Abstract
In dose-response and structure-activity studies, human hepatic HepG2 cells were exposed for 3 days to nano Cu, nano CuO or CuCl2 (ions) at doses between 0.1 and 30 ug/ml (approximately the no observable adverse effect level to a high degree of cytotoxicity). Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function, and oxidative stress. With nano Cu and nano CuO, few indications of cytotoxicity were observed between 0.1 and 3 ug/ml. In respect to dose, lactate dehydrogenase and aspartate transaminase were the most sensitive cytotoxicity parameters. The next most responsive parameters were alanine aminotransferase, glutathione reductase, glucose 6-phosphate dehydrogenase, and protein concentration. The medium responsive parameters were superoxide dismutase, gamma glutamyltranspeptidase, total bilirubin, and microalbumin. The parameters glutathione peroxidase, glutathione reductase, and protein were all altered by nano Cu and nano CuO but not by CuCl2 exposures. Our chief observations were (1) significant decreases in glucose 6-phosphate dehydrogenase and glutathione reductase was observed at doses below the doses that show high cytotoxicity, (2) even high cytotoxicity did not induce large changes in some study parameters (e.g., alkaline phosphatase, catalase, microalbumin, total bilirubin, thioredoxin reductase, and triglycerides), (3) even though many significant biochemical effects happen only at doses showing varying degrees of cytotoxicity, it was not clear that cytotoxicity alone caused all of the observed significant biochemical effects, and (4) the decreased glucose 6-phosphate dehydrogenase and glutathione reductase support the view that oxidative stress is a main toxicity pathway of CuCl2 and Cu–containing nanomaterials.
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Abbreviations
- ALP :
-
alkaline phosphatase
- ALT :
-
alanine aminotransferase
- AOP :
-
adverse outcome pathway
- AST :
-
aspartate transaminase
- BET :
-
specific surface area/porosity as determined by the Brunauer, Emmett, Teller test
- CAT :
-
catalase
- DLVO :
-
Derjaguin, Landau, Verwey, and Overbeek theory
- DPBS :
-
Dulbecco’s phosphate-buffered saline
- EDX :
-
energy-dispersive x-ray analysis
- FTIR :
-
Fourier transform infrared spectroscopy
- GGT :
-
gamma glutamyltranspeptidase
- G6PDH :
-
glucose 6-phosphate dehydrogenase
- GPx :
-
glutathione peroxidase
- GRD :
-
glutathione reductase
- GSH :
-
reduced glutathione concentration
- HepG2 :
-
human hepatocellular carcinoma cells, ATCC catalog number HB-8065
- LDH :
-
lactate dehydrogenase
- MIA :
-
microalbumin
- MTS :
-
4-[5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-1,3-thiazol-2-yl)tetrazol-3-ium-2-yl]benzenesulfonate
- MTT :
-
3-[4,5-dimethyl-2-thiazol]-2,5-diphenyl-2H-tetrazolium bromide
- PBS :
-
phosphate buffered saline
- ROS :
-
reactive oxygen species
- SEM :
-
scanning electron microscopy
- SOD :
-
superoxide dismutase
- TBARS :
-
thiobarbituric acid reactive substances
- T BIL :
-
total bilirubin
- TEM :
-
transmission electron microscopy
- THRR :
-
thioredoxin reductase
- TRIG :
-
triglycerides
- XRD :
-
X-ray diffraction
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Acknowledgements
We are grateful for the participation of many individuals in this study. Particularly we thank Will Boyes and Maribel Bruno for reviewing this manuscript as part of EPA clearance procedures.
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Supplementary file1 (PPTX 92 kb) Supplementary Figure 1 Media, cellular and total LDH enzyme activity following Cu treatment of HepG2 cells
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Supplementary file3 (PPTX 103 kb) Supplementary Figure 3 Media, cellular and total AST enzyme activity following Cu treatment of HepG2 cells
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Supplementary file5 (PPTX 95 kb) Supplementary Figure 5 Media, cellular and total ALT enzyme activity following Cu treatment of HepG2 cells
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Supplementary file7 (PPTX 79 kb) Supplementary Figure 7 Effects of 3 Cu containing materials on HepG2 cellular protein content
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Supplementary file10 (DOC 659 kb) Supplementary Table 1. Cytotoxic and Biochemical effects from 3 different Cu containing materials: experimental values useful for modelers
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Kitchin, K.T., Richards, J.A., Robinette, B.L. et al. Biochemical effects of copper nanomaterials in human hepatocellular carcinoma (HepG2) cells. Cell Biol Toxicol 39, 2311–2329 (2023). https://doi.org/10.1007/s10565-022-09720-6
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DOI: https://doi.org/10.1007/s10565-022-09720-6