Liver Postmitochondrial Fraction Research Articles

Comparative effects of galactose-induced aging on mitochondrial permeability transition in rat liver and testis

This study investigated the status and sensitivity of mitochondrial permeability transition (mPT) pore in testis and liver of rats exposed to doses of galactose, an acceptable model used to mimic natural aging. Male albino rats were divided into five groups of eight animals in each group for in vivo studies and administered distilled water, 50,100, 200 and 500 mg galactose/kgbdwt, respectively, for six consecutive weeks. Mitochondria were isolated from liver and testis by differential centrifugation. Mitochondrial permeability transition (mPT) was assessed as mitochondrial swelling and was monitored spectrophotometrically. Mitochondrial lipid peroxidation, ATPase activity, antioxidant enzymes, caspase activation, interleukins, and sperm functional characteristics were also assessed. Administration of galactose (50–500 mg/kg) to male Wistar albino rats had no effect whatsoever on the testicular mPT pore. However, liver mPT pore was significantly opened. Furthermore, the enhancement of mitochondrial ATPase activity and malondialdehyde generation were observed in the liver of galactose-exposed rats. Significant alterations in antioxidant enzymes were observed in post-mitochondrial fraction (PMF) of liver and testis. There were also increases in serum levels of IL-1β and 6. In addition, caspases 9 and 3 were significantly elevated in PMF of the liver with evidence of DNA fragmentation. However, there was no significant difference in levels of caspases in PMF of testis in model groups of galactose when compared with control. These results provide evidence that testis mitochondria do not readily undergo permeability transition pore upon exposure to doses of D-galactose that induce the opening of the pore in the liver.HighlightsTesticular mitochondria are less sensitive to induction of permeability transition than liver mitochondria in rats exposed to D-galactose for 6 weeks, despite the occurrence of alterations in the antioxidant defense system and generation of ROS in sperm cells as in hepatocytes.The occurrence of mitochondrial permeability transition in liver of galactose-exposed rats is consistent with malondialdehyde production, alteration in antioxidant levels, enhanced ATPase activity, caspases-9 and 3 activation, immune dysfunction, and DNA fragmentation.The study of biochemical basis of reduced sensitivity of testis to permeability transition under conditions which the liver is extremely susceptible may become useful in age associated-neurodegenerative diseases where apoptosis is upregulated and has to be properly managed to achieve downregulation.

Hepatoprotective Properties of Aqueous and Ethanolic Stem Bark Extracts of Hura crepitans Linn. Against CCl4− Induced Acute Liver Damage in Rats

Medicinal plants are gaining wide acceptance even amongst the literate population of the world. Secondary metabolites obtained from herbal sources contain bioactive phytochemicals, many of which have little or no side effects. The hepatoprotective properties of Hura crepitans against CCl4‐induced acute hepatoxicity were investigated, using 48 male Sprague‐Dawley rats. Liver damage was induced with administration of a 1:1 (v/v) CCl4 and olive oil after pre‐treating for 7 days with ethanolic stem bark extract (EES) and aqueous stem bark extract (AES). Hepatoprotective effects of Hura crepitans were investigated by estimating the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin levels (TBIL). Effects of Hura crepitanson biomarkers of lipid peroxidation (oxidative damage) and antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S‐transferase (GST) and the non‐enzymic antioxidant, reduced glutathione (GSH) were determined in liver post mitochondrial fraction (PMF). EES, AES and Silymarin displayed significant hepatoprotective activities by reducing the activities of ALT, AST and ALP on one hand, and also decreased the level of TBIL on the other hand. Pre‐treatment with Hura crepitans extracts and the standard drug Silymarin led to reduction in the activities of antioxidant enzymes, reduction in levels of malondialdehyde (MDA) and reduction in serum levels of GSH in a significant manner. The histopathological studies also demonstrate the protective effect of pre‐treatment with Hura crepitans and Silymarin against oxidative damage by CCl4. Our investigations show that Hura crepitans extracts possess significant hepatoprotective effect against CCl4‐induced hepatoxicity, an endowment arising from the constituent phytochemicals. The present study has demonstrated that EES and AES protected the liver from the detrimental effects of CCl4 through their effects on the antioxidant defence system and their abilities to mitigate against lipid peroxidation.Support or Funding InformationTertiary Education Trust Fund (TETFund), Nigeria/ASPET.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Toxicity of some broad-spectrum antibacterials in normal rat liver: the role of mitochondrial membrane permeability transition pore

Ciprofloxacin (CIP) and Amoxycillin/Clavulanate (AC) are broad-spectrum antibiotics that are commonly administered for treatment of various bacterial infections. Studies have reported the antiproliferative and apoptotic activities of CIP in several cancer cell lines while AC has been implicated in drug–induced liver injury. We investigated the influence of CIP and AC on mitochondrial Permeability Transition (mPT) pore, ATPase activity, and cytochrome C release of normal Rat Liver Mitochondria (RLM) spectrophotometrically. In vitro, CIP and AC induced the opening of the mPT pore in a concentration-dependent manner with evidence of cytochrome C release maximally at 70 µg/ml by 13 and 10 folds, respectively. In vivo, CIP (100, 200 mg/kgbw) significantly induced mPT pore opening with induction folds of 2.4 and 2.6, respectively. However, low dose of AC (10 mg/kgbw) had no effect whatsoever on the mPT pore while higher dose (30 mg/kgbw) significantly induced pore opening by 3.4 folds. Similarly, CIP(100 mg/kgbw) and AC (30 mg/kgbw), significantly enhanced RLM ATPase activity, induced cytochrome C release and increased levels of RLM malondialdehyde generation and triggered the activation of caspases-9 and 3 in liver post-mitochondrial fraction. There were also significant (p<0.05) elevation in levels of serum aminotransferases and white blood cell count. Our results show that prolonged use of Ciprofloxacin and Amoxicillin Clavulanate could result in mitochondrial membrane breakdown via induction of opening of mPT pore leading to expulsion of cytochrome C, lipid peroxidation and decrease in energy content in healthy liver cells. These drugs should therefore be used with caution.

Effect of reduced glutathione on the indexes of oxidative stress and heme metabolism in liver and blood of rats under hemin chloride injection in vivo

Heme (iron-protoporphyrin IX) is involved in various cellular functions. The release of heme under hemolysis or under the damage of intracellular hemeproteins leads to its accumulation in tissues and, as a result, to the activation of free radical processes. Reduced glutathione (GSH) functions as an endogenous water-soluble antioxidant and a regulator of cells redox status, but its effect on the development of oxidative stress under hemin action in mammals remains not investigated. The aim of this work was to study the effect of hemin chloride on some hemeproteins activity and a number of prooxidant-antioxidant status indexes in rat liver and blood under GSH level modulation in vivo. White male rats weighing 170–280 g were taken for investigation. Hemin chloride and GSH were injected intraperitoneally. Blood plasma, homogenate, and postmitochondrial fraction of liver were the objects of study. Hemin chloride injection (50 mg/kg body weight) caused the increase in heme-containing products level in blood and free heme level in liver of rats, which was accompanied by the activation of free radical processes in these tissues. The accumulation of free heme in liver was proved by an increase in tryptophan 2,3-dioxygenase (TDO) holoenzyme activity and heme saturation. The pretreatment by GSH (500 mg/kg body weight) 0.5 h before hemin chloride injection normalized GSH content, but did not prevent heme accumulation, the decrease in triglycerides level and the increase in lipid hydroperoxides content in rat blood plasma under hemin action. In liver, GSH injection prevented the increase in lipid hydroperoxides and protein carbonyl derivatives concentration as well as in TDO holoenzyme activity, and decreased the degree of TDO heme saturation. All these changes occurred under GSH content increase in liver. Catalase activity in liver did not differ from the control values after hemin chloride injection as well as after glutathione and hemin coadministration. The analysis of relationship between parameters studied in this work revealed the strong positive correlation between GSH content in plasma and liver (r=0.85; p&lt;0.001), which was consistent with literature data on the significant role of liver in supplying other tissues with reduced glutathione. A negative correlation was found between lipid peroxidation products and triglycerides content in plasma (r=–0.52; p&lt;0.05), which indicated the participation of triglycerides unsaturated fatty acids as substrates in the peroxidation processes under hemin action. No significant correlation between GSH and hydroperoxides content, as well as between GSH and heme-containing products levels in blood plasma was revealed. Thus, the water-soluble antioxidant glutathione was not effective enough to prevent damage of lipid components in blood under hemin chloride action in the selected dose. In the liver, on the contrary, GSH injection prevented heme accumulation and oxidative stress development under hemin action, which was obviously associated with an increase in the GSH content in this organ.

Eugenia caryophyllus Extract Exerts Hypocholesterolemic and Antioxidant Effects in High-Cholesterol-Fed Rats

Background: Eugenia caryophyllus (clove) is an important aromatic household spice. The plant is believed to possess medicinal properties and is commonly used in herbal preparations by traditional healers in the treatment of different ailments and diseases. Objectives: We investigated the lipid-lowering and antioxidant effects of aqueous extract of Eugenia caryophyllus on high-cholesterol-fed rats. Materials and Methods: Cholesterol (40 mg/0.3 mL) was administered to induce hypercholesterolemia in rats by oral gavage, and Eugenia caryophyllus (100 or 200 mg/kg) and Questran (0.26 g/kg) were administered five times a week for eight consecutive weeks. Serum lipid-profiles, lipid peroxidation (LPO), and antioxidant parameters were examined in liver and heart post mitochondrial fraction (PMF). Aspartate and alanine aminotransferase (ALT and AST) activities and liver tissue histology were used to evaluate tissue toxicity. Results: Hypercholesterolemia produced a noticeable decrease in serum HDL-c, whereas a concurrent elevation in serum LDL-c, total cholesterol, and triglycerides as well as serum ALT and AST was observed. Furthermore, hypercholesterolemia remarkably decreased antioxidant status, but LPO content was increased. These indices were significantly attenuated in hypercholesterolemic rats treated with E. caryophyllus extract (100 or 200 mg/kg of body weight). Specifically, reduced glutathione (GSH) concentration was increased in a dose dependent manner in liver and heart PMF. Conclusions: These results demonstrate that the hypolipidemic and antioxidative effects of aqueous extract of E. caryophyllus might be due to its ability to ameliorate lipid profiles, enhance antioxidant activities, and delay the lipid peroxidation process. This confirms the previously identified protective roles of E. caryophyllus in human health.

A 3-hydroxy %-end group in xanthophylls is preferentially oxidized to a 3-oxo ε-end group in mammals

We previously found that mice fed lutein accumulated its oxidative metabolites (3'-hydroxy-ε,ε-caroten-3-one and ε,ε-carotene-3,3'-dione) as major carotenoids, suggesting that mammals can convert xanthophylls to keto-carotenoids by the oxidation of hydroxyl groups. Here we elucidated the metabolic activities of mouse liver for several xanthophylls. When lutein was incubated with liver postmitochondrial fraction in the presence of NAD(+), (3'R,6'R)-3'-hydroxy-β,ε-caroten-3-one and (6RS,3'R,6'R)-3'-hydroxy-ε,ε-caroten-3-one were produced as major oxidation products. The former accumulated only at the early stage and was assumed to be an intermediate, followed by isomerization to the latter. The configuration at the C3' and C6' of the ε-end group in lutein was retained in the two oxidation products. These results indicate that the 3-hydroxy β-end group in lutein was preferentially oxidized to a 3-oxo ε-end group via a 3-oxo β-end group. Other xanthophylls such as β-cryptoxanthin and zeaxanthin, which have a 3-hydroxy β-end group, were also oxidized in the same manner as lutein. These keto-carotenoids, derived from dietary xanthophylls, were confirmed to be present in plasma of normal human subjects, and β,ε-caroten-3'-one was significantly increased by the ingestion of β-cryptoxanthin. Thus, humans as well as mice have oxidative activity to convert the 3-hydroxy β-end group of xanthophylls to a 3-oxo ε-end group.

Influence of NAADP and bafilomycine A1 on activity of ATPase in liver postmitochondrial fraction

Influence of NAADP and bafilomycine A1 on activity of ATPase in liver postmitochondrial fraction

Toxicokinetics and tissue depletion of Fusarenon-X and its metabolite nivalenol in piglets

Fusarenon-X (FX) is one of the trichothecene mycotoxins mainly produced by Fusarium crookwellense, which naturally occurs in agricultural commodities such as wheat and barley. To investigate the toxicokinetics of FX and its metabolite nivalenol (NIV), FX was then administered intravenously or orally to piglets at a dosage of 1mg/kg body weight. The concentrations of FX and NIV in the plasma and various tissues were measured using LC-MS/MS. The plasma concentrations of FX in the piglets were determined up to 24h and 48h after iv and po administration, respectively, and the concentration of NIV was detected up to 12h after both types of administration. The Cp0 of FX was 580.28±140.81ng/ml after iv administration. The values of t1/2β, Vss and Foral were 1.71±0.74h, 0.009±0.002ml and 74.40±18.96%, respectively. FX and NIV were detectable in the vital organs up to 24h after po administration. The peak level of FX in the liver, the kidney, and the spleen, respectively, were 165.95±9.68ng/g, 66.29±8.48 and 7.35±0.69ng/g at 3h following po administration. In vitro of liver postmitochondrial fractions with FX demonstrated that the liver and kidney are capable of FX-to-NIV metabolism.

Effects of Oral Administration of Non-genotoxic Hepato-hypertrophic Compounds on Metabolic Potency of Rat Liver

It remains uncertain why non-genotoxic compounds that result in liver hypertrophy cause liver tumors. In an effort to resolve this issue, we examined whether liver post-mitochondrial fraction (S9) prepared from rats treated with non-genotoxic compounds affected the genotoxicity of pro-mutagens. Known hepatotoxic compounds, such as piperonyl butoxide (PBO), decabromodiphenyl ether (DBDE), beta-naphthoflavone (BNF), indole-3-carbinol (I3C) and acetaminophen (AA), were orally administered to male and female F344 rats at doses sufficient to cause liver hypertrophy. Rats received diets containing each test compound for 3 days, 4 weeks or 13 weeks, and were then kept for 4 weeks without the test chemical. S9 prepared from the livers of each group was used for the Ames test with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), benzo[a]pyrene (BaP) and N-nitrosodimethylamine (NDMA). In both sexes, liver hypertrophy was observed following administration of all test compounds, and was then reversed to the control state when administration ceased. The mutagenicity of MeIQx, BaP and NDMA increased with the use of S9 derived from rats treated with non-genotoxic compounds other than AA. DBDE administration had a marked effect on the mutagenicity of BaP (over a 30-fold increase in females) and NDMA (about a 20-fold increase in males). To estimate the involvement of metabolic enzymes in the alteration of mutagenicity, we measured the activity of ethoxyresorufin-O-deethylase (EROD) and methoxyresorufin-O-demethylase (MROD) (phase I enzymes), and UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) (phase II enzymes) in each S9 sample. The activity of phase I enzymes increased, even at the 3rd day following administration, and then decreased gradually, except in the case of AA, while the activity of phase II enzymes increased slightly. These results suggest that non-genotoxic hepato-hypertrophic compounds may be partly involved in carcinogenesis by modulating the metabolism of pre-carcinogens incorporated from the environment, in a manner that is dependent on sex and pre-incorporated chemicals.

Evaluation of Level of Precursors of N-Nitrosamine in Vitro in Wistar Rats Fed Different Levels of Dietary Protein

This study compares the level of Nitrite in urine, protein concentration and nitrite concentration in post mitochondrial fraction of rats fed different levels of dietary protein with concurrent administration of precursors of N-nitrosamine; dimethylamine hydrochloride (DMA-HCl) and sodium nitrite (NaNO2). Thirty Male Wistar rats were divided into three groups and were kept for four weeks. Group one was given high protein diet (64%), group two was given a normal protein diet (27%) and group three was given low protein diet (3.5%). All the groups were administered with 3mg NaNO2 and 20mg DMA-HCL/kg, using the application of spectrophotometric analysis, centrifugation, as well as colorimetric methods. Following administrations of the chemicals to the test animal groups, the concentration of 24 hours urinary excretion of nitrite was 7.417µg/ml in high protein fed rats, 2.063µg/ml in normal protein fed rats and 0.569 µg/ml in low protein fed rats. There was a significant difference (p<0.05) in the nitrite concentration in the group fed high protein diet with concurrent administration of NaNO2 and DMA-HCl. The wistar rats fed with high protein diet, excreted 5.8 to 7 times more nitrite in urine than the severely protein deprived animals. The protein and nitrite concentration of the post mitochondrial fraction of liver was highest in rats that were fed high protein diet. This study has revealed that nutrition status affects metabolism of foreign compounds including nitrites and dimethylamine hydrochloride.

Curcumin and vitamin E modulate hepatic antioxidant gene expression in PTU-induced hypothyroid rats

In the present study, regulatory role of vitamin E and curcumin on antioxidant gene (AOG) expression in hypothyroid rat liver is reported. Adult male rats were rendered hypothyroid by administration of 0.05 % 6-propyl-thiouracil in their drinking water, while vitamin E (200 mg/kg body weight) and curcumin (30 mg/kg body weight) were supplemented orally for 30 days. Expression of antioxidant genes (Cu/Zn-superoxide dismutase; SOD1, Mn superoxide dismutase; SOD2, catalase; CAT, glutathione peroxidase; GPx1 and glutathione reductase; GR) was evaluated using RT-PCR and Western blot analyses. The activities of antioxidant enzymes were measured in mitochondrial fraction (MF) and post-mitochondrial fraction (PMF) of rat liver. In addition measurement of glutathione redox status was also carried out in both the fractions. The enhanced transcripts of CAT, GPx1 and GR in hypothyroid rat liver were alleviated by administration of vitamin E and curcumin. Elevated levels of translated product of all AOGs in hypothyroid group were remained unchanged after antioxidant administration. However, enhanced SOD1, GPx1 and decreased GR activities in PMF were normalized by vitamin E and curcumin. Similarly the increased SOD2, GPx1 and decreased CAT activities in MF were also normalized by vitamin E and curcumin supplementation. Administration of vitamin E and curcumin enhanced mitochondrial GSH level; whereas the enhanced GSH level in PMF of hypothyroid rats was alleviated by vitamin E. Thus it can be concluded that besides the antioxidant role of vitamin E and curcumin, they also regulate hepatic antioxidant gene expression in hypothyroid rats.

The activity of the first line enzymes of the antioxidant defence in the liver of pubertal rats during stress

The purpose of the work was to study the activity of the first line antioxidant defence enzymes in postmitochondrial fraction of liver of pubertal rats during immobilization stress. During short-term immobilization the activity of catalase and glutathione peroxidase (GPx) decreased. Long-term immobilization was accompanied by activation of GPx and superoxide dismutase in the liver postmitochondrial fraction of late pubertal and adult animals, but not early pubertal rats.

Activity of the first line antioxidant defense enzymes in the liver of pubertal rats during stress

Activities of catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD) have been investigated in the liver postmitochondrial fraction of pubertal rats exposed to stress. Short-term immobilization of pubertal rats caused a decrease of catalase and GPx activities. Long-term immobilization was accompanied by activation of GPx and SOD in the liver postmitochondrial fraction of late pubertal and adult animals, but not early pubertal rats.

In Vitro Metabolism of Oxymetazoline: Evidence for Bioactivation to a Reactive Metabolite

Oxymetazoline (6-tert-butyl-3-(2-imidazolin-2-ylmethyl)-2,4-dimethylphenol) has been widely used as a nonprescription nasal vasoconstrictor for >40 years; however, its metabolic pathway has not been investigated. This study describes the in vitro metabolism of oxymetazoline in human, rat, and rabbit liver postmitochondrial supernatant fraction from homogenized tissue (S9) fractions and their microsomes supplemented with NADPH. The metabolites of oxymetazoline identified by liquid chromatography (LC)/UV/tandem mass spectrometry (MS/MS), included M1 (monohydroxylation of the t-butyl group), M2 (oxidative dehydrogenation of the imidazoline to an imidazole moiety), M3 (monohydroxylation of M2), M4 (dihydroxylation of oxymetazoline), and M5 (dihydroxylation of M2). Screening with nine human expressed cytochromes P450 (P450s) identified CYP2C19 as the single P450 isoform catalyzing the formation of M1, M2, and M3. Glutathione conjugates of oxymetazoline (M6) and M2 (M7) were identified in the liver S9 fractions, indicating the capability of oxymetazoline to undergo bioactivation to reactive intermediate species. M6 and M7 were not detected in those liver S9 incubations without NADPH. Cysteine conjugates (M8 and M9) derived from glutathione conjugates and hydroxylated glutathione conjugates (M10 and M11) were also identified. The reactive intermediate of oxymetazoline was trapped with glutathione and N-acetyl cysteine and identified by LC/MS/MS. M6 was isolated and identified by one-dimensional or two-dimensional NMR as the glutathione conjugate of a p-quinone methide. We have shown the tendency of oxymetazoline to form p-quinone methide species via a bioactivation mechanism involving a CYP2C19-catalyzed two-electron oxidation. Nevertheless, we conclude that the formation of this reactive species might not be a safety concern for oxymetazoline nasal products because of the typical low-dose and brief dosage regimen limited to nasal delivery.

DNA-damaging ability of isoprene and isoprene mono-epoxide (EPOX I) in human cells evaluated with the comet assay

Isoprene is produced in combustion processes and is widely used as an industrial chemical. It is a natural product emitted by plants and endogenously produced by humans and other mammals. Therefore, exposure to isoprene from both endogenous and exogenous sources is unavoidable and occurs during the entire human life. Based on evaluations of the International Agency for Research on Cancer (IARC), isoprene has been classified in Group 2B ( possibly carcinogenic to humans). In the present work, we have demonstrated, by use of the single-cell gel electrophoresis assay (SCGE or comet assay), that isoprene is able to induce DNA damage in peripheral blood mononuclear cells (PBMCs) in the presence of metabolic activation. In addition, treatment of cells with the main isoprene mono-epoxide (EPOX I) induced time- and dose- dependent DNA damage in both PBMCs and human leukaemia cells (HL60). The metabolic activation system, represented by rat liver post-mitochondrial fractions (S9), was obtained from rats that had been treated – or not – with inducing agents such as phenobarbital and ethanol. The inclusion of S9 fractions (4 mg protein/mL) from non-induced or phenobarbital-induced rats resulted in a statistically significant enhancement of isoprene genotoxicity. A different pattern was obtained by the addition of ethanol-induced S9, which appeared highly genotoxic by itself even in the absence of isoprene. Reducing the concentration of ethanol-induced S9 to 0.25 mg protein/mL resulted in a considerable enhancement of isoprene genotoxicity. In the absence of clear epidemiological evidence of the carcinogenicity of isoprene in humans, the results of this study seem to be particularly important since they add new findings to support the classification of this chemical as possibly carcinogenic to humans.

An in vitro study on metabolism of 17β-boldenone and boldione using cattle liver and kidney subcellular fractions

17β-Boldenone (17β-BOLD) and Boldione (ADD) are steroid compounds with androgenic activity, likely to be used as growth promoters in cattle. Different studies still on-going aiming to distinguish between “natural” occurrence or illegal BOLD source had already indicated that their metabolism in cattle is of relevant significance. To identify metabolites as in vivo markers to support the thesis of exogenous administration, a further approach to the in vitro biotransformation of 17β-BOLD and ADD was performed using different subcellular fractions obtained from both liver and kidney of untreated cattle. Polar and non-polar metabolites obtained from incubated parent compounds were formerly separated by high performance liquid chromatography (HPLC) elution and successively identified by liquid chromatography tandem mass spectrometry (LC–MS/MS) detection.The bovine liver was the target tissue of the main metabolic reaction transforming 17β-BOLD to ADD and vice versa. The presence of 6β-hydroxy-17β-BOLD, produced from both compounds when NADPH was added as cofactors to liver post mitochondrial and microsomal fractions suggests that cytochrome P450-dependent enzymes could be involved in the biotransformation, as it occurs for 6β-hydroxylation of 17β-testosterone. The results indicated that the urinary excretion profile in vivo of 6β-hydroxy-17β-BOLD and 16α-hydroxy-17β-BOLD could be studied together with 17α- and 17β-BOLD as putative markers of BOLD treatment in cattle.

Neurotoxicity of the pentabrominated diphenyl ether mixture, DE-71, and hexabromocyclododecane (HBCD) in rat cerebellar granule cells in vitro

Polybrominated diphenyl ethers (PBDE) and hexabromocyclododecane (HBCD) are compounds used as additive flame retardants in plastics, electronic equipment, and textiles. The aim of the present study was to investigate the in vitro effects of the pentabrominated diphenyl ether mixture, DE-71, and HBCD on cerebellar granule cells (CGC). Both DE-71 and HBCD induced death of CGC in low micromolar concentrations. The NMDA receptor antagonist MK801 (3 microM), and the antioxidant alpha-tocopherol (50 microM) significantly reduced the cell death. Incubation of the compounds together with the rat liver post-mitochondrial (S9) fraction reduced cell death by 58 and 64% for DE-71 and HBCD, respectively. No ROS formation and no elevation in intracellular calcium were observed. We further demonstrated apoptotic morphology (Hoechst straining) after exposure to low levels of the two brominated flame retardants and signs of DNA laddering were found after DE-71 exposure. However, other hallmarks of apoptosis, like caspase activity, were absent indicating an atypical form of apoptosis induced by DE-71. After intraperitoneal injection of the two compounds both DE-71 and HBCD were found in significant amounts in brain (559 +/- 194 and 49 +/- 13 microg/kg, respectively) and liver (4,010 +/- 2,437 and 1,248 +/- 505 microg/kg, respectively) 72 h after injection. Our results indicate that the lower brominated PBDEs have a higher potency of bioaccumulation than HBCD, and that both compounds have a neurotoxic potential in vitro.

Free radical scavenging activity of Achillea alexandri-regis extracts

The antioxidative potential of Achillea alexandri-regis has been studied by measuring the influence of herb extracts on hydroxyl and superoxide radicals' quantity in different in vitro systems. The ethyl acetate extract exhibited hydroxyl radical scavenging activity in all tested biological system (liver homogenate, hemolyzed blood, serum and postmitochondrial liver fraction), whereas butanol extract reduced hydroxyl radicals significantly only in the postmitochondrial liver fraction. Both extracts showed superoxide radical scavenging effect only in hemolyzed blood.

Antioxidant enzyme activity and lipid peroxidation in liver of female rats co-exposed to lead and cadmium: Effects of vitamin E and Mn2+

The oxidative status of liver of female rats exposed to lead acetate and cadmium acetate either alone or in combination at a dose of 0.05 mg/kg body wt intraperitoneally for 15 days was studied. After the administration of lead alone, the activity of superoxide dismutase (SOD) decreased in liver, whereas no changes were observed in catalase (CAT) activity, and glutathione (GSH) and thiobarbituric acid (TBARS) levels. Cadmium exposure and combined exposure to lead and cadmium led to decrease in GSH content and increased TBARS levels. Moreover, animals exposed to either cadmium alone or in combination with lead showed a decrease in SOD activity and an increase in CAT activity. The in vitro experiments showed that vitamin E failed to restore the antioxidant enzyme activities in metal treated postmitochondrial supernatant fraction of liver. But Mn2+ ions protected the mitochondria from lipid peroxidation and could completely restore Mn-superoxide dismutase (Mn-SOD) activity following metal intoxication. The results of this study indicate that despite the ability of lead and cadmium to induce oxidative stress the effect in liver is not intensified by combined exposure to both lead and cadmium. The observed changes in various oxidative stress parameters in the liver of rats co-exposed to lead and cadmium may result from an independent effect of lead and /cadmium and also from their interaction such as changes in metal accumulation and content of essential elements like Cu, Zn and Fe. These results suggest that when lead and cadmium are present together in similar concentrations, cadmium mediates major effects due to its more reactive nature.

Involvement of oxygen free radicals in the serum‐mediated increase of benzoquinone genotoxicity

The genotoxicity of benzoquinone (BQ), a toxic benzene metabolite, is greatly enhanced by the presence of fetal calf serum (FCS) in the incubation medium. The FCS effect is abolished by heat denaturation of serum proteins and is slightly decreased by dialysis. In the present study, we have further investigated the serum effect on BQ genotoxicity by measuring DNA damage produced in peripheral blood mononuclear cells (PBMCs) using the Comet assay. We have also evaluated the effect of human serum and rat liver post-mitochondrial fraction (S9) on the DNA damage produced by BQ. Both human serum and a rat liver S9 enhanced the genotoxicity of BQ in a manner similar to FCS. Gel filtration experiments showed that all the enhancing activity of the serum eluted with the high molecular weight fractions, suggesting that low molecular weight serum constituents do not play an important role in modulating genotoxicity. The genotoxicity-enhancing activity of serum was inhibited by the iron chelator deferoxamine and by superoxide dismutase and catalase. Incubating PBMCs with BQ in the presence of FCS also resulted in the accumulation of intracellular peroxides as demonstrated by loading the cells with 2',7'-dichlorofluorescin diacetate and analyzing for peroxide formation by flow cytometry. These results indicate that oxygen free radicals are involved in the enhancement of BQ-induced DNA damage by serum. We hypothesize that enzyme activities that reduce BQ by transferring single electrons could be the source of the oxygen free radicals.