Potentiation of acetaminophen hepatotoxicity by doxapram in mouse primary cultured hepatocytes

The augmentation by doxapram (DOP) of the reduction in viability and of the apoptosis of cells induced by acetaminophen (AA) was examined in mouse primary cultured hepatocytes. Loss of viability on exposure to AA and/or DOP in cultured hepatocytes was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and the apoptosis of cultured hepatocytes was detected by nuclear morphologic observation and from a ladder-like DNA fragmentation pattern. The combination of AA (5 mM) and DOP (10, 20, 50 or 100 microM) potentiated the reduction in cell viability and increased the oxidative stress. Hepatocytes exposed for 24 h to AA (5 mM) plus DOP (100 microM), showed atrophy of nuclei, including chromatin condensation and a ladder-like DNA fragmentation pattern, characteristic of apoptosis. Benzyl-oxycarbonyl-Asp-CH2OC (O)-2,6-dichlorobenzene (Z-Asp-CH2-DCB, 50 microM), an inhibitor for caspases, improved the viability and ladder-like DNA fragmentation in cells exposed to DOP (200 or 500 microM) alone or AA ( 5 mM) plus DOP (100 microM). However, loss of viability on exposure to a high concentration of AA (10 mM) and ladder-like DNA fragmentation were not affected by Z-Asp-CH2-DCB. These results indicated that the synergistic increase in oxidative stress, activation of caspases and DNA fragmentation induced by DOP potentiated the hepatotoxicity of AA.     

Phenobarbital and phenytoin increased acetaminophen hepatotoxicity due to inhibition of UDP-glucuronosyltransferases in cultured human hepatocytes.

Here we present a preclinical model to assess drug-drug interactions due to inhibition of glucuronidation. Treatment with the antiepileptics phenobarbital (PB) or phenytoin (PH) has been associated with increased incidence of acetaminophen (APAP) hepatotoxicity in patients. In human hepatocytes, we found that the toxicity of APAP (5 mM) was increased by simultaneous treatment with phenobarbital (2 mM) or phenytoin (0.2 mM). In contrast, pretreatment with PB for 48 h prior to APAP treatment did not increase APAP toxicity unless both drugs were present simultaneously. Cells treated with APAP in combination with PB or PH experienced decreases in protein synthesis as early as 1 h, ultrastructural changes by 24 h, and release of liver enzymes by 48 h. Toxicity correlated with inhibition of APAP glucuronidation. PB or PH also inhibited APAP glucuronidation in rat and human liver microsomes and expressed human UGT1A6, 1A9, and 2B15. As with intact hepatocytes, PB and PH were neither hydroxylated nor glucuronidated, suggesting the direct inhibition of UGTs. Our findings suggest that, in multiple drug therapy, an inhibitory complex between UGT and one of the drugs can lead to decreased glucuronidation and increased systemic exposure and toxicity of a coadministered drug.     

酮康唑对原代培养大鼠肝细胞的毒性作用及其机制

目的观察酮康唑（ketoconazole，KCZ）对原代培养大鼠肝细胞的毒性效应，探讨其可能的毒性机制。方法采用胶原酶二步灌流法分离制备雄性SD大鼠肝细胞，进行原代培养。量效关系研究中KCZ设56、75、94、113和188μmol／L，染毒时间4h；时效关系研究中KCZ设188μmol／L，染毒时间分别为0．5、1、2和4h；同时设溶剂对照。检测染毒后肝细胞活力、胞内LDH泄漏情况以及巯基状态的变化。结果（1）量效关系研究中，随着KCZ剂量的升高，肝细胞活力逐渐下降，培养液中LDH活力逐渐增高，肝细胞内巯基也呈下降趋势，上述指标均有明确的剂量．反应关系（γLDH=0．906，P〈0．01）；（2）时效关系研究中，KCZ在188μmol／L剂量下染毒后肝细胞活力呈时间依赖性下降；培养液中LDH活力随染毒时间延长逐渐增高；细胞内巯基出现相应性下降。结论KCZ可以导致肝细胞活力下降，胞内LDH泄漏，巯基含量减少，并有明确的量效和时效关系，提示酮康唑对肝细胞的毒作用可能与细胞内巯基状态改变有关。     

Interspecies differences in acetaminophen sensitivity of human, rat, and mouse primary hepatocytes

Most of the experiments studying acetaminophen (APAP) induced hepatotoxicity were performed using moue as model specie, right because its high sensitivity. While the toxic responses can be called forth easily in mice, the human relevancy of these results is questionable. In this study human, rat, and mouse primary hepatocytes were treated with increasing concentrations of APAP, and cell viability was measured by MTT cytotoxicity assay. Pronounced interspecies differences were obtained in cell viability following 24 h of APAP treatment starting at 24 h after seeding (EC₅₀: 3.8 mM, 7.6 mM, and 28.2 mM, in mouse, rat, and human hepatocyte culture, respectively). The longer time of culturing highly increased the resistance of hepatocytes of all species investigated. In rat hepatocyte culture EC₅₀ values were 6.0 mM, 12.5 mM, and 18.8 mM, when starting APAP treatment after 24, 48, and 72 h of seeding. Although N-acetylbenzoquinoneimine, a minor metabolite of APAP, which is mainly formed by CYP2E1 at high APAP concentration in every species studied, is thought to initiate the toxic processes, no correlation was found between CYP2E1 activities and hepatocyte sensitivity of different species. We conclude that the toxicity induced by APAP overdose highly depends on the animal model applied.     

Characterization of hydrogen peroxide-induced apoptosis in mouse primary cultured hepatocytes

The influence of oxidative stress by hydrogen peroxide (H2O2) was examined in mouse primary cultured hepatocytes. A change in morphology was observed in hepatocytes incubated for 30 min in saline A containing H2O2. The percentage of dead cells, as measured by the fluorescence method, was increased in a dose-dependent manner. In addition, a ladder-like DNA fragmentation pattern was detected by agarose gel electrophoresis 1 h after exposure to 3 mM H2O2. This phenomenon was prolonged for 24 h. Hydrogen peroxide-induced cell viability reduction and DNA fragmentation were dose-dependently protected by the addition of antioxidants (N-acetylcysteine, L-ascorbic acid), a metal-chelator (1,10-phenanthroline), iron-chelator (deferoxamine) and intracellular calcium ion chelator (quin 2-AM). No influence, however, was detected by endonuclease inhibitors (zinc, aurintricarboxylic acid) and poly (ADP-ribose) polymerase inhibitors (3-aminobenzamide, theophylline). These results following H2O2-induced cell viability reduction suggested that oxidative stress by H2O2 itself or H2O2-derived changes involved in ferrous or intracellular calcium ions resulted in apoptosis in mouse primary cultured hepatocytes. These phenomena are not likely to be associated with endonuclease or poly (ADP-ribose) polymerase.     

Protective effect of dieckol against chemical hypoxia-induced cytotoxicity in primary cultured mouse hepatocytes

Hepatic ischemic injury is a major complication arising from liver surgery, transplantation, or other ischemic diseases, and both reactive oxygen species (ROS) and pro-inflammatory mediators play the role of key mediators in hepatic ischemic injury. In this study, we examined the effect of dieckol in chemical hypoxia-induced injury in mouse hepatocytes. Cell viability was significantly decreased after treatment with cobalt chloride (CoCl₂), a well-known hypoxia mimetic agent in a time- and dose-dependent manner. Pretreatment with dieckol before exposure to CoCl₂ significantly attenuated the CoCl₂-induced decrease of cell viability. Additionally, pretreatment with dieckol potentiated the CoCl₂-induced decrease of Bcl-2 expression and attenuated the CoCl₂-induced increase in the expression of Bax and caspase-3. Treatment with CoCl₂ resulted in an increased intracellular ROS generation, which is inhibited by dieckol or N-acetyl cysteine (NAC, a ROS scavenger), and p38 MAPK phosphorylation, which is also blocked by dieckol or NAC. In addition, dieckol and SB203580 (p38 MAPK inhibitor) increased the CoCl₂-induced decrease of Bcl-2 expression and decreased the CoCl₂-induced increase of Bax and caspase-3 expressions. CoCl₂-induced decrease of cell viability was attenuated by pretreatment with dieckol, NAC, and SB203580. Furthermore, dieckol attenuated CoCl₂-induced COX-2 expression. Similar to the effect of dieckol, NAC also blocked CoCl₂-induced COX-2 expression. Additionally, CoCl₂-induced decrease of cell viability was attenuated not only by dieckol and NAC but also by NS-398 (a selective COX-2 inhibitor). In conclusion, dieckol protects primary cultured mouse hepatocytes against CoCl₂-induced cell injury through inhibition of ROS-activated p38 MAPK and COX-2 pathway.     

Cytotoxicity of acetaminophen and papaverine in primary cultures of rat hepatocytes

Primary cultures of hepatocytes obtained from postnatal Sprague-Dawley rats were grown in arginine-deficient medium to inhibit fibroblastic overgrowth and to selectively isolate relatively pure cultures of parenchymal hepatocytes. The cultures were grown on collagen-coated lens paper which freely floated in the medium and thus facilitated the exchange of nutrients and waste products between the cells and the culture medium. This system of primary hepatocytes was used to study the cytotoxicity of acetaminophen, papaverine, nitrofurantoin, and sodium salicylate. Cytotoxicity was monitored by measurement of leakage of intracellular enzymes into the culture medium: argininosuccinate lyase (ASAL), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and acid phosphatase (AP). Cultures were treated with each of the agents in concentrations ranging from 5 × 10^?6 to 5 × 10^?3m and for durations from 1 to 24 hr. ASAL was found to be most sensitive in predicting early cell injury and AP the least sensitive. GPT, GOT, and LDH were intermittent in value and equally sensitive in evaluating cytotoxicity. No sign of cytotoxicity was observed within 3 hr of treating cultures with different concentrations of acetaminophen and papaverine. At 6 hr a significant increase in leakage of enzymes into the culture medium was observed relative to controls. After the first 3 hr the hepatotoxic agents demonstrated a time- and dose-dependence of cytotoxicity. Treatment of the cultures with acetaminophen and papaverine (5 × 10^?5m) for 12 hr resulted in ASAL leakage that was 360 and 260% of control values, respectively. In contrast, nitrofurantoin and sodium salicylate were only minimally toxic to the cultured cells. The time lag observed in the cytotoxicity of acetaminophen and papaverine may be related to the period required for metabolite(s) accumulation and saturation of detoxifying systems of the cultured liver cells.     

Exposure to hydrogen peroxide induces cell death via apoptosis in primary cultured mouse hepatocytes

To investigate whether direct oxidant damage induces hepatotoxicity via an apoptotic cell suicide pathway, we exposed primary cultured mouse hepatocytes to pro-oxidant hydrogen peroxide. We demonstrate that brief exposure to a concentration of hydrogen peroxide (3 mM) can induce hepatocyte cell death via apoptosis as shown by toxicity assays, specific DNA staining, and the appearance of DNA laddering on agarose gels. When hepatocytes were treated with N-acetylcysteine 15 min prior to hydrogen peroxide exposure, the cells were found to be protected from cytotoxicity and apoptosis. These results suggest that direct oxidative injury serves as a general trigger for apoptosis in the liver, and that other apoptotic stimuli, such as exposure to acetaminophen, also involve oxidative injury. Hydrogen peroxidase-induced apoptosis may serve as a valuable model for further studies of apoptosis in the liver.     

Protective effect of S-adenosyl-L-methionine against CCl4-induced hepatotoxicity in cultured hepatocytes

Effect of S-adenosyl-L-methionine disulfate tosylate salt (SAMe-ST) and L-methionine (L-Met) on primary cultured rat hepatocytes were studied. In cultured hepatocytes treated with CCl4, SAMe-ST and L-Met suppressed the decrease in urea-nitrogen secretion as well as the leakages of GOT and GPT. The membrane-protective action of these two compounds was verified by the histological data. Failure of SAMe-ST to counteract CCl4-induced reduction of radioactive leucine incorporation into the trichloroacetic acid-insoluble materials in hepatocytes indicates that the observed effects of SAMe-ST or L-Met do not involve acceleration of protein synthesis. The present results indicate that SAMe-ST remarkably protects hepatocytes from CCl4-induced hepatotoxicity, probably by either changing the structure or compositions of membrane phospholipids or by modifying the interaction of CCl4 with the intracellular drug-metabolizing enzyme systems.     

The potential use of cultured hepatocytes in predicting the hepatotoxicity of xenobiotics

1. A protocol is proposed for screening for hepatotoxicity of xenobiotics in vitro in which hepatocytes exposed to the compounds are evaluated for both cytotoxic and metabolic effects. Four established hepatotoxins have been studied. 2. alpha-Amanitin at 1.5 pg/mg cell protein inhibited RNA synthesis by 93% and reduced albumin synthesis to 56% of the control after 13 h treatment. 3. D-Galactosamine at 40 microM inhibited glycogen synthesis by 31%, glucuronidation of p-nitrophenol by 13% and albumin synthesis by 10%, and produced an increase in cytosolic enzyme leakage. 4. Thioacetamide decreased ureogenesis after 24 h of treatment at 230 microM (31% inhibition) and after 48 h at 2.3 microM (25% inhibition). 5. Ultrastructural alterations of hepatocytes were found after 48 h exposure to 1 mM acetaminophen and were preceded by extensive leakage of the enzymes GOT and LDH. Membrane damage was observed after 24 h exposure to 0.1 mM acetaminophen.     

Disulfiram prevents acetaminophen hepatotoxicity in rats

Hepatic necrosis due to an oral acetaminophen overdose (4.25 g/kg b.wt.) was prevented by pretreatment with disulfiram 100 mg/kg, given for 3 weeks or as a single dose. Twenty-four hours after acetaminophen the impairment of hepatic function, measured as prothrombin index, and the depletion of hepatic glutathione were prevented. Hepatic cytochrome P-450 levels were unchanged but cytochrome P-450 mediated p-nitroanisole demethylation was reduced by disulfiram pretreatment. Disulfiram pretreatment reduced 24 hour urinary excretion of acetaminophen-mercapturate and- cysteine while excretion of -sulfate and -glucuronide was unchanged. After 72 hours acetaminophen induced hepatic necrosis were prevented. Identical observations were made in animals pretreated with disulfiram for 3 weeks. Five hours after acetaminophen overdose its irreversible binding to hepatic proteins was not changed. After 24 hours, however, it was increased in animals pretreated with a single disulfiram dose and unchanged in animals pretreated for 3 weeks. The protective mechanism of disulfiram after acetaminophen overdose is not mediated via a change in overall irreversible binding of acetaminophen to hepatic protein.     

Level of cytosolic free calcium during acetaminophen toxicity in mouse hepatocytes.

It has been suggested that elevated cytosolic free calcium plays a key role in acetaminophen-induced cell death. The present study has examined the effect of a toxic concentration of acetaminophen on cytosolic free calcium in single mouse hepatocytes, using the dye fura-2 and video imaging fluorescence microscopy. Cytosolic free calcium was calculated from the ratio of emitted fluorescence at greater than 475 nm produced by excitation at 340 and 380 nm, using a double-intensified silicon target camera and digital image processing. In the presence of 5 mM acetaminophen, cell death did not occur for 2 hr, but the toxic lesion that ultimately killed the cells occurred as early as 1 hr. If cytosolic free calcium plays an important role in these toxic events, it would be expected to increase during this period. However, during a 2-hr exposure, cytosolic free calcium concentration in cells exposed to acetaminophen was not different from control. In hepatocytes incubated for longer than 2 hr, the calcium concentration increased shortly before loss of cell viability (i.e., as a late event), consistent with an influx of calcium through a damaged cell membrane. This late increase in calcium occurred well after the appearance of cell surface blebs. The data suggest that there is no sustained change in cytosolic free calcium in acetaminophen injury either before or during the time when irreversible toxic events occur in hepatocytes.     

大鼠肝实质细胞原代培养模型的研究及其功能鉴定

目的:为肝细胞功能及相关的研究提供一种客观的、简便的大鼠肝细胞原代培养模型,并鉴定肝细胞的功能。方法:在传统的两步灌流法的基础上进行改良分离培养肝实质细胞;流式细胞术测定原代肝细胞的细胞周期和细胞凋亡情况;SRB法测定不同培养基对原代肝细胞的生长和功能的影响。结果:分离得到的肝细胞成活率可达90%以上,纯度可达95%以上;流式细胞术结果表明原代肝细胞大多处于G0/G1期,且基本无凋亡;SRB法和白蛋白分泌量检测结果显示,低糖DMEM组原代肝细胞生长和白蛋白分泌功能在短期内(1~5 d)与高糖DMEM组没有明显差异;RPMI1640组肝细胞的生长和功能则明显低于前两组(P<0.05)。结论:体外培养的肝细胞活力和纯度均较高,体外培养后细胞功能正常,是一种实用的体外研究肝细胞良好的细胞学模型。     

Gene expression in primary cultured mouse hepatocytes with a cationic liposomal vector, TFL-3: comparison with rat hepatocytes

We recently reported that a cationic liposomal vector, TFL-3, could be used to achieve significant gene expression in primary cultured rat hepatocytes (Nguyen et al., Biol. Pharm. Bull., 26, 880-885 (2003)). A combination of hepatocyte transplantation and hepatocyte-targeted gene transfer represents a potentially important strategy for expanding treatment options for liver disease. A widely applied approach to support cross-species is necessary before human applications can be realized. Therefore, in this study, we examined the utility of TFL-3 in another species of rodent hepatocytes, namely mouse hepatocytes. Gene expression in mouse hepatocytes by TFL-3 was successful and the level was higher than those in rat hepatocytes that we recently reported on. Interestingly, it appears that both the degree and rate of gene expression were dependent on the incubation time prior to lipofection as well as on the density of cells per dish, but these parameters were independent of the amount of pDNA associated with the cells. These significantly suggest that the culture time prior to and following lipofection, which are related to the biological condition of the cells, may be one of major factors that affect gene expression in hepatocytes and non- or less dividing cells.     

Mitochondrial dysfunction in paracetamol hepatotoxicity: in vitro studies in isolated mouse hepatocytes

The effect of paracetamol intoxication on mitochondrial function was studied in isolated mouse hepatocytes. Inhibition of cellular respiration as well as a lowering of cellular ATP contents and ATP/ADP ratios was associated with exposure to toxic concentrations of paracetamol. Significantly, inhibition of 3-hydroxybutyrate- and lactate/pyruvate-supported respiration, as well as the reduction in cellular ATP levels and ATP/ADP ratios, preceded the appearance of plasma membrane damage, as assessed by LDH leakage. N-Acetylcysteine reduced the extent of plasma membrane damage induced by paracetamol and protected against the impairment of cellular respiration. This suggests that respiratory dysfunction was a consequence of the oxidation of paracetamol to its reactive metabolite within the liver cell. These findings indicate that paracetamol toxicity results in an impairment of mitochondrial function which precedes the loss of plasma membrane integrity.     

Selective alterations in the patterns of newly synthesized proteins by acetaminophen and its dimethylated analogues in primary cultures of mouse hepatocytes.

Alterations in protein synthesis following exposure to and recovery from hepatotoxic doses of acetaminophen (APAP) and its analogues, 3,5-dimethyl acetaminophen (3,5-DMA) and 2,6-dimethyl acetaminophen (2,6-DMA), were investigated in primary cultures of mouse hepatocytes. The rates of protein synthesis decreased within 4 hr after administration of 10 mM APAP and occurred after significant depletion of intracellular glutathione and covalent binding of APAP to proteins, but preceded the leakage of lactate dehydrogenase into the media. The inhibition of protein synthesis was reversible only if APAP exposure did not exceed 8 hr. Electrophoretic analysis of 35S-labeled proteins by one-dimensional SDS-PAGE revealed two consistent alterations in the patterns of newly synthesized proteins. First was a progressive diminution in the de novo synthesis of a protein migrating at approximately 58 kDa (p58). This was observed with APAP (10 mM) and 3,5-DMA (5 mM) but not with 2,6-DMA (10 mM). If exposure to APAP exceeded 8 hr, the biosynthesis of this protein was not only further decreased but was also no longer detectable during the recovery period. The second major alteration was an increase in the relative rate of biosynthesis of a 32-kDa protein (p32) following exposure and recovery from APAP and 3,5-DMA but not 2,6-DMA. Exposure to heme or arsenite induced the synthesis of a protein of similar molecular weight but did not result in the inhibition of p58 biosynthesis. The fact that the reactive metabolites of both APAP and 3,5-DMA, but not 2,6-DMA, possess oxidative properties suggests that the alterations in the synthesis of p32 and p58 may be related to an oxidative component induced by these compounds.     

Cellular disposition of arabinogalactan in primary cultured rat hepatocytes

To characterize a targeting property of arabinogalactan (AG) as a carrier to the liver, we examined cellular disposition, such as binding and internalization in primary cultured rat hepatocytes, comparing them to those of asialofetuin (AF). A tyramine derivative of AG was synthesized to allow labeling with ¹²⁵I. Binding of AG to the cells was concentration-dependent and saturable. The number of binding sites (n) of AG on the cell surface was 4.0 × 10⁵ ± 0.1 × 10⁵ sites per cell which was about similar to that of AF. The value of K_a of AG was 2.2 × 10⁸ ± 0.1 × 10⁸ M⁻¹ being seven-fold higher than that of AF. The binding of AG was competitively inhibited by AF and was decreased by calcium depletion. These results indicate that AG can bind strongly to hepatocytes probably through the recognition by the asialoglycoprotein receptor (ASGP-R). Both ¹²⁵I-labeled AG and fluorescein-labeled AG were internalized into the cells. The rate of internalization of AG was faster than that of AF, indicating that AG is effectively endocytosed. Microscopic observations showed that FITC labeled AG accumulated in granules within the primary cultured rat hepatocytes. Subcellular fractionation indicated that the internalized AG was mainly associated with the lysosomal fraction. However, the internalized AG seemed to remain intact in the hepatocytes. In conclusion, we found that AG is effectively internalized in primary cultured rat hepatocytes. Although AG seems a good candidate for targeting to the liver due to its high affinity binding and rapid internalization, it remains to be established whether the apparent lack of biodegradation will result in cytotoxic effects at chronic administration in vivo.     

Autoprotection against acetaminophen toxicity in cultured rat hepatocytes: the effect of pretreatment and growth factors

To evaluate the effect of acetaminophen pretreatment and growth factors on acetaminophen hepatotoxicity in cultured rat hepatocytes, rat hepatocytes in primary culture were exposed to acetaminophen 8 mM after pretreatment with either acetaminophen 1 mM, treatment with growth factors (EGF and HGF), or no treatment. Growth response was measured by changes in DNA, [3H]thymidine incorporation and mRNA of growth related proteins, cell damage by leakage of LDH to the medium and changes in ATP, and protection against toxicity by changes in glutathione, cytochrome p450 and the expression of glutathione-S-transferase and Cyp1A2. Pretreatment with acetaminophen induced growth response, weaker than that of growth factors, but pretreatment and growth factors reduced cell damage equally effectively. Glutathione and glutathione-S-transferase increased more by growth factors than by pretreatment, but both conditions reduced Cyp1A2 to near zero. Pretreatment and growth factors protect against acetaminophen toxicity by suppressing the expression of Cyp1A2, thereby reducing the production of the intermediate N-acetyl-p-benzoquinone imine (NAPQI). Suppression of Cyp1A2 expression by pretreatment is assumed to be due to a growth-stimulating effect of low concentrations of acetaminophen.     

Role of UDPGA flux in acetaminophen clearance and hepatotoxicity

Factors which determine the acetaminophen glucuronidation capacity in the male rat have been examined. Conditions previously shown to increase (streptozotocin diabetes) or decrease (a 24 h fast) the glucuronidation capacity in vivo did not alter the microsomal glucuronyl transferase activity, indicating that the amount of enzyme is not rate-limiting. Acetaminophen caused a rapid depletion of hepatic levels of the co-substrate, UDPGA; both the extent of depletion and the time required for recovery back to pre-drug levels were dependent on the dose of acetaminophen administered. The amount of UPDGA required for the glucuronidation of a therapeutic dose was nearly equal to the total content of UDPGA in the liver; after a toxic dose, the UDPGA demand was over 100-fold greater than the normal basal level. It is concluded that the glucuronidation capacity of the animals is determined by their capacity to synthesize UDPGA, which in turn is dependent on flux through the glucuronic acid pathway.