Site-specific mutagenesis in Escherichia coli by N2-deoxyguanosine adducts derived from the highly carcinogenic fjord-region benzo[c]phenanthrene 3,4-diol 1,2-epoxides.

Although there have been numerous studies of site-specific mutagenesis by dGuo adducts of benzo[a]pyrene diol epoxides (B[a]P DEs), the present study represents the first example of site-specific mutagenesis by dGuo adducts of the highly carcinogenic benzo[c]phenanthrene 3,4-diol 1,2-epoxides (B[c]Ph DEs). The eight adducts that would result from cis- and trans-opening at C-1 of four optically active isomers of B[c]Ph DEs by the N(2)-amino group of dGuo were incorporated into 5'-TTCGAATCCTTCCCCC (context III) and 5'-GGGGTTCCCGAGCGGC (context IV) at the underlined site. These modified oligonucleotides along with unmodified controls were ligated into single-stranded M13mp7L2, which were then used to transfect SOS-induced Escherichia coli.

Protease inhibitor-induced stabilization of p21(waf1/cip1) and cell-cycle arrest in chemical carcinogen-exposed mammary and lung cells.

In previous studies, we have shown that human breast and lung carcinoma cells and mouse nontransformed type II lung cells fail to undergo cell-cycle arrest in G(1) phase in response to treatment with hydrocarbon carcinogens but rather accumulate in the S phase with damaged DNA. This situation may lead to replication of DNA on a damaged template and enhance frequency of mutations. The mechanism of this G(1) arrest failure was examined.

Hydrocarbon carcinogens induce p53 activity in normal mouse tissue.

Previous studies led to the suggestion that a sub class of hydrocarbon carcinogens have the capacity of damage DNA and evade normal cellular defence mechanisms. To examine the ability of hydrocarbon carcinogens to induce p53 transcriptional activity in normal tissues, we have used transgenic mice in which LacZ transgene is driven by a p53 response element. We demonstrate that transcriptionally active p53 is induced in normal tissue after exposure to putative stealth carcinogens in an identical manner to non-stealth agents.

Effect of benzo-ring hydroxyl groups on site-specific mutagenesis by tetrahydrobenzo[a]pyrene adducts at N(6) of deoxyadenosine.

We have previously investigated the mutations induced on replication in Escherichia coli of the M13mp7L2 genome containing each of the eight possible adducts derived from the four optically active 7,8-diol 9,10-epoxide metabolites of benzo[a]pyrene (B[a]P) by alkylation of a specific deoxyadenosine (dAdo) residue at N(6). Observed mutational frequencies depended in part on the relative spatial orientations of the three hydroxyl groups in these adducts. To determine how the presence or absence of these hydroxyl groups affects mutational response, we have synthesized 16-mer oligonucleotides with the same sequence as one of those previously studied with the diol epoxide adducts, but containing B[a]P-dAdo adducts in which two or all three of the adduct hydroxyl groups were replaced by hydrogen.

Differences between the mutational consequences of replication of cis- and trans-opened benzo[a]pyrene 7,8-diol 9,10-epoxide-deoxyguanosine adducts in M13mp7L2 constructs.

The four adducts at N(2) of deoxyguanosine derived from cis-opening at C-10 of four optically active isomers of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene were incorporated into 5'-TTCGAATCCTTCCCCC [context III(G)] and 5'-GGGGTTCCCGAGCGGC [context IV(G)] at the underlined site. The mutagenic consequences of these lesions in each of the two sequence contexts were examined after ligation of the modified oligonucleotides into single-stranded M13mp7L2 and replication of the vector in SOS-induced Escherichia coli. Total frequencies of base substitution mutations ranged between 14 and 48%.