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 . 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 . 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 .
Summary of cytotoxic indicators affected by select doses of CT
Figure 1 (A) Liver histopathology (100×) following carbon tetrachloride (CT) treatment (0.6 mL/kg). Areas of necrosis are recognized by the reddish color of hemorrhage associated with lack of tissue avidity for dyes for hematoxylin due to breakdown of (more ...)
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 . 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.
Indicators of hepatotoxicity and PARP activity for PARP-1 inhibitor treatment groups and treatment groups
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 . 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 Mean alanine transaminase levels at select times following N-(6-oxo- 5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl (PJ-34) treatment of 3 mg/kg and carbon tetrachloride (CT) treatment 0.6 mL/kg: CT+PJ(–1): 1 h before CT administration; (more ...)
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 . 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).
Figure 3 Mean lipid peroxidation levels following N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide HCl (PJ-34) treatment of 3 mg/kg and CT treatment 0.6 mL/kg: CT +PJ(–1): 1 h before CT administration; CT+PJ(0): concomitant administration (more ...)
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 Mean glutathione levels following N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl (PJ-34) treatment of 3 mg/kg and carbon tetrachloride (CT) treatment 0.6 mL/kg: CT +PJ(–1): 1 h before CT administration; CT+PJ(0): concomitant (more ...)
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 . 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).
Figure 5 Mean carbonyl levels following N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl (PJ-34) treatment of 3 mg/kg and carbon tetrachloride (CT) treatment 0.6 mL/kg: CT +PJ(–1): 1 h before CT administration; CT+PJ(0): concomitant administration (more ...)
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 . 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.