Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl hydrocarbon receptor

Abstract

The contribution of the aryl hydrocarbon receptor (AhR) in induction of a battery of xenobiotic-metabolizing enzymes has been studied extensively. However, no direct proof has been obtained that it plays a role in modulating carcinogenesis. To address the question of whether AhR is required for tumor induction, we have investigated the response of AhR-deficient mice to benzo[a]pyrene (B[a]P), a widely distributed environmental carcinogen. B[a]P treatment induced expression of the cytochrome P450 gene Cyp1a1 in the skin and liver of AhR-positive mice bearing +/+ and +/- genotypes and did not induce expression of the cytochrome P450 gene Cyp1a1 in AhR-null mice in either skin or liver. In contrast, Cyp1a2 gene expression was positive in liver irrespective of the presence or absence of the AhR gene, or B[a]P treatment, although its inducibility was lost in the AhR(-/-) mouse. All AhR-positive male mice of both +/+ and +/- genotypes that received subcutaneous injection of B[a]P (2 mg) on the first and the eighth days had developed subcutaneous tumors at the site of injection at the end of the 18-week experiment. In contrast, no tumors were apparent in any of the AhR-deficient mice. Likewise, topical application of B[a]P (200 microg) at weekly intervals to the skin of female mice for 25 weeks produced skin tumors only in the AhR-positive mice. Thus the carcinogenic action of B[a]P may be determined primarily by AhR, a transcriptional regulator of the gene for CYP1A1. The results of the present study provide direct evidence that AhR is involved in carcinogenesis.

Figure 1

Cyp1a1, Cyp1a2, and AhR gene expression in the skin and liver of AhR(+/+), AhR(+/−), and AhR(−/−) mice, with and without B[a]P treatment. One-microgram aliquots of RNA extracted from skin and liver of control and B[a]P-treated mice of the three genotypes were reverse-transcribed and analyzed by PCR using specific primers for the Cyp1a1, Cyp1a2, and AhR and β-actin genes.

Figure 2

Subcutaneous tumor induction in wild-type (▵) and AhR-deficient male mice (+/−, □; −/−, ○) injected with B[a]P.

Figure 3

Gross appearance of flank skins in AhR-wild-type mice (+/+), AhR-heterozygous mice (+/−), and AhR-deficient mice (−/−) injected subcutaneously with B[a]P.

Figure 4

(A–C) Histological appearance of a fibrosarcoma (A), a rhabdomyosarcoma (B), and a squamous cell carcinoma (C) induced in AhR(+/+) male mice by subcutaneous injection of B[a]P. (Hematoxylin/eosin staining, ×100.) (D–F) Histological appearance of a squamous cell carcinoma (D), papilloma (E), and keratoacanthoma (F) induced in AhR(+/+) female mice by topical application of B[a]P. (Hematoxylin/eosin staining, ×100 for D, ×5 for E and F.)

Figure 5

Skin tumor induction in AhR-wild-type (+/+) (▵) and AhR-deficient female mice (+/−, □; −/−, ○) by repeated topical application of B[a]P.

Figure 6

Gross appearance of skin tumors in AhR-deficient (−/−), AhR-heterozygous (+/−), and AhR-wild-type (+/+) female mice after repeated topical application of B[a]P.