[Effects of garlic oil, age and sex on n-hexane metabolism in rats].

OBJECTIVE: To investigate effects of garlic oil (GO), age and sex on n-hexane metabolism in rats. METHODS: The Wistar rats were used as experimental animals. (1) Intragastric administration: n-hexane group (3000 mg/kg n-hexane), GO treated group (80 mg/kg GO ig. an hour earlier than 3000 mg/kg n-hexane), then blood was taken from tails of rats at 8, 12, 16, 20, 24, 28, 32 h points after n-hexane administration. (2) Intraperitoneal injection: n-hexane group (1000 mg/kg n-hexane), GO treated group (80 mg/kg GO ig. an hour earlier than 1000 mg/kg n-hexane), then took blood was taken from tails of rats at 8, 12, 16, 20, 24, 28 h points after n-hexane injection. (3) 7 rats each group of 6, 8, 10 weeks age were administrated by 3000 mg/kg n-hexane intragastrically, then were taken blood from tails at 16, 20, 24 h points after administration. (4) 7 male and 7 female rats of 8 weeks age were administrated by 3000 mg/kg n-hexane intragastrically, then were taken blood from tails at 16, 20, 24, 28 h points after administration. The gas chromatography was used to determine the metabolite 2, 5-hexanedione concentration of n-hexane in serum and 2, 5-hexanedione concentration was compared between GO and no GO treated rats, different ages and different sexes. RESULTS: (1) Intragastric administration: 2, 5-hexanedione concentrations in serum of n-hexane group and GO treated group had the peak 19.2 and 12.3 µg/ml at 20h and 24 h points. Compared with n-hexane group, the serum 2, 5-hexanedione concentration of GO treated group was lower at time points prior to peak and 2, 5-hexanedione eliminating process was slower after peak. (2) Intraperitoneal injection: effects of GO on the serum 2, 5-hexanedione concentrations was very similar to intragastric administration, 2, 5-hexanedione concentrations in serum of n-hexane group and GO treated group had the peak 15.0 and 6.7 µg/ml at 12 h and 16 h points. (3) Comparison of the serum 2, 5-hexanedione concentrations of different weeks age rats: The serum 2, 5-hexanedione concentrations of 6, 8, 10 weeks age rats were 25.5, 15.0, 12.8 µg/ml each (8, 10 weeks age significantly lower than 6 weeks age) at 16 h point; at 20 h point, they were 24.7, 18.3, 15.0 µg/ml each (10 weeks age significantly lower than 6 weeks age); at 24 h point, they were 11.0, 14.7, 8.1 µg/ml each (10 weeks age significantly lower than 8 weeks age). (4) Comparisons of the serum 2, 5-hexanedione concentrations of different sex rats: the serum 2, 5-hexanedione concentrations of male and female rats were 22.5, 17.2 µg/ml each at 16 h point (different significantly); at 20, 24, 28 h points, they were 27.6, 22.9 µg/ml, 24.6, 19.1 µg/ml, 19.1, 13.8 µg/ml each (different non-significantly). CONCLUSION: GO reduces production of 2, 5-hexanedione in serum generated by n-hexane in rats; the metabolic capacity of low age rats on n-hexane is stronger than high age ones.

[Effects of garlic oil on n-hexane metabolized to 2, 5-hexanedione in mice serum].

OBJECTIVE: To investigate the effects of garlic oil (GO) on n-hexane metabolized to 2, 5-hexanedione (2, 5-HD) in mice. METHODS: Adult healthy Kunming-mice were treated with n-hexane and GO. The serum was obtained and extracted with ethyl acetate, and the levels of the serum 2, 5-HD were determined by gas chromatography. RESULTS: (1) The concentration of 2, 5-HD in serum increased firstly after a single exposure to n-hexane (4 000 mg/kg). The peak value occurred at 10 hours after n-hexane treatment, but could hardly be detected at 20 h. (2) There was no 2, 5-HD in serum of control mice. The content of 2, 5-HD in serum increased along with the exposure dose of n-hexane. The serum 2, 5-HD contents of the 2000, 4000 and 6000 mg/kg groups mice were 8.04, 16.68 and 22.38 microg/ml at 8 h in pretreated mice, respectively, and showed significant dose-effect relationship. (3) When the different age mice were exposed to the same dose of n-hexane, the contents of 2, 5-HD in serum were significantly different after 8 hours (P<0.05). The serum 2, 5-HD level of the 5 weeks old mice (22.83 microg/ml) was much higher than the 4 (19.59 microg/ml) and 6 (16.42 microg/ml) weeks old mice. (4) When the different gender mice were exposed to the same dose of n-hexane, the concentration of 2, 5-HD in serum of female mice (13.22 microg/ml) was higher than that of the female mice (10.34 microg/ml, P<0.05). (5) GO significantly inhibited the increase of the serum 2, 5-HD levels of both the pretreatment and post-treatment groups treated with 80 mg/kg n-hexane respectively, but the pretreatment with GO exhibited the more suppressive effects than the post-treatment (P>0.05). Compared with the n-hexane group, the concentrations of serum 2, 5-HD in GO-pretreated groups mice decreased by 16.2%, 20.8%, 22.8% (P<0.05) and 32.1% (P<0.01), respectively, and showed significant dose-effect relationship. CONCLUSION: The serum content of 2, 5-HD, the metabolite of n-hexane, is different in different genders and age mice after exposed to the same dose of n-hexane. GO can effectively inhibit the production of n-hexane metabolized to 2, 5-HD in mice serum.

Ketone Body Acetoacetate Buffers Methylglyoxal via a Non-enzymatic Conversion during Diabetic and Dietary Ketosis.

The α-oxoaldehyde methylglyoxal is a ubiquitous and highly reactive metabolite known to be involved in aging- and diabetes-related diseases. If not detoxified by the endogenous glyoxalase system, it exerts its detrimental effects primarily by reacting with biopolymers such as DNA and proteins. We now demonstrate that during ketosis, another metabolic route is operative via direct non-enzymatic aldol reaction between methylglyoxal and the ketone body acetoacetate, leading to 3-hydroxyhexane-2,5-dione. This novel metabolite is present at a concentration of 10%-20% of the methylglyoxal level in the blood of insulin-starved patients. By employing a metabolite-alkyne-tagging strategy it is clarified that 3-hydroxyhexane-2,5-dione is further metabolized to non-glycating species in human blood. The discovery represents a new direction within non-enzymatic metabolism and within the use of alkyne-tagging for metabolism studies and it revitalizes acetoacetate as a competent endogenous carbon nucleophile.

The role of pyrrole formation in the alteration of neurofilament transport induced during exposure to 2,5-hexanedione.

Exposure to the gamma-diketone, 2,5-hexanedione (HD), results in the accumulation of neurofilaments within the distal axon and is associated with acceleration of neurofilament transport within the proximal axon. The epsilon-amino groups of lysyl residues react with HD forming pyrrole adducts, followed by pyrrole-mediated protein crosslinking. Both reaction steps have been proposed as mechanisms causing neurofilament accumulation and acceleration of transport. In order to assess the importance of these steps on neurofilament transport, we compared transport in the optic system of rats exposed to HD and 3-acetyl-2,5-hexanedione (AcHD), a non-toxic analog of HD which forms pyrroles but does not crosslink proteins. Control, HD-treated, and AcHD-treated rats received intraoptic injections of [35S]-methionine and were exposed to saline, HD, or AcHD by intraperitoneal injections before and during the period of neurofilament transport. Neurofilament triplet proteins in the optic nerve and tract were identified by polyacrylamide gel electrophoresis followed by fluorography. The rate of neurofilament transport was accelerated in HD-treated animals over that of controls. However, despite higher levels of protein-bound pyrroles in AcHD-treated animals, the rate of transport was indistinguishable from that of controls. These findings indicate that pyrrole formation alone is not sufficient to cause acceleration of neurofilament transport.

Interaction of dacarbazine and imexon, in vitro and in vivo, in human A375 melanoma cells.

AIM: We evaluated mechanisms of interaction between the alkyating agent dacarbazine (DTIC) and the pro-oxidant, imexon, in the human A375 melanoma cell line. MATERIALS AND METHODS: The effect of DTIC and imexon, alone and in combination, was evaluated for growth inhibition (MTT), radiolabeled drug uptake, cellular thiol content (HPLC), and DNA strand breaks (Comet assay). Pharmacokinetic and antitumor effects were evaluated in mice. RESULTS: Growth inhibition in vitro was additive with the two drugs. There was no effect on drug uptake or on the number of DNA strand breaks. There was a >75% reduction in cellular glutathione and cysteine with imexon but not DTIC. Co-administration of the two drugs in mice caused an increase in the area under the curve of both drugs, but the combination was not effective in reducing human A375 melanoma tumors in vivo. CONCLUSION: Imexon and dacarbazine show additive effects in vitro but not in vivo in human A375 melanoma cells.

Biochemical and physiological aspects of 2,5-hexanedione: endogenous or exogenous product?

This article reports results regarding two different physiological aspects of 2,5-hexanedione (2,5-HD). The first is the relationship between "free" 2,5-HD (the fraction of "real" 2,5-HD) and "total" 2,5-HD (2,5-HD obtained from acid hydrolysis) in urine and blood of workers exposed to n-hexane. The second part of the study is an attempt to clarify "physiological" excretion of 2,5-HD in subjects not occupationally exposed to n-hexane. The concentration of free 2,5-HD in urine of workers exposed to n-hexane is about 8% of total urinary 2,5-HD. In blood, free 2,5-HD is about 50% of the total. The serum concentration range of total and free 2,5-HD in workers from whom blood was taken was 33-418 micrograms/l and 14-283 micrograms/l respectively. In subjects not exposed to n-hexane, urinary concentration of 2,5-HD ranged between 0.17 and 0.98 mg/l, the urinary excretion rate between 0.23 and 0.57 microgram/min, and renal clearance between 14 and 66 ml/min. The blood concentration of 2,5-HD in nonexposed subjects was 6-30 micrograms/l. Fluctuations typical of a circadian rhythm were not observed for 2,5-HD in blood or urine. We think that 2,5-HD is mainly a product of intermediate metabolism in the human body. Only a minimal part could derive from n-hexane as a ubiquitous micropollutant.

Volatile metabolites in sera of normal and diabetic patients.

The profiles of volatile metabolites in serum samples from normal individuals and from individuals with diabetes mellitus with varying degrees of polyneuropathy have been studied. The transevaporator procedure was used to obtain sample extracts which were chromatographed on a highly efficient glass column coated with Silar 10C (106 m x 0.25 mm I.D.). Differences in profiles between normal subjects and diabetic subjects on no drug therapy were noticed. However, correlations between the severity of the neuropathy and the concentrations of certain ketones could not be established. Compounds present both in diabetic and normal sera have been identified by mass spectrometry.

Relation between schedules of exposure to hexane and plasma levels of 2,5-hexanedione.

Male Fischer rats were exposed repeatedly to high concentrations of hexane for 10 minutes in a pattern resembling human solvent abuse or to low concentrations of hexane continuously for 8 or 24 hours daily. Concentrations of hexane in blood and brain were linearly related to the concentrations of hexane in the chamber after a 10-minute exposure, and declined thereafter, with half lives of about 2 1/2 and 4 minutes in blood and brain, respectively. Despite the rapid elimination of hexane, neurotoxic levels of 2,5-hexanedione (2,5-HD) were formed from repeated 10-minutes exposures to a high concentration of hexane when the interexposure interval was 20 minutes. Neurotoxic levels of 2,5-HD also resulted from continuous exposure to much lower concentrations of hexane. Both exposure schedules caused an increase in 2,5-HD concentrations in blood after repeated daily treatments, suggesting induction of liver microsomal enzymes synthesizing 2,5-HD from hexane. The minimal sustained plasma 2,5-HD concentration that will result in neurotoxicity appears to be less than 50 micrograms/ml in the rat.

Effects of MEK on kinetics of n-hexane metabolites in serum.

The neurotoxicity of n-hexane is thought to be caused ultimately by 2,5-hexanedione (2,5-HD), one of the n-hexane metabolites. The potentiation of n-hexane neurotoxicity by co-exposure with MEK, therefore, is suspected to be related to kinetics of 2,5-HD in blood. To clarify the kinetics of n-hexane metabolites in the mixed exposure of n-hexane and MEK, rats were exposed to 2000 ppm n-hexane or a mixture of 2000 ppm n-hexane and 2000 ppm MEK, and the time courses of serum n-hexane metabolites were determined. 2,5-HD in serum increased until 2 h after the end of exposure, when serum 2,5-HD concentration reached a peak of 16.35 micrograms/ml in the n-hexane-alone group. In contrast, 2,5-HD in the mixed exposure group increased much more slowly during and after exposure than in the n-hexane-alone group. It reached a peak of 2.12 micrograms/ml at 8 h after the end of exposure. Serum MBK, a precursor of 2,5-HD in the co-exposure group, was about half in the n-hexane-alone group during exposure. However, MBK decreased more slowly in the co-exposure group than in the n-hexane-alone group after the end of the exposure. The results suggest that co-exposed MEK might inhibit oxidation of n-hexane and decrease clearance of n-hexane metabolites. Co-exposed MEK did not increase serum 2,5-HD, which was considered a main neurotoxic metabolite. Therefore the enhancement of neurotoxicity could not be attributed to increased serum 2,5-HD in the co-exposed group.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicity and metabolism of methyl n-butyl ketone.

MEK (2-butanone) when combined with MBK (2-hexanone) markedly enhanced MBK neurotoxicity. MBK in rat plasma after exposure to MBK/MEK increased with time. Metabolites of MBK identified in blood and urine of rats and guinea pigs were 2-hexanol and 2,5-hexanedione.

Decreased levels of the high molecular weight subunit of neurofilaments and accelerated neurofilament transport during the recovery phase of 2,5-hexanedione exposure.

The neurotoxicant 2,5-hexanedione (HD) causes the accumulation of neurofilaments in the distal axon and an acceleration of neurofilament transport proximal to the site of their accumulation. It has been proposed that the acceleration of transport is due to the direct reaction of HD with neurofilament proteins and, conversely, that this acceleration is a secondary response to the axon to injury. The objective of this study was to determine whether the response of axons to HD intoxication includes acceleration of neurofilament transport. Pulse labelling was used to analyze neurofilament transport in age-matched rats exposed to HD or PBS. The animals receiving HD were exposed either throughout the period of radiolabel transport, or prior to the pulse labeling of neurofilament proteins. If acceleration of the rate of neurofilament transport was due to the direct reaction of HD with proteins, then neurofilaments synthesized after the exposure period should travel at control rates, since these proteins would not have been exposed to the toxicant. After 28 days of transport, optic nerve proteins were examined using SDS-PAGE, fluorography, and computerized densitometry. In both HD-treated groups, neurofilament transport was accelerated relative to age-matched control animals. In addition, the amount of NFH was decreased relative to other neurofilament subunits. The combination of accelerated transport and a diminished proportion of NFH is similar to the observations of neurofilament axonal transport during growth and development. These observations suggest that this persistent, secondary effect is a reparative response to injury that recapitulates axonal growth and development.

Potentiation of 2,5-hexanedione neurotoxicity by methyl ethyl ketone.

Chronic oral administration of a combination of 2.2 mmol methyl ethyl ketone (MEK) and 2.2 mmol 2,5-hexanedione (2,5-HD)/kg/day, 5 days/week resulted in more rapid onset of motor deficits than did chronic dosing with 2.2 mmol 2,5-HD/kg/day alone. In kinetic studies blood time courses of 2,5-HD were determined in rats in the presence and absence of MEK. Concomitant administration of MEK reduced blood 2,5-HD clearance and increased the area under the curve (AUC) for the blood 2,5-HD. In companion experiments with 2,5-[1,6-14C]HD as a tracer, neural and nonneural tissues were examined 72 hr following the last treatment at Weeks 1, 2, and 3 of chronic administration of 2,5-HD alone or in combination with an equimolar dose of MEK. Rats treated with 2,5-[14C]HD alone or in combination with MEK demonstrated no difference in total or trichloroacetic acid-precipitable radioactivity in blood, in liver homogenates, or in neurofilament-enriched fractions from sciatic nerve and spinal cord. The data support a suggestion that the potentiation of hexacarbon neurotoxicity by MEK is the result of the persistence of the neurotoxic metabolite in the blood and not the enhanced metabolism of parent hexacarbon to 2,5-HD.

A comparison of the rates of development of functional hexane neuropathy in weanling and young adult rats.

Chronic inhalation exposure of adult rats to hexane causes neural toxicities to develop over a period of several weeks. Because the developing organism is in many cases more vulnerable to toxic insult than the adult, and children make up a substantial proportion of the population of solvent abusers, we compared the effects of exposing weanling and young adult rats to 1000 ppm of hexane for 24 hrs/day, 6 days/week, for 11 weeks. Within two weeks of exposure, significance decreases in body weight and grip strength were observed in rats of both ages. However, the subsequent effects of the treatment on these indices of toxicity were greater in the young adults than in the rats first exposed as weanlings. The older rats also exhibited earlier and more severe signs of hindlimb flaccid paralysis. In contrast, the effects of hexane on tail nerve conduction time and on the brainstem auditory-evoked response were about the same in rats of both ages, with latencies increasing compared to controls over the exposure period. The relative resistance of the weanling rats to hexane neuropathy may be due to shorter, smaller-diameter axons, or to a greater rate of growth and repair in their peripheral nerves compared to those of adults.

Relationship between 2,5-hexanedione concentrations in nerve, serum, and urine alone or under co-treatment with different doses of methyl ethyl ketone, acetone, and toluene.

To ascertain the relationship among 2,5-hexanedione (2,5-HD) concentrations in nerve, serum and urine, rats were injected subcutaneously with 2.6 mmol/kg 2,5-HD alone, or together with 2.6 or 13.0 mmol/kg of methyl ethyl ketone, acetone and toluene. 2,5-HD concentrations in sciatic nerve (NC), serum (SC) and urine (UC) were determined, and the linear regression between each two of NC, SC, and UC were calculated. There was good correlation between NC and SC, SC and UC in the 2,5-HD alone group, and good correlation between NC and SC in the co-treated groups. Co-treatment solvent had little effect on the relationship between SC and NC. 13.0 mmol/kg co-treated solvent tended to decrease the regression coefficients compared with 2.6 mmol/kg co-treated solvent. These results show that SC can be used in estimating NC in the 2,5-HD alone or co-treated groups, and UC can be used in estimating SC in the 2,5-HD alone group.