iPSC-derived cortical neurons to study sporadic Alzheimer disease: A transcriptome comparison with post-mortem brain samples.

Alzheimer's disease (AD) is the most common cause of dementia, characterized by the progressive impairment of cognition and memory loss. Sporadic AD (sAD) represents approximately 95 % of the AD cases and is induced by a complex interplay between genetic and environmental factors called "Alzheimerogens". Heavy metals (e.g. copper) and pesticides (e.g. fipronil) can affect many AD-related processes, including neuroinflammation (considered as AD-inducing factor). Research would benefit from in vitro models to investigate effects of Alzheimerogens. We compared transcriptomics changes in sAD induced pluripotent stem cell (iPSC) derived cortical neurons to differentially expressed genes (DEGs) identified in post-mortem AD brain tissue. These analyses showed that many AD-related processes could be identified in the sAD iPSC-derived neurons, and furthermore, could even identify more DEGs functioning in these processes than post-mortem AD-brain tissue. Thereafter, we exposed the iPSCs to AD-inducing factors (copper(II)chloride, fipronil sulfone and an inflammatory cytokine cocktail). Cytokine exposure induced expression of immune related genes while copper-exposure affected genes involved in lipid and cholesterol metabolism, which are known AD-related processes. Fipronil-exposure did not result in significant transcriptomic changes, although prolonged exposures or higher doses may be necessary. Overall, we show that iPSC-derived cortical neurons can be beneficial in vitro models to identify Alzheimerogens and AD-related molecular mechanisms.

Human 3D multicellular microtissues: An upgraded model for the in vitro mechanistic investigation of inflammation-associated drug toxicity.

Inflammation is one of the factors that may increase the sensitivity of hepatic cells to acetaminophen (APAP) induced toxicity. To investigate the mechanisms, we exposed 3-dimensional (3D) Human Liver Microtissues, a co-culture of primary human hepatocytes (PHH) and Kupffer cells (KCs), to 0, 0.5 (low), 5 (median) and 10 mM (high dose) APAP for 24 h, with/without lipopolysaccharide (LPS). Microarray-technology was used to evaluate the transcriptome changes. In the presence of LPS, the median-dose of APAP is sufficient to inhibit the expression of respiratory chain- and antioxidant-related genes, suggesting the involvement of reactive oxygen species (ROS) and oxidative stress. Furthermore, the median- and high-dose of APAP inhibited the expression of Fc fragment receptor (FcγR)-coding genes, regardless of the presence of LPS. The toll-like receptor 4 (TLR4) expression, however, was continuously elevated after the LPS/APAP co-exposures, which may result in reduced KC-phagocytosis and unbalanced cytokine patterns. Compared to the treatment with LPS only, LPS/APAP co-exposures induced the production of interleukin (IL)-8, a pro-inflammatory cytokine, but suppressed the secretion of IL-6, a cytokine regulating hepatic regeneration, along with the increase in APAP dosages. In addition to the disrupted mitochondrial functions, the presence of LPS exacerbated APAP toxicity. These findings suggest that 3D Microtissues are a suitable model for the mechanistic exploration of inflammation-associated drug toxicity.

Benzo[a]pyrene-induced transcriptomic responses in primary hepatocytes and in vivo liver: toxicokinetics is essential for in vivo-in vitro comparisons.

The traditional 2-year cancer bioassay needs replacement by more cost-effective and predictive tests. The use of toxicogenomics in an in vitro system may provide a more high-throughput method to investigate early alterations induced by carcinogens. Recently, the differential gene expression response in wild-type and cancer-prone Xpa (-/-) p53 (+/-) primary mouse hepatocytes after exposure to benzo[a]pyrene (B[a]P) revealed downregulation of cancer-related pathways in Xpa (-/-) p53 (+/-) hepatocytes only. Here, we investigated pathway regulation upon in vivo B[a]P exposure of wild-type and Xpa (-/-) p53 (+/-) mice. In vivo transcriptomics analysis revealed a limited gene expression response in mouse livers, but with a significant induction of DNA replication and apoptotic/anti-apoptotic cellular responses in Xpa (-/-) p53 (+/-) livers only. In order to be able to make a meaningful in vivo-in vitro comparison we estimated internal in vivo B[a]P concentrations using DNA adduct levels and physiologically based kinetic modeling. Based on these results, the in vitro concentration that corresponded best with the internal in vivo dose was chosen. Comparison of in vivo and in vitro data demonstrated similarities in transcriptomics response: xenobiotic metabolism, lipid metabolism and oxidative stress. However, we were unable to detect cancer-related pathways in either wild-type or Xpa (-/-) p53 (+/-) exposed livers, which were previously found to be induced by B[a]P in Xpa (-/-) p53 (+/-) primary hepatocytes. In conclusion, we showed parallels in gene expression responses between livers and primary hepatocytes upon exposure to equivalent concentrations of B[a]P. Furthermore, we recommend considering toxicokinetics when modeling a complex in vivo endpoint with in vitro models.

Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn).

Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO-BP) were identified after 24h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO-BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO-BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO-BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.

A sandwich-cultured rat hepatocyte system with increased metabolic competence evaluated by gene expression profiling.

A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Consequently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved hepatocyte-based in vitro system was developed with special focus on metabolic competence. Therefore, a mixture of CYP450 inducers, phenobarbital, dexamethasone and beta-naphthoflavone, was added to culture medium of sandwich-cultured rat hepatocytes. The resulting modified model was evaluated by comparing its genome-wide expression profiles with liver and a standard model without the inducer mixture. Metabolic capacity for CYP450 enzymes showed that the modified model resembled more closely the in vivo situation. Gene expression results revealed large differences between in vivo and both in vitro models. The slight differences between the two sandwich models were predominantly represented by gene expression changes in CYP450s. Importantly, in the modified model, expression ratios of the phase I and the majority of phase II genes more closely resembled liver in vivo. The CYP450 enzyme activities corresponded with gene expression data. In conclusion, for toxicological applications using sandwich-cultured hepatocytes, the modified model may be preferred.

Induction of oral tolerance to oxidized low-density lipoprotein ameliorates atherosclerosis.

BACKGROUND: Oxidation of low-density lipoprotein (LDL) and the subsequent processing of oxidized LDL (oxLDL) by macrophages results in activation of specific T cells, which contributes to the development of atherosclerosis. Oral tolerance induction and the subsequent activation of regulatory T cells may be an adequate therapy for the treatment of atherosclerosis. METHODS AND RESULTS: Tolerance to oxLDL and malondialdehyde-treated LDL (MDA-LDL) was induced in LDL receptor-/- mice fed a Western-type diet by oral administration of oxLDL or MDA-LDL before the induction of atherogenesis. Oral tolerance to oxLDL resulted in a significant attenuation of the initiation (30% to 71%; P<0.05) and progression (45%; P<0.05) of atherogenesis. Tolerance to oxLDL induced a significant increase in CD4+ CD25+ Foxp3+ cells in spleen and mesenteric lymph nodes, and these cells specifically responded to oxLDL with increased transforming growth factor-beta production. Tolerance to oxLDL also increased the mRNA expression of Foxp3, CTLA-4, and CD25 in the plaque. In contrast, tolerance to MDA-LDL did not affect atherogenesis. CONCLUSIONS: OxLDL-specific T cells, present in LDL receptor-/- mice and important contributors in the immune response leading to atherosclerotic plaque, can be counteracted by oxLDL-specific CD4+ CD25+ Foxp3+ regulatory T cells activated via oral tolerance induction to oxLDL. We conclude that the induction of oral tolerance to oxLDL may be a promising strategy to modulate the immune response during atherogenesis and a new way to treat atherosclerosis.

Genome-wide differential gene expression in children exposed to air pollution in the Czech Republic.

The Teplice area in the Czech Republic is a mining district where elevated levels of air pollution including airborne carcinogens, have been demonstrated, especially during winter time. This environmental exposure can impact human health; in particular children may be more vulnerable. To study the impact of air pollution in children at the transcriptional level, peripheral blood cells were subjected to whole genome response analysis, in order to identify significantly modulated biological pathways and processes as a result of exposure. Using genome-wide oligonucleotide microarrays, we investigated differential gene expression in children from the Teplice area (n=23) and compared them with children from the rural control area of Prachatice (n=24). In an additional approach, individual gene expressions were correlated with individual peripheral blood lymphocyte micronuclei frequencies, in order to evaluate the linkage of individual gene expressions with an established biomarker of effect that is representative for increased genotoxic risk. Children from the Teplice area showed a significantly higher average micronuclei frequency than Prachatice children (p=0.023). For considerable numbers of genes, the expression differed significantly between the children from the two areas. Amongst these genes, considerable numbers of genes were observed to correlate significantly with the frequencies of micronuclei. The main biological process that appeared significantly affected overall was nucleosome assembly. This suggests an effect of air pollution on the primary structural unit of the condensed DNA. In addition, several other pathways were modulated. Based on the results of this study, we suggest that transcriptomic analysis represents a promising biomarker for environmental carcinogenesis.

Effects of polyunsaturated fatty acids on prostaglandin synthesis and cyclooxygenase-mediated DNA adduct formation by heterocyclic aromatic amines in human adenocarcinoma colon cells.

Dietary heterocyclic aromatic amines (HCA) and polyunsaturated fatty acids (PUFA) are both believed to play a role in colon carcinogenesis, and are both substrate for the enzyme cyclooxygenase (COX). In HCA-7 cells, highly expressing isoform COX-2, we investigated the effects of PUFA on prostaglandin synthesis and DNA adduct formation by the HCA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Furthermore, we studied the role of COX, COX-2 in particular, and cytochrome P4501A2 (CYP1A2) by using the enzyme inhibitors indomethacin (IM), NS-398, and phenethyl isothiocyanate (PEITC), respectively. COX-mediated formation of prostaglandin E2 (PGE2) from linoleic acid (LA) showed that HCA-7 cells can convert LA into arachidonic acid (AA). Alternatively, eicosapentaenoic acid (EPA) was found to compete with AA for COX. Strongly decreased PGE2 levels by addition of IM demonstrated involvement of COX in PUFA metabolism. Both IM and NS-398 inhibited adduct formation by HCA to nearly the same extent, indicating involvement of COX-2 rather than COX-1, while CYP1A2 activity in HCA-7 cells was demonstrated by addition of PEITC. Overall, inhibiting effects were stronger for PhIP than for IQ. HCA-DNA adduct formation was stimulated by addition of PUFA, although high PUFA concentrations partly reduced this stimulating effect. Finally, similar effects for n-3 and n-6 fatty acids suggested that adduct formation may not be the crucial mechanism behind the differential effects of PUFA on colon carcinogenesis that have been described. These results show that COX, and COX-2 in particular, can play a substantial role in HCA activation, especially in extrahepatic tissues like the colon. Furthermore, the obvious interactions between PUFA and HCA in COX-2 expressing cancer cells may be important in modulating colorectal cancer risk.

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.

Biological monitoring the exposure to polycyclic aromatic hydrocarbons of coke oven workers in relation to smoking and genetic polymorphisms for GSTM1 and GSTT1.

UNLABELLED: Occupational exposure to polycyclic aromatic hydrocarbons (PAH) increases the risk of developing lung cancer. Human exposure is often demonstrated by increased internal levels of PAH metabolites and of markers for early biological effects, like DNA adducts and cytogenetic aberrations. OBJECTIVE: This study aimed to assess whether the current exposure to PAH of coke oven workers in a Dutch plant induced biological effects, and to determine if these effects are influenced by tobacco smoking and by genetic polymorphisms for the glutathione S-transferase genes GSTM1 and GSTT1. METHODS: Urinary 1-hydroxypyrene (1-OHpyr) levels were used to monitor the internal dose, while the internal effective dose was assessed by monitoring PAH-DNA adducts, DNA strand breaks (Comet assay), sister-chromatid exchanges (SCE) and cells with a high frequency of SCE (HFC) in lymphocytes together with micronuclei (MN) in exfoliated urothelial cells. RESULTS: Occupational exposure to PAH resulted in statistically significant increased 1-OHpyr levels (P<0.001), but it did not cause a significant induction of SCE, HFC, MN, DNA strand breaks or DNA adducts. Smoking caused a significant increase of 1-OHpyr (P<0.05), SCE (P<0.001), HFC (P<0.001) and DNA adducts (P<0.05), but not of MN or DNA strand breaks. Following correction for the smoking-related effects, no occupational induction of the effect biomarkers could be discerned. Multi-variate analysis did not show a significant influence of GSTM1 and GSTT1 polymorphisms on any biomarker. Also no significant interactions were observed between the various biomarkers. CONCLUSION: This study shows that in the examined plant, the occupational exposure to PAH does not result in measurable early biological effects

A multi-biomarker approach to study the effects of smoking on oxidative DNA damage and repair and antioxidative defense mechanisms.

We investigated the effects of smoking-induced oxidative stress in healthy volunteers (21 smokers versus 24 non-smokers) by quantifying various markers of oxidative DNA damage and repair, and antioxidative defense mechanisms. Lymphocytic 7-hydroxy-8-oxo-2'-deoxyguanosine (8-oxo-dG) levels measured by high performance liquid chromatography with electrochemical detection, were significantly lower in smokers as compared with non-smokers (38.6 +/- 5.2 versus 50.9 +/- 4.6/10(6) dG, P = 0.05). The levels of oxidized pyrimidine bases in lymphocytes of smokers quantified by the endonuclease III-modified comet assay were non-significantly lower than those of non-smokers (% DNA in tail: 13 +/- 3 versus 14 +/- 2; tail length: 69 +/- 13 versus 96 +/- 10; tail moment: 6416 +/- 1220 versus 7545 +/- 1234). Urinary excretion levels of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) assessed by enzyme-linked immunosorbent assay did not differ significantly between smokers and non-smokers (197 +/- 31 versus 240 +/- 33 ng/body mass index, P = 0.3). Overall DNA repair activity expressed as unscheduled DNA synthesis in blood leukocytes, was not significantly different between smokers and non-smokers (2.9 +/- 0.3 versus 3.3 +/- 0.3, P = 0.4). Plasma antioxidative capacity measured by the Trolox equivalent antioxidant capacity assay was slightly higher in smokers as compared with non-smokers (440 +/- 16 versus 400 +/- 15 microM Trolox equivalent, P = 0.09), and it was significantly related to lymphocytic 8-oxo-dG levels (r = 0.4, P = 0.001). Genotyping of human 8-OH-dG glycosylase/apurinic lyase and glutathione S-transferase M1 showed that a polymorphism in either or both of the two genes does not affect any of the quantified biomarkers. We conclude that oxidative stress imposed by cigarette smoking has a low impact upon certain pathways involved in DNA damage and the antioxidative defense system.

In vitro effects of coal fly ashes: hydroxyl radical generation, iron release, and DNA damage and toxicity in rat lung epithelial cells.

Oxygen radical generation due to surface radicals, inflammation, and iron release has been suggested as the mechanism of adverse effects of quartz, such as emphysema, fibrosis, and carcinogenic effects. Therefore, we measured iron release, acellular generation of hydroxyl radicals, and oxidative DNA damage and cytotoxicity in rat lung epithelial (RLE) cells by different coal fly ashes (CFA) that contain both quartz and iron. Seven samples of CFA with different particle size and quartz content (up to 14.1%) were tested along with silica (alpha-quartz), ground coal, and coal mine dust (respirable) as positive control particles, and fine TiO(2) (anatase) as a negative control. Five test samples were pulverized fuel ashes (PFA), two samples were coal gasification (SCG) ashes (quartz content <0.1%), and one sample was a ground coal. No marked differences between SCG and PFA fly ashes were observed, and toxicity did not correlate with physicochemical characteristics or effect parameters. Stable surface radicals were only detected in the reference particles silica and coal mine dust, but not in CFA. On the other hand, hydroxyl radical generation by all fly ashes was observed in the presence of hydrogen peroxide, which was positively correlated with iron mobilization and inhibited by deferoxamine, but not correlated with iron or quartz content. Also a relationship between acellular hydroxyl radical generation and oxidative DNA damage in RLE cells by CFA was observed. Differences in hydroxyl radical generation and oxidative damage by the CFA were not related to iron and quartz content, but the respirable ashes (MAT023, 38, and 41) showed a very extensive level of hydroxyl radical generation in comparison to nonrespirable fly ashes and respirable references. This radical generation was clearly related to the iron mobilization from these particles. In conclusion, the mechanisms by which CFA and the positive references (silica, coal mine dust) affect rat lung epithelial cells seem to be different, and the data suggest that quartz in CFA does not act the same as quartz in silica or coal mine dust. On the other hand, the results indicate an important role for size and iron release in generation and subsequent effects of reactive oxygen species caused by CFA.

Oxidative DNA damage and cytogenetic effects in flight engineers exposed to cosmic radiation.

This study set out to analyze biomarkers for genotoxic events, e.g., oxidative DNA damage, chromosomal damage and hprt mutations, among flight personnel, who are known to be occupationally exposed to ionizing radiation of cosmic origin. Twenty-three flight engineers were recruited while ground personnel served as a matched control group. Cumulative radiation doses during flight were calculated on the basis of subjects' flight records assuming an exposure rate of 6 microSv per hour of flight. Oxidative DNA damage in peripheral lymphocytes from flight engineers appeared significantly increased in comparison with controls and was associated with cumulative exposure to cosmic radiation. Frequencies of peripheral lymphocyte chromosome aberrations, micronuclei and hprt mutations appeared also to be increased in flight engineers, but not significantly. It was also observed that DNA damage was higher in flight engineers with a relatively shorter flight history in comparison with flight engineers with higher cumulative exposures to radiation, suggesting adaptation to DNA damage caused by ionizing radiation. DNA repair activities measured as unscheduled DNA synthesis were clearly increased in the higher-exposed subgroup of flight engineers, and appeared significantly correlated with cumulative radiation dose, as well as inversely with oxidative DNA damage. The implications for cancer risk assessment in relation to exposure to cosmic radiation are discussed.

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.