The incorporation of drugs into hair: relationship of hair color and melanin concentration to phencyclidine incorporation

Rodents with different hair pigmentation patterns were studied to evaluate the role of melanin in the incorporation of phencyclidine (PCP) into hair. There are two types of melanin in hair and other tissues: eumelanin, a brown-black pigment and pheomelanin, a reddish-yellow pigment. Sprague Dawley (SD; nonpigmented), Dark Agouti (DA; brown), Copenhagen (CP; brown hooded), Long Evans (LE; black hooded), and LBNF1 (deep brown) rats and Swiss-Webster (SW; nonpigmented), C57BL6 (black), and C57BL6 Ay/a (yellow) mice were administered PCP at 10 mg/kg/day for 5 days (n = 5 for each strain). Hair was collected either 14 (rats) or 35 (mice) days (mice) after beginning drug administration and analyzed for PCP, eumelanin, and pheomelanin. PCP concentrations in ng/mg (mean +/- SEM) were as follows: SD, 0.46 +/- 0.13; DA, 12.25 +/- 1.24; CP nonpigmented, 0.12 +/- 0.004; CP pigmented, 9.16 +/- 2.8; LE nonpigmented, 0.66 +/- 0.07; LE pigmented, 21.2 +/- 1.4; LBNF1, 21.64 +/- 3.8; SW, 0.48 +/- 0.36; C57 black, 11.0 +/- 4.03; and C57 yellow, 2.26 +/- 0.55. Eumelanin concentrations in microg/mg (mean +/- SEM) were as follows: DA, 20.50 +/- 1.58; CP pigmented, 19.43 +/- 0.40; LE pigmented, 17.56 +/- 0.61; LBNF1, 27.26 +/- 2.52; C57 black, 37.33 +/- 3.61; and C57 yellow, 1.76 +/- 0.02. Eumelanin was not detected in nonpigmented hair. Pheomelanin concentrations in microg/mg (mean +/- SEM) were as follows: DA, 0.09 +/- 0.00; CP pigmented, 0.20 +/- 0.03; LBNF1, 0.06 +/- 0.01; C57 black, 0.16 +/- 0.05; and C57 yellow, 29.16 +/- 0.97. Pheomelanin was not detected in nonpigmented or LE pigmented hair. These data demonstrate that PCP is incorporated into black hair to a greater extent than yellow or nonpigmented hair. There appears to be a linear relationship between the PCP concentration in hair and the ratio of eumelanin to pheomelanin. Our data suggest that despite variations in PCP concentration because of hair color, they may be normalized by using the ratio of eumelanin to pheomelanin rather than hair weight.     

(Pheo)melanin photosensitizes UVA-induced DNA damage in cultured human melanocytes

The question of whether melanins are photoprotecting and/or photosensitizing in human skin cells continues to be debated. To evaluate the role of melanin upon UVA irradiation, DNA single-strand breaks (ssb) were measured in human melanocytes differing only in the amount of pigment produced by culturing at two different concentrations, basic (0.01 mM) or high (0.2 mM), of L-tyrosine, the main precursor of melanin. In parallel, pheo- and total melanin contents of the cells were determined. Identical experiments were performed with two melanocyte cultures derived from a skin type I and a skin type VI individual. For the first time the correlation between UVA-induced genotoxicity and pheo-/total melanin content has been investigated. We observed that cultured in basic medium, the skin type VI melanocytes contained 10 times more total melanin and about seven times more pheomelanin than the skin type I melanocytes. Elevation of tyrosine level in the culture medium resulted in an increase of both pheo- and total melanin levels in both melanocyte cultures; however, the melanin composition of skin type I melanocytes became more pheomelanogenic, whereas that of skin type VI melanocytes remained the same. The skin type VI melanocytes cultured in basic medium demonstrated a very high sensitivity (1.18 ssb per 10(10) Da per kJ per m2) toward UVA that is probably related to their high pheo- and total melanin content. Their UVA sensitivity, however, did not change after increasing their melanin content by culturing at high tyrosine concentration. In contrast, the skin type I melanocytes demonstrated a low sensitivity (0.04 ssb per 10(10) Da per kJ per m2) toward UVA when cultured in basic medium, but increasing their melanin content resulted in a 3-fold increase in their UVA sensitivity (0.13 ssb per 10(10) Da per kJ per m2). These results demonstrate that UVA-irradiated cultured human melanocytes are photosensitized by their own synthesized chromophores, most likely pheomelanin and/or melanin intermediates.     

The concept of delirium in Spanish psychiatry (19th-20th century)

Important regulatory controls of melanogenesis that operate at the subcellular level to modulate the structural and/or the functional nature of the melanins and melanin granules produced in melanocytes are reviewed. Melanocyte stimulating hormone and agouti signal protein have antagonistic roles and possibly opposing mechanisms of action in the melanocyte. In the mouse, melanocyte stimulating hormone promotes melanogenic enzyme function and elicits increases in the amount of eumelanins produced, while agouti signal protein reduces total melanin production and elicits the synthesis of pheomelanin rather than eumelanin. We are now beginning to understand the complex controls involved in regulating this switch at the molecular and biochemical levels. The quality and quantity of melanins produced by melanocytes have important physiological consequences for melanocyte function and undoubtedly play important roles in the various functions of the melanins per se, including hair and skin coloration and photoprotection.     

Free radicals, oxidative stress and antioxidant vitamins

Free radicals having oxidizing properties are produced in vivo. The monoelectronic reduction of dioxygen generates the superoxide radical (.O2-) which, according to the experimental conditions, behaves as a reducing or an oxidizing agent. Its dismutation catalyzed by superoxide dismutases (SODs) produces hydrogen peroxide. The latter reacting with .O2- in the presence of "redox-active" iron produces highly aggressive prooxidant radicals, such as the hydroxyl radical (.OH). This production is prevented through intracellular enzymes (catalase and glutathione peroxidases) which destroy the hydrogen peroxide involved in the biosynthesis of .OH. An increase in SODs activity without parallel enhancement of the enzymes destroying H2O2 may lead to important cellular disturbances. Other enzymes acting with glutathione as substrate (especially glutathione S-transferases) contribute to the antioxidant defence. The same holds true for selenium and zinc which act mainly through their involvement in the structure of both antioxidant enzymes and nonenzymatic proteins. Another line of antioxidant defence is represented by substrates acting as chain-breaking antioxidants in destructive processes linked to prooxidant free radicals, such as lipid peroxidation. The main membranous antioxidant is alpha-tocopherol which is able to quench efficiently lipid peroxyl radicals. Its efficiency would be quickly exhausted if the tocopheryl radical formed during this reaction wouldn't be retransformed into alpha-tocopherol through the intervention of ascorbate and/or glutathione. Ubiquinol and dihydrolipoate also contribute to the membranous antioxidant defence, whereas carotenoids are mainly responsible for the prevention of the deleterious effects of singlet oxygen. An oxidative stress is apparent when the antioxidant defence is insufficient to cope with the prooxidant production.     

Markers of melanogenesis in malignant melanoma

Biochemical specifity of malignant melanoma is represented in part by the formation of specific cytoplasmatic particles of the pigment cell--melanosomes--in which the synthesis of pigment eumelanin and pheomelanin takes place and in part by the presence of a specific enzyme--tyrosinase--which catalyzes the formation of pigment eumelanin and pheomelanin and even the formation of specific metabolites (so called melanogens) which are excreted in increased amounts in the course of the disease. Tyrosinase and melanogens are specific for pigment cell and therefore can be used for monitoring of melanogenesis in malignant melanoma. When comparing our results and the results of other authors we can conclude that following of specific markers of melanogenesis in malignant melanoma should serve for the evaluation of prognosis of the disease. The study of melanoma markers is by far not finished. We do hope that nearly future will be able to give a sufficient answer to the question, whether melanogenuria is actually an expression of expected different biochemical or metabolic types of malignant melanoma on the one hand and/or biochemically or immunologically conditioned responses of the host organism on the other.     

Morphological and chromosomal descriptions of new species in the Anopheles subpictus complex

In the present work, the role of lipid peroxidation in cellular lethal injury induced by various types of oxidative stress has been studied in both normal and tumor thymocytes. The prooxidants included either a xanthine/xanthine oxidase system, which is an exogenous source of oxyradicals, or tert-butyl hydroperoxide (t-BOOH), which enters the cell and endogenously produces free radicals. Our data demonstrate that: (A) Using xanthine/xanthine oxidase system as a prooxidant, normal thymocytes are more sensitive than thymoma cells to oxidative damage, as their lactate dehydrogenase (LDH) and malondialdehyde (MDA) release is higher than that of tumor cells. By varying Fe3+/ADP ratios, a positive correlation can be established between LDH and MDA release only in normal thymocytes. While thymoma cells still show a very high level of vitamin E (80%) after 15 min of incubation with this prooxidant, normal thymocytes lose it after the same incubation time. (B) Using t-BOOH as a prooxidant, normal thymocytes release a higher amount of MDA but a lower amount of LDH than thymoma cells. In agreement with the results obtained with the xanthine/xanthine oxidase system, by varying the concentrations of the prooxidant, a correlation between LDH and MDA release can be established only in normal thymocytes. Although high levels of the antioxidant are still present in both kinds of cells after 15 min of incubation with t-BOOH, normal thymocytes consume vitamin E faster than thymoma cells. These data suggest that the role of lipid peroxidation in cell lethal injury is influenced by the source and the site of radical production as well as by the cell type. With t-BOOH as a prooxidant in normal thymocytes, lipid peroxidation is only partially involved in the induction of irreversible cell injury, but it plays a crucial role when the xanthine/xanthine oxidase system is used as a prooxidant. Moreover, whatever the prooxidant used in tumor thymocytes, membranes are more resistant to lipid peroxidation, suggesting that this mechanism is not causally related to cell death.     

Inhibitory effect of ethanolamine plasmalogen on iron- and copper-dependent lipid peroxidation

The effect of ethanolamine plasmalogen (EtnPm) on lipid peroxidation was investigated in liposomal suspension of egg yolk phosphatidylcholine. EtnPm inhibited iron- and copper-dependent peroxidation in the presence of preformed hydroperoxides, although it was not effective for radical initiator mediated lipid peroxidation. EtnPm resulted in complete binding of iron to liposomal lipids, suggesting that EtnPm exerts its inhibitory effect on lipid peroxidation through inhibiting preformed peroxide decomposition by trapping transition metal ions.     

The protective role of plasmalogens in iron-induced lipid peroxidation

The role of plasmalogens in iron-induced lipid peroxidation was investigated in two liposomal systems. The first consisted of total brain phospholipids with and without plasmalogens, and the second of phosphatidylethanolamine/phosphatidylcholine liposomes with either diacyl- or alkenylacyl-phosphatidylethanolamine. By measuring thiobarbituric acid reactive substances, oxygen consumption, fatty acids and aldehydes, we show that plasmalogens effectively protect polyunsaturated fatty acids from oxidative damage, and that the vinyl ether function of plasmalogens is consumed simultaneously. Furthermore, the lack of lag phase, the increased antioxidant efficiency with time, and the experiments with lipid- and water-soluble azo compounds, indicate that plasmalogens probably interfere with the propagation rather than the initiation of lipid peroxidation, and that the antioxidative effect cannot be related to iron chelation.     

Neuromelanin of the human substantia nigra: a mixed-type melanin

Model melanins, synthesized with different cysteinyldopamine/dopamine ratios in the incubates, were oxidized with KMnO4 and the resulting compounds were analyzed by HPLC. The ratios between a phaeomelanin-derived compound, thiazole-4,5-dicarboxylic acid (TDCA), and a compound derived from eumelanin, pyrrole-2,3,5-tricarboxylic acid (PTCA), reflected the composition of the model melanins. The neuromelanin of the human substantia nigra was isolated, and the pigment, as well as intact brain tissue from human substantia nigra was oxidized with KMnO4 and the TDCA/PTCA ratios were determined. Analysis of the isolated neuromelanin showed it to contain 2.3% sulfur and 8.1% nitrogen. The sulfur content indicates the pigment is a mixed-type melanin, and the TDCA/PTCA ratio indicates that it consists of units derived from benzothiazines and from indoles in about equal amounts.     

Prooxidant effects of ferrous iron, hemoglobin, and ferritin in oil emulsion and cooked-meat homogenates are different from those in raw-meat homogenates

Oil emulsion and raw and cooked tissue homogenates were used to determine the mechanisms of various iron forms on the catalysis of lipid peroxidation. Flax oil (0.25 g) was blended with 160 mL maleate buffer (0.1 M, pH 6.5) to prepare an oil emulsion. Raw or cooked turkey leg meat was used to prepare meat homogenates. Samples were prepared by adding iron from each of the various sources, reactive oxygen species, or enzyme (xanthine oxidase and superoxide dismutase) systems into the oil emulsion or meat homogenates. In oil emulsion and cooked-meat homogenates, ferrous iron and hemoglobin had strong prooxidant effects, but ferritin became prooxidant only when ascorbate was present. Hemoglobin and ferritin had no prooxidant effect in raw-meat homogenates. The status of heme iron and the released iron from hemoglobin had little effect on the prooxidant effect of hemoglobin in oil emulsion and cooked meat homogenate systems. The prooxidant effect of ferrous iron in oil emulsion and cooked-meat homogenates disappeared in the presence of superoxide (.O2-), H2O2, or xanthine oxidase systems. In raw-meat homogenates, however, ferrous had strong prooxidant effects even in the presence of .O2-, or H2O2. The status of free iron was the most important factor in the oxidation of oil emulsion and cooked-meat homogenates but the impact in raw-meat homogenates was small.     

Ascorbic acid inhibits lipid peroxidation but enhances DNA damage in rat liver nuclei incubated with iron ions

In this report we studied DNA damage and lipid peroxidation in rat liver nuclei incubated with iron ions for up to 2 hrs in order to examine whether nuclear DNA damage was dependent on membrane lipid peroxidation. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) and DNA damage was measured as 8-OH-deoxyguanosine (8-OH-dG). We showed that Fe(II) induced nuclear lipid peroxidation dose-dependently but only the highest concentration (1.0 mM) used induced appreciable 8-OH-dG. Fe(III) up to 1 mM induced minimal lipid peroxidation and negligible amounts of 8-OH-dG. Ascorbic acid enhanced Fe(II)-induced lipid peroxidation at a ratio to Fe(II) of 1:1 but strongly inhibited peroxidation at ratios of 2.5:1 and 5:1. By contrast, ascorbate markedly enhanced DNA damage at all ratios tested and in a concentration-dependent manner. The nuclear DNA damage induced by 1 mM FeSO4/5 mM ascorbic acid was largely inhibited by iron chelators and by dimethylsulphoxide and mannitol, indicating the involvement of OH. Hydrogen peroxide and superoxide anions were also involved, as DNA damage was partially inhibited by catalase and, to a lesser extent, by superoxide dismutase. The chain-breaking antioxidants butylated hydroxytoluene and diphenylamine (an alkoxyl radical scavenger) did not inhibit DNA damage. Hence, this study demonstrated that ascorbic acid enhanced Fe(II)-induced DNA base modification which was not dependent on lipid peroxidation in rat liver nuclei.     

Action spectrum for UV-induced lipid peroxidation in cultured human skin fibroblasts

Lipid peroxidation was measured by release of thiobarbituric acid-reactive substances (TBARS) into the supernatant of cultured human skin fibroblasts. This process is triggered by ultraviolet A (UVA) and ultraviolet B (UVB) radiations. For UVA irradiances and irradiation times up to 40 W.m-2 and 90 min, respectively, the peroxidation response is linear and obeys the reciprocity law. Corresponding values for UVB are 12 W.m-2 and 30 min, respectively. The action spectrum of the peroxidation process shows a continuously increasing response from about 425 to 275 nm. Whereas the UVB to UVA effectiveness ratio lies in the range of 10(3) to 10(4) for most in vitro or in vivo UV-induced responses, the ratio is only 10 to 100 for the peroxidation process. Given the solar spectral distribution, solar UVA radiation is by far the most effective in triggering the peroxidation response.     

gamma-Glutamyl transpeptidase-dependent lipid peroxidation in isolated hepatocytes and HepG2 hepatoma cells

Gamma-glutamyltranspeptidase (GGT), a plasma membrane-bound enzyme, provides the only activity capable to effect the hydrolysis of extracellular glutathione (GSH), thus favoring the cellular utilization of its constituent amino acids. Recent studies have shown however that in the presence of chelated iron prooxidant species can be originated during GGT-mediated metabolism of GSH, and that a process of lipid peroxidation can be started eventually in suitable lipid substrates. The present study was undertaken to verify if a GGT-dependent lipid peroxidation process can be induced in the lipids of biological membranes, including living cells, and if this effect can be sustained by the GGT highly expressed at the surface of HepG2 human hepatoma cells. In rat liver microsomes (chosen as model membrane lipid substrate) exposed to GSH and ADP-chelated iron, the addition of GGT caused a marked stimulation of lipid peroxidation, which was further enhanced by the addition of the GGT co-substrate glycyl-glycine. The same was observed in primary cultures of isolated rat hepatocytes, where the lipid peroxidation process did not induce acute toxic effects. GGT-stimulation of lipid peroxidation was dependent both on the concentration of GSH and of ADP-chelated iron. In GGT-rich HepG2 human hepatoma cells, the exposure to GSH, glycyl-glycine, and ADP-chelated iron resulted in a nontoxic lipid peroxidation process, which could be prevented by means of GGT inhibitors such as acivicin and the serine-boric acid complex. In addition, by co-incubation of HepG2 cells with rat liver microsomes, it was observed that the GGT owned by HepG2 cells can act extracellularly, as a stimulant on the GSH- and iron-dependent lipid peroxidation of microsomes. The data reported indicate that the lipid peroxidation of liver microsomes and of living cells can be stimulated by the GGT-mediated metabolism of GSH. Due to the well established interactions of lipid peroxidation products with cell proliferation, the phenomenon may bear particular significance in the carcinogenic process, where a relationship between the expression of GGT and tumor progression has been envisaged.     

Inhibition of class-3 aldehyde dehydrogenase and cell growth by restored lipid peroxidation in hepatoma cell lines

Hepatoma cells have a below-normal content of polyunsaturated fatty acids; this reduces lipid peroxidation and the production of cytotoxic and cytostatic aldehydes within the cells. In proportion to the degree of deviation, hepatoma cells also show an increase in the activity of Class-3 aldehyde dehydrogenase, an enzyme important in the metabolism of lipid peroxidation products and also in that of several drugs. When hepatoma cells with different degrees of deviation were enriched with arachidonic acid and stimulated to peroxidize by ascorbate/iron sulphate, their growth rate was reduced in proportion to the quantity of aldehydes produced and to the activity of aldehyde dehydrogenase. Therefore, 7777 cells, less deviated and with low Class-3 aldehyde dehydrogenase activity, were more susceptible to lipid peroxidation products than JM2 cells. It is noteworthy that repeated treatments with prooxidant also caused a decrease in mRNA and activity of Class-3 aldehyde dehydrogenase, contributing to the decreased growth and viability. Thus, Class-3 aldehyde dehydrogenase could be considered relevant for the growth of hepatoma cells, since it defends them against cell growth inhibiting aldehydes derived from lipid peroxidation.     

Relations between tocopherol depletion and coenzyme Q during lipid peroxidation in rat liver mitochondria

In order to evaluate different mitochondrial antioxidant systems, the depletion of alpha-tocopherol and the levels of the reduced and oxidized forms of CoQ were measured in rat liver mitochondria during Fe++/ascorbate and NADPH/ADP/Fe++ induced lipid peroxidation. During the induction phase of malondialdehyde formation, alpha-tocopherol declined moderately to about 80% of initial contents, whereas the total CoQ pool remained nearly unchanged, but reduced CoQ9 continuously declined. At the start of massive malondialdehyde formation, CoQ9 reaches its fully oxidized state. At the same time alpha-tocopherol starts to decline steeply, but never becomes fully exhausted in both experimental systems. Evidently the oxidation of the CoQ9 pool constitutes a prerequisite for the onset of massive lipid peroxidation in mitochondria and for the subsequent depletion of alpha-tocopherol. Trapping of the GSH by addition of dinitrochlorbenzene (a substrate of the GSH transferase), results in a moderate acceleration of lipid peroxidation, but alpha-tocopherol and ubiquinol levels remained unchanged when compared with the controls. Addition of succinate to GSH depleted mitochondria effectively suppressed MDA formation as well as alpha-tocopherol and ubiquinol depletion. The data support the assumption that the protective effect of respiratory substrates against lipid peroxidation in the absence of mitochondrial GSH is mediated by the regeneration of the lipid soluble antioxidants CoQ and alpha-tocopherol.     

Carvedilol inhibition of lipid peroxidation. A new antioxidative mechanism

To define the molecular mechanism(s) of carvedilol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Carvedilol inhibits the peroxidation of sonicated phosphatidylcholine liposomes triggered by FeCl2 addition whereas atenolol, pindolol and labetalol are ineffective. The inhibition proved not to be ascribable (a) to an effect on Fe2+ autoxidation and thus on the generation of oxygen derived radical initiators; (b) to the scavenging of the inorganic initiators O2*- and *OH; (c) to an effect on the reductive cleavage of organic hydroperoxides by FeCl2; (d) to the scavenging of organic initiators. The observations that (a) carvedilol effectiveness is inversely proportional to the concentration of FeCl2 and lipid hydroperoxides in the assay; (b) the drug prevents the onset of lipid peroxidation stimulated by FeCl3 addition and; (c) it can form a complex with Fe3+, suggest a molecular mechanism for carvedilol action. It may inhibit lipid peroxidation by binding the Fe3+ generated during the oxidation of Fe2+ by lipid hydroperoxides in the substrate. The lag time that carvedilol introduces in the peroxidative process would correspond to the time taken for carvedilol to be titrated by Fe3+; when the drug is consumed the Fe3+ accumulates to reach the critical parameter that stimulates peroxidation. According to this molecular mechanism the antioxidant potency of carvedilol can be ascribed to its ability to bind a species, Fe3+, that is a catalyst of the process and to its lipophilic nature that concentrates it in the membranes where Fe3+ is generated by a site specific mechanism.     

Effects of consecutive thiouracil exposures in the juvenile and adult single comb White Leghorn chicken on body weight and reproductive performance

To develop a novel potent radical-scavenging antioxidant, the ideal structure of a phenolic compound was designed considering the factors that determine antioxidant potency. 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4, 6-di-tert-butylbenzofuran (BO-653) was thus synthesized and its antioxidant activity was evaluated against lipid peroxidations in vitro. The electron spin resonance study showed that the phenoxyl radical derived from BO-653 was more stable than alpha-tocopheroxyl radical. BO-653 reduced alpha-tocopheroxyl radical rapidly, but alpha-tocopherol did not reduce the phenoxyl radical derived from BO-653. However, the chemical reactivity of BO-653 toward peroxyl radical was smaller than that of alpha-tocopherol. This was interpreted as the steric effect of bulky tert-butyl groups at both ortho positions which hindered the access of peroxyl radical to the phenolic hydrogen. However, the tertbutyl substituents increased the stability of BO-653 radical and also lipophilicity, and its antioxidant potency against lipid peroxidation in phosphatidylcholine liposomal membranes was superior to that of alpha-tocopherol. Ascorbic acid reduced the phenoxyl radical derived from BO-653 and spared BO-653 during the oxidation of lipid in the homogeneous solution. On the other hand, ascorbic acid did not spare BO-653 in the oxidation of liposomal membranes. It was concluded that BO-653 is a potent novel radical-scavenging antioxidant.     

Lipid peroxidation in rat lung induced by neuroleptanalgesia and its components

The aim of the present work was to determine the likelihood of lipid peroxidation in the lungs of rats subjected to neuroleptanalgesia and its components. In particular, the effect of fentanyl, droperidol, a nitrous oxide/oxygen mixture when used separately or in combination, on the lung level of lipid peroxidation was investigated. The in vitro antioxidant properties of fentanyl and droperidol were also tested. Lipid peroxidation was evidenced by the endogenously generated conjugated dienes and fluorescent products of lipid peroxidation and the decrease in lung vitamin E content. It was found that fentanyl and droperidol, used separately or in combination, did not induce lipid peroxidation in the rat lung, while the exposure of rats for 120 min to a nitrous oxide/oxygen mixture (2:1 v/v) led to well-expressed peroxidation. The (N2O + O2)-pro-oxidant action was significantly inhibited in rats previously injected with fentanyl and/or droperidol. The results show that the application of fentanyl, droperidol and (N2O + O2), as in neuroleptanalgesia, ensures minimal lipid peroxidation in the lung. In addition, we found that fentanyl and droperidol were able to inhibit the Fe(2+)-catalysed lipid peroxidation in lung homogenate. We speculate that the inhibitory effect of fentanyl and/or droperidol on the (N2O + O2)-induced lipid peroxidation in the rat lung may be caused directly by their antioxidant properties. However, another explanation seems to be possible. The free radicals that are produced during the metabolism of fentanyl and droperidol may react with the radicals generated during the one-electron reduction of nitrous oxide. Such reactions will obviously reduce the free radical concentration in the organism and, hence, the likelihood of initiating lipid peroxidation.     

Test-tube simulated lipofuscinogenesis. Effect of oxidative stress on autophagocytotic degradation

Cysteine-stimulated oxidation of a rat liver lysosomal-mitochondrial fraction (LMF) was studied. The process would simulate oxidative stress-related events during the degradation of autophagocytosed material within secondary lysosomes, which may contribute to the formation of lipofuscin or age pigment. Millimolar concentration of cysteine was needed to stimulate LMF lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS). The amount of endogenous LMF iron was 545 micrograms/l and was enough to initiate peroxidation, probably through the reduction of ferric to ferrous iron by cysteine with induction of Fenton chemistry. Peroxidation could be completely inhibited by the addition of the iron chelator desferal or the antioxidant BHT. A substantial amount of the formed TBARS was associated with trichloroacetic acid (TCA) precipitable proteins. Elevated protein carbonyls was observed 1-2 h after the increase of TBARS. The tryptophan-tyrosine related protein autofluorescence (280/335 nm) decreased sharply during the first few hours of incubation. In contrast, a lipofuscin-type autofluorescence (345/430 nm) appeared only after a few days, suggesting that the latter fluorophore is not an immediate product of protein oxidation. The sequential formation of TBARS, protein carbonyls and lipofuscin-type autofluorescence as well as their dependence on iron and reducing agent add further support to the concept that lipofuscin forms in secondary lysosomes as a result of iron-catalyzed oxidative reactions involving autophagocytosed materials.