Reduction of the cerebrovascular volume in a transgenic mouse model of Alzheimer's disease

Combined evidence from neuroimaging and neuropathological studies shows that signs of vascular pathology and brain hypoperfusion develop early in Alzheimer's disease (AD). To investigate the functional implication of these abnormalities, we have studied the cerebrovascular volume and selected markers of blood-brain barrier (BBB) integrity in 11-month-old 3×Tg-AD mice, using the in situ brain perfusion technique. The cerebrovascular volume of distribution of two vascular space markers, [³H]-inulin and [¹⁴C]-sucrose, was significantly lower (−26% and −27%, respectively; p < 0.01) in the brain of 3×Tg-AD mice compared to non-transgenic littermates. The vascular volume reduction was significant in the hippocampus (p < 0.01), but not in the frontal cortex and cerebellum. However, the brain transport coefficient (Clup) of [¹⁴C]-d-glucose (1 μM) and [³H]-diazepam was similar between 3×Tg-AD mice and controls, suggesting no difference in the functional integrity of the BBB. We also report a 32% increase (p < 0.001) in the thickness of basement membranes surrounding cortical microvessels along with a 20% increase (p < 0.05) of brain collagen content in 3×Tg-AD mice compared to controls. The present data indicate that the cerebrovascular space is reduced in a mouse model of Aβ and tau accumulation, an observation consistent with the presence of cerebrovascular pathology in AD.     

Tellurium tetrachloride and diphenyl ditelluride cause cytotoxicity in rat hippocampal astrocytes

Tellurium tetrachloride (TeCl₄) and diphenyl ditelluride (DPDT) cytotoxicity, was investigated in rat astrocytes. Concentrations of 0.24-250 μM (24 h) were tested for viability using MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and trypan blue exclusion. MTT showed significant decreases at all concentrations tested for both compounds. Significant decreases in viability were seen in 1.95-250 μM of DPDT and 0.97-250 μM of TeCl₄ with trypan blue exclusion. The LC₅₀ for both compounds was 62.5 μM. Light and scanning microscopy confirm toxicity observed at higher concentrations. Thiobarbituric acid reactive substances (TBARs) assay, TUNEL, cytochrome c and caspase release were carried out. No significant increase in TBARS with either agent was observed (15.625-62.5 μM). TUNEL and cytochrome c assays demonstrated apoptosis in TeCl₄ treated cells (31.25-125 μM). Non-apoptotic cells were observed in DPDT treated cells. Studies of caspase 3/7 and caspase 9 indicated increased activity in TeCl₄ but not in DPDT treated cells. Optical Emission Spectroscopy of DPDT and TeCl₄ treated cells demonstrated significant accumulation of elemental tellurium in all treatment groups (31.25-125 μM). We conclude that DPDT and TeCl₄ are cytotoxic to astrocytes. TeCl₄ treated cells die via the intrinsic apoptotic pathway. Accumulation of tellurium occurs with both compounds, but results in different mechanisms of cell death.     

Expression and functional characterization of cytochrome P450 26A1, a retinoic acid hydroxylase

Retinoic acid (RA) is a critical signaling molecule that performs multiple functions required to maintain cellular viability. It is also used in the treatment of some cancers. Enzymes in the CYP26 family are thought to be responsible for the elimination of RA, and CYP26A1 appears to serve the most critical functions in this family. In spite of its importance, CYP26A1 has neither been heterologously expressed nor characterized kinetically. We expressed the rCYP26A1 in baculovirus-infected insect cells and purified the hexahistidine tagged protein to homogeneity. Heme incorporation was determined by carbon monoxide difference spectrum and a type 1 spectrum was observed with RA binding to CYP26A1. We found that RA is a tight binding ligand of CYP26A1 with low nM binding affinity. CYP26A1 oxidized RA efficiently (depletion K_m 9.4 ± 3.3 nM and V_max 11.3 ± 4.3 pmoles min⁻¹ pmole P450⁻¹) when supplemented with P450 oxidoreductase and NADPH but was independent of cytochrome b5. 4-Hydroxy-RA (4-OH-RA) was the major metabolite produced by rCYP26A1 but two other primary products were also formed. 4-OH-RA was further metabolized by CYP26A1 to more polar metabolites and this sequential metabolism of RA occurred in part without 4-OH-RA leaving the active site of CYP26A1. The high efficiency of CYP26A1 in eliminating both RA and its potentially active metabolites supports the major role of this enzyme in regulating RA clearance in vivo. These results provide a biochemical framework for CYP26A1 function and offer insight into the role of CYP26A1 as a drug target as well as in fetal development and cell cycle regulation.     

Sex moderates stress reactivity in heavy drinkers

Psychological stress and alcohol use disorders have a well-known connection. Individual differences in stress reactivity have been an area of interest in alcohol research, particularly given the relationship between craving and stress reactivity to later relapse. The present study examines the role of sex on stress-induced alcohol craving and emotional reactivity using a guided imagery stress paradigm. Participants were 64 non-treatment seeking heavy drinkers from the community who completed a two-session protocol that included two guided imagery exposures, Stress and Neutral. Participants reported their mood and craving before and after each exposure using the Differential Emotions Scale and the Alcohol Urge Questionnaire respectively. Analyses revealed a significant Stress × Sex × Trial effect on craving [F (1,61) = 5.35; p < .05] after controlling for AUDIT scores [F (1,61) = 8.16; p < .01] such that females reported greater increases in craving from baseline to post-imagery during the stress imagery versus the neutral imagery condition, than did males. Mood reactivity analysis showed similar patterns. Specifically, there was a significant Stress × Sex × Trial effect on the anxiety subscale of the DES [F (1,61) = 15.81; p < .001] such that females reported greater increases in anxiety from baseline to post-imagery during the stress imagery versus the neutral conditions, than did males. These results suggest that female heavy drinkers were more sensitive to the effects of the stress-induction on alcohol craving and mood reactivity than males. If supported by future studies, these initial findings may help advance understanding of the mechanisms of stress and mood regulation as central to alcoholism liability and recovery in females.     

5-Year trends in the intention to quit smoking amidst the economic crisis and after recently implemented tobacco control measures in Greece

Objectives

The objective of the present study was to explore the trends in the intention to quit smoking among adults in Greece between 2006 and 2011, a period characterized by financial instability and newly endorsed tobacco control initiatives.

Methods

Trend analysis of 3 representative national and cross-sectional surveys, ‘Hellas Health I’ (2006), “Hellas Health III” (2010) and Hellas Health IV (2011).

Results

Since 2006, the intention to quit smoking has significantly increased among both genders (33.3% [in 2006] to 42.4% [in 2011], p = 0.002), among respondents aged > 54 years (26.9% [in 2006] to 45.1% [in 2011], p = 0.019) and among residents of rural areas (26.4% [in 2006] to 46.7% [in 2011], p = 0.001). Both highest (32.1% [in 2006] to 49.4% [in 2011], p = 0.036) and lowest (31.7% to 46.0%, p = 0.021) socioeconomic (SE) strata showed an increase in the proportion of smokers who intend to quit. However, in 2011, quit attempts were more frequent (35.3%, p = 0.009) in smokers of high socioeconomic status. Moreover, smoking prevalence has significantly decreased (43.1% [in 2006] to 38.1% [in 2011], p = 0.023), mainly among men (52.4% to 45.7%, p = 0.037), respondents of low socioeconomic status (38.9% to 29.4%, p = 0.008) and residents of urban areas (45.2% to 37.9%, p = 0.005).

Conclusions

Over the past 5 years and possibly as a combined result of the implemented tobacco control policies and austerity measures, the intention to quit smoking has increased among all SE strata, however actual quit attempts were higher among those less disadvantaged. Further effort should be made to support quit attempts, especially among vulnerable populations.     

Genetic variations of CYP2B6 gene were associated with plasma BPDE-Alb adducts and DNA damage levels in coke oven workers

Polycyclic aromatic hydrocarbons (PAHs), the main components of coke oven emissions, can induce activation of cytochrome P450 (CYP) enzymes, which metabolize PAHs and result in DNA damage by forming adducts. This study was designed to know whether genetic variants of CYP genes are associated with plasma benzo[a]pyrene-7,8-diol-9,10-epoxide-albumin (BPDE-Alb) adducts and DNA damage in coke oven workers. In this study, 298 workers were divided into four groups according to the environmental PAHs exposure levels. The concentrations of plasma BPDE-Alb adducts were detected by reverse-phase high-performance liquid chromatography and the DNA damage levels were measured using comet assay. Twelve tag single nucleotide polymorphisms (tagSNPs) of 4 CYP genes were selected and genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the top group, workers with CYP2B6 rs3760657GA genotype have lower BPDE-Alb adducts and DNA damage levels than those with rs3760657GG genotype (P < 0.05). In the control group, the DNA damage levels of subjects with CYP1A1 rs4646421AA or GA + AA genotypes were lower than those with GG genotype (P < 0.05). However, no such effects were shown for the other tagSNPs. These results suggested that genetic variations of CYP2B6 might be associated with low BPDE-Alb adducts and DNA damage levels in worker with high exposure to PAHs.     

Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH₂ substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH₂ metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE₂, thromboxane (TX)B₂, PGF_2α and 6-keto-PGF_1α] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE₂ in human recombinant mPGES-1 with an IC₅₀ of 0.06 μM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE₂ biosynthesis with an IC₅₀ of 0.41 μM. At 1 μM, AF3442 caused maximal selective inhibitory effect of PGE₂ biosynthesis by 61 ± 3.3% (mean ± SEM, P < 0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB₂, PGF_2α and 6-keto-PGF_1α). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE₂ biosynthesis with an IC₅₀ of 29 μM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100 μM (38 ± 14%, P < 0.05, and 69 ± 5%, P < 0.01, respectively). Up to 100 μM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE₂ generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH₂ metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.     

Dimethyl sulfoxide (DMSO) attenuates the inflammatory response in the in vitro intestinal Caco-2 cell model

This study aimed to investigate dose effects of dimethyl sulfoxide (DMSO) (0.05-1%) on the intestinal inflammatory response in confluent- and differentiated-Caco-2 cells stimulated with interleukin (IL)-1β or a pro-inflammatory cocktail for 24 h. Cyclooxygenase-2 (COX-2) activity was assayed by incubating inflamed cells with arachidonic acid and then measuring prostaglandin-E₂ (PGE₂) produced. Soluble mediators (IL-8, IL-6, macrophage chemoattractant protein-1 (MCP-1), and COX-2-derived PGE₂) were quantified by enzyme immunoassays and mRNA expression of 33 proteins by high throughput TaqMan Low Density Array. Data showed that DMSO decreased induced IL-6 and MCP-1 secretions in a dose-dependent manner (P < 0.05), but not IL-8; these effects were cell development- and stimulus- independent. Moreover, in IL-1β-stimulated confluent-cells, DMSO dose-dependently reduced COX-2-derived PGE₂ (P < 0.05). DMSO at 0.5% decreased significantly mRNA levels of 14 proteins involved in the inflammatory response (including IL-6, IL-1α, IL-1β, and COX-2). Thus, DMSO at low concentrations (0.1-0.5%) exhibits anti-inflammatory properties in the in vitro intestinal Caco-2 cell model. This point is important to be taken into account when assessing anti-inflammatory properties of bioactive compounds requiring DMSO as vehicle, such as phenolic compounds, in order to avoid miss-interpretation of the results.     

Ameliorative effect of sesame lignans on nicotine toxicity in rats

Nicotine causes oxidative and genotoxic damages in the tissues leading to several diseases. Any strategy through natural diet that prevents or slows the progression and severity of nicotine toxicity has a significant health impact. This work is designed to investigate natural antioxidants that play effective protective role against nicotine-induced toxicity. Experiments were conducted on male albino rats by injecting nicotine tartrate (3.5 mg/kg body wt./day for 15 days) subcutaneously and thereby supplementing sesame lignans (0.1 g/100 g diet and 0.2 g/100 g diet) orally to them. Significant (P < 0.01) increase of total cholesterol, triglyceride, LDL-cholesterol, VLDL-cholesterol, decrease of HDL-cholesterol, decrease in antioxidant enzymes and increase in concentration of lipid peroxidative product has been observed in plasma due to nicotine toxicity. Significant (P < 0.01) decrease of total DNA contents and highly significant (P < 0.001) DNA damage of liver tissue is also observed on nicotine treatment. Sesame lignans minimizes the above mentioned effects. The nicotine-induced oxidative and genotoxic damages on the tissues can be effectively attenuated by sesame lignans supplemented diet.     

Impact of 7,12-dimethylbenz[a]anthracene exposure on connexin gap junction proteins in cultured rat ovaries

7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles in a concentration-dependent manner. The impact of DMBA on connexin (CX) proteins that mediate communication between follicular cell types along with pro-apoptotic factors p53 and Bax were investigated. Postnatal day (PND) 4 Fisher 344 rat ovaries were cultured for 4 days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and cultured for 4 or 8 days. RT-PCR was performed to quantify Cx37, Cx43, p53 and Bax mRNA level. Western blotting and immunofluorescence staining were performed to determine CX37 or CX43 level and/or localization. Cx37 mRNA and protein increased (P < 0.05) at 4 days of 12.5 nM DMBA exposure. Relative to vehicle control-treated ovaries, mRNA encoding Cx43 decreased (P < 0.05) but CX43 protein increased (P < 0.05) at 4 days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P < 0.05) but no changes in Bax expression were observed after 4 days of DMBA exposures. In contrast, after 8 days, DMBA decreased Cx37 and Cx43 mRNA and protein but increased both p53 and Bax mRNA levels. CX43 protein was located between granulosa cells, while CX37 was located at the oocyte cell surface of all follicle stages. These findings support that DMBA exposure impacts ovarian Cx37 and Cx43 mRNA and protein prior to both observed changes in pro-apoptotic p53 and Bax and follicle loss. It is possible that such interference in follicular cell communication is detrimental to follicle viability, and may play a role in DMBA-induced follicular atresia.     

Potential of long-term dietary administration of rosemary in improving the antioxidant status of rat tissues following carbon tetrachloride intoxication

In this study, 24 Wistar rats were allocated to 4 groups of 6 animals each. Groups 1 and 2 were fed a basal diet, while groups 3 and 4 were fed the basal diet supplemented further with ground rosemary at 1% level. Following 6-weeks feeding, groups 2 and 4 were injected 1 ml CCl₄/kg bw and after six hours all animals were sacrificed. Results showed that feeding rosemary before CCl₄ treatment resulted in decline (P < 0.05) of the increased aspartate transaminase, alanine transaminase and alkaline phosphatase activities and increase (P < 0.05) of the reduced cholesterol and triacylglycerols in serum. It also decreased (P < 0.05) lipid peroxidation and increased (P < 0.05) the reduced hydroxyl anion radical and hydrogen peroxide scavenging activities in serum, liver, kidney and heart tissues. In addition, it increased (P < 0.05) the reduced ABTS radical cation and the superoxide anion scavenging activities in all tissues except in heart and in kidney and heart tissues, respectively. These results suggest that dietary rosemary has the potential to become a promising functional food component.     

Effect of argemone oil and argemone alkaloid,sanguinarine on Sertoli–germ cell coculture

Several incidences of reduction in the fertility (sperm count) have been reported in India and worldwide as well. Adulteration of food and consumption of adulterated mustard oil with argemone oil (AO) are presumed to be the factors for reduction in sperm count. In the present study we have studied the exfoliation of germ cells from Sertoli cell, its viability after detachment, cytotoxicity and execution of apoptosis via mitochondrial pathway for different concentration of AO, argemone alkaloid (AA) and its major constituent sanguinarine (SA). A dose dependent increase in germ cell detachment and decrease in viability of detached germ cells were observed (P < 0.05). A significant inhibition was observed via 3-(4,5-dimethylthiazol-2-yl)-2,5-dipehyl tetrazolium bromide (MTT) assay in the proliferative activity of germ cell and leakage of cytosolic enzyme was observed via Lactate dehydrogenase(LDH) assay (P < 0.05). A time and dose dependent inhibition of mitochondrial membrane potential was observed (P < 0.05). Treatment of Sertoli–germ cells with the lowest concentration of AO/AA and SA for 24 h resulted in 5.2- , 4.4- and 3.6-fold increase in the percentage of early apoptotic cells, respectively. This increase was enhanced to 8.3, 4.75 and 5.81-fold, respectively at 48 h in detached germ cells undergoing early apoptosis. These results suggest that alterations in germ cell apoptosis by a disruption in contact mediated communication between the Sertoli cells and germ cells, may subsequently lead to testicular impairment.     

Biochemical disorders induced by cytotoxic marine natural products in breast cancer cells as revealed by proton NMR spectroscopy-based metabolomics

Marine plants and animals are sources of a huge number of pharmacologically active compounds, some of which exhibit antineoplastic activity of clinical relevance. However the mechanism of action of marine natural products (MNPs) is poorly understood. In this study, proton NMR spectroscopy-based metabolomics was applied to unravel biochemical disorders induced in human MCF7 breast cancer cells by 3 lead candidate anticancer MNPs: ascididemin (Asc), lamellarin-D (Lam-D), and kahalalide F (KF). Asc, Lam-D, and KF provoked a severe decrease in DNA content in MCF7 cells after 24-h treatment. Asc and Lam-D provoked apoptosis, whereas KF induced non-apoptotic cell death. Metabolite profiling revealed major biochemical disorders following treatment. The response of MCF7 tumor cells to Asc involved the accumulation of citrate (×17 the control level, P < 0.001), testifying enzyme blockade in citrate metabolism, and the accumulation of gluconate (×9.8, P < 0.005), a metabolite never reported at such concentration in tumor cells, probably testifying glycolysis shutdown. The response to Lam-D involved the accumulation of aspartate (×7.2, P < 0.05), glutamate (×14.7, P < 0.05), and lactate (×2.3, P < 0.05), probably in relation with the targeting of the malate-aspartate shuttle, as discussed. The response to KF involved increased lipid accumulation (polyunsaturated fatty acids ×9.8, P < 0.05), and phospholipid and acetate derivative alterations. Altogether, this study demonstrates the potential of proton NMR spectroscopy-based metabolomics to help uncover metabolic targets and elucidate the mechanism of cytotoxicity of candidate antineoplastic MNPs.     

Fucoidan, a sulfated polysaccharide from brown algae, against myocardial ischemia-reperfusion injury in rats via regulating the inflammation response

The aim of the study was to determine the effects of fucoidan on rat myocardial ischemia-reperfusion (I/R) model and elucidate the potential mechanisms. Myocardial I/R injury was induced by the occlusion of left anterior descending coronary artery for 30 min followed by reperfusion for 2 h. After 2 h reperfusion, hemodynamics parameters were detected. Blood samples were collected to determine serum levels of tumor necrosis factor-α (TNF-α) and interleukin 6, 10 (IL-6, 10). Hearts were harvested to assess histopathological changes, infarct size (IS), and the content of myeloperoxidase (MPO). The expression of high-mobility group box 1 (HMGB1), phosphor-IκB-α and phosphor-nuclear factor kappa B (NF-κB) were assayed by western blot. Compared with control group, treatment with fucoidan improved left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and the contractility index (P < 0.05, P < 0.01). Fucoidan reduced the myocardial IS, the levels of TNF-α and IL-6, and the activity of MPO (P < 0.05, P < 0.01). Fucoidan down-regulated the expression of HMGB1, phosphor-IκB-α and NF-κB, but increased the content of IL-10 when compared with control (P < 0.05, P < 0.01). Besides, the infiltration of polymorph nuclear leukocytes (PMNs) and histopathological damages in myocardium were decreased in fucoidan treated groups (PMNs, P < 0.05, P < 0.01). These findings revealed that the administration of fucoidan could regulate the inflammation response via HMGB1 and NF-κB inactivation in I/R-induced myocardial damage.     

Anti-glycative and anti-inflammatory effects of protocatechuic acid in brain of mice treated by d-galactose

Protocatechuic acid (PCA) at 0.5%, 1% or 2% was supplied to d-galactose (DG) treated mice for 8 week. PCA intake at 2% increased its deposit in brain. DG treatment increased brain level of reactive oxygen species, protein carbonyl, carboxymethyllysine, pentosidine, sorbitol, fructose and methylglyoxal (P < 0.05). PCA intake, at 1% and 2%, lowered brain level of these parameters (P < 0.05). DG treatments enhanced activity and protein expression of aldose reductase (AR) and sorbitol dehydrogenase, as well as declined glyoxalase I (GLI) activity and protein expression (P < 0.05). PCA intake at 1% and 2% reduced activity and protein expression of AR (P < 0.05), and at 2% restored GLI activity and expression (P < 0.05). DG injection also elevated cyclooxygenase (COX)-2 activity and expression, and increased the release of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha and prostaglandin E₂ in brain (P < 0.05). PCA intake decreased these cytokines (P < 0.05), and at 1% and 2% suppressed COX-2 activity and expression (P < 0.05). PCA intake at 1% and 2% also lowered DG-induced elevation in activity, mRNA expression and protein production of nuclear factor kappa B p65 (P < 0.05). These findings suggest that the supplement of protocatechuic acid might be helpful for the prevention or alleviation of aging.     

The relative importance of CYP26A1 in hepatic clearance of all-trans retinoic acid

All-trans retinoic acid (RA) is a critical signaling molecule and its concentration is tightly regulated. Several P450 enzymes including CYP26A1, CYP2C8, and CYP3A4 have been proposed to be responsible for RA clearance in the liver but their quantitative importance has not been demonstrated. To determine the contribution of CYP26A1 to hepatic clearance of RA, CYP26A1 protein was quantified in 37 human liver microsomes (HLMs). CYP26A1 expression ranged from not detectable to 2.80 pmol/mg microsomal protein. RA clearance by P450 enzymes abundant in human liver was measured in Supersomes^®. CYP2C8, CYP3A4, CYP3A5 and CYP3A7 metabolized RA with unbound K_m values of 3.4-7.2 μM and V_max values of 2.3-4.9 pmol/min/pmol P450, but were less efficient than CYP26A1 in clearing RA. Simulations performed for livers with varying P450 expression levels over a range of RA concentrations demonstrated that at both endogenous and therapeutic concentrations of RA, CYP26A1 is the primary enzyme responsible for 4-OH RA formation clearance. HLM incubation data showed that 4-OH RA formation velocity varied from 0.2 to 15.3 pmol/min/mg microsomal protein and velocity in HLMs was significantly correlated (p < 0.01) to CYP26A1, CYP3A4, and CYP3A5 protein content, but not to CYP2C8. When experimental data were scaled to in vivo clearances, the predicted hepatic clearance of RA (0.07 L/min using combined Supersome^® data) was similar to the published in vivo clearance of RA. These findings suggest that CYP26A1 is the P450 isoform that should be targeted when designing RA metabolism blocking agents.     

Chain-dependent photocytotoxicity of tricationic porphyrin conjugates and related mechanisms of cell death in proliferating human skin keratinocytes

Photodynamic therapy (PDT) is a poor treatment option for nodular basal cell carcinomas and squamous cell carcinomas. As a result, the search for new photosensitizers with better effectiveness is of current interest. The photocytotoxicity of conjugates (P-R) of a water-soluble tri-cationic porphyrin (P-H) having similar efficiency of production of singlet oxygen, the PDT cytotoxin, has been assessed in vitro. Links between uptake, intracellular localization, photooxidative stress, photocytotoxicity and ability to induce programmed cell death are established. Conjugates bearing methyl (P-Me), Di-O-isopropylidene-(-d-galactopyranosyl (P-OGal) or N,N′-dicyclohexylureidooxycarbonyl (P-DDC) chains are efficiently taken-up by proliferating NCTC 2544 keratinocytes. The relative order of photocytotoxicity is P-OGal >P-DDC = P-Me ? P-H. The photocytotoxic potential of P-Me, P-OGal and P-DDC equals that of endogenous protoporphyrin IX induced by δ-aminolevulinic acid or its esters, the pro-drugs currently employed for PDT of skin lesions. Microfluorometry shows that P-Me, P-OGal, and P-DDC localize in endocytotic or pinocytotic vesicles but not in mitochondria or nucleus. Absence of annexin V binding, caspase activation or chromatin condensation suggests that cell photosensitization by P-R does not induce apoptosis. On the other hand, P-OGal photocytotoxicity correlates with appearance of multiple vesicles that have hallmarks of autophagy compartments, being decorated with the marker LC3 in cells transfected with an expression vector encoding GFP-LC3. p38 and JNK phosphorylation and inhibition of ERK1/2 phosphorylation suggest close relationship between mortality of NCTC 2544 keratinocytes and MAPK pathway impairment. Given their potentially easy formulation, water-soluble P-R are promising powerful photosensitizers for PDT of skin lesions.     

Synthesis and pharmacological characterization of [I]MRS5127, a high affinity, selective agonist radioligand for the A3 adenosine receptor

A recently reported selective agonist of the human A₃ adenosine receptor (hA₃AR), MRS5127 (1′R,2′R,3′S,4′R,5′S)-4′-[2-chloro-6-(3-iodobenzylamino)-purine]-2′,3′-O-dihydroxy-bicyclo-[3.1.0]hexane, was radioiodinated and characterized pharmacologically. It contains a rigid bicyclic ring system in place of a 5′-truncated ribose moiety, and was selected for radiolabeling due to its nanomolar binding affinity at both human and rat A₃ARs. The radioiodination of the N⁶-3-iodobenzyl substituent by iododestannylation of a 3-(trimethylstannyl)benzyl precursor was achieved in 73% yield, measured after purification by HPLC. [¹²⁵I]MRS5127 bound to the human A₃AR expressed in membranes of stably transfected HEK 293 cells. Specific binding was saturable, competitive, and followed a one-site binding model, with a K_d value of 5.74 ± 0.97 nM. At a concentration equivalent to its K_d, non-specific binding comprised 27 ± 2% of total binding. In kinetic studies, [¹²⁵I]MRS5127 rapidly associated with the hA₃AR (t_1/2 = 0.514 ± 0.014 min), and the affinity calculated from association and dissociation rate constants was 3.50 ± 1.46 nM. The pharmacological profile of ligands in competition experiments with [¹²⁵I]MRS5127 was consistent with the known structure-activity-relationship profile of the hA₃AR. [¹²⁵I]MRS5127 bound with similar high affinity (K_d, nM) to recombinant A₃ARs from mouse (4.90 ± 0.77), rabbit (2.53 ± 0.11), and dog (3.35 ± 0.54). For all of the species tested, MRS5127 exhibited A₃AR agonist activity based on negative coupling to cAMP production. Thus, [¹²⁵I]MRS5127 represents a new species-independent agonist radioligand for the A₃AR. The major advantage of [¹²⁵I]MRS5127 compared with previously used A₃AR radioligands is its high affinity, low degree of non-specific binding, and improved A₃AR selectivity.