Molecular and Histological Responses to AOM in Apex1 Haploinsufficient Mice Reveal a Protective Role of Ape1 in Liver Tissue

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Molecular and Histological Responses to AOM in Apex1 Haploinsufficient Mice Reveal a Protective Role of Ape1 in Liver Tissue

https://doi.org/10.21203/rs.3.rs-4965990/v1

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Strong evidence indicates that in liver diseases such as cirrhosis and hepatocellular carcinoma, there is a reduction of mitochondrial components, particularly mitochondrial DNA (mtDNA), thus suggesting that loss of mtDNA integrity plays an essential role in the development of liver pathologies. However, little is known about mtDNA repair's contribution to maintaining mtDNA and the prevention of liver-related diseases. We hypothesize that APE1, the main apurinic/apyrimidinic endonuclease that participates in base excision repair-mediated mtDNA repair, is required for the maintenance of mtDNA integrity after liver injury. To test this hypothesis, we used a mouse model with a heterozygous null mutation in the Apex1 gene, which encodes APE1. Liver tissue was evaluated for molecular and histological effects after treatment with the alkylating agent azoxymethane (AOM), a carcinogen widely used for the induction of colorectal cancer in rodents. AOM is bioactivated in the liver, thus this organ could represent a primary target of AOM action and a model for liver injury. We treated WT and Apex1^+/− mice with AOM (10 mg/kg body weight) once a week for 4 weeks and liver tissue was harvested 24 weeks after the first dose. Using a PCR-based approach, we observe a 3.2-fold increase in mtDNA damage and a concomitant 55% decrease in mtDNA abundance only in tissue from Apex1^+/− mice. To study the bioenergetics status of liver tissues after AOM treatment, we determined the relative protein levels of ATP5β (an oxidative phosphorylation marker) and GAPDH (a glycolysis marker). We observed a 1.5-fold increase and a 2.5-fold increase in the ATP5β/GAPDH in WT and Apex1^+/− mice, respectively, indicative of increased oxidative phosphorylation in response to AOM-induced alkylation damage. The noted alterations occur within significant histological transformations, including increased nuclear inclusions and ductular proliferation in liver tissue triggered by AOM in both strains of mice. In contrast, indicators of inflammation and hepatocyte injury, such as portal inflammation and fibrosis, were attenuated only in Apex1^+/− mice. In summary, these findings underscore the pivotal role of APE1 in the response of liver tissue to AOM-induced liver damage.

Biological sciences/Molecular biology/Dna damage and repair/Base excision repair

Biological sciences/Cancer/Gastrointestinal cancer/Liver cancer

Mitochondria

Base Excision Repair

Liver

The pathogenesis of liver diseases is a complex process that involves multiple environmental and genetic factors that make the liver unable to perform its biological functions ¹. Chronic liver disease and cirrhosis remain the most important risk factors for the development of hepatocellular carcinoma (HCC) ². The metabolic alterations that occur in chronic liver disease and their relationship with liver carcinogenesis are not fully understood. The impact of mitochondrial DNA (mtDNA) damage and mutations on mitochondrial function, energy metabolism, reactive oxygen species (ROS) production, and regulation of apoptosis has been the focus of recent research efforts ^{3 –6}. Several types of mtDNA alterations, such as point mutations, deletions, insertions, and copy number changes, have been identified in human cancers, including HCC. In addition, many studies have revealed that a decrease in mtDNA copy number is common in HCC ^7–9. Other studies have discovered that HCC has a high frequency of somatic mutations in the D loop and the coding region of mtDNA ^3,10. Therefore, mtDNA mutations, decreased mtDNA copy number, and mitochondrial dysfunction can modify the progression of HCC. The role of these mtDNA alterations in HCC progression could be related to increased mitochondrial ROS production, which could drive cancer initiation and progression ¹¹.

Mutations in mtDNA can occur due to exposure to carcinogens, oxidative stress, aging, or other factors. Azoxymethane (AOM) is a carcinogen widely used for the induction of colorectal cancer (CRC) in rodents ^12–15. AOM is converted in the liver to its active metabolite, methylazoxymethanol (MAM). Then, MAM can undergo further metabolic activation, leading to the production of highly reactive alkylating agents, which can add alkyl groups to DNA bases, causing DNA damage. Repair of DNA damage induced by AOM involves various cellular mechanisms, and base excision repair (BER) is particularly important to repair small base modifications, such as those induced by AOM ¹⁶. Also, AOM has been used to study the effects of a high-fat diet in the development of liver cancer. The liver pathology developed in mice from some studies provided important insights into the effects of the high-fat diet and azoxymethane treatment on the liver ^17,18.

Apurinic/apyrimidinic endonuclease 1 (APE1), encoded by the Apex1 gene, is a central component of BER due to its multifunctional enzymatic activities¹⁹. These encompass endonuclease activity targeted at abasic sites, 3’ phosphodiesterase function, and 3’ to 5’ exonuclease activities. Additionally, APE1 has a redox function, which influences the activity of transcriptional factors such as AP-1, p53, and NF-κB, thus contributing to cellular responses to oxidative stress ²⁰. Therefore, understanding the multifunctional roles of APE1 has created new possibilities for potential interventions to address several human diseases²¹

Previously, we showed that APE1 is required for the maintenance of mtDNA integrity in colorectal tissue after short- and long-term exposure to AOM²². Because AOM is bioactivated in the liver, this organ represents a primary target of AOM action and a model of liver injury ^17,18. In this work, we studied the role of APE1 in the response to AOM-induced liver injury, highlighting its role in maintaining mtDNA integrity, markers of bioenergetics status, and histological features associated with liver injury.

The alkylating agent AOM causes a decrease in mtDNA abundance and an increase in mtDNA lesions in mouse liver tissue.

Previously, we showed that the alkylating agent AOM induces mtDNA lesions and decreases the abundance of mtDNA in mouse colonocytes and that these effects are exacerbated in a mouse strain with a null allele of the Apex1 gene²². Since AOM is metabolized and activated in the liver, we hypothesized that mtDNA lesions would be greater in Apex1^+/− mice than in WT mice due to deficient DNA repair. We extracted DNA from liver tissue from the WT strain and Apex1^+/− mice chronically treated (1 injection per week for 4 weeks and sacrificed 24 weeks after the first injection) with AOM. We then used a PCR-based assay that can detect a variety of DNA lesions, such as base modifications, abasic sites, and single and double-strand breaks, to measure mtDNA damage. The results show a 55% significant decrease (p = 0.0003) in mtDNA abundance in Apex1^+/− mice with AOM treatment, which is not observed in WT mice (Fig. 1A). In terms of mtDNA damage, a significant 3.2-fold increase in mice (p = 0.0037) lesions was observed in the Apex1^+/− AOM-treated (from 0.30 lesions/10kb/strand to 0.96 lesions/10kb/strand) (Fig. 1B). No significant differences were observed in the frequency of mtDNA lesions in WT AOM-treated mice (0.44 lesions/10kb/strand) compared to WT Not Treated mice (0.20 lesions/10 kb/strand) (p = 0.4335). Therefore, the liver from Apex1^+/− mice chronically treated with AOM shows a significant increase in mtDNA lesions and a concomitant decrease in mtDNA abundance.

Alkylation damage increases the β-F1-ATPase/GAPDH ratio in liver tissue

Relative protein expression levels of β-F1-ATPase (involved in oxidative phosphorylation) and GAPDH (associated with glycolysis) have been used as a proxy of the cell bioenergetics signature ^8,23. Thus, the bioenergetics signature represents the relative contribution of oxidative phosphorylation and glycolysis in energy metabolism. Studies in hepatocellular carcinomas and colon cancer have shown that the β-F1-ATPase/GAPDH ratio is low in cancer cells ²³. To determine the effect of AOM-induced DNA damage on the β-F1-ATPase/GAPDH ratio in the liver, we performed a Western Blot analysis of protein samples isolated from liver tissue from mice chronically treated with AOM. Our results show a significant 1.5-fold (p = 0.0098) increase and a 2.5-fold (p = 0.0134) increase in the β-F1-ATPase/GAPDH ratio in WT mice and Apex1^+/− mice, respectively, after AOM treatment (Fig. 2). These results indicate that AOM causes an increase in the β-F1-ATPase/GAPDH ratio in both mouse strains, being more pronounced in Apex1^+/− mice.

Liver histological changes induced by AOM

To assess the long-term effects of AOM treatment for histopathological liver lesions, we analyzed liver tissue for several markers of injury: nuclear inclusions, bile ductular proliferation, portal inflammation, and fibrosis. Nuclear inclusions are invaginations of the cytoplasm into the nucleus that may contain degenerative cell organelles or glycogen deposits ²⁴. They have been observed during aging, in neoplastic hepatocytes, and virus-infected cells ²⁵. The average number of nuclear inclusions per 200x field were 0.1, 7.1, 0.2, and 9.2 for WT, WT AOM-treated, Apex1^+/− and Apex1^+/− AOM-treated mice, respectively (Fig. 3). Statistically significant differences were observed between WT vs WT AOM-treated (p = 0.0125) and between WT vs Apex1^+/− AOM-treated (p = 0.0008) mice. There was no statistically significant difference in nuclear inclusions between WT AOM-treated and Apex1^+/− AOM-treated mice (p = 0.3317).

Ductular reaction or proliferation refers to the appearance of biliary epithelial cells in the portal tracts of diseased livers and is associated with oval cell activation during liver regeneration²⁶. Scores for bile ductular proliferation were 0.0, 2.6, 0.0, and 1.9 for WT, WT AOM-treated, Apex1^+/− and Apex1^+/− AOM-treated mice, respectively (Fig. 4). Statistically significant differences were observed between WT vs WT AOM-treated (p = 0.0001) and WT vs Apex1^+/− AOM-treated mice (p = 0.0005). There was no statistically significant difference in bile ductular proliferation between WT AOM-treated and Apex1^+/− AOM-treated mice (p = 0.0961).

Portal inflammation is characterized by inflammatory infiltrates of lymphocytes, plasma cells, and occasionally eosinophils and monocytes ²⁷. The portal inflammation scores for the WT, WT AOM-treated, Apex1^+/−, and Apex1^+/− AOM-treated mice were the following: 0.5, 2.0, 0, and 1.6, respectively (Fig. 5). Statistically significant differences were observed between WT vs WT AOM-treated mice (p = 0.0025) and WT NT vs Apex1^+/− AOM-treated mice (p = 0.0425). The p-value for the comparison between WT AOM-treated and Apex1^+/− AOM-treated mice was not statistically significant at the 0.05 threshold (p = 0.0554).

Liver fibrosis consists of the accumulation of extracellular matrix proteins, including collagen. It may lead to other chronic liver diseases such as cirrhosis and cancer ²⁸. Fibrosis scores were 0.0, 2.1, 0.0, and 0.9 for WT, WT AOM-treated, Apex1 ^+/−, and Apex1 ^+/− AOM-treated mice, respectively (Fig. 6). Statistical analysis shows a significant difference between WT vs WT AOM-treated mice (p = 0.0188). However, contrary to the histopathological parameters mentioned above, there is no significant increase in the score of the WT NT and Apex1^+/− AOM-treated mice (p = 0.0956). The p-value for the comparison between WT AOM-treated and Apex1^+/− AOM-treated mice was not statistically significant at the 0.05 threshold (p = 0.0711).

In this work, we studied the contribution of APE1 in maintaining mtDNA integrity after long-term exposure to AOM in the liver of mice haploinsufficient for Apex1 (Apex1^+/−). We report a significant increase in mtDNA damage and a decreased abundance of mtDNA in liver tissue from Apex1^+/− mice treated with AOM. We also detected a stronger increase in the β-F1-ATPase/GAPDH ratio in Apex1^+/− mice compared to WT mice. All these changes are in the context of notable histological changes, such as increased nuclear inclusions and ductular proliferation in AOM-induced liver tissue in both mouse strains, while markers of inflammation and chronic injury, such as portal inflammation and fibrosis, were damped in the Apex1^+/− mice. Taken together, these results show that APE1 plays a role in the response of liver tissue to AOM-induced alkylation damage.

Wild-type mice treated with AOM do not show a significant increase in liver mtDNA lesions, while a significant increase was observed in the Apex1^+/− AOM-treated mice (Fig. 1). Concomitantly to mtDNA damage, we also observe a significant decrease in relative mtDNA abundance in Apex1^+/− AOM-treated mice, but not in WT AOM-treated mice. We interpret these results as evidence of deficient mtDNA repair of AOM-induced mtDNA lesions in the liver of Apex1^+/−. Our previous work in colonocytes from mice treated with AOM also shows that AOM treatment leads to loss of mtDNA abundance in both WT and Apex1^+/− mice²². These studies underscore the role of APE1 in maintaining mtDNA integrity after AOM-induced liver and colorectal injury. Studies focusing on HCC have shown that APE1 expression is significantly higher in HCC tissues and cells compared to normal liver cells ²⁹. APE1 levels correlate with TNM staging and histopathological grading, indicating its role in cancer progression. Furthermore, APE1 down-regulating in HCC cells reduces their proliferative activity and increases apoptosis, suggesting APE1's potential as a target for molecular therapy in HCC. A plausible mechanism for APE1’s role in cancer progression could be the recent observation that its inhibition leads to increased lipid peroxidation and ferroptosis in HCC cells³⁰. This process involves the NRF2/SLC7A11/GPX4 axis, where inhibition of APE1 results in oxidative stress and ferroptosis through the degradation of NRF2. Taken together, a scenario emerges in that APE1 is pivotal not only in the early stages after liver injury but also in advanced carcinogenic stages such as HCC.

Studies in hepatocellular carcinomas have revealed significant reductions in protein levels of mitochondrial components such as β-F1-ATPase, providing cancer cells with a bioenergetics phenotype characterized by enhanced glycolysis and concomitant down-regulation of OXPHOS ^23,31,32. However, our study shows an increase in the β-F1-ATPase/GAPDH ratio in both WT and Apex1^+/− mice treated with AOM (Fig. 2). The difference in the direction of the response to AOM in our study may represent the status of the non-cancerous state of the liver tissue used in our study versus the cancerous state of the tissues in the studies referenced above. In addition to inducing apoptosis, cell cycle arrest, and DNA damage response, the p53 protein also regulates metabolism by repressing the GLUT1 and GLUT4 transporters ^7,33. Therefore, in preneoplastic tissue, where p53 mutations have not occurred, the bioenergetics response may reflect an enhanced OXPHOS over glycolysis, such as the one we report in this work. Furthermore, recent work shows that the DNA damage response protein CHK2, regulates both glycolysis and oxidative metabolism; thus, in advanced stages of liver damage, the combination of the effects of p53 effects on the GLUT transporters mentioned above and increased energy demand could be reflected in the increase in the β-F1-ATPase/GAPDH ratio reported in our study⁵.

Long-term treatment with AOM resulted in significant liver histological changes in both WT and Apex1^+/− mice (Fig. 3). Specifically, we observed increased nuclear inclusions in AOM-treated mice. Nuclear inclusions can occur as a consequence of oxidative stress induced by AOM ³⁴ These results concur with previous studies reporting intranuclear inclusions in hepatocytes of rodents treated with AOM^17,35. Interestingly, we observed similar increases in intranuclear inclusions in the livers of AOM-treated WT and Apex1^+/− mice. A possible explanation for these results is that at the time of tissue harvesting, liver damage had reached its peak and the differences between the two strains of mice were not noticed.

We observed increased ductular proliferation, also known as ductular reactions, in mice treated with AOM (Fig. 4). Ductular proliferation involves the expansion of biliary epithelial cells and other cell types that are an integral component of processes such as liver regeneration and carcinogenesis ³⁶. Similarly, in terms of nuclear inclusions, an increase in ductular proliferation was observed in the livers of WT and Apex1^+/− mice treated with AOM.

Interestingly, Apex1^+/− mice showed less portal inflammation and fibrosis after long-term

treatment with AOM (Figs. 5 and 6), suggesting a damped inflammatory response. This can be explained in terms of a possible role for APE1 in modulating the liver inflammatory response after DNA damage. Support for this hypothesis comes from studies showing that APE1 endonuclease and redox activities are involved in innate immunity²⁰. Danger-associated molecular patterns (DAMPs) are molecules released by damaged or dying cells in response to various stressors, including genotoxic stress, which activate the cGAS-STING signaling cascade and finally induce the innate immune response³⁷. We proposed that the cGAS-STING signaling cascade may be reduced in the livers of Apex1^+/− mice. This proposal is supported by the fact that pharmacological inhibition of APE1's redox function prevents the activation of NK-kB, IL-6, and IL-8 induced by THF-α and free fatty acids in the human hepatocellular carcinoma cell line JHH-6 ³⁸. The role of APE1 in modulating the cGAS-STING pathway may be tissue-specific, considering recent findings showing that in lung adenocarcinoma, APE1 contributes to radiation resistance by inhibiting this pathway ³⁹. More studies are needed to pinpoint which of the several biochemical functions of APE1 (and its tissue specificity) are most relevant for the regulation of the cGAS-STING signaling cascade.

In summary, our findings reveal that Apex1^+/− mice exhibit increased mtDNA damage, reduced mtDNA abundance, and altered mitochondrial function markers compared to WT mice after exposure to AOM. Noteworthy histological changes were observed in both mouse strains, with Apex1^+/− mice showing dampened inflammation markers despite exhibiting increased nuclear inclusions and ductular proliferation. Collectively, these results emphasize the role of APE1 in protecting liver tissue against genotoxic stress.

Animals and Azoxymethane (AOM) treatment

Liver tissues were obtained from male 6-month-old wild type (WT) and Apex1 heterozygous (Apex1^+/−) mice in the C57BL/6J background as previously described²². Dr. Christi Walter (University of Texas Health Science Center at San Antonio) provided the original breeding pair. Mice were treated with 10 mg/kg of AOM weekly for 4 weeks. Six months after the first AOM injection, the animals were euthanized by cervical dislocation by trained personnel according to American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. Liver tissue was then removed, frozen or fixed in 10% formaldehyde, embedded in paraffin, and stored for subsequent analysis. Mice were housed in two per cage with a 12-h light-dark cycle and ad libitum access to food and water. All animal procedures and experiments were conducted within the international guidelines for the care and handling of experimental animals. The study is reported in accordance with ARRIVE guidelines. The Institutional Animal Care and Use Committee and the Institutional Biosafety Committee at the University of Puerto Rico Medical Sciences approved the research protocols.

DNA isolation and quantification from frozen liver tissue.

DNA from liver samples was isolated using the DNeasy Blood & Tissue Kit as previously published ²². The integrity of the genomic DNA was verified by running a 1% ethidium bromide (EtBr) stained agarose gel and visualizing it under UV light. Molecular weight was determined using a Lambda DNA Hind III digest. DNA was quantified using the PicoGreen dsDNA Quantitation Kit (Molecular Probes) which employs a fluorescent dye that interacts specifically with dsDNA. Sample fluorescence was detected using a microplate reader (Wallac 1420 VICTOR F or SpectraMax M3) with a 485-nm emission filter. DNA from 6-month-old C57BL6 WT control mice was pooled and used as reference DNA after being diluted to 3 ng/µl as previously described ²².

PCR-based assays to determine mtDNA abundance and mtDNA damage in mouse liver tissue after AOM treatment

To determine the relative abundance of mtDNA, we performed real-time PCR and evaluated the data using the 2^−ΔΔCT method described previously ⁴⁰. The nucleotide sequences for the PCR primers (Integrated DNA Technologies) were 5’-TCG GAG CCC ATA TAG CA-3’ (forward) and 5’-TTT CCG GCT AGA GGT GGG TA-3’ (reverse). These primers generated a 144 bp amplicon encompassing a mtDNA region from position 5587 to 5731. The nucleotide sequences of the primers for an nDNA amplicon were 5’-TTT GCT GTC CCA GTA GAG TAA G-3’ (forward) and 5’-CTC ACT AGA GTT GGT GGG AAT AG-3’ (reverse). The primers generated a 102-bp amplicon that encompassed a region of the hypoxanthine phosphoribosyl transferase (HPRT) gene. To determine the amplification efficiency of our primers, a standard curve was performed (data not shown) ⁴¹. The primer efficiency was determined to be 92.21% and 94.64% for mtDNA and nDNA, respectively. PCR reactions (10 ng of DNA) were prepared using iQ™ SYBR® Green Supermix following the manufacturer's instructions. Briefly, the PCR reactions were performed with an initial denaturation for 10 min at 95°C, 24 cycles of denaturation for 15 seconds at 95°C, annealing/extension at 62°C for 15 min, and a final melt curve of a single cycle with a denaturation for 15 seconds at 95°C and annealing/extension at 62°C for 1 minute. Real-time PCR data was evaluated using QuantStudio™ Real-Time PCR Software v1.3.

DNA samples (3 ng) were diluted to obtain 1 ng of DNA/sample and tested in triplicate following iQ™ SYBR® Green Supermix protocol in a 20 µl reaction. Both a No-DNA sample (in duplicate) and a reference DNA/calibrator in triplicate (from pooled DNA from the 6-month-old C57BL6 WT control mice) were included in the reaction. The assay was performed using mtDNA and nuclear DNA (nDNA) PCR primers separately. The mouse mtDNA (144 bp) and mouse nDNA (102 bp) fragments were amplified using an initial denaturation for 10 min at 95°C, followed by 24 cycles (mtDNA amplicon) and 32 cycles (nDNA amplicon) of denaturation for 15 seconds at 95°C, and annealing/extension at 62°C and 55°C for 15 min, respectively. Data were evaluated using the ΔΔCT (comparative CT) method using QuantStudio Software v1.3.

To detect mtDNA damage in mouse liver tissue, we performed qPCR assays as previously described ^42,43. The primer nucleotide sequences for the amplification of the 10 kb mtDNA amplicon were: 5’-GCC AGC CTG ACC CAT AGC CAT AAT AT -3’ (forward) and 5’-GAG AGA TTT TAT GGG TGT AAT GCG G -3’ (reverse). The PCR amplification was performed using the Master Amp XL Polymerase and Epicentre Premix E. The amplification profile for the 10 kb mtDNA fragment was: initial denaturation for 45 seconds at 94°C, followed by 24 cycles and annealing/extension at 64°C for 12 min, and a final extension at 72°C for 10 min. The amplification data were expressed relative to the amplification of a reference DNA prepared from pooled DNA obtained from 6-month-old C57BL6 WT mice without AOM treatment. Lesions per 10 kilobases (Kb) pairs were calculated using the Poisson equation as previously described ^42,44. Briefly, the average lesion frequency per DNA strand can be calculated as λ= -ln AD/AO, where AD is the amount of amplification of the damaged template and AO represents the amount of amplification product from undamaged DNA. The results are expressed as a relative amplification ratio (AD/AO) and, as lesion frequency per DNA strand per 10 Kb. The levels of mtDNA lesions in liver tissue from AOM-treated mice were calculated by comparing nontreated mice with treated mice from each strain separately. We normalized the amplification of the 10 kb mtDNA fragment with the data obtained using Real-Time PCR to determine relative mtDNA abundance.

Western blot analysis

For protein extraction, about 35 mg of liver tissue was minced to a fine powder using a mortar and pestle while keeping cryogenic conditions with liquid nitrogen. The minced liver tissue was transferred to a Potter-Evelhjem homogenizer tube and homogenized using 300 µl ice-cold Lysis Buffer (1% NP-40, Aprotinin (10µg/ml), Antipain dihydrochloride (2 µg/ml), Benzamidine hydrochloride (5mM), DL-Dithiothreitol (1mM), Ethylenediaminetetraacetic Acid (1mM), Ethylene glycol-bis [β-aminoethyl ether]-N,N, NʹNʹ-tetraacetic acid (1mM), Leupeptin hydrochloride (10µg/ml), Sodium Chloride (150mM), Sodium Fluoride (5mM), Sodium orthovanadate (1mM), Phenylmethanesulfonyl fluoride (1mM), Tris HCL (20mM), Trypsin inhibitor II-O (10µg/ml). Following homogenization, the sample was transferred to a microcentrifuge tube, incubated for 45 minutes at 4^oC, and centrifuged for 90 min at 14,000 rpm at 4°C. The supernatant containing the whole protein lysate was collected, and proteins were quantified and kept at − 30°C until analyzed.

Protein quantification (5 µl/sample) was performed using the colorimetric DC™ protein assay and the microplate reader SpectraMax M3. Twenty micrograms (20 µg) of protein were resolved by sodium dodecyl sulfate (SDS) polyacrylamide gel (12% separating gel and 4% stacking gel) electrophoresis for 60 min at 200V. The proteins were transferred to a 0.2 µm Immuno-Blot polyvinylidene difluoride (PVDF) membrane, using the tank transfer method overnight at 12V, 4°C.

Membranes were blocked in 5% Bovine Serum Albumin (BSA) for 1 hr at room temperature, followed by overnight incubation with the primary antibodies ATP5β (Abcam #ab14730; 1:30,000), β-Actin (Antibodies-online #ABIN1742508; 1:5000), and GAPDH (Abcam #ab8245; 1:1000) at 4°C. Membranes were incubated with secondary antibodies IRDye® 800CW Goat anti-Rabbit IgG (Li-cor # 926-32211; 1:25,000) and IRDye® 680RD Goat anti-Mouse IgG (Li-cor # 926-68070; 1:25,000) for 1 hour at room temperature protected from light. The Odyssey® DLx Imaging System was used to visualize the digital image of protein bands in the membranes. The intensity of the protein bands was quantified by the Image Studio™ Lite Software.

Histopathological analysis

Livers from control and AOM-treated animals were fixed in 10% formaldehyde, embedded in paraffin, and stained using standard procedures. Specifically, paraffin blocks were sectioned at a thickness of 5 µm and stained with hematoxylin and eosin (H&E) to evaluate nuclear inclusions, portal inflammation, and ductular reaction, and Masson trichrome stain for fibrosis analysis. Slides with 3 liver sections per mouse were analyzed. Histological analyses were performed blindly by a pathologist.

Nuclear inclusions were defined as round intranuclear forms with sharp borders, a diameter > 1/3 that of the nucleus (to distinguish them from nucleoli), and the same tinctorial quality as the adjacent cytoplasm. Five fields at 200x magnification were scanned, and the average number of nuclear inclusions per field was reported.

Bile ductular proliferation, portal inflammation, and fibrosis were evaluated at 100x magnification. Bile ductular proliferation was scored using the following scale: none = 0; moderate ductular proliferation, not extending to the adjacent portal tract = 1; severe ductular proliferation, extending to the adjacent portal tract = 2; severe ductular proliferation, extending to the adjacent portal tract, with nodularity of the hepatic parenchyma = 3.

Portal inflammation was scored semi-quantitatively by evaluating lymphocyte and plasma cell infiltration as follows: none = 0; minimal inflammation confined to the portal areas = 1; mild inflammation extending into the parenchyma = 2.

Fibrosis scoring, which is a measure of the extent of chronic liver damage, was performed using the Batts-Ludwig scoring system ⁴⁵. Briefly, none = 0; portal expansion of scar tissue = 1; extension of scar into the periportal region with rare bridges of scar tissue = 2; bridging of scar tissue from portal tract to central vein = 3.

Statistical analyses were performed for the following groups: Wild type (WT) Not Treated (NT) vs WT AOM; WT NT vs Apex1^+/− AOM; and WT AOM vs Apex1^+/− AOM. No statistical analysis was performed using the Apex1^+/− NT because the tissue was available from a single animal. Slices from this animal show scores very similar to WT NT animals.

Statistical analysis

Data are expressed as mean ± the standard error (SEM). The Student's t-test was used to determine the statistical difference between AOM-treated and untreated mice in wild type and Apex1^+/− mice. GraphPad PRISM version 10 was used for the statistical analyses.

Author Contribution

Pérez-Pérez, C., Rodríguez Muñoz, A., Ayala-Peña, S, and Torres-Ramos, C.A participated in the conceptualization of the work. Pérez-Pérez, C., Rodríguez Muñoz, A. and Castro-Achi, M., participated in data collection. and analyzed data. Mackenzie, G.G., Matsukuma K., participated in data analysis. Pérez-Pérez, C. wrote the original draft. Torres-Ramos, C.A and Ayala-Peña, S, oversaw the work and provided guidance to the research team. All the authors contributed to the refinement of the writing or revisions.

Data Availability

The datasets used during the current study are available from the corresponding author on reasonable request.

Conflict of Interest Statement: None declared.

Financial Support: This research project was supported in part by grants from NIH (SC1 NS127764, U54 MD00760, and T32GM148406).

Auger, C., Alhasawi, A., Contavadoo, M. & Appanna, V. D. Dysfunctional mitochondrial bioenergetics and the pathogenesis of hepatic disorders. Front. Cell. Dev. Biol. 3, 40. https://doi.org/10.3389/fcell.2015.00040 (2015).
Fahrer, J. & Christmann, M. D. N. A. Alkylation Damage by Nitrosamines and Relevant DNA Repair Pathways. Int. J. Mol. Sci. 24, 4684. https://doi.org/10.3390/ijms24054684 (2023).
Ayala-Torres, S., Chen, Y., Svoboda, T., Rosenblatt, J. & Van Houten, B. Analysis of gene-specific DNA damage and repair using quantitative polymerase chain reaction. Methods. 22, 135–147. https://doi:10.1006/meth.2000.1054 (2000).
Ballista-Hernández, J. et al. Mitochondrial DNA integrity is maintained by APE1 in carcinogen-induced colorectal cancer. Mol. Cancer Res. 15, 831–884. https://doi.org/10.1158/1541-7786.mcr-16-0218 (2017).
Balogh, J. et al. Hepatocellular carcinoma: a review. J. Hepatocell Carcinoma. 3, 41. https://doi.org/10.2147/JHC.S61146 (2016).
Bataller, R. & Brenner, D. a. Science in medicine Liver fibrosis. J. Clin. Invest. 115, 209–218. https://doi.org/10.1172/JCI24282 (2005).
Bissahoyo, A. et al. Azoxymethane is a genetic background-dependent colorectal tumor initiator and promoter in mice: Effects of dose, route, and diet. Toxicol. Sci. 88, 340–345. https://doi.org/10.1093/toxsci/kfi313 (2005).
Brunt, E. M. et al. Portal chronic inflammation in nonalcoholic fatty liver disease (NAFLD): A histologic marker of advanced NAFLD - Clinicopathologic correlations from the nonalcoholic steatohepatitis clinical research network. Hepatology. 49, 809–820. https://doi.org/10.1002%2Fhep.22724 (2009).
Caston, R. A. et al. The multifunctional APE1 DNA repair–redox signaling protein as a drug target in human disease. Drug Discov Today. 26, 218. https://doi.org/10.1016/j.drudis.2020.10.015 (2021).
Cesaratto, L., Codarin, E., Vascotto, C., Leonardi, A. & Kelley, M. R. Specific Inhibition of the Redox Activity of Ape1/Ref-1 by E3330 Blocks Tnf-A-Induced Activation of Il-8 Production in Liver Cancer Cell Lines. PLoS One. 8, 70909. https://doi.org/10.1371/journal.pone.0070909 (2013).
Chen, J. & Huang, X. F. The signal pathways in azoxymethane-induced colon cancer and preventive implications. Cancer Biol. Ther. 8, 1313–1317. https://doi.org/10.4161/cbt.8.14.8983 (2009).
Chen, R., Du, J., Zhu, H. & Ling, Q. The role of cGAS-STING signalling in liver diseases. JHEP Rep. 3, 100324. https://doi.org/10.1016%2Fj.jhepr.2021.100324 (2021).
Cuezva, J. M. et al. The bioenergetic signature of cancer: A marker of tumor progression. Cancer Res. 62, 6674–6681 (2002). https://aacrjournals.org/cancerres/article-abstract/62/22/6674/509418
Cuezva, J. M. et al. The tumor suppressor function of mitochondria: Translation into the clinics. Biochim. Biophys. Acta - Mol. Basis Dis. 1792, 1145–1158. https://doi.org/10.1016/j.bbadis.2009.01.006 (2009).
Du, Y. et al. APE1 inhibition enhances ferroptotic cell death and contributes to hepatocellular carcinoma therapy. Cell. Death Differ. 31, 431–446. https://doi.org/10.1038/s41418-024-01270-0 (2024).
Furda, A., Santos, J. H., Meyer, J. N. & Van Houten, B. Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods Mol. Biol. 1105, 419–437. https://doi.org/10.1007/978-1-62703-739-6_31( (2014).
Goodman, Z. D. Grading and staging systems for inflammation and fibrosis in chronic liver diseases. J. Hepatol. 47, 598–607. https://doi.org/10.1016/j.jhep.2007.07.006 (2007).
Gouw, A. S. H., Clouston, A. D. & Theise, N. D. Ductular reactions in human liver: Diversity at the interface. Hepatology. 54, 1853–1863. https://doi.org/10.1002/hep.24613 (2011).
Hsu, C. C., Lee, H. C. & Wei, Y. H. Mitochondrial DNA alterations and mitochondrial dysfunction in the progression of hepatocellular carcinoma. World J. Gastroenterol. 19, 8880. https://doi.org/10.3748%2Fwjg.v19.i47.8880 (2013).
Lee, H. C., Wei, Y. H. & Mitochondrial, D. N. A. Instability and Metabolic Shift in Human Cancers. OPEN. ACCESS. Int. J. Mol. Sci. 10, 10. https://doi.org/10.3390/ijms10020674 (2009).
Liang, Y., Liu, J. & Feng, Z. The regulation of cellular metabolism by tumor suppressor p53. Cell. Biosci. 3, 1 (2013). http://www.cellandbioscience.com/content/3/1/9
Livak, K. J. & Schmittgen, T. D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 C T Method. METHODS 25, 402–408. (2001). https://doi.org/10.1006/meth.2001.1262
Loureiro, D. et al. Mitochondrial stress in advanced fibrosis and cirrhosis associated with chronic hepatitis B, chronic hepatitis C, or nonalcoholic steatohepatitis. Hepatology 77, 1348–1365. DOI: 10.1002/hep.32731 (2023).
Lulli, M. et al. DNA damage response protein CHK2 regulates metabolism in liver cancer. Cancer Res. 81, 2861–2873. https://doi.org/10.1158/0008-5472.CAN-20-3134 (2021).
Matkowskyj, K. A. et al. Azoxymethane-induced fulminant hepatic failure in C57BL/6J mice: Characterization of a new animal model. Am. J. Physiol. - Gastrointest. Liver Physiol. 277 https://doi.org/10.1152/ajpgi.1999.277.2.G455 (1999). G455 - G462.
Oliveira, T. T. et al. APE1/Ref-1 Role in Inflammation and Immune Response. Front. Immunol. 13, 726. https://doi.org/10.3389/fimmu.2022.793096 (2022).
Rahman, K. M. W., Sugie, S., Tanaka, T., Mori, H. & Reddy, B. S. Effect of types and amount of dietary fat during the initiation phase of hepatocarcinogenesis. Nutr. Cancer. 39, 220–225. https://doi.org/10.1207/S15327914nc392_10 (2001).
Rao, X., Lai, D. & Huang, X. A new method for quantitative real-time polymerase chain reaction data analysis. J. Comput. Biol. 20, 703–711 (2013). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280562/
Rosenberg, D. W., Giardina, C. & Tanaka, T. Mouse models for the study of colon carcinogenesis. Carcinogenesis. 30, 183–196. https://doi.org/10.1093/carcin/bgn267 (2009).
Sánchez-Aragó, M., Chamorro, M. & Cuezva, J. M. Selection of cancer cells with repressed mitochondria triggers colon cancer progression. Carcinogenesis. 31, 567–576. https://doi.org/10.1093/carcin/bgq012 (2010).
Santos, J. H., Meyer, J. N., Mandavilli, B. S. & Van Houten, B. Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods Mol. Biol. 314, 183–199. https://doi.org/10.1385/1-59259-973-7:183 (2006).
Schwartzenberg-Bar-Yoseph, F., Armoni, M. & Karnieli, E. The tumor suppressor p53 down-regulates glucose transporters GLUT1 and GLUT4 gene expression. Cancer Res. 64, 2627–2633. https://doi.org/10.1158/0008-5472.CAN-03-0846 (2004).
Schwertheim, S. et al. Characterization of two types of intranuclear hepatocellular inclusions in NAFLD. Sci. Rep. 10. (2020). https://doi.org/10.1038/s41598-020-71646-y
Sun, Z. et al. Differential expression of APE1 in hepatocellular carcinoma and the effects on proliferation and apoptosis of cancer cells. Biosci. Trends. 12, 456–462. https://doi.org/10.5582/bst.2018.01239 (2018).
Thoolen, B. et al. Proliferative and nonproliferative lesions of the rat and mouse hepatobiliary system. Toxicol. Pathol. 38. https://doi.org/10.1177/0192623310386499 (2010).
Waggie, K. S. et al. Unexpected Liver and Kidney Pathology in C57BL/6J Mice Fed a High-fat Diet and Given Azoxymethane to Induce Colon Cancer. Comp. Med. 72, 330–335. https://doi.org/10.30802%2FAALAS-CM-22-000040 (2022).
Waly, M. I., Al Alawi, A. A., Marhoobi, A., Rahman, M. S. & I. M. & Red Seaweed (Hypnea Bryodies and Melanothamnus Somalensis) Extracts Counteracting Azoxymethane-Induced Hepatotoxicity in Rats. Asian Pac. J. Cancer Prev. 17, 5071–5074. https://doi.org/10.22034%2FAPJCP.2016.17.12.5071 (2016).
Ward, J. M. Dose response to a single injection of azoxymethane in rats. Induction of tumors in the gastrointestinal tract, auditory sebaceous glands, kidney, liver and preputial gland. Vet. Pathol. 12, 165–177. https://doi.org/10.1177/030098587501200302 (1975).
Whitaker, A. M. & Freudenthal, B. D. APE1: A skilled nucleic acid surgeon. DNA Repair. 71, 93–100. https://doi.org/10.1016%2Fj.dnarep.2018.08.012 (2018).
Xuan, W., Song, D., Yan, Y., Yang, M. & Sun, Y. A Potential Role for Mitochondrial DNA in the Activation of Oxidative Stress and Inflammation in Liver Disease. Oxid. Med. Cell. Longev. 1–10. (2020). https://doi.org/10.1155/2020/5835910 (2020).

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Molecular and Histological Responses to AOM in Apex1 Haploinsufficient Mice Reveal a Protective Role of Ape1 in Liver Tissue

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Abstract

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Introduction

Results

Alkylation damage increases the β-F1-ATPase/GAPDH ratio in liver tissue

Liver histological changes induced by AOM

Discussion

Materials and Methods

Animals and Azoxymethane (AOM) treatment

Western blot analysis

Histopathological analysis

Statistical analysis

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References

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