1 Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036, India
2 Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, S. A. S. Nagar, Punjab 160 002, India
Correspondence
Anand K. Bachhawat
akbachhawat{at}hotmail.com
anand{at}imtech.res.in
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ABSTRACT |
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INTRODUCTION |
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Despite extensive studies of the mechanisms of acetaminophen toxicity, the exact mechanism by which acetaminophen is toxic is surprisingly still controversial. Currently, two major theories have been proposed to explain the cytotoxicity. Although in both hypotheses the first step is the generation of the reactive intermediate NAPQI, the glutathione depletion theory states that an excess of NAPQI (generated from acetaminophen by cytochrome P450) leads to depletion of glutathione, followed by oxidative stress, ultimately leading to cell death. The second theory, the covalent binding theory or the macromolecular inhibition theory, considers that the major cause of cell death by acetaminophen is not the result of glutathione depletion per se, but the result of direct binding to macromolecules and inhibition of their function by NAPQI, eventually leading to cell death (Mitchell et al., 1973; Dahlin et al., 1984
; Ruepp et al., 2002
).
A second issue complicating studies of acetaminophen toxicity is the significant differences seen in the susceptibility of different species and even strains to acetaminophen toxicity (Potter et al., 1974; Hinson, 1980
; Ioannides et al., 1983
). It is not clear whether the increased drug resistance profiles are due to enhanced/reduced metabolism or other factors, hitherto unconsidered, such as increased efflux. Furthermore, the possible role of multidrug resistance associated proteins (MRPs) in these processes is unclear, although the involvement of MRPs has been indicated by one study (Xiong et al., 2000
).
The yeast Saccharomyces cerevisiae is an excellent model system to investigate mechanisms of drug resistance and toxicity at the cellular level. Not only are most of the enzymic and cellular structures conserved, but the maintenance of the redox balance and oxidative stress response is also highly conserved, with glutathione being the major non-protein thiol compound present in yeasts as well as in higher eukaryotes. In addition, the family of drug resistance pumps found in mammalian cells (the multidrug resistance proteins, MDRs, and MRPs) are also present as a family of pumps in yeasts (Decottignies & Goffeau, 1997).
An earlier report investigating the effects of aniline and its metabolites in yeasts also investigated acetaminophen (an aniline derivative), and under the conditions in which it was investigated it was found to be non-toxic (Brennan & Schiestel, 1997). Considering the potential importance of yeast in resolving some of the controversial issues relating to acetaminophen toxicity, such as those described above, we considered it important to reinvestigate the toxicity of acetaminophen more rigorously and compare the mechanisms of toxicity and resistance with those of mammalian cells. We decided to investigate this by initially examining pleiotropically drug-sensitive mutants (certain erg mutants defective in ergosterol biosynthesis). Acetaminophen was found to be toxic in these yeast mutants. This allowed one to examine the possible mechanisms of drug detoxification as well as test the existing models concerning the mechanisms of acetaminophen-induced cell death. The results suggested that acetaminophen toxicity in yeast is not due to the generation of reactive oxygen species (ROS) and is also not dependent on the intracellular glutathione status. Resistance was conferred by the MRPs Snq2p and Flr1p and was mediated by Yap1p, Yrr1p and Pdr1p/Pdr3p. The hierarchy of these factors in the resistance to acetaminophen was also determined and found to be distinct from the existing knowledge about their hierarchies. Together, these findings demonstrate that acetaminophen can exert its toxicity at the cellular level by pathways completely different from those considered previously. The implications of these studies for mammalian cells are also presented.
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METHODS |
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Strains, strain construction and growth conditions.
The yeast strains used in this study are listed in Table 1. The cells were routinely grown in YPD at 30 °C. For selection of transformants and
-galactosidase assays, minimal medium (SD media) with supplements was used. Acetaminophen stock solutions were prepared in 30 % methanol, and the appropriate amounts were added to the media just prior to pouring the plates. Control plates contained equivalent amounts of 30 % methanol. The strain ABC681 (snq2
) was constructed by PCR-mediated gene disruption of the SNQ2 gene of ABC154 using the KanMX2 module (Wach et al., 1994
). The disruption cassette was amplified using the primers SNQ2-DEL1: 5'-AAGGTATTAAGGCTAAGAGGCATCAAAAGATGAGACAGCTGAAGCTTCGTA-3' and SNQ2-DEL2: 5'-TTTCGAATTCCTCAGCGGTTCTTGGTACTTTATTTTCATAGGCCACTAGTGGATC-3'. The disruption of the desired locus was confirmed by PCR using the primers SNQ2-FOR: 5'-GATGCGAGTGCCCTAGAAGG-3' and SNQ2-REV: 5'-CTTGTTCCCAATATGACACT-3'. The pdr15
strain (ABC668) was also constructed by PCR-mediated gene disruption using the KanMX2 module, using the primers PDR15-D1: 5'-GTCAGAGGTGTTTCTGGTGGTGAAAGAAAGCGTGTATCCAGCTGAAGCTTCGTACGC-3' and PDR15-D2: 5'-TAAGGCAGTCAAAGTGCCTGGTTTTACCCAACCATCTACGTTCATAGGCCACTAGTGGAT-3'. Disruptions were confirmed by PCR. The ABC670 (pdr10
) strain was constructed by first cloning a 1·6 kb BglIIBamHI fragment of PDR10 that was amplified by PCR into pGEM7Z. A 4·5 kb LYS2 fragment containing the LYS2 gene was excised by PstI and cloned into the PstI site of PDR10 in pGEM7Z. A NsiIScaI digestion of this pPDR10 : : LYS2 disruption plasmid was excised and used to transform ABC154. Transformants were selected on SD media without lysine, and disruptions were confirmed by PCR.
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The SKN7 gene was disrupted in the ABC949 (wild-type) strain and ABC950 (yap1) to yield ABC1041 (skn7
) and ABC1042 (skn7
yap1
) by transforming a linearized fragment obtained by restriction digestion of an SKN7 disruption plasmid (skn7
: : TRP1) (Brown et al., 1993
) with SacI. Disruptions were confirmed by PCR and by t-butylhydroperoxide sensitivity.
Plasmids.
PDR5 on a multicopy plasmid (PDR5/Yeplac195) has been described earlier (Kaur & Bachhawat, 1999); YAP1 on a multicopy plasmid and YEp351-YAP1 were obtained from Dr S. Moye-Rowley; FLR1 on multicopy plasmid p425GPD-FLR1 and the control plasmid (p425 GPD) from Dr M. Raymond; ATR1 on multicopy plasmid Yrp74-Sc4018 and the corresponding control plasmid (Yrp74) from Dr K. Struhl; YOR1 on multicopy plasmid YEpYRS1 was sent by Dr T. Miyakawa. The plasmid bearing pdr3-9 (hyperactive allele of Pdr3p) was obtained from Dr J. Subik. The plasmid bearing GADYRR1* (a gene encoding the hyperactive allele of Yrr1p) was obtained from Dr C. Jacq.
The pSKN7 plasmid was isolated by library screen in the lab. (Sharma et al., 2003). pYCF-LacZ and pBPT-LacZ plasmids have been described previously (Sharma et al., 2002
).
The ERG5 and ERG11 genes were amplified by PCR using vent DNA polymerase and cloned into the BamHIXhoI sites of the yeast expression vector pTEF-416, a single copy centromeric vector. The primers used for amplification were ERG5-gen-Bam-F (5'-ACA AAA ggA TCC ATgAgT TCT gTC gCA gAA AAT ATA-3') as well as ERG5 gen-Eco-R (5'-AAgACTgAATTCTCTCCAgTAATTgggTCTCTC-3') for ERG5, and ERG11-gen-Bam-F (5'-ACA Agg ggA TCC ATg TCT gCT ACC AAg TCA ATC-3') as well as ERG11-gen-R1-R (5'-TTA CAA gAA TTC ACC TTA gAT CTT TTg TTC Tgg AT-3') for ERG11. The genes were cloned downstream of the strong and constitutive TEF promoter and confirmed by sequencing.
Growth experiments.
Cells from overnight cultures of strains ABC154, ABC936 and ABC591 were reinoculated to an OD600 of 0·1 (2x106 cells ml1) and allowed to grow to an OD600 of 0·5 to 0·6, and, at this stage, drug was introduced (16 mg ml1). In control experiments, an equivalent amount of 30 % methanol solution was added. Growth of these cultures was followed by measuring OD600 values at different time points (at 3 h intervals). At 12 and 24 h time points, a known number of cells was plated on YPD plates to check the number of viable cells.
Drug sensitivity experiments.
Strains were transformed with the plasmids, and the transformants were grown in SD media with appropriate selection until they reached exponential phase, and then equal numbers of cells were harvested and resuspended in sterile water to a density of 1x107 cells ml1. Portions (10 µl) of undiluted cell suspension, 1 : 10, 1 : 100 and 1 : 1000 dilutions were then spotted onto YPD plates containing different concentrations of acetaminophen. Growth was observed after 2 to 4 days at 30 °C.
Glutathione estimation.
The overnight cultures of strains ABC154, ABC591 (gsh1) and ABC936 (strain overexpressing GSH1 gene) were reinoculated at OD600=0·1 and acetaminophen was added when the OD600 reached 0·50·6. Glutathione estimation was carried out using the DTNB-glutathione reductase assay (Anderson & Meister, 1983
) at different time points, as described earlier (Sharma et al., 2000
). Oxidized glutathione levels were measured by using 2-vinyl pyridine to block the reduced glutathione (Anderson & Meister, 1983
).
Induction conditions and -galactosidase assays.
ABC154 was transformed with plasmids pYCF-LacZ and pBPT-LacZ, and the transformants were assayed for -galactosidase in the presence or absence of acetaminophen (14 mg ml1).
-Galactosidase assays were carried out on permeabilized cells, as previously described (Guarente, 1983
; Sharma et al., 2002
).
Detection of intracellular acetaminophen.
Cells of strain ABC154 were inoculated in YPD media and allowed to grow overnight, reinoculated at OD600=0·2 and allowed to grow for 34 h. To this growing culture, acetaminophen was added at 8 mg ml1, and the incubation continued for 46 h. The cells from this culture were harvested, washed thoroughly (twice) with sterile distilled water, and lysed using 5 % sulfosalicyclic acid and glass beads. The lysate was centrifuged at 5000 r.p.m. for 5 min to settle the unbroken cells and the cell debris. The supernatant was mixed with two volumes of ethyl acetate and vortexed vigorously for 1 min, the mixture was allowed to settle and the ethyl acetate layer was separated. The aqueous and ethyl acetate extract fractions were lyophilized, and the residues redissolved in small amounts of water and ethyl acetate, respectively. An aliquot of each of the ethyl acetate and aqueous extracts was subjected to mass spectroscopy (100600 a.m.u.) through direct infusion under positive atmospheric pressure chemical ionization (APCI). A separate aliquot of the ethyl acetate fraction was used for LC-MS experiments (100600 a.m.u., positive APCI) using a PDA detector.
Fluorescence assays using 2',7'-dichorodihydrofluorescein diacetate.
Overnight cultures of ABC154 (wild-type strain) and ABC681 (snq2 strain) were each subcultured into eight different flasks with 5 ml fresh medium at a concentration of 0·5x107 cells ml1 and incubated at 30 °C at 200 r.p.m. for 2 h. The test chemicals H2O2 and acetaminophen (at final concentrations of 4 mg ml1 and 18 mg ml1, respectively) were added to two flasks each of the wild-type and snq2
strain. The cultures were incubated for about 1 h and then 2',7'-dichlorodihydrofluorescein diacetate (DCFHDA) was added from a fresh 5 mM stock (prepared in ethanol) to a final concentration of 10 µM and the incubation was continued for a further 2 h. This permitted deacetylation of the dye and rendered it susceptible to oxidation in the presence of any ROS. About 1·5 ml of sample was removed from each of the above cultures and cells were harvested by centrifugation, washed twice with sterile water and resuspended in 100 µl 50 mM Tris/HCl buffer (pH 7·5). The cells were permeabilized by adding 50 µl chloroform and 20 µl 0·1 % SDS and by vortexing at high speed for 20 s. The tubes were left to stand for 10 min to allow the dye to diffuse into the buffer. Cells were pelleted in a microcentrifuge, and the fluorescence of the supernatant was measured using a Shimadzu fluorimeter (excitation, 502; emission, 521).
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RESULTS |
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Identification of intracellular acetaminophen (but no other metabolites of acetaminophen) in yeast cells grown in the presence of the drug
To determine if the acetaminophen-induced cell death was a result of acetaminophen or some other metabolite accumulating intracellularly, it was necessary to establish the accumulation of acetaminophen (or its metabolites) within the cell.
Whole-cell lysates of cells grown in the presence of acetaminophen were extracted with ethyl acetate (Methods) and the ethyl acetate and aqueous fractions were subjected to direct infusion mass spectroscopy. In each occasion, the peak at m/z 152 in the respective mass chromatogram indicated the presence of acetaminophen. However, the mass chromatogram did not reveal any new peaks of significant intensity. In order to investigate the accumulation of the drug inside the cell and to examine the possibility of any new peaks we further carried out LC-MS studies of the ethyl acetate fraction using a PDA detector. A major component with a retention time comparable to that of standard acetaminophen exhibited a peak at m/z 152 in the MS, revealing the presence of the drug in the ethyl acetate extract. The LC-MS did not reveal the presence of any new peak relative to the control, suggesting that the principal compound accumulating in these cells was acetaminophen and that no other transformed products of this drug were being generated. However, the possibility that other metabolic products (such as NAPQI) were being formed and rapidly removed from the cell, or conjugated to proteins preventing their extraction, still existed.
Overexpression of yeast-cytochrome-P450-encoding Erg5p (C22 sterol desaturase) and Erg11p (lanosterol demethylase) does not alter the acetaminophen resistance profiles in yeast
The inability to detect any other metabolites of acetaminophen suggested that acetaminophen was exerting its toxicity independently of a biotransformation step. This was in apparent contrast to mammalian cells, in which the activation of acetaminophen to the reactive intermediate has been shown to be dependent on the presence of specific cytochrome p450 enzymes. NAPQI is a very short-lived intermediate and, in the studies with mammalian cells, only 1 % of the acetaminophen is converted into NAPQI through cytochrome P450. The possible involvement of the yeast cytochrome P450s in toxicity needed more thorough investigation. S. cerevisiae has three P450 enzymes which play important metabolic roles in the cell. Erg5p (Skaggs et al., 1996) and Erg11p (Aoyama et al., 1981
) are involved in ergosterol biosynthesis, and homologues of these proteins are widely distributed in other yeasts as well. The third protein, Dit2p, is involved in the spore wall formation of S. cerevisiae, and is unique to S. cerevisiae (Briza et al., 1990
). Among these different P450 enzymes in yeast, only Erg5p has been implicated in also contributing to the detoxification pathway of some metabolites. To examine the possible role of Erg5p and Erg11p in the toxicity of (or resistance to) acetaminophen, we cloned and overexpressed these genes from a strong constitutive promoter. Both Erg5p and Erg11p overexpression could confer increased resistance to fluconazole, but we could not find any increased sensitivity or resistance to acetaminophen upon either Erg5p or Erg11p overexpression (data not shown).
Acetaminophen toxicity in yeast: absence of a role for glutathione
The inability to detect any intracellular metabolites other than acetaminophen in acetaminophen-treated cells and the lack of involvement of the yeast cytochrome P450s strongly suggested a toxicity mechanism that differed from the primary mechanism of toxicity observed in mammalian cells, in which reactive metabolites are generated through the action of specific cytochrome P450s. We decided to examine more rigorously whether the yeast cells were in fact subjected to an oxidative stress response in the presence of acetaminophen, and also if the glutathione status of the cell was important in the cellular response to acetaminophen.
We decided to initially examine this using 2',7'-dichlorodihydrofluorescein diacetate, a fluorogenic compound which has been used by several workers as a marker for oxidative stress and which is suggested to reflect the overall oxidative stress status of the cells, although its use as a marker of overall oxidative status is still controversial. Experiments were carried out as described in Methods. Cells exposed to H2O2 displayed a significant increase in fluorescence intensity, but no increase in fluorescence intensity was observed when cells were treated with acetaminophen concentrations from 4 to 18 mg ml1 for a period of 1 to 2 h (Fig. 2). This confirmed that the cells were not being subjected to oxidative stress. However, the limitations of the assay in being responsive to, and therefore suitable for, only some oxidants (Myhre et al., 2003
; Chignell & Sik, 2003
) prompted us to investigate more carefully the role of glutathione, since glutathione depletion has been implicated in the acetaminophen toxicity of mammalian cells.
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Yeast cell response to acetaminophen: role of Yap1p in resistance to acetaminophen
Yap1p is a transcription factor known to play a central role in the oxidative stress response of yeast (Moye-Rowley et al., 1988) as well as in the response to several drugs that generate an oxidative stress response.
Acetaminophen toxicity was initially examined in strains deleted for YAP1. Our results clearly indicated that yap1 strains displayed an increased sensitivity to acetaminophen (Fig. 4a
). Furthermore, overexpression of Yap1p in wild-type cells conferred increased resistance to acetaminophen (Fig. 4b
), confirming the role of Yap1p in the response to cellular injury by acetaminophen. We also investigated if Skn7p, a second transcription factor also implicated in the cellular oxidative stress response (Morgan et al., 1997
), might also be involved in the response to acetaminophen. However, neither the deletion of SKN7 nor the overexpression of SKN7 from a multicopy plasmid led to any discernable phenotype in the presence of acetaminophen (Fig. 4a, b
). A deletion of SKN7 in a yap1
background was also constructed to see if the phenotypes of an skn7
deletion might be seen in this background. However, no further increase in acetaminophen sensitivity was observed in the skn7
yap1
strains compared to yap1
strains. This indicated that, of the two oxidative-stress-responsive transcription factors, only Yap1p played a role in the response to acetaminophen. Although it has been widely used as an indicator of oxidative stress response in yeasts, recent studies have indicated that there are two independent mechanisms of Yap1p activation, one dependent on oxidative free radicals, and another which acts at an independent site of Yap1p which is activated by electrophiles (Azevedo et al., 2003
). The lack of ROS suggested that the acetaminophen response of Yap1p was occurring through the latter mechanism. To further confirm this, we examined the effects of gpx3
(orp1
) on acetaminophen sensitivity. The oxidative response of Yap1p has been shown to be dependent on the presence of Gpx3p (Delaunay et al., 2002
). We could not observe any effect of the presence of Gpx3p on the sensitivity to acetaminophen, further underlining that Yap1p was not functioning through this pathway and that it was being activated through an electrophilic compound which was not dependent on an oxidative stress response.
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Overexpression of Yap1p leads to a greater than 10-fold induction of YCF1 (Wemmie et al., 1994). We therefore decided to examine the role played by the yeast glutathione-conjugate pumps Ycf1p and Bpt1p. Bpt1p is a close homologue of Ycf1p, which has recently been also shown to function as a glutathione-conjugate pump but is not regulated by Yap1p (Sharma et al., 2002
; Klein et al., 2002
; Chaudhuri et al., 1997
). These pumps have also been shown to transport unconjugated compounds (Pascolo et al., 2001
; Petrovic et al., 2000
). However, in contrast to what we expected, we observed that deletion of both YCF1 and BPT1 led to an increase in resistance to acetaminophen (Fig. 5
). This resistance to acetaminophen was observed in a very narrow range of drug concentrations. The results were unexpected and were also in apparent conflict with the fact that Ycf1p is upregulated by Yap1p, as well as the observation, described above, that Yap1p leads to increased resistance to acetaminophen. To examine how acetaminophen affected the induction of YCF1 and BPT1, we checked the expression pattern of YCF1 and BPT1 using promoterLacZ fusions in the presence of acetaminophen. Only YCF1 (and not BPT1 or the other members of the group) is known to be induced by Yap1p (Wemmie et al., 1994
; Sharma et al., 2002
, 2003
). However, in the presence of acetaminophen we observed only a negligible (1·5-fold) increase in
-galactosidase activity in both YCF1 and BPT1 (Table 2
). These results indicate that, although Yap1p does play a role in resistance to acetaminophen, the response of YCF1 (a target of Yap1p) might be influenced by other unknown regulatory factors in addition to Yap1p. Furthermore, it suggests that the H2O2-activated Yap1p and the thiol-compound-activated Yap1p show differential activation responses. The increased resistance of ycf1
bpt1
strains to acetaminophen, though small, possibly suggests some involvement of Ycf1p and Bpt1p in the process, although the exact manner in which this might be occurring is not clear. We also overexpressed the YCF1 gene from a multicopy plasmid, but no phenotypes on acetaminophen-containing plates could be discerned upon overexpression of YCF1 (data not shown).
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DISCUSSION |
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The study clearly demonstrated the toxicity of acetaminophen in yeasts, although it became apparent only at higher concentrations of the drug or in ergosterol biosynthetic mutants.
Unlike mammalian cells, however, acetaminophen failed to induce an oxidative stress response in yeasts. The studies clearly show the involvement of Yap1p in acetaminophen resistance. However, Yap1p can be activated either by ROS or by electrophiles (Azevedo et al., 2003), and in the case of acetaminophen it appears that it is the generation of electrophiles, but not ROS, that is activating Yap1p. Acetaminophen itself is not considered an electrophile, while NAPQI, a metabolic product of acetaminophen, is an electrophile. The inability to detect any metabolites of acetaminophen probably explains the relative lack of toxicity of this compound in yeast. Since the toxicity was also not enhanced by increasing the levels of the two cytochrome P450s (Erg5p and Erg11p), there are two possible explanations for the toxicity of acetaminophen in yeast:
(i) acetaminophen itself (independent of its activation to NAPQI) can act as a weak electrophile;
(ii) the activation of acetaminophen occurs at exceedingly low levels in yeast, by a mechanism independent of the cytochrome P450s.
Both possibilities are intriguing, since they have not been considered in mammalian cells, and in the light of the results described here, these possibilities need to be seriously examined in mammalian cells too.
The experiments designed to evaluate the role of glutathione depletion or glutathione redox status on acetaminophen toxicity clearly argue against a role for glutathione depletion per se being the causative agent in acetaminophen toxicity.
Deletion of the genes for the glutathione conjugate pumps YCF1 and BPT1 surprisingly led to resistance to acetaminophen. This was an unexpected observation, since Ycf1p levels are actually enhanced by Yap1p. While a possible explanation is that the GSH conjugates in this case are more toxic, as has been suggested for some drugs (Monks & Lau, 1998), an alternative explanation is that accumulation of toxic intermediates and other cellular metabolic intermediates in a ycf1
bpt1
deletion strain may be causing a feedback inhibition of the enzymes responsible for the production of the toxic intermediate.
One of the surprising observations that was made in this study is that acetaminophen could be effluxed by the yeast multidrug resistance pump Snq2p (and to a lesser extent by Flr1p). In addition to the relative lack of formation of reactive acetaminophen metabolites (such as NAPQI), the efflux of acetaminophen by multidrug resistance transporters might be a second reason for the relative lack of toxicity of these drugs to wild-type yeasts and the consequent toxicity of the drug only at elevated concentrations. Furthermore, the findings would suggest that one should examine the role of these pumps in mammalian cells more carefully. Although the relative difference in tissue and species specificity of the effects of acetaminophen have been attributed to differences in the metabolism of the drug, the possibility that differences in direct drug efflux are a cause also needs to be examined more carefully.
Interestingly, in addition to YAP1, deletions in YRR1 and PDR1 led to an increased sensitivity to acetaminophen, indicating the involvement of the drug-resistance regulatory network in acetaminophen resistance. While the Yap1p response required a functional Pdr1p or Pdr3p protein, the resistance conferred by Pdr1p/Pdr3p as seen through a hyperactive pdr39 allele could occur independently of either Yap1p or Yrr1p, suggesting a hierarchy of these transcription factors in the resistance to acetaminophen. A link between Yap1p and Pdr1p/Pdr3p has previously been shown for diazaborine resistance (Wendler et al., 1997; Jungwirth et al., 2000
) and for benomyl resistance (Tenreiro et al., 2001
). In the case of diazaborine resistance it was observed that the resistance due to Yap1p was dependent on a functional Pdr1p or Pdr3p protein, but in this case the pumps conferring resistance were Ycf1p and Flr1p. More recently, it has been shown that the pdr333 mutation (a gain of function allele of PDR3) could specifically mediate resistance to diazaborine through Snq2p and Pdr5p, while a pdr112 mutant (a gain of function of PDR1) mediated resistance to the same drug through Ycf1p and Flr1p (Wehrschutz-Sigl et al., 2004
). In the case of benomyl resistance, Flr1p appeared to be the primary pump involved in resistance, and was dependent on Yap1p and partially on a functional Pdr1p or Pdr3p (Tenreiro et al., 2001
). Our investigations, while describing a quite different hierarchy in the resistance to acetaminophen, has also opened up several other interesting issues and considerations on the toxicity of acetaminophen in relation to the pathways and networks that mediate resistance.
In conclusion, our studies investigating acetaminophen toxicity in yeast have demonstrated that acetaminophen can exert its toxicity in these unicellular eukaryotes by mechanisms quite distinct from those otherwise observed and described in mammalian systems. The possibility that toxic effects of acetaminophen through these pathways might also be operating (at a secondary level, perhaps) in mammalian cells thus needs to be examined, especially so in the light of the drug's wide usage. The studies described here also throw light on important aspects of the resistance to the drug in yeast which might also help in resolving some of the conflicting issues regarding the toxicity in humans of this widely used drug.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Anderson, M. E. & Meister, A. (1983). Transport and direct utilization of gamma-glutamylcyst(e)ine for glutathione synthesis. Proc Natl Acad Sci U S A 80, 707711.[Abstract]
Aoyama, Y., Okikawa, T. & Yoshida, Y. (1981). Evidence for the presence of cytochrome P-450 functional in lanosterol 14 alpha-demethylation in microsomes of aerobically grown respiring yeast. Biochim Biophys Acta 665, 596601.[Medline]
Azevedo, D., Tacnet, F., Delaunay, A., Rodrigues-Pousada, C. & Toledano, M. B. (2003). Two redox centers within Yap1 for H2O2 and thiol-reactive chemicals signaling. Free Radic Biol Med 35, 889900.[CrossRef][Medline]
Balzi, E. & Goffeau, A. (1995). Yeast multidrug resistance: the PDR network. J Bioenerg Biomembr 27, 7176.[Medline]
Balzi, E., Wang, M., Leterme, S., Van-Dyck, L. & Goffeau, A. (1994). PDR5, a novel yeast multidrug resistance conferring transporter controlled by the transcription regulator PDR1. J Biol Chem 269, 22062214.
Bourbouloux, A., Shahi, P., Chakladar, A., Delrot, S. & Bachhawat, A. K. (2000). Hgt1p, a high affinity glutathione transporter from the yeast Saccharomyces cerevisiae. J Biol Chem 275, 1325913265.
Brennan, R. J. & Schiestel, R. H. (1997). Aniline and its metabolites generate free radicals in yeast. Mutagenesis 12, 215220.[Abstract]
Briza, P., Breitenbach, M., Ellinger, A. & Segall, J. (1990). Isolation of two developmentally regulated genes involved in spore wall maturation in S. cerevisiae. Genes Dev 10, 17751789.
Bróco, N., Tenreiro, S., Viegas, C. A. & Sá-Correia, I. (1999). FLR1 gene (ORF YBR008c) is required for benomyl and methotrexate resistance in Saccharomyces cerevisiae and its benomyl-induced expression is dependent on Pdr3 transcriptional regulator. Yeast 15, 15951608.[CrossRef][Medline]
Brown, J. L., North, S. & Bussey, H. (1993). SKN7, a yeast multicopy suppressor of a mutation affecting cell wall beta-glucan assembly, encodes a product with domains homologous to prokaryotic two-component regulators and to heat shock transcription factors. J Bacteriol 175, 69086915.[Abstract]
Chaudhuri, B., Ingavale, S. & Bachhawat, A. K. (1997). apd1+, a gene required for red pigment formation in ade6 mutants of Schizosaccharomyces pombe, encodes an enzyme required for glutathione biosynthesis: a role for glutathione and a glutathione-conjugate pump. Genetics 145, 7583.
Chignell, C. F. & Sik, R. H. (2003). A photochemical study of cells loaded with 2',7'-dichlorofluorescein: implications for the detection of reactive oxygen species generated during UVA irradiation. Free Radic Biol Med 34, 10291034.[CrossRef][Medline]
Dahlin, D. C., Miwa, G. T., Lu, A. Y. & Nelson, S. D. (1984). N-acetyl-p-benzoquinoneimine: a cytochrome P-450 mediated oxidation product of acetaminophen. Proc Natl Acad Sci U S A 81, 13271331.[Abstract]
Davidson, D. G. D. & Eastham, W. N. (1966). Acute liver necrosis following overdose with acetaminophen. Br Med J 2, 497.
Decottignies, A. & Goffeau, A. (1997). Complete inventory of the yeast ABC proteins. Nat Genet 15, 137145.[Medline]
Decottignies, A., Lambert, L., Catty, P., Degand, H., Epping, E. A., Moye-Rowley, W. S., Balzi, E. & Goffeau, A. (1995). Identification and characterization of SNQ2, a new multidrug ATP binding cassette transporter of the yeast plasma membrane. J Biol Chem 270, 1815018157.
Delaunay, A., Pflieger, D., Barrault, M. B., Vinh, J. & Toledano, M. B. (2002). A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation. Cell 111, 471481.[Medline]
DeRisi, J. L., Iyer, V. R. R. & Brown, P. O. (1997). Exploring the metabolic and genetic control of gene expression on a genome scale. Science 278, 680686.
Grant, C. M., Collinson, L. P., Roe, J. H. & Dawes, I. W. (1996). Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP-1 transcriptional regulation. Mol Microbiol 21, 171179.[Medline]
Guarente, L. (1983). Yeast promoters and LacZ fusions designed to study expressions of cloned genes in yeast. Methods Enzymol 101, 181191.[Medline]
Herzenberg, L. A., De Rosa, S. C., Dubs, J. G., Roederer, M., Anderson, M. T., Ela, S. W., Deresinski, S. C. & Herzenberg, L. A. (1997). Glutathione deficiency is associated with impaired survival in HIV disease. Proc Natl Acad Sci U S A 94, 19671972.
Hinson, J. A. (1980). Biological toxicology of acetaminophen. Rev Biochem Toxicol 2, 103129.
Howie, D., Asriaenssens, P. & Prescott, L. F. (1977). Paracetamol metabolism following overdose: application of high performance liquid chromatography. J Pharm Pharmacol 29, 235237.[Medline]
Ioannides, C., Steele, C. M. & Parke, D. V. (1983). Species variation in the metabolic activation of paracetamol to toxic intermediates: role of cytochrome P450 and P-448. Toxicol Lett 16, 5561.[CrossRef][Medline]
Jollow, D. J., Thorgeirsson, S. S., Potter, W. Z., Hashimoto, M. & Mitchell, J. R. (1974). Acetaminophen-induced hepatic necrosis. VI. Metabolic disposition of toxic and nontoxic doses of acetaminophen. Pharmacology 12, 251271.[CrossRef][Medline]
Jungwirth, H., Wendler, F., Platzer, B., Bergler, H. & Hogenauer, G. (2000). Diazaborine resistance in yeast involves the efflux pumps Ycf1p and Flr1p and is enhanced by a gain-of-function allele of gene YAP1. Eur J Biochem 267, 48094816.
Kanazawa, S., Driscoll, M. & Struhl, K. (1988). ATR1, a Saccharmoyces cerevisiae gene encoding a transmembrane protein required for aminotriazole resistance. Mol Cell Biol 8, 664673.[Medline]
Kaur, R. & Bachhawat, A. K. (1999). The yeast multidrug resistance protein, pdr5p, confers reduced drug resistance in the erg mutants of Saccharomyces cerevisiae. Microbiology 145, 809819.[Medline]
Klein, M., Mamnun, Y. M., Eggmann, T., Schuller, C., Wolfger, H., Martinoia, E. & Kuchler, K. (2002). The ATP-binding cassette (ABC) transporter Bpt1p mediates vacuolar sequestration of glutathione conjugates in yeast. FEBS Lett 520, 6367.[CrossRef][Medline]
Kozovska, Z., Maraz, A., Magyar, I. & Subik, J. (2001). Multidrug resistance as a dominant molecular marker in transformation of wine yeast. J Biotechnol 92, 2735.[CrossRef][Medline]
Le Crom, S., Devaux, F., Marc, P., Zhang, X., Moye-Rowley, W. S. & Jacq, C. (2002). New insights into the pleiotropic drug resistance network from genome-wide characterization of the YRR1 transcription factor regulation system. Mol Cell Biol 22, 26422649.
Mamnun, Y. M., Pandjaitan, R., Mahe, Y., Delahodde, A. & Kuchler, K. (2002). The yeast zinc finger regulators Pdr1p and Pdr3p control pleiotropic drug resistance (PDR) as homo- and heterodimers in vivo. Mol Microbiol 46, 14291440.[CrossRef][Medline]
Mitchell, J. R., Jollow, D. J., Potter, W. Z., Davies, C. C., Gillette, J. R. & Brodie, B. B. (1973). Acetaminophen induced hepatic necrosis. I. Role of drug metabolism. J Pharmacol Exp Ther 187, 195202.[Medline]
Monks, T. J. & Lau, S. S. (1998). The pharmacology and toxicology of polyphenolic-glutathione conjugates. Annu Rev Pharmacol Toxicol 38, 229255.[CrossRef][Medline]
Morgan, B. A., Banks, G. R., Toone, W. M., Raitt, D., Kuje, S. & Johnston, L. S. (1997). The Skn7 response regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae. EMBO J 16, 10351044.
Moye-Rowley, W. S., Harshman, K. D. & Parker, C. S. (1988). YAP1 encodes a yeast homolog of mammalian transcription factor AP-1. Cold Spring Harbor Symp Quant Biol 53, 711717.[Medline]
Myhre, O., Andersen, J. M., Aarnes, H. & Fonnum, F. (2003). Evaluation of the probes 2',7'-dichlorofluorescein diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem Pharmacol 65, 15751582.[CrossRef][Medline]
Pascolo, L., Petrovic, S., Cupelli, F., Bruschi, C. V., Anelli, P. L., Lorusso, V., Visigalli, M., Uggeri, F. & Tiribelli, C. (2001). ABC protein transport of MRI contrast agents in canalicular rat liver plasma vesicles and yeast vacuoles. Biochem Biophys Res Commun 282, 6066.[CrossRef][Medline]
Petrovic, S., Pascolo, L., Gallo, R., Cupelli, F., Ostrow, J. D., Goffeau, A., Tiribelli, C. & Bruschi, C. V. (2000). The products of YCF1 and YLL015w (BPT1) cooperate for the ATP-dependent vacuolar transport of unconjugated bilirubin in Saccharomyces cerevisiae. Yeast 16, 561571.[CrossRef][Medline]
Potter, W. Z., Thorgeirsson, S. S., Jollow, D. J. & Mitchell, J. R. (1974). Acetaminophen-induced hepatic necrosis. V. Correlation of hepatic necrosis, covalent binding and glutathione depletion in hamsters. Pharmacology 12, 129143.[Medline]
Prescott, L. F. (1983). The treatment of acetaminophen poisoning. Drugs 25, 290314.[Medline]
Ray, S. D., Kamendulis, L. M., Gurule, M. W., Yorkin, R. D. & Corcoran, G. B. (1993). Ca2+ antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen. FASEB J 7, 453463.
Ruepp, S. U., Tonge, R. P., Shaw, J., Wallis, N. & Pognan, F. (2002). Genomics and proteomics analysis of acetaminophen toxicity in mouse liver. Toxicol Sci 65, 135150.
Sevos, J., Hasse, E. & Brendel, M. (1993). Gene SNQ2 of Saccharomyces cerevisiae, which confers resistance to 4-nitroquinoline-N-oxide and other chemicals, encodes a 169 kDa protein homologous to ATP-dependent permeases. Mol Gen Genet 236, 214218.[Medline]
Sharma, K. G., Sharma, V., Bourbouloux, A., Delrot, S. & Bachhawat, A. K. (2000). Delayed growth stasis upon glutathione depletion in Saccharomyces cerevisiae: evidence for an overlapping role played by thioredoxin. Current Genetics 38, 7177.[CrossRef][Medline]
Sharma, K. G., Mason, D. L., Liu, G., Rea, P. A., Bachhawat, A. K. & Michaelis, S. (2002). Localization, regulation and substrate transport properties of Bpt1p, a Saccharomyces cerevisiae MRP-type ABC transporter. Eukaryot Cell 1, 391400.
Sharma, K. G., Kaur, R. & Bachhawat, A. K. (2003). The glutathione-mediated detoxification pathway in yeast: an analysis using the adenine pigment phenotype that accumulates in certain adenine biosynthetic mutants of yeasts reveals the involvement of novel genes. Arch Microbiol 180, 108117.[CrossRef][Medline]
Skaggs, B. A., Alexander, J. F., Pierson, C. A., Schweitzer, K. S., Chun, K. T., Koegel, C., Barbuch, R. & Bard, M. (1996). Cloning and characterization of the Saccharomyces cerevisiae C-22 sterol desaturase gene, encoding a second cytochrome P-450 involved in ergosterol biosynthesis. Gene 169, 105109.[CrossRef][Medline]
Tenreiro, S., Fernandes, A. R. & Sá-Correia, I. (2001). Transcriptional activation of FLR1 gene during Saccharomyces cerevisiae adaptation to growth with benomyl: role of Yap1p and Pdr3p. Biochem Biophys Res Commun 280, 216222.[CrossRef][Medline]
Wach, A., Brachat, A., Pohlman, R. & Philippsen, P. (1994). New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 17931808.[Medline]
Wehrschutz-Sigl, E., Jungwirth, H., Bergler, H. & Hogenauer, G. (2004). The transporters Pdr5p and Snq2p mediate diazaborine resistance and are under the control of the gain-of-function allele PDR1-12. Eur J Biochem 271, 11451152.
Wemmie, J. A., Szezypka, M. S., Thiele, D. J. & Moye-Rowley, W. S. (1994). Cadmium tolerance mediated by the yeast AP-1 protein requires the presence of an ATP-binding cassette transporter-encoding gene, YCF1. J Biol Chem 269, 3259232597.
Wendler, F., Bergler, H., Prutej, K., Jungwirth, H., Zisser, G., Kuchler, K. & Hogenauer, G. (1997). Diazaborine resistance in the yeast Saccharomyces cerevisiae reveals a link between YAP1 and the pleiotropic drug resistance genes PDR1 and PDR3. J Biol Chem 272, 2709127098.
Wu, Y., Zhang, X., Bardag-Gorce, F. & 8 other authors (2004). Retinoid X receptor alpha regulates glutathione homeostasis and xenobiotic detoxification processes in mouse liver. Mol Pharmacol 65, 550557.
Xiong, H., Turner, K. C., Ward, E. S., Jansen, P. L. & Brouwer, K. L. (2000). Altered hepatobiliary disposition of acetaminophen glucuronide in isolated perfused livers from multidrug resistance-associated protein 2-deficient TR() rats. J Pharmacol Exp Ther 295, 512518.
Received 3 June 2004;
revised 3 August 2004;
accepted 24 September 2004.
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