Cytotoxic Lipid Peroxidation Product Research Articles

A Monocarbonyl Curcuminoid Derivative Inhibits the Activity of Human Glutathione Transferase A4-4 and Chemosensitizes Glioblastoma Cells to Temozolomide.

Human glutathione transferase A4-4 (hGSTA4-4) displays high catalytic efficiency towards 4-hydroxyalkenals and other cytotoxic and mutagenic products of radical reactions and lipid peroxidation. Its role as a target for the chemosensitization of cancer cells has not been investigated so far. In this study, the inhibitory potency of twelve selected natural products and ten monocarbonyl curcumin derivatives against hGSTA4-4 was studied. Among natural products, ellagic acid turned out to be the strongest inhibitor with an IC50 value of 0.44 ± 0.01 μM. Kinetic analysis using glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as variable substrates showed that ellagic acid behaved as a competitive inhibitor towards both GSH and CDNB, with Ki values of 0.39 ± 0.02 and 0.63 ± 0.03 μM, respectively. Among the curcumin derivatives studied, three proved to be the most potent inhibitors, in the order DM151 > DM101 > DM100, with IC50 values of 2.4 ± 0.1 μM, 12.7 ± 1.1 μΜ and 16.9 ± 0.4 μΜ, respectively. Further kinetic inhibition analysis of the most active derivative, DM151, demonstrated that this compound is a mixed inhibitor towards CDNB with inhibition constants of Ki = 4.1 ± 0.5 μM and Ki' = 0.536 ± 0.034 μM, while it is a competitive inhibitor towards GSH with a Ki = 0.98 ± 0.11 μM. Molecular docking studies were performed to interpret the differences in binding of ellagic acid and curcumin derivatives to hGSTA4-4. The in silico measured docking scores were consistent with the obtained experimental data. Hydrogen bonds appear to be the main contributors to the specific binding of monocarbonyl curcumin derivatives, while π-π stacking interactions play a key role in the enzyme-ellagic acid interaction. In vitro cytotoxicity assessment of the worst (DM148) and the best (DM151) inhibitors was performed against glioblastoma cell lines U-251 MG and U-87 MG. The results revealed that DM151 displays considerably higher cytotoxicity against both glioblastoma cell lines, while the glioblastoma cytotoxicity of DM148 was very limited. Furthermore, low and non-toxic doses of DM151 sensitized U-251 MG cells to the first-line glioblastoma chemotherapeutic temozolomide (TMZ), allowing us to propose for the first time that hGSTA4-4 inhibitors may be attractive therapeutic partners for TMZ to optimize its clinical effect in glioblastoma chemotherapy.

Carbonyl reduction of 4-oxonon-2-enal (4-ONE) by Sniffer from D. magna and D.pulex

The α, β-unsaturated aldehydes 4-oxonon-2-enal (4ONE) and 4-hydroxynon-2-enal (4HNE) are products of unsaturated fatty acids and ROS, and can be formed in lipid-rich tissues such as neurons. As strong electrophiles, both compounds react with DNA and proteins, and are capable of inactivating enzymes. However, both the human carbonyl reductase and the carbonyl reductase Drosophila melanogaster Sniffer are known to reduce 4ONE, a major lipid peroxidation product, to a less or non-toxic form. In this study, products formed during carbonyl reduction of 4ONE and 4HNE by recombinant Sniffer proteins from Daphnia magna and Daphnia pulex were investigated. A high-performance liquid chromatography analysis showed that Sniffer from D. magna converted 35.6% of 4ONE to 11.9% HNO and 23.7% 4HNE, while D. pulex converted 34.5% of this substrate to 14.8% HNO and 19.7% 4HNE. Thus, 4HNE is the main product formed from the sniffer-mediated reduction of 4ONE. The kinetic parameters obtained from the reduction of 4ONE were Km = 13.9 ± 2.1 μM, kcat = 1.53 s−1, kcat/km = 0.11 s−1 μM−1 for D. magna Sniffer and Km = 29.2 ± 4.3 μM, kcat = 0.64 s−1, kcat/km = 0.02 s−1 μM−1 for D. pulex Sniffer. These results demonstrate that Sniffer from D. magna and D. pulex are important enzymes involved in the carbonyl reductive biotransformation of 4ONE, a cytotoxic lipid peroxidation product. Noteworthy, the catalytic properties of both Daphnia Sniffer enzymes reflect previous findings with Sniffer from Drosophila melanogaster.

Lipid peroxidation and oxidative modification of proteins in tick-borne encephalitis and ixodic tick-borne borreliosis

BACKGROUND: In the South Ural region of Russia, the group of ITTB and TBE occupies the leading rank positions in the structure of infections that are ecologically associated with ixodic ticks. At the present stage, studies aimed at studying non-specific pathological biochemical processes that determine the degree of oxidative damage to cell membranes and organelle membranes, the reactivity of the body, its reserve-adaptive potential under the influence of a number of endogenous and exogenous factors are becoming particularly relevant. One of the most significant metabolic processes of this kind is the free radical oxidation of lipids and the oxidative modification of proteins.
 AIMS: To give a clinical and epidemiological characteristic and to study the role of oxidative stress in the pathogenesis of various clinical forms of tick-borne infections in an endemic region (on the example of the Chelyabinsk region).
 MATERIAL AND METHODS: The study was conducted at the clinical base of the Department of Infectious Diseases of the Southern State Medical University in the MAUZ GKB No. 8 in Chelyabinsk. The study randomly included 105 patients with tick-borne encephalitis, ixodic tick-borne borreliosis and mixed infection who were hospitalized in the hospital in the period from May to October 20182019. The diagnosis was verified by standard immunological methods (ELISA). Modern molecular genetic methods (PCR-RV) were used in the etiological diagnosis of ICD. Material for the study: leukocyte fraction and blood serum. The content of carbonyl products of oxidative modification of proteins was estimated by their reaction with 2.4-dinitrophenylhydrazine, followed by spectrophotometric registration of the interaction products-dinitrophenylhydrazones. When determining the content of primary and secondary products of lipid peroxidation, a method based on the phenomenon of rearrangement of double bonds into diene conjugates during the peroxidation of polyunsaturated fatty acids was used. Statistical processing of the obtained results was carried out by standard methods of nonparametric statistics using the Statistica 8.0 for Windows software package.
 RESULTS: In the structure of infections transmitted by ixodic ticks, ixodic tick-borne borreliosis was most often registered (50.5%), tick-borne encephalitis was observed in 44.8% of cases, mixed infection ― in 4.7%. Borrelia miyamotoi DNA was detected by PCR-RV in 8.6% of biological samples (blood serum, leukocyte fraction). When analyzing the results of oxidative modification of proteins and lipid peroxidation, a reliable picture of oxidative stress is observed, which is more pronounced in patients with ixodic tick-borne borreliosis.
 CONCLUSION: The revealed changes in the study of oxidative stress in patients with tick-borne encephalitis and ixodic tick-borne borreliosis not only indicate the prospects of developing new approaches to the pathogenetic therapy of tick-borne infections, providing for the use of antioxidants in complex therapy, but also suggest a greater effectiveness of direct-acting lipophilic antioxidants that limit diene conjugation, and thereby prevent the accumulation of secondary cytotoxic products of lipid peroxidation.

Radiation-Induced Bystander Effect: Loss of Radioprotective Capacity of Rosmarinic Acid In Vivo and In Vitro.

In radiation oncology, the modulation of the bystander effect is a target both for the destruction of tumor cells and to protect healthy cells. With this objective, we determine whether the radioprotective capacity of rosmarinic acid (RA) can affect the intensity of these effects. Genoprotective capacity was obtained by determining the micronuclei frequencies in in vivo and in vitro assays and the cell survival was determined by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) (MTT) assay in three cell lines (PNT2, TRAMPC1 and B16F10), both in direct exposure to X-rays and after the production of radiation-induced bystander effect. The administration of RA in irradiated cells produced a decrease in the frequency of micronuclei both in vivo and in vitro, and an increase in cell survival, as expression of its radioprotective effect (p < 0.001) attributable to its ability to scavenge radio-induced free radicals (ROS). However, RA does not achieve any modification in the animals receiving serum or in the cultures treated with the irradiated medium, which expresses an absence of radioprotective capacity. The results suggest that ROS participates in the formation of signals in directly irradiated cells, but only certain subtypes of ROS, the cytotoxic products of lipid peroxidation, participate in the creation of lesions in recipient cells.

Silica nanoparticle minimizes aluminium imposed injuries by impeding cytotoxic agents and over expressing protective genes in Cicer arietinum

Nanoparticles (NPs) and nano-technological applications in varied economic sectors including agriculture, accomplished great attention in last decades, worldwide. In several instances, NPs has been applied as pre-treatment or toxicant scavenging agent for promoting seed germination/ plant growth responses or for abiotic stress amelioration. In order to promote this propensity, the development of sustainable eco-friendly processes for NP production is of utmost importance. In this study, silica NPs (SiNP) were synthesized following both chemical and biological (green) procedures, and were tested for their ameliorative efficacies against aluminium (Al)-induced toxicity in Cicer arietinum. Synthesized NPs were initially characterized following standard methods such as dynamic light scattering/ zetasizer, fourier transform infrared spectroscopy and UV–vis spectroscopy. Experimental results revealed that upon Al-exposure, growth traits and plasma membrane stability of C. arietinum were severely repressed along with increased accumulations of reactive oxygen species and malondialdehyde (an outcome of lipid peroxidation reaction), with consequent decline in the activities/ expression profiles of key defensive genes. However, exogenously applied SiNPs provided tolerance to growing C. arietinum against Al-toxicity by compensating the cellular redox homeostasis via enhancing the levels/ expression patterns of antioxidants genes and reducing cytotoxic products of lipid peroxidation. Both chemical and green manufactured SiNPs were ascertained as efficient ameliorating agents against Al-stress, at least for C. arietinum, but the green synthesized were proven to be comparatively more proficient in mitigating injury symptoms, even in relatively low concentration than the chemically manufactured particles.

Acute hepatic effects of low-dose fumonisin B(1) in rats.

Adult male Wistar rats were enrolled in a study to test the acute hepatic effects of 50 mg/kg fumonisin B1 in feed for 5 days. Fumonisin B1 depressed growth and feed intake, and absolute and relative liver weight showed a significant increase. The proportions of C17:0, C18:3 n3, C22:5 n3 and C22:6 n3 fatty acids decreased in the hepatic phospholipid fraction. All proportional decreases modified the hepatocellular membrane lipids into a more rigid state. The fatty acid profile modifications were partly compensated for by endogenous glutathione (preventing the formation of conjugated dienes and trienes as initial phase lipid peroxidation indicators), while the enzymatic antioxidant defence system (glutathione peroxidase) was unaltered. In contrast, hepatic malondialdehyde, the cytotoxic product of end-phase lipid peroxidation showed a concentration increase even after 5 days of feeding. The results indicate a rather strong and rapid hepatic effect of FB1, immediately impairing membrane phospholipids, even before the enzymatic antioxidant defence is activated.

Интенсивность развития эмбрионов кур при трансовариальном введении некоторых естественных метаболитов

При промышленном инкубировании яиц зачастую возникают различные стрессовые воздействия, негативно отражающиеся на количестве выведенного здорового молодняка. Это происходит за счет чрезмерной активации свободно-радикальных частиц, что приводит к деструктивным изменениям в мембране клеток, поэтому использование экологически безопасных биологически активных веществ способно снизить интенсивность свободно-радикального окисления и увеличить жизнеспособность организма развивающихся эмбрионов. Трансовариальная обработка инкубационных яиц композицией естественных метаболитов — коламина, янтарной кислоты и серина — нивелировала воздействие липопероксидации липидов за счет увеличения антиоксидантного потенциала организма развивающихся эмбрионов. Это выразилось в увеличении содержания наиболее важных показателей антиоксидантной защиты организма: супероксиддисмутазы и пероксидазы и снижении цитотоксичных продуктов перекисного окисления липидов: малонового диальдегида и оснований Шиффа. При этом содержание транспортного белка α-фетопротеина находилось на необходимом уровне, что определило оптимальную динамику развития куриных эмбрионов. При использовании естественных метаболитов отмечено увеличение числа яиц первой по степени замыкания аллантоиса по сравнению с контролем, что является показателем правильного и полноценного развития эмбрионов кур на 11-е сутки. Вышеуказанное позволило снизить число отходов инкубации, за счет чего увеличились такие показатели, как вывод цыплят и выводимость яиц. При применении коламина, янтарной кислоты и серина отмечена оптимизация обменных процессов в организме, что выразилось в достоверном увеличении содержания глюкозы, общего белка и общих липидов, а также отмечен рост бактерицидной активности сыворотки крови и лизоцима, что является доказательством увеличения неспецифической резистентности в организме цыплят суточного возраста.

4-Hydroxynonenal induces Nrf2-mediated UCP3 upregulation in mouse cardiomyocytes

4-Hydroxy-2-nonenal (HNE) is a highly cytotoxic product of lipid peroxidation. Nevertheless, at low concentrations, it is able to mediate cell signaling and to activate protective pathways, including that of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). In addition, HNE activates uncoupling proteins (UCPs), mitochondrial inner membrane proteins that mediate uncoupling of oxidative phosphorylation and have been proposed to protect against oxidative stress. It is not known, however, whether HNE might induce UCP expression via Nrf2 to cause mitochondrial uncoupling. We investigated the effects of HNE on UCP3 expression in mouse cardiomyocytes and the involvement of Nrf2. HNE induced the nuclear accumulation of Nrf2 and enhanced UCP3 expression, effects prevented by the antioxidant N-acetylcysteine. ChIP assays indicated that Nrf2 bound to the Ucp3 promoter after HNE treatment, increasing its expression. Cardiomyocytes treated with Nrf2- or UCP3-specific siRNA were less tolerant to HNE as reflected by increased cell death, and Nrf2 siRNA prevented HNE-induced UCP3 upregulation. The treatment with HNE greatly altered cardiomyocyte bioenergetics, increasing the proton leak across the inner mitochondrial membrane and severely decreasing the maximal respiratory capacity and the respiratory reserve capacity. These findings confirm that low HNE doses activate Nrf2 in cardiomyocytes and provide the first evidence of Nrf2 binding to the Ucp3 promoter in response to HNE, leading to increased protein expression. These results suggest that the upregulation of UCP3 mediated by Nrf2 in response to HNE might be important in the protection of the heart under conditions of oxidative stress such as ischemia–reperfusion.

4-Hydroxy-2-nonenal Induces Apoptosis by Inhibiting AKT Signaling in Human Osteosarcoma Cells

The onset of lipid peroxidation within cellular membranes is associated with changes in their physiochemical properties and enzymatic dysfunction of the membrane environment. There are increasing bodies of evidence indicating that aldehydic molecules generated endogenously during the process of lipid peroxidation are causally involved in most of the pathophysiological effects associated with oxidative stress in cells and tissues. 4-Hydroxy-2-nonenal (4-HNE), among them, is believed to be largely responsible for cytopathological effects observed during oxidative stress in vivo and has achieved the status of one of the best recognized and most studied of the cytotoxic products of lipid peroxidation. Here, we reported that 4-HNE treatment may induce cell death in MG63 human osteosarcoma cells. The 4-HNE treatment could activate caspase-3 and alter the Bax/Bcl-2 apoptotic signaling. All these changes are due to the inhibition of AKT activity by 4-HNE treatment, and we also found that the p70S6K activity, downstream factors of AKT, was also blocked by 4-HNE. Our results revealed the molecular mechanism of how 4-HNE induces cell death in MG63 human osteosarcoma cells, which contributes to the clinical treatment of cancer therapy.

Design and evaluation of a photoprotective compound able to release taurine under conditions of oxidative stress

Citation: IFSCC Magazine, 11 (2008) (2) 105–112Abstract: A novel photoprotective ingredient with anti‐oxidant and electrophilic scavenging properties was designed. A structure–activity relationship study was conducted in order to optimize its bioavailability, specifically its ability to reach deep layers of the skin that may be affected by ultraviolet radiation‐induced oxidative stress. A particular characteristic of this compound is its ability to release taurine upon reaction with some reactive oxygen species. In situ taurine formation may contribute to photoprotection. Preliminary in vitro experiments provide some evidence for effective protection against UVA‐induced oxidative stress. Scavenging of the electrophilic species 4‐hydroxynonenal, a highly cytotoxic end product of membrane lipid peroxidation, seems to be 2‐oxo‐1,3‐thiazolidine major photoprotective mechanism.Keywords: antioxidant, electrophilic species scavenging, photoprotection, structure‐activity relationship, taurinePaper presented at the IFSCC Conference 2007, Amsterdam, The Netherlands

Molecular Cloning and Expression of Several New Anopheles cracens Epsilon Class Glutathione Transferases

Glutathione transferases, GSTs, are detoxification proteins that are found in most organisms. The acGSTE3-3 had the ability to conjugate 4-hydroxynonenal, a cytotoxic lipid peroxidation product. Although other Epsilon GSTs showed roles in insecticide metabolism, the acGSTE3-3 appeared to have a major role in detoxifying lipid peroxidation products conferring protection against oxidative damage.

Characterization of glutathione S-transferase of the rice leaffolder moth, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae): Comparison of its properties of glutathione S-transferases from other lepidopteran insects

An enzyme that possesses the glutathione S-transferase (GST) activity was found in the rice leaffolder moth, Cnaphalocrocis medinalis. The enzyme was purified to homogeneity for the first time by ammonium sulfate fractionation and affinity chromatography. The resultant enzyme revealed a single band with a molecular mass of 24 kDa by SDS–polyacrylamide gel electrophoresis under reduced conditions. When assayed with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, the purified GST had an optimum pH at 8.0, and was fairly stable at pH 3–10 and at temperatures below 50 °C. The enzyme was also able to conjugate glutathione to 4-hydroxynonenal, a cytotoxic lipid peroxidation product. The present GST was inhibited by fenitrothion, permethrin, and deltamethrin, suggesting that the GST could be involved in metabolizing these organophosphorus and pyrethroid insecticides.

Low-Density Lipoprotein Has an Enormous Capacity To Bind (E)-4-Hydroxynon-2-enal (HNE): Detection and Characterization of Lysyl and Histidyl Adducts Containing Multiple Molecules of HNE

(E)-4-Hydroxynon-2-enal (HNE), an electrophilic bifunctional cytotoxic lipid peroxidation product, forms covalent adducts with nucleophilic side chains of amino acid residues. HNE-derived adducts have been implicated in many pathophysiological processes including atherosclerosis, diabetes, and Alzheimer’s disease. Tritium- and deuterium-labeled HNE (d4-HNE) were used orthogonally to study adduction with proteins and individual nucleophilic groups of histidyl, lysyl, and cysteine residues. Using tritium-labeled HNE, we detected the binding of 486 molecules of HNE per low-density lipoprotein (LDL) particle, significantly more than the total number of all reactive nucleophiles in the LDL particle. This suggests the formation of adducts that incorporate multiple molecules of HNE with some nucleophilic amino acid side chains. We also found that the reaction of a 1:1 mixture of d4-HNE and d0-HNE with N-acetylhistidine, N-acetyl-Gly-Lys-OMe, or N-acetyl cysteine generates 1:1, 2:1, and 3:1 adducts, which exhibit unique mass spectral signatures that aid in structural characterization. A domino-like reaction of initial 1:1 HNE Michael adducts of histidyl or lysyl nucleophiles with multiple additional HNE molecules forms 2:1 and 3:1 adducts that were structurally characterized by tandem mass spectrometry.

Differences in Lipid Peroxidation and Cu, Zn-Superoxide Dismutase in the Hippocampal CA1 Region Between Adult and Aged Dogs

Reactive oxygen species have been long associated with oxidative stress relevant to many pathological damages. In brain, 4-hydroxy-2E-nonenal (HNE), a major cytotoxic end product of lipid peroxidation, is produced. In contrast, superoxide dismutase (SOD), one of the major antioxidant enzymes, protects neurons from oxidative stress. The aim of this study is to observe differences in the distribution of HNE and Cu,Zn-superoxide dismutase (SOD1) in the hippocampal CA1 region of adult (2-3 years of age) and aged (10-12 years of age) dogs. The HNE immunoreactivity and protein level in the CA1 region were significantly high in the aged dogs compared to those in the adult dogs. SOD1 immunoreactivity and its protein level were also higher in the aged dogs than those in the adult dogs. However, there were not significant differences in NeuN (a neuron-specific soluble nuclear antigen) immunoreactivity in CA1 neurons between the adult and aged dogs. These differences may be associated with oxidative stress in aged dogs compared to that in adult dogs.

The Cytotoxic Lipid Peroxidation Product 4-Hydroxy-2-nonenal Covalently Modifies a Selective Range of Proteins Linked to Respiratory Function in Plant Mitochondria

Plants encounter a variety of environmental stresses that affect their cellular machinery and that they adapt to on a daily basis. Lipid peroxidation is one consequence, at the cellular level, of such stresses and yields cytotoxic lipid aldehydes, including 4-hydroxy-2-nonenal (HNE), that react with specific sites in proteins, leading to diverse changes in protein function and/or stability. We have assessed the sensitivity of plant mitochondrial proteins to HNE modification, using one-dimensional and two-dimensional denaturing PAGE and blue native-PAGE coupled to immunological detection and tandem mass spectrometry identification. A select range of proteins was modified by exogenous application of HNE to mitochondria isolated from Arabidopsis cell cultures. These included a number of proteins that directly interact with the ubiquinone pool, as well as a number of soluble matrix proteins. Mitochondria isolated from cell cultures following hydrogen peroxide, antimycin A, or menadione treatment had significantly reduced respiratory capacity and elevated levels of HNE adduction to specific subsets of proteins. Targets identified included the proteins affected by direct application of HNE but also some new proteins, including a number of matrix dehydrogenases, the inner membrane adenine nucleotide translocator, and the outer membrane voltage-dependent anion channel. Degradation products of some proteins were also found to be HNE adducted, suggesting a link between HNE adduction and protein turnover. Some of the major enzyme complexes that were HNE adducted did not show demonstrable changes in their maximal activity measured with artificial acceptors, but changes did occur in associations between respiratory chain complexes following stress treatments.

The effect of intermittent and continuous heating of soybean oil at frying temperature on the formation of 4‐hydroxy‐2‐trans‐nonenal and other α‐, β‐unsaturated hydroxyaldehydes

Abstract4‐Hydroxy‐2‐trans‐nonenal (HNE) is a cytotoxic secondary lipid peroxidation product of linoleic acid. Previous investigations in this laboratory showed that HNE is formed in thermally oxidized soybean oil, which is high in linoleic acid. Continuous exposure of the oil to frying temperature (185°C) for up to 6 h graduallyincreased the formation of HNE and other polar lipophilic aldehydes. Additional investigations in this laboratory showed that HNE is absorbed into food fried in thermally oxidized oil in the same concentration as was found in the oil. In the present experiment, the effect of intermittent heating on the formation of HNE in soybean oil was compared with continuous heating. Soybean oil samples were heated either for 1 h each day for five sequential days or for 5 h continuously at 185±5°C. The thermally oxidized soybean oil samples were analyzed by HPLC for the presence of HNE and three other polar lipophilic α‐,β‐unsaturated hydroxyaldehydes: 4‐hydroxy‐2‐trans‐hexanal, 4‐hydroxy‐2‐trans‐octenal, and 4‐hydroxy‐2‐trans‐decenal. Under intermittent and continuous heating over a total of 5 h, the concentration of these compounds increased similarly. These results indicate that the formation of HNE and other hydroxyaldehydes at frying temperature is a cumulative result of oxidation of PUFA over time.

High-resolution Crystal Structure of AKR11C1 from Bacillus halodurans: An NADPH-dependent 4-Hydroxy-2,3- trans-nonenal Reductase

Aldo-keto reductase AKR11C1 from Bacillus halodurans, a new member of aldo-keto reductase (AKR) family 11, has been characterized structurally and biochemically. The structures of the apo and NADPH bound form of AKR11C1 have been solved to 1.25 A and 1.3 A resolution, respectively. AKR11C1 possesses a novel non-aromatic stacking interaction of an arginine residue with the cofactor, which may favor release of the oxidized cofactor. Our biochemical studies have revealed an NADPH-dependent activity of AKR11C1 with 4-hydroxy-2,3-trans-nonenal (HNE). HNE is a cytotoxic lipid peroxidation product, and detoxification in alkaliphilic bacteria, such as B.halodurans, plays a crucial role in survival. AKR11C1 could thus be part of the detoxification system, which ensures the well being of the microorganism. The very poor activity of AKR11C1 on standard, small substrates such as benzaldehyde or DL-glyeraldehyde is consistent with the observed, very open active site lacking a binding pocket for these substrates. In contrast, modeling of HNE with its aldehyde function suitably positioned in the active site suggests that its elongated hydrophobic tail occupies a groove defined by hydrophobic side-chains. Multiple sequence alignment of AKR11C1 with the highly homologous iolS and YqkF proteins shows a high level of conservation in this putative substrate-binding site. We suggest that AKR11C1 is the first structurally characterized member of a new class of AKRs with specificity for substrates with long aliphatic tails.

Immunodetection of aldose reductase in normal and diseased human liver.

Aldose reductase is an NADPH-dependent aldo-keto reductase best known as the rate-limiting enzyme of the polyol pathway that is implicated in the complications of diabetes. Aldose reductase appears to be involved in a variety of disease states other than diabetes, presumably due to its ability to catalyze the reduction of a broad spectrum of aldehydes, including some cytotoxic products of lipid peroxidation. Although the data regarding expression of aldose reductase in normal liver are conflicting, prior studies have suggested that the enzyme may be induced in diseased liver. The goal of these studies was to characterize expression of aldose reductase in normal and diseased human liver, using RT-PCR, Western analysis and immunohistochemistry. Aldose reductase transcripts and protein were detected at low levels in control human livers. In contrast, levels of aldose reductase mRNA and protein were increased in chronically diseased human livers. Immunohistochemistry demonstrated localization of aldose reductase in sinusoidal lining cells; dual immunofluorescence confocal microscopy with the macrophage marker, CD68, confirmed that the aldose reductase-positive sinusoidal lining cells were Kupffer cells. Abundant aldose reductase-positive, CD68-positive cells were present in the fibrous septa of cirrhotic livers, accounting for the increase in immunoreactive aldose reductase in diseased livers. Immunostaining of human lung, spleen and lymph node revealed that macrophages in those tissues also express aldose reductase. These data are the first to demonstrate that aldose reductase is expressed by human macrophages in various tissues and suggest that this enzyme may play a role in immune or inflammatory processes.

Protein-Bound 4-Hydroxy-2-Nonenal as a Marker of Oxidative Stress

There is increasing evidence that aldehydic molecules generated endogenously during the process of lipid peroxidation are causally involved in most of the pathophysiological effects associated with oxidative stress in cells and tissues. 4-Hydroxy-2-nonenal (HNE), among them, is believed to be largely responsible for cytopathological effects observed during oxidative stress in vivo and has been achieved the status of one of the best recognized and most studied of the cytotoxic products of lipid peroxidation. In the present review, I provide a comprehensive summary on HNE as a marker of oxidative stress.

Moderation of morphogenetic and oxidative stress responses in flax in vitro cultures by hydroxynonenal and desferrioxamine

Hypocotyl segments of 7-day old seedlings of flax (Linum usitatissimum L.) cultivars Atalante, Flanders, Jitka, Szegedi 30 and Super were screened for organogenesis (shoot and root induction) and embryo-like structure production. A non-destructive assay for hydroxyl radicals (*OH), utilising DMSO as a radical trap, was used to determine *OH formation during tissue culture and morphogenesis. Desferrioxamine, an inhibitor of Fenton reaction, and 4-hydroxy-2-nonenal, a cytotoxic Lipid peroxidation product, were exogenously applied to flax cultures to determine the effect of antioxidative and prooxidative status on morphogenetic responses induced through the exogenous application of plant growth regulators. Flax genotypes varied in their response to treatments after exposure to different plant hormones. Hydroxyl radical (*OH) formation correlated with morphogenetic responses and this was affected by plant hormones. Desferrioxamine and 4-hydroxy-2-nonenal also moderated morphogenetic responses and influenced hydroxyl radical formation during in vitro propagation.