Benzene, a ubiquitous environmental contaminant that is widely used in industry, is an established human carcinogen. It undergoes extensive metabolism in humans and thus the molecular mechanisms underlying its toxicity remain elusive despite extensive study. To gain insight into these mechanisms we have performed genome-wide functional profiling of yeast, in order to identify non-essential genes required for tolerance to treatment with three phenolic benzene metabolites, hydroquinone (HQ), catechol (CAT) and 1,2,4-benzenetriol (BT). Assessing these metabolites individually has identified overlapping and synergistic toxicities, consistent with previously proposed mechanisms of carcinogenesis of benzene in which multiple metabolites act in concert 
. Comparison of the genes associated with sensitivity to these compounds suggests a mechanistic basis for toxicity and has identified conserved toxicity genes in humans ().
Selected yeast genes required for benzene metabolite tolerance and their orthologous human genes.
We determined that yeast cells are more resistant to HQ, CAT and BT than human cells, based on comparison of yeast IC20
doses with treatment doses used in studies with human cells in culture 
. This increased resistance could be due to the absence of peroxidase enzymes in yeast (such as MPO in humans), which rapidly catalyze the oxidation of HQ, CAT and BT to more toxic quinone species. We identified both distinct and overlapping cellular processes and proteins between metabolites as important for tolerance, including the oxidative stress response, lipid/fatty acid transport, and the pentose phosphate pathway (). Data also indicate that metabolites (particularly HQ) damage proteins, leading to both cytoskeletal and ER stress, and a requirement for ER-associated protein degradation. The vacuole, particularly the vacuolar ATPase, is important for tolerance. Benzene metabolites, especially CAT, appear to disrupt intracellular vesicle trafficking as an indirect result of lipid peroxidation caused by oxidative stress and/or cytoskeletal disruption. CAT may affect trafficking more than HQ and BT due to a bias in the species of ROS produced upon exposure, discussed below. These shared functional categories indicate that some molecular targets may be common to HQ, CAT and BT.
Proteins identified as being required for tolerance to benzene metabolites.
In agreement with observations from previous studies in other organisms 
, our results show that the primary mechanism of toxicity of these metabolites is induction of oxidative stress; we did not identify a requirement for the mitochondrial electron transport chain, suggesting that the level of hydrogen peroxide produced by HQ, CAT and BT does not significantly contribute to oxidative toxicity. The DSSA profiles identified in our study for HQ, CAT and BT are similar to that seen following cumene hydroperoxide treatment, an oxidant that is structurally similar to the phenolic metabolites of benzene 
, and requires the vacuole and vacuolar ATPase for tolerance. The profiles of HQ and BT from DSSA are more similar to each other than to that of CAT, perhaps reflecting differential ROS generation due to the difference between the para
(1,4) position of the hydroxyl groups of HQ and BT and the ortho
(1,2) hydroxyl groups of CAT. HQ, CAT and BT likely produce differing proportions of a wide range of ROS, which require multiple oxidative stress responses for tolerance. The lack of significant sensitivity of yeast glutathione pathway mutants to HQ, CAT and BT reflects either a difference in detoxification pathways between yeast and humans, or functional redundancy between glutathione pathway enzymes in yeast. The lack of significant sensitivity to decreased GSH levels suggest that the yeast thioredoxin system may be more important than the glutaredoxin system for benzene metabolite tolerance; previous studies have suggested it also has a more prominent role in hydrogen peroxide metabolism 
. Based on these results, it may be prudent to reevaluate the role of the thioredoxin pathway in the human response to benzene metabolites.
Our data implicate the previously uncharacterized bZIP transcription factor Yap3p as being involved in a cellular response specific to HQ. Yap3p may respond to ER stress, as yap3
Δ is sensitive to tunicamycin, a compound that causes ER stress through induction of the unfolded protein response 
. This is consistent with the identification of more ER stress sensitive mutants in the HQ data compared to the CAT and BT data, and suggests that HQ causes more extensive damage to proteins than CAT or BT. yap3
Δ was also identified in a similar screen as being sensitive to arsenic (As) treatment 
, suggesting that As and HQ (but not CAT or BT) share some cellular targets; analysis of transcription factor binding sequences has previously shown that Yap3p sites co-occur with those of Arr1p (Yap8p), the major arsenic response transcription factor 
. Tubulin may be such a shared cellular target, as both HQ and As are able to bind to tubulin 
. This could indicate that Yap3p is involved in the response to cytoskeletal stress, though our hypothesis is complicated by the fact that BT also inhibits microtubule polymerization 
Δ cells being sensitive to it. Overexpression of Candida albicans YAP3
) in S. cerevisiae
results in increased transcription of PDR5
, an ABC transporter that confers resistance to a range of xenobiotic compounds 
, and Yap3p also interacts with Pdr5p 
Δ was not identified as significantly sensitive to HQ in this study (though it is sensitive to CAT) and is also not sensitive to As 
, indicating that although PDR5
is a Yap3p target, it is not involved in the cellular response to HQ or As. High-throughput studies of the transcriptional targets of the total complement of yeast transcription factors are currently being undertaken by several groups 
, and these data will soon elucidate Yap3p targets and provide insight into the Yap3p stress response.
Several other processes important for benzene metabolite tolerance such as the vacuole, iron homeostasis, and lipid peroxidation, were likely identified due to the induction of oxidative stress by these compounds. Loss of the V-ATPase results in chronic oxidative stress 
and sensitizes cells to exogenous oxidative stress; previous data show that vacuolar function is required for oxidative stress resistance 
. The requirement of the V-ATPase may also be due to a need to maintain soluble (i.e. bioavailable) iron in the vacuole. The known decrease in available cellular iron caused through chelation by benzene metabolites 
is potentiated by a disrupted V-ATPase 
, leading to extremely low iron levels. HQ, CAT and BT can also cause iron deficiency through the disruption of iron-sulfur (Fe-S) clusters by ROS. Fe-S clusters are vital reactive centers of a large number of proteins and are vulnerable to attack and disruption by ROS. Loss of Fe2+
from Fe-S clusters can lead to further ROS production through Fenton chemistry 
, resulting in amplified disruption; increased synthesis and assembly of new Fe-S clusters then depletes available iron. Evidence to support the disruption of Fe-S clusters by benzene metabolites comes from the identification by DSSA of nfu1
Δ (HQ). Nfu1p is a scaffold protein required for correct assembly of Fe-S clusters in the mitochondria 
. Additionally, MRS4
(CAT, BT) encodes a mitochondrial iron transporter required for efficient mobilization of iron into the mitochondria, and is known to be sensitive to low iron 
; under conditions requiring the increased synthesis of Fe-S clusters, mrs4
Δ cells are unable to transport sufficient iron into mitochondria. The identification of sulfur metabolism (map00920) by KEGG pathway analysis supports a requirement for increased Fe-S synthesis in response to HQ, CAT and BT treatment.
We identified Pst2p and Ycp4p as putative mitochondrial NAD(P)H:quinone oxidoreductases that are capable of reducing para
(1,4) quinones (such as those produced through autoxidation of HQ and BT), but not ortho
(1,2) quinones (e.g. 1,2-BQ from autoxidation of CAT). The absence of Pst2p or Ycp4p is therefore deleterious following HQ treatment due to increased levels of more toxic 1,4-BQ. The resistance of both pst2
Δ and ycp4
Δ to BT can be explained by the autoxidation rate of BT, which is higher than that of HQ or CAT 
. HQ, CAT and BT all autoxidize by two successive one-electron oxidations, producing an extremely reactive semiquinone intermediate; these semiquinones are the most reactive and most toxic of the quinone species. Constant reduction of 2-OH-1,4-BQ back to BT results in elevated levels of transient semiquinones through repeat autoxidation of BT. Therefore, preventing reduction of 2-OH-1,4-BQ is beneficial to the cell as increased 2-OH-1,4-BQ is less harmful than increased transient semiquinone levels; concordantly, strains overexpressing PST2
have increased sensitivity to BT. The HQ resistance and BT sensitivity of the PST2
overexpression strain is a reversal of the pst2
Δ phenotype, and provides mechanistic support of the hypothesis that Pst2p and Ycp4p are quinone oxidoreductases. It is unclear why YCP4
overexpression causes increased sensitivity to HQ, CAT and BT, but it could be due to unintended general cellular consequences; the C-terminal region of Ycp4p, which is not present in Pst2p, may aggregate at high abundance, causing cells to become sensitized to any stressor. The lack of sensitivity of pst2
Δ and ycp4
Δ to CAT () can be explained if Pst2p and Ycp4p are only able to reduce para
quinones. Based on homology to the bacterial oxidoreductase WrbA, we propose that they function by direct two-electron reduction of quinones with the same mechanism as human NQO1 and the characterized cytoplasmic yeast NAD(P)H:quinone oxidoreductase Lot6p. Lot6p is not required for tolerance to HQ or BT, despite Lot6p being able to reduce 1,4-BQ in vitro
Δ is, however, moderately sensitive to CAT, indicating that Lot6p may be capable of reducing 1,2-BQ in vivo
. HQ and BT may target the mitochondria more than CAT, as the CAT sensitivity profile has more representation of cytoplasmic targets, such as vesicular trafficking. We propose Pst2p and Ycp4p to be novel yeast orthologs of NQO1 that are required for HQ tolerance.
Both homologous recombination (map03440) and non-homologous end joining (map03450) DNA repair pathways were identified by KEGG analysis as associated with sensitivity to benzene metabolite treatment (), but the absence of strains identified by DSSA suggests genotoxicity is limited. However, DNA damage caused by these metabolites has been reported in many other studies and they have also been shown to bind DNA 
. As our study shows very strong evidence for oxidative stress induction but only limited evidence of DNA damage, we suggest that a significant proportion of damage measured in other studies is an indirect effect resulting from reactive oxygen intermediates and/or topoisomerase inhibition 
, though we cannot rule out species differences in damage repair.
Oxidative DNA damage occurs in several ways: Hydrogen peroxide generated by CAT exposure increases the amount of 8-oxodG 
, while lipid peroxidation results in the production of malondialdehyde, which reacts with dA and dG in DNA. Lipid damage also produces etheno adducts in DNA that rearrange to form crosslinks between strands 
. Yeast cells lacking the DNA helicase Sgs1p, required for the maintenance of genomic stability, have increased sensitivity to high doses of HQ 
, and RNAi knockdown of WRN
, the human ortholog of SGS1
, increases HQ-generated DNA damage 
Many of the genes identified in this study have human orthologs (). Only two (PRDX1
) are currently associated with HQ in the Comparative Toxicogenomics Database (http://ctd.mdibl.org
) and none are associated with CAT or BT. All could be novel targets or modulators of benzene toxicity in humans. Though oxidative stress appears to be the primary means through which these metabolites exert their toxicity, many secondary processes were also identified that could be informative in terms of human susceptibility. The deleterious health effects caused by benzene in humans are likely due to multiple modes of toxicity, induced by multiple metabolites, acting synergistically 
Benzene is thought to cause leukemia through metabolite-induced oxidative stress, via mitochondrial imbalance in hematopoietic cells 
. The presence of phenolic benzene metabolites in bone marrow is likely to generate more oxidative stress than observed in yeast due to the presence of enzymes such as myeloperoxidase, which enhances the production of benzoquinones through direct oxidation 
. There may be many as-yet unidentified SNPs in human oxidative stress response genes that could increase susceptibility to benzene toxicity and that should be studied.