Coordinate Regulation of Drug Metabolising Enzymes and Transporters in Response to Xenobiotic Exposure.

The body is constantly exposed to many different external chemicals (xenobiotics). Cytochrome P450 enzymes and drug transporters form part of the core protection system for the body, regulating entry of xenobiotics, and increasing clearance rates of chemicals that do enter the body. It is therefore important to know how these systems will respond to novel chemicals, with human cell lines commonly used in both drug metabolism and gene expression studies. In this study we have determined the transcript levels for several cytochrome P450s or drug transporter from human liver (Huh7), intestine (CaCo-2) and lung (A549) cell lines, and compared them to adult and foetal levels in the respective tissues. Basal expression of the selected CYP genes and drug transporters is significantly lower in these cell lines than in either adult or foetal primary tissue. We have also examined the effect of xenobiotic exposure on the expression of these transcripts, showing that whereas some cell lines have maintained their ability to show genome-base activations of gene expression, others are refractory - a fact that has important implications for drug studies carried out in these cell lines. Finally, we have examined how two of these genes, CYP3A4 and MDR1, are regulated in different tissues and cell types. Current evidence suggests that in certain tissues these genes show positive coordination of their response to xenobiotic exposure, whereas in other negative or no coordination is seen. Using reporter gene constructs for the proximal promoters of these two genes we examined the molecular mechanisms underlying these complex responses to chemical exposure to individual polychlorinated biphenyls (PCBs). The polychlorinated biphenyls (PCBs) comprise a group of 209 congeners varying in the number of chlorine atom and substitution patterns. These compounds have been shown to induce an array of effects in exposed organisms and tend to be biomagnified in food webs. The structural characteristics of the PCBs influence their potency as well as their mechanism of action. PCB congeners with zero or one ortho-chlorine substituent can bind to the aryl hydrocarbon receptor (AhR receptor), thus mimicking the action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The effects of these PCB congeners (such as PCB 77, 126 and 169) appear to be mediated primarily via the AhR receptor. PCB congeners with ortho chlorine substituents can bind to the constitutive androstane receptor (CAR) and/or the pregnane X receptor (PXR) and they are called phenobarbital (PB)-type PCBs (for example PCB 87, 99, 153, 180, 183 and 194). Other ortho substituted PCB congeners interact with both the aryl hydrocarbon (AhR) and the CAR receptor and they are called mixed type PCBs (for example, PCB 128, 138, 170). Six PCBs (77, 99, 138, 153, 180 and 194) had been selected from the three categories and their properties were examined by looking at the possible induction of CYP3A4 and MDR1 genes through the human pregnane X receptor (hPXR) as well as their effects on the regulation of the human PXR gene expression itself in transiently transfected Huh7, Caco-2 and A549 cell lines. In summary, this work demonstrate that non-coplanar PCBs may act as direct activators of the nuclear receptor PXR and they are able to activate target gene expression in a tissue and substituent dependent manner. Such work will help improve prediction of body responses to PCBs exposure, as well as increase the accuracy of current risk assessment methodologies