Expression profiling of selected genes of toxication and detoxication pathways in peripheral blood lymphocytes as a biomarker for predicting toxicity of environmental chemicals

Abstract

To develop a rapid and sensitive tool for determining gene expression profiles of peripheral blood lymphocytes (PBL) as a surrogate for predicting toxicity associated with environmental exposures, studies were initiated using Taqman Low Density Array (TLDA), a medium throughput method for real time PCR (RT-PCR), for selected genes involved in toxication and detoxication processes. Total RNA was prepared from PBL and liver samples isolated from young rats treated with inducers of drug metabolizing enzymes, e.g. phenobarbital (PB, 80 mg/kg i.p. X5 days) or methylcholanthrene (30 mg/kg, i.p. X5 days) or ethanol (0.8 ml/kg, i.p. X1 day). TLDA data showed that PBL expressed drug metabolizing enzymes (DMEs), though the level of expression was several folds lower when compared to liver. Treatment with different inducers of DMEs produced a similar pattern of an increase in the expression of various phase I and phase II DMEs and their respective transcription factors in liver and PBL. While treatment with MC increased the expression of MC inducible cytochrome P450 (CYP) 1A1, 1A2, 1B1, 2A2 & 3A1 and their associated transcription factors in PBL, an increase in the expression of CYP2B1, 2B2, 2C11 & 3A1 and their transcription factor was observed in PBL after PB treatment. Similarly, treatment of ethanol increased the expression of CYP2E1 and 3A1 along with transcription factors in PBL. These inducers were found to increase the expression of various phase II enzymes such as glutathione S-transferases, GSTs (GSTM1, GSTA1, GSTP1 and GSTK1), NQO1, Ephx1 and Sod1, genes involved in inflammation and apoptosis (p53, BCl2, Apaf1 and Caspase9) in both PBL and liver. The data suggests that the low-density array of selected genes in PBL has the potential to be developed as a rapid and sensitive tool for monitoring of individuals exposed to environmental chemicals as well as in clinical studies.

Introduction

Humans are exposed to a variety of chemicals present in the environment, several of which are known to produce serious toxic manifestations, including cancer. Majority of these chemicals, including environmental carcinogens require metabolic activation by cytochrome P450 (CYPs), the phase I drug metabolizing enzymes (DMEs), to produce toxicity and carcinogenicity (Bozina et al., 2009, Enoch and Cronin, 2010). Alternatively, the reactive intermediates generated by CYPs are detoxified by the phase II drug metabolizing enzymes (Testa and Kramer, 2006, Crettol et al., 2010). These environmental chemicals are also known to induce the expression of DMEs including CYPs that catalyze their metabolism and induction of individual CYPs is often indicative of the exposure of a particular class of drug or chemicals (Thum et al., 2006, Totlandsdal et al., 2010). The regulation of these DMEs is moreover, under genetic control. Genetic polymorphism is reported for CYPs and other DMEs with even enzyme deletion or multiple copies of the gene being generated in individuals because of gene polymorphism that may modify the susceptibility of an individual to the toxic effects of these environmental chemicals or adverse health effects of drugs (Ma et al., 2004, Li et al., 2011).

The responses of the CYPs along with other genes relevant to xenobiotic metabolism have been demonstrated and validated in rat liver using DNA arrays and several novel gene regulatory pathways have been identified including induction of markers that leads to toxicological manifestations (Gerhold et al., 2001, de Longueville et al., 2002). It is also well established that induction of the individual CYP families is often indicative of the exposure of specific class of chemicals, e.g., induction in the expression of CYP1 family is indicative of polycyclic aromatic hydrocarbon (PAH) exposure (Dey et al., 2001, Uppstad et al., 2010). Similarly CYP2B family is induced primarily by phenobarbital (PB) or PB-like drugs or chemicals while CYP2E1 is primarily induced by ethanol. Any alterations in the expression profiles of these CYPs can thus be use as an indicative of exposure to specific class of chemicals (Muñoz and Albores, 2010). In recent years there has been interest to develop assays that can be used to predict exposure and toxicity of environmental toxicants. Gene expression profiling in PBL could be particularly useful for predicting exposure as well as the mechanism of action of drugs & environmental chemicals (McHale et al., 2010). Peripheral blood lymphocytes (PBL) have been shown to express various members of CYP super-family (Raucy et al., 1997, Krovat et al., 2000, Furukawa et al., 2004). Previous studies from our laboratory and elsewhere have demonstrated similarities in the expression of PAH and ethanol metabolizing CYPs in rat blood lymphocytes and similarities in the regulation of these CYPs with the tissue enzymes (Dey et al., 2001, Dey et al., 2002, Dey et al., 2006, Saurabh et al., 2010, Sharma et al., 2012, Srivastava et al., 2012). Significant increase in the expression of CYP1A1 or CYP2E1 in freshly isolated blood lymphocytes isolated from patients suffering from lung cancer and that of CYP2E1in patients of alcoholic liver cirrhosis have suggested that blood lymphocyte CYP expression profiles could help in identifying individuals susceptible to lung cancer or alcohol-induced diseases (Shah et al., 2009, Khan et al., 2011). Expression of the subset of CYPs along with their respective transcription factors and transporters has also been demonstrated in PBL suggesting that CYP expression in PBL could be utilized in a variety of clinical studies (Nguyen et al., 2000, Siest et al., 2008). Gladkevich et al. (2005) have suggested that expression profile in PBL could be used for monitoring and possible treatment in internal diseases such as asthma, rheumatoid arthritis and systemic lupus erythematosus. However, validation of gene expression profiles of PBL is needed for predicting toxicity associated with environmental exposures.

The present study was aimed to develop a rapid bioassay using PBL as a surrogate to simultaneously monitor the mRNA expression of selective genes for predicting toxicity in individuals exposed to drugs and environmental chemicals. Attempts were therefore made to develop a Taqman Low Density Array (TLDA) based assay of selected genes involved in metabolic activation and detoxification processes using PBL isolated from adult rats treated with different inducers of DMEs such as PB or MC or ethanol. As compared to DNA array, which is time-consuming and labor intensive, TLDA is a RT-PCR based assay where like DNA array expression of selected genes can be monitored simultaneously and the assay being RT-PCR based requires no further validation.