CR-LAAO causes genotoxic damage in HepG2 tumor cells by oxidative stress

Tássia R Costa; Martin K Amstalden; Diego L Ribeiro; Danilo L Menaldo; Marco A Sartim; Alexandre F Aissa; Lusânia M G Antunes; Suely V Sampaio

doi:10.1016/j.tox.2018.05.005

CR-LAAO causes genotoxic damage in HepG2 tumor cells by oxidative stress

Toxicology. 2018 Jul 1:404-405:42-48. doi: 10.1016/j.tox.2018.05.005. Epub 2018 May 5.

Authors

Tássia R Costa¹, Martin K Amstalden², Diego L Ribeiro³, Danilo L Menaldo², Marco A Sartim², Alexandre F Aissa², Lusânia M G Antunes², Suely V Sampaio⁴

Affiliations

¹ Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida do Café s/no, CEP 14040-903, Ribeirão Preto, São Paulo, Brazil. Electronic address: tassiarc@fcfrp.usp.br.
² Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida do Café s/no, CEP 14040-903, Ribeirão Preto, São Paulo, Brazil.
³ Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, Avenida Bandeirantes, 3900, CEP 14049-900, Ribeirão Preto, São Paulo, Brazil.
⁴ Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida do Café s/no, CEP 14040-903, Ribeirão Preto, São Paulo, Brazil. Electronic address: suvilela@usp.br.

PMID: 29738841
DOI: 10.1016/j.tox.2018.05.005

Abstract

Snake venom L-amino acid oxidases (SV-LAAOs) are enzymes of great interest in research due to their many biological effects with therapeutic potential. CR-LAAO, an L-amino acid oxidase from Calloselasma rhodostoma snake venom, is a well described SV-LAAO with immunomodulatory, antiparasitic, microbicidal, and antitumor effects. In this study, we evaluated the genotoxic potential of this enzyme in human peripheral blood mononuclear cells (PBMC) and HepG2 tumor cells, as well as its interaction with these cells, its impact on the expression of DNA repair and antioxidant pathway genes, and reactive oxygen species (ROS)-induced intracellular production. Flow cytometry analysis of FITC-labelled CR-LAAO showed higher specificity of interaction with HepG2 cells than PBMC. Moreover, CR-LAAO significantly increased intracellular levels of ROS only in HepG2 tumor cells, as assessed by fluorescence. CR-LAAO also induced genotoxicity in HepG2 cells and PBMC after 4 h of stimulus, with DNA damages persisting in HepG2 cells after 24 h. To investigate the molecular basis underlying the genotoxicity attributed to CR-LAAO, we analyzed the expression profile (mRNA levels) of 44 genes involved in DNA repair and antioxidant pathways in HepG2 cells by RT² Profiler polymerase chain reaction array. CR-LAAO altered the tumor cell expression of DNA repair genes, with two downregulated (XRCC4 and TOPBP1) and three upregulated (ERCC6, RAD52 and CDKN1) genes. In addition, two genes of the antioxidant pathway were upregulated (GPX3 and MPO), probably in an attempt to protect tumor cells from oxidative damage. In conclusion, our data suggest that CR-LAAO possesses higher binding affinity to HepG2 tumor cells than to PBMC, its genotoxic mechanism is possibly caused by the oxidative stress related to the production of H₂O₂, and is also capable of modulating genes related to the DNA repair system and antioxidant pathways.

Keywords: DNA damage; DNA repair; L-amino acid oxidase; Oxidative stress; Snake venom.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
DNA Damage / drug effects*
DNA Damage / physiology
Dose-Response Relationship, Drug
Hep G2 Cells
Humans
L-Amino Acid Oxidase / isolation & purification
L-Amino Acid Oxidase / toxicity*
Leukocytes, Mononuclear / drug effects
Leukocytes, Mononuclear / metabolism
Oxidative Stress / drug effects*
Oxidative Stress / physiology
Snake Venoms / isolation & purification
Snake Venoms / toxicity*

Substances

Snake Venoms
L-Amino Acid Oxidase