Comparative neurotoxicity and pyrrole-forming potential of 2,5-hexanedione and perdeuterio-2,5-hexanedione in the rat

2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, reacts with protein amines to form alkylpyrrole adducts. Pyrrolylation of neurofilament protein may be the initiating molecular event in 2,5-HD neuropathy. The present study compares the neurotoxic and pyrrole-forming potentials of 2,5-HD with those of perdeuterio-2,5-HD ([D10]-2,5-HD) in the rat. Due to a requirement for C-H bond breaking in the reaction mechanism, the latter derivative was expected to exhibit a primary isotope effect, thus forming the pyrrole at a slower rate. In vitro studies confirmed that [D10]-2,5-HD pyrrolylated protein at only one-third of the initial rate seen with native 2,5-HD. Prolonged incubation resulted in similar pyrrole concentrations with both derivatives. Adult, male Wistar rats were administered daily (5 days/week) ip doses of either 3.5 mmol 2,5-HD or [D10]-2,5-HD/kg/day for 17 days or 2.5 mmol/kg/day for 38 days. At termination, animals administered 2,5-HD and [D10]-2,5-HD exhibited 27 and 8% body weight loss, respectively. Moderate to severe hindlimb paralysis was present in the 2,5-HD groups while only mild effects were seen in [D10]-2,5-HD-dosed rats. Neuropathological changes were prominent in spinal cord sections from 2,5-HD-treated animals, while no effects were present in rats given the deuterated derivative. Pyrrole adduct concentrations in serum and axonal cytoskeletal proteins from 2,5-HD-treated animals were two- to threefold higher than in rats given equimolar doses of [D10]-2,5-HD. Levels of covalent crosslinking of axonal cytoskeletal proteins (assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) appeared to correlate with pyrrole concentrations. Tissue concentrations of each diketone isomer were not significantly different, indicating similar uptake of native and deuterated 2,5-HD. Mass spectrometry revealed rapid back exchange of the terminal (methyl) but not of the internal (methylene) deuteriums of [D10]-2,5-HD in vivo. These findings support an absolute requirement for pyrrole formation in gamma-diketone neurotoxicity.     

In vitro administration of sodium arsenite in mouse prepubertal testis induces germ cell loss and apoptosis

High levels of arsenic contamination in drinking water pose serious health risks in numerous countries. The documentation reporting arsenic toxicity on reproduction and development is increasing, with evidence of arsenic inducing fertility and developmental issues. Nonetheless, the impact of arsenic exposure on the development of the male reproductive system is not fully elucidated. In the present study, we have investigated the direct effects of arsenic on prepubertal mouse testis using an in vitro testicular organ culture system. Culture medium was supplemented with a range of concentrations of sodium arsenite, examining effects of low (0.5 and 1 μM) and high (10, 50, 100 μM) concentrations, in cultures of post-natal day 5 CD1 mouse testis. In vitro exposure of low arsenic concentrations (0.5 or 1 μM) for 6 days did not cause any change in the testicular morphology, germ cells density, or apoptotic marker cleaved caspase 3 (CC3) expression. In contrast, exposure of prepubertal testis to high arsenic concentrations (10, 50 or 100 μM) induced drastic changes: severe destruction of testicular morphology, with loss of seminiferous tubule integrity; a dose-dependent decrease in germ cell density, and a hundred-fold increase in CC3 expression after 50 μM arsenic exposure. In conclusion, high arsenic treatment induced a dose-dependent induction of apoptosis and germ cell loss in prepubertal mouse testis.     

Neurotoxicity and protein binding of 2,5-hexanedione in the hen

Previous studies in this laboratory have demonstrated 2,5-dimethylpyrrole adduct formation during in vitro exposure of protein amino groups to the neurotoxic n-hexane metabolite 2,5-hexanedione (2,5-HD). The present investigation reports in vivo pyrrole adduct formation in neural and nonneural protein from 2,5-HD-treated animals. Adult, White-Leghorn hens were given daily doses of either 200 or 70 mg 2,5-HD/kg, po, for up to 55 or 135 days, respectively. Additional animals were given 70 mg/kg for 63 days and then allowed to recover for 72 more days. Protein separation by gel electrophoresis followed by staining with a pyrrole-specific reagent yielded evidence of widespread adduct formation in protein from serum, liver, kidney, brain, and purified myelin. Binding was particularly strong in serum albumin nd myelin basic protein. Quantitation of the adduct in these tissues revealed that its formation reached peak levels at 20 days in high dose and 30 days in low-dose animals. Levels subsequently declined, suggesting the presence of a clearance mechanism capable of removing altered protein during continuing 2,5-HD exposure. Protein from animals on the recovery regimen contained no detectable pyrrole adduct. Pyrrole adduct formation was also detected in neurofilament protein preparations, although protein yields were too low to allow assessment of clearance. Hens at both dosages displayed clinical signs indicative of CNS and PNS neuropathy. Histologic findings included axonal swelling and degeneration in peripheral nerve and some spinal cord nerve tracts. A hypothesis is proposed involving differential clearance of pyrrole adduct from neural vs nonneural tissue to explain the mechanism of action and target organ specificity of 2,5-hexanedione.     

Effects of 2,5-hexanedione on the ovary and fertility. An experimental study in mice.

Sixty-day-old virgin female Swiss CD1 mice were treated with 1.5% 2,5-hexanedione in their drinking water; control mice received tap water; duration of treatment was either 4 or 6 weeks. Under these conditions the treated mice did not show any clinical symptoms although electromyography revealed some signs of polyneuropathy. Protein and DNA content per mg of ovarian tissue in treated mice were not significantly different from controls. Histological examination of ovarian sections at the light microscope level showed no significant alterations after exposure. A morphometric study revealed a statistically significant reduction in the number of growing oocytes after 6 weeks of treatment. For fertility studies three groups of 15 female mice each were treated for 0, 4 or 6 weeks as above and then permanently housed with untreated proven breeder male mice (one male per female); cages were checked daily for newly born mice. All litters appeared normal by gross examination. During the first 14 weeks of continuous mating the mean litter size (number of newborns per litter) remained about 11.4 in all groups; this number subsequently began to decrease. Control and 4-week treatment regression curves did not differ statistically, while the slope of the 6-week line was significantly steeper, indicating a faster decrease in litter size over time and a shortening of fertile life in the latter group of treated females.     

The rate of 2,5-hexanedione intoxication, not total dose, determines the extent of testicular injury and altered microtubule assembly in the rat

Charles River CD rats (220 g) were intoxicated with 1.0, 0.5, or 0.25% 2,5-hexanedione (2,5-HD) in the drinking water for a total of 21, 35, or 69 days, respectively. All rats received a total dose of 131 +/- 2 mmol/kg 2,5-HD at dose rates ranging from 1.9 to 6.1 mmol/kg/day. Rats were sacrificed 4 weeks after ending intoxication to evaluate the extent of testicular injury. An exposure rate of 6.1 mmol 2,5-HD/kg/day produced uniformally low testis weights (49% of control) and severe germ cell depletion, while exposure at 1.9 mmol/kg/day gave normal testis weights and histology. Exposure at the intermediate dose rate of 3.8 mmol 2,5-HD/kg/day produced an intermediate degree of testicular injury. In a separate experiment, testis pyrrole content and microtubule assembly behavior were measured in rats exposed to 2,5-HD at the various dose rates for 3 weeks. The rate of intoxication determined the extent of biochemical abnormality. Rats exposed to 1.0, 0.5, or 0.25% 2,5-HD had microtubule nucleation times 55, 63, and 72% of control and pyrrole contents equivalent to 2.14, 1.40, and 1.18 nmol 2,5-dimethylpyrrole/mg testis protein. These data demonstrate that 2,5-HD-induced testicular injury, unlike the nervous system toxicity, is dependent upon the rate of intoxication independent of total dose.     

Effects of in utero and postnatal sodium saccharin exposure on the nutritional status of the young rat. II. Dose response and reversibility

A previous study in our laboratory demonstrated that 30-day-old Sprague-Dawley rats exposed to 7.5% sodium saccharin (NaS) since conception differ from untreated rats in several physiological parameters. In the present study, to determine the dose response of the changes associated with NaS treatment, animals were evaluated at 30 days post-birth, after treatment with dietary levels of 0, 1, 3 or 7.5% NaS since conception. Most physiological consequences of NaS treatment in the weanling rat, including anaemia and reductions in serum folate and vitamin A concentrations, were dose dependent. Serum vitamin E, cholesterol and triglyceride concentrations were decreased at the two lower doses of NaS but were significantly increased with 7.5% NaS. The no-effect level (NOEL) was similar for physiological effects and for bladder tumour production in two-generation studies (1% NaS in the diet). The reversibility of the effects of 7.5% NaS was examined in 90-day-old rats. The increases in lipids and vitamin E were reversible. Although values for haematological parameters and serum vitamin A remained significantly reduced at 90 days, changes were less severe than at 30 days. Histological examinations revealed that the effects of 7.5% dietary NaS on the bladder were negligible, indicating that the physiological changes observed in the young rat are probably not directly related to the production of bladder tumours.     

Molecular alterations underlying the enhanced disruption of spermatogenesis by 2,5-hexanedione and carbendazim co-exposure

The current study investigated the co-exposure effects of 2,5-hexanedione (HD) and carbendazim (CBZ) on gene expression underlying the enhanced pathology previously observed. Adult male rats were exposed to HD (0.33 or 1%) followed by CBZ (67 or 200 mg/kg), and testis samples were collected after 3 and 24 h. Microarray analysis at 3 h revealed that CBZ and HD interact in an agonistic, or synergistic, way at the gene level. Further analysis of candidate genes by qRT-PCR at both 3 and 24 h after co-exposure, revealed that Loxl1 and Clca2/Clca4l were both decreased in expression. Immunohistochemical analysis of Loxl1 at 24 h revealed that Loxl1 is localized to the seminiferous tubules, with the most intense staining in the basement membrane, blood vessels, and acrosomes, with the relative intensity reflecting the gene level changes at 3 h. These findings provide candidate genes for further investigation of the testicular response to damage.     

Total number and mean cell volume of neocortical neurons in rats exposed to 2,5-hexanedione with and without acetone

The toxicological effects of 2,5-hexanedione (2,5-HD) alone and combined with acetone on the number and size of neurons in the cerebral cortex of rats were evaluated with stereological techniques. Thirty rats were equally divided into three groups: One control, one receiving 0.5% 2,5-HD, and one receiving 0.5% 2,5-HD and 0.5% acetone in the drinking water for seven weeks. Unbiased estimates of the total number of neocortical neurons, as well as the mean neuronal nuclei and cell body volumes were obtained from systematically sampled 3.5-microns sections. The total number of neurons in the 2,5-HD group was significantly smaller than the control group (p less than 0.05, one-tailed t-test). Both test groups showed significant changes in the mean cell body volume: Compared with the control group, animals exposed to 2,5-HD had 11% smaller cell body volumes while animals exposed to 2,5-HD and acetone had 13% larger cell body volumes. These data represent the first unbiased estimation of mean cell volume in toxicology. We propose the nucleator method as an efficient and accurate tool for estimating quantitative changes in toxicological research.     

Neurotoxicity of acrylamide and 2,5-hexanedione in rats evaluated using a functional observational battery and pathological examination.

The clinical effects of two neurotoxicants, acrylamide and 2,5-hexanedione, were compared in rats using a functional observational battery (FOB), which includes a series of home cage and open-field observations, sensorimotor measurements, and physiological parameters. Neurotoxicity was assessed weekly in adult male Long-Evans rats after initiation of IP administration of 9 doses of acrylamide (12, 15, or 50 mg/kg given 3 times a week) and 28 doses of 2,5-hexanedione (150, 225, and 350 mg/kg given daily). Using the FOB, it was possible to detect differences in neurotoxic effects of these two chemicals. Acrylamide significantly affected home cage posture, foot splay and time on the rotarod, whereas 2,5-hexanedione altered hindlimb grip strength and the approach response. Both compounds caused changes in ability to walk, right, and maintain agility on a rotarod within 21 days from initiation of toxicant administration. In addition, both compounds caused dose-dependent decreases in weight gain. Neuropathic changes were detectable at the highest dosages at 21 days in acrylamide-treated rats and at 28 days in rats treated with 2,5-hexanedione. Administration of acrylamide also decreased activities of neural esterases. This study indicated that the FOB could be used to detect evidence of neurotoxicity in rats treated with acrylamide and 2,5-hexanedione, with alterations evident even before pathological changes were induced by 2,5-hexanedione.     

Alterations of dendritic cells in the rat thymus without epithelial cell loss during cyclosporine treatment and recovery

After cyclosporine treatment, dendritic cells disappear from the rat thymic medulla. The present study was undertaken to examine the ultrastructural alterations in the dendritic cell population during 14-day cyclosporine treatment and subsequent 6-week recovery. Four dendritic cell subtypes were defined ultrastructurally by a newly developed classification system. In addition, the potential effect of cyclosporine on six medullary epithelial cell subtypes was studied. During cyclosporine treatment, a prominent reduction of dendritic cells was seen at the ultrastructural level, whereas the total number of medullary epithelial cells remained largely unchanged. These findings were confirmed by immunohistochemistry. The number of mature dendritic cells declined later than the number of immature ones. A decrease in the antigen-processing capacity of remaining dendritic cells was suggested by the disappearance of Birbeck granules and the reduced number of tubulovesicular complexes. These findings support a disturbance of clonal deletion during cyclosporine treatment. The dendritic cell alterations appeared reversible 4 weeks after the restoration of the original architecture. During recovery, dendritic cells displaying lysosomal elements outnumbered those found in the normal uninvoluted thymus. This phenomenon probably reflects an enhanced turnover of cell organelles. No treatment-related effect on epithelial cell subtypes was seen.     

Novel 2,5-hexanedione analogues. Substituent-induced control of the protein cross-linking potential and oxidation susceptibility of the resulting primary amine-derived pyrroles

The neurotoxic gamma-diketone, 2,5-hexanedione (2,5-HD), induces neurofilamentous swellings at prenodal sites in proximal axons as a consequence of pyrrolation of lysine epsilon-amino groups on neurofilament proteins. However, there is disagreement as to whether pyrrole formation and the associated alteration of noncovalent interactions is sufficient to cause neurofilament accumulation, or whether pyrrole autoxidation and subsequent protein-protein cross-linking is an obligatory event. To investigate gamma-diketones that might form pyrroles inert to autoxidative-induced cross-linking, we synthesized 1,1,1-trifluoro-2,5-hexanedione, 3-(trifluoromethyl)-2,5-hexanedione (3-TFMHD), and two 3-(dialkylaminocarbonyl)-2,5-diketones and assessed their rates of pyrrole formation with amines, the oxidation susceptibility of the resulting pyrroles, and the protein cross-linking potential in vitro, relative to those of 3-methyl-2,5-hexanedione. 1,1,1-Trifluoro-2,5-hexanedione does not form pyrroles, but the three 2,5-HD analogues with an electron-withdrawing 3-substituent all rapidly formed pyrroles that were inert to autoxidation. Although 3-TMFHD nonetheless still induced cross-linking of ribonuclease A, by a nonoxidative mechanism independent of the pyrrole, the two 3-(dialkylaminocarbonyl)-2,5-diketones did not exhibit any protein cross-linking. As these two gamma-diketones possess aqueous-organic partitioning properties similar to those of 2,5-HD, they should serve as useful mechanistic probes in further studies.     

Toxic neurofilamentous axonopathies and fast anterograde axonal transport. IV. In vitro analysis of transport following acrylamide and 2,5-hexanedione.

Recent investigations into the mechanisms of neurotoxicity of acrylamide and gamma-diketones have demonstrated reductions in the delivery of radiolabelled proteins to the distal axon. To differentiate a toxicant-induced compromise in the capacity of the fast anterograde axonal transport system from a neuron cell body processing effect, selective exposure of either the L5 dorsal root ganglion or sciatic nerve to 0.7 mM acrylamide (ACR) or 4 mM 2,5-hexanedione (2,5-HD) was performed during in vitro transport. Nerve exposure to ACR decreased the quantity of transport by 32%, 2,5-HD reduced the quantity by 44%. Ganglion exposure produced no significant changes. We conclude that both toxicants penetrate the nerve barriers and act directly and/or indirectly on the axonal transport mechanisms to cause the reductions in transport.     

2,5-Hexanedione exposure alters the rat Sertoli cell cytoskeleton. I. Microtubules and seminiferous tubule fluid secretion.

2,5-Hexanedione (2,5-HD) is a testicular and nervous system toxicant with an unknown mechanism of action. In this study, the effects of 2,5-HD on seminiferous tubule fluid (STF) secretion, testis morphology, and tubulin distribution were examined. Charles River CD rats (200 g) were exposed to 1% 2,5-HD in the drinking water for 5 weeks followed by a 3-week recovery period. STF secretion was measured by efferent duct ligation, and testis cross sections were prepared at 2, 3, 3.43, 3.57, 3.86, 4, and 8 weeks after beginning exposure. A dramatic inhibition of STF secretion was observed between Weeks 3 and 4. The inhibition of STF secretion occurred following simultaneous changes in Sertoli cell and elongate spermatid morphology but prior to changes in round spermatid morphology. Also, alterations in seminiferous tubule tubulin distribution were observed with kinetics similar to those for changes in seminiferous tubule morphology. This temporal sequence suggests a model of 2,5-HD-induced injury in which populations of germ cells are differentially sensitive to impairment of Sertoli cell function.     

Free and total 2,5-hexanedione in biological monitoring of workers exposed to n-hexane in the shoe industry

OBJECTIVES: To analyse the role of total 2,5-hexanedione (2,5-HD) compared with free 2,5-HD as a biological indicator of exposure to n-hexane at work. METHODS: One-hundred and thirty two workers in contact with this solvent during their occupation in the shoe industry in the province of Alicante (Spain) were studied. Environmental and biological tests were carried out analysing variations of the concentration of the metabolite in urine corresponding to different working conditions. Environmental exposure was evaluated in each work place using active personal monitors and measured by gas chromatography (GC). Dichloromethane extracts of the urine samples collected at the end of the working shifts were analysed, before (determining free 2,5-HD, the toxic metabolite) and after acid hydrolysis (pH 0.1) (yielding the total 2,5-HD) and also by GC. The concentration of conjugated metabolite 4,5-dihydroxy-2-hexanone was calculated from the difference between total and free 2,5-HD. RESULTS: Free 2,5-HD represented an average of 14.2% of the total 2,5-HD determined in urine, and this percentage increased significantly (P<0.01) with higher environmental levels of acetone. Other factors, such as absorption through the skin (depending on the use of gloves) and the day on which samples were taken also significantly affected the relation between the two indicators and their respective relationships with environmental concentrations of n-hexane. CONCLUSION: Although analyses of the relationship between the levels of atmospheric n-hexane and those of metabolites in urine show a greater correlation for total 2,5-HD than for free 2,5-HD, our results suggest that free 2,5-HD could be a better indicator in evaluating risk of exposure to n-hexane, since the concentration is directly related to the neurotoxic effect.     

The effects of 2,5-hexanedione on reproductive hormones and testicular enzyme activities in the F-344 rat

The chronic administration of 2,5-hexanedione (2,5-HD) to experimental animals can cause azoospermia and morphologic changes in central nervous system (CNS) areas related to visual and motor function. The present experiments were designed to determine the degree of CNS involvement in the testicular lesions seen after 2,5-HD administration. Additionally, activity measurements were made of some enzymes found in specific cell types in the testes. The 2,5-HD was administered as a 1% solution in the drinking water to adult male F-344 rats. Treated rats, pair fed controls, and ad libitum controls were killed after 1, 3, and 6 weeks of 2,5-HD treatment. The circulating levels of testosterone and the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), were not depressed at any time measured. After 6 weeks, the testes were azoospermic; this coincided with a rise in LH and FSH. After 3 weeks of 2,5-HD treatment, when the testes were morphologically normal, testicular activity of the Sertoli-cell-specific enzymes, β-glucuronidase and γ-glutamyl transpeptidase, were decreased. The testicular enzyme profile after 6 weeks was similar to that seen in the azoospermic, cryptorchid testis. Activities of hepatic β-glucuronidase and acid phosphatase were decreased at all time points. The data indicate that 2,5-HD does not act via the central gonadotropin control systems to induce azoospermia, and that demonstrable changes in Sertoli cell biochemistry occur prior to visible morphologic changes in the testis.     

Lack of evidence for the size principle of selective vulnerability of axons in toxic neuropathies. I. The effects of subcutaneous injections of 2,5-hexanedione on behavior and muscle spindle function

2,5-Hexanedione (HD) produces a central-peripheral distal axonopathy. It has been suggested that agents which produce this type of axonopathy show a predilection to the largest diameter fibers. This has been based primarily on morphological data. However, electrophysiological evidence and some clinical and morphological data suggest that this may not be the case. In particular, in acrylamide neuropathy, muscle spindle primary afferents do not show this selectivity, as well as autonomic fibers. This study was carried out to determine whether the largest diameter axons were selectively vulnerable to HD. We found that subcutaneous injections of HD in cats produced a dose-dependent increase in behavioral deficits such as contact placing, stepping, and locomotion. There was also a dose-dependent decrease in the position sensitivity of muscle spindle primary and secondary endings. However, the secondary endings, which are innervated by smaller axons, were affected prior to the primary endings. Also, the velocity sensitivity of the primary endings was depressed at a similar time frame as the position sensitivity of these same endings. These data are consistent with the hypothesis that the caliber of the axon is not the only determinant of the selective vulnerability of axons in distal axonopathy.     

Rat model to investigate the treatment of acute nitrogen dioxide intoxication.

1. The pulmonary toxic events induced by acute nitrogen dioxide (NO)2 exposure were studied in the rat to develop an inhalation model to investigate therapeutic measures. 2. A good correlation was observed between the lung weights and severity of the atypical pneumonitis. The pulmonary effects observed, became more pronounced with increasing NO2 concentrations (0, 25, 75, 125, 175 or 200 ppm, 1 ppm NO2 = 1.88 mg m-3 NO2) and exposure times (5, 10, 20 or 30 min). 3. An adequate NO2 concentration is 175 ppm, because it can induce a severe lung injury without mortality. This makes it possible to investigate suitable therapeutic interventions for several days. 4. Following acute inhalatory NO2 intoxication, transformation of NO2 to nitrate is presumably more notable than transformation to nitrite. 5. The transformation of NO2 to nitrate in lung tissue causes a slight increase in the serum nitrite concentration, which does not induce measurable formation of methaemoglobin. 6. Presumably, methaemoglobin does not contribute to the toxicity of NO2 intoxication.     

二乙氧基乙醇诱导大鼠生精细胞凋亡及其分子机制研究

目的观察二乙氧基乙醇(2-EE)诱导大鼠生精细胞凋亡及Bcl-2家族、Fas系统和Caspase-3与细胞凋亡过程的作用，探讨2-EE诱导大鼠生精细胞凋亡的分子调控机制。方法选用SD大鼠(8只，分3个实验组和1个对照组，每组2只)以800mg／kg剂量分别灌胃1、3和5d。提取一侧睾丸组织DNA用于DNA片段分析，另一侧睾丸组织RNA用于半定量RT-PCR分析Bax、Bcl-2、Fas、Fas-L和Caspase-3基因mRNA水平变化。用Quantity One4．1．1软件定量各条带亮度，以GAPDH RT-PCR定量结果为内对照得出各个基因mRNA相对定量值。结果2-EE染毒后第1天，大鼠睾丸细胞DNA片段出现明显的“DNA Ladder”。RT-PCR电泳结果结合Quantity One4．1．1定量结果显示：Bax与Fas mRNA水平在染毒后每天均升高，以第3天最为显著，并且Bax与Bcl-2 mRNA水平的比值也在染毒后上升。结论二乙氧基乙醇可以诱导生精细胞凋亡，其分子途径可能与Bax家族和Fas系统有关。     

The effect of 3,4-dimethyl substitution on the neurotoxicity of 2,5-hexanedione: II. Dimethyl substitution accelerates pyrrole formation and protein crosslinking

3,4-Dimethyl-2,5-hexanedione and 2,5-hexanedione were reacted with model amines to yield N-substituted 2,3,4,5-tetramethylpyrroles and 2,5-dimethylpyrroles, respectively. When compared to the unsubstituted parent compound 2,5-hexanedione, 3,4-dimethyl-2,5-hexanedione was found to cyclize approximately eight times as rapidly on a molar basis at 37°C, with an activation energy of 3290 cal/mole less than 2,5-hexanedione. In addition, 1-benzyl-2,3,4,5-tetramethylpyrrole oxidized more readily than 1-benzyl-2,5-dimethylpyrrole with a difference in the half-wave potentials of 0.29 V. Both γ-diketones led to progressive crosslinking of proteins in vitro, with the dimethyl substitution accelerating this process by a factor of 40. The formation of pyrrolyl derivatives in vivo was demonstrated by the characteristic absorption spectra obtained following reaction of erythrocyte proteins from intoxicated rats with Ehrlich's reagent. There was progressive formation of protein-bound dimethylpyrroles following exposure to 2,5-hexanedione and formation of tetramethylpyrroles following exposure to 3,4-dimethyl-2,5-hexanedione in vivo. Preparations of axonal pads also demonstrated pyrrole derivatization in vivo. In addition, spectrin preparations of erythrocytes from intoxicated rats showed a large amount of high molecular weight protein (400,000 Da), corresponding to dimerized spectrin. Thus, 3,4-dimethyl-2,5-hexanedione, which is 20 to 30 times more potent on a molar basis than 2,5-hexanedione in leading to a neurofilamentous neuropathy, is associated with more rapid pyrrole formation and protein crosslinking in vitro, and it has been demonstrated that these processes occur in vivo. These observations support the hypothesis that pyrrole formation and autoxidation occur following exposure to γ-diketones, leading to covalent crosslinking of proteins in vivo, a process which may explain the pathogenesis of neurofilament accumulation in these neuropathies.     

Toxic neurofilamentous axonopathies and fast anterograde axonal transport. III. Recovery from single injections and multiple dosing effects of acrylamide and 2,5-hexanedione

Fast anterograde axonal transport has been advanced as a potential site of action of acrylamide (ACR) and the neurotoxic gamma-diketones in producing nerve degeneration. The segmental analysis method of axonal transport was used to measure the rate and quantity of protein transport in the rat sciatic nerve from 1 to 24 hr after a single injection of 50 mg/kg (0.7 mmol/kg) ACR or 4 mmol/kg 2,5-hexanedione (2,5-HD). The single injection of ACR or 2,5-HD resulted in an immediate reduction in transport quantity of 48 and 43%, respectively. Transport remained depressed for 16 hr; recovery occurred from 16 to 24 hr reaching control levels at 24 hr postinjection for both toxicants. Protein transport, measured immediately after the 2nd, 4th, 7th, and 10th injections, was reduced 36-38% by 50 mg/kg ACR and 30-43% by 4 mmol/kg 2,5-HD. Therefore, both ACR and 2,5-HD produce a transient and repeated compromise of fast anterograde transport during the dosing regimen which results in distal nerve degeneration. Assuming a rate of recovery after each subsequent dose similar to the first, the protein delivery to the axon was calculated to be reduced 29% by ACR and 22% by 2,5-HD. Current evidence supports the hypothesis that a toxicant-induced reduction in protein delivery to the axon by ACR and 2,5-HD contributes to development of axonal degeneration.