Carl Johan Calleman

Formation of hemoglobin adducts of acrylamide and its epoxide metabolite glycidamide in the rat

A method was developed for the determination of hemoglobin (Hb) adducts formed by the neurotoxic agent acrylamide and its mutagenic epoxide metabolite glycidamide. The method was based on simultaneous measurements of the cysteine adducts formed by these two agents by means of gas chromatography/mass spectrometry in hydrolyzed hemoglobin samples. Rats were injected ip with acrylamide or glycidamide in doses ranging from 0 to 100 mg/kg body wt, and the hemoglobin adduct levels were determined. The hemoglobin binding index of acrylamide to cysteine was found to be 6400 pmol (g Hb)-1/mumol (kg body wt)-1, higher than for any other substance studied so far in the rat, and 1820 pmol (g Hb)-1/mumol (kg body wt)-1 for glycidamide. In rats injected with acrylamide, formation of adducts of the parent compound was approximately linear with dose (0-100 mg/kg), whereas adducts of the epoxide metabolite glycidamide generated a concave curve, presumably reflecting the Michaelis-Menten kinetics of its formation. On the basis of the rate constants for cysteine adduct formation determined in vitro, the first-order rates of elimination of acrylamide and glycidamide from the blood compartment of rats were estimated to be 0.37 and 0.48 hr-1, respectively, using a linear kinetic model. It was further estimated that the percentage of acrylamide converted to glycidamide in the rat decreased from 51% following administration of 5 mg/kg to 13% after a dose of 100 mg/kg. Subchronic treatment of rats with acrylamide (10 mg/kg/day for 10 days or 3.3 mg/kg/day for 30 days) confirmed that the conversion rate of acrylamide to glycidamide, as determined from hemoglobin adduct formation, is higher at low-administered doses. These findings suggest that dose-rate effects may significantly affect risk estimates of this compound and that different low-dose extrapolation procedures should be employed for effects induced by the parent compound acrylamide and those induced by the metabolite glycidamide.

Quantitative measurements of vibration threshold in healthy adults and acrylamide workers.

SummaryThe early detection of impaired of vibration sensation is necessary in order to monitor the adverse effects in workers occupationally exposed to neurotoxic chemicals such as acrylamide. The conventional neurological examination which assesses vibration sensation by utilizing a tuning fork is relatively insensitive for this purpose. In the present study, the Vibratron II, a new device for the quantitative measurement of vibration thresholds, was used in 105 healthy Chinese adults. A new testing procedure combining the “two-alternative forced-choice procedure” and the “yes-or-no method-of limits procedure” showed good reliability and was less time consuming. The results indicate that significant differences in the vibration threshold of index fingers and great toes were found neither between males and females, nor between the left and the right side. However, there was an age-dependent increase in vibration threshold in nonexposed healthy subjects. The vibration thresholds of 41 workers exposed to acrylamide detected by the Vibratron II were significantly higher than those of the healthy adults in the same age group. The quantitative measurement of vibration threshold seems to be potentially useful for screening peripheral nerve dysfunction in field studies.

Covalent binding of acetaminophen to mouse hemoglobin. Identification of major and minor adducts formed in vivo and implications for the nature of the arylating metabolites

When hepatotoxic doses of [ring-U-14C]acetaminophen ([ring-U-14C]APAP) were administered to mice, radioactivity became bound irreversibly to hemoglobin as well as to proteins in the liver and kidney. The covalent binding to hemoglobin was dose-dependent, and in phenobarbital-pretreated mice occurred to the extent of approximately 8% of the corresponding binding to liver proteins. Degradation of the modified globin by acid hydrolysis yielded 3-cystein-S-yl-4-hydroxyacetanilide as the major radioactive product, accounting for approximately 70% of protein-bound drug residues. This finding is consistent with the view that the majority of covalent binding of APAP to proteins is mediated by N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite which preferentially arylates cysteinyl thiol residues. However, after administration of [acetyl-3H]APAP to mice, it was found that approximately 20% of the drug bound to hemoglobin had lost the N-acetyl side-chain, indicating the existence of a second type of APAP-protein adduct. One minor component of the globin hydrolysate was identified as S-(2,5-dihydroxyphenyl)-cysteine, which most likely arises from binding to hemoglobin of p-benzoquinone, a hydrolysis product of NAPQI. The two adducts reported represent the first identified examples of arylating drugs binding to hemoglobin. Experiments on the influence of different cytochrome P-450 inducing agents on the ratio of drug bound to hemoglobin versus hepatic proteins suggested that the reactive metabolites of APAP are formed in the liver and migrate to the erythrocyte, rather than being produced by hemoglobin-catalyzed oxidation of APAP. These findings imply that the reactive metabolites of APAP escape from hepatocytes in some latent forms, which then participate in the arylation of protein thiols in red blood cells and, possibly, at other remote sites.