Alan A. Horton

Lipid Peroxidation and Mechanisms of Toxicity

Aerobic organisms by definition require oxygen, and the importance of iron in aerobic respiration has long been recognized, but despite their beneficial roles, these elements can pose a real threat to the organism. During oxygen reduction, reactive species such as O2-. and H2O2 are formed readily. Iron can combine with these species, or with molecular oxygen itself, to generate free radicals which will attack the polyunsaturated fatty acids of membrane lipids. This oxidative deterioration of membrane lipids is known as lipid peroxidation. To protect itself against this form of attack, the organism possesses several types of defense mechanisms. Under normal conditions, these defenses appear to offer adequate protection for cell membranes, but the possibility exists that certain foreign compounds may interfere with or even overwhelm these defenses, and herein could lie a general mechanism of toxicity. This possible cause of toxicity is discussed in relation to other suggested causes.

Mechanism of apoptosis in HL-60 cells induced by n-3 and n-6 polyunsaturated fatty acids

The biochemical properties and specificity of n-3 and n-6 polyunsaturated fatty acids (PUFAs) are not well known. Because PUFAs induce apoptosis of different cells, we studied the effect of various PUFAs, such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA), on the fate of cultured human promyelocytic leukemia cells (HL-60) to elucidate the mechanism of apoptosis and the difference in action between n-3 and n-6 PUFAs. Fairly low concentrations of PUFAs inhibited the growth of HL-60 cells and induced their apoptosis by a mechanism that is sensitive to DMSO, an antioxidant, and z-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor. PUFAs stimulated the generation of reactive oxygen species (ROS) and activated various types of caspase-like proteases, such as caspase-3, -6, -8, and -9, but not caspase-1. In addition, PUFAs triggered the reaction leading to the cleavage of Bid, a death agonist member of the Bcl-2 family, and also released cytochrome c from mitochondria into the cytosol. PUFAs also decreased the mitochondrial membrane potential of intact HL-60 cells. All of these actions of n-3 PUFAs were stronger than those of AA, an n-6 PUFA, although the mechanism is not known. PUFAs stimulate swelling and membrane depolarization of isolated mitochondria in a cyclosporin A-sensitive manner. The results indicated that PUFA-induced apoptosis of HL-60 cells may be caused, in part, by direct action on the cells and by activation of the caspase cascade through cytochrome c release coupled with mitochondrial membrane depolarization.

Age-dependent changes in rat liver microsomal and mitochondrial NADPH-dependent lipid peroxidation

Abstract NADPH-dependent lipid peroxidation was measured in foetal, neonatal, 6 month old and 26 month old rat liver microsomal and mitochondrial fractions, and shown to increase substantially with age in both fractions.

The subcellular localization of aldehyde dehydrogenase in rat liver.

Abstract Evidence supporting the existence of three aldehyde dehydrogenases, betaine aldehyde dehydrogenase and two aldehyde dehydrogenases ALDH-I and ALDH-II, in rat liver has been confirmed. Subcellular fractionation indicates that betaine aldehyde dehydrogenase is predominantly in the cytosol with about 5% in the mitochondria. Of the aldehyde dehydrogenase activity (ALDH-I plus ALDH-II) 80% is found in the mitochondria and 20% in the microsomal fraction. Of the two enzymes ALDH-I is exclusively mitochondrial and ALDH-II is distributed between mitochondria and microsomes. Submitochondrial fractionation indicates that betaine aldehyde dehydrogenase and ALDH-I are located in the matrix, and that ALDH-II is chiefly located in the outer membrane.

Studies on paracetamol-induced lipid peroxidation

Post-mitochondrial supernatants isolated from livers of rats given a single large oral dose of paracetamol (800 mg/kg) showed rapid rates of lipid peroxidation when incubated in vitro. As a result of paracetamol administration the level of reduced glutathione (GSH) declined to approx. 20-25% of the peak physiological value. Addition of reduced GSH to the supernatant inhibited the peroxidation. Paracetamol-induced lipid peroxidation was inhibited in vitro by antioxidants (e.g. vitamin E) but was unaffected by superoxide dismutase and mannitol. N-acetyl cysteine and cysteamine inhibited lipid peroxidation in vitro in a cytosol-dependent manner in the absence of glutathione. Lipid peroxidation probably occurs simultaneously with the proposed covalent binding of the active metabolite of paracetamol. Since the former process is known to cause severe and extensive membrane damage, it may be a very important factor in paracetamol-induced liver necrosis.

Caspase activation and cytochrome c release during HL-60 cell apoptosis induced by a nitric oxide donor

Nitric oxide (NO) from (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (NOC-18) induces apoptosis in human leukemia HL-60 cells. This effect was prevented by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), thereby implicating caspase activity in the process. NOC-18 treatment resulted in the activation of several caspases including caspase-3, -6, -8, and -9(-like) activities and the degradation of several caspase substrates such as nuclear lamins and SP120 (hnRNP-U/SAF-A). Moreover, release of cytochrome c from mitochondria was also observed during NOC-18-induced apoptosis. This change was substantially prevented by Z-VAD-FMK, thereby suggesting that the released cytochrome c might function not only as an initiator but also as an amplifier of the caspase cascade. Bid, a death agonist member of the Bcl-2 family, was processed by caspases following exposure of cells to NOC-18, supporting the above notion. Thus, NO-mediated apoptosis in HL-60 cells involves a caspase/cytochrome c-dependent mechanism.

Inhibition of stimulus-specific neutrophil superoxide generation by alpha-tocopherol.

Alpha-tocopherol but not 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (trolox or CTMC) and 2,2,5,7,8 pentamethyl-6-hydroxy chromane (PMC), derivatives of alpha-tocopherol, inhibited the superoxide (O2-.) generation of rat peritoneal neutrophils (RPMN) induced by phorbol 12-myrisate 13-acetate (PMA). ID50 for neutrophils obtained from the peritoneal cavity of rat and guinea pig was about 1microM. This concentration, however, was much lower than that for the inhibition of PMA-activated phospholipid-dependent protein kinase (PKC) (ID50 = 30 microM). The alpha-tocopherol sensitive O2-. generation was also observed in neutrophils induced by dioctanoylglycerol (diC8) and calcium ionophore A23187 but not by formylmethionyl-leucyl-phenylalanine (FMLP), opsonized zymosan (OZ) and sodium dodecyl sulfate (SDS). The pattern of inhibition by alpha-tocopherol was quite similar to that of staurosporine, a specific inhibitor of PKC. The alpha-tocopherol content of RPMN was 12 ng/10(6) cells and a linear increase to 200 ng/10(6) cells by addition of alpha-tocopherol to the cell suspension corresponded with an increased inhibition of O2-. generation. These results indicate that both the chemical structure and the content of alpha-tocopherol might be important factors in O2-. generation by neutrophils.

Molecular Mechanisms of Apoptosis in HL-60 Cells Induced by a Nitric Oxide-Releasing Compound

Nitric oxide (NO) generated from 1-hydroxy-2-oxo-3, 3-bis(2-aminoethyl)-1-triazene (NOC 18), an NO-releasing compound, induced monocytic differentiation of human promyelocytic leukemia HL-60 cells as assessed by expression of nonspecific esterases and morphologic maturation. Simultaneously, DNA fragmentation and morphological alterations typical of apoptosis were also induced. To investigate the mechanisms of apoptosis during differentiation of HL-60 cells induced by NO, the endogenous levels of Bcl-2 and Bax were assessed by immunoblotting. Treatment of cells with NOC 18 slightly reduced the level of Bcl-2 followed by Bax. These changes might be involved in the induction of apoptosis. The involvement of the activation of the interleukin-1 beta converting enzyme (ICE) family of proteases (caspases), such as ICE and CPP32, in the pathways was also investigated. CPP32, but not ICE, was strongly activated in response to NOC 18 stimulation, thereby implicating CPP32-like activity in the induction of apoptosis. Moreover, the possible involvement of tyrosine phosphorylation in apoptosis was investigated. Pretreatment of cells with herbimycin A, an inhibitor of tyrosine kinases, suppressed DNA fragmentation and CPP32-like activity, whereas pretreatment with vanadate, an inhibitor of tyrosine phosphatases, enhanced both parameters, suggesting that tyrosine phosphorylation might be involved in the pathways of apoptosis in HL-60 cells induced by NO.

INHIBITION OF NEUTROPHIL-SUPEROXIDE GENERATION BY ALPHA -TOCOPHEROL AND COENZYME Q

Effects of various derivatives of alpha-tocopherol (VE) and coenzyme Q (CoQ) on superoxide (O2.-) generation of neutrophils and protein kinase C (PKC) activity were examined. VE and CoQ8 inhibited O2.- generation of neutrophils stimulated by a protein kinase C mediated process monitored by cytochrome c reduction and spin trapping methods. The inhibitory action was observed not only with alpha-tocopherol, but also with beta-, gamma-, delta-tocopherols and with tocol which is a chemical similar to VE but lacking methyl groups on the chromanol ring structure and which is not a radical scavenger. By contrast, no inhibition was observed with 2-carboxy-2, 5, 7, 8-tetramethyl-6-chromanol (CTMC, trolox) or 2, 2, 5, 7, 8,-pentamethyl-6-chromanol (PMC) which are water soluble VE derivatives having radical scavenging activity. Compounds having a similar isoprenoid chain, such as CoQ, also have inhibitory activity on PKC-dependent O2.- generation of neutrophils. The inhibitory activity of CoQ derivatives is dependent on the length of the unsaturated isoprenoid chain. CoQ derivatives having 16, 24 and 32 carbon isoprenoid chains corresponding to CoQ4, 6, and 8 inhibited O2.- generation but 4 and 40 carbon isoprenoid chains corresponding to CoQ2 and 10 had no inhibitory activity on O2.- generation. Alpha-tocopherol and CoQ inhibited PKC activity but the ID50 for O2.- generation and PKC activity was different for each compound. However, no direct relationship between VE content and O2.- generation of neutrophils was observed. These results suggest that isoprenoids of VE and CoQ participate in the inhibition of the NADPH oxidase activation system through modulation of the neutrophil membrane probably by the inhibition of PKC.

Effect of the antiretroviral agent hypericin on rat liver mitochondria

The photosensitizing effect of hypericin (HY), an antiretroviral agent, on the functions of isolated rat liver mitochondria has been investigated. The respiratory control ratio (RCR), ADP/O and membrane potential of mitochondria were decreased by HY in a light-dependent manner. Uncoupled respiration of mitochondria in the presence of succinate was also inhibited by HY in a light-dependent manner. The ID50 of hypericin for these inhibitions was approximately 0.5 microM. These inhibitory effects of HY were not observed when photosensitization was conducted under anaerobic conditions and were not affected by desferrioxamine (DSF) or superoxide dismutase (SOD). Upon photosensitization of HY, mitochondria consumed oxygen in the absence of respiratory substrate with concomitant formation of thiobarbituric acid reactive substance (TBARS). The amount of oxygen consumed was 100-times greater than that of TBARS formed. The oxygen uptake was partially inhibited by NaN3, and formation of TBARS was inhibited by DSF. Upon photosensitization of HY in the presence of mitochondrial membranes, the electron spin resonance (ESR) signal of 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO/.OH) was increased by a mechanism which was suppressed by DSF. An ESR signal for singlet oxygen bound to 2,5-dimethylfuran, 2,2,6,6-tetramethyl-4-piperidone (TEMP) was also detected under light in the presence of mitochondria. This signal of the TEMP-N-oxyl radical (TEMPO) was decreased by azide, which physically quenches singlet oxygen, but was increased by DSF. These results indicate that HY might inhibit mitochondrial functions by a type II photodynamic mechanism but that lipid peroxidation of biological membranes through an active oxygen-mediated photodynamic mechanism is not involved.