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Sabine Rohrmann, Jakob Linseisen
2.2. Food-chemical-specific biomarkers
2.2.1. Heterocyclic aromatic aminesSabine Rohrmann and Jakob Linseisen
Division of Clinical Epidemiology, German Cancer Research Center, Heidelberg, Germany
Biomarkers of heterocyclic aromatic amines
Heterocyclic aromatic amines (HCAs) are compounds formed in meat and fish (from creatinine, sugar, amino acids) at temperatures exceeding 130°C [1,2]. The most common HCAs in the human diet are 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8--dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quin-oxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) [3]. The highest amounts have been found in foods cooked at high temperatures by methods such as barbecuing, grilling and frying [3]. Heterocyclic aromatic amines bind covalently to DNA after metabolic activation [4]. Different HCAs have shown carcinogenic effects in animal studies [1,4] but, for humans, the results of epidemiological studies are less clear. Some case–control studies reported positive associations between the intake of HCAs, assessed using dietary questionnaires, and the risk of cancer at different sites [5–11], whereas others did not [12–15]. For exposure assessment in humans, questionnaires are used in combination with data on the HCA content of foods to estimate dietary HCA intake in epidemiological studies. However, this method may give inaccurate results because of differences in the way cooking methods and the preferred degree of browning of meat and fish are assessed, and the use of HCA data from different meats. Thus, the actual exposure might deviate from the estimated intake based on dietary questionnaires [16]. Biomarkers of HCA exposure and metabolism might be better indicators of HCA intake. Several types of biomarkers have been evaluated so far [17], e.g. urinary excretion of HCA and metabolites; DNA and protein adducts; however, they reflect different time frames of HCA exposure and are, therefore, not equally appropriate for all research questions.
Long-term biomarkers such as DNA adducts and blood albumin and haemoglobin adducts reflect intake over weeks or months [17,18]; they can also be considered as bio-markers of internal exposure since various metabolic steps precede the formation of these adducts. As a further possibility for long-term biomarkers, measurement of the HCA content in hair has been suggested.
Validation of biomarkers of heterocyclic aromatic amines
Animal studies
Several studies have examined the kinetics of DNA adduct formation and dose–response relationships of HCA exposure with DNA adduct levels. We report on studies that examined these relationships using accelerator mass spectrometry
49 State of validation of biomarkers: Food-chemical-specific biomarkers – Heterocyclic aromatic amines
(AMS), which measures isotope ratios with high selectivity and sensitivity and is, thus, suitable for studies that examine the effects of the low doses of HCA that are typical of the human diet.
Dose-dependent associations between the administered amount of MeIQx and PhIP and the formation of DNA adducts have been observed in liver [19–21], colon [22], breast and prostate [23] of rats, even at low doses. In rats, PhIP- and MeIQx-albumin and -haemoglobin adducts are also formed in a dose-dependent manner [22,24]. So far, no results have been published on whether blood protein adduct levels of MeIQx or PhIP correlate with DNA adduct levels in organs such as colon or liver. However, based on the observations that PhIP and MeIQx generally form greater levels of both blood protein and colon DNA adducts in humans relative to rodents [24,25], it is questionable whether animal models are a good model for the human response to HCA exposure.
A few studies have examined whether dietary compounds can modify DNA adduct formation in different organs of rodent models. Feeding of phenethylisothiocyanate and chlorophyllin were associated with decreased PhIP DNA adduct levels in liver, colon, prostate and blood in rats [26]. However, rats fed with additional genistein and lycopene had increased PhIP DNA adduct levels in liver, colon, prostate and blood [26]. Dietary indole-3-carbinol has been shown to inhibit PhIP- and IQ-DNA adduct formation in mammary gland in mice [27] and dietary ˆ-3 fatty acids inhibited PhIP DNA adduct formation in spleen in mice [28].
Human studies
In humans, urinary HCA excretion, at present the type of biomarker of HCA exposure most studied, only reflects very recent exposure. HCAs (and mutagenic activity) in the urine were usually detectable only up to 12 hours after consumption of a meal high in HCAs [29,30]. Thus, urinary HCAs may not be the ideal measures of ‘usual’ intake in etiological studies, especially if there is substantial day-to-day variability in HCA excretion due to differing consumption of foods high or low in HCAs. Furthermore, urinary free or total (acid-hydrolysed urine samples that include free HCAs, and N2-glucuronide and sulphate metabolites) HCAs correlated modestly with
the amount of HCA consumed in controlled human feeding trials in which volunteers consumed standard cooked meat meals [30–34], but there was large inter-individual variation in the excretion of HCA and HCA metabolites. Nevertheless, with a large sample size, they could still be useful for validating the intake of HCAs as estimated by questionnaires [18]. Moreover, determination of urinary HCA metabolites makes it possible to take into account the role of different phase I and phase II biotransforming enzymes (activation or detoxification). This could be useful for inter-vention and chemopreinter-vention studies aiming at reducing biologically active HCA species in order to prevent DNA damage and cancer in target tissues. Encouraging results have been reported in a small study, in which the levels of urinary N-hydroxy--PhIP-N2-glucuronide were negatively correlated to colon DNA adduct levels [35].
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Sabine Rohrmann, Jakob Linseisen
MeIQx-DNA adducts have been detected in various human tissues such as rectum, sigmoid colon, and kidney at levels of ~1 per 109 nucleotides using 32P-postlabelling [36]. Using gas chromatography/mass spectrometry, PhIP-DNA
adducts have been detected in human colon tissue at levels of ~1 per 109nucleotides
[37]. Blood protein adducts and DNA adducts from various tissues have been analysed by means of AMS in a few controlled human feeding studies in which one, two or a range of HCA doses were administered singly. These studies have shown that both PhIP [22,26] and MeIQx [20,38] DNA adducts were formed in a dose-dependent manner in human colon.
Blood albumin and haemoglobin are easily obtainable and have a relatively long half-life. Albumin adducts appear to be more sensitive marker of exposure to MeIQx or PhIP than are haemoglobin adducts: higher levels of adduct formation per dose of MeIQx and PhIP, respectively, were noted for albumin than for haemoglobin after oral administration to humans [25]. MeIQx-albumin and–haemoglobin adducts were formed in a dose-dependent manner [24]. Interestingly, it was shown in an un-controlled trial including 35 volunteers with different dietary habits that levels of PhIP-serum albumin adducts (measured by means of liquid chromatography-tandem mass spectrometry) were significantly higher in meat consumers than in vegetarians. The globin adduct pattern was quantitatively lower but paralleled that of serum albumin [39]. These adduct patterns represent, in contrast to the previously mentioned feeding studies, the integral value of repeated exposure over the lifespan of the blood proteins.
DNA adducts of HCAs have been observed in human tumour tissue [37,38]. In controlled feeding studies in colon cancer patients, PhIP [40] but not MeIQx [38] adducts have been observed in higher quantities in tumour than in normal tissue, perhaps reflecting differences in metabolic activation, detoxification or DNA repair capacity between the normal and tumour tissue in the former case. However, no epidemiological studies have yet been conducted that have examined the association between HCA exposure assessed by means of any biomarker and the risk of cancer.
The levels of HCA in hair [41,42] probably reflect long-term exposure, and hair is easy to sample and store even in large-scale epidemiological studies. However, validation of this suggested biomarker of exposure in humans is still lacking [17].
Conclusions
Although it is reasonable to expect that the limitations in sensitivity and speci-ficity of existing analytical methods can be removed, it is doubtful whether the required amount of material and the time and costs of the analysis could be decreased sufficiently to allow analysis of biological samples in large-scale epidemiological studies. As long as these problems are not solved, epidemiological studies on the role of HCA intake and cancer risk must rely on HCA exposure estimates based on questionnaires.
51 State of validation of biomarkers: Food-chemical-specific biomarkers – Heterocyclic aromatic amines
References
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3. Skog KI, Johansson MA, Jagerstad MI. Carcinogenic heterocyclic amines in model systems and cooked foods: a review on formation, occurrence and intake. Food Chem Toxicol 1998;36:879–96.
4. Adamson RH, Thorgeirsson UP, Snyderwine EG, Thorgeirsson SS, Reeves J, Dalgard DW, et al. Carcinogenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in nonhuman primates: induction of tumors in three macaques. Jpn J Cancer Res 1990;81:10–4.
5. Sinha R, Kulldorff M, Swanson CA, Curtin J, Brownson RC, Alavanja MC. Dietary heterocyclic amines and the risk of lung cancer among Missouri women. Cancer Res 2000;60:3753–6. 6. Sinha R, Gustafson DR, Kulldorff M, Wen WQ, Cerhan JR, Zheng W.
2-amino-1-methyl-6--phenylimidazo[4,5-b]pyridine, a carcinogen in high-temperature-cooked meat, and breast cancer risk. J Natl Cancer Inst 2000;92:1352–4.
7. Sinha R, Kulldorff M, Chow WH, Denobile J, Rothman N. Dietary intake of heterocyclic amines, meat-derived mutagenic activity, and risk of colorectal adenomas. Cancer Epidemiol Biomarkers Prev 2001;10:559–62.
8. De Stefani E, Ronco A, Mendilaharsu M, Deneo-Pellegrini H. Case-control study on the role of heterocyclic amines in the etiology of upper aerodigestive cancers in Uruguay. Nutr Cancer 1998;32:43–8.
9. De Stefani E, Boffetta P, Mendilaharsu M, Carzoglio J, Deneo-Pellegrini H. Dietary nitrosamines, heterocyclic amines, and risk of gastric cancer: a case-control study in Uruguay. Nutr Cancer 1998;30:158–62.
10. De Stefani E, Deneo-Pellegrini H, Mendilaharsu M, Ronco A. Meat intake, heterocyclic amines and risk of colorectal cancer: a case-control study in Uruguay. Int J Oncol 1997;10:573–80. 11. De Stefani E, Ronco A, Mendilaharsu M, Guidobono M, Deneo-Pellegrini H. Meat intake,
heterocyclic amines, and risk of breast cancer: a case-control study in Uruguay. Cancer Epidemiol Biomarkers Prev 1997;6:573–81.
12. Delfino RJ, Sinha R, Smith C, West J, White E, Lin HJ, et al. Breast cancer, heterocyclic aromatic amines from meat and N-acetyltransferase 2 genotype. Carcinogenesis 2000;21:607–15. 13. Norrish AE, Ferguson LR, Knize MG, Felton JS, Sharpe SJ, Jackson RT. Heterocyclic amine
content of cooked meat and risk of prostate cancer. J Natl Cancer Inst 1999;91:2038–44. 14. Augustsson K, Skog K, Jagerstad M, Dickman PW, Steineck G. Dietary heterocyclic amines and
cancer of the colon, rectum, bladder, and kidney: a population-based study. Lancet 1999;353:703–7.
15. Lyon JL, Mahoney AW. Fried foods and the risk of colon cancer. Am J Epidemiol 1988;128:1000–6.
16. Skog K. Problems associated with the determination of heterocyclic amines in cooked foods and human exposure. Food Chemical Toxicol 2002;40:1197–203.
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17. Alexander J, Reistad R, Hegstad S, Frandsen H, Ingebrigtsen K, Paulsen JE, et al. Biomarkers of exposure to heterocyclic amines: approaches to improve the exposure assessment. Food Chem Toxicol 2002;40:1131–7.
18. Sinha R. An epidemiologic approach to studying heterocyclic amines. Mutat Res 2002;506–507:197–204.
19. Frantz CE, Bangerter C, Fultz E, Mayer KM, Vogel JS, Turteltaub KW. Dose-response studies of MeIQx in rat liver and liver DNA at low doses. Carcinogenesis 1995;16:367–73. 20. Turteltaub KW, Mauthe RJ, Dingley KH, Vogel JS, Frantz CE, Garner RC, et al.
MeIQx-DNA adduct formation in rodent and human tissues at low doses. Mutat Res 1997;376:243–52.
21. Dingley KH, Roberts ML, Velsko CA, Turteltaub KW. Attomole detection of 3H in biological samples using accelerator mass spectrometry: application in low-dose, dual-isotope tracer studies in conjunction with 14C accelerator mass spectrometry. Chem Res Toxicol 1998;11:1217–22.
22. Garner RC, Lightfoot TJ, Cupid BC, Russell D, Coxhead JM, Kutschera W, et al. Comparative biotransformation studies of MeIQx and PhIP in animal models and humans. Cancer Lett 1999;143:161–5.
23. Dingley K, Curtis K, Turteltaub KW. Distribution and DNA adduct formation of 2-ami-no-1-methyl-6-phenylimidazo[4,5-b]pyridine in F344 rats: a comparison of males and females. Am Assoc Cancer Res 1998;39:635.
24. Dingley KH, Freeman SP, Nelson DO, Garner RC, Turteltaub KW. Covalent binding of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline to albumin and hemoglobin at environ-mentally relevant doses. Comparison of human subjects and F344 rats. Drug Metab Dispos 1998;26:825–8.
25. Turteltaub KW, Dingley KH, Curtis KD, Malfatti MA, Turesky RJ, Colin Garner R, et al. Macromolecular adduct formation and metabolism of heterocyclic amines in humans and rodents at low doses. Cancer Lett 1999;143:149–55.
26. Dingley KH, Ubick EA, Chiarappa-Zucca ML, et al. Effect of dietary constituents with chemopreventive potential on adduct formation of a low dose of the heterocyclic amines PhIP and IQ and phase II hepatic enzymes. Nutr Cancer 2003;46:212–21.
27. He Y-H, Schut HAJ. Inhibition of DNA adduct formation of 2-amino-1-methyl-6--phenylimidazo[4,5-b]pyridine and 2-amino-3-methylimidazo[4,5-f]quinoline by dietary indole-3-carbinol in female rats. J Biochem Mol Toxicol 1999;13:239–47.
28. Josyula S, Schut HAJ. Dietary omega-3 fatty acids as potential inhibitors of carcinogenesis: effect on DNA adduct formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mice and rats. Food ChemToxicol 1999;37:287–96.
29. Murray S, Gooderham NJ, Boobis AR, Davies DS. Detection and measurement of MeIQx in human urine after ingestion of a cooked meat meal. Carcinogenesis 1989;10:763–5.
30. Strickland PT, Qian Z, Friesen M, Rothman N, Sinha R. Measurement of 2-amino-1-methyl-6--phenylimidazo[4,5-b]pyridine (PhIP) in acid-hydrolyzed urine by high performance liquid chromatography with fluorescence detection. Biomarkers 2001;6:313–25.
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31. Lynch AM, Knize MG, Boobis AR, Gooderham NJ, Davies DS, Murray S. Intra- and inter-individual variability in systemic exposure in humans to 2-amino-3,8-dimethylimi-dazo[4,5-f]quinoxaline and 2-amino-1-methyl- 6-phenylimidazo[4,5-b]pyridine, carcinogens present in cooked beef. Cancer Res 1992;52:6216–23.
32. Stillwell WG, Kidd LC, Wishnok JS, Tannenbaum SR, Sinha R. Urinary excretion of unmetabolized and phase II conjugates of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in humans: relationship to cytochrome P4501A2 and N-acetyltransferase activity. Cancer Res 1997;57:3457–64.
33. Stillwell WG, Turesky RJ, Sinha R, Skipper PL, Tannenbaum SR. Biomonitoring of heterocyclic aromatic amine metabolites in human urine. Cancer Lett 1999;143:145–8.
34. Stillwell WG, Sinha R, Tannenbaum SR. Excretion of the N(2)-glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine in urine and its relationship to CYP1A2 and NAT2 activity levels in humans. Carcinogenesis 2002;23:831–8.
35. Malfatti MA, Dingley KH, Nowell-Kadlubar S, Ubick EA, Mulakken N, Nelson D, et al. The urinary metabolite profile of the dietary carcinogen 2-amino-1-methyl-6--phenylimidazo[4,5-b]pyridine is predictive of colon DNA adducts after a low-dose exposure in humans. Cancer Res 2006;66:10541–7.
36. Totsuka Y, Fukutome K, Takahashi M, Takahashi S, Tada A, Sugimura T, et al. Presence of N2-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-C8-MeIQx) in human tissues. Carcinogenesis 1996;17:1029–34.
37. Friesen MD, Kaderlik K, Lin D, Garren L, Bartsch H, Lang NP, et al. Analysis of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in rat and human tissues by alkaline hydrolysis and gas chromatography/electron capture mass spectrometry: validation by comparison with 32P-postlabelling. Chem Res Toxicol 1994;7:733–9.
38. Mauthe RJ, Dingley KH, Leveson SH, Freeman SP, Turesky RJ, Garner RC, et al. Comparison of DNA-adduct and tissue-available dose levels of MeIQx in human and rodent colon following administration of a very low dose. Int J Cancer 1999;80:539–45.
39. Magagnotti C, Orsi F, Bagnati R, Celli N, Rotilio D, Fanelli R, et al. Effect of diet on serum albumin and hemoglobin adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in humans. Int J Cancer 2000;88:1–6.
40. Dingley KH, Curtis KD, Nowell S, Felton JS, Lang NP, Turteltaub KW. DNA and protein adduct formation in the colon and blood of humans after exposure to a dietary-relevant dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Cancer Epidemiol Biomarkers Prev 1999;8:507–12.
41. Reistad R, Nyholm SH, Haug LS, Becher G, Alexander J. 2-Amino-1-methyl--6-phenylimidazo[4,5-b]pyridine (PhIP) in human hair as biomarker for dietary exposure. Biomarkers 1999;4:263–71.
42. Hegstad S, Reistad R, Haug LS, Alexander J. Eumelanin is a major determinant for 2-amino--1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) incorporation into hair of mice. Pharmacol Toxicol 2002;90:333–7.
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David H. Phillips, Albrecht Seidel, Stan Venitt
2.2.2. Polycyclic aromatic hydrocarbons
David H. Phillips1, Albrecht Seidel2and Stan Venitt1 1 Institute of Cancer Research, London, UK
2 Biochemical Institute of Environmental Carcinogens Prof. Dr Gernot Grimmer-Foundation, Grosshansdorf, Germany
Because polycyclic aromatic hydrocarbons (PAHs) are widespread in the environment, human exposure can occur from a variety of sources, including food. As discussed in the earlier ECNIS review [1], diet contributes at least 70% of PAH exposure for non-occupationally exposed non-smokers. PAHs are present in food as a result of pyrolysis during cooking (especially barbecuing), or smoke curing (e.g. of fish and meat), but the major contribution to dietary intake is usually from more frequently consumed cereals and vegetables, whose contamination with PAHs arises primarily from atmospheric deposition of the compounds. Recently, occurrence data of PAHs in food from 11 European countries were collected in a report from the DG Health and Consumer Protection [2].
Several individual PAHs are classified by IARC as Group 2A (probably carcinogenic to humans) or 2B (possibly carcinogenic to humans), although benzo[a]pyrene (B[a]P) was recently elevated to Group 1 (carcinogenic to humans) [3].
Urinary biomarkers
The measurement of 1-hydroxypyrene (or its glucuronide conjugate) in urine has been widely used for biomonitoring exposure to PAHs [4]. Although pyrene is not car-cinogenic, it is a major component of many PAH mixtures. Consumption of PAH-rich food has been shown to result in elevated levels of urinary 1-hydroxypyrene [4]. However, it is a relatively short-lived biomarker of exposure (less than 3 days). In 2005 the German Federal Environmental Agency [5] set a reference value for 1-hydroxypyrene of 0.5 µg/l or 0.3 µg/g creatinine for the general nonsmoking population (exposed to PAHs through diet and ambient air). This was based on environmental survey data [6]. More recent data indicate that 1,6- and 1,8-dihydroxypyrenes are potential biomarkers for the general population (with low PAH exposure levels) as these two metabolites account for about 50% of the total excreted pyrene metabolites [7,8]. Larger, more carcinogenic, PAHs and their metabolites are excreted predominantly in faeces, and are present at only low levels in urine, and this has hindered the development of assays sensitive enough for human biomonitoring. However, some recent studies have suggested that urinary biomonitoring could be improved by the determination of metabolites of another small PAH, namely phenanthrene [4]. An increasing amount of data indicate that several classes of phenanthrene metabolites can serve as valuable biomarkers for PAH exposure, including the isomeric 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrenes [9], the dihydrodiols [9,10] and phenanthrene tetraol [7,11]. The latter two are of particular interest because they reflect the metabolic activation pathways of more carcinogenic PAHs [11].
55 State of validation of biomarkers: Food-chemical-specific biomarkers – Polycyclic aromatic hydrocarbons
DNA adducts
It has been shown in mice for a series of PAHs that the relationship between ip administered dose, tumour formation and DNA adduct formation, assayed by 32P-postlabelling, is linear [12]. In a study of DNA adduct formation in target
and surrogate tissues, rats were treated with B[a]P by intratracheal, dermal or oral admin-istration. Correlations were found between B[a]P-DNA adducts, assayed by P1-enriched
32P-postlabelling, in white blood cells and lung, skin or stomach, respectively [13]. Urinary
excretion of 3-hydroxy-B[a]P, was correlated with DNA adduct levels at the site of administration of of B[a]P [13].
The principal methods for detecting carcinogen-DNA adducts in human tissues are postlabelling analysis, immunochemical methods, fluorescence spectroscopy and mass spectrometry [1]. Analysis of DNA adducts in white blood cells in humans has been used to monitor dietary exposure to PAHs, although attributing the exposure detected by these methods solely to diet may not be justified, since formation of DNA adducts in blood will be a reflection of the overall PAH body burden, arising from uptake by inhalation and percutaneous absorption, as well as by ingestion (the same is true for urinary excretion of metabolites). However, in some studies of occupational exposure to PAHs, dietary sources of exposure appear to have exerted a stronger influence [14,15].
Only a few studies have specifically monitored dietary exposure to PAHs by mea-suring DNA adducts [16]. In a study in which adducts and urinary metabolites were both measured, 10 volunteers fed barbecued beef for 5 days exhibited elevated 1-hydroxy-pyrene glucuronide levels (10- to 80-fold) during the feeding period, but their levels returned to baseline levels 24–72 hours after the feeding period [17]. Only 4 of the subjects had markedly elevated levels of PAH-DNA adducts (measured by the enzyme-linked immunosorbent assay, ELISA) in their white blood cells. Nevertheless, there was a significant correlation between the two biomarkers.
Detection of PAH-DNA adducts in biopsy specimens has been explored by semi-quantitative immunohistochemistry. An example of a probable diet-related exposure is the detection of PAH-DNA adducts in oesophageal biopsies from residents of Linxian province, China, an area noted for its high incidence of oesophageal cancer and high levels of PAHs in food [18].
Conclusions
In conclusion, it is evident that measurement of urinary excretion of metabolites (of pyrene and, potentially, of phenanthrene) and of PAH-DNA adducts in white blood cells can be valid biomarkers of dietary exposure to PAHs, provided other potential sources of exposure (e.g. from smoking, occupation and atmospheric pollution) are taken into consideration.
Prospects for large-scale prospective studies involving mass screening of DNA adducts are restricted at present because of the lack of high-throughput techniques for DNA
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David H. Phillips, Albrecht Seidel, Stan Venitt
adduct analysis. Currently application has been limited to nested case–control studies within prospective cohorts, involving the analysis of a several hundred samples (rather than tens of thousands). Examples of such studies are mentioned in Section 2.1.1. Any move towards the routine screening of cohorts for DNA adducts will require development of DNA adduct assays to a state of considerably higher throughput than has been achieved hitherto.
References
1. Farmer PB, Emeny JM, editors. Biomarkers of carcinogen exposure and early effects. ¸ódê, Poland: ECNIS Publications, Nofer Institute of Occupational Medicine; 2006.
2. Experts participating in SCOOP Task 3.2.12. Collection of occurrence data on polycyclic aromatic hydrocarbons in food. Brussels, Belgium: European Commission, DG Health and Consumer Protection; 2004. http://ec.europa.eu/food/food/chemicalsafety/contaminants/scoop_3-2--12_final_report_pah_en.pdf.
3. IARC. Some non-heterocyclic polycyclic aromatic hydrocarbons and some related exposures. IARC Monogr Eval Carcinog Risks Hum 92. In press 2006.
4. Seidel A. Biomonitoring of polycyclic aromatic hydrocarbons — human exposure. In: Luch A, editor. The carcinogenic effects of polycyclic aromatic hydrocarbons. Singapore: Imperial College Press; 2005. p. 97–136.
5. German Federal Environmental Agency (Umweltbundesamt, UBA). 1-Hydroxypyren im Urin als Indikator einer inneren Belastung mit polyzyklischen aromatischen Kohlenwasserstoffen (PAK) — Referenzwert für 1-Hydroxypyren im Urin. Bundesgesundheitsbl-Gesundheits-forsch–Gesundheitsschutz 2005;48:1194–206. DOI 10.1007/s00103-005-1160-60.
6. Becker K, Kaus S, Krause C, et al. Umwelt-Survey 1998, Band III: Human-Biomo-nitoring. Stoffgehalte in Blut und Urin der Bevölkerung der Bundesrepublik Deutschland. WaBoLu-Heft 01/02. Berlin: Institut für Wasser-, Boden- und Lufthygiene des Umweltbundesamtes; 2002.
7. Seidel A, Dettbarn G, John A, Jacob J. Differences in the urinary excretion of PAH metabolites in nonsmoking cohorts of eastern and western Europe. Toxicol Lett 2005;158:S205.
8. Ruzgyte A, Bouchard M, Viau C. Development of a high-performance liquid chromato-graphic method for the simultaneous determination of pyrene-1,6- and 1,8-dione in animal and human urine. J Anal Toxicol 2005;29:533–8.
9. Grimmer G, Dettbarn G, Jacob J. Biomonitoring of polycyclic aromatic hydrocarbons (PAH) in highly exposed coke plant workers by measuring urinary phenanthrene and pyrene metabolites (phenols and dihydrodiols). Int Arch Occup Environ Health 1993;65:189–99. 10. Jacob J, Grimmer G, Dettbarn G. Profile of urinary phenanthrene metabolites in smokers
and non-smokers. Biomarkers 1999;4:319–27.
11. Hecht SS, Chen M, Yagi H, Jerina DM, Carmella SG. r-1,t-2,3,c-4-Tetrahydroxy-1,2,3,4--tetrahydrophenanthrene in human urine: a potential biomarker for assessing polycyclic aromatic hydrocarbon metabolic activation. Cancer Epidemiol Biomarkers Prev 2003;12:1501–8.
57 State of validation of biomarkers: Food-chemical-specific biomarkers – Polycyclic aromatic hydrocarbons
12. Ross JA, Nelson GB, Wilson KH, Rabinowitz JR, Galati A, Stoner GD, et al.. Adenomas induced by polycyclic aromatic hydrocarbons in strain A/J mouse lung correlate with time-integrated DNA adduct levels. Cancer Res 1995;55:1039–44.
13. Godschalk RW, Moonen EJ, Schilderman PA, Broekmans WM, Kleinjans JC, Van Schooten FJ. Exposure-route-dependent DNA adduct formation by polycyclic aromatic hydrocarbons. Carcinogenesis 2000;21:87–92.
14. Rothman N, Correa-Villaseñor A, Ford DP, Poirier MC, Haas R, Hansen JA, et al. Contribution of occupation and diet to white blood cell polycyclic aromatic hydro-carbon-DNA adducts in wildland firefighters. Cancer Epidemiol Biomarkers Prev 1993;2:341–7.
15. Poirier MC, Weston A, Schoket B, Shamkhani H, Pan CF, McDiarmid MA, et al. Biomonitoring of United States Army soldiers serving in Kuwait in 1991. Cancer Epidemiol Biomarkers Prev 1998;7:545–51.
16. Phillips DH. Macromolecular adducts as biomarkers of human exposure to polycyclic aromatic hydrocarbons. In: Luch A, editor. The carcinogenic effects of polycyclic aromatic hydrocarbons. Singapore: Imperial College Press; 2005. p. 137–69.
17. Kang DH, Rothman N, Poirier MC, Greenberg A, Hsu CH, Schwartz BS, et al. Interindividual differences in the concentration of 1-hydroxypyrene-glucuronide in urine and polycyclic aromatic hydrocarbon-DNA adducts in peripheral white blood cells after charbroiled beef consumption. Carcinogenesis 1995;16:1079–85.
18. Van Gijssel HE, Schild LJ, Watt DL, Roth MJ, Wang GQ, Dawsey SM, et al. Polycyclic aromatic hydrocarbon-DNA adducts determined by semiquantitative immunohisto-chemistry in human esophageal biopsies taken in 1985. Mutat Res 2004;547:55–62.
2.2.3. N-nitroso compounds
Panos Georgiadis
National Hellenic Research Foundation, Athens, Greece
Biomarkers of NNOC exposure and carcinogenesis
N-nitroso compounds (NNOCs), and methyl nitrosamines in particular, have been well-known experimental carcinogens since the late 1960s. However, it is still unclear whether they are involved in human carcinogenesis, mainly due to the difficulty in estimating the extent of external exposure and, even more so, of endogenous formation. Adducts generated in DNA by methylating carcinogenic NNOCs are primarily N7-methylguanine (N7-meG, ~70% of all adducts formed), O6-methylguanine (O6-meG, ~7%), N3-methyladenine (~3%), O4-methylthymine
(< 0.1%), methylphosphotriesters (~12%) as well as a number of other minor base modifications [1].
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Panos Georgiadis
O6-methylguanine
Animal studies
Several methods have been developed for the detection of the highly mutagenic and highly carcinogenic adduct O6-meG in DNA of experimental animals and humans. The most recent sensitive methods are the competitive repair assay [2] or methods based on multi-step processes involving high performance liquid chromatography (HPLC) or immunoaffinity enrichments, and adduct detection by 32P-postlabelling,
radioimmunoassay or electrochemical transmission [3–5].
Several studies have shown that O6-meG accumulates linearly in a dose-dependent fashion in multiple organs, irrespective of the route of administration or the type of methylating agent. In some cases there is a good correlation between O6-meG accumulation and specific species and tissue sensitivity to tumour induction [6-8]. A major carcinogenicity study involving near life-time exposure of rats to dimethylnitrosamine (NDMA) in drinking water showed a carcinogenic effect at doses as low as 0.01 ppm, a linear dose–response at doses lower than 1 ppm and an exponential increase in carcinogenicity at higher doses [9]. In a similarly designed experiment [10], O6-meG levels were measured in both liver and white blood cells and were found to increase linearly over a wide range of doses (0.2–2.5 ppm NDMA). Furthermore, an excellent correlation between adduct levels in liver and white blood cells (WBCs) was found, indicating that WBCs are a good surrogate tissue for monitoring exposure to NDMA at biologically relevant levels.
After treatment with methylonitrosourea (MNU), transgenic mice overexpressing or completely lacking O6-alkylguanine-DNA alkyltransferase (AGT), the enzyme that
repairs O6-meG, had a substantial decrease or increase of cancer incidence, respectively, relative to controls, in various tissues [11,12], indicating that modulation of O6-meG levels has a direct impact in cancer development. In addition, extracts of lemon grass (Cymbopogon citratus Stapf), or bitter melon administered by intragastric gavage in azoxymethane (AOM)-treated rats dramatically reduce, in a dose–response manner, formation of both early and late aberrant crypt foci (ACF) in the colon, as well as the levels of O6-meG in the DNA [13,14].
Human studies
In an early study, increased levels of O6-meG were observed in oesophageal DNA of individuals in China who habitually consume nitrosamine-rich foods and are at high risk for oesophageal cancer [15]. Since then the presence of this adduct has been reported in human DNA in a variety of tissues including gastrointestinal tract, lung, urinary bladder and liver [16–18]. A strong association has also been observed between the presence of increased O6-meG levels in blood leukocytes of asymptomatic individuals and the geographic prevalence of gastric cancer [19]. In colorectal cancer patients, high inter-individual variability in O6-meG levels was observed (2 adducts/109
59
in the distal colon and rectum where cancers were more frequently observed [20]. A recent investigation of non-smoking, asymptomatic Greek women, revealed O6-meG in maternal blood cell DNA and cord blood DNA of nearly all of the 35 individuals examined (range 3.5–42 adducts/109nucleotides (2). The significance of the widespread
presence in human DNA of O6-meG can only be explored in the context of large-scale molecular epidemiology studies, which are currently lacking due to the lack of high-throughput, high sensitivity assays for this adduct and the difficulties in determining external and endogenous exposure to NNOCs.
N7-methylguanine Animal studies
The quantitatively most important DNA alkylation lesion is N7-meG. It is not directly premutagenic, but it can undergo spontaneous or enzymatic depurination to form mutagenic apurinic sites. These adducts have been detected in animal and human tissues using various methods, including immunological assays, fluorescence techniques, mass spectrometry, 32P-postlabelling and electrochemical detection.
Bianchini et al. [21] observed dose-dependent formation of N7-meG in all tissues studied in rats after treatment with a variety of methylating agents. However, substantial background levels were observed probably due to contamination with RNA of which N7-meG is a natural component. More recently in a study using highly purified DNA, the background levels of N7-meG in both rat liver and kidney were found to be approximately 2.5 adducts/107nucleotides [22]. It was also found that
in rats fed with NDMA in drinking water, at all time points and all NDMA concentrations examined, levels of N7-meG in the liver and WBCs were proportional to dose but 3–4 times higher in liver than in WBCs [10]. N7-meG can also be detected in the urine of rats and mice using liquid chromatography; however, the background is high (34 mg/g creatinine) due to RNA degradation [23].
Extracts of lemon grass (Cymbopogon citratus Stapf) administered by intragastric gavage in AOM-treated rats dramatically reduce, in a dose–response manner, formation of both early and late aberrant crypt foci in the colon as well as the levels of N7-meG in the DNA [13].
Human studies
N7-Methylguanine is also frequently found in DNA of humans not known to have been exposed to methylating agents [24–26]. The background levels of N7-meG in human tissues range from 1 adduct/107 to 2 adducts/106 depending upon the tissue
and exposure. However, there are significant differences in the adduct levels in the same tissue obtained by different analytical methods. Such discrepancies might be mainly due to contamination with RNA. Nonetheless, all studies on N7-meG in various tissues showed a notable accumulation of N7-meG in smokers relative to nonsmokers, and a good correlation exists between the levels in bronchial and lymphocyte DNA [27]. State of validation of biomarkers: Food-chemical-specific biomarkers – N-nitroso compounds
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In addition, N7-meG adduct levels were found to be correlated with the number of cigarettes smoked per day, indicating that this biomarker could be used as an internal dosimeter of exposure to nitrosamines [28]. While the levels of N7-meG in mucosal adenoma and control samples were similar, they were higher in bladder tumour than adjacent normal epithelium, suggesting that methylating agents of unknown origin may play a role in tumour initiation and promotion [29,30].
Recently a liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with solid-phase extraction was developed to measure N7-meG in urine and it was shown that the urinary levels of N7-meG in smokers were higher than in nonsmokers [31]. However, the background is high (3 mg/g creatinine) due to RNA degradation and the consequent production of N7-meG, which is excreted by the urine. The relevance of N7-meG in urine has been shown by a prospective study indicating a higher risk of lung cancer in smokers with higher excretion [32].
Phosphate adducts
In addition to reacting with DNA bases, many genotoxic agents, including N-alkyl-nitrosoureas, react with the oxygen of the internucleotide phosphodiester linkages to form phosphotriester adducts (PTEs) [33]. Methyl phosphate triesters in DNA may be detected by electrospray ionization tandem mass spectrometry (ESI-MS/MS) coupled on-line to reversed-phase HPLC [34]. Another group has subsequently [35] developed a 32P-postlabelling method for detection of DNA alkylphosphotriesters in vivo. However,
phosphotriester adducts need to be further validated as biomarkers of NNOC exposure.
O6-carboxymethyl-dG
A range of nitrosated glycine derivatives react with DNA to form O6
-carboxy-methylguanine (O6-CmeG) and O6-methylguanine DNA adducts. Nitrosated glycine
derivatives may be formed in the gastrointestinal tract from the reaction of dietary glycine with nitrosating agents [36]. O6-CmeG can be detected with great sensitivity and specificity using an immuno-slot-blot assay [37] and was detected in human leukocytes [38]. However, O6-CmeG needs to be further validated if it is to be a suitable biomarker of endogenous nitrosation and potential cancer risk.
Protein adducts
Through the N-alkyl Edman method [39], adducts to the N-terminal valines of the globin chains of haemoglobin can be measured mass spectrometrically with high sensitivity. An average background level of about 200 pmol/g globin of methylvaline has been observed in unexposed humans. Bader et al. [40] reported significantly higher levels of N-methylvaline in smokers than in controls from the general population. However, the biomarker needs to be further validated.
61
Biomarkers of exposure to tobacco-specific nitrosamines
Tobacco-specific nitrosamines (TSNAs) such as 4-(nitrosomethylamino)-1-(3-pyri-dyl)-1-butanone (NNK), are formed from tobacco-specific alkaloids owing to the presence of nitrite generated from nitrate during curing and processing [36]. NNK is metabolised in mammals via reduction of the carbonyl group to 4-(methylnitrosamino)-1-(3-pyri-dyl)-1-butanol (NNAL), which is also carcinogenic. The latter is either conjugated with glucuronic acid and excreted in urine, or further metabolised by hydroxylation, giving rise to covalently bound hydroxypyridyloxobutyl (HPB) DNA and protein adducts [41,42], or, via a second pathway, mainly to N7-meG and O6-meG [42,43].
Biomarkers of exposure to NNK currently in use are: HPB, detected by gas chromatography (GC)-MS after alkali release from haemoglobin (Hb) adducts or after acidic release from HPB-DNA adducts; and the urinary NNK metabolites NNAL and its NNAL-glucuronide, determined by gas chromatography with nitrosamine-selective detection by thermal energy analysis (GC-TEA).
HPB-Hb and HPB-DNA adducts Animal studies
Murphy et al. [44] have demonstrated that the levels of HPB-Hb and HPB-DNA adducts derived from NNK are linearly correlated with exposure over a wide dose range in the rat. A significant correlation exists between lung cancer incidence in rats after treatment with NNK and HPB-DNA adduct levels in epithelial II cells (target cells for NNK carcino-genesis) [45]. Furthermore, rats fed throughout NNK treatment with the anti-carcinogen phenethyl isothiocyanate have 50% lower HPB-DNA adduct levels in epithelial II cells and are more resistant, by a similar extent, to lung carcinogenesis [46].
Human studies
Levels of both HPB-Hb and HPB-DNA adducts are significantly higher in smokers than in non-smokers. However, conspicuous inter-individual differences are found. Only about 20% of smokers exhibit increased adduct levels [47]. These results are in marked contrast to those obtained for urinary biomarkers of NNK uptake, NNAL and its O- and N-glucuronides, which show a more than 100-fold difference between smokers and nonsmokers (see below). In smokers the levels of HPB-releasing Hb adducts of NNK are very low compared with Hb adducts of other tobacco-specific carcinogens, probably because of the high reactivity of the alkylating HPB intermediate [48]. In addition, the method for the detection of HPB-releasing DNA adducts is not adduct specific and some of the more unstable pyridyloxybutyl adducts can react with buffer components and form artifacts, leading to false measurements [49]. Recently developed LC-MS/MS methods enable detection of individual pyridyloxybutyl adducts, especially those with O6-pyridyloxybutyldG, and represent a substantial improvement in both
specificity and sensitivity over the previous methods [50,51].
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Urinary adducts
The urinary NNK metabolites NNAL and its NNAL-glucuronide are absolutely specific for tobacco exposure and have not been detected in the urine of non-tobacco users [52–54]. However, they were consistently detected in individuals exposed to environ-mental tobacco smoke [55]. In addition a linear correlation exists between the levels of cotinine and the sum of NNAL and NNAL-glucuronide [56]. The NNAL-glucuro-nide/NNAL ratio varies at least 10-fold in smokers and, since the first is a detoxification product while the second is carcinogenic, the ratio has been used as an indicator of carcinogenic risk [57,58]. Recently a method has been developed that detects the above-mentioned metabolites in blood, which could be a very useful tool in molecular epidemiology studies [59]. In conclusion, NNAL and its glucuronide is the most relible biomarker for individuals exposed either actively or passively to tobacco smoke.
Conclusions
As described above there are a variety of NNOC biomarkers of exposure, some of them well validated in animal studies. However, very few studies have addressed the exposure–response relationship, inter-individual variation and background levels of DNA and protein adducts in humans, mainly because (a) it is difficult to monitor endogenous formation of NNOCs, (b) the assays for their detection are tedious and not sensitive enough and (c) tissue samples are rare and insufficient for adduct analysis.
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2.2.4. Acrylamide
Dan Segerbäck
Karolinska Institutet, Sweden
Some years ago it was shown that acrylamide is present in many commonly consumed food items and in some products, e.g. potato chips, at very high levels (mg per kg), resulting in intakes by the general public of the order of 0.3–0.8 µg per kg body weight per day [1]. Soon afterwards the mechanism of formation of acrylamide was shown to be the heating of certain amino acids in the presence of carbohydrates [2,3]. Since acrylamide
67 State of validation of biomarkers: Food-chemical-specific biomarkers – Acrylamide
is a known mutagen and animal carcinogen these findings caused concern among regulators as well as scientists. There was therefore a need to find out the extent of human exposure and biomarkers would be particularly suitable for obtaining that information. The biomarkers used so far to assess human exposure to acrylamide are haemoglobin adducts and urinary metabolites. The standard methods are gas chromatography (GC)- or liquid chromatography (LC)-tandem mass spectrometry (MS/MS) based and these assays are robust, highly reproducible and give high recoveries [4].
Animal studies
Dose–response
Linear dose–response relationships have been established for induction of DNA adducts [5] and micronuclei in the mouse [6] by acrylamide administered intraperitoneally (ip). More recently a linear dose–response relationship was shown for DNA as well as haemo-globin adducts using very low doses (down to 75 ng/kg bw ip) of acrylamide in mice [7], which is much lower than the average daily intake of persons eating a typical European diet. Only two animal studies have been published on dietary exposure to acrylamide. The first was the original study showing that acrylamide could be formed during cooking, i.e. an increase in haemoglobin adduct levels was found after rats were fed fried food [8]. In the other study it was observed that normal animal feed contains enough acrylamide to cause an increase in DNA adduct levels [9]. The conclusion that can be drawn from those two studies is that dietary exposures will increase levels of DNA and haemoglobin adducts.
Surrogate vs target tissue
Acrylamide causes tumours in several different tissues in the mouse and rat and there are species differences. In the mouse acrylamide (given ip or orally) gives rise to lung adenomas [10] and it was shown to be a tumour initiator in a skin painting experiment [11]. There have been two bioassays carried out in the rat following administration in drinking water [12,13]. Both studies showed induced peritesticular mesotheliomas, thyroid follicular cell tumours, and mammary gland tumours, as well as primary brain tumours. The reasons for these tissue specificities are unknown. Acrylamide and its genotoxic metabolite glycidamide are small, uncharged, water-soluble molecules and they have a relative long life-span in vivo, i.e. they are expected to be fairly evenly distributed to different tissues. Indeed several studies on DNA adducts from glycidamide (after administration of acrylamide) have shown that the adduct levels were relatively similar in different tissues [5,14–16]. The differences found did not indicate any direct correlation between DNA adduct formation and tissue specificity of tumour induction [14].
Modulation of biomarker/disease
No studies on modulation of formation of biomarkers or disease induced by acrylamide exposure have been carried out to date.
68
Dan Segerbäck
Human studies
Exposure–response
In a study of Chinese workers where the occupational exposure to acrylamide was very high there was a poor correlation between air levels of acrylamide and levels of haemoglobin adducts [17] and dermal exposure was hypothesised to have influenced adduct levels. A more recent study with lower exposures (below the UK Maximum Exposure Limit of 300 µg/m3)
showed a good correlation between haemoglobin adducts and air levels or glove contamination [18]. In another study a good correlation was found between air levels of acrylamide (< 300 µg/m3) and urinary metabolites [19]. For smokers, a correlation has
been established between levels of urinary metabolites of acrylamide or haemoglobin adducts and markers of smoking habits [4]. It has been estimated that smokers have an uptake of about 3 µg per kg body weight per day [20]. This is 5–10 times more than a non-smoker is expected to receive from the diet, but not far from what a consumer of large amounts of acrylamide-containing food products might receive. Hagmar and co-workers grouped individuals into high and low consumers of acrylamide-containing food (without estimating relative or absolute exposure levels) and found significantly higher haemoglobin adduct levels in men, but not women [21]. A problem with these types of studies is that levels of acrylamide are known to vary between brands of the same type of food and even between batches, making it very difficult to use questionnaires for exposure assessment [22]. Inter-individual variation
Among 70 non-smokers the level of the haemoglobin adduct from acrylamide itself varied by a factor of 5 [21]. Other studies, including both smokers and non-smokers, have found similar or smaller inter-individual variations, for haemoglobin adducts [23] or urinary metabolites [4]. Larger differences have been observed following occupational exposure [18], but in such settings exposure assessment is more problematic than, e.g., for smokers, so individual based variations (e.g. genetic polymorphisms in metabolising genes) are more difficult to assess. Background levels
Background levels of haemoglobin adducts from acrylamide and glycidamide can be detec-ted in non-smokers who are not occupationally exposed to acrylamide. The main source of this exposure is expected to be from consumption of heat-treated carbohydrate-rich food. There are many sources of acrylamide in dietary products and in addition there are large differences in levels between manufacturer and between batches. It is therefore difficult to establish a relationship between exposure (through diet) and biomarker response [22]. Case–control studies
The epidemiological studies carried out so far have not been able to show an association between exposure to acrylamide and cancer risk [24–29]. Epidemiological studies have limited power to detect small increases in cancer incidence and if acrylamide is not causing one specific type of cancer such studies might not be sensitive enough [1,30,31].
69 State of validation of biomarkers: Food-chemical-specific biomarkers – Acrylamide
A risk assessment by the FAO/WHO Joint Committee on Food Additives (JECFA) [32], using a Margin of Exposure (MOE) approach, took an estimated intake of acryla-mide from food of 0.001–0.004 mg/kg bw/day and a carcinogenicity estimate of 0.30 mg/kg bw/day from animal studies. The Committee concluded that the MOEs obtained for acrylamide in food may indicate a human health concern and that efforts to reduce levels in foodstuffs should continue.
Conclusions
It is difficult to assess dietary exposure to acrylamide from questionnaire data. Inter-vention studies would therefore be useful for establishing the relationship between exposure and biomarker response. Furthermore, no DNA adduct data are available in humans; these would be valuable for assessing the biologically significant dose and tissue-specific effects.
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