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J A Swenberg

Publications and source records attributed to J A Swenberg.

At least 37 records · Page 2Linked to original sources
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

Genotoxic markers among butadiene polymer workers in China.

While 1,3-butadiene is carcinogenic in rodents, cancer causation in humans is less certain. We examined a spectrum of genotoxic outcomes in 41 butadiene polymer production workers and 38 non-exposed controls, in China, to explore the role of butadiene in human carcinogenesis. Because in vitro studies suggest that genetic polymorphisms in glutathione S-transferase enzymes influence genotoxic effects of butadiene, we also related genotoxicity to genetic polymorphisms in GSTT1 and GSTM1. Among butadiene-exposed workers, median air exposure was 2 p.p.m. (6 h time-weighted average), due largely to intermittent high level exposures. Compared with unexposed subjects, butadiene-exposed workers had greater levels of hemoglobin N-(2,3,4-trihydroxybutyl)valine (THBVal) adducts (P < 0.0001) and adduct levels tended to correlate, among butadiene-exposed workers, with air measures (P = 0.03). Butadiene-exposed workers did not differ, however, from unexposed workers with respect to frequency of uninduced or diepoxybutane-induced sister chromatid exchanges, aneuploidy as measured by fluorescence in situ hybridization of chromosomes 1, 7, 8 and 12, glycophorin A variants or lymphocyte hprt somatic mutation. Also among the exposed, greater THBVal levels were not associated with increases in uninduced sister chromatid exchanges, aneuploidy, glycophorin A or hprt mutations. Butadiene-exposed workers had greater lymphocyte (P = 0.002) and platelet counts (P = 0.07) and lymphocytes as a percentage of white blood cells were moderately correlated with greater THBVal levels (Spearman's phi = 0.32, P = 0.07). Among butadiene-exposed workers, neither GSTM1 nor GSTT1 genotype status predicted urinary mercapturic acid butanediol formation, THBVal adducts, uninduced sister chromatid exchanges, aneuploidy or mutations in the glycophorin A or hprt genes. Overall, the study demonstrated exposure to butadiene in these workers, by a variety of short-term and long-term measures, but did not show specific genotoxic effects, at the chromosomal or gene levels, related to that exposure.

Adult↗
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

Expression of base excision repair enzymes in rat and mouse liver is induced by peroxisome proliferators and is dependent upon carcinogenic potency.

Elevated and sustained cell replication, together with a decrease in apoptosis, is considered to be the main mechanism of hepatic tumor promotion due to peroxisome proliferators. In contrast, the role of oxidative stress and DNA damage in the carcinogenic mechanism is less well understood. In view of possible induction of DNA damage by peroxisome proliferators, DNA repair mechanisms may be an important factor to consider in the mechanism of action of these compounds. Here, the ability of peroxisome proliferators to induce expression of base excision repair enzymes was examined. WY-14,643, a potent carcinogen, increased expression of several base excision DNA repair enzymes in a dose- and time-dependent manner. Importantly, expression of enzymes that do not repair oxidative DNA damage was not changed. Moreover, less potent members of the peroxisome proliferator group had much weaker or no effects on expression of DNA repair enzymes when compared with WY-14,643. Collectively, these data suggest that DNA base excision repair may be an important factor in peroxisome proliferator-induced carcinogenesis and that induction of DNA repair might provide further evidence supporting a role of oxidative DNA damage by peroxisome proliferators.

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

Endogenous apurinic/apyrimidinic sites in genomic DNA of mammalian tissues.

Apurinic/apyrimidinic (AP) sites are one of the most frequent lesions in DNA. Using a highly sensitive slot blot assay, we determined the number and condition of endogenous AP sites in normal tissues of rats and human liver. The number of AP sites (50,000-200,000 per mammalian cell) was greatest in brain, followed by colon and heart, and then liver, lung, and kidney. The majority of endogenous AP sites were cleaved 5' to the AP site. These data suggest that removal of the deoxyribosyl phosphate moiety is the rate-limiting step in base excision and AP site repair in vivo.

Animals↗
PubMed1999

Gentle organ manipulation during harvest as a key determinant of survival of fatty livers after transplantation in the rat.

Both in situ organ manipulation during harvest and steatosis reduce survival after liver transplantation via mechanisms involving Kupffer cells; thus, their effect on survival was compared here. Moderate steatosis was induced by a single dose of ethanol to Lewis rats, while long-term administration of ethanol yielded severe steatosis in donor animals. After minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 min subsequently. Orthotopic liver transplantation was performed after 1 h of cold storage in UW solution. Ethanol increased hepatic lipid content to a level of moderate or severe steatosis that reduced survival after transplantation from 100% to approximately 70% (P < 0.05). However, gentle manipulation decreased survival to approximately 30% (P < 0.05) in livers from normal, saline-treated rats and in livers from rats fed a high-fat control diet. Moreover, after short- or long-term ethanol administration, manipulation of fatty livers decreased survival from 70% to approximately 13% (P < 0.05). Further, manipulation elevated serum transaminases, total bilirubin, and necrosis significantly about 2- to 20-fold in fatty grafts after transplantation. At the end of harvest, trypan blue distribution time and hypoxia assessed from 2-nitroimidazole binding were elevated significantly about two- to fourfold by manipulation of fatty grafts. Gadolinium chloride, a Kupffer cell toxicant, blocked the detrimental effects of manipulation. These data demonstrate for the first time that, while steatosis is detrimental for survival, organ manipulation plays a much greater role than fat in mechanisms of primary nonfunction.

Animals↗
PubMed1999

Molecular dosimetry of N-7 guanine adduct formation in mice and rats exposed to 1,3-butadiene.

1,3-Butadiene (BD) is a high-volume chemical used in the production of rubber and plastic. BD is a potent carcinogen in mice and a much weaker carcinogen in rats, and has been classified as a probable human carcinogen. Upon metabolic activation in vivo, it forms DNA-reactive metabolites, 1,2-epoxy-3-butene (EB), 1,2:3, 4-diepoxybutane (DEB), and 3,4-epoxy-1,2-butanediol (EBD). The molecular dosimetry of N-7 guanine adduct formation by these metabolites of BD in liver, lung, and kidney of B6C3F1 mice and F344 rats exposed to 0, 20, 62.5, or 625 ppm BD was studied. The adducts, racemic and meso forms of N-7-(2,3,4-trihydroxybut-1-yl)guanine (THB-Gua), N-7-(2-hydroxy-3-buten-1-yl)guanine (EB-Gua I), and N-7-(1-hydroxy-3-buten-2-yl)guanine (EB-Gua II), were isolated from DNA by neutral thermal hydrolysis, desalted on solid-phase extraction cartridges, and quantitated by LC/ESI(+)/MS/MS. The number of adducts per 10(6) normal guanine bases for a given adduct was higher in mice than rats exposed to 625 ppm BD, but generally similar at lower levels of exposure. The THB-Gua adducts were the most abundant (6-27 times higher than EB-Gua) and exhibited a nonlinear exposure-response relationship. In rats, the exposure-response curves for the formation of THB-Gua adducts reached a plateau after 62.5 ppm, suggesting saturation of metabolic activation. The number of THB-Gua adducts continued to increase in mice between 62.5 and 625 ppm BD. In contrast, the less common EB-Gua adducts had a linear exposure-response relationship in both species. Combining the information from this study with previous data on BD metabolism, we were able to estimate the number of THB-Gua that resulted from DEB and EBD, and conclude that most of the THB-Gua is formed from EBD. We hypothesize that most of the EBD arises from the immediate conversion of DEB to EBD within the endoplasmic reticulum. This study highlights the need for measurements of the levels of EBD in tissues of rats and mice and for the development of a unique biomarker for DEB that is available for binding to DNA.

Administration, Inhalation↗
PubMed1999

Comparison of (32)P-postlabeling and high-resolution GC/MS in quantifying N7-(2-Hydroxyethyl)guanine adducts.

This study compares (32)P-postlabeling and high-resolution gas chromatography/mass spectrometry (GC/MS) in the quantification of N7-(2-hydroxyethyl)guanine adducts (7-HEG) in DNA obtained from the same tissue samples of control rats and rats exposed to ethene. The samples were obtained from two independent studies. In one study, male Sprague-Dawley rats were exposed to 300 ppm ethene for 12 h/day for 3 days ("Euro samples"). In the other study, male F-344 rats were exposed to 3000 ppm ethene for 6 h/day for 5 days ("U.S. samples"). DNA from liver and kidney from the European study was isolated in the European laboratory, and DNA from liver and spleen from the U.S. study was isolated in the U.S. laboratory. The DNA samples were coded, divided into two portions, and exchanged between the two laboratories. All DNA samples from both laboratories were analyzed with respect to 7-HEG adducts by (32)P-postlabeling and high-resolution GC/MS in the European and U.S. laboratories, respectively. However, the U.S. samples were repurified in the European laboratory before the postlabeling analysis. The data from the Euro and the U.S. samples were therefore treated separately in the regression analysis of the (32)P-postlabeling versus GC/MS data. The slope of the regression line for the Euro samples was 1.19 (r = 0.97), implying that the GC/MS data were slightly lower than the postlabeling data (one possible outlier was excluded). The slope of the regression line for the U.S. samples was 0.61 (r = 0.94), implying that the GC/MS data were somewhat higher than the postlabeling data. The main conclusion from this study is that there is very good agreement between the (32)P-postlabeling and high-resolution GC/MS methods in quantifying 7-HEG adducts to DNA, particularly when identical DNA samples are analyzed and the RNA content is <2%. The paper also discusses the background levels of adducts, the interorgan distribution, comparison between different strains, and exposure conditions.

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

Kupffer cell oxidant production is central to the mechanism of peroxisome proliferators.

Increased cell proliferation most likely plays a key role in peroxisome proliferator-induced liver cancer. Recently, Kupffer cells were shown to be responsible for Wy-14,643-induced cell proliferation. However, the mechanism by which peroxisome proliferators activate Kupffer cells is unknown. Since gut-derived endotoxin is a known activator of Kupffer cells, the hypothesis that it is involved was evaluated. Increased cell proliferation and peroxisome induction were unaffected by gut sterilization. Moreover, endotoxin was not detectable in portal blood following treatment with Wy-14,643. Therefore, it is concluded that gut-derived endotoxin is not responsible for Kupffer cell activation. To test the hypothesis that Kupffer cells are activated by Wy-14,643 directly, Kupffer cell superoxide production was measured following treatment in vitro. Wy-14,643 increased superoxide production in a dose-dependent manner (0.1 and 50 microM) with half-maximal stimulation at 2.5 microM. Diethylhexylphthalate (DEHP) and ethylhexanol did not increase superoxide production even at doses 50 times higher than Wy-14,643; however, monoethylhexylphthalate (MEHP) activated superoxide production as effectively as Wy-14,643 with half-maximal stimulation at 5 microM. Treatment with Wy-14,643 for 21 days caused a 2-fold increase in Kupffer cell superoxide production while DEHP did not. Pretreatment of Kupffer cells with staurosporine (0.01-10 pM) completely blocked generation of superoxide demonstrating that protein kinase C is required. Moreover, Wy-14,643 increased Kupffer cell protein kinase C activity 3-fold. Pretreatment of Kupffer cells with the amino acid glycine (0.01-3 mM), which blunts calcium signaling, inhibited Wy-14,643-stimulated superoxide production and increased protein kinase C activity completely. These data are consistent with the hypothesis that potent peroxisome proliferators (Wy-14,643 and MEHP) directly activate Kupffer cell production of oxidants via mechanisms involving protein kinase C. Further, peroxisome proliferator treatments that sustain elevated rates of cell proliferation (e.g. Wy-14,643) activate Kupffer cell superoxide production following long-term dietary treatment supporting the hypothesis that Kupffer cell-derived oxidants are involved in peroxisome proliferator-induced neoplasia.

Animals↗
PubMed1999

Corn oil rapidly activates nuclear factor-kappaB in hepatic Kupffer cells by oxidant-dependent mechanisms.

N-6 polyunsaturated fatty acids (N-6 PUFAs), major constituents of corn oil and natural ligands for peroxisome proliferator-activated receptors, increase the rate of growth of established tumors. It has been proposed that chemical peroxisome proliferators increase hepatocyte proliferation by mechanisms involving activation of nuclear factor-kappaB (NF-kappaB) and production of low levels of tumor necrosis factor alpha (TNFalpha) by Kupffer cells; however, how N-6 PUFAs are involved in increased cell proliferation in liver is not well understood. Here, the hypothesis that N-6 PUFAs increase production of mitogens by activation of Kupffer cell NF-kappaB was tested. A single dose of corn oil (2 ml/kg, i.g.), but not olive oil or medium-chain triglycerides (saturated fat), caused an approximately 3-fold increase in hepatocyte proliferation. Similarly, when activity of NF-kappaB in whole rat liver or isolated hepatocytes and Kupffer cells was measured at various time intervals for up to 36 h, only corn oil activated NF-kappaB. Corn oil increased NF-kappaB activity approximately 3-fold 1-2 h after treatment exclusively in the Kupffer cell fraction. In contrast, increases were small and only occurred after approximately 8 h in hepatocytes. The activation of NF-kappaB at 2 h and increases in cell proliferation at 24 h due to corn oil were prevented almost completely when rats were pretreated for 4 days with either dietary glycine (5% w/w), an agent that inactivates Kupffer cells, or the NADPH oxidase inhibitor, diphenyleneiodonium (s.c., 1 mg/kg/day). Furthermore, arachidonic acid (100 microM) activated superoxide production approximately 4-fold when added to isolated Kupffer cells in vitro. This phenomenon was not observed with oleic or linoleic acids. Interestingly, a single dose of corn oil increased TNFalpha mRNA nearly 2-fold 8 h after treatment. It is concluded that corn oil rapidly activates NF-kappaB in Kupffer cells via oxidant-dependent mechanisms. This triggers production of low levels of TNFalpha which is mitogenic in liver and promotes growth of hepatocytes.

Animals↗
PubMed1999

Elevated HPRT mutation frequencies in aflatoxin-exposed residents of daxin, Qidong county, People's Republic of China.

Molecular biomarkers are becoming increasingly important tools to identify people who are at highest risk of developing cancer. For many years we have been studying residents of Qidong County, People's Republic of China, to examine the combined impact of aflatoxin exposure with other risk factors as contributors to the high liver cancer incidence rates in this region. This study was conducted to determine the effects of aflatoxin exposure, as measured by serum aflatoxin-albumin adduct levels, on somatic mutation frequency in the human hypoxanthine guanine phosphoribosyl transferase gene (HPRT). Subjects were assigned as low or high according to a dichotomization around the population mean of aflatoxin-albumin adducts. HPRT mutant frequency was determined in individuals by a T cell clonal assay and the samples were categorized as low or high according to mean values. Separate analyses were also conducted for the small set of hepatitis B virus surface antigen (HBsAg)-positive and the larger set of HBsAg-negative individuals, known risk factors for liver cancer. An odds ratio of 19.3 (95% confidence interval 2.0, 183) was demonstrated for a high HPRT mutation frequency in individuals with high aflatoxin exposure compared with those with low aflatoxin exposure. This association indicates that aflatoxin-induced DNA damage in T lymphocytes, assessed using the validated surrogate albumin adduct markers, leads to increased mutations reflected as elevated HPRT gene mutations. This cross-sectional study suggests the potential use of mutation frequency of the HPRT gene as a long-term biomarker of aflatoxin exposure in high risk populations.

Adult↗
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

Dose-specific production of chlorinated quinone and semiquinone adducts in rodent livers following administration of pentachlorophenol.

Production of chlorinated quinoid metabolites was investigated in the livers of Sprague-Dawley rats and B6C3F1 mice following single oral administration of pentachlorophenol (PCP) (0-40 mg/kg body weight) and in male Fischer 344 rats, following chronic ingestion of PCP at 1,000 ppm in the diet for 6 months (equivalent to 60 mg PCP/kg body weight/day). Analyses of the rates of adduction in the livers of Sprague-Dawley rats and B6C3F1 mice suggested that the production of tetrachloro-1,2-benzosemiquinone (Cl4-1,2-SQ) adducts was proportionally greater at low doses of PCP (less than 4-10 mg/kg body weight) and was 40-fold greater in rats than in mice. Production of tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ) adducts, on the other hand, was proportionally greater at high doses of PCP [greater than 60-230 mg/kg body weight] and was 2- to 11-fold greater in mice than in rats over the entire range of dosages. A mathematical model employed these data to predict the rates of daily adduct production and steady state levels of PCP-derived quinone and semiquinone adducts in rats and mice. To evaluate predictions of the model, levels of PCP-derived adducts at steady state were investigated in the livers of male Fischer 344 rats chronically ingesting 60 mg PCP/kg body weight/day. Levels of total Cl4-1,4-BQ-derived adducts in liver cytosolic proteins (Cp) (22.0 nmol/g) and in liver nuclear proteins (Np) (3.07 nmol/g) were comparable to those of model predictions (15.0 and 3.02 nmol/g for Cp and Np, respectively). Overall, these results suggest that species differences in the metabolism of PCP to semiquinones and quinones were, in part, responsible for the production of liver tumors in mice but not rats in chronic bioassays.

Animals↗