Cancer Letters

Cancer Letters

Volume 207, Issue 1, 15 April 2004, Pages 9-17
Cancer Letters

Determination of acrylamide and glycidamide serum toxicokinetics in B6C3F1 mice using LC–ES/MS/MS

https://doi.org/10.1016/j.canlet.2003.10.017Get rights and content

Abstract

Acrylamide (AA) is a well-studied industrial toxicant; however, recent findings of AA at ppm levels in cooked starchy foods have refocused attention on the potential for neurotoxicity, germ cell mutagenicity, and carcinogenicity from AA. Oxidative metabolism of AA to glycidamide (GA) in experimental animals has previously been linked with many toxic effects of AA exposure. We report a new sensitive and selective analytical method, based on LC with electrospray tandem mass spectrometry, for the quantification of AA and GA in serum and its application to a preliminary toxicokinetic evaluation of AA and GA in adult B6C3F1 mice following oral administration of AA.

Introduction

Acrylamide (AA, Fig. 1) is a high-production volume chemical (>200 Gg/yr worldwide) whose polymeric forms are widely used in water treatment, crude oil processing, pulp-paper processing, concrete, and grouts [1], [2]. Monomeric AA is used extensively in biochemical applications for the polyacrylamide gel electrophoresis (PAGE) analysis of proteins; it is also a component of cigarette smoke (1–2 μg/cigarette) [3]. More recently, AA has been measured in baked and fried starchy foods, notably french fries (up to 0.4 ppm), potato chips (up to 1.5 ppm), and bread (0.04–1.7 ppm) [4], [5]. Further studies have shown that Maillard browning reaction products derived from glucose and asparagine, the latter being a major amino acid present in potatoes and cereals, are responsible for the formation of AA in these foods [6], [7], [8]. The classification of AA as a probable human carcinogen [2], based on findings of rodent carcinogenicity in several organs [9] and the characterization of GA-derived DNA adducts (see Fig. 1) [10], [11], underscore the significance of human exposures. In addition, AA produces central-peripheral distal axonopathy in rats [12], human neuropathy associated with occupational exposures [13], and germ cell mutagenicity in male rodents [14].

AA is clastogenic and mutagenic in vivo, although direct evidence for genotoxicity of AA is limited. AA is not mutagenic in Salmonella assays, either in the presence or absence of a microsomal activating system; however, glycidamide (GA, Fig. 1), the reactive epoxide metabolite of AA, is mutagenic under similar conditions [15]. The metabolism of AA to GA has been demonstrated in rodents and humans [16], [17], [18], and by comparing parental and cytochrome P450 (CYP) 2E1 knockout mice, CYP 2E1 was identified as an important mediator of AA oxidation to GA [19]. Furthermore, the comparable induction of micronuclei in rodents treated with either GA or AA suggests that GA is the predominate genotoxin derived from AA [20].

The toxicokinetics of AA have been determined previously in rats using either total [14C]-radioactivity [21] or after LC fractionation to determine both [14C]-labeled AA and GA [22]. These studies formed the basis for a physiologically based pharmacokinetic (PBPK) model for AA and GA in rats [23]. We now report a new sensitive and specific high throughput method, based on isotope dilution LC–ES/MS/MS, for the determination of AA and GA in mouse serum and its application to a preliminary assessment of the toxicokinetics of AA and GA in mice.

Section snippets

Reagents

Sigma Chemical Co. (St Louis, MO) supplied the AA and all biochemical reagents. Labeled 13C3-AA (99 atom %) was obtained from Cambridge Isotope Laboratories (Andover, MA) and 13C3-GA was synthesized from labeled AA by oxidation using dimethyldioxirane [20]. All solvents were HPLC grade and Milli-Q water was used throughout.

Liquid chromatography

A Waters Alliance 2795 Separation Module (Waters Co., Milford, MA) was used at a flow rate of 300 μl/min to deliver a mobile phase step gradient beginning at 2% (v:v)

Method performance

The SPE-LC/MS/MS method was first optimized with respect to the SPE cartridge, solvents, and LC column. Step gradient conditions were found to produce the highest ES/MS/MS responses for AA and GA (i.e. minimal ion suppression and peak width) when compared to an isocratic separation (not shown). The limits of quantification (LOQ) in serum for AA and GA were estimated to be 10 and 100 nM, respectively, based on a 100 μl serum sample size and 90 μl injected on-column (signal/noise ratio=10); the

Discussion

The present toxicokinetic study in mice extends our knowledge about the metabolism and disposition of AA and permits interspecies comparisons and predictions. Two previous studies of AA toxicokinetics in rats [21], [22] showed >2-fold longer elimination half-lives for AA than we observed in mice. In contrast, the mice had an elimination half-life for GA (1.9 h) identical to that measured in rats [22]. In mice, essentially identical rates were observed for linear phases corresponding to

Acknowledgements

This research was supported in part by Interagency Agreement #224-93-0001 between NCTR/FDA and the National Institute for Environmental Health Sciences/National Toxicology Program. G.G.C. thanks Fundacão Para a cıêncıa e Tecnologia, Portugal, for a postdoctoral fellowship.

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