Maurice J. Sauer

Oestrogenic activity of parabens in MCF7 human breast cancer cells

Parabens (4-hydroxybenzoic acid esters) have been recently reported to have oestrogenic activity in yeast cells and animal models. Since the human population is exposed to parabens through their widespread use as preservatives in foods, pharmaceuticals and cosmetics, we have investigated here whether oestrogenic activity of these compounds can also be detected in oestrogen-sensitive human cells. We report on the oestrogenic effects of four parabens (methylparaben, ethylparaben, n-propylparaben, n-butylparaben) in oestrogen-dependent MCF7 human breast cancer cells. Competitive inhibition of [3H]oestradiol binding to MCF7 cell oestrogen receptors could be detected at 1,000,000-fold molar excess of n-butylparaben (86%), n-propylparaben (77%), ethyl-paraben (54%) and methylparaben (21%). At concentrations of 10(-6)M and above, parabens were are able to increase expression of both transfected (ERE-CAT reporter gene) and endogenous (pS2) oestrogen-regulated genes in these cells. They could also increase proliferation of the cells in monolayer culture, which could be inhibited by the antiestrogen ICI 182,780, indicating that the effects were mediated through the oestrogen receptor. However, no antagonist activity of parabens could be detected on regulation of cell proliferation by 17 beta-oestradiol at 10(-10)M. Molecular modelling has indicated the mode by which paraben molecules can bind into the ligand binding pocket of the crystal structure of the ligand binding domain (LBD) of the oestrogen receptor alpha (ERalpha) in place of 17beta-oestradiol; it has furthermore shown that two paraben molecules can bind simultaneously in a mode in which their phenolic hydroxyl groups bind similarly to those of the meso-hexoestrol molecule. Future work will need to address the extent to which parabens can accumulate in hormonally sensitive tissues and also the extent to which their weak oestrogenic activity can add to the more general environmental oestrogen problem.

Absolute bioavailability and dose-dependent pharmacokinetic behaviour of dietary doses of the chemopreventive isothiocyanate sulforaphane in rat

Sulforaphane is a naturally occurring isothiocyanate with promising chemopreventive activity. An analytical method, utilising liquid chromatography-MS/MS, which allows the determination of sulforaphane in small volumes of rat plasma following exposure to low dietary doses, was developed and validated, and employed to determine its absolute bioavailability and pharmacokinetic characteristics. Rats were treated with either a single intravenous dose of sulforaphane (2.8 micromol/kg) or single oral doses of 2.8, 5.6 and 28 mumol/kg. Sulforaphane plasma concentrations were determined in blood samples withdrawn from the rat tail at regular time intervals. Following intravenous administration, the plasma profile of sulforaphane was best described by a two-compartment pharmacokinetic model, with a prolonged terminal phase. Sulforaphane was very well and rapidly absorbed and displayed an absolute bioavailability of 82 %, which, however, decreased at the higher doses, indicating a dose-dependent pharmacokinetic behaviour; similarly, Cmax values did not rise proportionately to the dose. At the highest dose used, the rate of absorption constant k(ab), biological half-life t(1/2) and apparent volume of distribution decreased significantly. It is concluded that in the rat orally administered sulforaphane is rapidly absorbed, achieving high absolute bioavailability at low dietary doses, but dose-dependent pharmacokinetics was evident, with bioavailability decreasing with increasing dose.

Trends in the identification of organic residues and contaminants: EC regulations under revision

The use of identification points (IPs) is a new approach to set up quality criteria for the identification of organic residues and contaminants: a laboratory is allowed to use any molecular spectrometric technique or combination of techniques in order to earn a minimum number of points. The system of IPs balances the identification power of the different analytical techniques and has the advantage that new techniques can be introduced very easily.

Modulation of hepatic cytochromes P450 and phase II enzymes by dietary doses of sulforaphane in rats: Implications for its chemopreventive activity.

The principal objectives of our study were to ascertain whether sulforaphane, at dietary levels of intake, modulates rat hepatic cytochrome P450 and phase II enzyme systems and to evaluate the impact of such changes in the chemopreventive activity of this isothiocyanate. Animals were exposed to sulforaphane in their drinking water for 10 days, equivalent to daily doses of 3 and 12 mg/kg. Depentylation of pentoxyresorufin decreased and was paralleled by a decline in CYP2B apoprotein levels. At the higher dose, erythromycin N‐demethylase activity declined and was accompanied by a similar decrease in CYP3A2 apoprotein levels. However, sulforaphane treatment upregulated CYP1A2 levels, determined immunologically, but the dealkylations of methoxy‐ and ethoxyresorufin were not similarly increased. Hepatic S9 preparations from sulforaphane‐treated rats were less effective than control preparations in converting IQ (2‐amino‐3‐methylimidazo‐[4,5‐f]quinoline) to mutagenic intermediates in the Ames test. To clarify the underlying mechanism, in vitro studies were undertaken. In β‐naphthoflavone‐treated rats, the inhibition by sulforaphane of the O‐dealkylations of methoxy‐ and ethoxyresorufin was enhanced if the isothiocyanate was preincubated in the presence of NADPH. It may be inferred that sulforaphane induces hepatic CYP1A2 but the enzyme is not catalytically competent because of bound sulforaphane metabolite(s). Finally, sulforaphane stimulated, in a dose‐dependent fashion, quinone reductase but failed to influence glutathione S‐transferase, epoxide hydrolase and glucuronosyl transferase activities. It is concluded that, even at dietary doses, sulforaphane can modulate the xenobiotic‐metabolising enzyme systems, shifting the balance of carcinogen metabolism toward deactivation, and this may be an important mechanism of its chemopreventive activity.

Absolute bioavailability of [14C] genistein in the rat; plasma pharmacokinetics of parent compound, genistein glucuronide and total radioactivity

SummaryThe systemic plasma pharmacokinetics of genistein were determined in rats to evaluate the absolute oral bioavailability and make comparison with similar data in the literature derived from humans subjects. The plasma concentrations of genistein, genistein glucuronide and carbon-14 were determined by LC-MS/MS and liquid scintillation counting following oral and intravenous dosing with [14C]genistein (4 mg kg−1 body weight). The absorption of total radioactivity from the gut, (parent compound and metabolites), was 56 and 111% in male and female rats, respectively. In contrast, the absolute oral bioavailability of genistein in male and female rats was 7 and 15%. There was a significant (P<0.001) difference between Cmax of genistein after intravenous (6921 and 4392 ng/ml) and oral (21 and 22 ng/ml) dosing in male and female rats, respectively. After oral administration, the concentration profile of genistein glucuronide in plasma greatly exceeded that of parent compound during the absorption/distribution phase suggesting extensive first pass metabolism, and provided evidence of entero-hepatic circulation. Selective plasma analysis by LC-MS/MS, without prior enzymatic hydrolysis, enabled ready discrimination between parent and conjugated metabolites and prevented gross overestimation of genistein bioavailability. Pharmacokinetic parameters Cmax, Tmax and AUC were similar to those reported in humans, which supports the use of the rat model for genistein toxicity studies.

A study of the expression of the xenobiotic-metabolising cytochrome P450 proteins and of testosterone metabolism in bovine liver.

The expression of xenobiotic-metabolising cytochrome P450 proteins in the liver of cattle was determined using substrate probes and immunologically by Western blot analysis. Compared to the rat, cattle displayed much higher coumarin 7-hydroxylase (CYP2A) and ethoxyresorufin O-deethylase (CYP1) activity but, in contrast, it exhibited much lower debrisoquine 4-hydroxylase (CYP2D) and lauric acid hydroxylase activities (CYP4A). The ethoxyresorufin O-deethylase activity was markedly inhibited by furafylline and a-naphthoflavone, and coumarin 7-hydroxylase by 8-methoxypsoralen. Immunoblot analysis employing antibodies to rat CYP1A1 recognised two immunorelated proteins in bovine liver whose expression appeared to be higher compared with rat. Kinetic studies indicated that a single enzyme is likely to be responsible for the O-deethylation of 7-ethoxyresorufin in bovine liver. When bovine microsomes were probed with antibodies to rat CYP2A2, a single protein was detected in cattle liver. Kinetic analysis followed by construction of Eadie-Hofstee plots indicated that more than one enzyme contributes to the 7-hydroxylation of coumarin. Immunoblot analysis employing antibodies to human CYP2D6 and rat CYP4A1 revealed in both cases a single, poorly expressed immunoreacting band in bovine microsomes. Similar immunoblot studies detected proteins in cattle liver immunorelated to the CYP2B, CYP2C, CYP2E, and CYP3A subfamilies. Bovine microsomes metabolised testosterone but, in contrast to the rat, failed to produce 2alpha- and 16alpha-hydroxytestosterone. On the other hand, bovine microsomes produced levels of another hydroxylated metabolite, possibly 12-hydroxytestosterone. In conclusion, results emanating from this study indicate the presence of proteins in the cattle liver belonging to all the xenobiotic-metabolising families of cytochrome P450.

Repeated intake of broccoli does not lead to higher plasma levels of sulforaphane in human volunteers

The plasma pharmacokinetic characteristics of the chemopreventive isothiocyanate sulforaphane were determined in six human volunteers following single and repeated intake of raw broccoli. Initially, an analytical method utilising LC-MS/MS, capable of determining low levels of sulforaphane in human plasma was developed and validated. The plasma profile of the isothiocyanate best fitted a two-compartment pharmacokinetic model. Sulforaphane was rapidly absorbed with peak plasma levels being attained within 1.5h, and was characterised by a long terminal elimination phase. Repeated intake of broccoli had no impact on the pharmacokinetic behaviour or plasma levels of sulforaphane, and there was no evidence of accumulation.

Modulation of rat pulmonary carcinogen-metabolising enzyme systems by the isothiocyanates erucin and sulforaphane.

The objective of this study was to evaluate the potential of the structurally related aliphatic isothiocyanates erucin and sulforaphane to modulate the pulmonary carcinogen-metabolising enzyme systems in rat lung, a target organ of their chemopreventive activity. Precision-cut rat lung slices were prepared and incubated for 24 h with a range of concentrations of either erucin or sulforaphane, up to 50microM. Neither compound modulated the O-deethylation of ethoxyresorufin whereas they elevated markedly CYP1A1 and, to a lesser extent, CYP1B1 apoprotein levels. Neither compound influenced the O-depentylation of pentoxyresorufin or CYP2B apoprotein levels, but sulforaphane caused a modest increase in CYP3A2 apoprotein levels. Pulmonary quinone reductase activity, monitored using 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide as substrate, was markedly up-regulated by both compounds and was paralleled by a similar rise in protein levels. Both compounds increased cytosolic glutathione S-transferase activity, measured using 1-chloro-2,4-dinitrobenzene as the accepting substrate; a modest rise was seen in GSTalpha protein levels, determined immunologically, whereas GSTpi levels were un-affected by the same treatment. Finally, both erucin and sulforaphane increased total glutathione concentration in lung cytosol. It is concluded that these aliphatic isothiocyanates have the potential to antagonise the carcinogenicity of pulmonary carcinogens by stimulating the in situ detoxication of their DNA-binding genotoxic metabolites.

Melanin as an adsorbent for drug residues

The binding of seven veterinary drugs (clenbuterol, chlorpromazine, diethylstilbestrol, 19-nortestosterone, salbutamol, salicylic acid and trenbolone) to melanin from Sepia officinalis was investigated. Basic and hydrophobic drugs were the most strongly bound. Desorption by ethanol was complete for neutral drugs but only partial for the basic drugs, which suggests that binding of the latter involves an ionic component. A method of synthesizing melanin in an immobilized form (melanin-PS) on the surface of porous silica was developed. When the drug binding properties of melanin-PS were investigated, its capacity to bind the basic drug clenbuterol was found to be higher (5.9 nmol mg-1) than that for the neutral hydrophobic drug 19-nortestosterone (0.56 nmol mg-1); for both drugs the attainment of binding equilibrium with melanin-PS was relatively rapid (< 5 min). By virtue of its binding kinetics, high capacity and mechanical robustness, melanin-PS offers potential for use in chromatography or solid-phase extraction and may additionally enable modelling of drug-melanin interactions.

Multi-residue analysis of avermectins and moxidectin by ion-trap LC-MSn

A multi-residue method was developed and validated for the quantitation and confirmation of avermectins and moxidectin residues in bovine liver. Target analytes were extracted from liver homogenate using C8 solid phase cartridges, chromatographed under basic pH conditions in order to promote the formation of analyte anions, and detected by ion-trap mass spectrometry (MS) in negative ion mode using an atmospheric pressure chemical ionization interface (APCI). The method provided detection capabilities (CC beta, where beta = 0.05) for eprinomectin, abamectin, doramectin, moxidectin and ivermectin of 3.1, 3.2, 2.2, 4.0 and 3.2 ng g-1 liver respectively, well below their respective maximum residue limits (MRLs). The critical concentrations for MRL compliance (CC alpha, where alpha = 0.01) were 840, 28, 130, 130 and 130 ng g-1 respectively. Analysis of liver fortified at the appropriate MRLs gave recoveries (% +/- RSD) of 70.9 +/- 11.6 (n = 14), 69.1 +/- 3.9 (n = 13), 65.9 +/- 6.4 (n = 19), 69.7 +/- 9.3 (n = 19) and 73.2 +/- 10.5 (n = 19), respectively, for each analyte. Calibration curves fitted a second order polynomial function (R2 > or = 0.9978) over a wide range of concentrations (0 to 10,000 ng ml-1). The detection of two daughter-ions for each analyte allowed for quantitation and the confirmation of identity. The method is suitable for application in European Union statutory veterinary drug residue surveillance programmes, since it fulfills appropriate analytical criteria, and has the particular advantage of enabling high throughput multi-residue quantitation and confirmation of the target analytes.