Pharmaceuticals and personal care products (PPCPs) are emerging contaminants that have been found ubiquitously in wastewater and surface waters around the world. A major source of these compounds is incomplete metabolism in humans and subsequent excretion in human waste, resulting in discharge into surface waters by wastewater treatment plant (WWTP) effluent. One pharmaceutical found in particularly high abundance in recent WWTP effluent and surface water studies is metformin, one of the world's most widely prescribed antidiabetic drugs. Interactions between insulin signaling and steroidogenesis suggest potential endocrine-disrupting effects of metformin found in the aquatic environment. Adult fathead minnows (Pimephales promelas) were chronically exposed to metformin for 4 wk, at 40 µg/L, a level similar to the average found in WWTP effluent in Milwaukee, Wisconsin, USA. Genetic endpoints related to metabolism and endocrine function as well as reproduction-related endpoints were examined. Metformin treatment induced significant up-regulation of messenger ribonucleic acid (mRNA) encoding the egg-protein vitellogenin in male fish, an indication of endocrine disruption. The present study, the first to study the effects of environmentally relevant metformin exposure in fathead minnows, demonstrates the need for further study of the endocrine-disrupting effects of metformin in aquatic organisms. Environ Toxicol Chem 2014;9999:1–6. © 2014 The Authors. Published by Wiley Periodicals, Inc. on behalf of SETAC.

The cardioprotective effects of 17β-estradiol (E2) in women are hypothesized to be partially mediated by its metabolites, 2-hydroxyestradiol (2-HOE) and 2-methoxyestradiol (2-MeOH). Therefore, the purpose of our study was to determine the acute effects of E2, 2-HOE, and 2-MeOH to inhibit coronary arterial constriction. Right coronary arteries were dissected out of hearts obtained from breeding sows, cut into 4 mm rings, and suspended in organ baths. Incubation of the rings with E2, 2-HOE, and 2-MeOH (10 μmol/L) for 60 min attenuated a subsequent KCl-induced contraction by ~ 50%; the protein synthesis inhibitor, cycloheximide, and the estrogen receptor antagonists, ICI 182,780 and tamoxifen, did not effect the attenuation. Moreover, E2, 2-HOE, and 2-MeOH antagonized the contraction induced by the vasospasm agonist, endothelin-1 (0.1μmol/L) by ~ 36%; when the L-type Ca2+ channel blocker, nifedipine, was added at the conclusion of the experiment no additional contractile attenuation was present. Our results suggest that E2, 2-HOE, and 2-MeOH demonstrate a similar nongenomic inhibition of agonist-induced extracellular Ca2+-dependent contractions.

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Phagocytosis and exocytosis are two cellular processes involving membrane dynamics. While it is easy to understand the purpose of these processes, it can be extremely difficult for students to comprehend the actual mechanisms. As membrane dynamics play a significant role in many cellular processes ranging from cell signaling to cell division to organelle renewal and maintenance, we felt that we needed to do a better job of teaching these types of processes. Thus, we developed a classroom-based protocol to simultaneously study phagocytosis and exocytosis in Tetrahymena pyriformis. In this paper, we present our results demonstrating that our undergraduate classroom experiment delivers results comparable with those acquired in a professional research laboratory. In addition, students performing the experiment do learn the mechanisms of phagocytosis and exocytosis. Finally, we demonstrate a mathematical exercise to help the students apply their data to the cell. Ultimately, this assay sets the stage for future inquiry-based experiments, in which the students develop their own experimental questions and delve deeper into the mechanisms of phagocytosis and exocytosis.

The cardioprotective benefit of estrogen (E2) has been well documented; however, its use in hormone replacement therapy has accentuated the development of breast cancer in some women. The purpose of this study was to compare the E2 metabolite, 2‐methoxyestradiol (2‐MeOH), with E2 on coronary arterial tone. Coronary arteries were dissected out of hearts obtained from female pigs, sectioned into 4 mm rings, and suspended in organ baths. A 60‐minute pre‐incubation using 10 [micro]M E2 and 2‐MeOH similarly inhibited a KCl and endothelin‐1‐induced contraction by 50% and 36%, respectively; thus, demonstrating inhibition of Ca2+ influx. Also, the K+ channel blocker, TEA (1 mM), enhanced the KCl‐induced contraction by 20 % after the pre‐incubation with 1 [micro]M 2‐MeOH, but not 10 [micro]M 2‐MeOH, this suggests 2‐MeOH stimulates Ca2+‐activated K+ channels. Breast cancer (MCF‐7) cells were incubated over a 6 day period with 1 nM E2 and 2‐MeOH in a normoxic and hypoxic environment. In contrast to E2, cell counts indicated that 2‐MeOH decreased the cell number by 39% and 32% in normoxic and hypoxic conditions, respectively. Overall, our data suggest that 2‐MeOH may be a therapeutic alternative in hormone replacement therapy because of its apoptotic effect on breast cancer and its similarity to the E2 cardioprotective effect on coronary tone. Support: NCRR of the NIH, Grant #P20 RR‐16460

Calcium channel blockers (CCBs) exert their antihypertensive effect by reducing cardiac afterload but not preload, suggesting that L‐type Ca2+ (CaL) channels contribute to arterial tone but are silent in resistance veins. Using rat small mesenteric arteries (MA) and veins (MV) perfused at their physiological pressures, we confirmed that the CCB nifedipine prevents KCl‐induced constriction of MA but not MV. We hypothesized that MVs rely on intracellular Ca2+ ([Ca]i) rather than CaL channels for constriction and that [Ca]i silences CaL channels in MVs. Indeed, the SERCA inhibitor, thapsigargin (1 μM), constricted MV but not MA (88±2% vs 3±3% relative to 60mM KCl constriction, respectively), suggesting that SR Ca2+ may fuel MV constriction. When [Ca]i was depleted by thapsigargin and extracellular Ca2+ ([Ca]o) omitted, KCl failed to constrict MVs. After depletion of [Ca]i by thapsigargin in MV and subsequent restoration of 1.5 mM [Ca]o, KCl (60 mM) contractions were partially (61±1%) restored but these contractions were blocked (61%) by 1 μM nifedipine. These data suggest: 1) a link between SR Ca2+ and constriction in MV and 2) that depletion of [Ca]i in MV disinhibits CaL channels, restoring CaL‐mediated Ca2+ influx as a source of venoconstriction. Thus, drugs that selectively deplete venous [Ca]i may represent novel therapies for the treatment of hypertension and other diseases that exhibit elevated venous tone.

Purple Martins (Progne subis) are insectivorous birds that spend their breeding season in North America before migrating to South America, where they molt. Many individuals migrate to the Amazon basin, a region of high mercury (Hg) contamination, which raises the possibility that observed declines in Purple Martins could be linked to mercury exposure. Hg is a toxic heavy metal; exposure to and bioaccumulation of Hg can result in numerous and systemic negative health outcomes, including disruption of hormones like Corticosterone (B) and triiodothyronine (T3). Both play important roles in metabolic regulation in migratory birds. Feathers, tissues that accumulate Hg and hormones during growth and can thus be used as a proxy to assess a bird's Hg load and endocrine profile. The objective of this study is to assess interrelationships among total Hg (THg), B and T3 concentrations in feathers grown in the Amazon and reproductive (productivity) and body condition (mass, fat score) endpoints. We assayed tail feathers from 80 Purple Martins from their breeding grounds in Florida, Virginia, and Wisconsin. The concentration of THg in Purple Martins’ feathers ranged from 1.103 ug/g dw to 8.740 ug/g dw, which is above the level at which physiological impacts have been observed in studies of other avian species. Hg concentration did not significantly differ by sampling locations or correlate with either B, T3 or nest productivity. However, THg concentration negatively correlated with both mass and fat score. Both B and T3 concentrations were significantly higher in Florida than in the other two states, but neither showed correlations with biometric data or nest productivity. Furthermore, B concentrations in three birds from Florida were much higher than the basal range and may indicate a high stress condition during feather‐growth in South America. Taken together, our findings suggest that concentrations of THg in Purple Martins’ feathers are high enough to be a cause of concern. The results show that THg concentration negatively impacts the ability of Purple Martins to accumulate fat, which can present a deleterious effect for a migratory bird. This result also indicates carryover effects of Hg contamination at the wintering grounds to the summer breeding grounds, where fat score was measured. The impact on fat accumulation, however, did not appear to be caused by Hg disruption of T3 or B at the time of feather‐growth. Nevertheless, endocrine disruption by Hg could still have happened at a later moment. These physiological impacts suggest a possible mechanism by which high THg exposure in the nonbreeding grounds could negatively impact purple martins and have contributed to observed population declines over the last several decade.