Knockdown of PCBER1, a gene of neolignan biosynthesis, resulted in increased poplar growth.

MAIN CONCLUSION: Poplar trees displayed an increased plant height due to the transgenic knockdown of PCBER1, a gene of lignan biosynthesis. The wood composition was slightly altered in both overexpression and knockdown lines. The gene PHENYLCOUMARAN BENZYLIC ETHER REDUCTASE1 (PCBER1) is well known as an important gene in the synthesis of lignans, a group of diverse phenylpropanoid derivatives. They are widely distributed in the plant kingdom and may have a role in both plant defense and growth regulation. To analyze its role in biomass formation and wood composition in poplar, both overexpression and knockdown approaches have been performed. Transgenic lines were analyzed on genetic and phenotypic levels, and partly in regard to their biomass composition. While the PCBER1 overexpression approach remained unremarkable concerning the plant height, biomass composition of obtained transgenic lines was modified. They had a significantly increased amount of ethanol extractives. The PCBER1 knockdown resulted in significantly deviating plants; after 17 months of greenhouse cultivation, transgenic plants were up to 38% higher compared to non-transgenic wild type. Most examined transgenic lines did not reveal a significantly enhanced stem diameter after three vegetation periods in the greenhouse. Significant changes were not obtained with regard to the three major wood components, lignin, cellulose and hemicelluloses. As a slight but not significant reduction in ethanol extractives was detected, the hypothesis arises that the lignan content could be influenced. Lignans become important in the pharmaceutical industry and clinical studies concerning cancer and other diseases, thus further investigations on lignan formation in poplar and its connection to biomass formation seem promising.

7-Hydroxymatairesinol improves body weight, fat and sugar metabolism in C57BJ/6 mice on a high-fat diet.

7-Hydroxymatairesinol (7-HMR) is a plant lignan abundant in various concentrations in plant foods. The objective of this study was to test HMRLignan™, a purified form of 7-HMR, and the corresponding Picea abies extract (total extract P. abies; TEP) as dietary supplements on a background of a high-fat diet (HFD)-induced metabolic syndrome in mice and in the 3T3-L1 adipogenesis model. Mice, 3 weeks old, were fed a HFD for 60 d. Subgroups were treated with 3 mg/kg body weight 7-HMR (HMRLignan™) or 10 mg/kg body weight TEP by oral administration. 7-HMR and TEP limited the increase in body weight (-11 and -13 %) and fat mass (-11 and -18 %) in the HFD-fed mice. Epididymal adipocytes were 19 and -12 % smaller and the liver was less steatotic (-62 and -65 %). Serum lipids decreased in TEP-treated mice (-11 % cholesterol, -23 % LDL and -15 % TAG) and sugar metabolism was ameliorated by both lignan preparations, as shown by a more than 70 % decrease in insulin secretion and insulin resistance. The expression of several metabolic genes was modulated by the HFD with an effect that was reversed by lignan. In 3T3-L1 cells, the 7-HMR metabolites enterolactone (ENL) and enterodiol (END) showed a 40 % inhibition of cell differentiation accompanied by the inhibited expression of the adipogenic genes PPARγ, C/EBPα and aP2. Furthermore, END and ENL caused a 10 % reduction in TAG uptake in HEPA 1-6 hepatoma cells. In conclusion, 7-HMR and TEP reduce metabolic imbalances typical of the metabolic syndrome and obesity in male mice, whereas their metabolites inhibit adipogenesis and lipid uptake in vitro.

Chemical Composition and Content of Lipophilic Seed Extractives of Some Abies and Picea Species.

The chemical content and composition of the lipophilic extracts from seeds of some fir species: Abies alba, A. cephalonica, A. concolor, and A. koreana, as well as of a few spruce species: Picea abies, P. orientalis, and P. pungens, were examined. The amount of lipophilic extractives is diverse among the tree species and it varies from 9.8% to 41% of seeds. The chemical characterization showed significant differences, not only in the content, but also in the composition of extractives. However, most of the identified compounds like resin alcohols, -aldehydes, and -acids, as well as fatty acids, were detected in the seed extracts of all the examined tree species. The dominating identified compound group was esterified fatty acids (2.5 - 55.4% w/w of dry extract), occurring mainly as tri- and diglycerides, as well as free acids. The main representatives of this group were linoleic and oleic acids. The resin acids, among which the main were abietic, neoabietic, dehydroabietic, and palustric acids, were also detected at high levels, from 1.8% to 16.9% of the dry seed extracts. Phytosterols, tocopherols, resin hydrocarbons, and resin esters, as well as fatty alcohols were also identified. The coniferous tree seeds, as a renewable natural material, could represent a prospective raw material for producing valuable chemicals.

Coniferyl alcohol hinders the growth of tobacco BY-2 cells and Nicotiana benthamiana seedlings.

MAIN CONCLUSION: Externally added coniferyl alcohol at high concentrations reduces the growth of Nicotiana cells and seedlings. Coniferyl alcohol is metabolized by BY-2 cells to several compounds. Coniferyl alcohol (CA) is a common monolignol and a building block of lignin. The toxicity of monolignol alcohols has been stated in the literature, but there are only few studies suggesting that this is true. We investigated the physiological effects of CA on living plant cells in more detail. Tobacco (Nicotiana tabacum) Bright yellow-2 cells (BY-2) and Nicotiana benthamiana seedlings both showed concentration-dependent growth retardation in response to 0.5-5 mM CA treatment. In some cases, CA addition caused cell death in BY-2 cultures, but this response was dependent on the growth stage of the cells. Based on LC-MS/MS analysis, BY-2 cells did not accumulate the externally supplemented CA, but metabolized it to ferulic acid, ferulic acid glycoside, coniferin, and to some other phenolic compounds. In addition to growth inhibition, CA caused the formation of a lignin-like compound detected by phloroglucinol staining in N. benthamiana roots and occasionally in BY-2 cells. To prevent this, we added potassium iodide (KI, at 5 mM) to overcome the peroxidase-mediated CA polymerization to lignin. KI had, however, toxic effects on its own: in N. benthamiana seedlings, it caused reduction in growth; in BY-2 cells, reduction in growth and cell viability. Surprisingly, CA restored the growth of KI-treated BY-2 cells and N. benthamiana seedlings. Our results suggest that CA at high concentrations is toxic to plant cells.

Oilseeds ameliorate metabolic parameters in male mice, while contained lignans inhibit 3T3-L1 adipocyte differentiation in vitro.

PURPOSE AND BACKGROUND: The focus was directed to the study of two of the most lignan-rich food sources: sesame and flaxseeds. Recent epidemiological and experimental evidences suggesting that these foods may improve metabolic functions underlying metabolic syndrome (MetS). METHODS: To characterize the effect of these oilseeds on metabolic functions, we conducted an experimental study aimed at preventing adiposity and metabolic imbalance in a mouse model of high-fat diet (HFD)-induced MetS. Statistical analysis was performed by two-way analysis of variance test followed by post hoc Bonferroni analysis. RESULTS: We studied the effect of the oilseeds sesame and flaxseed on metabolic parameters in mice on a HFD. When the HFD was integrated with 20% of sesame or flaxseed flours, the mice showed a decrease in body fat, already at day 15, from time 0. The size of the adipocytes was smaller in epididymal fat, liver steatosis was inhibited, and insulin sensitivity was higher in mice on the supplemented diets. The supplemented diets also resulted in a significant increase in the serum levels of the lignan metabolites enterodiol and enterolactone compared with the controls. The expression of genes associated with the inflammatory response, glucose metabolism, adipose metabolism and nuclear receptor were altered by the oilseed-supplemented diets. Some of the most abundant lignans in these oilseeds were studied in 3T3-L1 preadipocyte cells and were effective in inhibiting adipocyte differentiation at the minimal dose of 1 nM. CONCLUSIONS: The consumption of sesame and flaxseed may be beneficial to decrease metabolic parameters that are generally altered in MetS.

Pinaceae Pine Resins (Black Pine, Shore Pine, Rosin, and Baltic Amber) as Natural Dielectrics for Low Operating Voltage, Hysteresis-Free, Organic Field Effect Transistors.

Four pinaceae pine resins analyzed in this study: black pine, shore pine, Baltic amber, and rosin demonstrate excellent dielectric properties, outstanding film forming, and ease of processability from ethyl alcohol solutions. Their trap-free nature allows fabrication of virtually hysteresis-free organic field effect transistors operating in a low voltage window with excellent stability under bias stress. Such green constituents represent an excellent choice of materials for applications targeting biocompatibility and biodegradability of electronics and sensors, within the overall effort of sustainable electronics development and environmental friendliness.

Effect of dietary intervention on serum lignan levels in pregnant women - a controlled trial.

BACKGROUND: Mother's diet during pregnancy is important, since plant lignans and their metabolites, converted by the intestinal microflora to enterolignans, are proposed to possess multiple health benefits. Aim of our study was to investigate whether a dietary intervention affects lignan concentrations in the serum of pregnant women. METHODS: A controlled dietary intervention trial including 105 first-time pregnant women was conducted in three intervention and three control maternity health clinics. The intervention included individual counseling on diet and on physical activity, while the controls received conventional care. Blood samples were collected on gestation weeks 8-9 (baseline) and 36-37 (end of intervention). The serum levels of the plant lignans 7-hydroxymatairesinol, secoisolariciresinol, matairesinol, lariciresinol, cyclolariciresinol, and pinoresinol, and of the enterolignans 7-hydroxyenterolactone, enterodiol, and enterolactone, were measured using a validated method. RESULTS: The baseline levels of enterolactone, enterodiol and the sum of lignans were higher in the control group, whereas at the end of the trial their levels were higher in the intervention group. The adjusted mean differences between the baseline and end of the intervention for enterolactone and the total lignan intake were 1.6 ng/ml (p = 0.018, 95% CI 1.1-2.3) and 1.4 ng/mg (p = 0.08, 95% CI 1.0-1.9) higher in the intervention group than in the controls. Further adjustment for dietary components did not change these associations. CONCLUSION: The dietary intervention was successful in increasing the intake of lignan-rich food products, the fiber consumption and consequently the plasma levels of lignans in pregnant women. TRIAL REGISTRATION: ISRCTN21512277, http://www.isrctn.org.

Hydrophobic and Hydrophilic Extractives in Norway Spruce and Kurile Larch and Their Role in Brown-Rot Degradation.

Extractives found in the heartwood of a moderately durable conifer (Larix gmelinii var. japonica) were compared with those found in a non-durable one (Picea abies). We identified and quantified heartwood extractives by extraction with solvents of different polarities and gas chromatography with mass spectral detection (GC-MS). Among the extracted compounds, there was a much higher amount of hydrophilic phenolics in larch (flavonoids) than in spruce (lignans). Both species had similar resin acid and fatty acid contents. The hydrophobic resin components are considered fungitoxic and the more hydrophilic components are known for their antioxidant activity. To ascertain the importance of the different classes of extractives, samples were partially extracted prior to subjection to the brown-rot fungus Rhodonia placenta for 2-8 weeks. Results indicated that the most important (but rather inefficient) defense in spruce came from the fungitoxic resin, while large amounts of flavonoids played a key role in larch defense. Possible moisture exclusion effects of larch extractives were quantified via the equilibrium moisture content of partially extracted samples, but were found to be too small to play any significant role in the defense against incipient brow-rot attack.

Recovery of Bioactive Compounds from Hazelnuts and Walnuts Shells: Quantitative-Qualitative Analysis and Chromatographic Purification.

Hazelnut (HS) and walnut (WS) shells, an abundant by-product of the processing industries of these edible nuts, are traditionally considered as a low-value waste. However, they are a source of valuable compounds with an interesting chemical profile for the chemical and pharmaceutical sectors. In this study, the lipophilic and hydrophilic extracts present in HS and WS were quantified and identified, then the polar fractions were chromatographically separated, and their antioxidant capacity was studied. The experimental work includes the isolation of crude lipophilic and hydrophilic extracts by an accelerated extraction process, chromatographic analysis (gas chromatography-flame ionization (GC-FID), GC-mass spectroscopy (GC-MS), high-performance size-exclusion chromatography (HPSEC), thin-layer chromatography (TLC)), and quantification of the components. In addition, a thorough compositional characterization of the subgroups obtained by flash chromatography and their antioxidant capacity was carried out. The gravimetric concentrations showed different lipophilic/hydrophilic ratios (0.70 for HS and 0.23 for WS), indicating a higher proportion of polar compounds in WS than in HS. Moreover, the lipophilic extracts were principally composed of short-chain fatty acids (stearic, palmitic, and oleic acid), triglycerides, and sterols. The polar fractions were screened by thin-layer chromatography and then separated by flash chromatography, obtaining fractions free of fatty acids and sugar derivatives (97:3 in HS and 95:5 in WS), and mixtures richer in phenolic compounds and flavonoids such as guaiacyl derivatives, quercetin, pinobanksin, and catechin. The most polar fractions presented a higher antioxidant capacity than that of the crude extracts.

Dietary lariciresinol attenuates mammary tumor growth and reduces blood vessel density in human MCF-7 breast cancer xenografts and carcinogen-induced mammary tumors in rats.

Lariciresinol is a dietary lignan that accounts for a significant portion of the total phytoestrogen intake from Western foods. Recent epidemiological studies suggest that high dietary intake of lignans and lariciresinol is associated with reduced breast cancer risk. However, no causal relationship between lariciresinol intake and breast cancer development has been established. In this study, we investigated for the first time the effects and possible mechanisms of action of lariciresinol on hormone responsive mammary cancer in vivo in dimethylbenz[a]anthracene induced mammary cancer in rats, and in human MCF-7 breast cancer xenografts in athymic mice. For tumor bearing rats, lariciresinol (3 or 15 mg/kg of body weight) or vehicle was administered p.o. daily for 9 weeks. For E2-maintained ovariectomized athymic mice bearing orthotopic MCF-7 tumors, control diet (AIN-93G) or lariciresinol containing diet (AIN-93G supplemented with 20 or 100 mg of lariciresinol/kg of diet) was administered for 5 weeks. In both models, lariciresinol administration inhibited the tumor growth and tumor angiogenesis. In MCF-7 cells, enterolactone significantly inhibited the E2-stimulated VEGF secretion. Moreover, in MCF-7 xenografts, lariciresinol administration enhanced tumor cell apoptosis and increased estrogen receptor beta expression. Lariciresinol and its further metabolites secoisolariciresinol, enterodiol and enterolactone were found in serum of both rats and athymic mice confirming a similar lignan metabolism pattern as in humans. These findings indicate conceivable importance of dietary lignan lariciresinol in inhibition of breast cancer development.

Identification of lignans by liquid chromatography-electrospray ionization ion-trap mass spectrometry.

The fragmentation pattern of 30 compounds belonging to different classes of the lignan family was studied by liquid chromatography-electrospray ionization ion-trap mass spectrometry. On the basis of the observed fragmentation patterns, identification of different types of lignans was achieved. For example, dibenzylbutyrolactone lignans showed a characteristic fragmentation pathway by the loss of 44 Da (CO(2)) from the lactone moiety, whereas dibenzylbutanediols showed a loss of 48 Da by a combined loss of formaldehyde and water from the 1,4-butanediol moiety. Lignan glycosides readily lost the sugar residue to give the parent lignan as their primary product ion. In addition, several compound-specific fragmentations were observed and used for identification of individual compounds.A versatile method for analyses of lignans was developed using LC separation on a C8 column followed by fragmentation and detection of ions produced in the ion trap.

Role of dietary lignans in the reduction of breast cancer risk.

Lignans are a large group of fiber-associated phenolic compounds widely distributed in edible plants. Some of the ingested plant lignans are converted by intestinal microbiota to enterolignans, enterodiol (END) and enterolactone (ENL), the latter of which has been thought to be the major biologically active lignan, and suggested to be associated with low risk of breast cancer. In line with this, administration of plant lignans which are further metabolized to ENL, or ENL as such, have been shown to inhibit or delay the growth of experimental mammary cancer. The mechanism of anticarcinogenic action of ENL is not yet fully understood, but there is intriguing evidence for ENL as a modulator of estrogen signaling. These findings have generated interest in the use of lignans as components of breast cancer risk reducing functional foods. Identification of target groups, who would benefit most, is of pivotal importance. Therefore, further identification and validation of relevant biomarkers, which can be used as indicators of lignan or ENL action and breast cancer risk reduction at different stages of the disease, are of importance.

Maternal flaxseed diet during pregnancy or lactation increases female rat offspring's susceptibility to carcinogen-induced mammary tumorigenesis.

Flaxseed contains several dietary components that have been linked to low breast cancer risk; i.e., n-3 polyunsaturated fatty acids (PUFAs), lignans and fiber, but it also contains detectable levels of cadmium, a heavy metal that activates the estrogen receptor (ER). Since estrogenic exposures early in life modify susceptibility to develop breast cancer, we wondered whether maternal dietary intake of 5% or 10% flaxseed during pregnancy or lactation (between postpartum days 5 and 25) might affect 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumorigenesis in the rat offspring. Our data indicated that both in utero and postnatal 5% and 10% flaxseed exposures shortened mammary tumor latency, and 10% flaxseed exposure increased tumor multiplicity, compared to the controls. Further, when assessed in 8-week-old rats, in utero 10% flaxseed exposure increased lobular ER-alpha protein levels, and both in utero and postnatal flaxseed exposures dose-dependently reduced ER-beta protein levels in the terminal end buds (TEBs) lobules and ducts. Exposures to flaxseed did not alter the number of TEBs or affect cell proliferation within the epithelial structures. In a separate group of immature rats that were fed 5% defatted flaxseed diet (flaxseed source different than in the diets fed to pregnant or lactating rats) for 7 days, cadmium exposure through the diet was six-fold higher than allowed for humans by World Health Organization, and cadmium significantly accumulated in the liver and kidneys of the rats. It remains to be determined whether the increased mammary cancer in rats exposed to flaxseed through a maternal diet in utero or lactation was caused by cadmium present in flaxseed, and whether the reduced mammary ER-beta content was causally linked to increased mammary cancer risk among the offspring.

Quantification of a broad spectrum of lignans in cereals, oilseeds, and nuts.

Twenty-four plant lignans were analyzed by high-performance liquid chromatography-tandem mass spectrometry in bran extracts of 16 cereal species, in four nut species, and in two oilseed species (sesame seeds and linseeds). Eighteen of these were lignans previously unidentified in these species, and of these, 16 were identified in the analyzed samples. Four different extraction methods were applied as follows: alkaline extraction, mild acid extraction, a combination of alkaline and mild acid extraction, or accelerated solvent extraction. The extraction method was of great importance for the lignan yield. 7-Hydroxymatairesinol, which has not previously been detected in cereals because of destructive extraction methods, was the dominant lignan in wheat, triticale, oat, barley, millet, corn bran, and amaranth whole grain. Syringaresinol was the other dominant cereal lignan. Wheat and rye bran had the highest lignan content of all cereals; however, linseeds and sesame seeds were by far the most lignan-rich of the studied species.

The plant lignans matairesinol and secoisolariciresinol administered to Min mice do not protect against intestinal tumor formation.

The lignans matairesinol (MAT) and secoisolariciresinol (SECO) were fed to Min mice at 0.02% (w/w) in diet to study their effects on intestinal tumor development. The mean number (67 vs. 51, P=0.052) and size (1.4 vs. 1.2 mm, P=0.011) of tumors in the MAT group was elevated when compared with the control group. Tumor formation of the SECO group did not differ from the control group. Intake of MAT increased the level of both MAT and enterolactone in the plasma while SECO feeding increased SECO, enterodiol, and enterolactone (P=0.001). These results showed that MAT or SECO do not prevent intestinal carcinogenesis in Min mice and that MAT may have adverse effects.

Determination of plant and enterolignans in human serum by high-performance liquid chromatography with tandem mass spectrometric detection.

An HPLC-MS/MS method was validated for the determination of the plant lignans 7-hydroxymatairesinol (HMR), matairesinol (Mat), secoisolariciresinol (Seco), lariciresinol (Lar), and cyclolariciresinol (CLar) and for the enterolignans 7-hydroxyenterolactone (HEL), enterodiol (ED), and enterolactone (EL) in human serum. The method included sample enzymatic hydrolysis, solid-phase extraction, and lignan analysis using a triple quadrupole mass spectrometer with electrospray ionisation in the multiple-reaction monitoring mode. The serum lignans were quantified using deuterated Mat or EL as internal standards. The method met the validation criteria for selectivity, intra- and inter-assay precision, and accuracy. The method was applied to ten serum samples collected from healthy individuals (five men and five women) consuming their habitual Finnish diet. All lignans except HMR and Seco were found in quantifiable amounts in the samples. All serums contained EL; the average concentration was 34 nM. In three individuals, the serum concentration of plant lignans was higher than that of enterolignans. Using the method, common dietary plant lignans and their major metabolites can be reliably quantified in human serum at low-nanomolar concentrations in a simple and rapid way.

Chemical characterization of high-molar-mass fractions in a Norway spruce knotwood ethanol extract.

The low-molar-mass (LMM) fraction, only, i.e., the GC-eluting compounds, which are mainly lignans, has been characterized in Norway spruce knotwood hydrophilic extracts previously. Of this fraction, many lignans and sesquilignans and all GC peaks supposedly representing dilignans remain unidentified. In this work, dilignans and the GC non-eluting compounds (the high-molar mass fractions, HMM) were characterized in a 7-hydroxymatairesinol-reduced knotwood ethanol extract of Norway spruce by using several fractionation and analytical techniques. A methyl tert-butyl ether (MTBE) insoluble fraction of the extract contained mainly HMM material, of which the main part was shown to consist of lignan oligomers. The oligolignans (with a molar mass up to approximately 3700 Da) seemed to be linked by 55' bonds, some of them containing one or two guaiacylglycerol ether units linked to the lignan by ßO4 or ß5 bonds. Several oligolignans were identified or tentatively identified. The MTBE soluble fraction, which accounted for the major part (81%) of the extract, contained mainly LMM material (lignans, sesqui- and dilignans). The part of the HMM material in the MTBE soluble fraction that was easily isolable (2%) seemed to contain polymers of fatty acids and alcohols, resin acids, and sterols.

New lignan metabolites in rat urine.

Ten potential lignan metabolites were quantified in rat urine extracts using liquid chromatography-tandem mass spectrometry. The rats were orally administered with the plant lignans 7-hydroxymatairesinol, matairesinol, lariciresinol or secoisolariciresinol, or with the mammalian lignan enterolactone. The samples were enzymatically hydrolysed and solid-phase extracted before analysis. Of the analysed compounds, only trace amounts of 7-oxoenterolactone could be detected in the urine extracts before administration, but after administration of any of the lignans, the excretion of 7-oxoenterolactone increased and monodemethylated matairesinol and 4,4'-dihydroxyenterolactone could be detected. In addition, other novel lignan metabolites were detected, i.e., 7-oxomatairesinol, alpha-conidendrin, and alpha- and beta-conidendric acid.

Pharmaceuticals and Surfactants from Alga-Derived Feedstock: Amidation of Fatty Acids and Their Derivatives with Amino Alcohols.

Amidation of renewable feedstocks, such as fatty acids, esters, and Chlorella alga based biodiesel, was demonstrated with zeolites and mesoporous materials as catalysts and ethanolamine, alaninol, and leucinol. The last two can be derived from amino acids present in alga. The main products were fatty alkanol amides and the corresponding ester amines, as confirmed by NMR and IR spectroscopy. Thermal amidation of technical-grade oleic acid and stearic acid at 180 °C with ethanolamine were non-negligible; both gave 61% conversion. In the amidation of stearic acid with ethanolamine, the conversion over H-Beta-150 was 80% after 3 h, whereas only 63% conversion was achieved for oleic acid; this shows that a microporous catalyst is not suitable for this acid and exhibits a wrinkled conformation. The highest selectivity to stearoyl ethanolamide of 92% was achieved with mildly acidic H-MCM-41 at 70% conversion in 3 h at 180 °C. Highly acidic catalysts favored the formation of the ester amine, whereas the amide was obtained with a catalyst that exhibited an optimum acidity. The conversion levels achieved with different fatty acids in the range C12-C18 were similar; this shows that the fatty acid length does not affect the amidation rate. The amidation of methyl palmitate and biodiesel gave low conversions over an acidic catalyst, which suggested that the reaction mechanism in the amidation of esters was different.

Screening of some anti-androgenic endocrine disruptors using a recombinant cell-based in vitro bioassay.

The present work describes the development and optimization of a cell-based androgen reporter assay using the Chinese hamster ovarian cell line (CHO K1) in the 96-well format. The recent reports on increasing exposure of humans and wild-life to environmental endocrine disrupting chemicals (ED) prompt the need for high throughput screening systems for such compounds in environmental and biological samples. To this end, CHO cells were cotransfected with plasmids encoding mouse mammary tumour virus-neomycin-luciferase and human androgen receptor (hAR), and a stable cell line was established. After selection with neomycin, a highly active clone was obtained which stably expressed both the hAR and the androgen-responsive luciferase reporter. Stimulation of the cells with androgens for 24 h resulted in about 15-fold stimulation of luciferase activity, with the minimum effective dose of testosterone being 0.1 nmol/l. Potent steroidal and non-steroidal anti-androgens, such as hydroxyflutamide and cyproterone acetate, significantly inhibited the androgen-induced transactivation. Non-androgenic steroids like estradiol, progesterone, dexamethasone and cortisol showed weak activity at high concentrations. RT-PCR and western blot confirmed proper transcription and translation as well as stable expression of the AR gene in the cells. About 60 different chemicals (mostly pesticides or their metabolites, and common industrial chemicals) were screened with the cell line for their ability to stimulate luciferase activity or inhibit that evoked by 0.1 nmol/l R1881, used as a positive androgenic control. About 10 highly potent anti-androgenic chemicals were identified. The most potent anti-androgenic compounds identified in our assay included bisphenol A, alpha-hexachlorocyclohexane, vinclozolin and 4,4-DDE. These compounds had alone either no effect or were weak agonists (with cytotoxic effects at very high concentrations), but none showed any significant agonistic activity. In conclusion, we demonstrate that the bioassay based on this cell line provides a reliable test for detecting androgenic and anti-androgenic compounds. The 96-well plate format makes the assay suitable for high throughput screening.