Biomedical subjects

R H Stierum

Publications and source records attributed to R H Stierum.

13 recordsLinked to original sources

PubMed2005

Expression profiling of colon cancer cell lines and colon biopsies: towards a screening system for potential cancer-preventive compounds.

Interest in mechanisms of colon cancer prevention by food compounds is strong and research in this area is often performed with cultured colon cancer cells. In order to assess utility for screening of potential cancer-preventive (food) compounds, expression profiles of 14 human cell lines derived from colonic tissue were measured using cDNA microarrays with 4000 genes and compared with expression profiles in biopsies of human colon tumours and normal tissue. Differences and similarities in the gene expression profiles of the cell lines were analysed by clustering and principal component analysis (PCA). Cytoskeleton genes and immune response genes are two functional classes of genes that contributed to the differences between the cell lines. A subset of 72 colon cancer-specific genes was identified by comparing expression profiles in human colon biopsies of tumour tissue and normal tissue. A separation of the cell lines based on the tumour stage of the original adenocarcinoma was observed after PCA of expression data of the subset of colon cancer-specific genes in the cell lines. The results of this study may be useful in the ongoing research into mechanisms of cancer prevention by dietary components.

Adenocarcinoma↗

PubMed2001

Niacin, poly(ADP-ribose) polymerase-1 and genomic stability.

Nicotinic acid (NA) and nicotinamide (NAM), commonly called niacin, are the dietary precursors for NAD(+) (nicotinamide adenine dinucleotide), which is required for DNA synthesis, as well as for the activity of the enzyme poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) for which NAD(+) is the sole substrate. The enzyme PARP-1 is highly activated by DNA strand breaks during the cellular genotoxic stress response, is involved in base excision repair, plays a role in p53 expression and activation, and hence, is thought to be important for genomic stability. In this review, first the absorption, metabolism of niacin to NAD(+), as well as the assessment of niacin status are discussed. Since NAD(+) is important for PARP-1 activity, various aspects of PARP-1 in relation to DNA synthesis and repair, and regulation of gene expression are addressed. This is followed by a discussion on interactions between dietary methyl donor deficiency, niacin status, PARP-1 activity and genomic stability. In vitro studies show that PARP-1 function is impaired and genomic stability decreased when cells are either depleted from NAD(+) or incubated with high concentrations of NAM which is a PARP-1 inhibitor. In vitro as well as animal studies indicate that niacin deficiency increases genomic instability especially in combination with genotoxic and oxidative stress. Niacin deficiency may also increase the risk for certain tumors. Preliminary data suggest that niacin supplementation may protect against UV-induced tumors of the skin in mice, but data on similar preventive effects in humans are not available. NAM has been shown in vitro to have an antioxidant activity comparable to that of ascorbic acid. Data on niacin status and genomic stability in vivo in humans are limited and yield ambiguous results. Therefore, no firm conclusions with respect to optimal niacin intake are possible. As a consequence of oral niacin supplementation, however, NAM levels in the body may increase, which may result in inhibition of PARP-1 and increased genomic instability. More studies are needed to define an optimal level of niacin nutriture in relation to genomic stability and tumorigenesis.

Animals↗

PubMed1999

Single-nucleotide patch base excision repair of uracil in DNA by mitochondrial protein extracts.

Mammalian mitochondria contain several 16.5 kb circular DNAs (mtDNA) encoding electron transport chain proteins. Reactive oxygen species formed as byproducts from oxidative phosphorylation in these organelles can cause oxidative deamination of cytosine and lead to uracil in mtDNA. Upon mtDNA replication, these lesions, if unrepaired, can lead to mutations. Until recently, it was thought that there was no DNA repair in mitochondria, but lately there is evidence that some lesions are efficiently repaired in these organelles. In the study of nuclear DNA repair, the in vitro repair measurements in cell extracts have provided major insights into the mechanisms. The use of whole-cell extract based DNA repair methods has revealed that mammalian nuclear base excision repair (BER) diverges into two pathways: the single-nucleotide replacement and long patch repair mechanisms. Similar in vitro methods have not been available for the study of mitochondrial BER. We have established an in vitro DNA repair system supported by rat liver mitochondrial protein extract and DNA substrates containing a single uracil opposite to a guanine. Using this approach, we examined the repair pathways and the identity of the DNA polymerase involved in mitochondrial BER (mtBER). Employing restriction analysis of in vitro repaired DNA to map the repair patch size, we demonstrate that only one nucleotide is incorporated during the repair process. Thus, in contrast to BER in the nucleus, mtBER of uracil in DNA is solely accomplished by single-nucleotide replacement.

Animals↗

PubMed1999

Mitochondrial DNA repair pathways.

DNA repair mechanisms are fairly well characterized for nuclear DNA while knowledge regarding the repair mechanisms operable in mitochondria is limited. Several lines of evidence suggest that mitochondria contain DNA repair mechanisms. DNA lesions are removed from mtDNA in cells exposed to various chemicals. Protein activities that process damaged DNA have been detected in mitochondria. As will be discussed, there is evidence for base excision repair (BER), direct damage reversal, mismatch repair, and recombinational repair mechanisms in mitochondria, while nucleotide excision repair (NER), as we know it from nuclear repair, is not present.

Aging↗

PubMed1999

Purification and characterization of a mitochondrial thymine glycol endonuclease from rat liver.

Mitochondrial DNA is exposed to oxygen radicals produced during oxidative phosphorylation. Accumulation of several kinds of oxidative lesions in mitochondrial DNA may lead to structural genomic alterations, mitochondrial dysfunction, and associated degenerative diseases. The pyrimidine hydrate thymine glycol, one of many oxidative lesions, can block DNA and RNA polymerases and thereby exert negative biological effects. Mitochondrial DNA repair of this lesion is important to ensure normal mitochondrial DNA metabolism. Here, we report the purification of a novel rat liver mitochondrial thymine glycol endonuclease (mtTGendo). By using a radiolabeled oligonucleotide duplex containing a single thymine glycol lesion, damage-specific incision at the modified thymine was observed upon incubation with mitochondrial protein extracts. After purification using cation exchange, hydrophobic interaction, and size exclusion chromatography, the most pure active fractions contained a single band of approximately 37 kDa on a silver-stained gel. MtTGendo is active within a broad KCl concentration range and is EDTA-resistant. Furthermore, mtTGendo has an associated apurinic/apyrimidinic-lyase activity. MtTGendo does not incise 8-oxodeoxyguanosine or uracil-containing duplexes or thymine glycol in single-stranded DNA. Based upon functional similarity, we conclude that mtTGendo may be a rat mitochondrial homolog of the Escherichia coli endonuclease III protein.

Animals↗

PubMed1998

Homogenous repair of singlet oxygen-induced DNA damage in differentially transcribed regions and strands of human mitochondrial DNA.

Photoactivated methylene blue was used to damage purified DNA and the mitochondrial DNA (mtDNA) of human fibroblasts in culture. The primary product of this reaction is the DNA lesion 7-hydro-8-oxo-deoxyguanosine (8-oxo-dG). The DNA damage was quantitated using Escherichia coli formamidopyrimidine DNA glycosylase (Fpg) in a gene-specific damage and repair assay. Assay conditions were refined to give incision at all enzyme-sensitive sites with minimal non-specific cutting. Cultured fibroblasts were exposed to photoactivated methylene blue under conditions that would produce an average of three oxidative lesions per double-stranded mitochondrial genome. Within 9 h, 47% of this damage had been removed by the cells. This removal was due to repair rather than to replication, cell loss or degradation of damaged genomes. The rate of repair was measured in both DNA strands of the frequently transcribed ribosomal region of the mitochondrial genome and in both strands of the non-ribosomal region. Fpg-sensitive alkali-resistant oxidative base damage was efficiently removed from human mtDNA with no differences in the rate of repair between strands or between two different regions of the genome that differ substantially with regard to transcriptional activity.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1998

Nicotinic acid supplementation: effects on niacin status, cytogenetic damage, and poly(ADP-ribosylation) in lymphocytes of smokers.

As a substrate for poly(ADP-ribose) polymerase (PARP; EC, 2.4.2.30), an enzyme that is activated by DNA strand breaks and is thought to facilitate efficient DNA repair, NAD+ and its precursor nicotinic acid (niacin) are involved in the cellular defense against DNA damage by genotoxic compounds. In this study, the effect of nicotinic acid supplementation on cytogenetic damage and poly(ADP-ribosylation) was evaluated in a human population that is continuously exposed to genotoxic agents, e.g., smokers. By use of a placebo-controlled intervention design, 21 healthy smokers received supplementary nicotinic acid at 0-100 mg/day for 14 weeks. An increased niacin status, as assessed from blood nicotinamide concentrations and lymphocyte NAD+ concentrations, was observed in groups supplemented with 50 and 100 mg/day. This effect was most pronounced in subjects with lower initial NAD+ levels. An increased niacin status did not result in decreased hypoxanthine guanine phosphoribosyltransferase variant frequencies and micronuclei induction in peripheral blood lymphocytes (PBLs). Sister chromatid exchanges in PBLs, however, were increased after supplementation with nicotinic acid. This increase was positively associated with the daily dose of nicotinic acid. No effects of nicotinic acid supplementation were found for ex vivo (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-induced poly(ADP-ribosylation), although the small number of samples that could be analyzed (n = 12) does not allow firm conclusions. Because no evidence was found for a decrease in cigarette smoke-induced cytogenetic damage in PBLs of smokers after nicotinic acid supplementation of up to 100 mg/day, it is concluded that supplemental niacin does not contribute to a reduced genetic risk in healthy smokers.

Adult↗

PubMed1997

Age-related negative associations between parameters of cytogenetic damage and ex vivo (+/-)-anti-benzo(a)pyrene diolepoxide-induced unscheduled DNA synthesis in smoking humans.

Chemical or physical modification of DNA may cause an increase in genomic mutations or other genetic alterations, which may ultimately result in the onset of cancer. To avoid these deleterious effects of DNA damage, humans possess DNA repair mechanisms. Decreased DNA repair, induced ex vivo by UV light or ionizing radiation in human peripheral blood lymphocytes (PBLs), has been associated with aging. The aim of this study was to investigate whether repair of DNA damage, after ex vivo exposure of PBLs obtained from smokers (n = 20) to (+/-)-anti-benzo(a)pyrene diolepoxide [(+/-)-anti-BPDE], which is a mixture of reactive metabolites from the environmental carcinogen benzo(a)pyrene, is also associated with age. Furthermore, age-related associations between ex vivo (+/-)-anti-BPDE-induced DNA repair and the frequency of endogenous cytogenetic damage (sister chromatid exchange frequencies and micronuclei frequencies) in PBLs were evaluated. A statistically significant negative association was observed between ex vivo (+/-)-anti-BPDE-induced unscheduled DNA synthesis and age of the donors. Also, parameters of endogenous lymphocytic cytogenetic damage were negatively associated with ex vivo (+/-)-anti-BPDE-induced unscheduled DNA synthesis and positively associated with age in this population. It is concluded that, with increasing age, a decrease in lymphocytic excision repair capacity may be responsible for increased lymphocytic DNA damage in smokers.

Adult↗

PubMed1995

Inhibition of poly(ADP-ribose) polymerase increases (+/-)-anti-benzo [a]pyrene diolepoxide-induced micronuclei formation and p53 accumulation in isolated human peripheral blood lymphocytes.

In response to DNA damage, in particular DNA strand breaks, the proposed roles for normal tumour suppressor protein p53 are to increase the period of time available for DNA repair prior to replication, or to direct damaged cells into programmed cell-death. Since treatment of mammalian cells with (+/-)-anti-benzo[a]pyrene diolepoxide [(+/-)-anti-BPDE] --a mixture of metabolites comprising the most reactive (+)-anti-enantiomer of the full environmental carcinogen benzo[a]pyrene--has been shown to result in induction of DNA repair processes and consequently in DNA strand break formation, the aim of the present study was to investigate whether p53 accumulation is induced in (+/-)-anti-BPDE-treated phytohaemagglutinin-stimulated human peripheral blood lymphocytes (PBLs). Both immunocytochemical and immunoblot analysis indicated that treatment of PBLs with (+/-)-anti-BPDE results in p53 accumulation. Optimal accumulation was observed at 2.5 microM, while no increase of p53 levels was observed at concentrations < 2.5 microM and > 10 microM. Further, (+/-)-anti-BPDE-induced p53 accumulation in PBLs was found to be time-dependent with accumulation up to 24 h after the onset of treatment. Treatment of PBLs with 2.5 microM of (+/-)-anti-BPDE and 1 mM of 3-aminobenzamide, an inhibitor of the DNA strand break-dependent enzyme poly(ADP-ribose) polymerase, resulted in increased p53 levels, in comparison to cells treated with (+/-)-anti-BPDE alone. This combination also potentiated the frequency of (+/-)-anti-BPDE-induced micronuclei. These findings suggest that (+/-)-anti-BPDE-induced DNA strand break formation is responsible for the observed p53 accumulation. It is unlikely that poly(ADP-ribose) polymer formation is a prerequisite in the process of p53 accumulation, as triggered by DNA strand-break inducing agents like (+/-)-anti-BPDE. It is hypothesized that p53-dependent pathways may be activated in phytohaemagglutinin-stimulated human peripheral blood lymphocytes exposed ex vivo to (+/-)-anti-BPDE.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1994

Measurement by 32P-postlabeling of (+/-)anti-benzo[a]pyrene-diolepoxide-N2-deoxyguanosine adduct persistence in unstimulated human peripheral blood lymphocytes.

In order to study the relative importance of endogenous and environmental factors for the individual relation between DNA damage and DNA excision repair, a method was developed for measuring quantitatively the persistence of N2-deoxyguanosine adducts formed in non-stimulated isolated human peripheral blood lymphocytes after in vitro incubation with 0.2 microM (+/-)anti-BPDE, applying 32P-postlabeling. Total binding of radiolabeled (+/-)anti-BPDE to DNA and its removal has been studied previously in human peripheral blood lymphocytes, but the method presented here enables the direct investigation of repair of the main (+/-)anti-BPDE-DNA adduct, which is implicated in benzo[a]pyrene-induced mutagenesis. Using this method, it was found that in lymphocytes, obtained from 5 individuals, most (+/-)anti-BPDE-N2-dG adducts are removed within the first 24 h after treatment, while interindividual differences appear to exist in both adduct formation and rate and extent of removal.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1994

Increased poly(ADP-ribose) polymerase activity during repair of (+/-)-anti-benzo[a]pyrene diolepoxide-induced DNA damage in human peripheral blood lymphocytes in vitro.

Poly(ADP-ribose) polymerase, which catalyzes the formation of poly(ADP-ribose) polymers, is an enzyme involved in cell proliferation, differentiation and transformation as well as in recovery from DNA damage. Poly(ADP-ribose) polymers are rapidly synthesized from the ADP-ribose moieties from intracellular NAD+, which, as a consequence, is depleted. It has been shown that DNA strand breaks are required for enzyme activation and it is suggested that one of the functions of poly(ADP-ribosylation) is to improve accessibility of damaged sites to other DNA repair enzymes. The aim of this study was to investigate whether poly(ADP-ribosylation) is involved in repair of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE]-induced DNA damage in human lymphocytes in vitro. Results show that (+/-)-anti-BPDE is capable of inducing poly(ADP-ribosylation), NAD+ depletion and inhibition of proliferation in phytohemagglutinin-stimulated human peripheral blood lymphocytes. Also, repair of (+/-)-anti-BPDE induced DNA damage was confirmed by both unscheduled DNA synthesis and (+/-)-anti-BPDE-deoxyguanosine adduct removal. Based on these findings, it is concluded that poly(ADP-ribosylation) is involved in (+/-)-anti-BPDE-induced DNA repair in these cells. In addition, these results confirm the possible relation between poly(ADP-ribosylation), NAD+ depletion and inhibition of proliferation, after induction of DNA damage.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1993

Evaluation of exposure reducing measures on parameters of genetic risk in a population occupationally exposed to coal fly ash.

In a previous study we found increased SCE frequencies in peripheral blood lymphocytes (PBLs) of workers occupationally exposed in a coal fly ash processing industry, as compared to a non-exposed control population. Shortly after this study, measures were taken in this plant to reduce fly ash levels. The objective of the present study, conducted 2 years later in the same plants, was to evaluate the effect of these measures with respect to genotoxic risk. A group of 18 male workers of the coal fly ash processing industry agreed to participate in the study. The control population consisted of 18 male workers from a flour processing industry, who were matched for age and smoking behavior. In contrast to our previous study, no increased SCE frequencies were found in PBLs of workers potentially exposed to coal fly ash when compared to the control group (mean SCEs: 6.4 +/- 1.2 and 7.0 +/- 0.9, respectively). In addition, no differences were observed between the exposed and control groups for frequencies of gene mutations at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus in PBLs, for micronucleus frequencies using the cytokinesis block method, or for urinary mutagen excretion measured with Salmonella typhimurium tester strains TA98 and TA97 with and without metabolic activation. In smokers, however, SCE frequencies in PBLs were significantly increased in comparison to non-smokers (7.1 +/- 1.1 vs. 6.1 +/- 0.5; P < 0.005), as was 24-h urinary mutagen excretion measured with strain TA98 with S9 mix (2373 +/- 1870 vs. 156 +/- 211; P < 0.001) and with TA98 with S9 mix and beta-glucuronidase/arylsulfatase (2361 +/- 1958 vs. 538 +/- 396; P < 0.005). In addition, hprt variant frequencies in PBLs were higher in smokers than in non-smokers (15.0 +/- 23.5 x 10(-6)6 vs. 2.6 +/- 2.8 x 10(-6); P < 0.05). No differences were observed for micronucleus induction between smokers and non-smokers. It is concluded that the protective measures taken in the coal fly ash processing plant appear to have been sufficient, since an effect of exposure to coal fly ash on parameters of genetic risk was not found any longer.

Adult↗

PubMedDate not supplied

Safety evaluation of chemical mixtures and combinations of chemical and non-chemical stressors.

Recent developments in hazard identification and risk assessment of chemical mixtures are reviewed. Empirical, descriptive approaches to study and characterize the toxicity of mixtures have dominated during the past two decades, but an increasing number of mechanistic approaches have made their entry into mixture toxicology. A series of empirical studies with simple chemical mixtures in rats is described in some detail because of the important lessons from this work. The development of regulatory guidelines for the toxicological evaluation of chemical mixtures is discussed briefly. Current issues in mixture toxicology include the adverse health effects of ambient air pollution; the application of such modern, sophisticated methodologies as genomics, bioinformatics, and physiologically based pharmacokinetic modeling; and databases for mixture toxicity. Finally, the state of the art of our knowledge on the potential adverse health effects of combined exposures to chemicals and non-chemical stressors (noise, heat/cold, microorganisms, immobilization, restraint, or transportation), research initiatives in these fields, and the development of an indicator for the cumulative health impact of multiple environmental exposures are discussed.

Dose-Response Relationship, Drug↗