Calcium dependence of Eugenol tolerance and toxicity in Saccharomyces cerevisiae

Eugenol is a plant-derived phenolic compound which has recognised therapeutical potential as an antifungal agent. However little is known of either its fungicidal activity or the mechanisms employed by fungi to tolerate eugenol toxicity. A better exploitation of eugenol as a therapeutic agent will therefore depend on addressing this knowledge gap. Eugenol initiates increases in cytosolic Ca2+ in Saccharomyces cerevisiae which is partly dependent on the plasma membrane calcium channel, Cch1p. However, it is unclear whether a toxic cytosolic Ca2+elevation mediates the fungicidal activity of eugenol. In the present study, no significant difference in yeast survival was observed following transient eugenol treatment in the presence or absence of extracellular Ca2+. Furthermore, using yeast expressing apoaequorin to report cytosolic Ca2+ and a range of eugenol derivatives, antifungal activity did not appear to be coupled to Ca2+ influx or cytosolic Ca2+ elevation. Taken together, these results suggest that eugenol toxicity is not dependent on a toxic influx of Ca2+. In contrast, careful control of extracellular Ca2+ (using EGTA or BAPTA) revealed that tolerance of yeast to eugenol depended on Ca2+ influx via Cch1p. These findings expose significant differences between the antifungal activity of eugenol and that of azoles, amiodarone and carvacrol. This study highlights the potential to use eugenol in combination with other antifungal agents that exhibit differing modes of action as antifungal agents to combat drug resistant infections

Current benzodiazepine issues

This article deals with some of the recent evidence bearing on the issues of the liability of benzodiazepines to lead to abuse, dependence, and adverse behavioral effects. Reviews of epidemiological, clinical and experimental literature indicated that the previous conclusion about abuse of these drugs still holds: the vast majority of the use of benzodiazepines is appropriate. Problems of nonmedical use arise nearly exclusively among people who abuse other drugs. Nevertheless, there are reasons for concern about patients who take benzodiazepines regularly for long periods of time. These drugs can produce physiological dependence when taken chronicaly, and although this does not appear to result in dose escalation or other evidence of “psychological dependence,” physiological dependence can result in patient discomfort if drug use is abruptly discontiniued. Also, physicians are currently prescribing shorter-acting benzodiazepines in preference to longer-acting benzodiazepines. The shorter-acting drugs can produce a more intense withdrawal syndrome following chronic administration. Furthermore, rates of use of benzodiazepines increase with age, and elderly patients are more likely than younger ones to take the drug chronically. The clearest adverse effect of benzodiazepines is impairment of memory. This, too, may be particular concern in older patients whose recall in the absence of drug is typically impaired relative to younger individuals, and who are more compromised following drug administration.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46347/1/213_2005_Article_BF02245824.pd

Dependence of eugenol tolerance on Cch1p-mediated Ca²⁺ influx.

<p>A) Dilution assays of eugenol activity versus <i>Jk9-3da</i> growth in SCM-leu-Ca supplemented with 100 µM CaCl₂ (▪), 50 µM BAPTA (•) and 200 µM BAPTA (♦). Absorbance was recorded after 48 hours incubation and is shown as growth (absorbance) relative to growth exhibited by yeast in eugenol-free control media. Data are fitted with the dose-response function min + (max-min)/1+ ((x/IC50)^−p)) where p is the slope, IC50 is the eugenol concentration inhibiting 50% growth and min and max represent minimum and maximum relative absorbance values respectively. Mean values (± SEM) from 4 experiments are shown. <i>Inset</i>: Dilution assays showing growth of <i>jk9-3da</i> (solid symbols) and <i>cch1Δ</i> (open symbols) cells in SCM-leu-Ca supplemented with BAPTA. Growth is shown as optical density (600 nm) after 48 hours and data are fitted with the dose-response function used in ‘A’. IC50 for BAPTA is 359±35.9 µM and 97.7±12.4 µM for <i>jk9-3da</i> and <i>cch1Δ</i> cells respectively. Mean values (± SD) from 3 experiments are shown. B) as ‘A’ except growth of <i>cch1Δ</i> strain in SCM-leu-Ca supplemented with 100 µM CaCl2 (▪), 50 µM BAPTA (•). C) IC50 for eugenol plotted as a function of CaCl₂ and BAPTA added to SCM-leu-Ca growth media for <i>Jk9-3da</i> (•) and <i>cch1Δ</i> (▪) strains. IC50 values where obtain from fits of data as shown in parts ‘A’ and ‘B’. Data are fitted with linear regression fits for <i>Jk9-3da</i> (r² = 0.8775) and <i>cch1Δ</i> (r² = 0.8694).</p

Eugenol toxicity is not dependent on Ca²⁺ influx.

<p>A) Time dependence of eugenol toxicity. Viability of <i>JK9-3da</i> cells after exposure to 6.4 mM eugenol suspended in Ca buffer for 1, 2, 4, 8 and 10 minutes. Yeast cultures are spotted on to SCM-leu media containing 2% agar; left most spots are growth after 2 days following inoculation with 5 µl of culture. Serial 10-fold dilution of the left most inoculum is shown to the right. B) as A except cells were exposed to varying concentrations of eugenol (as indicated) for 10 minutes in Ca buffer. C) as B except cells were exposed to eugenol in BAPTA buffer. D) Eugenol-induced cytosolic Ca²⁺ elevation in the presence of extracellular Ca²⁺ in mid log growth phase yeast cells. Ca²⁺-dependent aequorin luminescence from <i>Jk9-3da</i> cells in response to 3.2 and 9.6 mM eugenol in Ca buffer. Eugenol was added at 40 seconds (indicated by arrow). Traces represent mean (± SEM) from at least 5 independent experiments. SEM values are illustrated using grey shading. Luminescence was recorded every 0.2 seconds and is expressed in arbitrary units (AU). Inset is data from the main figure on an expanded y axis. E) As D except eugenol was in BAPTA buffer.</p

Extracellular Ca²⁺ is necessary for eugenol tolerance.

<p>Yeast cultures were spotted onto YPD, YPD supplemented with 10₂ agar plates containing either 0 (1% ethanol), 1.3, 2.6 or 3.2 mM eugenol. Left-most spots on each plate are growth after 2 days at 30 C after inoculation with 5 µl culture at approximately 0.5×10⁸ cells/ml. Serial 10-fold serial dilution of the left-most inoculum is shown to the right.</p