Detection of 2-amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP)–DNA adducts in human pancreatic tissues

Recent epidemiological investigations have observed an association between the consumption of grilled or barbecued meat and an increased risk of pancreatic cancer, suggesting that dietary exposure to heterocyclic aromatic amines (HCA) may contribute to the development of this disease. 2-Amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP) is the most abundant HCA found in well-done and grilled meats. To determine whether HCA-induced DNA damage is present in the human pancreas, immunohistochemistry and computer-assisted image analysis were used to measure PhIP–DNA adducts in 54 normal pancreatic tissues (N) from persons without pancreatic cancer and in 38 normal adjacent pancreatic tissues (A) and in 39 cancer tissues (T) from 68 patients with pancreatic adenocarcinoma. PhIP–DNA adducts were detected in 53 N, 34 A and 39 T samples. Mean values (±SD) of the absorbency for PhIP staining were 0.22±0.04, 0.24±0.04, and 0.24±0.03 for N, A, and T samples, respectively (p=0.004). Using the median absorbency (0.21) of the samples from normal controls as the cut-off, 71% of A and 77% of T tissues, compared with 48% of N tissues, were distributed in the higher range (p=0.009). The odds ratio of pancreatic cancer was 3.4 (95% confidence interval 1.5–7.5, p=0.002) for individuals with a higher level of PhIP–DNA adducts. This is the first report of the detection of PhIP–DNA adducts in human pancreatic tissue samples obtained from patients with unknown exposure to HCA. Although limited by the small sample size, these preliminary results suggest that PhIP exposure may contribute to human pancreatic cancer development.     

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) level in human hair as biomarkers for dietary grilled/stir-fried meat and fish intake

Several case-control studies have reported possible associations between heterocyclic amine (HCA) intake and the risk of cancer. However, the validity of a questionnaire to assess HCA intake has hardly been examined. In particular, no biomarker which could serve as an independent measure of habitual HCA intake has been established. Therefore, the validity of a questionnaire to assess HCA intake by means of a biomarker remains to be investigated. In this study, we examined the availability of hair HCAs as a biochemical indicator of dietary intake of HCAs. Study subjects were 20 volunteers (7 men and 13 women) aged 25-57 years, either residents of Tokyo or the neighboring cities in Japan. We collected individual weighed dietary records (DR) over 28 consecutive days. Approximately 3-5 g of hair was collected twice from all subjects before and after DR at intervals of 1-3 months. The mean (S.D.) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) level of hair was 1376.0 pg/g hair (928.9) and 16.6 ng/g melanin (12.3). A steady increase in the mean PhIP level in hair from the lowest to the highest tertile of the grilled/stir-fried meat intake was observed (P = 0.009), but not in the grilled/stir-fried fish intake (P = 0.461). The PhIP level in hair was highly correlated with the grilled/stir-fried meat intake (r = 0.68) but not with the grilled/stir-fried fish intake (r = 0.28). These observations were made of hair with and without melanin adjustment. The present study indicates that the PhIP level in hair can be used as a biological indicator of dietary intake of HCAs.     

A new approach to risk estimation of food-borne carcinogens--heterocyclic amines--based on molecular information

Identification of causative agents for human cancers is the goal of our studies. We analyzed ordinary foods for mutagenicity, using the well-established Salmonella test. Heating fish and meat yielded mutagens that require metabolic activation for exhibition of mutagenicity. Structural determination revealed these mutagens to be heterocyclic amines (HCAs), their precursors in some cases being creatin(in)e, sugars and amino acids. Ten HCAs so far examined have all proved carcinogenic in mice and rats, inducing cancers in various organs such as in the mammary glands, prostate, lung, colon, skin, bladder and liver. Human exposure to HCAs is 0.1-12 microg/day, predominantly to 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). For these types of genotoxic carcinogens, DNA-adduct formation is crucially important and PhIP-DNA adducts have been detected in human tissues. However, the amounts of individual HCAs ingested by humans may not be sufficient to induce cancers by themselves and many environmental factors have also been implicated in neoplasia in man, with other considerable inter-individual variation in susceptibility, e.g., to colon carcinogenesis. This is in line with results obtained by feeding different strains of rats with HCA. Studies using lacI transgenic mice and rats have revealed that DNA adducts do not directly correlate with mutant frequencies at the organ level, or cancer incidence. However, sequencing of the Apc gene of rat colon tumors induced by PhIP revealed that it induces a signature mutation of G deletion from the GGGA sequence. This type of mutation is found in the p53 gene of 0.3% human cancers having p53-somatic mutations, and it has been calculated that 3%-10% of the p53 mutations detected in human cancers could be ascribable to PhIP. Although there remains the possibility that other carcinogens involved in human carcinogenesis cause the same signature mutation, the available data point to an important role for PhIP.     

Benzo(a)pyrene diolepoxide adducts to albumin in workers exposed to polycyclic aromatic hydrocarbons: association with specific CYP1A1, GSTM1, GSTP1 and EHPX genotypes

We investigated whether the presence of (+)-anti-benzo(a)pyrene diolepoxide adducts to serum albumin (BPDE-SA) among workers exposed to benzo(a)pyrene (BaP) and unexposed reference controls was influenced by genetic polymorphisms of cytochrome P4501A1 (CYP1A1), microsomal epoxide hydrolase (EHPX), glutathione S-transferases M1 (GSTM1) and P1 (GSTP1), all involved in BaP metabolism. Exposed workers had significantly higher levels of adducts (0.124 ± 0.02 fmol BPTmg^-1 SA, mean ± SE) and a higher proportion of detectable adducts (40.3%) than controls (0.051 ± 0.01 fmol BPT mg^-1 SA; 16.1%) (p = 0:014 and p = 0:012). Smoking increased adduct levels only in occupationally exposed workers with the GSTM1 deletion (GSTM1 null) (p = 0:034).

Smokers from the exposed group had higher adduct levels when they were CYP1A1 *1/*1 wild-type rather than heterozygous and homozygous for the variant alleles (CYP1A1 *1/*2 plus *2/*2) (p = 0:01). The dependence of BPDE-SA adduct levels and frequency on the CYP1A1 *1/*1 genotype was most pronounced in GSTM1-deficient smokers. Exposed workers with GSTM1 null/GSTP1 variant alleles had fewer detectable adducts than those with the GSTM1 null/GSTP1*A wild-type allele, supporting for the first time the recent in vitro finding that GSTP1 variants may be more effective in the detoxification of BPDE than the wild-type allele. Logistic regression analysis indicated that occupational exposure, wild-type CYP1A1*1/*1 allele and the combination of GSTM1 null genotype+EHPX genotypes associated with predicted low enzyme activity were significant predictors of BPDE-SA adducts. Though our findings should be viewed with caution because of the relatively limited size of the population analysed, the interaction between these polymorphic enzymes and BPDE-SA adducts seems to be specific for high exposure and might have an impact on the quantitative risk estimates for exposure to polycyclic aromatic hydrocarbons.     

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) level in human hair as biomarkers for dietary grilled/stir-fried meat and fish intake

Several case-control studies have reported possible associations between heterocyclic amine (HCA) intake and the risk of cancer. However, the validity of a questionnaire to assess HCA intake has hardly been examined. In particular, no biomarker which could serve as an independent measure of habitual HCA intake has been established. Therefore, the validity of a questionnaire to assess HCA intake by means of a biomarker remains to be investigated. In this study, we examined the availability of hair HCAs as a biochemical indicator of dietary intake of HCAs. Study subjects were 20 volunteers (7 men and 13 women) aged 25–57 years, either residents of Tokyo or the neighboring cities in Japan. We collected individual weighed dietary records (DR) over 28 consecutive days. Approximately 3–5 g of hair was collected twice from all subjects before and after DR at intervals of 1–3 months. The mean (S.D.) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) level of hair was 1376.0 pg/g hair (928.9) and 16.6 ng/g melanin (12.3). A steady increase in the mean PhIP level in hair from the lowest to the highest tertile of the grilled/stir-fried meat intake was observed (P = 0.009), but not in the grilled/stir-fried fish intake (P = 0.461). The PhIP level in hair was highly correlated with the grilled/stir-fried meat intake (r = 0.68) but not with the grilled/stir-fried fish intake (r = 0.28). These observations were made of hair with and without melanin adjustment. The present study indicates that the PhIP level in hair can be used as a biological indicator of dietary intake of HCAs.     

Inhibition of cell death in human mammary epithelial cells by the cooked meat-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary gland carcinogen present in the human diet in cooked meat. To examine if PhIP and its reactive metabolite N-hydroxy-PhIP inhibit apoptosis in human mammary epithelial MCF-10A cells, confluent cultures deprived of serum and growth factors were incubated for 24 h with either compound. The percentages of dead cells (mean +/- SEM, n = 3) as measured by trypan blue exclusion were 5.7 +/- 0.6, 3.4 +/- 0.3, 2.7 +/- 0.3, and 0.2 +/- 0.003%, in control, 1 microM N-hydroxy-PhIP-, 5 microM N-hydroxy-PhIP-, and 100 microM PhIP-treated dishes, respectively. The expression of Bcl-2 and Bcl-x(L) as quantitated by Western blotting was 1.2- to 1.9-fold higher in the treated groups. PhIP-DNA adducts induced by N-hydroxy-PhIP in MCF-10A cells measured by the (32)P-postlabeling assay were low (<1 x 10(7), relative adduct labeling). No adducts were detected after incubation with PhIP. Western blot analysis indicated that PhIP increased ERK2 phosphorylation concomitant with Bcl-2. The results suggest that the inhibition of cell death in mammary epithelial cells by PhIP occurs independently of PhIP-DNA adducts and may involve enhanced signaling through the MAP kinase pathways.     

Improved high-performance liquid chromatography analysis of P-postlabeled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA adducts using in-line precolumn purification

An improved HPLC-based ³²P-postlabeling assay has been developed for the analysis of DNA modified with the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Postlabeled samples are loaded onto a C₁₈ precolumn and adducted bases are retained while excess radioactivity and unmodified DNA bases are eluted directly to waste through a switching valve. The use of this HPLC in-line precolumn purification (HIPP) technique allows entire postlabeled samples to be analyzed without prior removal of inorganic phosphate and unmodified DNA bases. The method has a sample to sample precision of 15% and accuracy of 20%, at adduct levels of 2 adducts/10⁷ bases and shows a linear relationship between signal and adduction levels from 1 adduct per 10⁴ to ≈ 2±1 adducts per 10⁹ bases. Individual postlabeled DNA samples can be analyzed by HPLC in less than 1 h, allowing high throughput. The use of calf-thymus DNA (CT-DNA), highly modified with PhIP, or DNA isolated from mice chronically fed a PhIP-modified diet shows two major PhIP-DNA adduct peaks and three additional minor adduct peaks when labeled under ATP-limiting conditions. Isolation of the HPLC purified peaks and analysis by thin layer chromatography (TLC) matches the five HPLC peaks to the spots typically seen by TLC, including N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP). Variations in digestion techniques indicate a potential resistance of the PhIP-DNA adducts to the standard enzymatic digestion methods. Attempts at adduct intensification by solid phase extraction, nuclease P1 enrichment or 1-butanol extraction decreased PhIP-DNA adduct peaks and introduced a large early eluting peak. Removal of the 3′-phosphate with nuclease P1 following the kinase labeling reaction simplifies the HPLC profile to one major peak (dG-C8-PhIP monophosphate) with several minor peaks. In addition to the high resolution provided by HPLC separation of the PhIP-DNA adducts, this method can be adjusted for analysis of other DNA adducts and is readily automated for high throughput.     

Age at exposure and Apc status influence the levels of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts in mouse intestine and liver

We have previously shown that C57BL/6J-Min/+ (multiple intestinal neoplasia) mice, heterozygous for the Min mutation in the adenomatous polyposis coli gene, were more susceptible to intestinal tumorigenesis and had higher intestinal PhIP-DNA adduct levels after exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) on day 12 than on day 36 after birth [I.-L. Steffensen, H.A.J. Schut, J.E. Paulsen, A. Andreassen, J. Alexander, Intestinal tumorigenesis in multiple intestinal neoplasia mice induced by the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine: perinatal susceptibility, regional variation, and correlation with DNA adducts, Cancer Res. 61 (200l) 8689-8696]. In the present study, we have evaluated further whether this difference in susceptibility is related to adduct formation/removal, cell proliferation, apoptosis or expression of the nucleotide excision repair protein Xeroderma pigmentosum group A (XPA) in the intestines. Min/+ and +/+ (wild-type) mice were given a subcutaneous injection of 50 mg/kgbw PhIP on day 12 or 36, and the levels of PhIP-DNA adducts after 8, 12, 24 h, 3 or 7 days were quantified by use of 32P-postlabelling. In Min/+ mice, adduct levels were significantly higher after exposure on day 12 than on day 36 in the middle (1.5- to 8.5-fold) and distal (1.3- to 6.5-fold) small intestine from 8h to 3 days after administration of PhIP, but not in the colon and proximal small intestine. In the liver - a non-target organ for PhIP - adduct levels were 2.0- to 7.5-fold higher after exposure on day 12 than on day 36 from 8 to 24h after exposure. Adduct levels were generally higher in the middle (1.1- to 1.8-fold) and distal (1.1- to 2.0-fold) small intestines of Min/+ compared with +/+ mice after PhIP exposure on day 12, i.e. in the area of the intestines previously found also to have the highest number of tumors in Min/+ mice. PhIP increased cell proliferation and the number of apoptotic cells in the intestine and liver. However, the higher susceptibility to intestinal tumorigenesis in Min/+ mice exposed to PhIP at early age, or in Min/+ mice compared with +/+ mice, could not be explained by differences in cell proliferation, apoptosis or expression of the XPA repair protein.     

Detection and quantitation of N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine adducts in DNA using online column-switching liquid chromatography tandem mass spectrometry

The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed by the grilled cooking of certain foods such as meats, poultry and fish. PhIP has been shown to induce tumours in the colon, prostate and mammary glands of rats and is regarded as a potential human dietary carcinogen. PhIP is metabolically activated via cytochrome P450 mediated oxidation to an N-hydroxylamino-PhIP intermediate that is subsequently converted to an ester by N-acetyltransferases or sulfotransferases and undergoes heterolytic cleavage to produce a PhIP-nitrenium ion, which reacts with DNA to form the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) adduct. Thus far, the detection and quantification of PhIP-DNA adducts has relied to a large extent on ³²P-postlabelling methodologies. In order to expand the array of available techniques for the detection and improved quantification of PhIP-C8-dG adducts in DNA we have developed an online column-switching liquid chromatography (LC)–electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) method incorporating an isotopically [¹³C₁₀]-labelled PhIP-C8-dG internal standard for the analysis of DNA enzymatically hydrolysed to 2′-deoxynucleosides. A dose-dependent increase was observed for PhIP-C8-dG adducts when salmon testis DNA was reacted with N-acetoxy-PhIP. Analysis of DNA samples isolated from colon tissue of mice treated by oral gavage daily for 5 days with 50 mg/kg body weight of PhIP resulted in the detection of an average level of 14.8 ± 3.7 PhIP-C8-dG adducts per 10⁶ 2′-deoxynucleosides. The method required 50 μg of hydrolysed animal DNA on column and the limit of detection for PhIP-C8-dG was 2.5 fmol (1.5 PhIP-C8-dG adducts per 10⁸ 2′-deoxynucleosides). In summary, the LC–ESI-MS/MS SRM method provides for the rapid automation of the sample clean up and a reduction in matrix components that would otherwise interfere with the mass spectrometric analysis, with sufficient sensitivity and precision to analyse DNA adducts in animals exposed to PhIP.     

Carcinogen DNA adducts and the risk of colon cancer: case-control study

Colorectal cancer represents 8.5% of all tumours at the King Faisal Specialist Hospital & Research Centre. Environmental and dietary carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs) have long been suspected to play a prominent role in colon cancer aetiology. We designed a case-control study to test the hypothesis of whether or not the presence of DNA adducts can play a role in the aetiology of colon cancer. DNA adducts were measured in 24 cancerous and 20 non-cancerous tissue samples of newly diagnosed colon cancer patients by ³²P-post-labelling technique. Normal tissue from 19 hospital patients served as controls. The mean levels of adducts per 10¹⁰ nucleotides in cancerous and non-cancerous tissue were 151.75±217.27 and 114.81±186.10, respectively; however, only adducts in cancerous tissue were significantly higher than controls (32.78±57.51 per 10¹⁰ nucleotides) with p-values of 0.017. No BPDE-DNA adducts were found. No relationship was found between urinary cotinine as a marker of tobacco smoke and 1-hydroxypyrene as an indicator of an individual's internal dose of PAHs and DNA adducts. In a logistic regression model, only adducts in cancerous tissue were associated with the subsequent risk of colon cancer, with an odds ratio of 3.587 (95% confidence interval 0.833-15.448) after adjustment for age and the duration of living in the current region, but of a borderline significance (p=0.086). Although it is difficult to arrive at a definite conclusion from a small dataset, our preliminary results suggest the potential role of DNA adducts in the colon carcinogenesis process. Additional studies with larger sample sizes are needed to confirm our preliminary finding. It is also important to identify the structural characterization of these unknown DNA adducts in order to have a better understanding of whether or not environmental carcinogens play a role in the aetiology of colon cancer.     

Association between nucleotide excision repair gene polymorphisms and chromosomal damage in coke-oven workers

The associations between several genetic polymorphisms of nucleotide excision repair genes (NER) and chromosome damage level were studied among 140 coke-oven workers exposed to a high level of polyaromatic hydrocarbons (PAHs) and 66 non-exposed workers. Seven polymorphisms with functional potential in five NER genes (ERCC1, ERCC2, ERCC4, ERCC5 and ERCC6) were genotyped in the 206 study subjects. Multivariate analysis of covariance revealed that coke-oven workers with the ERCC1 19007 CC genotype had significantly higher cytokinesis-block micronucleus frequency (CBMN) (10.5±6.8‰) than those with CT (8.1±6.6‰, p=0.01) or TT (6.6±3.7‰, p=0.05) or CT+TT genotypes (7.5±6.3‰, p=0.004). The ERCC6 A3368G polymorphism was also associated with CBMN frequency among coke-oven workers. Subjects with the AA genotype have a significantly higher CBMN frequency (10.0±6.9‰) than those with AG (6.7±4.2‰, p=0.05) or AG+GG genotypes (6.6±4.1‰, p=0.02). Stratification analysis revealed the significant associations between ERCC1 C19007T and ERCC6 A3368G, and the CBMN frequencies were only found among older workers. In addition, a significant association between ERCC2 G23591A polymorphism and CBMN frequencies was also found among older coke-oven workers. The results suggest that polymorphisms of ERCC1 C19007T, ERCC6 A3368G and ERCC2 G23591A are associated with the CBMN frequencies among coke-oven workers     

Analysis of 8-hydroxydeoxyguanosine among workers exposed to diesel particulate exhaust: comparison with urinary metabolites and PAH air monitoring

Oxidative DNA damage and repair, as measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine and DNA samples were studied in association with work-related diesel exhaust exposure among garage and waste collection workers. Seasonal variations of the urinary 8-OHdG levels in pre- and two post-workshift urine samples of 29 exposed workers and 36 control persons were evaluated. The mean±SE levels of post-workshift 8-OHdG (μmol/mol crea) were 1.52±0.44 in winter and 1.61±0.33 in summer for the exposed workers, and 1.56±0.61 in winter and 1.43±0.49 in summer for the controls, respectively. No significant difference in the urinary 8-OHdG levels between exposed workers and control subjects in winter (p=0.923) and summer (p=0.350) was observed. A linear mixed model, adjusted for years of employment, age, ex/non-smoking and BMI, indicated no significant dose exposure-relationships between the urinary 8-OHdG and 15 PAH air concentrations nor between the 8-OHdG and 7 PAH monohydroxy-metabolites analyzed in the same workers. 8-OHdG was also analyzed in the mononuclear cell DNA of 19 exposed and 18 control subjects. The mean value of 8-OHdG/non-modified 2'-deoxyguanosine (8-OHdG/105 dG±SE) were 4.89±0.17 for the exposed and 4.11±0.16 for the control persons, which showed no correlation with the urinary 8-OHdG levels (r=0.01, n=28, P=0.96). The PAH exposure at workplaces was mainly composed of volatile compounds, particularly naphthalene, suggesting low exposure through the respiratory tract and a low effect of PAH in ROS induction.     

Influence of GSTM1, GSTT1, GSTP1, and EPHX gene polymorphisms on DNA adduct level and HPRT mutant frequency in coke-oven workers

To evaluate the influence of individual susceptibility factors on the level of polyaromatic (PAH) hydrocarbon DNA adducts and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants in peripheral lymphocytes, 70 coke-oven workers exposed to PAH were genotyped for four metabolic enzyme polymorphisms of potential importance in PAH metabolism. The examined genetic polymorphisms concerned glutathione S-transferases M1 (GSTM1; gene deletion; 96 workers), T1 (GSTT1; gene deletion), P1 (GSTP1; Ile→Val substitution at codon 104 or Ile→Val at codon 104 and Val→Ala at codon 113), and microsomal epoxide hydrolase (EPHX; Tyr→His substitution at codon 113 and His→Arg at codon 139). The workers were classified in a high- and low-exposure group on the basis of urinary concentration of 1-pyrenol. The GSTM1 null genotype increased the number of DNA adducts in smoking coke-oven workers with high PAH exposure. DNA adducts were affected by PAH-exposure in non-smokers and in GSTM1 null smokers and by smoking in GSTM1 null individuals. In a multiple linear regression analysis, the interaction of the GSTM1 genotype was statistically significant (p=0.04) with smoking (yes/no) and of borderline significance (p=0.06) with PAH-exposure (high/low). As smoking also increased urinary 1-pyrenol, the genotype modification seemed to concern DNA adducts due to smoking rather than occupational exposure. GSTT1 positive individuals showed an elevated level of DNA adducts in comparison with GSTT1 null subjects (p=0.04), and EPHX genotypes associated with slow hydroxylation reaction yielded a higher (p=0.05) HPRT mutant frequency than fast EPHX genotypes; these findings were, however, based on small numbers of subjects and need to be clarified in further studies. In conclusion, our findings indicate that homozygous deletion of GSTM1 results in an increased sensitivity to genotoxic PAHs in tobacco smoke, which is seen as an increase in aromatic DNA adducts in blood mononuclear cells.     

More prominent reactivity in mood than activity and sleep induced by differential light exposure due to seasonal and local differences

The aim of this study was to investigate how mood, work time, light exposure, activity, and sleep indices are affected by the differences of latitude and season in healthy volunteers. Twenty-four subjects (38.92±11.32 yrs) in Rochester, Minnesota, USA (latitude 44°1'N) and 30 subjects (47.03±16.32 yrs) in San Diego, California, USA (latitude 32°43'N) completed a 1 yr protocol measuring daily logs including daily work time and sleep diary; the Center for Epidemiologic Studies Depression Scale (CES-D); the eight-item atypical subscore of the Structured Interview Guide for the Hamilton Depression Rating Scale, Seasonal Affective Disorder version (SIGH-SAD); and actigraphic measures of light exposure and sleep for one-week in each solstice of summer and winter. Higher scores of CES-D (p=0.038) and the eight-item atypical subscore of SIGH-SAD (p=0.009), and longer indoor (p=0.001) and shorter outdoor (p=0.002) work times were observed during winter than summer only in Rochester, with no differences in San Diego. The mesor of light was decreased in both Rochester (p≤0.001) and San Diego (p=0.004) during winter compared to summer, with no differences in the mesors (24 h means) of activity and sleep. The eight-item atypical subscore of SIGH-SAD was significantly negatively correlated with the mesor of light (p=0.034). Sleep indices showed no significant differences between two locales or two seasons. A more prominent depressive mood in Rochester than San Diego during winter can be explained by decreased light exposure of healthy subjects in Rochester. Despite a significant difference of mood and light exposure between the two seasons in Rochester, there were no differences in activity or sleep. Therefore, mood might be more reactive than activity and sleep in the seasonal variation induced by differential light exposure.     

The effects of coffee on enzymes involved in metabolism of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in rats

The effects of coffee on the metabolism and genotoxicity of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were investigated. Coffee diminished the bacterial mutagenicity of PhIP in the Ames reversion assay through inhibition of cytochrome P450 1A2 (CYP1A2), a key enzyme involved in the metabolic activation of PhIP. When given as part of the diet (0, 1 or 5% w/w) to male Fischer-344 rats for 2 weeks, coffee affected the expression of hepatic enzymes involved in PhIP metabolism. Coffee increased the expression of CYP1A2 by 16-fold in the 5% coffee-treated group, and approximately half of this inductive effect was attributed to caffeine. Coffee also increased the expression of enzymes involved in the detoxication of PhIP. A 2-fold increase in expression of glutathione S-transferase alpha was observed, UDP-glucuronosyl transferase (UGTs) activities of p-nitrophenol increased 2-fold, while N(2)-and N3-glucuronidation of the genotoxic metabolite 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP) increased by 1.3-fold in the 5% coffee-treated over the control group. The amount of PhIP (0.75 mg/kg, 24 h) eliminated in urine as the N(2)-and N3-glucuronide conjugates of HONH-PhIP increased by 1.8- and 2.5-fold, respectively, in the 5% coffee-treated group over control rats, suggesting either increased rates of N-oxidation of PhIP or N-glucuronidation of HONH-PhIP. Despite the strong induction of CYP1A2, there was no increase in PhIP-DNA adduct formation in colon and pancreas while liver adducts decreased by 50% over control animals. These data suggest that the effect of coffee on inhibition of PhIP N-oxidation and ensuing DNA damage is more important in vivo than its effect on induction of PhIP N-hydroxylation.     

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PK_cs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs.     

The grapefruit flavanone naringin protects against the radiation-induced genomic instability in the mice bone marrow: a micronucleus study

To test the hypothesis that carcinogen exposure and oxidative stress are involved in pancreatic carcinogenesis in susceptible individuals, aromatic DNA adducts and 8-hydroxyguanosine (8-OH-dG) were measured by (32)P-postlabeling and HPLC-EC, respectively, in 31 pancreatic tumors and 13 normal tissues adjacent to the tumor from patients with pancreatic cancer. Normal pancreatic tissues from 24 organ donors, from six patients with non-pancreatic cancers, and from five patients with chronic pancreatitis served as controls. It was found that tissue samples from patients with pancreatic cancer had significantly higher levels of both aromatic DNA adducts and 8-OH-dG compared with control samples. The mean (+/-S.D.) levels of aromatic DNA adducts were 101.8+/-74.6, 26.9+/-26.6, and 11.2+/-6.6 per 10(9) nucleotides in adjacent tissues, tumors, and controls, respectively. The mean (+/-S.D.) levels of 8-OH-dG were 11.9+/-9.6, 10.8+/-10.6, and 6.7+/-4.6 per 10(5) nucleotides in adjacent tissues, tumors, and controls, respectively. Polymorphisms of the CYP1A1, CYP2E1, NAT1, NAT2, GSTM1, MnSOD, and hOGG1 genes were determined in these patients. The level of aromatic DNA adducts was significantly associated with polymorphism of the CYP1A1 gene. No significant correlation was found between the level of 8-OH-dG and the MnSOD, GSTM1, and hOGG1 polymorphisms. However, one novel polymorphism/mutation of the hOGG1 gene was found in a pancreatic tumor. Mutation at codon 12 of the K-ras gene was found in 25 (81%) of 31 pancreatic tumors, including three G-to-A transitions and 22 G-to-T transversions. Patients with the G-to-T mutation had a significantly higher level of aromatic DNA adducts than those with G-to-A or wild-type codon (P=0.02). On the other hand, the K-ras mutation profile was not related to the level of 8-OH-dG. Given the limitation of sample size, these preliminary data lend further support the hypothesis that carcinogen exposure and oxidative stress are involved in pancreatic carcinogenesis.     

Chromosomal aberrations in environmentally exposed population in relation to metabolic and DNA repair genes polymorphisms

The capital city of Prague is one of the most polluted localities of the Czech Republic. Therefore, the effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) adsorbed onto respirable air particles (<2.5 μm) on chromosomal aberrations was studied in a group of policemen (males, aged 22–50 years) working in the downtown area of Prague and spending daily >8 h outdoors (N = 53). Age- and sex-matched healthy volunteers spending > 90% daily time indoors were chosen as controls (N = 52). Ambient air particles (PM10, PM2.5) and c-PAHs were monitored using versatile air pollution sampler (VAPS), and personal exposure was evaluated using personal samplers during working shift. Chromosomal aberrations were analyzed by conventional cytogenetic analysis and fluorescent in situ hybridization (FISH). Urinary cotinine plasma levels of vitamins A, E and C, folate, total cholesterol, HDL, LDL cholesterols and triglycerides were also analyzed as possible effect modifiers. Genotypes CYP1A1*2A, CYP1A1*2C, GSTM1, GSTP1, GSTT1, EPHX1, NAT2, hOGG1, XRCC1, XPD, p53 BstI, p53 MspI, MTHFR677, and MS2656 were determined by PCR-based RFLP assays. The following levels of air pollution were recorded during the study period (mean from HiVol sampling): PM10 62.6 μg/m³, c-PAHs 24.7 ng/m³, B[a]P 3.50 ng/m³. The conventional cytogenetic analysis did not reveal any differences between the group of policemen exposed to the ambient air pollution and the control group. The cytogenetic analysis by FISH analysis used the whole chromosome painting probes for chromosomes #1 and #4 (Cambio, UK). It detected a significant increase in all studied endpoints in the policemen compared to controls (% AB.C. = 0.33 ± 0.25 versus 0.24 ± 0.18, p < 0.05, F_G/100 = 1.72 ± 1.57 versus 1.25 ± 1.11, p < 0.05, AB/1000 (aberrations/1000 cells) = 5.58 ± 4.62 versus 3.90 ± 3.06, p < 0.05). CYP1A1*2C (Ile/Ile), XPD 23 (Lys/Lys), and XPD 6 (CC) genotypes were associated with an increase of aberrant cells by conventional method. Factors associated with an increased level of translocations by FISH included age, smoking, B[a]P-like DNA adducts (corresponding to the exposure of c-PAHs), folate, polymorphisms of CYP1A1*2C, GSTP1, EPHX1, p53 MspI and MTHFR. Ambient air exposure to c-PAHs significantly increased FISH cytogenetic parameters in nonsmoking policemen. We may conclude that FISH indicates that the city policemen in Prague represent a group of increased genotoxic risk. This is the first study that has reported a relationship between DNA adducts (biomarker of exposure) and chromosomal aberrations by FISH (biomarker of effect).     

Clara cell protein (CC16) in serum and bronchoalveolar lavage fluid of subjects exposed to asbestos

The Clara cell protein (CC16) is a small and readily diffusible protein of 16kDa secreted by bronchiolar Clara cells in the distal airspaces. These epithelial cells are altered in several pulmonary pathological processes induced by various lung toxicants. In the search for a new biomarker of asbestos-induced lung impairment, we used a sensitive immunoassay to determine the levels of CC16 in bronchoalveolar fluid (BALF) and serum of subjects exposed to asbestos compared with a group of healthy controls. In the BALF of asbestos-exposed subjects there was an insignificant trend towards CC16 elevation compared with controls, with a (mean ±SD of 0.81 ±0.65mg l^-1 for asbestos-exposed subjects (n = 23) versus 0.39 ±0.19mg l^-1 for controls (n = 11) (p = 0.09). In serum, CC16 concentration was significantly increased among asbestos-exposed subjects, with values of 27.2 ±24.0 µg l^-1 for asbestos-exposed subjects (n = 34) versus 16.1 ±7.6 µg l^-1 for controls (n = 34) (p = 0.01). Regarding the effects of smoking, there were significant differences between generally lower CC16 levels in serum and BALF (p = 0.05 and 0.001, respectively) of smokers compared with the higher levels in non-smokers. Serum CC16 levels positively correlated with those in BALF, which is consistent with a diffusional transfer of CC16 from the bronchoalveolar space into the serum. No association, however, emerged between the levels of CC16 in serum or BALF and either the duration of asbestos exposure or the severity of the lung impairment as assessed by chest X-ray. These findings suggest that exposure to asbestos elicits early changes in the local and, importantly, also the systemic levels of CC16. This pneumoprotein therefore appears as a promising non-invasive biomarker of asbestos-induced lung injury and occupational disease in both smoking and non-smoking exposed subjects.     

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE-Hb) and serum albumin (BPDE-SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m^-3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry. BPDE-Hb adducts were detected in 16% of the population and BPDE-SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE-Hb and BPDE-SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE-Hb and BPDE-SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced the results to some degree.