Effect of buthionine sulfoximine on orthophenylphenol-induced hepato- and nephrotoxic potential in male rats

A single oral administration of orthophenylphenol (OPP, 1400 mg/kg; about half the LD₅₀) to male Fischer 344 rats produced an elevation of serum transaminase activity 24 h later. Pretreatment with l-buthionine-S,R-sulfoximine (BSO, 900 mg/kg) in the OPP-treated rats potentiated the hepatic and renal damage which was accompanied by necrosis. Six hours after the administration of OPP (700 or 1400 mg/kg), hepatic and renal glutathione (GSH) levels decreased with increasing dosage. Hepatic GSH depletion with OPP was enhanced with BSO pretreatment and the recovery of GSH in both organs was slow in the high-dose OPP group. These results suggest that hepatic and renal damage is associated with a serious and prolonged GSH depletion. When either phenyl-p-benzoquinone (PBQ) or phenylhydroquinone (PHQ), which are intermediates of OPP, was administered orally to rats at 700 or 1400 mg/kg, the mortality with the high dose of PBQ was 75% at 24 h. The serum transaminase activity and UN level increased with the low dose of PBQ, accompanied by necrotic hepatocytes. The toxic effects of PHQ on kidney or liver were less than those on PBQ. These observations suggest that the liver and kidney may be target organs for toxic actions of a large dose of OPP and its intermediate, PBQ.Part of this work was presented at IInd International ISSX Meeting Xenobiotic Metabolism and Disposition, May 16–20, 1988, Kobe, Japan     

几种物质对汞致大鼠肾脏氧化损伤的影响

目的探讨1次染汞致大鼠肾脏氧化损伤作用并观察2-氨基4-(S-丁基磺酰亚氨)丁酸(BSO)、还原型谷胱甘肽(GSH)、维生素C(Vit C)和二巯丙磺钠(DMPS)预处理对汞致肾脏氧化损伤的影响. 方法 Wistar大鼠64只,随机分成8组:对照组1组,染汞组3组,分别皮下注射0.75、1.5或2.5 mg/kg的氯化汞溶液,预处理干预组4组.BSO预处理组先腹腔注射BSO 0.5 mmol/kg体重,4 h后皮下注射0.75 mg/kg HgCl2溶液.其他3个预处理组中,先分别腹腔注射GSH 3 mmol/kg,Vit C 4 mmol/kg或DMPS 200 μmol/kg体重,2 h后皮下注射2.5 mg/kg HgCl2溶液.测定肝脏、肾皮质和尿汞含量以及肾皮质中GSH、丙二醛(MDA)、蛋白含量和谷胱甘肽过氧化物酶(GSH-Px)活性. 结果染汞后肝、肾皮质和尿汞含量随染汞剂量加大而增加.BSO预处理组肾皮质MDA含量和GSH-Px活性显著高于对照组和0.75 mg/kg HgCl2组,而GSH含量则与此相反.GSH、Vit C和DMPS预处理组肾皮质中:MDA含量均显著低于2.5 mg/kg HgCl2组;GSH含量均高于对照组;GSH-Px活性显著高于对照组和2.5 mg/kg HgCl2组.Vit C和DMPS预处理组肾皮质GSH含量也显著高于2.5 mg/kg HgCl2组. 结论肝脏、肾皮质和尿汞含量随染汞剂量而增加.BSO预处理可增强汞致肾脏氧化损伤作用,而GSH、Vit C和DMPS预处理则对汞致肾脏氧化损伤具有一定的拮抗作用.     

Genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells

The recent finding that acrylamide (AA), a carcinogen in animal experiments and a probable human carcinogen, is formed in foods during cooking raises human health concerns. The relevance of dietary exposure for humans is still under debate. The purpose of the study was to evaluate the possible genotoxicity of acrylamide in human hepatoma G2 (HepG2) cells, a cell line of great relevance to detect genotoxic/antigenotoxic substances, using single cell gel electrophoresis (SCGE) assay and micronucleus test (MNT). In order to clarify the underlying mechanism(s) we evaluated the intracellular generation of reactive oxygen species (ROS) and the level of oxidative DNA damage by immunocytochemical analysis of 8-hydroxydeoxyguanosine (8-OHdG). The involvement of glutathione (GSH) in the AA-induced oxidative stress was examined through treatment with buthionine sulfoximine (BSO) to deplete GSH. The results indicate that AA caused DNA strand breaks and increase in frequency of MN in HepG2 cells in a dose-dependent manner. The possible mechanism underlies the increased levels of ROS, depletion of GSH and increase of 8-OHdG formation in HepG2 cells treated with AA. We conclude that AA exerts genotoxic effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS and depletion of GSH.     

Enhancement of Antitumor Activity of Cisplatin on Human Gastric Cancer Cells in vitro and in vivo by Buthionine Sulfoximine

An attempt was made to evaluate the enhancement of the antitumor activity of cisplatin (DDP) by buthionine sulfoximine (BSO) in vitro and in vivo. In the in vitro study, pre-treatment with BSO (5, 10 and 25 mM ) increased the antitumor activity of DDP against the gastric cancer cell lines MKN-28 and MKN-45, whereas BSO alone exhibited only slight antitumor activity (inhibition rate, 20–30%). In the in vivo study, the antitumor effects of DDP against human gastric cancer xenografts St-15 and SC-l-NU in BALB/c nu/nu mice were enhanced pretreatment with BSO, which was administered intraperitoneally at a dose of 500 mg/kg according to a schedule of qd × 3. BSO alone showed no antitumor effects against these tumors in nude mice. The side effects (assessed in terms of death rate and body weight loss) associated with the maximum tolerated dose of DDP (9 mg/kg) were not increased by BSO pretreatment. As BSO increased the antitumor activity of DDP without a corresponding increment of its toxicity, BSO appears to be a promising agent for further study.     

The effect of in utero administration of buthionine sulfoximine on rat development

Glutathione (GSH) is a tripeptide that is thought to be an essential cell component playing an important role as a cellular antioxidant and scavenger of free radicals. GSH depletion has been shown to render cells more sensitive to various insults. GSH has a protective effect. GSH levels can be decreased by inhibition of its synthesis with buthionine sulfoximine (BSO), which inhibits γ-glutamylcysteine synthetase. Several studies have shown that treatment with BSO enhances the toxicity of some drugs and radiation. A previous study indicated that the effects of BSO on the developing embryo were short lived and did not persist to birth. In the above-mentioned study, mothers were treated with BSO only on days 10 and 11 of gestation. The objective of the present study was to determine the effects of BSO administration on GSH depletion throughout pregnancy on the developing rat. Timed pregnant Sprague-Dawley rats were placed on a liquid BioServ diet containing BSO starting on day 1 of pregnancy. The mothers received a daily dose of BSO ranging from 2 to 6 mmol/kg/24 h. The mothers were maintained on the diet until gestation day 21 when they were anesthetized with sodium pentobarbital and the pups delivered by Cesarean section. GSH levels were measured in brain and liver, and various parameters relating to development were assessed. A dose-response curve showed that a maximum depletion (86%) of GSH in the mother's liver was produced by the 6 mmol/kg dose of BSO. However, no change was seen in brain GSH levels of the mothers. GSH levels in brain and liver of the offspring were decreased by 60% and 66%, respectively. No significant effect of treatment with BSO was observed on growth-related parameters, such as body or brain weight. A significant decrease in neuron-specific enolase (NSE) activity in cerebellum and an increase in liver γ-glutamyl transpeptidase activity were observed in pups born to mothers treated with BSO. A decrease in NSE activity consistent with delayed development was observed. Therefore, we conclude that although a decrease in GSH may not produce obvious or observable teratogenic effects, it may produce a delay in development and may have a permissive role in teratogenic effects produced by other drugs by virtue of GSH depletion.     

Effects of exogenous factors on the cerebral glutathione in rodents

Since glutathione is thought to be involved in cerebral functions, changes in the glutathione level imply modulations of the neurotransmission in addition to all the known effects of GSH. It was investigated whether alterations of the cerebral glutathione can be induced by consumption of GSH, by inhibition or stimulation of the synthesis of GSH, or by an inhibition of the re-reduction of the oxidized glutathione. Aminophenazone, propyphenazone, acetaminophen, phenytoin, morphine and nitrofurantoin, known to deplete hepatic GSH, had no effects on cerebral GSH. Diethyl maleate (0.6 ml/kg) decreased the cerebral content of GSH and GSSG in adult rats as well as in fetuses. The depletion of the cerebral GSH caused by diethyl maleate treatment for 4 days was followed by an increase up to 125% and a subsequent return to the normal level after 1 week. In rats starved up to 71 h deficiency of exogenous amino acids caused only a minimal or no decrease in cerebral GSH. The specific inhibitor of the gamma-glutamylcysteine synthetase BSO only depleted GSH in the brain of young mice following the repeated s. c. administration of a high dose (890 mg/kg). After cobaltous chloride (20 mg/kg; twice a day for 2 or 4 days) the GSH level in the brain was unchanged. In vivo inhibition of the cerebral glutathione reductase was caused by ammonium metavanadate (12.5 mg/kg; three times a week for 6 weeks). Nitrofurantoin (150 mg/kg) had no effect. After lomustine (10 mg/kg) a minimal increase in glutathione reductase was found, but simultaneously also an increase in GSSG and of the ratio GSSG/total glutathione.     

丁胱亚磺酰亚胺对胶质瘤细胞谷光甘肽及放射增敏作用的实验研究

研究巯基修饰剂ＢＳＯ对胶质瘤细胞ＧＳＨ含量的修饰作用和放射增敏作用。方法利用Ｔｉｅｔｚｅ还原酶法观察ＢＳＯ对体外培养的胶质瘤细胞株及裸小鼠移植瘤模型胶质瘤细胞ＧＳＨ的作用。利用ＭＴＴ法观察ＢＳＯ对胶质瘤细胞放射敏感性的影响。结果经ＢＳＯ作用后胶质瘤细胞的ＧＳＨ含量显著下降，放射敏感性增加。结论无论是离体还是整体用药，ＢＳＯ均能降低胶质瘤细胞的ＧＳＨ含量。ＢＳＯ对胶质瘤细胞有放射增敏作用。     

Accumulation and toxicity of monophenyl arsenicals in rat endothelial cells

Clark 1 (diphenylarsine chloride) and Clark 2 (diphenylarsine cyanide) were used as chemical weapon agents (CWA), and the soil contamination by these CWA and their degraded products, diphenyl and phenyl arsenicals, has been one of the most serious environmental issues. In a series of comparisons in toxicity between trivalent and pentavalent arsenicals we investigated differences in the accumulation and toxicity of phenylarsine oxide (PAO³⁺) and phenylarsonic acid (PAA⁵⁺) in rat heart microvascular endothelial cells. Both the cellular association and toxicity of PAO³⁺ were much higher than those of PAA⁵⁺, and LC₅₀ values of PAO³⁺ and PAA⁵⁺ were calculated to be 0.295 µM and 1.93 mM, respectively. Buthionine sulfoximine, a glutathione depleter, enhanced the cytotoxicity of both PAO³⁺ and PAA⁵⁺. N-Acetyl-l-cysteine (NAC) reduced the cytotoxicity and induction of heme oxygenase-1 (HO-1) mRNA in PAO³⁺-exposed cells, while NAC affected neither the cytotoxicity nor the HO-1 mRNA level in PAA⁵⁺-exposed cells. The effect of NAC may be due to a strong affinity of PAO³⁺ to thiol groups because both NAC and GSH inhibited the cellular accumulation of PAO³⁺, but PAA³⁺ increased tyrosine phosphorylation levels of cellular proteins. These results indicate that the inhibition of protein phosphatases as well as the high affinity to cellular components may confer PAO³⁺ the high toxicity.     

Preclinical evaluation of azathioprine plus buthionine sulfoximine in the treatment of human hepatocarcinoma and colon carcinoma

AIM: To evaluate the efficacy and the safety of azathioprine (AZA) and buthionine sulfoximine (BSO) bylocalized application into HepG2 tumor in vivo.METHODS: Different hepatoma and colon carcinoma cell lines (HepG2, HuH7, Chang liver, LoVo, RKO, SW-48, SW-480) were grown in minimal essencial medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution and maintained in a humidified 37 ℃ incubator with 5% CO2. These cells were pretreated with BSO for 24 h and then with AZA for diffe...     

Ferulic acid attenuated cognitive deficits and increase in carbonyl proteins induced by buthionine-sulfoximine in mice

beta-Amyloid peptide (Abeta), the major constituent of the senile plaques observed in the brains of Alzheimer's disease patients, is cytotoxic to neurons and plays a central role in the pathogenesis of this disease. Previous studies have suggested that oxidative stress is involved in the mechanisms of Abeta-induced neurotoxicity in vivo. Here, we used a mouse model of brain dysfunction induced by dl-buthionine-(S,R)-sulfoximine (BSO: 3micromol/3microL/mouse, i.c.v.), an inhibitor of glutathione synthesis. In the novel object recognition test, we found impairments of exploratory preference in the retention trial but not the training trial 24h after BSO treatment, suggesting that BSO produces cognitive dysfunction in mice. In the forebrain of this model, we observed increase in carbonyl protein levels, an index of biochemical oxidative damage of proteins, compared to vehicle-treated mice. Pretreatment with ferulic acid (5mg/kg, s.c.) once a day for 6 days inhibited the induction of deficits in memory and increase in carbonyl protein levels by BSO. These findings suggest that pretreatment with FA may attenuate the memory deficits and increase the carbonyl protein levels induced by BSO in mice.