Probiotic Lactobacillus rhamnosus reduces organophosphate pesticide absorption and toxicity to ...

38 Pages·2016·1.28 MB·English

AEM Accepted Manuscript Posted Online 12 August 2016
Appl. Environ. Microbiol. doi:10.1128/AEM.01510-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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1 Probiotic Lactobacillus rhamnosus reduces organophosphate pesticide absorption and toxicity to
2 Drosophila melanogaster
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4 Mark Trinder1,2, Tim W. McDowell3, Brendan A. Daisley1,2, Sohrab N. Ali4, Hon S. Leong2,4, o
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5 Mark W. Sumarah3, Gregor Reid1,2,4#. lo
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7 Centre for Human Microbiome and Probiotic Research, Lawson Health Research Institute, o
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8 London, Ontario, Canada1; Department of Microbiology and Immunology, The University of h
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9 Western Ontario, London, Ontario, Canada2; London Research and Development Center, //
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10 Agriculture and Agri-Food Canada, London, Ontario, Canada3, Department of Surgery, St.
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11 Joseph's Health Care London, London, Ontario, Canada4 m
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12 g
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13 Running title: Lactobacilli mitigate organophosphate exposure.
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15 # Address correspondence to Dr. Gregor Reid, [email protected]. b
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20 ABSTRACT
21 Organophosphate pesticides used in agriculture can pose health risks to humans and wildlife. We
22 hypothesized that dietary supplementation with Lactobacillus, a genus of commensal bacteria,
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23 would reduce absorption and toxicity of consumed organophosphate pesticides (parathion and w
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24 chlorpyrifos). Several Lactobacillus species were screened for tolerability of 100 ppm of a
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25 chlorpyrifos (CP) or parathion in MRS broth using 24 h growth curves. Certain Lactobacillus
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26 strains were unable to reach stationary phase culture maxima and displayed abnormal culture m

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27 morphology in response to pesticide. Further characterization of commonly used, pesticide- tp
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28 tolerant and pesticide-susceptible, probiotic L. rhamnosus GG (LGG) and L. rhamnosus GR-1 e
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29 (LGR-1) revealed that both strains could significantly sequester organophosphate pesticides from a
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30 solution after 24 h co-incubations, respectively. This effect was independent of metabolic .o
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31 activity, as LGG did not hydrolyze CP and no difference in organophosphate sequestration was o
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32 observed between live and heat-killed strains. Furthermore, LGR-1 and LGG, reduced the
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33 absorption of 100 µM parathion or CP in a Caco-2 Transwell model of the small intestinal m
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34 epithelium. To determine the effect of sequestration on acute toxicity, newly eclosed Drosophila r

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35 melanogaster were exposed to food containing 10 µM CP with or without supplementation of 2
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36 live LGG. Drosophila supplemented with LGG simultaneously, but not 3 d prophylactically,
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37 mitigated CP-induced mortality. In summary, the results suggest that L. rhamnosus may be g
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38 useful for reducing toxic organophosphate pesticide exposure via passive binding. These findings t
39 could be transferable to clinical and livestock applications due to the affordable and practical
40 ability to supplement products with food grade bacteria.
41 IMPORTANCE
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42 The consequences of environmental pesticide pollution due to widespread usage in agriculture
43 and soil leaching are becoming a major societal concern. Although the long-term effects of low
44 dose pesticide exposure to humans and wildlife remain largely unknown, logic suggests these
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45 chemicals are not aligned with ecosystem health. This observation is most strongly supported by o
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46 the agricultural losses associated with honeybee population declines known as colony collapse lo
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47 disorder, in which pesticide usage is a likely trigger. Lactobacilli are bacteria used as beneficial e
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48 microorganisms in fermented foods and have shown potential to sequester and degrade o
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49 environmental toxins. This study demonstrated that commonly used probiotic strains of h
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50 lactobacilli could sequester, but not metabolize, organophosphate pesticides (parathion and //
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51 chlorpyrifos). This lactobacilli-mediated sequestration was associated with decreased intestinal
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52 absorption and insect toxicity in appropriate models. These findings hold promise for m
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53 supplementing human, livestock, or apiary foods with probiotic microorganisms to reduce g
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54 organophosphate pesticide exposure.
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56 INTRODUCTION
57 Organophosphate pesticides are a class of insecticide under scrutiny for being linked to toxic
58 effects in both humans and wildlife (1). However, these compounds are still commonly used in
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59 agriculture and pest control programs. Organophosphate pesticides include parathion, malathion, w
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60 methyl parathion, chloropyrifos (CP), diazinon, dichlorvos, phosmet, fenitrothion, a
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61 tetrachlorvinphos, azamethiphos, and azinphos-methyl. Organophosphate pesticides irreversibly
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62 inhibit acetylcholinesterase to induce excessive cholinergic stimulation (2, 3). The potential off- m

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63 target health consequences of organophosphate pesticide exposure to humans include: neonatal tp
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64 developmental abnormalities (4, 5), endocrine disruption (6), neurodegeneration, cancer (7, 8), e
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65 metabolic disruption (9), heart disease (10), chronic kidney disease, and other less common a
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66 pathologies. In addition, organophosphate pesticides are reported to have negative impacts on .o
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67 honey bee colonies, which are critical pollinators for numerous agricultural products (11- o
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68 13). Although the evidence is mixed and often correlative, the original use of these
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69 organophosphates as nerve agents strongly suggests that these pesticides are not aligned with m
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70 human or wildlife health. r

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71 Despite negative consequences, the affordability and need to prevent crop losses associated with 1
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72 insect infestations suggest that organophosphate pesticide use will continue in the near future. y

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73 One long-considered counter to these effects is the use of microbes to detoxify organophosphate e
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74 pesticide-contaminated environments (14-20). Classical bioremediation efforts have identified
75 numerous strains of soil bacteria that contain genetically diverse phosphotriesterases capable of
76 organophosphate degradation (15). Moreover, the symbiotic relationship between Burkholderia
77 strains and the bean bug, Riportus pedestris, have been shown to confer these insects with
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78 resistance to organophosphate insecticides (21). Taken together, these observations suggest that
79 microorganisms could be employed to reduce the toxic effects of organophosphate insecticides in
80 vivo.
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81 There has been development within bioremediation research to investigate the potential of w
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82 transitory food-grade bacteria to prophylactically prevent the absorption of environmental toxins a
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83 such as pesticides (22), heavy metals (23), and aflatoxin (24, 25). Many lactobacilli are
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84 commonly used in fermented foods such as yogurt, cheese, sauerkraut, pickles, beer, wine, cider, m

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85 kimchi, cocoa, and kefir (26). Lactobacilli have also been shown to be a natural and beneficial tp
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86 member of numerous organisms relevant to humans such as livestock, honeybees (27), and fish e
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87 (28). Notably, lactobacilli have been shown to reduce organophosphate pesticide contamination a
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88 of dairy products (29, 30). The mechanism of action used by lactobacilli against .o
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89 organophosphate pesticides remains unclear and is commonly attributed to phosphatase o
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90 capabilities. However, one kimchi isolate, Lactobacillus brevis WCP902, was shown to contain
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91 the opdB gene that conferred the active ability to degrade CP (31). Similarly, the common m
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92 probiotic, Lactobacillus rhamnosus GG (LGG), has a predicted hydrolase/phosphotriesterase r

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93 (LGG_RS02045) with sequence similarity to the experimentally-validated parathion hydrolase 2
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94 (opd) found in Brevundimonas diminuta.
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95 The aim of this study was to better characterize the in vivo bioremediation potential of probiotic e
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96 food-grade bacteria interacting with organophosphate pesticides. We hypothesized that the direct
97 binding or metabolism of organophosphate pesticides by L. rhamnosus strains would reduce
98 toxin absorption and downstream organismal toxicity.
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99 MATERIALS AND METHODS
100 Chemicals
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101 CP (catalog number: 45395) and parathion (catalog number: 45607) were obtained from Sigma-
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102 Aldrich. Stock solutions were prepared at 100 mg/mL or 10 mg/mL dimethyl sulfoxide (Sigma- lo
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103 Aldrich) and stored frozen at -20°C until usage. e
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104 Bacterial strains and culture m

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105 Lactobacilli strains used were: L. rhamnosus GG, L. rhamnosus GR-1, L. casei ATCC 393, L. :
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106 delbrueckii DSM 20074, L. plantarum ATCC 14917, L. crispatus ATCC 33820, L. fermentum m
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107 ATCC 11739, L johnsonii DSM 20053, L. reuteri ATCC 2773, L. rhamnosus ATCC 7469. s
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108 A pET20b vector (EMD Millipore), containing the organophosphate insecticide-degrading PTE /

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109 (phosphotriesterase) gene inserted between NdeI and EcoRI (20), was obtained as a gift from Dr. D
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110 Frank M. Raushel (Texas A&M University, United States). Three-hundred ng of pET20b/PTE e
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111 vector was transformed into 50 µL of chilled (ice for 15 min) chemically competent Escherichia e
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112 coli BL21(DE3) cells by 2 min heat shock at 42°C followed by incubation on ice for 2 min. The ,
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113 transformation mixture was serially-diluted and plated for positive selection on Luria-Bertani 8

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114 (LB; BD Difco, catalog number: DF0446173) agar plates containing 300 µg ampicillin/mL.
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115 Plasmids were isolated from transformants grown at 37°C aerobically overnight using the s
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116 PureLink Quick Plasmid Miniprep Kit according to the manufacturer’s instructions (Invitrogen,
117 catalog number: K210010). Positive transformants were confirmed by sequencing plasmids
118 using the T7 promoter (TAATACGACTCACTATAGGG) and T7 terminator
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119 (GCTAGTTATTGCTCAGCGG) primers with the Applied Biosystems 3730 Analyzer platform
120 at the London Regional Genomics Centre (Robart’s Research Institute, London, Canada).
121 E. coli BL21(DE3) pET20b/PTE were inoculated from LB agar plates into LB broth, both
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122 supplemented with 300 µg ampicillin/mL of culture media and incubated overnight (18 h) at w
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123 37°C aerobically. Lactobacilli were inoculated from lactobacilli de Man, Rogosa and Sharpe a
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124 (MRS) agar (BD Difco, catalog number: 288130) plates into MRS broth. Inoculated MRS broth
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125 cultures were subcultured and incubated overnight (18 h) at 37°C anaerobically and statically for m

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126 experimental procedures. Bacteria were heat-killed by incubation at 56°C for 90 min when tp
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127 necessary. Bacterial killing was confirmed by spread plating 100 µL of bacterial culture on MRS e
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128 agar plates and verifying the absence of colony growth after 3 d (37°C anaerobically). a
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129 Pesticide tolerance assay rg
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130 Overnight broth cultures (stationary phase, 109 CFU/mL) were subcultured (1:100 dilution) into D
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131 96 well plates (Falcon, catalog number: 351177) containing MRS broth with or without the m
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132 addition of 100 ppm of CP or parathion or vehicle vehicle (DMSO). Plates were sealed with
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133 optically clear multi well plate sealing films and incubated at 37°C and read every 30 min for 24 2
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134 h at a wavelength of 600 nm using a Labsystems Multiskan Ascent microplate reader. 8

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135 Pesticide hydrolysis assay u
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136 Semi-qualitative assessment of bacterial CP hydrolysis was determined based on a modified
137 protocol previously described (21). Briefly, 1 μL of overnight broth cultures (106CFU) of LGG

138 or E. coli BL21(DE3) pET20b/PTE (positive control) were spot plated onto brain-heart infusion
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139 (BD Difco, catalog number: B11059) agar plates containing 1000 ppm (1 mg/mL) of emulsified
140 CP. After 48 h of anaerobic or aerobic incubation at 37°C for LGG and E. coli BL21(DE3)
141 pET20b/PTE, respectively, the radius of halo formation (zone of clearing) was determined.
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142 Pesticide binding and metabolism assay w
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143 Overnight bacterial cultures were pelleted at 5000 g, washed and resuspended in 50 mM HEPES e
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144 (pH 6.8, 109 CFU/mL). Bacterial-buffer or buffer-alone solutions were incubated with 100 ppm fr
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145 of parathion or CP protected from light at 37°C for 24 h with 200 rpm gentle shaking. Cultures h
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146 were pelleted and supernatant collected. Pellets were washed in 50 mM HEPES, resuspended in :/
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147 methanol, and sonicated for 15 min. Supernatants were again collected for pellet assessment of m
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148 organophosphate levels. m
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149 Pesticide levels were determined by high performance-liquid chromatography (HPLC) using a /
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150 Poroshell 120 column (100 × 4.6 mm, 2.7 μm; Agilent, Mississauga, Ontario) and a UV detector D
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151 based on a modified protocol (32). CP and parathion were detected at 288 and 275 nm m
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152 wavelengths, respectively. Solvent flow rate was 1 mL/min of A (H O + 0.1% trifluoroacetic
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153 acid) and B (acetonitrile + 0.1% trifluoroacetic acid). Solvent gradient was set to 25, 25, 100, 2
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154 100, 25, and 25 percent B at 0, 2, 9, 11, 11.5, and 13 min, respectively. Sample peak area units 8

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155 were compared to a linear relationship of appropriate standards of known concentrations. The g
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156 limit of detection for parathion and CP was 0.001 ppm and 0.02 ppm, respectively. Percent s
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157 pesticide remaining was calculated as: ppm of biological replicate/ppm of mean pesticide-only
158 control replicates X 100.
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159 Caco-2 cell culture and Transwell experimentation
160 Caco-2 cells were obtained as a gift from Dr. Brad Urquhart (Western University, London,
161 Canada). Caco-2 cells were routinely maintained at 37°C in atmospheric conditions with 5%
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162 CO . Cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco, catalog number: w
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163 11960044) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 1 mM sodium a
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164 pyruvate (Gibco, catalog number: 11360070), 1% MEM non-essential amino acids (Gibco,
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165 catalog number: 11140050), 4 mM L-glutamine (Gibco, catalog number: 25030081), and 100 m

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166 U/mL penicillin-streptomycin (Gibco, catalog number: 15140122). Cells were used tp
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167 experimentally between passages 35-45. e
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168 Cells were seeded onto 12mm Transwell (12-well with 0.4µm pore polyester membrane insert, m
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169 Corning, catalog number: 3460) plates at a concentration of 1.5 x 105 cells/insert. Cells were rg
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170 differentiated by culturing cells for 21 d as previously described (33). Prior to absorption n

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171 experimentation, apical and basolateral compartments were washed 3x and resuspended with c
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172 Hank’s Balanced Salt Solution (Gibco, catalog number: 14025092). Cells were treated apically b
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173 with 100 μM of parathion or CP in the presence or absence of 109 CFU/mL of LGG or LGR-1. 6
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174 Basolateral sampling (100 μL) with replacement was performed at 30 min, 1 h, and 2 h. 1
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175 Following absorption experimentation, Caco-2 monolayer integrity was confirmed by lucifer y

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176 yellow (lucifer yellow CH lithium salt, Biotium, catalog number: 80015) rejection according to e
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177 manufacturer’s instructions.
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178 Drosophila melanogaster husbandry
179 Wild type Canton-S (stock number: 1) stocks were obtained from Bloomington Drosophila Stock
180 Center at Indiana University. Drosophila were maintained using media containing 1.5% agar
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181 (w/v), 1.73 % yeast (w/v), 7.3% cornmeal (w/v), 7.6% corn syrup (v/v), and 0.58% acid mix w
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182 (v/v) at 22°C with 12 h light/dark cycles. For experimental procedures, media were a
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183 supplemented with or without CP at concentrations of 1, 10, or 100 μM prior to solidification.
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184 Food was stored at 4°C and used within a week. Experiments with adult Drosophila used newly m

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185 eclosed flies. All experiments were performed in polypropylene Drosophila vials (Diamed Lab tp
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186 Supplies Inc., GEN32-121 and GEN49-102) containing 10 mL of food with 15-25 flies/tube. e
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187 Adult Drosophila survival assays m
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188 Fifteen to twenty-five newly eclosed Drosophila were anesthetized with CO2 and randomly o/
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189 transferred (mixed sex) into standard vials at mid light cycle (9 AM EST) for each replicate. D
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190 Drosophila were confirmed to be alive following anesthetization and subsequently monitored m
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191 daily (9 AM EST) for survival. Media was supplemented with a single dose of 100 µL (109 e
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192 CFU) of washed and concentrated LGG or phosphate-buffered saline vehicle when 2
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193 experimentally appropriate and allowed to air dry before use. 8

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194 Drosophila negative geotaxis assay u
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195 Negative geotaxis assays were performed as previously described (34). Mean distance climbed
196 (after 3 s) of 5 replicates from 3 independent experiments were determined.