Comparative metabolism of benzo[a]pyrene by human keratinocytes infected with high-risk human papillomavirus types 16 and 18 as episomal or integrated genomes

Abstract

Background: Infection with human papillomavirus (HPV) is a critical factor in the development of cervical cancer. Smoking is an additional risk factor. Tobacco smoke carcinogens, such as benzo[a]pyrene (B[a]P), and their cytochrome P450-related metabolites are present in significantly higher levels in the cervical mucus of women smokers than in nonsmokers. We determined the metabolism and P450 expression of B[a]P-treated human keratinocytes infected with HPV-16 or -18.

Materials and methods: Monolayer cultures of uninfected primary human foreskin keratinocytes, human vaginal and cervical keratinocytes carrying episomal genomes of HPV-16 and -18, respectively, and invasive cervical carcinoma cell lines carrying either HPV-16 or -18 genomes integrated into the host DNA, were incubated with 0.1 μM [(3)H]B[a]P. The resulting oxidative metabolites were analyzed and quantified by radioflow high-performance liquid chromatography. Additionally, all cell lines were incubated with unlabeled 0.1 μM B[a]P for Western blot analysis of cytochrome P450 1A1 and 1B1.

Results: Significant enhancement in levels of both detoxification and activation metabolites was found in incubations with all types of HPV-infected cells compared with control incubations (P < 0.05). The highest capacity to metabolize B[a]P was observed with cells containing integrated HPV-18 genomes. Induction of cytochrome 1B1 was observed in HPV-16 and -18 integrated, and in HPV-16 episomal cell types.

Conclusions: Both viral genotype and genomic status in the host cell affect B[a]P metabolism and cytochrome P450 1B1 expression. An increase of DNA-damaging metabolites might result from exposure of HPV-infected women to cigarette smoke carcinogens.

Keywords: Benzo[a]pyrene metabolism; benzo[a]pyrene; cervical cancer; cigarette smoke carcinogen; cytochrome P450 1A1; cytochrome P450 1B1; human papillomavirus.

Figure 1

Metabolic oxidation of benzo[a]pyrene. Metabolites identified in this study are depicted in boxes

Figure 2

High-performance liquid chromatography elution profiles of [³H]benzo[a]pyrene metabolites. (a) synthetic standards. (b) uninfected primary human foreskin keratinocytes. (c) human vaginal keratinocytes infected with episomal human papillomavirus (HPV-16). (d) human cervical keratinocytes infected with episomal HPV-18. (e) invasive cervical carcinoma keratinocytes with HPV-16 integrated into the genome. (f) invasive cervical carcinoma keratinocytes with HPV-18 integrated into the genome. Arrows have been used to clarify the positions of closely eluting peaks in the chromatograph

Figure 3

Levels of benzo[a]pyrene (B[a]P) metabolites, expressed as a percent of total radioactivity, from cell cultures after exposure for 24 h with 0.1 μM [³H]B[a]P

Figure 4

Western blot analysis of cell extracts. Lanes 1 and 2: uninfected primary foreskin keratinocytes. Lanes 3 and 4: cells infected with episomal human papillomavirus (HPV-16) DNA. Lanes 5 and 6: integrated HPV-16 DNA. Lanes 7 and 8: episomal HPV-18 DNA. Lanes 9 and 10: integrated HPV-18 DNA. The minus sign signifies untreated cells and the plus sign signifies treatment with 0.1 μM benzo[a]pyrene