Potential adverse outcome pathway of neurodevelopmental toxicity, inflammatory response, and oxidative stress induction mediated by three alkyl organophosphate flame retardants in zebrafish larvae.

Following restrictions on polybrominated flame retardants, trimethyl phosphate (TMP), triethyl phosphate (TEP), and tris(2-butoxyethyl) phosphate (TBEP) have been frequently used as plasticizers for fire-resistant plastics. This study investigated the neurodevelopmental effects, inflammatory response, and oxidative stress induction of three alkyl organophosphate flame retardants using a zebrafish embryo/larvae model. After exposure of zebrafish embryos to TMP, TEP, and TBEP (0, 0.02, 0.2, 2, 20, and 200 µg L-1) for 96 h, survival, development, swimming behavior, changes in acetylcholinesterase (AChE) activity, dopamine, tumor necrosis factor-alpha (TNF-α), interleukin (IL), reactive oxygen species (ROS), and antioxidant enzyme activities were observed. Concentrations of TMP, TEP, and TBEP were also measured in the whole body of exposed larvae. Our results showed that exposure to 200 µg L-1 TEP and ≥20 µg L-1 TBEP significantly reduced larval body length; however, TMP had no significant effects on developmental parameters up to 200 µg L-1. After 96 h of exposure to TBEP, total distance moved, mean velocity, AChE, and dopamine concentrations were significantly decreased. Exposure to TEP and TBEP decreased the expression of genes that regulate central nervous system development (e.g. gap43 and mbpa), whereas ROS, antioxidant enzymes, TNF-α, and IL-1ß concentrations were significantly increased. Notably, pretreatment with an antioxidant N-acetylcysteine reduced neurotoxicity and oxidative stress caused by TEP and TBEP. The results of this study demonstrated that exposure to TEP and TBEP causes oxidative stress and has adverse effects on the neurobehavioral and immune system of zebrafish, leading to hypoactivity and ultimately impairing development.

The combined effects of preservative chemicals in consumer products: An analysis using embryonic and adult zebrafish.

Benzisothiazolinone (BIT) and propyl paraben (PP) are preservatives in cleaning products; however, their toxicities are not well understood. In this study, zebrafish embryos were exposed to BIT, PP, and mixtures of both for 96 h to investigate the effects on growth hormone (GH), insulin-like growth factor-1 (IGF-1), and the transcription of 19 genes related to the GH/IGFs axis. Concentrations of BIT and PP were measured in the whole body of larvae. Zebrafish pairs were also exposed to BIT, PP, and mixtures for 21 d to evaluate the effects on sex hormones, histology in gonad, and transcription of 22 genes related to the hypothalamus-pituitary-gonad axis and vitellogenin. The mixtures had potentiation effects on development, reproduction, hormones, and gene transcripts than individual exposure. Larvae exposed to 229 µg L-1 BIT, 64.5 µg L-1 PP, and mixtures showed reduced growth. Decreased GH and IGF-1 levels were supported by gene regulation associated with the GH/IGFs axis. In larvae, reactive oxygen species, superoxide dismutase, catalase, and glutathione peroxidase levels were increased under all exposures. The gonadosomatic index in males and number of eggs decreased after mixture exposure. In females exposed to mixtures, the percentage of atretic follicle in ovary was significantly increased. The significant decrease in testosterone in males and significant decrease in 17ß-estradiol in females exposed to mixtures suggest anti-estrogenic and anti-androgenic potential. Thus, preservative mixtures in consumer products may be more toxic than the individual substances, which is important for managing the risks of mixing preservatives.

Effects of two alternative plasticizers on the growth hormone-related endocrine system, neurodevelopment, and oxidative stress of zebrafish larvae.

In response to the restriction of phthalate plasticizers, acetyl tributyl citrate (ATBC) and acetyl triethyl citrate (ATEC) have been used in medical devices and food packaging. In the present study, the effects of ATBC and ATEC on the development, behavior, growth hormone (GH)-related endocrine system, neurotransmitters, and oxidative stress of zebrafish embryo or larvae were investigated. After exposure of zebrafish to ATBC and ATEC (0, 0.03, 0.3, 3, 30, and 300 µg/L) for 96 h, developmental toxicity, behavioral changes under light/dark condition, changes in hormones and genes involved in GH/insulin-like growth factors (IGFs) axis, changes in hormone, enzyme, and genes related to neurodevelopment, antioxidant enzymes activities were determined. Larvae exposed to 30 or 300 µg/L ATBC showed significant reductions in body length and moving distance and speed, whereas no significant effects on development and locomotor behavior were observed in larvae exposed to ATEC. The contents of GH and IGF-I were significantly reduced in larvae exposed to 3, 30, and 300 µg/L ATBC. Hormonal changes in fish exposed to ATBC are well supported by regulation of genes related to GH (gh1) and the activity of IGF-I (igf1). In fish exposed to ATBC, reduced acetylcholinesterase activity and down-regulation of genes related to the central nervous system development (ache, gap43, mbpa, and syn21) were observed. ATBC increased the production of reactive oxygen species and the levels of superoxide dismutase, catalase, and glutathione peroxidase. Notably, pre-treatment with the classic antioxidant N-acetylcysteine alleviated ATBC-induced GH-related endocrine disruption and neurotoxicity. Our observations showed that exposure to low levels of ATBC could disturb the regulatory systems of GH/IGFs axis and neurobehavior, ultimately leading to developmental inhibition and hypoactivity, and that increased oxidative stress plays a major role in these toxicities.

Single and mixture toxicity evaluation of avobenzone and homosalate to male zebrafish and H295R cells.

Avobenzone and homosalate are widely used in sunscreens to provide ultraviolet (UV) protection, either as single compounds or in combination. Some UV filters exhibit estrogenic or anti-androgenic activities, however, studies regarding their interactions and toxicity in mixtures are limited. In this study, the effect of the toxicity of a binary mixture comprising avobenzone (0.72 µg L-1) and homosalate (1.02 and 103 µg L-1) on steroid hormone biosynthesis were investigated using male zebrafish and human adrenocortical carcinoma (H295R) cells. In fish exposed to homosalate, a significant decrease in the gonadosomatic index, testosterone level, and transcription of several genes (e.g, hsd3b2, cyp17a1, and hsd17b1) and a significant increase in the hepatosomatic index, liver steatosis, 17ß-estradiol level, and transcription of vtg gene were observed. These results suggest that estrogenic and anti-androgenic effects of homosalate were mediated by the steroidogenic pathway. The presence of 0.72 µg L-1 of avobenzone augmented the anti-androgenic responses in male fish. The testosterone level in the H295R cells were significantly decreased after they were exposed to homosalate alone or in combination with avobenzone, which is consistent with observations in male zebrafish. Further studies need to be conducted to understand the endocrine disrupting properties of long-term exposure to substances typically used in sunscreens.

Effects of di-(2-ethylhexyl) terephthalate on hypothalamus-pituitary-gonad axis in adult zebrafish.

Di-(2-ethylhexyl) terephthalate (DEHTP) is frequently used in food packaging and medical devices as an alternative to di-(2-ethylhexyl) phthalate (DEHP). In this study, zebrafish pairs were exposed to DEHTP for 21 d and the effects on fertility, sex hormone levels, vitellogenin levels, and transcription of genes along the hypothalamic-pituitary-gonad axis were evaluated. Results showed that mean egg numbers were significantly reduced in the 30 and 300 µg/L DEHTP groups. The adverse effects of DEHTP on hormones and gene transcripts were more prominent in males than in females. In male fish, the gonadosomatic index, hepatosomatic index, and vitellogenin concentration were significantly increased. The results of a significant decrease in testosterone (T) and an increase in the 17ß-estradiol (E2)/T ratio in males exposed to 3-300 µg/L DEHTP suggest that the endocrine potential of DEHTP is similar that of DEHP. In females, genes related to gonadotropin-releasing hormone and gonadotropin were up-regulated while E2 was significantly down-regulated. These findings suggest that positive E2 feedback mechanisms in the hypothalamus and pituitary gland are activated to balance sex hormones. The effects of chronic exposure to DEHTP on the neuroendocrine system require further investigation.

Thyroid hormone disrupting potentials of benzisothiazolinone in embryo-larval zebrafish and rat pituitary GH3 cell line.

Benzisothiazolinone (BIT), one of the most widely used antimicrobial agents in consumer products, has frequently been detected in the water environment. The present study was conducted to determine the adverse effects of BIT on the thyroid neuroendocrine system of zebrafish embryos/larvae. Rat pituitary (GH3) cell line was employed to support the underlying mechanism of thyroid hormone disrupting effects. Significant coagulation and hatching delay were observed in embryos exposed to 30 µg/L of BIT, which in turn remarkably decreased hatchability and larval survival. In BIT-exposed larvae, tshß, tshr, and trh genes were significantly upregulated along with a decrease in thyroxine and triiodothyronine content, indicating that BIT decreased thyroid hormones and increased thyrotropin-releasing hormone and thyroid stimulating hormone secretion through a feedback circuit. The downregulation of trα and deio2 genes in the zebrafish larvae suggests the inhibition of thyroid hormone receptors and deiodination. Similar to the results in zebrafish, upregulation of tshß and downregulation of trα, trß, deio1, and deio2 genes were observed in GH3 cells. Our observations suggest that BIT can decrease the level of thyroid hormones by influencing central regulation, receptor binding, and deiodination.

Toxicological signature for thyroid endocrine disruption of dichlorooctylisothiazolinone in zebrafish larvae.

Dichlorooctylisothiazolinone (DCOIT), which is one of the isothiazolinone preservatives, is applied to water-based adhesives in food packaging. This study investigated the effects of DCOIT on the embryonic growth and thyroid endocrine system using zebrafish. Organism-level (hatchability, survival, and growth), hormone-level (triiodothyronine (T3) and thyroxine (T4)), gene-level (genes associated with the hypothalamus-pituitary-thyroid axis), and microRNA-level (microRNAs related to thyroid endocrine disruption) endpoints were measured. Significant rise in embryonic coagulation and delayed hatching (≥0.3 µg/L), and decreased larval length (30 µg/L) were observed in fish exposed to DCOIT. Lower contents of T3 and T4 were observed after exposure to DCOIT, which was accompanied by the upregulation of genes associated with the thyrotropin releasing hormone and thyroid stimulating hormone and the downregulation of genes associated with the thyroid hormone receptors and deiodination. Strong influence of DCOIT on dre-miR-193b and -499 may be a critical mechanism to inhibit transcription of trαa and trß, which in turn may affect thyroid hormones and development of the organism. Our findings suggest that hypothyroidism induced by the exposure to DCOIT is potentially associated with genetic and microRNA-level changes, which eventually affects development.

Concentrations of blood and urinary arsenic species and their characteristics in general Korean population.

Arsenic (As) exposure has been extensively studied by investigating As species (e.g., inorganic arsenic (iAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA)) in urine, yet recent research suggests that blood could be a possible biomarker of As exposure. These investigations, however, were conducted on iAs-contaminated areas, and evidence on populations exposed to low levels of iAs is limited. This study aimed to describe the levels and distributions of As species in urine and blood, as well as to estimate methylation efficiency and related factors in the Korean population. Biological samples were obtained by the Korean Ministry of Food and Drug Safety. A total of 2025 urine samples and 598 blood samples were utilized in this study. Six As species were measured using ultra-high-performance liquid chromatography with inductively coupled plasma mass spectrometry (UPLC-ICP-MS): As(V), As(III), MMA, DMA, arsenobetaine (AsB), and arsenocholine (AsC). Multiple linear regression models were used to examine the relationship between As species (concentrations and proportions) and covariates. AsB was the most prevalent species in urine and blood. The relative composition of iAs, MMA, DMA, and AsC in urine and blood differed significantly. Consumption of blue-backed fish was linked to higher levels of AsB in urine and blood. Type of drinking water and multigrain rice consumption were associated with increased iAs concentration in urine. Except for iAs, every species had correlations in urine and blood in both univariate and multivariate analyses. Adolescents and smokers presented a lower methylation efficiency (higher %MMA and lower %DMA in urine) and females presented a higher methylation efficiency (lower %iAs, %MMA, and higher %DMA in urine). In conclusion, blood iAs concentration cannot represent urinary iAs; nonetheless, different compositions of urine and blood might reflect distinct information about iAs exposure. Further investigations on exposure factors and health are needed using low-exposure groups.

Identification of combinations of endocrine disrupting chemicals in household chemical products that require mixture toxicity testing.

People are exposed to various chemicals contained in consumer products for which the risks are poorly characterized. There is growing evidence that exposure to endocrine disrupting chemicals (EDCs) through product use potentially affects development, behavior, and reproduction. However, limited information is available about common combinations of chemicals based on their appearance and potential health effects. The present study listed the ingredients contained in 11064 household chemical products from a publicly available database, and identified EDCs related to estrogenicity, androgenicity, thyroid hormone disruption, and changes in steroidogenesis. Association rule mining was applied to the dataset to identify frequent combinations of chemicals or commonly occurring EDCs contained in a single product. Among the target products, ingredient names were matched with 1241 chemical identifiers. A total of 293 chemicals were related to endocrine disruption, and nearly two-thirds of the products contained more than one of these chemicals. Cleaning products, synthetic detergents, fabric softeners, air fresheners, and deodorants have several hotspots for fragrances, isothiazolinones, glycol ethers, and parabens. The three most prevalent EDCs in household chemical products were added to act as fragrances and preservatives. The present study demonstrated that commonly occurring chemical combinations can be derived using an association rule mining algorithm. The results of this study will be useful in prioritizing chemical combinations and developing management plans for EDC mixture in consumer products.

Waterborne exposure to avobenzone and octinoxate induces thyroid endocrine disruption in wild-type and thrαa-/- zebrafish larvae.

Avobenzone and octinoxate are frequently used as organic ultraviolet filters, and these chemicals are widely detected in water. This study evaluated the potential of avobenzone and octinoxate to disrupt thyroid endocrine system in wild-type and thyroid hormone receptor alpha a knockout (thrαa-/-) zebrafish embryo/larvae. Following a 120 h exposure to various concentrations of avobenzone and octinoxate, larvae mortality and developmental toxicity in wild-type and thrαa-/- fish were assessed. Triiodothyronine (T3) and thyroxine (T4) levels as well as transcriptional levels of ten genes associated with the hypothalamus-pituitary-thyroid (HPT) axis were measured in wild-type fish. Significantly lower larvae survival rate in thrαa-/- fish exposed to ≥3 µM avobenzone and octinoxate suggests that the thyroid hormone receptor plays a crucial role in the toxic effects of avobenzone and octinoxate. A significant increase in the deio2 gene level in avobenzone-exposed zebrafish supports the result of an increased ratio of T3 to T4. Significant decrease of T4 level with upregulation of trh, tshß, and tshr genes indicates feedback in the hypothalamus and pituitary gland to maintain hormonal homeostasis. Our observation indicates that exposure to avobenzone and octinoxate affects the thyroid hormone receptor and the feedback mechanisms of the HPT axis. CLINICAL TRIALS REGISTRATION: Not applicable.

Effects of tetramethyl bisphenol F on thyroid and growth hormone-related endocrine systems in zebrafish larvae.

Trimethyl bisphenol F (TMBPF) has recently been used as a bisphenol A substitute in polymer coatings for metal cans containing beverages or food. This study investigated whether TMBPF disrupts the endocrine system associated with thyroid hormones and growth hormones employing zebrafish embryos and larvae. After 14 days of exposure, body weight was significantly reduced when zebrafish were exposed to a TMBPF concentration greater than 50 µg/L. The triiodothyronine levels were significantly increased, while growth hormone levels were significantly decreased in larvae exposed to 5 µg/L TMBPF. The transcription of genes associated with thyroid hormone production (trα, tpo, tg, and nis), deiodination (deio2), growth hormone production (gh1, ghrh, and ghra), and insulin-like growth factor (igf2a, igf2b, igf2r, igfbp1a, igfbp1b, igfbp2a, igfbp2b, and igfbp5a) was significantly upregulated, whereas the transcription of genes association with thyrotropin-releasing hormone (trh and trhr1) was significantly downregulated. These results suggest that hyperthyroidism, decrease in growth hormone, and regulation of genes involved in the hypothalamus-pituitary-thyroid and growth hormone/insulin-like growth factor might be responsible for the observed growth inhibition in larvae exposed to TMBPF. The bioaccumulation of TMBPF and its effects on the endocrine system after chronic exposure requires further investigation.

Effects of methylisothiazolinone and octylisothiazolinone on development and thyroid endocrine system in zebrafish larvae.

Methylisothiazolinone (MIT) and octylisothiazolinone (OIT) are used as preservatives and biocides to prevent product decay or deterioration. In the present study, developmental toxicity and the effect on the thyroid endocrine system were investigated in zebrafish embryos exposed to MIT and OIT for 96 h. Coagulation was significantly increased when zebrafish embryos were exposed to a concentration of 300 µg/L MIT and ≥ 0.3 µg/L OIT, resulting in a significant decrease in hatchability and larvae survival. The body length in zebrafish larvae exposed to 30 µg/L OIT was significantly shorter than that of the control group. The whole-body levels of triiodothyronine and thyroxine were significantly decreased in larvae exposed to MIT and OIT. Significant upregulation of crh, trh, tshß, and tshr genes and downregulation of trαa, tg, ttr, and deio2 genes were observed in fish exposed to two isothiazolinones. The expression of dre-miR-193b and dre-miR-499 was significantly increased in zebrafish larvae exposed to MIT and OIT, indicating that epigenetic deregulation of miRNAs modulated genes involved in thyroid hormone regulation. OIT has a higher magnitude of toxicity than MIT, corresponding to the observed changes in thyroid hormones and developmental toxicity.

Occurrences of benzalkonium chloride in streams near a pharmaceutical manufacturing complex in Korea and associated ecological risk.

Benzalkonium chloride (BKC) is a commonly used preservative in personal care products and pharmaceutical preparations. However, its ecological risks are not well understood because of lack of monitoring data and ecotoxicological information. In the present study, occurrence of BKC was investigated in the waters near a pharmaceutical manufacturing complex of South Korea and its acute and chronic ecotoxicities were evaluated using Daphnia magna and Japanese medaka (Oryzias latipes). Associated ecological risks were estimated by calculating hazard quotients (HQs). In addition, endocrine disruption potency of BKC was compared with those of other frequently used preservatives using human adrenal (H295R) and rat pituitary (GH3) cells. High concentration of BKC was detected at locations near the pharmaceutical manufacturing plants, i.e., 35.8 µg/L for dodecyl benzyl dimethyl ammonium chloride (BKC-C12), and 21.6 µg/L tetradecyl benzyl dimethyl ammonium chloride (BKC-C14). In Daphnia, 48 h immobilization EC50 and 21 d reproduction NOEC were determined at 41.1 µg/L and ≥10.8 µg/L, respectively. For O. latipes, 96 h LC50 was determined at 246 µg/L while the growth inhibition NOEC was ≥113.4 µg/L following early life stage exposure. BKC significantly up-regulated vitellogenin gene of juvenile fish, indicating its endocrine disrupting potential in fish. Exposure to BKC increased steroid hormone level in H295R cells, and induced cytotoxicity in GH3 cells. HQ values of BKC were determined at greater than one in the ambient water near pharmaceutical manufacturing facilities. Considering high ecological risk and endocrine disrupting potential, long-term consequences of BKC contamination in aquatic ecosystem need to be examined.

Influence of Vegetarian Dietary Intervention on Urinary Paraben Concentrations: A Pilot Study with 'Temple Stay' Participants.

Personal care products and cosmetics have been identified as major sources of paraben exposure among humans. However, the contribution of dietary factors has not been well understood. We recruited temple stay participants (n = 25) who followed a strict Buddhist vegetarian diet during a five-day period, and assessed the influence of this lifestyle change, employing their urine samples collected before and after the temple stay. Before the temple stay, methylparaben (MeP) was detected at the highest levels, followed by ethylparaben (EtP), propylparaben (PrP), butylparaben (BuP), and benzophenones (BPs) in the urine samples. Following the temple stay, the urinary EtP concentrations remarkably increased from 14.0 to 105 µg/L, and were around two orders of magnitude higher than those reported from other countries. Dietary factors associated with the temple diet may partly explain the increase, because EtP is allowed in Korea for seasoning and condiments, which are frequently added in vegetarian diets. Following the temple stay, however, MeP, PrP, and BPs did not show significant decreasing trends. In contrast, BuP levels decreased significantly, especially in male urine samples, that is, from 3.60 to 1.03 µ/L, suggesting a reduced use of certain personal care products during the temple stay. Our observations outline the potential importance of dietary factors on EtP exposure, and might help explain its high exposure levels among Korean population.

Consumer exposure and risk assessment to selected chemicals of mold stain remover use in Korea.

Mold stain remover (MSR) is used to clean mold and mildew spots from surfaces and contains a variety of chemical substances. In this study, we estimated the inhalation and dermal exposures associated with the use of trigger spray MSRs, and performed screening-level risk assessments for the use of this type of product in Korea. Inhalation and dermal exposures were estimated using exposure algorithms based on exposure factors obtained from a nationwide survey of 10,000 participants and chemical analyses of the four most popular trigger spray MSRs. The hazard quotients (HQs) for noncancer risk and excess cancer risk (ECR) were calculated for each chemical. The mean inhalation exposure estimates for formaldehyde, benzene, chloroform, and carbon tetrachloride were 6.9 × 10-7, 1.7 × 10-7, 5.4 × 10-6, and 2.7 × 10-5 mg/kg/day, respectively. Dermal exposures of the chemicals were 5.7-6.5 times higher than inhalation exposures. The HQs for total exposure were all below 1, which indicated little noncancer risk from the use of MSRs. The safe ECR value of 1 × 10-6, was exceed in one subject for inhalation exposure of benzene and four subjects for dermal exposure of formaldehyde, while 19.8% for dermal exposure of benzene were above this value. Therefore, use of trigger spray MSRs in Korea should require more detailed exposure and risk assessment, especially for benzene.

Exposure to humidifier disinfectants induces developmental effects and disrupts thyroid endocrine systems in zebrafish larvae.

Humidifier disinfectants have been widely used in Korea to prevent the growth of microorganisms in humidifier water. However, their use has been banned since 2011 after epidemiological studies reported humidifier disinfectant induced lung injury. In the present study, the developmental effects of exposure to two humidifier disinfectants (Oxy® and Wiselect) and their main component, polyhexamethylene guanidine (PHMG)-phosphate, were investigated in zebrafish embryos/larvae for seven days. The effects on triiodothyronine (T3) and thyroxine (T4) hormones, reactive oxygen species (ROS) generation, antioxidant enzyme activities, and changes in expression of the genes related to the hypothalamus-pituitary-thyroid (HPT) axis and oxidative stress were also investigated. Zebrafish embryos exposed to the highest concentration (amounts recommended for use by the manufacturers) of all tested humidifier disinfectants showed an increase in embryo coagulation, leading to death without hatching. Exposure to Oxy® and Wiselect resulted in significantly decreased body length, increased ROS generation and antioxidant enzyme activities, decreased T4, and up-regulated genes related to the HPT axis (trh, trß, and tpo) and oxidative damage (sod2 and gpx1b). The humidifier disinfectants and PHMG-phosphate could induce oxidative stress and disrupt thyroid hormone systems in zebrafish, leading to developmental retardation when used at sub-lethal concentrations. Potential effects of long-term exposure to humidifier disinfectants and mixture effects of several major components deserve further investigation.

Chronic effects of bisphenol S and bisphenol SIP on freshwater waterflea and ecological risk assessment.

Bisphenol S (BPS) and 4-hydroxyphenyl 4-isoprooxyphenylsulfone (BPSIP) have been used as substitutes for bisphenol A (BPA) owing to increased regulation of BPA in plastics. In this study, long-term toxicity tests of BPS and BPSIP were performed using Daphnia magna and Moina macrocopa. The predicted no-effect concentration (PNEC) of BPA, BPS, and BPSIP were derived by the assessment factor (AF) method and the species sensitivity distribution (SSD) method. An ecological risk assessment was performed based on the measured environmental concentrations of BPA in surface water worldwide and the derived PNECs. The chronic NOEC of D. magna was 2.5 mg/L for BPS and 0.5 mg/L for BPSIP, and that of M. macrocopa was 0.03 mg/L for BPS and 0.1 mg/L for BPSIP. The PNECAF was generally one order of magnitude less than the PNECSSD, and the PNEC of BPS was 10 times lower than that of BPA. The hazard quotients of BPA and BPS exceeded 1, indicating that concentrations in ambient water conditions could pose a potential risk to aquatic organisms. Since the use of alternative compounds is increasing, further monitoring data of the water environment and chronic toxicity in various aquatic organisms appears to be necessary.

Co-exposure to ketoconazole alters effects of bisphenol A in Danio rerio and H295R cells.

Chemicals are present in combination in ambient water, however toxicities of their mixtures are not well understood. This study investigated the effects of ketoconazole (KCZ) on the responses induced by bisphenol A (BPA) in zebrafish and in human adrenocarcinoma (H295R) cells. After exposure to BPA alone or mixed with KCZ for 21 d, egg production, relative tissue weights, sex hormone levels, cytochrome P450 (CYP)3a activity, and transcriptions of genes related to CYP metabolism, vitellogenesis, and steroidogenesis were determined in zebrafish. Male fish were more sensitive to the adverse effects of BPA than females, and the presence of KCZ potentiated the BPA-induced estrogenic responses in the male and anti-estrogenic responses in the female fish. In male zebrafish exposed to BPA, a significant reduction in egg number and relative gonad weight, an increase in 17ß-estradiol (E2) to testosterone (T) ratio, and an upregulation of vtg, erα, and cyp19a genes were observed. Under KCZ, BPA exposure resulted in a significant downregulation of cyp3a65 and pxr genes and an increase in estrogenic responses in males. In female fish, anti-estrogenic effects, such as a decrease in E2 concentration, were observed following the combined exposure. These results indicate that KCZ could increase the toxicity of the chemicals that depend on the given CYP metabolism for their elimination or other crucial functions such as steroidogenesis. Co-exposure to BPA and KCZ in H295R cells also increased E2 and decreased T production. Release and presence of this azole compound warrant caution, because it could modify adverse effects of BPA.

Pharmaceutical residues in streams near concentrated animal feeding operations of Korea - Occurrences and associated ecological risks.

Concentrated animal feeding operations (CAFOs) have been suggested to be the most significant source of pharmaceutical release into the environment. However, limited information is available on the occurrence of veterinary pharmaceutical residues and the associated ecological risks to the aquatic environment near CAFO areas. In this study, ten commonly used veterinary antibiotics, including sulfonamides, tetracyclines, and cephalosporins, along with three analgesics, were measured in water samples collected from the streams that run near two CAFOs in Korea in 2013 (n = 16) and 2014 (n = 10). In addition, the associated ecological risks were estimated by calculating risk quotient. The pharmaceuticals were detected in a higher amount in the samples collected downstream from the CAFO than in those collected upstream. Acetaminophen, sulfamethazine, sulfathiazole, and oxytetracycline were detected at maximum concentrations of 38.8 µg/L, 21.3 µg/L, 17.4 µg/L, and 16.9 µg/L, respectively. Relatively higher concentrations of pharmaceuticals were observed in locations adjacent to the CAFO and the downstream area, suggesting the influence of the CAFO. Except for acetaminophen, lower concentrations of the target pharmaceuticals were detected in the samples collected during the high-flow season. The concentrations of most of the target pharmaceuticals exceeded the risk quotient of one, suggesting potential ecological effects in the areas affected by CAFOs. Our observations show that the water environment near a CAFO could be heavily affected by veterinary pharmaceuticals and analgesic drugs that are also frequently used among humans. Hence, the ecological consequences of pharmaceutical residues in the water bodies near CAFOs warrant further investigation.