CYP1A1 Gene Expression Research Articles

Biochanin a modulates steroidogenesis and cellular metabolism in human granulosa cells through TAS2Rs activation: a spotlight on ovarian function

BackgroundEndocrine-disrupting chemicals (EDCs) interfere with the endocrine system and negatively impact reproductive health. Biochanin A (BCA), an isoflavone with anti-inflammatory and estrogen-like properties, has been identified as one such EDC. This study investigates the effects of BCA on transcription, metabolism, and hormone regulation in primary human granulosa cells (GCs), with a specific focus on the activation of bitter taste receptors (TAS2Rs).MethodsPrimary human GCs from 60 participants were treated with 10 µM BCA, and selective antagonists were used to block TAS2R activation. The study assessed the expression of TAS2R14 and TAS2R43, and analyzed the impact on StAR and CYP17A1 gene expression. Intracellular calcium levels, lipid droplet size, and mitochondrial network complexity were measured to evaluate cellular metabolism and energy dynamics.ResultsBCA treatment significantly upregulated TAS2R14 and TAS2R43 expression, leading to a 70% increase in StAR mRNA levels and a twofold increase in CYP17A1 expression (p < 0.05). These effects were reversed by TAS2R antagonists. Additionally, BCA treatment decreased intracellular Ca2+ levels (p < 0.01) and reduced lipid droplet size (p < 0.001), both of which were counteracted by antagonists. Enhanced mitochondrial network complexity (p < 0.001) was also observed, suggesting increased mitochondrial fusion and improved cellular energy dynamics.ConclusionThe findings indicate that BCA modulates transcriptional and metabolic processes in GCs through the activation of TAS2Rs, highlighting their role in endocrine regulation. The statistically significant results emphasize the relevance of further exploring the effects of EDCs like BCA on reproductive health. Collaborative research efforts are essential to address and mitigate the adverse impacts of EDCs on fertility.

Pentoxifylline ameliorates carbamazepine-induced hepatotoxicity via down expression of CYP3A4 and NF-kB gene expression in the rat: in vivo study

Drug-induced liver injury (DILI) is a serious complication of many drugs, including carbamazepine; this study investigates the protective effect of pentoxifylline (PTX) against DILI induced by carbamazepine in rat models by attenuating CYP3A4 and NF-κB gene expression. Forty rats were divided into five groups: the control group received no treatment, the induction group received 50 mg/kg carbamazepine orally for 28 days, and three groups received PTX (100, 200, and 300 mg/kg) once daily for one hour before carbamazepine induction for 28 days. Then, the rats were euthanized, and blood and liver tissue were collected for biochemical, gene expression, and histopathology. PTX attenuates the carbamazepine-induced increased CYP3A4 and NF-κB gene expression, with the highest dose showing the best result. PTX also reduced aspartate aminotransferase, alanine aminotransferase, and malondialdehyde, while glutathione levels increased. In conclusion, PTX is highly efficacious in preventing and restoring the liver cells’ normal morphology and cellular function caused by carbamazepine hepatotoxicity. A possible mechanism of the PTC effect is hindering oxidative stress by scavenging free radicals and enhancing the body’s natural defense antioxidant system. Additionally, it exerts an anti-inflammatory impact by modulating NF-κB gene expression.

Evaluation of the Decreased Cholesterol Potential of Levilactobacillus brevis M-10 Isolated from Spontaneously Fermented Sour Porridge in Mice with High-Cholesterol Levels.

Excessive cholesterol levels can lead to hypercholesterolemia, which is related to cardiovascular diseases (CVDs), and CVDs are a serious threat to human health. Therefore, lowering cholesterol levels is necessary, and diet intervention is safer than drugs are. The cholesterol-lowering effect of Levilactobacillus brevis M-10 isolated from spontaneously fermented millet sour porridge was investigated in fifty C57BL/6N male mice. After a 4-week intervention, the food intake, weight gains and organ indices were calculated; the lipid contents in the serum, liver, and feces were determined; the histopathology of the liver tissues was observed; the expression of metabolism-related genes was determined; and short-chain fatty acid (SCFA) levels in the droppings were monitored. The results showed that administration of a high dose of L. brevis M-10 (1 × 1010CFU/mL) significantly reduced food intake, suppressed weight gain; prevented excessive liver growth; and reduced the total serum cholesterol, triglycerides, low-density lipoproteins; and total hepatic cholesterol and triglyceride contents (P < 0.05) in high-cholesterol mice. Moreover, a high dose of L. brevis M-10 significantly promoted the fecal excretion of cholesterol and triglycerides (P < 0.05) and alleviated liver damage induced by a high-cholesterol diet. Furthermore, a high dose of L. brevis M-10 significantly downregulated the cholesterol metabolism-related gene expression of NPC1L1, ACAT2, HMG-CoA, and SREBP2 but upregulated the gene expression of ABCG5, CYP7A1, and LXR-α (P < 0.05). Additionally, a high dose of L. brevis M-10 significantly increased SCFA contents, including those of acetic acid, propionic acid and n-butyric acid (P < 0.05). These findings could provide support for the use of L. brevis M-10 in the application of functional foods to alleviate hypercholesterolemia.

Ethyltoluenes Regulate Inflammatory and Cell Fibrosis Signaling in the Liver Cell Model.

Crude oil naphtha fraction C9 alkylbenzenes consist of trimethylbenzenes, ethyltoluenes, cumene, and n-propylbenzene. The major fraction of C9 alkylbenzenes is ethyltoluenes (ETs) consisting of three isomers: 2-ethyltoluene (2-ET), 3-ethyltoluene (3-ET), and 4-ethyltoluene (4-ET). Occupational and environmental exposure to ETs can occur via inhalation and ingestion and cause several health problems. Exposure to ETs causes eye and upper respiratory tract irritation, coughing, gagging, vomiting, griping, diarrhea, distress, and depressed respiration. Previous studies suggest that ETs target the respiratory tract and liver and produce several lesions in the nose, lungs, and liver areas. In the current study, we investigated the impact of low concentrations of ETs on cell metabolism, cell inflammation, steatosis, and fibrosis signaling in liver cell models in vitro. Dose-dependent exposure of 2-ET, 3-ET, and 4-ET to HepaRG and hepatocellular carcinoma (HCC) HepG2 and SK-Hep1 cells affects cell survival/real-time proliferation and increases ROS production. ETs induce inflammatory CAT, SOD1, CXCL8, IL1B, HMOX1, NAT1 (3), and STAT3 gene expression. Exposure of 2-ET, 3-ET, and 4-ET to HepaRG and HCC HepG2 and SK-Hep1 cells affects mitochondrial respiration/cellular energetics and upregulates metabolic CYP1-A1, CYP1-A2, CYP2-D6, CYP2-E1, CYP3-A4, CYP3-B4, and VEGFA gene expression. However, no significant change in lipogenesis-related gene expression and modulation of cell steatosis was observed after ET exposure. Acute exposure to induvial ETs and in combination or chronic 2-ET exposure alone modulates cell fibrosis markers such as AST, FGF-23, Cyt-7 p21, TGFβ, TIMP2, and MMP2 in liver cell models, suggesting that ETs target liver cells and may dysregulate liver function.

Replacement of fish meal with full fat Hermetia illucens modulates hepatic FXR signaling in juvenile rainbow trout (Oncorhynchus mykiss): Exploring a potential role of ecdysteroids

The present study was conducted to investigate the effects of fish meal (FM) replacement with full fat Hermetia illucens (HI) on the molecular mechanisms regulating lipid and bile salt (BA) homeostasis in rainbow trout (Oncorhynchus mykiss) juveniles. We thus explore the presence of 20-hydroxyecdysone (20E) in an insect meal-based diet and evaluate its potential involvement in regulating the molecular mechanisms/basis of FXR:RXR axis signaling. Ecdysteroids are a category of steroid hormones which bind a nuclear–receptor complex composed of ecdysone receptor (EcR) and ultraspiracle protein (USP) and regulate insect molting and metamorphosis. In all vertebrates, including fish, EcR-USP homologs are the Farnesoid X receptors (FXR) and the Retinoid X receptors (RXR), which are known to regulate crucial physiological and metabolic aspects, including BA synthesis and cholesterol homeostasis. In silico prediction indicates that 20E binds the heterodimeric complex with a binding affinity constant Kd equals to 610 ± 60 nM and affects positively the dimerization process. Results also demonstrated the coordinated increased expression of FXR and RXR, as well as their downstream target genes (i.e. short heterodimer partner 1 and 2) in rainbow trout fed diets containing HI meal. This latter finding was paralleled by a significant down-regulation of CYP7a1 and CYP8b1 gene expression together with a decrease in hepatic total cholesterol, triglyceride, and BA levels. Overall, our study suggested that FXR is a potential target for 20E content in insect meal and provided preliminary data on the potential role of ecdysteroids in regulating the metabolic status of teleost fish through modulation of FXR signaling in the enterohepatic system.

Gene expression of MTATP6 and cytochrome P450 in MCF-7 and MDA-MB -231 breast cancer cell lines with juglone and curcumin supplemented

It is aimed to determine the effects of naphthoquinones as juglone and curcumin application on cell viability and expression analyzes of CYP3A4 and MTATP6 genes in MCF-7 and MDA-MB-231 human breast cancer cell lines. MCF-7 and MDA-MB-231 cells were incubated, were replaced with containing various concentrations of 5, 10, 15 μM curcumin and 5, 10, 15 μM juglone for MCF-7 and 1, 5, 10 μM curcumin and 1, 2, 3 μM juglone for MDA-MB-231 for 24 h. CYP3A4 and MTATP6 gene expression levels in both cell lines were determined by quantitative real-time polymerase chain reaction (qPCR) method and western blot method. IC50 values for 24 h were found as 22.41 μM for curcumin, and 16.27 μM for juglone in MCF-7, and 10.43 μM for curcumin, and 3.42 μM for juglone in MDA-MB-231 cells. Curcumin showed anti-proliferative, and antioxidant effects. CYP3A4 and MTATP6 gene expressions were decreased in MCF-7 breast cancer cell line when the cells treated with juglone or curcumin. CYP3A4 and MTATP6 gene expressions were decreased at all application doses of juglone in MDA-MB-231 cells whereas CYP3A4 and MTATP6 protein levels were only decreased at 10 μM curcumin compared with the control group.

Structural characterization and hypolipidemic activity of a natural polysaccharide from Suaeda salsa L.

Herein, the identification and analysis of a newly discovered hypolipidemic polysaccharide extracted from Suaeda salsa L., SS3-N1, is reported. The weight-average molecular weight (Mw), number-average molecular weight (Mn), and dispersity (Ð) of SS3-N1 were determined to be 45.50 kDa, 34.21 kDa, and 1.33, respectively. This polysaccharide primarily consists of galactose (50.80 %) and arabinose (30.70 %), with lower proportions of xylose, mannose, guluronic acid, rhamnose, glucuronic acid, ribose, and fucose. Methylation and NMR analyses indicated that its backbone was primarily composed of R → 3,6)-β-D-Galp-(1 → R and R → 5)-α-L-Araf-(1→ residues. The sugar units at the reducing and nonreducing ends were identified as R → 4)-β-D-Xylp-(1 → R and R → 3)-β-D-Galp-(1 → R, respectively. In addition, α-L-Araf (1 → R side branches were incorporated at the C-3 position of R → 3,6)-β-D-Galp-(1 → R. At 100 μg/mL, SS3-N1 surpassed the lipid-lowering efficacy of the positive control, atorvastatin (0.4 μM), in an egg yolk powder (EYP)-induced hyperlipidemic zebrafish model. This effect may be attributed to the modulation of cholesterol metabolism due to the upregulation of nrf2, ho-1, ampk, ppara, and cyp7a1 gene expression and the downregulation of acaca and hmgcr gene expression. Such dual gene regulation inhibits fatty acid and cholesterol synthesis, suggesting potential applications for the natural hypolipidemic polysaccharide derived from S. salsa L.

Human Multi-Lineage Liver Organoid Model Reveals Impairment of CYP3A4 Expression upon Repeated Exposure to Graphene Oxide.

Three-dimensional hepatic cell cultures can provide an important advancement in the toxicity assessment of nanomaterials with respect to 2D models. Here, we describe liver organoids (LOs) obtained by assembling multiple cell lineages in a fixed ratio 1:1:0.2. These are upcyte® human hepatocytes, UHHs, upcyte® liver sinusoidal endothelial cells, LSECs, and human bone marrow-derived mesenchymal stromal cells, hbmMSCs. The structural and functional analyses indicated that LOs reached size stability upon ca. 10 days of cultivation (organoid maturation), showing a surface area of approximately 10 mm2 and the hepatic cellular lineages, UHHs and LSECs, arranged to form both primitive biliary networks and sinusoid structures, alike in vivo. LOs did not show signs of cellular apoptosis, senescence, or alteration of hepatocellular functions (e.g., dis-regulation of CYP3A4 or aberrant production of Albumin) for the entire culture period (19 days since organoid maturation). After that, LOs were repeatedly exposed for 19 days to a single or repeated dose of graphene oxide (GO: 2-40 µg/mL). We observed that the treatment did not induce any macroscopic signs of tissue damage, apoptosis activation, and alteration of cell viability. However, in the repeated dose regimen, we observed a down-regulation of CYP3A4 gene expression. Notably, these findings are in line with recent in vivo data, which report a similar impact on CYP3A4 when mice were repeatedly exposed to GO. Taken together, these findings warn of the potential detrimental effects of GO in real-life exposure (e.g., occupational scenario), where its progressive accumulation is likely expected. More in general, this study highlights that LOs formed by many cell lineages can enable repeated exposure regimens (suitable to mimic accumulation); thus, they can be suitably considered alternative or complementary in vitro systems to animal models.

Expression of steroidogenesis pathway genes in cumulus cells from women with diminished ovarian reserve after gonadotropin administration: A case-control study.

Women with diminished ovarian reserve (DOR) respond differently to gonadotropin medications. This study investigates the relationship between effective gene expression in the steroidogenesis pathway and gonadotropin responsiveness in DOR. In this case-control study, cumulus cells were obtained from women with DOR after gonadotropin administration (n = 20) and normal ovarian reserve (n = 20). They were divided into the following groups, oocyte number 3 and oocyte number 3. After RNA extraction and cDNA synthesis, quantitative polymerase chain reaction was performed to assess the expression levels of cytochrome P450 aromatase (CYP19A1), protein kinase A(PKA), and glycogen synthase kinase 3 beta (GSK3B)genes. The women with DOR had statistically significant lower expression of CYP19A1 and PKA genes in their cumulus cells compared to control group (p = 0.04, and p 0.001, respectively). There was also lower expression of the GSK3B gene in DOR compared to control group, but it was not significant. Although the expression of the CYP19A1, PKA, and GSK3B genes was lower in women with 3 oocytes compared to women with more oocytes, this difference was not statistically significant. In conclusion, DOR may be associated with lower expression of CYP19A1 and PKA genes. Also, considering the decrease in the expression of these genes in people with DOR, the expression of these genes can be used as a tool to predict the treatment.

Influence of gut microbiome composition on effect variations of anti-cholesterol treatment among individual mice

This study investigated if the variation in the effect of anti-cholesterol (AC) treatment on individual mice are related to gut microbiome composition. The bile salt hydrolase (BSH) activity of 23 commercial fermented milk products was examined to select a fermented milk product for AC treatment. Mice were fed to different diets for 6 weeks: high-fat (660 % of total calories from fat; D1), high-dietary fibre (20 % cellulose; D2), and low-fat (17.2 % of total calories from fat; D3) diets to change their gut microbiomes. Subsequently, faecal microbiome was transplanted (FMT) into mice treated with high cholesterol diet contained 2 % cholesterol, followed by AC or non-AC (sterile tap water, STW) treatments. Control groups with normal (NC) and high-cholesterol diets (PC) were prepared for both AC and STW treatment. All experimental groups were subjected to serum and liver cholesterol, cholesterol metabolism-related (CMR) gene expression, and intestinal microbiome analyses. D3-FMT mice showed the most significant enhancements in cholesterol ratio and decreased hepatic cholesterol levels with AC treatment. Moreover, upregulation of the Cyp7a1 gene expression was observed in this group. Furthermore, the intestinal microbiome analysis indicated higher abundances of BSH-producing Eubacterium, Bifidobacterium, and Parabacteroides in the D3-FMT + AC group compare to others, potentially contributing to increased bile acid synthesis.

Involvement of ahr-dependent Cyp1a detoxification activity, oxidative stress and inflammatory regulation in response to graphene oxide exposure in rainbow trout (Oncorhynchus mykiss)

Graphene oxide (GO) is a very attractive material for use in a vast number of applications. However, before its widespread use, it is important to consider potential issues related to environmental safety to support its safe application. The aim of this study was to investigate effects on fish (rainbow trout) following GO exposure. Using both an in vitro approach with the RTL W1 rainbow trout liver cell line, and in vivo exposures, following OECD TG 203, disturbances at the cellular level as well as in the gills and liver tissue of juvenile trout were assessed. In RTL W1 cells, a time and concentration-dependent loss in cell viability, specifically plasma membrane integrity and lysosomal function, was observed after 96 h of exposure to GO at concentrations ≥18.75 mg/L. Additionally, increased reactive oxygen species (ROS) levels were evidenced at concentrations ≥18.75 mg/L, and an enhancement of metabolic activity was noted with concentrations ≥4.68 mg/L. In vivo exposures to GO did not provoke mortality in rainbow trout juveniles following 96 h exposure but led to histological alterations in gills and liver tissues, induction of enzymatic detoxification activities in the liver, as well as aryl hydrocarbon receptor (ahr)–cytochrome P450 1a (cyp1a) gene expression downregulation, and upregulation of pro-inflammatory cytokines il1b and il8 at GO concentrations ≥9.89 mg/L.

Comparison of the effects of copper oxide nanoparticles (CuO-NPs) and copper (II) sulfate on oxidative stress parameters in rainbow trout hatchlings (Oncorhynchus mykiss)

Nanoparticles of copper oxide, released uncontrollably into the environment, pose a threat to living organisms. One of the mechanisms of their toxicity is the induction of oxidative stress, contributing to the disruption of antioxidant mechanisms. The aim of the present study was to assess the degree and toxic dose of copper oxide nanoparticle (CuO-NPs) associated with the induction of oxidative stress and compare them to the toxicity of copper sulfate (VI) (CuSO4) by measuring the expression of genes encoding some key enzymes involved in protection against oxidative stress: glutathione peroxidase (gpx), cytochrome P450 (cyp1a), heat shock protein (hsp70) and superoxide dismutase (sod). Hatchlings of rainbow trout (Oncorhynchus mykiss) were used as biological material. The test individuals were exposed for 2 hours to CuO-NPs and CuSO4 in the concentration range of 4–256 mg dm−3. Individuals exposed to all concentrations of CuO-NPs showed an increase in the expression of gpx, cyp1a and hsp70 genes, and a decrease in sod gene expression, while the hatchlings exposed to CuSO4 exhibited an increase in the expression of all genes. The expression of hsp70 and sod genes in the hatchlings exposed to CuSO4 was higher than in the CuO-NP treatment, while the expression levels of gpx and cyp1a were similar. Based on the obtained results, potentially higher toxicity of CuSO4 was observed in the hatchlings of Oncorhynchus mykiss. However, the elevated expression of gpx, cyp1a and hsp70 after the exposure to CuO-NPs, indicates toxicity via oxidative stress. The decrease (sod in hatchlings exposed to CuO-NPs) or increase of gene expression was not dose-dependent. The presented study confirms a need for further research regarding the toxicity of metal nanoparticles to aquatic organisms and the necessity for monitoring the release of CuO-NPs into the aquatic environment.

The hepatically less toxic metabolites of aflatoxin B1 versus the parent toxin: an in vivo bioremediation study

Aflatoxin B1 (AFB1) is a well-established potent food mycotoxin. We previously evidenced that laccase could restrain AFB1’s genetic and oxidative toxicity in vitro conditions; nevertheless, there is no report about the in vivo toxicity of the laccase-mediated metabolites of aflatoxin B1 (LM-AFB1). In this investigation, we evaluated the in vivo safety of LM-AFB1 in comparison with the parent toxin using serum liver function tests (LFTs), oxidative stress biomarkers, histopathological examinations, and CYP1A2 and GSTA3 gene expression. Adult male mice were divided into four groups: (1) Control; (2) 4 mg/kg AFB1; (3) 4 mg/kg LM-AFB1; (4) 4 mg/kg intermediate transformational metabolites of AFB1 (CA-AFB1). In this study, AFB1’s liver toxicity was confirmed but LM-AFB1 did not significantly change the values. Gene expression analysis indicates that the utilization of laccase for bioremediation does not result in the suppression of glutathione S-transferase, in contrast to AFB1. Our research findings demonstrate that laccase has the potential to mitigate AFB1-induced liver toxicity in mice by averting oxidative stress caused by AFB1.

Ursodeoxycholic Acid Modulates the Interaction of miR-21 and Farnesoid X Receptor and NF-κB Signaling.

(1) Background: This study investigates the effects of Ursodeoxycholic acid (UDCA) on NF-κB signaling, farnesoid X receptor (FXR) singling, and microRNA-21 in HepG2 cells. (2) Methods: HepG2 cells were treated with lipopolysaccharide (LPS) to simulate hepatic inflammation. The investigation focused on the expression of NF-κB activation, which was analyzed using Western blot, confocal microscopy, and Electrophoretic Mobility-shift Assays (EMSA). Additionally, NF-κB and farnesoid X receptor (FXR) singling expressions of micro-RNA-21, COX-2, TNF-α, IL-6, cyp7A1, and shp were assessed by RT-PCR. (3) Results: UDCA effectively downregulated LPS-induced expressions of NF-κB/65, p65 phosphorylation, and also downregulated FXR activity by Western blot. Confocal microscopy and EMSA results confirmed UDCA's role in modulating NF-κB signaling. UDCA reduced the expressions of LPS-induced COX-2, TNF-α, and IL-6, which were related to NF-κB signaling. UDCA downregulated LPS-induced cyp7A1 gene expression and upregulated shp gene expression, demonstrating selective gene regulation via FXR. UDCA also significantly decreased micro-RNA 21 levels. (4) Conclusions: This study demonstrates UDCA's potent anti-inflammatory effects on NF-κB and FXR signaling pathways, and thus its potential to modulate hepatic inflammation and carcinogenesis through interactions with NF-κB and FXR. The decrease in micro-RNA 21 expression further underscores its therapeutic potential.

The Effects of Aflatoxin B1 on Liver Cholestasis and Its Nutritional Regulation in Ducks.

The aim of this study was to investigate the effects of aflatoxin B1 (AFB1) on cholestasis in duck liver and its nutritional regulation. Three hundred sixty 1-day-old ducks were randomly divided into six groups and fed for 4 weeks. The control group was fed a basic diet, while the experimental group diet contained 90 μg/kg of AFB1. Cholestyramine, atorvastatin calcium, taurine, and emodin were added to the diets of four experimental groups. The results show that in the AFB1 group, the growth properties, total bile acid (TBA) serum levels and total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and glutathione (GSH) liver levels decreased, while the malondialdehyde (MDA) and TBA liver levels increased (p < 0.05). Moreover, AFB1 caused cholestasis. Cholestyramine, atorvastatin calcium, taurine, and emodin could reduce the TBA serum and liver levels (p < 0.05), alleviating the symptoms of cholestasis. The qPCR results show that AFB1 upregulated cytochrome P450 family 7 subfamily A member 1 (CYP7A1) and cytochrome P450 family 8 subfamily B member 1 (CYP8B1) gene expression and downregulated ATP binding cassette subfamily B member 11 (BSEP) gene expression in the liver, and taurine and emodin downregulated CYP7A1 and CYP8B1 gene expression (p < 0.05). In summary, AFB1 negatively affects health and alters the expression of genes related to liver bile acid metabolism, leading to cholestasis. Cholestyramine, atorvastatin calcium, taurine, and emodin can alleviate AFB1-induced cholestasis.

Transcriptome profile of sex-related gene expression and effects of cyp19a1 siRNA-silencing on sex differentiation and determination in yellow perch

In this study, we first sequenced the whole genome of the yellow perch (YP, Perca favencens) and screened transcripts of brain, liver, and gonadal tissues from fish of different sexes. By comparing known female sex-related genes in fish such as cyp19a1 and dmrt1 with YP genome, we obtained full-length sequences through genomic homology analysis. Then an experiment was conducted to evaluate the effects of cyp19a1-siRNA interference on gonadal development and sex differentiation and determination in YP. Transcriptome analysis revealed that the gonadal tissue of female YP exhibited predominantly and differentially up-regulated expression of genes than males, and cyp19a1 was predominantly expressed in the gonads while cyp19a2 showed predominant expression in the brain. KEGG pathway enrichment analysis illustrated the metabolic pathways of the top 25 enriched genes and confirmed CYP19 (P450) pathway in YP. For the cyp19a1-siRNA interference experiment, four intermittent in vivo injections of siRNA were administered to interfere with cyp19a1 gene expression in an all-female group of YP. The results collected at 180 dph revealed that the siRNA interference resulted in an incomplete masculinization and hermaphroditism of all-female monosex YP, displaying the presence of male spermatocytes along with female oocytes. In contrast, tissues from the blank control and negative control groups only contained oocytes. The appearance of the gonads in the experimental group appeared more irregular, lacking the smooth surface and typical histoarchitecture observed in the blank control and negative control groups. In addition, the cyp19a1-siRNA treatment group had significantly (P < 0.05) lower body weight than the control groups, indicating that the interference with cyp19a1 might have affected the expression of estrogen, leading to a trend similar to the growth of males; similarly, the gonad weight and GSIs of the cyp19a1 group were significantly (P < 0.05) lower than those in the control groups, suggesting that the cyp19a1-siRNA interference might have led to a trend of male type of gonadal development. Meanwhile, the treatment group had significantly (P < 0.05) higher HSI and VSI levels compared to control groups, indicating that stimulation of external siRNA might have resulted in more vigorous liver metabolism processes. Furthermore, the expression level of the dmrt1 in the treatment group was significantly (P < 0.05) upregulated compared to the control groups, suggesting the correlation roles between these two genes in the regulation of sex development. The findings provide valuable insights for future research into the underlying mechanisms involved and hold significant promise for sex control in aquaculture.

Regulatory Effect of L-Cysteine on Testosterone Biosynthesis Genes in Leydig Cells and THP-1 Monocytes

Background: Spermatogenesis involves a series of complicated cellular events resulting in the production of mature spermatids. Leydig cells of the testis are essential to forming a spermatozoon. Leydig cells are the interstitial cells that reside between seminiferous tubules and their key role is in the biosynthesis of testosterone. Leydig cells also regulate testicular macrophage and are stocked with innate defense systems. CYP11A1 is a major enzyme in the biosynthesis pathway of testosterone which converts the cholesterol to pregnenolone in the mitochondria of Leydig cells. L-Cysteine (L-Cys) acts as a precursor of glutathione, an antioxidant that plays a vital role against oxidative stress damage. Previous animal studies report the protective effect of L-Cys against chemical/radiation-induced-testicular damage. This study examined the biochemical mechanisms by which L-Cys stimulates testosterone biosynthesis and relative expression of blood-testis barrier (BTB) genes in Leydig cells and THP-1 monocytes. Methodology: The THP-1 monocytes and Leydig cells were treated with L-Cys for 24 hours. Following treatment, RNA was extracted, and the expression of the testosterone biosynthesis gene and BTB genes was analyzed using quantitative RT-PCR. Data (n=4) is presented as mean ± SEM and p-values ≤0.05 are considered as significant. Results: Expression of CLDN1 (p=0.006), CLDN11 (p=0.03), and TJP1 (p=0.04) genes was significantly higher in Leydig cells than in the THP-1 monocytes. There is no significant change in the expression of testosterone-regulatory genes between Leydig cells and THP-1 monocytes. In THP-1 monocytes, L-Cys treatment significantly upregulated the expression of CYP11A1 (p=0.03), CYP19A1 (p=0.002), and BTB genes: CLDN1 (p=0.05), CLDN2 (p=0.001), CLDN4 (p=0.0006), CLDN11 (p=0.006), and TJP1 (p=0.03). Similarly, L-Cys (500uM) treatment significantly upregulated (p=0.03) the CYP11A1 gene expression in HLC, further, the expression of BTB genes: CLDN1 (p=0.03), CLDN11 (p=0.02), and TJP1 (p=0.02) was also upregulated. Conclusion: This study shows that L-Cys increases the expression of testosterone-related genes in both HLC and THP-1. The expression profile of the BTB genes was higher in HLC than in THP-1 monocytes. L-Cys supplementation upregulates the expression of the testosterone biosynthesis pathway gene which can increase spermatogenesis and reduce the male infertility risk. Funding source: NIH/NCCIH (5R33AT010637) and the Malcolm Feist Endowed Chair in Diabetes. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Polycyclic aromatic hydrocarbon (PAH) activation of AhR inhibits glutathione S-transferase (GST) activity in primary human small airway epithelial cells (hSAECs)

Background: PAH represents the organic phase of particulate matter (PM) in air pollution emitted via the incomplete combustion of various compounds (i.e. diesel fuel, wood, and plastics). It is widely regarded that PAH activation of aryl hydrocarbon receptor (AhR) plays an important role in pro-inflammatory signaling, however the mechanisms remain unknown. In contrast- endogenous AhR agonists, such as 6-Formylindolo[3,2-b]carbazole (FICZ), exert pleiotropic protective effects against inflammation, fibrosis, and oxidative stress. The objective of our research program is to delineate the differences between activation of AhR by PAH (with adverse effects) compared to its beneficial homeostatic activation by endogenous FICZ. PM may induce oxidative stress in part through inhibition of antioxidant defense mechanisms. GSTs are a superfamily of enzymes that plays an important role in protecting cells from oxidative damage (by conjugating reduced glutathione to substrates under pro-oxidizing conditions). We hypothesize that PAH activation of AhR contributes to oxidative stress via inhibition of GST activity in primary hSAECs. Methods: Primary hSAECs were subjected to varying dosages of PAH [0, 250, 500, 1000, 5000 ng/mL] to model airborne PM exposure levels, and to increasing doses of FICZ [0, 0.50, 10, 100, 1000 nM] to model normal and excessively high endogenous ligand activation of AhR. We determined the relative potencies of PAH and FICZ by measuring CYP1A1 and CYP1B1 gene expression. Oxidative stress was measured using DHE-labeling and GST activity was measured with enzymatic kits (Sigma Aldrich). AhR activity was pharmacologically inhibited using 10μM CH223191. Statistical analysis was conducted using SigmaPlot 14.0 (Systat Software). Results: PAH and FICZ maximally stimulate hSAEC CYP1A1 and CYP1B1 expression at 1000ng/mL and 100nM, respectively, and are therefore defined as equipotent concentrations used in our studies. In a representative experiment in hSAECs, PAH (1000ng/mL) significantly increased hSAEC oxidative stress vs FICZ (100nM) n=6, p&lt;0.01), while significantly decreasing GST specific activity vs equipotent FICZ activation of AhR in primary hSAECs from a 66-year-old female without lung disease (26.6±0.874 vs 28.3±.611 nmol/mL/min); n=6 from 2 independent assays; p=0.032. Similar differential effects of PAH vs FICZ activation of AhR on GST were seen in additional experiments with male hSAECs and Calu-3 cells (male). Importantly, CH223191 inhibition of GST enzymatic activity suggests that AhR plays an important role in protecting cells from oxidative stress. Conclusions: Our studies indicate that airborne PAH exposure can have detrimental health effects on the lung via aberrant AhR signaling. Significance: Understanding activity of AhR signaling can leads to novel therapeutic options for redox disorders in the lung. Funding: VA 5I01BX001777-05 (RP) and R01HL137033 (MH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Advanced Glycation End-Products of Follicular Fluid are Associated with Embryo Morphokinetic Parameters and ART Outcomes.

Advanced glycation end products (AGEs) can disrupt antioxidant system and steroidogenesis, resulting in detrimental effects on assisted reproductive technology (ART) outcomes. This study aimed to investigate the association of AGEs in follicular fluid (FF) with morphokinetic parameters of embryos and ART outcomes. Fifty women undergoing ART treatment were studied. AGEs, glucose, 25(OH) vitamin D, malondialdehyde (MDA) levels and catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) activities were evaluated in FF. The expression of 3βHSD, CYP11A1, and CYP19A1 genes were analyzed in granulosa cells (GCs) by qRT-PCR technique. Morphokinetic parameters were evaluated using time-lapse technology. The FF level of AGEs was reversely associated with CAT, SOD, and GPX activities, and total and mature oocytes number, blastocyst formation rate, and high-grade embryos number, while it showed positive correlations with the FF MDA levels, the expression of steroidogenesis genes, number of immature oocytes, morphokinetic parameters, and number of low-grade embryos. Furthermore, the level of vitamin D in FF had an inverse association with AGEs and positive correlations with ART outcomes and morphokinetic parameters. Comparison between the those with positive and negative biochemical pregnancy showed no significant differences in terms of FF factors and just the expression of 3βHSD, CYP11A1, and CYP19A1 genes were higher in pregnant women (p < 0.05). AGEs could delay blastomere division and lead to an increase in the number of low-quality embryos, while vitamin D have an adverse effect on AGEs and a protective function against AGEs negative effects.

Co-treatment with melatonin and ortho-topolin riboside reduces cell viability by altering metabolic profiles in non-small cell lung cancer cells

Lung cancer is a highly prevalent and lethal malignancy worldwide, with non-small cell lung cancer (NSCLC) accounting for 85% of cancer-related deaths. In this study, the effects of co-treatment with melatonin and ortho-topolin riboside (oTR) on the cell viability and alteration of metabolites and transcripts were investigated in NSCLC cells using gas chromatography-mass spectrometry (GC-MS) and next-generation sequencing (NGS). The co-treatment of melatonin and oTR exhibited synergistic effects on the reduction of cell viability and alteration of metabolic and transcriptomic profiles in NSCLC cells. We observed that the co-treatment inhibited glycolytic function and mitochondria respiration, and downregulated glycine, serine and threonine metabolism alongside tyrosine metabolism in NSCLC cells. In the glycine, serine and threonine metabolism pathway, the co-treatment resulted in a significant 8.4-fold reduction in the expression level of the SDS gene, which encodes the enzyme responsible for the breakdown of serine to pyruvate. Moreover, co-treatment decreased the gene expression of TH, DDC, and CYP1A1 in tyrosine metabolism. Additionally, we observed that the co-treatment resulted in a significant 146.9-fold reduction in the expression of the DISC1 gene. The alteration in metabolites and transcript expressions might provide information to explain the cytotoxicity of co-treatment of melatonin and oTR in NSCLC cells. Our study presents insights into the synergistic anticancer effect of the co-treatment of melatonin and oTR, which could be a potential future therapeutic strategy for the treatment of NSCLC patients.