- Email: [email protected]
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COMPARISON OF DIFFERENT OXIDATIVE STRESS BIOMARKERS IN VIVO Maria Kadiiska, NIEHS/NIH, USA Investigation of oxidative stress biomarkers is a subject of continuously increasing interest, both in science and in commerce. The field is evolving and there is a great need to validate and to understand biomarkers at a chemical and enzymatic molecular-mechanism level. Antioxidants and oxidized lipids, proteins and DNA levels in plasma and urine of experimental animals exposed to varied environmental oxidants were measured as a method-validation study. The goal of this international and multi-laboratory research is to study biomarkers generated by CCl4-, ozone- or LPS -induced oxidative stress and to assess whether inconsistent results often reported in the literature might be due to the limitations of the available methods. This presentation is centered on comparing data from measurements with more than 30 different techniques to estimate plasma and urinary oxidative products and antioxidants in all three models of oxidant injuries. Results will conclude which oxidized molecules or antioxidants fulfilled the oxidative stress biomarker criterion of significant effects measured in biological fluid and seen at two doses at more than one time point. It is determined that measurements of lipid oxidation products MDA and 8-iso-PGF2α in plasma and urine are potential candidates for general biomarkers of oxidative stress. However, further investigation showed that solely measuring an increase in 8-iso-PGF2α levels does not necessarily reflect increases in oxidative stress due to confounding enzymatic generation. We established that the 8-iso-PGF2α/PGF2α ratio can be used to distinguish chemical from enzymatic lipid peroxidation and therefore the ratio of 8-iso-PGF2α to prostaglandin F2α (PGF2α) is recommended as a quantitative measure of increased oxidative stress in vitro, in animal models, and in humans. Antioxidants and all other oxidative products were not changed or showed fewer significant effects. Current negative findings are of great importance because they provide evidence that in complex systems, as established here, the assays used may not provide meaningful data of oxidation, especially in oxidative stress related human studies.
Maria Kadiiska
doi: 10.1016/j.freeradbiomed.2016.10.017
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STEROL OXIDATION: ERRORS IN CHOLESTEROL BIOSYNTHESIS Ned Porter, Vanderbilt University, USA Severe mutations of the enzymes in the pathway from lanosterol to cholesterol are frequently embryonically lethal while the phenotype of mildly-affected individuals includes intellectual disabilities and developmental disorders. For example, 7-Dehydrocholesterol (7-DHC) accumulates in tissues and fluids of patients with Smith-Lemli-Opitz syndrome (SLOS), a recessive neurodevelopmental disorder caused by mutations in the gene encoding 3-hydroxysterol-reductase (DHCR7; EC 1.3.1.21). Although the exact mechanism of the syndrome is not understood, 7-DHC is the most oxidizable lipid known and its derived oxysterols may be critical to the pathobiology of SLOS. 7-DHC derived oxysterols have been isolated from brain, liver and serum from the Dhcr7-null mouse that has been used extensively to study the phenotype and pathogenesis of SLOS and these oxysterols have significant biological activities. Studies in cell culture described in this lecture have identified dozens of small molecules, many of which are in the US and European Pharmacopoeias, that inhibit specific biosynthetic transformations in the cholesterol biosynthesis pathway. The consequence of exposure of cells to an “active” compound is a perturbation of sterol homeostasis similar to the change in the sterol profile found for a known genetic disorder. Active small molecules inhibit the conversion of 7-DHC to cholesterol, the consequence of exposure being the accumulation of cholesterol biosynthetic precursors and their toxic oxysterols in a way that parallels SLOS, the genetic disorder. doi: 10.1016/j.freeradbiomed.2016.10.018
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Ned Porter
COMPARISON OF DIFFERENT OXIDATIVE STRESS BIOMARKERS IN VIVO Maria Kadiiska, NIEHS/NIH, USA Investigation of oxidative stress biomarkers is a subject of continuously increasing interest, both in science and in commerce. The field is evolving and there is a great need to validate and to understand biomarkers at a chemical and enzymatic molecular-mechanism level. Antioxidants and oxidized lipids, proteins and DNA levels in plasma and urine of experimental animals exposed to varied environmental oxidants were measured as a method-validation study. The goal of this international and multi-laboratory research is to study biomarkers generated by CCl4-, ozone- or LPS -induced oxidative stress and to assess whether inconsistent results often reported in the literature might be due to the limitations of the available methods. This presentation is centered on comparing data from measurements with more than 30 different techniques to estimate plasma and urinary oxidative products and antioxidants in all three models of oxidant injuries. Results will conclude which oxidized molecules or antioxidants fulfilled the oxidative stress biomarker criterion of significant effects measured in biological fluid and seen at two doses at more than one time point. It is determined that measurements of lipid oxidation products MDA and 8-iso-PGF2α in plasma and urine are potential candidates for general biomarkers of oxidative stress. However, further investigation showed that solely measuring an increase in 8-iso-PGF2α levels does not necessarily reflect increases in oxidative stress due to confounding enzymatic generation. We established that the 8-iso-PGF2α/PGF2α ratio can be used to distinguish chemical from enzymatic lipid peroxidation and therefore the ratio of 8-iso-PGF2α to prostaglandin F2α (PGF2α) is recommended as a quantitative measure of increased oxidative stress in vitro, in animal models, and in humans. Antioxidants and all other oxidative products were not changed or showed fewer significant effects. Current negative findings are of great importance because they provide evidence that in complex systems, as established here, the assays used may not provide meaningful data of oxidation, especially in oxidative stress related human studies.
Maria Kadiiska
doi: 10.1016/j.freeradbiomed.2016.10.017
P2
STEROL OXIDATION: ERRORS IN CHOLESTEROL BIOSYNTHESIS Ned Porter, Vanderbilt University, USA Severe mutations of the enzymes in the pathway from lanosterol to cholesterol are frequently embryonically lethal while the phenotype of mildly-affected individuals includes intellectual disabilities and developmental disorders. For example, 7-Dehydrocholesterol (7-DHC) accumulates in tissues and fluids of patients with Smith-Lemli-Opitz syndrome (SLOS), a recessive neurodevelopmental disorder caused by mutations in the gene encoding 3-hydroxysterol-reductase (DHCR7; EC 1.3.1.21). Although the exact mechanism of the syndrome is not understood, 7-DHC is the most oxidizable lipid known and its derived oxysterols may be critical to the pathobiology of SLOS. 7-DHC derived oxysterols have been isolated from brain, liver and serum from the Dhcr7-null mouse that has been used extensively to study the phenotype and pathogenesis of SLOS and these oxysterols have significant biological activities. Studies in cell culture described in this lecture have identified dozens of small molecules, many of which are in the US and European Pharmacopoeias, that inhibit specific biosynthetic transformations in the cholesterol biosynthesis pathway. The consequence of exposure of cells to an “active” compound is a perturbation of sterol homeostasis similar to the change in the sterol profile found for a known genetic disorder. Active small molecules inhibit the conversion of 7-DHC to cholesterol, the consequence of exposure being the accumulation of cholesterol biosynthetic precursors and their toxic oxysterols in a way that parallels SLOS, the genetic disorder. doi: 10.1016/j.freeradbiomed.2016.10.018
S2
Ned Porter