Exposure-route-dependent DNA adduct formation by polycyclic aromatic hydrocarbons

Roger W.L. Godschalk1, Edwin J.C. Moonen, Pauline A.E.L. Schilderman, Wendy M.R. Broekmans2, Jos C.S. Kleinjans and Frederik J. Van Schooten3

Department of Health Risk Analysis and Toxicology, Maastricht University, Universiteitssingel 50, PO Box 616, 6200 MD, Maastricht, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Understanding the kinetics of aromatic–DNA adducts in target tissues and white blood cells (WBC) would enhance the applicability of DNA adducts in WBC as surrogate source of DNA in biomonitoring studies. In the present study, rats were acutely exposed to benzo[a]pyrene (B[a]P; 10 mg/kg body wt) via intratracheal (i.t.), dermal and oral administration. DNA adducts were analyzed in relevant target organs and WBC by nuclease P1 enriched 32P-post-labeling at 1, 2, 4, 11 and 21 days after exposure. Additionally, the internal dose was assessed by measurement of urinary excretion of 3-hydroxy-B[a]P (3-OH-B[a]P). Total B[a]P–DNA adduct levels in WBC were highest after i.t. and oral administration, whereas DNA adducts were hardly detectable after dermal exposure. Highest adduct levels were reached at 2 days after exposure. In lung tissue, DNA adduct levels reached maximal values at 2 days and were highest after i.t., oral and dermal exposure, respectively. DNA adduct levels were significantly lower in WBC as compared with lung. Nonetheless, overall B[a]P–DNA adduct levels in WBC were significantly correlated with those in lung. In target organs, highest DNA adduct levels were observed in skin after topical application, and lowest in stomach after oral administration of B[a]P. Furthermore, DNA adduct levels in WBC were correlated with DNA adduct levels in skin after dermal exposure and stomach after oral administration of B[a]P. Two-fold higher levels of 3-OH-B[a]P were excreted after i.t. administration of B[a]P as compared with dermal or oral exposure. Urinary 3-OH-B[a]P concentrations were correlated with DNA adduct levels at the site of B[a]P application. Overall, it can be concluded that aromatic–DNA adduct levels in WBC can be applied as a surrogate source of DNA for the site of application of B[a]P and reflect binding to lung DNA, independently of the exposure route.

Abbreviations: 3-OH-B[a]P, 3-hydroxy-benzo[a]pyrene; B[a]P, benzo[a]pyrene; i.t., intratracheal instillation; NP1, nuclease P1; PAHs, polycyclic aromatic hydrocarbons; WBC, white blood cells.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment in complex mixtures, such as automobile exhaust, cigarette smoke, foods, water and urban air. Human body burden occurs via inhalation, ingestion and dermal absorption, and probably contributes to the overall human cancer risk (1). PAHs are thought to elicit cancer via covalent interactions with DNA, so-called PAH–DNA adducts (1), which offer a potential dosimeter for the body load of exposed individuals by these environmental genotoxins. PAHs exert their carcinogenic activities at the site of application (1). Also, systemic effects have been observed and the respiratory tract seems to be the most predominant site of B[a]P induced cancers. Unfortunately in humans, most target tissues for genotoxic agents are not readily accessible for monitoring DNA adduct formation and therefore peripheral white blood cells (WBC) most often served as surrogate source of exposed DNA (26). Still, the reliability of WBC as surrogate for target organs needs further research. In particular it is not clear how DNA adduct formation in WBC and relevant target organs may differ dependently on the exposure routes. Inhalatory exposure to PAHs in smokers or in occupationally exposed workers does not seem to be the only predominant route (7,8). Other exposure routes, especially via food, appear important for adduct formation in WBC as well. For instance, several studies showed that the consumption of PAH-containing foods enhanced DNA adduct levels in lymphocytes and the excretion of 1-hydroxypyrene in urine (79). Additionally, cutaneous application of coal-tar based ointments may result in the formation of measurable amounts of PAH–DNA adducts in total WBC (10) or separated WBC subpopulations (6). Therefore, an understanding of the influences of exposure routes on DNA adduct formation in WBC is relevant for exposure monitoring studies. Ideally, non-target cells should have similar properties as compared with tissues that are targets for tumor induction, with regard to carcinogen metabolism, DNA binding and repair and cell turnover. Detailed knowledge of these parameters for both target tissue and WBC might improve the utility of the latter as surrogate for target tissues.

In the present study, we investigated the relationship between DNA adduct levels in WBC and internal organs in rats acutely exposed to B[a]P via three different exposure routes; gavage, dermal administration or intratracheal (i.t.) instillation. Since in animal experiments, carcinogenic PAH become effective predominantly at the site of application (11), B[a]P–DNA adduct levels were studied in lung, stomach and skin by the 32P-post-labeling assay. WBC were analyzed by 32P-post-labeling as surrogate tissue. Additionally, excretion of the major B[a]P metabolite 3-hydroxy-B[a]P (3-OH-B[a]P) was assessed in 24 h urine to assess the whole body dose of B[a]P.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
All solutions used were sterile. B[a]P was purchased from Sigma (St Louis, MO). Tricaprylin was obtained from ICN Biochemicals Inc., OH. All other chemicals were purchased from Merck (Darmstadt, Germany). Water was purified by means of a milli-Q purification system.

Preparation of B[a]P-solutions for animal treatment
B[a]P was initially dissolved in acetone at a concentration of 10 mg/ml, which was used for the dermal application of B[a]P as described below. Six milliliters of this solution were added to an equal volume of tricaprylin and subsequently the acetone was evaporated under nitrogen, resulting in a concentration of 10 mg B[a]P/ml tricaprylin. This solution was used for oral administration of B[a]P as described below. Identically, 6 ml of B[a]P in acetone was added to 3 ml tricaprylin. Evaporation of the acetone resulted in 20 mg B[a]P/ml, which was used for the i.t. administration of B[a]P. To prevent heterogenous distributions of B[a]P within these solutions, all were vortexed for 24 h before use.

Animal treatment
Forty-five (n = 45) male Lewis rats weighing 250–320 g were acutely exposed to B[a]P (10 mg/kg body weight) via oral (n = 15), i.t. (n = 15) or dermal administration (n = 15). Rats were randomly allocated over the three exposure groups. Nine rats were used as controls (three rats per exposure route; received vehiculum only). Oral exposure: B[a]P was administered through a gavage needle (100 µl/100 g body wt). Immediately thereafter, rats were anesthetized by a s.c. injection of ketamin/xylazin (0.1 and 0.05 ml per 100 g body wt, respectively). Anesthetization was applied to prevent differences in experimental conditions as compared with i.t. and dermal exposure. Intratracheal exposure: rats were anesthetized by a s.c. injection of ketamin/xylazin, and subsequently a plastic tube (2 mm diameter) was placed into the trachea. B[a]P solutions were injected into the lung through this plastic tube (50 µl/100 g body wt) and artificial respiration was applied briefly. Dermal exposure: rats were anesthetized by a s.c. injection of ketamin/xylazin. Approximately 4 cm2 (2x2 cm) of the skin was shaved (on the back, between shoulders) and B[a]P solutions were pipetted on this area (100 µl/100 g body wt). The first 2 days after treatment of rats with an acute dose of B[a]P, the eating and drinking behavior was influenced as determined by reweighing the supplied food and water. However, relative weights of lung and stomach (organ weight/total body weight) were constant during the experiment.

The animals were housed individually in metabolic cages for collection of urine, in a room maintained at 25°C, 50% humidity and a 12 h light–dark cycle. Standard rodent lab chow (diet no. RSM-A; Hope Farms, Woerden, The Netherlands) and water were provided ad libitum. Each morning, 24 h urine was collected individually. The samples were weighed and stored at –20°C until analysis. To examine the time-course of adduct formation, the rats were killed 1, 2, 4, 11 and 21 days after treatment (three rats per time point). Lung, stomach and skin were removed, washed with PBS and quickly frozen at –20°C until DNA isolation. Blood was collected by aortic puncture and WBC were isolated by lysis of erythrocytes with 3 vol lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 10 mM EDTA, pH 7.4) at 4°C and subsequently collected by centrifugation at 4°C for 10 min at 800 g.

DNA isolation
Approximately 1 g of tissue was washed with 0.25 M sucrose/0.1 M Tris–HCl (pH 7.4), cut into small pieces and subsequently homogenized with a potter (1000 r.p.m.) in 1% SDS/1 mM EDTA. The solutions were incubated overnight at 37°C with 0.5 mg Proteinase K. WBC were lysed with 2.5 ml SDS/NEP (75 mM NaCl, 25 mM EDTA, 50 mg/ml Proteinase K, 1% SDS) and incubated for 4 h at 37°C. DNA was extracted with phenol–chloroform–isoamyl alcohol [25:24:1 (v/v/v)] and chloroform–isoamyl alcohol [24:1 (v/v)] respectively. The DNA was precipitated with 2 vol cold ethanol after addition of 1/30 vol 3 M sodium acetate, pH 5.3, and washed with 70% ethanol. Subsequently, DNA was dissolved in 5 mM Tris/1 mM EDTA, pH 7.4. RNase T1 (50 U/ml) and RNase A (100 µg/ml) were added, followed by 30 min incubation at 37°C. The solutions were extracted with chloroform–isoamyl alcohol [24:1 (v/v)] and DNA was precipitated from the aqueous phase with cold ethanol, washed with 70% ethanol and dissolved in 2 mM Tris, pH 7.4. Concentration and purity were determined spectrophotometrically by absorbances at 230, 260 and 280 nm. In all cases, A260/A280 were ~1.8 and A230/A260 were ~0.43. The final volume was adjusted to achieve a DNA concentration of 2 mg/ml.

32P-post-labeling
The 32P-post-labeling assay was performed as described by Reddy and Randerath (12) with some modifications. Briefly, DNA (~10 µg) was digested using micrococcal endonuclease (0.4 U) and spleen phosphodiesterase (2.8 µg) for 3 h at 37°C. Subsequently, half of the digest was treated with nuclease P1 (6.3 µg) for 40 min at 37°C. The modified nucleotides were labeled with [{gamma}-32P]ATP (50 µCi/sample) by incubation with T4-polynucleotide kinase (5.0 U) for 30 min at 37°C. NP1-efficiency and ATP-excess were checked with an aliquot of the NP1 treated fraction by one-dimensional chromatography on poly(ethylenimine) (PEI)-cellulose sheets (solvent: 0.12 M NaH2PO4 pH 6.8 on Merck-sheets). Radiolabeled adduct nucleotide biphosphates were separated by chromatography on PEI-cellulose sheets (Machery Nagel, Germany). The following solvent systems were used: D1, 1 M NaH2PO4 pH 6.5; D2, 8.5 M urea, 5.3 M lithium formate pH 3.5; D3, 1.2 M lithium chloride, 0.5 M Tris, 8.5 M urea, pH 8.0; D4, 1.7 M NaH2PO4 pH 6.0. The remaining half of the digest was used to determine the final amount of DNA in the assay; the normal nucleotides were labeled with [{gamma}-32P]ATP (15 µCi/sample) by incubation with T4-polynucleotide kinase (2.5 U) for 30 min at 37°C. Nucleotides were separated by one-dimensional chromatography on PEI-cellulose sheets (solvent: 0.12 M NaH2PO4 pH 6.8 on Merck-sheets). A dAp standard (27.5 pmol/µl) was labeled in each experiment for quantitation purposes. In each experiment, three standards of [3H]BPDE-modified DNA with known modification levels (1 per 107, 108 and 109 nucleotides) were run in parallel for quantitation purposes. These standards were made by in vitro incubation of [3H]anti-BPDE with calf-thymus DNA. The modification level was determined, using the radioactivity of DNA-bound BPDE and the specific activity of [3H]BPDE. Anti-BPDE will predominantly bind to deoxyguanosine (BPDE-dG), but adducts with other nucleotides (e.g. deoxyadenosine) will also be formed. Quantitation was performed using phosphor imaging technology (Molecular Dynamics, Sunnyvale, CA) with which detection limits of 1 adduct per 109 nucleotides can be obtained. Interassay variation was <25%.

3-OH-B[a]P in rat urine
Detection of conjugated and unconjugated 3-OH-B[a]P in 24 h urine was performed as described by Jongeneelen et al. (13). The method consisted of enzymatic hydrolysis with ß-glucuronidase and arylsulfatase, solid-phase extraction on a Sep-pak C-18 cartridge and elution with methanol. Reversed-phase HPLC (Kratos solvent delivery system; Column: Hypersil 5 ODS) and fluorescence detection (Perkin-Elmer LS-30) were applied, using excitation and emission wavelengths of 253 and 432 nm. Peak area was integrated for quantification. The detection limit was 200 ng 3-OH-B[a]P/l urine. Inter- and intra-assay variation were 14 and 6%, respectively.

Statistics
Results are presented as means ± SD (data were normally distributed). Two-tailed Student's t-tests were applied to assess statistical differences between DNA adduct levels in various tissues, and linear regression was applied to examine the quantitative relationship between DNA adducts in WBC and tissues. P < 0.05 is considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Qualitative results of adduct analysis in B[a]P-treated rats
In WBC DNA of exposed rats, two DNA adduct spots were observed independently of the exposure route, of which the predominant one comigrated the BPDE-dG adduct standard. The second adduct spot migrated less as compared with the BPDE-dG adduct standard in both chromatographic directions. In lung, stomach and skin, two similar adduct spots were observed, but in B[a]P-treated skin an additional third DNA adduct spot was found (Figure 1Go). Although exact adduct identification is not possible by NP1-enriched 32P-post-labeling, adduct formation in WBC seemed to be qualitatively in agreement with DNA adducts found in organs.



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Fig. 1. Adduct profiles obtained by 32P-post-labeling. Origins are located at the lower left corner and were excised before analysis. (A) WBC DNA of a non-treated rat. (B) WBC DNA of a rat exposed to B[a]P by gavage (t = 2 days after exposure). (C) Lung DNA of a rat i.t. exposed to B[a]P (t = 2 days). (D) Stomach DNA of a rat orally exposed to B[a]P (t = 1 day). (E) Skin DNA of a rat dermally exposed to B[a]P (t = 2 days). (F) BPDE–DNA adduct standard (modification level: 1 adduct per 107 nucleotides).

 
Quantitative results of adduct analysis in rat organs and WBC
Since adduct identification is not possible, quantitative analysis of DNA adducts is presented as total DNA adduct levels only (results using the spot that co-migrated with the BPDE-dG standard are essentially the same). Total DNA adduct levels were calculated by adding the levels of individual DNA adduct spots. In WBC, maximal adduct levels after i.t. (4.3 ± 0.5 adducts per 108 nucleotides), oral (3.6 ± 1.8 adducts) or dermal (0.3 ± 0.2 adducts) application of B[a]P, respectively, were found at ~2 days after exposure (Figure 2Go). The estimated half-life of DNA adducts in WBC was 2 weeks, calculated on basis of a semi-logarithmic plot. In lung tissue of exposed rats, highest adduct levels were observed at day 2 after exposure via i.t. (20.5 ± 5.1 adducts per 108), oral (6.9 ± 2.3 adducts) and dermal (0.5 ± 0.2 adducts) administration of B[a]P. The removal of DNA adducts in lung tissue over a period of 21 days appeared to be inversely related to the maximal DNA adduct level. Highest DNA adduct levels in lung were observed after i.t. exposure, but adduct levels decreased relatively quickly (at day 21, only 34% of the maximal adduct level was detected). Oral administration of B[a]P resulted in maximal adduct levels in lung that were ~3-fold lower as compared with i.t. exposure, but adduct persistence was higher (at day 21, 74% of the maximal adduct level was still detectable). Highest persistence of DNA adducts in lung was observed after dermal application of B[a]P (no decrease of adducts from day 2 to day 21), whereas maximal DNA adduct levels were only 0.5 ± 0.2 adducts per 108 nucleotides (Figure 2Go). Since carcinogenic PAH become effective predominantly at the site of application, also DNA adduct formation in skin after dermal application and stomach after oral administration of B[a]P was studied. High adduct levels were found in skin DNA at 2 days after exposure by dermal application of B[a]P (70.3 ± 14.0 adducts per 108 nucleotides). Total DNA adduct levels in stomach after oral administration of B[a]P were surprisingly low. In stomach, maximal DNA adduct levels were observed at day 1 after exposure (1.6 ± 0.5 adducts per 108 nucleotides), and thereafter adduct levels gradually decreased (Figure 3Go). Adduct persistence appeared to be identical for both tissues, with calculated half-lives of 16 days. DNA adduct levels in WBC after i.t., oral and dermal exposure were significantly lower as compared with DNA adducts in lung (~5-, 3- and 4-fold, respectively; P < 0.001). If DNA adducts in WBC were compared with adduct levels at the site of application, significantly higher adduct levels were found in lung (P = 0.0001) and skin (P = 0.0004) after i.t. and dermal application, respectively. On the other hand, exposure of rats to B[a]P by gavage resulted in ~2.5-fold higher adduct levels in WBC as compared with the stomach; the site of entry of B[a]P (P = 0.0012).



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Fig. 2. Kinetics of DNA adduct levels in lung tissue (A) and WBC (B) after acute exposure to B[a]P via i.t. (filled circles), oral (open circles) and dermal (open squares) application of B[a]P (10 mg/kg body wt).

 


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Fig. 3. Kinetics of DNA adducts in rat lung after i.t. exposure (filled circles), in stomach after oral administration (open circles) and in skin after dermal application (open squares) of B[a]P (10 mg/kg body wt).

 
Regression analysis of DNA adducts in WBC versus tissues
To determine whether DNA adducts in WBC quantitatively represent DNA adducts in target organs, linear regression models were applied. The relationship between DNA adduct levels in WBC (as independent variable x) and total DNA adduct levels in lung tissue (as dependent variable y) was influenced by the exposure route. The slopes of the regression lines after i.t., oral and dermal application of B[a]P were 5.0 [95% confidence interval (CI): 3.7–6.4, P < 0.001], 1.4 (95% CI: 0.7–2.0, P < 0.001) and 0.7 (95% CI: –0.3–1.7, P = 0.18), respectively (Figure 4AGo). The slope of the regression line mainly depends on the ratio between DNA adduct levels in lung and WBC. The overall relationship between DNA adduct levels in WBC and lung was highly significant (r = 0.83, P = 0.0001) (Figure 4BGo), but large scattering was observed. If the exposure route was taken into account (multiple regression analysis), the fraction of explained variance (R2) was only slightly increased from 0.67 to 0.76. Correspondingly, highly significant relationships between DNA adducts in WBC and stomach after oral application of B[a]P (r = 0.80, P = 0.0001) and between WBC and skin after dermal application of B[a]P (r = 0.61, P = 0.0077) were observed.



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Fig. 4. Relationship between total DNA adduct levels in WBC (x) and lung tissue (y) after three different exposure routes. (A) Intratracheally (filled circles), orally (open circles) and dermally exposed rats (open squares). (B) Overall relationship (r = 0.83, P = 0.0001) with 95% CI.

 
3-OH-B[a]P excretion in 24 h urine
To assess the internal dose, we measured a major B[a]P metabolite, 3-OH-B[a]P, in 24 h urine (the conjugated as well as the unconjugated form of 3-OH-B[a]P). Highest excretion levels were found after i.t. exposure, which reached a `peak' value of 11.5 µg/24 h at day 2 after exposure. Highest levels of urinary 3-OH-B[a]P after dermal and oral exposure were 4.8 µg/24 h (at day 2) and 4.5 µg/24 h (at day 1), respectively. Thereafter, the excretion quickly decreased to background levels (Figure 5Go). Low levels of 3-OH-B[a]P were found in rats receiving vehiculum only (12.6 ± 6.8 ng/24 h).



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Fig. 5. Kinetics of 3-OH-B[a]P excretion in 24 h urine of rats after acute i.t. exposure (filled circles), oral administration (open circles) and dermal application (open squares) of B[a]P (10 mg/kg body wt).

 
Cumulative excretion was assessed by the area under the curve over a period of 21 days, and added up to ~31 µg after i.t. exposure, i.e. 1% of the total administered dose, whereas dermal and oral exposure to B[a]P resulted in a cumulative excretion of 3-OH-B[a]P that added up to a total of 18 and 12 µg (0.6 and 0.4% of the administered dose, respectively).

Urinary 3-OH-B[a]P concentrations at single time-points and DNA adducts in lung after i.t. exposure were highly correlated (r = 0.85, P = 0.0001). Similar results were found for 3-OH-B[a]P in urine versus DNA adducts in skin after dermal exposure (r = 0.85, P = 0.0001), and 3-OH-B[a]P in urine versus DNA adducts in stomach after oral administration of B[a]P (r = 0.84, P = 0.0001). On the other hand, the exposure routes seemed to influence the relationship between 3-OH-B[a]P levels in urine and DNA adduct levels in lung, which was only highly correlated after i.t. exposure, but not after oral (r = 0.48, P = 0.05) or dermal (r = 0.29, P = 0.237) treatment of rats with B[a]P. Generally, the excretion kinetics of 3-OH-B[a]P coincided with the rate of DNA adduct formation in tissues at the site of B[a]P application.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Determining adduct kinetics in both target and non-target tissue will help in selecting tissues or fluids most appropriate for biomonitoring of PAH exposure, and will help in the interpretation of data obtained from target and surrogate tissue (14). In this study, we investigated whether DNA adducts in WBC can be applied as surrogate source of DNA to assess the biologically effective dose in major target organs after different routes of exposure to B[a]P. In rats, exposed to B[a]P by gavage, i.t. or dermal application, DNA adduct formation and elimination curves were determined in WBC, lung, skin and stomach. The various exposure routes had different impacts on DNA adduct formation and removal in WBC and target organs. Still, a significant overall relationship was observed between DNA adducts in WBC and lung. DNA adduct levels in WBC were consistently lower as compared with those in lung of the same animal, which indicates that DNA adduct levels in WBC might be an underestimate of the biologically effective dose in this target tissue. Similarly, significant relationships were observed between WBC and stomach after oral exposure and between WBC and skin after dermal exposure to B[a]P. Moreover, urinary excretion of 3-OH-B[a]P seemed to coincide with DNA adduct levels at the site of B[a]P application at the same time point, and the cumulative excretion of 3-OH-B[a]P over a period of 21 days added up to <1% of the total administered dose.

In humans, the relationship between aromatic–DNA adduct levels in target tissues and adduct levels in WBC or subpopulations of WBC remains a matter of debate, because contradicting results were reported. Some studies found that adducts in WBC DNA did not reflect binding to lung DNA (15,16). On the other hand, Wiencke et al. (17) did find a relationship between DNA adducts in mononucleated WBC and lung tissue of smokers. Additionally, Szyfter et al. (18) demonstrated a high correlation between the level of aromatic–DNA adducts in larynx cells and leukocytes of humans with primary larynx tumours, and recently Godschalk et al. (6) showed that adduct levels in isolated monocytes and lymphocytes were related with adduct levels in skin of individuals topically treated with coal-tar ointments. In humans, studies to further elucidate this relationship between WBC (subpopulations) and potential target organs are difficult to perform, and the use of animal models is therefore a valuable approach. In laboratory rodents, several such studies have been done using various classes of chemical carcinogens, including heterocyclic amines (19,20), methylating agents (21) and PAHs (2224). Nesnow et al. (23) observed a significant relationship between DNA adducts in WBC and lung tissue of rats over a period of 56 days after i.p. injection of B[a]P. Identically, Qu and Stacey (24) found DNA adducts in WBC to reflect the binding of B[a]P in lung after single and multiple injections of B[a]P. However, i.p. injections of B[a]P are not relevant for the human situation. In the present study, it was found that DNA adducts in WBC and target organs may vary due to differences in exposure routes with more relevance for the human situation. Nonetheless, an overall relationship was observed between DNA adducts in WBC and lung. Additionally, DNA adduct concentrations in WBC were related to those at the site of B[a]P application. Thus, studies using animals exposed to a single carcinogenic compound, including this one, indicate that DNA adducts in WBC might be of use as a molecular dosimeter for DNA adducts in remote tissues. However, the human situation is of course much more complex; exposure to chemical mixtures via multiple routes over prolonged periods of time. It is questionable whether DNA adducts in WBC still reflect the binding of carcinogens in target organs under these circumstances. Some studies in laboratory rodents tried to address this issue. For example, Genevois et al. (25) treated rats dermally with bitumen and coal-tar fume (complex mixtures containing high concentrations of aromatic compounds) and it was concluded that some of the detected adducts in lymphocytes may be used as a marker of exposure to bitumen. It remained necessary, however, to determine the persistence of these adducts in the lymphocytes and to compare it to their persistence in the various organs and no other routes of exposure were investigated. It can be concluded that more studies are needed.

Most carcinogenic PAH become effective predominantly at the site of application (11). As expected, DNA adduct levels in exposed skin were high. However, DNA adduct levels in stomach were surprisingly low after oral exposure to a relatively high dose of B[a]P. This is in line with the local carcinogenic effect of B[a]P, since considerably higher doses are required after oral exposure as compared with dermal application of B[a]P to reach a similar incidence of tumours (11). Under normal circumstances, the mucous layer of the gastrointestinal tract hampers the absorption of the lipophilic B[a]P. Nonetheless, DNA adduct levels in remote tissues and the urinary excretion of 3-OH-B[a]P indicated that considerable amounts of B[a]P were absorbed after oral exposure. Dermal exposure resulted in relatively high DNA adduct levels at the site of application, but the systemic effect (i.e. adduct formation in WBC and lung) appeared to be much lower. On the other hand, 3-OH-B[a]P excretion in dermally exposed animals was not lower as compared with oral exposed rodents. These observations suggest that the metabolic conversion of B[a]P to (non-)reactive derivatives in skin itself is of importance. However, dermal exposure to complex chemical mixtures that contain PAHs may result in higher systemic effect as compared with dermal treatment with single PAH (data not shown). Intratracheal instillation of B[a]P resulted in considerable systemic and local effects. Furthermore, analysis of 24 h urine showed that ~1% of the administered dose was excreted as 3-OH-B[a]P after i.t. exposure, whereas after oral and topical administration of B[a]P the cumulative excretion added up to only 0.4 and 0.6 %, respectively. Also, Jacob et al. (26) found higher levels of monohydroxylated metabolites of B[a]P in urine after i.t. instillation as compared with oral exposure. Furthermore, Jongeneelen et al. (27) reported that the cumulative excretion of 3-OH-B[a]P in rat urine was only 0.22 to 0.35% of the orally administered dose after three consecutive high doses of B[a]P.

The urinary excretion of 3-OH-B[a]P correlated significantly with adduct formation at the site of application of B[a]P. In a recent study, we found 3-OH-B[a]P concentrations in urine of individuals topically treated with ointments that contained B[a]P also correlated with BPDE–DNA adduct levels in skin DNA (6). On the other hand, most other studies in PAH-exposed humans used 1-hydroxypyrene in urine as dosimeter for the internal dose of PAH (5,8,28,29), because it can be detected more easily as it is excreted in high levels. However, the parent compound (pyrene) or its derivatives do not react with DNA. Furthermore, since the changes in urinary concentrations of PAH metabolites after exposure to PAHs are relatively fast, the sampling timing and/or period is of major importance. In the present study, levels of 3-OH-B[a]P were back to baseline levels within 11 days after the exposure; thus, measurements of urinary 3-OH-B[a]P seem to represent recent exposures only.

Overall, it can be concluded that aromatic–DNA adduct levels in WBC can be applied as surrogate source of DNA at the site of exposure and reflect binding of B[a]P derivatives to lung DNA, independently from the exposure route. Furthermore, the urinary excretion of 3-OH-B[a]P coincided with DNA adduct formation, but measurements of urinary PAH metabolites represent recent exposures only. Our results may form a basis for further studies, using more complex situations that are more relevant for human exposures.


    Notes
 
1 Present address: Department of Toxicology and Cancer Risk Factors, German Cancer Research Center, PO Box 101949, D-69009 Heidelberg, Germany Back

2 Present address: Department of Physiology, TNO Voeding, 3700 AJ, Zeist, The Netherlands Back

3 To whom correspondence should be addressed Email: f.vanschooten{at}grat.unimaas.nl Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 31, 1999; revised September 6, 1999; accepted September 22, 1999.