Hematotoxicity of Bacillus thuringiensis as Spore-crystal Strains Cry1Aa, Cry1Ab, Cry1Ac or Cry2Aa

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Hematotoxicity of Bacillus thuringiensis as Spore-crystal Strains Cry1Aa, Cry1Ab, Cry1Ac or Cry2Aa

Tag: # Bacillus thuringiensis,  #δ-endotoxins, #Cry, #Biosafety, #Nontarget organisms, #Bioinsecticide

Hematotoxicity of Bacillus thuringiensis as Spore-crystal Strains Cry1Aa, Cry1Ab, Cry1Ac or Cry2Aa in Swiss Albino Mice

 

Bt  is a microbial control agent (MCA) that produces a range of entomopathogenic toxins [10,11]. The most prominent feature of  Bt is that during sporulation it synthesizes δ-endotoxins or insecticidal crystal proteins (ICPs), which are parasporal crystalline protein inclusions containing crystal proteins (Cry proteins or Cry toxins) as their major constituent [12-15]. These are toxic to larvae of susceptible insects and small invertebrates [1,16], and their use in combating predators from the Hymenoptera, Homoptera, Orthoptera, Coleoptera, Diptera and Lepdoptera Orders, the main cause of damage to agriculture, has been effective [7,17].

Apart from the wide use of formulated and sporulated cultures of Bt as foliar sprays, forming part of integrated pest management strategies against insect pests of agricultural crops [11,18], advances in biotechnology have allowed the development of many genetically modified plants expressing Bt δ-endotoxins [8,19,20]. Consequently, this gene has been widely cloned in different crops and then large amounts of such toxins are released into the environment. However, its adverse effects on non-target organisms are poorly understood [7,9,20].

The primary threat to the effectiveness of long-term use of  Bt  toxins is the evolution of resistance by pests [21], and one of the strategies to delay the emergence of resistant pests is the combined use of Cry toxins that are effective for the same target species. The simultaneous expression of binary combinations of Cry toxins minimizes the chance of insect resistance to  Bt -plants [22]. In addition to the binary combinations, advances in genetic engineering promise the expression of multiple Cry toxins in  Bt -plants, known as gene pyramiding [23]. Therefore, studies on non-target species are requirements of international protocols to verify the adverse effects of these toxins, ensuring human and environmental biosafety [8].

 

Abstract

Formulated and sporulated cultures of Bacillus thuringiensis (Bt) have been widely used against insect pests, but after the advent of genetically modified plants expressing δ-endotoxins, the bioavailability of Cry proteins has been increased. For biosafety reasons their adverse effects should be studied, mainly for non-target organisms. Thus, we evaluated, in Swiss albino mice, the hematotoxicity and genotoxicity of four Bt spore-crystals genetically modified to express individually Cry1Aa, Cry1Ab, Cry1Ac or Cry2A, administered alone by gavage with a single dose of 27 mg/Kg, 136 mg/Kg or 270 mg/Kg, 24 h, 72 h or 7 days before euthanasia. Binary combinations of these four spore-crystal proteins were also assayed at 270 mg/Kg with a single administration 24 h before euthanasia. Control mice received filtered water or cyclophosphamide at 27 mg/kg. For hematotoxicity evaluations, blood samples were drawn by cardiac puncture and processed in a multiple automated hematology analyzer; for genotoxicity analyses, micronucleus test was carried out in mice bone marrow cells. Spore-crystal administrations provoked selective hematotoxicity for the 3 exposure times, particularly for erythroid lineage. A significant reduction in bone marrow cell proliferation demonstrated cytotoxic but not genotoxic effects. These effects persisted for all exposure times, becoming more evident at 7 days.  Similar results were observed for binary combinations at 24 h, suggesting that further studies are required to clarify the mechanism involved in the hematotoxicity found in mice, and to establish the toxicological risks to non-target organisms, especially mammals, before concluding that these microbiological control agents are safe for mammals.

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