Carbonyl-scavenging drugs & protection against carbonyl stress-associated cell injury.

This mini-review highlights the chemical and cytoprotective properties of various hydralazine analogues that block the induction of cell death by acrolein, a highly toxic contributor to "carbonyl stress" during a diverse range of human diseases. Recent work on the action of hydralazine against various carbonyl-mediated pathologies is also reviewed.

The ability of thiourea to scavenge hydrogen peroxide and hydroxyl radicals during the intra-coronal bleaching of bloodstained root-filled teeth.

BACKGROUND: Hydrogen peroxide, an agent used in the intra-coronal bleaching of root-filled teeth for over a century, has been shown to diffuse from the pulp chamber to the outer root surface. Furthermore, it has been demonstrated that destructive hydroxyl radicals, the by-products of the bleaching process, have been detected on the external root surface. The control of such diffusion may be of importance in minimizing the risk of invasive cervical resorption (ICR) which has been linked to intra-coronal bleaching of discoloured root-filled teeth using hydrogen peroxide. The aims of the present in vitro study are to quantify the diffusion of hydrogen peroxide and hydroxyl radicals to the outer root surface following intra-coronal bleaching, and to evaluate the ability of thiourea incorporated into the bleaching protocol to scavenge residual hydrogen peroxide and hydroxyl radicals. METHODS: Thirty-five single rooted premolar teeth with intact cementum at the cemento-enamel junction were used in this project. Thirty teeth were stained with red blood cells and root-filled with gutta-percha and AH26. The five unstained teeth were root-filled and constituted a negative control (Group 1). The stained teeth were divided equally into the following experimental groups and subjected to various intra-coronal bleaching regimes: Group 2--'walking bleach' with 20 microl 30 per cent w/w hydrogen peroxide; Group 3--20 microl 30 per cent w/w hydrogen peroxide and thermocatalytically activated; Group 4--20 microl acidified thiourea; Group 5--20 microl acidified thiourea and 20 microl 30 per cent w/w hydrogen peroxide; Group 6--20 microl acidified thiourea and 20 microl one per cent sodium hypochlorite; Group 7--20 microl acidified thiourea, 20 microl one per cent sodium hypochlorite and 20 microl 30 per cent w/w hydrogen peroxide. The reaction products of the bleaching process were quantified at the outer root surface using high performance liquid chromatography and electrochemical detection (HPLC-ECD). RESULTS: Results showed that hydrogen peroxide used alone in Groups 2 and 3 was able to be detected at the outer root surface in 100 per cent of the samples, and that the presence of the hydroxyl radical generated in both groups was detected in equal amounts (P < 0.05). When thiourea was incorporated into the bleaching protocols in Groups 5-7, it was shown to scavenge both hydrogen peroxide and hydroxyl radicals to a significant degree (P < 0.05). CONCLUSIONS: Acidulated thiourea is an effective scavenger of residual hydrogen peroxide and hydroxyl radicals generated during the intra-coronal bleaching of bloodstained root-filled teeth.

Moderate acute intake of de-alcoholized red wine, but not alcohol, is protective against radiation-induced DNA damage ex vivo -- results of a comparative in vivo intervention study in younger men.

Moderate intake of wine is associated with reduced risk of cardiovascular disease and possibly cancer however it remains unclear whether the potential health benefits of wine intake are due to alcohol or the non-alcoholic fraction of wine. We therefore tested the hypothesis that the non-alcoholic fraction of wine protects against genome damage induced by oxidative stress in a crossover intervention study involving six young adult males aged 21-26 years. The participants adhered to a low plant phenolic compound diet for 48 h prior to consuming 300 mL of complete red wine, de-alcoholized red wine or ethanol on separate occasions 1 week apart. Blood samples were collected 0.5, 1.0 and 2.0 h after beverage consumption. Baseline and radiation-induced genome damage was measured using the cytokinesis-block micronucleus assay and total plasma catechin concentration was measured. Consumption of de-alcoholized red wine significantly decreased the gamma radiation-induced DNA damage at 1 and 2 h post-consumption by 20%. In contrast alcohol tended to increase radiation-induced genome damage and complete wine protected against radiation-induced genome damage relative to alcohol. The observed effects were only weakly correlated with the concentration of total plasma catechin (R=-0.23). These preliminary data suggest that only the non-alcoholic fraction of red wine protects DNA from oxidative damage but this effect cannot be explained solely by plasma catechin.

Optimisation of the comet genotoxicity assay in freshly isolated murine hepatocytes: detection of strong in vitro DNA damaging properties for styrene.

While the comet assay is used to detect DNA damage in isolated cells following exposure to chemicals in vitro, few publications report the use of the procedure in liver cells isolated from mice. Our initial efforts to use the assay to assess DNA damage in mouse hepatocytes maintained on collagen-coated dishes were hampered by high levels of baseline damage in controls, which appeared to result from mechanical damage sustained during the dislodgement of adherent cells in the early stages of the assay protocol. Here we describe an efficient version of the comet assay in cultured mouse hepatocytes that involves careful recovery of cells using a "scraping" buffer supplemented with 10% high purity grade DMSO. Use of this buffer strongly diminished the frequency of false positives. Using the industrial reagent styrene as a positive control in the optimised procedure, non-cytotoxic concentrations of this substance (2.5-10 mM) significantly increased mean comet tail length, area, and moment. Co-incubation with the CYP inhibitor SKF-525A strongly attenuated these effects of styrene. Collectively, these findings confirm this method is highly suitable for the detection of DNA damage by bioactivation-dependent compounds in freshly isolated mouse hepatocytes.

Cell-free protein synthesis inhibition assay for the cyanobacterial toxin cylindrospermopsin.

The cyanobacterial toxin cylindrospermopsin (CYN) is known to be a potent inhibitor of protein synthesis. This paper describes the use of a rabbit reticulocyte lysate translation system as a protein synthesis inhibition assay for CYN. A dose response curve for protein synthesis inhibition by CYN was constructed and was modeled to a sigmoidal dose response curve with variable slope (R2 = 0.98). In this assay, CYN has an IC50 of 120 nM [95% confidence limits (Cl) = 111-130 nM] with a detection limit in the region of 50 nM in the assay solution. Application of the assay allows quantification of toxin samples within the range 0.5-3.0 microM (200-1200 micrograms/L) CYN. To assess the usefulness of this assay, a range of toxic and nontoxic Cylindrospermopsis raciborskii extracts, including both laboratory strains and environmental samples, were assayed by protein synthesis inhibition. These CYN quantifications were then compared to quantifications obtained by high performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLCMS-MS). The results demonstrate that the protein synthesis inhibition assay correlates well with both HPLCMS-MS (r2 = 0.99) and HPLC (r2 = 0.97) quantifications. We conclude that this is an accurate and rapid assay for the measurement of cylindrospermopsin in cyanobacterial extracts.

Extensive protein carbonylation precedes acrolein-mediated cell death in mouse hepatocytes.

Allyl alcohol hepatotoxicity is mediated by an alcohol dehydrogenase-derived biotranformation product, acrolein. This highly reactive alpha,beta-unsaturated aldehyde readily alkylates model proteins in vitro, forming, among other products, Michael addition adducts that possess a free carbonyl group. Whether such damage accompanies acrolein-mediated toxicity in cells is unknown. In this work we established that allyl alcohol toxicity in mouse hepatocytes involves extensive carbonylation of a wide range of proteins, and that the severity of such damage to a subset of 18-50 kDa proteins closely correlated with the degree of cell death. In addition to abolishing cytotoxicity and glutathione depletion, the alcohol dehydrogenase inhibitor 4-methyl pyrazole strongly attenuated protein carbonylation. Conversely, cyanamide, an aldehyde dehydrogenase inhibitor, enhanced cytotoxicity and protein carbonylation. Since protein carbonylation clearly preceded the loss of membrane integrity, it may be associated with the toxic process leading to cell death.

Clofibrate pretreatment in mice confers resistance against hepatic lipid peroxidation.

Pretreatment with peroxisome proliferators protects mice against various hepatotoxicants. Since our previous work suggested that the hepatoprotection may involve an increased ability to cope with oxidative stress, the present work directly addressed this possibility. Several observations indicated a heightened defense against oxidative stress accompanies the hepatoprotection produced by clofibrate. Firstly, the carbonyl content of hepatic proteins from clofibrate-pretreated mice was 40% lower than those from vehicle-treated controls. Secondly, liver homogenates from clofibrate-pretreated mice produced less thiobarbituric acid reactive substances upon incubation under aerobic conditions or exposure to ferrous sulfate. This effect was not due to lower levels of peroxidation-prone polyunsaturated fatty acids in clofibrate-treated livers. Thirdly, in vitro experiments indicated that the antioxidant factor in liver homogenates from clofibrate-pretreated mice was not glutathione. Rather, since it was inactivated by proteases and heat treatment, we concluded that a protein is involved. Collectively, our results suggest that a resistance to lipid peroxidation develops in mouse liver during exposure to clofibrate. The identity of the putative antioxidant protein and its contribution to the protection against liver toxicity observed in this and other laboratories awaits future investigation.

The antihypertensive hydralazine is an efficient scavenger of acrolein.

Recent work indicates the highly toxic alpha,beta-unsaturated aldehyde acrolein is formed during the peroxidation of polyunsaturated lipids, raising the possibility that it functions as a 'toxicological second messenger' during oxidative cell injury. Acrolein reacts rapidly with proteins, forming adducts that retain carbonyl groups. Damage by this route may thus contribute to the burden of carbonylated proteins in tissues. This work evaluated several amine compounds with known aldehyde-scavenging properties for their ability to attenuate protein carbonylation by acrolein. The compounds tested were: (i) the glycoxidation inhibitors, aminoguanidine and carnosine; (ii) the antihypertensive, hydralazine; and (iii) the classic carbonyl reagent, methoxyamine. Each compound attenuated carbonylation of a model protein, bovine serum albumin, during reactions with acrolein at neutral pH and 37 degrees C. However, the most efficient agent was hydralazine, which strongly suppressed carbonylation under these conditions. Study of the rate of reaction between acrolein and the various amines in a protein-free buffered system buttressed these findings, since hydralazine reacted with acrolein at rates 2-3 times faster than its reaction with the other scavengers. Hydralazine also protected isolated mouse hepatocytes against cell killing by allyl alcohol, which undergoes in situ alcohol dehydrogenase-catalysed conversion to acrolein.

Role of cytochrome P450 3A in the metabolism of mefloquine in human and animal hepatocytes.

We studied mefloquine metabolism in cells and microsomes isolated from human and animal (monkey, dog, rat) livers. In both hepatocytes and microsomes, mefloquine underwent conversion to two major metabolites, carboxymefloquine and hydroxymefloquine. In human cells and microsomes these metabolites only were formed, as already demonstrated in vivo, while in other species several unidentified metabolites were also detected. After a 48 hr incubation with human and rat hepatocytes, metabolites accounted for 55-65% of the initial drug concentration, whereas in monkey and dog hepatocytes, mefloquine was entirely metabolized after 15 and 39 hrs, respectively. The consumption of mefloquine was less extensive in microsomes, and unchanged drug represented 60% (monkey) to 85-100% (human, dog, rat) of the total radioactivity after 5 hr incubations. The involvement of the cytochrome P450 3A subfamily in mefloquine biotransformation was suggested by several lines of evidence. Firstly, mefloquine metabolism was strongly increased in hepatic microsomes from dexamethasone-pretreated rats, and also in human and rat hepatocytes after prior treatment with a cytochrome P450 3A inducer. Secondly, mefloquine biotransformation in rifampycin-induced human hepatocytes was inhibited in a concentration-dependent manner by the cytochrome P450 3A inhibitor ketoconazole and thirdly, a strong correlation was found between erythromycin-N-demethylase activity (mediated by cytochrome P450 3A) and mefloquine metabolism in human microsomes (r=0.81, P < 0.05, N=13). Collectively, these findings concerning the role of cytochrome P450 3A in mefloquine metabolism may have important in vivo consequences especially with regard to the choice of agents used in multidrug antimalarial regimens.

Clofibrate-induced in vitro hepatoprotection against acetaminophen is not due to altered glutathione homeostasis.

Prior induction of peroxisome proliferation protects mice against the in vivo hepatotoxicity of acetaminophen and various other bioactivation-dependent toxicants. The mechanisms underlying such chemoresistance are poorly understood, although they have been suggested to involve alterations in glutathione homeostasis. To clarify the role of glutathione in this phenomenon, we isolated hepatocytes from mice in which hepatic peroxisome proliferation had been induced with clofibrate. The cells were incubated with a range of acetaminophen concentrations and the extent of cell killing after up to 8 h was assessed by measuring lactate dehydrogenase leakage from the cells. Hepatocytes from clofibrate-pretreated mice were much less susceptible to acetaminophen than cells from vehicle-treated controls. However, the extent of glutathione depletion during exposure to acetaminophen was similar in both cell types, as were rates of excretion of the product of glutathione-mediated detoxication of acetaminophen's quinoneimine metabolite, 3-glutathionyl-acetaminophen. The glutathione-replenishing ability of clofibrate-pretreated cells after a brief exposure to diethyl maleate also resembled that of control cells. More importantly, prior depletion of glutathione by diethyl maleate did not abolish the resistance of clofibrate-pretreated cells to acetaminophen. Taken together, these findings indicate that although glutathione-dependent pathways may contribute to hepatoprotection during peroxisome proliferation, the resistance phenomenon is not due exclusively to this mechanism.

Mutations at G:C base pairs predominate after replication of peroxyl radical-damaged pSP189 plasmids in human cells.

The mutagenicity of peroxyl radicals, important participants in lipid peroxidation cascades, was investigated using a plasmid-based mutational assay system. Double-stranded pSP189 plasmids were incubated with a range of concentrations of the water-soluble peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). Following replication in human Ad293 cells, the plasmids were screened for supF mutations in indicator bacteria. Exposure to peroxyl radicals caused strand nicking and a decrease in transfection efficiency, which was accompanied by a significant increase in supF mutants. Each of these effects was abolished in the presence of the water-soluble vitamin E analogue Trolox. Automated sequencing of 76 AAPH-induced mutant plasmids revealed that substitutions at G:C base pairs were the most common changes, accounting for 85.5% of all identified mutations. Of these, most comprised G:C-->T:A transversions (53.5%), with lesser contributions by G:C-->A:T transitions (23.9%) and G:C-->C:G transversions (22.5%). Collectively, these data confirm our previous findings concerning the spectrum of mutations produced upon bacterial replication of peroxyl radical-damaged phage DNA and extend them by showing that such damage has mutagenic consequences during replication in more complex eukaryotic systems.

Internal hazards: baseline DNA damage by endogenous products of normal metabolism.

Recent improvements in the ability to detect chemically modified bases in DNA have revealed that not only does the genetic material incur damage by foreign chemicals, but that it also sustains injury by reactive products of normal physiological processes. This review summarises current understanding of the DNA-damaging potential of various substances of endogenous origin, including oxidants, lipid peroxidation products, alkylating agents, estrogens, chlorinating agents, reactive nitrogen species, and certain intermediates of various metabolic pathways. The strengths and weaknesses of the existing database for DNA damage by each class of substance are discussed, as are future strategies for resolving the difficult question of whether endogenous chemicals are significant contributors to spontaneous mutagenesis and cancer development in vivo.

Genotoxic lipid peroxidation products: their DNA damaging properties and role in formation of endogenous DNA adducts.

The peroxidation of polyunsaturated lipids generates a range of substances that possess DNA damaging potential. This includes lipid hydroperoxides and various species that contain unpaired electrons, such as the alkoxyl and peroxyl radicals. In addition, a range of genotoxic carbonyl-containing compounds are formed, such as malondialdehyde, various 4-hydroxy-2-alkenals such as 4-hydroxynonenal and a number of 2-alkenals. It has previously been assumed that the antioxidants and electrophile scavenging enzymes existing in mammalian cells effectively protect the genetic material against these substances. However, thanks to recent analytical advances in the detection of low levels of DNA adducts, it is now evident that DNA adducts formed from a range of lipid peroxidation products are abundant in both rodent and human genomes. This suggests that the cellular defence system is not 100% efficient and that a proportion of endogenously produced lipid peroxidation products escape detoxification and cause DNA damage. This review surveys the genotoxic properties of the major classes of lipid peroxidation products, focusing on their chemistry of DNA adduction, the mutagenic properties of such damage and the evidence that it occurs in intact biological systems. Furthermore, avenues of future research that will clarify the significance of such damage to spontaneous mutagenesis and carcinogenesis are proposed and discussed.

Formation of novel C1-oxidised abasic sites in alkylperoxyl radical-damaged plasmid DNA.

We have recently shown that peroxyl radicals react with DNA to form alkali-labile sites. To further characterise these lesions, we studied their susceptibility to digestion by repair endonucleases that recognise different types of abasic sites. We found that peroxyl radical-damaged pSP189 plasmids were resistant to cleavage by T4 endonuclease V, an enzyme that incises DNA at "regular" and C4-oxidised abasic residues. In contrast, the DNA was digested by exonuclease III, an enzyme that recognises "regular" and C1-oxidised abasic sites. The presence of Trolox during exposure to peroxyl radicals reduced subsequent DNA cleavage by exonuclease III, while prior incubation of damaged plasmids with methoxyamine potentiated digestion by this enzyme. These findings suggest that peroxyl radical-induced DNA damage involves the generation of novel C1-oxidised deoxyribose residues.

Role of G-->T transversions in the mutagenicity of alkylperoxyl radicals: induction of alkali-labile sites in bacteriophage M13mp19.

The mutagenicity of peroxyl radicals, ubiquitous products of lipid peroxidation, was assessed using an in vitro M13 forward mutational assay. Single-stranded M13mp19 plasmids were incubated with a range of concentrations of the azo initiator 2,2'-azobis(2-amidinopropane) hydrochloride, and then transfected into competent, SOS-induced Escherichia coli JM105 cells. Incubation with peroxyl radicals produced a concentration-dependent decrease in phage survival, with a 500 microM concentration of the azo initiator reducing the transfection efficiency by more than 90% while inducing a corresponding 6-fold increase in lacZ alpha mutation frequencies. Peroxyl radical-induced mutagenesis was completely prevented by the peroxyl radical scavenger Trolox. Automated DNA sequence analysis of the lacZ alpha gene of 100 peroxyl radical-induced mutants revealed that the most frequent sequence changes were base pair substitutions (92/95), with G-->T transversions predominating (73/92). Alkaline treatment prior to transfection diminished the mutagenicity of damaged plasmids to a level resembling that of unmodified DNA. While abasic sites might account for the sensitivity to alkaline cleavage, the possibility that unidentified nonabasic alkaline-labile lesions also contribute to peroxyl radical mutagenesis cannot be excluded. Collectively, these findings raise the possibility that DNA damage caused by a major class of endogenous radicals contributes to one of the most common spontaneous mutational events, the G-->T transversion.

Diminished susceptibility to proteolysis after protein modification by the lipid peroxidation product malondialdehyde: inhibitory role for crosslinked and noncrosslinked adducted proteins.

The lipid peroxidation product malondialdehyde forms adducts with proteins that are detected during routine assays for protein carbonylation. To test whether this damage alters the susceptibility of a protein to proteolysis, we treated bovine serum albumin with various concentrations of malondialdehyde and examined its susceptibility to digestion by alpha-chymotrypsin. In keeping with findings concerning the consequences of protein damage by other carbonyl products of lipid peroxidation, we found that malondialdehyde-modified protein was resistant to proteolysis. Since significant protein crosslinking occurred during modification with malondialdehyde, we investigated the possibility that crosslinked proteins were acting as proteolytic inhibitors. Malondialdehyde-modified proteins were resolved into crosslinked and noncrosslinked forms and the effectiveness of both species as proteolytic antagonists was examined. While both forms of malondialdehyde-adducted proteins were more potent proteolytic inhibitors than unmodified albumin, there were no significant differences in inhibitory potency between crosslinked and noncrosslinked proteins. Our findings suggest that malondialdehyde-modification produces protease-resistant proteins without an obligatory role for crosslinking.

Genotoxicity of acyl glucuronide metabolites formed from clofibric acid and gemfibrozil: a novel role for phase-II-mediated bioactivation in the hepatocarcinogenicity of the parent aglycones?

Glucuronides formed from carboxylate-containing xenobiotics are more chemically reactive than most Phase II conjugates. However, while they have been shown to form protein adducts, their reactions with DNA have received little attention. We thus used the M13 forward mutational assay to assess the genotoxicity of acyl glucuronides formed from two widely used fibrate hypolipidemics, clofibric acid and gemfibrozil. Single-stranded M13mp19 bacteriophage DNA was incubated in pH 7.4 buffer for 16 h in the presence of 0, 1, 2.5, and 5 mM concentrations of each glucuronide as well as the respective aglycones. The modified DNA was then transfected into SOS-induced competent Escherichia coli JM105 cells and the transfection efficiency was determined after phage growth overnight at 37 degrees C. Significantly, both acyl glucuronides, but not the aglycones, caused a concentration-dependent decrease in the transfection efficiency of the DNA, with a greater than 80% decrease in phage survival produced by the 5 mM concentrations of the glucuronides. No increase in lacZa mutations accompanied the loss of phage survival. We propose that these genotoxic effects involve reactions with nucleophilic centers in DNA via a Schiff base mechanism that is analogous to the glycosylation of DNA by endogenous sugars. Since strand nicking is known to accompany such damage, we also analyzed glucuronide-treated pSP189 plasmids for strand breakages via agarose gel electrophoresis. Both clofibric acid and gemfibrozil glucuronides produced significant concentration-related strand nicking and exhibited over 10-fold greater reactivity than the endogenous glycosylating agent, glucose 6-phosphate. On the basis of these findings, the possibility that this novel bioactivation route participates in the carcinogenicity of the fibrate hypolipidemics deserves investigation.

Introduction of carbonyl groups into proteins by the lipid peroxidation product, malondialdehyde.

Incubation of model proteins with the toxic lipid peroxidation product malondialdehyde resulted in a time- and concentration-dependent increase in carbonyl contents. Carbonyl groups were detected either spectrophotometrically or immunochemically after derivatization with 2,4-dinitrophenylhydrazine. Although significant adduction occurred when modifications were performed at pH 7.0, carbonyl formation was most extensive when modifications were carried out at pH 4.0 or pH 5.0. Similarly, formation of intermolecular crosslinks was most extensive when reactions were carried out under mildly acidic conditions. Our results raise the possibility that malondialdehyde adducts contribute to the carbonyl content of proteins recovered from mammalian tissues.

Abasic sites stimulate double-stranded DNA cleavage mediated by topoisomerase II. DNA lesions as endogenous topoisomerase II poisons.

Several clinically relevant anticancer drugs induce genomic mutations and cell death by increasing topoisomerase II-mediated DNA breakage. To determine whether endogenous DNA damage also affects this cleavage event, the effects of abasic sites (the most commonly formed spontaneous DNA lesion) on topoisomerase II activity were investigated. The presence of 3 abasic sites/plasmid stimulated enzyme-mediated DNA breakage > 6-fold, primarily by enhancing the forward rate of cleavage. This corresponds to a potency that is > 2000-fold higher than that of the anticancer drug, etoposide. These findings suggest that abasic sites represent endogenous topoisomerase II poisons and imply that anticancer drugs mimic the cleavage-enhancing actions of naturally occurring DNA lesions.