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An indigenously developed human whole blood assay for pyrogenicity: A comparative assessment
Abstracts / Toxicology Letters 196S (2010) S37–S351
of combining a biological and a chemical assay: (i) The KeratinoSens assay is a new highly standardized assay which measures Nrf2-dependent gene activation and cytotoxicity of chemicals in parallel in a high throughput format. For each chemical full doseresponse curves are measured. Sensitizer-induced gene activity is not directly related to cytotoxicity, as gene induction by skin sensitizers occurs at subtoxic concentrations. (ii) An LC–MS based peptide reactivity measurement monitors peptide depletion and peptide-adduct formation to characterize the potential for covalent protein binding. We summarize results in both assays on a list of 67 reference chemicals. By combining evidence of both assays an overall accuracy of 89.6% is achieved. In addition, we present data on the use of this approach for specific applicability domains such as terpenes acting as pre-haptens and photosensitizers. We also discuss and present case studies showing the possibility to refine sensitisation potency assessments by making predictions and comparisons within such chemical reactivity and applicability domains. doi:10.1016/j.toxlet.2010.03.456
P201-002 An indigenously developed human whole blood assay for pyrogenicity: A comparative assessment P. Mohanan, B. Siddharth, C. Geetha Sree Chitra Tirunal Institute for Medical Sciences and Technology, BMT Wing, Thiruvananthapuram, Kerala, India An indigenous in vitro method is developed using human whole blood for the assessment of pyrogenicity. The methodology include, development of pure antibody against Interleukin 1 (IL 1), which was coated on polystyrene plates. The sample to be tested was incubated at 37 ◦ C in the presence of a small volume of blood taken from a healthy donor. Any pyrogen present, independent of its chemical nature, induces the formation of IL 1 which can be determined by ELISA. The main aim of the present study is a comparative assessment of pyrogenicity of five polymer (gelatin) materials (intended for capsule manufacturing) using the indigenously developed human whole blood assay, rabbit pyrogen and LAL tests. 0.4 g of the material was soaked in 10 ml of physiological saline at 60 ◦ C until a clear solution was obtained and then autoclaved. This solution was used for human whole blood assay, rabbit pyrogen and LAL tests. It was found that the rise in temperature in all the animals of rabbit pyrogen test (five materials) was above 0.5 ◦ C and is above the acceptable level. The materials (samples 4 and 5) were found positive in LAL test. International standard has indicated that the rise in temperature above 0.5 ◦ C to be considered as pyrogenic in rabbit pyrogen test. The requirement of LAL was not met by any of the two materials (all above 0.5EU) and considered as pyrogenic. Similarly, the amount of IL1 released on challenge with all the five materials were above the threshold limit and is pyrognic as per WHO classification. Hence it can be declared that the new in vitro alternative method is an accurate quantitative assay (the lowest limit of detection is 1 ng) for pyrogenicity. This will be a total replacement of animal experimentation, less expensive and readily available to the health care industry. doi:10.1016/j.toxlet.2010.03.457
S131
P201-003 pH dependence on the hemolysis induced by anionic surfactants D. Rubert, M. Mitjans, M.P. Vinardell Universitat de Barcelona, Spain Cell lysis by surfactants is a process of great fundamental and practical importance. Much research has been done to understand the mechanism underlying this process, mostly using erythrocytes as a convenient model system. However, despite the meaningful advances in understanding the lysis process, its mechanism is still unclear and the dependence on the surfactant type, initial surfactant concentration, and experimental conditions (osmolarity, pH, temperature) is not fully understood. Better understanding of the process of cell lysis by surfactants may assist in developing surfactants with enhanced selectivity, and in widening its range of applications. To increase our knowledge underlying the pH effect on hemolysis induced by surfactants, we investigated the actions of several anionic amino acid-based surfactants from the type Nalpha,Nepsylon-dioctanoyl lysine with different counterions: lysine salt, tris(hydroxymethyl)aminomethane salt, sodium salt, lithium salt and potassium salt, previously synthesized in our laboratory. Erythrocytes were obtained from rat blood after washing three times in phosphate buffered saline (PBS), containing: 6.78 g NaCl, 1.42 g Na2 HPO4 and 0.4 g KH2 PO4 in 1 L distilled water (pH 7.4). PBS was prepared at different pH (8, 7.4, 6.5 and 5.4). For the hemolysis study, aliquots of 25 l of erythrocyte suspension were pipetted into polystyrene tubs containing PBS at different pH and various surfactants concentrations, and were incubated at room temperature for different times. Following incubation, the tubes were centrifuged and the percentage of hemolysis was determined by comparing the absorbance (540 nm) of the supernatant with that of control samples totally hemolysed with distilled water. Electronic microscopy was performed to see possible alteration of cell membrane previously to hemolysis induced by surfactants. Hemolysis was affected by pH, showing an increase with reducing pH, being more affected when surfactant presents small counterions. The most hemolytic at acidic pH was the surfactant with lithium as counterion. doi:10.1016/j.toxlet.2010.03.458
P201-004 Antioxidant and anticancer activity of essential oil from Origanum onites (Lamiaceae) A. Ozkan, A. Erdogan, N.G. Trak Akdeniz University, Turkey The antioxidant and anticancer properties of a medicinal plant, Origanum onites (O. onites) (Lamiaceae) essential oil were investigated. In Hepatoma G2 culture cells, pretreatment with essential oil (<100 g/ml) from Origanum onites significantly decreased the cytotoxicity of H2 O2 in a dose-dependent manner. The essential oil also showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 80 g/ml) and lipid peroxidation inhibitory activity (40%). Treatment with higher essential oil concentration (>100 g/ml) induced cytotoxicity in the cells. The essential oil significantly affected Hepatoma G2 cell proliferation, with a reduction down to 50% (IC50; 149.12 g/ml).
of combining a biological and a chemical assay: (i) The KeratinoSens assay is a new highly standardized assay which measures Nrf2-dependent gene activation and cytotoxicity of chemicals in parallel in a high throughput format. For each chemical full doseresponse curves are measured. Sensitizer-induced gene activity is not directly related to cytotoxicity, as gene induction by skin sensitizers occurs at subtoxic concentrations. (ii) An LC–MS based peptide reactivity measurement monitors peptide depletion and peptide-adduct formation to characterize the potential for covalent protein binding. We summarize results in both assays on a list of 67 reference chemicals. By combining evidence of both assays an overall accuracy of 89.6% is achieved. In addition, we present data on the use of this approach for specific applicability domains such as terpenes acting as pre-haptens and photosensitizers. We also discuss and present case studies showing the possibility to refine sensitisation potency assessments by making predictions and comparisons within such chemical reactivity and applicability domains. doi:10.1016/j.toxlet.2010.03.456
P201-002 An indigenously developed human whole blood assay for pyrogenicity: A comparative assessment P. Mohanan, B. Siddharth, C. Geetha Sree Chitra Tirunal Institute for Medical Sciences and Technology, BMT Wing, Thiruvananthapuram, Kerala, India An indigenous in vitro method is developed using human whole blood for the assessment of pyrogenicity. The methodology include, development of pure antibody against Interleukin 1 (IL 1), which was coated on polystyrene plates. The sample to be tested was incubated at 37 ◦ C in the presence of a small volume of blood taken from a healthy donor. Any pyrogen present, independent of its chemical nature, induces the formation of IL 1 which can be determined by ELISA. The main aim of the present study is a comparative assessment of pyrogenicity of five polymer (gelatin) materials (intended for capsule manufacturing) using the indigenously developed human whole blood assay, rabbit pyrogen and LAL tests. 0.4 g of the material was soaked in 10 ml of physiological saline at 60 ◦ C until a clear solution was obtained and then autoclaved. This solution was used for human whole blood assay, rabbit pyrogen and LAL tests. It was found that the rise in temperature in all the animals of rabbit pyrogen test (five materials) was above 0.5 ◦ C and is above the acceptable level. The materials (samples 4 and 5) were found positive in LAL test. International standard has indicated that the rise in temperature above 0.5 ◦ C to be considered as pyrogenic in rabbit pyrogen test. The requirement of LAL was not met by any of the two materials (all above 0.5EU) and considered as pyrogenic. Similarly, the amount of IL1 released on challenge with all the five materials were above the threshold limit and is pyrognic as per WHO classification. Hence it can be declared that the new in vitro alternative method is an accurate quantitative assay (the lowest limit of detection is 1 ng) for pyrogenicity. This will be a total replacement of animal experimentation, less expensive and readily available to the health care industry. doi:10.1016/j.toxlet.2010.03.457
S131
P201-003 pH dependence on the hemolysis induced by anionic surfactants D. Rubert, M. Mitjans, M.P. Vinardell Universitat de Barcelona, Spain Cell lysis by surfactants is a process of great fundamental and practical importance. Much research has been done to understand the mechanism underlying this process, mostly using erythrocytes as a convenient model system. However, despite the meaningful advances in understanding the lysis process, its mechanism is still unclear and the dependence on the surfactant type, initial surfactant concentration, and experimental conditions (osmolarity, pH, temperature) is not fully understood. Better understanding of the process of cell lysis by surfactants may assist in developing surfactants with enhanced selectivity, and in widening its range of applications. To increase our knowledge underlying the pH effect on hemolysis induced by surfactants, we investigated the actions of several anionic amino acid-based surfactants from the type Nalpha,Nepsylon-dioctanoyl lysine with different counterions: lysine salt, tris(hydroxymethyl)aminomethane salt, sodium salt, lithium salt and potassium salt, previously synthesized in our laboratory. Erythrocytes were obtained from rat blood after washing three times in phosphate buffered saline (PBS), containing: 6.78 g NaCl, 1.42 g Na2 HPO4 and 0.4 g KH2 PO4 in 1 L distilled water (pH 7.4). PBS was prepared at different pH (8, 7.4, 6.5 and 5.4). For the hemolysis study, aliquots of 25 l of erythrocyte suspension were pipetted into polystyrene tubs containing PBS at different pH and various surfactants concentrations, and were incubated at room temperature for different times. Following incubation, the tubes were centrifuged and the percentage of hemolysis was determined by comparing the absorbance (540 nm) of the supernatant with that of control samples totally hemolysed with distilled water. Electronic microscopy was performed to see possible alteration of cell membrane previously to hemolysis induced by surfactants. Hemolysis was affected by pH, showing an increase with reducing pH, being more affected when surfactant presents small counterions. The most hemolytic at acidic pH was the surfactant with lithium as counterion. doi:10.1016/j.toxlet.2010.03.458
P201-004 Antioxidant and anticancer activity of essential oil from Origanum onites (Lamiaceae) A. Ozkan, A. Erdogan, N.G. Trak Akdeniz University, Turkey The antioxidant and anticancer properties of a medicinal plant, Origanum onites (O. onites) (Lamiaceae) essential oil were investigated. In Hepatoma G2 culture cells, pretreatment with essential oil (<100 g/ml) from Origanum onites significantly decreased the cytotoxicity of H2 O2 in a dose-dependent manner. The essential oil also showed high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 80 g/ml) and lipid peroxidation inhibitory activity (40%). Treatment with higher essential oil concentration (>100 g/ml) induced cytotoxicity in the cells. The essential oil significantly affected Hepatoma G2 cell proliferation, with a reduction down to 50% (IC50; 149.12 g/ml).