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The effect of mammalian steroid hormones (estradiol, estrone, progesterone, and testosterone) in environmentally relevant concentrations on a soil free-living nematode community was examined. Steroids were applied in 2.5 L water on 0.25 m 2 plots in triplicate, and comparison was made with plots receiving water alone. Soil samples were taken from the 0 to 5, 5 to 10, and 10 to 20-cm soil layers. The soil free-living nematode populations were examined at zero time, 12 h, 72 h, 96 h, 120 h, and 25 d post-treatment. Testosterone, progesterone, and estrone were persistent in the soil while the estradiol rapidly dissipated. The total number of nematodes was decreased by the presence of testosterone, progesterone, or estrone but not estradiol. Testosterone, progesterone, and estrone increased the number of males in relationship to females (60:40) compared to the control and estradiol-treated plots (50:50). The presence of steroids had no consistent effect on the distribution of bacteria-feeders, plant-feeders, and omnivore-predators. We conclude that the addition of steroid hormones in the soil can reduce abundance and change the sex ratio in a free-living nematode community. This would be the first demonstration of an effect of an endocrine-active agent excreted by mammals on a free-moving terrestrial organism.

Natural (estradiol, estrone, testosterone, estriol) and synthetic hormones (ethinylestradiol) are constantly excreted into the environment from human and animal sources but little is known of their transport. The purpose of this study was to determine how far along a 100 km river course that hormones could be detected after contamination with sewage effluent or fishpond effluent. Fourteen sites in the Lower Jordan River drainage were sampled (two sites above the sewage effluent contamination, eight sites below the contamination and four tributaries) before and after the dry season of 2002 (Spring and Fall). Samples were tested for testosterone, estrogen (estrone and estradiol combined), estriol, ethinylestradiol, ammonia and fecal coli. It was found that the fecal coli count dropped exponentially (from 250,000 to 60/100 ml3) and the ammonia dropped from 15 to less than 1mg/l over the initial 25 km stretch. Over the same stretch, the hormone values declined by half from their maximum values for testosterone (3.3 ng/l), estriol (8.8 ng/l), ethinylestradiol (6.1 ng/l), and estrogen (4.9 ng/l). From 67 to 100 km mark, testosterone (4.8 ng/l) and estrogen (2.4 ng/l) were still elevated while ethinylestradiol and estriol were >or=1.5 ng/l. The high level of testosterone and estrogen between 67 and 100 km marks was probably due to major discharge from fishponds between 23 and 27 km marks. Levels of ethinylestradiol above 1 ng/l, a level which can affect fish, was seen in 70% (12/16) of the samples tested. The data suggest that hormones in readily measured quantities can be transported considerable distances from the source of pollution.

Testosterone is constantly excreted into the environment by both human and animal sources but little is known about how it is transported in the environment. In this study, testosterone was measured in 15 sites in the Upper Jordan Valley after major rain events (238 samplings) for two consecutive rain seasons. The area consists of small farms, cattle pasture, fish ponds with some urban development. One liter samples were extracted on solid phase columns and the eluates measured using specific radioimmunoassay for testosterone and estrogen (estradiol+estrone). The first rain season was the first above average season after a 3-year period of well below average rainfall. It was found in the rain season of 2001/2002, that following a rain sequence of 131 mm/week there was an initial large increase in the concentration of testosterone (maximum 6 ng/l) accompanied by high estrogen (maximum 6 ng/l), which then gradually declined to non-detectable levels (

The present study was conducted to utilize a double-stranded RNA (dsRNA) specific for cyclooxygenase (COX) II and demonstrate inhibition of the expression of COX II protein and its product PGE. A 21-dsRNA specific for COX II was introduced by lipofectamine into a primary cell culture of bovine aortic coronary endothelial cells (BAECs). BAECs basally express COX I but not COX II, and COX II expression is only apparent after stimulation with phorbol 12-myristate acetate (PMA). We first demonstrated that the lipofected fluorescent dsRNA-COX II is accumulated and localized within the cultured cells. We then demonstrated gene silencing of PMA-induced COX II protein expression by dsRNA-COX II using immuno-histochemistry. Western blot analysis and radioimmunoassay were used to quantitate the percent of inhibition. It was found that lipofected dsRNA-COX II reduced the percent of PMA-induced COX II enzyme by 36% and PGE production by 40%. There was no demonstrable effect of dsRNA-COX II or PMA on COX I expression.

Steroidal hormones produced by humans and animals are constantly excreted into the environment in their active forms. The primary steroid hormones are progesterone, es- trone, estradiol, testosterone, and cortisol, all of which are lipophilic and poorly soluble in water. The steroids of major concern are estrone and estradiol-17β, since they exert their physiological effects at a lower concentration than other steroids and can be found in the en- vironment in concentrations above their lowest observable effect level for fish and plants (10 ng/l). The steroid hormones can be readily measured in run-off, soil, and groundwater, but each steroid has its distinct pathway of transport. Since the major source of steroids in the environment appears to be cattle and chickens, the hormonal steroid input into the envi- ronment could be drastically reduced by well-established techniques such as buffer strips and composting.

We have previously reported that bovine endometrium contains LH/human CG binding receptors and LH induces cyclooxygenase and prostaglandin production in the bovine endometrium. The present study investigated 1) whether bovine uterine vein and artery contain LH receptor messenger RNA (mRNA) and receptor protein and 2) whether LH can regulate the formation of vasoactive eicosanoids by the uterine vein. The uterine vein endothelium, but not the uterine artery, contained LH receptor mRNA transcript essentially identical to that found in the bovine corpus luteum. The uterine vein endothelium also contained a 95-kDa immunoreactive receptor protein that bound to rat anti-LH receptor antibody in Western blots. The LH receptor mRNA and LH receptor were maximally expressed in the uterine vein from cows in proestrus/estrus compared with cows in luteal or postovulatory phases. Incubation of endothelial minces of uterine vein with LH resulted in a 2-fold increase in cyclooxygenase concentration as determined by Western blot using an antibody to ram seminal vesicle cyclooxygenase. The increase in cyclooxygenase was maximal in cows in proestrus/estrus compared with postovulatory and luteal phase cows. Incubation of proestrous/estrous uterine vein or artery minces with LH or mellitin (a phospholipase A2 stimulator) caused increased production of eicosanoids. In the uterine vein, LH caused a significant increase in both PGF2alpha (basal 4.1 +/- 0.4 vs. 5.7 +/- 0.4 ng/100 mg x 6 h, P < 0.01; N = 9 cows) and PGE2 (basal 5.7 +/- 0.3 vs. 7.7 +/- 0.8 ng/100 mg x 6 h, P < 0.01; N = 6 cows) but had no effect on prostaglandin production by the artery. Mellitin increased PGF2alpha production by both uterine vein and artery minces but had no effect on PGE2 production in either tissue. Addition of steroids (progesterone, estradiol) or cytokines (tumor necrosis factor-alpha, IL-6) to the uterine vascular tissues had essentially no effect on prostanoid production. In summary, bovine uterine vein from proestrous/estrous cows expressed the LH receptor and its mRNA. Expression of the receptor may have physiological significance as LH induces cyclooxygenase and increases prostaglandin release in the uterine vein. The maximal stimulation of the receptor and its mRNA at proestrus/ estrus may serve to increase the amounts of prostanoids reaching the regressing corpus luteum either directly by increasing prostanoid production or indirectly by increasing the blood flow to the ovary.

The phytoestrogenic activity of alfalfa irrigated with sewage water was determined by radioreceptor assay (RRA) and high pressure liquid chromatography (HPLC). It was found that irrigation of alfalfa fields increased the phytoestrogenic activity measured in the leaves from 10 ng estrogen equivalents (est. eqv.) to 137 ng est. eqv. g−1, which was principally due to a rise in coumestrol (

Progesterone (P 4 ) production by the bovine placenta differs from that of other steroidogenic tissue in two important respects: 1) it is calcium-dependent but cyclic nucleotide-independent and 2) it is suppressed by an endogenous inhibitor for most of the life span of the placenta. This natural refractory state of the placenta can be overcome in in vitro incubations of fetal cotyledon cells by agents that increase intracellular calcium (3-isobutylmethylxanthine [MIX], calcium ionophore (A23187)), addition of substrate (pregnenolone, hydroxycholesterol), and stimulators of protein kinase C (PKC) such as phorbol ester (TPA). We therefore tested, in cultures of cotyledonary cells, two compounds that have been reported to inhibit protein kinases: 1) staurosporine (STA), an inhibitor of PKC, cAMP-dependent kinase, tyrosine kinase (TK), and the epidermal growth factor (EGF) receptor TK, and 2) genistein, an inhibitor of TK It was found that STA stimulated steroidogenesis in a dose-dependent manner in both the absence and presence of added calcium. STA (10 -9 M) stimulated at least a twofold increase in P 4 production by cultured fetal cotyledon cells throughout the first half of gestation (50-130 days). EGF was also found to cause a twofold stimulation of P 4 production, and the effect was additive to that of STA. Both basal and EGF- or STA-stimulated production were inhibited by genistein. In contrast, two inhibitors of PKC and PKA (H-7, H-8) had no effect on P 4 production. We conclude that STA-induced steroidogenesis in the bovine placenta is not related to its reported ability to inhibit PKC, TK, or EGF receptor TK

In the last decade interest has increased in the possible effects of endocrine disruptor compounds. Numerous papers have appeared as to their possible effects on humans, but definitive effects are hard to demonstrate. In the field of animal husbandry, however, the effects of the endocrine disruptors are well documented. This paper discussed the effects of the endocrine disruptors seen in animals and the possible implications for humans. The areas considered are reproductive disorders, premature udder/breast develop.nent, prolapsed oviduct/uterus, scrotal atrophy, and skewed sex ratio.

Compounds with estrogenic activity are ubiquitous in nature. Phytoestrogens and steroidal estrogens are found naturally in the food, in particular legumes and milk products. Naturally occurring steroidal estrogens and synthetic estrogens such as ethinylestradiol are constantly excreted into the environment as the result of the release of animal and human waste. As the result of this constant exposure, testosterone, estrogens, and ethinylestradiol are readily detectable in every stream examined in Israel. The concentrations observed of the estrogenic compounds are physiological, i.e., at these concentrations fish reproductive function can be affected. Estrogenic compounds do not usually reach the groundwater but testosterone percolates through the soil and is frequently found in the groundwater. In the dry season, there are no natural or synthetic steroids in the streams of the Jordan River watershed. However, at the beginning of the rainy season, both estrogen and testosterone reach the surface waters in the runoff from fields with manure, whereas ethinylestradiol reaches the streams in overflows from oxygenation ponds used to treat sewage prior to irrigation.

Effluents containing pharmaceuticals and hormones from CAFOs are of concern because they are directly applied to the soil without any pre-treatment. To evaluate the potential affect of these compounds, an understanding of soil ecology is required. Soil ecology is a daunting topic as there is so much soil mass and diversity in the world. However, some parameters are consistent for the study of the factors of soil ecology, like nematode numbers and bacterial/fungal biomass. The biomass can by characterized by using arrays of distinct substrates used or amounts of specific chemicals produced, e.g. ergosterol is produced primarily by fungi.

Although concentrated animal feeding operations constantly generate physiologically active steroidal hormones, little is known of their environmental fate. Estrogen and testosterone concentrations in groundwater and their distribution in sediments below a dairy-farm wastewater lagoon were therefore determined and compared to a reference site located upgradient of the farm. Forward simulations of flow as well as estrogen and testosterone transport were conducted based on data from the sediment profile obtained during drilling of a monitoring well belowthe dairy-farm waste lagoon. Testosterone and estrogen were detected in sediments to depths of 45 and 32 m, respectively. Groundwater samples were directly impacted by the dairy farm, as evidenced by elevated concentrations of nitrate, chloride, testosterone, and estrogen as compared to the reference site. Modeling potential transport of hormones in the vadose zone via advection, dispersion, and sorption could not explain the depths at which estrogen and testosterone were found, suggesting that other transport mechanisms influence hormone transport under field conditions. These mechanisms may involve interactions between hormones and manure as well as preferential flow paths, leading to enhanced transport rates. These types of interactions should be further investigated to understand the processes regulating hormone transport in the subsurface environment and parametrized to forecast long-term fate and transport of steroidal hormones.

Ram and bull seminal plasma, respectively, contain 0.5-20 microg PGE/ml and 5-10 ng PGE/ml. To demonstrate that PGE concentrations in the seminal plasma are related to sperm quality and could be affected by hormonal stimulation in vivo, four rams were injected with 500 IU hCG, in and out of season. The rams responded 1 week after hCG with a 1.5- to 4-fold increase in seminal plasma PGE. The PGE peak was temporally separate from the hCG-induced rise in seminal plasma testosterone which was observed after 1 day. Using a simulated cryptochid ram, peaks in seminal fluid PGE were found to be associated with increased sperm velocity and sperm counts. In bulls, PGE concentrations in the seminal plasma of good bulls were significantly higher than that found in poor and cryptorchid bulls.

Bovine myometrium and cervix contain luteinizing hormone/human chorionic gonadotropin (LH/hCG) binding sites, LH receptor (LH-R) messenger RNA (mRNA), and LH-R protein. Expression of LH-R is dependent on the stage of the cycle. LH-R expression is high during the luteal phase but weak during the follicular phase. In both myometrium and cervix, LH activates both the adenylate cyclase and phospholipase C pathways, and the effect of LH on both pathways at each stage of the cycle is correlated with the amount of LH-R present in the tissue. Because activation of cyclic AMP (cAMP) is associated with myometrial quiescence, we suggest that LH activation of uterine cAMP could serve to keep the uterus quiescent during the luteal phase. On the other hand, in the uterine vein LH-R mRNA and LH-R are maximal during preestrus/estrus as opposed to the luteal phase. In the uterine vein, LH increases the expression of cyclooxygenase and production of both prostaglandin E2 (PGE2) and PGF2 alpha. Because PGF2 alpha is the physiological luteolytic signal in the cow, we suggest that this increase in prostaglandin production by the uterine vein is part of the physiological events leading to luteolysis. In addition to uterine LH-R, the bovine cervix at preestrus/estrus has high levels of follicle-stimulating hormone receptor (FSH-R) and its corresponding mRNA. As with LH-R, activation of FSH-R by FSH is associated with activation of a G protein-coupled receptor family that mediates the cAMP and inositol phosphate signaling pathways. Activation of these signaling pathways is associated with an increase in the expression of cyclooxygenase and production of PGE2. Because expression of the FSH receptor was maximal at the time of the FSH peak in the blood, we suggest a physiological role for FSH in the cervix relaxation and opening at estrus.

Tumor necrosis factor (TNF-alpha), a 17 kDa cytokine, is a product of activated macrophages which was recently shown to be produced by rat and bovine granulosa cells. In the present work, human granulosa cells derived from preovulatory follicles were used. It was demonstrated that human granulosa cells produce TNF-alpha (5-10 units/300,000 cells per 15 h). This production was increased by addition of follicle-stimulating hormone or by a combination of human chorionic gonadotrophin and CSF to the culture media. TNF was also found in bovine follicular fluid and the concentration was higher in the periovulatory than mid-cycle follicles. TNF-alpha was found to increase prostaglandin F-2 alpha production by human granulosa cells (P less than 0.001). We conclude that granulosa cells are both a source and target organ for TNF-alpha.

It was previously reported that dispersed bovine placentome secretes progesterone and that the steroidogenic activity of these cells is stimulated by a calcium-mediated, cyclic nucleotide independent mechanism. In the present study, the influence of substrate availability was explored and the roles of calmodulin and protein kinase C in progestin production examined. Incubation of dispersed fetal cotyledon cells with 25-hydroxycholesterol (25-OH-C), a soluble sterol which readily enters cells and is metabolized to steroid hormones, increased progesterone secretion in a dose-dependent manner. The response to 25-OH-C was dependent on the extracellular calcium concentration. Methyl isobutyl xanthine (MIX) alone also increased pregnenolone as well as progesterone secretion, and the combination of 25-OH-C and MIX stimulated progesterone secretion was inhibited by trifluoperazine. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), caused no major effects on steroidogenesis but the stimulatory effects of MIX or the ionophore A23187 were enhanced in its presence. These findings suggest that (1) basal progesterone secretion by fetal cotyledon cells is limited by cholesterol availability; (2) MIX increases steroidogenesis in part by increasing the synthesis of pregnenolone, but its actions are expressed independently of cholesterol availability; (3) both calmodulin and protein kinase C may participate in the modulation of bovine placental steroidogenesis.

The effect of exogenously administered somatostatin (SRIF) on meal-stimulated secretions of the exocrine pancreas was studied in dogs with chronic pancreatic Fistulas. Dogs were fed 600 gm. of raw meat, and pancreatic output of water, bicarbonate, and protein was measured. Bicarbonate and protein secretions rose markedly postfeeding in all control animals. Four hundred micrograms or 100 μg. of SRIF infused for one hour together with a meal completely prevented the postfeeding rise in pancreatic secretions. SRIF (100 μg./hr.) infused one hour after a meal suppressed pancreatic secretions to basal levels within 30 minutes. Pancreatic secretions rose promptly after discontinuation of SRIF in all dogs. These data indicate (1) SRIF completely prevents pancreatic bicarbonate and enzyme responses when given together with a meal; (2) it completely suppresses already initiated pancreatic responses when given one hour after a meal; (3) 100 μg. of SRIF is as effective as 400 μg. in suppressing the postprandial rise in pancreatic secretions. We conclude that SRIF severely interferes with pancreatic secretions during normal alimentation and that this observation should be considered if SRIF is to be used as a therapeutic agent.

We have previously reported that progesterone synthesis in the bovine placenta is regulated by Ca2+ dependent and cyclic nucleotide independent mechanism. In studies conducted to further define the role of Ca2+ in the synthesis of progestins in bovine placental tissue, it was found that both protein kinase C (PKC), as determined by phosphorylation, and cytochrome P-450 side chain cleavage, as determined by Western blot analysis, were detectable in the steroidogenetically active portion of the placentome. To determine the site of action of PKC, fetal cotyledon cells were incubated in media containing 25-hydroxycholesterol in the absence or or presence of 10 ng/ml 12-O-tetradecanoyl-phorbol-13-acetate (TPA). It was found that TPA significantly (P less than 0.05) increased the conversion of the exogenous cholesterol analog to progesterone. To determine if the TPA could act synergistically with calcium activators, fetal cotyledon cells were incubated with either methyl isobutyl xanthine (MIX), an activator of intracellular calcium, or the calcium ionophore, A23187, which increases extracellular calcium influx, or both of these agents, in the presence or absence of TPA. It was found that TPA synergistically increased the conversion of sterol to progestins induced by submaximal concentrations of either MIX or A23187. In the presence of both compounds, TPA induced an even more dramatic increase in progestin synthesis. In experiments in which cyanoketone, an agent that inhibits the conversion of pregnenolone to progesterone, was added, TPA addition resulted in increased pregnenolone production, indicating that side chain cleavage of cholesterol is the site of action. The data, therefore, suggest that: (a) Ca2+ affects mechanisms regulating placental steroidogenesis; (2) one locus of Ca2+ is the cholesterol side chain cleavage reaction; and (3) PKC found in this tissue has a role in the Ca activated progestin production.

The effect of nicotine (100 mug/kg hr-1) on serum secretin and pancreatic secretions was studied in dogs with chronic pancreatic fistulas. Release of immunoreactive secretin (IRS) was stimulated by intraduodenal infusion of HCl (9.6 mEq/30 min). Pancreatic flow rate and bicarbonate and protein secretions were stimulated either by intestinal acidification or infusion of exogenous secretin (1.0 IU/kg hr-1). It was found that nicotine delayed the appearance of peak IRS concentrations in response to intraduodenal HCl by about 20 min. However, nicotine had no effect on the total amount of IRS released nor was this delay accompanied by a similar delay in the appearance of peak bicarbonate output. Furthermore, nicotine did not affect pancreatic secretory function stimulated by either HCl or exogenous secretin. These data do not support the thesis that nicotine plays an important role in the pathogenesis of duodenal ulcers in smokers by inhibiting the pancreatic secretion of bicarbonate.