Oestrogenic activity of p-hydroxybenzoic acid (common metabolite of paraben esters) and methylparaben in human breast cancer cell lines

This paper addresses the question of whether p-hydroxybenzoic acid, the common metabolite of parabens, possesses oestrogenic activity in human breast cancer cell lines. The alkyl esters of p-hydroxybenzoic acid (parabens) are used widely as preservatives in consumer products to which the human population is exposed and have been shown previously to possess oestrogenic activity and to be present in human breast tumour tissue, which is an oestrogen-responsive tissue. Recent work has shown p-hydroxybenzoic acid to give an oestrogenic response in the rodent uterotrophic assay. We report here that p-hydroxybenzoic acid possesses oestrogenic activity in a panel of assays in human breast cancer cell lines. p-Hydroxybenzoic acid was able to displace [H-3]oestradiol from cytosolic oestrogen receptor of MCF7 human breast cancer cells by 54% at 5 x 10(6)-fold molar excess and by 99% at 10(7)-fold molar excess. It was able to increase the expression of a stably integrated oestrogen responsive reporter gene (ERE-CAT) at a concentration of 5 x 10(-4) M in MCF7 cells after 24 h and 7 days, which could be inhibited by the anti-oestrogen ICI 182 780 (Faslodex, fulvestrant). Proliferation of two human breast cancer cell lines (MCF7, ZR-75-1) could be increased by 10(-5) M p-hydroxybenzoic acid. Following on from previous studies showing a decrease in oestrogenic activity of parabens with shortening of the linear alkyl chain length, this study has compared the oestrogenic activity of p-hydroxybenzoic acid where the alkyl grouping is no longer present with methylparaben, which has the shortest alkyl group. Intrinsic oestrogenic activity of p-hydroxybenzoic acid was similar to that of methylparaben in terms of relative binding to the oestrogen receptor but its oestrogenic activity on gene expression and cell proliferation was lower than that of methylparaben. It can be concluded that removal of the ester group from parabens does not abrogate its oestrogenic activity and that p-hydroxybenzoic acid can give oestrogenic responses in human breast cancer cells. Copyright (C) 2005 John Wiley & Sons, Ltd.

A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria

We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.

A comparative study on the relationship between acetyl cholinesterase activity and acute toxicity in Daphnia magna exposed to anticholinesterase insecticides

Acetylcholinesterase (AChE) activity was measured in Daphnia magna that had been exposed to four organophosphates (OPs; parathion, chlorpyrifos, malathion, and acephate) and one carbamate (propoxur) for 48 h. These results were related to acute toxicity (median effective concentration [EC50] for immobility). For the four OPs, the EC50s were 7.03 pM, 3.17 pM, 10.56 pM, and 309.82 muM, respectively. The EC50 for propoxur was 449.90 pM. Reduction in AChE activity was directly related to an increase in immobility in all chemicals tested. However, the ratio between the EC50 and the AChE median inhibiting concentration ranged from 0.31 to 0.90. A 50% reduction in AChE activity generally was associated with detrimental effects on mobility. However, for acephate, high levels of AChE inhibition (70%) were observed in very low concentrations and were not associated with immobility. In addition, increasing the concentration of acephate further had a slight negative effect oil AChE activity but a Strong detrimental effect on mobility. Binding sites other than AChE possibly are involved in acephate toxicity to D. magna. Our findings demonstrate different associations between AChE inhibition and toxicity when different chemicals are compared. Therefore, the value of using AChE activity as a biomarker in D. magna will be dependent on the chemical tested.

Haem peroxidase activity in Daphnia magna: A biomarker for sub-lethal toxicity assessments of kerosene-contaminated groundwater

A novel biomarker was developed in Daphnia magna to detect organic pollution in groundwater. The haem peroxidase assay, which is an indirect means of measuring oxidase activity, was particularly sensitive to kerosene contamination. Exposure to sub-lethal concentrations of kerosene-contaminated groundwater resulted in a haem peroxidase activity increase by dose with a two-fold activity peak at 25%. Reproduction in D. magna remained unimpaired when exposed to concentrations below 25% for 21 days, and a decline in fecundity was only observed at concentrations above the peak in enzyme activity. The measurement of haem peroxidase activity in D. magna detected sublethal effects of kerosene in just 24 h, whilst offering information on the health status of the organisms. The biomarker may be useful in determining concentrations above which detrimental effects would occur from long-term exposure for fuel hydrocarbons. Moreover, this novel assay detects exposure to chemicals in samples that would normally be classified as non-toxic by acute toxicity tests.

Oestrogenic activity of benzylparaben

Previous work has demonstrated that the alkyl esters of p-hydroxybenzoic acid (parabens) possess oestrogenic activity, which increases with length of alkyl chain from methylparaben to n-butylparaben and with branching in the alkyl chain from n-butylparaben to isobutylparaben. This study reports on the oestrogenic activity of benzylparaben in a variety of assays in vitro and in vivo. Benzylparaben was able to displace [H-3]oestradiol from cytosolic oestrogen receptor (ER) of MCF7 human breast cancer cells by 22% at 1000-fold molar excess, by 40% at 10000-fold molar excess, by 57% at 100000-fold molar excess and by 100% at 1000000-fold molar excess. It was able to increase expression of a stably transfected oestrogen responsive reporter gene (ERE-CAT) in MCF7 cells after 24 h at 10(-5)M/10(-4)M and after 7 days at 10(-6)M/10(-5)M/10(-4)M. Proliferation of MCF7 cells could be increased by 10(-6)M/10(-5)M benzylparaben and this could be inhibited by 10(-7)M pure anti-oestrogen ICI 182,780, indicating that growth effects were ER mediated. Further evidence for ER-mediation was provided from the ability of benzylparaben to increase the growth of a second oestrogen-dependent human breast cancer cell line ZR-75-1, but not the oestrogen-insensitive NIDA-MB-231 cell line. When tested in the presence of 10(-10)M 17beta-oestradiol, benzylparaben gave no antagonist response on the growth of either MCF7 or ZR-75-1 cells. Finally, benzylparaben could increase uterine weight in the immature mouse following topical application of three daily doses of 33 mg to dorsal skin. These results demonstrate that the oestrogenicity of methylparaben can be increased by the addition of an aryl group as well as by lengthening or branching the alkyl grouping. Copyright (C) 2003 John Wiley Sons, Ltd.

Functional coupling of the human dopamine D-2 receptor with G alpha(i1), G alpha(i2), G alpha(i3) and G alpha(o) G proteins: evidence for agonist regulation of G protein selectivity

1 The human dopamine D-2long (D-2L) receptor was expressed with four different G proteins in Sf9 cells using the baculovirus expression system. When co-expressed with G(i)/G(o) G proteins (G(i1)alpha, G(i2)alpha, G(i3)alpha, or G(o)alpha, plus Gbeta(1) and Ggamma(2)) the receptor displayed a high-affinity binding site for the agonists (dopamine and NPA), which was sensitive to GTP (100 mum), demonstrating interaction between the receptor and the different G proteins. 2 The receptor to G protein ratio (R: G ratio) was evaluated using [H-3]-spiperone saturation binding (R) and [S-35]-GTPgammaS saturation binding (G). R: G ratios of 1: 12, 1: 3, 1: 14 and 1: 5 were found for G(i1), G(i2), G(i3), and Go preparations, respectively. However, when R:G ratios of 1:2 and 1: 12 were compared for G(i2) and G(o), no difference was found for the stimulation of [S-35]-GTPgammaS binding. 3 Several agonists were tested for their ability to stimulate [S-35]-GTPgammaS binding to membranes co-expressing the receptor and various G proteins. All the compounds tested showed agonist activity in preparations expressing G(i3) and G(o). However, for G(i2) and G(i1) preparations, compounds such as S-(-)-3-PPP and p-tyramine were unable to stimulate [S-35]-GTPyS binding. 4 Most of the compounds showed higher relative efficacies (compared to dopamine) and higher potencies in the preparation expressing G(o). Comparison of the effects of different agonists in the different preparations showed that each agonist differentially activates the four G proteins. 5 We conclude that the degree of selectivity of G protein activation by the D-2L receptor can depend on the conformation of the receptor stabilised by an agonist.

Characterization of the endokinins: Human tachykinins with cardiovascular activity

We report four human tachykinins, endokinins A, B, C, and D (EKA-D), encoded from a single tachykinin precursor 4 gene that generates four mRNAs (alpha, beta, gamma, and delta). Tachykinin 4 gene expression was detected primarily in adrenal gland and in the placenta, where, like neurokinin B, significant amounts of EKB-like immunoreactivity were detected. EKA/B 10-mers displayed equivalent affinity for the three tachykinin receptors as substance P (SP), whereas a 32-mer N-terminal extended form of EKB was significantly more potent than EKA/B or SP. EKC/D, which possess a previously uncharacterized tachykinin motif, FQGLL-NH2, displayed low potency, EKA/B displayed identical hemodynamic effects to SP in rats, causing short-lived falls in mean arterial blood pressure associated with tachycardia, mesenteric vasoconstriction, and marked hindquarter vasodilatation. Thus, EKA/B could be the endocrine/paracrine agonists at peripheral SP receptors and there may be as yet an unidentified receptor(s) for EKC/D.

Intraclonal variability in Daphnia acetylcholinesterase activity: The implications for its applicability as a biomarker

The relationship between individual growth and acetylcholinesterase (AChE).activity was evaluated for Daphnia magna. Analysis on the influence of two different culture media on baseline AChE activity was performed with Daphnia similis. The results indicated an inverse relationship between D. magna body length and AChE activity. An increase in total protein, which was not proportional to an increase in the rate of the substrate hydrolysis (Delta absorbance/min), seems to be the reason for this inverse size versus AChE activity relationship. Therefore, toxicants such as phenobarbital, which affect protein and size but not AChE activity directly, have an overall affect on AChE activity. In contrast, the AChE inhibitor parathion altered AChE activity but not protein. Culture medium also had a significant affect on AChE activity in D. similis. Changes in total protein seem to be the main reason for the variations in baseline AChE activity in Daphnia observed in the different evaluations performed in this work. Therefore, AChE activity in Daphnia must be interpreted carefully, and variations related to changes in total protein must be taken into account when applying this enzyme as a biomarker in biological monitoring.

Antioxidant and free radical scavenging activity of isoflavone metabolites

1. Soy isoflavones have been extensively studied because of their possible health-promoting effects. Genistein and daidzein, the major isoflavone aglycones, have received most attention; however, they undergo extensive metabolism in the gut and liver, which might affect their biological properties. 2. The antioxidant activity, free radical-scavenging properties and selected cellular effects of the isoflavone metabolites equol, 8-hydroxydaidzein, O-desmethylangiolensin, and 1,3,5 trihydroxybenzene were investigated in comparison with their parent aglycones, genistein and daidzein. 3. Electron spin resonance spectroscopy indicated that 8-hydroxydaidzein was the most potent scavenger of hydroxyl and superoxide anion radicals. Isoflavone metabolites also exhibited higher antioxidant activity than parent compounds in standard antioxidant (FRAP and TEAC) assays. However, for the suppression of nitric oxide production by activated macrophages, genistein showed the highest potency, followed by equol and daidzein. 4. The metabolism of isoflavones affects their free radical scavenging and antioxidant properties, and their cellular activity, but the effects are complex.

Synthesis and Biological Evaluation of New Ozonides with Improved Plant Growth Regulatory Activity

The iron oxyallyl carbocation generated from 2,7-dibromocycloheptanone was induced to undergo [4 + 3] cycloaddition reactions with various furans, affording a series of 12-oxatricyclo-[4.4.1.1(2,5)]-dodec-3-en-11-one adducts. Similar methodology was used to prepare two additional cycloadducts using menthofuran and two homologous aliphatic dibromoketones. Dipolar cycloaddition of ozone to the adducts afforded the corresponding secondary ozonides (i.e., 1,2,4-trioxolanes) in variable yields. Ozonides were investigated by quantum mechanics at the B3LYP/6-31+G* level to study structural features including close contacts which may be responsible for enhancing ozonide stability. The effect of these ozonides and their corresponding precursor cycloadducts upon radicle growth of both Sorghum bicolor and Cucumis sativus was evaluated at 5.0 x 10(-4) mol L-1. The most active cycloadducts and ozonides were also evaluated against the weed species Ipomoea grandifolia and Brachiaria decumbens, and the results are discussed. Compared to ozonides previously synthesized in our laboratory, the new ozonides described herein present improved plant growth regulatory activity.

Synthesis and structure-activity relationship studies of CD4 down-modulating cyclotriazadisulfonamide (CADA) analogues

HIV attachment via the CD4 receptor is an important target for developing novel approaches to HIV chemotherapy. Cyclotriazadisulfonamide (CADA) inhibits HIV at submicromolar levels by specifically down-modulating cell-surface and intracellular CD4. An effective five-step synthesis of CADA in 30% overall yield is reported. This synthesis has also been modified to produce more than 50 analogues. Many tail-group analogues have been made by removing the benzyl tail of CADA and replacing it with various alkyl, acyl, alkoxycarbonyl and aminocarbonyl substituents. A series of sidearm analogues, including two unsymmetrical compounds, have also been prepared by modifying the CADA synthesis, replacing the toluenesulfonyl sidearms with other sulfonyl groups. Testing 30 of these compounds in MT-4 cells shows a wide range of CD4 down-modulation potency, which correlates with ability to inhibit HIV-1. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) approaches. The X-ray crystal structures of four compounds, including CADA, show the same major conformation of the central 12-membered ring. The solid-state structure of CADA was energy minimized and used to generate the remaining 29 structures, which were similarly minimized and aligned to produce the 3D-QSAR models. Both models indicate that steric bulk of the tail group, and, to a lesser extent, the sidearms mainly determine CD4 down-modulation potency in this series of compounds.

X-ray crystallographic study of DNA duplex cross-linking: simultaneous binding to two d(CGTACG)(2) molecules by a bis(9-aminoacridine-4-carboxamide) derivative

Acridine-4-carboxamides form a class of known DNA mono-intercalating agents that exhibit cytotoxic activity against tumour cell lines due to their ability to inhibit topoisomerases. Previous studies of bis-acridine derivatives have yielded equivocal results regarding the minimum length of linker necessary between the two acridine chromophores to allow bis-intercalation of duplex DNA. We report here the 1.7 angstrom resolution X-ray crystal structure of a six-carbon-linked bis(acridine-4-carboxamide) ligand bound to d(CGTACG)(2) molecules by non-covalent duplex cross-linking. The asymmetric unit consists of one DNA duplex containing an intercalated acridine-4-carboxamide chromophore at each of the two CG steps. The other half of each ligand is bound to another DNA molecule in a symmetry-related manner, with the alkyl linker threading through the minor grooves. The two crystallographically independent ligand molecules adopt distinct side chain interactions, forming hydrogen bonds to either O6 or N7 on the major groove face of guanine, in contrast to the semi-disordered state of mono-intercalators bound to the same DNA molecule. The complex described here provides the first structural evidence for the non-covalent cross-linking of DNA by a small molecule ligand and suggests a possible explanation for the inconsistent behaviour of six-carbon linked bis-acridines in previous assays of DNA bis-intercalation.

Correlation-type structure activity relationships for the kinetics of gas-phase RO2 self-reactions and reaction with HO2

Structure activity relationships (SARs) are presented for the gas-phase reactions of RO2 with HO2, and the self- and cross-reactions of RO2. For RO2+HO2 the SAR is based upon a correlation between the logarithm of the measured rate coefficient and a calculated ionisation potential for the molecule R-CH=CH2, R being the same group in both the radical and molecular analogue. The correlation observed is strong and only for one RO2 species does the measured rate coefficient deviate by more than a factor of two from the linear least-squares regression line. For the self- and cross-reactions of RO2 radicals, the SAR is based upon a correlation between the logarithm of the measured rate coefficient and the calculated electrostatic potential (ESP) at the equivalent carbon atom in the RH molecule to which oxygen is attached in RO2, again R being the same group in the molecule and the radical. For cases where R is a simple alkyl-group, a strong linear correlation observed. For RO2 radicals which contain lone pair-bearing substituents and for which the calculated ESP<-0.05 self-reaction rate coefficients appear to be insensitive to the value of the ESP. For RO2 of this type with ESP>-0.05 a linear relationship between log k and the ESP is again observed. Using the relationships, 84 out of the 85 rate coefficients used to develop the SARs are predicted to within a factor of three of their measured values. A relationship is also presented that allows the prediction of the Arrhenius parameters for the self-reactions of simple alkyl RO2 radicals. On the basis of the correlations, predictions of room-temperature rate coefficients are made for a number of atmospherically important peroxyl-peroxyl radical reactions. (C) 2003 Elsevier Ltd. All rights reserved.

Gas-phase rate coefficients for reactions of NO3, OH, O-3 and O(P-3) with unsaturated alcohols and ethers: correlations and structure-activity relations (SARs)

Experimental difficulties sometimes force modellers to use predicted rate coefficients for reactions of oxygenated volatile organic compounds (oVOCs). We examine here methods for making the predictions for reactions of atmospheric initiators of oxidation, NO3, OH, O-3 and O(P-3), with unsaturated alcohols and ethers. Logarithmic correlations are found between measured rate coefficients and calculated orbital energies, and these correlations may be used directly to estimate rate coefficients for compounds where measurements have not been performed. To provide a shortcut that obviates the need to calculate orbital energies, structure-activity relations (SARs) are developed. Our SARs are tested for predictive power against compounds for which experimental rate coefficients exist, and their accuracy is discussed. Estimated atmospheric lifetimes for oVOCs are presented. The SARs for alkenols successfully predict key rate coefficients, and thus can be used to enhance the scope of atmospheric models incorporating detailed chemistry. SARs for the ethers have more limited applicability, but can still be useful in improving tropospheric models. (C) 2008 Elsevier Ltd. All rights reserved.