Biosensor zebrafish provide new insights into potential health effects of environmental estrogens

Background: Environmental estrogens alter hormone signaling in the body that can induce reproductive abnormalities in both humans and wildlife. Available testing systems for estrogens are focused on specific systems such as reproduction. Crucially, however, the potential for significant health impacts of environmental estrogen exposures on a variety of body systems may have been overlooked.Objective: Our aim was to develop and apply a sensitive transgenic zebrafish model to assess real-time effects of environmental estrogens on signaling mechanisms in a whole body system for use in integrated health assessments.Methods: We created a novel transgenic biosensor zebrafish containing an estrogen-inducible promoter derived with multiple tandem estrogen responsive elements (EREs) and a Gal4ff-UAS system for enhanced response sensitivity.Results: Using our novel estrogen-responsive transgenic (TG) zebrafish, we identified target tissues for environmental estrogens; these tissues have very high sensitivity even at environmentally relevant concentrations. Exposure of the TG fish to estrogenic endocrine-disrupting chemicals (EDCs) induced specific expression of green fluorescent protein (GFP) in a wide variety of tissues including the liver, heart, skeletal muscle, otic vesicle, forebrain, lateral line, and ganglions, most of which have not been established previously as targets for estrogens in fish. Furthermore, we found that different EDCs induced GFP expression with different tissue response patterns and time trajectories, suggesting different potential health effects.Conclusion: We have developed a powerful new model for understanding toxicological effects, mechanisms, and health impacts of environmental estrogens in vertebrates.     

Photodegradation of common environmental pharmaceuticals and estrogens in river water

Photodegradation rates of five pharmaceuticals (gemfibrozil, ibuprofen, ketoprofen, naproxen, and propranolol) and of four estrogens (estriol, estrone [E1], 17beta-estradiol [E2], and 17alpha-ethinylestradiol [EE2]), which are common contaminants in the aquatic environment, were measured in both purified and river water at environmentally relevant concentrations (1-2 microg/L) and different oxygen concentrations. Solutions were irradiated with a xenon arc lamp (765 W/m2; 290 nm < lambda < 700 nm) and analyzed using a high-performance liquid chromatography-tandem mass spectrometry method with electrospray ionization for pharmaceuticals and atmospheric pressure photoionization for estrogens. In river water, half-lives were 4.1 min [corrected] for ketoprofen, 1.1 h [corrected] for propranolol, 1.4 h for naproxen, 2 to 3 h for estrogens, and 15 h for gemfibrozil and ibuprofen. In air-saturated purified water, rates generally were slower except for that of ketoprofen, which reacted with a half-life of 2.5 min. Naproxen, propranolol, and E1 reacted with half-lives of 1.9, 4.4, and 4.7 h, respectively. The EE2, estriol, E2, gemfibrozil, and ibuprofen reacted with half-lives of 28.4, 38.2, 41.7, 91.4, and 205 h, respectively. The presence of oxygen doubled the direct photolysis rates of naproxen and propranolol. In nonautoclaved river water, 80% of E2 rapidly biotransformed to E1 within less than 20 min, whereas all other compounds remained stable over 22 h.     

Occurrence and fate of pharmaceuticals and alkylphenol ethoxylate metabolites in an effluent-dominated river and wetland

The occurrence of pharmaceuticals, nonylphenol ethoxylate metabolites, and other wastewater-derived contaminants in surface waters is a potential environmental concern, especially since the discovery of contaminants with endocrine-disrupting properties. The present study investigated the discharge of emerging contaminants into the Santa Ana River (CA, USA) and their attenuation during river transport and passage through a constructed wetland. Contaminants studied included pharmaceuticals (gemfibrozil, ibuprofen, naproxen, ketoprofen, and carbamazepine) and their metabolites, hormones, the metabolites of alkylphenol polyethoxylates (APEMs), N-butyl benzenesulfonamide (NBBS), and chlorinated tris-propylphosphates (TCPPs). The APEMs included alkylphenols (APs), short-chain AP polyethoxylates (APEOs), AP polyethoxycarboxylates (APECs), and carboxylated APECs (CAPECs). In wastewater treatment plant effluent, APECs and CAPECs represented the dominant APEM fraction (1.8-18.7 microg/L), whereas APEOs and APs contributed only small amounts to the overall APEM concentrations (0.10-0.92 and < or =0.1 microg/L, respectively) except where the effluent was infiltrated into soil (5.2 microg/L). In effluents, ibuprofen and its metabolites, TCPPs, and NBBS were detected regularly (<0.5 microg/L), and the other pharmaceuticals were detected occasionally. Transport in the Santa Ana River for 11 km resulted in the significant attenuation of all contaminants, from 67% for gemfibrozil to 100% for others. Wetland treatment (residence time, 2-4 d) resulted in partial removal of ibuprofen, gemfibrozil, and TCPPs and transformed APEOs to APECs.     

Cysteic Acid in Dietary Keratin is Metabolized to Glutathione and Liver Taurine in a Rat Model of Human Digestion

Poultry feathers, consisting largely of keratin, are a low-value product of the poultry industry. The safety and digestibility of a dietary protein produced from keratin (KER) was compared to a cysteine-supplemented casein-based diet in a growing rat model for four weeks. KER proved to be an effective substitute for casein at 50% of the total dietary protein, with no changes in the rats’ food intake, weight gain, organ weight, bone mineral density, white blood cell counts, liver glutathione, or blood glutathione. Inclusion of KER in the diet reduced total protein digestibility from 94% to 86% but significantly increased total dietary cysteine uptake and subsequent liver taurine levels. The KER diet also significantly increased caecum weight and significantly decreased fat digestibility, resulting in a lower proportion of body fat, and induced a significant increase in blood haemoglobin. KER is therefore a safe and suitable protein substitute for casein, and the cysteic acid in keratin is metabolised to maintain normal liver and blood glutathione levels.     

Whey protein, as exclusively nitrogen source, controls food intake and promotes glutathione antioxidant protection in Sprague-Dawley rats

The inclusion of whey protein concentrates (WPC) in the diet can lead to a decrease in food intake. Considering that excessive food intake and weight gain are correlated with increased oxidative stress and other risk factors, the anorectic action of WPC may have important clinical implications. The aims of the current study were to verify the effects of WPC in comparison with those of casein on food intake, weight, and oxidized glutathione (GSSG) and total glutathione (GSH) concentrations in the blood and liver with or without oxidative stress induced by oral carbon tetrachloride intoxication. Male Sprague-Dawley rats were fed a balanced liquid diet for 3 weeks. Half of the rats received WPC (group P), while the control group received casein (group C). Group P rats ate significantly less than group C rats (p < 0.0001), and their weights decreased significantly. After carbon tetrachloride intoxication, there was a significant increase in GSH in rats of group P compared with the levels in rats of group C both in the liver (GSH group P 4,994 ± 652.6, group C 2,196 ± 323.2 nmol/mg, p < 0.01) and in the blood (GSH group P 1,368 ± 69.56, group C 1,088 ± 48.35 nmol/ml, p < 0.05). These findings indicate that WPC is effective in reducing food intake and preventing weight gain, and it may also play a protective role against oxidative stress by increasing glutathione synthesis in the liver.     

Relative contribution of cysteine and methionine to glutathione content and thyroid hormone levels in the rat

1. For a period of 24 d rats were given diets containing either casein or pea (Pisum sativum) protein at two different concentrations (180 and 120 g/kg) without or with cysteine or cysteine + methionine supplementation. 2. The effects of these diets on levels of blood and liver reduced glutathione (GSH) and serum thyroid hormones were studied. 3. When compared with the 180 g casein/kg diet, the 120 g casein/kg diet decreased liver GSH and serum thyroid hormone concentrations. These changes were related to dietary cysteine supply since supplementation induced an increase in these variables. 4. When compared with 180 g pea protein/kg diet, the 120 g pea protein/kg diet decreased liver GSH and serum thyroid hormone concentrations. These changes could not be corrected by cysteine or cysteine + methionine supplementation.     

Uptake from water, biotransformation, and biliary excretion of pharmaceuticals by rainbow trout

An urgent need exists to assess the exposure of fish to pharmaceuticals. The aim of the present study was to assess the uptake and metabolism of waterborne pharmaceuticals in rainbow trout (Oncorhynchus mykiss). A further objective was to determine the possibility of monitoring exposure to low levels of pharmaceuticals by bile assays. Rainbow trout were exposed for 10 d under flow-through conditions to mixtures of five pharmaceuticals (diclofenac, naproxen, ibuprofen, bisoprolol, and carbamazepine) at high and low concentrations. The low concentration was used to mimic the conditions prevailing in the vicinity of the discharge points of wastewater treatment plants. The uptake and the bioconcentration were determined by blood plasma and bile analyses. The average bioconcentration factor in plasma ranged from below 0.1 for bisoprolol to 4.9 for diclofenac, the values being approximately similar at low and high ambient concentrations. The biotransformation of diclofenac, naproxen, and ibuprofen was considered efficient, because several metabolites could be detected in concentrations clearly exceeding those of the unmetabolized compounds. The glucuronides were the dominant metabolites for all three pharmaceuticals. The total bioconcentration in the bile was two to four orders of magnitude higher than in the plasma. The results of this work show that the exposure of fish to pharmaceuticals in environmentally relevant concentrations may be monitored by blood plasma and bile analyses, the latter allowing detection at markedly lower ambient concentration.     

Effects of a Rubus coreanus Miquel supplement on plasma antioxidant capacity in healthy Korean men

Korean raspberry, Rubus coreanus Miquel (RCM), contains high concentrations of phenolic compounds, which prevent oxidative stress. To determine the effect of RCM on antioxidant capacity in humans, we assessed in vivo lipid oxidation and antioxidant enzyme activities from plasma in 15 healthy men. The subjects ingested 30 g of freeze-dried RCM daily for 4 weeks. Blood was taken at baseline and at the end of the study to determine blood lipid profiles, fasting plasma glucose, liver function, lipid peroxidation, and antioxidant enzyme activities. RCM supplementation had no effect on blood lipid or fasting plasma glucose concentrations but decreased alkaline phosphatase activity. RCM supplementation increased glutathione peroxidase activities (P < 0.05) but had no effect on lipid peroxidation. These results suggest that short-term RCM supplementation may offer health benefits by enhancing antioxidant capacity in a healthy population.     

Transgenic Zebrafish Reveal Tissue-Specific Differences in Estrogen Signaling in Response to Environmental Water Samples

Background: Environmental endocrine disruptors (EEDs) are exogenous chemicals that mimic endogenous hormones such as estrogens. Previous studies using a zebrafish transgenic reporter demonstrated that the EEDs bisphenol A and genistein preferentially activate estrogen receptors (ERs) in the larval heart compared with the liver. However, it was not known whether the transgenic zebrafish reporter was sensitive enough to detect estrogens from environmental samples, whether environmental estrogens would exhibit tissue-specific effects similar to those of BPA and genistein, or why some compounds preferentially target receptors in the heart.Methods: We tested surface water samples using a transgenic zebrafish reporter with tandem estrogen response elements driving green fluorescent protein expression (5xERE:GFP). Reporter activation was colocalized with tissue-specific expression of ER genes by RNA in situ hybridization.Results: We observed selective patterns of ER activation in transgenic fish exposed to river water samples from the Mid-Atlantic United States, with several samples preferentially activating receptors in embryonic and larval heart valves. We discovered that tissue specificity in ER activation was due to differences in the expression of ER subtypes. ERα was expressed in developing heart valves but not in the liver, whereas ERβ2 had the opposite profile. Accordingly, subtype-specific ER agonists activated the reporter in either the heart valves or the liver.Conclusion: The use of 5xERE:GFP transgenic zebrafish revealed an unexpected tissue-specific difference in the response to environmentally relevant estrogenic compounds. Exposure to estrogenic EEDs in utero was associated with adverse health effects, with the potentially unanticipated consequence of targeting developing heart valves.Citation: Gorelick DA, Iwanowicz LR, Hung AL, Blazer VS, Halpern ME. 2014. Transgenic zebrafish reveal tissue-specific differences in estrogen signaling in response to environmental water samples. Environ Health Perspect 122:356–362; http://dx.doi.org/10.1289/ehp.1307329     

Biological effect monitoring of occupational exposure to 1,3-dichloropropene: effects on liver and renal function and on glutathione conjugation

A prospective study was performed in the Dutch flower bulb culture to investigate the possible effects of subchronic exposure to the soil fumigant 1,3-dichloropropene (DCP) on liver and kidney function and on glutathione conjugation capacity in blood. Urine spot samples and venous blood samples from 14 workers applying DCP (applicators) were taken at the start of the season in July, and after the season in October. The parameters of liver function measured were: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, gamma-glutamyltranspeptidase, and total bilirubin (conjugated and unconjugated). Total bilirubin was significantly decreased from 9.5 before to 7.0 mumol/l after the season. In combination with an increase in serum gamma-glutamyltranspeptidase activity from 12.5 to 19.5 U/l this indicates moderate hepatic enzyme induction. To study renal function, creatinine and beta 2-microglobulin in serum, and beta 2-microglobulin, albumin, alanine aminopeptidase, beta-galactosidase, and retinol binding protein in urine were measured. The glomerular function parameters albumin in urine and creatinine in serum changed significantly during the season: albumin concentration increased from 5.2 to 7.6 mg/l, whereas creatinine concentration [corrected] decreased from 93.0 to 87.5 mumol/l. The tubular function parameter retinol binding protein also increased in concentration from 20.0 to 26.9 micrograms/l. Therefore, a subclinical nephrotoxic effect of subchronic exposure to DCP cannot be excluded. Effects on glutathione conjugation capacity were studied by measuring erythrocyte glutathione S-transferase activity and blood glutathione concentrations. The activity of glutathione S-transferase in erythrocytes was significantly decreased from 4.7 before to 3.3 U/g haemoglobin after the season. The same was true for the blood glutathione concentrations, which decreased from 0.93 to 0.82 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological effect monitoring of occupational exposure to 1,3-dichloropropene: effects on liver and renal function and on glutathione conjugation.

A prospective study was performed in the Dutch flower bulb culture to investigate the possible effects of subchronic exposure to the soil fumigant 1,3-dichloropropene (DCP) on liver and kidney function and on glutathione conjugation capacity in blood. Urine spot samples and venous blood samples from 14 workers applying DCP (applicators) were taken at the start of the season in July, and after the season in October. The parameters of liver function measured were: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, gamma-glutamyltranspeptidase, and total bilirubin (conjugated and unconjugated). Total bilirubin was significantly decreased from 9.5 before to 7.0 mumol/l after the season. In combination with an increase in serum gamma-glutamyltranspeptidase activity from 12.5 to 19.5 U/l this indicates moderate hepatic enzyme induction. To study renal function, creatinine and beta 2-microglobulin in serum, and beta 2-microglobulin, albumin, alanine aminopeptidase, beta-galactosidase, and retinol binding protein in urine were measured. The glomerular function parameters albumin in urine and creatinine in serum changed significantly during the season: albumin concentration increased from 5.2 to 7.6 mg/l, whereas creatinine concentration [corrected] decreased from 93.0 to 87.5 mumol/l. The tubular function parameter retinol binding protein also increased in concentration from 20.0 to 26.9 micrograms/l. Therefore, a subclinical nephrotoxic effect of subchronic exposure to DCP cannot be excluded. Effects on glutathione conjugation capacity were studied by measuring erythrocyte glutathione S-transferase activity and blood glutathione concentrations. The activity of glutathione S-transferase in erythrocytes was significantly decreased from 4.7 before to 3.3 U/g haemoglobin after the season. The same was true for the blood glutathione concentrations, which decreased from 0.93 to 0.82 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

3,4-dichloroaniline-induced oxidative stress in liver of crucian carp (Carassius auratus)

3,4-dichloroaniline (DCA) and its analogs are widely used as chemical intermediates in the synthesis of herbicides, azo dyes, and pharmaceuticals. They bring danger to growth, development, and propagation of aquatic organisms. The purpose of this study was to show DCA-induced oxidative stress response in liver of crucian carp (Carassius auratus). Superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), nitric oxide (NO), and NO synthase (NOS) in liver were measured in crucian carp after exposure to DCA solution (0.2, 0.4, and 0.8 mg/L, respectively) for 15 days and compared with the control. DCA significantly induced SOD activity and enhanced MDA concentration in liver of crucian carp. Compared with the control, GSH and NO concentrations decreased and NOS activity was inhibited in crucian carp liver 15 days after DCA treatment. The data suggested that DCA-induced free radical generation and antioxidant depletion, and caused oxidative stress and lipid peroxidation in liver of crucian carp.     

Bioaccumulation, oxidative stress, and neurotoxicity in Danio rerio exposed to different isotopic compositions of uranium

Experiments were carried out on adult male zebrafish (Danio rerio) to assess early changes induced by waterborne exposure to different isotopic compositions of uranium (depleted uranium associated or not with 233U). Oxidative stress and neurotoxicity were selected as effect endpoints to characterize uranium chemo- and radiotoxicity. Catalase, glutathione peroxidase, and superoxide dismutase activities and total glutathione content of hepatic extracts, as well as brain acetylcholinesterase activity and uranium bioaccumulation, were measured. Oxidative stress induced by uranium exposure led to decreases in superoxide dismutase and catalase activity levels as well as total glutathione content in liver extracts. These perturbations were significantly more marked in 233U-exposed fish. Furthermore, significant increase in acetylcholinesterase activity was observed in brain extracts at the same level, whatever the isotopic composition of uranium.     

Whole blood and mononuclear cell glutathione response to dietary whey protein supplementation in sedentary and trained male human subjects

Sedentary male subjects (n=9) on a controlled diet were fed two doses (0.8 or 1.6 g/kg body mass) of a whey protein isolate (WPI), in addition to an isocaloric placebo; blood samples were drawn over a 4-h period and glutathione concentration determined. There was no effect of the supplementation at either level over the 4-h sampling period. The effects of a WPI supplemented diet on glutathione concentrations in whole blood as well as peripheral mononuclear cell populations were also investigated over a 6-week period in male subjects (n=18) involved in arduous aerobic training; blood was collected prior to and following a 40 km simulated cycling trial. The aerobic training period resulted in significantly lower glutathione concentrations in whole blood, an effect that was mitigated by WPI supplementation. A significant increase in mononuclear cell glutathione was also observed in subjects receiving the WPI supplement following the 40 km simulated cycling trial.     

Acute ingestion of dietary proteins improves post-exercise liver glutathione in rats in a dose-dependent relationship with their cysteine content

Dietary sulfur amino acids affect glutathione synthesis, but their acute effect under conditions of oxidative stress is unknown. We assessed the effect of the selective ingestion of alpha-lactalbumin, a cysteine-rich protein, on glutathione homeostasis before a single bout of exhaustive exercise. One hour before a 2-h run on a treadmill, untrained rats ingested a meal enriched with either milk protein (TMP), alpha-lactalbumin-enriched milk protein (alpha-LAC), glucose (GLUC) or milk protein plus 150 mg N-acetyl-L-cysteine, a pharmacologic cysteine donor (NAC). Glutathione status was monitored in the blood and measured postexercise in the liver and heart. A group of fed sedentary rats was used as a control (CON). Blood total glutathione levels declined over time in all test groups. Although postexercise heart glutathione did not differ among groups, postexercise liver glutathione was curvilinearly related to prior cysteine intake (R2=0.999, P<0.05). In alpha-LAC rats, liver glutathione was 60-80% higher than in GLUC or CON rats (P<0.05) and did not differ from that of NAC rats. Cysteine from dietary proteins exhibits a considerable, dose-dependent and acute stimulatory effect on liver glutathione during exercise but does not immediately benefit whole-body glutathione homeostasis, presumably because of an overlap between the postprandial and exercise-related states.     

Oxidative stress on domestic ducks (Shaoxing duck) chronically exposed in a Mercury-Selenium coexisting mining area in China

The Wanshan mercury mine is the largest mercury deposit in Guizhou Province, China. Few attempts have been made to study the toxic effects of mercury on biota in this mining area. This study was the first to investigate the oxidative stress on domestic ducks (Shaoxing duck) chronically exposed to mercury in the Wanshan mining area. Chemical analyses revealed higher concentrations of both, mercury and selenium in samples from the Wanshan area. Total mercury and selenium concentrations in duck tissues varied from 0.073 to 4.465 mg/kg and from 1.073 to 6.35 mg/kg, respectively. Analysis of covariance revealed that there were significant effects of zone on accumulation of mercury and selenium in all duck tissues (P<0.01). Moreover, analysis of covariance indicated that mercury content significantly affected the accumulation of selenium in duck muscle, brain, and liver (P<0.01). Mercury and selenium were also highly correlated in Wanshan duck liver, muscle, brain, and lung. The statistical analysis suggested that selenium might be an interactive factor in mercury toxicity. As for the biochemical analyses, it was observed that selenium-dependent glutathione peroxidase and superoxide dismutase activities and glutathione (GSH) content were significantly increased in the livers and brains of Wanshan ducks (P<0.01, 0.05). However, no significant changes were observed in malondialdehyde content (P>0.05). Although the results indicated that adaptive responses of the redox-defense system are associated with the increased enzyme activities and GSH content, the most likely explanation is that selenium plays a critical role. Therefore, the effects of the interaction environmentally occurring selenium and mercury on public health in the Wanshan area should be examined in further studies.     

Effects of ammonium perchlorate on the reproductive performance and thyroid follicle histology of zebrafish

Adult zebrafish were reared up to eight weeks in control water or in water containing ammonium perchlorate (AP) at measured perchlorate concentrations of 18 (environmentally relevant, high) and 677 ppm. Groups of eight females were paired with four males on a weekly basis to assess AP effects on spawned egg volume, an index of reproductive performance. All treatments were applied to four to five spawning replicates. At 677 ppm, spawn volume was reduced within one week and became negligible after four weeks. At 18 ppm, spawn volume was unaffected even after eight weeks. Also, perchlorate at 18 ppm did not affect percentage egg fertilization. Fish were collected at the end of the exposures (677 ppm, four weeks; control and 18 ppm, eight weeks) for whole-body perchlorate content and thyroid histopathological analysis. Fish perchlorate levels were about one-hundredth of those of treatment water levels, indicating that waterborne perchlorate does not accumulate in whole fish. At 677 ppm for four weeks, perchlorate caused thyroid follicle cell (nuclear) hypertrophy and angiogenesis, whereas at 18 ppm for eight weeks, its effects were more pronounced and included hypertrophy, angiogenesis, hyperplasia, and colloid depletion. In conclusion, an eight-week exposure of adult zebrafish to 18 ppm perchlorate (high environmentally relevant concentrations) affected the histological condition of their thyroid follicles but not their reproductive performance. The effect of 677 ppm perchlorate on reproduction may be due to extrathyroidal toxicity. Further research is needed to determine if AP at lower environmentally relevant concentrations also affects the thyroid follicles of zebrafish.     

Detection of pharmaceutical contaminations of river,pond, and tap water from Cologne (Germany) and surroundings

A method for the simultaneous determination of polar pharmaceutical compounds in water is presented. Samples from 27 rivers and ponds were investigated for contents of Gemfibrocil, a lipid blood regulating compound, Clofibric acid, a metabolite of lipid regulating compounds (Clofibrat, Etofibrat, Etofyllinclofibrat), antirheumatics (Diclofenac, Ibuprofen, Ketoprofen, Indomethacin, Fenoprofen) and Sarkosin-N-(phenylsulfonyl) (SPS), a metabolite of a corrosion inhibiting agent. In addition water samples were investigated for some main metabolites of Ibuprofen, 2-[4-(2-hydroxy-2-methylpropyl)phenyl]propionic acid (hydroxy-ibuprofen) and 2-[4-(2-carboxypropyl)phenyl]propionic acid (carboxy-ibuprofen), which are excreted after oral intake. For gas chromatographic analysis the drugs were converted into their methyl-derivatives by "on column" reaction with TMSH, TMAH and "pre column" with diazomethane. The detection limits with TMSH were in the same range or lower than those with diazomethane or TMAH. The compound most frequently found was SPS which was only absent in waters with natural surroundings. Diclofenac could be detected in 10 out of 27 water samples in concentrations of up to 15 micrograms/l. In contrast, none of the pharmaceuticals investigated were present in 8 drinking water samples from Cologne and surroundings. Though concentrations measured were far below pharmacological doses, the data suggest a steady flux of these drugs into the environment.