Androgens modulate gene expression and specific DNA methylation pattern of steroid 5α-reductases in the frog Silurana tropicalis.

In vertebrates, androgens are essential in many biological functions, including reproduction, immune system, metabolism, cardiovascular function, and the central nervous system. The most potent androgen 5α-dihydrotestosterone (5α-DHT), which is actively involved in sexual differentiation and development, is converted from testosterone (T) by the steroid 5α-reductases type 1, 2, and 3 (Srd5α1, Srd5α2, and Srd5α3). Alternatively, steroid 5ß-reductase (Srd5ß) converts T to 5ß-dihydrotestosterone (5ß-DHT), a metabolite believed to be involved in steroid clearance. Recent studies suggested that Srd5 isoforms are targets for endocrine disruption. Thus, understanding the regulation of Srd5 is important to expand our knowledge on how exogenous compounds can interfere with these enzymes. In this study, we exposed frog brain, liver, and gonads ex vivo to T, 5α-DHT, and 5ß-DHT in order to investigate the regulation of srd5 in response to androgens as a simulation of endocrine disrupting chemicals with androgenic properties. Androgens did not modulate srd5α2, suggesting that this isoform is not regulated by T and 5α-DHT in frogs. However, the DNA methylation of srd5α2 increased following 5α-DHT treatment suggesting that androgens can modulate epigenetic mechanisms in amphibians. In contrast, the DNA methylation of srd5α1 and srd5α3 remained stable after androgen exposure, but the mRNA levels of srd5α1 and srd5α3 were modulated by T, 5α-DHT, and 5ß-DHT in a sex- and tissue-specific manner. While T positively regulates srd5α1 and srd5α3 in testes, T negatively regulates srd5α3 in ovaries. Moreover, exposure to T also increased the mRNA level of srd5ß in the male brain suggesting a mechanism to protect the brain from androgen action by elimination of T into 5ß-DHT. Thus, exogenous compounds with androgenic properties potentially interact with srd5 transcription and DNA methylation pattern, which could adversely affect biological functions of vertebrates during development and reproduction.

Transcriptomic profiling in Silurana tropicalis testes exposed to finasteride.

Investigations of endocrine disrupting chemicals found in aquatic ecosystems with estrogenic and androgenic modes of action have increased over the past two decades due to a surge of evidence of adverse effects in wildlife. Chemicals that disrupt androgen signalling and steroidogenesis can result in an imbalanced conversion of testosterone (T) into 17ß-estradiol (E2) and other androgens such as 5α-dihydrotestosterone (5α-DHT). Therefore, a better understanding of how chemicals perturb these pathways is warranted. In this study, the brain, liver, and testes of Silurana tropicalis were exposed ex vivo to the human drug finasteride, a potent steroid 5α-reductase inhibitor and a model compound to study the inhibition of the conversion of T into 5α-DHT. These experiments were conducted (1) to determine organ specific changes in sex steroid production after treatment, and (2) to elucidate the transcriptomic response to finasteride in testicular tissue. Enzyme-linked immunosorbent assays were used to measure hormone levels in media following finasteride incubation for 6 h. Finasteride significantly increased T levels in the media of liver and testis tissue, but did not induce any changes in E2 and 5α-DHT production. Gene expression analysis was performed in frog testes and data revealed that finasteride treatment significantly altered 1,434 gene probes. Gene networks associated with male reproduction such as meiosis, hormone biosynthesis, sperm entry, gonadotropin releasing hormone were affected by finasteride exposure as well as other pathways such as oxysterol synthesis, apoptosis, and epigenetic regulation. For example, this study suggests that the mode of action by which finasteride induces cellular damage in testicular tissue as reported by others, is via oxidative stress in testes. This data also suggests that 5-reductase inhibition disrupts the expression of genes related to reproduction. It is proposed that androgen-disrupting chemicals may mediate their action via 5-reductases and that the effects of environmental pollutants are not limited to the androgen receptor signalling.

Reprint of "Current perspectives on the androgen 5 alpha-dihydrotestosterone (DHT) and 5 alpha-reductases in teleost fishes and amphibians".

The androgen 5 alpha-dihydrotestosterone (DHT) is a steroidogenic metabolite that has received little attention in non-mammalian species. DHT is produced by the reduction of the double-bond of testosterone by a group of enzymes called 5 alpha-reductases of which there can be multiple isoforms (i.e., srd5a1, srd5a2, and srd5a3). Data from amphibians suggest that the expression of the srd5a genes occurs in early development, and continues until adulthood; however insufficient data exist in fish species, where DHT is thought to be relatively biologically inactive. Here, we demonstrate that fathead minnow (FHM; Pimephales promelas) developing embryos and adults express srd5a enzyme isoforms. During FHM embryogenesis, both srd5a1 and srd5a3 mRNA levels were significantly correlated in expression levels while srd5a2 showed a more unique pattern of expression. In adult FHMs, males had significantly higher levels of srd5a2 in the liver and gonad compared to females. In the male and female liver, transcript levels for srd5a2 were more abundant compared to srd5a1 and srd5a3, suggesting a prominent role for srd5a2 in this tissue. Interestingly, the ovary expressed higher mRNA levels of srd5a3 than the testis. Thus, data suggest that srd5a isoforms can show sexually dimorphic expression patterns in fish. We also conducted a literature review of the biological effects observed in embryonic and adult fish and amphibians after treatments with DHT and DHT-related compounds. Treatments with DHT in teleost fishes and amphibians have resulted in unexpected biological responses that are characteristic of both androgens and anti-androgens. For example, in fish DHT can induce vitellogenin in vitro from male and female hepatocytes and can increase 17ß-estradiol production from the teleost ovary. We propose, that to generate further understanding of the roles of DHT in non-mammals, studies are needed that (1) address how DHT is synthesized within tissues of fish and amphibians; (2) examine the full range of biological responses to endogenous DHT, and its interactions with other signaling pathways; and (3) investigate how DHT production varies with reproductive stage. Lastly, we suggest that the Srd5a enzymes can be targets of endocrine disruptors in fish and frogs, which may result in disruptions in the estrogen:androgen balance in aquatic organisms.

A positive correlation between mercury and oxidative stress-related gene expression (GPX3 and GSTM3) is measured in female Double-crested Cormorant blood.

Mercury (Hg) is a widespread contaminant that has been shown to induce a wide range of adverse health effects in birds including reproductive, physiological and neurological impairments. Here we explored the relationship between blood total Hg concentrations ([THg]) and oxidative stress gene induction in the aquatic piscivorous Double-crested Cormorants (Phalacrocorax auritus) using a non-lethal technique, i.e., blood gene expression analysis. P. auritus blood was sampled at five sites across the Great Lakes basin, Ontario, Canada and was analyzed for [THg]. To assess cellular stress, the expression of glutathione peroxidases 1 and 3 (GPX1, GPX3), superoxide dismutase 1 (SOD1), heat-shock protein 70 kd-8 (HSP70-8) and glutathione S-transferase µ3 (GSTM3) were measured in whole blood samples using real-time RT-PCR. Results showed a significantly positive correlation between female blood [THg] and both GPX3 and GSTM3 expression. Different levels of oxidative stress experienced by males and females during the breeding season may be influencing the differential oxidative stress responses to blood [THg] observed in this study. Overall, these results suggest that Hg may lead to oxidative stress as some of the cellular stress-related genes were altered in the blood of female P. auritus and that blood gene expression analysis is a successful approach to assess bird health condition.

Current perspectives on the androgen 5 alpha-dihydrotestosterone (DHT) and 5 alpha-reductases in teleost fishes and amphibians.

The androgen 5 alpha-dihydrotestosterone (DHT) is a steroidogenic metabolite that has received little attention in non-mammalian species. DHT is produced by the reduction of the double-bond of testosterone by a group of enzymes called 5 alpha-reductases of which there can be multiple isoforms (i.e., srd5a1, srd5a2, and srd5a3). Data from amphibians suggest that the expression of the srd5a genes occurs in early development, and continues until adulthood; however insufficient data exist in fish species, where DHT is thought to be relatively biologically inactive. Here, we demonstrate that fathead minnow (FHM; Pimephales promelas) developing embryos and adults express srd5a enzyme isoforms. During FHM embryogenesis, both srd5a1 and srd5a3 mRNA levels were significantly correlated in expression levels while srd5a2 showed a more unique pattern of expression. In adult FHMs, males had significantly higher levels of srd5a2 in the liver and gonad compared to females. In the male and female liver, transcript levels for srd5a2 were more abundant compared to srd5a1 and srd5a3, suggesting a prominent role for srd5a2 in this tissue. Interestingly, the ovary expressed higher mRNA levels of srd5a3 than the testis. Thus, data suggest that srd5a isoforms can show sexually dimorphic expression patterns in fish. We also conducted a literature review of the biological effects observed in embryonic and adult fish and amphibians after treatments with DHT and DHT-related compounds. Treatments with DHT in teleost fishes and amphibians have resulted in unexpected biological responses that are characteristic of both androgens and anti-androgens. For example, in fish DHT can induce vitellogenin in vitro from male and female hepatocytes and can increase 17ß-estradiol production from the teleost ovary. We propose, that to generate further understanding of the roles of DHT in non-mammals, studies are needed that (1) address how DHT is synthesized within tissues of fish and amphibians; (2) examine the full range of biological responses to endogenous DHT, and its interactions with other signaling pathways; and (3) investigate how DHT production varies with reproductive stage. Lastly, we suggest that the Srd5a enzymes can be targets of endocrine disruptors in fish and frogs, which may result in disruptions in the estrogen:androgen balance in aquatic organisms.

Influence of Plant Species on Microbial Activity and Denitrifier Population Development in Vegetated Denitrifying Wood-Chip Bioreactors.

The microbial characteristics of four vegetated and one unplanted wood-chip bioreactors treating greenhouse effluent were investigated in a continuous experiment operated for over 2.5 years. The bioreactors were designed to reduce nitrate concentrations via naturally induced microbial denitrification. The vegetation type and reactor depth were both found to be significant factors in defining the mixed microbial activity. However, a consistent correlation between the abundance of the denitrifying communities and reactor depth could not be found across all reactors. The media samples from the unit planted with Typha angustifolia displayed higher microbial activities compared with the other reactors. This plant's root-associated bacteria also demonstrated the greatest copies of the denitrifying genes nirK and nosZ. The most abundant denitrifier communities and those encoding the nosZ gene were found in the unplanted reactor, followed by the T. angustifolia unit. The T. angustifolia reactor demonstrated greater microbial activity and denitrification capacity at the depth of 20 cm, while the greatest denitrification capacity in the unplanted reactor was found at the depth of 60 cm. These findings indicated the importance of the T. angustifolia rhizosphere to support microbial community establishment and growth in the vicinity of the plant's roots, although those populations may eventually develop in an unplanted environment.

Interstitial water microbial communities as an indicator of microbial denitrifying capacity in wood-chip bioreactors.

The discharge from food production greenhouses (greenhouse effluent) contains high nutrient and salt concentrations, which, if directly released, can have adverse effects on the environment. Wood-chip bioreactors are increasingly popular passive water treatment systems favoured for their economical denitrification in treating agricultural field tile drainage. Microbial communities are central to denitrification; however little is known about the maturation of microbial communities in wood-chip bioreactors treating greenhouse effluents. In this study, multiple subsurface flow wood-chip bioreactors, each vegetated with a different plant species, together with an unplanted unit, received synthetic greenhouse effluent with elevated nitrate concentrations. The hybrid bioreactors were operated for over 2 years, during which time water samples were collected from the inlet, outlet and within the reactors. The increasing denitrification rate in the bioreactor planted with Typha angustifolia (narrowleaf cattail) correlated with increasing microbial activity and metabolic richness, measured by the carbon utilization patterns in Biolog® EcoPlates. Increased denitrifying gene (nirS) copies (determined by quantitative polymerase chain reaction, qPCR), and near-complete nitrate removal were observed in the T. angustifolia and unplanted reactors after 16 and 23 months of operation respectively. The findings suggested that an acclimation period of at least one year can be expected in unseeded bioreactors planted with T. angustifolia, while bioreactors without vegetation may require a longer time to maximize their denitrification capacity. These results are important for the design and operation of wood-chip bioreactors, which are expected to be more commonly applied in the future.

Phthalates modulate steroid 5-reductase transcripts in the Western clawed frog embryo.

Phthalates are used worldwide in the manufacturing of plastics, added to cosmetic products, personal care products, pharmaceuticals, medical devices, and paints; and are widely detected in soil, surface water, and organism tissues. Phthalate esters have been previously shown to interfere with the endocrine system in vertebrates. However, few studies have investigated the effects of phthalates on testosterone-converting enzymes that affect hormone levels and reproduction. In the present study, we exposed the Western clawed frog (Silurana tropicalis) to 0.1, 1, and 10 µM diethylhexyl phthalate (DEHP), dibutyl phthalate (DBP), and diethyl phthalate (DEP) during early amphibian embryonic development. Additional DBP exposures were conducted ex vivo using mature frog testes. Malformations and mRNA levels of genes associated to reproduction and oxidative stress were evaluated. 0.1 µM DEHP, DBP, and DEP induced an array of malformations, including incomplete gut coiling, edemas, and eye malformations. Moreover, all three phthalates increased the expression of androgen-related genes, such as steroid-5α-reductase 1, 2, 3, steroid-5ß-reductase, and androgen receptor at concentrations ranging from 0.1 to 10 µM depending on the phthalate and gene. Data suggest that the phthalate esters tested are teratogens to the amphibian embryo and that these phthalates exhibit an androgenic activity in amphibians.

Effect of gold nanoparticles and ciprofloxacin on microbial catabolism: a community-based approach.

The effect of gold nanoparticles (AuNPs) and ciprofloxacin on the catabolism of microbial communities was assessed. This was accomplished through an ex situ methodology designed to give a priori knowledge on the potential for nanoparticles, or other emerging contaminants, to affect the catabolic capabilities of microbial communities in the environment. Microbial communities from a variety of sources were incubated with 31 prespecified carbon sources and either National Institute of Standards and Technology reference material 10-nm AuNPs or ciprofloxacin on 96-well microtiter plates. From the ciprofloxacin study, dose-response curves were generated and exemplified how this method can be used to assess the effect of a toxicant on overall catabolic capabilities of microbial communities. With 10-nm AuNPs at concentrations ranging from 0.01 µg/mL to 0.5 µg/mL, rhizosphere communities from Typha roots were only slightly catabolically inhibited at a single concentration (0.05 µg/mL); no effects were seen on wetland water communities, and a minor positive (i.e., enhanced catabolic capabilities) effect was observed for loamy soil communities. This positive effect might have been because of a thin layer of citrate found on these AuNPs that initiated cometabolism with some of the carbon sources studied. Under the conditions considered, the possible adverse effects of AuNPs on the catabolic capabilities of microbial communities appears to be minimal.