Daniela Gradinaru

Drug Transport into the Mammalian Brain: The Nasal Pathway and its Specific Metabolic Barrier

It is generally accepted that the rate of entry into and distribution of drugs and other xenobiotics within the central nervous system (CNS) depends on the particular anatomy of the brain microvessels forming the blood-brain barrier (BBB), and of the choroid plexus forming the blood-cerebrospinal fluid barrier (CSF), which possess tight junctions preventing the passage of most polar substances. Drug entry to the CNS also depends on the physicochemical properties of the substances, which can be metabolised during this transport to pharmacologically inactive, non-penetrating polar products. Finally, the entry of drugs may be prevented by multiple complex specialized carriers, which are able to catalyse the active transport of numerous drugs and xenobiotics out of the CNS. Nasal delivery is currently considered as an efficient tool for systemic administration of drugs that are poorly absorbed via the oral route, and increasing evidence suggests that numerous drugs and potentially toxic xenobiotics can reach the CNS by this route. This short review summarizes recent knowledge on factors controlling the nasal pathway, focusing on drug metabolising enzymes in olfactory mucosa, olfactory bulb and brain, which should constitute a CNS metabolic barrier.

MARK-AGE biomarkers of ageing

Many candidate biomarkers of human ageing have been proposed in the scientific literature but in all cases their variability in cross-sectional studies is considerable, and therefore no single measurement has proven to serve a useful marker to determine, on its own, biological age. A plausible reason for this is the intrinsic multi-causal and multi-system nature of the ageing process. The recently completed MARK-AGE study was a large-scale integrated project supported by the European Commission. The major aim of this project was to conduct a population study comprising about 3200 subjects in order to identify a set of biomarkers of ageing which, as a combination of parameters with appropriate weighting, would measure biological age better than any marker in isolation.

An inter-laboratory validation of methods of lipid peroxidation measurement in UVA-treated human plasma samples

Abstract Lipid peroxidation products like malondialdehyde, 4-hydroxynonenal and F2-isoprostanes are widely used as markers of oxidative stress in vitro and in vivo. This study reports the results of a multi-laboratory validation study by COST Action B35 to assess inter-laboratory and intra-laboratory variation in the measurement of lipid peroxidation. Human plasma samples were exposed to UVA irradiation at different doses (0, 15 J, 20 J), encoded and shipped to 15 laboratories, where analyses of malondialdehyde, 4-hydroxynonenal and isoprostanes were conducted. The results demonstrate a low within-day-variation and a good correlation of results observed on two different days. However, high coefficients of variation were observed between the laboratories. Malondialdehyde determined by HPLC was found to be the most sensitive and reproducible lipid peroxidation product in plasma upon UVA treatment. It is concluded that measurement of malondialdehyde by HPLC has good analytical validity for inter-laboratory studies on lipid peroxidation in human EDTA-plasma samples, although it is acknowledged that this may not translate to biological validity.

Vitamin D status and oxidative stress markers in the elderly with impaired fasting glucose and type 2 diabetes mellitus

Background and aims: Vitamin D deficiency has been identified in the elderly as a potential risk factor for cardiovascular disease development, possibly through its association with other risk factors, such as type 2 diabetes mellitus (T2DM), hypercholesterolemia and hypertension. The aim of this cross-sectional study was to evaluate the vitamin D status in elderly subjects with impaired fasting glucose (IFG) or T2DM, and to examine its relationships to systemic oxidative stress and biochemical markers of endothelial dysfunction. Methods: Serum 25-hydroxyvitamin D [25(OH)D], fasting glucose, insulin, lipid profile, advanced glycation end products (AGEs), advanced oxidation protein products (AOPPs), low-density lipoprotein susceptibility to oxidation (oxLDL) and nitric oxide metabolic pathway products (NOx) were analyzed in elderly subjects with IFG (n=30) and T2DM (n=35) compared with aged-matched controls (n=25). Results: 25(OH)D levels in the IFG and T2DM groups were significantly lower than in controls (31.9±1.9 and 28.5±1.9 vs 39.4±2.4 ng/mL, p<0.001), and associated with significantly (p<0.001) higher levels of the oxidative stress parameters AGEs, AOPPs, oxLDL and NOx. Hypovitaminosis D [25(OH)D)>-30 ng/ml] markedly enhanced the oxidative stress and cardiovascular risk in hyperglycemic subjects compared with sufficient vitamin D [25(OH)D)=30 ng/mL] status subjects. In subjects with IFG and T2DM (n=65), the vitamin D status was significantly inversely correlated both with oxLDL (r=−0.413, p=0.001) and AOPPs (r=−0.475, p<0.001), and strongly positively associated with highdensity lipoprotein cholesterol (r=0.609, p<0.001). Conclusions: In the elderly with impaired glucose metabolism the vitamin D status is inversely associated with levels of circulating markers of oxidative stress and endothelial dysfunction, especially in subjects with hypovitaminosis D.

Glucuronidation of odorant molecules in the rat olfactory system. Activity, expression and age-linked modifications of UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT2A1, and relation to mitral cell activity

The aim of the present study was to examine the glucuronidation of a series of odorant molecules by homogenates prepared either with rat olfactory mucosa, olfactory bulb or brain. Most of the odorant molecules tested were efficiently conjugated by olfactory mucosa, whereas olfactory bulb and brain homogenates displayed lower activities and glucuronidated only a few molecules. Important age-related changes in glucuronidation efficiency were observed in olfactory mucosa and bulb. Therefore, we studied changes in expression of two UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT2A1, in 1-day, 1- and 2-week-, 3-, 12- and 24-month-old rats. UGT1A6 was expressed at the same transcriptional level in the olfactory mucosa, bulb and brain, throughout the life period studied. UGT2A1 mRNA was expressed in both olfactory mucosa and olfactory bulb, in accordance with previous results [Mol. Brain Res. 90 (2001) 83], but UGT2A1 transcriptional level was 400-4000 times higher than that of UGT1A6. Moreover, age-dependent variations in UGT2A1 mRNA expression were observed. As it has been suggested that drug metabolizing enzymes could participate in olfactory function, mitral cell electrical activity was recorded during exposure to different odorant molecules in young, adult and old animals. Age-related changes in the amplitude of response after stimulation with several odorant molecules were observed, and the highest responses were obtained with molecules that were not efficiently glucuronidated by olfactory mucosa. In conclusion, the present work presents new evidence of the involvement of UGT activity in some steps of the olfactory process.

Validation of protein carbonyl measurement: a multi-centre study.

Protein carbonyls are widely analysed as a measure of protein oxidation. Several different methods exist for their determination. A previous study had described orders of magnitude variance that existed when protein carbonyls were analysed in a single laboratory by ELISA using different commercial kits. We have further explored the potential causes of variance in carbonyl analysis in a ring study. A soluble protein fraction was prepared from rat liver and exposed to 0, 5 and 15 min of UV irradiation. Lyophilised preparations were distributed to six different laboratories that routinely undertook protein carbonyl analysis across Europe. ELISA and Western blotting techniques detected an increase in protein carbonyl formation between 0 and 5 min of UV irradiation irrespective of method used. After irradiation for 15 min, less oxidation was detected by half of the laboratories than after 5 min irradiation. Three of the four ELISA carbonyl results fell within 95% confidence intervals. Likely errors in calculating absolute carbonyl values may be attributed to differences in standardisation. Out of up to 88 proteins identified as containing carbonyl groups after tryptic cleavage of irradiated and control liver proteins, only seven were common in all three liver preparations. Lysine and arginine residues modified by carbonyls are likely to be resistant to tryptic proteolysis. Use of a cocktail of proteases may increase the recovery of oxidised peptides. In conclusion, standardisation is critical for carbonyl analysis and heavily oxidised proteins may not be effectively analysed by any existing technique.

Oxidized LDL and NO synthesis--Biomarkers of endothelial dysfunction and ageing.

Oxidized LDL (oxLDL) and nitric oxide (NO) exert contradictory actions within the vascular endothelium microenvironment influencing key events in atherogenesis. OxLDL and NO are so far regarded as representative parameters of oxidative stress and endothelial dysfunction, new targets in prevention, diagnosis and therapy of cardiovascular diseases, and also as candidate biomarkers in evaluating the human biological age. The aim of this review is to explore recent literature on molecular mechanisms and pathophysiological relationships between LDL oxidation, NO synthesis and vascular endothelium function/dysfunction in ageing, focusing on the following aspects: (1) the impact of metabolic status on both LDL oxidation and NO synthesis in relation with oxidative stress, (2) the use of oxidized LDL and NO activity as biomarkers in human studies reporting on cardiovascular outcomes, and (3) evidences supporting the importance of oxidized LDL and NO activity as relevant biomarkers in vascular ageing and age-related diseases.

Effect of oxidative stress on UDP-glucuronosyltransferases in rat astrocytes.

The present work reports data regarding effects of an induced oxidative stress on the mainly expressed isoforms of UDP-glucuronosyltransferases (UGTs) in the brain. UGT1A6 and UGT1A7 expression and enzymatic activities toward the 1-naphthol were analyzed in rat cultured astrocytes following the exposure for 48 h to redox-cycling xenobiotic compounds such as quinones and bipyridinium ions. The expression of NADPH:cytochrome P450 reductase and NAD(P)H:quinone oxidoreductase 1 (NQO1) was also investigated. Oxidative stress induced significant deleterious changes in astrocyte morphology, decreased cell viability and inhibited catalytic function of UGTs as a result of protein oxidation. Alternatively, in the surviving impaired astrocytes, oxidative conditions induced a significant overactivity and overexpression of xenobiotic detoxification enzymes, as adaptive response. These effects were significantly prevented by the presence of melatonin, suggesting its direct antioxidant action on reactive oxygen species, reflected further on the glucuronidation activity and transcriptional regulation of both UGT1A6 and UGT1A7. Results show that both catalytic properties of UGTs and the expression of UGT1A6, UGT1A7, NQO1 and NADPH:cytochrome P450 reductase in rat astrocytes are greatly influenced by the pro-oxidative environment. In conclusion, an experimental increase in oxidative cellular status could have both immediate and long term consequences on detoxification enzymatic system activity and expression.

Quercetin and epigallocatechin gallate effects on the cell membranes biophysical properties correlate with their antioxidant potential.

Quercetin and epigallocatechin gallate are two of the most abundant polyphenols in dietary plants, including apples, onions, red wine and green tea. The bioactivity of polyphenols is linked to their ability to interact with cell membranes without being internalized. The aim of the present study was to assess the short-time effect of these polyphenols on membrane anisotropy and transmembrane potential of U937 monocytes and Jurkat T lymphoblasts. Results showed that quercetin and epigallocatechin gallate induced, after 20 minutes cell exposure, a dose-dependent increase of membrane anisotropy and polarization. Anisotropy increase was correlated with the reduction of lipid peroxidation. Our results could indicate that the antioxidant capacity of the tested polyphenols is due to their stabilizing effect on the cell membranes, thus contributing to cell protection in various pathologies and as adjuvant therapy in highly toxic treatment regimens.

Lipid peroxidation due to in vitro and in vivo exposure of biological samples to nanoparticles.

The increasing use of nanomaterials in biological applications raises numerous concerns about the dangers they might pose to living organisms. The rise in oxidative stress is usually the most readily observed effect induced by nanoparticles, with the measurement of lipid peroxidation levels being one of the most frequently used biological markers for its evaluation. Here, we describe the spectrophotometric and fluorimetric methods for determining the modifications of the malondialdehyde (MDA) level induced by many types of nanoparticles in in vitro and in vivo biological systems.