Seasonal variations in road runoff quality in Luleå, Sweden.

In regions with cold climate the urban drainage and highway runoff processes become much more complex, compared to temperate regions. Therefore, climatic conditions should be taken into account in planning and design of BMPs and snow handling strategies. In order to increase the knowledge of road runoff quality during melt and rain periods, respectively, measurements were carried out at a field site during a two-month period. The field site was situated at Södra Hamnleden, a road with 7,400 vehicles/day, in the central part of Luleå. Runoff samples were analysed for suspended solids and heavy metals (Pb, Cu, Cd, Ni and Zn). The results showed that the concentrations of suspended solids, lead, copper and cadmium were higher for the melt period, compared to rain generated runoff on the catchment without snow, and the highest concentrations were found during the rain-on-snow events. The results indicate a flow dependent increase in the concentration of suspended solids during the melt period. A comparison of the total mass of suspended solids over a one-month period showed that the melt period produced about 3 times more suspended solids. Metal elements during melt period were more particulate bound as compared to the rain period characterised by a higher percentage of the dissolved fraction.

N,N'-diacetyl-L-cystine (DiNAC), the disulphide dimer of N-acetylcysteine, inhibits atherosclerosis in WHHL rabbits: evidence for immunomodulatory agents as a new approach to prevent atherosclerosis.

Oxidation of lipoprotein-derived lipids is generally accepted to be important in atherogenesis, and lipophilic antioxidants have been suggested as potential antiatherosclerotic agents. The antiatherogenic effects observed by certain antioxidants, especially probucol, in different animal models support this suggestion. There are however also cases where other lipophilic antioxidants have not been able to support this hypothesis. This has raised the question whether the effects of probucol and similar compounds are mainly due to some other property, unrelated to their antioxidant efficacy. For example, probucol is shown to possess immunomodulatory properties. Immune reactions are known to occur during atherogenesis. We therefore tested the dimer of N-acetylcysteine, DiNAC, which is a disulfide with immunomodulating properties and enhances oxazolone-induced contact sensitivity (CS) reactions in mice, for effects on atherosclerosis. When given to male heritable hyperlipidemic rabbit (WHHL) rabbits from 10 to 22 weeks of age, this compound reduced by 50% thoracic aorta atherosclerosis (p < 0.05), without affecting plasma lipid levels. Here we also show that probucol and a close chemical analog, both known to prevent atherosclerosis in WHHL rabbits, enhance the CS reaction in mice, while two other related antioxidants did not affect the CS reaction. At least one of these is also without effect on atherosclerosis in WHHL rabbits. The results show that DiNAC might represent a new treatment modality for atherosclerosis-related disease, and suggest that some antioxidants may have antiatherosclerotic properties more related to "immunomodulatory" properties than to antioxidant properties in general.

Dissociation of atherogenesis from aortic accumulation of lipid hydro(pero)xides in Watanabe heritable hyperlipidemic rabbits.

Antioxidants can inhibit atherosclerosis, but it is unclear how inhibition of intimal lipid oxidation relates to atherogenesis. Here we tested the effect of probucol and its metabolite bisphenol on aortic lipid (per)oxidation and atherogenesis in Watanabe heritable hyperlipidemic (WHHL) rabbits. LDL and aortas from rabbits fed probucol contained bisphenol at concentrations comparable to those in bisphenol-treated animals. Bisphenol treatment increased plasma cholesterol slightly, and plasma and aortic alpha-tocopherol more substantially; these parameters were unaffected by probucol. Bisphenol and probucol treatment both enhanced the resistance of circulating LDL to peroxyl radical-induced lipid peroxidation; this was due to bisphenol, not probucol. Only probucol enhanced LDL's resistance to Cu(2+)-induced oxidation. Both bisphenol and probucol treatment strongly inhibited aortic accumulation of hydroperoxides and hydroxides of cholesteryl esters and triglycerides [LO(O)H]. Despite this, however, probucol had a modestly significant effect on the extent of lesion formation; bisphenol had no inhibitory effect. In addition, the extent of atherosclerosis did not correlate with amounts of aortic LO(O)H present, but, as expected, it did correlate with aortic alpha-tocopherol and cholesterol. Together, these results suggest that aortic accumulation of LO(O)H is not required for, nor is alpha-tocopherol depleted during, the initiation and progression of atherogenesis in WHHL rabbits.

Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice.

Antioxidants can inhibit atherosclerosis in animals, though it is not clear whether this is due to the inhibition of aortic lipoprotein lipid (per)oxidation. Coantioxidants inhibit radical-induced, tocopherol-mediated peroxidation of lipids in lipoproteins through elimination of tocopheroxyl radical. Here we tested the effect of the bisphenolic probucol metabolite and coantioxidant H 212/43 on atherogenesis in apolipoprotein E and low density lipoprotein (LDL) receptor gene double knockout (apoE-/-;LDLr-/-) mice, and how this related to aortic lipid (per)oxidation measured by specific HPLC analyses. Dietary supplementation with H 212/43 resulted in circulating drug levels of approximately 200 microM, increased plasma total cholesterol slightly and decreased plasma and aortic alpha-tocopherol significantly relative to age-matched control mice. Treatment with H 212/43 increased the antioxidant capacity of plasma, as indicated by prolonged inhibition of peroxyl radical-induced, ex vivo lipid peroxidation. Aortic tissue from control apoE-/-;LDLr-/- mice contained lipid hydro(pero)xides and substantial atherosclerotic lesions, both of which were decreased strongly by supplementation of the animals with H 212/43. The results show that a coantioxidant effectively inhibits in vivo lipid peroxidation and atherosclerosis in apoE-/-;LDLr-/- mice, consistent with though not proving a causal relationship between aortic lipoprotein lipid oxidation and atherosclerosis in this model of the disease.

Synthesis and biological activity of potent, low molecular weight renin inhibitors.

A series of renin inhibitors containing the dipeptide transition state mimics (2R,4S,5S)-5-amino-4-hydroxy-2-methyl-6-cyclohexyl hexanoic acid (Cha-psi[CH(OH)CH2]Ala) and (2R,4S,5S)-5-amino-4-hydroxy-2-isopropyl-6-cyclohexyl hexanoic acid (Cha-psi[CH(OH)CH2]Val) were prepared. A structure-activity study, using pseudopeptide (Boc-Phe-His-Leu-psi[CH(OH)CH2]Val-Ile-His-OH) as our lead structure, led to a new series of inhibitors, which correspond to tripeptides and contain no natural amino acids. For example, R,S-Bpma-Ape-Cha-psi[CH(OH)CH2]Ala-NH2 (IC50 = 1.26 nM against human plasma renin at pH 6.0; molecular weight = 564) has only two thirds of the molecular weight but twice the potency of our original lead. This new class of low molecular weight renin inhibitor displays excellent specificity toward human renin versus the related aspartic proteinase pepsin and angiotensin-1-converting enzyme. Examples are given of selected inhibitors showing encouraging evidence for intestinal absorption after intracolonic and oral administration in male Sprague-Dawley rats.

Design, synthesis, and biological activities of four angiotensin II receptor ligands with gamma-turn mimetics replacing amino acid residues 3-5.

Disulfide cyclization is a powerful method for reducing the conformational space of a peptide. This in turn may enable the study of its bioactive conformation. Several analogues of angiotensin II (Ang II) containing a disulfide bridge between amino acids 3 and 5 have been reported. Among these the cyclic octapeptides c[Hcy3,5]-Ang II, c[Cys3,5]-Ang II, and c[Pen 3,5]-Ang II showed significant activity at Ang II receptors. We have performed conformational analysis studies using theoretical calculations and 1H-NMR spectroscopy on tripeptide model compounds of these cyclic octapeptides which show that the cyclic moieties of c[Cys3,5]-Ang II and c[Pen3,5]-Ang II preferentially assume an inverse gamma-turn conformation. On the basis of these results, we substituted amino acid residues 3-5 in Ang II with two different gamma-turn mimetics giving four diastereomeric Ang II analogues. Interestingly, two of these are equipotent to Ang II in binding to AT1 receptors. In the contractile test using rabbit aorta rings, one of the analogues is an agonist with full contractile activity approximately equipotent to c[Pen3,5]-Ang II but 300-fold less potent than Ang II. This low potency may suggest that Ang II does not adopt a gamma-turn in the 3-5 region when interacting with the receptor.

Characterization of novel indenoindoles. Part I. Structure-activity relationships in different model systems of lipid peroxidation.

Structure-activity relationships are presented for some representative compounds from a novel series of potent inhibitors of lipid peroxidation. The compounds are indenoindole derivatives with oxidation potentials in organic solvents of between 0.2 and 1.5 V. Two of these compounds, cis-5,5a,6,10b-tetrahydro-9-methoxy-7-methylindeno[2,1-b]indole (H 290/51) with an oxidation potential of 0.32 V and cis-4b,5,9b,10- tetrahydro-8-methoxy-6-methylindeno[1,2-b]indole (H 290/30) with an oxidation potential of 0.30 V, have been tested more extensively and compared with reference compounds in several pharmacological models of lipid peroxidation. The inhibitory potencies (pIC50) of the compounds in respect to Fe/Ascorbate-induced production of thiobarbituric acid-reactive substances (TBARS) in a suspension of purified soybean lecithin were calculated. These data are 8.2 for H 290/51; 8.0 for H 290/30; 5.6 for vitamin E; and 6.6 for butylated hydroxytoluene (BHT). In isolated rat renal tissue subjected to hypoxia and reoxygenation, the potency for inhibition of TBARS formation is 6.9 for H 290/51, 6.9 for H 290/30, and <5 for vitamin E. In oxidative modification of low-density lipoproteins (LDL) induced by mouse peritoneal macrophages, the corresponding pIC50 values for TBARS inhibition for each compound are: 8.7, 8.3, <5, and 6.9, respectively. It is concluded that the synthetic indenoindoles are potent antioxidants. The results suggest that indenoindoles such as H 290/51 and H 290/30 could be useful as therapeutic agents in pathophysiological situations where lipid peroxidation plays an important role.

Characterisation of novel indenoindoles. Part II. Redox-recycling with ascorbate.

In the accompanying paper it was shown that the new antioxidant, H 290/51 (cis-5,5a,6,10b-tetrahydro-9-methoxy-7-methylindenol[2,1-b]indole) , is a powerful antioxidant in several pharmacological models of lipid peroxidation and could be useful as a therapeutic agent in pathophysiological situations where lipid peroxidation plays an important role. In the present study, we characterised H 290/51 as an inhibitor of peroxidation of pure methyl linoleate. H 290/51 almost completely inhibited peroxidation induced by a lipid-soluble initiator at 37 degrees C during the induction period, both in an aqueous solution of micelles in the presence of detergents and in a homogeneous ethanol solution. In both systems, the time of the induction period was linearly related to the concentration of H 290/51. In the ethanol solution, ascorbic acid had a sparing effect on H 290/51, indicating effective interference with radical chain propagation. In aqueous solution with micelles of methyl linoleate made with the nonionic detergents Triton X-100 or Lubrol PX, ascorbic acid did not inhibit peroxidation. However, in these micelles, H 290/51 showed a concentration-dependent extension of the induction period by ascorbic acid, suggesting recycling. In the presence of the zwitterionic detergent CHAPS, although a clear induction period is seen with H 290/51, no recycling by ascorbic acid was found. The ability of H 290/51 to recycle in aqueous solutions, thus depends on the micellar composition.

A rapid and simple screening test for potential inhibitors of tocopherol-mediated peroxidation of LDL lipids.

We report a rapid and convenient method for screening potential inhibitors of the initiation of low density lipoprotein (LDL) lipid peroxidation. The method uses positively and negatively charged micelles of either cetyltrimethyl ammonium chloride or sodium dodecyl sulfate with added alpha-tocopherol. It is based on the capacity of an antioxidant to attenuate alpha-tocopheroxyl radicals, generated by irradiation of the alpha-tocopherol-containing micelles with UV light, and measured directly by electron spin resonance spectroscopy. To establish the reliability of the method, we compared the alpha-to-copheroxyl radical attenuating ability (TRAA) of 53 natural and synthetic potential antioxidants with their respective ability to inhibit the early stages of LDL lipid peroxidation initiated by a low flux of water-soluble peroxyl radicals. The relationship between the measured TRAA and corresponding LDL antioxidation activity was highly significant (P < 0.00005, Rank test). Thus, the potency of a co-antioxidant for LDLs alpha-tocopherol could be predicted with > 98% probability by the TRAA test alone. The results suggest that this relatively simple method represents an effective and simple screening test that can be used for large numbers of potential inhibitors of the early stages of LDL lipid oxidation.

Lipoprotein fractionation in deuterium oxide gradients: a procedure for evaluation of antioxidant binding and susceptibility to oxidation.

Oxidative modifications of lipoproteins appear to contribute to their atherogenicity. Very low and low density lipoproteins (VLDL and LDL) are protected against these modifications by antioxidants that can be incorporated in vivo or in vitro into the particles. We describe here ultracentrifugal procedures for isolation of VLDL and LDL that do not require subsequent dialysis or buffer equilibration. Lipoproteins were isolated in buffers with physiological ionic composition prepared in D2O (deuterium oxide). This allowed measurements of the content of antioxidants and of the susceptibility to oxidation of the isolated LDL without further manipulations. Conventional ultracentrifugal methods use high salt concentrations and require additional steps to eliminate them. This introduces uncertainties in the evaluation of antioxidant binding and on measurements of their effect on VLDL and LDL oxidation. With the method described, the composition of the isolated VLDL and LDL was indistinguishable from that of fractions isolated with KBr gradients. Also, the content of alpha-tocopherol was similar. LDL isolated with KBr solutions appeared to bind 20-45% more of the probucol present in serum than LDL isolated in isotonic solutions prepared with D2O. This was the case with probucol incorporated into plasma or serum in vivo or in vitro. Five out of seven LDL isolated with the D2O procedure from different human sera appeared more resistant to Cu(2+)-catalyzed oxidation than those obtained with KBr gradients from the same serum. In addition to the gradient procedure, we also describe a preparative version of the method that can be used with multiple samples.

Molecular basis of the association of arterial proteoglycans with low density lipoproteins: its effect on the structure of the lipoprotein particle.

Modifications of low density lipoproteins (LDL) that enter the arterial intima appear to be responsible for their eventual extracellular and intracellular accumulation during atherogenesis. Some of these modifications seem to be the result of LDL association with intimal chondroitin sulphate-rich proteoglycans (CSPG). We have used frontal elution affinity chromatography, binding and competition experiments with synthetic segments of apoB-100 to better define the ligand regions for the LDL-CSPG complexes. The minimum structural requirement for recognition by the CSPG appears to be a hydrophilic nine-residue amino-acid segment with five lysine and arginine residues. Analysis of other similar regions in apoB-100 and other glycosaminoglycan-binding proteins suggest that besides a cluster of positively charged amino-acids, the presence of hydroxyl-containing residues favours the association with sulphated proteoglycans. With controlled proteolytic hydrolysis, we found that the interaction of LDL with CSPG modifies the surface accessibility of a apoB-100 segments containing arginine and lysine. Because these apoB-100 domains may also be involved in cell-receptor binding, the CSPG-induced modifications could be the structural explanation for the observed increase in cellular uptake of proteoglycan-modified LDL.

A 62 kDa protein is photoaffinity labelled by [3H]felodipine in vascular smooth muscle, but not in cardiac and skeletal muscle.

Irradiation of a cytosolic fraction from vascular smooth muscle in the presence of [3H]felodipine resulted in the labelling of a protein with an apparent molecular weight of 62 kDa. The labelling was seen on UV-irradiation at 360 nm, but not at 254, 278 or at wavelengths above 410 nm. The photolabelling was enhanced in the absence of oxygen. In cytosolic fractions prepared from porcine liver, cardiac and skeletal muscle no photoaffinity labelling of proteins between 90 and 45 kDa could be demonstrated. The results suggest that felodipine is a photoaffinity ligand and that felodipine binds to a soluble protein present in vascular smooth muscle but not in the other tissues tested.

Binding of a dihydropyridine felodipine-analogue to calmodulin and related calcium-binding proteins.

A dihydropyridine-affinity column was prepared by coupling a physiologically active and vasoselective amino-derivative of felodipine to divinylsulfone-activated Trisacryl GF2000. Calmodulin (CaM) as well as the homologous calcium-binding proteins skeletal and cardiac Troponin C (sTnC and cTnC) and S100b bound to this resin in a calcium-dependent manner. In contrast, other homologous proteins such as parvalbumin and the intestinal calcium-binding protein did not bind. Competition studies showed that CaM had a higher affinity for the felodipine-column than sTnC or cTnC. Through studies with a series of proteolytic fragments of CaM and sTnC, it was found that the felodipine binding site is located in the amino-terminal domain of the protein. These results illustrate the utility of affinity-chromatography for the study of dihydropyridine-binding proteins.

Felodipine analogs: structure-activity relationships.

Several 3,5-pyridinedicarboxylic acid [4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-] esters, which are analogs to felodipine, were synthesized and tested for peroral activity and vascular selectivity. Structure-activity relationships demonstrate that felodipine has biological properties (selectivity and oral activity) close to the optimum for this class of compounds.

Photoaffinity labeling of felodipine-binding proteins in vascular smooth muscle.

[3H]-Felodipine and high-intensity ultraviolet irradiation were used in the photoaffinity labeling of soluble proteins prepared from porcine mesenteric vascular smooth muscle. Irradiation of the soluble proteins in the presence of [3H]-felodipine resulted in the labeling of a protein with an apparent molecular weight of 62 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Labeling of the protein did not occur without ultraviolet irradiation. An [3H]-azido analog of felodipine was found to show less specificity than felodipine in its protein labeling when irradiated, since proteins with apparent molecular weights of 44, 29, and 14, as well as 62 kDa, were labeled. The photoaffinity labeling of the proteins were inhibited by excess of unlabeled felodipine.

Quantitative relationships between structure and microsomal oxidation rate of 1,4-dihydropyridines.

The oxidation rate of a series of 4-phenyl substituted 1,4-dihydropyridine esters was investigated in dog liver microsomes and was evaluated in relation to physiochemical properties as well as to antihypertensive effect. The relative rate of microsomal oxidation was mainly dependent on the substituents of the aromatic ring and almost unaffected by small changes in the ester substituents. A 20-fold variation in the microsomal oxidation rate was observed within a group of dichloro-substituted analogues. Generally, the highest oxidation rate was found with 2',6'-disubstituted derivatives, while compounds with substituents in position 4' exhibited longer half-lives. The oxidation rate increased with increased steric bulk, increased lipophilicity and increased electron withdrawal of the substituents in position 2'. The energies of the highest occupied molecular orbital (HOMO) were calculated and correlated with the oxidation rate of some of the dihydropyridine analogues. The antihypertensive effect appeared to be restricted to compounds with oxidation rates within a narrow range, indicating the unlikelihood of increasing the duration of the pharmacologic effect by stabilisation of the dihydropyridine system.

The enantiomers of the dihydropyridine derivative H 160/51 show opposite effects of stimulation and inhibition.

H 160/51 is a calcium agonistic 1,4-dihydropyridine. Its optical isomers were now found to have opposing actions in the cat papillary muscle and rat portal vein. The inhibitory (+)-enantiomer had potencies of the same order as those of the stimulatory (-)-enantiomer. However the racemate and the (-)-enantiomer had approximately the same potencies and intrinsic activities. A model is presented to account for the fact that the effects of the inhibitory (+)-enantiomer can be concealed in the effects of the racemate of H 160/51.