Chlorpyrifos (CPF) and aflatoxin B1 (AFB1) are each known to adversely affect hepatic tissue individually, but their combined hepatic effects have never been previously investigated. HepG2 cell viability, oxidative status, and genetic impairment were examined after exposing HepG2 cells to: (1) CPF alone, (2) AFB1 alone, and (3) CPF and AFB1 combined (20:1). CPF exposure decreased cell viability, reduced glutathione (GSH) content, and superoxide dismutase (SOD) activity but increased both glutathione peroxidase (GPx) and paraoxonase 1 activity. AFB1 exposure decreased cell viability and GSH content but increased reactive oxygen species (ROS) production. CPF and AFB1 combined exposure decreased GSH content (p < 0.05) further over individual CPF and AFB1 exposures. Induction of micronucleus formation was detected in AFB1-treated cells but undetected in both CPF and combination-treated cells. In conclusion, cytotoxic effects caused by combined exposure were antagonistic, as shown by a combination index value of 1.67. Although no change in ROS production was observed in CPF groups, the overall results confirmed the occurrence of oxidative stress through the alterations of GSH content, GPx, and SOD activity. Only intracellular GSH was evidently changed upon exposure to CPF and AFB1 combined. Thus, this study suggested cellular GSH as a potential indicator for detecting the combined effects of CPF and AFB1 in HepG2 cells, the detection of which could be adapted to estimate the potential toxicity of additional multiple toxicant exposures.
AdeyemiJAMartins-juniorACBarbosaFJr (2015) Teratogenicity, genotoxicity and oxidative stress in zebrafish embryos (Danio rerio) co-exposed to arsenic and atrazine. Comparative Biochemistry and Physiology172–173: 7–12.
2.
BayramSRencüzoğullarıEAlmasAM, et al. (2016) Effect of p53 Arg72Pro polymorphism on the induction of micronucleus by aflatoxin B1 in in vitro in human blood lymphocytes. Drug and Chemical Toxicology39(3): 331–337.
3.
BuranratBMairuaeNKanchanarachW (2017) Cytotoxic and antimigratory effects of cratoxy formosum extract against HepG2 liver cancer cells. Biomedical Reports1: 441–448.
4.
CartwrightIMSuCHaskinsJS, et al. (2018) DNA repair deficient Chinese hamster ovary cells exhibiting differential sensitivity to charged particle radiation under aerobic and hypoxic conditions. International Journal of Molecular Sciences19: 2228.
5.
ChenJBhandarBKavdiaM (2015) Interaction of ROS and RNS with GSH and GSH/GPx systems. The FASEB Physiology Journal29: 1.
6.
ChouT (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Research70(2): 440–447.
7.
CorcueraLAVettorazziAArbillagaL, et al. (2015) Genotoxicity of aflatoxin B1 and ochratoxin A after simultaneous application of the in vivo micronucleus and comet assay. Food and Chemical Toxicology76: 116–124.
8.
EyerPPodhradskyD (1986) Evaluation of the micromethod for determination of glutathione using enzymatic cycling and Ellman’s reagent. Analytical Biochemistry153: 57–66.
9.
EzziLSalahIBHaouasZ, et al. (2016) Histopathological and genotoxic effects of chlorpyrifos in rats. Environmental Science and Pollution Research23(5): 4859–4867.
10.
GoelADaniVDhawanDK (2005) Protective effects of zinc on lipid peroxidation, antioxidant enzymes and hepatic histoarchitecture in chlorpyrifos-induced toxicity. Chemico-Biological Interactions156: 131–140.
11.
HanifHAMuradNAWan-NgahWZ, et al. (2005) Effects of Zingiber officinale on superoxide dismutase, glutathione peroxidase, catalase, glutathione and malondialdehyde content in HepG2 cell line. Malaysian Journal of Biochemistry and Molecular Biology11: 36–41.
12.
InanFPalaMDoymazI (2007) Use of ozone in detoxification of aflatoxin B1 in red pepper. Journal of Stored Products Research43(4): 425–429.
13.
International Agency for Research on Cancer (IARC) (2012) IARC monographs on the identification of carcinogenic hazard to humans. Available at: www.monographs.iarc.fr/list-of-classifications-volumes (accessed 9 March 2019).
14.
JaipieamSVisuthismajarnRSiriwongW, et al. (2009) Inhalation exposure of organophosphate pesticides by vegetable growers in the Bang-Rieng subdistrict in Thailand. Journal of Environmental and Public HealthEpub ahead of print 7 February 2010. DOI: 10.1155/2009/452373.
15.
Karami-MohajeriSAhmadipourARahimiH, et al. (2017) Adverse effects of organophosphorus pesticides on the liver: a brief summary of four decades of research. Archives of Industrial Hygiene and Toxicology68(4): 261–275.
16.
MahakunakornPTohdaMMurakamiY, et al. (2005) Effects of choto-san and its related constituents on endogenous antioxidant systems. Biological and Pharmaceutical Bulletin28(1): 53–57.
17.
MarinDETaranuI (2012) Overview on aflatoxins and oxidative stress. Toxin Reviews31(3–4): 32–43.
18.
MarinhoATDiasCGPinheiroPF, et al. (2016) Nevirapine modulation of paraoxonase-1 in the liver: an in vitro three-model approach. European Journal of Pharmaceutical Sciences82: 147–153.
19.
MehtaAVermaRSSrivastavaN (2009) Chlorpyrifos induced alterations in the levels of hydrogen peroxide, nitrate and nitrite in rat brain and liver. Pesticide Biochemistry and Physiology94(2–3): 55–59.
20.
MizunoCSAmpomaahWMendonçaFR, et al. (2017) Cytotoxicity and genotoxicity of stilbene derivatives in CHO-K1 and HepG2 cell lines. Genetics and Molecular Biology40(3): 656–664.
21.
MoonERheeDPyoS (1999) In vitro suppressive effect of aflatoxin B1 on murine peritoneal macrophage functions. Toxicology133: 171–179.
22.
ParkSKimDKimJ, et al. (2015) Effects of mycotoxins on mucosal microbial infection and related pathogenesis. Toxin7: 4484–4502.
23.
PatnaikRPadhyRN (2016) Evaluation of geno-toxicity of methyl parathion and chlorpyrifos to human liver carcinoma cell line (HepG2). Environmental Science and Pollution Research23(9): 8492–8499.
24.
SalyhaYT (2013) Chlorpyrifos leads to oxidative stress-induced death of hippocampal cells in vitro. Neurophysiology45(3): 193–199.
25.
SapbamrerRHongsibsongS (2014) Organophosphorus pesticide residues in vegetables from farms, markets, and a supermarket around Kwan Phayao lake of Northern Thailand. Archives of Environmental Contamination and Toxicology67(1): 60–67.
26.
ShenHMShiCYShenY, et al. (1996) Detection of elevated reactive oxygen species level in cultured rat hepatocytes treated with aflatoxin B1. Free Radical Biology and Medicine21(2): 139–146.
27.
TangJCaoYRoseRL, et al. (2001) Metabolism of chlorpyrifos by human cytochrome p450 isoforms and human, mouse, and rat liver microsomes. The American Society for Pharmacology and Experimental Therapeutics29(9): 1201–1204.
28.
TotejaGSMukherjeeADiwakarS, et al. (2006) Aflatoxin B1 contamination in wheat grain samples collected from different geographical regions of India: a multicenter study. Journal of Food Protection69(6): 1463–1467.
29.
TripathiSSrivastavAK (2010) Liver profile of rats after long-term ingestion of different doses of chlorpyrifos. Pesticide Biochemistry and Physiology97(1): 60–65.
30.
UchenduCAmbaliSFAyoJO, et al. (2018) Chronic co-exposure to chlorpyrifos and deltamethrin pesticides induces alterations in serum lipids and oxidative stress in Wistar rats: mitigating role of alpha-lipoic acid. Environmental Science and Pollution Research25: 19605–19611.
31.
UzunFGKalenderY (2013) Chlorpyrifos induced hepatotoxic and hematologic changes in rats: the role of quercetin and catechin. Food and Chemical Toxicology55: 549–556.
32.
WongCKYeungHYWooPS, et al. (2001) Specific expression of cytochrome P4501A1 gene in gill, intestine and liver of tilapia exposed to coastal sediments. Aquatic Toxicology54: 69–80.
33.
YoonJHamHSungJ, et al. (2014) Black rice extract protected HepG2 cells from oxidative stress-induced cell death via ERK1/2 and Akt activation. Nutrition Research and Practice8(2): 125–131.
