Lactobacillus Casei Decreases Organophosphorus Pesticide Diazinon Cytotoxicity in Human HUVEC Cell Line

2016 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2016, 6(2), 201-210 doi: 10.15171/apb.2016.028 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
Pesticides are widely used to increase the agricultural production through the control of the harmful insects' populations.
Among these pesticides, the organophosphorus pesticides accounted for 50 percent of all insecticide applications. 1,2Diazinon, (O, Odiethyl O -2-isopropyl-6-methylpyrimidin-4-yl phosphorothioate), a broad-spectrum organophosphorus pesticide, is used on a wide range of crops such as rice, fruits, wine grapes, sugarcane, corn, and potatoes. 3The main concern about the utilization of pesticides such as diazinon (DZN) is their residual amount in agricultural products, soil and water. 4,5Pesticides are stored in various tissues of plants that enter the human food chain through the consumption of the edible segments.According to the commission of the European communities report, 45 percent of fruits and vegetables contain maximum residue levels of pesticides (MRLs). 6onitoring of pesticide residue in Brazilian fruits has indicated that 14.3% of samples exceeded European Union MRLs. 7 DZN inhibits the acetylcholinesterase enzyme activity that hydrolyzes the neurotransmitter acetylcholine in cholinergic synapses and neuromuscular junctions. 3,8][11] The organophosphorus pesticides significantly induce DNA damage and pro-apoptotic effects in many different healthy cells. 12,13nvironmental protection agency (EPA) classifies DZN as "not likely a human carcinogen", but experimental studies have confirmed its carcinogenicity such as leukemia, non-Hodgkin's lymphoma besides lung, brain and prostate cancers. 14robiotic bacteria are living microorganisms and when are administered in adequate amounts, confer health benefits on the host. 15Probiotics may have positive effects on toxic substances and performance or on toxicity of transferred drugs or toxins into the body. 16umerous studies have previously shown the probiotics beneficial effects on human health through their antimicrobial effects, antitoxin effects, the improvement of intestinal barrier function, the modulation of immune responses, the impacts on apoptosis and cell proliferation, and anti-oxidant function. 17,18Several studies have assayed the viability, the colonization ability, and the binding capacity of probiotics to toxic substances. 19Lactobacillus casei strain DN114001 can bind to heterocyclic aromatic amines in vitro and can decrease the concentration and the genotoxicity of these amines. 19Various strains of bifidobacteria hand in hand with Lactobacillus reuteri strain NRRL14171 and Lactobacillus casei strain Shirota were able to bind to aflatoxin B 1 that can be attributed to the presence of these bacteria in the gastrointestinal tract which may prevent the absorption of aflatoxins. 20,21Lactobacillus kefir strains (CIDCA 83115, 8321, 8345 and 8348) were able to bind to Clostridium difficile toxins by surface layer (slayer) proteins. 22By considering the aforementioned issues, this study aimed to evaluate the effects of lactobacillus casei secretion metabolites on the toxicity of agricultural organophosphorus pesticide (diazinon) through investigating the metabolites effects on diazinon treated/untreated human normal cell line, HUVEC in vitro.

Bacteria isolation
Lactobacillus casei was isolated from the traditional yogurt samples collected from East Azarbayjan, the northwest province in Iran. 5 g of each sample was suspended in 2% w/v sodium citrate solution and homogenized using the Stomacher 400 Circulator (Seward Laboratory Systems Inc, USA) for 2 min.Afterwards, 1 ml of the samples was added to 24 ml of de Man Rogosa and Sharpe (MRS, Merck, Germany) broth medium and incubated at 37 °C for 24 h.After the incubation time, the bacteria were isolated by spreading them on a de Man Rogosa and Sharpe (MRS, Merck, Germany) agar plate similar to the previous condition.Many single colonies were randomly selected and again incubated in 5 ml MRS broth for 24 h.The individual colonies were subjected to morphological evaluation.Gram positive and catalase negative bacilli colonies were stored at -80°C in MRS supplemented with glycerol 25% (v/v).

Molecular identification by 16S rDNA
Total genomic DNA was extracted from the cultures inoculated with a single colony using the previously described procedure by Drisko. 23For this purpose, 1.5 ml of the bacterial culture, a single sub-cultured colony in MRS broth for 24 h at 37°C, was centrifuged at 10000×g for 5 min and the cell pellet was used to isolate the DNA.All the extracted genomic DNAs of the samples, resuspended in 50 µl distilled water, were then checked and visualized via 0.8% agarose gel electrophoresis.Subsequently, the gel monitoring apparatus (Biometra, Gottingen, Germany) and spectrophotometric method were used to evaluate the quality and quantity of the extracted DNA, respectively.The PCR amplification was conducted in a thermal cycler PTC 200 (MJC research, Waltham, USA) by using a pair of LAB-specific universal primers (LABF 5´-AGATTTTGATCMTGGCTCAG-3´ and LABR 5´-TACCTTGTTAGGACTTCACC-3´).PCR amplification was performed using the following temperature profile: an initial denaturation at 94°C for 4 min, followed by 32 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 1 min, and a final extension step at 72°C for 5 min. 24The PCR products were determined by electrophoresis in a 1% (w/v) agarose gel and were visualized through ethidium bromide staining.The PCR products were sequenced at Sinaclone Corporation, Tehran, Iran.The sequences were then analyzed using the BLAST program of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST).

Acid and bile salt tolerance
The isolated cells were harvested from the cultures incubated overnight followed by centrifugation for 10 min at 6000×g and 4°C .The cell pellets were then resuspended in PBS (80 mM Na2HPO4, 1.5 M NaCl, 20 mM KH2PO4, 30 mM KCl, pH 3.0) and were incubated at 37°C for 3 h in MRS broth.The viable cells after low pH treatment were subjected to PBS (80 mM Na2HPO4, 1.5 M NaCl, 20 mM KH2PO4, 30 mM KCl, pH 7.2) containing 0.3% (w/v) of bile salt (Sigma Chemical Co., St. Louis, Mo., USA) then were incubated at 37°C for 4 h in MRS broth.Proper dilutions based on 1 h time intervals were performed, and the dilutions were centrifuged for 10 min at 6000×g and 4°C then the cell plates (acid and bile salt resistance bacteria) were incubated at 37°C in anaerobic conditions for 24 h.The survival of the bacterial cells was evaluated using log phase cultures (8 log 10 CFU ml -1 ) by plating them on MRS agar after 0, 1, 2, and 3 h of incubation in acidic (pH 3.0)/bile supplemented MRS broth at 37°C via the standard pour plate technique.The survival rate (for both acid/bile resistance) was calculated using the following equation: survival rate (%) = (log cfu N 1 /log cfu N 0 ) × 100, where N 1 corresponds to the total clones treated with extra bile salts or acids and N 0 corresponds to the total clones before they were incubated under harsh conditions.

Antimicrobial activity
Antibacterial activity assessments were conducted against clinically important human pathogens, including native isolate of E. coli (026), Candida albicans (PTCC 5027), Escherichia coli (057) (PTCC 1276), Salmonella ).The overnight cultured isolated strains in MRS broth medium at 37 °C were filtered through 0.2 µm filter, and then 50 µl of each filtrate was added to 7 mm diameter wells on the indicator growth medium agar, which were previously incubated overnight by indicator pathogens at 37 °C.The pH of the isolated fresh supernatants was adjusted to the pH of the each indicator pathogens growth media (Table 1).After the overnight incubation of plates at 37 °C, the clear zones around of each well were measured and considered as positive antibacterial activity.Based on the diameter of the inhibition zone, anti-pathogen activities were divided to strong (diameter ≥ 20 mm), moderate (20 mm ≤ diameter ≥ 10 mm), and weak (diameter ≤ 10 mm). 25
Based on the areola size, the isolates were classified to sensitive, intermediate and resistant groups through the analysis of data.

Cell culture
The Human umbilical vein endothelial cells (HUVEC) were obtained from Pasteur Institute, National Cell Bank of Iran.The cells were cultured into 25 cm 2 T-flask with cell density (1×10 6 cell/ml) in RPMI 1640 (GIBCO, Uxbridge, UK) medium containing 10% fetal bovine serum (HyClone, Logan, UT, USA) inactivated at 45 °C for 1 h, 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma, St. Louis, MO, USA) were incubated at 37˚C in a humidified atmosphere with 5% CO2.The media of tflasks were renovated each 48 h intervals.For the cell passage, all cells were detached using 0.025% trypsin-0.02%EDTA (Sigma, St. Louis, MO, USA).

Cell treatment
Lactobacillus casei was cultured overnight in de Man Rogosa (MRS) broth medium (Merck, Darmstadt, Germany).To prepare cell free supernatant, the culture was centrifuged at 4000 rpm for 5 min and the supernatant's pH was adjusted to 7.2 before sterilizing through filtering using 0.

DAPI staining
All treated/untreated HUVEC cells' culture medium was removed from the 6 well plates and the attached cells were washed twice by PBS (pH=7.2).The washed cells were fixed by 500 µl of 1% paraformaldehyde for 5 min.
To increase the permeability of fixed cells, paraformaldehyde was replaced with 500 µl of 0.1% Triton X-100 and incubated for 10 min at room temperature.The permeabilized cells were subjected to 100 µl of 4', 6-Diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) solution (250 ng/ml for each well) and then were incubated for 10 min at room temperature.The morphological changes were analyzed using a fluorescent microscope (Olympus BX64, Olympus, Japan) equipped with a U-MWU2 fluorescence filter (excitation filter BP 330e385, dichromatic mirror DM 400, and emission filter LP 420).

Flow cytometric analysis
To remove RPMI medium, the detached all treated/untreated control HUVEC cells were centrifuged at 335 ×g for 10 min and the cell plates were washed with PBS (pH=7.2) then were centrifuged again similar to previous condition.During the next phase, the cells were re-suspended in 100 µl binding buffer (1×10 6 cells/ ml) of Annexin V-FITC kit (eBioscience, San Diego, CA).The binding buffer containing cells were mixed with 5 µl Annexin V-FITC, next 10 µl propidium iodide solution was added to the cell suspension and were kept in a 5 ml culture tube for 15 min at room temperature in dark conditions.Binding buffer (400 µl) was again added to each culture tube and assessments were conducted using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA).The analysis on 100,000 cells was accomplished at a rate of 1000 cells/s.Quadrant setting was conducted using the untreated cell line as the negative control.Data analysis was performed using CellQuest Pro software (BD Biosciences, San Jose, CA, USA).Flow cytometry assessments were conducted thrice with three repetitions for each time.

Statistical Analysis
The statistical analysis was performed by SPSS software version 18.0 (SPSSInc, Chicago, IL, USA).The normal distribution of data was tested by Kolmogorov-Smirnov test.ANOVA and Tukey's post hoc test were used for analyzing data and multiple mean comparisons, respectively.Statistical significance was considered a value of P≤ 0.05 and quantitative data were reported as mean ± SD.All experiments were repeated three times with six replicates for each experiment.

Isolation and identification
A total of 22 grown hemispherical white or achromatic colonies was separately propagated for further assessments.The presence of lactic acid bacteria strains in the isolated samples were confirmed by amplifying the 16S rDNA gene using gene-specific primers.Sequences of 16S rDNA gene 1500 bp fragments were blasted with the deposited sequences in GenBank.Isolates with 99% to 100% homology were identified by considering the threshold values of taxonomical studies (97%). 26These 22 colonies belonged to Lactobacillus casei strain YSH, Lactobacillus paracasei strain YJ, Lactobacillus rhamnosus strain YI, Lactobacillus fermentum strain YAL, Lactobacillus plantarum strain YSH1, and Lactobacillus delbrueckii strain YJI.

Resistance to acid and bile salt
The survival rates of the six isolated LAB strains after incubating for 3 h at pH 3 and in 0.3% bile salt (oxgall; Sigma Chemical Co., St. Louis, Mo., USA) are shown in Table 2. Based on the results, all six selected strains retained their viability at the mentioned harshness condition where the tolerance to acidic/high bile salt conditions was strain specific.The survival rates ranging from 73% to 85% were observed in Lactobacillus strains at acidic condition, whereas the survival rates, ranging from 92% to 98%, were observed in bile salt condition.
The strains with the most efficient tolerance to acidic conditions were L. plantarum strain YSH1, L. rhamnosus strain YI, L. delbrueckii strain YJI, and L. casei strain YSH with survival rates of 85%, 82%, 81%, and 78%, respectively.Meanwhile, the six isolates showed high survival rates with >90% under high bile conditions.The strains with the highest tolerance to 0.3% oxgall were L. casei strain YSH, L. fermentum strain YAL, L. rhamnosus strain YI with the survival rates of 98%, 98%, and 96%, respectively.

Antimicrobial activity
Table 3 shows the 6 isolated strains, including L. casei strain YSH, L. paracasei strain YJ, L. rhamnosus strain YI, L. fermentum strain YAL, L. plantarum strain YSH1, and L. delbrueckii strain YJI displayed significant antipathogenic activities against indicator microorganisms.Lactobacillus species, particularly L. casei strain YSH, showed the most efficient antagonistic activity and inhibited the growth of 13 indicator pathogens among the isolated bacteria.Meanwhile, L. rhamnosus strain YI, and L. paracasei strain YJ exhibited an overall good antagonistic activity and inhibited the growth of indicator pathogens.Notes: values are mean ± standard error S (strong r ≥20 mm), M (moderate r<20 mm and >10 mm), and W (weak ≤10 mm) Lactobacillus casei strain YSH, Lactobacillus paracasei strain YJ, Lactobacillus rhamnosus strain YI, Lactobacillus fermentum strain YAL, Lactobacillus plantarum strain YSH1, and Lactobacillus delbrueckii strain YJI

Antibiotic susceptibility
The antibiotic susceptibility of the isolated bacteria against the high consumption antibiotics was evaluated using the measurements of inhibition zone diameter.The antibiotic susceptibility results of the six isolated LAB against clinically important antibiotics are presented in Table 4. Based on our findings, all six isolated bacteria were sensitive or semi-sensitive to tetracycline and clindamycin.Lactobacillus strains generally displayed the highest susceptibility to the majority of antibiotics.L.
casei strain YSH displayed the best results and was sensitive or semi-sensitive to all antibiotics.

The effect of L. casei on diazinon toxicity
The effect of different concentrations of DZN on cell proliferation was measured by MTT assay.DZN at concentration of 70 µg/ml reduced the viability of HUVEC cell lines by 50.97% in a dose-response manner, so, this concentration was selected as IC50 of diazinon in this study and used in other treatments.Among the other strains, L. casei cell free supernatant (50 µg/ml) significantly increased the cell viability compared with control (p≤0.01) while DZN IC 50 increased cell death at a concentration of 70 µg/ml (p≤ 0.01).The treatment of HUVEC cell line with IC50 of diazinon and L. casei secreted metabolites showed a significant decrease in the cytotoxicity of DZN on HUVEC cell lines (Figure 1).

Apoptosis assessment
To analyze the apoptosis incidence in DZN treated cells and prove the effect of L. casei on the viability of HUVEC cells, the latter was exposed to 50 µg/ml of filtered supernatant of late stationary phase growth of L. casei after 24 h incubation to visualize the apoptosis using Dapi staining and observation by fluorescent microscopy (Olympus BX64, Olympus, Japan).The intact viable cells displayed a plenary health nucleus (Figure 2B), whereas the apoptotic cells were characterized by shrinking cells with condensed (early apoptosis) or fragmented (late apoptosis) nuclei (Figure 2A).The DZN treated HUVEC cells illustrated the very distinctive signs of apoptosis, including the formation of micronuclei, cell shrinkage, membrane blebbing, nucleus fragmentation, and apoptotic bodies (Figure 2A).None of these signals were observed in untreated HUVEC cells (Figure 2C).The L. casei secretion metabolites could decrease the apoptosis-related signals in DZN-treated HUVEC cells (Figure 2D).cells was induced at early and late stages of apoptosis after 24 of incubation.These results revealed that significant differences existed in apoptotic induction by DNZ.The cells stained by PI alone (V -/PI + ) underwent necrosis.As shown in Figure 3, the DZN-treated cells demonstrated an increase in the necrotic population (6.2%) shown in the upper left quadrant whereas the toxin treated cells exposed to L. casei supernatant showed a slight increase in necrotic cells (0.32%) compared to the untreated cells (0.04%).These findings proved that L. casei secretion metabolites could decrease the DZN cytotoxicity effects on human normal cells (HUVEC) at in vitro.

Discussion
The results of the present study indicated the treatment of human poisoned cells with L. casei showed the protective effects against DZN induced cytotoxicity at in vitro.Human and animal studies have shown the toxicity of DZN in different tissues such as hematological disorders, 27 cardiotoxicicty, 27 hepatotoxicity, 28 nephrotoxicity, 29 neurotoxicity, 30 and both female and male reproductive toxicity. 29The exposure of the NTera2/D1 (NT2) cell line to diazinon at concentrations ranging between 10 -4 and 10 -5 M enhanced cell death with a number of special features of apoptosis including membrane and mitochondrial potential changes. 31Slotkin and Seidler indicated that the target genes of the organophosphates including chlorpyrifos and diazinon are the cell cycle and apoptosis-regulating genes in the developing brain and in neuronotypic cells in vitro. 32n our study, the co-treatment of the HUVEC cells with L. casei supernatant and diazinon decreased cell death and apoptosis induced by diazinon.The previous studies have suggested that some probiotics may regulate apoptosis. 33The activation of the antiapoptotic Akt/protein kinase B and inhibition of the activation of proapoptotic p38/mitogen-activated protein kinase by probiotic bacteria were suggested to prevent apoptosis in the colon epithelial cells. 34,35Also, probiotics possess the antitoxin effects through binding to the toxins ;thus, can protect cells against both membrane and DNA-damaging toxins. 36lthough the main mechanism of DZN toxic effects on target and non-target organisms is the inhibition of acetylcholinesterase, 37 the researchers have shown that it is not in charge of all of toxic effects and several studies suggest that DZN induces oxidative stress and produces free radicals in biological systems which is the main mechanism of chronic organophosphates (Ops) toxicity. 38The chronic Ops elevate the level of reactive oxygen species which is a major apoptotic stimulant in different organs. 31Indeed, apoptosis is a common outcome for the exposures to toxicant that evoke oxidative stress. 2,5,28,39Diazinon increase lipid peroxidation and decrease antioxidant biomarkers including reduced glutathione, glutathione peroxidase, superoxide dismutase, catalase and total antioxidant capacity in male wistar albino rats. 29The injection of DZN at high doses has increased the level of malondialdehyde, superoxide dismutase and glutathione S-transferase activities and has decreased glutathione (GSH) level, lactate dehydrogenase, and cholinesterase activities in the brain, heart, and spleen of female Wistar and Norway rats. 38Sub-acute exposure to DZN has induced oxidative stress-mediated apoptosis in rat liver through the activation of caspases-9 and -3, and increasing Bax/Bcl-2 expression ratio. 28robiotics with antioxidant effects, Lactobacillus acidophilus, could decrease malondialdehyde and could increase the levels of antioxidants, glutathione reductase, superoxide dismutase, and glutathione peroxidase in Sprague-Dawley rats. 40Therefore, it seems that antiapoptotic effects of L. casei in HUVEC cells treated with DZN probably due to a decrease in oxidative stress and anti-oxidant effects.

Conclusion
The results of the present study reveal that diazinon has cytotoxic effects on normal cells and apoptosis is the main cytotoxic mechanism of diazinon; however, L. casei secretions is able to decrease its cytotoxic effects.Although the exact mechanism of anti-toxic effects of L. casei is not clear but the reduction of DZN cytotoxicity on human normal cells is because of its anti-apoptotic mechanisms, antitoxin effects and/or via decreasing the oxidative stress.

Figure 1 .
Figure 1.The effect of L. casei on diazinon toxicity.Error bares represent standard deviations.*P≤0.05

Figure 2 .
Figure 2. Nuclear morphology analysis using DAPI of HUVEC cell line, A) HUVEC cells treated with IC50 dose of diazinon, B) Untreated HUVEC cells, C) HUVEC cells treated with L. casei and D) HUVEC cells treated with L. casei and IC50 dose of diazinonAnnexin V-FITC/PI flow cytometry analysis was performed to confirm the metabolite secreted from selected L. casei strain effects on apoptosis of HUVEC cells.These cells were treated with 50 µg/ml L. casei supernatant for 24 h.The dual parameter fluorescent dot blots revealed the viable cell population in the lower left quadrant (annexin V -/PI -), the cells at the early apoptosis are in the lower right quadrant (annexin V + /PI -), and the cells at the late apoptosis are in the upper right quadrant (annexin V + /PI + ).Figures 3A to 3C show that 0.53 percent of the cells was annexin V + /PI -in untreated cells after 24 h of post seeding.Our findings indicated that the cytotoxicity of DNZ on HUVEC cells occurred during apoptosis.A total of 18.3% and 11.6% of the treated

Figure 3 .
Figure 3.The flow cytometry assessment on HUVEC cell line, A) Untreated HUVEC cells, B) HUVEC cells treated with L. casei and IC50 dose of diazinon.C) HUVEC cells treated with IC50 dose of diazinon, Lower left column: Annexin V/PI (viable cells), lower right column: Annexin V + /PI (early apoptotic cells), upper right column: Annexin V + /P + (late apoptotic cells) and upper left column Annexin V/P + (necrotic cells).

Table 2 .
The survival rates of isolated LAB after 3 h incubation at pH 3 and 0.3% bile salts

Table 3 .
The inhibitory effect of isolated strains against pathogenic microorganisms

Table 4 .
Antibiotic susceptibility of isolated LAB against the high consumption antibiotics by disc diffusion assay