Biomedical subjects

A Ranasinghe

Publications and source records attributed to A Ranasinghe.

At least 19 recordsLinked to original sources

PubMed2001

Hemoglobin adducts as biomarkers of 1,3-butadiene in occupationally low exposed Italian workers and a few diesel-exposed miners.

Hemoglobin adducts were determined as biomarkers of 1,3-butadiene (BD) in 30 workers and 10 controls from an Italian BD plant and in 14 diesel-exposed miners. N-(2,3,4-trihydroxybutyl)valine (THBVal), an N-terminal valine globin adduct of reactive butadiene metabolites, was analyzed by gas chromatography/high resolution mass spectrometry after a modified Edman degradation and further acetylation. The BD exposure for the plant workers was 31 microg/m(3) (personal sampling). Whereas there was no detectable difference in hemoglobin adduct levels (range 17.7-61.4 pmol/g globin) between the total group of exposed and controls, slight but significant differences could be found between two subgroups of workers from different production units as well as one subgroup and controls (P<0.05), between smoking (n=13) and non-smoking exposed workers (n=17; P=0.066) as well as between smoking exposed workers and controls (P=0.055). Adduct levels of the miners (all non-smokers) were in the same range as those of the Italian BD-workers and controls. The internal exposure and strain measured by THBVal levels resulting from a very low occupational BD exposure was in the range of the contribution of moderate smoking.

Biomarkers↗

PubMed2001

Using DNA and hemoglobin adducts to improve the risk assessment of butadiene.

The purpose of this paper is to review what we know about various biomarkers of butadiene in animal, human and in vitro studies, and to draw inferences from these data that impact on the accurate assessment of human risks for cancer. Studies comparing the DNA and hemoglobin adducts of butadiene with exposure, metabolism and genotoxicity have provided a great deal of insight that is applicable to biologically based risk assessment. First, the DNA and hemoglobin adduct data strongly support the conclusion that 3,4-epoxy-1,2-butanediol is the major electrophile available for binding to these macromolecules. Biomarker studies have also provided insight into the possibility of a sensitive population associated with the GSTT1 null genotype. While it is clear that lymphocytes from GSTT1 null individuals are more sensitive for the induction of sister chromatid exchanges (SCE) following in vitro exposure to 1,2,3,4-diepoxybutane, there was no such increase in SCE or other biomarkers of genotoxicity in workers exposed to 1-3 p.p.m. butadiene, regardless of GST genotype. The globin adduct data also demonstrate that there is roughly a tenfold range for interindividual differences in the metabolism of butadiene. This type of analysis represents an excellent means for providing scientific data for this critical determinant. Another useful application of hemoglobin adducts in risk assessment was demonstrated by regressing data for various endpoints for genotoxicity against that individual's biologically effective dose, thereby providing an independent mechanism for evaluation that excludes any possible confounding by inappropriate controls. Finally, biomarker studies have identified critical gaps in our knowledge that are needed for the accurate assessment of butadiene. Most notable of these is the lack of diepoxide-specific biomarkers in mice, rats and humans.

Animals↗

PubMed2001

A new face on apoptosis: death-associated protein 3 and PDCD9 are mitochondrial ribosomal proteins.

Two proteins known to be involved in promoting apoptosis in mammalian cells have been identified as components of the mammalian mitochondrial ribosome. Proteolytic digestion of whole mitochondrial ribosomal subunits followed by analysis of the peptides present using liquid chromatography-tandem mass spectrometry revealed that the proapoptotic proteins, death-associated protein 3 (DAP3) and the programmed cell death protein 9, are both components of the mitochondrial ribosome. DAP3 has motifs characteristic of guanine nucleotide binding proteins and is probably the protein that accounts for the nucleotide binding activity of mammalian mitochondrial ribosomes. The observations reported here implicate mitochondrial protein synthesis as a major component in cellular apoptotic signaling pathways.

Amino Acid Sequence↗

PubMed2001

Simultaneous quantitation of N(2),3-ethenoguanine and 1,N(2)-ethenoguanine with an immunoaffinity/gas chromatography/high-resolution mass spectrometry assay.

We have previously described an immunoaffinity/gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry (IA/GC/ECNCI-HRMS) assay for quantitation of the promutagenic DNA adduct N(2),3-ethenoguanine (N(2),3-epsilonGua) in vivo. Here we present an expanded assay that allows simultaneous quantitation of its structural isomer, 1,N(2)-ethenoguanine (1,N(2)-epsilonGua), in the same DNA sample. 1,N(2)-epsilonGua and N(2),3-epsilonGua were purified together from hydrolyzed DNA using two immobilized polyclonal antibodies. GC/ECNCI-HRMS was used to quantitate the 3,5-bis(pentafluorobenzyl) (PFB) derivative of each adduct against an isotopically labeled analogue. Selected ion monitoring was used to detect the [M - 181](-) fragments of 3,5-(PFB)(2)-N(2),3-epsilonGua and 3,5-(PFB)(2)-[(13)C(4),(15)N(2)]-N(2),3-epsilonGua and the [M - 201](-) fragments of 3,5-(PFB)(2)-1,N(2)-epsilonGua and 3,5-(PFB)(2)-[(13)C(3)]-1,N(2)-epsilonGua. The demonstrated limits of quantitation in hydrolyzed DNA were 7.6 fmol of N(2),3-epsilonGua and 15 fmol of 1,N(2)-epsilonGua in approximately 250 microg of DNA, which corresponded to 5.0 N(2),3-epsilonGua and 8.7 1,N(2)-epsilonGua adducts/10(8) unmodified Gua bases, respectively. 1,N(2)-epsilonGua was found to be the predominant ethenoguanine adduct formed in reactions of lipid peroxidation products with DNA. The respective ratios of 1,N(2)-epsilonGua to N(2),3-epsilonGua were 5:1 and 38:1 when calf thymus DNA was treated with ethyl linoleate or 4-hydroxynonenal, respectively, under peroxidizing conditions. Only N(2),3-epsilonGua was detected in DNA treated with the vinyl chloride (VC) metabolite 2-chloroethylene oxide and in hepatocyte DNA from rats exposed to 1100 ppm VC for 4 weeks (6 h/day for 5 days/week). These data suggest that 1,N(2)-epsilonGua plays a minor role relative to N(2),3-epsilonGua in VC-induced carcinogenesis, but that 1,N(2)-epsilonGua may be formed to a larger extent from endogenous oxidative processes.

Animals↗

PubMed2000

DNA adducts: effects of low exposure to ethylene oxide, vinyl chloride and butadiene.

Dose-response relationships of genotoxic agents differ greatly depending on the agent and the endpoint being evaluated. Simple conclusions that genotoxic effects are linear cannot be applied universally. The shape of the molecular dose of DNA adducts varies from linear, to supralinear, to sublinear depending on metabolic activation and detoxication, and repair of individual types of DNA adducts. For mutagenesis and other genotoxicity endpoints, the dose-response reflects the molecular dose of each type of DNA adduct, cell proliferation, as well as endogenous factors that lead to mutagenesis such as the formation and repair of endogenous DNA adducts. These same factors are important when interpreting the shape of dose-response data for carcinogenesis of genotoxic agents, however, tumor background variability adds additional complexity. Endogenously formed DNA adducts may be identical to those formed by chemicals, as in the case of vinyl chloride and ethylene oxide, or they may be those associated with oxidative stress. Data presented in this paper demonstrate that the exogenous number of adducts induced by 5 days of exposure to 10 ppm vinyl chloride is only 2. 2-fold greater than that present as a steady-state amount in unexposed control rats. Similar data are shown for ethylene oxide. Extremely sensitive methods have been developed for measuring the molecular dose of genotoxins. These methods can detect DNA adducts as low as 1 per 10(9) to 10(10). However, in view of the high number of endogenous DNA adducts that are present in all cells, it is unlikely that causal relationships can be attributed to very low numbers of such DNA adducts. Effects of both exogenous and endogenous DNA adducts need to be factored into the interpretation of chemical exposures.

Animals↗

PubMed2000

Formation of quinonoid-derived protein adducts in the liver and brain of Sprague-Dawley rats treated with 2,2',5, 5'-tetrachlorobiphenyl.

A possible role for metabolic activation of 2,2',5, 5'-tetrachlorobiphenyl (TCB) to quinonoid metabolites was investigated in vitro in rat liver microsomes and in vivo in male Sprague-Dawley rats. Incubation of TCB with phenobarbital-induced rat liver microsomes resulted in metabolism of TCB to 3-hydroxy-TCB (3-OH-TCB) and 3,4-dihydroxy-TCB (3,4-diOH-TCB), which were further oxidized to form a reactive intermediate that bound to liver proteins. The predominant species observed in the Raney nickel assay for cysteinyl adducts was identified as 3,4-diOH-TCB, consistent with an adduct having the structure 5-cysteinyl-3,6-dichloro-4-(2', 5'-dichlorophenyl)-1,2-benzoquinone. This adduct may arise via the Michael addition of the sulfhydryl group of cysteine to 3, 6-dichloro-4-(2',5'-dichlorophenyl)-1,2-benzoquinone (Cl(4)PhBQ). Metabolism of 3-OH-TCB by phenobarbital-induced microsomes in the presence of either NADPH or cumene hydroperoxide as a cofactor resulted in the formation of adducts. Dose-dependent formation of cysteinyl adducts was observed in liver cytosolic protein from rats treated with a single dose of TCB (0-200 mg/kg) by gavage. By regression analysis, the TCB adducts decayed with a half-life of 2. 03 +/- 0.131 days (mean +/- SE), which is approximately 2.5-fold shorter than the endogenous half-life for liver cytosolic protein in rat liver, suggesting adduct instability. Saturable formation of TCB adducts was observed in liver cytosolic protein of rats receiving multiple doses of TCB over 5 days. The levels of Cl(4)PhBQ-derived adducts were 2.1-fold greater than the estimated steady-state levels predicted by the single-dose treatment [97.7 +/- 13.2 vs 45.7 +/- 3. 73 (pmol/g)/(mg/kg of body weight)], suggesting induction of metabolism. A single cysteinyl adduct, inferred to be 5-cysteinyl-3, 6-dichloro-4-(2',5'-dichlorophenyl)-1,2-benzoquinone, was detected in brain cytosolic protein of rats treated with multiple doses of TCB with levels of 15.2 (pmol/g)/(mg/kg of body weight). Implied involvement of a reactive quinone in the liver and brain of TCB-treated rats supports the idea that quinonoid metabolites may be important contributors to PCB-derived oxidative damage to genomic DNA.

Animals↗

PubMed2000

4-Hydroxy-2-nonenal and ethyl linoleate form N(2),3-ethenoguanine under peroxidizing conditions.

In these studies, we demonstrate that N(2),3-ethenoguanine (N(2), 3-epsilonGua) is formed from lipid peroxidation as well as other oxidative reactions. Ethyl linoleate (EtLA) or 4-hydroxy-2-nonenal (HNE) was reacted with dGuo in the presence of tert-butyl hydroperoxide (t-BuOOH) for 72 h at 50 degrees C. The resulting N(2), 3-epsilonGua was characterized by liquid chromatography/electrospray mass spectroscopy and by gas chromatography/high-resolution mass spectral (GC/HRMS) analysis of its pentafluorobenzyl derivative following immunoaffinity chromatography purification. The amounts of N(2),3-epsilonGua formed were 825 +/- 20 and 1720 +/- 50 N(2), 3-epsilonGua adducts/10(6) normal dGuo bases for EtLA and HNE, respectively, corresponding to 38- and 82-fold increases in the amount of N(2),3-epsilonGua compared to controls containing only t-BuOOH. Controls containing t-BuOOH but no lipid resulted in a >1000-fold increase in the level of N(2),3-epsilonGua over dGuo that was not subjected to incubation. EtLA and HNE, in the presence of t-BuOOH, were reacted with calf thymus DNA at 37 degrees C for 89 h. The amounts of N(2),3-epsilonGua formed in intact ctDNA were 114 +/- 32 and 52.9 +/- 16.7 N(2),3-epsilonGua adducts/10(6) normal dGuo bases for EtLA and HNE, respectively. These compared to 2.02 +/- 0. 17 and 2.05 +/- 0.47 N(2),3-epsilonGua adducts/10(6) normal dGuo bases in control DNA incubated with t-BuOOH, but no lipid. [(13)C(18)]EtLA was reacted with dGuo to determine the extent of direct alkylation by lipid peroxidation byproducts. These reactions resulted in a 89-93% level of incorporation of the (13)C label into N(2),3-epsilonGua when EtLA and dGuo were in equimolar concentrations, when EtLA was in 10-fold molar excess, and when deoxyribose (thymidine) was in 10-fold molar excess. Similar reactions with ctDNA resulted in an 86% level of incorporation of the (13)C label. These data demonstrate that N(2),3-epsilonGua is formed from EtLA and HNE under peroxidizing conditions by direct alkylation. The data also suggest, however, that N(2),3-epsilonGua is also formed by an alternative mechanism that involves some other oxidative reaction which remains unclear.

Aldehydes↗

PubMed2000

Quantitation of DNA and hemoglobin adducts and apurinic/apyrimidinic sites in tissues of F344 rats exposed to propylene oxide by inhalation.

Propylene oxide (PO) is a relatively weak mutagen that induces nasal tumor formation in rats during long-term inhalation studies at high exposures (> or =300 p.p.m.), concentrations that also cause cytotoxicity and increases in cell proliferation. Direct alkylation of DNA by PO leads mainly to the formation of N:7-(2-hydroxypropyl)guanine (7-HPG). In this study, the accumulation of 7-HPG in tissues of male F344 rats exposed to 500 p. p.m. PO (6 h/day, 5 days/week for 4 weeks) by the inhalation route was measured by gas chromatography-high resolution mass spectrometry (GC-HRMS). In animals killed up to 7 h following the end of the last exposure the levels of 7-HPG (pmol/micromol guanine) in nasal respiratory tissue, nasal olfactory tissue, lung, spleen, liver and testis DNA were 606.2 +/- 53.0, 297.5 +/- 56.5, 69.8 +/- 3.8, 43.0 +/- 3.8, 27.5 +/- 2.4 and 14.2 +/- 0.7, respectively. The amounts of 7-HPG in the same tissues of animals killed 3 days after cessation of exposure were 393.3 +/- 57.0, 222.7 +/- 29.5, 51.5 +/- 1.2, 26.7 +/- 1.0, 18.0 +/- 2.6 and 10.4 +/- 0.1. A comparable rate of disappearance of 7-HPG was found among all tissues. DNA from lymphocytes pooled from four rats killed at the end of the last exposure was found to have 39.6 pmol adduct/micromol guanine. Quantitation of DNA apurinic/apyrimidinic sites, potentially formed after adduct loss by chemical depurination or DNA repair, showed no difference between tissues from control and exposed rats. The level of N:-(2-hydroxypropyl)valine in hemoglobin of exposed rats was also determined using a modified Edman degradation method followed by GC-HRMS analysis. The value obtained was 90.2 +/- 10.3 pmol/mg globin. These data demonstrate that nasal respiratory tissue, which is the target tissue for carcinogenesis, has a much greater level of alkylation of DNA than non-target tissues.

Animals↗

PubMed2000

Biomarkers of exposure and effect as indicators of potential carcinogenic risk arising from in vivo metabolism of ethylene to ethylene oxide.

The purposes of the present study were: (i) to investigate the potential use of several biomarkers as quantitative indicators of the in vivo conversion of ethylene (ET) to ethylene oxide (EO); (ii) to produce molecular dosimetry data that might improve assessment of human risk from exogenous ET exposures. Groups (n = 7/group) of male F344 rats and B6C3F1 mice were exposed by inhalation to 0 and 3000 p. p.m. ET for 1, 2 or 4 weeks (6 h/day, 5 days/week) or to 0, 40, 1000 and 3000 p.p.m. ET for 4 weeks. N:-(2-hydroxyethyl)valine (HEV), N:7-(2-hydroxyethyl) guanine (N7-HEG) and HPRT: mutant frequencies were assessed as potential biomarkers for determining the molecular dose of EO resulting from exogenous ET exposures of rats and mice, compared with background biomarker values. N7-HEG was quantified by gas chromatography coupled with high resolution mass spectrometry (GC-HRMS), HEV was determined by Edman degradation and GC-HRMS and HPRT: mutant frequencies were measured by the T cell cloning assay. N7-HEG accumulated in DNA with repeated exposure of rodents to 3000 p.p.m. ET, reaching steady-state concentrations around 1 week of exposure in most tissues evaluated (brain, liver, lung and spleen). The dose-response curves for N7-HEG and HEV were supralinear in exposed rats and mice, indicating that metabolic activation of ET was saturated at exposures >/=1000 p.p.m. ET. Exposures of mice and rats to 200 p.p.m. EO for 4 weeks (as positive treatment controls) led to significant increases in HPRT: mutant frequencies over background in splenic T cells from exposed rats and mice, however, no significant mutagenic response was observed in the HPRT: gene of ET-exposed animals. Comparisons between the biomarker data for both unexposed and ET-exposed animals, the dose-response curves for the same biomarkers in EO-exposed rats and mice and the results of the rodent carcinogenicity studies of ET and EO suggest that too little EO arises from exogenous ET exposure to produce a significant mutagenic response or a carcinogenic response under standard bioassay conditions.

Animals↗

PubMed2000

1,3-butadiene: cancer, mutations, and adducts. Part V: Hemoglobin adducts as biomarkers of 1,3-butadiene exposure and metabolism.

1,3-Butadiene (BD) is an important chemical used largely in the manufacture of synthetic rubber and thermoplastic resins. In addition, it has been identified in cigarette smoke, automobile exhaust, and gasoline vapor. The objective of this research was to develop highly sensitive and specific assays for the detection and quantitation of hemoglobin adducts of three BD metabolites: 1,2-epoxy-3-butene (BDO), 1,2,3,4-diepoxybutane (BDO2), and 1,2-dihydroxy-3,4-epoxybutane (BDO-diol). We have successfully developed an assay for both N-(2-hydroxy-3-butenyl)valine (HBVal) and N-(2,3,4-trihydroxybutyl)valine (THBVal) in hemoglobin. The six adducts measured were the two diastereomers (isomers I and II) of HBVal and the four diastereomers of THBVal (isomers I through IV, which were eluted as three peaks, 1, 2, and 3). HBVal and THBVal were measured in control and exposed B6C3F1 mice and Sprague-Dawley rats (1,000 ppm BD for 13 weeks at 6 hours/day, 5 days/week). In a second set of animal exposures, total THBVal was determined in B6C3F1 female mice (n = 5) exposed to 1,250 ppm BD for 1, 5, or 10 days (6 hours/day, 5 days/week). THBVal adducts were also monitored in occupationally exposed Chinese workers and nonoccupationally exposed U.S. laboratory workers. This study utilized the modified Edman degradation method of Törnqvist and colleagues (1986). Briefly, the samples were subjected to Edman degradation, Centricon-30 ultrafiltration, washing on C18 columns, and acetylation for isomers of THBVal only, followed by gas chromatography-mass spectrometry (GC-MS) quantitation. For the HBVal assay, an authentic internal standard globin alkylated with [2H6]BDO was used; for the THBVal assay, a synthesized external standard, THB[13C5]Val, was used after Edman degradation. The mean +/- SD amounts of total HBVal measured in exposed mice (in pmol/g globin) were 16,560 +/- 3,910 for female mice (n = 4) and 12,400 +/- 2,030 for male mice (n = 5). The corresponding values for rats were 8,690 +/- 930 for female rats (n = 5) and 5,480 +/- 2,880 for male rats (n = 3). The total amount of THBVal (eluted peaks 1, 2, and 3) in male mice (n = 5) was 78,900 +/- 13,700; and in females (n = 2) was 56,100 +/- 100. In male rats (n = 3), the detected value was 9,650 +/- 1,620 and in females (n = 3) the value was 21,600 +/- 6,780. In control male mice (n = 4), the total level of THBVal isomers was approximately 27 pmol/g globin. In a control male rat, total THBVal was approximately 15 pmol/g globin. In the time course study, the amount of THBVal adducts increased linearly with exposure, resulting in values of 4,200 +/- 830, 19,760 +/- 1,780, and 35,940 +/- 3,460 pmol/g globin following 1, 5, or 10 days of exposure to 1,250 ppm BD, respectively. Detection of HBVal in human samples was difficult due to low concentrations of adducts and a high background in the chromatograms. In a pooled sample from 4 individuals, we performed multiple separations with high-pressure liquid chromatography (HPLC) of the derivatized adducts and detected 4.6 pmol/g globin (that is, 2.7 and 1.9 pmol/g globin for isomers I and II, respectively). We measured the amounts of THBVal in both nonoccupationally exposed U.S. laboratory workers and occupationally exposed workers from a polybutadiene plant in China. The mean total amount of THBVal among the U.S. laboratory workers was 36 +/- 23 pmol/g globin for nonsmokers (n = 7) and 40 +/- 9 for smokers (n = 4), compared with a mean total amount of 39 +/- 13 pmol/g globin in a control set of Chinese workers (n = 25). These control values are overestimations of the true values because the amounts of THBVal in globin samples from other unexposed individuals (15 of 51) were below our limit of detection. BD-exposed Chinese workers had a total amount of 88 +/- 59 pmol/g globin THBVal. The difference between smokers and nonsmokers was not significant, whereas the difference between control and exposed Chinese workers was highly significant (p < 0.001).

Animals↗

PubMed1999

A gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry method for analysis of endogenous and exogenous N7-(2-hydroxyethyl)guanine in rodents and its potential for human biological monitoring.

A gas chromatography/electron capture/negative chemical ionization high-resolution mass spectrometry (GC/EC/NCI-HRMS) method was developed for quantitating N7-(2-hydroxyethyl)guanine (N7-HEG) with excellent sensitivity and specificity. [4,5,6,8-(13)C(4)]-N7-HEG was synthesized, characterized, and quantitated using HPLC/electrospray ionization mass spectrometry (HPLC/ESI-MS) so it could serve as an internal standard. After being converted to its corresponding xanthine and derivatized with pentafluorobenzyl (PFB) bromide twice, the PFB derivative of N7-HEG was characterized using GC/EC/NCI-HRMS carried out at full scan mode. The most abundant fragment was at m/z 555, with a molecular formula of C(21)H(9)N(4)O(3)F(10), resulting from the loss of one PFB group. By monitoring m/z 555.0515 (analyte) and m/z 559.0649 (internal standard), this assay demonstrated a linear relationship over a range of 1 fmol to 1 pmol of N7-HEG versus 20 fmol of [(13)C(4)]-N7-HEG on column. The limit of detection (LOD) for the complete assay was 600 amol (S/N = 5) injected on column. The variation of this assay was within 15% from 1 to 20 fmol of N7-HEG versus 2 fmol of [(13)C(4)]-N7-HEG with four replications for each calibration standard. Two hundred to three hundred micrograms of spleen DNA of control rats and mice and 100 microg of spleen DNA of rats and mice exposed to 3000 ppm ethylene for 6 h/day for 5 days were analyzed using GC/EC/NCI-HRMS. The amounts of N7-HEG varied from 0.2 to 0.3 pmol/micromol of guanine in tissues of control rats. Ethylene-exposed animals had 5-15-fold higher N7-HEG levels than controls. This assay was able to quantitate N7-HEG in 25-30 microg of DNA from human lymphocytes with excellent specificity. This was due in part to human tissues having 10-15-fold higher amounts of endogenous N7-HEG than rodents. These results show that this GC/EC/NCI-HRMS method is highly sensitive and specific and can be used in biological monitoring and molecular dosimetry and molecular epidemiology studies.

Animals↗

PubMed1999

Immunoaffinity/gas chromatography/high-resolution mass spectrometry method for the detection of N(2),3-ethenoguanine.

Etheno adducts are formed after exposure to a number of carcinogens, including vinyl chloride, as well as endogenously as a result of lipid peroxidation. A sensitive and selective assay for N(2), 3-ethenoguanine (epsilonGua) was developed using immunoaffinity (IA) columns made with polyclonal antibodies to epsilonGua followed by gas chromatography/electron capture negative chemical ionization/high-resolution mass spectrometry (GC/ECNCI/HRMS) analysis of its pentafluorobenzyl derivative. These IA columns were specific for epsilonGua and did not bind guanine, deoxyguanosine, 1, N(6)-ethenoadenine, or 1,N(2)-ethenoguanine. The level of recovery of standards from the IA columns was 107 +/- 7% and throughout the entire method (using nucleoside enzymatic digestion) with or without DNA was 72 +/- 6%. Four different hydrolysis/digestion procedures were compared, nucleoside enzymatic (EZ), neutral thermal hydrolysis (NT), formic acid hydrolysis (FA), and HCl hydrolysis. All hydrolysis methods with subsequent IA chromatography produced linear standard curves with r(2) values of 0.999 or better. The level of epsilonGua in chloroethylene oxide-treated calf thymus DNA (CEO-ctDNA) was 38 +/- 2, 42 +/- 3, and 49 +/- 2 fmol of epsilonGua/microg of DNA using EZ, NT, and FA, respectively. These numbers remained consistent when the amount of DNA processed was doubled or tripled. These numbers were comparable to the previously published value of 55 +/- 8 fmol of epsilonGua/micrograms of DNA for the same DNA using HCl hydrolysis, cation exchange cleanup, and LC/MS analysis [Yen, T. Y., et al. (1996) J. Mass Spectrom. 31, 1271-1276]. Additionally, HCl hydrolysis of rat liver DNA from control and vinyl fluoride-exposed rats gave similar epsilonGua results when compared to those from enzymatic digestion using this method. This method gave a detection limit of 5 epsilonGua adducts/10(8) normal dGuo nucleosides in 150 micrograms of DNA using EZ and somewhat lower detection limits using NT and HCl hydrolysis. The method is more sensitive and selective than previously used methods for the quantitation of this adduct.

Animals↗

PubMed1999

Molecular dosimetry of endogenous and ethylene oxide-induced N7-(2-hydroxyethyl) guanine formation in tissues of rodents.

The formation of N7-(2-hydroxyethyl)guanine (7-HEG) in DNA was investigated previously in target and non-target tissues of F-344 rats and B6C3F1 mice exposed to >/=ISOdia>/=10 p.p.m. concentrations of ethylene oxide (EO) using fluorescence-linked high-performance liquid chromatography [V.E. Walker et al. (1992) Cancer Res., 52, 4238-4334]. In order to study the dose-responses for 7-HEG at lower exposures, a highly sensitive and specific gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS) assay was developed. DNA was extracted from liver, brain, lung and spleen of B6C3F1 mice and F-344 rats exposed to 0, 3, 10, 33 or 100 p.p.m. EO for 4 weeks (6 h/day, 5 days/week). Analysis of DNA from control rodents showed that endogenous 7-HEG varied from 0.2 +/- 0.1 to 0.3 +/- 0.2 pmol/micromol guanine in tissues of rats and mice. 7-HEG exhibited tissue- and species-specific dose-response relationships in EO-exposed animals. Linear dose-response relationships were evident in mouse liver, brain and spleen at exposures between 3 and 100 p.p.m. Mouse lung exhibited a slightly sublinear response between 33 and 100 p.p.m. EO. The relationships were linear in liver and spleen of rats between 3 and 100 p.p.m. EO, but were slightly sublinear in brain and lung between 33 and 100 p.p.m. EO. The number of 7-HEG adducts present in rats exposed to 3 p.p.m. EO was 5.3-12.5 times higher than endogenous 7-HEG in unexposed controls. In contrast, mice exposed to 3 p.p.m. EO only had 1.3- to 2.5-fold greater numbers of 7-HEG adducts. The factors driving the exposure-response relationships are also likely to affect carcinogenic and mutagenic responses of rodents to EO. Likewise, a better understanding of the relationships between 7-HEG derived from low exposures to EO and endogenously formed 7-HEG may be important for the accurate extrapolation of risk to humans.

Animals↗

PubMed1999

Formation and repair of DNA adducts in vinyl chloride- and vinyl fluoride-induced carcinogenesis.

Vinyl chloride is a known human and animal carcinogen that induces angiosarcomas of the liver. We review here studies on the formation and repair of DNA adducts associated with vinyl chloride and vinyl fluoride in exposed and control rodents and unexposed humans. These vinyl halides induce etheno (epsilon) adducts that are identical to those formed after lipid peroxidation. Of these adducts, N2,3-ethenoguanine (epsilon G) is present in greatest amounts in tissues of exposed animals. After exposure to vinyl chloride for four weeks, epsilon G levels attain steady-state concentrations, such that the amount of newly formed adducts equals the number of adducts that are lost each day. We report the first dosimetry of epsilon G in rats exposed to 0, 10, 100 or 1100 ppm vinyl chloride for five days or four weeks. The number of adducts increased in a supralinear manner. Exposure to 10 ppm vinyl chloride for five days caused a two- to threefold increase in epsilon G over that of the controls, while four weeks' exposure resulted in a fivefold increase. This was confirmed with [13C2]vinyl chloride and by measuring exogenous and endogenous adducts in the same animals. Exposure to 100 ppm vinyl chloride for four weeks caused a 25-fold increase in epsilon G levels over that found in control rats, while exposure to 1100 ppm resulted in a 42-fold increase. The amount of endogenous epsilon G was similar in liver DNA from rats and humans. A comparable response to exposure was seen in rats and mice exposed to 0, 25, 250 or 2500 ppm vinyl fluoride for 12 months. There was a very high correlation between epsilon G levels in rat and mouse liver at 12 months and the incidence of haemangiosarcoma at two years. We were able to demonstrate that the target cell population for angiosarcoma, the nonparenchymal cells, contained more epsilon G than hepatocytes, even though nonparenchymal cells are exposed by diffusion of vinyl halide metabolites formed in hepatocytes. The expression of N-methylpurine-DNA glycosylase mRNA was induced in rat liver after exposure to either 25 or 2500 ppm vinyl fluoride. When this induction was investigated in hepatocytes and nonparenchymal cells, it was found that the latter had only 20% of the N-methylpurine-DNA glycosylase mRNA of hepatocytes, and that only the hepatocytes had induction of this expression after exposure to vinyl fluoride. Thus, the target cells for vinyl halide carcinogenesis have much lower expression of this DNA repair enzyme, which has been associated with etheno adduct repair.

Animals↗

PubMed1998

Application of gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry for analysis of DNA and protein adducts.

The analytical potential of gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry (HRMS) for characterization and quantitation of DNA and hemoglobin adducts was demonstrated using three model compounds: N2, 3-ethenoguanine (EG), 7-(2-hydroxyethyl)guanine (7-HEG), and N-(2-hydroxyethyl)valine (HEV). At a resolving power of 10 000, the signal-to-noise (S/N) ratios obtained from quantitative selected ion monitoring (SIM) experiments using biological samples were comparable to or better than existing unit mass resolution experiments due to the reduction of chemical noise from the use of narrower mass windows. The specificity gained by HRMS was essential for quantitation of ultratrace amounts near the limit of detection since coeluting interferences of the analyte or internal standard can lead to inaccurate measurement of response factors. The limit of detection (LOD) was 100 amol (S/N = 5) using a pure standard of TTB2-EG. The LOD for complete assays using spiked samples was 500 amol (S/N = 5) for EG and 600 amol (S/N = 5) injected for 7-HEG. The standard deviation (SD) for the HRMS quantitative measurements was typically less than 10%. The SD for the complete biological assays as determined by spiking replicate samples was less than 15%. This method has adequate sensitivity and specificity to accurately measure DNA and protein adducts as low as endogenous concentrations in rodent and human tissues.

Animals↗

PubMed1998

Street children--a preliminary study.

OBJECTIVE: To study the background, life styles, health and prevalence of abuse of street children in Colombo. STUDY SAMPLE: 50 street children receiving temporary day care or night shelter from community service centres were serially recruited. DESIGN: A retrospective descriptive survey. Demographic, experiential and clinical data were collected by an interviewer, based on a semistructured questionnaire, followed by physical examination and laboratory investigation. RESULTS: The children were aged between 4 and 17 years. (Mean 10.5 years.) 64% were males and 18% belonged to ethnic minorities. The majority (84%) had family ties. Family disintegration was mentioned as the cause for life on the streets by 36%. Child labour was reported in 38%. 16% admitted to being sexually abused. 20% were tobacco smokers. Respiratory and skin infections, and injuries incurred in road traffic accidents were the main causes of morbidity. CONCLUSION: Street children in Colombo are an abused and exploited group. Road traffic accidents, and physical and sexual abuse are the main hazards of the street they are exposed to.

Adolescent↗

PubMed1997

Propylene oxide: mutagenesis, carcinogenesis and molecular dose.

The results from mutagenic and carcinogenic studies of propylene oxide (PO) and the current efforts to develop molecular dosimetry methods for PO-DNA adducts are reviewed. PO has been shown to be active in several bacterial and mammalian mutagenicity tests and induces site of contact tumors in rodents after long-term administration. Quantitation of N7-(2-hydroxypropyl)guanine (7-HPG) in nasal and hepatic tissues of male F344 rats exposed to 500 ppm PO (6 h/day; 5 days/week for 4 weeks) by inhalation was performed to evaluate the potential of high concentrations of PO to produce adducts in the DNA of rodent tissues and to obtain information necessary for the design of molecular dosimetry studies. The persistence of 7-HPG in nasal and hepatic tissues was studied in rats killed three days after cessation of a 4-week exposure period. DNA samples from exposed and untreated animals were analyzed for 7-HPG by two different methods. The first method consisted of separation of the adduct from DNA by neutral thermal hydrolysis, followed by electrophoretic derivatization of the adduct and gas chromatography-high resolution mass spectrometry (GC-HRMS) analysis. The second method utilized 32P-postlabeling to quantitate the amount of this adduct in rat tissues. Adducts present in tissues from rats killed immediately after cessation of exposure were 835.4 +/- 80.1 (respiratory), 396.8 +/- 53.1 (olfactory) and 34.6 +/- 3.0 (liver) pmol adduct/mumol guanine using GC-HRMS. Lower values, 592.7 +/- 53.3, 296.5 +/- 32.6 and 23.2 +/- 0.6 pmol adduct/mumol guanine were found in respiratory, olfactory and hepatic tissues of rats killed after three days of recovery. Analysis of the tissues by 32P-postlabeling yielded the following values: 445.7 +/- 8.0 (respiratory), 301.6 +/- 49.2 (olfactory) and 20.6 +/- 1.8 (liver) pmol adduct/mumol guanine in DNA of rats killed immediately after exposure cessation and 327.1 +/- 21.7 (respiratory), 185.3 +/- 29.2 (olfactory) and 15.7 +/- 0.9 (liver) pmol adduct/mumol guanine after recovery. Current methods of quantitation did not provide evidence for the endogenous formation of this adduct in control animals. These studies demonstrated that the target tissue for carcinogenesis has much greater alkylation of DNA than liver, a tissue that did not exhibit a carcinogenic response.

Alkylation↗

PubMed1995

Metabolism of fenbufen by cultured 3T3-L1 adipocytes: synthesis and metabolism of xenobiotic glycerolipids.

The storage of xenobiotic compounds as glycerolipids and their subsequent mobilization was studied using fenbufen and differentiated 3T3-L1 cells in culture. Fenbufen was taken up from the incubation medium and incorporated into triacylglycerol, diacylglycerol, and phospholipids. The triacylglycerol was susceptible to digestion by pancreatic lipase. The xenobiotic phospholipid contained three species, one of which behaved as fenbufenoyl phosphatidylcholine as judged by TLC, HPLC, choline analysis, and mass spectroscopy. After incubation with radioactive fenbufen for 18 h, the cells were transferred to a chase medium where radioactivity was lost from the cells and appeared in the medium. The rate was three times higher when 10 microM isoproterenol was present; insulin had no effect. Non-esterified fenbufen and analogues of mono- and di-acylglycerol were secreted. Monofenbufenoylglycerol was characterized by its ability to be used as a substrate by purified monoacylglycerol acyltransferase. When oleic acid was used in place of fenbufen, the majority of the radioactivity released in a chase experiment was the non-esterified acid (over 90%) and neither mono- nor di-acylglycerol was detected. These data indicate that 3T3-L1 adipocytes can synthesize fenbufen-containing lipids and release them into the medium on hormonal stimulation. The secretion of mono- and di-acylglycerols may have unforeseen pharmacological or toxicological implications.

3T3 Cells↗