In Vitro toxicity of 2- and 4-chloroaniline: comparisons with 4-amino-3-chlorophenol, 2-amino-5-chlorophenol and aminophenols

Chloroanilines have been associated with renal and hepatic toxicity. This study (a) examined the in vitro hepatic and renal toxicity of 2-chloroaniline and 4-chloroaniline, (b) further examined whether aromatic ring hydroxylation would increase toxicity of the parent compound and (c) compared toxicity between respective aminochlorophenol and aminophenol. Renal and hepatic slices were exposed to varying concentrations of 2-chloroaniline, 4-chloroaniline. 4-amino-3-chlorophenol, 2-amino-5-chlorophenol, 2-aminophenol or 4-aminophenol. Toxicity was monitored by measurement of pyruvate-directed gluconeogenesis and leakage of lactate dehydrogenase (LDH). Hepatic tissue was less susceptible to toxicity than kidney tissue for all compounds since LDH leakage was elevated only in renal tissue. Gluconeogenesis was reduced in renal cortical slices exposed to 0.1 μ aminochlorophenols or 4-aminophenol, whereas a concentration of 0.5 μ was necessary for the chloroanilines and 2-aminophenol. LDH release was increased in renal slices by aminochlorophenols and aminophenols but not by the chloroanilines. The nephrotoxic potential in renal cortical slices was 4-aminophenol > 2-amino-5-chlorophenol > 4-amino-3-chlorophenol > 2-aminophenol > 2-chloroaniline = 4-chloroaniline. These results suggest that aromatic ring hydroxylation increased in vitro toxicity of the chloroanilines. Comparison of aminophenols with aminochlorophenols indicated that addition of a halogen can have variable effects on toxicity.     

Immortalized hepatocytes as in vitro model systems for toxicity testing: the comparative toxicity of menadione in immortalized cells, primary cultures of hepatocytes and HTC hepatoma cells

Immortalized differentiated liver cell lines capable of continuous proliferation, and expressing stable liver-specific functions, would be valuable for in vitro toxicity testing in the pharmaceutical, chemical, food and cosmetics industries. Immortalized rat hepatocyte cell lines have been produced by transfection of SV40 DNA by electroporation or calcium phosphate precipitation. Their utility has been assessed by studying the toxicity of a model compound, menadione, and by measuring the activities of DT-diaphorase and NADPH cytochrome c reductase. Enzyme activities and toxicity were compared in freshly isolated hepatocytes, the immortalized cell lines and dedifferentiated HTC hepatoma cells. In HTC cells DT-diaphorase activity was 100-fold elevated compared with freshly isolated hepatocytes. In only one cell line, C2.1.2. (produced by calcium phosphate precipitation), was DT-diaphorase activity increased (twofold) compared with freshly isolated hepatocytes. Menadione caused loss of viability at similar concentrations (40–80 μ ) in the immortalized cell lines and 24-hr primary cultures of hepatocytes, whereas HTC cells showed loss of viability only with menadione concentrations above 200 μ . The immortalized lines therefore appear to have potential for predicting toxicity and for menadione this can be correlated with the expression of DT-diaphorase.     

Myeloperoxidase-mediated bioactivation of 5-hydroxythiabendazole: A possible mechanism of thiabendazole toxicity

Thiabendazole (TBZ), an antihelminthic and antifungal agent, is associated with a host of adverse effects including nephrotoxicity, hepatotoxicity, and teratogenicity. Bioactivation of the primary metabolite of TBZ, 5-hydroxythiabendazole, has been proposed to yield a reactive intermediate. Here we show that this reactive intermediate can be catalyzed by myeloperoxidase (MPO), a neutrophil-bourne peroxidase. Using a cell viability endpoint, we examined the toxicity of TBZ, 5OH-TBZ, and MPO-generated metabolites in cell-based models including primary rat proximal tubule epithelial cells, NRK-52E rat proximal tubule cells, and H9C2 rat myocardial cells. Timecourse experiments with MPO showed complete turnover of 5OH-TBZ within 15 min and a dramatic leftward shift in dose-response curves after 12 h. After a 24 h exposure in vitro, the LC₅₀ of this reactive intermediate was 23.3 ± 0.2 μM reduced from greater than 200 μM from 5OH-TBZ alone, an approximately 10-fold decrease. LC₅₀ values were equal in all cell types used. Comparison of lactate dehydrogenase leakage and caspase 3/7 activity revealed that cell death caused by the reactive intermediate is primarily associated with necrosis rather than apoptosis. This toxicity can be completely rescued via incubation with rutin, an inhibitor of MPO. These results suggest that MPO-mediated biotransformation of 5OH-TBZ yields a reactive intermediate which may play a role in TBZ-induced toxicity.     

Evaluation of drug interactions in intact hepatocytes: Inhibitors of terfenadine metabolism

Terfenadine has been associated with several adverse drug interactions and it was of interest to develop in vitro systems to explain and predict such interactions. The metabolism of terfenadine was studied using intact hepatocytes from primary human and rat hepatocyte cultures, and the immortalized human hepatoma cell line HepG2. Rates and routes of biotransformation were analysed by HPLC. Terfenadine was extensively metabolized by all three cell culture systems during exposure periods ranging from 4 to 24 hr. Human and rat hepatocytes and HepG2 cells formed products of C-oxidation (an acid metabolite and its precursor alcohol metabolite). Human hepatocytes also formed the N-dealkylation product azacyclonol. Several cytochrome P4503A (CYP3A) substrates and inhibitors were evaluated for their ability to inhibit terfenadine biotransformation. In rat hepatocytes, ketoconazole, erythromycin and troleandomycin failed to inhibit; in HepG2 cells, only ketoconazole potently inhibited terfenadine metabolism. In human hepatocytes, ketoconazole, itraconazole, erythromycin, troleandomycin, cyclosporin and naringenin inhibited terfenadine metabolism. The results suggest that human hepatocytes may be a useful system for screening for inhibitors of terfenadine metabolism.     

Spontaneous apoptosis, necrosis, energy status, glutathione levels and biotransformation capacities of isolated rat hepatocytes in suspension: effect of the incubation medium

Isolated hepatocytes in suspension express most of the functional activities of the intact liver and offer an easy-to-handle in vitro system for investigating both the biotransformation and damaging effects induced after a single exposure to xenobiotics upto 3-4h. There is, however, a general lack of consensus with respect to the choice of a suitable suspension medium. This motivated us to perform a comparative study of the effects of five frequently used bicarbonate-based media (Ca(2+)-containing Krebs-Henseleit buffer (KHB) with or without 25mM HEPES, 10mM glucose and 2% (g/v) BSA supplements, and Williams' E culture medium) on the viability (LDH leakage, caspase-3 processing and activity, Bid/Bax expression) and functionality (energy status, glutathione content, phases I and II biotransformation) of freshly isolated rat hepatocytes in suspension upto 3h. Also included was the bicarbonate-free HEPES buffer that does not require carbogen gassing, and is therefore handled more easily. The results clearly demonstrated that the type of incubation medium profoundly affected the functionality of the suspended hepatocytes, changing their sensitivity and response to exogenous damaging effects. While HEPES buffer and Williams' E medium offered the lowest background of spontaneous cell death, bicarbonate-based buffers and media seemed more suitable for obtaining both phases I and II biotransformation. Williams' E medium ensured a constant glutathione content of the cells and a lower level of oxidative stress.     

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.     

Role of phospholipids in the inhibitory action of DDT and permethrin on the nerve ATPases of lobster, homarus americanus

Efforts were made to understand the nature of the site of 1,1-bis-(p-chlorophenyl)-2,2,2-trichloroethane (DDT) inhibition of nerve ATPase. The phospholipid content of nerve preparations from the walking leg of the lobster was reduced by treating them with phospholipase A, or with a chloroform-methanol mixture at ?75°. By these treatments the enzymes lost approximately 70 or 95% of their phospholipids and 50–80% of their Na, K? and Ca-ATPase activities. The lost ATPase activities could be partially restored by the addition of phospholipids, either the ones extracted from the lobster nerves or those from commercial sources. ATPase inhibition by DDT and permethrin was found to be highest in preparations where the phospholipids were removed by the above treatments, next highest with the untreated original enzyme, and least with the reconstituted ATPase regardless of the source of phospholipids used for reconstitution. This tendency was more pronounced in the case of Ca-ATPase. The effects of DDT and permethrin on inhibition of reconstituted Ca-ATPase were higher when the insecticide was first added to the protein portion and the enzyme was then reconstituted with the phospholipids, than when the same amount of insecticide was first added to the phospholipids which were then used for reconstitution.     

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.     

Cytotoxic and genotoxic effects of megazol,an anti-Chagas' disease drug,assessed by different short-term tests

Cyto- and genotoxicity induced by drugs can limit the dose and duration of treatment, can adversely affect patient quality of life, and may be life-threatening. Two drugs are currently being used for treatment of the acute phase of Chagas' disease and both have serious undesirable effects. In this research, cyto- and genotoxic activity of the nitroimidazole-tiadiazole derivative CL 64855 2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole (megazol), a promising alternative drug, was evaluated in vitro with different short-term tests: (a) induction of recombination events and mutation in the yeast Saccharomyces cerevisiae D7 strain, with and without induction of cytochrome P-450; DNA damage (single and double strand breaks, alkali-labile sites, etc.) by the Comet assay in different mammalian cells. S. cerevisiae did not show a significant increase of mutant and recombinant event frequency, both with and without cytochrome P-450. On the other hand, the cytochrome complex appeared to detoxify the drug with respect to cytotoxicity. Results in rat and mouse fresh leukocytes showed a dose-response relation of drug-induced DNA damage. Findings in treated VERO cells suggested a complex treatment time-DNA damage relationship and the possible induction of repair mechanisms. Furthermore, bleomycin effects were increased in rat cells by simultaneous administration of megazol. Megazol shows different biological activity in relation to cellular types and experimental conditions (with or without cytochrome P-450, short/long time of exposure, with or without other genotoxins), thus suggesting a modulation of effectiveness by different physiological/biochemical conditions of cells. The findings could be useful to evaluate new megazol-derived compounds and to assess the risks/benefits relationship for each drug.     

Role of N-methyl-D-aspartate receptors in polychlorinated biphenyl mediated neurotoxicity

Polychlorinated biphenyls (PCBs) are widespread persistent environmental pollutants. Chronic human and animal exposure to PCBs results in various harmful effects including neurotoxicity. This study investigates the effects of the PCB mixture Aroclor 1254 (A1254) and two PCB congeners (coplanar, non-ortho PCB 126, and non coplanar PCB 99) on the expression of N-methyl-D-aspartate receptors (NMDARs) and the subsequent toxic effects using a human SHS5-SY neuroblastoma cell line. NMDAR was measured using a radiolabeled phencyclidine receptor ligand [³H]-MK801, apoptosis was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and cell death using lactate dehydrogenase (LDH) release. After treatment, a positive dose–response relationship of increasing NMDARS, increasing caspase-3 activity and cell death was observed in all PCB compounds. The non-coplanar PCB compounds were found to be significantly more toxic than the coplanar congener and the PCB mixture A1254. PCB-mediated cell death was attenuated with 10 μM NMDAR antagonists: 1-amino-3,5-dimethyladamantane hydrochloride (memantine) and (+)-5-methyl-10,11-dihydro-5H-debenzocyclhepten-5,10-imine maleate ((+)-MK-801), thus demonstrating the importance of NMDAR in PCB neurotoxicity. Intracellular calcium [Ca²⁺]_i chelator BAPTA-AM (1 μM) partially attenuated the neurotoxic effect of the PCBs suggesting a role of calcium homeostasis disruption in the neurotoxicity of PCBs. These results suggest that the neurotoxicity of PCBs can be mediated through activation of NMDARs.     

Cytotoxic effects of incense particles in relation to oxidative stress,the cell cycle and F-actin assembly

Epidemiological studies have suggested that combustion-derived smoke, such as that produced during incense burning, is a deleterious air pollutant. It is capable of initiating oxidative stress and mutation; however, the related apoptotic processes remain unclear. In order to elucidate the biological mechanisms of reactive oxygen species (ROS)-induced respiratory toxicology, alveolar epithelial A549 cells were exposed to incense particulate matter (PM), with and without antioxidant N-acetyl-l-cysteine (NAC). The cross-linking associations between oxidative capacity, cell cycle events, actin cytoskeletal dynamics and intracellular calcium signals were investigated. An incense PM suspension caused significant oxidative stress in A549 cells, as shown by inhibition of the cell cycle at G1 and G2/M check-points, and the induction of apoptosis at Sub-G1. At the same time, alterations in the F-actin filamentous assemblies were observed. The levels of intracellular Ca²⁺ were increased after incense PM exposure. Antioxidant NAC treatment revealed that oxidative stress and F-actin remodelling was significantly mitigated. This suggests that ROS accumulation could alter cell cycle regulation and anomalous remodelling of the cortical cytoskeleton that allowed impaired cells to enter into apoptosis. This study has elucidated the integral patho-physiological interactions of incense PM and the potential mechanisms for the development of ROS-driven respiratory impairment.     

Induction of liver microsomal cytochrome P-450 And associated monooxygenases by octachlorostyrene in inbred strains of mice: Lack of correlation with the murinE Ah locus

Single intraperitoneal injections of octachlorostyrene (OCS) and hexachlorobenzene in genetically polycyclic aromatic hydrocarbon ‘responsive’ C57/BL/6 (B6) mice led to a time- and dose-dependent increase in the levels of liver microsomal cytochromes P-450 and b₅ as well as in the activities of NADPH cytochrome P-450 (cytochrome c) reductase, ethylmorphine (EM) N-demethylase, 4-nitroanisole (PNA) O-demethylase and acetanilide 4-hydroxylase (AcA hydroxylase). No, or only a very moderate, increase in the activity of aryl hydrocarbon hydroxylase was seen after OCS and HCB, respectively. Pretreatments with phenobarbital (PB) or 3-methylcholanthrene (MC) both increased AcA hydroxylase activity to a similar degree, whereas pretreatment with polychlorinated biphenyls (Aroclor 1254) had an effect equal to the sum of PB and MC. Judged from sodium dodecylsulfate polyacrylamide gel electrophoresis studies, OCS and HCB predominantly increased a microsomal polypeptide of apparent mol. wt 52,000, similar to PB. A reduced response was seen after OCS or HCB treatment of aromatic hydrocarbon ‘non-responsive’ DBA/2 (D2) mice compared to B6 mice, both with respect to AcA hydroxylase as well as EM demethylase and PNA demethylase activities. OCS treatment of B6D2F1 mice resulted in a doubling of AcA hydroxylase activity, but in mice of the (B6D2)D2 backcross no distinct subgroupings of individual AcA hydroxylase activities were apparent. These results demonstrate that OCS is an inducer of the PB-type in mice and that induction of AcA hydroxylase by OCS is not regulated by the Ah locus.     

Functional properties and substrate characterization of human CYP26A1, CYP26B1, and CYP26C1 expressed by recombinant baculovirus in insect cells

Introduction

The cytochrome P450 CYP26 family of retinoic acid (RA) metabolizing enzymes, comprising CYP26A1, CYP26B1, and CYP26C1 is critical for establishing patterns of RA distribution during embryonic development and retinoid homeostasis in the adult. All three members of this family can metabolize all trans-RA. CYP26C1 has also been shown to efficiently metabolize the 9-cis isomer of RA.

Methods

We have co-expressed each of the CYP26 enzymes along with the NADPH-cytochrome P450 oxidoreductase using a baculovirus/Sf9 insect cell expression system to determine the enzymatic activities of these enzymes in cell free preparations and have established an in vitro binding assay to permit comparison of binding affinities of the three CYP26 enzymes.

Results

We demonstrated that the expressed enzymes can efficiently coordinate heme, as verified by spectral-difference analysis. All CYP26s efficiently metabolized all-trans-RA to polar aqueous-soluble metabolites, and in competition experiments exhibited IC₅₀ values of 16, 27, and 15 nM for CYP26A1, B1, and C1 respectively for all-trans-RA. Furthermore, this metabolism was blocked with the CYP inhibitor ketoconazole. CYP26C1 metabolism of all trans-RA could also be effectively competed with 9-cis RA, with IC₅₀ of 62 nM, and was sensitive to ketoconazole inhibition.

Discussion

CYP26 enzymes are functionally expressed in microsomal fractions of insect cells and stably bind radiolabeled RA isomers with affinities respecting their substrate specificities. We demonstrated that compared to CYP26A and CYP26B, only CYP26C1 was able to bind with high affinity to 9-cis-RA. These assays will be useful for the screening of synthetic substrates and inhibitors of CYP26 enzymes and may be applicable to other cytochrome P450s and their respective substrates.     

Cytotoxic effects of metal protoporphyrins in glioblastoma cells: roles of albumin, reactive oxygen species, and heme oxygenase-1

We investigate the cytotoxic effect of metal protoporphyrins including ferric protoporphyrin (FePP; hemin), cobalt protoporphyrin (CoPP), and tin protoporphyrin (SnPP) in glioblastoma cells C6 and GBM8401. Data of MTT assay show that FePP and CoPP, but not SnPP, significantly reduce the viability of glioma cells C6 and GBM8401 in the absence of serum. In the condition with fetal bovine serum (FBS) or bovine serum albumin (BSA), the cytotoxic effect of FePP and CoPP was completely inhibited. Binding of FePP, CoPP, and SnPP with BSA was examined via spectrophotometer analysis, and the protective effect of serum against FePP and CoPP-induced cell death was abolished by BSA depletion. A loss in the integrity of DNA with an occurrence of apoptotic events including DNA ladders, caspase 3 and PARP protein cleavage, and chromatin-condensed cells is observed in FePP-treated or CoPP-treated C6 cells. An increase in intracellular peroxide level was examined in FePP, but not CoPP, -treated C6 cells, and N-acetyl-l-cysteine (NAC) addition significantly protected C6 cells from FePP, but not CoPP, -induced cell death with reducing FePP-stimulated reactive oxygen species (ROS) production. Activation of extracellular regulated kinases (ERKs) and c-Jun-N-terminal kinases (JNKs) with an increase in the heme oxygenase-1 (HO-1) protein was observed in FePP-treated or CoPP-treated C6 cells in the absence of FBS or BSA, and adding JNKs inhibitor SP600125 (SP), but not ERKs inhibitor PD98059 (PD), significantly attenuated FePP-induced or CoPP-induced HO-1 protein expression in accordance with reducing JNKs protein phosphorylation. However, PD98059, SP600125, or transfection of C6 cells with antisense HO-1 oligonucleotides show no effect on the cytotoxicity elicited by FePP and CoPP in C6 cells. Effect of serum and BSA on the cytotoxicity of metal protoporphyrins in glioma cells is first demonstrated in the present study, and the roles of ROS, MAPKs, and HO-1 were elucidated.     

Effect of taurine on advanced glycation end products-induced hypertrophy in renal tubular epithelial cells

Mounting evidence indicates that advanced glycation end products (AGE) play a major role in the development of diabetic nephropathy (DN). Taurine is a well documented antioxidant agent. To explore whether taurine was linked to altered AGE-mediated renal tubulointerstitial fibrosis in DN, we examined the molecular mechanisms of taurine responsible for inhibition of AGE-induced hypertrophy in renal tubular epithelial cells. We found that AGE (but not non-glycated BSA) caused inhibition of cellular mitogenesis rather than cell death by either necrosis or apoptosis. There were no changes in caspase 3 activity, bcl-2 protein expression, and mitochondrial cytochrome c release in BSA, AGE, or the antioxidant taurine treatments in these cells. AGE-induced the Raf-1/extracellular signal-regulated kinase (ERK) activation was markedly blocked by taurine. Furthermore, taurine, the Raf-1 kinase inhibitor GW5074, and the ERK kinase inhibitor PD98059 may have the ability to induce cellular proliferation and cell cycle progression from AGE-treated cells. The ability of taurine, GW5074, or PD98059 to inhibit AGE-induced hypertrophy was verified by the observation that it significantly decreased cell size, cellular hypertrophy index, and protein levels of RAGE, p27^Kip1, collagen IV, and fibronectin. The results obtained in this study suggest that taurine may serve as the potential anti-fibrotic activity in DN through mechanism dependent of its Raf-1/ERK inactivation in AGE-induced hypertrophy in renal tubular epithelial cells.     

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.     

Taurine protects rat testes against NaAsO2-induced oxidative stress and apoptosis via mitochondrial dependent and independent pathways

Arsenic (As) is a well known toxicity inducer. Recent investigations, however, showed that it might have some therapeutic application in cancer treatment. These dual roles of arsenic have attracted a renewed research in organ pathophysiology. In this study, we report that As administration (in the form of NaAsO₂ at a dose of 10 mg/kg body weight for 2 days, orally) induces apoptosis in testicular tissue of the experimental rats by the activation of caspase-3 and reciprocal regulation of Bcl-2/Bad with the concomitant reduction of mitochondrial membrane potential and increased level of cytosolic cytochrome C. Arsenite has also been shown to induce activation of mitogen-activated protein kinases (MAPKs), Akt as well as NF-κB (p65) in testicular tissue. In addition, As significantly decreased testicular Δ⁵-3β-HSD and 17β-HSD activities and reduced the plasma testosterone level, testicular sperm count and sperm motility. Besides, arsenite exposure increased the levels of reactive oxygen species (ROS), serum TNF-α, As accumulation and lipid peroxidation and decreased the activities of the antioxidant enzymes and glutathione in the testicular tissue. Oral administration of taurine (at a dose of 100 mg/kg body weight for 5 days) was found to be effective in counteracting As-induced oxidative stress, attenuation of testicular damages and amelioration of apoptosis in testicular tissue by controlling the reciprocal regulation of Bcl-2/Bad, phospho-ERK1/2, phospho-p38, phospho-Akt and NF-κB. Taurine was also found to play similar beneficial role via mitochondrial dependent pathways in As-induced testicular damages leading to apoptotic cell death.     

Elimination,distribution and metabolism of di-(2-ethylhexyl)adipate (deha) in rats

The excretion, retention, distribution and metabolism of di-(2-ethylhexyl)adipate (DEHA) have been studied in the rat.After oral administration of [¹⁴C]DEHA, almost all the dose was excreted within 48 h, predominantly in the urine and as respiratory carbon dioxide. The faecal excretion was low. There was no evidence of the accumulation of radioactivity in any organs or tissues. Adipic acid (AA) was found to be the main urinary metabolite; it was also detected in the digestive tract, blood and liver.In vitro, DEHA was hydrolyzed at a significant rate by tissue preparations prepared from liver, pancreas and small intestine of the rat.These results suggest that orally administered DEHA is rapidly hydrolyzed in the body to form AA without any accumulation of mono-(2-ethylhexyl)adipate (MEHA).     

Crotonaldehyde induces oxidative stress and caspase-dependent apoptosis in human bronchial epithelial cells

Crotonaldehyde is a widespread environmental pollutant and lipid peroxidation product. Crotonaldehyde is a risk factor for many diseases (e.g., chronic pulmonary inflammation). However, its toxicity and its mechanism of action have not been thoroughly investigated. The purpose of this study is to investigate crotonaldehyde-induced oxidative stress and mechanism of cell death in BEAS-2B cells. Crotonaldehyde caused decreases of intracellular reduced glutathione levels and increases of reactive oxygen species in a dose-dependent manner. Crotonaldehyde induced cell death by apoptosis, and gradually transitioned to necrosis at high dose of crotonaldehyde, as demonstrated by Annexin V-FITC/PI staining and cell morphology analysis. Crotonaldehyde-induced ATP decline observed in the study might partially account for the switch from apoptosis to necrosis. Mitochondria membrane potential, cytochrome c release, caspase-9, and caspase-3/7 activity were investigated, and the results suggest that crotonaldehyde-induced apoptosis was activated in a caspase-dependent way. Collectively, these results demonstrate crotonaldehyde induces cell oxidative stress and caspase-dependent apoptosis.     

Characterization of major phytocannabinoids, cannabidiol and cannabinol, as isoform-selective and potent inhibitors of human CYP1 enzymes

Inhibitory effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN), the three major constituents in marijuana, on catalytic activities of human cytochrome P450 (CYP) 1 enzymes were investigated. These cannabinoids inhibited 7-ethoxyresorufin O-deethylase activity of recombinant CYP1A1, CYP1A2, and CYP1B1 in a competitive manner. CBD most potently inhibited the CYP1A1 activity; the apparent K_i value (0.155 μM) was at least one-seventeenth of the values for other CYP1 isoforms. On the other hand, CBN more effectively decreased the activity of CYP1A2 and CYP1B1 (K_i = 0.0790 and 0.148 μM, respectively) compared with CYP1A1 (K_i = 0.541 μM). Δ⁹-THC less potently inhibited the CYP1 activity than CBD and CBN, and showed low selectivity against the CYP1 inhibition (K_i = 2.47-7.54 μM). The preincubation of CBD resulted in a time- and concentration-dependent decrease in catalytic activity of all the recombinant CYP1 enzymes and human liver microsomes. Similarly, the preincubation of Δ⁹-THC or CBN caused a time- and concentration-dependent inhibition of recombinant CYP1A1. The inactivation of CYP1A1 by CBD indicated the highest k_inact/K_I value (540 l/mmol/min) among the CYP1 enzyme sources tested. The inactivation of recombinant CYP1A1 and human liver microsomes by CBD required NADPH, was not influenced by dialysis and by glutathione, N-acetylcysteine, and superoxide dismutase as trapping agents. These results indicated that CBD and CBN showed CYP1 isoform-selective direct inhibition and that CBD was characterized as a potent mechanism-based inhibitor of human CYP1 enzymes, especially CYP1A1.