Methylseleninic Acid Prevents Patulin-Induced Hepatotoxicity and

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Methylseleninic acid prevents patulin-induced hepatotoxicity and nephrotoxicity via inhibition of oxidative stress and inactivation of p53 and MAPKs Xiaotong Lu, Enxiang Zhang, Shutao Yin, Lihong Fan, and Hongbo Hu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 08 Jun 2017 Downloaded from http://pubs.acs.org on June 9, 2017

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Journal of Agricultural and Food Chemistry

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Methylseleninic acid prevents patulin-induced hepatotoxicity and

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nephrotoxicity via inhibition of oxidative stress and inactivation

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of p53 and MAPKs

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Xiaotong Lu# , Enxiang Zhang# , Shutao Yin#, Lihong Fan* and Hongbo Hu#

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#

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of Food Science and Nutritional Engineering, China Agricultural University, Beijing

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Key Laboratory for Food Non-thermal Processing

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No17 Qinghua East Road, Haidian District, Beijing 100083, China

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Beijing Advanced Innovation Center for Food Nutrition and Human Health, College

*

College of Veterinary Medicine, China Agricultural University,

No2 Yuanmingyuan West Road, Haidian District, Beijing 100193, China

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Corresponding author:

Hongbo Hu, Ph.D., Phone: 86-10-6273-8653, Email: [email protected]

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and Lihong Fan, Phone :86-10-62733322, Email : [email protected]

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ACS Paragon Plus Environment


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Abstract

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Patulin is one of the common food-borne mycotoxins. Previous studies have

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demonstrated that patulin can cause diverse toxic effects in animals including

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hepatotoxicity and nephrotoxicity. In the present study, we have addressed the

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protective effect of two forms of selenium compounds methylseleninic acid (MSeA)

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and sodium selenite, on patulin-induced nephrotoxicity and hepatotoxicity using both

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in vitro and in vivo models. Results showed that MSeA at concentrations of 3-5μM,

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not sodium selenite at the same concentrations is capable of protecting against

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patulin-induced cytotoxicity in cell culture model. Moreover, the hepatoprotective and

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nephroprotective effects of MSeA (2mg/kg body weight, oral administration) on

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patulin-induced toxicity (10mg/kg body weight, intraperitoneal injection) were also

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achieved in animal model. Further mechanistic study revealed that the protective

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effect of MSeA on patulin-mediated toxicity is attributed to its ability to inhibit

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patulin-mediated ROS generation and inactivate p53 and MAPKs signaling pathways.

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Our findings support a possible usefulness of MSeA as a novel detoxicant to mitigate

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toxicities of patulin.

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Keywords: Patulin; methylseleninic acid; hepatotoxicity; nephrotoxicity; ROS; p53;

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MAPKs;

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Introduction

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Patulin (PAT) is a fungal secondary metabolite made by Penicillium, Aspergillus,

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Paecylomtces and Byssochlamys 1, 2. It has been shown that patulin can cause diverse

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toxic effects at concentrations of 0.1-48mg/kg body weight in animals including

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neurotoxicity, hepatotoxicity and nephrotoxicity and with highly potential impact on

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human health

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contributed to the pleiotropically toxic effects of patulin

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pathways have been identified to be involved in patulin-induced apoptosis such as

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ROS generation and activation of p53 and MAPKs 12-14.

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3-6

. Apoptosis induction is suggested to be the key cellular event 7-11

. A number of signaling

Selenium is an essential micronutrient with certain pharmacological activities for

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humans and animals

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diseases such as cancer, virus infections and abnormal immune responses

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beneficial role of selenium in human health is mainly attributed to its biological

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functions in the redox regulation and defense against oxidative stress 21. Several

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selenocysteine-based selenoproteins have been identified to be involved in the

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regulation of redox signaling. Selenium exists in two forms: inorganic and organic.

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Sodium selenite is a representative of inorganic form selenium, and its biological

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activity has been well documented. Methylseleninic acid (CH3SeO2H, MSeA), a

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monomethylated selenium compound, has been shown encouraging anticancer

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activity in a number of preclinical models 22, 23. MSeA acts as an immediate precursor

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of methylselenol which is a major excretory form of selenium, acts as a precursor for

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. Selenium deficiency possibly contributes to a number of

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17-20

. The


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the synthesis of biologically important selenoproteins 24. Selenoenzyme thioredoxin

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reductase (TrxR) is involved in MSeA reduction to methlyselenol. Methylselenol can

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be further methylated to dimethylselenide and trimethylselenonium, which are

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eventually excreted via the breath or urine. It has been shown that methylselenol can

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efficiently reduce hydrogen peroxide25, 26.

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designed to evaluate the possibility of MSeA or sodium selenite protecting against

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patulin-mediated nephrotoxicity and hepatotoxicity using both in vitro and in vivo

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models. Results showed that MSeA, but not sodium selenite significantly inhibited

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patulin-induced apoptosis in cultured kidney and liver cells. Mechanistic investigation

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demonstrated that MSeA is able to suppress patulin-mediated ROS generation and to

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inactivate p53 and MAPKs signaling pathways. Moreover, the protective effect of

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MSeA on patulin-induced kidney and liver toxicity was observed in vivo. The

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findings of the present study suggest that MSeA holds potential as a novel detoxicant

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to protect against nephrotoxicity and hepatotoxicity of patulin.

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Materials and Methods

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Chemicals and reagents

The purpose of the present study was

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Patulin, Methaneseleninic acid, sodium selenite and DCFH-DA were purchased

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from Sigma-Aldrich (St. Louis, MO, USA). Antibodies specific for cleaved

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PARP(9546S), Caspase-9(9502), Cleaved Caspase-3(Asp175) (9664s), Bax(5023S),

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Bcl2(2872S),

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phospho-ERK(4094S), phospho-JNK1/2(9255), phospho-p38(4511S) and β-actin

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were purchased from Cell Signaling Technology (Beverly, MA, USA).

p21(2947S),

phospho-p53(ser15),

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phospho-H2AX(Ser139),

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Cell culture and treatments

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Human Embryonic Kidney (HEK) 293 (HEK293) cells and AML-12 mouse liver

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cells were obtained from the American Type Culture Collection (ATCC) and cultured

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in Dulbecco’s Modification of Eagle’s Medium (DMEM) ( HyClone, Logan, USA)

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supplemented with 10% fetal bovine serum. When cells were grown to approximate

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50% confluence, the treatments with MSeA and/or other agents were given. The

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doses of MSeA or patulin used in the present study are based on literature and our

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previous studies11-14.

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Western blotting

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HEK293 or AML12 cells were lysed with ice-cold RIPA buffer. SDS-PAGE

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electrophoresis was performed to resolve the proteins and which was further

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transferred to a polyvinylidene fluoride (PVDF) membrane. After incubation with

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specific primary and secondary antibodies, the immunoreactive blots were detected by

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enhanced chemiluminescence and X-ray film was used to record the signal.

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western blot images were quantitatively analyzed by ImageJ software.

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Apoptosis measurement

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The

Apoptosis was measured by methods of Annexin V/PI staining and

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immunoblotting analysis of caspases and PARP cleavages.

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Crystal violet staining

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Crystal violet staining was used to assess the influences of patulin and/or MSeA

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on cell viability. HEK293 or AML12 cells were exposed to patulin and/or MSeA for 5



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the indicated times. After the treatments, 1% glutaraldehyde solution was added to fix

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the cells. After fixation, the cells were stained with 0.02% crystal violet solution. 70%

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ethanol was used to solubilize the stained cells. The absorbance was examined at 570

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nm with the reference filter 405 nm using a microplate reader.

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Measurement of reactive oxygen species

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At

30

min

before

harvesting

the

cells,

20µM

DCFH-DA

(2',

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7'-dichlorodihydrofluorescein diacetate) was added to the cultured medium.

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DCFH-DA can be hydrolyzed to nonfluorescent dichlorofluorescein (DCFH) by

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intracellular esterases, which is then oxidized to produce fluorescent DCF in the

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presence of hydrogen peroxides. The enhanced intracellular fluorescence was

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measured using 530 nm bandpass filter with a Becton Dickinson flow cytometer.

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Animals and treatments

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BALB/c mice weighing 20.5±1.0g were obtained from Vital River (Beijing,

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China). Animal Care and procedures were approved by the Institutional Animal Care

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and Use Committee (China Agricultural University). The mice were received a

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commercial standard mouse cube diet (Beijing Keaoxieli Feed Company, China).

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After 5 days of acclimatization, the mice were randomly divided into 4 groups

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containing 8 mice each. Group 1: vehicle control, received physiological saline.

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Group 2: received MSeA (2 mg/kg). Group 3: received patulin (10 mg/kg). Group 4:

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received MSeA (2mg/kg) and patulin (10mg/kg). Mice were treated orally with

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MSeA with a single dose of 2 mg/kg body weight (B.W.) for four days continuously. 6


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patulin (10 mg/kg/i.p.) was injected 3 hours after the last MSeA administration.

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patulin and MSeA were both dissolved in physiological saline. The doses of MSeA or

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patulin are determined based on literature and our dose-finding experiments. The

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mice were sacrificed 24 h after the patulin administration and blood samples were

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collected. Kidney and liver tissues were collected and frozen immediately in liquid

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nitrogen and stored at −80 °C.

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Histopathology assessment

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Neutral-buffered formalin-fixed liver and kidney samples were processed to

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paraffin sections and stained with hematoxylin and eosin (H&E). Histopathological

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alterations were analyzed and scored by a pathologist (Dr. Fei Pei, a pathologist in the

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Department of Pathology at Peking University). Stained kidney sections were

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analyzed under a microscope using a 20× objective lens. A numeric grading scale

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from 0 to 4 was used to evaluate the changes of pathology including swelling, protein

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casts and vacuolar degeneration. Grade 0 indicated that pathology was not present.

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Grade 1 indicated minimal focal pathology. Grade 2 indicated mild multifocal

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pathology. Grade 3 indicated moderate pathology. Grade 4 indicated severe

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pathology.

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Biochemical assay

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Serum ALT(C009-2), AST(C010-2), UREA(C013-2) and LDH(A020-2) activity

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was assessed by a commercially available kit (Alanine aminotransferase Assay Kit,

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Aspartate aminotransferase Assay Kit, Urea Assay Kit, and Lacate dehydrogenase

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assay kit, Nanjing Jiancheng Institute of Biotechnology, Nanjing, China).

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Statistical analysis

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Data are presented as mean ± SD. These data were evaluated using one-way

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ANOVA followed by appropriate post-hoc comparisons among means. p

Methylseleninic Acid Prevents Patulin-Induced Hepatotoxicity and

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