Anesthetic pretreatment confers thermotolerance on Saccharomyces cerevisiae yeast
Saccharomyces cerevisiae yeast, when pretreated with elevated temperatures, undergo adaptive changes that promote survival after an otherwise lethal heat stress. The heat shock response, a cellular stress response variant, mediates these adaptive changes. Ethanol, a low-potency anesthetic, promotes thermotolerance possibly through heat shock response activation. Therefore, we hypothesized other anesthetic compounds, like ethanol, may invoke the heat shock response to promote thermotolerance. To test this hypothesis, we pretreated yeast with a series of non-volatile anesthetic and anesthetic-related compounds and quantified survival following lethal heat shock (52\xc2\xb0C for 5 min). Most compounds invoked thermoprotection and promoted survival with a potency proportional to hydrophobicity: tribromoethanol (5.6 mM, peak survival response), trichloroethanol (17.8 mM), dichloroethanol (100 mM), monochloroethanol (316 mM), trifluoroethanol (177.8 mM), ethanol (1 M), isopropanol (1 M), propofol (316 \xce\xbcM), and carbon tetrabromide (32 \xce\xbcM). Thermoprotection conferred by pretreatment with elevated temperatures was \xe2\x80\x9cleft shifted\xe2\x80\x9d by anesthetic co-treatment from (in \xc2\xb0C) 35.3\xc2\xb10.1 to 32.2\xc2\xb10.1 with trifluoroethanol (177.8 mM), to 31.2\xc2\xb10.1 with trichloroethanol (17.8 mM), and to 29.1\xc2\xb10.3 with tribromoethanol (5.6 mM). Yeast in postdiauxic shift growth phase, relative to mid-log, responded with greater heat shock survival; and media supplementation with tryptophan and leucine blocked thermoprotection, perhaps by reversing the amino acid starvation response. Our results suggest S. cerevisase may serve as a model organism for understanding anesthetic toxicity and anesthetic preconditioning, a process by which anesthetics promote tissue survival after hypoxic insult.
Introduction<!>Yeast cultures<!>Anesthetics and related compounds preparation<!>Yeast pretreatment and heat shock<!>Yeast viability<!>Statistical analysis<!>Results<!>Discussion
<p>General anesthetic drugs are widely-used for their hypnotic, amnestic, immobilizing, and analgesic properties to facilitate medical procedures. Some anesthetics, particularly halogenated ones (e.g., isoflurane or sevoflurane), through anesthetic preconditioning, provide a clinical benefit in protecting tissues/organs from hypoxic, ischemic, and other noxious insults[1]. The mechanisms of anesthetic preconditioning are not completely understood. We hypothesize anesthetics elicit a cellular stress response, present in most cells including those of bacterial, yeast, and mammalian origins, that protects against diverse insults[2]. An understanding of the cellular stress response and the role of anesthetics in invoking this process may guide use of anesthetics or other drugs to protect tissues from insults and/or to identify and minimize anesthetic toxicities.</p><p>The cellular stress response is a conserved, adaptive response by cells or organisms to a mild environmental stress that protects them from a subsequent, more severe, and otherwise-lethal stress. The response causes cell cycle arrest, metabolic reprogramming, altered cell wall and membrane dynamics, and recruitment of multiple heat shock proteins (e.g., molecular chaperone proteins that guide protein folding or degradation); and, importantly, provides cross protection to a range of environmental stresses. The heat shock response, which confers thermotolerance on cells/organisms, is a variant of the cellular stress response[3].</p><p>S. cerevisiae yeast has been used in studies of the cellular stress and heat shock responses and of anesthetic mechanisms[4–6]. Ethanol, a low-potency anesthetic, at molar concentrations, confers resistance to heat shock (i.e., thermotolerance) upon yeast[7]. Notably, ethanol confers thermotolerance[8] and resistance to oxidative stress[9] on mammalian cells and activates Hsf1, a key heat shock regulatory protein, in yeast and in mouse cerebral cortex[10], suggesting ethanol may act upon a process that is conserved. With regards to anesthetic mechanisms, halogenated anesthetics at supraclinical concentrations slow S. cerevisiae growth through effects in part on the amino acid starvation response[4]. Importantly, no one has studied halogenated anesthetic and related halocarbon effects on the S. cerevisiae cellular stress/heat shock response. Such studies may provide a foundation for S. cerevisiae use as a model organism in genetic dissection of anesthetic toxicity or preconditioning and/or in high-throughput chemical compound screens for identification of novel preconditioning drugs.</p><p>Mammalian cell studies suggest an overlap between anesthetic preconditioning by clinically-relevant halogenated anesthetics (e.g., isoflurane and sevoflurane) and the cellular stress response. For example, isoflurane treatment is associated with upregulation of chaperone genes (i.e., heat shock proteins)[11, 12] and Hsp90 inhibition abolishes anesthetic preconditioning[13]. Conversely, preconditioning with sevoflurane is enhanced by an Hsp70 inducer[14]. Finally, Urner et al. investigated the effects of different halocarbons on protection in LPS-treated mammalian cells and determined that fluorinated compounds protect cells from death[15].</p><p>In this study, we tested the hypothesis that anesthetics will confer thermotolerance on S. cerevesiae through effects on the amino acid starvation response. We used tribromoethanol(TBE), since volatile agents are difficult to maintain in solution. TBE is a non-volatile, injectable, veterinary anesthetic previously used in human medicine. We also studied the effects of a TBE-related drug/compound panel to better define the physicochemical requirements necessary for thermotolerance and cellular stress response activation.</p><!><p>S. cerevisiae, strain w303-1b (MATalpha ade2-1 ura3-1 his3-11 trp1-1 leu2-3 leu2-112 can1-100) from ATCC (Manassas, VA; ATCC No. 2083530), was streaked on a YPD agar plate and expanded in YPD liquid media. All yeast media supplies were from United States Biological (Salem, MA). SC media was prepared by supplementing uracil drop-out media with uracil (74 mg/L). For high tryptophan and leucine conditions, tryptophan and leucine were added to SC media to a final concentration of 150mg/L and 300mg/L[4]. Yeast were grown at 30°C or as specified. Liquid cultures were mixed on a "Roto-Torque" (Model 7837; Cole-Parmer, Vernon Hills, IL) at a rate sufficient for yeast suspension (~30 RPM).</p><!><p>Anesthetics and anesthetic-related compounds were from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). CBr4 and propofol were dissolved in DMSO prior to SC media addition; a final DMSO concentration of 0.1 % was present in CBr4 and propofol samples and relevant controls. All other compounds were solubilized in SC media. All compound SC media preparations were sterile-filtered (0.1 μM pore; Millipore, Burlington, MA).</p><!><p>A freshly-picked w303-1b yeast colony was grown overnight in SC media at 30°C. Optical density at 600 nm (OD600) was measured (P330 Nanophotometer, Implen, Westlake Village, CA), and cultures were diluted to an OD600 of 0.2 in SC media and further grown to an OD600 of 0.8–1.2 (mid-log phase) or 6–10 (postdiauxic shift phase) at 30°C. Postdiauxic shift cultures were diluted, again, to an OD of ~1 immediately prior to pretreatment. One ml yeast culture aliquots were pelleted in a table top centrifuge (13,000 RPM) and resuspended in 1 ml SC media with or without study compound in an Eppendorf tube. Prior to heat shock, study compounds were removed by pelleting with centrifugation and by resuspension in SC media. All heat preconditioning and lethal heat shock were done on yeast aliquoted into thin-walled PCR tubes (in 200 μl volumes) using a PCR thermocycler (PCRMax Alpha Cycler, Cole-Parmer).</p><!><p>Yeast viability following heat shock was measured in two ways: 1) OD650 measurements in liquid culture at a time point after heat shock, and 2) colony forming unit count by plating on SC agar plates just after heat shock. For OD650 measurements after heat shock, yeast were diluted 1:10 and aliquoted (200 μl volumes) into wells of a 96 well plate and incubated on a titer plate shaker (Model 4625, Lab-Line Instruments/ThermoFisher Scientific, Waltham, MA) at 30°C. After plating, OD650 measurements were taken using a SpectraMAX 190 plate reader (Molecular Devices, Sunnyvale, CA) at 20 or 24h, as noted. For colony forming unit estimation, yeast were diluted (1:100 for heat shock studies or 1:10,000 for toxicity studies) and plated (100 μl volumes) on SC agar plates and incubated for 72h at 30°C; colonies were counted manually.</p><!><p>Microsoft Excel (Microsoft Inc., Redmond, WA) and Prism 7 (Graphpad, La Jolla, CA) were used for data and statistical analysis, as described in the text. cLogP values were calculated using the XLOGP v2.0 algorithm in ChemDoodle software (iChemLabs, Somerset, NJ).</p><!><p>To determine if TBE confers thermotolerance on yeast, we exposed S. cerevisiae in mid-log growth phase to a TBE concentration range for 60 min at 30 °C, washed the yeast to remove TBE, and heat shocked at 52 °C for 5 min (Fig. 1A). We determined that TBE protects yeast from an otherwise-lethal heat stress in a concentration-dependent manner (Figs. 1B and 1C). The TBE concentration-response for protection was hormetic in shape with a peak at 5.6 mM TBE (Fig. 1C). Protection occurred with TBE (5.6 mM) using exposure times ranging from 30 to 240 min (Fig. 1D). Effects were reversible and subsided by 60 min following TBE removal (Fig. 1E). TBE (1 mM) provided no significant protection after exposure times of 30 to 240 min, suggesting time can not compensate for concentration-dependent effects (Figs. 1D). TBE exposure (5.6 mM for 60 min), with no heat shock, reduced yeast colony forming units (69±0.1% of control; n=4; Supplementary Fig. 1), consistent with mild toxicity and/or slowed growth during the 60 min incubation period.</p><p>We next tested a series of compounds to better understand the physicochemical requirements necessary to confer yeast thermotolerance (Fig. 2A and 2B). The compounds differed in their type and degree of halogenation and in their predicted lipophilicity (cLogP). All were non-volatile, and most were alcohols. Like TBE, most compounds provided enhanced yeast thermotolerance in a concentration-dependent manner with a concentration-response, like TBE, hormetic in shape (Fig 2). The peak effect concentration correlated, linearly, with lipophilicity on a double logarithmic plot (Fig. 2C). CBr4 was the most potent with peak thermotolerance at ~30 μM. Fluorinated compounds TFE and HFP, a sevoflurane anesthetic metabolite, and propofol, an intravenous anesthetic, relative to the other compounds, had limited efficacy (Figs. 2A and 2B). Finally, TCE (17.8 mM over 60 min) and TFE (177.8 mM over 60 min) treatment, with no heat shock, similar to TBE, impaired colony unit formation (71±0.1% and 72±0.1% of control, respectively; n=4; Supplementary Fig. 1).</p><p>We next addressed the mechanism by which TBE confers thermotolerance. Halogenated anesthetics slow S. cerevisiae growth, and Palmer et al. had determined that S. cerevisiae is resistant to anesthetic growth-inhibition when media is tryptophan and leucine supplemented[4]. Hypothesizing a mechanism of action common between TBE-induced thermotolerance and slowed yeast growth, we studied tryptophan and leucine supplementation effects on heat shock survival. Tryptophan and leucine supplementation (150 mg/L and 300 mg/L, respectively) during pretreatment relative to standard SC media (20 mg/L and 100 mg/ml) caused a > 50% decrease in TBE-mediated thermotolerance efficacy (Fig. 3).</p><p>Mild, sublethal heat pretreatment, like TBE pretreatment, confers thermotolerance on yeast[7]. To determine if these processes interact, we combined mild heat pretreatment with TBE, TCE, or TFE pretreatment. When yeast were pretreated with mild heat (25–42 °C), increased thermotolerance was conferred with temperatures ranging from 35–42°C (Fig. 4A). TBE, TCE, and TFE "left shifted" the heat pretreatment temperature range towards lower temperatures and broadened the range (25–39°C, 27–42°C, and 30–42°C, respectively; Fig. 4A).</p><p>Postdiauxic shift and stationary yeast cultures (i.e., cultures grown to high confluency beyond the log phase) are resistant to heat and other stresses compared to earlier phase cultures, possibly due to a shift in carbon source from glucose to ethanol[16]. In yeast obtained from postdiauxic shift cultures, relative to those from mid-log phase cultures, TBE, TCE and TFE were more effective in conferring thermotolerance in a wider concentration range (Fig. 4B). Peak protective concentrations were shifted by approximately a quarter log towards higher concentrations in postdiauxic shift cultures (Fig. 4B).</p><!><p>In this study, we demonstrated that several non-volatile anesthetics (TBE, TCE, ethanol, isopropranol, carbon tetrabromide, propofol), and anesthetic-related compounds (TFE, HFP), in a concentration-dependent manner, protect S. cerevisiae yeast from a lethal heat shock. Halogenation, a common property of anesthetics, was not essential, although the fluorinated compounds, TFE and HFP, had less efficacy. Test compound potency correlated in a log-linear manner with predicted lipophilicity (cLogP): CBr4 (cLogP = 3.42) was the most potent providing peak effects at ~30 μM compared to ethanol (cLogP = −0.31), the least potent, with peak effects at ~1M. Interestingly, the concentration-response was biphasic (or hormetic) in shape for all compounds, suggesting commonalities in their mechanisms of action. TBE-induced thermotolerance was reversible and antagonized by leucine and tryptophan media supplementation, which prevents the amino acid starvation response. Yeast confluency modified TBE-induced thermotolerance, decreasing efficacy in mid-log relative to postdiauxic shift cultures. Finally, TBE treatment modified the thermotolerance conferred by mild heat pretreatment, shifting the mild heat-induced tolerance to lower temperatures, and consistent with an overlap and/or an interaction between the yeast responses to these disparate stressors.</p><p>Anesthetic preconditioning is an important, but poorly understood anesthetic property, that confers protection from ischemia and other insults on tissues[1]. A better understanding of this process might yield drugs that protect tissues during a variety of insults (e.g., heart attack, stroke, cardiac arrest, hypoxia). S. cerevisiae is a eukaryotic organism, amenable to high-throughput studies and to genetic manipulation; and many fundamental processes present in S. cerevisiae are evolutionarily conserved[17]. Our study purpose was to provide a foundation for S. cerevisiae use in dissecting the genetic and molecular mechanisms of anesthetic preconditioning. For example, gene knockout or gene overexpression libraries may identify unique and essential proteins or pathways involved in anesthetic preconditioning. In our current studies, we identified and determined the potency and efficacy of multiple non-volatile anesthetics that confer thermotolerance. For one compound, TBE, an injectable veterinary anesthetic, we determined kinetics, culture conditions, and interactions with other stressors (e.g., heat, starvation, and culture confluency). Our data will guide future S. cerevisiae studies.</p><p>However, there are limitations to our approach. Anesthetic preconditioning conferring resistance to ischemia on a mammalian cell/tissue and anesthetic-induced thermotolerance in yeast may be mediated by unrelated mechanisms. For reasons of parsimony, we speculate both phenomena occur via an evolutionarily conserved and generalized cellular stress response, but this cannot be assumed. Therefore, any discovery in yeast will require validation in a relevant cell or tissue. Another limitation are the high concentrations required for thermotolerance induction relative to that required for anesthesia (i.e., clinically-relevant concentrations where preconditioning likely occurs), which challenges our data's clinical relevance. For example, in tadpoles, TBE causes loss of righting at 0.3 mM, whereas peak yeast thermotolerance effects occur at 5.6 mM. Similar concentration differences have been observed in studies of anesthetic effects on yeast growth[4, 5]. The high concentrations suggest differing pharmacodynamics, however it is unclear if two organisms, one multicellular and the other unicellular and rapidly dividing, of such different scale, can be directly compared. Yeast may also be resistant to organic solvent-imposed stress as it produces ethanol and grows in its presence; 4 to 6% ethanol (~1 M) is required to invoke a yeast heat shock response [7, 18, 19]. Finally, propofol conferred thermotolerance on yeast, but, clinically, is sometimes avoided in favor of an inhaled agent (e.g., sevoflurane), because it lacks preconditioning properties[1]. We speculate this may be due to enhanced anesthetic potency, and a more selective mechanism of action (i.e., GABAA chloride channel activation), relative to an inhaled agent. Propofol causes loss of righting in tadpoles at ~1 μM, whereas peak yeast thermotolerance effects occur at ~300 μM[20]. Therefore, propofol has a ~300-fold concentration difference between anesthetic and thermotolerance concentrations relative to the ~10-fold difference observed with TBE.</p><p>Several observations in our study highlight potential mechanisms by which the compounds confer thermotolerance. We observed a log-linear relationship between potency in conferring thermotolerance and predicted lipophilicity (cLogP), a calculated physical property relatively independent of chemical structure. This observation is similar to the Meyer-Overton relationship, which predicts anesthetic potency, and which is often interpreted as evidence of a non-specific, hydrophobic interaction between anesthetic drugs and the lipid membrane and/or a hydrophobic protein binding site[21]. A log-linear relationship between alcohol potency in inhibiting yeast glucose utilization and anaerobic fermentation has been reported[22, 23].</p><p>Thermotolerance was tested on yeast after washing and removal of test compound, and TBE effects persisted for at least 30 min (Fig. 1E). Although we acknowledge small amounts of TBE may remain despite washing, we speculate persistence may be caused by TBE-invoked changes in gene expression related to stress survival. For example, ethanol induces multiple yeast genes, some involved in stress resistance[24]. However, a multitude of effects are also possible (e.g., changes in protein phosphorylation, etc.).</p><p>We studied a range of compounds that differ in chemical structure. Although potency and efficacy for invoking thermotolerance varied, interestingly, the biphasic (hormetic) shape of the concentration-response curves was remarkably similar. We speculate the curve shape is caused by concentration-dependent changes in stress or toxicity. Below a certain threshold, test compounds have no overt effect. However, with increasing concentration, elevated stress invokes adaptive changes in yeast that confers thermotolerance, and high concentrations may confer toxicity. The interaction between TBE-induced thermotolerance and that provided by mild heat pretreatment is consistent with this idea; TBE-induced stress and mild heat-induced stress may be additive in invoking an adaptive response and in causing toxicity. A similar interaction between heat and ethanol has been described in yeast[18], in mammalian cells[25], and in rats[26], where heat stress increases ischemic preconditioning in the spinal cord. A hormetic concentration-response curve has been observed in other stress responses, including pre- and postconditioning[27, 28]. An alternative interpretation is that test compounds engage with two different processes/binding sites, one augmenting thermotolerance and one causing toxicity/impaired thermotolerance. However, it seems unlikely that the relative potency for these two theoretical processes would be preserved over such a large number of test compounds.</p><p>We determined that tryptophan and leucine supplementation antagonizes TBE-induced thermotolerance. We undertook these studies because Palmer et al. had determined tryptophan and leucine supplementation decreases sensitivity to anesthetic-induced growth inhibition in S. cerevisiae[4]. The mechanism may involve the "general amino acid control" (GCN) network, a signaling pathway activated during amino acid starvation that inhibits translation of most mRNA, but augments transcription of amino acid biosynthetic genes[29]. Anesthetics decrease uptake of leucine and tryptophan, and, notably, the w303-1b strain is auxotrophic for both amino acids. These data suggest there may be similarities in the mechanisms by which anesthetics inhibit yeast growth and by which they confer thermotolerance on yeast. As studied by Palmer et al.[29], it will be interesting to determine if yeast lacking factors in the GCN pathway are resistant to TBE-induced thermotolerance.</p><p>Finally, we observed that yeast grown in culture conditions of high confluency (i.e., postdiauxic shift phase), relative to low confluency (i.e., mid-log phase), are more receptive to TBE-induced thermotolerance (Fig. 3). Similar to the interaction with mild heat, we speculate confluency may impose stress on the yeast that is additive with that imposed by TBE. This stress might be due to pH changes, nutrient depletion (e.g., amino acid starvation), and/or changes in nutrient source (e.g., from dextrose to ethanol with confluency). Such ideas can be addressed in future studies. However, these observations demonstrate the importance of culture conditions and growth phase in studies of yeast thermotolerance.</p><p>In summary, we have identified a range of anesthetic and anesthetic-related compounds that confer thermotolerance on S. cerevisiae. Moreover, we identified at least two factors that antagonize this process, leucine and tryptophan supplementation, and low confluency culture conditions. Our observations may have relevance to anesthetic preconditioning, which, like thermotolerance, is related to the generalized and evolutionarily-conserved cellular stress response. Our studies will provide a foundation for S. cerevisiae use in dissecting genetic and molecular details of anesthetic preconditioning.</p>