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The xenobiotic metabolizing enzymes Cyp1a1 and Cyp1b1 can be induced by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-rho-dioxin (dioxin) via the aryl hydrocarbon receptor (AhR). These genes are differentially induced by dioxin in different mouse cell lines. In the mouse hepatoma cell line Hepa1c1c7 (Hepa-1), the Cyp1a1 gene is induced to very high levels by dioxin, but the levels of Cyp1b1 mRNA are extremely low and are not inducible by dioxin. The reverse is the case for the mouse embryonic fibroblast cell line C3H10T1/2, in which Cyp1b1 is induced to very high levels by dioxin, but the levels of Cyp1a1 mRNA are extremely low and not inducible by dioxin. However, dioxin treatment leads to the recruitment of AhR to the enhancer regions of both genes in both cell lines. Somatic cell hybrid clones generated between the two cell lines display high levels of induction of both genes in response to dioxin. Strong reactivation of the Cyp1a1 gene was also observed in C3H10T1/2 cell line after treatment with the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine (5-AzadC) and the histone deacetylase inhibitor, trichostatin-A (TSA). However, only modest reactivation of Cyp1b1 was observed in Hepa-1 cells after 5-AzadC or TSA treatment. These data demonstrate that the presence or absence of binding of AhR to regulatory regions is not responsible for determining the differences in levels of induction of the two genes in these cell lines and indicate that DNA methylation plays a major role in silencing of Cyp1a1 gene expression in C3H10T1/2 cells, but appears to play only a minor role in silencing Cyp1b1 gene expression in Hepa-1 cells, which likely occurs principally because Hepa-1 cells lack a factor required for high levels of induction of this gene., (Published by Elsevier Ireland Ltd.)

The CYP1A family of cytochrome P450s (CYPs), comprising CYP1A1, CYP1A2, and CYP1B1, plays a role in bioactivation of several procarcinogens to carcinogenic derivatives, and also in detoxification of several xenobiotic compounds. Resveratrol (3,4,5-trihydroxystelbine) is a naturally occurring compound that has been shown in a number of studies to inhibit the induction of CYP1A1 and CYP1B1 by dioxin (2,3,7,8-tetrachloro-dibenzo-p-dioxin), but the mechanism(s) of resveratrol inhibition is controversial. In the current study, 100nM dioxin treatment for 24, 48, and 72 h induced CYP1A1, CYP1A2, and CYP1B1 mRNA levels in the human breast cancer cell line MCF-7, and CYP1A1 and CYP1A2 mRNA levels in the human hepatocellular carcinoma cell line, HepG2. Simultaneous treatment with 10 microM resveratrol significantly inhibited dioxin-induced mRNA expression levels of these genes in both cell lines. Our studies are novel in that we used the chromatin immunoprecipitation assay to assay dioxin-induced recruitment of the aryl hydrocarbon receptor (AHR), and aryl hydrocarbon nuclear translocator (ARNT) to the enhancer regions and recruitment of RNA polymerase II to the promoter regions, of the CYP1A1 and CYP1B1 genes in their natural chromosomal settings. These recruitments were significantly inhibited in cells cotreated with resveratrol. Our studies thus indicate that resveratrol inhibits dioxin induction of the CYP1 family members either by directly or indirectly inhibiting the recruitment of the transcription factors AHR and ARNT to the xenobiotic response elements of the corresponding genes. The reduced transcriptional factor binding at their enhancers then results in reduced pol II recruitment at the promoters of these genes.

Two benzo[a]pyrene-resistant mutant clones (c1 and c37) of the mouse hepatoma Hepa-1 wild-type (wt) cell line were examined for their lack of P(1)450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)] activity. From lambda gt11 cDNA libraries, the nearly full-length P(1)450 cDNAs of wt, c1 and c37 were isolated and sequenced. The c1 cDNA was found to have a single mutation leading to premature termination of the protein after Asn-414; a rapidly migrating band corresponding to this truncated protein was found on Western immunoblots. The c37 cDNA was found to have two point mutations, leading to Leu-118----Arg-118 and Arg-245----Pro-245, but otherwise to encode the normal (524-residue) protein; the mature protein was confirmed by Western blot analysis. P(1)450 cDNA from wt, c1 and c37 and chimeric cDNAs between wt and c37 were inserted into the expression vector pAAH5 and expressed in Saccharomyces cerevisiae strain 50.L4. The Leu-118----Arg-118 mutation alone was found to have negligible effect on AHH activity, while the Arg-245----Pro-245 mutation alone leads to a 2- to 3-fold decrease in enzyme activity. The two mutations together totally abrogate AHH activity. The biologic mutant c37 provides the first evidence for the importance of Arg-245, and the complementary function of Leu-118, in normal P(1)450 enzymic function. This alteration in a single amino acid from arginine to proline might block electron flow directly, or change secondary structure of the protein, such that normal monooxygenation of benzo[a]pyrene cannot occur.

High mol. wt genomic DNA from a genetically dominant aryl hydrocarbon hydroxylase (AHH)-deficient mutant cell line derived from the mouse hepatoma cell line Hepa-1 was used to transfect the parent cell line. AHH-deficient transfectants were recovered following single-step selection in medium containing the carcinogen benzo[a]pyrene. The transfectants arose at a frequency of 2 x 10(-7). This frequency was at least 4-fold greater than the frequency of spontaneous forward mutation in this cell line. In another set of experiments, dominant mutant DNA was co-transfected along with the selectable plasmid pSV2ecogpt into parental Hepa-1 cells. The frequency of co-transfection was determined to be 3 x 10(-8). This frequency was approximately 150 times greater than that expected on the basis of coincident but unrelated spontaneous mutation and plasmid uptake. Both types of transfectants were judged, following somatic cell hybridizations, to possess the dominant phenotype of the mutant cell line, demonstrating that a trans-acting dominant negative regulator of AHH was transferred in these experiments. DNA transfection should therefore provide a means for the molecular cloning of the gene that encodes the dominant regulator.

An aryl hydrocarbon hydroxylase (AHH)-deficient gene A- mutant of the mouse line Hepa-1 was treated with calcium phosphate precipitates of DNA from Hepa-1, the rat line H4IIEC3, or an A- -human hybrid in which the A- mutation is complemented by the corresponding human gene. AHH+ transfectants were isolated by selection with benzo[ghi]perylene plus near UV. In addition, a gene A- mutant which also carries a mutation for hypoxanthine phosphoribosyltransferase deficiency was treated with the above genomic DNAs together with pSV2-gpt DNA, and cotransfectants were isolated after treatment with both benzo[ghi]pereylene and HAT. All transfectants and cotransfectants were inducible for AHH by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Both transfectants and cotransfectants were unstable during culture, rapidly losing AHH activity. Rat DNA-derived transfectants were probed in Southern blots with a cDNA probe to mouse cytochrome P1-450 that cross-hybridizes to the corresponding rat gene. All rat DNA-derived transfectants contained the rat P1-450 gene. In half of the transfectants, the rat gene was amplified four- to sevenfold. In one transfectant, the rat gene was truncated at the 3' end. The proportion of rat DNA in different transfectants, as determined by hybridization to a rat repetitive sequence, ranged from less than 1% to 5%. AHH activity and the rat P1-450 gene segregated together in subclones of one of the transfectants. These results demonstrate that the A gene is either the structural gene for cytochrome P1-450, or another very closely linked gene. Previous results (O. Hankinson et al., J. Biol. Chem. 260:1790-1795, 1985) favor the former alternative.

Revertants were selected from aryl hydrocarbon hydroxylase (AHH)-deficient recessive mutants belonging to three complementation groups and from a dominant mutant of the Hepa-1 cell line. The recessive mutants had low spontaneous reversion frequencies (less than 4 X 10(-7] that were increased by mutagenesis. The majority of these revertants also had reacquired only partial AHH activity. Revertants of group A mutants were identical to the wild type with respect to both in vivo and in vitro enzyme stability and the Km for the substrate, benzo [alpha]pyrene, and therefore failed to provide evidence that gene A is the AHH structural gene. Group B and group C mutants are defective in the functioning of the Ah receptor required for AHH induction. Revertants of these groups were normal with respect to in vivo temperature sensitivity for AHH induction and for the 50% effective dose for the inducer, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and thus provided no evidence that the B and C genes code for components of the receptor. Two rare group C revertants possessed AHH activity in the absence of induction. The phenotype of one of these was shown to be recessive to the wild type. Spontaneous revertants of the dominant mutant occurred at a frequency 300-fold greater than those of the recessive mutants, and this frequency was not increased by mutagenesis. These revertants all displayed complete restoration of AHH activity to wild type levels. These observations and the results from cell hybridization studies suggest that the dominant revertants arose by a high frequency event leading to functional elimination of the dominant mutation.

The extent of phenotypic derangement was investigated in representative aryl hydroxylase-deficient mutants of Hepa-1. One mutant is dominant while the others are recessive and are mutated in 3 different genes. All the mutants lacked ethoxyresorufin-O-deethylase activity as well as aryl hydrocarbon hydroxylase. However all had normal activities of NADPH-cytochrome P-450 reductase, epoxide hydrolase and ornithine decarboxylase. They also retained 2 liver-specific functions possessed by the parental line, namely albumin secretion and transferrin secretion. The phenotypic alterations in the mutants were therefore restricted to the cytochrome P-450 activities measured.

The H-4-II-E-C3 line has high aryl hydrocarbon hydroxylase (AHH) activity. It was also found to be very sensitive to benzo(a)pyrene (BP) toxicity, and a single-step selection procedure for isolating BP-resistant (BPr) clones was designed. All the BPr variants tested had reduced AHH activities under both inducing and noninducing conditions. As H-4-II-E-C3 was propagated, it gradually declined in AHH activity and increased in cloning efficiency in BP. The spontaneous rate of origin of the BPr variants was approximately 3 x 10(-5) events per cell generation. However, the variants gradually increased in AHH activity and decreased in cloning efficiency in BP as they were propagated. Partially resistant variants also generated, at an appreciable rate, cells with even greater degrees of resistance to BP. The instabilities of the wild-type and variant cultures suggest that the variants arose by an epigenetic mechanism or by changes in gene dosage, rather than by point mutations.

The mouse hepatoma line Hepa-1 has high and inducible aryl hydrocarbon (benzo[a]pyrene) hydroxylase[benzo[a]pyrene, reduced-flavoprotein:oxygen oxidoreductase (3-hydroxylating), EC 1.14.14.2] activity. Fourteen subclones of a clonal derivative of Hepa-1 were isolated and shown not to display heterogeneity in aryl hydrocarbon hydroxylase activity beyond what could be ascribed to experimental error. Hepa-1 was found to be very sensitive to benzo[a]pyrene (BaP) toxicity and a single-step selection procedure for isolating clones resistant to BaP at 4 microgram/ml was designed. Those BaP-resistant variants tested had much reduced aryl hydrocarbon hydroxylase activities under both inducing and noninducing conditions and they retained their resistance to BaP and low aryl hydrocarbon hydroxylase activities over considerable periods of time of culture in the absence of BaP. The spontaneous rate of origin of the BaP-resistant clones was estimated, by Luria-Delbrück fluctuation analysis, to be 2 X 10(-7) events per cell per generation. It is concluded that the properties of the variants are consistent with the notion that they are mutational in origin and that this system is a promising one for carrying out a somatic cell genetic analysis of aryl hydrocarbon hydroxylase.

The development of a selection procedure is described which utilizes the fact that certain polycyclic aromatic hydrocarbons are rendered highly cytotoxic when illuminated with near-ultraviolet light. Twenty polycyclic aromatic hydrocarbons were screened for phototoxicity and toxicity in the absence of light in the mouse hepatoma line Hepa1c1c7, which has high inducible aryl hydrocarbon hydroxylase (AHH) activity, and in AHH-deficient mutants derived from this line. In the assessment of phototoxicity, a period of time was allowed for the metabolism of these compounds prior to illumination. Benzo(g,h,i)perylene had the greatest phototoxicity in cells lacking AHH but was not toxic to cells possessing AHH either in the presence or absence of light. We show that, under conditions of our selection procedure, cells which possess AHH and thus are capable of metabolizing, and therefore of eliminating, benzo(g,h,i)perylene (as determined by the magnitude of the fluorescence of the compound in the cells) are resistant to subsequent exposure to near-ultraviolet light, while cells which lack AHH and are thus unable to eliminate the compound are killed by subsequent illumination. We show, furthermore, that cells possessing AHH can be selected from a majority population of cells lacking the enzyme by this procedure.

In order to study the induction mechanism of aryl hydrocarbon hydroxylase (AHH), non-inducible mutants have previously been isolated from the mouse hepatoma cell line, Hepa-1. With the ultimate goal of isolating the corresponding genes, restoration of AHH inducibility to representative mutants by means of DNA-mediated gene transfer has been set out. The successful transfection of a C- mutant, which is defective in nuclear translocation of the Ah receptor-inducer complex, is described here, using rat genomic DNA as donor material. Primary and secondary rat transfectants were obtained, and they were assayed for hydroxylase activity, receptor translocation, and homology with a rat repetitive DNA sequence.

Fifty-four benzo[a]pyrene (BP)-resistant, aryl hydrocarbon hydroxylase (AHH)-deficient mutants were found to be recessive, while five were dominant. Hybrids between the former mutants and the wild-type were killed by BP, and possessed AHH activities of at least 0.5 (relative to the wild-type). Dominant-mutant--wild-type hybrids were resistant to BP and had activities of about 0.05. Additional experiments assigned the recessive mutants to three complementation groups, designated A through C. Group-B--group-C hybrids were exceptional in possessing a mean AHH activity (0.36), less than the value (0.5) expected from gene dosage. This deficiency was probably due, in part, to instability of AHH activity in these hybrids. However, all hybrids tested retained stable DNA complements, equal to the sum of those of their parents, for 140 days in culture. Previous studies have shown that group B and group C mutations both affect the functioning of a cytosolic receptor required for AHH induction (1).