Dose-dependent DNA adduct formation by cinnamaldehyde and other food-borne α,β-unsaturated aldehydes predicted by physiologically based in silico modelling

Genotoxicity of α,β-unsaturated aldehydes shown in vitro raises a concern for the use of the aldehydes as food flavourings, while at low dose exposures the formation of DNA adducts may be prevented by detoxification. Unlike many α,β-unsaturated aldehydes for which in vivo data are absent, cinnamaldehyde was shown to be not genotoxic or carcinogenic in vivo. The present study aimed at comparing dose-dependent DNA adduct formation by cinnamaldehyde and 18 acyclic food-borne α,β-unsaturated aldehydes using physiologically based kinetic/dynamic (PBK/D) modelling. In rats, cinnamaldehyde was predicted to induce higher DNA adducts levels than 6 out of the 18 α,β-unsaturated aldehydes, indicating that these 6 aldehydes may also test negative in vivo. At the highest cinnamaldehyde dose that tested negative in vivo, cinnamaldehyde was predicted to form at least three orders of magnitude higher levels of DNA adducts than the 18 aldehydes at their respective estimated daily intake. These results suggest that for all the 18 α,β-unsaturated aldehydes DNA adduct formation at doses relevant for human dietary exposure may not raise a concern. The present study illustrates a possible use of physiologically based in silico modelling to facilitate a science-based comparison and read-across on the possible risks posed by DNA reactive agents.     

Evaluation of serum and liver toxicokinetics for furan and liver DNA adduct formation in male Fischer 344 rats

Furan is a food processing contaminant found in many common cooked foods that induces liver toxicity and liver cancer in animal models treated with sufficient doses. The metabolism of furan occurs primarily in the liver where CYP 2E1 produces a highly reactive bis-electrophile, cis-2-butene-1,4-dial (BDA). BDA reacts with nucleophilic groups in amino acids and DNA in vitro to form covalent adducts. Evidence for BDA-nucleoside adduct formation in vivo is limited but important for assessing the carcinogenic hazard of dietary furan. This study used controlled dosing with furan in Fischer 344 rats to measure serum and liver toxicokinetics and the possible formation of BDA-nucleoside adducts in vivo. After gavage exposure, furan concentrations in the liver were consistently higher than those in whole blood (∼6-fold), which is consistent with portal vein delivery of a lipophilic compound into the liver. Formation of BDA-2′-deoxycytidine in furan-treated rat liver DNA was not observed using LC/MS/MS after single doses as high as 9.2 mg/kg bw or repeated dosing for up to 360 days above a consistent background level (1-2 adducts per 10⁸ nucleotides). This absence of BDA-nucleoside adduct formation is consistent with the general lack of evidence for genotoxicity of furan in vivo.     

Physiologically based kinetic modeling of bioactivation and detoxification of the alkenylbenzene methyleugenol in human as compared with rat

This study defines a physiologically based kinetic (PBK) model for methyleugenol (ME) in human based on in vitro and in silico derived parameters. With the model obtained, bioactivation and detoxification of methyleugenol (ME) at different doses levels could be investigated. The outcomes of the current model were compared with those of a previously developed PBK model for methyleugenol (ME) in male rat. The results obtained reveal that formation of 1′-hydroxymethyleugenol glucuronide (1′HMEG), a major metabolic pathway in male rat liver, appears to represent a minor metabolic pathway in human liver whereas in human liver a significantly higher formation of 1′-oxomethyleugenol (1′OME) compared with male rat liver is observed. Furthermore, formation of 1′-sulfooxymethyleugenol (1′HMES), which readily undergoes desulfonation to a reactive carbonium ion (CA) that can form DNA or protein adducts (DA), is predicted to be the same in the liver of both human and male rat at oral doses of 0.0034 and 300 mg/kg bw. Altogether despite a significant difference in especially the metabolic pathways of the proximate carcinogenic metabolite 1′-hydroxymethyleugenol (1′HME) between human and male rat, the influence of species differences on the ultimate overall bioactivation of methyleugenol (ME) to 1′-sulfooxymethyleugenol (1′HMES) appears to be negligible. Moreover, the PBK model predicted the formation of 1′-sulfooxymethyleugenol (1′HMES) in the liver of human and rat to be linear from doses as high as the benchmark dose (BMD₁₀) down to as low as the virtual safe dose (VSD). This study shows that kinetic data do not provide a reason to argue against linear extrapolation from the rat tumor data to the human situation.     

The mycotoxin patulin reacts with DNA bases with and without previous conjugation to GSH: implication for related α,β-unsaturated carbonyl compounds?

The α,β-unsaturated carbonyl group is recognized as alert for mutagenicity, attributed to (1) its direct reaction with DNA, counteractable by glutathione (GSH), and (2) oxidative stress caused indirectly by GSH depletion. Accordingly, the α,β,γ,δ-unsaturated lactone patulin (PAT), a mycotoxin detected in fruits and products derived thereof, is known to induce gene, chromosome, and genome mutations in vitro, its mutagenicity correlating inversely with intracellular GSH levels. Thus, the reactivity of PAT against DNA bases and nucleosides in the absence and presence of GSH and glutathione S-transferases (GSTs) was investigated under cell-free conditions using HPLC mass spectrometry techniques for identification of reaction products. Adduct formation with all four nucleobases as well as with purine base nucleosides occurred even in the presence of GSH, revealing several adducts of PAT, mono- and disubstituted with nucleobases/nucleosides as well as novel GSH–PAT adducts. In addition, novel mixed GSH–PAT–nucleobase adducts were observed. These adducts exhibited a ketohexanoic acid-type structure of the PAT molecule, C6 substituted with GSH and linking C1 of PAT with nitrogens of nucleobases/nucleosides via an amide bond. Formation of GSH–PAT–adenine adducts was not prevented by GSTs, and excess of GSH needed to reduce their formation was higher than for PAT–adenine adducts. The formation of mixed GSH–DNA base adducts has not been described for PAT or any other α,β-unsaturated carbonyl before, although the reaction mechanism seems to be applicable to a variety of α,β-unsaturated carbonyls occurring in food and in the environment.     

Overcoming the genotoxicity of a pyrrolidine substituted arylindenopyrimidine as a potent dual adenosine A(2A)/A(1) antagonist by minimizing bioactivation to an iminium ion reactive intermediate

2-Amino-4-phenyl-8-pyrrolidin-1-ylmethyl-indeno[1,2-d]pyrimidin-5-one (1) is a novel and potent selective dual A(2A)/A(1) adenosine receptor antagonist from the arylindenopyrimidine series that was determined to be genotoxic in both the Ames and Mouse Lymphoma L5178Y assays only following metabolic activation. Compound 1 was identified as a frame-shift mutagen in Salmonella typhimurium tester strain TA1537 as indicated by a significant dose-dependent increase in revertant colonies as compared to the vehicle control. The metabolic activation-dependent irreversible covalent binding of radioactivity to DNA, recovery of 1 and its enamine metabolite from acid hydrolysis of covalently modified DNA, and protection of covalent binding to DNA by both cyanide ion and methoxylamine suggest that the frame-shift mutation in TA1537 strain involved covalent binding instead of simple intercalation to DNA. Compound 1 was bioactivated to endocyclic iminium ion, aldehyde, epoxide, and α,β-unsaturated keto reactive intermediates from the detection of cyano, oxime, and glutathione conjugates by data-dependent high resolution accurate mass measurements. Collision-induced dissociation of these conjugates provided evidence for bioactivation of the pyrrolidine ring of 1. The epoxide and α,β-unsaturated keto reactive intermediates were unlikely to cause the genotoxicity of 1 because the formation of their glutathione adducts did not ameliorate the binding of compound related material to DNA. Instead, the endocyclic iminium ions and amino aldehydes were likely candidates responsible for genotoxicity based on, first, the protection afforded by both cyanide ion and methoxylamine, which reduced the potential to form covalent adducts with DNA, and, second, analogues of 1 designed with low probability to form these reactive intermediates were not genotoxic. It was concluded that 1 also had the potential to be mutagenic in humans based on observing the endocyclic iminium ion following incubation with a human liver S9 preparation and the commensurate detection of DNA adducts. An understanding of this genotoxicity mechanism supported an evidence-based approach to selectively modify the structure of 1 which resulted in analogues being synthesized that were devoid of a genotoxic liability. In addition, potency and selectivity against both adenosine A(2A) and A(1) receptors were maintained.     

Development of a 32P-postlabeling method for the detection of 1, N2-propanodeoxyguanosine adducts of 2-hexenal in vivo

2-Hexenal is an alpha,beta-unsaturated carbonyl compound which forms cyclic 1,N2-propano adducts in vitro. The adduct formation in vivo was not reported by others to date. Because this type of adduct is considered promutagenic (2-hexenal is actually mutagenic and genotoxic) and humans are permanently exposed to this compound via vegetarian food, 2-hexenal may play a role in carcinogenicity. To improve the cancer risk assessment, we developed a new 32P-postlabeling technique for this compound and optimized the different steps of the postlabeling procedure. The results of the postlabeling methods are shown. A labeling efficiency of 35%, a recovery of 10% for the synthesized standards, and a detection limit of three 2-hexenal adducts per 10(8) nucleotides was achieved. After gavage of 500 mg/kg of body weight to male Fischer 344 rats, the respective DNA adducts were detected in rat liver DNA. With this study, we demonstrate in vivo adduct formation of 2-hexenal for the first time. Highest adduct levels were found 2 days after gavage, and after 4 days, the level was even higher than after 1 day. No adducts were detected 8 h after gavage. The respective adducts could not be found as a background in tissues of untreated rats or in calf thymus DNA at the limit of detection.     

Effects of solvent on DNA adduct formation in skin and lung of CD1 mice exposed cutaneously to benzo(a)pyrene

The effect of solvent polarity and lipophilicity on DNA adduct formation by polycyclic aromatic hydrocarbons in skin and lung has been studied in CD1 mice exposed cutaneously in vivo to benzo(a)pyrene (∼0.01–7.0 μg/animal) in either tetrahydrofuran or n-dodecane. The nature and amounts of DNA adducts, measured as 7R,8S,9R-trihydroxy-10S-(N²-deoxyguanosyl-3′-phosphate)-7,8,9,10-tetrahydrobenzo(a)pyrene, in relation to exposure dose and treatment regime was determined by ³²P-postlabelling. In skin DNA there was a linear relationship between exposure dose and adduct formation with both solvents, though the amount of adduct formed was significantly lower from treatment with benzo(a)pyrene in n-dodecane than in tetrahydrofuran. The amounts of adducts measured in skin DNA ranged from 67 amol adducts/μg DNA at the lowest exposure dose of benzo(a)pyrene in n-dodecane to 3.5 fmol adducts/μg DNA (1 adduct in 5 × 10⁷ nucleotides to 1 adduct in 9 × 10⁵ nucleotides) at the highest dose. In tetrahydrofuran the corresponding levels were 89 amol adducts/μg DNA (1 adduct in 3 × 10⁷ nucleotides) to 16.9 fmol adducts/μg DNA (1 adduct in 2 × 10⁵ nucleotides). DNA adducts could not be detected in lung tissue following cutaneous treatment of animals with benzo(a)pyrene in n-dodecane. Cutaneous treatment of animals with benzo(a)pyrene in tetrahydrofuran, however, resulted in adducts in lung DNA at a level of 88 amol/μg DNA from exposures only at the highest dose (6.72 μg/animal). The difference in octanol-water partition coefficient, log P_ow between n-dodecane compared to tetrahydrofuran is considered to be the most likely reason for the reduction in the bioavailability of benzo(a)pyrene and/or its metabolites and hence the degree of genotoxicity in tissues. The results suggest that other paraffinic hydrocarbon solvents may moderate the genotoxicity of polycyclic aromatic hydrocarbons in vivo. The assessment of the genotoxicity in vivo of mixtures of compounds should be carried out on complete mixtures of substances of interest in order to take account of these possible antagonistic or synergistic effects. Received: 10 February 1999 / Accepted: 10 May 1999     

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.     

Inhibition of methyleugenol bioactivation by the herb-based constituent nevadensin and prediction of possible in vivo consequences using physiologically based kinetic modeling

Methyleugenol (ME) occurs naturally in a variety of spices, herbs, including basil, and their essential oils. ME induces hepatomas in rodent bioassays following its conversion to a DNA reactive metabolite. In the present study, the basil constituent nevadensin was shown to be able to inhibit SULT-mediated DNA adduct formation in HepG2 cells exposed to the proximate carcinogen 1′-hydroxymethyleugenol in the presence of nevadensin. To investigate possible in vivo implications of SULT inhibition by nevadensin on ME bioactivation, the rat physiologically based kinetic (PBK) model developed in our previous work to describe the dose-dependent bioactivation and detoxification of ME in male rat was combined with the recently developed PBK model describing the dose-dependent kinetics of nevadensin in male rat. The resulting binary ME–nevadensin PBK model was used to predict the possible nevadensin mediated reduction in ME DNA adduct formation and resulting carcinogenicity at the doses of ME used by the NTP carcinogenicity study. Using these data an updated risk assessment using the Margin of Exposure (MOE) approach was performed. The results obtained point at a potential reduction of the cancer risk when rodents are orally exposed to ME within a relevant food matrix containing SULT inhibitors compared to exposure to pure ME.     

Protein Adducts Generated from Products of Lipid Oxidation: Focus on HNE and ONE

Modification of proteins in conditions of oxidative stress can contribute to protein dysfunction or tissue damage and disease progression. Bifunctional, most often α,β-unsaturated carbonyl compounds such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and acrolein, generated from oxidation of polyunsaturated fatty acids (PUFAs), readily bind to protein nucleophiles. Modification by bifunctional aldehydes can also lead to intramolecular or intermolecular protein crosslinking. Model studies are revealing the structure of adducts that can then be more readily identified in mass spectrometric studies on proteins exposed to the various pure aldehydes or to peroxidized PUFAs. Although simple Michael and Schiff base adducts are often formed initially, only some of these adducts, such as the HNE- and ONE-derived Michael adducts on Cys and His residues, are found to survive the conditions of proteolysis and HPLC-MS analysis. Reversibly formed adducts, such as the HNE-Lys Michael adduct, can be found on proteolytic peptides only if a NaBH₄-reduction step is used prior to proteolysis. Initial adducts can evolve by tautomerization, oxidation, cyclization, dehydration, and sometimes condensation with a second aldehyde molecule (the same or different), to give stable advanced lipoxidation end products (ALEs) that can be found by mass spectrometry. These include the HNE-Lys-derived 2-pentylpyrrole, the ONE-Lys-derived 4-ketoamide, the ONE-derived His-Lys pyrrole crosslink, and a Lys-derived 3-formyl-4-pentylpyrrole that results from combined action of ONE and acrolein. Michael adducts of α,β-unsaturated aldehydes such as HNE and ONE can be derivatized by 2,4-dinitrophenylhydrazine (DNPH) and can thus constitute significant DNPH-detectable protein-bound carbonyl activity that serves as a key indicator of oxidative stress in tissues. It appears that lipid oxidation is a more important contributor to such activity than metal-catalyzed oxidation of protein side-chains.     

DNA damage induced in mouse tissues by organic wood preserving waste extracts as assayed by 32P-postlabeling

 Numerous wood preserving waste (WPW) sites in the United States pose genotoxic hazards. WPWs consist of complex mixtures containing toxic, including genotoxic, compounds which are derived from the preservatives coal tar creosote and pentachlorophenol (PCP) and other polychlorinated aromatics. The genotoxicity of WPW extracts, which has not been tested in mammals, cannot be evaluated on the basis of data for individual components because of possible compound interactions. Therefore, whole extracts need to be assayed. ³²P-postlabeling represents a powerful tool to determine DNA adduct formation by complex genotoxic mixtures, such as cigarette smoke, diesel exhaust, and coke oven and foundry emissions in experimental animals and humans. In the present study, a mouse bioassay was used in combination with ³²P-postlabeling to determine DNA adduct formation induced by hexane/acetone extracts of two samples from a WPW site. Female ICR mice were treated dermally with extract corresponding to 3 mg residue or vehicle control once per day for 2 days and killed 24 h later. Skin, lung, liver, kidney, and heart DNA preparations were assayed by nuclease P1-enhanced postlabeling. Adduct profiles were tissue-specific and displayed a multitude of non-polar DNA adducts with levels amounting to one adduct in 1.6×10⁶ DNA nucleotides in skin (both extracts) and one adduct in 3.2×10⁷ or 1.2×10⁷ DNA nucleotides in liver (extract 1 or extract 2). Based on their chromatographic properties, these adducts appeared largely derived from polycyclic aromatic hydrocarbons (PAHs) present in the extracts. One of the major adducts was identified as the ³²P-labeled derivative of the reaction product of 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) with N ² of deoxyguanosine. Total non-polar DNA adduct levels were highest in skin and lung, amounting to 17.4 and 24.0% of the skin values for extracts 1 and 2, respectively, in lung while the corresponding levels in liver were 5.0 and 12.6%. These results were in accord with the carcinogenic potencies of PAHs in these organs. Extract 2 induced higher adduct levels in internal organs, although its PAH concentrations were lower than those of extract 1, i.e. lung, liver, kidney, and heart had 1.4, 2.5, 1.9, and 1.7 times higher total adduct levels and 1.6, 3.3, 1.6, and 1.9 times higher benzo[a]pyrene adduct levels. With the exception of total adducts in lung, the differences between the two extracts were all significant, suggestive of compound interactions. The benzo[a]pyrene adduct levels in the five tissues correlated linearly with total adduct levels and thus represented a surrogate for the latter. Overall, the results suggest that DNA adducts in mouse tissues, as analyzed by ³²P-postlabeling, are suitable biomarkers and dosimeters of the genotoxicity of WPW extracts. Received: 26 September 1995/Accepted: 7 December 1995     

Formation of deoxyguanosine cross-links from calf thymus DNA treated with acrolein and 4-hydroxy-2-nonenal

Acrolein (AC) and 4-hydroxy-2-nonenal (HNE) are endogenous bis-electrophiles that arise from the oxidation of polyunsaturated fatty acids. AC is also found in high concentrations in cigarette smoke and automobile exhaust. These reactive α,β-unsaturated aldehyde (enal) covalently modify nucleic acids, to form exocyclic adducts, where the three-carbon hydroxypropano unit bridges the N1 and N(2) positions of deoxyguanosine (dG). The bifunctional nature of these enals allows them to undergo reaction with a second nucleophilic group and form DNA cross-links. These cross-linked enal adducts are likely to contribute to the genotoxic effects of both AC and HNE. We have developed a sensitive mass spectrometric method to detect cross-linked adducts of these enals in calf thymus DNA (CT DNA) treated with AC or HNE. The AC and HNE cross-linked adducts were measured by the stable isotope dilution method, employing a linear quadrupole ion trap mass spectrometer and consecutive reaction monitoring at the MS(3) or MS(4) scan stage. The lower limit of quantification of the cross-linked adducts is ～1 adduct per 10(8) DNA bases, when 50 μg of DNA is assayed. The cross-linked adducts occur at levels that are ～1-2% of the levels of the monomeric 1,N(2)-dG adducts in CT DNA treated with either enal.     

Possible involvement of genotoxic mechanisms in estragole-induced hepatocarcinogenesis in rats

Estragole (ES) is a natural organic compound used frequently as a flavoring food additive. Although it has been reported to be tumorigenic and induce DNA adducts in the mouse liver, there have been no reports regarding ES hepatocarcinogenicity in rats. In the current study, we therefore examined potent carcinogenicity, DNA adduct formation and in vivo genotoxicity of ES in the livers of wild and reporter gene-carrying F344 rats. Males were administered 600?mg/kg bw ES by gavage and sequentially sacrificed at weeks 4, 8 and 16 for GST-P and PCNA immunohistochemistry and measurement of ES-specific DNA adducts by LC-MS/MS in the livers. GST-P-positive foci increased with time in ES-treated rats from week 4, PCNA-labeling indices being similarly elevated at both weeks 4 and 8. ES-specific DNA adducts such as ES-3'-N (2)-dG, 3'-8-dG and 3'-N (6)-dA were consistently detected, particularly at week 4. In a second study, male F344 gpt delta rats were administered 0, 22, 66, 200 or 600?mg/kg bw ES for 4?weeks. Gpt mutant frequency in the liver was increased in a dose-dependent manner, with significance at 200 and 600?mg/kg bw in good correlation with PCNA-labeling indices. Mutation spectra analysis showed A:T to G:C transitions to be predominantly increased in line with the formation of ES-3'-N (6)-dA or 3'-8-dG. These results indicate that ES could be a possible genotoxic hepatocarcinogen in the rat, at least when given at high doses.     

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.     

Potential Application of Benzo(a)Pyrene-Associated Adducts (Globin or Lipid) as Blood Biomarkers for Target Organ Exposure and Human Risk Assessment

In order to investigate the potential application of blood biomarkers as surrogate indicators of carcinogen–adduct formation in target-specific tissues, temporal formation of benzo[a]pyrene (BaP)-associated DNA adducts, protein adducts, or lipid damage in target tissues such as lung, liver, and kidney was compared with globin adduct formation or plasma lipid damage in blood after continuous intraperitoneal (ip) injection of [³H]BaP into female ICR mice for 7 d. Following treatment with [³H]BaP, formation of [³H]BaP–DNA or –protein adducts in lung, liver, and kidney increased linearly, and persisted thereafter. This finding was similar to the observed effects on globin adduct formation and plasma lipid damage in blood. The lungs contained a higher level of DNA adducts than liver or kidneys during the treatment period. Further, the rate of cumulative adduct formation in lung was markedly greater than that in liver. Treatment with a single dose of [³H]BaP indicated that BaP–globin adduct formation and BaP–lipid damage in blood reached a peak 48 h after treatment. Overall, globin adduct formation and lipid damage in blood were significantly correlated with DNA adduct formation in the target tissues. These data suggest that peripheral blood biomarkers, such as BaP–globin adduct formation or BaP–lipid damage, may be useful for prediction of target tissue-specific DNA adduct formation, and for risk assessment after exposure.     

Molecular mechanism of glyceraldehyde-3-phosphate dehydrogenase inactivation by α,β-unsaturated carbonyl derivatives

α,β-Unsaturated carbonyls make up an important class of chemicals involved in environmental toxicity and disease processes. Whereas adduction of cysteine residues on proteins is a well-documented reaction of these chemicals, such a generic effect cannot explain the molecular mechanism of cytotoxicity. Instead, more detailed information is needed regarding the possible specificity and kinetics of cysteine targeting and the quantitative relationship between adduct burden and protein dysfunction. To address these data gaps, we incubated purified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with acrylamide (ACR), acrolein, or methylvinyl ketone (MVK). Results show that these α,β-unsaturated carbonyl toxicants inhibited GAPDH activity in a concentration- and time-dependent manner. The rank order of enzyme inhibition (K(I)) (i.e., ACR ? MVK < acrolein) was related to the calculated electrophilic reactivity of each compound and to the corresponding kinetics of cysteine adduct formation. Tandem mass spectrometry revealed that adduct formation was selective at lower concentrations; i.e., ACR preferentially formed adducts with Cys152 (residues 146-162). At higher concentrations, ACR also formed adducts with Cys156 and Cys247 (residues 235-248). Adduct formation at Cys152 was correlated to enzyme inhibition, which is consistent with the regulatory role of this residue in enzyme function and its location within the GAPDH active site. Further analyses indicated that Cys152 was present in a pK(a)-lowering microenvironment (pK(a) = 6.03), and at physiological pH, the corresponding sulfhydryl group exists in the highly reactive nucleophilic thiolate state. These data suggest a general cytotoxic mechanism in which electrophilic α,β-unsaturated carbonyls selectively form adducts with reactive nucleophilic cysteine residues specifically associated with the active sites of proteins. These specialized cysteine residues are toxicologically relevant molecular targets, because chemical derivatization causes loss of protein function.     

DNA adducts and oxidative DNA damage induced by organic extracts from PM2.5 in an acellular assay

The genotoxic activities of complex mixtures of organic extracts from the urban air particles collected in various localities of the Czech Republic, which differed in the extent and sources of air pollution, were compared. For this purpose, PM2.5 particles were collected by high volume samplers in the most polluted area of the Czech Republic - Ostrava region (localities Bartovice, Poruba and Karvina) and in the locality exhibiting a low level of air pollution - Trebon - a small town in the non-industrial region of Southern Bohemia. To prepare extractable organic matter (EOM), PM2.5 particles were extracted by dichloromethane and c-PAHs contents in the EOMs were determined. As markers of genotoxic potential, DNA adduct levels and oxidative DNA damage (8-oxo-7,8-dihydro-2′-deoxyguanosine, 8-oxodG, levels) induced by EOMs in an acellular assay of calf thymus DNA coupled with ³²P-postlabeling (DNA adducts) and ELISA (8-oxodG) in the presence and absence of microsomal S9 fraction were employed. Twofold higher DNA adduct levels (17.20 adducts/10⁸ nucleotides/m³ vs. 8.49 adducts/10⁸ nucleotides/m³) were induced by EOM from Ostrava-Bartovice (immediate proximity of heavy industry) compared with that from Ostrava-Poruba (mostly traffic emissions). Oxidative DNA damage induced by EOM from Ostrava-Bartovice was more than fourfold higher than damage induced by EOM from Trebon (8-oxodG/10⁸ dG/m³: 0.131 vs. 0.030 for Ostrava-Bartovice vs. Trebon, respectively). Since PM2.5 particles collected in various localities differ with respect to their c-PAHs content, and c-PAHs significantly contribute to genotoxicity (DNA adduct levels), we suggest that monitoring of PM2.5 levels is not a sufficient basis to assess genotoxicity of respirable aerosols. It seems likely that the industrial emissions prevailing in Ostrava-Bartovice represent a substantially higher genotoxic risk than mostly traffic-related emissions in Ostrava-Poruba. B[a]P and c-PAH contents in EOMs are the most important factors relating to their genotoxic potential.     

Isolation and structural characterization of acrylamide-pyridoxamine adducts

Pyridoxamine (PM) is an effective inhibitor of the formation of the carcinogen acrylamide (AA) from its precursors in low-moisture model systems. Although AA is widely assumed to act by scavenging carbonyl compounds, no alternative pathways have to date been explored. In this work, we found AA to directly react with PM in a low-moisture acrylamide-pyridoxamine model system heated at 140 °C for up to 40 min. The reaction products gave four major chromatographic peaks that were assigned to acrylamide-pyridoxamine adducts. Two of the adducts (AA-PM-1 and AA-PM-3) were selected for isolation and structural characterization with various spectroscopic (UV, fluorescence, IR, and NMR) and mass spectrometric techniques (MS, MS/MS). As shown by the proposed reaction scheme, PM can directly react with AA via Michael addition. The reaction involves a nucleophilic attack of the PM amine group on AA (an α,β-unsaturated carbonyl compound) to give adduct AA-PM-3, which was identified as 3-(((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methyl)amino)propanamide. However, AA-PM-3 further reacts with any additional AA present in the medium to give adduct AA-PM-1 identified as 3,3'-(((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methyl)azanediyl)dipropanamide. The time courses of these adduct formation reactions were studied in cookies supplemented with PM, where AA-PM-3 was found to be the predominant structure.