Short Culture of Bovine Hepatocytes Biopsied from Dairy Cows as a Model for Toxicological Studies-CYP 1A1 Activity Response to Zearalenone Treatment.

This study presents a simple and cost-effective method for isolating hepatocytes from liver biopsies obtained from healthy and ketotic dairy cows, which can be utilized for studying cellular metabolism, drug toxicity, and hepatocyte-specific gene function and regulation. The expression of hepatocyte marker genes (G6PC, ALB, CYP1A2) was measured and found to be highest at 6 h post-isolation, with a subsequent decrease over time. Cells isolated from ketotic livers exhibited lower expression levels than those from healthy livers. Furthermore, for the functional characterization of ketotic hepatocytes, the cells were exposed to varying doses of zearalenone (ZEA). While doses of 10-50 µM did not affect cell viability, the highest dose of ZEA (100 µM) significantly decreased cell viability, as measured using XTT assay. Additionally, the potential induction of cytochrome P450 A1 (CYP1A1) by ZEA was found. Despite limitations such as a short-term culture, this model provides a useful tool for conducting toxicological research.

Changes in Expression of the Genes for the Leptin Signaling in Hypothalamic-Pituitary Selected Areas and Endocrine Responses to Long-Term Manipulation in Body Weight and Resistin in Ewes.

Both long-term undernutrition and overnutrition disturb metabolic balance, which is mediated partially by the action of two adipokines, leptin and resistin (RSTN). In this study, we manipulated the diet of ewes to produce either a thin (lean) or fat (fat) body condition and investigated how RSTN affects endocrine and metabolic status under different leptin concentrations. Twenty ewes were distributed into four groups (n = 5): the lean and fat groups were administered with saline (Lean and Fat), while the Lean-R (Lean-Resistin treated) and Fat-R (Fat-Resistin treated) groups received recombinant bovine resistin. Plasma was assayed for LH, FSH, PRL, RSTN, leptin, GH, glucose, insulin, total cholesterol, nonesterified fatty acid (NEFA), high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol and triglycerides. Expression levels of a suppressor of cytokine signaling (SOCS-3) and the long form of the leptin receptor (LRb) were determined in selected brain regions, such as the anterior pituitary, hypothalamic arcuate nucleus, preoptic area and ventro- and dorsomedial nuclei. The results indicate long-term alterations in body weight affect RSTN-mediated effects on metabolic and reproductive hormones concentrations and the expression of leptin signaling components: LRb and SOCS-3. This may be an adaptive mechanism to long-term changes in adiposity during the state of long-day leptin resistance.

Nutrient Content of Different Wheat and Maize Varieties and Their Impact on Metabolizable Energy Content and Nitrogen Utilization by Broilers.

The study aimed to determine the effect of nutrients of wheat (nine cultivars) and maize (nine cultivars) grain on nitrogen balance and apparent metabolizable energy (AMEN) content for broiler chickens. In vivo digestibility and balance trials were carried out with 90 Ross 308 chickens (2 × 9 groups with 5 birds per group) aged from 42 to 49 days, separately for each cultivar. Considerable variation within each cereal species in fiber and non-fiber carbohydrate fractions and nutrient digestibility of grain were demonstrated. Additionally, regression equations were proposed which allow the estimation of AMEN content of wheat and maize grain varieties based on simple analytical procedures, including cell wall components, starch, and sugars. For practical purposes, these equations seem to be the best solution while reducing time, labor, and cost of analytical procedures.

Differences in the action of lower and higher chlorinated polychlorinated naphthalene (PCN) congeners on estrogen dependent breast cancer cell line viability and apoptosis, and its correlation with Ahr and CYP1A1 expression.

There are data showing that exposition to PCNs mixture increased incidence of gastrointestinal and respiratory neoplasms, but data regarding incidence of hormone-dependent cancer so far not shown. The objective was to determine if exposure to single lower and higher chlorinated PCN congeners is associated with altered proliferation and apoptosis of estrogen dependent breast cancer cells, and whether such effects are related to induction of AhR and CYP1A1 protein expression. MCF-7 cells were exposed to PCN 34, 39, 42, 46, 48, 52, 53, 54, 66, 67, 70, 71, 73 and 74 at concentrations of 100-10,000pg/ml. We evaluated the action of these PCN congeners on cell proliferation, DNA fragmentation and caspase-8,-9 activity. AhR and CYP1A1 protein expression and CYP1A1 activity was evaluated at a concentration of 1000pg/ml. An opposite action of tri- to tetraCNs than of penta-to heptaCNs on cell proliferation and apoptosis was evident. Tetra PCNs increased cell proliferation, but had no effect on DNA fragmentation nor caspase activity. Fast induction of CYP1A1 protein expression under the influence of lower chlorinated PCNs suggests faster metabolism and a possible stimulatory action of locally formed metabolites on cell proliferation. None of the higher chlorinated PCNs affected cell proliferation but all higher chlorinated PCNs increased caspase-8 activity, and hexa PCNs also increased caspase-9 activity. The rapid activation of the Ah receptor and CYP1A1 protein expression by higher chlorinated PCNs point to their toxicity; however, it is not sufficient for potential carcinogenicity. Action of lower chlorinated naphthalenes metabolites should be explored.

Halowax 1051 affects steroidogenesis by down-regulation of aryl hydrocarbon and estrogen receptors and up-regulation of androgen receptor in porcine ovarian follicles.

Polychlorinated naphthalenes (PCNs) are thought to interact with the aryl hydrocarbon receptor (AHR) and to have enzyme-inducing properties comparable to polychlorinated dibenzo-p-dioxins, therefore activation of steroid hormone receptors in endocrine tissues is also possible. The aim of the present study was to examine the effects of PCNs mixture, Halowax 1051 on gene and protein expression of receptors: estradiol (ERα/ß), androgen (AR) and AHRGene expression was evaluated by real-time PCR after 6 h of exposition and protein expression by Western blot after 24 h. Levels of sex steroids: androstenedione (A4), estradiol (E2) and testosterone (T) were measured by enzyme immunoassays. Results of the data show down-regulation of AHR gene expression after 6 h in parallel with an inhibition in AHR protein expression at doses 10 pg-10 ng/mL, down-regulation of ER at all doses used, and up-regulation of AR gene expression at doses 1 and 10 ng/mL without affecting their protein expression. To indicate the involvement of AHR, ERs and AR in the impact of PCNs on steroidogenesis, we used their specific blockers. Blocker of AHR reversed the inhibitory effect of Halowax 1051 on A4 secretion, and strengthened its effect on T secretion. Blockers of both ER and AR had no effect on Halowax 1051 action on steroids secretion. The results of this study suggest that AHR is involved in the effect of PCNs on steroidogenesis in the ovary. Additionally, we propose that cross-talk between AHR-ER and AHR-AR receptors mediates the effects of Halowax 1051 on ovarian follicles.

Bisphenol A induce ovarian cancer cell migration via the MAPK and PI3K/Akt signalling pathways.

Bisphenol A (BPA), is present in a multitude of products, including food and water containers, food can linings, dentistry sealants, and thermal paper. BPA can induce the growth of human ovarian cancer cell lines. Reduction of adhesion and the initiation of metastasis are important events in cancer progression; therefore, this study investigated the effects of BPA (0.1-100nM) on the migration of OVCAR-3 ovarian cancer cells and the expression levels of metalloproteinases (MMPs) and cadherins. The oestrogenic compound 17ß-estradiol (40nM) was used as a positive control for estrogenic properties of bisphenol A. BPA stimulated cell migration, and the effect of BPA was similar to that of 17ß-estradiol. BPA-induced cell migration was accompanied by up-regulation of the migration-related factors MMP-2, MMP-9 and N-cadherin, but E-cadherin expression and activity was unaffected. The stimulatory effects of BPA on cell migration were abolished by pre-treatment of the cells with inhibitors of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase pathways (PI3K). In conclusion, the results presented here show that BPA induces OVCAR-3 cells migration by activating MAPK and PI3K/Akt signalling pathways.

Effects of individual polychlorinated naphthalene (PCN) components of Halowax 1051 and two defined, artificial PCN mixtures on AHR and CYP1A1 protein expression, steroid secretion and expression of enzymes involved in steroidogenesis (CYP17, 17ß-HSD and CYP19) in porcine ovarian follicles.

In this study we tried to answer a question which component of Halowax 1051 is responsible for, observed in previously published study, androgenic effects of the mixture, and whether it is possible to draw conclusions about the action of mixtures by examining the effect of an indicator congener. Ovarian follicles were incubated with individual congeners of an artificial mixture for 6-24h. At the end of the incubation period, media were collected for determination of progesterone (P4), androstenedione (A4), testosterone (T) and estradiol (E2) levels by enzyme immunoassay, and follicles were retained for an examination of aryl hydrocarbon receptor (AHR), cytochrome p450 enzymes (CYP1A1, CYP17, CYP19), and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) protein expression by Western blotting. CN73 in dose 50pg/ml after 6h had no effect and decreased AHR expression after 24h, while at dose 400pg/ml increased AHR protein expression after 6h of exposure which remained elevated after 24h. CN74 and CN75 at both concentrations tested (25 and 50pg/ml) stimulated AHR protein expression after 6h and decreased it after 24h of exposure. Individual congeners induced a rapid increase in CYP1A1 protein expression, with a rank order of efficacy of CN73>CN74=CN75. All congeners increased P4/A4 and T/E2 secretion ratios in association with a decrease in the A4/T ratio, pointing to androgenic and anti-estrogenic properties of PCNs in ovarian follicles. The most potent congener in this context was CN73. The effects of mixtures were comparable to those of CN74 and CN75, and were not as strong as those observed for CN73. Collectively, these data suggest antagonistic actions of single congeners in a mixture, indicating that the actions of a mixture cannot be predicted based on the actions of individual congeners.

Action of Halowax 1051 on Enzymes of Phase I (CYP1A1) and Phase II (SULT1A and COMT) Metabolism in the Pig Ovary.

Polychlorinated naphthalenes (PCNs) are a group of organochlorinated compounds exhibiting dioxin-like properties. Previously published data showed the direct action of PCN-rich Halowax 1051 on ovarian follicular steroidogenesis. Taking into consideration that the observed biological effects of PCNs may be frequently side effects of metabolites generated by their detoxification, the aim of this study was to determine the activity and expression of enzymes involved in phase I (cytochrome P450, family 1 (CYP1A1)) and phase II (sulfotransferase (SULT1A) and catechol-O-methyltransferase (COMT)) detoxification metabolism. Cocultures of granulosa and theca interna cells collected from sexually mature pigs were exposed to 1 pg/mL to 10 ng/mL of Halowax 1051 for 1 to 48 hours, after which levels and activities of CYP1A1, SULT1A, and COMT were measured. Dose-dependent increases of CYP1A1 activity and expression were observed. High doses of Halowax 1051 were inhibitory to COMT and SULT1A activity and reduced their protein levels. In conclusion, fast activation of phase I enzymes with simultaneous inhibition of phase II enzymes indicates that the previously observed effect of Halowax 1051 on follicular steroidogenesis may partially result from metabolite action occurring locally in ovarian follicles.

The 2,2',4,4'-tetrabromodiphenyl ether hydroxylated metabolites 5-OH-BDE-47 and 6-OH-BDE-47 stimulate estradiol secretion in the ovary by activating aromatase expression.

The aim of the present study was to assess the effect of two hydroxylated BDE-47 metabolites, 5-OH-BDE-47 and 6-OH-BDE-47, on steroidogenesis in the ovary. Both metabolites failed to affect the production of androstenedione and testosterone but increased the secretion of estradiol at all concentrations tested. The increased secretion of estradiol was due to the stimulation of aromatase gene and protein expression. Direct assessment of aromatase activity by dibenzylfluorescein assay and indirect assessment of aromatase activity by measurement of the conversion of testosterone to estradiol confirmed that 5-OH-BDE-47 and 6-OH-BDE-47 stimulate aromatase activity. The aromatase inhibitor CGS 16949A abolished this stimulatory activity and reduced estradiol levels in the control and treatment groups.