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. 2019 Nov 20;8(6):1035–1041. doi: 10.1039/c9tx00139e

Benzoquinone alters the lipid homeostasis in Saccharomyces cerevisiae†

Abhishek Raj a, Vasanthi Nachiappan a,✉
PMCID: PMC7067238  PMID: 32190295

graphic file with name c9tx00139e-ga.jpgBQ exposure inhibited cell growth, increased the ROS-mediated apoptosis, and altered the phospholipids that resulted in membrane proliferation.

Abstract

Objective: To elucidate the impact of benzoquinone (BQ) on lipid homeostasis and cytotoxicity in Saccharomyces cerevisiae. Methods: The impact of BQ exposure on wild-type and knockouts of PC biosynthesizing genes revealed the alterations in the lipids that were analyzed by fluorescence microscopy, thin layer chromatography, and gene expression studies. Results: In yeast, BQ exposure reduced the growth pattern in wild-type cells. The gene knockout strains of the phospholipid metabolism altered the mRNA expression of the apoptosis genes – both caspase-dependent and independent. The BQ exposure revealed an increase in both the phospholipids and neutral lipids via the CDP:DAG and the Kennedy pathway genes. The accumulation of both neutral lipids and phospholipids during the BQ exposure was discrete and regulated by different pathways. Conclusions: BQ exposure inhibited cell growth, increased the reactive oxygen species (ROS), and altered membrane proliferation. The CDP:DAG and Kennedy pathway lipids also discretely altered by BQ, which is required for the membrane functions and energy purposes of life.

1. Introduction

Benzene is haemotoxic and its exposure decreases the circulating blood cells even at low concentrations1 and benzoquinone (2,6-dichloro-1,4-benzoquinone), a benzene derivative, is potentially toxic and carcinogenic.2 In yeast, the apoptosis proteins Yca1p, encoding for metacaspase, and Nuc1p, a mitochondrial death effector with endonuclease G activity, are involved in cytochrome c release. The Ysp1p, a mitochondrial protein, is required for thread-grain transition, de-energization, and cell death in yeast. The apoptosis-inducing factor, Aif1p, translocates from the mitochondria to the nucleus, where it leads to chromatin condensation and DNA degradation, and these genes are orthologous to the mammalian system.3 ROS are continuously generated during cellular metabolism in all aerobic animals, and are potentially dangerous due to their high reactivity and ability to interact virtually with all cellular components including carbohydrates, proteins, lipids, and nucleic acids.4 The toxicity of 4-nitroquinoline 1-oxide (4-NQO) treatment resulted in MDA production and increased lipid peroxidation, resulting in the rearrangement of the unsaturated lipids.5 Exposure of BQ increased oxidative stress and altered lipid metabolism.6 In eukaryotes, the phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylcholine (LPC), play a vital role in membrane structure, transport, and signaling.7 PC is an abundant phospholipid and serves as a structural component and as a source for bioactive lipids.8 In S. cerevisiae, PC is synthesized by two pathways, the Kennedy and the CDP-DAG pathways. In the CDP-DAG pathway, the enzymes PS synthase, PS decarboxylases (Psd1p and Psd2p), and PE methyltransferases (Cho2p & Opi3p) are all involved in PC synthesis. In the Kennedy pathway, PC is synthesized from choline via the reactions catalyzed by choline kinase Cki1p, phosphocholine cytidyltransferase Pctp1p, and choline phosphotransferase Cpt1p9 (Fig. 1). The expression of genes that are involved in phospholipid biosynthesis (INO1, OPI3, and CHO1) depends on Ino2p/Ino4p transcriptional regulation, which is under the control of a PA-sensor protein, Opi1p. Storage molecules such as triacylglycerol (TAG) and steryl esters (SE) are clustered to form a hydrophobic core called lipid droplets (LDs).10

Fig. 1. Cell viability. (a). The growth and viability of wild-type (WT) control cells in the presence or absence of BQ were monitored by measuring the OD (A600 nm) at frequent time intervals. (b). Viability of the PC knockout cells was also confirmed by spotting the serially diluted cells (10–3) on a YPD agar plate in the presence or absence of BQ.

Fig. 1

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We chose S. cerevisiae as a model system because the study of lipid metabolism is very well characterized, biochemical pathways are very well-known, and gene knockout strains are well established in S. cerevisiae. The genetic characteristics have similarities with the mammalian system, and hence, it is easy to understand the pathologies at both the organismal and cellular levels in yeast. The main objective of the present study is to explain the mechanisms of benzene-induced membrane modification leading to lipid alterations during BQ exposure.

2. Materials and methods

2.1. Chemicals

1-4-p-Benzoquinone, yeast extract, peptone, and dextrose were purchased from Hi-media, India. Silica gel 60F254 thin layer chromatography (TLC) plates were purchased from Merck, India. Phospholipid standards were procured from Avanti Polar Lipids, USA. All the chemicals and solvents were purchased from Sigma unless specifically mentioned. A stock solution of BQ was prepared with dimethyl sulphoxide (DMSO), and the final concentration of DMSO in the growth medium was 0.02% (v/v), which had no effect on the parameters that we analyzed in the yeast cells (unpublished data).

2.2. Strains and culture conditions

The strains of S. cerevisiae, wild-type BY4741 [+MATα his3 Δ1 leu2 Δ0 met15 Δ0 ura3 Δ0], and the single knockouts cki1Δ, pct1Δ, cpt1Δ, cho2Δ, and opi3Δ were procured from Euroscarf by Prof. Ram Rajasekharan, Central Food Technological Research Institute (CFTRI), India and they also gifted them to us. Prof. De Kroon (Department of Membrane Biochemistry & Biophysics, Utrecht University, The Netherlands) gifted the cho2opi3Δ double mutant. The yeast cells were grown in the YPD liquid medium (1% yeast extract, 2% peptone, and 2% dextrose) up to the stationary phase, inoculated to an optical density (OD) of 0.1 at A600 nm in fresh YPD liquid medium and grown to the late logarithmic phase. The growth of different strains in liquid media was followed by measuring the OD at A600 nm. To analyze the growth phenotype differences, cells were cultured in YPD medium to the mid-logarithmic phase in the absence or presence of different BQ concentrations (10–50 μM) (data not shown). From the growth curve and spot test analysis, we found that 40 μM BQ significantly retarded growth, so 40 μM BQ was chosen for further studies. Cells were washed three times with sterile double distilled water, centrifuged and adjusted to 1.0 OD (A600 nm). Next, 3 μl aliquots of 1 : 10 serial dilutions were added to the solid YPD medium for spot test analysis.

2.3. Lipid extraction and quantification

Wild-type cells were grown in YPD medium for 12 h in the presence or absence of BQ, and the total lipids were extracted by the Bligh & Dyer method.11 Briefly, chloroform, methanol, and 2% of orthophosphoric acid were added to the cell pellets in the ratio of 1 : 2 : 1 and vortexed. The organic (upper) phase contained the lipids and was separated by centrifugation and dried under nitrogen. Phospholipids were separated by TLC using chloroform/methanol/acetone/acetic acid/water (50 : 10 : 20 : 15 : 5, v/v). The separated phospholipids were identified by comparing unknown spot Rf values with the standard Rf values. The individual phospholipid spots in the TLC were stained with iodine vapor; phospholipid spots were scraped off from the plate and subjected to phosphorus estimation.12 Neutral lipids were separated by single-dimensional TLC using petroleum ether/diethyl ether/acetic acid (70 : 29 : 1, v/v). Densiometric values were obtained using ImageJ software. Data are mean ± SD from three independent experiments.

2.4. The mRNA expression studies by qPCR

The wild-type cells were grown in YPD medium for 12 h with/without BQ, and the total RNA was isolated using the RNeasy kit from Qiagen. Total RNA (1 μg) was used to synthesize cDNA (First-strand cDNA Synthesis Kit, Applied Biosystem, India) with random hexadeoxynucleotide and reverse transcriptase for 2 h at 37 °C. Quantitative polymerase chain reaction (qPCR) was performed with the Light Cycler FastStart DNA Master PLUS SYBR Green I kit with a light cycler (Roche Applied Science, Meylan, France), according to the manufacturer's instructions, and the data were analyzed with ICycler IQ5 software. Gene expressions were studied by qPCR using the cycle threshold (CT)-based method.13 Actin was chosen as the reference gene as its expression is relatively invariant. The relative expression of each target gene was normalized to that of the reference gene, actin (see primers list in ESI Table 1†).

2.5. Laser scanning fluorescence microscopy [only rhodamine B in confocal microscopy (Fig. 2a) and DCFH-DA for the graph (Fig. 2b)]

Fig. 2. The link between apoptosis and ROS evaluation. Wild-type control cells (equal number) were grown in the absence or presence of BQ. (a). ROS generation was assessed by using a laser scanning fluorescence microscope using rhodamine B staining ( 490 nm Ex and 516 nm Em). Arrow marks (↑) indicate the cells that were stained with rhodamine B. (b). ROS estimation – equal amount of cells were taken, and the pmol of DCF (min per mg protein) formed was calculated using a spectrophotometer. (c). The expression of apoptosis genes was studied by qRT-PCR for YCA1, NUC1, YSP1, and AIF1.

Fig. 2

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The yeast cells were grown in YPD medium for 12 h in the presence or absence of BQ and cells were washed and re-suspended in phosphate-buffered saline (PBS), stained with rhodamine B/Nile Red/DiOC6 solution (0.0005% in PBS) for 15 min at room temperature in the dark. The cells were washed six times with PBS to remove excess dye and re-suspended in 50 μl of PBS and observed with a laser scanning confocal microscope.

ROS were quantified using dichlorodihydro-fluorescein diacetate (DCFH-DA), a non-polar compound which reacts with ROS to form the highly fluorescent dichlorofluorescein (DCF),14 and the protein sample (100 mg) was incubated with Locke's solution (NaCl 154 mM, KCl 5.6 mM, NaHCO3 3.6 mM, HEPES 5.0 mM, CaCl2 2.0 mM, glucose 10 mM mL–1, pH 7.4 containing DCF-DA (5 mM mL–1)) for 30 min at 37 °C. Fluorescence was measured with excitation (Ex) and emission (Em) wavelength at 480 nm and 530 nm. ROS levels were quantified from a DCF standard curve and expressed as pmol DCF (min per mg protein).

2.6. Statistics

Data were analyzed using the programs of SPSS 10.0 (Statistical Package for the Social Sciences for Windows 10.0). All the values reported in this work are the mean of three replicates. Statistical analysis was carried out by the analysis of variance (ANOVA) test and values are the mean ± SD of three separate experiments. Wild-type/knockout control cells were compared with the BQ exposed group with a statistical significance of ***P < 0.001, **P < 0.01,*P < 0.05.

3. Results and discussion

3.1. Benzoquinone inhibits cell growth

The yeast growth was affected by the exposure of chemicals in a dose-dependent manner.15,16 Preliminary studies were carried out to determine the minimum concentration required to reduce the growth rate (data not shown), and it was determined to be 40 μM BQ and this concentration was used in this study. We examined the cell growth with BQ exposure in wild-type and PC knockout cells and BQ notably retarded the cell growth in wild-type cells (Fig. 1). The spot test analyzed the cell-tolerance assay in wild-type cells and PC knockouts (Fig. 1b). BQ exposure reduced cell growth in wild-type cells (Fig. 1a), and earlier reports from our lab and others revealed increased ROS levels, increased oxidative stress and lowered antioxidant level leading to a decline in cell growth or death.6,17

3.2. Exposure of benzoquinone increased apoptosis via increased ROS generation

The exposure to diverse environmental stress conditions such as the presence of oxidants, metal ions, heat shock, ethanol, etc. increased ROS generation.18 The cell growth was reduced with BQ exposure; hence, we wanted to know the impact of BQ on ROS generation in yeast. ROS generation is a redox determinantal status, and the BQ exposure increased ROS generation by ∼60% (Fig. 2a and b) and reduced cell growth (Fig. 1).

We found reduced cell growth and an increase in ROS generation with BQ exposure.6 Hence we studied the effect of BQ on apoptosis gene expression in yeast. Numerous exogenous and endogenous toxic agents such as acetic acid, hydrogen peroxide, plant antifungal peptides, and sugar or salt-stress induce yeast apoptosis.19 The cells were cultured exponentially in the presence of BQ, and the total RNA was extracted by the Trizol method, and the cDNA was constructed. An expression of the desired genes was measured by using gene-specific primers by real-time PCR. We quantified the mRNA expression of yeast apoptosis inducible genes YCA1, NUC1, YSP1, and AIF1. The mRNA expression of all apoptotic genes was significantly increased in wild-type cells, namely YCA1 (∼0.54 fold), NUC1 (∼0.70 fold), YSP1 (∼1.79 fold), and AIF1 (∼1.18 fold) with BQ exposure (Fig. 2c). Similarly, osmotin and pradimicin (PRM) were found to induce cell death in yeast with increased apoptotic features.20

3.3. Benzoquinone altered lipid levels and mRNA expression of lipid synthesizing genes

We determined the effect of BQ on lipid homeostasis associated with cell death and oxidative stress, particularly in PC biosynthetic pathway knockouts. The wild-type cells were treated with 40 μM BQ for 12 h, and the phospholipids were quantified. There was a significant increase in all the phospholipids PC, PE, and PI/PS, but a decrease was observed with LPC (Fig. 3a and b). The CDP:DAG pathway knockouts synthesize PC using LPC as a substrate.21 The mRNA expression studies revealed an up-regulation of the Kennedy pathway genes and OPI3 gene in the CDP:DAG pathway but not CHO2 (Fig. 3c). Positive regulators of a phospholipid, IN02, and IN04,22 were also up-regulated by ∼1 and ∼0.8 fold, respectively in BQ exposed cells. OPI3 and INO2 genes regulate phospholipid biosynthesis, and BQ exposure up-regulated these genes (Fig. 3c). The results showed an increased expression of CKI1 (∼1.9 fold), CPT1 (∼1.6 fold) and PCT1 (∼1.5 fold) genes. On the other hand, the CDP:DAG pathway gene CHO2 was down-regulated (∼0.58 fold), and OPI3 (∼0.4 fold) up-regulated (Fig. 3c).

Fig. 3. Effect of BQ on lipid composition. Wild-type control cells were grown in YPD medium for 12 h in the presence or absence of BQ, and the lipids were extracted and resolved on TLC (a). The phospholipid profile of wild-type cells was shown by the graphical representation (b). Wild-type cells were grown for 12 h with or without BQ and the mRNA was extracted and used for cDNA construction. The qPCR has been carried out using specific primers for the following phospholipid synthesizing genes CKI1, CPT1, PCT1, CHO2, OPI3, INO2 and INO4 (c) and neutral lipid synthesizing genes DGA1, LRO1, ARE1 and ARE2 (d). The phospholipid pattern of the PC knockouts (cho2Δ, opi3Δ, cho2opi3Δ, cki1Δ, cpt1Δ and, pct1Δ) was studied by growing the cells in the presence or absence of BQ for 12 h. The PC knockouts in yeast were grown in the presence or absence of BQ, and the lipids were extracted and separated using thin-layer chromatography (g); the phospholipids and neutral lipids were quantified. Panels (e, f, h, i) show the quantity of PC, PE, TAG, and SE, respectively in all the PC knockouts.

Fig. 3

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PC is the most abundant phospholipid and serves as a structural membrane component.7 PC synthesis is mainly accomplished by CDP:DAG pathway genes in the Kennedy pathway knockouts.23 The PC levels were higher in the Kennedy pathway knockouts with or without BQ exposure. With BQ exposure the PC levels were increased in wild-type cells (∼30%) and also in both the CDP:DAG pathway knockouts [opi3Δ (∼46%) and cho2opi3Δ (∼38%)] and the Kennedy pathway knockouts [cki1Δ (∼32%), cpt1Δ (∼41%), and pct1Δ (∼48%) ], but decreased in cho2Δ (∼20%) (Fig. 3e). Hence BQ increased PC in the knockouts of both the CDP:DAG and the Kennedy pathway genes.

PE is the second most abundant phospholipid in yeast and is distributed across the plasma membrane. Environmental stress can cause membrane asymmetry. PE is accumulated by both the pathways and might account for the membrane asymmetry and disturbs the membrane homeostasis.24 PE accumulation was high in CDP:DAG knockouts with and without BQ exposure (Fig. 3f). Genes of both the pathways were involved in PE accumulation. PE was increased in the wild-type (∼55%) cells, the CDP:DAG pathway knockouts [cho2Δ (∼21%), opi3Δ (∼26%), and cho2opi3Δ (∼38%)] and the Kennedy pathway knockouts [cki1Δ (∼17%), cpt1Δ (∼14%), and pct1Δ (∼14%)] as well (Fig. 3f). The PE level was much higher in CDP:DAG pathway knockouts compared to the Kennedy pathway knockouts, suggesting BQ accelerates PE synthesis mainly via the Kennedy pathway genes. BQ disrupts the membrane structure by altering the phospholipid levels and was supported by DiOC6 staining (ESI Fig. 1†).

In S. cerevisiae, the acyltransferases Lro1p and Dga1p convert DAG to TAG and the acyl-CoA sterol acyltransferases Are1p and Are2p are involved in steryl ester (SE) formation.25 The expression of DGA1 (∼0.51 fold), LRO1 (∼0.63 fold), ARE1 (∼0.94 fold), and ARE2 (∼1.63 fold) was significantly up-regulated in wild-type cells upon BQ exposure (Fig. 3d).

TAG was increased in wild-type (∼0.38 fold), and the Kennedy pathway knockouts [cki1Δ (∼0.21 fold), cpt1Δ (∼0.16 fold) and pct1Δ (∼0.18 fold)], and cho2Δ (∼0.27 fold) during BQ exposure, but decreased in opi3Δ (∼0.27 fold) and cho2opi3Δ (∼0.6 fold) knockouts (Fig. 3g and h). BQ accumulates TAG with CHO2 gene in the CDP:DAG pathway. There are enhanced expressions of the acyltransferases with BQ in the wild-type cells.

Similarly, SE levels were quantified with/without BQ. SE was decreased in the CDP:DAG pathway [cho2Δ (∼0.16 fold), opi3Δ (∼0.18 fold), and cho2opi3Δ (∼0.12 fold)] and interestingly increased in the wild-type (∼0.23 fold), and Kennedy pathway knockouts [cki1Δ (∼0.22 fold), cpt1Δ (∼0.11 fold), and pct1Δ (∼0.24 fold)] (Fig. 3g and i). These results also suggest that BQ increased SE synthesis in wild-type and CDP:DAG pathway genes.

Sterol content also influences the fluidity and permeability of the cell membrane and thus is transported through the membrane to the cell surface and allocated to cell poles.26 Sterol was increased in wild-type cells (∼0.12 fold) and cho2Δ (∼0.31 fold), but decreased in opi3Δ (∼0.53 fold) and cho2opi3Δ (∼0.44 fold) and not much alteration was observed in the Kennedy pathway knockouts [cki1Δ (∼0.10 fold), cpt1Δ (∼0.12 fold) and pct1Δ (∼0.05 fold)] with BQ exposure (Fig. 3g, ESI Fig. 2†). Hence, the increased neutral lipids are through the upregulation of four acyltransferases in wild-type cells, and TAG increased via the CDP:DAG pathway genes with the BQ exposure, and was supported by SE and sterol levels. Similar results were observed along with growth retardation and loss of cell permeability with BQ exposure (ESI Fig. 1†).

3.4. Lipid droplet formation

TAG was increased in wild-type cells, and the Kennedy pathway knockouts (Fig. 3g and h). In the CDP:DAG pathway knockouts, the LD numbers were increased in cho2Δ whereas they decreased in opi3Δ and cho2opi3Δ (Fig. 3g and h, ESI Fig. 3†) with BQ exposure. Laser scanning fluorescence microscopy studies and TLC results confirmed the conclusion. With BQ exposure the average LD numbers per cell increased in wild-type (from 4.1 to 6.9), in cki1Δ (from 5.6 to 6.5), cpt1Δ (from 4.6 to 7.7), and pct1Δ (from 3.8 to 5.7), whereas TAG was increased in the CDP:DAG pathway in cho2Δ (from 5.4 to 6.4), whereas not much change was observed in opi3Δ (Fig. 3g, ESI Fig. 3a, b,† and Table 1).

Table 1. Number of LDs in wild-type cells and PC knockouts with BQ exposure.

Average number of LDs per cell
Strains Control BQ exposure
Wild-type 4.1 ± 0.78 6.9 ± 0.50**
cho2Δ 5.4 ± 0.36 6.4 ± 0.50*
opi3Δ 4.4 ± 0.47 4.2 ± 0.30
cho2opi3Δ 7.8 ± 0.35 5.3 ± 0.24**
cki1Δ 5.6 ± 0.25 6.5 ± 0.45
cpt1Δ 4.6 ± 0.2 7.7 ± 0.45**
pct1Δ 3.8 ± 0.30 5.7 ± 0.25**
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4. Conclusion

BQ is a toxic benzene metabolite which causes alteration at the cellular level, stimulates the ROS levels17 (Fig. 3) and covalently binds in the nucleophilic regions of DNA.27 The cell growth was inhibited (Fig. 1) with increased expression of apoptosis genes (Fig. 2) in wild-type cells. Cells with BQ exposure altered the membrane potential (ESI Fig. 1†), and membrane lipid composition.28

BQ exposure increased the lipid compositions (PC, PE, TAG, and SE) in wild-type cells (Fig. 3a, b, e–i, ESI Fig. 2†). In wild-type cells and the CDP-DAG pathway knockouts (all the Kennedy pathway genes are present) PC synthesis was increased, and the mRNA expression studies also supported the TLC data (Fig. 3g). These results suggest that in WT cells in the absence of BQ exposure, the PC synthesis is mainly accomplished by CDP-DAG pathway genes, whereas upon BQ exposure it is routed through both the CDP-DAG and the Kennedy pathways. In the presence of BQ, the neutral lipids were increased in the wild-type cells, the Kennedy pathway knockouts (all the CDP-DAG pathway genes are present) and the CDP-DAG pathway (specially, cho2Δ) knockouts (Fig. 3g,h and i, ESI Fig. 2 and 4†), and is supported by the mRNA expression (Fig. 3e), and LD morphology (ESI Fig. 3,† Table 1). The DiOC6 measures the membrane potential29 and BQ exposure affected the membrane lipid bilayer permeability and fluidity.30 In the current study, we observed that BQ exposure altered the membrane permeability (ESI Fig. 1†).

To conclude, the present study demonstrates that BQ decreased cell growth, increased apoptosis, increased phospholipids and neutral lipids, leading to disturbance in the membrane potential (Fig. 4). The current study revealed the BQ exposure has an effect on lipid disorder with the enhancement of TAG production by altering the lipid metabolic pathways in S. cerevisiae.

Fig. 4. Graphical representation of benzoquinone induced lipid alteration associated apoptosis.

Fig. 4

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Abbreviations

PA

Phosphatidic acid

DAG

Diacylglycerol

CDP:DAG

Cytidine diphosphate diacylglycerol

PS

Phosphatidylserine

PE

Phosphatidylethanolamine

PC

Phosphatidylcholine

PMME

Phosphatidyl monomethylethanolamine

PDME

Phosphatidyl dimethylethanolamine

P

Phosphate

Cho

Choline

Pah1p

Phosphatidate phosphatases

Are1

Acyl-coenzyme A: cholesterol acyltransferase 1

Are2

Acyl-coenzyme A: cholesterol acyltransferase 2

Dga1p

Diacylglycerol acyltransferase

Lro1p

Lecithin cholesterol acyltransferase

Cho2p

Phosphatidylethanolamine methyltransferase

Opi3p

Phosphatidyl monomethylethanolamine methyltransferase

Cki1p

Choline kinase1

Pct1p

Choline phosphate cytidylyltransferase1

Cpt1p

Choline phosphotransferase1

Conflicts of interest

The authors declare no conflict of interest.

Supplementary Material

Click here for additional data file. (558KB, pdf)

Acknowledgments

The authors sincerely thank Prof. Ram Rajasekharan, Dept. of Biochemistry, Indian Institute of Science (Bangalore-India), for generously providing the reagents. We also thank the infrastructure facilities from the DST-FIST of Biochemistry Department, DST-PURSE and Life Sciences facilities of Bharathidasan University.

Footnotes

†Electronic supplementary information (ESI) available: Table 1 List of gene-specific primers used in this study for qPCR analysis. Fig. 1 DiOC6 staining. Fig. 2 Sterol level in PC knockouts. Fig. 3 Lipid droplet morphology studies with/without benzoquinone exposure. See DOI: 10.1039/C9TX00139E

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