Cytochrome P450 (CYP)1A1 and CYP1B1, which are under the regulatory control of the aryl hydrocarbon (Ah) receptor (AhR), catalyze the metabolic activation of numerous procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. There is evidence of cross-talk between estrogen receptor alpha (ERalpha)- and AhR-mediated signaling in breast and endometrial cells. To further examine these interactions, we investigated the short- and long-term effects of E2 exposure on Ah responsiveness in MCF-7 human breast cancer cells. Short-term exposure to 1 nM E2 elevated the ratio of the 4- to 2-hydroxylation pathways of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced E2 metabolism and the ratio of the induced CYP1B1 to CYP1A1 mRNA levels, as determined by real-time PCR. Cells maintained long-term (9-12 months) in low-E2 medium progressively lost Ah responsiveness, as indicated by diminished rates of TCDD-induced E2 metabolism and ethoxyresorufin O-deethylase activity, and the reduced expression of the CYP1A1 and CYP1B1 mRNAs and proteins levels. These E2-deprived cells showed elevated levels of ERalpha mRNA, depressed levels of AhR mRNA, and unchanged levels of the AhR nuclear translocator mRNA. Transient transfection studies using a CYP1B1-promoter-luciferase reporter construct showed that reduced CYP1B1 promoter activity in E2-deprived cells could be restored by co-transfection with an AhR expression construct, indicating that AhR expression was limiting in these cells. The reduced Ah responsiveness of E2-deprived cells was reversed by culture for four passages in medium supplemented with 1 nM E2; ERalpha and AhR mRNAs returned to near-normal levels and the inducibility of the CYP1A1 and CYP1B1 mRNAs, proteins, and E2 metabolic activities by TCDD was restored. These studies indicate that the continued presence of estrogen is required to maintain high levels of AhR expression and inducibility of the procarcinogen-bioactivating enzymes, CYP1A1 and CYP1B1, in MCF-7 cells.

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Agrobacterium tumefaciens biovar 3 (AT 3) was isolated from grapevine typical colonies of Agrobacterium were selected from dilution plates from galls, sap of "bleeding" vines, and from vineyard soil by using a selective 30 vineyard soil samples. Five of these strains were pathogenic, three being medium. AT 3 was the predominant biovar isolated from galls. The AT 3 and two similar to A. tumefaciens biovar I. Almost all of the AT 3 bacterium was recovered from sap from seven of 24 infected vines and one strains from galls, sap, and soil caused galls on tomato, sunflower, and of 17 apparently healthy vines. Ten sap isolates were identified as AT 3 and grapevine inoculated in greenhouse tests. all were pathogenic on grapevine and sunflower. Two hundred forty-four

ABSTRACT:Polycyclic aromatic hydrocarbons (PAHs) and metals are often environmental cocontaminants, yet there have been relatively few studies of combined effects of PAHs and metals on cytochrome P450 (P450)-catalyzed metabolism. We examined the effects of NaAsO 2 in combination with benzo[a]pyrene (BAP) on CYP1A1 and CYP1B1 in T-47D human breast cancer cells by using estrogen metabolism as a probe of their activities. Exposure to BAP caused elevated rates of the 2-and 4-hydroxylation pathways of estrogen metabolism, indicating induction of both CYP1A1, an estradiol 2-hydroxylase, and CYP1B1, an estradiol 4-hydroxylase. BAP-induced metabolism peaked 9 to 16 h after exposure and returned to near-basal levels by 48 h. Concentration-response studies showed maximal induction of the 2-and 4-hydroxylation pathways at 3 M BAP; higher levels caused reduced rates of metabolism due to inhibition of CYP1A1 and CYP1B1. NaAsO 2 caused pronounced decreases in the induction of CYP1A1 and CYP1B1 by 3 M BAP because cotreatment with 10 M NaAsO 2 inhibited the rates of the 2-and 4-hydroxylation pathways by 86 and 92%, respectively. Western immunoblots showed diminished levels of BAP-induced CYP1A1 by coexposure to NaAsO 2 . The levels of the CYP1A1 and CYP1B1 mRNAs induced by BAP were not significantly affected by coexposure to NaAsO 2 ; however, heme oxygenase 1 mRNA levels were markedly induced by coexposure to BAP and NaAsO 2 . These results indicate a post-transcriptional inhibitory effect of arsenite on the expression of CYP1A1 and CYP1B1 in T-47D cells, possibly resulting from reduced heme availability.Among the environmental contaminants of most concern due to their toxicity, including carcinogenicity, are heavy metals, such as arsenic, lead, and mercury, and polycyclic aromatic hydrocarbons (PAHs 1 ) typified by benzo[a]pyrene (BAP). PAHs and heavy metals are often cocontaminants in the environment, yet there have been relatively few studies of the combined toxic and particularly the carcinogenic effects elicited by PAHs and heavy metals. The carcinogenicity of BAP and other PAHs is a consequence of their metabolic activation catalyzed by cytochromes P450 (P450) and epoxide hydrolase (Wood et al., 1976;Kapitulnik et al., 1978). Covalent adducts formed by the reaction of PAH diol epoxide metabolites with guanine in mutational hotspots of critical genes such as that of the p53 tumor suppressor (Denissenko et al., 1996), if not efficiently repaired, may initiate tumorigenesis.Several members of the P450 superfamily in conjunction with epoxide hydrolase have been shown to catalyze the metabolism of BAP to carcinogenic intermediates. In extrahepatic tissues, CYP1A1 and CYP1B1 are thought to be the most important enzymes in catalyzing the formation of mutagenic intermediates from BAP and a number of other PAHs, including several that are potent mammary gland carcinogens in rodents. CYP1B1 appears to be more active than CYP1A1 in the conversion of a number of PAHs to genotoxic intermediates (Shimada et al., 1996). In the presence...

Sulfate conjugates of the B-ring unsaturated estrogens, equilin, equilenin, and 8-dehydroestrone, and their 17alpha- and 17beta-dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most commonly prescribed estrogen formulation in estrogen replacement therapy. Despite the wide clinical use of Premarin, there have been very few studies on the metabolism of the B-ring unsaturated estrogens in humans and there is no information regarding the fate of these compounds in breast tissue or tumors. In this study, we investigated the metabolism of equilenin in two lines of human breast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment with Ah-receptor agonists with induction of cytochromes P450 1A1 and 1B1, whereas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of equilenin were identified and quantified by GC/MS utilizing a series of synthetic metabolite standards and deuterium-labeled analogues as internal standards. In the two cell lines, the same pathways of equilenin metabolism were observed. Equilenin was reduced at C-17 to the 17beta-dihydro form, with minimal production of the 17alpha-dihydro isomer. Both equilenin and 17beta-dihydroequilenin were hydroxylated at the C-4 position, and the resultant catechol metabolites were methylated to form 4-methoxyequilenin and 4-methoxy-17beta-dihydroequilenin. Rates of equilenin metabolism were markedly elevated in cultures exposed to the Ah-receptor agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,4,4',5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation pathways of equilenin and 17beta-dihydroequilenin metabolism were not observed. In microsomal reactions with cDNA-expressed human enzymes, both P450s 1A1 and 1B1 catalyzed the 4-hydroxylation of 17beta-dihydroequilenin, whereas with 17beta-estradiol as substrate P450 1A1 catalyzes predominantly 2-hydroxylation and P450 1B1 predominantly 4-hydroxylation. Since P450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indicate the potential for 4-hydroxylation of equilenin and 17beta-dihydroequilenin in extrahepatic, estrogen-responsive tissues.

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In a previous study of nine human breast-derived cell lines, rates of metabolism of 17beta-estradiol (E(2)) were greatly enhanced when cultures were exposed to the aromatic hydrocarbon receptor agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin. Elevated rates of E(2) hydroxylation at the C-2, -4, -6alpha and -15alpha positions were observed concomitant with the induction of cytochromes P450 1A1 and 1B1. In each cell line, 2- and 4-hydroxyestradiol (2- and 4-OHE(2)) were converted to 2- and 4-methoxyestradiol (2- and 4-MeOE(2)) by the action of catechol O:-methyltransferase. In this study, conjugation of these estrogen metabolites was investigated. A comparison of the levels of metabolites determined with and without prior treatment of the media with a crude beta-glucuronidase/sulfatase preparation showed that most of the 2-MeOE(2) present was in conjugated form, whereas 4-MeOE(2), 6alpha-OHE(2) and 15alpha-OHE(2) were minimally conjugated. Inhibitor studies suggested that it was the sulfatase activity of the preparation that hydrolyzed the 2-MeOE(2) conjugates in MCF-7 cell media; the presence of 2-MeOE(2)-3-sulfate in MCF-7 culture media was confirmed by electrospray ion-trap mass spectrometry. To identify the enzyme catalyzing this conjugation, the expression of mRNAs encoding five sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1E1 and SULT2A1) was evaluated in the nine cell lines by use of the reverse transcription-polymerase chain reaction. Only expression of SULT1A1 mRNA correlated with the observed conjugation of nanomolar levels of 2-MeOE(2) in these cell lines. Cloning and sequencing of SULT1A1 cDNA from MCF-7 cells revealed that mRNAs encoding two previously identified allelic variants, SULT1A1*1 ((213)Arg) and SULT1A1*2 ((213)His), were expressed in these cells. Heterologous cDNA-directed expression of either variant in MDA-MB-231 cells, which do not normally express SULT1A1, conferred 2-MeOE(2) sulfonation activity. The SULT1A1 allelic variants were also expressed in SF:9 insect cells, from which post-microsomal supernatants were used to determine K:(m) values of 0.90 +/- 0.12 and 0.81 +/- 0.06 microM for SULT1A1*1 and SULT1A1*2, respectively, with 2-MeOE(2) as substrate. These results show that SULT1A1 is an efficient and selective catalyst of 2-MeOE(2) sulfonation and, as such, may be important in modulating the anticarcinogenic effects of 2-MeOE(2) that have been described recently.

A monoclonal antibody, designated AbF21-1D3G7C8, reacts in useful for laboratory diagnosis of A. tumejaciens biovar 3, and thus for enzyme-linked immunosorbent assay with biovar 3 strains of detection and diagnosis of grapevine crown gall, indexing of propagation Agrobacterium tumefaciens, causal agent of grapevine crown gall, from wood, and quarantine testing. A. tumeJaciens biovar 3 was identified North America, Europe, and Asia, regardless of tumorigenicity.