Formation Of Hemoglobin Adducts Research Articles

The hemoglobin adduct N-(2,3-dihydroxypropyl)-valine as biomarker of dietary exposure to glycidyl esters: a controlled exposure study in humans

Fatty acid esters of glycidol (glycidyl esters) are heat-induced food contaminants predominantly formed during industrial deodorization of vegetable oils and fats. After consumption, the esters are digested in the gastrointestinal tract, leading to a systemic exposure to the reactive epoxide glycidol. The compound is carcinogenic, genotoxic and teratogenic in rodents, and rated as probably carcinogenic to humans (IARC group 2A). Assessment of exposure from occurrence and consumption data is difficult, as lots of different foods containing refined oils and fats may contribute to human exposure. Therefore, assessment of the internal exposure using the hemoglobin adduct of glycidol, N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val), may be promising, but a proof-of-principle study is needed to interpret adduct levels with respect to the underlying external exposure. A controlled exposure study was conducted with 11 healthy participants consuming a daily portion of about 36 g commercially available palm fat with a relatively high content of ester-bound glycidol (8.7 mg glycidol/kg) over 4 weeks (total amount 1 kg fat, individual doses between 2.7 and 5.2 µg/kg body weight per day). Frequent blood sampling was performed to monitor the 2,3-diHOPr-Val adduct levels during formation and the following removal over 15 weeks, using a modified Edman degradation and ultrahigh performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Results demonstrated for the first time that the relatively high exposure during the intervention period was reflected in corresponding distinct increases of 2,3-diHOPr-Val levels in all participants, following the expected slope for hemoglobin adduct formation and removal over time. The mean adduct level increased from 4.0 to 12.2 pmol 2,3-diHOPr-Val/g hemoglobin. By using a nonlinear mixed model, values for the adduct level/dose ratio (k, mean 0.082 pmol 2,3-diHOPr-Val/g hemoglobin per µg glycidol/kg body weight) and the adduct lifetime (τ, mean 104 days, likely the lifetime of the erythrocytes) were determined. Interindividual variability was generally low. 2,3-DiHOPr-Val was therefore proven to be a biomarker of the external dietary exposure to fatty acid esters of glycidol. From the background adduct levels observed in our study, a mean external glycidol exposure of 0.94 µg/kg body weight was estimated. This value is considerably higher than current estimates for adults using occurrence and consumption data of food. Possible reasons for this discrepancy are discussed (other oral or inhalational glycidol sources, endogenous formation, exposure to other chemicals also forming the adduct 2,3-diHOPr-Val). Further research is necessary to clarify the issue.

Methemoglobin Formation and Characterization of Hemoglobin Adducts of Carcinogenic Aromatic Amines and Heterocyclic Aromatic Amines.

Arylamines (AAs) and heterocyclic aromatic amines (HAAs) are structurally related carcinogens formed during the combustion of tobacco or cooking of meat. They undergo cytochrome P450 mediated N-hydroxylation to form metabolites which bind to DNA and lead to mutations. The N-hydroxylated metabolites of many AAs also can undergo a co-oxidation reaction with oxy-hemolgobin (HbO2) to form methemoglobin (met-Hb) and the arylnitroso intermediates, which react with the β-Cys(93) chain of Hb to form Hb-arylsulfinamide adducts. The biochemistry of arylamine metabolism has been exploited to biomonitor certain AAs through their Hb arylsulfinamide adducts in humans. We examined the reactivity of HbO2 with the N-hydroxylated metabolites of 4-aminobiphenyl (ABP, HONH-ABP), aniline (ANL, HONH-ANL), and the HAAs 2-amino-9H-pyrido[2,3-b]indole (AαC, HONH-AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, HONH-PhIP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx, HONH-MeIQx). HONH-ABP, HO-ANL, and HONH-AαC induced methemoglobinemia and formed Hb sulfinamide adducts. However, HONH-MeIQx and HONH-PhIP did not react with the oxy-heme complex, and met-Hb formation and chemical modification of the β-Cys(93) residue were negligible. Molecular modeling studies showed that the distances between the H-ON-AA or H-ON-HAA substrates and the oxy-heme complex of HbO2 were too far away to induce methemoglobinemia. Different conformational changes in flexible helical and loop regions around the heme pocket induced by the H-ON-AA or H-ON-HAAs may explain the different proclivities of these chemicals to induce methemoglobinemia. Hb-Cys(93β) sulfinamide and sulfonamide adducts of ABP, ANL, and AαC were identified, by Orbitrap MS, following the proteolysis of Hb with trypsin, Glu-C, or Lys-C. Hb sulfinamide and sulfonamide adducts of ABP were identified in the blood of mice exposed to ABP, by Orbitrap MS. This is the first report of the identification of intact Hb sulfinamide adducts of carcinogenic AAs in vivo. The high reactivity of HONH-AαC with HbO2 suggests that the Hb sulfinamide adduct of AαC may be a promising biomarker of exposure to this HAA in humans.

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.

Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring

A method is described for profiling putative adducts (or other unknown covalent modifications) at the Cys34 locus of human serum albumin (HSA), which represents the preferred reaction site for small electrophilic species in human serum. By comparing profiles of putative HSA-Cys34 adducts across populations of interest it is theoretically possible to explore environmental causes of degenerative diseases and cancer caused by both exogenous and endogenous chemicals. We report a novel application of selected-reaction-monitoring (SRM) mass spectrometry, termed fixed-step SRM (FS-SRM), that allows detection of essentially all HSA-Cys34 modifications over a specified range of mass increases (added masses). After tryptic digestion, HSA-Cys34 adducts are contained in the third largest peptide (T3), which contains 21 amino acids and an average mass of 2433.87 Da. The FS-SRM method does not require that exact masses of T3 adducts be known in advance but rather uses a theoretical list of T3-adduct m/z values separated by a fixed increment of 1.5. In terms of added masses, each triply charged parent ion represents a bin of ±2.3 Da between 9.1 Da and 351.1 Da. Synthetic T3 adducts were used to optimize FS-SRM and to establish screening rules based upon selected b- and y-series fragment ions. An isotopically labeled T3 adduct is added to protein digests to facilitate quantification of putative adducts. We used FS-SRM to generate putative adduct profiles from six archived specimens of HSA that had been pooled by gender, race, and smoking status. An average of 66 putative adduct hits (out of a possible 77) were detected in these samples. Putative adducts covered a wide range of concentrations, were most abundant in the mass range below 100 Da, and were more abundant in smokers than in nonsmokers. With minor modifications, the FS-SRM methodology can be applied to other nucleophilic sites and proteins.

Effects of Ethanol and Phenobarbital on Hemoglobin Adducts Formation in Rats Exposed to Benzidine and Direct Black 38

The objective of this study was to evaluate the effects of pretreatment of ethanol (EtOH) and phenobarbital (PB), which are known to affect the metabolism of xenobiotics, in the formation of hemoglobin adducts in rats administered with benzidine (BZ) and Direct Black 38 (DB38). The experimental rats were divided into BZ and DB38 groups; each group was subdivided into control, EtOH, and PB groups. Blood samples were separated into hemoglobin and plasma immediately after obtaining and basic hydrolysis was done to convert the adducts into aromatic amines. Hydrolyzed BZ, monoacetylbenzidine (MABZ), and 4-aminobiphenyl (4ABP) were separated by reversed-phase liquid chromatography without derivatization. Then, quantitative analyses were performed using a high performance liquid chromatograph equipped with an electrochemical detector. The amount of metabolites was expressed in the hemoglobin binding index (HBI). As a result, the formations of hemoglobin in BZ-, MABZ-, and 4ABP-HBI of BZ-EtOH and BZ-PB groups were increased compared with those of BZ-control group. In DB38 group, all of HBIs except for BZ-HBI were increased more than those of DB38-control group regardless of the pretreatment. These results are attributable to the fact that EtOH and PB induced N-hydroxylation is related to the formation of hemoglobin adducts. They indicate that EtOH not only increases the adduct formation by inducing N-hydroxylation but also induces N-acetylation. PB induced N-hydroxylation and increased the adduct formation in BZ group, but decreased the adduct formation in DB38 group due to decreasing azo reduction. These results suggest that the effects of EtOH or PB should be considered in biochemical monitoring of BZ and DB38 for the assessment of intermittent exposure to BZ and DB38.

Formation of Hemoglobin Adducts of Acrylamide after Its Ingestion in Rats Is Dependent on Age and Sex

The effect of fiber and fat contents of food and of age and sex of animals on the formation of hemoglobin adducts of acrylamide (AA-Hb) in blood has been studied. The results suggest that the absorption of acrylamide (AA) present in food is not affected by the fiber or fat contents of food. However, AA-Hb resulting from the intake of an aqueous solution of AA is dependent on the age and sex of rats: AA-Hb levels were higher in females than in males (3.53- and 2.55-fold higher, respectively, for AA doses of 25 and 100 mg/kg) and in younger than in older rats (30.1% higher in 1.5 month old as compared to 14 month old rats). In males, AA-Hb levels found after the oral administration of AA in an aqueous solution were significantly lower than those found after dietary or intravenous administration. In conclusion, these results show the existence of significant differences in AA bioavailability from an aqueous solution depending on the sex and age of animals. If similar differences also occur in humans, they would be relevant to assess the exposure of different subpopulations to AA.

Genotoxicity and Potential Carcinogenicity of 2,4,6-Trinitrotoluene: Structural and Toxicological Considerations

Environmental contamination with 2,4,6-TNT (trinitrotoluene) represents a worldwide problem. Concern for carcinogenicity can be derived from chemically related compounds, especially the dinitrotoluenes. In the metabolism of TNT, the reductive routes are preponderant. The main urinary metabolites of TNT are 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene. In humans exposed to TNT, the formation of hemoglobin adducts of the amino-dinitrotoluenes is in general concordance with the ratio of urinary excretion. The variations in quantities of excreted metabolites among the different occupational cohorts studied are likely explained by the different routes of exposure to TNT, including dermal uptake. Most studies show that urinary excretion of the amino-dinitrotoluenes (4-amino-dinitrotoluene plus 2-amino-dinitrotoluene) in a range of 1 to 10 mg L(-1) (5-50 microM) are not uncommon--for instance in persons employed with the disposal of military waste. Trinitotoluene is mutagenic in Salmonella typhimurium strains TA98 and TA100, with and without exogenous metabolic activation. Mutagenic activity has been found in urine from workers who were occupationally exposed to TNT. An unpublished 2-year study was reported in 1984 by the IIT Research Institute, Chicago, IL. Fischer 344 rats were fed diets containing 0.4, 2.0, 10, or 50 mg/kg TNT per day. In the urinary bladder, hyperplasia (12 of 47 animals p < .01) and carcinoma (11 of 47 animals, p < .05) were observed at significant levels in high-dose (50 mg kg(-1)) females and in one or two females, respectively, at 10 mg kg(-1). Taking all the available evidence together, the appropriate precautions should be taken.

Role of CYP2E1 in the Epoxidation of Acrylamide to Glycidamide and Formation of DNA and Hemoglobin Adducts

Acrylamide (AA) is an animal carcinogen, neurotoxin, and reproductive toxin. AA is formed in baked and fried carbohydrate-rich foods. Metabolism of AA occurs via epoxidation to glycidamide (GA) or direct conjugation with glutathione. Using CYP2E1-null mice, recent studies in this laboratory demonstrated that induction of somatic and germ cell mutagenicity in AA-treated mice is dependent on CYP2E1. We hypothesized that AA metabolism to GA is a prerequisite for the induction of AA-induced mutagenicity. Current studies were undertaken to assess the role of CYP2E1 in the epoxidation of AA to GA and the formation of DNA and hemoglobin (HGB) adducts. AA was administered to CYP2E1-null or wild-type mice at 50 mg/kg ip. Mice were euthanized 6 h later and blood and tissues were collected. Using LC-ES/MS/MS, AA, GA, and DNA- and HGB-adducts were measured. While the plasma levels of AA and GA were 115 +/- 14.0 and 1.7 +/- 0.31 microM in CYP2E1-null mice, they were 0.84 +/- 0.80 and 33.0 +/- 6.3 microM in the plasma of AA-treated wild-type mice. Administration of AA to wild-type mice caused a large increase in N7-GA-Gua and N3-GA-Ade adducts in the liver, lung, and testes. While traces of N7-GA-Gua adducts were measured in the tissues of AA-treated CYP2E1-null mice, these levels were 52- to 66-fold lower than in wild-type mice. Significant elevation of both AA- and GA-HGB adducts was detected in AA-treated wild-type mice. In AA-treated CYP2E1-null mice, levels of AA-HGB adducts were roughly twice as high as those in wild-type mice. In conclusion, current work demonstrated that CYP2E1 is the primary enzyme responsible for the epoxidation of AA to GA, which leads to the formation of GA-DNA and HGB adducts.

Metabolism and Hemoglobin Adduct Formation 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 objectives of this study were to evaluate the metabolism of AM in humans following oral administration, to compare hemoglobin adduct formation on oral and dermal administration, and to measure hormone levels. The health of the people exposed under controlled conditions was continually monitored. Prior to conducting exposures in humans, a low-dose study was conducted in rats administered 3 mg/kg (1,2,3-13C3) AM by gavage. The study protocol was reviewed and approved by Institute Review Boards both at RTI, which performed the sample analysis, and the clinical research center conducting the study. (1,2,3-13C3) 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 (3 mg/kg oral dose) were analyzed for AM metabolites using 13C NMR spectroscopy. Approximately 86% of the urinary metabolites were derived from GSH conjugation and excreted as N-acetyl-S-(3-amino-3-oxopropyl)cysteine and its S-oxide. Glycidamide, glyceramide, and low levels of N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine were detected in urine. On oral administration, a linear dose response was observed for N-(2-carbamoylethyl)valine (AAVal) and N-(2-carbamoyl-2-hydroxyethyl)valine (GAVal) in hemoglobin. Dermal administration resulted in lower levels of AAVal and GAVal. This study indicated that humans metabolize AM via glycidamide to a lesser extent than rodents, and dermal uptake was approximately 6.6% of that observed with oral uptake.

Evaluation of the clinical, immunologic, and biochemical effects of nitroso sulfamethoxazole administration to dogs: a pilot study

Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated in humans with hypersensitivity reactions, to include fever, skin eruptions, hepatotoxicity, and blood dyscrasias. These reactions also occur in dogs, the only non-human species known to develop a similar spectrum of sulfonamide hypersensitivity. Sulfonamide hypersensitivity is not well understood, but has been hypothesized to be due to the generation of the reactive oxidative metabolite, nitroso sulfamethoxazole (SMX-NO). SMX-NO, unlike the parent sulfonamide, is cytotoxic in vitro, haptenizes tissue proteins, and is immunogenic in rodents. The purpose of this pilot study was to determine whether SMX-NO, when administered to dogs, would lead to drug-tissue adducts, anti-drug antibodies, antioxidant depletion, or clinical evidence of drug hypersensitivity. Four dogs were randomized to one of four treatments: SMX-NO 1 mg/kg; SMX-NO 3 mg/kg; SMX-NO 10 mg/kg; or vehicle control. Dosing was by the intraperitoneal route, once daily for four consecutive days per week, for 2 weeks total, followed by a third week of observation. Following this, all dogs were challenged with trimethoprim-sulfamethoxazole, 25 mg/kg for 12 h for 2 weeks. No dog developed clinical or biochemical evidence of drug hypersensitivity. Plasma cysteine and leukocyte reduced glutathione were not depleted during dosing; however, ascorbate was significantly depleted by week 2 following SMX-NO at 10 mg/kg. Anti-SMX antibodies (IgG or IgM by ELISA) were not detected in any dogs at any time points. SMX-hemoglobin adducts were detected in the spleen in SMX-NO dosed dogs; however, these adducts were not accompanied by an immunologic or systemic response. The results of this pilot study indicate that SMX-NO dosing in dogs, using a dosing protocol shown to be immunogenic in other species, produces modest ascorbate depletion and hemoglobin adduct formation, but is insufficient to produce an immunologic response or a clinical syndrome of sulfonamide hypersensitivity in this susceptible species.

Dose responses for the formation of hemoglobin adducts and urinary metabolites in rats and mice exposed by inhalation to low concentrations of 1,3-[2,3- 14C]-butadiene

Blood and urine were obtained from male Sprague–Dawley rats and B6C3F1 mice exposed to either a single 6 h or multiple daily (5×6 h) nose-only doses of 1,3-[2,3- 14 C ]-butadiene at atmospheric concentrations of 1, 5 or 20 ppm. Globin was isolated from erythrocytes of exposed animals and analyzed for total radioactivity and also for N-(1,2,3-trihydroxybut-4-yl)-valine adducts. The modified Edman degradation procedure coupled with GC–MS was used for the adduct analysis. Linear relationships were observed between the exposures to 1,3-[2,3- 14 C ]-butadiene and the total radioactivity measured in globin and the level of trihydroxybutyl valine adducts in globin. A greater level of radioactivity (ca. 1.3-fold) was found in rat globin compared with mouse globin. When analyzed for specific amino acid adducts, higher levels of trihydroxybutyl valine adducts were found in mouse globin compared with rat globin. Average levels of trihydroxybutyl valine adduct measured in globin from rats and mice exposed for 5×6 h at 1, 5 and 20 ppm 1,3-[2,3- 14 C ]-butadiene were, respectively, for rats: 80, 179, 512 pmol/g globin and for mice: 143, 351, 1100 pmol/g globin. The profiles of urinary metabolites for rats and mice exposed at the different concentrations of butadiene were obtained by reverse phase HPLC analysis on urine collected 24 h after the start of exposure and were compared with results of a previous similar study carried out for 6 h at 200 ppm butadiene. Whilst there were qualitative and quantitative differences between the profiles for rats and mice, the major metabolites detected in both cases were those representing products of epoxide hydrolase mediated hydrolysis and glutathione (GSH) conjugation of the metabolically formed 1,2-epoxy-3-butene. These were 4-( N-acetyl- l-cysteine- S-yl)-1,2-dihydroxy butane and ( R)-2-( N-acetyl- l-cystein- S-yl)-1-hydroxybut-3-ene, 1-( N-acetyl- l-cystein- S-yl)-2-( S)-hydroxybut-3-ene, 1-( N-acetyl- l-cystein- S-yl)-2-( R)-hydroxybut-3-ene, ( S)-2-( N-acetyl- l-cystein- S-yl)-1-hydroxybut-3-ene, respectively. The former pathway showed a greater predominance in the rat. The profiles of metabolites were similar at exposure concentration in the range 1–20 ppm. There were however some subtle differences compared with results of exposure to the higher 200 ppm concentrations. Overall the results provide the basis for cross species comparison of low exposures in the range of occupational exposures, with the wealth of data available from high exposure studies.

Binding of 3-hydroxybenzo[a]pyrene to bovine hemoglobin and albumin.

Previous studies examined the bioavailability and first-pass biotransformation of 3-hydroxy[(3)H]benzo[a]pyrene ([(3)H]-3-OHBaP) in an isolated perfused catfish intestinal model. This work showed that 3-OHBaP, or a metabolite formed in intestine, bound covalently to blood protein. In this study, the blood adducts were characterized in vitro by incubating bovine ferric hemoglobin or albumin with [(3)H]-3OHBaP under various conditions. Incubation of 2 microM [(3)H]-3-OHBaP with hemoglobin for 1 h resulted in 7.49 pmol bound/mg protein, while albumin binding was 1.37 pmol/mg protein. Mild acid hydrolysis released only 5% of the radioactivity from 3-OHBaP-hemoglobin adducts. After gel filtration, the 3-OHBaP-hemoglobin adducts were examined by HPLC analysis. A single peak of radioactivity was detected at the same retention time as the heme component of hemoglobin. Unbound 3-OHBaP was oxidized to BaP-3,6-dione during incubation with ferric hemoglobin. Treatment of hemoglobin with ascorbic acid decreased the formation of hemoglobin adducts by 33%, while hydrogen peroxide treatment increased adduct formation by 44%. Incubation of [(3)H]-BaP-3-beta-D-glucuronide (BaP-3G) with hemoglobin and beta-glucuronidase resulted in greater binding to hemoglobin than incubation with [(3)H]-3-OHBaP alone. The hemoglobin adduct obtained from [(3)H]-BaP-3G also co-migrated with heme. These results indicate that an oxidative process is involved in formation of the heme adduct and that 3-OHBaP or BaP-3G might be a precursor of the bound metabolite.

Primaquine-induced hemolytic anemia: effect of 6-methoxy-8-hydroxylaminoquinoline on rat erythrocyte sulfhydryl status, membrane lipids, cytoskeletal proteins, and morphology.

Previous studies have shown that 6-methoxy-8-hydroxylaminoquinoline (MAQ-NOH), an N-hydroxy metabolite of the antimalarial drug, primaquine, is a direct-acting hemolytic agent in rats. To investigate the mechanism underlying this hemolytic activity, the effects of hemotoxic concentrations of MAQ-NOH on rat erythrocyte sulfhydryl status, membrane lipids, skeletal proteins, and morphology have been examined. Treatment of rat erythrocytes with a TC(50) concentration of MAQ-NOH (350 microM) caused only a modest and transient depletion of reduced glutathione (GSH) (~30%), which was matched by modest increases in the levels of glutathione disulfide and glutathione-protein mixed disulfides. Lipid peroxidation, as measured by thiobarbituric acid-reactive substances and F(2)-isoprostane formation, was induced in a concentration-dependent manner by MAQ-NOH. However, the formation of disulfide-linked hemoglobin adducts on membrane skeletal proteins and changes in erythrocyte morphology were not observed. These data suggest that hemolytic activity results from peroxidative damage to the lipid of the red cell membrane and is not dependent on skeletal protein thiol oxidation. However, when red cell GSH was depleted (>90%) by titration with diethyl maleate, hemolytic activity of MAQ-NOH was markedly enhanced. Of interest, exacerbation of hemotoxicity was not matched by increases in lipid peroxidation, but by the appearance of hemoglobin-skeletal protein adducts. Collectively, the data are consistent with the concept that MAQ-NOH may operate by more than one mechanism; one that involves lipid peroxidation in the presence of normal amounts of erythrocytic GSH, and one that involves protein oxidation in red cells with low levels of GSH, such as are seen in individuals with glucose-6-phosphate dehydrogenase deficiency.

DOSE-DEPENDENT EXCRETION OF UNCONJUGATED 3-BUTENE-1,2-DIOL MEASURED IN URINE WITH A GC/MS AFTER 1,3-BUTADIENE EXPOSURE

1,3-Butadiene is clearly carcinogenic, and it has a complex pathway of metabolic transformation, as it forms several reactive intermediates. We have previously shown that butadiene diolepoxide is a key metabolite in DNA and hemoglobin adduct formation. Here we report the analysis of 3-buten-1,2-diol—the precursor of butadiene diolepoxide—in urine samples from rats exposed to butadiene by means of inhalation. Urine samples were extracted with isopropanol, and extracts were analyzed using a gas chromatograph and mass spectrometer. Selected ion monitoring was performed by using ion 57 m/z; a retention time allowed reliable analysis. The analysis showed a linear excretion of 3-buten-1,2-diol during the 5 days of 6-h exposures and 18-h recovery times between exposures. The daily correlation coefficient (r) values varied from 0.9945 to 0.9999. A 6-h exposure of rats to 1000 ppm 1,3-butadiene resulted in a mean urinary concentration of 3-buten-1,2-diol of approximately 38 mg/L. Urine samples were also collected during the recovery times, and the extracts were analyzed. The linearity of the excretion during the recovery times showed r values ranging from 0.6932 to 0.9813; 0.5 mg/L of 3-butene-1,2-diol was detected in urine samples excreted after exposure to 1000 ppm 1,3-butadiene. The data demonstrated that butadiene monoepoxide was converted into 3-buten-1,2-diol which, to some extent, was excreted in urine in a nonconjugated form. The excretion of 3-buten-1,2-diol was prompt, with about 98% of the compound being excreted during exposure.

DOSE-DEPENDENT EXCRETION OF UNCONJUGATED 3-BUTENE-1,2-DIOL MEASURED IN URINE WITH A GC/MS AFTER 1,3-BUTADIENE EXPOSURE

1,3-Butadiene is clearly carcinogenic, and it has a complex pathway of metabolic transformation, as it forms several reactive intermediates. We have previously shown that butadiene diolepoxide is a key metabolite in DNA and hemoglobin adduct formation. Here we report the analysis of 3-buten-1,2-diol—the precursor of butadiene diolepoxide—in urine samples from rats exposed to butadiene by means of inhalation. Urine samples were extracted with isopropanol, and extracts were analyzed using a gas chromatograph and mass spectrometer. Selected ion monitoring was performed by using ion 57 m/z; a retention time allowed reliable analysis. The analysis showed a linear excretion of 3-buten-1,2-diol during the 5 days of 6-h exposures and 18-h recovery times between exposures. The daily correlation coefficient (r) values varied from 0.9945 to 0.9999. A 6-h exposure of rats to 1000 ppm 1,3-butadiene resulted in a mean urinary concentration of 3-buten-1,2-diol of approximately 38 mg/L. Urine samples were also collected during the recovery times, and the extracts were analyzed. The linearity of the excretion during the recovery times showed r values ranging from 0.6932 to 0.9813; 0.5 mg/L of 3-butene-1,2-diol was detected in urine samples excreted after exposure to 1000 ppm 1,3-butadiene. The data demonstrated that butadiene monoepoxide was converted into 3-buten-1,2-diol which, to some extent, was excreted in urine in a nonconjugated form. The excretion of 3-buten-1,2-diol was prompt, with about 98% of the compound being excreted during exposure.We acknowledge Mr. Yrjö Peltonen for his very skillful assistance in maintaining a stable BD atmosphere during animal exposure, and we thank Ms. Marja Pihlaja for the animal care.

Metabolism, Toxicokinetics and Hemoglobin Adduct Formation in Rats Following Subacute and Subchronic Acrylamide Dosing

Long-term, low-dose (subchronic) oral acrylamide (ACR) exposure produces peripheral nerve axon degeneration, whereas irreversible axon injury is not a component of short-term, higher dose (subacute) i.p. intoxication [Toxicol Appl Pharmacol 1998;151:211]. It is possible that this differential axonopathic expression is a product of exposure-dependent differences in ACR biotransformation and/or tissue distribution. Therefore, we determined the toxicokinetics and metabolism of ACR following subchronic oral (2.8 mM in drinking water for 34 days) or subacute i.p. (50 mg/kg per day for 11 days) administration to rats. Both dosing regimens produced moderate levels of behavioral neurotoxicity and, for each, ACR was rapidly absorbed from the site of administration and evenly distributed to tissues. Peak ACR plasma concentrations and tissue levels were directly related to corresponding daily dosing rates (20 or 50 mg/kg per day). During subchronic oral dosing a larger proportion (30%) of plasma ACR was converted to the epoxide metabolite glycidamide (GLY) than was observed following subacute i.p. intoxication (8%). This subchronic effect was not specifically related to changes in enzyme activities involved in GLY formation (cytochrome P450 2E1) or metabolism (epoxide hydrolases). Both ACR and GLY formed hemoglobin adducts during subacute and subchronic dosing, the absolute quantity of which did not change as a function of neurotoxicant exposure. Compared to subacute i.p. exposure, the subchronic schedule produced approximately 30% less ACR adducts but two-fold more GLY adducts. GLY has been considered to be an active ACR metabolite and might mediate axon degeneration during subchronic ACR administration. However, corresponding peak GLY plasma concentrations were relatively low and previous studies have shown that GLY is only a weak neurotoxicant. Our study did not reveal other toxicokinetic idiosyncrasies that might be a basis for subchronic induction of irreversible axon damage. Consequently the mechanism of axon degeneration does not appear to involve route- or rate-dependent differences in metabolism or disposition.

Reactive metabolites of 1,3-butadiene: DNA and hemoglobin adduct formation and potential roles in carcinogenicity.

1,3-Butadiene (BD), a petrochemical widely used in the manufacture of synthetic rubber and plastics, has recently been added to the list of chemicals Known To Be Human Carcinogens based upon epidemiological and mechanistic data indicating a causal relationship between occupational exposure to BD and excess mortality from lymphatic and/or hematopoietic cancers (U.S. Department of Health and Human Services, 2000). Long-term BD inhalation studies in mice and rats have also been associated with genotoxicity and carcinogenicity, with mice being much more sensitive to BD-induced carcinogenicity than rats (Melnick et al, 1990; Owen et al, 1987). Because BD toxicities are believed to be mediated by reactions of BD metabolites with nucleophilic sites on macromolecules, studies in our laboratory have focused on the characterization of potential metabolic pathways of BD bioactivation, in terms of the enzymes involved, the metabolites formed, and the kinetics of the reactions. Bioactivation reactions of several primary and secondary BD metabolites were also examined. In addition, the reactions of butadiene monoxide (BMO), a primary metabolite of BD, with macromolecules were characterized.KeywordsHuman Liver MicrosomeEpoxide HydrolaseReactive MetaboliteGlobin ChainFast Atom Bombardment Mass SpectrumThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Characterization of Hemoglobin Adducts of 1,3-Dinitrobenzene and 1,3,5-Trinitrobenzene

1,3-dinitrobenzene (DNB) and 1,3,5-trinitrobenzene (TNB) are used primarily in explosive compositions and munitions and have been detected as environmental contaminants of surface waters as well as ground waters near production waste disposal sites. Hemoglobin (Hb) adducts have recently been proposed as biological markers of exposure assessment for various environmental compounds, including nitroaromatics. In the present study, we have investigated the formation of DNB and TNB hemoglobin adducts in vivo and in vitro in the blood of shrew (Cryptotis parva). DNB and TNB hemoglobin adducts were detected by GC/MS after either basic (0.1N NaOH) or acid (2N HCl) hydrolysis followed by organic solvent extraction and derivatization of the corresponding amines. The levels of DNB-Hb adducts detected after basic hydrolysis (238.7 ± 50.2 pg/mg Hb) are higher than the corresponding levels detected after acid hydrolysis (52.5 ± 16.2 pg/mg Hb). For the TNB-Hb the levels after acid hydrolysis (132.2 ± 37.8 pg/mg Hb) are higher than the levels detected after basic hydrolysis (44.7 ± 15.3 pg/mg Hb.) These results demonstrate the effectiveness of the hemoglobin adduct model for monitoring exposure to nitroaromatics.

Chemoprevention of DMBA-induced Mammary Carcinogenesis: Relationship Between Induction of Phase II Enzymes, Effects on DMBA-induced Hemoglobin Adducts and Decreases in Mammary Tumor Multiplicity

The chemopreventive potential of 1,2-dithiole-3-thione, ethoxyquin, butylated hydroxanisole (BHA) and β-naphthoflavone (BNF) was investigated in the 7,12-dimethylbenz(a)anthracene (DMBA) rat mammary tumor model. Female Sprague-Dawley rats received a single dose of DMBA (15 mg) at 50 days of age. Chemopreventive agents were administered via the diet, beginning one week prior to DMBA dosing and continuing until one week following DMBA. Tumor latency, multiplicity and incidence were not affected by BHA (2.5 or 5.0 g/kg diet), but administration of low or high doses of β-napthflavone (1.65 and 3.3 g/kg diet), or high doses of ethoxyquin (5.0 g/kg diet) or 1,2-dithiole-3-thione (0.6 g/kg diet), effectively inhibited all parameters of tumorigenesis. It is presumed that many of these agents which decrease the tumorigenicity act by altering the metabolism of the carcinogen resulting in diminished numbers of reactive ultimate carcinogens. We examined both for the induction of enzymes that might alter carcinogen metabolism as well as alterations in levels of DMBA adducts to hemoglobin. A strong correlation was observed between the effect of these agents on carcinogenesis and the induction of phase I1 enzymes (quinone reductase and glutathione S-transferase) and glutathione in the mammary gland. We also examined the effects of BNF, 1.2- dithiol-3-thione and BHA on formation of hemoglobin adducts by DMBA. Similarly to the results on carcinogenesis the relative efficacy of compounds in inhibiting DMBA adduct formation and DMBA induced mammary tumorigenesis was BNF > 1,2 Dithiolthione >> BHA. These results support the hypothesis that these agents work by altering metabolism of DMBA to reactive intermediates but further offer the possibility that modulation of hemoglobin adducts may serve as a surrogate marker of efficacy.

Gas Chromatographic and Mass Spectrometric Determination of Hemoglobin Adducts of 1,3-Dinitrobenzene and 1,3,5-Trinitrobenzene in Shrew Cryptotis Parva

1,3-Dinitrobenzene (DNB) and 1,3,5-trinitrobenzene (TNB) are used primarily in explosive compositions and munitions and have been detected as environmental contaminants of surface waters as well as ground waters near production waste disposal sites. Hemoglobin (Hb) adducts have recently been proposed as biological markers of exposure assessment for various environmental compounds, including nitroaromatics. In the present study, we have investigated the formation of DNB and TNB hemoglobin adducts in vivo and in vitro in the blood of shrew (Cryptotis parva). DNB and TNB hemoglobin adducts were detected by gas chromatography/mass spectrometry (GC/MS) after either basic (0.1 N NaOH) or acid (2 N HCl) hydrolysis followed by organic solvent extraction and derivatization of the corresponding amines. The levels of DNB-Hb adducts detected after basic hydrolysis (238.7 &amp; pm; 50.2 pg/mg Hb) are higher than the corresponding levels detected after acid hydrolysis (52.5 &amp; pm; 16.2 pg/mg Hb). For the TNB-Hb the levels after acid hydrolysis (132.2 &amp; pm; 37.8 pg/mg Hb) are higher than the levels detected after basic hydrolysis (44.7 &amp; pm; 15.3 pg-mg Hb). These results demonstrate the effectiveness of the hemoglobin adduct model for monitoring exposure to nitroaromatics.