Oocyte quality and heat shock proteins in oocytes from bovine breeds adapted to the tropics under different conditions of environmental thermal stress.

In order to evaluate the influence of thermal stress on physiological parameters, and the oocyte quality of Girolando (n = 12) and adapted Pantaneira (n = 12) cattle, twelve sessions of ultrasound guided follicular aspiration (OPU) were performed, between January and November 2014 (during dry (May-September) and rainy season (October-April) in Brazil). The recovered cumulus-oocyte complexes (COCs) were selected and classified, according to quality, immediately after OPU. The oocytes were then stored in 3% paraformaldehyd before conducting immunofluorescence analysis under confocal microscopy to identify HSP70 and 90 proteins. Before each OPU session, the rectal temperature (RT) and respiratory frequency (RF) of each animal were measured. The black globe humidity index (BGHI) was calculated on the day of the OPUs and 90 days before each OPU session, and related to the thermal stress of the animals. The quality of oocytes from Girolando cattle, but not Pantaneira, showed a negative relationship with BGHI of 90 days before OPU. RT of both breeds did not exceed normal values for cattle below BGHI 95. BGHI variation on the day of OPU did not affect RF of the adapted Pantaneira breed (p = 0.3221). On the other hand, Girolando cattle showed a positive relationship between RF and BGHI (p = 0.0103). With increasing BGHI, the amount of HSP70 increased in Girolando oocytes, however, decreased in the Pantaneira breed. We have not observed a relationship between HSP 90 and BGHI, however Girolando cattle produced a greater amount of this protein in relation to the Pantaneira breed. In conclusion, higher BGHIs, 90 days before OPU session, negatively affect oocyte quality of Girolando cattle and positively affect oocyte quality of the Pantaneira breed. Higher BGHIs on the day of the OPU session negatively affected the respiratory frequency of the Girolando breed, and lead to a higher recruitment of HSP70 to protect oocyte maturation. The opposite pattern was observed for Pantaneira. In addition, Pantaneira cattle produced twice as much as HSP70 as Girolando cattle, suggesting that a natural higher production of this protein could be involved in the mechanisms of adaptation to heat conditions.

The margin of safety of a single application of transdermal fentanyl solution when administered at multiples of the therapeutic dose to laboratory dogs.

Previous studies have demonstrated that a single, topical application of a novel, long-acting transdermal fentanyl solution provides analgesic fentanyl concentrations for at least 4 days. The objective of this study was to describe the margin of safety following application at multiples of the therapeutic dose. Twenty-four laboratory dogs were administered a single placebo or 1×, 3×, or 5× multiple of the dose of 2.6 mg/kg (50 µL/kg) to the ventral abdominal skin and observed for 14 days. Plasma fentanyl concentrations increased in proportion to dose. Adverse reactions in the 1× group were transient and included a low prevalence (≤ 33%) of mild sedation, reduced food intake, modest weight loss, and minimal reductions in heart rate and rectal temperature. Moderate to severe sedation emerged in the 3× and 5× groups, which was associated with a dose-limiting reduction in food and water intake, necessitating maintenance fluid replacement for the first 2 days following application. Also observed in the higher-dose groups were an increased prevalence of abnormal stools and transient lens opacities. All abnormal health observations were completely resolved prior to necropsy on day 14, and there were no histological abnormalities identified. These data support the safe use of the 1× dose and describe the outcome of an overdose of up to 5× dose in the absence of opioid reversal.

The mycotoxins alternariol and alternariol methyl ether negatively affect progesterone synthesis in porcine granulosa cells in vitro.

Mycotoxins as contaminants of animal food can impair fertility in farm animals. In the regulation of female fertility the ovarian steroid hormone progesterone (P(4)) plays an important role. In the present study we have investigated the influence of the mycotoxins alternariol (AOH), alternariol mono-methyl ether (AME), and tenuazonic acid (TeA) on cell viability, P(4) synthesis, abundance of the key enzymes of P(4) synthesis, P450 cholesterol side-chain cleavage enzyme (P450SCC) and 3-beta-hydroxysteroid dehydrogenase (3-beta-HSD), and of the corresponding Cyp11a1 and Hsd3b transcripts in cultured pig granulosa cells. Already 0.8 microM, AOH and AME inhibited P(4) secretion and 1.6 microM also significantly reduced cell viability. The abundance of P450scc protein but not of Cyp11a1 or Hsd3b transcripts was already significantly reduced by 0.8 microM AOH and AME. 1.6 microM AOH but not AME significantly reduced the abundance of alpha-tubulin and also clearly affected actin protein concentrations. TeA neither impaired viability nor P(4) secretion. Also mycotoxin extracts isolated from naturally occurring Alternaria strains by HPLC purification inhibited cell viability and P(4) synthesis, however at higher concentrations compared to AOH and AME. In conclusion, AOH and AME, but not TeA specifically inhibited P(4) secretion in cultured porcine granulosa cells. Alternaria toxin contaminated food may therefore affect reproductive performance in pig and other mammalian species.

The effect of feeding a diet naturally contaminated with deoxynivalenol (DON) and zearalenone (ZON) on the spleen and liver of sow and fetus from day 35 to 70 of gestation.

Pregnant sows were fed a control diet (CON, 0.15 mg deoxynivalenol (DON) and 0.0035 mg zearalenone (ZON) per kg diet) or diet containing 15% of Fusarium toxin contaminated triticale (MYCO, 4.42 mg DON and 0.048 mg ZON per kg diet) during days 35-70 of gestation. All sows were fed in a restricted feeding regimen with the same amount of feed (2000 g/d) over the whole study. At the end of the experiment, fetuses were delivered by Caesarian section and samples of spleen and liver of euthanized sows and fetuses were analyzed. At terminal necropsy, no macroscopic lesion was observed in any organ of either sows or fetuses. The histopathological data indicated significant alteration only in elevated iron staining in the red pulp of spleens in sows of MYCO group after 35 days of feeding. The presence of hemosiderin particles in the spleen sections was confirmed by transmission electron microscopical investigation and by an enhanced Fe2+ concentration in spleen. A glycogen increase (p<0.05) was found in liver cells of fetuses in the experimental group. Together, the results provide evidence of spleen dysfunction (hemosiderosis) in sows fed a Fusarium toxin-contaminated wheat, however, with absence of clinical signs. Enhanced glycogen and an impairment of mitochondria in liver of fetuses was present when their mothers consumed the MYCO diet.

Changes in the spleen and liver of pregnant sows and full-term piglets after feeding diets naturally contaminated with deoxynivalenol and zearalenone.

Wheat contaminated naturally with the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) was fed to pregnant Landrace sows for 35days. On day 110, caesarean section was carried out, the offspring were killed immediately after birth, and their livers and spleens examined. At necropsy there were no macroscopic lesions observed in any organ of either sows or piglets. Histopathological evaluation of tissues from sows of the treated group revealed changes in liver and spleen tissues, whereas no significant changes were observed in these tissues in their piglets. Liver damage, as measured by prominent elevated transaminase activities, was not detected in the serum of the sows. In pregnant sows there were individual variations in sensitivity to the Fusarium toxins. In conclusion, it can be assumed that there are no adverse effects on the liver and spleen of full-term piglets when their mothers consumed diets containing up to 9570 and 358mug DON/ZON per kg diet.

Influence of diets with cereal grains contaminated by graded levels of two Fusarium toxins on selected enzymatic and histological parameters of liver in gilts.

Feeding experiments with diets containing Fusarium toxin-contaminated wheat were conducted to clarify the pathogenesis of enzymatic and histopathological effects of Fusarium toxins on porcine liver cells. A total of 36 prepuberal gilts were divided into four groups and fed diets with increasing proportions of Fusarium toxin-contaminated wheat at a total wheat proportion of 40% over a period of 35 days. The concentrations of the indicator toxins deoxynivalenol (DON) and zearalenone (ZON) which were analyzed by HPLC methods were 210/4, 3070/88, 6100/235, and 9570/358 microg/kg in the diets fed to groups I-IV, respectively. The feeding of mycotoxin-contaminated diets did not cause gross pathological findings in the livers of the animals. Liver tissues were subjected to enzymatic, histological, and ultrastructural examinations. The percentages of the stained areas in periodic acid-Schiff (PAS), Berlin-Blue, and Masson Goldner's trichrome stainings were calculated using the AnalySIS 3.4-system. Significant histopathological findings of alterations with varying degrees in glycogen reduction and increase of hemosiderin particles were found in the liver cells of groups II, III and IV. The thickness of interlobular connective tissue septum in liver cells was significantly increased in groups III and IV. Qualitative ultrastructural alterations were observed in hepatocytes of gilts in groups III and IV. Dependent upon the mycotoxin concentration in the diet, the hepatocytes developed a dose-dependent, extensive, smooth endoplasmic reticulum, exhibited loss of ribosomes, and acquired an increased number of fatty and autophagic vacuoles. However, liver damage as measured by prominent elevated transaminase activities in serum was not detected. Together, the histopathological results provide evidence of liver dysfunction in the absence of clinical signs, especially in pigs fed higher concentrations of Fusarium toxin-contaminated wheat.

Effects of diets with cereal grains contaminated by graded levels of two Fusarium toxins on selected immunological and histological measurements in the spleen of gilts.

Feeding experiments with diets containing Fusarium toxin-contaminated wheat were conducted to clarify the pathogenesis of immunological effects of Fusarium toxins to porcine spleen cells. Contaminated diets were fed to 36 Landrace prepubertal gilts for 35 d. Concentrations (as-fed basis) of the indicator toxins deoxynivalenol (DON) and zearalenone (ZON), respectively, were 210 and 4 (control--group I), 3,070 and 88 (group II), 6,100 and 235 (group III), and 9,570 and 358 microg/kg (group IV). No signs of hyperestrogenism or uterotrophic effects were observed because of dietary treatments. The feeding of mycotoxin-contaminated diets did not cause gross pathological findings in the spleens of animals. In vivo, no inhibitory effects were detected on concanavalin A-stimulation of blood lymphocytes; however, the proliferation rate of splenocytes was inhibited (P < 0.05) in pigs fed the diet with the highest DON/ZON concentration. With in vitro studies, lower proliferation rates of blood lymphocytes and splenocytes preexposed to DON were detected. Serum IgA concentrations of pigs in group II were increased (P < 0.05) compared with the baseline value before feeding the DON/ZON diet. The histopathological data indicated elevated (P < 0.05) iron staining in the red pulp of spleens in gilts from groups I to IV after 35 d of feeding. The presence of hemosiderin particles in the spleen sections was confirmed by transmission electron microscopic investigation. Together, the results provide evidence of spleen dysfunction (hemosiderosis) in the absence of clinical signs, especially in pigs fed higher concentrations (groups III and IV) of Fusarium toxin-contaminated wheat.

Influence of ovarian steroid hormones or platelet-activating factor on mRNA of platelet-activating factor receptor in endometrial explant perfusion cultures from ovariectomized bovine.

Platelet-activating factor (PAF) and its receptors are involved in inflammatory-like processes of the uterus associated with increased vascular permeability. PAF is supposed to be influenced by ovarian steroid hormones. The present study was undertaken to examine whether progesterone (P(4)), estradiol (E(2)) or PAF influence the PAF receptor gene expression in perfused endometrial explants derived from ovariectomized bovine. Furthermore, we identified the cell types in which the PAF receptor gene and protein are expressed. In endometrial explants, applications of 10 nM P(4) or 10nM P(4) plus 10 nM E(2) for 24 h induced elevated transcript levels of PAF receptor in comparison to the controls or after treatment with 1 nM E(2). When explants were administered 10 nM E(2), a slight decrease in the transcript level was recorded. After treatment of explants with PAF, no significant changes in PAF receptor mRNA expression was observed compared to the control group. We demonstrate that PAF receptor immunoreactivity and mRNA are detected mainly in the luminal epithelium, epithelial cells of the superficial glands and to a lesser degree in stroma. Levels of PAF receptor mRNA in bovine endometrial explants were correlated with PAF receptor protein localization assessed by immunohistochemistry. The regulation of PAF receptor by progesterone in bovine endometrial explants suggests that PAF is involved in the physiological process of reproduction.

Promoter-2-derived Cyp19 expression in bovine granulosa cells coincides with gene-specific DNA hypo-methylation.

Cyp19, the key gene of oestrogen biosynthesis, is expressed at very different concentrations and from different promoters in bovine granulosa cells (GCs) and in pregnant corpora lutea (CL), respectively. The present study was aimed to investigate if DNA methylation and thus epigenetic mechanisms might play a potential role in the regulation of Cyp19 expression and promoter-specific activity in GCs of cycling versus CL of pregnant cows. It was demonstrated that GCs express high concentrations of promoter-2-derived Cyp19 transcripts whereas CL samples isolated before and after implantation, and at the end of the first trimester, showed very low Cyp19 transcript concentrations, all of them derived from promoter 1.1. Two genomic regions including promoter 1.1 and promoter 2 were largely unmethylated in GCs but methylated in all CL samples. The data suggest that promoter-2-derived high-level expression but not promoter-1.1-derived low-level expression of Cyp19 might be controlled by cell-type-specific DNA methylation.

Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation.

In this study, we evaluated the distribution and oxidative activity of mitochondria in ex vivo pre-ovulatory porcine oocytes using the fluorescence probe MitoTracker CMTM Ros Orange. Cumulus-oocyte complexes (COCs) were classified according to cumulus morphology and time from hCG administration. The meiotic configuration of the oocytes and the degree of apoptosis in the surrounding cumulus cells were also evaluated. Estrus was synchronized in 45 crossbred Landrace gilts by feeding altrenogest for 15 days and administering 1000 IU PMSG on Day 16. The LH peak was simulated by treatment with 500 IU hCG, given 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before or 10, 22, or 34 h after hCG administration. Altogether 454 COCs were aspirated from follicles with a diameter of more than 5 mm. Cumulus morphology in the majority of COCs recovered 2 h before and 10 h after hCG was compact (60.4 and 52.7%, respectively; P<0.05). At 22 h after hCG, COC morphology changed significantly from 10 h dramatically: 74% of COCs had an expanded cumulus (P<0.01). At 34 h after hCG, 100% of recovered COCs had an expanded cumulus. The percentage of oocytes with a mature meiotic configuration differed among COC morphologies and increased as the interval after hCG administration increased (P<0.05). The type of mitochondrial distribution in the oocytes (n=336) changed from homogeneous to heterogeneous as the interval after hCG administration increased (P<0.01) and was associated with the cumulus morphology. Representative mitochondrial distributions were found as follows: -2 h: fine homogeneous in compact and dispersed COCs; 10 h: granulated homogeneous in compact and dispersed COCs; 22 h: granulated homogeneous in expanded COCs; and 34 h: granulated heterogeneous and clustered heterogeneous in expanded COCs (P<0.01). The oxidative activity of mitochondria measured by fluorescence intensity (Em: 570 nm) per oocyte after Mitotracker CMTM Ros Orange labeling increased in the oocyte as the post-hCG interval increased (P<0.01) and depended on the type of mitochondrial distribution. Lowest oxidative activity of mitochondria was found in oocytes with fine homogeneous distribution (253.1+/-9.4 microA). The oxidative activity increased (334.4+/-10.3 microA) in oocytes with granulated homogeneous distribution of mitochondria, and reached highest level in oocytes with granulated heterogeneous (400.9+/-13.0 microA) and clustered heterogeneous distributions (492.8+/-13.9 microA) (P<0.01). Mitochondrial activity in oocytes coincided with apoptosis in surrounding cumulus cells which increased in a time-dependent manner during pre-ovulatory maturation in vivo (P<0.01). These results indicate that there is a relationship between meiotic progression, cumulus expansion and mitochondrial redistribution and their oxidative activity during final pre-ovulatory maturation in pig oocytes. It appears that increased levels of mitochondrial activities in oocytes are correlated to increased levels of apoptosis in surrounding cumulus cells, in which mitochondria may play a role.

Forskolin-induced cyclic AMP signaling in single adherent bovine oviductal cells: effect of dichlorodiphenyltrichloroethane (DDT) and tris(4-chlorophenyl)methanol (TCPM).

The influence of tris(4-chlorophenyl)methanol (TCPM) and dichlorodiphenyltrichloroethane (o,p'DDT) on forskolin induced cAMP signalling in single adherent bovine oviductal cells was investigated. An increase in the intracellular cAMP levels was measured indirectly by an increase in the 520/580 nm fluorescence emission ratio of the protein kinase A fluorosensor (FICRhR). FICRhR was microinjected into single cells, and the 520/580 nm fluorescence emission ratio was monitored by image cytometry with an image analysis system as a measure of intracellular cAMP concentration ([cAMP](i)). Applications of dibutyryl cAMP and forskolin caused time- and dose-dependent effects on [cAMP](i) in single oviductal cells. The addition of 16 or 32 microM TCPM or DDT for 1 h to the culture medium decreased the intracellular cAMP concentration significantly, whereas 8 microM was not able to influence the [cAMP](i). In the presence of both pesticides at 16 microM the forskolin (30 microM)-induced [cAMP](i) was significantly reduced after 1 h of incubation. It is suggested that TCPM can have the same influence compared with DDT on cells responsible for reproduction.

Influence of tris(4-chlorophenyl)methanol (TCPM) on gap junction-mediated intercellular communication of cultured bovine granulosa cells.

Tris(4-chlorophenyl)methanol (TCPM) is a by-product in the manufacture of technical grade DDT, which is known to alter properties and functions of the female reproductive system. We investigated whether in vitro TCPM has an influence on the function of gap junction-mediated intercellular communication (GJIC) and gap junction protein expression of connexin 43 (Cx43) in cultured bovine granulosa cells. GJIC was assessed by fluorescent dye microinjection (dye-coupling). After a 1-h exposure to TCPM at a concentration of 32 microM, a significant (P<0.05) reduction in dye coupling occurred. The same result was obtained with o,p'-DDT. At a concentration of 32 microM both pesticides were cytotoxic as indicated by significant (P<0.05) increased propidium iodide staining of the cell nuclei. Little or no effect on the stainable pattern of connexons occurred after 1 h incubation time, while after 3 h treatment from 16 to 64 microM TCPM, a significant inhibition in the immunostaining resulted and the concentrations of 32 and 64 microM TCPM were cytotoxic for the granulosa cells. The freeze-fracture electron microscopy resulted in small differences in the morphology of gap junction plaques of cell cultures treated for 3 h with 8 or 16 microM TCPM in comparison to untreated cells. After treatment with 32 microM TCPM, gap junction plaques were very rarely detected and the lateral intramembraneous particles (IMP) distribution of many plasma membranes was strongly altered. Estimation of the cellular parameters may lead to an enhanced understanding of the mechanism of chemically induced toxicity by TCPM, that causes a general toxic effect on granulosa cells. We can conclude that TCPM is a toxic risk in the same manner as DDT.

Fluorometric detection of platelet activating factor receptor in cultured oviductal epithelial and stromal cells and endometrial stromal cells from bovine at different stages of the oestrous cycle and early pregnancy.

During the oestrous cycle and early pregnancy, the oviduct and uterus undergo a variety of morphological and physiological modifications in which the platelet activating factor receptor (PAF-R) plays an important role. PAF-R levels were quantified in bovine oviductal epithelial and stromal cells and endometrial stromal cells at days 2 to 4, 12, and 20 of the estrous cycle and during early pregnancy. Cells were grown in vitro and their intracellular PAF-R concentration was measured by flow cytometry using a polyclonal anti-PAF-R antibody system. A significant increase (P < 0.05) in the portion of PAF-R-positive oviductal epithelial and stromal cells was detected in both non-pregnant and pregnant cattle on days 2 to 4 in comparison to day 12 and 20. In endometrial stromal cells derived from day 20 pregnant bovine, a significant increase (P < 0.05) in PAF-R staining was observed in comparison to the day 20 non-pregnant and days 2 to 4 or 12 pregnant and non-pregnant animals. The PAF-R was detected in oviductal cells by using immunoblotting and immuno-gold postembedding method. Positive binding of the anti-PAF-R antibody was found on the cell membrane and in the cytoplasm. We concluded that the increased PAF-R concentration measured in cultured oviductal epithelial and stromal cells of cyclic and pregnant heifers on days 2 to 4 was hormonally regulated. The increased PAF-R in endometrial stromal cells on day 20 of pregnant heifers was a pregnancy-specific effect and may mediate a local increase in endometrial vascular permeability known to precede the implantation.

Platelet-activating factor (PAF)-like activity, localization of PAF receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) activity in bovine endometrium at different stages of the estrous cycle and early pregnancy.

PAF-like activity in the endometrium increased from days 2-4 to day 12 and day 20 in both cyclic and pregnant cows. There was an increase in platelet aggregation induced by PAF-like activity in the endometrium of pregnant animals on day 20 as compared to cyclic animals at the same point in time. Two major bands of PAF-R protein at 67 kDa and 97 kDa were detected by Western blot analysis. PAF-R was localized mainly in luminal and glandular epithelium of the endometrium, but the staining was markedly increased in the endometrium of pregnant cows on day 20 compared to cyclic animals on the same day. The purified PAF-AH from the endometrium is similar to in plasma. In cyclic cattle, no changes in PAF-AH activity of endometrium were observed, whereas a decrease in enzyme activity occurred in pregnant cows on day 20 as compared to cyclic animals on the same day. We suggest that the bovine endometrium produces PAF-like activity, expresses the PAF-R and possesses a PAF-AH activity which varies during pregnancy.

Influence of inhibitors on increase in intracellular free calcium and proliferation induced by platelet-activating factor in bovine oviductal cells.

Oviductal endosalpingeal cells were isolated mechanically from heifers and cultured until there was 100% confluency. The cells were loaded with the Ca(2+)-sensitive fluorochrome, fura-2/acetoxymethylester, and cytosolic free calcium ([Ca2+]i) was monitored by spectrofluorimetry. Platelet-activating factor, at a concentration of 30 nmol l-1, induced an intracellular Ca2+ increase in cultured bovine oviductal cells, mainly via influx from the extracellular space. In fura-2-loaded oviductal cells, different Ca2+ channel blockers were investigated to characterize the pathways responsible for the Ca2+ influx. The negative effects of Ni(2+)-, La(3+)-activated K+ channel blockers, such as apamin and charybdotoxin, and Ca2+ channel blockers, such as dotarizine, on the platelet-activating factor-induced [Ca2+]i increase indicate the minor participation of the voltage-gated Ca2+ channels. TMB-8 and flufenamic acid blocked the platelet-activating factor-induced Ca2+ increase directly on non-selective cationic channels or acted via a Ca2+ release-triggered Ca2+ influx. Platelet-activating factor, at concentrations of 1.25 mumol l-1 and 2.5 mumol l-1, significantly stimulated the proliferation and depolarization of oviductal cells, but 10 mumol l-1 significantly decreased both parameters and exerted a cytotoxic effect on cells. After incubation with TMB-8 or flufenamic acid, the cell proliferation was inhibited in a concentration-dependent manner, with IC50 values of 26.57 mumol l-1 and 95.29 mumol l-1, respectively. The depolarization was significantly inhibited at 50 mumol l-1 for both TMB-8 and flufenamic acid. The results of the present study may contribute to further understanding of the mechanism behind the actions of platelet-activating factor on oviductal cells.

Inhibitory effects of organochlorine pesticides on intercellular transfer of Lucifer Yellow in cultured bovine oviductal cells.

The present study investigated the effects of dichlorodiphenyltrichloroethane (DDT), methoxychlor (MXC), and gamma-hexachlorocyclohexane (gammaHCH, lindane) on gap junctional intercellular communication (GJIC) in cultured bovine oviductal cells. GJIC was evaluated by microinjecting fluorescent dye Lucifer Yellow and observing the inhibition of the spreading of dye into adjacent cells. After incubation for 1 h at 37 degrees C, a dose-dependent inhibition of GJIC was observed over a concentration range of 16 to 128 microM DDT, MXC, or gammaHCH compared with nonexposed controls. A significant inhibition began at 32 microM DDT, MXC, or gammaHCH. After incubation for 5 h, a dose-dependent inhibition of GJIC was obtained in the concentration range from 8 to 64 microM of the pesticides. The first significant inhibitory effect on GJIC was caused by 8 microM DDT, 16 microM MXC, and 32 microM gammaHCH. The 128 microM concentration of the pesticides was toxic. At pesticide concentration of 64 microM, the decrease in dye-coupling observed was not due to lethal cell injury, as is indicated by the use of trypan blue dye exclusion. After removal of 64 microM DDT from the culture medium, intercellular communication was reestablished within 3 h. Measurement of cytosolic free Ca2+ concentration [Ca2+]i in fura-2/AM-loaded oviductal cells showed that the inhibition of GJIC by addition of DDT, MXC, or gammaHCH was not associated with a detectable increase in [Ca2+]i. Coincubation of the DDT with dibutyryl-cAMP prevented the 64 microM DDT-induced inhibition of intercellular communication in adherent oviduct cells. It is suggested that organochlorine pesticides can influence cells responsible for reproduction.

Detection and functional characterisation of the transcription factor peroxisome proliferator-activated receptor gamma in lutein cells.

A prominent functional change during differentiation of lutein cells from follicular thecal and granulosa cells is an enhanced production and secretion of progestins. The regulation of this process is not fully understood but may be associated with the expression of transcription factors which activate genes, products of which are involved in pathways of the cholesterol and lipid metabolism. As peroxisome proliferator-activated receptors (PPARs) play a role in both pathways, we were interested in the expression of PPARgamma, a PPAR form which is involved in adipogenic differentiation. First, we were able to show the expression of PPARgamma in bovine lutein cells (day 12 of the ovarian cycle) at the mRNA and protein level by imaging, flow cytometry and blot analysis, and secondly a role of PPARgamma in the secretion of progesterone. The cells (24 h culture) responded dose dependently by increasing progesterone secretion (up to 1.5-fold of the basal level) to an endogenous ligand of PPARgamma, 15-deoxy-delta12,14 prostaglandin J2 (15-dPGJ2) and to the thiazolidinedione ciglitizone. Aurintricarboxylic acid (ATA) was found to reduce the intracellular PPARgamma level and to promote cell cycle progress, indicating that ATA can be used as a tool for experimental changes of PPARgamma proteins in intact cells and for studying the physiological consequences. The ATA-mediated decrease of PPARgamma was accompanied by reduced progesterone production and a progression of the cell cycle, suggesting a function of PPARgamma in both processes. The response to ATA was abrogated by a high dose (>490 nM) of 15-dPGJ2, suggesting that 15-dPGJ2 exerts its effect on steroidogenic activity via PPARgamma and that the 15-dPGJ2-PPARgamma system plays a role in the maintenance of a differentiated quiescent stage in lutein cells.

Influence of organochlorine pesticides on transmembrane potential, oxidative activity, and ATP-induced calcium release in cultured bovine oviductal cells.

The present study investigated the effects of the pesticides DDT, MXC, and gammaHCH on transmembrane potential, oxidative activity, cytotoxicity and ATP-induced intracellular Ca2+ release in cultured bovine oviductal cells. Transmembrane potential, oxidative activity, and cytotoxicity were assessed using the fluorescent dyes bis-oxonol, dihydrorhodamine 123, and propidium iodide (PI), respectively, and measured spectrofluorometrically in a microplate reader. The cultured cells were loaded with Ca2+-sensitive fluorochrome fura-2-AM, and cytosolic free calcium ([Ca2+]i) was monitored by a microscope image analysis system. A dose-dependent increase in depolarization and changes of oxidative activity were observed over a concentration range of 8 to 128 microM DDT and MXC compared to nonexposed controls. At a concentration of 16 microM DDT or MXC, the oxidative activity and depolarization of cells were significantly enhanced compared to controls, but most of the cells were intact as indicated by the fact that PI-staining was not significantly increased. Trypan-blue staining indicated that the viability of oviductal cells decreased significantly when exposed to concentrations of 64 and 128 microM DDT or MXC. ATP-mediated enhancement of [Ca2+]i in cells was almost completely inhibited after incubation with 128 microM DDT for 3 h at 37 degrees C. This response was reduced to approximately 50% after incubation of the cells with MXC at 128 microM; lindane did not significantly interfere with the above physiologic parameters.

Whole-body disposition and polyglutamate distribution of the GAR formyltransferase inhibitors LY309887 and lometrexol in mice: effect of low-folate diet.

PURPOSE: The whole-body autoradiographic distribution of two radiolabeled antifolate inhibitors of GAR formyltransferase, lometrexol and LY309887, were compared in tumor-bearing mice maintained on standard diet (SD) and a low-folate diet (LFD) in order to determine the total amounts of drug that accumulated in blood, tumor, liver and kidney. The time-dependent changes in tissue distribution were evaluated over a 7-day period in order to compare the pharmacokinetic properties of both inhibitors and to assess the influence of dietary folate on this distribution. In addition, the effect of dietary folate on polyglutamation of compound accumulating in the liver was measured. The results have bearing on the potential of these two clinical agents to produce delayed toxicity in cancer patients and the use of dietary folate to modulate or prevent the development of this toxicity. METHODS: Single equimolar i.v. doses of [14C]LY309887 and [14C]lometrexol were administered to C3H mammary tumor bearing mice on SD or LFD, and the disposition of these compounds was quantitated using whole-body autoradiography. Livers were also harvested and extracted for determination of polyglutamate distribution. Animals were sacrificed both early and late (7 days) after dosing to determine the long-term retention of these compounds. RESULTS: Whole-body autoradiography revealed that the highest concentrations of both compounds were in liver and kidney. Concentrations of both compounds were two-fold higher in livers from LFD mice than in livers from SD mice. Lometrexol concentrations in liver averaged 2.8- and 2.2-fold higher than LY309887 in SD and LFD livers, respectively. In SD livers, the polyglutamate profiles of both compounds were similar, with hexaglutamates being the longest chain species detected. In LFD livers, hexaglutamates of LY309887 were observed, while hepta- and octaglutamates of lometrexol were detected after 168 h. CONCLUSIONS: The reduced hepatic retention and biochemical profile of LY309887 compared to lometrexol suggest that it may be less likely to produce delayed cumulative toxicity while still retaining antitumor activity. However, the increased hepatic accumulation observed in LFD mice emphasizes the importance of assessing and supplementing folate in cancer patients treated with this class of compounds.

Postmortem serum and tissue redistribution of fluoxetine and norfluoxetine in dogs following oral administration of fluoxetine hydrochloride (Prozac).

Antemortem serum and postmortem serum and tissues were evaluated to determine if postmortem redistribution of the antidepressant, fluoxetine (Prozac) and its major active metabolite, norfluoxetine, occurred in dogs following oral administration of fluoxetine hydrochloride. Beagle dogs (four males) received daily oral doses of 10 mg fluoxetine/kg for five days. Antemortem serum concentrations of fluoxetine and norfluoxetine were determined 3 and 24 h following administration of the first four daily doses of fluoxetine and 3 h after the fifth dose in order to monitor for steady-state serum concentrations of parent and metabolite prior to postmortem serum concentration determinations. Antemortem serum concentrations of fluoxetine and norfluoxetine 3 h postdose on Day 5 ranged from 530 to 1210 ng/mL and 1460 to 1980 ng/ mL, respectively. Immediately following the 3 h blood sample on Day 5, each dog was euthanized. Serum concentrations of fluoxetine and norfluoxetine were determined from blood samples collected from the vena cava, heart, and clotted blood within the heart at 2 h after death in two dogs and 12 h after death in the remaining two dogs. Similarly, tissue concentrations of fluoxetine and norfluoxetine in heart, liver, and lung were determined 2 and 12 h postmortem. Serum concentrations of fluoxetine and norfluoxetine from the vena cava and heart 2 h postmortem were 2.2- to 6.0-fold and 2.3- to 3.6-fold higher, respectively, than antemortem serum concentrations. Similarly, serum concentrations of fluoxetine and norfluoxetine from vena cava and heart 12 h postmortem were 1.3- to 3.5-fold and 1.7- to 3.3-fold higher, respectively, than antemortem serum concentrations. However, 2-h and 12-h postmortem serum concentrations of fluoxetine and norfluoxetine from clotted blood within the heart were equal to or less than levels determined in antemortem serum. Results from 2-h and 12-h postmortem average tissue concentrations of fluoxetine and norfluoxetine in heart, liver, and lung demonstrated that fluoxetine and norfluoxetine concentrations in myocardium were similar 2 h and 12 h postmortem. However, in liver, fluoxetine concentrations decreased 39% and norfluoxetine concentrations decreased 23% from 2 h to 12 h postmortem. Even greater postmortem decreases in fluoxetine and norfluoxetine concentrations were observed in lung. Fluoxetine and norfluoxetine concentrations in lung decreased 49% and 39%, respectively, from 2 h to 12 h postmortem. In conclusion, this study demonstrated that fluoxetine and norfluoxetine undergo postmortem redistribution in the dog. Furthermore, postmortem serum concentrations appear to be dependent on the sample site and the degree of coagulation of the blood. Finally, postmortem decreases in concentrations of fluoxetine and norfluoxetine in liver and lung may, in part, explain the observed increase in serum concentrations at 2 and 12 h postmortem.