Food Additives as Inhibitors of Intestinal Drug Transporter OATP2B1.

Food additives are compounds that are added to food and beverage to improve the taste, color, preservation, or composition. Generally, food additives are considered safe for human use due to safety evaluations conducted by food safety authorities and high safety margins applied to permitted usage levels. However, the interaction potential of food additives with simultaneously administered medication has not received much attention. Even though many food additives are poorly absorbed into systemic circulation, high concentrations could exist in the intestinal lumen, making intestinal drug transporters, such as the uptake transporter organic anion transporting polypeptide 2B1 (OATP2B1), a possible site of food additive-drug interactions. In the present work, we aimed to characterize the interaction of a selection of 25 food additives including colorants, preservatives, and sweeteners with OATP2B1 in vitro. In human embryonic kidney 293 (HEK293) cells transiently overexpressing OATP2B1 or control, uptake of dibromofluorescein was studied with and without 50 µM food additive at pH 7.4. As OATP2B1 displays substrate- and pH-dependent transport functions and the intraluminal pH varies along the gastrointestinal tract, we performed the studies also at pH 5.5 using estrone sulfate as an OATP2B1 substrate. Food additives that inhibited OATP2B1-mediated substrate transport by ≥50% were subjected to dose-response studies. Six colorants were identified and validated as OATP2B1 inhibitors at pH 5.5, but only three of these were categorized as inhibitors at pH 7.4. One sweetener was validated as an inhibitor under both assay conditions, whereas none of the preservatives exhibited ≥50% inhibition of OATP2B1-mediated transport. Extrapolation of computed inhibitory constants (Ki values) to estimations of intestinal food additive concentrations implies that selected colorants could inhibit intestinal OATP2B1 also in vivo. These results suggest that food additives, especially colorants, could alter the pharmacokinetics of orally administered OATP2B1 substrate drugs, although further in vivo studies are warranted to understand the overall clinical consequences of the findings.

Dual cancer targeting using estrogen functionalized chitosan nanoparticles loaded with doxorubicin-estrone conjugate: A quality by design approach.

In this study, estrone was used as targeting functionality in chitosan nanoparticles (DoxEs-CSEsNPs) carrying doxorubicin-estrone conjugate for dual targeted intracellular delivery to breast cancer cells. Estrone was conjugated with Dox and CS and characterized by FTIR and FT-NMR spectroscopy. Dox/DoxEs containing CSEsNPs were prepared with ionic gelation method and for the effect of formulation variables a 3-factor, 3-level Box-Behnken design (BBD) was explored, which predict the responses like particle size (Y1) and percent entrapment efficiency (%EE) (Y2) when CSEs: TPP ratio (X1), sonication time (X2) and stirring speed (X3) were selected as independent variables. The Dox-CSEsNPs and DoxEs-CSEsNPs were characterized for size, shape, PDI, surface charge and thermal analysis. The drug entrapment efficiency was 66.33 ± 2.82% and 62.25 ± 2.63% for Dox-CSEsNPs and DoxEs-CSEsNPs formulation respectively. The in vitro release, haemolytic toxicity, and fluorescent microscopy studies were also assessed. Anticancer activity on the MCF-7 cell line indicated the higher potency of DoxEs-CSEsNPs as compared to Dox-CSEsNPs, DoxEs, and Dox solution. The findings are decisive for selective targeting of antineoplastic agents to the ERs, which indicate that the DoxEs loaded CSEsNPs were able to significantly improve the efficacy of Dox.

Aspartate racemase and D-aspartate in starfish; possible involvement in testicular maturation.

D-Aspartate, aspartate racemase activity, and D-aspartate oxidase activity were detected in tissues from several types of starfish. Aspartate racemase activity in male testes of Patiria pectinifera was significantly elevated in the summer months of the breeding season compared with spring months. We also compared aspartate racemase activity with the gonad index and found that activity in individuals with a gonad index ≥6% was four-fold higher than that of individuals with a gonad index <6%. The ratio of the D-form of aspartate to total aspartate was approximately 25% in testes with a gonad index <6% and this increased to approximately 40% in testes with a gonad index ≥6%. However, such changes were not observed in female ovaries. Administration of D-aspartate into male starfish caused testicular growth. These results indicate the possible involvement of aspartate racemase and D-aspartate in testicular maturation in echinoderm starfish.

Turtle hatchlings show behavioral types that are robust to developmental manipulations.

There can be substantial variation among individuals within a species in how they behave, even under similar conditions; this pattern is found in many species and across taxa. However, the mechanisms that give rise to this behavioral variation are often unclear. This study investigated the influence of environmental manipulations during development on behavioral variation in hatchlings of the red-eared slider turtle (Trachemys scripta). First, we examined the effects of three manipulations during incubation (estrone sulfate exposure, corticosterone exposure, and thermal fluctuations) on hatchling righting response and exploration. Second, we determined whether hatchlings showed consistent differences (i.e. behavioral types) in their righting response and exploration across days and months, and whether these behaviors were correlated with one another. Finally, we examined whether righting response was predictive of ecologically relevant behaviors such as habitat choice and dispersal. Hatchling behavior was robust to our early manipulations; none of the pre-hatch treatments affected later behavior. There were significant clutch effects, which due to the split-clutch design suggests genetic underpinnings and/or maternal effects. We found evidence for behavioral types in turtles; both righting response and exploration were strongly repeatable and these behaviors were positively correlated. Righting response was not predictive of dispersal ability in the field, necessitating a revision in the general interpretations of righting response as a proxy for dispersal ability in turtles. Thus, turtle hatchlings show consistent behavioral differences that are robust to early developmental manipulations, and while not necessarily predictive of dispersal, these behavioral types can have important consequences throughout ontogeny.

Transdermal estradiol treatment during breastfeeding: maternal and infant serum concentrations.

We examined estradiol (E2) and estrone (E1) concentrations in breastfeeding mother-infant dyads. The mothers had postpartum depression and were participants in a randomized clinical trial with three treatments (transdermal E2, sertraline, and placebo). Neither infant E1 and E2 concentrations nor infant growth differed across the treatments. Transdermal E2 administration of 50 to 200 mcg/day for breastfeeding women did not affect infant E1 or E2 concentrations or infant growth.

Salutary Effects of Estrogen Sulfate for Traumatic Brain Injury.

Estrogen plays an important role as a neuroprotector in the central nervous system (CNS), directly interacting with neurons and regulating physiological properties of non-neuronal cells. Here we evaluated estrogen sulfate (E2-SO4) for traumatic brain injury (TBI) using a Sprague-Dawley rat model. TBI was induced via lateral fluid percussion (LFP) at 24 h after craniectomy. E2-SO4 (1 mg/kg BW in 1 mL/kg BW) or saline (served as control) was intravenously administered at 1 h after TBI (n=5/group). Intracranial pressure (ICP), cerebral perfusion pressure (CPP), and partial brain oxygen pressure (pbtO2) were measured for 2 h (from 23 to 25 h after E2-SO4 injection). Brain edema and diffuse axonal injury (DAI) were assessed by diffusion tensor imaging (DTI), and cerebral glycolysis was measured by (18)F-labeled fluorodeoxyglucose (FDG) positron emission tomography (PET) imaging, at 1 and 7 days after E2-SO4 injection. E2-SO4 significantly decreased ICP, while increasing CPP and pbtO2 (p<0.05) as compared with vehicle-treated TBI rats. The edema size in the brains of the E2-SO4 treated group was also significantly smaller than that of vehicle-treated group at 1 day after E2-SO4 injection (p=0.04), and cerebral glycolysis of injured region was also increased significantly during the same time period (p=0.04). However, E2-SO4 treatment did not affect DAI (p>0.05). These findings demonstrated the potential benefits of E2-SO4 in TBI.

Synergistic dual-targeting hydrogel improves targeting and anticancer effect of Taxol in vitro and in vivo.

A synergistic dual-targeting molecular self-assembly hydrogel was designed with estrone (Et) and Arg-Gly-Asp (RGD) peptide. Confocal imaging in vitro and real-time visualization in vivo researches were carried out to evidence enhanced targeted delivery and anticancer effect of Taxol.

Separation of dansylated 17ß-estradiol, 17α-estradiol, and estrone on a single HPLC column for simultaneous quantitation by LC-MS/MS.

We show here that baseline separation of dansylated estrone, 17ß-estradiol, and 17α-estradiol can be done, contrary to previous reports, within a short run time on a single RP-LC analytical column packed with particles bonded with phenyl-hexyl stationary phase. The chromatographic method coupled with isotope dilution tandem MS offers a simple assay enabling the simultaneous analysis of these analytes. The method employs (13)C-labeled estrogens as internal standards to eliminate potential matrix effects arising from the use of deuterated estrogens. The assay also offers adequate accuracy and sensitivity to be useful for biological samples. The practical applicability of the validated method is demonstrated by the quantitative analyses of in vivo samples obtained from rats treated with Premarin®.

Continuous estrone treatment impairs spatial memory and does not impact number of basal forebrain cholinergic neurons in the surgically menopausal middle-aged rat.

CEE (conjugated equine estrogens) is the most widely prescribed estrogen-only menopausal hormone therapy in the United States, and is comprised of over 50% estrone (E1) sulfate. Following CEE administration, E1 is the principal circulating estrogen. However, the cognitive and neurobiological effects of E1 in a middle-aged rodent model have not yet been evaluated. We assessed cognitive effects of continuous E1 treatment in middle-aged surgically menopausal rats using a maze battery. We also quantified number of choline acetyltransferase-immunoreactive (ChAT-IR) neurons in distinct basal forebrain regions known in earlier studies in to be impacted by the most potent naturally-circulating estrogen in rodents and women, 17ß-estradiol (17ß-E2), as well as CEE. On the spatial working memory delayed-match-to-sample water maze, the highest E1 dose impaired memory performance during acquisition and after delay challenge. E1 did not impact ChAT-IR neuron number in the medial septum (MS) or horizontal/vertical diagonal bands. In a comparison study, 17ß-E2 increased MS ChAT-IR neuron number. Findings indicate that E1 negatively impacts spatial working memory and memory retention, and does not increase ChAT-IR neuron number in basal forebrain, as does 17ß-E2. Thus, data from prior studies suggest that 17ß-E2 and CEE can enhance cognition and increase number of ChAT-IR basal forebrain neurons, while here we show that E1 does not induce these effects. Findings from preclinical basic science studies can inform the design of specific combinations of estrogens that could be beneficial to the brain and cognition. Accumulating data suggest that E1 is not likely to be among these key beneficial estrogens.

Development of an LC-MS/MS method to quantify sex hormones in bovine milk and influence of pregnancy in their levels.

Hormones work in harmony in the body, and this status must be maintained to avoid metabolic disequilibrium and the subsequent illness. Besides, it has been reported that exogenous steroids (presence in the environment and food products) influence the development of several important illnesses in humans. Endogenous steroid hormones in food of animal origin are unavoidable as they occur naturally in these products. The presence of hormones in food has been connected with several human health problems. Bovine milk contains considerable quantities of hormones and it is of particular concern. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, based on hydroxylamine derivatisation, has been developed and validated for the quantification of six sex hormones in milk [pregnenolone (P5), progesterone (P4), estrone (E1), testosterone (T), androstenedione (A) and dehydroepiandrosterone (DHEA)]. This method has been applied to real raw milk samples and the existence of differences between milk from pregnant and non-pregnant cows has been statistically confirmed. Basing on a revision of existing published data, it could be concluded that maximum daily intakes for hormones are not reached through milk ingestion. Although dairy products are an important source of hormones, other products of animal origin must be considered as well for intake calculations.

Effects of unintentional exposure of children to compounded transdermal sex hormone therapy.

Gynecomastia and rapid growth progressed in twin brothers and pubic hair in one, over a period of 2 years. A combination of contra- and isosexual development was induced by transdermal exposure to compounded estradiol, estrone, and testosterone creams applied to their mother's body as part of a hormone replacement regimen.

Oleoyl-estrone is a precursor of an estrone-derived ponderostat signal.

Oleoyl-estrone (OE) is a powerful anti-obesity compound that decreases food intake, decreases insulin resistance and circulating cholesterol. OE stimulates a severe loss of body fat by decreasing adipose tissue lipid synthesis and maintaining lipolysis. Therefore, the body economy loses lipid energy because energy expenditure is maintained. This study analyses the discrepancy between OE effects and the distribution of labelled OE in plasma. Estrone radioimmunoassay of organic solvent plasma extracts of rats treated with OE showed the massive presence of acyl-estrone, but saponification did not release estrone, but containing similar unknown compound. Analysis of label distribution in plasma after oral gavages of (3)H-OE showed the presence of a more hydrophilic compound than OE or any estrogen as well as (3)H(2)O, formed from (3)H-OE in the acidic stomach medium. OE was not attached to a specific transporter in plasma. Through serum HPLC analysis we found W, a labelled derivative more hydrophilic than OE or estrone. The results were confirmed using (14)C-OE. HPLC-MS/MS studies showed that plasma OE levels were one order of magnitude lower than those of W. When liver cell cytosols from rats laden with (3)H-OE were incubated with nuclei from untreated rats, the OE-derived label (i.e., Ws) was found attached to nuclear DNA. Neither estradiol nor estrone interfered with its binding. W is a fairly hydrophilic compound of low molecular weight containing the estrone nucleus, but it is not an ester because saponification or esterases do not yield estrone as OE does. It is concluded that OE acts through its conversion to W, its active form; which binds to a nuclear receptor different from that of estrogen. The estimated W serum levels are proportional to the pharmacological OE effects in vivo. We postulate W as a new type of hormone that exerts the full range of in vivo effects thus far attributed to OE. The full identification of W is anticipated to open the way for the development of new OE-like anti-obesity drugs.

Site-specific modulation of white adipose tissue lipid metabolism by oleoyl-estrone and/or rosiglitazone in overweight rats.

In spite of their shared decrease of insulin resistance, oleoyl-estrone [OE], and rosiglitazone show diverging effects on body fat mass and distribution. In this study, we studied whether their effects on white adipose tissue [WAT] were due to a shared or synergistic mechanism of action. Combined effects of OE and rosiglitazone 10-day treatment on WAT lipid, cell mass/number, and the expression of key lipid metabolism and regulatory agents were studied using an adult male overweight rat model. OE decreased WAT cell mass and lipids, parameters not changed by rosiglitazone. The effects of OE and--specially--rosiglitazone were more marked in small-cell WAT (i.e., mesenteric and subcutaneous sites) than in larger cell WAT (retroperitoneal and perigonadal). OE decreased the expressions in WAT of lipogenic enzymes, lipoprotein lipase, PPARs, and SREBP1c, effects symmetrically reversed by rosiglitazone. OE showed no effects on hormone-sensitive lipase expression, which was increased by rosiglitazone. OE strongly inhibited WAT lipogenesis, leaving lipolysis unchanged, thus unbalancing (and helping mobilize) WAT lipid stores. Rosiglitazone acted practically only on small-cell WAT sites, where it favored lipogenesis, but also stimulated lipolysis, which resulted in limited changes in lipid stores. Combination of OE and rosiglitazone induced less fat loss than OE alone.

Oleoyl-estrone increases adrenal corticosteroid synthesis gene expression in overweight male rats.

Oleoyl-estrone (OE) induces a marked loss of body fat in rats by maintaining energy expenditure, body protein and blood glucose despite decreasing food intake. OE increases glucocorticoids, but they arrest OE lipid-mobilization. We studied here whether OE induces a direct effect on adrenal glands function as part of this feedback regulation. Dietary overweight male rats were given oral 10nmol/g OE gavages for ten days. A group (PF) of pair-fed to OE rats, and controls received vehicle-only gavages. OE rats lost slightly more body than PF, but had larger adrenal glands. Tissue corticosterone levels, and gene expressions for glucocorticoid-synthesizing enzymes were increased in OE versus controls and PF; thus, we assumed that adrenal growth affected essentially its cortex since OE also lowered the expression of the medullar catecholamine synthesis enzyme genes. Serum corticosterone was higher in PF than in OE and controls, but liver expression of corticosteroid-disposing steroid 5alpha-reductase was 3x larger in OE than PF and controls. Circulating glucocorticoids changed little under OE, in spite of higher adrenal gland and liver content, hinting at modulation of glucocorticoid turnover as instrumental in their purported increased activity. In conclusion, we have observed that OE considerable enhanced the expression of the genes controlling the synthesis of glucocorticoids from cholesterol in the rat and increasing the adrenal glands' corticosterone, size and cellularity, but also the liver disposal of corticosteroids, suggesting that OE increases corticosterone synthesis and degradation (i.e. serum turnover), a process not driven by limited energy availability but directly related to the administration of OE.

Evaluation of estrogen treatment in an immunodeficient mouse endometriosis model.

BACKGROUND/AIMS: Endometriosis is known to be an estrogen-dependent disease. However, only a few studies have analyzed the effect of estrogen treatment in mice xenotransplanted with human endometrium. The objective of this study was to adapt a previously developed heterologous murine model to the study of estrogens and test the impact of estrone treatment on endometriosis development. METHODS: Human proliferative endometrium was xenotransplanted into the peritoneal cavity of castrated immunodeficient mice. These mice were treated with estrogens by means of subcutaneous estrone-releasing pellets. The effect of estrone on estradiol level, uterine histology and endometriosis development was evaluated after 21 days. RESULTS: Bioactivity of estrone pellets and their metabolization into estradiol were demonstrated. However, there was no impact on endometriosis development (no difference in lesion number, weight, size or fluorescence). This lack of response was not due to absence of estrogen receptor expression, since strong expression was found in all lesions harvested. Surprisingly, castrated nontreated mice presented with lesions showing high proliferative activity, similar to lesions found in treated mice (around 30%). CONCLUSION: The high proliferation observed in lesions recovered from ovariectomized nontreated mice questions the utility of using estrogens in heterologous murine models.

Molecular clustering of endometrial carcinoma based on estrogen-induced gene expression.

Identification of biomarkers potentially provides prognostic information that can help guide clinical decision-making. Given the relationship between estrogen exposure and endometrial cancer, especially low grade endometrioid carcinoma, we hypothesized that high expression of genes induced by estrogen would identify low risk endometrioid endometrial cancers. cDNA microarray and qRT-PCR verification were used to identify six genes that are highly induced by estrogen in the endometrium. These estrogen-induced biomarkers were quantified in 72 endometrial carcinomas by qRT-PCR. Unsupervised cluster analysis was performed, with expression data correlated to tumor characteristics. Time to recurrence by cluster was analyzed using the Kaplan-Meier method. A receiver operating characteristic (ROC) curve was generated to determine the potential clinical utility of the biomarker panel to predict prognosis. Expression of all genes was higher in endometrioid carcinomas compared to non-endometrioid carcinomas. Unsupervised cluster analysis revealed two distinct groups based on gene expression. The high expression cluster was characterized by lower age, higher BMI, and low grade endometrioid histology. The low expression cluster had a recurrence rate 4.35 times higher than the high expression cluster. ROC analysis allowed for the prediction of stage and grade with a false negative rate of 4.8% based on level of gene expression in endometrioid tumors. We have therefore identified a panel of estrogen-induced genes that have potential utility in predicting endometrial cancer stage and recurrence risk. This proof-of-concept study demonstrates that biomarker analysis may play a role in clinical decision making for the therapy of women with endometrial cancer.

Impact of carriers in oral absorption: Permeation across Caco-2 cells for the organic anions estrone-3-sulfate and glipizide.

Carriers may mediate the permeation across enterocytes for drug substances being organic anions. Carrier mediated permeation for the organic anions estrone-3-sulfate (ES) and glipizide across Caco-2 cells were investigated kinetically, and interactions on involved carriers evaluated. Initial uptakes (P(UP)) at apical and basolateral membranes, apparent permeabilities (P(APP)) and corresponding intracellular end-point accumulations (P(EPA)) of radioactive labeled compounds were studied. Possible effects of other anionic compounds were investigated. Apical P(UP) and absorptive P(APP) for ES were inhibited and its absorptive P(EPA) prevented in presence of the investigated organic anions and apical P(UP) was saturable with K(m) 23microM. Basolateral P(UP) and exsorptive P(APP) were inhibited, its exsorptive P(EPA) was prevented, and basolateral P(UP) and exsorptive P(APP) were saturable with K(m) 44microM and 38microM, respectively. BCRP inhibition affected both absorptive an exsorptive P(EPA) and P(APP) for ES. Glipizide apical P(UP) and absorptive P(APP) were not inhibitable. Basolateral P(UP) for glipizide was inhibitable, its P(EPA) prevented, and P(UP) was saturable with K(m) 56microM, but exsorptive P(APP) was not affected. Carrier mediated exsorption kinetics for ES are seen at both apical and basolateral membranes, resulting in predominant exsorption despite presence of absorptive carrier(s). Carrier mediated basolateral P(UP) for glipizide was observed, but glipizide P(APP) was not described by carrier kinetics. However, glipizide is affecting exsorption for ES, due to interactions on basolateral carrier. The study confirms that estrone-3-sulfate can be used to characterize anionic carrier kinetics. Furthermore it is suggested that estrone-3-sulfate may be used to identify compounds which may interact on anionic carriers.

Oleoyl-estrone affects lipid metabolism in adrenalectomized rats treated with corticosterone through modulation of SREBP1c expression.

Oleoyl-estrone (OE) elicits a decrease in body fat, which is blocked by glucocorticoids. In order to analyze this counterregulatory effect, we studied the effects of oral OE on adrenalectomized female rats simultaneously receiving corticosterone (subcutaneous pellets). Circulating corticosteroids, liver glycogen, lipids and the expressions in whole liver, soleus muscle, interscapular brown adipose tissue (BAT), and the inguinal and periovaric white adipose tissue (WAT) of genes controlling lipid metabolism were analyzed. Corticosterone reversed OE lipid mobilization, storing fat in liver and subcutaneous WAT. This was not simply the predominance of corticosteroid enhancement of lipogenesis against OE inhibition, but a synergy to enhance lipogenesis. Periovaric WAT showed a different effect, with corticosterone inhibiting OE arrest of lipogenic gene expressions. The data presented suggests that interaction of OE and glucocorticoids (and the metabolic response) depends on the organ or WAT site; there was a direct relationship on the direction and extent of change of SREBP1c expression with those of important energy and lipid handling genes. Our results confirm that corticosterone blocks - and even reverses - OE effects on body lipids in a dose-dependent way, a process mediated, at least in part, by modulation of SREBP1c expression.

Short-term oral oleoyl-estrone decreases the expression of ghrelin in the rat stomach.

Oleoyl-estrone (OE) mobilizes body fat and decreases food intake. The precise mechanism of its modulation of appetite is unknown. Since the effects of OE on food intake appear early, here we studied the effect of OE on the expression of gut peptides that affect short-term ingestive behavior: ghrelin, leptin, CCK, PYY, and GLP-1. Two hours after a single OE dose, adult male rats were killed and their stomach fundus and intestine sections were dissected and processed for real-time PCR amplification. Semi-quantitative estimation of gene mRNA tissue levels showed that OE markedly decreased ghrelin expression in the stomach; leptin mRNA was unchanged; CCK mRNA decreased in the proximal intestine while PYY and GLP-1 expression in the intestine was not altered. Our results indicate that the short-term decrease in food intake induced by OE may be essentially the consequence of a marked decrease in the expression of ghrelin in the stomach.

Sensitivity of brown trout reproduction to long-term estrogenic exposure.

A decline in brown trout (Salmo trutta fario) catches has been reported in Switzerland, but at present the causative factors have not been clearly identified. Estrogen-active endocrine disrupters (EEDs) have been suggested as one possible explanation, since they are widespread in the aquatic environment and often found at elevated concentrations. In the present study the effects of long-term estrogenic exposure on the reproductive capability of brown trout were investigated. Adult fish were continuously exposed to an environmentally relevant mixture of the natural estrogens estrone (E1), 17beta-estradiol (E2) and the xenoestrogen 4-nonylphenol (NP); the average measured concentrations over the entire exposure time (n=9) were 14.0 ng/l (Min 8.1 and Max 20.6) for E1, 2.1 ng/l (Min 1.3 and Max 4.1) for E2 and 111.0 ng/l (Min 106.7 and Max 115.9) for NP. A solvent control served as negative control, and up to 10-fold higher mixture concentration than the environmentally relevant concentration served as positive control. The fish were exposed for 150 days from the onset of gonadal recrudescence until sexual maturation. Plasma vitellogenin (Vtg) was significantly induced by both concentrations of the estrogenic mixture, whereas effects on growth and fertility were only observed in fish exposed to the high mixture treatment. Fertilization success and offspring hatchability in brown trout exposed to the high mixture treatment were significantly reduced to 9% and 6%, respectively. Developmental time from fertilization until hatching, the percentage of larvae with malformations and survival of larvae, however, were not affected. The results suggest that a combination of estrogen-active compounds at environmentally relevant concentrations would not adversely affect those parameters of brown trout reproductive capability measured in this study. Plasma Vtg in male brown trout appeared to be more sensitive to (xeno)estrogen exposure than the measured reproductive effects.