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Functional Profiling Discovers the Dieldrin Organochlorinated Pesticide
Affects Leucine Availability in Yeast
Brandon D. Gaytán,* Alex V. Loguinov,* Stephen R. Lantz,* Jan-Michael Lerot,* Nancy D. Denslow,† and Chris D. Vulpe*,1
*Department of Nutritional Science and Toxicology, University of California, Berkeley, California 94720; and †Department of Physiological Sciences and
Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida 32611
1To whom correspondence should be addressed at University of California, Berkeley, Nutritional Science and Toxicology, 317 Morgan Hall, Berkeley,
CA 94720. Fax: (510) 642-0535. E-mail: email@example.com.
Received December 4, 2012; accepted January 22, 2013
Exposure to organochlorinated pesticides such as dieldrin has
been linked to Parkinson’s and Alzheimer’s diseases, endocrine
disruption, and cancer, but the cellular and molecular mecha-
nisms of toxicity behind these effects remain largely unknown.
Here we demonstrate, using a functional genomics approach
in the model eukaryote Saccharomyces cerevisiae, that dieldrin
alters leucine availability. This model is supported by multiple
lines of congruent evidence: (1) mutants defective in amino acid
signaling or transport are sensitive to dieldrin, which is reversed
by the addition of exogenous leucine; (2) dieldrin sensitivity of
wild-type or mutant strains is dependent upon leucine concen-
tration in the media; (3) overexpression of proteins that increase
intracellular leucine confer resistance to dieldrin; (4) leucine
uptake is inhibited in the presence of dieldrin; and (5) dieldrin
induces the amino acid starvation response. Additionally, we
demonstrate that appropriate negative regulation of the Ras/pro-
tein kinase A pathway, along with an intact pyruvate dehydro-
genase complex, is required for dieldrin tolerance. Many yeast
genes described in this study have human orthologs that may
modulate dieldrin toxicity in humans.
Key Words: dieldrin; organochlorine; yeast; functional genom-
ics; alternative models.
The persistent and bioaccumulative nature of organochlorin-
ated pesticides (OCPs), combined with their widespread use
during the mid to late 20th century, resulted in pervasive envi-
ronmental contamination that exists to the present day. Dieldrin
and aldrin (which is converted to dieldrin in biological systems)
were two of the most heavily applied cyclodiene OCPs in the
United States, utilized to control insects on corn, cotton, and cit-
rus and to prevent or treat termite infestations (ATSDR, 2002).
Dieldrin has been detected in soil, water, air, wildlife, and
human samples (reviewed in Jorgenson, 2001) and is found at
159 of the 1363 current or proposed Environmental Protection
Agency National Priorities List (NPL) hazardous waste sites.
It is currently ranked 18th on the Agency for Toxic Substances
and Disease Registry (ATSDR) Priority List of Hazardous
Substances, a list of compounds that possibly threaten human
health on account of their toxicity and potential for exposure
at NPL sites. Although the use of dieldrin has been banned or
restricted in many countries, concern remains over its persis-
tence in sediment and potential to bioaccumulate in wildlife
and humans. Acute exposure to dieldrin results in antagonism
of the GABAA receptor, prompting excessive neurotrans-
mission and convulsions (ATSDR, 2002). In both animals
and humans, dieldrin has been linked to cancer (reviewed in
ATSDR, 2002; Jorgenson, 2001), Alzheimer’s and Parkinson’s
diseases (Richardson et al., 2006; Singh et al., 2013; Weisskopf
et al., 2010), and endocrine modulation (reviewed in Jorgenson,
2001), but the cellular and molecular mechanisms behind these
effects remain largely unknown.
The conservation of basic metabolic pathways and
fundamental cellular processes to humans, as well as unmatched
genetic resources, makes the eukaryotic yeast Saccharomyces
cerevisiae an ideal model system for identifying potential
cellular and molecular mechanisms of toxicity. Sequence
comparison has identified a close human homolog for much
of the yeast genome, with several hundred of the conserved
genes implicated in human disease (Steinmetz et al., 2002).
The development of a yeast deletion library (Giaever et al.,
2002) enables the use of functional toxicogenomics (also
known as functional profiling) to determine the importance
of individual yeast genes for toxicant susceptibility. Unlike
typical gene expression experiments that correlate toxicant
exposure to changes in mRNA levels, this approach identifies
genes functionally involved in toxicant response. Functional
profiling has been utilized to discover yeast genes required
for tolerance to a broad array of toxicants, including arsenic,
iron, benzene, and more (reviewed in dos Santos et al., 2012).
In addition, human homologs or functional orthologs of yeast
genes uncovered by this approach have been associated with
sensitivity to the same toxicant in human cells (Jo et al., 2009a).
toxicological sciences 132(2), 347–358 2013
Advance Access publication January 28, 2013
In this study, a genome-wide functional screen identified the
genetic requirements for tolerance to the dieldrin OCP in yeast.
To our knowledge, it is the first comprehensive report inves-
tigating the yeast genes necessary for growth in the presence
of a persistent organic pollutant, with the results demonstrat-
ing that dieldrin toxicity can be primarily attributed to altered
leucine availability. Additionally, both proper regulation of the
Ras/protein kinase A (PKA) pathway and components of the
pyruvate dehydrogenase (PDH) complex are required for diel-
drin tolerance. Many yeast genes involved in dieldrin resist-
ance have human homologs that may also play a role in dieldrin
MATERiAls And METHods
Yeast strains, culture, and plasmids. The diploid yeast deletion strains
used for functional profiling and confirmation analyses were of the BY4743
background (MATa/MATα his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2
MET15/met15Δ0 ura3Δ0/ura3Δ0, Invitrogen). The haploid yeast LEU2 MORF
overexpression strain was of the Y258 background (MATa, pep4-3, his4-580,
ura3-53, leu2-3,112, Open Biosystems). The BAP2 HIP FlexGene expression
vector, the B180 plasmid (GCN4-lacZ), and linearized pRS305 plasmid con-
taining the LEU2 gene were transformed into the BY4743 background. For
deletion pool growth, cells were grown in liquid rich media (1% yeast extract,
2% peptone, 2% dextrose, YPD), whereas confirmation assays were performed
in YPD or liquid synthetic complete media lacking leucine (0.68% yeast
nitrogen base without amino acids, 0.077% CSM-Leu dropout mixture, 2%
dextrose, SC-Leu) at 30°C with shaking at 200 revolutions per minute (rpm).
Starvation media (SD-N) was composed of 0.68% yeast nitrogen base without
amino acids and 2% dextrose. Protein overexpression was induced as in North
et al. (2011) using liquid rich media containing 2% galactose and 2% raffinose
Dose-finding and growth curve assays. Dose-finding and growth curve
experiments were performed as in North et al. (2011). Briefly, cells were grown
to mid-log phase, diluted to an optical density at 600 nm (OD600) of 0.0165,
and dispensed into nontreated polystyrene plates. Dieldrin (a gift from N.D.)
stock solutions were prepared in dimethyl sulfoxide (DMSO) and added to the
desired final concentrations (1% or less by volume) with at least two techni-
cal replicates per dose. Based upon literature searches for dieldrin toxicity in
human cells (Ledirac et al., 2005), the dieldrin yeast dose-response curve was
narrowed to 200–800μM, which was examined in three independent experi-
ments. Plates were incubated in Tecan microplate readers set to 30°C with
shaking and OD595 measurements were taken every 15 min for 24 h. The raw
absorbance data were averaged, background corrected, and plotted as a func-
tion of time. The area under the curve was calculated with Apache OpenOffice
Calc and expressed as a percentage of the untreated control.
Functional profiling of the yeast genome. Growth of the deletion pools,
genomic DNA extraction, barcode amplification, Affymetrix TAG4 array
hybridization, and differential strain sensitivity analysis (DSSA) were per-
formed as described (Jo et al., 2009b). Briefly, pools of homozygous diploid
deletion mutants (n = 4607) were grown in YPD at various dieldrin concentra-
tions for 15 generations and genomic DNA was extracted using the YDER kit
(Pierce Biotechnology). The DNA sequences unique to each strain (barcodes)
were amplified by PCR and hybridized to TAG4 arrays (Affymetrix), which
were incubated overnight, stained, and then scanned at an emission wavelength
of 560 nm with a GeneChip Scanner (Affymetrix). Data files are available at the
Gene Expression Omnibus database.
Overenrichment and network mapping analyses. Significantly over-
represented Gene Ontology (GO) and MIPS (Munich Information Center
for Protein Sequences) categories within the DSSA data were identified by
a hypergeometric distribution using the Functional Specification resource,
FunSpec, with a p value cutoff of 0.001 and Bonferroni correction. For the
network mapping, fitness scores for strains displaying sensitivity to at least
two doses of dieldrin were mapped onto the BioGrid S. cerevisiae functional
interaction network using the Cytoscape software. The jActiveModules plugin
then identified subnetworks of genes enriched with fitness data, and the BiNGO
plugin assessed overrepresentation of GO categories within these subnetworks.
Analysis of relative strain growth by flow cytometry. Assays were per-
formed as in North et al. (2012), with slight modifications. Briefly, green
fluorescent protein (GFP)-tagged wild-type and untagged mutant strains were
grown overnight in YPD, diluted to 0.5 OD600, and mixed in approximately
equal numbers. Cells were inoculated into YPD or SC-LEU at 0.00375 OD600
in microplate format, treated with dieldrin, and grown for 24 h at 30°C with
shaking at 200 rpm. Approximately 20,000 cells per culture were analyzed at
T = 0 and T = 24 h using a FACSCalibur flow cytometer. GFP-expressing wild-
type cells were distinguishable from untagged mutant cells. The percentages
of wild-type GFP and untagged mutant cells present in the cultures were used
to calculate a ratio of growth for untagged cells in treated versus untreated
samples. Statistically significant differences between the means of three inde-
pendent DMSO-treated and dieldrin-treated cultures were determined using
Student’s t-test. Raw p values were corrected for multiplicity of comparisons
using Benjamini-Hochberg correction.
Leucine uptake assays. Leucine transport was measured as in Heitman
et al. (1993), with slight modifications. Briefly, overnight cultures were diluted,
incubated for 4 h at 30°C to mid-logarithmic phase, and washed with wash
buffer (10mM sodium citrate, pH 4.5). Cells were resuspended in 10mM
sodium citrate (pH 4.5)-20mM (NH4)2SO2-2% glucose and the OD600 was
measured. Import reactions contained resuspended cells, DMSO or dieldrin at
a final concentration of 460μM, and 15.9 μl of L-[14C]leucine (53 mCi/mmol,
5μM final concentration) in a total of 3 ml. Aliquots (0.5 ml) of the import reac-
tion were taken at 0, 2, 5, 10, 30, and 60 min and vacuum filtered through
25 mm Whatman GF/C glass microfiber filters presoaked in wash buffer. Filters
were washed four times with 0.5 ml wash buffer containing 2mM unlabeled
L-leucine and bound radioactivity was quantified in Safety-Solve counting
cocktail using a Beckman LS-6000IC liquid scintillation counter.
Amino acid starvation analyses. Cells harboring the B180 plasmid (con-
taining a GCN4-lacZ reporter) were cultured overnight in SC-ura 2% dextrose,
diluted to 0.25 OD600, and spun down at 3500 rpm following a 5-h period of
growth. A wash and resuspension occurred in either SC-ura 2% dextrose or
starvation media (SD-N), upon which cells were aliquoted to a microplate and
treated with DMSO or the dieldrin IC20 (460μM) for 3 h. β-Galactosidase activ-
ity was assayed with the yeast β-galactosidase assay kit (ThermoScientific)
and Miller units were calculated with the equation (1000 × A420)/(minutes of
incubation × volume in milliliters × OD660).
A Genome-Wide Screen Identifies Mutants With Altered
Growth in the Presence of Dieldrin
Growth curve assays were performed to determine the toxic-
ity of dieldrin to yeast (Fig. 1B), based upon knowledge that
(1) dieldrin causes physiological effects in human cell culture
systems at 25–50μM (Ledirac et al., 2005) and (2) the cell wall
and abundant multidrug transporters often lend yeast resistance
to chemical insult. From the growth curves, the IC20, a con-
centration determined as appropriate for use in the functional
screen (Jo et al., 2009b), was calculated as 460μM (Fig. 1C). To
discover genes important for growth in dieldrin, pools of yeast
homozygous diploid deletion mutants (n = 4607) were grown
348 GAYTÁN ET AL.
for 15 generations at the IC20 (460μM), 50% IC20 (230μM), and
25% IC20 (115μM). A DSSA identified 427 mutants as sensi-
tive and 320 mutants as resistant to at least one dose of dieldrin
(Supplementary table 1), with the top 25 sensitive strains at the
IC20 shown in Table 1. Strains sensitive to dieldrin were the
focus of this study.
Enrichment Analyses and Network Mapping Identify
Attributes Necessary for Dieldrin Tolerance
A list of mutant strains displaying sensitivity to at least two of
the dieldrin treatments (n = 219) was analyzed for significantly
overrepresented biological attributes using FunSpec. Both GO
and MIPS categories were enriched for various classifications
Fig. 1. Dose determination of dieldrin IC20 for functional profiling. (A) The chemical structure of dieldrin. (B) Representative growth curves for the BY4743
wild-type strain treated with dieldrin in YPD media. Curves were performed for 200, 300, 400, 500, 600, and 800μM dieldrin, but for clarity, only the 200, 500,
and 800μM doses are shown. (C) The area under the curve (AUC) at each dose was expressed as the mean and SE of three independent experiments and plotted
as a percentage of the untreated control. Dashed lines indicate the dose (460μM) at which growth was inhibited by 20% (IC20).
Fitness scores for the Top 25 Mutants identified as significantly sensitive to the dieldrin iC20 (460μM) After
15 generations of growth
Deleted geneDescription25% IC20
GTPase-activating protein; negatively regulates Ras
GTPase-activating protein; involved in vesicle docking
High-affinity leucine permease
Major of three pyruvate decarboxylase isozymes
Dubious ORF; partially overlaps the verified gene RPE1
Putative integral membrane protein
Regulates PtdIns(4,5)P2 levels and autophagy
Regulator of PKA signal transduction pathway
MAPK involved in cell wall integrity and cell cycle progression
Unknown function; structurally similar to plant storage proteins
Component of the mitochondrial TIM22 complex
Negative regulatory subunit of protein phosphatase 1
Homolog of bacterial chaperone DnaJ
Tyr phosphatase; involved in actin organization and endocytosis
Calcineurin B; the regulatory subunit of calcineurin
Oxysterol-binding protein; functions in sterol metabolism
Ubiquitin chain assembly factor (E4)
Transcriptional activator; maintains ion homeostasis
Ubiquitin protease cofactor; forms complex with Ubp3p
Protein required for function of the Sit4p protein phosphatase
Nonessential protein of unknown function
GPI inositol deacylase; negatively regulates vesicle formation
Note. Fitness scores quantify the requirement of a gene for growth and are defined as the normalized log2 ratio of the deletion strain’s growth in the presence
versus absence of dieldrin. A total of 427 genes were important for fitness (i.e., had negative fitness scores) in at least one dieldrin treatment. Supplementary table 1
contains a list of all genes identified as significant by DSSA.
DIELDRIN ALTERS LEUCINE STATUS IN YEAST
at a corrected p value of 0.001, including nitrogen utilization,
protein phosphorylation, the PDH complex, negative regula-
tion of Ras signaling, and sensitivity to amino acid analogs
(Table 2). For additional insight into the attributes required for
dieldrin tolerance, network mapping was performed with the
Cytoscape visualization tool and plugins identifying enriched
categories. Similar to the FunSpec evaluation, nitrogen pro-
cesses and phosphorylation were overrepresented within the
network data (Fig. 2). These enrichment analyses guided the
selection of candidate cellular processes and components for
Mutants Defective in Amino Acid Uptake and Nitrogen
Signaling Are Sensitive to Dieldrin
Overrepresentation analyses with FunSpec and Cytoscape
implicated nitrogen processes as important for dieldrin tolerance.
Therefore, we used flow cytometry to assay relative growth of
a wild-type strain to mutants deficient in amino acid signaling
and uptake, as well as nitrogen utilization. Both DSSA and flow
cytometry identified bap2Δ, which lacks a gene encoding for a
high-affinity leucine permease, as one of the strains most sensi-
tive to dieldrin (Fig. 3A). Additional amino acid signaling genes
were confirmed to be required for dieldrin tolerance, including
genes Required for growth in the Presence of dieldrin and Their Associated MiPs or go Categories
GO biological process
p valueGenes identifiedka
Protein folding in endoplasmic reticulum
Regulation of nitrogen utilization
Protein phosphorylation (GO:0006468)
EMC1, JEM1, EMC2, SCJ1
VID30, NPR1, URE2
SAT4, GCN2, SLT2, IRE1, CKA1,
PBS2, YAK1, PTK2, RCK2,
NPR1, SSK2, MKK1, DBF20
GPB2, IRA2, GPB1
Negative regulation of Ras protein signal
MIPS functional classification
p valueGenes identifiedka
Regulation of nitrogen metabolism
Modification by phosphorylation, dephospho-
rylation, autophosphorylation (14.07.03)
GAT1, VID30, NPR1, URE2
SAT4, GCN2, SAP4, SLT2, IRE1,
CKA1, PBS2, YAK1, PTK2,
CNB1, RCK2, SIW14, OCA1,
NPR1, SSK2, MKK1, DBF20
SAT4, BCK2, SAP4, CKA1,
VHS2, PTK2, SIS2
RMD5, UBC8, VID30, PFK26,
PDB1, LPD1, PDX1
GCV3, LPD1, SHM2
SAT4, RBK1, GCN2, PPN1,
YND1, SAP1, SAP4, SLT2,
INM1, IRE1, CKA1, PFK26,
PBS2, YAK1, FBP26, PTK2,
CNB1, RCK2, SIW14, OCA1,
NPR1, PEX6, SSK2, LCB4,
G1/S transition of mitotic cell cycle
Regulation of glycolysis and gluconeogenesis
PDH complex (02.08)
Degradation of glycine (01.01.09.01.02)
Phosphate metabolism (01.04)
p valueGenes identifiedka
Sensitivity to other aminoacid analogs and other
RVS161, PDR1, LST7, PBS2,
PTK2, HMG2, LEU3, TAT2,
RVS161, VID30, SLT2, IRA2,
Starvation sensitivity (62.10)1.64E-004626
Note. A list of strains exhibiting sensitivity to at least two out of the three doses of dieldrin was entered into the FunSpec tool and analyzed for overrepresented
biological attributes (see Materials and Methods section).
aNumber of genes in category identified as sensitive to dieldrin.
bNumber of genes in GO or MIPS category.
350 GAYTÁN ET AL.
NPR1 (a kinase that prevents degradation of several amino acid
transporters), STP1 (a component of the Ssy1p-Ptr3p-Ssy5p SPS
system that transduces extracellular amino acid status), and LEU3
(a transcription factor regulating branched-chain amino acid bio-
synthesis and amino acid permeases) (Fig. 3A). The NPR2, NPR3,
and URE2 genes involved in the cellular response to nitrogen
were also identified as necessary for growth in dieldrin (Fig. 3A).
Leucine Availability Is Linked to Dieldrin Toxicity
With the determination that bap2Δ (which lacks a high-
affinity leucine permease) was sensitive to dieldrin, we
hypothesized that supplementation of YPD-dieldrin medium
with excess leucine might mitigate the toxicity of dieldrin.
Indeed, addition of 5mM leucine reversed the sensitivity not
only of bap2Δ but also leu3Δ, npr1Δ, and stp1Δ to dieldrin
Fig. 2. Cytoscape network mapping identifies biological attributes required for dieldrin tolerance. Fitness scores (the difference in the mean of the log2 hybrid-
ization signal between DMSO and dieldrin treatment) for strains displaying sensitivity to at least two dieldrin treatments were mapped onto the Saccharomyces
cerevisiae BioGrid interaction data set using Cytoscape. A smaller subnetwork (235 genes) was created containing genetic and physical interactions between the
sensitive, nonsensitive, and essential genes. Significantly overrepresented (p value cutoff of 0.03) GO categories were visualized as a network in which the green
node color corresponds to significance, whereas node size indicates the number of genes present in the category. Edge arrows indicate hierarchy of GO terms. Gene
networks corresponding to various GO categories are shown, where node color signifies the deletion strain fitness score (fitness not determined for white nodes)
and edge styling defines the interaction between nodes.
DIELDRIN ALTERS LEUCINE STATUS IN YEAST
(Fig. 3A). Although leucine moderately rescued the dieldrin
sensitivity of the ure2Δ mutant, it did not rescue npr2Δ or
npr3Δ (Fig. 3A). Bap2p can also transport isoleucine, valine,
and tryptophan (Regenberg et al., 1999), but supplement-
ing YPD medium with these amino acids did not reverse the
sensitivity of bap2Δ to dieldrin (Fig. 3B). Deletion of addi-
tional amino acid transporter genes (AGP1, BAP3, GAP1,
and GNP1) known to facilitate uptake of leucine (Regenberg
et al., 1999) or other amino acids did not result in sensitiv-
ity to dieldrin (Supplementary fig. 1). To further demonstrate
that dieldrin toxicity in yeast was linked to leucine availability,
wild-type BY4743 and bap2Δ strains were grown in media
containing defined concentrations of leucine. Dieldrin sensi-
tivity was exacerbated at low concentrations of leucine and
remediated by increased leucine levels in the media (Figs. 4A
and B). The deletion strains utilized in this study were leucine
auxotrophs of the BY4743 background that lacks LEU2, the
β-isopropylmalate deyhydrogenase responsible for catalyz-
ing the penultimate step in leucine biosynthesis. Restoration
of leucine prototrophy through knock-in of the LEU2 gene
resulted in resistance to dieldrin at decreased concentrations
of leucine (Fig. 5A). Finally, overexpression of Bap2p (the
high-affinity leucine permease) or Leu2p (an enzyme involved
in leucine biosynthesis) conferred resistance to dieldrin (Figs.
5B and C). Collectively, these data suggest that leucine avail-
ability plays a key role in the response to dieldrin.
Dieldrin Inhibits Leucine Uptake and Causes Amino Acid
BY4743 strains are leucine auxotrophs dependent on leucine
import from the external environment. It was therefore hypoth-
esized that dieldrin did not affect leucine availability by inter-
fering with leucine biosynthesis but instead altered the uptake
of leucine from the media. To test this, mid-log phase wild-type
cells were incubated with radiolabeled leucine in the presence
or absence of the dieldrin IC20 (460μM). Results show that diel-
drin significantly inhibited leucine import at various time points
as compared with untreated controls (Fig. 6A). With leucine
uptake inhibited, it was anticipated that dieldrin would starve
the cell for leucine and induce the general control response, a
signaling cascade in which increased GCN4 mRNA levels pro-
mote transcription of amino acid biosynthetic machinery and
permeases (Hinnebusch, 1990). To examine amino acid starva-
tion, a plasmid harboring a GCN4-lacZ fusion gene was trans-
formed into yeast cells and assayed for β-galactosidase activity
following dieldrin exposure. As shown in Figure 6B, dieldrin
induces β-galactosidase expression from the GCN4-lacZ fusion
in both wild-type and bap2Δ cells, demonstrating that amino
Fig. 3. Dieldrin sensitivity of mutants involved in amino acid or nitrogen processes is reversed by leucine. Deletion mutants were tested for sensitivity to the
dieldrin IC25 (690μM) by flow cytometry, in which relative growth of each mutant was compared with a wild-type GFP strain after 24 h. Means of the growth ratios
(treatment vs. control—T/NT) to wild-type GFP are shown with SE for three independent YPD cultures. Significance values were calculated by Student’s t-test,
where ap < 0.001 and bp < 0.01 for dieldrin-treated wild-type versus mutant, whereas ***p < 0.001, **p < 0.01, and *p < 0.05 for dieldrin versus dieldrin-leucine
treatment. (A) Amino acid uptake and signaling mutants, as well as those involved in nitrogen utilization, are sensitive to dieldrin, with most mutants rescued by
addition of 5mM leucine. (B) Amino acids related to leucine or transported by Bap2p cannot reverse dieldrin sensitivity in bap2Δ. Leucine, isoleucine, valine, and
histidine were added to YPD media at a final concentration of 5mM, whereas tryptophan was present at 2.5mM.
352 GAYTÁN ET AL.
acid starvation occurs in the presence of dieldrin. Interestingly,
although autophagy mutants (which are involved in the star-
vation response) were identified as sensitive by DSSA at the
highest dose of dieldrin, we were unable to confirm growth
defects in YPD (after 24 or 48 h) or leucine deficient media
(Supplementary table 2 and data not shown).
Ras/PKA Signaling, But Not the Target of Rapamycin
Pathway, Is Implicated in Dieldrin Toxicity
In S. cerevisiae, the two main nutrient signal transduction
pathways are Ras/PKA and target of rapamycin (Tor); signal-
ing through either may be affected during starvation for amino
acids such as leucine. Rapamycin inhibits the yeast Tor path-
way and induces a nitrogen starvation–like phenotype via
formation of a toxic complex with Fpr1p (FKBP12) (Lorenz
and Heitman, 1995). We hypothesized that dieldrin was acting
similarly to rapamycin, as rapamycin also affects amino acid
availability (Beck et al., 1999) and various amino acid sign-
aling mutants sensitive to dieldrin (Fig. 3A) have been con-
firmed as sensitive to rapamycin (Xie et al., 2005). However,
multiple lines of evidence suggest otherwise. Deletion of
FPR1 or TOR1 confers resistance or sensitivity, respectively, to
rapamycin (Xie et al., 2005) but neither of these mutants was
affected by dieldrin (Fig. 7A). Moreover, removal of compo-
nents downstream of Tor (SIT4, SAP4, SAP155, TIP41, RRD1,
and RRD2) and peptidyl-prolyl cis-trans isomerases related to
Fpr1p (FPR2, FPR3, and FPR4) did not affect growth in diel-
drin (Supplementary table 2 and data not shown). In contrast,
mutants known to exhibit altered Ras/PKA signaling were sen-
sitive to dieldrin, including those unable to negatively regulate
Ras (gpb1Δ, gpb2Δ, and ira2Δ) or PKA (bcy1Δ, deleted for
the PKA regulatory subunit), with most unable to be rescued
by leucine addition (Fig. 7B). Other mutants (pfk26Δ, fpb26Δ,
rmd5Δ, and vid30Δ) lacking genes involved in the regulation
Fig. 4. Limiting leucine exacerbates dieldrin sensitivity. Cells were cul-
tured in media containing defined concentrations of leucine. (A) The BY4743
wild-type strain is dependent on leucine for dieldrin tolerance. Growth curves
were performed for the indicated doses of dieldrin and the area under the curve
(AUC) was calculated. Graphs express AUC as a percentage of untreated wild-
type with SE for three independent cultures. Statistical significance between
the 2mM leucine AUC and the 0.5, 0.75, and 1mM leucine AUCs was deter-
mined by Student’s t-test, where ***p < 0.001 and *p < 0.05. (B) The bap2Δ
strain exhibits increased sensitivity to the dieldrin IC25 (690μM) at decreased
leucine concentrations. Flow cytometry confirmed altered growth ratios, with
data displayed as the mean and SE of three independent cultures. Statistical
significance between the corresponding leucine doses in wild-type and bap2Δ
was calculated by Student’s t-test, where ***p < 0.001 and **p < 0.01.
Fig. 5. Increasing intracellular leucine results in dieldrin resistance. All data shown represent the mean and SE for three independent cultures. (A) Knock-in
of the LEU2 gene into BY4743 wild-type increases dieldrin resistance. Cells were cultured in media (SC-LEU) containing defined concentrations of leucine along
with the dieldrin IC25 (690μM) and assayed for relative growth to the GFP-expressing BY4743 wild-type strain, which lacks LEU2. Resistance was not seen in
YPD media (data not shown). Data were analyzed with two-way ANOVA with a Bonferroni posttest, where ***p < 0.001, compared with the corresponding leucine
dose in wild-type. (B) Wild-type or bap2Δ strains overexpressing Bap2p exhibit increased resistance to dieldrin. Cells harboring empty vector or the HIP FlexGene
BAP2 ORF were cultured in SC-LEU media containing 1mM leucine and the dieldrin IC25 (690μM). Relative growth to a wild-type GFP strain was assayed by flow
cytometry and statistical significance was determined by Student’s t-test, where ***p < 0.001 and **p < 0.01. (C) Overexpression of Leu2p imparts resistance to
dieldrin in the Y258 haploid wild-type strain. Growth curve analyses were performed in YPD for dieldrin-treated (IC25: 690μM) Y258 cells overexpressing Leu2p.
The area under the curve (AUC) is expressed as a percentage of the untreated strain. Statistical significance was calculated with Student’s t-test, with *p < 0.05.
DIELDRIN ALTERS LEUCINE STATUS IN YEAST
of glucose metabolism, a process under the control of PKA,
were sensitive to dieldrin (Supplementary fig. 2A), with
rmd5Δ displaying leucine-dependent sensitivity to dieldrin
(Supplementary fig. 2B). Together, these data show that proper
Ras/PKA regulation is required for dieldrin tolerance, but the
Tor pathway is not.
The PDH Complex Is Necessary for Resistance to Dieldrin
The mitochondrially localized PDH complex catalyzes the
oxidative decarboxylation of pyruvate to acetyl-CoA, thus link-
ing glycolysis to the citric acid cycle. Our DSSA and meta-
analysis identified four out of the five subunits of PDH (Lpd1p,
Lat1p, Pdx1p, and Pdb1, but not Pda1p) as sensitive to dieldrin,
which was confirmed by the flow cytometry relative growth
assay (Fig. 8A and Supplementary table 2). Surprisingly,
exogenous leucine moderately reversed the sensitivity of these
mutants to dieldrin. In addition, similar to BY4743 wild-type,
bap2Δ, and rmd5Δ (Fig. 4 and Supplementary fig. 2B), the
pdb1Δ and lat1Δ PDH mutants were more sensitive to diel-
drin when the leucine concentration in the media was decreased
Dieldrin is a bioaccumulative and persistent pollutant with
the potential to cause adverse effects on both the environment
and human health (ATSDR, 2002). In this study, we performed
a functional screen to identify nonessential yeast deletion
mutants experiencing growth defects in dieldrin. Functional
profiling of yeast mutants has not been reported for dieldrin or
any other environmentally persistent halogenated contaminant.
Confirmation of the yeast genes required for dieldrin tolerance,
many of which are conserved in humans (Table 3), suggested
a mechanism of toxicity—altered leucine availability—that
was validated by further experimentation. In yeast, the toxic
mechanism of dieldrin is different from that of the toxaphene
OCP (Gaytán et al., in preparation). Overlapping GO catego-
ries were not identified between our study and gene expression
profiles in dieldrin-exposed largemouth bass (Martyniuk et al.,
2010), but specific transcriptional responses do not always cor-
relate with genes required for growth under a selective condi-
tion (Giaever et al., 2002).
Yeast functional genomics, although an invaluable tool in the
field of toxicology, is not without its limitations. First, achieving
toxicity in yeast often requires high concentrations of xenobiotic;
considering the presence of a cell wall and multidrug resistance
machinery, this is not surprising. To increase toxicant sensitivity,
a strain deleted for important drug resistance transporters could
potentially serve as the deletion library background. Dieldrin
concentrations used in this study (115–690μM) are higher than
those reported as toxic to human cells (25–50μM) (Ledirac
et al., 2005). Since the ban of dieldrin, National Health and
Nutrition Examination Surveys (NHANES) describe a steady
decrease in mean human blood serum concentrations (maxi-
mum 1.4 ppb in the 1976–1980 data); therefore, our results may
be most relevant to (1) those with a history of dieldrin exposure;
(2) populations living in termiticide-treated homes; and (3) com-
munities consuming fish or other bioaccumulating aquatic spe-
cies caught adjacent to dieldrin-contaminated hazardous waste
sites. Second, although endogenous P450 enzymes can mediate
Fig. 6. Dieldrin inhibits leucine uptake and induces the starvation response.
(A) Leucine uptake is inhibited in the presence of dieldrin. Radiolabeled leu-
cine was incubated with yeast cells with or without the dieldrin IC20 (460μM)
and uptake was measured by counting radioactivity bound to the filter. Each
time point was normalized for cell number and expressed as a percentage of
combined total measured radioactivity over the time course for the control.
The means and SEs for three independent experiments are shown. Statistical
significance between corresponding time points was determined by Student’s
t-test, where *p < 0.05. (B) Dieldrin induces amino acid starvation. GCN4-lacZ
expression was measured via β-galactosidase activity after treating wild-type
or bap2Δ cells with the dieldrin IC20 (460μM) in SC-ura or SD-N media. The
means and SEs for two to three independent cultures are shown. Data were
analyzed with Student’s t-test. ***p < 0.001, **p < 0.01, and *p < 0.05.
354 GAYTÁN ET AL.
xenobiotic biotransformation in yeast (Käppeli, 1986), differ-
ences in metabolism complicate direct comparison with humans.
To address these concerns, human S-9 liver microsomes may be
added to catalyze toxicant activation. Third, one cannot identify
target organs or adverse systemic effects, such as perturbations
of the endocrine, immune, or circulatory systems. The discovery
of nonobvious equivalent mutant phenotypes between different
species (i.e., orthologous phenotypes) may prove useful in this
arena. For example, McGary et al. (2010) identified five genes
shared between studies examining yeast deletion strain sensi-
tivity to the hypercholesterolemia drug lovastatin and abnormal
angiogenesis in mutant mice, suggesting that despite the lack
of blood vessels, yeast can model the genetics of mammalian
vasculature formation. Similar analyses of yeast functional toxi-
cogenomics data, although not performed within this study, may
reveal potential mechanisms of action related to more complex
biological processes not present in yeast.
Compounds other than dieldrin alter amino acid availability
in yeast, including the immunosuppressant drugs rapamycin
(Beck et al., 1999), FK506 (Heitman et al., 1993), and FTY720
(Welsch et al., 2003), the antimalarial drug quinine (Khozoie
et al., 2009), the anesthetic isoflurane (Palmer et al., 2002),
and the orphan drug phenylbutyrate (Grzanowski et al., 2002).
The chemical structure of dieldrin does not exhibit similarity
to any of these compounds. Portions of our data suggested that
the mechanism of action for dieldrin is similar to rapamycin,
but removal of the rapamycin targets Fpr1p or Tor1p, which
results in rapamycin resistance and sensitivity, respectively,
does not affect growth in the presence of dieldrin (Fig. 7A).
FK506 and FTY720 inhibit uptake of leucine and trypto-
phan (Heitman et al., 1993; Welsch et al., 2003); however, a
mutant lacking TAT2, a high-affinity tryptophan and tyrosine
permease, was not sensitive to dieldrin (Supplementary fig. 1).
Moreover, inhibition of amino acid uptake is dependent upon
a 4–5 h preincubation of yeast cells with FK506 or FTY720,
indicating that time is needed for the drug’s effects, possibly
because transporter folding, assembly, or transport is altered. In
contrast, dieldrin inhibited amino acid uptake without preincu-
bation, a result similar to that for the antimalarial drug quinine,
which competitively inhibits tryptophan uptake via the Tat2p
permease (Khozoie et al., 2009). Further studies are needed to
determine if dieldrin inhibits amino acid transport by binding
directly to a leucine permease.
Although our results do not indicate that dieldrin’s toxic
mechanism in yeast is conserved to humans, studies have dem-
onstrated that dieldrin and other OCPs can alter availability of
amino acids or their derivatives in mammalian systems. An oral
dose of dieldrin to rhesus monkeys depressed leucine uptake
in the intestine (Mahmood et al., 1981), whereas a series of ip
injections of lindane, a related OCP, decreased leucine trans-
port in chicken enterocytes (Moreno et al., 1994). Leucine
uptake was decreased in rat intestine after a single oral dose to
endosulfan, another OCP (Wali et al., 1982). Of greater con-
cern is the potential for dieldrin, a known neurotoxicant linked
to Alzheimer’s and Parkinson’s diseases, to affect the levels
of amino acids or their derivatives in the brain. Several neu-
rotransmitters are amino acids (glutamate, aspartate, and gly-
cine) or amino acid derivatives (tryptophan is the precursor for
serotonin, tyrosine for dopamine, and glutamate for GABA).
Leucine is neither a neurotransmitter nor a precursor, but it fur-
nishes α-NH2 groups for glutamate synthesis via the branched-
chain amino acid aminotransferase, thus playing a major role
in regulating cellular pools of the glutamate neurotransmitter
(Yudkoff et al., 1994). Further experimentation is necessary to
determine whether dieldrin inhibits leucine uptake in human
cells or more complex organisms.
Genes encoding proteins that negatively regulate Ras (the
GPB1/GPB2 paralogs and IRA2) or PKA (BCY1) are required
Fig. 7. Altered Ras/PKA, but not Tor signaling, causes dieldrin sensitivity. Relative growth ratios (treatment vs. control) to the GFP-expressing wild-type
strain were obtained. All data represent the mean and SE for three independent YPD cultures treated with the dieldrin IC25 (690μM). Statistical significance
between dieldrin-treated wild-type and mutant strains were determined with Student’s t-test, where ap < 0.001 and bp < 0.01. Statistical differences between a
dieldrin-treated strain versus the same strain treated with dieldrin and leucine are shown as *p < 0.05. (A) Dieldrin does not affect strains lacking components
involved in Tor signaling. (B) Strains unable to negatively regulate Ras or PKA are sensitive to dieldrin.
DIELDRIN ALTERS LEUCINE STATUS IN YEAST
for dieldrin tolerance (Fig. 7B). Combined with evidence
that gpb1Δgpb2Δ ira2Δ and bcy1Δ strains are intolerant to
nitrogen starvation (Harashima and Heitman, 2002; Tanaka
et al., 1990), these data are consistent with dieldrin’s ability to
decrease leucine (nitrogen) availability (Fig. 6A) and induce
nitrogen starvation via Gcn4p (Fig. 6B). Double GPB1/2 as
well as single IRA2 or BCY1 deletion mutants also display phe-
notypes consistent with hyperactive Ras or PKA (Harashima
and Heitman, 2002), phenomena linked to cancer in humans.
A constitutively active Ras2Val19 protein reduces the response
of leucine transport to a poor nitrogen source (Sáenz et al.,
1997), but experiments with a Ras2Val19 allele did not alter
sensitivity to dieldrin (data not shown). Dieldrin has tumor-
promoting properties in rodents (ATSDR, 2002), possibly
via inhibition of intracellular gap junction channels (Matesic
et al., 2001). Chaetoglobosin K, a compound with Ras tumor
suppressor activity, alleviates dieldrin inhibition of gap junc-
tion channels (Matesic et al., 2001). Dieldrin also affects
signaling downstream of Ras, increasing phosphorylated Raf,
MEK1/2, and ERK1/2 in human keratinocytes (Ledirac et al.,
2005). Human homologs of the Ras/PKA signaling genes
required for dieldrin resistance in yeast are posited tumor sup-
pressors, including the IRA2 homolog neurofibromin 1 and the
GPB1/2 functional ortholog ETEA (Phan et al., 2010). If our
data are validated in higher organisms, it may suggest that an
individual with altered Ras/PKA signaling is more susceptible
Components of the highly conserved mitochondrial PDH
complex were also identified as necessary for dieldrin tolerance
(Fig. 8 and Table 3). PDH links glycolysis to the citric acid cycle
by catalyzing the oxidative decarboxylation of pyruvate to acetyl-
CoA (reviewed in Pronk et al., 1996). Leucine modestly reversed
the sensitivity of PDH mutants to dieldrin, indicating that leucine
is necessary for or a product of a PDH-mediated process needed
for dieldrin tolerance. Except for LAT1, all PDH genes contain
putative recognition sites for the master regulator of the amino
Fig. 8. The PDH complex is required for dieldrin tolerance. Relative
growth ratios (treatment vs. control) to the GFP-expressing wild-type strain were
obtained for three independent YPD cultures, for which the means and SEs are
shown. Dieldrin was added at a final concentration of 690μM (IC25). (A) Four
PDH subunits are necessary for dieldrin resistance in YPD. (B) The lat1Δ and
pdb1Δ strains exhibit dieldrin sensitivity that is dependent on leucine concentra-
tion. Strains were grown in media containing defined concentrations of leucine
and assayed for relative growth to a wild-type GFP strain. Statistical significance
between corresponding leucine doses in wild-type and mutant strains was deter-
mined by Student’s t-test, where ***p < 0.001, **p < 0.01, and *p < 0.05.
selected Yeast genes Required for dieldrin Tolerance and Their Human orthologs
Human orthologHuman protein
Cationic amino acid transport permease
cAMP-dependent protein kinase regulatory subunit
Eukaryotic translation initiation factor 2-alpha kinase
Functional ortholog of GPB1/2; inhibits neurofibromin 1
Neurofibromin 1, tumor suppressor protein
Dihydrolipoamide acetyltransferase component of PDH complex
Dihydrolipoamide dehydrogenase component of PDH complex
Nitrogen permease regulator-like 2, tumor suppressor candidate
PDH, E1 component
Anchors DLD to the DLAT core in the PDH complex
v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
Required for meiotic nuclear division 5 Saccharomyces cerevisiae homolog
Ran-binding protein 10
aDeletion of any of these genes caused sensitivity to dieldrin (listed in alphabetical order).
356 GAYTÁN ET AL.
acid starvation response, GCN4 (Wenzel et al., 1992), with LPD1
under the control of Gcn4p during amino acid starvation (Zaman
et al., 1999). This suggests that PDH is involved in the starvation
response, which we show is induced by dieldrin (Fig. 6B). In
addition, the pda1Δ strain demonstrates a partial leucine require-
ment for growth (Wenzel et al., 1992). Reduced PDH activity has
been associated with various neurodegenerative diseases, includ-
ing Alzheimer’s and Huntington’s diseases (Sorbi et al., 1983),
and mice deficient in dihydrolipoamide dehydrogenase (DLD—
the LPD1 homolog) show increased vulnerability to 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a dopaminergic
neurotoxicant used as a model for Parkinson’s disease (Klivenyi
et al., 2004).
We hypothesize a simple model connecting leucine uptake,
Ras/PKA signaling, and PDH. Our results suggest that dieldrin
likely affects leucine uptake at the amino acid transporter level.
Leucine auxotrophs, along with strains lacking leucine trans-
porters or requisite amino acid signaling, cannot adequately cope
with the resulting leucine starvation and therefore experience
growth defects in dieldrin. Leucine starvation caused by dieldrin
would also be detrimental to strains unable to negatively regu-
late Ras/PKA, as activation of this signaling pathway promotes
cell growth, a cellular process incompatible with the starva-
tion response. Finally, the sensitivity of PDH mutants may be
explained by their inability to activate starvation pathways essen-
tial for the response to dieldrin-induced leucine depletion. Our
results underscore the value of functional profiling in yeast and
provide data useful for further gene or pathway-specific studies.
Supplementary data are available online at http://toxsci.
National Institute of Environmental Health Sciences
Superfund Research Program (P42ES004705 to C.D.V.,
3P42ES004705-22S1 to N.D.D. and C.D.V.).
The content is solely the responsibility of the authors and does
not necessarily represent the official views of NIEHS or NIH.
We thank Aaron Welch of the Koshland laboratory (University
of California, Berkeley) for the BAP2 HIP FlexGene, Alan
Hinnebusch (National Institutes of Health) for the B180 plasmid
(GCN4-lacZ), Akemi Kunibe of the Drubin-Barnes laboratory
(University of California, Berkeley) for the pRS305 plasmid
and technical advice on the LEU2 knock-in, and Vanessa De
La Rosa and Tami Swenson for critical reading of the manu-
script. B.D.G. is a trainee in the Superfund Research Program
(University of California, Berkeley).
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