Quantitative structure toxicity relationships for phenols in isolated rat hepatocytes

Quantitative structure toxicity relationship (QSTR) equations were obtained to predict and describe the cytotoxicity of 31 phenols using logLD(50) as a concentration to induce 50% cytotoxicity of isolated rat hepatocytes in 2 h and logP as octanol/water partitioning: logLD(50) (microM)=-0.588(+/-0.059)logP+4.652(+/-0.153) (n=27, r(2)=0.801, s=0.261, P<1 x 10(-9)). Hydroquinone, catechol, 4-nitrophenol, and 2,4-dinitrophenol were outliers for this equation. When the ionization constant pK(a) was considered as a contributing factor a two-parameter QSTR equation was derived: logLD(50) (microM)=-0.595(+/-0.051)logP+0.197(+/-0.029)pK(a)+2.665(+/-0.281) (n=28, r(2)=0.859, s=0.218, P<1 x 10(-6)). Using sigma+, the Brown variation of the Hammet electronic constant, as a contributing parameter, the cytotoxicity of phenols towards hepatocytes were defined by logLD(50) (microM)=-0.594(+/-0.052)logP-0.552(+/-0.085)sigma+ +4.540(+/-0.132) (n=28, r(2)=0.853, s=0.223, P<1 x 10(-6)). Replacing sigma+ with the homolytic bond dissociation energy (BDE) for (X-PhOH+PhO.-->X-PhO.+PhOH) led to logLD(50) (microM)=-0.601(+/-0.066)logP-0.040(+/-0.018)BDE+4.611(+/-0.166) (n=23, r(2)=0.827, s=0.223, P<0.05). Hydroquinone, catechol and 2-nitrophenol were outliers for the above equations. Using redox potential and logP led to a new correlation: logLD(50) (microM)=-0.529(+/-0.135)logP+2.077(+/-0.892)E(p/2)+2.806(+/-0.592) (n=15, r(2)=0.561, s=0.383, P<0.05) with 4-nitrophenol as an outlier. Our findings indicate that phenols with higher lipophilicity, BDE, or sigma+ values or with lower pK(a) and redox potential were more toxic towards hepatocytes. We also showed that a collapse of hepatocyte mitochondrial membrane potential preceded the cytotoxicity of most phenols. Our study indicates that one or a combination of mechanisms; i.e. mitochondrial uncoupling, phenoxy radicals, or phenol metabolism to quinone methides and quinones, contribute to phenol cytotoxicity towards hepatocytes depending on the phenol chemical structure.     

Quantitative structure toxicity relationships for catechols in isolated rat hepatocytes

One- and two-parameter quantitative structure toxicity relationship (QSTR) equations were obtained to describe the cytotoxicity of isolated rat hepatocytes induced by 23 catechols in which LD(50) represents the catechol concentration required to induce 50% cytotoxicity in 2 h. A QSTR equation logLD(50) (microM = - 0.464(+/-0.065) log P + 3.724(+/-0.114) (n = 20, r(2) = 0.740, s(y,x) = 0.372, P < 1 x 10(-6), outliers: 4-methoxycatechol, 3-methoxycatechol, L-dopa) was derived where logP represents octanol/water partitioning. Outliers were determined by adopting a statistical method to standardize the identification of outliers. When pK(a1), the first ionization constant, was considered as a contributing parameter a two-parameter QSTR equation was derived: logLD(50) (microM = - 0.343(+/-0.058) log P - 0.116(+/-0.041) pK(a1)+4.389 (+/-0.315) (n = 22, r(2) = 0.738, s(y,x) = 0.375, P < 0.01, outlier: 4-methoxycatechol). Replacing logP with logD(7.4), the partition coefficient at pH 7.4, improved the first correlation by limiting the outlier to 4-methoxycatechol: logLD(50) (microM)=-0.252(+/-0.039) logD(7.4)+3.168(+/-0.090) (n = 22, r(2) = 0.671, s(y,x) = 0.420, P < 1 x 10(-5). In this study, 4-methoxycatechol (readily autooxidizable) was found to be an outlier for all QSTR equations derived. These findings point to lipophilicity and pK(a1) as two important characteristics of catechols that can be used to predict their cytotoxicity towards isolated rat hepatocytes. The catechols with the higher lipophilicity/distribution coefficient, the lower degree of ionization and the higher pK(a(catechol)) were more toxic towards hepatocytes than the other catechols.     

Prooxidant toxicity of polyphenolic antioxidants to HL-60 cells: description of quantitative structure-activity relationships

Polyphenolic antioxidants exhibited a dose-dependent toxicity against human promyelocytic leukemia cells (HL-60). Their action was accompanied by malondialdehyde formation, and was partly prevented by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine. This points to a prooxidant character of their cytotoxicity. A quantitative structure-activity relationship (QSAR) has been obtained to describe the cytotoxicity of 13 polyphenolic antioxidants belonging to three different groups (flavonoids, derivatives of gallic and caffeic acid): log cL50 (microM) = (2.7829+/-0.2339)+(1.2734+/-0.4715) Ep/2 (V)-(0.3438+/-0.0582) log P (r2 = 0.8129), where cL50 represents the concentration for 50% cell survival, Ep/2 represents the voltammetric midpoint potential, and P represents the octanol/water partition coefficient. Analogous QSARs were obtained using enthalpies of single-electron oxidation of these compounds, obtained by quantum-mechanical calculations. These findings clearly point to two important characteristics determining polyphenol cytotoxicity, namely their ease of oxidation and their lipophilicity.     

Role of single-electron oxidation potential and lipophilicity in the antiplasmodial in vitro activity of polyphenols: comparison to mammalian cells

In spite of extensive studies, the structure-activity relationships in the action of polyphenols against the malaria parasite Plasmodium falciparum are poorly understood so far. As the mammalian cell cytotoxicity of polyphenols shows a negative dependence on the potential of the phenoxyl radical/phenol redox couple (E(2)(7)), due to the involvement of prooxidant events, and a positive dependence on the octanol/water distribution coefficient at pH 7.0 (log D), we examined the role of these parameters in their antiplasmodial in vitro activity. We found that the concentrations of hydroxybenzenes causing 50% inhibition of the growth of P falciparum strain FcB1 (IC50) are described by the regression log IC50 (microM) = 0.36 + 1.81 E(2)(7) (V) - 0.10 log D [n = 11, r2 = 0.760, F(2.8) = 12.03]. The IC50 values of flavonoids (n = 5), comprising a separate less active series, did not depend on their E(2)(7) values, 0.33 V-0.75 V. These findings were similar to the mammalian cell cytotoxicity data. However, the mammalian cell cytotoxicity of hydroxybenzenes showed more pronounced dependence on their E(2)(7) values [delta log CL50/delta E(2)(7) = (6.9 - 5.1) V(-1), where CL50 is the compound concentration for 50% cell survival] than on their antiplasmodial activity. Although it is unclear whether the prooxidant action is the main factor in the antiplasmodial action of polyphenols or not, our data showed that the ease of their oxidation (decrease in E(2)(7)) may enhance their activity. On the other hand, the different sensitivity of the mammalian cell cytotoxicity and the antiplasmodial activity of the hydroxybenzenes to their E(2)(7) values implied that compounds with high oxidation potential may be used as relatively efficient antiplasmodial agents with low mammalian cell cytotoxicity.     

Quantitative structure-activity relationships of phenolic compounds causing apoptosis

A study of a variety of phenolic compounds (simple phenols, estradiol, bisphenol A, diethylstilbesterol) on their action on L1210 leukemia cells led to the formulation of the following QSAR for apoptosis:log 1/C=-3.16 Clog P+2.77 CMR-3.76n=11, r(2)=0.939, s=0.630, q(2)=0.892C is the molar concentration causing 25% apoptosis, Clog P is the calculated octanol/water partition coefficient and CMR is the calculated molecular refractivity. Our results imply the significance of characterization of the phenolic compounds with apoptotic activity and the development of new agents for cancer therapy.     

Quantitative structure-activity relationships in prooxidant cytotoxicity of polyphenols: role of potential of phenoxyl radical/phenol redox couple

The aim of this work was to characterize the role of the potential of phenoxyl radical/phenol redox couple, E(7)(2), in the cytotoxicity of polyphenols. The cytotoxicity of polyphenols in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK), and human promyelocytic leukemia cells (line HL-60) was partly inhibited by catalase, by the antioxidant N,N'-diphenyl-p-phenylene diamine and desferrioxamine, and potentiated by 1,3-bis-(2-chloro-ethyl)-1-nitrosourea, thus showing its prooxidant character. Dapsone, an inhibitor of myeloperoxidase, did not affect the cytotoxicity of polyphenols in HL-60 cells, whereas dicumarol, an inhibitor of DT-diaphorase, showed controversial effects on their cytotoxicity in FLK cells. Inhibitors of cytochromes P-450, alpha-naphthoflavone and izoniazide, decreased the cytotoxicity of several polyphenols, whereas 3,5-dinitrocatechol, an inhibitor of catechol-o-methyltransferase (COMT), increased it. The cytotoxicity of 13 polyhydroxybenzenes was described by the equations: logcL50 (microM) = -0.67 + 5.46E(7)(2) (V) - 0.16 logD (FLK), and logcL50 (microM) = -1.39 + 6.90E(7)(2) (V) - 0.20logD (HL-60), where cL50 is compound concentration for 50% cell survival, and D is octanol/water distribution coefficient at pH 7.0. The flavonoids comprise a separate series of compounds with lower cytotoxicity. The correlations obtained quantitatively confirm the parallelism between the polyphenol cytotoxicity and the rates of their single-electron oxidation, and point to the leading role of formation of the reactive oxygen species in their cytotoxicity. Depending on the examined system, this parallelism may be distorted due to the cytochrome P-450 and COMT-catalyzed transformation of polyphenols.     

QSAR studies for the inhibition of the transmembrane isozymes XII and XIV of human carbonic anhydrase with a series of sulfonamides

A diverse series of aromatic/heterocyclic sulfonamides possessing inhibitory action against the human transmembrane isoforms XII (cancer-associated) and XIV of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) has been used to develop QSAR models. Including all the 55 investigated sulfonamides in the calibration set, the predictive qualities of the QSAR equations were weak (r(2)=0.1771, F=5.70) for CA XII and good for CA XIV inhibition (r(2)=0.8222, F=57.04 before eliminating the outliers, and r(2)=0.8911, F=67.07 after eliminating them). The obtained models suggest a slightly different inhibition mechanism for the two isoforms. 3-Halogeno-4-amino-benzenesulfonamides were outliers for scaffold hopping for the inhibition of CA XIV. CA XIV inhibitory activity was proportional to the degree of molecular surface rugosity. For compounds of the type X-Ar-SO(2)NH(2) and Ar'-Ar-SO(2)NH(2) type, best inhibitors were detected when Ar/Ar' incorporates a heterocyclic moiety. These studies may be helpful for the design of more specific CA XII/XIV inhibitors, since this is the first QSAR model investigating them.     

The isolated anterior stomach of larval mosquitoes (Aedes aegypti): voltage-clamp measurements with a tubular epithelium

The anterior stomach of larval Aedes aegypti was isolated and perfused via two pipettes. For transepithelial voltage (V(te)) measurement, the inflow pipette and the bath were connected via agar bridges to calomel electrodes. For voltage-clamping, the lumen of the tissue contained an Ag/AgCl wire held by the outflow pipette, and the preparation was placed in a bath within a spiral of Ag/AgCl wire. After equilibrating the tissue in mosquito saline on both sides, a V(te) of -8+/-1 mV was measured (+/-S.E.M., N=32). Current-voltage curves (+/-100 mV) demonstrated ohmic behaviour of the epithelium. Short-circuiting resulted in a current (I(sc)) of 103+/-16 microA cm(-2) and a mean transepithelial conductance (G(te)) of 11.8+/-1.3 mS cm(-2) (+/-S.E.M., N=32). A Yonath-Civan plot of G(te) of individual preparations over the corresponding I(sc) resulted in a straight line (r(2)=0.8422), indicating that the difference in I(sc) of individual preparations is mainly based on different transcellular conductances (G(c)). This analysis allowed to estimate the mean leak conductance (G(l) approximately 3.9 mS cm(-2)) and the mean transcellular electromotive force (E(c) approximately 13 mV). After administering 0.2 micromol L(-1) serotonin, I(sc) and G(te) significantly increased, to 457+/-49 microA cm(-2) and to 21.3+/-2.3 mS cm(-2) (+/-S.E.M., N=31, P<0.05), respectively. The Yonath-Civan plot after serotonin resulted again in a straight line (r(2)=0.8219), indicating a mean G(l) of about 1 mS cm(-2) and a mean E(c) of about 22 mV. Dinitrophenol (2.5 mmol L(-1)) almost abolished I(sc) and significantly reduced G(te) (N=6). Concanamycin A (100 micromol L(-1)) reduced I(sc) by more than 90% without significantly affecting G(te).     

Comparative QSAR evidence for a free-radical mechanism of phenol-induced toxicity

Phenol and 14 substituted-phenols were tested for their ability to impair epithelial cell membrane integrity in WB rat liver cells as determined by an increase in lactate dehydrogenase release. Two quantitative structure-activity relationship (QSAR) regression equations were developed which showed that separate mechanisms of phenolic cytotoxicity are important - nonspecific toxicity due to hydrophobicity and formation of phenoxyl radicals. The equations most predictive of phenol toxicity are denoted as log1/C=-0. 98sigma(+)+0.77logP+0.23 or log1/C=-0.11BDE+0.76logP+0.21, respectively, where C is the minimum concentration of substituted-phenol required for a toxic response. P is the octanol-water partition coefficient, sigma(+) is the electronic Hammett parameter and BDE is the OH homolytic bond dissociation energy. In the literature, phenol toxicity correlated to sigma(+) is rare, but there is strong evidence that phenols possessing electron-releasing groups may be converted to toxic phenoxyl radicals. A common feature in a variety of cells is generation of elevated amounts of reactive oxygen species (ROS) associated with a rapid growth rate. The slightly elevated cancer risk associated with the use of Premarin may be due to phenoxyl-type radicals derived from one or more of its components.     

Analysis of soluble interleukin 6 receptor in cerebrospinal fluid in inflammatory and non-inflammatory conditions

The objective of this study was to investigate the pathophysiological roles of soluble interleukin 6 receptor (sIL-6R) in cerebrospinal fluid (CSF). CSF was obtained from patients suspected with meningitis. Eight patients without any meningeal signs or symptoms were enrolled as controls. An additional 34 CSF samples were collected to measure both biologically active and immunoreactive sIL-6R. All CSF samples were proven to be aseptic. IL-6 and sIL-6R were measured using specific ELISAs. Patients were divided into three groups on the basis of cell number in CSF; inflammatory group (cell number >5 microl, mean 241+/-363.1, n=61); non-inflammatory group (cell number < or =5 microl, mean=2.1+/-1.7, n=12) and controls (cell number < or =5 microl, mean=0.3+1.7, n=8). Among these three groups, the differences in protein (F (2,78)=8.274, P<0.0001) and IL-6 concentration (F (2,78)=6.475, P<0.001) were statistically significant but those of sIL-6R concentration were not. There were only weak correlations between log (sIL-6R) versus log (cell number) (r=0.23, P=0.0375), log (protein) (r=0.239, P=0.0358) and log (IL-6) (r=0.27, P=0.0167). Amounts of immunoreactive and biologically active sIL-6R were closely correlated (r=0.62, n=34, P<0.005). It was concluded that sIL-6R is present constitutively in CSF and its level may not increase significantly in inflammatory conditions; infiltrating cells in CSF are not the main source of sIL-6R; and sIL-6R in CSF can bind IL-6.     

QSAR studies and pharmacophore identification for arylsubstituted cycloalkenecarboxylic acid methyl esters with affinity for the human dopamine transporter

Data from a series of 29 monoamine transport inhibitors were used to generate 2D and 3D QSAR models for their binding affinity to the human dopamine transporter (hDAT). Among the inhibitors were many non-nitrogen containing compounds. The 2D QSAR analysis resulted in the equation -logK(i)=4.00-3.93E(LUMO)-0.67E(HOMO)-3.24sigma(p), which predicted the importance of electron withdrawing groups in the aromatic moiety. However, the model failed to predict the observed poor binding of nitro-substituted compounds. In contrast, a derived 3D QSAR model was capable of predicting these more correctly.     

Modeling ATP protonation and activity coefficients in NaClaq and KClaq by SIT and Pitzer equations

The acid-base properties of Adenosine 5'-triphosphate (ATP) in NaCl and KCl aqueous solutions at different ionic strengths (0相似文献   

A rearranging ligand enables allosteric control of catalytic activity in copper-containing nitrite reductase

In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axial methionine ligand of the type-1 site was replaced (M150G) to make the copper ion accessible to external ligands that might affect the enzyme's catalytic activity. The type-1 site optical spectrum of M150G (A(460)/A(600)=0.71) differs significantly from that of the native nitrite reductase (A(460)/A(600)=1.3). The midpoint potential of the type-1 site of nitrite reductase M150G (E(M)=312(+/-5)mV versus hydrogen) is higher than that of the native enzyme (E(M)=213(+/-5)mV). M150G has a lower catalytic activity (k(cat)=133(+/-6)s(-1)) than the wild-type nitrite reductase (k(cat)=416(+/-10)s(-1)). The binding of external ligands to M150G restores spectral properties, midpoint potential (E(M)<225mV), and catalytic activity (k(cat)=374(+/-28)s(-1)). Also the M150H (A(460)/A(600)=7.7, E(M)=104(+/-5)mV, k(cat)=0.099(+/-0.006)s(-1)) and M150T (A(460)/A(600)=0.085, E(M)=340(+/-5)mV, k(cat)=126(+/-2)s(-1)) variants were characterized. Crystal structures show that the ligands act as allosteric effectors by displacing Met62, which moves to bind to the Cu in the position emptied by the M150G mutation. The reconstituted type-1 site has an otherwise unaltered geometry. The observation that removal of an endogenous ligand can introduce allosteric control in a redox enzyme suggests potential for structural and functional flexibility of copper-containing redox sites.     

Polyphenols as potential indicators for drought tolerance in tea (Camellia sinensis L.)

Plant polyphenols have gained prominence in quality of plant products and in human health. An experiment was conducted to determine the association of tea polyphenols with water stress and their suitability as indicators for drought tolerance. The experiment was conducted in a 'rain-out' shelter, and consisted of six tea clones (BBK 35, TRFK 6/8, TRFK 76/1, TRFK 395/2, TRFK 31/30, and TRFK 311/287) and four levels of soil water contents (38, 30, 22, and 14% v/v), which were maintained for a period of 12 weeks. The treatments were arranged in a completely randomized design and replicated three times. Plant growth was monitored over 6 weeks, and a water stress index was calculated to determine water-stress tolerant clones. Total polyphenols in tea shoots was analyzed and a regression analysis done. The results indicate that declining soil water content (SWC) reduced both growth and content of polyphenols in tea. Tolerant clones maintained a high polyphenol content at low SWC, and also showed less fluctuation in phenolics when subjected to changes in SWC. There was significant (P<0.001) correlation of total polyphenol content with shoot growth and WSI of tea, and a linear relationship (r2=0.97) between SWC for tea and both, water stress index and shoot polyphenol content. We report that there is a potential to use polyphenols as indicators for selection of drought-tolerant tea cultivars.     

Structure-activity relationships for halobenzene induced cytotoxicity in rat and human hepatocytes

Halobenzenes are ubiquitous environmental contaminants, which are hepatotoxic in both rodents and humans. The molecular mechanism of halobenzene hepatotoxicity was investigated using Quantitative structure-activity relationships (QSAR) and accelerated cytotoxicity mechanism screening (ACMS) techniques in rat and human hepatocytes. The usefulness of isolated hepatocytes for prediciting in vivo xenobiotic toxicity was reassessed by correlating the LC(50) of 12 halobenzene congeners in phenobarbital (PB) induced rat hepatocytes in vitro determined by ACMS to the hepatotoxicities reported in vivo in PB-induced male Sprague-Dawely (SD) rats. A high correlation (r(2)=0.90) confirmed the application of hepatocytes as a "gold standard" for toxicity testing in vitro. QSARs were derived to determine the physico-chemcial variables that govern halobenzene toxicity in PB-induced rat, normal rat and human hepatocytes. We found that toxicity in normal rat and normal human hepatocytes both strongly correlate with hydrophobicity (logP), ease of oxidation (E(HOMO), energy of the highest molecular orbital) and on the asymmetric charge distribution according to arrangement of halogen substituents (dipole moment, mu). This suggests that halobenzene interaction with cytochrome P450 for oxidation is the metabolic activating path for toxicity and is similar in both species. In PB-induced rat hepatocytes the QSAR derivation is changed, where halobenzene toxicity strongly correlates to logP and dipole moment, but not E(HOMO). The changed QSAR suggests that oxidation is no longer the rate-limiting step in the cytotoxic mechanism when CYP2B/3A levels are increased, confirming CYP450 oxidation as the metabolic activating step under normal conditions.     

Effects of redox buffer properties on the folding of a disulfide-containing protein: dependence upon pH, thiol pKa, and thiol concentration

Aliphatic thiols are effective as redox buffers for folding non-native disulfide-containing proteins into their native state at high pH values (8.0-8.5) but not at neutral pH values (6-7.5). In developing more efficient and flexible redox buffers, a series of aromatic thiols was analyzed for its ability to fold scrambled ribonuclease A (sRNase A). At equivalent pH values, the aromatic thiols folded sRNase A 10-23 times faster at pH 6.0, 7-12 times faster at pH 7.0, and 5-8 times faster at pH 7.7 than the standard aliphatic thiol glutathione. Similar correlations between thiol pK(a) values and folding rates at each pH value suggest that the apparent folding rate constants (k(app)) are a function of the redox buffer properties (pH, thiol pK(a) and [RSH]). Fitting the observed data to a three-variable model (logk(app)=-4.216(+/-0.030)+0.5816(+/-0.0036)pH-0.233(+/-0.004)pK(a)+log(1-e(-0.98(+/-0.02)[RSH]))) gave good statistics: r2=0.915, s=0.10.