Biomonitoring the intake of garlic via urinary excretion of allyl mercapturic acid.

Allium vegetables (onions, leeks, chives) and in particular garlic have been claimed to have health-promoting potential. This study was conducted to get insight into the perspectives for monitoring the intake of garlic by a biomarker approach. Chemically, the biomarker results from exposure to gamma-glutamyl-S-allyl-l-cysteine, which is first hydrolysed by gamma-glutamine-transpeptidase resulting in the formation of S-allyl-l-cysteine. The latter compound is subsequently N-acetylated by N-acetyltransferase into S-allyl-mercapturic acid (ALMA) and excreted into urine. The mercapturic acid was measured in urine using gaschromatography with mass spectrometry. Thus the intake of garlic was determined to check the compliance of garlic intake in a placebo-controlled intervention study. Results indicate that S-allyl-mercapturic acid could be detected in 15 out of 16 urine samples of garlic supplement takers, indicating good compliance. In addition, the intake of garlic was also monitored in a cross-section study of vegans versus controls in Finland, in which no differences in garlic consumption nor in ALMA output were recorded between vegans and controls. These data indicate good possibilities for further studies in the field of biomarkers to investigate the putative chemopreventive effects of garlic and garlic-containing products.

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.

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.

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.

Reducing effects of garlic constituents on DNA adduct formation in human lymphocytes in vitro.

A water extract of raw garlic (RGE) and two organosulfur compounds, diallyl sulfide and S-allylcysteine (SAC), were evaluated for their relative effectiveness in reducing benzo[a]pyrene (BaP)-DNA adduct formation in stimulated human peripheral blood lymphocytes in vitro. In replicate experiments, RGE significantly inhibited BaP-DNA adduct formation at concentrations of 0.001, 0.01, and 0.1 mg/ml. SAC also significantly decreased BaP-DNA adduct formation at concentrations of 0.01 and 0.1 mg/ml. For diallyl sulfide, no significant reduction in BaP-DNA adduct formation was found. BaP-DNA adduct formation was not associated with cell viability or proliferation of peripheral blood lymphocytes after the various treatments. No clear scavenging activity was detected for the garlic constituents. Aryl hydrocarbon hydroxylase activity was not decreased, nor was formation of sulfate and glucuronide conjugates of 3-hydroxy-BaP increased in the presence of RGE and SAC, indicating that increased glutathione S-transferase activity or a more efficient repair of BaP-DNA adducts may explain the observed effects. In addition, reactive oxygen species-induced 8-oxodeoxyguanosine in DNA was reduced in the presence of SAC. It is concluded that raw garlic and SAC may be useful in the prevention of BaP-associated tumorigenesis and that further evaluation of their preventive potential in humans at risk appears feasible.

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.

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.

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.

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.

Genotoxicity of coal fly ash, assessed in vitro in Salmonella typhimurium and human lymphocytes, and in vivo in an occupationally exposed population.

Fly ash as a product of coal combustion is known to contain various mutagenic substances, but genotoxic properties, especially of the particular (larger-size) fly ash fraction which is electrostatically precipitated (ESP) in the energy plant, have hardly been investigated. While smaller-size fly ash particles escape through the stack during powder coal combustion, the ESP fraction is collected and used for the manufacturing, for instance according to the Lytag process, of secondary products which can serve several construction purposes. Since fly ash as well as fly ash products are generally introduced into the human environment, a study of possible genotoxic effects to human DNA is indicated. Mutagenic properties of ESP fly ash, as well as of the Lytag product, were investigated by means of the Salmonella microsome assay. The capacity to cause human chromosome damage of both ESP fly ash and Lytag dust was studied in vitro by application of the sister-chromatid exchange (SCE) test using human lymphocytes. Furthermore, effects of ESP fly ash/Lytag dust on the incidence of SCE in peripheral lymphocytes in vivo were measured in an occupationally exposed, male population, using individually matched employees from a flour-processing industry as the control population. It is demonstrated that ultrasonically treated DMSO extracts of ESP fly ash are slightly mutagenic to Salmonella tester strains TA97 and TA102. Lytag dust is effective in inducing reversions in all tester strains. Furthermore, it appeared that both compounds significantly increase the SCE frequency of human lymphocytes after incubation in vitro in comparison to non-exposed cells. Also, peripheral lymphocytes of the occupationally exposed population show a considerably higher incidence of SCE than the control population. Major disturbing factors in assessing the effects of occupational exposure to fly ash/Lytag dust on lymphocyte SCE frequency appeared to be smoking behavior and alcohol consumption. It is concluded that exposure to fly ash from powder coal combustion implies a moderate genotoxic risk to man.

Stabilization and quantitative analysis of fecapentaenes in human feces, using synthetic fecapentaene-12.

Naturally occurring fecal mutagens, called fecapentaenes, are hypothesized to contribute to large bowel carcinogenesis. To understand health risks it is necessary to relate fecal mutagen concentrations to intestinal pathologies, i.e. colon cancer. However, fast and reliable methods for analysis of stool for fecapentaene levels are not available. This study presents an evaluation of stabilizing effects on synthetic fecapentaene-12 of various antioxidants, indicating tri-ethylamine to give the best results. Furthermore, it describes a fast extraction procedure for human stool and a subsequent HPLC-analysis which produces quantitative data on fecapentaene-12 concentrations within 30 min.

Butylated hydroxyanisole, butylated hydroxytoluene and tert.-butylhydroquinone are not mutagenic in the Salmonella/microsome assay using new tester strains.

The phenolic antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert.-butylhydroquinone (TBHQ) were reassessed for mutagenic activity using the recently developed Salmonella tester strains TA97, TA102 and TA104, and in addition TA100. None of the phenolic antioxidants showed mutagenic activity, either with or without metabolic activation. At doses of 100 micrograms/plate and higher all 3 phenolic antioxidants exhibited toxic effects. A modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Combinations of BHA and BHT, tested to detect possible synergistic effects, did not exert mutagenic activity.