Absence of acrylamide-induced genotoxicity in CYP2E1-null mice: evidence consistent with a glycidamide-mediated effect.
ABSTRACT Acrylamide, an animal carcinogen and germ cell mutagen present at low (ppm) levels in heated carbohydrate-containing foodstuffs, is oxidized by cytochrome P4502E1 (CYP2E1) to the epoxide glycidamide, which is believed to be responsible for the mutagenic and carcinogenic activity of acrylamide. We recently reported a comparison of the effects of acrylamide on the genetic integrity of germ cells of male wild-type and CYP2E1-null mice [B.I. Ghanayem, K.L. Witt, L. El-Hadri, U. Hoffler, G.E. Kissling, M.D. Shelby, J.B. Bishop, Comparison of germ-cell mutagenicity in male CYP2E1-null and wild-type mice treated with acrylamide: evidence supporting a glycidamide-mediated effect, Biol. Reprod. 72 (2005) 157-163]. In those experiments, dose-related increases in dominant lethal mutations were detected in uterine contents of female mice mated to acrylamide-treated wild-type males but not CYP2E1-null males, clearly implicating CYP2E1-mediated formation of glycidamide in the induction of genetic damage in male germ cells. We hypothesized that acrylamide-induced somatic cell damage is also caused by glycidamide. Therefore, to examine this hypothesis, female wild-type and CYP2E1-null mice were administered acrylamide (0, 25, 50mg/kg) by intraperitoneal injection once daily for 5 consecutive days. Twenty-four hours after the final treatment, blood and tissue samples were collected. Erythrocyte micronucleus frequencies were determined using flow cytometry and DNA damage was assessed in leukocytes, liver, and lung using the alkaline (pH>13) single cell gel electrophoresis (Comet) assay. Results were consistent with the earlier observations in male germ cells: significant dose-related increases in micronucleated erythrocytes and DNA damage in somatic cells were induced in acrylamide-treated wild-type but not in the CYP2E1-null mice. These results support the hypothesis that genetic damage in somatic and germ cells of mice-treated with acrylamide is dependent upon metabolism of the parent compound by CYP2E1. This dependency on metabolism has implications for the assessment of human risks resulting from occupational or dietary exposure to acrylamide. CYP2E1 polymorphisms and variability in CYP2E1 activity associated with, for example, diabetes, obesity, starvation, and alcohol consumption, may result in altered metabolic efficiencies leading to differential susceptibilities to acrylamide toxicities in humans.
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ABSTRACT: This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.Environmental and Molecular Mutagenesis 02/2001; 38(1):59-68. · 3.71 Impact Factor
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ABSTRACT: Monomeric acrylamide is an important industrial chemical primarily used in the production of polymers and copolymers. It is also used for producing grouts and soil stabilizers. Acrylamide's neurotoxic properties have been well documented. This review will focus on pertinent information concerning other, non-neurotoxic, effects observed after exposure to acrylamide, including: its genotoxic, carcinogenic, reproductive, and developmental effects. It will also cover its absorption, metabolism, and distribution. The data show that acrylamide is capable of inducing genotoxic, carcinogenic, developmental, and reproductive effects in tested organisms. Thus, acrylamide may pose more than a neurotoxic health hazard to exposed humans. Acrylamide is a small organic molecule with very high water solubility. These properties probably facilitate its rapid absorption and distribution throughout the body. After absorption, acrylamide is rapidly metabolized, primarily by glutathione conjugation, and the majority of applied material is excreted within 24 h. Preferential bioconcentration of acrylamide and/or its metabolites is not observed although it appears to persist in tests and skin. Acrylamide can bind to DNA, presumably via a Michael addition-type reaction, which has implications for its genotoxic and carcinogenic potential. The available evidence suggests that acrylamide does not produce detectable gene mutations, but that the major concern for its genotoxicity is its clastogenic activity. This clastogenic activity has been observed in germinal tissues which suggest the possible heritability of acrylamide-induced DNA alterations. Since there is 'sufficient evidence' of carcinogenicity in experimental animals as outlined under the U.S. EPA proposed guidelines for carcinogen risk assessment, acrylamide should be categorized as a 'B2' carcinogen and therefore be considered a 'probable human carcinogen.' The very limited human epidemiological data do not provide sufficient evidence to enable one to judge the actual carcinogenic risk to humans. Acrylamide is able to cross the placenta, reach significant concentrations in the conceptus and produce direct developmental and post-natal effects in rodent offspring. It appears that acrylamide may produce neurotoxic effects in neonates from exposures not overtly toxic to the mothers. Acrylamide has an adverse effect on reproduction as evidenced by dominant lethal effects, degeneration of testicular epithelial tissue, and sperm-head abnormalities.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 02/1988; 195(1):45-77. · 3.90 Impact Factor
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ABSTRACT: Acrylamide (AM), which is used to manufacture polymers, is carcinogenic and a reproductive and neurological toxicant. The objective of this study was to compare the metabolism of AM administered orally (po), dermally, intraperitoneally (ip), or by inhalation, and to measure the hemoglobin adducts produced. Rats and mice were exposed to 2.9 ppm [1,2,3-13C] and [2,3-14C]AM for 6 h. [2,3-14C]AM (162 mg/kg) or [1,2,3-13C]AM (13 8 mg/kg) in water was administered dermally to rats for 24 h, and [1,2,3-13C]AM was administered ip (47 mg/kg). Urine and feces were collected for 24 h. Urine was the major elimination route in rats (ip, 62% and po, 53% of the dose; dermal, 44% of the absorbed dose; inhalation, 31% of the recovered radioactivity) and mice (inhalation, 27% of the recovered radioactivity). Signals in the 13C-NMR spectra of urine were assigned to previously identified metabolites derived from AM glutathione conjugation (AM-GSH) and conversion to glycidamide (GA). AM-GSH was a major metabolic route in rats accounting for 69% (ip), 71% (po), 52% (dermal), and 64% (inhalation). In mice, AM-GSH accounted for only 27% (inhalation) of the total urinary metabolites. The remaining urinary metabolites were derived from GA. Valine hemoglobin adducts of AM and GA were characterized using liquid chromatography-mass spectrometry. The ratio of AM to GA adducts paralleled the flux through pathways based on urinary metabolites. This study demonstrates marked species differences in the metabolism and internal dose (Hb-adducts) of AM following inhalation exposure and marked differences in uptake comparing dermal with po and ip administration.Toxicological Sciences 11/2003; 75(2):260-70. · 4.33 Impact Factor