Chemical characteristics for optimizing CYP2E1 inhibition.

Cytochrome P450 2E1 (CYP2E1) expression and activity in the liver is associated with the degree of liver damage in patients with alcoholic steatohepatitis (ASH) as well as non-alcoholic steatohepatitis (NASH). CYP2E1 is known to generate reactive oxygen species, which leads to oxidative stress, one of the hallmarks of both diseases. Apart from ROS, toxic metabolites can be formed by CYP2E1 metabolism, further potentiating liver injury. Therefore, CYP2E1 is implicated in the pathogenesis of ASH and NASH. The aim of this study was to determine the chemical characteristics of compounds that are important to inhibit CYP2E1. To this end, structurally related analogs that differed in their lipophilic, steric and electronic properties were tested. In addition, homologues series of aliphatic primary alcohols, secondary alcohols, aldehydes, ketones and carboxylic acids were tested. It was found that inhibition of the CYP2E1 activity is primarily governed by lipophilicity. The optimal log D7.4 (octanol/water distribution coefficient at pH 7.4) value for inhibition of CYP2E1 was approximately 2.4. In the carboxylic acids series the interaction of the carboxylate group with polar residues lining the CYP2E1 active site also has to be considered. This study sketches the basic prerequisites in the search for inhibitors of CYP2E1, which would strengthen our therapeutic armamentarium against CYP2E1 associated diseases, such as ASH and NASH.

The olive oil antioxidant hydroxytyrosol efficiently protects against the oxidative stress-induced impairment of the NObullet response of isolated rat aorta.

The Mediterranean diet, which is abundant in antioxidants, is associated with a relatively low incidence of coronary heart disease. Olive oil and olives, which contain the antioxidants hydroxytyrosol, oleuropein, and tyrosol, are important components of this diet. In this study, the effects of oxidative stress on the nitric oxide radical (NO(*))-mediated relaxation of rat aorta and the protection by these antioxidants were determined. Cumene hydroperoxide (CHP) was used to mimic oxidative stress induced by lipid hydroperoxides, which is mediated by the formation of hydroxyl radicals (OH(*)). CHP (300 microM) impaired the NO(*)-mediated relaxation of rat aorta by the acetylcholine receptor agonist carbachol (P < 0.05). This was due to a reduction in NO(*) production. A diminished NO(*)-mediated relaxation disturbs the vascular tone and leads to a rise in blood pressure, which is a well-established risk factor for coronary heart disease. Hydroxytyrosol (10 microM) efficiently protected the aorta against the CHP-induced impairment of the NO(*)-mediated relaxation (P < 0.05). Oleuropein, tyrosol, and homovanillic alcohol, a major metabolite of hydroxytyrosol, did not show protection. Moreover, hydroxytyrosol was found to be a potent OH(*) scavenger, which can be attributed to its catechol moiety. Because of its amphiphilic characteristics (octanol-water partitioning coefficient = 1.1), hydroxytyrosol will readily cross membranes and provide protection in the cytosol and membranes, including the water-lipid interface. The present study provides a molecular basis for the contribution of hydroxytyrosol to the benefits of the Mediterranean diet.

Protectors against doxorubicin-induced cardiotoxicity: flavonoids.

Doxorubicin is a widely used anthracycline anticancer agent. Its use may cause cardiomyopathy: in fact, the development of cumulative dose-related cardiotoxicity forms the major limitation of clinical doxorubicin use. We therefore searched for protective agents that combine iron-chelating and oxygen radical-scavenging properties. Moreover, any novel protector should not interfere with the cytostatic activity of doxorubicin. After extensive in vitro screening we found that flavonoids could serve this purpose. In particular 7-monohydroxyethylrutoside almost completely protected against the negative inotropic action of doxorubicin in the electrically paced mouse left atrium model. In vivo it gave full protection at 500 mg/kg intraperitoneally against the doxorubicin-induced ST-interval lengthening in the ECG. Moreover, this protector did not influence the antitumor effect of doxorubicin either in vitro using the human ovarian cell lines A2780 and OVCAR-3 and the human breast cancer cell line MCF-7 or in vivo in A2780 and OVCAR-3 subcutaneous xenografts in nude mice. Comparison of various iron chelators suggest that iron, in contrast to the general assumption, might not play a crucial role in the oxidative stress-induced toxicity of doxorubicin. Moreover, incubation of vascular endothelial cells with doxorubicin produced overexpression of adhesion molecules, which could be inhibited by 7-monohydroxyethylrutoside. From a study in human volunteers, we conclude that an intravenous dose of 1500 mg/m(2) of 7-monohydroxyethylrutoside is feasible and is safe to be investigated as protection against doxorubicin-induced cardiotoxicity.

Analysis of oxidative DNA damage after human dietary supplementation with linoleic acid.

It has been hypothesized that oxygen radicals generated by peroxidation of dietary linoleic acid may induce genetic damage and thereby increase cancer risk. We examined the effect of dietary supplementation with linoleic acid on the levels of oxidative DNA damage in peripheral lymphocytes and on the blood plasma antioxidant potential. Thirty volunteers received during 6 weeks either a high dose of linoleic acid (15 g/day), an intermediate dose of linoleic acid (7.5 g/day) or an isocaloric supplement without linoleic acid (15 g palmitic acid/day). After the intervention, no significant increase in oxidative DNA damage, measured as relative amounts of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) in DNA from peripheral lymphocytes, was observed in both high and intermediate linoleic acid-supplemented groups (increase of respectively 13 and 21%; P>0.05). Also, the differences between levels of oxidative DNA damage in the high or intermediate linoleic acid-supplemented group and the control group receiving palmitic acid (23% decrease) were not significant. Furthermore, no statistically significant differences were found between the total antioxidant capacities of blood plasma from the different experimental groups. Plasma levels of malondialdehyde, an important end-product of lipid peroxidation, were not increased after supplementation, nor were effects found on the plasma concentrations of retinol, alpha-tocopherol and beta-carotene. Despite the experimental design that excludes several forms of bias introduced in studies based on modulation of dietary composition, our results provide no indication of increased oxidative stress or genetic damage as a result of increased dietary intake of linoleic acid. Therefore, we see no scientific basis to reconsider the public health policy to stimulate the intake of polyunsaturated fatty acids aimed at the reduction of coronary heart diseases.

The use of vitamin supplements in self-medication.

Supplements, frequently containing vitamins, are often taken to try to compensate for an unhealthy life style or in an attempt to maximise health or performance. As illustrated by vitamin E, fundamental issues on both the efficacy and safety of the chronic use of relatively high dosages of vitamins still need to be resolved. To recommend the application of vitamin supplements, firstly the gaps in our knowledge on these fundamental issues should be filled. Secondly, objective and scientifically sound information should be given to the public. Via diverse sources there is an uncontrolled supply of vitamins. Educating the consumer on vitamins is therefore the only way to come to a rational use of vitamin supplements in self medication.

Protection against nitric oxide toxicity by tea.

It is found that green tea and black tea are able to protect against nitric oxide (NO(*)) toxicity in several ways. Both green tea and black tea scavenge NO(*) and peroxynitrite, inhibit the excessive production of NO(*) by the inducible form of nitric oxide synthase (iNOS), and suppress the LPS-mediated induction of iNOS. The NO(*) scavenging activity of tea was less than that of red wine. The high activity found in the polyphenol fraction of black tea (BTP) could not be explained by the mixed theaflavin fraction (MTF) or catechins [epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate (EGCG)], which were tested separately. Synergistic effects between the compounds, or the presence of a potent, unidentified NO(*) scavenger, may explain the high activity of BTP. The peroxynitrite scavenging of tea was comparable to that of red wine. The main activity was found in the polyphenol fraction. MTF and the catechins were found to be potent peroxynitrite scavengers. Tea and tea components were effective inhibitors of iNOS. Of the tea components tested, only MTF had an activity higher than that of the tea powders. The polyphenol fractions of tea were much more active than the tea powders in suppressing the induction of iNOS. On the basis of its abundance and activity, EGCG was the most active inhibitor. The protective effect of tea on NO(*) toxicity is discussed in relation to the beneficial effect of flavonoid intake on the occurrence of cardiovascular heart disease.

Thiazoloindans and thiazolobenzopyrans: a novel class of orally active central dopamine (partial) agonists.

The 2-aminothiazole moiety has proven its value in medicinal chemistry as a stable and lipophilic bioisosteric replacement of a phenol group. This approach has provided dopamine (DA) agonists with good oral availability. To further explore its use in the development of DA agonists, we have combined the 2-aminothiazole moiety with 2-aminoindans and 3-aminobenzopyrans, which are known templates for DA agonists. In this study we have synthesized 6-amino-3-(N,N-di-n-propylamino)-3,4-dihydro-2H-thiazolo[5, 4-f]-[1]benzopyran (12) and 6-amino-2-(N, N-di-n-propylamino)thiazolo[4,5-f]indan (20) and several analogues (13, 17, and 21). The affinity of the thiazolobenzopyrans and thiazoloindans for DA receptors was evaluated, which revealed compound 20 to have high affinity for DA D(3) receptors. In addition, the compounds were screened for their potential to inhibit lipid peroxidation, to determine their radical scavenging properties. Compounds 12, 20, and 21 were subjected to further pharmacological evaluation in a functional assay to determine intrinsic activity. Compound 20 was also studied with microdialysis (to determine effects on DA turnover in striatum) and in unilaterally 6-OH-DA lesioned rats (to determine their potential as DA agonists). These studies selected compound 20 (GMC 1111) as particularly interesting. Compound 20 caused a rotation activation in unilaterally 6-OH-DA lesioned rats and an increase in DA turnover in rat striatum. This dual agonist/antagonist action is best accounted for by its partial agonism at striatal DA D(2) receptors. Interestingly, 20 displayed long-lasting activity and excellent oral availability in 6-OH-DA lesioned rats, making this compound potentially useful for the treatment of Parkinson's disease.

In vitro screening of antitumour agents for cardiotoxicity by means of isolated mouse left atria.

Cardiotoxicity, a side-effect that can occur after treatment with an anticancer drug, has severe clinical implications. Therefore, a model is desired to screen new anticancer drugs or drug combinations for possible cardiotoxic side-effects. In the present study we tested the applicability of the electrically stimulated isolated mouse left atrium model using a wide range of anticancer drugs with known cardiotoxicity. It appeared that the cardiotoxicity observed in our model, i.e. the negative or positive inotropic effects of the drugs on the isolated atrium, corresponded with the observed cardiotoxicity in animals and/or humans. It is therefore concluded that our model can be used to wam for possible cardiotoxic side-effects of anticancer drugs in vivo.

Antioxidant effects of carotenoids.

Surprisingly, neither the precise pharmacological effect nor the toxicological profile is usually established for food components. Carotenoids are no exception in this regard. Only limited insight into the pharmacology and toxicology of carotenoids exists. It is known that the antioxidant action of carotenoids is determined by 1. electron transfer reactions and the stability of the antioxidant free radical, 2. the interplay with other antioxidants and 3. the reaction with active oxygen. Numerous metabolites of carotenoids are formed upon their action as an antioxidant. Most of these metabolites have an unknown biological activity. It is concluded that a severe lack of knowledge hampers adequate suggestions for human supplementation.

Mechanism of the reaction of ebselen with endogenous thiols: dihydrolipoate is a better cofactor than glutathione in the peroxidase activity of ebselen.

The therapeutic effect of ebselen has been linked to its peroxidase activity. In the present study, the peroxidase activity of ebselen toward H2O2 with the endogenous thiols GSH and dihydrolipoate [L(SH)2] as cofactors was determined. When GSH was used, peroxide removal was described by a ter uni ping pong mechanism with Dalziel coefficients for GSH and H2O2 of 0.165 +/- 0.011 and 0.081 +/- 0.005 mM min, respectively. When L(SH)2 was used, peroxidase activity was independent of the concentration of L(SH)2 in the concentration range studied (5 microM to 2 mM) and peroxide removal was only dependent on the concentration of H2O2 and ebselen, with the second-order rate constant being 12.3 +/- 0.8 mM-1 min-1. To elucidate the difference between GSH and L(SH)2, the molecular mechanism of the peroxidase activity of ebselen was investigated, using UV spectrophotometry, high pressure liquid chromatography, 77Se NMR, and mass spectrometry. GSH was found to react quickly with ebselen to give a selenenyl sulfide, an adduct of GSH to ebselen. Subsequently, the GSH-selenenyl sulfide is converted into the diselenide of ebselen. Finally the diselenide reacts with a peroxide and ebselen is regenerated. The formation by GSH of the diselenide from the GSH-selenenyl sulfide of ebselen is slow and linearly dependent on the concentration of free thiol; however, no net consumption of GSH was observed. Furthermore, it is likely that a selenol is an intermediate in diselenide formation. After reaction between ebselen and L(SH)2 the diselenide of ebselen was immediately detected. The fast formation of the diselenide with L(SH)2 versus the slow formation of the diselenide with GSH accounts for our observation that L(SH)2 is a better cofactor than GSH in the peroxidase activity of ebselen. Our results suggest that the interaction between ebselen and L(SH)2 might be of major importance in the mechanism by which ebselen exerts its therapeutic effect.