pubMedId
stringlengths 5
1.75k
| title
stringlengths 1
2.03k
⌀ | abstract
stringlengths 1
15.7k
⌀ |
|---|---|---|
20702122 | Personality traits and social behaviors predict the psychological adjustment of Chinese people with epilepsy. | Very little is known about the psychosocial correlates of psychological morbidity among Chinese people with epilepsy (PWE). No studies have investigated the association between social relationships and psychological morbidity, while most studies examined only the negative impact of maladaptive personalities on psychological adjustment in PWE. This study examined the association of psychological morbidity with a broad array of personality traits and social skills in a sample of 54 Chinese PWE. pleted the Temperament and Character Inventory (TCI), the Social Performance Survey Schedule (SPSS), and the Hospital Anxiety and Depression Scale (HADS) via semi-structured interview. Regression analyses revealed that, independent of demographic and medical variables and perceived impact, Harm Avoidance was positively associated with anxiety and depression whereas Self-Directedness was negatively associated with anxiety and depression; that Cooperativeness was inversely associated with anxiety. Social skills were inversely associated with depression whereas negative social skills were inversely associated with anxiety. Clinical implications of adaptive personality traits and social skills functioning are discussed. |
20702123 | Evolution in VNS therapy for refractory epilepsy, experience with Demipulse devices at Ghent University Hospital. | Vagus nerve stimulation (VNS) is a frequently used treatment for patients with refractory epilepsy who are unsuitable candidates for epilepsy surgery. There has been a steady evolution in VNS technology, as generators' volumes have e smaller and battery life expectancy longer. This pilot study is an open-label retrospective study that describes our experience with the mercially available generator, i.e. the VNS Therapy Demipulse Model 103. Treatment efficacy and side effects, as well as technical and practical enhancements useful for the patient and for the medical staff are discussed in this study. |
20702125 | PERFIDI filters to suppress and/or quantify relaxation time components in multi-component systems: an example for fat-water systems. | Parametrically Enabled Relaxation FIlters with Double and multiple Inversion (PERFIDI) is an experimental NMR/MRI technique devised to analyze samples/voxels characterized by multi-exponential longitudinal relaxation. It is based on a bination of NMR sequences with suitable posed of inversion pulses. Given any standard NMR/MRI sequence, it permits one to modify it in a way which will attenuate, in a predictable manner and before data acquisition, signals arising ponents with different r rates (r=1/T1). Consequently, it is possible to define relatively simple protocols to suppress and/or to quantify signals of ponents. This article describes a simple way to construct low-pass, high-pass and band-pass PERFIDI filters. Experimental data are presented in which the method has been used to separate fat and water proton signals. We also present a novel protocol for very fast determination of the ratio between the fat signal and the total signal which avoids any time-consuming magnetization recovery multi-array data acquisition. The method has been validated also for MRI, producing well T1-contrasted images. |
20702126 | [The primordial follicle: outline of a portrait]. | Ovarian primordial follicles present original features of quiescence and long survival. Follicular growth is triggered by withdrawal of inhibitory mechanisms maintaining the quiescence and supported by a finely tuned molecular dialogue between the oocyte and its surrounding granulosa cells. The reserve of primordial follicles is oversized and functionally heterogeneous. Different ponents can account for this heterogeneity. |
20702127 | Scleral buckling biomaterials and implants for retinal detachment surgery. | Scleral buckling is a widely used surgical procedure that aims at repairing retinal detachments. Many materials and procedural techniques have been variously proposed and tested in an attempt to find the bination for providing optimal results to the patient. This review highlights the evolution of scleral buckling implants and chronicles the main advances that have been made in such a context. Specifically, the limitations of the materials and implants fallen in disuse, as well as the advantages of currently adopted devices are critically examined and discussed. Future directions for the research are considered, underlining in particular the great potential carried by the development of accurate mathematical models for describing the postoperative evolution of buckled eye. These analytical models, supported by prehensive data set provided by advanced techniques of medical investigations, may e useful tools for helping surgeons to choose, and to design if necessary, the best buckling material and configuration to be used in each specific clinical case. |
20702128 | Mitral valve dynamics in structural and fluid-structure interaction models. | Modelling and simulation of heart valves is a challenging biomechanical problem due to anatomical variability, pulsatile physiological pressure loads and 3D anisotropic material behaviour. Current valvular models based on the finite element method can be divided into: those that do model the interaction between the blood and the valve (fluid-structure interaction or 'wet' models) and those that do not (structural models or 'dry' models). Here an anatomically sized model of the mitral valve has been used pare the difference between structural and fluid-structure interaction techniques in two separately simulated scenarios: valve closure and a cardiac cycle. Using fluid-structure interaction, the valve has been modelled separately in a straight tubular volume and in a U-shaped ventricular volume, in order to analyse the difference in the coupled fluid and structural dynamics between the two geometries. The results of the structural and fluid-structure interaction models have shown that the stress distribution in the closure simulation is similar in all the models, but the magnitude and closed configuration differ. In the cardiac cycle simulation significant differences in the valvular dynamics were found between the structural and fluid-structure interaction models due to difference in applied pressure loads. Comparison of the fluid domains of the fluid-structure interaction models have shown that the ventricular geometry generates slower fluid velocity with increased pared to the tubular geometry. In conclusion, structural heart valve models are suitable for simulation of static configurations (opened or closed valves), but in order to simulate full dynamic behaviour fluid-structure interaction models are required. |
20702129 | Autonomic failures in Perry syndrome with DCTN1 mutation. | Perry syndrome is a familial parkinsonism associated with central hypoventilation, mental depression, and weight loss. Previously, this very rare syndrome has been reported in only 7 families worldwide including in one Japanese family. We recently identified an additional family with Perry syndrome with DCTN1 mutation residing in Japan. The pedigree contains 19 family members spanning three generations, with four affected individuals. Affected members with early stage disease in this family presented with marked autonomic dysfunction including orthostatic hypotension and decreased cardiac uptake with [123]I-metaiodobenzylguanidine scintigram features that have not been described in previous cases. Because of central hypoventilation, all affected members need ventilation assistance, which is thought beneficial for prolongation of survival time as well as improving quality of life in this syndrome. |
20702130 | FMR1 gene expansion and scans without evidence of dopaminergic deficits in parkinsonism patients. | To determine if patients with parkinsonism and fragile X mental retardation 1 (FMR1) gene expansions have a striatal dopamine deficit similar to Parkinson disease (PD) patients. |
20702132 | Synthesis, crystal structure and photoelectric property of two new coordination polymers constructed by longer-spanning suberic acid and 4,4'-bipyridine ligands. | Two-dimensional coordination polymers, [M(C₈H₁₂O₄)(C₁₀H₈N₂)]·H₂O [M=Co (1), Cd (2); C₁₀H₈N₂ = 4,4-bipyridine, C₈H₁₄O₄=subaric acid] were obtained from the reaction of the metal salts, bipy and subaric acid at 180°C and characterized by elemental analysis, infrared spectrum, and single-crystal X-ray diffraction and surface photovoltage spectrum (SPS). The single-crystal X-ray diffraction showed that the subaric ligand in the plexes exhibits two types of modes coordinating to transition metal ions, resulting in the formation of a 1D infinite chain along the c-axis. In addition, the results of SPS plexes 1 and 2 indicate that these plexes exhibit positive surface photovoltage responses in the range of 300-800 nm, which can be assigned to LMCT and MLCT, respectively. And the SPS plex 1 also can be assigned to the d→d* electronic transition. The SPS spectra of the plexes are consistent with their UV-vis spectra. |
20702133 | Anisotropic media effect on the dipole moment of some coumarin dyes. | The ground state (μ(g)) and the excited state (μ(e)) dipole moments of two coumarin laser dyes, C500 and C503, were studied at room temperature in various solvents, viz., aprotic solvents, alcohols and liquid crystals at 298 K. We report dipole moment of laser dyes in different anisotropic (liquid crystals) and isotropic environments. The dipole moments values in different media help to investigate environment effects on the molecular dipole moment and provide a straightforward method paring their properties. Ground and excited state dipole moments of coumarin dyes were evaluated by means of solvatochromic shift method. It was observed that dipole moment values of excited states (μ(e)) were higher than corresponding ground state values (μ(g)), indicating a substantial redistribution of the π-electron densities in a more polar excited state for the dyes investigated. |
20702134 | Prevalence and clinical characteristics of human CoV-HKU1 in children with acute respiratory tract infections in China. | Human CoV-HKU1 (HCoV-HKU1) has been isolated from a 71-year-old man with pneumonia; however, the impact and role of emerging HCoV-HKU1 have not been defined in children with acute respiratory tract infection (ARTI). |
20702135 | More prolonged brain activity related to gaze cueing in schizophrenia. | The ability to use the gaze direction of another person to guide attention is important for social functioning, but behavioral reports on this topic among individuals with schizophrenia are inconclusive. Event Related Potentials (ERPs) can very accurately pinpoint the shifting of attention, and can therefore shed more light on cueing abilities in schizophrenia. |
20702136 | Comparing potential COI and SSU rDNA barcodes for assessing the diversity and phylogenetic relationships of cyphoderiid testate amoebae (Rhizaria: Euglyphida). | The mitochondrial Cytochrome Oxidase Subunit 1 gene (COI) has been promoted as an ideal "DNA barcode" for animal species and other groups of eukaryotes. However, the utility of the COI marker for species level discrimination and for phylogenetic analyses has yet to be tested within the Rhizaria. Accordingly, we analysed mitochondrial COI gene sequences and nuclear small subunit rDNA (SSU) sequences from several morphospecies of euglyphid testate amoebae (Cercozoa, Rhizaria) in order to evaluate the utility of these DNA markers for species discrimination and phylogenetic reconstructions. Sequences were obtained from eleven populations belonging to sixCyphoderiamorphospecies that were isolated from field samples in North America and Europe. Mean inter-population COI sequence dissimilarities were on average 2.9 times greater than in the SSU, while the intra-population sequence dissimilarities were higher in the SSU (0-0.95%) than in the COI (0%); this suggests that the COI fragment is valuable for discriminating Cyphoderiidae isolates. Our study also demonstrated that COI sequences are useful for inferring phylogenetic relationships among Cyphoderiidae isolates. COI and SSU tree topologies were very similar even though the COI fragment used in these analyses (500bp) was much shorter than the SSU sequences (1600bp). Altogether, these results demonstrate the utility of the COI as a potential taxonomic DNA barcode for assessing cyphoderiid species diversity and for inferring phylogenetic relationships within the group. |
20702137 | Endovascular treatment of superior vena cava syndrome by percutaneous venoplasty. | Thrombosis of the superior vena cava leads to obstruction of venous outflow of the head and upper extremities and causes severe clinical symptoms. The management of SVC syndrome depends on aetiology and acuity at clinical presentation and ranges from conservative medical treatment to bypass surgery. Endovascular treatment can provide rapid relief of symptoms and substantial clinical improvement independent of aetiology. We report a case of successful interventional treatment in a patient with catheter-induced SVC thrombosis and present a review of the literature. |
20702138 | Bevacizumab with FOLFOXIRI (irinotecan, oxaliplatin, fluorouracil, and folinate) as first-line treatment for metastatic colorectal cancer: a phase 2 trial. | The FOLFOXIRI (irinotecan, oxaliplatin, fluorouracil, and folinate) regimen has been shown to be better than FOLFIRI (fluorouracil, folinate, and irinotecan) in a phase 3 trial in patients with metastatic colorectal cancer. Results of various studies have shown that the addition of bevacizumab to chemotherapy increases treatment efficacy. We therefore assessed the safety and activity of bination of FOLFOXIRI plus bevacizumab in patients with colorectal cancer. |
20702139 | The platelet storage defect as measured in Costa Rica. | The platelet storage defect passes all untoward effects on platelet morphology structure and function with storage and the mechanisms responsible are not fully understood but are clearly multifactorial. The presence of swirling may correlate with acceptable pH values and the volume of suspending plasma to be effective to maintain a pH greater than 6,4 is between 75-85 g. |
20702140 | Stable feature selection for biomarker discovery. | Feature selection techniques have been used as the workhorse in biomarker discovery applications for a long time. Surprisingly, the stability of feature selection with respect to sampling variations has long been under-considered. It is only until recently that this issue has received more and more attention. In this article, we review existing stable feature selection methods for biomarker discovery using a generic hierarchical framework. We have two objectives: (1) providing an overview on this new yet fast growing topic for a convenient reference; (2) categorizing existing methods under an expandable framework for future research and development. |
20702141 | Food-related attitudes and behaviors at home, school, and restaurants: perspectives from racially diverse, urban, low-income 9- to 13-year-old children in Minnesota. | This qualitative study explored e children's food-related attitudes and behaviors, and current weight status. |
20702142 | High-dose rate brachytherapy in the treatment of penile carcinoma--first experience. | Interstitial low-dose rate brachytherapy (BRT) allows a conservative treatment of T1-T2 penile carcinoma. High-dose rate (HDR) BRT is often considered as a dangerous method for interstitial implants because of higher risk plications. However, numerous reports suggest that results of HDR-BRT may parable to low-dose rate BRT. There are no data available in the literature regarding HDR interstitial BRT for carcinoma of the penis. |
20702143 | Anemia after bariatric surgery cannot be explained by iron deficiency alone: results of a large cohort study. | We sought to identify the frequency and mechanisms of anemia after bariatric surgery in a bariatric surgery program at the Medical College of Wisconsin, (Milwaukee, WI). Anemia after bariatric surgery has often been attributed to iron deficiency, although an ponent might be present, making the anemia after surgery plex. |
20702144 | Improvement of esophageal dysmotility after conversion from gastric banding to gastric bypass. | Patients undergoing adjustable gastric banding can develop clinically apparent alterations in esophageal motility. There is little data on how such patients do after band removal and revision to other bariatric operations. One article in the literature describes long term manometric evidence of dysmotility in a band patient converted to gastric bypass. |
20702151 | [Thyroid dysfunction in patients with advanced renal cell carcinoma treated with sunitinib: a multifactorial issue]. | Several studies have reported the substantial prevalence of sunitinib-induced thyroid dysfunction. However, the underlying mechanism and the benefit of thyroid hormone replacement therapy remain to be determined. To evaluate the effect of sunitinib on thyroid function, we carried out a descriptive study in patients with advanced renal cell carcinoma. |
20702150 | Measurement of urinary oxypurinol by high performance liquid chromatography-tandem mass spectrometry. | Oxypurinol is the active metabolite of allopurinol which is used to treat hyperuricaemia associated with gout. Both oxypurinol and allopurinol inhibit xanthine oxidase which forms uric acid from xanthine and hypoxanthine. Plasma oxypurinol concentrations vary substantially between individuals and the source of this variability remains unclear. The aim of this study was to develop an HPLC-tandem mass spectrometry method to measure oxypurinol in urine to facilitate the study of the renal elimination of oxypurinol in patients with gout. Urine samples (50 microL) were prepared by dilution with a solution of acetonitrile/methanol/water (95/2/3, v/v; 2 mL) that contained the internal standard (8-methylxanthine; 1.5 mg/L), followed by centrifugation. An aliquot (2 microL) was injected. Chromatography was performed on an Atlantis HILIC Silica column (3 microm, 100 mm x 2.1mm, Waters) at 30 degrees C, using a mobile prised of acetonitrile/methanol/50 mM ammonium acetate in 0.2% formic acid (95/2/3, v/v). Using a flow rate of 0.35 mL/min, the analysis time was 6.0 min. Mass spectrometric detection was by selected reactant monitoring (oxypurinol: m/z 150.8-->108.0; internal standard: m/z 164.9-->121.8) in negative electrospray ionization mode. Calibration curves were prepared in drug-free urine across the range 10-200 mg/L and fitted using quadratic regression with a weighting factor of 1/x (r(2) > 0.997, n=7). Quality control samples (20, 80, 150 and 300 mg/L) were used to determine intra-day (n=5) and inter-day (n=7) accuracy and imprecision. The inter-day accuracy and imprecision was 96.1-104% and <11.2%, respectively. Urinary oxypurinol samples were stable when subjected to 3 freeze-thaw cycles and when stored at room temperature for up to 6h. Samples collected from 10 patients, not receiving allopurinol therapy, were screened and showed no significant interferences. The method was suitable for the quantification of oxypurinol in the urine of patients (n=34) participating in a clinical trial to optimize therapy of gout with allopurinol. |
20702149 | Determination of the gamma-secretase inhibitor MK-0752 in human plasma by online extraction and electrospray tandem mass spectrometry (HTLC-ESI-MS/MS). | A sensitive and rapid HTLC-ESI-MS/MS method with an advanced online sample preparation was developed for determination of the gamma-secretase inhibitor MK-0752 in human plasma using an internal standard. Plasma samples (100 microL) were diluted and injected directly onto an online extraction column (Cohesive Cyclone MAX 0.5 mm x 50 mm, > 30 microm), the sample matrix was washed out with an aqueous solution, and retained analytes were eluted out and transferred directly to the analytical column (Phenomenex Gemini 3 micron C18 110A, 50 mm x 2.0 mm at 50 degrees C) for separation using a gradient mobile phase. The eluted analytes were then detected on an API-3000 LC-MS/MS System with ESI and a negative multiple reaction monitoring mode. The monitored ion transitions were m/z 441-->175 for MK-0752 and 496-->175 for the internal standard. Online extraction recoveries were 81%. The method was validated and was linear in the range of 0.05-50 microg/mL. Within-day and between-day precisions were<8.6%, and accuracies were 0.7 and 7.1%. This method was applied to the measurement of plasma MK-0752 levels in a Phase I study of pediatric patients with recurrent or refractory brain tumors. |
20702152 | Cholangiocarcinoma: A position paper by the Italian Society of Gastroenterology (SIGE), the Italian Association of Hospital Gastroenterology (AIGO), the Italian Association of Medical Oncology (AIOM) and the Italian Association of Oncological Radiotherapy (AIRO). | The incidence of Cholangiocellular carcinoma (CCA) is increasing, due to a sharp increase of the intra-hepatic form. Evidence-ascertained risk factors for CCA are primary sclerosing cholangitis, Opistorchis viverrini infection, Caroli disease, congenital choledocal cist, Vater ampulla adenoma, bile duct adenoma and intra-hepatic lithiasis. Obesity, diabetes, smoking, abnormal biliary-pancreatic junction, bilio-enteric surgery, and viral cirrhosis are emerging risk factors, but their role still needs to be validated. Patients with primary sclerosing cholangitis should undergo surveillance, even though a survival benefit has not been clearly demonstrated. CCA is most often diagnosed in an advanced stage, when therapeutic options are limited to palliation. Diagnosis of the tumor is often difficult and multiple imaging techniques should be used, particularly for staging. Surgery is the standard of care for resectable CCA, whilst liver transplantation should be considered only in experimental settings. Metal stenting is the standard of care in inoperable patients with an expected survival >4 months. Gemcitabine or platinum analogues are mended in advanced CCA whilst there are no validated neo-adjuvant treatments or second-line chemotherapies. Even though promising results have been obtained in CCA with radiotherapy, further randomized controlled trials are needed. |
20702146 | Laparoscopic Roux-en-Y gastric bypass versus laparoscopic adjustable gastric banding: five years of follow-up. | Bariatric surgery is an effective treatment for morbid obesity. Laparoscopic Roux-en-Y gastric bypass (LRYGB) and laparoscopic adjustable gastric banding (LAGB) monly performed procedures. The aim of the present study was to evaluate pare the long-term es after LRYGB and LAGB. |
20702147 | Revisional bariatric surgery: who, what, where, and when? | Revisional bariatric surgery (RBS) es have been poorly characterized. pared the RBS and primary bariatric surgery (PBS) es at the Penn State Milton S. Hershey Medical Center in the United States. |
20702154 | Acute disassembly of a bipolar radial head arthroplasty. | The GUEPAR(®) implant is a metallic bipolar radial head prosthesis designed to minuted radial head fractures when anatomic realignment of the articular surface of the radiocapitellar joint is not possible. We report herein the rare case of an plete disassembly of this implant, discuss the reason for this occurrence and review the literature. In the presented case, plete removal of the prosthesis provided a satisfactory e with an excellent Mayo Elbow Performance Score at 12months follow-up. |
20702148 | Roux-en-Y gastric bypass alters tumor necrosis factor-α but not adiponectin signaling in immediate postoperative period in obese rats. | Adiponectin has anti-inflammatory properties and is increased with weight loss. Tumor necrosis factor (TNF)-α is a pro-inflammatory cytokine that negatively regulates adiponectin. Previously, we have demonstrated that Roux-en-Y gastric bypass (RYGB) induces weight loss and improves steatosis in obese rats. We hypothesized that RYGB would alter the interplay of TNF-α and adiponectin signaling in the postoperative period. |
20702158 | Primary cultures of rabbit renal proximal tubule cells: II. Selected phase I and phase II metabolic capacities. | Specific characteristics of cells vary as a function of time in culture. We have determined the stability of selected Phase I and Phase II biotransformation capacities in rabbit renal proximal tubule cells in primary culture. When grown in hormonally-defined medium, proximal tubule cells lost Phase I metabolic capacity. Cytochrome P-450 content and associated mixed-function oxidase activities present in kidney cortex microsomes were not detectable after 14 days in culture. Phase II glutathione-dependent metabolic functions were well retained in cultured pared with freshly isolated proximal tubules (FIPT). Cellular total glutathione content was 2.8 mug/mg protein in pared with approximately 10 mug/mg protein in stable confluent cultures. A higher total glutathione content of 20.6 mug/mg was noted in preconfluent cultures. The glutathione redox state was initially perturbed in FIPT with 37% of the total glutathione present found in its oxidized form. Tubule cells recovered to a normal ratio (6-13% of total glutathione in the oxidized form) while in culture. The glutathione S-transferase activity in 4-day-old cells in culture was reduced to 50% of the 4 U/mg protein level found in FIPT. No appreciable further decline in glutathione S-transferase activity was detected during 15 days in culture. The level of gamma-glutamyl-transpeptidase (a brush-border enzyme necessary for glutathione uptake into proximal tubule cells) declined from 1499 mU/mg protein in homogenates of FIPT to 636 mU/mg in homogenates of 8-day-old cultured cells. A further decline in activity occurred during the next 7 days in culture. In conclusion, although Phase I metabolic functions were diminished in primary cultured rabbit proximal tubule cells, Phase II metabolic functions were retained at parable with FIPT and well above those found in several established kidney cell lines. |
20702155 | A higher prediagnostic insulin level is a prospective risk factor for incident prostate cancer. | A higher insulin level has been linked to the risk of prostate cancer promotion. However, several reports claim that there is no association between a higher insulin level and the risk of incident prostate cancer. In the present report, the insulin hypothesis was tested once more prospectively in men with a benign prostatic disorder. Three hundred and eighty-nine consecutive patients referred with lower urinary tract symptoms without clinical prostate cancer were included during 1994-2002. Follow-up was performed in 2006. Data were obtained from the Swedish National Cancer Register and the Regional Cancer Register, Oncological Centre, Göteborg, Sweden. At this follow-up, 44 of the patients included had developed prostate cancer. Men with prostate cancer diagnosis had a higher systolic (P<0.001) and diastolic blood pressure (P<0.000), were more obese as measured by BMI (P=0.010), waist (P=0.007) and hip measurements (P=0.041) than men who did not have prostate cancer diagnosis at follow-up. These men also had a higher uric acid level (P=0.040), and a higher fasting serum insulin level (P=0.023) than men who did not have prostate cancer diagnosis at follow-up. Following exclusion of T1a/b prostate cancer cases, the difference of the fasting serum insulin level between the groups was still significant (P=0.038). Our data support the hypothesis that a higher insulin level is a promoter of prostate cancer. Moreover, our data suggest that the insulin level could be used as a marker of the risk of developing prostate cancer. The present findings also seem to confirm that prostate cancer is ponent of the metabolic syndrome. Finally, our data generate the hypothesis that the metabolic syndrome conceals early prostate cancer. |
20702156 | Intake of dietary fats and colorectal cancer risk: prospective findings from the UK Dietary Cohort Consortium. | Epidemiologic evidence for an association between colorectal cancer (CRC) risk and total dietary fat, saturated fat (SF), monounsaturated fat (MUFA) and polyunsaturated fat (PUFA) is inconsistent. Previous studies have used food frequency questionnaires (FFQ) to assess diet, but data from food diaries may be less prone to severe measurement error than data from FFQ. |
20702159 | Induction of micronuclei in cultured human bronchial epithelial cells by direct-acting carcinogens. | The sensitivity of human bronchial epithelial cells to induction of micronuclei was determined in cultures derived from seven different donors. Two direct-acting carcinogens, dl-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and N-methyl-N'-nitro-N-nitrosoguanidine were used to induce micronuclei. Both agents increased the incidence of micronuclei in a concentration-dependent fashion in cells from most donors, event at concentrations that did not produce appreciable cytotoxicity; there were considerable variations in the responses of different donors. Cytokinesis was blocked with cytochalasin B so that micronuclei were counted only in binucleate cells, thereby decreasing the total number of cells that needed to be examined and also eliminating variations due to possible differences in cell growth rates. The results demonstrate the potential usefulness of the micronucleus assay as a sensitive measure of genetic damage in human epithelial cells from the lung. |
20702160 | Implication of alterations in intracellular calcium ion homoeostasis in the advent of paracetamol-induced cytotoxicity in primary mouse hepatocyte monolayer cultures. | We have examined the fluctuation of free cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) using the fluorescent probe quin-2 during the cytotoxic response induced by low concentrations (100-250 mum) of the model hepatotoxin paracetamol (APAP) in primary mouse hepatocyte cultures over 5 days. APAP-associated increases in [Ca(2+)](i) were recorded prior to APAP-associated cytotoxicity, and correlated with the subsequent loss of cell viability as measured by intracellular lactate dehydrogenase and K(+) efflux. Co-incubation with promethazine (1 mum) or ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic 0215 acid (4 mm) attenuated both the APAP-associated [Ca(2+)](i) changes and cytotoxicity. These results support the hypothesis that mobilization of intracellular Ca(2+) may be an important early event in APAP-induced hepatotoxicity. |
20702161 | Comparative assessment of in vitro toxicity of xenobiotics using flow cytometry and spectrophotometry. | Cell viability and cell proliferation are endpoints that can be used to identify cytotoxic effects. In a study of the cytotoxicity of four biomaterials and drugs, these two criteria were determined by different techniques. There were notable similarities and differences among the different methods used. Cell viability, which was determined by the trypan blue exclusion test, spectrophotometric microtitration (neutral red) and flow cytometry (fluorescein diacetate) gave similar results. However, the neutral red assay was found to be the most sensitive method for determining the cytotoxicity of these biomaterials and drugs. Cell proliferation measurement, by cell counts and quantitative protein estimation (coomassie blue), revealed important variations between the two methods and indicated poor sensitivity for the protein assay. A slight variability in the determination of the inhibitory concentration 50 (IC(50)) for the two drugs was observed for all the techniques. |
20702162 | The effects of ethylene oxide sterilization on the in vitro cytotoxicity of a bone replacement material. | The effect of degassing on the cytotoxicity of an ethylene oxide (EtO)-sterilized copolymer constituent of a bone replacement material, was determined using the (51)Cr release assay. An initial experiment used mouse L929 cells and a copolymer with an ambient pressure and temperature (APT) degassing time of 24 hr. Both copolymer and nylon control discs caused a significantly (P </= 0.05) increased release of (51)Cr from the cells pared with discs that had not been EtO sterilized. In a second experiment using L929 cells, copolymer and nylon discs were EtO-sterilized and degassed (APT) for 1-14 days. After 24 hr in culture, discs degassed for up to 14 days caused significantly increased cytotoxicity. A third experiment used L929 cells and copolymer/demineralized bone matrix blocks with APT degassing times of 2, 4 and 6 wk, or vacuum degassing for 48 hr. A decrease in cytotoxicity was observed from 2 to 6 wk of degassing, but there was still a significant increase over the untreated discs at 2 and 4 wk and a non-significant increase at 6 wk. Vacuum degassing parable to 4 wk of APT. In the fourth experiment, vacuum degassing times of 1, 7 and 14 days were studied using both L929 and human fibroblast cells. Vacuum degassing for up to 14 days did not reduce the cytotoxicity to the levels observed for samples that had not been EtO sterilized. All four experiments demonstrated that EtO sterilization of the copolymer used in bone replacement materials caused increased in vitro pared with non-sterilized copolymer, and that degassing either at APT for up to 6 wk or vacuum degassing for up to 14 days reduced, but did not eliminate, the cytotoxicity observed. |
20702163 | Effects of lead on haem biosynthesis during erythroid differentiation in vitro. | Murine erythroleukaemia cells (MELC) are erythroid precursor cells that undergo erythroid differentiation in the presence of the inducer hexamethylene bisacetamide (HMBA). The effects of lead on haem biosynthesis in MELC following HMBA-induced differentiation were studied. MELC were induced with HMBA in the presence of 20, 40 and 80 mum-lead acetate and cell density, haem content, incorporation of (14)C-labelled delta-aminolaevulinic acid (ALA) into haem, and the activities of the enzymes delta-aminolaevulinic acid dehydratase (ALA-D), uroporphyrinogen I synthetase (URO-S) and ferrochelatase (FERRO) were determined. MELC exposed to 80 mum-lead showed significant erythroid hypoplasia (40-50%) and a significant decrease (30-50%) in haem content at 2, 4 and 6 days after induction parison with the controls. Significant inhibition of ALA-D, the most sensitive index, was noted at 20 mum-lead, and at 80 mum-lead ALA-D activity was decreased by 60-80% parison with the controls. URO-S and FERRO showed significant decreases of 34% and 50%, respectively, at 80 mum-lead. A decrease of 50% in the incorporation of [(14)C]ALA into haem at 80 mum-lead indicated an impairment in haem synthesis. The results suggest that the impairment of haem formation by lead is coincident with the production of severe erythroid hypoplasia. |
20702165 | The effects of morphine, cocaine, amphetamine and hashish on the phagocytosis of the protozoon Tetrahymena pyriformis strain W. | Cells of the ciliated protozoon Tetrahymena pyriformis, strain W, grown in a peptone-yeast medium usually contain many phagocytic vacuoles. The phagocytic activity of this protozoon was studied in vivo using heat-killed yeast stained with carmine dye and after exposing the cultures for 2 hr to morphine (20 mug/ml), cocaine (20 mug/ml), amphetamine (0.5 mug/ml) or hashish (0.1 mug/ml). The number of vacuoles formed indicated the phagocytic activity after treatment with the drugs of abuse. Amphetamine caused a slight increase (P < 0.05) in the phagocytic activity of the protozoon, whereas morphine, cocaine and hashish each caused a significant (P < 0.01) decrease in this activity. |
20702164 | Specific teratogenic action of 2,4,5-trichlorophenoxyacetic acid on mouse and rat whole-embryo cultures. | The effects of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) were studied at concentrations ranging from 0.17 to 3.52 mm on post-implanted mouse and rat embryos cultured for 48 hr, either in the absence of any metabolic activation system or in the presence of mouse and rat S-9 mix. 2,4,5-T proved to be a potential teratogen, at 0.88 and 1.76 mm, on mouse embryos in the absence of any metabolic activation system. In the presence of mouse S-9 mix, 2,4,5-T showed a high teratogenic potential at 0.17, 0.88 and 1.76 mm. In contrast, in the presence of rat S-9 mix, 2,4,5-T induced structural defects only at 1.76 mm. At 3.52 mm, 2,4,5-T was 100% embryolethal with or without S-9 mix. On rat embryos, 2,4,5-T was potentially teratogenic only in the presence of mouse S-9 mix, causing a significant increase in dysmorphogenic effects at 0.17, 0.88 and 1.76 mm. With or without rat S-9 mix, 2,4,5,-T was only embryolethal to rat embryos at 1.76 and 3.52 mm. The abnormalities mainly involved the forebrain, the midbrain and the branchial arches. These results are consistent with the known in vivo embryotoxic action of pound. |
20702166 | The liver slice system: A rapid in vitro acute toxicity test for primary screening of hepatotoxic agents. | A simple method for the rapid screening of hepatotoxic agents is described. Liver slice systems were prepared from rats and mice, and incubated in Krebs-Ringer-Hepes medium with different concentrations of the pounds. Hepatotoxicity was monitored by determination of liver enzymes in the slice medium. Enzyme leakage was dose- and time-dependent. Histopathological changes in the hepatotoxin-treated slices were well correlated with the extent of enzyme leakage. Species differences in susceptibility to various hepatotoxins could be easily detected by this in vitro system: the dose-toxicity curves revealed that the mouse is more vulnerable than the rat to acetaminophen and furosemide. These findings are well correlated with those of in vivo experiments. A preliminary study showed that, in the same species, the relative toxicities of various chemicals in the liver slice system were similar to those reported in vivo. In summary, these results on the tissue slice system are encouraging. However, much more work will have to be done before the system can be considered sufficiently well validated for routine use. |
20702167 | Cadmium-induced metallothionein synthesis in the rat liver slice system. | In vivo exposure of a rat to cadmium results in elevation of the hepatic metal-binding protein, metallothionein (MT). The present work describes the induction of MT in the in vitro liver slice system. Incubation of rat liver slices with CdCl(2) resulted in a dose-dependent elevation of tissue MT. The cadmium-binding protein was increased by 140 and 220% in the presence of 10 and 20 mum CdCl(2), respectively. A lower level (5 mum) of the inducer had only a slight effect. A time-course study showed a gradual increase in the MT level following incubation of the liver slices for 4 and 6 hr with 10 mum-cadmium. Characterization of the metal-binding protein by Sephadex G-75 gel filtration revealed that it posed of MT and also of a high-molecular-weight fraction that might be either a polymerized or aggregated form of MT, or another type of cadmium-binding protein. These findings indicate that the response of the liver slice system to toxic agents is similar to that of the intact animal. These are encouraging results which need to be extended before the system can be introduced into the routine screening of hepatotoxic agents. |
20702170 | Inflammatory mediators applied to in vitro toxicology: Studies on mediator release and two-cell systems. | Release of mediators is a manifestation of tissue stimulation or damage applicable to in vitro toxicological investigations. Intercell mediators, or cytokines, are released on tissue stimulation or as a result of mild damage. Intracell mediators, or enzymes of respiration and metabolism, released from damaged cells, participate in inflammatory change and are the usual mediator determinants in in vitro tests. Cells examined for in vitro mediator release are mast cells, which are applicable to anaphylactic allergy and acute inflammation, macrophages, which are particularly applicable to toxicity of inhalable dusts, and fibroblasts/fibrocytes, which are widely used as a general model for cytotoxicity. In tests on organs, lungs may be perfused to identify cytokines released by test substances, which may be used to distinguish between effects due to allergy and those resulting from chemically-induced inflammation. Skin, both human and animal, as full depth or keratome slices of epidermis, has an application in the detection of irritant and corrosive substances. Weak irritants are identified by release of enzymes, for example acid phosphatase, neutral protease and lactate dehydrogenase. Strong irritants and corrosive substances inhibit release or destroy the enzymes. There are inconsistencies in the identification of moderate irritants, both chemicals and bacterial toxins, in which the rank order of severity of effect does not correspond to other in vitro features or in vivo potency. Detection of cytokines (prostaglandins, leukotrienes and interleukin-1) is possible with appropriate conditions of culture, but has been little applied to toxicology. Studies on two-organ/cell systems are briefly reported in which biological mediator release is detectable without specific identification. Cultured skin preparations were treated in vitro with irritants. The culture media were transferred to cultures of normal skin (examined for stimulus to DNA synthesis), to fibroblasts (examined for DNA and polysaccharide/heparan sulphate synthesis) and to suspensions of neutrophils in Boyden chambers (for chemotaxis). The media were also examined for histamine (none found), acid protease cathepsins, acid phosphatase and lactate dehydrogenase. As before, weak chemical irritants were detectable by stimulation of normal skin and fibroblasts to DNA synthesis, fibroblasts to increased heparan sulphate-like synthesis, and analytically by release of enzymes. Moderate irritants and Clostridium perfringens type C toxins depressed these features. Reference is made to the possibility to identify substances likely to induce progressive pulmonary fibrosis, by transfer to fibrocyte cultures of media from macrophages ingesting the dusts or co-culturing macrophages and fibrocytes, to detect a sustained stimulus to fibrogenesis. |
20702171 | In vitro predictive tests for eye irritants. | The developments and attitudes towards in vitro testing since the first major workshop on irritation testing five years ago (Reinhardt et al., 1985) are summarized. Many test systems have been described and an increasing number pounds tested. However, the in vivo data basis used parison is still heterogeneous and a proper analysis of most in vivo/in vitro correlations is difficult. Some progress has been achieved with various controlled validation programmes on a national and international level, all of which have not pleted. In order to improve this slow progress the following measures are proposed: (1) expectations for a uniform test need to be discouraged when heterogeneous chemical actions may occur, and rigid test guidelines have to be replaced by flexible ones (differentiated approach); (2) many practical small steps need to be taken rather than one theoretical large step, that is by accepting simple in vitro tests for restricted groups of chemicals such as severe irritants (adaptive approach); and (3) the threshold for acceptance of in vitro tests needs to be lowered by regulatory bodies and routine laboratories (optimistic approach). |
20702172 | In vitro tests with sensitized lymphocytes-Relevance for predictive allergenicity testing. | The main obstacle to routine in vitro tests to identify contact sensitizers is the inappropriate application of the allergens in vitro, even when sufficient antigen-presenting cells are available. Some allergens may need the skin tissue for adequate conjugation with carrier molecules, whereas others may be poorly solubilized or toxic for the cultures. Methods, therefore, which make use of the in vivo system for allergen binding and presentation, but which assess in vitro the subsequent lymphocyte activation are most promising. For this reason, the local lymph node assay deserves more attention as a predictive assay in allergenicity testing and should be validated in more laboratories in the near future. The value of allergen-stimulated in vitro tests for predictive allergenicity screening will remain limited. This type of in vitro test is, however, invaluable for mechanistic studies on contact sensitivity in well defined allergen models. Also for clinical diagnosis of allergic contact sensitivity to selected allergens, the lymphocyte transformation test and macrophage migration inhibition test may be useful, particularly when skin tests fail to be conclusive. |
20702173 | Microtubule disassembly induced by sensitizing halogenated nitrobenzene derivatives. | The potency of certain halogenated nitrobenzene derivatives to induce allergic contact dermatitis has been demonstrated in guinea-pigs and in man. As the first step towards understanding the mechanisms of cellular injury induced by these sensitizers, we have studied by fluorescence microscopy the effects of 13 halogenated (nitro)benzene derivatives on microtubule organization in mouse 3T3 fibroblasts and human AG1522 skin fibroblasts. Untreated cells have numerous microtubules distributed in a network fashion throughout the cytoplasm and extending to the cell periphery. Exposing cells for 3 hr to micromolar doses of halogenated nitrobenzene derivatives, which induce allergic contact dermatitis in guinea-pigs and man, resulted in a dose-dependent disassembly of microtubules. In contrast, incubation with 10-70 times higher doses of seven halogenated (nitro)benzene derivatives that do not sensitize guinea-pigs or man, had no discernible effect on microtubule organization of both cell types under identical assay conditions. Thus, for the 13 halogenated (nitro)benzene derivatives tested in this study, a 100% positive correlation exists between the sensitizing capacity as determined by in vivo tests on guinea-pigs and man (Landsteiner and Jacobs, 1936) and the ability to cause microtubule disassembly in cultured cells. These results may provide the basis for developing an in vitro screening assay for identifying other potential halogenated nitrobenzene sensitizers. In addition, these studies represent a new approach to investigating the mechanisms of contact sensitivity induced by simple chemicals. |
20702174 | Neutral red release from pre-loaded cells as an in vitro approach to testing for eye irritancy potential. | Many potential alternatives to the Draize eye irritation test are general cytotoxicity tests, which require modification if they are to e ponents of an acceptable battery of tests. Preliminary studies were made on the exposure of fibroblast-like cells, pre-loaded with a vital dye (neutral red), to high concentrations of test materials for brief periods. Measurement of loss of neutral red from damaged cells provides a readily quantifiable endpoint. When the results obtained with eight detergent formulations pared with maximum Draize eye scores, a better correlation was obtained with conjunctival and iris effects than with corneal scores or average Draize scores. |
20702175 | Eye irritancy screening for classification of chemicals. | A screening method was applied to determine the eye irritation potential of industrial chemicals. Bovine eyes (BE) were used to predict corneal damage and chicken egg chorioallantoic membranes (CAM) to estimate the irritancy potential of chemical substances towards the conjunctivae. Exposure of the BE to a test substance is followed by grading of the corneal opacity and epithelial injury. The CAM is inspected for signs of capillary injection, haemorrhages and coagulation. The tests are collectively called the BECAM assay. So far, almost 150 substances have been evaluated in this test system. A good correlation was observed between the BECAM assay and in vivo data; less than 5% of chemicals showed a clear disagreement. Also the assay is promising for labelling requirements according to the EEC criteria. |
20702176 | Induction of heat shock proteins as a measure of chemical cytotoxicity. | Exposure of human keratinocytes to non-lethal heat shock treatment (43 degrees C for 90 min) followed by a recovery period of 2 hr at 37 degrees C resulted in the rapid accumulation of two proteins with polypeptide molecular weights of 72 and 90 kDa. Exposure of human keratinocytes to sodium arsenite (10-200 mug/ml) for 90 min at 37 degrees C resulted in the synthesis of proteins with polypeptide molecular weights of 110, 90, 72, 46 and 28 kDa. The 72 kDa heat- or sodium arsenite-induced protein was identified by immunoprecipitation as the 72 kDa heat shock protein. In contrast, the human epithelial tumour cell line (A431) synthesized only the 72 and 28 kDa heat shock proteins in response to arsenite treatment with all other stress proteins being expressed constitutively. General protein synthesis was inhibited in cells exposed to elevated temperature or sodium arsenite. Using immunofluorescence a rapid and reversible accumulation of the 72 kDa heat shock protein was demonstrated within the nucleolus of heat stressed human fibroblasts and keratinocytes. |
20702177 | Evaluation of normal human epidermal keratinocyte cultures as a test system for the assessment of the dermal irritancy of pesticides. | Dermal irritation studies are an essential part of the regulatory assessment of potential occupational and environmental health hazards of pesticides. The in vivo methods now widely used have not been improved substantially since the late 1940s, despite several ings. These include: the subjectivity of the assessment of irritation; interlaboratory variability; and differences between animal and human skin permeability, thickness and sensitivity to irritants. Relatively little research has thus far been undertaken in Canada to determine the suitability of cell culture assays as alternatives for the assessment of dermal irritancy. Recently, cultured normal human epidermal keratinocytes (NHEK) have been proposed as a good model system for the assessment of dermal irritancy and toxicity. Epidermal keratinocytes are useful for this because they participate in dermal inflammation, and therefore constitute ponents of the reactions of human skin to toxins. The usefulness of this approach for dermal irritancy/toxicity evaluation of pesticide ingredients and formulations will be assessed by measuring the release of [(3)H]arachidonic acid from prelabelled NHEK cultures in response to pesticides added to the tissue culture medium. Dose-response relationships for pesticides of well characterized in vivo dermal irritancy will be determined and a rank-order correlation of the in vitro/in vivo data will also be performed. |
20702178 | Comparison of cultured keratinocytes and fibroblasts as models for irritancy testing in vitro. | Surfactants are widely used and often cause irritation to human skin. Three groups of surfactants, the trimethylammonium bromides (cationic), sodium dodecyl sulphate (anionic) and the polyoxyethylene sorbitans (Tweens, nonionic) were tested on a rat keratinocyte line (RTE) and an established fibroblast line (3T3-L1) to assess their potential as models for skin irritancy testing. Acid phosphatase (AP) release seems to parallel the development of signs of irritation in vivo and therefore AP activity was assayed after 4 hours' treatment to give an early indication of toxicity. AP activity in RTE keratinocytes rose to a peak and fell sharply, whereas in 3T3-L1 it did not change with treatment. Therefore AP may be a specific indicator of toxicity in keratinocytes. Neutral red (NR) uptake and kenacid blue (KB) staining were both assayed after 3 days' treatment as an indicator of cell proliferation. RTE and 3T3-L1 were equally sensitive in terms of NR and KB-ID(50) values for the anionic and pounds; however, 3T3 was more sensitive to the pounds. |
20702179 | Differential effects of mineral dusts on the in vitro activation of alveolar macrophage eicosanoid and cytokine release. | We investigated the in vitro effects of the pneumotoxic agents, silica and asbestos, and the relatively innocuous materials, aluminium oxide (Al(2)O(3)) and titanium dioxide (TiO(2)), on alveolar macrophages (AM) using endpoints reflecting the cytotoxic and AM activating properties of the dusts. Rat AM were exposed in vitro (24 hr) to 10-1000 mug/ml of the dusts. AM conditioned media was analysed for lactate dehydrogenase (cytotoxicity), beta-glucuronidase (lysosomal enzyme), leukotriene B4 (LTB4), prostaglandin E(2) (PGE(2)), tumour necrosis factor alpha (TNF) and interleukin-1 (IL-1). AM LTB4 and TNF release were increased by silica and asbestos but not by Al(2)O(3) or TiO(2). IL-1 release was not affected, and changes in PGE(2) release were minimal, after dust exposure. Cytotoxic activity was not consistently associated with LTB4, TNF or beta-glucuronidase release. The ability of silica and asbestos, but not Al(2)O(3) and TiO(2), to activate AM to release the pro-inflammatory mediators, LTB4 and TNF, may be responsible, at least in part, for the greater inflammation and pneumotoxicity associated with silica and asbestos exposure. These findings suggest that assessment of AM mediator secretion in vitro can provide information to understand better the potential of a material to cause respiratory toxicity. |
20702180 | An optimized lymphocyte blastogenesis assay for detecting the response of contact sensitized or photosensitized lymphocytes to hapten or photohapten modified antigen presenting cells. | In vitro assays for predictive identification of contact sensitizers of photosensitizers are more quantifiable than current skin test methods and have the potential to reduce animal use. Mice were sensitized to various strong contact allergens, then an optimized lymphocyte blastogenesis assay was used for detecting the proliferation of lymph node lymphocytes from those mice in response to soluble antigen or hapten-modified antigen presenting cells in culture. In vivo sensitization to oxazolone (OXAZ), a prototype sensitizer, resulted in poor in vitro lymphocyte blastogenesis to a solubilized OXAZ preparation. To increase the assay sensitivity, dendritic cells from the draining lymph nodes of OXAZ-painted mice or OXAZ-modified Langerhans cell-enriched cultured epidermal cells (EC) were used as antigen presenting cells. This increased the OXAZ-specific response greater than 10-fold and 50-fold, respectively. This approach has since been used to demonstrate significant, albeit smaller, responses to weaker contact allergens such as nickel sulphate. EC were also used as antigen-presenting cells for in vitro lymphocyte blastogenesis assays designed to detect photoallergens. Lymphocytes from mice photosensitized to tetrachlorosalicylanilide (TCSA) showed a significantly increased response to EC previously photohapten-modified by treatment with TCSA plus ultraviolet A (UVA), pared with lymphocytes cultured with EC treated with TCSA but no UVA. The lymphocyte blastogenesis assay may have potential as a predictive screen to identify contact and photocontact allergens. |
20702181 | Skin organ culture for the study of skin irritancy. | Skin explants maintained in culture may represent a reliable model for in vitro tests of the irritancy of chemicals. During the process of skin irritation intracellular enzymes migrate into the culture medium. The amount of released enzyme activity corresponds to the degree of skin damage. Skin of hairless mice (hr/hr) has been found to be especially useful for this model. Histomorphology demonstrated that the explants were almost identical to the in vivo situation. Skin explants of hairless mice of 50 mm(2) were used for the tests. The dermal side of the skin is in contact with the medium whereas the substance is applied to the epidermal side and incubated for 24 hr. As parameters for the membrane-damaging effect, the enzymes lactate dehydrogenase and glutamic-oxaloacetate transaminase were measured. The determination of the glucose utilization during the incubation period gave additional information about the viability of the cultured skin. Various chemicals were used. Histological plemented the biochemical results and differentiated epidermal lesions, but was limited by the absence of inflammatory reactions of the dermal part of the skin. Overall, in vitro skin culture tests seem to be useful as screening tests prior to in vivo studies and for the development of new formulations. |
20702182 | Attempts to identify the causative allergen in cases of allergic contact dermatitis using an in vitro lymphocyte transformation test. | The use of an in vitro lymphocyte transformation test for the identification of the causative allergen in cases of human allergic contact dermatitis is described. Peripheral blood mononuclear cells (PBMC) were prepared from the venous blood of seven nickel-sensitized (patch test-positive) patients and ten non-sensitized control donors, and induced proliferation was measured by [(3)H]thymidine incorporation following culture for 7 days in the presence of various concentrations of nickel sulphate. Nickel sulphate is non-specifically mitogenic for human lymphocytes and although PBMC from all donors exhibited a significant increase in proliferation with nickel sulphate concentrations of 20 mug/ml, those isolated from Ni-sensitive patients responded to lower concentrations of the allergen. Employing a stimulation index of 3 following culture with 5 mug NiSO(4)/ml as an arbitrary indicator of specific proliferative responses to nickel, it was possible, using the lymphocyte transformation test, to separate Ni-sensitive from non-sensitized donors. In a limited series of experiments it was observed that lymphocytes prepared from each of two patients sensitized to neomycin sulphate mounted significant proliferative responses to this allergen in vitro; neomycin sulphate failed to induce similar changes in PBMC from non-sensitized donors. Finally, PBMC from one of two 2,4-dinitrochlorobenzene-sensitized donors were found to respond to autologous cells bearing the relevant dinitrophenol hapten. |
20702183 | Photochemical binding of photoallergens to human serum albumin: A simple in vitro method for screening potential photoallergens. | An in vitro screening procedure is described whereby potential photoallergens are irradiated with ultraviolet (UV) light in the presence of monomeric human serum albumin. UV spectroscopy before and after irradiation and after passage of the reaction mixture through Sephadex G-10, is used to determine whether or not the pound has bound to the albumin. Using this method and others employing pounds, nine photoallergens have been shown to bind to protein under the influence of UV light. In contrast, a cinnamate sunscreen, which absorbs UV light but is not a photoallergen, does not bind under these conditions. The method is proposed as an in vitro screening procedure for potential photoallergens. |
20702184 | Evaluation of an agarose overlay assay to determine the eye irritation potential of detergent-based products. | The in vitro agarose overlay method (using L929 mouse fibroblast cells) has been evaluated and modified as an in vitro alternative to the Draize test for the ranking of ocular irritancy of detergent-based products. Modifications examined include the use of shorter incubation times, dilution of test materials and use of thiazolyl blue reduction as an alternative endpoint. Several cationic and anionic detergent-based products have been tested and ranked in order of their toxicity in this assay. Good correlations were found between these ranks and the appropriate in vivo ranks for irritancy. Thus an in vitro agarose overlay assay may be useful as a screening assay in irritancy prediction. The advantages of the agarose-overlay method over other similar in vitro tests include speed, lower cost, simple equipment and the ability to test formulations as well as pure substances. |
20702185 | Applications of the EYTEX method. | The EYTEX method is an in vitro test to predict the ocular irritation of chemicals and formulations based on alterations in a protein matrix. Results of a multi-laboratory study of 130 samples have previously been published. Four independent studies were conducted to evaluate further the accuracy, reproducibility and applicability of the EYTEX method. This method was performed according to the previously published procedures. Dose-response curves were produced at concentrations of samples similar to those used in the Draize test. Spectrophotometric data were used to establish irritancy classifications, and an EYTEX/Draize equivalent was calculated. These studies included a wide range of chemicals and formulations with varying degrees of irritation and mechanisms of ocular toxicity. Nine hundred and twenty-one cosmetic, household, petroleum and agricultural products were analysed by the EYTEX method. Seven hundred and thirty-six EYTEX results were within one classification of the Draize result. The EYTEX results exhibited an overall substantial equivalence of 91% to the Draize results with a range of 89 to 93% depending on the particular product group. The inter-assay coefficient of variation was 8.1% and the intra-assay coefficient of variation was 10.7%. These studies indicate that the EYTEX method exhibits a high correlation with the Draize test for the four diverse product groups studied and validates the predictive potential of the EYTEX method as an in vitro screen for ocular irritation. |
20702186 | Screening test for phototoxins using solutions of simple biochemicals. | The feasibility of using solution photochemistry for in vitro phototoxicology testing was examined. Solutions of substrate and test chemical, in binary aqueous/organic solvent to ensure solubility, were exposed to ultraviolet/visible radiation. Quantum efficiencies for photosensitized oxidation of substrate were calculated. The phototoxins rose bengal, anthracene and acridine were good photooxidizers of histidine, in contrast to the other chemicals studied. Solution photochemistry using histidine may be a useful screening test for molecules that photooxidize by the singlet oxygen pathway. Other indications of in vivo activity may be gained from the molecular weight, ionic nature, lipophilicity and photodegradation (photoinactivation). The photoallergens tetrachlorosalicylanilide, tribromosalicylanilide and musk ambrette, and the photomutagen 8-methoxypsoralen, were detected qualitatively by these studies, but in vivo potency could not be predicted. |
20702187 | The chorioallantoic membrane in the prediction of eye irritation potential. | It has been suggested that the chick chorioallantoic membrane (CAM) could be used as an alternative to the rabbit eye test for irritation. Various authors have examined different methods for investigating the irritant potential of materials to the CAM. We used the CAM method as described by Luepke, to assess the effect of 34 materials of known in vivo eye irritancy, including fully formulated products ranging from skin creams to industrial detergents. Test materials were applied undiluted to the CAM on day 10 of incubation with rinsing after 20 seconds of contact. Effects to the CAM including vasodilation and haemorrhage were assessed for up to 5 min after application. parison was made between the rank order of effect on the CAM and the known ranking for in vivo eye irritancy. There was in general only a limited correlation between in vitro and in vivo results but a reasonable distinction between irritant materials and those of only very slight irritancy was observed. Among products of a similar type there was a better correlation between results from the CAM and in vivo eye results than when all materials were considered together. These data indicate that this method would only have a limited application as a screening procedure for eye irritation potential or as part of a battery of in vitro tests but so far has not been found to be superior to cell culture tests in our laboratory. |
20702188 | Immaturity of the inflammatory response of the chick chorioallantoic membrane. | Previous tests on the chick embryo chorioallantoic membrane (CAM) had shown the 24- to 48-hr response to irritants to be necrosis, with a primitive granulation proliferation at the periphery. There were few heterophils (avian neutrophils). The present investigation confirmed the immaturity of blood leucocyte development. Few heterophils were seen at 14 days but were found in significant numbers on the 19th and 20th days just before hatching. The numbers of blood heterophils in 14-day-old embryos were almost doubled and the number of immature granulocytes slightly reduced when the CAM was treated with zymosan or N-formylmethionyl peptide, which are chemo-attractants for mammalian neutrophils. Treatment with chemicals that do not have selective activity for neutrophils, for example a surfactant or alcohol, did not stimulate this change. It was not feasible to pretreat 14-day-old embryos to increase the number of heterophils or to use 19- to 21-day-old embryos as a model to detect irritants inducing an acute inflammatory response, or to make the CAM more relevant as a substitute for the in vivo eye irritation test. No difference was found in the phagocytic ability of macrophages of 14-day-old embryos and 16-wk-old adults. Macrophages from both sources contained lactate dehydrogenase and beta-glucuronidase revealed by histochemical and fluorometric examination, and non-specific esterase by histochemistry, though staining was stronger in cells from the adults. |
20702189 | The chick embryo yolk-sac blood vessel system as an experimental model for irritation and inflammation. | Experimental studies on the incubated egg, for example the embryo and the chorioallantoic membrane (CAM), are carried out frequently. The yolk-sac blood vessel system, the first system supplying the embryo, has not yet been used in toxicological studies. This paper describes experiments with ethanol and sodium hydroxide to test the application of this blood vessel system in the assessment of toxic effects. The experiments were carried out at day 4 of incubation. Different concentrations of the test substances were applied directly on yolk-sac. The acute reaction, mainly haemorrhages, was recorded semi-quantitatively up to 5 min post-application (p.a.). The results revealed a clear concentration dependency and marked differences between the two substances. During the late reaction up to 3 days p.a., repair processes occurred showing signs of a granulation tissue, including angiogenesis and the development of collagenous fibres. These experiments show the functional ability of the yolk-sac blood system for toxicological tests. Additionally, this system has some pared with the CAM, for example the immaturity of the embryo nervous system and the possibility to evaluate findings on the embryo itself. |
20702190 | An in vitro method for estimating ocular irritation. | Three variations of an in vitro assay system called EYTEX were evaluated paring maximum in vivo Draize scores for 70 experimental consumer product formulations. The EYTEX in vitro assay (EIA) was chosen for in-house evaluation because it is an economical, objective system that is easy to learn. In the present study, double-blind samples were tested at National Testing Corporation (Lab 1) and an in-house laboratory (Lab 2). All products were tested in the standard, membrane, and rapid membrane (RMA) EIA. Most samples qualified for the membrane and RMA EIA, whereas approximately half qualified for the standard EIA. Results from both Labs 1 and 2 were highly correlated. The RMA assay gave the best overall performance of the three assays. The RMA EIA demonstrated the best correlation with in vivo data and qualified the highest percentage of formulations. RMA showed a 7.5% false positive identification of irritants. False negatives or irritants not identified were 6.1%. The predictive value for identifying irritants was 89%. Specificity or the ratio of non-irritants giving negative results to the total was 84%. The ratio of positive irritants to the total or sensitivity was 93%. Based on these results, EIA has demonstrated value as a screening tool for a broad variety of consumer products. Advantages of EIA include the opportunity to test undiluted products, reduce animal use, and lower costs. |
20702191 | In vitro systems for nephrotoxicity studies. | A variety of in vitro systems, including the perfused kidney, microperfused nephron(s), cultured renal slices, renal tubule suspensions/cultures, isolated ponents (glomeruli and tubule segments) and renal cell suspensions/cultures can be utilized to evaluate chemicals for their potential nephrotoxicity and mechanisms of action. These systems have varying degrees of relevance to the intact kidney or portions thereof; which model to use depends on the specific question asked. If maintenance of physiological flow conditions is important then only the perfused kidney (or tubule) can be used. If the toxicity can be studied in the absence of normal fluid flow, then the other systems may be utilized. Kidney subfractions also have the advantage of multiple biological units that can be examined simultaneously and separately. Closely defined renal slice systems allow for enrichment and evaluation of multiple target cells within the slice. Suspensions or cultures of nephron subfractions are now frequently used to assess the potency and/or mechanism of renal toxins. Disadvantages include the utilization of collagenase, which scars cell and basement membranes and affects membrane transport proteins, which leads to inadequate maintenance of transport mechanisms. Isolation of tubule segments (P(1), P(2), P(3)), allows site-specific toxicity studies. Lastly, there has been a dramatic increase in renal cell culture to study toxic events in the kidney. Because of the multiple cell types, minimal availability of specific cell markers, and de-differentiation phenomena, this technique should be used with caution. These in vitro systems have allowed us to examine the role of transport and biotransformation, and pare potency and mechanism of nephrotoxic chemicals. Although in vitro systems cannot totally duplicate or replace in vivo toxicity studies, they are a very useful and plement. |
20702192 | Neurotoxicity in vitro: Model systems and practical applications. Comparative studies with the cholinergic neurotoxin in primary brain cultures and in rabbit retina in vivo. | In vitro neural systems can be predictive for central nervous system neurotoxicity, except where xenobiotics primarily affect the blood-brain barrier directly. The wide range of systems now used in neurobiological studies is available for mechanistic neurotoxicological investigations although the choice of system is generally arbitrary and for this reason a more rational 'stepwise' approach may now be justified. There are many culture systems available, including neural cell lines, organotypic explant or reaggregation cultures, and primary monolayer cultures of individual, or mixtures of, neural cell types: neurones, astrocytes and oligodendrocytes. Of these models much success has recently been achieved using the organotypic culture. Using rat whole-brain reaggregate cultures, good in vitro/in vivo correlations for the cholinergic neurotoxicant, ethylcholine mustard aziridinium (ECMA) have been demonstrated paring its actions with those in the rabbit retina in vivo. Low concentrations of ECMA (12.5 mum) cause a biphasic loss of choline acetyltransferase activity (ChAT) in foetal rat brain reaggregate cultures. Initial direct inhibition is followed by an apparently selective loss of cholinergic neurones. More widespread cytotoxicity occurs at higher ECMA concentrations. Similarly, intravitreal injection of ECMA at a final concentration of 12.5 mum into the rabbit eye in vivo for 96 hr produced a 36% loss of retinal ChAT activity with no change in the electroretinogram (ERG), whereas 600 mum-ECMA produced a 55% loss of ChAT with a significant loss of the ERG b-wave function, which is suggestive of a more generalized cellular toxicity. Exogenous nerve growth factor applied to the brain reaggregates 48 hr post-ECMA lesioning reverses the neurotoxin-induced loss of ChAT activity. By supplementing neurotoxicological information gained in the in vitro brain reaggregate culture system with tests using primary monolayer cultures of neurones or astrocytes a stepwise 'screening' system is proposed for potential neurotoxicants in vitro. In its simplest form this is: (1) screening initially by using tumour-derived neural cell line and/or primary mixed neural culture such as the 'Micromass' system; (2) testing of pounds in whole-brain reaggregates and (3) supplementing this information if necessary with effects on primary monolayer cultures of individual neural cell types. |
20702193 | Species-specific toxic effects of alpha-chlorohydrin and 6-chloro-6-deoxyglucose on astrocytes. | In 1969 alpha-chlorohydrin was reported to have a reversible antifertility effect in male rats. It has since been reported to have a reversible antifertility effect in many other species but not in mice and rabbits. In chronic high-dosage regimens it was reported to be neurotoxic to mice. At high doses the 6-chloro-6-deoxysugars were found to have a plete antifertility effect. 6-Chloro-6-deoxyglucose had an antifertility effect in rats and marmosets but not in mice, hamsters, guinea-pigs or rabbits. However, repeated high doses were found to be neurotoxic in the mouse and marmoset, the most prominent pathological feature being vacuolation of astrocytes. In the studies reported here the ability of alpha-chlorohydrin and 6-chloro-6-deoxyglucose to induce vacuolation of mouse astrocytes has been confirmed in vitro. The failure of either agent to induce vacuolation of rat astrocytes suggests that both the antifertility effect of these agents and their neurotoxic effects are species-specific. The lack of correlation between neurotoxic and antifertility effects of such agents suggests that in vitro techniques may aid the development of a safe, reversible male antifertility agent and also aid in the design of further agents for rodent control. |
20702194 | An in vitro test for immunomodulators? | Certain sublines of EL-4 thymoma cells can be stimulated to produce interleukin-2 (IL-2) by the mitogens phorbol-12-myristate-13-acetate and concanavalin A. EL-4 cells were cultured under conditions found to be most effective for stimulating the release of IL-2 into the culture medium. The resultant conditioned medium (CM) was used to maintain the IL-2-dependent T-cell line, CTLL-2. The response of the CTLL-2 cells to the CM was assessed by measuring [(3)H]thymidine incorporation. Since IL-2 is one of the major mediators in immune responses, it was felt that inhibition of the response of CTLL-2 cells might provide a means of screening for chemicals with the potential to affect immune responsiveness in vivo. Fourteen chemicals, including known pounds, were tested in the CTLL-2 responding system and ID(50) values determined. The basic cytotoxicities of the same chemicals were established by measuring growth inhibition in 3T3-L1 fibroblast-like cells. Significant differences between these two indices of toxicity for some chemicals suggested that the CTLL-2 responding system might be demonstrating effects other than basic cytotoxicity. As a result, this system is undergoing further examination to assess whether it could be developed as an assay for evaluating the potential immunomodulatory effects of chemicals. |
20702195 | Depression of contractility in cultured cardiac myocytes from neonatal rat by carbon tetrachloride and 1,1,1-trichloroethane. | The cardiac depressant effects of carbon tetrachloride (CCl(4)) and 1,1,1-trichloroethane (CH(3)CCl(3)) were evaluated in cultured heart cells from neonatal rats. Heart cells were grown on glass coverslips and formed a confluent monolayer that beat spontaneously, rhythmically and in synchrony. Contractility was assessed by video-motion analysis. Stock solutions of CCl(4) or CH(3)CCl(3) were prepared in dimethylsulphoxide (DMSO) and aliquoted (final DMSO concentration 0.2%) into medium (M199 supplemented with 5% serum) immediately prior to perfusion across myocytes in an environmentally controlled chamber. CCl(4) and CH(3)CCl(3) had a negative chronotropic effect on myocytes by prolonging the relaxation phase of beating. Duration of the contraction phase of beating, and peak velocity of cell wall movement were not affected by these halocarbons. Beating was stopped by 2.5 mm-CCl(4) or 5 mm-CH(3)CCl(3), and washout of pounds resulted in a resumption of beating activity. Increasing (3.6 mm) or decreasing (0.6 mm) the calcium concentration of the medium (normal = 1.8 mm) significantly affected the duration of contraction and relaxation phases of beating, but did not alter the concentration-dependent action of CCl(4). A positive chronotropic effect of isoproterenol was evident from 10(-9) to 10(-6)m, but contractility was depressed by isoproterenol concentrations greater than 10(-8)m in the presence of 750 mum-CCl(4). This study demonstrates the usefulness of cultured heart cells for assessing the cardiac depressant and sensitizing actions of halogenated hydrocarbons. |
20702196 | A comparison of cultured rat FRTL-5 and porcine thyroid cells for predicting the thyroid toxicity of xenobiotics. | monly used cell culture systems are currently available for testing the effects of xenobiotics on thyroid cell function and growth. These are the rat FRTL-5 thyroid cell line and the primary, cultured porcine thyrocyte. Both systems have inherent advantages and disadvantages. The FRTL-5 line has proved successful for testing growth stimulators, directly acting cytotoxic agents, pounds directly affecting membrane transport processes (e.g. perchlorate anion: iodide transporter, ouabain: Na(+)K(+)-ATPase, amiloride: Na(+)-H(+) antiporter). These cells can be produced in large quantities in monolayers and permit the necessary interlaboratory standardization procedures. However, these cells have a limited ability to iodinate thyroglobulin and synthesize thyroid hormones and thus, for testing for direct inhibitors of the peroxidases (e.g. sulphonylurea-like antithyroid agents), cultured porcine cells can be used. These cells can be 'manipulated' in culture to form 'pseudo-follicular' structures under the correct conditions. |
20702197 | In vitro toxicity of antibiotics to LLC-RK(1) rabbit kidney cells. | The renal toxicity of cephalosporin and aminoglycoside antibiotics has been studied in an in vitro system using the rabbit kidney cell line LLC-RK(1). The effect of a rabbit kidney S-9 metabolizing system on the toxicity the antibiotics was also studied. Cultures of the LLC-RK(1) cells were established in microtitre plates and exposed to pounds in the concentration range 0 to 2000 mug/ml, either in the presence or absence of an S-9 metabolizing system. pounds were in contact with the cultures for a period of 48 hr before viability of the cells was determined. Assay of cell viability was based on the mitochondrial conversion of 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide to a coloured formazan product (MTT Assay). Duplicate cultures were also tested with a neutral red staining assay. Cephalosporins tested were cephaloridine, cephalothin, cephapirin, cefotaxime, ceftazidime, cephaloglycin, cefoperazone and cefazolin. The aminoglycoside antibiotics tested were gentamicin, neomycin and kanamycin. A good correlation was seen between in vitro toxicity and in vivo effects for pounds. Further techniques were investigated to assess sub-cytolethal impairment of renal function in the LLC-RK(1) cells. Both in vivo clearance studies and in vitro kidney slice methods have used transport of the organic ions tetraethyl ammonium and p-aminohippurate as indices of renal function. Therefore the ability of LLC-RK(1) cells to transport these ions was investigated. Where ion transport was demonstrated the effect of nephrotoxic antibiotics on this kidney function was studied. |
20702198 | Comparison of in vitro and in vivo haemotoxic effects of aminoglutethimide and glutethimide. | The aromatase inhibitor, aminoglutethimide, causes a blood dyscrasia in about 1% of women taking the drug for the treatment of advanced breast cancer. The pound, glutethimide, is known not to possess this toxic action. The effect of both drugs on bone (femur) marrow cells of mice and rats was examined by means of standard methods for the colony forming assay for granulocyte-macrophage (GM-CFC) and erythroid cells (CFU-E). There were no significant effects on the cell colonies up to drug concentrations of at least 10 mug/ml. However, above this level concentration-dependent effects were observed. For mouse GM-CFC, 50 and 30% of the cell colonies died when incubated with glutethimide (33 mug/ml) and aminoglutethimide (100 mug/ml), respectively. Similar results for the two drugs were obtained with the mouse CFU-E and with both cell types from the rat. The results indicate that the inhibitory effects were most likely to be non-specific. For the in vivo study, glutethimide and aminoglutethimide were given orally (50 mg/kg, daily). After 3 wk of this regimen in mice, there was a significant fall (about 50%) in both white blood cell and platelet counts. This is similar to the blood picture changes induced by aminoglutethimide in women. No haematological effects were observed in rats receiving this drug and glutethimide was without effect in either species. Thus, the toxic effects of aminoglutethimide appear to be more selective in vivo than in vitro. It is proposed that N-hydroxylation of aminoglutethimide, which proceeds in mouse and man but not rat, is an important step in the toxic mechanism. Overall the results indicate that the in vitro assessment used in this study was not a suitable predictor of the in vivo haemotoxic effects of aminoglutethimide. |
20702199 | In vitro cytotoxicity of tetracyclines and aminoglycosides in LLC-PK(1), MDCK and Chang continuous cell lines. | The objective of this study was to determine if cell lines isolated from particular organs retain their sensitivity to xenobiotic toxicity in vitro. The toxicity of chlortetracycline, demeclocycline and tetracycline was examined in the proximal kidney (LLC-PK(1)), distal kidney (MDCK) and human liver (Chang) cell lines. The toxicity of amikacin, gentamicin and neomycin was studied in the LLC-PK(1) and MDCK lines. Cytotoxicity was assessed by cytoplasmic LDH leakage. Kidney cells treated with tetracyclines displayed minimal toxic response. The Chang cell line showed greater sensitivity to pounds and ranked them as follows: demeclocycline > chlortetracycline > tetracycline. The kidney lines produced the rankings as follows: LLC-PK(1): amikacin > neomycin > gentamicin; MDCK: neomycin > amikacin > gentamicin. The results from the tetracyclines are consistent with the expectation that pounds would not be nephrotoxic in vitro since in vivo investigations suggest hormonal mediation is required, and would be hepatotoxic in vitro because of direct action of these xenobiotics on liver cells in vivo. Similarly, the aminoglycosides were more toxic to the proximal kidney cells than to the distal cells as seen in vivo. These results suggest that continuous cell lines may provide important information in the assessment of xenobiotic cytotoxicity. |
20702200 | The neurotoxic effect of carbon disulphide, N-hexane and its metabolites studied with erythrocyte and synaptosome membranes in vitro. | The aim of the present study was to find a method for in vitro studies of the neurotoxic mechanism of industrial solvents. The effects of carbon disulphide and n-hexane and its metabolites were studied. Cell membrane changes were studied by measuring changes in the activity of the integral cell membrane enzymes acetylcholinesterase (AChE) and adenosinetriphosphatase (ATPase). The erythrocyte membranes were isolated from human peripheral blood samples and the synaptosome membranes from rat brain by using the non-toxic iso-osmotic Percoll gradient system. The cell membrane samples were incubated at +37 degrees C in an incubation mixture with known solvent concentrations. In erythrocyte membranes, n-hexane decreased the activity of AChE more than did carbon disulphide. In synaptosome membranes, 2-hexanone and 2,5-hexanedione inhibited AChE significantly more than did n-hexane. On ATPase, n-hexane, as well as the metabolites of n-hexane, had a slightly activating effect in erythrocyte membranes, and no or a slightly inactivating effect in synaptosome membranes. Carbon disulphide had a clear inactivating effect on ATPase in synaptosome membranes. The action of the neurotoxic organic solvents may be mediated by way of the integral membrane proteins, especially AChE. Thus, in neural synaptosome membranes, the metabolites of n-hexane had a greater effect on AChE activity than did n-hexane. The in vitro membrane model can be applied in studies of the neurotoxicity of organic solvents, and in predicting their neurotoxic potency. |
20702203 | Respiratory tract epithelium in primary culture: Effects of ciliotoxic compounds. | Acrolein ciliotoxicity was studied on primary cultures from rabbit tracheal epithelium. The inhibition of ciliary beat was chosen as a criterion for ciliotoxicity. The measurement of ciliary beat was plished using an original image analysis process. |
20702204 | Studies of nephrotoxic agents in an improved renal proximal tubule system. | Renal proximal tubule fragments (RPT) were prepared from young-adult, male F-344 rats by deferoxamine/collagenase perfusion and evaluated as a potential model for mechanistic studies and screening, using known nephrotoxins. Chloroform and S-(1,2- dichlorovinyl )- l - cysteine (DCVC) produced depressed O(2) consumption rates (basal and/or nystatin-stimulated) and lactate dehydrogenase (LDH) release during 8-hr incubations at 0.5 mg RPT protein/ml. Cytochrome P-450 inhibitors piperonyl butoxide and metyrapone were either without effect or potentiated chloroform-induced toxicity. DCVC was more cytotoxic to RPT than to rat hepatocytes. The cytotoxic potency for cephalothin relative to cefazolin decreased as RPT content in the medium was increased to 3.0 mg protein/ml, giving a rank order more in accord with results reported in vivo. Cephalosporins markedly depressed brush border alkaline phosphatase (ALP) activity, without affecting gamma-glutamyltranspeptidase activity; the effect on ALP was less sensitive to the RPT level. Acetaminophen (25 mm) and p-aminophenol (1.0 mm) induced LDH release without ALP depression and inhibited mitochondrial respiration. These results in general corresponded well with in vivo responses and indicate that this RPT system may be valuable for studies of chemical-induced nephrotoxicity. |
20702205 | Nephrotoxicity in vitro: Role of ion deregulation in signal transduction following injury-Studies utilizing digital imaging fluorescence microscopy. | Over the years, many approaches have been utilized for studying in vitro toxicity in the kidney. These have included the use of isolated perfused kidneys, renal slices, isolated nephron explants and cultured tubular epithelium. Currently, in vitro systems of either primary cultures or cell lines make it possible to use newly developed fluorescent probes and digital imaging fluorescence microscopy coupled with image analysis to quantify various ions (e.g. [Ca(2+)](i), [Mg(2+)](i), [Na(+)](i) and [H(+)](i)) in individual live cells. Methods have been developed for the primary culture of rat, rabbit and human proximal tubular epithelium and these cultures are being utilized for the study of acute cell injury and for parison of animal cell data with that of human cells. In the current studies, the fluorescent probe, Fura 2, was used to observe changes in [Ca(2+)](i) as they relate to cell injury. The results show that changes in [Ca(2+)](i) begin very early after treatments with a variety of agents that produce lethal and sublethal toxic injuries. These injuries can increase [Ca(2+)](i) from its normal 100 nm level to a 2 mum level. The rise in [Ca(2+)](i) precedes early bleb formation and cell death. Elevated [Ca(2+)](i) may also activate enzymes such as lipases and proteases that lead to cell death. |
20702206 | Long-term culture of functional hepatocytes. | Recent studies have clearly demonstrated that the hepatocyte requires plex and well defined environment to survive and maintain differentiated functions in vitro. Soluble factors as well as cell-matrix and cell-cell interactions have been found to affect markedly hepatocyte functions. Thus co-culturing hepatocytes with another rat liver cell type results in a prolonged expression of liver functions including phase I and phase II drug-metabolizing enzymes. Addition of corticosteroids to the co-culture medium is a prerequisite, and accumulation of insoluble ponents is observed within a few days primarily between the two cell types. Hepatocyte cultures have been widely used for pharmacology and toxicology studies during recent years, but most studies deal with short-term investigations. Although specific functions are pletely stabilized the use of long-term hepatocyte cultures represents a promising tool to investigate enzyme induction and inhibition, and drug chronic toxicity. |
20702207 | The significance of in vitro studies on peroxisome proliferation. | Peroxisome proliferation in rodents is associated with hepatocarcinogenicity. This association has led to increased interest in the phenomenon and to the search for in vitro tests to detect peroxisome proliferators and to study the mechanisms by which proliferation occurs. Primary cultures of adult rat hepatocytes provide such a system: peroxisomal enzymes, cytochrome P-452 and replicative DNA synthesis may all be induced and the response to treatment with many peroxisome proliferators is observed in a manner similar to that in the liver in vivo. Cultured hepatocytes, therefore, provide an optimal system: (i) to screen for potential peroxisome proliferators; (ii) to study structure-activity relationships; (iii) to investigate species differences in the effects of peroxisome proliferators on hepatocytes; and (iv) to study the molecular mechanisms underlying peroxisome proliferation and its relationship to tumour formation. |
20702208 | Comparison of cultured human hepatocytes isolated from surgical biopsies or cold-stored organ donor livers. | The purpose of this work was pare yield, attachment rate and specific metabolic functions (stimulation of ketone body production by glucagon) of human hepatocytes isolated from surgical biopsies and from organ donor livers cold stored with a modified University of Wisconsin (MUW) solution. A significantly greater number of hepatocytes was isolated from MUW-stored livers than from surgical biopsies. On average, 60% of hepatocytes isolated from surgical biopsies attached to uncoated flask whereas the attachment rate of hepatocytes isolated from MUW-stored livers was inconsistent and always below 40%. Glucagon significantly enhanced the rate of ketone body production of hepatocytes isolated from surgical biopsies; in contrast, glucagon had marginal effects on the rate of ketone body production in hepatocytes isolated from MUW-stored livers. These results demonstrate that human hepatocytes isolated from surgical biopsies maintain liver-specific and non-specific functions better than hepatocytes isolated from MUW-stored livers. Human hepatocytes isolated from surgical biopsies should be preferentially used for the study of metabolism in human liver. |
20702209 | Adult rat liver slices as a model for studying the hepatotoxicity of vincaalkaloids. | There are certain disadvantages associated with the use of isolated or cultured cells including the need to use proteolytic enzymes for their isolation and loss of tissue organization. In order to provide an in vitro system for toxicological studies that preserves tissue integrity, a method for preparation and incubation of adult rat liver slices has been developed. Fresh ultra-thin liver slices were produced in large quantities at a rapid rate under conditions that cause minimal tissue trauma. They were incubated in a system that has been designed to allow optimum gas and nutrient diffusion. Several biochemical parameters (lactate dehydrogenase (LDH) leakage, ATP content, protein synthesis and secretion) were monitored during a 20-hr incubation period. This liver slice model was used to study the toxicity of four vincaalkaloids: vincristine, vindesine, vinblastine and navelbine. Treatment with the vincaalkaloids resulted in an inhibition of protein synthesis and secretion without any effect on LDH leakage. |
20702210 | Effects of dimethylsulphoxide on phase I and II biotransformation in cultured rat hepatocytes. | Addition of 2% dimethylsulphoxide (DMSO) to the medium has been described to retain biochemical and morphological differentiation of cultured rat hepatocytes. The maintenance of differentiated hepatocytes for a long time with stable phase I and II biotransformation capacities provides an important model for pharmacological and toxicological studies. This work has been undertaken to study the effects of DMSO on phase I and II parameters in cultured hepatocytes. For phase I cytochrome P-450 content, 7-ethoxycoumarin O-deethylase and aldrin epoxidase activities and for phase II glutathione S-transferase (GST) activity and its isoenzyme profile have been measured. In control cells cytochrome P-450 content and the enzymic activities measured declined as a function of culture time. Addition of DMSO partially prevented the loss of all parameters measured. The changes in GST activity were related to changes in its isoenzyme pattern. In control cells subunits 1 and 2 decreased, 3 and 4 increased and de novo expression of 7 was observed. When DMSO was added, the subunit profile better resembled that observed in vivo and the expression of subunit 7 was suppressed. In conclusion DMSO protects cultured hepatocytes from dedifferentiation and stabilizes the cells. |
20702211 | Toxicity of paracetamol and cyclophosphamide in monolayer cultures of rat and human hepatocytes. | Hepatocyte structural and functional integrity was characterized in short-term primary monolayer cultures. Ethoxycoumarin-O-deethylase activity, cellular glutathione, ATP and ADP content, protein synthesis and lactate dehydrogenase (LDH) leakage were monitored. Cytotoxicity and perturbation of hepatocyte function involving measurement of these biochemical parameters was investigated following exposure to paracetamol and cyclophosphamide. Evaluation of several biochemical parameters appears to be a more appropriate assay of a perturbation in cellular integrity than the use of a single parameter. In the case of paracetamol the response of human hepatocyte cultures was broadly similar to that observed in rat hepatocyte cultures. An increase in LDH leakage occurred following exposure to high dose levels and was associated with depletion of cellular glutathione levels. At both toxic and non-toxic dose levels protein synthesis was impaired and a decrease in the cellular ATP/ADP ratio was evident. A decrease in protein synthesis and cellular glutathione was observed in both rat and human hepatocytes following exposure to cyclophosphamide. The data highlight the potential use of hepatocyte cultures for investigation of specific cytotoxic events and also emphasize the incorporation of human tissue in such systems. |
20702212 | The liver slice system: An in vitro acute toxicity test for assessment of hepatotoxins and their antidotes. | A simple method for rapid and reliable assessment of hepatotoxic agents is described. Liver slices from rats and mice of two age groups were incubated with the test hepatotoxins. Exposure of liver slices from 3-month-old mice to acetaminophen (6.8 mg/ml) resulted in 80% leakage of lactate dehydrogenase into the incubation medium, whereas liver slices from one-day-old mice showed only 12% leakage. Similar results were obtained with rat liver slices. The relative lack of response by livers of newborn rats was also demonstrated with carbon tetrachloride. The in vitro liver slice system has also been used to test the potency of N-acetylcysteine (NAC) as an antidote to acetaminophen toxicity. NAC protected mouse liver slices against acetaminophen toxicity in a dose-dependent manner. Addition of the antidote (10 mm) 20 min following hepatotoxin application reduced enzyme leakage by 75% pared with the system with acetaminophen only. These findings demonstrate that the liver slice system provides the same type of information about hepatotoxins that is usually obtained by the use of acute in vivo tests on a large number of animals. It can be used for testing potential antidotes against hepatotoxins as well as for demonstration of species and age differences in the toxicity of various substances. |
20702213 | Biliary excretion of fluorescent cholephiles in hepatocyte couplets: An in vitro model for hepatobiliary and hepatotoxicity studies. | Hepatocyte couplets (26.8% of cell preparation with >85% viability) were prepared from rat livers. The preparation maintained the activity of 7-ethoxycoumarin-O-deethylase throughout an 8-hr incubation. Couplets extensively secreted the fluorescent cholephiles, fluorescein and cholyl-lysyl-fluorescein isothiocyanate, into sealed canalicular spaces within 2 hr and 15-30 min, respectively, which is indicative of functional tight junctions. The time scale for biliary secretion of the cholephiles correlated well with the relative kinetics of biliary excretion reported in vivo. Hepatocyte couplets may prove useful for studies on toxicity directed towards the hepatobiliary system. |
20702214 | Effects of various inducers on the expression of cytochromes P-450 IIC8, 9, 10 and IIIA in cultured adult human hepatocytes. | Primary cultures of adult human hepatocytes were used to determine the capability of the human liver to respond to phenobarbital, 3-methylcholanthrene, troleandomycin and rifampicin, pounds known to be potent inducers of hepatic cytochrome P-450 in various animal species. Both mRNA and corresponding protein of two major isoenzymes, that is, P-450 IIC8, 9, 10 and P-450 IIIA, were measured after daily exposure to the drugs for 3 days. Phenobarbital and rifampicin were found to increase the levels of P-450 IIC8, 9, 10 mRNA and protein while troleandomycin and 3-methylcholanthrene were ineffective. Different effects were obtained for P-450 IIIA. Both mRNA and related protein were markedly increased by troleandomycin and rifampicin and decreased by 3-methylcholanthrene. mRNAs were slightly increased by phenobarbital. The results demonstrate that human hepatocytes retain drug-inducible P-450 isoenzymes in primary culture and represent a unique approach to investigate regulation of human liver drug metabolizing enzymes. |
20702215 | Prolonged expression of biotransformation activities of rat hepatocytes co-cultured with established cell lines. | Co-culture of hepatocytes with fibroblastic cells (C3H/10T1 2 and 3T3) allowed the expression of phase I and II biotransformation enzymes for a prolonged time in culture. After 7 days in co-culture, cytochrome P-450 and monooxygenase activities were still 20-30% of the initial value, while in pure hepatocyte cultures they were undetectable. NADPH-cytochrome c reductase and conjugation activities remained at nearly initial levels for at least 7 days in co-cultured hepatocytes. Finally, in co-cultures a clear and prolonged induction of monooxygenase activities by 3-methylcholanthrene and phenobarbital was observed. |
20702216 | Competing pathways in metabolism-mediated cytotoxicity in vitro. | The metabolism of most xenobiotics to reactive metabolites is panied petition with inactivation pathways, and the balance of these activation/inactivation pathways is critical in determining the precise circumstances in which metabolism-mediated cytotoxicity may be detected in vitro. Discussion of the importance of this balance must also take into account the nature of the question posed at the start of an in vitro study, and the choice of metabolizing system used. In quantitative terms, liver microsomal cytochrome P-450-mediated monooxygenase activity and conjugation with glutathione (GSH) are the major routes of activation and inactivation, respectively. Results of recent studies on the cytotoxicity of cyclophosphamide, bromobenzene and paracetamol measured in a liver homogenate fraction-cultured cell co-incubation have confirmed the importance of activation/inactivation balance and stability of the reactive metabolite as determinants of metabolism-mediated cytotoxicity in vitro. Competition between metabolism by P-450-mediated enzyme activity and conjugation with GSH may be used as the basis of a screen for detection of reactive metabolites by measurement of GSH depletion or conjugation with GSH. The use of cells in culture for studies of metabolism-mediated cytotoxicity is confounded by the time-dependent alterations in various enzyme activities that occur and so distort the balance of activation/inactivation. This, in turn, reduces the reliability of such systems as predictors of the events likely to occur in vivo; the lack of a ponent in the in vitro system further distorts this in vivo/in parison. Rational development of in vitro systems for detection of reactive metabolites can only occur with an understanding of the role peting pathways in metabolism. |
20702217 | A study of the metabolism and kinetics of SK&F 94120 and two structural analogues in rat hepatocytes and in vivo. | The major route of metabolism of 5-(4-acetamidophenyl)pyrazin-2(1H)-one (SK&F 94120) was by glucuronidation at the oxygen of the pyrazinone ring. Other metabolites originated from metabolism by gut microflora with subsequent hepatic metabolism (Ross et al., 1988). SK&F 94120 was metabolized by cultures of rat hepatocytes to a glucuronide conjugate and was rapidly cleared in the hepatocyte system (Cl(int) = 0.320 ml/hr/mg protein). A structural analogue (SK&F 94467) was not glucuronidated by rat hepatocytes and therefore its clearance in vitro could not be detected. In vivo SK&F 94120 was rapidly cleared from the plasma. Plasma clearance was greater for SK&F 94120 than for SK&F 94467, which is consistent with data from the hepatocyte system. Thus the mean retention time of SK&F 94467 in vivo is longer than for SK&F 94120. |
20702218 | Xenobiotic metabolism in the isolated conceptus. | The development of culture systems using either pre- or post-implantation embryos has made it possible to study the metabolizing capacity of the isolated conceptus in vitro. In the rodent pre-implantation embryo and post-implantation conceptus (embryo and its membranes), constitutive levels and inducibility of different enzyme systems involved in drug metabolism have been shown in vitro to lead to the formation of embryotoxic metabolites of different xenobiotics. This indicated the presence of enzyme systems during early organogenesis. For example, using the rat post-implantation embryo culture, we could show that incubation with the lipoxygenase inhibitor N-hydroxy-N-methyl-7-propoxy-2-naphthalenethanamine (QAB) led to high levels of the main in vivo metabolite 7-propoxy-naphthalene-2-ylacetic acid (QAA) and two as yet unidentified products, M5 and M6, in the conceptus. QAB was not found in tissues and QAA itself did not enter the partments. In addition, accumulation in tissue was dependent on the time and duration of exposure. It started at 10.5 days of development. A similar metabolite pattern was obtained after yolk-sac tissue had been cultured alone, which suggests metabolizing capacity of mainly the yolk-sac tissue. The enzyme reactions involved might have included oxidative N-demethylation and oxidative deamination, probably also including the formation of reactive intermediate metabolites. In conclusion, our data demonstrate that not only maternal metabolism may play an important role in the toxic action of xenobiotics, but also the metabolizing capacity of the conceptus itself may be crucial, since the formation of (intermediate, highly reactive) metabolites takes place at the target site. |
20702219 | Reactive metabolite formation catalysed by cytochrome P-450j. | Cytochrome P-450j is induced by ethanol, isoniazid, fasting and in diabetes mellitus; the hepatotoxicity of paracetamol and N,N-dimethylnitrosamine is increased following such induction. We have assessed the potential of liver microsomes isolated from control and isoniazid-treated rats to convert some hepatotoxins to reactive metabolites using loss of glutathione, added to microsomal incubations, as an index of reactive metabolite generation. No change or a decrease in glutathione pared with controls, was found when 4-ipomeanol, cyclophosphamide, toluene, coumarin and butylated hydroxytoluene were incubated with liver microsomes from isoniazid-treated rats. No glutathione depletion was caused by trichloroethylene. Increased glutathione depletion was shown for paracetamol and bromobenzene and their non-hepatotoxic analogues 3-hydroxyacetanilide and p-bromophenol. This confirmed that the glutathione depletion assay fails to distinguish between toxic and non-toxic reactive metabolites. Increased glutathione depletion was also observed for 2-methyl furan, aniline, allyl alcohol, thiophene and chloroform. Analysis of glutathione depletion/concentration relationships demonstrated that P-450j has high affinity for chloroform, as judged by reactive metabolite formation. |
20702220 | The susceptibility of various cultured cells to induction of clear cytoplasmic vacuoles by disobutamide. | Cultured cells were found to be highly useful for investigating intracellular storage of pounds using disobutamide as a model agent. To select types of cultured cells most suitable for investigations, cells of dog coronary artery muscle, rabbit aorta muscle, rat urinary bladder carcinoma, rat basophilic leukaemia, human skin fibroblasts, bovine aorta endothelium, Chinese hamster ovary tumour and mouse fibroblasts were incubated with 0, 1, 2, 4, 6, 8 and 10 x 10(-4)m-disobutamide for 24 hr. Cultures were examined in situ by phase light microscopy for the presence of clear cytoplasmic vacuoles, cell death (cell detachment), and for drug effect on confluency/cell count. Disobutamide induced vacuoles in all cell types except rat leukaemia. The drug induced cell death and reduction in confluency or cell count in cultures of all cell types except rat carcinoma and rabbit aorta muscle. Release of lactic dehydrogenase from cells confirmed the relative resistance of the rat carcinoma and rabbit cells, and susceptibility of rat leukaemia, to drug-induced cell death. By means of electron microscopy of rat carcinoma and rabbit cells, it was established that vacuoles were membrane-bound and their content was predominantly electron-lucent. |
20702221 | Variations in the response of cell lines to metabolism-mediated toxicity. | One major criticism of cytotoxicity tests is the negligible capacity for metabolism of pounds, which exists in most cell lines. We have investigated the use of an exogenous metabolizing prising 9000 g supernatant of liver from Aroclor 1254-pretreated rats (S-9) and an NADPH regenerating system, in the kenacid blue test. BCL-D1 cells (a human embryonic lung finite cell line) appeared to be sensitive to the toxicity of active metabolites of several model toxins. Other cell lines appeared to be less sensitive but the effects varied with the cell line and the pound. These differences may, in part, be due to differences in the intrinsic ability of different cell lines to detoxify the active metabolites. It is therefore possible to use this type of test system for mechanistic studies but results with pounds should be treated with caution. |
20702222 | The in vitro effects and metabolism-mediated cytotoxicity of phorone, a glutathione-depleting agent. | The use of S-9 liver fractions to examine metabolism-mediated cytotoxicity in vitro has the inherent problem that not only are activating enzyme systems added, but also deactivation pathways, such as that involving glutathione. The in vivo manipulation of these activating and deactivating systems prior to S-9 preparation is possible with animals, but not with humans. Hence, the possibility of depleting glutathione in target cells and S-9 fractions was evaluated using phorone, a known glutathione-depleting agent. 3T3-L1 mouse fibroblast-like cells and V79 Chinese hamster lung fibroblasts were used as the target cells, and cytotoxicity was assessed by the FRAME (Fund for the Replacement of Animals in Medical Experiments) kenacid blue method. Phorone was found to have a moderate intrinsic cytotoxicity and to effectively deplete cellular glutathione. When phorone was used in the ponent S-9 fraction/target cell system, its toxicity to 3T3-L1 cells was markedly increased, which suggests transformation to a toxic metabolite. The use of S-9 fractions from animals pretreated with phenobarbitone and beta-naphthoflavone resulted in greatly increased phorone toxicity, which indicates the involvement of cytochrome P-450 enzymes in its metabolism. The metabolism-mediated toxicity of phorone was reduced by the addition of exogenous glutathione. |
20702223 | Glutathione conjugation and cytochrome P-450 metabolism of methyl chloride in vitro. | Possible carcinogenic properties of methyl chloride (CH(3)Cl) have been under discussion since an increase of renal tumours was observed in male B6C3F(1) mice after a 2-yr inhalation exposure to the substance. This was, however, only observed following exposure of male mice to the highest concentration level and not after exposure of females or F344 rats of both sexes. Accumulation of formaldehyde in the kidneys was thought to be responsible for tumour production. In the experiments presented here, cytosolic enzymes from the liver and kidneys of different mouse strains and F344 rats were incubated in head-space vials with methyl chloride or methyl bromide. Following equilibration, the decrease in the concentration of the gases was monitored by gas chromatography as a parameter for metabolic elimination. The metabolic turnover of the methyl halides was found to be significantly higher in female animals than in the males. In parallel experiments, the glutathione content of the liver and kidneys of mice exposed by inhalation to 1000 ppm methyl chloride was determined. In both organs, the glutathione content diminished rapidly after exposure to the methyl halide. The glutathione depletion was slightly greater in females than in males. Finally, the content of cytochromes P-450, P-420 and b5 was determined in liver and kidneys of different mouse strains by difference spectroscopy. Female mice were found to have a lower content of P-450 and b5 than males in the kidneys; there was no such sex difference in liver tissue. The results show that a sex difference in metabolism is unlikely to be responsible for the unique kidney tumour production in male B6C3F(1) mice. Other possible explanations are discussed. |
20702224 | Studies on the mechanism of coumarin-induced toxicity in rat hepatocytes. | Rat hepatocyte suspensions have been used as a model system for some studies on the mechanism of coumarin-induced hepatotoxicity. Hepatocytes were isolated from male Sprague-Dawley rats and subjected to Percoll centrifugation to obtain preparations with >/=92% viability. Coumarin produced time- and concentration-dependent cytotoxic effects in rat hepatocytes as indicated by loss of cell viability and glutathione depletion. [3-(14)C]Coumarin was metabolized by rat hepatocytes to polar metabolites including o-hydroxyphenylacetic acid and to metabolites that became covalently bound to hepatocyte proteins. The addition of 10 mum-ellipticine significantly reduced coumarin cytotoxicity, coumarin metabolism and covalent binding in rat hepatocytes. These results demonstrate that coumarin-induced liver injury in the rat can be modelled in hepatocyte suspensions and that toxicity appears to be due to one or more cytochrome P-450 generated metabolites. |
20702225 | Continuous culture of human faecal bacteria as an in vitro model for the colonic microflora. | To investigate the role of human gut bacteria in the metabolism of potentially pounds we have developed an in vitro model of the human faecal microflora using a two-stage continuous culture inoculated with human faeces. The cultured bacterial population retained many of the bacteriological and biochemical characteristics of the flora present in the faecal sample used for inoculation. Obligate anaerobes were the predominant bacterial types found in vitro and included Bacteroides ovatus and Bifidobacterium adolescentis. parison of in vivo (faeces) and in vitro bacterial enzyme activities that are known to be involved in the biotransformation of potentially pounds found the activities of hydrolytic enzymes to be similar but reductive enzymes exhibited higher activities in the continuous culture model. When substrates of the enzymes were added to the culture vessel, the enzymes were induced to varying extents. The short-chain fatty acid profile in the culture was almost identical to that in faeces with the order of abundance being the same in two systems. These results indicate that the continuous culture of faecal bacteria can provide a suitable model for studying bacterial interactions and biotransformation of the human colonic flora. |
20702226 | Toxicokinetic-toxicodynamic models describing the relation of plasma and red blood cell potassium with plasma digitalis in acute human digitalis poisoning. | Toxicity of cardiac glycosides involves the inhibition of the Na(+)-K(+) ATPase pump. As a consequence, extracellular K(+) concentration rises and intracellular K(+) concentration strongly decreases. Red blood cell (RBC) K(+) is a practical marker of ATPase inhibition. In a group of 15 patients intoxicated by digitoxin and lanatoside C, correlations between the calculated digitoxin ingested dose or plasma digitoxin levels and the kinetics of plasma K(+) and RBC K(+) have been assessed using kinetic-effect modelling. A correlation between the calculated ingested dose of digitoxin with RBC K(+) was found (r = 0.64). A direct relation based on the linear model fitted the relation between extracellular K(+) and digitalis concentration. An indirect relation based on the Emax sigmoid model fitted the relation between RBC K(+) and digitoxin concentrations. Specific parameters were obtained from the linear model with a = 0.0196 +/- 0.0272 and b = 0.455 +/- 0.035. Specific parameters were derived from the Emax sigmoid model with k(eo) = 0.0139 +/- 0.0052/hr and EC(50) = 91.95 +/- 20.55 ng/ml, where k(eo) = first-order rate constant of the disappearance of the toxic effect and EC(50) = digitoxin concentration decreasing the RBC K(+) concentration by 50%. These data showed that the in vitro assays of plasma K(+) and RBC K(+) are convenient and predictive assays for evaluating the severity of human digitoxin poisoning. |
20702227 | An apparatus to simulate metabolism of ingested substances. | A prototype in vitro, closed in-line apparatus was designed to simulate the passage of a substance through the gastro-intestinal tract and absorption by internal body organs with appropriate metabolism at each stage, puter-controlled culture conditions to resemble normal in vivo physiology. In tests to define conditions of parisons of tests using water-bath and metabolic simulator techniques showed formation of similar metabolites of sodium lauryl sulphate. Comparison of in vivo and in vitro results showed the advantage of the in vitro system to identify metabolites. The end-product butyric acid-4-sulphate excreted in the urine of rats after a single oral dose of [(14)C]sodium lauryl sulphate was also formed by hepatocytes in vitro in a water-bath vessel, which indicates the relevance of the intermediary metabolites detected by sequential sampling over 24 hr in the metabolic simulator. The intermediary metabolites identified were 11- and 12-hydroxy dodecyl sulphate, 12-carboxy dodecyl sulphate and probably 11-keto dodecyl sulphate. This evidence was not readily obtainable from the in vivo test. Once limitations of this prototype design are e, the system should parison of the metabolism of test substances between species, including man, and eventually the use of laboratory-adapted cultures of relevant organs to replace primary in vivo sources of tissues. |
20702228 | Trichloroethylene biotransformation in human and rat primary hepatocytes. | The biotransformation of trichloroethylene (TCY) was studied in male Sprague-Dawley rats and in human hepatocyte suspensions to aid in estimating the potential for hepatocarcinogenesis in humans. The major metabolites were qualitatively identical in both species, but rat hepatocytes metabolized about four times more TCY than did human hepatocytes under the same experimental conditions. The quantities of chloral hydrate, trichloroethanol (free plus conjugated) and trichloroacetic acid (TCA) were 15, 5 and 20 times greater, respectively, in rat hepatocyte suspensions. Since the TCA metabolite has been implicated in TCY-induced peroxisomal proliferation and hepatocarcinogenesis and rats form less TCA than do mice, which are susceptible to these effects, the results suggest that humans are at low risk from TCY exposure. |
20702230 | Development of in vitro tests of human sperm function: A diagnostic tool and model system for toxicological analyses. | The mammalian spermatozoon is a highly specialized cell which, during the process of evolution, has developed a partmentalized structure in order to express the diverse array of biological properties (movement, cell recognition, secretion, membrane fusion) required to fertilize the egg. This paper describes an integrated battery of tests that can be used to obtain information on certain key aspects of human sperm function in vitro. These tests puterized digital image analysis systems to record the movement characteristics of the spermatozoa, in vitro assays of sperm-zona recognition based on human ova stored in high ionic strength salt solutions, the use of fluorescein-conjugated lectins to detect the acrosome reaction and inter-species in vitro fertilization procedures to assess the ability of human spermatozoa to fuse with the vitelline membrane of the oocyte. bination these assays provide information that is predictive of the fertilizing capacity of human spermatozoa in vitro and in vivo, and, as such, should find application in assessing the influence of xenobiotics on male fertility. In addition, such tests may be of value in developing model in vitro systems employing human spermatozoa for the analysis of toxicity at the cellular level, particularly in relation to the influence of xenobiotics on the properties of biological membranes. |
20702231 | In vitro embryotoxicity and teratogenicity studies. | During the past decade many publications have appeared describing test methods for in vitro toxicological research and emphasizing their desirability, appropriations and necessity. One reason for this might be the pressure imposed on regulatory, industrial and munities by society to reduce the number of animals used in research and testing strategies. In addition, sophisticated analytical techniques have been developed that allow the measurement of small quantities of biologically important material. Moreover, the present knowledge gained in the area of tissue culture and in vitro embryo culture allows the application of these techniques for more routine studies on the one hand, and studies on mechanisms of action of teratogens in model systems of isolated developmental processes on the other hand. With respect to reproductive toxicity, embryotoxicity and teratogenicity there are now diverse systems available ranging in plexity from bacteria, insects, invertebrates, lower vertebrates, avian embryos and mammalian cells, tissues and organs to whole rodent embryos. This presentation serves only as an introduction to plex issues raised by the many methods available. The itant application of both in vivo and in vitro methodologies will improve the quality of teratological research, and therefore will contribute to a critical evaluation of developmental hazards. |
Subsets and Splits