An immunoblot assay for cysteine oxidation by reactive oxygen species allows detection of novel thioprotective efficacy of black tea extracts.

INTRODUCTION: While cysteine thiol groups help to maintain the redox status of many proteins, they can be very susceptible to damaging oxidants. Despite broad interest in their antioxidant properties, whether tea polyphenols protect against protein thiol damage of this kind is unclear. This study sought to develop a simple immunoassay for use in screening tea extracts and other antioxidants for thioprotective efficacy at protein thiol groups. METHODS: Fresh aqueous extracts were prepared from commercially sourced green, white, black and red teas. Traut's reagent (2-iminothiolane) was used to prepare surface-thiolated bovine serum albumin for use as assay substrate in the protein oxidation assay. Oxidative damage was induced during a 15 min incubation with hydrogen peroxide (H2O2) in the presence of tea extracts and reference antioxidants. The substrate protein was then derivatised with dimedone before samples were loaded onto a nitrocellulose membrane housed within a Slot-Blot apparatus. After blocking nonspecific protein binding a commercially available antibody was used to detect dimedone-labelled groups. RESULTS: While the total phenol content of tea extracts typically correlated with their activity in lipid peroxidation and galvinoxyl radical-trapping assays, the former did not fully predict their abilities to suppress H2O2-induced cysteine oxidation, with black tea extracts displaying greater activity than the other teas and an apparent ability to reverse pre-existing cysteine oxidation. Among the model antioxidants tested, quercetin displayed a heightened ability to suppress cysteine oxidation. DISCUSSION: This slot-blot immunoassay is a convenient method that facilitates standardised comparisons between the thioprotective properties of structurally- and constitutively-diverse antioxidants.

Carbonyl scavengers as pharmacotherapies in degenerative disease: Hydralazine repurposing and challenges in clinical translation.

During cellular metabolism, spontaneous oxidative damage to unsaturated lipids generates many electrophilic carbonyl compounds that readily attack cell macromolecules, forming adducts that are potential drivers of tissue dysfunction. Since such damage is heightened in many degenerative conditions, researchers have assessed the efficacy of nucleophilic carbonyl-trapping drugs in animal models of such disorders, anticipating that they will protect tissues by intercepting toxic lipid-derived electrophiles (LDEs) within cells. This Commentary explores recent animal evidence for carbonyl scavenger efficacy in two disparate yet significant conditions known to involve LDE production, namely spinal cord injury (SCI) and alcoholic liver disease (ALD). Primary emphasis is placed on studies that utilised hydralazine, a clinically-approved "broad-spectrum" scavenger known to trap multiple LDEs. In addition to reviewing recent studies of hydralazine efficacy in animal SCI and ALD models, the Commentary reviews new insights concerning novel lifespan- and healthspan-extending properties of hydralazine obtained during studies in model invertebrate organisms, since the mechanisms involved seem of likely benefit during the treatment of degenerative disease. Finally, noting that human translation of the histoprotective properties of hydralazine have been limited, the final section of the Commentary will address two obstacles that hamper clinical translation of LDE-trapping therapies while also suggesting potential strategies for overcoming these problems.

Taking Pharmaceutical Innovation to the Masses.

General levels of "pharmaceuticals literacy" are not high in contemporary societies. To address this educational need, in 2012 the University of Western Australia introduced an innovative multidisciplinary course for undergraduates within any degree program entitled PHAR1101: Drugs that Changed the World. Now ranking among the largest courses at the institution, PHAR1101 enrollments will likely approach 1000 students in 2017.

Functional characterisation and application of an ex vivo perfusion-superfusion system in murine airways.

INTRODUCTION: The aim of this study was to develop two dynamic ex vivo airway explant systems, a perfusion-superfusion system and a ventilation-superfusion system, for the study of toxic airborne substances, such as the prevalent smoke constituent acrolein. METHODS: Mouse isolated tracheal segments were perfused with physiological media or ventilated with humidified air at 37°C to mimic dynamic flow conditions, and superfused with media over the exterior surface. At selected time points, the histological and functional integrity of segments was evaluated. The perfusion-superfusion system was subsequently used to examine mucin secretory responses elicited by acrolein in airways in which mucous metaplasia had been induced with lipopolysaccharide (LPS; 1µgml-1) prior to 24h of media perfusion, followed by stimulation with acrolein or ATP for 15min. Epithelial mucin levels were determined by quantitative analysis of periodic acid-Schiff's reagent (PAS)-stained sections. RESULTS: Epithelial morphology was successfully preserved in the perfusion-superfusion and ventilation-superfusion systems for at least 24h and up to 18h, respectively. At these time points, the contractile and relaxation responses of perfused and ventilated tracheal segments to carbachol, the neuropeptide substance P, and the prostanoid PGE2 were also preserved. Using the perfusion-superfusion system, acute exposure to acrolein caused a dose-dependent reduction in the levels of PAS-positive mucin stores induced by LPS, consistent with mucin secretion. DISCUSSION: Both the perfusion-superfusion and ventilation-superfusion systems successfully preserved the viability of mouse isolated tracheal segments on a histological and functional level, and the perfusion-superfusion system was used to characterise the mucin secretory responses elicited by acrolein. Thus, this system may be a useful model through which to conduct further toxicological studies in mammalian airways.

Acrolein and Human Disease: Untangling the Knotty Exposure Scenarios Accompanying Several Diverse Disorders.

Acrolein is a highly toxic electrophile that participates in many diseases, yet efforts to delineate its precise mechanistic contributions to specific conditions are complicated by its wide distribution within human environments. This Perspective develops the proposal that due to its mixed status as environmental pollutant, metabolic byproduct, and endotoxicant which forms via ubiquitous pathophysiological processes, many diseases likely involve acrolein released from multiple sources. Although the category boundaries are indistinct, at least four identifiable exposure scenarios are identifiable. First, in some syndromes, such as those accompanying chronic or acute intoxication with smoke, whatever role acrolein plays in disease pathogenesis mainly traces to exogenous sources such as the combustion of tobacco or other organic matter. A second exposure category involves xenobiotics that undergo metabolism within the body to release acrolein. Still other health conditions, however, involve acrolein that forms via several endogenous pathways, some of which are activated upon intoxication with xenobiotics (i.e., Exposure Category 3), while still others accompany direct physical trauma to body tissues (Exposure Category 4). Further complicating efforts to clarify the role of endogenous acrolein in human disease is the likelihood that many such syndromes are complex phenomena that resemble "chemical mixture exposures" by involving multiple toxic substances simultaneously. This Perspective contends that while recent decades have witnessed much progress in describing the deleterious effects of acrolein at the cellular and molecular levels, more work is needed to define the contributions of different acrolein sources to "real-world" health conditions in human subjects.

Influenza A infection attenuates relaxation responses of mouse tracheal smooth muscle evoked by acrolein.

The airway epithelium is an important source of relaxant mediators, and damage to the epithelium caused by respiratory tract viruses may contribute to airway hyperreactivity. The aim of this study was to determine whether influenza A-induced epithelial damage would modulate relaxation responses evoked by acrolein, a toxic and prevalent component of smoke. Male BALB/c mice were inoculated intranasally with influenza A/PR-8/34 (VIRUS-infected) or allantoic fluid (SHAM-infected). On day 4 post-inoculation, isometric tension recording studies were conducted on carbachol pre-contracted tracheal segments isolated from VIRUS and SHAM mice. Relaxant responses to acrolein (30 µM) were markedly smaller in VIRUS segments compared to SHAM segments (2 ± 1% relaxation vs. 28 ± 5%, n=14, p<0.01). Similarly, relaxation responses of VIRUS segments to the neuropeptide substance P (SP) were greatly attenuated (1 ± 1% vs. 47 ± 6% evoked by 1 nM SP, n=14, p<0.001). Consistent with epithelial damage, PGE2 release in response to both acrolein and SP were reduced in VIRUS segments (>35% reduction, n=6, p<0.01), as determined using ELISA. In contrast, exogenous PGE2 was 2.8-fold more potent in VIRUS relative to SHAM segments (-log EC50 7.82 ± 0.14 vs. 7.38 ± 0.05, n=7, p<0.01) whilst responses of VIRUS segments to the ß-adrenoceptor agonist isoprenaline were similar to SHAM segments. In conclusion, relaxation responses evoked by acrolein were profoundly diminished in tracheal segments isolated from influenza A-infected mice. The mechanism through which influenza A infection attenuates this response appears to involve reduced production of PGE2 in response to SP due to epithelial cell loss, and may provide insight into the airway hyperreactivity observed with influenza A infection.

Airborne acrolein induces keratin-8 (Ser-73) hyperphosphorylation and intermediate filament ubiquitination in bronchiolar lung cell monolayers.

The combustion product acrolein is a key mediator of pulmonary edema in victims of smoke inhalation injury. Since studying acrolein toxicity in conventional in vitro systems is complicated by reactivity with nucleophilic culture media constituents, we explored an exposure system which delivers airborne acrolein directly to lung cell monolayers at the air-liquid interface. Calu-3 lung adenocarcinoma cells were maintained on membrane inserts such that the basal surface was bathed in nucleophile-free media while the upper surface remained in contact with acrolein-containing air. Cells were exposed to airborne acrolein for 30 min before they were allowed to recover in fresh media, with cell sampling at defined time points to allow evaluation of toxicity and protein damage. After prior exposure to acrolein, cell ATP levels remained close to controls for 4h but decreased in an exposure-dependent manner by 24h. A loss of transepithelial electrical resistance and increased permeability to fluorescein isothiocyanate-labeled dextran preceded ATP loss. Use of antibody arrays to monitor protein expression in exposed monolayers identified strong upregulation of phospho-keratin-8 (Ser(73)) as an early consequence of acrolein exposure. These changes were accompanied by chemical damage to keratin-8 and other intermediate filament family members, while acrolein exposure also resulted in controlled ubiquitination of high mass proteins within the intermediate filament extracts. These findings confirm the usefulness of systems allowing delivery of airborne smoke constituents to lung cell monolayers during studies of the molecular basis for acute smoke intoxication injury.

Acrolein relaxes mouse isolated tracheal smooth muscle via a TRPA1-dependent mechanism.

Airway sensory C-fibres express TRPA1 channels which have recently been identified as a key chemosensory receptor for acrolein, a toxic and highly prevalent component of smoke. TRPA1 likely plays an intermediary role in eliciting a range of effects induced by acrolein including cough and neurogenic inflammation. Currently, it is not known whether acrolein-induced activation of TRPA1 produces other airway effects including relaxation of mouse airway smooth muscle. The aims of this study were to examine the effects of acrolein on airway smooth muscle tone in mouse isolated trachea, and to characterise the cellular and molecular mechanisms underpinning the effects of acrolein. Isometric tension recording studies were conducted on mouse isolated tracheal segments to characterise acrolein-induced relaxation responses. Release of the relaxant PGE2 was measured by EIA to examine its role in the response. Use of selective antagonists/inhibitors permitted pharmacological characterisation of the molecular and cellular mechanisms underlying this relaxation response. Acrolein induced dose-dependent relaxation responses in mouse isolated tracheal segments. Importantly, these relaxation responses were significantly inhibited by the TRPA1 antagonists AP-18 and HC-030031, an NK1 receptor antagonist RP-67580, and the EP2 receptor antagonist PF-04418948, whilst completely abolished by the non-selective COX inhibitor indomethacin. Acrolein also caused rapid PGE2 release which was suppressed by HC-030031. In summary, acrolein induced a novel bronchodilator response in mouse airways. Pharmacologic studies indicate that acrolein-induced relaxation likely involves interplay between TRPA1-expressing airway sensory C-fibres, NK1 receptor-expressing epithelial cells, and EP2-receptor expressing airway smooth muscle cells.

Chaperone heat shock protein 90 mobilization and hydralazine cytoprotection against acrolein-induced carbonyl stress.

Toxic carbonyls such as acrolein participate in many degenerative diseases. Although the nucleophilic vasodilatory drug hydralazine readily traps such species under "test-tube" conditions, whether these reactions adequately explain its efficacy in animal models of carbonyl-mediated disease is uncertain. We have previously shown that hydralazine attacks carbonyl-adducted proteins in an "adduct-trapping" reaction that appears to take precedence over direct "carbonyl-sequestering" reactions, but how this reaction conferred cytoprotection was unclear. This study explored the possibility that by increasing the bulkiness of acrolein-adducted proteins, adduct-trapping might alter the redistribution of chaperones to damaged cytoskeletal proteins that are known targets for acrolein. Using A549 lung adenocarcinoma cells, the levels of chaperones heat shock protein (Hsp) 40, Hsp70, Hsp90, and Hsp110 were measured in intermediate filament extracts prepared after a 3-h exposure to acrolein. Exposure to acrolein alone modestly increased the levels of all four chaperones. Coexposure to hydralazine (10-100 µM) strongly suppressed cell ATP loss while producing strong adduct-trapping in intermediate filaments. Most strikingly, hydralazine selectively boosted the levels of cytoskeletal-associated Hsp90, including a high-mass species that was sensitive to the Hsp90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin. Biochemical fractionation of acrolein- and hydralazine-treated cells revealed that hydralazine likely promoted Hsp90 migration from cytosol into other subcellular compartments. A role for Hsp90 mobilization in cytoprotection was confirmed by the finding that brief heat shock treatment suppressed acute acrolein toxicity in A549 cells. Taken together, these findings suggest that by increasing the steric bulk of carbonyl-adducted proteins, adduct-trapping drugs trigger the intracellular mobilization of the key molecular chaperone Hsp90.

Prior exposure to acrolein accelerates pulmonary inflammation in influenza A-infected mice.

The combustion product acrolein contributes to several smoke-related health disorders, but whether this immunomodulatory toxicant alters pulmonary susceptibility to viruses has received little attention. To study the effects of prior acrolein dosing on the severity of influenza A viral infection, male BALB/c mice received acrolein (1mg/kg) or saline (control) via oropharyngeal aspiration either 4- or 7-days prior to intranasal inoculation with either influenza A/PR/8/34 virus or vehicle. At 0, 2, 4 and 7 days post-inoculation, lung samples were assessed for histological changes while pulmonary inflammation was monitored by estimating immune cell numbers and cytokine levels in bronchoalveolar lavage fluid (BALF). After viral challenge, animals that were exposed to acrolein 4 days previously experienced greater weight loss and exhibited an accelerated inflammatory response at 2 days after viral inoculation. Thus compared to saline-pretreated, virus-challenged controls, BALF recovered from these mice contained higher numbers of macrophages and neutrophils in addition to increased levels of several inflammatory cytokines, including IL-1α, IL-1ß, IL-6, TNF, IFN-γ, KC, and MCP-1. The acrolein-induced increase in viral susceptibility was suppressed by the carbonyl scavenger bisulphite. These findings suggest acute acrolein intoxication "primes" the lung to mount an accelerated immune response to inhaled viruses.

Intermediate filament carbonylation during acute acrolein toxicity in A549 lung cells: functional consequences, chaperone redistribution, and protection by bisulfite.

Extensive protein carbonylation accompanies cellular exposure to acrolein, a ubiquitous smoke constituent implicated in life-threatening pulmonary edema in fire victims, a condition involving rapid erosion of the "watertight" properties of respiratory epithelium. Since the identities of lung epithelial proteins that sustain carbonylation by acrolein are unknown, we sought to identify significant targets in subcellular fractions from A549 cells after 30 min exposure to either subtoxic or acutely toxic acrolein concentrations (60 or 360 fmol acrolein/cell). The lower concentration mainly modified cytosolic proteins while the higher concentration also damaged nuclear, membrane, and cytoskeletal proteins. The multifunctional intermediate filament proteins vimentin, keratin-18, keratin-7 and keratin-8, were conspicuous targets. Consistent with their mechanical functions, a loss of cellular adhesive strength accompanied adduction of the two most abundant intermediate filaments in A549 cells, keratins-8 and -18. Acrolein also elicited redistribution of several chaperones (Hsp40, -70, -90, and -110) to intermediate filament fractions, suggesting chaperone-mediated autophagy contributes to the triage of acrolein-adducted proteins. The carbonyl scavenger bisulfite suppressed acrolein toxicity, intermediate filament adduction, vimentin cross-linking, Hsp90 redistribution, and loss of cellular adhesive strength, while also suppressing vimentin hyperphosphorylation. These novel observations identify intermediate filaments as key targets for the reactive smoke constituent acrolein.

Toxicity of smoke extracts towards A549 lung cells: role of acrolein and suppression by carbonyl scavengers.

The noxious 3-carbon electrophile acrolein forms on combustion of diverse organic matter including synthetic polymers such as polyethylene. While known to play a key role in smoke inhalation injury (SII), the molecular basis for the pulmonary toxicity of high dose acrolein-containing smoke is unclear. As a result, drug interventions in SII are poorly directed against pathogenetic smoke toxicants such as acrolein. The first aim of this study was to confirm a role for acrolein in the acute toxicity of smoke extracts towards A549 lung cells by monitoring adduction of known acrolein targets and the expression of acrolein-inducible genes. A second aim was to evaluate carbonyl scavengers for their abilities to protect cell targets and block smoke extract toxicity. Extracts were prepared by bubbling smoke released by smouldering polyethylene through a buffered saline-trap. Acrolein levels in the extracts were estimated via HPLC after derivatisation with 2,4-dinitrophenylhydrazine. Extracts were highly toxic towards A549 cells, eliciting greater ATP depletion than an equivalent concentration of acrolein alone. The toxicity was accompanied by pronounced carbonylation of several cytoskeletal targets, namely vimentin and keratins-7, -8 and -18. Western blotting revealed that polyethylene combustion products also upregulated several acrolein-responsive protein markers, including GADD45beta, NQO1, HMOX, Hsp70, Nur77 and Egr1. Several carbonyl scavengers (bisulfite, d-penicillamine, hydralazine and 1-hydrazinoisoquinoline) strongly attenuated smoke extract toxicity, with bisulfite suppressing both the adduction and cross-linking of intermediate filament targets. Bisulfite also suppressed the cytotoxicity of smoke extracts when detected using real-time monitoring of cellular impedance. These findings confirm a key role for acrolein in smoke cytotoxicity and suggest drugs that block acrolein toxicity deserve further investigation as possible interventions against SII.

Protein alkylation, transcriptional responses and cytochrome c release during acrolein toxicity in A549 cells: influence of nucleophilic culture media constituents.

Acrolein is a toxic combustion product that elicits apoptotic and/or necrotic cell death depending on the conditions under which exposure occurs. As a strong electrophile, side-reactions with nucleophilic media constituents seem likely to accompany study of its toxicity in vitro, but these reactions are poorly characterized. We have thus examined the effect of media composition on the toxicity of acrolein in A549 cells. Cells were exposed to acrolein in either Dulbecco's buffered saline (DBS) or F12 supplemented with various concentrations of fetal bovine serum. Cell viability was assessed using the MTT assay, while heme oxygenase-1 (HO-1) and cytoplasmic cytochrome c were measured as respective markers of transcriptional response and apoptosis. Protein damage was evaluated using the protein carbonyl assay. Compared to F12 media (with or without serum), maximal cell death as evaluated using the MTT assay, as well as adduction of intracellular proteins, occurred when cells were exposed to acrolein in DBS. In contrast, cytochrome c release was maximal in cells exposed to acrolein in serum-containing F12, conditions which inhibited protein modification and overt cell death. These findings highlight the need for careful attention to experimental conditions when conducting in vitro toxicological studies of reactive substances.

Potentialities and pitfalls accompanying chemico-pharmacological strategies against endogenous electrophiles and carbonyl stress.

The use of powerful analytical technologies to detect endogenous carbonyls formed as byproducts of oxidative cell injury has revealed that these species contribute to many human diseases. As electrophiles, they are attacked by reactive centers in cell macromolecules to form adducts, the levels of which serve as useful biomarkers of oxidative cell injury. Because the pathobiological significance of such damage is often unclear, the possibility of using low molecular weight drugs as exploratory sacrificial nucleophiles to intercept reactive carbonyls within cells and tissues is appealing. This perspective highlights the potential benefits of using carbonyl scavengers to evaluate the significance of endogenous carbonyls in particular diseases but also canvasses a number of challenges confronting this therapeutic strategy. Chief among the latter is the task of confirming that carbonyl sequestration underlies any suppression of disease symptoms elicited by these multipotent reagents, an issue needing clarification if these compounds are to command consideration as drug interventions in humans. Other problems include adverse consequences of reactions between carbonyl scavengers and important endogenous carbonyls (e.g., neurotoxicity due to pyridoxal depletion), as well as the potential for drugs to form ternary complexes with carbonylated cell proteins, raising the prospect of immunotoxicological outcomes. Strategies for moving carbonyl sequestering reagents from the laboratory bench to a clinical testing environment are discussed within the context of the search for new treatments for spinal cord injury, one of the most debilitating medical conditions sustainable by humans. This condition seems an appropriate test case for assessing carbonyl sequestering drugs given growing evidence for noxious carbonyls in the wave of neuronal cell death that follows traumatic injury to the spinal cord.

Genome-wide transcriptional responses to acrolein.

The lipid peroxidation product and environmental pollutant acrolein participates in many diseases. Because of its formation during tobacco combustion, its role in various smoking-related respiratory conditions including lung cancer has received increasing attention. As a reactive electrophile, acrolein seems likely to disrupt many biochemical pathways, but these are poorly characterized on a genome-wide basis. This study used microarrays to study short-term transcriptional responses of A549 human lung cells to acrolein, with cells exposed to 100 microM acrolein for 1, 2, or 4 h prior to RNA extraction and transcription profiling. Major pathways dysregulated by acrolein included those involved in apoptosis, cell cycle control, transcription, cell signaling, and protein biosynthesis. Although HMOX1 is a widely used marker of transcriptional responses to acrolein, this gene was the sole upregulated member of the Nrf2-driven family of antioxidant response genes. Transcript levels of several members of the metallothionein class of cytoprotective metal-chelating proteins decreased strongly in response to acrolein. Other novel findings included strong and persistent upregulation of several members of the early growth response (EGR) class of zinc finger transcription factors. Real-time PCR and Western blotting confirmed strong upregulation of a key member of this family (EGR-2), the DNA damage response gene GADD45beta, the heat shock response participant Hsp70, and also HMOX1. Consistent with changes in Nur77 mRNA levels during the microarray study, Western blotting confirmed strong Nur77 induction at the protein level, raising the possibility that this death-inducing protein contributes to the loss of cell viability during acrolein exposure. Collectively, the transcriptional response to acrolein is complex and dynamic, with future work needed to determine whether acrolein-responsive genes identified in this study contribute to cell and tissue injury in the smoke-exposed lung.

Intermolecular protein cross-linking during acrolein toxicity: efficacy of carbonyl scavengers as inhibitors of heat shock protein-90 cross-linking in A549 cells.

The smoke-borne electrophile acrolein reacts extensively with proteins, forming carbonyl-retaining Michael adducts that may be attacked by adjacent protein nucleophiles to form cross-links. Because little information is available concerning the extent of intermolecular protein cross-linking during acrolein toxicity in cells, we used an antibody against a known target for toxic carbonyls, the chaperone protein Hsp90, to detect the formation of high-mass protein complexes in acrolein-exposed A549 cells. A 3 h exposure to acrolein (0 to 200 microM) resulted in concentration-dependent formation of a single high-mass band (approx. 180 kDa). This species was detected in cells exposed to just 50 microM acrolein, a concentration that did not elicit acute cell death as assessed by measurements of cell ATP levels. The formation of cross-linked Hsp90 coincided with a rapid loss of carbonyl adducts within cells that had been subjected to a brief "pulse" exposure to a subtoxic concentration of acrolein, suggesting Michael adducts are short-lived within cells due in part to consumption during reactions with protein nucleophiles. Cross-linked Hsp90 persisted following an overnight recovery incubation, suggesting the cellular ability to repair or degrade these species is limited. Two known carbonyl scavengers, hydralazine and bisulfite, strongly protected against the ATP depletion accompanying acrolein exposure, but only the latter suppressed protein adduction and Hsp90 cross-linking. As previously shown for hydralazine, mass spectrometry studies using a model peptide indicated that bisulfite traps carbonyl groups possessed by Michael addition adducts, and such adduct-trapping reactivity appeared to contribute to the blockade of Hsp90 cross-linking in acrolein-preloaded cells. Collectively, these findings establish that formation of stable intermolecular protein cross-links accompanies exposure to acrolein. Future clarification of the chemistry underlying this damage may provide novel biomarkers of acrolein exposure.

Carboxylic acid drug-induced DNA nicking in HEK293 cells expressing human UDP-glucuronosyltransferases: role of acyl glucuronide metabolites and glycation pathways.

Glucuronidation of carboxylic-acid-containing drugs can yield reactive acyl (ester-linked) glucuronide metabolites that are able to modify endogenous macromolecules. Previous research has shown that several carboxylic acid drugs are genotoxic in isolated mouse hepatocytes, and that DNA damage is prevented by the glucuronidation inhibitor, borneol. Whether these species induce comparable genetic damage in human cells is unknown. In this study, we investigated the mechanisms of clofibric acid-induced genotoxicity in HEK293 cells expressing the human UDP-glucuronosyltransferases UGT1A3, UGT1A9, or UGT2B7, and screened three other carboxylic acid drugs for UGT-dependent genotoxicity. DNA damage was detected using the alkaline version of the comet assay. HEK293 cells were incubated for 18 h with vehicle (2.5 mM NaOH), 0.1-2.5 mM clofibric acid or 0.1-1.0 mM benoxaprofen, bezafibrate, or probenecid. To identify mechanisms underlying any observed genotoxicity, we treated UGT2B7 transfectants with 10 mM aminoguanidine, 1 mM borneol, or 2 mM desferrioxamine mesylate prior to co-incubation with 1 mM clofibric acid for 18 h. Compared to vehicle, clofibric acid, benoxaprofen, and probenecid produced significant DNA damage in all three UGT-transfected HEK293 cell lines, detectable from the lowest concentration tested. Bezafibrate caused DNA damage only at higher concentrations (1.0 mM) in UGT2B7- and UGT1A9-, but not UGT1A3-transfected cells. No drug-induced DNA damage was detected in untransfected cells, consistent with the limited glucuronidation capacity of these cells. The glycation/glycoxidation inhibitor aminoguanidine and the glucuronidation inhibitor borneol significantly decreased clofibric-acid-mediated DNA damage in UGT2B7 transfected cells by 73.5 and 94.8%, respectively. The inhibitor of transition-metal-catalyzed oxidation, desferrioxamine mesylate, had no significant effect on DNA damage. This study demonstrates the substrate-dependent role of human UGTs in the bioactivation of carboxylic acid drugs to genotoxic acyl glucuronide metabolites that are able to damage nuclear DNA via glycation and/or glycoxidation mechanisms.

Inactivation of glutathione peroxidase activity contributes to UV-induced squamous cell carcinoma formation.

Cutaneous squamous cell carcinomas (CSCC) are a common malignancy of keratinocytes that arise in sites of the skin exposed to excessive UV radiation. In the present study, we show that human SCC cell lines, preneoplastic solar keratoses (SK), and CSCC are associated with perturbations in glutathione peroxidase (GPX) activity and peroxide levels. Specifically, we found that two of three SKs and four of five CSCCs, in vivo, were associated with decreased GPX activity and all SKs and CSCCs were associated with an elevated peroxide burden. Given the association of decreased GPX activity with CSCC, we examined the basis for the GPX deficiency in the CSCCs. Our data indicated that GPX was inactivated by a post-translational mechanism and that GPX could be inactivated by increases in intracellular peroxide levels. We next tested whether the decreased peroxidase activity coupled with an elevated peroxidative burden might contribute to CSCC formation in vivo. This was tested in Gpx1(-/-) and Gpx2(-/-) mice exposed to solar-simulated UV radiation. These studies showed that Gpx2 deficiency predisposed mice to UV-induced CSCC formation. These results suggest that inactivation of GPX2 in human skin may be an early event in UV-induced SCC formation.

Modified protein carbonyl assay detects oxidised membrane proteins: a new tool for assessing drug- and chemically-induced oxidative cell injury.

INTRODUCTION: The carbonyl content of cell proteins is a useful indicator of oxidative protein damage during drug- and chemically-induced toxicities. Since a range of lipophilic carbonylating agents is produced during the membrane peroxidation that accompanies chemically-induced oxidative stress, integral membrane proteins seem especially vulnerable to adduction by these species. To develop tools for assessing such damage, this work refined a popular spectrophotometric assay so that hydrophobic and hydrophilic proteins are separated prior to carbonyl analysis. METHODS: The low cloud point properties of Triton X114 were used to resolve mouse liver extracts into fractions comprising hydrophilic and hydrophobic proteins. Following phase separation, protein precipitation and removal of unwanted detergent was achieved via extraction with toluene containing 5% trichloroacetic acid. Carbonyl groups were derivatised using 2,4-dinitrophenylhydrazine and then quantified via spectrophotometry. RESULTS: Postmitochondrial fractions from mouse liver were incubated with azo initiators that generate peroxyl radicals in respective phases of biphasic systems, namely the water-soluble initiator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) and the lipophilic initiator 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). Following resolution using Triton X114, the carbonyl content of hydrophilic proteins in controls was 1.5+/-0.1 nmol/mg protein, compared to 6.7+/-0.57 nmol/mg protein in the corresponding hydrophobic proteins (N=3, p<0.05). Exposure to AAPH for 2 h at 37 degrees C increased the carbonyl content of proteins in the hydrophilic and hydrophobic phases by 4.6- and 3.5-fold, respectively (p<0.05). AMVN produced a 3.3-fold increase in the carbonyl content of hydrophobic proteins (p<0.05) but did not alter that of aqueous phase proteins. DISCUSSION: This method allows facile resolution of proteins into hydrophobic and hydrophilic fractions prior to carbonyl determination. This modified assay method may facilitate studies of the role of oxidative protein damage in drug- and chemically-induced toxic syndromes.