluated through the analysis of 10 different natural samples.

Mass Spectrometry Imaging of the Hypoxia Marker Pimonidazole in a Breast Tumor Model.

Although tumor hypoxia is associated with tumor aggressiveness and resistance to cancer treatment, many details of hypoxia-induced changes in tumors remain to be elucidated. Mass spectrometry imaging (MSI) is a technique that is well suited to study the biomolecular composition of specific tissue regions, such as hypoxic tumor regions. Here, we investigate the use of pimonidazole as an exogenous hypoxia marker for matrix-assisted laser desorption/ionization (MALDI) MSI. In hypoxic cells, pimonidazole is reduced and forms reactive products that bind to thiol groups in proteins, peptides, and amino acids. We show that a reductively activated pimonidazole metabolite can be imaged by MALDI-MSI in a breast tumor xenograft model. Immunohistochemical detection of pimonidazole adducts on adjacent tissue sections confirmed that this metabolite is localized to hypoxic tissue regions. We used this metabolite to image hypoxic tissue regions and their associated lipid and small molecule distributions with MALDI-MSI. We identified a heterogeneous distribution of 1-methylnicotinamide and acetylcarnitine, which mostly colocalized with hypoxic tumor regions. As pimonidazole is a widely used immunohistochemical marker of tissue hypoxia, it is likely that the presented direct MALDI-MSI approach is also applicable to other tissues from pimonidazole-injected animals or humans.

Prognostic significance of hypoxic PET using (18)F-FAZA and (62)Cu-ATSM in non-small-cell lung cancer.

OBJECTIVES: Tumor hypoxia is believed to have a strong correlation with the resistance to chemoradiotherapy. Noninvasive evaluation of hypoxic status in tumors using molecular imaging has the potential to characterize the tumor aggressiveness. We evaluated the clinical usefulness of newly-developed tumor hypoxic positron emission tomography (PET) tracers in localized non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Forty-seven patients with localized NSCLC received either or both hypoxic PETs using the tracers: (18)F-fluoroazomycin arabinoside ((18)F-FAZA) (n=45) and/or (62)Cu-diacetyl-bis (N4)-methylsemithiocarbazone ((62)Cu-ATSM) (n=22). All received (18)F-fluorodeoxyglucose ((18)F-FDG) PET tracer (n=47). We examined the correlation between uptake of three PET tracers and clinicopathological factors, and evaluated their impacts on survival after treatment retrospectively. RESULTS: A couple of commonly-identified unfavorable factors such as presence of vascular invasion and pleural invasion was significantly correlated with higher uptake of these hypoxic agents as well as that of (18)F-FDG. Larger tumor diameter, high neutrophil-to-lymphocyte ratio and advanced pathological stage were also associated with accumulation of hypoxic PETs ((18)F-FAZA, p<0.01; (62)Cu-ATSM, p<0.04), but not with that of (18)F-FDG. The patients with a higher accumulation had significantly poorer overall survival [(18)F-FAZA, HR (hazard ratio), 9.50, p<0.01; (62)Cu-ATSM, HR, 4.06, p<0.05] and progression free survival ((18)F-FAZA, HR, 5.28, p<0.01, (62)Cu-ATSM, HR, 2.72, p<0.05). CONCLUSION: Both (18)F-FAZA and (62)Cu-ATSM PET provide useful information regarding tumor aggressiveness and prediction of survival among NSCLC patients. We believe these hypoxic PETs could contribute to the establishment of the optimally individualized treatment of NSCLC.

Dynamic contrast-enhanced magnetic resonance imaging of the metastatic potential of melanoma xenografts.

PURPOSE: Gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA)-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been suggested as a useful noninvasive method for characterizing the physiologic microenvironment of tumors. In the present study, we investigated whether Gd-DTPA-based DCE-MRI has the potential to provide biomarkers for hypoxia-associated metastatic dissemination. METHODS AND MATERIALS: C-10 and D-12 melanoma xenografts were used as experimental tumor models. Pimonidazole was used as a hypoxia marker. A total of 60 tumors were imaged, and parametric images of K(trans) (volume transfer constant of Gd-DTPA) and v(e) (fractional distribution volume of Gd-DTPA) were produced by pharmacokinetic analysis of the DCE-MRI series. The host mice were killed immediately after DCE-MRI, and the primary tumor and the lungs were resected and prepared for histologic assessment of the fraction of pimonidazole-positive hypoxic tissue and the presence of lung metastases, respectively. RESULTS: Metastases were found in 11 of 26 mice with C-10 tumors and 14 of 34 mice with D-12 tumors. The primary tumors of the metastatic-positive mice had a greater fraction of hypoxic tissue (p = 0.00031, C-10; p < 0.00001, D-12), a lower median K(trans) (p = 0.0011, C-10; p < 0.00001, D-12), and a lower median v(e) (p = 0.014, C-10; p = 0.016, D-12) than the primary tumors of the metastatic-negative mice. CONCLUSIONS: These findings support the clinical attempts to establish DCE-MRI as a method for providing biomarkers for tumor aggressiveness and suggests that primary tumors characterized by low K(trans) and low v(e) values could have a high probability of hypoxia-associated metastatic spread.

pO2 fluctuation pattern and cycling hypoxia in human cervical carcinoma and melanoma xenografts.

PURPOSE: Blood perfusion in tumors is spatially and temporally heterogeneous, resulting in local fluctuations in tissue oxygen tension (pO(2)) and tissue regions showing cycling hypoxia. In this study, we investigated whether the pO(2) fluctuation pattern and the extent of cycling hypoxia differ between tumor types showing high (e.g., cervical carcinoma xenograft) and low (e.g., melanoma xenograft) fractions of connective tissue-associated blood vessels. METHODS AND MATERIALS: Two cervical carcinoma lines (CK-160 and TS-415) and two melanoma lines (A-07 and R-18) transplanted into BALB/c nu/nu mice were included in the study. Tissue pO(2) was measured simultaneously in two positions in each tumor by using a two-channel OxyLite fiber-optic oxygen-sensing device. The extent of acute and chronic hypoxia was assessed by combining a radiobiological and a pimonidazole-based immunohistochemical assay of tumor hypoxia. RESULTS: The proportion of tumor regions showing pO(2) fluctuations, the pO(2) fluctuation frequency in these regions, and the relative amplitude of the pO(2) fluctuations were significantly higher in the melanoma xenografts than in the cervical carcinoma xenografts. Cervical carcinoma and melanoma xenografts did not differ significantly in the fraction of acutely hypoxic cells or the fraction of chronically hypoxic cells. However, the ratio between fraction of acutely hypoxic cells and fraction of chronically hypoxic cells was significantly higher in melanoma than in cervical carcinoma xenografts. CONCLUSIONS: Temporal heterogeneity in blood flow and tissue pO(2) in tumors may depend on tumor histology. Connective tissue surrounding microvessels may stabilize blood flow and pO(2) and, thus, protect tumor tissue from cycling hypoxia.

Dynamic contrast-enhanced-MRI of tumor hypoxia.

Patients with highly hypoxic primary tumors show increased frequency of locoregional treatment failure and poor survival rates and may benefit from particularly aggressive treatment. The potential of gadolinium diethylene-triamine penta-acetic acid-based dynamic contrast-enhanced-MRI in assessing tumor hypoxia was investigated in this preclinical study. Xenografted tumors of eight human melanoma lines were subjected to dynamic contrast-enhanced-MRI and measurement of the fraction of radiobiologically hypoxic cells and the fraction of pimonidazole-positive hypoxic cells. Tumor images of K(trans) (the volume transfer constant of gadolinium diethylene-triamine penta-acetic acid) and v(e) (the fractional distribution volume of gadolinium diethylene-triamine penta-acetic acid) were produced by pharmacokinetic analysis of the dynamic contrast-enhanced-MRI data, and K(trans) and v(e) frequency distributions of the non-necrotic tumor tissue were established and related to the extent of hypoxia. Tumors showing high K(trans) values and high v(e) values had low fractions of hypoxic cells, whereas tumors showing both low K(trans) values and low v(e) values had high hypoxic fractions. K(trans) differentiated better between tumors with low and high hypoxic fractions than did v(e). This study supports the current attempts to establish dynamic contrast-enhanced-MRI as a method for assessing the extent of hypoxia in human tumors, and it provides guidelines for the clinical development of valid assays.

HIF-VEGF pathways are critical for chronic otitis media in Junbo and Jeff mouse mutants.

Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF-mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF-mediated pathways, and we conclude that targeting molecules in HIF-VEGF signaling pathways has therapeutic potential in the treatment of chronic OM.

Evaluation of [125I]IPOS as a molecular imaging probe for hypoxia-inducible factor-1-active regions in a tumor: comparison among single-photon emission computed tomography/X-ray computed tomography imaging, autoradiography, and immunohistochemistry.

To image hypoxia-inducible factor-1 (HIF-1)-active tumors, we previously developed a chimeric protein probe ([(123/125) I]IPOS) that is degraded in the same manner as HIF-1α under normoxic conditions. In the present study, we aim to show that the accumulation of radioiodinated POS reflects the expression of HIF-1. In vivo single-photon emission computed tomography (SPECT)/X-ray CT (CT) imaging, autoradiography, and double-fluorescent immunostaining for HIF-1α and pimonidazole (PIMO) were carried out 24 h after the injection of [(125) I]IPOS. Tumor metabolite analysis was also carried out. A tumor was clearly visualized by multi-pinhole, high-resolution SPECT/CT imaging with [(125) I]IPOS. The obtained images were in accordance with the corresponding autoradiograms and with the results of ex vivo biodistribution. A metabolite analysis revealed that 77% of the radioactivity was eluted in the macromolecular fraction, suggesting that the radioactivity mainly existed as [(125) I]IPOS in the tumors. Immunohistochemistry revealed that the HIF-1α-positive areas and PIMO-positive areas were not always identical, only some of the regions were positive for both markers. The areas showing [(125) I]IPOS accumulation were positively and significantly correlated with the HIF-1α-positive areas (R = 0.75, P < 0.0001). The correlation coefficient between [(125) I]IPOS-accumulated areas and HIF-1α-positive areas was significantly greater than that between the [(125) I]IPOS-accumulated areas and the PIMO-positive areas (P < 0.01). These findings indicate that [(125) I]IPOS accumulation reflects HIF-1 expression. Thus, [(123/125) I]IPOS can serve as a useful probe for the molecular imaging of HIF-1-active tumors.

Indirect competitive enzyme-linked immuno-sorbent assay (ELISA) for nitroimidazoles in food products.

Ronidazole was used as the starting material to prepare an immunogen and coating antigen. An anti-nitroimidazole monoclonal antibody was produced and an indirect competitive ELISA was established to detect nitroimidazole compounds in food products. The IC(50) values were determined to be 0.20 ng/ml for metronidazole, 4.0 ng/ml for tinidazole, 0.17 ng/ml for dimetridazole and 0.24 ng/ml for ornidazole. Considering that nitroimidazoles were commonly used as veterinary drugs, nitroimidazole residues in food products of animal origin were detected by the method. The coefficient of variation for nitroimidazoles determination in contaminated chicken, chicken liver and shrimp were all <14% and the recovery rate was in the range 74.0-90.6%. The results proved that the developed method was successful in detecting nitroimidazoles in food products.

Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia.

Oxygen supply and diffusion into tissues are necessary for survival. The oxygen partial pressure (pO(2)), which is a key component of the physiological state of an organ, results from the balance between oxygen delivery and its consumption. In mammals, oxygen is transported by red blood cells circulating in a well-organized vasculature. Oxygen delivery is dependent on the metabolic requirements and functional status of each organ. Consequently, in a physiological condition, organ and tissue are characterized by their own unique 'tissue normoxia' or 'physioxia' status. Tissue oxygenation is severely disturbed during pathological conditions such as cancer, diabetes, coronary heart disease, stroke, etc., which are associated with decrease in pO(2), i.e. 'hypoxia'. In this review, we present an array of methods currently used for assessing tissue oxygenation. We show that hypoxia is marked during tumour development and has strong consequences for oxygenation and its influence upon chemotherapy efficiency. Then we compare this to physiological pO(2) values of human organs. Finally we evaluate consequences of physioxia on cell activity and its molecular modulations. More importantly we emphasize the discrepancy between in vivo and in vitro tissue and cells oxygen status which can have detrimental effects on experimental outcome. It appears that the values corresponding to the physioxia are ranging between 11% and 1% O(2) whereas current in vitro experimentations are usually performed in 19.95% O(2), an artificial context as far as oxygen balance is concerned. It is important to realize that most of the experiments performed in so-called normoxia might be dangerously misleading.

Potential drug delivery system: study of the association of a model nitroimidazole drug with aggregates of amphiphilic polymers on aqueous solution

This study evaluated the association of N-hexyl-2-methyl-4-nitroimidazol, a model drug, to aggregates formed by anionic polyelectrolytes on aqueous solution. The alternating copolymers of maleic anhydride and N-vinyl-2-pyrrolidone were synthesized and then modified by reaction of the anhydride groups with aliphatic amines and alcohols of varying length of the alkyl chain. The partition of the model drug between water and the hydrophobic microdomains provided by the copolymers was studied using the pseudo-phase model to determinate the distribution coefficient K S, and the standard free energy of transfer ∆µ°t. The results indicate that all copolymers assessed are potential pharmaceutical reservoirs of the model drug. Nevertheless, the solubility of N-hexyl-2-methyl-4-nitroimidazol on the polymeric solutions is independent from the length of the alkyl chain of the copolymer.

Realizou-se estudo sobre a associação da N-hexil-2-metil-4-nitroimidazol, fármaco modelo, aos agregados formados por polieletrólitos aniônicos em solução aquosa. Os copolímeros alternados de anidrido maléico e N-vinil-2-pirrolidona foram sintetizados e, em seguida, modificados pela reação dos grupos de anidrido com aminas e álcoois alifáticos de duração variável da cadeia alquílica. A partição do fármaco modelo entre a água e os microdomínios hidrofóbicos fornecido pelos copolímeros foi estudada usando o modelo de pseudo-fase, a fim de determinar a distribuição do coeficiente K S e a energia livre padrão de transferência ∆µ°t. Os resultados indicam que todos os copolímeros avaliados são potenciais reservatórios farmacêuticos do fármaco. No entanto, a solubilidade do N-hexil-2-metil-4-nitroimidazol sobre as soluções poliméricas é independente do comprimento da cadeia alquílica do copolímero.

Analytical methodologies for the determination of nitroimidazole residues in biological and environmental liquid samples: a review.

Nitroimidazoles (NDZs) are antiprotozoal drugs that are typically used in veterinary and human medicine. NDZs and their metabolites are believed to possess genotoxic, carcinogenic and mutagenic properties, and this is (one reason) why their use has been banned within the European Union. Hence, the determination of trace residues of these substances in edible animal tissues has been of growing concern over the past few years. Even, though there has been a need to develop sensitive and reliable analytical methods to study the residues of these compounds in different matrices, available methodologies in environmental samples are rather limited. These and other pharmaceutical compounds have become one of the most important new classes of environmental pollutants that have been detected in wastewater-treatment-plant (WWTP) effluents, receiving waters, drinking water and groundwater. A compilation of the most representative analytical methodologies for the determination of NDZ residues during the last decade is presented in this paper. Its scope is the two main areas which require their determination, namely biological and environmental matrices. A detailed explanation of both areas, including sample treatment and detection systems, and future trends is presented, focusing on the difficulties of confirming analytes at low concentration levels.

Metabolization of nifurtimox and benznidazole in cellular fractions of rat mammary tissue.

Two nitroheterocyclic drugs, nifurtimox (NFX) and benznidazole (BZ), used in the treatment of Chagas' disease have serious side effects attributed to their nitroreduction to reactive metabolites. Here, we report that these drugs reach the mammary tissue and there they could undergo in situ bioactivation. Both were detected in mammary tissue from female Sprague-Dawley rats after their intragastric administration. Only NFX was biotransformed by pure xanthine-oxidoreductase and from tissue cytosol. These activities were purine dependent and were inhibited by allopurinol. Also, only NFX was biotransformed by microsomes in the presence of ß-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and was inhibited by carbon monoxide and partially by diphenyleneiodonium. NFX treatment produced significant decrease in protein sulfhydryl content after 1, 3 and 6 hours; no increases in protein carbonyl content at any time tested and significantly higher levels of lipid hydroperoxides at 3 and 6 hours; besides, ultrastructural observations after 24 hours showed significant differences in epithelial cells compared to control. These findings indicate that NFX might be more deleterious to mammary tissue than BZ and could correlate with early reports on its ability to promote rat mammary tissue toxicity.

Detection of different hypoxic cell subpopulations in human melanoma xenografts by pimonidazole immunohistochemistry.

This study aimed at developing immunohistochemical assays for different subpopulations of hypoxic cells in tumors. BALB/c-nu/nu mice bearing A-07 or R-18 tumors were given a single dose of 90 mg/kg body weight or three doses (3 h apart) of 30 mg/kg body weight of pimonidazole hydrochloride intravenously. The fraction of pimonidazole-labeled cells was assessed in paraffin-embedded and frozen tumor sections and compared with the fraction of radiobiologically hypoxic cells. The staining pattern in paraffin-embedded sections indicated selective staining of chronically hypoxic cells. Frozen sections showed a staining pattern consistent with staining of both chronically and acutely/repetitively hypoxic cells. Fraction of pimonidazole-labeled cells in paraffin-embedded sections was lower than the fraction of radiobiologically hypoxic cells (single-dose and triple-dose experiment). In frozen sections, fraction of pimonidazole-labeled cells was similar to (single-dose experiment) or higher than (triple-dose experiment) fraction of radiobiologically hypoxic cells. Three different subpopulations of hypoxic cells could be quantified by pimonidazole immunohistochemistry: the fraction of cells that are hypoxic because of limitations in oxygen diffusion, the fraction of cells that are hypoxic simultaneously because of fluctuations in blood perfusion, and the fraction of cells that are exposed to one or more periods of hypoxia during their lifetime because of fluctuations in blood perfusion.

Correlation of [18F]FMISO autoradiography and pimonidazole [corrected] immunohistochemistry in human head and neck carcinoma xenografts.

PURPOSE: Tumour cell hypoxia is a common feature in solid tumours adversely affecting radiosensitivity and chemosensitivity in head and neck squamous cell carcinomas. Positron emission tomography (PET) using the tracer [(18)F]fluoromisonidazole ([(18)F]FMISO) is most frequently used for non-invasive evaluation of hypoxia in human tumours. A series of ten human head and neck xenograft tumour lines was used to validate [(18)F]FMISO as hypoxia marker at the microregional level. METHODS: Autoradiography after injection of [(18)F]FMISO was compared with immunohistochemical staining for the hypoxic cell marker pimonidazole in the same tumour sections of ten different human head and neck xenograft tumour lines. The methods were compared: first, qualitatively considering the microarchitecture; second, by obtaining a pixel-by-pixel correlation of both markers at the microregional level; third, by measuring the signal intensity of both images; and fourth, by calculating the hypoxic fractions by pimonidazole labelling. RESULTS: The pattern of [(18)F]FMISO signal was dependent on the distribution of hypoxia at the microregional level. The comparison of [(18)F]FMISO autoradiography and pimonidazole immunohistochemistry by pixel-by-pixel analysis revealed moderate correlations. In five tumour lines, a significant correlation between the mean [(18)F]FMISO and pimonidazole signal intensity was found (range, r(2)=0.91 to r(2)=0.99). Comparison of the tumour lines with respect to the microregional distribution pattern of hypoxia revealed that the correlation between the mean signal intensities strongly depended on the microarchitecture. Overall, a weak but significant correlation between hypoxic fractions based on pimonidazole labeling and the mean [(18)F]FMISO signal intensity was observed (r(2)=0.18, p=0.02). For the three tumour models with a ribbon-like microregional distribution pattern of hypoxia, the correlation between the hypoxic fraction and the mean [(18)F]FMISO signal intensity was much stronger and more significant (r(2)=0.73, p<0.001) than for the tumours with a more homogenous, patchy, microregional distribution pattern of hypoxia. CONCLUSION: Different patterns of [(18)F]FMISO accumulation dependent on the underlying microregional distribution of hypoxia were found in ten head and neck xenograft tumours. A weak albeit significant correlation was found between the mean [(18)F]FMISO signal intensity and the hypoxic fraction of the tumours. In larger clinical tumours, [(18)F]FMISO-PET provides information on the tumour oxygenation status on a global level, facilitating dose painting in radiation treatment planning. However, caution must be taken when studying small tumour subvolumes as accumulation of the tracer depends on the presence of hypoxia and on the tumour microarchitecture.

Testosterone-stimulated growth of the rat prostate may be driven by tissue hypoxia and hypoxia-inducible factor-1alpha.

Testosterone-stimulated growth of the ventral prostate (VP) in castrated rats is preceded by angiogenesis, but the mechanisms coordinating vascular and tissue growth are unknown. Adult rats were castrated and some treated with testosterone. Tissue hypoxia was studied morphologically using the hypoxia marker pimonidazole (Hypoxyprobe), hypoxia-inducible factor-1 (HIF-1) alpha, vascular endothelial growth factor (VEGF), and carbonicanhydrase 9 (CA-9) levels by western blotting and quantitative RT-PCR. In the intact untreated prostate, most glands were unstained by the hypoxia marker but already 1 day after castration most epithelial cells in the VP were stained. Seven days after castration prostate glands were apparently normoxic again, and HIF-1alpha, VEGF, and CA-9 were decreased. Treatment of 7-day castrated rats with testosterone resulted in increased epithelial hypoxyprobe staining and increased HIF-1alpha, VEGF, and CA-9 levels. The transient increase in tissue hypoxia after testosterone treatment is probably caused by a temporary mismatch between oxygen consumption and supply. Treatment of prostate epithelial cells in vitro under normoxic conditions also increased HIF-1alpha, and this could be blocked if epidermal growth factor receptor (EGFR) signaling was blocked with gefitinib. In vivo gefitinib could, however, not block the testosterone induced increase in HIF-1alpha. Testosterone may thus induce HIF-1alpha and its downstream angiogenesis promoting genes by at least two mechanisms, hypoxia and EGFR signaling. Transient epithelial cell hypoxia could by rapidly increasing HIF-1alpha and VEGF be an essential coordinator of testosterone-stimulated vascular and glandular growth.