MT Concentrations Research Articles

Molecular and Physiological Effects of 17α-methyltestosterone on Sex Differentiation of Black Rockfish, Sebastes schlegelii.

It is widely known that all-female fish production holds economic value for aquaculture. Sebastes schlegelii, a preeminent economic species, exhibits a sex dimorphism, with females surpassing males in growth. In this regard, achieving all-female black rockfish production could significantly enhance breeding profitability. In this study, we utilized the widely used male sex-regulating hormone, 17α-methyltestosterone (MT) at three different concentrations (20, 40, and 60 ppm), to produce pseudomales of S. schlegelii for subsequent all-female offspring breeding. Long-term MT administration severely inhibits the growth of S. schlegelii, while short term had no significant impact. Histological analysis confirmed sex reversal at all MT concentrations; however, both medium and higher MT concentrations impaired testis development. MT also influenced sex steroid hormone levels in pseudomales, suppressing E2 while increasing T and 11-KT levels. In addition, a transcriptome analysis revealed that MT down-regulated ovarian-related genes (cyp19a1a and foxl2) while up-regulating male-related genes (amh) in pseudomales. Furthermore, MT modulated the TGF-β signaling and steroid hormone biosynthesis pathways, indicating its crucial role in S. schlegelii sex differentiation. Therefore, the current study provides a method for achieving sexual reversal using MT in S. schlegelii and offers an initial insight into the underlying mechanism of sexual reversal in this species.

Melatonin treatment enhances bioactive compound retention, antioxidant activity and shelf‐life of bell pepper (Capsicum annuum L.) during cold storage

SummaryMelatonin (MT), a natural signalling compound derived from tryptophan, has gained significant attention for delaying postharvest ripening and senescence in various fruits and vegetables. In the present study, the effect of MT treatment was evaluated on the postharvest shelf‐life of bell pepper. Fruits were dipped in various MT concentrations (70, 120, 170, 220 μmol L−1) for 20 min and stored at 10 ± 1 °C for 20 days. The bell pepper treated with 120 μmol L−1 MT significantly suppressed respiration, weight loss and delayed chlorophyll degradation. Additionally, MT (120 μmol L−1) treatment reduced loss of firmness, titratable acidity and retained total phenol, flavonoid and ascorbic acid content. It hindered malondialdehyde accumulation by enhanced DPPH radical scavenging and antioxidant enzyme activity like superoxide dismutase, catalase and peroxidase, over the control. Conclusively, 120 μmol L−1 MT prolongs the postharvest shelf‐life of bell pepper by 20 days with superior sensory quality, surpassing the control by 10 days. Therefore, MT treatment is a commercially useful technology for maintaining quality and prolonging the postharvest shelf‐life of bell pepper.

Pancreatic Antioxidative Defense and Heat Shock Proteins Prevent Islet of Langerhans Cell Death After Chronic Oral Exposure to Cadmium LOAEL Dose.

Cadmium, a hazardous environmental contaminant, is associated with metabolic disease development. The dose with the lowest observable adverse effect level (LOAEL) has not been studied, focusing on its effect on the pancreas. We aimed to evaluate the pancreatic redox balance and heat shock protein (HSP) expression in islets of Langerhans of male Wistar rats chronically exposed to Cd LOAEL doses, linked to their survival. Male Wistar rats were separated into control and cadmium groups (drinking water with 32.5 ppm CdCl2). At 2, 3, and 4 months, glucose, insulin, and cadmium were measured in serum; cadmium and insulin were quantified in isolated islets of Langerhans; and redox balance was analyzed in the pancreas. Immunoreactivity analysis of p-HSF1, HSP70, HSP90, caspase 3 and 9, and cell survival was performed. The results showed that cadmium exposure causes a serum increase and accumulation of the metal in the pancreas and islets of Langerhans, hyperglycemia, and hyperinsulinemia, associated with high insulin production. Cd-exposed groups presented high levels of reactive oxygen species and lipid peroxidation. An augment in MT and GSH concentrations with the increased enzymatic activity of the glutathione system, catalase, and superoxide dismutase maintained a favorable redox environment. Additionally, islets of Langerhans showed a high immunoreactivity of HSPs and minimal immunoreactivity to caspase associated with a high survival rate of Langerhans islet cells. In conclusion, antioxidative and HSP pancreatic defense avoids cell death associated with Cd accumulation in chronic conditions; however, this could provoke oversynthesis and insulin release, which is a sign of insulin resistance.

Effects of dietary supplementation with 17β-estradiol and 17α-methyltestosterone on growth performance and gonadal development of the juvenile oriental river prawn (Macrobrachium nipponense)

17β-Estradiol (E2) and 17α-methyltestosterone (MT) are steroid hormones with dramatic regulatory effects on the growth performance and gonadal development of aquatic species. The aim of the present study was to investigate the regulatory effects of E2 and MT on the growth performance and gonadal development of juvenile prawns (Macrobrachium nipponense). Male and female juvenile prawns were separately maintained and fed different concentrations of E2 and MT, respectively. Treatment with both E2 and MT at 500 mg/kg dramatically promoted the growth of male and female juvenile prawns, respectively (p < 0.05). The growth rate of M. nipponense treated with E2 and MT at 1000 mg/kg at 30 days were 2.59 ± 0.31 and 2.21 ± 0.37, which were almost two- and five-fold greater than that of the control group, respectively. E2 showed better effects on the promotion of growth performance than MT. Histologically, testicular and ovarian development were delayed at E2 and MT concentrations of ≥ 500 mg/kg. Sperm had normally developed after 15 and 30 days of E2 treatment at ≤ 250 mg/kg, but were rarely observed in the testis at E2 concentrations greater than 500 mg/kg. The number of prawns at ovarian stages Ⅳ and V was increased with decreased MT concentrations. The qPCR results revealed that the expression levels of sperm gelatinase and insulin-like androgenic gland hormone were significantly decreased after E2 treatment from 100 mg/kg as compared to the control group (p < 0.05), while the expression levels at different E2 concentrations remained stable (p > 0.05). The expression levels of vitellogenin and vitellogenin receptor were dramatically decreased with increased MT concentrations, and the lowest expression levels were observed in the group treated with 1000 mg/kg (p < 0.05). Testosterone levels decreased with increased E2 concentrations (p < 0.05). In addition, lower MT concentrations (≤ 250 mg/kg) stimulated the secretion of E2 (p < 0.05), while higher concentrations (500 and 1000 mg/kg) had an opposite effect (p < 0.05). In summary, dietary supplementation with high doses of E2 and MT (500 and 1000 mg/kg) promoted the growth and inhibited gonadal development of juvenile M. nipponense. These findings provide important evidence of the effects of MT and E2 on the growth performance and gonadal development of juvenile M. nipponense, thereby establishing an artificial technique to regulate gonadal development.

Ecological risk of 17α-methyltestosterone contaminated water discharged from a full water recirculating earthen masculinization pond

17α-methyltestosterone (MT) is an exogenous anabolic steroid hormone used widely in aquafarming. This study aimed to emphasize the environmental hazards of MT originated from a continuously operating masculinization pond with an intensive water recirculation system in Samut Prakan, Thailand. Risk quotient (RQ) and concentration factor (CF) were used to examine the ecological risk of MT in the pond environment. The results revealed relatively high MT concentrations in the water and sediments. These findings concurred the benefit of masculinization ponds with a water recirculation system in the way that it can effectively save the water and reduce the MT dosage that needs to be added into the feed. However, this could bring serious environmental concerns since releasing water containing MT residues and androgenic active metabolites through water overflowing from the masculinization pond to the environment during the wet season is inevitable. This is the first report indicating that CF relates to the androgenic potential activity and can be used to explain the ecological risk of MT contaminated water discharged from a full water recirculating earthen masculinization pond. This study provides important information indicating that CF values in a range of 7 to 16 are high enough to cause adversely affect the reproductive performance of tested fish (i.e., Probarbus jullieni, Barbonymus gonionotus, and Anabas testudineus).

Phytochemical analysis and in vitro anti-proliferative activity of Viscum album ethanolic extracts

BackgroundViscum album L. (Santalaceae), commonly known as mistletoe, is a hemiparasitic plant traditionally used in complementary cancer treatment. Its antitumor potential is mostly attributed to the presence of aqueous soluble metabolites; however, the use of ethanol as solvent also permits the extraction of pharmacological compounds with antitumor potential. The clinical efficacy of mistletoe therapy inspired the present work, which focuses on ethanolic extracts (V. album “mother tinctures”, MT) prepared from different host trees.MethodsSamples from three European subspecies (album, austriacum, and abietis) were harvested, and five different V. album-MT strains were prepared. The following phytochemical analyses were performed: thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and liquid chromatography-high resolution mass spectrometry (LC-HRMS). The proliferation assay was performed with WST-1 after incubation of tumor (Yoshida and Molt-4) and fibroblast cell lines (NIH/3 T3) with different MT concentrations (0.5 to 0.05% v/v). The cell death mechanism was investigated by flow cytometry (FACS) using Annexin V-7AAD.ResultsChemical analyses of MT showed the presence of phenolic acids, flavonoids and lignans. The MT flavonoid and viscotoxin contents (mg/g fresh weight) were highest in Quercus robur (9.67 ± 0.85 mg/g) and Malus domestica (3.95 ± 0.58 mg/mg), respectively. The viscotoxin isoform proportions (% total) were also different among the VA subspecies with a higher content of A3 in V. album growing on Abies alba (60.57 ± 2.13). The phytochemical compounds as well as the viscotoxin contents are probably related to the antitumor effects of MT. The cell death mechanisms evaluated by colorimetric and FACS methodologies involved necrotic damage, which was host tree-, time- and dose- dependent, with different selectivity to tumor cells. Mother tincture from V. album ssp. abietis was the most effective at inducing in vitro cellular effects, even when incubated at the smallest concentration tested, probably because of the higher content of VT A3.ConclusionOur results indicate the promising antitumor potential of Viscum album ethanolic extracts and the importance of botanical and phytochemical characterization for in vitro anti-proliferative effects.

Suppression of osteogenic differentiation and mitochondrial function change in human periodontal ligament stem cells by melatonin at physiological levels.

N-Acetyl-5-methoxytryptamine (melatonin, MT) at pharmacological concentrations promotes the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells; however, its role at physiological concentrations (1 pM–10 nM) remains unclear. We explored the effects of 1 pM–1 µM MT on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and its underlying mitochondrial dynamics-mediated mechanisms. T he PDLSC phenotype was detected by flow cytometry and evaluated for three-line differentiation. Alkaline phosphatase activity assay and Alizarin red staining were used to evaluate osteogenic differentiation. Osteogenesis-related gene and protein expression levels were measured by quantitative reverse transcription -polymerase chain reaction and western blotting. Mitochondrial function assays were performed using reactive oxygen species, ATP and NAD+/NADH kits and molecular mechanisms of mitochondrial dynamics-related proteins were assessed by western blotting. Our results have shown that physiological MT concentrations induced differentiation of hPDLSCs and down-regulated osteopontin (OPN) and osteocalcin (OCN) expression levels, which were restored or even up-regulated by 1 µM MT (lowest pharmacological concentration). Compared to the osteogenic induction alone, this treatment decreased the intracellular ATP content, whereas the intracellular reactive oxygen species level and NAD+/NADH ratio were increased. Mitochondrial function- and dynamics-related protein expression levels were consistent with those of osteogenic genes following osteogenic induction and MT treatment of hPDLSCs at various physiological concentrations. Physiological MT concentrations inhibited the osteogenic differentiation of hPDLSCs and simultaneously altered mitochondrial function. These findings provide insights into the stem cell tissue engineering and functions of MT.

Metallic Film Modified Screen-Printed Carbon Electrode for Determination of 17α-Methyltestosterone

17α-methyltestosterone (MT) is a synthetic androgen. It is used widely for inducing an all-male population of Nile tilapia (Oreochromis niloticus). In this work, the detection of MT was conducted using screen-printed carbon electrodes (SPCE). These were a bare electrode, a bismuth modified electrode (Bi-SPCE) and an antimony modified electrode (Sb-SPCE). The successful electrode modification was confirmed by scanning electron microscopy. The electroanalytical performance of the SPCE modified electrodes for MT detection was examined by cyclic voltammetry. The highest active surface area of 1.073x10-4 cm2 was obtained on Sb-SPCE. This indicates that Sb-SPCE can enhance the sensitivity of MT detection better than the bare-SPCE and the Bi-SPCE. The Sb-SPCE showed a linear response for MT concentrations ranging from 2 to 8 mg.L-1. The sensitivity obtained from the slope of a calibration curve was -0.452 mA.mol-1.L-1 in a Britton-Robinson buffer pH 4.0 containing Sb 16 mg.L-1 with deposition potential and deposition time of 1 V and 90 seconds, respectively. A linear relationship between the square root of the scan rate and the peak current revealed that mass transfer of MT to the electrode was driven by a diffusion mechanism. The limit of detection was found to be 1 mg.L-1.

Exogenous melatonin confers salt stress tolerance to Chlamydomonas reinhardtii (Volvocales, Chlorophyceae) by improving redox homeostasis

:Melatonin (MT; N-acetyl-5-methoxy-tryptamine), a natural derivative of tryptophan, is a central biomolecule synthesised by almost all organisms, including animals and plants. As an antioxidant, MT is involved in many cell responses associated with the protection of plants against abiotic stress. However, there are few studies on the function of MT in microalgae. Our work focused on the relationship between exogenous MT and salt stress in the single-celled eukaryote Chlamydomonas reinhardtii, evaluating for growth, cell morphology, malondialdehyde (MDA) content (the peroxidation product of the cell membrane), activity of antioxidant enzymes and MT concentration under 100 mM NaCl treatment. Growth of Chlamydomonas was promoted at MT concentrations of 0.1 and 1.0 μM, but at 10 and 100 μM, MT was associated with negative effects on growth. MT at 1 μM maintained intact cell morphology and cellular aggregation in an unfavourable salt environment. MT concentrations of 0.1, 1 and 10 μM MT led to increased activity of the antioxidants superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) but reduced MDA. In conclusion, by increasing relative enzyme activity and maintaining a balanced redox state, exogenous MT may improve salt tolerance of C. reinhardtii to allow normal growth. A suitable concentration of exogenous MT improved the growth of C. reinhardtii in a salt environment and increased the activity of antioxidant system–related enzymes to protect cells. These results provide insight into the function of MT in a model green alga.

Long-term measurements of volatile organic compounds highlight the importance of sesquiterpenes for the atmospheric chemistry of a boreal forest

Abstract. The concentrations of terpenoids (isoprene; monoterpenes, MTs; and sesquiterpenes, SQTs) and oxygenated volatile organic compounds (OVOCs; i.e. aldehydes, alcohols, acetates and volatile organic acids, VOAs) were investigated during 2 years at a boreal forest site in Hyytiälä, Finland, using in situ gas chromatograph mass spectrometers (GC-MSs). Seasonal and diurnal variations of terpenoid and OVOC concentrations as well as their relationship with meteorological factors were studied. Of the VOCs examined, C2–C7 unbranched VOAs showed the highest concentrations, mainly due to their low reactivity. Of the terpenoids, MTs showed the highest concentrations at the site, but seven different highly reactive SQTs were also detected. The monthly and daily mean concentrations of most terpenoids, aldehydes and VOAs were highly dependent on the temperature. The highest exponential correlation with temperature was found for a SQT (β-caryophyllene) in summer. The diurnal variations in the concentrations could be explained by sources, sinks and vertical mixing. The diurnal variations in MT concentrations were strongly affected by vertical mixing. Based on the temperature correlations and mixing layer height (MLH), simple proxies were developed for estimating the MT and SQT concentrations. To estimate the importance of different compound groups and compounds in local atmospheric chemistry, reactivity with main oxidants (hydroxyl radical, OH; nitrate radical, NO3; and ozone, O3) and production rates of oxidation products (OxPRs) were calculated. The MTs dominated OH and NO3 radical chemistry, but the SQTs greatly impacted O3 chemistry, even though the concentrations of SQT were 30 times lower than the MT concentrations. SQTs were also the most important for the production of oxidation products. Since the SQTs show high secondary organic aerosol (SOA) yields, the results clearly indicate the importance of SQTs for local SOA production.

17α-Methyltestosterone implants accelerate spermatogenesis in common snook, Centropomus undecimalis, during first sexual maturation

The common snook, Centropomus undecimalis, is an emerging species for intensive fish culture, however, some reproductive aspects of this species, especially the development of the testes and the action of androgen hormones on spermatogenesis have not been studied. The objective of this study was to evaluate the effects of 17α-methyltestosterone (MT) on spermatogenesis and steroidogenesis during the first sexual maturation of the common snook. The fish, which were reproduced in captivity, had a body weight of 305.80 ± 35.60 g and a total length of 34,11 ± 1,08 cm. We used ethylene-vinyl-acetate (EVAc) implants with four concentrations of the hormone MT: T1 (0.3 mg/kg); T2 (3.0 mg/kg); T3 (15.0 mg/kg) and T4 (30.0 mg/kg), and a control group that did not receive the hormone. The gonads increased (P < 0.05) in relation to the concentrations of MT. Histological analysis revealed a progression of spermatogenesis in the MT treatments, especially in T3 and T4. Sperm release was attained in some fish treated with MT. However, there was a partial suppression of the levels of testosterone (T) and 11-ketotestosterone (11-KT) in plasma in the MT treatments, indicating a negative feedback on steroidogenesis. However, this suppression of T and 11-KT in plasma did not prevent an increase in the gonadosomatic index and the progression of gametogenesis. There was also an increase of estradiol (E2) in plasma in the treatments with the highest MT concentrations. The results suggest that the application of EVAc implants with MT at concentrations of 15 and 30 mg/kg stimulates the development and growth of the testes and accelerates spermatogenesis.

Detection of a synthetic sex steroid in the American crocodile (Crocodylus acutus): Evidence for a novel environmental androgen

Endocrine-disrupting contaminants (EDC's) are well known to alter sexual differentiation among vertebrates via estrogenic effects during development, particularly in organisms characterized by temperature-dependent sex determination. However, substances producing androgenic effects typically lack potency when tested in laboratory settings and are virtually unstudied in field settings. Here, we assay levels of a synthetic androgen, 17α-methyltestosterone (MT), in a heavily male-biased population of American crocodiles in the Tempisque River Basin of Costa Rica based on the recent hypothesis that this chemical is an EDC in developing crocodilian embryos. The presence of MT was documented in all field-collected samples of egg yolk and in plasma of all age classes in among population of crocodiles. Hatchlings exhibited higher plasma MT concentrations (102.1 ± 82.8 ng/mL) than juveniles (33.8 ± 51.5) and adults (25.9 ± 20.8 ng/mL). Among populations, crocodiles captured in the Tempisque River (62.9 ± 73.7 ng/mL) were higher in MT concentration than those from Tarcoles (13.3 ± 11.4 ng/mL) and negative controls (0.001 ± 0.0002 ng/mL). A mechanism for the bio-transport of MT and its subsequent effects is proposed.

Effects of model aromatizable (17α-methyltestosterone) and non-aromatizable (5α-dihydrotestosterone) androgens on the adult mummichog (Fundulus heteroclitus) in a short-term reproductive endocrine bioassay

Androgens originating from pulp mill processing, sewage treatment facilities and agricultural activities have the potential for discharge into aquatic receiving environments. To assess androgen effects on reproductive endocrine status in fish in estuarine environments, male and female adult northern mummichog (Fundulus heteroclitus macrolepidotus) were exposed to an aromatizable androgen (17α-methyltestosterone; MT) and a non-aromatizable androgen (5α-dihydrotestosterone; DHT) in a short-term reproductive endocrine bioassay. Fish were nominally exposed to 10μg/L or 100μg/L DHT, or 0.1μg/L or 1μg/L MT for 14days during gonadal recrudescence. Actual concentrations of androgens, as measured by enzyme immunoassay (EIA), were 10–49% of nominal MT 0.1, 3–6% of nominal MT 1, 5–10% of nominal DHT 10 and 3–25% of nominal DHT 100. Female mummichog were impacted to a greater degree by androgen exposure, with increased plasma testosterone (T) at 100μg/L DHT, depressed plasma 17β-estradiol (E2) at both DHT concentrations and at 1μg/L MT, as well as depressed in vitro E2 at both MT concentrations and 100μg/L DHT. Males had depressed plasma T in the 10μg/L DHT treatment and depressed in vitro 11-ketotestosterone production for both MT concentrations and 10μg/L DHT. Ovarian aromatase gene expression was induced in females exposed to 1μg/L MT. DHT at 100μg/L increased hepatic vitellogenin-1 (VTG1) expression in males and depressed VTG1 expression in females. The range of responses between sexes and among species provides evidence for modes of actions and potential impacts of androgens in aquatic receiving environments.

Miltefosine Increases Lipid and Protein Dynamics in Leishmania amazonensis Membranes at Concentrations Similar to Those Needed for Cytotoxicity Activity

Miltefosine (MT) is a membrane-active alkylphospholipid licensed for the topical treatment of breast cancer skin metastases and the oral treatment of leishmaniasis, although its mechanism of action remains unclear. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled lipid and a thiol-specific spin label in the plasma membrane of Leishmania promastigotes showed that MT causes dramatic increases in membrane dynamics. Although these alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Cell lysis was also detected by analyzing the supernatants of centrifuged samples for the presence of spin-labeled membrane fragments and cytoplasmic proteins. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment under the same range of MT concentrations that cause cell growth inhibition. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ∼70% cell death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. After this initial impact on the number of viable parasites, the processes of cell death and growth continued during the first 4 h of incubation. The EPR spectra of spin-labeled membrane-bound proteins were consistent with more expanded and solvent-exposed protein conformations, suggesting a detergent-like action. Thus, MT may form micelle-like structures around polypeptide chains, and proteins with a higher hydrophobicity may induce the penetration of hydrophilic groups of MT into the membrane, causing its rupture.

Metallothioneins pattern during ontogeny of coastal dolphin, Pontoporia blainvillei, from Argentina

Metallothioneins are signals of metal exposure and widely used in biomonitoring. Franciscana dolphin is an endemic cetacean from the Southwestern Atlantic Ocean, classified as Vulnerable A3d by the IUCN. Metallothionein, copper and zinc in Franciscana were assessed in two geographic groups; one inhabits La Plata River estuary, anthropogenically impacted, and the other inhabits marine coastal ecosystems, with negligible pollution. Despite the environment, hepatic and renal MT concentrations were similar, but there was a declining trend from early to later developmental stages. Metallothionein K/L, Cu and Zn levels corresponded to normal reported ranges. MT was not related with Cd. Fetal concentrations were higher than its mother. These results and the health status of dolphins are suggesting that MT correspond to physiological ranges for the species, and they are closely to homeostasis of Zn and Cu, according to its ontogenetic changes. The information constitutes the first MT information on Franciscana dolphin and can be considered as baseline for the species conservation.

Effects of 17α-methyltestosterone on the reproduction of the freshwater snail Biomphalaria glabrata

17-α-methyltestosterone (MT) is a synthetic hormone used in fish hatcheries to induce male monosex. Snails hold promise as possible test models to assess chemicals acting on the endocrine system. Biomphalaria glabrata is an aquatic gastropod mollusk (Pulmonata, Planorbidae) that can be easily maintained in aquaria, predisposing the species for use in ecotoxicological testing. This study evaluated the reproductive effects of MT on B. glabrata by examining histological changes and its reproductive performance. Ten snails per group were exposed for 4 weeks to different concentrations of MT (0.01, 0.1, and 1.0 mg/L). The total number of laid eggs, egg mass per group, size of type V oocytes, and production of spermatozoids were determined. Reproduction of B. glabrata was affected by MT. At the lowest concentration (0.01 mg/L), MT caused a statistically significant increase in the number of egg mass per snail compared with controls unexposed to MT. Histopathology analyses showed an increase in the sperm production at the higher MT concentrations of 0.1 and 1.0 mg/L. Chromatographic analyses of water samples showed that MT concentrations rapidly declined within a 96-h period. These results highlight the importance of giving more support to regulatory authorities, since MT is not registered for use on fish hatcheries in many countries around the world. Wastewater from fish farms discharged into aquatic ecosystems should be monitored for MT residues, since its presence could compromise the reproduction of other native snail species.

Biomarkers in &lt;i&gt;Nereis diversicolor&lt;/i&gt; (Polychaeta: Nereididae) as management tools for environmental assessment on the southwest Iberian coast

The environmental quality of the southwest Iberian coast was assessed in different areas (Ria Formosa Lagoon, Guadiana Estuary and Cadiz Bay) focusing on metal contamination (Cd, Cu, Ni, Pb and Zn) in Nereis diversicolor whole tissues. In addition, associated toxicological effects were assessed using a multibiomarker approach combining several conventional biomarkers. The set of biomarkers selected included antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase), metallothionein and lipid peroxidation. N. diversicolor from the Ria Formosa Lagoon, Guadiana River and Cadiz Bay responded differently to metal contamination with different biomarker responses showing a clear site trend, suggesting different sources and/or magnitudes of contamination. Cadmium was a source of oxidative stress in polychaetes, mainly in Cadiz Bay, with a significant influence on antioxidant enzymes and enhancement of lipid peroxidation. The highest MT concentrations were in the Ria Formosa Lagoon and in the Guadiana River, where there was a direct relationship with high nickel concentrations. Biomarker responses of N. diversicolor are useful tools for environmental quality assessment on the southwest Iberian coast, and more specific metal biomarkers should be included in future assessments.

Integrated assessment of estuarine sediment quality based on a multi-biomarker approach in the bivalve Scrobicularia plana

The bivalve Scrobicularia plana has been proposed as a sentinel species for the assessment of estuarine sediment quality. The aim of this study was to test the responsiveness of a set of biomarkers in bivalves originating from a moderately contaminated site (Loire estuary, France) and a comparatively cleaner site (Bay of Bourgneuf, France) used as reference. Temporal fluctuations were examined in animals collected on eight occasions from March 2008 to October 2009 for the determination of biochemical (MTs, GST, AChE), physiological (energy reserves as glycogen and lipids, condition, hepato-somatic and gonado-somatic indices), and behavioural biomarkers (burrowing). Metals in clams were examined with the aim of understanding the variations in MT concentrations. The current findings indicate that among biochemical markers MTs must be used in a precautionary fashion, and comparison with a reference site remains indispensable together with an appreciation of any salinity effects. Clearer responses were obtained for GST activity which contributes to defence against organic compounds. However, any such defence was not sufficient to ensure total protection since a number of impairments were observed at the individual level (burrowing behaviour, condition index, gonado-somatic index). The integrated biomarker response (IBR) illustrated higher levels of stress in clams collected from the Loire estuary compared to those gathered from the reference site at most of the sampling periods. The interpretation of responses involved in the cascade of energetic events (from available food, digestive enzyme activities, energy reserves to condition and reproductive status) is complex but did reveal disturbances in the Loire estuary which is far from being the most contaminated estuary in France and over the world. However, no links can be established between effects at individual and supra-individual levels in organisms in the Loire estuary, even in the case of a small oil spill which occurred at the beginning of this study.

Degradation of 17α-methyltestosterone by Rhodococcus sp. and Nocardioides sp. isolated from a masculinizing pond of Nile tilapia fry

17α-Methyltestosterone (MT), a synthetic anabolic androgenic steroid, is widely used in aquafarming for the production of an all male fish population such as Nile tilapia. This study isolated, identified and characterized MT-degrading bacteria in the sediment and water from a masculinizing pond of Nile tilapia fry. Based on the phylogeny, physiological properties and cell morphology, the three isolated MT-degrading bacteria were related closely to Rhodococcus equi, Nocardioides aromaticivorans, and Nocardioides nitrophenolicus. Growth of the three isolated strains was found to be inhibited for MT concentrations in the range of 1.0–10mg/L. The inhibition of cell growth was found to be modeled using the Haldane's substrate inhibition model. The kinetic constants ranged from 0.13 to 0.19h−1 for μmax, 0.7–24.8mg/L for Ks and 19.6–76.2mg/L for Ki. Androgenic activity using β-galactosidase assay showed that all strains degraded MT to the products with no androgenic potency.

Determination of Cd-MT in Pteria penguin with indirect non-competitive ELISA

Cadmium-metallothionein (Cd-MT) of Pteria penguin was used to immunize domestic rabbit in order to obtain polyclonal antibody against Cd-MT. Using the anti-Cd-MT antibody, a method of indirect non-competitive enzyme-linked immunosorbent assay (non-competitive ELISA) was established to detect shellfish Cd-MT. In this case, the minimum detectable concentration of P. penguin Cd-MT was (4.563±0.051) ng/mL. The linear range of detection was 9.75–2.0×104 ng/mL. Intra-assay relative standard deviation (RSD) was less than 11.6%, and inter-assay RSD less than 6.7%. The recovery rates ranged from 83.7% to 119.0%. The polyclonal antibody against Cd-MT of P. penguin can also be used to detect MT of other four types of shellfish. In the crude extracts from different organs of P. penguin, the MT concentrations determined by this method matched well with the concentrations of cadmium detected by the atomic absorption spectroscopy.