Hepatic CYP1A1 mRNA Levels Research Articles

Polychlorinated biphenyls (PCBs) enhance the neoplastic progression of human breast epithelial cells in a xenograft model

PCBs have long been suspected as environmental breast carcinogens but the epidemiological evidence has not strongly supported this concern. As an experimental approach to investigate the role of PCBs in breast cancer development, the preneoplastic MCF10AT1 human breast epithelial cell line was used in xenograft experiments. After implantation, MCF10AT1 cells form tubular structures, some of which progress to lesions with the histological features of atypical hyperplasia, ductal carcinoma in situ (DCIS), and invasive carcinoma (IC). MCF10AT1 cells were injected into female Ncr nude mice, and the mice were treated with corn oil, 0.3 or 1 μg/kg/day PCB126 (a coplanar PCB), or 1 or 3 mg/kg/day PCB153 (a non‐coplanar PCB) by gavage for 10 weeks. As indices of PCB exposure, 0.3 and 1 μg/kg/day PCB126 increased hepatic Cyp1a1 mRNA levels by ~40‐and ~1000‐fold while 1 and 3 mg/kg/day PCB153 increased hepatic Cyp2b10 mRNA levels by ~12‐and ~125‐fold. The MCF10AT1 lesions were graded histologically on a scale of 0 (simple tubules) to 5 (IC). 17% of the lesions in the corn oil group were graded at 4 (DCIS) or 5, while 45% and 36% in the 0.1 and 1 μg/kg/day PCB126 groups and 67% and 50% in the 1 and 3 mg/kg/day PCB153 groups were graded at 4 or 5. These results suggest that relatively low doses of PCBs can enhance the neoplastic progression of human breast epithelial cells. Supported by NIH grant (ARRA) 1R21ES016373.

Phenethyl isothiocyanate, a naturally occurring phytochemical, is an antagonist of the aryl hydrocarbon receptor

The aryl hydrocarbon (Ah) receptor is a ligand-activated transcription factor that is activated by many carcinogens, and its target gene products play a major role in tumour development, so that antagonists of the Ah receptor represent potential chemopreventive agents. Experimental evidence is presented herein that phenethyl isothiocyanate (PEITC), a phytochemical present in cruciferous vegetables, is such an antagonist. PEITC was a very weak ligand to the Ah receptor, as assessed using the chemical-activated luciferase expression (CALUX) assay, and a poor inducer of CYP1A1 mRNA levels when incubated in precision-cut rat liver slices for 24 h. It antagonised effectively, however, the interaction of benzo[a]pyrene to the receptor, being capable of preventing its binding as well as displacing it from the receptor. Moreover, PEITC suppressed in concentration-dependent manner the benzo[a]pyrene-mediated rise in rat hepatic CYP1A1 mRNA levels in rat slices. Finally, PEITC antagonised the benzo[a]pyrene-mediated increase in the O-deethylation of ethoxyresorufin in both rat and human precision-cut liver slices. It is concluded that PEITC is an effective antagonist of the Ah receptor in rat and human liver, and this potential may contribute to its established chemopreventive activity.

INTERACTION OF 2,2',4,4',5,5'-HEXACHLOROBIPHENYL WITH HEPATIC CYTOCHROME P-4501A IN RAINBOW TROUT ( ONCORHYNCHUS MYKISS )

Di- ortho polychlorinated biphenyls (PCBs) are prominent environmental contaminants and their biological activity in fish may be more significant than previously thought. Four weeks after intraperitoneal (ip) injection with 50 or 250 mug 2,2',4,4',5,5'-hexachlorobiphenyl (2HxCB)/ g fish, rainbow trout livers were removed and frozen at-80 C or microsomes were prepared. Microsomal ethoxyresorufin O -deethylase (EROD) activity was approximately one and two orders of magnitude greater than controls in fish treated with 50 and 250 mug 2HxCB/g fish, respectively. Cytochrome P4501A (CYP1A) Western immunoblot relative optical density increased with 2HxCB dose. Hepatic CYP1A1 mRNA levels were approximately threefold greater in fish treated with 250 mug 2HxCB/g fish than in controls, while hepatic CYP1A1 mRNA levels in fish treated with 50 mug 2HxCB/g fish were not significantly induced. There was no increase of CYP1A3 mRNA in 2HxCB-treated fish. The study showed 2HxCB induced hepatic EROD activity, CYP1A protein, and CYP1A1 mRNA content in rainbow trout.

Expression of CYP1A1 in livers and gonads of Pacific salmon: quantitation of mRNA levels by RT-cPCR

A reverse-transcriptase-competitive-polymerase-chain-reaction (RT-cPCR) assay was developed to accurately quantitate CYP1A1 mRNA levels in various tissues of Pacific salmon. Juvenile chinook salmon were injected intraperitoneally with the polynuclear aromatic hydrocarbon β-naphthoflavone (BNF), and levels of CYP1A1 mRNA were measured in livers and gonads over a 4-week period using RT-cPCR. There was a significant increase in hepatic CYP1A1 mRNA levels in the fish injected with BNF over the 28-day experiment, from undetectable levels (< 0.83 × 10 −3 fmol/μg RNA) at time zero to a mean level of 133.56 × 10 −3 fmol/ μg RNA, an increase of at least 160-fold. Although low levels of CYP1A1 expression were detected in the livers of control fish, hepatic CYP1A1 mRNA levels in BNF-treated juvenile chinook were always very significantly elevated above control values throughout the 4-week experiment. CYP1A1 mRNA levels were also significantly elevated (to a maximum of 18-fold above the assay detection limit) in the gonads of BNF-treated fish, whereas no detectable levels were observed in the gonads of control fish at any time. To examine the response of gonadal tissue in more detail, ovarian follicles isolated from maturing coho salmon were exposed to various doses of BNF or 20-methylcholanthrene (20-MC) in vitro, and CYP1A1 mRNA levels in the follicular cells quantitated. Exposure to either BNF or 20-MC for 18 h in vitro induced CYP1A1 in coho ovarian follicles. A dose response for both PAHs was obtained (between 50.0 nM and 5.0 μM) and revealed a greater sensitivity of ovarian follicle cells to induction by 20-MC. This study shows that RT-cPCR can be used to detect and quantify CYP1A1 gene expression in various tissues of fish in response to xenobiotic exposure. The induction of CYP1A1 in the ovaries and testes of juvenile chinook salmon, and in isolated ovarian follicles of mature coho salmon, following exposure to PAHs indicates that xenobiotics may impact on reproductive processes directly at the gonadal level.

Synergistic activity of polynuclear aromatic hydrocarbon mixtures as aryl hydrocarbon (Ah) receptor agonists

The relative potencies of benzo[ a]pyrene and a complex mixture of polynuclear aromatic hydrocarbons (PAHs) produced as by-products of manufactured gas plant (MGP) residues as inducers of hepatic microsomal ethoxyresorufin O-deethylase (EROD) activity were determined in the B6C3F1 mouse. The ED 50 values for the induction response were 78 and 65 mg/kg for benzo[ a]pyrene and the MGP-PAH mixture, respectively. Analysis of the MGP-PAH mixture indicated that benzo[ a]pyrene and other compounds containing four or more rings and which are known to induce EROD activity were only present as trace components of this mixture. A comparison of the EROD induction potencies of benzo[ a]pyrene and the MGP-PAH mixture showed that the mixture was approximately 706 times more potent than expected based on its benzo[ a]pyrene content (0.17%). This induced P-450 activity could significantly increase the metabolism of the carcinogenic PAHs and thereby modulate the overall carcinogenicity of the mixture. The apparent synergistic activity of the MGP-PAH mixture was further investigated by comparing the activities of this mixture and benzo[ a]pyrene for several other aryl hydrocarbon (Ah) receptor-mediated responses including (i) induction of hepatic CYP1A1 mRNA levels, (ii) transformation of the rat cytosolic Ah receptor to a complex which binds to a dioxin responsive element, (iii) induction of EROD activity and (iv) antiestrogenicity in MCF-7 human breast cancer cells, and (v) inhibition of the splenic plaque-forming cell (PFC) response to both T cell-dependent and independent antigens in B6C3F1 mice. For the EROD and CYP1A1 mRNA induction and cytosolic transformation activities and immunosuppressive effects, the MGP-PAH mixture was approximately 100–900 times more potent as an Ah receptor agonist than expected based on its benzo[ a]pyrene content. The synergistic activity was lower (19-fold) for the antiestrogenic response in MCF-7 cells. The reason for the synergistic effects of the MGP-PAH mixture were not due to contamination of the mixture by 2,3,7,8-tetrachlorodibenzo- p-dioxin and related compounds and the results suggest that the enhanced potency of the mixture is due to unknown interactions between the individual PAHs present in the mixture.