Polycyclic aromatic hydrocarbons as skin carcinogens: comparison of benzo[a]pyrene, dibenzo[def,p]chrysene and three environmental mixtures in the FVB/N mouse

Abstract

The polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP), was compared to dibenzo[def,p]chrysene (DBC) and combinations of three environmental PAH mixtures (coal tar, diesel particulate and cigarette smoke condensate) using a two stage, FVB/N mouse skin tumor model. DBC (4nmol) was most potent, reaching 100% tumor incidence with a shorter latency to tumor formation, less than 20 weeks of 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion compared to all other treatments. Multiplicity was 4 times greater than BaP (400 nmol). Both PAHs produced primarily papillomas followed by squamous cell carcinoma and carcinoma in situ. Diesel particulate extract (1 mg SRM 1650b; mix 1) did not differ from toluene controls and failed to elicit a carcinogenic response. Addition of coal tar extract (1 mg SRM 1597a; mix 2) produced a response similar to BaP. Further addition of 2 mg of cigarette smoke condensate (mix 3) did not alter the response with mix 2. PAH-DNA adducts measured in epidermis 12 h post initiation and analyzed by ³²P post-labeling, did not correlate with tumor incidence. PAH-dependent alteration in transcriptome of skin 12 h post initiation was assessed by microarray. Principal component analysis (sum of all treatments) of the 922 significantly altered genes (p<0.05), showed DBC and BaP to cluster distinct from PAH mixtures and each other. BaP and mixtures up-regulated phase 1 and phase 2 metabolizing enzymes while DBC did not. The carcinogenicity with DBC and two of the mixtures was much greater than would be predicted based on published Relative Potency Factors (RPFs).

Fig. 1

Metabolic pathways for bioactivation of PAHs using the fjord containing dibenzo[def,p]chrysene as an example. From left to right, the peroxidase pathway resulting in radical cations which may be capable of forming DNA adducts. The most well characterized P450 CYP dependent epoxygenation hydrolysis by epoxide hydrolase shows one of two (+) and (-) trans- DBC-11,12 DHD products followed by epoxygenation to one of four DBCDE products capable of adducting to macromolecules such as DNA. Also shown is the AKR pathway producing semi-quinones and quinones. *These reversible reactions can produce radical oxygen species resulting in additional oxidative stress.

Fig.2

Kaplan-Meier estimation of percent tumor bearing animals throughout 25 wk of promotion with TPA (6.5 nmol/200 μl), twice weekly starting two weeks post initiation. Initiation doses were 200 μl toluene (Ctrl), 400 nmol (100 μg) BaP, 4 nmol (1.2 μg) DBC, 1 mg DPE (mix 1), 1 mg DPE + 1 mg CTE (mix 2), and 1 mg DPE + 1 mg CTE + 2 mg CSC (mix 3). Individuals that died tumor free before the end of 25 weeks and those that were tumor free at the end of 25 weeks were censored and are indicated by an *.

Fig. 3

Tumor multiplicity in tumor-bearing animals (TBA, female FVB/N mice) with at least one tumor after initiation with a carcinogen followed by 25 wk of promotion with TPA. Dots represent individual animals with at least one tumor at the end of promotion; mean numbers of tumors in a given treatment represented by line. The number of mice surviving to 10 months in each group from the initial 35 is indicated below the treatments.

Fig. 4

Proportional incidence of hyperplasia, dysplasia, papillomas, carcinoma in situ (CIS), and squamous cell carcinoma (SCC) in each treatment group, determined by histopathology as described in Materials and Methods.

Fig. 5

Histopathology of epidermal hyperplasia, squamous papilloma and squamous cell carcinoma of the skin. A. Low (left) and high (right) magnification of skin with epidermal hyperplasia. In the low magnification, normal thickness of the epidermis is shown on the left (arrow head) and increased thickness of the hyperplastic epidermis on the right (arrow). While the number of cell layers is increased, keratinocytes progress in the same orderly manner from basal cells (arrow head) at bottom to fully keratinized cells (arrow) at top as they do in normal epidermis. B. Low (left) and high (right) magnification of skin with squamous papilloma. At lower magnification, the small, protruding mass is bordered by epidermis of normal (left) to slightly increased thickness (right). It comprises a fibrovascular core (asterisk) covered with very thick epithelium that, at high magnification, recapitulates orderly keratinization of basal cells (arrow head) to fully keratinized cells (arrow) of normal and hyperplastic epidermis shown in A. C. Low (left) and high (right) magnification of skin with squamous cell carcinoma. At low magnification, the plaque-like mass has a flat to umbilicated top (asterisk) and deeply infiltrates into the subcutis (arrow heads). At higher magnification, keratinocytes are arranged in solid nests. Individual keratinocytes (arrow) and groups of keratinocytes (arrow heads) haphazardly keratinize without orderly progression. A concentrically layered keratin pearl is present at the upper right (asterisk in center). Note that high magnification is not the same as in A and B.

Fig. 6

DNA adduct formation in FVB mouse skin tissue 12 h post-treatment with 200 μl toluene (Ctrl), 400 nmol (100 μg) BaP, 4 nmol (1.2 μg) DBC, 1 mg DPE (mix 1), 1 mg DPE + 1 mg CTE (mix 2), or 1 mg DPE + 1 mg CTE + 2 mg CSC (mix 3). DNAadducts were measured by the nuclease P1 enrichment version of ³²P-postlabeling method. (A) Bars represent mean ± SD, N=5 pools, 2 mice/pool. Total adducts were measured across the DRZ, diagonal radioactive zone of the TLC autoradiogram. (B) Representative autoradiograms showing DNA adduct profiles.

Fig. 7

Principal components analysis of gene expression data by treatment. Each data point represents a biological replicate (N=4 per treatment). All genes differentially regulated (p<0.05) between treated and toluene control were included in analysis. Replicates cluster based on treatment group. Toluene control = pink; Mix 1 = brown; Mix 2 = gray; Mix 3 = green; B[a]P = red; DBC = blue.

Fig. 8

Comparison of differentially expressed genes among PAH mixture treatments by Venn diagram (A). Values represent genes significant (p<0.05) versus toluene control. Genes common between mix 2 and mix 3, but unique from mix 1 are shown as a heatmap (B). Values in heatmap are fold-change (Log₂) compared to toluene control (red is up regulated, green is down regulated and black is no change). GO biological processes significantly enriched (p<0.05) for up or down regulated genes are shown.

Fig. 9

Gene expression of select xenobiotic metabolizing enzymes, including Phase 1 CYP enzymes and Phase 2 enzymes, regulated by PAH mixtures 2 and 3. Values are average Log₂ fold-change (± SE) for each treatment group (N=4 biological replicates) measured by Agilent microarray. *Indicates p<0.05 compared to toluene control.