Developing a high-throughput system for the screening of cytochrome P450 1A1--inhibitory polyphenols.

Polycyclic aromatic hydrocarbons (PAH) are established procarcinogens that can be found in our environment. The carcinogenicity of these compounds is initiated by their metabolic intermediates, and the extent of biotransformation determines the amount of reactive intermediates generated. CYP1A1 is a major enzyme that metabolizes PAH into carcinogenic moieties. Since previous studies have shown that increased CYP1A1 activity is associated with a higher cancer risk. Identifying CYP1A1-inhibitory compounds in diet or natural products are of genuine interest for chemoprevention studies. In this project, a stable cell line expressing human CYP1A1 was established for the screening of potential chemopreventive agents. Because of a lacking cellular transport mechanism in assays performed on recombinant protein, an 'in-cell' assay system might be a better estimate at the tissue level. Theaflavins were strong inhibitors of ethoxyresorufin-O-deethylase (EROD) activity when assayed on recombinant human CYP1A1 protein. However, this inhibition was not observed in the CYP1A1-expressing cells. The 'in-cell' IC50 values determined for compounds such as genistein, quercetin, chalcone, etc. were comparable to the values determined in recombinant protein. This 'in-cell' assay has the additional advantages of short sample processing time, and the tedious procedures of protein expression and purification can be waived.

Screening of chemopreventive tea polyphenols against PAH genotoxicity in breast cancer cells by a XRE-luciferase reporter construct.

Polycyclic aromatic hydrocarbons (PAHs) are established cancer initiators that can be found in our food and environment. Some dietary plant polyphenols are strong inhibitors to PAH-induced mutagenesis, whereas others may not be as effective. To identify the chemopreventive compounds from a huge volume of dietary components, the development of an efficient screening method is required. In this study, a xenobiotic response element (XRE)-luciferase reporter plasmid was constructed to screen for some potential chemopreventive agents in tea against PAH-induced DNA damage. Tea is one of the most consumed beverages worldwide, and its beneficial effects on health have been documented. Previous studies have claimed that tea polyphenols could be protective against various cancers, and the rich database can be a source for comparison. Among the green and black tea polyphenols, the XRE-luciferase reporter assays suggested that only epigallocatechin gallate (EGCG) was effective in reducing XRE-driven luciferase assay in MCF-7 cells at the concentrations tested. Further study indicated EGCG could reduce CYP1A1 and CYP1B1 mRNA abundances and decrease the DMBA-DNA lesions. The results of DNA covalent binding of all tea polyphenols tested were consistent with the XRE-reporter assays. This study illustrated that the XRE-reporter assay was a viable screening test for dietary chemopreventive agents against PAH-initiated breast mutagenesis. It has the advantages of shorter sample processing time and producing no radioactive waste over directly measuring the CYP1A1/1B1 expressions, DNA lesion, or gel mobility shift assay.

A potential protective mechanism of soya isoflavones against 7,12-dimethylbenz[a]anthracene tumour initiation.

Epidemiological studies indicate that Asian women have a lower breast cancer incidence compared with their counterparts in the West, and the difference has been related to soya consumption. Animal studies have suggested that soya may prevent dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis in the breast. In the present study a cell culture model was developed to address the effect of soya isoflavones on the DMBA-induced DNA damage. DMBA is metabolized into a DNA-attacking moiety by two phase I cytochrome P450 (CYP) enzymes CYP1A1 and CYP1B1. DNA mutation caused by this genotoxic agent is a crucial step in cancer initiation. Substances that interfere with the CYP1 enzyme activities can affect the initiation. In the present study, genistein was found to be an effective inhibitor of recombinant human CYP1A1 and CYP1B1 with Ki of 15.35 and 0.68 micromol/l. The other soya isoflavone daidzein, on the other hand, did not demonstrate any significant inhibition of the enzyme activities. At the transcriptional level, DMBA induced the CYP1 enzyme expressions by stimulating the xenobiotic response element (XRE)-dependent transactivation pathway. When genistein (25 micromol/l) was co-administered with DMBA, the XRE-Luc activity the CYP1 mRNA abundances were significantly suppressed. The present study illustrated that the soya isoflavone genistein, but not daidzein, protected against DMBA genotoxicity.

The red clover (Trifolium pratense) isoflavone biochanin A modulates the biotransformation pathways of 7,12-dimethylbenz[a]anthracene.

Several flavonoids have shown their anti-carcinogenic effects in various models. The soyabean isoflavone genistein was demonstrated earlier in our laboratory to be an effective inhibitor of dimethylbenz[a]anthracene (DMBA)-induced DNA damage in MCF-7 cells by curbing cytochrome P450 (CYP) 1 enzymes. The red clover (Trifolium pratense) isoflavone biochanin A is a methylated derivative of genistein, and its anti-mutagenic effect in bacterial cells has been shown previously. Because of its protection against chemical carcinogenesis in an animal model, biochanin A was selected for testing in our established MCF-7 cell system. From the results obtained in the semi-quantitative reverse transcription-polymerase chain reaction and xenobiotic response element (XRE)-luciferase reporter assays, biochanin A could reduce xenobiotic-induced CYP1A1 and -1B1 mRNA abundances through the interference of XRE-dependent transactivation. Enzyme kinetic studies also indicated that biochanin A inhibited both CYP1A1 and -1B1 enzymes with inhibition constant (Ki) values 4.00 and 0.59 microm respectively. Since the biotransformation of DMBA was dependent on CYP1 enzyme activities, biochanin A was able to decrease the DMBA-DNA lesions. The present study illustrated that the red clover isoflavone could protect against polycylic aromatic hydrocarbon-induced DNA damage.

Are herbal medicinal products less teratogenic than Western pharmaceutical products.

AIM: To determine the use and teratogenicity of herbal medicinal products (HMP). METHODS: A retrospective study was conducted in a University hospital to compare the difference in the pattern of use and fetal outcomes between pregnant women who took HMP and Western pharmaceutical products (WPP). RESULTS: From 1995 Jan to 2001 Dec, 61 and 372 women took HMP and WPP one month before or during their current pregnancies respectively. There was an increase in the prevalence of pregnant women who took HMP from 0 % in 1995 to 0.8 % in 2001. Among HMP users, 51.6 %, 82.8 % and 58.6 % of them had low monthly family income (<15 000), low education level (secondary education or below) and were unemployed respectively. In comparison to WPP, pregnant women used smaller number of HMP (1.4 vs 3.0, P < 0.01) at a later gestation (4.8 weeks vs 3.1 weeks, P <0.01) and within a shorter duration (11.1 d vs 47.9 d, P < 0.01). The prevalence of congenital fetal abnormalities in the group of women who took HMP (3.3 %) was not significantly higher than that who took WPP (0.8 %). There were no and two abnormal fetal karyotypes in the former and latter group respectively. No and ten women in the former and latter group underwent termination of pregnancy for anxiety respectively. The proportions of silent miscarriage in the former and latter group were similar (6.6 % vs 5.4 %). CONCLUSION: Pregnant users of HMP were from lower socio-economical status. There was no significant difference in the teratogenicity between HMP and WPP.

Baicalein inhibits DMBA-DNA adduct formation by modulating CYP1A1 and CYP1B1 activities.

Flavonoids are phenolic compounds isolated from plants, and several of them like genistein and quercetin, have been documented to be effective in preventing cancer. Baicalein, a flavonoid extracted from the root of Scutellaria species, is widely used as a health supplement and herbal medicine in Asian countries. In this study, the chemopreventive effect of baicalein on 7,12-dimethylbenz[a]anthracene (DMBA)-induced DNA damage was evaluated in an established cell culture model. In a preliminary screening, baicalein was identified to be a strong inhibitor to EROD activities induced by DMBA in MCF-7 cells. Subsequent enzyme kinetic analysis revealed that baicalein was a competitive inhibitor to EROD, and CYP1A1 and CYP1B1 gene expressions were also determined. Baicalein could reduce the CYP1A1/1B1 mRNA expressions induced by DMBA, and the mRNA abundance of CYP1A1 appeared to be more responsive than that of CYP1B1. A XRE-luciferase gene reporter assay indicated that AhR transactivation was suppressed. Since CYP1A1/1B1 were responsible for the biotransformation of polycyclic aromatic hydrocarbons, baicalein also demonstrated its ability to reduce DMBA-DNA adduct formation in MCF-7 cells. This study suggested that the natural occurring baicalein could be an agent preventing carcinogen-DNA adduct formation.