EB-treated Groups Research Articles

TREATMENT OF EXTRA-VIRGIN OLIVE OIL ON ETHANOL- AND BENZENE-INDUCED ACINAR CELLS DAMAGE IN ADULT WISTAR RATS

Objective: The study is focused on the effect ethanol and benzene on the acinar cells and extra- virgin olive oil (EVOO) as an ameliorative agent. Methods: Forty-eight Wistar rats were divided into eight groups of six animals per group: (1) Control group (a placebo of water), (2) EVOO, (3) 25% ethanol, (4) 200 mg/kg benzene, (5) 25% ethanol+200 mg/kg benzene (EB), (6) 25% ethanol+EVOO (EO), (7) 200 mg/kg benzene+EVOO (BO), and (8) 25% ethanol+200 mg/kg benzene+EVOO (EBO). The ethanol, benzene, and EVOO were administered orally. Results: All the treatment groups lost body weight except Groups 1 and 2. The histology of the pancreas showed significant (p≤0.05) damage to the acinar cells of groups exposed to 25% ethanol, 200 mg/kg benzene, and EB when compared to the control, EVOO, EO, BO, and EBO. The enzymatic activities of amylase and lipase evaluated showed significant (p≤0.05) decrease in EO and BO as compared to 25% ethanol, 200 mg/kg benzene, and EB-treated groups. Conclusion: There was an elevated glucose concentration in 25% ethanol and EBO as compared to control and EVOO has anti-inflammatory potential across the ameliorated groups.

Endometrial Histomorphometry of Anestrous Mares Under the Influence of Different Embryo Transfer Hormonal Protocols

Aiming to investigate the effects of different hormonal protocols on the endometrium morphometry of anestrous mares, 26 animals were assigned to four different treatment groups: (1) EB2.5LAP4: single dose of 2.5 mg of estradiol benzoate (EB); (2) EB5LAP4: 5 mg of EB in 2 consecutive days; (3) EB10LAP4: 10 mg of EB in three consecutive days, considering that all EB-treated groups received a single dose of 1,500 mg of long-acting progesterone (LA P4) after the single/last EB dose; and (4) LAP4: only 1,500 mg of LA P4. Results were also compared with those found in cyclic mares (control group). Endometrial biopsies were collected before and after the hormonal treatments in anestrous mares, and during estrus and at 5 days after ovulation in cyclic mares (D5). Samples were prepared for histological and histomorphometric analysis. Tissue sections were examined to determine luminal epithelium height (LEH), glandular epithelium height (GEH), endometrial thickness (ET), and glandular density (GD). Similar morphometric changes were observed after EB and P4 were administered to groups EB5LAP4 and EB10LAP4. Five days after LA P4 administration (D5), all the assessed variables were similar between all EB-treated groups. In addition, all variables of the EB-treated groups were similar to the control group on D5. Although most of the LAP4 group variables on D5 were similar to the EB-treated groups (except GD), reduced GD and GEH were found when compared with the control group, demonstrating the importance of estradiol priming before P4 on glandular activity and density.

Induction of spermatogenic cell apoptosis in prepubertal rat testes irrespective of testicular steroidogenesis: a possible estrogenic effect of di(n-butyl) phthalate

Although di(n-butyl) phthalate (DBP), a suspected endocrine disruptor, induces testicular atrophy in prepubertal male rats, whether it exerts estrogenic activity in vivo remains a matter of debate. In the present study, we explored the estrogenic potency of DBP using 3-week-old male rats, and then examined the relationship between estrogen-induced spermatogenic cell apoptosis and testicular steroidogenesis. Daily exposure to DBP for 7 days caused testicular atrophy due to loss of spermatogenic cells, whereas testicular steroidogenesis was almost the same with the control values. A single exposure of DBP decreased testicular steroidogenesis in addition to decreasing the level of serum LH at 3 h after DBP treatment, with an extremely high incidence of apoptotic spermatogenic cells at 6 h after administration. To elucidate the estrogenic activity of DBP, we carried out an inhibition study using pure antiestrogen ICI 182,780 (ICI) in a model of spermatogenic cell apoptosis induced by DBP or estradial-3-benzoate (EB). Although both the DBP- and EB-treated groups showed a significant increase in spermatogenic cell apoptosis, ICI pretreatment significantly decreased the number of apoptotic spermatogenic cells in these two groups. In contrast, testicular steroidogenesis and serum FSH were significantly reduced in all the treated groups, even in the DBP+ICI and EB+ICI groups. Taken together, these findings led us to conclude that estrogenic compounds such as DBP and EB induce spermatogenic cell apoptosis in prepubertal rats, probably by activating estrogen receptors in testis, and that reduction in testicular steroidogenic function induced by estrogenic compounds is not associated with spermatogenic cell apoptosis.

Genes Associated with Membrane-Initiated Signaling of Estrogen and Energy Homeostasis

During the reproductive cycle, fluctuations in circulating estrogens affect multiple homeostatic systems controlled by hypothalamic neurons. Two of these neuronal populations are arcuate proopiomelanocortin and neuropeptide Y neurons, which control energy homeostasis and feeding. Estradiol modulates these neurons either through the classical estrogen receptors (ERs) to control gene transcription or through a G protein-coupled receptor (mER) activating multiple signaling pathways. To differentiate between these two divergent ER-mediated mechanisms and their effects on homeostasis, female guinea pigs were ovariectomized and treated systemically with vehicle, estradiol benzoate (EB) or STX, a selective mER agonist, for 4 wk, starting 7 d after ovariectomy. Individual body weights were measured after each injection day for 28 d, at which time the animals were euthanized, and the arcuate nucleus was microdissected. As predicted, the body weight gain was significantly lower for EB-treated females after d 5 and for STX-treated females after d 12 compared with vehicle-treated females. Total arcuate RNA was extracted from all groups, but only the vehicle and STX-treated samples were prepared for gene microarray analysis using a custom guinea pig gene microarray. In the arcuate nucleus, 241 identified genes were significantly regulated by STX, several of which were confirmed by quantitative real-time PCR and compared with EB-treated groups. The lower weight gain of EB-treated and STX-treated females suggests that estradiol controls energy homeostasis through both ERalpha and mER-mediated mechanisms. Genes regulated by STX indicate that not only does it control neuronal excitability but also alters gene transcription via signal transduction cascades initiated from mER activation.

Effects of estradiol on gonadotrophin release, estrus and ovulation in CIDR-treated beef cattle

The effects of estradiol-17β (E-17β) or estradiol benzoate (EB) on gonadotrophin release, estrus and ovulation in beef cattle were evaluated in two experiments. In experiment 1, 16 ovariectomized cows received a previously used CIDR insert from days 0 to 7 and 1 mg of EB on day 8; they also received 5 mg of E-17β on days 0 or 1, or 5 mg of E-17β + 100 mg of progesterone on day 0. There was only an effect of time ( P < 0.0001) on plasma concentrations of progesterone, estradiol, FSH, and LH. Following treatment with E-17β, plasma FSH concentrations were suppressed for approximately 36 h, whereas plasma LH concentrations were reduced ( P < 0.05) for 6 h, but surged within 24 h. Injecting 1 mg of EB 24 h after CIDR removal decreased ( P < 0.02) plasma LH concentrations for 6 h, followed by an LH surge at 18 h. In experiment 2, ovary-intact heifers ( n = 40) received a used CIDR and 5 mg of E-17β + 100 mg of progesterone on day 0. On day 7, CIDR were removed, PGF given, and heifers received nothing (control) or 1 mg of EB 12, 24, or 36 h later. In these groups, plasma LH peaked (mean ± SEM) 78.0 ± 23.0, 37.8 ± 8.5, 44.4 ± 10.3, and 51.0 ± 5.1 h after CIDR removal (means, P < 0.001; variances, P < 0.001) and intervals from CIDR removal to ovulation were 102.0 ± 6.7, 63.6 ± 3.6, 81.6 ± 3.5, and 78.0 ± 4.1 h ( P < 0.05). The interval from CIDR removal to ovulation was shorter and less variable in EB-treated groups; the interval from EB to ovulation was shortest ( P < 0.05) in the 12-h group. In summary, E-17β or EB decreased both FSH and LH, but LH increased after 6 h (despite elevated progesterone concentrations). Following CIDR removal, 1 mg of EB effectively synchronized LH release, and ovulation (in intact cattle), but the interval from CIDR removal to EB treatment affected the time of ovulation.

Estrogen increases locomotor activity in mice through estrogen receptor alpha: specificity for the type of activity.

Estrogens are known to increase running wheel activity of rodents primarily by acting on the medial preoptic area (mPOA). The mechanisms of this estrogenic regulation of running wheel activity are not completely understood. In particular, little is known about the separate roles of two types of estrogen receptors, ERalpha and ERbeta, both of which are expressed in mPOA neurons. In the present study the effects of continuous estrogen treatment on running wheel activity were examined in male and female mice specifically lacking either the ERalpha (alphaERKO) or the ERbeta (betaERKO) gene. Mice were gonadectomized and 1 wk later implanted with either a low dose (16 ng/d) or a high dose (160 ng/d) of estradiol benzoate (EB) or with a placebo control pellet. Home cage running wheel activity was recorded for 9 d starting 10 d after EB implants. The same mice were also tested for open field activity before and after EB implants. In both female and male alphaERKO mice, running wheel activity was not different from that in corresponding wild-type (alphaWT) mice in placebo control groups. In both females and males it was increased by EB only in alphaWT, not alphaERKO, mice. In betaERKO mice, on the other hand, both doses of EB equally increased running wheel activity in both sexes just as they did in betaWT mice. Absolute numbers of daily revolutions of EB-treated groups, however, were significantly lower in betaERKO females compared with betaWT females. Before EB treatment, gonadectomized alphaERKO female were significantly less active than alphaWT mice in open field tests, whereas betaERKO females tended to be more active than betaWT mice. In male mice there were no effect of ERalpha or ERbeta gene knockout on open field activity. Unlike its effect on running wheel activity, EB treatment induced only a small increase in open field activity in female, but not male, mice. These findings indicate that 1) in both sexes estrogenic regulation of running wheel activity is primarily mediated through the ERalpha, not the ERbeta; and 2) hormone/genotype effects are specific to the type of locomotor activity (i.e. home cage running wheel activity and open field activity) measured.

Posthatch Oral Estrogen Exposure Impairs Adult Reproductive Performance of Zebra Finch in a Sex-Specific Manner

We determined whether short-term, posthatch oral exposure to estradiol benzoate (EB) or the industrial surfactant octylphenol (OP) could impair the reproductive performance of zebra finches. If so, naturally occurring phytoestrogens and xenoestrogens might influence reproduction in wild populations. Chicks were given oral administration of 10 or 100 nmol EB per gram of body mass (earlier work showed the latter to be the minimum oral dose required to maximally masculinize female song nuclei) or an equimolar amount of OP daily from 5 through 11 days of age. Canola oil was used as a vehicle and control. Reproductive testing was done either in individual pair cages or in communal cages that permitted self-selection of mates, N = 10 pairs per group. Pairs consisted of EB-treated males and females, EB-treated males paired with canola-treated females, vice versa, and canola-treated males and females. Posthatch EB treatment produced sex-specific impairments in reproduction that, in some instances, were additive when both sexes were treated. Egg production was reduced and egg breakage was increased in 100 nmol/g EB-treated male and female pairs. The incidence of missing eggs was increased in 10 nmol/g EB-treated male and female pairs. Candled fertility was reduced in both groups containing 100 nmol/g EB-treated males. The number of hatched chicks was severely reduced in all EB-treated groups. No adverse effects of OP treatment were detected. These significant treatment effects (all P < 0.05) show that posthatch EB treatment profoundly disrupts the reproductive performance of zebra finches, suggesting that exposure to estrogens in the wild could impair the reproductive performance of wild populations.

Ethylbenzene Induces Microsomal Oxygen Free Radical Generation: Antibody-Directed Characterization of the Responsible Cytochrome P450 Enzymes

Small aromatic hydrocarbons cause changes in oxidative metabolism by modulating the levels of cytochrome P450 enzymes, with the changes in these enzymes being responsible for qualitative changes in aromatic hydrocarbon metabolism. The goal of this study was to determine if exposure to the small alkylbenzene ethylbenzene (EB) leads to an increase in hepatic free radical production. Male F344 rats were treated with ip injections of EB (10 mmol/kg) and compared to corn oil controls. Hepatic free radical production was examined by measuring the conversion of 2′,7′-dichlorofluorescin diacetate (DCFH–DA) to its fluorescent product 2′,7′-dichlorofluorescein (DCF). A significant elevation of fluorescent DCF production was observed after treatment with EB, despite the lack of effect on overall cytochrome P450 levels. This process was shown to be inhibitable by metyrapone, an inhibitor of P450. DCF production was also inhibited by catalase, suggesting that hydrogen peroxide (H2O2) is one of the reactive oxygen intermediates involved in EB-mediated reactive oxygen species (ROS) formation. Interestingly, superoxide dismutase (SOD) did not inhibit DCF production in corn oil-treated rats but was an effective inhibitor in the EB-treated groups. In an effort to determine if the increase in ROS production was related to changes in specific P450 enzymes, DCF production was measured in the presence of anti-CYP2B, anti-CYP2C11, anti-CYP2E1, and anti-CYP3A2 inhibitory antibodies. Anti-CYP2B antibodies inhibited DCF production in EB-treated, but not corn oil groups, which is consistent with the low constitutive levels of this enzyme and its induction by EB. The data also demonstrate that CYP2B contributes to ROS production. Anti-CYP2C11 did not influence DCF production in either group. ROS formation in corn oil-treated rats as well as in ethylbenzene-treated rats was also inhibited with antibodies to anti-CYP2E1 and anti-CYP3A2. These results suggest that CYP2C11 does not appear to influence free radical production and that the increase in free radical production in EB treated rats is consistent with the EB-mediated elevation of CYP2B, CYP 2E1, and CYP3A2. Such alterations in free radical generation in response to hydrocarbon treatment may contribute to the toxicity of these compounds.

Effects of estradiol on aldosterone secretion in ovariectomized rats.

The effects and action mechanisms of estradiol on aldosterone secretion in female rats were studied. Replacement of estradiol benzoate (EB) increased the levels of plasma estradiol and aldosterone in ovariectomized (Ovx) rats. The aldosterone release from zona glomerulosa (ZG) cells was higher in EB-treated rats than in oil-treated animals. EB treatment potentiated the responses of aldosterone release to adrenocorticotropic hormone (ACTH), forskolin (FSK), and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Administration of EB in vivo did not alter cAMP production in response to ACTH or FSK. Although angiotensin II (Ang II) increased aldosterone secretion by rat ZG cells, the stimulatory effect of Ang II on the release of aldosterone was not altered by EB treatment. The conversions of [3H]-deoxycorticosterone to [3H]-corticosterone and [3H]-corticosterone to [3H]-aldosterone in EB-treated groups were greater than those in the oil-treated group. These results suggest that estradiol increases aldosterone secretion in part through the mechanisms involving the activation of the post-cAMP pathway, 11beta-hydroxylase and aldosterone synthase activity.

Ovarian steroid modulation of [ 3H]muscimol binding in the spinal cord of the rat

[ 3H]Muscimol binding was measured in the lumbar spinal cord of female rats by in vitro quantitative autoradiography. Ovariectomized rats were treated subcutaneously with either oil, estradiol benzoate (EB) or EB plus progesterone (P) in a regime known to reliably induce sexual receptivity. The level of [ 3H]muscimol binding was highest in laminae I–III and in the region around the central canal. Binding was lower in laminae IV–VI and was frequently undetectable in the ventral horn. There was a significant increase in the level of binding in laminae I–III after EB treatment. There was also a significant increase after treatment with EB+P in comparison to both the ovariectomized and EB-treated groups in this same region of the spinal cord.

Synaptic changes in the hypothalamus of the prepuberal female rat administered estrogen.

Subcutaneous administration of estradiol benzoate (EB) to prepuberal female rats can advance vaginal opening, phasic pituitary gland luteinizing hormone (LH) secretion, and ovulation, presumably through a neural mechanism. This study investigated whether these effects are associated with changes in synaptic profiles in the arcuate nucleus of the hypothalamus (ARC) and the preoptic area (POA). Twenty-five-day-old female rats were administered EB, EB followed by progesterone on day 27, or oil vehicle alone; or they received no treatment. Blood was collected by jugular venipuncture at 1600 hr, on day 27, and plasma was assayed for LH by radioimmunoassay. Rat brains were immediately perfused for electron microscopy, and the ARC and POA were dissected out. Tissue blocks from these areas were processed with phosphotungstic acid for selective staining of neural synapses. Serum LH was markedly elevated in the EB-treated rats compared with controls. In the treated groups, LH values in serum were above 1,000 ng/ml, whereas the control values were less than 50. This acute rise of serum LH was accompanied by an acute increase of synaptic volume percent, area density, and numerical density in the ARC of EB-treated rats. The numerical density of the control groups was approximately 800 million observed synapses per cubic millimeter, whereas in the EB-treated groups, there were approximately 1.8 billion synapses per cubic millimeter. We found no differences in synaptic profiles of the POA in EB-treated animals as compared to the controls. We conclude from this study that estrogens act through neural mechanisms to accelerate maturation of neuroendocrine processes that govern phasic pituitary gland LH release and that this maturation process entails synaptogenesis in the ARC.

Ovarian hormones influence brown adipose tissue

Adult female rats were ovariectomized (OVX) or sham-operated and 4–5 weeks later OVX groups were treated with estradiol benzoate (EB), progesterone, both hormones, or the oil vehicle. All rats were sacrificed on the 4th day of hormone following an overnight fast and a terminal meal. Interscapular brown adipose tissue (BAT) pads of EB-treated groups were heavier and contained more lipid than those of the other OVX groups. Lipid content of adipose tissue differed according to site (BAT < inguinal < parametrial = retroperitoneal), but only BAT exhibited differential responsiveness to hormonal treatments. There was also a trend for increased oxygen consumption by BAT from EB-treated rats. It is concluded that BAT may be involved in the process of increased energy expenditure by estrogen-treated rats.

Sexual behavior in male rats following intracerebral estrogen application.

The effects on sexual behavior and morphology of estradiol benzoate (EB) applied directly to the medial preoptic nucleus were assessed in three groups of male rats with varying sexual behavior potentials. High levels of lordotic behavior were observed in all EB-treated groups. Cholesterol was uniformly ineffective. Central EB failed to facilitate male copulatory behavior in any group. However, there was evidence that cannula implantation may have impaired the potential for male behavior. Significant reductions in testis and epididymis weights were evident in some but not all EB-treated males. It was concluded that EB can activate lordotic behavior consistently in male rats if applied in sufficient amounts to the medial preoptic region.