4. Specific biomarkers
related to food
4.1. Biomarkers for intake of heterocyclic aromatic amines
Sabine Rohrmann and Jakob Linseisen
Division of Clinical Epidemiology, German Cancer Research Center, Heidelberg, Germany
4.1.1. Introduction
Thirty years ago, certain mutagenic and carcinogenic compounds were found in fried meat and fish and named heterocyclic aromatic amines (HCAs) because of their chemical structure [1,2]. Heterocyclic aromatic amines are formed from precursors (creatinine, sugar, amino acids) in meat and fish at temperatures exceeding 130°C [2,3]. The amount of HCAs produced depends mainly on the cooking method, temperature, and the meat or fish. The highest amounts have been found in foods cooked at high temperatures by methods like barbecuing, grilling and frying [1]. Besides meat itself, meat drippings and gravy made from these drippings also contain considerable amounts of HCAs [4]. Human exposure to HCAs has been estimated to range from nanograms per person per day to a few micrograms per person per day [5–7]. The most common HCAs in human nutrition (and, thus, those on which research concentrates) are 2-amino-3-methylimidazo[4,5-f]qui-noline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-tri-methylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimi-dazo[4,5-b]pyridine (PhIP). PhIP is found in highest concentrations in cooked foods, followed by MeIQx [1].
Heterocyclic aromatic amines must be metabolically activated to bind covalently to DNA. Metabolic activation occurs primarily by hepatic cytochrome P450 1A2 (CYP1A2)-and, to a lesser extent, by CYP1A1- and CYP1B1-mediated N-oxidation in extrahepatic tissues to form N-hydroxy-HCA derivates. The N-hydroxy-HCA metabolites formed in the liver may enter the general circulation to react with DNA in various target tissues or undergo phase II metabolism reactions to form highly sensitive esters that bind to DNA [8]. Further activation of the N-hydroxylamine metabolite generally occurs by means of enzymatic esterification reactions catalysed by acetyltransferases (NAT enzymes) or sulfotransferases. These more reactive metabolites can covalently bind to DNA. Alternatively, the N-hydroxylamine metabolites of the HCAs may undergo phase II conjugation reactions via UDP-glucuronosyltransferase(s) to form the corresponding N-glucuronide conjugates. This is considered to be a significant detoxification pathway for N-hydroxy HCAs and was found in in vitro studies to limit the extent of HCA-induced DNA damage. In addition to this enzymatic pathway, glutathione S-transferases and glutathione can inhibit the covalent reaction of the
84
activated derivative of HCAs with DNA. Thus, the balance between these two processes is thought to play a central role in the potency of a HCA and thereby determine its genotoxicity.
Although the carcinogenicity of different HCAs has been proven in animal studies [2,9], conflicting results have arisen from epidemiological studies. Although some case-control studies conducted in Uruguay and the USA showed a possible relationship between the intake of HCA and the risk of breast, colon, lung and gastric cancer [10–16], other groups were not able to detect any association between HCA intake and cancer risk at different sites [6,17–19].
Usually, dietary questionnaires are used to estimate the intake of HCAs. However, assessing exposure to HCAs by means of a questionnaire may be inaccurate because of differences in cooking methods, meat quality, consumption of meat with other foods, e.g. cruciferous vegetables, or inconsistent reporting. These factors are likely to have an impact on the actual exposure compared with the calculated intake from dietary questionnaires [20]. Thus, biomarkers of HCA exposure (and metabolism) are thought to reflect better the burden due to HCA intake. Several types of biomarkers have been evaluated so far (Table 4.1.) [21]. However, they reflect different aspects and time frames of HCA exposure and are, thus, not equally appropriate for all research questions. Urinary excretion, at present the biomarker of HCA exposure most studied, only reflects very
Table 4.1. Biomarkers of exposure to heterocyclic amines
Biomarker Advantages Disadvantages
Urine, parent compound and metabolites of HCAs
Protein adducts of HCAs in blood Hair, parent compound of HCAs
Assessment of bioactivation and detoxication capacity, bioactive dose
Bioactive dose, reflects exposure up to 1 month Reflects exposure up to several months. Easy to sample and store
Reflects only recent exposure
Needs validation Needs validation
See [21] for details.
recent exposure, i.e. 24 hours after intake. However, determination of metabolites makes it possible to assess bioactivating and detoxification capacity and the role of different phase I and phase II biotransforming enzymes. This could be useful for studies on dietary intervention and chemoprevention aiming at reducing biologically active species in order to prevent DNA damage and cancer in target tissues. Short-term markers may also be useful for validating questionnaires. However, in order for biomarkers to be a viable exposure assessment tool for epidemiological studies, long-term biomarkers, such as DNA, protein or haemoglobin adducts and HCA in hair, that reflect intake over weeks or months are needed [21,22] (Fig. 4.1.).
85 Specific biomarkers related to food: HCAs
Adapted from [22] with permission from Elsevier.
CYP1A2, cytochrome P450 1A2; HCA, heterocyclic aromatic amine; NAT1 & 2, N-acetyltransferase 1 & 2.
Fig. 4.1. Biomarkers of HCAs and polymorphic enzymes involved in HCA metabolism.
4.1.2. Parent compounds and metabolites in urinary samples
Several studies have examined the excretion of unmetabolised HCAs, especially PhIP and MeIQx, in urine (a summary of human studies is given in Table 4.2.). In different studies, a defined amount of HCA was added to the participants’ meals. After these meals, urine was collected for 0–24 hours and HCA concentration was measured. Concen-trations of PhIP and/or MeIQx have been determined using ion exchange chromato-graphy and multiple HPLC purification steps [23], gas chromatochromato-graphy [24–28], liquid chromatography-mass spectrometry [27,29,30], or HPLC with fluorescence detection after immunoaffinity chromatography [31].
PhIP as well as MeIQx are excreted unmetabolised in urine. Murray et al. [32] deter-mined MeIQx in human urine by gas chromatographic-mass spectrometric assay (GC-MS) and observed that 1.8–4.9% of the ingested MeIQx was excreted. Lynch
et al. [33] reported that 2.1% (mean, range 1.2–4.3%) of MeIQx and 1.1% (mean,
range 0.6–2.3%) of PhIP were excreted unmetabolised. They reported that, irrespective of dose, urinary excretion of unchanged MeIQx or PhIP remained constant for each individual subject. The intraindividual coefficients of variation for MeIQx (28.4%) and PhIP (23.7%) were low. In contrast, intersubject variation was greater, with pooled coefficients of variation of 145% for MeIQx and 71% for PhIP. Similar results were noted in other studies [26,34]. Reistad et al. [26] determined PhIP and MeIQx by a series of liquid/liquid extractions, followed by blue cotton adsorption and finally by a novel derivatization technique for GC-MS and reported that concentrations were higher in
hy-86
Table 4.2. Determination of HCA and HCA metabolites in human urinary samples
Author Compound Intervention Method Results
Murray et al. MeIQx 6 subjects consumed GC-MS 1.8–4.9% of the ingested dose 1989 [32] a fried beef meal Limit of detection 5 pg (mean 3.25±1.1%) was excreted 0–12 h
MeIQx/ml urine after intake;
Recovery rate ∼25% no MeIQx in 12–24 h urine
Boobis et al. MeIQx 6 healthy males received GC-MS CYP1A2-catalysed metabolism accounted 1994 [25] PhIP either placebo or 125 mg for 91% of the elimination of ingested
furafylline 2 h prior to MeIQx and 70% of ingested PhIP a test meal of fried beef (most likely via N-hydroxylation) with a known amount
of HCA
Lynch et al. DiMeIQx 10 normal, healthy males GC-MS No DiMeIQx was detected in urine 1992 [33] MeIQx consumed a standard Recovery rates ∼ 30% MeIQx mean 2.1% (1.2–4.3%)
PhIP cooked meat meal Precision was PhIP mean 1.1% (0.6–2.3%) (containing 2.2±0.2 ng determined by analysing Irrespective of dose, % excreted PhIP MeIQx/g meat, 65 ml aliquots of one and MeIQx remained constant for each 0.7±0.1 ng DiMeIQx/g subject’s urine: subject
meat, and 16.4±2.1 ng coefficients of variation Intraindividual coefficients of variation were PhIP/g meat) on four MeIQx = 3.2%, 28.4% (MeIQx) and 23.7% (PhIP) separate occasions over PhIP = 9.8% Interday (intrasubject) variation: pooled a period of 14 months coefficients of variation for both
compounds = 19% (MeIQx) and 3.4% (PhIP) Intersubject (intraday) variation: pooled coefficients of variation = 145% (MeIQx) and 71% (PhIP)
Urinary levels correlated with amount ingested: MeIQx r = 0.64, PhIP r = 0.69 Stillwell et al. MeIQx (free) 7 subjects consumed GC-MS after HPLC Unconjugated (free) MeIQx 0.5–4.7% 1994 [34] Sulfamate & panfried fish, beef separation and acid 0–12 h after exposure
glucuronide or bacon hydrolysis Hydrolysed: 1–14% (mean 8.7±5.1%)
conjugates Coefficient Sulfamate 34–45%
of variation < 5% Glucuronide 55–66%
Reistad et al. MeIQx 8 subjects consumed GC-MS with a novel Not hydrolysed: 1–6% MeIQx, 0.5–2% PhIP 1997 [26] PhIP fried minced beef patties derivatization procedure Hydrolysed: 13–22% MeIQx, 2–8.5% PhIP
DiMeIQx (295 g containing Limits of detection DiMeIQx was below the limit of detection 4.0±2.6 PhIP, MeIQx = 10 pg/ml
3.5±0.9 MeIQx, PhIP = 2.5 pg/ml 0.3±0.1 ng/g DiMeIQx)
Stillwell et al. MeIQx 57 subjects in the study MeIQx: LC-MS/MS Hydrolysed MeIQx: mean 10.5±3.5% 1997 [27] PhIP ate meat containing PhIP: ESI-MS/MS (3.2–22.7%), no MeIQx in 12–24 h urine
known amounts of MeIQx Limit of detection = detected
and PhIP = 4 pg/ml Hydrolysed PhIP: mean 4.3±1.7%
Controlled feeding trials
Specific biomarkers related to food: HCAs
87
Table 4.2. Determination of HCA and HCA metabolites in human urinary samples — cont.
Author Compound Intervention Method Results
Coefficient of variation (1.9–9.8%) in 0–12 h urine, 0.9±0.4% (pooled urine samples): in 12–24 h urine
MeIQx = 16%, Correlation with intake: MeIQx r = 0.40,
PhIP = 31% PhIP r = 0.50
Kulp et al. PhIP 8 volunteers were fed LC-MS/MS N2-OH-PhIP-N2-glucuronide is the most
2000 [30] N2-OH-PhIP- 200 g of cooked chicken Repeatability: Each abundant urinary metabolite, followed
-N2-glucuroni- containing 27 µg PhIP urine sample was by PhIP-N2-glucuronide
de, PhIP-N2- injected three times: Both account for 92–98% of the total
-glucuronide, Variation with a sample metabolite excreted
4’-PhIP-sulfate ranged from 20–30% Ratio N2-OH-PhIP-N2-glucuronide:
and N2-OH- PhIP-N2-glucuronide = 1:1–8:1
-PhIP-N-3-glu-curonide
Sinha et al. MeIQx (free) 66 healthy subjects ate GC/MS Mean 1.31% (0.13–4.13% of ingested dose)
1995 [24] lean ground beef cooked CYP1A2 activity was inversely associated
at low temperature for 7 with free MeIQx concentration in urine days; on day 8, subjects
ate lean ground beef cooked at high temperature (9.0 ng MeIQx/g meat)
Stillwell et al. N-OH-MeIQx- See Sinha et al. NCI-GC-MS Mean 9.4±3.0% (2.2–17.1%) 0–12 h 1999 [36] -N2-glucuro- 1995 [24] Analysis of replicate after exposure
nide samples (n = 8) over Significant correlation between the level a period of 4 months: of N-OH-MeIQx-N2-glucuronide in urine and
intrasample CV = 8.5% the amount of MeIQx ingested (r = 0.44, p = 0.0002)
Excretion level of N-OH-MeIQx-N2
-glucuro-nide in urine was not associated with the enzyme activities of NAT2 or CYP1A2 Stillwell et al. N2-(beta-1- See Sinha et al. NCI-GC-MS Mean N2
-(beta-1-glucosiduronyl)-2-hydroxy-2002 [60] -glucosiduro- 1995 [24]
amino-1-methyl-6-phenylimidazo[4,5-b]pyri-nyl)-2-hydroxy- dine measured as the acid hydrolysis
amino-1-me- product 2-OH-PhIP in the 0–12 h urine was
thyl-6-phenyli- 20.2±8.0% of the ingested dose; median
midazo[4,5-b] 18.8% (5.4–39.6%)
pyridine Excretion of N2
-(beta-1-glucosiduronyl)-2-hy- droxyamino-1-methyl-6-phenylimidazo-[4,5-b]pyridine in the 0–12 h urine was significantly related to the quantity of PhIP ingested for all subjects (r = 0.52, p < 0.0001)
Sabine Rohrmann, Jakob Linseisen 88
Table 4.2. Determination of HCA and HCA metabolites in human urinary samples — cont.
Author Compound Intervention Method Results
Controlled feeding trials
Strickland PhIP See Sinha et al. HPLC with fluorescence Significant correlation between the amount
et al. 1995 [24] detection after immuno- of fried meat ingested and urinary PhIP:
2001 [31] affinity chromatography r = 0.61 (p < 0.0001)
Friesen et al. PhIP 10 healthy non-smoking NCI-GC-MS After broiled beef consumption, urinary 2001 [28] males consumed identical Limit of detection concentration of PhIP increased 14–38-fold
amounts of broiled beef 0.1 pmol/ml above mean pre-feed concentration on five consecutive days Following cessation of broiled beef
con-sumption, urinary PhIP concentration decli- ned to near pre-feed levels within 48–72 h Ratio total alkali-labile PhIP metabolites: unmetabolised PhIP = 18:1–48:1 Strickland PhIP See Friesen et al. HPLC with fluorescence Overall correlation between the two assays:
et al. 2001 [28] detection after immuno- r = 0.87 (p < 0.0001)
2001 [31] affinity chromatography 89% of PhIP detected in urine was
and GC-MS excreted within the first 12 h Studies not applying a controlled diet
Ushiyama MeIQx 10 healthy volunteers HPLC after partial No HCA in urine of patients with parenteral
et al. PhIP eating normal diet purification by treatment nutrition
1991 [23] (5 males, 5 females) with blue cotton and ion % excreted could not be calculated b/c and 3 inpatients (2 males exchange column HCA intake was not known
and 1 female) receiving chromatography parenteral alimentation Recovery rates:
MeIQx 55%, PhIP 55%
Ji et al. MeIQx (free) 47 black, 41 Asian GC-MS Geometric mean level in blacks was
1994 [61] (Chinese or Japanese), 1.3- and 3.0-fold higher than that in Asians
and 43 non-Hispanic and whites, respectively
white (white) males who Urinary level of MelQx was positively
consumed an associated with intake frequencies
unrestricted diet of bacon, pork/ham and sausage/luncheon
meats among study subjects
Kidd et al. PhIP 45 African-American, ESI-LC-MS Geometric mean levels of PhIP in
Asian-1999 [29] 42 Asian-American LC-MS/MS Americans and African-Americans were
(Chinese or Japanese), approximately 2.8-fold higher than in whites and 42 non-Hispanic Urinary excretion levels of PhIP were not
white males who associated with intake frequencies of any
consumed an cooked meat based on a self-administered
unrestricted diet dietary questionnaire
CYP1A2, cytochrome P450 1A2; ESI, electrospray ionization; GC-MS, gas chromatography-mass spectrometry; HPLC, high performance liquid chromatography; NCI, negative ion chemical ionization; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; NAT2, N-acetyltransferase 2; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.
Specific biomarkers related to food: HCAs
89
drolysed urine than in unhydrolysed urine and that the ratio for hydrolysed/ /unhydrolysed HCA concentration varied between individuals. Similarly, Stillwell
et al. [34] observed that unconjugated MelQx excreted in urine ranged between 0.5
and 4.7% of the ingested dose. In acid-treated urine the amount of MelQx varied between 1 and 14% of the ingested dose. No MelQx was found in urine samples 12–24 hours after the meal was consumed.
Strickland et al. [31] determined PhIP in hydrolysed urine of 66 subjects who had consumed fried meat, using HPLC with fluorescence detection following immunoaffinity chromatography. Their immunoaffinity chromatography/HPLC fluorescence method was validated using GC-MS. The urinary PhIP concentrations determined with both methods were statistically significantly correlated (Pearson correlation coefficient r = 0.87, Spearman correlation coefficient r = 0.75). Using the HPLC fluorescence method, urinary PhIP was highly correlated with dietary PhIP intake (Pearson correlation coefficient r = 0.61, Spearman correlation coefficient r = 0.54).
Besides the measurement of unmetabolised HCAs, several oxidised metabolites of PhIP and MeIQx have been determined in urine. Major metabolites of PhIP detected in human urine are N2-OH-PhIP-N2-glucuronide, PhIP-N2-glucuronide, N2 -OH-PhIP-N-3-glu-curonide, and 4’-PhIP-sulfate, with the first two accounting for most of the PhIP excre-ted [30,35]. Stillwell et al. [34] confirmed the presence of sulfamate and glucuronide con-jugates of MelQx in human urine.
Bioactivation of HCAs occurs in two steps: N-oxidation followed by esterification of the exocyclic hydroxylamine. CYP1A2 catalyses the N-oxidation of several HCAs, including IQ and PhIP. This enzyme is induced in humans by ingestion of charbroiled beef and specific inhibition of this enzyme in humans results in reduced metabolism of PhIP and MeIQx. Detoxification may take place prior to bioactivation, but bioactive intermediates may also undergo detoxification, primarily by conjugation and reduction. The glucuronyl derivates of N-hydroxy MeIQx and N-hydroxy PhIP, detoxification products of the proximate metabolites of these two compounds, are major urinary metabolites in humans [30,36,37]. It has been suggested that the amount of these meta-bolites could serve as a biomarker of activation and the active dose [37]. These metaboli-tes represent the first step of activation. Another metabolite, 2-amino-1-methyl-6-(5-hy-droxy-)phenylimidazo[4,5-b]pyridine (5-OH-PhIP), is formed through several steps as a by-product when N-acetoxy PhIP reacts with DNA, protein, and thiols [26,38]. It has been proposed that this metabolite and its conjugates might serve as a biomarker for the ultimate genotoxic dose, since its formation correlated with that of PhIP-DNA adducts in in vitro experiments [39,40]. In an animal study, urinary excretion of 5-OH-PhIP showed a linear dose-response relationship in rats dosed orally with PhIP. This shows that 5-OH-PhIP is also formed in vivo and that 91% is excreted with the urine in 24 h, indicating the possible use of 5-OH-PhIP as a urinary biomarker for the bioactive dose of PhIP [39]. In a preliminary study, 5-OH-PhIP was identified in a 24 h urine sample from a male volunteer who had ingested fried beef [39]. This indicates that urinary 5-OH-PhIP could be used as an easily obtainable marker for the genotoxic dose of PhIP in human
Sabine Rohrmann, Jakob Linseisen 90
biomonitoring studies. However, it has the same disadvantage as other urinary markers in that it only represents recent exposure.
Malfatti et al. [37] confirmed the interindividual differences in metabolism of HCA in human using accelerator mass spectrometry (AMS) after administering [14C]PhIP prior to surgery to three volunteers with colon cancer. Urine and blood were analysed for PhIP and PhIP metabolites by HPLC. The excretion of [14C]PhIP with urine varied from 50–90%. PhIP accounted for < 1% of the excreted radiolabel, while 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-N2-glucuronide (N-hydroxy-PhIP-N2-glucuronide) accoun-ted for 47–60% of urinary metabolites in 24 h. Other metabolites detecaccoun-ted in the urine in significant amounts were 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate, N-hydroxy-PhIP-N-3-glucuronide and PhIP-N2-glucuronide. However, the relative amount of each metabolite varied by subject, and the amounts of each metabolite within subjects changed over time. In the plasma, unchanged PhIP accounted for 17–56% and N-hydroxy-PhIP-N2-glucuronide for 18–60% of the recovered plasma radioactivity at 1 h post PhIP dose. Because of its high concentration N-hydroxy-PhIP-N2-glucuronide might be a good biomarker of PhIP intake. Interindividual differences in PhIP metabolites might be useful as predictors of an individual’s susceptibility to carcinogenic risk from PhIP.
In summary, urinary free HCAs or total HCAs (acid-hydrolysed urine samples that include free, N2-glucuronide, and sulfate metabolites) correlate modestly with the amount of HCA consumed in the metabolic study (see Table 4.2.). HCAs and mutagenic activity in urine were usually not detectable more that 12 hours after consuming a high HCA meal. Thus, HCAs in urine have short half-lives and may not be ideal measures of ‘usual’ intake in aetiological studies, especially if there is substantial day-to-day variability. However, with a large sample size, HCAs in urine could still be used to validate intake of HCAs as estimated by questionnaires [22].
4.1.3. Blood protein and DNA adducts
For IQ, MeIQx, and PhIP, the major adduct formed in vitro is the N-(deoxyguanosin-8-yl) derivate [41]. MeIQx-DNA adducts have been detected in various human tissues such as rectum, sigmoid colon, and kidney at levels of ~10–9 using 32P-postlabelling [42]. Using GC-MS, PhIP-DNA adducts have been detected in human colon at levels of ~10–9[43]. Besi-des DNA adducts in organs (including lymphocyte DNA), blood protein adducts, i.e. of HCA, have been examined. Blood protein, i.e. haemoglobin and albumin, products are easily obtainable and adducts with these proteins may have lifetimes as long as those of the prote-ins (e.g. 120 days for haemoglobin) and are, thus, prone to reflect long-term exposu-re/accumulated exposure to certain carcinogens such as HCAs. A summary of human studies of HCA intake and formation of DNA and protein adducts is given in Table 4.3.
32P-Postlabelling has been widely used because of its high sensitivity (1 adduct in 109 unmodified nucleotides) and because this sensitivity can be reached with small quantities of DNA (2–15 µg) [44]. Yamashita et al. [45] analysed HCA-DNA adducts in rat liver. They demonstrated that the N-hydroxy derivative of MeIQx was reactive toward DNA
Specific biomarkers related to food: HCAs
91
Table 4.3. HCA intake and formation of DNA and protein adducts — human studies
Author What is measured? Intervention Method Results
Controlled feeding trials
Dingley et al. MeIQx Oral administration AMS Dose-dependent levels of MeIQx-albumin 1998 [54] of a single dose of either and MeIQx-Hb adducts (regression coefficient
21.3 µg (n = 5) of the dose–response curve was approximately 1) or 228.0 µg (n = 2) Covalent binding of [14C]MeIQx to albumin [14C]MeIQx and Hb was detectable in all subjects 3.5–6 h
after administration
Albumin 3.79 (95% CI 1.95–7.01), Hb 0.18 (95% CI 0.04–0.77) fmol MeIQx/g protein per ng MeIQx/kg body weight
Mauthe et al. MeIQx Oral administration AMS No difference existed between the levels detected
1999 [55] of a single dose of in normal (97±26 pg MeIQx/g) and tumour
21.3 µg [14C]MeIQx (101±15 pg/g) tissue
(n = 5) No difference existed in adduct levels between
normal (25.4±4.2 adducts/1012nucleotides) and
tumour tissue (28.0±2.5 adducts/1012nucleotides)
Major adduct MeIQx-dG-C8
Dingley et al. PhIP Oral administration AMS WBC DNA adducts (up to 1.4 adducts/109 1999 [56] of a single dose of 70 µg nucleotides) were unstable & declined
(n = 2) or 84 µg (n = 3) substantially over 24 h
[14C]PhIP 48–72 h before Albumin 11.52 (±9.29), Hb 0.22 (±0.13) fmol
surgery for removal PhIP/g protein per ng PhIP/kg body weight of colon tumours PhIP is bioavailable to the colon: 42–122 pg
PhIP/g tissue
DNA adduct levels in the normal colon (35–135 adducts/1012nucleotides), were
significantly lower than in tumour (up to 308 adducts/1012 nucleotides) tissue
No correlation between PhIP levels in tissue and DNA adduct levels in that tissue Garner et al. Oral administration AMS 1.57–143.7 DNA adducts/1012nucleotides in
1999 [62] of [14C]MeIQx (20 µg) normal tissue; 6.63–167.9 DNA adducts/1012
and [14C]PhIP 20, 50, nucleotides in tumour tissue
or 200 µg
Studies not applying a controlled diet
Totsuka et al. MeIQx 38 DNA samples 32P-postlabelling N2
-(deoxyguanosin-8-yl)-2-amino-3,8-dimethyli-1996 [42] obtained from surgical midazo- [4,5- f]quinoxaline 5’-monophosphate and autopsy specimens (dG-C8-MeIQx) at levels down to 1/1010
nucleotides
DNA samples from colon and rectum surgical spe-cimens and a kidney contained an adduct spot of 14,18 and 1.8 per 1010 nucleotides, respectively
Sabine Rohrmann, Jakob Linseisen 92
Table 4.3. HCA intake and formation of DNA and protein adducts — human studies — cont.
Author What is measured? Intervention Method Results
Studies not applying a controlled diet
Friesen et al. PhIP 24 individual human Alkaline Both methods detected dG-C8-PhIP adducts 1994 [43] tissue DNA samples: hydrolysis-GC-MS in two of the colon samples, but not in the
pancreas (n = 12), colon 32P-postlabelling samples from human pancreas or urinary bladder
mucosa (n = 6), urinary bladder epithelium (n = 6)
Magagnotti PhIP 35 volunteers with LC-MS/MS PhIP-albumin adducts were significantly higher
et al. different dietary habits in meat consumers than in vegetarians: 6.7±1.6
2000 [50] and 0.7±0.3 fmol/mg albumin; respectively,
mean ±SE; p = 0.04
Globulin adducts: 3±0.8 fmol/mg in meat consumers and 0.3±0.1 fmol/mg in vegetarians Magagnotti PhIP Lymphocytes from 76 LC-MS/MS High vegetable intake significantly reduced
et al. incident colorectal PhIP-DNA adducts (Mann-Whitney U, p = 0.044) 2003 [51] cancer patients likely to None of the genetic polymorphisms studied
be exposed to dietary [N-acetyltransferase (NAT1 and NAT2),
PhIP sulfotransferase (SULT1A1) and glutathione
S-transferase (GSTM1 and GSTA1) genes] significantly affected PhIP-DNA adducts AMS, accelerator mass spectrometry; GC/MS, gas chromatography mass spectrometry; Hb, haemoglobin; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; WBC, white blood cell.
in vitro, forming adducts. Addition of acetic anhydride to N-OH-MeIQx increased its
reactivity to DNA. 32P-Postlabelling analysis revealed that the MeIQx-DNA adducts formed in vivo and in vitro were identical. Thus, MeIQx is metabolised in vivo to the N-hydroxy form and further esterified to produce more reactive species, such as the N-acetoxy form, which modify DNA to form adducts. Another Japanese group observed, using 32P-labelling, that N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (dG-C8-PhIP) was the major adduct in DNA of rats given PhIP [46].
Turesky et al. [47] published results of two trials conducted in cynomolgus monkeys. In the first trial, the animals received a single dose of IQ prior to death. The pattern and distribution of DNA adducts examined by 32P-postlabelling were similar in all tissues 24 h after a single oral dose of IQ (20 mg/kg). The highest DNA adduct levels were found in the liver (3.67–11.19 adducts per 107 bases), followed by kidney colon, heart, and pancreas. N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ) was the major adduct and accounted for approximately 50–80% of the adducts, followed by 5-(deoxyguanosin-N2-yl)-amino-3-methylimidazo[4,5-f]quinoline (dG-N2-IQ), which accounted for 20-40% of the adducts. In the second trial, IQ was administered at 10 or 20 mg/kg, 5 days per week for up to 9.2 years. In these chronically treated animals, the DNA adduct levels in pancreas, kidney, and heart increased on average by 40- to 90-fold
Specific biomarkers related to food: HCAs
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over those observed in animals given a single dose, while only 3- to 10-fold increases in adducts were observed in colon and liver. A sharp increase in the contribution of dG-N2-IQ to total DNA adducts occurred in all slowly dividing tissues during chronic treatment, and dG-N2-IQ became the predominant lesion. However, there was no preferential accumulation of dG-N2-IQ in the colon, a tissue with a high rate of cell division, where dG-C8-IQ remained the predominant adduct.
IQ-DNA adducts (dG-C8-IQ and dG-N2-IQ) have been detected in kidney tissue [48] and in pancreatic tissue [49] of cynomolgus monkeys using capillary liquid chromatography/microelectrospray mass spectrometry. These results were consistent with previously reported results in the same tissue by means of 32P-postlabelling [46]. Friesen et al. [42] used gas chromatography/electron capture mass spectrometry (GC-EC-MS) to determine PhIP DNA adducts in rat and human tissue and to validate these results using 32P-postlabelling. Results from these two methods were highly correlated (Pearson correlation coefficient r = 0.83) when adducts were determined in rat pancreas, colon, lung, heart, and liver. In human colon tissue, Friesen et al. [42] were able to determine adducts with both methods. An Italian group used gas chromato-graphy-mass spectrometry with negative ion chemical ionization and selected ion recording (GC-NICI-SIR) as well as liquid chromatography-tandem mass spectrometry (LC-MS/MS) to examine the formation of serum albumin and globin adducts in rats and humans [50]. In rats that had received defined oral doses of PhIP, PhIP-serum albumin and -globin adducts increased linearly with dose. Both methods gave comparable results, but LC-MS/MS was both more specific and more sensitive and was, thus, selected for the human studies. In 35 volunteers with different dietary habits, levels of PhIP-serum albumin adducts were significantly higher in meat consumers than in vegetarians (6.7±1.6 and 0.7±0.3 fmol/mg serum albumin, respectively, mean±SE; p = 0.04). The globin adduct pattern was quantitatively lower but paralleled serum albumin (3±0.8 fmol/mg in meat consumers and 0.3±0.1 fmol/mg in vegetarians). PhIP-serum albumin adducts did not differ in smokers and in non-smokers. These results confirm what has already been reported via AMS, i.e. that albumin adducts seems to be better biomarkers than globin adducts and that the formation of protein adducts seems to be dose dependent (assuming a higher intake of HCAs in meat than in non-meat eaters). The same method was used in another study to examine the effects of genetic polymorphisms on PhIP-DNA adducts in human lymphocytes [51].
Accelerator mass spectrometry measures isotope ratios with high selectivity, attomole sensitivity, and precision of 0.5–10% depending on isotope level and preparation method [52]. Turteltaub et al. [53] used AMS to determine human and rodent MeIQx adduct formation in the colon after oral administration of [14C]MeIQx. About 90% of the MeIQx-DNA adduct in human and rat colon appears to be the dG-C8-MeIQx adduct. In chronically fed animals, adduct levels were generally linear with administered dose, although adduct levels began to plateau slightly at high chronic doses. The authors concluded from this plateau that linear extrapolation from high-dose studies might underestimate the amount of DNA damage present in the tissues following low dose.
Sabine Rohrmann, Jakob Linseisen 94
Dingley et al. [54] examined the effects of oral administration of [14C]MeIQx on albumin and haemoglobin adduct levels in humans and rats by means of AMS. The results of their studies indicate that the formation of albumin and haemoglobin adducts is dose dependent and that a trend exists for higher adduct levels per unit dose in humans, compared with F344 rats. MeIQx-albumin adducts appear to be a more sensitive marker of exposure to MeIQx than are MeIQx-haemoglobin adducts because higher levels of adduct formation per dose were noted for albumin than for haemoglobin. They were also able to demonstrate that MeIQx forms DNA adducts in the human colon after oral administration of a single dose in volunteers undergoing surgery for colon cancer [55]. In a different study, participants received a single dose of [14C]PhIP prior to colon cancer surgery [56]. The results demonstrated that PhIP is activated to a form that will bind to albumin, haemoglobin, and white blood cell (WBC) DNA in peripheral blood. However, WBC DNA adducts were unstable and declined substantially over 24 hours. Additionally, PhIP was determined in the colon and both protein and DNA adducts were observed. DNA adduct levels in the normal tissue were significantly lower than in tumour tissue.
Using AMS, Dingley et al. [57] simultaneously measured 3H-labelled PhIP and 14C-labelled MeIQx in rat liver tissue and bound to liver DNA and protein. Levels of PhIP and MeIQx in whole tissue and bound to liver protein were dose dependent. MeIQx-protein and -DNA adduct levels were higher than PhIP adduct levels. No synergistic effects could be detected for co-administration of PhIP and MeIQx compared with the individual administration of these compounds.
4.1.4. Hair
The use of HCAs as a biomarker in hair has the potential of reflecting exposure over a much longer period than other biomarkers, and hair is easy to sample and store for later analysis in prospective studies on the relationship between HCA exposure and cancer. This biomarker still needs validation as a biomarker of exposure in humans as well as improvement of the chemical analytical method [21].
Reistad et al. [58] measured the concentration of PhIP in human hair by GC-MS and compared the concentration with the results of a questionnaire about consumption of fried/grilled meat and smoking habits. Twelve out of 14 hair samples contain-ed 50–5000 pg PhIP/g hair. Grey and white hair straws had an approximately 50% lower PhIP concentration compared with coloured hair straws of the same person, which may indicate binding to melanin and possibly keratin. Only some agreement between intake of fried/grilled meat and PhIP concentration in hair was seen. In a recent study in mice, a linear relationship (r2 = 1.00, p < 0.0001) was found between relative PhIP incorpo-ration and eumelanin concentincorpo-ration in hair [59].
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4.1.5. HCA biomarkers and cancer risk
DNA adducts of HCA have been observed in human tumour tissue [42,43]. In controlled feeding studies, PhIP [55] but not MeIQx [54] adducts have been observed in higher quantities in tumour than in normal tissue. However, so far, no epidemiological studies have been conducted in humans that have examined the association between HCA exposure assessed by means of any biomarker and the risk of cancer.
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26. Reistad R, Rossland OJ, Latva-Kala KJ, Rasmussen T, Vikse R, Becher G, et al. Heterocyclic aromatic amines in human urine following a fried meat meal. Food Chem Toxicol 1997;35:945–55.
27. 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.
28. Friesen MD, Rothman N, Strickland PT. Concentration of 2-amino-1-methyl-6-phenylimida-zo(4,5-b)pyridine (PhIP) in urine and alkali-hydrolyzed urine after consumption of charbroiled beef. Cancer Lett 2001;173:43–51.
29. Kidd LC, Stillwell WG, Yu MC, Wishnok JS, Skipper PL, Ross RK, et al. Urinary excretion of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in White, African-American, and Asian-American men in Los Angeles County. Cancer Epidemiol Biomarkers Prev 1999;8:439–45. 30. Kulp KS, Knize MG, Malfatti MA, Salmon CP, Felton JS. Identification of urine metabolites of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine following consumption of a single cooked chicken meal in humans. Carcinogenesis 2000;21:2065–72.
31. 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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34. Stillwell WG, Turesky RJ, Gross GA, Skipper PL, Tannenbaum SR Human urinary excretion of sulfamate and glucuronide conjugates of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx). Cancer Epidemiol Biomarkers Prev 1994.;3:399–405.
35. Strickland PT, Qian Z, Friesen MD, Rothman N, Sinha R. Metabolites of 2-amino-1-methyl-6--phenylimidazo(4,5-b)pyridine (PhIP) in human urine after consumption of charbroiled or fried beef. Mutat Res 2002;506–7:163–73.
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40. Frandsen H, Alexander J. N-acetyltransferase-dependent activation of 2-hydroxyamino-1-me-thyl-6-phenylimidazo[4,5-b]pyridine: formation of 2-amino-1-methyl-6-(5-hydroxy)pheny-limidazo [4,5-b]pyridine, a possible biomarker for the reactive dose of 2-amino-1-methyl-6-phe-nylimidazo[4,5-b]pyridine. Carcinogenesis 2000;21:1197–203.
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4.2. Polycyclic aromatic hydrocarbons in food
David Phillips
Institute of Cancer Research, London, UK
4.2.1. Introduction
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants formed by the incomplete combustion of organic matter and are thus generated when-ever fossil fuels or vegetation is burned; they are one of swhen-everal classes of carcinogenic chemicals present in tobacco smoke. They are also present in food. Reviews of the literature on PAHs in food have been written by Howard and Fazio [1], by Bartle [2] and by Phillips [3].
4.2.2. Methods of analysis
There have been two main approaches to measurement of PAHs in complex matrices such as food. The first is the determination of around 15–20 PAHs, including both carcinogenic compounds, such as benzo[a]pyrene, dibenz[a,h]anthracene, benzo[b]fluo-ranthene and indeno[1,2,3-cd]pyrene, and compounds considered to be non-carcino-genic, such as phe-nanthrene, anthracene, pyrene and benzo[ghi]perylene. The other approach has used measurement of benzo[a]pyrene as a surrogate for all PAHs. The first approach gives a truer picture of the overall burden of PAHs in food, because the relative amounts of each can vary widely. However, often more than half of the total PAHs is accounted for by pyrene and fluoranthrene, which are non-carcinogenic and very weakly carcinogenic, respectively. Thus it can be argued that benzo[a]pyrene, because of its carcinogenic potency in experimental animals, represents a biologically significant measure of PAH contamination. Nevertheless, because benzo[a]pyrene is only one of many possible carcinogenic PAHs present, it is often preferable to determine a number of different PAHs.
4.2.3. Uncooked food
Contamination of the atmosphere with PAHs results in the compounds becoming deposited in the aquatic environment. Once there they are readily taken up by aquatic organisms [4] and so enter the food chain. Polycyclic aromatic hydrocarbons become concentrated in marine sediments, especially in coastal waters, where bottom-feeding fish and filter-feeding invertebrates are particularly prone to exposure and accumulation of the compounds. Sources of contamination can include oil spills and run-off from land of industrial effluent. Another example is the use of creosote-treated wood to support the cultivation of mussels [5], or to impound lobsters [6]. To some extent, the profile of different PAHs detected can give a clue as to the major source of the contamination, i.e. oil spill PAHs or combustion PAHs [4]. In general, fish have Specific biomarkers related to food: PAHs
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David Phillips
a greater ability to metabolise PAHs than do molluscs, so the compounds tend to persist more in the latter [4].
The presence of PAHs in a wide variety of plants has been demonstrated [7]. Three possible sources of contamination have been considered: uptake as a result of atmospheric exposure, uptake from the soil, and endogenous biosynthesis. Most investigators now consider that the most important route of contamination is via atmospheric exposure of plants to PAHs, with the other routes playing lesser, possibly negligible, roles [8,9].
Leaf vegetables can be a significant source of PAHs in the human diet, the main source of contamination being the deposition of airborne particles containing the compounds [10]. Those vegetables grown close to roads are likely to be contaminated with both PAHs and nitro-PAHs. Broad-leaved vegetables such as lettuce can have particularly high levels of PAHs [10]. Many other fresh vegetables contain relatively high levels of PAHs; as high, or higher, than the levels found in cooked meat (see below).
Vegetable oils are a source of dietary PAHs, and dairy products often contain lower concentrations of PAHs than their non-dairy equivalents [11–13]. Human breast milk has been reported to contain benzo[a]pyrene at levels of only parts per trillion (6.5 ng/kg) [14].
4.2.4. Processed food
The preservation of food by curing it with wood smoke is a process that has been used since antiquity. Since the production of wood smoke is an example of incomplete com-bustion, PAHs are generated. In a detailed analysis of smoked food, total PAH concen-trations in smoked meat ranged from 2.6–29.8 ppb, while in smoked fish the range was 9.3–86.6 ppb [15]. Concentrations of five carcinogenic PAHs (benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene, dibenz[a,h]anthracene and indeno[1,2,3-cd]py-rene) reached levels of 16.0 ppb (in salmon). Polycyclic aromatic hydrocarbons were also measured in liquid smoke seasonings, the total levels ranging up to 43.7 ppb, and the levels of the five selected compounds up to 10.2 ppb.
A study of smoked food commercially available in Canada produced similar findings [16]. Polycyclic aromatic hydrocarbons were detected in 19/43 smoked meat samples at levels of up to 13 ppb total PAHs. The compounds were also detected in 18/25 smoked fish samples, the levels ranging up to 141 ppb (in smoked oysters).
4.2.5. Cooked food
When food, particularly meat, is cooked over an open flame, PAHs are formed. Charred food of almost any composition will contain PAHs; however, normal roasting or frying of food does not produce copious quantities of PAHs [1]. Some of the highest levels of PAHs reported in foods have been detected in food cooked over open flames. For example, in barbecued meat total PAHs were found to be present at levels up to 164 ppb [16], with benzo[a]pyrene being present at levels as high as 30 ppb.
101 Specific biomarkers related to food: PAHs
4.2.6. PAHs in the total human diet
In an analysis of PAHs in the UK diet, it was found that the major contributions came from cereals (about one-third) and from oils and fats (also one-third) [17]. Fruits, vegetables and sugars contributed much of the remainder; however, the contributions of meat, fish, milk and beverages were comparatively minor. Based on a total daily consumption of 1.46 kg food and beverages, the total daily dietary load of PAHs was calculated to be 3.70 µg. A similar study of the Dutch diet estimated an average daily intake of PAHs at between 5 and 17 µg/day. Compounds for which there is at least some evidence for carcinogenic activity accounted for about half of the total. The Dutch study concluded that cereal products were a major source of PAHs, and that vegetable oils and their products were also major contributors.
Although there are numerous differences between the diets of northern and southern Europe, analysis of the Italian diet has shown similar levels and sources of PAHs to those described above [18]. Total daily intake of PAHs was estimated to be 3 µg/day and, as with the Dutch study, nearly half of this was contributed by carcinogenic compo-nents of the class. In New Zealand, it has been estimated that mean dietary intake of PAHs amounts to 1.2 µg daily [13]. A figure of 3 µg/day was also estimated by Menzie
et al. [19] to be the median intake level of PAHs, and these authors concluded that
a vegetarian diet, if high in leafy vegetables and unrefined grains, can result in elevated PAH intake compared with the average diet. Exposure is also elevated in diets high in smoked and barbecued meat and fish.
High levels of PAHs have been detected in both raw and cooked cereal samples from the Linxian region of China [20]. Levels of up to 50 ppb of benzo[a]pyrene were detected, comparable with levels found in well-done barbecued meat in other studies. The probable source is the extensive use of soft coal for both heating and cooking purposes in unventilated dwellings. This region of China has a high incidence of oesophageal cancer. In comparison with PAHs, nitro-PAHs, a major environmental source of which is diesel engine exhaust particulates, were not found to be present at detectable levels in most food samples analysed [21].
4.2.7. Diet versus other sources of human exposure
In Italy, dietary exposure to PAHs (3 µg/day) was estimated to be significantly higher that respiratory intake of PAHs from polluted urban air (370 ng/day) [18]. In a US study that used benzo[a]pyrene as a surrogate for all PAHs, estimates of the proportion of weekly exposure to benzo[a]pyrene that was derived from food were commonly about 70% in the homes of non-smokers [22]. Similarly, Menzie et al. [19] estimated that the median dietary intake of 3 µg/day (total PAHs) represents around 96% of the total daily exposure for non-smokers. Tobacco smoke adds around 2–5 µg/day for a 1 pack/day smoker, and up to 15 µg/day for a heavy smoker (3 packs/day).
These representative studies indicate that of all the possible routes of human exposure to environmental PAHs, ingestion is predominant. This is borne out by biomonitoring studies of human exposure to PAHs.
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4.2.8. Biomarkers of human exposure to PAHs
Diets that are rich in PAHs have been reported to result in the excretion of urine with mutagenic activity [23]. It has been shown that cultured human colon tissue can meta-bolise benzo[a]pyrene to DNA-binding products via diol-epoxide formation [24] and, more recently, direct evidence has been obtained for the formation of benzo[a]py-rene-DNA adducts in human colon in vivo [25].
In a small controlled experiment, four volunteers abstained from eating barbecued or smoked meat for one month and then ate barbecued hamburgers daily for a week [26]. In two individuals the levels of PAH-DNA adducts in their white blood cells increased 3- to 6-fold over the levels during the preceding month, while in the other two volunteers there was no increase in adduct levels. In a subsequent study ten subjects were monitored [27]. DNA adduct levels increased in only four of the subjects; however, urinary levels of 1-hydroxypyrene-glucuronide increased by a factor of between 10 and 80 over baseline levels in all subjects, although the elevation was short-lived (in some cases only 24 h). Thus both methods of analysis revealed considerable interindividual variations.
Similar findings have been reported by van Maanen et al. [28]. They studied 21 individuals, measuring their DNA adducts in mononuclear cells by 32P-postlabelling and their urinary 1-hydroxypyrene excretion by high performance liquid chromatography (HPLC)--fluorescence. After a period of abstinence from eating barbecued or smoked food, the subjects consumed barbecued hamburgers containing 8.6 ppb benzo[a]pyrene for 5 days. Excreted levels of 1-hydroxypyrene were higher after the commencement of the meat consumption compared with before, and DNA adducts characteristic of those for-med by benzo[a]pyrene were detected in 8/21 subjects, at levels varying from 3–103 adducts/1010nucleotides.
There have also been a number of studies in which diet has been shown to play a confounding role in attempts to biomonitor occupational exposure to PAHs. These include studies of forest fire-fighters in the USA [29,30] and US army personnel working in Kuwait in 1991 [31].
In a comparison of an urban polluted area of the Czech Republic with a non-polluted mountainous area, the atmospheric concentrations of PAHs were measured in winter and summer and the urinary levels of 1-hydroxypyrene measured in selected individuals [32]. Although seasonal and locational variations in urinary 1-hydroxypyrene were seen in the volunteers, they did not reflect the large changes in atmospheric levels of PAHs. The authors concluded that dietary intake of PAHs probably masked the influence of air pollution on urinary 1-hydroxypyrene levels. This conclusion is in line with that of an earlier study of the influences on urinary excretion of 1-hydroxypyrene, which concluded that diet and tobacco smoking were the major determinants of levels, with inhalation of PAHs from ambient air relatively unimportant [33].
Immunohistochemical analysis of human oesophageal biopsies from the Linzian region of China, where the incidence of oesophageal cancer is high, has revealed the presence of PAH-DNA adducts [34]. As noted above, food samples from this area have been found to contain high levels of PAHs.
103 Specific biomarkers related to food: PAHs
Thus it is apparent that biomonitoring of human subjects reveals evidence of dietary exposure to PAHs, that there are large variations between individuals even in circum-stances where attempts have been made to make the levels of exposure similar, and that dietary exposure can be a significant effect in studies designed to determine occupational exposure to PAHs, or exposure due to urban pollution.
4.2.9. Conclusions
Because of the widespread distribution of PAHs in the environment, most types of food contain measurable levels of PAHs, generally in the parts per billion, or micrograms per kilogram, range. Although foods such as smoked or barbecued meat and fish may contain relatively high levels, unless these foods are consumed frequently, it is cereals and vegetables, and their fats and oils, that make the major contributions to human dietary exposure, not meat [2,17]. Nitro-PAHs do not appear to be present at significant levels in most foods.
It is clear that diet contributes substantially to non-occupational exposure to PAHs. For non-smokers, more than 70% of exposure is attributable to diet.
Epidemiological studies have indicated that a large proportion of human cancers is attributable, at least in part, to dietary factors [35]. Human exposure to environmental carcinogens, such as PAHs, is predominantly from dietary sources.
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