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Safety evaluation of chinese medicine injections with a cell imaging-based multiparametric assay revealed a critical involvement of mitochondrial function in hepatotoxicity.

Wang M, Liu CX, Dong RR, He S, Liu TT, Zhao TC, Wang ZL, Shen XY, Zhang BL, Gao XM, Zhu Y - Evid Based Complement Alternat Med (2015)

Bottom Line: The safety of herbal medicine products has been a widespread concern due to their complex chemical nature and lack of proper evaluation methods.Our results suggested that the reported hepatotoxicity by one of the drugs, Fufangkushen injection, could be attributed at least in part to the interference of mitochondrial function in human HepG2 cells by some of its constituents.This method should be useful for both preclinical screen in a drug discovery program and postclinical evaluation of herbal medicine preparations.

View Article: PubMed Central - PubMed

Affiliation: Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China ; Research and Development Center of TCM, Tianjin International Joint Academy of Biotechnology & Medicine, 220 Dongting Road, TEDA, Tianjin 300457, China.

ABSTRACT
The safety of herbal medicine products has been a widespread concern due to their complex chemical nature and lack of proper evaluation methods. We have adapted a sensitive and reproducible multiparametric cell-based high-content analysis assay to evaluate the hepatic-safety of four Chinese medicine injections and validated it with classical animal-based toxicity assays. Our results suggested that the reported hepatotoxicity by one of the drugs, Fufangkushen injection, could be attributed at least in part to the interference of mitochondrial function in human HepG2 cells by some of its constituents. This method should be useful for both preclinical screen in a drug discovery program and postclinical evaluation of herbal medicine preparations.

No MeSH data available.


Related in: MedlinePlus

Representative images of HCA for positive drugs induced hepatotoxicity on Hoescht 33342, Mitotracker Deep Red FM, PI iodide, and Rhodamine 123 channels. HepG2 cells treated with blank (a, e, i, m, and q), 3 μM FCCP (d, h, l, p, and t), 3 mM acetaminophen (b, f, j, n, and r), and 3 μM doxorubicin hydrochloride (DOX) (c, g, k, o, and s) are shown. The increased intensity of Mitotracker Deep Red FM indicated an increased mitochondrial mass (MS) (g, h). The decreased intensity of Rhodamine 123 indicated a decreased mitochondrial membrane potential (MMP) (j, k, and l). The MMP decrease was indicated by white arrow (g, k, and o). The intensity of PI was increased when plasma membrane permeability (PMP) increased. The PMP increase was indicated by white arrow (o). The cell nucleuses stained by Hoechst 33342 shrunk and CN decreased by FCCP, indicated by white arrow (d).
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fig1: Representative images of HCA for positive drugs induced hepatotoxicity on Hoescht 33342, Mitotracker Deep Red FM, PI iodide, and Rhodamine 123 channels. HepG2 cells treated with blank (a, e, i, m, and q), 3 μM FCCP (d, h, l, p, and t), 3 mM acetaminophen (b, f, j, n, and r), and 3 μM doxorubicin hydrochloride (DOX) (c, g, k, o, and s) are shown. The increased intensity of Mitotracker Deep Red FM indicated an increased mitochondrial mass (MS) (g, h). The decreased intensity of Rhodamine 123 indicated a decreased mitochondrial membrane potential (MMP) (j, k, and l). The MMP decrease was indicated by white arrow (g, k, and o). The intensity of PI was increased when plasma membrane permeability (PMP) increased. The PMP increase was indicated by white arrow (o). The cell nucleuses stained by Hoechst 33342 shrunk and CN decreased by FCCP, indicated by white arrow (d).

Mentions: The sensitivity of the multiparametric HCA assay was first validated by three known hepatotoxic compounds. Representative images of Hoescht 33342, Mitotracker Deep Red FM, PI iodide, and Rhodamine 123 channels captured by the HCA established typical cytotoxic effects caused by FCCP (3 μM), acetaminophen (3 mM), and doxorubicin hydrochloride (3 μM). As shown in Figure 1, an increased intensity of Mitotracker Deep Red FM indicated MS increase caused by FCCP and doxorubicin hydrochloride (Figures 1(g) and 1(h)); a decreased intensity of Rhodamine 123 indicated the MMP decrease caused by all three positive drugs (Figures 1(j), 1(k), and 1(l)). The cell nucleuses shrunk were founded by FCCP, Figure 1(d). As shown in Figure 2, FCCP at 3 μM, 10 μM, and 30 μM (P < 0.01), acetaminophen at 1 mM, 3 mM, and 10 mM (P < 0.01), and doxorubicin hydrochloride at 0.1 μM to 10 μM (P < 0.01) dramatically decreased the CN by 35.8%–70.4% compared with blank group. FCCP at 30 μM (P < 0.05), acetaminophen at 100 μM (P < 0.01), and doxorubicin hydrochloride at 3 μM and 10 μM (P < 0.01) significantly increased the PMP by 17.5-, 61.4-, 19.0-, and 44.9-fold, respectively. Acetaminophen significantly increased the NA by 25.7% at 3 mM (P < 0.01), while it increased the MMP by 26.7% at 100 μM (P < 0.01). In addition, for FCCP, acetaminophen, and doxorubicin hydrochloride, the IC 50 of CN was 5.18 μM, 1.84 mM, and 1.67 μM, respectively. For NA, the IC 50 was 0.60 μM, 111.63 mM, and 0.17 μM, respectively. For MS, the IC 50 was 7.02 μM, 9.78 mM, and 1.94 μM, respectively. For MMP, the IC 50 was 0.77 μM, 113.18 mM, and 0.63 μM, respectively. For FCCP, acetaminophen and doxorubicin hydrochloride, the IC 50 of PMP was 25.69 μM, 0.01 mM, and 10.73 μM.


Safety evaluation of chinese medicine injections with a cell imaging-based multiparametric assay revealed a critical involvement of mitochondrial function in hepatotoxicity.

Wang M, Liu CX, Dong RR, He S, Liu TT, Zhao TC, Wang ZL, Shen XY, Zhang BL, Gao XM, Zhu Y - Evid Based Complement Alternat Med (2015)

Representative images of HCA for positive drugs induced hepatotoxicity on Hoescht 33342, Mitotracker Deep Red FM, PI iodide, and Rhodamine 123 channels. HepG2 cells treated with blank (a, e, i, m, and q), 3 μM FCCP (d, h, l, p, and t), 3 mM acetaminophen (b, f, j, n, and r), and 3 μM doxorubicin hydrochloride (DOX) (c, g, k, o, and s) are shown. The increased intensity of Mitotracker Deep Red FM indicated an increased mitochondrial mass (MS) (g, h). The decreased intensity of Rhodamine 123 indicated a decreased mitochondrial membrane potential (MMP) (j, k, and l). The MMP decrease was indicated by white arrow (g, k, and o). The intensity of PI was increased when plasma membrane permeability (PMP) increased. The PMP increase was indicated by white arrow (o). The cell nucleuses stained by Hoechst 33342 shrunk and CN decreased by FCCP, indicated by white arrow (d).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4352439&req=5

fig1: Representative images of HCA for positive drugs induced hepatotoxicity on Hoescht 33342, Mitotracker Deep Red FM, PI iodide, and Rhodamine 123 channels. HepG2 cells treated with blank (a, e, i, m, and q), 3 μM FCCP (d, h, l, p, and t), 3 mM acetaminophen (b, f, j, n, and r), and 3 μM doxorubicin hydrochloride (DOX) (c, g, k, o, and s) are shown. The increased intensity of Mitotracker Deep Red FM indicated an increased mitochondrial mass (MS) (g, h). The decreased intensity of Rhodamine 123 indicated a decreased mitochondrial membrane potential (MMP) (j, k, and l). The MMP decrease was indicated by white arrow (g, k, and o). The intensity of PI was increased when plasma membrane permeability (PMP) increased. The PMP increase was indicated by white arrow (o). The cell nucleuses stained by Hoechst 33342 shrunk and CN decreased by FCCP, indicated by white arrow (d).
Mentions: The sensitivity of the multiparametric HCA assay was first validated by three known hepatotoxic compounds. Representative images of Hoescht 33342, Mitotracker Deep Red FM, PI iodide, and Rhodamine 123 channels captured by the HCA established typical cytotoxic effects caused by FCCP (3 μM), acetaminophen (3 mM), and doxorubicin hydrochloride (3 μM). As shown in Figure 1, an increased intensity of Mitotracker Deep Red FM indicated MS increase caused by FCCP and doxorubicin hydrochloride (Figures 1(g) and 1(h)); a decreased intensity of Rhodamine 123 indicated the MMP decrease caused by all three positive drugs (Figures 1(j), 1(k), and 1(l)). The cell nucleuses shrunk were founded by FCCP, Figure 1(d). As shown in Figure 2, FCCP at 3 μM, 10 μM, and 30 μM (P < 0.01), acetaminophen at 1 mM, 3 mM, and 10 mM (P < 0.01), and doxorubicin hydrochloride at 0.1 μM to 10 μM (P < 0.01) dramatically decreased the CN by 35.8%–70.4% compared with blank group. FCCP at 30 μM (P < 0.05), acetaminophen at 100 μM (P < 0.01), and doxorubicin hydrochloride at 3 μM and 10 μM (P < 0.01) significantly increased the PMP by 17.5-, 61.4-, 19.0-, and 44.9-fold, respectively. Acetaminophen significantly increased the NA by 25.7% at 3 mM (P < 0.01), while it increased the MMP by 26.7% at 100 μM (P < 0.01). In addition, for FCCP, acetaminophen, and doxorubicin hydrochloride, the IC 50 of CN was 5.18 μM, 1.84 mM, and 1.67 μM, respectively. For NA, the IC 50 was 0.60 μM, 111.63 mM, and 0.17 μM, respectively. For MS, the IC 50 was 7.02 μM, 9.78 mM, and 1.94 μM, respectively. For MMP, the IC 50 was 0.77 μM, 113.18 mM, and 0.63 μM, respectively. For FCCP, acetaminophen and doxorubicin hydrochloride, the IC 50 of PMP was 25.69 μM, 0.01 mM, and 10.73 μM.

Bottom Line: The safety of herbal medicine products has been a widespread concern due to their complex chemical nature and lack of proper evaluation methods.Our results suggested that the reported hepatotoxicity by one of the drugs, Fufangkushen injection, could be attributed at least in part to the interference of mitochondrial function in human HepG2 cells by some of its constituents.This method should be useful for both preclinical screen in a drug discovery program and postclinical evaluation of herbal medicine preparations.

View Article: PubMed Central - PubMed

Affiliation: Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China ; Research and Development Center of TCM, Tianjin International Joint Academy of Biotechnology & Medicine, 220 Dongting Road, TEDA, Tianjin 300457, China.

ABSTRACT
The safety of herbal medicine products has been a widespread concern due to their complex chemical nature and lack of proper evaluation methods. We have adapted a sensitive and reproducible multiparametric cell-based high-content analysis assay to evaluate the hepatic-safety of four Chinese medicine injections and validated it with classical animal-based toxicity assays. Our results suggested that the reported hepatotoxicity by one of the drugs, Fufangkushen injection, could be attributed at least in part to the interference of mitochondrial function in human HepG2 cells by some of its constituents. This method should be useful for both preclinical screen in a drug discovery program and postclinical evaluation of herbal medicine preparations.

No MeSH data available.


Related in: MedlinePlus