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Bacillus thuringiensis Cry1Ac toxin and protoxin do not provoke acute or chronic cytotoxicity on macrophages and leukocytes

Abstract

The bioinsecticidal Cry1Ac proteins (protoxin and toxin) are potent immunogens that can activate macrophages by inducing upregulation of costimulatory molecules, pro-inflammatory cytokines, and mitogen-activated protein kinase (MAPK) signaling pathways. Besides, by the oral route, Cry1Ac toxin is mildly allergenic and induces intestinal lymphoid hyperplasia in mice. Given the potential utility of Cry1Ac protoxin as an adjuvant, as well as the human consumption of Cry1Ac toxin in transgenic crops, it is necessary to more deeply evaluate the toxicological potential of these proteins in mammalian immune cells. Here, were used in vitro evaluations in leukocyte and macrophage cell lines to test the potential toxicity of various doses of Cry1Ac proteins, by means of Alamar Blue, MTT, Annexin V, and JC1 assays. Our results indicated that neither Cry1Ac protoxin nor toxin elicited acute toxic effects, after monitoring the cell activity for 4, 8, 10, and 24 h of exposure. By flow cytometry and confocal microscopy analysis, it was observed that neither Cry1Ac toxin nor protoxin generated mitochondrial damage or depolarization or induced apoptosis or necrosis. In conclusion, despite their immunostimulatory effects, it was demonstrated that Cry1Ac proteins did not have cytotoxic effects, even at high concentrations, in primary leukocytes or macrophages or cell lines.

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Acknowledgments

We acknowledge Edith González-González, PhD, for support in JC1 assays. Rubio-Infante acknowledges CONACYT for posdoctoral fellowships granted 2019 – 1 (740975) and 2020 – 1 (769256).

Funding

This work was supported by CONACYT (CB283319) and UNAM PAPIIT (IN223319).

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Correspondence to L. Moreno-Fierros.

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Editor: Tetsuji Okamoto

Supplementary Information

Supplementary Fig. 1
figure 6

Cry1Ac toxin or protoxin did not have post-stimulus cytotoxic effects in splenocytes. To determine potential post-stimulus cytotoxic effects the lymphocytes and macrophages were incubated with Cry1Ac (A) toxin or (B) protoxin for 24 h. Subsequently, fresh media was added plus 10% fetal bovine serum without Cry1Ac proteins and metabolic functions were evaluated with Alamar Blue reagent. There was no toxicity after 4, 8, 10, or 24 h (p < 0.0 (PNG 607 kb)

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Bautista-Jacobo, I., Rubio-Infante, N., Ilhuicatzi-Alvarado, D. et al. Bacillus thuringiensis Cry1Ac toxin and protoxin do not provoke acute or chronic cytotoxicity on macrophages and leukocytes. In Vitro Cell.Dev.Biol.-Animal 57, 42–52 (2021). https://doi.org/10.1007/s11626-020-00525-7

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  • DOI: https://doi.org/10.1007/s11626-020-00525-7

Keywords

  • Cry proteins
  • Cytotoxicity
  • Mammals
  • Effects on mammals
  • Immune cells