4-Methylbenzylidene camphor triggers estrogenic effects via the brain-liver-gonad axis in zebrafish larvae.

4-Methylbenzylidene camphor (4-MBC), an emerging contaminant, is a widely-used ultraviolet (UV) filter incorporated into cosmetics because it protects the skin from UV rays and counters photo-oxidation. Despite the well-established estrogenic activity of 4-MBC, the link between this activity and its effects on neurobehavior and the liver remains unknown. Thus, we exposed zebrafish larvae to environmentally relevant concentrations of 4-MBC with 1.39, 4.17, 12.5 and 15.4 µg/mL from 3 to 5 days postfertilization. We found that 4-MBC produced an estrogenic effect by intensifying fluorescence in the transgenic zebrafish, which was counteracted by co-exposure with estrogen receptor antagonist. 4-MBC-upregulated estrogen receptor alpha (erα) mRNA, and an interaction between 4-MBC and ERα suggested ERα's involvement in the 4-MBC-induced estrogenic activity. RNA sequencing unearthed 4-MBC-triggered responses in estrogen stimulus and lipid metabolism. Additionally, 4-MBC-induced hypoactivity and behavioral phenotypes were dependent on the estrogen receptor (ER) pathway. This may have been associated with the disruption of acetylcholinesterase and acetylcholine activities. As a result, 4-MBC increased vitellogenin expression and caused lipid accumulation in the liver of zebrafish larvae. Collectively, this is the first study to report 4-MBC-caused estrogenic effects through the brain-liver-gonad axis. It provides novel insight into how 4-MBC perturbs the brain and liver development.

Sub-lethal Camphor Exposure Triggers Oxidative Stress, Cardiotoxicity, and Cardiac Physiology Alterations in Zebrafish Embryos.

Camphor is a terpene ketone with aromatic and volatile properties in nature derived from the bark of Cinnamomum camphora or synthesized from turpentine. Camphor exhibits various biological properties such as anti-microbial, anti-viral, anti-coccidial, and anti-cancer. It is also used as a form of topical medication for skin irritation, joint pain, and as a relief for itching from insect bites. However, even though the high dose of camphor has been documented to be toxic/lethal in humans in different studies, camphor's developmental toxicity has not yet been explored, and its extensive mechanism of action is still unclear. In the present study, we aimed to assess the toxic effects of camphor in zebrafish embryos in the initial developmental stages. The obtained results demonstrated that a sub-lethal dose of camphor caused a decrease in hatching rate, body length, and substantial elevation in malformation rate on zebrafish embryos. On further observation, in the following time frame, curved body and pericardial edema of zebrafish were also observed. Furthermore, exposure to a sub-lethal dose of camphor was also able to trigger cardiotoxicity in zebrafish larvae. Later, on subsequent biochemical analysis, it was found that the antioxidant capacity inhibition and oxidative stress elevation that occurred after camphor exposure might be associated with the inhibition of total superoxide dismutase (SOD) activity and an increase in reactive oxygen species (ROS) and malondialdehyde (MDA) concentration. In addition, compared to the control group, several apoptotic cells in treated zebrafish were also found to be elevated. Finally, after further investigation on marker gene expressions, we conclude that the developmental toxicity of camphor exposure might be associated with apoptosis elevation and oxidative stress. Taken together, the current study provides a better understanding of the developmental toxicity of camphor on zebrafish, a promising alternative animal model to assess the developmental toxicity of chemical compounds.

4-Methylbenzylidene-camphor inhibits proliferation and induces reactive oxygen species-mediated apoptosis of human trophoblast cells.

4-Methylbenzylidene-camphor (4-MBC) is an estrogenic compound used in a variety of personal care products and is associated with water pollution. In this study, we verified that exposure to 4-MBC suppresses the proliferation and invasiveness of the HTR8/SVneo human trophoblast cell line. Moreover, HTR8/SVneo cells treated with 4-MBC underwent apoptosis with increased DNA fragmentation. 4-MBC also activated the PI3K/AKT and ERK1/2 signaling pathways in HTR8/SVneo cells. Furthermore, 4-MBC induced oxidative stress mediated by reactive oxygen species production, which was associated with HTR8/SVneo cell death. 4-MBC promoted lipid peroxidation and loss of mitochondrial membrane potential in HTR8/SVneo cells and activated the expression of genes encoding a protein expressed on the surface of human trophoblast cells, including the EPH receptor B4 and G protein-coupled receptor 56 genes. Therefore, 4-MBC may retard the normal growth and survival of human trophoblast cells and may hamper normal placental formation during early pregnancy.

An environmental risk assessment of three organic UV-filters at Lac Bay, Bonaire, Southern Caribbean.

Although organic UV filters (OUVFs) benefit human health by preventing skin burns and cancer, several studies revealed that organic UV filters can induce developmental and reproductive toxicity to aquatic organisms. Discharge of OUVFs occurs predominantly at marine recreational hotspots, such as Lac Bay, Bonaire, and is predicted to increase significantly due to growing tourism worldwide. Unfortunately, there is no insight what the current and future discharge of OUVF at Lac Bay is. Therefore, this study aimed to 1) measure concentrations and estimate the risk of specific OUVFs to different nursery habitats at Lac Bay, and 2) compare measured and predicted concentration based risk assessment outcome. Results showed that at least one of the three nurseries at Lac Bay had a potential for adverse effects. Furthermore, predicted environmental concentrations of UV filter discharge can be applied to gain more insight in the order of extent of OUVF discharge by marine tourism.

Genotoxic and mutagenic potential of camphorquinone in L5178/TK+/- mouse lymphoma cells.

OBJECTIVES: Camphorquinone (CQ) is the most important photoinitiator used in dental composite resins. Sparse data indicate a mutagenic potential of CQ. Therefore, it was aim of this study to evaluate the cytotoxicity, genotoxicity, and mutagenicity of CQ in L5178Y TK+/- mouse lymphoma cells. METHODS: L5178Y/TK+/- cells were exposed to different concentrations of non-irradiated CQ (0.25-2.5mM). Cytotoxicity was evaluated by propidium iodide assay, determination of suspension growth rate, relative total growth and the mitotic index. Intracellular levels of reactive oxygen/nitrogen species (ROS/RNS) were quantified by 2',7'-dichlorofluoresceine diacetate (DCFH-DA). Early induction of DNA strand breaks and oxidative DNA base lesions was assessed using the 8-hydroxyguanine DNA-glycosylase 1 (hOGG1)-modified alkaline comet assay, whereas mutagenicity of CQ was determined in the mouse lymphoma TK assay (MLA), according to OECD Guideline No. 490. RESULTS: CQ (0.5-2.5mM) induced concentration- and time-dependent inhibition of cell growth associated with increased ROS/RNS production, amounting to 2342%±1108% of controls after 90min at 2.5mM. Additionally, CQ concentration-dependently caused direct DNA-damage, i.e. formation of DNA strand breaks and 8-hydroxy-2'-deoxyguanosine. Whereas the MLA indicated lack of mutagenicity of CQ after a 4h of treatment, CQ concentration-dependently increased total mutant frequency (MF) after 24h (about 2-fold at 2.5mM). But, based on the global evaluation factor concept, increase in MF did not reach biologically relevance. SIGNIFICANCE: CQ induced concentration-dependent, cytotoxic and genotoxic effects in L5178Y/TK+/- cells, most likely due to oxidative stress, but without mediating obvious biological relevant mutagenicity.

Use of Camphor and Essential Oil Balms for Infants in Cambodia.

Balms and oils containing terpenic compounds, such as camphor, menthol and eucalyptus, are potentially toxic, and numerous reports of adverse events stemming from their use in infants and young children have been published. During qualitative research on newborn practices in rural Cambodia, these products were found to be commonly applied to the skin of newborns and infants and available in most households. Parents and caregivers of infants in Cambodia and other settings where use of camphor- and menthol-containing products are common should be educated on the risks of these to prevent child morbidity and potential mortality.

Comparative study of genotoxic, antigenotoxic and cytotoxic activities of monoterpenes camphor, eucalyptol and thujone in bacteria and mammalian cells.

Genotoxic/antigenotoxic, mutagenic/antimutagenic and cytotoxic effects of monoterpenes camphor, eucalyptol and thujone were determined in bacteria and mammalian cells using alkaline comet assay, Escherichia coli K12 reversion test and MTT assay, respectively. When applied in low doses (up to 200 µM in bacterial assay and 50 µM in comet assay) monoterpenes protected repair proficient E. coli and Vero cells against UV-induced mutagenesis and 4NQO-induced DNA strand breaks, respectively. Antimutagenic response was not detected in nucleotide excision repair (NER) deficient bacteria. When monoterpenes were applied in higher doses, a weak mutagenic effect was found in mismatch repair (MMR) and NER deficient E. coli strains, while induction of DNA strand breaks was evident in human fetal lung fibroblasts MRC-5, colorectal carcinoma HT-29 and HCT 116 cells, as well as in Vero cells. Moreover, the involvement of NER, MMR and RecBCD pathways in repair of DNA lesions induced by monoterpenes was demonstrated in E. coli. Camphor, eucalyptol and thujone were cytotoxic to MRC-5, HT-29 and HCT 116 cells. The most susceptible cell line was HCT 116, with IC50 values of 4.5 mM for camphor, 4 mM for eucalyptol and 1 mM for thujone. Observed effects of monoterpenes are consistent with hormesis response, characterized by a low dose beneficial effect and a high dose adverse effect of a stressor agent, and provide a basis for further study of both chemopreventive and chemotherapeutic potential of camphor, eucalyptol and thujone.

Genotoxic effects of camphorquinone and DMT on human oral and intestinal cells.

OBJECTIVE: Released components of oral biomaterials can leach into the oral cavity and may subsequently reach the gastrointestinal tract. Camphorquinone (CQ) is the most common used photoinitiator in resinous restorative materials and is often combined with the co-initiator N,N-dimethyl-p-toluidine (DMT). It has been shown that CQ exerts cytotoxic effects, at least partially due to the generation of reactive oxygen species (ROS). Objective of this study was to examine the cytotoxic and genotoxic potential of CQ in human oral keratinocytes (OKF6/TERT2) and immortalized epithelial colorectal adenocarcinoma cells (Caco-2). Furthermore, the effects of visible-light irradiation and the co-initiator DMT were investigated as well as the generation of ROS, the potential protective effect of glutathione (GSH) and a recovery period of CQ-treated Caco-2 cells. METHODS: The alkaline comet assay was used to determine DNA damage. Additionally, an enzyme modified comet assay was applied, which detects 7,8-dihydro-8-oxoguanine (8-oxoguanine), a reliable marker for oxidative stress. RESULTS: Our data revealed that high concentrations of CQ induced DNA lesions in OKF6/TERT2 cells. This DNA damage is at least partly caused by the generation of 8-oxoguanine. In addition, CQ and DMT increased ROS formation and induced DNA damage in Caco-2 cells. CQ-treatment resulted in generation of 8-oxoguanine. The antioxidant GSH efficiently prevented CQ-associated DNA damage. Furthermore, a recovery following CQ-treatment significantly reduced DNA damage. SIGNIFICANCE: We conclude that CQ-induced DNA damage is caused by oxidative stress in oral and intestinal cells. These lesions can be prevented and possibly repaired by GSH-treatment and recovery of cells after the photoinitiator is removed from cultures.

Bioactivity of essential oil from Artemisia stolonifera (Maxim.) Komar. and its main compounds against two stored-product insects.

Artemisia stolonifera, a perennial herb, is widely distrbuted in China. The aim of this study was to analyze the essential oil from the aerial parts of Artemisia stolonifera, as well as to evaluate the bioactivity of the oil and its main constituents. The essential oil was analyzed by gas chromatography-flame ionization detector and gas chromatography-mass spectrometry that allowed characterizing 22 compounds. The main components were eucalyptol (32.93%), ß-pinene (8.18%), camphor (6.12%) and terpinen-4-ol (6.11%), and obtained from the essential oil after a further isolation. During the contact toxicity tests, the essential oil (LD50 = 8.60 µg/adult) exhibited stronger toxicity against Tribolium castaneum adults than those isolated constituents, however, camphor and terpinen-4-ol showed 1 and 2 times toxicity against Lasioderma serricorne adults than the essential oil (LD50 = 12.68 µg/adult) with LD50 values of 11.30 and 5.42 µg/adult, respectively. In the fumigant toxicity tests, especially on Tribolium castaneum, the essential oil (LC50 = 1.86 mg/L air) showed almost the same level toxicity as positive control, methyl bromide (LC50 = 1.75 mg/L air). Moreover, the essential oil and its four isolated constituents also exhibited strong repellency against two stored-product insects.

Reduced glutathione prevents camphorquinone-induced apoptosis in human oral keratinocytes.

OBJECTIVES: Camphorquinone (CQ) is a widely used photoinitiator in dental visible light (VL)-cured resinous materials. However, little is known about the toxicity of CQ in human cells. This study was designed to investigate CQ induced oxidative strain and apoptosis in cultured human oral keratinocytes (OKF6/TERT 2). Furthermore, the effects of visible-light (VL)-irradiation and the reducing agent N,N-dimethyl-p-toluidine (DMT) were investigated. In addition, the preventive potential of the antioxidant glutathione (GSH) against CQ induced toxicity was analyzed as well. METHODS: The fluorescent DNA-staining dye Hoechst 33342 was used to quantify total cell numbers. Intracellular levels of reactive oxygen species (ROS) were measured by the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). Apoptosis was determined by FACS analysis (Annexin V-FITC/propidium iodide), by measuring caspase-3/7 activity (ELISA) and by DNA laddering. RESULTS: Our data show that CQ was dose-dependent cytotoxic and caused oxidative stress by inducing reactive oxygen species (ROS). The redistribution of phosphatidylserine (PS) to the outer layer of the plasma membrane, induction of caspase-3 enzyme activity and DNA fragmentation were also observed in CQ exposed cells. Interestingly, CQ-induced ROS generation enhanced by VL irradiation or a simultaneous treatment with DMT showed no quantitative effect on apoptosis. However, co-exposure of cells with GSH significantly reduced the intracellular ROS generation as well as apoptosis caused by CQ. SIGNIFICANCE: This is the first report showing that ROS-induced apoptosis, which is caused by CQ, is prevented by GSH.

Late-life effects on rat reproductive system after developmental exposure to mixtures of endocrine disrupters.

This study examined late-life effects of perinatal exposure of rats to a mixture of endocrine-disrupting contaminants. Four groups of 14 time-mated Wistar rats were exposed by gavage from gestation day 7 to pup day 22 to a mixture of 13 anti-androgenic and estrogenic chemicals including phthalates, pesticides, u.v.-filters, bisphenol A, parabens, and the drug paracetamol. The groups received vehicle (control), a mixture of all 13 chemicals at 150-times (TotalMix150) or 450-times (TotalMix450) high-end human exposure, or 450-times a mixture of nine predominantly anti-androgenic chemicals (AAMix450). Onset of puberty and estrous cyclicity at 9 and 12 months of age were assessed. Few female offspring showed significantly regular estrus cyclicity at 12 months of age in the TotalMix450 and AAMix450 groups compared with controls. In 19-month-old male offspring, epididymal sperm counts were lower than controls, and in ventral prostate an overrepresentation of findings related to hyperplasia was observed in exposed groups compared with controls, particularly in the group dosed with anti-androgens. A higher incidence of pituitary adenoma at 19 months of age was found in males and females in the AAMix450 group. Developmental exposure of rats to the highest dose of a human-relevant mixture of endocrine disrupters induced adverse effects late in life, manifested as earlier female reproductive senescence, reduced sperm counts, higher score for prostate atypical hyperplasia, and higher incidence of pituitary tumors. These delayed effects highlight the need for further studies on the role of endocrine disrupters in hormone-related disorders in aging humans.

New quaternary ammonium camphor derivatives and their antiviral activity, genotoxic effects and cytotoxicity.

The synthesis and biological evaluation of a novel series of dimeric camphor derivatives are described. The resulting compounds were studied for their antiviral activity, cyto- and genotoxicity. Compounds 3a and 3d in which the quaternary nitrogen atoms are separated by the C5H10 and С9H18 aliphatic chain, exhibited the highest efficiency as an agent inhibiting the reproduction of the influenza virus A(H1N1)pdm09. The cytotoxicity data of compounds 3 and 4 revealed their moderate activity against malignant cell lines; compound 3f had the highest activity for the CEM-13 cells. These results show close agreement with the data of independent studies on toxicity of these compounds, in particular that the toxicity of compounds strongly depends on spacer length.

Camphorquinone inhibits odontogenic differentiation of dental pulp cells and triggers release of inflammatory cytokines.

INTRODUCTION: Camphorquinone (CQ) is a photoinitiator that triggers polymerization of light-curing materials such as dental adhesives and composites. CQ does not become a part of the polymer network, suggesting that CQ can be leached out into surrounding environment including dental pulp and exert adversary effects on tissues. In order to understand the mechanisms of CQ-induced side effects, we investigated the effect of CQ on cell viability, cytokine secretion, and odontogenic differentiation of dental pulp stem cells in vitro. METHODS: Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after CQ exposure. Western blotting was performed for p16(INK4A), p21(WAF1), and p53. Secretory cytokines were evaluated using the membrane-enzyme-linked immunosorbent assay as well as conventional and quantitative reverse-transcription polymerase chain reaction. The effects of CQ on odontogenic differentiation were evaluated using alkaline phosphatase and alizarin red S staining methods. RESULTS: CQ treatment suppressed the proliferation of DPSCs and induced the expression of p16(INK4A), p21(WAF1), and p53. Levels of proinflammatory cytokines (eg, interleukin 6, interleukin 8, and matrix metalloproteinase-3 [MMP3]) were increased by CQ treatment. CQ also inhibited odontogenic differentiation and mineralization capacities of DPSC and MC3T3-E1 cells. CONCLUSIONS: Our study showed that CQ may trigger pulpal inflammation by inducing proinflammatory cytokine production from the pulpal cells and may impair odontogenic differentiation of dental pulp cells, resulting in pulpal irritation and inflammation.

Chemical composition, antibacterial activity and cytotoxicity of essential oils of Tanacetum parthenium in different developmental stages.

CONTEXT: Tanacetum parthenium Schultz Bip. (Asteraceae) is an aromatic perennial plant, widely distributed in the northern hemisphere. This species traditionally has been used in insecticides, cosmetics, balsams, dyes, medicines and preservatives. MATERIAL AND METHODS: The essential oil of T. parthenium was obtained by hydrodistillation in three developmental stages and analyzed by gas chromatography-mass spectrometry. The antibacterial activity of the oils was investigated against four Gram-positive and four Gram-negative bacteria. The oil was tested for cytotoxicity against THP-1 cells using the Trypan blue assay. RESULTS: Twenty-nine components were identified in the essential oil; the highest amount was extracted at the flowering stage. The main component, in the flowering stage, was camphor (18.94%) and other major components were bornyl acetate (18.35%), camphene (13.74%), bornyl isovalerate (3.15%), borneol (10.93%), juniper camphor (6.23%) and ß-eudesmol (2.65%). Minimum inhibitory concentration of essential oil was evaluated from 4 µL mL(-1) against Staphylococcus subtilis to 38 µL mL(-1) against Entrobacter aerogenes. Toxicity assay showed that the oil has no significant toxicity at 5-15% v/v concentrations on THP-1 cells. DISCUSSION AND CONCLUSION: This study demonstrates the occurrence of camphor/bornyl acetate chemotype of T. parthenium in western regions of Iran. The finding showed also the studied oils have relatively good antibacterial activity without significant toxicity, thus have great potentiality to be used as natural health product.

Phytotoxic volatiles in the roots and shoots of Artemisia tridentata as detected by headspace solid-phase microextraction and gas chromatographic-mass spectrometry analysis.

In the vicinity of big sagebrush (Artemisia tridentata), the growth of Nicotiana attenuata is negatively affected, in part due to the alleopathic effect of methyl jasmonate (MeJA) which is produced in large quantities by the aerial parts of sagebrush. Preliminary experiments suggested that growth-inhibiting substances were being emitted from the sagebrush roots. To identify the allelochemical secondary metabolites, we tested different root extracts in seedling growth bioassays with the naturally co-occurring native tobacco, Nicotiana attenuata, in a two-chamber Petri dish assay, optimized for tests of volatiles. Fractions rich in volatile compounds were particularly phytotoxic. We analyzed the volatiles emitted from the roots of intact Artemisia tridentata plants grown in soil, sand, and hydroponic cultures by using dynamic headspace extraction, headspace solvent-microextraction (HSME) and headspace solid-phase microextraction (HSPME), and GC-MS. Camphor, 1,8-cineol, nerol, and neryl isovalerate were phytotoxic and released as the major constituents. In addition to the phytotoxic monoterpenes, himachalenes, longifolene, caryophyllene, and acetylenic spiroethers, were found as characteristic components in the root's volatiles. The allelopathic potential of these root volatiles was compared with that of methyl jasmonate (MeJA), one of the most active compounds emitted from above-ground parts of the plant.

Estrogenic activity of ternary UV filter mixtures in fish (Pimephales promelas) - an analysis with nonlinear isobolograms.

Numerous estrogenic compounds are present in aquatic environments, but currently it is not well understood how compounds that differ in maxima and slope of their individual dose-response curves contribute to the overall mixture effect. In order to better understand such interactions we investigated 3 commonly used UV filters, for their estrogenic mixture activity and analysed their joint effects by using the concentration addition (CA) concept. Thereby, we extended the method of isoboles for analysis of 3 compounds that differ in maxima and slopes of their dose-response curves. 3-Benzylidene camphor (3BC), benzophenone-1 (BP1) and benzophenone-2 (BP2) are estrogenic in fish and act as pure- or partial estrogen receptor alpha agonists. First we exposed juvenile fathead minnows for 14 days to six concentrations of each UV filter alone to determine vitellogenin (VTG) induction curves, calculate equi-effective mixture concentrations and predict mixture effects. For 3BC, BP1 and BP2 significant VTG-induction occurred at 420, 2668, and 4715 microg/L, respectively. BP2 displayed a full dose-response curve, whereas 3BC and BP1 showed submaximal activity of 70 and 78%, respectively. Second, we exposed fish to 6 equi-effective mixtures (EC-NOEC, EC1, EC5, EC10, EC20, EC30) of these UV filters. Significant VTG-induction occurred at EC5 and higher. Submaximal activity of 67% as compared to the control EE2 (100 ng/L) was reached. The curves for the observed and predicted mixture activity agreed for mixture levels (EC10 to EC30), however, at EC-NOEC, EC1 and EC5, lower activity was observed than predicted by CA. Detailed isobolographic analysis indicate additivity at EC10 to EC30, and antagonism at low levels (EC-NOEC to EC5). Our data show for the first time, that for compounds with differences in maxima and slope, considerably more mixture combinations are additive than previously thought. This should be taken into account for hazard and risk assessment of UV filters and xenoestrogens.

Developmental toxicity of UV filters and environmental exposure: a review.

Several ultraviolet (UV) filters exhibit estrogenic, some also anti-androgenic activity. They are present in waste water treatment plants, surface waters and biosphere including human milk, suggesting potential exposure during development. Developmental toxicity was studied in rats for the UV filters 4-methylbenzylidene camphor (4-MBC, 0.7, 7, 24, 47 mg/kg/day) and 3-benzylidene camphor (3-BC, 0.07, 0.24, 0.7, 2.4, 7 mg/kg/day) administered in chow to the parent generation before mating, during pregnancy and lactation, and to the offspring until adulthood. Neonates exhibited enhanced prostate growth after 4-MBC and altered uterine gene expression after both chemicals. 4-MBC and 3-BC delayed male puberty and affected reproductive organ weights of adult offspring. Effects on the thyroid axis were also noted. Expression and oestrogen sensitivity of oestrogen-regulated genes and nuclear receptor coregulator levels were altered at mRNA and protein levels in adult uterus, prostate and brain regions involved in gonadal control and sexual behaviour. Female sexual behaviour was impaired by both filters; 3-benzylidene camphor caused irregular cycles. Classical endpoints exhibited lowest observed adverse effect levels (LOAELs) and no observed adverse effect levels (NOAELs) of 7/0.7 mg/kg for 4-MBC and 0.24/0.07 mg/kg for 3-BC. Molecular endpoints were affected by the lowest doses studied. Our data indicate that the potential risk posed by endocrine active UV filters warrants further investigations.

Endocrine modulation and toxic effects of two commonly used UV screens on the aquatic invertebrates Potamopyrgus antipodarum and Lumbriculus variegatus.

The two UV screens 3-benzylidene-camphor (3-BC) and 3-(4'-methylbenzylidene)-camphor (4-MBC) were tested regarding their toxicity and estrogenic activity. The Yeast Estrogen Screen (YES) and two sediment assays with the freshwater invertebrates Lumbriculus variegatus and Potamopyrgus antipodarum were performed. In the YES, both substances activated the human estrogen receptor alpha with EC50 values of 44.2 microM for 3-BC and 44.3 microM for 4-MBC, whereby 4-MBC attained only 8% of the maximal response of 17beta-estradiol. For P. antipodarum embryo production increased after exposure to both substances (EC50 of 4.60 microM 4-MBC=1.17 mg kg(-1)dw) while mortality increased at high concentrations. The reproduction of L. variegatus was decreased by 3-BC with an EC50 of 5.95 microM (=1.43 mg kg(-1)dw) and also by 4-MBC, where no EC50 could be calculated. While reproduction decreased, the worms' weight increased after exposure to 3-BC with an EC50 of 26.9 microM (=6.46 mg kg(-1) dw), hence the total biomass remained unaffected.

Sexually dimorphic gene regulation in brain as a target for endocrine disrupters: developmental exposure of rats to 4-methylbenzylidene camphor.

The developing neuroendocrine brain represents a potential target for endocrine active chemicals. The UV filter 4-methylbenzylidene camphor (4-MBC) exhibits estrogenic activity, but also interferes with the thyroid axis. We investigated effects of pre- and postnatal exposure to 4-MBC in the same rat offspring at brain and reproductive organ levels. 4-MBC (7, 24, 47 mg/kg/day) was administered in chow to the parent generation before mating, during gestation and lactation, and to the offspring until adulthood. mRNA of estrogen target genes involved in control of sexual behavior and gonadal functions was measured by real-time RT-PCR in ventromedial hypothalamic nucleus (VMH) and medial preoptic area (MPO) of adult offspring. 4-MBC exposure affected mRNA levels of ER alpha, progesterone receptor (PR), preproenkephalin (PPE) and insulin-like growth factor-I (IGF-I) in a sex- and region-specific manner. In order to assess possible changes in sensitivity of target genes to estrogens, offspring were gonadectomized on day 70, injected with estradiol (E2, 10 or 50 microg/kg s.c.) or vehicle on day 84, and sacrificed 6 h later. The acute induction of PR mRNA, and repression (at 6 h) of PPE mRNA by E2 was enhanced by 4-MBC in male and female VMH and female MPO, whereas male MPO exhibited reduced responsiveness of both genes. Steroid receptor coactivator SRC-1 mRNA levels were increased in female VMH and MPO. The data indicate profound sex- and region-specific alterations in the regulation of estrogen target genes at brain level. Effect patterns in baseline and E2-induced gene expression differ from those in uterus and prostate.

Estrogen sensitivity of target genes and expression of nuclear receptor co-regulators in rat prostate after pre- and postnatal exposure to the ultraviolet filter 4-methylbenzylidene camphor.

BACKGROUND AND OBJECTIVES: In previous studies, we found that the ultraviolet filter 4-methyl-benzylidene camphor (4-MBC) exhibits estrogenic activity, is a preferential estrogen receptor (ER)-beta ligand, and interferes with development of female reproductive organs and brain of both sexes in rats. Here, we report effects on male development. METHODS: 4-MBC (0.7, 7, 24, 47 mg/kg/day) was administered in chow to the parent generation before mating, during gestation and lactation, and to offspring until adulthood. mRNA was determined in prostate lobes by real-time reverse transcription-polymerase chain reaction and protein was determined by Western blot analysis. RESULTS: 4-MBC delayed male puberty, decreased adult prostate weight, and slightly increased testis weight. Androgen receptor (AR), insulin-like growth factor-1 (IGF-1), ER-alpha, and ER-beta expression in prostate were altered at mRNA and protein levels, with stronger effects in dorsolateral than ventral prostate. To assess sensitivity of target genes to estrogens, offspring were castrated on postnatal day 70, injected with 17beta-estradiol (E(2); 10 or 50 microg/kg, sc) or vehicle on postnatal day 84, and sacrificed 6 hr later. Acute repression of AR and IGF-1 mRNAs by E(2), studied in ventral prostate, was reduced by 4-MBC exposure. This was accompanied by reduced co-repressor N-CoR (nuclear receptor co-repressor) protein in ventral and dorsolateral prostate, whereas steroid receptor coactivator-1 (SRC-1) protein levels were unaffected. CONCLUSIONS: Our data indicate that 4-MBC affects development of male reproductive functions and organs, with a lowest observed adverse effect level of 0.7 mg/kg. Nuclear receptor coregulators were revealed as targets for endocrine disruptors, as shown for N-CoR in prostate and SRC-1 in uterus. This may have widespread effects on gene regulation.