Impairment of human polymorphonuclear leukocyte chemotaxis by 2,5-hexanedione

The effect of n-hexane metabolites on human polymorphonuclear leukocyte chemotaxis and luminol-dependent chemiluminescence was investigated. No effect was detected when 2-hexanol, 2-hexanone and -valerolactone were used, 2,5-hexanedione at 75 g/ml inhibited chemotaxis and a direct correlation between increasing the xenobiotic concentration and the degree of inhibition was found. Chemotactic peptide-induced chemiluminescence was not affected by 2,5-hexanedione. In order to clarify the phenomenon, plasma membrane fluidity was investigated by fluorescence polarization of the fluorescent probe trimethylammonium diphenylhexatriene. 2,5-hexanedione increased the membrane fluidity, while the other n-hexane metabolites did not change the degree of flourescence polarization. Results suggest that the cellular functions modulated by membrane-cytoskeletal organization are affected by 2,5-hexanedione also at the low concentrations.Abbreviations 2,5-Hxdn 2,5-hexanedione - 2-Hxl 2-hexanol - 2-Hxn 2-hexanone - PMN polymorphonuclear leukocyte - TMA DPH trimethylammonium diphenylhexatriene - -V1 -valerolactone     

Microbial metabolism of some 2,5-substituted thiophenes

2,5-Dialkylthiophenes are found in bitumens and crude oils, and previous studies showed that bacterial metabolism of some with a methyl substituent lead to the formation of 5-methyl-2-thiophenecarboxylic acid, which persisted in the culture medium (Fedorak PM & Peakman TM 1992 Biodegradation 2: 223–236). The objectives of this investigation were to study the further metabolism of this acid, and of two dialkylthiophenes, 2,5-diundecylthiophene and 2-(3,7-dimethyloctyl)-5-methylthiophene. Undefined, oil-degrading mixed cultures were used. 5-Methyl-2-thiophenecarboxylic acid was oxidized to 2,5-thiophenedicarboxylic acid which was identified by gas chromatography-mass spectrometry (GC-MS). This dicarboxylic acid was degraded and supported the growth of a mixed microbial population, and approximately 50% of the sulfur in this substrate was detected as sulfate in the medium at the end of the 15-day incubation time. Mixed cultures were incubated with 2,5-diundecylthiophene or 2-(3,7-dimethyloctyl)-5-methylthiophene as their sole carbon source, and at various times some of these were freezedried and the residues were treated to form methyl esters of any carboxylic acids produced. GC-MS analyses showed the presence of several dicarboxylic acids, indicating that both alkyl groups were oxidized. A small amount of the dimethyl ester of 2,5-thiophenedicarboxylic acid was detected in the culture grown on 2,5-diundecylthiophene, and 37% of the sulfur from this dialkylthiophene was detected as sulfate in the medium after 35 days of incubation.     

1,4-diazepine-2,5-dione ring formation during solid phase synthesis of peptides containing aspartic acid beta-benzyl ester.

The Fmoc-based SPPS of H-Xaa-Asp(OBzl)-Yaa-Gly-NH(2) sequences results in side reactions yielding not only aspartimide peptides and piperidide derivatives, but also 1,4-diazepine-2,5-dione-peptides. Evidence is presented to show that the 1,4-diazepine-2,5-dione derivative is formed from the aspartimide peptide. The rate of this ring transformation depends primarily on the tendency to aspartimide and piperidide formation, which is influenced by the nature of the amino acid following the aspartic acid beta-benzyl ester (Xaa). However the bulkiness of the amino acid side chain preceeding the aspartic acid beta-benzyl ester (Yaa) is also important. Under certain conditions the 1,4-diazepine-2,5-dione peptide derivative may even be formed dominantly, which is a highly undesirable side reaction in peptide synthesis, but which provides a new way for the synthesis of diazepine peptide derivatives with targeted biological or pharmacological activity.     

Effect of 2,5-hexanedione and 3,4-dimethyl-2,5-hexanedione on retrograde axonal transport in sciatic nerve

The effects of systemically introduced neurotoxic solvents 2,5-hexanedione (2,5-HD) and 3,4-dimethyl-2,5-hexanedione (DMHD) on retrograde axonal transport (RT) of¹²⁵I-labeled tetanus toxin (TT) was studied in rat and mouse sciatic nerves. The rate of retrograde transport of TT in control rat sciatic nerves was slightly higher (6.8±0.4 mm/h) than in mouse sciatic nerves (5.4±0.5 mm/h). A single high dose of 2,5-HD (1,000 mg/kg, i.p.) produced a time-dependent effect on RT in mouse sciatic nerves. 2,5-HD caused a gradual decrease in the velocity of RT (approximately 65% inhibition between 2.0–2.5 h) with a reversal to normal rate 3–5 h after the toxin administration. The effect of DMHD on RT was examined following semi-chronic treatment in rats. DMHD caused a significant decrease (approximately 50%) in the rate of TT transport, in addition, it produced weight loss and hind-limb paralysis.I had the good opportunity of being a member of Professor Alan N. Davison' research team during 1971–1977. This research paper is dedicated to his retirement.     

Urinary 2,5 hexanedione as a biomarker of n-hexane exposure

n-Hexane is a saturated aliphatic hydrocarbon widely used in industry. In most cases it is used as a mixture with hexane isomers and various others solvents in the form of commercial hexane. n-Hexane is metabolized oxidatively to a number of compounds, including 2,5-hexanedione (2,5-HD), which is eliminated through the urine and is implicated in the neurotoxic effect of this solvent. The main objective of this study was to evaluate urinary 2,5-HD as a biomarker of n-hexane exposure. The study was carried out in seven industrial units. Post-shift urine samples from 111 workers who handled commercial hexane were collected and analysed for 2,5-HD by capillary gas chromatography. Air sampling was performed in the breathing zones of the workers, and the air samples were analysed using validated methods. Monitoring individual exposures showed that n-hexane exposure varied from 5 to 70 p.p.m. (mean±SD = 15.24±2.98 p.p.m.). Significant correlation was observed between exposure to n-hexane and urinary 2,5-HD levels, with high correlation coefficients (ρ= 0.81, p = 0.000), suggesting that urinary 2,5-HD is a good biomarker of occupational exposure to n-hexane. Urinary 2,5-HD is recommended as a better tool than air monitoring in the assessment of health risk, namely the early detection of n-hexane neurotoxicity.     

Synthesis and anticonvulsant activity of new N-mannich bases derived from benzhydryl- and isopropyl-pyrrolidine-2,5-dione

Synthesis and anticonvulsant properties of 26 new N-Mannich bases of 3-benzhydryl-(5–17) and 3-isopropyl-pyrrolidine-2,5-diones (18–30) have been described. Initial anticonvulsant screening for these compounds was evaluated in mice after intraperitoneal administration in the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizures tests. The acute neurological toxicity was determined by applying the rotorod test. The in vivo results in mice showed that the majority of 3-benzhydryl-pyrrolidine-2,5-dione derivatives revealed effectiveness, while 3-isopropyl-pyrrolidine-2,5-dione derivatives were practically devoid of activity. The quantitative evaluation in both tests revealed that the most active were N-[{4-(3-chlorophenyl)-piperazin-1-yl}-methyl]-3-benzhydryl-pyrrolidine-2,5-dione (9) with ED_{5 0} value?=42.71?mg/kg (MES), ED_{5 0} value?>150?mg/kg (scPTZ), and N-[{4-(3-trifluoromethylphenyl)-piperazin-1-yl}-methyl]-3-benzhydryl-pyrrolidine-2,5-dione (13) with ED_{5 0} value?=101.46?mg/kg (MES) and ED_{5 0} value?=72.59?mg/kg (scPTZ). These molecules showed higher potency and lower neurotoxicity than the reference antiepileptic drugs (ethosuximide and valproic acid). To explain the probable mechanism of action of selected active derivatives (9 and 13), their influence on Na_v1.2 and l-type calcium channel was evaluated in vitro.     

Inhibition of glycogenolysis by 2,5-anhydro-D-mannitol in isolated rat hepatocytes

2,5-Anhydro-D-mannitol, an analog of D-fructofuranose, inhibited basal and glucagon-stimulated glycogenolysis and glucose production in hepatocytes isolated from fed rats. Glucose formation from galactose was unaffected by the inhibitor. 2,5-Anhydro-D-mannitol-1-phosphate inhibits phosphorylase alpha with a Ki value of 2.4 mM. This same phosphorylated metabolite accumulates to the extent of 9.2 mumol/g wet wt in treated hepatocytes suggesting that phosphorolysis is the locus of the inhibition of glucose production from glycogen. Our results suggest that 2,5-anhydro-D-mannitol can be used to produce a model of hereditary fructose intolerance and that it merits further study as a hypoglycemic agent.     

Morphologic changes induced in vitro by 2,5 hexanedione

Summary The effects of 2,5 hexanedione (2,5 HD), a metabolite of n-hexane, were investigated in different in vitro systems. A human mammary carcinoma cell line, a human melanoma cell line, and fetal mouse neuronal cells in primary culture were considered. Light and electron microscopic observations demonstrated clearly that changes in cell proliferation can be detected. Furthermore, morphologic differentiative phenomena characterized by a noticeable increase in cell protrusions and dendriticlike processes can occur. Differences in the features of these processes were also detected between the different cell lines. These data can indicate non-neuronal cells as possible further targets of the toxicant. The possibility could be hypothesized that toxic neuropathies are generalized disorders, being neuronal system exceptionally vulnerable to 2,5 HD. Moreover, results obtained suggest that the sensitivity of in vitro systems could represent a useful tool in studying the mechanisms of action of the neurotoxicant 2,5 HD.     

Synthesis of poly-O-sulfated glycosides of 2,5-anhydro-D-mannitol

2,5-Anhydro-3-O-beta-D-glucopyranosyl-; -3-O-alpha-L-idopyranosyl-; -3-O-alpha-D-arabinopyranosyl-; -3-O-alpha-L-arabinopyranosyl-; -3-O-beta-D-maltopyranosyl-; -3-O-beta-D-gentiobiopyranosyl-; -1,6-di-O-beta-D-glucopyranosyl-; -1,6-di-O-alpha-L-idopyranosyl-; -1-O-beta-D-maltopyranosyl-; -1,3,6-tri-O-beta-D-glucopyranosyl-; -1,6-di-O-beta-maltopyranosyl- and -1,6-di-O-beta-D-gentiobiopyranosyl-2,5-anhydro-D-mannitol as well as their poly-O-sulfated derivatives were synthesized. The IP3-IC50 values of their sodium and/or potassium salts were determined for structure-activity studies aiming at the synthesis of new, orally active antiasthmatic compounds.     

Degradation of 2,5-dichlorobenzoic acid byPseudomonas aeruginosa JB2 at low oxygen tensions

From long-term chemostat experiments, variants ofPseudomonas aeruginosa JB2 were obtained which exhibited altered properties with respect to the metabolism of 2,5-dichlorobenzoic acid (2,5-DBA). Thus, unlike the original strain JB2-WT, strain JB2-var1 is able to grow in continuous culture on 2,5-DBA as the sole limiting carbon and energy source. Yet, at a dilution rate of 0.07 h^–1 and a dissolved oxygen concentration of 12 µM, even with this strain no steady states with 2,5-DBA alone could be established in continuous cultures. Yet another strain was obtained after prolonged continuous growth of JB2-var1 in the chemostat. It has improved 2,5-DBA degrading capabilities which become apparent only during growth in continuous culture: a lower apparent K _m for 2,5-DBA and lowered steady-state residual concentrations of 2,5 DBA. Although with this strain steady states were obtained at oxygen concentrations as low as 11 µM, at further lowered concentrations this was no longer possible. In C-limited continuous cultures of JB2-var1 or JB2-var2, addition of benzoic acid (BA) to the feed reduced the amounts of 2,5-DBA degraded, which was most apparent at low oxygen concentrations (< 30 µM). At higher dissolved oxygen concentrations the addition of BA resulted in increasing cell-densities but did not affect the residual steady state concentration of 2,5-DBA. Indeed, whole cell suspensions from chemostat cultures grown on BA plus 2,5-DBA did show a lower apparent affinity for 2,5-DBA than those from cultures grown on 2,5-DBA alone. These results indicate that in environments with low oxygen concentrations and alternative, more easily degradable, substrates the degradation rates of chloroaromatic compounds by aerobic organisms may be negatively affected.Abbreviations BA benzoic acid - 2,5-DBA 2,5-dichlorobenzoic acid - QO ₂ ^max maximum specific respiration rate     

Action of Nitrile Hydratase from Rhodococcus rhodochrous IFO 15564 on Derivatives of 2,5-Anhydro-d-allononitrile

The conversion of 2,5-anhydro-d-allononitrile derivatives by a nitrile hydratase from Rhodococcus rhodochrous IFO 15564 was studied. The activity of the enzyme was strongly effected by the steric bulkiness of the substituents at the 3-position of the substrates, and the corresponding amides were obtained in high yields from the nitriles with free hydroxyl groups at the 3- and 4-positions.     

Redox status of the testes and sperm of rats following exposure to 2,5-hexanedione

Objectives: Exposure to 2,5-hexanedione (2,5-HD) is well known to be associated with reproductive dysfunctions in both humans and animals. However, the role of oxidative stress in 2,5-HD-induced toxicity in testes and sperm has not yet been studied.

Methodology: The present study investigated the influence of 2,5-HD on antioxidant systems in the testes and epididymal sperm of rats following exposure to 0, 0.25, 0.5, and 1% 2,5-HD in drinking water for 21 consecutive days.

Results: Administration of 0.5% 2,5-HD significantly (P?<?0.05) decreased epididymis weight, whereas 1% 2,5-HD-treated rats showed significantly decreased body weight, testis, and epididymis weights compared with the control group. Exposure to 2,5-HD caused a significant dose-dependent increase in the activities of superoxide dismutase, catalase, and glutathione peroxidase in both testes and sperm compared with the control group. Moreover, 2,5-HD-exposed rats showed significant decrease in glutathione-S-transferase activity and glutathione level with concomitant significant elevation in the levels of hydrogen peroxide and malondialdehyde in both testes and sperm. Testicular and epididymal atrophy with significant, dose-dependent, decrease in epididymal sperm number, sperm motility, and viability were observed in 2,5-HD-treated rats.

Conclusion: 2,5-HD exposure impaired testicular function and sperm characteristics by disruption of the antioxidant systems and consequently, increased oxidative stress in the treated rats.     

Design, synthesis, and anticonvulsant activity of N-phenylamino derivatives of 3,3-dialkyl-pyrrolidine-2,5-diones and hexahydro-isoindole-1,3-diones

In the present study on the development of new anticonvulsants, the library of differently substituted N-phenylamino pyrrolidine-2,5-dione and hexahydro-isoindole-1,3-dione derivatives was synthesized. The anticonvulsant activity of all the compounds was evaluated using the maximal electroshock (MES) and pentylenetetrazole (scPTZ) screens, which are the most widely employed seizure models for early identification of candidate anticonvulsants. Their neurotoxicity was determined applying the rotorod test. The pharmacological results revealed that the majority of compounds were effective in electrical (MES) and/or pentylenetetrazole induced seizure (scPTZ) models. The quantitative in vivo anticonvulsant evaluation of N-phenylamino-3,3-dimethyl-pyrrolidine-2,5-dione (15), conducted at the time of peak pharmacodynamic activity (TPE), showed the MES ED₅₀ value of 69.89 mg/kg in rats. The median toxic dose (TD₅₀) was 500 mg/kg, providing compound 15 with a protective index (TD₅₀/ED₅₀) of 7.15 in the MES test.     

Derivatives of 2,5-anhydroallitol and 2,5-anhydroaltritol

Two routes to protected derivatives of 2,5-anhydroallitol were investigated. The first route, involving a two-step reduction of 2,5-anhydro-6-O-benzoyl-3,4-O-isopropylidene-D-allonitrile (4), gave a mixture of 2,5-anhydro-6-O-benzoyl-3,4-O-isopropylidene-D-altritol (7) and a lesser amount of the desired 2,5-anhydro-6-O-benzoyl-3,4-O-isopropylidene-D-allitol (6). Isomerization was shown to occur in the first reduction step—treatment of the nitrile 4 with Raney nickel, sodium hypophosphite, and acetic acid. The second route gave isomerically pure 2,5-anhydro-3,4,6-tri-O-benzyl-D-allitol (21) via reduction of the corresponding ethyl allonate (18).     

In vitro binding of [14C]2,5-hexanedione to rat neuronal cytoskeletal proteins

2,5-Hexanedione (2,5-HD) induces central-peripheral axonpathy characterized by the accumulation of 10-nm neurofilaments proximal to the nodes of Ranvier and a Wallerian-type degeneration. It has been postulated that neurofilament crosslinking may be involved in the production of this axonopathy. A potential initiating event in this neurotoxic process may be the direct binding of 2,5-HD to neurofilament and microtubule proteins. In this study, the in vitro binding of [¹⁴C]2,5-HD to neurofilament and microtubule proteins was examined. Neurofilament proteins isolated from rat spinal cord or microtubule proteins isolated from rat brain were incubated in the presence of 2,5-HD at concentrations ranging 25 to 500 mM. Quantitative analysis of sodium dodecyl sulfate (SDS) polyacrylamide gels revealed a dose- and time-dependent binding of 2,5-HD to both neurofilament proteins and microtubule proteins. Expressed as pmol 2,5-HD bound per g protein, the observed relative binding was MAP2>NF160>NF200>NF68>tubulin. These data demonstrate the direct binding of 2,5-HD to cytoskeletal proteins including both neurofilaments and microtubules.     

Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase

Corynebacterium 2,5-Diketo-D-gluconic acid reductase (2,5-DKGR) catalyzes the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-Keto-L-gulonic acid (2-KLG). 2-KLG is an immediate precursor to L-ascorbic acid (vitamin C), and 2,5-DKGR is, therefore, an important enzyme in a novel industrial method for the production of vitamin C. 2,5-DKGR, as with most other members of the aldo-keto reductase (AKR) superfamily, exhibits a preference for NADPH compared to NADH as a cofactor in the stereo-specific reduction of substrate. The application of 2,5-DKGR in the industrial production of vitamin C would be greatly enhanced if NADH could be efficiently utilized as a cofactor. A mutant form of 2,5-DKGR has previously been identified that exhibits two orders of magnitude higher activity with NADH in comparison to the wild-type enzyme, while retaining a high level of activity with NADPH. We report here an X-ray crystal structure of the holo form of this mutant in complex with NADH cofactor, as well as thermodynamic stability data. By comparing the results to our previously reported X-ray structure of the holo form of wild-type 2,5-DKGR in complex with NADPH, the structural basis of the differential NAD(P)H selectivity of wild-type and mutant 2,5-DKGR enzymes has been identified.     

Modification of the cell surface expression of histocompatibility antigens induced by the neurotoxin 2,5 hexanedione

Class I histocompatibility antigens (HLA) are expressed on the surface of almost all nucleated mammalian cells; the expression of this surface antigenic molecule may be changed or abrogated by several factors. In this paper, a modification in HLA expression in a human carcinoma cell line following exposure to the neurotoxicant 2,5 hexanedione is reported. This compound is known to produce a wide spectrum of subcellular pathological events; in this study, we describe an effect on the surface and cytoplasmic distribution of both light and heavy subunits of HLA antigens, demonstrated by immunocytochemical and immunoelectron microscopy techniques. Human carcinoma cells, which under normal growing conditions express the HLA, abrogate the surface expression of this glycoprotein after exposure to 2,5 hexanedione and an intracytoplasmic accumulation seems to occur. Several possibilities are discussed, such as an effect of the toxicant on the transport of the nascent glycoprotein.Abbreviations DMEM Dulbecco's Modified Eagle's Medium - 2,5-HD 2,5 hexanedione - HLA Class I histocompatibility antigen - IU international unit     

Different Administration Schedules of the Same Dose of 2,5-Hexanedione Influence the Development of Neuropathy and the Toxicokinetics

The same total dose (1.2 g/kg/week) of 2,5-hexanedione (2,5-HD) was administered subcutaneously at 100 mg/kg/12 hr, 200 mg/kg/24 hr, and 400 mg/kg/48 hr to three groups of Donryu rats. The peripheral neuropathy induced by 2,5-HD was confirmed by clinical observation every day, and neurophysiological measurements every 4 weeks. During the 15th week of this experiment, 2,5-HD concentrations in plasma 0.5 to 24 hours after injection were determined. It was found that the greater the dose of 2,5-HD per treatment injected, the earlier peripheral neuropathy developed. Toxicokinetic analysis showed that both the values of the area under the plasma concentration versus time curve and the half life of 2,5-HD were increased, but the excretion parameters (Ke) were decreased, in animals treated with 200 mg/kg/24 hr and 400 mg/kg/48 hr 2,5-HD.     

Synthesis,in-vitro α-glucosidase inhibition,antioxidant, in-vivo antidiabetic and molecular docking studies of pyrrolidine-2,5-dione and thiazolidine-2,4-dione derivatives

α-Glucosidase is considered as a therapeutic target for the treatment of type 2 diabetes mellitus (DM2). In current study, we synthesized pyrrolidine-2,5-dione (succinimide) and thiazolidine-2,4-dione derivatives and evaluated for their ability to inhibit α-Glucosidase. Pyrrolidine-2,5-dione derivatives (11a–o) showed moderate to poor α-glucosidase inhibition. Compound 11o with the IC₅₀ value of 28.3 ± 0.28 µM emerged as a good inhibitor of α-glucosidase. Thiazolidine-2,4-dione and dihydropyrimidine (TZD-DHPM) hybrids (22a–c) showed excellent inhibitory activities. The most active compound 22a displayed IC₅₀ value of 0.98 ± 0.008 µM. Other two compounds of this series also showed activity in low micromolar range. The in-vivo antidiabetic study of three compounds 11n, 11o and 22a were also determined using alloxan induced diabetes mice model. Compounds 11o and 22a showed significant hypoglycemic effect compared to the reference drug. In-vivo acute toxicity study showed the safety of these selected compounds. In-silico docking studies were carried out to rationalize the in-vitro results. The binding modes and bioassay results of TZD-DHPM hybrids showed that interactions with important residues appeared significant for high potency.     

Time-dependent Alteration of Cytoskeletal Proteins in Cerebral Cortex of Rat During 2,5-Hexanedione-induced Neuropathy

To investigate the mechanisms of the axonopathy induced by 2,5-hexanedione (2,5-HD), male Wistar rats were administered at a dosage of 400 mg/kg/day 2,5-HD (five times per week). The rats produced a slightly, moderately, or severely abnormal neurological changes, respectively, after 2, 4, or 8 weeks of treatment. The cerebrums were Triton-extracted and ultracentrifuged to yield a pellet fraction and a corresponding supernatant fraction. The relative levels of six cytoskeletal proteins (NF-L, NF-M, NF-H, α-tubulin, β-tubulin, and β-actin) in both fractions were determined by immunoblotting. The results showed that NFs content in HD-treated rats demonstrated a progressive decline as the intoxication of HD continued. As for microtubule proteins, the levels of α-tubulin and β-tubulin demonstrated some inconsistent changes. The content of α-tubulin kept unchangeable, while the content of β-tubulin increased significantly at the late stage of HD exposure. Furthermore, the content of β-actin in both fractions remained unaffected throughout the study. These findings suggest that HD intoxication resulted in a progressive decline of NFs, which was highly correlated with the development of HD-induced neuropathy.