Glyoxal.

The Expert Panel for Cosmetic Ingredient Safety reviewed information that has become available since their year 2000 assessment, along with updated information regarding product types, and frequency and concentrations of use, and reaffirmed their original conclusion that Glyoxal is safe for use in products intended to be applied to the nail at concentrations ≤1.25% and that the available data are insufficient to support the safety for other uses.

Investigating the molecular mechanisms of glyoxal-induced cytotoxicity in human embryonic kidney cells: Insights from network toxicology and cell biology experiments.

Glyoxal, a reactive carbonyl species, can be generated both endogenously (glucose metabolism) and exogenously (cigarette smoke and food system). Increasing evidence demonstrates that glyoxal exacerbates the development and progression of diabetic nephropathy, but the underlying mechanisms of glyoxal toxicity to human embryonic kidney (HEK293) cells remain unclear. In this work, the molecular mechanisms of glyoxal-induced cytotoxicity in HEK293 cells were explored with network toxicology and cell biology experiments. Network toxicology results showed that oxidative stress and advanced glycation end products (AGEs)/RAGE signaling pathways played a crucial role in glyoxal toxicity. Next, further validation was performed at the cellular level. Glyoxal activated the AGEs-RAGE signaling pathway, caused the increase of cellular ROS, and activated the p38MAPK and JNK signaling pathways, causing cellular oxidative stress. Furthermore, glyoxal caused the activation of the NF-κB signaling pathway and increased the expression of TGF-ß1, indicating that glyoxal caused cellular inflammation. Moreover, glyoxal caused cellular DNA damage accompanied by the activation of DNA damage response pathways. Finally, the mitochondrial apoptosis pathway was activated. The results that obtained in cell biology were consistent with network toxicology, which corroborated each other and together indicated that glyoxal induced HEK293 cells damage via the process of oxidative stress, the AGEs-RAGE pathway, and their associated signaling pathways. This study provides the experimental basis for the cytotoxicity of glyoxal on HEK293 cells.

An in vitro cell model to study microglia activation in diabetic retinopathy.

Microglial activation has been studied extensively in diabetic retinopathy. We have previously detected activation and migration of microglia in 8-week-old diabetic rat retinas. It is widely acknowledged that microglia-mediated inflammation contributes to the progression of diabetic retinopathy. However, existing cell models do not explore the role of activated microglia in vitro. In this study, microglia were subject to various conditions mimicking diabetic retinopathy, including high glucose, glyoxal, and hypoxia. Under high glucose or glyoxal treatment, microglia demonstrated only partially functional changes, while under hypoxia, microglia became fully activated showing enlarged cell bodies, enhanced migration and phagocytosis as well as increased production of pro-inflammatory factors such as cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), and inducible nitric oxide synthase (iNOS). The data indicate that hypoxia-treated microglia is an optimal in vitro model for exploration of microglia activation in diabetic retinopathy.

Glyoxal damages human aortic endothelial cells by perturbing the glutathione, mitochondrial membrane potential, and mitogen-activated protein kinase pathways.

BACKGROUND: Exposure to glyoxal, the smallest dialdehyde, is associated with several diseases; humans are routinely exposed to glyoxal because of its ubiquitous presence in foods and the environment. The aim of this study was to examine the damage caused by glyoxal in human aortic endothelial cells. METHODS: Cell survival assays and quantitative fluorescence assays were performed to measure DNA damage; oxidative stress was detected by colorimetric assays and quantitative fluorescence, and the mitogen-activated protein kinase pathways were assessed using western blotting. RESULTS: Exposure to glyoxal was found to be linked to abnormal glutathione activity, the collapse of mitochondrial membrane potential, and the activation of mitogen-activated protein kinase pathways. However, DNA damage and thioredoxin oxidation were not induced by dialdehydes. CONCLUSIONS: Intracellular glutathione, members of the mitogen-activated protein kinase pathways, and the mitochondrial membrane potential are all critical targets of glyoxal. These findings provide novel insights into the molecular mechanisms perturbed by glyoxal, and may facilitate the development of new therapeutics and diagnostic markers for cardiovascular diseases.

Lovastatin attenuates glyoxal-induced toxicity on rat liver mitochondria.

Alpha-dicarbonyls such as glyoxal (GO) trigger mitochondrial dysfunction resulting in the development of different diabetic complications. The present study investigated the effects of lovastatin against GO-induced toxicity on rat liver mitochondria. The rat liver mitochondria (0.5 mg protein/mL) were treated with various concentrations of lovastatin (1, 5, 10 µM) at 37°C for 30 min and then exposed to GO (3 mM) at 37°C for 30 min. Oxidative stress markers including MDA, reactive oxygen species (ROS), glutathione (GSH) and protein carbonylation (PC) level were measured. Mitochondrial complex II activity and mitochondrial membrane potential (MMP) were assessed for evaluating mitochondrial function. Glyoxal significantly increased the level of ROS, PC and MDA. This effect was associated with the reduction of MMP, complex II activity and GSH content. Pre-treatment with lovastatin potentially reversed GO-induced mitochondrial toxicity. These results suggest that lovastatin have a protective effect against GO-induced toxicity in isolated rat liver mitochondria.

Protective effects of dissolved molecular hydrogen against hydrogen peroxide-, hydroperoxide-, and glyoxal-induced injuries to human skin keratinocytes.

Molecular hydrogen (H2) is recognized as a gaseous antioxidant, and it is expected to ameliorate various disorders related to oxidative stress and inflammation. However, there are still many unclear points regarding its effectiveness in the skin. Therefore, the purpose of this study was to examine the protective effect of H2 against ultraviolet (UV) irradiation-related stress injury in human epidermal HaCaT cells. We investigated the effects of H2 against three types of UV-derived oxidative stress using human skin keratinocytes: hydrogen peroxide (H2O2)-induced oxidative stress, tert-butyl hydroperoxide (t-BuOOH)-induced lipid peroxidation stress, and glyoxal-induced carbonyl stress. Our results showed that H2 exerted cytoprotective effects against stress induced by H2O2, t-BuOOH, and glyoxal. Furthermore, our results also revealed that H2 suppressed H2O2-induced increases in intracellular peroxide and H2O2 levels, and suppressed the progression of lipid peroxidation. Taken together, our results demonstrate that H2 can exert protective effects against oxidative stress-, lipid peroxidation-, and carbonyl stress-induced cellular injuries in human keratinocytes, partly mediated via suppression of intracellular oxidative stress and peroxide generation. Therefore, H2 is expected to be utilized as an effective and attractive component in cosmetic formulations in the future.

Protective actions of bioactive flavonoids chrysin and luteolin on the glyoxal induced formation of advanced glycation end products and aggregation of human serum albumin: In vitro and molecular docking analysis.

The post-translational modification of proteins by nonenzymatic glycation (NEG) and the accumulation of AGEs are the two underlying factors associated with the long-term pathogenesis in diabetes. Glyoxal (GO) is a reactive intermediate which has the ability to modify proteins and generate AGEs at a faster rate. Human serum albumin (HSA) being the most abundant serum protein has a higher chance to be modified by NEG. The key objective of the present study is to investigate the potency of chrysin and luteolin as antiglycating and antifibrillating agents in the GO-mediated glycation and fibril formation of HSA. AGEs formation were confirmed from the absorption and fluorescence spectral measurements. Both the flavonoids were able to quench the AGEs fluorescence intensity in vitro indicating the antiglycating nature of the molecules. The formation of fibrils in the GO-modified HSA was confirmed by the Thioflavin T (ThT) fluorescence assay and the flavonoids were found to exihibit the antifibrillation properties in vitro. Docking results suggested that both the flavonoids interact with various amino acid residues of subdomain IIA including glycation prone lysines and arginines via non-covalent forces and further stabilized the structure of HSA, which further explains their mechanisms of action as antiglycating and antifibrillating agents.

The transcription factor NemR is an electrophile-sensing regulator important for the detoxification of reactive electrophiles in Acinetobacter nosocomialis.

NemR is an electrophile-sensing regulator which controls two enzymes required for the detoxification of reactive electrophiles: N-ethylmaleimide (NEM) reductase and glyoxalase I in Escherichia coli. Both enzymes are essential for bacterial survival in the presence of toxic reactive electrophiles, such as N-ethylmaleimide and methyl glyoxal. Here, we report the identification and characterization of NemR from Acinetobacter nosocomialis, a nosocomial pathogen. We confirmed that nemR and the nemA gene which encodes N-ethylmaleimide reductase form a single operon, which is in accordance with the reports from E. coli. Bioinformatic analysis revealed the presence of an NemR binding motif in the promoter regions of nemRA operon and gloA (encoding glyoxalase I) and the binding was confirmed by gel mobility shift assay. The deletion of nemR resulted in increased biofilm/pellicle formation in A. nosocomialis. mRNA expression analysis revealed that NemR acts as a repressor of the nemRA operon and gloA, and that the repressor function is inactivated by the addition of toxic Cys modification agents, contributing to bacterial survival. In addition, it was demonstrated that the nemRA operon is positively regulated by the quorum sensing regulator, AnoR and the operon plays a role in biofilm/pellicle formation in A. nosocomialis.

Opposing roles of Y-family DNA polymerases in lipid peroxide mutagenesis at the hisG46 target in the Ames test.

DNA polymerases play a key role in mutagenesis by performing translesion DNA synthesis (TLS). The Y-family of DNA polymerases comprises several evolutionarily conserved families, specializing in TLS of different DNA adducts. Exocyclic etheno and propano DNA adducts are among the most common endogenous DNA lesions induced by lipid peroxidation reactions triggered by oxidative stress. We have investigated the participation of two enterobacterial representatives of the PolIV and PolV branches of Y-family DNA polymerases in mutagenesis by two model lipid peroxidation derived genotoxins, glyoxal and crotonaldehyde. Mutagenesis by the ethano adduct (glyoxal-derived) and the propano adduct (crontonaldehyde-derived) at the GC target in the Ames test depended exclusively on PolV type DNA polymerases such as PolRI. In contrast, PolIV suppressed glyoxal and, even more, crotonaldehyde mutagenesis, as detected by enzyme overexpression and gene knockout approaches. We propose that DNA polymerase IV, which is the mammalian DNA polymerase κ ortholog, acts as a housekeeper protecting the genome from lipoxidative stress.

Glyoxal-induced exacerbation of pruritus and dermatitis is associated with staphylococcus aureus colonization in the skin of a rat model of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a highly pruritic, chronic inflammatory skin disease associated with hyperreactivity to environmental triggers. Among those, outdoor air pollutants such as particulate matter (PM) have been reported to aggravate pre-existing AD. However, underlying mechanisms of air pollution-induced aggravation of AD have hardly been studied. OBJECTIVE: To investigate the molecular mechanisms by which glyoxal, a PM-forming organic compound, exacerbates the symptoms of AD induced by neonatal capsaicin treatment. METHODS: Naïve and AD rats had been exposed to either fresh air or vaporized glyoxal for 5 weeks (2 h/day and 5 days/week) since one week of age. Pruritus and dermatitis were measured every week. The skin and blood were collected and immunological traits such as Staphylococcus aureus skin colonization, production of antimicrobial peptides and immunoglobulin, and mRNA expression of inflammatory cytokines were analyzed. RESULTS: Exposure to glyoxal aggravated pruritus and dermatitis in AD rats, but did not induce any symptoms in naïve rats. Staphylococcus aureus skin colonization was increased in the skin of both naïve and AD rats. Expression of antimicrobial peptides such as LL-37 and ß-defensin-2 was also increased by exposure to glyoxal in the skin of both naïve and AD rats. The mRNA expression of Th1-related cytokines was elevated on exposure to glyoxal. However, serum immunoglobulin production was not significantly changed by exposure to glyoxal. CONCLUSION: In AD rats, exposure to glyoxal exacerbated pruritus and cutaneous inflammation, which was associated with increased colonization of S. aureus and subsequent immunological alterations in the skin.

Glyoxal toxicity in isolated rat liver mitochondria.

Glyoxal is a physiological metabolite formed by lipid peroxidation, ascorbate autoxidation, oxidative degradation of glucose, and degradation of glycated proteins. Glyoxal has been linked to oxidative stress and can cause a number of cellular damages, including covalent modification of amino and thiol groups of proteins to form advanced glycation end products. However, the mechanism of glyoxal toxicity has not been fully understood. In this study, we have focused on glyoxal toxicity in isolated rat liver mitochondria. Isolated mitochondria (0.5 mg protein per milliliter) were prepared from the Wistar rat liver using differential centrifugation and incubated with various concentrations of glyoxal (1, 2.5, 5, 7.5, and 10 mM) for 30 min. The activity of mitochondrial complex II was determined by measurement of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) conversion. The mitochondrial membrane potential (MMP), lipid peroxidation (MDA), reactive oxygen species (ROS) formation, glutathione (GSH) content, and protein carbonylation were also assessed. After an incubation of isolated liver mitochondria with glyoxal, disrupted electron transport chain, increased mitochondrial ROS formation, lipid peroxidation, mitochondrial membrane damage, GSH oxidation, and protein carbonylation ensued as compared to the control group ( p < 0.05). Glyoxal toxicity in isolated rat liver mitochondria was dose-dependent. In conclusion, glyoxal impaired the electron transport chain, which is the cause of increased ROS and MDA production, depletion of GSH, and disruption of MMP. Mitotoxicity of glyoxal might be related to the pathomechanisms involved in diabetes and its complications.

An Eco-Safety Assessment of Glyoxal-Containing Cellulose Ether on Freeze-Dried Microbial Strain, Cyanobacteria, Daphnia, and Zebrafish.

The objective of this study was to investigate the aquatic-toxic effects of glyoxal-containing cellulose ether with four different glyoxal concentrations (0%, 1.4%, 2.3%, and 6.3%) in response to global chemical regulations, e.g., European Union Classification, Labeling and Packaging (EU CLP). Toxicity tests of glyoxal-containing cellulose ether on 11 different microbial strains, Microcystis aeruginosa, Daphnia magna, and zebrafish embryos were designed as an initial stage of toxicity screening and performed in accordance with standardized toxicity test guidelines. Glyoxal-containing cellulose ether showed no significant toxic effects in the toxicity tests of the 11 freeze-dried microbial strains, Daphnia magna, and zebrafish embryos. Alternatively, 6.3% glyoxal-containing cellulose ether led to a more than 60% reduction in Microcystis aeruginosa growth after 7 days of exposure. Approximately 10% of the developmental abnormalities (e.g., bent spine) in zebrafish embryos were also observed in the group exposed to 6.3% glyoxal-containing cellulose ether after 6 days of exposure. These results show that 6.3% less glyoxal-containing cellulose ether has no acute toxic effects on aquatic organisms. However, 6.3% less glyoxal-containing cellulose ether may affect the health of aquatic organisms with long-term exposure. In order to better evaluate the eco-safety of cellulosic products containing glyoxal, further studies regarding the toxic effects of glyoxal-containing cellulose ether with long-term exposure are required. The results from this study allow us to evaluate the aquatic-toxic effects of glyoxal-containing cellulosic products, under EU chemical regulations, on the health of aquatic organisms.

Glyoxal administration induces formation of high molecular weight aggregates of hemoglobin exhibiting amyloidal nature in experimental rats: An in vivo study.

Glyoxal, a highly reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs). In the present study, we have investigated the effect of glyoxal on experimental rat hemoglobin in vivo after external administration of the α-dicarbonyl compound in animals. Gel electrophoretic profile of hemolysate collected from glyoxal-treated rats (32mg/kg body wt. dose) after one week exhibited the presence of some high molecular weight protein bands that were found to be absent for control, untreated rats. Mass spectrometric and absorption studies indicated that the bands represented hemoglobin. Further studies revealed that the fraction exhibited the presence of intermolecular cross ß-sheet structure. Thus glyoxal administration induces formation of high molecular weight aggregates of hemoglobin with amyloid characteristics in rats. Aggregated hemoglobin fraction was found to exhibit higher stability compared to glyoxal-untreated hemoglobin. As evident from mass spectrometric studies, glyoxal was found to modify Arg-30ß and Arg-31α of rat hemoglobin to hydroimidazolone adducts. The modifications thus appear to induce amyloid-like aggregation of hemoglobin in rats. Considering the increased level of glyoxal in diabetes mellitus as well as its high reactivity, the above findings may be physiologically significant.

The food processing contaminant glyoxal promotes tumour growth in the multiple intestinal neoplasia (Min) mouse model.

Glyoxal is formed endogenously and at a higher rate in the case of hyperglycemia. Glyoxal is also a food processing contaminant and has been shown to be mutagenic and genotoxic in vitro. The tumourigenic potential of glyoxal was investigated using the multiple intestinal neoplasia (Min) mouse model, which spontaneously develops intestinal tumours and is susceptible to intestinal carcinogens. C57BL/6J females were mated with Min males. Four days after mating and throughout gestation and lactation, the pregnant dams were exposed to glyoxal through drinking water (0.0125%, 0.025%, 0.05%, 0.1%) or regular tap water. Female and male offspring were housed separately from PND21 and continued with the same treatment. One group were only exposed to 0.1% glyoxal from postnatal day (PND) 21. There was no difference in the number of intestinal tumours between control and treatment groups. However, exposure to 0.1% glyoxal starting in utero and at PND21 caused a significant increase in tumour size in the small intestine for male and female mice in comparison with respective control groups. This study suggests that glyoxal has tumour growth promoting properties in the small intestine in Min mice.

Identification of methylglyoxal as a major mutagen in wood and bamboo pyroligneous acids.

To identify the major mutagen in pyroligneous acid (PA), 10 wood and 10 bamboo pyroligneous acids were examined using the Ames test in Salmonella typhimurium strains TA100 and TA98. Subsequently, the mutagenic dicarbonyl compounds (DCs), glyoxal, methylglyoxal (MG), and diacetyl in PA were quantified using high-performance liquid chromatography, and the mutagenic contribution ratios for each DC were calculated relative to the mutagenicity of PA. Eighteen samples were positive for mutagens and showed the strongest mutagenicity in TA100 in the absence of S9 mix. MG had the highest mutagenic contribution ratio, and its presence was strongly correlated with the specific mutagenicity of PA. These data indicate that MG is the major mutagen in PA.

Mass Spectrometric Analysis of Glyoxal and Methylglyoxal-Induced Modifications in Human Hemoglobin from Poorly Controlled Type 2 Diabetes Mellitus Patients.

Glyoxal and methylglyoxal are oxoaldehydes derived from the degradation of glucose-protein conjugates and from lipid peroxidation, and they are also present in the environment. This study investigated the site-specific reaction of glyoxal and methylglyoxal with the amino acid residues on human hemoglobin using a shot-gun proteomic approach with nanoflow liquid chromatography/nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS). In human hemoglobin incubated with glyoxal, modification on 8 different sites, including lysine residues at α-Lys-11, α-Lys-16, α-Lys-56, ß-Lys-17, ß-Lys-66, ß-Lys-144, and arginine residues at α-Arg-92 and ß-Arg-30, was observed using a data-dependent scan. In methylglyoxal-treated hemoglobin, there were specific residues, namely, α-Arg-92, ß-Lys-66, ß-Arg-30, and ß-Lys-144, forming carboxyethylation as well as the dehydrated product hydroimidazolone at α-Arg-92 and ß-Arg-30. These lysine and arginine modifications were confirmed by accurate mass measurement and the MS(2) and MS(3) spectra. The most intensive signal of each modified peptide was used as the precursor ion to perform the product ion scan. The relative extent of modifications was semiquantified simultaneously relative to the native reference peptide by nanoLC-NSI/MS/MS under the selected reaction monitoring (SRM) mode. The extent of these modifications increased dose-dependently with increasing concentrations of glyoxal or methylglyoxal. Six out of the eight modifications induced by glyoxal and three out of the six modifications induced by methylglyoxal were detected in hemoglobin freshly isolated from human blood samples. The relative extent of modification of these post-translational modifications was quantified in poorly controlled type 2 diabetes mellitus patients (n = 20) and in nondiabetic control subjects (n = 21). The results show that the carboxymethylated peptides at α-Lys-16, α-Arg-92, ß-Lys-17, ß-Lys-66, and the peptide at α-Arg-92 with methylglyoxal-derived hydroimidazolone are significantly higher in diabetic patients than in normal individuals (p value <0.05). This report identified and quantified glyoxal- and methylglyoxal-modified hemoglobin peptides in humans and revealed the association of the extent of modifications at specific sites with T2DM. Only one drop (10 µL) of fresh blood is needed for this assay, and only an equivalent of 1 µg of hemoglobin was analyzed by the nanoLC-NSI/MS/MS-SRM system. These results suggest the potential use of these specific post-translational modifications in hemoglobin as feasible biomarker candidates to assess protein damage induced by glyoxal and methylglyoxal.

Structure- and concentration-specific assessment of the physiological reactivity of α-dicarbonyl glucose degradation products in peritoneal dialysis fluids.

In peritoneal dialysis (PD), glucose degradation products (GDPs), which are formed during heat sterilization of dialysis fluids, lead to structural and functional changes in the peritoneal membrane, which eventually result in the loss of its ultrafiltration capacity. To determine the molecular mechanisms behind these processes, the present study tested the influence of the six major α-dicarbonyl GDPs in PD fluids, namely, glyoxal, methylglyoxal, 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxyglucosone-3-ene (3,4-DGE), and glucosone with respect to their potential to impair the enzymatic activity of RNase A as well as their effects on cell viability. For comprehensive risk assessment, the α-dicarbonyl GDPs were applied separately and in concentrations as present in conventional PD fluids. Thus, it was shown that after 5 days, glucosone impaired RNase A activity most distinctly (58% remaining activity, p < 0.001 compared to that of the control), followed by 3,4-DGE (62%, p < 0.001), 3-DGal (66%, p < 0.001), and 3-DG (76%, p < 0.01). Methylglyoxal and glyoxal caused weaker inactivation with significant effects only after 10 days of incubation (79%, 81%, p < 0.001). Profiling of the advanced glycation end products formed during the incubation of RNase A with methylglyoxal revealed predominant formation of the arginine modifications imidazolinone, CEA/dihydroxyimidazoline, and tetrahydropyrimidine at Arg10, Arg33, Arg39, and Arg85. Particularly, modification at Arg39 may severely affect the active site of the enzyme. Additionally, structure- and concentration-specific assessment of the cytotoxicity of the α-dicarbonyl GDPs was performed. Although present at very low concentration, the cytotoxic effect of PD fluids after 2 days of incubation was exclusively caused by 3,4-DGE (14% cell viability, p < 0.001). After 4 days of incubation, 3-DGal (13% cell viability, p < 0.001), 3-DG (24%, p < 0.001), and, to a lower extent, glyoxal and methylglyoxal (both 57%, p < 0.01) also reduced cell viability significantly. In conclusion, 3,4-DGE, 3-DGal, and glucosone appear to be the most relevant parameters for the biocompatibility of PD fluids.

Cytotoxicity of α-dicarbonyl compounds submitted to in vitro simulated digestion process.

α-Dicarbonyl compounds (α-DCs), such as glyoxal, methylglyoxal and 2,3-butanedione, are highly reactive substances occurring in thermally treated and fermented foods, that may react with amino and sulphydryl groups of side chains of proteins to form Maillard reaction end products, inducing a negative impact on the digestibility and on nutritional value of protein. In recent years the role of food derived α-DCs in gastroduodenal tract is under investigation to understand whether excess consumption of such dietary compounds might be a risk for human health. In this study the interactions between a mixture of glyoxal, methylglyoxal and 2,3-butanedione and the digestive enzymes (pepsin and pancreatin) were studied. The results showed that during gastroduodenal digestion α-DCs react with digestive enzymes to produce carbonylated proteins. Moreover, undigested and digested α-DC cytotoxicity against human cells, as well as their ability to inhibit the function of human enzymes responsible for DNA repair were shown.

Mechanisms of methimazole cytotoxicity in isolated rat hepatocytes.

Methimazole is an antithyroid drug widely used in the treatment of hyperthyroidism. Administration of this drug, often in a chronic manner, is associated with several adverse drug reactions in humans, including life-threatening hepatotoxicity. This study attempted to investigate the cytotoxic mechanism(s) of methimazole toward isolated rat hepatocytes. In addition, the role of proposed methimazole intermediary metabolites, such as N-methylthiourea and glyoxal, in the toxicity induced by this drug was evaluated. Isolated hepatocytes were prepared by the collagenase enzyme perfusion method. Cells were treated with methimazole, N-methylthiourea, and other chemicals and markers, such as cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) formation, lipid peroxidation (LPO), and cellular glutathione (GSH) content, were measured. Methimazole-induced cytotoxicity was accompanied by collapse in MMP, increase in ROS formation, and LPO. Further, methimazole caused reduction in GSH reservoirs, and the cytotoxic effect of the drug was much more severe in GSH-depleted cells. N-methylthiourea caused toxicity in lower concentrations than methimazole and reduced hepatocytes glutathione content. The specific flavin-containing monooxygenase inhibitor, N,N-dimethylaniline, attenuated toxicity induced by N-methylthiourea. Administration of glyoxal trapping agents, such as metformin, hydralazine, or N-acetyl cysteine, effectively prevented methimazole toxicity in intact or GSH-depleted rat hepatocytes. This study indicates that methimazole reactive metabolites are responsible for the cytotoxicity induced by this drug, but the role of glyoxal as a metabolite, which causes ROS formation, LPO, and mitochondrial injury, is predominant because the glyoxal-trapping agents diminished these adverse effects.

Differences in glyoxal and methylglyoxal metabolism determine cellular susceptibility to protein carbonylation and cytotoxicity.

Chronic hyperglycemia in diabetic patients often leads to chronic side effects associated with protein glycation and the formation of reactive carbonyl species, such as methylglyoxal (MGO) and glyoxal (GO). We have shown that both MGO and GO carbonylated bovine serum albumin (BSA) in vitro to the same degree and stability. The carbonylated BSA formed initially could be a reversible Schiff base as the UV absorbance formed after the addition of 2,4-dinitrophenylhydrazine was decreased when sodium borohydride was added. MGO and GO also carbonylated hepatocyte protein rapidly with similar dose and time dependence. In contrast to BSA carbonylation, the amount of carbonylated proteins in hepatocytes decreased over time, much more rapidly for hepatocytes treated with MGO than with GO. This could be attributed to the rapid hepatocyte metabolism of MGO with glyoxalase I, the predominant detoxification enzyme for MGO. Protein carbonylation and the associated toxicity caused by GO and MGO were studied in the following hepatocyte models: (1) control hepatocytes, (2) glutathione (GSH)-depleted hepatocytes, (3) mitochondrial aldehyde dehydrogenase (ALDH2)-inhibited hepatocytes, (4) hepatocyte inflammation model, and (5) catalase-inhibited hepatocyte model. Carbonylation and cytotoxicity caused by MGO or GO was markedly increased in GSH-depleted hepatocytes as compared to control hepatocytes. Hepatocytes exposed to non-toxic concentrations of H(2)O(2) or hepatocytes treated with catalase inhibitors also showed a marked increase in GO-caused cytotoxicity and protein carbonylation, whereas there were only minor increases with MGO. The GO effect was attributed to potential radical formation and the inhibition effect of H(2)O(2) on aldehyde dehydrogenase, a major GO metabolising enzyme. GO-caused cytotoxicity and protein carbonylation were also increased with ALDH2-inhibited hepatocytes whereas such an increase was only observed with MGO in GSH-depleted hepatocytes.