An examination of the antibiotic effects of cylindrospermopsin on common gram-positive and gram-negative bacteria and the protozoan Naegleria lovaniensis

The importance of the toxin cylindrospermopsin to the function and fitness of the cyanobacteria that produce it remains a matter of conjecture. Given that the structure of cylindrospermopsin has commonalities with other antibacterial protein synthesis inhibitors, such as streptomycin, authors tested the possibility that the toxin might act as an antibacterial compound that can kill competing microbes. Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa were tested by the minimal inhibitory concentration method and significant antibacterial activity was only observed at a cylindrospermopsin concentration of 300 microg mL(-1) after exposure for 5 days. No effect on log phase growth of E. coli was observed for this same toxin concentration. Protein synthesis was inhibited by cylindrospermopsin in E. coli 70S extracts, reduced by 25% compared with controls when treated with 41.5 microg mL(-1) of the toxin; however, a much greater reduction of 97% was observed for chloramphenicol in the same experiment. Naegleria lovaniensis, a phagotrophic protozoan, was more susceptible to cylindrospermopsin, with a decrease in the number of N. lovaniensis plaques after 24-h treatment with 5-50 microg mL(-1) of toxin and an LC(50) of approximately 60 microg mL(-1). Given these results, cylindrospermopsin is clearly not antibacterial at concentrations found in environmental waters, nor will it adversely affect N. lovaniensis at these concentrations. For organisms that are able to ingest cylindrospermopsin-producing cells, the response of N. lovaniensis to the toxin suggests that only a few ingested cells would be enough to kill predatory organisms with similar susceptibility.     

Impaired glutathione-conjugating capacity by cryopreserved human and rat hepatocytes

The activity of glutathione transferase was measured in sonicates of fresh rat hepatocytes and of cryopreserved rat, human and dog hepatocytes in the presence of added glutathione and by using 1-chloro-2,4-dinitrobenzene (CDNB) as non-selective substrate. The glutathione-conjugating capacity was also investigated in the presence of CDNB alone (without glutathione) with intact fresh rat hepatocytes and cryopreserved rat and human hepatocytes. Finally, the intracellular level of glutathione was measured in these hepatocytes. The specific activity of glutathione transferase in sonicates of fresh rat hepatocytes (in the presence of added GSH and CDNB) was about 415 nmol/min/10(6) cells. The corresponding activities in cryopreserved rat, human and dog hepatocytes were approximately 320, 440 and 540 nmol/min/10(6) cells, respectively. In contrast, glutathione conjugation by the intact cryopreserved human and rat hepatocytes in the presence of CDNB alone was less than 10% of the corresponding conjugation by fresh rat hepatocytes, indicating that glutathione was depleted in these cryopreserved hepatocytes. Glutathione depletion was confirmed after analytical measurement of the glutathione levels in fresh and cryopreserved hepatocytes. In fresh rat hepatocytes the level of glutathione was 44 nmol/10(6) cells, whereas it was 2.5 and 4.4 nmol/10(6) cells in cryopreserved rat and human hepatocytes, respectively. In summary, glutathione transferase was active in these cryopreserved hepatocytes but the cryopreservation procedure likely causes depletion in the intracellular level of glutathione, resulting in an overall reduced glutathione conjugating capacity.     

Impaired glutathione-conjugating capacity by cryopreserved human and rat hepatocytes

The activity of glutathione transferase was measured in sonicates of fresh rat hepatocytes and of cryopreserved rat, human and dog hepatocytes in the presence of added glutathione and by using 1-chloro-2,4-dinitrobenzene (CDNB) as non-selective substrate. The glutathione-conjugating capacity was also investigated in the presence of CDNB alone (without glutathione) with intact fresh rat hepatocytes and cryopreserved rat and human hepatocytes. Finally, the intracellular level of glutathione was measured in these hepatocytes. The specific activity of glutathione transferase in sonicates of fresh rat hepatocytes (in the presence of added GSH and CDNB) was about 415?nmol/min/10⁶ cells. The corresponding activities in cryopreserved rat, human and dog hepatocytes were approximately 320, 440 and 540?nmol/min/10⁶ cells, respectively. In contrast, glutathione conjugation by the intact cryopreserved human and rat hepatocytes in the presence of CDNB alone was less than 10% of the corresponding conjugation by fresh rat hepatocytes, indicating that glutathione was depleted in these cryopreserved hepatocytes. Glutathione depletion was confirmed after analytical measurement of the glutathione levels in fresh and cryopreserved hepatocytes. In fresh rat hepatocytes the level of glutathione was 44?nmol/10⁶?cells, whereas it was 2.5 and 4.4?nmol/10⁶?cells in cryopreserved rat and human hepatocytes, respectively. In summary, glutathione transferase was active in these cryopreserved hepatocytes but the cryopreservation procedure likely causes depletion in the intracellular level of glutathione, resulting in an overall reduced glutathione conjugating capacity.     

Conjugated bile acids regulate hepatocyte glycogen synthase activity in vitro and in vivo via Galphai signaling

The regulation of glycogen synthase activity by bile acids in primary hepatocytes and in the intact liver was investigated. Bile acids (deoxycholic acid, DCA; taurocholic acid, TCA) activated AKT and glycogen synthase (GS) in primary rat hepatocytes. Incubation with a phosphatidyl inositol-3 kinase inhibitor or expression of dominant-negative AKT in primary rat hepatocytes abolished activation of AKT and GS by DCA and TCA. TCA, but not DCA, activated Galpha(i) proteins in primary rat hepatocytes. Treatment of cells with pertussis toxin or expression of dominant-negative Galpha(i) blocked TCA-induced activation of AKT and of GS but did not alter AKT or GS activation caused by DCA. TCA caused activation of AKT and GS in intact rat liver. Expression of dominant-negative Galpha(i) reduced TCA-induced activation of AKT and of GS in intact rat liver. Together, our findings demonstrate that bile acids are physiological regulators of glycogen synthase in rat liver and that conjugated bile acids use a Galpha(i)-coupled G protein-coupled receptor to regulate GS activity in vitro and in vivo.     

Inhibition of protein synthesis in liver and kidney of mice by bolesatine: mechanistic approaches to the mode of action at the molecular level.

Protein synthesis was assayed in liver and kidney of mice treated with bolesatine, a toxic glycoprotein from the mushroom Boletus satanas (Lenz) which was previously shown to be an inhibitor of protein synthesis by cell-free systems in vitro and by cultured cell-lines. Protein synthesis in vivo (Swiss mice) is inhibited in a dose-dependent manner in liver and kidney. The mechanism of action does not appear to be due to RNA-N-glycosidase activity of bolesatine or a RNAase activity of this toxin on the ribosomal RNAs. Ribosomes do not appear to be damaged by pretreatment with bolesatine as judged by a poly(U) translation system. Thus bolesatine cannot be included in the group of protein synthesis inhibitors of plant origin known as ribosome-inactivating proteins (RIPs).     

丹参对GSH-Px/MDA比值的影响及其在人肝细胞胶原合成中的作用

利用四氯化碳（CCl4）造成肝细胞脂质过氧化损伤，测定原代培养人胚肝细胞匀浆中羟脯氨酸（Hyp）含量及培养液中Ⅲ型前胶原（PCⅢ）、脂质过氧化物（MDA）、谷胱甘肽过氧化物酶（GSH－Px）活性水平．观察丹参对GSH－Px／MDA比值的影响及其与PCⅢ、Hyp水平的关系。结果：与损伤模型组比较，丹参（1mg／ml）预作用4h，可明显减少受损肝细胞产生PCⅢ、Hyp及MDA，提高GSH－Px活性，GSH－Px／MDA比值增加非常显著（P＜0．01）。提示丹参可抑制CCl4所致人胚肝细胞胶原合成。     

Synthesis and biological evaluation of antibody conjugates of phosphate prodrugs of cytotoxic DNA alkylators for the targeted treatment of cancer

The synthesis and biological evaluation of phosphate prodrugs of analogues of 1 (CC-1065) and their conjugates with antibodies are described. The phosphate group on the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) portion of the compounds confers enhanced solubility and stability in aqueous solutions. In the presence of phosphatases, these compounds convert into active DNA-alkylating agents. The synthesis of the prodrugs was achieved sequentially through coupling of CBI with a bis-indolyl moiety, followed by attachment of a thiol-containing linker, and conversion of the hydroxyl group of CBI into a phosphate prodrug. The linkers incorporated into the prodrugs enable conjugation to an antibody via either a stable disulfide or thioether bond, in aqueous buffer solutions containing as little as 5% organic cosolvent, resulting in exclusively monomeric and stable antibody-cytotoxic prodrug conjugates. Two disulfide-containing linkers differing in the degree of steric hindrance were used in antibody conjugates to test the effect of different rates of intracellular disulfide cleavage and effector release on biological activity. The prodrugs can be converted to the active cytotoxic compounds through the action of endogenous phosphatases. Antibody-prodrug conjugates displayed potent antigen-selective cytotoxic activity in vitro and antitumor activity in vivo.     

Responses of antioxidant systems in the hepatocytes of common carp (Cyprinus carpio L.) to the toxicity of microcystin-LR.

The freshwater, bloom-forming cyanobacterium (blue-green alga) Microcystis aeruginosa produces a peptide hepatotoxin, which causes the damage of animal liver. Recently, toxic Microcystis blooms frequently occur in the eutrophic Dianchi Lake (300 km2 and located in the South-Western of China). Microcystin-LR from Microcystis in Dianchi was isolated and purified by high performance liquid chromatography (HPLC) and its toxicity to mouse and fish liver was studied (Li et al., 2001). In this study, six biochemical parameters (reactive oxygen species, glutathione, superoxide dismutase, catalase, glutathione peroxide and glutathione S-transferase) were determined in common carp hepatocytes when the cells were exposed to 10 microg microcystin-LR per litre. The results showed that reactive oxygen species (ROS) contents increased by more than one-time compared with the control after 6 h exposure to the toxin. In contrast, glutathione (GSH) levels in the hepatocytes exposed to microcystin-LR decreased by 47% compared with the control. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxide (GSH-Px) increased significantly after 6 h exposure to microcystin-LR, but glutathione S-transferase (GST) activity showed no difference from the control. These results suggested that the toxicity of microcystin-LR caused the increase of ROS contents and the depletion of GSH in hepatocytes exposed to the toxin and these changes led to oxidant shock in hepatocytes. Increases of SOD, CAT and GSH-Px activities revealed that these three kinds of antioxidant enzymes might play important roles in eliminating the excessive ROS. This paper also examined the possible toxicity mechanism of microcystin-LR on the fish hepatocytes and the results were similar to those with mouse hepatocytes.     

Futile cycling of a sulfate conjugate by isolated hepatocytes

The sulfate conjugate of the model compound 4-methylumbelliferone was taken up and hydrolyzed considerably more rapidly by isolated hepatocytes than was the glucuronide conjugate. Using intact hepatocytes or homogenates of hepatocytes, compounds were identified that either inhibited 4-methylumbelliferyl sulfate hydrolysis via arylsulfatase or impaired its uptake into cells. For example, sodium sulfate inhibited hydrolysis of 4-methylumbelliferyl sulfate by intact hepatocytes (half-maximal inhibition, 0.1 mM) but not by homogenates, suggesting a selective action on organic sulfate uptake at the plasma membrane. In contrast, cholesterol sulfate inhibited hydrolysis of 4-methylumbelliferyl sulfate by homogenates but not by hepatocytes, consistent with the hypothesis that cholesterol sulfate does not readily enter intact cells. Compounds that inhibited hydrolysis of 4-methylumbelliferyl sulfate by both isolated hepatocytes and microsomes include sodium sulfite (half-maximal inhibition, 0.1 mM), pregnenolone sulfate (half-maximal inhibition, 1 microM), and estrone sulfate (half-maximal inhibition, 10 microM). To test whether production of sulfate conjugates could be modified by agents affecting arylsulfatase in intact hepatocytes, we examined the effects of pregnenolone sulfate on the production of 4-methylumbelliferyl sulfate from 4-methylumbelliferone. Addition of pregnenolone sulfate (100 microM) to intact cells increased rates of 4-methylumbelliferone sulfate production and decreased the fraction of 4-methylumbelliferone converted into the glucuronide. Hydrolysis of 4-methylumbelliferyl sulfate by isolated microsomes was inhibited in a dose-dependent manner by adenosine 3'-phosphate 5'-phosphosulfate (PAPS) when cytosol, a source of sulfotransferase was present. Furthermore, addition of low concentrations of PAPS (0.5 microM) to a reconstituted system of microsomes and cytosol impaired the formation of fluorescent product from 4-methylumbelliferyl sulfate until PAPS was consumed, indicating that futile cycling via arylsulfatase and sulfotransferase occurred. Subsequent futile cycling of free 4-methylumbelliferone and 4-methylumbelliferyl sulfate occurred upon repeated additions of PAPS and was prevented by sodium sulfite, an inhibitor of arylsulfatase. These results argue strongly that sulfate conjugate production within hepatocytes is regulated by futile cycling via sulfotransferase and arylsulfatase. Thus, drugs and endogenous substances that affect arylsulfatase may have marked effects on sulfate conjugate production by the liver.     

The effect of synthetic ceramide analogues on gastritis and esophagitis in rats

The effects of ceremide analogues on esophagitis and gastritis in rats were examined. Gastritis induced by indomethacin was significantly reduced after CY3325 and CY3723 treatment, whereas other analogues had no effect. The amount of malondialdehyde in gastritis was significantly reduced by CY3325 or CY 3723. CY3325 or CY 3723 decreased the glutathione levels in gastritis. The myeloperoxidase level in gastritis is increased, and its increment was decreased by CY3325 and CY3723. In reflux esophagitis, the ulceration was decreased by CY3325, CY3723. The gastric volume and acid output are reduced, whereas the pH value is increased by CY3325 or CY3723 after esophagitis. These results suggest that ceramide analogues, CY3325 and CY3723, can prevent the development of gastritis and reflux esophagitis in rats.     

Synthesis and antitumor activity of conjugates of muramyldipeptide, normuramyldipeptide, and desmuramylpeptides with acridine/acridone derivatives

The synthesis of two groups (Chart 1, types A and B) of conjugates of MDP (muramyldipeptide) and nor-MDP (normuramyldipeptide) with acridine/acridone derivatives and the synthesis of analogues of desmuramylpeptides (Chart 1, types C and D) containing acridine/ acridone derivatives have been described. In type A conjugates, the hydroxyl group at C6 of the sugar moiety was acylated with acridine/acridone N-substituted omega-aminoalkanocarboxylic acids (Scheme 1), whereas the conjugates of type B (Table 2) and three analogues of type C or D (Scheme 2) have an amide bond formed between the carboxylic group of isoglutamine and the amine function of the respective acridine/acridone derivatives. The preliminary screening data indicate that the analogues of groups A, C, and D exhibit small cytotoxic activity, whereas several analogues of type B, 4b, 4c, 4e, 4g, 4h, 4i, and 4l, exhibiting potent in vitro cytotoxic activity against a panel of human cell lines (Table 4), have been selected by the National Cancer Institute (NCI) Evaluation Committee for further testing. Analogues 4b and 4h were active in the in vivo hollow fiber assay (Table 5). Analogue 3a shows an immunostimulating effect on the cytotoxic activity of the NK cells obtained from the spleen of healthy and Ab melanoma bearing animals.     

Mechanisms of in vitro immunosuppression by hepatocyte-generated cyclophosphamide metabolites and 4-hydroperoxycyclophosphamide

Cyclophosphamide (CY) is metabolized to 4-hydroxy-CY which spontaneously breaks down to the reactive intermediates, phosphoramide mustard (PAM) and acrolein. The alkylating property of PAM is thought to mediate the anti-proliferative and cytotoxic actions of CY. Acrolein is known to bind sulfhydryl groups of cellular molecules and may contribute to the action of CY. However, the role of acrolein in the CY-induced immunosuppression remains unclear. The results of studies in which a hepatocyte co-culture system was used suggest that acrolein may play an important role in the cytotoxic action of CY, whereas those investigations using activated derivatives of CY indicate that acrolein is not an important factor. Thus, both approaches of CY exposure were utilized in the present study. Splenocytes of B6C3F1 mice were incubated with syngeneic isolated hepatocytes and CY or with 4-hydroperoxycyclophosphamide (4-HC) (which spontaneously decomposes to 4-hydroxy-CY). The in vitro antibody forming cell (AFC) response was found to be suppressed with both methods of exposure to CY metabolites. The addition of DNA to bind extracellular PAM was ineffective in preventing the suppression produced by hepatocyte-activated CY. However, it was also observed that DNA was able to attenuate the PAM-induced suppression. The sulfhydryl compounds 2-mercaptoethanesulfonate (MESNA) (15 microM) or reduced glutathione (GSH) (1 mM) inhibited the suppression of the AFC response of splenocytes incubated with CY and mouse hepatocytes. The suppression produced by 4-HC, however, was not affected by MESNA and only slightly inhibited by GSH. Similarly, the PAM-induced suppression was not affected by MESNA and slightly attenuated with GSH. In contrast, both MESNA and GSH were very effective in abrogating the acrolein-induced suppression, whereas DNA was ineffective. The findings of this study suggest that in the hepatocyte co-culture system, the immunosuppressive actions of CY are mediated by acrolein generated outside of the splenocyte, whereas the 4-HC induced suppression is not mediated by extracellular acrolein. Thus, this difference may explain the contradictory findings of previous studies that used different means of exposing cells to activated CY.     

Cylindrospermopsin-induced protein synthesis inhibition and its dissociation from acute toxicity in mouse hepatocytes

The toxicology of the cyanobacterial alkaloid cylindrospermopsin (CYN), a potent inhibitor of protein synthesis, appears complex and is not well understood. In exposed mice the liver is the main target for the toxic effects of CYN. In this study primary mouse hepatocyte cultures were used to investigate the mechanisms involved in CYN toxicity. The results show that 1-5 microM CYN caused significant concentration-dependent cytotoxicity (52%-82% cell death) at 18 h. Protein synthesis inhibition was a sensitive, early indicator of cellular responses to CYN. Following removal of the toxin, the inhibition of protein synthesis could not be reversed, showing behavior similar to that of the irreversible inhibitor emetine. In contrast to the LDH leakage, protein synthesis was maximally inhibited by 0.5 microM CYN. No protein synthesis occurred over 4-18 h at or above this concentration. Inhibition of cytochrome P450 (CYP450) activity with 50 microM proadifen or 50 microM ketoconazole diminished the toxicity of CYN but not the effects on protein synthesis. These findings imply a dissociation of the two events and implicate the involvement of CYP450-derived metabolites in the toxicity process, but not in the impairment of protein synthesis. Thus, the total abolition of protein synthesis may exaggerate the metabolite effects but cannot be considered a primary cause of cell death in hepatocytes over an acute time frame. In cell types deficient in CYP450 enzymes, protein synthesis inhibition may play a more crucial role in the development of cytotoxicity.     

In vitro effect of methanol on folate-deficient rat hepatocytes

Methanol is primarily metabolized by oxidation to formaldehyde and then to formic acid. These processes are accompanied by formation of superoxide anion and hydrogen peroxide. This paper reports the in vitro antioxidant effect of vitamin E on isolated hepatocytes of folic acid deficient rats rendered so as to emulate a human hepatocyte model. These hepatocytes were treated with 320 microM of methanol per million cells and incubated for 30 min. The microsomal fraction of these hepatocytes showed a decreased level of superoxide dismutase (SOD), with increase in lipid peroxidation (LPO) shown by increase in recorded levels of malondialdehyde (MDA). Catalase activity was shown to be increased. Levels of reduced glutathione (GSH) were decreased and the activity of glutathione peroxidase (GSH-Px) and of glutathione reductase (GSSG-R) were not altered. The hepatocytes of folate deficient rats pretreated with vitamin E, when subjected to methanol treatment, showed no significant change in SOD levels and a significant decrease in MDA levels. The catalase activity in this group of animals showed a highly significant decrease. These animals had normal levels of GSH, while a significant fall in GSH-Px and GSSG-R levels were observed. These results suggest that Vitamin E exerts a protective effect on hepatocytes by acting as a free radical scavenger, proving its usefulness in treating methanol toxicity.     

Semisynthetic macrolide antibacterials derived from tylosin. Synthesis and structure-activity relationships of novel desmycosin analogues

A series of 20-O-substituted and 3,20-di-O-substituted derivatives of desmycosin were synthesized and their biological properties were evaluated. In particular, we have synthesized numerous side chain modified analogues of desmycosin as well as some analogues possessing a combination of modified side chain and alternative C-3 substituents. Thus, alpha,beta-unsaturated analogues of desmycosin (2), tylosin (1), 10,11,12,13-tetrahydrotylosin (11), and 2,3-didehydrodesmycosin (13) were prepared from the corresponding aldehydes by a Wittig reaction with the stabilized ylides (a-d), generating a trans-double bond, followed by modified Pfitzner-Moffat oxidation of the C-3 hydroxyl group. To evaluate the importance of the C-3 position of desmycosin for biological activity, the C-3 substituted derivatives were synthesized by a standard sequence of protective group chemistry followed by Wittig reaction and esterification as the key steps. For the attachment of the C-3 ester functionality, a mixed anhydride protocol was adopted. Reaction proceeded smoothly to give corresponding esters in yields ranging from 70 to 80%. Base- and acid-catalyzed rearrangement products including desmycosin 8,20-aldols (24a and 24b) and desmycosin 3,19-aldol (25) are also described. Parallel array synthesis and purification techniques allowed for the rapid exploration of structure-activity relationships within this class and for the improvement in potency. In vitro evaluation of these derivatives demonstrated good antimicrobial activity against Gram-positive bacteria for most of the compounds. The present derivatives of 16-membered macrolides were active against MLS(B)-resistant strains that were inducibly resistant, but not those constitutively resistant to erythromycin.     

Remaxol restores the liver antioxidant protection system in experimental cyclophosphan-induced injury

It is established that remaxol influences the main links of cells antioxidant system in case of experimental drug-induced liver damage. The injection of remaxol to animals with hepatotoxic damage increases the level of restored glutathione and maintains the concentration of SH groups of proteins in liver tissues on the level typical of intact animals. Remaxol helps to keep intact the energetic substrates of hepatocytes by saving the activity of glucose-6-phosphatedehydrogenase (which increases restored NADPH and glutathione enzymes), thus preventing the oxidative destruction of glutathione reductase and glutathione S-transferase. The antioxidant effect has been also confirmed by the study of catalase and glutathione peroxidase activity, the concentration of which under the action of remaxol increased above the normal level. Additional evidence is a remaxol-induced decrease in concentration of the final products of lipid peroxidation.     

Expression of glutathione S-transferase and phenol sulfotransferase, but not of UDP-glucuronosyltransferase, in the human lung tumor cell lines NCI-H322 and NCI-H358

The expression of xenobiotic-metabolizing enzymes was studied in the human lung tumour cell lines NCI-H322 and NCI-H358. These cells are derived from adenocarcinomas and exhibit features of Clara cells and alveolar type II cells, respectively. Examination of the in vitro activities showed that both cell lines lack UDP-glucuronosyltransferase against the substrates 3-hydroxybenzo[a]pyrene (3-OH-BaP) and 4-hydroxybiphenyl (4-OH-Bph) and that in vitro conjugation of sulfate with 3-OH-BaP was only just detectable. In contrast, both cell lines showed fairly high levels of glutathione-S transferase activity with the substrate 1-chloro-2,4-dinitro-benzene (54.4 and 83.0 nmol/min X mg protein, respectively) and of glutathione (81 and 41 nmole/mg protein, respectively). The metabolic capacity of intact NCI-H322 and NCI-H358 cells was examined using benzo[a]pyrene (BaP) and 3-OH-BaP as substrates. The cell lines formed sulfate conjugates from 3-OH-BaP (4.5 and 0.4 pmol/min X mg protein, respectively) but did not produce any detectable glucuronides. When cultures of the two cell lines were exposed to BaP, phenolic products accumulated in the growth medium. NCI-H322 cells also formed some sulfate conjugates, whereas such conjugates were barely detectable in the medium of NCI-H358 cells. In contrast A549, a human pulmonary adenocarcinoma cell line known to contain UDP-glucuronosyltransferase activity, efficiently conjugated 3-OH-BaP to glucuronic acid and converted the primary phenolic products formed from BaP to glucuronides. Thus the NCI-H322 and NCI-H358 cells are exceptional in that they possess no or very little glucuronosyltransferase activity but exhibit appreciable monooxygenase activity. The cell lines may therefore be of interest for examining the biological effects of potentially toxic chemicals which are otherwise detoxified by glucuronic acid conjugation. The cells may also be useful as test systems for evaluating the potential cytotoxicity and genotoxicity of chemicals to human lung.     

Taurine, a conditionally essential amino acid, ameliorates arsenic-induced cytotoxicity in murine hepatocytes

Arsenic is a potent environmental toxin. Present study has been designed to evaluate the protective role of taurine (2-aminoethanesulfonic acid) against arsenic induced cytotoxicity in murine hepatocytes. Sodium arsenite (NaAsO₂) was chosen as the source of arsenic. Incubation of hepatocytes with the toxin (1 mM) for 2 h reduced the cell viability as well as intra-cellular antioxidant power. Increased activities of alanine transaminase (ALT) and alkaline phosphatase (ALP) due to toxin exposure confirmed membrane damage. Toxin treatment caused reduction in the activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx). In addition, the same treatment reduced the level of glutathione (GSH), elevated the level of oxidized glutathione (GSSG) and increased the extent of lipid peroxidation. Incubation of hepatocytes with taurine, both prior to and in combination with NaAsO₂, attenuated the extent of lipid peroxidation and enhanced the activities of enzymatic as well as non enzymatic antioxidants. Besides, taurine administration normalized the arsenic-induced enhanced levels of the marker enzymes ALT and ALP in hepatocytes. The cytoprotective activity of taurine against arsenic poisoning was found to be comparable to that of a known antioxidant, vitamin C. Combining all, the results suggest that taurine protects mouse hepatocytes against arsenic induced cytotoxicity.     

Inhibitory effect of flavonoids on sulfo- and glucurono-conjugation of acetaminophen in rat cultured hepatocytes and liver subcellular preparations

A large group of flavonoids was investigated for inhibitory effects on sulfo- and glucurono-conjugation of acetaminophen when added to rat cultured hepatocytes and liver subcellular preparations. The flavonoids inhibited the production of both sulfate and glucuronide conjugates in the cultured cells, with potencies that depended on the specific flavonoid. Among the flavonols, quercetin, kaempferol and galangin were much more effective than myricetin and morin. Flavones including luteolin, apigenin and chrysin were as effective as the corresponding three flavonols above. The inhibition of conjugation by other simple flavones such as 3-, 5-, 7- and 3',4'-OH flavones, and by catechins such as epicatechin and epigallocatechin, was very weak. These data suggest that the presence of both C5 and 7 hydroxyl substitutions on the A-ring in the flavone structure is required for effective inhibitory activity. The effect of flavonoids on sulfo- and glucurono-conjugation was also examined by incubating acetaminophen with isolated liver cytosolic and microsomal preparations, respectively. The active flavonoids in the cells remarkably inhibited the sulfation, but not glucuronidation, in cell-free enzymatic preparations in vitro. The mechanism of inhibition of conjugation by flavonoids in cultured hepatocytes is not likely to depend on the direct inhibition of sulfo- and glucurono-transferase activity by flavonoids.