George B. Corcoran

Mechanism of Action of N-Acetylcysteine in the Protection Against the Hepatotoxicity of Acetaminophen in Rats In Vivo

N-Acetylcysteine is the drug of choice for the treatment of an acetaminophen overdose. It is thought to provide cysteine for glutathione synthesis and possibly to form an adduct directly with the toxic metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine. However, these hypothese have not been tested in vivo, and other mechanisms of action such as reduction of the quinoneimine might be responsible for the clinical efficacy of N-acetylcysteine. After the administration to rats of acetaminophen (1 g/kg) intraduodenally (i.d.) and of [(35)S]-N-acetylcysteine (1.2 g/kg i.d.), the specific activity of the N-acetylcysteine adduct of acetaminophen (mercapturic acid) isolated from urine and assayed by high pressure liquid chromatography averaged 76+/-6% of the specific activity of the glutathione-acetaminophen adduct excreted in bile, indicating that virtually all N-acetylcysteine-acetaminophen originated from the metabolism of the glutathione-acetaminophen adduct rather than from a direct reaction with the toxic metabolite. N-Acetylcysteine promptly reversed the acetaminophen-induced depletion of glutathione by increasing glutathione synthesis from 0.54 to 2.69 mumol/g per h. Exogenous N-acetylcysteine did not increase the formation of the N-acetylcysteine and glutathione adducts of acetaminophen in fed rats. However, when rats were fasted before the administration of acetaminophen, thereby increasing the stress on the glutathione pool, exogenous N-acetylcysteine significantly increased the formation of the acetaminophen-glutathione adduct from 57 to 105 nmol/min per 100 g. Although the excretion of acetaminophen sulfate increased from 85+/-15 to 211+/-17 mumol/100 g per 24 h after N-acetylcysteine, kinetic simulations showed that increased sulfation does not significantly decrease formation of the toxic metabolite. Reduction of the benzoquinoneimine by N-acetylcysteine should result in the formation of N-acetylcysteine disulfides and glutathione disulfide via thiol-disulfide exchange. Acetaminophen alone depleted intracellular glutathione, and led to a progressive decrease in the biliary excretion of glutathione and glutathione disulfide. N-Acetylcysteine alone did not affect the biliary excretion of glutathione disulfide. However, when administered after acetaminophen. N-acetylcysteine produced a marked increase in the biliary excretion of glutathione disulfide from 1.2+/-0.3 nmol/min per 100 g in control animals to 5.7+/-0.8 nmol/min per 100 g. Animals treated with acetaminophen and N-acetylcysteine excreted 2.7+/-0.8 nmol/min per 100 g of N-acetylcysteine disulfides (measured by high performance liquid chromatography) compared to 0.4+/-0.1 nmol/min per 100 g in rats treated with N-acetylcysteine alone. In conclusion, exogenous N-acetylcysteine does not form significant amounts of conjugate with the reactive metabolite of acetaminophen in the rat in vivo but increases glutathione synthesis, thus providing more substrate for the detoxification of the reactive metabolite in the early phase of an acetaminophen intoxication when the critical reaction with vital macromolecules occurs.

Predicting creatinine clearance and renal drug clearance in obese patients from estimated fat-free body mass

Existing methods for predicting creatinine clearance provide accurate estimates for normal-weight patients but not for patients who are obese. Studies into this problem began with an animal model of obesity, the obese overfed rat. Mean creatinine clearance was found to vary in direct proportion to fat-free body mass, determined in both obese and normal animals. The relevance of this observation to renal function in humans was evaluated by analyzing published studies reporting creatinine clearance and creatinine excretion rates in obese and normal persons. Measured creatinine clearance correlated well with estimated fat-free body mass (r = 0.772, p less than 0.02), and urinary excretion of creatinine normalized to fat-free mass correlated impressively with age (r = 0.960). Formulas derived from these observations allow for the prediction of creatinine clearance at steady state: (formula; see text) In initial tests of these formulas, their predictions appeared to be as accurate as existing methods for the normal-weight population and far superior to these methods when applied to the obese population. Therefore, when creatinine clearance is not measured in obese patients, the estimation of this parameter with the proposed formulas should improve the ability to select the appropriate dose for drugs that are cleared principally by renal filtration.

Ca2+ antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen.

Ca2+ accumulates in the nucleus and DNA undergoes enzymatic cleavage into internucleosomelength fragments before acetaminophen and dimethylnitrosamine produce hepatic necrosis in vivo and toxic cell death in vitro. However, Ca2+‐endonuclease fragmentation of DNA is characteristic of apoptosis, a type of cell death considered biochemically and functionally distinct from toxic cell death. The present studies investigate DNA fragmentation as a critical event in toxic cell death by testing whether the Ca2+‐calmodulin antagonist chlorpromazine and the Ca2+ channel blocker verapamil prevent acetaminophen‐induced hepatic necrosis by inhibiting Ca2+ deregulation and DNA damage. Acetaminophen overdose in mice produced accumulation of Ca2+ in the nucleus (358% of control) and fragmentation of DNA (250% of control) by 6 h, with peak release of ALT occurring at 12–24 h (58,000 U/l). Pretreatment with chlorpromazine prevented increases in nuclear Ca2+ and DNA fragmentation and nearly abolished biochemical evidence of toxic cell death. Verapamil pretreatment also decreased Ca2+ accumulation and DNA damage while attenuating liver injury. The Ca2+ antagonists did not protect against toxic cell death through hypothermia because neither produced the delay in toxicity that is customarily associated with hypothermia. Nor did chlorpromazine or verapamil protect through inhibiting acetaminophen bioactivation. Chlorpromazine failed to diminish glutathione depletion in whole liver and isolated nuclei. Verapamil (250 μM) also failed to alter glutathione depletion in whole liver and had no effect on acetaminophenglutathione adduct formation by mouse liver microsomes and by cultured mouse hepatocytes. Collectively, these results support the hypothesis that Ca2+‐induced DNA fragmentation plays a significant role in cell necrosis produced by acetaminophen and may contribute to toxic cell death caused by other alkylating hepatotoxins.— Ray, S. D., Kamendulis, L. M., Gurule, M. W., Yoridn, R. D., Corcoran, G. B. Ca2+ antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen. FASEB J. 7: 453‐463; 1993.

Early loss of large genomic DNA in vivo with accumulation of Ca2+ in the nucleus during acetaminophen-induced liver injury

Hepatotoxic doses of acetaminophen cause early impairment of Ca2+ homeostasis in the liver. This in vivo study considers the nucleus as a possible site of lethal Ca2+ action by evaluating whether acetaminophen raises Ca2+ in this compartment, whether DNA becomes altered, and whether DNA changes occur early enough during injury to contribute causally to necrosis. Fed Swiss mice were treated with 600 mg/kg acetaminophen ip and livers and blood samples were collected over time. Total nuclear Ca2+ accumulation and fragmentation damage to DNA showed modest parallel increases between 2 and 6 hr, followed by greater than 200% rises at 12 hr mirroring the appearance of frank liver injury (ALT greater than 10,000 U/liter). However, agarose gel electrophoresis revealed extensive loss of large genomic DNA from 2 hr onward, accompanied by the appearance of periodic DNA fragments. Thus, acetaminophen raised nuclear Ca2+ concentrations and promoted DNA fragmentation in vivo. The considerable cleavage of DNA seen at late times probably resulted from cell death, whereas loss of large genomic DNA from 2 hr onward appeared at an early enough point in time to be a contributing factor in acetaminophen-induced liver necrosis.

Resveratrol-induced apoptotic death in human U251 glioma cells

Resveratrol (trans-3,4′,5-trihydroxystilbene) is a naturally occurring polyphenolic compound highly enriched in grapes, peanuts, red wine, and a variety of food sources. Resveratrol has antiinflammatory and antioxidant properties, and also has potent anticancer properties. Human glioma U251 cells were used to understand the molecular mechanisms by which resveratrol acts as an anticancer agent, since glioma is a particularly difficult cancer to treat and eradicate. Our data show that resveratrol induces dose- and time-dependent death of U251 cells, as measured by lactate dehydrogenase release and internucleosomal DNA fragmentation assays. Resveratrol induces activation of caspase-3 and increases the cleavage of the downstream caspase substrate, poly(ADP-ribose) polymerase. Resveratrol-induced DNA fragmentation can be completely blocked by either a general caspase inhibitor (Z-VAD-FMK) or a selective caspase-3 inhibitor (Z-DEVD-FMK), but not by a selective caspase-1 inhibitor. Resveratrol induces cytochrome c release from mitochondria to the cytoplasm and activation of caspase-9. Resveratrol also increases expression of proapoptotic Bax and its translocation to the mitochondria. Resveratrol inhibits U251 proliferation, as measured by MTS assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], and induces G0/G1 growth arrest, as determined by flow cytometry. The cyclin-dependent kinase inhibitor, olomoucine, prevents cell cycle progression and resveratrol-induced apoptosis. These results suggest that multiple signaling pathways may underlie the apoptotic death of U251 glioma induced by resveratrol, which warrants further exploration as an anticancer agent in human glioma.

Silymarin modulates doxorubicin-induced oxidative stress, Bcl-xL and p53 expression while preventing apoptotic and necrotic cell death in the liver

The emergence of silymarin (SMN) as a natural remedy for liver diseases, coupled with its entry into NIH clinical trial, signifies its hepatoprotective potential. SMN is noted for its ability to interfere with apoptotic signaling while acting as an antioxidant. This in vivo study was designed to explore the hepatotoxic potential of Doxorubicin (Dox), the well-known cardiotoxin, and in particular whether pre-exposures to SMN can prevent hepatotoxicity by reducing Dox-induced free radical mediated oxidative stress, by modulating expression of apoptotic signaling proteins like Bcl-xL, and by minimizing liver cell death occurring by apoptosis or necrosis. Groups of male ICR mice included Control, Dox alone, SMN alone, and Dox with SMN pre/co-treatment. Control and Dox groups received saline i.p. for 14 days. SMN was administered p.o. for 14 days at 16 mg/kg/day. An approximate LD(50) dose of Dox, 60 mg/kg, was administered i.p. on day 12 to animals receiving saline or SMN. Animals were euthanized 48 h later. Dox alone induced frank liver injury (>50-fold increase in serum ALT) and oxidative stress (>20-fold increase in malondialdehyde [MDA]), as well as direct damage to DNA (>15-fold increase in DNA fragmentation). Coincident genomic damage and oxidative stress influenced genomic stability, reflected in increased PARP activity and p53 expression. Decreases in Bcl-xL protein coupled with enhanced accumulation of cytochrome c in the cytosol accompanied elevated indexes of apoptotic and necrotic cell death. Significantly, SMN exposure reduced Dox hepatotoxicity and associated apoptotic and necrotic cell death. The effects of SMN on Dox were broad, including the ability to modulate changes in both Bcl-xL and p53 expression. In animals treated with SMN, tissue Bcl-xL expression exceeded control values after Dox treatment. Taken together, these results demonstrated that SMN (i) reduced, delayed onset, or prevented toxic effects of Dox which are typically associated with hydroxyl radical production, (ii) performed as an antioxidant limiting oxidative stress, (iii) protected the integrity of the genome, and (iv) antagonized apoptotic and necrotic cell death while increasing antiapoptotic Bcl-xL protein levels and minimizing the leakage of proapoptotic cytochrome c from liver mitochondria. These observations demonstrate the protective actions of SMN in liver, and raise the possibility that such protection may extend to other organs during Dox treatment including the heart.

The role of the nucleus and other compartments in toxic cell death produced by alkylating hepatotoxicants

Hepatocellular necrosis occurs under a wide range of pathological conditions. In most cases, toxic cell death takes place over a finite span of time, delayed from the point of initial injury and accompanied by homeostatic counterresponses that are varied and complex. The present strategies for discovering critical steps in cell death recognize that (1) different toxins produce similar morphologic changes that precede killing in widely varied cell types, and that (2) lethal events are likely to involve one or more compartmentalized functions that are common to most cells. Investigations of the plasma membrane, endoplasmic reticulum, cytoplasm, mitochondrion, and nucleus have greatly advanced our understanding of acute hepatocellular necrosis. This report examines each compartment but emphasizes molecular changes in the nucleus which may explain cell death caused by alkylating hepatotoxicants. Accumulating knowledge about two distinct modes of cell death, necrosis and apoptosis, indicates that loss of Ca2+ regulation and subsequent damage to DNA may be critical steps in lethal damage to liver cells by toxic chemicals.

Pain sensitivity in dietary-induced obese rats

Previous literature indicates possible interrelationships between the endogenous opioids or endorphins, pain response, and obesity or eating behaviour. The pain response was, therefore, examined in a rat model of obesity induced by palatable food high in unsaturated fats. Pellet-fed control and energy-dense obese and nonobese rats were tested for latency of response to a thermal stimulus using the tail flick test. Obese rats exhibited a statistically significant increase in tail flick latency compared to controls. In addition, the observed latencies were significantly correlated to the body weight of the rats (r = 0.52, p < 0.01). These data suggest that dietary-induced obese rats are similar to obese humans in being less sensitive to painful stimuli, consistent with an increase in endogenous opioids in obesity.

Induction of P4502E1 by acetone in isolated rabbit hepatocytes: Role of increased protein and mRNA synthesis

The molecular mechanism(s) underlying induction of the hepatic microsomal cytochrome P4502E1 (2E1) by xenobiotics (e.g. ethanol and acetone) is controversial. Proposed mechanisms include increased rates of enzyme synthesis due to elevated 2E1 mRNA levels, enhanced translation of pre-existing mRNA, or stabilization of 2E1 protein. To further assess which, if any, of these events predominates during the initial stages of 2E1 protein induction, we investigated the effects of acetone treatment on 2E1 content in cultured rabbit hepatocytes, an in vitro system that allows for precise control of the cellular mileau. Hepatocytes harvested from female rabbits and plated on plastic dishes with serum-supplemented medium were 90-100% viable for at least 48 hr in culture. Analysis of immunoreactive 2E1 content and aniline hydroxylase activity in microsomes isolated from hepatocytes cultured for up to 24 hr revealed that 2E1 expression was equal to that of microsomes from unplated cells and by 48 hr of culture, 2E1 levels decreased by only 35%. Moreover, microsomes isolated from cells exposed to 17 mM acetone for 24 hr exhibited a 53 and 62% increase in aniline hydroxylase activity and 2E1 content, respectively, compared to untreated cells. To explain these increases, the rate of 2E1 protein synthesis was determined in untreated cells or in cells treated with 17 mM acetone by first exposing hepatocytes to medium supplemented with 35S-labeled methionine and cysteine ([35S]Met/Cys) and subsequently assessing radiolabel incorporation into 2E1 protein. While no difference was found between untreated and acetone-treated cells in the incorporation of [35S]Met/Cys into trichloracetic acid-precipitable microsomal proteins, immunoaffinity purification of 2E1 revealed that incorporation of 35S-labeled amino acids specifically into 2E1 was elevated by acetone to 200% of control values. Treatment of hepatocytes with the transcriptional inhibitor, alpha-amanitin, markedly inhibited this acetone-mediated increase in [35S]Met/Cys incorporation into 2E1. Analysis of hepatocyte RNA revealed that acetone increased 2E1 mRNA to 130 and 160% of control levels at 6 and 24 hr, respectively, and that these increases were prevented by pretreatment with alpha-amanitin. Our results indicate that acetone increases 2E1 protein levels in cultured rabbit hepatocytes by stimulating its rate of de novo synthesis. Since this increase in 2E1 synthesis stems, at least in part, from the acetone-mediated enhancement of hepatocyte 2E1 mRNA content and is inhibitable by alpha-amanitin, transcriptional activation of the rabbit CYP2E1 gene is apparently involved in the induction of 2E1 protein by acetone.

Obesity as a risk factor for drug-induced organ injury. VI. Increased hepatic P450 concentration and microsomal ethanol oxidizing activity in the obese overfed rat.

The obese overfed rat effectively models many of the pharmacological changes in human obesity. Recent data show that the obese rat is unusually susceptible to liver damage by several metabolically activated drugs that may be more toxic in obese humans. Results of the present study suggest a specific molecular locus for this interaction. In obese rats, P450 content of liver and the microsomal concentration of P450 were elevated 88% and 31%, respectively, over nonobese controls. Increases in microsomal ethanol oxidation were of identical magnitude. The ethanol-inducible form of P450 that is responsible for microsomal ethanol oxidation, P450IIE1, bioactivates several drugs that are shown to cause increased injury in obese rats. Collectively, these findings indicate that specific forms of P450 may become up-regulated in obesity, increasing the risk of a biochemically defined spectrum of drug-induced organ injuries.