ABSTRACT
Yinzhihuang oral liquid (YZH) is a traditional Chinese medicine that has been widely used in Asia to prevent and treat neonatal hyperbilirubinemia, but the published preclinical studies on its anti-hyperbilirubinemia effect are conducted in adult animals, partly due to the lack of preclinical neonatal hyperbilirubinemia animal models. In the present study, we tested six reagents to induce hyperbilirubinemia in neonatal rats, and established two appropriate neonatal hyperbilirubinemia rat models by subcutaneous injection of δ-Aminolevulinic acid (ALA, 200â mg/kg) or novobiocin (NOVO, 200â mg/kg). Oral treatment of YZH (80, 160 and 320â mg/kg) significantly decreased serum conjugated bilirubin levels in ALA-treated neonatal rats and serum unconjugated bilirubin levels in NOVO-treated neonatal rats, respectively. Additionally, pre-treatment of YZH also prevented the increase of serum bilirubin levels in both ALA- and NOVO-treated rats. Mechanistically, YZH significantly up-regulated the mRNA expression of genes involved in hepatic bilirubin disposition (organic anion-transporting polypeptide 1b2, Oatp1b2; multidrug resistance-associated proteinâ 2, Mrp2) and bilirubin conjugation (UDP-glucuronosyltransferase 1a1, Ugt1a1). Additionally, YZH up-regulated the mRNA expression of cytochrome P450 1A1 (Cyp1a1), the target gene of aryl hydrocarbon receptor (AhR), and increased the nuclear protein levels of AhR in livers of neonatal rats. YZH and its two active ingredients, namely baicalin (BCL) and 4'-hydroxyacetophenone (4-HT), up-regulated the mRNA expression of AhR target genes (CYP1A1 and UGT1A1) and increased nuclear protein levels of AhR in HepG2 cells. In conclusion, the present study provides two neonatal hyperbilirubinemia animal models and evaluates the anti-hyperbilirubinemia effect and mechanisms of YZH in neonatal animals.
Subject(s)
Drugs, Chinese Herbal/chemistry , Administration, Oral , Aminolevulinic Acid/toxicity , Animals , Animals, Newborn , Bilirubin/blood , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A1/metabolism , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/therapeutic use , Hep G2 Cells , Humans , Hyperbilirubinemia/chemically induced , Hyperbilirubinemia/drug therapy , Hyperbilirubinemia/pathology , Liver/drug effects , Liver/metabolism , Liver/pathology , Medicine, Chinese Traditional , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Novobiocin/toxicity , RNA Interference , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Aryl Hydrocarbon/antagonists & inhibitors , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Up-Regulation/drug effectsABSTRACT
The constant release of pharmaceuticals products to aquatic environment even at low concentrations (ng L-1 to µg L-1) could lead to unknown chronic effects to non-target organisms. The aim of this study was to evaluate neurotoxic responses, inflammation, gametogenic activity and energy status on the fresh water clam C. fluminea after exposure to different concentrations of caffeine (CAF), ibuprofen (IBU), carbamazepine (CBZ), novobiocin (NOV) and tamoxifen (TMX) for 21 days under laboratory conditions. During the assay, water was spiked every two days with CAF (0; 0.1; 5; 15; 50µgL-1), IBU (0; 0.1; 5; 10; 50µgL-1), CBZ, NOV, and TMX (0.1, 1, 10, 50µgL-1). After the exposure period, dopamine levels (DOP), monoamine oxidase activity (MAO), arachidonic acid cyclooxygenase activity (COX), vitellogenin-like proteins (VTG), mitochondrial electron transport (MET), total lipids (TLP), and energy expenditure (MET/TLP) were determined in gonad tissues, and acetyl cholinesterase activity (AChE) was determined in digestive gland tissues. Results showed a concentration-dependence response on biomarkers tested, except for MAO. Environmental concentrations of pharmaceuticals induced significant changes (p < 0.05) in the neurotoxic responses analyzed (CAF, CBZ and NOV increased DOP levels and CBZ inhibited AChE activity), inflammation (CAF induced COX), and energy status (MET and TLP increased after exposure to CBZ, NOV and TMX). Responses of clams were related to the mechanism of action (MoA) of pharmaceuticals. Biomarkers applied and the model organism C. fluminea constituted a suitable tool for environmental risk assessment of pharmaceutical in aquatic environment.
Subject(s)
Caffeine/toxicity , Carbamazepine/toxicity , Corbicula/physiology , Ibuprofen/toxicity , Novobiocin/toxicity , Tamoxifen/toxicity , Water Pollutants, Chemical/toxicity , Animals , Biomarkers/metabolism , Corbicula/metabolism , Energy Metabolism , Germ Cells , Humans , Inflammation , NeurotoxinsABSTRACT
Inhibition of the heat shock protein 90 (Hsp90) C-terminus represents a promising therapeutic strategy for the treatment of cancer. Novobiocin, a coumarin antibiotic, was the first Hsp90 C-terminal inhibitor identified, however, it manifested poor anti-proliferative activity (SKBr3, IC50 ≈700â µm). Subsequent structure-activity relationship (SAR) studies on novobiocin led to development of several analogues that exhibited improved anti-proliferative activity against several cancer cell lines. Recent studies demonstrate that the biphenyl core could be used in lieu of the coumarin ring system, which resulted in more efficacious analogues. In continuation of previous efforts, the work described herein has identified the phenyl cyclohexyl core as a novel scaffold for Hsp90 C-terminal inhibition. Structure-activity relationship (SAR) studies on this scaffold led to the development of compounds that manifest mid-nanomolar activity against SKBr3 and MCF-7 breast cancer cell lines through Hsp90 inhibition.
Subject(s)
Amidines/chemistry , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Amidines/chemical synthesis , Amidines/toxicity , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Design , HSP90 Heat-Shock Proteins/metabolism , Humans , MCF-7 Cells , Novobiocin/chemistry , Novobiocin/toxicity , Protein Domains , Structure-Activity RelationshipABSTRACT
The marine amphipod Ampelisca brevicornis was used as model organism of benthic macrofauna to assess the possible adverse effects of pharmaceuticals bound to sediments. Organisms were exposed to sediment spiked with novobiocin (NOV) and methotrexate (MTX) for 10 days in order to estimate the acute toxicity (lethal effects) produced by the two compounds. The surviving organisms were pooled and analyzed to determine their sublethal responses associated with different phases of metabolism (enzyme activities in phases I and II), oxidative stress (antioxidant enzyme activities and lipid peroxidation), and genotoxicity (DNA damage in the form of strand breaks). No lethal or sublethal effects were observed in the amphipods exposed to NOV. For organisms exposed to sediments spiked with MTX the results were found to calculate the concentration that was lethal to 50% of the organisms exposed in the toxicity tests (LC50 of 30.36 ng/g). MTX also induced the metabolism of enzyme detoxification activities in phases I and II. Oxidative stress and DNA damage in particular were also observed, indicating responses associated with MTX's mechanism of action. Both mortality and the set of applied biomarkers allowed for the assessment of bioavailability, oxidative stress, and genotoxicity of NOV and MTX. The information obtained in this investigation can assist in ecological risk assessment of marine sediments contaminated by pharmaceuticals.
Subject(s)
Amphipoda/physiology , Methotrexate/toxicity , Novobiocin/toxicity , Water Pollutants, Chemical/toxicity , Animals , Biomarkers/metabolism , DNA Damage , Environmental Monitoring , Geologic Sediments/chemistry , Lipid Peroxidation/drug effects , Oxidative Stress , Toxicity TestsABSTRACT
A battery of biomarkers was evaluated on Ruditapes philippinarum exposed during 14 days to caffeine, ibuprofen, carbamazepine and novobiocin (0.1, 1, 5, 10, 15, and 50µgL(-1)). The battery included general stress (lysosomal membrane stability - LMS) analysed in the hemolymph, and biochemical biomarkers analysed in digestive gland tissues including: biomarkers of phase I (etoxyresorufin O-deethylase - EROD, dibenzylfluorescein dealkylase - DBF), phase II (gluthathione-S-transferase - GST), oxidative stress (gluthathione reductase - GR, gluthathione peroxidase - GPX, lipid peroxidation - LPO), neurotoxicity (acetylcholinesterase activity - AChE), and genotoxicity (DNA damage). Pharmaceuticals tested induced the sublethal responses (even at the environmental range 0.1µgL(-1)). At this low concentration; caffeine, ibuprofen and carbamazepine decreased the LMS significantly compared with controls (p<0.05). The four compounds induced significantly the detoxification metabolism and oxidative stress (p<0.05). Neurotoxicity was noticed in clams exposed to caffeine and carbamazepine (p<0.05). Ibuprofen, carbamazepine and novobiocin produced genotoxic effects (p<0.05). Results from this research validate the use of biomarkers when assessing the effects of pharmaceuticals within a marine environmental risk assessment framework, using as a laboratory bioassay model the species R. philippinarum.
Subject(s)
Bivalvia/drug effects , Caffeine/toxicity , Carbamazepine/toxicity , Ibuprofen/toxicity , Novobiocin/toxicity , Water Pollutants, Chemical/toxicity , Acetylcholinesterase/metabolism , Animals , Biomarkers/metabolism , Bivalvia/metabolism , Cytochrome P-450 CYP1A1/metabolism , DNA Damage , Gastrointestinal Tract/drug effects , Gastrointestinal Tract/metabolism , Glutathione Peroxidase/metabolism , Glutathione Reductase/metabolism , Glutathione Transferase/metabolism , Hemolymph/drug effects , Hemolymph/metabolism , Inactivation, Metabolic , Lipid Peroxidation/drug effects , Oxidative Stress/drug effects , Water Pollutants, Chemical/pharmacokineticsABSTRACT
We cloned and sequenced the waaL (rfaL) gene from Bradyrhizobium japonicum, which infects soybean and forms nitrogen-fixing nodules on soybean roots. waaL has been extensively studied in the lipopolysaccharide (LPS) biosynthesis of enteric bacteria, but little is known about its function in (brady)rhizobial LPS architecture. To characterize its role as O-antigen ligase in the LPS biosynthesis pathway, we constructed a waaL knock-out mutant and its complemented strain named JS015 and CS015, respectively. LPS analysis showed that an LPS structure of JS015 is deficient in O-antigen as compared to that of the wild type and complemented strain CS015, suggesting that WaaL ligates the O-antigen to lipid A-core oligosaccharide to form a complete LPS. JS015 also revealed increased cell surface hydrophobicity, but it showed decreased motility in soft agar plates. In addition to the alteration in cell surface properties, disruption of the waaL gene caused increased sensitivity of JS015 to hydrogen peroxide, osmotic pressure, and novobiocin. Specifically, plant tests revealed that JS015 failed to nodulate the host plant soybean, indicating that the rhizobial waaL gene is responsible for the establishment of a symbiotic relationship between soybean and B. japonicum.
Subject(s)
Adaptation, Physiological , Bacterial Proteins/genetics , Bradyrhizobium/genetics , Genes, Bacterial , Glycine max/microbiology , Hydrophobic and Hydrophilic Interactions , Stress, Physiological , Symbiosis , Adaptation, Physiological/drug effects , Bacterial Proteins/metabolism , Bradyrhizobium/drug effects , Bradyrhizobium/physiology , Cell Membrane/metabolism , Electrophoresis, Polyacrylamide Gel , Flagella/metabolism , Flagella/ultrastructure , Lipopolysaccharides/metabolism , Molecular Sequence Data , Movement , Mutation , Novobiocin/toxicity , O Antigens , Osmotic Pressure/drug effects , Oxidative Stress/drug effects , Plant Root Nodulation/drug effects , Plant Root Nodulation/genetics , Root Nodules, Plant/drug effects , Root Nodules, Plant/microbiology , Glycine max/drug effects , Stress, Physiological/drug effects , Stress, Physiological/genetics , Symbiosis/drug effectsABSTRACT
Reports indicating the presence of pharmaceutical in fresh water environment in the ngL(-1) to µgL(-1) range are occurring with increasing frequency. It is also a fact that pharmaceuticals may produce adverse effects on aquatic organisms. Nevertheless, there is still a lack of knowledge regarding how these emergent contaminants may affect aquatic biota. The goal of this research was to evaluate the sublethal responses in Corbicula fluminea such as, general stress (lysosomal membrane stability [LMS]), biomarkers of phase I and II (etoxyresorufin O-deethylase [EROD], dibenzylfluorescein dealkylase [DBF], gluthathione-S-transferase [GST]), oxidative stress (gluthathione reductase [GR], gluthathione peroxidase [GPX], lipid peroxidation [LPO]), and biomarkers of effect (DNA damage) after 21 days of exposure to caffeine, ibuprofen, carbamazepine, novobiocin and tamoxifen at 0.1, 1, 5, 10, 15, 50µgL(-1). Environmental concentrations tested in this study caused general stress and produced changes on biomarkers tested. LMS, responses from phase I and II enzymatic activity, oxidative stress, and biomarker of effect represent important ecotoxicological information, and will provide a useful reference for the assessment of selected drugs and the effects which these compounds may have on aquatic invertebrates, using C. fluminea as a bioindicator species.
Subject(s)
Caffeine/toxicity , Carbamazepine/toxicity , Corbicula/drug effects , Ibuprofen/toxicity , Novobiocin/toxicity , Tamoxifen/toxicity , Animals , Biomarkers/metabolism , DNA Damage/drug effects , Environmental Monitoring , Glutathione Peroxidase/metabolism , Glutathione Reductase/metabolism , Glutathione Transferase/metabolism , Lipid Peroxidation/drug effects , Oxidative Stress/drug effects , Toxicity Tests , Water Pollutants, Chemical/analysis , Water Pollutants, Chemical/toxicityABSTRACT
Although pharmaceuticals have been detected in the environment only in the range from ng/L to microg/L, it has been demonstrated that they can adversely affect the health status of aquatic organisms. Lysosomal membrane stability (LMS) has previously been applied as an indicator of cellular well-being to determine health status in bivalve mussels. The objective of this study is to evaluate LMS in Ruditapes philippinarum haemolymph using the neutral red retention assay (NRRA). Clams were exposed in laboratory conditions to caffeine (0.1, 5, 15, 50 microg/L), ibuprofen (0.1, 5, 10, 50 microg/L), carbamazepine and novobiocin (both at 0.1, 1, 10, 50 microg/L) for 35 days. Results show a dose-dependent effect of the pharmaceuticals. The neutral red retention time measured at the end of the bioassay was significantly reduced by 50% after exposure to environmental concentrations (p < 0.05) (caffeine = 15 microg/L; ibuprofen = 10 microg/L; carbamazepine = 1 microg/L and novobiocin = 1 microg/L), compared to controls. Clams exposed to these pharmaceuticals were considered to present a diminished health status (retention time < 45 min), significantly worse than controls (96 min) (p < 0.05). The predicted no environmental effect concentration (PNEC) results showed that these pharmaceuticals are very toxic at the environmental concentrations tested. Measurement of the alteration of LMS has been found to be a sensitive technique that enables evaluation of the health status of clams after exposure to pharmaceuticals under laboratory conditions, thus representing a robust Tier-1 screening biomarker.
Subject(s)
Bivalvia , Lysosomes/drug effects , Water Pollutants, Chemical/toxicity , Animals , Biomarkers , Caffeine/toxicity , Carbamazepine/toxicity , Hemocytes , Ibuprofen/toxicity , Lysosomes/metabolism , Neutral Red/metabolism , Novobiocin/toxicity , Wastewater/toxicityABSTRACT
One of the main consequences of the constant input of pharmaceuticals to the aquatic environment is that biota might develop unknown chronic effects, thus affecting their health even at low concentrations. The aim of this study is to evaluate the health status of Carcinus maenas employing a 2-tier approach, after 28 days of exposure to carbamazepine (CBZ) and novobiocin (NOV) at 0.1, 1, 10 and 50µgL(-1). Lysosomal membrane stability (LMS) is employed in tier 1. In tier 2 was applied a battery of biomarkers of exposure and effect (ethoxyresorufin O-deethylase (EROD), dibenzyl flourescein dealkylase (DBF), glutathione S-transferase (GST), glutathione peroxidase (GPx), lipid peroxidation (LPO) and DNA adducts) measured in gill, hepatopancreas, muscle and gonad tissues. Results show a dose-dependent effect. LMS in crabs exposed to environmental concentrations of pharmaceuticals was significantly lower compared to controls (p<0.05), indicating their stressed status. EROD activity was induced significantly (p<0.05) in all tissues by NOV (10-50µgL(-1)). DBF activity was induced significantly (p<0.05) in gill and hepatopancreas tissues by CBZ (10-50µgL(-1)). GST activity was activated in all tissues of crabs exposed to the highest concentrations tested (p<0.05). All tissues showed induction of GPX activity after exposure to selected drugs (p<0.05). LPO was activated in gill and hepatopancreas tissues by the pharmaceuticals at 50µgL(-1) (p<0.05). Crabs exposed to NOV (50µgL(-1)) presented DNA damage in gill and hepatopancreas tissues (p<0.05). Environmental concentrations of these pharmaceuticals have a measurable effect on the biomarkers studied. The 2-tier approach applied might be a suitable tool for the assessment of sublethal responses in crabs exposed to pharmaceuticals in the marine environment.
Subject(s)
Brachyura/drug effects , Carbamazepine/toxicity , Novobiocin/toxicity , Water Pollutants, Chemical/toxicity , Animals , Biomarkers/analysis , Brachyura/enzymology , Brachyura/metabolism , Cytochrome P-450 CYP1A1/metabolism , Environmental Exposure , Enzymes/metabolism , Gills/drug effects , Gills/enzymology , Glutathione Peroxidase/metabolism , Glutathione Transferase/metabolism , Hepatopancreas/drug effects , Hepatopancreas/metabolism , Lipid PeroxidationABSTRACT
Recent studies have shown that novobiocin (NB), a member of the coumermycin (CA) family of antibiotics with demonstrated DNA gyrase inhibitory activity, inhibits Heat shock protein 90 (HSP90) by binding weakly to a putative ATP-binding site within its C-terminus. To develop more potent HSP90 inhibitors that target this site and to define structure-activity relationships (SARs) for this class of compounds, we have synthesized twenty seven 3-amido-7-noviosylcoumarin analogues starting from NB and CA. These were evaluated for evidence of HSP90 inhibition using several biological assays including inhibition of cell proliferation and cell cycle arrest, induction of the heat shock response, inhibition of luciferase-refolding in vitro, and depletion of the HSP90 client protein c-erbB-2/HER-2/neu (HER2). This SAR study revealed that a substantial increase in biological activity can be achieved by introduction of an indole-2-carboxamide group in place of 4-hydroxy-isopentylbenzamido group at C-3 of NB in addition to removal/derivatization of the 4-hydroxyl group from the coumarin ring. Methylation of the 4-hydroxyl group in the coumarin moiety moderately increased biological activity as shown by compounds 11 and 13. Our most potent new analogue 19 demonstrated biological activities consistent with known HSP90-binding agents, but with greater potency than NB.
Subject(s)
Antineoplastic Agents/chemical synthesis , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Novobiocin/analogs & derivatives , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , HSP90 Heat-Shock Proteins/metabolism , Humans , MCF-7 Cells , Novobiocin/chemical synthesis , Novobiocin/toxicity , Structure-Activity RelationshipABSTRACT
KU-32 is a novel, novobiocin-based Hsp90 inhibitor that protects against neuronal glucotoxicity and reverses multiple clinical indices of diabetic peripheral neuropathy in a rodent model. However, any drug with potential for treating diabetic complications must also have no adverse effects on the function of pancreatic islets. Thus, the goal of the current study was to assess the effect of KU-32 on the in vitro viability and function of human islets. Treating human islets with KU-32 for 24 hours showed no toxicity as assessed using the alamarBlue assay. Confocal microscopy confirmed that with a minimum of 2-day exposure, KU-32 improved cellular viability by blocking apoptosis. Functionally, isolated human islets released more glucose-stimulated insulin when preincubated in KU-32. However, diabetic BKS-db/db mice, a model for type 2 diabetes, administered KU-32 for 10 weeks did not show any significant changes in blood glucose and insulin levels, despite having greater insulin staining/beta cell in the pancreas compared to untreated BKS db/db mice. In summary, KU-32 did not harm isolated human islets and may even be protective. However, the effect does not appear significant enough to alter the in vivo metabolic parameters of diabetic mice.
Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Diabetic Neuropathies/drug therapy , Insulin/metabolism , Islets of Langerhans/drug effects , Neuroprotective Agents/pharmacology , Novobiocin/analogs & derivatives , Adult , Animals , Apoptosis/drug effects , Blood Glucose/drug effects , Blood Glucose/metabolism , Cell Survival/drug effects , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/pathology , Diabetic Neuropathies/blood , Diabetic Neuropathies/pathology , Disease Models, Animal , Dose-Response Relationship, Drug , Female , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/metabolism , Humans , Insulin/blood , Insulin Secretion , Islets of Langerhans/metabolism , Male , Mice , Microscopy, Confocal , Middle Aged , Novobiocin/pharmacology , Novobiocin/toxicity , Time Factors , Tissue Culture TechniquesABSTRACT
Glycosylated analogues of novobiocin, discovered using a broad library of enzymes, have 100-fold improved activity against breast, brain, pancreatic, lung and ovarian cancers and ablated associated off-target activity leading to an up to 2.7 × 10(4) fold increase in selectivity.
Subject(s)
Enzyme Inhibitors/chemistry , Enzymes/chemistry , Novobiocin/chemistry , Cell Line, Tumor , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/metabolism , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/toxicity , Enzymes/metabolism , Glycosylation/drug effects , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/metabolism , Humans , Novobiocin/chemical synthesis , Novobiocin/toxicityABSTRACT
Heat shock proteinâ 90 (Hsp90) is a significant target in the development of rational cancer therapy, due to its role at the crossroads of multiple signaling pathways associated with cell proliferation and viability. Here, a novel series of Hsp90 inhibitors containing a quinolein-2-one scaffold was synthesized and evaluated in cell proliferation assays. Results from these structure-activity relationships studies enabled identification of the simplified 3-aminoquinolein-2-one analogue 2 b (6BrCaQ), which manifests micromolar activity against a panel of cancer cell lines. The molecular signature of Hsp90 inhibition was assessed by depletion of standard known Hsp90 client proteins. Finally, processing and activation of caspases 7, 8, and 9, and the subsequent cleavage of PARP by 6BrCaQ, suggest stimulation of apoptosis through both extrinsic and intrinsic pathways.
Subject(s)
Antineoplastic Agents/chemistry , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Quinolones/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/toxicity , Apoptosis , Cell Line, Tumor , Drug Evaluation, Preclinical , HSP90 Heat-Shock Proteins/metabolism , Humans , Novobiocin/toxicity , Proteasome Endopeptidase Complex/metabolism , Protein Folding , Quinolones/chemical synthesis , Quinolones/toxicityABSTRACT
Since porphyrins are regarded as endogenous substrates for the ATP-binding cassette (ABC) transporter ABCG2, it is hypothesized that functional impairment owing to genetic polymorphisms or inhibition of ABCG2 by drugs may result in a disruption of cellular porphyrin homeostasis. In the present study, we expressed ABCG2 genetic variants, i.e., V12M, Q141K, S441N, and F489L, as well as the wild type (WT) in Flp-In-293 cells to examine the hypothesis. Cells expressing S441N and F489L variants exhibited high levels of both cellularly accumulated pheophorbide a and photosensitivity, when those cells were incubated with pheophorbide a and irradiated with visible light. To further elucidate the significance of ABCG2 in cellular porphyrin homeostasis, we observed cellular accumulation and compartmentation of porphyrin and pheophorbide a by means of a new fluorescence microscopy technology, and found that accumulation of porphyrin and pheophorbide a in the cytoplasm compartment was maintained at low levels in Flp-In-293 cells expressing ABCG2 WT, V12M, or Q141K. When ABCG2 was inhibited by imatinib or novobiocin, however, those cells became sensitive to light. Based on these results, it is strongly suggested that certain genetic polymorphisms and/or inhibition of ABCG2 by drugs can enhance the potential risk of photosensitivity.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Light , Membrane Transport Modulators/toxicity , Neoplasm Proteins/metabolism , Photosensitivity Disorders/etiology , Photosensitizing Agents/toxicity , Polymorphism, Single Nucleotide , Porphyrins/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/genetics , Animals , Benzamides , Cell Line , Cell Survival/drug effects , Cell Survival/radiation effects , Chlorophyll/analogs & derivatives , Chlorophyll/toxicity , Dose-Response Relationship, Drug , Drug Interactions , Homeostasis , Humans , Imatinib Mesylate , Inhibitory Concentration 50 , Insecta , Microscopy, Fluorescence , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Novobiocin/toxicity , Photosensitivity Disorders/chemically induced , Photosensitivity Disorders/genetics , Photosensitivity Disorders/metabolism , Photosensitizing Agents/metabolism , Piperazines/toxicity , Pyrimidines/toxicity , Quercetin/pharmacology , Quercetin/toxicity , Risk Assessment , TransfectionABSTRACT
Municipal wastewaters are recognized as a major source of pharmaceutical and personal care products to the aquatic environment, thereby exposing biota to unknown chronic effects. This study sought to examine the immunotoxic effects of pharmaceutical and urban waste products on the freshwater mussel Elliptio complanata. Hemolymph samples were collected and treated in vitro with increasing concentrations of various drugs (bezafibrate, carbamazepine, fluoxetine, gemfibrozil, morphine, naproxen, novobiocin, oxytetracycline, sulfamethazole, sulfapyridine and trimethoprim) and urban waste related chemicals (coprostanol, caffeine, cotinine) for 24 h at 15 degrees C. In a parallel experiment, mussels were caged and placed in two final aeration lagoons for the treatment of domestic wastewaters. At the end of the exposure period, hemolymphs were tested for phagocytic activity, intracellular esterase activity, cell adherence and lipid peroxidation (LPO). The products that most increased phagocytosis were bezafibrate, gemfibrozil and trimethoprim, while novobiocin and morphine reduced its activity. Intracellular esterase activity was reduced most strongly with sulfamethazole, novobiocin, gemfibrozil, bezafibrate and carbamazepine. Cell adherence was decreased by oxytetracycline, novobiocin and naproxen, and increased by gemfibrozil, bezafibrate and sulfapyridine. Exposure to these products also modulated LPO in hemocytes. Coprostanol and naproxen were more potent to reduce LPO while novobiocin and sulfapyridine were the most potent to induce LPO. The potential to induce LPO was positively correlated with the number of functional groups on the molecule (i.e., its nucleophilicity). Mussels exposed to domestic wastewater treatment plant aeration lagoons had decreased intracellular esterase and phagocytic activity as well, suggesting immunosuppression. PPCPs (pharmaceuticals and personal care products) that are recognized to disrupt cytokine signalling network by the nitric oxide pathway and cell permeability were generally the most potent ones. The data suggest that PPCPs have the potential to cause adverse effects on the immune system of bivalves.
Subject(s)
Bivalvia/drug effects , Phagocytosis , Water Pollutants, Chemical/toxicity , Animals , Bivalvia/enzymology , Bivalvia/immunology , Carbamazepine/chemistry , Carbamazepine/toxicity , Cell Adhesion , Esterases/metabolism , Gemfibrozil/chemistry , Gemfibrozil/toxicity , Hemocytes/drug effects , Hemocytes/enzymology , Hemocytes/immunology , Lipid Peroxidation , Novobiocin/chemistry , Novobiocin/toxicity , Phagocytosis/drug effects , Structure-Activity Relationship , Waste Disposal, FluidABSTRACT
PURPOSE: The coumarin antibiotic novobiocin potentiates the activity of etoposide (VP-16) in vitro by increasing intracellular accumulation of VP-16. The drug efflux pump inhibited by novobiocin appears to be distinct from both of the major proteins associated with the multidrug resistance phenotype in human cancers, the 170-kDa P-glycoprotein and the 190-kDa multidrug resistance protein. In a recent study, we found that novobiocin augmented VP-16 accumulation ex vivo in 16 of 24 fresh tumor samples at concentrations that could be achieved in vivo. Therefore, we conducted a clinical trial to determine the maximum tolerated dose and the pharmacokinetics of novobiocin when given in combination with VP-16. PATIENTS AND METHODS: Patients with refractory cancer were treated with VP-16 on days 1, 3, and 5. Antiemetics, consisting of ondansetron and dexamethasone, were given 60 minutes before the VP-16 was administered. Novobiocin was given orally 30 minutes before the VP-16, and the dose was escalated in successive groups of patients according to a standard dose escalation design. Treatment cycles were repeated every 4 weeks. Plasma concentrations of novobiocin were determined during the first treatment cycle by high-performance liquid chromatography. RESULTS: Thirty-three patients were treated for a total of 69 cycles. Eleven patients were treated with a starting dose of VP-16 of 120 mg/m2, and three of these patients experienced neutropenic fever. The dose of VP-16 was reduced to 100 mg/m2, and an additional 22 patients were enrolled. The dose of novobiocin ranged from 3 to 9 g. At a novobiocin dose of at least 5.5 g, plasma concentrations of at least 150 microM were sustained for 24 hours. Dose-limiting toxicities consisted of neutropenic fever and reversible hyperbilirubinemia. Nausea, which was a limiting toxicity in other trials of novobiocin, was well controlled with the use of serotonergic antiemetics. Diarrhea was common but mild in most patients. DISCUSSION: In previously treated patients, the recommended dose of novobiocin in this schedule is 7 g/m2/day. Novobiocin does not appear to augment the toxicity of VP-16 to the bone marrow or the gastrointestinal mucosa. Plasma concentrations of novobiocin equivalent to the levels required to modulate VP-16 in vitro are readily achievable for total but not unbound free drug.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Etoposide/therapeutic use , Novobiocin/administration & dosage , Novobiocin/pharmacokinetics , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/toxicity , Drug Administration Schedule , Etoposide/toxicity , Female , Humans , Male , Maximum Tolerated Dose , Middle Aged , Neoplasm, Residual/drug therapy , Neoplasm, Residual/metabolism , Novobiocin/toxicityABSTRACT
A simple, sensitive, and reliable "DNA diffusion" assay for the quantification of apoptosis is described. Human lymphocytes and human lymphoblastoid cells, MOLT-4, were exposed to 0, 12.5, 25, 50, or 100 rad of X-rays. After 24 h of incubation, cells were mixed with agarose, microgels were made, and cells were lysed in high salt and detergents. DNA was precipitated in microgels by ethanol. Staining of DNA was done with an intense fluorescent dye, YOYO-1. Apoptotic cells show a halo of granular DNA with a hazy outer boundary. Necrotic cells, resulting from hyperthermia treatment, on the other hand, show an unusually large homogeneous nucleus with a clearly defined boundary. The number of cells with apoptotic and necrotic appearance can be scored and quantified by using a fluorescent microscope. Results were compared with other methods of apoptosis measurement: morphological estimations of apoptosis and DNA ladder pattern formation in regular agarose gel electrophoresis. Validation of the technique was done using some known inducers of apoptosis and necrosis (hyperthermia, hydrogen peroxide, mitoxantrone, novobiocin, and sodium ascorbate).
Subject(s)
Apoptosis , Lymphocytes/cytology , Adult , Animals , Apoptosis/drug effects , Apoptosis/radiation effects , Ascorbic Acid/toxicity , Benzoxazoles , Brain/cytology , Cell Nucleus/ultrastructure , Cells, Cultured , Coloring Agents , DNA Fragmentation , Electrophoresis, Agar Gel/methods , Fluorescent Dyes , Humans , Hydrogen Peroxide/toxicity , Lymphocytes/physiology , Lymphocytes/radiation effects , Male , Microscopy/methods , Mitoxantrone/toxicity , Novobiocin/toxicity , Quinolinium Compounds , Rats , Rats, Inbred BN , Sensitivity and Specificity , Thymus Gland/cytology , Tumor Cells, CulturedABSTRACT
Antifolates have been shown to increase the DNA strand breaks produced by the topoisomerase inhibitor etoposide. PT523 is a potent new antifolate that cannot be polyglutamated. Human SCC-25 squamous carcinoma cells were exposed to methotrexate, trimetrexate or PT523 at a concentration of 5 microM for 24 h along with various concentrations of etoposide or novobiocin during the final 2 h. Isobologram analysis of the treatment combinations indicated that exposure of the cells to PT523/etoposide, methotrexate/etoposide, PT523/novobiocin, methotrexate/novobiocin and trimetrexate/novobiocin resulted in greater than additive cytotoxicity. DNA alkaline elution studies with the same drug combinations indicated that there were three- to four-fold increases in the radiation equivalent (rad equivalent) strand breaks in the cellular DNA with etoposide or novobiocin along with the antifolate compared with the topoisomerase II inhibitors alone. Tumor growth delay studies were carried out in the murine SCC VII squamous carcinoma. PT523 (0.5 mg/kg) and methotrexate (2 mg/kg) were administered by 7-day continuous infusion while trimetrexate (3.75 mg/kg) was administered intraperitoneally daily on days 7-9. Etoposide (10 mg/kg) and novobiocin (100 mg/kg) were administered intraperitoneally on alternate days (7, 9, 11). The combinations of PT523 with etoposide or novobiocin were significantly more effective than methotrexate and etoposide or novobiocin, producing tumor growth delays of 8.4 days and 6.9 days, respectively. Overall, the antifolate/topoisomerase II inhibitor treatment combinations produced tumor growth delays that were apparently additive to greater than additive.
Subject(s)
Antineoplastic Agents/toxicity , Carcinoma, Squamous Cell/drug therapy , DNA Damage , Folic Acid Antagonists/toxicity , Ornithine/analogs & derivatives , Pterins/toxicity , Pterins/therapeutic use , Topoisomerase II Inhibitors , Animals , Antineoplastic Agents/therapeutic use , Cell Division/drug effects , Cell Line , Cell Survival/drug effects , DNA, Neoplasm/drug effects , Drug Synergism , Etoposide/therapeutic use , Etoposide/toxicity , Folic Acid Antagonists/therapeutic use , Head and Neck Neoplasms , Humans , Male , Methotrexate/therapeutic use , Methotrexate/toxicity , Mice , Mice, Inbred C3H , Novobiocin/therapeutic use , Novobiocin/toxicity , Ornithine/therapeutic use , Ornithine/toxicity , Trimetrexate/therapeutic use , Trimetrexate/toxicity , Tumor Cells, CulturedABSTRACT
Chicken embryo brain and liver cells in vitro exhibited spontaneous DNA fragmentation as determined by viscometry of alkaline cell lysates. Ca2+ and Mg2+ enhanced, while Zn2+, the Ca2+ chelator ethylenglycol-bis(beta-aminoethyl-ether)-N,N,N'-tetraacetic acid (EGTA), spermine and--to a lesser extent--spermidine and Hoechst 33,258 inhibited spontaneous DNA fragmentation. Under the same conditions chromatin condensation, as assessed by nucleoid sedimentation, increased. Exposure of chicken embryo cells to various genotoxic agents, i.e. doxorubicin, bleomycin, methyl methanesulfonate, thiyl radicals, H2O2, UV light, and X-rays, increased DNA fragmentation in a dose dependent manner. Zn2+ or EGTA diminished DNA fragmentation in cells exposed to bleomycin, thiyl radicals, H2O2 and UV light. An apparent sensitisation to X-irradiation has been observed in Zn2+ or EGTA-pretreated cells. It is suggested by the present investigations that, with agent specific peculiarities, apoptotic phenomena are implicated when nucleotoxicity is assessed in chicken embryo cells by physico-chemical short-term tests in vitro.
Subject(s)
DNA/drug effects , Deoxyribonuclease I/antagonists & inhibitors , Animals , Brain/cytology , Cations, Divalent/toxicity , Cell Nucleus/chemistry , Cell Nucleus/ultrastructure , Cells, Cultured , Chemical Phenomena , Chemistry, Physical , Chick Embryo , Chromatin/chemistry , Chromatin/ultrastructure , DNA/radiation effects , Egtazic Acid/toxicity , Intercalating Agents/toxicity , Novobiocin/toxicity , Spermidine/pharmacology , Spermine/pharmacology , Suramin/toxicity , Ultraviolet Rays , Viscosity , X-RaysABSTRACT
Various compounds were evaluated for their ability to induce prophage lambda in the Escherichia coli WP2s(lambda) microscreen assay. The inability of a DNA gyrase subunit B inhibitor (novobiocin) to induce prophage indicated that inhibition of the gyrase's ATPase was insufficient to elicit the SOS response. In contrast, poisons of DNA gyrase subunit A (nalidixic acid and oxolinic acid) were the most potent inducers of prophage among the agents examined here. This suggested that inhibition of the ligation function of subunit A, which also has a DNA nicking activity, likely resulted in DNA breaks that were available (as single-stranded DNA) to act as strong SOS-inducing signals, leading to prophage induction. Agents that both intercalated and produced reactive-oxygen species (the mammalian DNA topoisomerase II poisons, adriamycin, ellipticine, and m-AMSA) were the next most potent inducers of prophage. Agents that produced reactive-oxygen species only (hydrogen peroxide and paraquat) were less potent than adriamycin and ellipticine but more potent than m-AMSA. Agents that intercalated but did not generate reactive-oxygen species (actinomycin D) or that did neither (teniposide) were unable to induce prophage, suggesting that intercalation alone may be insufficient to induce prophage. These results illustrate the variety of mechanisms (and the relative effectiveness of these mechanisms) by which agents can induce prophage. Nonetheless, these agents may induce prophage by producing essentially the same type of DNA damage, i.e., DNA strand breaks. The potent genotoxicity of the DNA gyrase subunit A poisons illustrates the genotoxic consequences of perturbing an important DNA-protein complex such as that formed by DNA and DNA topoisomerase.