Mechanism of ketone-induced protection from acetaminophen hepatotoxicity in the rat

The effects of ketones on acetaminophen metabolism and hepatotoxicity were investigated in male rats. Ketosis was produced by oral administration of either acetone or 1,3-butanediol. Histologic studies revealed that both ketogenic agents conferred protection from acetaminophen-induced liver necrosis. Pharmacokinetic studies indicated that both acetone and 1,3-butanediol: a) increased the blood half-life of acetaminophen, b) markedly decreased the apparent rate constant for formation of acetaminophen mercapturate, and c) modestly decreased the capacities for acetaminophen sulfate formation and renal elimination of the drug. Neither acetone nor 1,3-butanediol had any effect on either the apparent rate constant for formation of acetaminophen glucuronide or on the predrug levels of hepatic glutathione. However, after a large dose of acetaminophen, the rate and percentage of glutathione depletion were markedly less in 1,3-butanediol-treated rats and modestly less in acetone-treated rats as compared with controls. These data indicate that acetone- or 1,3-butanediol-induced ketosis confers protection from hepatic necrosis due largely to decreased formation of the reactive metabolite. The effects of ketosis and of diabetes on acetaminophen metabolism and hepatotoxicity are compared.     

Mechanism of protection by metallothionein against acetaminophen hepatotoxicity

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the US. Metallothionein (MT) expression attenuates APAP-induced liver injury. However, the mechanism of this protection remains incompletely understood. To address this issue, C57BL/6 mice were treated with 100 μmol/kg ZnCl₂ for 3 days to induce MT. Twenty-four hours after the last dose of zinc, the animals received 300 mg/kg APAP. Liver injury (plasma ALT activities, area of necrosis), DNA fragmentation, peroxynitrite formation (nitrotyrosine staining), MT expression, hepatic glutathione (GSH), and glutathione disulfide (GSSG) levels were determined after 6 h. APAP alone caused severe liver injury with oxidant stress (increased GSSG levels), peroxynitrite formation, and DNA fragmentation, all of which were attenuated by zinc-induced MT expression. In contrast, MT knockout mice were not protected by zinc. Hydrogen peroxide-induced cell injury in primary hepatocytes was dependent only on the intracellular GSH levels but not on MT expression. Thus, the protective effect of MT in vivo was not due to the direct scavenging of reactive oxygen species. Zinc treatment had no effect on the early GSH depletion kinetics after APAP administration, which is an indicator of the metabolic activation of APAP to its reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). However, MT was able to effectively trap NAPQI by covalent binding. We conclude that MT scavenges some of the excess NAPQI after GSH depletion and prevents covalent binding to cellular proteins, which is the trigger for the propagation of the cell injury mechanisms through mitochondrial dysfunction and nuclear DNA damage.     

Analysis of the aryl hydrocarbon hydroxylase assay

The assay method and the properties of aryl hydrocarbon hydroxylase were studied with rat liver microsomes. The assay could be carried out by two methods: (1) the microsomes (initially at 15–20°) were pre-incubated at 37° and then benzo[a]pyrene and NADPH were added to initiate the assay; and (2) benzo[a]pyrene was added to microsomes at 15–20°, pre-incubated at 37°, and then NADPH was added to initiate the reaction. The aryl hydrocarbon hydroxylase activity obtained by method 1 was usually 60–120 per cent higher than that obtained by method 2. Several lines of study suggested that the microsomes can exist in two states. Binding of benzo[a]pyrene at temperatures lower than 20° would limit the microsomes to a low activity state whereas binding at temperatures above 25° would enable the microsomes to exist in a high activity state. The molecular basis of these activity states remains to be investigated. The effects of acetone and methanol, both having been widely used as the solvents for benzo[a]pyrene, on aryl hydrocarbon hydroxylase have also been studied. The aryl hydrocarbon hydroxylase activities of control and phenobarbital-induced microsomes were higher with acetone as the solvent for benzo[a]pyrene as compared with methanol. The hydroxylase activity of 3-methylcholanthrene-induced microsomes, however, was slightly higher with methanol.     

The induction of rat lung aryl hydrocarbon hydroxylase by diluted smoke from commercial cigarettes

Induction of pulmonary aryl hydrocarbon hydroxylase (AHH) by cigarette smoke in small rodents is well established. However, studies on dose-response relationships particularly at the lower ends of such relationships are few indeed. Availability of the British-American Tobacco Co. (B.-A.T.)-Mason inhalation system, in which precisely-calibrated dilutions of cigarette smoke can be released into an inhalation chamber, enables excellent dose-response relationships to be established. Using this inhalation system and cigarettes delivering tar (total particulate matter, water and nicotine free) equal to 1, 4 and 20 mg/cigarette, it has been shown that induction of AHH in male Sprague-Dawley rat lung is an extremely sensitive system to inhaled cigarette smoke. AHH induction was observed at a 200-s exposure to 40 puffs of a 1 : 5 dilution of smoke from an ultra mild cigarette delivering 1 mg tar. At this low concentration, it is not even possible to accurately weigh the TPM from diluted smoke in the exposure chamber. This extremely sensitive rat pulmonary enzyme system may prove valuable in the biological testing of modern low tar cigarettes.     

The mechanisms of cobalt chloride-induced protection against acetaminophen hepatotoxicity

The mechanism by which cobalt chloride protects hamsters from acetaminophen-induced hepatotoxicity has been reexamined. In agreement with earlier studies, cobalt chloride treatment produced a 1.5-fold increase in hepatic glutathione levels and a decrease in the cytochrome P-450-dependent oxidative metabolism of acetaminophen. Cobalt chloride treatment also suppressed the sulfation while enhancing the glucuronidation of acetaminophen. The suppression of sulfation was most marked at low, non-hepatotoxic doses, whereas the enhancement of glucuronidation was greatest at higher hepatotoxic doses of acetaminophen. Collectively, the data suggest that the protective effect of cobalt chloride treatment on acetaminophen hepatotoxicity results from a combination of the suppression of the toxic pathway of cytochrome P-450 oxidation, an increase in the protective capacity of glutathione, and an enhancement of the nontoxic pathway of glucuronidation.     

Exacerbation of acetaminophen hepatotoxicity by thalidomide and protection by nicotinic acid amide

• 1.1. The effects of racemic thalidomide (d[ + ]/l[ - ] α-phthalimido-glutarimide) on acetaminophen (AAP)-induced hepatitis were tested in male NMRI mice (n = 133) and quantified as serum activities of glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT).
• 2.2. A 2.1-fold increase of GOT and a 1.9-fold increase of GPT activities (P < 0.001) were observed in mice treated perorally with 500 mg/kg of AAP plus 150 mg/kg of thalidomide (Tha1). In the absence of AAP, Thal did not display any detectable hepatotoxic effects.
• 3.3. The Thal-induced exacerbation of AAP hepatotoxicity was completely inhibited by nicotinic acid amide, a selective inhibitor of poly(ADP-ribose) polymerase (PARP) (P < 0.0001), suggesting a possible influence of Thal on the hepatic metabolism of NAD-adenoribosylation.
• 4.4. We see the main application of nicotinic acid amide as for the combinational use in pharmaceutical preparations of AAP in order to avoid hepatic damage in patients treated with AAP and Thal.

     

Acetaminophen hepatotoxicity: studies on the mechanism of cysteamine protection

Inhibition of the cytochrome P-450-dependent formation of the acetaminophen-reactive metabolite was investigated as a possible mechanism for cysteamine protection against acetaminophen hepatotoxicity. Studies in isolated hamster hepatocytes indicated that cysteamine competitively inhibited the cytochrome P-450 enzyme system as represented by formation of the acetaminophen-glutathione conjugate. However, cysteamine was not a potent inhibitor of glutathione conjugate formation (Ki = 1.17 mM). Cysteamine also weakly inhibited the glucuronidation of acetaminophen (Ki = 2.44 mM). In vivo studies were in agreement with the results obtained in isolated hepatocytes; cysteamine moderately inhibited both glucuronidation and the cytochrome P-450-dependent formation of acetaminophen mercapturate. The overall elimination rate constant (beta) for acetaminophen was correspondingly decreased. Since cysteamine decreased both beta and the apparent rate constant for mercapturate formation (K'MA), the proportion of the dose of acetaminophen which is converted to the toxic metabolite (K'MA/beta) was not significantly decreased in the presence of cysteamine. Apparently, cysteamine does inhibit the cytochrome P-450-dependent formation of the acetaminophen-reactive metabolite, but this effect is not sufficient to explain antidotal protection.     

The activity of aryl hydrocarbon hydroxylase in adult human skin.

1 Aryl hydrocarbon hydroxylase (AHH), a mixed-function oxidase system, has been identified in microsomal preparations of adult breast skin and foreskin. 2 Separation of dermis from epidermis by stretching, showed that AHH activity in human skin is almost exclusively located within the epidermis. 3 Preincubation of whole chopped skin with benzanthracene (5 muM to 100 muM) using a tissue culture system was accompanied by a concentration dependent increase in AHH activity. 4 Although there was no significant difference in AHH activity between the two sites, individuals differed markedly from one another in activity at any one site. Activity was observed to be positively correlated with age.     

Induction of aryl hydrocarbon hydroxylase in rat lung by marijuana smoke.

Rats were exposed to the smoke produced by burning four cigarettes of marijuana or of marijuana placebo material. Six hours later, maximally stimulated aryl hydrocarbon hydroxylase (AHH) activity was observed in the lung. Marijuana placebo material also induced pulmonary AHH activity. Actinomycin D (1 mg/kg) and cycloheximide (2 mg/kg), given ip 1 hr before exposure to marijuana smoke, partially blocked AHH induction. If rats were exposed repeatedly to marijuana smoke, they showed higher activities of pulmonary AHH within 6 hr of the last exposure than did animals exposed for the first time. Inhalation of marijuana smoke increased the number and size of debris-filled vacuoles in alveolar macrophages and in the ciliated cells of the bronchiolar epithelium. Smoke condensate from the marijuana material used in these studies contained 0.45 ng/mg of benzo(a)pyrene.     

The heme oxygenase system: its role in liver inflammation

Heme Oxygenase is the rate-limiting enzyme in the degradation of heme into carbon monoxide (CO), iron and bilirubin. To date, three heme oxygenase isozymes have been identified: HO-1, HO-2 and HO-3. While HO-1 is structurally different from its counterparts, HO-2 and HO-3 are very similar (90% homology), with HO-3 being a poor heme catalyst. Of the three isozymes, HO-1 is believed to be the only inducible form. Constitutively expressed HO-2 has been identified in several organs including kidney and vascular smooth muscle, with the most abundant sources (and activity) being in the liver, brain, spleen and testes. Within the normal liver, HO-2 is constitutively expressed within hepatocytes, Kupffer cells, endothelial cells and Ito cells. Until recently, products of the HO reaction were regarded as potentially toxic waste destined only for excretion. However, this view is changing as evidence suggests that HO activity plays an important protective role against cellular stress during inflammatory diseases. Biliverdin is reduced to bilirubin, which has been shown to possess potent antioxidative properties. CO, which is produced in equimolar concentrations to biliverdin and ferrous iron during heme oxidation by HO, may function as a second messenger stimulating soluble guanylate cyclase (sGC) and regulating vascular tone in combination with the free radical gas NO. CO may also possess anti-inflammatory properties such as the capacity to inhibit platelet aggregation, or the expression of pro-inflammatory cytokines. Recently, it has been shown that CO regulates bile formation and bile flow. We review the functional role of HO in liver and the potential application of HO-1 in therapeutic approaches to the treatment of inflammation.     

The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity

Acetaminophen (AAP) overdose can cause severe hepatotoxicity and even liver failure in experimental animals and humans. Despite substantial efforts over the last 30 years, the mechanism of AAP-induced liver cell injury is still not completely understood. It is widely accepted that the injury process is initiated by the metabolism of AAP to a reactive metabolite, which first depletes glutathione and then binds to cellular proteins including a number of mitochondrial proteins. One consequence of this process may be the observed inhibition of mitochondrial respiration, ATP depletion and mitochondrial oxidant stress. In the presence of sufficient vitamin E, reactive oxygen formation does not induce severe lipid peroxidation but the superoxide reacts with nitric oxide to form peroxynitrite, a powerful oxidant and nitrating agent. Peroxynitrite can modify cellular macromolecules and may aggravate mitochondrial dysfunction and ATP depletion leading to cellular oncotic necrosis in hepatocytes and sinusoidal endothelial cells. Thus, we hypothesize that reactive metabolite formation and protein binding initiate the injury process, which may be then propagated and amplified by mitochondrial dysfunction and peroxynitrite formation. This concept also reconciles many of the controversial findings of the past and provides a viable hypothesis for the mechanism of hepatocellular injury after AAP overdose.     

Hemopexin and aryl hydrocarbon hydroxylase induction in the mouse and in cell culture

Plasma hemopexin levels in mice treated with polycyclic aromatic compounds such as 3-methylcholanthrene or β-naphthoflavone are increased 71–160 per cent in three inbred strains known to be “responsive” at the Ah locus (C57BL/6N. C3H/HeN and Balb/cAnN) and ranged from 14 to 60 per cent in three inbred strains known to be “nonresponsive” at the Ah locus (DBA/2N, NZB/BLN and NZW/BLN). Phenobarbital treatment causes increases in plasma hemopexin of similar magnitude in both aromatic hydrocarbon responsive and nonresponsive mice. In F₂ progeny from crosses between the C57BL/6N and NZW/BLN progenitor strains, a correlation between inducible hepatic hydroxylase and plasma hemopexin concentrations could not be demonstrated, principally because of the small magnitude in hemopexin induction. Data in this study show that plasma hemopexin rises later than the initiation of the aryl hydrocarbon hydroxylase induction process and, therefore, presumably in response to increased heme synthesis—whether it is the genetically regulated hemoprotein P₁-450 induction by polycyclic aromatic compounds or hemoprotein P450 induction by pheno-barbital. An association between hemopexin and hydroxylase induction is not seen in H-4-II-E Reuber rat hepatoma cultures. Hemopexin synthesis and secretion into the growth medium occurs in H-4-II-E cells but is not detectable in HTC or Hepa-1 cultures. The rate of hemopexin appearance in the culture medium is the same in bcnz[a]anthracene-treated as in control H-4-II-E cells, whereas the hydroxylase induction occurs primarily in benz[a]anthracene-treated and not in control cultures. Daily changes in growth medium result in greater intracellular hemopexin levels and a greater rate of hemopexin secretion into the medium, compared with cells whose medium was changed less frequently or not at all.     

Increased aryl hydrocarbon hydroxylase activity in hepatic microsomes from streptozotocin-diabetic female rats.

In streptozotocin-induced diabetic male rats, hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activity was depressed to less than control values, but was increased in microsomes from diabetic female rats. Insulin treatment of diabetic animals returned the altered AHH activity to control values in both sexes of rats. 2. Hepatic microsomal AHH activity was increased over control values in both sexes of diabetic mice. 3. Protection of female rats from the diabetogenic effects of streptozotocin by nicotinamide pretreatment also prevented the increase in AHH activity observed in unprotected animals. 4. Treatment of control and diabetic female rats with 3-methylcholanthrene resulted in larger increases in hepatic AHH activity in control animals, but similar increases in cytochrome P-448 content occurred in both treatment groups. 5. Differential stimulatory or inhibitory effects on AHH activity were observed after the addition of SKF 525-A, metyrapone, and rotenone to hepatic microsomes in vitro from control and diabetic female rats. However, similar stimulatory responses in AHH activity were observed after addition of alpha-naphthoflavone to microsomes from both treatment groups.     

Therapeutic role of telmisartan against acetaminophen hepatotoxicity in mice

The therapeutic potential of telmisartan was investigated in mice exposed to acute hepatotoxicity induced by a single dose of acetaminophen (500mg/kg, p.o.). Telmisartan treatment (two i.p. injections, 10mg/kg, each) was given at 1 and 12h following acetaminophen administration. Telmisartan significantly reduced the level of serum alanine aminotransferase, and suppressed lipid peroxidation, prevented the depletion of the antioxidant defenses (reduced glutathione level, and catalase and superoxide dismutase activities), and attenuated the elevation of nitric oxide resulted from acetaminophen administration. Also, telmisartan ameliorated the histopathological liver tissue damage induced by acetaminophen. Immunohistochemical analysis revealed that telmisartan significantly decreased the expression of inducible nitric oxide synthase, tumor necrosis factor-α, cyclooxygenase-2, nuclear factor-κB and caspase-3 in liver tissue of mice received acetaminophen overdose. In conclusion, telmisartan can be considered as a potential therapeutic option to protect against acute acetaminophen hepatotoxicity commonly encountered in clinical practice.     

血红素氧化酶在软骨藻酸对细胞DNA损伤中的作用

目的　　研究血红素氧化酶 (hemeoxygenase ,HO)系统在软骨藻酸 (domoicacid ,DA)对H4细胞DNA损伤中的作用。方法　应用单细胞凝胶电泳法 (SCGE)分别测定 0、0 0 64、0 64和 6 4μmol/LDA单独及与HO系统的活性阻断剂ZnPP 9(10 - 5mol/L)联合作用于H4细胞 6、12、2 4和 48h后彗星拖尾细胞率及拖尾尾长。结果　彗星拖尾细胞率 :中剂量和高剂量组各时间点均比对照组高 ,差异有显著性 (P <0 0 5 )。中剂量 +阻断剂组各时间点均相应高于中剂量组 ,差异有显著性 (P <0 0 5 )。彗星拖尾尾长 :中剂量组 12、2 4和 48h比对照组长 ,2 4、48h比低剂量组长 ,差异均有显著性 (P<0 0 5 )。高剂量组各时间点均比对照组、低剂量组和中剂量组长 ,差异有显著性 (P <0 0 5 )。高剂量 +阻断剂组和中剂量 +阻断剂组 2 4、48h分别高于高剂量和中剂量组 ,差异有显著性 (P <0 0 5 )。尾长 时间作Regression分析 ,各剂量组r差异值均有显著性 (P <0 0 1)。结论　软骨藻酸能诱导H4细胞DNA损伤 ,HO系统对这种损伤有一定的保护作用。