Rattlesnake Crotalus molossus nigrescensvenom induces oxidative stress on human erythrocytes

 

David Meléndez-Martínez¹, Juan Manuel Muñoz¹, Guillermo Barraza-Garza¹, Martha Sandra Cruz-Peréz², Ana Gatica-Colima¹, Emilio Alvarez-Parrilla¹, Luis Fernando Plenge-Tellechea¹

 

1 Departamento de Ciencias Químico Biológicas, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Anillo Envolvente del PRONAF y Estocolmo s/n, C. P. 32310. A. P. 1595-D Ciudad Juárez, Chihuahua, Mexico.

2 Herpetario de la Universidad Autónoma de Querétaro, Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Juriquilla, Querétaro, Mexico.

 

ABSTRACT

Background

Globally, snake envenomation is a well-known cause of death and morbidity. In many cases of snakebite, myonecrosis, dermonecrosis, hemorrhage and neurotoxicity are present. Some of these symptoms may be provoked by the envenomation itself, but others are secondary effects of the produced oxidative stress that enhances the damage produced by the venom toxins. The only oxidative stress effect known in blood is the change in oxidation number of Fe (from ferrous to ferric) in hemoglobin, generating methemoglobin but not in other macromolecules. Currently, the effects of the overproduction of methemoglobin derived from snake venom are not extensively recorded. Therefore, the present study aims to describe the oxidative stress induced by Crotalus molossus nigrescensvenom using erythrocytes.

Methods

Human erythrocytes were washed and incubated with different Crotalus molossus nigrescens venom concentrations (0–640 μg/mL). After 24 h, the hemolytic activity was measured followed by attenuated total reflectance-Fourier transform infrared spectroscopy, non-denaturing PAGE, conjugated diene and thiobarbituric acid reactive substances determination.

Results

Low concentrations of venom (<10 μg/mL) generates oxyhemoglobin release by hemolysis, whereas higher concentrations produced a hemoglobin shift of valence, producing methemoglobin (>40 μg/mL). This substance is not degraded by proteases present in the venom. By infrared spectroscopy, starting in 80 μg/mL, we observed changes in bands that are associated with protein damage (1660 and 1540 cm⁻¹) and lipid peroxidation (2960, 2920 and 1740 cm⁻¹). Lipid peroxidation was confirmed by conjugated diene and thiobarbituric acid reactive substance determination, in which differences were observed between the control and erythrocytes treated with venom.

Conclusions

Crotalus molossus nigrescens venom provokes hemolysis and oxidative stress, which induces methemoglobin formation, loss of protein structure and lipid peroxidation.

 

Key words: Attenuated total reflectance-Fourier transform infrared spectroscopy; Crotalus molossus nigrescens; Venom; Snake venom; Methemoglobin; Oxidative stress; Oxyhemoglobin

 

Funding

This project was supported by Programa Integral de Fortalecimiento Institucional, SEP (PIFI) for Academic Groups, Universidad Autónoma de Ciudad Juárez.

 

Received: November 5, 2016.

Revised: April 12, 2017.

Accepted: April 21, 2017.

 

Correspondence: fplenge@uacj.mx

 

Authors’ contributions

DMM and GBG performed the experiments; MSCP contributed with the C. m. nigrescens venom used in this research; DMM, GBG, JMM, AGC, EAP and LFPT analyzed the data and wrote the paper. All the authors read and approved the final manuscript.

 

Competing interests

The authors declare that they have no competing interests.

 

Consent for publication

Not applicable

 

Ethics approval and consent to participate

The C. m. nigrescens specimens used were maintained in captivity under permit of the Direccion General de Vida Silvestre of Secretaria de Medio Amiente y Recursos Naturales de México (INE/CITES/DGVS-CR-IN-0619-QRO-00) at the Universidad Autónoma de Queretaro.

 

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