Mutagenesis induced by benzo[a]pyrene in lacZ mouse mammary and oral tissues: comparisons with mutagenesis in other organs and relationships to previous carcinogenicity assays

Wieslawa Kosinska1, Marcia d.M. von Pressentin1 and Joseph B. Guttenplan1,2,3

1 Division of Basic Sciences/Biochemistry, New York University, Dental Center, 345 East 24th Street, New York, NY 10100 and
2 Department of Environmental Medicine, New York University, Medical Center, 550 1st Avenue, New York, NY 10016, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thus far, in vivo mutagenic assays have detected organ-specific effects of benzo[a]pyrene (B[a]P) in a number of organs, but not in oral tissues and breast. Previous studies have shown that the mouse tongue is a target for tumorigenesis induced by B[a]P when incorporated into feed, and polycyclic aromatic hydrocarbons are carcinogens in mouse mammary tissue. In order to evaluate the capacity of the lacZ mouse in vivo mutagenesis assay to detect mutations in these target tissues, we have measured mutagenesis induced by B[a]P in breast and oral tissues. The oral tissue consisted of either tongue or a mixture of oral tissues from several sites in the oral cavity. B[a]P was more mutagenic in breast tissue than in most other organs tested (liver, lung and kidney) when administered at relatively high dose by gavage, and more mutagenic than in liver, but not lung, at low dose. When administered in an emulsion in drinking water, B[a]P was more mutagenic in oral tissues than in liver, and somewhat less mutagenic than in lung. Regardless of dose, the mutagenic activity was greatest in colon where it was much higher than in other organs. A reasonable correlation was observed between mutagenesis observed here and carcinogenesis in previous studies although some differences were noted. To our knowledge, this represents the first report of in vivo mutagenesis in non-tumor mammary and oral tissue, and the results indicate these organs can efficiently metabolize B[a]P to genotoxic products, although some transport of active metabolites from the liver cannot be ruled out. The lacZ mouse mutagenesis assay may represent a shorter term alternative to carcinogenesis assays for investigations of factors affecting initiation of carcinogenesis in mammary and oral tissues. However, it is less predictive of actual tumor formation.

Abbreviations: B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene 7,8-dihyro-7,8-dihydroxy-9,10-dihydro-9,10 epoxide; PAH, polycyclic aromatic hydrocarbon.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Benzo[a]pyrene (B[a]P) is a highly prevalent environmental carcinogen, a model for carcinogenic polycyclic aromatic hydrocarbons (PAHs), and a carcinogen in a number of organs in mice (17). It is reported to induce tumors in mice of the lung, forestomach, liver, spleen, esophagus, tongue and skin (by topical application) (17). The related PAH, 7,12-dimethylbenz[a]anthracene has been extensively employed in studies on carcinogenesis in rat mammary tissue, and is also a carcinogen in mouse mammary tissue (8). The actual sites of the tumors observed after administration of B[a]P depends on the strain and sex employed and the route and protocol of administration. B[a]P is believed to lead to genotoxic products via its metabolism to a 7,8-dihydrodiol-9,10 epoxide, which reacts with macromolecules, including DNA, to yield premutagenic adducts (7). Because there is a large database on the carcinogenic effects of B[a]P it has been utilized as a positive control in the evaluation of in vivo mutagenic assay systems (6,9,10).

The lacZ transgenic mouse (MutaTMMouse) was developed to detect mutations in any organ of the mouse (11,12). This system and the related lacI system (13) contain a reporter gene incorporated into a shuttle vector which can be rescued from the tissue of interest and analyzed for mutations. Presumably, tissues with enhanced levels of mutagenesis relative to background levels are initiated and are at increased risk of developing tumors. However, these assays monitor mutagenesis in a reporter gene and not in critical growth control genes and, additionally, factors affecting promotion and progression in particular cell types of organs of interest may lead to differences between organ specificities in mutagenesis and tumorigenesis. Although B[a]P has been reported to induce mutagenesis in lac mice in a number of target organs (6,9,10), there are no reports that it induces mutagenesis in tongue or mammary tissue. Indeed, we are aware of no reports on mutagenesis induced in vivo in these tissues by any carcinogens. We report here that B[a]P is mutagenic in tongue, other pooled oral tissues and mammary tissue of lacZ mice and that the mutagenic potency of B[a]P in these tissues is comparable with, and under some conditions greater than, that in other target organs.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Female mice (MutaTMMouse; 6 weeks old) were purchased from Covance Research Products (Denver, PA) and acclimatized for 1 week before the start of treatments. The mice were maintained on an AIN-76 diet (ICN Pharmaceuticals, Aurora, OH) and were allowed food and water ad libitum.

Treatment protocols
After the acclimatization period B[a]P (Sigma, St Louis, MO) was administered by gavage in corn oil (0.2–0.3 ml of 2 or 10 mg/ml on days 1, 3, 5, 8 and 12, for a total of five doses). The individual doses administered were 25 and 125 mg/kg. For all groups, after the last dose, the mice were left 2 weeks for expression of mutations, and then killed. Additionally, in one experiment designed to measure the effectiveness of B[a]P at inducing mutations in the oral cavity with other organs, B[a]P was added to the drinking water in a 20 mg/ml suspension of soybean lecithin (ICN Pharmaceuticals) resulting in a concentration of B[a]P of 0.1 mg/ml. In order to ensure homogeneity, the lecithin–water mix (500 ml in a 1 l Erlenmeyer flask) was first stirred vigorously with a magnetic stirrer at 50°C for 1 h and powdered B[a]P was slowly added under a fume hood to a concentration of 0.1 mg/ml. The mixture was sonicated at a setting of six on a Branson Sonicator for five 30 s pulses (under a fume hood, with a mask of aluminium foil over the top of the flask). The mixture was then shaken vigorously in an orbital air shaker at 37°C for 2 h and then sonicated as above. Any unsuspended material was allowed to settle overnight and the supernatant was decanted. The concentration of B[a]P in the mix was determined by diluting a small aliquot of the mixture 1 in 10 000 in methanol and comparing its fluorescence (excitation, 380 nM; emission 410 nM) with that of standard solutions of B[a]P in methanol. The water bottle was shaken vigorously at the beginning of each day to ensure homogeneity, although only a trace amount of residue was seen. Although B[a]P had been administered in feed (5), it was felt that smokers have an increased risk for cancer of the oral cavity and therefore the tobacco-derived PAHs retained in the oral cavity are not necessarily simultaneously present in the oral cavity along with dietary components. Such components might result in competitive inhibition of activation, or of detoxification processes. The B[a]P was administered for 12 days. At the end of this period, the mice received a single dose of 125 mg/kg by gavage, as above. Based on an estimated water consumption of 4 ml/day and an average weight of 32 g per mouse the total dose was estimated as 273 mg/kg, including the gavaged dose. The mice were then left for 2 weeks before being killed. Five mice treated only with corn oil were utilized as controls.

DNA isolation
DNA was isolated by two methods. The first was a phenol–chloroform ethanol precipitation method and the second a phenol–chloroform dialysis method. The first method was used mainly for tongue and other pooled oral tissues. This latter tissue was comprised of palatal, gingival, buccal and sublingual tissue. In both methods, a portion of the tissue was gently homogenized by hand in a ground glass homogenizer using 3 ml of 10 mM Tris–HCl (pH 8.0), 10 mM EDTA and 150 mM NaCl per gram of tissue. To this mix, SDS at 1% and protease K (Sigma) to a concentration of 1 mg/ml, were added. The mixture was incubated overnight at 37°C or 2–3 h at 50°C, and then for 30 min at 37°C with 0.1 mg/ml RNase A (Sigma). After this, the mixture was extracted once with an equal volume of 50% phenol–chloroform, centrifuged in the presence of Phase Lock gel (5'->3'; Boulder, CO) and the aqueous phase was removed. The aqueous phase was layered onto a 0.25 µ pore-size nylon membrane (MSI, Westboro, MA) in a multichannel dialyzer and dialyzed overnight against 1 mM Tris–HCl (pH 7.4), 1 mM EDTA. The DNA solutions were removed and brought to 5 mM Tris (pH 7.4) with 1/10 vol of 50 mM Tris–HCl and stored at 4°C. In the case of liver, the tissue was homogenized in 3 vol 50 mM Tris–HCl (pH 8.0), 3 mM MgSO4, 150 mM NaCl, 250 mM sucrose and 0.1% SDS per gram of tissue. The homogenate was centrifuged for 6 min at 800 g to sediment nuclei. The supernatant was decanted and the nuclei were processed as above for the dialysis method. In the ethanol precipitation method, after the phenol–chloroform extraction, the aqueous layer was brought to 0.5 M NaCl and DNA was precipitated with 2 vol ethanol, and washed once with 70% ethanol.

Mutagenesis assay
Phage packaging was carried out using a Transpack packaging mix (Stratagene, La Jolla, CA), and the positive selection (galE) mutation assay was performed according to published techniques (14,15). The necessary bacterial strain, Escherichia coli C lac galE, was obtained from Ingeny (Leiden, The Netherlands).


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
At the low dose by gavage, B[a]P was mutagenic in the organs examined with a potency order of: lung > breast > liver (Figure 1Go). When administered by drink and gavage, B[a]P was mutagenic in most of the tissues examined, with the absolute differences between the tissues (with the exception of colon) relatively small (Figure 1Go). Essentially, no mutagenesis was observed in the kidney (Figure 2Go). When background levels of mutagenesis were subtracted, mutagenesis was highest in lung followed by breast and tongue and other pooled oral tissues and then by liver, where it was barely above background levels. Relative to other tissue, mutagenesis in breast tissue was most strongly dependent on the route of administration, being relatively higher when administered by gavage than in drinking water. Mutagenesis was much greater in the colon than in any of the other tissues. The mutant frequency was 132 ± 47 mutants/pfu.



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Fig. 1. Mean mutant frequencies in several organs of MutaTMMice treated with 5x25 mg/kg B[a]P in corn oil by gavage, or in corn-oil only controls. Controls are described in Materials and methods. Five mice per group were used. Results are expressed as group means ± SD. P < 0.05 in Student's t-test versus corresponding controls in all groups. The range of pfus and mutants in the B[a]P-treated mice were, 323 000–945 000 and 42–103, respectively. B, B[a]P-treated; Bre, breast; C, control; Li, liver; Lu, lung; pfu, plaque forming unit.

 


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Fig. 2. Mean mutant frequencies in several organs of MutaTMMice treated with 0.1 mg/kg B[a]P in a lecithin emulsion in drinking water followed by a single dose of 125 mg/kg by gavage in corn oil, or in untreated controls. Not shown are the results for colon (see text) which would be well off the scale for this figure. Four mice per group were used. Results are expressed as group means ± SD. P < 0.05 in Student's t-test versus corresponding controls for all groups except kidney. The range of pfus and mutants in the B[a]P-treated mice were, 173 000–761 000 and 11–56, respectively. B, B[a]P-treated; Bre, breast; C, control; Ki, kidney; Li, liver; Lu, lung; OT, pooled oral tissue (not including tongue); pfu, plaque forming unit; To, tongue.

 
At the high dose, B[a]P was highly mutagenic in most of the tissues examined (Figure 2Go). Compared with low doses, mutagenesis was particularly elevated in mammary tissue (~50% greater in mammary tissue than in liver and lung). As observed at the low dose, the mutagenic activity in the colon was much greater than that in other organs. Unlike the situation with mammary tissue, in the case of colon tissue, the route of administration of B[a]P was not a factor in its potency relative to that in other organs. B[a]P was relatively weakly mutagenic in kidney at the high dose. For liver, lung, colon and kidney at the high dose, the results here are very similar to those in a previous study on mutagenesis induced by B[a]P in lacZ male mice under similar conditions (6), although the mutant frequencies in liver and lung were ~2-fold greater here than reported previously. That study (6) employed the same five doses of B[a]P, but it was administered over 5 consecutive days, rather than over 12 days, and to male rather than female mice.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study compared B[a]P-induced mutagenesis in mammary and oral tissues with mutagenesis in several other organs of the lacZ mouse. At the lower doses, in addition to these other organs, B[a]P was mutagenic in mammary tissue, tongue and other pooled oral tissues. At the high dose, B[a]P was extremely potent in several organs including mammary tissue, with mutant frequencies one to two orders of magnitude above spontaneous levels. As mammary and oral tissues have not been investigated previously, this expands the number of organs reported to be targets for B[a]P-induced mutagenesis.

One factor presumably important in the genotoxicity of B[a]P is its bioactivation. B[a]P and other PAHs are known to be metabolized to genotoxic products by the cytochrome P450 system and, in particular, the CYP1 superfamily of cytochrome P450 isozymes (16,17). Lung and liver have significant ability to metabolize B[a]P to DNA-binding products after a single dose or multiple doses in rodents (18,19) and metabolism is greatly enhanced by pre-treatment with PAHs and certain other agents in these and other organs (17,20). The 2 week time period over which the mice were treated should have been more than sufficient to allow induction of cytochrome P450, and the induction should result in enhanced biotransformation of later doses. At the high dose metabolism by extrahepatic organs may have been favored because of a saturation of hepatic metabolism of B[a]P (see above), although this would not explain the increased mutagenic potency in mammary tissue relative to lung as compared with the lower doses. Also, a smaller fraction of B[a]P may have been metabolized by the liver when administered by gavage, because the bolus injection resulted in hepatic concentrations of B[a]P well above saturating levels for the metabolizing enzyme(s) in the liver. The high mutant frequency in mammary tissue suggests that B[a]P or a proximate mutagen, possibly 7,8-dihyro-7,8-dihydroxy-B[a]P can be metabolized to an ultimate mutagen in mammary tissue. Although transport of the probable ultimate mutagen, 7,8-dihydro-7,8-dihydroxy-9,10-dihydro-9,10 epoxide (BPDE), from other organs to mammary tissue cannot be ruled out (21) BPDE is very short lived, and it seems unlikely that transport of this metabolite from other organs would be so efficient that mutagenic activity in mammary tissue would be greater than that in other organs, such as liver, which are capable of producing the ultimate mutagen. It has been reported that BALB/c mouse mammary minces metabolize B[a]P to BPDE–DNA-binding products in vitro (22) and this result is consistent with in vivo results (22).

With respect to correlations between mutagenic and carcinogenic activity of B[a]P at any of the doses, several observations can made. In carcinogenesis studies, the treatment protocol, and the strain and sex of the mice all influence the tumor outcome and, therefore, a number of different sites of tumor formation have been reported. Notwithstanding the disparity in outcomes of carcinogenicity studies, all of the target organs assayed here and previously (6,9,10) in studies of in vivo mutagenesis are also susceptible to the mutagenic effects of B[a]P. This observation suggests that, generally, organs where B[a]P-induced mutagenesis occurs are potential sites for tumorigenesis. Factors such as the rate of cell proliferation, the cell types initiated and possibly the effects of specific genes in certain strains of mice (23,24) presumably play important roles in the eventual tumor outcome. It does appear then that in vivo mutagenesis in lacZ is a reasonably general marker for initiation of carcinogenesis, but not all sites of mutagenic activity are sites of tumor formation. For example, the lung was not a site for tumorigenesis in lacZ mice (6) although it was a major site for mutagenesis. However, it has been pointed out that early mortality from forestomach tumors in mice may preclude the development of lung tumors (5). On the other hand, the lung is a major site for tumorigenesis in the A/J mouse (3) and a minor site in infant B6C3F1 mice (4). A/J mice are particularly suceptible to lung carcinogenesis. It may also be significant that mutagenesis was observed in both tongue and other pooled sites in the oral cavity, but thus far only the tongue is a target organ for carcinogenesis (5). The colon, which thus far is not a reported target for B[a]P induced carcinogenesis, had the highest level of mutagenesis, consistent with a previous report (6). Probably the high rate of cellular proliferation resulted in very efficient fixation of mutations. On the other hand, since tumorigenesis is believed to involve the accumulation of mutations and chromosome abnormalities in multiple genes, the short lifetime of the target cells for mutagenesis may preclude such accumulation (23,25). In addition, the DNA isolated for the mutagenesis assay is bulk DNA, and may not reflect mutagenesis in stem cells.

Also significant was the observation that the non-target organ, kidney, exhibited weak or negative mutagenic responses. This result is also consistent with a previous report (6), and in that study mutagenesis in heart (a non-target organ) was also weak. Thus, it appears that a negative response in lacZ mutagenesis is a strong predictor of a negative response for carcinogenesis, although, as discussed above, the converse is not necessarily true. It is noteworthy that B[a]P is reported to lead to similar or higher levels of BPDE–DNA adducts in heart than in liver or lung (18), which are target organs. Based on this example, in vivo mutagenesis is a more relevant marker of initiation than DNA adduct levels, a result consistent with the fact that mutation fixation occurs subsequent to adduct formation.

In conclusion, this study reports for the first time that B[a]P is mutagenic in lacZ mouse tongue and mammary tissue. The mutagenic activity in mammary tissue was among the highest in the tissues monitored, with the exception of colon. The mutagenic activity of B[a]P in the oral cavity when administered in drinking water was similar to, or greater than, that in liver, and slightly lower than in lung. This result is consistent with results of studies using cultured rat cells, where oral tissue was of similar activity as other tissues (26). The observation of mutagenic activity of B[a]P in mouse tongue is also consistent with recent long-term feeding studies in mice, which have shown that the tongue is a major target organ (5). It has been previously reported that B[a]P is carcinogenic in B6C3F1 mouse esophagus (5), and the results of this and a previous study (6) have shown B[a]P is mutagenic in lacZ mouse lung. Preliminary results (not shown) indicate that B[a]P is very mutagenic in lacZ esophagus. Taken together, these observations lend support to the hypothesis that the upper aerodigestive tract is active in the biotransformation of procarcinogens and susceptible to their genotoxic effects.



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Fig. 3. Mean mutant frequencies in several organs of MutaTMMice treated with 5x125 mg/kg B[a]P in corn oil by gavage, or in corn oil only controls. Five mice per group were used. Results are expressed as group means ± SD. P < 0.05 in Student's t-test versus corresponding controls for all groups. The range of pfus and mutants in the B[a]P-treated mice were, 13 000–953 000 and 4–527, respectively. B, B[a]P-treated; Bre, breast; C, control; Ki, kidney; Li, liver; Lu, lung; pfu, plaque forming unit.

 

    Acknowledgments
 
This work was supported by grant no. 95B104-rev from the American Institute for Cancer Research and NIH grant no. CA/ES76281.


    Notes
 
3 To whom correspondence should be addressed Email: joseph.guttenplan{at}nyu.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 30, 1998; revised February 17, 1999; accepted March 1, 1999.