Evaluation of acetamiprid and azoxystrobin residues and their hormonal disrupting effects on male rats using liquid chromatography-tandem mass spectrometry.
ABSTRACT: Endocrine-disrupting compounds as pesticides affect the hormonal balance, and this can result in several diseases. Therefore, the analysis of representative hormones with acetamiprid (AC) and azoxystrobin (AZ) was a good strategy for the investigation of the endocrine-disrupting activity of pesticides. Hence, a sensitive and rapid analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The method was validated for the analysis of AC, AZ, estriol, estrone, progesterone, and testosterone in the serum, testis, and liver of rats. The correlation between the residues of pesticides and the disturbance of the endocrine system was evaluated. The different mass parameters, mobile phase types, analytical columns, injection volumes, and extraction solvents were compared to get the lowest limit of detection of the studied compounds. The detection limits of AC, AZ, estriol, estrone, progesterone, and testosterone were 0.05, 0.05, 1.0, 10, and 1.0 ng/ml, respectively. The method developed was applied to evaluate the changes in these hormones induced by the duration of exposure to AC and AZ in rat testis and serum. The hormones level in rat serum and testis had a significant decrease as they were oral gavage treated with different high concentrations of studied pesticides. Both pesticides were distributed in the body of rats by the multi-compartment model (liver, testis, and serum).
Project description:Quantification of steroid hormones in fish is an important step for toxicology and endocrinology studies. Among the hormone analysis techniques, liquid chromatography tandem mass spectrometry (LC-MS/MS) has widely been used for measuring hormones in various biological samples. Despite all improvements in the technique, detection of several hormones in a low volume of serum or plasma is still challenging. We developed a robust method for simultaneous quantification of 14 steroid hormones including corticosterone, cortisol, 11-ketotestosterone, progesterone, testosterone, 17OH-progesterone, aldosterone, dihydrotestosterone, estrone, 17β-estradiol, estriol, ethinylestradiol, levonorgestrel and equilin from volumes as low as 10 µL serum or plasma in a short run by LC-MS/MS. The lowest limit of detection in 10 µL serum was 0.012 ng/mL measured for cortisol, progesterone, testosterone, 17OH-progesterone and estrone. Use of high (25 times more) serum volume improved detection limit of hormones by 2-40 times. The method was compared with the radioimmunoassay technique in which testosterone and 17β-estradiol were highly correlated with R<sup>2</sup> of 0.95 and 0.96, respectively. We validated the method by measuring four selected hormones, in low and high plasma volumes of largemouth bass (Micropterus salmoides). In addition, we developed a method to quantify hormones in whole body fish homogenates of small fish and compared the values to plasma concentrations, using fathead minnow (Pimephales promelas). Calculated concentrations of the hormones in plasma were consistent with those in the homogenate and 11-ketotestosterone and 17β-estradiol were significantly different in males and females. The ability to measure hormones from whole body homogenates was further evaluated in two model small fish species, zebrafish (Danio rerio) and juvenile silverside (Menidia beryllina). These results suggest that whole tissue homogenate is a reliable alternative for hormone quantification when sufficient plasma is not available.
Project description:Monitoring complex endocrine pathways is often limited by indirect measurement or measurement of a single hormone class per analysis. There is a burgeoning need to develop specific direct-detection methods capable of providing simultaneous measurement of biologically relevant concentrations of multiple classes of hormones (estrogens, androgens, progestogens, and corticosteroids). The objectives of this study were to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for multi-class steroid hormone detection using biologically relevant concentrations, then test limits of detection (LOD) in a high-background matrix by spiking charcoal-stripped fetal bovine serum (FBS) extract. Accuracy was tested with National Institute of Standards and Technology Standard Reference Materials (SRMs) with certified concentrations of cortisol, testosterone, and progesterone. 11-Deoxycorticosterone, 11-deoxycortisol, 17-hydroxypregnenolone, 17-hydroxyprogesterone, adrenosterone, androstenedione, cortisol, corticosterone, dehydroepiandrosterone, dihydrotestosterone, estradiol, estriol, estrone, equilin, pregnenolone, progesterone, and testosterone were also measured using isotopic dilution. Dansyl chloride (DC) derivatization was investigated maintaining the same method to improve and expedite estrogen analysis. Biologically relevant LODs were determined for 15 hormones. DC derivatization improved estrogen response two- to eight-fold, and improved chromatographic separation. All measurements had an accuracy ?14 % difference from certified values (not accounting for uncertainty) and relative standard deviation ?14 %. This method chromatographically separated and quantified biologically relevant concentrations of four hormone classes using highly specific fragmentation patterns and measured certified values of hormones that were previously split into three separate chromatographic methods.
Project description:BACKGROUND: Studies relating long-term exposure to persistent organochlorine pollutants (POPs) with endocrine activities (endocrine disrupting chemicals) on circulating levels of steroid hormones have been limited to a small number of hormones and reported conflicting results. OBJECTIVE: We examined the relationship between serum concentrations of dehydroepiandrosterone, dehydroepiandrosterone sulphate, androstenedione, androstenediol, testosterone, free and bioavailable testosterone, dihydrotestosterone, estrone, estrone sulphate, estradiol, sex-hormone binding globulin, follicle-stimulating hormone, and luteinizing hormone as a function of level of exposure to three POPs known to interfere with hormone-regulated processes in different way: dichlorodiphenyl dichloroethene (DDE), polychlorinated biphenyl (PCB) congener 153, and chlordecone. METHODS: We collected fasting, morning serum samples from 277 healthy, non obese, middle-aged men from the French West Indies. Steroid hormones were determined by gas chromatography-mass spectrometry, except for dehydroepiandrosterone sulphate, which was determined by immunological assay, as were the concentrations of sex-hormone binding globulin, follicle-stimulating hormone and luteinizing hormone. Associations were assessed by multiple linear regression analysis, controlling for confounding factors, in a backward elimination procedure, in multiple bootstrap samples. RESULTS: DDE exposure was negatively associated to dihydrotestosterone level and positively associated to luteinizing hormone level. PCB 153 was positively associated to androstenedione and estrone levels. No association was found for chlordecone. CONCLUSIONS: These results suggested that the endocrine response pattern, estimated by determining blood levels of steroid hormones, varies depending on the POPs studied, possibly reflecting differences in the modes of action generally attributed to these compounds. It remains to be investigated whether this response pattern is predictive of the subsequent occurrence of disease.
Project description:C. testosteroni is a research topic that can degrade steroid hormones into water and carbon dioxide through a series of enzymes in the body. Short-chain dehydrogenase (SDR) are a class of NAD (P) H-dependent oxidoreductases in C. testosteroni. Its main function is catalyzing the redox of the hydroxyl/ketone group of the hormone. In this paper, a SDR gene(SDRx) is cloned from C. testosteroni ATCC11996 and expressed. The polyclonal antibody was prepared and the SDRx gene knocked out by homologous recombination. Wild type and mutant C. testosteroni induced by testosterone, estradiol, estrone and estriol. The growth curves of the bacteria were measured by spectrophotometer. ELISA established the expression of SDRx protein, and high-performance liquid chromatography(HPLC) detected the contents of various hormones. The results show that the growth of wild type was faster than mutant type induced by testosterone. The concentration of SDRx is 0.318?mg/ml under testosterone induction. It has a great change in steroid hormones residue in culture medium measured by HPLC: Testosterone residue in the mutant type group was 42.4 % more than the wild type in culture medium. The same thing happens with induced by estrone. In summary, this SDRx gene involved in the degradation of testosterone and estradiol, and effects the growth of C. testosteroni.
Project description:A new extraction phase based on hydrogel disks of polyvinyl alcohol (PVOH) and pectin was proposed, characterized and evaluated for the extraction of six steroidal hormones (estriol, estrone, 17?-estradiol, 17?-ethinylestradiol, progesterone, and testosterone) in aqueous samples with subsequent determination by gas chromatography-tandem mass spectrometry (GC-MS/MS) after the derivatization procedure. The developed extraction procedure was based on the solid phase extraction (SPE) technique, but employed hydrogel as the sorbent phase. The effects of several parameters, including the amount and composition of the sorbent phase, pH, sample volume, flow rate, and gel swelling over the extraction efficiency, were evaluated. Gels with lower swelling indexes and larger amounts of sorbent ensured higher extraction yields of analytes. The main benefits of using the PVOH/pectin-based hydrogel as the extraction phase are the ease of synthesis, low-cost preparation, and the possibility of reusing the extraction disks. Limits of quantification of 0.5 ?g L<sup>-1</sup> for estrone and 17?-estradiol, and 1 ?g L<sup>-1</sup> for testosterone, 17?-ethinylestradiol, progesterone, and estriol were obtained. Accuracy values ranged from 80% to 110%, while the inter-assay precision ranged from 0.23% to 22.2% and the intra-assay from 0.55% to 12.3%. Since the sorbent phase has an amphiphilic character, the use of hydrogels is promising for the extraction of medium-to-high polarity compounds.
Project description:Objectives To identify factors predicting maternal sex steroid hormone concentrations in early pregnancy. Methods The Infant Development and the Environment Study recruited healthy pregnant women from academic medical centers in four US cities. Gold standard liquid chromatography-tandem mass spectrometry was used to measure maternal sex steroids concentrations (total testosterone [TT], free testosterone [FT], estrone [E1], estradiol [E2], and estriol [E3] concentrations) in serum samples from 548 women carrying singletons (median?=?11.7 weeks gestation). Women completed questionnaires on demographic and lifestyle characteristics. Results In multivariable linear regression analyses, hormone concentrations varied in relation to maternal age, body mass index (BMI), race, and parity. Older mothers had significantly lower levels of most hormones; for every year increase in maternal age, there was a 1-2% decrease in E1, E2, TT, and FT. By contrast, each unit increase in maternal BMI was associated 1-2% lower estrogen (E1, E2, E3) levels, but 1-2% higher androgen (TT, FT) concentrations. Hormone concentrations were 4-18% lower among parous women, and for each year elapsed since last birth, TT and FT were 1-2% higher (no difference in estrogens). Androgen concentrations were 18-30% higher among Black women compared to women of other races. Fetal sex, maternal stress, and lifestyle factors (including alcohol and tobacco use) were not related to maternal steroid concentrations. Conclusions for Practice Maternal demographic factors predict sex steroid hormone concentrations during pregnancy, which is important given increasing evidence that the prenatal endocrine environment shapes future risk of chronic disease for both mother and offspring.
Project description:Abnormal sex hormone levels in utero have been associated with child behavioral problems, but it is unclear if normal variation in prenatal sex hormones is associated with subsequent behavior in childhood. We assessed maternal sex hormones, including serum estrone (E1), estradiol (E2), estriol (E3), free testosterone (FT), and total testosterone (TT), during early pregnancy (gestational week 6-21 (mean = 11.1)) and evaluated child behavior at ages 4-5 using the Behavioral Assessment System for Children (BASC-2) and Social Responsiveness Scale (SRS-2) in 404 mother/child pairs (211 girls, 193 boys) within The Infant Development and Environment Study, a multi-site pregnancy cohort study. Associations between hormones and composite scores were evaluated using multiple linear regressions in both sexes combined, and separate models assessed effect modification by sex with the addition of interaction terms. A 10-fold increase in maternal FT or TT was associated in both sexes with a 4.3-point (95 % CI: 0.5, 8.2) or 4.4-point (0.8, 8.0) higher BASC-2 internalizing composite T score, respectively. In addition, a 10-fold increase in FT or TT was associated with a 3.8-point (0.04, 7.5) or 4.0-point (0.5, 7.5) higher behavioral symptoms index composite score. In models evaluating effect modification by sex, a 10-fold increase in E1 was associated with a 4.3-point (1.2, 7.4) decrease in adaptive skills composite score in girls only (interaction p = 0.04). We observed associations between testosterone and internalizing behaviors and behavioral symptoms index in both sexes, as well as a female-specific association between E1 and adaptive skills. Sex hormones during pregnancy may play a key role in influencing later-life behavior, and additional studies should further examine different periods of susceptibility to hormonal signals.
Project description:Breast cancer resistance protein (BCRP) is known for its protective function against the toxic effects of exogenous compounds. In addition to this, a role in the transport of endogenous compounds has been described. Since BCRP in the plasma membrane was shown to be regulated by sex steroids, we investigated the presence and possible role of BCRP in steroid hormone-producing organs. Therefore, the presence and localization of Bcrp was investigated in endocrine organs of wild-type mice. Furthermore, the interaction of various steroid hormones with human BCRP activity was studied. Quantitative PCR revealed Bcrp mRNA in the pituitary and adrenal glands, pancreas, ovary, testis and adipose tissue. Immunohistochemistry revealed the presence of Bcrp in the cortex of the adrenal gland and in plasma membranes of adipocytes. In the pituitary gland, pancreas, ovary and testis, Bcrp was mainly located in the capillaries. The interaction between BCRP and 12 steroid hormones was studied using membrane vesicles of HEK293-BCRP cells. Estradiol, testosterone, progesterone and androstenedione inhibited BCRP-mediated uptake of (3)H-estrone sulphate (E(1)S) most potently, with calculated inhibitory constant (Ki) values of 5.0 ± 0.2, 36 ± 14, 14.7 ± 1.3 and 217 ± 13 μM, respectively. BCRP function was attenuated non-competitively, which implies an allosteric inhibition of BCRP-mediated E(1)S transport by these steroids. In conclusion, localization of Bcrp in endocrine organs together with the efficient allosteric inhibition of the efflux pump by steroid hormones are suggestive for a role for BCRP in steroid hormone regulation.
Project description:Menstrual cycle characteristics are markers of endocrine milieu. However, associations between age at menarche and adulthood sex steroid hormone levels have been inconsistent, and data on menstrual characteristics and non-sex steroid hormones are sparse.We assessed the relations of menstrual characteristics with premenopausal plasma sex steroid hormones, sex hormone binding globulin (SHBG), prolactin, and growth factors among 2,745 premenopausal women (age 32-52) from the Nurses' Health Study II. Geometric means and tests for trend were calculated using multivariable general linear models.Early age at menarche was associated with higher premenopausal early-follicular free estradiol (percent difference <?12 vs.?>?13 years?=?11%), early-follicular estrone (7%), luteal estrone (7%), and free testosterone (8%) (all p trend?<?0.05). Short menstrual cycle length at age 18-22 was associated with higher early-follicular total (<?26 vs.?>?39 days?=?18%) and free estradiol (16%), early-follicular estrone (9%), SHBG (7%), lower luteal free estradiol (-?14%), total (-?6%), and free testosterone (-?15%) (all p trend?<?0.05). Short adult menstrual length was associated with higher early-follicular total estradiol (<?26 vs.?>?31 days?=?14%), SHBG (10%), lower luteal estrone (-?8%), progesterone (-?9%), total (-?11%) and free testosterone (-?25%), and androstenedione (-?14%) (all p trend?<?0.05). Irregularity of menses at 18-22 was associated with lower early-follicular total (irregular vs. very regular?=?-?14%) and free estradiol (-?14%), and early-follicular estrone (-?8%) (All p trend?<?0.05). Irregularity of adult menstrual cycle was associated with lower luteal total estradiol (irregular vs. very regular?=?-?8%), SHBG (-?3%), higher total (8%), and free testosterone (11%) (all p trend?<?0.05).Early-life and adulthood menstrual characteristics are moderately associated with mid-to-late reproductive year's hormone concentrations. These relations of menstrual characteristics with endogenous hormone levels could partially account for associations between menstrual characteristics and reproductive cancers or other chronic diseases.
Project description:Natural and synthetic steroid hormones, excreted by humans and farmed animals, have been considered as important sources of environmental endocrine disruptors. A suite of estrogens, androgens and progestogens was measured in the wastewater treatment plant outfall (WWTPO) of Chascomús city (Buenos Aires province, Argentina), and receiving waters located downstream and upstream from the WWTPO, using solid phase extraction and high-performance liquid chromatography mass spectrometry. The following natural hormones were measured: 17β-estradiol (E<sub>2</sub>), estrone (E<sub>1</sub>), estriol (E<sub>3</sub>), testosterone (T), 5α-dihydrotestosterone (DHT), progesterone (P), 17-hydroxyprogesterone (17OHP) and the synthetic estrogen 17α-ethinylestradiol (EE<sub>2</sub>). Also, in order to complement the analytical method, the estrogenic activity in these surface water samples was evaluated using the in vitro transactivation bioassay that measures the estrogen receptor (ER) activity using mammalian cells. All-natural steroid hormones measured, except 17OHP, were detected in all analyzed water samples. E<sub>3</sub>, E<sub>1</sub>, EE<sub>2</sub> and DHT were the most abundant and frequently detected. Downstream of the WWTPO, the concentration levels of all compounds decreased reaching low levels at 4500 m from the WWTPO. Upstream, 1500 m from the WWTPO, six out of eight steroid hormones analyzed were detected: DHT, T, P, 17OHP, E<sub>3</sub> and E<sub>2</sub>. Moreover, water samples from the WWTPO and 200 m downstream from it showed estrogenic activity exceeding that of the EC<sub>50</sub> of the E<sub>2</sub> standard curve. In sum, this work demonstrates the presence of sex steroid hormones and estrogenic activity, as measured by an in vitro assay, in superficial waters of the Pampas region. It also suggests the possibility of an unidentified source upstream of the wastewater outfall.