Mutagenesis, Vol. 16, No. 4, 333-337,
July 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Studies of dose distribution, premutagenic events and mutation frequencies for benzo[a]pyrene aiming at low dose cancer risk estimation
1 Department of Genetic and Cellular Toxicology, 2 Department of Molecular Genome Research and 3 Department of Environmental Chemistry, Stockholm University, S-106 91 Stockholm and 4 Unit for Molecular Epidemiology, Novum, Karolinska Institute, S-141 57 Huddinge, Sweden
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Abstract |
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Cancer risk assessment of polycyclic aromatic hydrocarbons (PAH) is complicated by several of these compounds exerting a promoter action leading to high tumour incidences at high doses. Cancer risks at low doses corresponding to the uptake from air and food in the general environment would best be estimated on the basis of measurement of in vivo target doses of genotoxic (mutagenic) intermediates and a determination of mutation frequency per unit of dose. In experiments ultimately aiming at a risk assessment of environmental PAH from in vivo doses benzo[a]pyrene (BaP) was chosen as a model.
-Radiation has earlier been used as a reference standard in cancer risk estimation of genotoxic chemicals where dose equivalents (rad-equivalents) have been shown to give reliable risk estimates for several alkylating agents. Variation in dose of BaP diolepoxide between organs was studied by measurement of deoxyguanosine-N2 adducts in DNA after administration of BaP by gavage to mice of a strain with reduced DNA repair (Xpa–/–). The adduct levels in spleen, forestomach, stomach and small intestine were approximately the same; with the adduct level in spleen as reference it was twice as high in liver and lung and about half as high in colon tissue. A chemical or radiation dose is proportional to the cumulative frequency of putatively premutagenic changes (premutagenic hits) in DNA. The mutation frequency per premutagenic hit (genotoxic chemicals) and per unit of dose (-radiation) were calculated from acutely exposed V79 cells in order to determine the mutagenic effectiveness of each agent. Based on the mutagenic effectiveness determined in this study 10–4 Gy can be regarded equally effective in causing phenotypically expressed HPRT mutations as the dose of BaP which causes the formation of one deoxyguanosine-N2 adduct per cell. |
Introduction |
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The present study concerns pieces of knowledge required in the development of reliable methods for cancer risk estimation of polycyclic aromatic hydrocarbons (PAH). These compounds are general pollutants in air and food (Beckman Sundh et al., 1998; World Health Organization, 1998, and also occur in certain occupational environments (World Health Organization, 1998
). Preliminary estimates indicate that PAH in urban air contributes considerably to the cancer risk, but due to absence of adequate methods these estimates are very uncertain (Törnqvist and Ehrenberg, 1994) and call for improvement.
In the cancer-risk increasing action of genotoxic agents mutations in somatic cells play a key role (Fearon and Vogelstein, 1990; McCormick and Maher, 1994; Herrero-Jimenez et al., 2000). An increase of the mutation rate caused by exposure to a genotoxic agent will, in multiplicative interaction with inherited or acquired factors promoting growth and clonal expansion, lead to an increased probability (risk) of cancer. At low doses this may be described by a linear multiplicative model in which the risk increment is proportional to the background incidence multiplied by the dose of the genotoxic agent or metabolite (Granath et al., 1999).
A cancer risk estimation by extrapolation of incidence or mortality data from animal experiments is rendered inappropriate because of the promoter action of many PAH leading to convex dose–response curves with very high tumour incidences at high experimental doses. This promoter action, i.e. an increment of propensity for proliferation, is probably due to interaction of the unmetabolized hydrocarbons with the Ah receptor (Sjögren et al., 1996; Enan et al., 1998). At the often low exposure levels in the general environment the cancer risk increments due to PAH will be mainly determined by the induced incremental mutation frequencies rather than this promoter action. These mutation frequencies should be assumed to follow linear dependencies on doses of genotoxic metabolites, in many cases diolepoxides, of the PAH. For the achievement of a reliable risk estimation of a genotoxic factor it is necessary to know the target doses of the genotoxic factor and the relationship between induced mutation frequency and target dose.
In the present study, benzo[a]pyrene (BaP) was used as a model and indicator of PAH. A benzo[a]pyrene diol epoxide (BPDE) was assumed to be the major mutagenic metabolite through reaction with deoxyguanosine-N2 (dGuo-N2) in DNA and the formation of dGuo-N2–BPDE adducts being considered as putatively premutagenic events (`premutagenic hits'). The level of this adduct per unit amount of DNA could be used as a measure of dose, provided losses due to repair etc. could be avoided. To minimize such losses the inter-organ distribution of dose was studied in mice deficient in excision repair, and the relationship between frequencies of HPRT mutation and premutagenic hits were studied in V79 cells following acute exposure of short duration, where it could be assumed that the measured levels of dGuo-N2-DNA adducts reflect the cumulative levels of premutagenic events. With respect to mutants per premutagenic hit BaP was compared with -radiation and, using literature data, other PAH and simple alkylators.
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Materials and methods |
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Animals and treatment
An Xpa deficient and p53 heterozygous transgenic mouse strain with a C57Bl/6 genetic background crossbred as described previously (van Oostrom et al., 1999
) was used in the experiment. From the age of 17 weeks the mice (two males and three females) were treated by gavage with 13 mg BaP (Fluka, Switzerland) per kg body weight three times per week. After 7 weeks the treated mice and control mice (one male and one female) were killed and tissues from liver, lung, spleen, forestomach, stomach, small intestine and colon were collected and stored at –70°C.
Cell line
Cells of a V79 derived cell line, XEM2, expressing rat cytochrome P4501A1 were used. This cell line was kindly provided by Dr J.Doehmer (Technical University, Munich). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (supplemented with 10 mM HEPES, 10 IU/ml penicillin, 10 µg/ml streptomycin and 1.5 µg/ml fungizone with 10% fetal calf serum) according to Dogra et al. (1990).
Treatments of cells
Cells were reseeded in DMEM using either (A) 2x106 cells per 75 cm2 culture flask or (B) 1x106 cells per 25 cm2 culture flask. After one day of growth the medium was changed to 20 ml/flask of Hank's balanced salt solution (HBSS) containing Ca2+ and Mg2+ (supplemented with HEPES, antibiotics and calf serum, see above).
Acute intracellular exposure of the XEM2 cells to ±(anti)r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was obtained by treatment with ± trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BPD) (obtained from NCI Chemical Carcinogen Repository, Midwest Research Institute, Kansas City, MO). The cells from group A were exposed to BPD at five concentrations (in the range 0.05–0.7 µM) added from a stock solution of 0.1 mM BPD in dimethylsulfoxide (DMSO). The treatment was carried out in groups of three flasks per BPD concentration; three flasks were used as controls (addition of 140 µl DMSO). After addition of BPD and DMSO the media were thoroughly mixed and kept in the CO2 incubator for 3 h.
The cells from group B were exposed to 137Cs -radiation doses in the range 0.5–5 Gy in groups of two flasks per dose and two non-exposed flasks were used as controls. The dose rate was 0.54 Gy/min.
HPRT mutagenicity assay
After exposure the cells were washed with HBSS, trypsinized and 500 000 cells were added to 9 cm Petri dishes in DMEM and reseeded every third day. After a total expression time of 9 days cells were plated in DMEM containing 6-thioguanine (6-TG, 5 µg/ml), 200 000 cells/plate (five plates/dose), and in 6-TG-free medium, 100 cells/plate, for calculation of cloning efficiency (CE) and mutant frequency.
Isolation and digestion of DNA from animal tissue and XEM2 cells
Mouse tissues (0.1–0.2 g) were cut into small pieces and then homogenized. A major part of the BPD-treated, HBSS washed and trypsinized XEM2 cells (8–12x106) were spun down at 3000 g. The pellets from homogenized mouse tissue and XEM2 cells were washed in 10 ml buffer (0.15 M NaCl, 10 mM Tris–HCl pH 7.4) and retrieved after centrifugation at 2000 r.p.m. for 10 min. The isolation and digestion of DNA were then done according to Yang et al. (1996).
32P post-labelling
The DNA concentration was determined by measurement of absorbance at 260 nm and DNA purity by the A260/A280 ratio according to Szyfter et al. (1994). The 32P post-labelling was done according to Yang et al. (1996). All the adduct spots were excised together for Cerenkov counting while keeping the excised areas from different plates separate. The radioactivity measured at the adduct spot area in the negative control plate was used as background value when the adduct levels were calculated. In all experiments positive BPDE-dGMP and BPDE-treated DNA standards, see Phillips and Castegnaro (1999), and negative standards (digestion solution without DNA) were used. The recovery of the BPDE-treated DNA (1.11 adducts/108 nucleotides) was used to adjust the adduct levels in the in vitro samples. The average recovery from four analyses was 35% and in compatibility with earlier quantitative 32P-post-labelling studies (Segerbäck and Vodicka, 1993). The measured amounts of adducts were divided by 0.35 to obtain total adduct level. In order to express adduct levels per cell the calculated levels of adducts per nucleotide were multiplied by 12x109 nucleotides per mammalian cell (Lehninger, 1975).
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Results |
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Levels of observed BPDE adducts to dGuo-N2 of DNA in different tissues from Xpa deficient mice are given in Table I
. The data were collected from control mice and mice treated with BaP per os (total dose in 7 weeks 273 mg/kg). The adduct levels were in the range 55–63 dGuo-N2 adducts/108 nucleotides in spleen, forestomach, stomach and small intestine; with the adduct level in spleen as reference it was about twice as high in liver and lung (115 and 146) and about half as high in colon tissue (26).
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Treatment of the XEM2 cells with 50–200 nM BPD led to mutant frequency increments of 30–100 mutants per 105 cells with the mutant frequency increasing proportionally to dose (Table II
). Survival decreased with dose to 33% at the highest dose. The cloning efficiency (CE) was 100–78% in the dose range 0–700 nM BPD. BPD treatment with 50–700 nM gave adduct levels in the range 60–300x103 adducts per cell. The adducts were identified as deoxyguanosine-N2 (dGuo-N2) adducts from Rf values (TLC analysis) of a 32P post-labelled dGuo-N2 adduct standard (BPDE–dGMP). The number of adducts reached a steady state level above 400 nM BPD (possibly due to saturation of CYP1A1).
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Treatment of the XEM2 cells with 0.5–5 Gy
-radiation led to mutant frequency increments of 3–14 mutants per 105 cells with the mutant frequency increasing proportionally with dose (Table III). Survival decreased in approximately linear dependence on dose to 27% at the highest dose. Cloning efficiency (CE) was 100–77% in the dose interval 0–5 Gy.
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Discussion |
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Doses, premutagenic hits and mutants per premutagenic hit
Cancer risk estimation of radiation or reactive chemicals has to be based on assessed in vivo doses (particularly target doses, i.e. doses at tumour sites). Doses of chemically reactive compounds/metabolites are defined as the integrals over time of concentration (e.g. in mMh), which is comparable with radiation doses expressed as energy absorbed per unit of mass (Gy = Jxkg–1) (Ehrenberg et al., 1983
). It should be stressed that effects of inherited or acquired factors that modify the dose, i.e. changes in rates of formation or disappearance of reactive intermediates of chemicals, are included in the measured dose. The frequencies of primary putatively premutagenic changes (`premutagenic hits') formed in DNA are proportional to the measured dose. An observed frequency of mutants per premutagenic hit, in the genome or the coding sequence of a gene, reflects the probability that a primary lesion leads to mutation after repair, apoptosis and other processes that modify the manifestation of a mutation after the event considered as the premutagenic hit.
The present paper deals with the distribution of dose over target organs, the frequency of premutagenic hits in DNA per unit of dose, as well as the probability that a premutagenic hit leads to mutation, i.e. in principle, data of importance in cancer risk estimation of a genotoxic agent.
DNA adducts as a measure of dose
In the present study, doses, D, of the reactive diolepoxide of BaP (BPDE) were assessed from the levels of its adducts to dGuo-N2 in DNA. Since a dose, defined as the integral over time of concentration, is proportional to the cumulative level of adducts formed and since relevant data for the kinetics of the decrease of the adduct level through repair (and other processes) were not available, the experimental conditions were chosen with the aim that measured DNA adduct levels should approach the cumulative levels of adducts formed. Thus, an acute exposure of V79 cells was gained by treatment with BPD (the 7,8-trans-dihydrodiol of BaP) for 3 h using a cell line, XEM2, expressing CYP1A1, and preparing DNA for adduct analysis directly after treatment. From available data (MacLeod et al., 1991; Chen et al., 1992; Celotti et al., 1993) it was estimated that a loss of adducts through repair could be 20% at most under these conditions.
In the study of dose distribution in vivo a mouse strain deficient in excision repair (Xpa–/–) was used. In a previous experiment with Xpa–/– mice under the same conditions (de Vries et al., 1997) it was shown that adduct levels in studied organs increased linearly with time of BaP treatment for up to 9 weeks. It may therefore be assumed that, unless fast cell division occurs, the adduct levels after 7 weeks exposure in the present study will be representative of the cumulative levels of the adducts formed and thus of the doses. In the previously mentioned study (de Vries et al., 1997) the adduct levels were 2–3-fold lower in repair proficient mice (Xpa+/+).
For computation of dose (e.g. in mMh) the rate constant, kdGuo, for adduct formation should be known. However, it is possible to use the cumulative adduct level, AdGuo = kdGuoxD, as a relative dose measure.
Target dose and variation between tissues
BaP fed to rats is to a large extent excreted in faeces directly or after entero-hepatic circulation (Stavric and Klassen, 1994). The absorbed part is transported (to a great extent via the lymph vessels) to adipose and breast tissues where it is accumulated (Rees et al., 1971). BaP is then gradually released into the blood and to the different tissues; its distribution between tissues has so far only been studied to a limited extent. It should be remembered that BaP gives rise to metabolites other than BPDE that are reactive towards DNA (Penning et al., 1999). However, the diol epoxides of BaP exhibit the strongest genotoxic action observed among known BaP metabolites. This is in acceptable agreement with data for several other diol epoxides of PAH (World Health Organization, 1998).
In the present study the adduct levels were measured in seven different tissues in order to learn more about the distribution of BaP and its metabolites after chronic exposure. The found adduct levels were highest in liver and lung. The levels in spleen, forestomach, stomach and small intestine being 2–3-fold lower (Table I
). The higher adduct levels in liver and, possibly, lung may reflect contributions from induced CYP1A activities, single doses of 13 mg/kg BaP being slightly above the threshold for induction of ethoxyresorufin deethylase (EROD) activity in liver of C57Bl/6 mice (Vaca et al., 1992). Relatively high systemic adduct levels in the lung after oral treatment have also earlier been observed in an Xpa–/– mouse strain (de Vries et al., 1997) and in a study with rats (Godschalk et al., 2000). In the present study the adduct level in the colon was exceptionally low, probably due to a fast decrease of the DNA adduct level through high cell division rate in this tissue. The observed adduct level in the forestomach does not disclose an enhanced rate of bioactivation of BaP, expected for this tissue, which in administration by gavage or via the feed may be seen as the site of application. The high incidence of tumours in the forestomach which predominates relative to other tumours (Chen and Chu, 1991; Culp et al., 1998) might therefore be provoked mainly by a promoter action of the unmetabolized hydrocarbon. The cancer-risk/dose relationship in this organ calls for further experimental elucidation.
Putatively premutagenic adducts formed by PAH
The in vitro study was focused on DNA damages caused by metabolites from BPD, where formation of BPDE is favoured. The adducts formed in DNA by diol epoxides of PAH are mainly adducts bound to the exocyclic amino groups dGuo-N2 and deoxyadenosine-N6 (dAdo-N6) (Szeliga and Dipple, 1998) and are regarded as putatively premutagenic events of major importance. Studies on cells treated with PAH DE in vitro have given relevant information about the genotoxic action of such adducts and the adduct patterns are well correlated with mutational spectra. The manifest structural changes seen in DNA after PAH DE exposure are mainly point mutations (only 1–3% are deletions and insertions) (Bigger et al., 2000). After exposure to bay region PAH DE (such as BPDE), which give a comparatively high proportion of dGuo-N2 adducts, substitutions at GC base pairs (especially GC
TA transversions) are seen (Lambert et al., 1994; Wei et al., 1994; Szeliga and Dipple, 1998; Bigger et al., 2000). After exposure to fjord region PAH DE (e.g. benzo[c]phenanthrene), which give mixtures of dGuo-N2 and Ado-N6 adducts, substitutions at GC and AT base pairs are seen (Szeliga and Dipple, 1998; Bigger et al., 2000).
Mutants per putatively premutagenic hit
From the HPRT mutation frequencies (mutants per cell) and dGuo-N2 adduct levels (adducts per cell) in the BPD treated XEM2 cells observed in the present experiment, the mutants per premutagenic hit in the genome (MPH) were calculated (Table II). The average value was 4.4 ± 1.4 (SD)x10–9 mutants per adduct. For alkylating agents giving rise mainly to point mutations, MPH may be referred to as the calculated number of premutagenic hits in the coding regions (provided that premutagenic hits occur at random in the genome). The sum of the coding regions (exons) is 654 bp in HPRT (Jansen et al., 1992) out of the 6x109 bp in the genome. MPH for the coding regions of the gene would thus (from data in Table II) be computed to 0.04 for BPD, following acute exposure with preparation and analysis of dGuo-N2 adducts directly after treatment, the fraction lost by repair being negligable (cf. above).
From earlier studies in V79 cells (HPRT mutants and dGuo-N2 and Ado-N6 adducts) for four PAH (syn and anti diol epoxides of BaP, benzo[c]chrysene, benzo[g]chrysene and benzo[c]phenanthrene) (Phillips et al., 1991) values in the range of 0.01–0.04 per premutagenic hit in the HPRT exons were calculated; these values are consistent with the present values for V79 cells treated by BPD.
An in vivo value for MPH could be obtained from data (de Vries et al., 1997; Boerrigter, 1999; Swiger et al., 1999) for BaP-induced frequencies of phenotypically expressed mutation per lacZ gene (3000 bp) and DNA adduct levels in transgenic C57Bl/6 mice (Xpa deficient and proficient). MPH are in the range 0.01–0.02 (Xpa–/–) and 0.01–0.05 (Xpa+/+) in liver, lung and spleen, i.e. a range overlapping with the one found for HPRT mutations in vitro.
For a few simple alkylating agents where dGuo-O6 alkylations may be considered putatively premutagenic hits similar MPH values were obtained. Ethylene oxide gives 0.03, computed from rad-equivalence determination (Granath et al., 1999) and ethylating agents 0.08 computed from data derived from van Zeeland (1988).
There is thus a conspicuous similarity of mutagens with respect to the probability that a premutagenic hit leads to manifest mutation. For point mutations this probability is in the order of a few percent for premutagenic hits in the coding regions of the HPRT gene. This similarity may very well be fortuitous but is to some extent expected, at least for low molecular mass alkylators, which exhibit a predictability of mutagenic potency from rates of reactions towards DNA oxygens (Vogel et al., 1996).
As for ionizing radiation, too little is known about primary effects to permit a proper definition of premutagenic hits. If we generously define premutagenic hits as primary ionizations in hydrated DNA or as the sum of measurable structural changes (single- and double-strand breaks and base damages according to Ward (1994), the number of HPRT mutants per premutagenic hit in the genome turns out to be about 5-fold higher than the corresponding values for the chemicals discussed. This is due to the occurrence of genotoxically effective clusters of primary events (Goodhead, 1994). The genetic effects comprise various kinds of chromosomal rearrangements including deletions of single or several loci and was recognized early by Lea (1946) to be due to ionization clusters produced by 
-rays and track-ends of ionizing particles.
Viewpoints on cancer risk estimation
From the comparison of BPDE and -radiation with respect to HPRT mutant frequencies in V79 cells (Tables II and III) it follows that one dGuo-N2 BPDE adduct per cell is associated with about 4x10–9 mutants per cell and that 1 Gy -radiation induces 3–6x10–5 mutants per cell, i.e. that a BPDE dose leading to the formation of one adduct per cell has approximately the same mutagenic effectiveness as 10–4 Gy. In view of the demonstration that the ratio of the in vitro mutagenic potencies of ethylene oxide and -radiation is approximately the same as the ratio of carcinogenic potencies of these agents (Granath et al., 1999) it is probable that the found ratio for BaP and -radiation is valid for their relative carcinogenic potencies as well.
To the extent that the mouse model is relevant to man the data indicate that, following intake via the gastro-intestinal tract, i.e. the predominant route for human uptake of PAH (Hemminki and Pershagen, 1994; Törnqvist and Ehrenberg, 1994; Beckman Sundh et al., 1998) the inter-organ variation in dose, as shown in a mouse model, may amount to a factor about 2 around a mean value. A variation in target doses within such a relatively narrow range is advantageous with regard to estimation of cancer risk at low doses of PAH.
Since extrapolation from animal cancer tests would exaggerate the risk due to promoter action at higher doses, the radiation-dose equivalents of chemical doses appears at present to be a practicable way to estimate the risk coefficient in combination with a multiplicative model (Granath et al., 1999). For example, a dose of BaP leading to an incremental adduct level of 1x10–8 dGuo-N2 BPDE adducts per nucleotide would, according to the above comparison of mutagenic potencies, correspond to the risk increment caused by about 1 cGy.
However, when it comes to the risk estimation of an exposure situation in humans or for extrapolation from animal experiments, DNA adducts as a measure of dose is rendered questionable because of the modification of initially established adduct levels at varying rates, particularly by repair. For this reason the measurement of adducts to blood proteins with well-characterized turnover kinetics has been preferred as a safer dose monitor, and methods for analysis of PAH adducts to proteins from mutagenic PAH metabolites are being developed (Helleberg and Törnqvist, 2000).
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Acknowledgments |
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We thank Dr K.Yang, Unit for Molecular Epidemiology, Novum, Karolinska Institute, and Ms Y.Eklund, Department of Genetic and Cellular Toxicology, Stockholm University, for valuable discussions and technical assistance. The study was supported financially by Swedish Radiation Protection Institute, Foundation for Strategic Environmental Research, the Swedish National Board for Laboratory Animals and the Commission of the European Communities Biotechnology Programme (BI02-CT92-0004-NL).
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Notes |
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5 Present address: China Institute for Radiation Protection, Taiyuan, Shanxi 030006, China
6 To whom correspondence should be addressed. Email: margareta.tornqvist{at}mk.su.se
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Received on January 17, 2001; accepted on March 8, 2001.
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