In This Issue
In This Issue
Rouzer Carol
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Spotlight
Carol A. Rouzer
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Reviews
Small Molecule Signaling Agents: The Integrated Chemistry and Biochemistry of Nitrogen Oxides, Oxides of Carbon, Dioxygen, Hydrogen Sulfide, and Their Derived Species
Jon M. Fukuto *- ,
Samantha J. Carrington - ,
Dean J. Tantillo - ,
Jason G. Harrison - ,
Louis J. Ignarro - ,
Bruce A. Freeman - ,
Andrew Chen - , and
David A. Wink
Several small molecule species formally known primarily as toxic gases have, over the past 20 years, been shown to be endogenously generated signaling molecules. The biological signaling associated with the small molecules NO, CO, H2S (and the nonendogenously generated O2), and their derived species have become a topic of extreme interest. It has become increasingly clear that these small molecule signaling agents form an integrated signaling web that affects/regulates numerous physiological processes. The chemical interactions between these species and each other or biological targets is an important factor in their roles as signaling agents. Thus, a fundamental understanding of the chemistry of these molecules is essential to understanding their biological/physiological utility. This review focuses on this chemistry and attempts to establish the chemical basis for their signaling functions.
Use of Hazard Indices for a Theoretical Evaluation of Cigarette Smoke Composition
Hans-Juergen Haussmann *
The chemical composition of cigarette mainstream smoke (MS) has been quantitatively analyzed in multiple studies, often with the objective to toxicologically evaluate and compare various types of MS. Increases and decreases in yields of constituents between MS types can only be consolidated if these yields are compared on the basis of toxicological properties of the individual constituents. For the risk assessment of various complex mixtures including MS, a hazard index (HI) approach has been used that requires weighing of the exposure to individual MS constituents by cancer and noncancer potency values. The objective of the current study is to review the past uses of the HI concept for MS and smokeless tobacco and discuss strengths and limitations of using this concept. Published information as well as information made available on the Web was used. The HI concept has been applied to MS for determining and comparing theoretical lifetime risks, for consumer communication, for the prioritization of constituents for reduction, for ingredient assessment, and for the selection of constituents for regulation. The limitations of this approach are associated with the limited number of MS constituents with available yield data, the gaps and uncertainties in available potency values, the application to relatively high exposure concentrations, and the default assumption of additivity. The derived theoretical noncancer index is dominated by acrolein to an extent that there seems to be not much advantage in using the HI concept for noncancer assessments. The derived theoretical cancer index is dominated by genotoxic carcinogens of the MS vapor phase and may thus complement currently used toxicological assays in a tiered evaluation approach. As is the case for every other assay and interpretation model, the HI concept needs to be applied with its limitations and weaknesses in mind. Its best application is for comparative purposes. It should be kept in mind that the HI concept is a theoretical concept and does not provide actual risk information.
Circadian Regulation of the Hepatic Endobiotic and Xenobitoic Detoxification Pathways: The Time Matters
Ursula Prosenc Zmrzljak - and
Damjana Rozman *
Metabolic processes have to be regulated tightly to prevent waste of energy and to ensure sufficient detoxification. Most anabolic processes operate in a timely manner when energy intake is the highest, while catabolism takes place in energy spending periods. Endobiotic and xenobiotic metabolism are therefore under circadian control. Circadian regulation is mediated through the suprachiasmatic nucleus (SCN), a master autonomous oscillator of the brain. Although many peripheral organs have their own oscillators, the SCN is important in orchestrating and entraining organs according to the environmental light cues. However, light is not the only signal for entrainment of internal clocks. For endobiotic and xenobitoic detoxification pathways, the food composition and intake regime are equally important. The rhythm of the liver as an organ where the major metabolic pathways intersect depends on SCN signals, signals from endocrine tissues, and, importantly, the type and time of feeding or xenobiotics ingestion. Several enzymes are involved in detoxification processes. Phase I is composed mainly of cytochromes P450, which are regulated by nuclear receptors. Phase II enzymes modify the phase I metabolites, while phase III includes membrane transporters responsible for the elimination of modified xenobiotics. Phases I–III of drug metabolism are under strong circadian regulation, starting with the drug-sensing nuclear receptors and ending with drug transporters. Disturbed circadian regualtion (jet-lag, shift work, and dysfunction of core clock genes) leads to changed periods of activity, sleep disorders, disturbed glucose homeostasis, breast or colon cancer, and metabolic syndrome. As many xenobiotics influence the circadian rhythm of the liver, bad drug administration timing can worsen the above listed effects. This review will cover the major hepatic circadian regulation of endogenous and xenobiotic metabolic pathways and will provide examples of how good timing of drug administration can change drug failure to treatment success.
Rapid Reports
A Major Glucuronidated Metabolite of JWH-018 Is a Neutral Antagonist at CB1 Receptors
Kathryn A. Seely *- ,
Lisa K. Brents - ,
Anna Radominska-Pandya - ,
Gregory W. Endres - ,
Gregory S. Keyes - ,
Jeffery H. Moran - , and
Paul L. Prather
Recently, hydroxylated metabolites of JWH-018, a synthetic cannabinoid found in many K2/Spice preparations, have been shown to retain affinity and activity for cannabinoid type 1 receptors (CB1Rs). The activity of glucuronidated metabolites of JWH-018 is not known; hence, this study investigated the affinity and activity of a major metabolite, JWH-018-N-(5-hydroxypentyl) β-d-glucuronide (018-gluc), for CB1Rs. The 018-gluc binds CB1Rs (Ki = 922 nM), has no effect on G-protein activity, but antagonizes JWH-018 activity at CB1Rs. The data suggests that hydroxylation by cytochrome P450s and subsequent glucuronidation by UDP-glucuronosyltransferases produces a metabolite, 018-gluc, which possesses antagonistic activity at CB1Rs.
Articles
Oxidative Stress Induced by Pure and Iron-Doped Amorphous Silica Nanoparticles in Subtoxic Conditions
Dorota Napierska - ,
Virginie Rabolli - ,
Leen C. J. Thomassen - ,
David Dinsdale - ,
Catherine Princen - ,
Laetitia Gonzalez - ,
Katrien L. C. Poels - ,
Micheline Kirsch-Volders - ,
Dominique Lison - ,
Johan A. Martens - , and
Peter H. Hoet *
Amorphous silica nanoparticles (SiO2-NPs) have found broad applications in industry and are currently intensively studied for potential uses in medical and biomedical fields. Several studies have reported cytotoxic and inflammatory responses induced by SiO2-NPs in different cell types. The present study was designed to examine the association of oxidative stress markers with SiO2-NP induced cytotoxicity in human endothelial cells. We used pure monodisperse amorphous silica nanoparticles of two sizes (16 and 60 nm; S16 and S60) and a positive control, iron-doped nanosilica (16 nm; SFe), to study the generation of hydroxyl radicals (HO·) in cellular-free conditions and oxidative stress in cellular systems. We investigated whether SiO2-NPs could influence intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, increase lipid peroxidation (malondialdehyde (MDA) and 4-hydroxyalkenal (HAE) concentrations), and up-regulate heme oxygenase-1 (HO-1) mRNA expression in the studied cells. None of the particles, except SFe, produced ROS in cell-free systems. We found significant modifications for all parameters in cells treated with SFe nanoparticles. At cytotoxic doses of S16 (40–50 μg/mL), we detected weak alterations of intracellular glutathione (4 h) and a marked induction of HO-1 mRNA (6 h). Cytotoxic doses of S60 elicited similar responses. Preincubation of cells being exposed to SiO2-NPs with an antioxidant (5 mM N-acetylcysteine, NAC) significantly reduced the cytotoxic activity of S16 and SFe (when exposed up to 25 and 50 μg/mL, respectively) but did not protect cells treated with S60. Preincubation with NAC significantly reduced HO-1 mRNA expression in cells treated with SFe but did not have any effect on HO-1 mRNA level in cell exposed to S16 and S60. Our study demonstrates that the chemical composition of the silica nanoparticles is a dominant factor in inducing oxidative stress.
Benzo[a]pyrene-Induced Changes in MicroRNA–mRNA Networks
Daneida Lizarraga - ,
Stan Gaj - ,
Karen J. Brauers - ,
Leen Timmermans - ,
Jos C. Kleinjans - , and
Joost H. M. van Delft *
Toxicological studies assessing the safety of compounds for humans frequently use in vitro systems to characterize toxic responses in combination with transcriptomic analyses. Thus far, changes have mostly been investigated at the mRNA level. Recently, microRNAs have attracted attention because they are powerful negative regulators of mRNA levels and, thus, may be responsible for the modulation of important mRNA networks implicated in toxicity. This study aimed to identify possible microRNA–mRNA networks as novel interactions on the gene expression level after a genotoxic insult. We used benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, as a model genotoxic/carcinogenic compound. We analyzed time-dependent effects on mRNA and microRNA profiles in HepG2 cells, a widely used human liver cell line that expresses active p53 and is competent for the biotransformation of BaP. Changes in microRNA expression in response to BaP, in combination with multiple alterations of mRNA levels, were observed. Many of these altered mRNAs are targets of altered microRNAs. Using pathway analysis, we evaluated the relevance of such microRNA deregulations to genotoxicity. This revealed eight microRNAs that appear to participate in specific BaP-responsive pathways relevant to genotoxicity, such as apoptotic signaling, cell cycle arrest, DNA damage response, and DNA damage repair. Our results particularly highlight the potential of microRNA-29b, microRNA-26a-1*, and microRNA-122* as novel players in the BaP response. Therefore, this study demonstrates the added value of an integrated microRNA–mRNA approach for identifying molecular mechanisms induced by BaP in an in vitro human model.
Hematite Nanoparticles Larger than 90 nm Show No Sign of Toxicity in Terms of Lactate Dehydrogenase Release, Nitric Oxide Generation, Apoptosis, and Comet Assay in Murine Alveolar Macrophages and Human Lung Epithelial Cells
Francesca Stefania Freyria - ,
Barbara Bonelli - ,
Maura Tomatis - ,
Mara Ghiazza - ,
Elena Gazzano - ,
Dario Ghigo - ,
Edoardo Garrone - , and
Bice Fubini
Three hematite samples were synthesized by precipitation from a FeCl3 solution under controlled pH and temperature conditions in different morphology and dimensions: (i) microsized (average diameter 1.2 μm); (ii) submicrosized (250 nm); and (iii) nanosized (90 nm). To gain insight into reactions potentially occurring in vivo at the particle–lung interface following dust inhalation, several physicochemical features relevant to pathogenicity were measured (free radical generation in cell-free tests, metal release, and antioxidant depletion), and cellular toxicity assays on human lung epithelial cells (A549) and murine alveolar macrophages (MH-S) were carried out (LDH release, apoptosis detection, DNA damage, and nitric oxide synthesis). The decrease in particles size, from 1.2 μm to 90 nm, only caused a slight increase in structural defects (disorder of the hematite phase and the presence of surface ferrous ions) without enhancing surface reactivity or cellular responses in the concentration range between 20 and 100 μg cm–2.
γH2AX, pChk1, and Wip1 as Potential Markers of Persistent DNA Damage Derived from Dibenzo[a,l]pyrene and PAH-Containing Extracts from Contaminated Soils
Magdalena Niziolek-Kierecka - ,
Kristian Dreij *- ,
Staffan Lundstedt - , and
Ulla Stenius
Polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete combustion of organic material and are ubiquitous environmental contaminants. High levels of PAHs are commonly found in soils at industrial sites, thereby constituting a risk for humans and the environment. However, this risk is often difficult to estimate due to the complexity of the contamination. In the present study, we investigated the cellular DNA damage response induced by extracts of PAH-contaminated soils collected at various industrial sites in Sweden. The results show that interactions of PAHs in the soil extracts caused activation of DNA damage signaling consistent with persistent DNA damage. Signaling in HepG2 cells exposed to soil PAH extracts corresponding to 1 μM benzo[a]pyrene was similar to that of 0.1 μM dibenzo[a,l]pyrene, a highly carcinogenic PAH known to produce persistent DNA damage. The response involved prolonged activation of DNA damage marker (H2AX), check point kinase (Chk1), and phosphatases (Wip1). Furthermore, blocking DNA damage signaling using specific inhibitors and siRNA showed the important role of signaling through Chk1 for the level of DNA damage. We conclude that the combination of prolonged Chk1 phosphorylation and induced expression of Wip1 might serve as potential markers for persistent DNA damage induced by complex mixtures of environmental PAHs. Discrepancies between mRNA and protein levels of Wip1 in response to soil extracts, in parallel with increased microRNA (miR)-16 levels, suggest a role of miR-16 in the regulation of DNA damage signaling in response to PAHs. Taken together, our data indicate that PAH extracts induce irreparable DNA damage and that this is consistent with the prolonged activation of DNA damage signaling.
Delivery Method, Target Gene Structure, and Growth Properties of Target Cells Impact Mutagenic Responses to Reactive Nitrogen and Oxygen Species
Min Young Kim - ,
Chang Hoon Lim - ,
Laura J. Trudel - ,
William M. Deen - , and
Gerald N. Wogan *
Dysregulated production of nitric oxide (NO•) and reactive oxygen species (ROS) by inflammatory cells in vivo may contribute to mutagenesis and carcinogenesis. Here, we compare cytotoxicity and mutagenicity induced by NO• and ROS in TK6 and AS52 cells, delivered by two methods: a well-characterized delivery system and a novel adaptation of a system for coculture. When exposed to preformed NO•, a cumulative dose of 620 μM min reduced the viability of TK6 cells at 24 h to 36% and increased mutation frequencies in the HPRT and TK1 genes to 7.7 × 10–6 (p < 0.05) and 24.8 × 10–6 (p < 0.01), 2.7- and 3.7-fold higher than background, respectively. In AS52 cells, cumulative doses of 1700 and 3700 μM min reduced viability to 49 and 22%, respectively, and increased the mutation frequency 10.2- and 14.6-fold higher than the argon control (132 × 10–6 and 190 × 10–6, respectively). These data show that TK6 cells were more sensitive than AS52 cells to killing by NO•. However, the two cell lines were very similar in relative susceptibility to mutagenesis; on the basis of fold increases in MF, average relative sensitivity values [(MFexp/MFcontrol)/cumulative NO• dose] were 5.16 × 10–3 and 4.97 × 10–3 μM–1 min–1 for TK6 cells and AS52 cells, respectively. When AS52 cells were exposed to reactive species generated by activated macrophages in the coculture system, cell killing was greatly reduced by the addition of NMA to the culture medium and was completely abrogated by combined additions of NMA and the superoxide scavenger Tiron, indicating the relative importance of NO• to loss of viability. Exposure in the coculture system for 48 h increased mutation frequency in the gpt gene by more than 9-fold, and NMA plus Tiron again completely prevented the response. Molecular analysis of gpt mutants induced by preformed NO• or by activated macrophages revealed that both doubled the frequency of gene inactivation (40% in induced vs 20% in spontaneous mutants). Sequencing showed that base-substitution mutations dominated the spectra, with transversions (30–40%) outnumbering transitions (10–20%). Virtually all mutations took place at guanine sites in the gene. G:C to T:A transversions accounted for about 30% of both spontaneous and induced mutations; G:C to A:T transitions amounted to 10–20% of mutants; insertions, small deletions, and multiple mutations were present at frequencies of 0–10%. Taken together, these results indicate that cell type and proximity to generator cells are critical determinants of cytotoxic and genotoxic responses induced by NO• and reactive species produced by activated macrophages.
Surface Reactivity and Cell Responses to Chrysotile Asbestos Nanofibers
Francesco Turci - ,
Massimiliano Colonna - ,
Maura Tomatis - ,
Stefano Mantegna - ,
Giancarlo Cravotto - ,
Giulia Gulino - ,
Elisabetta Aldieri - ,
Dario Ghigo - , and
Bice Fubini *
High aspect-ratio nanomaterials (HARNs) have recently attracted great attention from nanotoxicologists because of their similarity to asbestos. However, the actual risk associated with the exposure to nanosized asbestos, which escapes most regulations worldwide, is still unknown. Nanometric fibers of chrysotile asbestos have been prepared from two natural sources to investigate whether nanosize may modulate asbestos toxicity and gain insight on the hazard posed by naturally occurring asbestos, which may be defined as HARNs because of their dimensions. Power ultrasound was used to obtain nanofibers from two different chrysotile specimens, one from the dismissed asbestos mine in Balangero (Italian Western Alps) and the other from a serpentine outcrop in the Italian Central Alps. Electron microscopy, X-ray diffraction, and fluorescence spectroscopy revealed that the procedure does not affect mineralogical and chemical composition. Surface reactions related to oxidative stress, free radical generation, bioavailability of iron, and antioxidant depletion, revealed a consistent reduction in reactivity upon reduction in size. When tested on A549 human epithelial cells, the pristine but not the nanosized fibers proved cytotoxic (LDH release), induced NO production, and caused lipid peroxidation. However, nanofibers still induced some toxicity relevant oxidative stress activity (ROS production) in a dose-dependent fashion. The reduction in length and a lack of poorly coordinated bioavailable iron in nanochrysotile may explain this behavior. The present study provides a one-step procedure for the preparation of a homogeneous batch of natural asbestos nanofibers and shows how a well-known toxic material might not necessarily become more toxic than its micrometric counterpart when reduced to the nanoscale.
Differential Oxidation of Two Thiophene-Containing Regioisomers to Reactive Metabolites by Cytochrome P450 2C9
Peter M. Rademacher *- ,
Caleb M. Woods - ,
Qingbiao Huang - ,
Grazyna D. Szklarz - , and
Sidney D. Nelson
The uricosuric diuretic agent tienilic acid (TA) is a thiophene-containing compound that is metabolized by P450 2C9 to 5-OH-TA. A reactive metabolite of TA also forms a covalent adduct to P450 2C9 that inactivates the enzyme and initiates immune-mediated hepatic injury in humans, purportedly through a thiophene-S-oxide intermediate. The 3-thenoyl regioisomer of TA, tienilic acid isomer (TAI), is chemically very similar and is reported to be oxidized by P450 2C9 to a thiophene-S-oxide, yet it is not a mechanism-based inactivator (MBI) of P450 2C9 and is reported to be an intrinsic hepatotoxin in rats. The goal of the work presented in this article was to identify the reactive metabolites of TA and TAI by the characterization of products derived from P450 2C9-mediated oxidation. In addition, in silico approaches were used to better understand both the mechanisms of oxidation of TA and TAI and/or the structural rearrangements of oxidized thiophene compounds. Incubation of TA with P450 2C9 and NADPH yielded the well-characterized 5-OH-TA metabolite as the major product. However, contrary to previous reports, it was found that TAI was oxidized to two different types of reactive intermediates that ultimately lead to two types of products, a pair of hydroxythiophene/thiolactone tautomers and an S-oxide dimer. Both TA and TAI incorporated 18O from 18O2 into their respective hydroxythiophene/thiolactone metabolites indicating that these products are derived from an arene oxide pathway. Intrinsic reaction coordinate calculations of the rearrangement reactions of the model compound 2-acetylthiophene-S-oxide showed that a 1,5-oxygen migration mechanism is energetically unfavorable and does not yield the 5-OH product but instead yields a six-membered oxathiine ring. Therefore, arene oxide formation and subsequent NIH-shift rearrangement remains the favored mechanism for formation of 5-OH-TA. This also implicates the arene oxide as the initiating factor in TA induced liver injury via covalent modification of P450 2C9. Finally, in silico modeling of P450 2C9 active site ligand interactions with TA using the catalytically active iron-oxo species revealed significant differences in the orientations of TA and TAI in the active site, which correlated well with experimental results showing that TA was oxidized only to a ring carbon hydroxylated product, whereas TAI formed both ring carbon hydroxylated products and an S-oxide.
Prooxidant and Proinflammatory Potency of Air Pollution Particulate Matter (PM2.5–0.3) Produced in Rural, Urban, or Industrial Surroundings in Human Bronchial Epithelial Cells (BEAS-2B)
Mona Dergham - ,
Capucine Lepers - ,
Anthony Verdin - ,
Sylvain Billet - ,
Fabrice Cazier - ,
Dominique Courcot - ,
Pirouz Shirali - , and
Guillaume Garçon *
Compelling evidence indicates that exposure to air pollution particulate matter (PM) affects human health. However, how PM composition interacts with PM-size to cause adverse health effects needs elucidation. In this study, we were also interested in the physicochemical characteristics and toxicological end points of PM2.5–0.3 samples produced in rural, urban, or industrial surroundings, thereby expecting to differentiate their respective in vitro adverse health effects in human bronchial epithelial cells (BEAS-2B). Physicochemical characteristics of the three PM2.5–0.3 samples, notably their inorganic and organic components, were closely related to their respective emission sources. Referring also to the dose/response relationships of the three PM2.5–0.3 samples, the most toxicologically relevant exposure times (i.e., 24, 48, and 72 h) and doses (i.e., 3.75 μg PM/cm2 and 15 μg PM/cm2) to use to study the underlying mechanisms of action involved in PM-induced lung toxicity were chosen. Organic chemicals adsorbed on the three PM2.5–0.3 samples (i.e., polycyclic aromatic hydrocarbons) were able to induce the gene expression of xenobiotic-metabolizing enzymes (i.e., Cytochrome P4501A1 and 1B1, and, to a lesser extent, NADPH-quinone oxidoreductase-1). Moreover, intracellular reactive oxygen species within BEAS-2B cells exposed to the three PM2.5–0.3 samples induced oxidative damage (i.e., 8-hydroxy-2′-deoxyguanosine formation, malondialdehyde production and/or glutathione status alteration). There were also statistically significant increases of the gene expression and/or protein secretion of inflammatory mediators (i.e., notably IL-6 and IL-8) in BEAS-2B cells after their exposure to the three PM2.5–0.3 samples. Taken together, the present findings indicated that oxidative damage and inflammatory response preceeded cytotoxicity in air pollution PM2.5–0.3-exposed BEAS-2B cells and supported the idea that PM-size, composition, and origin could interact in a complex manner to determine the in vitro responsiveness to PM.
TiO2 Nanoparticles Induce Dysfunction and Activation of Human Endothelial Cells
Angélica Montiel-Dávalos - ,
José Luis Ventura-Gallegos - ,
Ernesto Alfaro-Moreno - ,
Elizabeth Soria-Castro - ,
Ethel García-Latorre - ,
José Gerardo Cabañas-Moreno - ,
María del Pilar Ramos-Godinez - , and
Rebeca López-Marure *
Nanoparticles can reach the blood and cause inflammation, suggesting that nanoparticles–endothelial cells interactions may be pathogenically relevant. We evaluated the effect of titanium dioxide nanoparticles (TiO2) on proliferation, death, and responses related with inflammatory processes such as monocytic adhesion and expression of adhesion molecules (E- and P-selectins, ICAM-1, VCAM-1, and PECAM-1) and with inflammatory molecules (tissue factor, angiotensin-II, VEGF, and oxidized LDL receptor-1) on human umbilical vein endothelial cells (HUVEC). We also evaluated the production of reactive oxygen species, nitric oxide production, and NF-κB pathway activation. Aggregates of TiO2 of 300 nm or smaller and individual nanoparticles internalized into HUVEC inhibited proliferation strongly and induced apoptotic and necrotic death starting at 5 μg/cm2. Besides, TiO2 induced activation of HUVEC through an increase in adhesion and in expression of adhesion molecules and other molecules involved with the inflammatory process. These effects were associated with oxidative stress and NF-κB pathway activation. In conclusion, TiO2 induced HUVEC activation, inhibition of cell proliferation with increased cell death, and oxidative stress.
Cyclohexyl Analogues of Ethylenediamine Dipropanoic Acid Induce Caspase-Independent Mitochondrial Apoptosis in Human Leukemic Cells
Sonja Misirlic Dencic - ,
Jelena Poljarevic - ,
Urosh Vilimanovich - ,
Andrija Bogdanovic - ,
Aleksandra J. Isakovic - ,
Tamara Kravic Stevovic - ,
Marija Dulovic - ,
Nevena Zogovic - ,
Andjelka M. Isakovic - ,
Sanja Grguric-Sipka - ,
Vladimir Bumbasirevic - ,
Tibor Sabo - ,
Vladimir Trajkovic *- , and
Ivanka Markovic *
We investigated the cytotoxicity of recently synthesized (S,S)-ethylendiamine-N,N′-di-2-(3-cyclohexyl)propanoic acid esters toward human leukemic cell lines and healthy blood mononuclear cells. Cell viability was assessed by acid phosphatase assay, apoptosis, and differentiation were analyzed by flow cytometry and electron microscopy, while intracellular localization of apoptosis-inducing factor (AIF) was determined by immunoblotting. It was demonstrated that methyl, ethyl, and n-propyl esters were toxic to HL-60, REH, MOLT-4, KG-1, JVM-2, and K-562 leukemic cell lines, while the nonesterified parental compound and n-butyl ester were devoid of cytotoxic action. The ethyl ester exhibited the highest cytotoxic activity (IC50 10.7 μM–45.4 μM), which was comparable to that of the prototypical anticancer drug cisplatin. The observed cytotoxic effect in HL-60 cells was associated with an increase in superoxide production and mitochondrial membrane depolarization, leading to apoptotic cell death characterized by phosphatidylserine externalization and DNA fragmentation in the absence of autophagic response. DNA fragmentation preceded caspase activation and followed AIF translocation from mitochondria to nucleus, which was indicative of caspase-independent apoptotic cell death. HL-60 cells treated with subtoxic concentration of the compound displayed morphological signs of granulocytic differentiation (nuclear indentations and presence of cytoplasmic primary granules), as well as an increased expression of differentiation markers CD11b and CD15. The cyclohexyl analogues of ethylenediamine dipropanoic acid were also toxic to peripheral blood mononuclear cells of both healthy controls and leukemic patients, the latter being more sensitive. Our data demonstrate that the toxicity of the investigated cyclohexyl compounds against leukemic cell lines is mediated by caspase-independent apoptosis associated with oxidative stress, mitochondrial dysfunction, and AIF translocation.
Glycophthalocyanines as Photosensitizers for Triggering Mitotic Catastrophe and Apoptosis in Cancer Cells
Ana R. M. Soares - ,
Maria G. P. M. S. Neves - ,
Augusto C. Tomé - ,
M. Carmen Iglesias-de la Cruz - ,
Alicia Zamarrón - ,
Elisa Carrasco - ,
Salvador González - ,
José A. S. Cavaleiro - ,
Tomás Torres - ,
Dirk M. Guldi - , and
Angeles Juarranz *
Photodynamic therapy (PDT) is a treatment modality for different forms of cancer based on the combination of light, molecular oxygen, and a photosensitizer (PS) compound. When activated by light, the PS generates reactive oxygen species leading to tumor destruction. Phthalocyanines are compounds that have already shown to be efficient PSs for PDT. Several examples of carbohydrate substituted phthalocyanines have been reported, assuming that the presence of carbohydrate moieties could improve their tumor selectivity. This work describes the photoeffects of symmetric and asymmetric phthalocyanines with d-galactose (so-called GPh1, GPh2, and GPh3) on HeLa carcinoma cells and their involvement in cell death. Photophysical properties and in vitro photodynamic activities for the compounds considered revealed that the asymmetric glycophthalocyanine GPh3 is very efficient and selective, producing higher photocytotoxicity on cancer cells than in nonmalignat HaCaT. The cell toxiticy after PDT treatment was dependent upon light exposure level and GPh3 concentration. GPh3 causes cell cycle arrest at the metaphase stage leading to multiple spindle poles, mitotic catastrophe, followed by apoptosis in cancer cells. These effects were partially negated by the pancaspase inhibitor Z-VAD-FMK. Together, these results indicate that GPh3 is an excellent candidate drug for PDT, able to induce selective tumor cell death.
Exposure and Kinetics of Polycyclic Aromatic Hydrocarbons (PAHs) in Cigarette Smokers
Gideon St.Helen - ,
Maciej L. Goniewicz - ,
Delia Dempsey - ,
Margaret Wilson - ,
Peyton Jacob III,- , and
Neal L. Benowitz *
Our study objectives were (1) to investigate the selectivity of polycyclic aromatic hydrocarbon (PAH) metabolites for tobacco smoke exposure and (2) to determine half-lives of PAH metabolites in smokers. There were 622 participants from the United States (US) and Poland, and of these, 70% were smokers. All subjects provided spot urine samples, and 125 smokers provided blood samples. Urinary PAH metabolite half-lives were determined in 8 smokers. In controlled hospital studies of 18 smokers, the associations between various measures of nicotine intake and urinary excretion of PAH metabolites were investigated. Plasma nicotine was measured by GC. LC-MS/MS was used to measure the plasma levels of cotinine and trans-3′-hydroxycotinine, and urine levels of nicotine and its metabolites, total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and PAH metabolites (2-naphthol, 1-, 2-, and 3-hydroxyfluorenes, 1-, 2-, 3-, and 4-hydroxyphenanthrenes, and 1-hydroxypyrene). Regardless of smoking status, PAH metabolite excretion was higher in Polish subjects than in US subjects (p-values <0.001). 1-Hydroxyfluorene exhibited the greatest difference between smokers and nonsmokers, with a 5-fold difference in Polish subjects and a 25-fold difference in US subjects, followed by 3- and 2-hydroxyfluorenes, 2-naphthol, and 1-hydroxypyrene. The differences for hydroxyphenanthrenes were small or nonsignificant. 1-Hydroxyfluorene had the highest correlation with urine nicotine equivalents (r = 0.77) and urine NNAL (r = 0.64). While the half-lives of PAH metabolites were <10 h in smokers, 1-hydroxyfluorene had the largest ratio of initial to terminal urine concentration (58.4 ± 38.6, mean ± SD) after smoking. Receiver Operating Characteristic (ROC) analysis of PAHs among Polish and US subjects further showed that hydroxyfluorenes are most highly discriminative of smokers from nonsmokers followed by 2-naphthol and 1-hydroxypyrene. In conclusion, hydroxyfluorenes, particularly 1-hydroxyfluorene, and 2-naphthol are more selective of tobacco smoke than 1-hydroxypyrene and hydroxyphenanthrenes. Characterization of hydroxyfluorene and 2-naphthol metabolites in urine may improve the characterization of PAHs from tobacco smoke and related disease risks among smokers and nonsmokers.
4-HNE Adduct Stability Characterized by Collision-Induced Dissociation and Electron Transfer Dissociation Mass Spectrometry
Kristofer S. Fritz - ,
Katherine A. Kellersberger - ,
Jose D. Gomez - , and
Dennis R. Petersen *
4-Hydroxynonenal (4-HNE) alters numerous proteomic and genomic processes. Understanding chemical mechanisms of 4-HNE interactions with biomolecules and their respective stabilities may lead to new discoveries in biomarkers for numerous diseases of oxidative stress. Collision-induced dissociation (CID) and electron transfer dissociation (ETD) MS/MS were utilized to examine the stability of a 4-HNE-Cys Michael adduct. CID conditions resulted in the neutral loss of 4-HNE, also known as a retro-Michael addition reaction (RMA). Consequently, performing ETD fragmentation on this same adduct did not result in RMA. Interestingly, 4-HNE adduct reduction via sodium borohydride (NaBH4) treatment stabilized against the CID induced RMA. In a direct comparison of three forms of 4-HNE adducts, computational modeling revealed sizable shifts in the shape and orientation of the lowest unoccupied molecular orbital (LUMO) density around the 4-HNE-Cys moiety. These findings demonstrate that ETD MS/MS analysis can be used to improve the detection of 4-HNE-protein modifications by preventing RMA reactions from occurring.