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Neural specificity of acrylamide action upon enzymes associated with oxidative energy-producing pathways. I. Histochemical analysis of NADH-tetrazolium reductase activity.

The mechanism by which acrylamide (ACR) produces a distal neurofilamentous axonopathy has not been determined. Our investigations are focused upon the effects of this toxicant on energy transformations; more specifically, on enzymes of oxidative metabolism. The current study determines the effects of ACR on NADH-tetrazolium reductase (TR) activity (composite of primarily lipoamide and NADH dehydrogenases) in a variety of neural and non-neural tissues in order to test for a correlation between the neural specificity of enzyme inhibition with specific neurotoxicity. Quantitative histochemical assays demonstrate a significant inhibition of NADH-TR activity in ligated axons (-9.5%), Purkinje neurons (-17.3%) and dorsal root ganglion neurons (-17.8%) following a single ACR injection. Non-neural tissue enzymes were not significantly inhibited. A non-neurotoxic analogue, methylene-bis-ACR, had no effect upon these enzymes except in Purkinje neurons. The data supports, in general, the hypothesis that specific inhibition of neural NADH-TR activity by acrylamide is the primary site of action in producing the neuropathy. The neural specificity is attributed to lower levels of glutathione in neural tissues rather than specific inhibition of neural isoenzymes of NADH-TR.

Acrylamide↗

Glucose-dependent lactate production by homogenates of neuronal tissues prepared from rats treated with 2,4-dithiobiuret, acrylamide, p-bromophenylacetylurea and 2,5-hexanedione.

Chronic treatment of rats with dithiobiuret (DTB) produces a delayed onset muscle weakness and, as suggested by a preliminary study, a distal axonopathy. An inhibition of glycolysis resulting in an energy deficit has been suggested as a possible mechanism of neurotoxin-induced distal axonopathies. To determine whether chronic DTB administration (1 mg/kg/day X 7 days; sacrificed on Day 7) might be associated with a perturbation of glucose metabolism in neuronal tissues, lactate production was measured in brain and spinal cord homogenates prepared from rats which exhibited hindlimb skeletal muscle weakness. Kinetic and statistical analysis showed that glucose-dependent lactate production in these homogenates was not different from that of controls. Lactate production was also determined in brain homogenates prepared from rats intoxicated with acrylamide (50 mg/kg/day X 9 days; sacrificed on Day 9), p-bromophenylacetylurea (200 mg/kg/day X 2 days; sacrificed on Day 14) or 2,5-hexanedione (400 mg/kg/day X 21 days; sacrificed on Day 21). The results indicated that whereas 2,5-hexanedione produced a slight decrease in Vmax, acrylamide and p-bromophenylacetylurea did not cause changes in glucose-dependent lactate production. These findings do not support the suggestion that an inhibition of glycolysis represents a common biochemical lesion associated with neurotoxins which cause delayed onset muscle weakness and distal axonopathy.

Acrylamide↗

Determination of acrylamide metabolite, mercapturic acid by high performance liquid chromatography.

A HPLC Assay was developed to identify and measure the metabolite of acrylamide, mercapturic acid, N-Acetyl-s-(propionamide)-cysteine (APC) in urine. O-phthalaldehyde (OPA) was utilized as a precolumn derivatizing agent in the assay. This derivative was isolated with a good selectivity by high performance liquid chromatography (HPLC) employing reversed phase ODS columns. The quantitation of the mercapturic acid derivative was reproducible and with a detection limit of 1 pmol. The average coefficient of variation for the runs carried out on the same day was approximately 4.6% at the range of 80-160 mumol.L-1 of APC, and the mean analytical recovery from urine samples was 94%. The metabolite of urine of workers exposed to acrylamide was identified as N-acetyl-s-(propionamide)-cysteine by HPLC chromatography and fluorescence scan and HPLC-Mass spectra. All results were identical with the authentic synthesized compound.

Acetylcysteine↗

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry for monitoring alkylation of beta-lactoglobulin B exposed to a series of N-substituted acrylamide monomers.

Delayed-extraction matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, in both linear and reflectron modes, has been used to examine the alkylation of bovine beta-lactoglobulin-bound cysteines exposed to various molar concentrations (0.5-30 mM) of acrylamide and a number of its N-substituted monomers. These measurements were conducted at pH approximately 9.5, and showed that at 0.5 mM all monomers (except N-acryloylaminopropanol) resulted in a measurable alkylation of at least one cysteine out of five. At higher concentrations (15 mM) all investigated monomers resulted in substantial alkylation which, for some, involved all five cysteine residues. Reflectron MALDI-TOF measurements of a number of alkylated protein digests revealed that, at low molar ratios, the alkylation site is influenced by the identity of the monomer. For example acrylamide and N, N-dimethylacrylamide attacked Cys(160) as well as one of the three cysteines within the tryptic fragment [102-124], while other investigated monomers did not involve Cys(160). The implications of the present data for two-dimensional (2D) gel electrophoresis, and their eventual correlation to the toxicity of the investigated monomers, are discussed.

Acrylamides↗

Effect of residual acrylamide monomer from two-dimensional gels on matrix-assisted laser desorption/ionization peptide mass mapping experiments.

Residual acrylamide can cause severe suppression of signal intensity during matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping experiments. This suppression phenomenon can compromise the ability to detect low picomole and subpicomolar amounts of peptides extracted from two-dimensional gels. A rapid and simple method that exploits the use of pipette tips incorporating C18 packing materials for the enhancement of MALDI signal intensity is presented. The utility of the method is demonstrated with peptide solutions incorporating residual acrylamide and/or gel monomer components.

Acrylamides↗

Performances of new sugar-bearing poly(acrylamide) copolymers as DNA sieving matrices and capillary coatings for electrophoresis.

We have synthesized two new sugar monomers, allylamine of gluconic and lactobionic acid, by opening the corresponding lactone ring with allylamine. These monomers were copolymerized with acrylamide leading to formation of copolymers with a relative molecular mass of 288000 and 180000 Da, respectively. Double-stranded DNA fragments were separated in entangled solutions of these linear polymers in capillary electrophoresis. Resolution, peak spacing and peak width were the parameters taken into account to evaluate the quality of the separation achieved with the new polymers. This work indicates that the copolymers of acrylamide and allyl gluconic acid have a high sieving capacity and provide a performance similar to that of hydroxyethylcellulose (HEC) of comparable viscosity. Unlike HEC, this copolymer selfcoats onto the capillary wall, allowing DNA fragments to be efficiently separated in an uncoated capillary.

Acrylamides↗

Surface modification based on Si-O and Si-C sublayers and a series of N-substituted acrylamide top-layers for capillary electrophoresis.

Two approaches were used to prepare a series of surface-modified capillaries. In the first, a sublayer was formed by coupling gamma-methacryloxypropyltrimethoxysilane to the surface silanol groups forming an SI-O bond; a top layer was then formed by polymerizing acrylamide in the capillary, which reacted with the sublayer. In the second approach, a sublayer was formed by silanol chlorination, followed by Grignard coupling of vinylmagnesium bromide to form an Si-C bond at the surface; a top layer was formed by polymerizing either acrylamide (AA), dimethylacrylamide (DMA), N-acryloylaminoethoxyethanol (AAEE), or N-acryloylaminopropanol (AAP) onto the sublayer. The Si-Cpoly(AA) capillaries were more stable and produced an approximately 10-fold lower electroosmotic flow compared to the Si-O-poly(AA) capillaries. The Si-C sublayer was used to compare the performance of all four top layers. Electroosmotic flow decreased in the order: Si-O-poly(AA), Si-C-poly(AA), Si-Cpoly(AAEE), Si-C-poly(DMA), and Si-C-poly(AAP). Si-C-poly(AA) showed evidence of irreversible degradation at pH 9 already after 40-50 runs. Si-C-polyAAP-coated capillaries demonstrated superior efficiency and migration time reproducibility for a number of alkaline proteins and for fluorescently labeled ovalbumin. Excellent performance was maintained, in the case of poly(AAP), for a least 300 runs (of 30 min duration) at pH 9.0.

Acrylamide↗

Simultaneous electrophoretic analysis of proteins of very high and low molecular weights using low-percentage acrylamide gel and a gradient SDS-PAGE gel.

To be able to separate and analyze giant proteins and small proteins in the same electrophoretic gel, we have used a continuous SDS-PAGE gel formed by the combination of a low-percentage acrylamide gel and a gradient SDS-PAGE gel that we have named LAG gel. To get a good resolution for proteins of more than 200 kDa, we used an acrylamide/bisacrylamide ratio of 80:1 in the low-percentage acrylamide gel. To successfully resolve proteins in the 5-200 kDa range, we used a conventional 6-15% SDS-PAGE gradient gel with the standard acrylamide/bisacrylamide ratio of 40:1. We show that the LAG system can be successfully used in general applications of SDS-PAGE electrophoresis such as proteomics and immunobloting techniques. Thus, using this continuous LAG gel, it is possible to simultaneously analyze giant proteins, such as HERC1 and dynein, big proteins like clathrin heavy chain and small proteins like ARF. The LAG system has a good resolution, low cost, and high reproducibility. Moreover, to simultaneously analyze all proteins saves time. All these characteristics, together with the use of a standard apparatus found in any biochemistry laboratory, make the LAG system an easy tool to use.

Acrylamides↗

Oxygen consumption by acrylamide polymerization: a method for rapid screening of anticancer agents.

We have developed a rapid and sensitive method to measure oxygen consumption of tumor cells by acrylamide polymerization. This method could be used as an in vitro screen for potential chemotherapeutic agents. Previous techniques have lacked either the sensitivity or the speed required for use as an effective clinical tool. This method was modified from a technique previously developed in our laboratory for measuring oxygen in blood. Standard anticancer agents were tested against Walker 256 ascites tumor. Ascites was harvested from 20 rats on each of 50 days, incubated briefly with drugs, and acrylamide polymerization time was measured hourly. Individual controls were established for each drug-treated group, and oxygen uptake measured after one hour of incubation. Actinomycin, cyclohexamide, and cytosine arabinoside inhibited tumor cell oxygen consumption by 80%, 40%, and 30%, respectively. These results correlated with the known in vivo effects of these drugs on Walker carcinosarcoma. Therefore, this sensitive method could potentially be used directly on tumor cells removed from biopsy material so that the testing of anticancer drugs could be completed on the day of biopsy.

Acrylamides↗

Acrylamide levels in Finnish foodstuffs analysed with liquid chromatography tandem mass spectrometry.

Sample clean-up and HPLC with tandem mass spectrometric detection (LC-MS/MS) was validated for the routine analysis of acrylamide in various foodstuffs. The method used proved to be reliable and the detection limit for routine monitoring was sensitive enough for foods and drinks (38 microg/kg for foods and 5 microg/L for drinks). The RSDs for repeatability and day-to-day variation were below 15% in all food matrices. Two hundred and one samples which included more than 30 different types of food and foods manufactured and prepared in various ways were analysed. The main types of food analysed were potato and cereal-based foods, processed foods (pizza, minced beef meat, meat balls, chicken nuggets, potato-ham casserole and fried bacon) and coffee. Acrylamide was detected at levels, ranging from nondetectable to 1480 microg/kg level in solid food, with crisp bread exhibiting the highest levels. In drinks, the highest value (29 microg/L) was found in regular coffee drinks.

Acrylamide↗

Acrylamide neuropathy and changes in the axonal transport and muscular content of the molecular forms of acetylcholinesterase.

Acetylcholinesterase (AChE) is present in nervous and muscular tissues of normal chickens in four main molecular forms (G1, G2, G4, and A12), distinguishable by sedimentation analysis. In the sciatic nerve of acrylamide-poisoned chickens, the anterograde axonal transport of A12 AChE was reduced by 60%, and that of G4 by 21%, compared to control values whereas the slow axoplasmic transport of G1 and G2 was unaffected. Regarding the leg muscles, only the tibialis anterior revealed dramatic alterations in the distribution of it AChE forms coinciding with a large reduction in the number of nerve endings. In acrylamide poisoning, the AChE molecular forms were considered as very sensitive markers of both axonal transport phases and of the innervation state. Our results support the hypothesis that a defect in the fast axonal transport of proteins might be involved in the degeneration process of the disease.

Acetylcholinesterase↗

Recent advances in capillary electrophoresis of DNA fragments and PCR products in poly(n-substituted acrylamides).

In this article, capillary electrophoresis of DNA fragments and polymerase chain reaction products in sieving liquid polymers is reviewed. The first part of the review is dedicated to the development of N-substituted acrylamides, which in general guarantee a greater resistance to hydrolytic attack. Among the various monomers developed, an outstanding molecule appears to be N-acryloyl aminopropanol (AAP), which combines an extreme resistance to alkaline hydrolysis (500 times greater than plain acrylamide) with a substantially higher hydrophilicity. Poly(AAP) matrices exhibit a unique behavior in DNA separations, especially at high temperatures (60 degrees C), where most other matrices fail. In separation of antisense oligonucleotides, isoelectric buffers allow very high field strengths (up to 800 V/cm) due to their extremely low conductivity, thus permitting separations on the order of a few minutes. Among such buffers, histidine (pH = pI = 7.47) and lysine (pH = pI = 9.74) appear to be the best ones. Detection of DNA point mutations is accomplished with a peculiar nonisocratic capillary zone electrophoresis (CZE), consisting of creating temporal thermal gradients, analogous to temperature-programmed gas chromatography. A unique approach is described, consisting of creating temperature gradients not with external thermostats, but with internal Joule effects produced by voltage programming (which results in temperature ramps). The review ends with some unique applications of CZE: the possibility of precise gene dosage, relying on on-line detection and quantitation of the separated peaks. Examples of such gene dosages are given, such as assessment of Down's syndrome and determination of homozygous and heterozygous states of RhD blood groups.

Acrylamides↗

Pressure/temperature effects on protein flexibilty from acrylamide quenching of protein phosphorescence.

Pressure is an effective modulator of protein structure and biological function. The influence of hydrostatic pressure (</=3 kbar, 10-50 degrees C) on conformational dynamics was assessed from the rate of migration of acrylamide through the protein interior. Migration rates in apoazurin, alcohol dehydrogenase and alkaline phosphatase were obtained from the phosphorescence quenching rate constant (kq) of the deeply buried Trp residues. The dominant effect of applied pressure is to slow the diffusion process, although at low temperature, high pressure may also accelerate it. For apoazurin, alcohol dehydrogenase and alkaline phosphatase the activation free volumes, DeltaV(obs), derived from the pressure-dependence of kq, ranges from +10, +16 and +20 ml mol(-1)at 50 degrees C to -20, +5 and 0 ml mol(-1)at 10 degrees C, respectively. Analysing DeltaV(obs) in terms of a positive contribution from cavity expansion and a negative one from peptide hydration, the results emphasise that whereas at warm temperature the formation of cavities plays a dominant role in the migration process, at low temperature the required flexibility may be conferred by internal protein hydration. The relatively small magnitude of both DeltaV(obs) and the activation enthalpy (DeltaH=10-20 kcal mol(-1)) indicates that acrylamide diffusion jumps inside these proteins appear to involve relatively small amplitude structural fluctuations not requiring major unfolding-like transitions. The implication of these findings for the thermodynamic stability of proteins under pressure is discussed.

Acrylamide↗

On line monitoring of acrylamide formation.

A system to monitor the formation of acrylamide in model systems and from real food products under controlled conditions of temperature, time and moisture content has been developed. By humidifying the gas that flows through the sample, some control over moisture content can be affected. Results are presented to show the validity and reproducibility of the technique and its ability to deliver quantitative data. The effects of different processing conditions on acrylamide formation and on the development of color, due to the Maillard reaction, are evaluated.

Acrylamide↗

Acrylamide reduction in processed foods.

The discovery of the formation of acrylamide in fried and baked foods containing high levels of starch and the amino acid asparagine, prompted widespread concern. Both processed and home cooked foods are affected and this has led to the increased study of variations in cooking and processing conditions to minimize formation. While changes in cooking protocols have been in part successful, particularly when lower frying and baking temperatures are used, pretreatments to reduce levels of acrylamide by prevention of formation or acceleration of destruction have been investigated. In this study, a range of pretreatments of grilled potato were investigated and compared with surface washing to remove asparagine and reducing sugars. Synergies were observed between different treatments, and reductions of up to 40% were achieved in a non-optimized system.

Acrylamide↗

Reproductive toxicity of acrylamide and related compounds in mice--effects on fertility and sperm morphology.

Acrylamide and its three neurotoxic analogues, which are known to induce testicular damage in animals, were tested for reproductive toxicity in male and female mice and for the effect on germ cells in males, after subchronic treatment. After mating the treated males with untreated females, a decrease in fertility rate for all analogues except for N-hydroxymethylacrylamide, and an increase in the number of resorptions/dam for all test compounds were found. When untreated males were mated with treated females, an increase in the number of resorptions/dam was seen only after acrylamide dosing without any effect on the fertility rate. The reproductive toxicity appeared with only slight or no testicular atrophy, and without any apparent neurotoxicity after treatment with all test compounds. High doses of all test compounds produced both a decreased sperm count and an increase in abnormal sperm morphology.

Acrylamides↗

Acrylamide and thiamine.

Acrylamide administration to rats caused clear alteration of blood thiamine content, which appeared to reflect suppressed intake and/or utilization of the vitamin in the body. Decreased utilization of thiamine does not seem to be induced by decreased binding of thiamine pyrophosphate (TPP) and apotransketolase, because acrylamide did not affect the Km of TPP for transketolase activity.

Acrylamides↗

Unmyelinated nerve fibres in feline acrylamide neuropathy.

Electronmicroscope studies have been performed on the greater splanchnic nerve and the nerve to the medial head of gastrocnemius muscle of control and acrylamide poisoned cats. Degeneration of unmyelinated as well as of myelinated fibres was observed in both nerves. In cats severely poisoned with acrylamide, some very large unmyelinated axons undergoing early degeneration were seen in the splanchnic nerve. In the nerve to medial head of gastrocnemius, there was a decrease in the proportion of large diameter unmyelinated axons.

Acrylamides↗