Relationships between biomarkers of exposure and toxicokinetics in Fischer 344 rats and B6C3F1 mice administered single doses of acrylamide and glycidamide and multiple doses of acrylamide

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells and carcinogenic in rodents. AA is also formed in many commonly consumed starchy foods during cooking. Our previous toxicokinetic investigations of AA and its important genotoxic metabolite, glycidamide (GA), in rodents showed that AA is highly bioavailable from oral routes of administration, is widely distributed to tissues and that the dietary route, in particular, favors metabolism to GA. Measurements of DNA adducts in many tissues supported the hypothesis that AA is carcinogenic in rodent bioassays through metabolism to GA. The current investigation describes the development and validation of methodology for measuring hemoglobin (Hb) adducts with AA and GA in the same rodents previously used for toxicokinetic and DNA adduct measurements. The goal was to investigate possible relationships between these circulating biomarkers of exposure and serum toxicokinetic parameters for AA and GA and tissue GA-DNA adducts in rodents from both single and repeated dosing with AA. Significant correlations were observed between GA-Hb and liver GA-DNA adducts for either single or multiple dosing regimens with AA. Using available GA-Hb adduct data, empirical and allometric relationships permitted estimation of liver DNA adducts in humans in the range of 0.06-0.3 adducts/10(8) nucleotides. This approach may prove useful in extrapolating human cancer risks from findings in rodent bioassays.     

Pre-diagnostic acrylamide exposure and survival after breast cancer among postmenopausal Danish women

Acrylamide is a probable human carcinogen, with industrial contact, tobacco smoking and foods processed at high temperatures as the main routes of exposure. In animal studies oral intake of acrylamide has been related to cancer development, with indications that the increased cancer occurrence especially regards endocrine related tumors. In human epidemiological studies, dietary exposure to acrylamide has also been suggested related to higher risk of endocrine related tumors, like estrogen sensitive breast cancer. The aim of the present study was to evaluate if pre-diagnostic acrylamide exposure, measured by acrylamide and glycidamide hemoglobin adducts (AA-Hb and GA-Hb), were associated to mortality in breast cancer cases. Among 24,697 postmenopausal women included into a Danish cohort between 1993 and 1997, 420 developed breast cancer before 2001 and 110 died before 2009. AA-Hb and GA-Hb concentrations measured in blood samples were related to mortality by Cox proportional hazard models. Estimates are given per 25 pmol/g globin higher levels. Among non-smokers, higher concentrations of GA-Hb were associated to a higher hazard rate of breast cancer specific mortality (HR (95% CI): 1.63 (1.06-2.51)), the hazard rate among women diagnosed with estrogen receptor positive tumors was (HR (95% CI): 2.23 (1.38-3.61)). For AA-Hb the tendency was similar, but only statistically significant among those with estrogen receptor positive tumors (HR (95% CI): 1.31 (1.02-1.69)). In conclusion, the present study indicates that pre-diagnostic exposure to acrylamide may be related to mortality among breast cancer patients and that this may especially concern the most endocrine related type of breast cancer.     

Hemoglobin adducts of ethylene oxide,propylene oxide,acrylonitrile and acrylamide-biomarkers in occupational and environmental medicine

In a chemical plant, ethylene oxide (EO) and propylene oxide (PO) were used for the production of surfactants for the textile industry. Within health supervision, we investigated the internal exposure of the workers using hemoglobin adducts as parameters of biochemical effects. The 95th percentile for N-2-hydroxyethylvaline (HEV) was 1280 pmol/g globin (=29.4 microg/l blood) in blood from exposed workers compared with 100 pmol/g globin (or 2.3 microg/l) in controls. N-(R,S)-2-hydroxypropylvaline (HPV) both in workers and controls was below the detection limit (80 pmol/g globin or 2 microg/l). The levels of the adducts of acrylonitrile (ACN) and acrylamide (AA) were also determined, though they were mainly accounted for by smoking and diet. Median values of N-2-cyanoethylvaline (CEV) were below 4 pmol/g globin (or 0.1 microg/l) in non-smokers (n=24) and 131 pmol/g globin (or 3.3 microg/l) in smokers (n=38). Median values of N-2-carbamoylethylvaline (AAV) were 22 pmol/g globin (or 0.6 microg/l) in non-smokers compared with 89 pmol/g globin (or 2.4 microg/l) in smokers. Correlations were found between smoking habits and adduct levels of CEV and AAV.     

Adduction of biomacromolecules with acrylamide (AA) in mice at environmental dose levels studied by accelerator mass spectrometry

Since 2002, WHO has strongly called scientists to investigate intensively the toxicity and potential carcinogenicity of acrylamide (AA), because humans are widely exposed to AA via various foodstuffs. In this study we measured the biomacromolecule adducts of [2,3-(14)C]AA (0, 7.5 x 10(-2), 7.5 x 10(-1), 7.5, 9.3 x 10(1), 2.4 x 10(2) and 1.0 x 10(3)microg/kg b.w.) in adult male mice by ultrasensitive accelerator mass spectrometry (AMS) technique. The aim is to evaluate the potential molecular toxicity of AA at human relevant dose levels, particularly towards the sperm cells. Hemoglobin (Hb), serum albumin (SA), protamine, sperm DNA, tails and heads were isolated 24h post dosing and the adduct levels were measured by AMS, respectively. Good log/log linear dose-response correlations were established. Moreover, the correlation of AA-protamine adducts, AA-sperm DNA adducts, as well as AA-sperm head/tail adducts with AA-Hb or AA-SA adducts presented a linear log/log mode. In sperm, the formation of AA-protamine adducts were predominating to AA-DNA adducts. The adducts on sperm heads/tails might both influence the fertility efficacy.     

Adducts of acrylonitrile with hemoglobin in nonsmokers and in participants in a smoking cessation program.

Hemoglobin adducts have been used to assess exposure to carcinogenic compounds in tobacco smoke. However, because of background levels in nonsmokers, most adducts that have been studied are not useful for monitoring low-level exposure. Bergmark [(1997) Chem. Res. Toxicol. 10, 78-84] showed that the level of adducts of acrylonitrile (AN) with N-terminal valine (ANVal) increases with increasing cigarette consumption, and the increment from 1 cigarette/day was estimated to be 8 pmol/g of globin. The background level of ANVal in nonsmokers was not quantified (<2 pmol/g of globin). The objective of this study was to determine the background level of ANVal in hemoglobin and to study the stability of this adduct in vivo. Globin samples previously analyzed by Bergmark from 17 nonsmokers and 2 smokers were reanalyzed in the study presented here. Globin samples from 7 additional nonsmokers and from 10 participants in a smoking cessation program were also analyzed. Smoking habits and exposure to environmental tobacco smoke (ETS) were assessed by interview. Only two of the participants completed the program. The levels of ANVal in these 2 subjects decreased after quitting and were at background level by 126 days. The time course of the decrease was compatible with removal of stable adducts. The levels of ANVal in the nonsmokers were 0.76 +/- 0.36 (mean +/- SD) (n = 18; reporting no exposure ETS), 1.1 +/- 0.6 (n = 3; reporting exposure to ETS), and 1. 2 +/- 0.5 pmol/g of globin (n = 3; snuff users). Thus, the adduct level in nonsmokers corresponds to the adduct increment from about 0. 1 cigarette/day. Measurements of the level of ANVal could be used to distinguish between nonsmokers and low-level smokers on an individual level, but larger groups of individuals would be required to detect a possible contribution to the background from passive smoking.     

Physiologically based pharmacokinetic/pharmacodynamic model for acrylamide and its metabolites in mice, rats, and humans

A physiologically based pharmacokinetic model was developed for acrylamide (AA) and three of its metabolites: glycidamide (GA) and the glutathione conjugates of acrylamide (AA-GS) and glycidamide (GA-GS). Liver GA-DNA adducts and hemoglobin (Hb) adducts with AA and GA were included as pharmacodynamic components of the model. Serum AA and GA concentrations combined with urinary elimination levels for all four components from male and female mice and rats were simulated from iv and oral administration of 0.1 mg/kg AA or 0.12 mg/kg GA. Adduct formation and decay rates were determined from a 6 week exposure to approximately 1 mg/kg AA in the drinking water and subsequent 6 week nonexposure period. Human urinary excretion data and Hb adduct data were utilized to extrapolate to a human model. The steady-state human liver GA-DNA adduct level from exposure to background levels of AA in the diet was predicted to be between 0.06 and 0.26 adducts per 10(8) nucleotides.     

Hemoglobin adducts as a measure of variations in exposure to acrylamide in food and comparison to questionnaire data

Measurement of haemoglobin (Hb) adducts from acrylamide (AA) and its metabolite glycidamide (GA) is a possibility to improve the exposure assessment in epidemiological studies of AA intake from food. This study aims to clarify the reliability of Hb-adduct measurement from individual single samples for exposure assessment of dietary AA intake.     

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.     

DNA adduct formation from acrylamide via conversion to glycidamide in adult and neonatal mice

Acrylamide (AA) is a high production volume chemical with many industrial uses; however, recent findings of ppm levels in starchy foods cooked at high temperature have refocused worldwide attention on the neurotoxicity, germ cell mutagenicity, and carcinogenicity of AA. Oxidative metabolism of AA to its epoxide metabolite, glycidamide (GA), has been observed in experimental animals and humans and may be associated with many of the toxic effects of AA exposure, including formation of N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) in vivo. This paper describes the characterization of two new GA-derived DNA adducts formed in vitro, N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade) and N1-(2-carboxy-2-hydroxyethyl)-2'-deoxyadenosine. A sensitive method for quantification of N7-GA-Gua and N3-GA-Ade, based on LC with tandem mass spectrometry and isotope dilution, was developed and validated for use in measuring DNA adduct formation in selected tissues of adult and whole body DNA of 3 day old neonatal mice treated with AA and GA. In adult mice, DNA adduct formation was observed in liver, lung, and kidney with levels of N7-GA-Gua around 2000 adducts/10(8) nucleotides and N3-GA-Ade around 20 adducts/10(8) nucleotides. Adduct levels were modestly higher in adult mice dosed with GA as opposed to AA; however, treatment of neonatal mice with GA produced 5-7-fold higher whole body DNA adduct levels than with AA, presumably reflective of lower oxidative enzyme activity in newborn mice. DNA adduct formation from AA treatment in adult mice showed a supralinear dose-response relationship, consistent with saturation of oxidative metabolism at higher doses. These results increase our understanding of the mutagenic potential of GA and provide further evidence for a genotoxic mechanism in AA carcinogenesis.     

Toxicokinetics of acrylamide and glycidamide in Fischer 344 rats

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in rodents. The recent discovery of AA at ppm levels in a wide variety of commonly consumed foods has energized research efforts worldwide to define toxic mechanisms, particularly toxicokinetics and bioavailability. This study compares the toxicokinetics of AA and its epoxide metabolite, glycidamide (GA), in serum and tissues of male and female F344 rats following acute exposure by intravenous, gavage, and dietary routes at 0.1 mg/kg AA or intravenous and gavage routes with an equimolar amount of GA. AA was rapidly absorbed after oral dosing, was widely distributed to tissues, was efficiently converted to GA, and produced increased levels of GA-DNA adducts in liver. GA was also rapidly absorbed, widely distributed to tissues, and produced increased liver DNA adduct levels. AA bioavailability after aqueous gavage was 60--98% and from the diet was 32--44%; however, first-pass metabolism or other kinetic change resulted in much higher internal exposures to GA (2- to 7-fold) when compared to the intravenous route. A similar effect on metabolism to GA following oral administration was previously observed under an identical exposure paradigm in mice. Furthermore, DNA adduct formation in rat liver showed the same proportionality with the respective GA AUC value as did mice in the previous study. These findings suggest that as the AA content in food is reduced, species-differences in GA formation and subsequent DNA adduct formation may be minimized. These findings provide additional information needed to assess genotoxic risks from the low levels of AA that are pervasive in the food supply.     

Genotoxic effects of acrylamide and glycidamide in mouse lymphoma cells.

In addition to occupational exposures to acrylamide (AA), concerns about AA health risks for the general population have been recently raised due to the finding of AA in food. In this study, we evaluated the genotoxicity of AA and its metabolite glycidamide (GA) in L5178Y/Tk(+/-) mouse lymphoma cells. The cells were treated with 2-18 mM of AA or 0.125-4 mM of GA for 4 h without metabolic activation. The DNA adducts, mutant frequencies and the types of mutations for the treated cells were examined. Within the dose range tested, GA induced DNA adducts of adenine and guanine [N3-(2-carbamoyl-2-hydroxyethyl)-adenine and N7-(2-carbamoyl-2-hydroxyethyl)-guanine] in a linear dose-dependent manner. The levels of guanine adducts were consistently about 60-fold higher across the dose range than those of adenine. In contrast, no GA-derived DNA adducts were found in the cells treated with any concentrations of AA, consistent with a lack of metabolic conversion of AA to GA. However, the mutant frequency was significantly increased by AA at concentrations of 12 mM and higher. GA was mutagenic starting with the 2mM dose, suggesting that GA is much more mutagenic than AA. The mutant frequencies were increased with increasing concentrations of AA and GA, mainly due to an increase of proportion of small colony mutants. To elucidate the underlying mutagenic mechanism, we examined the loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11 for mutants induced by AA or GA. Compared to GA induced mutations, AA induced more mutants whose LOH extended to D11Mit22 and D11Mit74, an alteration of DNA larger than half of the chromosome. Statistical analysis of the mutational spectra revealed a significant difference between the types of mutations induced by AA and GA treatments (P=0.018). These results suggest that although both AA and GA generate mutations through a clastogenic mode of action in mouse lymphoma cells, GA induces mutations via a DNA adduct mechanism whereas AA induces mutations by a mechanism not involving the formation of GA adducts.     

N7-glycidamide-guanine DNA adduct formation by orally ingested acrylamide in rats: a dose-response study encompassing human diet-related exposure levels

Acrylamide (AA) is formed during the heating of food and is classified as a genotoxic carcinogen. The margin of exposure (MOE), representing the distance between the bench mark dose associated with 10% tumor incidence in rats and the estimated average human exposure, is considered to be of concern. After ingestion, AA is converted by P450 into the genotoxic epoxide glycidamide (GA). GA forms DNA adducts, primarily at N7 of guanine (N7-GA-Gua). We performed a dose-response study with AA in female Sprague-Dawley (SD) rats. AA was given orally in a single dosage of 0.1-10?000 μg/kg bw. The formation of urinary mercapturic acids and of N7-GA-Gua DNA adducts in liver, kidney, and lung was measured 16 h after application. A mean of 37.0 ± 11.5% of a given AA dose was found as mercapturic acids (MAs) in urine. MA excretion in urine of untreated controls indicated some background exposure from endogenous AA. N7-GA-Gua adduct formation was not detectable in any organ tested at 0.1 μg AA/kg bw. At a dose of 1 μg/kg bw, adducts were found in kidney (around 1 adduct/10(8) nucleotides) and lung (below 1 adduct/10(8) nucleotides) but not in liver. At 10, respectively, 100 μg/kg bw, adducts were found in all three organs, at levels close to those found at 1 μg AA/kg, covering a range of about 1-2 adducts/10(8) nucleotides. As compared to DNA adduct levels from electrophilic genotoxic agents of various origin found in human tissues, N7-GA-Gua adduct levels within the dose range of 0.1-100 μg AA/kg bw were at the low end of this human background. We propose to take the background level of DNA lesions in humans more into consideration when doing risk assessment of food-borne genotoxic carcinogens.     

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.     

Characterization of hemoglobin adduct formation in mice and rats after administration of [14C]butadiene or [14C]isoprene

Occupational exposures to 1,3-butadiene or isoprene occur through their use in the manufacture of rubber and other related polymer products. The purpose of this study was to determine if butadiene or isoprene administration would result in the formation of adducts with blood hemoglobin (Hb), and if such adducts can be used as a measure of previous exposure(s). Male B6C3F1 mice and male Sprague-Dawley rats were injected intraperitoneally with 1, 10, 100, or 1000 mumol [14C]butadiene or 0.3, 3.0, 300, 1000, or 3000 mumol [14C]isoprene per kilogram body weight. Animals were killed 24 hr later. Globin was isolated from blood samples and was analyzed for 14C by liquid scintillation spectroscopy. Hb adduct formation was linearly related to administered doses up to 100 mumol [14C]butadiene or 500 mumol [14C]isoprene per kilogram body weight for mice and rats, respectively. For [14C]butadiene, the efficiency of Hb adduct formation in mice and rats within the linear response range was 0.177 +/- 0.003 and 0.407 +/- 0.019 (pmol of 14C-adducts/mg globin)/(mumol of retained [14C]butadiene/kg body wt), respectively (mean +/- SE; n = 18). For [14C]isoprene, these values for mice and rats were 0.158 +/- 0.035 and 0.079 +/- 0.016 (pmol of 14C-adducts/mg globin)/(mumol of retained [14C]isoprene/kg body wt), respectively (mean +/- SE; n = 12). Hb adducts also accumulated linearly after repeated daily administration of 100 mumol [14C]butadiene or 500 mumol [14C]isoprene per kilogram body wt to mice and rats, respectively, for 3 days. [14C]Butadiene-derived Hb adducts in blood showed lifetimes of approximately 24 and approximately 65 days for mice and rats, respectively, which correlate with the reported lifetimes for red blood cells in these rodent species. Thus, levels of butadiene- or isoprene-derived adducts on Hb in circulating blood may be a useful measure of prior repeated exposures to these compounds.     

Hemoglobin adducts of epoxybutene in workers occupationally exposed to 1,3-butadiene

1,3-Butadiene (BD) is an important chemical widely used in the synthetic rubber industry. Hemoglobin adducts of two of its reactive metabolites have been already investigated as possible parameters for exposure assessment. In this study hemoglobin adducts of epoxybutene (EB) were analyzed in blood samples from 17 workers in a BD monomer production unit and 19 controls in a heat production unit of a petrochemical plant near Prague, Czech Republic. BD exposure was determined by personal air sampling. The median level of exposure was 440 microg/m3 (range < 11-17 mg/m3) for the exposed workers and < 6 microg/m3 (< 5-150 microg/m3) for the controls. The adduct N-(2-hydroxy-3-butenyl)valine (HBVal) formed by the reaction of the N-terminal valine of globin with carbon-1 of EB was measured. The N-alkylated amino acid was analyzed by gas chromatography/mass spectrometry (GC/MS) after degradation by the modified Edman procedure. Using published methods problems arose with high background levels, especially in the negative ion chemical ionization (NCI) mode. In the present study a limit of detection of 0.2 pmol/g globin was achieved by using 400 mg globin, a variation in extraction solvents, an additional purification step and a widely extended GC temperature program. The median hemoglobin adduct level of the Czech BD monomer production workers (0.7 pmol/g globin; n = 17) was significantly higher than that of the controls (0.2 pmol/g globin; n = 19; P<0.05). Smoking controls showed higher hemoglobin adduct levels than nonsmoking controls (P<0.1) and significantly higher BD exposure levels (P<0.01).     

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.     

Measurement of glycidol hemoglobin adducts in humans who ingest edible oil containing small amounts of glycidol fatty acid esters

Hemoglobin (Hb) adducts are frequently used to address and/or monitor exposure to reactive chemicals. Glycidol (G), a known animal carcinogen, has been reported to form Hb adducts. Here, we measure G adduct levels in humans who daily ingest DAG oil, an edible oil consisting mainly of diacylglycerol. Since DAG oil contains a small amount of glycidol fatty acid esters (GEs), possible exposure to G released from GEs has been raised as a possible concern. For measurement of Hb adducts, we employed the N-alkyl Edman method reported by Landin et al. (1996) using gas chromatography-tandem mass spectrometry with minor modifications to detect G-Hb adducts as N-(2,3-dihydroxy-propyl)valine (diHOPrVal). Blood samples were collected from 7 DAG oil users and 6 non-users, and then G-Hb adduct levels were measured. G-Hb adducts were detected in all samples. The average level of diHOPrVal was 3.5 ± 1.9 pmol/g globin in the DAG oil users and 7.1 ± 3.1 pmol/g globin in the non-users. We conclude that there is no increased exposure to G in individuals who daily ingest DAG oil.     

Toxicokinetics of acrylamide in primary rat hepatocytes: coupling to glutathione is faster than conversion to glycidamide

Acrylamide (AA), classified as class 2A carcinogen (probably carcinogenic to humans) by the International Agency for Research on Cancer (IARC), is formed during heating of food from reducing carbohydrates and asparagine by Maillard reaction chemistry. After dietary uptake, AA is in part metabolically converted into the proximate genotoxic phase I metabolite glycidamide (GA). GA reacts with nucleophilic base positions in DNA, primarily forming N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) adducts. In a competing phase II biotransformation pathway AA, as well as its phase I metabolite GA, is coupled to glutathione (GSH). The GSH coupling products are further biotransformed and excreted via urine as mercapturic acids (MA), N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA). In the present study, hepatic biotransformation pathways and DNA adduct formation were studied in primary rat hepatocytes, incubated with AA (0.2–2,000 μM) for up to 24 h. Contents of AA-GSH, GA, AAMA, and GAMA were measured in the cell culture medium after solid phase extraction (SPE). N7-GA-Gua adducts in DNA of hepatocytes were determined by HPLC–ESI–MS/MS after lysis of the cells and neutral thermal hydrolysis. Formation of AA-GSH was linear with AA concentration and incubation time and became detectable already at 0.2 μM (4 h). In contrast to AA, GA was not detected before 16 h incubation at 10-fold higher AA concentration (2 μM). In summary, the rate of AA-GSH formation was found to be about 1.5–3 times higher than that of GA formation. N7-GA-Gua adducts were found only at the highest AA concentration tested (2,000 μM).     

Hydroxyethylvaline adduct formation in haemoglobin as a biological monitor of cigarette smoke intake

The ethylene oxide adduct formed on the N-terminal valine in haemoglobin was investigated as a biological monitor of tobacco smoke intake. The modified method developed for the determination of the hydroxyethylvaline adduct (HOEtVal) involved reaction of globin with pentafluorophenyl isothiocyanate, extraction of the HOEtVal thiohydantoin product, derivatization of this by trimethylsilylation and quantitation by capillary gas chromatography with selective ion monitoring mass spectrometry using a tetradeuterated internal standard. The method was applied to globin samples from 26 habitual cigarette smokers and 24 non-smokers. There was a significant correlation between cigarette smoke intake as measured by the average number of cigarettes smoked per day and HOEtVal levels (r=0.537, p<0.01). Background levels were found in non-smokers (mean 49.9 pmol/g Hb, range 22–106 pmol/g Hb). Smoking increased these levels by 71 pmol/g Hb/ 10 cigarettes per day.Cotinine levels in plasma of the smokers were determined by GC-NPD using 2-methyl-4-nitroaniline as internal standard. For non-smokers cotinine was determined by GC-MS selective ion monitoring using d₃-methylcotinine as internal standard. There was no correlation between number of cigarettes smoked per day and cotinine levels (r=0.297, p>0.05) although cotinine was correlated with HOEtVal (r=0.43, p<0.01).The HOEtVal adduct levels thus appear to be a suitable biomonitor for exposure to hydroxyethylating agents in cigarette smoke, reflecting an integrated dose over the erythrocyte lifetime. This is in contrast to plasma cotinine determinations which reflect only the previous day's exposure to nicotine in smoke.     

Acrylamide: a cooking carcinogen?

Exposure to acrylamide (AA) has been monitored by mass spectrometric detection of the adduct, N-(2-carbamoylethyl)valine (CEV), to the N-termini of hemoglobin (Hb), according to the N-alkyl Edman method. In these studies, a conspicuous background level, about 40 pmol/g of globin, of apparently the same adduct was regularly observed in Hb from persons without known exposure to AA. For testing of the hypothesis that this adduct originates from AA formed in cooking, rats were fed fried animal standard diet for 1 or 2 months. These animals exhibited a strong increase of the level of the studied Hb adduct, compared to control rats fed unfried diet. By gas chromatography/tandem mass spectrometry, the identity with CEV was confirmed by the concordance of the product ion spectrum of the studied adduct with that of a verified standard and by interpretation of the fragment ions. Further support of the chemical structure, at the same time pinpointing AA as the causative reactive factor, was obtained through the demonstration that AA is formed in the heating of the feed and that the level of AA in the fried feed is compatible with the measured levels of the CEV adduct. The raised CEV adduct levels observed in experimental animals are of a magnitude that is similar to the background level in nonsmoking humans. These data render it likely that cooking of food is a major source of the background dose of AA also in humans. An evaluation of cancer tests of AA and available data for its metabolism leads to the estimation that the background dose of AA is associated with a considerable cancer risk.