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Naphthalene-Induced Oxidative Stress and DNA Damage in Cultured Macrophage J774A.1 Cells
Abstract
Naphthalene is a bicyclic aromatic compound that is widely used in various domestic and commercial applications including lavatory scent disks, soil fumigants and moth balls. However, little information is available regarding the mechanism of naphthalene toxicity. We have assessed the concentration-dependent in vitro effects of naphthalene on increased lipid peroxidation, cytochrome c reduction, hydroxyl radical production, modulation of intracellular oxidized states by laser scanning confocal microscopy, and DNA fragmentation in cultured macrophage J774A.1 cells. The cells were incubated with 0-500 microM concentrations of naphthalene for 0, 12 and 24 h at 37 degrees C. Concentration- and time-dependent changes were observed. No significant changes were observed with concentrations of naphthalene up to 100 microM. At 24 h, lipid peroxidation increased by 1.8-, 2.4- and 2.9-fold at 200, 300 and 500 microM concentrations of naphthalene. Approximately 2.0-, 3.1- and 4.6-fold increases in cytochrome c reduction were observed at 200, 300 and 500 microM concentrations of naphthalene, respectively, at this time point demonstrating the production of superoxide anion, while under the same conditions approximately 2.4-, 3.2- and 4.9-fold increases in hydroxyl radical production were observed, respectively. Following incubation of these cells with 200 and 500 microM concentrations of naphthalene 2.3- and 4.7-fold increases in fluorescence intensity were observed, respectively, as compared to the untreated cells. At 24 h, approximately 1.8-, 2.3- and 3.0-fold increases in DNA fragmentation were observed following incubation with 200, 300 and 500 microM concentrations of naphthalene, respectively. Naphthalene also produced concentration- dependent decreases in cell viability. At the 12 h time point, significant changes were observed only with 300 and 500 microM concentrations of naphthalene. These results demonstrate that naphthalene may induce toxic manifestations by enhanced production of oxygen free radicals, resulting in lipid peroxidation and DNA damage.
... Acute toxicity: 1-ethylnaphthalene 48 h LC50 ¼ 0.295 mg L À1 glass shrimp Palaemonetes pugio (Unger et al., 2007); 2ethylnaphthalene 96 h LC50 ¼ 8.13 mg L À1 mussel Mytilus edulis (Bagchi et al., 1998). Oxidative stress responses, ROS production, lipid peroxidation and membrane damage (Bagchi et al., 1998;Stohs et al., 2002). ...
... Acute toxicity: 1-ethylnaphthalene 48 h LC50 ¼ 0.295 mg L À1 glass shrimp Palaemonetes pugio (Unger et al., 2007); 2ethylnaphthalene 96 h LC50 ¼ 8.13 mg L À1 mussel Mytilus edulis (Bagchi et al., 1998). Oxidative stress responses, ROS production, lipid peroxidation and membrane damage (Bagchi et al., 1998;Stohs et al., 2002). ...
... In addition, a PAH mixture (fluoranthene, pyrene and benzo[a]pyrene) supressed gene expression for photosynthetic pigments and silica-associated proteins that prevent cell division in diatoms (Bopp and Lettieri, 2007). Bagchi et al. (1998) hypothesise that genotoxicity resultant of PAH contamination is due to DNA damage caused by ROS formed during hydrocarbon degradation. ...
Oil spills of varying magnitude occur every year, each presenting a unique challenge to the local ecosystem. The complex, changeable nature of oil makes standardised risk assessment difficult. Our review of the state of science regarding oil's unique complexity; biological impact of oil spills and use of rapid assessment tools, including commercial toxicity kits and bioassays, allows us to explore the current issues preventing effective, rapid risk assessment of oils. We found that despite the advantages to monitoring programmes of using well validated standardised tests, which investigate impacts across trophic levels at environmentally relevant concentrations, only a small percentage of the available tests are specialised for use within the marine environment, or validated for the assessment of crude oil toxicity. We discuss the use of rapid tests at low trophic levels in addition to relevant sublethal toxicity assays to allow the characterisation of oil, dispersant and oil and dispersant mixture toxicity. We identify novel, passive dosing techniques as a practical and reproducible means of improving the accuracy and maintenance of nominal concentrations. Future work should explore the possibility of linking this tiered testing system with ecosystem models to allow the prediction and risk assessment of the entire ecosystem.
... This could be due to the very low concentrations of these compounds. Moreover, our analytical method only targeted the most nonpolar compounds present, whereas phototoxic manifestation induced by naphthalene and its analogs (the major components of the WSF used in the present investigation) may involve the rapid conversion of these compounds to the corresponding more polar oxidation products including naphtols, ketones and quinones (Bagchi et al. 1998). However, such new fluorescent materials formed during the course of the experiment might not have derived from the WSF used, but from fluorophores, located between 260 and 280 nm emission wavelengths, which are often prominent in marine waters (De Souza Sierra et al. 1994). ...
... Moreover, being relatively hydrophobic, dissolved PAHs accumulate in the photosynthetic membrane, where they could interfere with chl a synthesis ). More specifically, it has been demonstrated that the toxic manifestations induced by naphthalene (i.e. the main compound in the WSF used in the present study) may involve the conversion of this compound to the corresponding naphthoquinones, as well as hydroxylated products including naphthols (Bagchi et al. 1998). Such degradation products can potentially block photosynthesis, especially when plastoquinone (Q b ) is used as an electron acceptor or donor (Huang et al. 1997), because of their structural similarity with the Q b -binding niche in Photosystem II. ...
... Although major hydrocarbon compounds in our WSF (i.e. naphthalene and its analogs) are not considered to be genotoxic, they can induce the production of ROS (which may enhance lipid peroxidation and cause DNA damage: Bagchi et al. 1998), following their photosensibilization after bioaccumulation (Marwood et al. 1999, Choi & Oris 2000. The highly reactive quinone derivates may also be responsible for the hydrocarbon-induced oxidative stress and toxic manifestation, as they are know to bind covalently to biological membranes (Sikkema et al. 1995). ...
The present study demonstrates the effects of the water-soluble fraction (WSF) of a crude oil, enhanced ultraviolet-B radiation (UVBR: 280 to 320 nm), and the combination of WSF and enhanced UVBR on a natural plankton assemblage (< 150 mu m) isolated from the lower St. Lawrence Estuary. To study the separate and dual effects of WSF and UVBR, 12 microcosms (9 1) were immersed in the water column of larger mesocosms (polyethylene bags; 1800 1), providing 4 treatments, each in triplicate: (1) NUVBR + WSF (natural UVBR with WSF), (2) HUVBR + WSF (enhanced UVBR with WSF), (3) NUVBR (natural UVBR without WSF), and (4) HUVBR (enhanced UVBR without WSF). During 5 d we monitored the incident radiation, WSF and nutrient concentrations, abundance and production of heterotrophic bacteria and phytoplankton. Strong deleterious effects of WSF and lower effects of UVBR were observed on the phytoplankton assemblage, with d decrease in growth rates accompanied by an increase in mean cell size which reflected a perturbation of the cell division cycle. Using the NUVBR treatement as reference conditions, the above effects resulted in d reduction of 84, 79 and 60% of total abundance of the phytoplankton fraction < 20 mu m in the HUVBR + WSF, NUVBR + WSF and HUVBR treatments, respectively. Significant higher values of bacterial abundances were observed in the WSF-added treatments compared to NUVBR without WSF. However, bacterial thymidine incorporation exhibited diel variations, suggesting cumulative UVBR-induced DNA and/or PAH-induced DNA damages, and possible repair mechanisms with the co-occurrence of more available growth substrates from stressed phytoplankton. The absence of significant differences between both WSF-added treatments under the 2 different UVBR conditions suggests that there is no additive interaction between WSF and UVBR. This study provides therefore the first evidence of a non-synergistic interaction between both stresses, and suggests that UVBR-induced effects on marine microorganisms can be completely masked by the strong deleterious effects of soluble petroleum hydrocarbons.
... Both 1-naphthol and 2-naphthol compounds are oxidized either enzymatically or nonenzymatically to 1,4-naphthoquinone (1,4-NPQ) and 1,2-NPQ, respectively (Chichester et al., 1994;Lin et al., 2009;Wilson et al., 1996;Zheng et al., 1997). These compounds cause DNA damage by binding N3 and N7 position guanine and adenine in DNA and increasing oxidative stress (Bagchi et al., 1998(Bagchi et al., , 2001Saeed et al., 2009). ...
... However, naphthalene has no specific affinity of binding directly to DNA; more recently some researchers indicated that some naphthalene metabolites can react with the adenine and guanine in DNA to form adducts at the N3 and N7 positions of the two molecules, respectively (McCoull et al., 1999;Saeed et al., 2007). A number of studies, which have demonstrated that naphthalene administration results in DNA damage (Bagchi et al., 1998), reported that naphthalene has caused approximately 1.8-, 2.3-and 3.0-fold DNA fragmentation in hepatic and brain DNA fragmentation (Bagchi et al., 1998(Bagchi et al., , 2001. ...
... However, naphthalene has no specific affinity of binding directly to DNA; more recently some researchers indicated that some naphthalene metabolites can react with the adenine and guanine in DNA to form adducts at the N3 and N7 positions of the two molecules, respectively (McCoull et al., 1999;Saeed et al., 2007). A number of studies, which have demonstrated that naphthalene administration results in DNA damage (Bagchi et al., 1998), reported that naphthalene has caused approximately 1.8-, 2.3-and 3.0-fold DNA fragmentation in hepatic and brain DNA fragmentation (Bagchi et al., 1998(Bagchi et al., , 2001. ...
Naphthalene, a bicyclic aromatic hydrocarbon, has toxic effects on animals and humans. Although recent studies stressed on the genotoxic and cytotoxic effects of naphthalene and its metabolites on eukaryotic cells, there is a big controversy among the results of these studies. The aim of this study is to investigate the effects of naphthalene and its metabolites on the cytotoxicity and genotoxicity in the human lymphocytes in the culture. The genotoxic and cytotoxic effects of naphthalene and its metabolites, 1-naphthol and 2-naphthol, were studied using cytotoxicity test (lactate dehydrogenase and cell proliferation (WST-1) assays) and DNA fragmentation assay (terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay). Naphthalene and its metabolites had no significant cytotoxic effect on treated samples when compared with untreated ones. This result was also confirmed by WST-1 assay. In the TUNEL assay, DNA fragmentation was induced significantly by all concentrations of naphthalene and 2-naphthol and 50 and 100 µM concentrations of 1-naphthol (p < 0.05 or 0.001). In the DNA fragmentation, the most effective dose of 2-naphthol (63%) was 100 µM, when compared with negative control group (13%). These results suggest that naphthalene and its metabolites, 1-naphthol and 2-naphthol, may cause DNA damage on human lymphocytes.
... Naphthalene-1,2-oxide, 1-naphthol (1-NAO), 2-naphthol (2-NAO), 1,2-naphthoquinone (1,2-NQ), and 1,4-naphthoquinone (1, were typical metabolites of NAP (Brusick 2008;Cho et al. 2006;Kong et al. 2011). The toxicity of NAP and its metabolites have been studied at the cellular level such as in mouse hepatocytes and macrophage cell lines, and previous results confirmed that the metabolites exhibited large differences in cytotoxicity relative to the parent substance (Bagchi et al. 1998;Norikura et al. 2002). However, differential eco-toxicological responses related to oxidative stress of NAP and metabolites in earthworms have not been thoroughly investigated. ...
... Bagchi et al. (2000) have found DNA fragments in the brain and liver tissue of mice after oral administration of NAP. At the cellular level, NAP could induce DNA fracture in macrophage cell lines (Bagchi et al. 1998), and it was believed that the genotoxicity of NAP mainly depended on its metabolites such as naphthoquinone (Brusick 2008). NAP, 1-NAO, and 2-NAO at 10, 25, 50, and 100 μM all caused DNA fracture of lymphocytes, and the order of DNA damage level was 2-NAO > 1-NAO > NAP (Kapuci et al. 2014). ...
Naphthalene (NAP) was frequently detected in polycyclic aromatic hydrocarbons (PAHs)-contaminated soil, and its residues may pose an eco-toxicological threat to soil organisms. The toxic effects of NAP were closely tied to phenolic and quinone metabolites in biological metabolism. However, the present knowledge concerning the eco-toxicological impacts of NAP metabolites at the animal level is scanty. Here, we assessed the differences in the eco-toxicological responses of Eisenia fetida (E. fetida) in NAP, 1-naphthol (1-NAO) or 1,4-naphthoquinone (1,4-NQ) contaminated soils. NAP, 1-NAO, and 1,4-NQ exposure triggered the onset of oxidative stress as evidenced by the destruction of the antioxidant enzyme system. The lipid peroxidation and DNA oxidative damage levels induced by 1-NAO and 1,4-NQ were higher than those of NAP. The elevation of DNA damage varied considerably depending on differences in oxidative stress and the direct mode of action of NAP or its metabolites with DNA. All three toxicants induced different degrees of physiological damage to the body wall, but only 1, 4-NQ caused the shedding of intestinal epithelial cells. The integrated biomarker response for different exposure times illustrated that the comprehensive toxicity at the animal level was 1,4-NQ > 1-NAO > NAP, and the time-dependent trends of oxidative stress responses induced by the three toxicants were similar. At the initial stage, the antioxidant system of E. fetida responded positively to the provocation, but the ability of E. fetida to resist stimulation decreased with the prolongation of time resulting in provocation oxidative damage. This study would provide new insights into the toxicological effects and biohazard of PAHs on soil animals.
Graphical Abstract
... The covalent modification of PRO by these naphthalene metabolites can induce structural changes, potentially impacting their function. 28 Additionally, the binding of reactive metabolites to PRO may activate cellular stress response pathways, initiating mechanisms to counteract damage and facilitate repair, ultimately influencing the overall fate of the cell. 29 Additionally, the study aligns with previous research indicating the sensitivity of fish PRO to environmental contaminants, which can have adverse effects on fish physiology. ...
... In the stomach of A. testudineus, the activity of AST exhibited significant changes in response to naphthalene exposure. Under T1 conditions, the activity increased notably over 1, 5, 10, 15, and 21 days of exposure with increments of 12.77, 28 Table S2). In the liver of A. testudineus, AST activity was significantly affected by naphthalene exposure. ...
This study addresses the increasing concern about naphthalene, a polycyclic aromatic hydrocarbon (PAH), highlighting its growing threats to the environment and aquatic life. The research examines its impact on Anabas testudineus (Bloch) through a detailed dose-specific bioenzymological analysis. Experimental fish groups were exposed to T1 (0.71 mg/L) and T2 (1.42 mg/L) naphthalene concentrations, representing 25 and 50% of the LC50 value, respectively, over a 1–21 day period. Following the experiment, water samples underwent physicochemical analysis, while fish tissues were examined for diverse bioenzymological parameters. Among these parameters, aspirate aminotransferase (AST) and alanine aminotransferase (ALT) serve as crucial indicators for monitoring the physiological status of fish and addressing pollution induced by PAHs, especially naphthalene. Statistical significance was observed in morpho-pathological changes and erythrocyte alterations, particularly the presence of tear-drop appearance (Tr) positively interacting with swelled cells (Sc), vacuolated cells (Va), and sickle cells (Sk) (P < 0.05). These findings highlight tear-drop appearance (Tr) as a significant biomarker in response to naphthalene exposure. The observed changes in A. testudineus tissue bioenzymology, apoptosis, and erythrocytic alterations were exposure and dose-dependent. The research highlights the significance of overseeing and controlling PAH concentrations in aquatic ecosystems to ensure the well-being of A. testudineus (Bloch).
... Naphthalene produces excess of free oxygen radicals which results in lipid peroxidation and DNA damage [7]. When this activity occurs in red blood cells, it will result in cell lysis. ...
Background
Naphthalene is an aromatic hydrocarbon that potentially produces methemoglobinaemia but rarely causes hemolysis, especially in children with underlying glucose-6-phosphate dehydrogenase deficiency. Although ingestion of a single moth ball by an older child may not be life threatening, it can be fatal if ingested by a toddler.
Case presentation
A 2-year-old Singhalese boy developed acute severe hemolysis and methemoglobinaemia following ingestion of a mothball. On admission, the patient was ill and pale. The child was tachycardic and tachypnoiec with oxygen saturation of 76% on air. Blood investigations showed significant anemia, elevated reticulocytes, and evidence of hemolysis in a blood picture, along with elevated lactate dehydrogenase and indirect bilirubin. Child also had ST depressions on electrocardiogram examination with negative troponin-I. He was given four packed red blood cell (PRBC) transfusions and was successfully discharged in 3 days time following optimal supportive treatment. A glucose-6-phosphate dehydrogenase assay confirmed the diagnosis of glucose-6-phosphate dehydrogenase deficiency in this child: 0.9 U/gHb (4.0–13.0 U/gHb).
Conclusion
This case report highlights a rare life-threatening presentation of naphthalene ingestion in a child with previously undiagnosed glucose-6-phosphate dehydrogenase deficiency. Ingestion of even a single moth ball can be fatal in vulnerable children given the altered toxicokinetics of naphthalene in children.
... Naphthalene causes oxidative stress by enhancing the production of free oxygen radicals, which then results in lipid peroxidation and deoxyribonucleic acid damage to cells. [8] Hemolysis is thus particularly seen in patients who have a low tolerance to oxidative stress, such as patients with G6PD deficiency. [9] G6PD is essential in red cell metabolism due to its role in the pentose phosphate pathway. ...
... Naphthalene enhances the production of free radicals, which cause oxidative stress and subsequently damage to cells due to lipid peroxidation [5]. Individuals who are vulnerable to oxidative stress can present with hemolysis, such as people with G6PD deficiency [6]. ...
Naphthalene is a major component of mothballs. Domestically, people use mothballs as an insect repellent. Its deliberate or accidental ingestion leading to toxicity has rarely been reported in the medical literature, despite its widespread use in Southeast Asia. Naphthalene, or mothball poisoning, is a rare but serious condition that can have detrimental effects on human health. This case report presents the clinical course of a 22-year-old male who ingested six naphthalene balls, resulting in severe symptoms including fever, abdominal pain, vomiting, jaundice, and dark-colored urine. Laboratory investigations were suggestive of acute intravascular hemolysis and methemoglobinemia. The patient was promptly admitted to the hospital, where he received supportive care along with specific treatment in the form of red blood cell transfusions, intravenous methylene blue, ascorbic acid, and N-acetyl cysteine. Through this report, the importance of raising awareness about the dangers of naphthalene poisoning and the specific treatment options available is highlighted.
... Methemoglobinemia and hemolytic anaemia have both emerged as common findings through multiple case reports on naphthalene toxicity, and this correlation remains true in the index case as well [5,6]. Naphthalene poisoning induces oxidative stress by amplifying the generation of free radicals, leading to lipid peroxidation and subsequent damage to cellular membranes, culminating in cell lysis [7]. The exacerbation of these effects is particularly pronounced in patients with G6PD deficiency. ...
Acute-onset unexplained hypoxemia persisting despite 100% oxygen has a limited differential diagnosis but poses a challenging diagnostic dilemma. Methemoglobinemia, a hemolytic condition, may lead to significant complications if it goes undiagnosed during the critical golden hour of an emergency department (ED) presentation. This case report presents the clinical details of a 30-month-old child with acute intravascular hemolysis evident by severe pallor and hemoglobinuria and severe hypoxia documented on pulse oximetry. During the ABCDE (Airway, Breathing, Circulation, Disability, Exposure) of the primary survey, "exposure" revealed the parent's deliberate fastening of a mothball around the waist of the baby on the advice of a traditional healer, which was identified as the source of naphthalene toxicity. The swift intervention was undertaken for hypoxic respiratory compromise with 100% oxygen just after triage, and the naphthalene ball with the tied cloth was removed. Arterial blood gas and co-oximetry analysis confirmed the diagnosis of methemoglobinemia, and other laboratory tests suggested severe hemolytic anaemia as well as hemoglobinuria favouring intravascular hemolysis. With the exclusion of other common differentials for hemolytic anaemia, including sickle cell crisis, autoimmune hemolytic anaemia, hemolytic uremic syndrome, and G6PD deficiency, naphthalene exposure was considered the culprit for both hemolysis and methemoglobinemia. After obtaining the history of another similar episode of anaemia six months ago requiring blood transfusion, we retrospected on similar mothball exposure, but parents denied that, saying they were using the mothball only for the last 10 days on the advice of a local healer with intent to get rid of some evil power and sickness in their child. After analyzing the old records of prior hospitalization and getting assured of a normal report of G6PD level, intravenous methylene blue was administered. But in view of an inadequate response, a single blood volume exchange transfusion was performed during the ED stay only, which resulted in a notable reduction in subsequent methemoglobin levels and an improvement of the child's clinical condition by the second day. The child was discharged by the third day with no distress and no further episodes of hemoglobinuria, with detailed parental counselling and follow-up advice. This case underscores the imperative need for timely recognition and effective management of methemoglobinemia in the paediatric population while emphasizing the potential hazards associated with naphthalene exposure. Further comprehensive investigations are warranted to elucidate optimal treatment strategies and explore long-term outcomes in similar clinical scenarios.
... The majority of bivalves like P. viridis toxicity research has been focused on the animal's metal bio-accumulation mechanism and the organs involved Satheeswaran et al., 2019). There has been very little research on the impact of naphthalene on the physiological processes of bivalves like P. viridis (Bagchi et al., 1998). This is the first research to look at how biotransformation enzymes and DNA damage in P. viridis are affected by chronic long-term exposure to sublethal concentrations of naphthalene. ...
... In rats, the ROS production following the exposure to naphthalene may be result in lipid peroxidation in mitochondria of liver and brain cells, with glutathione depletion by 83% and 49%, respectively (Vuchetich et al., 1996). To further detail its relationship with oxidative stress, Bagchi et al. (1998) Glutathione (GSH) is a tripeptide, also called γ-L-glutamyl-L-cysteinylglycine, present in all vertebrates (Hermes-Lima et al., 2012;Srikanth et al., 2013;Xia & Møller, 2018;Dahms-Verster et al., 2020). In mammalian tissues, its concentration is between 1-10 mM (the highest concentration being found in the liver), being the most abundant non-protein thiol that defends the cells against oxidative stress (Lu et al., 2018). ...
In 2017 around 5 million people had premature deaths due to air pollution. Our objectives were to identify which atmospheric nanoparticles cause negative effects on human health, their mechanisms of action and how to prevent them. Two types of particles were tested, naphthalene (anthropogenic origin) and particles from biomass burning (from both anthropogenic and biogenic origin). Three different methodologies were used: real time cell analysis, a measure of cell proliferation and adhesion; quantitative phase contrast microscope (Holomonitor, PHI), that gives behavioral and structural parameters of cells (e.g. area, motility and migration) and bright field real time microscopy (Cytation, Biotek), for real time cell imaging. Fresh naphthalene nanoparticles were more toxic than aged. Glutathione at 1 mM partially restored cell proliferation. Four by-products of naphthalene nanoparticles were tested on lung cell model A549 :1.4-naphthoquinone (1.4-NQ); 2-hydroxy-1.4-naphthoquinone (2-OH-NQ), phthalic acid (PA) and phthaldialdehyde (OPA). According to their EC50, 1.4-NQ showed a toxicity 100 times higher than other compounds. 1.4-NQ and OPA decreased the optical volume, migration, motility and motility speed of cells. 1.4-NQ increased the oxidative stress (DHR staining). Addition of glutathione protected the cells against 1,4-NQ. The toxicity of particles from different mode of burning wood (flaming (FWS), smoldering (SWS) and pyrolysis (PWS)) were similarly toxic on A549 cell proliferation. In conclusion, toxicity of naphthalene nanoparticles depends on 1,4 naphthoquinone which is mediated through oxidative stress.
... The toxicity of naphthalene relates to increased production of oxygen free radicals and oxidative stress causing lipid peroxidation and damage to DNA. 7 Oxidative injuries can be more severe in those with sickle cell disease or sickle cell trait 8 due to the increased oxidative stress resulting from chronic hemolysis. Furthermore, toxicity can be more pronounced in infants and those with G6PD deficiency, 2,9 as this enzyme provides resistance against oxidative stress on the RBCs. ...
... Naphthalene is a polyaromatic hydrocarbon (PAH) and exposure to this environmental toxicant can promote ROS, DNA damage and disrupt cellular glutathione levels [20][21][22][23][24][25]. Naphthalene is found in cigarette smoke, gasoline, and mothballs [26][27][28] as well as being derived in a number of industrial processes. ...
The epitranscriptomic writer Alkylation Repair Homolog 8 (ALKBH8) is a transfer RNA (tRNA) methyltransferase that modifies the wobble uridine of selenocysteine tRNA to promote the specialized translation of selenoproteins. Using Alkbh8 deficient (Alkbh8def) mice, we have investigated the importance of epitranscriptomic systems in the response to naphthalene, an abundant polycyclic aromatic hydrocarbon and environmental toxicant. We performed basal lung analysis and naphthalene exposure studies using wild type (WT), Alkbh8def and Cyp2abfgs-null mice, the latter of which lack the cytochrome P450 enzymes required for naphthalene bioactivation. Under basal conditions, lungs from Alkbh8def mice have increased markers of oxidative stress and decreased thioredoxin reductase protein levels, and have reprogrammed gene expression to differentially regulate stress response transcripts. Alkbh8def mice are more sensitive to naphthalene induced death than WT, showing higher susceptibility to lung damage at the cellular and molecular levels. Further, WT mice develop a tolerance to naphthalene after 3 days, defined as resistance to a high challenging dose after repeated exposures, which is absent in Alkbh8def mice. We conclude that the epitranscriptomic writer ALKBH8 plays a protective role against naphthalene-induced lung dysfunction and promotes naphthalene tolerance. Our work provides an early example of how epitranscriptomic systems can regulate the response to environmental stress in vivo.
... Though it is poorly soluble in water, it is readily absorbed after systemic or topical exposure and metabolized into epoxide metabolites and quinone derivatives by cytochrome P450. 2 These metabolites act as oxidants and result in methemoglobinemia and hemolytic anaemia in susceptible indivisuals like G-6 PD defi cient persons. ...
... Though it is poorly soluble in water, it is readily absorbed after systemic or topical exposure and metabolized into epoxide metabolites and quinone derivatives by cytochrome P450. 2 These metabolites act as oxidants and result in methemoglobinemia and hemolytic anaemia in susceptible indivisuals like G-6 PD defi cient persons. ...
Mothballs are commonly used household product that contain an aromatic hydrocarbon named naphthalene. On systemic exposure it causes oxidant injury to hemoglobin molecules resulting in oxidized forms of hemoglobin like methaemoglobin and further hemoglobinuria. Most commonly naphthalene poisoning cases present as hemolytic anaemia with hemoglobinuria and methemoglobinemia following oral ingestion of moth balls. Here we are reporting an unusual case of methemoglobinemia without hemolytic anaemia following absorption of the aromatic hydrocarbon from skin after application of powdered mothballs mixed with coconut oil.
... Naphthalene (NA) is a polyaromatic hydrocarbon (PAH) and exposure to this environmental toxicant can promote ROS, DNA damage and disrupt cellular glutathione levels (ATSDR, 2005;D. Bagchi, Bagchi, Balmoori, Vuchetich, & Stohs, 1998;M. Bagchi, Bagchi, Balmoori, Ye, & Stohs, 1998;Carratt et al., 2019;Lin et al., 2005;Plopper et al., 2013a). NA is found in cigarette smoke, gasoline, and mothballs (Jia & Batterman, 2010; Kakareka & Kukharchyk, 2003;Sudakin, Stone, & Power, 2011) as well as being derived in a number of industrial processes. We postulated that ALKBH8 dependent epitranscriptomic marks would play an im ...
Background
The epitranscriptomic writer Alkylation Repair Homolog 8 (ALKBH8) is a tRNA methyltransferase that modifies the wobble uridine of selenocysteine tRNA to promote the specialized translation, via stop codon recoding, of proteins that contain selenocysteine. Corresponding selenoproteins play critical roles in protecting against reactive oxygen species and environmental stress. Using a novel animal model deficient in Alkbh8 , we have investigated the importance of epitranscriptomic systems in the response to naphthalene (NA), an abundant polycyclic aromatic hydrocarbon, glutathione depleter and lung toxicant found in tobacco smoke, gasoline and mothballs.
Objectives
Our goal was to define the molecular reprogramming of Alkbh8 deficient ( Alkbh8 def ) mice and evaluate the roles that the epitranscriptomic writer ALKBH8 and selenoproteins play in mitigating NA-induced toxicity and lung dysfunction.
Methods
We performed basal lung analysis and NA exposure studies using WT, Alkbh8 de f and Cyp2abfgs-null mice, the latter of which lack the cytochrome P450 enzymes required for NA bioactivation. We characterized gene expression, molecular markers of damage, viability and tolerance to NA.
Results
Under basal conditions, lungs from Alkbh8 def mice have increased oxidation-reduction potential (ORP) and 8-isoprostane levels, and have reprogrammed at the molecular level to display increased stress response transcripts. In addition, the ALKBH8 writer deficient lungs have a decreased GSH/GSSG ratio. Alkbh8 def mice are more sensitive to NA than WT, showing higher susceptibility to lung damage both at the cellular and molecular levels. WT mice develop a tolerance to NA after 3 days, defined as resistance to a high challenging dose after repeated exposures, which is absent in Alkbh8 def mice, with writer deficient not surviving NA exposure.
Discussion
We conclude that the epitranscriptomic writer ALKBH8 plays a protective role against NA-induced lung dysfunction and promotes NA tolerance. Our work provides an early example of how epitranscriptomic systems can regulate the response to environmental stress in vivo .
... scent disks, moth bails…). This compounds induce toxicity through its ability to increase lipid peroxidation, cytochrome c reduction and hydroxyl radical production (25). Previous studies identified 1,2-dihydrodiol naphthalene and 1-naphthol as the major metabolites of naphthalene in rats (4). ...
Background: Oxidative stress is a situation where the cell no longer
controls the excessive presence of toxic oxygen radicals. Many human
diseases have a strong relationship with oxidative stress due to
an imbalance between antioxidants and pro-oxidants. The objective of
this study is to evaluate the in vivo antioxidant capacity of Theobroma
cacao (T.cacao) beans extract and its acute toxicity. Methods: T. cacao
beans were collected in the Obala locality (Center Cameroon) and
then subjected to hydroethanolic extraction (70:30) at pH 3. The in vivo
oxidative stress induction was done using naphthalene at 110 mg / kg
and different doses of extracts (50 mg / kg, 100 mg / kg, and 200 mg /
kg) were orally administered to rats. Some oxidative stress parameters
helped to evaluate the antioxidant potential of the extract (superoxide
dismutase (SOD), reduced glutathione (GSH) and malondialdehyde
(MDA)). In addition, the acute toxicity of T. cacao was evaluated by the
methods recommended by the ODCE. Test groups received respectively
the extract at different doses (5000 mg / kg and 2000 mg / kg)
against 10% of DMSO and distilled water as neutral controls. Hepatic
function was assessed using transaminase assays (ASAT, ALAT), proteins
and histological sections. Also the blood count allowed to explore
the haematological function. Results: The administration of different
doses of extracts or vitamin C as standard significantly increased GSH
levels as well as antioxidant enzymes (SOD, CAT) and a significant
decrease in MDA in studied organs and serum of animals compared
to pro-oxidant control. ALAT and ASAT activities did not significantly
vary in rats compared to neutral controls. No deaths and hepatic injuries
were observed at different doses of the extracts. Conclusion:
The extract of T. cacao beans possess in vivo antioxidant capacities
capable of protecting tissues against oxidative stress and toxicity in
rats at 2000 mg / kg and 5000 mg / kg.
... scent disks, moth bails…). This compounds induce toxicity through its ability to increase lipid peroxidation, cytochrome c reduction and hydroxyl radical production (25). Previous studies identified 1,2-dihydrodiol naphthalene and 1-naphthol as the major metabolites of naphthalene in rats (4). ...
... Its toxic manifestations are mainly due to production of oxygen free radicals leading to lipid peroxidation and deoxyribonucleic acid (DNA) damage [5]. Hemolysis occurs usually in susceptible individuals such as in those who are G6PD deficient. ...
Background
Naphthalene (mothball) is a commonly used deodorizer in the Indian subcontinent, including Sri Lanka. Though it is freely available around this country, poisoning has never been reported in the literature. Ingestion, either accidental or by deliberate self-harm, can occur due to its abundance as well as its candy-resembling appearance.
Case Presentation
A 33-year-old Sri Lankan woman presented to us 2 days after the self-ingestion of 15 naphthalene balls. She had features of intravascular hemolysis without features of pigment nephropathy or methemoglobinemia. She was symptomatically managed with blood transfusion and adequate hydration.
Conclusion
Naphthalene ingestion can lead to severe intravascular hemolysis as well as methemoglobinemia. The resultant pigment nephropathy may also lead to acute kidney injury.
... Older studies performed on macrophages J774A.1 described increased hydroxyl radical production with increased lipid peroxidation, DNA fragmentation, and decreased viability of cells incubated for 24 h at concentrations of naphthalene greater than 200 µM [76,77]. Another report revealed a cell-specific effect of naphthalene on the viability of promyelocytic leukemia HL-60 cells and hepatocellular carcinoma Hep G2 cells. ...
We investigated the use of a supported silicalite-1 film (SF) as a promising coating for metallic materials used in the fabrication of prostheses. The role of carbonaceous residua present on high-temperature calcined-SF in generating singlet oxygen for future use as a sterilization method has also been addressed, and the potential genotoxicity of these residua in osteoblast-like cells has been investigated. Calcination of as-synthesized SF induced the appearance of a rather complicated mixture of aliphatic and aromatic species on its outer surface. A series of variously volatile polycyclic aromatic hydrocarbons (PAH), including naphthalene, fluorene, phenanthrene, anthracene, fluoranthene, and pyrene, were identified in micromole concentrations. Irradiation of these PAHs on calcined-SF immersed in air-saturated chloroform led to the formation of very low concentrations of singlet oxygen. However, an increased level of DNA damage was observed on calcined-SF by immunofluorescence staining of phosphorylated histone H2AX analyzed by flow cytometry.
... The increase in acetone in the BALF of the injured mice may reflect lipid peroxidation in the airways of the injured mice compared with the tolerant mice [14]. Previous studies have also reported the presence of oxidative damage and lipid oxidation after naphthalene treatment in the rodent models and cell cultures [43][44][45][46]. ...
Naphthalene causes mouse airway epithelial injury. However, repeated exposures of naphthalene result in mouse airway tolerance. Previous results showed that toxicity or tolerance was correlated with changes of phosphorylcholine-containing lipids. In this study, a mass spectrometry-based lipidomic approach was applied to examine the effects of naphthalene-induced injury or tolerance in the male ICR mice. The injury model was vehicle x 7 plus 300 mg/kg naphthalene while the tolerant one was 200 mg/kg daily x 7 followed by 300 mg/kg naphthalene on day 8. The lung, liver, kidney, and serum samples were collected for profiles of phosphorylcholine-containing lipids including phosphatidylcholines (PCs) and sphingomyelins (SMs). A partial least-square-discriminate analysis model showed different lung phosphorylcholine-containing lipid profiles from the injured, tolerant, and control groups. Perturbation of diacyl-PCs and plasmenylcholines may be associated with enhanced membrane flexibility and anti-oxidative mechanisms in the lungs of tolerant mice. Additionally, alterations of lyso-PCs and SMs may be responsible for pulmonary dysfunction and inflammation in the lungs of injured mice. Moreover, serum PC(16:0/18:1) has potential to reflect naphthalene-induced airway injuries. Few phosphorylcholine-containing lipid alterations were found in the mouse livers and kidneys across different treatments. This study revealed the changes in lipid profiles associated with the perturbations caused by naphthalene tolerance and toxicity; examination of lipids in serum may assist biomarker development with the potential for application in the human population.
... Severe neutrophilic leukocytosis and acute kidney injury is also observed [4]. Hyperbilirubinemia, with indirect predominance, elevated lactate dehydrogenase and decreased haptoglobin levels are seen [5]. in lipid peroxidation and deoxyribonucleic acid damage to cells [6]. Hemolysis is thus particularly seen in patients who have a low tolerance to oxidative stress, such as patients with G6PD deficiency [7]. ...
Naphthalene poisoning is a rare form of toxicity that may occur after ingestion, inhalation, or dermal exposure to naphthalene-containing compounds such as mothballs. Clinically, patients present with acute onset of dark brown urine, watery diarrhea, and non-bloody bilious vomiting 48-96 hours after exposure. Vital sign abnormalities include fever, tachycardia, hypotension, and persistent pulse oximetry readings of 84%-85% despite oxygen supplementation. Laboratory workup demonstrates hyperbilirubinemia with indirect predominance, hemolytic anemia, methemoglobinemia, and renal dysfunction. Treatment options include supportive care, red cell transfusion, ascorbic acid, methylene blue, and N-acetylcysteine. We present a case of naphthalene toxicity in a 20-year-old autistic male, who improved with supportive care, red blood cell transfusion, and ascorbic acid.
... The toxicity of NA compound and its by-products have been characterized by several studies (Environmental Protection Agency 2003a, b). In humans, acute exposition to NA causes liver and kidney damage, cataract formation, changes in hemoglobin oxidation, and neurological and DNA damage, among other affections (Bagchi et al. 1998a;Stohs et al. 2002;Bagchi et al. 1998b). ...
Naphthalene (NA) is a polycyclic aromatic hydrocarbon with toxic properties in aquatic systems. Ozonation (O3) and catalytic ozonation (O3-cat) processes are attractive alternatives of degradation for this kind of compound. NA (20 mg L(-1)) degradation by conventional and catalytic ozonation in the presence of a cosolvent (ethanol) was the aim of this study. This solution was proposed to simulate some aspects of real wastewaters where not only water acts as solvent. Two proportions of the mixture ethanol/water were selected (30:70 and 50:50) with the purpose of studying the cosolvent effect on NA degradation system by ozonation. O3-cat process used nickel oxide as catalyst (0.1 g L(-1)). The degradation analysis of NA by O3-cat in two different proportions of cosolvent showed that in the case of 30:70 (ethanol/water), a 95 % of NA elimination in 60 min was obtained, while in the case 50:50 (ethanol/water), only 55 % was achieved. The O3 process showed similar results of degradation to the initial compound in comparison with catalytic system. According to these results, there is an inhibition effect in pollutant removal by ethanol due to the higher ethanol concentration; the lower elimination rate of NA was obtained (by 40 % during the 60 min). The by-products analysis of ozonation process detected oxalic and formic acids. Treatments with NiO presented less production of organic acids in comparison with conventional ozonation process. The high concentration of ethanol has a relevant factor in the elimination of NA and formation of organic acids; samples with 50 % of cosolvent have showed a higher concentration of organic acids. X-ray photoelectron spectroscopy (XPS) study of O3-cat of diluent (O3-NiO control) and O3-NA-NiO showed the presence of -CO3 absorbed on catalyst due to ethanol decomposition.
... Naphthalene is readily absorbed in the systemic circulation by the skin or inhalation and is initially metabolized into a number of reactive metabolites by cytochrome P450 oxidation and then excreted in the urine. Following liver metabolism, naphthol-alpha, the most potent derivative of naphthalene, causes hemolysis with severe anemia and Heinz bodies' formation [2]. Patients with low tolerance to oxidative stress like those with G6PD deficiency are particularly at higher risk for hemolysis. ...
... Lipophilic oil compounds accumulate in the cell membrane and change its structural and functional properties, including the loss of cell permeability, and cause other types of irreversible damage at the cell surface (Sikkema et al. 1995). Furthermore, toxicity studies have demonstrated that hydrocarbons can cause loss of cell mobility (Soto et al. 1975), DNA damage (Bagchi et al. 1998;Tang et al. 2002), prevention of nutrient and CO 2 absorption (Koshikawa et al. 2007), inhibition of nucleic acid and protein synthesis , chloroplast shrinkage, and loss of pigments in phytoplankton (Smith 1968). Many of these processes involve reactive oxygen species (ROS) (Torres et al. 2008;Lushchak 2011). ...
Exposure of phytoplankton to the water-accommodated fraction of crude oil can elicit a number of stress responses, but the mechanisms that drive these responses are unclear. South Louisiana crude oil was selected to investigate its effects on population growth, chlorophyll a (Chl a) content, antioxidative defense, and lipid peroxidation, for the marine diatom, Ditylum brightwellii, and the dinoflagellate, Heterocapsa triquetra, in laboratory-based microcosm experiments. The transcript levels of several possible stress-responsive genes in D. brightwellii were also measured. The microalgae were exposed to crude oil for up to 96 h, and Chl a content, superoxide dismutase (SOD), the glutathione pool (GSH and GSSG), and lipid peroxidation content were analyzed. The cell growth of both phytoplankton species was inhibited with increasing crude oil concentrations. Crude oil exposure did not affect Chl a content significantly in cells. SOD activities showed similar responses in both species, being enhanced at 4- and 8-mg/L crude oil exposure. Only H. triquetra demonstrated enhanced activity in GSSG pool and lipid peroxidation at 8-mg/L crude oil exposure, suggesting that phytoplankton species have distinct physiological responses and tolerance levels to crude oil exposure. This study indicated the activation of reactive oxygen species (ROS) in phytoplankton under crude oil exposure; however, the progressive damage in cells is still unknown. Thus, ROS-related damage in nucleic acid, lipids, proteins, and DNA, due to crude oil exposure could be a worthwhile subject of study to better understand crude oil toxicity at the base of the food web.
... Therefore, it is proposed that the similarities of these two pathways cause a disruption in brevetoxin production, as crude oil can disrupt the biosynthesis mechanisms required for glycolipids and lipid pigments (Morales-Loo and Goutx 1990). On the other hand, hydrocarbon toxicity can cause damage to and alterations of DNA and RNA (Bagchi et al. 1998;El-Sheekh et al. 2000;Tang et al. 2002;Parab et al. 2008), reduction in the size of cell nuclei (Tukaj et al. 1998), and loss of CO 2 absorption (Koshikawa et al. 2007), all of which could directly or indirectly disrupt the PKS pathway. ...
Impacts of the Deepwater Horizon oil spill on phytoplankton, particularly, the tolerability and changes to the toxin profiles of harmful toxic algal species remain unknown. The degree to which oil-affected sympatric Karenia brevis, Prorocentrum minimum, and Heterosigma akashiwo, all of which are ecologically important species in the Gulf of Mexico, was investigated. Comparison of their tolerability to that of non-toxic species showed that the toxin-production potential of harmful species does not provide a selective advantage. Investigated toxin profiles for K. brevis and P. minimum demonstrated an increase in toxin productivity at the lowest crude oil concentration (0.66 mg L(-1)) tested in this study. Higher crude oil concentrations led to significant growth inhibition and a decrease in toxin production. Findings from this study could assist in the assessment of shellfish bed closures due to high risk of increased toxin potential of these phytoplankton species, especially during times of stressed conditions.
... The substances were obtained from Sigma Aldrich Inc., Austria, with purity greater than 99%. Naphthalene as a common xenobiotic chemical is listed as a priority pollutant by the USEPA (USEPA, 1992) and can damage DNA in cultured macrophage cells (Bagchi et al., 1998). ...
Summary This study provides insight into the relevance of the chemical functional groups of soil organic matter (aromatic, paraffinic, O-alkyl, carboxyl and carbonyl carbon), as determined by CPMAS 13C NMR, on adsorption processes. Batch adsorption experiments with eight naphthalene derivatives were conducted with soils from a long-term field experiment and model sorbents. Although the adsorption of some derivatives was mainly affected by the paraffinic organic carbon content in soil, the relation between the C-distribution and adsorption was complex. This casts doubt on the use of such NMR data to estimate sorption behaviour. Additionally, sorption experiments were performed with six model sorbents representing typical soil components. Considerable adsorption of naphthalene derivatives was observed for montmorillonite and lignin; the smallest values were for kaolinite and cellulose. A quantum chemical approach was used to calculate a local polarity parameter as a molecular property of the naphthalene derivatives. This parameter was correlated with the logarithm of the adsorption coefficients, logKd. Here, clear trends were observed for three of the model sorbents (kaolinite, montmorillonite and lignin).
... role in the presence of different cell death chemical inducers— etoposide, naphthalene, anti-Fas, and gliotoxin. Etoposide and naphthalene are DNA damaging agents that trigger mitochondrial ROS production, inducing cell death via the intrinsic pathway (Gorman et al., 1997; Bagchi et al., 1998 Bagchi et al., , 2001 Pham and Hedley, 2001 ). Treatment with anti-Fas antibody mimics activation of death receptor signaling by FasL through the extrinsic pathway. ...
Programmed cell death (PCD) can play a crucial role in tuning the immune response to microbial infection. Although PCD can occur in different forms, all are mediated by a family of proteases called caspases. Caspase-2 is the most conserved caspase, however, its function in cell death is ill-defined. Previously we demonstrated that live attenuated cattle vaccine strain Brucella abortus RB51 induces caspase-2-mediated and caspase-1-independent PCD of infected macrophages. We also discovered that rough attenuated B. suis strain VTRS1 induces a caspase-2-mediated and caspase-1-independent proinflammatory cell death in infected macrophages, which was tentatively coined “caspase-2-mediated pyroptosis”. However, the mechanism of caspase-2-mediated cell death pathway remained unclear. In this study, we found that caspase-2 mediated proinflammatory cell death of RB51-infected macrophages and regulated many genes in different PCD pathways. We show that the activation of proapoptotic caspases-3 and -8 was dependent upon caspase-2. Caspase-2 regulated mitochondrial cytochrome c release and TNFα production, both of which are known to activate caspase-3 and caspase-8, respectively. In addition to TNFα, RB51-induced caspase-1 and IL-1β production was also driven by caspase-2-mediated mitochondrial dysfunction. Interestingly, pore formation, a phenomenon commonly associated with caspase-1-mediated pyroptosis, occurred; however, unlike its role in S. typhimurium-induced pyroptosis, pore formation did not contribute to RB51-induced proinflammatory cell death. Our data suggest that caspase-2 acts as an initiator caspase that mediates a novel RB51-induced hybrid cell death that simulates but differs from typical non-proinflammatory apoptosis and caspase-1-mediated proinflammatory pyroptosis. The initiator role of the caspase-2-mediated cell death was also conserved in cellular stress-induced cell death of macrophages treated with etoposide, naphthalene, or anti-Fas. Caspase-2 also regulated caspase-3 and -8 activation, as well as cell death in macrophages treated with each of the three reagents. Taken together, our data has demonstrated that caspase-2 can play an important role in mediating a proinflammatory response and a hybrid cell death that demonstrates features of both apoptosis and pyroptosis.
... In the presence of UV radiation, compounds in oil may also degrade to form photoderivatives with increased toxicity (Mallakin et al. 1999). Furthermore, the formation of oxygen free radicals may damage DNA and reduce the viability of cultured cells (Bagchi et al. 1998). This is supported by the observation that certain algae, when exposed to oil pollution, have reduced DNA, RNA, and protein content (El-Sheekh et al. 2000). ...
While the ecological impacts of crude oil exposure have been widely studied, its sublethal effects on phytoplankton community structure in salt marsh estuaries have not been well documented. The purpose of this study was to simulate oil spill conditions using a microcosm design to examine short-term (2 day) changes in phytoplankton community composition and total biomass following exposure to crude oil obtained from the Deepwater Horizon oil spill and a mixture of Texas crude oils. Microcosm experiments were performed in situ in North Inlet Estuary near Georgetown, SC. A control and six replicated experimental treatments of crude oil additions at final concentrations of 10, 50, or 100 μl l−1 of either Deepwater Horizon spill oil or the Texas crude mixture were incubated under in situ conditions. Photopigments were analyzed using high-performance liquid chromatography and community composition was determined using ChemTax. Total phytoplankton biomass (as chl a) declined with increasing crude oil concentrations. Prasinophytes, the most abundant microalga in both experiments, showed no response to oil exposure in one experiment and a significant negative response in the other. Diatoms euglenophytes and chlorophytes appeared relatively resistant to oil contamination at the exposure levels used in this study, maintaining or increasing their relative abundance with increasing oil concentrations. Chlorophytes and cyanobacteria increased in relative abundance while cryptophyte abundance decreased with increasing oil concentrations. The results of these experiments suggest that low levels of crude oil exposure may reduce total biomass and alter phytoplankton community composition with possible cascade effects at higher trophic levels in salt marsh estuaries.
Naphthalene, an environmental pollutant classified as a polycyclic aromatic hydrocarbon (PAH), can induce toxicity in fish and other aquatic organisms. Through our investigation, we determined how Takifugu obscurus juveniles were affected by naphthalene (0, 2 mg L-1) exposure in terms of oxidative stress biomarkers and Na+/K+-ATPase activity in various tissues (gill, liver, kidney and muscle) under dissimilar salinities (0, 10 psu). Results suggest that naphthalene exposure significantly affects the survival of T. obscurus juveniles and leads to significant changes in the levels of malondialdehyde, superoxide dismutase, catalase, glutathione, and Na+/K+-ATPase activity, which are indicative of oxidative stress and emphasized the risks associated with osmoregulatory function. The higher salinity affected on the noxious effects of naphthalene can be observed, resulting in decreased biomarker levels and increased Na+/K+-ATPase activity. Salinity levels affected the uptake of naphthalene and its impact on different tissues, with high salinity conditions having mitigating effects on oxidative stress and naphthalene uptake in the liver and kidney tissues. Increased Na+/K+-ATPase activity was observed in all tissues treated with 10 psu and 2 mg L-1 naphthalene. Our findings deepen the understanding of T. obscurus juveniles' physiological responses to naphthalene exposure, and highlight the potential mitigating effects of salinity. These insights can inform the development of appropriate conservation and management practices to protect aquatic organisms from susceptibility.
Multi organ failure following naphthalene toxicity
Naphthalene is a chemical substance which is widely used as moth repellent, insecticide and deodorizer. Naphthalene mothballs are potent hemolytic agents specially for pediatric group and Glucose 6 phosphate dehydrogenase (G6PD) deficient individuals. Our patient, a 14-year-old boy got admitted in our institution with progressive pallor, jaundice, hematuria and oliguria. He used to chew naphthalene mixed flavored raw rice for the last six months. On investigation he was found to have features of intravascular hemolysis and AKI necessitating hemodialysis with blood transfusion. His G6PD activity was below normal. After seven sessions of Hemodialysis (HD) his renal function recovered and discharged accordingly. One month post discharge follow up was normal. J Dhaka Med Coll. 2021; 29(1): 123-126
Naphthalene exerts potential threats on the soil environment and soil-dwelling species. The toxic effects on soil organisms, the response of antioxidant enzymes and the underlying molecular mechanisms for naphthalene metabolites remain to be fully elucidated. Here we report the cytotoxicity and oxidative stress induced by naphthalene and a major metabolite (1-naphthol) on Eisenia fetida coelomocytes and the interaction mechanism between 1-naphthol and the critical antioxidant enzymes (CAT and SOD). Earthworm coelomocytes were more susceptible to 1-naphthol than to naphthalene. At the molecular level, 1-naphthol preferentially bound to the active site of CAT with catalytic residues (His 74, Tyr 357 and Asn147) and the surface of SOD via hydrophobic forces and hydrogen bonds. Direct exposure to 1-naphthol resulted in the unfolding of the CAT skeleton accompanied by secondary structure changes, though only changes in SOD skeleton were observed, without a change in the secondary structure. Static fluorescence quenching of CAT and SOD were initiated with 1-naphthol by the formation of a non-fluorescent complex. Direct 1-naphthol binding inhibited the activity of CAT but presented no adverse effects on the activity of SOD. This paper provides a theoretical basis for the elucidation and better understanding of the toxicity of naphthalene and its metabolites.
During the coffee beans roasting process, occurs the formation of polycyclic aromatic hydrocarbons, which are associated with the incidence of cancer in humans. This study aimed to evaluate the influence of coffee bean quality and roasting degree regarding mutagenicity, cytotoxicity and genotoxicity. Six samples of coffee drink made with roasted and ground Coffea arabica beans from different qualities and roast degrees were used after freeze-drying. Both commercial and special quality grains suffered light, medium and dark roasting. According to the Salmonella/microsome assay, the highest concentration of commercial grain sample (dark roast) significantly increased the number of revertants of the TA98 strain in the absence of metabolization. All the samples induced cytotoxicity to HepG2 cells. These effects can be ranked in the following order from most to least toxic: medium roast – special grain > light roast – special grain > dark roast - commercial grain > dark roast – special grain > light roast – commercial grain > medium roast – commercial grain. None of the samples induced genotoxicity in HepG2 cells. Our findings show that the harmful effects of coffee depend not only on the degree of roasting but also on the grain quality.
Background
Exposure to naphthalene, which is widely used in mothballs, does not usually produce adverse effects. However, naphthalene can be toxic, especially in individuals with underlying conditions such as glucose-6-phosphate-dehydrogenase (G6PD) deficiency.
Case Report
A 3-year-old boy was brought to our Emergency Department after accidentally ingesting naphthalene mothballs 3 days prior to presentation. Laboratory investigations revealed that he had severe hemolytic anemia and mild methemoglobinemia (6%), which were treated with ascorbic acid and N-acetylcysteine. The patient tested positive for G6PD deficiency after stabilization and completion of his treatment. All provided treatments were administered empirically; test results were available only after the patient was discharged.
Why Should an Emergency Physician Be Aware of This?
Naphthalene exposure is a common pediatric presentation with various complications that can occur in certain high-risk individuals, such as those with G6PD deficiency. Emergency physicians should be aware of this to anticipate and be able to treat worsening toxicity.
Naphthalene, a naturally-occurring polyaromatic hydrocarbon, pose potential threats to health for its wide exposures in environment. Naphthalene could disrupt the redox equilibrium resulting in oxidative damage. Antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) are considered to be the efficient defense barriers to protect organisms from negative impacts of toxicants. Limited information is available regarding the underlying molecular mechanism between antioxidant enzymes and naphthalene. In this paper, structural and functional alterations of CAT and SOD for low dose (1.6–25.6 mg/L) naphthalene exposure have been investigated at the molecular and cellular levels. The enzyme activity responses of CAT and SOD in hepatocytes for naphthalene were consistent with the molecular, in which the activity of CAT increased and the activity of SOD slightly inhibited. Spectroscopy methods and molecular docking were carried out to investigate the underlying binding mechanisms. Naphthalene exposure significantly changed the conformation of CAT with secondary structure alteration (α-helix increase) but only changed the skeleton structure of SOD without secondary structure alteration. Naphthalene could bind to CAT and SOD primarily via H-binding force accompanied with the particle size of CAT/SOD agglomerates decreasing. Naphthalene preferentially bound to the surface of CAT and SOD. Besides, naphthalene could also bind directly to the active center of CAT with the key residues Arg364 and Tyr 357 for activity. This paper provides a combined cellular and molecular strategy to research biomarker responses for toxicants exposure. Besides, this study offers detailed basic data for the comprehensive understanding of naphthalene toxicity.
Naphthalene is bicyclic aromatic compound that is widely used in various domestic and commercial applications including lavatory scent disks, soil fumigants and moth balls. Exposure to naphthalene results in the development of bronchiolar damage, cataracts and hemolytic anemia in humans and laboratory animals. However, little information is available regarding the mechanism of naphthalene toxicity. We investigated gene expression profiles and potential signature genes in human hepatocellular carcinoma HepG2 cells and human promyelocytic leukemia HL-60 cells after 3 h and 48 h incubation with the IC(20) and IC(50) of naphthalene by using 44 k agilent whole human genome oligomicroarray and operon human whole 35 k oligomicroarray, respectively. We identified 616 upregulated genes and 2,088 down-regulated genes changed by more than 2-fold by naphthalene in HepG2 cells. And in HL-60, we identified 138 up-regulated genes and 182 down-regulated genes changed by more than 2-fold. This study identified several interesting targets and functions in relation to naphthalene-induced toxicity through a gene ontology analysis method. Apoptosis and cell cycle related genes are more commonly expressed than other functional genes in both cell lines. In summary, the use of in vitro models with global expression profiling emerges as a relevant approach toward the identification of biomarkers associated with toxicity after exposure to a variety of environmental toxicants.
A cell bound red pigment was synthesized by an indigenous petroleum contaminated soil isolate, Comamonas testosteroni, during growth on naphthalene as a sole source of carbon and energy. Ferric ion supplementation in the medium inhibited the pigment production. UV-visible spectrum of the pigment in ethanol showed absorption maxima at 251, 289, 318, 328, 333 and 362 nm and a broad peak at 479 nm. Ethanolic solution of pigment showed development of crystals on storage. Chemical analysis, FTIR and NMR spectroscopy studies revealed it as a novel isoprenoid quinone, a 2-methyl naphthoquinone with phytyl side chain. Ferric reducing/antioxidant power assay proved its in vitro antioxidant activity. Both pigment and ferric ions are proposed to have protective role against superoxide radical generated through intense activity of flavin containing naphthalene dioxygenase during naphthalene metabolism.
Naphthalene is a widely used industrial and household chemical in the form of mothballs. But it has rarely been an agent of poisoning
worldwide. We describe a case of ingestional naphthalene poisoning with a good outcome after proper management. A 29-year-old girl
ingested 8 mothballs, and presented two days later with haemolysis and methaemoglobinaemia. She was given intravenous methylene
blue, N-acetylcysteine and ascorbic acid, besides supportive treatment. Renal replacement therapy in the form of SLED of 8 hours was
done on a daily basis. She was discharged after ten days on twice a week outpatient follow-up haemodialysis.
Inhalation of naphthalene causes olfactory epithelial nasal tumors in rats (but not in mice) and benign lung adenomas in mice (but not in rats). The limited available human data have not identified an association between naphthalene exposure and increased respiratory cancer risk. Assessing naphthalene's carcinogenicity in humans, therefore, depends entirely on experimental evidence from rodents. We evaluated the respiratory carcinogenicity of naphthalene in rodents, and its potential relevance to humans, using our Hypothesis-Based Weight-of-Evidence (HBWoE) approach. We systematically and comparatively reviewed data relevant to key elements in the hypothesized modes of action (MoA) to determine which is best supported by the available data, allowing all of the data from each realm of investigation to inform interpretation of one another. Our analysis supports a mechanism that involves initial metabolism of naphthalene to the epoxide, followed by GSH depletion, cytotoxicity, chronic inflammation, regenerative hyperplasia, and tumor formation, with possible weak genotoxicity from downstream metabolites occurring only at high cytotoxic doses, strongly supporting a non-mutagenic threshold MoA in the rat nose. We also conducted a dose-response analysis, based on the likely MoA, which suggests that the rat nasal MoA is not relevant in human respiratory tissues at typical environmental exposures. Our analysis illustrates how a thorough WoE evaluation can be used to support a MoA, even when a mechanism of action cannot be fully elucidated. A non-mutagenic threshold MoA for naphthalene-induced rat nasal tumors should be considered as a basis to determine human relevance and to guide regulatory and risk-management decisions.
Naphthalene ingestion is a rare cause of hemolysis.
Introduction
Naphthalene ingestion is a rare cause of hemolysis.
Case report
We report a 33-year-old woman, originating from the Comoros, hospitalized for intense fatigue associated with delirium, fever and jaundice, three days after ritual ingestion of naphthalene. Biochemical parameters showed marked hemolysis. Outcome was favorable after red cells transfusion and hydratation with intravenous fluids.
Conclusion
Diagnostic work-up of unexplained hemolysis should include the search for toxic exposition. Naphthalene poisoning can present with diagnostic challenge for physicians.
Cancer-associated fibroblasts (CAFs) have been described to play critical roles in initiation, progression and metastasis of various cancers. However, the involvement of CAFs in oral cancer (OC) has not been well addressed. In this study, we demonstrate that CAFs, when cocultured with OC cells (OCCs), produce high levels of chemokine (C-C motif) ligand 2 (CCL2) and, subsequently, enhance endogenous reactive oxygen species production in cells. Oxidative stress stimulates expression of cell cycle progression proteins in OCCs, leading to promotion of OCC proliferation, migration, invasion and, OC tumor growth. On the other hand, oxidative stress triggered the activation of nuclear factor-kappaB (NF-κB) and STAT3 in CAFs, resulting in accelerating CCL2 expression. In this way, CAFs-OCCs coculture creates a favorable cytokine-rich microenvironment, beneficial for both CAFs and OCCs. In addition, upregulation of CCL2 expression has been observed in oral squamous cell carcinoma tumors and patient plasma. We also showed that inhibition of CCL2 reduced OC tumor burden in mice. Therefore, our data suggested that CCL2 represents a potential therapeutic target for treatment of OC. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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Ophthalmic toxicology is a specialist area that deals with potentially adverse effects on the eye from chemicals and their metabolites, reaching the eye by local contact and from the systemic circulation, and also covers the effects that may be produced systemically by chemicals that have been absorbed into the circulation following topical contact with the eye. This chapter reviews, with illustrative examples, all these aspects of ophthalmic toxicology, with particular reference to causation, mechanisms of production of lesions, relevant in vivo and in vitro laboratory testing procedures, the significance and clinical relevance of the observed effects. Site-specific lesions and functional disturbances in the eye that are discussed in detail are those to the cornea, iris, ciliary body, aqueous humour production, the lens, retina, optic nerve and extraocular muscles. Systemic effects following topical contamination of the eye are discussed particularly for chemicals and drugs, and discussed in terms of the sites of absorption.
Thioredoxin from Escherichia coli was shown to catalyze the reduction of insulin disulfides by dithiothreitol. A quantitative assay was developed which measures the rate of insulin reduction spectrophotometrically at 650 nm as turbidity formation from the precipitation of the free insulin B chain. Thioredoxin, at 5 microM concentration, accelerated the reaction between 0.130 mM insulin and 1.0 mM dithiothreitol at pH 7 around 20-fold. The pH optimum of the reaction was 7.5. Thioredoxins from E. coli and calf liver showed similar specific activities. Stopped flow fluorescence measurements of the rate of reduction of thioredoxin-S2 by dithiothreitol showed a second order rate constant of 1647 M-1 s-1 at pH 7.2. This is between 10(2) to 10(3) times larger than the reaction between insulin or linear model disulfides and dithiothreitol. It is consistent with a ping-pong mechanism of thioredoxin catalysis since reduced thioredoxin is known to react very fast with insulin. Thioredoxin also catalyzed lipoamide-dependent reduction of the insulin disulfides in a coupled system with NADH, lipoamide, and lipoamide dehydrogenase. The fast spontaneous reaction between dihydrolipoamide and thioredoxin-S2 provides a mechanism for NADH or pyruvate-dependent disulfide reduction. The implication of the dithiol-disulfide oxidoreductase activity of thioredoxin for the regulation of enzyme activities by thiol oxidation-reduction control is discussed.
Intercellular adhesion molecule-1 (ICAM-1) is an inducible glycoprotein expressed on the surface of inflamed endothelium which mediates in part the extravasation of granulocytes into sites of infection or injury. ICAM-1 mRNA is not detected in unstimulated human umbilical vein endothelial cells (HUVECs), but accumulates transiently following tumor necrosis factor-alpha (TNF-alpha) or phorbol myristate acetate (PMA) treatment with maximal steady state levels occurring at 2 or 4 h, respectively. Pretreating HUVECs with PMA for 72 h down-regulates protein kinase C and inhibits the subsequent induction of ICAM-1 mRNA by PMA, but does not affect TNF-alpha-induced message accumulation. Nuclear run-on assays showed that the ICAM-1 gene is transcribed under basal conditions in HUVECs, and that TNF-alpha stimulates transcriptional activity 3- to 4-fold within 30 min of treatment. In contrast, PMA has little effect on ICAM-1 gene transcription up to 4 h following stimulation. Message stability studies established that ICAM-1 mRNA induced by PMA has a longer half-life than the TNF-alpha-induced message. These results suggest that PMA acts through protein kinase C to up-regulate ICAM-1 expression primarily at a post-transcriptional level by stabilizing ICAM-1 mRNA, whereas TNF-alpha transcriptionally regulates ICAM-1 gene expression through an undefined, protein kinase C-independent pathway.
The activation of nuclear factor kappa B (NF-kappa B) has been implicated in the regulation of transcription of a variety of genes and has been shown to be essential for the expression of genes controlled by the long terminal repeat of human immunodeficiency virus (HIV LTR). We show here that intracellular thiol levels play a key role in regulating this process. That is, stimulation with tumor necrosis factor alpha and/or phorbol 12-myristate 13-acetate activates NF-kappa B and markedly decreases intracellular thiols; N-acetyl-L-cysteine, an efficient thiol source, prevents this thiol decrease and blocks the activation of NF-kappa B; and the lack of activated NF-kappa B prevents the activation of the HIV LTR and the transcription of genes under its control. These findings reveal a previously unrecognized genetic regulatory mechanism in which cytokine-induced shifts in intracellular thiol levels are crucial in the control of NF-kappa B activity and thereby influence the spectrum of genes expressed by cytokine-stimulated cells.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
Male, weanling Blue-Spruce rats were treated with naphthalene (p.o.) in defined dose increments up to 750 mg/kg body weight over 9 weeks. At necropsy, treated rats showed a 20% decrease in body weight compared to controls. Naphthalene treatment resulted in enhanced peroxidation (p less than 0.001) only in the liver. This increased peroxidation was associated with reductions (p less than 0.05) in the activity of the selenoenzyme glutathione peroxidase in hepatic cytosolic fractions and an associated increase (p less than 0.05) in the selenium-independent glutathione peroxidase. No increase in peroxidation was observed in the lung, eye or heart of these rats and the activities of the selenoenzyme and the selenium-independent glutathione peroxidases were also unaffected by naphthalene in these organs. Naphthalene also did not affect superoxide dismutase activity in any of the organs examined. Thus, in addition to the known effects of naphthalene on tissue glutathione, naphthalene-induced reductions in the selenoenzyme glutathione peroxidase can also contribute to peroxidation in the liver and must be considered as a contributing factor in naphthalene toxicity in vivo.
Antioxidants have been proposed to be anti-atherosclerotic agents; however, the mechanisms underlying their beneficial effects are poorly understood. We have examined the effect of alpha-tocopherol (alpha-tcp) on one cellular event in atherosclerotic plaque development, monocyte adhesion to stimulated endothelial cells (ECs). Human umbilical vein ECs were pretreated with alpha-tcp before stimulation with known agonists of monocyte adhesion: IL-1 (10 ng/ml), LPS (10 ng/ml), thrombin (30 U/ml), or PMA (10 nM). Agonist-induced monocytic cell adhesion, but not basal adhesion, was inhibited in a time- and concentration-dependent manner by alpha-tcp. The IC50 of alpha-tcp on an IL-1-induced response was 45 microM. The inhibition correlated with a decrease in steady state levels of E-selectin mRNA and cell surface expression of E-selectin which is consistent with the ability of a monoclonal antibody to E-selectin to inhibit monocytic cell adhesion in this system. Probucol (50 microM) and N-acetylcysteine (20 mM) also inhibited agonist-induced monocytic cell adhesion; whereas, several other antioxidants had no significant effect. Protein kinase C (PKC) does not appear to play a role in the alpha-tcp effect since no suppression of phosphorylation of PKC substrates was observed. Activation of the transcription factor NF-kappa B is reported to be necessary but not sufficient for E-selectin expression in EC. Electrophoretic mobility shift assays failed to show an alpha-tcp-induced decrease in activation of this transcription factor after cytokine stimulation. It has been hypothesized that alpha-tcp acts as an anti-atherosclerotic molecule by inhibiting generation of oxidized LDL--a putative triggering molecule in the atherosclerotic process. Our results point to a novel alternative mechanism of action of alpha-tcp.
Transcription of endothelial-leukocyte adhesion molecule-1 (E-selectin or ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) is induced by the inflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha). The positive regulatory domains required for maximal levels of cytokine induction have been defined in the promoters of all three genes. DNA binding studies reveal a requirement for nuclear factor-kappa B (NF-kappa B) and a small group of other transcriptional activators. The organization of the cytokine-inducible element in the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human interferon-beta gene in that both promoters require NF-kappa B, activating transcription factor-2 (ATF-2), and high mobility group protein I(Y) for induction. Based on this structural similarity, a model has been proposed for the cytokine-induced E-selectin enhancer that is similar to the stereospecific complex proposed for the interferon-beta gene promoter. In these models, multiple DNA bending proteins facilitate the assembly of higher order complexes of transcriptional activators that interact as a unit with the basal transcriptional machinery. The assembly of unique enhancer complexes from similar sets of transcriptional factors may provide the specificity required to regulate complex patterns of gene expression and correlate with the distinct patterns of expression of the leukocyte adhesion molecules.
When macrophage-like J-774 cells are subjected to limited oxidative stress, such as exposure to hydrogen peroxide in a moderate bolus dose, some of their lysosomes rupture - as here assayed by the acridine orange relocalization test - secondary to intralysosomal, iron-catalysed, oxidative reactions. The resultant leakage into the cytosol of hydrolytic enzymes, such as cathepsin-D (as shown here), may initiate a slow degradation/fragmentation process of an apoptotic type within cells still having intact plasma membranes. In contrast, severe oxidative stress also results in extensive lysosomal rupture but leads to necrosis. The chelation of (normally occurring) intralysosomal low molecular weight iron, by endocytotic uptake of desferrioxamine, largely prevents oxidative stress-induced apoptosis whereas lysosomal iron-loading, by endocytotic uptake of complexed ferric iron, considerably enhances the process. We conclude that oxidant-mediated and iron-catalysed lysosomal rupture leads to decompartmentalization of lysosomal enzymes which in turn may initiate and promote the apoptotic process.
TGF-beta 1 controls the expression of numerous genes, including early response and cellular matrix genes. However, the signal-transducing mechanism underlying this regulation of gene expression is not fully understood. In this study, we investigated whether redox regulation plays a role in the TGF-beta 1 signal transduction in the mouse osteoblastic cell line (MC3T3-E1). The overall intracellular oxidized state of the cells, when measured using 2',7'-dichlorofluorescin diacetate by laser-scanning confocal microscopy, was increased transiently after the addition of TGF-beta 1. This increase was abolished by the addition of oxygen radical scavengers such as catalase and N-acetylcysteine. In a variant cell line lacking the TGF-beta 1 receptor, the intracellular oxidized state was not modulated by treatment with TGF-beta 1. We then examined the expression of early growth response-1 (egr-1) gene, which is inducible by TGF-beta 1 and H2O2. Radical scavengers inhibited the induction of egr-1 by TGF-beta 1, but not that by 12-O-tetradecanoylphorbol-13 acetate. A nuclear run-on assay indicated that this inhibition was at the transcriptional level. From transient expression experiments using chloramphenicol acetyltransferase gene linked to serially deleted egr-1 gene 5'-upstream region, the CArG element in the 5' flanking region of egr-1 was identified as an essential sequence in the transcriptional activation for both TGF-beta 1 and H2O2 stimulation. These findings suggest that H2O2 acts as a mediator for the TGF-beta 1-induced transcription of egr-1 gene.
Nuclear factor (NF)-κB is a redox sensitive cytosolic transcription factor. Redox regulation of NF-κB has been implicated in the activation of the human immuno-deficiency virus (HIV). Therefore, inhibition of NF-κB activation may be an effective strategy for acquired immunodeficiency syndrome therapy. Anetholdithiolthione (ADT, 5-[p-methoxyphenyl]-3H-1,2-dithiol-3-thione) is an antioxidant which has been used to protect against acetaminophen- and CCl4-induced hepatotoxicity, lipid peroxidation, radiation injury, and also has been used clinically as an anti-choleretic agent. The present study examined the effect of ADT pretreatment on NF-κB activation in response to a variety of stimuli such as H2O2, phorbol myrisitate acetate (PMA) or tumor necrosis factor α (TNFα). PMA and TNFα induced activation of (NF)-κB in human Jurkat T-cells was partially inhibited by ADT (0.1 mM) pretreatment. ADT (0.1 mM) also inhibited H2O2induced activation of the transcription factor in the peroxide sensitive human Wurzburg T-cells. Furthermore, ADT treated Wurzburg cells had significantly higher glutathione levels as compared with untreated cells. H2O2induced lipid peroxidation in Wurzburg cells was remarkably inhibited by ADT pretreatment. ADT, a pro-glutathione antioxidant, was observed to be capable of modulating NF-κB activation.
The pyruvate and α-ketoglutarate dehydrogenase complexes isolated from pig heart mitochondria promote the reduction of thioredoxin in the presence of their α-ketoacid substrates, coenzyme A, and free lipoate. Substrate-specific generation of reduced thioredoxin was established by two independent methods, viz. reduction of insulin and thioredoxin reductase-catalyzed NADPH formation. Dihydrolipoate accumulating in the absence of NAD+ is the likely intermediate. A redox function in α-ketoacid oxidation provides a potential role for the specific thioredoxins previously identified by us in mitochondria.
Anti-oxidant treatment has been shown to prevent nerve dysfunction in experimental diabetes mellitus, thus providing a rationale of potential therapeutic value for diabetic patients. The effects of the anti-oxidant α-lipoic acid (thioctic acid) were studied in a 3-week multicentre, randomized, double-blind placebo-controlled trial (Alpha-Lipoic Acid in Diabetic Neuropathy; ALADIN) in 328 non-insulin-dependent diabetic patients with symptomatic peripheral neuropathy who were randomly assigned to treatment with intravenous infusion of α-lipoic acid using three doses (1200, 600, or 100 mg ALA) or placebo (PLAC). Neuropathic symptoms (pain, burning, paraesthesiae, and numbness) were scored at baseline and at each visit (days 2–5, 8–12, and 15–19) prior to infusion. In addition, the Hamburg Pain Adjective List, a multidimensional specific pain questionnaire, and the Neuropathy Symptom and Disability Scores were assessed at baseline and day 19. According to the protocol 260 (65/63/66/66) patients completed the study. The total symptom score in the feet decreased from baseline to day 19 by −4.5±3.7 (−58.6%) points (mean ± SD) in ALA 1200, −5.0±4.1 (−63.5%) points in ALA 600, −3.3±2.8 (−43.2%) points in ALA 100, and −2.6±3.2 (−38.4%) points in PLAC (ALA 1200 vs PLAC: p=0.003; ALA 600 vs PLAC: pp=0.002). The total scale of the Pain Adjective List was significantly reduced in ALA 1200 and ALA 600 as compared with PLAC after 19 days (both p
Lipoic acid has been reported recently to be an effective antioxidant in biological systems. It may act in vivo through reduction to its dithiol form, dihydrolipoic acid. Using a dual Hg/Au electrode, and HPLC with electrochemical detection, a method was developed which allowed simultaneous measurement of lipoic acid and dihydrolipoic acid, at nanomolar levels. (RS)-α-Lipoic acid was added to human cells in tissue culture (Jurkat T-lymphocytes and primary neonatal diploid fibroblasts). Lipoic acid was converted rapidly by the cells to dihydrolipoic acid, which accumulated in the cell pellet. Monitored over a 2-hr interval, dihydrolipoic acid was released, and several-fold more dihydrolipoic acid could be found in the medium than in the pellet.
Modulation of cellular thiols is an effective therapeutic strategy, particularly in the treatment of AIDS. Lipoic acid, a metabolic antioxidant, functions as a redox modulator and has proven clinically beneficial effects. It is also used as a dietary supplement. We utilized the specific capabilities of N-ethylmaleimide to block total cellular thiols, phenylarsine oxide to block vicinal dithiols, and buthionine sulfoximine to deplete cellular GSH to flow cytometrically investigate how these thiol pools are influenced by exogenous lipoate treatment. Low concentrations of lipoate and its analogue lipoamide increased Jurkat cell GSH in a dose-dependent manner between 10 (25 μM for lipoamide) to 100 μM. This was also observed in mitogenically stimulated peripheral blood lymphocytes (PBL). Studies with Jurkat cells and its Wurzburg subclone showed that lipoate dependent increase in cellular GSH was similar in CD4+ and − cells. Chronic (16 week) exposure of cells to lipoate resulted in further increase of total cellular thiols, vicinal dithiols, and GSH. High concentration (2 and 5 mM) of lipoate exhibited cell shrinkage, thiol depletion, and DNA fragmentation effects. Based on similar effects of octanoic acid, the cytotoxic effects of lipoate at high concentration could be attributed to its fatty acid structure. In certain diseases such as AIDS and cancer, elevated plasma glutamate lowers cellular GSH by inhibiting cystine uptake. Low concentrations of lipoate and lipoamide were able to bypass the adverse effect of elevated extracellular glutamate. A heterogeneity in the thiol status of PBL was observed. Lipoate, lipoamide, or N-acetylcysteine corrected the deficient thiol status of cell subpopulations. Hence, the favorable effects of low concentrations of lipoate treatment appears clinically relevant. © 1997 Elsevier Science Inc.
In cellular, tissue, and organismal systems, exogenously supplied α-lipoic acid (thioctic acid) has a variety of significant effects, including direct radical scavenging, redox modulation of cell metabolism, and potential to inhibit oxidatively-induced injury. Because reduction of lipoate to dihydrolipoate is a crucial step in many of these processes, we investigated mechanisms of its reduction. The mitochondrial NADH-dependent dihydrolipoamide dehydrogenase exhibits a marked preference for R(+)-lipoate, whereas NADPH-dependent glutathione reductase shows slightly greater activity toward the S(−)-lipoate stereoisomer. Rat liver mitochondria also reduced exogenous lipoic acid. The rate of reduction was stimulated by substrates which increased the NADH content of the mitochondria, and was inhibited by methoxyindole-2-carboxylic acid, a dihydrolipoamide dehydrogenase inhibitor. In rat liver cytosol, NADPH-dependent reduction was greater than NADH, and lipoate reduction was inhibited by glutathione disulfide. In rat heart, kidney, and brain whole cell-soluble fractions, NADH contributed more to reduction (70–90%) than NADPH, whereas with liver, NADH and NADPH were about equally active. An intact organ, the isolated perfused rat heart, reduced R-lipoate six to eight times more rapidly than S-lipoate, consistent with high mitochondrial dihydrolipoamide dehydrogenase activity and results with isolated cardiac mitochondria. On the other hand, erythrocytes, which lack mitochondria, somewhat more actively reduced S- than R-lipoate. These results demonstrate differing stereospecific reduction by intact cells and tissues. Thus, mechanisms of reduction of α-lipoate are highly tissue-specific and effects of exogenously supplied α-lipoate are determined by tissue glutathione reductase and dihydrolipoamide dehydrogenase activity. Copyright © 1996 Elsevier Science Inc.
Glutathione (GSH) has emerged to be one of the most fascinating endogenous molecules virtually present in all animal cells often in quite high (mM) concentrations. In addition to the detoxicant, antioxidant, and cysteine-reservoir functions of cellular glutathione, the potential of this ubiquitous thiol to modulate cellular signal transduction processes has been recently evident. Lowered tissue GSH levels have been observed in several disease conditions. Restoration of cell GSH levels in a number of these conditions have proven to be beneficial. Thus, strategies to boost cell glutathione level are of marked therapeutic significance. Availability of cysteine, a precursor for glutathione synthesis, inside the cell is a critical determinant of cellular glutathione level. N-acetylcysteine and α-lipoic acid are two pro-glutathione agents that have remarkable clinical potential. The ability of these two clinical drugs to enhance cellular glutathione level, coupled with their favorable effect on the molecular biology of HIV infection may make them useful tools for AIDS treatment.
Publisher Summary This chapter discusses microsomal lipid peroxidation. Lipid peroxidation is a complex process known to occur in both plants and animals. It involves the formation and propagation of lipid radicals, the uptake of oxygen, a rearrangement of the double bonds in unsaturated lipids, and the eventual destruction of membrane lipids, producing a variety of breakdown products, including alcohols, ketones, aldehydes, and ethers. Biological membranes are often rich in unsaturated fatty acids and bathed in an oxygen-rich, metal-containing fluid. Lipid peroxidation begins with the abstraction of a hydrogen atom from an unsaturated fatty acid, resulting in the formation of a lipid radical. The formation of lipid endoperoxides in unsaturated fatty acids containing at least 3 methylene interrupted double bonds can lead to the formation of malondialdehyde as a breakdown product. Nonenzymic peroxidation of microsomal membranes also occurs and is probably mediated in part by endogenous hemoproteins and transition metals. The direct measurement of lipid hydroperoxides has an advantage over the thiobarbituric acid assay in that it permits a more accurate comparison of lipid peroxide levels in dissimilar lipid membranes.
The blood monocytes adhere to endothelial cells unstimulated and after stimulation by interleukin-1, tumor necrosis factor or other mediators. This process is mediated through specific molecules on both endothelial cells and monocytes. Using specific monoclonal antibodies and molecular cloning several families of molecules involved in leukocyte endothelial cell interaction have been defined. Leukocyte adhesion molecules include the three beta 2 integrins (CD11/CD18 molecules), VLA-4 and the L-Selectin. E-Selectin (ELAM-1), P-Selectin (GMP-140) and receptors of the immunoglobulin superfamily (ICAM-1, ICAM-2 and VCAM-1) are expressed on endothelial cells in basal conditions and after activation. It has been shown that these adhesive molecules are involved in blood monocyte adhesion to endothelial cells. Monocytes from patients with diabetes mellitus had an increased adhesion to endothelial cells in culture. As estimated by flow cytometry CD11b/CD18 expression on diabetic monocytes was increased. Pentoxifylline reduced CD11b/CD18 expression on normal and diabetic monocytes. This effect was associated to a decrease in monocyte adhesion to endothelial cells.
Polycyclic aromatic hydrocarbon (PAH) o-quinones are products of an NADP+ dependent oxidation of non-K-region trans-dihydrodiols catalyzed by dihydrodiol dehydrogenase (EC 1.3.1.20). Since these PAH o-quinones could be detoxified by non-enzymatic or enzymatic conjugation with cellular thiols, their reactivity with 2-mercaptoethanol, cysteine and glutathione (GSH) was examined by ion-pair reverse phase high pressure liquid chromatography (RP-HPLC). Second-order rate constants for the addition of these thiols to naphthalene-1,2-dione (NPQ) in water ranging from 4.9 x 10(3) - 1.1 x 10(4) min-1 M-1 and the reactions were complete within 10 min. When these reactions were conducted at near physiological pH (50 mM potassium phosphate buffer pH 7.0), the rate constants increased by 2-orders of magnitude. When benzo[a]pyrene-7,8-dione (BPQ) was substituted in these reactions the second-order rate constants decreased by 2-3 orders of magnitude and the reactions took several hours to reach completion. The decrease in reactivity can be explained by the presence of the bay region in BPQ. Methylation influenced the reactivity of PAH o-quinones with GSH and the following order of reactivity was observed: 7,12-dimethyl-benz[a]anthracene-3,4-dione (7,12-DMBAQ) > 12-methyl-BAQ, 7-methyl-BAQ and BAQ > BPQ. Of these quinones 7,12-dimethyl-BAQ was almost equi-reactive with NPQ. This suggests that methyl substitution in the bay and peri regions enhances reactivity with GSH. Using NPQ as a model for other PAH o-quinones, N-acetyl-L-cysteine, L-cysteine and GSH conjugates of NPQ were synthesized and characterized by [1H]- and [13C]NMR. Evidence for Michael type 1,4-addition products was obtained in which the resultant adduct could exist as either a catechol or o-quinone. By contrast, L-cysteine was able to form adducts via S- or N-attack and N-attack gave a purple p-iminoquinone. There was no evidence for the formation of bis-N-acetyl-L-cysteinyl-, bis-glutathionyl adducts or phenolic coupled products. The toxicity of thiol conjugates of NPQ remains to be explored.
Naphthalene is toxic to the eye and results in opacification of the lens. To investigate the metabolic events that may be occurring in the lens epithelial cells, a cell line of lens from a transgenic mouse was incubated with various metabolites of naphthalene. Naphthoquinone at 50 microM was toxic to most cells with a depletion of glutathione levels noted within 6 h of incubation. At 10 microM, naphthoquinone caused an increase in specific activity of the enzyme DT-diaphorase. This enzyme is thought to be a defense against quinones since semiquinone formation is thought to be lessened. Naphthalene-1,2-dihydrodiol at 50 microM also caused an increase in the specific activity of the DT-diaphorase, while at 10 microM no apparent change occurred in the cells. Although there was evidence of metabolic alterations in the cells with the metabolites of naphthalene, the protein profile by two-dimensional gel electrophoresis did not change and there was no indication of an increase in carbonyl formation in the soluble proteins of the cells. These experiments indicate that the metabolites of naphthalene can cause alteration in the metabolism of the lens cells but may not cause apparent changes in the major proteins within the lens epithelium.
We have examined the fate of glutathione conjugates derived from naphthalene metabolism at various dose levels (5-80 mg/kg) in an effort to explore the potential use of urinary mercapturic acids as biomarkers of exposure to naphthalene and as indicators of the activity and stereoselectivity of cytochrome P-450-dependent naphthalene epoxidation. This approach extends previous studies which demonstrated a high degree of stereoselectivity in the formation of (+)-1R,2S-naphthalene oxide from naphthalene in target tissue microsomes (mouse lung), but not in microsomal preparations isolated from nontarget tissues such as mouse liver. To validate the use of mercapturic acids as indicators of epoxide formation in vivo, individual naphthalene oxide glutathione adduct isomers were administered iv to mice, and urinary metabolites were identified and quantified. Mercapturates accounted for 69-75% of the administered dose in the 8-hr urines of animals treated with trans-1-(S)-hydroxy-2-(S)-glutathionyl-1,2-dihydronaphthalene (adduct 1) and 76-84% for trans-1-(R)-hydroxy-2-(R)-glutathionyl-1,2-dihydronaphthalene (adduct 2). Only 39-57% of the dose of trans-1-(R)-glutathionyl-2-(R)-hydroxy-1,2-dihydronaphthalene (adduct 3) administered to mice was excreted as the mercapturic acid derivative; however, two additional metabolites were detected which were not present in the urine of animals treated with adducts 1 or 2. The first metabolite, accounting for 2-4% of the dose of adduct 3, was not identified. The second metabolite, isolated by HPLC and identified by mass spectrometry as (hydroxy-1,2-dihydronaphthalenylthio)pyruvic acid, accounted for 14-25% of the administered dose of adduct 3.(ABSTRACT TRUNCATED AT 250 WORDS)
Pulmonary toxicity of naphthalene (NAP), 2-methylnaphthalene (2-MN), 2-isopropylnaphthalene (2-IPN) and 2,6-diisopropylnaphthalene (2,6-DIPN) was studied in mice. Twenty four h after the intraperitoneal (i.p.) administration of NAP (200 mg/kg (1.6 mmol] or 2-MN (400 mg/kg (2.8 mmol], pulmonary damage was detected. Prior treatment with diethyl maleate resulted in enhancement of NAP and 2-MN-induced bronchiolar damage. In contrast to the effects of NAP and 2-MN, injections of 2-IPN (3000 mg (17.6 mmol)/kg) and 2,6-DIPN (3000 mg (14.2 mmol)/kg) did not cause detectable pulmonary damage. Injections of NAP and 2-MN caused considerable depletion of pulmonary reduced glutathione (GSH), while injections of 2-IPN and 2,6-DIPN caused only a slight depletion. There were general decreases in the binding of the compounds to lung slices with increasing number of carbons of the alkyl substituent. Pretreatment with a cytochrome P-450 inducer (beta-naphthoflavone) increased the binding of NAP, 2-MN, and 2-IPN to lung slices. Treatments with NAP, 2-MN, 2-IPN and 2,6-DIPN did not affect the lipid peroxidation or phospholipid contents in the lung. These results suggest that the difference in pulmonary toxicity among NAP, 2-MN, 2-IPN, and 2,6-DIPN may be dependent on the ability of these compounds to irreversibly bind to lung tissue.
The feasibility of polymorphonuclear leucocytes as a potential source of free radicals during reperfusion of ischaemic myocardium was evaluated. Isolated rat heart was perfused in the presence of f-Met-Leu-Phe-activated and normal polymorphonuclear leucocytes for 30 min. To judge the degree of cellular injury which might result from activated polymorphonuclear leucocytes during perfusion, isolated hearts were also perfused with superoxide anions, hydroxyl radicals, and hypochlorous acid-generating systems in the absence or presence of their corresponding scavengers, superoxide dismutase plus catalase, dimethylthiourea, and allopurinol, respectively. Activated polymorphonuclear leucocytes stimulated the release of lactate dehydrogenase, a biological marker of cellular injury, and malondialdehyde, a presumptive marker for lipid peroxidation; increased tissue injury, as evidenced by morphologic examinations using light and electron microscopy; decreased dry/wet ratios of heart, signifying oedema formation; and reduced myocardial adenosine triphosphate and creatine phosphate content as well as coronary flow, indicating decreased myocardial performance. These biological, physiological, and morphologic parameters were reversed significantly, but not completely, by treating the heart with scavengers, superoxide dismutase plus catalase or allopurinol, but were reversed completely by simultaneous treatment with superoxide dismutase, catalase, and allopurinol. Comparable results were obtained when the hearts were treated with each of these free radical-generating systems and their corresponding scavengers. Generation of free radicals was confirmed either by cytochrome c reduction or by examining the chemiluminescence response using a luminometer. These results indicate that activated polymorphonuclear leucocytes can cause myocardial cellular injury equivalent to the damage caused by free radicals and oxidants which are present in an ischaemic-reperfused heart, suggesting that polymorphonuclear leucocytes may be a potential source of free radicals in the reperfused heart.
Activation of protein kinase C (PKC) in human T lymphocytes is an immediate consequence of mitogenic signalling via the antigen-receptor complex and CD2 antigen. In order to investigate further the signal-transduction pathways which result in PKC activation, we have established a novel PKC assay system using streptolysin-O-permeabilized T cells. Known peptide substrates of PKC were introduced into permeabilized cells in the presence of [gamma-32P]ATP, 3 mM-Mg2+ and 150 nM free Ca2+. The peptide found to have the lowest background phosphorylation had the sequence Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys (peptide GS), and the phosphorylation of the peptide was increased up to 6-fold by direct activation of PKC with phorbol 12,13-dibutyrate. Induction of PKC activation with the UCHT1 antibody against the CD3 antigen, or with phytohaemagglutinin (PHA) or guanosine 5'-[gamma-thio]triphosphate (GTP[S]), increased peptide-GS phosphorylation by 2-3 fold. The specificity of PKC action on peptide GS was demonstrated by blocking increases in phosphorylation with a pseudosubstrate peptide PKC inhibitor. PKC activation by this technique could be detected within 1 min of adding external ligand. Dose-response curves revealed that PHA-induced production of inositol phosphates correlated closely with PKC activities, whereas only a partial correlation between these parameters was observed with GTP[S]. Our data are consistent with the presence of more than one G-protein-mediated pathway of PKC regulation in T cells. The quantitative PKC assay system described is both simple and reproducible, and its potential application to a wide range of cell types should prove useful in further investigations of PKC activation mechanisms.
The cataractogenicity of naphthalene derivatives was investigated in a lens culture system that included the lens with an intact capsule and epithelium. The in vivo cataractogenicity of naphthalene, 1000 or 2000 mg/kg ip, also was evaluated in New Zealand white and Chinchilla pigmented rabbits. A dose-related brunescence was observed in lenses incubated with 1,4-naphthoquinone in concentrations from 31.6 to 316 microM. With 316 microM naphthoquinone, lenses were totally opaque within 24 hr. No lenticular opacities were observed with 1-naphthol or 2-naphthol in incubations lasting up to 96 hr. The bioactivation of naphthalene derivatives to reactive free radical intermediates by lenses in organ culture was investigated by electron spin resonance spectrometry (ESR) using the spin trap alpha-phenyl-N-t-butylnitrone (PBN). Lenses were incubated with 316 microM naphthoquinone and 100 mM PBN for 0.25, 4 or 7 hr. A spin trapped radical product with unresolved peaks was observed with 0.25 and 7 hr incubation. No radicals were detected in the 4 hr incubation, nor in control cultures lacking either the lens, naphthoquinone or PBN. In the in vivo studies, naphthalene was cataractogenic in both albino and pigmented rabbits. The in vitro results indicate that naphthoquinone can be bioactivated by rabbit lens to a reactive free radical intermediate, which may contribute to cataractogenicity.
The toxic effects of compounds which undergo redox cycling via enzymatic one-electron reduction are reviewed. First of all, the enzymatic reduction of these compounds leads to reactive intermediates, mainly radicals which react with oxygen, whereby superoxide anion radicals are formed. Further oxygen metabolites are hydrogen peroxide, singlet oxygen and hydroxyl radicals. The role of these oxygen metabolites in toxicity is discussed.
The occurrence of lipid peroxidation during redox cycling of quinonoide compounds, e.g., adriamycin, and the possible relationship to their toxicity is critically evaluated. It is shown that iron ions play a crucial role in lipid peroxidation induced by redox cycling compounds.
DNA damage by metal chelates, e.g., bleomycin, is discussed on the basis of findings that enzymatic redox cycling of a bleomycin-iron complex has been observed. The involvement of hydroxyl radicals in bleomycin-induced DNA damage occurring during redox cycling in cell nuclei is claimed. Redox cycling of other substances, e.g., aromatic amines, is discussed in relation to carcinogenesis.
Other chemical groups, e.g., nitroaromatic compounds, hydroxylamines and azo compounds are included. Other targets for oxygen radical attack, e.g., proteins, are also dealt with.
It is concluded that oxygen radical formation by redox cycling may be a critical event in toxic effects of several compounds if the protective mechanisms of cells are overwhelmed.
A high-performance liquid chromatographic method was developed for quantification of malondialdehyde (MDA) in human plasma. Deproteinized samples were injected onto a Waters carbohydrate analysis column which was eluted with 20% (v/v) 0.03 M Tris buffer, pH 7.4, in acetonitrile. Peak absorbancy was measured at 267 nm. In contrast to data already published, we did not detect any free MDA in normal human plasma. This suggests that the classical thiobarbituric acid test is not suitable for the determination of MDA in human plasma.
When naphthalene was administered at a daily dose of 1 g/kg body weight to Wistar strain rats, their serum lipid peroxide levels were increased on the 4th day after the first administration and reached a maximum on the 7th day. This seems to be due to lipid peroxidation in the liver, in which lipid peroxide levels were increased in a similar pattern as those in the serum. The content of reduced glutathione in lenses of naphthalene-administered rats decreased on the 4th day. These results suggest that in naphthalene-induced cataract in albino rats increased lipid peroxides in the bloodstream may play a role in cataractogenesis.
Previous studies have indicated that 1-naphthol is metabolised by the cytochrome P-450 mixed function oxidase enzyme system to form a reactive species capable of covalently binding to microsomal protein (1,2). Furthermore, Hesse and Mezger (1) suggested the involvement of quinones and/or semiquinones in 1-naphthol-dependent covalent binding. In more recent studies, 1,4-naphthoquinone formed from 1-naphthol has been directly measured with both rat liver microsomes (2,3) and a reconstituted cytochrome P-450 enzyme system (4). From this, it was hypothesised that the toxicity of 1-naphthol, previously reported in isolated hepatocytes (5), may be mediated by naphthoquinone metabolites of l-naphthol.
As a highly reactive substance produced in biological systems by the one-electron reduction of oxygen, superoxide (O(2) (-)) seemed a likely candidate as a bactericidal agent in leukocytes. The reduction of cytochrome c, a process in which O(2) (-) may serve as an electron donor, was found to occur when the cytochrome was incubated with leukocytes. O(2) (-) was identified as the agent responsible for the leukocyte-mediated reduction of cytochrome c by the demonstration that the reaction was abolished by superoxide dismutase, an enzyme that destroys O(2) (-), but not by boiled dismutase, albumin, or catalase. Leukocyte O(2) (-) production doubled in the presence of latex particles. The average rate of formation of O(2) (-) in the presence of these particles was 1.03 nmol/10(7) cells per 15 min. This rate, however, is only a lower limit of the true rate of O(2) (-) production, since any O(2) (-) which reacted with constituents other than cytochrome c would have gone undetected. Thus. O(2) (-) is made by leukocytes under circumstances which suggest that it may be involved in bacterial killing.
The addition of 2,3-dichloro-1,4-naphthoquinone (CNQ) to substrate-depleted, GSH-supplemented rat liver mitochondria resulted in a dose-dependent depletion of reactable suflhydryl groups and a concomitant increase in mitochondrial disulfide content at a ratio of 2 thiols depleted/disulfide generated. The molar ratio of thiol depleted/CNQ added approached 20 at low CNQ concentrations and was unity at higher doses. The addition of CNQ to substrate-depleted mitochondrial suspensions resulted in O2 consumption which increased with increasing concentrations of mitochondria and was sensitive to N-ethylmaleimide (NEM) which establishes the ability of CNQ to interact with mitochondrial thiol redox centers. The CNQ-mediated large amplitude swelling of rat liver mitochondria was exacerbated by thiol oxidizing agents and depressed by disulfide reducing agents. A redox cycling mechanism between mitochondrial thiol groups, CNQ and oxygen was proposed to lower the matrix glutathione pool and make the mitochondria more susceptable to toxic oxygen radicals which induce swelling in isolated mitochondrial suspensions. In support of this mechanism, alpha-tocopherol was shown to prevent the CNQ-mediated swelling process. Beef heart mitochondrial NADH was oxidized by CNQ in a 1/1 molar ratio anaerobically and in a 3/1 molar ratio under aerobic conditions, whereas the fully reduced quinone, CNQH2, oxidized NADH aerobically but not anaerobically. Thus, CNQ is capable of interacting with NADH of the mitochondrial electron transport chain in a redox cycling fashion.
Intraperitoneal administration of the volatile hydrocarbon, naphthalene, resulted in severe bronchiolar epithelial cell necrosis in mice, while hepatic or renal necrosis was not observed. Pulmonary damage and mortality by naphthalene were increased by prior treatment with diethyl maleate and decreased by prior treatment with piperonyl butoxide (1600 mg/kg). SKF 525A pretreatment had no effect on naphthalene-induced pulmonary damage. Administration of [14C]naphthalene resulted in the covalent binding of radiolabel to tissue macromolecules. Highest levels of binding occurred in lung, liver and kidney. Levels of covalent binding reached a maximum 2--4 h after treatment and corresponded to rapid glutathione depletion in lung and liver. Covalent binding was dose-dependent and showed a threshold between 200 and 400 mg/kg which coincided with almost total depletion of tissue glutathione levels. Covalent binding of reactive metabolites was increased 3--4-fold by prior treatment with diethyl maleate, and was decreased 3--4-fold by pretreatment with piperonyl butoxide. These studies support the view that naphthalene-induced pulmonary damage is mediated by the cytochrome P-450-dependent metabolism of naphthalene and that glutathione plays an important role in the detoxification of the lung damaging metabolite(s).
Previous studies have demonstrated that an aqueous smokeless tobacco extract (STE) administered in an acute oral dose to rats induces an enhanced induction of hepatic mitochondrial and microsomal lipid peroxidation, hepatic nuclear DNA single strand breaks, enhanced excretion of urinary lipid metabolites, including malondialdehyde, formaldehyde, acetaldehyde and acetone, and increased production of nitric oxide (NO) by peritoneal macrophage cells. These observations indicate that STE induces the production of oxygen free radicals. We have therefore examined the in vitro incubation of cultured J774A.1 macrophage cells with STE on the release of the enzyme lactate dehydrogenase (LDH) into the media as an indicator of cellular membrane damage and cytotoxicity. The amount of LDH released by STE was both concentration- and time-dependent. The cytotoxicity of STE to macrophage J774A.1 cells in culture was further determined from percent viability after various periods of incubation. The addition of 250 micrograms STE/ml to the cultured J774A.1 cells resulted in a 2.9-fold increase in the release of LDH. Individual coincubation with superoxide dismutase (SOD), catalase, mannitol, and allopurinol had no significant effect on the release of LDH into the culture medium, while a combination of the four free radical scavengers resulted in a 59% decrease in the STE-induced release of LDH. At 75 microM concentrations of viramine E and vitamin E succinate, approximately 28% and 41% inhibitions were observed in STE-induced LDH leakage, respectively. Taken together with previous studies, the results indicate that STE activates macrophage cells, resulting in the production of reactive oxygen species.(ABSTRACT TRUNCATED AT 250 WORDS)
There is evidence that vascular endothelium directs the accumulation of leukocytes in inflammation through various means, particularly by the expression of specific cell surface molecules which are adhesive for ligands on circulating leukocytes. Examples of such molecules are E-selectin and intercellular adhesion molecule 1 (ICAM-1). In an experimental model of various forms of inflammation, E-selectin and ICAM-I were induced in association with adhesion and emigration of circulating polymorphonuclear and mononuclear leukocytes. Further work in humans showed endothelium to express E-selectin in inflammation. In addition, the presence of a leukocyte ligand for E-selectin, sialyl-Lewis X, has been seen on cells accumulating in inflammation. Furthermore, sialyl-Lewis X was also unexpectedly seen on endothelium. The role of sialyl-Lewis X on endothelium is as yet uncertain, although it may function as an adhesion receptor for leukocytes. Other endothelial adhesion receptors, such as vascular cell adhesion molecule 1 (VCAM-1), are described. Atherosclerosis shows many features in common with inflammation. These are discussed, and the demonstrated and potential relevance of endothelial adhesive phenomena in routine inflammation to those in atherosclerosis are reviewed. For example, a VCAM-1 homologue has been described on the endothelium over evolving atherosclerotic lesions in rabbits.
We compared the effects of phorbol 12-myristate 13-acetate (PMA) and thrombin with those of nonlytic concentrations of reactive oxygen species (ROS) generated by hypoxanthine (HX)-xanthine oxidase (XO) on the adhesion properties of human umbilical cord vein endothelial cells (HUVEC) to resting polymorphonuclear neutrophils (PMN). PMN adherence to HX-XO-treated HUVEC was increased approximately twofold to 2.5-fold relative to untreated HUVEC, both immediately and after 2 hours. It was not additive to that induced by PMA or thrombin stimulation of HUVEC. ROS-induced adherence was not due to platelet-activating factor (PAF) or P-selectin expression, as it was neither antagonized by BN52021 (PAF receptor antagonist) nor inhibited by anti-P-selectin monoclonal antibody (MoAb), contrary to the increased adhesion of PMA- and thrombin-stimulated HUVEC. PMN preincubated with mannose-6-P or N-acetylneuraminic acid (sialic acid), but not mannose or galactose-6-P, showed reduced adherence to ROS-treated HUVEC, suggesting that carbohydrate molecules were expressed on the latter and served as the ligand for the PMN L-selectin. Intercellular adhesion molecule (ICAM-1), constitutively present on the surface of resting HUVEC, was involved in the PMN adherence to ROS-treated HUVEC, since this adherence was inhibited by anti-ICAM-1, anti-CD11a, anti-CD11b, and anti-CD18 MoAbs. A non-CD18, non-ICAM-1-dependent mechanism is also involved in this adherence, since effects of these MoAbs were not additive; moreover, combinations of anti-CD18 and anti-ICAM-1 MoAbs with mannose-6-P and sialic acid completely inhibited PMN adherence. The increased binding of PMN to HX-XO-exposed HUVEC observed here involved IC-AM-1, but was independent of its upregulation, and another non-ICAM-1-dependent mechanism, in which carbohydrates expressed on HUVEC recognize L-selectin on PMN.
The infiltration of leukocytes into the central nervous system is associated with many pathologic conditions of the brain. The mechanisms by which these immune cells can penetrate the blood-brain barrier and remain within the brain are not understood. However, elevated brain levels of the pro-inflammatory cytokine IL-1 appear to accompany pathogenesis. The present study provides the first evidence that IL-1 can induce the expression of adhesion molecules for leukocytes on glial cells and suggests a role for the transcription factor NF-kappa B in the induction process. Human rIL-1 alpha was found to induce the expression of the cell adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) but not E-selectin in human 1321N1 astrocytoma. Both VCAM-1 and ICAM-1 were detectable from 3 h and remained sustained for up to 72 h. Induction was inhibited by the IL-1 receptor antagonist. IL-1 alpha was also shown to induce the expression of VCAM-1 and ICAM-1 in a receptor-dependent fashion in human A172 glioblastoma. Activation of the transcription factor NF-kappa B was also observed in 1321N1 astrocytoma in response to IL-1 alpha treatment and was similarly abolished by pretreatment of cells with antagonist. Activated NF-kappa B was apparent from 20 min and remained for up to 24 h. N-acetylcysteine (NAC) and pyrollidinedithiocarbamate (PDTC), which were shown to inhibit activation of NF-kappa B in Jurkat E6.1 lymphoblasts and EL4.NOB-1 thymoma, failed to block IL-1 activation of NF-kappa B in 1321N1 astrocytoma. However, both of these antioxidants demonstrated complex modulatory effects on the induction of cell adhesion molecule expression by IL-1. The induction of VCAM-1 but not of ICAM-1 proved susceptible to inhibition by both PDTC and NAC. The expression of adhesion molecules for leukocytes on glial cells in response to IL-1 may represent an important mechanism for retention of immune cells in the central nervous system that may be a prologue to inflammatory conditions in the brain.
Cell adhesion to endothelial cells stimulated by tumor necrosis factor-alpha (TNF) is due to induction of surface receptors, such as vascular cell adhesion molecule-1 (VCAM-1). The antioxidant pyrrolidine dithiocarbamate (PDTC) specifically inhibits activation of nuclear factor-kappa B (NF-kappa B). Since kappa B motifs are present in VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) promoters, we used PDTC to study the regulatory mechanisms of VCAM-1 and ICAM-1 induction and subsequent monocyte adhesion in TNF-treated human umbilical vein endothelial cells (HUVECs). PDTC or N-acetylcysteine dose dependently reduced TNF-induced VCAM-1 but not ICAM-1 surface protein (also in human umbilical arterial endothelial cells) and mRNA expression (by 70% at 100 mumol/L PDTC) in HUVECs as assessed by flow cytometry and polymerase chain reaction. Gel-shift analysis in HUVECs demonstrated that PDTC prevented NF-kappa B mobilization by TNF, suggesting that only VCAM-1 induction was controlled by NF-kappa B. Since HUVECs released superoxide anions in response to TNF, and H2O2 induces VCAM-1, PDTC may act as a radical scavenger. Although ICAM-1 induction was unaffected, inhibitors of NADPH oxidase (apocynin) or cytochrome P-450 (SKF525a) suppressed VCAM-1 induction by TNF, revealing that several radical-generating systems are involved in its regulation. PDTC, apocynin, or SKF525a decreased adhesion of monocytic U937 cells to TNF-treated HUVECs (by 75% at 100 mumol/L PDTC). Inhibition by anti-VCAM-1 monoclonal antibody 1G11 indicated that U937 adhesion was VCAM-1 dependent and suppression by antioxidants was due to reduced VCAM-1 induction.(ABSTRACT TRUNCATED AT 250 WORDS)
Intercellular adhesion molecule-1 (ICAM-1), the ligand of lymphocyte function-associated antigen-1, plays an important role in the interactions of a variety of hemopoietic and nonhemopoietic cells, including leukocytes, fibroblasts, and endothelial cells. ICAM-1 is known to be involved in the onset of several diseases such as inflammation, allograft rejection, and so on. In this report, we investigated the effects of dexamethasone, cyclosporin A, FK506, and pyrrolidine dithiocarbamate (PDTC) on the induction of the ICAM-1 gene by cytokines in fibroblasts. PDTC, a potent inhibitor of NF-kappa B, was shown by ELISA and FACS analysis to prevent dramatically the expression of the ICAM-1 gene stimulated by IL-1 alpha, IFN-gamma, and PMA, although the other reagents inhibited it only slightly. Ribonuclease protection assay revealed that PDTC blocked the expression of the ICAM-1 gene at the mRNA level. To elucidate the mechanism of this inhibition, we constructed a series of ICAM-1 promoter deletion mutants linked to the chloramphenicol acetyl transferase gene and analyzed the effect of PDTC on their activities. Transient transfection analysis indicated that the critical region for inhibition by PDTC is an NF-kappa B binding site-like motif (GGGAGGATTCC, ICAM-1 kappa B) that is located at position-540. Electrophoresis mobility shift assay revealed that PDTC actually inhibits the binding of NF-kappa B (or NF-kappa B-like) protein to the ICAM-1 kappa B site. These findings suggest that PDTC inhibits ICAM-1 gene expression by inhibiting the association of NF-kappa B (or NF-kappa B-like) protein with the ICAM-1 kappa B site.
Loss and gain of cell surface molecules determines the mobilization, emigration and invasiveness of epithelial cancer cells. As a first approach to gain further insight into these processes, we have followed two strategies: (1) to identify tumour cells which have disseminated early from primary carcinomas and to obtain information about the phenotype and prognostic significance of these cells; and (2) to identify molecular changes occurring in primary tumour cells at the time they develop their metastatic potential. Our analyses indicate that changes in the adhesive properties of solid tumour cells, such as down-regulation of desmosomal proteins (e.g. plakoglobin) and neo-expression of ICAM-1 or MUC18, are important determinants of the metastatic capability of individual malignant cells. The expression pattern of these cell adhesion molecules during tumour progression appears to reflect a disturbance at the level of the molecular elements normally responsible for controlling their expression. The outlined current strategies for detection, characterization and antibody therapy of cancer micrometastasis can be applied to the secondary prevention of metastatic disease in patients with minimal residual cancer.
alpha-Lipoic acid, which plays an essential role in mitochondrial dehydrogenase reactions, has recently gained considerable attention as an antioxidant. Lipoate, or its reduced form, dihydrolipoate, reacts with reactive oxygen species such as superoxide radicals, hydroxyl radicals, hypochlorous acid, peroxyl radicals, and singlet oxygen. It also protects membranes by interacting with vitamin C and glutathione, which may in turn recycle vitamin E. In addition to its antioxidant activities, dihydrolipoate may exert prooxidant actions through reduction of iron. alpha-Lipoic acid administration has been shown to be beneficial in a number of oxidative stress models such as ischemia-reperfusion injury, diabetes (both alpha-lipoic acid and dihydrolipoic acid exhibit hydrophobic binding to proteins such as albumin, which can prevent glycation reactions), cataract formation, HIV activation, neurodegeneration, and radiation injury. Furthermore, lipoate can function as a redox regulator of proteins such as myoglobin, prolactin, thioredoxin and NF-kappa B transcription factor. We review the properties of lipoate in terms of (1) reactions with reactive oxygen species; (2) interactions with other antioxidants; (3) beneficial effects in oxidative stress models or clinical conditions.
rac-a-Lipoic acid (CAS 62-46-4, thioctic acid) is used in human therapy besides the parenteral route also orally in gastric juice soluble galenic formulations in patients suffering from diabetic polyneuropathy which also involves the gastrointestinal tract (GI) tract in about 20% of the diabetic population. In those patients the most common manifestation of the disease due to small intestine dysfunction is diarrhoea as a consequence of which malabsorption of orally administered drugs may result. Due to the importance of the knowledge on absorption characteristics, in preclinical studies on pharmacokinetics the extent of [14C]absorption from a solution of [7,8-14C]rac-a-lipoic acid was investigated in the rat after oral administration by means of comparison of the AUCs from the [14C]plasma concentrations vs those from the intravenous route, yielding 66%. An alternative evaluation by comparison of [14C]material excreted into the urine yielded 93% [14C]absorption. Despite this high and nearly complete absorption, due to the gastroenteral disturbances mentioned above, the question was investigated if the absorption is restricted to only a small area of the GI tract or is extended over a wider area. The latter is expected to make the absorption less sensitive against variations caused by gastrointestinal disturbances due to longer residence times in the GI tract. In order to approach most closely the physiological situation--as compared with different in vitro incubation techniques using isolated GI tract sacs--the in situ technique on 5 ligated areas of the GI tract of the aneasthetized rat (stomach, duodenum, jejunum, ileum, and colon with caecum) was established.(ABSTRACT TRUNCATED AT 250 WORDS)
Intercellular adhesion molecule-1 (ICAM-1) is strongly expressed by human epidermal keratinocytes during the course of inflammatory skin diseases. To test the possibility that reactive oxygen species produced in the skin during an inflammatory response affect ICAM-1 expression, cultured human epidermal keratinocytes were treated with H2O2 at concentrations that did not damage the cells, and cell-surface ICAM-1 expression was analyzed. Expression of ICAM-1 was induced on keratinocytes by treatment with 300 microM H2O2 for 1 h. The antioxidant N-acetyl-L-cysteine strongly inhibited H2O2-induced ICAM-1 expression, whereas the antioxidants pyrrolidine dithiocarbamate and alpha-tocopherol were less inhibitory. N-acetyl-L-cysteine also suppressed keratinocyte surface expression of ICAM-1 induced by the cytokines interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha), whereas pyrrolidine dithiocarbamate and alpha-tocopherol suppressed IFN-gamma-induced surface expression but not TNF-alpha-induced expression. We found that N-acetyl-L-cysteine treatment reduced ICAM-1 mRNA levels when keratinocytes were stimulated with either IFN-gamma or TNF-alpha; however, pyrrolidine dithiocarbamate and alpha-tocopherol had no effect on either IFN-gamma- or TNF-alpha-induced ICAM-1 mRNA levels. Our results indicate that reactive oxygen species may be involved in the skin inflammatory process by increasing epidermal ICAM-1 expression and that some antioxidants may be effective in suppressing the epidermal ICAM-1 expression induced by reactive oxygen species and cytokines in inflammatory skin diseases.
Reactive oxygen species are thought to be messengers for nuclear factor (NF)-kappa B activation because its activation can be abrogated by antioxidants. However, this study identifies, for the first time, NF-kappa B activators that are insensitive to antioxidants. NF-kappa B activation that is induced by either calyculin A or okadaic acid (inhibitors of serine/threonine protein phosphatases 1 and 2A) is not blocked by N-acetylcysteine or dihydrolipoate in Jurkat and U937 cells. Nonetheless, these antioxidants block induction by TNF-alpha, lymphotoxin, and PMA. Unlike okadaic acid and calyculin A, neither TNF-alpha, lymphotoxin, nor PMA inhibited activities of phosphatases 1 and 2A. NF-kappa B activation induced by okadaic acid or calyculin A was not blocked by a myosin light chain kinase inhibitor, but was prevented by a protease inhibitor. The mitochondrial inhibitor, rotenone, also inhibited NF-kappa B activation by calyculin A; however, this inhibition was accompanied by a depletion of cellular ATP. These results suggest that 1) phosphatase inhibitors either target a component of signal transduction, which occurs downstream to an antioxidant-sensitive step or use distinct signaling pathways; 2) inhibition of phosphatases 1 and 2A is not a step in the pathway of TNF-alpha-, lymphotoxin-, or PMA-induced NF-kappa B activation; 3) myosin light chain kinase does not participate in NF-kappa B activation; and 4) activation of NF-kappa B by phosphatase inhibitors is controlled by proteases.
The effects of hydrogen peroxide, d-α-tocopherol and of d-β-tocopherol on proliferation, protein kinase C and activator protein-1 (AP-1) activation have been studied in vascular smooth muscle cells. Cell proliferation, when activated by foetal calf serum, was inhibited by d-α-tocopherol. Protein kinase C activity was stimulated by hydrogen peroxide in a manner similar to phorbol myristate acetate; in the latter case, but not in the former, d-α-tocopherol inhibited the reaction. Hydrogen peroxide prevented phorbol-myristate-acetate-stimulated AP-1 binding to DNA but stimulated it if protein kinase C was down-regulated or inhibited. d-α-Tocopherol promoted AP-1 activation in quiescent cells but prevented its activation by phorbol myristate acetate.
None of the described effects of d-α-tocopherol were shared by d-β-tocopherol, suggesting a non-antioxidant mechanism as the basis of its action. The data show that hydrogen peroxide and d-α-tocopherol affect more than one element in the cell signal-transduction cascade.