Cytotoxic effects of hydroxylated fullerenes on isolated rat hepatocytes via mitochondrial dysfunction

The cytotoxic effects of hydroxylated fullerenes, also termed fullerenols or fullerols [C₆₀(OH) _n ], which are known nanomaterials and water-soluble fullerene derivatives, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to C₆₀(OH)₂₄ caused not only concentration (0–0.25 mM)- and time (0–3 h)-dependent cell death accompanied by the formation of cell blebs, loss of cellular ATP, reduced glutathione (GSH), and protein thiol levels, but also the accumulation of glutathione disulfide and malondialdehyde, indicating lipid peroxidation. Of the other analogues examined, the cytotoxic effects of C₆₀(OH)₁₂ and fullerene C₆₀ at a concentration of 0.125 mM were less than those of C₆₀(OH)₂₄. The loss of mitochondrial membrane potential and generation of oxygen radical species in hepatocytes incubated with C₆₀(OH)₂₄ were greater than those with C₆₀(OH)₁₂ and fullerene C₆₀. In the oxygen consumption of mitochondria isolated from rat liver, the ratios of state-3/state-4 respiration were more markedly decreased by C₆₀(OH)₂₄ and C₆₀(OH)₁₂ compared with C₆₀. In addition, C₆₀(OH)₂₄ and C₆₀(OH)₁₂ resulted in the induction of the mitochondrial permeability transition (MPT), and the effects of C₆₀(OH)₁₂ were less than those of C₆₀(OH)₂₄. Taken collectively, these results indicate that (a) mitochondria are target organelles for fullerenols, which elicit cytotoxicity through mitochondrial failure related to the induction of the MPT, mitochondrial depolarization, and inhibition of ATP synthesis in the early stage and subsequently oxidation of GSH and protein thiols, and lipid peroxidation through oxidative stress at a later stage; and (b) the toxic effects of fullerenols may depend on the number of hydroxyl groups participating in fullerene in rat hepatocytes.     

AMELIORIATION OF CHEMICALLY-INDUCED HEPATOCYTE INJURY BY CYCLOSPORINE A

Cyclosporine A, beside its current applications, possesses potential hepatoprotective effects. This study was directed to investigate the effect of Cyclosporine A pretreatment on hepatic injury due to carbon tetrachloride (CCl₄) and -galactosamine. Rats were injected by two successive doses of Cyclosporine A (5mgkg⁻¹day⁻¹). Six hours after the second dose, 1mlkg⁻¹of CCl₄was administered i.p. Effects associated with Cyclosporine A pretreatment were examined by using isolated hepatocytes and hepatocytes that were immobilized and continuously perfused. -Galactosamine (5m ) was added directly to the perfusion medium. After isolation, hepatocytes were examined histologically by light and electron microscopy, immobilized and perfused for further metabolic functional activity evaluation. Cyclosporine A pretreatmentin vivoproduced hepatoameliorative effects of various degrees which were statistically significant as manifested by: (1) an increased trypan blue exclusion after CCl₄; (2) an improved ureagenesis after CCl₄; (3) a reduction in the lipid droplets accumulation in the cytoplasm produced by CCl₄administration; (4) well preserved cytoplasmic organelles as mitochondria, endoplasmic reticulum ER, nuclear chromatin structures that were altered by CCl₄; and (5) an increased hepatocytes survival in the agarose gel matrix, reduction of LD leakage and improvement of ureagenesis after -galactosamine addition to the perfusion medium. The beneficial effect of Cyclosporine A pretreatment in modifying hepatotoxicity of chemical insults merits further studies.     

Lipid peroxidation and cell damage in isolated hepatocytes due to bromotrichloromethane

Small amounts of malondialdehyde (MDA, about 0.5 nmol/10⁶ cells) were produced during 120-min incubation of isolated rat hepatocytes indicating lipid peroxidation in the cells. Trypan blue uptake and lactate dehydrogenase release followed MDA formation. MDA increased to about 3.5 nmol/10⁶ cells in the presence of 2 mm bromotrichloromethane (CBrCl₃). A parallelism of MDA production with trypan blue uptake and lactate dehydrogenase release was observed. The MDA production depended on the CBrCl₃ concentration in the incubation medium. A higher oxygen consumption of cells incubated in the presence of CBrCl₃ was detectable compared to controls. During incubation no increase in glutamate dehydrogenase release could be observed, even if 2 mm CBrCl₃ was present. This led us to conclude that lipid peroxidation induced by CBrCl₃ destroys the plasma membrane of the hepatocytes without seriously damaging mitochondria.     

Propionyl- -Carnitine as protector against adriamycin-induced Cardiomyopathy

Propionyl- -carnitine (PLC) is a naturally occurring compound that has been considered for the treatment of many forms of cardiomyopathies. In this study, the possible mechanisms whereby PLC could protect against adriamycin (ADR)-induced cardiomyopathy were carried out. Administration of ADR (3 mg kg⁻¹i.p., every other day over a period of 2 weeks) resulted in a significant two-fold increase in serum levels of creatine phosphokinase, lactate dehydrogenase and glutamic oxaloacetic transaminase, whereas daily administration of PLC (250 mg kg⁻¹, i.p. for 2 weeks) induced non-significant change. Daily administration of PLC to ADR-treated rats resulted in complete reversal of ADR-induced increase in cardiac enzymes except lactate dehydrogenase which was only reversed by 66%. In cardiac tissue homogenate, ADR caused a significant 53% increase in malonedialdehyde (MDA) and a significant 50% decrease in reduced glutathione (GSH) levels, whereas PLC induced a significant 33% decrease in MDA and a significant 41% increase in GSH levels. Daily administration of PLC to ADR-treated rats completely reversed the increase in MDA and the decrease in GSH induced by ADR to the normal levels. In rat heart mitochondria isolated 24 h after the last dose, ADR induced a significant 48% and 42% decrease in¹⁴CO₂released from the oxidation of [1-¹⁴C]palmitoyl-CoA and [1-¹⁴C]palmitoylcarnitine, respectively, whereas PLC resulted in a significant 66% and 54% increase in the oxidation of both substrates, respectively. Interestingly, administration of PLC to ADR-treated rats resulted in complete recovery of the ADR-induced decrease in the oxidation of both substrates. In addition, in rat heart mitochondria, the oxidation of [1-¹⁴C]pyruvate, [1-¹⁴C]pyruvate and [1-¹⁴C]octanoate were not affected by ADR and/or PLC treatment. Moreover, ADR caused severe histopathological lesions manifested as toxic myocarditis which is protected by PLC. Worth mentioning is that PLC had no effect on the antitumour activity of ADR in solid Ehrlich carcinoma. Results from this study suggest that: (1) in the heart, PLC therapy completely protects against ADR-induced inhibition of mitochondrial β -oxidation of long-chain fatty acids; (2) PLC has and/or induces a powerful antioxidant defense mechanism against ADR-induced lipid peroxidation of cardiac membranes; and finally (3) PLC has no effect on the antitumour activity of ADR.     

Selenite causes cytotoxicity in rainbow trout (Oncorhynchus mykiss) hepatocytes by inducing oxidative stress

Selenium is an essential micronutrient to freshwater fish, but can be very toxic at slightly above the threshold level. The liver is known to be the major site of selenium accumulation and metabolism in fish. Recent evidence from mammalian systems suggests that oxidative damage is an important mechanism of selenium toxicity; however this phenomenon has not been investigated in-depth in fish, either in vivo or in vitro. Therefore, the present study was designed to investigate whether selenium (as selenite) exposure causes cytotoxicity in fish by inducing oxidative stress. We used isolated hepatocytes in primary culture from freshwater rainbow trout (Oncorhynchus mykiss) as the model in vitro experimental system. The 24 h LD₅₀ of selenite to trout hepatocytes was found to be 587 μM. In order to evaluate the dose-dependent response patterns of various oxidative stress parameters, the trout hepatocytes were exposed to three different doses of selenite [50, 100 and 200 μM (corresponding to approximately 10%, 20% and 35% of 24 h LD₅₀)] in addition to control (0 μM of selenite) for 24 h. We observed an induction of catalase (CAT) and superoxide dismutase (SOD) activities at 50 and 100 μM of selenite exposure, but not at 200 μM, relative to the control. In contrast, the induction of glutathione peroxidase (GPx) activity was recorded at 100 and 200 μM exposure doses, but not at 50 μM. We also demonstrated that selenite exposure (100–200 μM) increased intracellular ROS formation at an early stage (2 h). The reduced to oxidized glutathione ratio (GSH:GSSG) decreased sharply with increasing selenite dose, indicating the loss of cellular reducing capacity. The cellular lipid peroxidation tended to increase with increasing selenite exposure dose, indicating the occurrence of membrane damage. A 20–40% decrease in cell viability was observed at 100 and 200 μM of selenite exposure. The increase in cell death was associated with a significant increase of caspase-3/7 activity, suggesting the induction of apoptosis. Overall, the present study suggests that selenite exposure at high level causes oxidative damage to trout hepatocytes, probably by inducing the imbalance of intracellular glutathione (GSH) redox.     

The effect of L-cysteine on the portion-selective uptake of cadmium in the renal proximal tubule

Cadmium (Cd), co-administered with an excess of L-cysteine, accumulates rapidly in the kidneys of the rat. After subcutaneous (s. c.) injection of 3 mol CdCl₂/kg body wt the concentrations of Cd in the blood and kidneys increase with the dose of cysteine over the range 0.06–5.0 mmol/kg body wt. At cysteine doses of less than 1.5 mmol/kg body wt the ratio of the concentrations of Cd in the outer medulla and cortex of the kidney remains the same as that after the injection of Cd alone. This ratio, however, is more than doubled at dose levels of 5–10 mmol cysteine/kg body wt. Hepatic uptake of Cd is unaffected by doses of cysteine below 1.5 mmol/kg body wt but decreases markedly at higher doses. In animals that are dosed simultaneously with 5 mmol cysteine/kg body wt, renal uptake of ¹⁰⁹Cd is known to occur in the straight segments of the proximal tubules. At a dose level of less than 1.5 mmol cysteine/kg body wt the present autoradiographical studies show that ¹⁰⁹Cd is taken up predominantly by the proximal convoluted tubules of the kidney cortex. At the critical dose level (1.5 mmol/kg body wt), cysteine decreases the retention of Cd at the s. c. injection site, but probably has little effect on the distribution of Cd between protein and other carrier molecules in the blood. This distribution, however, is altered at higher cysteine dose levels. It is suggested that, under the latter conditions, stable Cd-cysteine complexes are formed in the blood and are filtered readily through the glomeruli. These complexes are taken up in the kidney at the sites of cysteine reabsorption which, by studies with L-[³⁵S]-cysteine, are identified as the straight segments of the proximal tubules.     

S-nitrosyl glutathione-mediated hepatocyte cytotoxicity

1. The addition of n-butyl nitrite (BN) to isolated rat hepatocytes caused rapid S-nitrosyl glutathione (GSNO) formation, then a concomitant decrease in protein thiols, followed by a marked ATP depletion. Cytotoxic concentrations of BN also caused lipid peroxidation after a long lag period but before cytotoxicity ensued.

2. Prior glutathione (GSH) depletion protected hepatocytes against the BN-induced decrease in protein thiols, ATP depletion, lipid peroxidation and cytotoxicity. Thus cytotoxic effects were thought to be mediated via GSNO formed by reaction of BN with GSH, a reaction catalysed by the cytosolic fraction.

3. Cytotoxicity and lipid peroxidation, but not depletion of GSH, protein thiols or ATP, could be averted by the subsequent addition of antioxidants or the iron chelator, desferoxamine.

4. Addition of the thiol reductant, dithiothreitol to BN-treated hepatocytes restored GSH and protein thiols and also prevented cytotoxicity.     

Metabolism of acrylamide to glycidamide and their cytotoxicity in isolated rat hepatocytes: protective effects of GSH precursors

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in rodents. The recent discovery of AA at ppm levels in a wide variety of commonly consumed foods has energized research efforts worldwide to define toxicity and prevention. Metabolism and cytotoxicity of AA and its epoxide glycidamide (GA) were studied in the hepatocytes freshly isolated from male Sprague–Dawley rats. The isolated hepatocytes metabolized AA to GA. The formation of GA followed Michaelis-Menten kinetic parameters yielded apparent K _m = 0.477 ± 0.100 and 0.263 ± 0.016 mM, V _max = 6.5 ± 2.1 and 26.4 ± 3.0 nmol/h/10⁶ cells, and CL_int = 14 ± 5 and 100 ± 12 μl/h/10⁶ cells for the hepatocytes from untreated and acetone-treated rats, respectively. There were lower K _m and marked increases in V _max (fourfold) and in CL_int (sevenfold) in acetone-treated rat hepatocytes. The data suggest that CYP2E1 played a major role in metabolizing AA to more toxic GA. Both AA and GA induced a concentration- and time-dependent glutathione (GSH) depletion of the hepatocytes. From decreasing rates of GSH contents in hepatocytes, the parameters of glutathione S-transferase (GST) in hepatocytes to AA and GA were calculated to be K _m = 1.4 and 1.5 mM, V _max = 21 and 33 nmol/h/10⁶ cells, and CL_int = 15 and 23 μl/h/10⁶ cells, respectively. GA 1.5-times more readily depleted GSH content than AA. GA decreased the viability of hepatocytes at 3 mM, but AA did not. These data indicate that GA is more toxic than AA as assessed by intracellular GSH depletion and loss of viability of hepatocytes. GSH precursors such as N-acetylcysteine and methionine provided significant anti-cytotoxic effects on the decrease of GSH content and cell viability of hepatocytes induced by GA and AA.     

Toxic effects of carvacrol, caryophyllene oxide, and ascaridole from essential oil of Chenopodium ambrosioides on mitochondria

Chenopodium ambrosioides have been used for centuries in the Americas as a popular remedy for parasitic diseases. The essential oil of this plant possesses anthelmintic activity and is still used in some regions to treat parasitosis and leishmaniasis. However, the Chenopodium oil caused also some fatalities, leading to its commercial disuse. In this work, we studied the mechanism of toxicity of the essential oil and its major pure ingredients (carvacrol, caryophyllene oxide, and ascaridole, which was synthesized from α-terpinene) with respect to mammalian cells and mitochondria. We observed that all products, but especially caryophyllene oxide, inhibited the mitochondrial electron transport chain. This effect for carvacrol and caryophyllene oxide was mediated via direct complex I inhibition. Without Fe²⁺, ascaridole was less toxic to mammalian mitochondria than other major ingredients. However, evidence on the formation of carbon-centered radicals in the presence of Fe²⁺ was obtained by ESR spin-trapping. Furthermore, it was shown that Fe²⁺ potentiated the toxicity of ascaridole on oxidative phosphorylation of rat liver mitochondria. The increase of the α-tocopherol quinone/α-tocopherol ratio under these conditions indicated the initiation of lipid peroxidation by Fe²⁺-mediated ascaridole cleavage. Further ESR spin-trapping experiments demonstrated that in addition to Fe²⁺, reduced hemin, but not mitochondrial cytochrome c can activate ascaridole, explaining why ascaridole in peritoneal macrophages from BALB/c mice exhibited a higher toxicity than in isolated mitochondria.     

诱导血红素氧化酶表达对乙醇所致人原代肝细胞氧化损伤的保护作用

目的　探讨乙醇暴露与诱导血红素氧化酶(HO 1)表达之间的关系。方法　经体外灌流、分离培养人原代肝细胞 ,观察 10 0mmol·L- 1乙醇暴露 9h后谷胱甘肽 (GSH) ,谷草转氨酶 (GOT) ,乳酸脱氢酶 (LDH)和丙二醛 (MDA)的变化以反应人原代肝细胞的氧化损伤 ,用RT PCR及Western印迹方法检测乙醇对人原代肝细胞HO 1mRNA及蛋白表达的影响。结果 10 0mmol·L- 1乙醇暴露 9h后可导致人原代肝细胞明显的氧化损伤 ,肝细胞中的GSH明显降低 ,而MDA明显升高 ,GOT和LDH ,此外 ,急性乙醇暴露下 ,HO 1表达开始应激升高 ,随后慢慢降低 ,肝细胞经HO 1诱导剂预处理后再与乙醇作用 ,能减轻乙醇对肝细胞的氧化损伤作用 ,并且这种作用可被HO 1抑制剂所抵消。结论　诱导HO 1对乙醇所致人原代肝细胞的氧化损伤有保护作用。     

Absence of cardiotoxicity of the experimental cytotoxic drug cyclopentenyl cytosine (CPEC) in rats

The experimental anticancer drug cyclopentenyl cytosine (CPEC) was associated with cardiotoxicity in a phase I study. The aim of the present study was twofold; first we investigated whether the observed effects could be reproduced in in-vitro and in-vivo rat models. Second, we intended to investigate the underlying mechanism of the possible cardiotoxicity of CPEC. Effects on frequency and contractility were studied on the isolated atria of 18 male Wistar rats. Atria were incubated with 0.1 mmol L^–1 (n=6) or 1 mmol L^–1 (n=6) CPEC for 1.5 h and compared with control atria (incubation with buffer solution, n=6). The cardiac apoptosis-inducing potential was studied in-vivo on 66 rats by ^99mTc-Annexin V scintigraphy, followed by postmortem determination of radioactivity in tissues, histological confirmation with the TUNEL assay (late-phase apoptosis), and immunohistochemical staining for cleaved caspase 3 and cytochrome C (early-phase apoptosis). Serum levels of the necrotic cardiomyopathy marker troponin T were also determined. No effect on heart frequency was found in the isolated atria after CPEC treatment. A trend towards a decrease of contraction force was observed. However, the differences were not statistically significant. ^99mTc-Annexin V scintigraphy showed no increase in cardiac uptake ratio upon CPEC treatment in the in-vivo rat model, which was confirmed by determination of radioactivity in heart versus blood ratios. At each section a few individual isolated late apoptotic cells (<5) could be identified by the TUNEL assay in the highest CPEC dose group (90 mg kg^–1) but not in controls or in rats treated with 60 mg kg^–1 CPEC. Staining for the early apoptosis markers cleaved caspase 3 and cytochrome C did not reveal any significant differences between treated and control rats. Cardiac troponin T levels were not increased after CPEC treatment. CPEC does not affect heart frequency or contraction force in our cardiotoxicity models. Moreover, we did not find an indication of CPEC-induced apoptosis in heart tissue.     

Cellular responses of Prochilodus lineatus hepatocytes after cylindrospermopsin exposure

Cylindrospermopsin is a potent toxicant for eukaryotic cells produced by several cyanobacteria. Recently, primary hepatocyte cultures of Neotropical fish have been established, demonstrating to be a quite efficient in vitro model for cellular toxicology studies. In the current study, a protocol for culture of Prochilodus lineatus hepatocytes was established and utilized to investigate the cellular responses to purified cylindrospermopsin exposure. Hepatocytes were successfully dissociated with dispase, resulting in a cell yield of 6.36 × 10⁷ cells g⁻¹ of liver, viability of 97% and attachment on uncoated culture flasks. For investigation of cylindrospermopsin effects, hepatocytes were dissociated, cultured during 96 h and exposed to three concentrations of the toxin (0.1, 1.0 or 10 μg l⁻¹) for 72 h. Cylindrospermopsin exposure significantly decreased cell viability (0.1 and 1 μg l⁻¹) and multixenobiotic resistance mechanism, MXR (all exposed groups), but increased reactive oxygen/nitrogen species levels (all exposed groups) and lipid peroxidation (10 μg l⁻¹). On the other hand no significant alterations were observed for other biochemical biomarkers as 2GSH/GSSG ratio, protein carbonyl levels and DNA strand breaks or glutathione S-transferase and glucose 6-phosphate dehydrogenase activities. In conclusion, hepatocytes might be made sensitive to cylindrospermopsin, at least in part, due to reduction of xenobiotics and endobiotics efflux capacity by MXR. Additionally, the toxin exposure suggests important issues regarding hepatocytes survival at the lowest cylindrospermopsin concentrations.     

The hydroxyl radical generation and oxidative stress for the earthworm Eisenia fetida exposed to tetrabromobisphenol A

Biochemical effects of tetrabromobisphenol A (TBBPA) on the earthworm Eisenia fetida, including superoxide dismutase, catalase, glutathione-S-transferase, reduced glutathione, oxidized glutathione, GSH/GSSG ratio and malondialdehyde (MDA) level, were measured to assess ecological toxicity of TBBPA. With OECD standard filter-paper contact test method, earthworms were exposed to TBBPA of a range of concentrations (0.00, 0.01, 0.05, 0.1, 0.5 and 1.0 mg L⁻¹). According to the electron paramagnetic resonance spectrum, reactive oxygen species (ROS) generated in the earthworm was identified as the hydroxyl radical (^•OH) which was significantly induced at all TBBPA concentrations. With the increasing of TBBPA concentration, the antioxidant enzymes, glutathione and MDA levels varied significantly. The results showed that TBBPA exerts its toxic effects on E. fetida by inducing the generation of ROS and resulting in oxidative damage. The results show that the ^•OH production leads to oxidative stress in the tissues of the earthworm E. fetida.     

Arsenic induces mitochondria-dependent apoptosis by reactive oxygen species generation rather than glutathione depletion in Chang human hepatocytes

This study was conducted to evaluate the possible involvement of mitochondrial pathway in NaAsO₂-induced apoptosis and the role of reactive oxygen species (ROS) and reduced glutathione (GSH) in the apoptotic effect in Chang human hepatocytes. The MTT assay demonstrated that sodium arsenite (NaAsO₂) treatment for 24 h caused a dose-dependent decrease of cell viability. NaAsO₂ treatment (0–30 μM) was also found to induce phosphatidylserine externalization, a hallmark of apoptosis; to disrupt the mitochondrial membrane potential (Δψ _m ); to cause the release of cytochrome c into the cytosol, and to trigger cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner. All these changes were accompanied with the enhanced generation of intracellular ROS and malondialdehyde (MDA). Increase of intracellular GSH also coincided unexpectedly. Moreover, the extracellular addition of N-acetyl-l-cysteine (NAC, 5 mM) effectively reduced the generation of ROS and MDA, and rescued the cells from NaAsO₂ induced apoptosis and related alteration of mitochondria. These data suggest that the arsenic-induced cell apoptosis occurs though the mitochondrial pathway, and is mostly dependent on generation of ROS rather than GSH depletion in Chang human hepatocytes.     

d-Amphetamine-induced hepatotoxicity: possible contribution of catecholamines and hyperthermia to the effect studied in isolated rat hepatocytes

Amphetamines are indirect-acting sympathomimetic drugs widely abused due to their physical and psychostimulating effects. However, the use of these drugs has been associated with numerous reports of hepatotoxicity. While glutathione depletion induced by amphetamines contributes to the exposure of hepatocytes to oxidative damage, other indirect effects attributed to amphetamines may have a role in cell injury. To examine this possibility, Wistar rats were used for plasma measurements of d-amphetamine and catecholamines (noradrenaline, adrenaline and dopamine) (15 min) after i.p. injection of d-amphetamine (5, 20 and 80 mg/kg). Freshly isolated rat hepatocytes were put into contact for 2 h with concentrations of d-amphetamine and catecholamines similar to those found in vivo. Since hyperthermia is a common consequence of acute amphetamine intake, the study using isolated hepatocytes was conducted at 37 °C and also at 41 °C in order to simulate high temperature levels. We found that hyperthermia was an important cause of cell toxicity: in vitro, a rise in incubation temperature from 37 to 41 °C causes oxidative stress in freshly isolated rat hepatocytes, as shown by a depletion of reduced glutathione (GSH; 23%), an increase of oxidized glutathione (GSSG; 157%), the induction of lipid peroxidation with 77% increase of thiobarbituric acid substances TBARS) and the consequent loss of cell viability (≤ 44%). Single treatment of isolated hepatocytes with catecholamines at 37 °C induced lipid peroxidation (29% increase of TBARS) but had no effect on glutathione or cell viability. Conversely, a single treatment with d-amphetamine induced glutathione depletion (≤ 24% depletion of GSH) with no effect on lipid peroxidation or cell viability. Also, d-amphetamine potentiated the induction by catecholamines of lipid peroxidation at 37 °C (≤ 48% increase of TBARS), while concomitant treatment of d-amphetamine and catecholamines potentiated cell death at 41 °C (≤ 56% of cell death) although no effect on viability was seen at 37 °C. It is concluded that the aforementioned modifications induced by d-amphetamine in vivo are cytotoxic to freshly isolated rat hepatocytes. Received: 30 October 1996 / Accepted: 13 January 1997     

Effects of N-nitrosofenfluramine, a component of Chinese dietary supplement for weight loss, on CD-1 mice

Many cases of hepatopathy including deaths have frequently occurred after ingestion of Chinese dietary supplements for weight loss containing N-nitrosofenfluramine (N-fen), a nitroso derivative of fenfluramine (Fen), which was used for the treatment of obesity in the United States. Since Fen decreases appetite by decreasing the serotonin level and exhibits an antibiotic effect, N-fen may have been added, expecting a similar effect. Thus, we synthesized N-fen and orally administered it to mice, and investigated its effect on the liver as well as on the cerebral serotonin nervous system to investigate whether N-fen exhibits an anorectic effect. Three doses of N-fen were orally administered once daily to mice for 1 week. No significant changes in body weight, food intake, and general condition were noted. The liver and kidney weights were significantly increased. On blood chemistry, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities were increased, and total bilirubin and albumin were slightly decreased. On histopathological examination, acidophilic changes and mild cellular swelling were noted in the liver. The liver drug-metabolizing enzyme (P-450) level was significantly higher. The effect of N-fen on the serotonin (5HT) nervous system was examined by quantitative autoradiography of the mouse brain, and it was found that N-fen did not decrease the 5HT nerve activity. Effects of reuptake and release of monoamine neurotransmitters [dopamine (DA), 5HT, and norepinephrine (NE)] were investigated. N-fen inhibited a little 5HT reuptake, and did not inhibit reuptakes of DA and NE. Moreover, N-fen did not affect release of the three monoamines. The above findings suggested that N-fen did not exhibit a serotonin nerve fiber-mediated anorectic effect in mice, but induced hepatopathy.     

新型含二硫键和硫酯键的乙酰半胱氨酸衍生物的合成与抗肝损伤活性评价

目的 设计、合成N-乙酰半胱氨酸衍生物,并评价目标化合物对H₂O₂诱导的LO2细胞氧化损伤的保护作用。方法 以L-半胱氨酸和N-乙酰半胱氨酸为起始原料,采用酰氯酯化法合成具有全新结构的乙酰半胱氨酸衍生物;以H₂O₂损伤LO2人肝细胞建立体外氧化损伤模型,利用CCK-8法检测不同浓度H₂O₂对LO2细胞存活率的影响,并检测细胞上清中MDA含量和SOD活性。结果 共合成了6个全新结构的N-乙酰半胱氨酸衍生物,其结构经¹H-NMR、¹³C-NMR、ESI-MS确证,目标化合物能够抑制H₂O₂诱导的LO2氧化损伤,并能够降低MDA含量和提高SOD活性（P<0.01或P<0.05）。结论 本研究快速、高效地合成了N-乙酰半胱氨酸系列衍生物,目标化合物对体外肝细胞损伤具有保护作用。     

The mechanism of acute cytotoxicity of triethylphosphine gold(I) complexes : I. Characterization of triethylphosphine gold chloride-induced biochemical and morphological changes in isolated hepatocytes

Triethylphosphine gold complexes are effective therapeutic agents used for the treatment of rheumatoid arthritis. Many of those molecules are also highly cytotoxic in vitro and can inhibit DNA and protein synthesis. Preliminary experiments have indicated that triethylphosphine gold chloride (TEPAu) may induce the peroxidative decomposition of cellular membrane lipids. The purpose of these investigations therefore was to evaluate the role of lipid peroxidation in the mechanism of acute cytotoxicity of a gold(I) coordination complex, TEPAu, and to examine the early morphological and biochemical changes induced by TEPAu in suspensions of freshly isolated rat hepatocytes. TEPAu caused a rapid loss of cell viability at concentrations above 25 microM which was significantly different from that of control by 60 min and complete by 180 min of incubation. TEPAu also depleted cells of reduced glutathione (GSH) and increased the formation of malondialdehyde (MDA) by 60 min. Incubation of cells with either of the antioxidants, N,N'-diphenyl-p-phenylenediamine (DPPD) or promethazine blocked the formation of MDA but did not alter the time course of cell death or GSH depletion induced by TEPAu. TEPAu also caused a decrease in cellular NADPH and NADH by 10 min. Electron microscopy of hepatocytes exposed to TEPAu revealed early (5 min) formation of flocculent electron-dense precipitates within condensed mitochondria. These changes characteristically preceded cell death. Energy-dispersive electron-probe microanalysis indicated that the electron-dense precipitates did not contain detectable amounts of gold. TEPAu also caused a concentration-dependent decrease in cellular ATP and oxygen consumption in isolated rat hepatocytes. These data suggest that lipid peroxidation, as indicated by the formation of MDA, is probably not a major mechanism by which triethylphosphine gold complexes lethally injure cells. These data, therefore, suggest that mitochondria may be target organelles in TEPAu-induced toxicity to isolated rat hepatocytes.     

Leucocidin from Pseudomonas aeruginosa and membrane functions

Summary Leucocidin from Pseudomonas aeruginosa (strain 158) induced loss of potassium from isolated hepatocytes. The (Na⁺–K⁺)-stimulated ATPase activity of isolated rat liver plasma membranes showed dose-dependent activation up to 56%. Electron-spinresonance (ESR) measurements gave no indication of toxin-induced changes in membrane fluidity. On isolated guinea pig heart auricles the toxin produced an increase in frequency from 180/min to about 300/min, with arrhythmia and transitory flutter. On isolated nerve-diaphragm preparations the toxin caused a contracture and a decline in twitch tension, with a loss of potassium into the bathing solution. The action potential of the electrically stimulated N. ischiadicus of rat or frog was not affected when leucocidin was added to the bathing solution up to a concentration of 10 g/ml.     

Calcium-antagonizing activity of S-petasin, a hypotensive sesquiterpene from Petasites formosanus, on inotropic and chronotropic responses in isolated rat atria and cardiac myocytes

Petasites formosanus, an indigenous species of Petasites, has been used to treat cardiovascular diseases such as hypertension for years. We have suggested recently that S-petasin, a major sesquiterpene from P. formosanus, inhibits vascular smooth muscle contraction through inhibition of voltage-dependent Ca²⁺ channels, a phenomenon possibly responsible for the hypotensive effect of P. formosanus. This study was designed to examine the chronotropic and inotropic actions of S-petasin in the heart in vivo and in vitro. Administration of S-petasin (0.1–1.5 mg/kg i.v.) in anesthetized rats reduced heart rate dose-dependently. This response was consistent with significant suppression of both contractile amplitude and spontaneous firing rate of isolated atria, responses that were not antagonized by atropine (1 µM). Mechanical evaluation in isolated ventricular myocytes showed that S-petasin (0.1 to 100 µM) depressed peak myocyte contraction and intracellular Ca²⁺ transients concentration-dependently. The duration of myocyte contraction was not affected. Whole-cell voltage clamp analysis revealed that S-petasin inhibited the L-type Ca²⁺ current (I_Ca,L) concentration-dependently and shifted the steady-state inactivation curve of I_Ca,L to more negative potentials. However, a receptor-binding assay failed to identify any significant interaction between S-petasin (0.1–300 µM) and the dihydropyridine binding sites of L-type voltage-dependent Ca²⁺ channels. Taken together, these data show that the negative chronotropic and inotropic properties of S-petasin that can be ascribed mainly to I_Ca,L inhibition, but not to blockade of dihydropyridine binding sites of L-type Ca²⁺ channel or to muscarinic receptor activation.