Ochratoxin A mediates cytotoxicity through the MAPK signaling pathway and alters intracellular homeostasis in bovine mammary epithelial cells

Environmental Pollution 246 (2019) 366e373

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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol

Ochratoxin A mediates cytotoxicity through the MAPK signaling pathway and alters intracellular homeostasis in bovine mammary epithelial cells* Jin-Young Lee a, 1, Whasun Lim b, 1, Soomin Ryu c, Jinyoung Kim d, Gwonhwa Song c, * a

Department of Pharmacy, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea Department of Biomedical Sciences, Catholic Kwandong University, Gangneung, 25601, Republic of Korea Institute of Animal Molecular Biotechnology and Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea d Department of Animal Resources Science, Dankook University, Cheonan, 330-714, Republic of Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 July 2018 Received in revised form 18 November 2018 Accepted 11 December 2018 Available online 12 December 2018

Ochratoxin A (OTA), a secondary metabolite of the genera Penicillium and Aspergillus, contaminates many types of food and causes apoptosis as well as immunosuppression in many animal species. However, a mechanistic analysis of OTA-mediated cytotoxicity in bovine mammary epithelial cells has not yet been performed. Hence, we investigated the effects of OTA on bovine mammary epithelial (MAC-T) cells using several mechanistic analyses. We report that OTA may induce cell cycle arrest and apoptosis via MAPK and JNK signaling pathways in MAC-T cells. Moreover, homeostasis of cellular components, such as that of the mitochondrial membrane, was disrupted by OTA, leading to a decrease in mitochondrial and cytosolic Ca2þ in MAC-T cells. In addition, we evaluated the effects of OTA on inflammatory responses and major tight junction regulators, such as occludin and claudin 3. In summation, we suggest that OTA contamination may adversely affect bovine mammary epithelial cells, leading to improper lactation and decreased milk quality. This article aims to improve the understanding of physiological mechanisms involved in lactation, in addition to providing a guideline for the stabilization of industrial milk production by countering exogenous contaminants in livestock. © 2018 Elsevier Ltd. All rights reserved.

Keywords: Ochratoxin A Mammary epithelial cell Cytotoxicity Contaminant Cow

1. Introduction OTA is a foodborne mycotoxin produced by some species of the genera Penicillium and Aspergillus as a toxigenic secondary metabolite. During dry processing of raw food under suboptimal conditions, these fungal species contaminate and produce OTA in a wide range of agricultural products, including fruits, cereal grains, coffee, and wine (Mitchell et al., 2017). However, inactivating OTA during general food processing has proven to be a difficult task, due to its structural stability (Bui-Klimke and Wu, 2015). Furthermore, OTA has been shown to be carcinogenic, immunotoxic, and teratogenic to a number of animal species (Bendele et al., 1985; Boorman et al.,

*

This paper has been recommended for acceptance by Dr. David Carpenter. * Corresponding author. Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea. E-mail address: [email protected] (G. Song). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.envpol.2018.12.032 0269-7491/© 2018 Elsevier Ltd. All rights reserved.

1992; Castegnaro et al., 1998; Sorrenti et al., 2013). OTA induces genetic toxicity in humans and animals. It has been reported to cause DNA damage, mutagenesis, and reduction of protein synthesis (Palma et al., 2007; Ringot et al., 2006). In humans, OTA plays a carcinogenic role by inducing covalent bond reactions between its metabolites and DNA, resulting in damaged DNA (Pfohl-Leszkowicz and Manderville, 2007). Moreover, OTA exposure disrupts intestinal barrier functions, leading to increased OTA absorption, which, in turn, causes rapid inflammation associated with desquamation and necrosis (Maresca et al., 2001). In addition, OTA stimulates the release of major inflammatory cytokines, tumor necrosis factor-alpha (TNF-a), interleukin-6 (IL-6), and IL-8 (Periasamy et al., 2016; Weidenbach et al., 2000). Mammary epithelial cells are mainly involved in the synthesis of milk components and the regulation of blood-milk barrier junctions (Anderson et al., 2007; Shennan, 1998; Truchet and OllivierBousquet, 2009). However, under mastitis, exogenous stimulimediated inflammation of mammary epithelial cells may disrupt

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tight junctions of the blood-milk barrier and cause it to collapse, resulting in the leakage of blood and milk into and out of the barrier (Lehmann et al., 2013; Nguyen and Neville, 1998). The blood-milk barrier is composed not only of mammary epithelial cells but also of tight junctions, which are a branching network of membrane proteins such as claudins, occludins, and junction adhesion molecules, sealed tightly to prevent milk leakage (Furuse et al., 1993; Tsukita and Furuse, 2000). Claudins and occludins, the two most important components in tight junctions, are modified by inflammation, resulting in the loss of tight junctions in mammalian models (Prasad et al., 2005; Tedelind et al., 2003). Therefore, a better understanding of the physiological interactions which occur between mammary epithelial cells and genes regulating tight junctions may contribute to improved stabilization of milk production. Despite the widespread knowledge in regard to the toxic effects of OTA on bovine mammary epithelial cells (Fusi et al., 2008), there is a lack of information about the mechanisms by which OTA induces cytotoxicity. Therefore, the effects of OTA on bovine mammary epithelial cells were investigated using several mechanistic analyses. We found that OTA may mediate mitotic arrest, induce cell death, and regulate related kinase signaling pathways. Moreover, we studied the effects of OTA on inflammatory responses and two major tight junction regulators, occludin and claudin-3. In totality, this study is expected to improve the understanding of physiological mechanisms involved in lactation, in addition to providing a guideline for the stabilization of industrial milk production by countering exogenous contaminants in livestock. 2. Materials and methods 2.1. Chemicals OTA (catalog number: O1877) was purchased from Sigma Aldrich (St. Louis, MO). Antibodies used in the present study were shown in Table 1. The inhibitor for ERK1/2 (U0126, catalog number: EI282), JNK MAPK (SP600125, catalog number: EI305), P38 (SB203580, catalog number: EI286) was obtained from Enzo Life Sciences, Inc. (Farmingdale, NY), and the PI3K/AKT inhibitor (wortmannin, catalog number: 9951) was obtained from Cell Signaling Technology, Inc. 2.2. Cell culture The bovine mammary epithelial (MAC-T) cells used in the Table 1 List of antibodies used in immunofluorescence and western blot analysis. Primary antibodies

Dilution

Supplier

Catalog No.

Phospho-AKT (SER473) AKT Phospho-P70S6K (Thr421/Ser424) P70S6K Phospho-S6 (Ser235/236) S6 Phospho-Cyclin D1 (Thr286) Cyclin D1 Phospho-ERK1/2 (Thr202/Tyr204) ERK1/2 Phospho-JNK (Thr183/Tyr185) JNK Phospho-P38 (Thr180/Tyr182) P38 Phospho-c-Jun (Ser73) c-Jun PCNA TUBA

1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:1000 1:100 1:2000

Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Cell Signaling Santa Cruz Santa Cruz

4060 9272 9204 9202 2211 2217 3300 2922 9101 4695 4668 9252 4511 9212 3270 9165 sc-56 sc-5286

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present in vitro studies were a gift from Dr. Hong Gu Lee (Konkuk University, Republic of Korea) and maintained in DMEM/High glucose Medium (catalog number: SH30243.01) with 10% fetal bovine serum (FBS) at 37  C in a 5% CO2 incubator. Based on preliminary dose-response experiments, 1 mM of OTA was used in all experiments. This design was replicated in 3 independent experiments. 2.3. Proliferation assay Proliferation assays were conducted using Cell Proliferation ELISA, BrdU kit (catalog number: 11647229001, Roche, Indianapolis, IN, USA) according to the manufacturer's recommendations as described previously (Lim et al., 2018). 2.4. Cell cycle analysis Cells were seeded in a 6-well plate, and incubated for 24 h in serum-free DMEM/High Glucose medium. Cells were then treated with OTA in a dose-dependent manner (0, 0.1, 0.5, 1.0 mM) for 24 h and cell cycle assay was conducted using propidium iodide (PI; BD Biosciences, Franklin Lakes, NJ, USA) as described previously (Lim et al., 2018). 2.5. Immunofluorescence analysis The effects of OTA on PCNA expression were determined via immunofluorescence microscopy. MAC-T cells (3  104 cells per 300 mL) were seeded on confocal dishes (catalog number: 100350, SPL Life Science, Republic of Korea), and incubated for 24 h in serum-free DMEM/High Glucose medium. For detection of PCNA protein, the serum-starved cells were treated with 1.0 mM of ochratoxin A for 24 h following which the cells were fixed using methanol and performed as described previously (Lim et al., 2018). 2.6. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay MAC-T cells (3  104 cells per 300 mL) were seeded on confocal dishes (Cat No. 100 350, SPL Life Science, Republic of Korea) and treated with ochratoxin A at a final concentration of 1.0 mM for 24 h at 37  C in a CO2 incubator. After incubation, the cells were air dried and fixed with 4% paraformaldehyde for 1 h at room temperature. The cells were briefly rinsed with PBS and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. Subsequently, the cells were subjected to TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining mixture by using the In Situ Cell Death Detection kit, TMR red for 1 h at 37  C in the dark according to the manufacturer's recommendations as described previously (Park et al., 2018). 2.7. Mitochondrial membrane potential assay Changes in mitochondrial membrane potential (MMP) were analyzed using a Mitochondria staining kit (Cat No: CS0390, SigmaAldrich) in ochratoxin A-treated bovine mammary epithelial cells (0, 0.1, 0.5 and 1.0 mM) for 24 h according to the manufacturer's recommendations as described previously (Park et al., 2018). 2.8. Measurement of intracellular and mitochondrial free Ca2þ concentration MAC-T cells (2  105 cells) were seeded on 6-well plates and incubated for 24 h in serum-free medium until 70e80% confluency was reached. Cells were then treated with OTA (0, 0.1 0.5 1.0 mM) for

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24 h at 37  C in a CO2 incubator and estimated intracellular and mitochondrial free Ca2þ concentration as described previously (Lim et al., 2017).

3. Results

2.9. Western blot analysis

In order to investigate the cytotoxic effects of OTA on MAC-T cells, we performed a viability and proliferation assay using different OTA concentrations (0, 0.1, 0.5, 1.0 mM) for 24 h. Results demonstrated inhibition of MAC-T cell proliferation by OTA in a dose dependent manner (Fig. 1A). Based on the results, 1.0 mM of OTA which inhibited cell proliferative activity by approximately 52% compared to control cells was considered the optimal concentration for further experimentation. The expression of proliferating cell nuclear antigen (PCNA), a co-factor of DNA polymerase, decreased by 68% in MAC-T cells treated with 1.0 mM OTA for 24 h, compared to non-treated MAC-T cells (Fig. 1B). To support these results, we conducted a cell cycle assay using flow cytometric estimation by propidium iodide (PI)-stained MAC-T cells and examined the expression of cyclins and cyclin-dependent kinases (CDK) using western blot and qualitative PCR. The Sub-G1 phase, especially the dead cell population, was increased by OTA treatment of MAC-T cells in a dose-dependent manner with concomitant reduction of G1 phase population (Fig. 2A). With respect to protein levels, OTA treatment of MAC-T cells decreased cyclin D1 phosphorylation in a dose- and time-dependent kinetic approach (Fig. 2BeC). In addition, the mRNA expression of cyclin D1 and CDK4 decreased in MAC-T cells treated with 1 mM OTA (Fig. 2D). These

Protein concentrations in whole-cell extracts were determined using the Bradford protein assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin (BSA) as the standard. Each protein (20 mg) were denatured, separated by using sodium dodecyl sulfate polyacrylamide gel electrophoresis, (SDSePAGE) and transferred to nitrocellulose as described previously (Lim et al., 2018). The information of primary antibodies was illustrated in Table 1.

2.10. Annexin V and PI staining Apoptosis of bovine mammary epithelial cells induced by ochratoxin A (1 mM) with or without inhibitors such as wortmannin (1 mM), U0126 (20 mM), SP600125 (20 mM) and SB203580 (20 mM) for 24 h incubation were analyzed by using an FITC Annexin V apoptosis detection kit I (BD Biosciences) as described previously (Park et al., 2018).

2.11. Quantitative RT-PCR analysis

3.1. Inhibitory effects of OTA on MAC-T cell proliferation and cell cycle progression

Complementary DNA was synthesized using total RNA extracted from each of the tissues and AccuPower RT Pre-Mix (Bioneer, Daejeon, Korea). Gene expression was measured using SYBR Green (Sigma, St. Louis, MO, USA) and a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's recommendations as described previously (Lim et al., 2018). Cyclin D1, CDK4, occludin, claudin 3, IL-8, TNF-a, IL-1b, and GAPDH were analyzed in triplicate. For Cyclin D1, the sense primer (50 -GCA CTT CCT CTC CAA GAT GC-30 ) and antisense primer (50 -GGT TGG AAA TGA ACT TCA CG-30 ) amplified a 110-bp product. CDK4, the sense primer (50 -ATT TCC TTC ATG CCA ACT GC-30 ) and antisense primer (50 -TCA GCC AGC TTG ACT GTC C-30 ) amplified an 88-bp product. For occludin, the sense primer (50 -GCC AGC ATA TTC CTT CTA CCC-30 ) and antisense primer (50 -AAG AGT GGA GGC AAC ACA GG-30 ) amplified a 139-bp product. For TNF-a, the sense primer (50 -CAT CAA GAG CCC TTG CCA CA-30 ) and antisense primer (50 -CGG CAT AGT CCA GGT AGT CC-30 ) amplified a 149-bp product. For IL-8, the sense primer (50 -CAG AAC TTC GAT GCC AAT GC-30 ) and antisense primer (50 -TTT AGG CAG ACC TCG TTT CC-30 ) amplified a 148bp product. For IL-1b, the sense primer (50 -CGA CGA GTT TCT GTG TGA CG-30 ) and antisense primer (50 -CCA CTT CTC GGT TCA TTT CC30 ) amplified a 148-bp product. For GAPDH, the sense primer (50 CAC AGT CAA GGC AGA GAA CG-30 ) and antisense primer (50 -TAC TCA GCA CCA GCA TCA CC-30 ) amplified a 108-bp product.

2.12. Statistical analyses All quantitative data were subjected to least squares ANOVA using the General Linear Model procedures of the Statistical Analysis System (SAS Institute Inc., Cary, NC). Western blot data were corrected for differences in sample loading using total protein data as a covariate. All tests of significance were performed using the appropriate error terms according to the expectation of the mean squares for error. A P-< 0.05 was considered significant. Data are presented as least-square means (LSMs) with SEs.

Fig. 1. Effects of OTA on MAC-T cell proliferation. [A] Dose-dependent effects of OTA on MAC-T cell proliferation were determined. Data are presented as percentage relative to vehicle (100%). Cells were treated with OTA for 24 h. [B] PCNA protein (green) was detected and nuclei were counterstained with DAPI (blue) in MAC-T cells treated with 1 mM OTA for 24 h. Data represent three independent experiments. Significance was determined as, *** ¼ p < 0.001 and * ¼ p < 0.05. White squares zoomed in the proliferating cells in MAC-T cells with or without ochratoxin A. Scale bar represents 40 mm (first and third horizontal panels) and 20 mm (second and fourth horizontal panels).

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Fig. 2. Effects of OTA on MAC-T cell cycle progression, cyclin D1 protein and cyclin D1, CDK4 mRNA expression. [A] Dose-dependent effects of OTA on the cell cycle progression of MAC-T cells were analyzed by flow cytometry. Data are presented as percentage relative to vehicle (100%). Cells were treated with OTA for 24 h. Changes in phospho- and total-cyclin D1 in dose-dependent effects [B] and time-dependent effects [C] of OTA treatment were analyzed by western blot. Blotting membranes were captured to calculate the normalized values for p-cyclin D1 and t-cyclin D1 relative to a-tubulin and the bar values are presented as fold changes relative to non-treated control MAC-T cells. [D] Differential expression of cyclin D1 and CDK4 in MAC-T cells treated with OTA was determined by quantitative RT-PCR. These experiments were performed in triplicate and normalized to GAPDH. Each bar in the graphs represents the mean ± SE of pooled data from three independent experiments. Significance was determined as, *** ¼ p < 0.001 and ** ¼ p < 0.01.

results indicate that OTA suppresses MAC-T cell proliferation through cell cycle abrogation via the degradation of cyclin D1 and CDK4. 3.2. OTA causes DNA fragmentation and mitochondrial membrane potential disruption in MAC-T cells Annexin V and PI staining was conducted to verify OTA-induced cell death modalities of MAC-T cells. Treatment of MAC-T cells with OTA (1 mM) increased apoptotic cells by approximately 1.76-fold compared to non-treated MAC-T cells (Fig. 3A). DNA fragmentation-mediated cell death induction in MAC-T cells treated with 1 mM OTA was detected by TUNEL assay and TMR red-stained apoptotic cells were visualized by red fluorescence. TMR red fluorescence was substantially expressed in the nuclei of OTA-treated MAC-T cells, compared to non-treated cells (Fig. 3B). Furthermore, JC-1 staining analysis was conducted to identify the effects of OTA on the mitochondrial membrane potential of MAC-T cells. Loss of mitochondrial membrane potential of MAC-T cells was induced by OTA treatment. In addition, quantification of JC-1-positive cells revealed an increase of over twofold in 1 mM OTA-treated cells compared to control cells (Fig. 3C). These results indicate that OTA may induce mitochondrial dysfunction followed by apoptotic cell death in MAC-T cells. 3.3. Effects of OTA on mitochondrial and cytosolic calcium concentration in MAC-T cells Calcium is an important ion, required to maintain cellular homeostasis under normal conditions. Hence, the reduction of calcium capacity is considered a relevant marker for detecting cellular homeostasis disruption-related cell death induction. Mitochondrial and intracellular calcium concentrations were measured by Rhod-2 and Fluo-4 staining analysis, respectively (Fig. 4A and B). OTA treatment decreased both intracellular and mitochondrial calcium concentrations in a dose-dependent manner in MAC-T cells. These results indicate that OTA may inhibit calcium homeostasis in MACT cells, leading to the induction of cell death via disruption of cellular metabolism and mitochondrial functions.

Fig. 3. Cytotoxic and mitochondrial membrane potential disrupting effects of OTA on MAC-T cells. [A] The apoptotic cell population in OTA treated MAC-T cells was sorted by flow cytometry after staining with annexin V and propidium iodide (PI) dye. The number of late apoptotic cells (upper right quadrant) was expressed as a percentage relative to vehicle-treated control cells (100%). [B] TMR red-stained apoptotic cells (red) and nuclei counterstained with DAPI (blue) were visualized in MAC-T cells treated with OTA. [C] Depolarization of mitochondrial membrane potential (MMP) by OTA in MAC-T cells was analyzed by flow cytometry via JC-1 staining. Significance was determined as *** ¼ p < 0.001. White squares zoomed in the proliferating cells in MACT cells with or without ochratoxin A. Scale bars represent 40 mm (first and third vertical panels) and 20 mm (second and fourth vertical panels).

3.4. OTA mediates signal transduction cascade in MAC-T cells To identify the OTA induced signaling pathway in MAC-T cells, we measured the activation of PI3K (phosphoinositide 3 kinase)/ AKT (protein kinase B) and MAPK (mitogen-activated protein kinase) signals using western blot. Treatments of 0, 0.1, 0.5, and 1 mM OTA increased phosphorylation of MAPK signal molecules in MAC-T cells, in a dose-dependent manner. Phosphorylation of ERK 1/2, JNK, c-JUN, P38 increased 2.6-fold, 9.4-fold, 8.1-fold, and 2.7-fold respectively (Fig. 5DeG). However, OTA treatment did not affect phosphorylation of AKT (Fig. 5A). By contrast, phosphorylation of P70S6K and S6 increased in a dose-dependent manner in response to OTA treatment (Fig. 5B and C). In order to further investigate OTA-associated signaling interrelations, pharmacological

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Fig. 4. OTA decreases cytosolic Ca2þ and mitochondrial Ca2þ concentration in MAC-T cells. [A] Flow cytometric detection of intracellular Ca2þ in response to OTA in a dose-dependent manner for 24 h using Fluo-4 staining analysis. [B] Rhod-2 fluorescence was identified by flow cytometry in response to OTA in a dose-dependent manner for 24 h in MAC-T cells to detect mitochondrial Ca2þ concentration. Data are representative of three independent experiments. Significance was determined as *** ¼ p < 0.001.

Fig. 5. Dose-dependent effects of OTA on the activation of intracellular signaling proteins. Changes in phosphorylation of [A] AKT, [B] P70S6K, [C] S6, [D] ERK1/2, [E] P38, [F] JNK and [G] c-Jun in response to treatment of OTA in MAC-T cells were determined using western blot. Blotting membranes were captured to calculate the normalized values for phosphorylated proteins (p-proteins) relative to total proteins (t-proteins). The graphical presentation indicates the abundance of p-proteins normalized with an abundance of t-proteins, and the bar values are presented as fold changes relative to non-treated (0 mM) control MAC-T cells. Each bar in the graphs presents a mean ± SE of pooled data from three independent experiments. Significance was determined as, *** ¼ p < 0.001; ** ¼ p < 0.01; * ¼ p < 0.05.

inhibitors, Wortmannin (PI3K inhibitor, 1 mM), U0126 (ERK 1/2 inhibitor, 20 mM), SP600125 (JNK inhibitor, 20 mM) and SB203580 (P38 inhibitor, 20 mM) were pre-treated prior to OTA (1 mM) treatment in MAC-T cells based on previous study using each inhibitor (Yang et al., 2018). Pretreatment of SP600125 and SB203580 decreased susceptibility of MAC-T cells to OTA, indicating that JNK and P38 may be direct target molecules leading to OTA-induced anti-proliferative activity (Fig. 6A). Wortmannin pre-treatment suppressed AKT phosphorylation (Fig. 6B) but not P70S6K and S6. Instead, the ERK 1/2 inhibitor, U0126, effectively suppressed phosphorylation of P70S6K and S6 (Fig. 6C and D). Phosphorylation of P70S6K, S6 and ERK 1/2 increased following pre-treatment with SB203580 (Fig. 6CeE). U0126 increased phosphorylation of AKT and p38 (Fig. 6B and F). SP600125 pre-treatment suppressed phosphorylation of JNK and c-JUN and increased phosphorylation of p38 (Fig. 6G and H). In line with proliferation results shown in Fig. 6A, pre-treatment with SP600125 and SB203580 decreased OTA-induced apoptotic cell death compared to OTA single

Fig. 6. Signaling cascades in response to OTA and their effect on OTA induced MAC-T cell proliferation. [A] The effects of inhibition of PI3K and MAPK on OTA induced cell proliferation were determined using a BrdU cell proliferation assay. Serum-starved MAC-T cells were incubated with OTA alone or OTA plus inhibitors for 24 h. Cell proliferation is shown as a percentage relative to non-treated cells (100%). The effects of inhibition of PI3K and MAPK on OTA-mediated signaling proteins were determined using western blot with MAC-T cells cultured in the presence of OTA alone or cotreatment of OTA with wortmannin (1 mM, PI3K inhibitor), U0126 (20 mM, ERK1/2 inhibitor), or SP600125 (20 mM, JNK inhibitor). The abundance of phosphorylated and total proteins of [B] AKT, [C] P70S6K, [D] S6, [E] ERK1/2, [F] P38, [G] JNK and [H] c-Jun were analyzed. After pre-incubation of MAC-T cells with each pharmacological inhibitor for 2 h, the cells were additionally incubated with OTA. Blotting membranes were captured to calculate the normalized values for p-proteins relative to t-proteins. The graphical presentation indicates the abundance of p-proteins normalized with the abundance of t-proteins, and the bar values are presented as fold changes relative to non-treated cells. [I] The apoptotic population related to treatment with OTA and pretreatment with pharmacological inhibitor-treated MAC-T cells was sorted by flow cytometry after staining with annexin V and PI dye. Each bar in the graphs presents a mean ± SE of pooled data from three independent experiments. Significant differences between the OTA only treatment and the other treatment groups was determined as, *** ¼ p < 0.001; ** ¼ p < 0.01; * ¼ p < 0.05.

treatment, which supports our hypothesis that JNK and P38 are major targets in OTA-induced triggering of apoptotic cell death in MAC-T cells (Fig. 6I). In totality, these results indicate that OTA may regulate MAPK and PI3K signaling in MAC-T cells and p38, and that the JNK-MAPK pathway is the major cell signaling pathway associated with OTA-triggered apoptosis.

3.5. OTA transforms functional characteristics of bovine mammary epithelial cells To investigate the effects of OTA on functional molecules of MAC-T cells, we conducted quantitative RT-PCR. mRNA expression of the two major milk-blood barrier tight junction markers, occludin and claudin 3 was decreased in MAC-T cells due to OTA treatment (Fig. 7A). Next, we assessed the mRNA expression of inflammatory cytokines which are negative regulators of occludin and claudin 3. Overall, cytokines, IL-8, IL-1b, and TNF-a in MAC-T cells were increased by OTA treatment (Fig. 7B). These results indicate that OTA disrupts tight junction functions in the bloodmilk barrier and induces inflammatory cytokine stimulations in MAC-T cells.

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Fig. 7. OTA decreases mRNA expression levels of blood-milk barrier genes and increases inflammatory cytokines. [A] Differential expression of tight junction genes, occludin and claudin 3 mRNA in MAC-T cells and [B] inflammatory cytokines, IL-8, IL-1b and TNF-a were determined by qualitative PCR. These experiments were performed in triplicate and normalized to GAPDH. Each bar represents a mean ± SEM of three independent experiments. Significance was determined as, *** ¼ p < 0.001 and ** ¼ p < 0.01.

4. Discussion Hazards posed by OTA to humans as well as livestock remains an active topic in both public health and scientific research circles, due to its role as a widespread contaminant, which has harmful effects in humans and animals under continuous exposure. Ingestion of OTA-contaminated feed is a major factor in the chronic accumulation of OTA in body fluids, such as the bloodstream and milk of livestock. Subsequently, OTA accumulated in animal organs such as the kidneys and adipose tissues, may eventually transfer to humans via the food chain (Breitholtz-Emanuelsson et al., 1993; Persi et al., 2014; Shreeve et al., 1979). As cow milk constitutes the major portion of human milk intake, an understanding of the physiological aspects associated with the harmful effects of OTA with particular reference to cow lactation may be vital. In cow lactation, proper regulation of the function of bovine mammary epithelial cells is crucial for the regular production of milk. Hence, it is evident that toxic effects caused by external contaminants such as mycotoxins may have a significant impact on bovine mammary cells and thereby affect cow milk production. Although toxic effects induced by OTA on bovine mammary epithelial cells have largely been characterized (Fusi et al., 2008), there remains a lack of information concerning mechanisms underlying OTA-induced cytotoxicity. Therefore, we investigated the mechanistic effects of OTA on MAC-T cells. In this study, we elucidated that OTA abrogates proliferation and stimulates apoptotic cell death in MAC-T cells. Furthermore, OTA reduces mitochondrial and cytosolic calcium levels, leading to late stage apoptosis. Moreover, we evaluated OTA-mediated PI3K and MAPK signal transduction, as well as the reduction in tight junction regulatory genes, which occur as a consequence of the inflammatory response. Cell cycle arrest may be evaluated with DNA replication (G1/S

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checkpoint), and mitosis (G2/M checkpoint), to allow DNA repair and decline of damaged DNA (Cui et al., 2010; Paulovich et al., 1997; Weinert, 1997). This substantial process is controlled by the interaction between cyclin proteins and associated cyclin-dependent kinases (Lee et al., 2012). Several mycotoxins, such as OTA, deoxynivalenol, sterigmatocystin, and zearalenone, are known to induce cell cycle arrest in leukemia, colon cancer, mouse embryo fibroblasts, human intestinal epithelial cells, and Chinese hamster lung fibroblasts (Kamp et al., 2005; Palma et al., 2007; Xie et al., 2000; Yang et al., 2008). OTA induced mitotic arrest in MAC-T cells, as observed in our results, is consistent with a previous study, indicating that OTA may be linked to chromosomal instability and mitosis abnormality in human epithelial cells (Adler et al., 2009). This suggests that OTA induced cytotoxicity in both humans and bovines may be due to cell cycle arrest, which, in turn, leads to the inhibition of proliferation and induction of apoptosis. In line with cell cycle arrest, expression of the anti-proliferative markers, PCNA, Cyclin D1, and CDK4 was reduced by OTA, revealing that OTA may possess an anti-proliferative function, in addition to causing mitotic arrest, and inhibiting cell cycle regulatory genes in bovine mammary epithelial cells. Along with cell cycle arrest, mycotoxin-induced apoptosis is another common toxic effect seen in cells (Ayed-Boussema et al., 2008; Minervini et al., 2004). OTA induced apoptosis was shown in HepG2 (Bouaziz et al., 2008), HeLa (Seegers et al., 1994), MDCK C7 (Gekle et al., 2000), and HEK293 cells (Kamp et al., 2005), where the mechanism underlying the induction of cell death was associated with intracellular calcium balance (Walter and Hajnoczky, 2005). Under stressful conditions, endoplasmic reticulum (ER) releases calcium via the RyR and IP3 receptors, resulting in the rapid elevation of cytosolic calcium levels. Furthermore, abnormal calcium accumulation or reduction is also caused by mitochondria, which plays a key role in cellular metabolism. Overloading of calcium through the mitochondrial permeability transition pore channel, leads to mitochondrial damage causing the release of cytochrome c, which activates apoptosis (Breckenridge et al., 2003; Di et al., 2012; Kadenbach et al., 2004; Lamb et al., 2006; Walter and Hajnoczky, 2005). OTA reduced cytosolic and mitochondrial calcium levels in MAC-T cells, suggesting that OTA may disrupt cellular homeostasis, resulting in apoptosis induction. Proper interaction between PI3K and MAPK signaling pathways is fundamental for many physiological processes, such as cell proliferation, metabolism, differentiation, cell death, and survival (Aksamitiene et al., 2012). Among the subgroups of MAPKs, c-Jun N-terminal kinase (JNK), P38 and extracellular signal-regulated kinase (ERK) are known to be components of apoptosis associated pathways (Chuang et al., 2000). JNK regulates apoptosis by suppressing anti-apoptotic proteins such as Bcl-2 and Bcl-xL, which disrupt mitochondrial membrane potential. Disruption of the mitochondrial membrane potential results in the release of cytochrome c, and the activation of caspase 3 and 9 to trigger apoptosis. In mammary epithelial cells, JNK signaling is required for mammary gland development, whereas mammary malignant tumor transformation was suppressed by this pathway (Cellurale et al., 2012). In addition, signal transduction of MAPK pathway triggers the elongation of mammary epithelial tubes (Huebner et al., 2016). Moreover, OTA mediated activation of MAPK and JNK, increased apoptotic cell death in human CD4þ T lymphoma cells and canine renal epithelial cells (Darif et al., 2016; Gekle et al., 2000). Our results reveal that OTA activated MAPKs (JNK, P38, and ERK), P706K, S6, and c-Jun pathways, although AKT was not affected by OTA in MAC-T cells. Pretreatment with SP600125 and SB203580 decreased susceptibility of MAC-T cells to OTA, indicating that JNK and P38 may be direct target molecules related to the OTA-induced antiproliferative activity. Furthermore, pretreatment with ERK

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inhibitor, U0126, sensitized MAC-T cells to OTA treatment leading to synergistic apoptosis, indicating that ERK may be one of the relevant pathways for OTA mediated apoptosis signaling. Considering the fact that there is only a limited number of mechanistic studies concerning OTA-triggered cell death induction in mammary epithelial cells, this study may help to obtain a better understanding of the mechanism underlying the induction of cell death by the prominent mycotoxin OTA. Mammary epithelial cells play a functional role in overall lactation, inclusive of stages such as milk protein synthesis, secretion, and blood-milk barrier control (Anderson et al., 2007; Larson, 1979; Shennan, 1998; Truchet and Ollivier-Bousquet, 2009). In lactating mammals, mammary tissues undergo a rapid reversible transition of structural regression and restoration according to their functional distribution (De Vries et al., 2010; Kang et al., 2018; Seeth et al., 2015). In order to maintain and establish appropriate lactation, the mammary epithelium is transformed, a process which requires a three-dimensional structure with complete tight junctions between extracellular matrix attachments and adjoining mammary epithelial cells (Janjanam et al., 2014). Such repeated cellular remodeling is accompanied by cell death and proliferation, which allow regulation of mammary lactation and blood-milk barrier junctions. The bovine lactation curve of mammary cell growth and differentiation indicates that the ascending portion is associated with increasing capacity for milk production, while the descending portion is associated with declining milk yield of mammary cells (Di et al., 2012). Therefore, stimulation of mammary cell proliferation and abrogation of cell death contributes to increased persistence of lactation and proper regulation of bloodmilk barrier tight junctions. However, as a consequence of inflammation due to exogenous contaminants such as mycotoxin, tight junctions of the blood-milk barrier collapse, leading to milk leakage inside and outside the barrier (Lehmann et al., 2013; Nguyen and Neville, 1998). In addition, the blood-milk barrier is regulated by the proper interaction between mammary epithelial cells and tight junction regulators which prevent abnormal outflow of milk and blood components (Furuse et al., 1993; Tsukita and Furuse, 2000). Moreover, the most relevant tight junction regulators of the blood-milk barrier are claudins and occludins, which are affected by inflammatory responses resulting in the loss of tight junctions in mammalian models (Prasad et al., 2005; Tedelind et al., 2003). Consistent with previous results, we found that OTA

Fig. 8. Hypothetical schematic illustration of OTA induced apoptosis in bovine mammary epithelial cells. OTA induced apoptosis by activating JNK MAPK, P38 MAPK pathways, inducing DNA fragmentation and inhibition of cell cycle progression in bovine mammary epithelial cells. Furthermore, OTA decreased both cytosolic and mitochondrial Ca2þ concentration and disrupted MMP in a dose-dependent manner in MAC-T cells. Gene expression of occludin and claudin 3, as well as the composition of tight junction proteins, was decreased by OTA treatment in MAC-T cells. Moreover, OTA increased gene expression levels of inflammatory cytokines.

increased the expression of IL-8, IL-1b, and TNF-a, while simultaneously overexpressing occludin, and claudin 3, indicating that OTA contamination may stimulate inflammatory responses, leading to the collapse of the blood-milk barrier junction. This may cause a leakage of milk and blood in the bovine mammary. In summation, our results suggest that OTA contamination may affect mammary functions of lactating cows via the remodeling of junctional adhesion molecules as illustrated in Fig. 8. This may be influenced by OTA-induced inflammatory responses, as well as by the disruption of homeostasis in bovine mammary epithelial cells due to calcium reduction and mitochondria permeabilization. Further functional investigation is needed in order to precisely determine how OTA affects the mammary lactation system and lactating qualities in cows. Therefore, further studies may bring forth a comprehensive understanding of the systematic effects of OTA in bovine mammary epithelial cells and thereby contribute to the enhancement of the quality of cow milk and the stabilization of industrial dairy production affected by exogenous contamination. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute funded by the Ministry of Health and Welfare (grant number: No. HI15C0810 awarded to G.S. and HI17C0929 awarded to W.L.) References Adler, M., Muller, K., Rached, E., Dekant, W., Mally, A., 2009. Modulation of key regulators of mitosis linked to chromosomal instability is an early event in ochratoxin A carcinogenicity. Carcinogenesis 30, 711e719. Aksamitiene, E., Kiyatkin, A., Kholodenko, B.N., 2012. Cross-talk between mitogenic Ras/MAPK and survival PI3K/Akt pathways: a fine balance. Biochem. Soc. Trans. 40, 139e146. Anderson, S.M., Rudolph, M.C., McManaman, J.L., Neville, M.C., 2007. Key stages in mammary gland development. Secretory activation in the mammary gland: it's not just about milk protein synthesis! Breast Cancer Res. 9, 204. Ayed-Boussema, I., Bouaziz, C., Rjiba, K., Valenti, K., Laporte, F., Bacha, H., Hassen, W., 2008. The mycotoxin Zearalenone induces apoptosis in human hepatocytes (HepG2) via p53-dependent mitochondrial signaling pathway. Toxicol. Vitro 22, 1671e1680. Bendele, A.M., Carlton, W.W., Krogh, P., Lillehoj, E.B., 1985. Ochratoxin A carcinogenesis in the (C57BL/6J X C3H)F1 mouse. J. Natl. Cancer Inst. 75, 733e742. Boorman, G.A., McDonald, M.R., Imoto, S., Persing, R., 1992. Renal lesions induced by ochratoxin A exposure in the F344 rat. Toxicol. Pathol. 20, 236e245. Bouaziz, C., Sharaf El Dein, O., El Golli, E., Abid-Essefi, S., Brenner, C., Lemaire, C., Bacha, H., 2008. Different apoptotic pathways induced by zearalenone, T-2 toxin and ochratoxin A in human hepatoma cells. Toxicology 254, 19e28. Breckenridge, D.G., Stojanovic, M., Marcellus, R.C., Shore, G.C., 2003. Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol. J. Cell Biol. 160, 1115e1127. Breitholtz-Emanuelsson, A., Olsen, M., Oskarsson, A., Palminger, I., Hult, K., 1993. Ochratoxin A in cow's milk and in human milk with corresponding human blood samples. J. AOAC Int. 76, 842e846. Bui-Klimke, T.R., Wu, F., 2015. Ochratoxin A and human health risk: a review of the evidence. Crit. Rev. Food Sci. Nutr. 55, 1860e1869. Castegnaro, M., Mohr, U., Pfohl-Leszkowicz, A., Esteve, J., Steinmann, J., Tillmann, T., Michelon, J., Bartsch, H., 1998. Sex- and strain-specific induction of renal tumors by ochratoxin A in rats correlates with DNA adduction. Int. J. Canc. 77, 70e75. Cellurale, C., Girnius, N., Jiang, F., Cavanagh-Kyros, J., Lu, S., Garlick, D.S., Mercurio, A.M., Davis, R.J., 2012. Role of JNK in mammary gland development and breast cancer. Cancer Res. 72, 472e481. Chuang, S.M., Wang, I.C., Yang, J.L., 2000. Roles of JNK, p38 and ERK mitogenactivated protein kinases in the growth inhibition and apoptosis induced by cadmium. Carcinogenesis 21, 1423e1432. Cui, J., Xing, L., Li, Z., Wu, S., Wang, J., Liu, J., Wang, J., Yan, X., Zhang, X., 2010. Ochratoxin A induces G(2) phase arrest in human gastric epithelium GES-1 cells in vitro. Toxicol. Lett. 193, 152e158. Darif, Y., Mountassif, D., Belkebir, A., Zaid, Y., Basu, K., Mourad, W., Oudghiri, M.,

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