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PubMed1996

Flavin motion in p-hydroxybenzoate hydroxylase. Substrate and effector specificity of the Tyr22-->Ala mutant.

The side chain of Tyr222 in p-hydroxybenzoate hydroxylase interacts with the carboxy moiety of the substrate. Studies on the Tyr222-->Phe mutant, [F222]p-hydroxybenzoate hydroxylase, have shown that disruption of this interaction hampers the hydroxylation of 4-hydroxybenzoate. Tyr222 is possibly involved in flavin motion, which may facilitate the exchange of substrate and product during catalysis. To elucidate the function of Tyr222 in more detail, in the present study the substrate and effector specificity of the Tyr222-->Ala mutant, [A222]p-hydroxybenzoate hydroxylase, was investigated. Replacement of Tyr222 by Ala impairs the binding of the physiological substrate 4-hydroxybenzoate and the substrate analog 4-aminobenzoate. With these compounds, [A222]p-hydroxybenzoate hydroxylase mainly acts as a NADPH oxidase. [A222]p-hydroxybenzoate hydroxylase tightly interacts with 2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate. Crystallographic data [Schreuder, H.A., Mattevi, A., Oblomova, G., Kalk, K.H., Hol, W.G.J., van der Bolt, F.J.T. & van Berkel, W.J.H. (1994) Biochemistry 33, 10161-10170] suggest that this is due to motion of the flavin ring out of the active site, allowing hydrogen-bond interaction between the 2-hydroxy group of the substrate analogs and N3 of the flavin. [A222]p-Hydroxybenzoate hydroxylase produces about 0.6 mol 2,3,4-trihydroxybenzoate from 2,4-dihydroxybenzoate/mol NADPH oxidized. This indicates that reduction of the Tyr222-->Ala mutant shifts the equilibrium of flavin conformers towards the productive "in' position. [A222]p-Hydroxybenzoate hydroxylase converts 2-fluoro-4-hydroxybenzoate to 2-fluoro-3,4-dihydroxybenzoate. The regioselectivity of hydroxylation suggests that [A222]p-hydroxybenzoate hydroxylase binds the fluorinated substrate in the same orientation as wild-type. Spectral studies suggest that wild-type and [A222]p-hydroxybenzoate hydroxylase bind 2-fluoro-4-hydroxybenzoate in the phenolate form with the flavin ring preferring the "out' conformation. Despite activation of the fluorinated substrate and in contrast to the wild-type enzyme, [A222]p-hydroxybenzoate hydroxylase largely produces hydrogen peroxide. The effector specificity of p-hydroxybenzoate hydroxylase is not changed by the Tyr222-->Ala replacement. This supports the idea that the effector specificity is mainly dictated by the protein-substrate interactions at the re-side of the flavin ring.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2001

Effects of mono-, di- and tri-hydroxybenzoic acids on the nitrosation of propranolol: structure-activity relationship.

Nitrosation of propranolol under standard conditions recommended by the World Health Organization (10mM propranolol hydrochloridre, 40mM sodium nitrite, pH 3.5) was performed in the absence and in the presence of benzoic acid and of twelve mono-, di- and tri-hydroxybenzoic acids, added to the nitrosation mixture in concentrations ranging from 2 to 40mM, in order to examine their effect on the nitrosation reaction. The yield of N-nitrosopropranol (NOP) was reduced by benzoic acid and, with potency decreasing in the following order, by 2,3,4-tri-hydroxybenzoic acid>/=3,4-tri-hydroxybenzoic acid>2,5-di-hydroxybenzoic acid>2,3-di-hydroxybenzoic acid>3-hydroxybenzoic acid>2-hydroxybenzoic acid>3,4,5-tri-hydroxybenzoic acid>4-hydroxybenzoic acid; their inhibiting effect was concentration-dependent. In contrast, 2,4-di-hydroxybenzoic acid, 2,6-di-hydroxybenzoic acid and 2,4,6-tri-hydroxybenzoic acid caused an increase in the yield of NOP that was inversely related to their concentration. 3,5-Di-hydroxybenzoic acid was substantially inactive. These findings indicate that, depending on the positions of carboxyl group and hydroxyl groups on the benzene ring, mono-, di- and tri-hydroxybenzoic acids may inhibit or hasten nitrosation reactions. As compared with benzenediols and benzenetriols [Mutat. Res. 398 (1998) 75], hydroxybenzoic acids inhibit the nitrosation of propranolol to a greater extent and have the advantage of being nonmutagenic and less toxic.

Area Under Curve↗
PubMed1989

Involvement of coenzyme A thioesters in anaerobic metabolism of 4-hydroxybenzoate by Rhodopseudomonas palustris.

The initial steps of anaerobic 4-hydroxybenzoate degradation were studied in whole cells and cell extracts of the photosynthetic bacterium Rhodopseudomonas palustris. Illuminated suspensions of cells that had been grown anaerobically on 4-hydroxybenzoate and were assayed under anaerobic conditions took up [U-14C]4-hydroxybenzoate at a rate of 0.6 nmol min-1 mg of protein-1. Uptake occurred with high affinity (apparent Km = 0.3 microM), was energy dependent, and was insensitive to external pH in the range of 6.5 to 8.2 Very little free 4-hydroxybenzoate was found associated with cells, but a range of intracellular products was formed after 20-s incubations of whole cells with labeled substrate. When anaerobic pulse-chase experiments were carried out with cells incubated on ice or in darkness, 4-hydroxybenzoyl coenzyme A (4-hydroxybenzoyl-CoA) was formed early and disappeared immediately after addition of excess unlabeled substrate, as would be expected of an early intermediate in 4-hydroxybenzoate metabolism. A 4-hydroxybenzoate-CoA ligase activity with an average specific activity of 0.7 nmol min-1 mg of protein-1 was measured in the soluble protein fraction of cells grown anaerobically on 4-hydroxybenzoate. 4-Hydroxybenzoyl-CoA was the sole product formed from labeled 4-hydroxybenzoate in the ligase reaction mixture. 4-Hydroxybenzoate uptake and ligase activities were present in cells grown anaerobically with benzoate, 4-hydroxybenzoate, and 4-aminobenzoate and were not detected in succinate-grown cells. These results indicate that the high-affinity uptake of 4-hydroxybenzoate by R. palustris is due to rapid conversion of the free acid to its CoA derivative by a CoA ligase and that this is also the initial step of anaerobic 4-hydroxybenzoate degradation.

Acyl Coenzyme A↗
PubMed1993

Enzymes of anaerobic metabolism of phenolic compounds. 4-Hydroxybenzoate-CoA ligase from a denitrifying Pseudomonas species.

The initial step of anaerobic 4-hydroxybenzoate and 3-hydroxybenzoate degradation was studied in a denitrifying Pseudomonas sp. 4'-Hydroxybenzoate and 3-hydroxybenzoate are converted into their coenzyme A (CoA) thioesters by two different specific coenzyme A ligases. 4-Hydroxybenzoate-CoA ligase (AMP-forming) was purified 350-fold. The ligase is active as a monomer of molecular mass 48 kDa, as determined by gel filtration and SDS/PAGE. At a pH optimum of 8.5, the apparent Km values for 4-hydroxybenzoate, ATP, and coenzyme A are 37 microM, 77 microM, and 125 microM, respectively. The enzyme reacts specifically with 4-hydroxybenzoate (100%) and 4-aminobenzoate (30%). Other analogues of benzoate, notably 3- or 2-hydroxybenzoate, are inactive, and 2,4-dihydroxybenzoate and 2-hydroxy-4-methylbenzoate act as competitive inhibitors (Ki = 1 microM). Polyclonal antibodies were raised and used in immunoblot assays to study the regulation of the expression of 4-hydroxybenzoate-CoA ligase. The ligase is synthesized when cells are grown anaerobically with 4-hydroxybenzoate, phenol, or p-cresol; phenol and p-cresol are degraded via 4-hydroxybenzoate. The enzyme is not present in cells grown aerobically with 4-hydroxybenzoate or anaerobically with benzoate or 4-hydroxyphenylacetate.

Amino Acid Sequence↗
PubMed1995

Repression of 4-hydroxybenzoate transport and degradation by benzoate: a new layer of regulatory control in the Pseudomonas putida beta-ketoadipate pathway.

Pseudomonas putida PRS2000 degrades the aromatic acids benzoate and 4-hydroxybenzoate via two parallel sequences of reactions that converge at beta-ketoadipate, a derivative of which is cleaved to form tricarboxylic acid cycle intermediates. Structural genes (pca genes) required for the complete degradation of 4-hydroxybenzoate via the protocatechuate branch of the beta-ketoadipate pathway have been characterized, and a specific transport system for 4-hydroxybenzoate has recently been described. To better understand how P. putida coordinates the processes of 4-hydroxybenzoate transport and metabolism to achieve complete degradation, the regulation of pcaK, the 4-hydroxybenzoate transport gene, and that of pcaF, a gene required for both benzoate and 4-hydroxybenzoate degradation, were compared. Primer extension analysis and lacZ fusions showed that pcaK and pcaF, which are adjacent on the chromosome, are transcribed independently. PcaR, a transcriptional activator of several genes of the beta-ketoadipate pathway, is required for expression of both pcaF and pcaK, and the pathway intermediate beta-ketoadipate induces both genes. In addition to these expected regulatory elements, expression of pcaK, but not pcaF, is repressed by benzoate. This previously unrecognized layer of regulatory control in the beta-ketoadipate pathway appears to extend to the first two steps of 4-hydroxybenzoate degradation, since levels of 4-hydroxybenzoate hydroxylase and protocatechuate 3,4-dioxygenase activities were also depressed when cells were grown on a mixture of 4-hydroxybenzoate and benzoate. The apparent consequence of benzoate repression is that cells degrade benzoate in preference to 4-hydroxybenzoate. These findings indicate that 4-hydroxybenzoate transport is an integral feature of the beta-ketoadipate pathway in P. putida and that transport plays a role in establishing the preferential degradation of benzoate over 4-hydroxybenzoate. These results also demonstrate that there is communication between the two branches of the beta-ketoadipate pathway.

Acetyl-CoA C-Acyltransferase↗
PubMed2000

[Competitive binding of some alkyl p-hydroxybenzoates to human estrogen receptor alpha and beta].

Alkyl p-hydroxybenzoates such as isobutyl p-hydroxybenzoate (PHBA-iBu), butyl p-hydroxybenzoate (PHBA-nBu), isopropyl p-hydroxybenzoate (PHBA-iPr), propyl p-hydroxybenzoate (PHBA-nPr), ethyl p-hydroxybenzoate (PHBA-Et), and methyl p-hydroxybenzoate (PHBA-Me) are widely used as preservatives, stabilizers and antiseptics for medical supplies, cosmetics, foodstuffs etc. We determined the binding affinity of alkyl p-hydroxybenzoates to human estrogen receptor alpha (ER alpha) and beta (ER beta) by non-RI receptor binding assays. PHBA-iBu had a high binding affinity for ER alpha (IC50: 6.0 x 10(-6) M, the relative binding affinity (RBA): 0.267) and ER beta (IC50: 5.0 x 10(-6) M, RBA: 0.340). These IC50 values and RBA were almost the same as those of bisphenol A. The ranking of the estrogenic potency of alkyl p-hydroxybenzoates for both ERs is different; that is, PHBA-iBu > PHBA-nBu[symbol: see text]PHBA-iPr[symbol: see text]PHBA-nPr > PHBA-Et >> PHBA-Me. Alkyl p-hydroxybenzoates bound with equal relative affinity to both ER alpha and beta proteins. Alkyl p-hydroxybenzoate having a long alkyl side-chain showed a high affinity for ER alpha and beta. These findings suggest that p-hydroxybenzoates may be endocrine disruptors.

Binding, Competitive↗
PubMed2001

Anaerobic metabolism of 3-hydroxybenzoate by the denitrifying bacterium Thauera aromatica.

The anaerobic metabolism of 3-hydroxybenzoate was studied in the denitrifying bacterium Thauera aromatica. Cells grown with this substrate were adapted to grow with benzoate but not with 4-hydroxybenzoate. Vice versa, 4-hydroxybenzoate-grown cells did not utilize 3-hydroxybenzoate. The first step in 3-hydroxybenzoate metabolism is a coenzyme A (CoA) thioester formation, which is catalyzed by an inducible 3-hydroxybenzoate-CoA ligase. The enzyme was purified and characterized. Further metabolism of 3-hydroxybenzoyl-CoA by cell extract required MgATP and was coupled to the oxidation of 2 mol of reduced viologen dyes per mol of substrate added. Purification of the 3-hydroxybenzoyl-CoA reducing enzyme revealed that this activity was due to benzoyl-CoA reductase, which reduced the 3-hydroxy analogue almost as efficiently as benzoyl-CoA. The further metabolism of the alicyclic dienoyl-CoA product containing the hydroxyl substitution obviously required additional specific enzymes. Comparison of the protein pattern of 3-hydroxybenzoate-grown cells with benzoate-grown cells revealed several 3-hydroxybenzoate-induced proteins; the N-terminal amino acid sequences of four induced proteins were determined and the corresponding genes were identified and sequenced. A cluster of six adjacent genes contained the genes for substrate-induced proteins 1 to 3; this cluster may not yet be complete. Protein 1 is a short-chain alcohol dehydrogenase. Protein 2 is a member of enoyl-CoA hydratase enzymes. Protein 3 was identified as 3-hydroxybenzoate-CoA ligase. Protein 4 is another member of the enoyl-CoA hydratases. In addition, three genes coding for enzymes of beta-oxidation were present. The anaerobic 3-hydroxybenzoate metabolism here obviously combines an enzyme (benzoyl-CoA reductase) and electron carrier (ferredoxin) of the general benzoyl-CoA pathway with enzymes specific for the 3-hydroxybenzoate pathway. This raises some questions concerning the regulation of both pathways.

Acyl Coenzyme A↗
PubMed1979

Kinetic studies on the reaction of p-hydroxybenzoate hydroxylase. Agreement of steady state and rapid reaction data.

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens is a NADPH-dependent, FAD-containing monooxygenase catalyzing the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate in the presence of NADPH and molecular oxygen. The mechanism of this three-substrate reaction was investigated in detail at pH 6.6, 4 degrees C, by steady state kinetics, stopped flow spectrophotometry, and equilibrium binding experiments. The initial velocity patterns are consistent with a ping-pong type mechanism which involves two ternary complexes between the enzyme and substrates. The first ternary complex is formed by random addition of p-hydroxybenzoate and NADPH to the enzyme, followed by the release of the first product (NADP+). The reduced enzyme . p-hydroxybenzoate complex now reacts with oxygen, the third substrate, to form the second ternary complex. The enzyme-bound p-hydroxybenzoate then reacts with the activated oxygen to give 3,4-dihydroxybenzoate which is released regenerating the oxidized enzyme for the next cycle. The binding of p-hydroxybenzoate to the oxidized enzyme to form a 1:1 complex causes large, characteristic spectral perturbations and fluorescence quenching. The dissociation constant for the enzyme . substrate complex was obtained by titrations in which absorbance and/or fluorescence quenching was measured. The binding constants of NADPH to the enzyme with and without p-hydroxybenzoate were determined kinetically by measuring the rate of reduction of the enzyme at different concentrations of NADPH. The reduction of the enzyme proceeds extremely slowly in the absence of p-hydroxybenzoate. The presence of the substrate causes a dramatic stimulation (140,000-fold) in the rate of enzyme reduction. The anaerobic reduction of the enzyme by NADPH in the presence of p-hydroxybenzoate produces a transient charge-transfer intermediate. On the basis of the proposed mechanism, the dissociation constants for p-hydroxybenzoate and NADPH as well as the Michaelis constants for all the three substrates were calculated from the initial velocity data. The agreement obtained between various kinetic parameters from the initial rate measurements and those calculated from the individual rate constants determined in rapid reactions, strongly supports the proposed mechanism for the p-hydroxybenzoate hydroxylase reaction.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1990

Catabolism of 3-hydroxybenzoate by the gentisate pathway in Klebsiella pneumoniae M5a1.

Growth of Klebsiella pneumoniae M5a1 on 3-hydroxybenzoate leads to the induction of 3-hydroxybenzoate monooxygenase, 2,5-dihydroxybenzoate dioxygenase, maleylpyruvate isomerase and fumaryl-pyruvate hydrolase. Growth in the presence of 2,5-dihydroxybenzoate also induces all of these enzymes including the 3-hydroxybenzoate monooxygenase which is not required for 2,5-dihydroxybenzoate catabolism. Mutants defective in 3-hydroxybenzoate monooxygenase fail to grow on 3-hydroxybenzoate but grow normally on 2,5-dihydroxybenzoate. Mutants lacking maleylpyruvate isomerase fail to grow on 3-hydroxybenzoate and 2,5-dihydroxybenzoate. Both kinds of mutants grow normally on 3,4-dihydroxybenzoate. Mutants defective in maleylpyruvate isomerase accumulate maleylpyruvate when exposed to 3-hydroxybenzoate and growth is inhibited. Secondary mutants that have additionally lost 3-hydroxybenzoate monooxygenase are no longer inhibited by the presence of 3-hydroxybenzoate. The 3-hydroxybenzoate monooxygenase gene (mhbM) and the maleylpyruvate isomerase gene (mhbI) are 100% co-transducible by P1 phage.

Chromatography, Ion Exchange↗
PubMed1991

Catalytic properties of phenol carboxylase. In vitro study of CO2: 4-hydroxybenzoate isotope exchange reaction.

Phenol is metabolized in a denitrifying bacterium in the absence of molecular oxygen via para-carboxylation to 4-hydroxybenzoate (biological Kolbe-Schmitt synthesis). The enzyme system catalyzing the presumptive carboxylation of phenol, tentatively named 'phenol carboxylase', catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate (specific activity 0.1 mumol 14CO2 incorporated into 4-hydroxybenzoate x min-1 x mg-1 cell protein) which is considered a partial reaction of the overall enzyme catalysis; 14C from [14C]phenol was not exchanged into 4-hydroxybenzoate ring positions to a significant extent. The 14CO2 isotope exchange reaction was studied in vitro. The reaction was dependent on the substrates CO2 and 4-hydroxybenzoate and required K+ and Mn2+. The actual substrate was CO2 rather than HCO3-. The apparent Km values were 1 mM dissolved CO2, 0.2 mM 4-hydroxybenzoate, 2 mM K+, and 0.1 mM Mn2+. The cationic cocatalysts could be substituted by ions of similar ionic radius: K+ could be replaced to some extent by Rb+, but not by Li+, Na+, Cs+, or NH4+; Mn2+ could be replaced to some extent by Fe2+ greater than Mg2+, Co2+, but not by Ni2+, Zn2+, Ca2+, or Cu2+. The exchange reaction was not strictly specific for 4-hydroxybenzoate, however it required a p-hydroxyl group; derivatives of 4-hydroxybenzoate with OH, CH3 or Cl substituents in m-position did react, whereas those with substitutions in the o-position were inactive or were inhibitory. The enzyme was induced when cells were grown on phenol, but not on 4-hydroxybenzoate. Comparison of SDS/PAGE protein patterns of cells grown on phenol or 4-hydroxybenzoate revealed several additional protein bands in phenol-grown cells. The possible role of similar enzymes in the anaerobic metabolism of phenolic compounds is discussed.

Carbon Dioxide↗
PubMed1994

Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate.

Pseudomonas putida PRS2000 is chemotactic to 4-hydroxybenzoate and other aromatic acids. This behavioral response is induced when cells are grown on 4-hydroxybenzoate or benzoate, compounds that are degraded via the beta-ketoadipate pathway. Isolation of a transposon mutant defective in 4-hydroxybenzoate chemotaxis allowed identification of a new gene cluster designated pcaRKF. DNA sequencing, mutational analysis, and complementation studies revealed that pcaR encodes a regulatory protein required for induction of at least four of the enzymes of the beta-ketoadipate pathway and that pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway. The third gene, pcaK, encodes a transporter for 4-hydroxybenzoate, and this protein is also required for chemotaxis to aromatic acids. The predicted PcaK protein is 47 kDa in size, with a deduced amino acid sequence indicative of membership in the major facilitator superfamily of transport proteins. The protein, expressed in Escherichia coli, catalyzed 4-hydroxybenzoate transport. In addition, whole cells of P. putida pcaK mutants accumulated 4-hydroxybenzoate at reduced rates compared with that in wild-type cells. The pcaK mutation did not impair growth at the expense of 4-hydroxybenzoate under most conditions; however, mutant cells grew somewhat more slowly than the wild type on 4-hydroxybenzoate at a high pH. The finding that 4-hydroxybenzoate chemotaxis can be disrupted without an accompanying effect on metabolism indicates that this chemotactic response is receptor mediated. It remains to be determined, however, whether PcaK itself is a chemoreceptor for 4-hydroxybenzoate or whether it plays an indirect role in chemotaxis. These findings indicate that aromatic acid detection and transport are integral features of aromatic degradation pathways.

Acetyl-CoA C-Acyltransferase↗
PubMed1996

4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3. Purification, characterization, gene cloning, sequence analysis and assignment of structural features determining the coenzyme specificity.

4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was purified by five consecutive steps to apparent homogeneity. The enrichment was 50-fold with a yield of about 20%. The enzyme is a homodimeric flavoprotein monooxygenase with each 44-kDa polypeptide chain containing one FAD molecule as a rather weakly bound prosthetic group. In contrast to other 4-hydroxybenzoate hydroxylases of known primary structure, the enzyme preferred NADH over NADPH as electron donor. The pH optimum for catalysis was pH 8.0 with a maximum turnover rate around 45 degrees C. Chloride ions were inhibitory, and competitive with respect to NADH. 4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 has a narrow substrate specificity. In addition to the transformation of 4-hydroxybenzoate to 3,4-dihydroxybenzoate, the enzyme converted 2-fluoro-4-hydroxybenzoate, 2-chloro-4-hydroxybenzoate, and 2,4-dihydroxybenzoate. With all aromatic substrates, no uncoupling of hydroxylation was observed. The gene encoding 4-hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was cloned in Escherichia coli. Nucleotide sequence analysis revealed an open reading frame of 1182 bp that corresponded to a protein of 394 amino acid residues. Upstream of the pobA gene, a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The deduced amino acid sequence of Pseudomonas sp. CBS3 4-hydroxybenzoate hydroxylase revealed 53% identity with that of the pobA enzyme from Pseudomonas fluorescens for which a three-dimensional structure is known. The active-site residues and the fingerprint sequences associated with FAD binding are strictly conserved. This and the conservation of secondary structures implies that the enzymes share a similar three-dimensional fold. Based on an isolated region of sequence divergence and site-directed mutagenesis data of 4-hydroxybenzoate hydroxylase from P. fluorescens, it is proposed that helix H2 is involved in determining the coenzyme specificity.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMedDate not supplied

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 [(3)H]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.

Binding, Competitive↗