Abstract
Terbuthylazine belongs to the chloro-s-triazine group of herbicides and acts primarily as a photosynthesis inhibitor. The mechanisms of action related to its exposure, relevant both in animals and humans, are still insufficiently investigated. This comprehensive study focused on the outcomes of terbuthylazine exposure at cell level in vitro, and a mice model in vivo. Experiments in vitro were conducted on whole human peripheral blood, isolated lymphocytes, and HepG2 cells exposed for 4 h to terbuthylazine at 8.00, 0.80, and 0.58 ng/mL, which is comparable with current reference values set by the European Commission in 2011. Terbuthylazine cytotoxicity was evaluated using dual fluorescent staining with ethidium bromide and acridine orange on lymphocytes, and CCK-8 colorimetric assay on HepG2 cells. The levels of DNA damage were measured using alkaline and hOGG1-modified comet assays. The potency of terbuthlyazine regarding induction of oxidative stress in vitro was studied using a battery of standard oxidative stress biomarkers. The in vivo experiment was conducted on Swiss albino mice exposed to terbuthlyazine in the form of an active substance and its formulated commercial product Radazin TZ-50 at a daily dose of 0.0035 mg/kg bw for 14 days. Following exposure, the DNA damage levels in leukocytes, bone marrow, liver, and kidney cells of the treated mice were measured using an alkaline comet assay. In vitro results suggested low terbuthylazine cytotoxicity in non-target cells. The highest tested concentration (8.00 ng/mL) reduced lymphocyte viability by 15%, mostly due to apoptosis, while cytotoxic effects in HepG2 cells at the same concentration were negligible. Acute in vitro exposure of human lymphocytes and HepG2 cells to terbuthylazine resulted in low-level DNA instability, as detected by the alkaline comet assay. Further characterization of the mechanisms behind the DNA damage obtained using the hOGG1-modified comet assay indicated that oxidative DNA damage did not prevail in the overall damage. This was further confirmed by the measured levels of oxidative stress markers, which were mostly comparable to control. Results obtained in mice indicate that both the active substance and formulated commercial product of terbuthylazine produced DNA instability in all of the studied cell types. We found that DNA in liver and kidney cells was more prone to direct toxic effects of the parent compound and its metabolites than DNA in leukocytes and bone marrow cells. The overall findings suggest the formation of reactive terbuthylazine metabolites capable of inducing DNA cross-links, which hinder DNA migration. These effects were most pronounced in liver cells in vivo and HepG2 cells in vitro. To provide a more accurate explanation of the observed effects, additional research is needed. Nevertheless, the present study provides evidence that terbuthylazine at concentrations comparable with current reference values possesses toxicological risk because it caused low-level DNA instability, both at cellular and animal organism level, which should be further established in forthcoming studies.
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Abbreviations
- ADI:
-
Acceptable daily intake
- AO:
-
Acridine orange
- AOEL:
-
Acceptable operator exposure level
- ARfD:
-
Acute reference dose
- AU:
-
Fluorescence arbitrary unit
- EMEM:
-
Eagle’s minimum essential medium
- EtBr:
-
Ethidium bromide
- FBS:
-
Foetal bovine serum
- GSH:
-
Glutathione
- GSH-Px:
-
Glutathione peroxidase
- hOGG1:
-
Human 8-hydroxyguanine DNA-glycosylase 1
- LMP:
-
Low melting point
- LPO:
-
Lipid peroxidation
- NC:
-
Negative control
- NMP:
-
Normal melting point
- ROS:
-
Reactive oxygen species
- SOD:
-
Superoxide dismutase
- TB:
-
Terbuthylazine
- TBARS:
-
Thiobarbituric acid reactive substances
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This work was financially supported by Project No. 8366 Organic Pollutants in Environment—Markers and Biomarkers of Toxicity (OPENTOX), funded by the Croatian Science Foundation.
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Želježić, D., Žunec, S., Bjeliš, M. et al. Effects of the chloro-s-triazine herbicide terbuthylazine on DNA integrity in human and mouse cells. Environ Sci Pollut Res 25, 19065–19081 (2018). https://doi.org/10.1007/s11356-018-2046-7
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DOI: https://doi.org/10.1007/s11356-018-2046-7