Biomedical subjects

J A Swenberg

Publications and source records attributed to J A Swenberg.

At least 19 recordsLinked to original sources

PubMed2006

Tandem mass spectrometry measurements of creatinine in mouse plasma and urine for determining glomerular filtration rate.

Endogenous creatinine clearance (Ccr) is widely accepted as an estimate of glomerular filtration rate (GFR), the best overall biomarker of kidney function. However, current common methods of measuring creatinine are not sensitive enough for mouse plasma. Accordingly, we here report a new method of measuring creatinine by liquid chromatography tandem mass spectrometry (LC-MS/MS) using deuterated [2H3]-creatinine as an internal standard. The assay requires 10 microl or less of plasma or urine, and is eight times more sensitive than high-performance liquid chromatography. The reproducibility of the assay of replicates is approximately +/-10%. The plasma creatinine levels of wild type male C57BL/6J mice obtained by LC-MS/MS are 0.076+/-0.002 mg/dl (n=65). To estimate daily urinary creatinine excretion for calculating Ccr, we collected urine from mice housed in metabolic cages, and combined this with washes from the cage internal surfaces. Creatinine in the wash varies from 4 to 67% of the total daily urinary creatinine excretion (typically approximately 400 microg/day). Ccr obtained by LC-MS/MS was 329+/-17 microl/min, which is indistinguishable from GFR measured by using fluorescein isothiocyanate-inulin. The LC-MS/MS method is sensitive, specific, simple, fast, and inexpensive; it is suitable for estimating GFR in conscious mice or other small animals. As it allows repeated measurements in the same animals, it facilitates detection of subtle differences or changes in renal function.

Animals↗

PubMed2005

Quantification of DNA and hemoglobin adducts of 3,4-epoxy-1,2-butanediol in rodents exposed to 3-butene-1,2-diol.

1,3-Butadiene (BD) is a confirmed rodent carcinogen and a suspect human carcinogen that forms mutagenic epoxide metabolites during biotransformation. Species differences in the roles of individual DNA reactive intermediates in BD mutagenicity and carcinogenicity are not completely understood. Evidence suggests that 1,2:3,4-diepoxybutane (DEB) is responsible for the mutagenic effect induced by exposures to low concentrations of BD in mice and that metabolites of 3-butene-1,2-diol (BD-diol) are involved in the mutagenicity at high exposures in both mice and rats. Two reactive metabolites, 3,4-epoxy-1,2-butanediol (EB-diol) and hydroxymethylvinyl ketone (HMVK), are formed during the biotransformation of BD-diol and could potentially be involved in BD-diol associated mutagenicity. To examine the role of EB-diol in BD-diol mutagenicity we have evaluated the dosimetry of N7-(2,3,4-trihydroxybutyl)guanine (THB-Gua) and N-(2,3,4-trihydroxybutyl)valine (THB-Val) in female B6C3F1 mice and female F344 rats exposed by inhalation to 0, 6, 18 and 36 p.p.m. BD-diol for 4 weeks (6 h/day x 5 days/week). Results showed higher levels of both THB-Gua and THB-Val in mice than in rats. An evaluation of THB-Gua adducts showed virtually no differences between liver and lung for either species, suggesting that EB-diol is stable and is freely circulated. The data also indicated that THB adduct formation began to plateau around 18 p.p.m. in both species. Most importantly, the shape of the dose-response curve for THB adduct formation mimicked the one observed for hypoxanthine-guanine phosphoribosyltransferase (Hprt) mutation frequency. This showed that THB adducts, which are not thought to be responsible for causing the mutations, are good quantitative indicators of mutagenicity in rodents exposed to BD-diol. Although the potential contribution of HMVK still needs to be evaluated, the data suggest that EB-diol is responsible, at least in part, for BD-diol associated mutagenicity in rodents.

Animals↗

PubMed2001

Biomarkers for assessing occupational exposures to 1,3-butadiene.

The overall objective of this study was to evaluate a continuum of biomarkers in blood and urine for their sensitivities as indicators of low level occupational exposures to 1,3 butadiene (BD). The study design was largely cross-sectional, with biological samples collected within a short timeframe. Personal 8-h BD exposure measures were made on several occasions over a 60-day period for each potentially exposed worker in order provide maximum accuracy for this independent variable and to accommodate the different expression intervals of the several biomarkers. Co-exposures to styrene, toluene and benzene were also measured. The study included 24 BD monomer production workers (mean BD exposure=0.642 mg/m(3)), 34 polymerization workers (mean BD exposure=1.794 mg/m(3)) and 25 controls (mean BD exposure=0.023 mg/m(3)). The several biomarkers were measured by a consortium of investigators at different locations in the US and Europe. These biomarkers included: (1) metabolic genotypes (CYP2E1, EH, GST M1, GST T1, ADH2, ADH3), determined in Prague and Burlington, VT; (2) urinary M1 and M2 metabolites (1,2-dihydroxy-4-[N-acetylcysteinyl]-butane and 1-hydroxy-2-[N-acetylcysteinyl]-3-butene, respectively), determined in Albuquerque, NM and Leiden; (3) hemoglobin adducts (N-[2-dihydroxy-3-butenyl]valine=HBVal and N-[2,3,4-trihydroxybutyl]valine=THBVal), determined in Amsterdam and Chapel Hill, NC, respectively; (4) HPRT mutations determined by autoradiographic assay in Galveston, TX, with slides re-read in Burlington, VT; (6) hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations determined by cloning assay in Leiden with mutational spectra characterized in Burlington, VT; (7) sister chromatid exchanges and chromosome aberrations determined by standard methods and FISH analysis in Prague. Urinary M1 and M2 metabolites and HBVal and THBVal hemoglobin adducts were all significantly correlated with BD exposure levels, with adducts being the most highly associated. No significant relationships were observed between BD exposures and HPRT mutations or any of the cytogenetic endpoints, regardless of method of assay.

Adult↗

PubMed2001

Markers for carcinogenicity among butadiene-polymer workers in China.

We examined a spectrum of genotoxic and other outcomes in 41 butadiene-polymer production workers and 38 nonexposed controls, in China, to explore the role of butadiene in human carcinogenesis. Among butadiene-exposed workers, median air exposure was 2 ppm (6-h TWA), due largely to intermittent high-level exposures. Compared to 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 percent of white blood cells were moderately correlated with greater THBVal levels (Spearman's rho=0.32, P=0.07). Among butadiene-exposed workers, several serum cytokines correlated with THBVal adduct levels. 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.

Air Pollutants, Occupational↗

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

Mutagenicity at the Hprt locus in T cells of female mice following inhalation exposures to low levels of 1,3-butadiene.

A study was conducted to test the hypothesis that repeated low level exposures to 1,3-butadiene (BD), approaching the OSHA occupational threshold for this chemical, produce a significant mutagenic response in mice. Female B6C3F1 mice (4-5 weeks of age) were exposed by inhalation for 2 weeks (6 h/day, 5 days/week) to 0 or 3 ppm BD, and then necropsied at 4 weeks after the cessation of exposures to measure the frequency of mutations (MF) at the Hprt locus using the T-lymphocyte clonal assay. At necropsy, T cells were isolated from spleen and cultured in the presence of mitogen, growth factors, and a selection agent. Cells were scored for growth on days 8-9 after plating to determine cloning efficiencies (CEs) and Hprt MFs. There was a marginal but significant reduction in the growth of splenic T cells from mice exposed to 3 ppm (n=27) compared with control mice (n=24) (P=0.004), suggesting the occurrence of BD-induced cytotoxicity at this low exposure concentration. In addition, the average Hprt MF in mice exposed to 3 ppm BD [1.54+/-0.82 (S.D.)x10(-6)] was significantly increased by 1.6-fold over the average control value of 0.96+/-0.51 (S.D.)x10(-6) (P=0.004). Comparisons of these data to earlier Hprt mutagenicity studies of mice exposed to high concentrations of BD (where significant mutagenic but not cytotoxic effects were observed) indicate that the ability to detect the cytotoxic and mutagenic responses of T cells to low levels of BD was enhanced by using a much larger sample size than usual for both the control and treatment groups. Additional analyses of the quantitative relationships between CE and MF demonstrated that CE had no significant effect upon MF values in sham-exposed control mice or mice exposed to low-level BD. Furthermore, the approaches for assessing the impact of CE and clonality on Hprt MFs in these control and BD-exposed mice were applied with the same rigor as in in vivo Hprt mutagenicity studies in human children. The overall study results support the conclusion that short-term low-level BD exposure is mutagenic in the mouse.

Administration, Inhalation↗

PubMed2001

Repeated inhalation exposure to octamethylcyclotetrasiloxane produces hepatomegaly, transient hepatic hyperplasia, and sustained hypertrophy in female Fischer 344 rats in a manner similar to phenobarbital.

Octamethylcyclotetrasiloxane (D4) has been described as a phenobarbital-like inducer of hepatic enzymes. Phenobarbital (PB) and phenobarbital-like chemicals induce transient hepatic and thyroid hyperplasia and sustained hypertrophy in rats and mice. The extent to which these processes are involved with D4-induced hepatomegaly is not known. The present study has evaluated the effects of repeated inhalation exposure to D4 vapors on hepatic and thyroid cell proliferation and hypertrophy with respect to time and exposure concentration. Female Fischer 344 rats were exposed via whole body inhalation to 0 ppm D4, 700 ppm D4 vapors (6 h/day; 5 days/week), or 0.05% PB in drinking water over a 4-week period. Incorporation of 5'-bromo-2-deoxyuridine (BrdU) and the abundance of proliferating cell nuclear antigen were used as indicators of cell proliferation. Designated animals from each treatment group were euthanized on study days 6, 13, and 27. The effect of D4 exposure concentration on hepatic cell proliferation was evaluated at 0, 7, 30, 70, 150, 300, or 700 ppm. Liver-to-body weight ratios in animals exposed to 700 ppm D4 were increased 18, 20, and 22% over controls while PB-treated animals showed increases of 33, 27, and 27% over controls on days 6, 13, and 27 respectively. Hepatic incorporation of BrdU following exposure to D4 was highest on day 6 (labeling index = 15-22%) and was at or below control values by day 27. This pattern of transient hyperplasia was observed in all hepatic lobes examined and was similar to the pattern observed following treatment with PB.

Animals↗

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↗

PubMed2001

Oxidative damage and direct adducts in calf thymus DNA induced by the pentachlorophenol metabolites, tetrachlorohydroquinone and tetrachloro-1,4-benzoquinone.

DNA damage induced by quinoid metabolites of pentachlorophenol (PCP), i.e. tetrachloro-1,4-benzoquinone (Cl(4)BQ) and tetrachlorohydroquinone (Cl(4)HQ), was investigated in calf thymus DNA. The (32)P-post-labeling assay revealed four major and several minor adducts (3.5 adducts per 10(5) total nucleotides) that were produced in calf thymus DNA treated with Cl(4)BQ (5 mM). These DNA adducts were chemically stable even after conditions that induce thermal depurination and are unlikely to undergo depurination/depyrimidination to form apurinic/apyrimidinic (AP) sites. In addition, increases in 8-hydroxy-deoxyguanosine (8-HO-dG) (5 8-HO-dG per 10(5) nucleotides) and AP sites (0.5 AP sites per 10(5) nucleotides) were observed in Cl(4)BQ-modified calf thymus DNA. Further investigation indicated that in the presence of Cu(II) and NADPH, low concentrations of Cl(4)BQ (1 microM) induced a doubling of 8-HO-dG (10 8-HO-dG per 10(5) nucleotides) and dramatic increases in AP sites (20 AP sites per 10(5) nucleotides) and DNA single-strand breaks. The types of DNA damage induced by Cl(4)HQ plus Cu(II) were similar to those by Cl(4)BQ plus Cu(II) and NADPH, whereas catalase inhibited the formation of DNA damage. These data suggest that oxidative damage is causally involved in the formation of AP sites. Concentration-dependent increases in 8-HO-dG induced by Cl(4)HQ plus Cu(II) and Cl(4)BQ plus Cu(II) and NADPH were correlated with the formation of AP sites (r(2) = 0.977) with a ratio of 8-HO-dG to AP sites at 1:1.6. The AP site-cleavage assay confirmed that approximately 85% of the AP sites induced by Cl(4)HQ and Cu(II) were detected as 5'-cleaved AP sites. Since hydrogen peroxide alone causes similar DNA damage, these results suggest the involvement of Cu(II) and hydrogen peroxide in the induction of oxidative DNA damage by Cl(4)HQ/Cl(4)BQ. The data demonstrate that PCP quinone and hydroquinone induce direct and oxidative base modifications as well as the formation of 5'-cleaved AP sites in genomic DNA. These lesions may have important implications for PCP clastogenicity and carcinogenicity.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2001

Induction of direct adducts, apurinic/apyrimidinic sites and oxidized bases in nuclear DNA of human HeLa S3 tumor cells by tetrachlorohydroquinone.

DNA damage induced by tetrachlorohydroquinone (Cl(4)HQ), the quinonoid metabolite of pentachlorophenol (PCP), was investigated in human HeLa S3 tumor cells. Formation of one major and two minor DNA adducts in cells treated with Cl(4)HQ (50-300 microM) was detected by (32)P-post-labeling assay and the adducts accumulated over the course of the experiment (0.5-2 h), with total adduct levels estimated to be 3-6 per 10(8) nucleotides. These adducts did not correspond to those derived from calf thymus DNA treated with tetrachloro-1,4-benzoquinone. Results from the apurinic/apyrimidinic (AP) sites assay indicated that the number of AP sites was 2-fold greater in cells exposed to Cl(4)HQ (300 microM) than the corresponding control. Further characterization of the AP sites confirmed that Cl(4)HQ induced predominantly (75%) putrescine-excisable AP sites in HeLa S3 cells. In parallel, the concentration of 8-hydroxy-2'-deoxyguanosine (8-HO-dG) in cells treated with Cl(4)HQ for 0.5 and 2 h was increased 2- and 5-fold, respectively, compared with the control. The extent of oxidative DNA damage induced by Cl(4)HQ was approximately two orders of magnitude greater than those of direct DNA adducts. Overall, it appears that reactive oxygen species mediate the parallel formation of AP sites and 8-HO-dG in HeLa S3 cells following treatment with Cl(4)HQ and that the contribution of depurination/depyrimidination of direct DNA adducts is relatively insignificant compared with the formation of oxidized AP sites. We conclude that putrescine-excisable AP sites represent a major type of ROS-mediated oxidative DNA damage in cellular DNA induced by Cl(4)HQ and may play a role in PCP-induced clastogenicity in mammalian cells.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2000

Oxidants from nicotinamide adenine dinucleotide phosphate oxidase are involved in triggering cell proliferation in the liver due to peroxisome proliferators.

It was shown that 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (Wy-14,643), a potent peroxisome proliferator, caused rapid oxidant-dependent activation of nuclear factor kappaB (NF-kappaB) in Kupffer cells in vivo and activated superoxide production by isolated Kupffer cells. Here, we tested the hypothesis that NADPH oxidase (NADPH OX) is the source of oxidants increased by Wy-14,643. Indeed, both activation of NF-kappaB and increases in cell proliferation due to a single dose of Wy-14,643 (100 mg/kg) were prevented completely when rats were pretreated with diphenyleneiodonium (1 mg/kg), an inhibitor of NADPH OX. p47phox is a critical subunit of NADPH OX; therefore, p47phox knockout mice were used to specifically address the hypothesis of NADPH OX involvement. In livers of wild-type mice, Wy-14,643 activated NF-kappaB, followed by an increase in mRNA for tumor necrosis factor a. Importantly, these changes did not occur in p47phox knockouts. Moreover, when Kupffer cells were treated with Wy-14,643 in vitro, superoxide production was increased in cells from wild-type but not p47phox-null mice. Finally, when mice were fed a Wy-14,643-containing (0.1%) diet for 7 days, the increase in liver weight and cell proliferation caused by Wy-14,643 in wild-type mice was blocked in p47phox-null mice. Combined, these results are consistent with the hypothesis that Wy-14,643 activates NADPH OX, which leads to NF-kappaB-mediated production of mitogens that causes hepatocellular proliferation characteristic of this class of nongenotoxic carcinogens.

Animals↗

PubMed2000

Deficiency of N-methylpurine-DNA-glycosylase expression in nonparenchymal cells, the target cell for vinyl chloride and vinyl fluoride.

The ability to repair promutagenic damage resulting from exposure to carcinogens is a critical factor in determining quantitative relationships in carcinogenesis, including the target cell for neoplasia. One major pathway for the repair of alkylating agent-induced DNA damage involves removal of alkylated bases by N-methylpurine-DNA-glycosylase (MPG), the first enzyme in base excision repair. We have measured the expression level of MPG mRNA in liver, lung, and kidney of Sprague-Dawley rats as a function of age. A quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) method was used to measure cellular MPG mRNA. MPG mRNA was readily detectable in each tissue analyzed and the age-dependent and tissue specific expressions were not statistically different. The lowest amount of mRNA was measured in preweanling liver and the highest amounts were found in preweanling lung and kidney. Since MPG is reported to be responsible for excision of 1,N(6)-ethenoadenine and N(2),3-ethenoguanine, two promutagenic DNA adducts of vinyl chloride (VC) and vinyl fluoride (VF), we examined the regulation of this enzyme after carcinogen exposure. Expression of MPG was induced in rat liver by these carcinogens. In order to determine the repair capacity in different cell populations of liver, we measured MPG gene expression in isolated hepatocytes and nonparenchymal cells (NPC). The amount of MPG mRNA was 4.5-5 times higher in hepatocytes than in NPC of control rats. Induction of MPG expression was observed in hepatocytes of VF exposed-rats but not in NPC. The expression of MPG in NPC was only 15% of that of the hepatocytes from exposed rats. Western blots of MPG protein confirmed the cell type differences, but did not show increased protein in exposed vs. control liver and hepatocytes. Since metabolism of VC and VF requires CYP2E1, an enzyme exhibiting much greater activity in hepatocytes, formation of etheno adducts preferentially occurs in hepatocytes. These data suggest that cellular differences in the repair of N-alkylpurines may be a critical mechanism in the development of cell specificity in VC carcinogenesis.

Aging↗

PubMed2000

Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancer-prone chronic inflammatory disease.

Ulcerative colitis (UC) is a chronic inflammatory disease that produces reactive oxygen and nitrogen species and increases the risk of colorectal cancer (CRC). The p53 tumor suppressor gene is frequently mutated in UC-associated dysplastic lesions and CRC. We are exploring the hypothesis that p53 mutations in the nontumorous colonic tissue in noncancerous UC cases indicate genetic damage from exposure to exogenous and endogenous carcinogens and may identify individuals at increased cancer risk. We are reporting, for the first time, the frequency of specific p53 mutated alleles in nontumorous colon tissue from donors either with or without UC by using a highly sensitive genotypic mutation assay. Higher p53 mutation frequencies of both G:C to A:T transitions at the CpG site of codon 248 and C:G to T:A transitions at codon 247 were observed in colon from UC cases when compared with normal adult controls (P = 0.001 and P = 0.001, respectively). In the UC cases, higher p53 codon 247 and 248 mutation frequencies were observed in the inflamed lesional regions when compared with the nonlesional regions of their colon (P < 0.001 and P = 0.001). The colonic nitric oxide synthase-2 activity was higher in UC cases than in non-UC adult controls (P = 0.02). Our data are consistent with the hypothesis that a higher frequency of p53 mutant cells can be generated under oxidative stress in people with UC. The increased frequency of specific p53 mutated alleles in noncancerous UC colon tissue may confer susceptibility to the development of CRC in an inflammatory microenvironment.

Adult↗

PubMed2000

Inhibition of chronic rejection of aortic allografts by dietary glycine.

BACKGROUND: Chronic rejection is influenced by a variety of risk factors, including histoincompatibility and ischemia. Glycine, a cytoprotective agent, has been shown to protect against ischemia-reperfusion injury in the liver, inactivate hepatic resident macrophages, minimize cyclosporin A-induced nephrotoxicity, and exhibit immunosuppressive properties in vitro. The aim of this study was to investigate whether dietary glycine could reduce development of chronic rejection. METHODS: Lewis recipients of Fisher-344 abdominal aortic allografts received diets that contained either 5% glycine plus 15% casein or 20% casein as control for 10 weeks. Vascular lesions of aortic isografts and allografts were evaluated quantitatively with image analysis and cell counting. RESULTS: No significant vascular changes were observed in isografts (mean medial areas of 3.3 +/- 0.3x0(5) microm2). However, dramatic intimal thickening (neointimal area 2.1+/-0.3) and medial thinning (1.5+/-0.3) were observed in allografts from rats fed control diet. In contrast, glycine significantly reduced the neointima by 45% (1.2+/-0.3) and protected the media (3.5+/-0.2). This led to intima to media area ratios almost twice as large in the control group as in glycine-fed rats (2.2+/-0.4 vs. 1.1+/-0.3, P<0.05). Moreover, infiltrating leukocytes, especially macrophages, were reduced significantly in the adventitia by glycine. In addition, glycine inhibited proliferation and migration of rat aortic smooth muscle cells in culture by 45 and 60%, respectively. CONCLUSION: These results indicate that dietary glycine minimizes histopathological changes of chronic rejection by reducing the immune response and, in part, by minimizing proliferation and migration of smooth muscle cells.

Animals↗

PubMed2000

5'-nicked apurinic/apyrimidinic sites are resistant to beta-elimination by beta-polymerase and are persistent in human cultured cells after oxidative stress.

Genomic DNA is continuously exposed to oxidative stress. Whereas reactive oxygen species (ROS) preferentially react with bases in DNA, free radicals also abstract hydrogen atoms from deoxyribose, resulting in the formation of apurinic/apyrimidinic (AP) sites and strand breaks. We recently reported high steady-state levels of AP sites in rat tissues and human liver DNA (Nakamura, J., and Swenberg, J. A. (1999) Cancer Res. 59, 2522-2526). These AP sites were predominantly cleaved 5' to the lesion. We hypothesized that these endogenous AP sites were derived from oxidative stress. In this investigation, AP sites induced by ROS were quantitated and characterized. A combination of H(2)O(2) and FeSO(4) induced significant numbers of AP sites in calf thymus DNA, which were predominantly cleaved 5' to the AP sites (75% of total aldehydic AP sites). An increase in the number of 5'-AP sites was also detected in human cultured cells exposed to H(2)O(2), and these 5'-AP sites were persistent during the post-exposure period. beta-Elimination by DNA beta-polymerase efficiently excised 5'-regular AP sites, but not 5'-AP sites, in DNA from cells exposed to H(2)O(2). These results suggest that 5'-oxidized AP sites induced by ROS are not efficiently repaired by the mammalian short patch base excision repair pathway.

8-Hydroxy-2'-Deoxyguanosine↗

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↗