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. 2019 Oct;32(4):261-274.
doi: 10.1293/tox.2019-0048. Epub 2019 Jul 28.

Differential Responses on Energy Metabolic Pathway Reprogramming Between Genotoxic and Non-Genotoxic Hepatocarcinogens in Rat Liver Cells

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Free PMC article

Differential Responses on Energy Metabolic Pathway Reprogramming Between Genotoxic and Non-Genotoxic Hepatocarcinogens in Rat Liver Cells

Yuko Ito et al. J Toxicol Pathol. .
Free PMC article

Abstract

To clarify difference in the responses on the reprogramming of metabolism toward carcinogenesis between genotoxic and non-genotoxic hepatocarcinogens in the liver, rats were repeatedly administered genotoxic hepatocarcinogens (N-nitrosodiethylamine, aflatoxin B1, N-nitrosopyrrolidine, or carbadox) or non-genotoxic hepatocarcinogens (carbon tetrachloride, thioacetamide, or methapyrilene hydrochloride) for 28, 84, or 90 days. Non-genotoxic hepatocarcinogens revealed transcript expression changes suggestive of suppressed mitochondrial oxidative phosphorylation (OXPHOS) after 28 days and increased glutathione S-transferase placental form-positive (GST-P+) foci downregulating adenosine triphosphate (ATP) synthase subunit beta, mitochondrial precursor (ATPB), compared with genotoxic hepatocarcinogens after 84 or 90 days, suggesting that non-genotoxic hepatocarcinogens are prone to suppress OXPHOS from the early stage of treatment, which is in contrast to genotoxic hepatocarcinogens. Both genotoxic and non-genotoxic hepatocarcinogens upregulated glycolytic enzyme genes and increased cellular membrane solute carrier family 2, facilitated glucose transporter member 1 (GLUT1) expression in GST-P+ foci for up to 90 days, suggesting induction of a metabolic shift from OXPHOS to glycolysis at early hepatocarcinogenesis by hepatocarcinogens unrelated to genotoxic potential. Non-genotoxic hepatocarcinogens increased c-MYC+ cells after 28 days and downregulated Tp53 after 84 or 90 days, suggesting a commitment to enhanced metabolic shift and cell proliferation. Genotoxic hepatocarcinogens also enhanced c-MYC activation-related metabolic shift until 84 or 90 days. In addition, both genotoxic and non-genotoxic hepatocarcinogens upregulated glutaminolysis-related Slc1a5 or Gls, or both, after 28 days and induced liver cell foci immunoreactive for neutral amino acid transporter B(0) (SLC1A5) in the subpopulation of GST-P+ foci after 84 or 90 days, suggesting glutaminolysis-mediated facilitation of cell proliferation toward hepatocarcinogenesis. These results suggest differential responses between genotoxic and non-genotoxic hepatocarcinogens on reprogramming of energy metabolic pathways toward carcinogenesis in liver cells from the early stage of hepatocarcinogen treatment.

Keywords: genotoxicity; glycolysis; hepatocarcinogenesis; liver; oxidative phosphorylation; rat.

Conflict of interest statement

All authors declare that there are no conflicts of interest that influenced the outcome of the present study.

Figures

Fig. 1.
Fig. 1.
Immunohistochemical cellular distribution of adenosine triphosphate (ATP) synthase subunit beta, mitochondrial precursor (ATPB), solute carrier family 2, facilitated glucose transporter member 1 (GLUT1), and glucose-6-phosphate 1-dehydrogenase (G6PD) in association with glutathione S-transferase placental form-positive (GST-P+) liver cell foci after treatment with genotoxic [N-nitrosodiethylamine (DEN), aflatoxin B1 (AFB1), or N-nitrosopyrrolidine (NPYR)] or non-genotoxic hepatocarcinogens [carbon tetrachloride (CCl4), thioacetamide (TAA), or methapyrilene hydrochloride (MP)] for 84 or 90 days. (A) Representative images of the expression of ATPB, GLUT1, and G6PD in GST-P+ foci in the DEN and CCl4 groups (×10 objective; GLUT1 ×20 objective; inset ×60 objective). Bar = 100 µm, 50 µm, or 10 µm (inset). (B) Incidences of ATPB foci in GST-P+ foci in genotoxic and non-genotoxic hepatocarcinogens. (C) Incidences of GLUT1+ foci in GST-P+ foci in genotoxic and non-genotoxic hepatocarcinogens. (D) Incidences of G6PD+ foci in GST-P+ foci in genotoxic and non-genotoxic hepatocarcinogens. Graphs in (B), (C), and (D) show incidences (% value, n=10) of GST-P+ foci showing altered expression of each molecule (open column, decreased; filled column, increased) in each group. **P<0.01, significantly different from the DEN or AFB1 group by Fisher’s exact test. P<0.01, significantly different from the NPYR group by Fisher’s exact test.
Fig. 2.
Fig. 2.
Immunohistochemical cellular distribution of pyruvate kinase L/R (PKLR) and pyruvate kinase isozyme M2 (PKM2) in association with glutathione S-transferase placental form-positive (GST-P+) liver cell foci after treatment with genotoxic [N-nitrosodiethylamine (DEN), aflatoxin B1 (AFB1), or N-nitrosopyrrolidine (NPYR)] or non-genotoxic hepatocarcinogens [carbon tetrachloride (CCl4), thioacetamide (TAA), or methapyrilene hydrochloride (MP)] for 84 or 90 days. (A) Representative images of the expression of PKLR and PKM2 in GST-P+ foci in the NPYR and TAA groups (×20 objective). Bar = 50 µm. (B) Incidences of PKLR foci in GST-P+ foci in genotoxic and non-genotoxic hepatocarcinogens. (C) Incidences of PKM2+ foci in GST-P+ foci in genotoxic and non-genotoxic hepatocarcinogens. Graphs in (B) and (C) show incidences (% value, n=10) of GST-P+ foci showing altered expression of each molecule (open column, decreased; filled column, increased) in each group. **P<0.01, significantly different from the AFB1 group by Fisher’s exact test. P<0.05, significantly different from the NPYR group by Fisher’s exact test. P<0.01, significantly different from the NPYR group by Fisher’s exact test.
Fig. 3.
Fig. 3.
Immunohistochemical cellular distribution of neutral amino acid transporter B(0) (SLC1A5) in association with glutathione S-transferase placental form-positive (GST-P+) liver cell foci after treatment with genotoxic [N-nitrosodiethylamine (DEN), aflatoxin B1 (AFB1), or N-nitrosopyrrolidine (NPYR)] or non-genotoxic hepatocarcinogens [carbon tetrachloride (CCl4), thioacetamide (TAA), or methapyrilene hydrochloride (MP)] for 84 or 90 days. (A) Representative images of the expression of SLC1A5 in GST-P+ foci in the DEN and CCl4 groups (×20 objective). Bar = 50 µm. (B) Incidences of SLC1A5+ foci in GST-P+ foci in genotoxic and non-genotoxic hepatocarcinogens. Graphs in (B) show incidences (% value, n=10) of GST-P+ foci showing altered expression of each molecule (filled column, increased) in each group. *P<0.05, significantly different from the DEN or AFB1 group by Fisher’s exact test. **P<0.01, significantly different from the DEN or AFB1 group by Fisher’s exact test. P<0.01, significantly different from the NPYR group by Fisher’s exact test.
Fig. 4.
Fig. 4.
Distribution of c-MYC+ cells in the liver of rats after treatment with genotoxic [N-nitrosodiethylamine (DEN) or N-nitrosopyrrolidine (NPYR)] or non-genotoxic hepatocarcinogens [carbon tetrachloride (CCl4) or thioacetamide (TAA)] for 28 days and distribution of c-MYC+ cells in association with glutathione S-transferase placental form-positive (GST-P+) liver cell foci after treatment with genotoxic (DEN or NPYR) or non-genotoxic hepatocarcinogens (CCl4 or TAA) for 84 or 90 days. (A) Representative images of the expression of c-MYC in the liver in the DEN and CCl4 groups (×40 objective). Bar = 20 µm. (B) Representative images of the expression of c-MYC of inside (IN) or outside (OUT) of GST-P+ foci in the DEN and CCl4 groups (×40 objective). Bar = 20 µm. Graphs in (A) and (B) show the number of c-MYC+ cells (/100 cells; value, mean + SD) IN or OUT of GST-P+ foci in each group. **P<0.01, significantly different from OUT of untreated controls by Tukey’s or Steel-Dwass test. P<0.01, significantly different from OUT of GST-P+ foci in the DEN or NPYR group by Tukey’s or Steel-Dwass test. §P<0.05, significantly different from OUT of GST-P+ foci in the CCl4 or TAA group by Tukey’s or Steel-Dwass test. §§P<0.01, significantly different from OUT of GST-P+ foci in the CCl4 or TAA group by Tukey’s or Steel-Dwass test.
Fig. 5.
Fig. 5.
Schematic summary of the responses on energy metabolic pathway reprogramming by genotoxic or non-genotoxic hepatocarcinogens in rat liver cells.

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