Protective effects of probiotic Lactobacillus plantarum BJ0021 on liver and kidney oxidative stress and apoptosis induced by endosulfan in pregnant rats

Abstract

Endosulfan (EDS) is one of the most widely organochlorine insecticide used in many parts of the world, although it is currently banned or severely restricted in use in some countries. EDS causes a variety of negative effects in non-target species including humans. Therefore, the aim of the present study was to investigate the possible protective effects of Lactobacillus plantarum BJ0021 on toxicity, oxidative stress, and apoptosis induced by EDS intoxication on liver and kidneys of pregnant rats. This pesticide induced a significant increase in total cholesterol, alanine-amino transferase (ALAT), aspartate-amino transferase (ASAT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), urea and creatinine in serum, while urinary urea and creatinine were lower than those of the control group. In the liver and kidney, lipid peroxidation increased significantly, the antioxidant levels, such as superoxide dismutase (SOD) and catalase (CAT) were markedly depressed and TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) revealed more apoptotic cells. In contrast, co-administration of L. plantarum BJ0021 to EDS-treated animals ameliorated most of these biochemical parameters, but the activity of antioxidant enzymes (SOD, CAT) did not modify and the number of apoptotic nuclei remained significantly raised in kidney compared to control. In conclusion, the administration of L. plantarum BJ0021 decreased apoptosis and might play a protective role in reducing toxicity of EDS in pregnant rats.

• Apoptosis
• Bacillus plantarum
• EDS
• oxidative stress
• pregnant rats

Introduction

Residues from some long-lived pesticides may build up in the food chain and therefore, cause a widespread contamination of the environment with some repercussion on human health and wildlife. Among these pesticides, organochlorines, a large class of multipurpose chlorinated hydrocarbon chemicals, break down slowly in the environment and are finally used as food and accumulate in fatty tissues.1

Endosulfan (EDS)-(6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hex-ahydro-6,9-methano-2,4,3-benzo-o-dioxa-thiepin-3-oxide) is a chlorinated cyclodiene insecticide.2 It has been widely used in a variety of food and non-food crops as a broad-spectrum insecticide in order to control numerous insect species. Consequently, EDS has been ubiquitously detected in the atmosphere, soils, sediments, surface water, and rain water, and food stuffs.3 A global monitoring network of some persistent pollutants revealed that EDS was abundant in the environment when its use increased.4 This molecule is easily absorbed through the skin, stomach, and lung.5

Due to its toxic effect, EDS is currently classified as Class II (moderately hazardous to human health) by the World Health Organization (WHO) and as category Ib (highly hazardous) by the United States Environmental Protection Agency.6 In spite of the effects that are negative on health, EDS is still employed, though many other organochlorine pesticides were banned.7

Populations usually sensible to endosulfan include the unborn and neonates, the elderly and people with liver, kidney, immunological, hematological, or neurological diseases.8 The most common manifestations of EDS intoxication are neurological, although other organ dysfunctions can also occur. In many clinical studies, it was shown that EDS affected many organs, such as brain, liver, kidney, and heart. Histopathological findings in these organs have also been shown with experimental animal studies.9

Pre- and post-natal exposures to EDS were confirmed by measuring residues in human milk placenta, cord blood, and adipose tissue showing it caused congenital malformations in both animal’s and human’s offspring exposed during pregnancy and/or lactation.10

Endosulfan caused several toxic effects in the organs by inducing oxidative stress, which is one of the most important causes of pesticide.11^,12 Among the suggested mechanisms of EDS-induced toxicities in biological systems, reactive oxygen species (ROS) and other free radicals were included during detoxification process of pesticide.13 Generated ROS may then provoke certain unwanted reactions in the cell, such as membrane damage, alterations in metabolic activity, and cell death. The action of these molecules alters the permeability of membrane, impairing the functionality of the plasma membrane causing the intracellular constituents to leak out into the extracellular compartment.14

The role of oxidative stress and lipid peroxidation in EDS toxicity has been shown in erythrocytes, blood mononuclear cells, liver, kidney,15 and heart,16 which reflected on the hematologic and biochemical parameters.

The negative effects of ROS on cell metabolism included roles in apoptosis,17 individual apoptosis being an important normal physiological function in any multicellular organism. Apoptosis not only plays a crucial role in tissue development and homeostasis, but is also involved in a wide range of pathological conditions.18 Apoptotic cell death is accompanied with a series of complex biochemical events and definite morphologic changes including cell shrinkage, chromatin condensation, DNA fragmentation, membrane budding, and presence of membrane-associated apoptotic bodies.19 Recently, much attention has been focused on the use of probiotics, which were defined, such as living microorganisms, principally bacteria that are safe for human consumption and have beneficial effects on human health if they are ingested in sufficient quantities.20 Lactic acid producing bacteria (LAB), particularly Lactobacilli and Bifidobacteria are considered, such as the most probable agents responsible of these effects. The potential effectiveness for both the prevention and the treatment of immune diseases draw the attention of public on probiotic, they may promote the balance between microbial community and enzymes, and stimulate the specificity and non-specificity of immunity mechanism in the body.21

The presence of Lactobacillus in sufficient amounts is associated with decreasing cholesterol levels in serum in experimental animals.22 Studies using in vitro, cell culture, and in vivo animals indicated probiotics can bind and prevent the absorption of aflatoxins, which have been demonstrated to be implicated in the etiology of liver cancer in humans,23^,24 a rat model of ethanol-induced liver damage, which has been used to demonstrate the protective effects of probiotics.25 A recent review supported the use of probiotics in at least those patients with an end-stage kidney disease.26

Several in vivo studies demonstrated that some Lactobacillus strains can improve the antioxidant status of rats and humans, and could decrease the risk of accumulation of ROS. Therefore, many investigations focused on antioxidant properties of LAB and their role in health and disease recently.27

Lactobacillus plantarum is a non-pathogenic gram-positive bacterium naturally living in human saliva and gastrointestinal tract. As a member of the LAB, it is commonly used in food fermentation and its biotherapeutic applications have been increasingly recognized28; L. plantarum is often used as a probiotic on its own for its beneficial properties and anti-inflammatory activity.29 To our knowledge, very few studies on the protective effects of lactobacilli against EDS toxicity have been carried out so far, and the mechanism of such protection has not been studied yet. Therefore, in the present study, an attempt was made to investigate whether the supplementation of Lactobacillus plantarum BJ0021, could provide a protective effect on toxicity, oxidative stress, and apoptosis induced by EDS intoxication in pregnant rats.

Materials and methods

Chemical

A commercial formulation of the organochlorine insecticide EDS (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-ben-zodioxathiepine-3-oxide, CAS number 115-29-7) was purchased from Labor Dr. Ehrenstorfer Schäfers (Augsburg, Germany). The test chemical was prepared in corn oil (vehicle) and the dose was 4 mg/kg body weights based on a previous report30 was selected for the study.

Bacteria strain

The bacterial strain used was a local strain isolated in our laboratory from cow’s butter according to the study of Idoui and Karam.31 This strain named Lactobacillus plantarum BJ0021 was able to survive in vitro at low pH and in the presence of bile salt, and demonstrated inhibitory activity against Gram+ and Gram− bacteria.

Animals and breeding procedure

Mature and healthy Wistar rats (females of 180–220 g weight and males of 220–250 g weight) were obtained from Pasteur Institute, Algiers, Algeria. Animals were housed in plastic cages and quarantined for one week before mating with free access to water and food. During the entire study, animals were kept at the following controlled conditions: 22 °C, 40–70% relative humidity, and light/dark cycle of 12 h/12 h. Two females were cohabited overnight with a male and examined for the presence of sperm in their vagina. The day when sperm was detected was defined as gestational day 0.

Experimental design

After successful mating, the pregnant rats were individually housed in polypropylene cages and randomly distributed into three groups of 10 animals each: group I was used such as a control group and received only corn oil by oral gavage during the experiment, group II received EDS (4 mg/kg body weight) and group III received EDS (4 mg/kg body weight) plus probiotic (0.1 mL per os during the experiment and one hour before the administration of EDS). The daily dose of EDS was administered by gavage from the 6th to 20th day of gestation.

Blood sampling

On the 20th day before delivering blood samples were collected from all rats (under ether anesthesia) into heparinized tubes by puncturing the retro-orbital venous plexus with fine sterilized glass capillary tubes. Blood samples were left for 20 min at room temperature then centrifuged at 3000 rpm for 10 min to separate the plasma. Samples of plasma were kept in a deep freezer (−20 °C) until analyzed within 1 week for the biochemical estimations.

Urine collection

At the end of the experiment, the rats were placed in metabolic cages and 24-h urine was collected for the measurement of urea and creatinine.

Biochemical analysis

The activities of alanine-amino transferase (ALAT), aspartate-amino transferase (ASAT), and alkaline phosphatase (ALP) were determined with commercial kits from Spinreact, Girona, Spain (refs: GOT-1001161, GPT-1001171, and ALP-1001131). Cholesterol and triglycerides concentrations were also measured using commercial kits (Spinreact, refs: cholesterol-1001091 and triglycerides-1001311). Creatinine and urea were also measured in both serum and urine with use of commercial kits (Spinreact, Girona, Spain, refs; creatinine-1001113 and urea-1001333).

Gamma-glutamyl transpeptidase

The enzymatic activity of gamma-glutamyl transpeptidase (GGT) in serum was measured in 0.1 mol/L Tris-HCl buffer (1 M Tris, 100 mM MgCl₂), pH 9.0, following the method given by Beck et al.32 Fifty microliters of serum were incubated at 25 °C in the presence of 5 mMol/L l-γ-glutamyl-p-nitroanilide. The enzymatic reaction was stopped by the addition of 1 mL of 1 N acetic acid and the rate of p-nitroaniline release was determined with a spectrophotometer at 405 nm. One unit of GGT activity was defined as the amount of enzyme releasing μmol of p-nitroanilide per minute. The specific activity of the enzyme was expressed as units/mg protein.

Antioxidant parameters estimations

Preparation of homogenates

About 1 g of liver or 0.5 g of kidney was homogenized in 2 mL of phosphate buffer saline solution 1:2 (w/v; 1 g tissue 2 mL TBS, pH = 7.4), homogenates were centrifuged at 10,000 g for 15 min at 4 °C, and the resultant supernatant was used for the determination of catalase (CAT) and superoxide dismutase (SOD) activities.

Assay of catalase activity

Catalase activity was assayed from the rate of H₂O₂ decomposition measured by the decrease of absorbance at 240 nm, following the modified procedure by Claiborne.33 The reaction mixture constituted 1 mL of 0.059 M H₂O₂ in 0.1 M sodium phosphate buffer (pH 7.4), 1.9 mL of distilled water and 0.1 mL of tissue homogenate (3 mL assay volume). Catalase activity was expressed as units/mg protein. One unit (U) of CAT is defined as the amount of enzyme catalyzing the formation of 1 µmole of product per min and per mg of protein.

Assay of superoxide dismutase activity

Measure of SOD activity was based on the ability of the enzyme to inhibit the auto-oxidation of pyrogallol. The measurement was based on the modified method of Marklund and Marklund.34 Reagent consisted of 0.252 M pyrogallol in 0.1 M sodium phosphate buffer (pH 7.4) and the appropriate volume of enzyme extract. The reaction was initiated by light illumination and the rate of oxidation was measured with a spectrophotometer at 420 nm. SOD activity is expressed as units/mg protein. The unit is defined as the amount of the enzyme which caused 50% inhibition of pyrogallol oxidation per min and per mg of protein.

Determination of malondialdehyde

Malondialdehyde (MDA) was measured according to the method described by Sastre et al.35 Liver and kidney homogenates 10% (w/v) were prepared in 0.1 Mol/L Tris-HCl buffer, pH 7.4. An aliquot of the homogenate is then precipitated with 10% trichloroacetic acid (w/v) and added with thiobarbituric acid 0.67% (w/v). Then, the mixture was centrifuged at 3000 rpm for 15 min, and the supernatant was heated at 100 °C for 15 min in a boiling water bath. After cooling, n-butanol was added to neutralize the mixture, and the absorbance was measured at 532 nm. The results were expressed as nMol of MDA/g tissue.

Protein determination

Liver and kidney homogenates were centrifuged at 10,000 g for 15 min at 4 °C, and the resultant supernatant was used for the determination of protein concentration with Bradford method,36 using bovine serum albumin (BSA) as a standard.

Paraffin embedded tissue sections

After paraffin processing 3 μm and 4 μm, thick tissue sections were prepared using microtome SM 2000R (Leica, Wetzlar, Germany) and mounted on poly-l-lysine-coated glass slides. The samples were deparaffinized and rehydrated through a graded series of ethanol and distilled water.

TdT-mediated dUTP-biotin Nick End Labeling analysis of apoptosis

The in-situ DNA fragmentations were visualized with TUNEL method.37 Briefly, dewaxed tissue sections were predigested with 20 mg/mL proteinase K for 20 min and incubated for 10 min in PBS containing 3% H₂O₂ in order to block the endogenous peroxidase activity. The sections were incubated with the TUNEL reaction mixture, fluorescein-dUTP (in situ Cell Death Detection, POD kit, Roche, Germany), for 60 min at 37 °C. The slides were then rinsed three times with PBS and incubated with secondary antifluorescein-POD-conjugate for 30 min. After washing three times in PBS, diaminobenzidine-H₂O₂ (DAB, Roche, Germany) chromogenic reaction was added on sections and was counterstained with hematoxylin. As a control for method specificity, the step using the TUNEL reaction mixture was omitted in negative control serial sections, and nucleotide mixture in reaction buffer was used instead. Apoptotic cells were quantified by image analysis.

Statistical analysis

Data are expressed as means ± SEM. The statistical GraphPad.Prism. V. 5.01 software (San Diego, CA) was used for the statistical analysis. Statistical comparison between different groups was done using one-way analysis of variance (ANOVA) followed by Student’s test. Values of p ≤ 0.05 were considered statistically significant.

Results

Blood and urine biochemical values

The data showing the level of liver and kidneys damages sustained following exposure to EDS and Lactobacillus plantarum Bj0021 supplementation are shown in Table 1. From the current data, it could be noticed that cholesterol, ASAT, ALAT, and GGT activities were increased significantly (p < 0.05) in serum of EDS treated pregnant female compared to control.

Table 1. Serum and urine biochemical parameters of control group, EDS-treated groups (EDS) and EDS treated animals supplemented with BJ0021 (EDS + BJ0021) at the end of experimental period.

	Experimental groups
	Control (n = 10)	Endo (n = 10)	Endo + BG021 (n = 10)
Parameters	Mean ± SEM	Mean ± SEM	Mean ± SEM
Serum
Cholesterol (g/L)	1.72 ± 0.77	2.78* ± 0.50	1.83^b ± 0,62
ASAT (U/L)	103.50 ± 0.50	1500* ± 5,20	117^b ± 05.02
ALAT (U/L)	73.70 ± 06.30	179.4* ± 7.4	57.8^a ± 5.7
ALP(U/L)	183.3 ± 16.9	190.3 ± 29.00	139.8 ± 18.4
GGT (U/mg protein)	0.03 ± 0.002	0.11* ± 0.04	0.08^a ± 0.01
Urea (g/L)	0.38 ± 0.003	0.42* ± 0.03	0.37^a ± 0.02
Creatinine (mg/L)	9,15 ± 0,29	11.27* ± 0.39	10.16 ± 0.43
Urine
Urea (mg/24 h)	1.08 ± 0.03	0.64** ± 0.05	1.24^a ± 0.13
Creatinine (mg/kg/24 h)	0.392 ± 0.10	0.36 ± 0.09	0.403 ± 0.14

Notes: Statistically significant differences from control: *p < 0.05, **p < 0.01; from EDS + BJ0021: ^ap < 0.05, ^bp < 0.01; Values are given as mean ± SEM, n = 10 number of animals.

In contrast, application of Lactobacillus plantarum Bj 0021 after treatment with EDS significantly reduced (p < 0.01) the cholesterol level and marked depletion (p < 0.05) of hepatic enzymes, their values remained slightly increased (p > 0.05) excepted for ALAT, when compared to control.

Administration of EDS increased concentration of blood urea (p < 0.05), serum creatinine (p < 0.05), and a decreased urinary urea (p < 0.01), even though EDS did not affect urinary creatinine. Although serum urea and creatinine decreased in animals supplemented with the probiotic, this improvement was not statistically significant, urinary urea significantly increased (p < 0.05) with supplementation.

Antioxidant enzymes and lipid peroxidation

The hepatic and renal MDA levels were significantly higher in animals treated with EDS than in the control group (p < 0.05). MDA concentration decreased in liver and kidney (p < 0.01 and p < 0.05, respectively) (Figure 1) after supplementation with Lactobacillus plantarum BJ0021, but remained no significant compared to control.

Figure 1. CAT, SOD activities and MDA level in liver and kidney tissues of control, EDS and EDS + BJ0021 groups at the end of treatment. Notes: Statistically significant differences from control: *p < 0.05, **p < 0.01; from EDS + BJ0021: ^ap < 0.05, ^bp < 0.01. Values are given as mean ± SEM, n = 10 number of animals.

The exposure to EDS caused a significantly decreasing (p < 0.05) CAT activity both in liver and kidney (Figure 1), the application of probiotic did not change these levels, yet the difference between the supplemented and control animals was highly significant (p < 0.01).

SOD activity was significantly altered (p < 0.05) in the liver and kidney (Figure 1) of rats treated with EDS, though the activities of antioxidant enzymes were not significantly improved by Lactobacillus plantarum Bj0021.

Liver and kidneys apoptosis

Induction of apoptosis was detected by immunohistochemical detection of fragmented DNA (TUNEL) in response to EDS and BJ0021 treatment. Representative tissue sections of each treatment are depicted in Figure 2(A and B). The number of apoptotic nuclei in liver of animals treated with EDS was significantly elevated (p < 0.05) when compared to control and significantly (p < 0.01) reversed with co-administration of Lactobacillus plantarum BJ0021 and remained no significantly higher (p > 0.05) than the control (Figure 2C).

Figure 2. TUNEL staining cells cross section counterstained with hematoxylin. Notes: Arrows point to brown stained apoptotic cells. (A) Kidney section. Magnification 40 × . (B) Liver section. Magnification 100×. (a) Negative control, (b) Control, (c) EDS treatment, (d) EDS + BJ0021 treatment. (C) Number of apoptotic nucleus obtained from liver and kidney by TUNEL method in contol, EDS and EDS + BJ0021 groups. Notes: Statistically significant differences from control: *p < 0.05, ***p < 0.001; from EDS + BJ0021: ^ap < 0.05, ^bp < 0.01. Values are given as mean ± SEM, Number of animals n = 10.

In kidneys of animals treated with EDS, number of apoptotic nuclei significantly increased (p < 0.001) compared to control, and significantly (p < 0.05) decreased with supplementation of Lactobacillus plantarum BJ0021 even the number of apoptotic nuclei remained higher (p < 0.05) than the control (Figure 2C).

Discussion

Liver is a target organ, which plays a major role in detoxification and excretion of many endogenous and exogenous compounds. It plays important role in metabolism and biotransformation of toxic compound.38

Therefore, any type of injury or impairment of its function produces hepatotoxicity and causes health complications. Liver enzymes activities were used as important biomarkers for detection of hepatotoxic nature of this pesticide. In this study, four serum hepatic marker enzymes ALAT, ASAT, ALP, and GGT were evaluated for hepatotoxicity. Results revealed that EDS treatment caused an increase in the activities of ASAT, ALAT, and GGT in serum of pregnant rats. The enhanced activities of these enzymes may be due to lysis/damage of hepatocytes resulting in the permeation of these enzymes into serum. ALP, ASAT, ALAT, and GGT have been commonly associated with liver dysfunction/damage and released into the circulation after cellular damage leading to their elevation in serum.39

Lactobacillus plantarum BJ0021 significantly reduced the parameters of liver function (ASAT, ALAT, GGT, and ALP) compared to females treated with EDS only, this may imply a better liver function as deduced from similar findings,40–42 this effect could be partly through decreasing bacterial translocation and by stimulatory effects of intestinal mucosa, and alteration of intestinal microflora, which thus influence the intestinal barrier.43

Imani Fooladi et al.44 reported that a symbiotic relationship between the liver and intestinal tract enables the healthy status of both organs. Microflora resident in intestinal lumen plays a significant role in hepatocytes function. Alterations to the type and amount of microorganisms that living in the intestinal tract can result in serious and harmful liver dysfunctions, such as cirrhosis, non-alcoholic fatty liver disease, alcoholic liver disease, and hepatic encephalopathy.

Hypercholesterolemia present in the current data may be attributed to increased synthesis and accumulation of cholesterol in hepatotoxicity liver and/or impaired biliary function,45 or it may indicates inhibitory action of pesticide on Cyt-p-450 enzymes.46

Our results showed that the serum cholesterol decreased significantly with co-administration of Lactobacillus plantarum BJ0021 when compared to controls; the relationship between lactic acid bacteria (LAB) and the serum cholesterol became a focus of great interest. Studies evaluating this relationship found that lactobacilli or bifidobacteria could exhibit hypocholesterolemic properties in animal models47^,48 and in humans.20

Goyal et al.49 proposed to explain these findings by: (1) the consumption of cholesterol by intestinal bacteria, thus reducing the amount of cholesterol available for absorption; (2) cholesterol may be bound to the bacterial cellular surface or incorporated into the bacterial cell membranes, thereby inhibiting the absorption of cholesterol back into the body; (3) the bacteria facilitated the elimination of cholesterol in feces; (4) the deconjugation of the cholesterol to bile acids, thereby reducing the total body pool; (5) the assimilation of lactic acid for their own metabolism.

The kidney is one of the organs responsible for the maintenance of constant extracellular environment through its involvement in the excretion of such catabolites as urea and creatinine, as well as electrolyte balance. Abnormal concentration of these catabolites and some electrolytes in the serum is a clear indication of renal function impairment.14 The significant increase in the concentration of creatinine in the serum reported in this study might have resulted from its decreased excretion which, in turn, is related to renal insufficiency; the concentration of creatinine in the blood is known to correlate inversely with the volume of glomerular filtration.50

Hence, creatinine is considered to be among the useful markers of the filtration function of kidneys, particularly for creatinine is excreted only via the kidneys.51 Also, increased serum urea concentrations confirmed the impaired renal function implicated in rats treated with EDS. The high serum urea levels may result from a decrease in the rate of secretion in urine, which may likely results from a renal insufficiency,52 this was confirmed by a significant decrease urea in urine in this study.

These renal parameters were improved in pregnant rats receiving L. plantarum. The supplemented group presented almost the same values than the control group, which could imply a better restoration of kidney function. The exact mechanisms of this phenomenon are unknown but may be a consequence of the metabolism of the probiotic, the site of action, or the individual variation.53 Probiotics supplement may also plausibly lead to the reductions in gastrointestinal tract (GIT) uremia and improve chronic kidney diseases (CKD) adverse outcomes.54

Several studies support the view that dietary supplements of probiotics will proffer beneficial application for uremia.55

Pesticides have been shown to enhance the production of reactive oxygen species (ROS), which in turn generate oxidative stress in different tissues. In liver, metabolism of EDS involves oxidases. Reactive metabolites occur,56 the toxic effects of which occurring probably through the generation of ROS causing damage to membranous components of the cell. Our results revealed that EDS exposure induced a oxidative stress in the liver of pregnant rats as indicated by decreased SOD, CAT activities and elevated MDA concentration, which would further induce lipid peroxidation, initiate free radicals damage to hepatocellular membrane, and lead to liver injury. The increasing of liver lipid peroxidation in EDS-intoxicated animals could act as a signal in maintaining low levels of antioxidant enzymes (SOD, CAT) in order to enhance the triggering of EDS detoxification process,57 so, the decrease of SOD and CAT activities could indicate an insufficient detoxification of EDS in rats. Our data were in agreement with other data.7^,45^,58

Comparable parameters indicating a stress were also altered in the kidney of pregnant rats; MDA level increased and CAT activity significantly decreased, which could be the result of its consumption counteracting the insecticide-induced oxidative stress.56

Several evidences showing some antioxidant properties of Lactobacilli were reported in literature.59^,60 The data obtained in this study showed that LAB induced a high significant decrease in lipid peroxidation in liver and kidney tissues as well as a decreasing in MDA production, proving that LAB presented effective antioxidative properties and could scavenge the excess of free radicals.

The ability of Lactobacillus to scavenge free radicals has been shown previously in in vitro studies.61^,62 The increased CAT and SOD activities we found in both liver and kidney were not statistically significant, consequently, we suggest that these enzymes would have a minor role in the inhibition of lipid peroxidation; therefore, the level of MDA was reduced probably with the increase of the scavenging rate of free radicals consequently to the enhancing of non-enzymatic defense, such as GSH (no determined here) or by other enzymes. Evidences have been shown that the antioxidant triad comprising SOD, CAT, and GPX constituted the first line of defense against the adverse effects of ROS.63 Besides, GSH redox system appears to be the main non-enzymatic antioxidant against ROS-mediated damage. GSH can be converted to GSSG through GPX, and converted back to GSH by GR. Thus, changes in the GSH, GSSG, and related enzymatic reactions (GPX and GR) may sensibly reflect the antioxidant status.64

Some kinds of LAB have an antioxidant system NADH oxidase/NADH peroxidase.21 Many studies supported our results.27^,65

Apoptosis is a form of programmed cell death allowing the elimination of irreversibly damaged cells. Preserving overall tissue function, this phenomenon plays a vital role in normal developmental processes. However, overwhelming activation of apoptosis may result in tissue failure. The TUNEL method clearly demonstrated the increased number of apoptotic cells in the liver and kidneys, confirming the occurrence of apoptosis after the administration of EDS. Apoptosis observed in hepatocytes and renal cells were previously described by Saraste and Pulkki.66

Several works indicated EDS also induces apoptosis in a human T-cell leukemic line67 and in adult rat testis68; this form of cell death could be mediated through alterations in redox homeostasis generated by a decrease of antioxidant defenses and by the accumulation of ROS seeing that EDS caused oxidative stress. The involvement of ROS in apoptosis has been suggested, Junn and Mouradian69 demonstrated that ROS serves as a signal for induction of apoptosis by activating an array of cell signaling molecules, such as caspases and kinases; also, the link between oxidative stress and apoptosis was recorded.70 The mechanism(s) by which EDS causes apoptosis has not been established. The current data showed that treatment with L. plantarum BJ0021 significantly decreased hepatic and renal apoptosis.

Zhang et al.71 reported that oral L. plantarum treatment in rats with obstructive jaundice significantly decreased apoptosis in liver.

Sunanliganon et al.72 revealed that L. plantarum B7 could perform an anti-Helicobacter pylori activity in vitro and some anti-inflammatory effects on H. pylori infection by improving stomach histopathology, and reducing serum TNF-α levels, gastric MDA, and epithelial cell apoptosis.

Lam et al.73 showed that pre-treatment of rats with L. rhamnosus GG markedly reduced ethanol-induced mucosal lesion area and gastric cell apoptosis. Recent study demonstrated that Bifidobacterium bifidum administration reduced intestinal apoptosis in necrotizing enterocolitis (NEC) in vitro and in vivo. In animal NEC model, probiotic decreased expression of proapoptotic protein (Bax) and enhanced the antiapoptotic Bcl-w and COX-2 expression and PGE-2 synthesis.74

In conclusion, the present data confirm the toxicity induced by EDS in which hepatotoxicity and nephrotoxicity are accompanied with an elevation in cholesterol, liver, and kidney functional parameters (ASAT, ALAT, GGT, ALP, urea, and creatinine) and LPO, along with a reduction in CAT and SOD activities. This toxicity also induced apoptosis. Oral administration of BJ0021 to pregnant rats significantly mitigated EDS-induced toxicity. However, the exact mechanism through which Lactobacillus plantarum BJ0021 is involved in the elimination of free radical remains still uncertain and further research is needed to fully determine the exact antioxidative mechanism of this strain through which the antioxidative effects are achieved. Also the benefit action on apoptosis is unclear.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.