Comparison of estimated dietary intake of acrylamide with hemoglobin adducts of acrylamide and glycidamide.

In a study comprising 50 subjects, we investigated the relationship between acrylamide (AA) intake from food using food frequency questionnaires and the concentration of hemoglobin (Hb) adducts of AA and its genotoxic metabolite glycidamide (GA) as a measure of the internal exposure. A method using solid-phase extraction and liquid chromatography with negative electrospray tandem mass spectrometric (MS/MS) detection for the determination of the Hb adducts as phenylthiohydantoin derivatives in human blood was developed. The limit of quantification for AA- and GA-Hb adducts were 2 and 6 pmol/g globin, respectively, and the between-assay precision was below 25%. The estimated dietary intake of AA was (median and range) 13.5 microg/day (4.1-72.6) in nonsmokers and 18.3 microg/day (7.8-32.0) in smokers. In nonsmokers, males had a higher intake than females, 16.6 microg/day (18.6-72.6) and 12.8 microg/day (4.1-30.2), respectively. Nonsmokers had a median AA and GA adduct concentration of 36.8 (range 17.9-65.5) and 18.2 (range 6.7-45.6) pmol/g globin, respectively. In smokers, the values were 165.8 (98.8-211) and 83.2 (29.1-99.0) pmol/g globin, respectively. Using multiple linear regression analysis, a significant positive correlation was found between the AA-Hb adduct concentration and the intake of chips/snacks and crisp bread. GA-Hb adduct did not correlate with consumption of any of the main food groups. Neither AA-Hb nor GA-Hb adduct concentration correlated with total dietary intake of AA as calculated from the reported food intake. Adduct concentrations did not correlate with 24 h urinary excretion of mercapturic acid metabolites of AA and GA in the same subjects reported previously.     

Comparison of the hemoglobin adducts formed by administration of N-methylolacrylamide and acrylamide to rats.

Acrylamide (AM) and N-methylolacrylamide (NMA) are used in the formulation of grouting materials. AM undergoes metabolism to a reactive epoxide, glycidamide (GA). Both AM and GA react with hemoglobin to form adducts that can be related to exposure to AM. The objective of this study was to evaluate the extent to which NMA could form the same adducts as AM. N-(2-carbamoylethyl)valine (AAVal derived from AM) and N-(2-carbamoyl-2-hydroxyethyl)valine (GAVal derived from GA) were measured following a single oral dose of AM (50 mg/kg) or NMA (71 mg/kg) in male F344 rats. AAVal and GAVal were measured by a modified Edman degradation to produce phenylthiohydantoin derivatives and liquid chromatography/tandem mass spectrometry. In AM-treated rats, AAVal was 21 +/- 1.7-pmol/mg globin (mean +/- SD, n = 4), and GAVal was 7.9 +/- 0.8 pmol/mg. In NMA-treated rats, AAVal was 41 +/- 4.9 pmol/mg, and GAVal was 1.4 +/- 0.1 pmol/mg. Whether AAVal was derived from reaction of NMA with globin followed by loss of the hydroxymethyl group, or loss of the hydroxymethyl group to form AM prior to reaction with globin, is not known. However, the higher ratio of AAVal:GAVal in NMA-treated rats (29 vs. 2.6 in AM-treated rats) suggests that reaction of NMA with globin is the predominant route to AAVal in NMA-treated rats. The detection of GAVal in NMA-treated rats indicates oxidation of NMA, either directly or following conversion to AM. The lower levels of GAVal on NMA administration suggest that a much lower level of epoxide was formed than compared with AM treatment.     

Kinetics of elimination of urinary metabolites of acrylamide in humans.

Acrylamide (AM), used in the manufacture of polyacrylamide and grouting agents, is produced during the cooking of foods. Workplace exposure to AM can occur through the dermal and inhalation routes. The objective of this study was to define the kinetics of elimination of AM and its metabolites following oral and dermal administration. This is the second part of a study in which metabolites and hemoglobin adducts of AM were determined in people (Fennell et al., 2005, Toxicol. Sci. 85, 447-459). (1,2,3-(13)C(3))AM was administered in an aqueous solution orally (single dose of 0.5, 1.0, or 3.0 mg/kg) or dermally (three daily doses of 3.0 mg/kg) to sterile male volunteers. Urine samples were collected at 0-2, 2-4, 4-8, 8-16, and 16-24 h following administration orally, or at 0-2, 2-4, 4-8, 8-16, and 16-24 h following each of three daily dermal doses. (13)C(3)-AM and its metabolites in urine, (13)C(3)-glycidamide, (13)C(3)-N-acetyl-S-(3-amino-3-oxopropyl)cysteine and its S-oxide, and (13)C(3)-N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine, were quantitated using liquid chromatography-tandem mass spectrometry. The recovered urinary metabolites accounted for 45.6, 49.9, and 39.9% of a 0.5, 1.0, and 3.0 mg/kg oral dose (0-24 h), respectively, and for 4.5% of the dose after 3 mg/kg was administered daily for 3 days dermally (0-4 days). These results indicate that after oral administration AM is rapidly absorbed and eliminated. The half-life estimated for elimination of AM in urine was 3.1-3.5 h. After dermal administration, AM uptake is slow. This study indicated that skin provides a barrier that slows the absorption of AM, and results in limited systemic availability following dermal exposure to AM.     

Cytogenetic damage induced by acrylamide and glycidamide in mammalian cells: correlation with specific glycidamide-DNA adducts.

Acrylamide (AA) is a suspected human carcinogen generated in carbohydrate-rich foodstuffs upon heating. Glycidamide (GA), formed via epoxidation, presumably mediated by cytochrome P450 2E1, is thought to be the active metabolite playing a central role in AA genotoxicity. In this work we investigated DNA damage induced by AA and GA in mammalian cells, using V79 Chinese hamster cells. For this purpose, we evaluated two cytogenetic end points, chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs), as well as the levels of specific GA-DNA adducts, namely, N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade) using high-performance liquid chromatography coupled with tandem mass spectrometry. GA was more cytotoxic and clastogenic than AA. Both AA and GA induced CAs (breaks and gaps) and decreased the mitotic index. GA induced SCEs in a dose-responsive manner; with AA, SCEs were increased at only the highest dose tested (2mM). A linear dose-response relationship was observed between the GA concentration and the levels of N7-GA-Gua. This adduct was detected for concentrations as low as 1 microM GA. N3-GA-Ade was also detected, but only at very high GA concentrations (>or= 250 microM). There was a very strong correlation between the levels of N7-GA-Gua in the GA- and AA-treated cells and the extent of SCE induction. Such correlation was not apparent for CAs. These data suggest that the induction of SCEs by AA is associated with the metabolism of AA to GA and subsequent formation of depurinating DNA adducts; however, other mechanisms must be involved in the induction of CAs.     

Investigation of the Low-Dose Response in the In Vivo Induction of Micronuclei and Adducts by Acrylamide

Acrylamide is an industrial chemical used in polymer manufacture.It is also formed in foods processed at high temperatures. Itinduces chromosome aberrations and micronuclei (MN) in somaticcells of mice, but not rats, and mutations in transgenic mice.This study evaluated the low-dose MN response in mouse bonemarrow and the shape of the dose-response curve. Mice were treatedorally with acrylamide for 28 days using logarithmically spaceddoses from 0.125 to 24 mg/kg/day, and MN were assessed in peripheralblood reticulocytes (RETs) and erythrocytes by flow cytometry.Liver glycidamide DNA adducts and acrylamide and glycidamideN-terminal valine hemoglobin adducts were also determined. Acrylamideproduced a weak MN response, with statistical significance at6.0 mg/kg/day, or greater, in MN-RETs and at 4.0 mg/kg/day orgreater in MN normochromatic erythrocytes (NCEs). The MN responsesat the lower doses were indistinguishable from the concurrentand historical controls. The adducts increased at a much differentrate than the MN. When the MN-NCE values were compared to administereddose, the response was consistent with a linear model. However,when hemoglobin or DNA adducts were used as the dose metric,the response was significantly nonlinear, and models that assumeda threshold dose of 1 or 2 mg/kg/day provided a better fit thana linear model. The MN-RET dose-response had greater variabilitythan the MN-NCE response and was consistent with linearity andwith a threshold at 1 or 2 mg/kg/day, regardless of the dosemetric. These data suggest a threshold for acrylamide in theMN test.     

Urinary metabolites as biomarkers of acrylamide exposure in mice following dietary crisp bread administration or subcutaneous injection.

Heat-treated carbohydrate-rich foods may contain high levels of acrylamide (AA). Crisp bread is a significant dietary AA source in the Nordic countries. We studied whether urinary metabolites of AA could be candidate biomarkers of AA intake and internal dose in mice following dietary crisp bread administration or sc injection. The crisp bread was experimentally baked to contain three different concentrations of AA: 0.19, 1.02, and 2.65 mg/kg, giving dietary exposures to AA of 0.024 +/- 0.002, 0.14 +/- 0.02, and 0.29 +/- 0.04 mg/kg bodyweight (bw)/day (mean +/- SD), respectively. A linear relationship was found between dietary AA exposure and urinary AA metabolites. On average, 55% of the ingested dose was recovered as urinary AA metabolites, and the molar proportions between the urinary metabolites showed similar proportions for the different doses. Urine AA metabolites were measured after sc injection of AA at doses of 0.05, 0.5, 5, and 50 mg/kg bw, and the urinary recovery for the three lowest doses was 54%. With the highest dose, 80% was recovered in urine, and the changed pattern of urinary metabolites indicated saturation of the metabolic conversion of AA to glycidamide. These results indicate that urinary metabolites of AA are good biomarkers of AA intake and internal dose up to 5 mg/kg bw/day. After sc injection of [(14)C]AA, 92% of the radioactivity was found in the urine and 2% in feces, liver, blood, and intestinal content (6% was not detected), demonstrating that sc AA was highly systemically available, that the major part AA metabolites was excreted, and that a significant portion of urinary AA metabolites (most likely glyceramide) was not accounted for by the present analytical method. Since the urinary recovery of AA after crisp bread feeding and sc injection was practically identical, an indicative "bioavailability" of AA from crisp bread was suggested to be approximately complete.     

Protein adduct formation as a molecular mechanism in neurotoxicity.

Chemicals that cause nerve injury and neurological deficits are a structurally diverse group. For the majority, the corresponding molecular mechanisms of neurotoxicity are poorly understood. Many toxicants (e.g., hepatotoxicants) of other organ systems and/or their oxidative metabolites have been identified as electrophiles and will react with cellular proteins by covalently binding nucleophilic amino acid residues. Cellular toxicity occurs when adduct formation disrupts protein structure and/or function, which secondarily causes damage to submembrane organelles, metabolic pathways, or cytological processes. Since many neurotoxicants are also electrophiles, the corresponding pathophysiological mechanism might involve protein adduction. In this review, we will summarize the principles of covalent bond formation that govern reactions between xenobiotic electrophiles and biological nucleophiles. Because a neurotoxicant can form adducts with multiple nucleophilic residues on proteins, the challenge is to identify the mechanistically important adduct. In this regard, it is now recognized that despite widespread chemical adduction of tissue proteins, neurotoxicity can be mediated through binding of specific target nucleophiles in key neuronal proteins. Acrylamide and 2,5-hexanedione are prototypical neurotoxicants that presumably act through the formation of protein adducts. To illustrate both the promise and the difficulty of adduct research, these electrophilic chemicals will be discussed with respect to covalent bond formation, suspected protein sites of adduction, and proposed mechanisms of neurotoxicity. The goals of future investigations are to identify and quantify specific protein adducts that play a causal role in the generation of neurotoxicity induced by electrophilic neurotoxicants. This is a challenging but critical objective that will be facilitated by recent advances in proteomic methodologies.     

关注某些医用产品中丙烯酰胺的潜在风险

根据世界卫生组织发布的有关文件。简要概述了丙烯酰胺以及代谢产物在动物方面的研究和有限的人体研究情况．主要涉及神经毒性、生育毒性、致癌性和遗传毒性及其可能发生的机制等。结果表明：丙烯酰胺引起的神经毒性可能与丙烯酰胺有关；导致动物多器官肿瘤的发生与丙烯酰胺的代谢产物环氧丙酰胺有关。     

Mutagenicity and DNA adduct formation by the urban air pollutant 2-nitrobenzanthrone.

2-Nitrobenzanthrone (2-NBA) has recently been detected in ambient air particulate matter. Its isomer 3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust. The highest mutagenic activity of 2-NBA tested in Salmonella typhimurium was exhibited in strain TA1538-hSULT1A1 expressing human sulfotransferase (SULT) 1A1. 2-NBA also induced mutations in Chinese hamster lung V79 cells expressing human N-acetyltransferase 2 or SULT1A1, but no mutagenicity was observed in the parental cell line. DNA adduct formation in vitro was examined in different human cell lines by thin-layer chromatography (32)P-postlabeling. Whereas 3-NBA formed characteristic DNA adducts in lung A549, liver HepG2, colon HCT116, and breast MCF-7 cells, 2-NBA-derived DNA adducts were only observed in A549 and HepG2 cells, indicating differences in the bioactivation of each isomer. The pattern of 2-NBA-derived DNA adducts in both cell lines consisted of a cluster of up to five adducts. In HepG2 cells DNA binding by 2-NBA was up to 14-fold lower than by 3-NBA. DNA adduct formation of 2-NBA was also investigated in vivo in Wistar rats treated with a single dose of 2, 10, or 100 mg/kg body weight (bw). No DNA adduct formation was detected at doses of up to 10 mg/kg bw 2-NBA, even though 3-NBA induced DNA adducts at a dose of 2 mg/kg bw. Only after administration of one high dose of 100 mg/kg bw 2-NBA was a low level of DNA adduct formation detected, and then only in lung tissue. Density functional theory calculations for both NBAs revealed that the nitrenium ion of the 3-isomer is considerably more stable ( approximately 10 kcal/mol) than that of the 2-isomer, providing a possible explanation for the large differences in DNA adduct formation and mutagenicity between 2- and 3-NBA.     

Risk assessment of acrylamide in foods.

Daily mean intakes of acrylamide present in foods and coffee in a limited Norwegian exposure assessment study have been estimated to be 0.49 and 0.46 microg per kg body weight in males and females, respectively. Testicular mesotheliomas and mammary gland adenomas have consistently been found in 2-year drinking water rat cancer studies with acrylamide. Acrylamide also shows initiating activity in mouse skin after systemic administration. Since acrylamide is converted to the mutagenic metabolite glycidamide and forms adducts to hemoglobin in rodents and humans, the tumorigenic endpoints in rats were assumed to be an expression of acrylamide genotoxicity. Using the default linear extrapolation methods LED10 and T25, the lifetime cancer hazard after lifelong exposure to 1 microg acrylamide per kg body weight per day, scaled to humans, was calculated to be, on average, 1.3 x 10-3. Using this hazard level and correlating it with the exposure estimates, a lifetime cancer risk related to daily intake of acrylamide in foods for 70 years in males was calculated to be 0.6 x 10-3, implying that 6 out of 10,000 individuals may develop cancer due to acrylamide. For females, the risk values were slightly lower. It must be emphasized that this risk assessment is conservative. A number of processes may result in nonlinearity of the dose-response relationships for acrylamide carcinogenicity in the low-dose region, including detoxication reactions, cell cycle arrest, DNA repair, apoptosis, and immune surveillance. Thus, the true risk levels related to acrylamide intake may be considerably lower.     

Proteomic analysis of acrylamide-protein adduct formation in rat brain synaptosomes

Evidence suggests that the neurological defects (gait abnormalities, foot splay, and skeletal muscle weakness) associated with acrylamide (ACR) intoxication are mediated by impaired neurotransmission at central and peripheral synapses. ACR can form adducts with nucleophilic residues on proteins and thereby alter corresponding structure and function. To evaluate protein adduction in nerve terminals as a possible mechanism of action, recombinant N-ethylmaleimide sensitive factor (NSF) was exposed in vitro to ACR (10 micromol) and mass spectrometry (MS) was used to identify adduct sites. MS analyses demonstrated that ACR formed adducts with sulfhydryl groups on cysteine residues (carbamoylethylcysteine, or CEC) of NSF. Ex vivo incubation of whole brain synaptosomes with ACR (0.001-1.0 M) produced concentration-dependent increases in CEC that were inversely correlated to reductions in neurotransmitter release that occurred over the same neurotoxicant concentration range. In synaptosomes isolated from rats intoxicated at a higher (50 mg/kg per day x 3, 5, 8, or 11 days) or a lower (21 mg/kg per day x 14, 21, or 28 day) ACR dose rate, CEC levels increased progressively up to a moderate level of neurotoxicity. To identify protein adducts, synaptosomal proteins labeled by ex vivo 14C-ACR exposure were separated by gel electrophoresis and probed by immunoblot analysis. Results showed that NSF and the SNARE protein, SNAP-25, were tentative ACR targets. Subsequent experiments indicated that ACR exposure increased synaptosomal levels of the 7S SNARE core complex, which is consistent with inhibition of NSF, SNAP-25 function, or both. These data suggest that adduction of cysteine residues on NSF and certain SNARE proteins might be causally involved in the nerve terminal dysfunction induced by ACR.     

Protein targets of acrylamide adduct formation in cultured rat dopaminergic cells

Acrylamide (ACR) is an electrophilic unsaturated carbonyl derivative that produces neurotoxicity by forming irreversible Michael-type adducts with nucleophilic sulfhydryl thiolate groups on cysteine residues of neuronal proteins. Identifying specific proteins targeted by ACR can lead to a better mechanistic understanding of the corresponding neurotoxicity. Therefore, in the present study, the ACR-adducted proteome in exposed primary immortalized mesencephalic dopaminergic cells (N27) was determined using tandem mass spectrometry (LTQ-Orbitrap). N27 cells were characterized based on the presumed involvement of CNS dopaminergic damage in ACR neurotoxicity. Shotgun proteomics identified a total of 15,243 peptides in N27 cells of which 103 unique peptides exhibited ACR-adducted Cys groups. These peptides were derived from 100 individual proteins and therefore ~0.7% of the N27 cell proteome was adducted. Proteins that contained ACR adducts on multiple peptides included annexin A1 and pleckstrin homology domain-containing family M member 1. Sub-network enrichment analyses indicated that ACR-adducted proteins were involved in processes associated with neuron toxicity, diabetes, inflammation, nerve degeneration and atherosclerosis. These results provide detailed information regarding the ACR-adducted proteome in a dopaminergic cell line. The catalog of affected proteins indicates the molecular sites of ACR action and the respective roles of these proteins in cellular processes can offer insight into the corresponding neurotoxic mechanism.     

The modifying effect of CYP2E1, GST,and mEH genotypes on the formation of hemoglobin adducts of acrylamide and glycidamide in workers exposed to acrylamide

This study assesses the association of acrylamide (AA) and glycidamide (GA) hemoglobin adducts (AAVal and GAVal) and their ratios with genetic polymorphisms of the metabolic enzymes cytochrome P450 2E1 (CYP2E1), exon 3 and 4 of microsomal epoxide hydrolase (mEH3 and mEH4), glutathione transferase theta (GSTT1), and mu (GSTM1) or/and the combinations of these polymorphisms, involved in the activation and detoxification of AA in humans. Fifty-one AA-exposed workers and 34 controls were recruited and provided a post-shift blood sample. AAVal and GAVal were determined simultaneously using isotope-dilution liquid chromatography-electronspray ionization/tandem mass spectrometry (LC-ESI–MS/MS). Genetic polymorphisms of CYP2E1, mEH3 and 4, GSTT1, and GSTM1 were also analyzed. Our results reveal that the GAVal/AAVal ratio, potentially reflecting the proportion of AA metabolized to GA, ranged from 0.13 to 0.45 with a mean at 0.27. Multivariate regression analysis demonstrates that the joint effect of CYP2E1, GSTM1, and mEH4 genotypes was significantly associated with AAVal and GAVal levels after adjustment for AA exposures. These results suggest that mEH4 and the combined genotypes of CYP2E1, GSTM1 and mEH4 may be associated with the formation of AAVal and GAVal. Further studies may be needed to shed light on the roles that phase I and II enzymes play in AA metabolism.     

阿西维辛或谷胱甘肽降低顺铂引起的肾脏毒性和顺铂-DNA加合物在鼠肾脏中的水平(英文)

目的　为了了解阿西维辛 (acivicin)和GSH预防肾脏毒性的机制 ,研究了顺铂 DNA加合物在大鼠肾脏中的水平。方法　顺铂 (6mg·kg- 1)从尾静脉注入大鼠 ,5d后处死。其他两组动物在给顺铂前2 .5h ,给予阿西维辛或者GSH。测量顺铂 DNA加合物在肾脏中的浓度、血中尿素氮 (BUN)和丝氨酸肌酸的浓度。结果　在给顺铂前 2 .5h ,给 10mg·kg- 1阿西维辛完全阻断了顺铂引起的肾脏毒性。具体表现是血氮和肌酸浓度降低 ,DNA加合物减少了17.1% (P <0 .0 5 )。在给顺铂前 ,给 5 0 0mg·kg- 1GSH ,肾脏毒性和顺铂 DNA加合物水平均显著性减低 (P <0 .0 5 )。另外 ,在DNA加合物和血氮之间存在着一个弱正相关关系。结论　DNA加合物在顺铂引起的肾脏毒性中引了一些作用。但是DNA加合物和血氮之间的弱相关关系提示DNA加合物与肾脏毒性只有较弱的联系 ,在顺铂引起的肾脏毒性中不是主要因素     

Stability of hemoglobin and albumin adducts of benzene oxide and 1,4-benzoquinone after administration of benzene to F344 rats.

The stability of cysteinyl adducts of benzene oxide (BO) and mono-S-substituted cysteinyl adducts of 1,4-benzoquinone (1,4-BQ) was investigated in both hemoglobin (Hb) and albumin (Alb) following administration of a single oral dose of 400 mg [U-14C/13C6]benzene/kg body weight to F344 rats. Total radiobound adducts to Hb were stable, as were adducts formed by the reaction of [13C6]BO with cysteinyl residues on Hb. In both cases adduct stability was indicated by zero-order kinetics with decay rates consistent with the lifetime of rat erythrocytes. Hb adducts of 1,4-BQ were not detected, possibly due to the production of multi-S-substituted adducts within the erythrocyte. Regarding Alb binding, total radiobound adducts decayed more rapidly than expected (half-life of 0.4 days), suggesting that uncharacterized benzene metabolites were noncovalently bound or formed unstable adducts with Alb. Although adducts from reactions of BO and 1,4-BQ with Alb both decayed with rates consistent with those of Alb turnover in the rat, the half-life for 1,4-BQ-Alb (2.5 days) was shorter than that for BO-Alb (3.1 days), suggesting some instability of 1,4-BQ-Alb. Assuming similar rates of adduct instability in humans and rats, the 1,4-BQ-Alb adducts would be eliminated with a half-life of approximately 8 days, compared with BO-Alb, which would be expected to turnover with Alb (half-life of approximately 21 days).     

An absorption study of dietary administered acrylamide in swine.

Acrylamide is a food toxicant suspected to be carcinogenic to humans. It is formed in the heat processing of carbohydrate-rich food. A current issue in food safety is whether acrylamide actually represents a risk for human health. At present, available information is insufficient to reach any conclusions. Inter alias, a still unclear matter is the fraction of acrylamide ingested by food that is absorbed and metabolized. This study compared the in vivo relative absorption of acrylamide formed in cooked food with that of the pure compound dissolved in drinking water using the pig (25 Italian Large White females) as the animal model. Acrylamide intakes of about 0.8 and 8 microg kg(-1) pig body wt day(-1) equal to one and ten times, respectively, the maximum average acrylamide daily intake for humans from the diet (expressed on a body wt basis) in industrialized countries, were chosen for the study. Adducts with the N-terminal valine of haemoglobin formed by acrylamide and its epoxide metabolite glycidamide, were used as exposure markers. Analyses were carried out by gas chromatography/mass spectrometry following in-house method validation. Both for the low and the high dose regimen, the glycidamide adduct levels in swine globins were lower of the limit of quantification of the method. As concerns acrylamide adducts, it was found that the relative absorption of acrylamide from feed and water was the same and that there is a direct proportionality between the adduct concentration and acrylamide intake.     

Hemoglobin and DNA adduct formation in Fischer-344 rats exposed to 2,4- and 2,6-toluene diamine

 Using gas chromatography/mass spectrometry for detection of hemoglobin adducts, and ³²P-postlabelling for DNA adducts, we examined macromolecular binding in Fischer-344 rats administered 2,4- or 2,6-toluene diamine (TDA). The dose-response and correlative relationship between the two macromolecules were investigated over a range of doses (0–250 mg/kg). The time course of adduct formation and removal was also examined. Both TDA isomers induced formation of hemoglobin adducts, but only the 2,4-isomer induced DNA binding. Maximum hemoglobin and DNA adduct levels were detected 24 h following administration. Both hemoglobin and DNA binding increased in a dose-dependent manner. Hemoglobin adduct clearance demonstrated a nonlinear decay, with adduct loss occurring faster than normal erythrocyte clearance. The effects of metabolic inhibitors on adduct formation were examined using piperonyl butoxide and pentachlorophenol to inhibit p450 isozymes and sulfotransferase, respectively. Microsomal enzymatic activation was critical to hemoglobin adduct formation with inhibition by piperonyl butoxide reducing adduct yields by over 90%. Sulfation did not appear to play a significant role in TDA-induced hemoglobin adduct formation. Received: 19 July 1995/Accepted: 9 January 1996     

Urinary mercapturic acids and a hemoglobin adduct for the dosimetry of acrylamide exposure in smokers and nonsmokers

Acrylamide, used in the manufacture of polyacrylamide and grouting agents, is also present in the diet and tobacco smoke. It is a neurotoxin and a probable human carcinogen. Analytical methods were established to determine the mercapturic acids of acrylamide (N-acetyl-S-(2-carbamoylethyl)-L-cysteine, AAMA) and its metabolite glycidamide (N-(R/S)-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine, GAMA) by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), as well as the N-terminal valine adduct of acrylamide (N-2-carbamoylethylvaline, AAVal) released by N-alkyl Edman degradation of hemoglobin by gas chromatography-mass spectrometry (GC-MS). Twenty-four-hour urine samples from 60 smokers and 60 nonsmokers were analyzed for AAMA and GAMA, and blood samples were analyzed for AAVal. Smokers excreted 2.5-fold higher amounts of AAMA and 1.7-fold higher amounts of GAMA in their urine and had 3-fold higher levels of AAVal in their blood. All three biomarkers of acrylamide exposure were strongly correlated with the smoking dose as determined by the daily cigarette consumption, nicotine equivalents (the molar sum of nicotine, cotinine, trans-3'-hydroxycotinine, and their respective glucuronides) in urine, salivary cotinine, and carbon monoxide in expired breath. In nonsmokers, a weak but significant correlation between AAMA and the estimated dietary intake of acrylamide was found. It is concluded that all three biomarkers of acrylamide are suitable for the determination of exposure in both smokers and nonsmokers.     

Effects of intermittent exposure to aflatoxin B1 on DNA and RNA adduct formation in rat liver: dose-response and temporal patterns.

We studied the effects of intermittent exposure to aflatoxin B1 (AFB1) on hepatic DNA and RNA adduct formation. Fisher-344 male rats were fed 0.01, 0.04, 0.4, or 1.6 ppm of AFB1 intermittently for 8, 12, 16, and 20 weeks, alternating with 4 weeks of dosing and 4 weeks of rest. Other groups of rats were fed 1.6 ppm of AFB1 continuously for 4, 8, 12, and 16 weeks. Control rats received AFB1-free NIH-31 meal diet. AFB1-DNA and -RNA adducts were measured by HPLC with fluorescence detection. The data are presented as total DNA or RNA adducts. The DNA and RNA adduct levels increased or decreased depending on the cycles of dosing and rest. Rats removed from treatment 1 month after 1 or 2 dosing cycles (8 and 16 weeks of intermittent exposure) showed approximately a twofold decrease in DNA adduct levels and a two- to elevenfold decrease in RNA adduct levels compared with rats euthanized immediately after the last dosing cycle (12 and 20 weeks of intermittent exposure). Our data indicate that DNA and RNA adducts increased linearly, from 0.01 ppm to 1.6 ppm of AFB1 after 12 and 20 weeks of intermittent treatment. A linear dose response was also apparent for DNA but not for RNA adducts after 8 and 16 weeks of treatment. As biomarkers of exposure, AFB1-RNA adducts were three to nine times more sensitive than AFB1-DNA adducts but showed greater variability. These results suggest that binding of AFB1 to hepatic DNA is a linear function of the dose, regardless of the way this is administered. The dose-response relationship for RNA adducts depends on the length of the no-dosing cycles and on the turnover rate of RNA.     

Determination and quantification of urinary metabolites after dietary exposure to acrylamide

It is known that heat-treated carbohydrate-rich foods may contain high levels of acrylamide (AA) and up to 4000?µg?kg^?1 in potato crisps and 2000?µg?kg^?1 in French fries have been reported. In order to obtain more information on the human exposure to and metabolism of AA, a method for the determination of known urinary metabolites from the dietary exposure of AA using solid-phase extraction and liquid chromatography with positive electrospray MS/MS detection was developed. The validated assay range was from 8.6 to 342.9?µg?l^?1. The urinary metabolites were synthesized and their structures determined by NMR and MS. To test the method, a pilot study was conducted in which all urine during 48?h starting with 24?h fasting was collected. The two urinary metabolites, N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine (MA-GA₃) and N-acetyl-S-(3-amino-3-oxopropyl)cysteine (MA-AA), were found to be above the detection limit. Fasting during 1?day caused about a 50% decrease in the total level of the metabolites, but after 1?day of a normal diet, the metabolite levels increased back to pre-fasting levels. The total amount of AA in the form of urinary metabolites excreted over the period was estimated to be about 40?µg?AA?day^?1 for the average non-smoker.