Binding and Detoxification of Insecticides by Potentially Probiotic Lactic Acid Bacteria Isolated from Honeybee (Apis mellifera L.) Environment-An In Vitro Study

Abstract

Lactic acid bacteria (LAB) naturally inhabiting the digestive tract of honeybees are known for their ability to detoxify xenobiotics. The effect of chlorpyrifos, coumaphos, and imidacloprid on the growth of LAB strains was tested. All strains showed high resistance to these insecticides. Subsequently, the insecticide binding ability of LAB was investigated. Coumaphos and chlorpyrifos were bound to the greatest extent (up to approx. 64%), and imidacloprid to a much weaker extent (up to approx. 36%). The insecticides were detected in extra- and intracellular extracts of the bacterial cell wall. The ability of selected LAB to reduce the cyto- and genotoxicity of insecticides was tested on two normal (ovarian insect Sf-9 and rat intestinal IEC-6) cell lines and one cancer (human intestinal Caco-2) cell line. All strains exhibited various levels of reduction in the cyto- and genotoxicity of tested insecticides. It seems that coumaphos was detoxified most potently. The detoxification abilities depended on the insecticide, LAB strain, and cell line. The detoxification of insecticides in the organisms of honeybees may reduce the likelihood of the penetration of these toxins into honeybee products consumed by humans and may contribute to the improvement of the condition in apiaries and honeybee health.

Keywords: Apis mellifera L.; Caco-2; Sf-9; chlorpyrifos; coumaphos; detoxification; imidacloprid; insecticides; probiotics; toxicity.

Figure 1

Effect of chlorpyrifos (A), coumaphos (B), and imidacloprid (C) on the growth of lactic acid bacteria strains during 48 h incubation, as evaluated with microtitration assay. Bars: green—20 µg/mL; red—100 µg/mL; blue—500 µg/mL. Each data point represents the mean from four individual wells. Results are presented as mean ± standard deviation (SD). Statistically significant compared to ^a A. kunkeei DSM 12361 and ^b L. rhamnosus GG at p ≤ 0.05.

Figure 1

Effect of chlorpyrifos (A), coumaphos (B), and imidacloprid (C) on the growth of lactic acid bacteria strains during 48 h incubation, as evaluated with microtitration assay. Bars: green—20 µg/mL; red—100 µg/mL; blue—500 µg/mL. Each data point represents the mean from four individual wells. Results are presented as mean ± standard deviation (SD). Statistically significant compared to ^a A. kunkeei DSM 12361 and ^b L. rhamnosus GG at p ≤ 0.05.

Figure 2

Decrease in cytotoxicity of chlorpyrifos, coumaphos, and imidacloprid after 24 h incubation with lactic acid bacteria (LAB) strains measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Each data point represents the mean of the absorbance values from cells from eight individual wells. Results are presented as mean ± standard deviation (SD). Statistical significance was calculated versus positive control (appropriate pesticide standard), * p ≤ 0.1, ** p ≤ 0.05, *** p ≤ 0.01.

Figure 3

Decrease in the genotoxicity of chlorpyrifos, coumaphos, and imidacloprid after 24 h incubation with lactic acid bacteria (LAB) strains expressed as the mean percentage of DNA in the comet tail in the alkaline comet assay. Fifty cells were analyzed for each treatment. Results are presented as mean ± standard error of the mean (S.E.M.). Statistical significance was calculated versus positive control (appropriate pesticide standard), * p ≤ 0.1, ** p ≤ 0.05, *** p ≤ 0.01.

Figure 4

Representative images of comets stained with 1 µ/mL DAPI, fluorescence microscopy (Nikon, Tokyo, Japan), 20× objective.