Benzo(a)pyrene-induced cytotoxicity, cell proliferation, DNA damage, and altered gene expression profiles in HT-29 human colon cancer cells.

In the US alone, around 60,000 lives/year are lost to colon cancer. In order to study the mechanisms of colon carcinogenesis, in vitro model systems are required in addition to in vivo models. Towards this end, we have used the HT-29 colon cancer cells, cultured in Dulbecco's Modified Eagle Medium (DMEM), which were exposed to benzo(a)pyrene (BaP), a ubiquitous and prototypical environmental and dietary toxicant at 1, 10, 100 nM and 1, 5, 10, and 25 µM concentrations for 96 h. Post-BaP exposure, growth, cytotoxicity, apoptosis, and cell cycle changes were determined. The BaP metabolite concentrations in colon cells were identified and measured. Furthermore, the BaP biotransformation enzymes were studied at the protein and mRNA levels. The BaP exposure-induced damage to DNA was assessed by measuring the oxidative damage to DNA and the concentrations of BaP-DNA adducts. To determine the whole repertoire of genes that are up- or downregulated by BaP exposure, mRNA transcriptome analysis was conducted. There was a BaP exposure concentration (dose)-dependent decrease in cell growth, cytotoxicity, and modulation of the cell cycle in the treatment groups compared to untreated or dimethylsulfoxide (DMSO: vehicle for BaP)-treated categories. The phase I biotransformation enzymes, CYP1A1 and 1B1, showed BaP concentration-dependent expression. On the other hand, phase II enzymes did not exhibit any marked variation. Consistent with the expression of phase I enzymes, elevated concentrations of BaP metabolites were generated, contributing to the formation of DNA lesions and stable DNA adducts, which were also BaP concentration-dependent. In summary, our studies established that biotransformation of BaP contributes to cytotoxicity, proliferation of tumor cells, and alteration of gene expression by BaP. • Benzo(a)pyrene (BaP) is an environmental and dietary toxicant. • BaP causes cytotoxicity in cultured HT-29 colon cancer cells. • mRNA transcriptome analyses revealed that BaP impacts cell growth, cell cycle, biotransformation, and DNA damage.

Metabolism of benzo(a)pyrene by subcellular fractions of gastrointestinal (GI) tract and liver in Apc(Min) mouse model of colon cancer.

Given the fact that increased dietary intake of polycyclic aromatic hydrocarbons (PAHs; a family of environmental toxicants) leads to the formation and development of colon tumors, the ability of the gastrointestinal tract to process these compounds is important from the viewpoint of toxicity/carcinogenesis. Benzo(a)pyrene (BaP), a prototypical PAH compound is released into the environment from automobile exhausts, cigarette smoke, and industrial emissions. Additionally, considerable intake of BaP is expected in people who consume barbecued foods and a diet rich in saturated fat. In exposed animals, BaP becomes activated to potent metabolites that interfere with target organ function and as a consequence cause toxicity and cancer. Therefore, knowledge of BaP metabolism in the digestive system will be of importance in the management of cancers of the digestive tract. The objective of our study was to study the metabolism of BaP by subcellular fractions (nuclear, cytosolic, mitochondrial, and microsomal) of the gastrointestinal tract and liver. Subcellular fractions were isolated by differential centrifugation from the stomach, jejunum, colon, and liver tissues of Apc(Min) mice that received a subchronic dose of 25 µg/kg BaP. The fractions were incubated with 1 and 3 µM BaP. Subsequent to incubation, samples were extracted with ethyl acetate and analyzed for BaP metabolites by reverse-phase HPLC equipped with fluorescence detection. Among the different fractions tested, microsomal BaP metabolism was higher than the rest of the fractions in all the samples analyzed. Additionally, a BaP exposure concentration-dependent effect on metabolite levels generated by the subcellular fractions was recorded. The BaP metabolites identified were the following: BaP-9,10-diol; BaP-4,5-diol; BaP-7,8-diol; 9(OH) BaP; 3(OH) BaP; BaP-3,6-dione; and BaP-6,12-dione. While the diol group of metabolites was frequently detected, among diones, the 3,6 and 6,12-dione metabolites were infrequently detected. Among the diol metabolites, the preponderance of BaP-7,8-dihydrodiol is interesting, since this metabolite is a precursor to the DNA-reactive BaP-7,8-dihydrodiol epoxide (BPDE) that has been linked to BaP-induced cancer.

Tumor microsomal metabolism of the food toxicant, benzo(a)pyrene, in ApcMin mouse model of colon cancer.

The present study was conducted to investigate whether colon tumors were capable of metabolizing benzo(a)pyrene (BaP), and fluoranthene (FLA), two toxicants that belong to the polycyclic aromatic hydrocarbon family of compounds. Microsomes were isolated from the colon tumors of Apc( Min ) mice that received subchronic doses of 50 µg/kg BaP and incubated with either BaP or FLA (3 µM each) alone or in combination and appropriate control groups that received nothing. Subsequent to incubation, samples were extracted with ethyl acetate and analyzed for BaP and FLA metabolites by reverse-phase HPLC equipped with fluorescence detection. Microsomes from tumor tissues were found to metabolize BaP to a greater extent than those from the non-tumor tissues. The rate of BaP metabolism (picomoles of metabolite per minute per milligram of protein) was found to be more when microsomes from BaP-pretreated mice were exposed to BaP alone and FLA in combination with BaP, compared to controls. The microsomes from BaP-preexposed mice generated greater proportion of BaP 7,8-diol and BaP 3,6- and 6,12-diones compared to other experimental groups. Additionally, microsomes from BaP-pretreated mice produced greater proportion of FLA 2, 3-diol and 2, 3 D FLA when microsomes were incubated with FLA alone or a combination of BaP and FLA. Our studies revealed that the tumor microsomes were competent to metabolize BaP and FLA either singly or in combination. The biotransformation of BaP and FLA as a consequence of prior and simultaneous exposure to BaP may influence the growth of tumors. Our findings may have relevance to human long-term dietary intake of these toxicants and the consequent acceleration of the colon carcinogenesis process.

Polycyclic aromatic hydrocarbons and digestive tract cancers: a perspective.

Cancers of the colon are most common in the Western world. In majority of these cases, there is no familial history and sporadic gene damage seems to play an important role in the development of tumors in the colon. Studies have shown that environmental factors, especially diet, play an important role in susceptibility to gastrointestinal (GI) tract cancers. Consequently, environmental chemicals that contaminate food or diet during preparation become important in the development of GI cancers. Polycyclic aromatic hydrocarbons (PAHs) are one such family of ubiquitous environmental toxicants. These pollutants enter the human body through consumption of contaminated food, drinking water, inhalation of cigarette smoke, automobile exhausts, and contaminated air from occupational settings. Among these pathways, dietary intake of PAHs constitutes a major source of exposure in humans. Although many reviews and books on PAHs and their ability to cause toxicity and breast or lung cancer have been published, aspects on contribution of diet, smoking and other factors toward development of digestive tract cancers, and strategies to assess risk from exposure to PAHs have received much less attention. This review, therefore, focuses on dietary intake of PAHs in humans, animal models, and cell cultures used for GI cancer studies along with epidemiological findings. Bioavailability and biotransformation processes, which influence the disposition of PAHs in body and the underlying causative mechanisms of GI cancers, are also discussed. The existing data gaps and scope for future studies is also emphasized. This information is expected to stimulate research on mechanisms of sporadic GI cancers caused by exposure to environmental carcinogens.

Comparative evaluation of different cell lysis and extraction methods for studying benzo(a)pyrene metabolism in HT-29 colon cancer cell cultures.

Lysis and extraction of cells are essential sample processing steps for investigations pertaining to metabolism of xenobiotics in cell culture studies. Of particular importance to these procedures are maintaining high lysis efficiency and analyte integrity as they influence the qualitative and quantitative distribution of drug and toxicant metabolites in the intra- and extracellular milieus. In this study we have compared the efficiency of different procedures viz. homogenization, sonication, bead beating, and molecular grinding resin treatment for disruption of HT-29 colon cells exposed to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound and a suspected colon carcinogen. Also, we have evaluated the efficiency of various procedures for extracting BaP parent compound/metabolites from colon cells and culture media prior to High Performance Liquid Chromatography (HPLC) analyses. The extraction procedures include solid phase extraction, solid-supported liquid- liquid extraction, liquid-liquid extraction, and homogeneous liquid- liquid extraction. Our findings showed that bead-beating in combination with detergent treatment of cell pellet coupled with liquid-liquid extraction yielded greater concentrations of BaP metabolites compared to the other methods employed. Our method optimization strategy revealed that disruption of HT-29 colon cells by a combination of mechanical and chemical lysis followed by liquid-liquid extraction is efficient and robust enough for analyzing BaP metabolites from cell culture studies.