Lou M. Maas

Putative susceptibility markers of coronary artery disease: association between VDR genotype, smoking, and aromatic DNA adduct levels in human right atrial tissue

Cancer and cardiovascular diseases share risk factors such as smoking, and the onset of both diseases have been suggested to have a common mechanistic basis. The binding of carcinogens to DNA (carcinogen‐DNA adducts), genetic polymorphisms in carcinogen‐detoxifying enzymes glutathione S‐transferases (GSTs), and genetic polymorphisms in the vitamin D receptor (VDR) are among the candidates for modifiers of cancer risk. We determined whether these biomarkers could be related to individual characteristics of patients suffering from cardiovascular diseases. For that purpose, DNA from the right atrial appendage of 41 patients who underwent open heart surgery was analyzed for smoking‐related DNA adducts and polymorphisms in GSTM1, GSTT1, and VDR genes. Statistical analysis was used to identify any patients characteristics associated with these molecular markers. Our results showed that heart tissue of cigarette smokers contained a variety of aromatic DNA adducts in significantly elevated levels compared to ex‐smokers (P<0.01) or nonsmokers (P<0.001). A linear relationship was observed between DNA adduct levels and daily cigarette smoking (rs=0.73; P=0.0003). Since cardiac myocytes are terminally differentiated cells that have lost their ability to divide and seemingly have limited DNA repair capacities, their levels might accumulate with time and thereby affect heart cell function or viability. Substantial interindividual differences between DNA adduct levels were observed, and persons with severe coronary artery disease (CAD), as assessed by coronary angiography, had higher DNA adduct levels than persons with no or mild CAD (P=0.04). As polymorphisms in GST genes have been shown to modulate DNA adduct levels and risk for lung cancer in smokers, we explored for the first time whether the GST polymorphisms could also explain deviating heart DNA adduct levels and CAD risk. However, no relation could be found between these covariants. In contrast, a VDR genotype, which has been associated with decreased serum levels of the active hormonal form of vitamin D and increased risk for certain cancers, seemed to be related to severity of CAD (P=0.025). Our findings support the hypothesis that smoking‐related DNA damage may be involved in the onset of cardiovascular diseases and suggest that VDR genotype may be a useful susceptibility marker of CAD.—van Schooten, F. J., Hirvonen, A., Maas, L. M., de Mol, B. A., Kleinjans, J. C. S., Bell, D. A., Durrer, J. D. Putative susceptibility markers of coronary artery disease: association between VDR genotype, smoking, and aromatic DNA adduct levels in human right atrial tissue. FASEB J. 12, 1409–1417 (1998)

32P-Postlabelling of aromatic DNA adducts in white blood cells and alveolar macrophages of smokers : saturation at high exposures

DNA adducts may serve as a molecular dosimeter of exposure to cigarette smoke-associated carcinogens such as polycyclic aromatic hydrocarbons (PAH). Target tissues for cigarette smoke-induced carcinogenesis are rarely accessible; therefore, peripheral blood cells or cells obtained by bronchoalveolar lavage (BAL) may be used as surrogate sources of exposed DNA. However, the relationship between cigarette smoke exposure and aromatic-DNA adducts in white blood cells and BAL cells is still unclear. In this study, we examined DNA adduct formation in lymphocytes and BAL cells in several populations of smoking individuals by means of 32P-postlabelling. Significant correlations between the amount of cigarettes smoked per day and the level of aromatic-DNA adducts were found in lymphocytes. In BAL cells, DNA adduct levels were associated with age (p = 0.05) and gender (p = 0.10) after adjustment for smoking behaviour. Adduct formation levelled off at higher exposure levels, suggesting less efficient adduct formation; decreases in the formation of adducts per unit of exposure were found in lymphocytes (r(s) = -0.80, p < 0.001) and BAL cells (r(s) = -0.72, p < 0.001). To assess intra-individual variation in adduct levels at constant smoking behaviour, sampling was repeated after a period of 2 and 6 months. In lymphocytes, repeated measurements with an interval of 2 months were highly correlated (r = 0.84, p = 0.009, n = 8), whereas repeated measurements with an interval of 6 months showed no correlation (r = 0.30, p = 0.27, n = 16). Repeated measurements in BAL cells showed a significant correlation after 6 months (r = 0.68, p = 0.03, n = 10). Furthermore, in a group of occupationally exposed aluminium workers, adduct levels in total white blood cells were correlated with the average concentrations of PAH in the ambient air of workers who smoked cigarettes, whereas in non-smokers, no such relationship was found. We conclude that cigarette smoking may directly or indirectly influence DNA adduct levels and saturation of DNA adduct formation may occur, leading to non-linear dose-response relationships.

Induction of DNA adducts by several polychlorinated biphenyls.

It is known that lower‐chlorinated biphenyls are metabolically activated to electrophilic quinoid species capable of binding to DNA. Also, certain metabolites are capable of redox cycling, thereby increasing oxidative stress in biological systems. In the present study, we tested mono‐, di‐, tri‐, tetra‐, penta‐, hexa‐, and heptachlorinated biphenyls for their ability to bind with DNA and to induce oxidative DNA damage. We present additional evidence that several PCB congeners form DNA adducts after metabolic activation, which can be detected by the nuclease P1‐ or butanol‐enrichment procedures of the 32P‐postlabeling technique. Butanol and nuclease P1 enrichments showed different adduct recoveries, depending on the level of chlorination of the biphenyls. Application of the nuclease P1 enrichment showed that the incubation of 2‐chloro‐; 3,4‐dichloro‐; 2,4,4′‐trichloro‐; 3,4,5‐trichloro‐; and 2,2′,5,5′‐tetrachlorobiphenyl with calf thymus DNA and liver microsomes from rats treated with phenobarbital, followed by oxidation with a peroxidase, produced five to eight different DNA adducts. For these lower‐chlorinated biphenyls, butanol enrichment generally showed a lower recovery. For some higher substituted congeners (3,3′,4,4′,5‐pentachloro‐, 2,2′,3,4,4′,5′‐hexachloro‐, 2,2′,4,4′,5,5′‐hexachloro‐, and 2,2′,3,4,4′,5,5′‐heptachlorobiphenyl), after butanol enrichment a single dominant spot was observed, which was absent in the nuclease P1 procedure. After incubation of calf thymus DNA with either higher‐ or lower‐chlorinated PCB congeners, we were not able to detect significantly increased levels of oxidative DNA damage above background levels, measured as 8‐oxo‐7,8‐dihydro‐2′deoxyguanosine. In view of the carcinogenicity of PCB mixtures in animals and the ability of PCB metabolites to bind covalently to DNA, rats were orally treated with a mixture of PCBs (Aroclor 1242). PCB‐DNA adduct levels were analyzed in PCB target organs: liver, thymus, glandular stomach, spleen, testes, seminal vesicles and prostate DNA. In vivo PCB‐DNA adducts could not be detected by either the butanol‐ or by the NP1‐enrichment procedure in rat target tissue DNA. Also, no differences in oxidative DNA damage could be observed between PCB‐treated rats and controls. These results indicate a lack of DNA reactivity of PCB mixtures in vivo. Environ. Mol. Mutagen. 36:79–86, 2000.

Evaluation of benzo(a)pyrene-induced gene mutations in male germ cells.

Polycyclic aromatic hydrocarbons (PAHs) are mutagenic in somatic cells, whereas it remains unclear whether PAHs induce mutations in male germ cells, subsequently increasing health risks in offspring. Although results from the classical specific locus test are negative or inconclusive, recent studies with environmentally exposed animals suggest that PAHs are mutagenic in sperm cells. Therefore, we studied whether benzo(a)pyrene (B[a]P) was able to induce gene mutations in testis and sperm cells of wild-type (Wt) and Xpc(-/-) mice containing the pUR288 lacZ reporter gene. Mice were exposed to B[a]P (13 mg/kg body weight, three times per week) during 1, 4, or 6 weeks and sacrificed 6 weeks after the final exposure to obtain mutations in sperm derived from B[a]P-exposed spermatogonial stem cells. The lacZ gene mutation assay was used to assess mutant frequencies in spleen, testis, and mature sperm, and (32)P-postlabeling was used for the detection of DNA adducts in testis. Successful exposure was confirmed by a dose-related higher mutant frequency in spleen of Xpc(-/-) mice as compared with Wt mice. Mutant frequencies were also increased in all ethyl nitrosourea-exposed samples, which were used as positive control. Although B[a]P-related DNA adducts were detected in testis, mutant frequencies were not increased. On the other hand, B[a]P increased mutant frequencies in sperm of Wt mice, but not in Xpc(-/-) mice, after 6 weeks exposure. Therefore, we conclude that B[a]P can induce gene mutations in spermatogonial cells of mice, but it remains to be elucidated whether these mutations can be transmitted to offspring.

Global Gene Expression Analysis Reveals Differences in Cellular Responses to Hydroxyl- and Superoxide Anion Radical-Induced Oxidative Stress in Caco-2 Cells

Reactive oxygen species-induced oxidative stress in the colon is involved in inflammatory bowel diseases and suggested to be associated with colorectal cancer risk. However, our insight in molecular responses to different oxygen radicals is still fragmentary. Therefore, we studied global gene expression by an extensive time series (0.08, 0.25, 0.5, 1, 2, 4, 8, 16, or 24 h) analyses in human colon cancer (caco-2) cells after exposure to H(2)O(2) or the superoxide anion donor menadione. Differences in gene expression were investigated by hybridization on two-color microarrays against nonexposed time-matched control cells. Next to gene expression, correlations with related phenotypic markers (8-oxodG levels and cell cycle arrest) were investigated. Gene expression analysis resulted in 1404 differentially expressed genes upon H(2)O(2) challenge and 979 genes after menadione treatment. Further analysis of gene expression data revealed how these oxidant responses can be discriminated. Time-dependent coregulated genes immediately showed a pulse-like response to H(2)O(2), while the menadione-induced expression is not restored over 24 h. Pathway analyses demonstrated that H(2)O(2) immediately influences pathways involved in the immune function, while menadione constantly regulated cell cycle-related pathways Altogether, this study offers a novel and detailed insight in the similarities and differences of the time-dependent oxidative stress responses induced by the oxidants H(2)O(2) and menadione and show that these can be discriminated regarding their modulation of particular colon carcinogenesis-related mechanisms.

Witch hazel (Hamamelis virginiana) fractions and the importance of gallate moieties--electron transfer capacities in their antitumoral properties.

Witch hazel (Hamamelis virginia) extracts are used in traditional medicine. They are particularly rich in gallate esters included in proanthocyanidins, hydrolyzable tannins (galloylated sugars), and methyl gallate. This study examines the response of human colon cancer cells to treatment with fractions obtained from a witch hazel polyphenolic extract. The results are compared with those obtained previously with homologous fractions from grape (less galloylated) and pine (nongalloylated). Witch hazel fractions were the most efficient in inhibiting cell proliferation in HT29 and HCT116 human colon cancer cell lines, which clearly shows that the more galloylated the fractions, the more effective they were at inhibiting proliferation of colon cancer cells. Witch hazel fractions were, in addition, more potent in arresting the cell cycle at the S phase and inducing apoptosis; they also induced a significant percentage of necrosis. Interestingly, the apoptosis and cell cycle arrest effects induced were proportional to their galloylation. Moreover, witch hazel fractions with a high degree of galloylation were also the most effective as scavengers of both hydroxyl and superoxide radicals and in protecting against DNA damage triggered by the hydroxyl radical system. These findings provide a better understanding of the structure-bioactivity relationships of polyphenolics, which should be of assistance in choosing an appropriate source and preparing a rational design for formulations of plant polyphenols in nutritional supplements.

B[a]P-DNA adduct formation and induction of human epithelial lung cell transformation

In this study we tested the suitability of the human epithelial lung cell line BEAS‐2B for in vitro studies of lung carcinogenesis. The human bronchial epithelial lung cell line BEAS‐2B, immortalized with an SV‐40/Ad‐12 hybrid virus construct, was treated for 24 hours with five different concentrations of the lung carcinogen benzo(a)pyrene (B[a]P) to assess the relationship between DNA adduct levels, cell cycle distribution, micronuclei formation (MN), colony forming efficiency (CFE), and anchorage independent growth (AIG).

Interactions between polycyclic aromatic hydrocarbons in binary mixtures: Effects on gene expression and DNA adduct formation in precision-cut rat liver slices

Although exposure to polycyclic aromatic hydrocarbons (PAHs) occurs mostly through mixtures, hazard and risk assessment are mostly based on the effects caused by individual compounds. The objective of the current study was to investigate whether interactions between PAHs occur, focusing on gene expression (as measured by cDNA microarrays) and DNA adduct formation. The effects of benzo[a]pyrene or dibenzo[a,h]anthracene (DB[a,h]A) alone and in binary mixtures with another PAH (DB[a,h]A, benzo[b]fluoranthene, fluoranthene or dibenzo[a,l]pyrene) were investigated using precision-cut rat liver slices. All compounds significantly modulated the expression of several genes, but overlap between genes affected by the mixture and by the individual compounds was relatively small. All mixtures showed an antagonistic response on total gene expression profiles. Moreover, at the level of individual genes, mostly antagonism was evident, with additivity and synergism observed for only a few genes. As far as DNA adduct formation is concerned, the binary mixtures generally caused antagonism. The effects in liver slices suggest a lower carcinogenic potency of PAH mixtures than estimated based on additivity of individual compounds.

Influences of DNA isolation and RNA contamination on carcinogen-DNA adduct analysis by 32P-postlabeling.

32P‐Postlabeling is a widely applied assay for the analysis of carcinogen‐DNA adducts. Optimization of most steps in this assay has been given attention, but influences of DNA isolation and DNA purity on adduct quantitation have not been investigated systematically. In this study, DNA was isolated from human lymphocytes exposed to benzo[a]pyrene (B[a]P, 10 μM) for 18 hr and from liver of rats i.p.‐treated with B[a]P (10 mg/kg body weight) using two different DNA isolation methods: a phenol‐extraction and a salting‐out procedure. Subsequently, DNA was analysed by nuclease P1 (NP1) or butanol‐enriched 32P‐postlabeling. Influences of RNA contamination were studied by labeling RNA isolated from in vitro exposed lymphocytes. In the in vitro experiment, DNA adduct levels were significantly higher using the salting‐out procedure (63.2 ± 13.7 adducts per 108 nucleotides, n = 9) as compared with the phenol‐extraction (14.3 ± 0.8). RNA was ∼4 times less efficiently labeled as compared to DNA. Nonetheless, RNA contamination of DNA samples may result in an overestimation of DNA adduct levels when butanol enrichment is used, because RNA adduct levels seemed to be substantially higher than DNA adduct levels in the same cells. DNA adduct analysis by nuclease P1 enrichment is probably less affected, since RNA adducts appeared to be NP1 sensitive. In vivo, three different adducts were found by NP1 enriched 32P‐postlabeling in the liver of B[a]P‐exposed rats. Again, DNA adduct levels were significantly higher using salting out as compared to phenol extraction for the adduct which comigrated with the BPDE‐DNA adduct standard (adduct 1) and an unknown adduct (adduct 2). However, the results were the opposite for another B[a]P‐derived DNA adduct (adduct 3). Our results suggest that differences in DNA isolation procedures as well as RNA contamination influence quantitative DNA adduct analysis by 32P‐postlabeling. Environ. Mol. Mutagen. 32: 344–350, 1998

Maternal intake of quercetin during gestation alters ex vivo benzo[a]pyrene metabolism and DNA adduct formation in adult offspring

Variation in xenobiotic metabolism cannot entirely be explained by genetic diversity in metabolic enzymes. We suggest that maternal diet during gestation can contribute to variation in metabolism by creating an in utero environment that shapes the offsprings defence against chemical carcinogens. Therefore, pregnant mice were supplemented with the natural aryl hydrocarbon receptor (AhR) agonist quercetin (1 mmol quercetin/kg feed) until delivery. Next, it was investigated whether the adult offspring at the age of 12 weeks had altered biotransformation of the environmental pollutant benzo[a]pyrene (B[a]P). In utero quercetin exposure resulted in significantly enhanced gene expression of Cyp1a1, Cyp1b1, Nqo1 and Ugt1a6 in liver of foetuses at Day 14.5 of gestation. Despite cessation of supplementation after delivery, altered gene expression persisted into adulthood, but in a tissue- and gender-dependent manner. Expression of Phase I enzymes (Cyp1a1 and Cyp1b1) was up-regulated in the liver of adult female mice in utero exposed to quercetin, whereas expression of Phase II enzymes (Gstp1, Nqo1 and Ugt1a6) was predominantly enhanced in the lung tissue of female mice. Epigenetic mechanisms may contribute to this adapted gene expression, as the repetitive elements (SINEB1) were hypomethylated in liver of female mice prenatally exposed to quercetin. Studies on ex vivo metabolism of B[a]P by lung and liver microsomes showed that the amount of B[a]P-9,10-dehydrodiol, B[a]P-7,8-dihydrodiol and 3-hydroxy-B[a]P did not change, but the amount of unmetabolised B[a]P was significantly lower after incubation with lung microsomes from offspring that received quercetin during gestation. Moreover, ex vivo B[a]P-induced DNA adduct formation was significantly lower for liver microsomes of offspring that were exposed to quercetin during gestation. These results suggest that prenatal diet leads to persistent alterations in Phase I and II enzymes of adult mice and may affect cancer risk.