Paraben esters: review of recent studies of endocrine toxicity, absorption, esterase and human exposure, and discussion of potential human health risks

This toxicology update reviews research over the past four years since publication in 2004 of the first measurement of intact esters of p-hydroxybenzoic acid (parabens) in human breast cancer tissues, and the suggestion that their presence in the human body might originate from topical application of bodycare cosmetics. The presence of intact paraben esters in human body tissues has now been confirmed by independent measurements in human urine, and the ability of parabens to penetrate human skin intact without breakdown by esterases and to be absorbed systemically has been demonstrated through studies not only in vitro but also in vivo using healthy human subjects. Using a wide variety of assay systems in vitro and in vivo, the oestrogen agonist properties of parabens together with their common metabolite (p-hydroxybenzoic acid) have been extensively documented, and, in addition, the parabens have now also been shown to possess androgen antagonist activity, to act as inhibitors of sulfotransferase enzymes and to possess genotoxic activity. With the continued use of parabens in the majority of bodycare cosmetics, there is a need to carry out detailed evaluation of the potential for parabens, together with other oestrogenic and genotoxic co-formulants of bodycare cosmetics, to increase female breast cancer incidence, to interfere with male reproductive functions and to influence development of malignant melanoma which has also recently been shown to be influenced by oestrogenic stimulation. Copyright (C) 2008 John Wiley & Sons, Ltd.

The extrapolation problem and how population modeling can help

We argue that population modeling can add value to ecological risk assessment by reducing uncertainty when extrapolating from ecotoxicological observations to relevant ecological effects. We review other methods of extrapolation, ranging from application factors to species sensitivity distributions to suborganismal (biomarker and "-omics'') responses to quantitative structure activity relationships and model ecosystems, drawing attention to the limitations of each. We suggest a simple classification of population models and critically examine each model in an extrapolation context. We conclude that population models have the potential for adding value to ecological risk assessment by incorporating better understanding of the links between individual responses and population size and structure and by incorporating greater levels of ecological complexity. A number of issues, however, need to be addressed before such models are likely to become more widely used. In a science context, these involve challenges in parameterization, questions about appropriate levels of complexity, issues concerning how specific or general the models need to be, and the extent to which interactions through competition and trophic relationships can be easily incorporated.

Oestrogenic and androgenic activity of triclosan in breast cancer cells

As a consequence of its widespread use as an antimicrobial agent in consumer goods, triclosan has become distributed ubiquitously across the ecosystem, and recent reports that it can cause endocrine disruption in aquatic species has increased concern. It is reported here that triclosan possesses intrinsic oestrogenic and androgenic activity in a range of assays in vitro which could provide some explanation for the endocrine disrupting properties described in aquatic populations. In terms of oestrogenic activity, triclosan displaced [H-3]oestradiol from oestrogen receptors (ER) of MCF7 human breast cancer cells and from recombinant human ER alpha/ER beta. Triclosan at 10(-5) M completely inhibited the induction of the oestrogen-responsive ERE-CAT reporter gene in MCF7 cells by 10(-10) M 17 beta-oestradiol and the stimulation of growth of MCF7 human breast cancer cells by 10(-10) M 17 beta-oestradiol. On its own, 1 mu M triclosan increased the growth of MCF7 cells over 21 days. Triclosan also had androgenic activity. It displaced [H-3]testosterone from binding to the ligand binding domain of the rat androgen receptor (AR). Triclosan was able to inhibit the induction of the androgen-responsive LTR-CAT reporter gene in S115 mouse mammary tumour cells by 10(-9) M testosterone and in T47D human breast cancer cells by 10(-8) M testosterone at concentrations of 10(-7) M and 10(-6) M, respectively. Triclosan at 2 x 10(-5) M antagonized the stimulation of the growth of S115+A mouse mammary tumour cells by 10(-9) M testosterone. The finding that triclosan has oestrogenic and androgenic activity warrants further investigation in relation to both endocrine disruption of aquatic wildlife and any possible impact on human health. Copyright (C) 2007 John Wiley & Sons, Ltd.

Chronic toxicity of ibuprofen to Daphnia magna: Effects on life history traits and population dynamics

The non-steroidal anti-inflammatory drug (NSAID) ibuprofen (IB) is a widely used pharmaceutical that can be found in several freshwater ecosystems. Acute toxicity studies with Daphnia magna suggest that the 48 h EC50 (immobilisation) is 10-100 mg IB l(-1). However, there are currently no chronic IB toxicity dataon arthropod populations, and the aquatic life impacts of such analgesic drugs are still undefined. We performed a 14-day exposure of D. magna to IB as a model compound (concentration range: 0, 20, 40 and 80 mg IB l(-1)) measuring chronic effects on life history traits and population performance. Population growth rate was significantly reduced at all IB concentrations, although survival was only affected at 80 mg IB l(-1). Reproduction, however, was affected at lower concentrations of IB (14-day EC50 of 13.4 mg IB l(-1)), and was completely inhibited at the highest test concentration. The results from this study indicate that the long-term crustacean population consequences of a chronic IB exposure at environmentally realistic concentrations (ng l(-1) to mu g l(-1)) would most likely be of minor importance. We discuss our results in relation to recent genomic studies, which suggest that the potential mechanism of toxicity in Daphnia is similar to the mode of action in mammals, where IB inhibits eicosanoid biosynthesis. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Comparison of the global gene expression profiles produced by methylparaben, n-butylparaben and 17 beta-oestradiol in MCF7 human breast cancer cells

Since the alkyl esters of p-hydroxybenzoic acid (parabens) can be measured intact in the human breast and possess oestrogenic properties, it has been suggested that they could contribute to an aberrant burden of oestrogen signalling in the human breast and so play a role in the rising incidence of breast cancer. However, although parabens have been shown to regulate a few single genes (reporter genes, pS2, progesterone receptor) in a manner similar to that of 17 beta-oestradiol, the question remains as to the full extent of the similarity in the overall gene profile induced in response to parabens compared with 17 beta-oestradiol. The GE-Amersham CodeLink 20 K human expression microarray system was used to profile the expression of 19881 genes in MCF7 human breast cancer cells following a 7-day exposure to 5 x 10(-4) m methylparaben, 10(-5) m n-butylparaben and 10(-8) m 17 beta-oestradiol. At these concentrations, the parabens gave growth responses in MCF7 cells of similar magnitude to 17 beta-oestradiol. The study identified genes which are upregulated or downregulated to a similar extent by methylparaben, n-butylparaben and 17 beta-oestradiol. However, the majority of genes were not regulated in the same way by all three treatments. Some genes responded differently to parabens from 17 beta-oestradiol, and furthermore, differences in expression of some genes could be detected even between the two individual parabens. Therefore, although parabens possess oestrogenic properties, their mimicry in terms of global gene expression patterns is not perfect and differences in gene expression profiles could result in consequences to the cells that are not identical to those following exposure to 17 beta-oestradiol. Copyright (c) 2006 John Wiley & Sons, Ltd.

Metalloestrogens: an emerging class of inorganic xenoestrogens with potential to add to the oestrogenic burden of the human breast

Many compounds in the environment have been shown capable of binding to cellular oestrogen receptors and then mimicking the actions of physiological oestrogens. The widespread origin and diversity in chemical structure of these environmental oestrogens is extensive but to date such compounds have been organic and in particular phenolic or carbon ring structures of varying structural complexity. Recent reports of the ability of certain metal ions to also bind to oestrogen receptors and to give rise to oestrogen agonist responses in vitro and in vivo has resulted in the realisation that environmental oestrogens can also be inorganic and such xenoestrogens have been termed metalloestrogens. This report highlights studies which show metalloestrogens to include aluminium, antimony, arsenite, barium, cadmium, chromium (Cr(II)), cobalt, copper, lead, mercury, nickel, selenite, tin and vanadate. The potential for these metal ions to add to the burden of aberrant oestrogen signalling within the human breast is discussed. Copyright (c) 2006 John Wiley & Sons, Ltd.

Expression of target and reference genes in Daphnia magna exposed to ibuprofen

Background: Transcriptomic techniques are now being applied in ecotoxicology and toxicology to measure the impact of stressors and develop understanding of mechanisms of toxicity. Microarray technology in particular offers the potential to measure thousands of gene responses simultaneously. However, it is important that microarrays responses should be validated, at least initially, using real-time quantitative polymerase chain reaction (QPCR). The accurate measurement of target gene expression requires normalisation to an invariant internal control e. g., total RNA or reference genes. Reference genes are preferable, as they control for variation inherent in the cDNA synthesis and PCR. However, reference gene expression can vary between tissues and experimental conditions, which makes it crucial to validate them prior to application. Results: We evaluated 10 candidate reference genes for QPCR in Daphnia magna following a 24 h exposure to the non-steroidal anti-inflammatory drug (NSAID) ibuprofen (IB) at 0, 20, 40 and 80 mg IB l(-1). Six of the 10 candidates appeared suitable for use as reference genes. As a robust approach, we used a combination normalisation factor (NF), calculated using the geNorm application, based on the geometric mean of three selected reference genes: glyceraldehyde-3-phosphate dehydrogenase, ubiquitin conjugating enzyme and actin. The effects of normalisation are illustrated using as target gene leukotriene B4 12-hydroxydehydrogenase (Ltb4dh), which was upregulated following 24 h exposure to 63-81 mg IB l(-1). Conclusions: As anticipated, use of the NF clarified the response of Ltb4dh in daphnids exposed to sublethal levels of ibuprofen. Our findings emphasise the importance in toxicogenomics of finding and applying invariant internal QPCR control(s) relevant to the study conditions.

Joint effects of density and a growth inhibitor on the life history and population growth rate of the midge Chironomus riparius

Results of previous laboratory studies suggest that high population density often buffers the effects of chemical stressors that predominately increase mortality. Mortality stressors act to release more resources for the survivors and, therefore, produce less-than-additive effects. By contrast, growth stressors are expected to have opposite results or more-than-additive effects. We investigated the effects of a growth inhibitor (lufenuron) on larval growth and survival of Chironomus riparius and examined its joint effects with density on population growth rate (PGR). Exposure to 60 mu g/kg sediment or greater inhibited larval growth, and exposure to 88 mu g/kg or greater often resulted in mortality before reaching emergence. The effects of lufenuron, however, differed with population density. At 88 mu g/kg, mortalities and, to a lesser extent, reduced fecundity resulted in a reduction in PGR at low density. Conversely, when populations were initiated at high density, PGR was similar to that of controls, because the few survivors reached maturity sooner and started producing offspring earlier. The effect of density as a growth stressor therefore was stronger than the effect of lufenuron, which had effects similar to those of a mortality stressor and produced less-than-additive effects. Longterm studies under field conditions, however, are needed before less-than-additive effects are considered to be the norm.

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.

Effects of dredging in Goteborg Harbor, Sweden, assessed by biomarkers in eelpout (Zoarces viviparus)

We used a battery of biomarkers in fish to study the effects of the extensive dredging in Goteborg harbor situated at the river Gota alv estuary, Sweden. Eelpout (Zoarces viviparus) were sampled along a gradient into Goteborg harbor, both before and during the dredging. Biomarker responses in the eelpout before the dredging already indicated that fish in Goteborg harbor are chronically affected by pollutants under normal conditions compared to those in a reference area. However, the results during the dredging activities clearly show that fish were even more affected by remobilized pollutants. Elevated ethoxyresorufin-O-deethylase activities and cytochrome P4501A levels indicated exposure to polycyclic aromatic hydrocarbons. Elevated metallothionein gene expression indicated an increase in metal exposure. An increase in general cell toxicity, measured as a decrease in lysosomal membrane stability, as well as effects on the immune system also could be observed in eelpout sampled during the dredging. The results also suggest that dredging activities in the Gota alv estuary can affect larger parts of the Swedish western coast than originally anticipated. The present study demonstrates that the application of a set of biomarkers is a useful approach in monitoring the impact of anthropogenic activities on aquatic environments.