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Inhibitors of the nuclear enzyme poly (ADP-ribose) polymerase (PARP-1) have been demonstrated to attenuate pathophysiologic conditions associated with oxidative stress, specifically with carbon tetrachloride (CT)-induced hepatotoxicity.
In this investigation, we evaluated 3 previously untested water-soluble PARP-1 inhibitors, namely, 3-aminobenzamide (ABA), 5-aminoisoquinolinone (AIQ), and N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl (PJ-34) to determine their efficacy in blocking or attenuating CT-induced hepatotoxicity in male imprinting control region (ICR) mice.
Indicators of hepatotoxicity were compared with F-tests among groups to determine statistically significant effects. Pearson's correlation coefficients were used to evaluate the correlation between PARP inhibition and the attenuation of hepatotoxicity.
CT treatment resulted in hepatic cytotoxicity, increased serum transaminase (ALT), lipid peroxidation (MDA), intracellular glutathione (GSH) depletion, increased carbonyl content, and substantially increased PARP-1 activity. CT treatment also produced profound observable hemorrhagic necrosis in the hepatic centrilobular region of ICR mice. Pretreatment with PJ-34, ABA, and AIQ before CT treatment significantly decreased PARP-1 activity in hepatocytes after CT treatment by 3.4, 2.0, and 1.9 times, respectively. Corresponding to this reduction in PARP-1 activity, a significant reduction in the ALT levels and MDA and a reduction in the GSH depletion were observed. Also, there were no visible tissue defects in the liver samples from animals pretreated with individual PARP-1 inhibitors before CT administration. These results demonstrate the efficacy of the 3 previously untested water-soluble PARP-1 inhibitors in attenuating CT-induced hepatocellular toxicity and further characterize the role of PARP-1 activation and oxidative stress among the cascade of events in hepatocellular necrosis induced by CT treatment.
The mechanism of action of carbon tetrachloride (CT)-induced hepatotoxicity has been linked to the metabolism of CT into the trichloromethyl radical and the trichloromethyl peroxy radical, and their subsequent reactions with lipids and proteins in the cell membrane, resulting in MDA, where the rate of MDA is mediated by reactive oxygen species, such as superoxide radicals and hydroxyl radicals.[1–12] Also, it has been suggested that CT-induced hepatotoxicity also involves the disruption of hepatic antioxidant defense systems by the depletion of reduced glutathione (GSH), superoxide dismutase (SOD), and catalase. Although a number of antioxidants have been evaluated for preventing CT-induced hepatotoxicity, efficacy has not been demonstrated. Recent findings indicate that free radicals produced by CT lead to mitochondrial damage and nuclear DNA damage, resulting in the activation of their respective repair mechanisms, including induction of the enzyme poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 represents the most abundant isoform of the poly (ADP-ribose) polymerase family, which responds to single- and double-strand nicks in nuclear DNA.[14–18] Upon binding to damaged DNA, PARP-1 forms homodimers and catalyzes the cleavage of NAD+ into nicotinamide and ADP-ribose to form long branches of ADP-ribose polymers on glutamic acid residues of a number of target proteins, including histones and the PARP enzyme itself. Under normal cellular operation, this activity provides protection against damage to nuclear DNA; however, when excessive activation of PARP-1 occurs, the result is a rapid depletion of NAD+ and ATP, eventually leading to necrotic cell death.
Previously, we have demonstrated the hepatoprotective effect against CT-induced centrilobular necrosis with concomitant treatment of the PARP inhibitor 6(5H)-phenanthridinone. However, due to poor water solubility, 6(5H)-phenanthridinone is required to be dissolved in dimethyl sulfoxide (DMSO) for effective administration, which may modify both effective delivery and bioavailability, potentially altering the attenuation of CT-induced toxicity. Recently, specific water-soluble PARP-1 inhibitors have become available, and some have been shown to confer protective effects in various models of toxic exposure. This investigation evaluates the ability of 3 water-soluble PARP-1 inhibitors, namely, 3-aminobenzamide (ABA), 5-aminoisoquinolinone (AIQ), and N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl (PJ-34) to block or attenuate centrilobular hepatotoxicity from CT treatment in imprinting control region (ICR) mice.
CT, malondialdehyde bis (dimethyl acetal), thiobarbituric acid, and 5,5′-dithiobis-2-nitrobenzoic acid were obtained from Sigma Chemical Co. (St. Louis, MO). The Alanine Amino Transferase (ALT) kit was obtained from TECO Diagnostics (Anaheim, CA). ApopTag® Plus In Situ Apoptosis Detection Kit was purchased from Biocompare, Inc. (San Francisco, CA). Cleaved Caspase-3 (Asp175) Rabbit Monoclonal Antibody was from Cell Signaling Technology (Danvers, MA). All other chemicals were of analytical grade or higher from Merck (Darmstadt, Germany).
Adult male ICR mice (30 ± 5 g) were used for all experiments. Animals were housed under controlled conditions (25 ± 2°C and a 12 h light/dark cycle) and allowed free access to food and water. The animals were divided into the following groups: the first group (basic control group) received no CT or PARP inhibitor, the second group (negative control) received no CT and received only individual PARP inhibitor, the third group (treatment) received CT and no PARP inhibitor, and the last group (experimental group) received CT and individual PARP inhibitors. PARP inhibitors were dissolved in normal saline (pH 7.4) and administered intraperitoneally (i.p.). CT was dissolved in corn oil and administered i.p. with doses ranging from 0.3 to 1.2 mL/kg. A time-dependent experiment followed, using 0.6 mL/kg PJ-34 in the following administration groups: 1 h before CT treatment, concomitantly with CT treatment, 1 h after CT treatment, and 3 h after CT treatment. All the experiments were conducted under controlled conditions according to the Guide to the Care and Use of Experimental Animals and the University of South Florida Institutional Animal Care and Use Committee (IACUC). The mice were sacrificed after 24 h and blood samples to determine alanine transaminase (ALT) activity were withdrawn by cardiac puncture. Livers were isolated, perfused with normal saline, and dissected for (1) histopathology and (2) for determination of total GSH, thiobarbituric acid-reactive substances (TBARS), SOD activity, and carbonyl content.
The colorimetric assay for PARP activity was performed in 96-well plates (Trevigen, Inc., Gaithersburg, MD) according to manufacturer's protocol. Serial dilutions of the PARP enzyme were distributed into wells to generate a standard curve. The clarified tissue homogenates (10 μL/well) were added to triplicate wells to determine cellular PARP activity. The reactions were allowed to proceed for 1 h at room temperature. The plate was washed 4 times with 1× phosphate-buffered saline (PBS) and then incubated for 20 min with 50 μL/well Streptavidin–horseradish peroxidase (Strep–HRP) diluted 1:500 in 1× Strep-Diluent (Trevigen). The plate was washed 4 times with 1× PBS prior to the addition of the HRP substrate. For colorimetric readout, 50 μL of TACS-Sapphire (Trevigen) was added to each well and incubated in the dark, at room temperature, for 15 min. Development of the colorimetric reaction was stopped by the addition of an equal volume of 0.2 M HCl. This generated a yellow color that was read at 450 nm. The results were expressed as units of PARP activity calculated per milligram of protein and normalized to equal concentrations of protein. Protein determination in all assays described here were determined by the bicinchoninic acid assay using bovine serum albumin as standard (Sigma).
Serum (0.2 mL) was obtained by centrifugation of cardiac puncture blood samples at 4000rpm for 15 min. Each serum sample was then transferred to a new sterile microcentrifuge tube and stored at 4°C until time of assay. ALT activity, expressed as IU/L, was determined by a previously described method using a commercially prepared reagent kit from TECO Diagnostics (Anaheim, CA).
Total GSH was determined as described previously by Sedlak et al. Individual liver samples (100 mg) were homogenized in 1 mL of 0.2 M PBS (pH 8.0), and the mixture was centrifuged at 12,000 rpm for 30 min. The supernatant (0.5 mL) was mixed with 0.5 mL of 4% sulfosalicylic acid, allowed to stand for 5 min at 4°C, and centrifuged again at 3000 rpm for 10 min. The supernatant obtained (0.5 mL) was mixed with 2 mL of 0.2 M PBS (pH 8.0) and 10 μL of 10 mM 5,5′-dithiobis-2-nitrobenzoic acid. The absorbance was measured at 412 nm and concentration of GSH was expressed as nmol/mg protein.
Formation of lipid peroxide derivatives was evaluated by measuring TBARS according to a method previously described by Cascio et al. Liver samples were individually homogenized in ice-cold 1.15% KCl (w/v). Homogenates (0.4 mL) were mixed with 1 mL of 0.375% TBA, 15% trichloroacetic acid (TCA) (w/v), 0.25 N HCl, and 6.8 mM butylated-hydroxytoluene, placed in a boiling water bath for 10 min, removed and allowed to cool on ice. Following centrifugation at 3000 rpm for 10 min, the absorbance in the supernatants was measured at 532 nm. The amount of TBARS produced was expressed as nmol TBARS/mg protein using malondialdehyde bis(dimethylacetal) for calibration.
Determination of SOD activity in mouse liver was based on inhibition of nitrite formation in the reaction of oxidation of hydroxylammonium with superoxide anion radical. Each sample was divided into 2 aliquots. The total SOD was determined in one aliquot and cytosolic SOD was determined in the second aliquot supplemented with 10 mM KCN. The concentration of mitochondrial SOD was calculated as the difference between the amount of total and cytosolic fraction of SOD. Nitrite formation was generated in a mixture that contained 25mL xanthine (15 mM), 25 mL hydroxylammonium chloride (10 mM), 250 mL PBS (65 mM, pH 7.8), 90 mL distilled water, and 100 mL xanthine oxidase (0.1 U/mL) to initiate the reaction. The inhibitory effect of the extant SOD was assayed at 25°C after 20 min of incubation with 10 mL of liver tissue extracts. The determination of the resulting nitrite was performed following the reaction (20 min at room temperature) with 0.5 mL sulfanilic acid (3.3 mg/mL) and 0.5 mL α-naphthylamine (1 mg/mL). Optical absorbance at 530 nm was measured on Ultrospec III spectrophotometer (Pharmacia LKB, Uppsala, Sweden). The results were expressed as units of SOD activity calculated per milligram of protein.
The procedure used was similar to that described by Levine et al (1990) with slight modifications. Two sets of 250 μL sample homogenates were labeled as “test” and “reference.” A 1 mL amount of 10mM 2,4-dinitrophenylhydrazine (DNPH) prepared in 2.5 M HCl was added to test samples and 2.5 M HCl alone was added to the reference. The contents were mixed and incubated in the dark for 1 h. Then 1 mL of 20% TCA was added to each tube. The tubes were centrifuged at 3500 rpm for 20 min and protein pellets were washed with 1 mL of 10% TCA. The precipitates were washed 3 times with 1 mL of ethyl acetate:ethanol (1:1, v/v) mixture to remove unreacted DNPH and lipids. Each pellet was then dissolved in 1 mL of 6 M guanidine hydrochloride, at 37°C, for 10 min. The insoluble matter was removed by centrifugation. Carbonyl content was determined with Ultrospec III spectrophotometer (Pharmacia LKB, Uppsala, Sweden). Each test sample was read against the corresponding control at 370 nm using an absorption coefficient of 22,000 M-1 cm-1. The protein carbonyl content was expressed in nmol/mg protein.
Liver samples were fixed in 10% neutral buffered formalin and embedded in paraffin following standard procedures. Tissue sections, 4 μm in thickness, were stained with hematoxylin and eosin (H and E) to assess parenchymal histopathologic changes. Histopathologic parameters evaluated in the centrilobular region were proliferation, apoptosis, necrosis, and fibrosis using the methodology described previously by Price et al.
Summary descriptive analysis for all cytotoxicity outcome data is reported as the mean levels of biomarkers of cytotoxicity ± SEM. F-test analysis was performed to evaluate the differences between CT dosage groups in the initial treatment experiment, and again for the select PARP inhibitor groups in the experiment that held the CT dosage constant for all the groups (P < 0.05 was considered to indicate a statistically significant difference). F-test analysis was then performed for the experiment that evaluated differences in measures of cytotoxicity between the time of treatment with PJ-34 relative to CT treatment among groups that held the CT and PJ-34 dosage constant for all groups (P < 0.05 was considered to indicate a statistically significant difference). Pearson's correlation coefficients were calculated for the combined data of PJ-34, ABA, and AIQ to determine the significance and magnitude of correlation between the degree of PARP-1 inhibition and measures of cytotoxicity (P < 0.05 considered to indicate a statistically significant correlation). The analysis was performed with SAS statistical software package 9.2, SAS Institute Cary, NC, USA.
Intraperitoneal injection of CT caused a dose-dependent elevation of ALT 24 h after treatment, with a statistically significant elevation at each increasing dose level [Table 1]. At a CT dose of 1.2 mL/kg, ALT was increased as much as 13.6 times compared with controls. A statistically significant increase of ALT was evident at doses of CT as low as 0.3 mL/kg, suggesting a significantly damaging effect to hepatocytes. Cellular damage was further confirmed by CT dose-dependent MDA. Hepatic GSH levels were depleted in treated mice, and SOD levels (both cytosolic and total) were elevated in response to CT treatment. CT treatment initiated substantial dose-dependent activation of the PARP-1 enzyme as indicated by a 2.4-fold increase at a CT dose of 1.2 mL/kg [Table 1]. Histologic examination of the livers from CT-treated mice revealed severe hepatocyte necrosis in the centrilobular area with an influx of inflammatory cells 24 h after CT treatment [Figure 1].
Three water-soluble PARP-1 inhibitors, namely, 3-aminobenzamide (ABA), 5-aminoisoquinolinone (AIQ), and N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl (PJ-34) were tested to determine their efficacy in attenuating CT-induced hepatotoxicity [Table 2]. The greatest reduction of ALT was produced by PJ-34 at a dose of 3 mg/kg, which resulted in a 2.9-fold decrease in the ALT levels compared with the CT-treated group. PJ-34 decreased MDA concentrations and reduced GSH depletion with greater efficacy compared with ABA or AIQ; PJ-34 also demonstrated the greatest inhibition of PARP-1 activity.
To evaluate the effect of differences in potency of ABA, AIQ, and PJ-34 as PARP-1 inhibitors and the correlation of the degree of PARP-1 inhibition to measures of cytotoxicity, the combined data of ABA, AIQ, and PJ-34 were analyzed to produce Pearson's correlation coefficients. ALT was strongly correlated to PARP-1 activity (r = 0.745, P < 0.001), and a moderate, inverse correlation was observed for GSH levels (r = –0.522, P = 0.007). The strongest correlation observed between the degree of PARP-1 activity and cytotoxicity was found with MDA (r = 0.840, P < 0.001).
As PJ-34 provided improved inhibition of PARP and protection against MDA at statistically significant levels over AIQ and ABA, PJ-34 was chosen to be used in the time of treatment experiments [Table 2]. The time of treatment with PJ-34 relative to CT administration was evaluated with the following treatment schedule: 1 h before CT (CT+PJ(–1)); concomitantly with CT (CT+PJ(0)); 1 h after CT (CT+PJ(+1)); and 3 h after CT (CT+PJ(+3)). The attenuation of CT-induced hepatotoxicity was demonstrated by PJ-34 treatment at 1 h before, concomitantly, and 1 h after CT treatment as indicated by ALT. These treatments reduced ALT elevation by more than 50% compared with mice receiving CT treatment alone. The protective effect was not observed with PJ-34 treatment 3 h after CT administration [Figure 2].
Substantial protection against MDA was conferred from PJ-34 treatment at 1 h before and concomitantly with CT treatment at statistically significant levels compared with mice treated with CT alone [Figure 3]. F-test analysis indicates that MDA levels were not statistically significantly different from untreated mice compared with mice pretreated with PJ-34 1 h before CT treatment (P = 0.332) and mice treated with PJ-34 and CT concomitantly (P = 0.168).
Treatment with PJ-34 1 h prior to CT treatment and concomitantly with CT treatment displayed the greatest protective effect against GSH depletion, with both treatment groups retaining levels not significantly different than untreated mice (P = 0.275 and P = 0.198, respectively). A statistically significant reduction in GSH depletion was observed for all treatment time intervals compared with mice treated with CT alone [Figure 4].
An observable component of cellular toxicity resulting from CT treatment is the disruption of both lipids and protein complexes, indicated by the observed increase of carbonyl content in hepatocytes. Treatment with PJ-34 at 1 h before, concomitantly with CT treatment, and 1 h after CT treatment resulted in statistically significant lower carbonyl levels compared with CT treatment alone [Figure 5]. The protective effect conferred to these groups kept carbonyl content to levels that did not differ significantly from untreated mice (P = 0.520, P = 0.932, and P = 0.439, respectively). However, treatment with PJ-34 did not result in any statistically significant reduction in total, cytosolic, or mitochondrial SOD activity (data not shown).
Necrosis and apoptosis in the centrilobular regions of the liver are consistent with CT-induced hepatotoxicity. A representative sample from the CT treatment group and the group that received PJ-34 1 h before CT treatment are presented for comparison in Figure 1. No necrosis or significant presence of inflammatory cells were observed in centrilobular hepatic tissues from the treatment group that received PJ-34 1 h prior to CT treatment, while substantial necrosis and inflammatory cell influx were observed in centrilobular hepatic tissue from mice treated with CT only. The protective effect observed in histopathology specimens is consistent with the protective effect observed in the biochemical indicators of cytotoxicity reported above from PJ-34 treatment 1 h prior to CT treatment.
In previous investigations, PARP-1 activity has been shown to mediate necrosis in centrilobular hepatocytes after treatment with select hepatotoxicants, which is consistent with the findings of the current investigation. Under normal cellular conditions, PARP-1 activity is very low. When activated in response to oxidative stress, however, PARP-1 initiates an excessive transference of ADP-ribose units from NAD+ to various nuclear proteins, including histones, several chromatin-binding proteins, and PARP-1 itself. The consequence of this response is a rapid depletion of intracellular NAD+, which prevents ATP synthesis and ultimately causes cellular apoptosis or necrosis. Excessive PARP-1 activation is clearly an important mechanism of hepatic tissue damage under conditions of increased oxidative stress, including chemical-induced toxicity, such as excessive CT exposure.
Several specific and nonspecific PARP inhibitors have been evaluated for their efficacy in blocking cellular necrosis and their ability to attenuate CT-induced hepatotoxicity.[32–34] Most commercially available PARP inhibitors (eg, 3-aminobenzamide or nicotinamide) have short cellular residence time and low potency in blocking CT-induced hepatotoxicity. It has been demonstrated that specific PARP-1 inhibitors, such as 6(5H)-phenanthridinone, may have a greater potency in attenuating CT-induced hepatotoxicity both in animal models and human HepG2 cell models.[20,35] However, 6(5H)-phenanthridinone is water insoluble, requiring the use of organic solvents, such as DMSO, as vehicles for administration. Although the use of DMSO as a vehicle for PARP-1 inhibitors may decrease hepatotoxicity from CT treatment, it has also been shown to increase the acute lethality from CT treatment.[36,37] As well, several studies have shown that DMSO can alter the activity of various isoforms of cytochrome P450 (CYP), including CYP2E1, the isoform that bioactivates CT to the trichloromethyl radical in human hepatocytes. The use of DMSO potentially confounds the effects of water-insoluble PARP inhibitors and increases the difficulty in characterizing bioavailability, dose–response, and adverse effects. The use of water-soluble PARP inhibitors eliminates the experimental issues associated with DMSO, providing more effective and transparent results.
The current investigation evaluated the efficacy of 3 novel, water-soluble PARP-1 inhibitors: the phenanthridinone-based PJ-34, ABA, and QIA in blocking or attenuating CT-induced hepatotoxicity and cellular necrosis. The results demonstrate that CT substantively increases PARP-1 activity in the centrilobular hepatic tissue of ICR mice during CT-induced hepatocellular necrosis. The measures of hepatocellular toxicity resulting from CT treatment, such as ALT, MDA, GSH depletion, and carbonyl content, were all significantly reduced with PJ-34 treatment, resulting in the preservation of hepatic tissue as observed through histopathology.
CT-induced hepatotoxicity is mediated through a cascade of events, beginning with the biotransformation and bioactivation of CT. An alternative explanation for the protective effect observed from treatment with PARP-1 inhibitors against CT-induced hepatotoxicity is that the inhibitors may alter CT metabolism and bioactivation. However, the water-soluble nature of the 3 PARP-1 inhibitors tested here makes interaction with a mixed function oxidase metabolism unlikely. Also, correlation coefficients demonstrated that measures of hepatocellular toxicity were strongly correlated to PARP activity, despite unaffected levels of intracellular free radical activity as indicated by a lack of statistically significant reduction in total, cytosolic, or mitochondrial SOD activity. If a significant alteration of metabolism was occurring as a result of PARP inhibitor treatment, a substantive difference in free radical production (and the resulting SOD depletion) would be expected. These results indicate that excessive PARP-1 induction is an important, pathologically relevant, event in the cascade of events leading to cellular necrosis from CT treatment, and that reduced PARP-1 activity is directly correlated with reduced hepatocellular toxicity.
The current investigation provides evidence that select water-soluble PARP-1 inhibitors confer potent hepatoprotective effects from the hepatocellular toxicity induced by CT treatment through reduced PARP-1 activation and the resultant preservation of cellular ATP. Future research should be conducted examining other models of chemically induced hepatotoxicity and the effect of water-soluble PARP-1 inhibitors to confirm the mechanistic role of excessive PARP-1 induction in chemically induced hepatocellular necrosis.
Source of Support: Nil
Conflict of Interest: None declared.