The toxicology and metabolism of chlorothalonil in fish. IV. Zinc coexposure and the significance of metallothionein in detoxication in Salmo gairdneri

Rainbow trout, Salmo gairdneri, were subjected to various regimes of exposure to zinc (0.36 mg/l) and ¹⁴C-chlorothalonil (¹⁴C-TCIN, 10 μg/l). Gel column chromatography of hepatic cytosol after exposure showed no binding of ¹⁴C-TCIN to metallothionein proteins with or without pre-exposure or with coexposure to zinc. ¹⁴C-TCIN did not induce the production of metallothionein with or without 96 h preexposure to 0.36 mg/l zinc, whereas induction did occur with zinc coexposure over 156 h. ¹⁴C-TCIN exposure did not affect cytosolic zinc levels, but exposure to zinc did reduce the level of cytosolic ¹⁴C-TCIN over the observed protein molecular weight range. It appears that metallothionein does not play a significant role in TCIN detoxication at this sublethal exposure level.     

The toxicology and metabolism of chlorothalonil in fish. II. Glutathione conjugates and protein binding

The existence of mono- and diglutathione conjugates of chlorothalonil (TCIN) in the bile of Salmo gairdneri exposed to ¹⁴C-TCIN is confirmed. A detailed analysis of the structural nature of the conjugates is described, using model thioether analogues. The degree of protein binding of TCIN in liver cytosol and bile is reported.     

In vitro metabolism of penicillic acid with mouse-liver homogenate fractions

The metaboism of penicillic acid (PA), a carcinogenic mycotoxin, was studied in vitro using subcellular fractions of mouse-liver homogenates. PA reacted with glutathione (GSH) both enzymatically and non-enzymatically. Each reaction was of equal importance. The in vitro metabolism of PA using different hepatic subcellular fractions was essentially non-enzymatic when GSH was absent, but metabolism was strikingly increased when GSH was available. In the microsomal preparation in the presence of GSH 75% of the added PA was biotransformed within 30 min to metabolite(s) that were not extractable with organic solvents. HPLC analysis indicated that the metabolite(s) were more polar than the parent compound.     

Favism: effect of divicine on rat erythrocyte sulfhydryl status, hexose monophosphate shunt activity, morphology, and membrane skeletal proteins.

Favism is an acute anemic crisis that can occur in susceptible individuals who ingest fava beans. The fava bean pyrimidine aglycone divicine has been identified as a hemotoxic constituent; however, its mechanism of toxicity remains unknown. We have shown recently that divicine can induce a favic-like response in rats and that divicine is directly toxic to rat red cells. In the present study, we have examined the effect of hemotoxic concentrations of divicine on rat erythrocyte sulfhydryl status, hexose monophosphate (HMP) shunt activity, morphology, and membrane skeletal proteins. In vitro exposure of rat red cells to divicine markedly stimulated HMP shunt activity and resulted in depletion of reduced glutathione with concomitant formation of glutathione-protein mixed-disulfides. Examination of divicine-treated red cells by scanning electron microscopy revealed transformation of the cells to an extreme echinocytic morphology. SDS-PAGE and immunoblotting analysis of the membrane skeletal proteins indicated that hemotoxicity was associated with the apparent loss of skeletal protein bands 2.1, 3, and 4.2, and the appearance of membrane-bound hemoglobin. Treatment of divicine-damaged red cells with dithiothreitol reversed the protein changes, which indicated that the observed alterations were due primarily to the formation of disulfide-linked hemoglobin-skeletal protein adducts. The data suggest that oxidative modification of hemoglobin and membrane skeletal proteins by divicine may be key events in the mechanism underlying favism.     

Styrene induces an inflammatory response in human lung epithelial cells via oxidative stress and NF-kappaB activation

Styrene is a volatile organic compound (VOC) that is widely used as a solvent in many industrial settings. Chronic exposure to styrene can result in irritation of the mucosa of the upper respiratory tract. Contact of styrene with epithelial cells stimulates the expression of a variety of inflammatory mediators, including the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1). To characterise the underlying mechanisms of the induction of inflammatory signals by styrene, we investigated the influence of this compound on the induction of oxidative stress and the activation of the nuclear factor-kappa B (NF-κB) signalling pathway in human lung epithelial cells (A549). The results demonstrate that styrene-induced MCP-1 expression, as well as the expression of the oxidative stress marker glutathione S-transferase (GST), is associated with a concentration dependent pattern of NF-κB activity. An inhibitor of NF-κB, IKK-NBD, and the anti-inflammatory antioxidant N-acetylcysteine (NAC) were both effective in suppressing styrene-induced MCP-1 secretion. In addition, NAC was capable of inhibiting the upregulation of GST expression. Our findings suggest that the activation of the NF-κB signalling pathway by styrene is mediated via a redox-sensitive mechanism.     

Antioxidant and prooxidant effects of quercetin on glyceraldehyde-3-phosphate dehydrogenase.

Anti- and prooxidant properties of quercetin under different conditions were investigated using glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme containing essential cysteine residues. Quercetin was shown to produce hydrogen peroxide in aqueous solutions at pH 7.5, this resulting in the oxidation of the cysteine residues of the enzyme. Quercetin significantly increased oxidation of GAPDH observed in the presence of ferrous ions, particularly when FeSO(4) was added to the solution containing GAPDH and quercetin. The results suggest the formation of hydroxyl radical in the case of the addition of FeSO(4) to a quercetin solution. At the same time, quercetin protects GAPDH from oxidation in the presence of ascorbate and Fe(3+). In the absence of metals, quercetin protects SH-groups of GAPDH from oxidation by the superoxide anion generated by the system containing xanthine/xanthine oxidase.     

黄酮类化合物对葡萄糖-6-磷酸脱氢酶缺乏者红细胞的体外氧化作用

目的:探讨黄酮类化合物对葡萄糖-6-磷酸脱氢酶(G6PD)缺乏者红细胞氧化还原状态的影响。方法:将低、中、高浓度的槲皮素、黄芩素、芹菜素、漆黄素、木犀草素、柚皮素、桑黄素、山奈酚、葛根素和芦丁分别与G6PD缺乏者及正常者红细胞在40%红细胞悬液和全血中进行体外孵育,测定红细胞还原性谷胱甘肽(GSH)和高铁血红蛋白(MetHb)的水平。结果:槲皮素、黄芩素、芹菜素、漆黄素、木犀草素、柚皮素、桑黄素、山奈酚具有较强的氧化作用,能明显降低G6PD缺乏者红细胞GSH水平,升高MetHb水平。葛根素仅降低G6PD缺乏者红细胞GSH水平,具有较弱的氧化作用。芦丁对G6PD缺乏者红细胞GSH和MetHb均无影响。较高浓度的槲皮素、芹菜素、桑黄素亦能使G6PD正常者MetHb水平升高。黄酮类化合物的氧化作用呈一定浓度依赖性,在中、高浓度时表现明显。结论:部分黄酮类化合物对G6PD缺乏者红细胞具有氧化作用,建议G6PD缺乏者慎用富含氧化性黄酮类化合物的中草药及其制剂。     

Effect of an inactivator of glyceraldehyde-3-phosphate dehydrogenase, a fortuitous arsenate reductase, on disposition of arsenate in rats.

The environmentally prevalent arsenate (AsV) is reduced in the body to the much more toxic arsenite (AsIII). Recently, we have demonstrated that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reduction of AsV in the presence of glutathione, yet the role of GAPDH in AsV reduction in vivo is unknown. Therefore, we examined the effect of (S)-alpha-cholorhydrin (ACH), which forms a GAPDH-inhibitory metabolite, on the reduction of AsV in rats. These studies confirmed the in vitro role of GAPDH as an AsV reductase, inasmuch as 3 h after administration of ACH (100 or 200 mg/kg, ip) to rats both the cytosolic GAPDH activity and the AsV-reducing activity dramatically fell in the liver, moderately decreased in the kidneys, and remained unchanged in the muscle. Moreover, the AsV-reducing activity closely correlated with the GAPDH activity in the hepatic cytosols of control and ACH-treated rats. Two confounding effects of ACH (i.e., a slight fall in hepatic glutathione levels and a rise in urinary AsV excretion) prompted us to examine its influence on the disposition of injected AsV (50 micromol/kg, iv) in rats with ligated bile duct as well as in rats with ligated bile duct and renal pedicles. These experiments demonstrated that the hepatic retention of AsV significantly increased, and the combined levels of AsV metabolites (i.e., AsIII plus methylated arsenicals) in the liver decreased in response to ACH; however, ACH failed to delay the disappearance of AsV from the blood of rats with blocked excretory routes. Thus, the GAPDH inactivator ACH inhibits AsV reduction by the liver, but not by the whole body, probably because the impaired hepatic reduction is compensated for by hepatic and extrahepatic AsV-reducing mechanisms spared by ACH. It is most likely that ACH inhibits hepatic AsV reduction predominantly by inactivating GAPDH in the liver; however, a slight ACH-induced glutathione depletion may also contribute. While this study seems to support the conclusion that GAPDH in the liver is involved in AsV reduction in rats, confirmation of the in vivo role of GAPDH as an AsV reductase is desirable.     

Preventive effects of a purified extract isolated from Salvia miltiorrhiza enriched with tanshinone I,tanshinone IIA and cryptotanshinone on hepatocyte injury in vitro and in vivo

Salvia miltiorrhiza is traditionally used to treat liver disease in Asia. In this study, we tested the ability of a purified extract of S. miltiorrhiza (PF2401-SF) and its constituents, tanshinone I, tanshinone IIA, and cryptotanshinone, to protect against acute and subacute liver damage induced by carbon tetrachloride by measuring serum transaminase levels, the reduced form of glutathione (GSH), antioxidant enzyme activities, and lipid peroxidation levels in the liver. We also evaluated their ability to protect primary cultured rat hepatocytes from tertiary-butylhydroperoxide (tBH) or d-galactosamine (GalN). PF2401-SF was protective at 50–200 mg/kg per day in acute liver injury and 25–100 mg/kg per day in subacute liver injury. Tanshinone I, tanshinone IIA, and cryptotanshinon (40 μM), inhibited lactate dehydrogenase leakage, GSH depletion, lipid peroxidation and free radical generation in vitro. PF2401-SF and its major constituents, tanshinone I, tanshinone IIA and cryptotanshinone, can protect against liver toxicity in vivo and in vitro due to its antioxidant effects.     

Hepatoprotective effect and antioxidant role of sun, sulphited-dried apricot (Prunus armeniaca L.) and its kernel against ethanol-induced oxidative stress in rats

The present study was carried to evaluate the hepatoprotective effect and antioxidant role of sun, sulphited-dried apricot and its kernel against ethanol-induced oxidative stress. The hepatopreventive and antioxidant potential of the plant’s supplementations were evaluated by measuring level of serum liver damage marker enzymes (AST, ALT, GGT and LDH), antioxidant defense systems (GSH, GR, SOD, GST and GPX) and MDA content in various tissues of rats. Eight experimental groups: I (control), II (20% ethanol), III (ethanol + 15% sun-dried apricot), IV (ethanol + 30% sun dried). V (ethanol + 15% sulphited-dried), VI (ethanol + 30% sulphited-dried), VII (ethanol + 15% kernel) and VIII (ethanol + 30% kernel). According to the results, the levels of serum enzymes increased significantly in the II group as compared to those of I group, but they decreased in the III, IV, V and VI groups as compared to those of II group. Also, administration of sun and sulphited-dried apricot supplementation restored the ethanol-induced imbalance between MDA and antioxidant system towards near normal particularly in tissues but not its kernel. It is concluded that apricot has a hepatoprotective effect in rats with ethanol, probably acting by promoting the antioxidative defense systems.     

益肝康及其拆方对肝星状细胞活化增殖及胶原代谢影响的研究

目的 探讨中药复方益肝康抗肝纤维化的作用机制及药效学配伍意义.方法 水蒸醇提法制备益肝康及其拆方-丹参小复方(丹参、黄芪、归尾)纯化浸膏.温育体外培养的肝星状细胞(HSC),3H-胸腺嘧啶核苷酸(3H-TdR)掺入法检测细胞增殖,免疫细胞化学法检测α-平滑肌肌动蛋白(α-SMA)表达,3H-脯氨酸(3H-Pm)掺入法检测HSC胶原合成.结果 益肝康及丹参小复方对HSC的3H-TdR掺入、α-SMA表达及3H-Pro掺入均有显著抑制作用,对3H-TdR掺入抑制作用全方与拆方差异无统计学意义,而α-SMA表达及3H-Pro掺入的抑制作用全方优于拆方.结论 益肝康及丹参小复方可抑制肝星状细胞活化增殖及胶原合成,全方作用优于拆方.     

Effects of dithiocarb and (+)-cyanidanol-3 on the hepatotoxicity and metabolism of vinylidene chloride in rats.

The acute hepatotoxic effects of vinylidene chloride (VDC) were evidenced by measurement of the increase in the serum levels of the aminotransferase (GPT) and sorbitol dehydrogenase (SDH), hepatic glutathione (GSH) depletion and histological examinations in rats. The hepatoprotective agents dithiocarb and (+)-cyanidanol-3 proved well able to antagonize these toxic effects of VDC. While dithiocarb inhibited the in vivo metabolism of VDC in a closed exposure system, (+)-cyanidanol-3 had no influence at all. These findings substantiate the role of the microsomal monooxygenase system in the metabolism and hepatotoxicity of VDC. The mechanisms by which dithiocarb and (+)-cyanidanol-3 act as antihepatotoxic agents are different: the inhibition of the metabolic activation by dithiocarb and free radical-scavenging by (+)-cyanidanol-3.     

Metabolism of 2, 6-dimethylnaphthalene in starry flounder (Platichthys stellatus) exposed to naphthalene and p-cresol

Juvenile starry flounder (Platichthys stellatus) were force-fed naphthalene, p-cresol, or a mixture of naphthalene and p-cresol in daily doses of 0.3–0.4 mg/kg body weight, for 6 consecutive days. On the 8th day, each fish was force-fed a dose of 12–15 mg/kg of 2,6-dimethyl[¹⁴C]naphthalene (DMN). Twenty-four hours later, the fish were killed and ¹⁴C-labelled metabolites in the bile were isolated and identified by thin-layer chromatography.Most of the biliary metabolites were recovered as conjugates, principally as glurosides and glucuronides. Analyses of the nonconjugated metabolites and metabolites resulting from enzymatic hydrolysis of the conjugates provided identification of four metabolites of DMN: 2,6-dimethyl-3-naphthol; 2,6-dimethyl-3,4-naphthoquinone; trans-3,4-dihydroxy-3,4-dihydro-2,6-dimethylnaphthalene (dihydrodiol); and b-methyl-2-naphthalenemethanol (alcohol). Enzymatic hydrolysis of the glucuronides yielded two metabolites: the alcohol, representing metabolism at a methyl substituent, and the dihydrodiol, representing oxidation of an aromatic ring. Exposure to naphthalene and/or p-cresol led to a significant reduction (P < 0.05) in the alcohol product and a corresponding increase in the dihydrodiol. This perturbation, which favors the formation of potentially damaging epoxides, may alter the nature of toxicological effects.     

In vitro and in vivo hepatoprotective effects of the aqueous extract from Taraxacum officinale (dandelion) root against alcohol-induced oxidative stress

The protective effects of Taraxacum officinale (dandelion) root against alcoholic liver damage were investigated in HepG2/2E1 cells and ICR mice. When an increase in the production of reactive oxygen species was induced by 300 mM ethanol in vitro, cell viability was drastically decreased by 39%. However, in the presence of hot water extract (TOH) from T. officinale root, no hepatocytic damage was observed in the cells treated with ethanol, while ethanol-extract (TOE) did not show potent hepatoprotective activity. Mice, which received TOH (1 g/kg bw/day) with ethanol revealed complete prevention of alcohol-induced hepatotoxicity as evidenced by the significant reductions of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase activities compared to ethanol-alone administered mice. When compared to the ethanol-alone treated group, the mice receiving ethanol plus TOH exhibited significant increases in hepatic antioxidant activities, including catalase, glutathione-S-transferase, glutathione peroxidase, glutathione reductase, and glutathione. Furthermore, the amelioration of malondialdehyde levels indicated TOH’s protective effects against liver damage mediated by alcohol in vivo. These results suggest that the aqueous extract of T. officinale root has protective action against alcohol-induced toxicity in the liver by elevating antioxidative potentials and decreasing lipid peroxidation.     

Variations in alcohol metabolism: influence of sex and age.

Alcohol-induced sleep time was measured subsequent to the intraperitoneal injection of a 3.5 g x kg-1 dose. The old male group had a sleep time approximately 4 times that of the young male group and approximately twice that of the old female group. Blood alcohol concentrations at time of awakening were nearly identical in all groups, indicating the difference in sleep time is not due to an altered CNS sensitivity. Measurement of in vivo alcohol disappearance rate indicates the old male group is different from the other groups because of a slower rate of alcohol metabolism. Although changes in hepatic alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities were seen, the changes do not explain the observed decrease in alcohol metabolism observed in the old male group. These data provide further evidence that hepatic ADH and ALDH activities are not rate limiting in alcohol metabolism.     

Hepatic metabolism of contaminants in the terapontid fish, yellowtail trumpeter (Amniataba caudavittata Richardson)

The yellowtail trumpeter (Amniataba caudavittata) is an estuarine-dependent omnivorous fish found in the Swan-Canning Estuary, Western Australia. Thirty five fish were injected with either the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P), the synthetic flavenoid beta-naphthoflavone (BNF), or used as controls. The fish were then sampled at 3 and 7 days postinjection. Induction of the enzyme ethoxyresorufin O-deethylase (EROD) activity was nonsignificant while ethoxycoumarin O-deethylase (ECOD) activity induction differed amongst treatments. A high interindividual variability in the EROD activity was observed. The measurement of sorbitol dehydrogenase in the serum (s-SDH) was elevated (BNF 2.2 times and B[a]P 3.2 times the control fish) demonstrating that liver cell damage had occurred. Increases in biliary metabolites of both B[a]P-type and pyrene-type (19 times and 3.4 times the controls respectively) indicated that detoxification of pyrene-type compounds had taken place. Fish of the Terapontidae family, such as the yellowtail trumpeter, were found to be suitable for biomonitoring the health of the Swan-Canning Estuary. A combination of ECOD activity, s-SDH, and the measurement of biliary metabolites represents a suitable suite of biomarkers for environmental monitoring of the sublethal effects of PAH pollution in these fish.     

The Metabolism of Biphenyl. III. Phenolic Metabolites in the Pig

Abstract The phenolic metabolites of biphenyl in both male and female pigs were qualitatively and quantitatively analysed as trimethylsilyl (TMS) ethers by combined gas chromatography/mass spectrometry and gas chromatography, respectively. This xenobiotic was metabolized mainly to mono–hydroxylated biphenyl, and in small amounts to di– and tri–hydroxylated biphenyls. Urinary excretion was the only route of elimination of these compounds from the body. The total urinary recovery in male pigs was 44.8 % of the dose 96 hrs after administration of the drug, and the corresponding value in female pigs was 27.6%. The main part of the urinary metabolites was excreted during the first 24 hrs after dosing in both sexes.     

Production,composition and toxicology studies of Enzogenol® Pinus radiata bark extract

Enzogenol® pine bark extract is a dietary supplement and food ingredient produced by water extraction of Pinus radiata. We present production method, composition, and safety data from rat and dog toxicological and human clinical studies. The dry powder contains proanthocyanidins (>80%), taxifolin (1–2%), other flavonoids and phenolic acids (up to 8%), and carbohydrates (5–10%). Reverse mutation assays showed lack of mutagenic activity. Single and 14-day repeat dosing in rats and dogs had no influence on body weight, feed consumption, blood chemistry, and haematology at any dose level. There were no treatment related findings on gross and detailed necroscopy, organ weights, organ weight ratios and histology. The only adverse events were emesis and diarrhoea in dogs occurring mainly in un-fed condition and at the highest dose level in a total of 18% of applications. The MTD and NOAEL in the present rat and dog studies were 2500 and 750 mg/kg/day, respectively. Consumption of 480 mg/day for 6 months and 960 mg/day for 5 weeks in two human studies showed Enzogenol® had no adverse influence on liver and kidney function, haematology, and did not cause any adverse events. Our studies indicate lack of toxicity of Enzogenol® and support safe use as a food ingredient.     

β-N-Methylaminoalanine (BMAA): Metabolism and metabolic effects in model systems and in neural and other tissues of the rat in vitro

The non-protein amino acid, β-N-methylaminoalanine (BMAA), is neurotoxic and has been implicated in the amyotrophic lateral sclerosis-Parkinsonism-dementia (ALS-PD) complex of Guam. This concept remains controversial, in part because of the lack of a convincing animal model. The neuropharmacology of BMAA is well established, but little is known of its metabolism. This paper reports aspects of the metabolism, and metabolic effects, of BMAA in rat tissues.BMAA changed the distribution of taurine, glycine and serine between rat brain slices and their incubation medium; the glutamate/glutamine cycle between neurones and glia was also compromised. In model experiments BMAA reacted non-enzymatically with pyridoxal-5′-phosphate, releasing methylamine. Rat liver and kidney homogenates, but not brain homogenates, also formed methylamine and 2,3-diaminopropanoic acid when incubated with BMAA.These results provide evidence that several biochemical mechanisms are involved in the neurotoxicity of BMAA. The novel discovery that methylamine is formed from BMAA in rat liver and kidney preparations may be significant since chronic administration of methylamine to rats causes oxidative stress. The extent to which this reaction occurs in different animal species might be a decisive factor in selecting an animal model.