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BOR - Papers in Press, published online ahead of print September 8, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.033308
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BIOLOGY OF REPRODUCTION 72, 157–163 (2005)
DOI: 10.1095/biolreprod.104.033308
© 2005 by the Society for the Study of Reproduction, Inc.

Comparison of Germ Cell Mutagenicity in Male CYP2E1-Null and Wild-Type Mice Treated with Acrylamide: Evidence Supporting a Glycidamide-Mediated Effect

B.I. Ghanayem1,2, K.L. Witt3, L. El-Hadri2, U. Hoffler2, G.E. Kissling4, M.D. Shelby5, and J.B. Bishop3

Laboratory of Pharmacology and Chemistry,2 Toxicology Operations Branch,3 Biostatistics Branch,4 Office of Program Development,5 National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acrylamide is an animal carcinogen and probable human carcinogen present in appreciable amounts in heated carbohydrate-rich foodstuffs. It is also a germ cell mutagen, inducing dominant lethal mutations and heritable chromosomal translocations in postmeiotic sperm of treated mice. Acrylamide's affinity for male germ cells has sometimes been overlooked in assessing its toxicity and defining human health risks. Previous investigations of acrylamide's germ cell activity in mice showed stronger effects after repeated administration of low doses compared with a single high dose, suggesting the possible involvement of a stable metabolite. A key oxidative metabolite of acrylamide is the epoxide glycidamide, generated by cytochrome P4502E1 (CYP2E1). To explore the role of CYP2E1 metabolism in the germ cell mutagenicity of acrylamide, CYP2E1-null and wild-type male mice were treated by intraperitoneal injection with 0, 12.5, 25, or 50 mg acrylamide (5 ml saline)–1 kg–1 day–1 for 5 consecutive days. At defined times after exposure, males were mated to untreated B6C3F1 females. Females were killed in late gestation and uterine contents were examined. Dose-related increases in resorption moles (chromosomally aberrant embryos) and decreases in the numbers of pregnant females and the proportion of living fetuses were seen in females mated to acrylamide-treated wild-type mice. No changes in any fertility parameters were seen in females mated to acrylamide-treated CYP2E1-null mice. Our results constitute the first unequivocal demonstration that acrylamide-induced germ cell mutations in male mice require CYP2E1-mediated epoxidation of acrylamide. Thus, CYP2E1 polymorphisms in human populations, resulting in variable enzyme metabolic activities, may produce differential susceptibilities to acrylamide toxicities.

acrylamide, CYP2E1-null mice, dominant lethals, environment, male reproductive tract, reproductive toxicant, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acrylamide is a rodent carcinogen and probable human carcinogen [1, 2], and a mutagen in mammalian somatic cells in vitro and in vivo, particularly in assays that detect induction of chromosomal damage [1, 3–7]. In addition, acrylamide is a mammalian germ cell mutagen, inducing high frequencies of dominant lethal mutations (generally associated with chromosomal alterations that result in death of embryos around the time of implantation) [8–12], heritable chromosomal translocations [12–14], and specific locus mutations [15] in postmeiotic sperm and spermatogonial stem cells [9] of male mice.

Human exposure to acrylamide occurs during manufacturing and through its use in polyacrylamide gels, as a grouting agent and soil conditioner, and in stabilization of tunnel and dam structures [2]. The recent discovery of acrylamide in grain-based and carbohydrate-rich foodstuffs subjected to high heat during processing [16–19] has added to concerns over the potential risks for detrimental health effects resulting from human exposure to this chemical. An FAO/WHO [19] panel estimated that the general public is exposed to 0.3–0.8 µg acrylamide kg–1 day–1 through food intake; children have 2–3 times the exposures of adults, when calculations are expressed on a bodyweight basis. The U.S. Food and Drug Administration issued preliminary exposure estimates of acrylamide from food sources of 0.43 µg kg–1 day–1 for adults and 1.06 µg kg–1 day–1 for children [20]. The review by the European Commission of dietary acrylamide exposure data (http://europa.eu.int/comm/food/fs/sc/scf/out131_en.pdf) reported estimated acrylamide intakes of 10–100 µg kg–1 day–1 by one group and lower intakes, ranging from 0.36 to 2.1 µg kg–1 day–1, were estimated by a second group. The recent discovery of acrylamide in food has increased the need for improved assessment of risk factors of human exposure to acrylamide, not only because of the possible cancer risk for populations or individuals who consume high amounts of acrylamide-containing foods but also for the potential risk of increased frequencies of germ cell mutations and resulting adverse pregnancy outcomes.

Dearfield et al. [7], in a review of the genotoxicity of acrylamide, discussed three possible metabolic pathways for acrylamide: radical-mediated polymerization, most efficiently conducted under anaerobic conditions and used to generate polyacrylamide; Michael-type reactions resulting from the alpha-beta unsaturated characteristics of the acrylamide molecule; and oxidative metabolism of the acrylamide double bond to yield the epoxide, glycidamide. Glycidamide is a relatively stable intermediate, having an in vivo half-life of 1.5 h in the rat and demonstrating a pattern of even distribution among tissues in mice and rats after acrylamide dosing [21].

The affinity of acrylamide for germinal tissues was illustrated by whole-body radiographic studies of the systemic distribution of radiolabeled acrylamide in male mice, which showed intense accumulation of acrylamide, or a metabolite, in testis, then epididymis, and finally, the glans penis, during a period of 1 h to 9 days posttreatment [22]. The mechanism(s) by which acrylamide induces germ cell mutagenic effects is not clear. Sega et al. [23] proposed that alkylation of the sulfhydryl groups of sperm protamine following acrylamide exposure produced the observed germ cell chromosomal damage in treated male mice. Subsequent studies of genetic effects, DNA adduct formation, and unscheduled DNA synthesis in spermiogenic cells after acrylamide exposure led to the proposal of a glycidamide-mediated mutagenic pathway via DNA or protamine alkylation [24–26]. Consistent with this hypothesis, glycidamide has been shown to induce dominant lethal mutations in male mice, with the most sensitive stages being spermatozoa of the testis and epididymis [27]; these are the same stages that show the highest sensitivity to dominant lethal induction by acrylamide.

Investigation of the oxidative metabolism of acrylamide in mice by Sumner et al. [28] using 13C-NMR to compare urinary metabolites in CYP2E1–/– (CYP2E1-null) versus CYP2E1+/+ (wild-type) mice treated with 13C-acrylamide demonstrated that oxidative metabolism of acrylamide in wild-type mice via CYP2E1 generated the reactive epoxide glycidamide. In contrast, they identified no urinary metabolites originating from the epoxidation of acrylamide in the urine of CYP2E1-null mice. Sumner et al. [28] concluded that metabolism of the parent compound was exclusively routed through direct glutathione conjugation, no detectable glycidamide was formed in the absence of CYP2E1, and there appeared to be no alternative pathways for the oxidative metabolism of acrylamide.

Our hypothesis centers on the premise that glycidamide is responsible for acrylamide-induced germ cell mutations in mice. The current investigations were undertaken to assess the role of acrylamide epoxidation to glycidamide in the induction of dominant lethal mutations in male mouse germ cells using CYP2E1-null and wild-type mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

Acrylamide (CAS no. 79-06-1; >99.5% pure) was manufactured by Fluka Chemie GmbH and purchased from Sigma-Aldrich Laborchemikalien GmbH (Milwaukee, WI). All dosing solutions were made daily by mixing acrylamide with normal saline (0.9%); dosing volume was 5 ml/ kg body weight.

Animal Husbandry

CYP2E1–/– (CYP2E1-null) and CYP2E1+/+ (wild-type) mouse strains were obtained from a colony developed in the laboratory of Dr. Frank Gonzalez (National Cancer Institute, Bethesda, MD) [29], and maintained by inbreeding at Charles River Laboratories (Wilmington, MA) (see Hoffler et al. [30] for details of stock development and maintenance). Nullizygosity of the CYP2E1-null mice was confirmed using Western blot analysis as previously described [31]. Male wild-type and CYP2E1-null mice were approximately 8 wk old and ranged in weight from 20 to 24 g at the beginning of each study. Female B6C3F1 (Taconic Laboratories, Germantown, NY) were also 8–9 wk old and weighed 20–24 g at the beginning of the study. All animals were housed in controlled environment facilities with a 12L:12D cycle and were fed National Institutes of Health (NIH) #31 diet and tap water. Both food and water were available ad libitum throughout the experiments. All animals were acclimated for a minimum of 1 wk before the start of the studies and all animal care and experimental procedures were conducted in strict accordance with NIH animal care and use guidelines (National Research Council's Guide for Care and Use of Laboratory Animals, 1996, National Academy of Sciences).

Experimental Design

For the three studies described below, acrylamide was administered to groups of CYP2E1-null and wild-type male mice by intraperitoneal (i.p.) injection at dose levels of 0, 12.5, 25, or 50 mg (5 ml saline)–1 kg–1 day–1 for 5 consecutive days. Matching vehicle controls were treated with 5 ml/ kg saline/day (i.p.), for 5 consecutive days. The i.p. injection was selected for the route of administration because this study was designed to investigate the role of CYP2E1 metabolism in the germ cell damage observed after acrylamide treatment, and i.p. injection was the route used in previous dominant lethal experiments with acrylamide [8] and glycidamide [27]. The number and distribution of mice varied among treatment groups and experiments and was dependent primarily on the availability of age-, weight-, and sex-matched CYP2E1-null and wild-type mice. In all experiments described below, male mice were mated to untreated virgin B6C3F1 female mice (3 females/male in study 1; 2 females/male in studies 2 and 3). Mating schemes are presented in Figure 1. The experimental procedures followed in the three studies are described below.



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  		FIG. 1. Dosing and mating schemes employed in studies 1–3, spermiogenic stages exposed, and spermatogenic stages sampled in each mating period. For example, the dosing and mating periods employed in study 1 sampled sperm a minimum of 7 days posttreatment (the shortest interval between the end of dosing and the initiation of cohabitation) up to a maximum of 17 days posttreatment (the longest interval, spanning the time from the beginning of dosing to the last day of cohabitation). Dark hatched areas on the spermatogenesis timeline indicate the spermiogenic period of greatest sensitivity to acrylamide (Days 5–8); light hatched areas indicate periods of lower sensitivity and nonhatched portions of the Stages sampled bars indicate nonsensitive spermiogenic stages [27]

Study 1

Untreated male CYP2E1-null and wild-type mice (30 per genotype) were each cohabited with three untreated B6C3F1 female mice for a period of 5 days. At 48 h after the end of this mating period, the 30 males within each genotype were subdivided into two groups. One group of 10 males/ genotype received saline (i.p., once daily for 5 consecutive days) and the other group of 20 males/genotype received 50 mg/kg acrylamide (i.p., once daily for 5 consecutive days). One week after treatments ended, males were cohabited with three females each, for a second round of mating, for a period of 5 days. Approximately 13 days after the last day of cohabitation, each female was humanely killed with CO2/O2 and uterine contents were carefully examined for the following endpoints: total number of implantation sites, number of live fetuses, number of resorption moles, number of early and late dead embryos, and number of dead fetuses [32]. Live fetuses were humanely killed after examination.

Study 2

Because the 50 mg/kg dose of acrylamide used in study 1 produced unacceptable levels of sterility in treated wild-type males, an investigation was conducted, using only wild-type mice, to identify lower doses of acrylamide that would induce dominant lethal mutations while maintaining fertility at consistently high levels. Thus, wild-type mice were treated with acrylamide at 0 (5 mice), 12.5 (12 mice), or 25 (13 mice) mg kg–1 day–1 for 5 consecutive days, as described above. Forty-eight hours after the last dose of acrylamide, each of the males was cohabited with two females for a period of 5 days. At the end of the 5-day mating period, females were removed and replaced with two females for a second 5-day mating period. Approximately 13 days after the last day of cohabitation (for each of the two mating periods), females were humanely killed with CO2/O2 and uterine contents were carefully examined as described above for study 1. Live fetuses were humanely killed after examination.

Study 3

Results of study 2 indicated that 12.5 and 25 mg/kg acrylamide were appropriate dose levels for use in a more definitive third study. Thus, CYP2E1-null and wild-type male mice were treated with acrylamide doses of 0, 12.5, or 25 mg kg–1 day–1 for 5 consecutive days, as described above for study 2. The saline-vehicle control groups consisted of 8 CYP2E1-null and 11 wild-type males; the two acrylamide dose groups consisted of 12 CYP2E1-null and 13 wild-type males each. Forty-eight hours after the last dose of acrylamide, each male was mated to two females for a period of 5 days. At the end of the 5-day mating period, females were removed and replaced with two females for a second 5-day mating period. Approximately 13 days after the end of each of the two cohabitation periods, females were humanely killed with CO2/O2 and uterine contents were carefully examined as described above for study 1. Live fetuses were humanely killed after examination.

Statistical Methods

Extrabinomial variability of proportions of adverse events was tested using the binomial variance test and Tarone test for binomial overdispersion [33]. Extrabinomial variability of proportions can arise when proportions vary substantially among females mated to each male, in addition to varying among males; when detected, it must be addressed in the statistical analyses. Extrabinomial variability was present only for resorptions in wild-type controls in the first mating period of study 3. In addition, number of implantations, percentage of resorptions, and percentage of live fetuses were not normally distributed. Transformations, such as the arcsine transformation, did not improve normality [34]. Therefore, nonparametric methods were used to make comparisons among dose groups and among genotype groups, and the litter was considered the unit of analysis. Kruskal-Wallis analysis of variance was applied to litter counts and percentages, and where significant differences across groups were found, pairs of groups were compared using the Mann-Whitney U-test [34]. Percentages of pregnant females were compared across groups using a chi-square test in study 1 [34]. In studies 2 and 3, dose-related trends were tested using the Jonckheere-Terpstra test [35]. Significant overall group differences in percentage of pregnant females were followed by Fisher exact test to identify which pairs of groups differed [36]. In studies 2 and 3, Week 1 outcomes were compared with Week 2 outcomes by considering the male as the unit of analysis. For each male, the total numbers of implantations, percentages of resorptions, and percentage of live fetuses were calculated for each week. These outcomes were then compared between Week 1 and Week 2 using the Wilcoxon signed-ranks test, which takes advantage of the paired nature of the data [34].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The fertility parameters most pertinent to the evaluation of the dominant lethality of acrylamide are percentage (%) pregnant females (Fig. 2), the mean number of implants per pregnant female (Fig. 3), percentage live fetuses per pregnant female (Fig. 4), and percentage resorptions per pregnant female (Fig. 5). None of these four fertility parameters were significantly altered in females mated to CYP2E1-null male mice treated with 12.5, 25, or 50 mg acrylamide kg–1 day–1 for 5 days. Thus, the combined results from the three independent studies that comprised this investigation clearly demonstrated that exposure to acrylamide for 5 consecutive days, at doses up to 50 mg kg–1 day–1, had no effect on the fertility of CYP2E1-null male mice. In contrast, acrylamide exposure induced marked reproductive effects in wild-type male mice.



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  		FIG. 2. Percentage (%) of pregnant females out of the total number of females mated by treatment group, genotype, dose of acrylamide, and mating period for each of the three studies. The numbers at the top of each bar are the actual numbers of pregnant females out of total number of females cohabited per treatment group. *, Significantly less than control at P ≤ 0.05; {dagger}, significantly different from same dose CYP2E1-null treatment group at P ≤ 0.05



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  		FIG. 3. Mean number of implantations per pregnant female (±SEM) by treatment group, genotype, dose of acrylamide, and mating period for each of the three studies. *, Significantly less than control at P ≤ 0.05; {dagger}, significantly less than same dose CYP2E1-null treatment group at P ≤ 0.05



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  		FIG. 4. Percentage (%) of live fetuses out of the total implantation sites per pregnant female (±SEM) by treatment group, genotype, dose of acrylamide, and mating period for each of the three studies. *, Significantly less than control at P ≤ 0.05; {dagger}, significantly less than same dose CYP2E1-null treatment group at P ≤ 0.05



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  		FIG. 5. Percentage (%) of resorption moles out of the total implantation sites per pregnant female (±SEM) by treatment group, genotype, dose of acrylamide, and mating period for each of the three studies. *, Significantly greater than control at P ≤ 0.05; {dagger}, significantly greater than same dose CYP2E1-null treatment group at P ≤ 0.05

Percentage Pregnant

In study 1, only 3 of 60 females mated to wild-type males treated with 50 mg/kg acrylamide for 5 days became pregnant (Fig. 2); the data from studies 2 and 3 showed that females mated to wild-type males treated with lower doses of acrylamide, 12.5 or 25 mg kg–1 day–1, did not show significant reductions in pregnancies, compared with the corresponding control groups (Fig. 2). The proportion of females that became pregnant after cohabitation with acrylamide-treated males was the least sensitive of the four indicators of the reproductive toxicity of acrylamide.

Implantations per Female

Treatment of wild-type male mice with acrylamide resulted in a reduction in the mean number of implantations per pregnant female in some dose groups or mating periods (Fig. 3). In study 1, the three pregnant females mated to wild-type males treated with 50 mg/kg acrylamide had a significantly reduced mean number of implantations (P < 0.01) compared with the two wild-type control groups as well as the acrylamide-treated CYP2E1-null group. In study 2, no effect on the number of implantations was noted in females mated to acrylamide-treated wild-type males during the first mating period; however, females mated to 12.5 or 25 mg/kg acrylamide-treated males during the second mating period had significant reductions (P < 0.01) in the number of implantation sites compared with females mated to the vehicle controls. These results indicate greater acrylamide-induced damage to sperm exposed during the earlier developmental stages (spermatids) sampled in the second mating period (Fig. 1). The same pattern of reduced implantation sites in females mated in the second mating period to wild-type males exposed to acrylamide was seen in study 3. Thus, acrylamide, at doses of 12.5–50 mg kg–1 day–1 for 5 consecutive days, produced dose-related decreases in the mean number of implantation sites per pregnant female in the second mating period (Fig. 3).

Percentage Live Fetuses per Female

Acrylamide treatment reduced the percentage live fetuses per pregnant female (percentage of live fetuses out of the total number of implantations) in particular dose groups and mating periods (Fig. 4). The percentage live fetuses per pregnant female was reduced from approximately 96% in the control groups to 44% in the females mated to wild-type males treated with 50 mg/kg acrylamide (P < 0.01). In study 2 (wild-type mice only), the reductions in percentage live fetuses were greater in matings that occurred during the first period, compared with those that occurred during the second mating period. This contrasts with the data on implantation reduction, which showed greater effects in the second mating period. However, in our experience with dominant lethal tests with a variety of chemicals, effects on implantation data tend to be more variable than effects on resorption and live fetus data, and thus the latter endpoints are more reliable indicators of the most sensitive treatment window. In general, the results for mating periods 1 and 2 are consistent with each other and with the literature and indicate critical sensitivity of condensed spermatids and early epididymal spermatozoa.

Percent Resorptions

The clearest indicator of dominant lethality is the percentage resorptions (mean proportions of implants/female that are resorptions) in females mated to treated males, and this parameter was dramatically altered in our studies: dose-related increases in percentage resorptions were seen in females mated to acrylamide-treated wild-type males (Fig. 5). No significant increases in resorptions were observed in females mated to CYP2E1-null males treated with 12.5–50 mg acrylamide kg–1 day–1 for 5 consecutive days in any of the three studies (Fig. 5). The inverse relationship between percentage resorptions and percentage live fetuses in the studies reported here clearly demonstrates that resorption replaced living fetuses in females mated to wild-type mice treated with acrylamide in a dose-dependent manner (Fig. 6).



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  		FIG. 6. Relationship between the percentage live fetuses (Fig. 4) and the percentage resorptions (Fig. 5). Error bars indicate SEM. Live fetuses are replaced by resorption moles (embryonic death) as dose of acrylamide increases. A similar pattern is seen in both mating periods, although the effects are slightly greater in mating period 1, which included the entire period of greatest sperm stage sensitivity


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The contrasting observations of increased levels of dominant lethal mutations in acrylamide-treated wild-type mice compared with the lack of reproductive effects in acrylamide-treated CYP2E1-null mice demonstrate that in vivo metabolism of acrylamide to the epoxide intermediate, glycidamide, is a prerequisite for the induction of dominant lethal mutations in postmeiotic germ cells of male mice. The CYP2E1 pathway is the only pathway by which acrylamide is oxidatively metabolized to glycidamide in wild-type mice [28]. In the absence of a functional CYP2E1 enzyme, metabolism of acrylamide in mice is directed primarily through glutathione conjugation [28]. Further confirmation of the requirement for a functional CYP2E1 enzyme in the metabolism of acrylamide to glycidamide comes from studies in which we treated CYP2E1-null and wild-type mice with acrylamide and used 13C NMR spectroscopy to identify urinary metabolites (urine collected over a 24-h period following a single i.p. dose of 50 mg acrylamide/kg). We detected no glycidamide or metabolites of glycidamide in the urine of CYP2E1-null mice, and all the detected urinary metabolites of acrylamide in these mice were derived from glutathione conjugation of the parent molecule [28, 37]. In contrast, a significant portion of acrylamide administered to wild-type mice was eliminated in the urine as glycidamide and other metabolites derived from this epoxide [28, 37]. Collectively, these results demonstrate that negligible formation of glycidamide occurs in CYP2E1-null mice treated with acrylamide.

Mature sperm through late spermatids are the spermiogenic stages sensitive to dominant lethal induction by acrylamide, with the period of greatest sensitivity being approximately 5–8 days after dosing [8, 12, 13, 38]. The mating schemes employed in this study overlapped in this period of greatest sensitivity (Fig. 1). This may be why comparative evaluations of some reproductive endpoints in the first and second mating periods in acrylamide-treated wild-type mice did not always reveal significant differences. The inverse relationship between percentage live fetuses and percentage resorptions expected in a positive dominant lethal assay was clearly apparent in the data from both the Week 1 and Week 2 matings in study 3 (Fig. 6). Both of these critical endpoints were more affected in the Week 1 matings that spanned the entire period of greatest sensitivity, consistent with previously published data [8, 27].

The present studies support the hypothesis that the epoxide intermediate of acrylamide, glycidamide, is the ultimate germ cell mutagen and may act by binding to nucleophilic sites in chromatin of late spermatids and early spermatozoa. Our experiments do not identify the location of the binding site, however. Whether glycidamide binds to the sulfhydryl groups of cysteine-rich protamines, to DNA, or both remains a question to be answered. The parent compound acrylamide should theoretically be able to directly alkylate DNA through Michael-type reactions, by virtue of the double bond linking the alpha and beta carbons [39], without a requirement for metabolism to the glycidamide intermediate. However, measurements of acrylamide adducts in sperm DNA and sperm protamine showed acrylamide to be only weakly effective in forming adducts in sperm DNA in vivo but quite efficient at alkylating cysteine-rich protamine in vitro [25]. Extending this observation, Sega [26] showed that the total amount of sperm head alkylation measured in mice after acrylamide exposure closely matched the amount of sperm protamine alkylation; alkylation of DNA after acrylamide dosing accounted for less than 5% of the total sperm head alkylation. Together, these adduct data strongly indicate a significant role for protamine alkylation in the induction of dominant lethal mutations in mice by acrylamide.

Acrylamide-induced genetic alterations in sperm might indeed arise through a mechanism that doesn't involve direct alkylation of DNA. Sega [26] suggested that DNA breakage in late spermatids and early spermatozoa could be induced by protamine alkylation and consequent chromatin strand distortion. Because these later spermiogenic stages are no longer DNA repair competent, any DNA damage induced at these stages would persist. It is during this period of spermatogenesis that histones are replaced by simpler, cysteine-rich protamines and the increasingly compacted DNA becomes rapidly inaccessible to interaction with chemicals. Thus, because acrylamide is most effective at inducing genetic damage in late spermatids and early spermatozoa, it appears more likely that it acts via formation of protamine adducts than through direct DNA alkylation.

On the other hand, although acrylamide is a weak alkylator of DNA in vivo [25], metabolism of acrylamide to glycidamide may enhance alkylation of the relatively weaker nucleophilic sites in DNA. Wang et al. [31] have shown that acrylonitrile, another CYP2E1 substrate, can alkylate the sulfhydryl groups of cysteine but only its epoxide metabolite, glycidonitrile, can react with nucleic acids. In experiments in our laboratory, we detected a significant increase in glycidamide-derived DNA adducts in whole testes from wild-type mice treated with acrylamide, confirming that glycidamide can form DNA adducts in mixed-cell testicular tissue of mice; negligible levels of these DNA adducts were detected in CYP2E1-null mice exposed to acrylamide (unpublished data). It is important to note that the testicular tissue we examined was from a mix of cell types, not just sperm. In addition, recent preliminary results from our laboratory showed that, while high levels of acrylamide were detectable in plasma of treated CYP2E1-null mice, negligible levels were found in the plasma of wild-type mice, suggesting that distribution and persistence of the parent molecule occurs (unpublished data). However, because we saw no induction of dominant lethal mutations in CYP2E1-null mice in the current study, it appears that even if acrylamide does alkylate DNA or protamines directly, this action is ineffective in inducing detectable germ cell damage.

To our knowledge, there has been no pharmacokinetic studies in CYP2E1-null mice designed to measure the distribution of unmetabolized acrylamide. The earlier studies of Marlowe et al. [22] in wild-type mice showed radiolabeled acrylamide moving from the site of administration to the testes and eventually to the glans penis in a fashion comparable with movement of sperm, but the radiolabel may have represented glycidamide rather than acrylamide; this experiment was not designed to distinguish the parent compound from the metabolites. Thus, the degree of direct testicular exposure to acrylamide as well as acrylamide's ability to alkylate protamine in vivo remains unresolved at this time. However, results from our comparative dominant lethal study using wild-type and CYP2E1-null mice are not contradictory to the hypothesis [25] that acrylamide is metabolized in the liver to glycidamide and that, although some metabolite might bind to liver DNA, some is transported to the testes, where it binds to accessible sperm DNA or, more likely, to sperm protamines, resulting in chromosomal damage that leads to death of early postimplantation conceptuses. It should be emphasized that CYP2E1 is present in many tissues, including the testes [40, 41], and that in situ metabolism of acrylamide to glycidamide in the testis may occur.

The confirmed presence of acrylamide in common carbohydrate foodstuffs, cooked at high temperatures, implies potential widespread exposure of the general population to acrylamide via the consumption of acrylamide-containing foods [19, 20, 42]. Estimates of human exposure range from 0.3 to 100 µg acrylamide (kg body weight)–1 day–1, as compiled from three large population studies in Europe and the United States [19, 20, http://europa.eu.int/comm/food/fs/sc/scf/out131_en.pdf]. The U.S. National Toxicology Program's Center for the Evaluation of Risks to Human Reproduction recently issued an expert panel report assessing acrylamide exposure in humans and the potential for acrylamide-induced genetic damage in germ cells [http://cerhr.niehs.nih.gov/news/acrylamide/final_report.pdf]. This report provides details on exposure levels in the general population as well as in occupationally exposed groups and smokers. Reviewing all the available data, panel members agreed that the general nonsmoking population is exposed to approximately 0.5–1.0 µg acrylamide (kg body weight)–1 day–1; estimated exposures in children 2–5 yr of age were 2–3 times the adult levels when expressed as a body weight ratio [page 6 of the report]. Although these estimated typical human exposure levels are markedly lower than the levels in treated laboratory animals, the panel cautioned that dose-response information for heritable effects in humans or animals is limited [page 151 of the report]. Additional evidence of human exposure comes from the identification of hemoglobin adducts of acrylamide in workers in surfactants production and the textile industry [43]. Most recently, glycidamide was identified in the urine of humans exposed to low levels of acrylamide [44], indicating that humans are capable of metabolizing acrylamide, presumably via CYP2E1, to glycidamide. Because our animal data and data from human studies [43] both implicate a critical role for CYP2E1 enzymes in conversion of acrylamide to the mutagenically active epoxide intermediate, glycidamide, a consideration of polymorphic enzyme variants in humans is integral to the determination of human risk of germ cell damage from low-dose chronic acrylamide exposure. Variable metabolic capacities linked to genetic polymorphisms in the CYP2E1 gene produce differences in the ability to metabolize a number of drugs, environmental pollutants, and other agents, and thus individual risks vary [45–48].

In conclusion, the current work confirmed that acrylamide is a potent inducer of dominant lethal mutations in male mice and that this effect is directly related to dose. Furthermore, our results provide the first direct demonstration that this dominant lethal effect of acrylamide is dependent on acrylamide epoxidation to glycidamide by CYP2E1.


   ACKNOWLEDGMENTS
 
The authors are grateful to Sue Edelstein of Image Associates for her patience and expert assistance in preparing the figures for this manuscript. In addition, we extend our sincere thanks to Drs. Paul Foster and Melissa Rhodes, NIEHS, and Dr. Sally Darney, EPA, for thorough and thoughtful reviews of this manuscript.


   FOOTNOTES
 
1 Correspondence: Burhan I. Ghanayem, National Institute of Environmental Health Sciences, P.O. Box 12233, MD B3-10, 111 Alexander Dr., Research Triangle Park, NC 27709. FAX: 919 541 4632; ghanayem{at}niehs.nih.gov Back

Received: 17 June 2004.

First decision: 8 July 2004.

Accepted: 27 August 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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