Anna Nynca

Effects of Acute and Chronic Exposure to the Aryl Hydrocarbon Receptor Agonist 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the Transition to Reproductive Senescence in Female Sprague-Dawley Rats

Abstract Activation of the aryl hydrocarbon receptor (AHR) can occur in polluted environments, either from smoking-related toxicants or from endogenous ligands. We tested whether acute or chronic exposure to the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the transition to reproductive senescence in female Sprague-Dawley rats. In experiment 1, rats (n = 6 per experimental group) received a single dose of 0 or 10 μg/kg of TCDD orally (p.o.) on Postnatal Day 29. Vaginal cytology was monitored for 1 wk each month until rats were killed at 1 yr of age. The single prepubertal exposure to TCDD hastened the transition to reproductive senescence in female rats and was associated with delayed puberty, abnormal cyclicity, and premature reproductive senescence. In a second experiment, rats were exposed to TCDD chronically through weekly dosing (0, 50, or 200 ng kg−1 wk−1 p.o., n = 7 each dose) beginning in utero. Lifelong exposure to these lower doses of TCDD induced a dose- and time-dependent loss of normal cyclicity and significantly hastened the onset of the transition to reproductive senescence (P < 0.05). This premature transition to reproductive senescence was associated with prolonged estrous cycles and, at the highest dose of TCDD, persistent estrus or diestrus. The number and size of ovarian follicles were not altered by TCDD. Diestrous concentrations of LH in rats exposed chronically to TCDD were similar to those in controls, whereas progesterone tended to be elevated at both doses of the dioxin (P < 0.08). Serum FSH was elevated in the group exposed to 50 ng/kg of TCDD (P < 0.02), whereas estradiol was decreased at both doses of dioxin (P < 0.01). Data thus far support endocrine disruption rather than depletion of follicular reserves as a primary mechanism of the premature transition to reproductive senescence following activation of the AHR pathway by TCDD in female rats.

In vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on ovarian, pituitary, and pineal function in pigs

The aims of the study were: (1) to examine 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and/or prolactin (PRL) effects on in vitro secretion of progesterone (P(4)) and estradiol (E(2)) by luteinized granulosa and theca cells from porcine preovulatory follicles; and (2) to determine the effects of TCDD on PRL, luteinizing hormone (LH), and melatonin luteal phase in pigs. We found that TCDD itself did not affect progesterone secretion, but it abolished the stimulatory effect of PRL in the follicular cells. TCDD stimulated PRL secretion during the luteal phase and inhibited during the follicular phase. Moreover, TCDD increased luteinizing hormone secretion by pituitary cells during the follicular phase. In contrast to protein and steroid hormones, melatonin secretion in vitro was not affected by TCDD. In conclusion, it was found that the pituitary-ovarian axis in pigs is sensitive to TCDD, and the dioxin exhibited a profound ability to disrupt the ovarian actions of prolactin.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and phytoestrogen genistein on the activity and the presence of steroidogenic enzyme proteins in cultured granulosa cells of pigs

Environmental estrogens such as dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) and phytoestrogens (e.g. genistein; G) are known to influence endocrine and reproductive processes in humans and animals. Because living organisms are usually exposed to small, non toxic, doses of dioxins and phytoestrogens, the aims of the study were to determine the effects of small, environmentally relevant doses of TCDD (100pM) and/or genistein (500nM) on: (1) the activity of steroidogenic enzymes (cholesterol side-chain cleavage enzyme, P450scc; 3β-hydroxysteroid dehydrogenase, 3β-HSD and aromatase, P450arom) and (2) amount of protein of the enzymes in granulosa cells isolated from medium and large ovarian follicles of pigs. To determine the activity of the enzymes, the incubation medium was supplemented with specific steroid substrates (25-hydroxycholesterol; pregnenolone; testosterone) of particular steroidogenic enzymes (P450scc, 3β-HSD and P450arom, respectively). Subsequently, the production of progesterone (P450scc and 3β-HSD) or estradiol (P450arom) was compared in the presence and absence of the appropriate steroid precursor. Neither genistein nor genistein combined with TCDD affected activity of P450arom and relative amounts of steroidogenic enzyme proteins in the examined granulosa cells of pigs. In contrast, genistein alone and in combination with TCDD decreased P450scc and 3β-HSD activity as well as progesterone production in granulosa cells isolated from medium and large follicles of pigs. Because TCDD alone did not affect steroid hormone production or enzyme activity, the above effects should be ascribed solely to genistein. It appears that the effects of the examined doses of TCDD and genistein on granulosal cell functions were not additive.

DAIDZEIN AFFECTS STEROIDOGENESIS AND OESTROGEN RECEPTOR EXPRESSION IN MEDIUM OVARIAN FOLLICLES OF PIGS

Daidzein, a phytoestrogen present in soybean products used in swine feed, has been demonstrated to affect both reproductive and endocrine functions. The aims of this study were to examine the in vitro effects of daidzein on (1) progesterone (P4) and oestradiol (E2) secretion by porcine luteinised granulosa cells harvested from medium follicles, and (2) the mRNA and protein expression of oestrogen receptors α and β (ERα and ERβ) in these cells. The influence of E2 on P4 secretion and ERα and ERβ expression in the granulosa cells of pigs was also investigated. It was found that daidzein inhibited progesterone secretion by luteinised granulosa cells isolated from medium follicles. In contrast, E2 did not affect progesterone production by these cells. Moreover, daidzein did not alter the granulosal secretion of E2. Both daidzein and E2 decreased mRNA expression of ERα in the cells examined. The expression of ERβ mRNA was not affected by daidzein but was inhibited by E2. ERα protein was not detected while ERβ protein was found in the nuclei of the cells. Daidzein and E2 upregulated the expression of ERβ protein in the cells. In summary, the phytoestrogen daidzein directly affected the porcine ovary by inhibiting progesterone production and increasing ERβ protein expression. Daidzein-induced changes in follicular steroidogenesis and granulosal sensitivity to oestrogens may disturb reproductive processes in pigs.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters steroid secretion but does not affect cell viability and the incidence of apoptosis in porcine luteinised granulosa cells.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a by-product of human industrial activity, was found to affect ovarian steroidogenesis in animals, but the mechanism of its action is still unclear. The aims of the study were to examine the effect of TCDD on (1) progesterone (P4) and oestradiol (E2) production by granulosa cells isolated from medium (3-6 mm) and preovulatory (≥ 8 mm) porcine follicles, (2) the viability of the cells, and (3) the incidence of apoptosis. Porcine granulosa cells were cultured (48 h) with or without TCDD (100 pM, 100 nM). Steroid hormone concentrations in the medium were determined by radioimmunoassay. The viability of granulosa cells was tested spectrophotometrically (alamarBlue™ assay). Apoptosis was evaluated by flow cytometry using Annexin V and by TUNEL assay. The higher dose of TCDD (100 nM) significantly inhibited P4 and stimulated E2 production by luteinised granulosa cells isolated from medium follicles. The lower dose of TCDD (100 pM) significantly stimulated P4 and inhibited E2 secretion by the cells isolated from preovulatory follicles. None of the two TCDD doses affected cell viability or induced apoptosis in granulosa cells. In conclusion, TCDD directly affected steroid production by granulosa cells obtained from mature pigs, but the effect of TCDD was not due to its cytotoxicity.

Biochanin A affects steroidogenesis and estrogen receptor-β expressionin porcine granulosa cells

Biochanin A, similar to other isoflavones, is present in soy and soy-based food, but predominantly in red clover. Red clover extract and biochanin A were reported to affect reproductive processes as well as to demonstrate menopause relief and anticancerogenic properties. Because porcine granulosa cells provide a suitable in vitro model for studying the intracellular mechanism of phytoestrogen action in the ovary, the objective of the study was to evaluate the in vitro effects of biochanin A on the following: (1) progesterone (P4) and estradiol (E2) secretion by granulosa cells, (2) viability of the granulosa cells, and (3) mRNA and protein expression of estrogen receptors α (ERα) and β (ERβ) in the granulosa cells harvested from both medium (3-6 mm) and large (≥8 mm) porcine ovarian follicles. RIA, alamarBlue assay, reverse transcriptase-polymerase chain reaction, and immunocytochemistry were used in the study to address the objectives. Biochanin A significantly inhibited P4 and did not affect E2 secretion by porcine granulosa cells regardless of the size of follicles that served as the source of the cells. Cell viability was not affected by the treatment. Biochanin A did not alter ERα and ERβ mRNA levels in the cultured porcine granulosa cells. In contrast, this isoflavone increased (P < 0.05) the immunoexpression of ERβ in the cells from both follicle types. In summary, biochanin A, similar to genistein and daidzein, affects follicular steroidogenesis and ER expression. Its effect on ERβ protein was more intense compared with other previously examined phytoestrogens.

Ploidy-dependent survival of progeny arising from crosses between natural allotriploid Cobitis females and diploid C. taenia males (Pisces, Cobitidae)

Crosses between 21 triploid hybrid Cobitis females and 19 C. taenia (2n = 48) males led to viable progeny; whereas no embryonic development was observed in crosses with tetraploid males (4n = 98). The ploidy status of 491 progenies randomly selected with flow cytometry (316) or chromosome analysis (175) revealed an average of 55.2 % triploids and 44.8 % tetraploids, but the ratio of 3n versus 4n fish did change during development. In the first 2 days after hatching, approximately 65.1 % of tetraploid larvae were observed. Their number decreased significantly to 30.8 and 6.2 % on average during 2–5 and 10–15 months of life, respectively. The karyotype of tetraploid progeny (4n = 98) included 3n = 74 chromosomes of the parental female and n = 24 of C. taenia male. The number of tetraploid progeny indicated indirectly that about 66 % of eggs from 3n females were fertilized with C. taenia. The rest of the eggs developed clonally via gynogenesis or hemiclonally via hybridogenesis into triploids of the same karyotype structure as parental females. We have documented for the first time that (at least under experimental conditions) tetraploids are commonly formed, but are less viable than triploids, and a ratio similar to what is found under natural conditions is finally attained. The current explanation concerning the ploidy and karyotype structure of the progeny confirms that the eggs of 3n Cobitis females are not only capable of maintaining all chromosomes but are also capable of incorporating the sperm genome, thus creating the potential to produce tetraploids.

Utilization of physiological and taxonomic fluorescent probes to study Lactobacilli cells and response to pH challenge

pH stress is recognized as an important feature for Lactobacillus in relation to lifestyle and commercial utility. Hence, this study aims to investigate the cell function of Lactobacilli cells subjected to pHs between 7.0 and 2.0. For this purpose, the Lactobacilli isolates of vegetable origin were first hybridized with fluorescent oligonucleotide rRNA probes for detecting Lactobacillus species. Then, cells were exposed to pH stress and labelled with fluorescent probes, carboxyfluorescein diacetate (CFDA) and propidium iodine (PI), which provided the insight into esterase activity and membrane integrity of cells. Among isolates, fluorescence in situ hybridization (FISH) enabled us to specifically detect L. plantarum and L. brevis. Interestingly, FCM analysis revealed that at pHs between 7.0 and 4.0 the cell membrane was intact, while after the exposure at pH 3.0, and 2.0 became perturbed or impaired. Finally, L. brevis and L. plantarum differed from each other in fluorescence labeling behaviour and culturability. However, the results showed that the same standard protocol for labeling enables discrimination of subpopulations of tested species. Depending on the species, the substantial culturability loss was observed at pH 3.0 and 2.0. These results suggest that the taxonomic and physiological fluorescent probes could be suitable for in situ detection of specific bacteria and rapid assessment of the physiological status of cells.

Structural-functional adaptations of porcine CYP1A1 to metabolize polychlorinated dibenzo-p-dioxins.

Polychlorinated dibenzo-p-dioxins (PCDDs) are widespread by-products of human industrial activity. They accumulate in tissues of animals and humans, exerting numerous adverse effects on different systems. In living organisms, dioxins are metabolized by enzymes of the cytochrome P450 family, including CYP1A1. Particular dioxin congeners differ in their toxicity level and ability to undergo biodegradation. Since the molecular mechanisms underlying dioxin susceptibility or resistance to biodegradation are unknown, in the present study the molecular interactions between five selected dioxins and porcine CYP1A1 protein were investigated. It was found that the ability of a dioxin to undergo CYP1A1-mediated degradation is associated mainly with the number and position of chlorine atoms in the dioxin molecule. Among all examined congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) demonstrated the highest affinity to CYP1A1 and, at the same time, the greatest distance to the active site of the enzyme. Interestingly, in contrast to other dioxins, the binding of the TCDD molecule to the porcine CYP1A1 active site resulted in a rapid and continuous closure of substrate channels. All the information may help to explain the extended half-life of TCDD in living organisms as well as its high toxicity.

The combined effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and the phytoestrogen genistein on steroid hormone secretion, AhR and ERβ expression and the incidence of apoptosis in granulosa cells of medium porcine follicles

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P4) and estradiol (E2) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor β (ERβ) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P4 or E2 production, but potentiated the inhibitory effect of genistein on P4 production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERβ and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.