Ashwagandha Leaf Derived Withanone Protects Normal Human Cells Against the Toxicity of Methoxyacetic Acid, a Major Industrial Metabolite

The present day lifestyle heavily depends on industrial chemicals in the form of agriculture, cosmetics, textiles and medical products. Since the toxicity of the industrial chemicals has been a concern to human health, the need for alternative non-toxic natural products or adjuvants that serve as antidotes are in high demand. We have investigated the effects of Ayurvedic herb Ashwagandha (Withania somnifera) leaf extract on methoxyacetic acid (MAA) induced toxicity. MAA is a major metabolite of ester phthalates that are commonly used in industry as gelling, viscosity and stabilizer reagents. We report that the MAA cause premature senescence of normal human cells by mechanisms that involve ROS generation, DNA and mitochondrial damage. Withanone protects cells from MAA-induced toxicity by suppressing the ROS levels, DNA and mitochondrial damage, and induction of cell defense signaling pathways including Nrf2 and proteasomal degradation. These findings warrant further basic and clinical studies that may promote the use of withanone as a health adjuvant in a variety of consumer products where the toxicity has been a concern because of the use of ester phthalates.


Introduction
Methoxyacetic acid (MAA, CH 3 OCH 2 COOH) is a primary metabolite of ester phthalates, a class of industrial chemicals used in a large variety of consumer products including household products (building materials, plastic objects, textiles, adhesives, paints and deodorants), food and personal care products including agricultural adjuvant, pesticides, cosmetics and perfumes, electronics (coatings, stabilizers and surfactants) and pharmaceuticals (enteric coating of oral pills, viscosity control agents, surfactants and stabilizers). The most widely-used phthalates are the di-2-ethyl hexyl phthalate (DEHP)-a low cost plasticizer used in PVC, benzylbutylphthalate (BBzP)-used in the manufacture of foamed PVC used as a flooring material, the diisodecyl phthalate (DIDP)-viscosity controlling reagent, the diisononyl phthalate (DINP) and small phthalates-used as solvents in perfumes and pesticides. Globally, more than 18 billion pounds of ester phthalates are used annually and toxicity of these chemicals occurs through ingestion, inhalation, intravenous injection and dermal exposure on a daily basis. In the United States and Canada, DEHP is no longer used to manufacture children's products intended for mouthing, such as pacifiers, but it is still used in larger toys. DEHP is approved for use in medical devices such as surgical vinyl gloves, blood bags, tubes and dialysis equipments. Dibutyl phthalate (DBP) is widely used in many cosmetic products including perfume, lotion, and nail polish putting women to a higher health-hazard risk category. Ester phthalates family, monoethyl phthalate (MEP), monobutyl phthalate (MBP), monobenzyl phthalate (MBzP), and mono(2ethylhexyl) phthalate (MEHP) have been detected in urine specimens, and their diester parent compounds in house dust samples, from pregnant and lactating women living in New York City [1]. Breast milk has been reported to contain phthalate metabolite monoesters in samples from Denmark/Finland [2], Sweden [3] and Italy [4].
Laboratory studies have shown that the toxicity of ester phthalates is caused by their metabolite, MAA. Gavage and intraperitoneal (i. p.) administration of MAA in rats resulted in their physical, reproductive, skeletal and hematopoietic abnormalities [5,6] and it was shown to be a proximal teratogenic metabolite of 2-methoxyethanol (ME) and 2-Dimethoxyethyl phthalate (DMEP) [5,7]. The toxicity of MAA has been shown to be mediated by multiple signaling pathways including oxidative stress, apoptosis, upregulation of heat shock proteins and deregulation of kinases, inhibition of histone deacetylase and disruption of estrogen receptor-mediated signaling [7][8][9][10][11][12][13][14][15][16][17][18]. In view of the extensive industrialization of present day lifestyle, there is a substantial urgency for molecular investigation to resolve the mechanism(s) of MAA toxicity and to invent novel ways and reagents to overcome its deleterious effects on human health.
In the present study, we report that a phytochemical, withanone, extracted from leaves of Ashwagandha protects human normal cells against the toxicity of MAA. We provide the very first molecular evidence showing the anti-toxic effects of withanone in normal human cells and propose its use as a MAA-antidote.

Results and Discussion
MAA causes senescence like growth arrest of normal human fibroblasts Normal human fibroblasts were cultured in the control medium for 24 h. Cells were then treated either with withanoneor MAA-supplemented medium for 48 h followed by recovery by placing them in either control normal medium or withanonesupplemented medium, respectively, for the next 24-48 h. As shown in Figure 1A, the treatment with MAA led to decrease in relative density of cells as detected by crystal violet DNA staining. Cell viability analysis by Alamar BlueH-cell proliferation assay revealed less number of viable cells in MAA-treated cultures. Cells cultured in the presence of withanone recovered from MAA-induced growth arrest and decline in viability. Of note, withanone treatment either before ( Figure 1B-E) or after ( Figure 1B-F) the exposure to MAA was effective in recovering the cells from MAA-induced cyto-toxicity. We next performed senescence-associated b-galactosidase staining assay in control and MAA-treated cells. Whereas MAA caused significant increase in senescence-associated b-galactosidase staining, the number of cells showing senescence like growth arrest ( Figures 1C and 1D) declined in the presence of withanone suggesting that the MAA-induced cellular damage caused premature senescence in normal fibroblasts, and withanone protected the cells from such deteriorative phenotype. In order to get further insights to the kind of growth arrest induced by MAA, we performed cell cycle analysis on the control and treated cells. As shown in Figure 1E, MAA-treated cultures showed significant increase in number of cells in G1 stage of the cell cycle and decrease in number of cells in S phase. Subsequent recovery of MAA-treated cells in withanone-supplemented culture medium reinitiated the cell cycle progression and entry into S phase ( Figure 1E).

Molecular pathways involved in MAA-induced premature senescence in normal human fibroblasts
In order to understand the molecular mechanism of MAAinduced growth arrest of normal human fibroblasts, we hypothesized that MAA may induce ROS resulting in oxidative damage to DNA and mitochondria, and that in turn induce DNA damage signaling and loss of mitochondrial membrane potential culminating in growth arrest of cells by pathways shown in Figure 2A. In order to test this hypothesis, we examined whether MAA caused ROS generation in cells. As shown in Figure 2B, cells treated with MAA for 6 and 12 h showed ROS generation, the amount of which was doubled in cells treated with 10 mM MAA for 12 h ( Figure 2C). Furthermore, we found that while the cells treated with MAA showed ROS induction, the recovery treatment with withanone caused significant reduction in ROS indicating the involvement of ROS pathway in the process ( Figures 2D and 2A).
Involvement of ROS and DNA damage response in MAA-induced premature senescence of normal human fibroblasts and its recovery by withanone We next examined the above hypothesis by undertaking the expression analysis of molecules involved in ROS-induced DNA damage signaling and growth arrest of cells. As shown in Figure 3A, MAA caused an induction of DNA damage as examined by cH2AX focus formation. Noticeably, recovery of cells in withanone-supplemented medium resulted in the reduction of cH2AX expression ( Figures 3A and 3B). These data demonstrated that the DNA damage caused by MAA was rectified by the addition of withanone. We next analyzed p53 and p21 WAF1 (the two key molecular mediators of DNA damage response and senescence pathway) expression in MAA-treated and withanonerecovered cells. As shown in Figure 3C, p53 and p21 were induced in MAA-treated cells in a dose-dependent manner. Furthermore, MAA-treated and withanone-recovered cells showed considerable normalization of both p53 and p21 WAF1 ( Figures 3A and 3D). These data demonstrated that the MAA-induced DNA damage and growth arrest of normal cells were blocked by withanone.
We next examined p16 INK4A -RB pathway, another major tumor suppressor pathway involved in senescence and has been shown to be upregulated in response to accumulation of DNA damage and replicative senescence of normal human fibroblasts. As shown in Figures 4A and 4B, p16 INK4A was upregulated in MAA-treated cells and its downstream effector RB ( Figure 2A) was unphosphorylated ( Figure 4A) leading to growth arrest of cells. These were consistent with the cell cycle, cell viability and p53-p21 WAF-1 status data as described above. Of note, in contrast to the decrease in phospho-RB ( Figure 4A), total RB expression was increased in MAA-treated cells ( Figures 4A and 4B). Furthermore, consistent with the recovery from growth arrest, MAA-treated and withanone-recovered cells showed decrease in p16 INK4A (p16), recovery of phospho-RB and decrease in total RB ( Figures 4A and  4B). These data revealed that the MAA-induced toxicity to normal cells involve p16 INK4A -RB pathway and the treatment with withanone leads to their recovery as hypothesized in our model ( Figure 2A).

Involvement of ROS and mitochondrial damage during MAA-induced premature senescence of normal human fibroblasts and its recovery by withanone
We next examined whether the ROS generated by MAA cause mitochondrial damage by analyzing mitochondrial membrane potential. We found that the cells when treated with MAA show loss of membrane potential as indicated by loss of both JC1-red and mitotracker staining. Most significantly, MAA-treated and withanone-recovered cells exhibited recovery of both JC-1 red and mitotracker staining suggesting that the mitochondrial membrane potential of these cells was comparable to the normal untreated cells ( Figures 5A and 5B). Furthermore, to test whether the treatment with withanone protected the normal cells against mitochondrial damage, we also exposed these cells to oxidative damage by treating with H 2 O 2 . As shown in Figure 5C, cell viability was significantly improved when H 2 O 2 -treated cells were recovered in withanone-supplemented medium demonstrating that whereas MAA caused ROS generation, DNA and mitochondrial damage, withanone protected the cells against such damage by suppressing ROS generation as hypothesized in our model ( Figure 2A).
Protection against ROS and MAA-induced premature senescence of normal human fibroblasts involve an induction of Nrf2-ARE signaling Keap1-Nrf2-ARE signaling plays a significant role in protecting cells from endogenous and exogenous stresses. Under normal conditions, the transcription factor Nrf2, a primary transcription factor required for the induction of a battery of phase II detoxification genes through activation of antioxidant response element (ARE), interacts with the actin-anchored protein Keap1 and is largely localized in the cytoplasm resulting in the low basal expression of Nrf2-regulated genes. However, upon recognition of chemical signals imparted by oxidative and electrophilic molecules that cause activation of MAP kinase signaling, Nrf2 is phosphorylated, released from Keap1, escapes proteasomal degradation, translocates to the nucleus, and transactivates the expression of several antioxidant and cytoprotective genes that increase resistance to oxidative stress and mitochondrial dysfunction leading to enhance cell survival (Figure 2A) [19]. We first examined Nrf2 in normal human fibroblasts treated with oxidative stress (H 2 O 2 ) and during recovery phase in withanone-supplemented medium. As expected, normal cells treated with cytotoxic Figure 2. MAA-induced ROS generation in normal human cells. A, Schematic presentation of the pathways predicted to be involved in MAAinduced growth arrest/senescence of normal cells and its recovery by the addition of withanone. B, MAA-induced ROS generation in normal human cells as early as 6-12 h of treatment time. C, ROS level in cells treated with 10 mM MAA for 12 h was double compared to the control. Increase in ROS generation with 5 mM and 10 mM MAA were statistically significant (*P,0.05). D, Cells treated with MAA but recovered in withanone-supplemented medium showed reduction in ROS as compared to the control that were recovered in normal medium. The MAA-induced increase and withanoneinduced recovery in ROS generation were statistically significant (*P,0.05). doi:10.1371/journal.pone.0019552.g002 doses of hydrogen peroxide and MAA showed reduction in Nrf2. However, the cells treated with withanone resulted in an induction of Nrf2 at much higher level than by genistein, a known inducer of Nrf2 pathway (Figures 6A and 6B). Whereas MAA-treated cells showed suppression of Nrf2, the MAA-treatment followed by addition of withanone showed recovery through induction of Nrf2. As shown in Figure 6C, withanone-recovered cells showed nuclear translocation of Nrf2, associated with expression of ARE-regulated gene glutathione S-transferase (GST) ( Figure 6D). There was a loss of 25-30% in GST activity in MAA-treated cells; withanonerecovered cells showed 15-20% GST recovery. We also examined the proteasomal activity in MAA-treated and withanone added recovered cells. Withanone caused recovery of the MAA-induced decrease in the proteasomal activity ( Figure 6E).
The present day life heavily depends on industrial products and hence it is a major challenge to protect human health against the toxic effects of industrial chemicals. Genetic and functional toxicity of MAA, a major metabolite of ester phthalates, has been shown in a variety of in vitro and in vivo model studies [9,16,[20][21][22][23]. It has been shown to cause change in gene expression pattern [8], cell environment [22] and mitochondrial function [24]. We had previously shown that Ashwagandha leaf extract (i-Extract) and withanone, a major constituent in the leaf, cause selective killing of cancer cells [25,26]. Normal human fibroblasts were benefited by treatment with i-Extract; these showed increase in lifespan, reduction in the molecular damage and protection against oxidative stress [27,28]. At this end, we investigated whether MAA-induced damage to normal human cells in culture could be recovered by the treatment with withanone. As shown above, we found that the MAA caused ROS-mediated DNA and mitochondrial damage to normal human cells. The cells showed significant recovery from such damages when subsequently treated with withanone ( Figures 2, 3, 4, 5). Furthermore, withanone stimulated the Nrf2 signaling resulting in the nuclear translocation of Nrf2 and an increase in GST activity ( Figure 6D) suggesting the increase in cellular defense mechanism to overcome the oxidative stress. Proteasomal function was also increased in cells that were treated with withanone ( Figure 6E). Taken together, these data suggested that the cells treated with withanone could be protected against MAA-toxicity by multiple mechanisms including reduction in the production of ROS, subsequent damage at DNA and mitochondrial level, and induction of cellular defense machinery including Nrf2 signaling and proteasomal degradation. As a result of these molecular effects of withanone, human normal cells were protected against MAA-induced toxicity indicated by growth arrest and premature senescence (Figure 1). These data suggest that withanone is a strong candidate for health adjuvant and could be recruited in multiple industrial products including cosmetics, toiletry, and medical products containing ester phthalates that are toxic and threat to human health.

Cell culture, treatments and viability assay
Normal human fibroblasts (TIG-1) were obtained from the Japanese Collection of Research Bio-resources (JCRB, Japan) and grown at 37uC in a 5% CO 2 incubator in low glucose Dulbecco's modified Eagle's minimal (DMEM; Gibco BRL, Grand Island, NY) essential medium supplemented with 10% fetal bovine serum. Cells (at about 50% confluency) were treated with 10 mM Methoxyacetic Acid (MAA) (WAKO Chemicals, Japan) for 24 h followed by culture in a medium-containing withanone 10 mg/mL for 24 h for recovery treatments. For pretreatment, withanone was added to the culture medium 24 h before the addition of MAA and was continued during the MAA-treatement. Control (MAA treatment for 24 h followed by recovery in normal culture medium) and treated cells were analyzed as described below.
Cell viability was examined using Alamar BlueH-cell proliferation assay kit (Invitrogen) following manufacturer's instructions. Briefly, cells (3610 3 cells) were plated in 96-well plates. After 24 h of incubation, cells were treated with MAA (10 mM, for 24 h) followed by culture in a fresh DMEM medium-supplemented with Withanone (10 mg/mL) for 24 h. Cells were then incubated with Alamar Blue (10% in DMEM) for 1 h. Change in Alamar Blue color from blue nonfluorescent to red fluorescent (proportional to the living and metabolically active cells) was recorded at 450 nm (absorbance is monitored at 570 nm and 600 nm) in a microplate reader. Cell viability was also assayed by Crystal violet staining. Control and treated cells were washed with PBS and fixed in 1% paraformaldehyde (15 mins at room temperature). After washing twice with PBS, the cells were incubated with Crystal violet dye (0.5% in H 2 O) at room temperature overnight. Cells were washed with H 2 O and plates were photographed with image scanner (EPSON GT-9800F).

Cell cycle analysis
The cells (about 50% confluent) were treated with MAA for 24 h followed by incubation in either control medium or in withanone-supplemented medium for another 24 h for recovery and then harvested by trypsin. Cells pellets were washed with cold PBS and fixed in 70% ethanol at 4uC for 12 h. The fixed cells were centrifuged at 2000 rpm for 10 min, washed with cold PBS twice and then re-suspended in 0.25 ml PBS. The cells suspensions were treated with RNAse A (5 ml, 1 mg/ml at 37uC for 1 h) to remove RNA followed by incubation with Propidium Iodide (PI, 10 ml, 1 mg/ml) for 30 min in dark. PI-stained cells were subjected to cell cycle analysis using Coulter Epics XL TM Flow Cytometer (Beckman).

Senescence-associated b-galactosidase assay
Control and treated cells were subjected to senescenceassociated b-galactosidase (SA-b-gal) staining using the standard protocol. In brief, cells were first treated with MAA and then incubated in either control medium or in withanone-supplemented medium for recovery. The cells were washed with PBS, fixed in 2% formaldehyde/0.2% glutaraldehyde in PBS for 10 min, and washed again with PBS, followed by an overnight incubation in staining solution (citric acid/phosphate buffer, pH 6.0, 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 , 2 mM MgCl 2 , 150 mM NaCl, supplemented with 1 mg/ml of 5-bromo-4-chloro-3-indolyl-b-Dgalactopyranoside (X-gal) at 37uC. Stained cells were examined under the microscope and photodocumented with a NIKON camera.

Proteasome activity assay
Control and treated cells were harvested with cell scraper and assayed for proteasome activity using Proteasome Activity Assay kit (Chemicon). The assay is based on the detection of the fluorophore 7-Amino-4-methylcoumarin (AMC) after cleavage from the labeled substrate LLVY-AMC. The free AMC fluorescence was quantitated using a 380/460 nm filter set in a fluorometer.

Glutathione-S-transferase assay
Control and treated cells were subjected for analysis using Glutahione-S-transferase Assay Kit (Cayman Chemical Company) following manufacturer's instructions. The cells (3610 4 ) were seeded in 100-mm plate and treated with MAA for 24 h followed by recovery either in normal or withanone-supplemented medium for 24 h. Cells were then washed with cold PBS, scraped and centrifuged at 20006 g for 10 min at 4uC. Cell pellets were sonicated in cold 1-2 ml of cold buffer (100 mM potassium phosphate, pH 7.0, containing 2 mM EDTA), centrifuged at 10,0006 g for 15 min at 4uC. The supernatant was subjected to assay. Reduced glutathione (GSH) content in supernatant was measured colorimetrically at 340 nm using plate reader.

Detection of reactive oxygen species
The reactive oxygen species were detected by fluorescent staining using the Image-iT TM LIVE Green Reactive Oxygen Species (ROS) Detection Kit (Molecular Probes Inc, USA). Cells were grown on glass coverslips placed in 12-well plate and were treated as described above. Cells were fixed and then stained for ROS by procedure recommended by the manufacturers.

JC1 staining and mitochondrial damage
Control and treated cells were incubated in JC-1 (Molecular probes) 10 mg/ml dye containing media at 37uC for 10 min. The JC-1 labeled cells were detected by fluorescent microscopy. Cells were also incubated with MitoTracker Deep Red (150 nM) for 30 min and examined under fluorescence microscope (Carl Zeiss).

Statistical analysis
Statistical significance of the data (viability, SA-ß-gal assay, ROS generation, mitochondrial membrane potential, GST and proteasomal activity) was calculated from three independent experiments using One-way ANOVA and Baferroni's test. The values were considered significant for P,0.05.