Search

Our working hypothesis states that DNA damage is a critical step in toxic cell death. The DNA hypothesis was tested in cultured mouse hepatocytes by examining whether inhibitors of DNA repair would increase dimethylnitrosamine toxicity and DNA damage in parallel. Inhibitors were chosen for selectivity toward DNA polymerase alpha (aphidicolin, myricetin), DNA ligase (ethidium bromide), or multiple repair enzymes (ara-C, doxorubicin). Dimethylnitrosamine caused concentration-dependent DNA damage at 6 hr and cell death at 24 hr (35% ALT release vs. 8.8% in control cultured hepatocytes). Each repair inhibitor increased dimethylnitrosamine-induced DNA damage and toxic cell death in parallel. Doxorubicin maximally elevated DNA fragmentation and toxicity (57% ALT release). Repair inhibitors alone failed to damage DNA or cause cell death in this model system. These data support the hypothesis that DNA damage is an early causal event in toxic cell death caused by alkylating hepatotoxicants.

Several hepatotoxic agents damage Ca++ regulation and produce toxic cell death in a manner consistent with a cause-and-effect relationship; however, vital targets of Ca++ remain unidentified. Recent results show that DNA may be the chief Ca++ target during apoptosis, a form of cell death considered distinct from toxic cell death or necrosis. The present studies explored whether nuclear Ca++ regulation is lost before dimethylnitrosamine-induced necrosis, whether DNA is attacked by Ca(++)-dependent endonucleases and whether inhibitors of Ca(++)-endonuclease activity and the DNA repair enzyme poly(ADP-ribose)polymerase affect necrosis. Adult male ICR mice received 100 mg/kg of dimethylnitrosamine i.p. By 2 to 4 hr, total nuclear Ca++ reached 150 to 180% of control and DNA fragmentation was 140 to 170% of control. Electrophoresis of DNA revealed a sharp decline in genomic DNA with the appearance of DNA fragments in a ladder-like pattern. Ca++ elevation and DNA fragmentation preceded toxic cell death by 4 hr or more and reached peak values at 18 to 24 hr, coincident with maximal alanine aminotransferase leakage. Aurintricarboxylic acid, a Ca(++)-endonuclease inhibitor, reduced toxicity 67%. 3-Aminobenzamide, nicotinamide adenine dinucleotide and theophylline, inhibitors of poly(ADP-ribose)polymerase-mediated DNA repair, potentiated liver damage 2-fold. These results support the hypothesis that DNA fragmentation plays a contributing role in toxic cell death induced by dimethylnitrosamine. Furthermore, the findings suggest that new opportunities may exist to moderate the toxicity of alkylating hepatotoxins by altering DNA regulation.

The overfed rat served as the animal model for examining the influence of obesity on the hepatotoxic and nephrotoxic potential of metabolically activated drugs, and acetaminophen served as the prototype drug. Weanling Sprague-Dawley rats were given a standard pellet diet or semisynthetic, energy-dense diet designed to produce obesity. After 24 weeks, when overfed rats outweighed controls by more than 50%, animals received 710 mg/kg of acetaminophen i.p., based on total body weight. Toxicity evaluation included biochemical signs of organ injury over the first 24 hr and histopathologic changes in tissue morphology at 48 hr. Both enzyme release (alanine aminotransferase into plasma, alkaline phosphatase into urine) and frank cellular necrosis in liver and kidney of obese rats greatly exceeded that in pellet-fed controls. Contributing to the potentiation of injury were higher peak plasma concentrations of acetaminophen in obese animals resulting from total body weight dosing. However, liver and kidney injury and mortality remained elevated when peak plasma concentrations were matched by fat-free mass dosing, indicating that increased toxicity also was related to obesity. Incomplete recovery of acetaminophen and metabolites from obese animals (45 vs. 71% in control rats) caused by a functional renal impairment made it impossible to determine the metabolic fate of acetaminophen in overfed animals from the analysis of urine collections. Drug products measured in urine were summed with amounts remaining in carcass at sacrifice, computed as terminal plasma concentrations times respective distribution volumes. These results showed obese rats to form more glucuronide and less sulfate conjugate than did pellet-fed controls, coinciding with clinical evidence for enhanced glucuronidation in obese humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Experiments were designed to test whether the protective effect of N-acetylcysteine against acetaminophen hepatotoxicity precedes arylation of tissue or whether protection occurs after arylation of tissue. Investigation of potential postarylation actions showed that N-acetylcysteine was unable to attenuate the liver necrosis caused by acetaminophen or several other hepatotoxins that act through chemically reactive metabolites. Furthermore, varying the time and route of N-acetylcysteine treatment indicated that the late protection against acetaminophen mortality probably was a consequence of pharmacokinetic factors rather than postarylation intervention in the process of cell death. The antidote was found to inhibit covalent binding of acetaminophen by about 70% when N-acetylcysteine protected against liver necrosis. Treatment regimens that had no effect upon covalent binding also had no effect on acetaminophen hepatotoxicity. Previous failures to detect this relationship apparently occurred because of a failure to consider biological events important in the pathophysiology of acetaminophen-induced necrosis, particularly the marked intrahepatic hemorrhage and vascular congestion with liver engorgement by protein and fluid. These results support the hypothesis that sulfhydryl nucleophiles such as N-acetylcysteine act primarily through prearylation mechanisms to decrease the amount of reactive metabolite available for initiation of hepatic injury.

N-acetylcysteine is the drug of choice for the treatment of acetaminophen poisoning, yet the mechanism of protection in vivo is unknown. Prevention of liver injury could result from decreased production of the toxic intermediate(s), from increased capacity to detoxify the toxic intermediate(s) or from increased ability of the tissue to withstand or even repair the molecular damage caused by the toxic species. Treatment of mice with N-acetylcysteine (1200 mg/kg p.o.) was found to prevent the hepatic damage caused by 1000 mg/kg p.o. of acetaminophen. Possible mechanisms for this hepatoprotective effect were examined by measurement at different time points of acetaminophen and its metabolites in plasma, urine, bile and whole-body homogenates and by evaluation of the changes in these parameters caused by treatment with N-acetylcysteine. A high-pressure liquid chromatographic method was developed to measure the majority urinary metabolites of acetaminophen and was validated by desorption chemical ionization mass spectral analysis of individual metabolites. Minimal differences in the concentration of unchanged acetaminophen and metabolites in whole-body homogenates at 4, 6 and 24 hr postdose were noted for N-acetylcysteine-treated vs. vehicle-treated mice. These results are incompatible with a decreased formation of the toxic species secondary to delayed acetaminophen absorption from the gastrointestinal tract or with an increased clearance of acetaminophen via nontoxic pathways such as sulfation as plausible mechanisms for the observed hepatoprotection.(ABSTRACT TRUNCATED AT 250 WORDS)

The revelation that many covalent binding estimates are falsely low due to flawed normalization discloses that protection by N-acetyl-L-cysteine against acetaminophen hepatotoxicity is accompanied routinely by a 50 to 80% decline in arylation. Elevated glutathione may be responsible for inhibiting covalent binding but above-normal concentrations have never been demonstrated in vivo after N-acetyl-L-cysteine treatment or separated adequately from other possible hepatoprotective actions including direct reduction of the toxic acetaminophen metabolite by the antidote. This led us to compare the conventional L-isomer of the antidote to its nonphysiologic stereoisomer, N-acetyl-D-cysteine, because the latter should be capable of reducing the toxic metabolite but not of stimulating glutathione biosynthesis. Oral coadministration of N-acetyl-D-cysteine (1200 mg/kg), however, failed in preventing the elevation of serum alanine aminotransferase activity, in decreasing hepatocellular necrosis, in interdicting covalent binding of the toxic metabolite to hepatocellular proteins and in preventing the depletion of liver glutathione caused by 500 mg/kg of acetaminophen. N-acetyl-L-cysteine succeeded in decreasing these measures of acetaminophen hepatotoxicity while driving liver glutathione concentrations 2-3 fold above control values. The L-isomer also increased urinary excretion of glutathione-derived acetaminophen metabolites whereas the D-isomer increased only acetaminophen sulfate excretion and reversed the customary predominance of acetaminophen cysteine over the mercapturic acid conjugate. Liver uptake of N-acetyl-D-cysteine was reflected in organ concentrations 7-fold higher than noted for the L-isomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Obese humans suffer from excessive organ dysfunction, altered drug pharmacokinetics and may be at increased risk of various drug toxicities. A recent report shows that gentamicin nephrotoxicity in critically ill patients is more frequent and more severe than usual in individuals who are substantially overweight. The present study utilizes an overfed rat model to examine the influence of obesity on the nephrotoxic potential of gentamicin. After 52 weeks on an energy-dense diet, obese animals outweighed pellet controls by more than 80% (913 +/- 86 vs. 507 +/- 52 g; X +/- S.D., n = 7). When animals were treated twice daily for 6 days with 30 mg/kg of gentamicin i.p. based on total body mass, obese rats sustained more cortical necrosis than control (median score 3+ vs. 0), higher serum creatinine (4.36 +/- 2.72 vs. 0.71 +/- 0.17) and greater creatinine adjusted N-acetyl hexosaminidase excretion. The impact of obesity on intrinsic susceptibility to gentamicin nephrotoxicity was assessed by dosing animals for 5 days to ideal body mass plus 40% of excess body mass, the current clinical practice for achieving normal gentamicin concentrations in obese patients. Obese rats again sustained more frequent and severe cortical necrosis (2+ vs. 0) and excreted more N-acetyl hexosaminidase than control animals. Urine pH averaged 1.7 U below normal in obese animals, but restoration to normal values by 2 weeks on the pellet diet did not diminish the toxicity increase. Results from the overfed rat closely resemble the recent clinical observation that obese patients sustain more frequent and severe kidney damage from aminoglycoside antibiotics.(ABSTRACT TRUNCATED AT 250 WORDS)