Nitrophenols suppress steroidogenesis in prehierarchical chicken ovarian follicles by targeting STAR, HSD3B1, and CYP19A1 and downregulating LH and estrogen receptor expression.

To assess the effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on steroidogenesis in the chicken ovary, white (WF, 1-4 mm) and yellowish (YF, 4-8 mm) prehierarchical follicles were incubated in a medium supplemented with PNP or PNMC (10-8-10-4 M), ovine LH (oLH; 10 ng/mL), and combinations of oLH with PNP or PNMC (10-6 M). Testosterone (T) and estradiol (E2) concentrations in media and mRNA expression for steroidogenic proteins (STAR, HSD3B1, and CYP19A1), and LH receptors (LHR), estrogen receptor α (ESR1) and ß (ESR2) in follicles were determined by RIA and real-time qPCR, respectively. PNP and PNMC decreased T and E2 secretion by the WF and YF, and oLH-stimulated T secretion from these follicles. PNP decreased basal STAR and HSD3B1 mRNA levels both in the WF and YF, and CYP19A1 mRNAs in the WF. PNP reduced oLH-affected mRNA expression of these genes in the YF. PNMC inhibited basal STAR, HSD3B1, and CYP19A1 mRNA expression in the WF, but not in the YF. PNMC reduced oLH-stimulated STAR and CYP19A1 expression in the YF and WF, respectively. PNP decreased basal mRNA expression of LHR, ESR1, and ESR2 in the WF, but it increased ESR1 and ESR2 mRNA levels in the YF. PNMC reduced both basal and oLH-affected LHR, ESR1, and ESR2 mRNA expression in the WF; however, it did not influence expression of these genes in the YF. We suggest that nitrophenols by influencing sex steroid synthesis and transcription of LH and estrogen receptors in prehierarchical ovarian follicles may impair their development and selection to the preovulatory hierarchy.

In vitro effects of PNP and PNMC on apoptosis and proliferation in the hen ovarian stroma and prehierarchal follicles.

This study aimed to examine the mRNA expression, activity, and immunolocalisation of apoptosis/proliferation regulating factors following in vitro exposure of the stroma, white (WFs), and yellowish (YFs) follicles of the chicken ovary to 4-nitrophenol (PNP) or 3-methyl-4-nitrophenol (PNMC). PNMC increased the mRNA expression of caspase-3, -8, Apaf-1, and cytochrome c in the ovarian stroma. The activity of caspase-3, -8, and -9 decreased in WFs in both nitrophenol-treated groups. PNP reduced the number of caspase-3-positive cells in the stromal connective tissue (CT) and the theca interna and externa layers of WFs. In the stroma, the proliferating index decreased in the wall of primary follicles in both nitrophenol-treated groups, however, in the CT, the effect of PNMC was opposite. In the theca interna of WFs, PNP diminished the proliferating index. These results suggest that nitrophenols might impact the development of chicken ovarian follicles by affecting cell death and proliferation.

Resorcinolic lipids improve the properties of sphingomyelin-cholesterol liposomes.

Alkylresorcinolic lipids isolated from cereal grains and their semi-synthetic myristoyl-sulphonyl derivatives (MSAR) were used to modify small long-circulating sphingomyelin-cholesterol liposomes. Those SM:Chol vesicles modified with 10-30 mol% resorcinolic lipids had stable size and low membrane permeability in vitro at 4 degrees C and 37 degrees C. Liposomes containing 30 mol% MSAR showed very fast solute release in the presence of human plasma at 37 degrees C, which was drastically diminished in heat-inactivated plasma. In vivo studies showed that unmodified SM:Chol liposomes and those modified with alkylresorcinols were eliminated from the circulation more slowly than liposomes with the highest concentration of MSAR in membrane and were located mostly in the liver and spleen.

Biological activity of phenolic lipids.

Phenolic lipids are a very diversified group of compounds derived from mono and dihydroxyphenols, i.e., phenol, catechol, resorcinol, and hydroquinone. Due to their strong amphiphilic character, these compounds can incorporate into erythrocytes and liposomal membranes. In this review, the antioxidant, antigenotoxic, and cytostatic activities of resorcinolic and other phenolic lipids are described. The ability of these compounds to inhibit bacterial, fungal, protozoan and parasite growth seems to depend on their interaction with proteins and/or on their membrane-disturbing properties.

Suppression of radical-induced lipid peroxidation in a model system by alkyl esters of cinnamate quaternary ammonium salts.

Three new groups of phenolic antioxidants, quaternary ammonium salts with a phenol ring and alkyl chains of different length (pyrrolidine ethyl esters of 3,5-di-t-butyl-4-hydroxydihydrocinnamic acid n-alkoxymethylchlorides (PYE-n) or n-alkylbromides (PYA-n) and 2-dimethylaminoethyl ester n-alkylbromides (PPA-n), were synthesized. Some of them were previously found to efficiently protect yeast cells against oxidants and to inhibit the production of thiobarbituric acid-reactive substances in whole yeast cells and in isolated membrane lipids. The new antioxidants (at 1-100 microM) abolished or diminished peroxidation of olive oil emulsions caused by the OH*-producing Fe2+ and RO* and ROO*-producing tert-butylhydroperoxide (TBHP) and the azo compounds 2,2'-azobis-(amidinopropane)dihydronitrile (AAPH) and 1,1'-azobis-(1-cyclohexanecarbonitrile) (ACHN): all present at 10 mM. The efficiency of individual both antioxidants was examined in relation to the type of lipid peroxidation inducer, the site of antioxidant incorporation into the emulsion lipid phase, the length of the alkyl chain, and the maximum concentration of effective antioxidant monomers given by its critical micelle concentration. PYA-n class compounds were highly efficient against all peroxidation inducers and their efficiency did not depend on the position of their molecules in the lipid phase and/or on the aliphatic chain length. In contrast, the efficiency of PYE-n and PPA-n class compounds depended both on the type of oxidant and on the length of their aliphatic chain. Their potency against Fe2+ and ACHN increased with increasing alkyl chain length whereas with AAPH it dropped with increasing alkyl chain length. A similar pattern was found with the action of PYE-n against TBHP whereas in the PPA-n group an extending alkyl chain reduced the anti-TBHP efficiency. These relationships may not be entirely straightforward and other factors (chemical nature of each compound, its possible interaction with fluorescent probes used for diagnostics, etc.) may play a considerable and not yet quite clear role. PPA-n class antioxidants have the lowest critical micelle concentration, which may limit their efficiency. Nevertheless, these phenolic antioxidants can be conveniently employed as highly efficient inhibitors of lipid peroxidation.

Formation of liposomes by resorcinolic lipids, single-chain phenolic amphiphiles from Anacardium occidentale L.

Resorcinolic lipids isolated from Anacardium occidentale nut oil extract (CNSL), unsaturated congeners of those isolated from bacterial and graminaceous sources, form at alkaline conditions liposomal structures alone as well as in the mixtures with cholesterol, fatty acids or phosphatidylethanolamine. Those vesicular structures show relatively high entrapment of the marker and stability of their size. The retention of the captured solute depends upon the type of resorcinolic lipid and on the temperature, but in general, is lower than control phospholipid liposomes.

Higher cardol homologs (5-alkylresorcinols) in rye seedlings.

The occurrence of alkylresorcinols, polyketide compounds that in the same homologous series as cardol isolated from Anacardium occidentale (cashew) or bilobol from Ginkgo biloba which are derivatives of 1,3-dihydroxy-5-alk(en)ylbenzene, have been demonstrated in developing rye (Secale cereale L.) kernels. The 3-day-old seedlings grown in sterile conditions already contain detectable amounts of phenolic compounds that were identified as alkylresorcinols. This fraction is the mixture of saturated and enoic homologs of various lengths of the aliphatic side chain. The composition of homologs is similar to that determined in mature grains. The relatively high level of alkylresorcinols in mitochondria and plastids (enhanced approximately twice in the absence of light) suggests that their synthetic pathway and/or biological function may be related to these cellular compartments. Resorcinolic lipids, when present in the external medium, are taken up by seedlings in the energy-dependent manner.

Alkylresorcinols in fruit pulp and leaves of Ginkgo biloba L.

These studies were undertaken to characterise resorcinolic lipids (5-n-alk(en)ylresorcinols) composition and to determine their seasonal fluctuations in fruit pulp and leaves of Ginkgo biloba L. Resorcinolic lipid concentrations were consistently higher in fruit pulp than in leaves. In pulp, several mono- and di-unsaturated homologs of alkylresorcinols were the predominant group of analysed lipids. Contrary to the fruit pulp, only 5-n-pentadecylresorcinol was demonstrated in leaves. Initially, the alkylresorcinol's content both in pulp and leaves increased until June-July and decreased following seeds ripening. This trend continued until senescence of leaves in late September and October.

Liposomal drug delivery, a novel approach: PLARosomes.

Almost from the time of their rediscovery in the 60's and the demonstration of their entrapment potential, liposomal vesicles have drawn attention of researchers as potential carriers of various bioactive molecules that could be used for therapeutic applications in humans and animals. Several commercial liposome-based drugs have already been discovered, registered and introduced with great success on the pharmaceutical market. However, further studies, focusing on the elaboration of more efficient and stable amphiphile-based vesicular (or non-viral) drug carriers are still under investigation. In this review we present the achievements of our group in this field. We have discovered that natural amphiphilic dihydroxyphenols and their semisynthetic derivatives are promising additives to liposomal lipid compositions. The presence of these compounds in lipid composition enhances liposomal drug encapsulation, reduces the amount of the lipid carrier necessary for efficient entrapment of anthracycline drugs by a factor of two, stabilizes liposomal formulation of the drug (both in suspension and in a lyophilized powder), does not influence liposomal fate in the blood circulation system and benefits from other biological activities of their resorcinolic lipid modifiers.

Dual effect of alkylresorcinols, natural amphiphilic compounds, upon liposomal permeability.

The effect of 5-n-alkylresorcinols, natural amphiphilic compounds, upon properties of phospholipid vesicles depends on their localization asymmetry. A significant increase of the bilayer permeability is observed when the title compounds are present only in the external medium. When these amphiphiles are preincorporated into the bilayer during its formation, the resulting liposomes effectively encapsulate water-soluble solutes which still remain in liposomes after 25 h. Additionally, the size of liposomes made of alkylresorcinol-phosphatidylcholine mixtures after eight cycles of freezing and thawing only (180-200 nm) is severalfold smaller than the size of vesicles prepared in a similar way from phospholipids only and the resulting liposomes are more homogeneous. These liposomes modified with alkylresorcinols are also stable during 40 day storage at both 4 degrees C and 20 degrees C, in contrast to control liposomes that already strongly aggregate after 10 days.

Quantum mechanical and experimental oxidation studies of pentadecylresorcinol, olivetol, orcinol and resorcinol.

Resorcinols (pentadecylresorcinol, olivetol, orcinol and resorcinol) exhibit antioxidant properties in liposomal systems. Antioxidant potency depends on the length of the alkyl chain. Pentadecylresorcinol has been demonstrated to be the most active antioxidant, indicating significance of its alkyl chain in a lipid bilayer. Quantum DFT computations demonstrated that hydroxyl group attached to the ring is the first target for the hydrogen abstraction after formation of the radical. However, the carbons of the side chain could also participate in the antioxidant properties of the alkylresorcinols. Formation of the radical at the hydroxyl oxygen initiates changes in the electron density which destabilise the whole system and subsequently leads to oxidation of the ring. The detailed study of lipophilicity and electrostatic properties of resorcinols is discussed.

Antimutagenic activity of alkylresorcinols from cereal grains.

Alkylresorcinols, natural amphiphilic compounds commonly found in cereal grains, markedly decreased mutagenic activity of four standard mutagens examined in the Ames test. The effect was the strongest in the case of indirect-acting mutagens, benzo[a]pyrene and 2-aminofluorene. In the case of direct-acting mutagens, daunorubicin and methyl methanesulfonate, the diminution of the mutagenic activity by the alkylresorcinols was smaller but still noticeable. In the Sister Chromatid Exchanges test (SCEs) with cultured in vitro human blood-derived lymphocytes, a significant decrease of SCEs frequency induced by benzo[a]pyrene was observed in the presence of alkylresorcinols. These preliminary results seem to be important in the aspect of possible antimutagenic and anticarcinogenic potency of alkylresorcinols found in cereal grains.

Alkylresorcinols are abundant lipid components in different strains of Azotobacter chroococcum and Pseudomonas spp.

The occurrence of various amounts of 5-n-alkylresorcinols was shown in lipids extracted from 14 bacterial strains of Azotobacter chroococcum as well as from strains of Pseudomonas aureofaciens, P. chlororapsis, and P. fluorescens. The amount of alkylresorcinols found varied from 2.3 to 56.2 microg/mg (dry weight) of cells in A. chroococum and from 0.2 to 0.8 microg/mg (dry weight) of cells in Pseudomonas spp. Strains of both genera produce saturated homologs with C13 to C27 side chains. C19, C21, and C23 homologs are predominant in and characteristic for A. chroococum strains, the C15 homolog is predominant in and characteristic for P. chlororapsis and P. fluorescens, and the C17 homolog is predominant in and characteristic for P. aureofaciens. The presence of 5-n-(2-ketoalkyl)resorcinols, not previously observed, was demonstrated in lipids isolated from the cells of A. chroococum Az5.

Fusiogenic activity of natural amphiphiles, 5-n-alkylresorcinols in a yeast protoplast system.

Two homologues of cereal grain resorcinolic lipids, 5-n-heptadecylresorcinol and 5-n-heptadecenylresorcinol studied in the system employing yeast cell protoplasts showed marked fusiogenic activity. The frequency of hybrid formation induced by studied amphiphiles was significantly higher than that obtained with the use of 40% (w/v) polyethylene glycol 4000. The resorcinolic lipids as fusion-inducing agents did not affect regeneration of the cellular wall. The fusiogenic activity of resorcinolic lipids lost when calcium ions were absent in the medium. Fusiogenic activity of studied amphiphiles is related to their ability to induce non-bilayer structures within the cellular membranes.

Modification of the lipid component modulates nuclear matrix nucleolytic activity.

It was shown that lipid composition of plant nuclear matrix depends on procedure of its isolation. The matrix isolated with the use of lithium diiodosalicylate (LiS) differs in its lipid composition from the preparation isolated with the use of nonionic detergent (Triton X-100). It was also shown that the nucleolytic activity of the matrix is related to its lipid component. Matrix depleted in lipids loses half of its nucleolytic activity which is recovered after supplementation with previously extracted lipids. The extent of recovery of the nucleolytic activity is also dependent on the presence of residual DNA in matrix preparation. The recoveries of nucleolytic activities were higher in matrices not depleted in their DNA content.

Modulation of some nuclear matrix enzymatic activities by free fatty acids.

It was shown that two of main enzymatic activities of plant nucleus and nuclear matrix, namely RNA-polymerasic and DNA-nucleolytic are susceptible to modulation with free fatty acids. The effects observed were dependent to both fatty acid length and degree of unsaturation. In nuclei a stimulation of nuclease activity was observed whereas in matrices short chain fatty acids inhibited the studied activity. The effect of fatty acids on RNA-polymerase was also different in nuclei and matrices. In nuclei all fatty acids studied inhibited polymerasic activity whereas in matrices short chain fatty acids stimulated this activity by up to 80% and the long chain fatty acids inhibited by up over 70%. The overall alteration of studied activities in nuclei and matrices by unsaturated fatty acids was similar. Nucleolytic activity was stronger inhibited and polymerasic activity was stimulated when the effects of linoleic and linolenic acids were studied. The results suggest possible importance of lipid component in nuclear matrix biological function.

Interaction of alkylresorcinols with proteins.

Alkylresorcinol homologs form stable monomolecular layers at air-water interface. Their interaction with proteins present in the subphase results in an increase of alkylresorcinol molecular packing in the extent dependent upon the protein studied. Strongest effects were observed for proteins with large hydrophobic regions, e.g. glycophorin or serum albumin. Interaction of proteins with alkylresorcinol monolayers is stronger than with phospholipids. A decrease and a shift of intrinsic protein fluorescence upon interaction with the compounds studied support their involvement in alteration of hydrophobic regions. For trypsin, 50% quenching was observed at the alkylresorcinol/trypsin ratio of 0.75. Concomitantly, an apparent inhibition of the enzymatic activity was noted. These results indicate that direct interaction of alkylresorcinols and modulation of enzymatic activities should be recognised as a significant part of the biological effect of these cereal bran components.

Determination of octanol/water partition coefficients for long-chain homologs of orcinol from cereal grains.

Polycratic reversed-phase high-performance liquid chromatography (RPHPLC) was used for estimation of the octanol/water partition coefficient for three highly hydrophobic long chain orcinol homologs. The homologs studied (C 15:0, C 17:0 and C 19:0) showed high preference for hydrophobic phase as evidenced by their high octanol/water partition coefficient (log Po/w) values of 7.02-7.74; 8.71-9.47 and 10.49-11.32 for the 95% prediction interval, respectively. Experimentally estimated values were compared with log P values calculated with the use of several fragmental systems. The experimental values of log Po/w are in best agreement to those calculated with the use of the Klopman system (Klopman, G., Namboodiri, K. & Schochet, M., 1985, J. Comput. Chem. 6, 28-38). The lack of appropriate standard compounds with known log Po/w in the range over 6 markedly affected the accuracy of experimental determinations.

Cereal grain resorcinolic lipids inhibit H2O2-induced peroxidation of biological membranes.

Cereal grain resorcinolic lipids (5-n-alk(en)ylresorcinols) at micromolar concentrations are able to protect the erythrocyte membrane against hydrogen peroxide-induced lipid oxidation. The antioxidative effect is dependent upon chain length of alkylresorcinol molecules. The C15:0 homolog (IC50 of 10 microM) exhibited strongest activity whereas for long chain homologs (C19:0 and C23:0) IC50 values were higher, 32.5 and 59 microM, respectively. The protective effect of alkylresorcinolic antioxidants was also dependent on their incorporation into the membrane, that is governed by their water-membrane partition coefficient. The results obtained show that alkylresorcinols should be recognized as hydrophobic, membrane-localised antioxidants.

The transit sequence mediates the specific interaction of the precursor of ferredoxin with chloroplast envelope membrane lipids.

The interaction of the precursor of the chloroplast protein ferredoxin with membrane lipids was studied in monolayer experiments in order to investigate the possible involvement of membrane lipids in the protein translocation process. The precursor efficiently and specifically inserts into a total lipid extract of its biological target the outer envelope membrane of chloroplasts. This interaction is mediated by the transit sequence as it can also be observed for the chemically prepared transit peptide of ferredoxin but neither for the ferredoxin apoprotein nor holoprotein. Interactions with the individual chloroplast lipids, monogalactosyl-diacylglycerol, sulfoquinovosyl-diacylglycerol, and phosphatidylglycerol are predominantly involved which corresponds to the results obtained for transit peptide fragments of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (van't Hof, R., Demel, R. A., Keegstra, K., and De Kruijff, B. (1991) FEBS Lett. 291, 350-354). No efficient interaction was obtained with digalactosyl-diacylglycerol and phosphatidylcholine, suggesting that a loose lipid headgroup packing due to small lipid headgroups and/or electrostatic repulsions facilitates efficient insertion. The observed preferences for interaction of the precursor and transit peptide of ferredoxin for the chloroplast outer envelope membrane lipid extract and the presequence of cytochrome c oxidase subunit IV for the mitochondrial outer membrane lipid extract indicate that targeting sequence-lipid interactions contribute to organelle-specific protein targeting.